J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 DOI 10.1007/s10545-013-9633-z

ABSTRACTS

ICIEM Abstracts

Workshop 1: Neurotransmitter defects

W-001 Impact of sapropterin dihydrochloride on mood and motor function in autosomal dominant DOPA-responsive dystonia (AD-DRD): a pilot study Newcomb TM1, Himle MB2, Smart AL1, Sharp AG1, Trussell DV1, Thulin PC1, Swoboda KJ1 Univ Utah Dept Neurology, Salt Lake City, United States; 2Univ Utah Dept Psychology, Salt Lake City, United States

1

Background: GTP cyclohydrolase deficiency results in diminished central nervous system BH4. Monotherapy with L-dopa/carbidopa treats dystonia, but not disease-related mood disorders, which are significantly under-reported. Design/Methods: Open-label dose-ranging study of sapropterin dihydrochloride in 12 patients with AD-DRD treated with 20-40 mg/kg/day for 12 weeks, +/- optional 12 week extension. Mood was assessed at baseline, 8, 12, +/- 24 weeks via the Hamilton Anxiety Questionnaire (HAM-A) and Hamilton Depression Rating Scale (HAM-D), with lumbar puncture performed to evaluate oral dosing on CSF BH4, HVA and 5-HIAA levels. Results: Twelve patients were enrolled. One subject was excluded for inappropriate diagnosis and two subjects withdrew. Nine subjects completed 12 weeks, and six subjects continued (to date) through 24 weeks. We have results on all subjects through 8 weeks. Mean HAM-D scores declined indicating improvement from moderate to mild depression; mean HAM-A scores decreased with uncertain significance. Mean CSF BH4 levels more than doubled, with 8 subjects reaching levels at least into the low-normal range. Conclusions: It appears oral sapropterin dihydrochloride in doses of 2030 mg/kg/day increases CSF BH4 levels in patients with AD-DRD. Improvement in HAM-D scores is encouraging, and indicates this medication may be a valuable adjunct treatment in patients with AD-DRD. Conflict of Interest declared. W-002 A novel Tyrosine Hydroxylase Knock-In (TH-KI) mouse as a model for brain catecholamine deficiency Korner G1, Noain D2, Scherer T1, Ying M3, Baumann C2, Martinez A3, Thöny B1 1

Div. of Metabolism, Dept. of Pediatrics, Zurich, Switzerland; 2Dept. of Neurology, University Hospital, Zurich, Switzerland; 3Dept. of Biomedicine, University Bergen, Bergen, Norway Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the synthesis of dopamine and a marker for dopaminergic neurons. Mutations in the

TH gene are associated to the rare neuropsychiatric recessive disorder TH deficiency (THD), which manifests as Dopa responsive dystonia (DRD), parkinsonism in infancy or progressive encephalopathy. DRD or THD have been treated with L-Dopa supplementation, which has secondary effects and low long-term effectiveness, since patients develop L-Dopa induced dyskinesia or intolerance. The most recurrent mutation in THD is TH1-p.R202H which is associated to a severe THD phenotype and a lower responsiveness to L-Dopa treatment. We have generated a constitutive mouse model called "Th-ki" expressing the Th-p.R203H mutation (equivalent to TH1-p.R202H). Whole brain extracts of homozygous Th-ki/ki mice showed in the brain normal serotonin and 5-hydroxyindoleacetic acid levels, but significantly and progressively reduced catecholamines, including low L-dopa, dopamine, norepinephrine and homovanillic acid. Preliminary behavior studies revealed that Th-ki/ki mice concomitantly have progressive locomotor and movement problems. Our studies with the Th-ki mouse model should elucidate the molecular mechanisms in THD, and to provide a platform for evaluation of novel therapeutics, including L-Dopa, BH4 and pharmachaperon treatments. W-003 A sibling study in attenuated non-ketotic hyperglycinemia provides direct evidence of therapeutic treatment effect of benzoate and dextromethorphan on neurocognitive outcome Swanson MA1, Bjoraker K1, Szerlong HJ1, Tan ES2, Christodoulou J2, Fergeson MA3, Dyack S4, Collard R1, Friederich MW1, Van Hove JLK1 1

Dept Pediatrics, Univ Colorado, Aurora, Colorado, United States; Western Sydney Genetics Prg. Westmead, Sydney, Australia; 3 Dept Pediatrics, Oklahoma Univ, Oklahoma City, Oklahoma, United States; 4Div Med Genetics, IWK Health Centre, Halifax, Nova Scotia, Canada 2

Treatment of non-ketotic hyperglycinemia consists of lowering glycine with benzoate and inhibiting overstimulated NMDA receptors with dextromethorphan. Lack of benefit in severely affected patients casted doubt on its therapeutic role. The large genetic heterogeneity of NKH impeded evaluation of treatment impact. We evaluate 4 sibling pairs with attenuated NKH where the oldest child was treated late, and the second child was treated from birth with effective dosing. All patients had at least one GLDC mutation conferring residual activity: A202V, A802V, and A389V having 18.5%, 18%, and 12% activity. Comparing neurocognitive outcome in siblings, the DQ increased from 37 to 51, from 10 to 48, and from 16 to 30, and in the first two families the Vineland adaptive score increased from < 45 to 52, from 40 to 67. The first patients had seizures for 4 years, 13 months, and 6 years, whereas the second child was seizure free or had rare seizures. The first child from the fourth family, homozygous C291Y, developed intractable seizures dying at 13 months. The second child has been seizure free with a DQ of around 75. Using siblings to control for genetic heterogeneity, we documented clear treatment effects on neurocognitive outcome in attenuated NKH.

S92 W-004 Improved motor function with 5-hydroxytryptophan in a family with systemic serotonin deficiency, hemiplegic migraines and neurodegenerative course Horvath GA1, Ye R2, Stockler-Ipsiroglu S1, Waters PJ3, Blakely RD2, Coulter-Mackie M4

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 was generally well tolerated. All except 4 adverse events were reported as unrelated to sapropterin. 3 occurred in the same pregnancy, were mild and reported as possibly related; sapropterin was not discontinued. The fourth was the pre-term birth. Mean (range) blood phenylalanine levels were 222 (121–681) μmol/L in women who completed pregnancies while on sapropterin, 775 (664–841) μmol/L in women who had miscarriages while on sapropterin, and 267 (84 – 614) μmol/L in the 3 women who completed pregnancies off sapropterin. Conflict of Interest declared.

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Biochem Dis, BC Children's Hospital, Vancouver, Canada; 2Pharmacol Psych, Vanderbilt Univ, Nashville, United States; 3Biochem Lab, Univ Sherbrooke, Sherbrooke, Canada; 4Pediatrics, UBC, Vancouver, Canada

Background: Serotonin's role is multiple: controlling mood and sleep, modifying vascular resistance, modulating spinal segmental reflexes and nociception amongst others. Case report: We present a family with three affected siblings, presenting with hemiplegic migraines, spinal cord atrophy, progressive lower limb weakness and spasticity, who were found to have profoundly low CSF 5HIAA levels (25, 14, 18 nmol/L, ref 87-189) and low platelet serotonin levels. Their motor function and strength greatly improved with 5-hydroxytrypotphan(5HTP)/carbidopa treatment. Platelet serotonin transporter function was diminished, and cytoskeletal aggregates were proven to keep membrane proteins in the non-soluble fractions. Whole exome sequencing revealed a novel variant in SRRM2 gene, supporting alternative splicing of many target genes. This protein is a component of the spliceosome and has multiple functions in the splicing process. It also plays an important role in pre-mRNA splicing and has a role in cell migration. Alternative splicing increases the complexity of mammalian transcriptomes since nearly all mammalian genes express multiple pre-mRNA isoforms. Full transcriptome analysis comparing RNA expression in two affected and one unaffected sibling revealed multiple differences in levels of RNA expression and splicing. Conclusion: Improvement of motor function with 5HTP/carbidopa proves yet another important role of serotonin as a signaling molecule.

Workshop 2: Phenylketonuria. Recent trends in therapy W-005 Use of sapropterin dihydrochloride in 20 pregnant women with phenylketonuria prior to or during pregnancy: an interim report of the PKU moms sub-registry Hillman R1, Peck D1, Grange DK2, Fong CT3, Burton BK4, Vockley J5, Yano S6, Violet S7, Cohen-Pfeffer J7 1

W-006 Effective minicircle-based naked-DNA gene therapy for hepatic diseases Viecelli HM1, Harbottle RP2, Wong SP2, Schlegel A3, Chuah M4, Vandendressche T4, Harding CO5, Thony B1 1 Dept Pediatrics, Univ Zurich, Zurich, Switzerland; 2Nat Heart and Lung Int, Imp Col London, London, United Kingdom; 3Swiss HPB Transpl Cent, Uni Hosp Zurich, Zurich, Switzerland; 4Free Uni Brussels, Div Gene Ther Reg Med, Brussels, Belgium; 5Dept Mol Med Gen Ped, OHSU, Portland, United States

Host immune response to viral vectors, persistence of non-integrating vectors, and sustained transgene expression are among the major limitations in gene therapy. To overcome these hurdles, we successfully used non-viral minicircle (MC)-DNA vectors devoid of bacterial sequences for long-term treatment of murine phenylketonuria (PKU), a mouse model for a genetic liver defect. A MC-DNA vector expressing the phenylalanine hydroxylase (PAH) under the control of a robust de novo liver-specific hybrid promoter-enhancer fragment (HYPERON) was constructed and delivered to the liver by a single hydrodynamic tail vein injection. While the parental plasmid did not result in any phenylalanine clearance, the corresponding MC-DNA vector normalized blood phenylalanine concomitant with reversion of hypopigmentation in a dose dependent manner up to at least six months. Upon analyzing the livers from sacrificed treated mice at different time points, we found MC vector persistence in episomal state concomitant with sustained transgene expression and stable hepatic PAH enzyme activity. Moreover, 14-20% of all hepatocytes expressed PAH, and expression was observed only in the liver and predominately in perivenous region while there was no transgene expression in periportal area. In conclusion, MC technology offers a favorable safety profile and has the potential for gene-therapeutic treatment of liver diseases. W-007

University of Missouri, Columbus, United States; 2Washington University, Columbus, United States; 3University of Rochester Medical Center, Rochester, United States; 4Ann and Robert H Lurie Children's Hospit, Chicago, United States; 5University of Pittsburgh School of Medic, Pittsburg, United States; 6 University of Southern California, Los Angeles, United States; 7BioMarin Pharmaceutical Inc., Novato, United States

Towards an alternative therapy for PKU: in vitro and in vivo assessment of a genetically modified probiotic (lactococcus lactis nz9000) expressing Phenylalanine Ammonia Lyase (PAL)

Twenty-five pregnancies from 20 women, ages 18-41 years, were enrolled in the PKUMOMS sub-registry of PKUDOS. Of these, 19 pregnancies (76%) occurred while women were taking sapropterin dihydrochloride; median dose 20 mg/kg/day. In the remaining 6 pregnancies, women were exposed to sapropterin >1 year prior to LMP date. 11/19 pregnancies on sapropterin were delivered at term, one delivered at 35 weeks gestation, 4 are ongoing, and 3 ended in first trimester spontaneous abortion (15.7%). Two of these three occurred in women with risk factors, both had prior history of miscarriages and were AMA (36 and 41 years). Of the 6 pregnancies off sapropterin; 1 ended in miscarriage (16.7%); 3 were delivered at term, and two are ongoing. Sapropterin

Met Res Unit, Kids Res Institute, Sydney, Australia; 2NSW Biochem Gen,The Child Hosp at West, Sydney, Australia; 3NSW NBS Prog, The Child Hosp at West, Sydney, Australia; 4Gen Ther Res Unit, The Child Hosp West, Sydney, Australia; 5Disc Paed & Child Health, Univ Sydney, Sydney, Australia

AL Hafid N1,5, Tong XZ1, Carpenter K2, Gold W1, Wiley V3, Alexander IE4, Christodoulou J1 1

Background: Phenylketonuria (PKU), a defect of phenylalanine hydroxylase (PAH), may cause severe mental retardation if untreated. Current treatment for most patients is a phenylalanine (Phe)-restricted diet. Dietary compliance is a major issue due to palatability of the diet. There is a critical need to develop novel approaches to therapy.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Objective: To evaluate a genetically modified probiotic that produces functional phenylalanine ammonia-lyase (PAL) for its capacity to catabolise phenylalanine in the gut prior to absorption. Methods: 1) The GM probiotic was exposed to gastric and small intestinal milieux in vitro, and the survival rate was examined using flow cytometry. 2) Gut phenylalanine absorption of probiotic-fed PKU and wild-type mice was measured using an in vivo stable isotope approach. Results: Probiotic survival was 90% after exposure to conditions mimicking the stomach, but decreased significantly after exposure to the duodenal environment. Following a labelled free Phenylalanine load, mice treated with the probiotic showed significantly lower labelled Phe blood levels than untreated mice. Conclusion: Our data provide initial proof of principle that the GM probiotic may be a viable alternative approach to therapy for PKU. We are currently investigating the effect of the probiotic on labelled phenylalanine contained within whole proteins in vivo. Conflict of Interest declared.

W-008 Preservation of the biological function and structural properties of human phenylalanine hydroxylase: a step towards an effective enzyme replacement therapy for phenylketonuria?

S93 Background: Coronary artery stenosis is a known fatal manifestation of mucopolysaccharidosis type I (MPS I) in patients both untreated and treated with hematopoietic stem cell transplantation or intravenous enzyme replacement therapy (ERT). Few studies have examined effects of MPS upon arterial gene expression to understand its pathogenesis. Methods: Gene expression in ascending and descending aortas from four non-tolerized, non-IV ERT-treated 19 month-old MPS I dogs was compared with expression from three normal, age-matched dog aortas. Data were analyzed using R and whole genome network correlation analysis. Genes were further categorized based on module-trait relationships. Results: Gene families with >five-fold increased expression involved lysosomal function (61 genes), proteasome function (27), and antigen presentation/cytokines (38). Downregulated gene classes included cellular adhesion/cytoskeletal (42) and calcium regulation (6). Toll-like receptor 4 (1.8-fold overexpression) and clusterin (7-fold overexpression) were confirmed via Western Blotting and in situ immunohistochemistry. Overexpressed clusterin was specifically located in aortic plaques. Conclusions: Overexpression of lysosomal and proteasomal-related genes are expected responses to cellular stress induced by lysosomal storage in MPS I. Upregulation of immunity-related genes (IL1,2,6,TLR4) implicates glycosaminoglycan-induced inflammation via TLR4 in pathogenesis of MPS arterial disease, for which clusterin represents a potential biomarker. Downregulated cellular adhesion genes merit additional investigation.

Lino PR1, Leandro J1, Amaro MP1, Almeida AJ2, Leandro P1 1

Met&Gen, iMed.UL, Fac Pharm, Univ Lisbon, Lisbon, Portugal; NanoDDS, iMed.UL, Fac Pharm, Univ Lisbon, Lisbon, Portugal

W-010

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The inborn error of metabolism phenylketonuria (PKU; OMIM# 261600) is caused by a dysfunction of the liver enzyme phenylalanine hydroxylase (hPAH; EC 1.14.16.1). If untreated, PKU patients present neurological damages due to the increased levels of phenylalanine (L-Phe). A life-long dietary restriction of L-Phe remains the most effective treatment for PKU, but compliance to this lifetime restriction diet is very difficult, and most of the patients would essentially not adhere to therapy. Some PKU patients respond to pterin cofactor (BH4/sapropterin) by a reduction in blood L-Phe concentration, a novel therapeutic approach mainly for mild forms of the disease. The search for alternative therapies includes enzyme substitution therapy with non-mammalian phenylalanine ammonia lyase enzyme and gene therapy. The ideal therapy would be enzyme replacement therapy with native human phenylalanine hydroxylase (hPAH). However, the intrinsic instability of hPAH and its complex structure/regulation have precluded its formulation. We reported here a lyophilization process of the human enzyme in the presence of several additives (sugars and aminoacids). We found that using a specific additive in the lyophilization process, we were able to prevent protein aggregation, to preserve enzymatic activity (100%), folding activation, and secondary and quaternary structure after protein reconstitution.

Workshop 3: Lysosomal diseases W-009 Aortic gene expression from the canine model of MPS I identifies upregulation of genes related to antigen presentation and inflammatory cytokines, and downregulation of cellular adhesion and cytoskeletal genes Wang R1, Vera M2, Khalid O3, Ellinwood NM4, Schwartz P3, Dickson P2 Div Metab Dis, CHOC Children's, Orange, United States; 2Div Med Genet, LA Biomed at Harbor, Torrance, United States; 3NHNSCR, CHOC Research Institute, Orange, United States; 4Col Agricult Cell Sci, Iowa State Univ, Ames, United States 1

Combined pharmacological chaperone therapy and enzyme replacement therapy in patients with Pompe disease Parenti G1, la Marca G2, Fecarotta S1, Donati MA3, Morandi LO4, Ravaglia S5, Danesino C6, Moglia A4, Ombrone D2, Della Casa R1, Ascione S1, Rosa M1, Carbone MT7, Concolino D8, Agovino T3, Porto C9, Rossi B9, Andria G1 1

Dip Sci Med Trasl, Federico II Uni, Naples, Italy; 2Div Clin Pharm, Osp Meyer, Firenze, Italy; 3UO Metab Union, Osp Meyer, Firenze, Italy; 4UO Musc Neuroimmun Pat, IRCCS Besta, Milano, Italy; 5Institute of Neurology C. Mondino, Pavia, Italy; 6Dep Public Health, University of Pavia, Pavia, Italy; 7Ospedale SS Annunziata, Naples, Italy; 8Dep Pediat "Magna Grecia", Catanzaro, Italy; 9TIGEM, Naples, Italy Background: Pompe disease (PD) is a metabolic myopathy, due to the deficiency of alpha-glucosidase (GAA) and characterized by intralysosomal glycogen storage. Enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) is approved for the treatment of PD, but its efficacy on the skeletal myopathy is variable. Objectives: To evaluate whether the combined treatment with ERT and a pharmacological chaperone, NB-DNJ, is more effective in correcting the enzyme deficiency in PD patients. Patients. Thirteen PD patients with various clinical phenotypes were enrolled in the study. Methods: GAA activity was assayed by tandem mass spectrometry in dried blood spots 24 hrs after ERT (T1) and every 48 hrs for 14 days, or every 12 hours after ERT for 3 days. Results: In all patients GAA activity at T1 with the combination treatment was higher than with ERT alone. The increase in activity ranged between 4- and 12-fold and was statistically significant (P<0.05) in 10 patients. GAA activity at T1 of the whole patients population with the combined treatment was 6.9 times higher than with ERT alone (P<0.00001). Conclusion: Our results suggest that the combined therapeutic approach can enhance the activity of rhGAA. Study supported by the Telethon Foundation (Grant GUP09017 to GA)

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Workshop 4: Fatty acid oxidations disorders and metabolism of ketone bodies W-011 Functional effects and conformational studies of 18 heterologous expressed medium-chain acyl-CoA dehydrogenase (MCAD) variants

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 mean number of events was observed compared to the year prior to treatment in these 6 patients. Rhabdomyolysis precipitated by infectious disease was the most frequently occurring event. A 66% reduction in the mean number of rhabdomyolysis events per year from 1.94 to 0.66 was observed. The data suggests that long-term triheptanoin treatment reduces the frequency of major medical events and mortality. Conflict of Interest declared.

Sturm M1, Koster K-L1, Herebian D1, Spiekerkoetter U2 1

Dept Gen Ped, Univ Child Hosp, Düsseldorf, Germany; 2Dept Gen Ped, Univ Child Hosp, Freiburg, Germany MCAD deficiency is the most common disorder of mitochondrial fatty acid oxidation. Since implementation of newborn screening, several new mutations with unknown clinical relevance have been identified. The functional analysis of newly identified mutations is necessary. Because genotypes of patients are predominantly compound heterozygous, C8-CoA oxidation in lymphocytes is inappropriate to determine the function of single mutations. In this study, 18 MCAD mutant proteins have been produced by sitedirected mutagenesis and expressed with and without the chaperones GroES and GroEL in E. coli. Afterwards octanoyl-CoA oxidation rates have been determined on both conditions. Three mutations (c.127G>A, c.92G>A, c.199T>C) presented with significantly higher (p<0.05) residual activities (47%, 44% and 22%) than the other 15 mutations (<10%). Co-overexpression with GroES and GroEL increased the residual activity of most mutants significantly while c.92G>A, c.127G>A, and wild type protein remained unaffected. In conclusion, three mutations could be identified as mild and possibly not disease-causing. For the prevalent mutation c.199T>C no clinical symptoms have been reported. Residual activities of c.92G>A and c.127G>A were even twice as high. Since chaperones did not improve residual activities of either c.92G>A or c.127G>A, both variants may still result in an appropriate protein folding and have no clinical relevance. W-012 The impact of triheptanoin treatment on the incidence of major medical events in patients with long-chain fatty acid oxidation defects (FAOD)

W-013 The use of 3-hydroxybutyrate in patients with fat oxidation disorders Dalkeith T1, Ellaway CJ1, Thompson S1, Dennison B1, Matar W2, Wilcken B1, Bhattacharya K1 1 Children's Hospital at Westmead, Sydney, Australia; 2The St. George Hospital, Sydney, Australia

Severe fat-oxidation disorders are associated with catastrophic illnesses including encephalopathy, liver dysfunction, rhabdomyolysis, and cardiac dysfunction. Insufficient ketone-body synthesis may be a contributing factor. Seven patients were treated with supplemental D-L-3-hydroxybutyrate (300–900mg/kg/day). Five commenced therapy in ICU. Underlying diagnoses included multiple acyl-CoA-dehydrogenase deficiency (MADD) (n=4), HMG CoA-Lyase deficiency (n=2) and carnitine-acylcarnitine translocase deficiency (n=1). Patients with MADD commenced treatment at 7 days (for hyperammonaemic encephalopathy), 3 months (following cardiac arrest), 5 months (for heart failure) and 5 months (for profound skeletal myopathy). All survived initial therapy but the first two died at 9 and 13 months, respectively, following recurrent acute life-threatening episodes. One patient with HMG CoA-Lyase deficiency commenced treatment empirically aged 3 months; the other at 16 years following acute encephalopathy, liver dysfunction and hyperammonaemia. Despite cerebellar tonsillar herniation, the only sequelae was cortical blindness. The patient with carnitine-acylcarnitine translocase deficiency presented with neonatal hypoglycaemic hyperammonaemic encephalopathy with seizures and cardiac dysfunction (fractional shortening 13%). Ninety-six hours of enteral ketones and intravenous dextrose led to complete resolution of cardiac findings. She had normal development at 3 years with improved brain MRI. Therapeutic use of ketones in hypo-ketotic disorders may resolve acute multi-organ dysfunction before institution of definitive therapy.

Vockley J1, DeWard SJ1, McCracken E1, Hsu K2 , Skrinar A2, Kakkis E2, Roe C3 W-014 1

University of Pittsburgh, Pittsburgh, PA, United States; 2Ultragenyx Pharmaceutical, Inc, Novato, CA, United States; 3Baylor Research Institute, Dallas, TX, United States Triheptanoin has been studied as an anaplerotic medium chain triglyceride therapy for FAOD and other diseases for more than a decade. A protocol-driven retrospective medical chart review was conducted for 24 subjects (12-VLCAD, 5-LCHAD, 4-CPT II, 2-TFP and 1-CACT) treated with triheptanoin for up to 13 years under a compassionate use protocol. The objective was to quantify major medical events prior to and after the initiation of triheptanoin treatment. Major medical events included hospitalizations for FAOD complications, such as rhabdomyolysis, hypoglycemia and cardiac disease. Data collected included precipitating events, total hospital days, CK levels and treatments administered. Six severely affected subjects experienced a 53% reduction in the mean number of events per year from 4.3 to 2.0 over a treatment period of 0.6 to 13.25 years. After 1 year of triheptanoin treatment, a 37% reduction in the

Response to steroid therapy in patients with long chain fatty acid oxidation disorders Arnold GL1 , Sanchez-Valle A2 , Vockley J1 1

Univ Pittsburgh Med Ctr, Pittsburgh, United States; 2Univ S Florida Morsani Coll of Med, Tampa, United States Background: Preliminary studies in mice and humans with VLCAD deficiency (VLCADD) demonstrate increases in some cytokines, suggesting an inflammatory component to the phenotype. Case Reports: Preliminary mouse and patient data suggest an unexpected atypical inflammatory response independent of glucose control in patients with VLCADD, with elevation of IL6, GM-CSF, INFγ, MCP-1, and TDFα in blood. Four patients with long chain FAO defects having repeated episodes of rhabdomyolysis in spite of normal blood glucose levels were given an oral prednisone burst of ~1 mg/kg/day to

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 determine if suppression of inflammation would ameliorate symptoms. Three of the patients had VLCADD and one had trifunctional protein deficiency. Age range was from 1.5-16 years. All patients experienced reduced CPK levels and fewer or no subsequent episodes of rhabdomyolysis. Two patients had a mild rebound in CPK following completion of steroids prior to stabilizing. No adverse clinical effects occurred. Conclusion: These preliminary data suggest that immune modulation is safe and may be beneficial for patients with long chain FAOD disorders and recurrent rhabdomyolysis, providing rationale for a formal clinical trial.

Workshop 5: Outcome of patients detected through expanded newborn screening W-015 Living with an inborn error of metabolism detected by newborn screening: parents’ perspectives on child development and impact on family life

S95 Urine Screening Programme. Allele frequency studies showed that CMAMMA is under-recognised. Its clinical significance, if any, requires clarification. Patients/Results: We have identified an additional 6 unrelated Quebec probands (aged 2-22 years) with characteristic biochemical profiles: persistent elevation of MMA and modest elevation of malonic acid (MMA/MA > 5) in urine and blood, with normal plasma homocysteine, B12 and acylcarnitines. Diagnosis was confirmed by identification of ACSF3 mutations in 4 (analysis pending for 2). All originally came to attention via urine newborn screening of MMA. None have clinical symptoms or signs suggestive of a metabolic disorder. Their ethnic backgrounds and mutations are heterogeneous. We will present detailed biochemical, molecular and clinical data. Conclusion/Discussion: Our experience suggests that ACSF3 deficiency is probably benign, though could be a risk factor for disease in a small minority. Natural history studies are ongoing. Unbiased ascertainment via urine newborn screening provides a unique perspective on this biochemical disorder.

W-017 Gramer G1, Haege G1, Glahn EM1, Hoffmann GF1, Lindner M1, Burgard P1 1

Div Metab Dis, Univ Child Hosp, Heidelberg, Germany

Background: Newborn screening is regarded as highly successful by health professionals. Little is known about parents' perspectives on child development and social impact on families. Methods: Parents of 187 patients with metabolic disorders detected by newborn screening rated child development, expectations for the future and burdens on child/family. Results: In 26.2% of patients, parents perceived delays in global development and/or in specific developmental domains. Parents expected normal future development in 95.7%, and an independent adult life for their child in 94.6%. Comparison with psychometric test results showed that parents of children with cognitive impairments tended to overrate their child's abilities. Mild/medium burden posed on the family (child) by the metabolic disorder was stated by 56.1% (48.9%) of parents, severe/very severe burden by 19.3% (8.6%). One third of families reported financial burdens. Dietary treatment and diagnoses with risk for metabolic decompensation despite treatment were associated with higher perceived burden. Disorders rated as potentially very burdensome by experts were not rated accordingly by parents, demonstrating different perspectives of professionals and parents. Conclusion: Although newborn screening leads to favourable physical and cognitive outcome, living with a metabolic disorder may cause considerable stress on families, emphasizing the need for comprehensive multidisciplinary care.

W-016 Combined Malonic and Methylmalonic Aciduria (CMAMMA) due to deficiency of ACSF3: the Quebec experience Waters PJ1, Lévesque S1, Clarke JTR1, Auray-Blais C1, Alfares A2, Braverman N2 CHUS / Université de Sherbrooke, Sherbrooke, Canada; 2McGill Univ / Montreal Children's Hosp, Montreal, Canada

1

Background: CMAMMA due to ACSF3 deficiency was identified in 2011 by two independent groups. One described 8 unrelated patients investigated because of 'symptoms suggestive of an intermediary metabolic disorder'. The other described 2 asymptomatic children, with elevated urine methylmalonic acid (MMA) identified by the Quebec Provincial Newborn

Glutaric Aciduria Type 1 (GA-1) – California newborn screening (NBS) program experience on detection, with and without derivatization, and long term clinical outcome Kanungo S1,2, Feuchtbaum L3, Sermba L2, Lorey F3, Abdenur JE2,4 1

UCLA Intercampus Genetics Training Prgm, Los Angeles, United States; 2CHOC Children's, Orange, United States; 3California Department of Public Health, Richmond, United States; 4University of California Irvine, Irvine, United States GA-1 is a potentially devastating disease detected on NBS by measuring acylcarnitines with MS/MS; however false negative results have been reported. Acylcarnitines can be measured as their butyl esters (derivatized) or without prior derivatization (underivatized). In July 2005, California began screening for GA-1 measuring derivatized glutarylcarnitine, C5DC(D). In December 2009, the methodology changed to the underivatized method C5DC(U). We reviewed the GA-1 NBS data from July 2005 to December 2012, and clinical follow-up reported information of 44 true GA-1 cases from 2007 to 2012. 2,435,199 and 1,520,452 newborns were screened by the C5DC(D) and the C5DC(U) method, respectively. The prevalence went from 1:93,661 to 1:89,438, positive predictive value was 0.95 and 0.96, and the false positive rate was 0.02 and 0.03 respectively. No false negatives have been reported. Demographic distribution was similar in the 2 groups and comparable to the general population. Out of the true positives, biochemical confirmatory data was available in 45% and molecular in 22%.Review of follow-up outcome data did not find macrocephaly. Hospitalizations as well as ER visits due to oral intolerance and dehydration seemed to be more common earlier in life. No significant difference was found in the detection of GA-1 using C5DC(D) versus C5DC(U). W-018 Fatty acid beta-oxidation disorders: acute decompensations in the expanded newborn screening era Jotta R1, Janeiro P1, Costa C1, Ramos R2, Tavares de Almeida I2, Vilarinho L3, Gaspar A1 1

Pediatric Depart, Hosp Santa Maria, CHLN, Lisbon, Portugal; 2Faculty of Pharmacy, Univ of Lisbon, Lisbon, Portugal; 3Genet Depart, National Inst Health, INSA, Oporto, Portugal

S96 Introduction: Fatty acid ß-oxidation disorders (FAO-disorders) are inherited disorders with a wide variety of symptoms which are not always evident when the patient is between episodes of metabolic crises. Implementation of expanded newborn screening (ENS) may account to prevent potentially life threatening events. Aim: Characterization of FAO-disorders acute decompensations. Methods: Observational study. Review FAO-disorders diagnosed by ENS since 2006. Demographic data, obstetric/neonatal background and acute decompensations were analyzed. Results: Sixty-seven clinical charts were reviewed, 50-MCAD, 5-CUD, 3-CPTI/CPTII, 5-VLCAD, 4-MADD. The median age of ENS was 6.33 days (SD+/-3.9) and treatment started at 21.39 days (SD+/-10.3). 71.6% of the patients are asymptomatic, 38.6% had poor compliance to therapeutic measures. Eighteen patients had acute decompensations (1-CPT1, 1-CPT2, 3-MADD, 13-MCAD) which were associated with vomiting in 44,4% of the patients. 33,3% developed symptoms previously to diagnosis by ENS. Severe clinical manifestations included obnubilation, Reye syndrome, cardiac failure and two sudden deaths (1-MCAD, 1-MADD). Discussion: Sudden deaths were not avoided besides precocity of treatment. Vomiting during acute illness, and the susceptibility of the neonatal period, were the main factors related to decompensation episodes. Despite early diagnosis in ENS era, noncompliance to treatment and follow-up, in silent diseases, seems to compromise the prognosis.

Workshop 6: The potential of model systems to understand inborn errors of metabolism W-019 Characterization of an ACAD10 deficient mouse model: pathological and biochemical analyses Kormanik K1, El Demellawy D2, Mohsen AW3, Karunanidhi A3, Reyes-Mugica M2, Vockley J3 1 Dept Human Genetics, Univ Pgh, GSPH, Pittsburgh, United States; 2 Dept Pathology, Univ Pgh School of Med, Pittsburgh, United States; 3Dept Pediatrics, Univ Pgh School of Med, Pittsburgh, United States Background: Acyl-CoA dehydrogenase 10 transcript (ACAD10) codes for 1059-aa peptide with a ~400-aa C-terminus domain homologous to members of the ACAD flavoenzymes family involved in oxidation of fatty acids and intermediates of amino acid metabolism. A polymorphism in the ACAD10 gene has been linked to obesity in Pima Indians, but its physiological role remains unknown. Objectives: To identify the role of ACAD10 in physiology and its link to diabetes. Methods: We have generated an ACAD10 deficient mouse and an anti-ACAD10 antibody to conduct pathological, biochemical, and molecular studies. Results: Deficient animals are viable and fertile, but accumulate excess abdominal adipose tissue. Pathological studies reveal that ACAD10 deficient mice manifest an early inflammatory liver process and secondary splenic extramedullary hematopoiesis. Although skeletal muscle is histologically normal, deficient mice have elevated creatine kinase when fasted indicative of rhabdomyolysis. Metabolomics analysis identifies elevated levels of malonylcarnitine, succinylcarnitine, methylmalonylcarnitine, glutaroylcarnitine, adipoylcarnitine in deficient mouse urine samples. Immunological studies reveals antigen detected in pancreas, lung and brain and localized to peroxisomes. RNAseq analysis reveals a broad spectrum of changes in gene expression including genes involved in the response to oxidative stress. Conclusions: We have made significant progress towards identifying the role of ACAD10 in physiology.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 W-020 Mice with IDH2(R140Q) knock-in mutation modeling human type II D-2-hydroxyglutaric aciduria Jin S1, Wang F1, Lin Z2, Silverman L1, Chen Y1, Lu Y2, Yang H1, Yen K1, Cang Y1, Su M1 1

Agios Pharmaceuticals Inc, Cambridge, United States; 2WuxiApptec Inc, Shanghai, China Background: A single amino acid substitution, R140Q, in isocitrate dehydrogenase 2 (IDH2) confers a novel enzymatic activity that converts α-ketoglutarate (αKG) to D-2-hydroxyglutarate (D-2-HG). This gain-of-function mutation has been identified in patients with type II D2-hydroxyglutaric aciduria (D-2-HGA), an ultra-rare and severe neurometabolic disorder that presents with a range of clinical findings, including seizures, hypotonia, developmental delay, cardiomyopathy, dysmophic features and early death. Material and Methods: A single amino acid replacement, R140Q, was introduced into IDH2 in mouse embryonic stem (ES) cell clones by homologous recombination. Correctly targeted ES cells were blastocyst-injected, and mice with germline mutation were identified and confirmed. 2HG levels, body weight, overall survival, and whole body pathology of IDH2(R140Q) mice were evaluated and compared to age-matched wildtype controls. Results: IDH2(R140Q) KI mice produce elevated levels of D-2-HG in all tissues tested, and exhibit multiple defects consistent with symptoms from D-2-HGA patients, including facial dysmorphism, early mortality. In addition, histologic brain and heart lesions are suggestive of central nervous system and cardiac dysfunction. Conclusion: The IDH2(R140Q) knock-in mouse model provides a valuable tool to elucidate the underlying disease mechanism of D-2HGA and to evaluate ways to mitigate the disease. Conflict of Interest declared.

Workshop 7: Metabolic leukodystrophies W-021 Natural history of X-linked adrenoleukodystrophy at the hospital for sick children and Toronto General Hospital: twenty-three-year experience Tran C1, Patel J1, Mamak E1, Hewson S1, Faghfoury H2, Raiman J1, Blaser S1, Clarke JTR1, Mercimek-Mahmutoglu SM1 1 Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 2Gen Med, Univ Health Network, Toronto, Canada

Background: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder associated with leukodystrophy, myeloneuropathy and adrenocortical insufficiency. Method: We retrospectively reviewed all patients diagnosed between 1989-2012. Information was entered into an excel database. Results: Forty-eight patients were included (14 female; 34 male). Thirteen patients had initial neurological presentation: 11 with childhood cerebral XALD (CC-ALD) and 2 with adrenomyeloneuropathy (AMN). Three patients had Addison disease only. Thirty-two patients had a positive family history and were asymptomatic at the time of the biochemical diagnosis. During follow-up of these 32 asymptomatic patients: 9 developed CCALD, 6 developed Addison disease, 2 developed AMN; 5 remained asymptomatic (current age 7-56 years); 10 had no follow-up. CC-ALD patients had significantly higher mean C26:0 levels compared to other phenotypes (p<0.05). The Moser-Raymond Severity Scoring System was significantly higher in patients with high C26:0 levels (correlation coefficient 0.477; p<0.05). Loes Score was between 1-5 in 7 and between 10-20 in 12 CC-ALD patients at the time of diagnosis.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Conclusion: This is a natural history of X-ALD phenotype varying from severe CC-ALD to asymptomatic adults in the largest metabolic center in Canada. CC-ALD was the most common phenotype in our cohort. The C26:0 levels correlated with disease severity and cerebral manifestation. W-022 Atypical alexander disease associated with a novel familial mutation diagnosed through whole-exome sequencing (WES): clinical, neuroimaging and molecular characterization

S97 patients. Treating X-ALD mice with resveratrol, a SIRT1 activator, normalises mitochondria number, preserves energetic homeostasis including NADH, ATP levels, and pyruvate kinase activity, and most importantly, halts axonal degeneration and related motor disability. These results link mitochondria depletion and energetic failure to axonal damage. Further, we underscore the Sirt1/PGCalpha axis as a pivotal therapeutic target for neurodegenerative disease. W-024

Iglesias A1

Toward newborn screening for Cerebrotendinous Xanthomatosis

1

DeBarber AE1, Steiner RD2

Div Med Genet, Columbia Univ Med Center, New York, United States

Mutations in the GFAP gene cause Alexander disease. Juvenile forms present with bulbar signs, ataxia, cognitive deficits, seizures and megalencephaly. Adult forms are variable. A 14 year old girl with an atypical phenotype is reported. Pregnancy, perinatal period and infancy were normal. At 6 delays were noted. Falls and ataxia started at 8. At 10 testing was done: metabolic testing, pyrimidines, purines, CSF studies, CGD, muscle biopsy, liver studies, eye exam, SNP-array and brain MRI were all nondiagnostic. At 13 she had hypotonia, dysarthria, ataxia, brisk reflexes, decreased strength in lower extremities with spasticity and cognitive deficit. Head size was normal. Diagnosis was made. Brain MRI at 14 showed diffuse volume loss in cerebrum, cerebellum and brain stem with ex vacuo dilation of ventricles and thinned corpus callosum. WES showed a heterozygote mutation, R105W, in the GFAP gene. Her father, asymptomatic, is a carrier. Although this is a novel mutation, it represents a non-conservative change from Arginine to Tryptophan and thus is likely pathogenic. Ongoing paternal formal neurological exam and brain MRI are in process. The possible role of modifier genes and incomplete penetrance can explain the variable phenotypes in this family and bring new insights into this fascinating disease. W-023 The role of SIRT1 in the physiopathology of X-linked adrenoleukodystrophy Morató L1,2, Galino J1,2, Ruiz M1,2, Naudí A3, Portero-Otín M3, Pamplona R3, Ferrer I4,5, Villarroya F6,7, Fourcade S1,2, Pujol A1, Pujol A2,8 1 Neurometabolic Diseases Lab (IDIBELL), Hospitalet de Llobregat, Barcelona, Spain; 2CIBERER, ISCIII, Spain; 3Exp Medicine Department (Univ of Lleida), Lleida, Spain; 4Institute of Neuropathology (UB), Hospitalet de Llobregat, Barcelona, Spain; 5CIBERNED, ISCIII, Spain; 6 Dep Bioq i Biol (UB), Barcelona, Spain; 7CIBEROBN, ISCIII, Spain; 8 ICREA, Barcelona, Spain

Oxidative stress and mitochondria dysfunction are noxious factors, intertwined in the ethiological core of most common neurodegenerative conditions, together with other various factors contributing to the pathology. To address the link between mitochondria malfunction and oxidative stress we have chosen the mouse model of adrenoleukodystrophy (X-ALD), which exhibits early oxidative stress leading to late onset axonal degeneration and disability. The disease is caused by loss of function of the ABCD1 transporter, involved in the import and degradation of very long-chain fatty acids (VLCFA) in peroxisomes. In this study, we demonstrate that excess of the VLCFA C26:0 generates mitochondrial radical oxygen species resulting in mitochondrial DNA and protein oxidation. This is concomitant with mitochondrial depletion in spinal cords due to an impairment of SIRT1/PGC-1alpha pathway. Similar results are observed in brain white matter of X-ALD

1 Oregon Health & Science University, Portland, Oregon 97239, United States; 2Marshfield Research Clinic Foundation, Marshfield, Wisconsin 54449, United States

Background: Cerebrotendinous xanthomatosis (CTX) is a rare autosomal recessive disorder of bile acid (BA) synthesis associated with progressive neurological damage and premature death. CTX is hard to diagnose; the mean age of first symptom onset is between 14-19 years old and of diagnosis 35-37 years old. Detection of CTX through newborn screening would be beneficial since a treatment for CTX is available to prevent disease complications. There is no suitable test to screen newborn dried bloodspots (DBS) for CTX. Blood screening for CTX is currently performed by GC-MS measurement of elevated cholestanol. Methods: We present here LC-ESI-MS/MS methodology utilizing keto derivatization to enable sensitive detection of ketosterol BA precursors that accumulate in CTX. Isotopically enriched derivatization reagent allowed tagging of multiple ketosterols to generate internal standards for quantification. Ketosterols in plasma or DBS were quantified and their diagnostic utility as markers for CTX compared to cholestanol. Results: Quantification of ketosterol BA precursor markers provided improved discrimination between CTX and unaffected samples, including DBS samples, to potentially enable screening of newborn DBS for CTX. Conclusion: We describe a proof-of-concept for screening DBS for CTX. Early detection and intervention through newborn screening would greatly benefit those affected with CTX. Conflict of Interest declared.

Workshop 8: Nuclear encoded mitochondrial diseases W-025 Mutation in IBA57 as new cause of iron-sulfur cluster biosynthesis defects: biochemical and clinical implications Vanlander A1, Wilbrecht C2, Ajit Bolar N3, Smet J1, De Paepe B1, De Latter E1, Van Laer L3, Loeys B3, Lill R2, Van Coster R1 1 Div Ped Neur and Metab, Univ Child Hos, Ghent, Belgium; 2Ins Zytobiol, Phil-Univ, Marburg, Germany; 3Dep Med Genet, Univ Hosp, Antwerp, Belgium

Background: Combined OXPHOS deficiencies involving complexes I and II have recently been detected in patients with deficient ironsulfur cluster (ISC) biogenesis. So far, patients were reported with pathogenic mutations in NFU1 and BOLA3 presenting with severe encephalomyopathy at young age. Objective: Two siblings with combined deficiency of complex I and II were investigated for possible defect in ISC. Patients and Methods: The siblings presented soon after birth with severe encephalomyopathy and died in the neonatal period. Biochemical

S98 investigations showed increased lactate in serum and increased glycine in CSF. Considering the consanguineous descent a search for genes in homozygous regions related to ISC metabolism was performed. Results: Isolating IBA57 as a strong candidate gene, sequencing detected a homozygous mutation (c.941A>C) in the two siblings and a heterozygous carrier status in both parents. Western blotting showed a severe decrease of CRM for the IBA57 protein. The protein amount in the complexes I and II was significantly decreased. Transfection experiments in HeLa cells demonstrated that the mutation was pathogenic and that excessive degradation of the IBA57 protein was responsible for the defective ISC biosynthesis. Conclusion: This is the first report of a pathogenic mutation in IBA57 in human. W-026 Mutations in FBXl4 cause mitochondrial encephalopathy and a disorder of mitochondrial DNA maintenance Bonnen P1, Yarham JW2, Besse A1, Wu P1, He L2, Smith F3, Yau S3, Simcox EL2, Miwa S2, Donti T1, Abu-Amero KK4, Faqeih E5, Eyaid W6, Rukban H6, Wong L-J1, Craigen WJ1, Graham BH1, Scott KL1, McFarland R2, Taylor RW2 1 Baylor College of Medicine, Houston, United States; 2Newcastle University, Newcastle upon Tyne, United Kingdom; 3Guy's and St Thomas' Serco Pathology, London, United Kingdom; 4King Saud University, Riyadh, Saudi Arabia; 5King Fahad Medical City, Riyadh, Saudi Arabia; 6 King Saud Bin Abdulaziz University, Riyadh, Saudi Arabia

Background: Nuclear genetic disorders causing mitochondrial DNA (mtDNA) depletion are clinically and genetically heterogeneous, and the molecular aetiology is undiagnosed in the majority of patients. Methods: Using whole exome sequencing we identified recessive nonsense and splicing mutations in the FBXL4 gene segregating in three unrelated consanguineous kindreds in which affected children present with a fatal encephalopathy, lactic acidosis and mtDNA depletion. Results: We show that FBXL4 is an F-box protein that co-localises with mitochondria, and that both loss-of-function and splice mutations in this protein results in a severe respiratory chain deficiency, loss of mitochondrial membrane potential and a disturbance of the dynamic mitochondrial network and nucleoid distribution. Over-expression of the wild-type FBXL4 transcript in patient cells rescued the levels of mtDNA copy number, correcting the biochemical deficit. Conclusions: Together our data establish FBXL4 as a new mitochondrial disease gene with a role in maintaining mtDNA integrity and stability. W-027 Mutations in ELAC2 cause a mitochondrial RNA processing defect associated with hypertrophic cardiomyopathy and psychomotor retardation Freisinger PJK1, Haack TB2, Kopajtich R3, Wieland T3, Rorbach J4, Nicholls TJ4, Baruffini E5, Walther A3, Dannhauser K2, Zimmermann FA6, Hussein RA7, Schumm J3, Mundy H8, Ferrero I5, Strom T3, Meitinger T2, Taylor RW9, Minczuk M4, Mayr JA6, Prokisch H2 1

Dep. Pediatics and Metab. Klinikum, Reutlingen, Germany; 2Inst. Human Genet, Techn. Univ. München, München, Germany; 3Inst. Human Genet., Helmholtz Center, München, Germany; 4MRC Mitochondrial Biol. Unit, Cambridge, United Kingdom; 5Dep. Life Sciences, Univ Parma, Parma, Italy; 6Dep. Pediatrics Paracelsus Med. Univ., Salzburg, Austria; 7Dep. Neuropediatrics Univ. Jena, Jena, Germany; 8C. Inh. Metab.Dis, Guys St. Thomas NHS, London, United Kingdom; 9WellcomeTrust C.for Mito. Research, Univ, Newcastle, United Kingdom

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Mitochondrial respiratory chain defects comprise a biochemically defined group of disorders associated with vast genetic and clinical heterogeneity. Still a large fraction of cases remains undiagnosed on the molecular genetic level and the set of genes involved awaits full definition. We identified mutations in ELAC2, coding for the 3'-end tRNA processing RNase Z ortholog, in five patients from 3 families with multisystem mitochondrial disease by exom sequencing. They all presented with infantile hypertrophic cardiomyopathy, psychomotor retardation, lactic acidosis and deficiency of respiratory chain complexes I or I/IV. The severity of the phenotype is highly variable even within the same family. 2 of 5 died in early infancy, the oldest is 14 years old and attends school. Mitochondrial transfer RNA (mt-tRNA) precursors were found increased in patients' skeletal muscle as well as fibroblasts. Although mature mt-tRNA, mt-mRNA, and mt-rRNA levels were not decreased in fibroblasts, the processing defect was associated with impaired mitochondrial translation. Rescue experiments in mutant cell lines and functional investigations in yeast provided additional evidence for the disease-causal role of defective ELAC2, thereby linking mtRNA processing to human disease.

W-028 Mutations in LYRM4, encoding iron-sulfur cluster biogenesis factor ISD11, cause deficiency of multiple respiratory chain complexes Thorburn DR1, Lim SC1, Friemel M2, Marum JE1, Tucker EJ3, Bruno DL4, Riley LG5, Christodoulou J6, Kirk EP7, Boneh A4, Rouault TA8, Leimkühler S2, Compton AG1 1

Murdoch Childrens Research Institute, Melbourne, Australia; 2Inst Biochem Biol, Univ of Potsdam, Potsdam, Germany; 3Paediatr Dept, University of Melbourne, Melbourne, Australia; 4Victorian Clinical Genetics Services, Melbourne, Australia; 5Children's Hospital at Westmead, Sydney, Australia; 6Discipline Paed & Genet Med, Univ Sydney, Sydney, Australia; 7Med Genet Dept, Sydney Children's Hosp, Sydney, Australia; 8Eunice Kennedy Shriver NICHHD, NIH, Bethesda, United States We previously reported sequencing ~1000 genes encoding the known mitochondrial proteome in 42 infants with definite biochemical OXPHOS disorders (Calvo et al., 2012 Sci Trans Med 4:118ra10). In addition to proven diagnoses, this identified mutations in a number of candidate disease genes. A patient with deficiency of complexes I, II and III in muscle and liver had a homozygous p.R68L mutation in LYRM4, encoding the ISD11 protein. ISD11 forms a complex with the sulfur donor NFS1 and stabilizes it. Complexes I, II and III all contain iron sulfur (Fe-S) clusters. Sanger sequencing identified the same mutation in his similarly affected cousin, who had a more severe phenotype and died in the neonatal period. Other Fe-S proteins were also affected in both patients, including the aconitases and ferrochelatase. In vitro studies showed that the L-cysteine desulfurase activity of NFS1 was barely present when co-expressed with mutant ISD11. LYRM4 mutations cause a novel defect in an early step of Fe-S cluster assembly, affecting a broad variety of Fe-S proteins. The differences in biochemical and clinical features between the two patients may relate to limited availability of cysteine in the newborn period and suggest a potential approach to therapy.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

Workshop 9: Transport defects W-029 Deficiency in SLC25A1, encoding the mitochondrial citrate carrier, causes combined D-2- and L-2-hydroxyglutaric aciduria Nota B1, Struys EA1, Pop A1, Jansen EE1, Kanhai WA1, Kranendijk M1, Bevova M2, vanDooren SJ1, Sistermans EA2, Niewint AW2, Barth M3, Ben-Omran T4, Hoffmann GF5, de Lonlay P6, McDonald MT7, Meberg A8, Muntau A9, Nuoffer JM10, Parini R11, Read MH12, Rennerberg A13, Santer R14, van Schaftingen E15, Strahleck T16, Fernandez Ojeda MR1, van der Knaap MS17, Jakobs C1, Salomons GS1 1

Metabolic Unit, VU University Med Center, Amsterdam, Netherlands; Dept of Clin Genetics, VUmc, Amsterdam, Netherlands; 3Génétique CHU Angers, Angers, France; 4Clinical and Metabolic Genetics, HMC WCMC, Doha, Qatar; 5Dept Kinderheilkunde I, Heidelberg, Germany; 6Inh Metabolic Diseases, Hôpital Necker, Paris, France; 7 Div of Med Genetics, Duke University, Durham, United States; 8 Neonatal Unit, Vestfold Hospital Trust, Txnsberg, Norway; 9Dept of Mol Paediatrics,Ludwig-Max. Univ, Munich, Germany; 10Div of Ped Endocrinology, Univ. of Bern, Bern, Switzerland; 11Dept of Pediatrics, Fondazione "MBBM", Monza, Italy; 12Laboratoire de biochimie, CHU de Caen, Caen, France; 13Dept of Neuropediatrics, Klin Bürgerpark, Bremerhaven, Germany; 14Ped Metabolic Med, Univ Childs Hospital, Hamburg, Germany; 15Lab of Physiol Chem, de Duve Institute, Brussels, Belgium; 16Dept of Neonatology, Olgahospital, Stuttgart, Germany; 17Dept of Child Neurology, VUmc, Amsterdam, Netherlands 2

The Krebs cycle is of fundamental importance for the generation of the energetic and molecular needs of both prokaryotic and eukaryotic cells. Both enantiomers of metabolite 2-hydroxyglutarate are directly linked to this pivotal biochemical pathway and are not only found elevated in several cancers, but also in different variants of the neurometabolic disease 2-hydroxyglutaric aciduria. Recently we showed that cancer-associated IDH2 germline mutations are the cause of one variant of 2-hydroxyglutaric aciduria (Kranenedijk et al, Science 2010). Complementary to this finding, we now report recessive mutations in the mitochondrial citrate carrier (CIC) gene (SLC25A1), in 12 out of 12 patients individuals with the severe variant combined D-2- and L-2-hydroxyglutaric aciduria. The impaired mitochondrial citrate efflux, demonstrated by stable isotope labeling experiments and the absence of SLC25A1 protein in fibroblasts harboring null alleles (SLC25A1-/-), confirmed the pathogenic nature of these mutations. Transfection of deficient fibroblasts with wild type SLC25A1 restored citrate efflux and decreased intracellular 2-hydroxyglutarate levels. Our results identify defects in SLC25A1 as a novel cause of 2-hydroxyglutarate elevation.

W-030 Neonatal intrahepatic cholestasis caused by citrin deficiency: variable genotypes and phenotypes Nguyen HAP1, Nguyen Huong M2, Kobayashi K3, Nguyen KG4, Nguyen NT5 1

Hepatology Dept, National hosp of Ped, Hanoi, Viet Nam; Human gen dept, National hosp of Ped, hanoi, Viet Nam; 3Univ Graduate Sch of Med and Dental Sci, Kagoshima, Japan; 4Medical college, Hanoi, Viet Nam; 5National hospital of pediatric, Hanoi, Viet Nam 2

S99 Background: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) is a novel metabolic disease caused by deficiency of citrin, which is encoded by the SLC25A13 gene. Aims of study: to find correlations between the phenotype and genotype of NICCD. Method: Prospective description study. 206 intrahepatic cholestasis patients were enrolled in this study. Diagnosis was made through SLC25A13 mutation analysis by PCR/ PCR-RFLP. Results: 85 NICCD cases were diagnosed with clinical manifestations: jaundice (96,5%), hepatomegaly (89,4%), steatorrhea (88,2%), chubby face (88,2%), splenomegaly (29,4%), faints (18,8%). Laboratory features: hyperbilirubinemia (100%), increase AST (100%), ALT (82,4%), AST/ALT ratio > 2,5 (89,4%), decreased prothrombin rate (88,2%), hypoproteinemia (85,9%), hypoalbuminemia (89,4%), hyperamonemia (91,8%). 100% of patients had elevation of AFP, 68,3% had increase of citrullin. 85 NICCD cases include 80 homozygous and 5 compound heterozygous patients. Four SLC25A13 gene mutations were found: 851del4 , 1638ins23, IVS6+5G>A and IVS16ins3kb, with 5 genotypes. No relation between phenotype and genotype was found. Prognosis was good in most NICCD patients (90,6%); the condition was fatal in 9,4% of cases, and 1 patient (1,2%) had a poor outcome with hepatic failure. Conclusions: The phenotype of NICCD is very variable but not always benign.

Workshop 10: Purine/pyrimidine and urea cycle disorders W-031 Mass screening system based on enzyme assay for adenosine deaminase deficiency in dried blood spots using ESI or DART ion sorce combined to triple quadruple mass spectrometer Nakajima H1, Miwa T1, Kida K1, Kosuga M1, Watanabe J2, Shiota T3, Okuyama T1, Onodera M1, Fujimoto J1 1

Natl Ctr for Child Health and Dev, Tokyo, Japan; 2Shimadzu Corp., Kyoto Japan; 3AMR Inc., Tokyo, Japan

Background: Adenosine deaminase (ADA) deficiency is an inherited disorder of purine metabolism characterized by severe combined immunodeficiency (SCID) and is fatal within the first months of life if left untreated. Thus, a quick and simple mass screening system is essential for early treatment to serve the patient. The enzyme catalyzes the reaction from adenosine to inosine. Then the enzyme product inosine is metabolized quickly to hypoxanthine by universally expressed purine-nucleoside phosphorylase. Materials and Methods: We use dried blood spots (DBS) from a healthy donor, the patient, and the patient treated by enzyme replacement therapy using ADAGEN. We incubated the reaction mixture of substrate and DBS extract as enzyme source, then products were analyzed using LC-ESI or DART ion source combined to triple quadruple mass spectrometer. Conclusion/Discussion: We developed a mass screening system for ADA deficiency by enzyme assay. Enzyme products increased in a linear fashion for 3 hours in the case of the healthy donor but no activity was detected from patient sample using LC-ESI-MS. DART ion source also showed same results without separation by chromatography. Our DART analysis consumed only 12 second per sample, thus it is preferable for the mass screening of ADA. Conflict of Interest declared.

S100 W-032 Novel insights into the pathogenesis of argininosuccinic aciduria Nagamani SC1, Jiang H2, Reddy A3, Campeau PM1, Kho J1, Madan S1, Premkumar MH3, Chen Y1, Zhang J1, Sun Q1, Bryan NS2, Cederbaum S4, Erez A5, Lee B1 1

Mol Hum Genet, Baylor Col of Medicine, Houston, United States; Univ Texas Health Scie Cent, Houston, United States; 3Baylor College of Medicine, Houston, United States; 4UCLA, Los Angeles, United States; 5Weizmann Institute, Rehovot, Israel 2

Background: Argininosuccinic aciduria (ASA), the second most common urea cycle disorder (UCD) is caused by deficiency of argininosuccinate lyase (ASL). Patients with ASA can develop hypertension and neurocognitive deficiencies even in the absence of hyperammonemia. The current therapy is geared towards the control of hyperammonemia and hence does not prevent these complications. Objectives: Recent studies show that in addition to ureagenesis, ASL is required for nitric oxide (NO) production. We explored the role of such non-ureagenic functions of ASL in causation of hypertension and neurocognitive deficits with the goal of developing new therapies. Methods: We developed novel mouse models with tissue-specific deficiency of ASL in vascular endothelium and neuronal cells by breeding Asl flox allele with mice expressing Cre-recombinase under Cadherin5 and nestin promoters, respectively. Results: Endothelium-specific deletion of Asl results in hypertension from the inability of vasculature to produce nitric oxide (NO). Importantly, hypertension is prevented by treatment with an exogenous NO source. Mice with the neuron-specific deletion of ASL have accumulation of argininosuccinate in the neurons and altered neurochemistry. Conclusion: Decreased NO and other non-ureagenic functions of ASL are involved in the pathogenesis of ASA. These findings may have therapeutic implications and help optimization of therapy for ASA.

W-033 Natural history of argininosuccinic aciduria from a British cohort of 53 patients Baruteau J1, Jameson E2, Morris AA2, Chakrapani A3, Saikat S3, Suresh V3, Grunewald S1, Murphy E4, Cleary M1, Mundy H5, Abulhoul LH1, Lachmann R4, Rahman Y5, Gissen P1, Davison JE1

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 status: EOF n=7, LOF n=1, unavailable data n=2). Treatment included protein restriction (100%), arginine supplementation (94%) and ammonia scavengers (54%). Neurodevelopmental delay was ubiquitous (EOF 81%, LOF 100%, SIB 100%). Epilepsy (EOF 42%, LOF 38%, SIB 37%) and liver impairment (EOF 84%, LOF 19%, SIB 75%) were frequent. Trichorrhexis nodosa was only reported in LOF (27%). Mean age of first seizure was 8 years. Hypokalaemia (n=10), renal enlargement suggestive of nephropathy (n=6), chronic diarrhoea (n=4) and hypertension (n=3) were also observed. Amongst 11 adult patients (age 18-52y), only one lived independently. This study highlights the severe multiorgan involvement in ASA, and need for novel treatment approaches.

W-034 Survival and outcome in neonatal onset urea cycle disorders: a metaanalysis of the literature over 30 years Burgard P1, Lindner M1, Rüdinger W2, Kölker S1, Hoffmann GF1 1

Div Metab Dis, Univ Child Hosp, Heidelberg, Germany; 2Cytonet Hannover GmbH, Hannover, Germany Background: Diagnosis and treatment of urea cycle disorder changed and improved markedly during the last decade. As randomized controlled trials are mostly lacking, historical comparison is an alternative and feasible methodological approach for benchmarking. Material and methods: Nine studies published between 1984 and 2013 including a total of 754 patients with OTC, CPS, ASS or ASA deficiency from Europe, USA and Japan were analyzed Results: From 754 reported patients 459 (61%) presented with neonatal onset (≤31 DOL); 252 of them (53%) survived. From those at one year 22 (9.1%) were dead, 139 (57.4%) were mentally retarded, and 80 (33.1%) were normal. Results were better for CPS and OTC than for ASS and ASA. Median years of publication were 2009 (US studies), 2001 (EU studies), and 1998 (Japanese study). Results for mortality improved with time periods. For normal development outcome improved from 1998 to 2001 but remained stable until 2009. Conclusions: A meta-perspective on early onset UCDs reveals better outcomes for proximal than for distal disorders. Outcomes seem to improve over time. However, interpretation of results should take into account selection bias, multiple reports of patients and confounding variables like metabolic parameters and type of treatment.

1

Metab Med Dep, Great Ormond Street Hosp, London, United Kingdom; Metab Med Dep, Royal Manch Children Hosp, Manchester, United Kingdom; 3Metab Med Depart, Children's Hosp, Birmingham, United Kingdom; 4Metab Unit, Nat Hosp Neurol & Neurosurg, London, United Kingdom; 5Metab Med Dep, Evelina Children's Hosp, London, United Kingdom 2

Argininosuccinic aciduria (ASA) caused by argininosuccinate lyase deficiency has a more complex phenotype than other urea cycle disorders. We report a multicentre retrospective study of 53 ASA patients. 44 were alive (mean age 13 years) after median follow-up of 12 years (range 4 days - 47 years). Death was caused by metabolic decompensation (n=4), infection (n=2), hepatocellular carcinoma (n=1), accidental (n=1), unknown (n=1). 43 patients were diagnosed after symptomatic presentation (21 with neonatal hyperammoniemia (early-onset form or EOF), and 22 as late-onset form (LOF)). 10 further siblings (SIB) were prospectively treated from birth (proband

Workshop 11: Countries with emerging inborn error of metabolism services W-035 Neonatal screening program among Arabs: Saudi experience Rahbeeni ZA1, Al-Hahshem A1, Shouki M1 1

KFSH and RC, Riyadh, Saudi Arabia

Introduction: Neonatal screening program proved without doubt that it is one of the best means for healthy children and community. In addition, it decreases the handicapped by early treatment. However the neonatal screening program should not be a test but a complete program.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Method: Blood samples were extracted from neonates during the first 3 days of life to be sent to KFSH and RC with biographic data including age, sex, weight, etc. The research involved more than 75 hospitals in Saudi Arabia.The period of research was August 2005-June 2012 Result: We screened 655733 samples and there was one neonate affected among 1003 neonates with one of the 16 disorders that we screened (654 positive neonates). Discussion: This study showed a high incidence of genetic disorders including inherited metabolic diseases (IMDs) in the Kingdom (and Arab worlds) compared with Western countries. Conclusion: This high incidence of IMDs in the nonatal screening program in the Kingdom (and Arab Countries) confirms the importance of the program. A complete program from diagnosis to treatment and follow up including genetic counselling is required in order to prevent the screened disorders in the future. Early diagnosis has a positive impact on developmental milestone of the affected children and compliance to special nutrition.

S101 A patient found at neonatal screening was classified as classical phenylketonuria according to phenylalanine level (>1200 μmol/l) and treated with low phenylalanine diet. However, thorough analysis of his PAH gene (13 exons/flanking intron regions sequencing and MLPA) didn't show any disease-causing mutations. Since tetrahydrobiopterin (BH4) loading test isn't available in Serbia, patient was considered for genetic analysis of genes responsible for BH4 biosynthesis/regeneration. We analyzed PTS gene, usually associated with higher phenylalanine concentration in patients. Genetic analysis of this patient (and his parents) showed that he was homozygote for p.Asp136Val mutation. This patient is the first detected case of tetrahydrobiopterin deficiency in Serbia. Coclusions: Our experience highlights the importance of performing BH4-loading test in order to differentiate PAH deficiency from BH4 deficiency. However, in countries where BH4-loading test is not available, we propose prompt analysis of PAH gene in order to target patients with possible BH4 deficiency. Thus, genetic analyses will point to treatment choice for patients with BH4 deficiency which will lead to a better disease outcome

W-036 W-038 Phenotypic expression and treatment outcome of biotin-responsive basal ganglia disease: a 15-years experience on 39 cases

Protocol based management of metabolic liver disease in children at a tertiary care specialized center in India

Al-Hassnan ZN1, Alsoqih N1, Al-Muhaizea MA1 Alam S1, Sood V1, Khanna R1, Rawat D1 1

King Faisal Specialist Hospital, Riyadh, Saudi Arabia 1

Institute of Liver and Biliary Sciences, New Delhi, India

Biotin-Responsive Basal Ganglia Disease (BRBGD) is a potentially treatable recessive disorder caused by mutated SLC19A3 gene. It was first described in 1998 with 10 patients from our center. Here we report our center experience on the diagnosis and treatment of 39 cases. Four patterns of clinical presentation were observed: (i) acute encephalopathy (51%); (ii) dystonia (46%); (iii) seizures (23%); and developmental delay (18%). The onset of symptoms ranged from 2 months to 15 years (median 30 months). Family screening identified 2 asymptomatic children. The MRI data showed abnormal signal changes on T2 and FLAIR images involving putamen in 94% and caudate in 86% of cases. Involvement of other deep gray matter structures, white matter, cerebellum and brain stem were uncommon and variable. All patients were treated with high doses of biotin and thiamine. Younger patients tended to have Leigh-like encephalopathy with poor outcome. Eight patients had full recovery and remained asymptomatic (the longest follow up 10 years). Homozygous p.T422A mutation was identified in 92% of patients. To our knowledge, our report represents the largest series of patients with BRBGD and describes the longest experience of treatment outcome. Our data highlight the crucial role of family screening and presymptomatic management.

Inborn errors of metabolism, with hepatomegaly and/ or abnormal liver function are referred to as "Metabolic liver disease (MLD)". Laboratory work up is exhaustive and not available in India. Screening with protocol based approach for MLDs is costeffective and could improve the identification rate. Methods: An age and presentation appropriate protocol based approach used to screen the children with encephalopathy, acute liver failure, cholestasis, hepatomegaly and chronic liver disease (Protocol 1-5 respectively). Results: Of the total 361 cases 74 (20.4 %) were MLD and cryptogenic/ indeterminate cases were 17 (4.7%). 2/7, 29.1%, 13.5%, 85.7% and 13% were MLDs respectively in protocols 1-5. Of 33 WD, 3 had LT, 9 died/ left and 21 survived on medical management. There were 10 glycogen storage disorder type 3, 8 PFIC 2, 6 lipid storage disorder and 3 galactosemia and 2 each of tyrosinemia and hereditary fructose intolerance. One child each had urea cycle defect, citrullenemia type II, fructose 1,6 biphosphatemia and phosphoenol pyruvate carboxykinase deficiency. Three siblings were found to have Chanarin Dorfman syndrome (nonlysosomal lipid storage disorder). Conclusion: Protocol based approach can effectively identify MLD in a developing world setting and would also reduce the cryptogenic liver disease.

W-037 Tetrahydrobiopterin deficiency in patients presenting with hyperphenylalaninemia - an experience from Serbia Stojiljkovic M1, Klaassen K1, Djordjevic M2, Sarajlija A2, Ugrin M1, Nikcevic G1, Zukic B1, Desviat LR3, Pavlovic S1, Perez B3 1

IMGGE, University of Belgrade, Belgrade, Serbia and Montenegro; 2 Mother and Child Healthcare Institute, Belgrade, Serbia and Montenegro; 3 UAM, CEDEM-CBMSO, CIBERER, Madrid, Spain To date, we have analyzed 62 unrelated patients with hyperphenylalaninemia from Serbia. By combining DGGE, PCR-RFLP and DNA sequencing methods, we identified disease-causing PAH mutations in 61 patients (99% detection rate).

Workshop 12: Disorders of complex lipids W-039 An ultra-rare defect due to glycerol-3-phosphate dehydrogenase deficiency redirects hepatic lipid metabolism causing hypertriglyceridemia and massive hepatic steatosis Newton SA1, Agrawal P1, Desai N1, deFerranti SD1, Joshi M1, Eagan J1, Rohr F1, Connolly M1, Bennett MJ2, Berry GT1 1

Boston Child Hosp, Harvard Med School, Boston, United States; Childrens Hosp Phila, UPenn Med School, Philadelphia, United States

2

S102 Glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible redox reaction whereby dihydroxyacetone phosphate (DHAP) and NADH is converted to glycerol-3-phosphate (G3P) and NAD+. It also plays an important role in the transport of reducing equivalents from the cytosol to the mitochondria. Previously, ten Israeli Arab individuals from 4 families were described with transient infantile hypertriglyceridemia, hepatomegaly, elevated transaminases, fatty liver and development of hepatic fibrosis due to homozygous mutations in GPD1 (Basel-Vanagaite et al, Am J Hum Genet 2012). We report the first caucasian patient with a compound heterozygous GPD1 defect, identified through whole exome sequencing. She is a 2 year old with massive hepatomegaly and steatosis, abnormal liver function tests, hypertriglyceridemia, and microcephaly. GPD1 protein was found to be absent in the patient's liver tissue sample. Interestingly, activity of carnitine palmitoyltransferase (CPT) I and CPT II was reduced in the patient's cultured skin fibroblasts. No mutations were identified in these genes, suggesting that the dual reduction in activities were secondary to dihydroxyacetone accumulation. Given these findings, we hypothesize that this accumulation leads to an imbalance between hepatic free fatty acid oxidation and free fatty acid incorporation into neutral lipid.

W-040 Spastic paraplegia and metabolic disorders: a focus on lipid metabolism Mochel F1, Lamari F2, Darios F3, Durr A1, Stevanin G3, Brice A3 1 Genetic dept, Hospital Pitié-Salpêtrière, Paris, France; 2Biochem dept, Hospital Pitié-Salpêtrière, Paris, France; 3INSERM UMR S975, Hosp Pitié-Salpêtrière, Paris, France

Spastic paraplegia is a common neurological symptom in adults. Affected individuals suffer from pyramidal motor neuron dysfunction such as spasticity, brisk reflexes, and pyramidal weakness of the lower limbs. Hereditary spastic paraplegias (HSP) are heterogeneous inherited neurodegenerative disorders. All modes of inheritance (autosomal dominant, autosomal recessive, or X linked) have been reported. Except in cases of clear dominant inheritance, metabolic causes of HSP should systematically be considered since some of them are treatable like cerebrotendinous xanthomatosis (CTX), or have an important impact for genetic counseling like X-linked adrenomyeloneuropathy (X-ALD). Likewise, plasma metabolic screening should comprise measurements of complex lipids such as very long chain fatty acids (X-ALD), cholestanol (CTX) and 25/27hydroxycholesterols (SPG5) - besides amino acids chromatography and homocystein for rare etiologies of HSP. Moreover, over the past year, our group has identified mutations in several genes involved in the metabolism of phospholipids and sphingolipids. DDHD1 (SPG28) and CYP2U1 (SPG49) are 2 functionally related genes involved in the metabolism of arachidonic acid; GBA2 (SPG46), encoding glucocerebrosidase type 2, catalyzes the conversion of glucosylceramide; and B4GALNT1 (SPG26) is the second human disease of gangliosides biosynthesis. The development of lipidomic approaches should allow the discovery of biomarkers in these newly identified HSP.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

Oral 1. Nutrition and dietetics O-001 Current dietary practice in adults with galactosaemia: a survey in 2013 Portnoi P 1 , Adam S 2 , Bernabei S 3 , Bollhalder S 4 , Boocock S 5 , Corthouts K6, Dalmau J7, Dawson S8, Defourny S9, De Meyer A10, Desloovere A11, Devlin Y12, Dianin A13, Diels M6, Dokoupil K14, Donald S15, Evans S16, Ferguson C12, Ford S17, Forga M18, Gallo G3, Grunert SC19, Heddrich-Ellerbrok M20, Heidenborg C21, Jonkers C22, Luyten K10, MacDonald A16, Meyer U23, Micciche A24, Muller E25, Ripley S26, Robert M9, Robertson L5, Rosenbaum-Fabian S19, Sahm K25, Schultz S20, Singleton K27, Sjoqvist E28, Stoelen L29, Terry A30, Timmer C31, Vande Kerckhove K6, van der Ploeg L32, Van Driessche M11, van Rijn M33, van Teeffelen-Heithoff A34, Vitoria I7, Voillot C35, Wenz J36, Wildgoose J37, Zweers H38 1

Galactosaemia Support Group, Sutton Coldfield, United Kingdom; Royal Hospital for Sick Children, Glasgow, United Kingdom; 3 Ospedale Pediatrico Bambino Gesù, Rome, Italy; 4UniversitätsSpital, Zurich, Switzerland; 5Univ Hospitals NHS Foundation Trust, Birmingham, United Kingdom; 6University Hosp, Center of Metab Disease, Leuven, Belgium; 7Hospital La Fe, Valencia, Spain; 8Royal Hospital for Sick Children, Edinburgh, United Kingdom; 9Hôpital Univers des Enfant Reine Fabiola, Brussels, Belgium; 10Center Metab Disease, Univ Hospital, Antwerp, Belgium; 11Universitair Ziekenhuis, Gent, Belgium; 12Royal Victoria Hospital, Newcastle, United Kingdom; 13 Division of Inher Metab Dis, Univ Hosp, Padova, Italy; 14Dr von Hauner Children's Hospital, Munich, Germany; 15Addenbrookes, Cambridge, United Kingdom; 16Birmingham Children's Hospital, Birmingham, United Kingdom; 17North Bristol NHS Trust Sthmead & French, Bristol, United Kingdom; 18Hospital Clinic, Barcelona, Spain; 19University Children's Hospital, Freiburg, Germany; 20Universitätsklinikum, HamburgEppendorf, Germany; 21Karolinska University Hospital, Stockholm, Sweden; 22Academic Medical Hospital, Amsterdam, Netherlands; 23 Clinic of Paed Kidney, Liver & Metab Dis, Hannover, Germany; 24 St Thomas' Hospital, London, United Kingdom; 25Children's Hospital, Heidelberg, Germany; 26Salford Royal, Manchester, United Kingdom; 27 University Hospital of Wales, Cardiff, United Kingdom; 28Children's Hospital, University Hospital, Skåne, Sweden; 29Oslo University Hospital Rikshospitalet, Oslo, Norway; 30Alderhey Children's Hospital, Liverpool, United Kingdom; 31UMC, Utrecht, Netherlands; 32University Hospital, Maastricht, Netherlands; 33Univ of Groningen, Univ Medical Center, Groningen, Netherlands; 34Univ Kinderklinik, Pädiatrische Diätetik, Munster, Germany; 35Hôpital Antoine Béclère, Clamart, France; 36CHU Bicëtre Hospital, Paris, France; 37Bradford Teaching Hospital NHS Trust, Bradford, United Kingdom; 38Radboud Univ Nijmegen Medical Centre, Nijmegen, Netherlands 2

Background: There are international uncertainties about dietary restriction in galactosaemia. Methods: In 2013, a questionnaire examining adult dietary practices in galactosaemia was sent to dietitians throughout Europe. Questionnaires were returned from 36 centres (10 countries) caring for 287 adult patients. Results: 73% were on a strict diet, 21% relaxed diet, 1% no diet and 5% uncertain. Most centres (n=30) did not calculate daily

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 galactose allowed. Reasons for diet relaxation included: mild mutations (28%), poor adherence (28%), patient request (19%), and strict diet unnecessary (22%). All centres restricted lactose; galactosides by 6 centres (Belgium, Italy, Spain); fruit and vegetables by 5 centres (Belgium, Italy, Spain); and offal/galactocerebrosides by 13 centres (Belgium, Italy, Sweden, Germany, Spain). For some centres, diet relaxation was defined as expanding the diet to lactose-free only; for others it meant the introduction of lactose mainly in manufactured foods only. Three centres did not allow low lactose cheese and 9 centres described at least one patient who had taken lactase treated milk in error. 18% (n=65) were lost to follow up. Conclusions: there is wide variation in adult dietary practice for galactosaemia. A lactose-free diet only was most common with many reporting reticence in further diet relaxation. O-002 Critical reappraisal of medical foods utilization in methylmalonic acidemia Manoli I1, Myles JC2, Sloan JL1, Shchelochkov OA3, Venditti CP1 1 GMBB, NHGRI, NIH, Bethesda, MD, United States; 2Nutrition, NIH, Bethesda, MD, United States; 3Div Genet, Univ Iowa Hosp, Iowa, IA, United States

Background: Medical foods free of propiogenic amino acids (valine, isoleucine, methionine and threonine), but containing leucine, are used in the management of methylmalonic acidemia (MMA) to provide calories and control the accumulation of toxic intermediates. However, evidence-based guidelines for their composition and use are lacking. Methods: Sixty-one patients with isolated MMA (46 mut, 9 cblA and 6 cblB; age range 2.5 to 35 years) were studied. Dietary intake was correlated with biochemical, anthropometric and body composition measures. Results: 85% of MMA patients consumed medical foods in addition to natural protein intake close to recommended-daily-allowance (RDA) (mut: 98.8 ± 6% RDA). In 30% of the patients medical foods exceeded the amount of natural protein administered. This resulted in higher leucine-to-valine intake ratio compared to patients not using medical foods (mut: 3.7 ± 1.6 vs. 1.5 ± 0.6, P<0.001) and lower plasma valine concentrations (P=0.01), and likewise for isoleucine, necessitating oral valine and isoleucine supplementation. Weight, height, and bone density correlated negatively with the leucine-to-valine intake ratio. Conclusions: Distorted branched chain amino acid composition of MMA diets can result in altered plasma amino acid ratios and adversely affect growth outcomes. The role of medical foods in MMA needs to be carefully re-evaluated. O-003 Dietary management and nutritional outcomes in children diagnosed with a long-chain fatty acid oxidation defect through newborn screening Nagy LL1, Saleh H2, Herd S1, Raiman JAJ1,2 1 The Hospital for Sick Children, Toronto, Canada; 2The University of Toronto, Toronto, Canada

S103 With early detection of long-chain fatty acid oxidation defects (LCFAOD) through newborn screening (NBS), infants are initiated on a high medium-chain triglyceride (MCT), low long-chain triglyceride (LCT) diet. However, little is known about nutritional outcomes in early life. The objective of this retrospective study was to examine growth and nutritional status of children diagnosed with a LCFAOD through NBS from 0-24 months of age. Charts (n=6) were reviewed for growth parameters, dietary intakes, supplementation, and biochemistry. Mild phenotypes were excluded. Diet therapy began at 12.6±6.6 days of life with 26.4 ±5.7% of energy from MCT and 13.7±5.7% of energy from LCT. Energy from LCT declined over 24 months while MCT remained stable. Normal growth was achieved. Iron (3/6) and vitamin D supplementation (5/6) was common. Inclusion of walnut and/or flax oils achieved mean intakes of 3.58±0.007% of energy from linoleic acid and 0.640±0.003% of energy from α-linolenic acid. There was no evidence of low plasma essential fatty acids (EFA). Mean plasma arachidonic acid (ARA; 15.7±0.05% fatty acids) was higher than reference values. We demonstrate a high-MCT diet initiated in early life can support growth with EFA supplementation. High plasma ARA may have resulted from the ARA content in the MCT-rich formula. O-004 Management and outcomes of very long chain acyl-CoA dehydrogenase deficiency (VLCADD) in a single centre in the United Kingdom Gribben J1, van Wyk K1, Walker R1, Champion M2, Mundy H2, Rahman Y2, Vara R2 1

N & D Dept, St. Thomas' Hosp, London, United Kingdom; 2Ctr Inherited Met Dis, St. Thomas' Hosp, London, United Kingdom Background: VLCADD, a long-chain fatty acid disorder is not part of newborn screening in the UK. Clinical presentations varies from severe early onset to late myopathic forms, some family screened patients are asymptomatic. Objectives: To review clinical presentation and tailoring of dietary management. Methods: A retrospective review of 8 patients treated from 1988-2013. Results: Infant group: 4 presented with classical biochemical and clinical phenotypes (median age 4 months, range 2 days - 2 years), 2 diagnosed following sibling death. All treated with minimal LCT diet (median 9g, range 3-12), EFA supplementation, MCT supplementation (median 14% of total energy, range 0-27%) and 1 uncooked cornstarch. 5 were fed overnight and 1 cornstarch alone. Later onset group: A 12 year old (rhabdomyolysis) treated with moderate fat restriction and MCT prior to exercise, and an asymptomatic 5 year old (previous sibling infantile presentation) with moderate fat restriction only. Outcomes: Median age at follow up 10 years (1-24 years): 66% of infantile onset developed recurrent rhabdomyolysis, median age 6 years (0.5-8 years) improved with MCT adjustment. None have cardiomyopathy or liver dysfunction. Conclusions: Dietary management tailored to age and mode of presentation improves clinical symptoms with a good long-term outcome.

S104

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

O-005

Oral 2. Organic acids and creatine disorders

Longterm use of a low saturated fat, carbohydrate free formula to manage elevated LDl cholesterol in a child with Glut 1 deficiency syndrome on treatment with a ketogenic diet

O-007

White FJ1, Gallagher J1, Jones SA1 1

Willink Unit, Genetic Med, St Marys Hosp, Manchester, United Kingdom

Novel biochemical findings in 3-hydroxyisobutyryl-CoA hydrolase deficiency: implications for screening Peters H1, Wanders R2, Ruiter J2, Ferdinandusse S2, Boneh A1, Pitt JJ1 Murdoch Childrens Research Institute, Melbourne, Australia; 2Academic Medical Centre, Amsterdam, Netherlands

1

Background: Glut 1 deficiency syndrome (Glut1DS), an autosomal dominant disorder caused by mutations in the SLC2A1 gene, reduces glucose transport across the BBB, causing low CSF: blood glucose ratio with consequent seizures, ataxia and movement disorders. Ketogenic diet (KD) treatment provides alternative fuel for brain cells. Hypercholesterolaemia is a risk factor with both classical and MCT based KD due to their high saturated fat (SFA) content. Case report: we describe the management of hypercholesterolaemia in a Glut1DS male (heterozygous c.1126G>C) following a modified MCT KD. Lipid profile normal pre diet (total cholesterol 4.4mmol/l, LDL 2.3mmol//l), elevated from 8 months with total cholesterol 5.9mmol/l, LDL 3.2mmol/l after 29 months (energy prescription 36% as MCT, 44% as LCT; 120g fat/day, 60% saturated). SFA intake was reduced, primarily substituting dairy products with a low SFA formula, CarbZero™ (Vitaflo International), high LCT (11% saturated, 60.5% monounsaturated, 28.5% polyunsaturated), protein and carbohydrate free. LDL levels normalised within 2 months of dietary changes. After 2 years total and LDL cholesterol remain in the normal range, with effective ketosis persisting. Conclusions: Hypercholesterolaemia in Glut1DS treated long term with KD can be managed by altering the LCT source. CarbZero™ was tolerable over a prolonged period. Conflict of Interest declared.

3-hydroxyisobutyryl-CoA hydrolase (HIBCH) deficiency (HIBCHD, OMIM 250620) is an inborn error of the valine catabolic pathway. Only two patients have been reported, having common features of hypotonia and neurological deterioration. We report an additional female patient who presented at 5 months of age with hypotonia, developmental delay and cerebral atrophy on MRI. PDHC activity in skin fibroblasts was deficient but molecular testing did not reveal any mutation. Urine tandem mass spectrometry screening showed increases in the cysteine and cysteamine conjugates of methacrylate previously described in HIBCHD. HIBCH activity in cultured skin fibroblasts was below the limit of quantitation of the assay (<2.6 nmol/(min.mg); reference range 5.3 – 10.5) and two novel HIBCH mutations were identified (c.[129dupA];[1033G>A]). Urine metabolite investigations showed increases in 3-hydroxyisobutyryl carnitine, methacryl glycine, 2-methyl-2,3-dihydroxybuyrate, and cysteine and cysteamine conjugates of acrylate. The last three metabolites are derived from a secondary propionate pathway that also uses HIBCH. The child's newborn screening OHC4 carnitine result (1.2 μmol/L) was just below the cutoff used at the time (<1.3) and led to a lowering of this cut-off to 1.0 μmol/L. Our findings demonstrate that urine tandem mass spectrometry and organic acid screening are useful diagnostic tools for HIBCHD.

O-006

Biochemical predictors of long-term outcome in patients with guanidinoacetate methyltransferase (GAMT) deficiency

Low protein intake in Maple Syrup Urine Disease (MSUD) mice improves branched chain amino acids (BCAAS) in blood but not in cerebellum or striata

O-008

Viau K1, Ernst SL1, Pasquali M2, Botto LD1, Hedlund GL3, Longo N1 1

Pediatric Medical Genetics, Univ Utah, Salt Lake City, United States; Dept Pathology, Univ Utah, Salt Lake City, United States; 3Dept Medical Imaging, PCMC, Univ Utah, Salt Lake City, United States

2

Vogel KR1, Arning E2, Wasek B2, Bottiglieri T2, Gibson KM1 1

Section of Clin Pharm, Wash St Univ, Spokane, WA, United States; Baylor Res Inst, Dallas, TX, United States

2

Therapy for MSUD centers on dietary protein restriction monitored via blood amino acids, yet how metabolic control in blood translates to brain, and the effect of elevated BCAAs (val, ile, leu) on transport of other large neutral amino acids (LNAAs: met, phe, tyr, trp) into brain, remain unknown. We examined these questions in murine MSUD mice via high (19%) and low (6%) protein feeding (8-18 days). Results for leucine were: blood (mmol/L); 966 +/- 520 (SD, n=6, 19%); 360 +/- 176 (n=4, 6%, p=0.058 vs 19%); 127 +/- 21 (n=12, wild-type, both diets); for comparison, human sera range 77-153 mmol/L); cerebellum (nmol/g): 1026 +/- 444 (n=5, 19%); 611 +/- 438 (n=5, 6%, p=ns vs 19%); 119 +/- 16 (n=13, wild type, both diets); striata (nmol/g): 1031 +/- 556 (n=6, 19%); 703 +/- 653 (n=5, 6%, p=ns vs 19%); 118 +/- 66 (n=13, wild type, both diets). With one exception (tyr, 19%), elevated BCAAs did not alter levels of met, phe, tyr or trp in either brain compartment. We conclude that: 1) elevated BCAAs do not prohibit transport of other LNAAs from the periphery to the brain; and 2) correction of blood BCAAs may not reflect correction in the brain.

Background: Guanidinoacetate methyltransferase (GAMT) deficiency causes cerebral creatine deficiency. Patients can have autistic behaviour, seizures, and severe speech delay. Therapy aims at increasing creatine while reducing guanidinoacetate that can be toxic to the brain. Objective: To determine how levels of amino acids correlate with plasma guanidinoacetate levels in patients with GAMT deficiency. Methods: Retrospective analysis of data from 5 patients with GAMT deficiency (4 with delays and seizures, one diagnosed at birth). Results: The 4 symptomatic patients had decreased brain creatine by MR spectroscopy and 3 also had abnormal globi pallidi by MRI. GAMT sequencing identified 4 previously reported mutations and one novel missense mutation (c.T233A/p.V78E). Creatine (250-1000 mg/kg/day), ornithine (100-800 mg/kg/day), and sodium benzoate (50-135 mg/kg/day) supplements with dietary protein restriction (0.8-1.5 g/kg/day) improved seizures and development. The patient treated at birth remains developmentally normal. Reduction in glycine (but not arginine) and increase in ornithine plasma levels significantly correlated with decreased plasma guanidinoacetate. Glycine levels were the best predictor. Conclusions: Effective therapy of GAMT deficiency should include supplemental creatine, sodium benzoate (to reduce glycine) and ornithine. Normal development with early therapy renders this condition an ideal candidate for newborn screening.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 O-009 Phenotype and genotype in 101 males with X-linked creatine transporter deficiency van de Kamp JM1, Betsalel OT2, Wamelink MMC2, Pouwels PJW3, van der Knaap MS4, Jakobs C2, Mancini GM5, Creatine Transporter Study Group x6, Salomons GS2 1

Dpt of Clin Genetics, VU Medical Center, Amsterdam, Netherlands; Dpt of Chem, VU Medical Center, Amsterdam, Netherlands; 3Dpt of Physics/Med Techno, VU Med Center, Amsterdam, Netherlands; 4Dpt of Child Neuro, VU Medical Center, Amsterdam, Netherlands; 5Dpt of Clin Genetics, Erasmus MC, Rotterdam, Netherlands; 6Study Group, Worldwide, Netherlands

2

Creatine transporter deficiency is a monogenic cause of X-linked intellectual disability. We present an overview of the condition based on a retrospective study of clinical, biochemical and molecular genetic data of 101 males from 85 families with a pathogenic mutation in the creatine transporter gene (SLC6A8). Most patients developed moderate to severe intellectual disability. Speech language development was especially delayed but almost one-third of the patients spoke in sentences. Besides behavioural problems and seizures, mild to moderate motor dysfunction, including extrapyramidal movement abnormalities, and gastrointestinal problems were frequent clinical features. Urinary creatine/creatinine ratio proved a reliable screening method besides magnetic resonance spectroscopy, molecular genetic testing and creatine uptake studies, allowing definition of diagnostic guidelines. One-third of patients had a de novo mutation in the SLC6A8 gene. Missense mutations with residual activity might be associated with a milder phenotype and large deletions extending beyond the 3' end of the SLC6A8 gene with a more severe phenotype. Evaluation of the biochemical phenotype revealed unexpected high creatine levels in cerebrospinal fluid suggesting that the brain is able to synthesize creatine and that the cerebral creatine deficiency is caused by a defect in the reuptake of creatine within the neurons. O-010 A clinical metabolomics approach to the study of inborn errors of metabolism: preliminary results on alkaptonuria Ross GA1, Curtis S2, Dutton J2, Gallagher J3, Ranganath L2, Roberts NB2

S105 Conclusions: High resolution scanning MS has great potential in highlighting changes in inherited metabolic disease. Conflict of Interest declared. O-011 Mutations in HCFC1, a transcriptional coregulator, causes a novel Xlinked cobalamin disorder (CBLX) with a severe neurological phenotype Sloan JL1, Yu H.-C.2, Scharer G2,3,4, Brebner A.5, Quintana A2, Achilly NP1, Manoli I1, Coughlin II CR2,3, Geiger EA2, Schneck U2, Watkins D5, VanHove JL2,3, Fowler B6,7, Baumgartner MR6,7, Rosenblatt DS5, Venditti CP1, Shaikh TH2,3,4 GMBB, NHGRI, NIH, Bethesda, United States; 2Dept Peds, Univ of CO School of Med, Aurora, United States; 3Genetics, Univ of CO School of Med, Aurora, United States; 4IDDRC, Univ of CO School of Med, Aurora, United States; 5Dept of Human Genetics, McGill Univ, Montreal, Canada; 6Div Metabolism, Univ Children's Hospital, Zurich, Switzerland; 7CRC, Univ Children's Hospital, Zurich, Switzerland 1

Combined methylmalonic acidemia and hyperhomocysteinemia cblC type (cblC) is one of the most common inborn errors of cobalamin metabolism, caused by mutations in MMACHC. However, several patients with cblC confirmed by complementation analysis lack mutations in MMACHC, suggesting genetic heterogeneity within this complementation group. We used exome sequencing to identify the genetic basis of a novel, X-linked form of combined methylmalonic acidemia and hyperhomocysteinemia, designated cblX. A missense mutation in a global transcriptional coregulator, HCFC1, was identified in the \index patient. Additional male subjects were ascertained through two international diagnostic laboratories and 14/18 had one of 5 distinct missense mutations affecting three highly conserved amino acids (Gln68, Ala73, Ala115) within HCFC1 Kelch domains. A common phenotype of severe neurological symptoms (intractable epilepsy, profound neurocognitive impairment) and mild biochemical manifestations compared to early onset cblC patients was observed in all affected subjects. In two patient fibroblast lines, MMACHC mRNA and protein expression was severely reduced while HCFC1 protein levels remained intact. Furthermore, siRNA knockdown of HCFC1 resulted in the coordinate down-regulation of MMACHC. The discovery of a functional relationship between HCFC1 and genes involved in cobalamin metabolism, represents the first inborn error of metabolism caused by transcriptional dysregulation. O-012

1

Agilent Technologies, Manchester, United Kingdom; 2Dept Clin Biochem, Royal Liverpool Hosp, Liverpool, United Kingdom; 3Inst Ageing Chr Dis, Univ Liverpool, Liverpool, United Kingdom Background: Alkaptonuria (AKU) is a rare autosomal recessive disorder with a frequency of 1 in 250,000, caused by a deficiency of homogentisate 1,2dioxygenase. This enzyme converts homogentisic acid (HGA) to maleylacetoacetate and has a major role in the catabolism of phenylalanine and tyrosine. We have applied high resolution mass spectrometry (QTOF) to investigate the implications of accumulation of HGA on other low molecular weight metabolites. Methods: 1μl of urine( n=6 normal and n=6 Alkaptonuria patients)injected into a QTOF LCMS ( AGILENT) with separation on 2.1mm x 100mm, Scherzo SM-C18 3μm / 2.1 x 100mm Eclipse Plus 1.8μm with gradient over 20 mins from 5mM Ammonium Acetate in water to 50mM Ammonium Acetate in Methanol at 0.4mls/min.The eluate scanned in ESI +ve and -ve mode over 40-1100 m/z, analysed and grouped by molecular feature extraction using AGILENT Mass Hunter and Mass Profiler professional software to create a compound spectra. Results: More than 2000 compounds were detected in each urine in positive mode.Analysing differences between AKU and healthy controls it could be concluded that 43 compounds including HGA were significantly higher (>2x) in AKU vs Healthy p(<0.05).

Update on transcobalamin deficiency: clinical presentation, treatment and outcome Trakadis YJ1, Alfares A1, Bodamer OA2, Buyukavci M3, Christodoulou J4, Connor P5, Glamuzina E6, Gonzalez- Fernandez F7, Bibi H8, Echenne B9, Manoli I10, Mitchell J1, Nordwall M11, Prasad C12, Scaglia F13, Schiff M14, Schrewe B1, Touati G15, Tchan MC4, Varet B15, Venditti CP10, Zafeiriou D16, Rupar T12, Rosenblatt DS1, Watkins D1, Braverman N1 1 Medical Genetics, McGill University, Montreal, Canada; 2Human Genetics, University of Miami, Miami, FL, United States; 3Atatürk University, Faculty of Medicine, Erzurum, Turkey; 4Western Sydney Genetics Program, Sydney, Australia; 5Children's Hospital for Wales Heath Park, Cardiff, United Kingdom; 6Starship Children's Hospital, Auckland City, New Zealand; 7Hospital Clínico San Carlos de Madrid, Madrid, Spain; 8Pediatric Department Barzilai medical Ce, Beer Sheva, Israel; 9Hôpital gui de Chauliac, Montpellier, France; 10National Human Genome Research Institute, Bethesda, United States; 11Vrinnevi Hospital, Norrköping, Sweden; 12London Health Sciences Centre, Western U, London, ON, Canada; 13Baylor College of Medicine, Houston, United States; 14Reference Center for Metabolic Disease, Paris, France; 15NeckerEnfants Malades Hospital, Paris, France; 16Hippokration General Hospital, Thessaloniki, Greece

S106 Background: Transcobalamin (TC) transports cobalamin from blood into cells. TC deficiency is a rare autosomal recessive disorder usually presenting in early infancy with failure to thrive, weakness, diarrhoea, pallor, anemia, pancytopenia or agammaglobulinemia and can sometimes resemble neonatal leukemia. Diagnosis of TC deficiency is suspected based on megaloblastic anemia and elevation of total plasma homocysteine, and blood or urine methylmalonic acid. It is confirmed by studying the synthesis of TC in cultured fibroblasts, or molecular analysis of the TCN2 gene. TC deficiency is treatable with supplemental cobalamin, but the optimal type, route and frequency of cobalamin administration and long term patient outcomes are unknown. Methods: Here we present a series of 30 patients with TC deficiency, including an update on multiple previously published patients, in order to evaluate the different treatment strategies and provide information about long term outcome. Results & Conclusions: Based on this series of patients, we conclude that early diagnosis and treatment with intramuscular (1 mg IM) hydroxyl- or (1 mg IM) cyanocobalamin injections, at least on a weekly basis, are associated with better outcomes and should be continued for life.

Oral 3. Mitochondrial disorders I O-013 Is there a molecular rationale for treating patients with riboflavinresponsive multiple acyl-CoA dehydrogenation deficiency (RR:MADD) with CoQ10 in addition to riboflavin? Olsen RKJ1, Cornelius N1, Corydon TJ2, Gregersen N1

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Inst of Hum Genetics, Technical Univ, Munich, Germany; 2Dep Pediatrics, Univ Child Hosp, Pittsburgh, United States; 3Dep of Pediatrics, Klinikum Reutlingen, Reutlingen, Germany; 4Mol Bioenergetik, Cen Biol Chem, Univ, Frankfurt, Germany; 5Inst of Hum Genetics, Helmholtz Zentrum, Munich, Germany; 6ELBLAB GmbH, Riesa, Germany; 7Dep Pediatrics, Paracelsus Med Univ, Salzburg, Austria; 8Metab Disease Unit, Technion Israel Inst, Haifa, Israel; 9Nuffield Dep Obst Gynec, Univ, Oxford, United Kingdom; 10Dep Pediatric Cardiology, Univ, Bonn, Germany; 11Dev Metabolism, Kinderspital Zuerich, Zuerich, Switzerland; 12Inst Nat de la Santé Recherche Médicale, Paris, France; 13Wellcome Trust Cent Mito Research, Univ, Newcastle, United Kingdom; 14Unit Mol Neurogen Fondazio, Inst Neurolo, Milano, Italy 1

Isolated complex I deficiency is the most common biochemical defect in patients with OXPHOS defects. ACAD9, a fatty acid oxidation enzyme, also functions as a complex I assembly factor. Here we describe a cohort of 26 patients with isolated complex I deficiency due to mutations in ACAD9. Patient phenotype varied from neonatal death to mild exercise intolerance. All patients presented with lactic acidosis and most developed hypertrophic cardiomyopathy (19/26) but also dilated cardiomyopathy was found in some patients. All but one (homozygous p.Arg518His) of 14 cell lines had reduced but present ACAD9 protein. E. coli expression of ACAD9 mutations showed that ACAD9 enzymatic and complex I assembly activities were independent functions (see companion abstract, Schiff, et al). Clinical phenotype was directly correlated with complex I activity but correlation with ACAD9 enzymatic activity remains under investigation. Supplementation with riboflavin or bezafibrate increased complex I activity in 8/14 and 11/14 cell lines, respectively. This increase was associated with increased ACAD9 protein levels (6/7 cell lines) and increased incorporation of complex I into respiratory chain supercomplexes in all 4 cell lines examined. These results suggest riboflavin and bezafibrate as treatment options for this specific subgroup of patients with complex I deficiency.

1

Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark; 2Department of Biomedicine, Aarhus Univ, Aarhus, Denmark

O-015 Diagnostic approaches for mitochondrial disorders

RR:MADD is most often caused by inherited deficiency of ETF-QO. ETF-QO uses FAD (derived from riboflavin) to transfer electrons from a number of mitochondrial acyl-CoA dehydrogenation reactions to CoQ10 in the respiratory chain. The clinical and molecular chaperone effect of riboflavin treatment in RR:MADD is well documented. Previously, ETF-QO deficiency in RR-MADD was shown to be associated with secondary CoQ10 deficiency and it was suggested to treat RRMADD patients with CoQ10 in addition to riboflavin, but a molecular rationale was lacking. We have recently shown that CoQ10 deficiency in fibroblasts from six RR-MADD patients was associated with increased mitochondrial ROS production, most likely being produced from misfolded variant ETF-QO proteins with impaired CoQ10 binding affinity. In the present study we use the same patient cells to test the hypothesis that CoQ10 treatment can decrease ROS and relieve a chronic state of mild oxidative stress. As compared to control fibroblasts, fibroblasts from the six patients show affection of the mitochondrial antioxidant and protein quality control system with increased mitophagy. Treating the cells with CoQ10, but not riboflavin, partially reverses these molecular and cellular signs of stress suggesting that RR-MADD patients could benefit from the combined treatment of riboflavin and CoQ10. O-014 ACAD9 mutations are the most frequent nuclear cause for complex I deficiency, which can be corrected by riboflavin and bezafibrate in cells Haberberger BM1, Schiff M2, Freisinger P3, Strecker V4, Wieland T5, Ahting U1, Rolinski B6, Mayr J7, Mandel H8, Poulton J9, Herberg U10, Haeberle J11, Reotig A12, McFarland R13, Taylor R13, Sperl W7, Strom T5, Ghezzi D14, Zeviani M14, Wittig I4, Meitinger T1, Vockley J2, Prokisch H5

Haack TB1, Kremer LS1, Kopajtich R2, Biagosch CA2, Haberberger B1, Wieland T2, Schwarzmayr T2, Walther A2, Strom TM2, Klopstock T3, Sperl W4, Konstantopoulou V5, Zeviani M6, Taylor R7, Rötig A8, Munnich A8, Smeitink J9, Mayr JA4, Freisinger P10, Meitinger T1, Prokisch H1 1

Technische Universität München, Munich, Germany; 2Helmholtz Zentrum München, Munich, Germany; 3Ludwig Maximilians Universität München, Munich, Germany; 4Paracelsus Medical University Salzburg, Salzburg, Austria; 5Universität Wien, Wien, Austria; 6Institute of Neurology Besta, Milan, Italy; 7Newcastle University, Newcastle, United Kingdom; 8 INSERM, Paris, France; 9Radboud University Nijmegen Medical Cent, Nijmegen, Netherlands; 10Klinikum Reutlingen, Reutlingen, Germany Mitochondrial disorders present with extreme genetic and clinical heterogeneity. More than 250 disease genes have been identified amongst the >1200 genes predicted to code for mitochondrial proteins. We performed exome sequencing followed by stepwise filtering of identified gene variants and functional complementation in 120 index cases with juvenile-onset mitochondrial disorders without pathogenic variants in the mtDNA. While we were able to detect mutations in known disease genes in 40% of cases, in another 10% novel disease causing genes were identified. The list of such newly identified genes increases steadily: recently MGME1 was found to be the first exonuclease involved in mitochondrial replication. With increasing numbers of patients being analyzed, we start to discover groups of unrelated individuals with mutations in the same gene. This approach disovers novel disease genes which have so far not been associated with a mitochondrial function. One such example is the identification of mutations in a gene coding for a protein of the F-box family which we currently analyze.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 In the unresolved patients main challenges are to annotate variants in non-coding regions, to identify indels and copy number variation, as well as considering how to tackle diseases caused by di- or oligogenic mutations via synergistic effects. O-016 Recessive mutations in LARS cause a multisystem disorder with infantile liver failure, recurrent hepatopathy, anaemia and epilepsy Casey J1, Lynam-Lennon N2, McGettigan P3, O'Sullivan J2, McDermott M4, Slattery SMC5, Forde K5, Monavari AA5, Knerr I5, Hughes J5, Bourke B6, Ennis S3, Lynch SA7, Crushell E5 1 Nat Child Res Centre, Dublin, Ireland; 2Inst Mol Med, Trinity Coll, Dublin, Ireland; 3Univ Coll Dublin, Dublin, Ireland; 4Dept Pathology, Our Lady's Child Hosp, Dublin, Ireland; 5Nat Cent Inherit Metab Dis, Dublin, Ireland; 6Dept Hepatology, Our Ladys Child Hosp, Dublin, Ireland; 7Nat Cent Med Genet, Dublin, Ireland

Our study involves 7 individuals (2-34 years) from 5 Irish Traveller families with intermittent hepatic dysfunction; 4 presented with recurrent acute liver failure in infancy. Additional features include chronic microcytic anaemia, developmental delay and poor growth. During intercurrent viral illnesses, hepatic decompensation (7/7) and complex seizures (5/7) are observed. One patient died following H1N1 infection; the eldest patient's worst decompensation was triggered by measles at age 7, his episodes have lessened with age. Liver biopsies show micro and macrosteatosis. Marrow erythroblasts have abnormal iron distribution. Extensive metabolic and mitochondrial investigations were negative. Exome sequencing identified leucyl-tRNA synthetase (LARS) as the causative gene. Subsequent testing identified another Irish Traveller patient who presented at 2 years with status epilepticus on a history of liver dysfunction and anaemia. He had acute Leigh-like changes on MRI brain which resolved on follow-up, thus expanding the phenotype associated with LARS. Knock-down of LARS in HEK293 cells did not impact on mitochondrial function suggesting that the hepatopathy is unlikely primarily due to mitochondrial dysfunction. Recently, LARS was shown to activate mTORC1 which regulates autophagy, a process implicated in liver disease and host response to infections. We hypothesise that defective autophagy may be the underlying disease mechanism. O-017 The MRC mitochondrial disease patient cohort UK. A national perspective of mitochondrial disease in the UK Nesbitt V1, Pitceathly R2, Grady J1, Cockell SJ1, Poulton J3, Rahman SJ2, Hanna M2, Chinnery PF1, Taylor RW1, Turnbull DM1, McFarland R1

S107 secondary aims of recording the natural history and breadth of clinical characteristics associated with each genotype. Methods: Recruitment was through 3 principal centres, with additional recruitment at 16 secondary centres and via national neurological surveillance units. Results: There are presently 1012 living patients recruited, each with a confirmed biochemical or genetic diagnosis. Clinical data is available from 951 (94%) patients, with 47% harbouring mtDNA mutations, 11% nDNA mutations and 42% with biochemical deficiencies but no established genetic aetiology. At present 58% exhibit neurological features, 35% ophthalmological involvement, 26% gastrointestinal disturbance, 19% haematological abnormalities, 18% cardiac involvement and 16% have endocrine dysfunction. Conclusion: This cohort provides a new perspective on clinical characteristics, genotypic diversity, epidemiology and progression of mitochondrial disease. It will prove a valuable resource in understanding mitochondrial disease, recruiting to clinical trials and in healthcare planning. O-018 Mitochondrial disease sequence data resource (MSEQDR) consortium: a global grass-roots effort to compile, organize, annotate, and analyze whole exome datasets from individuals with suspected mitochondrial disease Falk MJ1, Zuchner S2, Gonzalez M2, Wallace DC3, Parisi M4, Krotoski D4, Gai X5 1

Hum Gen Div, CHOP and Upenn SOM, Philadelphia, United States; Hum Get Dept, Univ of Miami, Miami, United States; 3Center for Mito Med, CHOP/UPenn, Philadelphia, United States; 4NICHD, NIH, Bethesda, United States; 5Center for Biomed Inf, Loyola Univ SOM, Chicago, United States

2

The success of whole exome sequencing (WES) for highly heterogeneous disorders, such as mitochondrial disease, is limited by substantial technical and bioinformatic challenges of identifying and prioritizing the extensive sequence variants present in each patient. Success rates can be greatly improved if a large cohort of patient data is assembled in which sequence variants can be systematically analyzed, annotated, and interpreted. Since Spring 2012, we have established a grass-roots effort facilitated by the United Mitochondrial Disease Foundation (UMDF) called the "Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium". We have engaged and united > 100 mitochondrial disease experts to identify and prioritize specific WES data analysis needs of the global mitochondrial disease community. We are moving forward toward our common goal of establishing a central data resource for the coherent compilation, organization, annotation, and analysis of WES data in dual genomes from suspected mitochondrial disease patients. Exome and phenotype data will be united in a publicly accessible, secure, and userfriendly web-based tool. MSeqDR prototype development is underway with investigators from 9 countries. A global MSeqDR will fill the existing void in bioinformatics tools and knowledge necessary for efficient WES data interpretation in the mitochondrial disease community. Oral 4. Miscellaneous

1

WTCMR, Newcastle University, Newcastle upon Tyne, United Kingdom; 2 University College London, London, United Kingdom; 3Oxford University, Oxford, United Kingdom Background: The heterogeneity of genotype and phenotype associated with mitochondrial disease has hindered clinical studies. The development of a large contemporaneous cohort of mitochondrial disease patients with defined genetic and/or biochemical defects offered an unprecedented opportunity for a national (UK) perspective on mitochondrial disease. Aims: The MRC Mitochondrial Disease Patient Cohort UK database was developed to facilitate preliminary clinical trials, with the

O-019 Elucidation of cerebral folate transport across the choroid plexus reveals a common mechanism shared by different metabolic disorders Steinfeld R1, Grapp M1, Schweizer M2, Gärtner J1 1

Dept of Pediatrics, Univ Med Cent, Goettingen, Germany; 2Centre Mol Neurobiology, Hamburg, Germany

S108 Loss of folate receptor alpha (FRα) function is associated with cerebral folate transport deficiency and childhood-onset neurodegeneration. To clarify the mechanism of cerebral folate transport at the blood-CSFbarrier, we investigated the transport of 5-methyltetrahydrofolate in polarized cells. We uncover FRα-positive intralumenal vesicles within multivesicular bodies and demonstrate the directional cotransport of human FRα and labelled folate from the basolateral to the apical membrane in rat choroid plexus cells. Both, the apical medium of FRα-transfected rat choroid plexus cells and human CSF contain FRα-positive membrane vesicles that express characteristic markers of exosomes. The importance of FRα-expressing exosomes was substantiated by Western blotting with human CSF from both healthy individuals and patients with cerebral folate transport deficiency. FRα could be detected in CSF of controls but was absent from patients that carry pathogenic FOLR1 mutations. Further, the CSF samples from patients with Kearns-Sayre syndrome, a mitochondrial disorder with disturbed blood-CSF transport of 5MTHF, showed severely reduced FRα expression in the CSF. Thus, deficiency of FRα-expressing exosomes correlates with loss of 5-methyltetrahydrofolate in the CSF. Our results unravel the mechanism of folate transport across the blood-CSF barrier and provide new insight into mitochondrial disorders that are associated with cerebral folate deficiency.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 oxidase (PNPO) deficiency, functional shortage of vitamin B6 leads to severe neonatal convulsions. Supplementation of PLP can be quite successful, but developmental delay still occurs despite early treatment. In CSF of healthy humans, pyridoxal (PL), PLP, pyridoxic acid and pyridoxamine are present. In CSF of PNPO deficient patients, decreased concentrations of PLP and PL have been reported. However, the consequences of PNPO deficiency inside brain cells are unknown. We investigated uptake and metabolism of B6 vitamers by Neuro2A (mouse neuroblastoma) cells and show that incubation with different vitamers results in different intracellular B6 vitamer profiles. Next, we generated an in vitro model of PNPO deficiency by RNA interference of the PNPO gene. By Western Blot, we demonstrate effective silencing (<30% protein expression). In PNPO knockdown cells, pyridoxine and pyridoxine phosphate accumulate, whereas formation of PL and PLP from pyridoxine is lower compared to control cells. In conclusion, PNPO deficiency not only results in shortage of PLP, but also in a disbalance between intracellular vitamers. Our continuing studies aim at finding the conditions to restore the brain cell B6 vitamer profile. O-022 PNPO mutations in patients with pyridoxine dependent epilepsy

O-020 Mechanisms underlying interallelic complementation: lesson learnt from phenylketonuria, glutaric aciduria type 1 and Alzheimer disease

Plecko B1, Paul K2, Paschke E2, Schmiedel G3, Maier O4, Hasselmann O4, Kanz S5, Connolly M6, Wolf N7, Clayton P8, Mills P8, Struys E9, Stockler-Ipsiroglu S6, Hofer D2 1

Danecka MK1, Reiss DD1, Nardecchia F2, Muntau AC1, Gersting SW1 1

Dep of Molec Peds, Hauner Child Hosp LMU, Munich, Germany; Dep of Child and Adol Neuro, Psych,Rehab, Rome, Italy

2

Interallelic complementation (IAC) is a well-known phenomenon in the field of inborn errors of metabolism making genotype-phenotype correlation ambiguous especially in compound heterozygosity. However, the underlying molecular and structural mechanisms are still unclear. To understand the mutual influence of interacting subunits in assembling heterooligomers, we analyzed proteins localizing to different subcellular compartments: phenylalanine hydroxylase (PAH, cytosol), glutaryl-CoA dehydrogenase (GCDH, mitochondria) and presenilin 1 (PS-1, membrane). We investigated protein stability, binding affinity, and composition of oligomers in living cells as well as the intracellular protein amount of each variant in homozygosity and compound heterozygosity. We showed positive and negative IAC and demonstrated that protein amount and function as endpoints of IAC in oligomeric proteins strongly depends on the composition of heterooligomers, which can be modulated by the individual differences in stability and affinity of the subunits. Moreover, alteration of the interaction between assembling subunits modulate the formation of higher protein complexes as seen for the γ-secretase complex. Taken together, IAC is based on different mechanisms depending on the genotype and the functional context of the protein. Therefore, in compound heterozygosity, heterogeneous populations of oligomers with varying stability and function are anticipated to have a significant impact on the patient's clinical phenotype. O-021

Childrens Univ. Hosp., Zurich, Switzerland; 2Metab Lab. Univ. Childrens Hosp., Graz, Austria; 3Childrens Hosp., Esslingen, Germany; 4 Kinderspit., St. Gallen, Switzerland; 5Childrens Hosp., Landshut, Germany; 6C&W Hosp., Vancouver, Canada; 7Dep. Child Neurol. VU, Amsterdam, Netherlands; 8Clin.&MOlec.Genet., London, United Kingdom; 9Dep. Clin. Chem. VU, Amsterdam, Netherlands So far resistance to pyridoxine but response to pyridoxal 5´-phosphate (PLP) was thought to distinguish between mutations of the antiquitin (ALDH7A1) or PNPO gene. We report on 9 individuals out of 5 families with partial or complete response to pyridoxine and proven mutations in the PNPO gene. Patients were products of 34th to 40th GWs and all had neonatal seizures. 8 patients received pyridoxine with prompt response in 4, delayed in 1 and initial unresponsiveness in 3. Biomarkers for antiquitin deficiency and sequencing of the ALDH7A1 gene was normal in all patients. Sequencing of the PNPO gene revealed a novel homozygous missense mutations in 8 patients/5 families and compound heterozygosity in 1 patient. Pathogenicity of the 3 novel mutations was suggested by affection of the PLP and FMN binding site and proven by expression studies in E.coli with residual enzyme activity ranging from 50% to 0% of wild type. Only 2 patients remained seizurefree on pyridoxine monotherapy. Switch to PLP lead to status epilepticus in 2 patients tested. Certain mutations of the PNPO gene show a clear pyridoxine response and unresponsive to PLP. Patients with PDE but normal AASA and/or PA should undergo mutation analysis of the PNPO gene. O-023 Regulation of insulin signaling by sialylation: can sialidase deficiency contribute to diabetes?

Albersen M1, Jans JJ1, Bosma M1, Knoers N1, Verhoeven-Duif N1

Pshezhetsky AV1, Dridi L1, Fougerat A1, Pan X1, Seyrantepe V1, Thibault P2, Mitchell GA1, Heveker N1, Cairo CW3, Issad T4, Hinek A5

1

1

Consequences of PNPO deficiency for vitamin B6 in Neuro2A cells

Dep Med Genet, Univ Med Center (UMC), Utrecht, Netherlands

Pyridoxal phosphate (PLP), the active form of vitamin B6, is crucial for normal brain development and functioning. In pyridox(am)ine phosphate

CHU Ste-Justine, Univ Hosp Res Center, Montreal, Canada; 2Institute of Immunology and Cancer, Montreal, Canada; 3Alberta Glycomics Cent, Univ of Alberta, Edmonton, Canada; 4Institut Cochin, Paris, France; 5The Hospital for Sick Children, Toronto, Canada

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Neuraminidases (sialidases) catalyze the removal of sialic acid residues from sialylated glycoconjugates. We now report that mammalian neuraminidase 1 (Neu1), in addition to its catabolic function in lysosomes, is transported to the cell surface where it is involved in regulation of insulin signaling. Insulin binding to its receptor rapidly induces interaction of the receptor with Neu1, which hydrolyzes sialic acid residues in the glycan chains of the receptor and, consequently, induces its activation. Cells from sialidosis patients with a genetic deficiency of Neu1 show impairment of insulin-induced phosphorylation of downstream protein kinase AKT, while treatment of these cells with purified Neu1 restores signaling. Genetically-modified mice with ~10% of the normal Neu1 activity exposed to a high-fat diet develop hyperglycemia and insulin resistance twice as fast as their wild type counterparts. Together, these studies identify Neu1 as a novel component of the signaling pathways of energy metabolism and glucose uptake. O-024 The frequencies of different inborn errors of metabolism in adult metabolic units: Report of 14 centres from the adult metabolic physicians group Tchan MC1, Sirrs S2, Merkel M3, Besson G4, Douillard C5, Glamuzina E6, Janssen M7, Lachmann R8, Langendonk J9, Maillot F10, Mochel F11, Scarpelli M12, Tawfeg B13, Hollak C14 Genetic Medicine, Westmead Hospital, Sydney, Australia; 2Vancouver Hospital, Vancouver, Canada; 3Asklepios Clinic, Hamburg, Germany; 4 CHU Grenoble, Grenoble, France; 5Hopital Jeanne de Flandre CHRU de Lille, Lille, France; 6Starship Childrens Hospital, Auckland, New Zealand; 7Nijmegen Medical Centre, Nijmegen, Netherlands; 8Charles Dent Metabolic Unit, London, United Kingdom; 9Erasmus Medical Centre, Rotterdam, Netherlands; 10Hôpital Bretonneau, CHU de Tours, Tours, France; 11 Hospitalier Pitié-Salpêtrière, Paris, France; 12University Hospital GB Rossi, Verona, Italy; 13Hamad Medical Corporation, Doha, Qatar; 14Academic Medical Centre, Amsterdam, Netherlands 1

Background: There are few centers which specialize in the care of adults with inborn errors of metabolism (IEM). To anticipate facilities needed at these centers, it is of interest to know the distribution of the different disorders. Methods: A survey was distributed through the list-serve of the Adult Metabolic Physicians Group asking clinicians for number of patients with confirmed diagnoses, types of diagnoses and age at diagnosis. Results: 14 adult clinics responded to our survey with information on 4884 patients. Of those 4884 patients, 538 were excluded for diagnoses not within the IEM spectrum (eg. genetic dyslipidemias, hemochromatosis etc). The most common diseases followed by the adult clinics were PKU (24%), lysosomal storage disorders (Fabry disease (9.8%),Gaucher disease (3.9%)) and mitochondrial disorders (9.7%). Among the disorders that can present with acute metabolic decompensation, the urea cycle disorders, specifically OTC deficiency, was most common (2%), followed by porphyria (2%) and MSUD (1%). Patients with mitochondrial disease, lysosomal storage disorders, X-ALD and porphyria were frequently diagnosed as adults. Conclusions: A wide spectrum of IEM's is followed at adult centers. Specific knowledge of these disorders is needed to provide optimal care including up-to-date knowledge of treatments and ability to manage acute decompensation.

S109 Oral 5. Mitochondrial disorders II O-025 Phenylbutyrate for therapy of pyruvate dehydrogenase complex deficiency Ferriero R1, Bourton A2, Bonafé L3, Baumgartner M4, Kerr D5, Manco G6, Brivet M2, Brunetti-Pierri N1 1

TIGEM, Naples, Italy; 2AP-HP Hôpital de Bicêtre, Le Kremlin Bicêtre, France; 3University of Lausanne, Lausanne, Switzerland; 4 Div Metab Dis, Univ Children's Hospital, Zurich, Switzerland; 5 CIDEM, Case Western Reserve Univ, Cleveland, OH, United States; 6 IBP, Naples, Italy Deficiency of pyruvate dehydrogenase complex (PDHC) is one of the most common inborn errors of mitochondrial metabolism presenting with neurological degeneration and lactic acidosis. Most cases result from mutations in the X-linked gene encoding for E1-alpha subunit (PDHA1) whereas fewer patients carry mutations in genes for E1-beta (PDHB), E2 (DLAT), E3 (DLD), E3BP (PDHX) or phosphatase (PDP1). PDHC activity is regulated by phosphorylation and four PDK isoforms (PDK1 to 4) phosphorylate E1-alpha thereby reducing enzyme activity. We have previously shown that phenylbutyrate increases PDHC residual activity by increasing unphosphorylated enzyme and reduces lactic acidosis. Phenylbutyrate and not its bio-product phenylacetate increases PDHC activity and by means of recombinant enzymes, we found that it prevents E1α phosphorylation through inhibition of PDK1, PDK2, and PDK3 but not PDK4. We found that most PDHA1 missense mutations are responsive to phenylbutyrate whereas mutations affecting Arg349-alpha, involved in interaction with E1-beta, and large deletions affecting PDHA1 resulting in undetectable protein, were consistently unresponsive to the drug. PDHB, PDHX, and DLD missense mutations were all responsive. The results of the present study may help predicting whether a patient with PDHC deficiency is responsive to phenylbutyrate based on affected gene, type, and location of the mutation. O-026 Clinical and molecular characterisation of PYCR1-related cutis laxa Fischer B1, Dimopoulou A1, Gardeitchik T2, Schlack C1, Fauler B3, Mundlos S1, Nijtmans L2, Wollnik B4, Morava E5, Kornak U1 1

Charité Universitätsmedizin, Berlin, Germany; 2St. Radboud University Medical Centre, Nijmegen, Netherlands; 3Max-Planck-Institut f. Molek. Genetik, Berlin, Germany; 4University of Cologne, Cologne, Germany; 5 Tulane University Medical Center, New Orleans, United States Autosomal recessive cutis laxa type 2B (OMIM #612940) is a segmental progeroid disorder characterized by intrauterine growth retardation, lax and wrinkled skin, a typical triangular face, and intellectual disability. This variable phenotype is often diagnosed as gerodermia osteodysplastica wrinkly skin, or De Barsy syndrome. Mutations in the PYCR1 gene encoding pyrroline-5-carboxylate reductase 1 were identified to be causative. This protein is part of a conserved metabolic pathway (proline-cycle) described to generate cytoplasmic NAD(P)+ via synthesis of proline. In this study, we analysed 32 patients from 26 families. In comparison with all patients identified so far, we could further delinate the genetic and clinical spectrum and found PYCR1 to be the second most frequent disease-causing gene in individuals with

S110

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autosomal recessive cutis laxa. A detailed analysis of the subcellular distribution revealed an exclusive mitochondrial localization of PYCR1. After in vitro RNAi-induced depletion of PYCR1 we found a severe fragmentation of the mitochondrial network, a decreased membrane potential and an increased apoptosis rate. Thus, we conclude a role of PYCR1 in the regulation of the mitochondrial redox state, which influences mitochondrial dynamics and possibly metabolic activity. This combination of defects is likely to be a key event in the pathogenesis of ARCL2B.

mitochondrial trifunctional protein interacts with the 75, 51, and 24kDa subunits of the NADH binding domain arm of complex I. ETF dehydrogenase, the common electron acceptor for all acyl-CoA dehydrogenases was seen to interact with the 75, 24 and 49kDa (NDUS2) subunits of complex I. NDUS2 is a membrane associated iron-sulfur protein constituting part of the QH2 electron transfer complex. ETFDH also interacts with complex III in supercomplexes. These findings substantiate the intimate physical and functional association of FAO and OXPHOS, and further define the molecular architecture of mitochondrial energy metabolism.

O-027

O-029

Defective mitochondrial RNA processing causes mitochondrial energy failure in HSD10 disease, also known as 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency

EPI-743 reduces seizure frequency in RARS2 defect syndrome

Friederich MW1, Chatfield KC1, Coughlin II CR1, Thomas JA1, Gallagher RC1, Lovell MA2, Wanders RJA3, Wartchow EP2, Van Hove JLK1

Martinelli D1, Catteruccia M1, Klein M2, Bevivino E1, Thoolen M2, Piemonte F1, Pastore A1, Tozzi G1, Pontrelli G1, Bertini E1, Miller G2, Dionisi-Vici C1 1

1

Dept Pediatrics, Univ Colorado, Aurora, Colorado, United States; Dept Pathology, Univ Colorado, Aurora, Colorado, United States; 3 Lab Genetic Metabolic Dis, Acad Med Ctr, Amsterdam, Netherlands

Bambino Gesù Children's Hospital, IRCCS, Rome, Italy; 2Edison Pharmaceuticals, inc, Mountain View, California, United States

2

The recognition of the HSD10 gene product as a component of mitochondrial RNAse-P, in addition to 2-methyl-3-hydroxybutyryl-CoA dehydrogenase activity, created a new hypothesis for the disease mechanism of HSD10 disease. Muscle, heart and liver tissues were analyzed from a male infant who succumbed to cardiac failure secondary to molecularly proven HSD10 disease. There was proliferation and ultrastructural disruption of the mitochondria in all tissues. Respiratory chain enzyme analysis and blue native PAGE showed disruption of the activities and assembly of complexes I, III, IV, and V. The mRNAs of all RNase P subunits, including MRPP2 encoded by HSD17B10, were preserved in heart and overexpressed in muscle. There were highly elevated amounts of unprocessed pre-tRNAs, and elevated levels of unprocessed transcripts encoding mitochondrial subunits of complexes I, III, IV and V, indicating deficient RNase P activity. The activities and protein amounts of other mitochondrial enzymes such as citrate synthase, complex II, and ANT1 were normal in heart, increased in muscle, and greatly increased in liver, indicating differential tissue upregulation which might explain differences in degrees of organ dysfunction. This study provides the first direct evidence of abnormal mitochondrial RNA processing causing mitochondrial energy failure in HSD10 disease.

Background: RARS2 defect syndrome is a neurodegenerative disorder caused by mutations in mitochondrial arginyl-transfer RNA synthetase, characterized by severe encephalopathy, intractable seizures, spasticdystonic quadriplegia. No specific treatment is available. EPI-743 is a novel molecule developed for the treatment of inherited mitochondrial diseases. In a phase 2A open-label trial in genetically-confirmed Leigh syndrome children, EPI-743 significantly improved neurological outcome. Our aim was to determine potential clinical benefit of EPI-743 in RARS2 patients. Patients and methods: Four RARS2 patients (2 males and 2 female, ages 5-13y) were treated with EPI-743 at the dose of 100mg TID for one year under compassionate use. Primary outcome measures were change in seizure frequency and improvement in redox state measured by glutathione levels in blood cells. Results: All children exhibited reversal of disease progression regardless of baseline disease severity. Two children showed resolution of epileptic status and two significant reduction of seizure frequency and duration. In addition, all showed significant improvement in standardized measures of neuromuscular function (Newcastle Pediatric Mitochondrial Disease Scale and PedsQL Neuromuscular Module) and redox state as assessed by glutathione levels. No drug-related adverse events were recorded. Conclusions: In our RARS2 patients EPI-743 reduced seizures frequency and improved neuromuscular function and redox state. Conflict of Interest declared.

O-028 O-030 Functional and physical mapping of the architecture of mitochondrial energy metabolism

Treating nutrient-sensing signaling network changes that modulate the cellular and metabolic sequelae of mitochondrial diseases

Wang Y1, Goetzman E1, Palmfeldt J2, Gregersen N2, Vockley J1 1

University of PIttsburgh School of Med, Pittsburgh, United States; Aarhus University Hospital, Aarhus, Denmark

Falk MJ1, Peng M2, Tsukikawa M1, Ostrovsky J1, Polyak E1, McCormack S3, Dingley S1, Nakamaru-Ogiso E4, Gasser DL2, Baur J5, Zhang Z6

2

1

Background: Fatty acid beta-oxidation (FAO) and oxidative phosphorylation (OXPHOS) are key pathways involved in mitochondrial energy metabolism. Reducing equivalents from FAO enter OXPHOS through complexes I and III. We have previously partially purified a multifunctional FAO complex that is physically associated with OXPHOS supercomplexes and promotes metabolic channeling. Here we characterize the effects of long chain acyl-CoA dehydrogenase deficiency in mice on this interaction. Results: Blue native polyacrylamide gel electrophoresis of mitochondria revealed a decrease of ETC supercomplexes in mutant animals, impacting catalytic efficiency. Similarly, the amount of FAO proteins and the flux of reducing equivalents from FAO to OXPHOS were decreased. Co-immunoprecipitation and cross linking showed that the

Hum Gen Div, CHOP and Upenn SOM, Philadelphia, United States; Genetics Dept, Univ of Penn SOM, Philadelphia, United States; 3 Endocrin Div, CHOP, Philadelphia, United States; 4Biochem and Biophys Dept, Upenn SOM, Philadelphia, United States; 5 IDOM and Physiology Dept, Upenn SOM, Philadelphia, United States; 6Bioinformatics Dept, Philadelphia, United States 2

The integrated nutrient-sensing signaling network (NSSN) plays a central role in modulating the cellular and metabolic sequelae of primary RC dysfunction in Caenorhabditis elegans, mouse, and human disease models. Key NSSN components include AMPK, PPAR, sirtuins, FOXO, and mTORC1, which sense energy and nutrient levels and mediate cellular and mitochondrial proliferation. Relative magnitude and direction of NSSN node changes in

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S111

RC disease are highly tissue and nutrient-constrained. Pharmacologically targeting central NSSN changes reverses 'maladaptive' physiologic sequelae of RC disease. Our data show that nicotinic acid, probucol, resveratrol, AICAR, and rapamycin can significantly ameliorate, or even reverse, physiologic sequelae of mitochondrial disease to restore cell and animal health. Notably, significant improvements in lifespan and respiratory capacity occur with nicotinic acid supplementation in RC mutant worms and human cells, respectively. This highlights a central signaling role of NAD+ deficiency in eliciting cellular responses to RC dysfunction. Further, a central role of mTORC1 signaling alterations in primary RC disease pathophysiology is supported both by rescue of NSSN signaling alterations and restoration of cellular and animal health with either probucol or rapamycin in both human cells and CoQ-deficient B6.Pdss2kd/kd mice. Systematic profiling and targeting of NSSN changes can enable personalized therapy for mitochondrial diseases.

Objective: Based on the demonstration that Phe acts as inhibitor of both CNS and peripheral tryptophan hydroxylase isoforms, our study was aimed to evaluate the possibility of inferring brain serotonergic deficit from measurement of a peripheral serotonin metabolite. Material and Methods: Mice with different degrees of HPA (normal controls, mild-HPA, mild-PKU and classic-PKU) were submitted to the concomitant biochemical assessment of brain serotonin and peripheral 5hydroxyindoleacetic acid (5-HIAA), the most important serotonin catabolite. Results: Data showed a negative correlation between blood Phe and brain serotonin levels in HPA mice and a positive correlation between blood 5-HIAA and brain serotonin concentrations. Conclusion/Discussion: We showed that blood 5-HIAA and brain serotonin are congruent in HPA animal model, and suggest 5-HIAA as a potential peripheral marker for the assessment of brain serotonin depletion in PKU patients.

Oral 6. Amino acid disorders

O-033

O-031

Diagnosis and management of hepatorenal tyrosinaemia: results of an international workshop

Enzyme replacement therapy for homocystinuria Mayorandan S1, Meyer U1, Das AM1 Kraus JP1, Bublil E1, Park IS1, Majtan T1, Salzman AL2 1

Clin Paed Metab Dis, Hannover Med School, Hannover, Germany

1

Dept. Peds, Univ. of Colorado Sch. Med., Aurora, United States; 2 Orphan Technologies Ltd., Zurich, Switzerland Cystathionine beta synthase (CBS) deficient homocystinuria is the most common inherited defect of sulfur amino acid metabolism. CBS condenses homocysteine and serine to cystathionine, which is then cleaved to cysteine by cystathionine gamma lyase. Homocystinuria is characterized by elevated levels of plasma Hcy and reduced concentrations of cystathionine and cysteine. These abnormalities are manifested in thrombotic vascular disease, connective tissue aberrations, and mental retardation. About 40% of the patients respond to pyridoxine therapy and the remaining ones are subjected to a low methionine diet supplemented with betaine to promote the re-methylation of homocysteine to methionine. This therapy is difficult to comply with and has little impact on downstream metabolites. We envisioned that administration of CBS to the circulation may alter the cellular equilibrium of sulfur amino acids. We found that administration of a PEGylated truncated human CBS enzyme to homocystinuric mice resulted in a ~80% decrease in Hcy, ~900% increase in cystathionine, and normalization of cysteine concentrations, indicative of reactivation of the intracellular transsulfuration pathway. The data strongly suggest that CBS enzyme replacement therapy is a promising approach for treatment of homocystinuria, and that ERT for metabolic deficiencies may not necessitate introduction of an enzyme into its native intracellular environment. Conflict of Interest declared. O-032 Identification of a peripheral marker of central serotonin alterations in phenylketonuria. 1,2,3

Pascucci T

4

5

1,2,3

, Carducci C , Leuzzi V , Puglisi-Allegra S

1 Dept Psychol, Univ Sapienza, Rome, Italy; 2Center Daniel Bovet, Univ Sapienza, Rome, Italy; 3IRCCS Santa Lucia Foundation, Rome, Italy; 4Dept Exper Med, Univ Sapienza, Rome, Italy; 5Dept Child Neurol Psychia, Univ Sapienza, Rome, Italy

Background: Cumulative evidences indicate that even mildly elevated blood Phenylalanine (Phe) levels increase the risk of neurocognitive impairment in phenylketonuria (PKU) patients. While our preclinical studies showed that serotonin is the most affected amine in brain of PKU mice, for obvious ethical reason, this result can not be confirmed in PKU patients.

Introduction: Hepatorenal Tyrosinaemia I (HT1) often presents with severe renal and liver dysfunction. HT1 lacks solid evidence-based recommendations for diagnosis, treatment, monitoring and follow-up. Methods: In our international workshop we collected retrospective data from 21 centres (Europe and Israel) about diagnosis, treatment, monitoring and outcome of HT1 based on data of 158 patients. Results: We found considerable differences among centres regarding diagnostic tools, treatment and monitoring. For example, dietary treatment differs among centres from simple protein restriction to calculation of phenylalanine and tyrosine intake. Target plasma levels of nitisinone and its monitoring were highly variable. For example, target peak values of nitisinone vary between 50 and 100 μmol/l. The frequency of outpatient visits, types of instrumental and laboratory examinations were inhomogeneous too. Follow-up procedures to monitor outcome such as ophthalmological examinations are performed in 15/21 centres, cardiac examinations in 14/21 centres only. Target values of laboratory tests, e.g. the maximal acceptable plasma level of tyrosine, were subject to discussion varying between 200 and 800 μmol/l. Conclusion: In order to optimize the handling and outcome of HT1 patients data-based recommendations on an international level are essential. This may help to standardize treatment and monitoring, thus optimizing outcome. O-034 A potentially treatable condition presenting with developmental delay and autistic behaviour in two patients with missense changes in the BCKDK gene Garcia-Cazorla A1, Oyarzabal A2, Fort J3, Robles C4, Bodoy S5, Palacín M3, Castejón E1, Merinero B2, Ruiz-Sala P2, Dopazo J6, Nunes V7, Ugarte M2, Artuch R1, Rodríguez-Pombo P2 1

Neurometabolic Unit, HSJD and CIBERER, Barcelona, Spain; 2CSICUAM, CIBERER U746, IDIPAZ, Madrid, Spain; 3Institute for Research in Biomedicine, Barcelona, Spain; 4Unidad Neuropediatria, HU San Cecilio, Granada, Spain; 5CIBERER U731, Barcelona, Spain; 6Instituto Príncipe Felipe, Valencia, Spain; 7CGMM-IDIBELL-CIBERER, Barcelona, Spain Inactivating mutations in the BCKDK gene encoding for the kinase responsible for negative regulation of the branched-chain keto-acid dehydrogenase complex, have been recently associated to a potentially treatable condition of autism. Neurological outcome after treatment has not been described.

S112 We report two unrelated patients with severe developmental delay, microcephaly, autistic traits, EEG discharges, growth retardation and markedly decreased levels of branched-chain amino acids (BCAA) in body fluids. They are carrying two distinct, novel missense, homozygous p.R174G and p.L389P BCKDK mutations. Functional consequences of those mutations were analyzed by tandem-mass spectrometry, cell-based experiments using wt-BCKDK-lentiviral constructs, recombinant human BCKDK expression and purification, and in vitro kinase activity. Fibroblasts derived from patients presented undetectable or fairly detectable levels of BCKDK protein and its phosphorylated substrate (phospho-E1α). Moreover, in vitro kinase assays showed loss-offunction of the recombinant BCKDK mutants.Patients started a baseline high-rich protein diet together with oral BCAA supplementation. After treatment, plasma BCAA levels were normalized. Growth parameters, developmental items and behaviour have notable improved in the patient with the longest follow-up (6 months). These data support the involvement of the identified changes in the pathological condition of both patients and describe a new potentially treatable disorder of the BCAA metabolism. O-035 New insights on mitochondrial alterations for Maple Syrup Urine Disease (MSUD) due to a deficiency in mitochondrial protein phosphatase 2C (PP2CM) Oyarzabal A1, Sánchez-Aragó M2, Ugarte M1, Rodríguez-Pombo P1 1

CEDEM, CBMSO-UAM, CIBERER, Madrid, Spain; 2CBMSO-UAM, CIBER, Madrid, Spain Branched-chain amino acid (BCAA) metabolism undergoes through a rate limiting decarboxylation catalyzed by BCKDH complex. Such complex is regulated by phosphorylation by a specific kinase and a protein phosphatase – PP2Cm, positive regulator of the complex activity. A defect in PP2Cm expression, besides causing a MSUD phenotype, is potentially related to stress signaling. In this work, we focus in greater depth on the relationship between PP2Cm deficiency and mitochondrial alterations by studying parameters related to mitochondrial function as network mitochondrial morphology, ROS generation and respiratory function, in presence or absence of α-ketoisocaproate (KIC) 5mM. In absence of KIC, even though PP2Cm-deficient patient fibroblasts' mitochondria do not show major morphological alterations –evidenced through Mitotracker® probing, an almost twofold increase in Reactive Oxygen Species (ROS) measured as H2O2 with H2DCFDA probe or O2- with MitoSOXTM by Flow Cytometry was detected. This correlates with a lowered OligomycinSensitive Respiration (OSR), observed by Seahorse Bioscience technology. Basal or maximal respiration capacity remained unaltered. Neither mitochondrial superoxide levels nor OSR changed from their basal condition in control cell lines or PP2Cm-deficient fibroblasts upon treatment with KIC. These data suggest mitochondrial alterations on PP2Cm-deficient fibroblasts are probably unrelated to BCAA levels, suggesting other roles for PP2Cm besides the regulation of BCKDHc. O-036 AMPK regulates urea cycle genes in response to dietary protein intake Haskins N1, Heibel SK2, McGuire PJ3, Rayavarapu S1, Nagaraju K1, Hathaout Y1, Tuchman M1, Brown K1, Caldovic L1 1

Children's National Medical Center, Washington DC, United States; USDA-ARS, Beltsville MD, United States; 3National Human Genome Research Institute, Bethesda MD, United States

2

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 The urea cycle converts ammonia, the toxic product of protein catabolism, into urea. Although urea cycle enzyme levels rise in response to a high protein diet, the signaling networks that sense dietary protein intake and trigger this increase have not been identified. The aim of this study was to identify signaling pathway(s) that respond to protein intake and regulate expression of urea cycle genes. Mice were placed on control or high (HP) protein diets. Liver protein and mRNA were isolated and an integrated proteomic and transcriptomic analysis was performed. HP diet led to increased expression of mRNA and enzymes in amino acid degradation pathways, and decreased expression of mRNA and enzymes in carbohydrate and fat metabolism. These changes implicated AMPK as a possible regulator. To test if AMPK also regulated the expression of urea cycle genes, primary human hepatocytes were treated with AICAR, an activator of AMPK. This led to an increased expression of urea cycle genes CPS1 and OTC at the mRNA level. This observation strongly suggests that AMPK signaling regulates the expression and activity of urea cycle enzymes and could lead to the development of new therapies for partial defects in urea cycle enzymes. Oral 7. Methods and treatment strategies O-037 Optimal methods for diagnosis of systemic primary carnitine deficiency suspected by newborn screening Gallant NM1, Wilnai Y2, Lee C3, Lorey F4, Feuchtbaum L4, Tang H4, Leydiker K5, Enns GM2, Packman S3, Lin HJ6, Wilcox W1, Cederbaum SD1, Abdenur JE5 Department of Pediatrics, UCLA, Los Angeles, United States; 2Div Med Genetics, Stanford Univ, Stanford, United States; 3Dep Peds, Inst Hum Genet, UCSF, San Francisco, United States; 4Genet Disease Branch, CA Dep Health Serv, Richmond, United States; 5Div Met Dis, CHOC Children's, Orange, United States; 6Div Med Genet, Harbor-UCLA, Torrance, United States 1

Background: This study uses California Newborn Screening (NBS) Program data to provide further information on optimal methods for diagnosis of systemic primary carnitine deficiency (PCD). Methods: California NBS data were collected (September, 2005-June, 2012). Medical records were reviewed for cases identified at 6 metabolic centers throughout California. Biochemical characteristics were analyzed for newborns identified with PCD (confirmed by molecular or fibroblast carnitine uptake analysis; 13 cases), carnitine deficiency secondary to a maternal disorder (SCD; 13 cases), and false-positive results (FP; 85 cases). Results: 3,618,698 newborns were screened. Of 1,030 positive screens, 48 were closed as PCD statewide (positive predictive value <5%). The confirmatory process was lengthy (median duration 3.5 months; range 0.75-13). Plasma carnitine was similar in PCD and SCD. Median newborn urine free carnitine (nmol/mg creatinine) was markedly elevated in PCD (401; range 45-1030) compared to SCD (14; range 5-61) or FP (28; range 7-516). Multivariate classification tree analysis using newborn and maternal plasma carnitine and newborn urine free carnitine correctly predicted 12/13 (92%) PCD cases, 12/13 (92%) SCD cases, and 81/85 (95%) FPs. Conclusion: Results support the utility of newborn spot urine free carnitine in addition to newborn and maternal plasma carnitine in diagnosing PCD suspected by NBS.

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O-038

O-040

Development of AAV8-mediated gene therapy clinical trial for Crigler-Najjar syndrome type I: optimization of liver-specific expression cassette

Antisense oligonucleotides as therapeutic agents in classic galactosemia

Pastore N1, Auricchio A1, Brunetti-Pierri N1

Coelho AI1, Lourenço S1, Trabuco M1, Silva MJ1, Tavares de Almeida I1, Vicente JB1, Rivera I1

1

1

Crigler-Najjar syndrome type I is a severe inborn error of metabolism due to mutations of liver-specific UGT1A1 gene resulting in severe hyperbilirubinemia. Affected patients are treated with phototherapy to prevent irreversible brain damage. Current treatment is unsatisfactory and patients often undergo liver transplantation. Alternative therapies are highly needed and gene therapy has the potential to provide a definitive cure for this disease. Our study focused on design and optimization of an AAV8 vector for gene therapy of Crigler-Najjar syndrome. We investigated two liver specific promoters expressing UGT1A1 inserted in AAV8 vectors in Gunn rats, an animal model for Crigler-Najjar syndrome. The vector containing the TBG promoter resulted in reduction of hyperbilirubinemia at the dose of 7.4x10e13 genome copies (gc)/kg whereas correction was achieved with the lower dose of 2.7x10e13 gc/kg of vector including LP1 promoter. The injection of lower dose of 1.1x10e13 gc/kg of a vector bearing codon optimized human UGT1A1 cDNA resulted in further improvement of efficacy. In summary, our study shows that AAV8 with codon optimized UGT1A1 gene under the control of LP1 results in sustained correction of hyperbilirubinemia in Gunn rats. These data form the preclinical basis for development of a gene therapy trial for Crigler-Najjar syndrome.

Introduction: Classic Galactosemia, an inborn error of metabolism (IEM) caused by galactose-1-phosphate uridylyltransferase (GALT) deficiency, results from mutations in GALT gene. The second most prevalent mutation in Portugal is the intronic variation IVS8+13A>G, suggesting its pathogenicity. Aim: Functional characterization of IVS8+13A>G mutation at three levels (in silico, in vitro and in vivo) and correction of its deleterious effect by antisense oligonucleotides. Methods: In silico approach was employed to predict mutation's effect (s). In vitro studies analyzed the transcription profile of wild-type and mutant minigenes expressed in eukaryotic cell lines. In vivo studies analyzed GALT transcription profile of a homozygous patient. Finally, cell lines expressing both minigenes were treated with locked nucleic acids (LNA), specifically designed to recognize the mutation. Results: In silico, in vitro and in vivo analyses concurred in confirming IVS8+13A>G as a pathogenic mutation. This transition activates a cryptic donor site, immediately downstream the mutation, leading to exonization of the first 13 intronic nucleotides, originating a frameshift with inclusion of a premature stop codon (p.D274GfsX291). Alternative splicing was reverted in vitro by a LNA, which forced the usage of the canonical site. Conclusion: Antisense therapy opens new therapeutic perspectives, not only for Classic Galactosemia but for all IEM. PEst-OE/SAU/UI4013/2011;FCT-SFRH/BD/48259/2008;SPDM

TIGEM, Naples, Italy

O-039 Helper-dependent adenoviral vectors for liver-directed gene therapy of primary hyperoxaluria type 1 Castello R1, D'Aria S1, Borzone R1, Piccolo P1, Annunziata P1, Brunetti-Pierri N1 1

TIGEM, Naples, Italy

Primary hyperoxaluria type 1 (PH1) is an inborn error of liver metabolism due to deficiency of peroxisomal enzyme alanine:glyoxylateaminotransferase (AGT), which catalyzes conversion of glyoxylate to glycine. AGT deficiency results in overproduction of oxalate which ultimately leads to end-stage renal disease. Combined liver/kidney transplantation is the only therapeutic strategy available to prevent disease progression. Gene therapy is an attractive alternative to provide a definitive cure for PH1. Towards this goal, we investigated helper-dependent adenoviral (HDAd) vectors for liver-directed gene therapy of PH1. We injected PH1 mice with an HDAd encoding the AGT under the control of a liver-specific promoter and observed long-term normalization of urinary oxalate excretion. In contrast to saline-injected controls, no evidence of oxalate stones was found in kidneys of mice injected with the HDAd vector after challenge with ethylene glycol, a precursor of oxalate. We previously developed a minimally invasive method to improve the therapeutic index of HDAd based on balloon occlusion catheter to achieve preferential delivery of the vector to the liver (BrunettiPierri et al., 2012). This method may permit the use of clinically relevant doses of HDAd in humans. Based on risk:benefit assessment, PH1 is an attractive disease for clinical application of this method.

Met&Gen – iMed, Fac Pharm, Univ Lisbon, Lisbon, Portugal

O-041 When silent DNA speaks up and interrupts the message pseudoexon activation in inborn errors of metabolism Doktor TK1, Sabaratnam R1, Larsen VS1, Kozich V2, Desviat LR3, Andersen HS1, Andresen BS1 1

Biochem & Mol Biol, Univ. South Denmark, Odense, Denmark; Inst.Inher. Metab. Dis., Charles Univ., Prague, Czech Republic; 3Cent. de Biologia Molecular Severo Ochoa, Madrid, Spain

2

Introns make up more than 90% of a gene sequence and harbor the vast majority of the sequence variation in the human genome. Introns also harbor pseudoexons, which are exons that are normally suppressed, and not spliced into the mRNA. Intronic mutations may cause inclusion of pseudoexons and thereby disease. We previously reported how a deep intronic single nucleotide variation activates a MTRR pseudoexon, resulting in aberrant splicing and homocysteinuria. We hypothesize that pseudoexon activation is more frequently involved in human disease than currently believed, but that this disease mechanism has been heavily underreported due to the misconception that intronic sequence variations are neutral, technological difficulties and difficulties in recognizing splicing regulatory motifs located outside the splice sites. We have employed patient cells, minigenes, RNA-affinitity purification and RNA-seq analysis to investigate in more detail the molecular mechanisms and motifs involved when single nucleotide changes located outside the splice sites cause activation of

S114 pseudoexons in three genes that cause inborn errors of metabolism (PCCA, MTRR and GLA) and in three other genes (FGB, COL4A5 and CFTR). We have identified general and unique characteristics, which are important for correct prediction of this molecular defect mechanism and for development of therapeutic methods. O-042 The metabolic phenotype guiding the discovery of novel and potentially treatable intellectual disability genes van Karnebeek CD1, Horvath G1, Salvarinova R1, Lehman A2, Shyr C2, Ye C2, Nosova E2, Eydoux P2, Vallance H3, Sinclair G3, Ross CJ2, Wasserman W2, Stockler S1 Div Biochem Dis, BC Child Hosp, Vancouver, Canada; 2Dept Med Genet, BC Child Hosp, Vancouver, Canada; 3Biochem Genet Lab, BC Child Hosp, Vancouver, Canada

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Materials and methods: The GALC expression vectors for infantile and late-onset mutations were constructed with pSVL by PCR based mutagenesis. The COS1 cells transfected with the mutant expression vectors were treated with NOEV and subjected to enzyme assay and western blotting. Results: In vitro NOEV treatment at 1 μM caused 50% inhibition in wild and late-onset mutants. NOEV treatment at 10 μM caused 80% heat stabilization for p.I66M+p.I289V and 60% for p.G270D and p.G569S. In vivo NOEV treatment of transfected COS1 cells showed a high enhancement of the enzyme activity in late-onset mutations up to 50-65% of the normal. 30kDa mature enzyme proteins increased remarkably with NOEV treatment only in late-onset mutations. Conclusions: Until now bone marrow transplantation in presymptomatic patients is the only treatment option available in the world, which is invasive with a high mortality rate. We hope that NOEV will become a therapeutic alternative also for Krabbe disease.

1

Introduction: Intellectual disability (ID) is a lifelong, debilitating condition affecting 2.5% of the population worldwide. Our TIDEX project aimed to identify novel (potentially treatable) ID genes employing the utility of the metabolic phenotype. Methods: Criteria were applied to select patients for whole exome sequencing (WES): patient with unexplained, Mendelian ID plus metabolic abnormalities. WES was performed for trio's with customized bio-informatics and subsequent validation. Results: In 10 families meeting selection criteria, we discovered 7 new gene defects (various phases of functional validation), including carbonic anhydrase VA deficiency (siblings with hyperammonemia /-lactatemia), Rabosyn5 deficiency (early endosomal recycling defect in female with intractable epilepsy), DSCAML1 deficiency (neuronal arborization defect in male with progressive white matter loss), DMBT1 deficiency (epithelial glycoprotein defect in boy with diarrhea and skin lesions), complex IV gene (boy with multi-organ involvement); as well as biotin responsive gene and neurotransmitter homeostasis genes. New phenotypes of known genes were detected in 2, including RMND1 defect in boy with kidney failure and encephalomyopathy. Conclusions: Our success rate (>70%) emphasizes the advantages of the metabolic phenotype for gene discovery including: facilitation candidate gene hypothesis, validation causality identified variants, and targets for improved management / treatment with direct translation into patient care. Oral 8. CDG, lysosomal and peroxisomal disorders O-043 Chaperone therapy for Krabbe disease; Japanese late-onset mutations can be treated effectively by NOEV Hossain MA1, Higaki K2, Nanba E2, Suzuki Y3, Ozono K1, Sakai N1

O-044 Arginine improves peroxisomal biogenesis in fibroblasts of mild Zellweger spectrum patients Berendse K1, Ebberink MS1, IJlst L1, Poll-The BT2, Wanders RJA1, Waterham HR1 1

Lab Genetic Metab Dis, AMC, Amsterdam, Netherlands; 2Dep Paediatric Neuro, Emma Child Hosp, Amsterdam, Netherlands Background: Zellweger spectrum disorders are multisystemic disorders due to a defect in peroxisome biogenesis. In contrast to severely affected patients, who lack peroxisomes in all cells, mild patients may have a mixed population of cells lacking peroxisomes and cells containing peroxisomes. This peroxisomal mosaicism has been observed for specific missense mutations in different PEX genes, which encode components of the peroxisomal protein import machinery. Peroxisome biogenesis in these cells is improved when they are cultured at 30°C, indicating that these mutations affect the folding/stability of the encoded PEX proteins. Objective: We investigated whether peroxisome biogenesis in fibroblasts of mild patients with a missense mutation in PEX-1, -6 or -12 can be improved by promoting protein folding with the chemical chaperone arginine. Methods: Cells were incubated with arginine and studied for the following parameters: peroxisomal protein import capacity by catalase immunofluorescence, PEX1 protein levels, intra peroxisomal processing of peroxisomal thiolase and β-oxidation of very long chain fatty acids and pristanic acid. Results: Peroxisome biogenesis and function in fibroblasts with mild missense mutations in PEX-1, -6 and -12 are improved by addition of arginine. Arginine may be an interesting compound to promote peroxisome function in patients with a mild peroxisomal biogenesis disorders. O-045 Translating ManNac into a novel therapeutic agent for patients with GNE myopathy

1

Osaka University Graduate School of Medi, Suita, Osaka, Japan; Tottori University, Yonago, Japan; 3International University of Health, Otawara, Japan

2

Celeste F1, Latham L1, DeDios J2, Ciccone C2, Robinson C1, McKew J1, Huizing M2, Gahl WA2, Carrillo-Carrasco N1

Background: Krabbe disease is an autosomal recessive leukodystrophy caused by deficiency of the lysosomal enzyme galactocerebrosidase (GALC). We reported that N-octyl-4-epi-βvalienamine (NOEV) was effective for GM1-gangliosidosis (Higaki et al. 2011). In this study we found its enzyme stabilization and effective intracellular transport of late-onset GALC mutant proteins.

1 TRND, NCATS, NIH, Bethesda, MD, United States; 2NHGRI, NIH, Bethesda, MD, United States

Background: GNE Myopathy is a rare muscular disease caused by mutations in GNE, which encodes the key enzyme in the sialic acid biosynthetic pathway. Patients have progressive muscle weakness leading to marked disability. Hyposialylation of muscle glycoproteins likely

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 contributes to the pathophysiology. ManNAc, a pathway intermediate, prevents muscle weakness in the mouse model of GNE myopathy. Objectives: Translate ManNAc into a therapy for patients with GNE myopathy. Methods: A first-in-human Phase 1a, randomized, placebo-controlled, double-blind, single-dose study (ClinicalTrials.gov NCT01634750; IND No.78,091) was conducted to evaluate the safety, pharmacokinetics (PK), and pharmacodynamics (PD) of ManNAc. GNE myopathy subjects (n=22) were enrolled into 3 cohorts (Cohorts A: 3,000mg, B: 6,000mg, C: 10,000mg) with a 3:1 ManNAc-to-placebo ratio. Results: Grade I and II (CTCAE) adverse events were encountered, mostly unrelated to study drug. PK data are currently been evaluated. We utilized potential plasma biomarkers to evaluate the onset and duration of the PD response. Conclusions: It is critical to perform high-quality clinical trials to develop approved therapies for rare disorders. ManNAc, a promising therapy for GNE myopathy, is safe and well-tolerated. PK and PD data will guide the selection of dose and frequency for upcoming Phase Ib and IIa trials. Conflict of Interest declared.

S115 to nephrocalcinosis and renal failure. Combined liver-kidney transplantation is currently the only available treatment and safer therapies are highly needed. Gene therapy has the potential to provide a definitive cure for this disorder and recent trial in hemophilia B patients highlighted the great potential of serotype 8 adenoassociated viral (AAV8) vector for long-term hepatic expression of a therapeutic gene. We investigated the efficacy of liver-directed, AAV8mediated gene therapy in the PH1 mouse model. PH1 mice injected intravenously with 3.2x10e12 genome copies (gc)/kg and 6.4x10e12 gc/kg of an AAV8 vector expressing AGT under the control of a liverspecific promoter (AAV8-AGT) showed sustained normalization of urinary oxalate levels for at least 6 months post-injection. In contrast to saline controls, AAV8-AGT injected mice did not develop calcium oxalate stones after challenge with an oxalate precursor. Taken together, these data show sustained biochemical and pathological correction in a murine model of PH1 by the AAV8-AGT vector and support the efficacy of AAV8 mediated gene therapy for PH1. O-048

O-046

MAN1B1-CDG: changing current beliefs in glycoprotein quality control

PGM1 deficiency: avoiding fatal outcome of a newly discovered inborn error of metabolism with many clinical faces.

Rymen D1, Péanne R1, Millón MB2, Jaeken J3, Foulquier F4, Matthijs G1

Tegtmeyer LC1, Rust S2, Reunert J1, Marquardt T1

1

1

Klinik f Kinder- & Jugendmed, Univ Klin, Münster, Germany; Leibniz-Inst for Arteriosclerosis Res, Münster, Germany

2

Background: Phosphoglucomutase 1 (PGM1) deficiency is one of the most recent discovered inborn errors of metabolism with more than 20 known patients. PGM1 plays a central role in sugar metabolism. The autosomal recessive disorder shows aspects of both, glycosylation disorders and glycogen storage diseases. Objectives: The diverse clinical characteristics and differential diagnoses need to be presented for clinicians of different subspecilaites to increase awareness and succeed in early diagnosis and therapy. Case Report: Patients present with uvula bifida and a variety of other symptoms varying from mild to severe development of hepatopathy, hypoglycemia, growth retardation, rhabdomyolysis, dilated cardiomyopathy and sudden cardiac arrest. Results: All patients show typical aspects of glycosylation disorders in isoelectric focusing, SDS-PAGE, HPLC and mass spectrometry. Frequency of PGM1 associated phenotypes in the patients is variable and showed diverse severity. Conclusion: PGM1 deficiency is characterized by a mixed CDG type and is also a glycogen storage disease especially of the muscle. The disease has multiple phenotypical faces that are important for the clinical workup of patients since early diagnosis and therapy may prevent fatal consequences.

Center for Hum Genetics, Univ of Leuven, Leuven, Belgium; 2Cent Est Metab Cong, Cordoba, Argentina; 3Cent Met Dis, Univ Hosp Gasthuisberg, Leuven, Belgium; 4Struct Funct Glycob Unit, Univ of Lilli, Lille, France Congenital Disorders of Glycosylation (CDG) is a group of genetic diseases due to impaired protein and lipid glycosylation. In the present study, exome sequencing revealed MAN1B1 to be the causative gene in an unsolved CDG-II case. Subsequently, 6 additional MAN1B1-CDG patients were identified. All patients presented mental retardation, facial dysmorphism and truncal obesity. MAN1B1 is generally believed to be an ER resident alpha (1,2) mannosidase that acts as a key factor in glycoprotein quality control. It targets terminally misfolded proteins for degradation by cleaving a terminal mannose residue from asparagine-linked Man9GlcNAc2, forming Man8GlcNAc2 isomer B. However, we demonstrated that the unfolded protein response (UPR) is not activated in MAN1B1 deficient fibroblasts, arguing against an accumulation of misfolded proteins within the ER. In contrast, an aberrant Golgi morphology was seen in all patients. These findings encouraged us to investigate the subcellular localization of MAN1B1. Indeed, MAN1B1 appeared to be confined to the Golgi complex instead of the ER, where quality control is assumed to occur. Hence, our results challenge the proposed role for MAN1B1 in glycoprotein quality control. However, more work is needed to elucidate the exact function of MAN1B1 and to explain the pathophysiology of its deficiency.

O-047 01. Phenylketonuria and BH4 AAV8-mediated liver gene therapy results in long-term correction of a murine model of primary hyperoxaluria type 1 Borzone R1, Castello R1, Auricchio A1, Brunetti-Pierri N1

P-001 Association of mean PHE-level with bone mineral status, D-vitamin level and with body fat in Hungarian adult patients with PKU

1

TIGEM, Naples, Italy

Primary hyperoxaluria type 1 (PH1) caused by deficiency of liverspecific alanine:glyoxylate aminotransferase (AGT) is a severe inborn error of metabolism that results in overproduction of oxalate, a toxic metabolite that accumulates primarily in the kidney leading

Reismann P1, Kiss E2, Simon E2, Szatmári I2, Bókay J2, Szőnyi L2, Rácz K1 1

2nd Depart. of Med., Semmelweis Univ, Budapest, Hungary; 21st Depart. of Pediat, Semmelweis Univ, Budapest, Hungary

S116 Objectives: Questions have been raised as to whether a contiguous strict diet in adult patients with PKU has any beneficial effect on bone metabolism and on body weight. Patients and Methods: In a retrospective monocentric study we analysed the data of 119 adult patients (mean age: 28.5 years) with PKU. Data were collected on age, sex, BMI, bone mineral density, D-vitamin status and mean phenylalanine level over the last year. Patients were divided in two groups depending on whether the mean Phe-level was under (Strict diet group: SD) or above (Moderate diet group: MD) 600 micromol/L. Results: The mean Phe was 397±122 micromol/L in the SD group (n=62), whereas in MD group the Phe level was 818±126 micromol/L (n=57) (p < 0.000). Neither BMI (SD: 25.3±4,8 kg/m2 vs. MD: 24.3±3,8 kg/m2, p=0.387), nor proportion of patients with decreased bone mineral density (SD: 37.9% vs. MD: 50%, p=0.2) was associated with lower Phe level. 25OH-D-vitamin level was similar in both groups (SD: 26.6±6.97 ng/ml vs. MD: 26.1 ±10.1 ng/ml, p=0.94 ). Conclusion: Our results suggest that a strict diet has not got any beneficial impact on body fat percentage and on bone mineral density in adult patients with PKU.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 by regular analysis of phenylalanine and tyrosine. The study aim was to develop a rapid method to routinely measure both metabolites in minimal bloodspot volume (1.5 mm X, 1.3 μL blood). Blood was spiked with phenylalanine and tyrosine at 24 different concentrations. DBS were prepared, after which punches of 1.5 mm X and 6 mm X (12.4 μL blood) were taken. Punches were prepared from DBS of PKU-patients (n = 77). Samples were analyzed by tandem mass-spectrometry and results were compared. A good correlation between phenylalanine and tyrosine in both punches was found (r2= 0.9917 and r2= 0.9892, respectively). Analysis of phenylalanine and tyrosine in punches of PKU-patients (n = 77) showed similar results and fitted within the procentual range of the between run variation. We developed an accurate method to analyze phenylalanine and tyrosine in a 1.5 mm X bloodspot punch. This technical improvement does not only result in a 10 fold reduction in required patients' material, but also in a 30 – 60 minutes time saving in sample preparation. P-005

P-002

Association of 10398A mtDNA variant with cognitive phenotype of patients with phenylketonuria

Tetrahydrobiopterin (BH4) deficiency in Lithuanian newborn screening

Klaassen K1, Djordjevic M2, Kotur N1, Srzentic S1, Stankovic B1, Glumac I1, Tosic N1, Spasovski V1, Pavlovic S1, Stojiljkovic M1

Cimbalistiene L1, Ambrozaityte L1, Meskiene R1, Smirnova M2, Blau N3, Utkus A1

IMGGE, University of Belgrade, Belgrade, Serbia and Montenegro; Mother and Child Healthcare Institute, Belgrade, Serbia and Montenegro

1

2

1

Dep of Hum and Med Genet of Vilnius Univ, Vilnius, Lithuania; Center for Med Genet, Vilnius Univ Hosp, Vilnius, Lithuania; 3Univ Child Hosp, Centre of Metabolic Dis, Heidelberg, Germany

2

Nationwide neonatal screening for PKU was introduced in Lithuania in 1975. During the period from 1975 to 2012, more than 1449783 neonates were screened for HPA in Center for Medical Genetics. Till now 170 patients with PKU, one case with DHPR deficiency and one case with PTPS deficiency have been detected. The first Lithuanian case of PTPS deficiency was presented with blood Phe concentration of 240 μmol/l in the neonatal period, which rose up to 1806 μmol/l on the 25th day. The Kuvan/BH4 loading test followed the standard procedure, the blood Phe concentration reduced by 88.3% after 4 hours. Elevated levels of neopterin and very low biopterin in urine of the patient and normal DHPR activity in dried blood spots, decrease of 5-HIAA and HVA in CSF were determined. Molecular analysis of PTS gene revealed a homozygous mutation (c.104A>G; p.E35G) – confirming a severe form of PTPS deficiency. Now at the age of 11 months patient is developing with a slight psychomotor retardation under treatment with sapropterin dihydrochloride (Kuvan), L-Dopa/Carbidopa and 5-OH tryptophan. CT of brain, EEG did not show any abnormality. P-004 Reliable analysis of phenylalanine and tyrosine in a minimal volume of blood Prinsen HCMT1, Holwerda-Loof NE1, de Sain-van der Velden MGM1, Visser G2, Verhoeven-Duif NM1

Phenylketonuria (PKU) is a metabolic disorder caused by phenylalanine hydroxylase gene (PAH) mutations. PAH genotype is the main determinant of metabolic phenotype severity. However, genotypecognitive phenotype correlation remains elusive. If left untreated, PKU patients develop severe mental retardation probably due to neurodegeneration. Having in mind the role of mitochondria in neurodegeneration, we investigated the presence of the mtDNA 10398A variant, reportedly associated with neurodegenerative diseases. We investigated 62 unrelated PKU patients and 50 healthy controls from Serbia. PKU patients were further categorized in groups according to time of diagnosis and compliance with low-phenylalanine diet. The IQ was determined according to age-appropriate scales. We detected 10398A variant by direct sequencing. Frequency of 10398A was the same in patients and healthy controls (82%) suggesting the same ethnic background. In a group of patients diagnosed at neonatal screening and treated with low-phenylalanine diet, no statistically significant difference in average IQ(97) was found between patients with 10398A and 10398G. The same was shown for PKU patients with low IQ(55-65) due to poorly controlled diet. Our study emphasized the importance of neonatal screening and good control of low-phenylalanine diet. Studies in larger cohorts will further elucidate the association between mt10398A and complex PKU cognitive phenotype. P-006 Maternal phenylketonuria:report from the Budapest registry 1975-2011. Bókay J1, Kiss E1, Reismann P2, Schuler A1, Simon E1, Szőnyi L1

1

Dept Med Genet, Metab Dis, UMC Utrecht, Utrecht, Netherlands; 2 Dept Metab and Endo Dis, UMC Utrecht, Utrecht, Netherlands Phenylketonuria (PKU) is an inborn error of phenylalanine metabolism. Treatment principle is to reduce phenylalanine concentration sufficiently to prevent neuropathological effects. Treatment effect is monitored

1 1st Dept of Pediatrics, Semmelweis Univ, Budapest, Hungary; 22nd Dept of Int Med, Semmelweis Univ,, Budapest, Hungary

The maternal phenylketonuria (MPKU) syndrome is caused by high blood phenylalanine (Phe) levels during pregnancy, leading to

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 intrauterine growth retardation, developmental delay, and several birth defects, microcephaly, congenital heart disease,etc. The goal of the retrospective study was to evaluate the impact of diet in pregnant women with PKU on their offspring. Data of all pregnancies in the northern half of Hungary between 1975 and 2011 were collected to Budapest, PKU Registry. The effect of the use and timing in relation to pregnancy of a phenylalanine restricted diet on the outcome of newborns was examined. A total of 29 pregnancies of 21 mothers (10 classic PKU, 11 HPA) resulted in live births. 19 mothers in 25 pregnancies started the diet before conception. All of them had healthy newborns. Two mothers with classic PKU refused the diet for several years. Despite intensive preconceptional counselling four newborns with classic MPKU syndrome were born from their pregnancies. MPKU is preventable by starting a Phe restricted diet even before conception and maintaining it during the whole pregnancy. The major factor in preventing Phe embryopathy is patient compliance in keeping the diet. We have to carry on our expansive action plan for prevention of MPKU.

S117 data about BH4 loading test includes patients given both the preparations. The real overlap between the two preparations is not known, no comparatvive data exists hence we wanted to compare the loading test performed with tetrahydrobiopterin and Kuvan® in the same subject. Nine patients, with different genotypes and with mild or classical phenotypes, that underwent a 24 h BH4 loading test with the preparation of the Schircks Laboratories were tested after an average of 9.4 years with Kuvan®. The loading test was consistence in 8 out of 9 tests. One unresponsive patient was responsive using Kuvan®. Mean Phenylalanine basal levels were 758.66 μM/L and 816.3 μM/L respectively for tetrahydrobiopterin and Kuvan® loading tests. Conclusion: We would suggest to repeat loading test with Kuvan® in case previously unresponsiveness to BH4. P-009 The resting energy expenditure of patients with phenylketonuria Kanufre VC1, Soares RDL1, Alves MRA2, Aguiar MJB3, Starling ALP3, Norton RC3

P-007 Maternal phenylketonuria international collaborative study revisited: evaluation of maternal nutritional risk factors besides phenylalanine for fetal congenital heart defects Yano S1, Moseley K1, Bottiglieri T2, Arning E2, Colleen A3 1

Genetics Ped, Univ Southern Cal, Los Angeles, United States; 2Inst Metab Dis, Baylor Res Inst, Dallas, United States; 3CTU, Univ Southern Cal, Los Angeles, United States Background: Maternal phenylketonuria (MPKU) is known to affect fetal outcome often with microcephaly and congenital heart defects (CHD) if diet is not appropriately managed. We hypothesized other nutrients aside from Phe may have significant effects on fetal outcome in MPKU pregnancies. Methods: The 416 pregnancies resulted in live births reported in the Maternal PKU Collaborative Study were grouped according to whether or not the offspring were diagnosed with CHD. The groups were compared on 1st trimester values of maternal data including weight gain, plasma amino acids, protein and Phe intake, and RBC folate. The subjects were also grouped by 1st trimester average blood Phe (≤910uM and >910uM) and divided by total natural protein and medical food intake. Results: The CHD group of 28 offspring had significantly higher blood Phe and lower Pro, Val, Met, Ile, Leu, Lys, Arg, and RBC folate. Conclusions: A significantly higher risk for CHD was found in the groups with lower natural protein and medical food intake, regardless of the blood Phe levels. Low RBC folate and plasma methionine levels in the CHD group may suggest involvement of global DNA hypomethylation.

1

Universidade Federal de Minas Gerais, Hc, Belo Horizonte, Brazil; Universidade Federal de Minas Gerais, Nú, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Fa, Belo Horizonte, Brazil 2

Objective: To evaluate the resting energy expenditure of individuals with PKU and its relationship to overweight. Methods: Cross-sectional study with 58 patients aged 4 to 15 years, divided into two groups: overweight (29) and eutrophic (29). Patients were fasted for ten hours prior to indirect calorimetry to determine the resting energy expenditure (REE)., FAO/WHO/UNU, 1985,Schofield WN, 1985. The REEs were compared with the results of the formulas. Measures of arm muscle area and fat mass were also estimated. Results: Differences were detected in the values of ratios REE / weight and REE/ arm muscle area between groups. The values obtained by REE were higher than those of the two prediction equations. There was a correlation between the REE and the two prediction equations, but there was no agreement of these variables between the groups. Eutrophic patients have a higher metabolic rate than PKU overweight, when proportionality of mass and weight were considered. Conclusions: The results of this study do not appear to differ from those found in individuals without the disease. Phenylketonuria by itself does not appear to alter the metabolism of individuals, even with indirect calorimetry results showed values higher than the ones of the prediction equations. P-010 Metabolic syndrome in children and teenagers with phenylketonuria Kanufre VC1, Soares RDL1, Alves MRA2, Aguiar MJB3, Starling ALP3, Norton RC3

P-008 Comparing tetrahydrobiopterin with sapropterin laoding tests

1

1

2

1

Nardecchia F , Carducci C , Leuzzi V

Universidade Federal de Minas Gerais, Hc, Belo Horizonte, Brazil; Universidade Federal de Minas Gerais, Nú, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Fa, Belo Horizonte, Brazil 2

1

Dept Ped and Child Neuro-Psych, Sapienza, Rome, Italy; 2Dept Experimental Med, Sapienza, Rome, Italy The only pharmacological alternative to diet for patients with Hyperphenylalaninemia due to Phenylalanine Hydroxylase deficiency is its natural cofactor tetrahydrobiopterin. The first drug on the market was the preparation from Schircks Laboratories, then, in December 2008 the sapropterin dihydrochloride preparation (Kuvan®) from Biomarin/Merck Serono was approved throughout Europe. Literature

Objective: To determine the factors involved in the metabolic syndrome (MS) in patients with phenylketonuria (PKU). Methods: Cross-sectional study with children and adolescents with PKU, constituted of patients with overweight (29) and eutrophic (29). We evaluated blood levels of phenylalanine (phe), total cholesterol (TC), HDL-c, triglycerides (TG), glucose and basal insulin. Was determined insulin resistance (HOMA) and measured waist circumference (WC).

S118 Results: The groups did not differ with respect to levels of blood phe. The patients in the overweight group had higher concentrations of TG, basal insulin and HOMA but lower HDL-c, compared to eutrophic. The overweight group had significantly higher values of the ratio of TC / HDL-c. The overweight group showed a positive correlation of basal insulin and HOMA with CC. Conclusions: Some patients were identified with MS according to the criteria of the International Diabetes Federation. The results of this study showed that individuals with PKU are more susceptible to MS. In addition to the genetic and environmental characteristics, add to, factors inherent to the disease, such as lipid profile and characteristic of power. Clinical and laboratory approaches are needed to prevent excessive weight gain and early cardiovascular damage in these individuals. P-011

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 knowledge, neurocognitive and neurochemical characteristics of murine models with milder levels of HPA have not been yet explored. Objective: This study was aimed to develop new mouse models with increasing severity of HPA and explore their neurocognitive and neurochemical characteristics as function of their different biochemical background. Material and Methods: Mice with different degrees of HPA (normal controls, mild-HPA, mild-PKU, and classic-PKU) have been submitted to behavioral (Plus Maze, Object Recognition Test, Delayed Alternation Task) and biochemical (blood Phe and brain biogenic amine concentrations) assay. Results: HPA mice showed an increasing impairment of neurocognitive functions, and a parallel derangement of brain aminergic system, as the severity of HPA increases. Conclusion/Discussion: Different murine models of HPA proved to be useful tools to elucidate the pathogenetic mechanism by which Phe impairs cognitive functioning and also to assess possible alternative treatments.

Lipid levels in children and adolescents with phenylketonuria P-013 Kanufre VC1, Soares RDL1, Alves MRA2, Neves M3, Aguiar MJB2, Starling ALP2, Norton RC2 1

Universidade Federal de Minas Gerais, Hc, Belo Horizonte, Brazil; Universidade Federal de Minas Gerais, Fa, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Belo Horizonte, Brazil

Reduced brain neurotransmitter concentrations and an impaired behavioral phenotype are improved with a low phenylalanine diet in PKU mice

2

Objective: To assess the levels of lipids in individuals with phenylketonuria (PKU). Methods: Cross-sectional study with children and adolescents with phenylketonuria: eutrophic (70) and overweight (31). We collected total cholesterol (TC), HDL-c and triglycerides (TG). We obtained the average blood phenylalanine (phe) to classify patients with adequate and inadequate levels. Results: Lipid levels were better when the blood phenylalanine levels were adequate, although no difference when compared to inadequate levels. The TC / HDL-c ratio was different between groups, adequate levels of phe (p=0.01), inadequate (p=0.001), with higher ratio in patients with overweight. No correlation was found between the levels of lipids and blood phe levels. Analyzing the inadequate values of lipids in the two groups, we find the following result: 45 (44.5%) of HDL-c, 40 (39.6%) of TC, and 52 (51.5%) of TG. Conclusion: Eutrophic patients have a better levels of lipids than overweight. Important results are the high ratio of TC / HDL-c, low levels of HDL-c and high TG levels found in patients with overweight. These results seem to be food related or due to a specificmetabolic cause.. This is a controversial issue in the literature, studies should be targeted in search of answers. P-012 Behavioral and neurochemical characterization of new mouse model of hyperphenylalaninemia

Sawin E1, Murali SG1, Ney DM1 1

University of Wisconsin-Madison, Madison, United States

Increased phe levels in untreated PKU alter brain neurotransmitters (NT) and impair cognitive development. Our objective was to determine the relationship between brain NT concentrations and behavioral phenotype in PKU (Pahenu2/enu2) and WT mice fed high-phe (casein) and low-phe (amino acid, AA; glycomacropeptide, GMP) diets between 3-23 weeks of age. NT concentrations were measured using HPLC with electrochemical detection system. Mouse behavioral phenotypes were characterized by the marble burying and open field testing for 30 minutes. Regardless of diet, brain concentrations of serotonin, dopamine and norepinephrine were significantly decreased in PKU vs WT mice. The low-phe diets increased brain serotonin to levels similar to WT casein. Marble burying, reflecting normal digging behavior, was significantly reduced in PKU mice fed high-phe casein, but normalized to WT levels with low-phe diets. Open field rest periods and vertical activity were significantly decreased suggesting hyperactivity and less vertical exploration, respectively, in PKU vs WT mice. The GMP diet normalized vertical activity to WT casein levels. In summary, PKU mice showed reduced brain NT concentrations in association with a behavioral phenotype of decreased environmental awareness, hyperactivity and reduced vertical exploration. The low-phe diet increased brain serotonin concentration and improved the behavioral phenotype of Pahenu2/enu2 mice. Conflict of Interest declared. P-014

Pascucci T1,2,3, Giacovazzo G3, Accoto A1, Carducci C4, Leuzzi V5, Puglisi-Allegra S1,2,3

Use of sapropterin: responsiveness & relevance

1 Dept Psychol, Univ Sapienza, Rome, Italy; 2Center Daniel Bovet, Univ Sapienza, Rome, Italy; 3IRCCS Santa Lucia Foundation, Rome, Italy; 4Dept Exper Med, Univ Sapienza, Rome, Italy; 5Dept Child Neurol Psychia, Univ Sapienza, Rome, Italy

Arnoux JB1, Valayannopoulos V1, Assoun M1, Le Verge S1, Bardet J1, Christa L1, de Lonlay P1

Background: Patients with a defect of phenylalanine (Phe) hydroxylase are classified according to the blood Phe levels ona free diet: classical phenylketonuria (PKU) (Phe >1200 microM), mild PKU (Phe 600-1200 microM) and persistent hyperphenylalaninemia (HPA; Phe 120-600 microM) (normal Phe value <120 microM). The ENU2 mouse is the most common animal model of classical PKU. To our

Background: Hyperphenylalaninemia includes phenylketonuria and pterins disorders. Treatment is a phenylalanine restricted diet, but treatment with sapropterin can alleviate the diet burden, in case of sapropterin responsiveness. Objective: to report our experience for the interpretation of the sapropterin loading test and to review the sapropterin use.

1

Necker Hosp, Paris Descartes Univ., Paris, France

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Methods: Sensitivity to sapropterin (20mg/kg)-loading test is defined as a > 30% decline of the phenylalaninemia within 24 hours in neonates or within 8 days later in life. Results: The 24h sapropterin loading test was positive in 23/41 neonates. Long term treatment with sapropterin was initiated since neonatal period in 8/23 and since childhood in 9/23 patients. This treatment was ended at 3 years old for 4 children (inefficient or useless). A 8 days sapropterin loading test was performed in 8 patients (5 children and 4 adults) and was positive in 7, among them one with DHPR deficiency. 1/4 children negative for the neonatal test, was finally a late responder on the 8 days loading test. All the sapropterin responsive patients are currently treated with sapropterin. Fatigability and motor disorder improved in the DHPR deficient patient. Conclusion: Responsiveness to sapropterin loading test and relevance of sapropterin treatment should be reassessed after the neonatal period. P-015 Neurocognitive and neuroimaging outcome of early treated PKU subjects. First longitudinal study Leuzzi V1, Pansini M1, Nardecchia F1, Nori M1, Santamaria-Palombo A1, Carducci C2 1

Dept Ped and Child Neuro-Psych, Sapienza, Rome, Italy; 2Dept Experimental Med, Sapienza, Rome, Italy Background: Despite favourable clinical outcome of early treated phenylketonuric (PKU) subjects, a lower IQ than expected, minor neuropsychological, psychiatric problems, and white matter (WM) alterations remain challenging aspects of the disease. We report on the first longitudinal study on both neurocognitive and neuroimaging outcome in PKU. Methods: 13 early treated PKU patients underwent neurocognitive (WISC/WAIS IQ and executive functions [EF]) and neuroimaging assessment at the age 8-14 years and about 12 years later (age 21-26 years) according to a similar protocol. In the time-gap between the two evaluations 8/13 patients discontinued the diet. Results: While the brain WM signal was normal in all the patients at the first examination, 12/12 re-examined subjects showed mild (10) to moderate(2) WM involvement. The severity of WM score was unrelated to the IDC between the two examinations and concomitant blood Phe. IQ was in the normal range in all subjects; 3/8 and 0/5 subjects, respectively off and on diet, experienced a IQ decline (>8 points); the remaining 10 subjects maintained or increased their IQ. EF generally improved in all subjects regardless of the severity of WM involvement, IQ variations, biochemical control. Conclusions: WM involvement, IQ, and EF are unrelated outcome measures in PKU patients. P-016 Inflammation and endothelial function in phenylketonuria (PKU) sapropterin responders and non-responders during meal induced oxidative stress

Douglas TD1, Quirk ME1, Coakley KE2, Ziegler TR3, Quyyumi AA4, Le NA5, Singh RH1 1

Metab Nutr Program, Emory Dept Human Gen, Atlanta, United States: Nutr and Health Sciences, Emory GDBBS, Atlanta, United States; 3 Div Endocrin, Metab & Lipids, Emory SOM, Atlanta, United States; 4 Div Cardiology, Emory School of Medicine, Atlanta, United States; 5 Biomarker Core Lab, Atl VA Med Center, Atlanta, United States 2

S119 Objective: To investigate inflammation and endothelial function in PKU patients with and without phenylalanine (Phe) response to sapropterin during an oxidative challenge. DESIGN: PKU subjects with documented Phe response to sapropterin are being recruited with healthy controls. Responders currently taking sapropterin (20mg/kg/d) and Controls complete one study visit. Non-responders attend two study visits: baseline without drug, and after two weeks on sapropterin. Blood Phe, brachial artery flow-mediated dilation (FMD), and inflammatory markers are measured at each visit. Preliminary mean outcomes are reported. Results: To date, 3 Non-responders, 5 Responders, and 2 controls have completed the study. Mean serum C-reactive protein (CRP)(mg/dL) in sapropterin Non-responders is higher than both Responders and Controls (NR: 1.46, R: 1.04, C: 0.61). Data to date suggest Non-responders have higher baseline FMD vs. Responders and Controls (NR: 9.8%, R: 6.9%, C: 8.2%). Nonresponders exhibit improved FMD response to an oxidative meal challenge after sapropterin (Visit 1: -2.1% decline post-challenge, 2 weeks on sapropterin: +1.9% increase post-challenge). Responders exhibited no change in FMD post-challenge, while Controls had a -0.8% FMD change. Conclusions: Early results indicate higher inflammation in patients with PKU, particularly sapropterin Non-responders. Non-responders appear to exhibit improved endothelial function after two weeks on sapropterin. Conflict of Interest declared. P-017 Protein nutrition status across one year in phenylketonuria (PKU) patients with and without PHE response to sapropterin Douglas TD1, Coakley KE2, Singh RH1 1

Metab Nutr Program, Emory Dept Human Gen, Atlanta, United States; 2 Nutr and Health Sciences, Emory GDBBS, Atlanta, United States Objective: To evaluate one year protein status in PKU patients with and without Phe response to sapropterin. Methods: After one month sapropterin(n=57), preliminary Responders (≥15% plasma Phe decrease) were given Phe challenge (previously described) and classified as Definitive(DR) or Provisional(PR) Responders. Non-responders(NR) discontinued drug. All participants provided 3-day diet records, anthropometrics, blood, and urine for one year. Protein status markers(mean+SD) were analyzed using regression techniques. Results: DR increased intact protein (g/kg)(Baseline:0.58±0.4, 1 year:0.75±0.3) but had net decrease in total protein intake (Baseline:1.4±0.7, 1 year:1.0±0.7; p<0.001) due to 75% decline in medical food(MF) intake. Total protein intake declined significantly in NR and PR without change in Phe tolerance or prescribed MF, indicating nonadherance. Biologic protein markers did not differ between groups or with increased intact protein intake in Responders. Baseline urine creatinine(mg/dL) was significantly lower in PKU than controls (74±40 vs. 141±61), but within normal ranges. BUN declined in DR and albumin, globulin, and total serum protein declined in NR over time(p<.05). Other markers did not change. Conclusions: Reduced MF requirement in sapropterin DR decreases total protein intake without affecting most biologic protein markers. Long-term declines in NR protein status and lower urinary creatinine in PKU subjects warrant further attention. Conflict of Interest declared.

S120 P-018 Breasr milk and plasma pterin levels and offspring outcomes of 25 pregnancies in women exposed to sapropterin dihydrochloride prior to or during pregnancy: an interim report of the PKU moms sub-registry Hillman R1, Peck D1, Grange DK2, Fong CT3, Burton BK4, Vockley J5, Yano S6, Violet S7, Cohen-Pfeffer J7 1

University of Missouri, Columbus, United States; 2Washington University, Columbus, United States; 3University of Rochester Medical Center, Rochester, United States; 4Ann and Robert H Lurie Children's Hospit, Chicago, United States; 5University of Pittsburgh School of Medic, Pittsburg, United States; 6University of Southern California, Los Angeles, United States; 7BioMarin Pharmaceutical Inc., Novato, United States

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 the last year. For the parents, QoL was good, but 3/14 reported anxiety, 2/14 depression, and 2/14 social difficulties. Conclusions: WHOQoL-100 is a comprehensive and valid instrument for PKU patients at different ages. Parents' perception should always be investigated. Conflict of Interest declared. P-020 Behaviour and social skills in adult phenylketonuria patients: preliminary results Jahja R1, Huijbregts SCJ2, De Sonneville LMJ2, Van der Meere JJ3, Bosch AM4, Hollak CEM4, Rubio-Gozalbo ME5, Brouwers MCGJ5, Hofstede FC6, De Vries MC7, Janssen MCH7, Van der Ploeg AT8, Langendonk JG8, Van Spronsen FJ1 1

Outcomes of 25 pregnancies from 20 women enrolled in the PKUMOMS sub-registry of PKUDOS are presented. To date 15 newborns have been born and recorded in the sub-registry. Of these, 12 (9 females, 3 males) were born to women with PKU exposed to sapropterin dihydrochloride during pregnancy; 11 at term, one pre-term (35 weeks 3 days). The other 3 were born at term to women exposed prior to pregnancy (1 female, 2 males). Median percentiles for weight, length and OFC at birth were 2550th, 50th and 50-75th, respectively. 7/15 offspring had ≥ 1 adverse events (AEs) reported at birth. 16 total events were reported, 5 were serious AEs and 11 were non-serious. Fifteen AEs were reported as unrelated and 1 was reported as possibly related to sapropterin. The possible drug related AE was hypophagia in the infant born prematurely; it resolved after 8 days. Plasma samples were collected in 4 women and their offspring and analyzed for biopterin, dihydro and tetrahydrobiopterin (BH4) at their 1-month visit; one infant's plasma sample was collected at birth. Breast milk samples of 3 women on sapropterin were collected and analyzed for BH4 content; levels were greater than that reported in human breast milk. Conflict of Interest declared. P-019 WHOQOL-100 for evaluation of quality of life in the long termtreatment PKU patients on tetrahydrobiopterin (BH4) therapy Burlina AB1, Burlina AP2 Cazzorla C1, Dianin A1, Bordugo A1, Massa P1, Giordano L1 1

Div Metab Dis,Univ Hosp, Padova, Italy, 2Neurological Un, St. Bassiano Hosp, Bassano del Grappa, Italy

Beatrix Child Hosp, Univ Med Cen Gron, Groningen, Netherlands; Clin Child Adol Studies, Leiden Univ, Leiden, Netherlands; 3Dev Clin Neuropsy, Univ Groningen, Groningen, Netherlands; 4Acad Med Center, Amsterdam, Netherlands; 5Acad Hosp Maastricht, Maastricht, Netherlands; 6Wilhemina Child Hosp, Univ Med Cen Utr, Utrecht, Netherlands; 7Univ Med Cen St Radboud Nijmegen, Nijmegen, Netherlands; 8 Erasmus Med Center, Rotterdam, Netherlands 2

Background: In Phenylketonuria (PKU) there is a clear relationship between blood phenylalanine (Phe) and cognitive outcomes, whereas less is known about behavioural problems and social functioning. Objective: To examine behavioural problems and social skills in earlyand continuously-treated adults with PKU. Methods: Thirty PKU patients (mean age 27.8±6.4) and 14 controls (mean age 26.9±5.9) filled out questionnaires measuring behavioural problems (Adult Self-Report, ASR) and social skills (Social Skills Checklist Self-Report, SSC), and completed the computer-task Identification of Facial Emotions. Independent-samples t-tests and Pearson correlations were used. Results: Compared to controls, PKU adults tended to report more 'Internalizing behaviour problems' (p = .062), and scored higher on 'Avoidant personality problems' (p = .018). Patients also scored worse on 'Relationships' (p = .048) and 'Self-care' (p = .025), and seemed to have more difficulty identifying the emotion 'Fear' (p = .075). Several internalizing problems were significantly related to childhood blood Phe levels (r's> .65). Conclusion: Behavioural and social problems probably have been underestimated in PKU. These data show that PKU adults experience behavioural and social problems despite early treatment and that these problems are correlated to childhood Phe levels. Taking into account the small sample size, these results should be considered preliminary. Conflict of Interest declared. P-021

Background: BH4 treatment for responsive patients is a therapy currently in practice for PKU patients. However, there are very few longterm studies concerning the patients' quality of life at different age. Methods: Our cohort comprised 21 patients: 7 adults, aged 18-35 years, 14 children, aged 5-16 years. All patients were on BH4 treatment (10 mg/kg/day) for at least one year. For the paediatric sample, the parents' perception was evaluated. We assessed the global QoL with the following scales: WHOQOL-100, PedsQL, PedsQL-Proxy Report, and, for specific aspects, TAD, BDI, SAT-P. Phenylalanine and Tyrosine levels were measured at the time of the visit and over the course of the previous 12 months. Results: Global QoL was normal for all patients. Evaluation of specific aspects highlighted: a) good satisfaction for the adults, better for sleep/food/free time and social well-being dimensions; b) children perceived a better physical well-being; 1/14 showed anxiety. We found a positive correlation between anxiety and Phenyalanine levels during

Cognitive functions evaluation in long-term treatment PKU patients on tetrahydrobiopterin (BH4) therapy Cazzorla C1, Massa P1, Burlina AP2, Bordugo A1, Giordano L1, Dianin A1, Burlina AB1, 1

Div Metab Dis,Univ Hosp, Padova, Italy; 2Neurological Un, St. Bassiano Hosp, Bassano del Grappa, Italy Background: BH4-responsive PKU patients benefit from BH4 treatment to reduce blood Phenyalanine levels. Few reports have been published on cognitive abilities in long-term BH4 -treated patients. Methods: We performed extensive cognitive evaluation in 21 PKU patients (7 adults, age range 18-35 years, and 14 children, age range

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5-16 years) treated with BH4 (10 mg/Kg/die) for at least one year. General Intelligence Quotient (IQ) was measured using age-related scales (WAIS-R, WISC-III, GMDS-ER). Attentive abilities and executive functions were measured. The tests results were correlated with Phenylalanine and Tyrosine levels at the time of visit and over the course of the previous 12 months. Results: General IQ, and executive functions were normal for all patients, except for one child with Verbal IQ in the borderline range. Attentive abilities measurement showed reduction in speed processing during attention tasks in 36% of patients. In addition, we found an inverse statistically significant correlation between the mean score of the Verbal IQ for the pediatric sample and the Phe levels at the time of the visit. Conclusion: Our study highlights that specific questionnaires are necessary for a complete cognitive assessment. Furthermore, specific abilities, like Verbal IQ, should be carefully investigated in relationship with Phe levels. Conflict of Interest declared.

Objective: To analyze Phe values' trend during BH4 loading test and correlate it with basal Phe levels, underlying PAH mutation and type of nutrition (breastfed, mixed fed or formula fed) as determinant influencing environmental factors. Patients and method: 76 PKU patients (with basal plasma Phe levels ≥360 μmol/L) were enrolled, analyzing genotype, BH4 loading test response and type of nourishment. Results: Only 43.63% of patients deemed as responsive genotype actually resulted positive to the loading test. Average basal Phe levels was lower in responders than in non-responders (p<0.001). No significant correlation was found with the different kind of nutrition. However, breastfed patients tended to have lower basal Phe levels compared to the others, confirming breast milk as the ideal PKU feed. Conclusions: Our data shed doubt on the value of genotype alone in predicting BH4 response, suggesting that all patients, should undergo BH4 test in order to not miss possible candidates for therapy.

P-022

First interim analysis of the French data from the Kuvan Adult Material Paediatric European Registry (KAMPER): patient characteristics and safety data

Tetrahydrobiopterin (BH4) was safe and effective in patients less than 4 years old with BH4-responsive PAH deficiency in Japan

P-024

Shintaku H

Barth M1, Fouilhoux A2, Mention K3, Arnoux JB4, Labarthe F5, Cano A6, De Parscau L7, Maillot F8, Solbes MN9, Feillet F10

1

1

1

Dept Ped, Osaka City Univ Grad School M, Osaka, Japan

Background: Tetrahydrobiopterin (BH4) therapy for BH4-responsive phenyalanine hydroxylase (PAH) deficiency (BPKU) began in 1996 in Japan. Objective: A longitudinal follow-up study is being conducted of all patients with BPKU throughout Japan. In this analysis, we evaluated the efficacy and safety of BH4 therapy initiated in patients under 4 years old. Patients: At the end of 2011, 43 patients were receiving BH4 therapy, of whom 21 were under 4 years of age when treatment began. The starting dose of BH4 was ≥10 mg/kg/day in 13 of the 21 patients. Follow-up was ≥ 4 years in 6 of the 13 patients; 3 of the 6 were followed ≥ 10 years. Nine patients were on BH4 monotherapy at the end of 2011. Results: The plasma Phe value was maintained within a favorable range in all 21 patients in whom BH4 therapy was started before 4 years of age. Only one non-serious adverse drug reaction (in one patient) occurred. No considerable abnormal behavior related to nerve disorders was reported. Conclusion: BH4 therapy initiated before 4 years of age was very effective to maintain favorable plasma phenylalanine levels in Japanese patients with BPKU and was safe. Conflict of Interest declared. P-023 Genotype, basal PHE levels and type of nourishment as predictive factors of the BH4 loading test response in phenylketonuria (PKU) affected patients Riva E1, Paramithiotti C1, Salvatici E1, Selmi R1, Vincenti S1, Paci S1, Giovannini M1 1

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy

Background: Tetrahydrobiopterin stimulates phenylalanine hydroxylase (PAH) activity in about 20% of PKU patients. PAH genotype is considered as the first factor responsible for BH4 responsiveness. Conflicting results in genotype-phenotype-responsiveness correlation reveal that this may not be the only determining factor but several others have to be evaluated.

CHU Hôtel Dieu, Angers, France; 2Hôpital Femme Mère Enfant, LyonBron, France; 3Hôpital Jeanne de Flandre, Lille, France; 4Hôpital Necker Enfants Malades, Paris, France; 5Hôpital Clocheville, Tours, France; 6 Hôpital de la Timone, Marseille, France; 7Hôpital Morvan, Brest, France; 8Hôpital Bretonneau, Tours, France; 9Merck Serono S.A.S, Lyon, France; 10Hôpital d'Enfants Brabois, Vandoeuvre les Nancy, France Objectives : KAMPER sub-analysis on French patients will provide information on the long-term treatment of patients with phenylketonuria (PKU) or BH4 deficiency treated with sapropterin. Methods : Observational, multicentre drug registry Results: This sub-analysis included data from 56 French patients (73% PKU; 27% BH4 deficiency; male/female ratio 55:45). Median(Q1-Q3) age was 7.9 (6.3-12.8) and 14.6(7.0-19.1) years in PKU and BH4-deficient patients, respectively. Median(Q1-Q3) phenylalanine concentration (μmol/l) at newborn screening and before sapropterin was 590(430-802;n=36) and 514(403663;n=32) in PKU patients and 460(381-1635;n=13) and 213(91-442;n=8) in BH4-deficient patients. Genotyping was reported in 66% of PKU and 0% of BH4-deficient patients. Median sapropterin daily dose was respectively 15.9 and 6.8mg/kg/day in PKU (n=33) and BH4-deficient (n=10) patients. To date, 8 adverse events (AEs) have been reported in 4 patients; 1 AE in 1 PKU patient and 7 AEs in 3 BH4-deficient patients. One event (BH4-deficiency: tic) was considered possibly related to sapropterin. One serious AE (BH4 deficiency: epistaxis), was mild in severity and unlikely related to sapropterin. Conclusion: KAMPER provides an opportunity to gather a large collection of long-term follow-up data related to BH4-responsive hyperphenylalaninemia. Consistent with clinical trial evidence, these preliminary results continue to demonstrate a favorable safety profile for sapropterin. Conflict of Interest declared. P-025 Incidence of overweight and obesity in PKU populations compared with the general population Castejon E1, Meavilla S1, Gutierrez A1, Garcia D1, Egea N1, Tondo M2 Ormazabal A2, Campistol J3, Lambruschini N3 1

Gastroenterology and Nutrition Department, Barcelona, Spain; Biochemistry Department, Barcelona, Spain; 3Neurology Department, Barcelona, Spain 2

S122 Aims: To determine the incidence of overweight and obesity in our PKU patients. To compare these results with the latest data for the general Spanish population. Methods: Patients visited in outdoors clinic during the last six months were reviewed Anthropometric measures (weight, height and BMI z-scores) and PKU treatment (protein restricted diet or BH4) were recorded Obesity was defined using age and sex cut-offs proposed by the International Obesity Task Force (ITOF) in children and using world Health Organization definitions in adults. Results: Complete anthropometric data were available for 110 patients, 58 children and 52 adults. Amongst them, 26 patients received treatment with BH4. The prevalence of overweight and obesity in the whole group was 22.5% and 6.3% respectively. In the BH4 group, (3%) 1/26 patients had obesity. In the pediatric group 18.9% had overweight and 5.1% obesity and in the adult group, 26% and 7%, respectively. In both groups (children and adults), obesity was more frequently observed in females, however, overweight was more frequent in males. Conclusions: The prevalence of obesity in children was lower than the general Spanish pediatric population (described as 9.5% obesity). For adults the results were similar to those described in the literature. P-026 Neuropsychiatric outcomes in PKU patients treated with sapropterin: results from the randomized, controlled PKU ascend (PKU 016) trial Prasad S1, Burton B 2, Feigenbaum A3, Grant M4, Hendren R5, Signh R6, Stahl S3, Zhang C1 1

BioMarin Pharmaceutical Inc., Novato, United States; 2Children's Memorial Hospital of Chicago, Chicago, United States; 3University California San Diego, San Diego, United States; 4St. Christopher's Hospital for Children, Philadelphia, United States; 5University California San Francisco, San Francisco, United States; 6Emory University, Decatur, United States

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Conclusions: Sapropterin was associated with improvement in inattention among children and adults with PKU who had a Phe response to sapropterin therapy. Conflict of Interest declared. P-027 Features of cellular immunity and intracellular enzyme activity in PKU patients Bushueva TV1, Kurbatova OV1, Semenova GF1, Samohina IV1, Borovik TE1, Petrichuk SV1 1

Scientific Center of Children's Health, Moscow, Russian Federation

The aim of the study was to determine the characteristics of cellular immunity and mitochondrial dehydrogenase activity in PKU patients. The activity of mitochondrial succinate dehydrogenase (SDH) and NADH-dehydrogenase (NADH-D) were investigated by quantitative cytochemical method in 38 PKU patients aged 6 months to 36 years, quantity of T-, B- and NK-cells were determined with a help of flow cytometry. The SDH activity in the general population of patients younger than 14 years was lower compared with healthy (p = 0,03), in patients over 14 years enzyme activity did not differ from normal values. In PKU patients the NADH-D activity maintained at 1.2-1.6 times higher as compared with healthy persons. The numbers of T and B lymphocytes did not differ from healthy individuals.In PKU patients there was a significant reduction in the absolute number of NK-cells, that was accompanied by a significant increase in their activity. Significant increased SDH was also found in activated T- lymphocytes (CD3+ HLA-DR+) and in activated Т-helpers (CD3+CD4+CD25+). The changes revealed evidence of the strength of the immune processes in PKU patients of different ages. P-028 Outcomes of material phenylketonuria in New Zealand Akroyd R1, Nicol R1, Webster D2, Glamuzina E1, Wilson C1

Background: Phenylketonuria (PKU) patients often exhibit attention deficits similar to attention deficit hyperactivity disorder (ADHD). Methods: PKU 016 is the largest health outcomes study in PKU. This randomized, placebo-controlled trial evaluated baseline neuropsychiatric impairment, specifically ADHD behaviors, symptoms of anxiety (HAM-A rating scale), depression (HAM-D rating scale) and executive dysfunction (BRIEF rating scale), and the effects of sapropterin dihydrochloride (Kuvan) on these impairments after 13 weeks of treatment in 206 children and adults with PKU. The primary endpoint was the total score on the ADHD Rating Scale (ADHD-RS) commonly used to evaluate symptoms of inattentiveness and hyperactivity. Results: Mean patient age was 22.5±11.6 years, with 42% aged <18 years. Baseline blood Phe was 841.4±473.1 μmol/L. 35% of the sample had symptoms of ADHD at baseline. 118/206 were sapropterin responders (defined as blood Phe level reduction ≥20%). Among sapropterin responders, ADHD-RS total score was improved in the sapropterin group compared with placebo (mean change from baseline -9.1±2.2 vs -4.9±2.0, P=0.085), driven by a statistically significant and clinically relevant change in the inattention subscale (-5.9±1.4 vs -2.5±1.3, P=0.036).

1

Metabolic Service, Auckl City Hosp, Auckland, New Zealand; Newborn Metab Screen, Auckl City Hosp, Auckland, New Zealand

2

Introduction: Maternal phenylketonuria (PKU) can result in neurological dysfunction and microcephaly. To prevent these complications women with PKU are encouraged to have planned pregnancies and aim for pre and post-conception phenylalanine levels of 100250μmol/L. Objective: To review pregnancies of women with PKU, managed by the National Metabolic Service. Method: Clinical and laboratory data was collected retrospectively for pregnancies between 1997 and 2013. Results: A total of 46 pregnancies resulted in 30 live births, six miscarriages and 10 terminations, of which five were medically advised. Thirteen pregnancies occurred in women on preconception diet, 7 were on an adult PKU diet and 26 were off diet preconception. Four pregnancies resulted in fetal anomalies, one with acrania requiring termination of pregnancy (TOP) and one with a meningocoele. The same mother had a subsequent medical TOP for megacystis secondary to posterior urethral valves. One twin pregnancy resulted in one healthy live birth and one

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 stillborn complicated by multiple fetal abnormalities. Three of these four cases were associated with poor maternal diet adherence. Conclusion: Diet management in maternal PKU is challenging, particularly when woman have been off diet prior to conception. Complications of poor diet control are preventable by maintaining tight phenylalanine restriction throughout pregnancy.

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Association of mean platelet volume with other vascular risk factors in phenylketonuria patients

(0.7%), IVS2+5G>C (0.7%), IVS10-14G>A (0.7%), R252W (0.7%). But in 30.9% we detected unknown mutations of PAH gene. We analyzed spectrum of PAH gene mutations in group of PKU patients only Kazakh nationality. The most common mutation was R243Q (21,4%). There were identified mutations R408W (19,6%), P281L (10,7%), IVS10-11G>A (3,6%), IVS12nt1 (3,6%), R281L (1,8%) , W187X (1,8%), IVS12+1G>A (1,8%), IVS10-14G>A (1,8%). In 33.9% we detected unknown mutations of PAH gene. Thus, PKU patients Kazakh nationality had specific spectrum of PAH gene mutations, which must be taken into account in molecular genetic diagnostics including prenatal diagnostics.

Gündüz M1, Cakar S2, Kuyum P2, Makay B2, Arslan Nur2

P-031

1

Cardiovascular risk factors in hyperphenylalaninemic children on dietary treatment: correlation between carotid intima-media thickness and docosaexahenoic acid supplementation

P-029

Div Metab Unit, Diskapi Child Hosp, Ankara, Turkey; 2Div Metab Unit, Dokuz Eylul Univ Pediatr, Izmir, Turkey Background: Risk of oxidative stress is increased in phenylketonuria (PKU)and may lead to atherosclerosis. Mean platelet volume (MPV) is a marker of atherosclerosis. Aim: To investigate the MPV levels in PKU patients and compare with controls. Methods: 29 PKU patients (mean age: 12.3±2.1 years) and 23 controls (11.7±2.0 years) were enrolled. PKU patients were divided into two groups: well-controlled (WCP) and poorly controlled patients (PCP). Obese patients were excluded. Results: MPV levels of WCP(10.1±1.8 fL) were higher than the PCP and controls (8.4±0.7 fL, p=0.036 and 7.7±0.5 fL, p=0.000, respectively). Besides, PCP had higher MPV levels than controls (p=0.007). The mean HDL-cholesterol levels of WCP(33.0±11.3 mg/dL) were lower than the PCP and controls (42.0±12.3 mg/dL, p=0.020 and 53.3±8.7, p=0.007, respectively). There were no significant differences among the groups regarding leukocyte and platelet count, triglyceride and LDL-cholesterol levels. Besides, PCP had significantly higher homocysteine levels than controls (8.8±4.5 vs 5.8±1.9, p=0.034). Conclusion: This study showed for the first time that well-controlled PKU patients have higher MPV and lower HDL-cholesterol levels compared to poorly-controlled PKU patients and healthy controls. Since MPV is a simple laboratory parameter, we suggest its use as a follow-up marker in PKU patients in relation to atherosclerosis. P-030 Spectrum of PAH gene mutation in phenylketonuria patients in Kazakhstan Salimbayeva DN1, Svyatova GS1, Berezina GM1, Polyakov AV2

Salvatici E1, Pederiva C1, Capra ME1, Vincenti S1, Paci S1, Giovannini M1 1

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy

Background: Hyperphenylalaninemic children follow a lifelong lowphenylalanine diet, which might effect cardiovascular disease (CVD) risk factors. Objective: Evaluate low-phe-diet's effects on CVD risk factors in a population of hyperphenylalaninemic children followed at our Metabolic Disease Center. Material and Methods: 50 hyperphenylalaninemic children (age 924y) were divided into two groups according to plasma phe levels and dietary treatment (dt): group-A (29 dt-compliant pku children) and group-B (21 no-dt-mild-hyperphenylalaninemic children). Anthropometric measures, dt-compliance, twelve-hour-fasting blood sample for total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C) and Triglycerides (TGC) were evaluated by enzimatic method. cIMT was measured by a single trained operator. Group-A-children were prescribed DHA (200 mg/die): 25 out of 29 complied with this therapy. Mann-Whitney test for independent samples. Results: Lipid profile (mg/dl, mean±ds) and cIMT (μm, mean±ds) in group A and B were, respectively: CT 148±29 vs 171±38, p=0.015, LDL-C 77±25 vs 98±32, p=0.012, HDL-C 55±12 vs 58±15, p>0.05, TGC 78±32 vs 79±37, p>0.05, cIMT 435±95 vs 512±190, p>0.05. cIMT in DHA supplementedchildren and not-supplemented was 425±70 vs 512±190, p=0.026. Conclusions: PKU children on dt have a better CVD risk profile, especially if supplemented with DHA. These preliminary data suggest a protective role for dt and need further investigation.

1

P-032

Phenylketonuria (PKU) is the most common inherited disorders of amino acid metabolism, the cause of which is mutation in PAH gene. Spectrum of PAH gene mutations has ethnic differences that is important for genetic counseling. We examined DNA of 68 PKU patients in Kazakhstan. Molecular genetic studies of PAH gene were carried out using PCR method (55 patients) and direct automated sequencing of PAH gene (13 Kazakh patients). In total group of PKU patients (Kazakh, Russian and other nationalities) mutation R408W was the most common (38.2%). Spectrum of PAH gene mutations was presented R261Q (5.4%), P281L (4.4%), IVS10nt546 (3.7%), R1580Q (1.5%), R281L (0.7%), W187X (0.7%), IVS12+1G>A

Characterization of tetrahydrobiopterin (BH4) metabolism in Zebrafish

SCOGP, Almaty, Kazakhstan; 2MGSC, Moscow, Russian Federation

Hemberger V1, Frese K2, Blau N1, Hoffmann GF1, Opladen T1, Sauer SW1 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany; 2Dep Int Med III, Univ Hosp, Heidelberg, Germany

Background: Tetrahydrobiopterin (BH4) is the essential cofactor of phenylalanine hydroxylase responsible for phenylalanine degradation as well as of tyrosine and tryptophan hydroxylase, rate-limiting

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enzymes in the biosynthesis of catecholamines and serotonin. Zebrafish has become an important model organism to study the role of genes in vertebrate brain development via mRNA specific morpholino knockdown. Objectives: The aim of this study was to characterize BH4 metabolism in zebrafish by analyzing the organ specific expression of the genes involved in the de-novo synthesis (GCH1, PTS and SPR) and recycling of BH4 (QDPR). Material and Methods: We visualized the expression of all four genes in the first 72 h of embryonic development via in-situ hybridization. Results and Discussion: Selection of potential mRNAs was based on the highest homology to the corresponding human genes. In-situ antisense probes for mRNAs showed tissue specific expression of the orthologues during zebrafish development at 24 hpf, 48 hpf and 72 hpf. For instance QDPR was highly expressed in hepatic tissue, whereas GCH1 was mainly detected in certain midbrain loci. Our results show that zebrafish express all genes of BH4 metabolism and, therefore, is a suitable model to study the role of BH4 in embryonic brain development.

targeted the 5'-splice sites of exons 11 or 12 of the PAH gene to produce exon skipping and thus a non-functional transcript resulting in hyperphenylalaninemia. This strategy was first assayed in a hepatoma cellular model, comparing two different chemistries (morpholino oligonucleotides and locked nucleic acids). After successful splice modulation resulting in the absence of PAH protein, we have tested Vivomorpholinos (VMO, morpholino oligonucleotides linked to an octaguanidine dendrimer) in both C57Bl/6 wild type and in C57Bl/6Pahenu2 heterozygote mice. Different amounts of VMO, dose frequency and mode of injection were compared. Consecutive intravenous injections of VMO resulted in transient hyperphenylalaninemia (> 1100 μmol/l) correlating with complete exon skipping and absence of PAH protein and activity in liver. These results provide the proof-of-concept for the in vivo use of VMO for splice modulation in a liver enzyme, as well as for the rapid and easy generation of murine models of disease, allowing relevant applications regarding the study of pathophysiological mechanisms and in vivo testing of future candidate therapeutics. P-035

P-033 Assesment of brain phenylalanine levels for different treatment modalities in patients with hyperphenylalaninemia Küçükçongar A1, Öner Y2, Cinasal Demir G1, Çelik B3, Tümer L1, Hasanoğlu A1, Ezgü FS1

Review of paediatric PKU patients of BH4 treatment with or without phenylalanine free formula Egea N1, Gutierrez A1, Garcia D1, Masferrer M1, Castejon E1, Meavilla S1, Fuste E2, Gassio R3, Lambruschini N1 1

1

2

Div Metab Dis, Univ Gazi Hosp, Ankara, Turkey; Div Radiology, Univ Gazi Hosp, Ankara, Turkey; 3Div Biostatiistics, Univ Gazi, Ankara, Turkey Objective: Magnetic resonance spectroscopy(MRS) is a novel noninvasive method to quantitate the brain metabolites. This study aimed to investigate the correlation between blood phenylalanine(Phe) ([Phe](blood)), brain [Phe](brain) in ten hyperphenylalaninemic(HPA) patients. Methods: Ten hyperphenylalaninemic patients were studied. The patients were divided into two groups: tetrahydrobiopterin (BH4 ) responsive (n = 4) and non-responsive (n = 6). [Phe](brain) were measured by absolute [Phe](brain) using creatine as an internal reference. Result: First procedure was done before treatment and the second procedure after 3 months treatment (Phe-restricted diet or BH4 ). There were significant differences, when compared to the blood Phe level between first and second procedure in all cases (p= 0,008). The brain Phe levels were decreased at the second procedure when compared to the first procedure in the diet group (p=0,027), there was no statistically significant difference between two procedures in the BH4 group (p=0,068). There was significant positive correlation in diet group between blood and brain Phe level, and there was positive correlation in BH4 group which was not statistically significant Conclusion: MRS can be used to quantitate intracerebral Phe concentrations non-invasively in HPA patients. MRS could provide proper objective standards for better diagnosis and treatment of HPA patients. P-034 In vivo effect of splice modulating antisense oligonucleotides: generation of hyperphenylalaninemia mice as model Gallego-Villar L1, Viecelli HM2, Perez B1, Ugarte M3, Thony B2, Desviat LR1 Centro de Biología Molecular, Madrid, Spain; 2University Children's Hospital, Zürich, Switzerland; 3CEDEM-UAM, MADRID, Spain

Gastroenterology and Nutrition Department;2Department of Psychology; Neurology Department, Hospital Sant Joan de Déu, Barcelona, Spain

3

Aim: To compare PKU patients on BH4 treatment and free diet (group A) with those on BH4 plus phenylalanine free formulae (group B). Methods: 32 patients: 21 in group A and 11 in group B. We compared: BMI (Body Mass Index), densitometry over 6 years old and biochemical parameters (Phe, Zn, Se, DHA, EPA, Omega3, Omega6). Results: Group A: 85% presented adequate Phe levels. Only one patient had obesity. 25% presented osteopenia. DHA and EPA were normal in all patients. 80% of them presented Omega6 within limits. 75% had normal W3. 53% presented with adequate Selenium. All patients presented normal Zinc values. Group B: 63.8% presented adequate Phe levels. All patients had normal BMI. Densitometry couldn't be studied due to insufficient data. 9% presented with low EPA values. 80% presented normal w6. All patients had Omega3 within reference values. Only 18% of patients presented adequate selenium. 9% presented a zinc deficiency. Conclusions: Group A presented with lower phe concentrations, and higher bone mass. Some patients also presented low Omega3 concentrations. Selenium was more frequently deficient in group B patients. This could be due to differences in Se bioavailability when comparing Se from natural sources and Se from formulae. P-036 Relationship between bone mineral density, dietary control and blood phenylalanine concentrations in adult patients with PKU Garcia D1, Gutierrez A1, Egea N1, Castejón E1, Meavilla S1, Artuch R2, Sierra C2, Gonzalez J3, Lambruschini N1 1

Gastroenterology and Nutrition; 2Biochemistry Department; 3Neurology Department, Hospital Sant Joan de Déu, Barcelona, Spain

1

Our groups have demonstrated the efficacy of antisense therapy for splicing defects in different cellular models of IEM. To date no specific animal models with these defects are available. As an alternative, we have

Introduction: A protein restricted diet is needed for the dietary control of phenylketonuria (PKU); adequate protein intake is achieved by adding low phenylalanine (Phe) formulae. Non-adherence to treatment, nutritional inadequacy, and high phenylalanine levels are associated with bone disease in several studies.

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Our aim was to evaluate and follow up bone mineral density (BMD) in PKU adult patients over the last year and to search for a possible relationship between BMD, dietary control, and blood Phe concentrations. Material and methods: Forty PKU subjects (40% male, 60% female), aged>18 years on dietary treatment were evaluated for bone mineral status using dual energy X-ray absorptiometry (DEXA), diet compliance and mean blood phenylalanine concentration for the last 12 months. Results: Mean blood phenylalanine concentration was 658 ± 230 μmol/L and significant association between diet-adherent and nonadherent groups were calculated (chi square, p=0.01). Osteopenia was detected in 18 patients (45%), while osteoporosis was detected in 3 (7.5%). A positive correlation was observed between low BMD and higher (>600 μmol/L) phenylalanine concentrations r=-0.63, p=0.01 Conclusions: The non-adherence to the diet and the consequent imbalance in the nutrient intake involved in bone metabolism, and high Phe levels suggest that these factors influence in reduced bone mineralization.

to explore the possible effect of BH4/Sapropterin treatment on both WM and neurocognitive outcome measures. Patients and methods: WMA score and WISC/WAIS IQ of 10 PKU subjects (12,28 years ± 4,11, age range : 7,83 – 22,25 years) under BH4/Sapropterin treatment for at least 1 year were compared with 10 PKU patients, who were treated only with the diet, matched for age, age of dietary onset, and concomitant and historical quality of dietary control. Results: No significant differences were found in severity of WMA score or cognitive profile between patients treated with BH4/sapropterin and patients under a phenylalanine-restricted diet. Conclusions: Our preliminary results suggest that under a similar quality of dietary control BH4/Sapropterin supplementation does not adjunctively improve the outcome measures in PKU. REFERENCE Leuzzi V, Gualdi GF, Fabbrizi F, Trasimeni G, Di Biasi C, Antonozzi I. Neuroradiological (MRI) abnormalities in phenylketonuric subjects: clinical and biochemical correlations. Neuropediatrics. 1993;24:302-6.

P-037

P-039

Sapropterin dihydrochloride treatment in Turkish patients under four years old with hyperphenylalaninemia

Influence of psycho-social and socioeconomic status on compliance in adult patients with phenylketonuria

Ünal Ö1, Gökmen-Özel H2, Coskun T1, Tokatlı A1, Hişmi B1, Dursun A1, Kalkanoğlu-Sivri HS1

Bosanska L1, Eckert U1, Moench E1, Tiling N1, Ploeckinger U1 1

Centre for Metab Dis, Charite Univ, Berlin, Germany

1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara; Turkey; 2 Hacettepe Univ, Dept Nutrition and Diet, Ankara, Turkey Background: Starting treatment with sapropterin in responsive patients early allows the use of a high-quality protein and micro- and macronutrient rich diet, when the brain development is fastest. However, it has not been given approval for use under four years of age in most of the countries, and studies on efficacy and safety of sapropterin under four years of age is limited in the literature. The purpose of the study is to evaluate efficacy and safety of sapropterin treatment in infants and children with sapropterin responsive hyperphenylalaninemia less than four years of age. Methods: 34 sapropterin responsive patients under four years of age were included in the study. Results: Median age of starting to the treatment was 2.3 years. Treatment period was median 17.8 months. Diet was liberalized in 27 patients (79.4%). In nine patients sapropterin treatment was started before instituting a phenylalanine restricted diet. Increasing Phe levels in the diet is going on in three patients. Phe levels could not to be controlled under treatment in four patients (11,74%), and treatment was stopped. Phe tolerance increased median 2.47-folds (1.21-5.63-folds). No adverse/side events were reported. Conclusion: Sapropterin treatment was found safe and efficacious under four years of age.

Objectives: The treatment of phenylketonuria (PKU) consists of a lifelong dietary phenylalanine restriction, an amino-acid supplementation as well as regular evaluation of metabolic control. We assume that psychosocial and socioeconomic status has a significant influence on the compliance of adult PKU-patients. Patients and Methods: 111 PKU-patients (18-42 yr, 46 % female), diagnosed and treated in the newborn period, were included. Telephone interviews were performed to collect the data. A scored questionnaire (0-8 points) for evaluation of compliance and psychosocial status addressed patients' knowledge about their disease, clinic visits, metabolic control and family support. Socioeconomic status was assessed by the educational classification Comparative Analyses of Social Mobility in Industrial Nations, the Hoffmeyer-Zlotnik-Scale and information about individual's net income. Results: High psychosocial status positively correlated with adherence to clinic visits, but not the metabolic control. No significant correlation was found between education, current employment status or individual's net income and compliance (defined as phenylalanine blood concentration below 20 mg/dl and regular clinic visits). The employment status corresponded with the general employment status in Berlin. Conclusions: Patients with high psycho-social status were more compliant regarding regular clinic visits, however no correlation was found between psycho-social or socioeconomic status and the metabolic control.

P-038 P-040 White matter involvement and neurocognitive outcome in phenylketonuric (PKU) patients with and without BH4/sapropterin dihydrochloride supplementation Mastrangelo M1, Caputi C1, Berillo L1, Manti F1, Caforio C1, Bertino S1, Carducci Cl2, Leuzzi V1 Dip Ped Neur Psi Inf, Sapienza Univ Rome, Rome, Italy; 2Dip Med Sper Sapienza Univ Rome, Rome, Italy

1

Background: White matter alterations (WMA) have been widely documented in early treated PKU patients, but the relationship between them and neurocognitive disorders is still debated. Our study was aimed

Rapid and sensitive determination of amino acids in serum and urine by aptamers Kyung-AE Y1, Worgall TS2, Stojanovic MN1 1 Dept. of Medicine, Div Exp, Therapeutics, New York City, United States; 2Dept. of Pathology and Pediatrics, New York City, United States

Background: Amino acids are measured by HPLC or mass spectrometry, methods not amenable to point-of-care testing (POCT). POCT is of interest for diagnosis and follow-up of for several inborn errors of amino acid metabolism, including phenylketonuria (PKU).

S126 Objective: We describe an aptamer (oligonucleotide-receptor)-based mix-and-measure test for sensitive and rapid determination of phenylalanine, tyrosine, and arginine in serum or urine. The method is robust, not dependent on temperature and expandable to any other amino acid. Success of our approach stems from a new vitro selection and amplification based selection protocol, yielding aptamers with a sensitivity that is two to three orders higher than previously reported aptamers. Conclusion: Novel amino acid specific aptamers enable rapid and easy-touse determination of serum and urine amino acids. The methodology can quantitatively measure phenylalanine concentrations above 200 micromolar and is thus suitable for surveillance of metabolic control in PKU. P-041 Predictable BH4-responsiveness: most south Portugal PKU patients are candidates for BH4 supplementation Pavlu-Pereira H1, Janeiro P2, Costa C2, Gaspar A2, Oliveira A3, Tavares de Almeida I1, Rivera I1 1

Met&Gen, iMed.UL, Fac Pharmacy, Lisbon, Portugal; 2Dept Paediatrics, Santa Maria Hospital, Lisbon, Portugal; 3Dept Medicine, Santa Maria Hospital, Lisbon, Portugal Hyperphenylalaninemia (HPA,OMIM #261600), which includes phenylketonuria (PKU), is caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). The great allelic heterogeneity of PAH gene, allied to the compound heterozigosity of most patients, leads to a wide spectrum of biochemical and clinical phenotypes. Genotypephenotype correlation is a useful tool to predict the metabolic phenotype, to establish the better tailored diet and, more recently, to assess the potential responsiveness to BH4 therapy, a current theme in the PKU field. The aim of this study was to evaluate the BH4-responsiveness among the South Portugal HPA population. PAH mutations were identified by direct sequence analysis of genomic DNA samples. The full characterization of 74 patients revealed the presence of 36 distinct mutations, five of which account for almost 60% of all mutant alleles. Notably, half of the mutations are BH4-responsive, according to BIOPKU database. Fifty-six different genotypic combinations were identified, most of them in single patients and involving a BH4-responsive mutation. In conclusion, a significant number of South Portugal PKU patients may potentially benefit from BH4 therapy which, combined with a less strict diet or eventually in special cases as monotherapy, may contribute to reduce nutritional deficiencies and minimize neurological and psychological dysfunctions. P-042 Using predictive modeling to estimate bone mineral density in children and adults with phenylketonuria Coakley KE1, Douglas TD2, Singh RH2 1 Ntrn and Health Sci Prgm, Emory Univ, Atlanta, United States; 2Dept Human Genetics, Emory University, Atlanta, United States

Background: Though bone abnormalities are prevalent in Phenylketonuria (PKU), bone is monitored infrequently and requires dual-energy x-ray absorptiometry(DXA), an expensive procedure involving radiation. Equations predicting total bone mineral density (TBMD) and z-scores using routine parameters were created to improve DXA-independent monitoring. Methods: Age, sex, body mass index(BMI), phenylalanine and medicalfood prescription, diet, fasting plasma phenylalanine and tyrosine, serum lipids, 1,25-dihyroxyvitamin-D, and genetic mutation severity (AV sum) were assessed in PKU patients 4 years and older (n=57). Variables significantly correlated with TBMD were candidates for models with

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 (1) all parameters, (2) routine parameters, and (3) routine and AV sum. Actual and model-derived TBMD and z-score category [normal, at-risk (1-2.5SD below normal), low (>2.5SD below normal)] were compared using t-tests and chi-square tests, assessing model fit. Results: In the sample (mean age+SD=17.5+11.0; 60% male), 16 (28%) had at-risk BMD; 3 (5%) had low BMD. TBMD correlated with age, BMI, medical-food prescription, cholesterol, triglycerides, vitamin D, and AV sum (p<0.05). R-square for models ranged from 0.75-0.86, indicating excellent fit. Actual and predicted TBMD strongly correlated (coefficients 0.87-0.93). Predicted and actual z-score categories agreed (kappa=1.00). Conclusions: One-third had reduced TBMD. Routinely collected parameters may predict TBMD in individuals with PKU, reducing reliance on DXA. P-043 BH4-responsive mouse models in phenylketonuria mutual impact of genotype, phenylalanine substrate and BH4 cofactor on enzyme function Eichinger A1, Danecka MK1, Reiß DD1, Woidy M1, Gersting SW1, Muntau AC1 1

Dep of Molec Peds, Hauner Child Hosp LMU, Munich, Germany

Homozygous Pahenu1/1(V106A/V106A) and compound heterozygous Pahenu1/2(V106A/F263S) have been identified as BH4-responsive mouse models in phenylketonuria (PKU). This allowed for better understanding of BH4-mode-of-action as a pharmacological chaperone exerting its stabilizing effect on phenylalanine hydroxylase (PAH) protein in vivo. To further understand the therapeutic BH4-effect as well as the interplay between phenylalanine, BH4 and murine genotypes we thoroughly investigated recombinantly expressed Pah mutations in vitro and homozygous and compound heterozygous genotypes in vivo and ex vivo. Bioluminescent resonance energy transfer (BRET) experiments revealed disturbed conformation for both homozygotes and decreased stability of V106A. Moreover the eukaryotic oligomerization pattern showed individual differences when comparing tetramer:dimer equilibria. Activity landscapes performed with recombinantly and eukaryotically expressed Pah variants showed differences in enzyme activity and also varying working optima. Additionally, in-depth ex vivo analysis revealed differences in the hepatic and plasmatic biopterin levels between genotypes. This has consequences for the availability of BH4, the metabolism of phenylalanine and therefore for the therapy of PAH-deficiency. Taken together, we were able to show that in murine PKU, as in human PKU, the mutual impact of substrate and cofactor should not be overlooked. Overall our findings elucidate the existing mouse models and open up further considerations for BH4-therapy. P-044 Towards European guidelines for PKU: how to speak a common language? van Wegberg AMJ1, Maillot F2, van Spronsen FJ1 1

Beatrix Child Hosp, Univ Med Centre, Groningen, Netherlands; CHRU de Tours, Université François Rabel, Tours, France

2

Background: At present, European PKU guidelines are being developed(1) urging the use of the same language. Objectives: To investigate whether consensus about the concepts of 'compliance' and 'off diet' and 'lost to follow-up' exists. Methods: A survey was conducted among 84 professionals at the workshop on European guidelines during the fifth European Phenylketonuria Group Symposium.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Results: Consensus for optimal versus poor compliance was considered as '>75% versus <50% of phe concentrations in target range' (45.2%;34.5%); '>75% versus <50% of agreed number of blood samples'(52.4%;58.3%); '>75% versus <50% of agreed number of outpatients controls' (54.8%;47.6%); '80-100% versus <50-66% taking protein substitutes' (82.1%;69.1%). There was also consensus for 'off diet', but not for 'lost to follow up'. Conclusion/Discussion: This survey showed that we can use the same language for important concepts used in day-to-day practice in PKU. A score table is proposed on the definition of poor, fair and optimal compliance independently of age and sex. Other concepts, as the definition of the severity of phenylalanine hydroxylase deficiency need to be worked out. 1)as done by 5 working groups with PKU professionals initiated by the ESPKU.

S127 control (IDC) (mean of median Phe levels) was calculated and tryptophan and platelet serotonin were determined. Results: Regarding health-related quality of life only one patient showed low scores in mental health scale, with significant results in anxious/depressed Achenbach scale, and normal tryptophan and serotonin levels. All patients showed good sleep quality. Regarding behavioural and emotional functioning two patients scored significantly, one in anxious/depressed scale and another in attention problems and intrusive scale, this one with an IDC of 1047 μmol/L. Mean patients' IDC was 634μmol/L. Biochemically, tryptophan values were normal, but serotonin was low in 6 patients, none with problems in any of the assessed items. Conclusions: The patients' satisfactory results were probably due to their good long-term dietetic control. P-047

P-045 Neurological outcome in early-treated phenylketonuric adult patients Vitamin B12 status by genotype and functional markers in phenylketonuria

González MJ1, Gassió R1, Tondo M1, Muchart J2, Fusté E1, Alonso I3, Lambruschini N1, Egea N1, Artuch R1, Campistol J1

Singh RH1, Coakley KE2, Douglas TD1 1

Dept Human Genetics, Emory University, Atlanta, United States; 2 Ntrn and Health Sci Prgm, Emory Univ, Atlanta, United States Background: Assess functional B12 deficiency with dietary intake and PAH mutation severity in individuals with PKU. Methods: Diet; plasma amino acids; serum vitamin B12, folate, methylmalonic acid(MMA), and total homocysteine were assessed in 26 females with PKU (mean age+SD:20.2+10.3). Functional B12 deficiency was indicated by MMA>0.37μmol/L, independent of serum B12. Correlations between nutrient intake and markers of B12 status (B12, MMA, homocysteine), and functional B12 deficiency by PAH mutation severity (classical, moderate, mild indicated by AV sum) were assessed. Results: One (4%) had elevated MMA but normal serum B12. Serum B12 was suboptimal(<350μmol/L) in eight(31%) and low(<180μmol/L) in one (4%). 18 (69%) had high serum folate(>24.8ng/mL). No differences in B12, MMA, or homocysteine were found by PAH mutation severity. Four (15%) consumed B12 below RDA, three of whom had suboptimal serum B12, though not significantly lower than those meeting RDA(p=0.0876). Serum B12 was positively correlated with dietary B12(p=0.058), tyrosine and energy(p<0.05) and negatively with dietary Phe:Tyr(r-square -0.52;p=0.01). Conclusions: Suboptimal B12 is related to lower dietary B12 and energy, and higher Phe:Tyr intake. Functional B12 deficiency is not prevalent in PKU or related to PAH mutation severity suggesting diets are sufficient, likely due to medical-food intake. High folate should be examined further.

1 PKU follow-up Unit. Hosp St Joan de Deu, Barcelona, Spain; 2Dep Radiology, Hosp St Joan de Deu, Barcelona, Spain; 3Dep Neurology, Hosp St Joan de Deu, Barcelona, Spain

Background: Elevated Phe levels have neurological effects even in early-treated PKU patients. Methods: To describe neurological outcome in early-treated PKU adult patients and its relationship with biochemical parameters. 25 adult patients were studied; the average of onset diet was 24 days. Neurological signs on examination, intellectual quotient (IQ), neuroradiological findings (NRF) in MRI, and visual evoked potentials (VEP) were evaluated. The Index of dietary control (IDC) was calculated in different periods of follow up. Results: The median of IDC in the last year was 588 μmol/L (48% with bad control). 71% had good control for the first 12 years of life. All had normal IQ (mean:102). MRI was performed in 23, being normal in 6. NRF were associated with IDC values higher than 383 μmol/L in the last year. The most frequent alteration was increased signal in periventricular white matter in T2 and DWI restriction. VEP was practiced in all patients, being abnormal in 7 of them, no correlation was observed with any IDC. Neurological examination revealed tremor in 12 and hyperreflexia in 8. Conclusions: Although neurological outcome was good in our patients, 74 % of them presented with abnormalities on MRI and 48% presented with tremor. P-048

P-046 Health-related quality of life, sleep and behavioural emotional functioning in early-treated phenylketonuric adult patients

Monitoring and treatment observance in patients with phenylketonuria (PKU) in a public hospital in argentina Cabrera AM1, Fain HE1, Blanco V1, Buiras VM1, Bonetto VV1, Fain C1

Gassió R1, González MJ1, Colomé R1, Sans O2, Sierra C1, Vilaseca MA1, Castejón E1, Gutiérrez A1, Artuch R1, Campistol J1 1

PKU follow-up Unit. Hosp St Joan de Deu, Barcelona, Spain; Dep Neurology, Hosp St Joan de Deu, Barcelona, Spain

2

Background: High Phe levels interfere with tryptophan transport through the blood-brain-barrier in PKU patients, resulting in decreased serotonin synthesis. Methods: To study the health-related quality of life, sleep, behavioural and emotional functioning in early-treated PKU adult patients and to search for their relationship with biochemical parameters. Short Form-36 Health Survey, Achenbach adult behavior checklist and Pittsburg sleep quality index were administered to 25 patients (12 females) with normal intellectual capacity. Last year's Index of dietary

1

Div Metab Dis, Vilela Children Hospital, Rosario, Argentina

Objective: To describe the outcome and treatment observance in patients with PKU. Materials and Methods: 13 patients with PKU were included. All of them were given a PHE restricted diet, with adequate formula requirements. The patients are assessed nutritionally, checked for calcium phosphorus metabolism, PKU levels and other parameters, following international recommendations. Results: Patients clinical and biochemical tests results and diet observance are satisfactory (85%/93%) but the formula intake is variable (45%). One of the patients is of short stature. He is the only early diagnosis and poor formula intake patient with erratic phenylalanine results and mild

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psychomotor retardation. Alterations in calcium / phosphorus metabolism (CPM) were detected in 6 patients, with good response to a calcium and vitamin D supplement.50% had a good diet and formula intake. A bone density scan was performed in 3 patients with a normal result in 1 case (good intake of formula) and a good improvement in another after treatment. Conclusions: Patients' fulfillment of the clinical, diet and lab tests is acceptable. The intake of the formula is not correct. This fact demands crafty responses from the dietitian. CPM is altered in a high percentage. It seems unrelated to the correct intake of the formula.

arginine/ADMA ratio were lower in BH4 treatment patients. Vitamin B12 levels were lower in BH4 patients in contrast with folic acid levels. Discussion: ADMA and SDMA were slightly higher and the Larginine/ADMA ratio was lower in BH4 treatment PKU children, therefore, NO synthesis could be altered in contrast with low-protein dietary patients. Metabolic profile and cardiovascular risk could be different among PKU patients according to their treatment.

P-049

Tetrahydrobiopterin therapy vs phenylalanine-restricted diet: impact on developmental outcomes of PKU patients

The effects of tetrahydrobiopterin (BH4) treatment on brain function in individuals with phenylketonuria Christ SE1, White DA2, Moffitt AJ1, MS Peck3 1 Univ of Missouri, Columbia, MO, United States; 2Washington Univ, St. Louis, MO, United States; 3Univ of Missouri School of Med, Columbia, MO, United States

Background: Recently, sapropterin dihydrochloride, a synthetic form of tetrahydrobiopterin (BH4), has been introduced as a supplemental treatment to dietary phe control for PKU. Very little is known regarding BH4 treatment and its effect on brain and cognition. The present study represents the first examination of potential changes in neural activity in patients with PKU during BH4 treatment. Methods: We utilized an n-back working memory task in conjunction with functional magnetic resonance imaging (fMRI) to evaluate functional brain integrity in a sample of 12 individuals with PKU at three timepoints: Just prior to BH4 treatment, after 4 weeks of treatment, and after 6 months of treatment. Neural activation patterns observed for the PKU treatment group were compared with those of a demographically-matched sample of 12 healthy non-PKU individuals who were assessed at identical time intervals (i.e., baseline, 4 weeks, and 6 months). Results & Conclusions: Consistent with past research, baseline evaluation revealed impaired working memory performance and atypical brain activation patterns in the PKU group as compared to the non-PKU group. Most importantly, BH4 treatment was associated with improvements in both brain activation and behavioral performance, with neural changes evident earlier (4week timepoint) as compared to the behavioral aspect (6-month timepoint). Conflict of Interest declared. P-050 Cardiovascular biomarkers in phenylketonuria patients with low-protein diet or BH4 treatment Andrade F1, Llarena M1, Sanjurjo P1, Aldámiz-Echevarría L1

P-051

Aldámiz-Echevarría L1, Bueno MA2, Couce ML3, Lage S1, Dalmau J4, Vitoria I4, Andrade F1, Blasco J5, Alcalde C6, Gil D7, García MC8, González-Lamuño D9, Ruiz M10, Ruiz MA11, González D12, SánchezValverde F13, Llarena M1 1

Cruces University Hospital, Barakaldo, Spain; 2Virgen del Rocio University Hospital, Sevilla, Spain; 3Santiago University Hospital, Santiago de Compostela, Spain; 4La Fe University Hospital, Valencia, Spain; 5Carlos Haya University Hospital, Málaga, Spain; 6Río Hortega University Hospital, Valladolid, Spain; 7Virgen de la Arrixaca Univ. Hospital, Murcia, Spain; 8Miguel Servet Univ. Hospital, Zaragoza, Spain; 9Marqués de Valdecilla Univ. Hosp., Santander, Spain; 10Nuestra Sña de la Candelaria Univ. Hosp., Tenerife, Spain; 11Son Espases Univ. Hosp., Mallorca, Spain; 12Maternal and Child Hospital, Badajoz, Spain; 13 Virgen del Camino Hospital, Pamplona, Spain Background: Tetrahydrobiopterin therapy has opened up new treatment options for some PKU patients, enabling them to eat normal diets. However, little is known about how this therapy affects their physical development. Methods: We conducted a retrospective longitudinal study examining anthropometric characteristics (height, weight, body mass index and growth speed Z-scores) in a cohort of PKU patients on tetrahydrobiopterin therapy (38 subjects) comparing their characteristics with those of a group of PKU patients on phenylalanine-restricted diets (76 subjects). Results: No improvement was observed in the anthropometric variables in the cohort on tetrahydrobiopterin therapy, from prior to starting treatment to when they had been taking the drug for two or five years. In almost all cases there was a fall in the mean Z-score for the variables during these periods. No association was found between higher protein intake and growth in individuals on tetrahydrobiopterin therapy. Growth impairment was also noted in the PKU patients on low-phenylalanine diets. Discussion: Our study identified growth impairment in patients with PKU on tetrahydrobiopterin, despite higher intakes of natural proteins. In fact, individuals undergoing long-term tetrahydrobiopterin treatment seemed to achieve similar anthropometric outcomes to those attained by individuals on more restricted diets.

1

P-052

Background: Protein-incorporated Arg residues can be methylated with subsequent proteolysis giving rise to asymmetric dimethylarginine (ADMA) that competes with L-arginine for binding to NOS. Several studies have suggested that increased ADMA levels can be used to assess cardiovascular risk. Patients with phenylketonuria (PKU) could suffer from high oxidative stress due to their low-protein dietary treatment or BH4 treatment. Free ADMA levels depend on both protein metabolism and oxidative stress, so ADMA levels may be different regarding their treatment. Patients and methods: Eighteen patients with PKU on low-protein diet or BH4 treatment participated in a cross sectional study. Biochemical variables as ADMA, SDMA, tHcys, CRP, folic acid, vitamin B12 and methylmalonic acid were analyzed. Results: ADMA, SDMA and CRP were lower in dietary treatment PKU patients compared to BH4 treatment. tHcy, methylmalonic acid and L-

A classical phenylketonuric infant with maternal phenylketonuria

Cruces University Hospital, Barakaldo, Spain

Küçükçongar A1, Özdemir Ö2, Dorum S3, Ekici A2 Şevket Yılmaz Child Hosp, Div Metab Dis, Bursa, Turkey; 2Şevket Yılmaz, Child Hosp, Div C Neurol, Bursa, Turkey; 3Şevket Yılmaz Child Hosp, Div Pediatr, Bursa, Turkey 1

Introduction: Elevated maternal phenylalanine (Phe) levels during pregnancy are teratogenic, and may result in embryo-foetopathy, which could lead to stillbirth, significant psychomotor handicaps and birth defects. This foetal damage is known as maternal phenylketonuria (MPKU). Reported here is an interesting case diagnosed classical PKU with maternal PKU.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Case report: A 17 days old boy patient, who is a ward of state living in a foster care facility, was diagnosed as phenylketonuria by neonatal screening with the level of phenylalanine 45 mg/dl. Physical examination showed microcephaly and generalized muscle hypertonicity. Because of unusual clinical findings for early diagnosed PKU patient (except for one with a tetrahydrobiopterin metabolism disorder), we queried the family history and learned that the biological mother is 29 years old and has mental retardation, and blonde hair. The diagnosis of PKU syndrome of the mother was determined following assessment of the serum Phe level, which was 22 mg/dl. Conclusion: According to the classical knowledge, generally, neurological abnormalities such as microcephaly,are not seen in early diagnosed and early treated classical PKU patients. For this reason, it is important to enquire in detail into the patient and family history. P-053 10-ARM radial maze: a new behavioural paradigm to unravel learning and memory deficits in a phenylketonuria mouse model Bruinenberg VM1, de Groot MJ2, Wubs M1, van Vliet D2, Anjema K2, van Spronsen FJ2, van der Zee EA1 1

Mol Neurobiology, Rijks Univ, Groningen, Netherlands; 2Beatrix Child Hosp, Univ Med Cent, Groningen, Netherlands Background: Immunohistochemical analysis of Pahenu2 C57Bl/6 mice (PKU) in our group, indicated disruptions in learning and memory related CREB/pCREB ratios in the hippocampal dentate gyrus and the dorsolateral striatum, respectively important for pattern separation and habit based learning. A specific paradigm to examine pattern separation is lacking. Therefore, the aim of this study was to generate a new paradigm to find the behavioural correlates of these PKU-specific neuromolecular change. Methods: Wild-type (WT: n=9) and PKU (n=9) mice were trained to locate food in a 10-arm radial maze (consisting of a starting arm which opened into a radial arena with nine arms). Key in this behavioural paradigm is the close proximity of arms which requires pattern separation to identify baited and non-baited arms. Results: Both PKU and WT mice mastered the task. However, a significant difference was found in strategy the mice used to master the task (p<0.0001). WT mice preferred a striatal strategy and PKU mice a hippocampal strategy. Conclusions: The preference of PKU mice for using a hippocampal strategy implies stronger PKU-specific difficulties on a striatal level than on the hippocampal level. Future studies will focus on verifying these findings by using specific striatum-based learning paradigms. P-054 Anthropometric characteristics and nutrition in a cohort of PAH-deficient patients

Aldámiz-Echevarría L1, Bueno MA2, Couce ML3, Lage S1, Dalmau J4, Vitoria I4, Andrade F1, Llarena M1, Sanjurjo P1, Blasco J5, Alcalde C6, Gil D7, Garcia MC8, González-Lamuño D9, Ruiz M10, Peña L11, Ruiz MA12, González D13, Sánchez-Valverde F14 1

Cruces University Hospital, Barakaldo, Spain; 2Virgen del Rocio University Hospital, Sevilla, Spain; 3Santiago University Hospital, Santiago de Compostela, Spain; 4La Fe University Hospital, Valencia, Spain; 5Carlos Haya University Hospital, Málaga, Spain; 6Río Hortega University Hospital, Valladolid, Spain; 7Virgen de la Arrixaca Univ. Hospital, Murcia, Spain; 8 Miguel Servet Univ. Hospital, Zaragoza, Spain; 9Marqués de Valdecilla Univ. Hosp., Santander, Spain; 10Nuestra Sña de la Candelaria Univ. Hosp., Tenerife, Spain; 11Mother and Child Hospital Complex, Las Palmas de Gran Canaria, Spain; 12Son Espases Univ. Hosp., Mallorca, Spain; 13Maternal and Child Hospital, Badajoz, Spain; 14Virgen del Camino Hospital, Pamplona, Spain

S129 Background: Treating phenylketonuria based upon strict vegetarian diets has occasionally been found to hamper physical development, presenting growth retardation and malnutrition. The present study aimed to evaluate anthropometric characteristics and nutrition in a cohort of patients with phenylketonuria and mildhyperphenylalaninaemia. Methods: We conducted a retrospective longitudinal study comparing anthropometric characteristics (weight, height, body mass index, and growth rate) in our patients and healthy subjects, with the measurements expressed as Z-scores. Data were collected every 6 months from birth to 18 years of age. Results: Growth impairment was observed in phenylketonuric patients. Specifically, there were two well-differentiated periods throughout which height fell well below Z-score = 0: from birth to two years of age, and on reaching adulthood. We also found height and weight to be positively correlated with phenylalanine intake. No growth retardation was seen in the patients with mildhyperphenylalaninaemia. Discussion: Phenylketonuric patients showed growth impairment in the early stages, with higher phenylalanine intakes being associated with attaining better developmental outcomes in this period. Therefore, prescribing very stringent diets in the early years might predispose phenylketonuric patients to retarded growth later in life, with developmental outcomes in adulthood being well below the 50th percentile for healthy subjects. P-055 In vivo evaluation of erythrocytes as carriers of the enzyme phenylalanine ammonia lyase (PAL) in ENU2 mice, an animal model of human phenylketonuria (PKU). Carducci C1, Rossi L2, Pascucci T3, Pierigé F2, Magnani M2,4, Rossi L4, Leuzzi V5 Dip. Med. Sperim, Sapienza Univ. di Roma, Rome, Italy; 2Dip. Scienze Biomolecol, Univ. di Urbino, Urbino, Italy; 3Dip. Psicol, Sapienza Univ. di Roma, Rome, Italy; 4EryDel SpA, Urbino, Italy; 5 Dip Ped Neuro Inf, Sapienza Univ di Roma, Rome, Italy

1

Aim: PKU is an autosomal recessive disease caused by PAH gene alterations. PAL converts Phe to the nontoxic t-cinnamate and proved to be an effective treatment for PKU. This study investigates erythrocytes (RBC) as a possible delivery system for PAL. Methods: Murine RBC were loaded with PAL through a hypotonic dialysis and isotonic resealing procedure (13.2 UI/ml RBC at 100% Hct; 8.8 mg of protein/ml RBC). ENU2 mice were treated with a intra-cardiac injection of PAL-loaded RBC (1.0, 0.5, or 0.25 IU). Blood Phe was monitored at 0, and 1, 2, 5, 8, 12, 16, and 21 days post-treatment. Results: Each of 3 doses markedly reduced blood Phe soon after injection; with 0.25 IU blood Phe fell (day 1) and slowly reached 50% of the pretreatment values on day 8; with 0.5 and 1 IU, blood Phe remained extremely low for 5 days and was still 21% and 25% of the basal values, respectively, on day 8. Conclusion: RBC is an effective vehicle for PAL and, potentially, for other enzymes acting on substrates free in the blood. Since the long life span of RBC, this approach assures a protracted enzymatic activity. We acknowledge Biomarine for providing rAvPAL Conflict of Interest declared.

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P-056

P-058

Multicenter study on growth in PKU patients: preliminary results

Third interim analysis of the Kuvan Adult Maternal Paediatric European Registry (KAMPER): sapropterin/BH4 responsiveness

Belanger-Quintana A1, Stanescu S1, Ahring K2, Dokoupil K3, Gokmen Ozel H4, Lammardo AM5, MacDonald A6, Robert M7, Rocha JC8, van Rijn M9 1 U. Enf. Metabolicas, Hosp Ramon y Cajal, Madrid, Spain; 2Center for PKU, Kennedy Institute, Glostrup, Denmark; 3Dp Metab Nutr, DrHauner Child Hosp, Munich, Germany; 4Dp Nutrition Dietetics, Hacettepe Univ, Ankara, Turkey; 5Dp Pediatric, San Paolo Hosp, Univ Milan, Milan, Italy; 6Inh Metab Disorders, Children's Hospital, Birmingham, United Kingdom; 7Dp Nutr Metab, Hosp Uv Enf Reine Fabiola, Brussels, Belgium; 8Centro Genética Médica Jacinto Magalhães, Porto, Portugal; 9 S. Metab Dis, Univ Med Centre Groningen, Groningen, Netherlands

Background: There is controversy as to whether PKU patients have an adequate physical development. Publications usually do not discuss dietary practices, metabolic control, and parental growth so it is unknown if any growth differences are due to the disorder or related to treatment practices. Objectives: To determine if PKU patients on dietary treatment have an adequate physical development and if different dietary practices relate to variation in physical development.. Material/patients and methods: Retrospective multicenter, multinational study collecting developmental and dietary data from birth to the end of puberty at 18 years of age in 101 PKU patients undergoing dietary treatment. Results: Growth Z-scores in PKU patients are within the normal range thought out the entire lifespan included in the study, and patients reach a final height as expected according to parental height with a normal distribution. However, mean Z-scores for body mass index show a tendency to high scores beginning in late infancy and becoming apparent in adolescence, predominantly in girls. Conclusions: The diet followed by PKU patients allows for an adequate physical development, but measures should be taken early in infancy to avoid overweight and obesity. P-057 At what FA-level shall we consider dietary intervention in the neonatal period? Pal A1, Lundqvist T1, Halldin Stenlid M1 1

Dept of Endo and Metab, Univ Child Hosp, Uppsala, Sweden

Following the expanded Swedish neonatal screening-program, a new group of patients with only slightly elevated phenylalanine (FA) levels has evolved. This has caused a discussion about the FA-level in which dietary interference should be considered. A boy had an elevated FA-level of 203 μmol/L (cut-off >180) and a FA/tyrosin ratio of 2.7 at 48 h. He was compound heterozygous with the mutations p.[Gly46Ser];[Ala403Val]. The boy was breast-fed. The infant period is an anabolic state of fast growth and increased protein tolerance. The protein content in breastmilk and infant formulas are low compared to food later in life. Growth velocity will subsequently decline. Catabolic situations will occur. There is a great probability that the child will need protein restriction in life. The aminoacid formulas are not well tolerated when introduced after infancy. The boy was started on a small dosage of aminoacid formula. Until seven months he had grown normally with an excellent metabolic control. At this time he had a cold and the FA-level increased to 430 μmol/L despite modestly increased protein restriction. This case shows that even with a small increase in FA-levels in the screening test, dietary modulation should be considered to meet different protein demand in life.

Burlina AB1, Feillet F2, Trefz FK3, Bélanger-Quintana A4, Lagler FB5, Stucker F6, Alm J7, Muntau AC8 University Hospital, Padova, Italy; 2Hôpital d'enfants Brabois, Vandoeuvre lès Nancy, France; 3School of Medicine Univ of Tuebingen, Reutlingen, Germany; 4Hospital Ramon y Cajal, Madrid, Spain; 5Paracelsus Medical University, Salzburg, Austria; 6Merck Serono S.A., Geneva, Switzerland; 7 Karolinska University Hospital, Stockholm, Sweden; 8LudwigMaximilians-University, Munich, Germany 1

Objectives: Evaluation of blood phenylalanine response to sapropterin/BH4 in patients with phenylketonuria (PKU) or BH4deficiency in KAMPER. Methods: Observational, multicentre drug registry, including a maternal sub-registry. Response test results were extracted from the 3-year interim analysis. Values presented as median(Q1,Q3). Results: Sapropterin/BH4 response test data were available for 291/296 (98.3%) PKU patients and 16/29(55.2%) BH4-deficient patients. PKU patient age (years) at testing was 4.7(2.8,5.4) (patients 4–<8 years; n=21); 8.1(2.7,8.8) (8–<12 years; n=12); 13.2(8.2,15.4) (12–<18 years; n=8); 16.9(15.5,19.7) (≥18 years; n=3). Sapropterin/BH4 test dose administered ranged 2–20 mg/kg over 8 hours to 1 month. Most frequent sapropterin/BH4 test dose administered and test duration were 20 mg/kg and 24 hours, respectively, in both indications. The lowest doses were used in BH4-deficient patients. Most PKU (n=282/296) and all BH4deficient (n=15) patients demonstrated ≥30% decrease in blood phenylalanine during the response test; 5 PKU patients were considered BH4responsive as per investigator's judgement. Maximum percentage phenylalanine decrease was 53%(41,68; n=125) in PKU and 90%(60,90; n=6) in BH4-deficiency. Maximum phenylalanine decrease was achieved later (12–24 hours) in PKU than in BH4-deficient patients (<12 hours). Conclusion: Evaluation of sapropterin/BH4 response test results confirm a ≥30% decrease in blood phenylalanine in most patients in KAMPER. Conflict of Interest declared. P-059 Body composition and phase angle by bioelectrical impedance analysis in patients with phenylketonuria (PKU) treated at hospital de clmnicas de Porto Alegre, Brazil Nalin T1, Tonon T1, Perry IDS2, Castro K3, Reyes AP3, Brandorff TI3, Busschers PMG4, Mainieri AS5, Derks TGJ6, van Rijn M6, van Spronsen FJ6, Schwartz IVD7 PPGBM - UFRGS, Porto Alegre, Brazil; 2Food and Nutr Res Centre HCPA, Porto Alegre, Brazil; 3Nutriotion course - UFRGS, Porto Alegre, Brazil; 4Prog Nut and Diet - Hanze Univ Groningen, Groningen, Netherlands; 5Depa of Ped and Child Care - UFRGS, Porto Alegre, Brazil; 6Sec of Metab Dis, Beatrix Child Hosp, Groningen, Netherlands; 7 Department of Genetics - UFRGS, Porto Alegre, Brazil 1

Background: PKU is caused by the enzyme deficiency of phenylalanine hydroxylase resulting in high blood and tissue concentrations of phenylalanine (Phe). The bioelectrical impedance analyses (BIA) determines both the body composition and phase angle (PA). Objective: To investigate body composition and PA in PKU patients compared to healthy controls. Methods: Twenty-three PKU patients and 17 healthy age and gendermatched controls were included. Both groups were assessed body mass index (BMI), body composition and PA by BIA.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Results: The sample presented a BMI mean of 19.8±3.7 and 20.9±4.6 for patients and controls, respectively. No differences were found in body composition of PKU patients compared to controls (% of fat free mass 80,9±7,7 vs 80,7±7,3; % of fat mass - 19,0±7,5 vs 19,2±7,3). Although no significant difference in PA was found in PKU patients compared to healthy controls (6,35 vs 6.89), a positive correlation was found between PA and phenylalanine levels in patients (r=0,457, p=0,032). Conclusions: Our data suggest PA does not differ between patients and controls, but shows correlation with Phe. In our study we did not find difference in BMI and body composition between patients and controls. An increase in the sample size is needed to confirm our findings. P-060 Brain levels of amines, tyrosine, and tryptophan in middle-aged PKU mice and the consequences of psychological challenges van Vliet D1, de Groot MJ1, Anjema K1, Bruinenberg VM2, Kema IP3, Heiner-Fokkema MR3, van Spronsen FJ1, van der Zee EA2

S131 Methods: Observational, multicentre drug registry, including a maternal sub-registry. Values are presented as median(Q1,Q3). Results: The third interim analysis included data from 51 adults with PKU (age 21.0[19.2,25.8] years; male:female, 41:59). Sapropterin/BH4 response test data were available for 48 patients. Daily sapropterin dose was 10.1 (10.0,17.7) mg/kg/day. Blood phenylalanine concentration (μmol/L) at baseline, 6 and 12 months was 609(450,869;n=34), 442(327,575;n=16) and 482 (270,678;n=12), respectively. Natural protein intake (g/day) at baseline, 6 and 12 months was 22(15.8,38.3;n=10), 44.8(43.0,50.5;n=8) and 72.0(62.0,77.5; n=4), respectively. At baseline, 3 patients had osteopenia, 1 had osteoporosis, 3 had mental retardation and 3 had neurological disorders. At follow-up, 12 mild/moderate adverse events (AEs) were reported in 10(20%) patients. AEs included hyperthyroidism, osteoporosis, rhinorrhoea, chronic sinusitis, upper respiratory tract infection and weight decrease; all were considered by investigators as unrelated to sapropterin. Conclusion: The third interim analysis of sapropterin treatment in adult PKU patients shows a substantial benefit with regard to natural protein intake that was close to a normal diet with good metabolic control. No safety issues were reported. Conflict of Interest declared.

1

Beatrix Child Hosp, Univ Med Center, Groningen, Netherlands; 2Center Behav Neurosci, Univ Groningen, Groningen, Netherlands; 3Dep Lab Med, Univ Med Center, Groningen, Netherlands

P-062 Molecular-genetic study of PTS gene in russian PKU patients

Background: Reduced brain amine, tyrosine, and tryptophan levels have been hypothesized to contribute to PKU pathophysiology. In young PKU mice, decreased brain levels of amines and their precursors, and impaired brain aminergic response to psychological challenges have been shown. Objective: This study investigates the brain levels of both amines and their precursors in middle-aged wild-type and PKU mice, and their response to psychological challenges. Results: In non-challenged mice, brain levels of amines, tyrosine, and tryptophan were clearly decreased in PKU compared to wild-type mice (p<0.01; p<0.05 for tyrosine). Psychological challenges normalized brain tyrosine and tryptophan levels in PKU mice, while they increased brain dopamine and serotonin (p<0.01), but not norepinephrine levels in wildtype and PKU mice (p=0.300 and p=0.089). After psychological challenges, brain serotonin and norepinephrine were still reduced in PKU compared to wild-type mice (p<0.01), but dopamine was not (p=0.234). Discussion: These results show that brain amine, tyrosine, and tryptophan deficiencies in PKU are still present at middle age, and that brain dopamine levels in PKU mice can be restored by adequate precursor amino acid availability, whereas serotonin and norepinephrine can be restored only in part. The underlying mechanisms may have consequences for the treatment of adult PKU patients. Conflict of Interest declared. P-061 Third interim analysis of the Kuvan Adult Maternal Paediatric European Registry (KAMPER): adult phenylketonuria characteristics at baseline and 1 year

Stepanova A1, Polyakov A1 1

Research Center for Medical Genetics, Moscow, Russian Federation

Hyperphenylalaninemia (HPA) may be caused by deficiency of phenylalanine hydroxylase or tetrahydrobiopterin (BH4). The most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyltetrahydropterin synthase (PTPS) activity. We report the molecular genetic study pyruvoyltetrahydropterin synthase (PTS) gene in agroup of PKU - patients from different Russian regions. The first we analyzed DNA of 915 PKU-patients for most frequent mutations in PAH gene using the multiplex system. Four patients diagnosed as BH4-deficient were analyzed for searching mutations in PTS gene. Mutations p.Asn72Lys and p.Thr106Met were detected in compound heterozygous state in two unrelated patients. The mutation p.Ser32Gly was found in two patients in a compound heterozygote state. Further for mutation p.Ser32Gly, p.Asn72Lys and p.Thr106Met in the PTS gene the multiplex system for MLPA PCR-analysis was created. We analyzed 152 PKU-patients without most common mutations in the PAH gene. Using this method, we detected mutations in 7 patients. A total 8 different mutations in PTS gene were identified. The p.Asn72Lys and p.Thr106Met mutations account for about 50% of PTS mutations detected in Russian patients with hyperphenylalaninemia owing to lack PTPS activity. HPA due to lack of PTPS was estimated around 1,3% of the all Russian PKU-patients. P-063

Alm J1, Burlina AB2, Trefz FK3, Muntau AC4, Lagler FB5, Feillet F6, Vincent C7, Bélanger-Quintana A8 1

Karolinska University Hospital, Stockholm, Sweden; 2University Hospital, Padova, Italy; 3School of Medicine Univ of Tuebingen, Reutlingen, Germany; 4Ludwig-Maximilians-University, Munich, Germany; 5 Paracelsus Medical University, Salzburg, Austria; 6Hôpital d'enfants Brabois, Vandoeuvre lès Nancy, France; 7Merck Serono S.A., Geneva, Switzerland; 8Hospital Ramon y Cajal, Madrid, Spain Objectives: KAMPER aims to provide information on long-term outcomes of approximately 625 sapropterin-treated patients with phenylketonuria (PKU) or BH4 deficiency.

Linking patients/genotypes database (BIOPKU) and locus-specific database (PAHvdb) with tools for prediction of phenotypes and BH4-responsiveness in phenylketonuria Wettstein S1, Yue WW2, Underhaug J3, Marsden BD2, Martinez A3, Honegger A4, Perez B5, Blau N1,6 1

Div Metab, Univ Child Hosp, Zurich, Switzerland; 2Struct Genom Consort, Univ Oxford, Oxford, United Kingdom; 3Dept Biomed, Univ Bergen, Bergen, Norway; 4Dept Biochem, Univ Zurich, Zurich, Switzerland; 5 Dept Mol Bio, CSIC, UAM, Univ Autonoma, Madrid, Spain; 6Div Inborn Metab Dis, Univ Child Hosp, Heidelberg, Germany

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Background: The variability in the metabolic phenotypes in PKU is caused by different mutations within the PAH gene. In addition, it has been shown that genotypes are useful in predicting BH4 responsiveness in PKU. Objectives: To analyze data from available PKU-associated databases and to establish algorithms for genotype-phenotype correlation and BH4-responsiveness prediction. Methods: Relative frequencies of variations, genotypes, affected gene region and PAH domain were calculated. PAH variations and genotypes were scored using data from FoldX, SIFT, Polyphen2 SNPs3D, and Rosetta ddG prediction tools. The 3D atomic environment of each mutation is visualized using the interactive iSee concept. The PAHvdb database (833 variations) and BIOPKU database (4181 PKU patients/genotypes) (www.biopku.org) were used. Results/Conclusions: Amongst the 4181 patients (15,1% HPA, 24,4% mild PKU, 41,3% classic PKU, 19,2% no information) we observed 463 different mutations. The most frequently affected sites were exon 7 (22,9%) and intron 10 (9,4%), and the most affected PAH region was the catalytic domain (60,8%), followed by the regulatory (14,0%) and tetramerization domain (4,9%). c.1066-11G>A/c.1066-11G>A was the most frequent genotype (3,3%). BH4-responsiveness data were available from 2128 patients (44,4% responders). Using genotype scoring both the phenotype and BH4-responsiveness can be estimated, offering a reliable method for patients' management. P-064 The maternal perception of the phenylketonuria in the family dynamic 1

2

1

3

Soares R , Kanufre V , Alves MRA , Goulart LMHF , Aguiar MJB

phenylalanine (Phe) concentration. Current European clinical practice uses 24–48-hour BH4 response tests, which may not detect late responders. This study evaluated the proportion of responders (≥30% reduction in blood Phe concentration from baseline) to synthetic BH4 (sapropterin), 20 mg/kg/day, measured at several time points over 28 days. Methods: Open-label, single-arm, cohort study in Norway and Denmark. Patients with PKU with known, suspected or unknown BH4responder mutations in the phenylalanine hydroxylase gene were recruited. Results: 59 patients recruited; mean(range) age 21.0(4.3–51.4) years. 75% of patients (44/59) responded to sapropterin treatment. Early response (≥30% reduction within 7 days) was achieved by 38/59(64%), late response (≥30% reduction within 8–28 days) by 6/59(10%) and partial response (10–30% reduction within 28 days) by 15/59(25%). Treatment-related adverse events (AEs) were recorded in 36(61%) patients; all were mild/moderate. One patient (2%) withdrew due to AEs (treatment-related nausea). Conclusion: Although most BH4-responsive PKU patients have an early response to sapropterin, 10% of patients respond beyond 7 days, highlighting the need for longer test periods. Partial response may be due to diet/lifestyle. Conflict of Interest declared. P-066

1

1

Center for Action and Research in Suppor, Belo Horizonte, Brazil; Clinics Hospital, Federal University of, Belo Horizonte, Brazil; 3 Department of Pediatrics, Medical School, Belo Horizonte, Brazil 2

Introduction: The treatment of Phenylketonuria (PKU) is based on diet and involves a severe reduction in protein intake. From the diagnosis, all the family activities are focused on the presence of disease, reinforced by the use of a restrictive and unpleasant diet, that should be maintained throughout life. Goal: To investigate the repercussion of the PKU diagnosis on the family dynamic. Methodology: This is a qualitative study of an exploratory descriptive nature, which uses semi structured interviews. Fourteen mothers of children from 2 to 6 years that were diagnosed early with PKU were interviewed. A content analysis was carried out for the evaluation of the data obtained. Results: The majority of the mothers reported that it was hard to accept and deal with the diagnosis for PKU. Guilt, fear and anxiety were the main feelings demonstrated. It was possible to observe changes in the family diet, as well as conflicts between the parents, difficulties inestablishing boundaries for the children, and worries about fitting in at school and exclusion in social events. Conclusion: The study allowed the mothers to express their anxieties, fears, facing the difficulties of dealing with PKU in family dynamics, expanding the understanding of the psychosocial and behavioral aspects of PKU children in their environment. P-065 Final results from endure: a phase iv, prospective, open-label, uncontrolled trial to assess the responsiveness of patients with phenylketonuria to treatment with sapropterin 20 mg/kg/day for 28 days Jxrgensen JV1, Mathisen P1, Lilje R1, Stxlen LH1, Jorgensen M-B2, Nielsen J3 1

Oslo University Hospital, Rikshospitalet, Oslo, Norway; 2Merck A/S, Hellerup, Denmark; 3The Kennedy Center, Glostrup, Denmark Background: A subset of patients with phenylketonuria (PKU) responds to tetrahydrobiopterin (BH4) with a reduction in blood

Third interim analysis of the Kuvan Adult Maternal Paediatric European Registry (KAMPER): patient characteristics at baseline and 1 year Trefz FK1, Bélanger-Quintana A2, Muntau AC3, Lagler FB4, Feillet F5, Vincent C6, Alm J7, Burlina AB8 1

School of Medicine Univ of Tuebingen, Reutlingen, Germany; 2Hospital Ramon y Cajal, Madrid, Spain; 3Ludwig-Maximilians-University, Munich, Germany; 4Paracelsus Medical University, Salzburg, Austria; 5 Hôpital d'enfants Brabois, Vandoeuvre lès Nancy, France; 6Merck Serono S.A., Geneva, Switzerland; 7Karolinska University Hospital, Stockholm, Sweden; 8University Hospital, Padova, Italy Objectives: KAMPER aims to track the long-term safety and outcomes of approximately 625 sapropterin-treated patients with hyperphenylalaninaemia due to phenylketonuria (PKU) or BH4 deficiency. Methods: Observational, multicentre drug registry, including a maternal sub-registry. Values presented as median(Q1,Q3) unless indicated otherwise. Results: Seven countries contributed 325 patients (91%[n=296] PKU; 9%[n=29] BH4-deficiency; male:female 51:49). Median age was 10.3 (7.2,15.0) years for PKU patients and 12.8(6.6,18.9) years for BH4deficiency patients. 51 PKU patients and 8 BH4-deficient patients were adults (≥18years). Phenylalanine concentration (μmol/L) before sapropterin and at 12 months, respectively, was 414(289,561;n=215) and 340(248,486;n=121) in PKU patients, and 91(67,313;n=20) and 89 (76,117;n=6) in BH4-deficient patients. In PKU patients, natural protein intake (g/day) before sapropterin and at 12 months was 19.7 (13.0,38.0;n=43) and 46.0(31.0,63.0;n=43), respectively; height Zscores were –0.1(–0.8,0.6;n=244) and –0.2(–0.9,0.6;n=115) and body mass index Z-scores were 0.4(–0.4,1.2;n=244) and 0.4(–0.3,1.3;n=115) at baseline and 12 months, respectively. At baseline, osteopenia was detected in 5/59 and osteoporosis in 2/59 PKU patients. Conclusion: The third interim analysis confirms that most patients in KAMPER have a mild phenotype. Median natural protein intake increased at 1 year while maintaining target phenylalanine levels and adequate somatic growth. Conflict of Interest declared.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 P-067 Growth and pubertal timing in patients with phenylketonuria (PKU) Bal MO1, Bettocchi I1, Cantasano A1, Zazzetta E1, Baronio F1, Rizzello A1, Mazzanti L1, Pession A1, Cassio A1 1

Neon Screen Center, Ped Endo Progr, Bologna, Italy

Background: While positive results of PKU dietary-treatment on neurological outcome are known, long term effect on growth and puberty have not been sufficiently documented. Objective: To study growth, puberty, BMI and final height in PKU patients. Methods: 48 treated PKU subjects (21 females-F,27 males-M) with different clinical severity and intake of synthetic proteins, diagnosed by newborn screening. Chronological age (CA) was >3 yrs in 35/48 subjects. Evaluations were taken at 3, 6 yrs, pubertal onset and at final height(FH). Results: 3 yrs evaluation in 26/35 subjects (14F,12M): mean female's height(H) was 95.15cm, BMI 15.85; mean male's H was 96.27cm, BMI 16.86. 6 yrs evaluation in 25/35 subjects (12F,13M): mean female's H was 120.18cm, BMI 17.33; mean male's H was 116cm, BMI 16.93. 24/35 subjects (10F,14M) were evaluated at pubertal onset according to Tanner: mean age of B2 was 10.71, mean female's H was 138.5cm, BMI 19.48, mean CA at menarche was 12.69; mean age of T4 was 11.11, mean male's H was 141,8cm, BMI 19.42. 22/24 subjects reached FH (10 F, 12 M): F FH/TH 0.99; M FH/TH 0.99. Conclusions: Despite poor natural protein intake growth, BMI, pubertal onset and menarche were normal and FH according to Target. P-068 Dihydrobiopterin reductase deficiency (DHPRD) consequences of late diagnosis Kusmierska K1, Szymanska K2, Demkow U3 1

Lab Newb Screening, Inst Mother Child, Warsaw, Poland; 2Dept Exp Clin Neurop, Polish Academy Sci, Warsaw, Poland; 3Dept Lab Diag Clin Imm Dev A, Med Univ, Warsaw, Poland Background: DHPRD is a genetic disorder of tetrahydrobiopterin regeneration causing hyperphenylalaninemia. DHPRD is associated with decreased biogenic amine metabolite, and low 5methyltetrahydrofolate (5-MTHF), levels in cerebrospinal fluid (CSF). Case report: A man with DHPRD was diagnosed at the age of 23. Neonatal screening revealed mild hyperphenylalaninemia and dietary treatment was started. At the age of two, a cerebral palsy due to abnormal development of motor skills was diagnosed. A systematic deterioration of motor and cognitive functions was documented. At 23 years, the patient was hospitalized because of a progressive extrapyramidal syndrome. Voluntary movements were severely impaired. Moreover bone densitometry revealed very advanced osteoporosis. Biogenic amine metabolites in CSF were below the detection limit, 5MTHF was decreased (25 nmol/L; N:40–180nmol/L). Pterin profile in urine and CSF was normal, while the enzyme activity in blood spot was very low (0,32 mU/mgprotein; N:1,65– 5,67). A significant clinical and biochemical improvement was observed after administration of l-dopa, 5-hydroxytryptophan and folic acid. Conclusion: Late diagnosis led to irreversible damage of the brain and other organs, nevertheless, a significant improvement was observed

S133 after implementation of the therapy. Therefore it is crucial to diagnose this syndrome as early as possible because the replacement therapy can prevent permanent damage. P-069 Low initial growth rate leaves phenylketonuria patients (PKU) shorter throughout childhood: longitudinal analysis using the crescnet database Thiele A1, Gausche R2, Vogel M2, Lindenberg C1, Mütze U1, Arelin M1, Rohde C1, Keller E2, Pfäffle R1, Mohnike K3, Kiess W1, Beblo S1 1

Div Metab Dis, Univ Child Hosp Leipzig, Leipzig, Germany; 2CrescNet gGmbH, Leipzig, Germany, Leipzig, Germany; 3Clinic for Pediatr, Univ of Magdeburg, Magdeburg, Germany Background: Newborn screening and early dietary treatment allow normal psychomotor development of PKU-patients. Data on suboptimal growth is controversial. The computerized national CrescNet database gathers detailed data of growth and development for the general population as well as specific patient groups. Patients and Methods: Retrospective longitudinal analysis of standardized, yearly measurements of weight, height and growth rate was performed for 128 PKU-patients (0-18 years, 63 girls, 65 boys). Data were compared to the reference values of healthy German children. Results: Mean height in PKU is lower compared to healthy children (height-SDS -0.760 to -0.088, during age 1-11 years, p<0.05; final height SDS at ≥17 years -0.5, p=0.002). In contrast, BMI is significantly higher during early childhood (1-2 years, BMI-SDS +0.34, p=0.05) and in adolescent girls (15-17 years, BMI-SDS +0.7, p=0.009). Growth rate (height velocity, HV) is significantly higher during childhood (2-7 years, HV-SDS +0.4, p=0.004) but significantly lower towards and during puberty (11-15 years, HV-SDS -0.2, p=0.025). Conclusion: PKU patients show significant growth differences compared to the reference values. They tend to be shorter but heavier. The possible relation to dietary treatment and metabolic control should be investigated. These differences may influence the incidence of BMIrelated disorders in adult PKU. P-070 Dispersion of the p wave and QT segment as a test for cardiac autonomic function in phenylketonuria Kose M1, Canda E2, Kagnıcı M3, Atik Altinok Y2, Kalkan Uçar S1, Levent E4, Çoker M2 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis, Ege Univ Child Hosp, İzmir, Turkey; 3Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 4Div Card Dis, Ege Univ Child Hosp, İzmir, Turkey

1

Background: Even if plasma Phenylalanine(Phe) is kept within normal limits;fluctuating concentrations of Phe could affect Tyrosine Hydroxylase activity and consequently catecholamines .This may provoke variations of electrochemical conduction system of the heart and cause cardiac autonomic dysfunction. Objective: Evaluating electrochemical conduction system changes in phenylketonuria(PKU) patients by the electrocadiographic(ECG) methods. Materials and Methods: We included 45 PKU patients older than 3 year old, seperated into three groups (groupI, groupII, groupIII) as

S134 patients whose Phe last year median levels were <600 μmol/l,600900μmol/l,>900 μmol/l respectively.We performed 12 derivation ECG and holter on all patients and control group(50 patients). Results: We found significantly increased p dyspersion and QT dyspersion in group III,compared with control group, groupI and groupII statistically.There is no difference between groupI, groupII and between control group.It is interesting that 4 patients in group III had vasovagal symptoms and heart rate variability of these 4 patients were increased,too. Conclusion: We demonstrated abnormal p dyspersion and QT dyspersion in PKU patients with high Phe levels for last one year.This finding could be significant for considering possibility of developing cardiac autonomic dysfuncton in PKU patients.It will benecessary to conduct further studies with a larger series and additive parametres to identify the relationship between Phe levels and autonomic dysfunction so that this can be considered as a follow-up issue.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 the disc, and phenylalanine and tyrosine determined by LCMS/MS. Results: Precision CV for PSD plasma phenylalanine and tyrosine concentrations is less than 6%. Values from fingerstick PSD plasma vs. venous-draw plasma (n=28) were well correlated: phenylalanine (Pearson r=0.978; 1/slope=0.973); tyrosine (Pearson r=0.983; 1/slope=0.996). Phenylalanine and tyrosine collected on the PSD is stable for at least 35 days at room temperature. Conclusion: The PSD collection method coupled to LC-MS/MS determination offers an accurate and "true" plasma concentration of phenylalanine and tyrosine. Values are comparable to those obtained by the regular venous draw method. Conflict of Interest declared. P-073

P-071

Early predictors of non-adherence to phenylketonuria (PKU) treatment

BH4 responsiveness: Ege university experience

Gibbons F1, Mumford N1, Skeath RH1, Abulhoul LH1, Cleary MA1

Kagnici M1, Atik Altinok Y2, Kose M1, Canda E1, Kalkan Ucar S1, Habif S1, Coker M1

1

1

Div Metab Dis,Ege Univ Child Hosp, Izmir, Turkey; 2Div Clin Biochem,Ege Univ Hosp, Izmir, Turkey

Background: Defects in either phenylalanine hydroxylase or the production or recycling of tetrahydrobiopterin (BH4) may cause hyperphenylalaninaemia(HPA). Pharmacological doses of BH4 can reduce blood phe levels in some patients with phenlyketonuria (PKU) and can be used solely as an adjunct to diet therapy in treatment of HPA. Objective: To evaluate patients with HPA in terms of BH4 responsiveness, rate of decline in phe levels and changes in daily consumption of phe. Results: In our study,we evaluated 45 patients, 33 of which is PKU and 12 of which is HPA. Mean age was 5.7 ±3.8 years in PKU group and 4.3±3.9 years in HPA group. Mean phe levels at diagnosis were 1258.2±486.7 in PKU group and 442.7±80.1 in HPA group (μmol/l). The response rate to BH4 was 45.5% in PKU group and 91.7% in HPA group. The mean rate of decline in phe levels is 28.6±30.3% in PKU group and 63.2±25.8% in HPA group. The mean daily consumption of phe rises from 344.1±81.7 to 518.7±644.3 after treatment in PKU group and from 387.6±89.4 to 1501±825.7 in HPA group. Conclusion: BH4 administration in HPA has increased phenylalanine tolerance in our BH4 responsive patients. P-072 PKU monitoring from a fingerstick using a plasma separator device

Great Ormond St Hosp for Children NHS FT, London, United Kingdom

Objective: To identify early predictors of non-adherence to recommended dietary regimen. Method: Retrospective review of clinical notes and database of 153 patients in our PKU clinic. Family demographics and behavioural factors such as clinic attendance rate and frequency of home PHE blood test were compared between adherent and non-adherent families. Results: Demographic factors associated with non-adherence included divorced/separated parents; both parents unemployed; non-English speaking parents; ethnic minority even if English spoken; and behavioural factors included the main carer being uncontactable by PKU team. Discussion: In our centre's experience managing non-adherence once established is difficult. Early intervention to provide individualised social and clinical support to families with identified risk factors may be beneficial in helping to prevent behaviours and beliefs that lead to poor compliance. Future research is required to establish a reliable and valid tool for early detection of the risk factors. P-074 The mutation spectrum of phenylalanine hydroxylase gene in Turkish patients with phenylketonuria: six novel mutations Yilmaz-Yucel D1, Ozgul RK1, Sivri S1, Unal O1, Coskun T1, Tokatli A1, Dursun A1

Arning E1, Hundley E1, Bottiglieri T1 1

Hacettepe University, Dep. of Metabolism, Ankara, Turkey

1

Baylor Research Institute, Metabolic Dis, Dallas, United States

Background: Regular measurement of blood phenylalanine and tyrosine levels is critical in patients with PKU. Measurements from dried blood spots (DBS) can often be inaccurate due to differences in hematocrit. Objective: To develop and validate a method to determine phenylalanine and tyrosine from plasma obtained from a fingerstick using a plasma separator device (PSD). Method: One or two drops of blood, obtained by fingerstick or spiked whole blood, were deposited on the test area of a PSD card. The PSD card contains two layers; a top layer retains blood cells while plasma diffuses to the second layer and is absorbed onto a small disc. Plasma (2.8μl) is extracted from

Phenylketonuria is an autosomal recessive disorder associated with deficient activity of hepatic phenylalanine hydroxylase (PAH) which converts phenylalanine to tyrosine in the presence of the essential cofactor tetrahydrobiopterin (BH4). The prevalence of PAH-deficient phenylketonuria (PKU) in Turkey is high (1 in 6500 births). Latediagnosed, untreated PKU leads to severe neurological impairment including mental retardation, microcephaly, autistic behavior, eczema, and seizures. Over 500 mutations has cataloged for the PAH gene. (www.pahdb.mcgill.ca/) In this study, mutation screening of thirteen exons of PAH gene was performed in 55 unrelated Turkish patients with PKU by using direct sequence analysis. Four different types of pathogenic mutations (6 splicing, 25 missense, 1 nonsense and 2 deletion) were detected in

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 patients suspected with PKU. The most common mutated alleles in our cohort from PKU patients are IVS10-11G>A (15%), p.Leu48Ser (12%) and IVS10-7C>A (8.5%), and p.Glu390Gly (7.5%), respectively. In addition, six novel mutations (p. Phe121Ser, p.Gly171Trp, p.Val262Gly, p. Asp296Gly, p. Phe331Ser, p.Tyr417Cys) was detected in six patients as heterozygous state. P-075 Russian experience in genotype-phenotype correlations assesment of hyperphenylalaninemia due to 6-pyruvoyltetrahydropterin synthase deficiency Latypov A1, Polyakov A.V.2, Stepanova A.A.2, Nazarenko L.P.3, Matulevich S.A.4, Golihina T.A.4, Lyazina L.V.5, Listopad G.G.6, Proskurina E.V.2, Romanenko O.P.5

S135 Results: 143 PKU patients were evaluated dermatologically. Their mean age was 10,02 years (median 7 years).Fair hair was observed in 17 patients (11,8%) and dark hair in 126 patients (n=88,2%). Colour eyes: 12,6% (n=18) had fair iris and 87,4% (n=125) dark iris. 23,1% (n=33) patients were diagnosed of atopic dermatitis (AD). It was observed that the group with lower Phe dietary tolerance included more patients with AD (29,8% vs 10,2%, p<0,05; Odds Ratio (OR)=2,64) and they have more frequent cutaneous criteria of AD (94,1% vs 66,7%, p<0,05; OR= 1,41). Xerosis (70,6% vs 33,3%) and pruritus (47,1% vs 22,2%) were the only two criteria observed significantly more frequently in the group with lower Phe dietary tolerance. Conclusions: Future studies would be necessary to determinate the reason for higher frequency of AD or atopiform dermatitis in patients with low Phe tolerance. P-077

1

Russian Med Postgraduate Acad, Moscow, Russian Federation; 2 Research Centre of Medical Genetics, Moscow, Russian Federation; 3 Research Institute of Medical Genetics, Tomsk, Russian Federation; 4 Medical genetic Centre, Krasnodar, Russian Federation; 5Medical Genetic Centre, St-Peterburg, Russian Federation; 6Medical genetic Centre, Volgograd, Russian Federation Background: Malignant phenylketonuria type III (HPABH4A) is caused by different mutations in 6-Pyruvoyl tetrahydropterin synthase (PTPS) gene. We analyzed the available data of HPABH4A cases for genotype-phenotype correlation. Method: In cohort involved patients with HPABH4A, confirmed by molecular genetic diagnostic (sequencing PTPS gene), evaluated data: age, sex, residence, weight, attending medical doctor clinical assessment and treatment efficacy. Results: PTPS mutations in 14 patients distributed: 1 homozygote, 13 compound heterozygotes. Expressed manifestation of neurological symptoms was registered from early age (during first y.o.) in 8 patients ("severe" group). The main symptoms were: physical and mental retardation, paresis, spasms, convulsions, weakness, and others. 7 patients were treated by commercially available synthetic form of tetrahydrobiopterin with positive outcome but the treatment was started in over 2 y.o. age in all cases. 7 patients ("mild group") were confirmed by mutation diagnostic and had comparatively mild form of neurological complications (with manifestation in puberty) or without symptoms (due to early age). Conclusions: Early diagnosis of mild and severe clinical forms of HPABH4A and timely treatment makes it possible to prevent neurological complications and disability for patients with PTPS deficiency. So, differential diagnosis of hyperphenylalaninemia needs to be developed in conjunction with newborn screening for phenylketonuria. P-076 Dermatological manifestations of phenylketonuria Dominguez-Cruz JJ1, Bueno-Delgado MA2, Delgado-Pecellin C2, Bernabeu-Wittel J1, Conejo-Mir J1 1 Pediatric Dermatologic Unit of HUV Rocío, Sevilla, Spain; 2Metabolic Disorders Unity, HUV Rocío, Sevilla, Spain

Background: No recent study has evaluated dermatological manifestations of early treated PKU patients. Method: Complete dermatological assessment was undertaken for every PKU patient of the Congenital Metabolic Disease Unit, Hospital Virgen del Rocío of Sevilla. PKU patients were classified in two groups: Low tolerance and mild-high tolerance of Phe.

Large neutral aminoacid supplementation improves executive functions in diet non-adherant phenlyketonuria adolescents Hişmi B1, Tüzün Z2, Gökmen Özel H3, Ünal Ö1, Sivri S1, Dursun A1, Coskun T1, Tokatlı A1 1

Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Div Adolescent, Hacettepe Uni Child Hosp, Ankara, Turkey; 3Fac Health Sci,Nutr and Diet,Hacettepe, Ankara, Turkey Background: A cognitive ability that is affected in phenylketonutria(PKU) patients is executive function(EF). Diet non-adherence becomes a big problem as PKU patients age. Large neutral amino acid(LNAA) supplementation is an alternative adjuvant treatment regimen for these patients.The aim of this study was to assess EF in LNAA supplemented PKU adolescents before and after the treatment, by both day-to-day and laboratory measures. Materials and Methods: All PKU subjects between age 12-18 years, for whom LNAA supplementation was to be introduced were invited to join the study. EF assessments were performed twice, before and 6 months after LNAA supplementation. Day-to-day EF was assessed with the Behavior Rating Inventory of Executive Function(BRIEF) parent and teacher forms. Wisconsin Card Sorting Test (WCST) and Stroop Test were introduced as laboratory measures. Blood phenylalanine levels and phenylalanine/tyrosine ratios were assessed. Results: Global Executive Composite demonstrated 19% and 23% improvement with LNAA supplementation on teacher and parent assesments respectively. Parents reported the best improvement on initiate, planning/organizing, monitoring subscales, while improvement of emotional control was more prominent according to teachers.There was some improvement of non-EF measures on WCST and no improvement on Stroop. The decrease in phenylalanine and increase in Phe/tyrosine levels were median 8% and 27.8% respectively. Conclusion: LNAA supplementation improves day-to-day EF in diet non-adherent PKU adolescents. P-078 Sapropterin as a chaperone in several patients with PKU Bueno-Delgado MA1, Delgado-Pecellín C1, Gonzalez-Meneses A1, Couce-Pico ML2, González-Lamuño D3, Aldámiz-Echevarría L4 1

Metabolic Disorders Unity, HUV Rocío, Sevilla, Spain; 2Metabolic Disorders Unity, SC Teaching H, Santiago de Compostela. Galicia, Spain; 3 Paediatric Nephrology and Metabolism Uni, Santander. Cantabria, Spain; 4 Div of Metab, D. Paediatrics Cruces H., Barakaldo. Vizcaya, Spain

S136 Background: Sapropterin could be a chaperone in patients with mild PKU. Methods: Observational study of patients with no protein restricted diet because of treatment with sapropterin, and progressive decrease of sapropterin up to total suppression. Selection of sensitive PKU patients to sapropterin with 24 hours BH4-loading test designed as a 24-h test based upon the 50 % criterion and consisted of a Phe load and the subsequent ingestion of the cofactor. Results: We observed 5 mild-PKU patients who have normal levels of Phe after 3 years of treatment with sapropterin, without restricted diet or sapropterin. Conclusion: Sapropterin could be a chaperone, stabilizing PAH. It would be necessary to conduct more studies with bigger samples to define genotypes associated with this function of sapropterin. Conflict of Interest declared. P-079

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 development, mainly in attention deficit, fine motor and cognitive skills. Pterines level analysis by Guthrie card was suggesting of PTPS deficiency, confirmed by molecular study. Currently he is in secondary school on treatment for ADHD and without BH4. Case 2: Fullterm newborn. PKU diagnosed by newborn screening. Phenylalanine levels 14.7 mg/dl (Ph/Tyr ratio 17,9). Early signs of neurological impairment were detected showing global developmental delay, extrapyramidal and pyramidal progressive deterioration, besides good metabolic control. Pterines deficiency (PTPS) was suspected and confirmed by Guthrie card and mutational analysis. After he started treatment (l-dopa), development and movement disorder improved. Conclusion: The road spectrum of clinical presentations makes it hard to suspected PTPS deficiency. In a child whit mild PKU and signs of neurological dysfunction pterines deficiency should be ruled out or treated early to improve their prognosis, even if access to BH4 is limited.

Point of care sensing of phenylketonuria P-081 Ayyub OB1, Cabrera-Luque J2, Natoli M1, Behrens AM1, Cunningham G2, Marugan J3, Simeonov A3, Summar M2, Kofinas P1 1

University of Maryland, College Park, United States; 2Children's National Medical Center, Washington, D.C., United States; 3National Institute of Health, Bethesda, United States

PKU embryofetopathy Chioukh FZ1, Ben Hamida H1, Ben Ameur K1, Bizid M1, Kaabachi N2, Monastiri K3 1

Background: Newborn infants with genetic metabolic disorders such as phenylketonuria may not express obvious symptoms. Current diagnostic methods require specialized and time intensive techniques such as tandem mass spectroscopy. An alternative method designed to have a fast response time, require little training and extended storage is needed Objectives: To develop an electrochemical method for determining phenylalanine levels, using the enzyme phenylalanine dehydrogenase which catalyzes a redox reaction that generates a detectable current. Materials and Methods: Initial electrochemical tests were performed using commercially available glutamate dehydrogenase using glutamate as substrate. Glutamate dehydrogenase was immobilized on a three carbon electrode system using an alginate hydrogel. The electrode was exposed to glutamate concentrations ranging from 0-2000micromoles in phosphate buffered saline(PBS) and plasma and the current generated was measured using a potentiostat. Discussion: The plasma and blood components that could interfere with the enzyme activity, such as uric acid, were excluded by the alginate hydrogel in which the enzyme was embedded allowing the electrode to detect a linear relationship between the glutamate concentration and the current generated in both matrices. Additionally, we have cloned and expressed a thermophilic form of phenylalanine dehydrogenase from Geobacillus thermoglucosidiasius for increasing the storage stability of the test strips.

NICU Teaching Hospital of monastir, Monastir, Tunisia; 2Departement of Biochemistry LaRabta, Tun, Tunis, Tunisia; 3Faculty of Medicine of Monastir, Monastir, Tunisia Background: Phenylketonuria related embryofoetopathy is frequent in poorly equilibrated affected mothers. Case 1: I born at term to non consanguinous parents. The mother had PKU and was not on diet since the age of 2 years. She has a history of two abortions. The pregnancy was not not followed correctly, PKU history was forgotten. In the second trimester the diagnosis of oligohydramnios was made in a fetus with growth retardation. Birth was by vaginal delivery, his Apgar scores was 9 at 5 min an 10 at 10 min. Birth weight 2100 g, Head circumference 30 cm and length 42 cm. He had no dysmorphy or cardiac murmur. Renal sonography revealed left kidney agenesis. by six years old, he had psychomotor growth retardation and marked microcephaly. Case 2: N, I's sister was born at term, her BW 2400 g, lenght 45 cm and HC 32 cm. She was followed for microcephaly and growth delay since the age of six months. By the age of 9 years she could not talk yet. Conclusions: PKU embryofoetopathy has severe consequences on the development of siblings. Prevention is possible through good surveillance of PKU mothers on diet before conception and during pregnancy. P-082

P-080 30 months follow up of a case of BH4 deficiency Clinical spectrum of PTPS in Chile, two case reports Arias C1, Bravo P1, Cabello JF1, Castro G1, Hamilton V1, Peredo P1, Valiente A1, Raimann E1, Cornejo V1

Lemes A1, Zabala C1, Queijo C1, Castro M1, Iraola I1, Fernández L1, Ferolla C1, Queiruga G1 1

Newborn Screening Laboratory, Montevideo, Uruguay

1

Inst Nut Food Tech, Univ of Chile, Santiago, Chile

6-Pyruvoyltetrahydropterin synthase (PTPS) deficiency is the most common cause of hyperphenyalalaninemia due to tetrahydrobiopterin (BH4) deficiency (1). We present two cases of PTPS deficiency with different clinical neurological impairment. Case 1: Fullterm newborn, PKU diagnosed by newborn screening. Phenylalanine level 14.7 mg/dl (Ph/Tyr ratio 9.8). Metabolic control within normal ranges. He presented a subtle decrease on neurological

Introduction: The deficiency of the cofactor tetrahydrobiopterin of the hepatic phenylalanine hydroxylase enzyme is cause of hyperphenylalaninemia in 1 to 3% of the cases. The cofactor is also active in hydroxylation of tyrosine and tryptophan, precursors of dopamine and serotonin. Clinic cofactor deficiency reflects the neurotransmitters deficit in the brain and may include intrauterine problems like growth restriction and microcephaly at birth.Confirmation requires study of DHPR and pterins in blood and CSF neurotransmitters. Treatment

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

S137

requires providing tetrahydrobiopterin, 5-hydroxytryptophan and Ldopa.The aim of this report is to present 30 months of follow up of a case of BH4 deficiency due to defect in the synthesis of tetrahydrobiopterin. Case report: Female, second pregnancy, no parental consanguinity, term birth, small for gestational age, microcephaly. During first months of life, we noticed poor visual fixation, feeding difficulties, salivation, sweating, and irritability. The diagnosis was confirmed at 3 months of age byanalysis of blood pterins, DHPR and CSF neurotransmitters. Treatment was started with neurotransmitter precursors and the cofactor. At present she has developmental delay, microcephaly, normal weight and length for age. Normal blood phenylalanine with unrestricted diet. No seizures. Conclusions: We highlight the importance of starting treatment as soon as possible to improve the prognosis. P-083

1

1

2

P-085 Evaluation of behavior, executive function, neurotransmitter function and genomic expression in PKU "nonresponders" to sapropterin Andersson HC1, Cunningham A1, Civelly K1, Chen TJ1

Adherence to treatment of children and adolescents with phenylketonuria 1

Results: Among all patients, 44 received breast milk. Of the subjects who were breastfed, 32 (72.7%) were classified as normal and 12 (27.3%), overweight or obese3. Among patients who were not breastfed 15 (75%) were classified as normal and 5 (25%), overweight or obese. Conclusion: It was observed that the prevalence of healthy individuals is greater, both in the group of those who were breastfed as that in which they were not. The number of cases of overweight / obesity was lower in both groups. One can not say that breastfeeding interferes with BMI.

1

Hayward Genet Cntr, Tulane Univ Med Sch, New Orleans, United States

1

Soares R , Kanufre V , Alves MRA , Magalhães L , Aguiar MJB , Starling ALP1, Norton R1

P-084

Background: Oral sapropterin was FDA-approved for PKU in 2007 and enhances residual metabolism of phenylalanine hydroxylase in some patients. Anecdotal reports exist of patients who have not experienced changes in plasma PHE but have been noted to, or have reported, improved attention and mental clarity. Behavior, metabolic effects and gene expression have not been studied in sapropterin nonresponder PKU patients. Methods: We studied 12 classical PKU patients (ages 8-39y) previously shown to be non-responders to Kuvan®. Patients completed behavioral inventories (BASC, BRIEF), submitted 24-hour urine for neurotransmitter analysis and had gene expression arrays performed at baseline and after 4 weeks of Kuvan® (20mg/kg). Results: On BASC, 4/9 patients had no improvement from "atrisk/clinically significantly abnormal"; 2/9 patients improved from "clinically significant" to "normal" range; 3/9 were in normal range before and after. On BRIEF, 8/12 patients were in "atrisk/clinically affected" range at baseline and 3 showed some improvement after treatment. Urine neurotransmitters showed no consistent pattern: 4 patients showed increased dopamine, 3 showed decreased dopamine. Many genes whose functions are poorly understood were observed to be up-/down-regulated in individual patients. Conclusions: No effect of Kuvan® was observed on behavior, urine transmitters or gene expression at the midpoint of this study. Conflict of Interest declared.

Assessment of nutritional status of patients with phenylketonuria breastfeeding and no breastfeeding

02. Urea cycle disorders

Soares R1, Kanufre V2, Alves MRA1, Magalhães L3, Neves M3, Aguiar MJB1, Starling ALP1, Norton R1

P-086

1

Center for Action and Research in Suppor, Belo Horizonte, Brazil; Clinics Hospital, Federal University, Belo Horizonte, Brazil

2

Introduction: Dietary treatment for phenylketonuria (PKU) is very effective in preventing clinical manifestations, and in particular, the irreversible mental retardation. However, the diet is very restrictive and monotonous, and compliance is difficult. Objective: To evaluate adherence to treatment of children and adolescents with PKU by means of phenylalanine (phe) blood. Methods: We evaluated the average phe blood of 79 patients, ages 2 < 6 years, > 6> 10 years and > 10 years. Were then classified into appropriate and inappropriate, according to age. Results: Most patients had inappropriate mean Phe levels, being 69.7% (53) within 2 < 6 years: 67.1% (53) in > 6 > 10 years, and 64% (48) in > 10. Conclusion: The literature describes the difficulties in compliance with the diet for PKU is, especially after the second year of life, associated with the autonomy acquired by a child, and progressive worsening in adolescence and adulthood. Adherence to treatment involves several factors. Among them, the family structure, and how phenylketonuric patients and their families deal with the disease, are of great importance and can define the success or failure of treatment.

1

Center for Action and Research in Suppor, Belo Horizonte, Brazil; 2 Center for Action and Research in Suppor, Belo Horizonte, Brazil; 3 Academic degrees in nutrition Federal Un, Belo Horizonte, Brazil Introduction: The treatment of phenylketonuria (PKU) is to limit the sources of dietary protein. Newborns start treatment combining breast milk with formula free of phenylalanine (phe). The positive effect of this treatment is described in several studies. Objective: To evaluate the nutritional status of patients with PKU with and without breastfeeding. Methodology: 64 patients 5-10 years of age, diagnosed early were evaluated. Classification of Body Mass Index (BMI) was performed using z - score, according to WHO.

Clinical, laboratory data, molecular features and outcome of nine patients, affected by citrullinemia type 1, Iranian experience Zaman T1, Moarefian S2, Nagel M3, Behnam B 4, Moradian R 2, Rahmanifar A2 1 1 IEM Department, Children HospitalMedic, Tehran, Iran, Islamic Republic of; 22 Research Unit,Iranian National Society, Tehran, Iran, Islamic Republic of; 3Molecular genetisches Lab Weisswasser, Weisswasser, Germany; 4 Akbarabadi,Medical Gen Lab,Tehran Univ, Tehran, Iran, Islamic Republic of

Background: Citrullinemia is an urea cycle disorder caused by argininosuccinate synthetase (ASS) deficiency. Initial symptoms frequently include neurological deterioration. It displays phenotypic

S138 variability. The most common mutation in early-onset /severe neonatal phenotype is p.G390R (c.1168G>A). Methods: A retrospective review of 9 cases( from 2008-2013). Sequence analysis of ASS1 gene confirmed diagnosis. They were started on standard & emergency treatment for hyperammonaemia. Results: 5 males, consanguinity; 5/9. 7/9 presented: poor feeding, lethargy at 0-3th day, citrulline, 1147-3871 μmol/L; ammonia, 3.4-9 times elevated (Severe neonatal). 4/7 died within a few days before treatment. 2/7 survived, developed, despite two attacks, one at birth and the second at 10 months; intractable vomiting , lethargy, ,kidney stones and developmental delay, ammonia; 6.5 times, citrulline;2717. She improved at 12 months. 2/9 were referred at ages: 2.5 year,15 months, case 1 for failing to gain weight ;(10 kg), (Citrulline;1298). Case 2 for delayed development and intractable convulsion following an attack at age of 8 months (citrulline;1147). Both responded well, regarding optimal growth & development. Sequence analysis; p.G390R (c.1168G>A) normally, but died in hyperammonemic attacks at 4 and 5 months despite aggressive therapy including hemofiltration . 1/ 7 survived with severe phenotype; 3/3 & p.R403A in late onset case 2. Conclusion: Citrullinemia type 1 has different phenotypic features.The most important prognostic factors are the mutation type, ammonia level and early treatment. P-087

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Case Summary: A previously healthy 6-year old girl presented with vomiting and drowsiness and found to have hyperammonemia (242 micromole/l) and orotic aciduria. Sequence analysis of OTC gene confirmed the diagnosis. Two weeks later, the patient presented with acute bilateral visual difficulties (visual acuity of 20/400 in right and 20/500 in left). Plasma ammonia was normal. MRI of brain showed post contrast cortical enhancement of posterior and medial occipital lobes. Her symptoms completely resolved within 72 hrs. Discussion: Acute reversible vision loss has never been described in OTC deficiency. Our patient's clinical presentation resembles posterior reversible encephalopathy syndrome (PRES). However, neuroimaging findings are distinct. PRES is thought to be associated with free radical damage. Oxidative stress is also considered to play an important role in pathogenesis of urea cycle disorders. Thus, we speculate that such mechanisms may have lead to symptoms and neuroimaging findings in our patient. Conclusion: OTC can manifest with acute cortical blindness even in the absence of hyperammonemia. OTC should be considered in differential diagnosis of acute neurological symptoms in children. P-089 Optimizing ammonia (NH3) control in Urea Cycle Disorder (UCD) patients: short and long-term implications

Walter JH1, Morris AA1, Jones SA1

Lee B1, Mokhtarani M2, Diaz GA3, Rhead W4, Lichter-Konecki U5, Feigenbaum A6, Berry SA7, Bartley J8, Longo N9, Berquist W10, Smith W11, Gallagher R12, Harding CO13, McCandless S14, Schulze A6, Nagamani S1, Le Mons C15, Dickinson K2, Coakley DF2, Moors TL2, Millikien D16, Marino M13, Scharschmidt BF2

1

1

Inter and intra familial variation in the clinical phenotype of arginase deficiency

Willink Biochemical Genetics Unit, Manchester, United Kingdom

Unlike other enzyme deficiencies of the urea cycle arginase deficiency is usually thought of as presenting with a progressive spastic diplegia rather than an acute hyperammonaemic encephalopathy. We report 8 patients from 3 consanguineous families, including one pair of twins, in which there was marked inter and intra familial variation in the clinical phenotype. All patients were diagnosed on the basis of raised blood arginine concentrations and confirmed by enzyme studies in erythrocytes. All patients are treated with nitrogen scavengers and a low ornithine diet. Cognitive outcome varies from normal development to severe learning difficulties. Some patients have developed a diplegia whereas others have brisk lower limb reflexes only. Significant hyperammonaemia (blood ammonia > 250μmol/l) has been evident in only 2 children at presentation. Most patients showed satisfactory wt and ht gain but poor head growth was evident in four. One child has a neuronal migration defect and one of the twins had diffuse white matter abnormalities at presentation. Two have developed epilepsy. There was no apparent correlation between blood arginine concentrations and outcome. The reason for the variation in phenotype is unclear. Of clinical importance, significant diplegia is not evident in a number of individuals. Conflict of Interest declared. P-088 Ornithine transcarbamylase deficiency presenting with acute reversible cortical blindness Prasun P1, Altinok D1, Misra V1 1

Children's Hospital of Michigan, Detroit, United States

Introduction: Ornithine transcarbamylase deficiency (OTC) is the most common urea cycle defect. We report a 6-year-old female with OTC presenting with acute reversible cortical blindness.

Baylor College of Medicine, Houston, TX, United States; 2Hyperion Therapeutics, Inc., South San Francisco, CA, United States; 3Mount Sinai School of Medicine, New York, NY, United States; 4Medical College of Wisconsin, Milwaukee, WI, United States; 5Children's National Medical Center, Washington, D.C., United States; 6The Hosp for Sick Child, Univ of Toronto, Toronto, ON, Canada; 7University of Minnesota, Minneapolis, MN, United States; 8Long Beach Memorial Hospital, Long Beach, CA, United States; 9The University of Utah, Salt Lake City, UT, United States; 10 Stanford University, Palo Alto, CA, United States; 11Maine Medical Center, Portland, ME, United States; 12Children's Hospital Colorado, Aurora, CO, United States; 13Oregon Health Sciences University, Portland, OR, United States; 14Case Western Reserve University, Cleveland, OH, United States; 15National Urea Cycle Disorders Foundation, Pasadena, CA, United States; 16Accudata Solutions, Inc., Lafayette, CA, United States Background: Ammonia (NH3) exhibits considerable fluctuation in UCD patients. Methods: Over 1000 NH3 values were analyzed from 80 UCD patients, including 26 children ≥2 months, who underwent 24hr blood sampling on sodium phenylbutyrate (NaPBA) or glycerol phenylbutyrate (GPB), and from 51 adult and 49 pediatric patients during 12-months of GPB dosing. Data on hyperammonemic crises (HACs) were collected for 12 months pre-enrollment (on NaPBA) and during GPB treatment. Fasting NH3 was analyzed as a predictor of daily NH3 exposure [AUC 0-24hr] as well as HACs during long term (median = 360.5 days; 93% >300 days) dosing. Results: During short term dosing, baseline fasting NH3 levels of 00.5xupper limit of normal (ULN), >0.5-1xULN and >1-1.5xULN were associated with a progressively decreasing probability of normal average daily NH3 (87%, 60%, 39%; p<0.001). Higher levels correlated significantly with decreased time to first HA crisis (p<0.01) during long-term dosing and, after controlling for age, gender and race, a baseline fasting NH3 >1.0 ULN vs. <1.0 ULN correlated with a ~15 x higher rate of HACs (p=0.0004). Conclusions: The findings suggest that UCD patients benefit from maintaining their fasting NH3 levels < 0.5 ULN. Conflict of Interest declared.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 P-090 Functional characterization and antisense therapy for the SPF/ASH splicing mutation in OTC deficiency in mice and man Sanchez-Alcudia R1, Rivera A1, Perez B1, Haberle J2, Thony B2, Ugarte M3, Desviat LR1 1

Centro de Biología Molecular, Madrid, Spain; 2University Children's Hospital, Zürich, Switzerland; 3CEDEM-UAM, MADRID, Spain

S139 and of glutamine was 1095 μmol/L (range: 189-4000). There were three deaths during the study period, all of them within the first days of life. The current age median is 9 years. Five patients were transplanted. 53 (51%) patients have impaired neurological outcome. Current ammonia level median is 33 μmol/L, and glutamine is 716 μmol/L. 91 patients are on low protein diet; 4 patients are on sodium benzoate treatment, 50 on sodium phenylbutyrate, 10 on combined treatment (sodium benzoate and sodium phenylbutyrate) and 1 on carglumic acid P-092

In this work we have analysed and compared in silico and in vitro the mutation identified in the spf/ash mouse model for OTC deficiency, also present in some OTC deficient patients. The mutation, located in the last nucleotide of exon 4, disrupts the 5' splice site and results in mice in the partial use of a cryptic splice site 48 bp into the adjacent intron. Different splice prediction programs were used to analyze the region revealing the presence of several cryptic splice sites albeit not the +48 site in the human sequence. Wild-type and mutant murine and human minigenes were constructed, transfected in hepatoma cells and the transcriptional profile analyzed. The mutation has different effects for mouse and human sequences, resulting in the latter mainly in the activation of a cryptic splice site at nucleotide +5. In the murine mutant minigene we observed several aberrant transcripts corresponding to the use of the +48 site, exon skipping and intron retention, as well as some normally spliced product. For the mutant minigenes we have used antisense oligonucleotides to block splicing at the murine cryptic site at +48, to confirm the efficacy of antisense treatment for specific splicing defects. P-091 Urea cycle disorders in Spain: a series of 104 patients Martín-Hernández E1, Castejón-Ponce E2, Pedrón-Giner C3, Couce Pico ML4, Serrano-Nieto J5, Sanjurjo Crespo P6, Pintos-Morell G7, VitoriaMiñana I8, Dalmau J8, Martínez-Pardo M9, Belanguer-Quintana A9, Lama-More R10, García-Silva MT1, Bueno Delgado M11, del ToroRiera M12, Quijada-Fraile P1, García-Jiménez MC13, Sierra-Córcoles C14, Ruiz-Pons M15, Peña-Quintana LJ16, Vives-Piñera I17, Moráis A10, Balmaseda-Serrano E18, Meavilla S2, Aldámiz-Echevarría L6 1

H.U. 12 de Octubre, Madrid, Spain; 2H. Sant Joan de Déu, Barcelona, Spain; 3H.U. Infantil del Niño Jesús, Madrid, Spain; 4C.H.U. de Santiago, Santiago de Compostela, Spain; 5H. Materno Infantil Carlos Haya, Málaga, Spain; 6H.U. de Cruces, Barakaldo, Spain; 7H.U. Germans Trias i Pujol, Badalona, Spain; 8H. Infantil La Fe, Valencia, Spain; 9H.U. Ramón y Cajal, Madrid, Spain; 10H.U. La Paz, Madrid, Spain; 11H.U. Virgen del Rocío, Sevilla, Spain; 12H. Vall d´Hebrón, Barcelona, Spain; 13H.U. Miguel Servet, Zaragoza, Spain; 14C.H. de Jaén, Jaén, Spain; 15Ntra. Sra. de la Candelaria, Santa Cruz de Tenerife, Spain; 16H.U. Materno Infantil de Las Palmas, Las Palmas de Gran Canaria, Spain; 17H.C.U. Virgen de la Arrixaca, Murcia, Spain; 18C.H.U. de Albacete, Albacete, Spain To expand the knowledge about the situation of patients with urea cycle disorders in Spain, clinical data of living patients were collected retrospectively in a non-interventional study. 104 cases have been reported: 49 males and 55 females (52.9%); 67 had ornithine transcarbamylase deficiency, 22 citrullinemia, 10 argininsuccinic aciduria, 2 carbamyl phosphate synthetase deficiency, 2 arginase deficiency and 1 N-acetylglutamate synthase deficiency. At diagnosis 30 (28.8%) patients presented with neonatal onset and 64 (61.5%) with late onset. The median of age at symptoms onset was 13 months (range: 1 day-45 years). 60.6% of the patients had neurological symptoms at diagnosis. The median of maximum level of ammonia was 243 μmol/L (range: 8-4584)

A multidisciplinary approach to the transition of adolescents with a urea cycle disorder from a pediatric to an adult health care centre Hewson S1, Mecija M1, Cordeiro D1, Nagy L1 1

Div Clin & Met Genet, SickKids, Toronto, Canada

We developed a protocol for transitioning adolescents with a urea cycle disorder (UCD) incorporating tools developed by the Good2Go program at our centre. UCD patients require dietary and medical therapy and are at risk for metabolic decompensation. A successful transition is important to ensure understanding of their condition, adherence to treatment and maintenance of health care engagement. A nurse, dietitian, and genetic counsellor team begin to engage the family in a discussion at age 15 about transition and eventual transfer. A knowledge questionnaire is completed by the adolescent and teaching to address gaps is provided. Teens complete transition readiness questionnaires and are engaged in topics relating to independent health care management, self advocacy, relationships, goals and reproductive risks. A wallet sized disease summary (MyHealth Passport) is prepared with the teen. Prior to the final visit, the patient is seen at the adult metabolic clinic and obtains an emergency letter. They are presented with a graduation certificate at their last pediatric appointment. A questionnaire provides feedback about the transition process. We have transitioned 5 UCD patients using this approach which has reduced family anxiety, improved engagement and understanding of the underlying UCD. This protocol can be applied to other metabolic conditions. P-093 Cognitive outcomes of ornithine transcarbamylase deficiency in children Crowe LM1, Anderson VA1, Wrennall J2, Boneh A1 1

Murdoch Childrens Research Institute, Melbourne, Australia; 2Royal Children's Hospital, Melbourne, Australia Background: Ornithine Trans-Carbamylase (OTC) deficiency is the most common disorder of the urea cycle. Research has been published on the cognitive outcomes of adults with these conditions, but detailed information on the neuropsychological outcome of children with this disorder is limited. Methods: A retrospective review of data of neuropsychological assessments over many years was conducted. Information was collected on the results of standardised, age-referenced measures of IQ, attention, memory and academic skills. Results: Generally, OTC deficiency was associated with lowered function in comparison to normative scores for IQ, attention, memory and academic skills. The cognitive outcomes varied by area. For example, Verbal IQ appeared to be more resilient to episodes of hyperammonaemia than Performance IQ and memory less affected than IQ. Other factors influencing outcome include illness severity and gender, which are contributing factors to age at diagnosis. Findings before and after liver transplantation demonstrate some improvements in cognition. The impact

S140 of the number of episodes of hyperammonaemia and of possible arginine deficiency was inconclusive. Conclusions: Taken together with previous findings on OTC deficiency, our results demonstrate that most neuro-cognitive damage occurs in the first years of life, suggesting that early liver transplantation may minimise cognitive impairment.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 This successful example of usage of "Pheburane" in one case of ASS is indicative of the advantages that a taste-free and odour-free formulation represent for the daily management of UCD patient. P-096

P-094

Evidence that ornithine and homocitrulline disturb redox homeostasis in cerebellum of young rats in vivo

Case report on a female patient transitioning from childhood to adolescence in KK Women's and Children's Hospital, Singapore

Zanatta A1, Viegas CM1, Busanello ENB1, Grings M1, Hickmann FH1, Monteiro WO1, Coelho DM2, Sitta A2, Leipnitz G1, Wajner M1

Lim SC1, Ong CBK1, Tan ES2

1

1

Dept of Nutrition & Dietetics, KK Women's and Children Hospital, Singapore; 2Dept of Paediatrics, Genetics Service, KK Women's and Children's Hospital, Singapore Background: Adolescence is a challenging time for patients and families whereby complacency and compliance issues arise. Method: We present a case report on a patient with Ornithine transcarbamylase deficiency (OTC) on follow-up from 18 months till 16 years of age. The objective of this case report is to track the outcome of our patient and highlight the needs of families of children with IEM, as ascertained from a self-administered questionnaire conducted at a support group session Results: Our patient has been managed on a low protein diet with sodium benzoate, arginine and multivitamin supplements. Except for major episodes of decompensation at 10 years of age (approaching puberty) and at 15 years of age (omission of chewable multivitamin supplement due to braces), patient has been well physically and intellectually. Eight families completed the questionnaire to ascertain their need for resources other than that provided during medical and dietetic consultations. Of the activities organised, families indicated that sharing of personal experiences, and talks conducted by the Rare Disorders Society of Singapore and the dietitian were the most useful. Conclusion: With on-going support and dietary adjustments, adolescents can transition from childhood to adulthood with minimal adverse effects as illustrated by our patient. P-095 A case report of the use of pheburane in one argininosuccinic aciduria patient Kalkan Ucar S1, Atik Altinok Y1, Köse M1, Canda E1, Kagnıcı M1, Coker M1 1

Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey

Argininosuccinic aciduria (ASS) is treated with dietary adjustment and nitrogen scavenging agents in order to avoid episodes of hyperammonemia. "Pheburane" is a new taste- and odour-free formulation of sodium phenylbutyrate, indicated in the treatment of urea cycle disorders (UCD) including ASS. A male patient presented at three months with symptoms of retarded development, loss of acquired skills, strabismus and vomiting. ASS was diagnosed based on elevated citrulline and argininosuccinate levels and treated accordingly. At the age of six the new formulation was started in replacement of the conventional sodium phenylbutyrate for a period of six months, at 250 – 500 mg/kg orally in 3 intakes/day. Plasma ammonia levels remained the same following the new formulation. Orotic acid, glutamine, arginine, essential amino acids, and potassium levels and liver and kidney function tests all remained unchanged. No hyperammonemia epısode occurred during treatment with Pheburane. While the initial doses needed some acceptance by the patient, successful posology adjustment was achieved at the end of the first week.

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil Cerebellar ataxia is commonly observed in hyperornithinemiahyperammonemia-homocitrullinuria (HHH) syndrome, an inherited metabolic disorder biochemically characterized by ornithine (Orn), homocitrulline (Hcit) and ammonia accumulation. Considering that the pathophysiology of the cerebellar symptoms in this disorder is unknown, the aim of the present work was to investigate the in vivo effects of Orn and Hcit on important parameters of redox homeostasis in cerebellum from 30-day-old rats. Animals received a single intracerebellar injection of Orn, Hcit or NaCl (control). Orn significantly increased thiobarbituric acid-reactive substances levels (lipid oxidative damage) and increased the activities of the antioxidant enzymes glutathione reductase and catalase. Furthermore, Hcit significantly decreased reduced glutathione levels and the activity of glutathione peroxidase (antioxidant defenses). Taken together, the present data show that Orn and Hcit disrupt cerebellum cellular redox homeostasis in vivo, a pathomechanism that may contribute to the pathophysiology of the cerebellar ataxia characteristic of the patients affected by HHH syndrome. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00, Instituto Nacional de Ciências e TecnologiaExcitotoxicidade e Neuroproteção (INCT-EN). P-097 Glycerol phenylbutyrate treatment in children with Urea Cycle Disorders (UCDS) Berry SA1, Lichter-Konecki U2, Diaz GA3, McCandless SE4, Rhead W 5 , Smith W 6 , Le Mons C 7 , Coakley DF 8 , Mokhtarani M 8 , Scharschmidt BF8, Lee B9 University of Minnesota, Minneapolis, MN, United States; 2Children's National Medical Center, Washington, D.C., United States; 3Mount Sinai School of Medicine, New York, NY, United States; 4Case Western Reserve University, Cleveland, OH, United States; 5Medical College of Wisconsin, Milwaukee, WI, United States; 6Maine Medical Center, Portland, ME, United States; 7National Urea Cycle Disorders Foundation, Pasadena, CA, United States; 8Hyperion Therapeutics, Inc., South San Francisco, CA, United States; 9 Baylor College of Medicine, Houston, TX, United States 1

Background: Glycerol phenylbutyrate (GPB) was recently approved for UCD patients ≥2 years. Methods: Data were pooled from 26 patients in 2 short-term, open label sodium phenylbutyrate (NaPBA) to GPB switchover protocols and from 49 patients in three 12-month GPB protocols. Results: NH3-AUC 0-24hr was lower on GPB vs. NaPBA (mean (SD) = 627 (302) vs. 878 (516) μmol/L x h; (p=0.001)). Glutamine (SD) averaged 710 (159) on NaPBA and 661 (164) (p=0.114) on GPB. During long-term GPB treatment, monthly NH3 averaged WNL (range = 17 to 26 μmol/L; normal <35 μmol/L) and 22% of patients (26% < 6 years) reported hyperammonemic crises (HACs) as compared with 39% (66% < 6 years) during the prior year on NaPBA. Mean glutamine ranged from 617-728

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 (μmol/L); BCAA were at the lower end of normal and unchanged vs. NaPBA. AEs (most commonly abdominal pain, rash, nausea, vomiting, diarrhea, decreased appetite, hyperammonemia, and headache) were generally mild to moderate in severity, transient and did not increase over time. Conclusions: GPB is well tolerated in pediatric UCD patients and effectively controls NH3 and glutamine. During long-term treatment, NH3 averaged well WNL and ~75% of patients experienced no HACs. Conflict of Interest declared. P-098 Intragenic deletion in ornithine transcarbamylase gene associated with nonhomologous recombination between an AluSx and MER68 repetitive sequneces Yoshino M1, Harada N2, Watanabe Y2, Soejima M3, Koda Y3, Okano Y4, Nakamura H5, Yorifuji T6

S141 Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic and regulative mechanisms. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K. The structural model of fulllength human NAGS has been derived based on the new structural information. New insights have been obtained into the arginine regulation mechanism and the potential impact of naturally occurring mutations in the human NAGS gene on enzyme function. P-100 A novel bisubstrate analog of N-acetyl-L-glutamate synthase Zhao G1, Allewell NM2, Tuchman M1, Shi D1

1

Cog & Mol Res Ins Brain Dis, Kurume Univ, Kurume, Japan; 2Dept Pediat, Kurume Univ, Kurume, Japan; 3Dept Forens Med & Hum Genet, Kurume Univ, Kurume, Japan; 4Dept Genet, Hyogo Med Col, Nishinomiya, Japan; 5Dept Ob & Gy, Osaka City Gen Hosp, Osaka, Japan; 6Dept Pediat, Osaka City Gen Hosp, Osaka, Japan Background: Large deletions constitute ~7% of mutations in ornithine transcarbamylase (OTC) gene. However, breakpoints and mechanism of such deletions have been barely studied. Objective: To clarify breakpoints of deletion and discuss its mechanism in a family with OTC deficiency. Case report: The subject was a 29 year-old woman with mild OTC deficiency. Methods: Analysis of nine SNP's, multiplex ligation-dependent probe amplification (MLPA) and long range PCR were performed according to respective methods. Results and discussion: Discordance in SNP in position 1 (SNP1) in the mutation-bearing allele between the patient (A) and mother (G) implied a deletion involving SNP1 (intron 1). MLPA analysis indicated that exons 2, 3 and 4 existed in single copy. The stepwise approach by long range PCR led to the detection of a 31.9 Kbp deletion. The breakpoints were located in an AluSx sequence in intron 1 and in an MER68 sequence in intron 4, respectively. This deletion appeared to be mediated by nonhomologous end rejoining between these two repetitive sequences. Conclusions: Analysis of SNP's can provide a clue to detection of a deletion. This is the first report of a deletion in OTC gene with breakpoints located in AluSx and MER68 repeats, respectively. P-099 Crystal structure of the n-acetyltransferase domain of human nacetyl-l-glutamate synthase in complex with n-acetyl-l-glutamate Shi D1, Zhao G1, Jin Z2, Allewell NM3, Tuchman M1 1

Children's National Medical Center, Washington, United States; Argonne National Laboratory, Argonne, United States; 3University of Maryland, College Park, United States

2

N-acetylglutamate synthase (NAGS) catalyzes the conversion of Lglutamate and AcCoA to N-acetyl-L-glutamate (NAG) an obligate cofactor for carbamyl phosphate synthetase I (CPSI), the rate limiting enzyme of the urea cycle. Since NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic, the structure of human NAGS is of clinical interest. However, the mammalian NAGS proteins has been proven challenging to crystallize due to their recalcitrant behavior in solution. We report herein the structure of the catalytic N-acetyltransferease (hNAT) domain of human NAGS with N-acetyl-L-glutamate bound at 2.1 Å resolution.

1

Children's National Medical Center, Washington, United States; University of Maryland, College Park, United States

2

N-Acetyl-L-glutamate synthase (NAGS) catalyzes the production of Nacetyl-L-glutamate (NAG) from AcCoA and L-glutamate. It is the first step of the arginine biosynthetic pathway in microorganisms whereas in mammals, NAG is an obligate cofactor of carbamoyl phosphate synthetase 1 (CPS1), the first and rate limiting step in the urea cycle. Deficiency of NAGS and consequentially, of NAG, causes hyperammonemia due to a secondary deficiency of CPS1 that results in a urea cycle block. We report herein the production of a novel bisubstrate analog of NAGS, CoA-S-acetyl-L-glutamate, which binds tightly to the active site of the enzyme and allowed to explore the catalytic mechanism. This specific analog was also used as an affinity ligand for purification of the recombinant NAGS from different species and for enrichment of native NAGS from mouse liver, allowing investigations of the post-translational processing of this critical enzyme of ureagenesis. P-101 The first continuous venous - venous hemofiltration with a Vietnamese newborn onset urea cycle disorder Nguyen KN1, Chi DV1, Can NBT1, Bui TP1, Yamaguchi S2 1 National Hospital of Pediatrics, Vietnam, Hanoi, Viet Nam; 2Shimane University School of Medicine, Shimane, Japan

UCD result in hyperammonemia leading to neurologic symptoms due to brain swelling. We report a case with newborn onset UCD who was treated by hemofiltration. Case report: A newborn boy was admitted with chief complaints of abnormal family history (2 older brothers died at 3 and 5 days of age with unknown coma). He presented irritability, poor feeding 8 days after birth. Coma appeared only few hours after the onset. The routine laboratory showed hyperammonemia (415 μg/dl, 1254 μg/dl). The results of Tandem mass and GC/MS showed normal blood amino acid profile and increased urine orotic and uracil acids. Immediately, he was treated with glucose infusion, stopped feeding for 24 hours, diet with special formula (WND1), arginine, continuous venous – venous hemofiltration. His cognition was better after hemofiltration: Glasgow score gained 8, 12 point after 2 days, 4 days, respectively. The blood ammonicalevel decreased gradually and was normalised after 2 days of hemofiltration. The hemofiltration was performed for 10 days. He was discharged after 28 days of treatment.

S142 Conclusions: This is first time, we have saved a patient with neonatal onset UCD from hyperammonemia by hemofiltration. It is an innovation in management of UCD as well as IEMs in Vietnam. P-102 Phenotypic variability in OTC deficiency: a large and paucisymptomatic family with late onset Rigoldi M1, Gasperini S1, Ravazzani V2, Alberti L2, Furlan F1, Pretese R1, Bertola F3, Citterio AM4, Boncimino A1, Parini R1 1

Metabolic Unit, S. Gerardo Hospital, Monza, Italy; 2Lab. Reg screening neonatale ICP Buzzi, Milano, Italy; 3Consorzio Genet .Molec. Um, Univ Bicocca, Monza, Italy; 4SC C.Ustioni, Chir.Plast. Niguarda Hosp, Milano, Italy Ornithine transcarbamylase deficiency(OTC), an X-linked urea cycle defect(UCD), affects males and females who may have neonatal(more frequent in males)or later onset. We diagnosed a family with 5 males(M)(15– 42years) and 7 females (F)(1.7–89y). The proband(M 42y),hospitalized for confusional state,died in hyperammonemic coma. A history of weight loss and chronic steroid therapy was reported in the previous months. Diagnosis was post-mortem: elevated urinary orotic acid(112.5mmol/molCreat.Ur., normal 0.14-2.35), gene mutation c.622G>A(p.Ala208Thr). His brother(41y) later resulted healthy, was immediately given instructions in case of symptoms. They saved a cousin(M 37y) who developed hyperammonemic coma few days later while hospitalized for a trauma and on steroid treatment. He is now on free diet and arginine treatment (10gr/day-125mg/kg). Five females had a total of 12 deliveries and, together with 3 males, underwent a total of 18 uneventful surgeries. Baseline ammonia, aminoacids, orotic acid was normal. Instructions for emergency were given. Conclusion: -even subjects with subclinical OTC defect are at risk of decompensation. -differential diagnosis of sudden neurological disorders associated to hyperammonemia includes UCD, at any age. -steroid treatment may be a decompensation trigger and metabolic monitoring is mandatory in any affected individual. -instructions for emergency must be given in the genetic counselling for UCD.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 phytohemagglutinin and cycloheximide. For control-RT-PCR, cDNAs derived from lymphocytes, liver and fibroblasts were used. Results: RT-PCR showed a predominant expression of a common exon 7-deleted ASL transcript in the patient. In contrast, only a small proportion of this transcript variant was expressed in controls. Conclusions: Predominant expression of a common splice variant, assumed to result in the unstable exon 7-deleted ASL transcript variant, may explain the ASA in this particular patient and may, in addition, contribute to the phenotypic variability of ASL deficiency. P-104 Short-term trial of N-carbamylglutamate in patients with partial CPS1 and OTC deficiency Ah Mew N1, Daikhin E2, Lichter U1, Nissim I2, McCandless S3, Konczal L3, Kerr D3, Yudkoff M2, Tuchman M1 1

Children's National Medical Center, Washington, DC, United States; Children's Hospital of Philadelphia, Philadelphia, PA, United States; 3 Rainbow Babies and Children's Hospital, Cleveland, OH, United States 2

Background: The first step of the urea cycle, the carbamyl phosphate synthase I (CPS1) reaction, requires an essential allosteric activator, Nacetylglutamate (NAG). Insufficiency of NAG causes decreased flux through CPS1, resulting in hyperammonemia. N-Carbamylglutamate (NCG), a stable NAG analog, can effectively treat hyperammonemia in NAG synthase deficiency. We hypothesized that individuals with partial CPS1 or Ornithine Transcarbamylase (OTC) deficiency might also benefit from NCG therapy. Methods: Identical studies were performed before and immediately after a 3-day trial of oral NCG in 7 patients with partial CPSD and 2 female patients with OTCD. An enteral bolus of [1-13C]sodium acetate, which is converted in-vivo to [13C]urea, was administered at the start of each study. Sequential blood samples were obtained to measure [13C] urea, ammonia, urea, and amino acids. Results: Four patients with CPSD and 1 with OTCD demonstrated an increase in peak [13C]urea, including 2 CPSD patients in whom there was a concurrent >50 micromolar decrease in mean ammonia. NCG was later clinically prescribed for one of these patients. On chronic NCG, ammonia remained stable at < 50 micromolar while ammonia scavengers were discontinued. Conclusions: NCG may augment ureagenesis and decreases plasma ammonia in some patients with partial CPSD and OTCD. P-105

P-103

Mutations in ASS1 gene in absence of hyperammonemia: case report

Predominant expression of exon 7-deleted argininosuccinate lyase in a patient with argininosuccinic aciduria

Djordjevic M1, Kecman B1, Sarajlija A1, Grkovic S1, Djuric M1, Klaassen K2, Desviat LR3, Ugarte M3, Pavlovic S2, Stojiljkovic M2, Perez B3

Hu L1, Wettstein V1, Pandey A2, Nuoffer JM3, Möslinger D4, Häberle J1

1

Mother and Child Health Institute, Belgrade, Serbia and Montenegro; IMGGE, University of Belgrade, Belgrade, Serbia and Montenegro; 3 CEDEM, CBMSO (CSIC-UAM), CIBERER, IDIPAZ, Madrid, Spain 2

1

Div Metab, Univ Child Hosp, Zurich, Switzerland; 2Ped Endocrin, Dept Clin Res, Univ, Bern, Switzerland; 3Univ Instit Clin Chem, Univ, Bern, Switzerland; 4Dept Ped Adolesc Med, Med Univ, Vienna, Austria Background: Argininosuccinic aciduria (ASA) is a rare autosomal recessive urea cycle disorder caused by deficiency of argininosuccinate lyase (ASL) with variable biochemical and clinical phenotype. In most patients, mutations on both alleles of the ASL gene can be found. Alternative exon skipping, mainly of exons 2 or 7, occurs frequently at the ASL gene. Methods: In a 18-year-old male patient, previously identified at age 13 with mild hyperammonemia (250 μmol/L) during an episode of gastroenteritis, DNA sequencing of the ASL gene had revealed only the heterozygous mutation c.566A>G. RT-PCR was performed using RNA derived from a 3-days full blood culture treated with

Background: Citrullinemia type I results from deficiency of argininosuccinate synthase (ASS), enzyme responsible for third step in urea cycle. Main biochemical characteristics of the disease are hyperammonemia, elevated plasma citrulline and low plasma arginine concentration. Results: The 11 months old boy of Tunisian-Serbian background was admitted to our Institute for generalized tonic-clonic seizures. Previous development was normal, with uneventful perinatal period. During hospitalization he had epileptic status. Ammonia level was normal (39-41 mcmol/L) with normal protein intake, but aminoacid analysis showed high concentration of plasma citrulline (305,8 mcmol/L). Plasma arginine was lower than normal while urinary orotic acid was mildly elevated. Treament with clobazam was instituted. During follow-up no further seizures were

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

S143

observed, ammonia level persisted normal (29 -42 mcmol/L), and plasma citrulline was consistently elevated (335-483 mcmol/L). Molecular testing for citrullinemia type 1 verified that patient is combined heterozygous for c.805G>A and c.1168G>A mutations in ASS1 gene (both previously described). At the age of 2,5 years, patient has mild cognitive delay without other clinical manifestations. Management includes l-arginine, anticonvulsants and protein dietary restriction. Conclusion: Citrullinemia is commonly characterized with significant hyperammonemia leading to clinical manifestations. Relations between genotype, biochemical phenotype and clinical manifestations in our patient remain unclear.

T1 and T2 MRI images may be normal. Multimodal neuroimaging using Fluid attenuated inversion recovery (FLAIR), Diffusion tensor imaging (DTI) and proton magnetic resonance spectroscopy (1HMRS) has potential impact on understanding altered cognitive function by interrogating neural networks, connectivity and biochemistry in a way where an understanding of individual patients and specialized treatment can be realized. We present imaging findings of a 59 year old patient with late-onset OTCD presenting with unilateral tremor initially diagnosed as Parkinson's disease (PD). However, FLAIR imaging, DTI and MRS showed findings typical of a UCD.

P-106

A key role for amniotic amino acid analysis in the prenatal diagnosis of citrullinemia type I

Neonatal carbamoyl phosphate synthetase 1 deficiency: a case of Slavic origin with novel mutations

P-108

Miller MJ1, Sutton VR1, Sun Q1, Elsea SH1

Bzduch V1, Behulova D2, Brennerova K1, Haberle J3

1

1 First Dept Pediat, Univ Child Hosp, Bratislava, Slovakia; 2Dept Labor Med, Univ Child Hosp, Bratislava, Slovakia; 3Univ Child Hosp Zurich, Zurich, Switzerland

The urea cycle disorder citrullinemia type I results from argininosuccinate synthetase 1 (ASS1) deficiency and can be diagnosed prenatally using fetal enzymatic and/or molecular testing. Additional analysis of amino acid levels in amniotic fluid (AF-AA) has been suggested to aid in the prenatal diagnosis of multiple urea cycle disorders but in the case of citrullinemia the published experience is limited (PMID# 6739433). To assess the clinical utility of AFAA testing we reviewed the 30 prenatal citrullinemia cases diagnosed with both enzyme and AF-AA assays in our laboratory over the last decade, 16 of which had further molecular testing and all involved mothers with prior affected offspring. Overall, AF-citrulline values strongly correlated with argininosuccinate synthetase enzyme activity in amniocytes. Consistent with this, AF-citrulline levels for affected fetuses (n=11) were significantly elevated above unaffected/carrier fetuses (n=34). Setting a cutoff of 50 μM AFcitrulline afforded a positive predictive value of 100% with 91% sensitivity. In two instances AF-citrulline confirmed a diagnosis in the face of ambiguous molecular and/or biochemical test results. Taken together our data demonstrate that amniotic citrulline levels serve as an important adjuvant in the interpretation of enzyme and DNA data in the prenatal diagnosis of citrullinemia.

Background: Neonatal form of carbamoyl phosphate synthetase 1 (CPS1)deficiency is a rare autosomal recessive disorder often leading to lethal hyperammonia. Case report: The girl,born to unrelated Slovak parents,presented hypotonia and respiratory distress at birth.During another two days respiratory alkalosis, vomiting, feeding problems and progression of neurological symptoms (lethargy,irritability) appeared. On the 5 day biochemical investigation revealed hyperammonemia (471,4 μmol/L) with low citrulline levels and undetectable orotic acid in the urine. This findings suggested a urea cycle disorder and we started prompt treatment with lowprotein high-caloric diet,sodium benzoate, sodium phenylbutyrate, arginine, L-carnitine, essential aminoacids and MCT oil.This leaded to clinical stabilization and normalization of ammonia levels.Molecular genetic investigation confirmed CPS1 deficiency by identification of two novel mutations of CPS1 gene:c.2611A>C(p.Thr871Pro)in exon 21 and c.3582A>C(p.Glu1194Asp)in exon 30.During next two years repeated hospitalizations were needed because of feeding problems and mild hyperammonemia. Percutaneous endoscopics gastrostomy was established at the age of 1 year and L-citrulline was added to the treatment. Conclusion: We consider the clinical course of our patient favourable. At the age of 4 years he is without psychomotor impairment. P-107 Flair imaging in urea cycle disorders defines structural etiology for unilateral tremor Sprouse C1, King J1, Ah Mew N1, Vezina LG1, Pacheco-Colon I2, Gropman AL1 1

Children's National Medical Center, Washington, DC, United States; 2Center for Mol Imaging, Georgetown Univ, Washington, DC, United States Urea-cycle disorders (UCDs) are a group of congenital enzyme deficiencies predisposing to hyperammonemia (HA), especially the proximal defects such as Ornithine Transcarbamylase Deficiency (OTCD). HA causes changes in the central nervous system (CNS) including alterations of neurotransmitter function, cell volume, and energy deprivation ultimately leading to cerebral edema, coma and possibly death. Neuropathological findings of UCDs primarily reflect changes in astrocyte morphology. Neurological features accompanying acute HA include changes in behavior and consciousness in the short term, and potential for impairments in memory and executive function as long-term effects. Plasma measures of ammonia and glutamine, although useful for clinical monitoring, prove poor markers of CNS function. Routine

Dept of Mol and Hum Gen, BCM, Houston, United States

P-109 Human recombinant carbamoyl phosphate synthetase 1 (CPS1) as a key tool for testing the impact of the mutations in CPS1 deficiency Diez-Fernández C1, Martinez AI2, Pekkala S2, Barcelona B1, PerezArellano I2, Guadalajara AM2, Summar M3, Cervera J4, Rubio V1 1

Inst. Biomedicina de Valencia CSIC, Valencia, Spain; 2Cent. Investigación Príncipe Felipe, Valencia, Spain; 3Childrens National Medical Center, Washington DC, Spain; 4Group 739 CIBERER-ISCIII, Valencia, Spain Background: The urea cycle disease carbamoyl-phosphate synthetase deficiency (CPS1D) associates with many CPS1 gene missense mutations for which the disease-causing potential remains largely unclarified, requring investigation. Objective: To develop a system for testing the effects of CPS1D missense mutations. Patient mutations, material and methods. His6-tagged recombinant human CPS1 (wild-type or carrying one of two polymorphisms or of eight neonatal CPS1D-reported mutations) was expressed in baculovirus/insect cells, affinity purified, and assayed for enzyme activity, kinetics and stability, molecular mass (gel filtration) and domain composition (limited proteolysis/SDS-PAGE). Results: Highly pure, active, mature CPS1 (1462-residue protein) was produced in large amount. It closely resembled natural CPS1 in kinetic and molecular properties, domain composition, and ability of glycerol to replace the CPS1 essential activator N-acetyl-L-glutamate (NAG). Five

S144 mutations decreased enzyme stability, two hampered catalysis and one impaired NAG activation. Both polymorphisms were without effects. NAG and its orphan drug analogue N-carbamoyl-L-glutamate protected CPS1 against proteolytic and thermal inactivation (in the presence of MgATP). Conclusions: Baculovirus/insect cell expression is a powerful tool for analyzing CPS1 mutation effects. Glycerol analogue CPS1 activation and carbamylglutamate chemical chaperoning herald potential avenues of CPS1D treatment. Grants. Fundación Alicia Koplowitz 2011, Prometeo 2009/051 (Valencian Government) and BFU2011-30407 (Spanish Government). P-110 Health-related quality of life in individuals with urea cycle disorders: a report from the urea cycle disorders consortium Gallant NM1, Simpson K2, Dorrani N1, McCarter R2, Cederbaum SD1, Hays RD3, Lichter-Konecki UL2 1 Department of Pediatrics, UCLA, Los Angeles, United States; 2Cent Genet Med, CNMC, Washington, DC, United States; 3Department of Medicine, UCLA, Los Angeles, United States

Background: Scant information is published on the impact of urea cycle disorders (UCD) on health-related quality of life (HRQOL). The aim of this study is to examine HRQOL in children and adults with UCD. Methods: Estimates of HRQOL are based on data collected in the Longitudinal Study of the Urea Cycle Disorders Consortium. Children ages 5-17 without cognitive impairment and parents of all children ages 2-17 completed the PedsQL 4.0 survey. Adults ages 18 and older completed the SF-36v2 survey. Results: 271 parents, 170 children and 217 adults with UCD participated. Parent-reported physical and psychosocial health scores were worse in children with neonatal-onset disease than late-onset disease. Both groups reported worse scores than healthy children. Children with neonatal-onset disease and their parents reported psychosocial health scores similar to children with cancer on chemotherapy. Adults with UCD, the majority of whom were asymptomatic, reported SF-36v2 physical and mental component summary scores similar to the general population. Conclusions: Children with UCD have impaired physical and psychosocial health. Children with neonatal-onset UCD have psychosocial health similar to children with cancer on chemotherapy. Further studies are needed to identify mechanisms that might ameliorate the negative impacts of UCD on HRQOL. P-111 Ornithine Transcarbamylase (OTC) deficiency: characterization of a portuguese population followed in an adult metabolic disease outpatient clinic Cardoso MT1, Castro Chaves P1, Rodrigues E2, Vasconcelos C2, Vilarinho L3, Azevedo L4, Leão Teles E2 1 Adults Metabolic Disease Unit, CH S Joao, Porto, Portugal; 2Metab Dis Unit, Paed Depart, CH S Joao, Porto, Portugal; 3Genetics Department – INSA, Porto, Portugal; 4IPATIMUP, Porto, Portugal

Ornithine transcarbamylase (OTC) deficiency is the most common urea cycle disorder. We describe 6 families (15 adult patients) identified from 5 index cases in infancy and 1 in adulthood. A family with seven patients was diagnosed after an infant index case of hyperammonaemic encephalopathy (HE) (IVS2+1G>T). Two mildly symptomatic hemizygous males with episodic headaches related to protein ingestion were diagnosed at 59 and 39 years-old. Two females

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 are asymptomatic carriers. Two heterozygous females with recurrent headaches and encephalopathy related to protein ingestion were diagnosed after the seventh decade. Another family with three heterozygous females (Asp196Tyr) was identified through an index female case diagnosed in late adulthood after recurrent episodes of nausea and HE. Two heterozygous females (Lys210Thr and Gly195Arg) diagnosed in infancy after HE and seizures are presently in their twenties being mildly symptomatic under diet. Finally two female heterozygous (Met1lle and Ile159Thr) with long history of proteinrelated headaches were identified through index cases in severely symptomatic newborn male offspring. In conclusion, our adult population has high phenotypic variability, high percentage of symptomatic female carriers and two mildly symptomatic hemizygous males. A definite diagnosis even in older patients allowed a specific management and offered the possibility of genetic counselling P-112 Acute neurological deterioration associated with moderate hyperammonemia and high intracerebral glutamine in urea cycle defect patients. Insights in pathophysiology and role of neuroprotective strategies Abi Warde MT1, Bonnemains C2, Kuster A3, Labarthe F4, Fallet C5, Lesage F6, Ottolenghi C7, Habarou F7, Feillet F2, de Lonlay P1, Valayannopoulos V1 1

Div Metab Dis, Necker Univ Hosp, Paris, France; 2Div Metab Dis, Nancy Univ Hosp, Nancy, France; 3Div Metab Dis, Nantes Univ Hosp, Nantes, France; 4Div Metab Dis, Tours Univ Hosp, Tours, France; 5Div Anat Path, Ste Anne Hosp, Paris, France; 6Div Ped Rea, Necker Univ Hosp, Paris, France; 7Div Metab Biochem, Necker Univ Hosp, Paris, France Acute neurological manifestations associated with hyperammonemia are usual complications in urea cycle defects (UCD), and a major cause of morbidity and mortality. In the current model, hyperammonemia triggers glutamine production in the brain causing edema. We present here five acute neurological episodes characterized by discrepancy between neurological symptoms and relatively low levels of ammonia. Patients: Four boys and one girl, born between 1996 and 2011, affected with various UCD. The age of the episodes varied between day 1 and 17 years. Results: All patients presented with severe neurological deterioration and coma; 4/5 displayed cerebral hypertension and brain edema. Mean NH3 at onset was 153 μmol/L (60-246). Plasma glutamine varied (7004000 μmol/L); CSF glutamine was found in all cases several-fold higher than plasma levels. All patients received treatment with ammonia scavengers and parenteral dietary treatment. Two main second-line treatments were used: hemodialysis (2/5) or neuroprotective strategies (2/5). Two patients were deceased from cerebral edema (one despite dialysis). All other patients recovered with no sequelae. Conclusion: these observations highlight the importance of glutamine toxicity in the brain that may also occur with relatively low ammonia levels and introduces the added value of neuroprotection in the acute management of patients with UCD. P-113 Understanding CPS1 deficiency (CPS1D): use of the recombinant enzyme to explore the effects of mutations affecting enzyme regions of poorly understood function Diez-Fernandez C1, Hu L2, Häberle J2, Rubio V1, Cervera J3 1

Inst. Biomedicina de Valencia-CSIC, Valencia, Spain; 2Kinderspital Zurich, Zurich, Switzerland; 3Group 739, CIBERER, ISCIII, Valencia, Spain

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

S145

Background: Many mutations reported in CPS1D fall on a CPS1 domain of unknown function (UFSD) while other mutations affect the C-terminal non-catalytic domain ("ASD"). Objective: To ascertain the impact of the CPS1D mutations mapping in UFSD and ASD domains, using recombinantly expressed CPS1. Patients, materials and methods. Thirty CPS1D mutations were tested using CPS1 site-directed mutagenesis, affinity-isolation, and kinetic and thermostability characterization. Results: Many of 18 UFSD mutations affected enzyme production, suggesting impaired protein folding. Surprisingly, although the catalytic and acetylglutamate-activating machinery fall outside this domain, CPS1 activity was importantly decreased by many of these mutations, with Km values for ATP and acetylglutamate being increased in some cases. Thermal stability was impaired in other cases. The 12 ASD domain mutations had particular impact on enzyme activity, mainly by interference with acetylglutamate activation, with increased Km values for ATP found sometimes. Conclusions: A disease-causing role of the studied mutations is supported, highlighting the value of recombinant CPS1 as a tool for CPS1D studies. A key conformational stability-giving role for the UFSD domain is suggested, and the ASD domain involvement in CPS1 activation is confirmed. Grants. Fundación Alicia Koplowitz2011, Prometeo 2009/051 (Valencian Government) and BFU2011-30407 (Spanish Government).

Background: Arginase 1 (Arg1) deficiency, a rare autosomal recessive disorder leads to hyperargininemia with progressive neurological impairment, growth retardation and infrequent episodes of hyperammonemia. We aim to construct a new mouse model of Arg1 deficiency to address the development of potential gene therapeutic protocols. Methods: Using a tamoxifen-inducible Cre/loxP conditional gene modification system, we induced Arg1-deficiency in mice at four different timepoints between the neonatal stage and 12 weeks of age. Results: At approximately two weeks after tamoxifen administration, regardless of the starting age of knockout induction, mice die. The knockout mice were nearly devoid of Arg1 activity, showing signs of elevated ammonia, hyperargininemia and increased plasma guanidinoacetate. Attempts to mitigate the biochemical consequences of Arg1 deficiency by supplementation of ornithine in the drinking water were unsuccessful. An Arg1-green fluorescent protein (GFP) construct that retains full catalytic activity is being introduced into hepatocytes and induced pluripotent stem cells obtained from knockout mice in vitro, as well as to animals in vivo to correct the biochemical and phenotypic abnormalities. Conclusions: Our model can be used to expedite further study of the hyperargininemia phenotype via modulation of tamoxifen dosing leading to insights for gene correction strategies for this disorder.

P-114

Low-dose vs high-dose arginine supplementation in argininosuccininc aciduria with liver disease: a case report after 6 years therapy

Elevated uridine as a biomarker for mild ornithine transcarbamylase deficiency

P-116

Ioannou HP1, Augoustides-Savvopoulou P1

Christensen M1, Lund AM1, Wibrand F1

1

1

Background: Increased incidence of liver disease is a unique feature of argininosuccinate lyase deficiency (ASLD), even in patients with good compliance. Current guidelines recommend the combination of lowdose L-arginine with nitrogen scavengers instead of the standard treatment with high-dose arginine. Case Report: We describe a boy with late-onset ASLD who presented at the age of 6m with hyperammonemia (peak 700 μmol/L) and mildly increased aminotransferases. Presence of argininosuccinic acid in plasma and urine was pathognomonic for ASL. Long-term treatment initially included protein restriction and L-arginine supplementation (~400mg/kg/d). Despite good compliance with satisfactory growth and normal neurocognitive development, the patient manifested mild hepatomegaly with elevated aminotransferases (3 to 5-fold upper normal) and triglycerides. Liver US was otherwise normal. At the age of 2y because of reports of possible argininosuccinate toxicity on hepatocytes, L-arginine was reduced to 120 mg/kg/d and sodium benzoate was initiated (200mg/kg/d). To date, aminotransferase levels in this patient, now 8y old, generally are ≤3-fold the upper normal limits. Conclusion: Our experience is in accordance with recent recommendations that combined low-dose arginine and sodium benzoate is preferable to monotherapy with high-dose arginine as regards hepatic involvement in ASLD.

Dept Clin Genet, Rigshospitalet, Copenhagen, Denmark

Ornithine transcarbamylase deficiency (OTCD, MIM 311250) is the most common inborn error of the urea cycle disorders (UCD). OTCD is inherited as an X-linked trait and male patients often present in the neonatal period with severe hyperammonemia, increased glutamine and orotic acid and low citrulline levels in plasma and urine. Severe OTCD is often distinguished from other UCDs due to accumulated carbamoylphosphate being metabolised through the pyrimidine biosynthetic pathway, yielding orotic acid and uridine monophosphate and eventually uridine and uracil. However, mild OTCD hemizygotes and female OTCD carriers may present suddenly with lethal hyperammonemia, highlighting the need for biomarkers before a metabolic crisis. In our laboratory, we have analysed pyrimidine and amino acid levels in plasma obtained from eight OTCD carriers and five male OTCD patients. By correlating plasma conc. of glutamine and uridine, most OTC carriers could be distinguished from the reference group. The concentration of uridine was higher in more severely affected OTCD patients, emphasized by higher plasma-uridine in severely affected OTCD carriers than in currently asymptomatic (mild) OTCD male patients. Furthermore, elevated uridine in plasma showed to be a useful marker for mild OTCD or OTCD carriers in cases with normal levels of glutamine and citrulline.

Dept Metab Dis, Hippokration Hosp, Thessaloniki, Greece

P-117

P-115

A zebrafish model of hyperammonemia

Hyperargininemia in an inducible arginase-1 deficient mouse model

Feldman B1, Tuchman M2, Caldovic L2

Sin YY1, Ballantyne LL1, Mukherjee K1, St. Amand T1, McCracken C1, Kyrikopoulou L2, Schulze A2, Funk CD1

1 NICHD, Bethesda MD, United States; 2Children's National Medical Center, Washington DC, United States

1

Hyperammonemia caused either by inborn errors of metabolism, liver failure or extreme catabolic stress can be life threatening and can cause

Dept Biomed & Mol Sci, Queen's Univ, Kingston, Canada; 2Div Clin Metab Genet, Hosp Sick Children, Toronto, Canada

S146

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

severe brain damage. Available therapies are targeted at reducing blood ammonia levels and although they can prevent death, they are inefficient insufficient at preventing brain damage. Our aim is to discover novel drugs that protect the brain from elevated levels of blood ammonia. We developed a zebrafish model of hyperammonemia by immersing 4 days old (dpf) fish in water containing ammonium acetate (AmAc). We then tested whether methionine sulfoximine (MSO) and/or MK-801, known to protect the mammalian brain from hyperammonemia prolong the survival of 4 dpf zebrafish exposed to lethal dose of AmAc. Treatment with either MSO or MK-801 prolonged the lives of 4 dpf fish exposed to a lethal dose of AmAc. Treatment with both drugs was more effective than either drug alone. These results demonstrate that zebrafish can be used in a high throughput screen to select ammonia-neuroprotective agents. If successful, drugs that result from this screen would complement current treatment approaches to improve the outcome of patients with hyperammonemia.

deranged with PT 70.3 sec; APTT 49.3 sec; ammonia 88μmol/L. Factors II, VII and IX were low. She received Vitamin K and FFP. Coagulopathy resolved with the addition of sodium benzoate and optimising her doses of phenylbutyrate, and L-arginine. Defects of coagulation should be considered in all UCD patients with deranged liver function. This should lead to extended clotting factor screening. The pattern of factor deficiencies can be atypical as in patient 1. Optimisation of metabolic control was necessary to resolve coagulopathy rather than the use of vitamin K and FFP alone.

P-118

1

Chronology of behavioral change during hyperammonemia

Background: Valproate acid (VPA) is a commonly used broadspectrum antiepileptic that is often avoided when there is a suspicion of a metabolic disease, given the risk of metabolic acidosis and hyperammonaemia. Case report: An 8-year-old male with no relevant family or personal history presented with an electro-clinical status epilepticus (SE). All investigation including brain MRI, serum and CSF studies and metabolic screening were normal. Over the next 3 years he had several hospitalizations with difficult seizure control. In the context of a refractory epilepsy, VPA (1000mg/day) was started but 3 months later the child was admitted with SE. Biochemical studies reveled rapidly progressive hyperammoniemia (378 mmol/L). VPA was stopped and carglumic acid (CA) was started (dose calculated according to body surface: loading dose of 1,5 g/m2 and maintenance dose of 3 g/m2/day) with a fast decrease of ammonia levels in less than 12 hours, reaching a normal range in 24 hours. Conclusion: There are few case reports about CA therapy in VPA induced hyperammonemia described in the literature, and the data is even more limited concerning the experience in adolescents/adults, in particular with regard to the prescribed dose. In this case the authors focus on their experience in an overweight patient with a body surface area overlapping with adults.

Senkevitch E1, Pumbo E2, Miller R2, Nagaraju K2, Morizono H2, Tuchman M2, Caldovic L2 1

National Cancer Institute, Frederick MD, United States; 2Children's National Medical Center, Washington DC, United States Hyperammonemia is the main consequence of urea cycle defects and liver failure. Exposure of the brain to elevated ammonia levels leads to a wide range of neuro-cognitive deficits, coma and death. We have created an N-acetylglutamate deficient (NAGSko) mouse model of inducible hyperammonemia. The NAGSko mice survive and reproduce when treated with N-carbamylglutamate (NCG) and L-citrulline (Cit). When NCG and Cit are withdrawn, the mice die within 24 hours of hyperammonemia. The aim of this study was to establish behavioral markers of hyperammonemia progression in the NAGSko mice and use them to screen drugs that could protect the brain from hyperammonemia. A home cage behavior monitoring system was used to determine the chronology of behavioral changes in NAGSko mice following the withdrawal of rescue chemicals. Seven behaviors: eating, drinking, hanging, walking, grooming, remaining low/sleeping, and rearing up, were monitored for 24 hrs before and after withdrawal of NCG and Cit. Cessation of eating, drinking and hanging occurred within 12 to 14 hrs after NCG and Cit withdrawal and 4-6 hrs thereafter NAGSko mice stopped walking, rearing up and grooming. Therefore, the onset of behavioral changes due to hyperammonemia can be precisely quantified and used for testing interventions that treat hyperammonemia.

P-120 Carglumic acid in hyperammoniemia induced by valproate França S1, Rodrigues E1, Pimenta J1, Sousa R1, Leão Teles E1 Metab Dis Unit Hospital S.João Hospital, Porto, Portugal

P-121 Urea cycle disorders: clinical, biochemical and genetic findings in argentinean patients Laróvere LE1, Silvera Ruiz SM1, Arranz JA2, Angaroni CJ1, Guelbert NB1, Antonozzi SL1, Bezard MB1, Dodelson de Kremer R1

P-119 1

CEMECO, Hosp Niños Córdoba, FCM, UNCor, Córdoba, Argentina; Unitat Metab, Hosp Vall d´Hebron, Barcelona, Spain

Coagulation factor deficiencies in urea cycle defects

2

Stepien KM1, Lee CY1, Jameson E1, Will A2, Wu HY1, Ramaswami U1

Urea cycle disorders (UCD) encompass several enzyme deficiencies with a wide clinical spectrum from asymptomatic to severe, mostly with cerebral damage. Objective: to communicate the autochthonous experience in the recognition of UCD. The diagnosis protocol included phenotype compatibility, metabolites analysis by HPLC, and genetic analysis by PCR, restriction assays, sequencing and MLPA. We recognised: i) Ornithine transcarbamylase deficiency, 11 patients: 2 males with neonatal onset (OTC mutations: delExon2-10, c.533C>T), 4 males with late onset (c.216+1G>A, c.386G>A, c.622G>A, c.829C>T), 5 females (delExon2-10, c.533C>T, c.452T>G, c.540+1G>A); ii) Argininosuccinate synthetase deficiency, Citrullinemia type I (CTLN1), 16 patients from 10 unrelated families from San Luis Province, all showed the same ASS1 mutation: c.1168G>A/c.1168G>A and died during neonatal period. This change was studied on their relatives and 172 healthy

1

Willink Biochemical Genetics Unit, CMFT, Manchester, United Kingdom; Paediatric Haematology, CMFT, Manchester, United Kingdom

2

Liver dysfunction is well recognised in Urea Cycle Defects (UCD). We present 2 cases presenting with a severe coagulopathy and proven clotting factor deficiencies. Patient 1, a 2.5-year-old girl with Ornithine Transcarbamylase deficiency presented with melaena, raised liver transaminases and coagulopathy. Serum ammonia was 150μmol/L. Factors II, V, VII and IX were low. Intravenous Vitamin K was unsuccessful. Citrulline and phenylbutyrate were added to sodium benzoate and L-arginine, resolving the coagulopathy. Patient 2, a 6-year-old girl with Citrullinaemia had a 7 month history of raised transaminases and routine coagulation screen was markedly

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 volunteers. The calculated carrier frequency in that population was 4.1%, suggesting the incidence of CTLN1 to be 1:2,427; iii) Argininosuccinate lyase, 1 patient with biochemical diagnosis, died during neonatal period. Our experience indicates: a) a high morbidity and mortality at least in our region, despite an early diagnosis and prompt treatment, b) OTC heterozygotes showed severe manifestations and mostly early onset, c) due to the high CTLN1 incidence in one group within ourpopulation (with a consequent high risk), we recommend a preconception carrier screening.

S147 their child's presentation ("it was in his/her eyes"). Longer-term effects were mixed: flourishing on the one hand ("I'm resurrecting my career"; "we're saving for a house"; "after what we've been through and survived, we know our relationship will last forever") and emotional difficulties on the other hand ("everyone in the family has moved on but me; There is nothing much to fill my days, I lost all my so-called friends long ago"). We conclude that in addition to medical advice, parents need on-going support and counselling following liver transplantation for OTC Deficiency.

P-122 P-124 Biochemical and molecular diagnosis of urea cycle enzyme defects in Indian population Bijarnia-Mahay S1, Puri RD1, Verma J1, Shigematsu Y2, Yamaguchi S3, Häberle J4, Kohli S1, Babbar D1, Gupta D1, Verma I C1 1 Center Med Genetics, Sir Ganga Ram Hosp, New Delhi, India; 2Dept of Ped Fukui Med Univ School of Med, Fukui, Japan; 3Dept of Ped, Shimane Univ School of Med, Shimane, Japan; 4Univ Children's Hosp Zurich Steinwiesstr, Zurich, Switzerland

Indian population is a unique mix of variety of cultures and sects and is expected to harbor all types of Urea cycle enzyme defects (UCED), although exact prevalence is not known. We present our experience at a referral centre from India. Over the last 10 years, we have seen many children presenting with hyperammonemia with high likelihood of urea cycle enzyme defect. A confirmative diagnosis was made in 37 children, 18 of which were Citrullinemia type 1, 9 OTC deficiency, 4 cases of Arginino-succinic aciduria (ASA), 3 cases each of Arginase and Citrin deficiency. Molecular studies have been performed in 20 families so far, including 12 since 2011. Nine prenatal diagnoses were performed in 7 families using molecular methods. The presentation in most children has been classic neonatal with progressive encephalopathy, seizures and death in majority of cases. Molecular analysis was done for ASS1 in 12 cases, ASL in 4 and OTC gene in 4 families, revealing 2 common mutations, p.G390R and p.R157H in ASS1 gene, and one recurring mutation, R213X in ASL gene amongst others. Very few reports exist from the Indian subcontinent of UCED. Increasing availability of gene sequencing has enabled increasing numbers of prenatal diagnosis for UCEDs.

Mutations in the human argininosuccinate synthetase (ASS1) gene with asymptomatic clinical course Gündüz Dr1, Bülbül P2, Okur Dr.1, Sürücü Dr.2, Häberle D3 1 Div Metab Dis, Ped&Ped Hem Onc Hosp, Ankara, Turkey; 2Div Metab Dis, Univ Kırıkkale Med School, Kırıkkale, Turkey; 3Div Metab&Resc Cent,Univ Zurich, Zurich, Switzerland

Background: Mild citrullinemia is an allelic variant of classical citrullinemia type I caused by deficiency of the urea cycle enzyme argininosuccinate synthetase (ASS). This autosomal recessive disorder is due to heteroge,neous ASS1 gene mutations leading to various clinical manifestations encompassing often severely affected patients with fatal neonatal hyperammonemia but also asymptomatic individuals with only a biochemical phenotype. Objective: The aim of this study was to describe three patients with mild citrullinemia and identify their molecular background. Cases: Three patients were identified at 15 days, 2 years and 8 years with elevated citrulline levels but without specific symptoms. Hyperammonemia was never documented and no clinical findings were observed. Analysis of the ASS1 gene revealed the homozygous mutations Ivs11+49C>T (r.773ins47;p.259X) in one patient and c.1085G>T (p.Gly362Val) in two patients as underlying the asymptomatic clinical course. Conclusions: An increasing number and variety of mutations in the ASS1 gene are known to cause type I citrullinemia and knowledge on some genotype-phenotype correlations is growing as increasing numbers of patients are investigated. The mutations found in this small series were both already associated with mild citrullinemia which is confirmed by the course in the patients reported here. Conflict of Interest declared.

P-123 P-125 OTC deficiency: three families' journey from diagnosis to post successful liver transplantation

Cranial Magnetic Resonance Imaging (MRI) in late diagnosed twins with citrullinemia type 1: recognize to diagnose

Upton H1, Boneh A2, Sahhar M2 Aktuglu Zeybek AC1, Kiykim E1, Cansever MS1, Altay S2, Aydin A1 1

Metab.Unit Vcgs, Royal Childrens Hosp, Parkville. Victoria, Australia; 2Metab Unit, Vcgs, Royal Children's Hosp., Parkville. Victoria, Australia

1 Div Ped Nutr and Met, Ist Univ Cer Med F, Istanbul, Turkey; 2Nutr Clin, Ist Univ Cer Med F, Istanbul, Turkey

In 2005, a boy and two girls, aged 13 and 17 months, and 5 years, respectively, were diagnosed with Ornithine Transcarbamylase(OTC) Deficiency. Over the following five years conservative treatment with diet and medications became difficult, with each child having dozens of hospital admissions due to hyperammonaemia. The option of liver transplantation was raised as each child's condition became more unstable and difficult to control. The recurrent admissions prior to transplantation led to a significant financial burden on each family as well as challenging behaviour of their children. All children had successful liver transplantation in 2009-2010. Immediately following the transplantation, parents identified a substantial difference in

Background: Here we report cranial MRI findings of twin brothers with citrullinemia type 1 (CT1) diagnosed at 8 months of age Cases: 8 months old male patient was admitted to hospital with tonic seizures. He was born as a twin brother of nonconsanguineous parents at 35th gestational week. Birth APGAR was 5/6, and was hospitalised for RDS for 10 days. He had truncal hypotonia, hyperreflexia in both lower extremities with ankle clonus. The plasma ammonia was 476 μmol/L. CT1 was diagnosed as his plasma citrulline concentration was markedly elevated (1205 μmol/L) with normal arginine-glutamine. His twin brother, who was diagnosed as hypoxic ischemic encephalopathy by another Pediatrics Unit, was also invited and found hypotonic with increased reflexes, he didn't make eye-to-eye contact. His plasma

S148 ammonia and citrulline levels were markedly increased. Reevaluation of the cranial MRI performed at the age of 5 months revealed increased signalling of frontoparietal subcortical, periventricular and cortical white matter in T2, diffusion, FLAIR which were consistent with cytotoxic eodema. His new MRI at admission revealed bilateral frontoparietooccipital corticosubcortical atrophy and ulegyric chances. Conclusion: Recognizing cytotoxic edema which may be a sign of hyperammonemia can help in early diagnosis and treatment of urea cycle disorders. P-126

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 difficulties swallowing and talking, which evolved into facial diplegia over a week. Brain MRI showed T2-weighted hyperintensities in the central pons evocative of central pontine myelinolysis (CPM). Only 2 patients with OTC deficiency have been previously reported with CPM. This case report emphasizes that CPM should be considered in case of secondary neurological deterioration in patients otherwise successfully treated for urea cycle disorders. Furthermore, the consequences of maternal hyperammoniemia on an 7-week-old fetus remain to be determined.

03. Sulphur aminoacid disorders

Double trouble in an encephalopathic newborn: citrullinemia and profound biotinidase deficiency

P-128

Müminoğlu Tahta N1, Öztürk Hişmi B2, Ünal Ö2, Sivri S2, Dursun A2, Tokatlı A2, Coskun T2

Homocysteine reduces energy metabolism in amygdala of rats: neuroprotective role of creatine

1

Kolling J1, Scherer EBS1, Siebert C1, Netto CA1, Wyse ATS1

Dep Pediatr, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey

1

UFRGS, Porto Alegre, Brazil

Coincidence of rare metabolic disorders is not a unexpected issue with consangineous marrieges.Such individuals were previously described who has phenylketonuria and hereditary fructose intolerance, methylmalonic aciduria and argininosuccinic aciduria, and etc. Here we decribe a girl with profound biotinidase deficiency and citrullinemia. Fortyday-old girl was referred from newborn screening being positive for biotinidase deficiency.She was the first child of first degree cousin parents and had poor sucking and jitterness for 10-15 days at admission. She had encephalopathy and alopecia, keratitis was prominent on physical examination. Plasma biotinidase enzyme activitiy was measured as 0.0 U/L fresh sample. She had no metabolic acidosis or alkalosis with only lactate elevation (8,3 mmol/L, urine organic acid analysis(UOAs) were normla then. She responded drammatically to 20 mg of biotin. At follow-up moderate sensory-neural hearing loss and developmental delay was prominent and metabolic tests performod at 17 months revealed citrullinemia with orotic acid excretion on repeated UOAs. P-127 Central pontine myelinolysis in a late-onset presentation of ornithine transcarbamylase deficiency

Objective: In the present study we investigate the effect of hyperhomocysteinemia on the cell mitochondrion viability and parameters of energy metabolism in amygdala of rats. The neuroprotective role of creatine was also evaluated. Methods: Wistar rats received daily subcutaneous injection of homocysteine (0.3-0.6 μmol/g body weight), and/or creatine (50 mg/Kg body weight) from their 6th to the 28th day of age. Rats were sacrificed 12 h after the last injection and the amygdala was dissected. Results: Homocysteine administration significantly altered mitochondrial mass, viability and membrane potential. This amino acid significantly decreased succinate dehydrogenase, complex IV (cytocrome c oxidase) and Na+,K±ATPase activities. In contrast, complex II activity was not changed. Creatine prevented the changes in the energy metabolism caused by Hcy or provoked other effects per se. Conclusion: These findings provide insights into the mechanisms by which Hcy exerts its effects. Creatine could be used with care as adjuvant therapy for improvement of symptoms related to an energy deficit in homocystinuric patients, since it causes effect per se. Supported by CNPq and FAPERGS. P-129

Mochel F1, Ottolenghi C2, Weiss N3, Thabut D4, Brisson H5, Lebray P4, Caillet S1, Bolgert F3, Dommergues M6, Bonnefont JP2, Ogier H7, Demeret S3 1

Genetic dept, Hospital Pitié-Salpêtrière, Paris, France; 2Biochemical dept, Necker Hospital, Paris, France; 3Neurological ICU, Hosp PitiéSalpêtrière, Paris, France; 4Gastrohepato dpt, Hosp Pitié-Salpêtrière, Paris, France; 5Multidisc ICU, Hosp Pitié-Salpêtrière, Paris, France; 6 OBGYN dept, Hosp Pitié-Salpêtrière, Paris, France; 7Ped Neurol dept, Hosp Robert Debré, Paris, France A 28-year old woman at 9 weeks amenorrhea presented with a prolonged episode of vomiting and confusion. Her neurological condition rapidly deteriorated with coma and asterixis requiring assisted ventilation. Her blood tests were remarkable for elevated blood ammonia (280 μmol/L) and low prothrombin time (31%) contrasting with a mild elevation of liver transaminases. Plasma amino acids and urine organic acids suggested an ornithine transcarbamylase deficiency, that was later confirmed by DNA analysis - c.919A>G heterozygous mutation. After 15 hours of hemodialysis and sodium phenylacetate, the patient markedly improved. During the 3 following days, the patient was able to start back eating and communicating while her ammonia levels and liver function normalized. However, at day 4 after hemodialysis, the patient started to manifest

Cystathionine beta-synthase deficient mice thrive on methionine restricted diet Kruger WD1, Gupta S1, Melnyk SB2, Fang Z1 1 Fox Chase Cancer Center, Philadelphia, United States; 2Univ Ark Med Sci, Little Rock, United States

Cystathionine beta-synthase (CBS) deficiency in humans is a recessive genetic disease characterized by elevated serum homocysteine . Dietary protein restriction is one of the strategies used to treat the disease in humans. Previously, we have created a mouse model of CBS deficiency, TgI278T Cbs-/- (Cbs-/-), which is characterized by low weight, low adiposity, facial alopecia, and osteoporosis. To investigate the effect of methionine restriction, we put Cbs-/- and control mice (Cbs+/-) on a methionine restricted diet (MRD) from weaning till 240 days of age. We found that Cbs-/- mice on MRD gained weight and had higher fat mass compared to age match mice on regular diet (RD). Surprisingly, Cbs-/- mice on MRD were not significantly different from Cbs+/- mice on RD with regards to weight or fat mass at the time of termination. Liver stearoyl CoA desaturase-1, a key fat biosynthesizing enzyme that is decreased in Cbs-/- mice on RD, was restored by MRD but was down-

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regulated in Cbs+/- mice on MRD. We also found that MRD mice entirely corrected facial alopecia and osteoporosis phenotypes in Cbs-/- mice. These studies show that a low methionine diet is highly effective in correcting the phenotypic consequences of loss of CBS in mice.

methionine. Homozyogosity-guided mutation analysis revealed a novel homozygous deletion of exon 3 in the ADK gene.

P-130

Sulfite and thiosulfate compromise brain mitochondrial energy homeostasis

P-132

Revising the psychiatric phenotype of homocystinuria 1

2

1

1

1

Jamuar SS , Burch S , Munir K , Waisbren S , Picker JD , Levy H

1

1 Boston Child Hosp, Boston, United States; 2Hosp Univ Pennsylvania, Philadelphia, United States

Objective: To evaluate the psychopathology found in well treated and poorly treated patients with homocystinuria presenting to Boston Children's Hospital (BCH) and relate them to an updated review of the literature. Methods: A chart review was carried out on all patients with homocystinuria presenting to BCH since the condition was first identified in 1962. The literature was also reviewed for psychopathology for all variants of homocystinuria and the data analyzed in conjunction with the described cases. Results: The proportion of patients identified as having psychopathology was 53%. Psychiatric problems were seen in all the patients with poor or variable treatment compliance, whereas no patients with good treatment compliance had psychiatric symptoms. The psychiatric phenotype varied and included mood disorder, psychosis and paranoia. Differences in the literature were found between the cystathionine βsynthase (CBS) deficient patients and those with methylenetetrahydrofolate reductase (MTHFR) deficiency, in which a schizophrenia like phenotype was additionally described. Conclusion: The decreased amount of psychopathology found in our cases appears to reflect consistently good treatment. Paranoia is almost ubiquitous, otherwise the psychopathology of homocystinuria may relate to the underlying biochemical disorder, the primary difference being whether methionine is elevated (as in CBS deficiency) or not.

Grings M1, Moura AP1, Amaral AU1, Parmeggiani B1, Marcowich GF1, Wyse AT1, Wajner M2, Leipnitz G1 1

Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; SGM, HCPA, Porto Alegre, Brazil

2

Background: Sulfite and thiosulfate accumulate in tissues and biological fluids of patients affected by sulfite oxidase deficiency, a disorder characterized by encephalopathy and neonatal seizures, whose pathophysiology is poorly established. Objective: We evaluated the in vitro effects of sulfite and thiosulfate on parameters of energy homeostasis in rat brain. Materials and Methods: The activities of the respiratory chain complexes and creatine kinase, as well as reactive species production and respiratory parameters were determined in the absence or presence of sulfite or thiosulfate (1-500μM) in cerebral cortex homogenates and brain mitochondrial preparations from young rats. Results: Sulfite inhibited complex IV activity, whereas sulfite and thiosulfate inhibited the activities of total creatine kinase (CK) and of its mitochondrial and cytosolic isoforms. Furthermore, antioxidants prevented sulfite- and thiosulfate-induced tCK inhibition and both metabolites increased 2',7'-dichlorofluorescin oxidation, suggesting the involvement of reactive species in these effects. Finally, sulfite decreased state 3 and RCR in mitochondria respiring with glutamate plus malate and inhibited the activities of glutamate and malate dehydrogenases. Conclusion: The data indicate that mitochondrial homeostasis disruption induced by sulfite and thiosulfate may be involved in the neurological symptoms of patients with sulfite oxidase deficiency. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP IBN-Net, INCT-EN.

P-131 P-133 Adenosine kinase deficiency in two sisters with a novel mutation in ADK gene

Mutation spectrum of the MAT1a g]ene in Korean patients with methionine adenosyltransferase deficiency

Makhseed N1, Anazi S2, Haas D3, Hoffmann G3, AlKuraya F2 1

2

Pediatric Department Jahra Hospital, Jahra, Kuwait; Dep Genetics, KFSHR, Riyadh, Saudi Arabia; 3Dep of Ped, Div of Inh Met Dis, Heidelberg, Germany Hypermethioninemia is known to be caused by four inborn errors of metabolism causing deficiency in enzymes that directly interfere with the methionine cycle. These enzymes are methionine adenosyltransferase (MAT) (1), glycine N-methyl transferase (GNMT) (2), Sadenosyl-homocysteine hydrolase (SAHH) (3), and CBS, cystathionine beta-synthase (4). In 2011 Bjursell et.al. described 6 patients, two from a Swedish family and four from two Malaysian families, with severe developmental delay, facial dysmorphism, liver dysfunction and hyper-methioninemia. Using whole exome sequencing, each family was found to have a deleterious homozygous missense mutation in the ADK gene encoding adenosine kinase, thus defining a novel form hypermethioninemia. Adenosine kinase deficiency causes hypermethioninemia by disrupting the futile cycle, which regulates the adenosine and adenine nucleotide levels. (5). We report two sisters born to double first degree cousin parents, who presented with severe global developmental delay, seizure disorder, facial dysmorphism, impaired liver function and high

Woo HI1, Ki CS1, Lee SY1, Kim JW1, Song J2, Lee DH3, Lee YH4, Park HD1 1

Samsung Medical Center, Seoul, Korea, Republic of; 2Seoul National Univ Bundang Hosp, Seongnam, Korea, Republic of; 3Soonchunhyang Univ Seoul Hosp, Seoul, Korea, Republic of; 4Soonchunhyang Univ Bucheon Hosp, Bucheon, Korea, Republic of Background: Methionine adenosyltransferase (MAT) deficiency is a rare metabolic disorder and the mutation distribution in the MAT1A gene varies among ethnic groups. We aimed to determine the molecular characteristics of MAT deficiency in Korean patients. Methods: Mutations in the MAT1A gene were identified using direct sequencing method in five patients with high methionine levels. We also reviewed previous genotypes reported for Korean patients with MAT deficiency. Results: We identified three mutations in six mutant alleles from the five patients. The most common mutation was the Arg264His mutation, which has an autosomal dominant trait and was present in 66.7% of the mutant alleles. The other mutations (Arg299Cys and Arg356Gln) were identified in one mutant allele each. A comprehensive review of previous Korean reports revealed that a total of 11 Korean patients,

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consisting of 3 males and 8 females with a median age of 10.8 months at diagnosis. The Arg264His, Arg299Cys, Arg356Gln, and Trp387* mutations accounted for 86.7% of the total mutations reported to date. Conclusion: We provided the mutational spectrum of MAT1A gene in Korean patients with MAT deficiency. We anticipate that this finding would be useful for the development of mutation detection technique targeted to common mutations.

sulfite is over 40mg/ml detected by sulfite test. Urinary excretion of sulfocysteine is very high (174μmol/mmol Cre.). The mutations are c.1040T>C and c.796G>A in SUOX, the gene that encodes the molybdohemoprotein sulfite oxidase. Conclusion: We reported first case of isolated sulfite oxidase deficiency in Japan. When we find multifocal leukoencephalopathy and uric acid in plasma is not low, it isnecessary to consider isolated sulfite oxidase deficiency.

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P-136

Urinary sulfites in infants with severe neurological impairment.

European network and registry for homocystinurias and methylation defects (E-HOD)

Luengo-Piqueras A1, Esteban-Oliva D2, Ocaña-Rico M2, Arias-Sáez K1, Coroleu-Lletget W2, Pintos-Morell G3, Monlleó-Neila L4 Pediatrics Department, HUGTiP, Badalona, Spain; 2Neonatal Unit, HUGTiP, Badalona, Spain; 3Genetics and Metab Dis, HUGTiP, Badalona, Spain; 4Ped Neurology, HUGTiP, Badalona, Spain Badalona, Spain , 4Ped Neurology, HUGTiP, Badalona, Spain 1

Background: Deficiency of molybdenum cofactor (MoCoD) and isolated sulfite oxidase (SOx) are cause of severe neurological alterations. Some biochemical markers include low serum urate and increased urine xanthine and hypoxanthine in MoCoD. Increased urinary sulfites and low homocysteine are present in both conditions. Case one: 9 days old boy, admitted for myoclonic epilepsy. Magnetic resonance (MRI) showed ventricular dilatation, cerebral and cerebelar atrophy, basal ganglia calcification and porencephaly. He required ventriculoperitoneal shunt due to progressive hydrocephalus. He was diagnosed with MoCoD with positive sulfites in urine, high thiosulfate and low uric acid in serum, and high urinary xanthines. He died from respiratory infection at the age of one year. Case two: 2 days old girl presenting with seizures. MRI showed corpus callosum dysgenesia, cystic leukoencephalopathy, ventricular enlargement and basal ganglia necrosis. She presented progressive spasticity and developmental delay. Urine sulfite test was strongly positive, with normal urinary xanthines and plasma sulfocysteine. She was therefore diagnosed with SOx deficiency. Discussion: In neonates with severe encephalopathy and important structural damage, the clinician must consider the possibility of these two inborn metabolic errors. The determination of urinary sulfites is the first and easy step in the diagnosis. P-135

Blom H 1, Kožich V2 , Huemer M3 , Parker S 4, Groenendijk M 1 , Meutgeert H5, Dionisi-Vici C6, Morris AA7, Hannigan S8, Baumgartner MR9, Kölker S10 1

VU University Medical Center, Amsterdam, Netherlands; 2Charles Univ in Prague - First Fac Med, Prague, Czech Republic; 3Landeskrankenhaus Bregenz, Bregenz, Austria; 4Orphan Europe, Paris, France; 5Vol Kind Stoffwisselingsziekten, Zwolle, Netherlands; 6Ospedale Pediatrico Bambino Gesu, Rome, Italy; 7University of Manchester, Manchester, United Kingdom; 8 Climb, Crewe, United Kingdom; 9Div Metab Dis, Univ Child Hosp, Zurich, Switzerland , 10University Children's Hospital, Heidelberg, Germany Background: Patients with homocystinurias (HCU), methylation defects (MD) and folate defects (FD) have an enormous need for improved medical awareness, optimisation of the diagnostic process and therapy, and improved networking between healthcare professionals and patients. Methods: An initiative named "European network and registry for homocystinurias and methylation defects (E-HOD)" funded by the European Commission DGSanco began 15/02/2013. The network has three major activities: 1/ collecting longitudinal data into a registry; 2/ developing evidence-based consensus diagnostic and clinical care protocols; 3/ evaluating different newborn screening programmes for HCU and producing a position paper. Results: E-HOD has 32 partners from 21 countries linking healthcare professionals, patient's representatives and industry. E-HOD adds value to the existing E-IMD project by using the existing registry technology and network and extending the disease spectrum. The registry will be available in mid 2013 and is expected to collect data on 600 individuals with an HCU, MD or FD over the next 3 years. Conclusion: The overall intended impact is to improve access to rapid diagnosis and optimal care for patients with HCU, MD and FD; the EHOD philosophy is that all patients in Europe should have an equal right to the best up-to-date care. Contact coordinator: [email protected]. Conflict of Interest declared.

First case of isolated sulfite oxidase deficiency in Japan Kurashige Y1, Takayanagi M1, Taku O1, Muta K1, Shiohama T1, Ichida K2 1

Chiba Children's Hospital, Chiba, Japan Pharmacy and Life S, TOKYO, Japan

2

Tokyo University of

P-137 Endoplasmic reticulum stress and autophagy activation in homocystinuria patients with genetic remethylation defects Richard E1, Martínez-Pizarro A1, Desviat LR1, Ugarte M1, Pérez B1

Background: Isolated Sulfite Oxidase (SO) Deficiency is a very rare and devastating inborn error of sulfur metabolism, characterized by refractory seizures, rapid progressive encephalopathy and dislocated ocular lenses. Case report: We report a case of infant boy with isolated sulfite oxidase deficiency who presented with generalized seizures at one month old. Brain CT scan showed bilateral multiple subcortical cystic lesions. Magnetic resonance imaging revealed severe cystic leukomalacia, cortical atrophy with ulegyric pattern ten days after onset. Serum uric acid level is 3.3mg/dl. Plasma level of cystine and serum levels of total homocysteine are very low too. Urinary excretion of

1

CEDEM, CBMSO-UAM, IDIPaz, CIBERER, Madrid, Spain

Remethylation disorders are inherited metabolic diseases of enzymes involved in the remethylation of homocysteine to methionine causing homocystinuria. Previously, we have shown an increased ROS and apoptosis content in homocystinuria patient-derived fibroblasts suggesting a role of homocysteine in these processes. Here we report higher expression levels of endoplasmic reticulum (ER) stress markers Grp78, CHOP, ATF6, p-IRE1 and p-PERK in patients´ fibroblasts with defects in MTRR, MTR and MTHFR genes compared to controls. Expression of Herp (homocysteine-

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 inducible ER stress protein) was also higher in patients´cells, and apoptosis rate was increased in fibroblasts from a cblE patient after shRNA-mediated silencing of Herp gene expression, indicating the protective function of Herp protein under conditions of ER stress. Patients´ cells also showed a marked increase in IP3R1 protein levels suggesting an accumulation of Ca2+ release channels that might lead to a disruption of ER Ca2+ homeostasis. Additionally, enhanced expression of proteins involved in the autophagic process, LC3B and LAMP, was observed in patients´cells compared to controls. Fluorescence microscopy studies showed an increase in autophagosomes in patient-derived fibroblasts and confirmed a massive degradation by autophagy. In conclusion, our results indicate that defects in the remethylation pathway of homocysteine elicit ER stress and an autophagy response. P-138 Slufite oxidase/molybdenum cofactor deficiency: a common neurometabolic disorder in Saudi Arabia

S151 Methods: Carnosinase was expressed in CHO cells and was purified by affinity chromatography. Enzyme activity of carnosinase was measured by fluorescence detection. Results: Homocysteine, cysteine, acetylcysteine, glutathione and methionine inhibit recombinant CN1 activity. Inhibition was noncompetitive and associated with Vmax reduction by 50%. Inhibition caused by GSH, a further thiol-containing compound, was less effective, while no inhibition was observed for GSSG and cystine, oxidized forms of glutathione and cysteine, respectively. The thiol-free compounds glutamate and glycine, components of GSH, did not affect CN1 activity. CN1 inhibition was reversible by addition of the bivalent metals, but not by Fe3+. Conclusions: Thiol-containing compounds, which form complexes with metal ions, inhibit CN1 most likely through removal of Zn2+ from the active site of the enzyme resulting in higher dipeptide concentrations in vitro. The role of homocysteine and other thiolcontaining compounds on CN1 metabolisms in vivo needs further investigations.

Dasouki M1, Jacob M1, Ahmad A1, Al Rashed B1, Amoudi M1, Alodaib A1 1

P-140

Department of Genetics, Kfshrc, RIYADH, Saudi Arabia

Background: Isolated sulfite oxidase (SUOX) and molybdenum cofactor (MOCO) deficiencies are rare autosomal recessive, clinically indistinguishable neurometabolic disorders involve cysteine metabolism, result in sulfite accumulation and present with neonatal intractable seizures, spherophakia, axial hypotonia and profound mental retardation. Objective: Clinical, biochemical and molecular characterization of SUOX and MOCO deficiencies in Saudi Arabian children. Case reports: Seven hundred seventy two symptomatic children suspected of having SUOX/MOCO deficiency were evaluated at our institution since 2005. Material (Patients) and Methods: We used LC-MSMS to measure SSC (sulfocysteine), Xanthine and Hypoxanthine concentrations in 772 urine samples. SNP autozygosity mapping followed by Ion Torrent Ampliseq and Sanger targeted sequencing are used for mutation analysis. Conclusion/Discussion: Seventy five (9.6%) patients were found to have abnormally elevated SSC with/without elevated Xanthine, Hypoxanthine levels (mean SSC excretion: 165; normal: <20 mmol/mol creatinine), consistent with the biochemical diagnosis of SUOX or MOCO deficiency including 5 previously reported cases (Rashed et al, 2005). 15/75 cases (20%) originated from one extended family with one child showing total truncation (delG1244) of the Molybdopterin/Dimerization Domains of SUOX (Seidahmed, 2005). Together, SUOX and MOCO deficiencies appear to be more common in the Saudi population due to consanguinity. The mutation spectrum is currently being analyzed. Conflict of Interest declared. P-139 Homocysteine increases the concentration of the cytoprotectants carnosine, homocarnosine and anserine Peters V1, Amberger A2, Lanthaler B2, Zschocke J2, Schmitt CP3 1 Div Met Dis, Univ Child Hosp, Heidelberg, Germany , 2Div Hum Gen, Med Univ, Innsbruck, Austria , 3Div Nephrol, Univ Child Hosp, Heidelberg, Germany

Background: Carnosinase 1 (CN1) degrades the cytoprotective dipeptides carnosine, anserine and homocarnosine. The Mannheim Allele of CN1 is associated with lower enzyme activity and reduced risk of diabetic nephropathy (DN). Carnosine supplementation in diabetic mice improves DN, vasculopathy and renal function. CN1 is a Zn2+ containing member of the M20 family of metalloproteases.

Folates, vitamin B12 and total homocysteine levels in Portuguese children and adolescents Caldeira Araújo H1, Pimenta A1, Rivera I2, Castro R2, Tavares de Almeida I2 1

Unit Medical Sciences, Univ Madeira, Funchal, Portugal; 2Met&Gen, iMed.UL, Faculty of Pharmacy, Lisbon, Portugal

Vitamins B status is well known to modulate total homocysteine (tHcy) plasma concentrations. Limited data are available concerning reference values for those biomarkers in children and adolescents. Therefore, we evaluated plasma tHcy, folate, vitamin B12 and holo-transcobalamin levels in a healthy population aged 9 (n=195; 107 males and 88 females) and 16 (n=128; 57 males and 71 females) from Madeira Island, Portugal. The results of both cohorts revealed the following plasma concentrations (mean±SD): vitamin B12 363.38±244.69 pmol/L; holotranscobalamin 90.95±33.29 pmol/L; folates 15.68±11.54 nmol/L and tHcy 5.74±3.53 μmol/L. The age effects were examined and statistically significant decreases (p<0.001) in plasma concentrations were observed across age categories (9 versus 16 years-old, respectively) for folates (22.87±9.25; 11.65 ±4.17 nmol/L), vitamin B12 (514.74±165.53; 302.51±118.32 pmol/L) and holo-transcobalamin (100.60±31.63; 66.20±25.10 pmol/L). Interestingly, a gender significant difference (p<0.001) in plasma tHcy levels within adolescents (males 10.24±4.01; females 7.51±2.21) was already observed. In conclusion, folates, vitamin B12 and holo-transcobalamin levels decrease with age while tHcy level increases two fold. Gender influences tHcy levels in the adolescents group. Portuguese adolescents showed lower folates and vitamin B12 than other European adolescents, probably due to the fact that food fortification is not obligatory. P-141 Mice deficient in cystathionine beta synthase display protein arginine hypomethylation Esse R1, Imbard A2, Kruger WD3, Teerlink T2, Castro R1, Tavares de Almeida I1, Blom HJ2 Metabolism and Genetics, iMed.UL, Lisbon, Portugal; 2Dep Clin Chem, VU Univ Med Center, Amsterdam, Netherlands; 3Fox Chase Cancer Center, Philadelphia, United States

1

S152 Cystathionine beta synthase (CBS) deficiency is an inborn error of the homocysteine (Hcy) metabolism characterized by severe hyperhomocysteinemia (HHcy) and a serious clinical outcome. The Hcy precursor S-adenosylhomocysteine (AdoHcy) is a methyltransferases inhibitor that accumulates in HHcy creating a cellular hypomethylation environment that may underlie the pathogenicity of Hcy. We aimed at determining whether protein arginine methylation, an essential post-translational modification, is impaired in CBS deficiency. We used a mouse model of CBS deficiency in which animals lack the endogenous cbs gene but express either the normal human protein or a mutant isoform with reduced activity (I278T). tHcy, S-adenosylmethionine (AdoMet), AdoHcy, and protein-incorporated asymmetric (ADMA) and symmetric (SDMA) dimethylarginines in several tissues were determined by adequate HPLC or LC-ESI-MS/MS methods. Tissue tHcy and AdoHcy levels in CBS deficient mice were at least 15fold and 6-fold higher than in controls, respectively, while AdoMet levels were increased in liver, kidney and brain (> 1.4-fold). Proteinincorporated ADMA was decreased in liver and brain (by 10% and 6%, respectively), as well as SDMA (by 9% and 35%, respectively), compared with controls. We conclude that CBS deficiency induces protein arginine hypomethylation in a tissue-specific manner. This study was supported by FCT grants SFRH/BD/48585/2008 and PTDC/SAUORG/112683/2009. P-142 Global protein and histone arginine methylation are affected in a tissue-specific manner in a rat model of diet-induced hyperhomocysteinemia

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 P-143 Classical homocystinuria in Argentinean patients. focus on an atypical response to betaine supplementation: in search of putative pathogenic mechanism Grosso CL1, Angaroni CJ1, Dodelson de Kremer R1 1

CEMECO, Hosp Niños Córdoba, FCM, UNCor, Córdoba, Argentina

Classical Homocystinuria (HCU) is caused by deficiency of Cystathionine b-synthase and characterized by multiple connective tissue disturbances, mental retardation and mainly, thromboembolic complications. Treatment for pyridoxine-nonresponsive HCU involves lowering homocysteine levels with a methionine-restricted diet and betaine supplementation. Betainehomocysteine S-methyltransferase (BHMT) catalyses the synthesis of methionine using homocysteine and betaine as methyl donor. We identified 8 HCU patients from 7 unrelated families, diagnosed by clinical, biochemical and molecular assessments. In this study we describe the finding of 3 pyridoxine-nonresponsive HCU, who showed partial or total unresponsiveness to betaine treatment together with an intriguing phenotype including lack of thromboembolic events and with mild intellectual disability. We amplified and sequenced the 8 exons and flanking regions of BHMT gene. No genomic change was found in any of the 3 patients that could explain the unexpected response to treatment. One recent research in HCU mice with the co-administration of taurine and betaine, showed relatively sharp threshold-effect between hyperhomocysteinemia and thrombotic risk. It was suggested that this innovative strategy would have a powerful antioxidant action on BHMT with an improvement of therapeutic effects in this disease. This focus is in progress in our Centre concomitantly with the genetic analysis of BHMT gene promoter region. 04. Other amino acid disorders

Esse R1, Florindo C1, Imbard A2, Rocha MS2, de Vriese AS3, Smulders YM4, Teerlink T2, Almeida IT1, Blom HJ2, Castro R1 Metabolism and Genetics, iMed.UL, Lisbon, Portugal; 2Dep Clin Chem, VU Univ Med Center, Amsterdam, Netherlands; 3Dept Intern Med, AZ Sint-Jan AV, Bruges, Belgium; 4Dept Intern Med, VU Univ Med Center, Amsterdam, Netherlands 1

P-145 Pregnancies in women with maple syrup urine disease Scott Schwoerer JA1, Rice GM1, Van Calcar S1 1

Univ of Wisconsin - Dept of Peds, Madison, United States

Accumulation of S-adenosylhomocysteine (AdoHcy), the homocysteine (Hcy) precursor and a potent methyltransferase inhibitor, may mediate the pathophysiology of elevated Hcy. We aimed at determining whether protein arginine methylation status, a crucial post-translational modification that generates monomethylarginine (MMA) and dimethylarginine (asymmetric, ADMA, and symmetric, SDMA) residues, is disturbed in diet-induced hyperhomocysteinemia (HHcy). HHcy was achieved by dietary manipulation of Wistar rats: methionineenrichment (HM), B vitamins deficiency (LV), or both (HMLV). tHcy, S-adenosylmethionine (AdoMet), AdoHcy, and methylarginines concentrations in plasma or tissues (heart, brain and liver) were determined by adequate HPLC or LC-ESI-MS/MS methods. In tissues from the HMLV group, histone arginine asymmetric dimethylation was evaluated by Western blotting, and the histone methylation marks H3R17me2a, H3R8me2a and H4R3me2a were studied. Compared to controls, AdoHcy concentrations were elevated in liver (LV and HMLV) and heart (HMLV), and plasma ADMA levels were lower in all groups. Protein-incorporated ADMA levels were decreased in brain and in heart (LV and HMLV groups). In brain of animals exposed to the HMLV diet, the H3R8me2a mark was decreased. In conclusion, diet-induced Hcy elevation disturbs global protein arginine methylation in a tissue-specific manner and affects histone arginine methylation in brain. This study was supported by grants FCTSFRH/BD/48585/2008 and FCT-PTDC/SAU-ORG/112683/2009.

Maple Syrup Urine Disease (MSUD) is a severe metabolic disorder where one cannot breakdown branched chain amino acids. With improvements in medical care, females with MSUD are now surviving to child-bearing years without significant neurologic sequelae. There is minimal literature about pregnancy in women with MSUD. The literature available describes three pregnancies (including one discussed here) with metabolic decompensation in the post-partum period including one leading to death. It appears the greatest risk to mother is in the early post-partum period where significant protein turnover occurs. Leucine, unlike phenylalanine does not appear to be a teratogen. Our clinic has followed 6 pregnancies in 3 mothers with classical MSUD including two pregnancies in a Mennonite woman. The pregnancies were closely monitored throughout the pregnancy and post-partum period for evidence of metabolic decompensation as well as to assure adequate nutrition for the developing fetus. All pregnancies led to delivery of healthy children. All pregnancies saw a significant increase in leucine tolerance during the second and third trimesters. Monitoring in the post-partum period showed elevated leucine in 2 pregnancies, including one with symptomatic decompensation. Women with MSUD can have successful pregnancies with close monitoring and metabolic therapies during the pregnancy and post-partum period. Conflict of Interest declared.

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S153

Dried blood spot nitisinone and succinylacetone monitoring in patients with tyrosinaemia type 1

Conclusions: These results confirm the relevance of performing functional studies for mutations potentially affecting the splicing process and open the possibility of therapeutical strategies based on splice modulating drugs.

Turner C1, Dalton N1, Walter JH2, Vara R3

P-148

1

Depressive-like behavior in rats submitted to chronic administration of branched-chain amino acids

P-146

WellChild Lab, King's College Lond, ECH, London, United Kingdom, Willink Biochemical Genetics Unit, Manchester, United Kingdom, 3 Evelina Children's Hospital, GSTFT, London, United Kingdom 2

Background: Treatment of tyrosinaemia type 1 (HT1) with Nitisinone (NTBC) has improved outcomes in these patients. Urine succinylacetone (SA) and plasma or dried blood spot (DBS) tyrosine measurement are used to assess adequacy of treatment/compliance. DBS assays for NTBC alone or in combination with other relevant biomarkers may improve patient monitoring. Method: We have validated a new LC-MSMS assay for NTBC & SA in patients with HT1. Following addition of stable isotopes (SA, methionine, tyrosine and phenylalanine), 3mm blood spots were extracted with 3% formic acid in methanol. SA was quantified in negative MRM mode; m/z157.1/99.1, 157.1/114.9, and polarity was switched for NTBC measurement (m/z330.3/218.2, 330.3/126.1), inject-inject time was 3.5min. Quantitation limits are 0.3μmol/L for SA and 0.5μmol/L for NTBC. Amino acids are quantitated from a second injection of supernatant. Results: In 33 DBS from 22 patients with SA levels below 0.3μmol/L the median NTBC was 16.2μmol/L (range 5.6-105.1). In 5 DBS from 3 patients with measurable SA levels (0.44, 0.50, 1.43, 1.73, 3.81), 2 due to non-adherence, one at diagnosis and immediately following institution of treatment, the corresponding NTBC levels were 34.9, 18.3, <0.5, <0.5 and <0.5. Conclusion: The assay allows assessment of NTBC/SA monitoring in optimizing NTBC therapy. Conflict of Interest declared. P-147 Functional analysis and in vitro correction of splicing FAH mutations causing tyrosinemia type I Perez-Carro R1, Sanchez-Alcudia R1, Navarrete R2, Perez B1, Ugarte M2, Desviat LR1

Scaini G1, Jeremias GC1, Morais MO1, Morais F1, Furlanetto C1, Dominguini D1, Comim C1, Quevedo J1, Schuck PF1, Ferreira G1, Streck EL1 1

Universidade do Extremo Sul Catarinense, Criciúma, Brazil

Background: Maple Syrup Urine Disease (MSUD) is a neurometabolic disorder that leads to accumulation of branched chain amino acids (BCAA) leucine, isoleucine, and valine. Because previous reports suggest that MSUD patients are at high risk for chronic neuropsychiatric problems, we investigated depressive-like parameters in rats submitted to chronic administration of BCAA during their development, treated or not with antioxidants and imipramine. Methods: Wistar rats (7 days) received a pool of BCAA or saline twice a day for 21 days, and were also supplemented with imipramine (10 mg/kg) once a day or N-acetylcysteine (20mg/kg) twice a day and deferoxamine (20mg/kg) every two days. Twelve hours after the last administration the rats were submitted to anhedonia and forced swimming tests; adrenal gland weight was also evaluated. Results: Our results demonstrated that animals subjected to chronic administration of BCAA presented decreased sucrose intake without significant changes in body weight. We also showed an increase in adrenal gland weight and in immobility time provoked by BCAA. However, the treatment with imipramine and N-acetylcysteine/deferoxamine reversed the depressive-like parameters. Conclusion: Our results supported the hypothesis that MSUD rats show depressive-like behavior. Moreover, we also demonstrated that the imipramine and N-acetylcysteine/deferoxamine prevented depressivelike behavior in MSUD rats. P-149 Mk-801 and antioxidants prevent glycine-induced bioenergetic dysfunction in rat brain: potential novel adjuvant therapies for nonketotic hyperglycinemia

1

Centro de Biología Molecular, Madrid, Spain; 2CEDEM-UAM, Madrid, Spain Background: Hereditary tyrosinemia type I is a rare metabolic disease caused by a deficient activity of the enzyme fumarylacetoacetate hydrolase (FAH) in the tyrosine catabolic pathway and resulting in hepatic alterations such as cirrhosis or hepatocarcinoma due to accumulation of the toxic metabolites fumarylacetoacetate, maleylacetoacetate and succinylacetone. Methods: We have characterized using minigenes four splicing mutations affecting exonic or intronic nucleotides of the FAH gene identified in two patients. We have analyzed the effect of compounds known to modulate splicing (valproic acid, phenyl butyrate, M344, EIPA, resveratrol) and the overexpression of SR splice factors on the transcriptional profile of the mutant minigenes. Results: Two mutations are novel, c.82-1G>A y c.913G>C and the other two have been previously associated with a splicing defect (c.836A>G and c.1062+5G>A). All mutations were confirmed to affect splicing in minigenes, resulting in exon skipping or activation of a cryptic splice site. For the c.836A>G mutation a partial recovery of the correctly spliced transcript was observed with some compounds and especially with the overexpression of SRp40 and SRp55.

Moura AP1, Grings M1, Parmeggiani B1, Marcowich GM1, Tonin AM1, Zanatta A1, Viegas CM1, Bumbel AP1, Ribeiro CAJ1, Wajner M2, Leipnitz G1 1

Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; SGM, HCPA, Porto Alegre, Brazil

2

Background: Nonketotic hyperglycinemia (NKH) is an inherited disorder caused by a defect in the glycine (GLY) cleavage system leading to high brain levels of GLY. Clinically, affected patients present severe neurological symptoms, whose pathophysiology is still unclear. Objective: In the present study we investigated the effects of intracerebroventricular GLY administration (5 μmol) on important parameters of energy metabolism in rat brain. Methods: Thirty-day-old Wistar rats were sacrificed 30 min after GLY administration, the cerebral cortex and striatum dissected, homogenized and used for the biochemical assays. Results: GLY significantly reduced CO2 production and the activities of citrate synthase and isocitrate dehydrogenase in striatum, as well as the activities of complexes I-III and IV of the respiratory chain, and of creatine kinase in cerebral cortex and striatum. Finally, the antioxidants N-acetylcysteine and creatine, and the NMDA receptor antagonist MK-

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801 prevented the inhibitory effects of GLY on CK and on the respiratory complexes, indicating the involvement of reactive species and of the NMDA receptor in these effects. Conclusion: The data indicate that bioenergetic dysfunction is strongly disrupted by GLY, a pathomechanism that may contribute to the brain injury in NKH. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP IBN-Net and INCT-EN. P-150 Acute and chronic administration of branched-chain amino acids decrease nerve growth factor in rat hippocampus Streck EL1, Scaini G1, Jeremias IC1, Morais MOS1, Mello-Santos LM1, Kist LW2, Pereira TCB2, Bogo MR2, Schuck PF3, Ferreira GC3

Results: Among those with normal activity, 2 siblings in group 1 showed mild elevation of blood leucine levels persistently. No mutation was found in the genes of E1α, E1β, E2, brabched-chain aminotransferase isozymes (BCAT1, BCAT2), BCKD phosphatase (BCKDP). Finally, we identified heterozygous base substitution in BCKD kinase (BCKDK); c.406C>A, p.L136M. Family analysis revealed that this variant allele derived from their father. Discussion: As BCKDK is the enzyme that inactivates BCKADC by phosphorylation, the variation 406C>A must be a "gain of function" mutation to cause elevation of blood leucine levels. Further functional analysis is proceeding to clarify its effect on phosphorylation of E1α. P-152 Citrin deficiency in Spain: a new caucasian case Vitoria I1, Dalmau J1, Rausell D2, García AM2, Lopez-Montiel J3, Rubio V4,5

1

2

Lab Bioenergetics, Univ Ext Sul Cat, Criciúma, Brazil; Lab Biol Gen Mol, Pontif Univ Cath PA, Porto Alegre, Brazil; 3Lab Err In Metab, Univ Ext Sul Cat, Criciúma, Brazil Background: Patients affected by Maple Syrup Urine Disease (MSUD) usually present neurological dysfunction. Thus, we investigated immunocontent and mRNA expression of nerve growth factor (NGF) in brain of rats submitted to an animal model of MSUD. Methods: For acute administration, Wistar rats (10 and 30 days-old) received three injections (1h interval) of a branched-chain amino acids (BCAA) pool or saline, subcutaneously. For chronic administration, Wistar rats (7 days-old) received a BCAA pool or saline twice a day for 21 days; the animals were supplemented with N-acetylcysteine (20mg/kg) twice a day and deferoxamine (20mg/kg) every two days. One (acute) or twelve hours (chronic) after the last injection, the animals were killed by decapitation; hippocampus, striatum and cerebral cortex were collected for assays. Results: NGF levels were decreased in hippocampus after acute and chronic administration of BCAA. The expression of ngf in hippocampus was also decreased only after acute administration in 10 days-old rats; antioxidants increased ngf expression. Conclusions: The results suggest that BCAA are involved in the regulation of NGF in the developing rat, and antioxidants are able to prevent these changes. Thus, it is possible that alteration of NGF could be of pivotal importance in cognitive impairment provoked by BCAA. P-151 Enzymatic diagnosis of maple syrup urine disease:genetic analysis of two siblings showing persistent elevation of leucine in blood with normal enzyme activities Kagawa R1, Tsumura M1, Hara K2, Okada S1, Tajima G1, Sakura N3, Tokuhara D4, Okano Y5, Kobayashi M1

1

Nutr Metab Unit,La Fe Hosp, Valencia, Spain; 2Nutr Metab Lab, La Fe Hosp, Valencia, Spain; 3M.G.C. Genetaq, Malaga, Spain; 4Inst Biomed, CSIC, Valencia, Spain; 5CIBERER-ISCIII, Valencia, Spain Background: The belief that deficiencies of the liver mitochondrial aspartate/glutamate antiporter citrin only affect East-Asians must be abandoned. We report a Caucasian infant with neonatal intrahepatic cholestasis due to citrin deficiency (NICCD), highlighting the value of dietary therapy. Case report: The 22 day-old male, third child of non-consanguineous Romanian parents, was seen with jaundice, failure to thrive, severe anaemia (requiring transfusion) and suspicion of sepsis. Intrahepatic cholestatic jaundice with modest hepatomegaly was documented, together with extremely high alfa-fetoprotein, hypoalbuminemia, hypocoagulability with low vitamin K-dependent coagulation factors, galactosuria, increased blood and urine citrulline, threonine, methionine and tyrosine, and increased urinary 4-hydroxyphenyl acids without succinylacetone. Blood ammonia was normal. Citrin deficiency was confirmed by finding in homozygosis the citrin-truncating nonsense mutation c.1078C>T (p.Arg360*) in the citrin gene SLC25A13. Breast milk replacement by lactose-free, low-carbohydrate, protein-enriched formula (2.5-3.5 gr protein/kg/day) caused immediate improvement. At 9 months the child has normal development and analytical values, craves for protein and avoids carbohydrate-rich foods Conclusion: Citrin deficiency must be considered in the differential diagnosis of infantile intrahepatic cholestasis regardless of ethnic origin, particularly since NICCD evolution is not always favorable and dietary treatment is highly efficacious. VR support:grants BFU2011-30407 and Prometeo 2009/051 (Spanish and Valencian Governments). P-153 L-tyrosine induces DNA damage in brain and blood of rats

1

Dept. Pediatrics, Hiroshima Univ hosp, hirosima, Japan; 2Dept. Pediatrics, Kure Medical Center, Kure, Japan; 3Nursing House Suzugamine, Hiroshima, Japan; 4Dept. Pediatrics, Osaka City Univ, Osaka, Japan; 5Okano Kodomo Clinic, Osaka, Japan

Ferreira G1, De Prá S1, Carvalho-Silva M1, Vieira J1, Leffa D1, Fagundes G1, Bristot B1, Borges G1, Andrade V1, Schuck P1, Streck E1 1

UNESC, Criciúma, Brazil

Background: We established a radioisotope-free enzymatic assay of branched-chain α-ketoacid dehydrogenase (BCKD) complex for the diagnosis of patients suspected with maple syrup urine disease (MSUD) (J Inherit Metab Dis 27: 633, 2004). In our series of patients, we found two siblings who showed mild but persistent elevation of blood leucine levels in spite of apparently normal activities of BCKD complex. Here we report on genetic analysis of these siblings and their family. Patients: Twelve newborn screening (NBS)-positive patients with clinical symptoms (group 1). Sixteen NBS-positive patients with asymptomatic (group 2). Eleven symptomatic patients with negative results in NBS.

Background: Mutations in TAT gene have been identified to cause tyrosinemia type II. Studies demonstrated that an excessive production of ROS can lead to reactions with macromolecules, such as DNA, lipids, and proteins. We evaluated whether acute and chronic administration of L-tyrosine cause transient DNA damage, determined by the alkaline comet assay, in brain and blood of rats. Methods: For acute administration, 30 days-old Wistar rats were killed one hour after a single intraperitoneal injection of L-tyrosine (500 mg/kg) or saline. For chronic administration, two injections (at 12h

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 interval) of L-tyrosine (500 mg/kg) or saline were given for 21 days, starting at postnatal day 7; twelve hours after the last injection, the animals were killed, brain and blood were collected. Results: We observed that acute administration of L-tyrosine increased DNA damage frequency and damage index in cerebral cortex and blood. Moreover, we observed that chronic administration of Ltyrosine increased DNA damage frequency and damage index in hippocampus, striatum, cerebral cortex and blood. Conclusions: These results demonstrate that DNA damage can be encountered in brain of animals submitted to hypertyrosinemia. DNA alterations may represent a further means to explain neurological dysfunction in this inherited metabolic disorder. P-154 Zinc and selenium deficiency in patients with maple syrup urine disease Kanufre VC1, Cruz AF2, Rocha CDN2, Valadares ER3 1

Universidade Federal de Minas Gerais, Hc, Belo Horizonte, Brazil; Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Fa, Belo Horizonte, Brazil

S155 Acidemias, MSUD, Homocystinuria, HMG-CoA Lyase, VLCAD and MCAD deficiencies, Chanarin-Dorfman Syndrome and Citrullinemia were included. According to height/age 31.25% presented stature deficit, probably due to late diagnosis or the disease per se. Weight/height deficit was lower (12.5%), indicating nutritional recovery. To children older than 5 years old, however, prevalence was the same: 30% for low height/age and 30% of low weight for BMI/age. Weight deficit is probably related to metabolic decompensation and catabolism or caregivers' insecurity in increasing the food quantity and, thus, triggering symptoms. Overweight risk in children above 5 years old was 12.5% (weight/height) and 18.75% (BMI/age) Conclusions: Even though no patient presents overweight, data show that is necessary to be aware of diet control in order to avoid excessive ingestion. The great majority of patients are eutrophic, which indicates the success of the nutritional treatment P-156 Discriminating aberrant excretion patterns: the role of relative concentrations of amino acids in urine of patients with Hartnup disorder

2

Jans JJ1, Prinsen HCM1, De Sain - van der Velden MGM1, VerhoevenDuif N1, van Hasselt PM1

Introduction: Due to dietetic restriction, crucial in the treatment of patients with MSUD, some micronutrient deficiencies can be observed, for example zinc and selenium, important antioxidants, essential for metabolic processes. Objective: To evaluate blood levels of zinc and selenium in patients with MSUD in treatment at the Ambulatory Clinic for Inborn Errors of Metabolism (HC - UFMG). Methods: Blood samples were taken from four patients to zinc and selenium quantification and the results were compared with the reference values from the laboratory. The food ingestion on the day before was recorded and the consumption of both micronutrients was estimated and compared to Dietary Reference Intakes. Results and discussion: All patients showed low blood levels of selenium and also low consumption compared to Recommend Dietary Allowances. All of them had normal blood levels of zinc, despite the fact that only two of them presented adequate intake. The average intake, however, was of 28% of the recommendation for selenium and 102% for zinc. Conclusions: The metabolic formula used to complement energy and protein intake in these patients is supplemented with zinc but not with selenium. Consequently, patients have selenium primary deficiency, due to restrictions of natural proteins intake, important sources of this micronutrient.

Hartnup disorder (OMIM#234500) is an autosomal recessive disorder biochemically characterized by a neutral amino aciduria. Although a pellagra-like rash, ataxia and abnormal (psychotic) behavior have been described in patients, many cases are asymptomatic. Hartnup disorder is caused by mutations in the SLC6A19 gene, encoding the amino acid transporter B0AT1 (B0Amino acid Transporter). B0AT1 functions as a neutral amino acid transporter and is primarily expressed in intestine and renal proximal tubule. While the aminoaciduria associated with Hartnup disorder is described to be mostly restricted to neutral amino acids, several patients in our laboratory presented with elevated excretion of almost all amino acids. In these cases it can be challenging to discriminate the excretion pattern of a Hartnup patient from that of patients with generalized aminoaciduria. To facilitate discrimination of Hartnup patients from patients with generalized aminoaciduria we developed a simple algorithm taking into account the degree of elevation of several amino acids. This algorithm distinguishes Hartnup patients diagnosed in our laboratory and Hartnup patients described in literature from patients with other amino acidurias.

P-155

P-157

Anthropometric evaluation of patients with inborn errors of metabolism

The regression of liver cirrhosis in tyrosinemia type 1 patients with NTBC treatment

Kanufre VC1, Cruz AF2, Rocha CDN2, Valadares ER3

Polyakova SI1, Jourkova N1, Varichkina M1, Sevostianov K1

1

1

Universidade Federal de Minas Gerais, Hc, Belo Horizonte, Brazil; Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; 3 Universidade Federal de Minas Gerais, Fa, Belo Horizonte, Brazil

1

University Medical Center Utrecht, Utrecht, Netherlands

Scientific Center of children's health, Moscow, Russian Federation

2

Objective: To evaluate the nutritional status of patients with IEM treated with restrict diets. Methods: Information of 26 patients was included and compared with OMS standards. The ratings utilized were weight/age, weight/height, BMI/age to evaluate children below 5 years old and height/age, BMI/age between 5 and 9 years old. Results and discussion: Patients with Glycogen storage disease, Galactosemia, Fructosemia, Methylmalonic, Methylglutagonic and Glutaric

Background: Effectiveness of NTBC treatment in hereditary Tyrosinemia type 1 patients (HT1) has been proved, especially in the group of patients who was diagnosed on the newborn screening basis. We started treating children on advanced stages of disease (cirrhotic liver and severe rickets). Material/patients The effectiveness of NTBC was evaluated in 11 children from Russia. HT1 was confirmed in 12 children (f/m: 5/7) at the age 37[6; 244] months by elevated succynilacetone level in the urine, aminoacid profile and 2 mutations in FAH gene. Cirrhosis presented in all children, one 10 y/o girl with late diagnosis has been administered to liver transplantation without

S156 NTBC treatment. Initial dose of NTBC was 1,5-2 mg/kg in 6 subacute HT1 patients of the age less than 12 months, 0,6-1 mg/kg - with chronic HT1 patients older than 36 mns. Regress of morphological signs of cirrhosis associated with AFP normalization was confirmed by various methods (MRI, CT), radioisotope scanning and fibroelastography. Excellent treatment results allowed us to avoid needle liver biopsy. Two boys after orthopedic bone reconstructive surgery started to walk without assistance. Results: The unexpected regression of liver cirrhosis was confirmed by different methods of visualization in 10 patients. Two incompliant patients demonstrated poor results. P-158

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 exclusively breastfed for six months. The mother received 1.3 g/kg/day protein in the lactation period. Mild proteinuria started at the forth month of the delivery. Placental complications were suggested to be related to either other prothrombotic risk factors or primary underlying disorder. P-160 Cysteine nitrosylation regulates carnosinase activity and specifity Amberger A1, Lanthaler B1, Zschocke J1, Schmitt C.P2, Peters V2 1

Dept.Human Genetics, Med.Uni.Innsbruck, Innsbruck, Austria; Div.Metabol.Dis., Med.Univ.Heidelberg, Heidelberg, Germany

2

Rickets and bone density in late diagnosed patienties with tyrosinemia type 1 Polyakova S1, Jerdev K1, Tcygina E1, Kurilenko M1, Vashurina T1 1

Scientific Center of children's health, Moscow, Russian Federation

Two boys with HT1 of 5 and 13 years who started nitisinone treatment in 2009 and 2011 respectively, had a growth deficit of more than 3SD. Both had severe phosphate diabetes, complete Fanconi syndrome (hypophosphatemia, hypocalcaemia, glycosuria, great bicarbonate deficiency, metabolic acidosis). Prior to initiation of therapy both children were immobile. Besides NTBC patients received calcitriol, phosphates, calcium and other adjuvant therapy. Bone mineral density and bone age was determined by densitometry. First child demonstrated normalization of bone mass (from initially BMD =0,56) and bone density (with initial Z-score = -3) in 1.5 years after NTBC treatment and second child showed significant improvement of BMD and Z-score after 2 years of NTBC treatment. Parathyroid hormone levels returned to normal. Patients stopped losing calcium and phosphorus with the urine. The patient's height increased in 4 and 2 years by 24 and 20 cm respectively. Two reconstructive operations on lower extremities have been performed in half year period: wedge resection of saber deformed bones of legs and hips with metal osteosynthesis.

Objectives: Diabetic nephropathy (DN) is the final stage in 40% of long term diabetic patients. A variant (Mannheim Allele) of carnosinase is associated with lower enzyme activity and reduced development of DN. Carnosinase catalyzes the degradation of cytoprotectants carnosine and anserine. Cysteine S-nitrosylation is an important regulatory modification of enzyme function. Here, we investigated the impact of cysteine Snitrosylation on carnosinase activity and specifity. Methods: Wild type and two cysteine mutants (Mut 1C102S and Mut 2 C229S) of carnosinase were expressed in CHO cells and recombinant proteins were purified by affinity chromatography. Nitrosylation was performed with 3-Morpholinosydnonimine and determined by biotin switch assay. Enzyme activity was determined by HPLC. Results: Carnosine degradation was about 10-fold higher than anserine degradation in wild type enzyme. Mut 2 showed a similar carnosine degrading activity than wild type, whereas this activity was completely inhibited in Mut 1. Interestingly, nitrosylation reduced carnosinase activity of wild-type and Mut 2 in a concentrationdependent manner. Importantly, nitrosylation didn't alter anserine degradation. Conclusion: Cysteine nitrosylation regulates carnosinase activity in a substrate-specific manner. These findings suggest a novel role of NO in the regulation of carnosinase activity. Therefore, modulation of NOproduction could be a novel therapeutic strategy to treat DN. P-161

P-159 Continuous age-corrected reference ranges of amino acids in CSF Pregnancy and lactation outcomes in a Turkish patient with lysinuric protein intolerance Coskun T1, Ünal Ö1, Orhan D2, Tokatlı A1, Dursun Ali1, Hişmi B1, Özyüncü Ö2, Kalkanoğlu-Sivri HS1

Mxrkrid L1, Woldseth B1, Matern D2, Rinaldo P2 1

Sect IEM Dep Med Biochem, Oslo Univ Hosp, Oslo, Norway; Biochem Genet Lab, Mayo Clinic, Rochester, Minneosta, United States 2

1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey; Hacettepe Univ, Dept Ped Pathology, Ankara, Turkey

2

Lysinuric protein intolerance (LPI) is an autosomal recessively inherited, rare, multisystem disorder affecting cationic amino acid transport. Maternal LPI is associated with increased risk of anemia, toxemia, and retarded growth in fetus during pregnancy and bleeding complications during delivery. Here we present pregnancy and lactation outcomes in a patient with LPI. In the patient who diagnosed with LPI, molecular genetic analysis revealed c.283insTGG/- (p.Glu95_Thr96insTrp) in the SLC7A7 gene. On follow-up she had hemophagocytic lymphohistiocytosis, osteoporosis, and hyperlipidemia, and was treated with a protein-restricted diet and citrulline treatment. When conception occured, she continued to be treated with protein restricted diet and citrulline. In the pregnancy and delivery period, metabolic status was stable. No complications occurred except for mild thrombocytopenia and severe anemia. The baby was born as full-term and healthy although, pathological examination of the placenta revealed multifocal placental infarcts. She was heterozygous for PAI-I 4G/5G and homozygous for MTHFR C677T. The baby was

Background: Age influences the concentration of several amino acids in CSF. This variable should be taken into account when establishing reference ranges and diagnostic decision limits. Objectives: To develop continuous, age-corrected reference ranges for CSF amino acids by mathematical modeling of large populations from multiple sites. Material and Methods: CSF amino acid profiles were obtained in two laboratories (Mayo Clinic and Oslo University) by analysis of 812 samples from individuals without known IEM. After Box-Cox transformations to obtain normal distributed and homoscedastic residuals, amino acid data fitted to a 3rd order polynomial regression model. E.g. for serine: Box-Cox lambda=-0.2; μ=2.588–0.1875 lg(age)+0.021 lg (age)^2 +0.0178 lg(age)^3; s=0.102.Thus a patient value can be converted to a Z-score = (transformed value- μ[age])/s[age]. Results: Data from the two sites exhibited similar age behavior and the model demonstrated that existing discrete reference limits carry a significant risk of misinterpreting results, for example a low concentration of serine in the newborn period.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Conclusion: Z-scores for patient values adapt better to the normal biological age variation and are equivalent to the use of percentiles in pediatric growth charts. International cooperation is indispensable to obtain an adequate number of reference individuals to estimate the percentiles with high precision. P-162 A 2-year old girl with methylmalonic acidemia and extremely restricted diet. Sdogou T1, Katsieri K1, Tsoukatou TH1, Garoufi A1, Georgouli H1, Kossiva L1 1

2nd Dep Ped P&AKyriakou Univ Child Hosp, Athens, Greece

Introduction: Children with methylamlonic acidemia may be healthy at birth and develop symptoms after commencing protein intake. Without treatment, the prognosis is extremely guarded. Case report: We present a toddler diagnosed with MMA (mut 0) shortly after her birth. Since then, she was placed on a low protein diet supplemented carnitine and vitamin B12. The initial evaluation was skin rash in the diaper area and was treated with local corticosteroroids but did not respond. The rash worsened after a gastroenteritis. She presented with erythematous lesions with erosions. Her scalp hair was sparse and she had foot edema. Skin changes resembled acrodermatitis enteropathica. Laboratory investigation revealed deficiency of essential amino acids, low zinc and copper plasma levels. Methylmalonic acid was increased. Skin cultures revealed pathogens. Her diet was enriched with the deficient aminoacids. Zinc, copper and biotin were added. The following days skin lesion worstened and her general condition deteriorated. Persistent oozing resulted in a considerable hypoalbuminemia. Despite supportive care, she died from sepsis. Postmortem blood cultures revealed Stenotrophomonas maltophilia. Conclusions: The close evaluation of the diet in children with MMA is important, since restricted regimes can lead to severe and life-threatening complications, such as acrodermatitis enteropathica. Some patients have an imoproved prognosis with early cobalamin treatment. P-163 Fibroblasts from Maple Syrup Urine Disease (MSUD) classic patients showed mitochondrial oxidative stress and protein damage Fernandez-Guerra P1, Palmfeldt J1, Merinero B2, Bross P1, RodriguezPombo P2

S157 antioxidant defense system we measured protein levels of manganese superoxide dismutase (SOD2), which catalyzes the conversion of mitochondrial superoxide into hydrogen peroxide. We observed no difference in SOD2 levels between fibroblasts from MSUD classic patients and controls. Conclusions: Fibroblasts from classic MSUD patients showed increased mitochondrial oxidative stress and increased protein carbonylation, but no detectable changes in SOD2 protein levels. This indicates that the mitochondrial oxidative stress is caused by an increase production of mitochondrial superoxide and not by decreased detoxification of mitochondrial superoxide. P-164 Neonatal intrahepatic cholestasis caused by citrin deficiency: an infant incidentally detected by phenylketonuria screening with a novel mutation in SLC25A13 gene Aktuglu Zeybek AC1, Kiykim E1, Ceylaner S2, Cansever MS1, Altay S1, Aydin A1 Div Ped Nutr and Met, Ist Univ Cer Med F, Istanbul, Turkey; 2Intergen Genetics Center, Ankara, Turkey

1

Citrin deficiency is an inborn error of metabolism due to mutations in SLC25A13 gene. The same mutations can lead three different clinical pictures: NICCD, developmental delay and dyslipidemia due to citrin deficiency (FTTDCD) and type 2 citrullinemia in adults. Most common finding in NICCD is intrahepatic cholestasis with fatty liver. Here we report a case with positive PKU screening test diagnosed as NICCD with a novel mutation. Case Report: 31 days old male patient was admittted with positive PKU screening test. Physical examination was normal except mild jaundice. Quantitative plasma aminoacid analysis revealed significant in citrulline and mild elevation in arginine and metionine levels. Blood ammonia was slightly increased. Serum AST, ALT, GGT, ALP, total bilirubin, alphafetoprotein levels were increased, with mild galactosuria. Prothrombin time was prolonged. Hepatic ultrasound was consistent with grade II hepatosteatosis. A compound heterozygous mutation of c.851854delGTAT/p.I290T(c.895T>C) was found in SCL25A13 gene. After two mild hyperammonemia attacts, the patient is now 14 months old and free of clinical symptoms. Conclusion: To our knowledge this is the first time SCL25A: c.851854delGTAT/p.I290T(c.895T>C) mutation is reported. Although the disease presented as NICCD, mild hyperammonemia attacts during the first 6 months may be an indicator for transforming into the adult form. P-165

1

Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark; 2 CEDEM, Dpto Biol.Mol, CBMSO, CIBERER, IDIPAZ, Madrid, Spain Background: MSUD is caused by the defective activity of branched-chain alpha-ketoacid dehydrogenase complex, a mitochondrial enzyme complex. Defects in mitochondrial enzymes can lead to oxidative stress. Aim: To analyze oxidative stress markers in fibroblasts from MSUD patients with classic phenotype and an enzymatic activity below 2.5% in comparison to controls: E1β[p.R216G+R216G], E1β[p.R285X+ p.R285X] and E1α[p.R40fs+p.R40fs]. Results: We measured mitochondrial superoxide levels with MitoSOX and we observed an up to 2.1-fold increase in fibroblasts from patients. Higher superoxide levels can lead to protein oxidative damage that was measured by protein carbonylation. We detected protein carbonylation by ELISA of DNPH-derivatized proteins. Fibroblasts from patients showed up to 1.5-fold higher carbonylation levels. To evaluate possible effects on the

Benefits of Na benzoate treatment for adult diagnosed non ketotic hyperglycinemia (NKH) Horvath GA1, Bishop CM2, Sirrs SM3 1

Biochem Dis, BC Children's Hospital, Vancouver, Canada; 2Pychol, Univ BC, Vancouver, Canada; 3Adult Metab Clin, Vancouver Gen Hosp, Vancouver, Canada

Background: Clinical symptoms of atypical NKH are heterogeneous for movement disorders, cognitive deficiencies and behavioural abnormalities. Most late onset form NKH patients have P-protein (GLDC gene) but rarely T-protein (AMT gene) mutations. Objective: To emphasize the importance of recognizing NKH in adulthood as a treatable disorder. Case report: A 33-year-old woman with remote history of encephalopathy, seizures at age 2 years and progressive, significant intellectual disability

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J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

was physically well until hospital admission for febrile illness with encephalopathy and severe myoclonus requiring intubation. Valproate treatment was initiated. Plasma glycine was moderately elevated. Symptoms slowly improved following valproate discontinuation. A previously reported homozygous missense mutation in the AMT gene c.139G>A (p.Gly47Arg) was identified. Treatment with Na benzoate and Dextromethorphan resulted in improvements in plasma glycine levels, myoclonus and behaviour. Successive neuropsychological evaluations demonstrated improved verbal recall but otherwise persistent impairments. Depression, anxiety, behavioural (agitation, irritability) symptoms and independent living skills improved. Supported independent living is now considered. Conclusions: It is important to suspect NKH in adults, even with mild to moderate elevations of glycine, as treatment in atypical cases can be beneficial. P-166 The ERNDIM external quality assurance (EQA) scheme for amino acids: lessons for treatment monitoring and multicentre studies in inherited metabolic disorders (IEM)

The newborn presented progressive encephalopathy from the 5th day of life (lethargy, feeding problems, somnolence) and coma; the acute cerebral damage was severe and the child died early after he was admitted to hospital. Rapid biochemical profile established by 1H NMR spectroscopy of the patient's urine showed the increase of 2-oxoisocaproic acid (4.2 mol/molCrn), 2-hydroxyisovaleric acid (2.8 mol/molCrn), 3-methyl-2-oxovaleric acid (1.7 mol/molCrn), establishing the diagnosis of MSUD. The molecular study done by DNA PCR-amplification and direct sequencing of the coding exons of BCKDHA gene revealed a mutation in the 2nd exon of this gene [c.190A>T (p.Lys64*)] in a homozygous state, leading to the stop of translation. This mutation has not yet been described in the literature. This report will describe the clinical and biochemical features of the patient and will highlight the importance of early confirmation of diagnosis for family counseling. The-biochemical-investigation-was-supported-by-Grant-CNCSUEFISCDI, PNII-ID-PCCE-2011-2-0045. P-168 Spectrum of mutations in prenatal hyperechoic colon and phenotype genotype correlations in cystinuria

Fowler B1, Weykamp CW2 Benoist JF1, Bourillon A2, Royer N2, Tostivint I3 1

2

University Children's Hospital,, Basel, Switzerland; Queen Beatrix Hospital, Winterswijk, Netherlands The ERNDIM aminoacid EQA scheme assesses accuracy, recovery, precision, linearity and inter-laboratory variance (CV) of quantitative measurement of 24 common and various special amino acids relevant to IEMs. Since our report in 2003 (JIMD 2003, 26: Issue 2 suppl. p135) participating laboratories increased from 160 (26 countries) to 241 (48 countries). Methods: used are (2002 values): ion exchange chromatography, 75% of laboratories (84.5%); reverse phase HPLC, 16% (11.7%); GC/MS, 1% (2.6%); others 2% (1.3%); tandem mass spectrometry, 6% (0). Overall performance appears to be satisfactory for 24 commonly measured amino acids with their average CV falling from 13.1% (7.6% for threonine – 36.6% for asparagine) in 2002 to 11.2% (7.3% for threonine – 24.2% for asparagine) in 2012. However for e.g. a consensus alanine value of 397μmol/L 8 laboratories reported values of >3 S.D. above /below the median ( <319 and >475). Inclusion of special amino acids can reveal methodological inadequacies in separation, standardisation or insufficient sensitivity e.g arginino succinic acid, pipecolic acid. Further improvement in performance in amino acid analysis is needed and the availability of performance assessment in an EQA scheme provides a valuable tool to test the validity of metabolite values in multicentre studies of treatment outcome.

1

Reference Centre Metab Dis, Hop R Debré, Paris, France; 2biochemistry, Hop R Debré, Paris, France; 3Nephrology, Hop Pitié Salpétrière, Paris, France Cystinuria is a are rare but important cause of recessive inherited kidney stone disease that affects luminal transport of cystine and dibasic amino acids in kidneys and small intestine. Two genes have been identified in cystinuria: SLC3A1 (genotype A) and SLC7A9 (genotype B). Antenatal hyperechoic colon (HC) is a non-pathological consequence of the intestinal transport defect observed in cystinuria. We determine the genotype of 43 cystinuric patients: 25 antenatal forms of HC and 18 patients with late onset (LO) diagnosed in infancy or adulthood. We analysed the genotype of those two groups. 76% of the patients have an AA genotype in HC group and only 44% in LO. T216M, a severe missense mutation, was very frequent in HC (30% of the alleles) whereas it was not observed in LO. Mutations responsible for a truncated or absent protein represent 60% of the alleles in HC (including 18% of large dup or del involving more than one exon) but only 30% in LO. This study shows a relation between the clinical presentation and the genotype in cystinuria. More studies are needed to confirm that HC occurs only in the most severe forms of cystinuria as suggested by their genotype. st severe forms of cystinuria as suggested by their genotype. P-169

P-167 Clinical and biochemical aspects in a newborn with classical MSUD, and report of a novel mutation in BCKDHA gene 1

2

3

Vulturar R , Nicolescu A , Deleanu C , Häberle J

4

1

Dept Cell and Molec Biol, Univ Med Pharm, Cluj-Napoca, Romania; 'P.Poni' Inst Macrom Chem, Romanian Acad, Iasi, Romania; 3 'Nenitescu' Inst Org Chem, Romanian Acad, Bucharest, Romania; 4 Div Metab, Univ Children Hospital, Zürich, Switzerland

Tyrosinemia type 1 during the first months of life: phenylalanine supplementation should really be considered van Vliet D1, van Dam E1, van Rijn G2, Derks TGJ1, de Boer F1, Venema G1, Lunsing RJ3, Heiner-Fokkema MR4, van Spronsen FJ1 1

Beatrix Child Hosp, Univ Med Center, Groningen, Netherlands; Beatrix Child Hosp, Univ Med Center, Groningen, Netherlands; 3 Dep Child Neurology, Univ Med Center, Groningen, Netherlands; 4 Dep Lab Med, Univ Med Center, Groningen, Netherlands

2

2

Maple Syrup Urine Disease (MSUD) is an IEM caused by the deficiency of branched-chain α-keto acid dehydrogenase complex (BCKDH), leading to the buildup of branched-chain aminoacids and their toxic byproducts causing cerebral damage. The most severe type of MSUD (classical form) accounts for 75% of patients. The disease is not yet included in the national-newborn-screening-program in Romania.

Background: Developmental delay in tyrosinemia type 1 (HT1) patients is observed, not knowing the relation to phenylalanine and tyrosine concentrations. Both hypophenylalaninemia and hypertyrosinemia have been observed. Objective: To investigate the effect of phenylalanine supplementation in two HT1 newborns, diagnosed by neonatal screening.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

S159

Methods: We compared two newborns with both Nitisinone: 1 mg/kg/day, natural protein intake: 0.5-1.0 g/kg/day, and total protein intake: 2.0-2.5 g/kg/day. The later child received phenylalanine supplementation (20 mg/kg/day). Results: In our first patient, we observed decreased growth and neurological problems, that later improved on phenylalanine supplementation. In the second child, we observed no clinical problems. Without supplementation, blood phenylalanine concentrations <30 μmol/l and <20 μmol/l were observed in 71.4% of measurements, while tyrosine remained <400 μmol/l and <600 μmol/l in 85.7% and 100% of measurements. On phenylalanine supplementation, these figures were 10.3% (<30 μmol/l) and 5.1% (<20 μmol/l) for phenylalanine, and 35.9% (<400 μmol/l) and 87.5% (<600 μmol/l) for tyrosine. Conclusion: Phenylalanine supplementation in HT1 newborns could prevent hypophenylalaninemia, but may cause hypertyrosinemia. The optimal phenylalanine supplementation regimen for HT1 newborns requires further investigation, but our cases suggest that phenylalanine supplementation could be clinically relevant. Conflict of Interest declared.

Case Report: The female patient born from healthy parents who were consanguineous, presented with delayed motor development, failure to thrive and hepatosplenomegaly at the age of 9 years. Laboratory studies revealed tyrosine concentration being mildly elevated. Diagnosis of tyrosinemia type I was made based on the findings of increased urinary excretion of succinylacetone. Alpha-1-fetoprotein concentration in plasma was increased (484000 ng/ml). Magnetic resonance imaging of upper abdomen showed multinodular lesions one of which is compatible with HCC in liver. Liver transplantation was not indicated because of the multiple lung metastases. Conclusion: We present a patient with tyrosinemia type I who developed HCC. Nitisinone (NTBC) treatment may prevent liver failure and may prolong life both in early and late presenting patients and may decrease the risk of development of HCC when compared to dietary treatment.

P-170

Ramos FJ1, Casado M1, Molero M1, Muchart J2, Rebollo M2, Castejon E1, Meavilla S1, Ormazabal A1, Rodriguez-Pombo P3, Artuch R1, Garcia-Cazorla A1

A unique presentation of skin findings in tyrosinemia type II Strenk ME1, Heese BA1, Atherton AM1, Smith LD1

P-172 Neurotransmission pathways in neonatal MSUD: a pathophysiological approach

1

Div Neurol and Metab,Univ SJD Hosp, Barcelona, Spain; 2Rad Dept,Univ SJD Hosp, barcelona, Spain; 3Mol Biol,Univ Aut, Madrid, Spain

1

Children's Mercy Hospitals and Clinics, Kansas City, United States

Tyrosinemia type II (Oculocutaneous tyrosinemia) is a rare inborn error of metabolism caused by tyrosine aminotransferase deficiency. Onset of symptoms is variable with early involvement of the eyes (corneal ulcerations, photophobia) and later involvement of the skin, particularly on the soles and palms. Skin symptoms generally occur after one year of age, and generally include nonpruritic, hyperkeratotic papules and plaques. Neurologic and/or intellectual disability can also be present. We report on a case of a male infant with tyrosinemia type II with early onset ocular and cutaneous findings. He was identified on expanded newborn screening with elevated tyrosine and subsequently persistent elevation of plasma tyrosine at one month of age. Molecular sequencing of the TAT gene identified a homozygous nonsense mutation c.169C>T (p.Arg57X) that has been previously reported. Despite initiation of treatment before 2 months of life with Tyrex-1 formula resulting in plasma tyrosine levels consistently less than 700 μmol/l, the patient exhibitied skin findings including the reported rash typical for Tyrosinemia type II. He also exhibited an atypical rash on his upper back that resulted in ulcerated lesions (urticaria pigmentosa) which, when irritated, resulted in an anaphylactoid reaction. This constellation of findings represents a unique presentation of the disorder.

Maple Syrup Urine Disease (MSUD) is an autosomal-recessive disorder caused by defects in the branched-chain alpha-ketoacid dehydrogenase complex. Bizarre boxing/pedaling-like movements and depressed consciousness are common in newborns. The mechanisms underlying these symptoms are poorly understood. We describe in 4 MSUD patients, the clinical, biochemical (including CSF neurotransmitters) and brain image data during the acute neonatal onset. They presented between 9 and 12 days with poor feeding, hypoactivity and progressive lethargy . Global hypokinesia associated with high amplitude slow movements of the extremities were present in all of them. Brain MRI showed diffuse cerebral edema and symmetric restricted diffusion in bilateral cerebellar white matter, dorsal brainstem, basal ganglia (3/4 thalamus, 2/4 globus pallidus), posterior limbs of internal capsules, and corona radiata. Leucine plasma levels ranged 1600 to 2700 μmol/L (Normal < 300). CSF GABA concentration tended to be in the lower range being notably low in 2/4 patients, whereas biogenic amine levels were normal. GABA and leucine values inversely correlated. Leucine is involved in GABA metabolism, which is abundant in the cortex and the basal ganglia, and has excitatory functions in the newborn. Our findings would suggest a role of GABA depletion in the pathophysiology of hypokinetic-bradykinetic-bizarre movements-lethargy complex.

P-171

P-173

Hepatocellular carcinoma in hereditary tyrosinemia, a case report

3-Methylcrotonyl-CoA carboxylase (3-MCC) deficiency detected by newborn screening: does early diagnosis influence phenotype?

Kagnici M1, Kose M1, Canda E1, Karakoyun M2, Kalkan Ucar S1, Ozgenc F2, Coker M1

Baronio F1, Baumgartner M2, Bettocchi I1, Righetti F1, Zazzetta E1, Cantasano A1, Pession A1, Cassio A1

1

Div Metab Dis,Ege Univ Child Hosp, Izmir, Turkey; 2Div Gastrol,Ege Univ Child Hosp, Izmir, Turkey Background: Hereditary tyrosinemia type I is an autosomal recessive inborn error of tyrosine catabolism caused by a deficiency of the enzyme fumarylacetoacetase that results in liver failure, hepatocellular carcinoma (HCC), renal tubular dysfunction and acute intermittent porphyria. Dietary treatment,results in a poor outcome and does not prevent the development of liver failure and HCC in all patients.

1

Neonat Screen, Ped Endo Program, Univ, Bologna, Italy; 2Div Metab, Univ Child Hosp, Zurich, Switzerland 3-Methylcrotonyl-CoA carboxylase (3-MCC) deficiency is an inborn error of metabolism in the catabolism of the amino acid leucine, with an extremely variable phenotype, ranging from severe to asymptomatic cases. We report two patients of Arab origin detected by tandem-massspectrometry newborn screening (NBS) in our centre.

S160 Our cases showed elevated levels of C5OH, reduced free carnitine levels and elevated urinary 3-methylcrotonylglycine (3-MCG) and 3hydroxyisovaleric acid (3-HIVA ). Treatment with L-carnitine and biotin was started early, with normal protein diet for age. They remained asymptomatic during follow up (16 and 10 months), with regular growth pattern and neurological development; excretion of 3-HIVA (>1000 mM/M creatinine ) and 3-MCG (>500 mM/M creatinine) remained massively elevated. Molecular analysis of MCCC1 and MCCC2 genes revealed a homozygous novel missense mutation on MCCC1 in both patients (c.1147G>A [p.Glu383Lys] and c.916G>A [p.Ala306Thr], respectively). Both cases are still asymptomatic despite the probably very low enzymatic residual activity. Our cases confirm the difficulty to correlate clinical outcome with biochemical phenotype in patients detected by NBS. In our patients, biotin does not seem to be effective on reducing acid excretion. Propstective studies are needed to determine whether early diagnosis and Lcarnitine treatment contribute to positive outcome in such patients.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 iminodipeptiduria. It can present at any age but it's rare in very small patients. We report a case of prolidase deficiency with a very early presentation. Case report: First daughter of consanguineous parents born after an uneventful pregnancy. From the first weeks of life she shows anemia and thrombocytopenia. At the age of 6 months splenomegaly, dysmorphic features and psychomotor retardation were detected. Extensive autoimmune, metabolic and genetic studies were performed. At the age of 15 months brownish papules appear in lower limbs, some of them with ulcerations. Prolidase deficiency is suspected and metabolic screening repeated: huge ammounts of glycine, proline and other aminoacids in urine acid hydrolysis pointed to iminodipeptiduria. Enzymatic activity was performed with positive results: prolidase activity in blood (1.9 μmol Pro/min/g Hb – controls 25.9 μmol Pro/min/g Hb) and fibroblasts (1.49 μmol Pro/h/ml/mg – controls 26.46 μmol Pro/min/g Hb). Conclusion: Prolidase deficiency should be ruled out in patients with splenomegaly from the first months of life.

P-174 P-176 Mutation analysis of SLC25A13 in Vietnamese patients: high frequency of mutation c.851del4

Rapid plasma amino acid analysis by UHPLC

Nguyen MH1, Nguyen PM1, NGO DN1, Nguyen PAH2, Nguyen TL1

Baggot M1, Ghansah N1, Prunty H1, Heales S1

1

1

Background: Neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) due to mutations in the SLC25A13 gene can present with transient intrahepatic cholestasis, low birth weight, growth retardation, hypoproteinemia, prolong jaundice, chronic liver disease, etc.. Objective: This study aimed to identify the frequency of the 4 SLC25A13 mutations c.851del4, IVS6+5G>A, c.1638ins23, and IVS16ins3kb in NICCD patients. Materials and Methods: 293 peripheral blood samples were collected for this study. PCR assays were used to performed to screen for the mutations, and the results were confirmed by direct sequencing. Results: c.851del4 was the major mutation, accounting for 18.77% and only found in homozygotes, followed by c.1638ins23, IVS16ins3kb, and IVS6+5G>A accounting for 0.68%. In 293 patients, 60 patients had identified mutations, including 31 patients who were c.851del4 homozygotes, 24 patients who were 851del4 heterozygotes, 1 patient who was heterozygous for the single mutation c.1638ins23, and 1 patients who was compound heterozygous for c.1638ins23 and c.851del4. 2 patients were compound heterozygous for [c.851del4+IVS16ins3kb], 2 patients were compound heterozygous for [c.851del4+ IVS6+5G>A. Conclusions: c.851del4 is the most common mutation in Vietnamese patients with NICCD.

Cation exchange chromatography with post-column ninhydrin derivatisation remains the gold standard for quantitative plasma amino acid analysis in the UK. However, the long run time of around three hours per sample means that this method is unsuitable for high throughput analysis. For this reason, in our laboratory we also utilise a reverse phase HPLC method with prior phenylisothiocyanate (PITC) derivatisation and UV detection (Waters Pico-Tag® method), which allows higher throughput, but has some limitations (eg. incomplete resolution of argininosuccinic acid and citrulline from surrounding peaks) and analysis time is still around 90 minutes per sample. We have adapted this method to develop a rapid ultra high performance liquid chromatography (UHPLC) method, using the Agilent 1290 infinity UHPLC system and Poroshell EC-C18 2.7 μm 2.1x150mm column, bringing the run time down to 25 minutes per sample. Validation work is still ongoing but preliminary results show equal or improved separation and resolution of all 21 amino acids reported by the Pico-Tag® method and additionally, complete resolution of argininosuccinic acid and citrulline. With ever-increasing workloads and demand for higher throughput, this method represents a simple and viable alternative/ addition to cation exchange chromatography for plasma amino acid anlaysis.

P-175

P-177

Prolidase deficiency: neonatal presentation

Measurement of homogentisic acid, tyrosine and nitisinone in urine and serum by LCMS and subsequent changes after treatment of alkaptonuria patients with nitisinone.

Hum Gen Dept, Vietnam Hosp of Pedia, Hanoi, Viet Nam; 2Hep Dept, Vietnam Hosp of Pedia, Hanoi, Viet Nam

Dapena JL1, Del Toro M2, García-Patos V3, Forlino A4, Domínguez C5, Corral N6, Riudor E6, Arranz JA6

Chemical Pathology, Gt Ormond St Hosp, London, United Kingdom

Ranganath L1, Roberts NB1, Curtis S1, Dutton J1 1

Pediatr Haematology, Hosp Vall d'Hebron, Barcelona, Spain; 2Pediatr Neurology, Hosp Vall d'Hebron, Barcelona, Spain; 3Pediatr Dermatology, Hosp Vall d'Hebron, Barcelona, Spain; 4Molec Genet Dep. Universita de Pavia, Pavia, Italy; 5CIBBIM Nanomed, Hosp Vall d'Hebron, Barcelona, Spain; 6Metabolic Lab, Hosp Vall d'Hebron, Barcelona, Spain Background: Prolidase deficiency (OMIM 170100) is a rare autosomal recessive disorder caused by mutations in PEPD gene with a wide spectrum of clinical features that include: characteristic facies, cognitive impairment, skin lesions, splenomegaly, recurrent infections and

1 Dept Clin Biochem, Royal Liverpool Hosp, Liverpool, United Kingdom

Background: In patients with alkaptonuria(AKU) it is important to measure homogentisic acid (HGA) and in treatment with nitisinone HGA and tyrosine production. An LCMS method has been established for measurement of urine and serum HGA, tyrosine and nitisinone(NTBC). Methods: Urine was diluted 1/1000 and deproteinised serum 1/100 in 0.06% formic acid containing deuterated internal standard(s) and 50 μl

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 injected into an LCMS Waters Acquity chromatography system with a Fortis H2O 3μm, I.D. 100 x 2.1 mm column and Waters Quatro Premier XE with MassLynx and QuanLynx packages. Mass detection was by electrospray ionization (ESI) tandem mass spectrometry in the negative ion mode with transitions: HGA167 > 123 167 > 108, 13C6-HGA173.1 > 129.1, TYR 180.1 > 163 180.1 > 119.2, D2-TYR 182.2>165.1182.2>119.1 and NTBC 328.2 > 281.4. Depending on urine or serum, calibrations were carried out between 10 – 5000 for HGA, tyrosine 10- 1000 and nitisinone 0-10 μmol/L. Results: In 21 AKU patients treated with nitisinone reduction in HGA urine(100 fold) and serum (10 fold) concomitant with 10 fold increases in serum tyrosine were observed. Increases in serum NTBC were seen but none in urine. Conclusions: The combined LCMS procedure is effective in routine monitoring of patients with AKU.

S161 A 11-year-old female with the complaints of bilateral palmoplantar hyperkeratosis was referred to our clinic. When she was 6 months, bilateral herpetiform lesions were detected during ocular examination due to complaints of pain and tearing in her eyes. Around the age of 1 year painful lesions of hands and feet had started. The patient who did not benefit from the treatments at dermatology clinic visits applied to our clinic for further examination. On physical examination of the patient; her growth and developement were appropriate for her age, there wasn't hepatosplenomegaly. There was severe palmoplantar hyperkeratotic skin lesions. Liver and kidney function tests were normal. Tyrosine levels were high in quantitative analysis of blood and urine amino acids, other amino acid levels were in normal range. Deposition of tyrosine was detected in the eye examination P-180

P-178 A hyperprolinemia type-2 case with a novel deletion on ALDH4A1 The first case of Turkish origin with neonatal intrahepatic cholestasis caused by citrin deficiency

Hişmi B1, Yiğit A2, Konuşkan B3, Tekşam Ö2, Tokatlı A1, Sivri S1, Dursun A1, Coskun T1

Okur I1, Gunduz M1, Ceylaner S2, Koc N1, Ozaydin E1, Kirsaclioglu C3 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Dep Pediatr, Hacettepe Univ Child Hosp, Ankara, Turkey; 3Div Ped Neurol, Hacettepe Univ Child Hosp, Ankara, Turkey

1 1

Div Ped Metab, Ankara Diskapi Child Hosp, Ankara, Turkey; 2 Intergen Gen Center, Ankara, Turkey; 3Div Ped Gastro,Ankara Diskapi Child Hosp, Ankara, Turkey Citrin deficiency resulted from mutations in the SLC25A13 gene is an autosomal ressesive inborn error of metabolism and it has two different age-dependent clinical phenotypes: Adult-onset type 2 citrullinemia (CTLN2) and neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD). Until recently, citrin deficiency was thought to be restricted to Japan; the finding of citrin mutations in patients of Arabic, Pakistani, French Canadian and Northern European origins supports the concept that citrin deficiency is a panethnic disease. To date, Turkish patient with citrin deficiency hasn't been reported in the literature. We presented 2 months-old boy of Turk origin who was diagnosed NICCD. Case report: The boy patient, was single child born from healthy parents who were consanguineous. He was admitted to our clinic with prolonged jaundice at 6 weeks of age. In laboratory examinations performed due to cholestasis, the aminoacid analysis in plasma and dried blood sample showed markedly high citrulline and threonine levels. He was found to carry the c.851854delGTAT mutation in SLC25A13 gene, which was reported previously in literature. Conclusion: This case was presented because he was first case of Turkish origin with NICCD. NICCD has become an additional differential diagnosis in patients with neonatal cholestasis. P-179 Tyrosinemia type 2: a case report Kiykim E1, Aktuglu Zeybek AC1, Cansever M1, Tekin B2, Lacinel S1, Dorkel Cetin I1, Aydin A1 Cerrahpaşa Med Faculty, Div Metab Dis, İstanbul, Turkey; 2Marmara Med Faculty, Dermatology Dep, İstanbul, Turkey

1

Richner-Hanhart syndrome is caused by mutations in the TAT gene . This disorder characterized by photophobia, red eye, painful palmoplantar hyperkeratosis. The diagnosis of the disease is made by the detection of high blood and urine levels of tyrosine. It may be especially confused with tyrosinemia type III so that enzymatic and/or genetic analysis should be performed.

Hyperprolinemia type II (HP II) is a rare inherited metabolic disease due to the deficiency of pyroline-5-carboxylate dehydrogenase and it is one of the four pyridoxine responsive childhood epileptic disorders. It is generally believed to be a benign condition although some patients have neurological problems such as refractory seizures. Here we report a nine-month-old girl with HP II who admitted to our hospital with recurrent mild seizures and normal developmental milestones. She was the second child of first degree cousin parents and she had normal neurological examination and cranial computed tomography findings. Urine and blood amino acid analysis revealed elevated levels of proline, glycine, and ornithine in serum and pyrroline-5-carboxylate and hydroxyproline in urine. When the ALDH4A1 gene was sequenced, a novel 11 bp deletion was found in exon 5. P-181 Epidemiological study of aminoacidopathies in Morocco: experience of biochemistry laboratory of University Hospital of Rabat, Morocco Dahri S1, Talbaoui H1, Benhammou B1, Kriouile Y1, Agadr O1, Meskini T1, Ouadghiri FZ1, Chabraoui L1 1

University Hospital of Rabat, Rabat, Morocco

Inborn errors of amino-acids metabolism seem to be common in the Middle East and North Africa. In our laboratory, diagnosis of aminoacidopathies (AA) was initiated since 1996 using TLC method followed by ion exchange determination or other specific assays. The laboratory undertook 9300 analysis for patients with symptoms suggestive of AA, covering mainly the region of Rabat. Our study has revealed 246 patients affected by AA with 10 different disorders. The most frequent were Phenylketonuria (115 patients), Tyrosinemia type I (63 patients) and Homocystinuria (38 patients). Only 10 MSUD patients were diagnosed representing a low rate compared to other Arabic countries. The age ranged from 24 hours to 20 years with a mean age at diagnosis of 6 years. Consanguinity was observed in 76 % of patients. The main clinical symptoms were mental retardation, hypotonia, hepatomegaly, seizures, failure to feed and coma. The incidence was estimated to be 1/12228 for PKU, 1/16853 for Tyrosinemia I. Aminoacidopathies seems to be highly frequent in Morocco. The AA profile showed a distinctive distribution compared to Mediterranean

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and Arabic countries. PKU was the most predominant amino-acid metabolism disorder followed by Tyrosinemia I. Thus, establishment of systematic neonatal screening is urgent for PKU in our country. P-182

Discussion: Although there was no complaint, atrophy of the left kidney was found in her mother. In all three children in this family in which there was no consanguinity, mutations were detected. Conclusion: Low uric acid should not be missed in the renal patients. In XDH, family of the index case should be screened even if there is no complaint.

Hereditary tyrosinemia type I: a severe disease with variable expression

P-184

Chioukh FZ1, Sakka R1, Ben Ameur K1, Ben Hamida H1, Kaabachi N2, Harbi A3, Monastiri K4

Effect of hypoxanthine, antioxidants and allopurinol on acetylcholinesterase and butyrylcholinesterase activities in rats

1

Wamser MN1, Ferreira VV1, Leite EF1, de Lima DD2, da Cruz JGP1, Wyse ATS3, Dal Magro DD1

NICU Teaching Hospital of monastir, Monastir, Tunisia; 2Departement of Biochemistry LaRabta, Tun, Tunis, Tunisia; 3Department of Pediatrics, CHU Sahloul, Sousse, Tunisia; 4Faculty of Medicine of Monastir, Monastir, Tunisia Background: Hereditary Tyrosinemia Type I is a severe metabolic disease with variable age of expression. The neonatal form is revealed by hepatocellular failure in the first 2 months but never before day 8 of life. subacute and chronic forms are frequent presenting with psychomotor delay, seizures, hepatomegaly, renal and bleeding disorders . Case reports: 4 childrens: 2 brothers, their sister and their cousin. They were the offspring of consanguinous marriages. The index case presented with umbilical bleeding in age 17 days, his sister died at 5 months with hepatomegaly and hypertrophic cardiomyopathy while the brother died at 4 years and half with hepatomas although he had NTBC late in his life. The cousin died at 6 months; he probably had hypertrophic cardiomyopathy. Biological tests: Prothrombin time < 12%; Cephalin Kaolin time no clotting; mild elevation of hepatic enzymes. The diagnosis of Tyrosinemia Type I was confirmed by abnormal presence of Succinyl acetone and Delta-amino-levulinelic acid in urine samples. He had NTBC and special formula feeding. Hepatic tests normalized by 4 weeks of treatment. Conclusions: These cases show how heterogenous the clinical expression of Tyrosinemia Type I may be, even in the same family.This may delay the diagnosis and the treatment in such patients. 05. Purine and pyrimidine disorders P-183 Novel mutation of a Turkish family with xanthine dehydrogenase deficiency: c.2224C>T homozygote mother, 2224C>T ; 3853C>T compound heterozygote daughters

1 FURB, Blumenau, Brazil; 2UNIVILLE, Joinville, Brazil; 3UFRGS, Porto Alegre, Brazil

Background: In the present study we investigate the in vitro effect of hypoxanthine on acetylcholinesterase and butyrylcholinesterase activities in the hippocampus, striatum, cerebral cortex and serum of 15, 30 and 60 day-old-rats. The influence of antioxidants, namely trolox and ascorbic acid and allopurinol were also evaluated. Methods: Acetylcholinesterase and butyrylcholinesterase activities were determined according to Ellman et al. (1961). Hypoxanthine was added to the incubation medium. Results: Hypoxanthine (10.0 μM ) enhanced acetylcholinesterase activity in hippocampus and striatum of 15 and 30-day-old rats and reduced butyrylcholinesterase in serum of 60-day-old rats. Allopurinol and/or antioxidants partially prevented the alterations in these enzymes. Conclusion: Our findings shown that hypoxanthine alters cholinesterase activities, probably through free radicals and uric acid production, since the alterations were prevented by the administration of allopurinol and antioxidants. It is presumed that the cholinesterase system may be associated, at least in part, with the neuronal dysfunction observed in patients affected by Lesch–Nyhan disease and although extrapolation of findings from animal experiments to humans is difficult, it is conceivable that these vitamins and allopurinol might serve as an adjuvant therapy to avoid progression of brain damage in patients affected by this disease. P-185 Identification of a novel synonymous mutation in the human βureidopropionase gene UPB1 affecting pre-mRNA splicing

Ozer I1, Candan C2, Turhan P2, Ichida K3 Nakajima Y1, Meijer J2, Zhang C3, Meinsma R2, Ito T1, Van Kuilenburg A2 1

2

Div Metab Dis, Goztepe Trai Res Hosp, Istanbul, Turkey; Div Ped Neph, Goztepe Trai Res Hosp, Istanbul, Turkey; 3Dept Internal Med, Jikei Univ Med Facult, Tokyo, Japan Introduction: Isolated xanthine dehydrogenase deficiency (type I XDH) is an autosomal recessive disorder that is characterized by decrease of uric acid in urine and blood. It can lead to renal failure. It is treatable disease with allopurinol. Case: When she was 8,5 year-old, she was admitted to the pediatric nephrology department with flank pain, nausea, vomiting, headache, and fatigue. In the patient, hypertension, atrophic right kidney, compensatory hypertrophy of the left kidney, pulmonary congestion, dilated cardiomyopathy was detected. Despite specific treatment, when she was 17 years old, peritoneal dialysis was started. She was consulted by a child metabolism specialist because of her low serum uric acid levels. In the patient, her mother and her sisters, low uric acid, high xanthine and hypoxanthine in the plasma, low uric acid, uracil, high xanthine and normal hypoxanthine in the urine were found. Molecular diagnosis was performed for XDH.

1

Div Pediatrics, Nagoya City Univ Hosp, Nagoya, Japan; 2Lab Gen Metab Dis, Academic Med Center, Amsterdam, Netherlands; 3Res and Dev, MILS International, Kanazawa, Japan Objectives: β-Ureidopropionase deficiency is an autosomal recessive condition caused by mutation in the UPB1 gene. We report the clinical, biochemical and molecular analysis of a newly identified patient with β-ureidopropionase deficiency. Patient and Method: Patient is a 17-month-old girl from Chinese non-consanguineous parents who presented with motordevelopmental delay. The concentrations of pyrimidine degradation metabolites in urine were determined by HPLC-MS/MS. Exons 1-10 of UPB1 and their flanking sequences were amplified. To investigate the function of mutant β-ureidopropionase, the mutations were introduced into pcDNA3.1-βUP and expressed in HEK293. Result: Urine analysis showed the characteristic pattern for βureidopropionase deficiency. Molecular analysis of UPB1 identified a

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 novel synonymous mutation c.93C>T (p.G31G) in exon 1 and a mutation c.977G>A (p.R326Q) in exon 9. The R326Q mutation yielded mutant proteins with a 0.71% of wild-type activity. A UPB1 minigene containing the entire exon1, intron1 and exon2 of human UPB1 gene was created. Introduction of the c.93C>T into the UPB1 minigene followed by transfection of HEK293 cells resulted in a shortened PCR fragment compared to that observed for the wild-type UPB1 minigene. Conclusion: Mutation R326Q yielded a markedly decreased βureidopropionase activity. Furthermore, we have identified the first synonymous mutation in the UPB1 gene affecting pre-mRNA splicing.

S163

Dihydropyrimidine dehydrogenase deficiency in two malaysian siblings

renal colic and nephrolithiasis. Stone analysis suggested APRT deficiency. He failed to attend outpatient services for follow up, then re-presented 11 years later with chronic kidney disease stage 4 and an atrophic left kidney. Urine, blood and DNA samples from the patient and family members were analysed for 2,8-DHA, APRT enzyme activity and mutation analysis of the APRT gene. Results: A novel homozygous variant genotype APRTc.543 A>T, p.181X>C segregated with APRT deficiency and the presence of 2,8DHA in the urine of four family members. This mutation abolishes the authentic protein stop codon resulting in an elongated protein and is consistent with APRT deficiency. Discussion: APRT deficiency remains an under-recognised but preventable cause of kidney failure. We suggest that this defect of purine metabolism should be considered in patients with recurrent renal stones.

Chen BC1, Rowani MR2, Meinsma R3, Meijer J3, van Kuilenburg ABP3

P-188

1

Genetics Dept, Kuala Lumpur Hospital, Kuala Lumpur, Malaysia; School of Medical, Uni Science Malaysia, Kelantan, Malaysia; 3Dept Pead, AMC, Univ Amstherdam, Amsterdam, Netherlands

Lead poisoning mimicking pyrimidine-5-nucleotidase deficiency

Dihydropyrimidine dehydrogenase (DPD) deficiency is an autosomal recessive pyrimidine metabolism disorder. DPD is the rate-limiting enzyme in pyrimidine catabolism and converts the pyrimidine bases thymine and uracil to dihydrothymine and dihydrouracil, respectively. A variable clinical phenotype has been described in paediatric patients ranging from asymptomatic to severe neurological manifestations including seizures, developmental delays, mental retardation, microcephaly, autistic and eye abnormalities. Most of the DPD patients are only incidentally detected when their urine showed persistently elevation of uracil and thymine in metabolic screening. Here, we report a pair of Malaysian siblings; a girl and her brother of 10 and 6 years old presented with developmental delay, mental retardation, microcephaly and hypotonia. Urine sample analysed by GCMS revealed severe thymine-uraciluria. Purine and pyrimidine analysis by HPLC detected the present of thymine and uracil ( 369 and 432 mmol/mol creatinine) further confirmed the diagnosis. Both patients were homozygous for mutation c.1651G>A (pAla551Thr) in the DPD gene. Analysis of the DPD crystal structure suggested that the A551T point mutation might prevent the binding of the prosthetic group FMN and affect the folding of the DPD protein. These two siblings appeared to be the third and fourth Malaysian children identified with DPD deficiency so far.

1 Purine Lab, GSTS Path, St Thomas' Hosp, London, United Kingdom; 2Clinical Toxicology, St Thomas' Hosp, London, United Kingdom

P-186

2

Fairbanks LD1, Dargan PI2, Marinaki A1, Escuredo E1

Introduction: Pyrimidine-5'-nucleotidase (P5N-I) deficiency results in a non-spherocytic anaemia with basophilic stippling and accumulation of pyrimidine nucleotides in erythrocytes. Acquired P5N-1 deficiency has been described in lead poisoning. Case Report: A 24 year old female presented with a two week history of nausea and vomiting, constipation, generalised abdominal and lower leg pain, lethargy, irritability and headaches. Laboratory investigations revealed that she was anaemic with Hb 7.3g/dl. A blood lead concentration and P5N-1 activity were also requested: blood lead was 3.19 μmol/L, P5N-1 activity was 3 nmol/h/mg Hb (control range 9-20). Pyrimidine nucleotides were present at low levels in the red cell nucleotide profile (normally absent). The source of lead poisoning was Ayurvedic medicines. The patient's symptoms have settled with removal from the source and over the last two years the patient's blood lead concentration has been 1.3 to 2.7 μmol/L and her anaemia has resolved. Conclusion: Lead poisoning should be considered in patients presenting with anaemia and P5N-1 deficiency. P-189

P-187 APRT deficiency in an Asian family Fairbanks LD1, Balasubramaniam G2, Almond MK2, Escuredo E1, Marinaki A1, Arenas MA1

A novel mutation in a Turkish case with molybdenum cofactor deficiency Kose M1, Canda E1, Kagnıcı M1, Kalkan Uçar S2, Ichida K3, Çoker M1

1

Purine Lab, GSTS Path, St Thomas' Hosp, London, United Kingdom; 2 Renal Unit, Southend University Hospital, Essex, United Kingdom

1 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis, Ege Univ Child Hosp, İzmir, Turkey; 3Div Pathophysiology, Univ Tokyo, Tokyo, Japan

Background: In adenine phosphoribosyltransferase (APRT) deficiency, adenine follows an alternate metabolic route and is oxidised by xanthine dehydrogenase to form 2,8-dihydroxyadenine (2,8-DHA) which is extremely insoluble. Treatment with allopurinol, a xanthine dehydrogenase inhibitor, can prevent the formation of 2,8-DHA. Patients and Methods: An 18 year old man of Pakistani origin presented to the urological service at Southend University Hospital with a history of

Molybdenum cofactor deficiency (MoCD) is a rare progressive neurodegenerative disorder with about 100 cases reported worldwide.The major clinical symptoms are intractable neonatal seizures,progressive encephalopathy, facial dysmorphic features. Most of the patients are misdiagnosed as hypoxic ischemic encephalopathy. Here we report an infant with MoCD with a novel mutation in MOCS1 gene. The patient, born to consanguineous Turkish parents,was admitted at the age of 10 months. He had mental,motor retardation, dystonic movements, spastisity.First convulsion was at the age of 8 months.Serum uric

S164 acid level was 0.7 mg/dl.There was increased urinary excretion of sulphocysteine and hypoxanthine. These laboratory findings suggested the diagnosis of MoCD.DNA analysis of the genes related with molybdenum cofactor synthesis was performed for the patient and his parents. Molecular genetic investigation revealed homozygously a novel mutation, c.949C > T in the MOCS1 gene from the infant.It was identified heterozygously in both of parents.Our patient had no typical MRI findings and dysmorphic features of MoCD. This disorder should be considered in the differential diagnosis of neonatal seizures. Blood uric acid measurement should be included in the battery of tests to be performed in all refractory neonatal seizures. We report this patient to emphasize the importance of early diagnosis.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Patients and methods: Our NMR method of L/D-enantiomeric distinction was applied to urine samples from three different patients, of age 1, 5, and 28 years, treated at Haceteppe University, Ankara, Turkey. D-2- and L-2-hydroxyglutaric acid were investigated in two different urine samples which were then treated with the samarium complex LSR. In all three cases, the L-enantiomer was found, confirming a diagnosis of L-2-HGA. Conclusions: NMR investigations for enantiomeric distinction between L-2- and D-2-hydroxyglutaric acid can now be routinely applied in the differential diagnosis of the L- and D-form of 2-hydroxyglutaric aciduria. P-192

P-190 Lesch-Nyhan syndrome: a case complicated by recurrent xanthine-hypoxanthine stones due to allopurinol

Use of carglumic acid in treatment of hyperammonemia during metabolic decompensation of patients with propionic acidaemia Abacan M1, Boneh A1

Hişmi B1, Ünal Ö1, Sivri S1, Dursun A1, Tokatlı A1, Coskun T1 1

Metab Genetics, VCGS, MCRI, RCH, Melbourne, Australia

1

Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey

06. Organic acidurias

Background: Propionic acidaemia (PA) is an organic acidaemia due to the absence of Propionyl-CoA carboxylase. During metabolic decompensation, the accumulation of propionyl-CoA causes hyperammonemia due to the inactivation of N-acetylglutamate synthetase. It is recommended to lower ammonia concentrations as fast as possible because of its neurologic toxicity. Cargulmic acid, a structural analogue of N-acetylglutamate, has been shown to decrease plasma ammonia levels in NAGS deficiency and CPS deficiency by replenishing the deficient carbamyl-phosphate and promoting ureagenesis. We aimed to assess the effect of Carglumic acid on hyperammonaemia in patients with PA during decompensation. Methods: Carglumic acid (Carbaglu® 70-100mg/kg/day in 2-4 divided doses) was given to patients with PA (n=3; all male) during episodes of metabolic decompensation (n=8, between the neonatal period to 4 years of age). Patients were treated with high energy and no- or low-protein enteral formulae and with carnitine. Plasma ammonia concentrations were regularly measured and recorded. Results: Treatment with Carbaglu was effective in significantly reducing plasma ammonia concentrations within two hours and ammonia concentrations dropped to <100mmol/L within 6-19 hours. Conclusion: Carglumic acid is an effective treatment of hyperammonemia in PA during episodes of metabolic crisis in conjunction with proper energy and protein intake and carnitine supplementation.

P-191

P-193

Differential diagnosis: distinction between L-2- and D-2-hydroxyglutaric acid

Clinical, laboratory data and outcome of four patients, affected by beta- ketothiolase deficiency 10 years follow up

Aygen S1, Dürr U1, Coskun T2, Dursun A2

Zaman T1, Goyens P2, Moarefian S3, Moradian R3

1

1 1 IEM Department, Children HospitalMedic, Tehran, Iran, Islamic Republic of; 2Pediatric Lab,Fabiola Hospital,ULB, Brussels, Belgium; 32 Research Unit,Iranian National Society, Tehran, Iran, Islamic Republic of

Congenital deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) results in a spectrum of clinical phenotypes.The most severe phenotype is known as Lesch-Nyhan disease, in which the phenotype also includes severe motor handicap, intellectual disability, and self-injurious behavior. It is reported that long-term use of higher doses of allopurinol may rarely cause xanthine-hypoxanthine stones.Here, we aim to emphasize on this rare treatment complication.A one-year old male patient was assessed for developmental delay,dystonia and choreoathetotic movements,hyperuricemia and nephrocalcinosis were evident.Allopurinol (10/kg/day) was started, then he was referred to our hospital. A novel hemizygote deletion at exon 1 and 2 of the gene HPRT1 was detected. At 9 months, an acute renal failure developed secondary to obstructive uropathy, a numerous calculi in both ureters and bladder, expansion in the collecting ducts were detected. Nephrostomy catheters were placed bilaterally.During the two-year follow-up period, a total of 13 urological procedures were needed for recurrent stones and which were xanthine/hypoxanthine stones when analyzed.When he was 3.5 year old, the plasma and urine uric acid levels were very low and xanthine/hypoxanthine levels were increased by 5-10 times and so allopurinol was discontinued.Then the patient was free of renal stones for the next 16 months.

Inst. f biomedical analysis NMR imaging, Cologne, Germany; Haceteppe University, Ankara, Turkey

2

Background: D-2- and L-2-hydroxyglutaric aciduria are rare clinically variable neurological forms of 2-hydroxyglutaric aciduria characterized biochemically by increased concentrations of D-2- or L-2hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid samples. Different enantiomeric forms of 2-hydroxyglutaric acid are related to different diseases. L-2-hydroxyglutaric aciduria (L-2-HGA) is related to the L-enantiomer of 2-hydroxyglutaric acid, while the less common D2-hydroxyglutaric aciduria (D-2-HGA) is related to the D-enantiomer. The prevalence of this disorder is not known, only 80 cases have been reported to date.

Background: Mitochondrial acetoacetyl-coA thiolase(T2)deficiency or beta ketothiolase deficiency is a rare inherited disease affecting isoleucine and ketone body catabolism, characterized by recurrent ketoacidotic crises and can result in neurological impairment and even death during crises. It is a result of mutations of both alleles of the ACAT gene. Methods: A retrospective study of four patients from 2002-2012. Results: Females 3, consanguinity 3/4, age at onset 7- 36months, age at referral&diagnosis 9 months-13 years, recurrent ketoacidotic attacks 4/4,

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

S165

convulsions followed by psychomotor regression 1/4. All had metabolic acidosis and ketonuria. Salicylate level measured by colourimetric method revealed detectable to therapeutic salicylate concentration (acetoacetate causes interference in salicylate assay.) 3/4. Acylcarnitine profile (MS/MS) revealed elevation of C5OH (3-hydroxyisovaleryl carnitine) 0.800-4.24 mean; 2.645 ( RI<0.800) & C5:1(tiglyl glycine) 0.200-10.66, mean; 3.45 μM/L,(RI< 0.250). As even during severe ketoacidotic attacks C5OH and C5:1 may remain within normal ranges, these results with increased amounts of tiglyl glycine & 2-methyl-3-hydroxybutyrate in urinary organic acid analyses (GC/MS) 4/4, confirmed the diagnosis. We established emergency treatment of ketoacidosis, mild protein and fat restricted diet and started L-carnitine supplementation.Ten years follow up; no further ketoacidotic episodes,they developed normally with very well school performance, intractable convulsion resolved 10 months after starting treatment. Conclusion: Beta ketothiolase deficiency should be considered in differential diagnosis of ketoacidotic attack. Early diagnosis and aggressive management can prevent further episodes and lead to normal development.

seizure control, he became lethargic and had severe metabolic acidosis with ketonuria. Urine organic acid analysis showed a large peak of 3OH-propionic acid. Acylcarnitine profile was significant for an elevated propionylcarnitine at 1.2 micromolar (age normal < 0.88). The patient was treated with natural protein restriction, carnitine, biotin, and thiamine and had subjective improvement reported by parents with increased level of alertness and improved seizure control. Patient and Methods: GLDC, PCCA and PCCB Sanger sequencing performed on PCR amplified DNA. Results: GLDC: paternal allele: c.1576_1577insC and c.1580delGinsCAA; maternal allele is G607S. A homozygous PCCB c.49 C>A, p.Leu17Met variation found in exon 1 which is not conserved across species and one expression report in fibroblasts for this variant was normal (Perez-Cerda et al. 2003). Conclusions: We present a patient with NKH and clinical PA with a homozygous PCCB variant not previously reported to be disease causing.

P-194

Genotoxicity following AAV gene therapy for methylmalonic acidemia (MMA) in mice

Severe chronic pancreatitis associated with propionic acidemia and treatment with liver transplantation

P-196

Chandler RJ1, Ashok AA1, Varshney GK1, LaFave MC1, Wu W1, Burgess SM1, Venditti CP1

Ammous Z1, Rajadhyaksha A2, Jayakar P2, Hernandez E2, Bodamer O1 1

National Institutes of Health, Bethesda, United States

1

Dpt of Human Genetics, Univ of Miami, Miami, United States; 2 Miami Children's Hospital, Miami, United States Background: Propionic Acidemia (PA) is an inherited disorder of propionate metabolism due to deficiency of propionyl-coenzyme A carboxylase (PCC). Severe chronic pancreatitis is only rarely seen in PA. Case Reports: We report two patients with PA and recurrent episodes of pancreatitis. The first patient is a 7 year-old boy who had a history of > 50 episodes of pancreatitis resulting in fibrosis, pancreatic cyst formation, and severe exocrine pancreatic insufficiency. He underwent orthotopic liver transplantation which significantly improved overall metabolic control. The second patient is a 10 year-old boy with > 10 acute episodes of pancreatitis, resulting in the formation of a hemorrhagic pseudocyst. He was successfully managed by replacing his high fat Pro-Phree formula with an elemental formula. Acute episodes of pancreatitis in both patients occurred with and without metabolic decompensation. Discussion: Pancreatitis is an important complication of PA that should be considered in any patient with gastrointestinal symptoms. Recurrence of symptoms may be avoided through liver transplantation in patients with poor metabolic control or with a low fat diet in others. The presence of yet to be identified genetic or environmental modifiers may explain why some patients with PA are more likely to develop pancreatitis than others.

We have previously demonstrated the pre-clinical efficacy of AAV gene delivery using a neonatal lethal MMA murine model. Although the evaluation of genotoxicity was not the intended focus of our studies, the long-term surveillance of both AAV-treated MMA mice and control littermates has revealed that 75% (n=48) of AAV8-CBA-Mut treated mice developed HCCs between 12 and 21 months compared to a 2% (n=41) HCC rate in untreated littermates. In addition, 50% of the mice similarly treated with a control vector, AAV8-CBA-GFP, also developed HCCs, indicating that overexpression of the Mut transgene is not responsible for the development of HCC. We next performed a dose escalation study by increasing the AAV8 dose from 7x10^11-12 GC/kg (n=16) to 1x10^14 GC/kg (n=19) and noted a corresponding increase in the incidence of HCC from 12% to 84%, respectively. As previously observed by Donsante et al., our preliminary data indicates that insertional mutagenesis of the vector at the Rian locus, with subsequent dysregulation of local miRNA expression, occurs in AAV-associated HCCs. Determining why AAV gene delivery is infrequently associated with tumorigenesis in mice and whether such toxicity is relevant to humans remain as unresolved questions for those using AAV in human gene therapy applications. P-197 Polyunsaturated fatty acid and vitamin B12 status in treated isovaleric acidemia patients

P-195 Concurrent non-ketotic hyperglycinemia and propionic acidemia in an eight year old boy

Dercksen M1, Kulik W1, Mienie LJ2, Reinecke CJ2, van Lenthe H1, Wanders RJA1, Duran M1 1

1

2

2

3

Kruszka PS , Kirmse B , Zand DJ , Van Hove JL , Chapman K

2

1 NHGRI, NIH, Bethesda, MD, United States; 2Dept Gen and Metab, CNMC, Washington, D.C., United States; 3Sec Clin Gen and Metab, Univ. of Colo, Denver, United States

Background: We describe the first reported case of a patient with both non-ketotic hyperglycinemia (NKH) and propionic acidemia (PA). Case report: A one month-old male with intractable seizures and encephalopathy was diagnosed with NKH after finding CSF glycine elevation. At 8 years of age after placed on ketogenic diet for better

Lab Gen Metab Dis, AMC, UvA, Amsterdam, Netherlands; 2Cen Hum Metabonomics, NWU, Potchefstroom, South Africa

Objectives: Nutritional deficiencies are frequently observed in treatable inborn errors of metabolism (IEMs) due to an exclusion dietary regimen. We recently indicated vitamin B12 deficiency in a metabolomics investigation of treated isovaleric acidemia (IVA) patients. Consequently, we investigated vitamin and essential fatty acid (EFA) deficiencies and subsequent biochemical consequences thereof in metabolically stable IVA patients. Methods: Plasma polyunsaturated fatty acids (PUFAs) of 10 IVA patients were analyzed with GC-FID. Markers of vitamin B12 deficiency (methylmalonic acid, total homocysteine and homocystine in urine and

S166 plasma) were assayed via GC-MS, MS/MS and UPLC-MS/MS. The results were compared with hospital-based control subjects. The possible correlation between PUFAs and platelet count was also investigated. Results: A significant reduction in omega-3- (all groups, p<0.001) and omega-6- (in particular 20:3n-6, p<0.001) fatty acids were indicative of an EFA deficiency. The PUFAs did not correlate significantly with blood platelet count, except for a strong negative correlation (r = 0.58) between the latter and 20:3n-6. In addition, markedly elevated methylmalonic acid, total homocysteine and homocystine levels were observed, but not indicative of life-threatening vitamin B12 deficiency. Conclusion: Nutritional intervention in treatable IEMs e.g. IVA, may result in the reduction of vital biomolecules necessary for neurological and/or immunological function. P-198 Propionic acidemia: two cases undetected by neonatal metabolic screening using tandem mass spectrometry Kim S1, Song W1, Jeon Y1

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 baseline (0.9 % lysine) or a high lysine (lysine 4.7 %) diet. When a baseline diet was given, we verified mild alterations of the activities of some respiratory chain complexes in cerebral cortex and hippocampus, but not in striatum from Gcdh-/- mice. Furthermore, the mitochondrial respiratory parameters and the activities of α-KGDH and CK were not modified in all brain structures from Gcdh-/- mice. In contrast, we found a significant reduction of Na+, K± ATPase activity associated with a lower degree of its expression in cerebral cortex from Gcdh-/- mice. Finally, a high lysine diet did not accentuate the biochemical alterations observed in Gcdh-/- mice fed a baseline diet. It is presumed that reduction of Na+, K± ATPase activity may represent a potential underlying mechanism involved in the brain swelling and cortical abnormalities (cortical atrophy with leukodystrophy) observed in patients affected by glutaric aciduria type I. Grants: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP, IBN-Net, INCT-TM P-200 Evidence that the major metabolites accumulating in 3-hydroxy-3methylglutaric aciduria provoke hypophosphorylation of cytoskeletal neural proteins in rat brain

1

Korea Genetics Research Center, Cheong Ju, Korea, Republic of

Background: Newborns with PPA have elevated propionylcarnitine tested by tandem mass spectrometry. Objective: Expanded newborn screening has failed to detect two cases of propionic acidemia. Analysis of clinical spectrum and biochemical metabolites of these unusual cases of propionic acidemia are important for the future references. Case report: Case I: Newborn screening found normal C3 carnitine. He was growing and developed normally until 23 month of age when he had URI and Rota gastroenteritis, he had seizure and became comatous. Biochemical analysis showed full brown characteristics of PPA during metabolic crisis. Now 10 years old, he grew and developed normally with low protein diet. Case 2: 2 year 4 month old boy with normal newborn screening findings had sinusitis and seizure followed by coma. There was another episode of coma after high protein food and respiratory infection. Biochemical studies revealed PPA with severe lactic acidosis. Results: Both cases showed normal or trace amounts of PPA metabolites but infection or high protein intake triggered metabolic decompensation. Case 2 has biotine responsiveness. Developmentally, Case I is normal but Case 2 has expressive language delay. Conclusion: Despite normal result of tandem newborn screening, we have to consider the possibility of late presentation of PPA. P-199 Na+, K+-ATPase activity is reduced in cerebral cortex of glutarylCoA dehydrogenase deficient mice: a possible mechanism for brain injury in glutaric acidurua type I Amaral AU1, Cecatto C1, Seminotti B1, Zanatta A1, Fernandes CG1, Busanello ENB1, Kist LW2, Bogo MR2, Leipnitz G1, Souza DOG1, Woontner M3, Koeller DM4, Goodman S3, Wajner M5 1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Lab de Biol Gen e Molec, PUC-RS, Porto Alegre, Brazil; 3School Med Univ of Colorado, Denver, United States; 4Dep of Ped, Mol and Med Gen,OHS Univ, Portland, United States; 5Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil We evaluated bioenergetics parameters (activities of the respiratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase- α-KGDH, creatine kinase - CK, Na+,K±ATPase and mitochondrial respiratory parameters) in cerebral cortex, striatum and hippocampus from 30-day-old glutaryl-CoA dehydrogenase deficient (Gcdh-/-) and wild type (WT) mice submitted to a

Fernandes CG1, Martell RW1, Pierozan P1, Ferreira F1, Wajner M1, Pureur RP1 1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil

3-Hydroxy-3-methylglutaric aciduria (HMGA) is an inherited metabolic disorder biochemically characterized by the tissue accumulation of 3hydroxy-3-methylglutaric (HMG), methylglutaconic (MGT) and 3methylglutaric (MGA) acids, and clinically by hypotonia, hepatopathy, convulsions and coma during metabolic decompensation. Since HMG, MGT and MGA are structurally similar to glutamate and NMDA receptors are responsible for the activation of several signaling pathways involving the homeostasis of the phosphorylating system, we evaluated the effects of HMG, MGT and MGA on cytoskeletal protein phosphorylation in rat striatum and cerebral cortex. Intermediate filaments (IFs) are important constituents of cytoskeleton and phosphorylation of their subunits is one of the main regulatory mechanisms of cellular functions. We therefore exposed brain slices to these metabolites and measured the in vitro phosphorylation of IF. HMG and MGA induced hypophosphorylation of the IFs in both cerebral structures, while MGT inhibited the phosphorylation of IFs only in the striatum. We propose that hypophosphorylation of IF subunits in astrocytes and neurons by the major metabolites accumulating in HMGA may provide new insights into brain damage of patients affected by HMGA. In this context, hypophosphorylation of these proteins has been previously related to neural dysfunction in other pathologies. Grant: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP, IBNNet, INCT-EN. P-201 Methylmalonic acid and ammonia act synergistically in vivo causing a redox imbalance in cerebral cortex and striatum of young rats Viegas CM1, Zanatta A1, Grings M1, Hickmann FH1, Monteiro WO1, Sitta A2, Leipnitz G1, Wajner M1 1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil Hyperammonemia is a common finding in children with methylmalonic acidemia and propionic acidemia, but its contribution to the neurological symptoms of the affected patients is poorly known. We investigated the effects of hyperammonemia induced by urease treatment together with an intracerebroventricular (ICV) injection of MMA or PA on important

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 parameters of redox homeostasis in cerebral cortex and striatum from 30-dayold rats. MMA decreased glutathione (GSH) concentrations, and sulfhydryl content in cerebral cortex and striatum of hyperammonemic rats, whereas MMA or ammonia per se did not alter these parameters. Furthermore, MMA combined with hyperammonemia decreased glutathione reductase activity and increased nitric oxide production in rat cerebral cortex, but. Furthermore, ICV PA administration solely or combined with hyperammonemia did not alter any of the evaluated parameters. Finally, various antioxidants prevented GSH reduction and sulfhydryl oxidation, as well as the increased formation of nitric oxide provoked by the MMA plus ammonia treatment, implying the participation of reactive species in MMA-induced effects. The data indicate a synergistic effect of MMA and ammonia reducing the antioxidant defenses and inducing reactive nitrogen species in rat brain. Financial support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP IBN-Net and INCT-EN. P-202 D-2-hydroxyglutaric and L-2-hydroxyglutaric acid disrupt redox homeostasis in vivo in striatum from young rats da Rosa MS1, Seminotti B1, Amaral AU1, Viegas CM1, Ribeiro CAJ1, Leipnitz G1, Wajner M1 1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil

Background: D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria are biochemically characterized by brain accumulation of D2-hydroxyglutaric acid (D-2-HG) and L-2-hydroxyglutaric acid (L-2HG), respectively. Affected patients usually present neurological symptoms and abnormalities, whose pathomechanisms are poorly known. Objectives: In the present work, we evaluated the effects of intrastriatal injection of D-2-HG and L-2-HG on important parameters of redox homeostasis in striatum of young rats. Materials and Methods: Thirty-day-old Wistar rats received a single injection (2.5 μmol) of D-2-HG, L-2-HG or NaCl. Thiobarbituric acidreactive substances (TBA-RS, lipid peroxidation), carbonyl formation (protein oxidative damage), reduced glutathione (GSH) levels and the activities of the antioxidant enzymes glutathione peroxidase (GPx), superoxide dismutase (SOD) and catalase (CAT) were assayed in striatum homogenates. Results: D-2-HG and L-2-HG induced lipid peroxidation (TBARS increase) and decreased GSH levels, as well as the activities of GPx and SOD, but did not alter CAT activity and carbonyl formation. Moreover, D-2-HG-induced increase of TBA-RS and decrease of GSH levels and GPx activity were prevented by the NMDA-receptor antagonist MK-801, indicating the involvement of the NMDA glutamate receptor in these effects. Conclusions/Discussion: D-2-HG and L-2-HG induce lipid oxidative damage and compromise antioxidant defenses in vivo. Grants: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP, IBN-Net, INCT-EN. P-203 Acute administration of 3-hydroxy-3-methylglutaric and 3methylglutaric acids induces oxidative stress in cerebral cortex and liver of young rats: a possible pathomechanism of brain and liver dysfunction Leipnitz G1, da Rosa MS1, Seminotti B1, Fernandes CG1, Amaral AU1, Fritch L1, Sitta A2, Coelho DM2, Wajner M2 1

Departamento de Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; SGM, HCPA, Porto Alegre, Brazil

2

S167 Background: 3-Hydroxy-3-methylglutaric aciduria (HMGA) is biochemically characterized by the accumulation of 3-hydroxy3-methylglutarate (HMG), 3-methylglutarate (MGA) and 3methylglutaconate. Affected patients present neurological dysfunction and hepatomegaly, whose pathophysiology is still unclear. Objectives: The ex vivo effects of HMG and MGA on oxidative stress parameters in rat brain and liver were evaluated. Materials and methods: Young Wistar rats were intraperitoneally administered with three injections of HMG, MGA or NaCl (the first injection of 10 μmol/g followed by two injections of 5 μmol/g body weight), with an interval of 1h30min, and sacrificed 1h after the last injection. Supernatants of cerebral cortex and liver were used for biochemical assays. Results: HMG and MGA increased carbonyl formation in cerebral cortex, but not in liver. Furthermore, HMG and MGA decreased glutathione concentrations and increased 2',7'-dichlorofluorescin oxidation and the activities of superoxide dismutase and catalase in cerebral cortex and liver. In contrast, thiobarbituric acidreactive substances and nitrate and nitrite levels were not modified by this treatment. Conclusion: It may be presumed that oxidative stress induced by HMG and MGA in vivo possibly contribute to the neurological and hepatic dysfunction found in HMGA. Financial support: CNPq, PROPESq/UFRGS, CAPES, FAPERGS, PRONEX, FINEP IBN-Net, INCT-EN. P-204 The cblC proteome: in vivo elucidation of altered cellular pathway in humans Caterino M1, Pastore A2, Strozziero MG3, Boenzi S2, Dionisi Vici C2, Ruoppolo M3 1

CEINGE Biotecnologie Avanzate, Naples, Italy; 2Div Metab Dis, Osp Ped Bambin Gesù, Rome, Italy; 3DMMBM, Univ. Naples "Federico II", Naples, Italy Methylmalonic Acidemia with Homocystinuria, cobalamin deficiency type C (cblC) (MMACHC) is the most common inborn error of cobalamin metabolism. Despite a multidrug treatment, the long-term follow-up of early-onset patients is often unsatisfactory, with progression of neurological and ocular impairment. Here, the proteome of control and MMACHC lymphocytes (obtained from patients treated with OHcbl, betaine, folate and carnitine) was quantitatively examined by two dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. 23 proteins were found up-regulated and 37 proteins were found down-regulated. Major changes were observed in the expression levels of proteins involved in cytoskeleton organization and assembly, in neurological function and in cellsignaling. Consistent with in vivo studies showing relevant disturbance of glutathione metabolism (Pastore et al., JIMD 2013), we found a disregulation in protein involved in cellular detoxification, especially in glutathione metabolism. Our study demonstrate in circulating lymphocytes of treated cblC patients relevant changes in the proteome profile, and confirms previous results observed in vitro in cultured fibroblasts (Hannnibal et al., MGM 2011). These observations may be helpful for better understanding the pathophysiology of the disease and in addressing future research and novel therapeutical strategies.

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P-205

P-207

Increased glutamate transporter gene expression in cerebral cortex and striatum from Gcdh-/- mice

Alterations of glutamate binding and uptake in striatum from glutarylCoA dehydrogenase deficient mice: the role of glutaric acid

Lagranha VL1, Fernandes CG1, Pereira CC1, Souza DOG1, Matte U2, Wajner M3

Busanello ENB 1 , Fernandes CG1 , Martell RW 1 , Lobato VGA 1 , Lagranha VL1, Souza DOG1, Woontner M2, Goodman S2, Koeller DM3, Wajner M4

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Centro de Terapia Gênica, HCPA, Porto Alegre, Brazil; 3Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil 1

Abnormalities of glutamatergic neurotransmission may contribute to the pathophysiology of glutaric acidemia type I (GA I). Therefore, we determined mRNA expression levels of the glutamate transporters GLAST and GLT1 in cerebral cortex and striatum from wild type (WT) and glutarylCoA dehydrogenase deficient (Gcdh-/-) mice aged 7, 30 and 60 days fed a normal chow (20% protein and 0.9% Lys). Thirty-day-old WT and Gcdh-/-mice were also submitted to a high Lys diet (20% protein and 4.7% Lys) for 60h. The mRNA was extracted from striatum and cerebral cortex of the animals and measured by qPCR using gene-specific TaqMan FAM/MGB invetoried assays. Significantly higher expression of GLAST and GLT1 transporters was observed in cerebral cortex and striatum from Gcdh-/- mice at 30 and 60 postnatal day, whereas GLAST was overexpressed only in striatum from 7day-old Gcdh-/-, as compared to WT mice. We also showed that high lysine intake induced a further increase of GLAST gene expression in the striatum of 30-day-old Gcdh-/-mice. The present data showing a differential expression of glutamate transporters in the Gcdh-/- mice may be possibly involved in the cortical and striatal abnormalities observed in GA-I patients. Grants: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede IBN-Net, INCT-EN, FIPE/HCPA P-206 Overexpression of glutamate receptors in cerebral cortex and striatum of Gcdh-/- mice: possible implications for the neuropathology of glutaric acidemia type I Lagranha VL1, Fernandes CG1, Pereira CC1, Matte U2, Wajner M3, Souza DOG1 1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Centro de Terapia Gênica, HCPA, Porto Alegre, Brazil; 3Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil The role of excitotoxicity in glutaric aciduria type I (GA-I) is controversial. Therefore, in the present work we evaluated the mRNA expression of ionotropic glutamate receptors (GLUR) in cerebral cortex and striatum of wild type (WT) and GCDH deficient (Gcdh-/-) mice aged 7, 30 and 60 days. The mRNA was extracted and measured by RT-qPCR. Overexpression of NR2A and NR2B in striatum and of GluR2 (AMPA) and GluR6 (kainate) in cerebral cortex were observed in 7-day-old Gcdh-/- mice. In addition, there was an increase of NMDA subunits in cerebral cortex and striatum from 30-day-old Gcdh-/- mice, whereas at 60 postnatal day all GLURs transcripts were markedly overexpressed in these animals. We also showed that a high lysine intake (4.7 %) induced a further increase of NR2A and NR2B transcripts in striatum and an overexpression of GluR2 and GluR6 in both cerebral structures from Gcdh-/- mice. These data revealing developmental regulated and tissue-specific expression of these proteins in Gcdh-/-)may induce a higher vulnerability of these cerebral structures to the effects of glutamate agonists, therefore contributing to the neuropathology of GA I.. Grants: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede IBN-Net, INCT-EN, FIPE/HCPA.

1

Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2School Med Univ of Colorado, Denver, United States; 3Dep of Ped, Mol and Med Gen,OHS Univ, Portland, United States; 4Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil Striatum damage is commonly observed in glutaric acidemia type I (GA I), a disorder biochemically characterized by predominant accumulation of glutaric acid (GA), which are structurally similar to glutamate. Since the pathogenesis of the striatal damage in GA I is not yet established, we determined important parameters of the glutamatergic system in striatum from 30-day-old normal (WT) and glutaryl-CoA dehydrogenase deficient mice (Gcdh-/-) in the presence or absence of GA. We measured Na± independent (receptors) and dependent (transporters) [3H] glutamate binding (GB) to synaptic plasma membranes and [3H]glutamate uptake into striatum slices. GB to receptors and transporters was reduced in Gcdh-/- mice, and this reduction was exacerbated by GA only in Gcdh-/- mice. On the other hand, glutamate uptake did not differ between Gcdh-/- and WT mice, but was decreased by GA in WT and Gcdh-/- mice. Our data point to a disturbance of glutamatergic neurotransmission in Gcdh-/- mice, that could be due to the variable expression of glutamate receptors and transporters and/or to competition between GA and glutamate for its receptors and transporters. Alterations of the glutamatergic system may underlie, at least in part, striatum injury in patients affected by GA I. Grants: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP, IBN-Net, INCTEN. P-208 A pilotstudy of hypothermia treatment for hyperammonemia and encephalopathy in urea cycle disorders and organic acidurias Lichter-Konecki U1, Nadkarni V2, Poeschel J3, Dimmock D4, Baumgart S5

1 Div Genet & Metab, Children's National MC, Washington, DC, United States; 2Div Ped Critical Care, CHOP, Philadelphia, PA, United States; 3 Div Neonatology, Univ Children's Hosp, Heidelberg, Germany; 4Div Genetics, Dept. Ped Medical College, Milwaukee, WI, United States; 5 Div Neonatology Children's National MC, Washington, DC, United States

Background: Children with urea cycle disorders (UCDs) or organic acidemias (OAs) acute hyperammonemia (HA) and encephalopathy are at great risk for brain injury. Nutritional support and dialysis are standard treatments. Neuroprotection during these interventions may improve intellectual outcome. Animal experiments and small clinical trials in acute liver failure indicate that hypothermia treatment (HT) is neuroprotective in HA. We report results of a feasibility and safety pilot study of whole body cooling for neonates with acute HA and encephalopathy. Methods: Hyperammonemic neonates were eligible for HT, if they required dialysis and had symptoms of UCDs or OAs. As in HT for neonatal hypoxic ischemic encephalopathy patients were cooled to 33.5 °C +/- 1°C. After 72h they were slowly rewarmed. Data of age-matched historic controls were also collected. Results: Seven patients were cooled and their data compared to data of seven historic controls. All patients survived dialysis and HT, 6 patients

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 were discharged home, five feeding orally. The main complication was hypotension. Conclusion: Hypothermia treatment was feasible and safe administered by teams experienced in cooling neonates and managing metabolic crises. HT however adds to the complexity of the treatment and should not be done unless proven efficacious in a randomized trial. P-209 Cobalamin C deficiency presenting with hemolytic uremic syndrome in two unrelated adults Garcia Segarra N1, Benoist JF2, Ogier de Baulny H1

S169 hyperkalaemia. He was successfully managed with intravascular volume expansion and urinary alkalinisation. The elder brother presented at birth with left congenital diaphragmatic hernia which was surgically repaired. He remained globally delayed with severe failure to thrive and presented with hypoglycaemic episodes at 4 months old. His CK levels remain between 5000-15,000 U/L with no episodes of rhabdomyolysis. Conclusion: The potential for severe rhabdomyolysis in complex GKD must be recognised so that timely intervention can be instituted. P-211 A second patient with a defect of the trifunctional methylene-thfdehydrogenase (MTHFD1) protein

1

Div Metab Dis, Robert Debré Hosp, Paris, France; 2Biochemistry, Robert Debré Hosp, Paris, France

Burda P1, Kuster A2, Suormala T1, Coelho D1, Bürer C1, Rosenblatt DS3, Baumgartner MR1, Fowler B1

CblC due to defective intracellular cobalamin metabolism usually presents in early infancy with multisystem deterioration among which hemolytic uremic syndrome has been regularly described. Conversely, this disorder in adults can be asymptomatic or presents with thrombotic events, neurological or psychiatric deterioration while renal involvement has never been reported. We describe two unrelated adults with late-onset cobalamin C disease who presented with hemolytic uremic syndrome and renal failure. The first is a 20 year-old man referred for malignant hypertension, mechanical hemolysis and renal insufficiency. The second is a 26 year-old woman with atypical preeclampsia leading to postpartum hemolytic uremic syndrome that recurred after kidney transplantation. Both had no past medical history. Initial diagnostic work-up failed to find etiology and renal failure progressed despite immunosuppressive treatment. Renal biopsy showed thrombotic microangiopathy. Because of the atypical evolution, etiological investigations were expanded and found hyperhomocysteinemia with hypomethioninemia and increased methylmalonic aciduria, suggesting a cobalamin disorder. Molecular analysis confirmed the diagnosis and specific treatment was started. These two unusual cases underlie that even in adults the investigation of unexplained hemolytic uremic syndrome should comprise metabolic investigations being aware that hyperhomocysteinemia by itself is insufficient to get the right diagnosis.

1

Div of Metabolism, Univ Child Hosp, Zurich, Switzerland; 2Dep of Pediatrics, CHU, Nantes, France; 3McGill Univ, Montreal, Canada

P-210

In the folate cycle MTHFD1 encoded by MTHFRD1 is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase. One patient with MTHFD1 deficiency with hyperhomocysteinaemia and surprisingly methylmalonic acidaemia was identified using exome sequencing (Watkins, 2011). We investigated fibroblasts of a patient with mild megaloblastic anemia and atypical hemolytic uraemic syndrome, hyperhomocystemia, low methionine and normal MMA suggesting a defect of methionine synthase (cblE and cblG). However, such a defect was excluded by somatic complementation analysis. Further fibroblast studies revealed mildly reduced synthesis of methylcobalamin and severely reduced formation of methionine from [14C]-formate which did not increase in cobalamin supplemented culture medium. However a mild increase was observed in the presence of 5-formyltetrahydrofolate. These observations led us to investigate MTHFRD1. Mutation analysis revealed heterozygosity for a missense mutation (c.806C>T, p.T296I) affecting a highly conserved amino acid and a splice site mutation (c.1674G>A, pT558T) located at the last nucleotide of exon 17, leading to exon skipping. MTHFD1 deficiency was confirmed by complementation analysis using MTHFD1 deficient fibroblasts from the previously published case. This second case adds to knowledge of this intriguing defect and indicates that increased MMA as reported in the first case is not a consistent finding.

Severe rhabdomyolysis in complex glycerol kinase deficiency

P-212

Tan ES1, Visruthan N1, Lee JH1, Lim J1, Hart C2

Functional characterization of 23 methylmalonyl-CoA mutase mutant alleles in a mammalian expression system

1

KK Women's and Children's Hospital, Singapore, Singapore; 2Willink Biochem Genetics,St Mary's Hosp, Manchester, United Kingdom Background: The genes for several X-linked disorders such as adrenal hypoplasia congenital (AHC), glycerol kinase (GKD) deficiency, and duchenne muscular dystrophy(DMD), are grouped together in the p21 region of the X chromosome. Complex GKD or Xp21 contiguous deletion occurs when there is a large deletion spanning several genes. We present a pair of siblings with complex GKD. This is the first report of severe rhabdomyolysis in this condition. Case Reports: The younger brother presented at 4 months with failure to thrive. His baseline creatine kinase (CK) levels were 20,000U/L and he was asymptomatic at these levels. During an episode of infection, he developed supraventricular tachycardia with rhabdomyolysis. The highest recorded concentration of CK was 263,583U/L. This was complicated by myoglobinuria and

Forny P1,2,3, Froese DS1,4, Suormala T1, Yue WW4, Fowler B1, Baumgartner MR1,2,3 Div of Metab, Univ Child Hosp, Zurich, Switzerland; 2radiz – Rare Disease Initiative Zurich, Zurich, Switzerland; 3Center for Integrative Human Physiology, Zurich, Switzerland; 4SGC Univ of Oxford, Oxford, United Kingdom

1

Deficiency of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM) leads to the accumulation of toxic intermediates and causes the severe disorder methylmalonic aciduria. Deficient MCM activity results either from defects of the MCM apoenzyme, which is encoded by MUT, or from defects of intracellular synthesis of its cofactor, 5-deoxyadenosylcobalamin (AdoCbl). The 100 MCM missense mutations reported to date can be classified according to whether the mutant enzyme exhibits either residual activity in the presence of high

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concentrations of AdoCbl (Km variants; mut-), or no detectable activity (mut0). To gain insight into the functional relevance and molecular basis of MCM mutations, 18 mut- and 5 mut0 missense mutations were characterized. Wild type and mutant constructs were transiently transfected into immortalized MCM-deficient fibroblasts with no detectable MCM expression by electroporation. We demonstrate (i) reduced enzyme activity of variable degree in all mutant proteins, (ii) 100 to 1300-fold increased Km values for AdoCbl in mut- mutants, (iii) decreased amount of 7 mut- and 3 mut0 mutant MCM proteins on Western blotting. This work using a novel mammalian expression system provides insight into the functional significance of 23 MCM mutant alleles as demonstrated by the reliable determination of residual enzyme activity and Km for AdoCbl.

MMAuria. Sequencing of the MCEE gene revealed homozygosity for the nonsense mutation that was first described by Bikker et al. (c.139C>T, p.Arg47*) in eight patients (two of them siblings). In addition, we identified one patient to be homozygous for a novel missense mutation (c.158T>G, p.Ile53Arg). Incorporation of [14C]propionate into fibroblast of the eight patients harbouring the nonsense mutation was reduced with no increase when grown in cobalamin supplemented culture medium (1.4 - 3.0 nmol/16h/mg; control range: 3.5 - 24.4 nmol/16h/mg). Propionate incorporation into fibroblasts carrying the missense mutation was inconsistent (2.9 - 6.1 nmol/16h/mg). This study substantially increases our knowledge of this rare disorder and long term follow up of these patients will help to define its clinical implications. P-215

P-213 Diabetes mellitus (T1D) and glutaric aciduria type I (GA-I): from one crisis to another Burlina AB1, Galderisi A2, Zschocke J3, Giordano L1, Bordugo A1, Monciotti CM2, Dianin A1, Polo G1, Burlina AP4

Characterization of functional domains of MMADHC, a protein responsible for intracellular cobalamin processing Jusufi J1, Froese DS1, Coelho D1, Suormala T1, Burda P1, Fowler B1, Baumgartner MR1 1

Div of Metabolism, Univ Child Hosp, Zurich, Switzerland

1

2

Div Metab Dis,Univ Hosp, Padova, Italy; Pediatric Dept, Univ Hosp, Padova, Italy; 3Div Hum Genet Clin Genet, Med Univ, Innsbruck, Austria; 4 Neurological Un, St. Bassiano Hosp, Bassano del Grappa, Italy Background: Based on Italian frequencies of 12 cases/100.000 for T1D and 1/100,000 for GA-I, the concomitant occurrence of both diseases, never reported before, may appear a pure coincidental association. Case: We report a 26-months-old boy who presented an hyperglycemic ketosis due to T1D and 6 months later, on occasion of an intercurrent illness, a devastating motor deterioration (acute crisis of dystonic movements, axial hypotonia) and severe dysphagia due to GA-I. T1D was characterized by high anti-GAD titre (>2000 KUI/L) and, after insulin therapy, many frequent episodes of hypoglycaemia. GA-I presented with elevated glutarylcarnitine (0.43; n.v. 0-0.25 μmol/L), increased 3-OH glutaric acid, but normal glutaric acid in urine. Brain MRI showed T2 hyperintensity in the putamina, globi pallidi and deep frontal white matter. Mutation analysis (heterozygosity for c.656C>A in exon 7 and c.1198G>A in exon 10) confirmed the diagnosis. The patient received age-adapted metabolic treatment including arginine (100 mg/kg/die) showing a rapid unexpected improvement of motor disabilities without further hypoglycaemic episodes. Conclusion: T1D, with a high level of anti-GAD antibodies, may determine an impairment of the activity of glutamic acid decarboxylase with a depletion of GABA and glutamate. This neurotransmitter imbalance may significantly affect the neurologic phenotype of GA-I. P-214 Methylmalonyl - CoA epimerase (MCE) deficiency: nine new cases

Vitamin B12 (cobalamin) must be converted into two coenzyme forms, methylcobalamin (MeCbl) and adenosylcobalamin (AdoCbl) in order to maintain intracellular homeostasis of homocysteine and methylmalonic acid, respectively. We hypothesized that in cblDdeficient patients, mutations in the N-terminus (p.C19-T152) of MMADHC cause isolated methylmalonic aciduria (cblD-MMA) due to AdoCbl deficiency, null mutations across the C-terminus (p.Y140R250) cause combined methylmalonic aciduria and homocystinuria (cblD-MMA/Hc) due to combined AdoCbl and MeCbl deficiency, while missense mutations in a conserved C-terminal region (p.D246L259) cause isolated homocystinuria (cblD-HC). To better understand the boundaries of each domain related to cobalamin processing, we tested the effect of selected point mutations and C-terminal truncations on MeCbl/AdoCbl synthesis in immortalized cblD-MMA/Hc patient fibroblasts. Testing 20 mutations (15 missense and 5 C-terminal truncations) across p.P154- D286 revealed: an extension to the known Nterminal region responsible for AdoCbl synthesis (p.C19-P154); an extension in both the N- and C-terminal directions (p.F204-R266) of the region responsible for MeCbl synthesis; and the identification of a region required for both (p.T182-R197). These results support our hypothesis and this approach helps to better define the sequence required for MeCbl/AdoCbl synthesis. P-216 Different types of cell death are involved in brain damage of methylmalonic aciduria and glutaric aciduria type I Zavadakova P1, Jafari P1, Cung HP1, Werner D2, Braissant O2, Bonafé L1, Ballhausen D1

Burda P1, Suormala T1, Bürer C1, Froese S1, Fowler B1, Baumgartner MR1 1

Div Mol Ped, Lausanne Univ Hosp, Lausanne, Switzerland; 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland

Methylmalonyl-CoA epimerase (MCE) catalyzes the isomerization of D-methylmalonyl-CoA to L-methylmalonyl-CoA, the substrate for methylmalonyl-CoA mutase. So far three cases of moderate methylmalonic aciduria (MMAuria) have been attributed to defects in the MCEE locus coding for MCE (Bikker 2006, Gradinger 2007) but it remains unclear whether this is a benign defect. We have re-investigated 150 cell lines from patients with mildly to moderately elevated levels of MMA who could not be assigned to any class of defect known to cause

We previously showed that exposure of 3D organotypic rat brain cell cultures to 1mM 2-methylcitrate (2-MCA) or 3-hydroxyglutarate (3OHGA) every 12h over three days (DIV11-DIV14) results in ammonium accumulation and cell death. The aim of this study was to define the time course (every 24h) of the observed effects. Ammonium in culture medium already increased at DIV12 staying stable on the following days under 3-OHGA exposure, while it increased consecutively up to much higher levels under 2-MCA

1

Div of Metabolism, Univ Child Hosp, Zurich, Switzerland

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 exposure. Lactate increase and glucose decrease were observed from DIV13 and DIV14, respectively. We conclude that ammonium accumulation precedes alterations of energy metabolism. As observed by immunohistochemistry glial cells were the predominant dying cells. Immunoblotting and immunohistochemistry with cell death specific markers (caspase-3, alpha-fodrin, LC3) showed that 2-MCA exposure significantly increased apoptosis on DIV14, but did not alter autophagy or necrosis. In contrast, 3-OHGA exposure substantially increased necrosis already from DIV13, while no change was observed for apoptosis and autophagy. In conclusion, ammonium accumulation, secondary disturbance of energy metabolism and glial cell death are involved in the neuropathogenesis of methylmalonic aciduria and glutaric aciduria type I. Interestingly, brain cells are dying by necrosis under 3-OHGA exposure and by apoptosis under 2-MCA exposure. P-217 Health-related quality of life and psychological adjustment in patients with intoxication-type inborn errors of metabolism: a systematic review

S171 levels has been proved in normal mice, in mouse model of Canavan disease, and in few patients. We report on a 28 months boy, who was conceived by consanguineous Libyan parents and presented with developmental delay, macrocephaly, generalized seizures and spastic tetraparesis. Biochemical, neuroimaging and genetic investigations leaded to Canavan disease diagnosis (the patient was homozygous for G503A mutation, Arg168His). Lithium carbonate treatment was started when he was 19 month old (10 mg/Kg for two weeks and 50 mg/kg thereafter). After five months, a 25% decrease of NAA levels was detected both in brain and urine. Even though the patient showed an improvement in alertness, eye-contact and rigidity, he remained severely impaired. So far eight patients have been treated with lithium citrate resulting in a lowering of N-acetylaspartate (NAA) brain levels, with feeble or any clinical improvement. The dissociation between clinical and biochemical effects of lithium may be due to the delay in starting the treatment or, alternatively, to the fact that NAA is not the only issue implied in disease pathogenesis. P-219 Location matters: Krebs cycle enzyme location and activity in cell lines from propionic acidemia patients and controls

Zeltner NA1, Huemer M1, Baumgartner MR2, Landolt MA3 Chapman KA1, Cunningham G2, Luque-Cabrera J2, Summar ML1 1

Div Metab, Univ Child Hosp, Zurich, Switzerland; 2radiz - Rare Disease Initiative Zurich, Zurich, Switzerland; 3Dep Psychosom Psych, Univ Child Hosp, Zurich, Switzerland Background: Health-related quality of life (HRQoL) and psychological adjustment (PA) have rarely been assessed in patients with "intoxication-type" (IT-) inborn errors of metabolism (IEM) such as urea cycle disorders or organic acidurias. IT-IEM require intense patient and caregiver's efforts in terms of strict dietetic and pharmacological treatment against the background of high risk for metabolic crises and impaired development. There is no consensus about whether and how HRQoL and PA are affected in patients with IT-IEM. Objectives: To systematically review the literature addressing HRQoL and PA in patients with IT-IEM. Methods: Relevant databases were searched and articles were selected based on predefined criteria. Methodological quality was assessed and data were extracted by two independent reviewers. Results: Studies investigating HRQoL and PA in IT-IEM are rare compared to the number of studies addressing other IEM - and of varying methodological quality. HRQoL and PA seem to be normal for some domains but impaired for the domains autonomy, physical and emotional well-being. Conclusion: Data on HRQoL and PA in IT-IEM are sparse. Since HRQoL and PA are highly relevant outcome parameters in this patient group, intense research using disease specific assessment tools is required.

1

CNMC, Genetics and Metabolism, Washington, United States; CNMC, Children's Research Institute, Washington, United States

2

Background: Kreb cycle enzymatic components were first identified in the 1930's, but their cellular localization in matrix or membrane of mitochondria are unknown. We sought to localize the components of this system and the enzymes leading to it in normal and propionic acidemia (PA) cell lines. Material and Methods: Fibroblasts and lymphoblastoid cell lines from age and sex-matched controls and PA patients were characterized for Kreb cycle enzyme activity and presence (by immunoblot) in both the membrane fraction and matrix fraction from isolated mitochondria. Results: Locations in mitochondrial matrix and membrane were identified for fumarate hydratase, methylmalonyl CoA mutase, Propionyl CoA carboxylase (subunits A and B), and oxoglutarate dehydrogenase by immunoblot. Enzyme activity for Malate dehydrogenase, oxoglutarate dehydrogenase, fumarate hydratase, and isocitrate dehydrogenase were all also done. We found that methylmalonyl CoA mutase and oxoglutarate dehydrogenase were concentrated in the mitochondrial membrane fraction. Discussion: These findings suggest a pathway organization for these enzymes that may result in substrate channeling and affect access to the pathway. This concept is important in considering the effects of defects in propionic acidemia and methylmalonic acidemia on energy metabolism through the Kreb cycle system.

P-218 P-220 The effect of lithium carbonate on clinical and biochemical picture of severe early onset Canavan disease

Canavan disease (CD) revisited: revealing the diagnosis in a mildly affected 24-year-old man

Celato A1, Tavazzi B2, Lazzarino G3, Colaiacomo MC4, Midulla F5, Mastrangelo M1, Leuzzi V1

Sass JO1, Bähr L1, Britschgi C1, Sommer A2, Ferreira P3

1

1

DivNeurPedDipPedNeurPsicInf UnivSapienza, Roma, Italy; 2Univ Cattolica Sacro Cuore, Roma, Italy; 3Dip Bio Geo Sci Amb Univ Catania, Roma, Italy; 4Rad Urg, DEA, Univ Sapienza, Roma, Italy; 5 PedUrgTerIntPedDipPedNeurPsicInfSapienza, Roma, Italy Canavan disease (OMIM #271900) is an early onset, severe, untreatable spongiform leukodystrophy due to mutations in aspartatoacylase gene (ASPA, OMIM*608034). The effect of lithium in lowering NAA brain

Div Clin Chem Biochem, Univ Child Hosp, Zürich, Switzerland; 2Lab Clin Biochem Metab, Univ Child Hosp, Freiburg, Germany; 3Div Med Genet, Alberta Child Hosp, Calgary, Canada CD is a neurodegenerative disease that often leads to death in early childhood. Ten years ago, a 13-year-old boy with mild developmental delay, partial cortical blindness, nystagmus, retinitis pigmentosa (RP), and macrocephaly was reported as a diagnostic dilemma for CD

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(Surendran et al., J Child Neurol 2003;18:809-13). Now aged 24, he is mildly cognitively handicapped with severe RP and cataracts, but shows no obvious regression. He continues to have only a slight increase in urinary N-acetylaspartic acid. Repeat biochemical and molecular investigations are at variance with the previous report. We found fibroblast aspartoacylase activity significantly decreased, and discovered that he is a compound heterozygote for two ASPA mutations: c.212G>A (p.Arg71His) and c.863A>G (p.Tyr288Cys), both previously reported in mild CD. Expression analyses of both mutants in HEK293 cells yielded loss of aspartoacylase activity. The intronic mutation reported by Surendran et al. was not confirmed. These results emphasise clinical heterogeneity in CD and demonstrate that even slight N-acetylaspartic aciduria should prompt a comprehensive diagnostic work-up in individuals with a compatible clinical presentation.

We developed a single injection SIM/Scan method on an Agilent 5975C gas chromatography-mass spectrometer (GC-MS). Total run time is 33 minutes allowing for a variable ramp rate to separate co-eluting peaks and accurately quantitate twenty compounds. A proper validation was performed in accordance with good laboratory practices. The assay passed all metrics including accuracy (samples from proficiency testing and individuals with known inborn errors of metabolism), precision, analytic sensitivity and specificity, and establishment of reference ranges for the normal population. A single SIM/Scan method allows for simultaneous collection of quantitative and qualitative data without increasing overall analytical time and decreasing dilapidation of the GC-MS. It also facilitates interpretation by allowing direct comparison of the SIM and TIC data making this method superior to all other methods including SIM, TIC, or independent SIM and TIC.

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P-223

Diagnostic difficulties in glutaryl-CoA dehydrogenase deficiency

Biochemical, bioinformatics and molecular analyses to diagnose 3methylglutaconic aciduria type I caused by a novel mutation in the AUH gene

Croft J1, Clark S1, Manning NJ1, Bonham JR1, Cleary M2, Scott C1, Alfadhel M3, Al Balwi M4, Allen KE5, Kirk R5, Catchpole A1, Hind H1, Sharrard M6, Yap S6, Olpin SE1 1

Dept Clin Chem, Sheffield Children's Hos, Sheffield, United Kingdom; Metabol Med, Great Ormond Street Hosp, London, United Kingdom; 3 Paed Bio Genet, King Saud Bin Abdul Uni, Riyadh, Saudi Arabia; 4Mol Path Genet, King Abdul Int Med Res, Riyadh, Saudi Arabia; 5Sheff Diagnostic Genet, Sheff Children's, Sheffield, United Kingdom; 6Paed Med, Sheffield Children's Hosp, Sheffield, United Kingdom 2

Over the past 23 years we have diagnosed >90 patients with glutarylCoA dehydrogenase deficiency expressing the results in fibroblasts as percentage of simultaneous controls. Most patients have low enzyme activity <5% (n=75), some have activities of 9-25% (n=11), a minority have high residual activity 26-45% (n=9). We have measured activity in obligate carriers 51+/-20% (n=6 range 25-83%). Many patients have been confirmed by mutation analysis. Some patients are low urinary excretors with only small amounts of 3-hydroxyglutarate and only relatively low C5DC in plasma <1.0 μmol/L. Enzyme analysis in many patients gives results that overlap with simple carriers. Several patients with high residual activity and low excretor status have been found to have novel mutation(s) of uncertain pathogenicity. We have demonstrated that these diagnostically very difficult patients can be more easily defined and separated from simple carriers by growing and assaying activity in patients and controls at 41oC. As an example a low excretor with changes of uncertain pathogenicity (homozygous c.278A>G plus homozygous c.1167G>A) gave activity of 45+/-15% (n=3) at 37oC vs 2% and 15% (n=2) at 41oC. We demonstrate that stressing mutant enzyme protein by increased temperature is an effective way of clarifying the diagnosis in diagnostically difficult patients.

Wierenga A1, Simon G1, Wierenga K1 1

OUHSC Dept, Peds, Section of Genetics, Oklahoma City, United States Background: 3-methylglutaconic acidurias, (3-MGAs) are a group of disorders in which increased urinary excretions of 3-methylglutaconic acid and 3-methylglutaric acid are common markers. Further distinction is based on molecular analysis. Methods:This case report involved an infant, born to a first cousin union, who presented with elevated C5OH acylcarnitine at newborn screening; and was well on physical examination. Confirmatory testing by urine organic acids (UOA) analysis and further investigation by molecular and bioinformatics analyses were carried out. Results: UOA showed elevated levels of 3-methylglutaconate, 3methylglutarate and 3-hydroxyisovalerate, biomarkers of 3-MGAs. SNP array revealed 226Mb of combined runs of homozygosity (ROH), consistent with consanguinity. The Genomic Oligoarray and SNP Array Evaluation Tool evaluated these ROH and revealed homozygosity for the AUH gene (3-MGA type I) and the OPA gene (3-MGA type III). The AUH gene was sequenced based on ethnicity. A novel homozygous mutation in AUH, (NM_001698, c.373C>T, p.R125W) was identified, adding to the list of mutations in patients with this condition. Oral L-carnitine was prescribed and the patient remains well, presently 2 years old. Conclusion:This case highlights that combined use of biochemical, bioinformatics and molecular analyses can be used for swift diagnosis of rare disorders and early implementation of appropriate treatment. P-224

P-222

Mevalonate kinase deficiency presenting as neonatal liver failure

Development of a GC-MS SIM/scan method for organic acid analysis

Regier DS1, Banks NA2, Leon EL1, Lichter-Konecki U1, CusmanoOzog KP3

Hofherr SE1, Cusmano-Ozog KP1 1 1

Lab Med CNMC, Washington, DC, United States

Urine organic acid analysis is typically performed by quantitative selected ion monitoring (SIM) and/or qualitative total ion chromatogram (TIC) scan. Most clinical laboratories choose to do SIM or TIC with few performing both independently for one sample. Reasons for this include cost and time for both the analytical and post-analytical processes as well as difficulty correlating between the SIM and TIC data.

Gene&Metab CNMC, Washington, DC, United States; 2NHGRI, NIH, Bethesda, MD, United States; 3Lab Med CNMC, Washington, DC, United States Mevalonic aciduria (MVA) is a rare autosomal recessive disorder of cholesterol biosynthesis caused by deficiency of mevalonate kinase (MVK). Although dysmorphism, ataxia and febrile episodes are usually the presenting signs, we describe two neonates who presented with severe liver failure.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 BG1 was born at term after an unremarkable pregnancy. Severe jaundice, transaminitis, hepatosplenomegaly, anemia and thrombocytopenia were identified shortly after birth and a diagnosis of hemochromatosis was entertained. BG2 was born prematurely; fetal ascites was noted at 28 weeks gestation. Liver failure with hepatosplenomegaly, cholestasis, anemia and thrombocytopenia were noted at birth. Upon urine organic acid analysis, both neonates had abnormal excretion of mevalonic acid and mevalonolactone consistent with a diagnosis of MVA. Molecular analysis of MVK revealed two variants (V22L/I64N) in BG1 and L297I homozygousity in BG2. Treatment with steroids following diagnosis resulted in improved clinical state including liver function. An interleukin-1 (IL-1) receptor antagonist was added in attempt to wean from steroids. Mevalonate kinase deficiency should be considered in all neonates who present with liver failure of unknown etiology. Urine organic acid anlaysis should be obtained in any neonate with abnormal or worsening liver function. Timely diagnosis can lead to early treatment improving symptomatology and facilitating genetic counseling. P-225 An infantile case of vitamin B12-responsive methylmalonic acidemia missed in newborn screening and diagnosed after presenting metabolic crisis. Hara K1, Ono H2, Tsumura M3, Kagawa R3, Okada S3, Tajima G3, Sakura N4, Hata I5, Shigematsu Y5, Kobayashi M3

S173 We previously showed in a 3D rat brain cell in vitro model for glutaric aciduria type-I that repeated application of 1mM 3-hydroxy-glutarate (3-OHGA) caused ammonium accumulation, morphologic alterations and induction of non-apoptotic cell death in developing brain cells. Here, we performed a dose-response study with lower concentrations of 3OHGA. We exposed our cultures to 0.1, 0.33 and 1mM 3-OHGA every 12h over three days at two developmental stages (DIV5-8 and DIV11-14). Ammonium accumulation was observed at both stages starting from 0.1mM 3-OHGA, in parallel with a glutamine decrease. Morphological changes started at 0.33mM with loss of MBP expression and loss of astrocytic processes. Neurons were not substantially affected. At DIV8, release of LDH in the medium and cellular TUNEL staining increased from 0.1mM and 0.33mM 3-OHGA exposure, respectively. No increase in activated caspase-3 was observed. We confirmed ammonium accumulation and non-apoptotic cell death of brain cells in our in vitro model at lower 3-OHGA concentrations thus strongly suggesting that the observed effects are likely to take place in the brain of affected patients. The concomitant glutamine decrease suggests a defect in the astrocyte ammonium buffering system. Ammonium accumulation might be the cause of non-apoptotic cell death. P-227 Altered expression and posttranslational modifications of neurofilaments in methylmalonic aciduria Cung HP1, Zavadakova P1, Braissant O2, Ballhausen D1

1

Dept. Pediatrics, Kure Medical Center, Kure, Japan; 2Dept. Pediat, Hiroshima Prefectural Hosp, Hiroshima, Japan; 3Dept. Pediatr, Hiroshima Univ Gradu Scho, Hiroshima, Japan; 4Nursing House Suzugamine, Hiroshima, Japan; 5Dept. Pediatr, University of Fukui, Fukui, Japan Background: Though newborn screening (NBS) is usually late for neonatal-onset methylmalonic acidemia (MMA), late-onset disease is expected to be a good target. To our experience, however, the frequency of such cases seems too low, suggesting inadequate sensitivity of present method for MMA. Case report: A one-year-old boy suffering from Noroviral gastroenteritis developed disturbance of consciousness accompanied by severe metabolic acidosis and mild hyperammonemia. Intensive care including continuous hemodiafiltation successfully rescued him without sequelae. Analysis of acute-phase specimens revealed highly elevated levels of C3-acylcarnitine and C3/C2 ratio in serum, and huge excretion of methylmalonate in urine. Nevertheless, methylmalonyl CoA mutase a c t i vi t y i n l y m p h o c y t e s m e a s u r e d u n d e r co e x i st e n c e o f adenosylcobalamin was not impaired, indicating vitamin B12responsive MMA, which was further confirmed by excellent effect of adenosylcobalamin administration in vivo. In NBS, C3-acylcarnitine in DBS elevated to 4.79 nmol/mL leading to false negative judgement. Discussion: Because of high frequency of mildest-form propionic academia (PA) in Japan, lower cutoff targeting late-onset MMA will supposedly increase undesirable detection of such PA cases. As a solution, we propose to use lower cutoff only for MMA by adopting GC/MS-analysis of methylmalonate in DBS as a 2nd-titer test. P-226

1

Div Mol Ped, Lausanne Univ Hosp, Lausanne, Switzerland; 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland Neurofilaments (NF), the main components of axonal cytoskeleton, are known to be involved in several neurodegenerative diseases. It has been reported that methylmalonate and propionate affect phosphorylation of NFs. In an in vitro model for methylmalonic aciduria our group has recently shown that 2methylcitrate (2-MCA) is the most toxic metabolite for developing brain cells. Here, we studied the effects of repetitive administration of 1mM 2MCA every 12 hours over 3 days on the development of NFs in 3D organotypic rat brain cell cultures. By immunohistochemistry with antibodies specific for the different NF subunits (light NFL, medium NFM, heavy NFH) as well as for phosphorylated (p) and glycosylated (g) forms of NFs, we observed a decrease of axonal labeling and a disorganized axonal pattern. Interestingly, signal retention of p-NFM and g-NFM was observed in neuronal soma. Western blotting showed the decrease of NFL and NFH subunits. Taken together, our data show that 2-MCA alters expression of the different NF subunits as well as their post-translational modifications. This likely results in disturbed NF assembly, abnormal accumulation of NF in neuronal cell bodies and impairment of axonal development. We conclude that NF are involved in 2-MCA-induced neurodegeneration in methylmalonic aciduria. P-228 Renal involvement in a patient with methylmalonic aciduria due to cblA deficiency: a 41-year follow-up

Low concentrations of 3-hydroxy glutarate leads to ammonium accumulation and non-apoptotic cell death in developing brain cells

Haarmann A1, Mayr M2, Koelker S3, Baumgartner ER4, Meienberg F5, Devuyst O6, Baumgartner MR1

Jafari P1, Zavadakova P1, Cung HP1, Braissant O2, Bonafe L1, Ballhausen D1

2

1

1

Div Mol Ped, Lausanne Univ Hosp, Lausanne, Switzerland; 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland

Div Metab Child Res Cen, Univ Child Hosp, Zurich, Switzerland; Clin Transpl Immunol, Univ Hosp, Basel, Switzerland; 3Div Inherit Metabol Dis, Univ Child Hosp, Heidelberg, Germany; 4Metabol Unit, Univ Child Hosp, Basel, Switzerland; 5Div Endocrinol, Univ Hosp, Basel, Switzerland; 6Inst Physiol, Univ Zur, Zurich, Switzerland

S174 Chronic kidney disease is a well-known long-term complication of methylmalonic aciduria (MMA-uria), occuring even under optimal metabolic management. The onset of renal dysfunction seems to be dependent on the type of defect and hydroxocobalamin-responsiveness. We report on a female patient with a hydroxocobalamin-responsive MMAuria due to a stop mutation (p.R145X) in the MMAA gene (cblA), diagnosed at age 8 month and initially treated with a low protein diet. At 12 years chronic kidney disease stage III was diagnosed. Following re-evaluation for hydroxocobalamin-responsiveness, she was treated with hydroxocobalamin, resulting in a significant decrease in the urine and plasma concentration of MMA by oral and more efficiently i.m. application. For the following 17 years glomerular filtration rate remained stable. Kidney biopsies showed non-specific manifestations of tubulointerstitial nephritis. Aged 29 years, however, the patient started hemodialysis for end-stage renal disease. She received a cadaveric renal transplant at age 35. She is still treated with hydroxocobalamin and a low protein diet. There is no evidence of relapsing tubulointerstitial nephritis or chronic kidney disease so far. This case report illustrates the longest follow-up of a patient with MMAuria due to CblA deficiency and highlights the importance of early and adequate hydroxocobalamin supplementation in responsive patients. P-229 Quality control of analytical performance by the ERNDIM quantitative organic acids scheme: highlights and recommendations

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 Methylmalonic aciduria cblB type is caused by mutations in t h e M M A B g e n e t h a t e n c o d e s t h e AT P : c o b ( I ) a l a m i n adenosyltransferase (ATR). ATR is responsible for the synthesis of adenosylcobalamin, the cofactor for the mitochondrial enzyme methylmalonyl-CoA mutase (MUT). Until now, no definitive treatment exists and although pharmacological B12 administration is used to ameliorate symptoms, only about 40% of the patients present a positive response. The presence of mutations causing destabilizing effects has been reported and hence, the main goal of our work was to seek pharmacological chaperones as a tailored therapy for stabilizing the ATR protein. High-throughput ligand screening of over 2000 compounds was performed and further studies using a bacterial system of recombinant ATR protein expression showed that compound V (N-{[(4-chlorophenyl)carbamothioyl]amino}-2phenylacetamide) increased stability and did not act as an inhibitor of the purified protein. It also increased ATR activity in patient-derived fibroblasts to control range. When cobalamin was co-administrated with compound V, mutant ATR activity further improved. Oral administration of compound V to C57BL/6J mice for 12 days increased steady-state levels of ATR protein in liver and brain. These results hold promise for the clinical use of pharmacological chaperones in MMA cblB type patients harboring chaperoneresponsive mutations. FCT-SFRH/BD/45753/2008

Martens GA1, Weykamp C2, Scott C3, Duran M4 Universitair Ziekenhuis Brussel (VUB), Brussels, Belgium; 2Stichtig Kwaliteitsbewaking Ziekenhuis L, Winterswijk, Netherlands; 3Sheffield Children's Hospital, Sheffield, United Kingdom; 4Academic Medical Center, Amsterdam, Netherlands

P-231

Background: Quantification of urinary organic acids is important for diagnosis and monitoring of many Inborn Errors of Metabolism. ERNDIM provides a quality control scheme that evaluates analytical proficiency for 20 clinically relevant organic acids Here we discuss the highlights of the 20082012 cycles. Highlights: from 2008-2012, participation increased by 40% to 107 labs (30 countries). Still, participation remains 2-fold lower than in the qualitative schemes, indicating that not all labs feel the need for quantification. Intra-lab imprecisions (CV%) declined from 20 to 16%, but variations remained large: CV% ranged from ±10% (3-methylglutaric, ethylmalonic) to >25% for 3hydroxyisovaleric, N-acetylaspartic, and 4-hydroxybutyric (4-HB) acids. 4-HB also showed poor recovery (73% versus average of 89%), which is compatible with its consistently poor identification in diagnostic proficiency schemes, potentially leading to missed diagnosis Also the inter-lab imprecisions declined (54% to 38%), but remained very high: for some compounds 4-fold (4hydroxybutyric, mevalonic, sebacic, glyceric acids) higher than their respective intra-lab CV%, indicating lack of standardization of sample extraction. Conclusions: (i) Analytical performance steadily improved, (ii) indicating the educational value of participation to this quantitative scheme. (iii) Inter-lab imprecisions remain disproportionately high, calling for further standardization.

Ruppert T1, Opp S1, Tagscherer KE2, L K3, Gröne HJ4, Hoffmann GF1, Kölker S1, Okun JG1, Morath MA1, Sauer SW1

1

P-230 Pharmacological chaperones as a potential therapeutic option in methylmalonic aciduria cblB type Brasil S1,2, Jorge-Finnigan A1,3, Underhaug J3, Ruíz-Sala P1, Merinero B1, Benerjee R4, Desviat RL1, Ugarte M1, Martinez A3, Pérez B1 1 CEDEM, CBMSO, UAM - CIBERER, IDIPAZ, Madrid, Spain; 2iMed, FFUL, Lisbon, Portugal; 3Dep Biomed, University of Bergen, Bergen, Norway; 4Dep. Biol. Chem., University of Michigan, Michigan, United States

Activation of autophagic and proinflammatory cascades in proximal tubule of methylmalonic aciduria patients

1 Div Inh Metab Dis, Univ Child Hosp, Heidelberg, Germany; 2Mol Tumor Path, DKFZ, Heidelberg, Germany; 3Tumor Immunology, DKFZ, Heidelberg, Germany; 4Dep Cell Mol Path, DKFZ, Heidelberg, Germany

Background: Patients with mut0 type methylmalonic aciduria often develop chronic renal failure involving chronic tubulointerstitial nephritis (cTIN). The mechanism underlying inflammation of proximal tubular epithelial cells (PTEC) is unknown. Therefore, we established a cell bank of PTEC from mut0 patients and controls and investigated different factors activating and promoting inflammation, namely autophagy markers, cytokines and electron microscopy. Methods: Purified and immortalized PTEC (iPTEC) of patients and controls were analyzed for autophagic markers (Western blots) and ROS production (FACS). Morphologic changes of iPTEC were studied by electron microscopy (EM). Chemokines and cytokines where investigated by ELISA. Results: In iPTEC of mut0 patients we found decreased phosphorylation and activation of mTOR complex 1 proteins. In line with this, EM revealed evidence of increased autophagic activity. Macroautophagic markers SQSTM1 and LC3-II were expressed. ELISA analysis showed a greater amount of secreted interleukin-8 and CCL2 in patient cells compared to controls highlighting proinflammatory activity and increased ROS production. iPTEC secrete proinflammatory cytokines attracting infiltrating macrophages and other mononuclear cells. Conclusion: Our data suggests that tubulointerstitial nephritis in mut0 patients involves enhanced macroautophagy and ROS

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 production resulting in chronic inflammation, cTIN and, finally, chronic renal failure. P-232 Detection of aminoacidopathies and organic acidopathies in a highly cansanguineous population; first study from Pakistan Ghani F1, Khan AH1, Dar FJ1, Jamil A1, Khan NA1, Maher R1, Afroze B1 1

The Aga Khan University, Karachi, Pakistan

Background: We report different types of aminoacidopathies and organic acidurias in children from parents who have a high rate of consangineuos marriages. Methods: Amino acids were quantified using Ion exchanged HPLC on Biochrom 30+ and organic acidurias were detected using gas chromatography-mass spectrometry on Agilent analayzer. Plasma amino acids and urine organic acidurias reported between month of January and April 2013 were analyzed. Results: Total of 89 patients, 49 males (45%) and 40 (55%) females were tested for Inborn error of metabolism (IEM). Age ranged from 1 day to 33 years with mean age around 4 years. Most common reason for testing these patients was developmental delay 47 (53%). History of consanguinity was found in 55 (62%). A total of 7 cases had low methionine level which may be due to cobalmin defect or deficiency, 3 had 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, 2 had hyperphenylalaninemia, 1 case each had cystathionine beta synthase deficiency, propionic academia, ethylmalonic aciduria, and isovaleric aciduria. Conclusion: Children born to parents with a high rate of consanguineous marriage are expected to have a higher rate of metabolic disorders. There is a need for more wide-spread screening for IEM in such a population. P-233 Low myocardial coenzyme Q10 status provides evidence that secondary mitochondrial dysfunction is an underlying mechanism for cardiomyopathy in propionic acidaemia. Baruteau J1, Hargreaves I2, Krywawych S3, Chalasani A2, Land JM2, Davison JE1, Kwok MK1, Christov G4, Karimova A4, Ashworth M5, Anderson G5, Rahman S6, Grunewald S1 1 Metab Med Dep, Great Ormond Street Hosp, London, United Kingdom; 2Metab Lab, Nat Hosp Neurol & Neurosurg, London, United Kingdom; 3Chemical Path, Great Ormond Street Hosp, London, United Kingdom; 4Cardiothor Dep, Great Ormond Street Hosp, London, United Kingdom; 5Pathology Lab, Great Ormond Street Hosp, London, United Kingdom; 6Clin & Mol Gen Unit, Inst Child Health, London, United Kingdom

Dilated cardiomyopathy is a rare complication in propionic acidaemia (PA). Underlying pathophysiological mechanisms are poorly understood. We present a child of Pakistani consanguineous parents, diagnosed with late-onset PA at 18 months of age. He presented a mild phenotype, showed no further decompensations, normal growth and psychomotor development on a low protein diet and carnitine supplementation. At 15 years, a mild dilated left ventricle was noticed. At 17 years, he presented after a short history of lethargy with severe decompensated dilated cardiomyopathy. He was stabilised on inotropic support and continuous

S175 hemofiltration; a bi-ventricular assist device (Berlin Heart Excor) was implanted. He received D,L-Hydroxybutyrate 200mg/kg/d, riboflavin and thiamine 200mg/d each and coenzyme Q10 (CoQ10). Myocardial biopsy showed endocardial fibrosis, enlarged mitochondria, with atypical cristae and slightly low respiratory chain complex IV activity relative to citrate synthase (0.012, N:0.014-0.034). Myocardial CoQ10 was markedly decreased (224pmol/mg, N:942-2738), with a marginally decreased white blood cell level (34 pmol/mg N:37-133). The dose of CoQ10 was increased from 1.5 to 25mg/kg/d. Cardiomyopathy slowly improved allowing removal of the external mechanical cardiac support after 67 days. We demonstrate for the first time low myocardial CoQ10 in cardiomyopathy in PA, highlighting secondary mitochondrial impairment as a relevant causative mechanism. P-234 D-lactic aciduria: an inborn error of metabolism after all? Jans JJ1, van Eerde A1, Lichtenbelt K1, Duran K1, Monroe G1, Gerrits J1, van Roosmalen M1, van Aalderen M1, Koot B2, Oostendorp M1, Visser G1, Nijman I1, Knoers N1, de Koning T3, Cuppen E1, van Haaften G1, Verhoeven-Duif N1 1

University Medical Center Utrecht, Utrecht, Netherlands; 2Academic Medical Center, Amsterdam, Netherlands; 3University Medical Center, Groningen, Netherlands We present a patient with mental retardation and blindness. Metabolic investigation revealed a consistently elevated excretion of D-lactate (1713mmol/mol kreatinine n<11) in urine, with a normal excretion of L-lactate. D-lactate concentration was also elevated in plasma (687uM, n<89). D-lactate in humans may originate from glyoxylate metabolism or from bacteria. Increased levels of D-lactate are usually regarded as having a bacterial origin. Here, there was no indication for bacterial overgrowth, and repeated antibiotic treatment did not alter D-lactate excretion. We hypothesized that a genetic mutation could underlie the observed D-lactic aciduria. We performed homozygosity mapping and identified large stretches of homozygosity, indicative of consanguinity. One homozygous stretch contained a gene with homology to D-lactate dehydrogenases from lower organisms. With Sanger sequencing we subsequently identified a homozygous missense variant Thr463Met in the putative human lactate dehydrogenase D (LDHD), which is predicted to have a deleterious effect on protein function. The phenotype associated with an LDHD mutation is unclear since this patient carries an additional chromosomal anomaly that can explain the clinical presentation. We conclude that humans do have endogenous D-lactate dehydrogenase activity. Whether LDHD deficiency is a biochemical anomaly or whether a clinical phenotype is associated with it remains to be elucidated. P-235 A case with hemolytic uremic syndrome related to cobalamin C defect Tokatlı A1, Ünal Ö1, Kalkanoğlu-Sivri HS1, Dursun A1, Hişmi B1, Düzova A2, Talim B3, Coskun T1 1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey; Hacettepe Univ Child Hosp, Div Nephrol, Ankara, Turkey; 3Hacettepe Univ, Dept Ped Pathology, Ankara, Turkey

2

S176 Cobalamin C defiency is one of the underlying etiologic factors of non-Shiga toxin associated hemolytic uremic syndrome (HUS). A two months old boy presented with hypotonia, irritability, diarrhea, edema of the eyelids, and failure to gain weight. He had been transfused with erythrocyte suspension for three times in another center. His parents were third cousins and her sister had died with hemolytic anemia, hematuria and renal failure. On his laboratory findings, pancytopenia, lactic acidemia, hematuria, proteinuria and hyperhomocysteinemia were detected. B12 level was normal. In the hospitalization period, hemolytic anemia worsened, homocysteine level elevated up to (298 μmol/L, N<15), and, first, renal failure then multiorgan failure occured. On tandem mass spectroscopy, carnitine was low, and, on urine organic acid analysis, 8-folds elevated methylmalonic acid was found. Cranial magnetic resonance imaging showed hydrocephalus. He followed with suspected B12 metabolism defect and HUS. Mechanical ventilation and dialysis treatment was started. Hydroxycobalamin, folic acid and betain treatments were started. With treatments, homocystein level and hemolysis were improved, but, he died in first month of hospitalization. Molecular genetic analysis showed 271dupA p.Arg91Lysfs mutation in the MMACHC gene, and cobalamin C defect was confirmed. Cobalamin C defect should be considered in atypic HUS patients. P-236 Functional assessment in patients with maple syrup urine disease with pediatric evaluation of disability inventory scale Ünal Ö1, Kerem-Günel M2, Mutlu A2, Kaya-Kara Ö2, Hişmi B2, Dursun A1, Tokatlı A1, Coskun T1, Kalkanoğlu-Sivri HS1 1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey; Hacettepe Univ, Dept Physiotherapy Rehab, Ankara, Turkey

2

Background: As like most of the inborn errors of metabolism, patients with maple syrup urine disease have risk of metabolic crisis in catabolic states. Accumulation of branched chain amino acids and corresponding keto acids leads to encephalopathy and brain injury. In patients who are not diagnosed and treated early, especially with classical MSUD, developmental delay occurs. MSUD may be considered one of the treatable causes of developmental delay and intellectual disability. Pediatric Evaluation of Disability Inventory (PEDI) is a scale that evaluates key functional capabilities and performance in children ages six months to seven years in the areas of selfcare, mobility and social function. PEDI provides resources to assess a child on functional skills, caregiver assessments and modifications. The scales can be used concurrently or independently of one another depending on the domain of interest for each child. Methods: Functional skills subdivisons of self care, mobility, and social functions were used in 36 children with MSUD. Results: In all age groups, the patients frequently have disability in functional skills of three different areas. Conclusion: Newborn screening program should be expanded to prevent disabilities in countries in which newborn screening programs are limited.

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 P-237 Protection of human methylmalonyl-CoA mutase from oxidative inactivation by MMAA and physiological evidence of their interaction Takahashi-Iñiguez T 1 , Gonzalez-Noriega A 2 , Michalak C 2 , Fernandez-Lainez C3 , Vela-Amieva M 3, Flores ME1 1

Dep Mol Biol Biotechnol, Inst Biomed Res, Mexico City, Mexico; Dep Cell Biol, Inst Biomed Res, Mexico City, Mexico; 3IEM Screen Lab, Nat Inst Pediatr, Mexico City, Mexico

2

Background: Human MMAA protein is a GTPase, which acts as a human adenosylcobalamin (AdoCbl) dependent Methylmalonyl-CoA mutase (MCM) chaperone. Mitochondrial location of MMAA and its in vivo interaction with MCM has not been demonstrated, neither the possible role of this interaction. Objetive: Demonstrate mitochondrial location of MMAA, its colocalization with MCM in human fibroblasts, and protection of AdoCbl-MCM against inactivation. Material and Methods: cDNA of both human genes was cloned and recombinant proteins were purified. These proteins were used to raise antibodies in rabbit; serum obtained was used for double immunofluorescence experiments in human fibroblasts. UV-Vis spectra of AdoCbl-MCM reaction were recorded in absence and presence of MMAA. Results: Results showed that MMAA protein was located both, in mitochondrial and cytoplasm as well as MCM. Only in mitochondria both proteins showed colocalization. UV-Vis spectra showed AdoCbl-MCM oxidative inactivation by increase in absorbance between 300-400 nm, which decreased in presence of MMAA. Conclusion: For the first time, localization of MMAA in both cytoplasm and mitochondria was demonstrated, as well as its colocalization with MCM in human fibroblasts. Furthermore UV-Vis spectra showed the in vitro oxidative inactivation of human MCM and the protective effect of MMAA during catalysis. P-238 Disturbance of glutathione metabolism precedes renal failure in patients with vitamin B12 non-responsive methylmalonic aciduria Kwok MK1, Oppenheim M2, Cleary M1, Abulhoul LH1, Gissen P1, Broomfield A1, Aitkenhead H3, Prunty H3, Van't Hoff W4, Shroff R4, Grunewald S1 1

Metab Dept, Great Ormond Street Hospital, London, United Kingdom; 2NMU, Nat Hosp Neurology & Neurosurgery, London, United Kingdom; 3Chem Path Dept, Great Ormond Street Hosp, London, United Kingdom; 4Nephro Dept, Great Ormond Street Hosp, London, United Kingdom Patients with vitamin B12 non-responsive (VitB12NR) methylmalonic aciduria (MMA) commonly develop chronic kidney disease (CKD). We

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342 examined the relationship between their glutathione status, an oxidative stress marker, and renal function. Methods: Data of 40 MMA patients of GOSH (1991-2013) were reviewed. 8 were excluded for <2 years' follow-up. Among 32 included (16 males, 16 females; median follow-up 9 years, range 2-19 years), 20 were VitB12NR; 7 died before end-point (median lifespan 7.9 years). Glutathione and renal function including glomerular filtration rate (GFR) were monitored during annual follow-up when they were well. Results: 31 patients had GFR monitoring. 14 VitB12NR patients developed CKD (GFR <60ml/min/1.73m2; mean age 4.2 years; range 1.7-8.3 years); 5 progressed to end-stage renal disease (mean age 6.2 years; range 2.7-8.6 years). Patients in CKD had low median glutathione of 473.50uM (reference: 525-1650uM) and reduced:oxidized glutathione ratio (GSH:GSSG) of 5.46 (reference: 38.09). Median glutathione of VitB12NR patients without CKD was much lower than that of the VitB12-responsive patients (575.25uM vs 771.00uM; p<0.05), but only slightly higher than that of CKD patients (p=0.275). Discussion: This study highlighted oxidative stress, preceding to development of CKD, in VitB12NR MMA. Further study is needed to examine its role in pathogenesis of CKD. P-239 Glycerol kinase over expression in mouse liver leads to increased fat deposition and obesity Dipple KM1, Badjatiya A1, Ho CK2 1

Dept Human Genetics, UCLA, Los Angeles, United States, 2Bioengineering IDP, UCLA, Los Angeles, United States

S177 P-240 Development of neuropsychologic functions in patients with glutaric aciduria type I Boy N1, Heringer J1, Hoffmann GF1, Haege G1, Glahn E1, Burgard P1, Kölker S1 1

Div Metab Dis, Univ Child Hosp, Heidelberg, Germany

Background: Treatment according to evidence-based guideline recommendations has significantly improved the neurological outcome in glutaric aciduria type I. However, cognitive functions in these patients have not yet been studied in detail. Patients and methods: We investigated 31 patients (age range 5-27 years) - detected by newborn screening (n=14), high-risk family screening (n=3) or after manifestation of neurologic symptoms (n=13) with tests for (1) simple reaction time/SRT, (2) divided attention/DT, (3) choice reaction time/CRT, (4) visual working memory/VWM, (5) tracking/TT, and (6) memory search/MS. Dystonia was present in 13 study patients (42%). Patients were compared to a control group of healthy probands (n=164) as well as to age-matched-pairs. Results: GA-I patients showed poorest results in simple motor reaction time. Although not always significant, patients revealed a consistently negative SD pattern in all other tests. Dystonic and non-dystonic patients did not differ. Across all age groups data of controls and patients (dystonic and asymptomatic) fitted equally well to an exponential function. Conclusions: Results indicate normal developmental functions in GA-I patients, however, with increased reaction times. P-241 The effect of glutaric acid derivatives on gutamate-glutamine metabolism in astrocytes Komatsuzaki S1, Ruppert T1, Opp S1, Okun J1, Kölker S1, Sauer S1 1

Glycerol kinase deficiency (GKD) is an X-linked inborn error of metabolism with no genotype-phenotype correlation. GK phosphorylates glycerol but also has other protein functions that may explain the pathogenesis of GKD. We developed a liver-specific Gyk transgenic mouse to understand the functions of GK, including its role in adipogenesis and obesity. Male wildtype (WT/WT), heterozygous (WT/T) and homozygous (T/T) transgenic mice were placed on chow or high fat (HF) diet for 12 weeks and monitored for weight gain, percentage body fat, and standard blood tests. WT/T and T/T mice on HF diet gained more weight, 2.8% and 11.3% respectively, than WT/WT mice (p<0.05). Nuclear magnetic resonance analysis revealed all mice had at least two-fold increase in percentage body fat on HF diet. T/T and WT/T mice on HF diet had greater body fat gains than WT, and this correlated with increased tissue mass [liver 12.5%, visceral fat 22.2%, perirenal fat 16.3%] (p<0.05). T/T mice also had elevated triglycerides, cholesterol, and fasting glucose levels implying high risk for obesity and type II diabetes mellitus. In conclusion, this transgenic Gyk mouse model demonstrates that GK is involved in fat deposition, adipogenesis, and increases risk of obesity and type II diabetes mellitus.

University Children' Hospital Heidelberg, Heidelberg, Germany

Background: Glutaric aciduria type I, D-2-, L-2- and D-2-/L-2hydroxyglutaric acidurias are cerebral organic acidurias which are characterized biochemically by accumulation of putatively neurotoxic five carbon dicarboxylic acids sharing the carbon backbone with glutamate and glutamine. We have investigated the effect of glutaric acid (GA), D-2and L-2-hydroxy-GA on glutamate-glutamine metabolism in astrocytes. Methods: Lactate dehydrogenase (LDH) levels in the medium were determined to detect the cytotoxic effect of glutaric acid (GA) on astrocytes with/without CoCl2, which induce chemical hypoxia. Purified glutamine synthetase and glutamate dehydrogenase (GDH) activity were measured with different concentration of GA, 3-hydroxy-GA, L2-hydroxy-GA and D-2-hydroxy-GA. Results and Conclusion: Cell viability was decreased by exposure to 2 mM GA, an effect enhanced by coincubation with CoCl2 which induces chemical hypoxia. Furthermore, GA, 3-hydroxy-GA, L-2-hydroxy-GA and D-2-hydroxy-GA inhibited GDH activity competitively. GDH catalyzes reversible oxidative deamination of glutamate to 2-oxoglutarate. Glutamate-glutamine metabolism is an important bioenergetic coupling between astrocytes and neurons which is impaired by GA and hydroxyGAs. Our results add to previously demonstrated inhibition of tricarboxylic

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acid cycle and dicarboxylic acid shuttle by these compounds thereby supporting the notion of impaired energy metabolism playing an important role in the pathophysiology of cerebral organic acidurias.

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Magnetic resonance spectroscopy before and after encephalopathic crises in glutaric aciduria type I patients homozygous for the glutaryl-coa dehydrogenase IVS-1+5G>T mutation

Impact of the chilean food complementary program (PNAC), in children with propionic (PA) and methylmalonic acidemia (MMA) first year results

Leung ECW1, Venugopal N2, Bunge M3, Ryner L4, Mhanni AA1, Greenberg CR1

Castro G1, Arias C1, Cabello JF1, Raimann E1, Hamilton V1, Valiente A1, Betta K1, De la Parra A1, Colombo M1, Cornejo V1 1

INTA, Univ de Chile, Santiago, Chile

Introduction: PA occurs due to a deficiency of Propionyl CoA carboxylase enzyme and MMA due to a deficit of Methylmalonyl CoA Mutase or their cofactors. The treatment is a protein-restricted diet, special formula without methionine, threonine, valine and isoleucine (MTVI), supplementation with L-Carnitine, biotin and B12. Objective: To determine the impact of formulas without MTVI subsidized by the PNAC. Methodology: We compared pre and post PNAC data regarding calorie intake, protein, MTVI, calcium, iron, zinc, anthropometry (weight and height), ammonium level and acylcarnitines. Statistical analysis was performed using SPSS. Results: 11 patients were admitted with PA and 4 MMA, age range between 2 and 18 years. There was a statistically significant difference in the contribution of special formula (average of 1 ± 0.7gr/kg/día increased) and a significant correlation with size. Also significant differences regarding supplementation of L-carnitine, MTVI and minerals. The size increased by 5.6 ± 4.4 cm and weight increased by 3.4 ± 5.4 kg, significant differences when compared with pre-admission. Conclusion: Admission to the PNAC has positively impacted children with PA and MMA, reflected in the size and weight gain. However longterm evaluations are required.

Dept Paediatrics, Univ Manitoba, Winnipeg, MB, Canada; Sunnybrook Health Sciences Centre, Toronto, ON, Canada; 3Dept Radiology, Univ Manitoba, Winnipeg, MB, Canada; 4CancerCare Manitoba, Winnipeg, MB, Canada Background: Magnetic resonance spectroscopy (MRS) has been described as a potential functional non-invasive neuroimaging technique to detect neurologic striatal injury in patients with glutaric aciduria type I (GA-1). Decreased N-acetyl-aspartate/creatinine ratio (NAA/Cr) has been seen as a marker for reduction in neuronal integrity and neuronal loss, seen in the majority of GA-1 patients with encephalopathic crises. Objectives: To understand the MRS abnormalities associated with striatal and white matter injury in GA-1 patients with the Manitoba variant of GA-1 Method: 8 Manitoba patients with GA-1 underwent a total of 11 singlevoxel hydrogen MRS. Results: 2 asymptomatic patients without encephalopathic crisis had normal MRS. Only 1 out of 3 patients in our cohort demonstrated decreased NAA/Cr on MRS during encephalopathic crisis. 3 more patients had a history of significant encephalopathic crises 7-15 years prior to their MRS and had normal NAA/Cr. Conclusion: Although decreased NAA/Cr can be seen in MRS of GA-1 patients during an encephalopathic crisis, it does not appear to be a consistent feature in our small cohort. Contrary to previous hypothesis, the absence of decreased NAA/Cr in chronic symptomatic patients suggests that decreased NAA/Cr is a marker for acute biochemical disturbance rather than differential neuronal loss in GA-1. P-245

P-243 Primary hyperoxaluria type 1: organic aciduria diagnosed in plasma Valongo C1, Rodrigues M2, Dias AJ1, Vilarinho L1 1

1 2

Clinical and genetic investigation of 6 Iranian cases of glutaric aciduria type1 Rahmanifar A1, Moarefian S1, Tehrani M1

2

Newb Screen, Metab, Gen Unit, INSA I.P., Porto, Portugal, Rheumatology Service, HUC, Coimbra, Portugal Background: Primary hyperoxaluria Type 1 (PH1) is a rare autosomal recessive inborn error of glyoxylate metabolism, caused by a deficiency of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase. The disorder results in overproduction and excessive urinary excretion of oxalate, causing recurrent urolithiasis and nephrocalcinosis. Patient and methods: The patient, a 38 year-old-woman, was referred to our lab with severe arthritis in the hips and knees, calcinosis and stage V chronic renal failure under hemodialysis. No urine samples were available to perform organic acids analysis so we studied patient plasma. Samples were extracted with ethylacetate and analyzed by GC-MS. Results: Plasmatic organic acids profile in two different samples revealed a marked concentration of oxalate (131 and 125 μmol/L; controls: 0-5) and glycolate (362 and 338 μmol/L; controls: 9-42). Glycerate concentration was normal (17 and 15 μmol/L; controls: 0-24). Conclusions: The usual biochemical indicator of PH1 is a persistently and markedly elevated urine oxalate. In the absence of urine samples, this biochemical diagnosis can also be done in plasma samples. PH1 is a treatable organic aciduria and an early and accurate diagnosis preserves renal function of the patients. So, it is important to screen for PH1 in patients with recurrent urolithiasis or unexplained renal insufficiency.

1

Pediatr. Metab. Clinic, Tehran, Iran, Islamic Republic of

Background: Glutaric Aciduria type l is an autosomal recessive disorder caused by mutations in glutaryl-Co A dehydrogenase gene(GCDH). Although newborn screening can reduce adverse outcomes, it is not done in Iran. We investigated clinical and genetic aspects of 6 Iranian GA-1 patients. Methods: The diagnosis was made by urinary organic acid and/or acylcarnitine analysis .Genomic DNA was extracted from peripheral blood lymphocytes. Results: Six Patients'(4 ♂,2♀) mean diagnosis age was 6.5±1.6 yrs. All 4 couples were first cousin. Presentations: Macrocephaly6/6, Developemental Delay /Regression & Dystonia 4/6, Normal Development 2/6. MRI: Hydrocephaly 6/6, Frontotemporal Atrophy4/6, Temporal fossa arachnoid cysts2/6 . Mean urine Glutaric acid ,3-OH Glutaric acid levels: 2051.5± 1026,29.5±19.4 mmol/molcr. respectively. DNA analysis: 2 siblings homozygote 181G>C, One homozygote 881G>A, One heterozygote 1204C>T & 707T>C, the other 2 sibling results are pending. Except one previously reported mutation (1204C>T by Bierly et al 1996) others were new in Iran. After therapy among 4 symptomatic patients the heterozygote one had repeated metabolic acidosis attacks and others remained unchanged. Conclusion: Our study indicates necessity of newborn screening to lower diagnosis age of GA-1 in Iran, also suggests that different presentation and treatment response may be due to mutation diversity which should be noted in therapy and genetic consultation.

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Comparison of gene therapy vectors in a new hypomorphic model of propionic acidemia

Cervical spondylosis and myelopathy: a complication of glutaric aciduria type 1

Guenzel AJ1, Hofherr SE2, Hillestad M1, Kraus JP3, Barry MA1

Cordeiro D1, Blaser S2, Clarke J2, Drake J2, Feigenbaum A2, Raiman J2, Siriwardena K2, Mecija M1

1 Mayo Clinic, Rochester, United States; 2National Children's Hospital, Washington DC, United States; 3University of Colorado, Aurora, United States

1

Clin & Met Gen, Hosp Sick Child, Toronto, Canada; 2Clin & Met Gen, Hosp Sick Child & U of T, Toronto, Canada

The development of new therapies for propionic acidemia (PA) is limited by the lack of an adult model of the disease. We generated a hypomorphic mouse model of PA using a transgene bearing an A138T mutant of the human PCCA protein in Pcca-/- null mice. Pcca-/-(A138T) mice have 2% of wild-type PCC activity, survive to adulthood, and have significant elevations in many biomarkers including propionyl-carnitine (C3), methylcitrate, glycine, and ammonia as well as cardiac and neurological defects. To test these mice as a model for PA gene therapy, adenovirus (Ad) and adeno-associated virus (AAV) vectors were engineered to express codon-optimized human PCCA. Ad5 mediated more rapid increases in PCCA protein and PCC activity in the liver than AAV; both vectors reduced C3 and methylcitrate levels. Phenotypic correction was transient with first generation Ad whereas AAV8 mediated long-lasting effects. With this model-vector system combination we hope to identify the vector most suited for PA gene therapy. Additionally, this model also has the potential to probe many features of PA and to elucidate the role of cell autonomous PCCA deficiency in relation to tissue-specific correction during gene therapy for the disease.

Glutaric aciduria Type 1 (GA-1) is an autosomal recessive organic aciduria resulting in toxic encephalopathy and brain damage, typically involving the basal ganglia. We present two patients diagnosed with symptomatic GA-1 in the first year of life with severe dystonia and spasticity that required intrathecal baclofen therapy who were later found to have incidental findings of cervical spondylosis and myelopathy during imaging in adolescence. Patient 1 was noted to have cervical abnormalities at 12 years of age on imaging to assess a decreased level of consciousness with an intercurrent illness. Patient 2 was noted to have abnormalities at 13 years of age on imaging to assess positional apnoea. We believe this complication has not been previously described in GA1 but is a noted complication in severe movement disorders involving the neck particularly in adulthood. It represents an extra potential source of morbidity and creates new challenges in managing these patients. We review the need to monitor individuals with GA-1 and other inherited metabolic disorders associated with severe dystonia.

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2-hydroxyglutarate is a strong serum biomarker for IDH1/2 mutations in de novo acute myeloid leukemia

Astrocytes influence survival and development of neurons obtained from wild type and GCDH KO mice Olivera S1, Ribeiro CAJ2, Isasi E1, Woontner M3, Goodman SI3, Wajner M2, Barbeito L4

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Ottolenghi C1,2, Janin M1, Mylonas E3, Saada V4, Koscielny S5, Renneville A6, Dombret H7, Barouki R1, Rabier D2, Bernard O3, Penard-Lacronique V3, de Botton S8 Metab Biochem, Descartes Univ. of Paris, Paris, France; 2Metab Biochem, Necker Hospital, AP HP, Paris, France; 3INSERM U985, Institut Gustave Roussy, Villejuif, France; 4Biol Pathol Med, Institut Gustave Roussy, Villejuif, France; 5Biostat Epidemiol, Inst. Gustave Roussy, Villejuif, France; 6Biol Hematol, CHRU Lille, ALFA group, Lille, France; 7Hematology, Saint-Louis Hosp, ALFA gp, Paris, France; 8 Hematology, Institut Gustave Roussy, Villejuif, France 1

1

2

NBCM, Instituto Clemente Estable, Montevideo, Uruguay; Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 3School Med Univ of Colorado, Denver, United States; 4Institut Pasteur, Montevideo, Uruguay Since glial role in glutaric acidemia I (GA-I) pathogenesis is mostly unknown, the present work investigated the effects of cultured astrocytes under basal conditions or exposed to GA or lysine on neuron survival. Confluent cortical astrocytes from newborn GCDH KO and wild type (WT) mice were exposed to 5 mM GA or 10 mM lysine during 72 h. Culture media was completely removed and 1 postnatal day WT or KO cortical neurons were seeded on top of astrocytes. Under basal conditions, KO neurons live 25% less than WT neurons, both in KO and WT astrocytes. In addition, KO neurons seeded on top of KO astrocytes are 50% smaller and 30% less ramified than those seeded on WT astrocytes. Furthermore, the viability of WT neurons decreased by 18 to 30% when co-cultured on KO or WT astrocytes submitted to 5 mM GA or 10 mM LYS. On the other hand, the number of living KO neurons co-cultured on GAtreated KO astrocytes decreased 30% related to those seeded on control KO astrocytes. Therefore, KO neurons seem to have an increased intrinsic vulnerability when compared to WT neurons and KO astrocytes seem to be less supportive to neurons, especially to KO neurons.

Background: Mutations in the genes encoding IDH1 and IDH2 produce high levels of 2-hydroxyglutarate (2HG). We asked whether, in acute myeloid leukemia (AML), serum 2HG predicts the presence of IDH mutations at diagnosis, and provide a marker of follow-up. Patients and Methods: Serum samples of 82 patients at diagnosis of de novo AML (IDH mutated n=53), 50 non-AML controls and 18 patients with inherited metabolic disorders, were analyzed for total 2HG and its L and D stereoisomers by gas chromatography/ mass spectrometry. Thirdy-six AML patients were studied during follow-up. Results: Total 2HG serum levels, the D stereoisomer and the D/L ratio were all significantly higher in AML patients with IDH1/2 mutations relative to IDH wild type AML patients. The area under curve [AUC] was 99%. Quantification of the D stereoisomer provided greater specificity (98%) at 100% sensitivity (p=0.02). Total serum 2HG levels showed significant positive correlation with molecular markers of tumor mass or minimal residual disease (IDH

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allele burden, WT1 expression, NPM1 mutation; p<0.01). Correlation increased with the D/L ratio (p<0.001). Conclusions: Serum 2HG is an inexpensive powerful predictor of IDH mutations and a strong marker of follow-up. Discrimination between the D and L stereoisomers markedly improved the test's performance. P-250 Aminoacylase I deficiency due to ACY1 mRNA exon skipping: the first Italian case Ferri L1, Funghini S2, Fioravanti A3, Biondi EG3, la Marca G1, Guerrini R1, Donati MA4, Morrone A1 1 Univ Florence and Meyer Child Hosp, Florence, Italy; 2Ped Neur Unit and Labs, Meyer Child Hosp, Florence, Italy; 3Int Res Instit, CNRS,Univ Lille Nord, Lille, France; 4Metab and Musc Unit, Meyer Child Hosp, Florence, Italy

Background: aminoacylase 1 (ACY1) deficiency is a rare hereditary metabolic disorder mainly associated to neurologic symptoms such as psychomotor delay, mental retardation or seizures. Muscular hypotonia, lack of speech development, growth delay and autistic behaviour have also been reported. Currently, less than twenty patients worldwide have been described so far. Case report and Methods: a six year old Italian female, first child of consanguineous parents, presenting with mild psychomotor retardation, severe speech delay and hypotonia. Urinary organic acids were analyzed as trimethylsilyl-derivatives by GC/MS and molecular analysis of ACY1 gene and mRNA. Results: The analysis of patient's urinary organic acids showed excretion of N-acetylated derivatives of glutamic acid, leucine, valine and isoleucine. Sequencing analysis of ACY1 gene identified, at the homozygous level, a new deletion c.1001_1001+5_del6 encompassing the intron 13/exon13 boundary. Sequencing of patient's ACY1 mRNA revealed exon-skipping of entire exon 13, causing the frameshift mutation p.Lys308Glufs*7. Conclusions: we report the first Italian case of ACY1 deficiency with a relatively severe phenotype. Due to the scarce number of ACY1 deficiency cases reported in the literature, the description of new patients is desirable. Moreover, more efforts are needed to improve understanding of this rare pathology and its patients' outcome. P-251 Small molecule inhibition of IDH2(R140Q) in lymphoblasts of D-2-hydroxyglutaric aciduria type II patients Salomons GS1, Straley KS2, Struys EA1, Travins J2, Gross S2, Agresta S2, Jakobs C1, Su M2, Yen K2 1

Metabolic Unit, Dept Clin Chem, VUMC, Amsterdam, Netherlands; 2 Agios Pharmaceuticals, Cambridge, United States

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one of these tool compounds (AGI-6780) in lymphoblasts of D-2HGA Type II patients. Methods: D-2-HGA Type II patient-derived lymphoblasts were incubated with increasing doses of the IDH2(R140Q) inhibitor compound, AGI-6780, for up to 168 hours. Newly generated [13C1]-D-2-HG was measured by an established enantiomer specific LC-MS/MS methodology after cells were fed [13C1]-D-glucose. Results: AGI-6780 lowered D-2-HG levels up to 98% in D-2-HGA Type II lymphoblasts in a dose-dependent manner. The IC50 was < 50nM following 96-168 hours of treatment. Conclusion: The significant reduction of D-2-HG by AGI-6780 in lymphoblasts of D-2-HGA Type II patients suggests that IDH2 inhibitors could play a role in mitigating Type II D-2-HGA disease. Further development of clinically viable inhibitors is warranted. Conflict of Interest declared. P-252 Generation of disease specific-iPS cell from glutaric acidemia type I patient Hamazaki T1, Hattori T1, Nishimura K2, Ohtaka M3, Nakanishi M3, Shintaku H1 Dpt Pediatrics, Osaka City Univ, Osaka, Japan; 2Labo Gene Reg, Univ of Tsukuba, Tsukuba, Japan; 3Res Cent for Stem Cell Engin, AIST, Ibaraki, Japan 1

Glutaric acidemia type 1 (GA1) is an inherited disorder of lysine and tryptophan metabolism caused by a severe deficiency of the mitochondrial glutaryl-CoA dehydrogenase activity. Acute metabolic decompensation occurs upon catabolic stress and leads to neuronal cell damage. Recent advances in induced pluripotent stem cell (iPSC) technology allow us to generate disease specific-iPS cells for disease modeling and drug discovery. Here we attempted to generate iPS cells from GA1 patient. Results: Skin fibroblast was obtained from GA1 patient under informed consent for reprogramming studies. Initially, we introduced reprogramming factors (Oct4, Sox2, Klf4, c-Myc) into the fibroblast by using conventional retroviral vectors. Although iPS colonies emerged with similar efficiency as healthy control fibroblast, immortalized epithelial-like cells dominantly grew on plate and GA1-iPS cells was failed to be maintained. We suspected that unbalanced expression of four exogenous genes caused transformation of fibroblast derived from GA1 patient. By using Sendai virus-based single vector system (SeVdp), GA1-iPS cells were successfully isolated without having abnormal cells. Supplementation of homoarginine into culture medium helped for maintenance of undifferentiated GA1-iPS cells. Conclusion: We have generated exogenous gene-free iPS cells from GA1 patient and further analysis of pathogenic mechanism of GA1 will be discussed. P-254 Maple Syrup Urine Disease (MSUD) : a case report diagnostic strategies in limited setting Nurani Neti1

Background: D-2 hydroxyglutaric aciduria (D-2-HGA) Type II is a rare and severe neurometabolic disorder that presents with a range of clinical findings including seizures, hypotonia, developmental delay, cardiomyopathy, dysmorphic features and early death. Increased levels of D-2-hydroxyglutarate (D-2-HG) in urine, serum and CSF are hallmarks of the disease. A de novo heterozygous mutation R140Q in isocitrate dehydrogenase 2 (IDH2) is responsible for the autosomal dominant gain-offunction causing overproduction of D-2-HG. We have developed potent and selective inhibitors of IDH2(R140Q) and sought to test

1

Pediatric Dept, Gadjah Mada Univ, Yogyakarta, Indonesia

Background: Maple syrup urine disease (MSUD) is caused by a deficiency of branched chain keto acid dehydrogenase. Objectives: To report a case of MSUD Case: A 52–days-old male child, with the chief complain of regurgitation and dyspnea. Family history: both parents are javanesse. Mother had 2 maternal aunts who died in the "first year" of life from unknown causes. One of his paternal aunt who also died at 3 months of age from unknown causes. There

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was consanguinity between his mother and father. He had been hospitalized 3 times when he was 9 days due to septic condition and other times with the diagnosis of pneumonia and gastroesophageal reflux. No history of "sweet smell of urine". Clinical examination revealed lethargy, chest indrawing, failure to thrive and delayed development. Laboratory examinations showed anemia, hypoglycemia, ketosis and high lactate. GC/MS and acylcarnitine analysis from Laboratory of Shimane university confirmed the diagnosis of MSUD. Protein restriction and Thiamine supplementation had been administered until MSUD special formula was available. Conclusion: Unspecific clinical symptoms made a difficulty to suspect MSUD. Complete history taking of family was a very important fact to support suspicion of MSUD especially in our setting with limited laboratory facilities.

We report two Cypriot sisters, born to healthy non-consanguineous parents, who were brought to our attention at the age of 26 and 30 years with a history of global developmental delay. Seizures manifested at the age of 10. Interestingly, both had a narrow forehead, mild hypertelorism and tapering fingers. Urinary organic acids analysis by GC-MS revealed increased excretion of 4-hydroxybutyric acid and di-hydroxyhexanoate lactone in both patients, a profile compatible with SSADH deficiency. Bi-directional sequencing of all exons of the ALDH5A1 gene confirmed the diagnosis. Both sisters were homozygous for the c.278G>T (p.Cys93Phe) mutation in exon 1. This mutation has been previously reported in four families of Spanish, Turkish, Indian and unknown origin and has been shown to be a disease-causing mutation by expression studies in HEK 293 cells (3% of wild type activity).

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P-257

Acyl-CoA analysis by UPLC/MS/MS to assay enzyme activity in a patient with methylmalonic acidemia

Identification of novel mutations in methylmalonic aciduria MUT type

1

2

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3

Maeda Y , Ito T , Gotoh K , Nakajima Y , Kato S , Sugiyama N , Saito S2, Hotta Y1, Kimura K1 Dep Hosp Pharmacy, Nagoya City Univ, Nagoya, Japan; 2Dep Pediatr, Nagoya City Univ, Nagoya, Japan; 3Dep Pediatr, Aichi-Gakuin Univ, Nagoya, Japan 1

Background: An assay of the methylmalonyl-CoA mutase has been carried out by measurement of succinyl-CoA generated by reaction of methylmalonyl-CoA with enzyme obtained from patient. Determination of succinyl-CoA by HPLC-UV lacks accuracy for influences such as matrices. We developed a correct measurement method of methylmalonyl-CoA mutase activity using acyl-CoA analysis by UPLC/MS/MS. Method: A mixture of methylmalonyl-CoA, lymphocyte and adenosylcobalamin in Tris-sulfate buffer (pH 7.5) was incubated for 20 min at 37 °C. After the suspension was centrifuged, the supernatant was injected onto Acquity UPLC BEH C18 column (2.0 x 150 mm). AcylCoAs were analyzed in positive ion MRM mode of Quattro Premier XE triple quadrupole mass spectrometer (Waters). Results: Although the enzyme reaction gave succinyl-CoA in healthy individual, succinyl-CoA decomposed to free-CoA gradually. In contrast, none or a small amount of succinyl-CoA and free-CoA was generated in a patient with methylmalonic acidemia. Therefore, methylmalonyl-CoA mutase activity was determined from the total amount of generated succinyl-CoA and free-CoA. Discussion: Not only succinyl-CoA but also the measurement of freeCoA is important in this enzyme reaction. The corrected determination of these acyl-CoAs was achieved by use UPLC/MS/MS. This analysis is very useful for the methylmalonyl-CoA mutase activity measurement.

Brasil S1, Desviat LR2, Ugarte M2, Tavares de Almeida I1, Leandro P1, Rivera I1, Perez B2 1 Met&Gen, iMed.UL, Fac Pharmacy, Lisbon, Portugal; 2 Centro Biologia Molecular Severo Ochoa, Madrid, Spain

Methylmalonic aciduria (MMA, MIM#251000) is an autosomal recessive metabolic disorder that results from the functional impairment of the mitochondrial enzyme methylmalonyl-CoA mutase (MCM, EC 5.4.99.2) (mut complementation group) or the synthesis of its cofactor, adenosylcobalamin (AdoCbl) (cblA, cblB and cblD variant2 complementation groups). The MCM is encoded by the MUT gene and at least 222 different mutations have been described, being most of them missense ones. We report the study of a Portuguese patient, born from nonconsanguineous parents and presenting a mild phenotype. Molecular characterization was achieved by direct sequence analysis of genomic DNA revealing two novel mutations. A small duplication in exon 1 (c.244dupA) is probably pathogenic and severe because it produces protein premature truncation (p.L81fs). The other mutation is a transition of A>G in intron 5 (c.1408-10A>G). Since no primary cells were available, we used an ex vivo minigene splicing assay to evaluate mutation's effect. The results suggest the activation of a novel splice site inside the intron but also normal mRNA which might retain some residual activity. Therefore, c.1408-10A>G probably induces an in-frame insertion of nine amino acids. The analysis of its underlying pathogenic mechanism opens the opportunity to rescue the splicing process using antisense therapy. FCT-SFRH/BD/45753/2008 P-258 Propionic academia and biotinidase deficiency in one patient

P-256 Succinic semialdehyde dehydrogenase (SSADH) deficiency in two adult cypriot sisters Mavrikiou G1, Petrou P1, Anastasiadou V1, Drousiotou A1, Tanteles G1 1

The Cyprus Inst. of Neurology & Genetics, Nicosia, Cyprus

Succinic semialdehyde dehydrogenase (SSADH) deficiency is a rare autosomal recessive metabolic disorder caused by a defect in the metabolism of γ-amino butyric acid (GABA). The resulting accumulation of 4-hydroxy butyric acid (GHB) in urine, blood and CSF is considered as the biochemical hallmark of the disease. The disease phenotype varies from mild global developmental delay to more severe manifestations including seizures, ataxia, hypotonia and hyperactivity.

Kilic M1, Yücel-Yılmaz D1, Ozbek MN2, Kandemirli G3, Onol S1, Ozgul RK1 1 Hacettepe Univ, Pediatric Metab Unit, Ankara, Turkey; 2Child Hosp, Pediatr Endoc and Metab Unit, Diyarbakır, Turkey; 3Hacettepe Univ, Faculty of Medicine, Ankara, Turkey

Background: Biotinidase deficiency and propionic acidemia are two different metabolic diseases that are autosomal recessively inherited with prevelances of 1/10.000 and 1/100.000, respectively. Case report: 5 months-old male patient presented with nausea, vomiting and tachypnea. He had been hospitalized two episodes with complaints of vomiting and tachypnea, one of which occurred after circumcision. At newborn period, he was diagnosed with severe biotinidase deficiency in newborn screening and treated with 5 mg/day biotin. Mild developmental delay and hypotonia was present in the patient. Metabolic tests suggested

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a diagnosis of severe biotinidase deficiency and propionic acidemia and his daily biotin was increased to 20 mg. Even with this treatment, the patient was hospitalized with similar symptoms and died while hospitalized. A known homozygous mutation p. Arg157His in BTD gene and a novel mutation p. Asp367Val in PCCB gene were detected. As a result, molecular analyses were confirmed the diagnosis of severe biotinidase deficiency and propionic acidemia together in this patient. Conclusion: Until molecular analysis results are available for definitive diagnosis, patients should be treated for the suspected metabolic diseases. P-259 Long-term neurological prognosis outcome of a cohort of 80 patients with classical organic acidurias Nizon M1, Ottolenghi C2, Valayannopoulos V1, Arnoux JB1, Barbier V1, Habarou F2, Desguerre I3, Boddaert N4, Bonnefont JP5, Acquaviva C6, Benoist JF7, Rabier D2, Touati G1, de Lonlay P1 Div Metab Dis, Univ Paris V, Hosp Necker, PARIS, France; 2Div Biochem, Univ V, Hosp Necker, PARIS, France; 3Div Neurol, Hosp Necker, PARIS, France; 4Div Ped Radio, Univ Paris V, Hosp Necker, PARIS, France; 5Div Med Genet, Univ Paris V, Hosp Necker, PARIS, France; 6Div Metab Dis, Hosp Est, BRON, France; 7Div Biochem Metab, Hosp Debré, PARIS, France 1

Background: Classical organic acidurias including methylmalonic aciduria (MMA), propionic aciduria (PA) and isovaleric aciduria (IVA) present with various severe complications. Methods: We investigated the long-term outcome of 80 patients with classical organic aciduria (38 with MMA, 24 with PA and 18 with IVA) to better delineate common and specific features by integrating clinical, radiological, biochemical and genetic data. Results: Patients were followed-up for 14 years [3.3-46.3 years old]. PA included a greater number of patients with abnormal neurological examination, lower psychometric scores and more frequent basal ganglia lesions; increased lactate peak was occasionally found by MRI. All patients with IVA presented a normal neurological examination and 1/3 presented cognitive troubles. Prognosis for MMA was intermediate. Biochemical metabolite analysis excluding acute decompensations revealed a significant increase of plasma glycine and alanine in patients with PA. A significant increase of plasma methylmalonic acid was found in MMA patients with intellectual deficiency (p<0.05). Urinary 3-hydroxypropionate (3-HP) levels were significantly higher in PA patients with intellectual deficiency (p<0.01). Conclusion: Propionic aciduria had the most severe neurological prognosis. Our radiological and biochemical data are consistent with a mitochondrial toxicity mechanism. Follow-up plasma MMA and urinary 3-HP levels may have prognostic significance.

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clinical features were global developmental delay, stereotypes, sleep disorders, and fluctuant awareness, social interaction and non-paretic hypotonia with hyporreflexia. Characteristic organic acids pattern. Molecular analysis of SSADH gene identified an homocygous 1226 G>A missense mutation. MRI at 4 years old showed an increased T2- weighted signal of globus pallidi, not observed in the previous study done at 8 months of age. In 8 years of follow-up, he developed a non-progressive severe autistic disorder with a global developmental delay, without seizures or other neuropsychiatric features, with severe constipation and urine retention due to bladder-sphincter dysinergia, autonomic symptoms not previously reported. Vigabatrin was administered without clinical effect. Conclusions: We report an early diagnosis of a SSADH patient with a longitudinal follow-up of 8 years that contributes, among other things, to widening the phenotype with the possible autonomic symptoms and highlighting that initial MRI can be non-typical. P-261 Secondary 3-methylglutaconic aciduria in glycogen storage disorders Rizzo C1, Inglese R1, Boenzi S2, Muraca M1, Goffredo BM1, Zelli E1, Semeraro M1, Deodato F2, Dionisi-Vici C2, Maiorana A2 Dep Lab Med, Bambino Gesù Child Hosp, Roma, Italy; 2Div Metab, Bambino Gesù Child Hosp, Roma, Italy 1

Introduction: 3-Methylglutaconic aciduria (3-MGA) is the biochemical marker of several inherited metabolic diseases that can be distinguished in 3 subgroups:"primary 3-MGA", "secondary 3-MGA" and "not otherwise specified 3-MGA". In the "primary 3-MGA" group, accumulation of 3-methylglutaconate is due to deficient activity of 3-methylglutaconyl-CoA hydratase, In the other two groups , 3-methylglutaconic acid is of unidentified origin, possibly derived from other metabolic pathways, such as mevalonate metabolism. We analyzed urinary organic acid by GC-MS of 14 patient with Glycogen Storage Disorders (GSD). We analyzed 35 urine samples:12 samples of 4 patients GSD1, 5 samples of 2 patients GSD3, 1 sample of a patient GSD6, and 17 samples of 7 patients GSD9. Results: Overall we found elevated 3-metilglutaconic acid (normal value <20 mmol/mol creat.) in 29/35 samples. Mild 3-MGA was found in GSD1 (mean 45.9; max value 167), GSD6 (75.8), GSD9 ( mean 49.1; max value 245) and slight 3-MGA was found in GSD3 (mean 20.2; max value 25). Conclusion: An imbalanced glycolysis, gluconeogenesis, lipogenesis and de novo cholesterol synthesis could result in increased 3methylglutaconic acid (1). We suggest to include Glycogen Storage Disorders as possible cause of "secondary 3-MGA". (1) Law et al. J Inherit Metab Dis (2003) 26:705-709 P-262

P-260 Eight years of longitudinal follow-up of a patient with succinic semialdehyde dehydrogenase (SSADH) deficiency

A large genomic deletion of PCCB gene in propionic acidemia patients Liu TT1, Chiu YH2, Liu YN3, Liao WL3, Chang YC1, Hsu CC4, Niu DM5, Chien YH6, Hsiao KJ7

Cerisola A1, González G1, Lemes A1 1

IEM Divison, School of Medicine, CHPR, Montevideo, Uruguay

Background: SSADH deficiency is an autosomal recessive inherited disorder of GABA metabolism. The early diagnosis is challenging due to the rather variable and non-specific phenotype of nonprogressive neurological dysfunction, including a variety of neurological and psychiatric symptoms. Clinical case: Infant of 7 months of age at diagnosis, born from a nonconsanguinuos couple. Uncomplicated pregnancy and delivery. Initial

1 Genome Res Center, Natl Yang-Ming Univ, Taipei, Taiwan; 2Dept Edu & Res, Taipei City Hospital, Taipei, Taiwan; 3Inst Genet, Natl YangMing Univ, Taipei, Taiwan; 4Dept Pediatr, TC Veter General Hospt, Taichung, Taiwan; 5Dept Pediatr, TPE Veter General Hospt, Taipei, Taiwan; 6Dept Med Genet, Natl Taiwan Univ Hospt, Taipei, Taiwan; 7 Preventive Medicine Foundations, Taipei, Taiwan

Background: Propionic acidemia (PA, MIM 232000, 232050) is caused by a deficiency of propionyl-CoA carboxylase (PCC, EC 6.4.1.3) which

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experience in PA is still limited. We describe a PA patient with severe neurologic complications after OLT. An 18-year-old female with PA, mental retardation and behavioural disorder, underwent OLT because of non-compliance concerning diet and intake of medication. Three months after OLT and further relaxation of the diet including termination of supplements, severe polyneuropathy developed. Consequently, tacrolimus was switched to cyclosporin. A severe change in mental status together with cardiorespiratory decompensation followed three weeks later. In blood, lactic acidosis was detected but hyperammonemia or a marked increase of typical metabolites of PA were not found. In CSF, however, an extreme elevation of methylcitrate was detected. Furthermore, severe thiamine deficiency had developed. Brain MRIs revealed marked signal alterations of basal ganglia. On thiamine, protein restriction, neuroleptics, and physiotherapy, dramatic clinical improvement was observed. In this case, OLT did not protect from neurometabolic decompensation, which was probably caused by many factors such as increased protein consumption with consecutive compartmental trapping of organic acids in CNS, thiamine deficiency, neurotoxicity of immunosuppression, and natural progression of PA.

Pyroglutamic aciduria : a neonatal case report

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Ben Ameur K1, Khemiss T1, Mnari A1, Chioukh FZ1, Ben Hamida H1, Bizid M1, Kaabachi N2, Monastiri K3

Expression, purification and in vitro kinase activity of the two new BCKDK mutants from developmental delay and autistic behaviour patients

consists of two subunits, alpha and beta, encoded by PCCA and PCCB, respectively. Several exonic deletions have been reported in PCCA, yet a large deletion in PCCB has not been found. One of our PA patients was found to be homozygous for a c.1301C>T mutation in the cDNA but shown a heterozygote of c.1301C>T at the genomic DNA suggesting the presence of exonic deletion. Materials and methods: Three patients with one PCCB mutation uncharacterized were rerolled. These three patients were confirmed as PCCdeficiency by enzyme assay. Southern blot analysis was applied to identify a large genomic deletion following by PCR and sequencing to characterize the breaking point. Results: A deletion of 8052 bp in the PCCB gene, namely c.1-4157_183 +3713del, was detected in these 3 patients. The deletions may be caused by a fusion between the direct repeat within two Alu sequences resulting in the depletion of the crucial transcriptional elements, including promoters and transcriptional start site, exon 1, and partial intron 1. Conclusion: This study describes for the first time a large genomic deletion in the PCCB gene.

NICU Teaching Hospital of monastir, Monastir, Tunisia; 2Departement of Biochemistry LaRabta, Tun, Tunis, Tunisia; 3Faculty of Medicine of Monastir, Monastir, Tunisia 1

Background: Pyroglutamic aciduria or 5-oxoprolinuria is a rare inborn error of metabolism caused by glutathione synthetase deficiency, usually revealed by a metabolic acidosis with hemolysis. Case report: We report an observation of a female newborn descended from consanguineous parents, with a family history of three cousins died in the neonatal period. Born at term without incident. In day 3, she developed a respiratory distress type Kussmaul breathing. Biology showed hemolysis, severe metabolic acidosis with no lactic acidosis and an elevated anion gap; suggesting a glutathione synthetase deficiency. This diagnosis was confirmed by elevation of urinary 5-oxoproline dosed by urinary organic acids chromatography. The treatment is based on correction of acidosis and supplementation with vitamins C and E. the course was marked by a transient period of hemolysis, acidosis and by deep electrolytes disorders. At 2 months-old she's doing well and tolerates treatment. Conclusions: This rare diagnosis should be evoked in case of recurrent metabolic acidosis with unexpected hemolytic anemia in a newborn infant. The prognosis is linked to the precocity of diagnosis and adequate treatment. Three others unrelated patients were described in our geographic area. A founding genetic event of this disease must be investigated.

Fort J1, Bodoy S2, Roig J2, Palacin M1 1 IRBBarcelona, U731 CIBERER, Barcelona, Spain ; 2IRBBarcelona, Barcelona, Spain

Two new branched-chain α-ketoacid dehydrogenase kinase (BCKDK) mutations were found in two unrelated patients. Other inactivating mutations in the BCKDK had been associated to a potentially treatable condition of autism (Novarino, Science, 2012). Human BCKDK wild type (wt) and both mutants were expressed with pMAL system vector in E. coli. After purification with amylose-sepharose resin, recombinant proteins were used for a kinase assay with γ32ATP and universal kinase substrate myelin basic protein (MBP). After an acrylamide gel, bands were revelled with autoradiography. Mutant R174G is able to autophosphorylate and phosphorylate MBP but with less activity than wt, whereas L389P doesn't show any activity. In a homology-based model from rat BCKDK structure (Davie, JBC,1995), we predict than R174G could affect catalytic mechanism whereas L389P could be implicated in oligomerization of kinase. Two new mutations have been characterized at molecular level to elucidate the implication of these residue changes in BCKDK kinase activity. Davie, J. R., R. M. Wynn, et al. (1995). J Biol Chem 270(34): 19861-19867. Novarino, G., P. El-Fishawy, et al. (2012). Science 338(6105): 394-397. P-266

P-264 Propionic acidemia (PA): neurometabolic decompensation after orthotopic liver transplantation (OLT)

Propionic acidemia: a case report of a successful pregnancy, labor, and lactation Önenli-Mungan N1, Kör D1, Büyükkurt S1, Atmış A1, Güleç Ü1

1

1

1

1

1

Tsiakas K , Denecke J , Briem-Richter A , Thies B , Mühlhausen C , Blom HJ2, Ullrich K1, Santer R1 1

Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany; 2Dept Clin Chem, VU Univ Med Cent, Amsterdam, Netherlands Under conservative treatment, PA has an unfavourable prognosis. OLT has therefore been recommended to avoid hyperammonemic crises, slow down mental deterioration, and improve quality of life but

1

Çukurova University, Adana, Turkey

Background: With early diagnosis and improved treatment, patients with organic acidemias will able to survive to childbearing ages. Both successful pregnancies with excellent outcomes and metabolic problems with eventful outcomes were reported. Here we report a pregnancy of a propionic acidemia (PA) patient as it is the first Turkish case, and to the best of our knowledge the third case of the literature.

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Case report: A 31 years old woman diagnosed as PA with a homozygous mutation c. C683T(p.P228L) evaluated for pregnancy. In addition to routine follow-up by gynecologist all biochemical, metabolic parameters and diet were frequently revised by us. A cesarean section with spinal anesthesia under the infusion of 10 % dextrose at 37. weeks of gestation have done without any complication. The baby has low birth weight and minimal facial asymmetry. She breast fed her baby two months, then formula added Conclusions: There were two cases in the literature with PA and pregnancy. One had severe hyperemesis and second had placenta previa. Although the genotype of our patient probably influenced the progression of the pregnancy and the labor, we believe that being together with the patient during the whole procedure and careful monitoring of metabolic status bring the success.

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differentiation revealed the patient is affected with L-2-HGA. Screening his brothers revealed that his 14 and 2 yr old brothers respectively are similarly affected, while their parents and their asymptomatic siblings had normal L2-hydroxyglutaric acid urinary excretion. Sequencing the L2HGDH gene revealed a homozygous c. 1107del (p.Thr370LeufsX13) mutation, that segregated in a recessive manner. The patients showed variable response to carnitine and riboflavin supplementation. The youngest affected was started on a lysine restricted diet. P-269 Urinary 2-oxoglutaric acid detected by NMR spectroscopy in the diagnosis of inborn errors of metabolism Usurelu N1, Nicolescu A2, Sacara V1, Garaeva S3, Szönyi L4, Deleanu C5

P-267 1

Three novel mutations in Turkish patients with isovaleric acidemia Yilmaz-Yucel D1, Ozgul RK1, Kilic M1, Sivri S1, Coskun T1, Tokatli A1, Dursun A1 1

Hacettepe University, Dep. of Metabolism, Ankara, Turkey

Isovaleric acidemia (IVA) is a rare autosomal recessive metabolic disease caused by a deficiency in isovaleryl-CoA dehydrogenase (IVD). Clinical features of IVA include poor feeding, recurrent vomiting, lethargy, seizures, mental retardation, coma and possibly death. IVD catalyzes the third step in leucine catabolism and is encoded by IVD. In this study, mutation screening of twelve exons in IVD gene was carried out in six patients with IVD deficiency from five unrelated families by using direct sequence analysis. Three different type of pathogenic mutations (one splicing, one deletion, three missense) were detected in patients suspected with IVA. Mutation analyses revealed that, detected homozygous nucleotide changes result in two previously reported mutations (IVS4+2T>C and p.Arg395Cys) and three novel mutations (c.154delC, p. Leu52Phe..fsX6, p.Glu411Lys, p. Glu117Gln) in the IVD gene. This study was supported by the Scientific and Technology Research Council of Turkey (TÜBİTAK-SBAG-111S217)

Institute of Mother and Child, Chisinau, Moldova, Republic of; "P.Poni" Inst Macromol Chem Rouman Acad, Iasi, Romania; 3Inst Phys Sanocreat Acad Scie Moldova, Chisinau, Moldova, Republic of; 4 1st Dept Pediatr Semmelweis Univ, Budapest, Hungary; 5 "C.D.Nenitescu" Inst Org Chem Roum Acad, Bucharest, Romania 2

Methods: We report on 4 children (two neonates, one each aged 7 mo and 8 yrs) suspected for IEM according to their clinical manifestations, all with liver disease. In the two older children the diagnosis of GSD1a was confirmed by DNA analysis. In all patients high urinary levels of 2oxoglutaric acid (321-2090 mmol/molCrn [ref.val.:1 5-200]) and high lactate (311-3352 mmol/molCrn [ref.val.:<0.06]) were found. One child had dysmorphic features resembling AFS, muscular hypotonia, apnoea, abnormal LFT from the birth, and died in the 3rd week of life without diagnosis. High urinary alanine and 2-oxoglutarate with elevated blood and urine were compatible with PDHC or KDHC deficiency. Another child had abnormal LFTs, nephromegaly, mild hypoglycemia, anemia from the birth, and high urinary 2oxoglutarate and Lactate; she was suspected to have GSD1a and a diet low in lactose was initiated. She developed fasting ketotic hypoglycemia with hyperlactatemia at the age of 1 year; mutation analysis is pending. All children were tested by MS/MS, excluding other metabolic conditions affecting the liver. Conclusion: NMR spectroscopy of body fluids is useful in diagnosis of IEM. 2-oxoglutarate could be considered as an early marker for IEM involving the TCA cycle.

P-268 P-270 L2-hydroxyglutaric aciduria in three omani siblings with a novel homozygous mutation of the L2HGDH gene

Mut(0)-methylmalonic acidemia: carglumic acid can help lower blood ammonia levels and allow for adequate protein intake

Al-Thihli K1, Al Habsi A2, Panteix G3, Koul R4, Al Mammary W4 Khan A1, Casey R1, Ferreira P1, Reeves M2 1

2

Genetic and Developmen Med. Clinic, SQUH, Al Khod, Oman; Directorate of Nursing, SQUH, Al Khod, Oman; 3Biomnis Laboratories, France, France; 4Department of Child Health, SQUH, Al Khod, Oman

Alberta Child Hosp, Univ Calgary, Calgary, Canada; 2Met Clinic, Alberta Child Hosp, Calgary, Canada

Background: L-2-Hydroxyglutaric aciduria (L-2-HGA) is a rare organic aciduria characterized by mild to severe intellectual disability, and variable neurological manifestations. We report the first three Omani siblings diagnosed with L-2-HGA. Case Report: A 7-yr-old boy with mild intellectual disability was evaluated for acute ataxia and spasticity. He had mild global developmental delay. He was born to double first cousins, and he has an older brother with seizures, ataxia, and severe intellectual disability. Examination revealed relative macrocephaly, bilateral pyramidal signs, and mild cerebellar ataxia. MRI brain showed diffuse bilateral white matter changes with cavitation particularly in the temporal regions. VLCFA, NCS, EMG, acylcarnitine profile, and arylsulfatase A enzyme assay were all normal. Urine organic acids revealed significantly elevated L2/D2- hydroxyglutaric acid. Chiral

Methylmalonyl-CoEnzyme A (MMA) due to complete mutase deficiency (mut0) is an autosomal recessive, heterogenous condition that can result in metabolic acidosis, hyperammonemia, developmental delay and renal insufficiency. This is the first reported case in a Canadian Dariusleut Hutterite child who presented at 3 weeks of age with a plasma ammonia of 599 μmol/L, anion gap metabolic acidosis and encephalopathy. His hyperammonemia was controlled using intravenous ammonia salvage therapy, but attempts to feed him more than 0.5 g/kg/day of protein while using sodium phenylbutyrate and L-arginine would still lead to hyperammonemic (>200 μmol/L) episodes controlled only by re-introduction of intravenous ammonia salvage therapy. After adding carglumic acid at 100 mg/kg/d, he tolerated up to 2.25 g/kg/d of protein, maintained normal blood ammonia levels, showed

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catch-up physical and developmental growth, was clinically well and only mild episodes of hyperammonemia during intercurrent illness . At 3 = years of age he developed a unrelated, severe respiratory illness and died. In our case, we concluded that carglumic acid can be a useful adjunct to control blood ammonia levels in MMA (mut0) so that dietary protein can be increased to promote adequate growth and nutrition.

Conclusions: Mut-/- fetuses exposed to high protein diet in utero display higher methylmalonic acid concentrations, suggesting that prenatal dietary modifications may be important to lower exposure to toxic (or harmful) metabolites during early development.

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Glutaric aciduria type I: clinical outcome of 4 cases detected through newborn screening in Alberta

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Infrequent organic acidurias in Colombia South America 1

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Echeverry OY , Guevara JM , Espinosa E , Pulido NF , Ardila YA , Cabarcas L3, Barrera LA1 Inst Inborn Errors Metab. Univ Javeriana, Bogotá, Colombia; 2Hosp Mil Central. Univ Mil Nueva Granada, Bogotá, Colombia; 3Hosp Univ San Ignacio, Bogotá, Colombia 1

Khan A1, Reeves M2, Bamforth F3, Spector E.B.4, Wei X-C5, Sinasac D6, Chan A7 1 Alberta Child Hosp, Univ Calgary, Calgary, Canada; 2Met Clinic, Alberta Child Hosp, Calgary, Canada; 3Univ Alberta, Edmonton, Canada; 4 Denver Genet Lab, Denver, United States; 5Diag Imag, Alberta Child Hosp, Calgary, Canada; 6Genet Lab Serv, Alberta Health Serv, Calgary, Canada; 7Univ Alberta, Edmonton, Canada

The Institute for the Study of Inborn Errors of Metabolism (IEIM) of Pontificia Universidad Javeriana is a national referral center for the diagnosis of organic acidurias (OA). In the last 5 years from a total of 1701 patients referred to exclude an OA by GC/MS, 2.29% were confirmed positive. Among 39 positive cases, 79.4% correspond to the most prevalent OA reported worldwide such as Glutaric aciduria type I (9-cases), PPA (6-cases), MMA (6-cases) and IVA (2-cases), 3Methylglutaconic aciduria (2-cases), MSUD (2-cases), and a high suspicion of Multiple Decarboxylase Deficiency (2-cases), Lactic aciduria (1-case), and Glutaric-aciduria type II (1-case). On the other hand it is remarkable that nearly 20.6% of the cases correspond to very rare OA such as Pyroglutamic-aciduria (1-case), 2Hydroxyglutaric-aciduria (3-cases), Deficiency of Succinic-semialdehydedehydrogenase (1-case), Mevalonic-aciduria (1-case), 3-Hydroxy-3Methylglutaric aciduria (2-case). This data does not agree with reports from other populations. This could be related to the high genetic heterogeneity in our population. However it is necessary to take into account that normally we analyze only high risk population and expanded newborn screening is being implemented.

Early detection and management of glutaryl-CoA dehydrogenase deficiency (GAI) through newborn screening has lead to improved outcomes. We report 4 cases out of 6 positive screens out of 293,104 births in Alberta from 2007 to 2012. There was not a single episode of decompensation during any illness. Case 1 had extensive cerebral injury, dystonia and developmental delay just after 6 months of age. She has 3 mutations in the GCDH gene: c.1173delG and c.1198G>A (p.V400M) in cis (based on parental testing) and a trans c.541G>A (p.E181K). Case 2 had mild dystonic posturing of his hands and feet at 1 = years of age with only nigrostriatal changes and is homozygous for the c.337T>C (p.Y113H) mutation. Case 3 is a 1 year-old girl with normal development and brain MRI and is a compound heterozygote for the p.R227P and a novel c.198del_C mutation. Case 4, is a normal 1 year 9 month-old boy with minimally enlarged anterior Sylvian fissures. All patients were maintained on a protein modified diet controlled in lysine and tryptophan with a specific GAI metabolic formula to optimize protein and caloric intake. Conclusion: early screening and treatment for GAI appears to result have improved outcomes.

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The ingestion of a high protein diet during gestation magnifies in utero production of metabolites in methylmalonyl-CoA mutase (MUT) knockout mice

Clinical outcome in glutaric aciduria type i, experience in center of metabolic disease in Chile

Brown D1, Senac J2, Sloan J2, Venditti C2

Arias C1, Bravo P1, Castro G1, Cabello JF1, Hamilton V1, Peredo P1, Valiente A1, Raimann E1, Cornejo V1

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1

Medstar Washington Hospital Center, Washington, United States; National Institutes of Health, Bethesda, United States

Inst Nut Food Tech, Univ of Chile, Santiago, Chile

2

Objective: To study the effects of dietary manipulation during gestation on affected fetuses using a knockout mouse model of methylmalonic acidemia (MMA). Background: There are reports examining the effects of prenatal vitamin B12 but not of the maternal diet on MMA embryos. Mut -/- mice, like patients, display increased methylmalonic acid in utero. We hypothesized that excess branched chain amino acid administration stimulates the accumulation of abnormal metabolites, potentially modifying the outcome of affected pregnancies. Methods: Mut +/- mice were bred with timed mating studies. Mice were assigned either regular- (20% by weight) or high protein-diet (70%). Pregnant mice were sacrificed on embryonic day 19 (E19). Methylmalonic acid was measured in amniotic fluid and plasma. Results: Methylmalonic acid levels of Mut -/- feti on E19 were 308.16 μmol/L (N=3) when mothers consumed a regular diet and 499.14 μmol/L (N=11) with high protein diet (P = 0.0018). Maternal serum levels were 7.24 and 13.08 μmol/L respectively.

Introduction: Glutaric aciduria type I (GA-1) is a metabolic disease caused by enzymatic deficiency of glutaryl-CoA dehydrogenase. Suspected clinically by a mild psychomotor retardation and macrocephaly. Generally have a severe encephalopathic crisis between 12-18 months of life, with devastating neurological sequelae. Objective: To perform a descriptive analysis of clinical manifestations in a series of cases with GA-1. Material and Methods: Retrospective review of 24 cases with GA-1, between the years 1997-2013. Results: Encephalopathic crisis occurred in 18/24 cases at an average of 9 months age, preceded by infection disease in 14/24. Patiens who had crisis developed serious consequences, such as abnormal movements, psychomotor developmental regression and failure to growth. Discussion: The diagnosis was made after encephalopathic crisis in the vast majority, all of this patients has serious consequence mainly movements disorders (BADS scale), malnutrition, epilepsy among others. Conclusion: We report a series of 24 patients diagnosed with GA-1 based on clinical criteria. In countries where no neonatal screening is

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available for the entire population, it is critical to increase the index of suspicion in order to avoid catastrophic consequences. 07. Mitochondrial fatty acid oxidation disorders

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W297L and P317R, were identified in cases 1 and 2, respectively. Our cases indicate that the abnormal findings are barely detectable by biochemical analyses in adult onset myopathic form of GA2. Pathological and/or molecular approaches are essential for the diagnosis. P-277

P-275 Differences between acylcarnitine profiles in plasma and bloodspots Responsiveness of bezafibrate for neonatal onset form of glutaric acidemia type II: comparison with milder form using in vitro probe assay

de Sain-van der Velden MGM1, Diekman EF2, Jans JJ1, van der Ham M1, Prinsen HCMT1, Visser G2, Verhoeven-Duif NM1

Yamada K1, Kobayashi H1, Takahashi T1, Hasegawa Y1, Purevsuren J1, Fukuda S1, Ito M2, Yamaguchi S1

Dep of Med Genet, Univ Med Centre, Utrecht, Netherlands; 2Dep of Metab Dis, Univ Med Centre, Utrecht, Netherlands

1 Dep Ped, Shimane Univ, Izumo, Shimane, Japan; 2Dep Ped, Natl Kagawa Child Hosp, Zentsuji, Kagawa, Japan

Quantification of acylcarnitines is used for screening and diagnosis of IEM. Information on the correlation between plasma and DBS acylcarnitine profiles is scarce. Acylcarnitine concentrations were measured in paired DBS and plasma samples. Additionally, we tested whether ratios of acylcarnitines in both matrices are helpful for diagnostic purpose. DBS and plasma were obtained from controls and patients with known IEM. (Acyl)carnitines were analyzed using HPLC/MS/MS. Free carnitine concentrations were 36 % higher in plasma compared to DBS. In contrast, in patients with CPT-1 deficiency free carnitine concentration in DBS was 4 times the concentration measured in plasma. In CPT-2 deficiency, primary diagnostic markers were abnormal in plasma but could be normal in DBS. The calculated ratios for CPT-1 (C0/(C16+ C18)) and CPT-2 ((C16+C18:1)/C2) revealed abnormal values in plasma. However, normal ratios were found in DBS of two (out of five) samples obtained from patients with CPT-2 deficiency. Relying on primary acylcarnitine markers, CPT-1 deficiency can be missed when analysis is performed in plasma, whereas CPT-2 deficiency can be missed when analysis is performed in DBS. Ratios of the primary markers to other acylcarnitines restore diagnostic recognition completely for CPT-1 and CPT-2 in plasma, while CPT-2 can still be missed in DBS.

Background: The neonatal onset form (severe form) of glutaric acidemia type II (GA2) is fatal regardless of any treatments. Bezafibrate is a promising drug for the management of fatty acid oxidation disorders. We previously reported a 2-year-old boy with a milder form of GA2, whose symptoms were markedly improved with bezafibrate treatment. In this study, we compared the effect of bezafibrate on the severe form with that on the milder form. Methods: Beta oxidation capacities of fibroblasts in severe and milder forms of GA2 were evaluated using a in vitro probe (IVP) assay with several concentrations of bezafibrate. Results: Without bezafibrate, the IVP assay revealed that C16 alone was markedly elevated in the severe form, whereas multiple acylcarnitines were increased in the milder form. Low dose bezafibrate (100 uM) showed little effect on C16 in the severe form; however, highdose bezafibrate (800 uM) significantly reduced C16. In contrast, even low-dose bezafibrate significantly decreased all accumulated acylcarnitines including C16 in the milder form. Conclusion: Although bezafibrate was more effective to reduce acylcarnitine in the milder compared to severe form, our data suggest that a higher dose of bezafibrate could improve the beta oxidation capacity even in severely affected patients.

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P-278 Cold intolerance in cardiac specific VLCAD-deficient mice

P-276 Khuchua Z1, Xiong D1, Powers C1, Strauss A1 Two japanese cases of adult onset myopathic form of glutaric acidemia type II Yamaguchi S1, Yamada K1, Kobayashi H1, Takahashi T1, Hasegawa Y1, Purevsuren J1, Ohkubo T2, Watanabe M2, Tsunemi T2, Ishii A3, Takuma H3, Tamaoka A3, Shigematsu Y4, Fukuda S1 1 Dep Pediatr, Shimane Univ Sch Med, Izumo, Shimane, Japan; 2Dep Neurol, Tokyo Med Dent Un, Tokyo, Japan; 3Dep Neurol, Univ Tsukuba Sch Med, Tsukuba, Japan; 4Dep Ped, Fukui Univ Med Sci, Fukui, Japan

Glutaric acidemia type II (GA2) can be clinically classified into 3 types: 1) Neonatal onset form with fatal outcome (severe form) , 2) Intermediate form with intermittent episodes of lethargy, hypoglycemia, or encephalopathy from infancy, and 3) Myopathic form with recurrent episodes of myalgia or myopathy-like illness. We report two cases of adult-onset myopathic form of GA2. Case 1 was a 58 year-old male. He had suffered from occasional shoulder stiffness, myalgia, hypotonia or liver dysfunction since he was 40's. Case 2 was a 31-year-old male, and was a baseball player. He also suffered from recurrence of hypotonia and myalgia for the last two years. Muscle biopsy revealed a mild fat deposit in both cases. Although GC/MS or MS/MS analyses showed very little abnormalities, immunoblot analyses failed to detect ETF dehydrogenase (ETFDH) protein. Mutations in ETFDH gene, P456L homozygote, and compound heterozygote of

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CCHRF and University of Cincinnati, Cincinnati, United States

Background: Very-long-chain acyl-CoA dehydrogenase (VLCAD) catalyzes the first step of mitochondrial beta-oxidation. Patients with VLCAD deficiency present with hypoketotic hypoglycemia, lactic acidosis, and cardiomyopathy, which can be exacerbated by physiological stress. Global VLCAD knockout mice recapitulate some of these phenotypes: mice develop cardiomyopathy and severe cold-intolerance and quickly die when exposed to cold. Objectives: We investigated the contribution of cardiac muscle in development of cold sensitive phenotype in VLCAD-deficiency. Methods: We generated conditional, cardiac-specific VLCAD-deficient (cVLCAD-/-) mice. Mice were subjected to cold stress at +4oC. Cardiac function was studied using echocardiography. Results: By 6 months of age cVLCAD-/- mice demonstrated increased end-diastolic and end-systolic left ventricular dimensions and decreased fractional shortening. Depression of cardiac function was associated with reduction of cardiac ATP production, although mitochondrial function and structure were largely preserved. Surprisingly, selective inactivation of VLCAD gene in cardiomyocytes was sufficient to evoke severe cold-intolerance in mice: following cold-exposure cVLCAD-/mice rapidly developed severe hypothermia, bradycardia and markedly depressed cardiac function and were moribund.

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Conclusion: Cardiac-specific VLCAD deficiency is sufficient to induce cold-intolerance and cardiac dysfunction. These results bring new knowledge about the essential role of fatty acid oxidation in cardiac muscle for body-core temperature maintenance.

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oxidation capacity. These results suggest that this technique may be useful to assess change in fatty acid oxidation capacity over time or with various clinical treatments such as triheptanoin. P-281

P-279 Sudden cardiac death among young adults with long-chain 3-hydroxy acyl-CoA dehydrogenase (LCHAD) deficiency Gillingham MB1, Strauss A2, Arch E3, Arnold GL4, Harding CO1

Carnitine-acylcarnitine translocase deficiency: two neonatal cases with common splicing mutation and in vitro bezafibrate response Vatanavicharn N1, Furui M2, Aoyama Y3, Fukao T4, Sathienkijkanchai A1, Wasant P1, Yamaguchi S1

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Dept Pediatr, Siriraj Hosp, Mahidol U, Bangkok, Thailand; 2Dept Pediatr, Shimane U School of Med, Shimane, Japan; 3Med Info Sci Div, Gifu U, Gifu, Japan; 4Dept Pediatr, Grad School Med, Gifu U, Gifu, Japan

Three LCHAD deficient young adults (19-21 years) presented with sudden cardiac arrest; one female died and two males were resuscitated. Subjects were homozygous or heterozygous for 1528G>C with a history of classical LCHAD deficiency presenting in infancy. Per family report all subjects had been relatively healthy with no hospitalizations for several years prior to the event; subjects were not taking oral carnitine and were consuming minimal or no medium chain triglyceride (MCT) at that time. Autopsy records of one subject indicate clear vessels and normal cardiac size, suggesting an arrhythmic event as the cause of death. She had a low free carnitine (7μM) with elevated straight and hydroxyl acylcarnitines measured 2 months prior to her death. Emergency room records indicate two subjects were transported to the hospital in ventricular tachycardia, were cardioverted, and had episodes of ventricular fibrillation before a normal sinus rhythm was established. One subject had a low free carnitine (9μM) with elevated straight and hydroxy acylcarnitines. No carnitine profile was measured in the other. Sudden cardiac death among young adults with LCHAD deficiency may be an unrecognized late complication of the disease. It is unknown if treatments such as carnitine and MCT supplements could prevent these events.

Background: Carnitine-acylcarnitine translocase (CACT) deficiency is a rare mitochondrial fatty acid oxidation (FAO) disorder. It is caused by mutations of the SLC25A20 gene. Most patients developed symptoms and died in the neonatal period. Objectives: We herein report the clinical findings of two unrelated cases, mutation confirmation, and in vitro bezafibrate response using in vitro probe acylcarnitine (IVP) assay. Case report: Patients 1 and 2 are products of nonconsanguineous parents. Both patients developed cardiac arrest at day 3 of life but survived the initial events. Their blood chemistry revealed hypoglycemia and metabolic acidosis. The acylcarnitine profiles in both patients demonstrated increased long-chain acylcarnitines, suggesting CACT deficiency or carnitine palmitoyltransferase 2 (CPT2) deficiency. Results: The mutation analysis identified homozygous IVS2-10T>G in the SLC25A20 gene in both patients, confirming the diagnosis of CACT deficiency. The IVP assay revealed increased C16, C16:1, and decreased C2 with improvement by bezafibrate in Patient 1's fibroblasts. The in vivo effect of bezafibrate is being evaluated. Conclusion: This splicing mutation has been identified in other Asian populations indicating the founder effect. If in vitro and in vivo effects of bezafibrate show responsiveness, it will be additional therapy for this severe FAO disorder.

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Oregon Health & Science University, Portland, OR, United States; University of Cincinnati, Cincinnati, OH, United States; 3AI duPont Hospital for Children, St George, UT, United States; 4University of Pittsburgh, Pittsburgh, PA, United States

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P-280 P-282 1-13C-Oleic acid recovery correlates with age of presentation in subjects with a long-chain fatty acid oxidation disorder Martin JM1, DeWard SJ2, DeLany JP2, Vockley J2, Harding CO1, Gillingham MB1

Changes in muscle fiber type in very-long-chain acyl-CoA dehydrogenase deficient (VLCAD-/-) mice Tucci S1, Pearson S2, Herebian D2, Spiekerkoetter U1

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Oregon Health & Science University, Portland, OR, United States; Children's Hosp of Pittsburgh of UPMC, Pittsburgh, PA, United States

Dep Gen Ped, Univ Child Hosp, Freiburg, Germany; 2Div Metab Dis, Univ Child Hosp, Düsseldorf, Germany

A novel stable isotope method was used to measure in vivo long-chain fatty acid oxidation among subjects (n=9) with carnitine palmitoyltransferase-2 (CPT2), very-long-chain acyl-CoA dehydrogenase (VLCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD), or trifunctional protein (TFP) deficiencies who were enrolled in a phase II trial comparing the efficacy of oral triheptanoin vs. medium chain triglyceride (MCT) in the management of long-chain fatty acid disorders. Subjects consumed 1-13C-Oleic acid (18:1, cis 9) at 17 mg/kg body weight with breakfast. Breath samples were collected hourly and analyzed for 13CO2 by isotope ratio mass spectrometry. Recovery of the label in breath represents one complete cycle of fatty acid oxidation, and further oxidation of acetyl-CoA to release 13CO2. Total label recovery over 12 hours positively correlated with age of presentation (r=0.78, p = 0.01). Age of symptomatic presentation ranged from birth to 39 years. Those with later onset of symptoms had increased fatty acid oxidation of the labeled oleic acid, presumably associated with higher residual enzyme activity and higher fatty acid

Dietary fat restriction and increased carbohydrate intake are part of treatment in very-long-chain-acyl-CoA-dehydrogenase-(VLCAD)-deficiency, the most common defect of long-chain fatty acid oxidation (FAO). We here characterize dietary effects on substrate selection and muscle fiber type in VLCADdeficient mice (VLCAD-/-). Wild-type and VLCAD-/- mice were fed one year either with a normal (5.1 % fat) or a low-fat (2.6 % fat), carbohydrate-enriched diet. Dietary effects on genes involved in lipogenesis, energy homeostasis and substrate selection were quantified by real-time-PCR. Impaired FAO was quantified by the degree of acylcarnitine accumulation. White skeletal muscle was characterized by muscle fiber-type as shown by the expression of the respective coding genes, glycogen and triacylglyceride content. Upon a normal diet genes coding for glycolytic muscle fibers were up-regulated in muscle of VLCAD-/- mice. However, the carbohydrate-enriched diet reversed these effects promoting an upregulation of genes coding for oxidative fibers-type I, in association with an enhanced accumulation of triacylglycerides and acylcarnitines.

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Skeletal muscle of VLCAD-/- mice undergoes metabolic modification by switch in muscle fiber-type reflecting adaptation by enhanced glucose oxidation under regular living conditions. These adaptive changes were reversed by the dietary interventions. The higher carbohydrate content was not able to meet the energy demands long-term.

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of ETFDH gene showed homozygosity for mutations p.Val451Leu (Patient1), p.Ile98Asn (Patient3) and compound heterozygosity for p.Val571Met/p.Asn582Lys (Patient2). MADD deficiency should be considered in patients with recurrent ketotic vomiting and normal/atypical acylcarnitine profile, even in the presence of normal in vitro FAO studies.

P-283 P-285 False positive rates in newborn screening of MCAD and VLCAD deficiency scheduled on 5th day after birth Kagawa R1, Tsumura M1, Hara K2, Satoshi O1, Tajima G1, Sakura N3, Hata I4, Shigematsu Y4, Kobayashi M1 Dept. Pediatrics, Hiroshima Univ hosp, hirosima, Japan; 2Dept. Pediatrics, Kure Medical Center, Kure, Japan; 3Nursing House Suzugamine, Hiroshima, Japan; 4Dept. Pediatrics, University of Fukui, Fukui, Japan 1

Background: In newborn screening (NBS) of fatty acid disorders, it is desirable to collect the first blood specimen before feeding is established. In Japan, however, it is still scheduled on 5th day after birth (5d-NBS) after introduction of tandem mass spectrometry-based system. Objectives: To evaluate potential risk of false negative decisions in 5d-NBS by comparing confirmatory diagnosis of newborns suspected with MCAD deficiency (MCADD) and VLCAD deficiency (VLCADD). Patients and Methods: During the period from 2001 to 2012, newborns with elevated levels of either C8- or C14:1-acylcarnitine in dried blood spots were diagnosed by measuring dehydrogenase activity toward n-octanoy-CoA or palmitoyl-CoA in lymphocytes. Abnormal results were further confirmed by genetic analysis. Results: Among the 24 newborns positive for C8, 16 were diagnosed with MCADD; 3 were judged to be heterozygous careers, and 5 had normal levels of enzyme activity. In contrast, all of the 6 newborns positive for C14:1 were diagnosed with VLCADD. Discussion: Considering previous reports on high false positive rates in NBS of VLCADD scheduled on 3rd day, our results suggest potential risk that some affected patients should be falsely missed in 5d-NBS. As for MCADD, 5d-NBS seems to keep enough sensitivity.

Short-chain acyl-CoA dehydrogenase deficiency (SCADD) in twin brothers presenting with non-alcoholic steatohepatitis Hewson S1, Nagy L1, Ling S2, Mercimek-Mahmutoglu S1 Div Clin & Met Genet, SickKids, Toronto, Canada; 2Div Gastro, Hepat & Nutrition, SickKids, Toronto, Canada 1

Background: Short-chain-acyl-CoA dehydrogenase deficiency (SCADD) is defined by: 1) increased plasma butyrylcarnitine (C4) and/or increased urine ethylmalonic acid (EMA) under non-stressed conditions; 2) biallelic mutations or susceptibility variants (c.511C>T and c.625G>A) in the ACADS gene. Case report: An eleven-year-old male presented with intermittent abdominal pain; found to have elevated liver enzymes and fatty liver changes on abdominal ultrasound. His twin brother was confirmed to have the same clinical, biochemical and imaging features. Liver biopsy confirmed mild to moderate steatosis, minimal portal inflammation and fibrosis in both. Acylcarnitine profile showed elevations of C4 (0.89-1.75; reference range <0.68) in both. One urine sample showed mild elevation of EMA in both. ACADS molecular testing showed homozygous c.625G>A; pGly209Ser susceptibility variant and a heterozygous c.1147C>T; p.Arg383Cys mutation in both. Mother was heterozygous for the variant. Father was unavailable for testing. There were no other risk factors for fatty liver changes such as obesity (body mass index of both brothers <95th percentile). Conclusion: An abnormal metabolite profile, one pathogenic mutation and homozygous susceptibility variant in the ACADS gene confirmed the diagnosis of SCADD in twin brothers. Abnormal metabolite profile suggestive of SCADD warrants genetic testing to prevent invasive liver biopsy in patients with steatohepatitis. P-286

P-284 Heterogenous clinical and laboratory presentations in multiple acyl-CoA dehydrogenase deficiency (MADD)

Octanoic acid administration causes behavioral and neurochemical alterations in rats

Boemer F1, Schoos R1, Weekers L1, Debray FG1

Schuck PF1, Ramos AC1, Rodrigues LB1, Comim CM1, Mafioleti RL1, Zapelini HG1, Scaini G1, Quevedo J1, Ferreira GC1, Streck EL1

1

1

We present 3 confirmed and 2 highly suspicious patients (molecular assay is pending) with Multiple Acyl-CoA Dehydrogenase Deficiency. The first patient presented at 4 years with recurrent ketotic vomiting without hypoglycemia. The second suffered at 1 year a severe hypoglycemic coma with ketoacidosis and hyperammonemia. The third presented an acute episode of rhabdomyolysis at 18, evolving to a chronic form of lipid storage myopathy. Her sister has a similar clinical presentation. The fifth patient, 16 years, presented an acute episode of rhadomyolysis at 5 years, without any recurrence. Acylcarnitines profiles were versatile: C4 and C5-carnitines were normal for all patients but one; Patient3 presented hugely elevated C6-carnitine. Patient1, 2 and 3 showed moderated to gross elevations of C8, C10, C12 and C14carnitines. Patient4 presented an unremarkable profile, while Patient5 showed unsettled borderline elevations of C6, C8 and C10–carnitines only. Additionally, Patient1 presented normal in vitro fatty acid oxidation (FAO) studies due to riboflavin responsiveness. Molecular analysis

Background: Patients affected by medium-chain acyl-CoA dehydrogenase deficiency (MCADD) present tissue accumulation of octanoic acid (OA). Clinically, progressive encephalopathy, behavioral alterations, drowsiness and lethargy that may develop into coma and death are found. Objective: The effects of intracerebroventricular OA administration on behavioral tasks and BDNF and NGF levels in rat brain were investigated. Methods: Animals received a single intracerebroventricular OA injection (1.66 μmol). Control animals received artificial cerebral spinal fluid at equivalent volumes. One hour after the administration, animals were submitted to behavioral tasks. Immediately after, animals were killed by decapitation and brain structures were isolated for BDNF and NGF levels determination. Results: OA administration impaired aversive memory, and the animals required twice more stimulus to reach the memory acquisition criteria in inhibitory avoidance task. They also showed memory impairment in the open-field task, without alteration of motor and exploratory activities.

CHU Liege, University of Liege, Liege, Belgium

Univ do Extremo Sul Catarinense, Criciúma, Brazil

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OA animals did not present depressive-like behavior. Furthermore, BDNF and NGF levels were decreased in OA animal striatum. NGF levels were also decreased in hippocampus. Conclusion: Considering that BDNF and NGF levels decrease alter synaptic plasticity, it is tempting to speculate that it could be related to learning/memory impairment observed in animals receiving OA administration. P-287 Hemodiafiltration in mitochondrial 3-hydroxy-3-methylglutarycoenzyme A synthase (HMG-CoA synthase) deficiency Sass JO1, Kühlwein E2, Klauwer D2, Rohrbach M3, Baumgartner MR3 1 Div Clin Chem Biochem, Univ Child Hosp, Zürich, Switzerland; 2Div Intens Care Neon, Univ Child Hosp, Zürich, Switzerland; 3Div Metab & CRC, Univ Child Hosp, Zürich, Switzerland

Ketone bodies can provide a major contribution to the brain's energy requirements. The initial step in ketogenesis is catalyzed by HMG-CoA synthase. Usually, HMG-CoA synthase deficiency presents with acute hypoketotic hypoglycemia. An excellent prognosis is assumed if prolonged fasting is avoided. We report Swiss siblings with infectiontriggered metabolic decompensations. They initially presented with hyperpnea with pronounced metabolic acidosis and low blood glucose, hepatopathy and encephalopathy. In spite of immediate correction of hypoglycemia, and hemodiafiltration started on day 3 after admission, the first patient (aged 10 months) died in brain edema. Notably, pronounced ketonuria was noted perimortally. When his sister became symptomatic at 12 months of age, hemodifiltration and L-carnitine administration were started immediately, resulting in stabilization within 5 days (with recurrent hyperpnea when hemodiafiltration was interrupted before) and subsequent normal development. In both children the diagnosis of HMG-CoA synthase deficiency was confirmed by identification of the homozygous mutation c.634A>G (p.Gly212Arg) in the HMGCS2 gene. Comparison of the two cases underlines that encephalopathy and hepatopathy are not simply linked to energy deficiency. Obviously, toxic processes play an important role in pathogenesis. They can be addressed by early onset of hemodiafiltration which serves as an important tool for stabilization of the patient. P-288 Evidence that fibroblasts from patients affected by medium-chain acyl-CoA dehydrogenase deficiency (MCADD) are under chronic oxidative stress

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media, the percentage of stressed cells in all groups increased, but the level of superoxide was still higher in the patient fibroblasts. When galactose plus palmitate media was used, patient fibroblasts with the c.[199T C] + [985A G] genotype, associated with mild disease, seem to have higher superoxide levels than patients carrying null mutations. Conclusion: These results indicate that fibroblasts of MCADD patients are under chronic oxidative stress. It could be speculated that such mild stress exposure could induce an adaptive response, protecting cells against oxidative damage during pathological situations of metabolic stress like feverish infections. P-289 Proteomic investigation of cultivated fibroblasts from patients with mitochondrial short-chain acyl-CoA dehydrogenase deficiency (SCADD) Edhager AV1, Gregersen N1, Palmfeldt J1 1

Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark

Introduction: Disease etiology mechanisms of the rare inherited metabolic disorder SCADD are not well established. In the present work, a large-scale label free proteomic approach was employed to investigate the proteome profiles in five patients carrying homozygous missense mutations in the ACADS gene coding for SCAD enzyme of mitochondrial β-oxidation. Material and methods: Fibroblasts from patients (c.319C>T (n=2) and c.1138C>T (n=3) in the ACADS gene) and controls (normal human dermal fibroblasts, n=3) were obtained through skin biopsies, cultivated, followed by enrichment of mitochondria. The mitochondrial protein extracts were then separated by SDS-PAGE, in-gel trypsin digested, and extracted peptides were purified and analyzed by nanoLC-MS/MS (ESILTQ Orbitrap, Thermo). Results: More than 400 proteins annotated to mitochondria were identified and quantified. Among these, approximately 20 proteins were significantly altered, with an overrepresentation of proteins involved in fatty acid β-oxidation, amino acid metabolism, protein quality control system and apoptosis. Conclusions: The results indicate that the cells, in response to the SCADD, decrease the levels of also other enzymes participating in the β-oxidation, in combination with additional metabolic re-programming. Apoptosis regulator BAX and Lon protease were up-regulated in the patient cells, indicating severe cell stress as a consequence of decreased SCAD and impaired βoxidation. P-290

Tonin AM1, Fernandez-Guerra P1, Cornelius N1, Olsen RKJ1, Wajner M2, Gregersen N1 1 Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark; 2Dept Biochem, Fed Univ Rio Grande do Sul, Porto Alegre, Brazil

Background: Mutations in the ACADM gene causes MCADD. The clinical features are variable and the physiopathology has been related to energy deficiency, accumulation of toxic metabolites and presence or lack of misfolded MCAD protein, resulting in oxidative stress. Objectives: To evaluate the extent of mitochondrial oxidative stress under different metabolic conditions in cultured skin fibroblasts of controls and MCADD patients carrying distinct ACADM mutations. Results: Fibroblasts were grown in standard glucose concentration (11mmol/L) and after 24hrs the media were replaced by galactose (11mmol/L) or galactose and palmitate (100μmol/L). At standard glucose concentration, patient fibroblasts presented higher levels of mitochondrial superoxide compared to controls fibroblasts. In galactose

The stress modulator protein p66Shc as the effector for lipotoxic damage in VLCADD patients. Lund M1, Gregersen N1 1

Res unit of Mole Med, Au Univ Hosp, Aarhus, Denmark

Very Long Chain Acyl-CoA Dehydrogenase Deficiency (VLCADD) present with severe symptoms such as cardiomyopathy or with acute inability to produce sufficient ATP – resulting in sudden death. Milder cases present with symptoms such as hypoglycemia and increased vulnerability to stresses such as fever. On a cellular scale the inability to oxidize long chain fatty acids (FA) in the mitochondria lead to FA accumulation and associated lipotoxic damage. Noting that 1; activation of the requisite regulator proteins (PKC and Rac1) via saturated long chain FAs has been shown in other contexts & 2; that p66Shc executes its damaging effect as a result

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of major stressors such as fever, we investigate whether the chronic lipoxtoxic stress induced p66Shc accumulation accounts for the disproportionate vulnerability to fever. Knockdown with siRNA against p66Shc may prevent the acute stress of fever affecting the mitochondria via p66Shc. This study is based on cultured fibroblasts from mild and severe VLCADD patients plus healthy controls. We assess the redox milieu and the overall mitochondria quality and immunoblotting is used to compare the metabolic and apoptotic status and evaluate the efficacy of the p66shc knockdown. P-291 Muscle MRI in patients with long-chain fatty acid oxidation disorders Diekman EF1, van der Pol WL2, Nievelstein RAJ3, Houten SM4, Wijburg FA5, Visser G1

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The activity of MCAD was measured in lymphocytes with a spectrofluorometric assay using an electron-transfer-flavoprotein assay with phenyl-propionyl-CoA as substrate. The activity was compared to genotype, acylcarnitines and acylglycines levels, and patient's clinical status. Since screening-start in November 2010, eight children have been diagnosed with MCADD in southwestern Sweden. Three novel mutations were identified. Patients harbouring known deleterious mutations showed a residual MCAD-activity <1% whilst one patient with c.199T>C in combination with a novel c.230del mutation had a residual activity of 21%. The median activity in carriers was 59% of normal mean. In newborn screening it is important to differentiate between patients with truly disease-causing mutations from those with benign variants. Our data indicate that our method can be used to evaluate the residual MCAD-activity in lymphocytes from patients with MCADD and possibly predict the clinical relevance for these children. P-293

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Dpt Metab Dis, Univ Med Center, Utrecht, Netherlands; 2Rudolf Magnus Inst of Neuroscience, UMCU, Utrecht, Netherlands; 3Dpt Radiology, Univ Med Center, Utrecht, Netherlands; 4Lab Gen Metab Dis, Academic Med Center, Amsterdam, Netherlands; 5Dpt Paediatrics, Academic Med Center, Amsterdam, Netherlands Background: Magnetic Resonance Imaging (MRI) is a non-invasive tool used for the diagnosis and follow-up of muscular dystrophies and congenital myopathies. Mitochondrial long-chain fatty acid oxidation (lcFAO) disorders may present as metabolic myopathies with weakness and episodic rhabdomyolysis. Objective: We investigated whether muscle MRI can be used for diagnosis, disease monitoring and understanding pathogenesis of myopathy in patients with lcFAO disorders. Methods: Lower body MRI was performed in 20 patients with lcFAO disease (3 patients with CPT2D, 12 with VLCADD, 3 with MTPD and 2 with isolated LCHADD). Results: Marked symmetrical abnormalities were found in the m. tibialis anterior, m. gastrocnemius and m. soleus in all MTPD patients and in 1 LCHADD patient. The majority of VLCADD patients showed mild muscle abnormalities of m. gluteus, m. biceps femoris and m. soleus. In CPT2D patients, no abnormalities were observed. Extensive abnormalities of almost all muscle groups were observed in 2 patients with very high CK (>11.000) levels. Conclusion: Muscle MRI in patients with lcFAO disorders show specific patterns of abnormalities with signs of lipid accumulation and oedema/inflammation. This technique may be used as a diagnostic tool, to monitor disease course, and to elucidate the pathogenesis of lcFAO related myopathy. P-292

Report of 23 cases with betaketothiolase deficiency in a Vietnam center Nguyen KN1, Chi DV1, Nguyen HT1, Can NBT1, Bui TP1, Yamaguchi S2, Fukao T3 1 National Hospital of Pediatrics, Vietnam, Hanoi, Viet Nam; 2Shimane University School of Medicine, Shimane, Japan; 3Graduate School of Medicine, Gifu Univ, Gifu, Japan

Betaketothiolase (T2) deficiency is a rare inherited metabolic disease. Objectives: to describe the phenotypes, genotypes and outcome of T2 deficiency in Vietnam. Methods: descriptive study of 23 patients with T2 deficiency at the National Hospital of Pediatrics - Hanoi - Vietnam from 2005 to 2012. 14 families were analyzed for mutations in the ACAT1 gene. Results: 23 patients were born to 21 unrelated families with nonconsanguineous parents. 22/23 patients presented with acute ketoacidotic crises, and 1 patient was asymptomatic at 3 days of age. Mean onset of the first crisis was 14.1 months. Clinical features of the crises were dehydration, tachypnea, and lethargy/coma; crises were often triggered by infections. Laboratory analyses during the crises showed 100 % severe ketoacidosis (pH: 6.5 – 7.05) and leukocytosis. 14/14 patients were found to have homozygous/compound heterozygous mutations of the ACAT1 gene. 4 different mutations were identified: R208X, IVS101g>c, A410V, and 163_167del5ins2. The R208X mutation is common in Vietnam (75% of mutant alleles). The prognosis was good: 81% showed normal development. Conclusions: A large cohort of patients with T2 deficiency was identified in a Vietnam center. Worldwide, no common mutations of the ACAT1 gene have been reported but the R208X mutation is common in Vietnam.

Genotype and lymphocyte electron-transfer-flavoprotein assay of medium chain acyl-CoA dehydrogenase for follow-up analysis of positive neonatal screening results

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Kollberg G1, Hellerud C1, Reims A2, Leckström K1, Holme E1

Carnitine palmitoyltransferase 2 deficiency - not always a straight forward diagnosis

Sahlgrenska University Hospital, Gothenburg, Sweden; 2The Queen Silvia Children's Hospital, Gothenburg, Sweden 1

Ferreira AC1, Vieira JP2, Sousa C3, Rocha H3, Vilarinho L3, Sequeira S1 Metab Unit, Hosp D Estefânia, Lisbon, Portugal; 2Serv Neuroped, Hosp D Estefânia, Lisbon, Portugal; 3Newb Screen, Met Gen, Nat Inst H, INSA, Oporto, Portugal 1

Children with medium-chain acyl-CoA dehydrogenase deficiency (MCADD) are now mostly identified by newborn screening. Genotyping, plasma-acylcarnitines and urinary acylglycines are in most case sufficient for confirmation of diagnosis. However, new genetic variants of unknown significance require further characterization. Thus, it is important to establish a reliable functional assay for risk assessment in asymptomatic newborns with uncertain genetic and metabolic findings.

Background: Carnitine Palmitoyltransferase 2(CPT2) deficiency is an autosomal recessive disorder of fatty oxidation presenting mainly in adolescents and young adults. The muscular form is characterized by attacks of myalgia and myoglobinuria while the infantile variety usually

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presents with recurrent attacks of hypoketotic hypoglycaemia, liver and, sometimes, concomitant cardiac involvement. Case Report: Two-year-old girl presenting a febrile episode followed by refusal to walk or stand, hepatomegaly, high CK levels (>140000IU/l), myoglobinuria and elevated transaminases (AST-4258U/L and ALT1753U/L). Glycaemia and cardiac evaluation were normal. She had a previous similar episode at 22 months of age. Despite the high CK levels muscle palpation seemed painless. Her father had recurrent episodes of muscular rigidity during his childhood and early adulthood with possible triggering events. Acylcarnitine cromatography diagnosed CPT2 deficiency and molecular studies showed that the child and her father were homozygous for the c.338C>T(p.S113L) mutation. Discussion: We describe a predominantly myopathic form of CPT2 deficiency with myoglobinuria in a very young child, an aspect that is very rarely described in the literature. It is also shows that the neonatal screening proved not useful for this diagnosis and, as the father was symptomatic and homozygous for the same mutations, that CPT2 deficiency is certainly underdiagnosed.

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metabolites in these individuals may not be detected on a standard urine organic acid test. However, as with other diagnostic methods involving multiple analytes and complex metabolic profiles, the clinical utility of urine AG analysis can be improved with postanalytical interpretation tools. The Collaborative Laboratory Integrated Reports (CLIR) is an initiative launched to reproduce in Clinical Biochemical Genetics the web-based products of the R4S (Region 4 Stork) newborn screening collaborative project. CLIR is based on multivariate pattern recognition software for developing diseasespecific computational tools that relies on dynamic disease-specific ranges and is capable of evaluating all possible markers provided within large data sets for any given disease. The tools can help discriminate between two or more conditions with overlapping biochemical phenotypes. One such condition is glutaric acidemia type II (GA2), in which there may be elevated urinary excretion of the same metabolic intermediates observed in other diseases such as MCAD deficiency and ethylmalonic encephalopathy. We present the first generation of post-analytical CLIR tools for the diagnosis of GA2, based on urine AG tests performed at Mayo Clinic from 1999-2012.

P-295 P-297 Maximal incremental and endurance exercise tests in a 13 year-old patient with long-chain acyl CoA dehydrogenase deficiency (LCHADD) and heptanoate treatment

Severe VLCAD with normal acylcarnitine profile. Is tandem ms still the best screening tool for vlcad?

Karall D1, Mair G2, Albrecht U1, Niedermayr K1, Scholl-Bürgi S1

Shuaib T1

1 Medical University, Pediatrics, Innsbruck, Austria; 2Institute for Sports & Alpine Medicine, Innsbruck, Austria

1

Pediatric Dep, King Abdulaziz University, Jeddah, Saudi Arabia

Background: Exercise and subsequent catabolism is a potential trigger for creatine kinase (CK) concentration increase (rhabdomyolysis) in patients with LCHADD. Objective: To evaluate clinical and biochemical stability under physical exertion conditions in a 13 year-old LCHADD patient treated with heptanoate. Case report: LCHADD was diagnosed during first decompensation at age 20 months. In the following two years, the patient had several episodes of rhabdomyolysis. Heptanoate 0.5-1 g/kg/day was started at 4 years, with no further CK elevations since. He is clinically stable, has retinopathy, but no vision impairment and no polyneuropathy. Maximal incremental and endurance exercise tests were performed to evaluate both clinical and metabolic stability during and under exertion. Results: Physical fitness was adequate for age (maximum blood lactate 7.0 mmol/l, appropiate lactate performance curve, maximum heart rate of 196 bpm, maximum power 139 Watt = 2.5 Watt/kg). There were no signs of clinical (muscle pain, dark urine) or metabolic derangement (stable CK, acyl carnitine profiles, blood gas analyses) – neither after maximal incremental nor endurance exertion. Conclusion: Both under maximal incremental and endurance exertion, clinical and biochemical parameters remained stable in this 13 year-old LCHADD patient receiving heptanoate treatment.

Background: Very long-chain acylCoA dehydrogenase deficiency is an autosomal recessive defect in mitochondrial fatty acid oxidation. Newborn screening can detect the disease early. Few reports address false negative cases. Our case was missed by newborn screening despite her severe phenotype. Method: 8m girl had normal newborn screening. At 3m and 6m her TMS was repeated. Urine GCMS and alpha-glucosidase assay on DBS done. Echo and MRI brain were performed. She had ACADVL gene sequencing. Results: 8m girl had normal newborn screening. At 3 months she was hypotonic with hypertrophic cardiomyopathy. Repeated Tandem MS revealed low free carnitine, and normal alpha-glucosidase. At 6m developed hypoglycemia, elevated CK, LFT. Echo revealed hypertrophic cardiomyopathy with pericardial effusion. She had increased C14:1 and dicarboxylic aciduria. Novel mutations were detected in ACADVL gene. She died at 8m with respiratory failure. Conclusion: Early onset VLCAD is a severe disease. Newborn screening can detect elevated C14:1 which is diagnostic. Few false negative cases been reported. Our case had a severe phenotype with negative screening. Her biochemical changes correlated poorly with her disease severity. Tandem MS is a useful screening tool with its limitation in VLCAD. Increasing false negative cases necessitate a new sensitive tool.

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Clinical utility of post-analytical interpretive tools for the diagnosis of glutaric acidemia type II by urine acylglycine analysis

A novel drug target site for medium chain acyl-CoA dehydrogenase deficiency: implications for future drug therapy

Hasadsri L1, Gavrilov D1, Matern D1, Oglesbee D1, Raymond K1, Tortorelli S1, McHugh D1, Marquardt G1, Rinaldo P1

Kang H1, Vockley J2, Mohsen A-W2

1

2

The analysis of urinary acylglycines (AG) by GC-MS/MS is especially useful in asymptomatic patients, as the excretion of key

Medium chain acyl-CoA dehydrogenase deficiency is one of the most common biochemical genetic disorders. A single point mutation in the ACADM gene (985A>G) has been identified in

1

Biochemical Genetics Lab, Mayo Clinic, Rochester, MN, United States

Dept Human Genetics, Univ of Pittsburgh, Pittsburgh, United States; Dept Pediatrics, Univ of Pittsburgh, Pittsburgh, United States

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90% of the alleles in deficient patients. This mutation results in a K304E replacement leading to reduced MCAD activity due to impaired folding and/or stability. To improve the MCAD K304E mutant activity, we hypothesized that binding of ligands to substrate binding sites can stabilize the mutant enzyme and hence rescue MCAD activity. The ETF docking site of the MCAD protein was investigated as a possible drug target site. 12-mer peptides, homologous to the ETF docking peptide, were synthesized. To test whether these peptides can influence the mutant MCAD stability, the MCAD K304E mutant protein was coincubated with these peptides and monitored for thermal stability. One of the peptides, DP193 enhanced the thermal stability of the mutant enzyme as manifested by Tm shifts of ~2.5:-4:C. CD spectra of protein melting assays and delayed proteolysis of specific peptide bonds in the presence of DP193 are also consistent with enhanced stability. These results suggest that ETF docking site is a viable target for MCADD drug design and that peptide DP193 can be used as a scaffold.

enroll up to 20 subjects (0.5 to 25 years) with VLCAD, CPT-II or LCHAD deficiency and severe manifestations of FAOD, including cardiomyopathy, hypoglycemia, rhabdomyolysis ,chronic elevated CK and/or frequent hospitalizations. Subjects will be monitored on their current therapy for 4 weeks and then cross-over to triheptanoin treatment. The short- (24 weeks) and long-term (78 weeks) effects of triheptanoin treatment will be evaluated. Subjects will be followed for changes in cardiac status, hypoglycemic events and muscle physiology/exercise intolerance as measured by ergometry, 12MWT, self-reported pain on exertion, and CK levels as well as longer term changes in major medical events. Changes occurring in the first 24 weeks will be compared with the prior 4 week run-in period and changes in major medical events over 78 weeks will be compared with the 78 weeks prior to therapy. The study will begin in 2013 as part of a broader clinical development program for triheptanoin in long-chain FAOD. Conflict of Interest declared.

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Very long chain acyl-CoA dehydrogenase deficiency (VLCAD) - a mimicker of infantile Pompe disease

Very long chain acyl-CoA dehydrogenase deficiency (VLCADD): management through puberty

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Hui J1, Kwok SY1, Yuen YP1, Law LK1, Tang NLS1

Regier DS1, MacLeod EL1, Greene CL2, Barshop BA3, Kirmse B1

1

Genetics and Metabolism, CNMC, Washington, DC, United States; 2Dept. of Pediatrics, U. of Maryland, Baltimore, United States; 3University of San Diego, SanDiego, CA, United States

Very long chain acyl-CoA dehydrogenase (VLCAD) deficiency is an autosomal recessive disorder characterized by impaired mitochondrial β-oxidation of fatty acids with a chain length between 14 and 18 carbons. Patients with the severe neonatal form present with cardiomyopathy, hepatopathy and skeletal myopathy. Case: We reported a 3 months girl of Chinese ethnic background presenting at 2 months of age with lethargy and poor feeding. There was family history of an older sibling dying at 2 months of age with pneumonia. Physical examination revealed a hypotonic lethargic baby with gross hepatomegaly. CXR revealed massive cardiomegaly. The initial clinical suspicion was infantile pompe disease. However, echocardiogram identified a pericardial effusion in addition to left ventricular hypertrophy. Subsequent acylcarnitine profile showed significantly elevated C14, C14:1, C16, and C18-carnitines. Sequence analysis of ACADVL gene revealed a heterozygous mutation S22X, a known disease causing mutation and another novel mutation K299E. Patient's pericardial effusion worsened with evidence of tamponade requiring urgent pericardial window. She succumbed two days after the operation with progressive pump failure. Conclusion: Very long chain acyl-CoA deficiency (VLCAD) rather than infantile pompe disease should be considered as a more likely clinical diagnosis when pericardial effusion is present in infants with hypotonia and cardiomyopathy.

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Case Report: Hormone fluctuations are anecdotally thought to affect metabolic homeostasis in those with inborn errors. AG was diagnosed with VLCADD in the neonatal period and was metabolically stable until her pre-pubertal years. In the year preceding menarche, she had 10 hospitalizations for rhabdomyolysis. During this time she required near-continuous parenteral and enteral caloric intake. In contrast, during the one year following menarche, she was hospitalized three times, all for gastric tube dysfunction/repairs and was weaned from continuous caloric intake. Pre-menarchal average CK levels during hospitalizations was higher (16,657 IU) than postmenarchal levels (8,517 IU). Titration of fatty acids, MCT supplementation and mitochondrial cofactors/supplements did not appear to affect frequency or severity of pre-menarchal decompensations. AG now enjoys riding her bike and attending school full-time, activities that were not possible in the pre-pubertal years. Conclusion: It is possible that hormonal fluctuations around puberty dynamically affect metabolic rate, thus making metabolic control difficult in some patients. Further systematic study of the relationship between pubertal changes and intermediary metabolism, especially fatty acid oxidation, is warranted.

The Chinese University of Hong Kong, New Territories, Hong Kong, China

P-302 P-300 Design of an open-label phase 2 study to assess safety and clinical effects of ux007 in subjects with long-chain fatty acid oxidation disorders (FAOD)

Mitochondrial proteome characterization in long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency. Rocha H1, Ferreira R2, Almeida V2, Vitorino R2, Lopes L1, Amado F2, Vilarinho L1

Kakkis E1, Skrinar A1, Hsu K1, Marsden D1 1

I&D unit, Genet Dep, Nat Inst of Heath, Porto, Portugal; 2Chemical Department, Aveiro University, Aveiro, Portugal

Triheptanoin is an anaplerotic medium chain triglyceride therapy that is expected to restore mitochondrial metabolism through replacement of both acetyl-CoA and TCA cycle intermediates. Although studied for more than a decade in investigator-sponsored trials, an industrysponsored multi-center Phase 2 study is now planned. The study will

Isolated long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) is a metabolic disorders affecting mitochondrial fatty acid beta-oxidation. Pathophysiological mechanisms leading to clinical phenotype are believed to rely mainly on the resulting energy deficiency and in the accumulation of toxic intermediates, namely long-chain 3-

1

Ultragenyx Pharmaceutical, Inc, Novato, CA, United States

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hydroxy fatty acids. However, its consequences on mitochondrial function are still poorly understood. In order to bring new insights, a proteome approach was adopted. We isolated mitochondria from cultured fibroblasts, from two patients with LCHADD (homozygous for the common mutation c.1528 G>C) and from normal controls. The used approach (nanoLC-MS/MS) allowed the positive identification of 729 proteins in both patients and controls, presenting 40 of them (based on iTRAQs data) significant differences in their relative abundance in the patients. Among the differentially expressed are several metabolic enzymes, proteins associated to binding/folding functions, mitochondrial antioxidant enzymes, proteases as well as proteins associated to apoptotic events. The overexpression chaperones like Hsp60, antioxidant enzymes and apoptotic proteins reflect the mitochondrial response to LCHADD. Our study provides a global perspective of the mitochondrial proteome plasticity in LCHADD and highlights the main molecular pathways involved in its pathogenesis. P-303 A synonymous polymorphic variation in ACADM exon 11 affects splicing efficiency and may affect fatty acid oxidation Bruun GH1, Doktor TK1, Andresen BS1 1

Biochem & Mol Biol, Univ. South Denmark, Odense, Denmark

In recent studies combining genome-wide association and metabolomics, a single-nucleotide polymorphism (SNP), rs211718C>T, located far upstream of the MCAD gene (ACADM) was found to be associated with improved beta-oxidation of medium-chain fatty acids. We examined the functional basis for this association and identified linkage between rs211718 and the intragenic synonymous SNP c.1161A>G in ACADM exon 11 (rs1061337). Employing ACADM minigenes we show that c.1161A is associated with aberrant splicing of exon 11, which is corrected by c.1161G. This may result in more full length MCAD from c.1161G alleles. Our RNA pull down analysis suggests that the improved splicing of the c.1161G allele is due to changes in the relative binding of the splicing regulatory proteins SRSF1 and hnRNP A1. Analysis of publicly available RNA-seq data from several hundred cell lines, show significantly more reads with c.1161G than with c.1161A in heterozygous individuals, supporting that c.1161A>G is a functional SNP, which leads to higher MCAD expression, perhaps due to improved splicing. This study is a proof of principle that synonymous SNPs are not neutral. By changing the binding sites for splicing regulatory proteins they can have significant effects on pre-mRNA splicing and thus protein function.

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Results confirming MCADD only became available after death. Case 2: included in this series as neonatal screening for LCHADD is currently being piloted in the UK. A baby girl died suddenly at home on day 3 after a period of poor feeding. Acylcarnitine analysis of bloodspots taken at post mortem gave a profile consistent LCHADD/MTP deficiency. Prior to this the cause of death was going to be attributed to coarctation of the aorta and a metabolic cause was not being highly considered. A diagnosis of MTP deficiency was later confirmed with enzyme and mutation analysis. We conclude that early death due to fat oxidation disorders are either under reported or under diagnosed in the UK. P-305 MCAD deficiency: insights on novel mutations in the ACADM gene Catarzi S1, Tonin R1, Caciotti A2, Thusberg J3, Malvagia S4, la Marca G4, Pasquini E5, Cavicchi C2, Ferri L1, Donati MA5, Baronio F6, Guerrini R1, Mooney SD3, Morrone A1 Dept NEUROFARBA, AOU Meyer, Un. Florence, Florence, Italy; 2Mol and Cell Biology Lab, AOU Meyer, Florence, Italy; 3Buck Institute for Research on Aging, Novato, California, United States; 4Newborn screen Biochem Pharm, AOU Meyer, Florence, Italy; 5Metabolic Disorders Unit, AOU Meyer, Florence, Italy; 6Dept of Pediatric, Un. Bologna, Bologna, Italy 1

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Background: Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common defect of mitochondrial β-oxidation, characterized by hypoglycemic crisis under fasting or stress conditions, leading to lethargy, seizures, brain damage or even death. Objectives: This report aims to improve the knowledge of this as dangerous as it is treatable metabolic disorder included in the expanded newborn screening (NBS) panel. Methods: Biochemical acylcarnitines data obtained through NBS by LC-MS/MS were confirmed by molecular analysis of the ACADM gene and by RT-PCR analysis and in silico predictions of altered MCAD protein structures. Results: Among 324.000 screened newborns we identified 14 MCADD patients bearing eight known and the following three new mutations: p.Gly85Arg, p.Val119Asp and p.Gly275*. In silico analysis predicted for p.Gly275* a high degree of potential alternative splicing for ACADM mRNA, confirmed by RT-PCR and sequencing analysis. Conclusions: These findings confirm the rising incidence of MCADD due to the efficacy of NBS. Our molecular data reveal that the "common" Lys329Glu mutation only accounts for 32% of the defective alleles identified by NBS, while in clinically diagnosed patients it accounted for 90% of them. Finally, in silico analysis proved to be useful for clarifying the pathogenetic mechanism of any new mutation including nonsense variants.

Early death due to fat oxidations disorders: an update

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Barski RR1, Henderson MJ1, Herrera DJ1, Olpin SE2, Walter JH3, Jameson E3

Evaluation of age-related variations in free carnitine and acylcarnitine concentrations from newborns to adults

Biochem Genet, LTHT, Leeds, United Kingdom; 2Dept Clin Chem, Sheff, Child Hosp, Sheffield, United Kingdom; 3Willink Biochem Genet Unit, Manchester, United Kingdom

Rausell D1, Bonet E1, Suescun M1, Ruiz S1, Garcia AM1

In 2010 we presented 5 cases over an 11 year period of infants who died of MCADD prior to newborn screening results being available. In this paper we provide an update with two further cases. Case 1: presented to her local district general hospital on day 2 with lethargy, hypoglycaemia and fitting. Despite treatment she went on to have recurrent cardiac arrhythmias which eventually led to her death on day 5. Bloodspots were taken early in the presentation but acylcarnitine analysis was not requested urgently.

Background: There are few studies published about age-related variations in the reference values of free carnitine (FC) and acylcarnitines (AC) obtained from dried blood spots. Objectives: Evaluate age-related variations in FC and AC concentrations. Methods: Dried blood spots on Whatman 903 paper were collected from 3966 healthy individuals in a period of a year. Exclusion criteria: very preterm newborns, special diet, acute or chronic illness, use of medications. No individual had fasted longer than 12 hours.

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1

Metab Dis & Newborn Scr Lab, La Fe Hosp, Valencia, Spain

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FC and AC concentrations were measured in an automated electrospray tandem mass spectrometry using NeoBase Non-derivatized MSMS kit (Perkin Elmer). The population was divided into 9 groups based on metabolic, growth and feeding status, from 48h to adulthood. For all analytes, mean and standard deviation were calculated. To enable comparison between groups, a normal distribution and a confidence interval of 99% was considered. Statistically significance was defined as a P<0.01 Results: Significant age-related variations were found for FC and AC in groups from 48h to 2years. No further changes from 2years to adolescence were presented, except for C18 to C18:2. Adult group showed significant lower concentrations for almost all AC. Conclusion: Age-related distribution of FC and AC should be taken into account in diagnosis. P-307 Two different clinical aspects of short chain acyl-CoA dehydrogenase deficiency (SCADD): one novel versus one common mutation Aktuglu Zeybek AC1, Kiykim E1, Ceylander S2, Cansever MS1, Soyucen E3, Demirbilek V4, Aydin A1 1 Div Ped Nutr and Met, Ist Univ Cer Med F, Istanbul, Turkey; 2Intergen Genetics Center, Ankara, Turkey; 3Div Metab Dis, Akdeniz Univ Child Hosp, Antalya, Turkey; 4Div Child Neurol, Ist Univ Cer Med F, Istanbul, Turkey

Background: SCADD is a rare fatty acid oxidation disorder, due to ACADS gene mutations. Phenotype varies from fatal metabolic decompensation to asymptomatic individuals. Here we report two cases with two different clinical and mutational findings. Cases: 1st patient is a male patient, product of an uneventful pregnancy of unrelated parents. Newborn screening revealed elevated C4 butyrylcarnitine, decreased free carnitine levels. Ethylmalonic (EMA), methylcitric acid (MCA) excretion was detected in urine. A novel, c.1157G>A/ c.625G>A compound heterozygote, mutation was found in ACADS gene. He is now 4 years old, free of symptoms with increased levels of C4 and EMA-MCA. 2nd patient is a female patient born to parents of 2nd degree consanguinity. Following a normal neurological development except clumsiness, at the age of 2.5 she was admitted to hospital with serious difficulty in walking. Pyramidal signs were detected. The MRI revealed hyperintensities in bilateral nucleus caudatus, putamen and basal ganglia. C4 was increased, EMA-MCA excretion was detected in urine. c.625G>A homozygote mutation was detected in ACADS gene. Conclusion: These two phenotypes confirm the wide range of clinical manifestations of the disease. Although, ACADS:c.625G>A is a commonly seen; to our knowledge ACADS:c.1157G>A is a novel variant not previously described in patients. P-308 Medium-chain acyl-CoA dehydrogenase deficiency: the particular picture of the Portuguese population Ventura FV1,2, Leandro P1,2, Luz A1,2, Ramos R1,2, Rocha H3, Diogo L4, Martins E5, Leão-Teles E6, Janeiro P7, Costa C7, Gaspar A7, Vilarinho L3, Tavares de Almeida I1 1

MetGen, iMed.UL, Fac Pharm Univ Lisbon, Lisbon, Portugal; 2Dp Bioch Hum Biol, Fac Pharm Univ Lisbon, Lisbon, Portugal; 3NBS Unit, Gen Dpt, NIH Dr Ricardo Jorge, Porto, Portugal; 4Metab Unit, Ped Hosp Coimbra, Coimbra, Portugal; 5Children's Hosp Maria Pia, Porto, Portugal; 6Metab Unit, Ped Serv, Hosp São João, Porto, Portugal; 7Metab Unit, Ped Clin Univ, Hosp St Maria, Lisboa, Portugal

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Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the commonest defect of mitochondrial fatty acid ß-oxidation. We present the first report on the biochemical and molecular features of MCADD in Portugal. From the 102 patients studied, 91% were diagnosed after newborn screening (NBS) being 79% of gypsy ancestry. Amongst this cohort, 17 individuals were identified through segregation studies which also revealed 75% heterozygous for the G985 allele. This allele was found in homozygosity in 96/102 and in compound heterozygosity in 5/102 patients. Three novel mutants were identified: p.Y48C, p.D143V and p.G377V. The G985 allele was exclusively associated with haplotype H112 of ACADM gene. Furthermore, new H212 and H122 haplotypes were revealed. The enlargement of NBS in 2004 changed the picture of the MCADD in Portugal reaching an incidence of 1:8,804 newborns. Interestingly, our study reports a peculiar high rate of MCADD patients with gypsy origin and the fact that although from a closed ethnic group, their genetic background is the same as in other populations: G985 allele and haplotype H112. This strongly supports a single origin for the c.985A>G mutation and the conclusion that MCADD is a true monogenic disorder not related to gender or ethnicity. Acknowledgement: SPDM; FCT/Portugal (PEst-OE/SAU/UI4013/2011) P-309 Functional and structural characterization of six novel medium-chain acyl-CoA dehydrogenase (MCAD) mutants Nunes J1,2, Louro F1,2, Lopes F1,2, Bonito CA1,2, Palir N1,2, Lino P1,2, Vilarinho L3, Waterham HR4, Wanders RJA4, Tavares de Almeida I1, Leandro P1,2, , Ventura FV1,2 1

MetGen, iMed.UL, Fac Pharm Univ Lisbon, Lisbon, Portugal; Dp Bioch Hum Biol, Fac Pharm Univ Lisbon, Lisbon, Portugal; 3 NBS Unit, Gen Dpt, NIH Dr Ricardo Jorge, Porto, Portugal; 4 Lab GMZ, Dp Clin Chem, AMC, Univ Amst, Amesterdam, Netherlands 2

MCAD is a homotetrameric protein whose deficiency is the primary genetic defect of mitochondrial fatty acid beta-oxidation (OMIM 201450). p.K304E is the most prevalent mutation but many other rare mutations with unknown relevance have been identified. We aimed to characterize six novel MCAD missense mutations identified in Portugal (p.Y48C, p.D143V, p.G377V) and in Netherlands (p.R55G, p.G224R, p.L238F). Recombinant purified proteins were investigated for enzymatic properties, protein yield/purity, oligomeric and proteolytic profiles and thermal/kinetic stability (DLS and DSF). The mutants are produced with high yield and present activities lower than Wt, except p.L238F which shows low yield but high residual activity (90%). The presence of FAD during purification only benefited Wt, p.Y48C and p.K304E activities. Oligomeric profiles indicate that like p.K304E, p.Y48C is highly unstable with elevated content of aggregates, high molecular weight and dimeric forms. This is reinforced by thermal aggregation kinetic data. All mutants showed lower resistance to proteolysis than the Wt. Thermal denaturation profiles suggest, in general, that FAD significantly raises mutants' in vitro stability. Our data demonstrate the disease-causing effect of the studied novel MCAD mutants and revealed that due to underlying instability some of them may be good candidates for chaperone therapy. Acknowledgement: SPDM; FCT/Portugal (PEst-OE/SAU/UI4013/2011)

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P-310 Genotype-phenotype correlation in MCADD detected by newborn screening and relevance of l-carnitine supplemention in patients homozygous for the common c.985A>G mutation Castiñeiras DE1, García-Nimo L1, Sánchez-Pintos P1, Diogo L2, LeãoTeles E3, Martins E4, Santos E5, Bueno MA6, Delgado-Pecellín C6, Bóveda MD1, Cocho JA1, García-Viloria J7, Ribes A7, Rocha H8, Couce ML1 1 Hospital Clínico Universitario, Santiago de Compostela, Spain; 2Hospital Pediátrico, Coimbra, Portugal; 3Centro Hospitalar de S. João, EPE, Porto, Portugal; 4Hospital de Crianças Maria Pia, Porto, Portugal; 5Centro Hospitalar Gaia/Espinho, Gaia/Espihno, Portugal; 6Hospital Universitario Virgen del Rocio, Sevilla, Spain; 7Hospital Clínic, Barcelona, Spain; 8 Instituto Nacional de Saúde Dr. Ricardo, Porto, Portugal

Medium-chain acyl-CoA dehydrogenase deficiency(MCADD) is a inherited defect of metabolism. The aim of this study was to evaluate the relationship between genotype-phenotype, clinical data and biochemical parameters at diagnosis and during follow-up to optimize monitoring of these patients. We carried out a multicenter study in southwest Europe, of MCADD patients detected by newborn screening(NSB), evaluated data included free carnitine(C0) and the acylcarnitines C8, C10, C10:1 together with C8/C2 and C8/C10, clinical presentation parameters and genotype, in 45 patients. Follow-up data included C0 levels, duration of carnitine supplementation and occurrence of metabolic crises. C8/C2 ratio and C8 were the most accurate biomarkers of MCADD in NBS. We found a high number of patients homozygous for the c.985A>G mutation (75%). Moreover, in these patients C8, C8/C10 and C8/C2 were higher than in patients with other genotypes, while median of C0 was significantly lower (23 μmol/L vs 36 μmol/L). Carnitine supplementation was required in 82% of patients (C0≥12μM), and necessary for a longer period in patients homozygous for the c.985A>G than in patients with other genotypes. Our data show a direct association between homozygosity for c.985A>G, lower carnitine values at diagnosis, and a higher dose of carnitine supplementation for maintenance within the normal range.

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profiles. Significant lipid accumulation in skeletal muscle (10/10), myogenic EMG (5/8) were present. Five patients with mild phenotype harboured homozygote L377P mutation. Clinical and biochemical parameters were either totally (n=17) or partially (n=8) corrected after riboflavin treatment. Conclusion: Since it is a treatable disorder, this diagnosis must be considered by performing an acylcarnitine profile in all patients presenting with an unexplained muscular weakness. P-312 Carnitine uptake defect presents with hypoglycemic coma with elevated ketone bodies Kuster A1, Hauet Q1, Piloquet H1, Picherot G1, Hubert G1, Boutron A2, Vianey-Saban C3, Acquaviva C3 1 Ped Int Care Unit, Univ Child Hosp, Nantes, France; 2Metabolic Biochemistry, Univ Hosp, Le Kremlin Bicêtre, France; 3Metabolic Biochemistry, Univ Hosp, Lyon, France

Clinical and biochemical evaluation of 27 patients with multiple acyl-CoA dehydrogenase deficiency

Carnitine is obligatory for mitochondria-directed long-chain fatty acid transport. Carnitine uptake defect (CUD) is potentially lethal, but highly responsive to L-carnitine therapy. Defect is due to carnitine transporter OCTN2 expressed in muscle, heart, intestine and kidney with renal carnitine wasting. Most CUDs first presented infantile-onset cardiomyopathy and muscle weakness due to impaired fatty acid oxidation. Rarely, fasting stress provokes hepatic presentation with hypoglycaemic encephalopathy where hypoketosis is reported due to diminished hepatic carnitine uptake impairing ketogenesis. A 5-years old girl presented unconsciousness after 24hour-fast, profound hypoglycaemia and elevated ketone bodies in plasma (3OHbutyrate 2, acetoacetate 5 mmol/l) and urine. Plasma carnitine levels were markedly reduced (free 2, total 4 μmol/l), but normal in urine. Organic acids confirmed high ketone- and dicarboxylic aciduria. Further evaluation revealed dilated cardiomyopathy. SLC22A5gene analysis found: c.67_69del(exon1) and c.1319C>T(exon8). On high dose carnitine, plasma levels normalized and urinary loss was unmasked. This case of rare hepatic presentation of CUD illustrates that hepatic beta-oxidation is independent of OCTN2 and that liver maintains sufficient carnitine levels to support ketogenesis as 1) it has a separate carnitine transporter (OCTN1) and 2) it provides carnitine by endogenous biosynthesis. CUD and early treatment should be considered in hypoglycemic coma even with ketosis.

Balci MC1, Ersoy M1, Demirkol M1, Gokcay G1

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1

Different clinical presentations in siblings with mitochondrial acetoacetyl-CoA thiolase deficiency

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Div Nutr Metab, Child Hosp, Ist Med Fac, Istanbul, Turkey

Background: Multiple acyl-CoA dehydrogenase deficiency (MADD,MIM 231680) is a disorder of fatty acid and amino acid metabolism, resulting from a deficiency of electron transfer flavoprotein (ETF) or ETF dehydrogenase that manifests with severe neonatal to late onset forms. Objectives: The aim was to evaluate presenting symptoms and clinical phenotypes of MADD. Results: At initial diagnosis, at the median age of (8.4+8.6 years, range:0.1-31), clinical and biochemical evaluation of 20/27 patients showed proximal myopathy and exercise intolerance (n=11), encephalopathy and hepatopathy (n=4), developmental delay (n=4), other neurologic symptoms (n=2) as a fatal neonatal form with congenital anomalies (n=1) and neonatal hypoglycemia (n=3). Seven asymptomatic patients were diagnosed with family history (n=5) and expanded neonatal screening (n=2).The diagnosis was made by pathologic acylcarnitines on tandem mass spectrometry, and urine organic acid

Kose M1, Canda E2, Kagnıcı M3, İşgüder R4, Kalkan Uçar S2, Bähr L5, Britschgi C5, Sass JO5, Çoker M1 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis, Ege Univ Child Hosp, İzmir, Turkey; 3Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 4Div Intensive Care,Behcet Uz Child Hosp, İzmir, Turkey; 5Div Clin Chem Biochem, Univ Child Hosp, Zürich, Switzerland 1

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency affects both isoleucine catabolism and ketone body metabolism. Here we report two siblings with this disorder. The proband is a Turkish boy born to first degree consanguineous parents. His initial neuromotor development was appropriate for age. At 8 months, he was admitted to the intensive care unit (ICU) with complaints including vomiting, somnolence, and excessive

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breathing. We detected deep metabolic acidosis, hyperammonemia and hyperglycemia. In urinary organic acid analysis, tiglyl glycine,2–methyl3-OH butyric acid and some 2-methylacetoacetate were noted. In plasma, elevated acylcarnitines C5:1 and C5:OH were found. After two months in the ICU spasticity and chronic lung disease arose. Brain imaging demonstrated hyperintensities involving basal ganglion and –surprisingly- brain stem. T2 activity was found to be deficient in fibroblasts of the proband. Genetic investigation demonstrated the homozygous missense mutation c.949G>A mutation (p.Asp317Asn) in the ACAT1 gene of both the proband and his elder sister, who had not suffered from a ketoacidotic episode, but also excreted characteristic organic acids. The parents were both heterozygous. Our cases illustrate the heterogeneity of clinical presentations in T2 deficiency. In families with a case of T2 deficiency, testing for this disease is important for early onset of preventive measures. P-314 Problems of acylcarnitine analysis when carnitine is depleted: insights from the ERNDIM qualitative acylcarnitine scheme

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consultation, samples were promptly sent to Temple St. Children's University Hospital (TSCUH). Plasma amino acids showed no evidence of a UCD and urinary organic acids showed marked dicarboxylic aciduria including elevated octenedioate, decenedioate, undecanedioate and dodecanedioate but no ketones. A FAOD was suspected so acylcarnitine profile was performed on the newborn screening card (NBS) showing an increase in C10 to C18:2 and a decrease in free and acetyl-carnitine suggestive of either CACT or carnitine-palmitoyl transferase II (CPTII) deficiency. The body was transferred to TSCUH for a paediatric post-mortem. Enzyme analysis on skin fibroblasts showed normal CPTII activity. Molecular genetics for CACT SLC25A20 gene identified homozygosity for a novel mutation c.[326+1delG]. Conclusion: This case emphasises the importance of immediate collection of appropriate samples, the potential use of NBS cards and the importance of full paediatric post-mortems, including skin biopsy, in individuals suspected of IEMs to enable genetic counselling. P-316 Abnormal fatty acid oxidation in HIV+, antriretroviral treated children

Turner C1, Dalton RN2 1

ERNDIM Qualitative Acylcarnitine Scheme, London, United Kingdom; 2 WellChild Lab, King's College Lond, ECH, London, United Kingdom

Kirmse B1, Hobbs CV2, Aaron L3, Montepiedra G3, Van Dyke R4, Yu C5, Summar M1, Borkowsky W6 1

Background: The ERNDIM qualitative acylcarnitine external QA scheme circulates dried blood spots from patients with metabolic disease detectable by acylcarnitine profiling. Secondary carnitine depletion has proved problematic for many participants. Results: Sample 13a (2009) was from a hyperammonaemic 8d old patient with propionyl CoA carboxylase deficiency. 55/73 respondents suggested the disorder but 18/73 did not, with 16/73 suggesting carnitine uptake disorder (CUD). The median(range) carnitine concentration was 4.2(2.015.0)μmol/L, 0.4 fold the median lower reference interval (MLRI). C3 carnitine was 3.4(2.2-9.4)μmol/L, 1.1 fold above the median upper reference interval (MURI) & C3/C2 ratio 1.2(0.5-2.9), 6.1 fold above MURI. Samples 2c (2003) and 19b (2012) were from patients with VLCADD. 16/32 respondents suggested VLCADD for sample 2c and 20/49 for sample 19b. 3/32 suggested CUD for 2c and 28/49 for 19b. The carnitine concentration of 19b was 6.3(2.8-15.0)μmol/L, 0.5 fold the MLRI. C14:1 carnitine was 0.26(0.18-0.34)μmol/L, 1.4 fold above MURI and C14:1/C16 ratio 0.65(0.38-0.86), 3.3 fold above MURI. 2c results were similar. Discussion: Carnitine depletion represents an interpretative challenge to many participants in the ERNDIM acylcarnitine scheme: a high index of suspicion is appropriate in qualitative analysis. Quantitatively, use of appropriate ratios provides better discrimination than simple reference intervals in this situation. P-315 Post- mortem diagnosis of carnitine-acylcarnitine translocase deficiency (CACT) Urbano Blanco G1, Fitzsimons PE1, Devaney D2, Murphy AM3, Kirk R4, Olpin S4, Mayne PD1

Children's National Medical Center, Washington, DC, United States; National Institutes of Health (NIAID), Bethesda, MD, United States; 3 Harvard School of Public Health, Boston, MA, United States; 4Tulane University School of Medicine, New Orleans, LA, United States; 5Ichan School of Medicine at Mount Sinai, New York, NY, United States; 6 NYU/Langone School of Medicine, New York, NY, United States 2

Background: Nucleoside reverse transcriptase inhibitor (NRTI)-associated mitochondrial toxicity can manifest as myopathy, but the mechanism remains unclear. Dysfunctional fatty acid oxidation (FAO) has been observed in antiretroviral (ARV)-exposed children. We hypothesized that myopathy in HIV+, ARV-treated children would be associated with abnormal acylcarnitine levels. Methods: Acylcarnitine profiles (ACP) for 74 HIV-infected children on NRTI-based ARV. 37 subjects with ≥2 two CK measurements >500 IU (n=18) or evidence of echocardiographic cardiomyopathy (n=19) were matched with 37 subjects without myopathy, for age, gender, race, and CD4% category. Abnormal ACP defined as at least one clinicallysignificant acylcarnitine >99th percentile of age-matched reference range. Results: The proportion of abnormal ACPs in myopathic group was 73%(95%CI=56-86%) and 62%(95%CI=45-78%) for the non-myopathic group. No significant association was found between presence of myopathy and having an abnormal acylcarnitine profile (OR=2.10, p=0.22). A oneyear increase in NRTI use was associated with a 17% increase in odds of having an abnormal acylcarnitine profile (p=0.08). Conclusions: Abnormal FAO does not fully explain myopathy in ARVexposed children. Duration of NRTI use was, however, associated with increased odds of having an abnormal ACP. The proportion of each group with at least 1 abnormal acylcarnitine is higher than the maximum expected of 36%. P-317

Dep Clin Bio, Temple St. Child Univ Hosp, Dublin, Ireland; 2Dep Clin Path,Temple St. Child Univ Hosp, Dublin, Ireland; 3Dep of Paed, MidWest Regional Hosp, Limerick, Ireland; 4Dep Clin Chem, Sheffield Child Hosp, Sheffield, United Kingdom 1

Case Report: A 6 week old infant attended the ED of an external hospital with poor feeding. On examination, she was hypotonic and had hepatomegaly. She collapsed soon after requiring resuscitation but died of ventricular tachycardia within 12 hours. Initial investigations revealed a blood glucose of 1.1mmol/L and an ammonia of 1100μmol/L. Following

Evidences that ethylmalonic acid modulates NA+, K+-ATPase activity and expression in rat cerebral cortex Ferreira GC1, de Assis DR1, Viegas CM2, Pereira TCB3, Machado JL1, Furlanetto CB1, Bogo MR3, Streck EL1, Schuck PF1 1 Univ Extremo Sul Catarinense, Criciúma, Brazil; 2Univ Fed do Rio Grande do Sul, Porto Alegre, Brazil; 3Pont Univ Cat do Rio Grande do Sul, Porto Alegre, Brazil

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Background: Ethylmalonic acid (EMA) accumulates in tissues of patients affected by SCAD deficiency and ethylmalonic encephalopathy, illnesses characterized by neurological symptoms. Objective: We investigated the in vitro and in vivo EMA effects on Na+, K±ATPase activity and mRNA levels in 30-day-old rat cerebral cortex. Methods: For in vitro studies, cerebral cortex homogenates were incubated in the presence of EMA at 0.5, 1 or 2.5 mM concentrations for 1 h. For in vivo experiments, animals received three subcutaneous EMA injections (6 μmol/g; 90 min interval) and were killed 1 h after the last injection. After that, Na+,K±ATPase activity and its mRNA expression were evaluated. Results: We observed that EMA did not affect Na+,K±ATPase activity in vitro. In contrast, EMA administration significantly increased this activity and decreased mRNA Na+,K±ATPase expression. Conclusion: Our results indicate that EMA induces alterations on Na+, K±ATPase, and they might be involved in the pathophysiology of brain damage found in patients in which EMA accumulates.

showed a significant elevation of 3-hydroxybutyrylcarnitine/3-hydroxyisobutyrylcarnitine. Lactate was marginally raised at 2.2 mmol/L. Urinary organic acid analysis showed no evidence of a beta-oxidation defect and only a very small peak of 4-hydroxy-6-methylpyrone. Sequencing of the HMGCS2 gene subsequently identified two compound heterozygous novel variants: c.553T>C (p.Trp185Arg) in exon 2 and c.1508A>G (p.Tyr503Cys)in exon 9. Functional assessment with prediction software indicates that both are likely to be deleterious mutations. Brain imaging initially showed cerebral oedema. Unfortunately severe neurological damage was sustained. In the subsequent months, the child showed marked global developmental delay and she died 5 months after presentation. This is the first reported ultimately fatal case of HMG-CoA synthase deficiency. The apparently "ketogenic" plasma acylcarnitine profile and the small amount of 4-hydroxy-6-methylpyrone could have detracted from the correct diagnosis which was clearly indicated by the marked discrepancy between highly elevated free fatty acids and only marginally elevated serum ketones.

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Schad deficiency with lethal hepatic phenotype may only be diagnosed through enzyme analysis.

Long chain 3-hydroxy acyl-CoA dehydrogenase deficiency in latvian patients

Berry GT1, Newton SA1, Bennett MJ2

Krumina Z1, Daneberga Z1, Kreile M2, Valaine S3, Brivmane I1

1

Boston Child Hosp, Harvard Med School, Boston, United States; Childrens Hosp Phila, UPenn Med School, Philadelphia, United States

1 Med.Genet.Clinic, Univ Child Hosp, Riga, Latvia; 2Riga Stradin's Univ., Riga, Latvia; 3Eye Clinic, Univ Child Hosp, Riga, Latvia

The majority of patients with short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency manifest hyperinsulinemic hypoglycemia and elevated plasma 3-hydroxybutyrylcarnitine. However, both may be absent in patients with the ultra-rare massive hepatic necrosis phenotype. We report a 7 year old male who presented to the emergency department in shock with coma, elevated transaminases and clotting abnormalities and died shortly thereafter. He had had 2 prior episodes of acute liver damage with peak AST and ALT of 13,060 and 4,360. During the first episode at 6 months of age, he was listed for liver transplantation but recovered spontaneously. The plasma acylcarnitine profile was normal. In a FAO flux assay the oxidation of labeled myristate was reduced. The SCHAD enzyme activity in skin fibroblasts was markedly reduced at 3.6 nmol/min/mg protein (normal: 40-200). Not unlike the previous reports in the literature, the SCHAD gene sequencing did not reveal any mutations. The molecular basis may be related to a protein interaction with SCHAD, similar to the SCHAD inhibition of GDH. This case greatly exemplifies the diagnostic dilemma that is SCHAD deficiency when it presents as hepatic failure. We suggest that patients with this phenotype undergo enzyme testing in fibroblasts or leukocytes to secure the diagnosis.

Background: Long chain 3–hydroxyacyl–CoA dehydrogenase (LCHAD) deficiency is one of the defects of mitochondrial β-oxidation. Objectives: To characterize clinical, biochemical and molecular data and outcome of 7 patients with LCHAD deficiency, found by selective screening in Latvia. Results: Five patients presented during early infancy (2.5 months – 6.5 months) with vomiting, poor feeding, lethargy, hepatopathy. Four of them developed hypoglycemic coma and three had seizures. One patient presented at the age of 21 months and one was diagnosed before clinical symptoms. Two patients were born preterm. During metabolic crisis patients had typical acylcarnitine profile, increased level of 3– hydroxydicarboxylic acids in urine, elevated liver enzymes, anemia, moderate lactic acidemia and hyperammonemia. All patients had homozygous mutation 1528G>C in HADHA gene. Acute hepatopathy during pregnancy was reported in three cases. Retinopathy occurred in four patients, two patients had episodes of severe muscle pain and rhabdomyolisis during intensive physical activities or illnesses. One patient died at the age of 6.5 months before diagnosis was made, other at the age of 10 years during acute gastroenterocolitis. Conclusion: LCHAD deficiency is the most frequent fatty acid oxidation disorder found in Latvian population. Newborn screening would be important for early detection of all patients.

2

P-319 Poor outcome in 3-hydroxy 3-methylglutaryl-CoA (HMG-CoA) synthase deficiency Loughrey CM1, Cundick J1, Olpin S2, Manning N2, Cardy D1, Zschocke J3 Belfast Health and Social Care Trust, Belfast, United Kingdom; 2Sheffield Children's Hospital, Sheffield, United Kingdom; 3Medical University Innsbruck, Innsbruck, Austria 1

P-321 Evidence for an association between infant mortality and homozygosity for a carnitine palmitoyltransferase 1a genetic variant Koeller DM1, Johnson MA2, Richards CS2, Sesser D3, Wood T4, Gessner BD4 1

A 13-month-old girl presented with profound hypoglycaemia (0.1 mmol/L). She had been unrousable one morning following a significant gastrointestinal upset (confirmed norovirus). Urinalysis showed only a trace of ketones. Poor ketogenic response was confirmed with serum 3-hydroxybutyrate 0.18mmol/L despite highly elevated free fatty acids (3.6 mmol/L). In contrast, plasma acylcarnitines

Pediatrics, Oregon Health & Science Univ, Portland, United States; Mol Med Gen, Oregon Health Sci Univ, Portland, United States; 3 NorthWest Regional Newborn Screening Pgm, Hillsboro, United States; 4Alaska Division of Public Health, Anchorage, United States 2

Background: Initiation of MS/MS based newborn screening in Alaska revealed a high incidence of carnitine palmitoyltransferase

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1A (CPT1A) deficiency, a fatty acid oxidation disorder that has been associated with infant death. All affected infants were Alaska Natives, and homozygous for a c.1436C→T sequence variant (p.P479L). Prevalence of the variant is highest (allele frequency, 0.7) in regions of Alaska with high infant mortality (11.4/1,000). Methods: We performed an unmatched case-control study of the relationship between genotype and infant mortality. Cases were 110 Alaska Native infant deaths; controls were 395 Alaska Native births from the same time period. We conducted two analyses: one limited to infants from the regions of highest prevalence, and one limited to homozygous or heterozygous infants. Results: In regions of high prevalence the odds ratio (OR) of homozygosity in normal birth weight infants that died was 3.0 (95% CI: 1.4-6.3). We also found that infants that died from infectious diseases were more likely to be homozygous (OR, 2.9; 95% CI: 1.0-8.0). Discussion: Homozygosity is associated with an increased risk of infant mortality, particularly in regions where prevalence of the variant and infant mortality rates are highest. Homozygosity may also impart an increased risk of death from infectious diseases.

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(GSTZ1), a bifunctional enzyme that, as maleylacetoacetate isomerase (MAAI), catalyzes the penultimate step in tyrosine catabolism. DCA inhibits GSTZ1/MAAI, leading to delayed plasma drug clearance. Haplotype variability in GSTZ1 also influences DCA kinetics. Fourteen children with CLA due to deficiency of the PDH complex or respiratory chain received 12.5 mg/kg/12h DCA during 6 m of a randomized controlled trial, followed by up to 24 additional months in an open-label safety study. Drug kinetics was studied with 1,213C-DCA. Plasma 13C-DCA half-life and trough levels at 6 and 30 m varied 10-fold among subjects, depending on GSTZ1/MAAI haplotype, and also correlated directly with urinary maleylacetone, a substrate for MAAI. Despite such marked kinetic differences, DCA exposure did not lead to progressive accumulation of plasma drug concentrations; instead, kinetics parameters plateaued, suggesting equipoise between the inhibitory effect of DCA on GSTZ1/MAAI expression and new enzyme synthesis. No adverse drug affects were observed. Conclusion: GSTZ1/MAAI haplotype variability markedly affects DCA kinetics and biotransformation. However, these differences are self-limiting and not associated with toxicity in young children with primary mitochondrial diseases.

08. Mitochondrial disorders P-324 P-322 Autism spectrum disorders associated to a deficiency of the enzymes of the mitochondrial respiratory chain Cauli O1, Puig C2, González-Guevara L3, Guevara-Campos J3 1 Dept. Nursing-University of Valencia, Valencia, Spain; 2Psychiatry Unit, Area 4, Sagunto-Valencia, Spain; 3"Felipe Guevara Rojas" Hospital, El Tigre-Anzoátegui., Venezuela

Background: Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders characterized by a combination of reciprocal social deficits, communication impairment, and rigid ritualistic interest and stereotypies. The etiology is generally multifactorial, including genetic, immunological and/or environmental factors. A group of ASD has been linked to mitochondrial dysfunction with subsequent deficiency in energy production. In patients with ASD and mitochondrial disease often appear signs and symptoms uncommon in idiopathic ASD such as pancreatic or liver dysfunction, cardiac, growth retardation, fatigability, but it's not always the case as reported here. Methods: Cases report Results: We show two clinical cases of ASD associated to a deficiency of the mitochondrial respiratory chain (complex I+III and IV) with very different clinical presentations. In one case patient's signs and symptoms of mitochondrial disorder were mild (developmental delay with delayed bowel and bladder control) and the second diagnosis was attained many years after that of ASD. Conclusions: These findings support the recent growing body of evidence that ASD can be associated to mitochondrial disorder.

MLASA (myopathy, lactic acidosis, sideroblastic anemia): case report Tümer L1, Kasapkara CS1, Hasanoglu A1, Zeviani M2 1 Div Metab Dis, Univ Hosp of Gazi, Ankara, Turkey; 2Div Mol Neurogenetics, Inst of Neurology, Milano, Italy

Myopathy, lactic acidosis, and sideroblastic anemia (MLASA) is a mitochondrial respiratory chain disorder characterized by progressive exercise intolerance and sideroblastic anemia. MLASA is associated with a mutation in pseudouridylate synthase 1(PUS1), resulting in decreased pseudouridylation of some cytoplasmic and mitochondrial tRNAs. We report here a patient with sideroblastic anemia and mitochondrial myopathy in whom long term red blood cell transfusion therapy was reduced and improvement of muscle strength was observed under coenzyme Q10 therapy. The proband, the first child of first-cousin parents of Turkish background, was born at term after an uncomplicated pregnancy and delivery. At around 14 years of age, pallor, progressive muscle weakness and lethargy became apparent and he was found to have severe sideroblastic anemia. He also had raised blood lactate of 6,1 mmol/l, and his urine organic acid analysis showed raised lactic acid, pyruvic acid levels and dicarboxylic aciduria. A muscle biopsy at 14 years of age showed many regions of subsarcolemmal abnormal mitochondrial aggregates in the gomori trichrome staining and total diffuse negative staining for cytochrome oxidase activity. The sequencing of the PUS1 gene demonstrated homozygous mutation in exon 2 of PUS1 gene c.302 A>G.Cp.Gln101Arg. P-325

P-323 Personalized dosing of dichloroacetate (DCA) based on haplotype variations glutathione transferase zeta 1

BCS1L gene mutation causing gracile syndrome and complex III deficiency: case report Kasapkara CS1, Tümer L1, Ezgü FS1, Kücükcongar A1, Hasanoglu A1

Shroads AL1, Langaee T1, Coats BS1, Stacpoole PW1 1

Div Metab Dis, Univ Hosp of Gazi, Ankara, Turkey

1

University of Florida, Gainesville, United States

DCA, an FDA-designated Orphan Product for Congenital Lactic Acidosis (CLA), is biotransformed by glutathione transferase zeta 1

GRACILE syndrome is a rare autosomal recessive disease characterized by fetal growth retardation, Fanconi type aminoaciduria, cholestasis, iron overload, profound lactic acidosis, and early death. It is caused

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by homozygosity for a missense mutation in the BCS1L gene. The BCS1L gene encodes a chaperone responsible for assembly of respiratory chain complex III. Here we report that a homozygous mutation c.296C>T (p.P99L), in the first exon of BCS1L gene found in an affected 2 month old boy of asymptomatic consanguineous parents results in GRACILE syndrome. The exons and immediately adjacent intronic regions of the BCS1L gene are PCR amplified and then sequenced in the forward and reverse directions using automated fluorescent dideoxy sequencing methods. The homozygous c.296C>T (p.P99L) mutation has been reported in two unrelated consanguineous families with Complex III deficiency and pathogenicity is supported by functional studies. These molecular results confirmed the clinical diagnosis. This genotype is associated with a severe clinical presentation. So far no available treatments have changed the fatal course of the disease, and the metabolic disturbance responsible is still not clearly identified. Therefore, providing prenatal diagnosis in families with previous affected infants is of major importance. P-326 Application of MR imaging and MR spectroscopy in mitochondrial encephalomyopathies in children Rogac M1, Suput D2, Pecaric-Meglic N3, Meznaric M4, Sirnik A3, Neubauer D1

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disorder in which nitric oxide (NO) deficiency can play roles in the pathogenesis of several complications including stroke-like episodes, myopathy, and lactic acidosis. Arginine and citrulline act as NO precursors and their administration can restore NO production in MELAS. Lactic acidemia in MELAS results from dysfunctional mitochondria. NO deficiency in MELAS can result in decreased blood perfusion and therefore aggravates lactic acidosis. Methods: We measured plasma lactate in 10 adults with MELAS before and after 48-hour supplementation of oral L-arginine. The study was subsequently repeated before and after L-citrulline. Results: The average plasma lactate was lower after arginine (3.16→2.99 mmol/L) and citrulline (3.17→2.94 mmol/L) supplementations. This reduction was statistically significant after citrulline (p<0.05). Conclusions: The reduction in lactate after arginine and citrulline supplementations add more evidence to their potential therapeutic utility in MELAS. Previous study showed that both arginine and citrulline supplementations increase NO production in MELAS with citrulline resulting in higher increment. In this study the lactate reduction was more significant and consistent after citrulline which can be due to the superiority of citrulline in increasing NO and suggest a better therapeutic effect for citrulline. P-328

Dept. of child neurol, Univ Child Hosp, Ljubljana, Slovenia; 2Inst. for pathophysiology,Medical Facult, Ljubljana, Slovenia; 3Dept. of radiology, Univ Med Cent, Ljubljana, Slovenia; 4Inst. for anatomy,Medical Faculty, Ljubljana, Slovenia 1

Background: The aim was to describe the importance of MR and MRS imaging for diagnostic evaluation of a group of children with mitochondrial encephalomyopathies. Methods: Out of 25 children with a diagnosis of definite mitochondrial disorder (either OXPHOS deficiency or PDHc deficiency), brain MRI and MRS in general anesthesia were performed in ten children. Eight healthy children comprised the control group for evaluation of metabolic changes. Results: Five children (20 %) had signs of Leigh syndrome. Morphological changes that might be specific for mitochondrial diseases were found in other children: 4 (16 %) children with delayed myelination, 6 (24 %) children with cerebral atrophy, 2 (8 %) children with cerebellum changes and in 5 (20 %) neuronal migration disorders were found. No statistically significant difference at relaxation time TE 35 ms was found in N-acetyl aspartate, myoinositol and cholin ratios between basal ganglia and white matter in children with mitochondrial diseases comparing to controls. Lactate peak was found in one child with Leigh syndrome. Conclusions: Morphological changes which can be in accordance with mitochondrial disorders in children were found in 71% of children. Brain MRS is not very sensitive method for evaluation of brain metabolic changes in unspecified mitochondrial encephalomyopathies. P-327 The effect of citrulline and arginine supplementation on lactic acidemia in MELAS syndrome El-Hattab AW1, Emrick LT2, Williamson KC2, Craigen WJ2, Scaglia F2 1 King Fahad Medical City, Riyadh, Saudi Arabia; 2Baylor College of Medicine, Houston, Texas, United States

Background: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a common mitochondrial

Incidence of mitochondrial disease in children presenting with acute liver failure under 2 years of age McKiernan PJ1, Ball S2, Santra S2, Pietrobattista A1, Poulton J3, Hickman K4, McFarland R4, Rahman S5, Gupte G1, Sharif K1, Fratter C3, Taylor RW4 Liver Unit, Children's Hospital, Birmingham, United Kingdom; 2IMD Department, Children's Hospital,, Birmingham, United Kingdom; 3 NSCT Rare Mitochondrial Disease Service, Oxford, United Kingdom; 4 NSCT Rare Mitochondrial Disease Service, Newcastle, United Kingdom; 5NSCT Rare Mitochondrial Disease Service, London, United Kingdom 1

Acute liver failure (ALF) is often fatal without liver transplantation (LT). Systemic mitochondrial disease (MD), where LT is contraindicated, should be excluded. Aim: To determine the incidence of MD in children with ALF under two years. Methods: 40 consecutive infants with ALF from 2009-11. Liver mitochondrial DNA (mtDNA) copy number were assessed & mtDNA maintenance genes sequenced. Results: 6 had proven MD. Five had pathogenic mutations detected: DGUOK (n=2), POLG (n=2) and MPV17 (1). Four of these died while one recovered. Another had equivocal mtDNA depletion in liver and muscle but no detected mutations. She underwent LT but died later from systemic disease. An additional 7 children had mtDNA depletion in liver tissue only. 4 underwent LT, 2 recovered and 1 died. None of these have developed systemic disease at up to 4 years later. Summary: MD is an important cause of ALF in children less than two. Where the genetic defect causing mtDNA depletion can be characterised, inappropriate LT can be avoided. Liver mtDNA depletion without a recognised genetic defect may be a secondary phenomenon. Conclusion: Screening mtDNA maintenance gene mutations may be the most efficient way to exclude MD in ALF in the first two years of life. Conflict of Interest declared.

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P-331

Hypoxia in HUVEC: alteration of sirtuins as regulatory elements

Mitochondrial dysfunction in immature rat model of epilepsy

Warneke S1, Potthast A1, von Versen-Höynck F2, Das AM1

Jesina P1, Folbergerova J2, Houstek J2

1

Metab Dis, Hann. Med School, Dept.. Paed, Hannover, Germany; Mol Perinatol, Hann. Medical School, Hannover, Germany

Inst Inher Metab Dis, Charles Univ, Prague, Czech Republic; 2Inst Physiol, Acad of Sciences, Prague, Czech Republic

Introduction: Hypoxia may occur during vascular compromise, perinatal complications and surgery. The first tissue exposed to hypoxic conditions is the endothelium. Hypoxia leads to an intracellular shift from NAD+ to NADH. As NAD+ is an essential cofactor for sirtuins activity of these regulatory elements may be compromised during hypoxia. Methods: Cultured HUVEC from uncomplicated term pregnancies were exposed to normoxia (21% o2) and hypoxia (2% O2) for 10, 60, 120, 180 minutes, and reoxygenated. Sirtuins 1 (cytosol) and 3 (mitochondrial) were analyzed at an enzymatic (fluorometry) level, sirtuins 1, 3 and 4 at protein and expression level, intracellular NAD+ levels were measured spectrophotometrically. Results: Activities of sirtuins 1 and 3 decreased in response to hypoxia and returned to normal in response to reoxygenation. At the protein level, sirtuin 1 did not change while sirtuins 3 and 4 increased. At the transcription level, hypoxia led to reversible decrease in sirtuin 1 only. NAD ±levels increased under hypoxia and reoxygenation. Conclusion: Decreased sirtuin activity is probably a protective mechanism as sirtuin 3 inhibits the mitochondrial respiratory chain thereby limiting production of free radicals and inactivating ATPsynthase thus conserving ATP. Increased NAD±levels are probably a result of sirtuin inhibition and support NADH-production after reoxygenation.

We demonstrated the marked decrease of respiratory chain complex I activity in the cerebral cortex of immature rats following seizures induced by DL-homocysteic acid. The inhibition persists during the 5 weeks periods of survival, corresponding to development of epileptogenesis in this model. The complex I activity decrease was not associated with changes in complex I content. The complex I inhibition was accompanied by significant increase of three independent mitochondrial markers of oxidative damage – 3-nitrotyrosine, 4hydroxynonenal and protein carbonyls. The decrease of complex I activity was substantially reduced by treatment with selected free radicals scavengers. In animals with seizures, the activity of superoxide dismutase (total SOD; SOD1 and SOD2) was significantly increased. Upregulation of SOD2 was also confirmed in mitochondria at the protein level by immunoblotting. The activities of other antioxidant enzymes including catalase and glutathione peroxidase did not differ. The pronounced and selective upregulation of SOD2 points to enhanced ROS levels in the mitochondrial matrix. The present findings suggest that oxidative stress occurring in the brain of immature rats during and following the seizures is apparently due to both the increased free radical production and the limited antioxidant defense. Supported by Ministry of Health, Czech Republic RVO-VFN64165.

2

P-330 A novel compound heterozygous mutation in NFU1 as cause of iron-sulfur cluster biosynthesis defects Van Coster R1, Smet J1, De Paepe B1, Vanlander A1, De Latter E1, De Meirleir L2, Lissens W2, Seneca S2 Div Ped Neur and Metab, Univ Child Hosp, Ghent, Belgium; 2Cent Med Gen, Univ Hosp, Brussels, Belgium 1

Background: Recently, mutations have been detected in NFU1 and BOLA3, two genes involved in iron-sulfur biogenesis. In affected patients, defects in the OXPHOS complexes I and II, in 2-oxoacid dehydrogenase and in lipoate synthesis were found. The objective was to screen our database of patients to find patients with similar clinical and biochemical findings and to sequence the two genes in suspected patients. Methods: Patients with encephalopathy, failure to thrive, hyperlactacidemia, increased glycine in cerebrospinal fluid and decreased activities of the complexes I and II were selected. BN-PAGE, western blotting and Sanger sequencing was performed. Results: A decreased protein amount in the complexes I and II was visualized using BN-PAGE. A decrease of structural subunits in complexes I and II and low signals of protein-bound lipoic acid were detected by western blotting. Mutations were detected in NFU1 in both patients. One patient carried the 'hotspot' mutation p.Gly208Cys and the other was a compound heterozygote for this mutation and for p.Arg21Pro. Conclusion: Only three papers were reported earlier in the literature describing patients with NFU1 deficiency. Our results are concordant with the assumed role of NFU1 in ISC biosynthesis and a novel compound heterozygous mutation is presented here.

1

P-332 Transcriptomic profiling of TK2 deficient human skeletal muscle reveals activation of the p53 signalling pathway and identifies GDF-15 as a potential novel biomarker for mitochondrial myopathies. Kalko SG1, Paco S2, Jou C3, Meznaric M4, Sciacco M5, De Paepe B6, Ferrer I7, Munell F8, Roig-Quilis M8, Montoya J9, López-Gallardo E9, Artuch R10, Montero R10, Torner F11, Nascimento A2, C Ortez2, Colomer J2, Jimenez-Mallebrera C2 Bioinformatics Core Facility. IDIBAPS., Barcelona, Spain; 2Neuromuscular Unit. HSJD, Barcelona, Spain; 3Pathology Dept. HSJD, Barcelona, Spain; 4 Inst. of Anatomy. University of Ljubljan, Ljubljana, Slovenia; 5Diagnostica Malattie Neuromuscolari, Milan, Italy; 6Neuropathology. Ghent University Hosp., Ghent, Belgium; 7Inst. Neuropathology. IDIBELL, Barcelona, Spain; 8 Neuropaediatrics Dept. Vall d'Hebron, Barcelona, Spain; 9University of Zaragoza, Zaragoza, Spain; 10Clinical Biochemistry. HSJD, Barcelona, Spain; 11Traumatology Dept. HSJD, Barcelona, Spain 1

Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome. In order to unveil some of the mechanisms involved in this pathology, to identify compensatory pathways and potential therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using microarrays. We have compared the whole transcriptome of skeletal muscle from patients with TK2 mutations and compare it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed

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changes in gene expression. Our data point towards p53 as the key factor at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, regeneration, apoptosis and oxidative stress. In addition, we have identified growth and differentiation factor 15 as a potential novel biomarker for mitochondrial dysfunction.

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only in 9%, and the majority of this group developed a multiorgan phenotype with a high frequency (44%) of heart involvement. P-335 Mitochondrial neurogastrointestinal encephalopathy: clinical, biochemical and molecular genetic study in three egyptian patients

P-333 Mitochondrial respiratory chain defects: alterations of sirtuins

Selim LAS1, Vancoster R2, Hassan S A1, Vanlanderb A2, Smet J2, De Latterb E2, Vandemeulebroeckeb K2, Mehaney D3, Abdo D3, Nakhla GA4, Mostafa M1, Habetse D5, Bakkere J5, Abdel Barry A6

Potthast A1, Warneke S1, Fitter A1, Skokowa J2, Das AM1 Univ Child Hosp, Div Neurometab Dis, Cairo, Egypt; 2Div ped neuro & metabol, Ghent Univ, Ghent, Belgium; 3Clin & Chem Path Dep, Cairo Univ Hosp, Cairo, Egypt; 4Pathology dep, Cairo Univ Hosp, cairo, Egypt; 5Dep clin Genetics, Maastricht Univ, Maastricht, Netherlands; 6 surgery Depart, Cairo Univ Hosp, Cairo, Egypt 1

1

Metab Dis, Hann. Med School, Dept.. Paed, Hannover, Germany; Molec.Hematopoiesis, Hannover, Germany

2

Introduction: Sirtuins are a group of proteins previously described as histone deacetylases (HDAC) class III. In humans, seven different members with specific subcellular localisations (nuclear: Sirt1, Sirt6, Sirt7; mitochondrial: Sirt3, Sirt4, Sirt5; cytoplasmic: Sirt2) are known. All Sirtuins require NAD+ as essential co-factor. Target proteins of the sirtuin family belong to signalling pathways, transcription factors, energy metabolism and ROS detoxification. Sirtuin pathways and the mitochondrial respiratory chain are linked by the NAD+/NADH-system. Methods: We studied Sirt1, Sirt3 and Sirt4 in cultured skin fibroblasts from patients with respiratory chain defects (complex IV and complex V) compared to cells from healthy subjects. Analysis was performed at transcriptional (qRT-PCR), protein (Western Blot), enzymatic (fluorimetric) and metabolite level (NAD±concentration). Results: We found decreased NAD+ levels in complex IV and complex V deficient cells. Enzyme activity of Sirt1 and Sirt3 in patient cells with complex IV defects as well as protein levels of sirtuins and transcription were reduced. These parameters remained unchanged in complex V deficiency. Conclusion: We found decreased sirtuin levels in fibroblasts with complex IV defect at enzymatic, protein and transcriptional level. In complex V deficiency Sirt 1 and Sirt 3 activities were reduced while these changes were not reflected at protein level. P-334 Mitochondrial diseases with neonatal onset: a study of 240 patients Ardissone A1, Lamantea E1, Genitrini S1, Invernizzi F1, Zeviani M1, Uziel G1

Background: Mitochondrial Neurogastrointestinal Encephalopathy (MNGIE) disease is characterized by gastrointestinal dysmotility, cachexia and episodic pseudoobstruction, ophthalmoplegia, hearing loss, and demyelinating peripheral neuropathy. The disease is caused by mutations in the thymidine phosphorylase (TP) gene. Subjects & methods: The patients were subjected to a thorough neurologic examination, Brain MRI, Muscle biopsy (1/3) and mitochondrial respiratory chain complexes were analysed in muscle homogenate, molecular testing for TP gene by direct sequencing (3/3) and TP enzyme activity performed in 2/3 patients. Results: All Patients presented symptoms of severe GIT dysmotility with progressive cachexia, neuropathy, sensory neural hearing loss, asymptomatic leukoencephalopathy (3/3) . Skeletal muscle biopsy revealed deficient Cytochrome C oxidase & Mitochondrial respiratory chain enzyme assay isolated complex I deficiency. TP enzyme activity revealed complete absence of enzyme activity in both index patients. Direct sequencing of TP gene revealed homozygous mutations c.3371A>C and c.4183G>A in both index patients. Molecular analysis of both families revealed compound heterozygous mutations in both parents and 4 siblings. Conclusion: MNGIE is a rare mitochondrial disorder with an important diagnostic delay. In case of pathogenic mutations in TP gene in the family, carrier testing and prenatal diagnosis of at risk members is recommended for early detection. P-336 Biochemical data are important clues for diagnosis of SUCLA2 defects

1

Fondazione IRCCS Besta, Milan, Italy

Background: Neonatal onset in Mitochondrial Disease (MD) is rather frequent, but early diagnosis is difficult. In this study we analyzed a large population with MD and neonatal onset to ascertain the presence and the quality of symptoms in prenatal and early postnatal life in order to address the biochemical and molecular diagnosis. Patients and methods: Among of a series of 557 patients with MD diagnosed according to defined biochemical or molecular criteria 43% presented symptoms before one year of age and were selected for a retrospective study. Results and conclusions: We identify five different clinical/ radiological phenotypes: Leigh disease (33%), Leukoencephalopathy (8%), Encephalopathy with or without involvement of other organs (43%), Isolated myopathy (2%) and multiorgan involvement sparing CNS (14%). Molecular diagnosis was achieved in 58% of cases. When available lactate was usually high and useful to address the diagnosis, but 12% had normal values either in blood and CSF. The presence of cardiomyopathy or hepatopathy with or without involvement of SNC was associated with the worst prognosis. Prenatal onset was documented

Nogueira C1, Garcia P2, Diogo L2, Valongo C1, Santorelli FM3, Vilarinho L1 R&D Unit, National Institute of Health, Porto, Portugal; 2Her Met Dis Unit, Children Hospital, HUC, Coimbra, Portugal; 3Mol Med & Neurogenetics, IRCCS Fondazion, Pisa, Italy 1

Background: Succinyl-CoA syntase (SCS) is a mitochondrial matrix enzyme that catalyzes the reversible synthesis of succinate and ATP from succinyl-CoA and ADP in the tricarboxylic acid cycle. This enzyme is made up of two subunits, α, and β, encoded by SUCLG1 and SUCLA2, respectively. Deficiencies in SCS subunits have been described in patients with encephalomyopathy, mitochondrial DNA depletion and mild methylmalonic aciduria. We report a two-year-old boy, born after a normal pregnancy and delivery, the first son of unrelated parents. He presented with feeding difficulties since birth. At 3 months of age, growth retardation, muscle atrophy, severe hypotonia, dystonia and mild hearing impairment were noticed. Brain MRI demonstrated mild cerebral atrophy. Psychomotor

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development evaluation (R.Griffiths Scale) revealed a mental age of 6 months at 2 years. The metabolic investigation disclosed hyperlactacidemia together with moderate excretion of methylmalonic acid and elevated C4dicarboxylic carnitine. These biochemical findings were important clues for diagnosis of SUCLA2 defect. Genetic study revealed a novel homozygous missense mutation (p.M329V) predicted to be pathogenic by bioinformatic tools. Our study is important for accurate genetic counseling and prenatal diagnosis to the affected family and contributes to expand the spectrum of patients with SUCLA2 mutations. P-337 Thiamine pyrophosphate defiency secondary to TPK-1 mutation presenting as leighs disease: diagnosis and management within a sibling pair Fraser JL1, Yang S2, Vanderver A3, Chang T3, Cramp L3, Smpokou P2, Chapman KA2, Zand D2 1

Nat Inst Health, Nat Hum Genome Inst, Bethesda, United States; 2 CNMC, Genetics and Metabolism, Washington, United States; 3 CNMC, Division of Neurology, Washington, United States Background: Thiamine pyrophosphate (TPP) is essential for pyruvate dehydrogenase complex, alpha-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase function. Five patients have been reported with thiamine pyrophosphokinase-1 (TPK1) deficiency, with encephalopathy, extrapyramidal, bulbar, and cerebellar signs, and metabolic decompensation leading to death. Some patients received high dose thiamine therapy. Case reports: We present a sibling pair with Leigh's disease, progressive hypotonia, regression, and chronic encephalopathy. Whole exome sequencing in the younger sibling demonstrated homozygous TPK1 mutations. The older sibling had died from progressive neurologic disease with metabolic strokes, and the younger sibling had progressive neurologic decline. MRI demonstrated putamen and thalamic abnormalities in the younger, with similar and progressive evolution in the older sibling. The patient was started on high dose thiamine, niacin, biotin, and alpha-lipoic acid; ketogenic diet was initiated to reduce metabolic burden. He subsequently demonstrated improvement in neurologic function, with re-attainment of lost milestones. Conclusions: TPK1 deficiency should be considered in children with Leigh's disease without identifiable mutations, and this gene should be evaluated as a candidate for testing in commercially available sequencing panels. Reducing metabolic burden in these children by addition of supplements and transition to ketogenic diet may reverse some of the neurologic progression and improve outcome. P-338

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four children and progressive axonal neuropathy, deafness and cognitive impairment were present in 7 patients (Cowchock syndrome; CMTX4). Here we describe another patient with neonatal onset of the disease with hypotonia, myoclonic epilepsy, hearing loss, visual impairment, severe encephalopathy, Leigh syndrome and lactic acidosis. In addition, the boy had hyporeflexia compatible with axonal neuropathy and died at the age of 18 months. Muscle biopsy demonstrated mild myopathic pattern with accumulation of SDH product. In tissues obtained at autopsy, the activity and amount of complex I and IV was decreased in muscle, brain and heart and the activity of complex II was upregulated. Targeted sequencing of mitochondrial exome revealed novel hemizygous mutation c.1391T>G (p.Leu464Trp) in F1 gene on chromosome X. Conclusions: X-linked F1 encephalomyopathy due to ApoptosisInducing Factor defect should be considered in the differential diagnostics in patients with Leigh syndrome, axonal neuropathy and myoclonic epilepsy. Supported by IGA NT 13114/4 and IGA NT 14156/3. P-339 Molecular screening and diagnosis of mitochondrial diseases: a two year study. Mehaney D1, Selim L2, Shaarawy M3 1 Chem Path dep,Faculty Med,Univ Cairo, Cairo, Egypt; 2Neurology dep,Cairo Uni Children's Hosp, Cairo, Egypt; 3Ophthalmol Dep, Faculty Med, Univ Cairo, Cairo, Egypt

Background: Mitochondrial diseases (MCDs) are an important cause of morbidity and mortality in pediatric patients. Objective: This report describes the screening and diagnostic molecular service for MCDs started at Cairo University Children's hospital (January 2011 - January 2013). Patients and Methods: DNA was extracted from whole blood samples of 62 Pediatric patients. Eight patients were clinically and radiologically suspected to have Myoclonus epilepsy and ragged-red fibers , 17 with mitochondrial encephalomyopathy , lactic acidosis and stroke-like episodes (MELAS), 31 with and Leigh syndrome/Neuropathy Ataxia Retinitis Pigmentosa syndromes (LS/NARP), 3 with Leber's hereditary optic neuropathy (LHON) and 2 with Mitochondrial Neurogastrointestinal Encephalopathy (MNGIE). Common mt DNA point mutations were screened using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism analysis. Sequencing analysis of the nuclear Thymidine Phosphorylase (TP) gene was done in MNGIE patients. Results: The A3243G and T14484C mutations were detected in one MELAS and in two LHON patients respectively. The c.3371A>C and c.4183G>An in TP gene were detected in MNGIE patients. Conclusion: As Egypt is a country with high rate of consanguineous marriage, the molecular pathogenesis of MCDs is suspected to be of nuclear genetic origin. Mitoexome and whole exome sequencing represents an appealing approach for detection of disease causing variants.

Leigh syndrome and myoclonic epilepsy caused by novel mutation in AIMF1 gene

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Honzik T1, Vondrackova A1, Stranecky V2, Rodinova M1, Kratochvilova H1, Hansikova H1, Magner M1, Mazurova S1, Zeman J1, Tesarova M1

Complex III deficiency in a Portuguese family: expanding the clinical phenotype

1 2

Dep Pediatrics, Charles Univ, Univ Hosp, Prague, Czech Republic; Inst Met Dis, Charles Univ, Univ Hosp, Prague, Czech Republic

Nogueira C1, Nesti C2, Meschini MC2, Barros J3, Sá MJ4, Azevedo L5, Carrozzo R6, Santorelli FM2, Vilarinho L1

Objective: Mutations in X-linked F1 gene, encoding the ApoptosisInducing Factor Mitochondrion-associated 1, cause disruption of the respiratory chain complexes, decrease stability of mtDNA and increase apoptogenic stimuli. Only 11 patients from 3 families were reported, so far. Severe encephalomyopathy and Leigh syndrome was present in

1 Genet Dep, National Institute of Health, Oporto, Portugal; 2Mol Med &Neurog, IRCCS Stella Maris, Pisa, Italy; 3Neurology Unit, Porto Hospital Center, Oporto, Portugal; 4Neurology Unit, Hospital S. João, Oporto, Portugal; 5Pop Genetics, IPATIMUP, Oporto, Portugal; 6Mol Med, IRCCS Bambino di Gesù, Rome, Italy

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Defects of mitochondrial complex III (CIII) are a relatively rare cause of mitochondrial dysfunction. Clinical phenotypes include mitochondrial encephalomyopathy, histiocytoid cardiomyopathy, neonatal onset of metabolic decompensation, and, more commonly, exercise intolerance with myoglobinuria. Recently, mutations in TTC19, a gene involved in CIII assembly, have been described in association with a severe neurodegenerative disease. We studied a consanguineous Portuguese family where a severe neurometabolic disorder occurred in four siblings in association with a slowly progressive disorder characterized by dystonia of hands and feet, ataxic gait, severe olivo-ponto-cerebellar atrophy, and relentless psychiatric manifestations. Variability in age at onset and disease course was observed. We identified a novel homozygous TTC19 mutation predicting frameshift and early protein truncation. The protein was undetectable in tissues by Western blot analyses. This is the fourth kindred presenting mutations in TTC19. The clinical phenotype of this family will contribute to a deeper understanding of the CIII-related disorders.

muscle abnormalities appear to improve both clinically and biochemically following CoQ10 supplementation, neurological symptoms are only partially ameliorated. At present, the reasons for the refractory nature of the neurological dysfunction remain unknown. In order to investigate this at the biochemical level we utilised a para-aminobenzoate (PABA) induced human SHSY5Y neuronal cell model of CoQ10 deficiency. 1mM PABA treatment resulted in a 54% decrease in cellular CoQ10 status which was accompanied by a global loss of mitochondrial electron transport chain (ETC) complex activities and a 4 fold increase in mitochondrial oxidative stress. CoQ10 treatment (2.5 μM) was effective in reducing oxidative stress to below control levels. However, CoQ10 supplementation (10 μM) was only partially effective at restoring ETC activity. Complex II/III activity was significantly (p < 0.05) increased to 82.5% of control levels. Complex I and IV activities were restored to 71.1% and 77.7%, respectively of control levels. In conclusion, the results of this study have indicated that a plasma CoQ10 status of > 10μM may be required to fully restore neuronal ETC activity.

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Gastroenterological symptoms of mitochondrial respiratory chain complex deficiency

Diagnostics of mitochondrial disorders by targeted sequencing of mitochondrial exome

Kawachi E1, Murayama K1, Fushimi T1, Ichimoto K1, Fujinami A1, Tsuruoka T1, Ajima M1, Harashima H2, Okazaki Y3, Takayanagi M1, Ohtake A2

Hansikova H1, Vondrackova A1, Stranecky V2, Kratochvilova H1, Rodinova M1, Langer J1, Honzik T1, Zeman J1, Tesarova M1

1 Dep of Metab, Chiba Child Hosp, Chiba, Japan; 2Dep of Pediatrics, Saitama Medical Univ, Saitama, Japan; 3Div of translational Res, Genomic med, Saitama, Japan

2

Background: Mitochondrial respiratory chain complex deficiency (MRCD) can affect any, in fact all, bodily systems including the gastrointestinal tract. Objectives: of this study is to reveal the manifestation of gastroenterological symptom in Japanese MRCD cases. Methods: A total 189 patients diagnosed as having MRCD in our hospital were enrolled in this study. Among them, 56 had some gastroenterological symptoms. Results: Of these 56 patients, 25 presented with a main problem of gastroenterological symptoms such as intractable diarrhea, bilious vomiting, gastroesophageal reflux and necrotizing enterocolitis. Mitochondrial gastrointestinal dysfunction occurred within the first year of life and more than half of the patients had a neonatal onset. Diagnosis was based mainly on enzyme assays performed using hepatic and muscle tissues. The other 31 patients had gastroenterological symptoms associated with severe respiratory distress, heart failure, seizures and lactic acidosis. Conclusions: In summary, based on these results, continuous poor sucking and gastroenterological symptoms in infancy are key features suggesting MRCD, necessitating evaluation of damage to other organs and the existence of lactic acidosis.

1

Dep Pediatr, Charles Univ, Gen Univ Hosp, Prague, Czech Republic; Inst Inher Metab Dis, Charles Univ, Prague, Czech Republic

Next generation sequencing (NGS) represents a significant milestone in the elucidation of the causes of inherited diseases. A major benefit is especially for mitochondrial diseases (MD) that are genetically very heterogeneous. More than 130 nuclear genes were described so far whose mutations lead to the MD. More than 25 genes were identified just by NGS in the last two years. Accurate targeting of the genetic analysis based on clinical symptoms or laboratory tests is possible for only a few types of MD (MNGIE syndrome, mitochondrial encephalopathy cardiomyopathy - TMEM70, Leigh syndrome due to SURF1 mutations). For most of MD, even specialized enzymatic and protein analysis does not allow unambiguously to narrow a group of candidate genes. Mitochondrial exome (> 1100 genes) was sequenced and analyzed in 26 patients with mitochondrial disease. Mutations in known MD-genes TSFM, COX10, AIFM1, TK2 and MGME1 were found in 6 patients. In three patients no gene was prioritized. In the other patients several candidate genes with previously unknown association with MD were selected and are further evaluated. Supported by PRVOUK P24/1.LF/3, IGA NT/11186 , IGA NT/13114, IGA NT/14156

P-344 P-342 A novel homozygous TMEM 70 mutation with additional features Effect of coenzyme Q10 supplementation on mitochondrial electron transport chain activity and mitochondrial oxidative stress in coenzyme Q10 deficient human neuronal cells.

Eminoglu FT1, Mayr JA2, Erdeve O3, Atasay B3, Arsan S3 Dept Pediatr Metab Dis, Ankara Univ Hosp, Ankara, Turkey; 2Dept of Pediatrics, Paracelsus Med Univ, Salzburg, Austria; 3Dept of Neonatol, Ankara Univ Hosp, Ankara, Turkey 1

1

2

3

3

4

Duberley K , Heales SJR , Chalasani A , Land JM , Rahman S , Hargreaves IP3 1

Dept Molec Neurosci, Institute of Neurol, London, United Kingdom; Dept Clin Path, Great Ormond St. Hosp, London, United Kingdom; 3 Neurometabolic Unit, National Hosp, London, United Kingdom; 4 Metabolic Unit, Great Ormond St., Hosp., London, United Kingdom 2

Primary Coenzyme Q10 (CoQ10) deficiency is an autosomal recessive disorder with an extremely heterogeneous clinical presentation. Both muscle and neurological dysfunction are a common presenting feature. Whilst

The TMEM70 gene defect was recently identified as a novel cause of autosomal recessive ATP synthase deficiency. The most of the 28 patients with TMEM70 disorder reported to date display a distinctive phenotype characterised by severe muscular hypotonia, hypertrophic cardiomyopathy, facial dysmorphism, profound lactic acidosis, and 3methylglutaconic aciduria. We report here a 7 month-old boy with ATP synthase deficiency who presents, besides the typical features, some additional features not described until now. This patient was the second

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child after third pregnancy of a healthy consanguineous couple of Turkish descent. His mother had suffered from HELLP syndrome in second pregnancy and in utero exitus happened during the 30th week of this pregnancy. This baby delivered prematurely and presented on day of life 1 with symptoms resembling Reye-like syndrome. He had dysmorphic features, hypospadia, hypertrophic cardiomyopathy, hypothyroidism, optic atrophy, muscular hypotonia, metabolic deterioration episodes with hyperlactatemia and mild hyperammonemia. His organic acids analysis showed 3-MGC-üria, increased excretion of lactat. We identified a new homozygous single base-pair deletion in the TMEM70 gene (c.359delC p.Thr120Asnfs*34). The hypothyroidism and optic atrophy has not been reported until now. The current report corroborates the previously described typical features of TMEM 70 deficiency. P-345 Suspected mitochondrial myopathies in the era of genomic medicine Atherton AM1, Smith LD1, Heese BA1, Soden SE1, Saunders CJ1, Farrow EG1, Willig L1, Miller NA1, Kingsmore SF1

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of life with electrolyte imbalances and hyperglycemia. His clinical course complicated by proteinuria and hypotonia. His genetic analysis revealed homozygosity for the same mutation. The amount of coenzyme Q 10 was clearly reduced to 35.1 nmol/g protein (control range: 111.2 - 517.9) in the muscle biopsy. At the age 2 months, the CoQ10 treatment was started. After the initiation of CoQ10, proteinüri was improved and insulin requirement was decreased. However, he developed nystagmus, and new-onset status epilepticus on treatment. This report showed futher and different phenotypical findings within a single family. Long-term follow-up is needed to define the ultimate prognosis of these patients. P-347 Two Turkish siblings with Megdel syndrome due to SERAC1 mutation Dursun A1, Ünal Ö1, Tokatlı A1, Özgül RK1, Yücel D1, Coskun T1, Hişmi B1, Yalnızoğlu D2, Kalkanoğlu-Sivri HS1 1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey; Hacettepe Univ Child Hosp, Div Neurology, Ankara, Turkey

2 1

Children's Mercy Hospitals and Clinics, Kansas City, United States

Mitochondrial myopathies (MM), a heterogeneous group of inherited disorders with multisystem involvement, can be caused by mutations in the nuclear or mitochondrial genome. Historically, individuals with a suspected MM were diagnosed based on blood and/or cerebral spinal fluid biochemical testing, neuroimaging or invasive muscle biopsy results. Nonspecific results often lead to the diagnosis of a "suspected mitochondrial myopathy". Extensive, generally expensive, molecular testing panels for genes causing MM are available but may also be uninformative. We explored the utility of exome sequencing for molecular diagnosis by analyzing whole exome sequences of 19 patients from 15 families with clinical symptoms suggestive of a mitochondrial disorder without a definitive diagnosis. Five of the patients (26%) were confirmed to have a primary MM, five patients (26%) were identified as having an unrelated disorder with secondary mitochondrial dysfunction, one patient (5%) has likely pathogenic variants in novel MM candidate genes and eight patients (42%) had negative testing results. Overall, 58% of patients received a molecular diagnosis utilizing whole exome sequencing. These results support the initial use of whole exome sequencing in patients suspected of having a MM as this testing modality is more practical, efficient, specific and cost effective.

Association of 3-methylglutaconic aciduria with impaired oxidative phosphorylation, deafness, encephalopathy, leigh-like lesions on brain imaging, progressive spasticity and dystonia defined as a distinct entity under the name of Megdel syndrome. It is an autosomal recessive disorder and, mutation in the serine active site-containing protein 1 (SERAC1) cause this syndrome. SERAC1 is localized at the interface between the mitochondria and the endoplasmic reticulum in the mitochondria-associated membrane fraction that is essential for phospholipid exchange. It was identified as a key player in the phosphatidylglycerol remodeling that is essential for both mitochondrial function and intracellular cholesterol trafficking. Here we report two new Turkish sibling patients affected with Megdel syndrome due to SERAC1 gene mutation. The patients were presented with 3-methylglutaconic acid and 3-methylglutaric aciduria, microcephaly, growth retardation, dysmorphic features, severe sensorineural deafness, progressive spasticity, dystonia, seizures, basal ganglia involvement and degeneration consistent with leigh-like syndrome. Metabolic acidosis, mild hyperammonemia and lactic acidemia were accompanying clinical findings in the newborn period. The patients were diagnosed with Megdel syndrome after molecular genetic analysis of SERAC1 gene The study supported by The Scientific and Technological Research Council of Turkey (TUBITAK)(Project No: 111S217).

P-346 P-348 A novel mutation in CoQ2 leading to phenotypic variability in a family Eminoglu FT1, Deda G2, Mayr JA3, Berberoglu M4, Ozcakar B5, Ozaltın F6 1 Dept Pediatr Metab Dis, Ankara Univ Hosp, Ankara, Turkey; 2Dept of Pediatr Neurol, Ankara Univ Hosp, Ankata, Turkey; 3Dept of Pediatrics, Paracelsus Med Univ, Salzburg, Austria; 4Dept Pediatr Endocrinol, Ankara Univ Hosp, Ankara, Turkey; 5Dept Pediatr Nephrol,Ankara Univ Hosp, Ankara, Turkey; 6Dept Pediatr Nephrol, Hacettepe Univ, Ankara, Turkey

Primary coenzyme Q10 (CoQ10) deficiency includes a group of rare autosomal recessive disorders primarily characterized by neurological and muscular symptoms. Until now, six patients have been described due to mutations in COQ2 gene. Here we report a novel homozygous mutation in two siblings from consanguineous Turkish parents.The old patient presented with steroid-resistant nephrotic syndrome at the age of 4 months without extrarenal symptoms. He died due to a renal failure 2 month after admission. Genetic analysis revealed a novel homozygous mutation in COQ2 (c.437 G>A, p.S146 N) gene. His brother presented at five days

A distinct mitochondrial myopathy, lactic acidosis and sideroblastic anaemia (MLASA) phenotype associates with YARS2 mutations Shahni R1, Wedatilake Y1, Cleary MA2, Lindley KJ2, Sibson KR2, Rahman S1 1 UCL Institute of Child Health, London, United Kingdom; 2Great Ormond Street Hospital, London, United Kingdom

Background: Nuclear-encoded defects of mitochondrial protein synthesis are increasingly recognised to cause complex childhood-onset multisystem disorders. Mutations of aminoacyl-tRNA synthetases such as YARS2 lead to failure of aminoacylation of mitochondrial tRNAs and consequently impair mitochondrial translation. Objective: To determine the cause of myopathy, lactic acidosis and sideroblastic anaemia (MLASA) in a 12-year-old male. Methods: The patient presented at 1 year with anaemia initially attributed to iron deficiency. Bone marrow aspirate at 5 years revealed ringed sideroblasts but transfusion dependency did not occur until 11 years. Other features included lactic acidosis, poor weight gain, hypertrophic cardiomyopathy and severe myopathy leading to respiratory failure

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necessitating ventilatory support. Long-range PCR of mitochondrial DNA excluded large-scale rearrangements. The YARS2 gene was sequenced in the patient and parents. Results: A homozygous known pathogenic YARS2 mutation, c.156C>G;p.F52L, affecting a highly conserved amino acid was identified. Comparison of our patient with previous cases revealed remarkable clinical homogeneity; all presented a highly specific form of MLASA. Conclusion: The combination of sideroblastic anaemia, myopathy and lactic acidosis is strongly suspicious of mitochondrial dysfunction. First line investigation of MLASA should include sequencing YARS2 and PUS1 (encoding a tRNA modification factor) which may preclude the need for muscle biopsy. P-349 Whole exome sequencing reveals novel mutations in mitochondrial elongation factor G1 causing Leigh syndrome with multiple respiratory chain enzyme deficiencies Shahni R1, Emmett W2, Saldanha JW3, Rahman S1 UCL Institute of Child Health, London, United Kingdom; 2UCL Genetics Institute, London, United Kingdom; 3National Institute for Medical Research, London, United Kingdom 1

Background: Multiple respiratory chain (RC) enzyme deficiencies are present in ~30% of childhood-onset mitochondrial disease and are phenotypically and genetically heterogeneous. Nuclear-encoded defects of mitochondrial translation are an increasingly recognised cause. Objective: To identify the genetic cause of Leigh syndrome in a singleton case with unrelated parents using whole exome sequencing (WES). Methods: The patient presented in early infancy with Leigh syndrome and combined RC deficiencies in muscle and fibroblasts. Bioinformatics analysis of WES data prioritised mitochondrially-targeted genes containing two novel predicted deleterious variants. Results: Two novel heterozygous mutations were identified in a single mitochondrially-targeted gene GFM1, encoding mitochondrial translation elongation factor mtEFG1. A splice mutation (c.55732A>C) inherited from the mother resulted in exon 5 skipping, whilst a missense mutation (c.1468G>A; p.E490K) inherited from the father affected a highly conserved amino acid. Western blot demonstrated low levels of EFG1 protein, and quantitative real-time PCR revealed reduced levels of GFM1 transcript but increased levels of transcripts of mitochondrial translation elongation factors mtEFTs and mtEFTu in the patient. Discussion: WES identified novel GFM1 mutations causing Leigh syndrome. We provide evidence that disturbance of the relative proportions of mitochondrial translation elongation factors contributes to disease pathogenesis in patients with GFM1 mutations. P-350 Mitochondrial ubiquinol-cytochrome C reductase core protein II defects may affect multiple metabolic pathways. Yano S1, Miyake N2, Watanabe Y3, Bartley J4, Abdenur JE5, Wang RY5, Chang R5, Goto Y6, Shiina M7, Ogata K7, Matsumoto N2 1

Genetics Ped, Univ Southern Cal, Los Angeles, United States; 2Dept Human Genet Yokohama City Univ, Yokohama, Japan; 3Med Genet Ped Kurume Univ, Kurume, Japan; 4Med Genet Children's Hp Los Angeles, Los Angeles, United States; 5Div Metab Dis Childrens Hp Orange County, Orange, United States; 6National Institute of Neuroscience, Tokyo, Japan; 7Dept Biochem Yokohama City Univ, Yokohama, Japan

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Background: Mitochondrial ubiquinol-cytochrome c reductase core protein II, encoded by the nuclear gene UQCRC2, is an essential component of respiratory chain complex III. Three patients (5y female, 4y male, and 18m female) with a homozygous UQCRC2 deleterious mutation (p.Arg183Trp) from a Hispanic family have recently been reported. Their clinical presentations were unique and characterized by neonatal/early infantile onset, recurrent metabolic acidosis, ketosis, hypoglycemia, lactic acidosis, and hyperammonemia. Methods: Metabolic profiles were studied in blood (NH3, lactate, pyruvate, amino acids, acylcarnitines8 hydroxy fatty acids) and urine (organic acids). Results: While decompensated, hyperammonemia, hypoglycemia, and lactic acidosis were present with abnormal acylcarnitine profiles suggestive of defective acyl-CoA dehydrogenases: SCHAD (C4OH), SBCAD (C5:1), and LCHAD (DC12OH, C14OH, C18OH). Plasma amino acid analysis showed high alanine and normal glutamine levels. Plasma hydroxyl fatty acid profiles showed abnormal elevations of 3-OH-C6 through 3-OH-C16, however, fatty acid oxidation probe assay was normal. Urine organic acids analysis showed high levels of acetoacetic, 3hydroxybutyric, 3-hydroxyadipic, dicarboxylic and 2-ethyl-3hydroxypropionic acids with increased citric acid cycle intermediates, lactate and pyruvate. Conclusions: Metabolic profiles of our kindred with UQCRC2 defect may suggest involvement of multiple metabolic pathways including fatty acid oxidation, branched chain amino acid metabolism, and urea cycle. P-351 Vacteral and mitchondrial dysfunction Cardoso C1, Correia J1, Martins M2, Grazina M3, Martins E1, Bandeira A1 1 Centro Hospitalar do Porto, Porto, Portugal; 2Centro Hosp Trás os Montes e Alto Douro, Vila Real, Portugal; 3Instituto de Bioquimica Faculd Medicina, Coimbra, Portugal

Background: VACTERL association is characterized by vertebral defects, anal atresia, cardiac defects, tracheo-esophageal fistula, renal anomalies, and limb abnormalities. Mitochondrial dysfunction is associated with congenital malformations. Case report: Female infant, two months old, second child of non consanguineous healthy parents. Born at term, Apgar 9/9. At birth she presented: dysplasic ears, microretrognathia, bilateral thumb agenesis, anal atresia. She also had esophageal atresia with tracheo-esophageal fistula. Surgical correction was performed on day one of life. Cardiac echocardiography showed ostium secundum atrial septal defect and persistent truncus arteriosus. Transfontanelar, abdominal and renovesical ultrasound were normal. Skeletal radiography showed hypoplastic thumb. At 2 months she presents failure to thrive, microcephaly and axial hypotonia. Analytically: normochromic and normocytic anemia, uncontrolled glycemia (hypoglycemia without hyperinsulinism and hyperglycemia) and tubulopathy with mild proteinuria (16,5 mg/m2/hour, without glycosuria). She also presented metabolic acidosis and hyperlactatemia. Karyotype and FISH of 22 cromossome were normal. A mitochondrial disease was suspected because of the multisystemic involvement, dysmorphic features, glycemic instability and hyperlactatemia. Respiratory chain defect was present in muscular biopsy. Conclusion: VACTERL association and mitochondrial dysfunction has been described but the genetic background is still unknown. Clinical signs and symptoms of mitochondrial dysfunction should be look for in VACTERL patients.

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Dual diagnoses the search beyond the obvious: trisomy 21 and a DGUOK mutation

Atypical presentation of Lebers hereditary optic neuropathy associated with the common 11778g>a mitochondrial DNA mutation: report of a rare case of LHON plus

Seprish ME1, Lanpher B1, Lam C2, Lichter U1, Chapman KA1 1

2

CNMC, Genetics and Metabolism, Washington, United States; Nat Inst Health, Nat Hum Genome Inst, Bethesda, United States Background: Dual genetic diagnoses are fairly rare in our field. Moreover, infantile liver failure from deoxyguanisine kinase (DGUOK) deficiency is also fairly rare. Although we think as more common, the incidence of Trisomy 21 in our population is estimated to be 1 in 1000. Objective: Discuss a patient with both trisomy 21 and DGUOK mutations that leads to infantile liver failure and death. Case report: We present a case of a 2-week-old female with trisomy 21 who presented in acute liver failure and a atrial-ventricular canal defect (AV canal). Newborn screen was consistent with liver failure showing an elevated tyrosine and the child had persistent hyperammonemia with levels >200, coagulopathy without bleeding, and significant hypoglycemia requiring IVF consistent with liver dysfunction. Infectious work-up, buccal biopsy for hemochromatosis, and bone marrow biopsy were all negative. The child died at 5 weeks of life secondary to multi-organ failure. DGUOK sequencing identified homozygous missense mutations (c.677A>G). This mutation has previously been associated with DGUOK deficiency. Conclusion: Mitochondrial DNA depletion syndrome should be considered in any child with trisomy 21 and acute liver failure. P-353 A drosophila model of complex I deficiency and leigh syndrome: tool for investigating disease mechanism and therapeutic role of antioxidants

Shuen AY1, Lefrancois M2, MacKay N3, Cameron J3, Abrecht S4, O'Ferrall E4, Saint-Martin C5, Al-Hertani W1 1 Dept Med Genet, MUHC, Montreal, Canada; 2Dept Clin Nutr, MUHC, Montreal, Canada; 3Dept Paed & Lab Med, Sickkids, Toronto, Canada; 4 Dept Path & Dep of Neuro/Neurosurg, MUHC, Montreal, Canada; 5 Dept Diag Radiology, MUHC, Montreal, Canada

Leber's hereditary optic neuropathy (LHON) is a maternally inherited mitochondrial disorder classically associated with bilateral, painless, subacute visual failure in young adult males predominantly. In most patients there are no other neurological manifestations and cerebral neuroimaging is normal, but some rare cases of ''LHON plus'' have been described. We describe a 3-year-old boy who presents with recurrent vomiting, failure to thrive, progressively worsening delayed gastric emptying, strabismus, hypotonia and elevations of CSF lactate, plasma alanine and alanine/lysine ratio levels. Brain MRI revealed progression of multiple T2 hyperintensities at subthalamic nuclei and periventricular ventral nuclei at the fourth ventricle as well as at the dorsal aspect of the upper medulla. Skeletal muscle histology and electron microscopy were normal. Activities of the respiratory chain complexes in muscle tissue were all within the normal range. Coenzyme Q10 levels in muscle were also normal. Whole genome sequence analysis of the mitochondrial DNA (mtDNA) revealed a homoplasmic 11778G>A mutation, in addition to two homoplasmic secondary LHON mtDNA mutations (4216T>C and 4917A>G), which in the presence of the primary LHON mutation, may have synergistic effects. This report expands on the clinical phenotype reported in patients with "LHON plus".

Li Z1, Zhang K1, Guan J1, Haueter C2, Bellen H3, Graham B1 P-355 Baylor College of Medicine, Houston, United States; 2Methodist Hospital, Houston, United States; 3Howard Hughes Medical Institute, Houston, United States 1

Background: Complex I (CI) deficiency as an inborn error of metabolism is a frequent cause of OXPHOS dysfunction and Leigh Syndrome (LS) with no effective treatments available. NDUFS3 is an Fe-S clustercontaining subunit of CI. Oxidative stress (OS) is an important pathogenic factor in OXPHOS deficiencies as well as in diseases with secondary defects of mitochondrial metabolism. Methods: P-element excision of CG12079, the Drosophila ortholog of NDUFS3, was used to generate mutants. Phenotype characterizations include molecular, biochemical, physiological and behavioral analyses as well as treatment with antioxidants. Results: Recessive mutations of NDUFS3 cause severe deficiency of CI activity with abnormal assembly. Most animals without NDUFS3 fail to pupate and die during larvogenesis, however, rare adult escapers survive and experience a shortened lifespan. NDUFS3-deficient flies exhibit increased levels of OS and neurological dysfunction manifested as increased sensitivity to mechanical stress, defective climbing ability and perturbed retinal function. The mutant NDUFS3 phenotypes are fully rescued by genetic construct of NDUFS3 and partially rescued by vitamin E analogs supplemented in food. Conclusion: Loss of NDUFS3 in Drosophila causes CI deficiency and increased OS. Drosophila is a useful model system for investigating the pathogenesis of mitochondrial disease as well as for exploring therapeutic strategies.

Case report: a novel previously unreported mitochondrial dna variant 7478G>A in a patient with acute metabolic encephalopathy, intractable seizures, and mitochondrial complex I and IV deficiency Kitzler T1, Lefrancois M2, Saint-Martin C3, Melancon SB1, Al-Hertani W1 1 Depart of Med Gen, McGill Univ HC, Montreal, Canada; 2Depart of Clin Nutr, McGill Univ HC, Montreal, Canada; 3Depart of Diagn Radiol, McGill Univ HC, Montreal, Canada

We report on a 13 year old girl adopted from Bangladesh at the age of 4 months, with unremarkable development until the age of 8 years, when she presented for the first time with sudden-onset generalized tonicclonic seizures. Subsequently, she suffered from acute metabolic encephalopathy with complex multifocal seizures and severe psychomotor retardation. She was started on anti-epileptic medications and due to the refractory nature of her seizures a vagal nerve stimulater was implanted for adjunctive therapy. MRI of the brain showed cerebral atrophy with progression of multiple T2 hyperintensities, primarily in the basal ganglia, without the presence of abnormal metabolites on MR spectroscopy (MRS). Activities of the respiratory chain complexes in muscle tissue revealed a complex I and IV deficiency by blue native polyacrylamide gel electrophoretic (BNPAGE) analysis. Subsequent, whole mitochondrial genome analysis demonstrated a novel, previously unreported, homoplastic variant of unknown

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clinical significance 7478G>A. As our patient was adopted, without any known family history or contact with the biological family, it was not possible to determine whether there are other affected matrilineal adult relatives and whether this previously unreported novel variant is homoplasmic in other affected vs. other unaffected matrilineal adult relatives. P-356 Phenotypic and genotypic variability in YARS2 mitochondrial myopathy, lactic acidosis and sideroblastic anaemia Riley LG1, Menezes MJ1,2, Rudinger-Thirion J3, de Lonlay P4, Rotig A4, Tchan MC5, Cooper ST2,6, Christodoulou J1,2,7 1 Genet Metab Dis Res Unit, Child Hosp, Westmead, Australia; 2Paed & Child Health, Uni Syd, Sydney, Australia; 3Arch React l'ARN, Uni Strasbourg, Strasbourg, France; 4Uni Paris Des & INSERM, Hosp N-E Mal, Paris, France; 5Dept Genet Med, Westmead Hosp, Westmead, Australia; 6INMR, Child Hosp, Westmead, Australia; 7Genet Med, Uni Syd, Sydney, Australia

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Results: All but two ACAD9 mutants were stable; three had a mildly decreased ACAD9 enzymatic activity while one had a normal activity; activity was partially decreased in 2 mutants and severely decreased in 7. ACAD9 protein stability in patient fibroblasts largely mirrored these findings though some discordance was apparent. Doubly deficient VLCAD/LCAD knockout mouse fibroblasts retained ACAD9 activity and supported significant capacity to oxidize long chain fatty acids. Conclusions: The moonlighting function of ACAD9 as a CI assembly factor is independent of its FAO enzymatic activity. Mutations in ACAD9 can affect either or both functions; this complicates characterization of genotype/phenotype correlations in affected patients. Conflict of Interest declared. P-358 Primary carnitine deficiency presenting as epilepsy in infant Widjaja NA1, Limanto L1 1

Div Nutr &Metab Dis,Airlangga Univ, Surabaya, Indonesia

Background: Mutations in the mitochondrial tyrosyl-tRNA synthetase (YARS2) gene have previously been identified as a cause of the mitochondrial respiratory chain (RC) disorder MLASA (Myopathy, Lactic Acidosis, Sideroblastic Anaemia). Methods and Results: We present three unrelated patients with MLASA symptoms. One mildly affected patient was compound heterozygous for two novel YARS2 mutations, p.Gly191Asp and p.Arg360X. The p.Gly191Asp mutation resulted in a 38-fold loss in YARS2 catalytic efficiency and the p.Arg360X mutation did not produce a stable protein. Two patients were found to be homozygous for the reported p.Phe52Leu mutation. One patient died at 3 months while the other was diagnosed in adulthood and is mildly affected. Together with three previously reported homozygous p.F52L YARS2 patients, mitochondrial haplogroups are being investigated as a potential modifier of the observed clinical variability. RNAseq has been undertaken on muscle samples taken before and after a remarkable spontaneous clinical improvement in one previously reported patient to identify potential compensatory mechanisms. Conclusion: We have identified 2 novel YARS2 mutations and noted marked phenotypic variability among YARS2 MLASA patients, with phenotypes ranging from mild to lethal in childhood. These findings have important implications for diagnosis and prognostication of the MLASA and related phenotypes

Background: Clinical manifestations of primary carnitine deficiency can vary widely with respect to age of onset, organ involvement, and severity of symptoms. Primary carnitine deficiency syndrome is an autosomal recessively inherited disease caused by a deficiency in the plasma membrane carnitine transporter, a rare and potentially fatal but treatable metabolic disorder. Objectives: This case report was to show the importance to research metabolic etiology, especially a carnitine deficiency in infant with recurrent seizure and hypotonia without unknown cause. Case Report: A forty five days old boy presented clinical features included generalized recurrent seizure, muscular hypotonia, acute encephalopathy. No apparent craniofacial dysmorphism were found. He had hypoketotic hypoglycemic encephalopathy with normal liver function. Electroencephalography showed epilepsy. Seizure still persisted after normal biochemical laboratory result and anti epileptic treatment. Chest x-ray and ECG result were normal. Tandem Mass Spectrometry result showed a decrease of free carnitine and total acylcarnitine (Shimane University Japan). After treatment with oral L carnitine (100 mg/kg/day) for 1 months, he was much improved clinically. Conclusion: Primary carnitine deficiency is a cause of epilepsy and hypotonia in infant. It must systematically be investigated when patients presents with recurrent seizures and hypotonia without known cause.The treatment with oral carnitine can improve the clinical outcome.

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P-359

Moonlighting in mitochondria: ACAD9 plays a dual role in energy metabolism

Diagnosis and molecular basis of mitochondrial respiratory chain disorders in Japan: exome sequencing for the disease gene identification

Schiff M1, Haberberger BM2, Goetzman ES1, Mohsen AW1, Prokisch H2, Vockley J1 1 Dep Pediatrics, Univ Pittsburgh, Pittsburgh, United States; 2Institute Hum Genet, Helmholtz Zentrum, Munich, Germany

Background: Liver ACAD9 deficiency was first described in 2 patients with liver failure. Subsequently, point mutations in ACAD9 were shown to cause respiratory chain complex I (CI) deficiency (companion abstract, Haberberger et al.) due to a moonlighting ACAD9 function as a CI assembly factor. However, the impact of these mutations on fatty acid oxidation (FAO) activity and the physiologic contribution of ACAD9 in eukaryotes to FAO remain unknown. Methods: Prokaryotic expression mutagenesis studies were performed for 13 missense ACAD9 mutations. Oxidation of long chain fatty acids was analyzed in VLCAD and LCAD double knockout mouse fibroblasts.

Fushimi T1, Murayama K1, Kawachi E1, Fujinami A1, Ajima M1, Kohda M2, Tokuzawa Y2, Mizuno Y2, Mori M3, Takayanagi M1, Okazaki Y2, Ohtake A4 1 Dep of Metab , Chiba Child Hosp, Chiba, Japan; 2Reser Cen for Gen Med, Saitama Med Univ, Saitama, Japan; 3Dep of Ped, Jichi Med Univ, Tochigi, Japan; 4Dep of Ped, Saitama Med Univ, Saitama, Japan

Background: Mitochondrial respiratory chain disorders (MRCD) are a group of the most common congenital metabolic disorders of energy production. Our aim is to make correct diagnoses of MRCD patients and to identify their causative genes. Methods: MRCD was diagnosed using both in vitro enzyme assay and BN-PAGE analysis. After sequencing the whole exome of the patient using high-speed sequencer, a filtering strategy included a screen for

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genes coding for mitochondrial proteins. Furthermore, we are performing an ab initio prediction approach for unresolved patients and complementation experiments to substantiate the pathogenic role of newly identified DNA variants. Results: Three hundred patients were diagnosed to have MRCD out of 890 candidate patients. Most frequent was complex I deficiency, and second was combined complexes deficiency. Forty nine out of 157 analyzed patients had mitochondrial DNA pathogenic mutations (31%). Among 104 patients analyzed with exome sequencing, we identified 18 known genes including SURF1, COX10, RARS2, TUFM, BOLA3 etc. and 27 unreported candidate genes for MRCD. Discussion: Using high-speed sequencers, we are now identifying the causative genes and the pathogenic mechanisms of MRCD. Our study will illustrate how large-scale sequencing, coupled with functional prediction and experimental validation, can identify disease mutations in MRCD patients. P-360 Segmental intestinal pseudo-obstruction responsive to pyridostigmine therapy in a patient with MELAS phenotype Horvath GA1, Siden H2, Cline L2, Lillquist Y1, Salvarinova-Zivkovic R1, Avinashi V3, Stockler-Ipsiroglu S1, Van Karnebeek C1

replication and repair. Mutations in POLG1 have been linked to a spectrum of clinical phenotypes, which can go from Alpers-Huttenlocher syndrome, Autosomal Dominant Progressive External Ophtalmoplegia to Childhood Myocerebrohepatopathy Spectrum Disorders. In these disorders is well known that individuals, who carry a mutation in compound heterozygosis, have a poorer survival rate compared to homozygotes. Objectives: Here, we report on a proband, carrying a novel POLG1 mutation in compound heterozygosis and has a severe neurologic involvement. Case report: The patient is a two months old girl, who since the newborn period had a severe axial hypotonia and recurrent vomiting. Unfortunately the girl had a fatal outcome due to her neurologic involvement. The metabolic work-up showed high serum lactic acid and hydroxylated acylcarnitines, suggestive of a mitochondrial disorder. The muscle biopsy disclosed a depletion of the 86% of the mtDNA and the molecular analysis a novel mutation of the POLG1 gene (H1134R) in compound heterozygosis. Conclusion: The addition of this mutation to the growing list of defects, confirms the importance of POLG1 mutations as the underlying abnormality in a wide range of severe neurological presentations. P-362 A homoplasmic mitochondrial t-RNA A3243G mutation as a cause of maternally-inherited mitochondrial myopathy in a Malaysian family Ch'ng GS1, Yakob Y2, Abd Azize NA2, Haniffa M1, Ngu LH1

1

2

Biochem Dis, BC Children's Hospital, Vancouver, Canada; Palliative Care, BC Child Hosp, Vancouver, Canada; 3Gastroenterol, BC Child Hosp, Vancouver, Canada

1

Genetics Dept, Kuala Lumpur Hospital, Kuala Lumpur, Malaysia; Specialized Diagnostics Centre, IMR, Kuala Lumpur, Malaysia

2

Background: The pathophysiology of intestinal pseudo-obstruction in mitochondrial disease remains unknown. Case report: A 14 year old girl with mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) (m.13513G>A) was admitted with symptoms of bowel obstruction. She continued with bilious vomiting and feeding intolerance even after her colon was clean. Symptoms were intermittent initially but over 3 weeks she became TPN dependent, not tolerating anything orally. Surgical causes of obstruction were ruled out. Abdominal CT was unremarkable. A gastrostomy tube was inserted for decompression and a jejunostomy tube for feeding. She continued to have large bilious emesis, containing no feeds. G-tube drainage was minimal. This suggests obstruction at jejunal level. Upper GI endoscopy found no fixed stenosis. She had prokinetics, proton pump inhibitors without relief. Introduction: of Pyridostigmine, an acetylcholinesterase inhibitor, improved her symptoms. No major side effects were noted. She continues to have pain, but less vomiting, and she takes small amounts of food orally. She was discharged home after almost 3 months of hospitalization. Conclusion: We suspect that a small segmental bowel dysfunction occurred in this patient with MELAS phenotype, which had a favorable response to acetylcholinesterase inhibition, suggesting cholinergic failure in pathology of pseudo-obstruction in mitochondrial diseases.

Background: Defects of mitochondrial genome cause a heterogenous group of clinical disorders. Mutations in mitochondrial transfer RNA (tRNA) are prevalent and m.3243A>G encoding mitochondrial tRNA for Leucine is commonest mutation causing MELAS syndrome. m3243A>G also causes myopathy,hypertrophic cardiomyopathy and others. Majority of pathogenic mutations are heteroplasmic and assigning pathogenicity of homoplasmic mutations must conform to specific pathogenic criteria. Methods: The proband exhibited myopathic symptoms from 8 years old. She had progressive reduced effort tolerance and developed type 2 respiratory failure at age 10. She has normal cognitive function and is BIPAP dependant. Her mother and 2 siblings were also affected. Results: Skeletal muscle showed numerous COX-positive ragged red fibers and homoplasmic m.3243A>G mutation which was not found in blood. Heteroplasmic m.3243A>G mutation load 50% and 64% was found in older sister and mother's blood respectively. A homoplasmic m.3290T>C mutation was also found in the blood of all three individuals. Conclusion : Clinical, histochemical and molecular genetic analysis confirmed diagnosis of mitochondrial myopathy. By assessing steady state levels of mutant mt-tRNA variants and rates of mitochondrial protein synthesis in tissue samples from these individuals may further establish the pathogenicity of homoplasmic m.3290T>C mutation.

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A novel mutation in POLG1 gene produces a severe neurological involvement

Extending the phenotype of AARS2 mutations

Roncalés-Samanes P1, Baquero-Montoya C 2 , Beltrán-García S1, Montoya-Villaroya J 3 , López-Pisón J 1 , Peña-Segura JL 1 , GilHernández I1, García-Jiménez MC1 1

Dep Pediatr, Hosp Miguel Servet, Zaragoza, Spain; 2Dep Pediatr. Hospital Pablo Tobón Uribe, Medellin, Colombia; 3Biochem Molec Cell Biol Depart, Univ Zar, Zaragoza, Spain Background: The POLG1 gene encodes the catalytic subunit of DNA polymerase gamma, essential for mitochondrial DNA (mtDNA)

Wedatilake Y1, Shahni R1, Fassone E1, Emmett W2, Saldanha J3, Manzur A4, Taanman JW5, Rahman S1 UCL Institute of Child Health, London, United Kingdom; 2University College London, London, United Kingdom; 3National Institute for Medical Research, London, United Kingdom; 4Great Ormond Street Hospital, London, United Kingdom; 5Institute of Neurology, UCL, London, United Kingdom 1

Background: Deficiencies in mitochondrial aminoacyl-tRNA synthetases cause defects in protein synthesis within mitochondria. Mitochondrial alanyl-tRNA synthetase (AARS2) mutations have been found in

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patients with multiple respiratory chain enzyme deficiency associated with severe infantile cardiomyopathy and death in infancy. Methods: Whole exome sequencing (WES) was performed in two affected siblings with suspected mitochondrial disease. We filtered for novel, mitochondria-targeted variants shared between the affected siblings. Results: WES revealed novel compound heterozygous mutations in AARS2. The two siblings presented with an early onset autistic spectrum disorder, developmental delay and upper motor neurone signs in the lower limbs. They were found to have dilated cardiomyopathy in adolescence and are currently alive at 18 and 16 years of age. Muscle biopsy demonstrated an isolated decrease in cytochrome c oxidase (COX) activity and pale COX histochemical staining in skeletal muscle. Mutation segregation within the family was confirmed and the pathogenicity of the mutations was validated in silico. Conclusions: We found two novel AARS2 mutations and demonstrate a new presenting clinical phenotype. In comparison to previous cases (n=3) the patients had an isolated decrease in COX activity and experienced longer survival. WES is a robust tool in the diagnosis of complex mitochondrial disorders.

Background: Patients with muscle mitochondrial respiratory chain (MRC) combined deficiencies without molecular lesions in mitochondrial DNA (mtDNA) are a priori good candidates for exome sequencing due to the clinical and genetic heterogeneity underlying these disorders. Objective: To identify a mutated nuclear gene as the causative of the disorder in a subset of patients with multiple deficiencies of MRC. Patients and Methods: Seven Spanish patients of age ranging 0.5–8 years old were included. Exome sequencing was performed on an Illumina NGS platform. To prioritize the candidate genes, the raw data was filtered according to following criteria: i) autosomal recessive inheritance, ii) variant frequency in 1000 genomes database <0.5%, and iii) genes annotated in MitoCarta database. Results and discussion: A homozygous mutation in complex I subunitNDUFB3 gene (NP_001244031, p.Trp22Arg) was identified in a patient presenting with neonatal lactic acidosis. This mutation was previously described in two unrelated patients with isolated complex I deficiency. In a child with cognitive impairment, cardiomyopathy, and lactic acidosis a novel homozygous missense mutation in the MTO1 gene (NP_598400, p.Arg489Cys) was found. MTO1 mutations were recently reported in patients with similar phenotypes. Presumably pathogenic variants were not prioritized in the remaining patients.

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Treatable leigh-like encephalopathy presenting in adolescence

Hashimoto thyroiditis: a respiratory chain disease?

Fassone E1, Wedatilake Y1, DeVile C2, Carr L2, Rahman S1

Zimmermann FA1, Neureiter D2, Sperl W1, Kofler B1, Mayr JA1

1

Mito Res Group, UCL Inst of Child Health, London, United Kingdom; Neuro Dep, Great Ormond Street Hospital, London, United Kingdom

Department of Pediatrics, PMU, Salzburg, Austria; 2Department of Pathology, PMU, Salzburg, Austria

Background: Mutations of thiamine transporter-2 cause biotin-responsive basal ganglia disease (BBGD), a potentially treatable neurological disorder linked to heterogeneous phenotypes ranging from neonatal lactic acidosis to late-onset Wernicke-like encephalopathy. We describe a late-onset case with dramatic clinical response to combined biotin and thiamine therapy. Methods: A previously healthy 15-year-old female presented with rapid onset ptosis and progressive ophthalmoplegia. Neuroimaging demonstrated bilateral basal ganglia and brainstem lesions suspicious of Leigh syndrome, but plasma and CSF lactate levels and skeletal muscle respiratory chain enzyme activities were normal. Oral biotin and thiamine supplementation were commenced, with dramatic improvement in symptoms by the following day. Therapeutic response suggested SLC19A3 as a strong candidate gene, and Sanger sequencing was performed. Results: A novel homozygous missense c.517A>G; Asn173Asp mutation in SLC19A3 was identified in the patient, which segregated with disease within the family. Conclusions: BBGD should be considered in the differential diagnosis of atypical Leigh syndrome. Since the time from presentation to biotin and thiamine supplementation largely influences disease progression and clinical outcome, prompt diagnosis is critically important. Delayed treatment may result in permanent neurological dysfunction or death; for this reason a high index of clinical suspicion and early supplementation with biotin +/- thiamine are essential.

Oncocytic cells (OC) are characterized by a large cytoplasm with intensive eosin staining due to an enormous accumulation of mitochondria. OC have been described as a sign of mitochondrial dysfunction in various tissues e.g. oncocytic tumors. The occurence of OC in the thyroid of patients with Hashimoto thyroiditis (HT) is known but poorly characterized. In the present study we investigated in patients with HT (n=12) respiratory chain complexes in oncocytic lesions of the thyroid gland by immunohistochemical staining and analysis of the mitochondrial DNA. In thyroid tissue samples from all 12 HT patients lesions with OC were identified. In 87% of 212 investigated oncocytic lesions a significant reduction of complex I was found compared to the adjacent normal tissue (P<0.0001). The other enzyme complexes of the oxidative phosphorylation were significantly increased. In two cases with large oncocytic lesion, we sequenced all mitochondrially encoded complex I subunits and found a nonsense mutation (m.3860G>A, p.Trp185*) in the ND1 subunit. We demonstrate for the first time a defect of respiratory chain complex I in OC in the thyroid of HT patients. Since OC are a characteristic finding in HT we postulate a substantial role of complex I deficiency in the pathomechanism of HT.

2

1

P-367 A case with mitochondrial seryl t-RNA synthetase gene (SARS2) defect: a novel mutation and a different phenotype from HUPRA syndrome

P-365 Exome sequencing reveals mutations in the NDUFB3 and MTO1 genes in mitochondrial respiratory chain combined defects. Delmiro A1,2, Blázquez A1,2, Martín-Hernández E1, Lara J3, Quijada P1, García-Silva MT1,2, Morán M1,2, García-Arumi E4,2, López E5,2, Bornstein B3,2, Arenas J1, Martín MA1,2 12 Octubre Hospital Research Institute, Madrid, Spain; 2Rare Diseases Research Centre (CIBERER), Madrid, Spain; 3Puerta de Hierro University Hospital, Madrid, Spain; 4Val d'Hebron Research Institute, Barcelona, Spain; 5Zaragoza University, Zaragoza, Spain 1

Öztürk Hişmi B1, Belostotsky R2, Utine GE3, Talim B4, Ünal Ö1, Dursun A1, Sivri S1, Coskun T1, Tokatlı A1, Özaltın F5, Gönç N6, Berberoğlu Ateş B7, Acar Arslan E8, Frishberg Y2 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Div Ped Nephrol,Shaare Zedek Med Cent, Jerusalem, Israel; 3Div Ped Genet, Hacettepe Univ Child Hosp, Ankara, Turkey; 4 Div Ped Pathol, Hacettepe Univ Child Hosp, Ankara, Turkey; 5Div Ped Nephro, Hacettepe Univ Child Hosp, Ankara, Turkey; 6 Div Ped Endoc, Hacettepe Univ Child Hosp, Ankara, Turkey; 7Div Ped Gastro, Hacettepe Univ Chil Hosp, Ankara, Turkey; 8Div Ped Neurol,Hacettepe Univ Child Hosp, Ankara, Turkey 1

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Mitochondrial aminoacyl-tRNA synthetases (mtARSs) are essential in the process of transferring genetic information from mitochondrial DNA to the complexes of the oxidative phosphorylation system. These synthetases perform an integral step in the initiation of mitochondrial protein synthesis by charging tRNAs with their cognate amino acids. All mtARSs are encoded by nuclear genes, nine of which have recently been described as disease genes for mitochondrial disorders. Homozygous missense mutation in SARS2 (p.Asp390Gly), encoding mitochondrial seryl-tRNA synthetase, was described in patients with tubulopathy (hyperuricemia, metabolic alkalosis), primary pulmonary hypertension (PHT), and progressive renal failure in infancy (HUPRA syndrome). They had high blood and CSF lactate levels, muscle biopsies showed signs of OXPHOS deficiency and all deceased within the first 14 months of their lives. Here we describe a novel SARS2 mutation (p.Arg402His) and a different phenotype with renal, endocrine and exocrine pancreas (recurrent pancreatitis), adrenal, muscle (COX deficiency), bone marrow (pancytopenia) and central nervous system (cerebral atrophy, hyperintensities and lactate peaks in cerebellar deep gray matter on MRI and MRS) involvement but without PHT. The patient is 3,5 years old now, thriving and developing well on peritoneal dialysis and supportive therapy. Renal transplantation is planned in the near future.

loss and cortical visual impairment. From 11 months of age, she has had seizures resembling infantile spasms. Head circumference was normal at birth but she has developed severe microcephaly. Muscle tone is normal. She has hypertension and chronic renal impairment. Isolated cytochrome oxidase deficiency was demonstrated in muscle and fibroblasts. Screening of the RMND1 coding region in fibroblast cDNA revealed an apparently homozygous missense mutation, c.631G>A, V211M, predicted to be highly damaging. However, this mutation was found to be heterozygous in genomic DNA. Suppression of nonsense-mediated decay revealed an additional transcript with skipping of exon 6 resulting in a frameshift and premature stop codon. This is due to a heterozygous base substitution, c.830+1G>A in the splice donor site of intron 6. RMND1 deficiency is a newly recognised defect in mitochondrial protein translation which particularly affects cytochrome oxidase. The clinical spectrum is yet to be properly defined, but includes neonatal lactic acidosis, seizures and progressive microcephaly.

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Broadening the genetic spectrum of mt-tRNACys mutations: m. 5766C>G a novel mutation

A new mutation in the gene encoding mitochondrial seryl-tRNA synthetase as a cause of HUPRA syndrome Rivera H1,2, Martín-Hernández E1, Delmiro A1, Delmiro A2, GarcíaSilva MT1,2, Martín MA1,2, Martínez-Azorín F1, Martínez-Azorín F2 1 12 Octubre Hospital Research Institute, Madrid, Spain; 2Rare Diseases Research Centre (CIBERER), Madrid, Spain

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Almeida LS1, Martins E2, Santorelli FM3, Vilarinho L1 R&D Unit, Nat Inst of Health, INSA, Porto, Portugal; 2Metab Dis Unit, Porto Hosp Center, Porto, Portugal; 3Mol Med & Neurogenetics, IRCCS, Pisa, Italy 1

Neonatal lactic acidosis and hypoglycaemia in a patient with RMND1 mutations

Mutations in mitochondrial tRNA genes are responsible for the majority of mitochondrial disease presentations being associated with marked clinical heterogeneity. We present an 11-year-old girl with vomiting episodes and migraine. The patient presented with epilepsy in the neonatal period that resolved upon valproate treatment (normal EEG). At age 18 months, she developed paroxysmal torticollis episodes; at the age of 4 years she showed vomiting episodes mainly during sleep and episodic migraine. Her psychomotor developmental milestones were within the normal range. Metabolic investigations were normal except for the presence of Krebs cycle metabolites in urinary organic acid profile. The mother is clinically normal. One of her sisters is deaf-mute, another sister presents with hypotonia and developmental delay, whereas the brother (who died at age 3 months) presented with severe developmental delay and epilepsy in the neonatal period. The whole mtDNA sequencing performed in blood from the index case revealed a heteroplasmic m.5766C>G mutation. The detected variant, meets the criteria for pathogenicity supported by in silico predictions. So far only three pathogenic mutations have been reported in the mt-tRNACys but none was associated with the clinical picture seen in our proposita. Our findings broaden the clinical and genetic spectrum of mt-tRNACys mutations.

Brown RM1, Morris AAM2, Taylor RW3, Brown GK1

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1 Dept Biochem, Univ Oxford, Oxford, United Kingdom; 2Willink Biochem Genet Unit, Manchester, United Kingdom; 3Wellcome Trust Centre Mitoch Res, Newcastle upon Tyne, United Kingdom

Pyruvate dehydrogenase complex deficiency: mutational spectrum of Portuguese patients

Background: HUPRA syndrome is a rare mitochondrial disease characterized by hyperuricemia, pulmonary hypertension, renal failure in infancy and alkalosis. This syndrome was previously described in three patients with a homozygous mutation c.1169A>G (p.D390G) in SARS2 (NG_031865.1), encoding the mitochondrial seryl-tRNA synthetase. Patients and Methods: We report two patients, a girl and her brother, clinically diagnosed with the HUPRA syndrome. Direct DNA sequencing by Sanger method of all the exons and the adjacent exon-intron boundaries of SARS2 gene was carried out. Results and Conclusion: Analysis of the pedigree identified a new homozygous mutation c.1205G>A (p.R402H) in SARS2 gene. This mutation is very rare in the population and it is located at the C-terminal globular domain of the homodimeric enzyme, very close to p.D390G. Several data support that p.R402H mutation in SARS2 is a new cause of HUPRA syndrome. P-369

A girl, now aged 15 months, presented on the first day of life with hypoglycaemia and severe lactic acidosis. Blood ammonia was normal and organic acid, amino acid and acylcarnitine profiles were unremarkable. Echocardiography and cranial ultrasound examination were normal. She has severe sensorineural hearing

Pavlu-Pereira H1, Janeiro P2, Costa C2, Gaspar A2, Oliveira A3, Tavares de Almeida I1, Silva MJ1, Rivera I1 1 Met&Gen - iMed, Fac Pharm, Univ Lisbon, Lisbon, Portugal; 2Dept of Paediatrics, Santa Maria Hosp, Lisbon, Portugal; 3Dept of Medicine, Santa Maria Hosp, Lisbon, Portugal

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Pyruvate dehydrogenase complex deficiency (PDCD) is associated with a broad variability at phenotype and genotype levels. Clinical spectrum ranges from fatal lactic acidosis or progressive neurological and neuromuscular degradation in the newborn period, to a chronic neurodegenerative condition. Most PDCD cases result from mutation in PDHA1, the X-linked gene encoding E1α subunit, while few cases reside in genes encoding other subunits: E1β (PDHB), E2 (DLAT), E3 (DLD), E3BP (PDHX) or the regulatory PDH-phosphatase (PDP1). This study involved 15 patients showing clinical, biochemical and enzymatic data compatible with this deficiency. Mutation were identified by direct sequence analysis of genomic and/or cDNA samples. We achieved the full characterization of 7 patients. Most of them, as expected, revealed mutations in PDHA1 gene (p.F205L, p.R253G, p.R378C and p.R378H). The remaining two patients carried mutations in PDHX gene: the p.R284X and the novel c.483delC (p.P161fsX184). These results confirm once more the low success rate achieved in molecular characterization of PDCD. Importantly, in all the studied genes a considerable number of silent mutations and intronic polymorphisms has been identified, many of them not listed in DNA variants databases. These DNA variations await further analysis for evaluation of potential effects on mRNA and/or protein deterioration. PEst-OE/SAU/UI4013/2011; FCT(SFRH/BD/91729/2012) P-372 A case report of COX-deficient leigh syndrome due to SURF1 gene mutation Kose M1, Kagnıcı M2, Canda E3, Kalkan Uçar S3, Diniz Ünlü G4, Taylor R5, Brown G6, Çoker M1

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P-373 Mitochondrial DNA depletion syndromes presenting with cholestasis in newborn Kilic M1, Ozgul RK1, Tokatli A1, Kalkanoglu-Sivri HS1, Dursun A1, De Meirleir L2, Seneca S3, Coskun T1 1

Hacettepe Univ, Pediatric Metab Unit, Ankara, Turkey; 2Dept of Pediatric Neurol, Brussels, Belgium; 3Centre for Medical Genetics, Brussels, Belgium Background: Mitochondrial DNA depletion syndromes are a group of genetically and clinically heterogeneous diseases that are autosomal recessively inherited with decreased mitochondrial copy number and respiratory chain complexes. Progressive liver failure, hypoglycemia, lactic acidemia and neurologic abnormalities are seen early in the hepatocerebral form. DGUOK mutations are the most common in mitochondrial hepatoencephalopathy and show a variety of phenotypic findings ranging from isolated liver failure to multisystemic disease. Patients: All 3 cases' symptoms started within the first few days after birth. Respiratory distress, liver failure, cholestasis, hypoglycemia, hyperferritinemia, coagulopathy, difficulty feeding and lactic acidemia were the major clinical and laboratory findings. In follow up, nystagmus, developemtal delay and hypotonia were seen. Liver biopsy showed hepatocellular damage, cholestasis and fibrosis. A novel homozygous mutation (c.34C>T) in DGUOK gene was found in one case, the other 2 cases were shown to be homozygous for previously reported mutations (c.707+3_707+6delAAGT and c.130G>A). Even with supportive treatment, all 3 cases died shortly after. Conclusion: Mitochondrial DNA depletion syndromes should be considered in the differential diagnosis in newborns and infants with treatment resistant liver failure and coagulopathy. P-374

Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis, Ege Univ Child Hosp, İzmir, Turkey; 3Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 4 Div Pathology,Behcet Uz Child Hosp, İzmir, Turkey; 5Wellc Trust Cent Mit Res,Univ Newcastle, Newcastle, United Kingdom; 6Div Biochem,Univ Oxford, Oxford, United Kingdom 1

Leigh Syndrome is a common presentation of paediatric mitochondrial disease and is characterised by severe neurodevelopmental delay, a subacute necrotising encephalopathy, basal ganglia involvement on MRI and marked biochemical and genetic heterogeneity. We decribe the clinical and laboratory findings in a 2 year old female patient who presented with growth retardation and mildly neurodevelopmental delay. Initital laboratory tests including serum lactate and cranial MRI-MRS were normal. In the third month of follow-up, she was found to have spasticity and delayed motor milestones; she also suddenly developed hyperventilation attacks. Brain MR imaging showed hyperintense lesions in the basal ganglia, brain stem and cerebellum, although there was no lactate peak on MRS. A diagnostic muscle biopsy revealed a diffuse, global loss of cytochrome c oxidase (COX) activity prompting molecular genetic studies which revealed a well-characterised, homozygous missense mutation (c.769G>A; p.Gly257Arg), in the SURF1 gene. Mutations in SURF1 are a recognised cause of COX-deficient Leigh syndrome, although missense variants are relatively uncommon. Of particular interest, our patient's initial symptoms did not show many clues to a diagnosis of mitochondrial disease, although the acutely changing character of her clinical presentation did prompt further investigation, aiding a genetic diagnosis.

Woodhouse-sakati syndrome with 3-methylglutaconic aciduria and coenzyme Q10 deficiency Hasadsri L1, Gavrilov DK1, Tortorelli S1, Oglesbee D1, Matern D1, Raymond K1, Rinaldo P1, Gavrilova RH1 1

Mayo Clinic, Rochester, MN, United States

Background: Woodhouse-Sakati syndrome (WSS) is an autosomal recessive disorder caused by mutations in DCAF17, a gene encoding a nucleolar protein. Clinical manifestations include hypogonadism, alopecia, diabetes mellitus, developmental delay, hearing loss, and extrapyramidal symptoms. The biochemical workup of previously reported, affected individuals has been described as benign. Objectives: We report a novel case of WSS with unusual biochemical findings. Case Report: Our patient is a 41 year-old Middle-Eastern female, progeny of a consanguineous marriage, who presented with WSS. Renal failure and leukodystrophy were also found. Results: Urine organic acids were significant for increased 3methylglutaric and 3-methylglutaconic acid excretion, while plasma coenzyme Q10 (CoQ) levels were decreased. Molecular testing revealed homozygosity for a c.436delC mutation in DCAF17. Sequencing of the mitochondrial genome was negative for mutations or deletions, as were several mitochondrial and CoQ biogenesis-associated nuclear genes. Her CoQ10 deficiency was attributed to be secondary to statin use and renal failure. Conclusions: In addition to phenotypic overlap that may exist between WSS and primary mitochondriopathies (i.e., deafness-dystonia), biochemical evidence of mitochondrial dysfunction can be observed, such

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as 3-methylglutaconic aciduria and coenzyme Q10 deficiency. Nevertheless, we cannot completely exclude co-inheritance of other genetic causes of 3-methylglutaconic aciduria or secondary mitochondrial dysfunction.

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heightened risk for accumulation of potentially toxic tyrosine metabolites, unless dose adjustments are made. P-377

P-375 Characterization of mitochondrial tRNA processing in HSD10 disease Developmental regression and congenital optic atrophy associated with OPA1 mutation

Deutschmann A1, Traunfellner P1, Amberger A1, Zschocke J1

Schlunz F1, O'Shea R1, Lynch B2, O'Keefe M3, Crushell E4

1

1 Nat Cent Inherit Metab Dis, Dublin, Ireland; 2Dept Neurol, Child Univ Hosp, Dublin, Ireland; 3Dept Ophthal, Child Univ Hosp, Dublin, Ireland; 4Nat Cent Med Genet, Dublin, Ireland

The OPA1 gene is involved in mtDNA maintenance and mutations in the OPA1 gene cause dominant optic atrophy which may be isolated or, when the GTPase domain of the gene is mutated, may result in adult onset neurological abnormalities such as dystonia and ataxia (dominant optic atrophy plus syndrome). We report a 5 year-old boy who had a period of rapid developmental regression at 3 years with almost complete loss of language, loss of cognitive and social skills and new onset of autistic features. Nystagmus was present from birth and bilateral optic nerve hypoplasia was diagnosed thereafter. He had had a normal neuro-developmental assessment at 2 years and 8 months. There was no family history of developmental or visual problems. MRI and MRS of brain were normal as was CGH microarray and biochemical screening for metabolic and mitochondrial disorders. Sequencing of the OPA1 gene confirmed heterozygosity for a sequence variant c.893G>A (p.Ser298Asn) which affects a conserved residue in the GTPase domain and is therefore likely to be pathogenic. Such early onset of optic atrophy and neurological features is unusual and to our knowledge this is the first association with childhood developmental regression thus expanding the neurological phenotype of OPA1 mutations.

Background: HSD10 disease is a rare X-chromosomal disorder mainly characterized by neurological abnormalities and progressive cardiomyopathy. Missense mutations of HSD17B10 reduce or eliminate enzymatic activity, but this defect is not disease causing. Additionally, HSD10 is an essential component of mitochondrial RNase P an enzyme required for mt-tRNAs processing. Therefore, we investigated mttRNA processing in HSD10 disease. Methods: Strand-specific qRT-PCR and Northern blot analysis were performed to quantify mt-tRNA processing in HSD10 knock-down cells, patient fibroblasts and control fibroblasts. Western blots were used to analyze expression of mitochondrial proteins. ROS production was determined by confocal microscopy and MitoSOX staining. Results: qRT-PCR revealed that HSD10 mutations result in the accumulation of precursor (pre)-tRNAMet on the heavy strand and pretRNAGln and pre-tRNATyr on the light strand. Northern blot analysis showed an enrichment of RNA precursors in patient fibroblasts which further indicate reduced RNase P activity. Western blots of respiratory chain proteins revealed a reduced expression of mt-ND1 and mt-COX III. Finally, patient fibroblasts exhibit 2.5 – 3 fold elevated ROS levels. Conclusion: Mt-tRNA processing of specific tRNAs is reduced in HSD10 disease. The accumulation of precursor transcripts is associated with reduced translation of selected mitochondrial encoded proteins, which subsequently causes respiratory chain dysfunction.

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P-378

Chronic DCA in children with mitochondrial diseases is safe, but alters phenylalanine/tyrosine metabolism relative to GSTZ1/MAAI haplotype

Mutations in the TRMU gene can cause acute liver failure-mind liver transplantation

Shroads AL1, Langaee T1, Felitsyn N1, Abdelmalak M1, Stacpoole PW1

Div Human Genetics, Innsbruck Med Univ, Innsbruck, Austria

Paležac L1, Mayr JA2, Ćuk M3, Sarnavka V3, Ćorić M4, Zekušić M5, Bilić K5, Vuković J3, Bogović M6, Zimmermann F2, Bogović TZ7, Fumić K5, Sperl W2, Barić I3

1

University of Florida, Gainesville, United States

Dichloroacetate (DCA) used to treat various acquired and congenital mitochondrial diseases, is dehalogenated by glutathione transferase zeta (GSTZ1)/ maleylacetoacetate isomerase (MAAI), the penultimate enzyme in the phenylalanine /tyrosine catabolic pathway. Polymorphic variations in GSTZ1/MAAI influence the pharmacokinetics of shortterm DCA administration to healthy adults. However, nothing is known of the pharmacogenetics of DCA administered chronically to patients. We determined the GSTZ1/MAAI haplotype in 12 children with primary mitochondrial diseases (age 5.2 ± 1.4 yrs). Patients received 25 mg/kg/day of DCA for 6 mos to 15 yrs. Those lacking the GSTZ1/MAAI wildtype EGT allele showed slowest clearance of DCA. Plasma DCA plateaued over time and did not accumulate. However, plasma DCA trough concentrations at 6 mo and 30 mo correlated directly with urinary accumulation of the heme precursor deltaaminolevulinate (δ-ala) (r2 ≤ 0.71) and the tyrosine intermediate maleylacetone (MA) (r2 ≤ 0.90), with the highest values corresponding to the non-EGT group. We conclude that long-term DCA exposure shows a plasma kinetic profile directly related to GSTZ1/MAAI polymorphism status and to accumulation of the reactive molecules MA and delta-ala. Individuals who biotransform DCA slowly may be at a

1 University of Zagreb, School of Medicine, Zagreb, Croatia; 2Dept of Pediatrics, PMU Salzburg, Salzburg, Austria; 3Department of Pediatrics, UHC Zagreb, Zagreb, Croatia; 4Dept of Path and Cyt, UHC Zagreb, Zagreb, Croatia; 5Clin Inst for Lab Diagnosis, UHC Zagreb, Zagreb, Croatia; 6Dept of Surgery, UHC Zagreb, Zagreb, Croatia; 7Dept of Anesth and IC,UHC Zagreb, Zagreb, Croatia

TRMU mutations cause a deficiency of an enzyme required for the 2thio modification of two mitochondrial tRNAs and lead to a combined respiratory chain deficiency due to a defect in mitochondrial translation. Clinically they present with acute liver failure in early infancy. Patients who survive the acute phase recover liver function and do not experience recurrence of disease. Our patient was a girl who presented with liver failure at an age of 2 months. She presented a severe phenotype with lactate peaked at 28.2 mmol/L and direct bilirubinemia at 362 μmol/L. She recovered after 8 weeks of intensive care when even liver transplantation was considered. In liver tissue a combined deficiency of respiratory chain complexes I, III and IV but normal mitochondrial DNA content was detected and TRMU sequencing revealed a novel homozygous mutation c.44T>G, p.Val15Gly. This case points to the necessity to consider TRMU gene mutations in infants with acute liver

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failure as it is probably more common than reported and to be cautious concerning liver transplantation because this can turn out to be an unnecessary risky procedure. Recognizing patients with TRMU deficiency and identifying factors which influence the prognosis is crucial for optimal management of these patients. P-379 Mutation load in different tissues in six pathogenic mitochondrial DNA (mtDNA) point mutations O'Callaghan MM1, López-Gallardo E2, Montero R2, Buján N3, Jou C1, Emperador S2, Garcia-Cazorla A1, Ruiz-Pesini E4, Briones P3, Artuch R2, Pineda M1, Montoya J4

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Results: All patients presented altered amino acids plasma levels. Multiple MRC deficiency was observed in all cases. Genetic analysis revealed 1 with mtDNA deletion, 1 with mtDNA point mutation, and 4 with mtDNA depletion. Discussion: Elevated plasma alanine and proline levels were observed in four patients. Tyrosine value was elevated in 83.3% of mtDNA depletion cases. According to the literature, mtDNA depletion disease should be considered in subjects with tyrosinemia and without urinary succinylacetone. Decreased arginine was observed in 66.7% of mtDNA depletion cases. This result could be associated with the need of ATP for the argininosuccinate synthase reaction. The findings are interesting, but confirmation of these results in a larger cohort is required. P-382

1

Neurometab dpt, Sant Joan de Déu Hosp, Barcelona, Spain; 2 CIBERER, Instituto de Salud Carlos III, Madrid, Spain; 3Institut Bioquímica Clinica- CSIC, Barcelona, Spain; 4Bioch and Biol Molec dpt, Zaragoza Univ, Zaragoza, Spain Background: Due to heteroplasmy, previously reports have studied m.A3243G mutation load in accessible samples and have found that urine mutation load is higher than mutation load in other tissues. Objectives: To demonstrate that urine is the best diagnosis method in six mtDNA point mutations. Material and methods: We have compared m.A3243G and m.3252A>G (in tRNALeu), m.15923A>G (in tRNAThr), m.13513G>A (in ND5), m.8993T>G and m.9176T>C (in ATPase6) mutation load in blood, urine and buccal mucosa in 7 patients and some relatives and with age. We have classified cases in: possible mitochondrial disorder group and no-possible mitochondrial disorder group (section I of Morava's Mitochondrial Disease Criteria) and we have compared mutation load in different tissues in the two groups. Results: Urine had the highest mutation load in all mtDNA mutations. In some relatives mutation load in blood was undetectable whereas mutation load in urine was detectable. Mutation load in urine had no significant correlation with age and was significantly higher in possible mitochondrial disorder group than in non-possible mitochondrial disorder group. Conclusion: Urine is tissue of choice in a non-invasive molecular diagnosis of mtDNA mutations and would be sample to study the attendant risk of recurrence in offspring of non-carrier-blood asymptomatic mothers. P-381 Relevance of plasma amino acids in mitochondrial respiratory chain disease with liver involvement Veríssimo C1, Simues M1, Mendes C1, Santos MJ1, Pratas J1, Ribeiro C1, Garcia P2, Diogo L2, Grazina M3 1 CNC - Lab of Biochemical Genetics, Coimbra, Portugal; 2CHUC Pediatric Hospital of Coimbra, Coimbra, Portugal; 3Fac Medicine & CNC, Univ of Coimbra, Coimbra, Portugal

Background: Hepatic involvement is a common feature of primary mitochondrial respiratory chain disorders (MRCD), particularly with neonatal presentation. Aim: Investigating the relevance of plasma amino acids in a group of patients with mitochondrial hepatopathies. Subjects and Methods: We reviewed MRC and DNA analysis (in liver tissue) of 6 patients with hepatic MRCD that underwent plasma amino acids evaluation, which was performed by ion exchange chromatography. MRC complexes activity was evaluated by double wavelength spectrophotometry; genetic analysis was performed according to clinic information and laboratory algorithm.

Exome sequencing identifies complex I NDUFV2 mutations as a novel cause of Leigh syndrome Cameron JM1, MacKay N2, Feigenbaum A3, Tarnopolsky M4, Blaser S5, Robinson BH1, Schulze A3 GGB, Sickkids, Toronto, Canada; 2DPLM, Sickkids, Toronto, Canada; Div Clin Met Genetics, Toronto, Canada; 4Dep Pediatrics, McMaster Univ, Hamilton, Canada; 5Dept Radiology, Sickkids, Toronto, Canada 1

3

Background: Two siblings with hypertrophic cardiomyopathy and brain atrophy were diagnosed with Complex I deficiency based on low enzyme activity in muscle and high lactate/pyruvate ratio in fibroblasts. Methods: 89,792 base pair changes or small insertions/deletions were identified by whole exome sequencing of fibroblast gDNA from one sibling. This was narrowed down to 190 candidate genes, by selecting only novel, non-synonymous coding sequence base pair changes. Results: Two compound heterozygous mutations were identified in both siblings in NDUFV2, encoding the 24kDa subunit of Complex I. One mutation is known (c.IVS2+1delGTAA) and one is novel (c.669_670insG, p.224Valfs*3), predicted to cause a pathogenic frameshift in the protein. Neither mutation was identified in control gDNA. The intronic mutation has been identified previously in homozygous form in a single family presenting with hypertrophic cardiomyopathy and encephalopathy. We subsequently identified the same homozygous mutation in a second family with three affected siblings with Complex I deficiency associated Leigh Syndrome, but no cardiac involvement. Conclusion: We have identified NDUFV2 mutations in two families with Complex I deficiency, including a novel mutation. The diagnosis of Leigh Syndrome expands the clinical phenotypes associated with mutations in this gene, which was previously reported in only one case. P-383 Mitochondrial disease phenotypes associated with m.8993t>g/c mutations Pratas J1, Diogo L2, Garcia P2, Martins C3, Mendes C1, Santos MJ1, Simues M1, Duarte J3, Grazina M4 1 CNC - Lab of Biochemical Genetics, Coimbra, Portugal; 2CHUC - Pediatric Hospital of Coimbra, Coimbra, Portugal; 3Garcia Orta Hospital, Almada, Portugal; 4Fac Medicine & CNC, Univ of Coimbra, Coimbra, Portugal

Introduction: Leigh Syndrome (LS) is a major clinical manifestation associated to mtDNA point-mutations. Our aim is to understand the phenotypic expression associated to m.8993T>G/C mutations.

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Description: We present four LS cases, one harbouring m.8993T>C and m.1555G>A mutation and three having m.8993T>G, one female carrier for OTC deficiency. Case 1 – Male, 3 years old, presenting postnatal failure to thrive, breathing problems, delayed developmental milestones, with normal hearing examination, ataxia and respiratory infection and MRI with LS-like pattern. Complex V deficiency (muscle) and m.1555A>G (homoplasmy) plus m.8993T>C (heteroplasmy) were detected. Case 2 – Female, aged 1.5 months, with vomiting and feeding refusal and coma due to sepsis, with persistent lactic acidosis. She was carrier for OTC deficiency (IVS8+1G>A) and had m.8993T>G mutation, with complex II, III (muscle) and V (liver) deficiency. Case 3 – Male, aged 5 months, hypotonic with seizures, hyperlactacidemia and MRI showing bilateral striatal necrosis. Mitochondrial disorder was confirmed with detection of m.8993T>G. Case 4 – Male, 3 years old suspected of NARP/LS. Laboratory screening revealed complex IV deficiency (muscle) and m.8993T>G (blood and muscle). Conclusion: LS presents with clinical variability and genetic heterogeneity. Regardless overlapping of mutations, the energetic deficiency is inevitable and disclosure is often fateful. P-384 Alpers-Huttenlocher syndrome: a case with cerebral folate deficiency and a novel POLG1 mutation 1

1

2

1

1

P-386

1

Öztürk Hişmi B , Ünal Ö , Genç Sel Ç , Dursun A , Sivri S , Coskun T , Ceylaner S3, Tokatlı A1 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Div Ped Neurol,Hacettepe Univ Child Hosp, Ankara, Turkey; 3İntergen Genetics Center, Ankara, Turkey

Alpers-Huttenlocher syndrome (AHS) is an uncommon mitochondrial disease associated with mutations in the mitochondrial DNA replicase, polymerase-gamma. Alterations in enzyme activity result in reduced levels or deletions in mitochondrial DNA. Classical clinical triad of seizures, liver degeneration, and progressive developmental regression helps define the disorder, but a wide range of clinical expression occurs. Here, we present an AHS case with epilepsia partialis continua, rapid psychomotor regression and cerebral folate deficiency but without hepatopathy. A 14-month-old previously healthy boy suddenly developed intractable seizures. In parallel with the developmental regression, progressive cerebral atrophy and lactate peaks in basal ganglia were evident on neuroimaging. CSF analysis revealed elevated protein (146 mg/dl), neopterin (139 μmol/L) and decreased folate (27 μmol/L) levels, folinic acid treatment was started for cerebral folate deficiency. The patient deceased without development of hepatopathy, A novel c.1590delC mutation in one allele and p.W748S and p.E1143G (c.[1590delC]+ p[W748S;E1143G]) mutations located in cis position on the other allele were detected in POLG1. P-385 A novel heteroplasmic frame shift mutation in the COX2 gene leading to severe recurrent rhabdomyolysis in association with viral illness Salvarinova R1, Al-Thihli K2, Dionne J3, Kollman TR4, Waters P5, Langley T6, Shoffner J6, Stockler S1, Vallance HD7 1

Background: Prolonged exercise and fasting are known triggers of acute rhabdomyolysis associated with a variety of metabolic disorders. Here we present a case of viral-induced rhabdomyolysis with an underlying mitochondrial DNA defect. Case report: A previously healthy 6 year old girl presented with severe rhabdomyolysis, acute renal failure, compartment syndrome treated with hemodyalisis and fasciotomies. The episode was attributed to parainfluenza-3 infection (1). After prolonged stay in ICU she recovered with normalization of her kidney function. At age 8, she had a second severe episode in association with influenza A infection. A third episode in association with influenza B was treated aggressively with intravenous fluids, carnitine, coenzyme Q10 and vitamins to which she responded well Results: A fatty acid oxidation defect was excluded with FAO flux in fibroblasts and molecular testing. Sequencing of PFKM, PYGMI, LPIN1 and cytochrome b genes were negative. Mitochondrial DNA sequencing revealed a novel heteroplasmic frame shift mutation in the COX2 gene, 8197-8198delCA, predicted to truncate the COX2 protein. Conclusion: Viral infection can trigger rhabdomyolysis in mitochondrial disease. Mitochondrial DNA sequencing should be considered in patients with recurrent rhabdomyolisis. 1.Ebbeson RL et al Ped Inf Dis 2009;28(9):850-51

Div Metab Dis,Dep Ped, BCCH, UBC, Vancouver, Canada; 2Genetic Developmental Med Clinic,SQUH, Muscat, Oman; 3Div Nephrology, Dep Ped, BCCH, UBC, Vancouver, Canada; 4Div Infect Dis, Dep Ped, BCCH, UBC, Vancouver, Canada; 5CHUS, U of Sherbrooke, Sherbrooke, Canada; 6Medical Neurogenetics, Atlanta, United States; 7 Dep Pathology Lab Med, UBC, Vancouver, Canada

Clinical and biochemical spectrum of six patients with NFU1 associated Multiple Mitochondrial Dysfunction Syndrome (MMDS) Ahting U1, Mayr J2, Santra S3, Rohrbach M4, Engelsberger I5, Spranger S6, Freisinger P7, Haack T8, Prokisch H8, Chakrapani AB 3 , Preece MA 9 , Alston CL 10 , Hempel M 1 , Seidel H 1 , Makowski C5, Plecko B11, Taylor R10, Sperl W2, Meitinger T1, Rolinski B12 Inst Hum Genetics, Technical Univ, München, Germany; 2Dept Pediatrics, Paracelsus Med Univ, Salzburg, Austria; 3Dept Clin Inh Metab Dis, Child Hosp, Birmingham, United Kingdom; 4Dept Metabolism, Univ Cild Hosp, Zürich, Switzerland; 5Dept Pediatrics, Klinikum Schwabing, München, Germany; 6Praxis für Humangenetik, Bremen, Germany; 7Dept Pediatrics, Kreisklinikum, Reutlingen, Germany; 8 Inst Hum Genetics, Helmholtz Center, München, Germany; 9Dept Newborn Screening, Child Hosp, Birmingham, United Kingdom; 10Wellcome Trust Centre for Mitochond Res, Newcastle, United Kingdom; 11Dept Child Neurol, Univ Child Hosp, Zürich, Switzerland; 1 2 Dept Clin Chem, Elblab, Elblandkliniken, Riesa, Germany 1

MMDS can be caused by defects in iron-sulphur-[Fe-S]-cluster synthesis. NFU1 is part of the [Fe-S]-cluster synthesis pathway. Here we present six patients from four families with mutations in NFU1 showing characteristic features. All patients presented with onset in the neonatal or early infantile period and with the following symptoms: muscle hypotonia, failure to thrive, respiratory failure and encephalopathy. In 3 of 4 families (3 of 6 patients) primary pulmonary hypertension was an additional leading symptom. All patients deceased before the age of three years. Severe lactic acidosis and mildly elevated glycine were consistent findings. Combined deficiencies of respiratory chain complexes and pyruvate dehydrogenase were measured in muscle biopsy or fibroblasts. Lipoic acid concentrations were decreased in fibroblasts of 3 patients. Mutation analysis of NFU1 revealed in one patient a known missense mutation in the metal binding motive CXXC. In the other patients a variety of here newly described missense, splicing or nonsense mutations was found.

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Mitochondriopathy due to NFU1 deficiency seems to be associated with a distinct and recognizable phenotype with multiorgan dysfunction, combined respiratory chain / pyruvate dehydrogenase defects and often primary pulmonary hypertension. MMDS should be taken into account in the work up of early onset pulmonary hypertension. P-387 Neonatal severe hypertropic cardiomiopathy and intestinal pseudo-obstruction in TMEM70 mutation: A very favourable course in a 3 years follow-up

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Muscle morphology was abnormal in 42% of MD , 23% of OD and 16% of controls, respectively. Muscle electron microscopy demonstrated abnormalities in 23% and light microscopy in 18% of patients with MD. Electron transport chain activity was defective in 34% (MD), 6.7% (OD) and 8.5% (control). mtDNA analysis revealed abnormalities in 43% (MD), 0% (OD) and 0% (control). Plasma 3-methyl glutaconic acid levels were elevated in 32.4% (MD), 4.2% (OD) and 7.7% (control). We conclude that muscle pathology is the most sensitive test and mtDNA analysis is the most specific test for mitochondrial disease. We conclude that multiple methods of testing may be necessary to make a diagnosis of mitochondrial disease. P-389

Cerutti M1, Sabatini C1, Manzoni F1, Bonarrigo F1, Lamantea E2, Ravazzani V3, Guez S1, Menni F1

Outcome for DGUOK associated liver failure 16.5 years postorthotopic transplantation

1

Feigenbaum A1, Raiman J2, Ling S3, Ng V3, Robinson B4, Siriwardena K2

Ped Dep, IRCCS F.Policlinico, Univ Stud, Milan, Italy; 2UO Mol Neurogenetic IRCCS C.Besta, Milano, Italy; 3Lab. Inborn Errors Metab, AO ICP, Milan, Italy We describe a female born at 33rd weeks ( BW 1,460 g) after a pregnancy complicated at 32 weeks by poor growth, encephalic dysmorphism and oligohydramnios. At delivery lactic acidosis (7 mmol/l), normoglycemia and hyperammonaemia were noted. Echocardiography showed a severe left hypertrophic cardiomyopathy. At 2 days: bowel pseudo-obstruction and pulmonary hypertension. A brain MRI showed frontal pseudocysts, vermis hypoplasia, enlargement of cortical spaces; neurological evaluation demonstrated diffuse hypertone. Plasma amino acids and acylcarnitine were essentially normal, urinary organic acids showed lactic aciduria and mild augmentation of urinary 3-methylglutaconic acid. A skin and muscle biopsy were performed in order to analyze activities of the mitochondrial respiratory chain complexes. In the meanwhile she started riboflavin. We found a low activity of complex V of respiratory chain on fibroblast and a TMEM70 gene mutation (heterozygous mutation IVS2-2 A>G and c.783A>G). This result, reported as a cause of autosomal recessive ATP synthase deficiency, fit for the clinical features of our patient. Contrary to the natural history of the disease the cardiomyopathy and pulmonary hypertension were promptly treated (diltiazem and sildenafil) with improvement. At 3 years we observed: mild left ventricle hypertrophy but normal deambulation and just a little delayed language with a good rehabilitation.

1

Dept of Ped Univ Calif, San Diego, United States; 2Div Clin & Metab Genet, SickKids, U of T, Toronto, Canada; 3Hepatology, SickKids, U of T, Toronto, Canada; 4Genome Biology, SickKids, U of T, Toronto, Canada Background: DGUOK related mitochondrial depletion syndrome can present as isolated liver failure in infancy or with extra-hepatic abnormalities, particularly neurological disease. Liver transplantation in the absence of neurological abnormalities has shown good outcome. To our knowledge only one patient developed neurological symptoms following orthotoptic liver transplantation (OLT) with the longest reported follow-up being 8 years. We report on a patient 16.5 years after OLT to highlight the long-term neurological outcome. Case Report: The male patient presented at 6 months of age with liver failure, underwent OLT at 17 months and was retrospectively confirmed to have mitochondrial depletion and DGUOK mutations. He was monitored regularly with clinical assessments supplemented by formal neurocognitive testing at 9 and 14.5 years of age that included WISC-IV. While patient has non-progressive deficits in attention and verbal skills, neurology and school achievement at 18 years is normal. Assessments including DTPA GFR and echocardiography show normal renal and cardiac functions. Cranial imaging showed a stable, uncomplicated arachnoid cyst. Conclusion: We report on the progress of a patient 16 .5 years following OLT for liver failure associated with DGUOK who remains neurologically well, highlighting this therapeutic option for DGUOK associated liver failure without neurological involvement.

P-388 P-390 Mitochondrial disease: a review of 292 investigated subjects to determine which tests will yield a diagnosis Khan A1, Khan M2, Aman S3, Sarnat H4, Hume S5, Marin-Garcia J6, Nakanishi S7, Snyder F8 1 Alberta Child Hosp, Univ Calgary, Calgary, Canada; 2Fac Sci, Biol Sci, Univ Calgary, Calgary, Canada; 3Univ Alberta, Edmonton, Canada; 4Dep Ped, Path, Clin Neurosci, Univ Calg, Calgary, Canada; 5Genet Lab Serv, Alberta Health Serv, Edmonton, Canada; 6Mol Cardiol Neuromusc Inst, Highland Park, United States; 7Dep Phys Pharm, Univ Calgary, Calgary, Canada; 8Genet Lab Serv, Alberta Health Serv, Calgary, Canada

Mitochondrial diseases are a heterogeneous group of disorders for which no single gold standard diagnostic test has been established. We reviewed the records on 2041 patients in the Metabolic Clinic at Alberta Children's Hospital and recovered 292 patients who had a clinical suspicion of mitochondrial disease. Of these patients, 49.5% were female and 65.8% were children. We compared 3 groups: those with a diagnosis of mitochondrial disease (MD) (n=102, 34.7%), those with another diagnosis (OD) (n=71, 23.8%) and controls that did not have a diagnosis (n=118, 40.8%).

ALDH18A1 mutations presents with severe generalized cutis laxa and retinitis pigmentosa van Asbeck EV1, Wolthuis DFGJ1, Mohamed M2, Gardeitchik T2, Morava E1 1

Dep of human genetics,Tulane University, New Orleans, United States; Dep of peds, Radboud UMC Nijmegen, Nijmegen, Netherlands

2

Autosomal recessive cutis laxa is characterized by wrinkled, inelastic skin, in association with a multisystem disease. Recently, the mitochondrial genes PYCR1 and ALDH18A1 were implicated in the etiology of different progeroid syndromes. Both are involved in mitochondrial proline synthesis and can cause De Barsy Syndrome, also labeled as autosomal recessive cutis laxa type III. Patients with ARCL3 have parchment-like skin, prominent veins, corneal opacities, joint abnormalities and athetoid movements. We performed targeted next generation sequencing in genetically unsolved patients with progeroid features, neurological and eye involvement to assess the underlying genetic etiology. One of our patients was diagnosed with a novel, homozygeous

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nonsense mutation in exon 18 of the ALDH18A1 gene. Our patient had dysmorphic facial features, generalized sagging skin instead of parchment-like skin, in association with joint contractures and severe neurological symptoms, comparable to the cutis laxa described in congenital glycosylation defects. Additionally the patient had spasticity, contractures, decreased vision, salt and pepper retinopathy and deceased before the first year of age. Contrary to the common presentation in patients with ALDH18A1 mutations, our patient showed no metabolic abnormalities. ALDH18A1 mutations have been only described in 7 patients. This is the first report of retinitis pigmentosa in ALDH18A1 defect.

09. Other disorders of energy metabolism (creatine, krebs cycle, pyruvate dehydrogenase, lipoic acid, thiamine) P-391 Impact of cardiolipin deficiency on cardiac mitochondria in a mouse model of Barth syndrome

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Malonyl-CoA decarboxylase (MLYCD, EC 4.1.1.9) is an enzyme that regulates cellular malonyl-CoA levels and maintains the balance between fatty acid and glucose metabolism. Inherited mutations in the MLYCD gene lead to a rare but severe metabolic disorder (OMIM 248360) characterized by malonic aciduria, developmental delay and cardiomyopathy. Current understanding of this crucial enzyme and its pathogenic mutations has been hampered by a lack of biochemical and structural characterization. Therefore, we have produced recombinant human MLYCD and solved its structure at 2.8 Å resolution. Mapping patient mutations on the structure reveals no localized hot-spot regions, but allows the categorization of each substitution into defect on protein localization (e.g. G3D and M40T), folding (e.g. A69V and L161P) or catalysis (e.g. S290F and Y456S). The catalytic mutations lie in the protein core that shows unexpected structural homology with the GCN5-related N-acetyltransferase (GNAT) family. Replication of these mutations in vitro for kinetic studies validates their proposed defects and further identifies Ser329 and His423 as essential catalytic residues. Further analysis illuminates catalytic and pantetheine-moiety binding regions which may be a starting point for structure guided therapeutic design. Together, these data reveal the molecular basis of malonic aciduria and point toward therapy.

Khuchua Z1, Huang Y1, Powers C1, Strauss A1 P-393 1

CCHRF and University of Cincinnati, Cincinnati, United States

Background: Cardiolipin is a unique mitochondrial phospholipid essential for maintaining proper cristae architecture and optimal organellar and enzymatic functions. Cardiolipin-deficiency in humans results in a multisystem pediatric disorder, Barth syndrome (BTHS), and is caused by mutations in the tafazzin gene on the X-chromosome. BTHS patients and tafazzin-deficient mice develop dilated cardiomyopathy and exercise intolerance. Objectives: To investigate molecular mechanisms that underlie the development of cardiomyopathy in BTHS, we analyzed metabolic rates, mitochondrial enzymatic activities and the mitochondrial proteome in tafazzin knockdown (Taz-KD) mice. Methods: Metabolic rates were assessed with indirect calorimetry. Two-dimensional difference gel electrophoresis was used to investigate changes of proteomic landscape in cardiac mitochondria. Enzymatic activities were measured in neonatal cardiomyocytes and isolated mitochondria. Results: Oxygen consumption rates during the exercise were markedly impaired in Taz-KD mice. Taz-deficient cardiomyocytes showed significantly diminished maximal mitochondrial respiration rates. Complex III activity was reduced by 45% in CL-deficient mitochondria. Tafazzin gene inactivation reduced abundances of Rieske and cytochrome c1 subunits of complex-III. CL-deficiency diminished binding of myoglobin to mitochondrial membrane. Conclusion: Mitochondrial complex-III deficiency in combination with diminished oxygen delivery to mitochondria by myoglobin may be major pathogenic factors in development of cardiomyopathy in BTHS patients.

The interplay between protein stability and folding co-operativity in loss-of-function genetic disease: The case of human PGK1 deficiency Pey AL1, Mesa-Torres N1, Chiarelli LR2, Valentini G2 1 Dept Phys Chem, Univ Granada, Granada, Spain; 2Dept Biol & Biotech, Univ Pavia, Pavia, Italy

Background: many loss-of-function genetic diseases are caused by defects in protein stability and foldability. Here, we present a strategy based on a combination of proteolysis, thermal and chemical denaturation studies to explore at the molecular level the structural and energetic bases of loss-offunction using human phosphoglycerate kinase 1 (hPGK1) as a model system. Results: Thermal denaturation analyses revealed that mutations causing hPGK1 deficiency induced kinetic destabilization of hPGK1 (i.e. up to 100000-fold lower denaturation half-lives). Kinetic destabilization is caused by strong effects on the structure/energetics of the (high-energy) denaturation rate-limiting states rather than on the native state. Chemical denaturation by urea and proteolysis studies support that the origin of this destabilization is a decrease in folding cooperativity (i.e. higher population of partially folded states prone to aggregation and proteolysis). Native state kinetic stabilization mediated by natural ligands is shown to be mutant- and ligand-selective. Conclusions: loss-of-function in hPGK1 deficiency can be explained by reduced protein kinetic stability and folding co-operativity, thus supporting therapeutic approaches targeting native (i.e. kinetic stabilizers) or partially folded (i.e. molecular chaperones) states for this disease. The strategy presented here can be useful to dissect mutational effects on protein foldability for other genetic loss-of-function diseases.

P-392 P-394 Molecular basis of malonic aciduria as explained by the crystal structure of malonyl CoA decarboxylase Froese DS1, Vollmar M1, Puranik S1, Cannone G2, Savitsky P1, Krojer T1, Pilka ES1, Lee WH1, Marsden BD1, von Delft F1, Allerston CK1, Spragnolo L2, Gileadi O1, Oppermann U3, Yue WW1 1

Structural Genomics Consort, Univ Oxford, Oxford, United Kingdom; 2 Inst Struct Molec Bio, Univ Edinburgh, Edinburgh, United Kingdom; 3 Botnar Res Cen, NIHR Oxford Biomed Res U, Oxford, United Kingdom

Targeting every cell type of cns by AAVS to develop a model of GAMT deficiency in 3D organotypic brain cell cultures Hanna-El-Daher L1, Béard E1, Henry H1, Tenenbaum L2, Braissant O1 1 Biomedicine, University Hospital, Lausanne, Switzerland; 2Clin Neurosci, University Hospital, Lausanne, Switzerland

Adeno-associated viruses (AAVs) are attractive for gene delivery to CNS due to low toxicity and stable expression properties. Several

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serotypes allow cell- and developmental stage-specific expression depending on their tropism. Aiming at developing a new GAMT deficiency model by RNAi, we determined which AAV serotype was able to transduce every brain cell type of 3D rat organotypic brain cell cultures, thereby improving creatine deficiency by minimizing the proportion of cells not affected by GAMT RNAi. 3D brain cell cultures were transduced at DIV0 by AAV-2, 5, 8 and 9 expressing EGFP. AAV2 was the most efficient, EGFP being increasingly expressed from DIV 8 to 28. EGFP co-labelling with cell-specific markers demonstrated that AAV2 was able to transduce neurons, astrocytes and oligodendrocytes with no toxic effect. AAV2 was further used to transduce a GAMT-specific shRNA, leading to 85% knock-down of GAMT protein, creatine deficiency and guanidinoacetate accumulation, and increase in brain cell apoptotic rate. AAV2 appeared the most efficient for RNAi delivery in 3D brain cell cultures, leading to stable and efficient expression of GAMT shRNA in neurons, astrocytes and oligodendrocytes. This new AAV2-RNAi model will help understand GAMT deficiency neuropathogenesis and creatine deficiency effects on developing brain cells. P-395

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BCKADH). E3 deficiency causes a combined defect, affecting pyruvate, BCAA and TCA cycle. The few reported patients presented hypotonia, Leigh's encephalopathy, psychomotor retardation, and microcephaly. Lactic acidosis along with elevated plasma leucine, isoleucine, valine and increased urinary excretion of lactic, pyruvic, 2-ketoglutaric, and branched-chain 2-hydroxy- and 2-keto acids are the biochemical hallmarks of E3 deficiency. One Ashkenazi-Jewish patient, carrying the founder mutation G229C, presented a riboflavin-, coenzymeQ-, biotin-, and carnitine-responsive Reye-like picture with muscle weakness and wasting. Case report/methods/results: A 15 year-old patient - presenting since infancy with hepatomegaly and recurrent episodes of drowsiness with ketonuria - showed progressive exertional fatigue (exercise-test interrupted after 1 minute). Metabolic investigations displayed a characteristic E3 deficiency profile, including persistent elevation of plasma alloisoleucine. Muscle biopsy showed ragged-red fibers; in isolated muscle mitochondria the pyruvate, 2-ketoglutarate and BCKAs oxidation was reduced to 30%. Two heterozygous mutation were found in the DLD gene [c.118+1G>A(p.I40LfsX4)/c.1382G>A(p.G461E)]. Riboflavin supplementation (220 mg/day) caused complete resolution of exercise intolerance and disappearance of muscle ragged-red fibers. Conclusions: This report highlights a new DLD mutation which reducing the E3 affinity for FAD causes a benign, riboflavin-responsive myopathy.

Whole exome sequencing reveals novel compound heterozygote mutations in SLC19A3 causing biotin-thiamine responsive basal ganglia disease (BTBGD)

P-397

Abdenur JE1, Sremba LJ1, Chang R1, Elbalalesy N2

Prevalence of Creatine Deficiency Syndromes (CDS) and impact of genotyphic variability of CDS genes in children with autism spectrum disorder (ASD)

Div Metab Dis, Child Hosp of Orange Co, Orange, United States; 2Div Neuro, Child Hosp of Orange Co, Orange, United States 1

Background: Biotin-Thiamine Responsive Basal Ganglia Disease (BTBGD) is a rare, under diagnosed autosomal recessive condition caused by mutations in the SLC19A3 gene, which encodes a thiamine transporter. BTBGD presents with subacute encephalopathy; progressing to dystonia, quadriparesis and death when not treated with biotin and/or thiamine. Objectives: To report clinical, biochemical, radiologic, and molecular findings on a patient with BTBGD. Case Report: Our patient was born to non-consanguineous, mixed ancestry parents and presented at 6 weeks with intractable seizures with burst-suppression EEG pattern. MRI findings were consistent with Leigh syndrome leading to a suspected diagnosis of mitochondrial disorder. Over time, the disease resulted in intellectual disability, absent speech, truncal hypotonia, spasticity and microcephaly with progression of basal ganglia involvement and cortical atrophy. Extensive workup failed to identify the aetiology of the Leigh syndrome, but subsequent whole exome sequencing revealed two heterozygous novel mutations, p.S26fs and p.M28fs, in the SLC19A3 gene. Discussion: We present a case of BTBGD with two novel deleterious mutations. This case highlights the clinical importance of considering SLC19A3 gene defects as part of the differential diagnosis for Leigh syndrome, given that early supplementation with biotin and/or thiamine can improve the devastating course of the disease. P-396

Schulze A1, Roberts W1, Scherer S1, Anagnostou E2, Tsai ACH3, Reynolds A3, Bauman M4, Loh A5 1

Hosp for Sick Children, Univ of Toronto, Toronto ON, Canada; 2Bloorview Research Institute, Toronto ON, Canada; 3Colorado Univ Medical School, Aurora CO, United States; 4Lurie Center for Autism MGH, Boston MA, United States; 5Surrey Place Centre, Univ of Toronto, Toronto ON, Canada Background: Creatine deficiency may play a role in the neurobiology of autism and may represent a treatable cause of autism. Methods: (I) Prospective enrolment of children from three autism centres after confirmed diagnosis of ASD. Random urine screening for creatine metabolites with LC-MS/MS, second tear test with HPLC, re-call testing in 24-hour urine, and confirmation testing including plasma metabolites, mutation analysis, and in vivo brain MR spectroscopy to ascertain the prevalence of CDS in ASD. (II) Sanger-based DNA sequencing of GAMT, GATM, and SLC6A8 in retrospective and prospective sample of children with ASD to assess genotypic variability in autism. Results: (I) The final result was negative in all of the 442 subjects enrolled; 418 were negative in screen (398) or second-tier test (20), 12 in recall test, and 12 in confirmation testing. (II) In 322 GATM- and GAMT- and 189 SLC6A8 alleles from 161 subjects (133 m, 28 f), we found novel variants/mutations in 7 alleles (2 GATM, 3 GAMT, 2 SLC6A8) and a novel GAMT haplotype in 16 individuals. Conclusion: The chance is low that a child primarily diagnosed with ASD has an underlying CDS. Preliminary investigation of genetic variability revealed no association between creatine metabolism genes and autism.

E3 deficincy a novel cause of riboflavin responsive myopathy P-398 Martinelli D1, Fiermonte G2, Rizzo C1, Carrozzo R1, Vozza A2, Torraco A1, Parisi G2, Goffredo BM1, Boenzi S1, Bertini ES1, Dionisi-Vici C1 1 Bambino Gesù Children's Hospital IRCCS, Rome, Italy; 2Dept. Pharmaco-Biology, Bari University, Bari, Italy

In vitro study on the responce of fibroblast cellular respiration to lipoic acid, thiamine and carnitine in patients with lipoamide dehydrogenase deficiency Al-Jasmi F1, Al Shamsi A1, Penefsky H1, Souid A1

Background: E3 (dihydrolipoamide dehydrogenase, DLD) is a flavoprotein common to all alpha-ketoacid dehydrogenases (PDH, 2-KGDH,

1

United Arab Emirates University, Al Ain, United Arab Emirates

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This in vitro study was designed to examine the response of cellular respiration to lipoic acid, thiamine and carnitine in patients with lipoamide dehydrogenase deficiency. Cultured fibroblasts from three patients were treated with these compounds for 24 hr and mitochondrial O2 consumption was measured by a phosphorescence oxygen analyzer. Patient 1 & 2 were severely symptomatic infant with novel homozygous c.1436A>T mutation in the DLD gene. The rate of respiration for patient 1 (kc, mean ±SD, in μM O2 min-1 per 107 cells) without additions was 14.9 ±1.0, with 10 μM lipoic acid was 13.8±0.7, with 50 μM lipoic acid was 16.3±0.4, and with 100 μM lipoic acid was 17.9±1.2. The value of kc for patient 2 without additions was 4.9±0.9, with 233-466 μM thiamine was 6.7±0.8 and with 100-200 μM carnitine was 7.7±0.07 μM O2 min-1 per 107 cells. Patient 3 was mildly symptomatic adolescent male with homozygous c.685G>T mutation. The fibroblast PDH activity was normal. The value of kc without additions was 15.3±1.5, with 466 μM thiamine was 15.3 and with 200 μM carnitine was 21.0μM O2 min-1 per 107 cells. Thus, fibroblast cellular respiration improved with thiamine, carnitine and lipoic acid treatments. P-399 A c.545+5G>A NFU1 splice site mutation leads to a particular biochemical phenotype Ferrer-Cortès X1, O'Callaghan M2, Pineda M2, Narbona J3, Montero R2, Bujan N1, Del Toro M4, Arranz JA4, Riudor E4, Alston CL5, Taylor RW5, Briones P6, Ribes A7, Tort F7 Hospital Clínic, IDIBAPS, Barcelona, Spain; 2Hospital Sant Joan de Deu, CIBERER, Barcelona, Spain; 3Clinica Univ Navarra, Fac Med, Pamplona, Spain; 4Hospital Vall d'Hebron, UAB, Barcelona, Spain; 5Wellc Trust Cent Mit Res,Newcastle Univ, Newcastle, United Kingdom; 6Hospital Clínic, IDIBAPS, CSIC, Barcelona, Spain; 7 Hospital Clínic,IDIBAPS,U737-CIBERER, Barcelona, Spain 1

Background: Pyruvate dehydrogenase complex(PDHc) activity and other lipoic acid-dependent enzymes were low in NFU1 patients, as well as in other defects involved in lipoic acid biosynthesis. Here we report two compound heterozygous individuals (c.[622G] >T;c.[545+5G>A]) with normal PDH activity and substrate oxidation rates. Our objective was to understand the molecular bases for the different biochemical phenotypes observed in NFU1 defects. Materials and Methods: Fibroblasts from patients carrying homozygous and compound heterozygous mutations were analyzed for protein-bound lipoic acid by immunoblotting. PCR was used to investigate mRNA levels and splicing defects. Substrate oxidation rates, PDH and respiratory chain activities were also analyzed. Results: Protein-bound lipoic acid was completely absent in homozygous patients. In contrast, compound heterozygous individuals showed very low, but detectable, levels of lipoylated proteins. Molecular studies demonstrate a peculiar regulation of the mutated splice site in compound heterozygous patients, which produces residual levels (up to 10%) of wild type NFU1 protein. This might be enough to partially lipoylate and restore the activity of some lipoic acid-dependent enzymes. Conclusion: Our study provides new insights into the molecular bases of NFU1-related disease. These observations should be considered to ensure a proper diagnosis and should be taken into account to understand the biochemical data in these patients.

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P-400 Continuous age-corrected reference ranges of urinary markers for cerebral Creatine Deficiency Syndromes (CDS) Mxrkrid L1, Elgstxen KB1, Woldseth B1, Olesen JH2, Ruiter GJG3, Tortorelli S4, Hall P4, Rinaldo P4 1

Sect IEM Dep Med Biochem, Oslo Univ Hosp, Oslo, Norway; 2Dept Clin Genet, Rigshospitalet, Copenhagen, Denmark; 3Dept Clin Genet, Erasmus Med Center, Rotterdam, Netherlands; 4Biochem Genet Lab, Mayo Clinic, Rochester, Minneosta, United States Background: Age strongly influences urine concentrations of creatine and guanidinoacetic acid, as well as their ratios to creatinine, which are used in screening for CDS. Objectives: To develop an age continuum of reference ranges by mathematical modeling after appropriate transformations of the disease markers (Box-Cox transformations). Material and Methods: Results from 8,897 reference individuals without known CDS were combined from four diagnostic laboratories. Quantile regression was used to estimate central tendencies and dispersions as a function of age, by which each patient value could be converted to a Z-score. Results: The different sites exhibited similar age behaviour and agreed that existing discrete reference limits result in frequent occurrences of both false positives and false negatives, e.g. in the screening of SLC6A8 deficiency we found false positive rates > 10% in the age range 1 – 4 years, and with GAMT defects negative false rates were likely to occur in newborns in the age range 2 – 14 years. Conclusion: The conversion to Z-scores of patient values is better adapted to the normal biological variation with age and is equivalent to the use of paediatric growth charts. Broad international cooperation is desirable to estimate reference percentiles with sufficiently high precision. P-401 Creatine derivatives in the treatment of creatine transporter deficiency in human cell lines Pajares S1, Arias A1, García-Villoria J1, Fons C2, Campistol J2, Ribes A1 1

Hospital Clinic, CIBERER, IDIBAPS, Barcelona, Spain; 2Hospital Sant Joan de Déu, CIBERER, Barcelona, Spain Background: Some creatine (Cr) derivatives have shown to improve the cellular Cr content in mouse brain slices which cross the cellular membranes in a transporter-independent way. Objectives: Our aim was to study the cellular Cr uptake of Cr derivatives in human cell lines. Materials and methods. The intracellular increase of Cr under Phosphocreatine-Mg-complex acetate (PCr-Mg), Cr benzyl ester (CrOBzl) and gold Cr nanoparticles (goldCrN) were tested and compared with Cr treatment. The material used was fibroblasts from 3 patients with Cr transporter deficiency, HUVEC and HBMEC cell lines with Cr transporter silenced with siRNA. Results and discussion; When compared with untreated fibroblasts, intracellular Cr increased 2-fold with gold CrN in all 3 patients, 7fold with PCr-Mg in 2 patients and 4-fold with CrOBzl in 1 patient. These increases were not significantly higher than those found with Cr treatment. The siRNA silencing in both HUVEC and HBMEC cell lines was successful; mRNA and protein inhibition were around 90% and 30%, respectively. Treatment with the same 3 compounds resulted in an intracellular Cr increase around 1

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fold in HBMEC cells, while in HUVEC cells the increase was 1.5fold after CrOBzl. Conclusion: Cr derivatives did not improve significantly Cr uptake.

Conclusion: Out of 1600 urine metabolic screens, we identified one patient with GAMT deficiency and two patients with SLC6A8 defect. Our method led to an earlier diagnosis of these patients.

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P-404

Biochemical and genetic data of thirteen patients with fatal infantile encephalopathy caused by mutations in NFU1 gene

Pyruvate carboxylase deficiency: an underappreciated cause of lactic acidosis ?

Pérez-Cerdá C1, Ferrer I1, Navarrete R1, García F1, Oyarzábal A1, Ugarte M1, Rodríguez- Pombo P1

Habarou F1,2,3, , Brassier A1, Rio M4, Chrétien D5, Monnot S45, Barouki R1,2,3, , Bonnefont JP4y5,6, Chadefeaux-Vekemans B,1,2,3, Le Moyec L7, Ottolenghi C1,2,3, de Lonlay P1,5

1

CEDEM.CBM.CIBERER. Univ Autonoma Madrid, Madrid, Spain

Mutations in NFU1 have been recently identified in patients with a multiple mitochondrial dysfunction syndrome. NFU1 encodes for a conserved protein involved in the pathway of the iron-sulfur cluster biogenesis, which acts as sulfur donor for the biosynthesis of lipoic acid. We present biochemical and genetic data of thirteen NFU1 deficient patients from nine families with different origins. First symptoms mostly appeared at 2-4 months of age. All patients showed failure to thrive, neurological deterioration and death occurred between 3-17 months after a fatal respiratory failure. In 7/ 13 a primary pulmonary hypertension was demonstrated. As biochemical hallmark of the disease in this cohort, increased levels of glycine in blood and LCR were detected. Other common biochemical data observed were lactic acidemia (9/12), elevated LCR lactate (4/8), and elevated urine glycine (10/13), 2-OHAdipic acid (10/13), 2-ketoadipic acid (8/13), lactic acid(6/13), alpha-aminoadipic acid (6/13) and alpha-ketoglutaric acid (6/13). Molecular analysis performed in 7/9 families identified 16 alleles carrying the previously described missense mutation p.Gly208Cys. Patient having the largest survival was a compound heterozygous for this common change and the splice variant c.545+5G>A. Our data expands the biochemical phenotype, increasing the understanding of this disease, whose pathophysiological mechanism remains to be unravelling. P-403 Extended urine metabolic screen including creatine and guanidinoacetate Wu JY1, Henman M1, McGill J2, Coman D2, Bowling FG3, Cowley DM1 1

Dept Clin Chem, Mater Hosp, South Brisbane, Australia; 2Dept Metab Med, Royal Children's Hosp, Herston, Australia; 3Dept Biochem Dis, Mater Children's Hosp, South Brisbane, Australia Background: At present our urine metabolic screen includes urinary dipstick, amino acids, organic acids and mucopolysaccharide quantification and electrophoresis. A large proportion of our screening population are being investigated for developmental delay and/or seizures. The creatine deficiency syndromes include the creatine synthesis defects, arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) deficiency and the creatine transporter (SLC6A8) defect. SLC6A8 alone is the cause of mental retardation in 1.4% of males. Objectives: Due to the significant prevalence of SLC6A8, we validated a LC/MSMS method to analyse creatine and guanidinoacetate as well as other metabolites currently not tested in our urine metabolic screen. Methods: Urine samples are diluted with 2% acetonitrile/0.05% formic acid. An isotope- labelled internal standard is added. Samples are vortexed and injected onto a reversed phase C18 HPLC column and monitored by ESI MS/MS using specific ion transitions (MRMs). All urine metabolic screen requests had this testing performed. Patients outside the literature reference intervals and the 99th percentile of our testing population were followed up.

1 IEM Reference Center, Necker Hosp, APHP, Paris, France; 2INSERM U747 Descartes Univ of Paris, Paris, France; 3Metab Biochemistry, Necker Hosp, APHP, Paris, France; 4Genetic Department, Necker Hosp, APHP, Paris, France; 5INSERM U781, Descartes Univ of Paris, Paris, France; 6Ped Radiol Department, Necker Hosp, APHP, Paris, France; 7INSERM U902, Univ of Evry, Evry, France

Background: Pyruvate carboxylase (PC) is a biotin-containing mitochondrial enzyme that is involved in gluconeogenesis and anaplerosis of the Krebs cycle. We report on a new patient affected by the moderate form (the American type A) of PC deficiency. Results: The patient presented with severe lactic acidosis and pronounced ketonuria, associated with lethargy at age 23 months. Mental retardation was noted at this time. Standard metabolic screens were not informative. Complex IV deficiency was initially detected in skeletal fibroblasts. Subsequently, we demonstrated PC deficiency with no detectable activity and normal biotinidase activity in skeletal fibroblasts. Another patient previously reported with the severe form of PC deficiency also had a secondary CIV deficiency in fibroblasts. Different anaplerotic treatments had no clinical nor biological effects in vivo or in vitro (citrate, aspartate and oxoglutarate). Conclusion: PC deficiency was revealed as the revision of an initial diagnosis of mitochondrial complex IV defect. This observation illustrates the difficulty to manage energy metabolism disorders in terms of diagnostic work-up and therapeutical strategy therapy. P-405 Severe lactic acidosis with mild methylmalonic aciduria and increased succinylcarnitine in body fluids points to a mitochondrial DNA depletion associated to a succinyl-CoA ligase deficiency Ruiz-Sala P1, Ferrer-López I1, Delgado-Pecellín C2, Martín MA3, Blázquez A3, Pérez B1, Pérez-Cerdá C1, Merinero B1, Ugarte M1 1 CEDEM, CBM, CIBERER, IDIPAZ, UAM, Madrid, Spain; 2Hospital Vírgen del Rocío, Sevilla, Spain; 3Hospital 12 de Octubre, Madrid, Spain

Succinyl-CoA ligase is a Krebs cycle enzyme that turns succinyl-CoA + GDP/ADP into succinate + GTP/ATP. These nucleotides are essential in the mitochondrial DNA (mtDNA) replication. In addition, succinylCoA is the product of methylmalonyl-CoA mutase activity in the metabolism of some branched-chain amino acids, odd-chain fatty acids and cholesterol. We present a patient admitted, at 6 days of age, with shock, severe metabolic acidosis and hyperammonemia. Expanded neonatal screening showed increase of acylcarnitines, especially C3. He died at 8 days. Organic acid analysis in urine showed elevated excretion of lactic, 2OH-butyric, methylmalonic and 4-OH-phenyllactic acids. In the analysis of serum and urine acylcarnitines, increases of C3 and C4DC were found. Separation of C4DC isomers by HPLC/MS/MS demonstrated the

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increase of succinylcarnitine levels with normal methylmalonylcarnitine. These biochemical and fatal multisystemic clinical profiles suggested a deficiency in succinyl-CoA ligase. Two mutations in SUCLG1 gene were identified: one previously reported (c.626C>A) and the other new but probably severe (c.825+ 1G>A). The study of mtDNA depletion demonstrated a significant decrease of the number of copies of mtDNA in brain and muscle. We want to emphasize the need to differentiate the C4DC isomers to demonstrate the increase of succinylcarnitine instead of methylmalonylcarnitine. P-406 Decreased thiamine monophosphate in cerebrospinal fluid is a diagnostic tool for SLC19A3 defects Molero-Luis M1, Ortigoza JD1, Hernández M1, Tondo M1, Serrano M1, Artuch R1, Pérez-Dueñas B1

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Results: The psychomotor development of this child was normal up to 14 months old; then she started to regress. The course of the disease was marked by two episodes of metabolic acidosis at 5 and 18 months of age. Brain magnetic resonance imaging (MRI) revealed progressive leukoencephalopathy with extensive signal abnormalities in the periventricular cerebral white matter and in the corpus callosum. Elevated levels of glycine in plasma, urine and CSF were consistent with a defect in lipoic acid metabolism. Support for the diagnosis was provided by pyruvate dehydrogenase (PDH) deficiency and multiple mitochondrial respiratory chain (MRC) deficiency in skin fibroblasts, as well as no expression in fibroblasts by western blot. Two mutations in the NFU1 gene confirmed the diagnosis. Conclusion: We confirm that specific radiological and biochemical symptoms are highly suggestive of dysfunctions involving lipoic acid metabolism. The p.Gly208Cys mutation has previously been reported suggesting a founder effect in Europe and a potential screening test.

1

P-408

Background: SLC19A3 defects cause acute and recurrent encephalopathy that is amenable to thiamine treatment. Determination of thiamine metabolites (thiamine-diphosphate (TDP) and thiamine-monophospate (TMP)) in biological fluids could be a sensitive biomarker for early diagnosis. Objectives: To establish reference values in whole-blood and cerebrospinal fluid (CSF) for TMP and TDP in a pediatric population and to compare them with those of children with SLC19A3 defects. Methods: 177 and 33 children (age range: 1 day-16 years) were analyzed by HPLC-fluorescence detection in whole blood and CSF, respectively. Two patients with confirmed SLC19A3 defects (Patient 1: 16 years-old; patient 2: one month-old) were also studied. Results: Whole-blood TDP and CSF TMP showed negative correlation with age. Therefore different reference intervals were established for both fluids. In CSF, median TMP concentrations were 34.3 nmol/L (1day-2years) and 23.1 nmol/L (>2years). Patient 1 did not show blood TDP and TMP deficiency, but CSF TMP concentrations disclosed low values (14.8 nmol/L). Patient 2 had also lower CSF TMP concentrations (10.35 nmol/L). Conclusion: Whole-blood thiamine analysis may show false negative results. However, the quantification of TMP in CSF seems a sensitive diagnostic tool for SLC19A3 defects. These findings support the hypothesis that SLC19A3 defects cause cerebral thiamine transport deficiency.

Case presentation: Sjvgren-Larsson syndrome

Backround: Sjögren–Larsson syndrome (SLS) is a neurometabolicneurocutaneous disease characterized by ichthyosis, mental retardation and spastic diplegia-quadriplegia. Case report: A 5 years old female presented with developmental delay. Since her birth, she had dry skin and severe developmental delay. No history of seizure was present. Physical examinations showed a body weight of 18,7 Kg(50-75p), height of 109cm (50p), head circumference of 52 cm (50-97p), generalized ichthyosis and spastic diplegia. She was able to sit without support, could step with help. Her routine laboratory results and cranial MRI at two years of age was normal. A prospective diagnosis of SLS was thought based on clinical findings. Cranial MRS showed a lipid peak suggestive of SLS. DNA sequencing analysis showed a homozygous mutation c.941-2A>G which results in splice site defect in FALDH gene. Conclusion: Clinical findings with developmental delay and ichthyosis, SLS should be thought for the differential diagnosis.

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P-409

Leukoencephalopathy with cysts and hyperglycinemia may result from NFU1 deficiency

A rapid sensitive underivatised msms method for determination of guanidinoacetate, creatine and creatinine in human fluids

Nizon M1, Boutron A2, Boddaert N3, Slama A2, Delpech H4, Sardet C4, Brassier A1, Habarou F5, Delahodde A6, Correia I2, Ottolenghi C5, de Lonlay P1

Krywawych S1, Ifederu A1, Wigley R1, Heales S1

Div Metab Dis, Univ Paris V, Hosp Necker, PARIS, France; 2Div Biochem, Hosp Bicêtre, LE KREMLIN BICETRE, France; 3Div Ped Radio, Univ Paris V, Hosp Necker, PARIS, France; 4Div Mol Genet, CNRS UMR 5535, MONTPELLIER, France; 5Div Biochem, Univ V, Hosp Necker, PARIS, France; 6Paris-Sud Univ, CNRS UMR 8621, ORSAY, France

Background: The two enzyme defects in the synthesis of creatine cause low creatine concentrations and are associated with neurological clinical presentations. These disorders are diagnosed biochemically by the abnormal relative concentrations of guanidinoacetate and creatine in plasma and urine. Generally LC-MSMS or MSMS is the favoured method for analysis of derivatives of the above metabolites or LCMSMS for the underivatised metabolites. Objectives: To develop a simple sensitive rapid MSMS method for the determination of guanidinoacetate, creatine and creatinine in plasma, blood, CSF and urine. Methods: Quantitation is based on a seven point calibration curve using stable isotope internal standards for each metabolite. Neat or aqueous

Hospital Sant Joan de Deu, Esplugues Llobregat, Spain

1

Background: Lipoic acid metabolism defects are new metabolic disorders that cause neurological, cardiomuscular or pulmonary impairment. We report on a patient that presented with progressive neurological regression suggestive of an energetic disease, involving leukoencephalopathy with cysts.

Kilic E1, Kilic M2, Utine GE1, Coskun T2, Nakano H3, Boduroglu K1 1 Hacettepe Univ, Pediatric Genetic Unit, Ankara, Turkey; 2Hacettepe Univ, Pediatric Metab Unit, Ankara, Turkey; 3Hirosaki Univ, Dept of Dermatol, Hirosaki, Japan

1

Dept Chem Path, Great Ormond Street Hosp, London, United Kingdom

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diluted biological specimens are analysed in positive ionisation mode without a separating column. Results: Analysis time was 3.5 minutes requiring a maximum total volume of 5 microlitres of urine or plasma of which 0.0375% was injected. Linearity for guanidinoacetate was shown up to 0.5 mmol/L, for creatine to 1.0 mol/L and for creatinine to 2.0 mmol/L. Recoveries ranged from 86% to 115% and intrabatch CV ranged from 1.7 to 11.7 over a series of concentrations. A comparison of the concentrations of the three metabolites was made between bloodspots, blood, plasma, CSF and urine.

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Materials and Methods: A six year-old boy with glutathione synthetase deficiency underwent an ophthalmological examination including full-field electroretinography (ERG) and optic coherence tomography imaging (OCT). Results: The complete ophthalmological evaluation revealed hyperopia as refractive error with a best corrected visual acuity of 20/40 bilaterally. Full field ERG had low rod and cone response with subnormal oscillatory potentials. Fundus autofluorescence obtained by Spectralis OCT showed perifoveal increased autofluorescence bilaterally. Conclusion: Glutathione synthetase deficiency may be associated with ophthalmic findings including hyperopia, retinal photoreceptor dysfunction and altered fundus autofluorescence pattern.

P-410 P-412 Fumarate hydratase deficiency in Ireland -the importance of carrier detection

An atypical form of creatine deficiency syndrome with dystonia

Fitzsimons PE1, Borovickova I1, Trench C1, Durkie M2, Tops B3, Hughes J4, Monavari AA4, Mayne PD1

Nasrallah F1, Khemir S1, Hammami M B1, Ben Rhouma H2, Feki M1, Khouja N2, Briand G3, Kaabachi N1

1 Dept Clin Bio, Temple St Child Univ Hosp, Dublin, Ireland; 2Dept Clin Genet, Sheff Child Hosp, Sheffield, Ireland; 3Lab Tumorgenetica, UMC St Radboud, Nijmegen, Netherlands; 4NCIMD, Temple St Child Univ Hosp, Dublin, Ireland

1 Lab Biochem, Rabta Hosp, Tunis, Tunisia; 2Dep Child Ado Neur, Nat Inst Neur, Tunis, Tunisia; 3Lab Biochem, Mol Bio Metab Dis, CHRU, Lille, France

Fumarate Hydratase (FH) deficiency, an autosomal recessive disorder is characterised by an isolated persistent increase in fumarate excretion. Carriers have an increased risk of developing Hereditary Leiomyomatosis, Renal Cell Cancer Syndrome (HLRCC). From ~18,000 urinary organic acid analyses performed in a 5y period, 3 cases of FH were identified. Case 1: 4y old girl presented with global developmental delay (GDD). She was heterozygous for the common mutation and a missense mutation previously reported in FH. Case 2: 3mth old female presented with poor feeding, GDD and profound hypotonia. FH activity in cultured fibroblasts confirmed severe deficiency. Mutation analysis showed bi-allelic mutations in FH gene. Maternal uncle diagnosed with renal cell carcinoma and mother has a history of uterine fibroids. Case 3: 10y old boy presented with metabolic acidosis secondary to recurrent UTI on a background history of lissencephaly. Mutational analysis showed bi-allelic mutations in FH gene. A missense mutation in exon 7:c.1020T>A(p.(Asn340Lys)) has only been reported in two patients with hereditary leiomyomatosis. His older brother died at 12y following a similar history. Mother and another brother are carriers. These cases indicate the importance of obtaining a detailed family history. Mutational analysis of asymptomatic at-risk relatives allows early surveillance and treatment.

Background: Guanidinoacetate methyltransferase (GAMT) deficiency is an autosomal recessive metabolic disorder that primarily affects the nervous system and muscles. It is the first observed disorder of creatine metabolism. Objectives: We report a case of GAMT deficiency in a Tunisian boy. Case report: An 18 years old boy presented with symptoms suggestive of Creatine deficiency syndrome (CDS), extrapyramidal movement disorders and dystonia. Amino and organic acids abnormalities also hypomethylation syndrome were excluded. Urinary creatine (Cr) and guanidinoacetate (GAA) were analyzed by gas chromatography–mass spectrometry method. The GAMT activity was determined in EpsteinBarr virus transformed lymphoblastoid cell lines by sensitive enzyme assay. Low levels of Cr and relatively high GAA concentration were observed, the Cr/GAA ratio was 0.45. The GAMT activity was 0.107 nmol/h/mg protein (GAMT activity in control were 0.29-0.31 nmol/h/mg protein). The low Cr/GAA ratio may be related to high endogen consumption of Cr. We also may speculate the diagnosis of an atypical form of CDS with non ubiquitous GAMT deficiency. Conclusion: Against this association of low Cr/GAA ratio with abnormal GAMT activity and these clinical features, many questions may be advanced to explain this profile after genetic analysis of dystonia. 10. Carbohydrate disorders P-413

P-411 Glut-1 deficiency presenting with hemiplegic migraine Fundus autofluorescence pattern in glutathione synthetase deficiency Mohammad SS1, Coman D1, Calvert S2 Taylan Şekeroğlu H1, Öztürk Hişmi B2, Kadayıfçılar S1, Coskun T2 Depart Ophtalm, Hacettepe Uni, Ankara, Turkey; 2Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey 1

Background: Glutatathione(GSH) is a tripeptide which plays essential roles in free radical scavenging, redox reactions, DNA synthesis, regulation of cell proliferation, apoptosis and neurotransmission.(1,2) Glutathione synthetase(GS) deficiency, a rare autosomal recessive disorder, is the most common type of hereditary enzyme defects in GSH metabolism Purpose: To report the fundus autofluorescence properties and retinal findings in a patient with glutathione synthetase deficiency. Design: Presentation of one case.

1 The University of Queensland, Brisbane, Australia; 2Royal Children's Hospital, Brisbane, Australia

Background: Glucose transporter-1 deficiency syndrome (GLUT1DS) is a treatable epileptic encephalopathy caused by mutations in the SLC2A1 gene causing impaired glucose transport into the brain. Paroxysmal dyskinesia and movement disorders have recently expanded the phenotype. Anecdotal evidence links typical and atypical migraine with mutations in SLC2A1. Methods: case study Results: An 8 year old boy with a label of ataxic cerebral palsy was seen with 3 year history of weekly headaches with hemiplegia. The episodes lasted 20-30 mins at a time associated with photopsia, pallor,

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nausea, phonophobia and photophobia. His mother, maternal uncle, two maternal aunts and maternal grandmother had similar episodes. He had separate absence seizures. He had lower limb spasticity and cerebellar signs with normal neuroimaging. At 8 years of age CSF glucose, tested for the first time, was 2.0 mmol/L with paired plasma glucose of 5.6 mmol/L (CSF: plasma ratio 0.36). A provisional diagnosis of GLUT1-DS was confirmed by a missense mutation in the SLC2A1 gene c.929C>T(p.(Thr310Ile)). Initiation of the Modified Atkins Diet (MAD) resulted in resolution of these episodes and seizures. Conclusions: The spectrum of GLUT-1 DS is expanding. We describe an uncommon presentation with hemiplegic migraine which resolved on the MAD.

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those distant from the active site can alter enzyme activity via a structural effect. Each reconstructed mutation resulted in unique alterations to the enzyme's stability against thermal denaturation and proteases, and ability to bind cofactors and substrates. These findings provide the first biochemical evidence that protein misfolding can be a contributing factor to galactosemia, and paves the way for investigating the use of proteostasis regulators and pharmacological chaperones as a potential treatment approach. P-416 Affected neural language production networks in patients with classic galactosemia Timmers I1,2, Jansma BM2, Rubio-Gozalbo ME1,3

P-414 Glycogen storage type IIIa: the effect of intensive dietary treatment on growth, skeletal myopathy and cardiomyopathy. Walter JH1, Hunjan I2, Rogozinski H2, Stainforth C2, Bradley J2

1 Dep Ped, MUMC, Maastricht, Netherlands; 2Dep Cogn Neurosc, UM, Maastricht, Netherlands; 3Lab Inh Metab Dis, MUMC, Maastricht, Netherlands

Glycogen Storage Disease type III is caused by mutations in AGL leading to a deficiency in glycogen debrancher enzyme (GDE). Mutations beyond exon 3 are associated with both hepatic and muscle manifestations (GSD IIIa) We report 3 siblings with GSD IIIa who are homozygous for a mutation in exon 7 [c.753_756del]. The older siblings were diagnosed and treated from 7 and 4 years of age respectively and the youngest from the newborn period. All children have short fasting tolerance, raised liver transaminases and raised creatine kinase. The two older siblings have been managed with frequent feeds and oral corn-starch while the younger sibling has had continuous overnight gastrostomy feeds from early infancy. The oldest child, who was extremely short for her age (ht<<0.2 centile), has shown remarkable catch up growth with intensive dietary treatment. The youngest, now age 2 yr, has grown normally from birth but, in contrast to her older siblings, has developed a hypertrophic cardiomyopathy. In this child the early intensive dietary regimen with continuous overnight feeds, high in carbohydrate, although providing protection from hypoglycaemia and allowing for normal growth, may have increased the risk of developing cardiac disease in early childhood.

Background: Language production impairments are prominent complications in classic galactosemia despite dietary treatment. Our objective is to study the neural language production network in these patients. Methods: We acquired functional Magnetic Resonance Imaging during active language performance in 11 patients (aged 14-20y) and 11 matched controls (aged 14-21y). During the task, participants either passively watched or overtly described visually animated scenes. After data pre-processing, a first-level fixed-effects analysis was performed, followed by a second-level random-effects analysis to examine group differences (at whole-brain level; when contrasting language production versus baseline). Results: Overall, patients and controls showed a similar network of regions involved in language production, such as inferior frontal, (supplementary) motor, angular and superior temporal regions. Nevertheless, the functional activity in several regions was significantly different between groups (statistical map tresholded at p = .05), most prominently in (bilateral) superior temporal, angular, superior frontal, motor, and posterior cingulate cortex. Conclusion: There are deviations in functional activity within the language production network in these patients, but other regions (not restricted to language functions) also show differences. This is in line with the cognitive profile seen in galactosemia. Further analyses will examine connectivity patterns (functional and structural) within this and other functional networks.

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P-418

Protein misfolding as a contributing factor to galactosemia

Congenital hyperinsulinism: retrospective study in a large cohort

McCorvie T J1, Gleason T J2, Frazer A3, Liu Y2, Fridovich-Keil J L2, Timson D J3

Maiorana A1, Barbetti F2, Faletra F3, Pizzoferro M4, Rufini V5, Francalanci P6, de Ville de Goyet J7, Rahier J8, Dionisi-Vici C1

Stru Gen Cons, Univ Oxf, Oxford, United Kingdom; 2Dep Hum Gen, Em Univ Sch Med, Atlanta, United States; 3Sch Biol Sci, Que Univ Bel, Med Biol Cen, Belfast, United Kingdom

Div Metab Dis, Bambino Gesù Child Hosp, Rome, Italy; 2Lab Mend Diab, Bambino Gesù Child Hosp, Rome, Italy; 3Med Genet, Burlo Garofalo Inst, Trieste, Italy; 4Nucl Med Dep, Bambino Gesù Child Hosp, Rome, Italy; 5Nucl Med, Catholic Univ of Sacred Heart, Rome, Italy; 6Pathology Dep, Bambino Gesù Chil Hosp, Rome, Italy; 7Liv Surg Transp, Bambino Gesù Child Hosp, Rome, Italy; 8 Pathol Endocrinol Metab, Univ of Louvain, Brussel, Belgium

1

Willink Biochemical Genetics Unit, Manchester, United Kingdom; Bradford Hosp NHS Foundation Trust, Bradford, United Kingdom

2

1

Galactosemia is an inborn error of sugar galactose metabolism, classified into three types depending on the enzyme affected. The most severe cases are type I and type III galactosemia, associated with impairment of galactose-1-phosphate uridylyltransferase (GALT) and uridine diphosphate galactose 4'-epimerase (GALE) respectively, and the majority of cases are caused by missense mutations. Using a combination of recombinant proteins, biophysical assays and a yeast model, we studied a number of disease associated mutations of GALE (p.K161N and p.D175N) and of GALT (p.D28Y, p.L74P, p.F171S, p.F194L and p.R333G ). These mutations are located throughout the structure of their respective protein and thus represent a diverse group with possible differing mechanisms of impairment. It was shown that each mutation leads to defects in protein stability and folding and

1

Congenital hyperinsulinism is a heterogeneous condition due to mutations in genes involved in the regulation of glucose metabolism. We retrospectively determined genotypic and phenotypic characteristics in a series of 86 patients. 18FDOPA-PET/TC was performed in heterozygous ABCC8/KCNJ11 and in patients without mutations in known genes. Genetic investigations allowed to characterize 30/51 (58,8%) patients: 20/30 had mutations in ABCC8, 41% diazoxide-responsive (including homozygous, focal and undetermined heterozygous), and

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12% octreotide-responsive. KCNJ11 mutations were found in 3 patients, GK in 2, and PMI in 1. Hyperinsulinism-hyperammonemia was found in 14% (1/7 without GLUD1 mutation), all were diazoxide-responsive, with 2 showing neurological impairment. The large majority of non-mutated patients were diazoxide-responsive. Merge of genetic and instrumental data, showed diffuse forms in 47/62 (69% diazoxide-responsive, 5% octreotide-responsive), focal forms in 10/62 (40% diazoxide/octreotide-responsive) and atypical forms in 5/62 of patients. At follow-up, 26% of patients manifested neurological outcomes (10% epilepsy). Twenty patients underwent pancreatectomy: 10 near-total (50% diabetes at followup), 10 partial (10/10 without neurological outcome). Spontaneous remission occurred in 19/86 (22%), with permanent neurological signs in 8 patients. This report highlights that congenital hyperinsulinism is a complex condition requiring a multidisciplinary approach based on clinical, instrumental and genetic investigations. P-419 Glycogen storage disease type III: atkins diet as a therapeutic option

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ALT and AST levels. Three GSD-IXc patients had hypoglycemia. Fifteen patients (14 GSD-IX; 1 GSD-VI) had RBC-phosphorylase kinase enzyme activity measurement (low 13 GSD-IX; low 1 GSD-VI; normal 1 GSD-IX). Liver histopathology was suggestive of GSD-III in two and of GSD-IV in one genetically confirmed GSD-IXa patients and of GSD-III/IV in one genetically confirmed GSD-IXb patient. One GSD-IXa patient developed a liver adenoma. One GSD-IXc patient developed liver cirrhosis. Another GSD-IXc patient had hypertrophic cardiomyopathy. Diagnosis was confirmed by molecular genetic testing in all patients (13 novel mutations: 3 PYGL; 7 PHKA2; 2 PHKB; 1 PHKG2). Eleven patients (5 GSD-IXa; 3 GSD-IXc; 3 GSD-VI) received uncooked cornstarch. One GSD-IXc patient required continuous NG-tube feeding for recurrent hypoglycemia. Conclusion: We report 21 GSD-VI/IX patients, 13 novel mutations and long-term out-come from a single metabolic centre. P-421 Isolated sedoheptulokinase deficiency diagnosed for the first time in two patients: is it a benign disorder? Wamelink MMC1, Ramos RJJF2, van den Elzen APM3, Ruijter GJG4, Bonte R4, Diogo L5, Garcia P5, Nota B1, Tavares de Almeida I2, Salomons GS1

Meyer U1, Mayorandan S1, Hartmann H1, Das AM1 1

1

Metab Dis, Hann. Med School, Dept. Paed, Hannover, Germany

Introduction: Frequent feeding with carbohydrate-rich meals or drip feeding is the therapy of choice in glycogen storage diseases(GSD) with liver involvement. In GSD IIIa there is both liver and muscle involvement, often muscle symptoms are the leading and life-limiting clinical feature. Administration of carbohydrates may lead to hyperinsulinism resulting in suppression of lipolysis, ketogenesis, gluconeogenesis and activation of glycogen synthesis. Thus, depletion of energy substrates for heart and skeletal muscle occurs. The Atkins diet leads to increased blood levels of ketone bodies and fatty acids thus improving the energetic state of muscles. Methods: We treated 2 boys with GSD IIIa aged 9 and 11 years with Atkins diet for over 12 months. Results: In both patients physical strength improved, cardiac function stabilised and creatine kinase levels in blood significantly decreased. When Atkins diet was withdrawn in one of the patients for 2 months he complained of chest pain, reduced physical strength and creatine kinase levels rapidly increased. All parameters improved upon reintroduction of the diet. Conclusions: Patients with GSD III benefit from an improved energetic state of heart and skeletal muscle by introduction of an Atkins diet as judged by physical strength, cardiac function and creatine kinase levels in blood. P-420 Retrospective review of all GSD type VI and IX patients at the hospital for sick children Roscher A1, Hewson S2, Feigenbaum A3, Kronick J2, Raiman J2, Schulze A2, Siriwardena K2, Mercimek-Mahmutoglu S2 1

Dp Ped, Med Univ Vienna, Vienna, Austria; 2Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 3Biochem Gen Dp Ped Univ California, San Diego, United States Background: Glycogen storage disorders (GSD) GSD-VI and -IX are caused by hepatic phosphorylase system enzyme deficiencies. Methods: All GSD-VI and -IX patients diagnosed between 1993-2012 were included. Information were entered into an excel database. Results: There were 21 patients: 4 GSD-VI; 11 GSD-IXa; 3 GSDIXb; 3 GSD-IXc. Nineteen patients presented with hepatomegaly and two were diagnosed due to positive family history. Twenty patients had elevated

Dept Clin Chem, VU Univ Medical Center, Amsterdam, Netherlands; Metab & Genet, iMed.UL, Fac. Pharm, Lisboa, Portugal; 3Dept of Paed, Reinier de Graaf Gasthuis, Delft, Netherlands; 4Dept Clin Genet, Erasmus Medical Center, Rotterdam, Netherlands; 5Paediatric Hospital of Coimbra, Coimbra, Portugal 2

Background: Sedoheptulokinase (SHPK) is an enzyme that is responsible for the phosphorylation of sedoheptulose to sedoheptulose-7-phosphate. SHPK deficiency has only been described as combined defect with Cystinosis caused by a 57-kb deletion. Patients with this deletion have infantile nephropathic cystinosis and the clinical relevance of SHPK deficiency is unclear. We have now diagnosed two patients with an isolated SHPK deficiency. Patients: Patient 1, a boy born January 2010, presented with genital and facial dysmorphisms, neonatal cholestatic hepatitis, failure to thrive, psychomotor retardation, anaemia, fasting hypoglycaemia, secondary hypocortisolism and diarrhoea. Patient 2, a girl born May 2012, presented with congenital artrogryposis multiplex, multiple dysmorfisms and severe feeding problems. Results: Strongly elevated excretion of erythritol and sedoheptulose were detected in both patients with normal excretion of sedoheptulose7-phosphate, biochemically indicating SHPK deficiency. In patient 1 and 2, a homozygous variant was detected (c.355C>T; p.Arg119X and c.211G>T; p.Glu71X respectively), both resulting in a predicted truncated non-functional protein. Conclusions: We present here two patients with severe, but different clinical presentations. Moreover, their symptoms have not yet been described in patients with cystinosis caused by the 57-kb deletion. Therefore it remains questionable if SHPK deficiency is the cause of the clinical phenotype in these patients. P-422 Excellent response to ketogenic diet (modified Atkins diet) in a case of alternating hemiplegia of childhood Roubergue A1, Philibert B2, Gautier A3, Kuster A3, Markowicz K4, Billette de Villemeur T1, Vuillaumier-Barrot S5, Nicole S6, Roze E7, Doummar D1 1

AP-HP, Neuropédiatrie, Trousseau Hosp, Paris, France; 2Neurologie, Centre Hosp Départemental, La Roche-sur-Yon, France; 3Clin Medic Pédiatrique, Hôp Mère-Enfant, Nantes, France; 4AP-HP, ServiceDiététique, Trousseau Hosp, Paris, France; 5AP-HP, Bioch, Hôp Bichat ClaudeBernard, Paris, France; 6INSERM U975, CRICM, Paris, France; 7AP-HP, Neurologie, GH Pitié-Salpétrière, Paris, France

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Background: Alternating Hemiplegia of Childhood (AHC) associates early onset of plegic and tonic/dystonic attacks and permanent movement disorders with mental retardation. It is caused by heterozygous mutations in the ATP1A3 gene. Treatment outcome is poor. To our knowledge, there are no reports on the response to Ketogenic diet/modified Atkins diet (KD/MAD). Objectives: To evaluate response to KD/MAD in a child with AHC. Case report: A 2 year-old girl displayed tonic/dystonic and plegic attacks from age 8 months. Attacks were mostly triggered by exercise and emotions. The mother, maternal uncle and grandmother displayed paroxysmal exercise-induced dystonia. All showed minimal permanent neurological involvement. The features of the attacks, similar to those of GLUT1 deficiency syndrome, led us to consider treatment with KD/MAD. MAD was chosen because it was easier to manage than KD. The 4 familial members harboured the ATP1A3 p.Asp923Asn mutation (Roubergue 2013). Result: MAD resulted in complete disappearance of the attacks (with one year of follow-up) and in improvement of the speech disorder. Discussion/Conclusion: The complete prophylactic effect of MAD on the attacks in our patient contrasts with the only partial effect obtained by the drugs usually administered in AHC. This suggests a new therapeutic approach in AHC. P-423 Fertility preservation in female classic galactosemia patients van Erven B1, Gubbels CS2, van Golde RJ3, Dunselman GA3, Derhaag JG3, de Wert G4, Geraedts JP2, Bosch AM5, Treacy EP6, Welt CK7, Berry GT8, Rubio-Gozalbo ME1 1 Div Pediatrics, MUMC, Maastricht, Netherlands; 2Div Clin Genet, MUMC, Maastricht, Netherlands; 3Div Obst Gyn, MUMC, Maastricht, Netherlands; 4 Health ethics society, Maastr Univ, Maastricht, Netherlands; 5Div Pediatrics, AMC, Amsterdam, Netherlands; 6 Inh Metab Disord, Child Univ Hosp, Dublin, Ireland; 7Repr Endocr Unit, Mass Gen Hosp, Boston, United States; 8Div Gen, Boston Child Hosp, Boston, United States

Almost every female classic galactosemia patient develops primary ovarian insufficiency (POI) as a diet-independent complication. This is a major concern for patients and their parents, and physicians are often asked about options to preserve fertility. Unfortunately, there are no recommendations on fertility preservation in this group. Several guidelines in other patient groups have been developed. However, the unique pathophysiology of classic galactosemia with a severely reduced follicle pool at an early age requires an adjusted approach. We propose recommendations for physicians based on current knowledge concerning galactosemia and fertility preservation. Fertility preservation is only likely to be successful in very young prepubertal patients, currently leaving cryopreservation of ovarian tissue the only available technique. However, this technique is not ready for clinical application, it is considered experimental and reduces the ovarian reserve. Fertility preservation in young girls also raises ethical questions that require consideration. In addition, spontaneous conception despite POI is well described in classic galactosemia. The uncertainty surrounding fertility preservation and the significant chance of spontaneous pregnancy warrant counseling towards conservative application of these techniques. We propose that fertility preservation should only be offered with appropriate institutional research ethics approval to classic galactosemia girls at a young pre-pubertal age.

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P-424 Sub-fertility and growth restriction in a new mouse model of galactose-1 phosphate uridylyltransferase-deficiency. Lai K1, Tang M1, Yin X1, Witt B2, Fraser N1, Rascon R1, Chen W1, Johnson B3, Bronner M2, Bodamer O3 1

Dept Pediatrics, Univ of Utah, Salt Lake City, United States; 2Dept Pathology, Univ of Utah, Salt Lake City, United States; 3Dept Human Genetics, Univ of Miami, Miami, United States Background: Early characterization of the world's first GalT geneknockout mouse revealed no disease phenotypes of human Classic Galactosemia. We recently constructed a new galactose-1 phosphate uridylyltransferase (GALT)-deficient mouse model, and upon expanded characterization, we uncovered phenotypes that could shed novel insights into this debilitating disorder. Methods: (1) We determined the RBC GSH/GSSG ratios in neonatal mice (mutant/normal) to assess for oxidative stress. (2) We measured the body weight of age- and sex- matched animals (mutant/normal) for 10 weeks since birth. (3) We followed age-matched females (mutant/normal) for six or more months since they reached puberty, and evaluated the number of litters, litter size and ovarian histology. Results: (1) Oxidative stress plays a role in the galactose-induced toxicity in neonatal mutant mice. (2) GALT-deficient mice fed with normal chow and galactose-fed (i.e., challenged) mice have significantly lower body weight than unchallenged normal littermates. (3) Unchallenged GALT-deficient females have smaller litter size (p<0.05) and longer time-to-pregnancy (TTP). These were corroborated by ovarian histological findings. (4) Galactose challenge of the GALT-deficient females led to a reversible infertility phenotype. Conclusions: Our new mouse model is a valuable research tool for the patho-physiological studies of Classic Galactosemia and development of improved therapies for this disorder. P-425 Tissue-specific mouse models for glycogen storage disease type 1a Clar J1, Mutel E1, Gri B1, Mithieux G1, Rajas F1 1

Inserm U855/University Lyon 1,, Lyon, France

Patients with glycogen storage disease type 1a (GSD1a) suffer from severe hypoglycaemia. With aging, they develop major and severe complications, such as liver cancer and end-stage renal failure. Until now, the molecular mechanisms involved in these pathologies are poorly understood and available animal models are not viable. To investigate the longterm complications of GSD1a, we have recently developed mice in which the catalytic subunit of glucose-6 phosphatase gene (G6pc) can be specifically and conditionally deleted in each glucose-producing organ. B6.G6pcex3lox/ex3lox mice are crossed with transgenic mice expressing an inducible CreERT2 recombinase under the control of the serum albumin, Kap or villin promoter to obtain liver, kidney and intestine G6pc-/- mice, respectively, after treatment with Tamoxifen. In contrast to total G6pc knockout mice, tissue-specific G6pc deficient mice are perfectly viable and maintain their blood glucose by induction of gluconeogenesis in untargeted organs. Liver G6pc-/- mice exhibit the same hepatic pathological features as GSD1 patients, including the late development of hepatocellular adenomas and carcinomas. Renal

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G6pc-/- mice also develop renal symptoms similar to the human pathology, including nephromegaly and functional alterations in the kidneys. These mice should allow us to improve the strategies of treatment on both nutritional and pharmacological viewpoints.

severe hypoglycemic and acidosis attacks. Although there is no genotype-phenotype correlation in fructose metabolism disorders, we think that this idea will change by investigating new mutations in different populations.

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P-428

Glut-1 deficiency syndrome: 3 case reports demonstrate clinical improvement due to a trial with glycosade, a high-amylopectin maize starch.

Use of fibroscan in monitoring liver impairment in glycogen storage disease type I and III Paci S1, Gasparri M1, Pieretti S1, Rovelli V1, Riva E1, Salvatici E1

1,2

3

3

3

Maes M , Van Coster R , Van Driessche M , Desloovere A , De Meirleir L4, Corthouts K4, Santens P5, Poppe B6, Verloo P3 Ctr Dev Disorders, Ghent, Belgium; 2Div of Ped, AZ Nikolaas, SintNiklaas, Belgium; 3Div of Ped Neur, Univ Hosp, Ghent, Belgium; 4Div of Ped Neur, Univ Hosp, Brussels, Belgium; 5Div of Neur, Univ Hosp, Ghent, Belgium; 6Div of Internal Med, Univ Hosp, Ghent, Belgium 1

Background: GLUT1 deficiency is a metabolic disorder characterized by epileptic encephalopathy, developmental delay and complex motor disorders caused by mutations in the SLC2A1 gene. Reduced function of the GLUT 1 lessens the availability of the brain's supply of glucose. Milder variants result in (exercise induced) movement disorders without epilepsy. Glycosade (Vitaflo ®) is a high-amylopectin maize starch physically modified using heat and moisture initially indicated for glycogen storage diseases. Through administration to the GLUT1 patient it is likely that a more continuous high amount of glucose is provided to the brain via the impaired glucose transporter. Methods: We present three patients (age 6 to 47) with proven GLUT1. Clinical picture varied from ataxia, epilepsy and mental retardation to exercise induced dystonia. Each patient was giving a dose of Glycosade (20g-125g/day). Results: For all patients the administration of Glycosade improved the symptoms, one became asymptomatic. Conclusion: Until now only a ketogenic diet was the only therapy. In our opinion administration of Glycosade is another therapeutic possibility without the risk of side effects and the discomfort of the ketogenic diet. Consequently, we suggest more trials with Glycosade in GLUT1 patients.

1

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy

Background: liver impairment in glycogen storage disease (GSD) I and III is characterized by steatosis/fibrosis due to progressive accumulation of glycogen and lipids. Fibroscan is a technique used in the monitoring of viral hepatitis based on the determination of stiffness in 10 standard measurements (normal ≤5,5kPa, high risk of cirrhosis ≥13kPa). Objectives: to study liver impairment and evaluate Fibroscan utility in GSDI and III follow up. Methods: 9 patients were recruited (5 GSDIa, 3 GSDIb, 4 GSDIII; 8♂:1♀; mean age 24 years). Liver impairment was evaluated by biochemical parameters (SGOT/SGPT, gammaGT, bilirubin, albumin, platelets, PT, APRi index), liver ultrasound and Fibroscan. Results: in GSDIII higher levels of SGOT/SGPT and lower levels of platelets, with consequent higher APRi index, were found. Ultrasound showed hepatomegaly with moderate-severe steatosis in all patients. Fibroscan showed higher stiffness in GSDIII (mean 11,4kPa) than in GSDI (mean 5,1kPa). Conclusions: our study confirms a more severe liver impairment in GSDIII. Furthermore, our data suggest that ultrasound alone seems not to be able to discriminate between steatosis and fibrosis and that steatosis in GSDI can bring to an underestimation of fibrosis at Fibroscan. Fibroscan may be useful in the monitoring of liver disease, especially in GSDIII. P-429 Glycosylation serum and gene biomarkers in classical galactosaemia

P-427 The clinical and molecular aspects of children with fructose-1, 6-biphosphatase deficiency and hereditary fructose intolerance

Coss KP1, Doran PP1, Knerr I2, Adamczyk B3, Murray DW4, RubioGozalbo ME5, Rudd PM3, Treacy EP6 1

1 Div Metab Dis,Ankara Dıskapı Child Hosp, Ankara, Turkey; 2Intergen Lab, Ankara, Turkey

University College Dublin, Dublin, Ireland; 2Metabolic Dept, Child Univ Hosp, Dublin, Ireland; 3NIBRT, Dublin-Oxford Glycobiology Lab, Dublin, Ireland; 4RCSI, Dept of Physiology and Med Physis, Dublin, Ireland; 5Maastricht Univ Medical Centre, Maastricht, Netherlands; 6 Child Uni Hos and Trinity College Dublin, Dublin, Ireland

Background: The aim of this study is to describe the clinical aspects and molecular genetic analysis of the patients with fructose-1,6-biphosphatase (FBPase) deficiency and hereditary fructose intolerance (HFI). Methods: Eleven patients with HFI and eight patients with FBPase deficiency were included in the study. The demographic and clinical characteristics were evaluated and genetic analysis was performed. Results: The patients with FBPase deficiency were diagnosed at smaller ages, the main complaints were recurrent hypoglycemia and acidosis attacks. Hepatomegaly was the major clinical finding but the patients with exon 1 deletion had developmental delay. Five patients had Arg218Lys mutation on exon 5 and three patients had exon 1 deletion. The patients with HFI had better prognosis and the aversion to sugar was the main complaint. One patient had mutations on both of the FBP1 and ALDOB genes. Discussion: The children with FBPase deficiency and especially exon 1 deletion seem to have a poor prognosis. One of the patients died and the other had neurodevelopmental abnormality which might be the result of

Background: We previously identified IgG N-glycan galactose incorporation ratios as informative markers in Gal adults (Coss et al, 2012). Objectives: To determine (a) if potentially modifiable N-glycan processing defects are identifiable in treated Gal children compared to PMM2-CDG and (b) to study related N-glycan genes. Materials and Methods: UPLC of whole serum and IgG N-glycans was applied to 9 treated Gal patients (Q188R/Q188R, 1-9 years), compared to 9 matched controls and 3 PMM2-CDG patients. Gratios (agalactosylated(G0), monogalactosylated(G1) and digalactosylated(G2) structures) estimated galactose incorporation into IgG N-glycans. T-lymphocyte microarray(Affymetrix U133a plus2.0) genes of interest were validated with qRT-PCR. In addition, 2 adult patient-derived dermal fibroblast cell-lines were exposed to 0.1% galactose (intoxication dose) and 0.01% galactose (treated dose) over time to monitor changes in gene expression.

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Gündüz M , Özaydın E , Koç N , Okur I , Ceylaner S

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Results: IgG N-glycan G-ratios differentiated processing defects in Gal vs. controls and PMM2-CDG, (G0/G1)/G2 ratios, p=0.003 and G0/G2, p=0.001respectively. Dysregulation of 3 CDG-linked genes was confirmed in T-lymphocytes (ALG9 p=0.005, COG1 p=0.0002, B4GALT1 p=0.01). ALG9 expression was also significantly decreased at 1hr 0.1% galactose exposure in fibroblasts (p=0.002 and p=0.03). Conclusion: We have identified serum and gene markers of on going Nglycosylation abnormalities in treated classical Gal which may serve as improved biomarkers for treatment. P-430 Lymphocytes intracellular enzymes activity in children with hepatic form of glycogen storage disease Kurbatova O1, Surkov A1, Polyakova S1, Miroshkina L1, Semenova G1, Samokhina I1, Izmailova T1, Kapustina E1, Dukhova Z1, Zakirov R1, Freydlin E1, Petrichuk S1

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and signs of chronic liver disease (jaundice and spider naevi). Muscle tone and motor development were normal. There was no history of fructose intolerance. Initial metabolic investigations showed no diagnostic abnormalities, specifically excluding galactosaemia, tyrosinaemia, Gaucher, Niemann Pick Type A/B and Wolman diseases. He developed varicella infection prior to a planned liver biopsy, subsequently developing fulminant hepatic failure. A transjugular liver biopsy demonstrated micronodular cirrhosis, globular pale PAS-D-resistant storage material and abundant foamy macrophages consistent with GSDIV. He was listed for superurgent liver transplantation, but developed a fatal pulmonary haemorrhage. DNA analysis confirmed compound heterozygosity for two pathogenic GBE mutations. Conclusion: Though usually associated with progressive cirrhosis, patients with GSDIV can develop fulminant hepatic failure. Varicella infection probably contributed to this infant's rapidly progressive course, demonstrating the need for timely transplantation. P-432 A zebrafish model for classic galactosemia

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Scientif Centre of Children Health,RAMS, Moscow, Russian Federation

Background. The aim is to evaluate the diagnostic value of the lymphocytes mitochondrial enzymes activity in children with hepatic form of glycogen storage disease(GSD). Methods: We examined 86 children with GSD. Distribution of children by disease types were:35 children-with type-I,23patients type-III,27children - type-VI of the disease, control group included 34 healthy children. As laboratory markers of energy metabolism intensity in the lymphocytes were chosen activities of mithochondrial enzymes: succinate dehydrogenase(SDH), glycerol-3-phosphate-dehydrogenase(GPDH), NADH-dehydrogenase(NADH-H). Dehydrogenases activity was measured by the quantitative cytochemical method, based on the n-nitrotetrazolium violet ability to form insoluble formazan granules during enzymic reduction with a help of cytomorphodensitometry.The statistical significance was evaluated according to the Kolmogorov-Smirnov criterion(P≤0.005). Results: We revealed significant decrease of SDH-activity in patients with GSD,most expressed in type-I. We found out a tendency of NADH-D activity increase in children with GSD, that can reflect respiratory chain I phase compensatory activation both II phase depression. Children with GSD had a significant decrease of GPDH-activity, compared with the control group that indicated disintegration of the coupling processes-glycolysis and the Krebs-cycle. Conclusions: Lymphocytes dehydrogenases activity criteria correlate with the main clinical and laboratory parameters and can be used as additional diagnostic data to assess the state of children diagnosed with GSD. P-431

Vanoevelen J1, van Erven B2, van der Wiel S2, Smeets H1, Bierau J3, Rubio-Gozalbo ME2 Div Clin Genet, MUMC, Maastricht, Netherlands; 2Div Pediatrics, MUMC, Maastricht, Netherlands; 3Gen Metab Dis Lab, MUMC, Maastricht, Netherlands 1

Classic galactosemia is a hereditary disorder of galactose metabolism caused by a deficiency in GALT (galactose-1-phosphate uridyl transferase). Patients show a potentially fatal phenotype after galactose intake in the first days of life. A galactoserestricted diet quickly resolves these acute symptoms. However, long-term complications, including cognitive impairment and, in females, primary ovarian insufficiency (POI) with subsequent infertility can arise. The exact mechanism(s) causing these complications and the time windows in which the damage occurs remain uncertain. Other animal models (mouse, fly) proved limited in providing answers. The aim of our study is to develop a galactosemia model in zebrafish to determine if damage to the brain and gonads has an early onset and to determine whether dysregulated apoptosis, induced by galactose or a metabolite, is a key mechanism in POI. To this end, a zebrafish GALT knockdown model was developed and characterized. We could show that GALT activity was severely reduced upon MO-mediated knockdown at 3dpf. The observed phenotypes are being compared to the human phenotype with special focus on the primordial germ cells (PGCs) and the brain. The time windows are chosen in order to determine when the onset of damage starts.

Fulminant hepatic failure in an infant with glycogen storage disease type IV

P-433

Santra S1, Chakrapani A1, Vijay S1, Mckiernan P1, Van Mourik I1, Kelly D1, Brundler M1, Allotey J1, Gupte G1

Hypoglycemia and seizure in hyperinsulinism/hyperammoniemia (HI/HA) syndrome due to a novel mutation in the GLUD 1 gene

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Tran C1, Perlman K2, Kronick JB1

Background: Patients with progressive hepatic Glycogen Storage Disease Type IV (GSDIV), an autosomal recessive deficiency of glycogen branching enzyme, usually present in early childhood with hepatomegaly, growth failure and progressive cirrhosis leading to death aged 4-5 years without liver transplantation. We report an infant with GSDIV who developed fatal acute-on-chronic liver failure. Case Details: A male infant, born to non-consanguineous Caucasian parents, presented aged eleven months with massive hepatosplenomegaly

Div of Clin and Metab Gen, Sickkids Hosp, Toronto, Canada; 2Div of Endocrinology, Sickkids Hosp, Toronto, Canada

Birmingham Children's Hospital, Birmingham, United Kingdom

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Background: Hyperinsulinism-hyperammoniemia syndrome (HI/HA) is a rare autosomal dominant disorder manifested by hypoglycemic symptoms and elevated ammonia. HI/HA is the second most common cause of hyperinsulinemic hypoglycemia of infancy and is caused by activating mutations in the GLUD 1 gene.

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Case report: A 14-month-old female presented with multiple episodes of seizures and a history of severe hypoglycemia responsive to diazoxide. Her mother also had a history of hypoglycemia and seizures. The patient had mild hyperammoniemia (81 μmol/L) and hyperinsulinism when she was hypoglycemic (2.2 mmol/L) thus suggesting a disorder of glutamate dehydrogenase (GDH). Molecular genetic testing was performed and a heterozygous mutation (c.1526G>C) in the GLUD 1 gene coding for GDH was identified. This mutation has not been reported before and involves the allosteric domain of GDH protein and appears to affect GTP inhibition which is associated with hyperinsulinism. To investigate a de novo occurrence testing of both parents for GLUD 1 gene mutation will be performed. Conclusion: We present a patient with HI/HA and a novel mutation in the GLUD 1 gene. Given the rarity of this mutation, the family history consistent with a dominant mode of inheritance we are confident that this novel mutation is indeed pathogenic. P-434 Glut1 deficiency syndrome with paroxismal dystonia. Clinicalelectroencephalography correlation Domínguez-Carral J1, Gonzalez-Lamuño Leguina D1, Sancho Gutiérrez R1, García Valle E1, Arteaga Manjón-Cabeza RM1, Orizaola Balaguer PJ1

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Background: GSD III autosomal recessive dis due to deficiency of glycogen debranching enzyme (AGL). AGL mutations are detected in exon-based sequencing In certain patients we failed to identify mutations. Objectives: to detect large rearrangements involving more than one exon. Methods: copy number of each exon analyzed by quantitative real-time PCR. Long-range PCR encompassing deleted exons used to characterize large deletions and breakpoints sequenced. Results: 4 large rearrangements were found: one novel,one novel large deletion-insertion and two known large deletions. The novel (NG_012865.1:g.9666_13063del3398) encompassing exons 4&5 found in 2 Turkish families, caused by Alu repetitive element mediated-homologous recombination. Another novel mutation in a Japanese patient was deletion-insertion involving exons 27 and 28 (g.49898_53860del3963insTG) due to non-homologous recombination between Alu and long interspersed nuclear element1.A known large deletion(g.59373_64940del5568) detected in 7 Egyptian families, caused by the same mechanism.1Egyptian family showed known deletion(g.50671_52195del1525),due to non-homologous end-joining. Conclusion: 4 large rearrangements in AGL and their potential mechanism were identified. Exon copy number assay by quantitative RPCR is useful for detection of large rearrangements. In Egyptian GSDIII patients, g.59373_64940del5568 is the most prevalent AGL mutation. This is the first report of Alu-mediated recombination in GSDIII patients. g.49898_53860del3963insTG is the first case of large deletion-insertion in AGL

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H. Universitario Marqués de Valdecilla, Santander, Spain P-436

Introduction: GLUT1 deficiency syndrome (GLUT1-DS) has a large clinical spectrum, including movement disorders without seizures. Case report: We report a three-year-old girl with mild developmental delay and abnormal gait episodes due to right lower limb dystonia that worsen with exercise. Later, abnormal gait makes continuous and an evident learning difficulty appears. Cerebral magnetic resonance imaging shows incipient parenchymal atrophy and two electroencephalograms show abnormal background-activity with poorly represented sleep elements and epileptiform discharges, without associated manifestations. GLUT1-DS is diagnosed by lumbar puncture, with cerebrospinal fluid glucose 46 mg/dl, normal lactate and CSF-to-blood glucose ratio 0,48. She starts ketogenic diet with rapid normalization of gait, progressive developmental improvement and, one month later, EEG backgroundactivity normalization and epileptiform abnormalities disappearance. No SLC2A1 gen mutations are found in the current-performing genetic studies. Three months later, gait disturbance relapses without developmental regression but with EEG worsening (recurrence of epileptiform activity and signs of EEG background-activity deterioration). Treatment with Levetiracetam is associated, with clinical improvement and EEG normalization in the next week. Conclusions: GLUT1-DS early diagnosis and treatment with ketogenic diet may improve patients outcome. EEG findings and clinical evolution seem to have an early correlation, so EEG could be useful in making therapeutic decisions. P-435 Alu-repetitive element mediated rearrangement and nonhomologous recombination in AGL in patients with glycogen storage disease type III

A third case of glycogen storage disease 1b and giant cell tumour of the mandible: a disease association or iatrogenic complication of therapy Estrella J1, Christodoulou J2, MacKellar G3, Tchan MC1 Genetic Medicine, Westmead Hospital, Sydney, Australia; 2WSGP, Childrens Hospital at Westmead, Sydney, Australia; 3Westmead Hospital, Sydney, Australia 1

Oral manifestations of Glycogen Storage Disease 1b (periodontitis, recurrent oral ulceration, bleeding diatheses and dental caries) are thought to be related to neutropenia and/or neutrophil dysfunction; similar oral manifestations are seen in other neutropenic states. Granulocyte-colony stimulating factor (G-CSF) in GSD 1b is indicated for persistent neutropenia, sepsis, inflammatory bowel disease and severe diarrhoea. We report a third case of GSD 1b whose oral manifestations included a giant cell tumour (GCT) of the mandible. Our patient was twelve years at GCT diagnosis and had been treated with GCSF for five years. He underwent therapy with interferon followed by local resection which was successful in initial control of the disease. Seven years later he required a repeat operation for local recurrence. GSD 1b and GCT of the mandible have previously been described in two other patients. Both were receiving G-CSF at time of tumour occurrence. GCSF (which stimulates osteoclastic differentiation) may be responsible for the development of these benign osteoclastic lesions. As GCTs are postulated to be reactive in nature, we hypothesize that the development of GCTs of the mandible in GSD 1b patients with concurrent G-CSF use, represents a disease association secondary to chronic oral mucosal inflammation. P-437 Assessment of safety and efficacy of extended release cornstarch therapy in glycogen storage disease

Fateen E.M.1, Okubo M2 Corrado MM1, Ross KM1, Brown LM1, Correia CE1, Weinstein DA1 Biochemical Genetics Dep National Res C, Cairo, Egypt; 2Endo&Met Dep Toranomon Hospital, Tokyo, Japan 1

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University of Florida, Gainesville, United States

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Background: Glycogen storage disease (GSD) is caused by mutations regulating glycogen synthesis or degradation. In 2012, an extendedrelease cornstarch (Glycosade) was released, but there are no studies on the long-term efficacy of Glycosade. Objective: Glycosade will allow patients longer uninterrupted sleep and improve quality of life without sacrificing metabolic control. Method: A prospective cohort study was performed. Glycosade was consumed in one nighttime dosage. Metabolic function tests were collected before and after 3 and 6 months of taking Glycosade. Results: Long-term follow-up data are available from 20 subjects treated with Glycosade (9 M, 11 F; average age 13.4 y). Fasting increased by an average of 4 hours. Markers of metabolic control remained stable on Glycosade trending towards improved control and lower laboratory studies: after 3 months (n=18), AST by 12%, ALT by 17%, triglycerides by 6%, and uric acid by 4%; after 6 months (n=8), AST by 3%, ALT by 18%, triglycerides by 0%, and uric acid by 5%. In the 1 GSD III patient, the CK decreased by 44% and there was a 21% decrease in ventricular mass. Conclusion: Glycosade has allowed patients with GSD to maintain normoglycemia for an extended period of time and improved quality of life without sacrificing metabolic control. P-438 Glycogen storage disease with phosphorylase kinase gamma (PHKG2) mutation (GSD IXc): a case report of novel clinical, molecular and pathological features and long term outcome post liver transplantation Kanungo S1,2, Aguilar J3, Lassman C3, McDiarmid S3, Abdenur J2,4

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P-439 Outcome of liver transplantation in a child with galactosaemia and progressive familial intrahepatic cholestasis type 3 McClean P1, Henderson M2, Prasad R1, Dobbie A3, Johnston C4, Brown A5, Walter JH6 1

Pead Hepatol, LTHT, Leeds, United Kingdom; 2Biochem Genet, LTHT, Leeds, United Kingdom; 3Clin Genet, LTHT, Leeds, United Kingdom; 4Neonatal Unit, LTHT, Leeds, United Kingdom; 5Biochem Genet, Southmead Hosp, Bristol, United Kingdom; 6Willink Biochem Genet Unit, Manchester, United Kingdom Liver transplantation is not usually necessary in galactosaemia. We report a child who received a liver transplant for PFIC type 3 but who also suffered from galactosaemia. An Asian boy presented with E.Coli septicaemia and liver failure at 7 days. Biochemical and genetic tests confirmed galactosaemia. He responded well to antibiotics and diet. Within the next few months his transaminases and GGT increased and he became pruritic. A liver biopsy showed marked fibrosis and copper associated protein that had progressed to micronodular cirrhosis by 18 months. He was confirmed to be homozygous for a nonsense mutation in ABCB4. He became jaundiced at 6 years. At 8 years he received a left lateral segment liver transplant for decompensated liver disease. Two years post transplant he is on a normal diet with normal liver function. Galactose 1 phosphate pre transplant was >1.48 μmol/g Hb (ref <0.6). Two years post transplant this was still elevated at 1.06. However, urinary galactitol has fallen by almost two thirds, from a pre transplant level of 369 μmol/mmol creat (ref 2-10) to 128 μmol/mmol creat. Galactitol may represent whole body galactose metabolism whereas the galactose 1 phosphate may be more indicative of that occurring in red cells.

1 UCLA Intercampus Genetics Training Prgm, Los Angeles, United States; 2CHOC Children's, Orange, United States; 3University of California Los Angeles, Los Angeles, United States; 4University of California Irvine, Irvine, United States

P-440

GSD IXc is caused by deficient activity of gamma subunit of phosphorylase kinase complex, encoded by PHKG2 gene. PHKG2 homozygous deleterious mutations are reported in patients with liver cirrhosis in adulthood. Long term outcomes post liver transplantation (LT) in GSD IXc - not known. We describe new PKHG2 deleterious heterozygous mutations in a Mexican adolescent patient, born to non-consanguineous parents. A missense mutation c.727 C>T leading to p.Arg243Cys on Exon 8 and a frameshift mutation c.843delA leading to p.Thr281ThrfsSTOP42 on Exon 9 were detected. Patient's clinical presentation included intellectual disability, seizure disorder, short stature, abnormal liver function, muscle weakness, hepatomegaly, but no reports of hypoglycemia. Over next few years, patient developed cirrhosis, progressing to end stage liver disease with portal hypertension and required a liver transplantation. Pre-LT biopsy demonstrated cirrhosis and variable hepatocellular morphology; some nodules had hepatocytes with GSD characteristics, whilst others had normal hepatocytes. Hepatectomy specimen demonstrated similar changes including additional findings of hepatocellular adenoma. Though, features suggesting carcinoma were absent. At 3.3 years post transplantation, patient had no transplant related complications, no seizures, liver functions normalized, significant improvement in muscle strength and tone, and improvement in cognitive ability and educational/ vocational performance. GSDIXc post-LT have good outcomes.

Mohnike K1, Wieland I2, Barthlen W3, Vogelgesang S4, Empting S1, Mohnike W5, Meissner T6, Zenker M2

Clinical and genetic evaluation of patients with katp-channel mutations from the German registry for congenital hyperinsulinism (CHI)

1 Dept of Pediatrics, OvG-University, Magdeburg, Germany; 2Inst Humam Genetics, OvG-University, Magdeburg, Germany; 3Clinic for Ped. Surgery, University, Greifswald, Germany; 4Institute of Pathology, University, Greifswald, Germany; 5DTZ Berlin Am Frankfurter Tor, Berlin, Germany; 6Dept. Gen Ped, University Child Hosp, Düsseldorf, Germany

Background: CHI is a disorder of dysregulated insulin secretion resulting in persistent hypoglycemia with potentially harmful consequences. Distinct types of CHI are described with diffuse, focal and atypical histology. The majority of mutations have been identified in ABCC8 and KCNJ11 genes encoding subunits of KATP-channel. Rapid genetic testing in infants has high impact on diagnostic strategy, i.e. referring for F18FDopa-PET/CT for localization of a circumscribed focus and individualized treatment with focus enucleation. Patients and Methods: Patients (146) from 136 unrelated families with clinical diagnosis of CHI were referred between 1/ 2005 and 11/ 2012. Coding and adjacent regions of ABCC8 and KCNJ11 exons were analyzed by Sanger sequencing. When no or only one heterozygous mutation in ABCC8 was detected multiplex ligation-dependent probe amplification (MLPA) was performed. Results: In 61 (45%) patients 64 different (incl. 40 novel) mutations in ABCC8 or KCNJ11 genes were identified. Biparental (recessive) inheritance occurred in 34% of mutation-positive patients, dominant inheritance

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in 11%. In 38% patients a paternally transmitted mutation was associated with focal type. Conclusion: Genetic analysis of ABCC8 and KCNJ11 genes revealed mutations in 45% unrelated patients of an unselected CHI patient cohort. No major recurrent mutation indicative of a founder effect was detected.

Alfa-glucosidase Inhibitors inhibit the conversion of carbohydrates to monosaccharides, and thereby slow postprandial glucose absorption. Kure et al. reported voglibose therapy improved hypoglycemia in a GSD Ib patient. The previous report and our result suggest that alfaglucosidase inhibitors are useful for management of GSD patients.

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P-443

Molecular diagnosis of glycogen storage diseases using next generation sequencing - detection of large scale deletions

Urine analysis and DNA sequencing as diagnostic tools for FBP1 gene mutations in costa rican patients with fructose-1,6bisphosphatase deficiency

Kirk RJ1, Allen KE1, Beauchamp NJ1, Dawe JH1, Niezen-Koning KE2, Smit GPA2, Grafham D1, Sharrard M3, Dalton A1

Badilla-Porras R1, Stockley T2, Saborio M.3, Schulze A1

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Sheffield Diagnostic Genetics Service, Sheffield, United Kingdom; 2Dept Metab Dis, Univ Med Cen Groningen, Groningen, Netherlands; 3Sheffield Children's NHS Foundtn Trust, Sheffield, United Kingdom

Dept Clin Metab Gen, Hosp for Sick Child, Toronto, Canada; 2Dept Ped Lab Med, Hosp for Sick Child, Toronto, Canada; 3Serv Gen Metab, Hosp Nac Niños, San José, Costa Rica

Massively parallel, or next-generation sequencing (NGS), is an efficient, accurate, and cost-effective method for simultaneous analysis of many genes. NGS is particularly useful for clinical diagnosis in genetically heterogeneous disorders such as the glycogen storage diseases (GSD). We have used NGS to sequence over thirty genes associated with GSD and related disorders. Validation work on patients diagnosed in-house by Sanger sequencing confirmed all previously reported mutations. NGS analysis was also able to detect large scale heterozygous deletions, a valuable addition to the current methodology for molecular diagnosis in autosomal recessive conditions. Patients referred with liver specific clinical symptoms and a possible diagnosis of GSD VI or IX were then examined with NGS. Pathogenic mutations were identified in all five patients. Mutations were detected in PHKA2 and, unexpectedly, G6PC. One patient has a previously unreported, hemizygous deletion encompassing the entire PHKA2 gene. Sanger sequencing could have required sequential analysis of at least four genes to determine the underlying cause of their GSD. These initial results illustrate the validity of NGS as a rapid, efficient and relatively inexpensive methodology for molecular diagnosis in clinically and genetically heterogeneous disorders. It also illustrates its ability to identify mutations not detectable by current sequencing technologies.

Background: Fructose-1,6-bisphosphatase is a key enzyme of gluconeogenesis. In acute crisis, the urinary profile is characterized by increased excretion of Lactate, 3-OH-Butyrate, Glycerol and Glycerol-3-Phosphate. Methods: the first phase of this project was a biochemical screening of patients with a previous hypoglycemic decompensation using gas chromatography-mass spectrometry (GC-MS) urine analysis. The second phase was DNA sequencing of the exons and flanking intronic regions of the FBP1 gene for all the patients that tested positive in the screen. Results: A total of 20 patients from 19 non related families screened positive; 14 out of 20 patients were homozygous for the same mutation in exon 8 (c.961insG; p. Ser321ValfsX13). The c.961insG mutation is a frameshift with predicted early termination, and in silico analysis revealed a probably deleterious effect (Polyphen score of 1.0 and SIFT score of 0.01) that was confirmed by 0% residual enzyme activity in patient's lymphocytes. Enzyme activity is pending for the remaining 6 screen positives without mutations in the FBP1 gene. Conclusions: this study is the first report to our knowledge of FBP1 gene analysis in a Latin American population. The findings suggest that there is a founder mutation in the Costa Rican population for the FBP1 gene

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Molecular genetics, phenotype and outcome of 41 Vietnamese patients with congenital hyperinsulinism

An alfa-glucosidase inhibitor improved hypoglycemia, hyperlactemia and growth retardation in a patient with type IA glycogen storage disease Yamamoto S1

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Vu D1, Nguyen L1, Nguyen K1, Can N1, Bui T1, Nguyen H1, Khu D1, Dang D1, Nguyen D1, Flanagan S2, Ellard S2 National Hospital of Pediatrics, Hanoi, Viet Nam; 2Peninsula Medical School, Exeter, United Kingdom 1

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Dept Ped Shimoshizu Natl Hosp, Yotsukaido, Japan

Affected individuals with Glycogen storage disease (GSD) type Ia exhibit growth retardation, lactic acidemia, etc. Treatment aims at preventing hypoglycemia in order to avoid neurological involvement and long-term complications (hepatic, renal, etc.) and to assure normal growth. Treatment is essentially dietary and consists of frequent meals and ingestion of uncooked cornstarch. However, growth retardation and other complications are often observed in spite of strict dietary therapy. A Japanese girl was diagnosed at age 4, and dietary treatment was started. The most prevalent mutation in Japanese, 727G-T, was detected in both alleles. At age 10, because control of the blood glucose levels became difficult and growth retardation was apparent (-1.5SD), trial therapy of an alfa-glucosidase inhibitor, voglibose, was added. Hypoglycemia and hyperlactemia were improved immediately. Improvement of hepatomegaly and catch-up growth were also achieved, and final height reached above the mean (+0.5SD) at age 18. The long-term complications were not observed at age 24.

Background: Hyperinsulinemic hypoglycemia (HH) is a consequence of unregulated insulin secretion by pancreatic β-cells. Objective: To identify mutations in ABCC8; KCNJ11, HNF4A and GLUD1 in Vietnamese patients with congenital HH, to describe phenotype – genotype correlation, and to evaluate outcome. Patients & Methods: 41 probands with congenital HH were analyzed for alterations in ABCC8; KCNJ11, HNF4A and GLUD1 using PCR & direct sequencing. Phenotype & outcome were reviewed. Results: 22/41 (54%) cases without mutations in ABCC8; KCNJ11, HNF4A and GLUD1 were stable with medical treatment. Sixteen probands (39%) had mutations in the ABCC8. Their blood glucose levels were normal after surgery. Altogether, 9 different ABCC8 mutations including three novel alterations (p.F686I, p.I395F and p.G1379S) and six reported mutations (p.F686S,IVS27-1G>A, p.R999X, c.1467+5G>A, p.R934X and p.S1387del) were identified. One case of responsive with diazoxide had parental inherited mutation in KCNJ11 [c.482C>T (p.A161V)] and one case of unresponsive with diazoxide and needed octreotide until 5 months

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of age who had homozygous mutation in KCNJ11 [c.185delC (p.T62fs)]. One case of responsive with diazoxide had a novel mutation and maternal inheritance in HNF4A [c.659T>C (p.L220P)]. Conclusions: Our results extend the knowledge of the molecular genetics, phenotype and outcome behind congenital HH in Vietnam.

early intervention, the youngest sibling has marked nephrocalcinosis and significant hepatomegaly. All four cases had significant osteopenia (DEXA Z score:-2.1 to 3.47), resulting in bone pain and immobility. This dramatically improved on intensified supplementation of electrolytes / vitamin D followed by orthopedic intervention. Continuous glucose monitoring showed predominately hyperglycaemia.

P-445 P-447 Two siblings with glycogen storage disease type IX; very early need of transplantation in the younger one Önenli-Mungan N1, Kör D1, Şeker Yılmaz B1, Ballı T1, Podskarbi Y.S2 1

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Çukurova University, Adana, Turkey; Molecular Genetics and Metabolism Lab., Munich, Germany Background: Glycogen storage disease type IX (GSD IX) due to the deficiency of hepatic phosphorylase kinase, occurs approximately 1 in 100,000 births and accounts for 25% of all GSDs. The enzyme is encoded by PHKA2, PHKB and PHKG2 genes. Clinical symptoms are hypoglycemia, hepatosplenomegaly, short stature, and hepatic dysfunction. The tissue specificity of the enzymatic activity causes high variability in clinical phenotype. Case Report: Here we report two siblings diagnosed as GSD type IX. Fasting hypoglycemia, hepatosplenomegaly, liver dysfunction and growth retardation are the main symptoms of both patients. Enzymology performed on blood samples of both patients revealed decreased levels of phosphorylase kinase activity. With mutation analysis the determined subtype was GSD type IX C. Although the younger brother showed a rapid progression towards liver cirrhosis and underwent a successful liver transplantation, elder brother is still handling with only supportive therapy. Conclusions: The different clinical progression of two siblings with GSD IX point out the phenotypic and probable allelic heterogeneity in GSD IX. It is evident that this disease exists in multiple forms even in the same family. The clinical variability of these siblings suggests us the phenotypic modulation by additional genetic and/or epigenetic factors. P-446 Further delineation of the clinical presentation of Fanconi-Bickel syndrome

Hyperinsulinism-hyperammonemia syndrome - a rare cause of hypoglycemia in childhood Konstantopoulou V1, Roscher A1, Ratschmann R1, Vodopiutz J1, Liepins E2, Santer R3, Moeslinger D1 1 Dept Ped Adol Med, Med Univ Vienna, Vienna, Austria; 2Dept Child Adol Med, Hosp Wr Neustadt, Wiener Neustadt, Austria; 3Dept Ped, Univ Med Cent Hamburg Ependorf, Hamburg, Germany

Background: The glucose homeostasis in blood depends on many physiological mechanisms. Based on the findings of ongoing research, new disorders causing hypoglycemia have been reported. Case report: We report a 2.5 years old child presenting with repeated shaking and fatigue at the age of 1.9 years. With the exception of a minor microcephaly, the psycho-motoric development and family history were inconspicuous. At age 2 the patient showed an afebrile generalized tonic-clonic seizure. At that time the plasma glucose measured 1.7 mmol/l. The patient showed normal EEG and brain MRI. After 18 hours fasting the plasma glucose measured 2.5 mmol/l and thirty minutes postprandial only 1.4 mmol/l. Thirty minutes after beginning the oral leucine tolerance test insulin levels raised to 48 μU/ml (Ref. < 2). The serum ammonia was slightly elevated at 70 mmol/l (Ref. < 48). The hypoglycemic episodes improved after treatment with diazoxide and a low protein diet. The patient was found to be heterozygous for the gainof-function mutation, p.Ser270Cys, in the GLUD1 gene. Discussion: The Hyperinsulinism-Hyperammonemia syndrome (HI/HA) is a rare condition in the differential diagnosis of hypoglycemia. Since rapid recognition prevents neurodevelopmental delay with the known associated complications, HI/HA syndrome should be included in the hypoglycemia work-up. P-448

Foley PA1, Kwok KM1, Stafford J1, McSweeney M1, Spoudeas H2, Trompeter RS3, Roposch A4, Al Eneze FH5, Alobaidy H6, Grunewald S1 1

Glut-1 deficiency syndrome: clinical and genetic study of five patients

Metabolic Dept Great Ormond St. Hos, London, United Kingdom; Endocrinology Dept. Gt Ormond St. Hosp, London, United Kingdom; 3 Nephrology Dept. Gt Ormond St. Hosp, London, United Kingdom; 4 Orthopaedic Dept. Gt Ormond St Hosp, London, United Kingdom; 5 Metabolic Dept, Al Jahra Hosp, Kuwait, Kuwait; 6Metabolic Dept. Elkhadra Hosp, Tripoli, Libyan Arab Jamahiriya

Flotats-Bastardas M 1, Vila-Pueyo M2 , Quiroz A1 , Domingo R3, Raspall-Chaure M1, Macaya A1, Macaya A2

Patients: We discuss four cases of Fanconi Bickel syndrome (FBS) of consanguineous parentage: three of nine siblings from Kuwait (16, 12, 8 years old) and an isolated case (8 yr) from Libya. Clinical Findings: All cases presented with typical features of FBS: renal tubulopathy, hypophosphataemic rickets and short stature. None of them had cataracts. The isolated case was originally diagnosed and treated as tyrosinaemia, however enzyme analysis of fumaryl-acetoacetase proved normal; a diagnosis of FBS was confirmed only at 7y (homozygosity for c.964-2A>C gene mutation intron 7 of the SLC2A2 gene). A liver biopsy of the eldest Kuwait sibling at 6 months suggested FBS; genetics showed homozygosity for c.474A>C exon 4, a novel missense mutation. All children had nephrocalcinosis and marked hepatomegaly initially. On recent review, the two elder Kuwait brothers show only borderline enlarged liver and subtle nephrocalcinosis. Despite a prompt diagnosis /

Background: Glut-1 deficiency syndrome is due to heterozygous mutations in SLC2A1. The classical phenotype comprises epilepsy, microcephaly and severe developmental delay that improves with ketogenic diet. However, the clinical spectrum has been extended to encompass less severe phenotypes, usually including paroxysmal or fluctuating clinical signs. Objective: To describe the clinical and molecular findings of five patients, including two monochorionic twins. Patients and methods: Patients were diagnosed at ages 8-14 years. All displayed mild-to-moderate cognitive dysfunction. Other symptoms were exercise-induced paroxysmal dyskinesia (n=3), epilepsy (n=4), migraine (n=2), tremor (n=1), and episodic ataxia (n=1). Three patients had a CSF/plasma glucose ratio <0,4 and two <0,6. Molecular studies confirmed the diagnosis in all patients and detected a previously

2

Ped Neurol Serv, Hosp Univ Vall Hebron, Barcelona, Spain; 2Ped Neurol Res Group, VHebron Res Inst, Barcelona, Spain; 3Neuroped Serv, Hal Virgen Arrixaca, Murcia, Spain 1

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reported mutation (p.Arg223Trp), two novel ones (p.Val165Ile and p.Val237Valfs*2), and a deletion of exon 1. Ketogenic diet or uncooked cornstarch partially improved some clinical manifestations Conclusion: The phenotypes in our present patients seem to be more frequent than the classical one. Thus, Glut-1 deficiency must be suspected in any patient with unexplained cognitive dysfunction and paroxysmal disorder at any age, even with mild hypoglycorrhachia. Appropriate dietary treatment can lead to an improvement of the paroxysmal manifestations although cognitive dysfunction is expected to be less responsive.

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paterns of apolipoprotein C-III IEF pointing to an O-glycosylation defect. At age of 16 months, he died suddenly at home after an epileptic seizure. P-451 GFPT1 mutations as an underlying cause of congenital myasthenic syndrome Zoltowska K1, Webster R1, Finlayson S1, Maxwell S1, Cossins J1, Beeson D1

P-449 1

Oxford University, Oxford, United Kingdom

An induced pluripotent stem cell model of GALT deficiency recapitulates the galactosemia biochemical phenotype Demirbas D1, Huang X 1, Cianci A1, Fitzgerald K1, DeVine A1, Schlaeger T1, Sahin M1, Daley GQ1, Berry GT1 1

Boston Child Hosp, Harvard Med School, Boston, United States

Classic galactosemia causes long term complications including cognitive deficits, language delays, speech defects and premature ovarian insufficiency. Our goal is to make induced pluripotent stem (iPS) cells from patients with galactose-1-phosphate uridyltransferase (GALT) deficiency and differentiate them and their TALEN gene edited control lines into neurons to study the molecular mechanisms of galactose stress on the transcriptome and metabolome. Two galactosemic fibroblast cell lines (GM1703 and GM727) and CD34+ cells from patients with Q188R/Q188R genotype were reprogrammed using oriP/EBNA1-based episomal vectors that derive the expression of human SOX2, KLF4, L-MYC, LIN28 and OCT3/4 along with suppression of p53. Isolated iPS cell colonies show distinct stem cell colony morphology and express pluripotency markers SSEA4, Tra1-60, OCT4 and Nanog as judged by immunoflorescence microscopy. Galactosemic iPS lines showed non-detectable GALT enzyme activity by LC-MS/MS. On day five of exposure to 5 mM galactose, galactosemic iPS cells accumulated galactose metabolites, suggesting that the biochemical phenotype seen in patients is recapitulated in these cells. Since nervous system and ovarian tissues from patients with galactosemia are not readily available for study, galactosemic iPS cells, as well as their neural progenitor and neuronal derivatives, may prove to be relevant cell types to uncover disease mechanisms.

Mutations in glutamine: fructose-6-phosphate transaminase 1 (GFPT1) - a rate-limiting enzyme in the hexosamine biosynthetic pathway providing building blocks for glycosylation of proteins and lipids - underlie a congenital myasthenic syndrome (CMS) characterised by a limb-girdle pattern of muscle weakness. It is not readily apparent why mutations in this ubiquitously expressed protein should cause a syndrome with symptoms restricted to the neuromuscular transmission. In order to elucidate the pathogenic mechanism of GFPT1 CMS we investigated acetylcholine receptor (AChR) expression in GFPT1mutated human skeletal myotubes and in a muscle cell line (TE 671 DB40) with GFPT1 silenced by siRNA. Cultured myotubes derived from two patients with GFPT1 mutations (Pt1 and Pt2) and TE 671 DB40 muscle cells with GFPT1 expression 'knocked down' showed a significant reduction in cell-surface AChR expression (Pt1 p<0.0001; Pt2 p=0.0097; TE 671 DB40 p<0.0001). This decrease appeared to be due to the reduced steady-state levels of AChR α, δ, ε, but not β subunits. The reduced AChR levels resulted from an increased protein degradation rather than diminished mRNA expression. Thus, the decreased levels of AChR at the motor endplate are likely to be a primary disease mechanism in the GFPT1 CMS. P-452 N-acetylmannosamine mitigates sialidase-induced podocyte injury in mice Malicdan MC1, Okafor O1, Leoyklang P1, Zerfas P2, Tardeni T3, Starost M2, Kopp JB4, Gahl WA5, Huizing M1 1

Medical Genetics Branch, NHGRI, NIH, Bethesda, United States; Office of Research Services, OD, NIH, Bethesda, United States; 3 Metab Dis Unit, Sheba Medical Center, Tel Aviv, Israel; 4Kidney Disease Section, NIDDK, NIH, Bethesda, United States; 5Office of Rare Dis Research, OD, NIH, Bethesda, United States 2

11. Glycosylation disorders P-450 A case of infant with brain malformations and suspected O-glycosylation defect Albahri Z1, Marklová E1, Stefáčková S2 1

Dep. Ped., Faculty Hosp, Charles Univ, Hradec Kralove, Czech Republic; Neurol. Dep., Faculty Hosp, Charles Univ, Hradec Kralove, Czech Republic

2

Defects in protein glycosylation have been linked to several forms of congenital muscular dystrophy that are frequently associated with brain abnormalities. We describe a 16 months old patient. At age of 2 months he presented with iritability, tremor, microcephaly, hypertonia and seizure, brain MRI showed hypoplasia of the corpus callosum and epidural hematoma. At age of 13 months he was addmited to the hospital for an episode of seizure, he presented with psychomotor retardation, strabismus and moderate elevation of creatine kinase, diagnostic work-up revealed abnormal

Background: Glomerular diseases are increasingly prevalent causes of end-stage-renal-disease. Current therapies are mostly targeted to reduce disease progression, but many carry side effects. Podocyte injury is present in a wide variety of glomerular diseases, and aberrant sialylation of glomerular glycans was found in sporadic cases of podocyte pathology. Methods: We explore the effects of hyposialylation on podocyte injury in mice and test the therapeutic effects of the sialic acid precursor Nacetylmannosamine (ManNAc). We induced podocyte injury in mice by intraperitoneal injection of Vibrio cholera sialidase, an enzyme that preferentially removes alpha 2-6-linked sialic acid endgroups from glycans. The resulting renal alterations were measured by documenting proteinuria, hypoalbuminemia, morphologic, proteomic, ultrastructural and biochemical changes. Results: Sialidase injection induced proteinuria, renal failure, flattening of podocytes, and hyposialylation of glomerular glycoproteins. Both prophylactic and therapeutic oral treatment with ManNAc significantly reduced proteinuria and podocyte injury.

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Conclusion: Our preclinical findings indicate that patients with unexplained glomerular disease may harbor a glycan sialylation defect that can be mitigated by increasing sialylation through oral ManNAc therapy. Since ManNAc is currently in Phase 1 clinical trial for the treatment of a hyposialylation disorder, GNE myopathy, this sugar could be repurposed for trials in patients with glomerular hyposialylation. P-454 Post-mortem magnetic resonance imaging (MRI) in a critically ill infant as a complementary tool for diagnosis of PMM2-CDG

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Our results demonstrate (i) the expression of NCX and NCKX proteins in human platelets, (ii) NCX1 and NCKX1 45Ca2±uptake, (iii) heavily N- and O-glycosylated proteins by lectin-affinity chromatography and by Western blot. Proteins were detected after the immunoprecipitation and, with different lectins, after enzymatic deglycosylation, (iv) lower levels of NCKX1 (n=3; P<0.05) and reduced expression of NCX1 in a PMM2-CDG patient (n=3; P< 0.0001) and (v) a significantly decreased Na±dependent 45Ca+2 uptake in a PMM2-CDG patient with abnormal platelet aggregation (t-test p< 0.05). Here we report that both exchangers are glycosylated and that the expression and activity of NCX1 are more dramatically reduced than NCKX1, suggesting a possible role for the abnormal platelet aggregation in CDG patients. CONICET-GI11220090100063/FONCyT/PICT2010-2824.

Haliloglu G1, Hismi B2, Oguz KK3, Dursun A2, Tokatli A2, Anlar B1 P-456 Ped Neurol, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 3Radiology, Hacettepe Univ Child Hosp, Ankara, Turkey 1

PMM2-CDG is the most frequent glycosylation disorder affecting the N-glycosylation pathway. Pediatric patients have a broad and variable clinical picture that affects nearly all systems. A 5.5-month-old girl, with dysmorphic features, inverted nipple, and abnormal fat distribution, admitted because of fever, gastroenteritis and focal seizures. She was born at 36th gestational week with a birthweight of 2790 g. There was a history of early membrane rupture, gestational diabetes, surfactant treatment, and neonatal hypoglycemia episodes. She presented with bilateral sensorineural deafness, abnormal liver function tests, pericardial effusion, and hypothyroidism at the age of 2 months. During the course of the disease she developed respiratory insufficiency, abnormalities in coagulation parameters, electrolyte disturbances, and died due to cardiac arrest. CDG screening revealed type 1 pattern. Postmortem MRI showed subgaleal bleeding in the parietal region, diffuse hpomyelinisation, middle cerebellar peduncle and severe cerebellar atrophy, increased cerebellar cortical intensity. Diagnosis of PMM2-CDG was confirmed by molecular diagnosis. Dysmorphic features, inverted nipple and abnormal fat distribution trigger diagnostic effort for CDG. In critically ill patients in whom the results are pending and/or a neuroimaging study is not suitable, postmortem MRI can serve as a complementary tool to define the CNS involvement and aid in differential diagnosis. P-455 NCX1 and Ca+2 exchangers in human platelets: a putative role in the thrombo-hemorrhagic events associated with congenital disorder of glycosylation Bistué Millón MB1, Siravegna M2, Spécola N3, Chacón S4, Dodelson de Kremer R5, Elso G2, Asteggiano CG6 CONICET-CEMECO (UNC), Córdoba, Argentina; 2CONICETINIMEC (IMMF), Córdoba, Argentina; 3Neurometabolism Child Hosp, La Plata, Buenos Aires, Argentina; 4Hospital Centenario de Gualeguaychu, Gualeguaychú, Entre Ríos, Argentina; 5CEMECOUNC Children´s Hospital, Córdoba, Argentina; 6CONICET-CEMECO UNC - UCC, Córdoba, Argentina 1

Congenital Disorders of Glycosylation (CDG) are genetic diseases due to defects in glycoproteins or glycolipids synthesis. The phenotype is multisystemic and thrombo-hemorrhagic events are frequently observed in these patients. In platelets, Ca+2 signaling is necessary to prevent inappropriate thrombus formation. The Na+/Ca2+ (NCX) and Na+/K±Ca2+ (NCKX) exchangers play a crucial role in controlling cytosolic [Ca2+]. The aim of this report is to contribute to the characterization of these exchangers in human platelets, with special emphasis in CDG patients with thrombo-hemorrhagic events.

Skeletal dysplasia due to congenital disorders of glycosylation Martinez Domenech EG1, Delgado MA1, Sarrión P2, Matthijs G3, Guelbert N1, Dodelson de Kremer R1, Balcells S2, Grinberg D2, Asteggiano CG4 1 CEMECO-UNC Children´s Hospital, Córdoba, Argentina; 2Dep Genética, Fac. Biol UB,CIBERER, IBU, Barcelona, Spain; 3Center for Human Genetics, Leuven Univ, Leuven, Belgium; 4CONICETCEMECO UNC - UCC, Córdoba, Argentina Defects in N-, O-glycosylation and combined glycosylation pathways have been identified as Congenital Disorders of Glycosylation (CDG). Most of them are autosomal recessive, but multiple osteochondromatosis (EXT1/EXT2-CDG) was described as a dominant disease restricted to the cartilage. A peculiar skeletal phenotype has been described in CDG patients and it has gained special relevance over the past few years. In patients exposed to the cell hypoglycosylation due to altered glycosylation pathway, numerous extracellular matrix proteins undergo glycosylation defects that lead to skeletal manifestations. The aim of this work is to communicate the first CDG research program in Argentina to study the glycobiology in skeletal dysplasia associated with CDG. Here we report the results of (i) a cohort of 42 patients diagnosed with clinical osteochondromatosis, most of them presenting a severe phenotype (83%), including condrosarcoma (11%), with mutations detected in EXT1 (72%) or EXT2 (28%), (ii) a patient with osteopetrosis-like phenotype, transferring-IEF type-II and altered fucosylation observed by mass-spectrometry, in which only a heterozygous variation was found in COG6, p.V631M (c.1891G>A); and (iii) the screening for: GALNT3-CDG (hyperfosfatemic-tumoral calcinosis); LFNG-CDG (spondylocostal-dysostosis); SLC35D1-CDG (Schneckenbecken-dysplasia); and B4GALT7-CDG (Progeroid-variant Ehlers-Danlos). Our results highlight the hypoglycosylation effect on the genesis of skeletal manifestation in CDG. FONCyT-PICT2010/2824-UCC2012-SAF2011-25431-PIB2010AR00473

P-457 Allogeneic bone marrow transplantation (BMT) ameliorates muscle atrophy, degeneration, and weakness in mice with GNE myopathy Malicdan MC1, Okafor OU1, Momma KK2, Funato F3, Hayashi YK3, Nonaka I3, Huizing M1, Gahl WA1, Nishino II3, Noguchi S3 1

Nat Human Genome Res Institute, NIH, Bethsda, United States; 2Dept of Neur, Nat Def Med Coll, Saitama, Japan; 3Nat Cntr of Neuroscience and Psychiatry, Kodaira, Japan Background: GNE myopathy is an autosomal recessive disease. Clinical features and pathology include progressive weakness and atrophy of the distal muscles, as well as the presence of myofiber vacuolation and

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degeneration. GNE myopathy is due to mutations in the GNE gene – this encodes a critical enzyme in the sialic acid biosynthesis. Hyposialylation has been shown to be an important element in the pathogenesis of GNE myopathy, although the pathomechanism remains elusive. Specifically, oral administration of sialic acid and its precursor, ManNAc, prevented myopathy in a mouse model. Though promising, these sugars are rapidly excreted which will require frequent dosing. We propose allogeneic bone marrow transplantation (BMT) as a rational therapy because of its continuous supply of sialic-acid producing cells. Methods: CAG-GFP mice were irradiated and their bone marrow cells subsequently injected into 30-week old GNE myopathy mice. We assessed changes monthly and noted an increase in cell surface sialylation correlating with chimerism. Results: BMT treatment markedly improved lifespan, motor performance, and muscle phenotype; sialylation of specific muscle glycoproteins and organs; and GNE enzymatic activities. Conclusion: Our results provide a proof-of-concept for BMT in GNE myopathy and show that hematopoietic cells can be a good source of sialic acid.

In a cohort of patients with PGM1-CDG1, three patients were treated with complex carbohydrates and galactose. Clinical characteristics included uvula bifida with or without cleft palate at birth, moderately elevated serum transaminases, low blood sugars, growth retardation, exercise intolerance and reduced coagulation factors. Two of the patients were diagnosed in their teens and had developed heart problems, whereas a young patient was recently diagnosed at 2.5 years of age with severe hypoglycemia and no heart involvement yet. By mass spectrometry of intact transferrin, a diagnostic profile was observed with a loss of complete glycans as well as the presence of truncated glycans lacking galactose. Low levels of blood glucose could be prevented by treatment with complex carbohydrates. The N-glycosylation profile remained severely affected under these conditions. After addition of galactose to the diet, the N-glycosylation profile of transferrin improved within two weeks time to near-normal. After prolonged periods of galactose treatment, the glycosylation profile became slightly more abnormal. Clinical symptoms related to the glycosylation problems improved with time. 1. Timal S, et al. Hum Mol Gen, 2012. P-460

P-458 Cellular models for functional confirmation in the dystroglycanopathies Riemersma M1, Jae L2, Buysse K1, Kamsteeg EJ1, Beusekom E1, Wevers RA1, Willemsen MA1, Brummelkamp T2, Van Bokhoven H1, Lefeber DJ1 1

2

Radboud University Medical Center, Nijmegen, Netherlands; The Netherlands Cancer Institute, Amsterdam, Netherlands In the muscular dystrophy-dystroglycanopathy syndromes, a specific type of O-mannose glycosylation is affected of the sarcolemmal protein alpha-dystroglycan. The clinical spectrum ranges from WalkerWarburg syndrome (WWS) with congenital muscular dystrophy and complex eye and structural brain defects to milder forms with limbgirdle muscular dystrophy and dilated cardiomyopathy. More than 10 genetic causes are known. Functional confirmation of novel genetic variants is complicated by a lack of suitable models. A functional genomics approach was developed in model cells and patient fibroblasts. Complementation of deficient alpha-dystroglycan glycosylation was performed by transfection with wild-type genes, followed by a functional read-out using a glycosylation-specific antibody and analysis by flow cytometry. An available cell line allowed confirmation of pathogenicity of B3GNT1 mutations1. In patient fibroblasts, lowered glycosylation efficiency was observed, however, not yet sufficiently robust for diagnostic application. Novel well-defined knock-out cells were created by use of transcription-activator like effector nuclease (TALEN) technology2 and used for the validation of new WWS genes, such as SGK196 and B3GALNT22. This provides a novel avenue for functional confirmation of novel genetic variants in the dystroglycanopathies. 1. Buysse K, et al, Hum Mol Genet 2013 2. Jae L, et al, Science 2013 P-459

The challenge of congenital disorders of glycosylation: nextgeneration sequencing as a powerful diagnostic-tool Medrano C1, Gómez-Puertas P2, Desviat LR1, Ugarte M1, Pérez-Cerdá C1, Pérez B1 1

CEDEM, CBMSO (CSIC-UAM), CIBERER, IDIPAZ, Madrid, Spain; BioMol-Informatics, S.L., UAM, Madrid, Spain

2

Congenital disorders of glycosylation (CDG) are a heterogeneous group of disorders caused by genetic defects in the glycosylation pathways. Most of them have a wide clinical spectrum and biochemical approaches cannot point out to a solely genetic defect, making diagnosis time consuming. Up to now, we have diagnosed over 40 CDG patients but there is still 23 CDG-suspected patients genetically unsolved. Therefore, our goal was to identify the gene and disease-causing mutations in that cohort of uncharacterized CDG-patients. We have addressed the genetic characterization by targeted exome sequencing (TES) and whole exome sequencing (WES). TES allowed us the detection of 3 out of 16 patients (RFT1, DPAGT1 and COG7). This low rate was probably due to limited number of 43 genes captured. For WES analysis we prioritized a list of 150 genes and detected 3 patients (DPAGT1, GALT and DPM1). Based on the clinical features of the GALT patient, we identified by Sanger sequencing two more GALT-deficient patients. Searching for new genes is still ongoing. In summary, we have detected disease-causing mutations in 8 patients using NGS. Besides, WES is a powerful technique for an efficient analysis when dealing heterogeneous disorders comprising a large cohort of genes such as CDG. P-461 Diagnostic mass spectrometry of intact transferrin reveals novel gene defects in CDG type II Van Scherpenzeel M1, Steenbergen G1, Morava E1, Wevers RA1, Lefeber DJ1

Follow-up on treatment response in PGM1-CDG 1

Morava E1, Van Scherpenzeel M1, Korsch E2, Wortmann S1, Maatman R3, Jonge Poerink A4, Sykut-Cegielska J5, Adamowicz M5, Socha P5, Wevers RA1, Lefeber DJ1 Radboud University Medical Center, Nijmegen, Netherlands; 2Pediatric Hospital, Koln, Germany; 3 Medlon BV, Enschede, Netherlands; 4 Medisch Spectrum Twente, Enschede, Netherlands; 5The Children's Memorial Health Institute, Warsaw, Poland 1

Radboud University Medical Center, Nijmegen, Netherlands

Subtype identification in the Congenital Disorders of Glycosylation (CDG) type II is a main challenge in CDG diagnostics. The clinical heterogeneity in this group is increasing rapidly with inclusion of skeletal dysplasia syndromes, non-syndromal liver disease, intellectual disability and neurological syndromes. In addition, the underlying biochemical mechanisms in this group are poorly understood, especially the Golgi trafficking defects.

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We applied a novel nanochip-C8-QTOF mass spectrometry method for the analysis of intact serum transferrin. The high resolution allowed direct structural annotation of peaks in as little as 5 microliter of serum within 1 h analysis time. Application in our cohort of patients with an abnormal CDG-II isofocusing profile revealed several subgroups with highly characteristic glycosylation profiles. In ten patients, a typical profile was observed with hybrid type N-glycans. Exome sequencing in a single patient revealed mutations in MAN1B1, which was confirmed in all other patients with the same profile. Clinical characteristics were intellectual disability, obesity and facial dysmorphisms. In several other patients, the same approach revealed mutations in SLC35A2, the X-linked UDP-galactose transporter. Our results show the promising addition of QTOF mass spectrometry to the diagnostic repertoire for fast identification of CDG-II subtypes. P-462

hypotonia during infancy. Since 3.5 years of age she developed limb-girdle congenital myasthenic syndrome accompanied by moderate intellectual disability and hepatopathy. LLO analysis in fibroblasts demonstrated normal profile. Exome sequencing revealed compound heterozygosity for novel mutations c.85A>T (p.I29F) and c.652C>T (p.R218W) in the DPAGT1 gene. In the second patient (7-year old girl) the cerebello-ocular syndrome has been diagnosed since infancy. Nystagmus, strabism, optic nerve hypoplasia, visual impairment, stereotypic movements and progressive ataxia and obesity are the hallmark of the disease. These findings are accompanied with severe coagulopathy and fluctuating hepatopathy. LLO analysis in fibroblasts proved incomplete LLO DolPP-GlcNAc2Man5 accumulation. By sequencing of "CDG-exome" identified SRD5A3 as a candidate gene. The causal role of the mutations found in SRD5A3 gene is being validated. We conclude that broad sequencing approaches enable efficient molecular diagnosis. Supported by RVO-VFN64165/2012, IGA NT 12166-5/2011 and GAUK 638512

Glycosylation defect caused by mutations in phosphoglucomutase 1 gene Ondruskova N1, Hansikova H1, Vondrackova A1, Tesarova M1, Honzik T1, Zeman J1 1

Dep Pediatr, Charles Univ, Gen Univ Hosp, Prague, Czech Republic

Background: Congenital disorders of glycosylation (CDG) are a complex group (> 70 types) of multisystemic and clinically heterogeneous diseases characterized by defective protein glycosylation. In this study, we report a case of a 10-year-old boy presenting with short stature, obesity, borderline mental retardation, hepatopathy, ketotic hypoglycemia, cholecystolithiasis and intermittent myopathy. Material and methods: Patient was screened for CDG by isoelectric focusing (IEF) of serum transferrin (TRF) and apolipoprotein C-III (ApoCIII), using PhastSystem workstation (GE Healthcare). Enzyme activity of phosphoglucomutase was measured in cultivated fibroblasts by spectrophotometric analysis. After PCR amplification, PGM1 gene was sequenced using ABI PRISM 3100 Avant Genetic Analyzer (Applied Biosystems). Results: Elevated levels of hyposialylated TRF isoforms were detected, while the ApoC-III glycoforms´ profile was normal. The patient´s clinical symptoms prompted us to investigate phosphoglucomutase activity in cultivated fibroblasts, where a marked decrease was found (<5 % of controls). Sequencing of PGM1 gene revealed the presence of two heterozygous mutations c.1010C>T (p.T337M) and c.1508G>A (p.R503Q). Their pathogenicity was confirmed by in silico analysis. Conclusion: We describe the first Czech patient with defective glycosylation due to mutations in phosphoglucomutase 1 gene. Acknowledgement This work was supported by GAUK 638512, 1.LF UK and IGA MZ NT/12166-5.

P-464 Promising results of oral D-mannose treatment of an ALG1-CDG (CDG-IK) child James PM1, Li X2, Zhang W2, Freeze HH3, He M2 Div Gen & Metab, Boston Child Hosp, Boston, United States; 2Dept Hum Gen, Emory Univ, Atlanta, United States; 3Sanford-Burnham Med Res Inst, La Jolla, United States 1

Patients with congenital disorder of glycosylation (CDG) type Ib show clinical improvement when given D-mannose. Our patient presented at 9 months of age with microcephaly, seizures, visual impairment and global developmental delay and a genetic diagnosis of ALG1-CDG (CDG-Ik). Like other ALG1-CDG cases, his cultured fibroblasts produced a specific abnormal tetrasaccharide biomarker. Addition of 500uM D-mannose to the fibroblast culture medium decreased the tetrasaccharide by 86% compared to cells grown without D-mannose. We extended this finding to the patient in a one-year treatment trial with 1.0 gm/kg/day of D-mannose beginning at age three years. CDG transferrin and N-Glycan analyses, coagulation factor levels, PT/PTT, CBC, ALT, HgbA1c, thyroid function, and immunoglobulin levels were tracked. He did not develop any coagulopathic, hematologic, endocrinologic, or septic complications. As in fibroblasts, the tetrasaccharide marker in plasma decreased with treatment. He improved clinically as well: there were no convulsive episodes; he gained global milestones including speaking phonemes, OFC increased, VEPs and visual acuity improved. We hope this CDG-Ik D-mannose treatment trial can be expanded, and possibly applied to other CDG subtypes. P-465

P-463 Next generation sequencing in diagnostics of two Czech CDG Ix patients: case reports of patients diagnosed with DPAGT1-CDG and SRD5A3-CDG 1

1

1

1

1

Honzik T , Ondruskova N , Tesarova M , Vesela K , Magner M , Hennet T2, Matthijs G3, Zeman J1, Hansikova H1 1

Dep Pediatrics, Charles Univ, Univ Hosp, Prague, Czech Republic; Inst Phys, Univ Zurich, Zurich, Switzerland; 3Lab Mol Diagn, Hum Gen, Univ Leuven, Leuven, Belgium 2

Next-generation sequencing is the method of choice for molecular diagnosis of disorders untraceable with conventional capillary sequencing due to the inability to prioritize few candidate genes. We used exome sequencing to identify the disease-causal variants in two CDG Ix patients. The first patient (5-year old girl) presented with mild craniofacial dysmorphy, inverted nipples, atypical fat pads, psychomotor delay and

Cutis laxa syndrome due to mutations in ATP6V0A2 gene: two new cases and two new mutations Valerio Hernandez E1, Hernandez Sanjuan I2, Ruiz Pons M2, Lopez Mendoza S2, Artuch R3,4, Casado M3, Perez B5, Perez Cerdá C5, Girós M6 1

Gerencia de Atencion Primaria. SCS, Santa Cruz de Tenerife, Spain; HUNSC. SCS, Santa Cruz de Tenerife, Spain; 3HSJD. Universitat de Barcelona, Barcelona, Spain; 4Instituto de Salud Carlos III (CIBERER), Barcelona, Spain; 5CEDEM. CBM-SO (CIBER-ER), Madrid, Spain; 6IBC, Hospital Clinic, Barcelona, Spain 2

Cutis laxa is a rare skin disorder characterized by excessive and redundant wrinkled skin. Congenital Disorders of Glycosylation (CDG) have recently been associated with cutis laxa, such as defects in Conserved Oligomeric Golgi Complex (COG7-CDG; #608779), autosomal recessive

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cutis laxa type 2A (ARCL2A; #219200) and Wrinkly Skin Syndrome (WSS; #278250) due to mutations in ATP6V0A2 gene, a H±ATPase pump localized in the Golgi System. We report two male siblings who presented at birth dysmorphic features: microretrognathia, a large anterior fontanel, big ears, wide nasal bridge, microcephaly, increased joint laxity and cutis laxa. The younger sibling presented a congenital chylothorax, which required thoracic drainage, MCT diet and octreotid treatment due to recurrent chylothorax in the first month. Serum transferrin isoelectrofocusing (TIEF) was abnormal with elevated mono- and tri- sialotransferrin and low di- sialotransferrin, a different pattern than previously described (type-2). Apoliprotein C-III (ApoC-III) showed abnormal values with higher ApoC-III1 over ApoC-III2. Molecular analyses demonstrated two new mutations in ATP6V0A2 gene: c.1249C>T (p.His417Tyr; PolyPhen-2 analysis suggests a pathological mutation) and c.2379dupT (p.Ile794fs; possibly a severe mutation). The presence of congenital chylothorax adds a new clinical feature not previously described in this syndrome, with a different pattern of TIEF and two new mutations.

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Congenital Disorders of Glycosylation (CDG) are a group of multisystemic, inherited, metabolic diseases resulting from defects in the protein glycosylation pathway. Phosphomanomutase 2 (PMM2) is the affected protein in CDG-PMM2 disorder, the most common CDG, for which there is currently not an effective treatment. The screening of target mutations for specific therapies was performed through the functional analysis of PMM2 mutations. Protein stability, studied by differential scanning fluorimetry and by a coupled transcription and translation system showed that in general, mutants are more unstable than wild-type. Furthermore, their specific activity presented a reduction from 50 to 100%. The oligomerization pattern showed a majority of dimeric forms for most of the mutants, whereas the mutation, p.P113L, did not present any oligomeric state. Based on these results and the mapping of residues in the PMM2 crystal structure, we have classified the mutations as catalytic, destabilizing and mutations affecting the dimerization. The destabilizing mutations pV44A, p.D65Y, p.R162W, p.C241S, p.F207S and p.T237M are promising candidates for testing the selected pharmacological chaperones by highthroughput screening from 100000 library´s compounds proved with the wild-type protein. Our preliminary results point out a promising compound which increases WT and some mutant's protein half life.

P-466 P-468 PGM1-CDG: a surprising congenital disorder of glycosylation Phosphoglukomutase-1 deficiency: a newly described CDG subtype Küçükçongar A1, Tümer L1, Ezgü FS1, Kasapkara CS1, Jaeken J2, Matthjis G3, Rymen D4, Dalgıç B5, Bideci A6, Hasanoğlu A1 1 Div Metab Dis, Univ Gazi Hosp, Ankara, Turkey; 2Div of Metab Dis, Univ Hosp Gasthuisberg, Leuven, Belgium; 3Div of Hum Genet, Univ Hosp Gasthuisberg, Leuven, Belgium; 4Div of Pediatr, Univ Hosp Gasthuisberg, Leuven, Belgium; 5Div of Gastroenterology, Univ Gazi Hosp, Ankara, Turkey; 6Div of Endocrinology, Univ Gazi Hosp, Ankara, Turkey

Congenital disorders of glycosylation (CDG) are a rapidly growing family of genetic diseases. Some 60 CDG are actually known. Phosphoglucomutase 1 deficiency, first reported in 1963. We report on another patient with this CDG type. Case Report: A 19-months-old girl was admitted for evaluation of feeding difficulties and recurrent hypoglycemia. She showed a short stature, prominent forehead, small face, depressed nasal bridge, unilateral-median cleft palate and hepatomegaly. Her weight was 7,1 kg (SDS: -3), height was 66 cm (SDS: -3,97). Serum transaminases were increased. On the follow-up hypoinsulinemic hypoglycemia was detected with increased serum cortisol, adrenocorticotropic hormone, growth hormone, and normal metabolic screening tests (urine glucose and organic acids, and serum lactic acid, ammonia, bicarbonate, amino acids and acylcarnitine levels. Serum insülin like growth factor 1 (IGF1) was low 16,15 ng/ml (nl: 55-110), but showed a normal response to growth hormone. Serum transferrin IEF showed a type 2 pattern. On the basis of these results and clinical features, mutation analysis of the PGM1 gene was performed. A homozygous mutation was found: c.551delT (p.F184Sfs*9). In conclusion, PGM1-CDG broadens the already very broad phenotypic spectrum of CDG. In particular, bifid uvula/cleft palate has not been reported before as a feature of CDG.

Eminoglu FT1, Cetinkaya S2, Aycan Z2, Ryman D3, Matthijs G3, Jaeken J3 Dept Pediatr Metab Dis, Ankara Univ Hosp, Ankara, Turkey; 2Dept Pediatr Endocrinol, SU Child Hosp, Ankara, Turkey; 3Center for Human Genetics,Univ of Leuven, Leuven, Belgium 1

Congenital disorders of glycosylation (CDG) are a group of genetic diseases characterized by defective protein glycosylation. To date, more than 40 defects have been identified in this rapidly growing disease family. We describe a female patient with PGM1 deficiency that is a new subtype of CDG. She was admitted to endocrinology clinic because of short stature and fasting hypoglycemia attacks 6 years ago. She was undergone surgery for the cleft palate and lip at the age of 1 year. The physical examination displayed dysmorphologic features. Liver function tests and creatine kinase levels were high. Hypertrophic cardiomyopathy was detected. GH treatment has been started, but the growth curve was borderline. She was referred to the metabolism clinic at the age of 14. The IEF of serum showed a type II pattern. PGM1 activity in the fibroblast was only 4%. Genetic analysis revealed a homozygous novel mutation (c.1508G>A) on genomic DNA. PGM1 is converting bidirectionally glucose-1-phoshate to glucose-6-phosphate. The deficiency will lead further onto the pathway to a deficiency of UDP-galactose. This will lead to lower levels of galactosylation. PGM1 deficiency should be considered in patients with no psycomotor retardation, fasting hypoglycemia, short stature, cleft lip and palate, muscle weakness or cardiomyopathy. P-469

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Congenital disorders of glycosilation: antisense therapy in TMEM165 deficiency

Characterization of PMM2 mutations: identification of target mutants for specific pharmacological chaperone therapy

Yuste-Checa P1, Medrano C1, Desviat LR1, Ugarte M1, Matthijs G2, Pérez-Cerdá C1, Pérez B1

Yuste-Checa P1, Sierra-González P1, Gámez A1, Underhaug J2, Martinell M3, Desviat LR1, Ugarte M1, Pérez-Cerdá C1, Martínez A2, Pérez B1

1

CBMSO-UAM, CEDEM, CIBERER, IDIPaz, Madrid, Spain; 2Center for Human Genetics, KU, Leuven, Belgium

1 CBMSO-UAM, CEDEM, CIBERER, IDIPaz, Madrid, Spain; 2University of Bergen, Biomedicine, Bergen, Norway; 3Minoryx therapeutics, Barcelona, Spain

Deficiency of glycosyltranferases, glycosidases or nucleotide-sugar transporters involved in protein glycosylation leads to Congenital Disorders of Glycosylation (CDG), a rare group of diseases characterized by

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multisystemic phenotype. Recently, other proteins implicated in Golgi trafficking and Golgi pH homeostasis have been identified as CDG causing genes. In this work, we describe the functional analysis of the intronic change c.792+182G>A identified in TMEM165 causing new CDG type II. Using a functional in vitro splicing assay based on minigenes and transcriptional profile studies in patient-derived fibroblasts, we have validated its pathogenic effect. We have applied a mutation specific therapy by design of a specific antisense oligonucleotide targeted to rescue the abnormal splicing process. Normal transcripts have been recovered which are efficiently translated into a well localized protein. The recovery was dose and sequence specific with no effect on patient-derived fibroblasts harboring missense changes. The results show the efficiency of the antisense therapy targeted to intronic changes, which are detectable by combined DNA and RNA studies. Up to now, five TMEM165-CDG patients have been described and three of them are homozygous for the intronic change c.792+182G>A which makes antisense morpholino therapy an excellent candidate treatment for this new CDG type II.

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Autosomal recessive cutis laxa type 2A (ARCL2A; MIM 219200) is a syndromal disorder involving connective tissue, skeleton and brain abnormalities caused by mutations in the ATP6V0A2 gene. It also includes a congenital glycoslyation defect type II, mainly due to impaired addition of sialic acid and galactose in the trans Golgi compartment. Accordingly, we showed that the a2 subunit of the V-type H±ATPase encoded by ATP6V0A2 resides in the Golgi compartment and is lost in fibroblasts from patients. Further investigations in ATP6V0A2-deficient fibroblasts and HeLa cells to uncover more details of the pathomechanism revealed altered Golgi morphology and delayed Golgi-to-ER transport after Brefeldin A treatment. However, we also found evidence for impaired receptor-mediated endocytosis. In order to assess which of these cellular dysfunctions contribute to the different aspects of the disorder we created an Atp6v0a2flox conditional mouse model. P-472

P-470

Characterization of CDG-IIc phenotype due to partial defect in fucosylation

A new polish SRD5A3-CDG patient: multisystemic abnormalities and feeding problems

James PM1, Dauber A2, Ercan A3, Jacobs P3, Wang S2, Miller T2, Ashine D4, Reinhold V4, Sackstein R3, Hirschhorn J2, Nigrovic P3

Adamowicz MA1, Bakula A2, Socha P2, Piwczynska K2, Bogdanska A1, Lefeber DJ3, Jurkiewicz E4

1 Div Gen & Metab, Boston Child Hosp, Boston, United States; 2Div of Endocrin, Boston Child Hosp, Boston, United States; 3Brigham & Women's Hosp, Boston, United States; 4Univ of New Hampshire, Durham, United States

Dept Biochem, Children Mem Health Inst, Warsaw, Poland; 2Dept Gastroent Children Mem Health Inst, Warsaw, Poland; 3Dept Neurol Radboud Univ Med Center, Nijmegen, Netherlands; 4Dept Diag Imag Children Mem Health Inst, Warsaw, Poland 1

Background: SRD5A3-CDG is caused by mutations in steroid 5αreductase type 3 gene which takes part in dolichol phosphate biosynthesis, a key molecule of multiple glycosylation pathways. 17 patients from Near East and Poland were characterized by multisystemic abnormalities and defective glycosylation. Case report: A 3 m. old male infant with feeding problems was referred to gastroenterology department. Physical examination revealed hypotonia, nystagmus, facial dysmorphism, shortening of shoulders and clavicles and loose, scaling skin. Brain MRI showed cerebellar hypoplasia, funduscopy pigmentary changes. Laboratory abnormalities included low cholesterol, albumin, ATIII, protein S and C. Transferrin isofocusing showed an abnormal profile suggesting CDG-I.Pylorostenosis or congenital flaccidity of the larynx were suspected as the cause of poor feeding but ultrasonographic gastrointestinal passage did not confirm it. The child was fed by noso-gastric tube and parenteral nutrition which was stopped because of venous thrombosis. Finally gastrostomy tube feeding was started which improved physical development (weight from 3rd to 10th cent.at 3 y). The clinical picture, transferrin profile an increase of plasma polyprenols (LCMS/MS) suggested a diagnosis of SRD5A3-CDG. Sequencing of SRD5A3 gene revealed a homozygous mutation c.424C>T (p.Arg142X) Conclusion: SRD5A3-CDG should be considered in children with cerebello-ocular-cutanous syndrome, dysmorphism, poor feeding and abnormal transferrin profile.

Congenital disorder of glycosylation type IIc is a very rare disorder caused by defects in SLC35C1 which encodes a GDP-fucose transporter present in the Golgi. CDG2c is characterized by short stature, developmental delay, Bombay blood type, and leukocytosis with recurrent infections due to impaired neutrophil rolling. Large scale candidate gene sequencing of 1077 genes was performed as part of a short stature research protocol. Functional effects of rare variants in SLC35C1 on fucosylation were explored. The two affected brothers were compound heterozygotes for two novel variants in SLC35C1, p.E31* and p.F168del. N-glycan analysis revealed a striking, but partial, impairment of serum protein fucosylation. Flow cytometry and western blot analysis showed decreased expression of the canonical selectin-binding glycan determinant, sLex. Leukocyte flow chamber assays showed that E-selectin-mediated rolling was present, with a decrease in the percentage of rolling cells in probands compared to the parents and normal controls. Flow cytometry demonstrated decreased presence of Eselectin ligand on leukocytes which could be corrected with exofucosylation. Functional studies demonstrate reduction but not absence of fucosylation, likely accounting for their milder phenotype compared to classical CDG2c, including the lack of Bombay blood type and clinical leukocyte adhesion deficiency. P-473 Congenital disorders of glycosylation (CDG) in six children diagnosis and management

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Horovitz DDG1, Lima MAFD2, Santos NCK1, Correia PS1

ATP6V0A2-CDG: a disorder due to impaired intracellular trafficking and Golgi function

2

Emmerich D1, Fischer B1, Gardeitchik T2, Mundlos S1, Morava E3, Kornak U1 1 Charité-Universitätsmedizin Berlin, Berlin, Germany; 2St. Radboud University Medical Center, Nijmegen, Netherlands; 3Tulane University Medical Center, New Orleans, United States

1

Instituto Fernandes Figueira / Fiocruz, Rio de Janeiro, Brazil; CERES-Genetica, Rio de Janeiro, Brazil

Aiming to discuss cardinal signs, diagnosis and management, six patients (two female, four male) with confirmed CDG-I are described. All were diagnosed by serum transferrin isoelectric focusing. Molecular analysis confirmed CDG-Ia in one patient with congenital nephrotic syndrome [mutations c.422G>A(p.R141H) and c.691G>A (p.V231M) in PMM2-CDG], whose similarly affected brother died undiagnosed at

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3 m. of age; and CDG-Ic in a sibling pair [compound heterozygotes for mutations c236A>G(p.H79R) and c.359A>G(p.D120G) in gene ALG6 not previously reported in the literature]. The main features shared by all patients were: hypotonia (6/6), developmental delay (6/6), abnormal fat deposition (6/6), inverted nipples (5/6), poor weight and height gain (4/6), gastroesophageal reflux (4/5), seizures (3/5), eye abnormalities. The brothers with ALG6-CDG (CDG-1c) had nystagmus, one had optic nerve pallor, both had short umbilical cord and seizures (one had myoclonic seizures that progressed to hypsarrhytmia); both died at one year. Three patients are on regular follow-up. Laboratory abnormalities were variable and intermittent throughout the clinical course, being the most striking changes in liver and muscle enzymes, and increased thyroid-stimulating hormone. Management has been directed to clinical and nutritional support, rehabilitation, multidisciplinary support, and continuous assessment of risk versus benefit regarding the need for surgery or medication. P-474 Discovery of abnormal N-glycan biomarkers for several subtypes of cdg He M1, Freeze HH2, Ng B2, Raymond K3, Matern D3, Jones M.A1, Hegde M1, Wood T4, Cummings R5, Burton BK6, Paras A6

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been reported from India, partly due to limited access to serum transferrin isoelectric focusing (IEF) Aim: To describe the phenotype of patients with CDG in India. Methods: A retrospective study of cases screened positive for CDG via transferrin IEF in the period 2004- 2013. Results: 86 unrelated patients with psychomotor disability (PD) were screened and 9 were positive. Seven had a type 1 pattern. Genotyping confirmed hereditary fructose intolerance (2), PMM2-CDG (1) and ALG3-CDG (1). The boy with PMM2-CDG showed the typical presentation including inverted nipples and abnormal fat distribution. Spasticity, ambiguous genitalia, unilateral iris coloboma and vermis hypoplasia were seen in ALG3-CDG. There were three patients with CDG-Ix: one with optic atrophy and seizures, the second with recurrent effusions and hypothyroidism, and the third with cerebellar hypoplasia, inverted nipples and growth failure. Two patients had a type 2 pattern, one with cutis laxa but negative ATP6V0A2 sequencing, the other with PD, hypotonia, epilepsy, abnormal fat distribution, inverted nipples, and thin corpus callosum. Conclusion: Screening for CDG is indicated in unexplained multisystem disease, particularly when the nervous system is involved. P-476 Exomes in CDG: solving the unsolvable?

1

Dept Hum Gene, Emory University, Atlanta, GA, United States; 2 Sanford Child, Burnham Inst for Med Res, La Jolla, United States; 3Mayo BGL, Mayo Med School, Rochester, MN, United States; 4 Greenwood Genetics Center, Greenwood, SC, United States; 5Dept of Biochemistry, Emory Univ, Atlanta, GA, United States; 6Children's Memorial Hospital, Chicago, IL, United States The congenital disorders of glycosylation (CDG) are a rapidly expanding group of inborn errors of metabolism that result from defects in the synthesis of glyco-conjugates. Majority of the known CDGs are related to genetic defects in N-linked protein glycosylation. N-linked glycan profile from serum total glycoproteins is known as important tool for diagnosis of CDG type II. We recently discovered the existence of a group of abnormal N-glycans on glycoproteins from the serum and cultured skin fibroblast from CDG type I patients that are completely deficient in mannose but act as primers for the addition of other monosaccharides. In combination of measuring these abnormal N-linked glycan levels and recognizing abnormal N-linked glycan pattern, several major CDG type I subtypes could be identified including CDG-Ia, CDG-Ib, CDG-Ik and CDG-Ig. These subtypes of CDG type I represent almost 60% of patients with CDG type I. In addition, these N-linked glycan biomarkers could also be used as therapeutic biomarkers for potential treatment for these affected patients. In summary, we found that N-linked glycan profiles are not only useful for the diagnosis of CDG type II subtypes, they also are important in the diagnosis and characterization of certain subtypes of CDG type I.

Rymen D1, Souche E1, Race V1, Matthijs G1 1

Center for Hum Genetics, Univ of Leuven, Leuven, Belgium

The group of Congenital Disorders of Glycosylation (CDG) has expanded tremendously since its first description in 1980. Over the last few years, exome sequencing established an important role in the diagnosis of novel CDG genes: 8 novel types were published over a period of less than 3 years. However, success is not a guarantee. In our series of exomes we identified mutations in an already known CDG gene in 3 families, and mutations in novel candidate genes in 2 families. One family was diagnosed with galactosemia. Seven cases remained unsolved. Although exome sequencing entailed the promise to solve the unsolvable, limitations are present. First of all, the target is limited to exons, so exome sequencing will overlook deep intronic mutations, as well as for example single exon deletions. Another issue is incomplete coverage. In our series (mean coverage 50-80X) only 80% of all exons of 65 CDG candidate genes were completely covered. Thirteen percent of the exons were missing in all the exomes, while the remaining 7% were partially covered. Hence, we conclude that indeed exome sequencing is a powerful technique; however combinations of different approaches will be needed to solve all CDG cases. P-477

P-475

Further characterization of congenital disorder of glycosylation IIb in siblings

The phenotypic spectrum of CDG in India: retrospective evaluation of cases examined at a tertiary referral center

Wolfe LA1, He M2, Rosenzweig S1, Boerkoel C1, Adams DR1, Gahl WA1

Kotecha D1, Bijarnia S1, Puri R1, Movva S1, Lingappa D2, Matthijs G3, Jaeken J4, Verma I1

National Institutes of Health, Bethesda, United States; 2Emory University, Decatur, United States

1 Cen of Med Gen, Sir Gangaram Hospital, New Delhi, India; 2Rainbow Children Hospital, Hyderabad, India; 3Cen for Hum Gen, Univ Hosp Gasthuisberg, Leuven, Belgium; 4 Cen of Met Dis, Univ Hosp Gasthuisberg, Leuven, Belgium

Background: Describe clinical and biochemical features of two siblings presenting to the Undiagnosed Diseases Program (UDP). Case report: Two siblings were born to non-consanguineous, Northern European parents with a negative family history. Birth measurements were normal. Comprehensive molecular and biochemical evaluations were unrevealing. Evaluations revealed generalized cerebral atrophy, delayed myelination and low NAA by brain MRI/MRS, dysmorphic facial features,

Congenital Disorders of Glycosylation (CDG), a very heterogeneous and largely underdiagnosed family of genetic diseases, have not yet

1

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cortical visual impairment with optic nerve atrophy, hypotonia and severe hypogammoglobulinemia without organomegaly in both siblings. Materials and Methods: Prospective evaluation and clinical biochemical testing were performed. Urine oligosaccharides on both sibs demonstrated a tetrasaccharide band, further identified as Hex4 with 3 glucose residues and a single mannose at the reducing end. DNA studies identified three mutations in the GCS1 gene and single mutations in the mother and unaffected sibling and two other mutations in the father. This gene encodes alpha-glucosidase Ia, and a single case report in 2000 describes loss of this enzymatic function as the cause of Congenital Disorder of Glycosylation (CDG) type IIB. Conclusion: This case underscores the difficulties of diagnosing complex multi-system disease especially when affected siblings have slightly different phenotypes. It also expands the phenotype of CDGIIb. P-478 Exploring dolichol as a diagnostic parameter of congenital disorders of glycosylation (CDG) Girós M1, Garcia-Pelegrí E2, Ballega E2, Briones P3 1

ECM /BGM Serv.Hospital Clinic Ciberer737, Barcelona, Spain; ECM/BGM Serv. Hosp. Clinic, Barcelona, Spain; 3ECM/BGM Serv Hosp Clinic Ciberer737.CSIC, Barcelona, Spain 2

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12 females with age from 9 months to 67 years. Eight sib pairs were found. Clinical data, genotype and MALDI-MS Transferrin glycosylation profiles were analyzed. Nineteen patients have a severe clinical phenotype based on inability to walk unaided, moderate to severe mental retardation and in all severe cerebellar atrophy with posterior fossa cyst. Thirteen patients have preserved gross-motor abilities, mild to moderate mental retardation and cerebellar atrophy of variable degree. Eleven patients with mild phenotype are compound heterozygous for p.L32R mutation. Follow-up reveals progression of cerebellar atrophy and possible brainstem involvement in twelve studied patients including five bearing p.L32R mutation. Serum Transferrin MALDI-MS analysis shows more pronounced glycosylation defects in the more severely affected patients. This survey comprises sixteen adults including two of 64 and 67 years respectively, that are among the eldest reported with this disorder. Based on our experience, PMM2-CDG may behave as a chronic disabling disease that at least in the mild phenotypes is compatible with a long life span. P-480 Profiling of N-linked glycans from human serum using microflow liquid chromatography and tandem mass spectrometry: a high throughput method for the diagnosis of congenital disorders of glycosylation Williams D1, Kyriakopoulou L1

Seven dolichol (Dol) metabolism dysfunctions linked to CDG have been described. Phenotypes range from isolated retinitis pigmentaria (RP) to multisystemic pathology. Our aim was to determine isoprenols, polyprenol (Pol) and dolichol (Dol) levels in control blood samples and in pathological conditions by HPLC after saponification and lipid extraction as described by Yasugi (1994). The levels of free plus esterified dolichol in 70 controls aged 1m-80y were 141,0 ng/ml ±29,9 (82,7-201,9). Isoprene units of Dol ranged from 17 to 21, being 18-Dol, 19-Dol and 20-Dol the most abundant. In controls, Pol levels were under detection limits but were detectable in two women toeards the end of pregnancy. As previously described, Dol levels were age independent and related to cholesterol content. They were high in hypercholesterolemic patients and low in Smith-Lemli-Opitz disorder. Low Dol levels were also found in alcohol abusers and in 2 undiagnosed patients with multiple malformations. In 12 RP studied, Dol levels were in the normal range, and just slightly decreased in one. In conclusion: blood Dol levels reflect the whole metabolism of this compound, particularly hepatic metabolism, and it is necessary to discern other factors of variability in order to use it as a marker of CDG.

1

The Hospital for Sick Children, Toronto, Canada

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Background: Human serum contains a diverse group of glycoproteins. Congenital disorders of glycosylation (CDG) are due to IEM of glycosylation. Serum glycan profile can reveal the etiology of CDG subtypes. Objectives: To develop a simple and sensitive method for the analysis and characterization of glycans in serum glycoproteins. Methods: 200 Âμl serum was used for the analysis. N-linked glycans were released by treatment with PNGaseF, and purified by solid phase extraction. The purified glycans were separated on a graphitized carbon column coupled to an ABSciex 400 QTRAP mass spectrometer operated in positive ion mode. Results: Glycans from normal serum were characterized on the basis of retention times and masses. Further characterization was obtained through analysis of the product ion spectra. Sera from patients with known CDG defects were used to characterize and establish glycan profiles with incomplete glycosylation. The glycan profile of each CDG sample was compared to normal serum. Glycan structures were determined based on ion spectra. Conclusions: The method obviates the need for purification of specific glycoproteins from serum. The purification is simple, inexpensive and requires instrumentation available in clinical laboratories. The method is applicable to large cohorts of CDG patients.

A survey on Italian patients with PMM2-CDG

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Barone R1, Carrozzi M2, Cosentini D2, Dionisi Vici C3, Di Rocco M4, Garozzo D5, Lilliu F6, Spada M7, Sturiale L5, Fiumara A8

Simultaneous transferrin and apolipoprotein cIII glycoforms analysis by online immuno-affinity chromatography electrospray ionization mass spectrometry

Child Neurol, Univ Child Hosp, Catania, Italy; 2Child Neurol, IRCCS Burlo Garofolo, Trieste, Italy; 3Div Metab Dis, IRCCS Bambino Gesù, Roma, Italy; 4Div Rare Dis, IRCCS Gaslini, Genova, Italy; 5CNRICTP, Catania, Italy; 6Div Metab Dis, Univ Child Hosp, Cagliari, Italy; 7 Div Metab Dis Univ Child Hosp, Torino, Italy; 8Div Metab Dis,, Univ Child Hosp, Catania, Italy 1

PMM2-CDG is the most common genetic disorder of protein Nglycosylation. Between 1996 and 2012 we collected data on 34 Italian patients with proven PMM2 gene mutations. They include 22 males and

Raymond K1, Porta F2, Turgeon C1, Magera MJ1, Liedtke K1, Gavrilov D1, Oglesbee D1, Tortorelli S1, Rinaldo P1, Matern D1 1

Biochem Genetics Laboratory, Mayo Clinic, Rochester, United States; Dept of Pediatrics, University of Torino, Torino, Italy

2

Background: The analysis of glycosylation of serum transferrin (Tf), an exclusively N-glycosylated protein, allows the differentiation of two main abnormal patterns, namely CDG type I and II, corresponding to

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disorders of assembly and disorders of processing of N-linked glycan, respectively. The addition of Apo CIII, an exclusively O-glycosylated protein, to the transferrin analysis serves to improve the diagnostic sensitivity and specificity for CDG testing. Methods: Using online LC-MS, serum is applied to a blended immuno affinity column, which sequesters Tf and Apo CIII isoforms. Tf and Apo CIII isoforms are then co-eluted from a C4 column and directly introduced to the MS which is operated in positive Q1 scan mode. Total analysis time is 7.5 minutes. Data: Controls (N=280, ApoCIII0/CIII2 range=0.05-0.80, ApoCIII1/CIII2 range=0.32-3.98), CDG II (N=8, ApoCIII0/CIII2 range=0.54-3.07, ApoCIII1/CIII2 range=1.73-6.24), and CDG I (N=5, ApoCIII0/CIII2 range=0.08-0.80, ApoCIII1/CIII2 range=1.00-1.62) serum specimens were analyzed. All CDG II specimens demonstrated elevations for the Tftrisialo/Tfdi-oligo ratio as well as elevations of the Apo CIII0/CIII2 and/or Apo CIII1/CIII2 ratios. CDG I specimens had elevated Tfa-oligo/ Tfdioligo and Tfmono-oligo/Tfdi-oligo ratios with no APO CIII abnormalities. Conclusion: This high-throughput assay provides the simultaneous analysis of transferrin and apolipoprotein CIII glycoforms in 8 minutes with minimal preparation.

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assembly or transfer of the dolichol-linked glycan, while type II involves glycan processing defects. More recently a third mixed type was recognized with new genetic defects. Since 2000, the Mayo Clinic Biochemical Genetics Laboratory has been utilizing liquid chromatography mass spectrometry (LC-MS) to analyze transferrin isoforms to detect CDGs. Recently, simultaneous analysis of apolipoprotein CIII isoforms has been added along with the utilization of post-analytical tools to further improve detection. One-hundred and thirtyseven confirmed CDG cases have been identified. The majority of these are PMM2-CDG (CDG-Ia) and 25 are additional types of CDGs. Abnormal transferrin isoforms have also been observed in cases of non-CDG related liver disease due to other genetic conditions including hereditary fructose intolerance, Zellweger syndrome, galactosemia, retinitis pigmentosa, pseudoachondroplasia, and Niemann-Pick disease, type B. While the specific type of CDG must be confirmed with molecular analysis, transferrin and apolipoprotein CIII analysis by LC-MS has proven a useful screening tool for CDGs. P-484 A new family with Dursun syndrome due to G6PC3 gene defect

P-482 The first COG1 deficient patient - nine years follow-up Oliveira R1, Garcia P2, Quelhas D3, Matthijs G4, Diogo L2

Dursun A1, Yilmaz-Yucel D1, Sagiroglu MS2, Erguner B2, Smits G3, Vilain C3, Ozgul RK1 1

Hacettepe University, Dep. of Metabolism, Ankara, Turkey; TÜBİTAK, BİLGEM-UEKAE, İzmit, Turkey; 3Queen Fabiola Brussels Children Hospital, Brussels, Belgium 2

1 Serviço de Genética, Hospital Pediátrico, Coimbra, Portugal; 2Centro Desenvolv LB Hospital Pediatrico, Coimbra, Portugal; 3CGMJM – INSA IP, Porto, Portugal; 4Center for Human Genetics, Leuven, Belgium

In recent years, congenital disorders of glycosylation (CDG) with a defect in both N and O-glycosylation due to conserved oligomeric Golgi (COG) subunits deficiency have been recognized. The clinical phenotypes are still poorly known. The first COG1 patient, a girl (DOB 13.06.03), was diagnosed based on early-onset generalized hypotonia, minor dysmorphisms, failure to thrive and a CDG type II pattern in IEF of serum transferrin. At the age of the first report (21 months), she had mildly enlarged liver and spleen, psychomotor retardation, microcephaly and a slight cerebral and cerebellar atrophy. Upon evolution, short stature (with normal weight, BMI and head circumference), myopathy (with myotatic arreflexia, muscle fatigue without myalgia, and persistently elevated creatine kinase and aminotransferases) have been registered. No cardiac problems or hepatosplenomegaly are currently noticed. Her cognitive development is significantly below the average expected for age (IQ: 66). She attends regular school with individual support. She has been well, with good tolerance to infrequent undercurrent infections, which caused three short inpatient admissions between 3 and 7 years of age. To our knowledge, this is the only COG1 deficient patient described to date. CDG, namely COG1 deficiency, should be searched in patients presenting myopathy and mild intellectual disability.

G6PC3 gene defects result in three main clinical spectrum; syndromic SCN4, Dursun syndrome and nonsyndromic SCN4. Major findings of Dursun syndrome are; primary pulmonary hypertension (PPH) developed in newborn period (NB), which is one of the most remarkable finding differentiating it from other types of SCN4 types, secundum-type ASD, neutropenia, thrombocytopenia, anemia showing fluctuating pattern, monocytosis, dysplasia in all cell lines in bone marrow and thymus hypoplasia associated with severe lymphopenia. Minor findings are; proximal localization of thumb, complete- incomplete simian line, broad nasal bridge, undescended testis, pectus carinatum and high palatal arch. PPH reported in three cases with syndromic SCN4 (9, 13, and 25 years old) was not developed in NB period and most probably secondary to frequent lung infection. The clinical findings of the presented cases were completely compatible with the Dursun syndrome. A novel homozygous c.765_766 delAG frameshift mutation was detected in G6PC3 gene in our patient. Exome sequencing of the patient was also performed to analyze the genes associated with PPH (BMPR2, ALK1, ENG, SMAD9, CAV1, KCNK3, CRHR-1, TSP1, GSK3B, ACVRL1) and any pathogenic mutation was detected. Dursun syndrome would make important contribution to understand the pathogenesis of PPH. This study was supported by TÜBİTAK-SBAG-111S217 P-485 Natural history study of patients with GNE myopathy

P-483 Congenital disorders of glycosylation: the Mayo Clinic experience

de Dios JK1, Latham L2, Shrader J3, Joe G3, Ciccone C1, Celeste F2, Robinson C2, Draper D1, McKew J2, Huizing M1, Gahl WA1, CarrilloCarrasco N2

Raymond K1, Hesemann J1, Anderson C1, Rinaldo P1, Matern D1 1

Nat'l Human Genome Research Ins, NIH, Bethesda, United States; Nat'l Ctr for Advancing Translational Sc, Bethesda, United States; 3 Clinical Center, NIH, Bethesda, United States

1

2

Congenital disorders of glycosylation (CDGs) are a group of inherited metabolic disorders caused by abnormal glycosylation of proteins. Almost 70 types have been described with multisystem disease and phenotypic variability. Type I CDG is characterized by defects in the

Background: GNE myopathy, a rare inborn error of sialic acid biosynthesis caused by GNE mutations, is characterized by progressive muscle atrophy, with onset in early adulthood.

Biochemical Genetics Lab, Mayo Clinic, Rochester, United States

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Methods: We evaluated 25 patients with molecularly confirmed GNE myopathy in a longitudinal, natural history study (ClinicalTrials.gov:NCT01417533). Results: Patients presented at a mean age of 29 years (17-40 years), typically with foot drop and gait disturbance. Mean diagnostic delay was 12 years from onset of symptoms. Negative muscle biopsies were seen in 71% of patients biopsied. Quantitative strength, functional and MRI evaluations supported slow progression, initially affecting the anterior tibialis, followed by posterior leg, hamstrings, gluteal and finally quadriceps muscles. Grip weakness was evident on average 9 years after onset, followed by progressive involvement of upper extremity muscles. The rate of progression was variable, but was increased in patients with earlier onset and muscle underuse or overuse. Genotype-phenotype correlations were not yet clear. Cardiac and respiratory manifestations were observed in a subset of patients. CPK ranged between 161-1152 U/L and values normalized as the disease progressed. Conclusions: There is a need to develop sensitive diagnostic tests for GNE myopathy. Understanding the natural history is critical for identifying outcome measures and designing clinical trials. P-486 Detection of glycosylation abnormalities among individuals with undiagnosed diseases: the NIH undiagnosed diseases program experience Boerkoel CF1, He M2, Adams DR1, Godfrey R1, Golas G1, Groden C1, Landis D1, Maduro V1, Nehrebecky M1, Tifft CJ1, Toro C1, Wang D1, Wahl C1, Wolfe L1, Gahl WA1 National Institutes of Health, Bethesda, United States; 2 Emory University School of Medicine, Atlanta, United States 1

Introduction: Carbohydrate-deficient glycoprotein (CDG) syndromes are a family of diseases due defects in the glycosylation pathway. Mutations in the Conserved Oligomeric Golgi (COG) complex is associated with CDG type II. Authors describe the evolution of the first described COG 4 CDG patient. Case report: 11 years old male, assyntomatic until four months of age when he developed a statuts epilepticus after immunization. Microcephaly, particular facies and slight hypotonia were present, and diagnosis of CDG IIx was made (2002). A development delay without speech, behavioral perturbation and hyperactivity were progressively noted. In 2008, COG4 deficiency was identified. At the age of 11 years in the context of fever he presented a sleep/wakefulness pattern change and visual hallucinations, mild systemic hypertension and then bilateral amaurosis. Electroencephalogram revealed bi-occipital epileptiform activity. CT brain scan was suggestive of posterior reversible vasoconstriction syndrome (PRES), confirmed by MRI. Cervical and transcranial ultrasound were normal. 48 h after starting treatment, he recovered visual acuity and epileptiform activity disappeared. All studies for PRES and hypertension, meanwhile normalized, were normal. Discussion: We report phenotype and evolution of the first described COG 4 CDG patient. Possible correlation between PRES and CDG syndromes has not been previously reported. P-488 Congenital disorder of glycosylation type Ia: neonatal presentation with generalized edema, hypertrophic cardiomyopathy and severe thrombocytopenia Pimenta J1, Rodrigues E1, Moura C1, Soares P1, Brandão O1, Quelhas D2, Rodrigues M1, Leão Teles E1 Centro Hospitalar de São João, Porto, Portugal; 2Centro de Genética Médica, Porto, Portugal 1

Background: Congenital disorders of glycosylation cause a pleiotropic group of disorders with variable expressivity and without consistent pathognomonic clinical features. This impedes diagnosis and directed testing for disorders of glycosylation. Methods: An agnostic screen of patient serum and urine for abnormalities of protein glycosylation using MALDI-TOF (N-linked glycan), LC-MSMS (O-linked glycan), and MALDI-TOF/TOF (urine free glycan). Results: 53 of 410 patients had glycosylation anomalies in the serum or urine. 8 had anomalies in both. Confirmed known disorders in synthesis and degradation of glycoproteins included CDG-IIb, GM1, GM2 and MPSIIIB. 3 patients had abnormal nondiagnostic N-linked and O-linked glycan profiles; 8 patients had isolated under sialylation of O-linked protein glycosylation, and 16 patients had isolated abnormal N-linked protein glycosylation. The remainder had isolated changes in urine free glycans. Studies of cultured skin fibroblasts from 50 individuals with non-diagnostic glycosylation abnormalities identified glycosylation abnormalities in 24. Compared to 8 individuals with serum or urine protein glycosylation abnormalities among 300 patients referred from a general genetics clinic, the relative risk for a disorder of glycosylation in the UDP cohort was 3. Conclusions: Comprehensive screening for protein glycosylation abnormalities is useful for biochemically phenotyping unknown diseases. P-487 COG4- CDG patient and posterior reversible vasoconstriction syndrome

Background: Congenital disorders of glycosylation (CDG) are a group of complex, genetic diseases, characterized by abnormal glycosylation of glycoproteins and glycolipids. CDG-Ia is the most common with estimated prevalence of 1:20000. Case report: Male, first child of healthy nonconsanguineous couple with prenatal diagnosis of high nuchal translucency and macrocephaly. Amniocentesis revealed 46 XY karyotype. Cesarean delivery at 40 weeks, apgar score 8/8, weight P25, length P10 and head circumference P95. At birth he presented generalized edema, several bruises and hemorrhagic suffusion, slight hypotonia, dismorfic facial features, inverted nipples, bilateral cryptorchidism and heart murmur. Echocardiography revealed biventricular hypertrophy. Since first day, anaemia, persistent thrombocytopenia and severe coagulation disorder were noted needing almost daily transfusions. The diagnosis of CDG as well as Noonan syndrome was considered. At 15th day he died due to a sudden bradycardia. Autopsy was performed with multiples abnormal morphological and histological data in different organs. Metabolic evaluation revealed abnormal CDT(47,3%) with type I isoelectric focusing transferrin. Molecular study confirms CDG-Ia (heterozygous for c.193G>T(p.D65Y) and c.470T>C(p.F157S) mutations). Conclusion: CDG-Ia has a broad sprectrum of clinical presentations ranging from severe neonatal presentation with hydrops fetalis to midly involved adults. Authors describe a very severe CDG Ia presentation with neonatal death. P-489 Abnormal fat distribution in PMM2-CDG

Rodrigues E1, França S2, Quelhas D3, Matthijs G4, Leão Teles E1 Wolthuis DFGJ1, van Asbeck EV1, Mohamed M2, Lefeber DJ2, Morava E1 1

2

Met Unit, Hosp Ped Int CH S João, Porto, Portugal; Neurology Depart CH S João, Porto, Portugal; 3Bioq Genet Unit, Centro Genetica Medica, Porto, Portugal; 4Lab Mol Diag, C Hum Gen, Univ Leuven, Leuven, Belgium

1

Dep of Med Genetcs, Tulane University, New Orleans, United States; Dep of Pediatrics, Radboud Med Centre, Nijmegen, Netherlands

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Abnormal fat distribution is almost pathognomic in patients with PMM2CDG at birth, however, absence of this symptom doesn't rule out the diagnosis. Patients have fat pads around the hips, genital area and upper thighs. Inverted nipples occur due to extra adipose breast tissue in the lack of glandular tissue. Many receptors and hormonal regulators, including receptors for insulin, IGF1, oestrogen and specific catecholamine receptors are glycosylated, leading to abnormal endocrine regulation in CDG. We evaluated the hypotheses that abnormal fat distribution is associated with perinatal abnormal hormone regulation in PMM2-CDG. We assessed 32 cases with PMM2-CDG, for the comorbidity of hypoglycemia/hyperinsulinism and fat pads and compared sex-related differences in fat distribution in 58 patients. We found that 90% of patients with hypoketotic hypoglycemia and/or hyperinsulinism had abnormal fat distribution, while normoglycemic patients showed this feature in only 50% of the cases (P=0.0302). We also found that a larger proportion of the female population showed abnormal fat distribution compared to the male population. The significant difference between normal and abnormal fat distribution in patients with normoinsulinemic normoglycemia and hyperinsulinaemic hypoglycemia at the other suggests the etiological role of the insulin receptor in de the development of abnormal fat distribution in PMM2-CDG.

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MRI is abnormal in males with neurologic symptoms. VLCFA levels are elevated in almost all males. In developing countries, the limitation of local facilities and financial support for VLCFA examination are the challenge to design a clinical screening that lead to diagnosis, Methods: Five previously healthy boys with school age onset psychomotor regression were evaluated. Detailed history, physical examination and brain MRI were studied; when brain MRI suggests X-ALD and if financially possible, basal cortisol and serum VLCFA were measured (Kennedy Krieger Institute, Baltimore). Results: All boys aged 5-7 years old presented with progressive impairment of cognition, behavior, vision, hearing, and motor function. Brain MRI showed characteristic bilateral demyelination in posterior brain especially in the parieto-occipital regions (Melhem et al., 1997). One of 2 patients showed deficiency of cortisol. Two patients showed significantly high concentration of VLCFA Conclusions: Brain MRI studies could be used as clinical screening tool for X-ALD among progressive impairment of cognition, behavior, vision, hearing, and motor function school age boys, before VLCFA confirmation diagnostic test. P-492

12. Peroxisomal, sterol and bile acid disorders P-490

Novel PEX3 mutations identified as the cause of a peroxisomal biogenesis disorder with moderate clinical phenotype

Chenodeoxycholic acid supplementation causes toxic hepatitis in infant with Cerebrotendinous Xantomathosis

Maxit C1, Denzler I1, Marchione D1, Agosta G1, Koster J2, Wanders RJA2, Ferdinandusse S2, Waterham HR2

Huidekoper HH1, Vaz FM2, Wijburg FA1, Bosch AM1

Depart Child Neurol Hospital Italiano, Buenos Aires, Argentina; 2Lab Gen Metab Dis Academic Med Centre, Amsterdam, Netherlands

Dept Ped, Acad Med Cent, Amsterdam, Netherlands; 2Lab Genet Metab Dis, Acad Med Cent, Amsterdam, Netherlands

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1

A girl born at term presented with neonatal convulsions at day 10. The diagnostic workup demonstrated a parecho virus infection to be the causative factor but, coincidently, metabolic evaluation demonstrated a raised plasma cholestanol and accumulation of bile alcohols consistent with Cerebrotendinous Xanthomatosis (CTX). Mutation analysis of CYP27A1 revealed two missense mutations confirming the diagnosis of CTX. The patient was started on chenodoxycholic acid supplementation at 15 mg/kg/day in three doses as advised in literature resulting in the normalisation of plasma cholestanol. Six weeks after initiation of therapy the patient presented with jaundice, pruritis and mild hepatomegaly without signs of infection. Bilirubin, liver transaminases and alkaline phosphatase were markedly elevated, gamma-glutamyltransferase was normal. An intercurrent viral infection (e.g. EBV, CMV) was excluded. Chenodoxycholic acid supplementation was stopped to rule out toxic hepatitis. Indeed, liver size normalised within 1 month and liver enzymes within 3 months. Chenodeoxycholic acid supplementation was restarted at a dosage of 5 mg/kg/day resulting in the normalisation of plasma cholestanol. This case illustrates that chenodeoxycholic acid supplementation at the advised dosage of 15 mg/kg/day may cause toxic hepatitis in infants and a lower dose can be sufficient to maintain metabolic control in infants with CTX. P-491 The challenge of diagnosis x-linked adrenoleukodystrophy (X-ALD) in developing countries Sjarif D R1

Background: Peroxisome biogenesis disorders (PBDs) may have variable clinical expression, from severe, lethal to mild phenotypes with progressive evolution. PBDs are caused by mutations in PEX genes, which encode proteins, called peroxins, involved in the assembly of the peroxisome. Objectives: To report a patient heterozygous for two novel mutations in the PEX3 gene with less severe phenotypic expression than reported previously for PEX3 patients. Case Report: A five years old boy, first child of unrelated parents, presented with psychomotor retardation, axial and peripheral muscular hypotonia and nephrocalcinosis at 3 months of life. He was born at term, and perinatal history was uneventful. At 18 months old he presented progressive spastic paraparesis, neurogenic bladder and nystagmus that evolved to bilateral cataract at 4 years old. Methods: Peroxisomal parameters were studied in cultured skin fibroblasts. PEX genes were sequenced in DNA isolated from fibroblasts. Results: Catalase immunofluorescence showed a peroxisomal mosaic pattern with all cells containing peroxisomal membrane structures. Immunoblotanalysis for acyl CoA oxidase and peroxisomal thiolase was normal. Sequencing identified two heterozygous, pathogenic mutations in the PEX3 gene. Conclusion: Mutations in PEX3 usually result in a severe and early lethal phenotype. The present report expands the clinical spectrum of this disease. P-493 Obstructive sleep apnea in patients with Smith-Lemli-Opitz syndrome

1

Haas D1, Gausepohl HJ2, Seipelt P3, Hoffmann GF1

Background: Childhood cerebral form of X-ALD are mostly seen in 48 years old males. Symptoms initially resembles ADD/ADHD followed by psychomotor regression lead to total disability within two years.

1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany; 2Div Neonatology, Univ Child Hosp, Heidelberg, Germany; 3Univ Child Hosp, Marburg, Germany

Dept Pediatrics Fac Med Univ Indonesia, Jakarta, Indonesia

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Background: Obstructive sleep apnea syndrome (OSA) is a common condition in children. Predisposing factors are craniofacial abnormalities, increased lymphoid tissue, upper airway inflammation and muscular hypotonia. Microretrognathia and muscular hypotonia are typical features in patients with Smith-Lemli-Opitz syndrome (SLOS). However, OSA has not been reported in this entity yet. Case report: We report on two SLOS patients with only slight microretrognathia, in whom overnight oximetry showed frequent oxygen desaturations. Severe OSA requiring non-invasive ventilation was confirmed by polysomnography. Both patients did not tolerate nasal CPAP by interface but oxymetry improved with PEEP high flow nasal canula. Conclusion: SLOS patients are at risk for OSA, which can lead to complications such as pulmonary hypertension, worsening of behavioural abnormalities and failure to thrive if left untreated. Therefore overnight oximetry should be done whenever an SLOS patient is hospitalized. However, noninvasive ventilation may be challenging in this patient group.

In order to be effective, the transplant must be done at an early stage of the disease. Thus, development of a simple screening method is important to enable the identification of pre-symptomatic ALD patients before showing symptoms of brain involvement. ALD is associated with an accumulation of VLCFA, particularly cerotic acid (26:0) and its metabolite lysophosphatidylcholine (Lyso PC26:0), which means that these molecules could be biomarker targets in the detection of ALD. We have developed a mass screening method for ALD through the quantification of Lyso PC or saturated fatty acid with different lengths of acyl chain using a liquid chromatography-mass spectrometry (LC-MS) selective reaction monitoring system. Those lipids could be extracted from dried blood spots. The analysis of each of the lipids by LC-MS was completed within 5 minutes per sample, thus it is an ideal method for the screening of ALD. P-496 10 years of follow up in a patient with Zellweger syndrome due to PEX 1 mutations in treatment with DHA and low phytanic diet

P-494 Mitochondrial oxidative phosphorylation and synaptic membrane Na+,K+-ATPase activity are strongly compromised by pristanic acid in cerebellum of rats

Girós M1, Vitoria I2, Moreno F3, Tomás M4, Cerón JA5, García-Hoyos M6, Dalmau J2 1

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1

1

1

Busanello ENB , Lobato VGA , Zanatta A , Viegas CM , Ribeiro CAJ1, Vargas CR2, Wajner M2 Dep Bioquímica, ICBS, UFRGS, Porto Alegre, Brazil; 2Serviço de Genética Médica, HCPA, UFRGS, Porto Alegre, Brazil 1

Background: Peroxisomal biogenesis disorders (PBD) are inherited disorders clinically manifested by neurological symptoms and brain abnormalities including the cerebellum. Biochemically, patients affected by some of these diseases accumulate pristanic acid (Prist) in the brain. Objectives and methods: We studied the in vitro influence of Prist on oxidative phosphorylation, by measuring the activities of the respiratory chain complexes I-IV and ATP production, as well as on creatine kinase and synaptic membrane Na+, K± ATPase activities in rat cerebellum. Results: Prist significantly decreased the activities of complexes I-III (65 %), II (40 %) and especially II-III (90 %), without altering complex IV activity of the respiratory chain and creatine kinase. Furthermore, ATP formation and synaptic Na+, K± ATPase activity were markedly inhibited (80- 90 %) by Prist. Conclusions: Considering the importance of oxidative phosphorylation for mitochondrial homeostasis and cell survival and of Na+, K± ATPase for the maintenance of cell membrane potential, the present data indicate that Prist compromises brain bioenergetics and neurotransmission in cerebellum. We postulate that these pathomechanisms may contribute to the cerebellar alterations observed in patients affected by PBD in which Prist is accumulated. Research grants from CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP, IBN-Net, INCT-EN. P-495 Development of a new method for diagnosis of adrenoleukodystrophy using liquid chromatography-mass spectrometry Kosuga M1, Kida K1, Nakajima H1, Fujimoto J1, Okuyama T1

ECM /BGM Serv.Hospital Clinic Ciberer737, Barcelona, Spain; Nutrition & Metab Unit .Hosp La Fe, Valencia, Spain; 3Pediatric Endocrinology Unit. Hosp La Fe, Valencia, Spain; 4Neuropediatric Unit. Hospl La Fe, Valencia, Spain; 5Genetic Unit, Hosp La Fe, Valencia, Spain; 6Instituto de Medicina Genómica, Valencia, Spain 2

Peroxisomal disorders of the Zellweger syndrome spectrum (ZSS) are caused at least by 13 different genes. Pex1 is the most affected gene among ZSS patients with a wide spectrum phenotype. We present 10 years follow up of a patient which was treated during 8 years with phytanic acid(PhA) restricted diet and one year on docosahexaenoic acid(DHA) supply. The main features at diagnosis, 2 years old, were failure to thrive, hypotonia, visual and hearing impairment, equinovarus foot deformities, hypertransaminasemia and mild facial dysmorphia. He has primary adrenal hypoplasia. Alteration of VLCFA and plasmalogen was inconstantly depending on cell types studied. Molecular analysis identified two PEX1 mutations, the common, c.2528G>A mutation and the variation, c.58_80del, not previously reported. During the 8 years of treatment, PhA levels were under 11μg/ml, VLCFA decreased at 50% of basal levels with occasional elevations. DHA was in the normal range in plasma and erythrocytes, but in one occasion, DHA decreased specially in erythrocytes in parallel with plasmalogen. Since then, the patient was on DHA supplement and the biochemical parameters as well as manual skills improved. This case supports the link between DHA, plasmalogens and peroxisome abundance in the ZSS cases with some preservation of peroxisomal functions. P-497 PEX6 mutations can be missed by screening of plasma very long-chain fatty acid profiles Visser G1, de Sain MGM2, Ferdinandusse S3, Waterham HR3 1

Wilhelmina Children's Hospital/ UMCU, Utrecht, Netherlands; Department of Medical Genetics, UMCU, Utrecht, Netherlands; 3 Lab Genetic Metabolic Diseases, AMC, Amsterdam, Netherlands 2

1

Nat Cent for Child Health and Dev, Tokyo, Japan

Adrenoleukodystrophy (ALD) is caused by a mutation in the ABCD1 gene and results in the apparent defect in peroxisomal beta oxidation and the accumulation of the saturated very long chain fatty acids (VLCFA). Hematopoietic stem cell transplantation is the only treatment that can stop the demyelination that is the hallmark of the cerebral forms of the disease.

Introduction: Zellweger syndrome (ZS) is a peroxisome biogenesis disorder and can, amongst others, be caused by mutations in the PEX6 gene. As a result of impaired peroxisomal function, patients with PEX6 mutations have elevated very long-chain fatty acids (VLCFA) in their plasma. In clinical practice this marker is used to screen for peroxisome

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biogenesis disorders. We present a family with 3 affected children in whom plasma VLCFA profiles normalized with aging. Cases: The index patient, a boy, youngest of 11 children, presented at day 5 with convulsions. MRI of the brain showed polymicrogyria. In plasma an elevated C26:0 level (2.88 μmol/l) and elevated ratios of C24/C22 and C26/C22 were found, which was also seen in fibroblasts. Furthermore, fibroblasts showed a diminished DHAPAT activity and an abnormal catalase immunofluorescence profile indicative of absence of peroxisomes. Molecular analysis of the PEX6 gene showed two pathogenic mutations c.2362G>A (p.Val788Met) and c.2734G>A (p.Ala912Thr). These mutations were also found in two older siblings with intellectual disability. Both have no speech, normal hearing and vision, and are wheelchair bound. Their plasma VLCFA profiles were normal, only pristanic acid and pipecolic acid were elevated. Conclusion: Peroxisomal defects can be missed by standard laboratory investigations in plasma.

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cholesterol in its more polar 24-hydroxycholesterol (24OHC), a blood-brain barrier permeable molecule. Objectives: Brain cholesterol synthesis depends on the mitochondrial formation of citrate serving as cytosolic acetyl-CoA donor. We investigate whether impaired brain cholesterol synthesis is a common denominator in inborn errors of metabolism by investigating the plasmatic levels 24OHC. Methods: 24OHC is measured in plasma samples by SID-GC-MS. The analytical procedure comprises a solid-phase extraction step to selectively isolate multiple-hydroxylated cholesterol derivatives. Conclusion: We have defined 24OHC reference values: a unique age dependent profile was found whereby 24OHC is low (50nM) in the neonatal period followed by a steep incline with its maximum at 1,5 yrs (450nM), followed by a slow decline to 50nM at 12 yrs, after which the levels of 24OHC are stable. Plasma samples from individuals affected with PDH deficiency, combined D/L-2HGA, SLOS, Niemann Pick C and PKU have been assessed. For the majority of these samples, the levels of 24OHC deviate from the average of the reference values.

P-498 P-500 Nonsense mediated mRNA decay affects nonsense transcripts levels and in vitro response to gentamicin and ataluren in X-ALD

Spinal cord anomalies in Smith-Lemli-Opitz syndrome (SLOS) new addition to the phenotype spectrum

Amorosi CA1, Kemp S2, Dodelson de Kremer R1, Argaraña CE3, Ramirez Oller AM1

Kanungo S1,2, Conley S3, Baker EH3, Vockley J2, Porter FD3

1 CEMECO ,Children's Hospital, UNC, Córdoba, Argentina; 2AMC, University of Amsterdam, Amsterdam, Netherlands; 3CIQUIBIC,School of Chemistry,UNC, Córdoba, Argentina

1 UCLA Intercampus Genetics Training Prgm, Los Angeles, United States; 2Children's Hospital of Pittsburgh - UPMC, Pittsburgh, United States; 3National Institutes of Health, Bethesda, United States

Background: X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene, characterized by increased concentrations of very long-chain fatty acids due to a defect in peroxisomal βoxidation. Aminoglycosides and PTC124 can readthrough premature termination codons (PTCs) allowing the translation of full length proteins. Response to drugs was found only in patients with the higher level of mRNA. Nonsense-mediated mRNA decay (NMD) is a mechanism which degrades transcripts carrying PTCs and has an important role in response to treatments to promote readthrough. UPF1 RNA helicase is involved in this pathway. Objectives: To prove aminoglycosides and PTC124 in fibroblast cultures from patients with X-ALD. To analyzed NMD efficiency in XALD fibroblasts. Materials and Methods: Fibroblasts from patients (p.Trp137*, p.Ser290*, p.Arg464*) were treated with different doses of gentamicin and PTC124. Protein expression was analyzed by Western blot. NMD was directly inhibited by using siRNA against UPF1 and indirectly by Cicloheximide. Levels of mRNA were determined by qPCR. Results: We didn't detect any increase in PALD expression after treatment with PTC124 or gentamicin. Downregulation of NMD increases the level of ABCD1 transcripts in two patients. Conclusions: Aminoglycoside therapy doesn't improve X-ALD for the mutations analyzed. NMD affects the level of many ABCD1 transcripts.

Background: Smith Lemli Opitz Syndrome (SLOS), the most common inborn error of sterol metabolism, includes a host of neurodevelopmental and behavioral findings. Cholesterol is necessary for sonic hedgehog signaling, thus, a potent morphogen influencing embryonic neurodevelopment. Known SLOS neurodevelopmental / CNS phenotype includes a variety of structural abnormalities of the cerebrum and cerebellum. Purpose: Our study expands the neurodevelopmental phenotype spectrum to report on spinal cord and spinal column malformations in SLOS. Methods: As part of the SLOS natural history protocol in progress at the NIH, medical records were reviewed for clinical symptoms potentially related to the spinal cord. Results: 6 patients with radiological evidence of spinal cord or spinal column abnormality were identified. The spine and spinal cord findings included: scoliosis, spina bifida occulta, spinal cord syrinx, type 1 Chiari malformation, caudal agenesis, and Klippel-Feil anomaly. Even after identification of these abnormalities, some patients had unexplained clinical neurological symptoms. Conclusion: We suspect the same neurodevelopmental pathogenesis involving sonic hedgehog signaling implicated with cerebrum and cerebellum malformation in SLOS, may also cause spinal cord and spinal column malformations in SLOS. Investigations of unexplained neurological symptoms with MRI and ultrasound of the spine can lead to identification of surgically correctable abnormalities.

P-499

P-501

Disturbed brain cholesterol homeostasis in inborn-errors of metabolism: a common denominator?

Mass spectrometric analysis of urinary steroids allows for the diagnoses of essential hypertension, resistant to classical treatment

Struys EA1, Jansen EEW1, Wamelink MMW1, Salomons GS1

Dumin E1, Knopf C1, Asadi S2, Chernikov M2, Torgeman K3

1

1

VUmc medical Center, Metabolic Laborator, Amsterdam, Netherlands

Background: Cholesterol is a key molecule needed for proper brain development and functioning and is locally synthesized since it cannot be imported from the blood. Brain cholesterol homeostasis comprises the brain-specific CYP46A1 enzyme, which can hydroxylate

Lab of Clin Biochem,Rambam hospital, Haifa, Israel; 2Dep of Nephrology,Rambam Hospital, Haifa, Israel; 3Dep of Endo,Sourasky Medical Center, Tel Aviv, Israel Hypertension is a ubiquitous health problem. A significant proportion of hypertensive patients are resistant to conventional therapy, and identification

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of the underlying cause of hypertension is particulary important. The principal aim of our study was to study the possible role of the 11 beta-hydroxydehydrogenase (11 beta-HSD) in the pathogenesis of essential hypertension, especially the type 2 deficiency, leading to a mild form of syndrome of apparent mineralocorticoid excess (AME). The syndrome is attributable to congenital deficiency of the enzyme 11 beta-HSD, which converts cortisol to biologically inactive cortisone. It is a heritable form of hypertension characterized by hypokalemia and low renin levels despite normal levels of aldosterone. Gas chromatography-mass spectrometry analysis was used to detect urinary steroid metabolites.The standard biochemical marker of the type1 disorder is an increased ratio of tetrahydrocortisol (THF) + alloTHF/tetrahydrocortisone (THE) in the urine, and it was only slightly increased in all three cases studied. Moreover, we calculated two additional ratios for THF+alloTHF/F and for allo-THF+THE/F, and they were slightly decreased and indicative of AME type 2. Conclusions: Analysis of specific urinary steroid metabolites can allow for the identification of candidate patients for AME type 2.

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oxidation system causes accumulation of VLCFA in blood and tissues. Determination of VLCFA levels mainly as C24:0/C22:0 and C26:0/C22:0 ratios are basic diagnostic criteria for peroxisomal diseases. Materials and Methods Case report The female child at the age of 4 -5 months presented increasing elevated serum transaminases, bilirubin, γGT, prolonged prothrombin time and portal hypertension. The hepatocirrhosis in course of biliary atresia was diagnosed. Measurements of serum VLCFA levels were also elevated, C24:0/C22:0 = 1.335 (n<0.960) and C26:0/C22:0 = 0.110 (n<0.020). At the age of 5 months the liver transplantation from family donor was performed. Serum VLCFA levels were analyzed by GC. Results and Conclusions: Strongly elevated VLCFA levels before liver transplantation, normalized afterwards. The cirrhosis of liver may result in elevated VLCFA levels and may cause false positive results in diagnosis of peroxisomal disorders. The C24:0/C22:0 and C26:0/C22:0 ratios are secondary markers of an ongoing pathological process of liver.

P-502 P-504 Peroxisomal disorders: five years experience Female child with X linked adrenoleukodystrophy Kilic M1, Tokatli A1, Kalkanoglu-Sivri HS1, Dursun A1, Topaloglu H2, Wanders RJ3, Waterham H3, Coskun T1 1 Hacettepe Univ, Pediatric Metab Unit, Ankara, Turkey; 2Hacettepe Univ, Pediatric Neurology Unit, Ankara, Turkey; 3Lab Genet Metab Dis, AMC, Univ of Amster, Amsterdam, Netherlands

Canda E1, Kose M1, Kagnici M1, Kurtgoz S1, Kitis O2, Ucar SK1, Coker M1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2Div Radiology, Ege Univ, Izmir, Turkey 1

Backround: Peroxisomal disorders are autosomal recessive inherited disorders with prevalence of 1/50.000 (except for X-ALD). 10 cases with a diagnosis of peroxisomal disorders are discussed in terms of clinical, biochemical, radiological and genetic findings. Patients: Total of 10 cases were diagnosed based on clinical, biochemical, radiological and genetic findings, ages between 7 days-7 years between 2008-2013 at Hacettepe University and Kecioren Pediatrics Metabolic Clinics. Plasma and fibroblast culture studies showed Dbifunctional protein deficiency (D-BP) in 3 patients, a patient with Zellweger syndrome (ZS), a patient with infantile Refsum disease (IRD), 3 patients with Zellweger spectrum diseases (ZSD), a patient with Contiguous ABCD1 DXS1357E deletion syndrome (CADDS) and a patient with X-adrenoleucodystrophy (X-ALD). Patients diagnosed with ZS, D-BP deficiency and CADDS died before one year of age. The others are in follow-up with severe mental-motor retardation. X-ALD patient underwent bone marrow transplantation without succeed. Conclusion: Although peroxisomal disorders are well established and discussed for diagnosis in terms of clinical, biochemical, radiological and genetic findings, still no definitive treatment exists to date. Currently, orthotropic liver transplantation (for IRD) and bone marrow transplantation (for X-ALD) are being used clinically, however more studies and clinical experience are needed.

X linked adrenoleukodystrophy(X-ALD) is the most common peroxisomal disorder. It's inherited neurodegenerative disorder caused by mutations in ABCD1 gene. Approximately 20% of X-ALD female carriers may develop neurological symptoms. Skewed X chromosome inactivation has been proposed to influence the manifestation of symptoms in X-ALD carriers. Six years old girl complained for loss of skills during last three months. History of neurological development was normal. During last year she had ataxia and behavioral changes. On physical examination, she had absence of head control, could not talk and had general hypertonicity. Peroral hyperpigmentation was also recognized. There was no consanguinity. Her father had Addison disease and died after a suicide. Her cranial MR showed signal hyperintensities in bilateral occipitoparietal white matter, corpus callosum and internal capsule. Her cervical MR investigation showed upper cervical hyperintensity. Very long chain fatty acids(VLCFA) levels were abnormal and C26 concentration was increased. Adrenal functions tests demonstrated insufficiency. Heterozygote p.W132X mutation was found in ABCD1 gene. She died at the age 6 years and 8 months of age. Our patient showed extremely rapid progression with devastating. We reported this case to emphasize the importance of the female carriers follow up and to discuss the phenotype-genotype correlation in X-ALD.

P-503

X-linked adrenoleukodystrophy: two manifesting heterozygotes in a cohort of Czech and Slovak patients

P-505

Very long-chain fatty acids as biomarkers of liver dysfunction Stradomska TJ1, Pawłowska J2, Jankowska I2 Dpt Biochem, Child Mem Heath Inst, Warsaw, Poland; 2Dpt Gastrol Hepat, Child Mem Heath Inst, Warsaw, Poland

Peskova K1, Dvorakova L1, Vlaskova H1, Treslova H1, Jahnova H1, Magner M2, Jesina P1, Hornik P1, Bartova P1, Waterham H3, Wanders R3, Hrebicek M1

1

1

Inst Inher Metab Disord, Gener Univ Hosp, Prague, Czech Republic; Depart Ped Adol Med, Gener Univ Hosp, Prague, Czech Republic; 3 Academic Medical Center, Amsterdam, Netherlands 2

Peroxisomal diseases are caused by inborn errors in the metabolic or biogenetic function of peroxisome. The main clinical symptoms are hepatosplenomegaly, liver dysfunction and also neurological disorders. Very long –chain fatty acids (VLCFA, > C22:0) are exclusively degraded in peroxisomes by β-oxidation pathway. Disturbance of peroxisomal β-

Introduction: The most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD), is a recessive neurodegenerative disease. The major phenotypic forms are

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childhood cerebral (ccALD) and adrenomyeloneuropathy (AMN). The diagnosis of X-ALD was established in 25 Czech and Slovak families. Results: All patients had elevated concentration of C26:0, elevated ratios of C26:0/C22:0 and C24:0/C22:0 and a mutation in ABCD1 gene. In the cohort there are two symptomatic females. The first is a carrier of previously published mutation c.887A>G. Sequencing of cDNA showed a strong prevalence of the peak corresponding to the mutated allele. The second female patient was a two-year-old girl with microcephaly, psychomotor delay, hearing and vision disorder, seizures, and leukodystrophy. Pathological VLCFA profile and a previously found pathogenic mutation c.1553G>A in a heterozygote state was identified. Sequencing of cDNA showed 70 – 80% prevalence of the mutated allele. However, the sequencing of patient's asymptomatic mother revealed very similar results. Conclusions: It may reflect at least three possible explanations: 1) The X-inactivation status does not correspond to the phenotype in X-ALD heterozygotes in all cases. 2) The genotype-phenotype correlation is poor. 3) Clinical picture of heterozygotes may result from a combination of X-ALD and other factors (severe perinatal risks). Support: RVO-VFN64165/2012, PRVOUK-P24/LF1/3

X-linked adrenoleukodystrophy (X-ALD) is the most common inherited leukodystrophy, a metabolic disorder of the nervous system characterized by axonopathy in spinal cords and/or cerebral demyelination, adrenal insufficiency and accumulation of very long-chain fatty acids (VLCFA) in plasma and tissues. The disease is caused by malfunction of the ABCD1 gene, which encodes a peroxisomal transporter of VLCFA or VLCFA-CoA. In the mouse, ABCD1 loss causes late onset axonal degeneration in the spinal cord, associated with locomotor disability resembling the most common phenotype in patients, adrenomyeloneuropathy. Using the mouse model, patients' fibroblasts, and patients' brain necropsies, we show that an excess of the VLCFA C26:0 leads to a multifaceted dysfunction of mitochondria by generating: i) mitochondrial radical oxygen species, ii) oxidative damage to mitochondrial DNA and proteins, iii) inefficient oxidative phosphorylation; and iv) reduced mitochondria biogenesis. Oral administration of the antidiabetic drug pioglitazone restored mitochondria biogenesis, neutralized oxidative damage, and reversed bioenergetic failure in the spinal cords of the X-ALD mouse model. Most importantly, the treatment halted locomotor disability and axonal damage. These results will be translated into a phase II clinical trial for adrenomyeloneuropathy patients.

P-506

Integrated biology reveals increased oxidative damage and circulating inflammation in adrenomyeloneuropathy (AMN)

Mutation of ABCD1 and phenotype of vietnamese patients with xlink adrenoleukodystrophy (X-ALD)

P-508

Fourcade S1, Ruiz M1, Jové M2, Schluter A1, Guilera C1, Casasnovas C3, Ortega FJ1, Ferreira B1, Pamplona R2, Portero-Otín M2, Pujol A4

Vu D1, Nguyen K1, Shimozawa N2 IDIBELL,CIBERER, Barcelona, Spain; 2IRBLleida, Lleida, Spain; Neuro Dep, Hosp Uni de Bellvitge, L'Hospitalet de Llobregat, Spain; 4 IDIBELL, CIBERER, ICREA, Barcelona, Spain 1

2

National Hospital of Pediatrics, Hanoi, Viet Nam; Gifu University School of Medicine, Gifu, Japan

3

Background: X-linked adrenoleukodystrophy (X-ALD) is caused by a defect in ABCD1 gene. This disease characterized by progressive neurologic dysfunction, occasionally associated with adrenal insufficiency. Objectives: To describe phenotype and to identify mutations of ABCD1 in Vietnamese patients with X-ALD. Patients and Methods: Clinical features, biochemical and cerebral MRI finding of 9 cases from 7 unrelated families were studied. Mutation analysis of ABCD1 was performed using PCR and DNA direct sequencing. Results: adrenal insufficiency were observed in 8/9 cases; 7/9 cases showed neurological symptoms; 2/9 cases had only symptoms of chronic adrenal insufficiency until 12 and 5 years of age, respectively. 8/8 cases showed increased plasma VLCFA. Neuroimaging studies (cerebral MRI) showed classical posterior pattern in 7 cases who had neurological symptoms. We identified 6 different mutations of ABCD1. Of which, four novel mutations [c.1202G>T (p.Arg401Trp); c.1208T>A (p.Met403Lys); IVS8+28-551bp del; and the extent of deletion included between IVS1+505 and IVS2+ 1501] were identified in four unrelated patients with neurological symptoms. The reported mutation c.1628C>T (p.Pro543Leu) was identified in two sibling. The reported mutation c.1553G>A (p.Arg518Gln) was found in a boy without neurogical symptoms at 5 years of age. Conclusions: the genotype-phenotype correlations have not been clarified in studied cases.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder characterized by axonopathy and demyelination in the central nervous system and adrenal insufficiency. Main X-ALD phenotypes are: (i) an adult adrenomyeloneuropathy (AMN) with axonopathy in spinal cords, (ii) cerebral AMN with brain demyelination (cAMN) and (iii) a childhood variant, cALD, characterized by severe cerebral demyelination. Loss of function of the ABCD1 peroxisomal fatty acid transporter and subsequent accumulation of very long-chain fatty acids (VLCFA) are the common culprits to all forms of X-ALD. The mouse model for XALD exhibits a late-onset neurological phenotype with locomotor disability and axonal degeneration in spinal cords. Recently, we have reported oxidative damage, inflammation and energetic failure as early events in the pathogenesis cascade. In order to identify new biomarkers and altered pathways for disease prognosis, but also for monitoring of therapeutic intervention, we have applied an integrative biology approach using untargeted, high throughput metabolomics and transcriptomics from mouse and human patients. Validation of altered pathways shows that low-grade inflammation, lipid peroxidation and DNA oxidative damage are disease hallmarks even in noninflammatory AMN patients, and have potential as disease biomarkers in plasma and peripheral mononuclear cells.

1

P-509 P-507 The peroxisomal disease adrenoleukodystrophy, a secondary mitochondriopathy?: translation to the clinics Lopez-Erauskin JL1, Ruiz M1, Morató L1, Naudi A2, Portero-Otín M2, Pamplona R2, Ferrer I3, Villarroya F4, Fourcade S1, Pujol A5

Atypical case of Smith-Lemli-opitz syndrome with a w326l/s397l mutation in DHCR7 gene Skoknova M1, Behulova D2, Pribilincova Z3, Fajkusova L4, Chandoga J5, Dolnikova D1, Bzduch V1 1st Dep Ped, Child Univ Hosp, Bratislava, Slovakia; 2Dep Lab Med, Child Univ Hosp, Bratislava, Slovakia; 32nd Dep Ped, Child Univ Hosp, Bratislava, Slovakia; 4Cent Mol Biol Gen Ther, Univ Hosp, Brno, Czech Republic; 5Cent Med Gen, Univ Hosp, Bratislava, Slovakia 1

1

2

3

IDIBELL,CIBERER, Barcelona, Spain; IRBLleida, Lleida, Spain; Institute of Neuropathology, CIBERNED, Barcelona, Spain; 4Dep Bioquim i Bio Mol, UB, Barcelona, Spain; 5IDIBELL, CIBERER, ICREA, Barcelona, Spain

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Background: Seriously affected newborns with Smith-Lemli-Opitz syndrome (SLOS) have high mortality. We are reporting a patient with a rare W326L/S397L mutation who was in critical state during the neonatal period but despite of that now has a favourable course of the disease. Such a case was not described before. Case report: A mature neonate (46,XY) was born with multiple anomalies (microcephaly, micrognathia, cleft defect, microglossia, polydactyly, multiple syndactyly with 2-3 toe syndactyly, micropenis with hypospadias). Immediately he required ventilatory support and on second day he incurred a heart failure, although no critical congenital heart defect was found. Diagnosis of SLOS was confirmed by GC-MS of serum lipids (cholesterol 0,79mmol/l, 7dehydrocholesterol 0,25mmol/l) and DNA analysis (p.W326L/p.S397L mutation in DHCR7 gene). During 3rd week we initiated cholestrol supplementation (50mg/kg/day). In two months the boy was successfully weaned from mechanical ventilation. Later he underwent operation of patent ductus arteriosus and cleft palate without complications. He began to thrive and since 1,5 years of age required no regular corticoid substitution of peripheral hypocorticism. In contrast to our other SLOS patients the changes in serum lipid profile didn`t show typical extremely low level of apolipoprotein A-I (Behulova D. et al., J.Inherit.Metab.Dis., 2000).

structurally related have been identified in databases but their function is unknown. A polymorphism in the ACAD10 gene has been linked to obesity in Pima Indians and hence the physiological role of both proteins draws interest. We cloned human ACAD10 and 11 transcripts in E. coli, but production of these proteins failed. Expression of inserts coding for ACAD10 S637-I1059 and ACAD11 S355-I780 sequences representing the ACAD domain was successful. The purified proteins have absorbance spectra typical of flavoproteins but are unstable. Antibodies were generated for each and eight mouse tissues were surveyed for presence of antigens using western blotting and immunofluorescence staining. Anti-ACAD10 detected ~100Kd and ~125Kd-bands in extracts from pancreas and brain, respectively. AntiACAD11 detected 75Kd-band was in all tissues with kidney and lung showing the strongest, and its signal was confined to peroxisomes. Removing a predicted ACAD10 N-terminus signal peptide resulted in production of an ~100Kd antigen in E. coli, while expressing the predicted 75Kd ACAD11 resulted in the production of a 65Kd antigen. Isolating the native forms of these proteins is now crucial to elucidating their structure and function. P-512 Bile acid synthesis defect misdiagnosed as autoimmune hepatitis

P-510 Duran G1, Gana C1 Smith-Lemli-Opitz syndrome diagnostic background for six Romanian cases Plaiasu V1, Kirov A2, Ochiana D1, Motei G1, Cretu R1, Brezan F1, Todorov T2, Todorova A2, Anca I1 1 IOMC Prof.dr.Alfred Rusescu, Bucharest, Romania; 2Genetic MedicoDiagnostic Lab Genica, Sofia, Bulgaria

The Smith-Lemli-Opitz syndrome (SLOS) is a rare autosomal recessive multiple malformation condition caused by a large spectrum of mutations in the DHCR7 gene and at the biochemical level by a deficiency of the enzyme 7-dehydrocholesterol reductase, the final enzyme of the cholesterol biosynthetic pathway. The aim of our study was to investigate DNA of the patients clinically diagnosed with SLOS to search for different mutations in the DHCR7 gene in Romanian patients and to study possible genotype-phenotype correlation. Six different cases were clinically suspected of SLOS. Phenotypic spectrum of our patients was broad: typical facial features, acral dysgenesis, neuro-developmental disability, gastrointestinal dysfunction, cardiac anomalies, genital-renal malformations. For two severely affected patients, life span was limited by lethal internal malformations. Sequencing of all exons and exon/intron boundaries of the DHCR7 gene was performed only for five patients and the diagnosis of SLOS was confirmed by the detection of pathogenic mutations. We identified 4 different mutations, in homozygous or compound heterozygous status, such as nonsense mutation p.Trp151*, missense mutations p.Thr93Met and p.Tyr432Cys and frameshift mutation c.1315delC. The Romanian mutational spectrum of DHCR7 gene is similar to that observed in other European countries, except c.1315delC, which is a novel mutation, unpublished so far.

1

Div Ped, P. Univ Cat Chile, Santiago, Chile

Inborn error of bile acids synthesis are rare hereditary defects presented as progressive cholestatic liver disease and fat soluble vitamin malabsorption secondary to the failure to produce normal bile acids and accumulation of unusual bile acids. Deficiency of 3β-hydroxy-Δ5-C27-steroid dehydrogenase(3β-HSD) is the most frequent defect. Autoimmune hepatitis is inflammatory disease associated to hypergammaglobulinemia, presence of autoantibodies, responsive to immunosuppressants. Hyperbilirubinemia, elevation of transaminases, normal γ-glutamyl transpeptidase (GGT) and nonspecific liver biopsy are common to both disorders. Objective: Report defect of bile acid misdiagnosed as an autoimmune hepatitis. Case Report: This is a previously healthy 4-year-old girl, first child of non-consanguineous parents that at 30 months presented coluria, jaundice, progressive increase of transaminases (AST378 IU/L, ALT262 IU/L), normal GGT, presence of anti-smooth muscle antibodies and unspecific liver biopsy diagnosed as autoimmune hepatitis, initially responsive to prednisone and azathioprine. Six months later, clinically worsening was evident and bile acid synthesis defect was suspected. The urine bile acids profile was consistent with 3β-HSD deficiency. Cholic acid was initiated followed of normalization of liver function. Conclusion: Bile acids synthesis defects must be included in the differential diagnosis of cholestatic liver disease beyond neonatal period, because it is a treatable defect with good prognosis. 13. Neurotransmitter disorders P-513 Mitochondrial proliferation and enlargement in succinic semialdehyde dehydrogenase (SSADH) deficiency

P-511 Vogel KR1, Schumaker JM1, Lakhani R2, Subramani S2, Gibson KM1 Cloning and expression of ACAD10 and ACAD11 proteins in E. coli 1 1

1

2

1

1

Section of Clin Pharm, Wash St Univ, Spokane, WA, United States; Dept Biol, Univ Calif San Diego, La Jolla, CA, United States

Mohsen A-W , Karunanidhi A , Kormanik K , Otsubo C , Vockley J

2

1 Dept of Pediatrics, Univ of Pittsburgh, Pittsburgh, United States; 2Dept Human Genetics, Univ of Pittsburgh, Pittsburgh, United States

Background: Oxidative damage in the murine model (aldh5a1-/- mice) of SSADH deficiency, a disorder of gamma-aminobutyric acid (GABA) degradation with gamma-hydroxybutyrate (GHB) accumulation, features increased superoxide dismutase and catalase and depleted glutathione. The latter has been confirmed in one patient. Here, we evaluated

Acyl-CoA dehydrogenases 10 (ACAD10) and 11 (ACAD11) transcripts encoding 1059-aa (118Kd) and 780-aa (87Kd) proteins predicted to be

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the hypothesis that altered antioxidant defenses would be reflected in abnormal mitochondrial number or structure. Methods: aldh5a1+/+ and aldh5a1-/- liver (median lobe; ML) and brain (cerebral cortex (CC); sagittal) were sectioned for mitochondrial (mito) analysis using transmission electron microscopy (TEM: FEI Tecnai G2; 9600X). Multiple micrographs from n=2 animals each were analyzed using ImageJ software. Results: TEM findings included ML mito number: aldh5a1-/-, 12.7 +/0.8 (39; micrographs counted); aldh5a1+/+, 9.7 +/- 0.8 (31) (p<0.01); ML mito area (nm2): aldh5a1-/-, 1215 +/- 71 (71); aldh5a1+/+, 855 +/54 (90) (p<0.0001); CC mito number: aldh5a1-/-, 7.8 +/- 0.7 (30); aldh5a1+/+, 4.9 +/- 0.4 (23) (p<0.01). Conclusions: To our knowledge, this is the first demonstration of mito proliferation in an inborn error of metabolism, suggesting new pathomechanisms and treatment possibilities for SSADH deficiency. The mechanism(s) underlying mitochondrial alterations in the murine model are under active investigation. Support: NIH HD58553; NS82286; the SSADH Foundation. P-514 Open-label trial of taurine in succinic semialdehyde dehydrogenase (SSADH) deficiency: preliminary outcomes Gibson KM1, Bottiglieri T2, Arning E2, Schreiber J3, Theodore WH3, McCarter RJ4, Wiggs E3, Yu Y5, He J5, Pearl PL5 1

Section of Clin Pharm, Wash St Univ, Spokane, WA, United States; 2 Baylor Res Inst, Dallas, TX, United States; 3Clin Epilepsy Section, NINDS, NIH, Bethesda MD, United States; 4Epidem/Biostat, Childrens Natl Med Ctr, Washington DC, United States; 5Child Neurol, Childrens Natl Med Ctr, Washington DC, United States An open-label trial of taurine in SSADH deficiency is underway in which subjects titrate to 200mg/kg/day taurine with assessment of adaptive functioning using age-normalized scales. Twenty-five patients (14M/11F, age 0.5-33yrs) have been recruited. Selected individuals provided additional data (neuropsychological testing, cerebrospinal fluid (CSF) amino acids and free/total GABA). Sixteen subjects (8M/8F) provided follow-up data (mean time elapsed 13 mo). Adaptive domains were unchanged with taurine (p=ns). Eight patients (6M/2F; age range 12-33 yrs) enrolled into the biomarker arm; baseline average full scale (FS) IQ was 44.1 (range 3455; 3.6 SD < mean). Four patients completed testing (2M/2F) at 6 month follow-up on taurine; average FS IQ was 43.4 (range 33-51; p=ns off/on treatment). CSF biomarkers were (n=4): taurine: 9 +/- 1 uM; SEM); 26 +/6 (p<0.05 off/on): free GABA (ref range, 32-170 nM): 394 +/- 88; 403 +/62 (p=ns, off/on): total GABA (ref range 3.3-12.1 uM): 14.3 +/- 1; 14.9 +/2.4 (p=ns, off/on). CSF beta-alanine and carnosine (histidine:beta-alanine dipeptide) were increased (10-30 uM; nl, undetected). Thus far, adaptive behaviours and CSF biomarkers are unimproved with taurine, and CSF amino acid data further support the concept that GABA and beta-alanine share identical transamination reactions. Support: NIH HD 58553 P-515

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homozygous IVS6+4A>T knock-in (KI) mutation grew poorly and exhibited severe dyskinesia and hindlimb clasping after birth. Objective: Understand the treatment effect on widespread gene therapy vector. Materials and Methods: In this study, we injected an AAV9-hAADC vector (2E+10 vg in 2 ul each side) into bilateral cerebral ventricles of newborn AADC-KI mice. Viral vector distribution, AADC enzyme activity, neurotransmitter levels, motor function and behaviours were evaluated. Results: The treated mice exhibited widespread but asymmetric expression of the vector especially over the cortex and septal nuclei. Brain dopamine levels returned to normal in treated mice, and the levels of serotonin also elevated. Both the growth speed and the survival increased in the treated mice. Improvements in hindlimb clasping and cardiovascular functions were also observed (p<0.05 between KI and KI-AAV9). The treated mice seemed to be more active than the wild-type mice, but other than that there were no complications from the treatment. Conclusion: Congenital deficiencies of neurotransmitters can be treated safely and efficiently by intracerebral ventricular injection of AAV9 vector in newborn mice. P-516 The natural history of L-aminoacid decarboxylase (AADC) deficiency: report of two never treated adult patients. Mastrangelo M1, Carducci Cl2, Polizzi A3, Artiola C2, Sofia V4, Barone R5, Carducci CA2, Zappia M3, Ruggieri M6, Leuzzi V1 Dip Ped Neur Psi Inf, Sapienza Univ Rome, Rome, Italy; 2Dip Med Sper Sapienza Univ Rome, Rome, Italy; 3Dip Sci Neurol, CNR, Catania, Italy; 4 Dip Neurol, Univ Catania, Catania, Italy; 5Div Neur Psi Inf, Univ Catania, Catania, Italy; 6Dip Sci Edu, Univ Catania, Catania, Italy 1

AADC deficiency results in a severe neurologic impairment in most of the patients. We report on two young adult Italian sisters with a history of early onset oculogyric crisis, ptosis, developmental and language delay, dysarthria, muscular hypotonia and hyposthenia, and orthostatic hypotension. On examination they showed: mild intellectual disability (without mental deterioration), bradyphrenia, bradykinesia, oculogyric crisis, multifocal spontaneous myoclonic jerks, and mild derangement of postural reactions. Blood Prolactin was high. CSF analysis revealed (nmol/L): HVA 124.37 and 169.349, respectively (r.v. 98-450), 5-HIAA 30.34 and 50.4, respectively (r.v. 45-135); MHPG 18.6 and 12.9, respectively (r.v. 28-60); 3 OMD 521.8 and 328.4, respectively (r.v. <50); 5-HTP 65.6 and 64.3, respectively (r.v. <10). Two novel pathogenic mutations were detected on AADC gene [p.P35FS (c.105delC) and p.F237S (c.710 T>C)]. The treatment (rotigotine, pyridoxine, escitalopram) was started when they were 22 and 32 years old, resulting in a relevant improvement of motor disorders which was paralleled by the normalization of blood prolactin. Present cases suggest that: a) after a progressive neurological and neurodevelopmental derangement during infancy and childhood, the clinical deficits associated with AADC deficiency stabilizes in adolescence and young adulthood; b) adult subjects are still remarkably responsive to the therapy.

Intracerebral ventricular injection of an AAV9 vector relieves symptoms and improves survival of newborn mice with a congenital neurotransmitter deficiency

P-517

Lee NC1, Chien YH1, Tzen KY1, Byrne BJ2, Hwu WL1

Aromatic L-amino acid decarboxylase (AADC) deficiency is a cause of long-fasting hypoglycaemia

1

Med Gent, Nat Taiwan Univ Hosp, Taipei, Taiwan; 2Powell Gene Therapy Center, University, Gainesville, United States

Arnoux JB1, Damaj L2, Napuri S2, Serre V3, Hubert L1, Cadoudal M1, Simard G4, Ceballos I1, Christa L1, de Lonlay P1

Background: Aromatic L-amino acid decarboxylase (AADC) is responsible for the syntheses of dopamine and serotonin. Children with AADC deficiency exhibit severe motor and autonomic dysfunctions. Mice with

1 Necker Hosp, Paris Descartes Univ., Paris, France; 2Paed Dept, Sud Univ Hosp, Rennes, France; 3Paris Diderot Univ, Paris, France; 4 Biochem Dep, Univ Hosp, Angers, France

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Objectives: Long-fasting hypoglycemia in children may be induced by neurotransmitter disorders. Case report: A 5 year-old girl with a medical history of chronic diarrhea presented 3 episodes of severe hypoglycemia (20 mg/dL) between 3 and 5 year-old. She became pale and sweaty with hypothermia (33.5°c), bradycardia (45/min), acidosis and she presented a generalized seizure. During the 17-h fast test, insulin, C-peptide, hGH, IGF1, cortisol, amino acids, and acylcarnitines were in the reference range for hypoglycemia. However, vanyllactic/vanylpyruvic acids in urine and neurotransmitters in CSF indicated an AADC deficiency. The low AADC enzyme activity in plasma (5 pmol/min/ml RV: 20-130) and the presence of two heterozygous mutations c.97G>C (p.V33>L, father) and c.1385G>C (R462>P, mother) in the DCC gene confirmed the diagnosis. She was supplemented with pyridoxine and raw corn starch (1g/kg) at evening diner to reduce the night fast. The episodes of hypoglycemia were suppressed and the diarrhoea improved. Conclusion: Here is the first case report of an isolated long-fasting hypoglycemia due to a non-typical AADC deficiency. Hypoglycemia was severe but the other neurological clinical hallmarks present in AADC deficient patients were moderate. Thus, neurotransmitter disorders should be considered in any patients presenting hypoglycemia with urine excretion of vanyllactic acid.

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recessive sepiapterin reductase (SR) deficiency. The hepatic phenylalanine hydroxylase system is compromised due to subnormal tetrahydrobiopterin (BH4) levels and hydroxylation of phenylalanine (Phe) to tyrosine (Tyr) is reduced, resulting in elevated Phe/Tyr ratios. Case report: We report on the Phe loading test results in a female adult with clinically suspected doparesponsive dystonia (DRD) before and under treatment with BH4. Results: During Phe challenge under BH4 treatment Phe concentration remained below the normal range while a transient mild hypertyrosinemia was observed, leading to an extremely low Phe/Tyr ratio. Total biopterin levels were, as expected, under substitution with BH4 elevated in all samples. After withdrawal of BH4, patterns of blood Phe, Tyr, and Phe/Tyr ratios on a repeat phenylalanine loading test resulted normal. The increase of biopterin levels supported the negative loading test and excluded a BH4 deficiency caused by a functional GTPCH or SR deficiency. Conclusion: These data indicates that therapy with BH4 should be avoided during Phe loading tests because of possible misinterpretation. P-520

P-518

Cerebrospinal fluid neopterin analysis in neuropediatric patients: establishment of a new cut-off value for identification of inflammatory and immune-mediated process

Hyperphosphatasia associated with markedly low CSF pyridoxal phosphate (PLP) but no impairment of monoamine metabolism.

Molero-Luis M1, Fernandez S1, Sierra C1, Ormazabal A1, Muñoz C1, Garcia-Cazorla A1, Pérez-Dueñas B1, Serrano M1, Jordán Y1, Artuch R1

Heales S1, Neergheen V2, Pope S2, Oppenheim M2, Dadhra J1, Eltze C1, Kurian M1, Gissen P1, Mills P3, Clayton P3

1

Great Ormond Street Hospital, London, United Kingdom; 2National Hospital, Queen Square, London, United Kingdom; 3UCL Institute of Child Health, London, United Kingdom 1

Disorders of PLP metabolism occur in conjunction with impaired dopamine and serotonin turnover, i.e. due to the PLP cofactor requirement of aromatic amino acid decarboxylase. Recently, we investigated a male infant who presented at 10 months with developmental delay and epileptic spasms. There was no evidence of liver or bone disease. CSF analysis revealed undetectable PLP. The CSF concentrations of homovanillic acid and 5hydroxyindoleacetic acid were within our age related reference ranges. A markedly elevated plasma alkaline phosphatase was noted; 1,500 (Ref Range; 60 – 330 U/L). Low serum PLP is reported to occur in hyperphosphatasia due to phosphohydrolysis of PLP to pyridoxal (PL). PL can cross the blood brain barrier and PLP regenerated intracellularly. Consequently, in conditions where extracellular alkaline phosphatase is elevated, the apparent paradox of decreased CSF PLP in the presence of normal dopamine and serotonin turnover can be explained. To investigate this further, we determined alkaline phosphatase activity in the CSF of our patient and 19 disease control samples. Activity was only demonstrable in the patient. PLP in the patient CSF was also unstable when compared to controls. Alkaline phosphatase activity should be considered when interpreting CSF profiles that indicate PLP deficiency and preserved monoamine metabolism.

Hospital Sant Joan de Deu, Esplugues Llobregat, Spain

Background: High cerebrospinal fluid (CSF) neopterin is a marker of central nervous system inflammatory-immune mediated processes. Objectives: To assess data from 606 neuropaediatric patients describing clinical and biochemical features of those neurological disorders with CSF neopterin values above a new cut-off value, which was previously defined. Methods: We selected two groups of patients (Group 1=68 patients with meningoenchepalithis and Group 2=52 children with peripheral infection) to establish the new CSF neopterin cut-off. Then, we studied 606 CSF samples from neuropediatric patients, classified in (A):genetic diagnosis, (B): acquired/unknown etiologic neurologic diseases and (C):inflammatory-immune mediated processes. Results: The CSF neopterin cut-off value was 61 nmol/L. Fifty-six out of 606 cases presented CSF neopterin above this value. Group C (n=23) had statistically higher CSF neopterin, proteins and leukocytes values. Sixteen of 23 patients had CSF neopterin above cut-off, while 3 and 7 patients presented increased leukocyte and protein values, respectively. A statistically significant association was found between CSF neopterin, proteins and leukocytes in the 606 patients. White matter disturbances were associated with high CSF neopterin concentrations. Conclusion: A representative percentage of children with neurological disorders presented increased CSF neopterin concentrations, showing the importance of CSF neopterin analysis for the identification of inflammatory-immune mediated processes. P-521

P-519 Impact of tetrahydrobiopterin (BH4) treatment on phenylalanine loading tests Opladen T1, Hoffmann GF1, Kühn AA2, Blau N1

Case report: french canadian male twins with pyridoxinedependent epilepsy and a single heterozygous variant of unknown significance in ALDH7A1 Shuen AY1, Lefrancois M2, Srour M3, Cyr D4, Waters PJ4, Spector E5, Hyland K6, Al-Hertani W1

1

Div Metab Dis, Univ Child Hosp, Heidelberg, Germany; 2Dep Neurology, C. Virchow, Charite, Berlin, Germany Background: Phenylalanine (Phe) loading test is a useful tool in the differential diagnosis of dopa-responsive dystonia (DRD) due to autosomal dominant or recessive GTP cyclohydrolase I (GTPCH) deficiency or autosomal

1 Dept Med Genet, MUHC, Montreal, Canada; 2Dept Clin Nutr, MUHC, Montreal, Canada; 3Dept Neuro/Neurosurg Ped, MUHC, Montreal, Canada; 4 Dept Ped, CHUS & Univ de Sherbrooke, Sherbrooke, Canada; 5Univ Colorado Sch Med DNA Diagnostic Lab, Aurora, United States; 6Medical Neurogenetics, Atlanta, United States

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We report on male twins with Pyridoxine-Dependent epilepsy (PDE), born to non-consanguineous French Canadian parents. They presented at 7 days of life with metabolic acidosis and seizures. EEG showed burst suppression refractory to anti-epileptic medications. α-Aminoadipic semialdehyde (α-AASA) in urine and plasma Pipecolic acid provided biochemical confirmation of the diagnosis. In addition, analysis of CSF monoamines via HPLC with electrochemical detection revealed the chromatographic pattern characteristic of PDE with the two peaks of unknown identity. The twins were started on Pyridoxine supplementation with resolution of electrographic and clinical seizures. Furthermore, a Lysine restricted diet was initiated, at 3 months of age, as part of the Vancouver based NOEL study and further clinical improvement is being noted on their neurological examination. Sequencing of the 18 coding exons and the intron donor/acceptor sequences of the ALDH7A1 gene identified a single heterozygous c.1292C>T variant of unknown significance, that is paternally inherited. This variant has been reported once previously in a Japanese patient with PDE who was found to be compound heterozygous for mutations in ALDH7A1. In an attempt to identify the second mutation in the probands, further analysis by targeted exonic aCGH was performed but was negative for an exonic deletion in ALDH7A1.

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SRD is a rare neurotransmitter disorder presenting with early developmental delay and axial hypotonia. We report on the presentation symptoms of SRD in a new case detected in the first months of life. This 7-month-old girl was born at term from healthy nonconsanguineous Italian parents. Occasional jerks of upper left limb were noticed since the first days of life and became continuous and generalised in the following few weeks, associated with irritability and gastrooesophageal reflux. On examination (5 months) she exhibited: neuromotor delay, extensor stiffening pattern of head and trunk with upward gaze deviation; hypokinesia; limb rigidity; almost continuous oscillatory movements of limb, trunk and head at rest, which could be interrupted by voluntary movements or when the examiner handled her. Social smile and reactivity were preserved. CSF examination revealed (r.v.): Neopterin 4.52 microg/L (2.30-10.10); Biopterin 21.05 microg/L (2.4011.80); Sepiapterin 17.5 nmol/L, (< 1); HVA 77 nmol/L (3241379); 5-HIAA nmol/L 18 (189-1380); 3-MHPG 42 nmol/L (98-168)]. SPR gene sequence (patient and parents) revealed the patient was compound heterozygote for two pathogenetic mutations (p.R150V, p.K251*). Levodopa/Carbidopa plus 5-OH-Tryptophan treatment resulted in a prompt clinical improvement. Conclusion: SRD is one of the few causes of early rest tremor in the infancy.

P-522 P-524 Development of a short assay for urinary vanillyllactate by GC-MS to aid in the diagnosis and monitoring of neurometabolic conditions affecting dopamine metabolism

Retrospective review of cerebrospinal fluid catecholamine and serotonin metabolites for indications and diagnosis of neurotransmitter disorders

Aitkenhead H1, Heales SJR1, Leakey J1, Winter R1 1

Chem Path, Great Ormond St Hospital, London, United Kingdom

Background: Cerebrospinal fluid (CSF) neurotransmitters are the most importance diagnostic biochemical test for inborn errors of dopamine biosynthesis. However there is also a need for more readily available non invasive biochemical tests. Urinary vanillyllactate (VLA) has been shown to be a suitable diagnostic marker for aromatic amino acid decarboxylase deficiency and potentially disorders of vitamin B6 metabolism. However, it is cumbersome to measure and usually semi-quantitative. Methods: Urinary VLA was quantitated by GC-MS in selective ion monitoring mode using deuterated vanillylmandelate as internal standard following extraction and derivatisation. Results: The VLA quantitation limit was 0.05 micromol/L, assay imprecision was 8.0 – 25.7 % and average recovery was 82 %. Urinary VLA reference intervals were determined. Urinary VLA was found to be stable at room temperature for 14 days. Conclusion: A short quantitative GC-MS method for urinary VLA has been developed and validated. It is envisaged that VLA in combination with prolactin will be measured as first line investigations in patients presenting with features of central dopamine deficiency. The results will assist in selecting patients for invasive lumbar puncture and CSF neurotransmitter analysis and have the potential for treatment monitoring in patients with diagnoses of inborn errors of dopamine biosynthesis. P-523 Rest tremor as very early presenting symptom of sepiapterin reductase deficiency (SRD) Leuzzi V1, Carducci Cl,2, Giannini MT1, Tolve M2, Pignattoni Cl,2, Pasquali A2, Danti R1, Carducci CA2 Dept Pediatrics and Child Neurol and Psy, Rome, Italy 2Dept Experimental Medicine, Rome, Italy 1

Mercimek-Mahmutoglu S1, Hyland K2, Kyriakopoulou L3, Sidky S1, Allam N1, Schulze A1, Siriwardena K1, Yoon G1, Logan W4, Soman T4 Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 2Med Neurogen, , LLC Neuromsc & Neurogen, Atlanta, United States; 3 Biochem Lab, DPLM, Univ Toronto, Toronto, Canada; 4Div Neurol, Dp Ped, Univ Toronto, Toronto, Canada 1

Background: Catecholamine, serotonin and pterine (CS&P) neurotransmitter deficiencies (CS&P-ND) result in global developmental delay (GDD), peripheral hypertonia, central hypotonia, movement disorder and autonomic dysfunction. Methods: To estimate the prevalence of CS&P-ND, we retrospectively reviewed all patients who underwent CSF neurotransmitter investigations (homovanillic acid (HVA); 5-hydroxyindol acetic acid (5-HIAA), tetrahydrobiopterin, neopterin, pyridoxal phosphate) at The Hospital for Sick Children between 2004-2012. All measurements were performed in the Medical Neurogenetics Laboratory. Results: 95 patients were included. Indications were: Group-1 (n=37): movement disorder or spasticity; Group-2 (n=46): GDD and epilepsy; Group-3 (n=7): GDD and hypotonia; Group-4 (n=5): positive newborn screening for phenylketonuria. In group-1; one patient had autosomal dominant GTP cyclohydrolase (GTP-CH) deficiency with low CS&P levels confirmed by molecular testing. In group-2; 3 patients had pyridoxine-dependent epilepsy (PDE) with an abnormal peak suggestive of PDE and one patient with pyridoxalphosphate dependent epilepsy by low HVA, 5-HIAA and pyridoxal phosphate levels. In group-4; 4 patients had dihydropteridine reductase deficiency (DHPR) and 1 patient had 6-pyruvoyltetrahydropterin synthase (PTPS) deficiency. Conclusion: The prevalence of confirmed CS&P-ND (4 DHPR; 1 PTPS; 1 GTP-CH) was 6.3% (6/95). Including three PDE and one pyridoxal-phosphate dependent epilepsy patients (4/95), the diagnostic yield of CSF CS&P analysis was 10.5% (10/95).

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P-525 Atypical clinical response and normal CSF neurotransmitters in a patient with pyridoxamine-5-phosphate oxidase deficiency caused by a novel deletion in the PNPO gene Guerin A1, Tran C1, Lewis J2, Go CY3, Salomons GS4, MercimekMahmutoglu S1

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organic acids were unremarkable. Pyridoxine was started at 250 mg/d; EEG showed no improvement. Plasmatic AASA was positive. At 24 days old the patient died. Analysis of the coding region and a Del-Dup targeted exonic microarray of ALDH7A1 demonstrated a single heterozygous duplication not previously reported in exon 6. The complex pathophysiology of antiquitin deficiency exposes difficulty in establishing an outcome as well as genotype-phenotype correlation. P-527

Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 2Div Neonataology, Dp Ped, Univ Toronto, Toronto, Canada; 3Div Neurol, Dp Ped, Univ Toronto, Toronto, Canada; 4Metab Lab, VU Med Center, Amsterdam, Netherlands 1

Background: Pyridox(am)ine-5-phosphate oxidase deficiency (PNPOD) is caused by mutations in the PNPO gene. Less than 20 patients have been reported. Case report: 2.5-month-old female was born at 35 weeks gestation. Apgars were 9 and 9. Hiccups were noticed from 32 weeks gestation. She presented with myoclonic seizures at age 1 hour. EEG showed focal and generalized myoclonic jerks and burstsuppression pattern. Despite clinical improvement on pyridoxalphosphate (40mg/kg/d), EEG showed frequent sharp waves and discontinues background at age 2 days. Lactate was moderately elevated (4.1mmol/L). Neuroimaging studies were normal. EEG showed prolonged background discontinuity, frequent sharp and polyspikes during burst activity at age 15 days. She was encephalopathic and hypotonic up to 3 weeks of age. She gradually started to improve from 3 weeks of age and was discharged home at age 5 weeks. PNPO-D was confirmed by homozygous novel pathogenic frameshift mutation (c.448_451del;p.Pro150Argfs*27) in the PNPO gene at age 2 months. She had marginally elevated 3-O-methyldopa (329nmol/L; reference<300) and low pyridoxal phosphate (27nmol/L; reference 30-80) in CSF on two days of pyridoxalphosphate therapy. Conclusion: We present a new patient with PNPO-D and a novel mutation. She had atypical response to pyridoxal-phosphate therapy with long-lasting encephalopathy despite well-controlled clinical seizures. P-526 Pyridoxine-dependent epilepsy: fatal outcome in a patient with early diagnosis and no response to pyridoxine. Monroy-Santoyo S1, Neria-Maguey E2, Ramirez-Navarrete E3, San Esteban-Sosa JE3, Martínez-Natera OC2 1 IEM and Screen Lab, Nat Inst Paed, Mexico City, Mexico; 2NICU, ABC Med Centre, Mexico City, Mexico; 3Ped Neu, ABC Med Centre, Mexico City, Mexico

Normal cerebrospinal fluid pyridoxal 5'-phosphate level in a patient with neonatal-onset epileptic encephalopathy and a homozygous deleterious PNPO mutation Levtova A1, Laberge AM1, Mills P2, Camuzeaux S2, Diadori P3, Clayton P2, Hyland K4, Rossignol E3, Mitchell G1 1 Medical Genetics, CHU Sainte-Justine, Montreal, Canada; 2Clin Mol Genet, UCL Inst Child Health, London, United Kingdom; 3Neurology, CHU Sainte-Justine, Montreal, Canada; 4Medical Neurogenetics, Atlanta, United States

Deficiency of pyridox(am)ine 5'-phosphate oxidase (PNPO, OMIM 610090) is a recently-discovered treatable autosomal recessive inborn error of metabolism. Neonatal epileptic encephalopathy and a low cerebrospinal fluid (CSF) pyridoxal 5'-phosphate level are the reported hallmarks of PNPO deficiency but its clinical and biochemical spectra are not fully known. Case presentation: A girl born at 33 3/7 weeks of gestation developed severe seizures in the first hours of life. The seizures initially responded to GABAergic agonists, but became refractory to all treatments in subsequent months. Brain MRI and infectious and metabolic evaluations at birth, including urinary alpha-aminoadipic semialdehyde (AASA), were normal. Lumbar puncture was unsuccessful until age three months, when it showed: pyridoxal 5'-phosphate, 52 nmol/L (normal, 23-64); homovanillic acid, 392 nmol/L (normal, 450-1132); 5-hydroxyindoleacetic acid, 341 nmol/L (normal, 179-711); and 3-O-methyl-dopa, 30 nmol/L (normal, below 300). The patient was not being treated with pyridoxine, nor with pyridoxal 5'-phosphate, at the time of the lumbar puncture. She died at age 14 months. PNPO gene sequencing revealed apparent homozygosity for c.674G>A (p.Arg225His), and both parents were p.Arg225His heterozygotes. Expression studies of mutant p.Arg225His PNPO revealed greatly reduced activity. Conclusion: A normal CSF level of pyridoxal 5'-phosphate does not rule out PNPO deficiency. P-528 Neuropsychiatric improvement with dopa agonist in PTPS deficiency Horvath GA1, Stockler-Ipsiroglu S1, Elbe D2, Ipsiroglu O3, Baer S4

Pyridoxine-dependent epilepsy (PDE) is a disorder associated with severe neonatal and perhaps, antenatal refractory epilepsy caused by antiquitin deficiency. Some patients respond to a single intravenous dose of pyridoxine, whereas others require multiple doses. Alphaaminoadipic semialdehide (AASA) and pipecolic acid serve as diagnostic markers of antiquitin deficiency. The objective of this work is to present a PDE case non responsive to pyridoxine with a novel mutation. First born to a non-related Mexican couple. Uneventful pregnancy until 36.5 weeks gestation when foetal hypo motility developed. Two hours after birth she became hypoxic with high-pitched cry and restlessness. Five days later hypo activity became severe, she developed several apnoea episodes and myoclonic movements. EEG showed burst suppression pattern; phenobarbital and vigabatrin were started. Brain MRI was normal. Lactate, pyruvate, acylcarnitines, aminoacids and urinary

1

Biochem Dis, BC Children's Hospital, Vancouver, Canada; 2Pharmacol, BC Children's Hospital, Vancouver, Canada; 3Dev Pediatrics, Univ BC, Vancouver, Canada; 4Psych, BC Children's Hospital, Vancouver, Canada Background: 6-pyruvoyl-tetrahydropterin synthase (PTPS) deficiency is treated with a combination of BH4, L-dopa/carbidopa, and 5-hydroxytryptophan, which is effective in controlling the hyperphenylalaninemia and neurological deficits, mainly the movement disorder. The behavioural and psychiatric aspects in many patients are difficult to control. Case report: premature, severe IUGR male newborn of consanguinous Afghani couple, with family history of multiple infant deaths. Started BH4 and neurotransmitter replacement therapy at 1 month of age.

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Dosages had to be increased very slowly because of side effects. At 9 months of age treatment with MAO inhibitor was added, but he didn't tolerate it. His motor development was delayed, and had irritability and severe feeding difficulties, requiring a gastrostomy-feeding tube. This was removed at the age of 3 = years. Currently at the age 7 years he has no motor difficulties, and he is in regular school, with average cognitive function. He still has severe behavioural problems, ADHD, aggressivity, and more recently severe stuttering. A dopa agonist, pramipexole was added to his treatment, and his behaviour and stuttering improved. His prolactin levels also stabilized. Conclusions: Treatment with dopa agonist in PTPS deficiency, could improve the behavioural and psychiatric symptoms, which are probably caused by dopamine deficiency P-529 Effect of treatment on neuropsychiatric outcome in an adult with autoimmune cerebral folate deficiency Horvath GA1, Bishop CM2, Sirrs SM3 1 Biochem Dis, BC Children's Hospital, Vancouver, Canada; 2Pychol, Univ BC, Vancouver, Canada; 3Adult Metab Clin, Vancouver Gen Hosp, Vancouver, Canada

Background: Cerebral folate deficiency (CFD) is a rare treatable cause of neurological dysfunction due to auto-antibody production to the folate receptor. Few adult cases with complex neuropsychiatric sequelae are described in the literature. Case report: A 48-year-old male presented with childhood-onset history of muscle weakness, persistent fatigue, cognitive deterioration and personality/behavioural problems. Depression, anxiety and severe psychiatric problems required several psychiatric admissions for psychosis. CSF investigations at age 47 years demonstrated low levels of HVA, 5HIAA and 5MTHF (29 nmol/L, ref 40-120), without signs of systemic folate deficiency. Comprehensive genetic testing was negative (DHFR, FOLR1). Serum folate receptor blocking antibody titers were high (1.43 pmoles/ml serum; ref >1high) and binding antibodies against the receptors titers were moderately high (2.04 pmoles IgG/ml serum; ref >1-5 medium). Neuropsychological/psychological evaluation demonstrated weak visual-spatial/perceptual, attention, higher-order language, working memory, mental processing speed and executive functioning skills, but high IQ. Treatment with L-dopa/carbidopa, 5-hydroxytryptophan and folinic acid normalized levels of CSF neurotransmitters and folate levels. Energy and neuropsychiatric symptoms improved for anxiety and depressive experience but not executive functioning deficits. Conclusions: Suspecting CDF in adult patients with complex neuropsychiatric symptoms might be rewarding, as treatment with folinic acid could be beneficial. P-530 Rat pheochromocytoma PC12 cell line as a useful model for the study of tyrosine hydroxylase deficiency Diez H1, Ortez C2, Fernandez-Castillo N3, Izquierdo M4, Gorostiza P4, Cormand B3, Llobet A5, Artuch R2, Garcia-Cazorla A2 FSJD, Barcelona, Spain; 2HSJD, Barcelona, Spain; 3Dep Gen, Fac Bio, UB, Barcelona, Spain; 4IBEC, Barcelona, Spain; 5IDIBELL-UB, Barcelona, Spain 1

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a group of neurotransmitters that include dopamine itself, norepinephrine and epinephrine and are necessary for the correct control of important neurological processes like motor control, movement, sympathetic nervous system response, motivation, attention or learning. PC12 is a widely studied catecholaminergic cell line that expresses the enzymes and the molecular machinery necessary for the synthesis and liberation of dopamine, making it a proper model to study at cellular level the steps of this process. We have focused on three patological mutations (R202H, L205P and Q381K) and developed plasmids for expression in mammals cells. Using transient transfection methodology, we have developed a cell culture system for the study of mutations of TH involved in THD. Our results indicate that our model is appropiate for the characterization of biochemical and cellular properties of altered TH and suggest that it could be a suitable tool for the development of in vitro preclinical assays of pharmacological compounds for treatment of TH. P-531 Sepiapterin reductase deficiency case report: a monoamine neurotransmitter disorder mimicking cerebral palsy Sivri S1, Öztürk Hişmi B1, Ünal Ö1, Konuşkan B2, Topçu M2, Dursun A1, Tokatlı A1, Thöny B3, Coskun T1 1 Div Metab Dis, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Div Ped Neurol,Hacettepe Univ Child Hosp, Ankara, Turkey; 3Div Metab Dis, Univ Child Hosp, Zürich, Switzerland

Sepiapterin reductase(SR) deficiency(SRD) is a rare but treatable metabolic disorder responsive to levodopa.Most patients present in infancy with non-specific features including developmental delay and axial hypotonia.When present, oculogyric crises may be a distinguishing sign. Over time there is development of limb hypertonia, hyper-reflexia, dystonia and more apparent diurnal fluctuation and sleep disturbance.The diagnosis is made upon abnormal CSF neurotransmitter and pterin profile, further supported by molecular and/or enzymatic analysis. Here,we present the first SRD case diagnosed upon abnormal CSF findings in Turkey.A 9-month-old girl with developmental delay,axial hypotonia and episodic upward gaze was referred to our hospital and she had normal EEG,brain MRI and routine metabolic screening tests.Axial hypotonisity, hypertonic extremities and oculogyric crisis suggested a neurotransmitter defect and lumbar puncture was performed.On CSF, HVA and 5-HIAA levels were extremely low, with high biopterine and normal neopterine levels which were all suggestive of SRD.CSF sepiapterin level was also in the upper high limit. SR enzyme levels were extremely low and a known stop codon mutation (p.K251X) was found in SPR. She dramatically responded to levodopa.SRD should be considered in patients with developmental delay and axial hypotonia,and in patients with unexplained or atypical cerebral palsy especially if hypotonia or dystonia are present. P-532 Unusual csf pyridoxal phosphate and pterin profiles in a subset of patients taking vitamin b6 supplementation Pope SAS1, Neergheen V1, Oppenheim M1, Bhairo C1, Mills P2, Footitt E2, Clayton P2, Land J1, Heales S3 1

Neurometabolic Unit, Nat Hosp Neurology, London, United Kingdom; Institute of Child Health, London, United Kingdom; 3Great Ormond Street Hospital, London, United Kingdom 2

Mutations on the Tyrosine Hydroxylase (TH) gene are the cause of a rare inborn metabolic disorder named TH Deficiency (THD). Tyrosine Hydroxylase is the enzyme responsible for the conversion of L-DOPA to dopamine, which is the limiting step of the production of cathecolamines,

Our laboratory receives over 800 CSF samples a year for the investigation of possible neurotransmitter disorders. As part of this service, essential

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P-533

Objectives: Biotin-responsive basal ganglia disease (BBGD) is an autosomal recessive neurometabolic disorder. It is characterized by subacute encephalopathy with seizure, dysarthria and dystonia following a history of febrile illness. The aim of this study was to summarize the long term outcome, biochemical, molecular and neuroradiological features and the importance of thiamine in the treatment regimen. Method: Chart review of 18 patients from two tertiary institutions. Result: 18 Saudi children presented with a clinical picture similar to that described above. 22% died,The frequency of acute crises, delayed diagnosis and the immediate initiation of biotin and thiamine treatment correlate directly with neurological outcome. Two of the patients are spastic quadriplegic, 6 patients are normal and the rest have neurological deficits affecting speech and motor function. One-third of the present patients showed the recurrence of acute crises while on biotin therapy alone, but after the addition of thiamine, crises did not recur. All of the patients have a homozygous missense mutation in SLC19A3 gene. Conclusion: Clinicians should suspect BBGD in any child presenting with the findings described above. Both biotin and thiamine are essential for disease management. Therefore, the disease name should be changed to biotin-thiamine-responsive basal ganglia disease (BTBGD).

Glut-1 deficiency: a clinical and genetical study of 5 patients

P-535

Flotats-Bastardes M1, Quiroz A1, Vila-Pueyo M2, Raspall-Chaure M1, Domingo R3, Macaya A1

Hypervitaminosis D due to 1,25-(OH)2D-24 hydroxylase (CYP24A1) deficiency causing nephrolithiasis

Pediatr Neurology, Hosp Vall d'Hebron, Barcelona, Spain; 2Neuroped Res Dep, Hosp Vall d'Hebron, Barcelona, Spain; 3Pediatr Neurology, Hosp Virgen Arrixaca, Murcia, Spain

Nesterova G1, Malicdan M1, Sakaki T2, Collins M3, Adams DR4, Boerkoel C4, Gahl W1

co-factors (pterins and pyridoxal phosphate (PLP)) are measured by HPLC. The pterin method uses electrochemical and fluorescence detection while the pyridoxal phosphate method uses fluorescence alone. We have noted a small group of patient CSF samples having a fluorescent compound (X) eluting very close to dihdrobiopterin in the pterin chromatograms - appearing at first glance similar to dihydropteridine reductase or sepiapterin reductase deficient patients. Patients with compound X in their pterin profiles also had a late running compound (Y), in addition to unusual unidentified compounds in their PLP chromatograms. On further investigation it was found that the majority of these patients were noted to be taking vitamin B6 supplementation. It was hypothesised that the unidentified compounds could be vitamin B6 derivatives. Pyridoxic acid was found to be compound Y in the pterin profile and one of the unidentified compounds in the PLP profile. However, compound X remains unidentified. Interestingly, not all patients taking vitamin B6 supplementation display these unidentified compounds. Therefore they may be potential biomarkers of a distinct biochemical phenotype with regards to vitamin B6 metabolism.

1

Background: Glut-1 deficiency syndrome is a disorder due to heterozygous mutations in SLC2A1. Classical phenotype comprises epilepsy, microcephaly and severe developmental delay which improve with ketogenic diet. However, clinical spectrum has been extended to encompass less severe phenotypes, usually including paroxysmal or fluctuating clinical signs. We describe clinical and molecular findings of five patients, including two monochorionic twins. Patients and methods: Patients were diagnosed at ages 8- 14 years All mild-to-moderate cognitive dysfunction. Other symptoms were exerciseinduced paroxysmal dyskinesia (n=3), epilepsy (n=4) migraine (n=2), tremor (n=1) and episodic ataxia (n=1). Three patients had a glucose LCR/plasmatic <0,4 and two <0,6. PCR and Sanger sequencing or MLPA of SLC2A1 revealed the variants p.Val165Ile, p.Arg223Trp, p.Val237Valfs*2 and exon 1 deletion, three of them novel. The ketogenic diet or uncooked cornstarch partially improved some clinical manifestations Conclusion: The described phenotypes of Glut-1 deficiency seem to be more frequent than the classical phenotype. Glut-1 deficiency must be suspected in patients with unexplained cognitive dysfunction and paroxysmal disorder at any age, even with mild hypoglycorrhachia. Clinical improvement is to be expected, at least of the paroxysmal signs, upon institution of appropriate therapy. 14. Disorders of vitamins and trace minerals P-534 Biotin-responsive basal ganglia disease should be renamed biotinthiamine-responsive basal ganglia disease: a retrospective review of the clinical, radiological and molecular findings of 18 new cases

1 National Human Genome Res Inst, NIH, Bethesda, United States; 2dep Biotech, Toyama prefecturial Univ, Toyama, Japan; 3Nat Inst Dental and Craniofac Res, NIH, Bethesda, United States; 4NIH Undiagnosed Diseases Prog, Bethesda, United States

Background/Objectives: An excess of 1α,25(OH)2D3 can result in nephrocalcinosis and nephrolithiasis. We evaluated the cause of increased 1α,25(OH)2D3 levels in the development of those disorders. Patients and Methods: We measured vitamin D metabolites and performed mutation analysis in CYP24A1 gene encoding 1,25(OH)2D24-hydroxylase in two patients with hypervitaminosis D nephrocalcinosis or nephrolithiasis enrolled in the National Institutes of Health Undiagnosed Diseases Program. CYP24A1 pathological variants were evaluated using the dbSNP database and the pathogenicity prediction programs. Results: Both patients exhibited elevated 1α,25(OH)2D3, and undetectable activity of 1,25(OH)2D-24-hydroxylase that inactivates 1α,25(OH)2D3. They had biallelic mutations in the CYP24A1 leading to the loss of function of this enzyme. Based upon dbSNP data, the frequency of deleterious CYP24A1 variants is estimated to be 1960 per 100,000 in the general population, or as high as 20% in all nephrolithiasis patients. Conclusion/Discussion: We found that CYP24A1 loss of the function mutations are associated with nephrocalcinosis and nephrolithiasis. Our assessment of CYP24A1 gene variants predicted that pathogenic defects in CYP24A1 may account for a significant fraction of nephrolithiasis. Nephrolithiasis represents a global health problem with a prevalence at 10%; recognition of CYP24A1 deficiency could prompt the recommendation that 1α,25(OH)2D3 levels be determined in such patients. P-536 Menkes disease caused by a novel frameshift mutation masquerading as an early onset mitochondrial encephalopathy

Alfadhel M1, Almuntashri M1, Bashiri FA2, Al Shalaan H1, Al Balwi M1, Al Rumayan A1, Eyaid W1, Al-Twaijri W1

Mecija M1, Gonzalez S1, Mercimek-Mahmutoglu S2

King Abdulaziz Medical City, Riyadh, Saudi Arabia; 2King Khalid University Hospital, Riyadh, Saudi Arabia

Clin & Met Gen, Hosp Sick Child, Toronto, Canada; 2Clin & Met Gen, Hosp Sick Child & U of T, Toronto, Canada

1

1

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Menkes disease is an X-linked inherited disorder of copper metabolism resulting in neurodegeneration, connective tissue disorder and failure to thrive. We report a 17-month-old boy presenting with focal seizures due to cerebral sinus venous thromobosis at age 2 months. Due to incerased seizure frequency cranial MRI was repeated at age 3 months showing bilateral symmetrical increased signal intensities in caudate nucleus and putamen together with elevated lactate peak in gray matter suggestive of a mitochondrial disorder. Muscle biopsy showed cytochrome c oxidase deficiency (0.13μmol/min/g; ref. range 0.8-6.03). His hair at age 4.5 months was steel-wool like and ceruloplasmin [<40 mg/L (264-473 mg/L)] and copper [1.2 umo/L (3.9-17.3 μmol/L)] levels were low. At time of diagnosis his seizures were well controlled with anti-epileptic medications and he had no failure to thrive. ATP7A genetic testing revealed a novel pathogenic frameshift mutation (c.3431delA; p.Asn1144MetfsStop20; maternally inherited). Despite starting copper-histidine therapy, he developed myoclonic seizures and bladder diverticulum on treatment. This case presented with atypical features masquerading as an early onset mitochondrial disorder which highlights the intitial diagnostic challenges. Menkes disease should be in the differential diagnsosis, even if there are no suggestive clinical features. P-537 Development of screening for pyridoxine-dependent epilepsy using newborn dried blood spots by LC-MS/MS

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Background: Pyridoxine dependent epilepsy (PDE) is an autosomal recessive disease caused by mutations in the ALDH7A1 gene resulting in alpha-amino adipic acid semialdehyde dehydrogenase enzyme deficiency in the lysine catabolic pathway. Patients: Case 1 is a 14-year-old male presented with neonatal seizures. Pyridoxine was started at age 2 weeks and seizures stopped. Since then he has been seizure free. Sequencing of the ALDH7A1 gene showed a novel homozygous c.1399G>A;pA467T mutation. Both parents were heterozygous. His urine alpha-amino adipic acid semialdehyde (alpha-AASA) was mildly elevated (2.0mmol/molcreatine; reference range<0.5) at age 14 years. Neuropsychological assessment revealed full Scale IQ of 81. Case 2 is a 12-year-old male presented with focal seizures at 9 weeks of age. He became seizure free after pyridoxine. Sequencing of the ALDH7A1 gene revealed c.500G>A (p.Asn167Ser), known disease causing (paternal) and c.1481+1G>T, novel splice site mutations (maternal). His urine alpha-AASA was moderately elevated (8.8mmol/molcreatinine). Neuropsychological assessment revealed full Scale IQ of 93. Conclusion: To best of our knowledge, there were about 5 patients with normal neurodevelopmental outcome reported so far. We presented two new patients with PDE-ALDH7A1 and two novel mutations in the ALDH7A1 gene. Both patients had normal cognitive outcome, but mild learning difficulties in other areas. P-539 Bilateral iliac arteries aneurysms in Menkes disease

Jung S1, Tran NTB1, Gospe Jr. SM2, Hahn SH3 Wichajarn K1, Auvichayapat N1, Techasatian L1 Seattle Children's Research Institute, Seattle, United States; 2Dept of Neurology, Univ of Washington, Seattle, United States; 3Dept of Pediatrics, Univ of Washington, Seattle, United States 1

Background: Early diagnosis and treatment for pyridoxine-dependent epilepsy (PDE) is important to minimize severe neurodevelopmental consequences. Objective: To evaluate the feasibility of PDE newborn screening by measuring the diagnostic markers (alpha-AASA and P6C) in DBS using LC-MS/MS. Methods: We analyzed original newborn DBS from three PDE patients and age-matched control retrieved from California DPH. These DBS had been stored at -20 °C. We also retrieved original newborn DBS from two PDE patients from Washington DOH, which had been stored at room temperature. Left-over blood samples from three PDE patients currently under our care were spotted on filter paper and stored at -80°C for positive control. Stability was analyzed. Results: Alpha-AASA and P6C in newborn DBS from three PDE patients retrieved from CA were elevated (A-AASA: 0.37~0.69 μmol/L; P6C: 0.35~2.29 μmol/L) while they were not detectable in controls. Newborn DBS stored at room temperature in WA did not show any detectable peaks. All positive control DBS from three PDE patients showed highly elevated biomarkers. A-AASA and P6C were fairly stable in DBS compared to plasma. Conclusions: The biomarkers for PDS are detectable in DBS from newborn patients with PDE which opens up the potential application of newborn screening for PDE. P-538 Two new patients with pyridoxine dependent epiliepsy caused by ALDH7A1 genetic defect: long-term follow-up and normal neurodevelopmental outcome

1

Dep Ped, Srinagarind Hosp, Khon Kaen U, Khon Kaen, Thailand

Background: Menkes disease is a rare x-linked recessive inherited disorder of abnormal Copper transportation, caused by ATP7A mutation. It's characterized by abnormal pigmentation of skin and hair, neurological deterioration and seizure. Most cases usually die by age 3 years. Objective: To demonstrate bilateral iliac arteries aneurysms as manifestation in Menkes disease. Case report: A 7-month-old Thai boy was referred with problems of seizure, global delayed development and abnormal skin and hair. Menkes disease was diagnosed by clinical characteristic pili torti, dysmorphic facies, hypotonia, feeding difficulty, delayed developmental milestone and seizure. Definite diagnosis was confirmed by low level of both serum copper and ceruloplasmin. From MRI brain, he had tortuous intracranial vessels and diffused brain atrophy. A large right internal iliac artery aneurysm and small left common iliac artery aneurysm were detected by MRA abdomen. Irregular tortuosity of abdominal aorta was also found in MRA; however, there was no bladder diverticulum. He received parenteral copper transfusion for a while and stop because of no significant clinical improvement. Seizure was controlled by vigabatrin and zonisamide. Genetic counseling was performed to his family. Conclusion: Anomalies of vessel especially aneurysm of iliac artery should become aware in Menkes disease. P-540 Development and validation of a simple LC-MS/MS method for the quantification of MTHF (N5-Mithyl-5, 6, 7, 8-titrahydrofolate) in human cerebrospinal fluid using deuterated MTHF as internal standard Dewulf J1, Vanderstocken C1, Nassogne MC2, Marie S1, Vincent MF1 1

Cordeiro D1, Mamak E2, Donner E3, Feigenbaum A4, Siriwardena K1, Mercimek-Mahmutoglu S1

Metab Dis Labo, Cliniques Univ St Luc, Brussels, Belgium; 2Ped Neuro & Metab, Cliniques Univ St Luc, Brussels, Belgium

1 Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 2Dp Psych Univ Toronto, Toronro, Canada; 3Div Neurol, Dp Ped, Univ Toronto, Toronto, Canada; 4Biochem Gen Dp Ped Univ California, San Diego, United States

Objective: Development and validation of a LC-MS/MS method for the quantification of MTHF in cerebrospinal fluid (CSF) using deuterated MTHF as internal standard (IS) for the diagnosis of cerebral folate deficiency.

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Materials and methods: No pre-treatment was required and artificial CSF was chosen for the validation. The CSF was first diluted = with ascorbic acid IS solution to reduce ion suppression effect. The analyte and IS were eluted (same retention time) on LiCrospher RP-18 5μ 150mm x 4,6mm (Grace) using an isocratic elution method. Detection and quantification were performed by electrospray ionization (ESI) in the positive ionization mode by multiple reaction monitoring (MRM). Results: The assay was linear over the concentration range of 10300nM (R2 >0,999) with the lower limit of quantification (LLOQ) of 10nM. The intra- and inter-day precision in terms of relative standard deviation (RSD %) was within 7, 2% and accuracy in terms of relative error (RE %) was within 13, 75%. Conclusion: This method fulfills all the regulatory requirements for specificity, linearity, LLOQ, precision and accuracy for the determination of MTHF in human CSF. P-541 Menkes disease in two females with characteristic clinical, biochemical, and molecular findings

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development of PH was not found and accepted as PPH and inhaler iloprost, furosemid,enalapril were initiated. Metabolic tests revealed high levels of urinary methylmalonic acid. Homocystein level was also found high and cobalamin was initiated. PPH regressed dramatically and disappeared within six months. At the last examination she was well and all the treatments were withdrawn except oral cobalamin. Mutation analysis identified c.484G>T (p.Gly162Trp) mutation in the cblC MMACHC gene which has not been described in the SNP database. Discussion: Hyperhomocysteinemia may cause PPH by complex mechanisms such as promoting vasoconstriction, endothelial dysfunction and enhancing in situ thrombosis. Cobalamin disorders should be screened in the etiology of PPH. P-543 CSF vitamin B6 levels and response to pyridoxal 5-phosphate in diverse epileptic encephalopathies Cortès-Saladelafont E1, Duarte S2, Molero M3, Casado M3, Sierra C3, O'Callaghan-Gordo M1, Sanmartí-Vilaplana X1, Fons C1, González-Álvarez V1, Pérez-Dueñas B1, Poo P1, Ramos F1, Artuch R3, García-Cazorla À1

Smpokou P1, Samanta M1, Lichter-Konecki U1 1

1

Div Genet, Ch National Med Center, Washington, DC, United States

Background: Menkes disease is an X-linked disorder of copper transport manifesting with neurologic, developmental, hair, cutaneous, and vascular findings. Females are generally thought to not be clinically affected. Objectives: We report two affected females with Menkes disease. Case report: An 11 month old girl presented at 8 months old with failure to thrive and hypotonia. Exam showed "kinky" hair with hypopigmented streaks, hypotonia, and cutis laxa. Labs showed low serum copper and ceruloplasmin levels, a heterozygous frameshift mutation in the ATP7A gene, and an Xq28 deletion (not including the ATP7A gene). Brain imaging showed vascular tortuosity. She is treated with copper injections. A 4 year old girl presented at 6 weeks old with seizures, then developed hypotonia and developmental delay. Exam showed white hair tips and microcephaly. Serum copper and ceruloplasmin levels were low normal and a heterozygous deletion in the ATP7A gene was found. Brain imaging showed vascular tortuosity. Conclusion: Menkes disease is important to consider in females with characteristic features due to treatment and management implications. In females, skewed X-inactivation likely plays a role in manifesting symptoms and clinical findings can vary owing to variable X-inactivation in different tissues. P-542 The co-existence of cobalamin C deficiency and primary pulmonary hypertension with a new mutation Gündüz M1, Ekici Filiz2, Özaydın Eda1, Ugarte M3, Ceylaner S4 1 Div Metab Dis,Ankara Dıskapı Child Hosp, Ankara, Turkey; 2Dep of Cardio,Ankara Dıskapı Chil Hosp, Ankara, Turkey; 3Dep Molec Biology, Madrid Auto Unv, Madrid, Spain; 4Intergen Lab, Ankara, Turkey

Dpt.Ped Neurol, Univ Hosp St.Joan de Déu, Esplugues de Llobregat (Barcelona), Spain; 2Mol Med Institute, Univ of Lisbon, Lisbon, Portugal; 3 Dpt. Biochem, Univ Hosp St.Joan de Déu, Esplugues de Llobregat (Barcelona), Spain PNPO gene mutations can produce neonatal refractory seizures that respond to pyridoxal phosphate (PLP). PLP is the active form of vitamin B6 and a cofactor of many enzyme reactions including neurotransmitter metabolism. We describe a series of 14 patients with CSF vitamin B6 deficiency and their response to PLP treatment. Patients (aged 1 day to 8 years) presented mainly with epileptic encephalopathy and low vitamin B6 in the CSF (4-31nmol/L; N:32-78). Some of them had a definite etiology (chromosomopathies, INAD, KCNQ2 mutations). 4 patients with unknown etiology had negative PNPO genetic study. Plasma PLP was decreased in 3 patients, plasma and CSF aminoacids were normal. 3-orthometyldopa was elevated in 1 case. 5-HIIA, HVA, and GABA were decreased in 8, 3 and 1 patients respectively. None had anemia or evidence of malabsortion syndrome. An extensive metabolic workup was negative. 12 received antiepileptic drugs being valproate the most frequently used. Response to treatment was globally positive for seizure control and cognitive performance. Other than PNPO mutations, PLP depletion in the CSF could be related to different clinical situations such as refractory epilepsy and the prolonged use of some particular antiepileptic drugs. PLP treatment may have positive effects regardless of the etiology. P-544 Cerebrospinal fluid selenium concentrations in pediatric patients with neurological disorders Tondo M1, Ormazabal A1, Casado M1, Pineda M1, Campistol J1, PérezDueñas B1, García-Cazorla A1, Serrano M2, Artuch R1 Div Metab Dis. Hosp St Joan Deu, Barcelona, Spain; 2Div Meab Dis, Universidad Autonoma, Madrid, Spain 1

Introduction: We present a case with cobalamin C (cblC) deficency and primary pulmonary hypertension (PPH) which has not been reported before according to our current knowledge. Case report: Fifteen month old girl was admitted to our hospital with dyspnea and edema in her legs. She was the fifth child of consanquineous parents. Physical examination revealed severe growth retardation, 1: systolic murmur, loaded S2 and marked hepatomegaly. Echocardiography showed the right ventricular and atrial dilatation. Pulmonary arterial systolic and diastolic pressures were high. Secondary etiology for the

Objectives: To study the cerebrospinal fluid (CSF) selenium (Se) status in pediatric patients with neurological disorders and to assess the relationship between CSF Se concentrations, biogenic amines and patient clinical data. Material and methods: Analysis of selenium, biogenic amine concentrations and glutathione peroxidase (GPx) activity in CSF of 179 patients with several neurological conditions was performed.

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Results: Twenty five patients presented with abnormal CSF Se concentrations associated with different clinical conditions. Eight had decreased CSF Se levels and 17 had increased levels. No association was found between Se values and biogenic amines concentrations by multiple linear regression analysis. A positive correlation between CSF Se and GPx (r=0.648; p<0.001) was observed. Amongst the patients with increased Se concentrations, 7 suffered from Kearns Sayre Syndrome (KSS) which is a mitochondrial DNA deletion syndrome that is thought to cause energy depletion in brain. Conclusions: Impaired Se concentrations affected 14% of patients with neurological dysfunction. The positive correlation observed between CSF Se values and GPx activity suggests that it may be a biomarker for antioxidant status in the CSF. High CSF Se concentrations found in KSS patients reinforce the idea of impaired transport (energy dependent process) through blood-CSFbarrier. P-545 Cobalamin (cbl) D deficiency - case report Ferreira AC1, Regala J1, Mira G2, Silva R3, Sequeira S1 Metab Unit, Hosp D Estefânia, Lisbon, Portugal; 2Ped Dep, Hosp Esp Santo, Évora, Portugal; 3Serv Neuroped, Hosp D Estefânia, Lisbon, Portugal 1

Introduction: Intracellular metabolism of cobalamin, comprehend several complementation groups, three of these affecting the common synthesis of Methylcobalamin and Adenosylcobalamin cofactors – cblC, cblD and cblF deficiencies. CblD deficiency is the rarest and can present with isolated methylmalonic aciduria or homocystinuria due to different mutations on the MMADHC gene. Case Report: A twenty-year-old gipsy girl presented at 18 months with seizures, developmental delay and frequent vomiting. She had axial hypotonia, decreased visual contact, poor hand manipulation, absent language and unsteady gait. Metabolic investigation revealed methylmalonic aciduria (1330mmol/mol Cr) and increased urine homocysteine (261μmol/gCr). Treatment showed improvement of biochemical parameters and neurological abnormalities. Complementation studies on cultured fibroblasts suggested a cblC/cblD deficiency and MMADHC sequencing detected homozygosity for c.748C>T mutation. She had two further episodes of metabolic decompensation at 13 and 20 years, with ataxia, tremor, slurred speech and decreased cognitive status related to discontinuation of medication. Presently, she has slight mental retardation, nystagmus on extreme gaze positions and brisk tendon reflexes. MRI is normal Comments: Only few cases of CblD defects have been reported. The description of the present patient may help in the recognition of apparent phenotype-genotype correlations and the association of mutations with specific ethnicities P-546 Biotinidase-deficient mice show cellular energy deficit and altered carbon metabolism gene expression, similar to that seen in nutritional biotin deprivation Hernández-Vázquez A1, Wolf B2, Pindolia K2, Hernández-González R3, Herédia-Antúnez A3, Ibarra-González I1, Velázquez-Arellano A1 IIB-UNAM. UGN-INP, Mexico; 2Dep Med Gen, Henry Ford Hosp, Detroit, MI, United States; 3INNSZ, Mexico 1

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Background: Nutritional biotin deficiency results in alterations of the expression of carbon metabolism genes, deficit of ATP and activation of the energy sensor, AMP-activated protein kinase (AMPK). To determine if similar changes occur in biotinidase deficiency (BD) we performed identical experiments in BD (homozygous knockout) mice compared to control mice. Material and methods: Determinations were performed on normal C57BL/6 mice and BD mice, with the same C57BL/6 genetic background, at days 5 and 10 on biotindeficient diet/22.1% VFTC (TD.98161 Harlan, Madison WI). These included serum glucose and insulin concentrations and, in liver, AMP and ATP, phosphorylated and total AMPK, mRNA concentrations (qRT-PCR) of genes involved in βoxidation (CPT1), gluconeogenesis (PEPCK), glycolysis (GK) and lipogenesis (FAS). Results/Discussion: BD mice had reduced ATP:AMP, activated AMPK, decreased concentrations of mRNAs of GK and FA, and increased concentrations for CPT1 and PEPCK. These results were not observed in control. In addition, there was reduced glucose with normal insulin concentrations, indicating increased insulin sensitivity in biotin deprivation. All of these findings are in agreement with our prior findings in nutritional of biotin deficiency. Therefore, these metabolic alterations likely play a role in the pathogenesis in BD. (Supported by PAPIIT-DGAPA-UNAM-IN206011, CONACYT-166857 and the Safra Research Foundation) P-547 Locus heterogeneity in riboflavin-responsive multiple acyl-CoA dehydrogenation deficiency Mosegaard S1, Olpin SE2, Sharrard MJ3, Manning NJ2, Boneh A4, Ryan K5, Andreasen C1, Kjeldsen M1, Gregersen N1, Olsen RKJ1 Res Unit Mol Med, Aarhus Univ Hosp, Aarhus, Denmark; 2Dept Clin Chem, The Children's Hospital, Sheffield, United Kingdom; 3Dept Paediatrics, The Children's Hosp, Sheffield, United Kingdom; 4Murdoch Childrens Research Institute, Melbourne, Australia; 5Dept of Clin Chem, Mater Hospital, Belfast, United Kingdom 1

Riboflavin-responsive multiple acyl-CoA dehydrogenation deficiencies (RR:MADD) are a heterogeneous group of disorders characterized by impaired function of several mitochondrial flavoprotein dehydrogenases that use FAD – derived from riboflavin – to transfer electrons to the electron transfer flavoprotein (ETF) and its dehydrogenase (ETFDH) for cellular ATP production in the respiratory chain. Genetic defects of ETFDH are the major cause of RR:MADD, where riboflavin-responsiveness seems to be mediated through FAD, which acts as a molecular chaperone stabilizing the structure and function of misfolded variant ETFDH proteins. Recently the clinical and genetic spectrum of RR:MADD was expanded by the identification of genetic defects of cellular riboflavin transporters (RFTs) in patients with Brown-Vialetto-Van Laere or Fazio Londe syndromes, showing RR:MADD metabolite-like profiles. In the present study we sequenced the coding regions of the SLC52A3 and SLC52A2 genes - encoding human RFT2 and RFT3, respectively - in six patients with clinical and biochemical indication of RR-MADD but without disease-causing variations in the genes encoding ETF or ETFDH. Besides a single SLC52A2 variation of unknown significance, no likely disease-causing variations were identified in the SLC52A3 and SLC52A2 genes, indicating that there are most probably undiscovered genes, which could lead to disturbed riboflavin metabolism and MADD.

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P-548

P-550

Implementation and verification of Wolf traditional method in patients with neurological problems associated with biotinidase deficiency in Obras Sociales del Santo Hermano Pedro, Antigua Guatemala

Expanding the phenotype of ATP7A-related copper transport diseases: response to cooper treatment Nascimento A1, Rego Sousa P1, Ortez C1, Boronat S 2, Rebollo M1, Jimenez-Mallabrera C2, Jou C1, Rovira C1, Colomer J1

Granados LN1, Barrientos C1, Silva G1, Silva L1 1

1

INVEGEM, Guatemala, Guatemala

Background: Biotinidase deficiency is an autosomal recessive disease affecting mainly the biotin metabolism, via alteration of the enzyme biotinidase, thereby causing neurological problems such as mental retardation, and skin conditions. In Guatemala currently scientific efforts are being done to implement the Neonatal Screening Program, which includes the test panel Wolf Traditional Method for the diagnosis of biotinidase deficiency. Objectives: implement the methodology for determining biotinidase deficiency by the traditional method of Wolf by UV-Vis spectrophotometry in the Laboratory of Metabolic Diseases INVEGEM. Evaluate the performance of UV-Vis spectrophotometer Glomax equipment. Verifying Wolf traditional method for determining biotinidase deficiency. Materials and Method: Blood spots on filter cards of 35 patients residents in Obras Sociales del Santo Hermano Pedro –OSSHP- Antigua Guatemala, with neurological problems, deafness and some of them with blindness were collected to evaluate the efficiency of the rmethod above described. The quantitative method was developed with controls and calibrators supplied by Kinderspital Zurich, Switzerland. Discussion and Results: Successful parameters such as specifity, linearity and quantitative range, validated our method with the analysis of variance ANOVA. These results, will be the base for our next metabolic program that will establish the cutoff values for the Guatemalan population.

Hospital Sant Joan de Deu, NMD Unit, Barcelona, Spain; 2Hospital Vall d' Hebron, Neurology Dpmt, Barcelona, Spain ATP7A-related copper transport disorders include classical Menkes disease and its milder forms, occipital horn syndrome (OHS), and ATP7A related distal motor neuropathy (DMN). DMN is described as a distinct clinical isolate phenotype of ATP7A mutations. Objectives: We describe two related patients with a novel ATP7A mutation, one of them with the association of OHS and DMN, and its response to copper treatment. Case Reports: Patient 1 was evaluated at one years-old for mild global developmental delay, hypermobile joints and pili torti. Serum copper and ceruloplasmin levels were normal. Imagiological evaluation showed cerebral vascular tortuosity. Abdominal ultrasound showed a giant bladder diverticulum. Neurophysiologic study was normal. Patient 2 is a sixteen-old male adolescent, maternal uncle of patient 1, with normal cognition, lax dry eczematous skin, hypermobile joints, pectus excavatum, distal lower limbs weakness with normal deep tendon reflexes and dysautonomia. Serum copper and ceruloplasmin levels were slightly diminished. Imagiological evaluations showed cerebral vascular tortuosity, occipital horns and abnormal bone densitometry. Neurophysiologic study showed axonal motor neuropathy. We started subcutaneous treatment with copper-histidinate, which failed to show clinical improvement. Conclusions: OHS can be associated with development of DMN, expanding the clinical spectrum of ATP7A-related copper transport disorders. P-551

P-549 Wernickes encephalopathy evoked by malnutrition in an adolescent Bimboese P1, Linden T1, Fiedler B1, Edelbusch C1, Schwartz O1, Schwindt W2, Rutsch F1, Omran H1, Kurlemann G1 1 Dep. of General Peds, Univ Child Hosp, Muenster, Germany; 2Inst Clin Radiology, Univ Hosp Muenster, Muenster, Germany

Wernicke's encephalopathy (WE) was first described as acute hemorrhagic superior polioencephalitis by Carl Wernicke in 1881. It is caused by thiamine (vitamin B1) deficiency. A clinical hallmark of WE is the classic triad of ataxia, somnolence and ophthalmoplegia/nystagmus, but only 10-16% of patients show this complete picture. The prevalence of WE in postmortem autopsy studies is much higher than expected from clinical diagnosis. A 17 year old girl was admitted to our emergency department with ophthalmoplegia and somnolence. A cranial magnetic resonance tomography (cMRT) showed a bithalamic signal enhancement in the T2 and FLAIR sequences. Blood thiamine level was initially low at 12,9 ng/ml (28,0 – 85,0). The clinical presentation improved rapidly after intravenous administration of thiamine. The past medical history revealed rapid weight loss because of an eating disorder. A common cause of WE in Europe is chronic alcohol abuse. In pediatrics, clinical settings involve states of malnutrition e.g. oncologic diseases or eating disorders. When a child is admitted to the emergency department with one symptom of the classic triad and evidence of malnutrition, thiamine deficiency and consecutive treatment has to be considered.

Low cerebrospinal fluid catecolamine, serotonin and 5methyltetrahydrofolate levels in a patient with infantile onset cblG deficiency Jobling R1, Mercimek-Mahmutoglu S1 1

Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada

Background: Cobalamin G (cblG) deficiency is an autosomal recessive cobalamin metabolism defect caused by mutations in the MTR gene, encoding methionine synthase. It is characterized by developmental delay, seizures, hyperhomocysteinemia, hypomethioninemia and low intracellular methylcobalamin. Case report: Twenty-three-month-old girl presented with developmental regression, microcephaly, central hypotonia and intractable epileptic encephalopathy at age 5 months. She had moderately elevated total homocysteine (100μmol/L; reference 0.5-11) and low methionine (9μmol/L; reference 1521) with normal methylmalonic acid in urine. Complementation analysis of the cultured skin fibroblasts showed low synthesis of methylcobalamin confirming cblG deficiency. She had known homozygous pathogenic intronic mutation (IVS-166 A>G) in the MTR gene (parents heterozygous). CSF neurotransmitters showed low levels of 5-methyltetrahydrofolate (28nmol/L; reference 40-240), homovanillic acid (245nmol/L; reference 450-1132) and 5 -hydroxyindolacetic acid (109nmol/L; reference 179-711). Conclusion: We presented a new patient with severe cblG deficiency and decreased CSF neurotransmitters levels. To our knowledge, this is the first patient with cblG disorder who had low levels of neurotransmitters and cerebral folate. These abnormalities are of particular interest given the known neurological and psychiatric manifestations of this condition. The

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findings in this case are a potential source of new insight into the pathophysiology of cblG.

Pugh's and New Wilson's index scores did not show significant association with poor outcome.

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P-554

First case of macular degeneration in cobalamin d deficiency

Mutation analysis of ATP7B gene in 12 Vietnameses Wilson patients

Ketteridge D1, Pater J2, Oates S1, Baumgartner M3, Bratkovic D1, Fletcher J1 SA Pathology at the WCH, North Adelaide, Australia; 2Womens and Childrens Hospital, North Adelaide, Australia; 3University Childrens Hospital, Zurich, Switzerland 1

Aim: We report the first case of macular degeneration in cobalamin D deficiency. Introduction: Retinal degeneration has been described in cobalamin C deficiency but not previously in cobalamin D deficiency. Case report: The patient was born to consanguineous parents and presented at 16 days with failure to thrive and poor feeding. Seizures began at day 18. Urinalysis showed elevated methyl malonic acid and homocystine. A provisional diagnosis of cobalamin C/D deficiency was made and treatment commenced with IM hydroxocobalamin and oral betaine by the 22nd day of age. Neurological status, feeding and weight improved. He was maintained on IM hydroxocobalamin and betaine. Cobalamin D deficiency was confirmed by molecular studies - MMADHC gene homozygous for c.696+1_4delGTGA, p.Phe204_Ala232del A squint repair was performed at 2 years 5 months with normal fundoscopy at that time. A mild squint subsequently recurred. Despite continued medical therapy his vision began to deteriorate and macular degeneration was evident on fundoscopy by 9 years of age. An increase in dose of IM hydroxocobalamin and oral betaine has not improved the maculopathy over the subsequent 12 months despite some biochemical improvement. Conclusion: The underlying mechanism of macular degeneration in cobalamin disorders remains to be elucidated. P-553 Prediction of poor outcome on medical therapy at the time of diagnosis in Wilsons Disease (WD) presenting as Chronic Liver Disease (CLD): is the new wilsons index helpful? Alam S1, Khanna R1, Sood V1, Rawat D1 1

Institute of Liver and Biliary Sciences, New Delhi, India

Twenty eight cases of WD presenting as CLD were managed with D Pencillamine and zinc. Univariate and multivariate analysis was done to see the association of the risk factors with poor outcome (liver transplantation or death) at the time of diagnosis. Four of 5 presenting as acute on chronic liver failure (ACLF), 3 of 17 as decompensated CLD had poor outcome. All 6 compensated CLD, 1 of 5 ACLF and 14 of 17 with decompensated CLD improved. On univariate analysis, bilirubin, AST, ALT, Child Pughs score and New Wilson's index score were significantly higher in those with poor outcome. On logistic regression analysis significant association with poor outcome were high ALT (adjusted OR 1.062, 95% CI 1.007-1.12, p= 0.015) and low albumin (adjusted OR 0.052, 95% CI 0.005-0.56, p= 0.028). With 20 IU increase in ALT the odd's ratio increased by 3.3(95% CI: 1.15 to 9.58), and 1 g decrease in albumin increases the odd's ratio by 0.95 (95% CI: 0.44-0.995). Conclusion: High ALT and low albumin are independently associated with poor outcome on medical therapy in WD presenting as CLD. Child

Nguyen M1, Nguyen P1, Ngo D1, Nguyen P2, Nguyen T1 Hum Gen Dept, Vietnam Hosp of Pedia, Hanoi, Viet Nam; 2Hep Dept, Vietnam Hosp of Pedia, Hanoi, Viet Nam 1

Background: Wilson disease (WD), an autosomal recessive disorder due to mutations of the ATP7B gene, is characterized by copper accumulation and toxicity in the liver and subsequently in other organs, mainly the brain and cornea. Objective: This study aimed to identify the spectrum of ATP7B gene mutations in Vietnamese Wilson patients and to report a novel common mutation in Vietnam. Material and method: Twelve unrelated WD patients were studied. Genomic DNA was extracted from peripheral blood samples. The R778L mutation, known as the most common mutation in Asia. was first screened by PCRRFLP, then all 21 exons of the ATP7B gene were analyzed by direct sequencing. Result: We identified 8 different mutations accounting for 95.8%, including one novel mutation, T850I. Mutation p.S105* was the most prevalent (54.2%). Others mutations were listed as following: R778L, L1371P, V176Sfs*28 (8.3%); P992L (4.2%), N1270S, M769Hfs*27, T850I (4.2%). Conclusion: This is the first report of WD in Vietnam. Mutation spectrum was different from other ethnics. S105* might be the most common mutation in Vietnam and we recommend that S105* should be screened first on those Vietnamese patients who have a higher risk for WD before sequencing the entire gene. P-555 Clinical course, biochemical profile and novel nonsense mutation in SLC46A1 gene in a 2 months old child with hereditary folate malabsorption Sorribes-Estorch J1, García-Jiménez MC1, Beltrán-García S1, BaqueroMontoya C2, González-Irazábal Y1, Castro M3, Navarrete R3, RodríguezPombo P3, Ugarte M3, Merinero B3 Univ Hosp Miguel Servet, IACS, Zaragoza, Spain; 2Pablo Tobón Uribe Hosp, Medellín, Colombia; 3CEDEM, Science Faculty, CIBERER, UAM, Madrid, Spain 1

Background: Hereditary folate malabsorption (HFM) is caused by mutations in the proton-coupled folate transporter (PCFT-SLC46A1). Among clinical manifestations, it can be found megaloblastic anemia, immunodeficiency and decreased serum and cerebrospinal fluid (CSF) levels of folate. Objectives: We report on two cases, homozygous for a novel SLC46A1 mutation and their clinical outcome. Case report: The patient is the second child of consanguineous parents, who was evaluated due to pancytopenia and sepsis in the context of a bronchiolitis. He has a family history of a deceased cousin due to sepsis and pancytopenia. The metabolic work-up showed hyperhomocysteinemia, B12 and folate levels in the lower normal range, hypomethioninemia and increased excretion of 5-hydantoin-propionic acid. Treatment with hydroxocobalamin and folic acid improved B12 and homocysteine levels as well as hematological parameters, but not folate levels. The undetectable levels of 5-methyltetrahydrofolate in CSF pointed to a defect of folate

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transporter. Molecular analysis of SLC46A1 identified a novel homozygous mutation (c.981_982delCT; p.Tyr327Term) in the proband and his cousin. Conclusions: Early recognition of HFM in individuals with pancytopenia, sepsis and persistent low serum levels of folate, despite of oral supplementation is crucial to avoid a fatal outcome. Parenteral folinic acid treatment may allow a good clinical course.

commenced day 9. Child 2 had clearly elevated MMA and tHcy day 24, treatment commenced day 37. Urine MMA was detectable by day 6 (40-60μmol/mmolcr) in both. The diagnosis could not be made on ENBS with the current cut-off. In children at risk, holotranscobalamin seemed most distinguishing for early postnatal diagnosis.

P-556

Cellular energy deficit may increase insulin sensitivity, as revealed by biotin deficiency

Novel mutations in patients with the cblC inborn error of cobalamin metabolism Brebner A1, Watkins D1, Rosenblatt DS1 1

Dept of Human Genetics McGill University, Montreal, Canada

The cblC complementation group, caused by mutations in the MMACHC gene, is the most common inborn error of vitamin B12 (cobalamin) metabolism. Mutation analysis of 38 cblC patients identified mutations in 36, including four novel mutations: two missense mutations, one nonsense mutation, a whole exon deletion and one presumed regulatory mutation. The missense mutation c.158T>C (p.L53P) was seen in four late-onset patients. In three cases it was in combination with known mutations. In the fourth, no second mutation was identified within the coding sequence, in the 5' and 3' UTRs, or in the MMACHC promotor. However, sequencing of the cDNA identified c.158T>C in apparent hemozygosity, suggesting an as yet unidentified mutation in a non-coding region that eliminated expression of the second allele. The c.566G>A (p.R189H) was identified in one late-onset patient. Both missense mutations affect conserved residues and were predicted to be probably damaging by Polyphen. The nonsense mutation c.292C>T (p.Q98X) was seen in one early onset patient. A deletion of exon 2 was discovered when cDNA amplification gave two products, one normal size and one 200bp shorter that was lacking exon 2. This brings the total number of mutations seen in cblC patients to 80.

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Salvador-Adriano A1, Vargas-Chávez S1, Hernández-Vázquez A1, Ortega-Cuellar D 2 , Carvajal-Aguilera K 2 , Tovar-Palacios A 3 , Velázquez-Arellano A1 1 Instituto Investigaciones Biomedic-UNAM., Mexico, Mexico; 2Instituto Nacional de Pediatría, Mexico; 3INNSZ, Mexico

Background: Glycemia decreases early in biotin deficiency (BtDEF), weeks before insulin changes. We therefore decided to study insulin sensitivity and its signaling pathway. Materials/Methods: Intraperitoneal glucose tolerance and insulin sensitivity and secretion tests and hyperinsulinemic-euglycemic clamps were performed in rats. ATP: AMP and AMPK phosphorylation, western blots of IR, IRS, AKT, S6K1 were determined in liver and muscular L6 cells, and flow cytometry of GLUT4. Results/Discussion: Increased glucose tolerance and insulin sensitivity, decreased insulin secretion and elevated glucose required for euglycemia in clamps fully demonstrate augmented insulin sensitivity in BtDEF. We propose this is caused by ATP deficit (due to reduced pyruvate carboxylase anaplerosis), AMPK activation and S6K1 reduction, increasing the insulin signaling pathway (IR, IRS, AKT) and cellular glucose uptake by GLUT4 translocation to the membrane. (Supported by research grants from PAPIIT-DGAPA-UNAM IN206011 and CONACYT 166857). P-559

P-557 Transcobalamin deficiency: diagnosis and expanded newborn screening de Luen C1, Knoll D1, Bettany B1, de Hora M1, Rosenblatt DS2, Rupar T3, Wilson C4, Glamuzina E4

Dramatic improvement of encephalopathy with thiamine is consistent with the diagnosis of SLC19A3 defects Pérez-Dueñas B1, Ortigoza JD1, Rebollo M2, Serrano M1, Muchart J2, Molero M3, Casado M3, Fons C1, Artuch R3 1

1

2

Biochemical Genetics, LabPlus, Auckland, New Zealand; McGill University, Montreal, Canada; 3University of Western Ontario, London ON, Canada; 4Starship Children's Hospital, Auckland, New Zealand Transcobalamin (TC) deficiency is a rare disorder of cobalamin absorption, transport and cellular uptake. Presentation is in early infancy with haematological and neurological abnormalities. Early diagnosis and treatment improves outcome. Expanded Newborn screening (ENBS) commenced in NZ in 2006. Notification cut-off for the marker of cobalamin related disorders propionylcarnitine is thought to be too high to diagnose TC deficiency (>10.0μmol/L at 48 hours). We present the diagnostic and ENBS data of four children from the same kindred affected with TC deficiency. The proband presented within one month with recurrent infections and pancytopaenia. The diagnosis was suspected just prior to his death at 7 months due to elevated urine MMA (69μmol/mmolcr) and plasma homocysteine (tHcy) (35.6μmol/L). TC deficiency was later confirmed via somatic cell studies and TCN2 sequencing (homozygous c.497_498delTC). Three subsequent pregnancies without prenatal diagnosis resulted in two affected children. Child 4 had undetectable holotranscobalamin at day 1, treatment

Child Neurol Dep, Sant Joan de Déu Hosp, Barcelona, Spain; 2Radiology Dep, Sant Joan de Déu Hosp, Barcelona, Spain; 3Clin Biochem Dep, Sant Joan de Déu Hosp, Barcelona, Spain Background: SLC19A3 deficiency causes acute/recurrent encephalopathy with symmetric lesions on MRI and lactic acid accumulation, being initially indistinguishable from mitochondrial encephalopathy, especially Leigh syndrome (LS). We aimed to compare clinicalradiological features between patients with SLC19A3 defects and LS, and to establish clinical clues for early diagnosis. Methods: Clinical and neuroimaging features, and biochemical parameters of mitochondrial dysfunction were compared between three children with SLC19A3 defects and eight children with LS (ATPase 6 (N=4), PDH-E1α (N=3), unknown genetic defect (N=1)). Results: Both groups of patients showed recurrent episodes of encephalopathy following febrile illness during infancy and early childhood, cranial nerve palsy, hypotonia, dystonia and ataxia. Both groups presented lactic acidosis and increased alanine concentrations, but excretion of alphaketoglutarate was only detected in a newborn with SLC19A3 deficiency. As compared to LS, SLC19A3 patients had lesions in the striatum (3/3), dorsal-medial thalamic nuclei (3/3) and cerebral cortex (2/3). Thiamine dramatically reversed the phenotype only in SLC19A3 children.

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Conclusion: In children with acute encephalopathy, an excellent response to thiamine is highly suggestive of SLC19A3 deficiency. High excretion of alpha- ketoglutarate and specific brain lesions affecting the striatum, dorsa-medial thalami and cerebral cortex are also helpful in the differential diagnosis with mitochondrial encephalopathies. 15. Lysosomal disorders P-560 Clinical course and safety in 13 Fabry Disease (FD) patients who switched from agalsidase-β to agalsidase-α Tsuboi K1, Yamamoto H1, Goto H1 1

Nagoya Central Hospital, Nagoya, Japan

Background: Therapy with agalsidase-α or agalsidase-β has revolutionized FD management. However, information is lacking regarding safety of switching from one enzyme to the other. We evaluated clinical course and safety in patients switching from agalsidase-β to agalsidase-α because of agalsidase-β supply problems. Methods: We studied 13 FD patients treated with agalsidase-β for ≥1year (pre-November 2009). 11 received the approved dosage (1.0mg/kg every other week), two reduced dosage, of agalsidase-β. Clinical and safety data following switch to agalsidase-α (0.2mg/kg every other week) were compared with minimum 1-year pre-switch data. Results: Plasma globotriaosylceramide, renal function, cardiac mass, pain and quality of life measurements revealed no clinical FD deterioration among "switch" patients. Echocardiography showed significant decrease in left ventricular mass after switching to agalsidase-α and a trend towards decreased brain natriuretic peptide levels in six patients. No adverse hepatic changes were observed in 11 "switch" patients previously on full-dose agalsidase-β. Following switch to algalsidase-α, one patient experienced improvement in allergic symptoms whilst another, experiencing worsening cardiac failure and renal failure symptoms on reduced dosage agalsidase-β, showed clinical improvement. Conclusions: These data support the safety of switching from agalsidase-β to agalsidase-α, whilst disease status measures indicate both products can be considered clinically equivalent when administered at approved dosage. P-561 Mucopolysaccharidosis IVa (Morquio A) molecular analysis: a review of the advantages and limitations of molecular testing of galns in the diagnosis of Morquio A Tylee K.L.1, Morrone A.2, Al Sayed M.3, Brusius-Facchin A.4, Church H.J.1, Coll M.J.5, Fietz M.6, Gort L.5, Kubaski F.4, Hegde M.7, Jin D.K.8, Lacerda L.9, Leistner-Segal S.4, Pajares S.5, Pollard L.10, Ribeiro I.9, Tsai F.J.11, Wiles J.12, Miller N.13 1 WillinkBiochemGen, StMarys Hosp OxfordRd, Manchester, United Kingdom; 2Univ of Florence & Meyer Child Hosp, Florence, Italy; 3 King Faisal Spec Hosp & Research Cen, Riyadh, Saudi Arabia; 4 Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 5Hospital Clínic, CIBERER, IDIBAPS, Barcelona, Spain; 6 SA Pathology, Women's & Children's Hosp, North Adelaide, Australia; 7Emory Gen Lab, Emory Univ School Med, Atlanta, United States; 8 Samsung Med Cen,Sunkyunkwan, Uni Sch Med, Seoul, Korea, Republic of; 9Centro Gen Méd Jacinto Magalhães do INSA, Porto, Portugal; 10Biochem Gen Lab, Greenwood Gen Center, Greenwood, United States; 11China Medical University Hospital, Taiwan, Taiwan; 12Facet Communications Inc., Toronto, Canada; 13BioMarin Pharmaceutical Inc., Novato, United States

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Mucopolysaccharidosis IVA (MPS IVA, Morquio A) is an autosomal recessive lysosomal storage disease. Mutations in the gene GALNS lead to deficient N-acetylgalactosamine-6-sulfatase (GALNS) activity. Laboratory diagnosis is commonly based on urinary excretion of keratan sulfate, decreased GALNS enzyme activity in vitro, which is the gold standard for diagnosis, and molecular analysis of the GALNS gene. Accurate and efficient diagnosis of Morquio A can be challenging. We focus our review specifically on the use of molecular testing/mutational analysis, as performed by authors' institutions, to elucidate the challenges of molecular testing for GALNS and we recommend potential solutions. Situations in which mutations were or can be missed are explored. Recommendations for reducing the rate of missed mutations include: parental testing to confirm biallelic inheritance, analysis to identify cases of partial/whole GALNS gene deletions or uniparental disomy, and use of advanced methods other than DNA sequencing to find otherwise undetectable mutations. To support accurate diagnoses, we recommend providing clear, interpretable molecular testing reports and suggest caution when using mutation prediction programs. A global, free-access, locus-specific database of GALNS mutations is also proposed to facilitate improved dissemination of information and to help reduce the number of mutations with unknown significance identified by GALNS sequencing. Conflict of Interest declared. P-562 Attenuated forms of Mucopolysaccharidosis type IVa in two Korean siblings Ko JM1, Cho TJ2, Choi IH2 Dept of Pediatrics, SNUCH, Seoul, Korea, Republic of; 2Dept of Orthopedics, SNUCH, Seoul, Korea, Republic of 1

Mucopolysaccharidosis type IVA (Morquio A, MPS IVA) is a rare autosomal recessive lysosomal storage disorder caused by a deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfate-sulfatase (GALNS). Phenotypes in MPS IVA vary from the classical form to the attenuated form. To date, only 10 patients have been reported in Korea and there was no report about attenuated forms of MPS IVA. Here, we present clinical, biochemical, and genetic characteristics of two Korean MPS IVA siblings. An 11-year-old boy presented with genu valgum and mild kyphosis. Similar skeletal findings were also observed in his 8-year-old younger brother. Their heights were 139 cm (-0.1 SD) and 122.5 cm (-0.3 SD), respectively. MPS urine tests were performed, and weak bands in keratan sulfate position were detected in both siblings. GALNS enzyme activities in leukocytes were 3.17 and 2.60 nmol/17hr/mg protein (reference range, 18.6-61.8), respectively. They had a homozygous mutation of the GALNS gene: p.Val488Met. There was no evidence of other organ involvements. Hemiepiphysiodesis of both knees were performed in both siblings. Now, they are 24 and 21 years with their heights of 153.8 cm (-3.47 SD) and 152.1 (-3.65 SD), respectively. They complain both hip and knee joints pain during long walks. P-563 Astrocytes in infantile neuronal ceroid lipofuscinosis Saravanabavan S1, Williams B1, Cooper J1, Milà M1 1

King's College London, London, United Kingdom

Background: INCL is the commonest pediatric neurdegenerative disorder, caused directly by mutation in CLN1 gene. This encodes for lysosomal enzyme palmitoyl protein thioesterase 1(Ppt1) which is a soluble protein that removes thioester-linked fatty acyl groups from

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cysteine residues. INCL is classified as neurodegenerative disease, yet astrocytes are activated prior to the neuronal death. Hence we aimed to observe PPT1-/- and WT astrocytes in vivo and in vitro. Aims: To determine if PPT1 deficient astrocytes behave differently to wild type astrocytes.To identify any difference in lysosomes of WT and PPT1 mice brain. Results: PPT1-/- Astrocytes were stimulated very early in development leading to increased cell death, variable morphology, altered protein expression and secretion. Initially some of these observations were localized to the thalamus and the cortex, indicating areas effected in the disease. Conclusion: PPT1-/- Astrocytes behave rather different to the WT astrocytes in all fundamental cellular behaviors. These alterations may result in secretion of substances which are harmful and halt secreting cytokines which are necessary leading to development of a toxic environment. Hence neuronal death is localized to these changes and observed soon after. P-564 Retrospective analysis of the clinical manifestations and survival of Korean patients with mucopolysaccharidosis type II: emphasis on the cardiovascular complication and mortality cases

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Background: MucopolysaccharidosisII (MPSII, Hunter syndrome) is a rare X-linked lysosomal storage disorder caused by the deficiency of iduronate-2-sulfatase (IDS). This phase I/II clinical study aimed to evaluate the efficacy and safety of recombinant human iduronate-2sulfatase (idursulfase beta, Hunterase®) in the treatment of MPSII. Methods: Thirty-one MPSII patients between 6 and 35 years of age were enrolled in a randomized, single-blinded, active comparator-controlled phase I/II trial for 24 weeks. Patients were randomized to active comparator infusions (N=11), 0.5 mg/kg idursulfase beta infusions (N=10), or 1.0 mg/kg idursulfase beta infusions (N=10). The primary efficacy variable was the level of urinary GAG excretion. The secondary variables were changes in the 6-minute walk test (6MWT), echocardiographic findings, pulmonary function tests, and joint mobility. Results: Urine GAG was significantly reduced in the two idursulfase beta groups compared to the active comparator group (P = 0.043, 0.002, respectively). Changes in 6MWT were significantly greater in the two idursulfase beta groups than the active comparator group (p= 0.003, 0.015, respectively). Idursulfase beta infusions were well tolerated, and elicited no serious adverse drug reactions. Conclusions: Idursulfase beta generates significant reduction of urinary GAG, improvements in endurance measured by 6MWT, and it has an acceptable safety profile. P-566

Sohn YB1, Choi EW2, Kim SJ3, Park SW3, Cho SY3, Jeong SI3, Huh J3, Lee HJ3, Paik KH3, Jin DK3

Assessment of bone mineral density by dual energy x-ray absorptiometry in patients with mucopolysaccharidoses

1

Dept Med Genet, Ajou Univ Hosp, Suwon, Korea, Republic of; Samsung Biomedical Research Institute, Seoul, Korea, Republic of; 3 Dept Pediatr, Samsung Medical Center, Seoul, Korea, Republic of 2

Lin H1, Lin S1, Chuang C1, Chen M1, Niu D2 Mackay Memorial Hospital, Taipei, Taiwan; 2Taipei Veterans General Hospital, Taipei, Taiwan 1

Background: Hunter syndrome (mucopolysaccharidosis II, MPS II) is a rare, X-linked disorder of glycosaminoglycan (GAG) catabolism caused by a deficiency of the lysosomal enzyme, iduronate-2sulfatase (I2S). Methods: In this study, the medical records of 75 Korean patients with Hunter syndrome (74 males, 1 female) were retrospectively reviewed to investigate the frequency of organ involvement and survival at a single center. Results: The three most common symptoms of organ involvement were hepatosplenomegaly (99%), facial dysmorphism (97%), and frequent otitis media (91%). Cardiovascular involvement was also common including valvular abnormalities (89%), left ventricular hypertrophy (68%), and hypertension (30%). The 19 patients who died had a median age of 16.8 years at the time of death. Four of them died within 1 year of the start of enzyme replacement therapy; autopsy showed myocardial infarction with severe coronary artery disease in one patient. Two other patients died due to pneumonia and sleep apnea. In one case, the cause of death was not investigated. Conclusions: The high incidence of hypertension, and the presence of valvular heart disease indicates that close cardiac monitoring is mandatory in all patients with Hunter syndrome, especially relatively older patients even if they are being treated with enzyme replacement therapy.

Background: Patients with mucopolysaccharidoses (MPS) have a skeletal dysplasia that is associated with poor bone growth and mineralization. Methods: We performed dual energy x-ray absorptiometry (DXA) to characterize lumbar spine bone mineral density (BMD) in 30 MPS patients. Results: The median age was 10.8 years (range 5.0 years to 23.7 years). MPS types included I (n=2), II (n=12), IIIB (n=2), IVA (n=9), and VI (n=5). For the 26 patients 19 years of age, standard deviation scores (Zscores) for height, weight, body mass index (BMI), and BMD were 4.53 ± 2.66, -1.15 ± 1.55, 0.74 ± 1.23, and -3.03 ± 1.62, respectively. There were 21 (81%), 8 (31%), 0 and 18 patients (69%) below -2 Zscore for these four parameters, respectively. However, after correction for height-for-age Z-score (HAZ), HAZ adjusted BMD Z-score was 0.7 ± 1.24. Eight patients (31%) met our center's criteria for osteopenia, and 4 patients (15%) had osteoporosis. Of 8 patients with MPS I, II or VI who underwent follow-up DXA after receiving ERT for 1.0 to 7.4 years, all showed an increase in absolute BMD values. Conclusions: These findings and the follow-up data can be used to develop quality of care strategies for patients with MPS. P-567

P-565

Biochemical and genetic cascade screening of family members of patients with Fabry disease found by selective screening in a population of stroke patients

Phase I/II clinical trial of enzyme replacement therapy with idursulfase beta in patients with mucopolysaccharidosis II (Hunter Syndrome)

Eyskens FJM1, Dedeyn PP2 1

Sohn YB1, Cho SY2, Park SW2, Kim SJ2, Ko AR3, Kwon EK2, Han SJ2, Jin DK2

Div Metab Dis, Dept of Paediatrics,UZA, Antwerp, Belgium; 2Borne Bunge, Univ Antwerp, Antwerp, Belgium

1 Dept Med Genet, Ajou Univ Hosp, Suwon, Korea, Republic of; 2Dept Pediatr, Samsung Medical Center, Seoul, Korea, Republic of; 3Samsung Biomedical Research Institute, Seoul, Korea, Republic of

Introduction: The results of the Belgian Fabry Study I (BeFaS I) suggested that Fabry disease may play a role in up to 1% of young patients with stroke, unexplained white matter lesions or vertebrobasilar dolichoectasia.

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Methods: A genetic family screening was performed in 4 index patients from BeFaS I. All subjects with an identified mutation by genetic cascade screening (N=18 carriers) were further investigated biochemically and clinically. Results: Genetic family screening revealed 18 additional carriers. Normal αGal A enzyme activity in plasma was detected in 6 male subjects, even in one with the known late-onset p.A143T mutation. Two patients had low α-Gal A enzyme activity in plasma. Plasma Gb3 and lyso-Gb3 levels were normal in most patients. Elevated Gb3 in urine was detected in 2 patients. Cardiac symptoms of Fabry disease were found in 6 out of 10 p.A143T carriers. Perceptive hearing loss was detected in 3 carriers of the p.A143T mutation. Conclusions: Only atypical variants of Fabry disease were detected in our cerebrovascular disease population. These variants can be missed by α-Gal A enzyme activity screening. Analysis of Gb3/lyso-Gb3 offered no added value in our population. The p.A143T mutation may be associated with a cardiovascular variant of the disease. P-568 The useful preliminary diagnosis of Niemann-Pick disease type C by filipin test in blood smear Takamura A1, Sakai N2, Shinpoo M2, Yamamoto M3, Narita A4, Ohno K4, Ohashi T5, Ida H6, Eto Y1

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is a rare inherited disease; therefore the incidence has not been obvious in Japan. The clinical trial with enzyme replacement therapy is in progress. Early diagnosis is important to get its therapeutic value sufficiently. In this study, we tried to elucidate the Japanese distribution of GALNS activities in dried blood spots (DBS) of normal controls and two patients, and to detect the mutations of GALNS gene in patients. Materials and Methods: GALNS activities in DBS of two MPS IVA patients and forty normal controls were determined using the fluorescent substrate, 4-MU-beta-Gal-6S. We also determined the proper conditions of GALNS assay. Direct sequencing of GALNS gene was performed to confirm mutations of Japanese patients. Results and Discussions:Normal GALNS activities in Japan were indicated normal distribution, whereas Japanese MPS IVA patients' were only about 20% of normal controls. And we also found novel GALNS mutations in Japanese patients. Now, we can apply our method for high risk screening of MPS IVA patients in nationwide. P-570 Cardiac manifestations and enzyme replacement therapy of Fabry disease Goto H1, Tsuboi K1, Yamamoto H1

1

Inst Neurol Dis, Adv Clin Res Cent, Fukushima and Kanagawa, Japan; 2 Dept Pediatr, Osaka Univ Sch Med, Osaka, Japan; 3Dept Neurol, Dokkyo Univ Sch Med, Tochigi, Japan; 4Div Child Neurol, Fac Med, Tottori Univ, Tottori, Japan; 5Dept Gene Ther, Inst Med Sci, Jikei Univ, Tokyo, Japan; 6Dept Pediatr, Jikei Univ Sch Med, Tokyo, Japan Background: Niemann-Pick disease type C (NPC) is an autosomal recessive lysosomal lipid storage disorder characterized with accumulation of cholesterol in endosomes and lysosomes. The diagnosis of NPC is difficult due to its heterogeneous group of diseases. Biochemical diagnosis of NPC is conducted by filipin staining with cultured skin fibroblasts and confirmed by the analysis of genetic mutations of NPC1 or NPC2 gene. Here, we report an easier biochemical diagnostic method with blood smears by filipin staining. Materials and Methods: All of five NPC patients have been confirmed NPC with both biochemical and genetic tests. Their blood smears were stained with filipin to examine cholesterol accumulations. Results: There were no morphological differences in blood cells between normal controls and NPC patients, but filipin signals were much higher in four NPC patients and slightly higher in one NPC patient than normal controls. Conclusions: It is important to diagnose in earlier stage of NPC because it shows progressive symptoms. Here, we showed an easier and a simpler screening method by staining cholesterol with filipin in blood smears before confirming diagnosis. P-569 Enzymatic screening in dried blood spots and gene analysis of Mucopolysaccharidosis IVa (MPS IVa) in Japanese

1

Nagoya Central Hospital, Nagoya, Japan

Introduction: Fabry disease is caused by deficiency or lack of the lysosomal enzyme α-galactosidase A activity. We investigated the effects of Enzyme replacement therapy (ERT) for cardiomyopathy in Fabry patients. Methods: Thirty six Fabry patients were enrolled. Patients were assigned 1 of 4 groups. Ia is all men and treated only Agalsidase alfa. Ib is all men and treated Agalsidase alfa after Agalsidase beta. IIa is all women and treated only Agalsidase alfa. IIb is all women and treated Agalsidase alfa after Agalsidase beta. All patients were performed Electrocardiography and Echocardiography before, at 12months and 24months after being treated Agalsidase alfa. Results: Left ventricular (LV) mass was reduced in all groups after ERT. Patients were assigned 2 groups according to with or without left ventricular hypertrophy (LVH). LV wall thickness at 24months and LV diastric dimension at 12months were significantly reduced in LVHgroup. LV mass was significantly reduced in 2 groups. Conclusion: In all groups LV mass was reduced at 12months and 24months and ERT may be effective for Fabry disease with or without LVH. We suggest that LV mass of Echocardiography is useful by estimating the effectiveness of Agalsidase alfa. P-571 Abnormal heart rate variability and left ventricular hypertrophy in patients with Fabry disease Goto H1, Tsuboi T1, Yamamoto H1

Fujisaki M1, Takamura A1, Dairaku T1, Ohashi T2, Ida H3, Eto Y1

1

1

Background: Fabry disease is an X-linked lysosomal strage disorder associated with cardiomyopathy and cardiac arrhythmia in middle age. Fabry patients also have symptoms and signs compatible with autonomic dysfunction. These symptoms and signs are considered to be due to impairment of the peripheral nervous system. We investigated cardiovascular autonomic function in Fabry patients. Methods: Seventeen Fabry patients (mean age 40years) and 32 sex- and age- matched controls (mean age 46years) were enrolled and performed 24-hour Holter ECG. Heart Rate Variability (HRV) was analyzed from

Inst Neurol Dis, Adv Clin Res Cent, Fukushima and Kanagawa, Japan; Dept Gene Ther, Inst Med Sci, Jikei Univ, Tokyo, Japan; 3Dept Pediatr, Jikei Univ Sch Med, Tokyo, Japan 2

Background: Mucopolysaccharidosis IVA (MPS IVA) is an autosomal recessive disease caused by a deficiency of the lysosomal Nacetylgalactosamine-6-sulfatase (GALNS). GALNS deficiency results in excess accumulations of keratan sulfate, progressively leading to mainly skeletal dysplasia and joint abnormalities. MPS IVA

Nagoya Central Hospital, Nagoya, Japan

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24-hour Holter ECG data using automatic measurement system. Fabry patients were divided into 2 groups with and without left ventricular hypertrophy (LVH). In Fabry patients echocardiography was performed. Results: There were not significant differences between Fabry patients and controls in HRV. Fabry patients include 7 LVH- and 8 LVH+ patients. In HRV parameter HF was significant high in LVH- (HF ;LVH- 316.3 ±162.4 vs LVH+ 154.8±26.2 ms2, p0.05). There was a strong correlation between HF and LV mass (correlation coefficient -0.71, p0.005). Conclusion: Parasympathetic nervous system was low in LVH+ patients. Fabry patients manifesting the cardiac disease may also be suffered from automatic dysfunction.

We conclude that the measurement of GALNS activity in DBS-H2O, after an incubation of 17 hours at 37:C, has a strong correlation (r=0.999) with the leukocyte activity. Further studies with the enzyme activity in affected individuals should be performed, but the technique adapted for this incubation period appears to be ideal for a screening test.

P-572

Rodríguez-Sureda V1, Domingo R2, Grinberg D3, Vilageliu Ll3, MartínezSalcedo E2, Sánchez O1, Domínguez C1

Histopathological findings of the nasal mucosa in 2 consanguineous patients with Fabry disease

P-574 Evidence of oxidative damage to proteins and increased activity of secretory acid sphingomyelinase in plasma of two patients with GM1-gangliosidosis

1

CIBERER, CIBBIM, Hosp Univ Vall Hebron, Barcelona, Spain; Neuropaediatrics Unit Hosp V Arrixaca, Murcia, Spain; 3Genetics Dept Univ Barcelona, CIBERER, Barcelona, Spain 2

Yamamoto H1, Tsuboi K1, Goto H1 1

Nagoya Central Hospital, Nagoya, Japan

Background/ Objective: Patients with Fabry disease (FD), caused by the deficiency of the lysosomal enzyme alpha-galactosidase A, have various clinical complications, such as progressive kidney disease, cardiomyopathy, cerebrovascular disease as well as head and neck abnormalities. Histopathological findings of characteristic glycosphingolipid accumulation in renal and cardiac tissues are important indicators for early diagnosis and severity of FD. However, biopsy of such vital organs is too invasive and stressful for patients. Patients: The 2 consanguineous patients with FD were a mother (heterozygote) and her son (hemizygote). Nasal mucosal biopsies were performed from them. Result: We confirmed the characteristic accumulation of glycosphingolipids in the form of lamellated structures or myelin-like bodies in the nasal mucosa of FD patients by transmission electron microscopy. The unique cellular depositions were observed in capillary endothelial cells of both patients, although the degree of deposition was more abundant and more striking in the hemizygous male than in the heterozygous female. Conclusion: We present the first report showing characteristic glycosphingolipid accumulation in the nasal mucosa of FD patients by transmission electron microscopy, suggesting that nasal mucosa biopsy may be a useful auxiliary diagnostic test for FD patients.

Background: GM1-gangliosidosis, an autosomal recessive lysosomal lipid storage disease caused by acid beta-galactosidase deficiency due to mutations in the GLB1 gene, results in the accumulation of GM1 and related glycolipids in the brain, bone and visceral organs. This work aimed to study possible new mechanisms involved in the pathogenesis of this disease. Methods: Two children with beta-galactosidase deficiency and typical features of infantile GM1-gangliosidosis (facial dysmorphism, hepatosplenomegaly, dysostosis multiplex and fatal progressive neurodegeneration) were studied. The plasma secretory variant of sphingomyelinase was determined. Protein oxidation was analysed through the quantification of protein carbonyl levels, protein-SH groups and immunoblot analysis of protein carbonylation in plasma. Results: Analysis of the patients' genomic DNA revealed that patient-1 was a compound heterozygote for mutations p.R68W (c.202C>T) and p.R590C (c.1768C>T). Patient-2 was homozygous for the p.R590C mutation. Both patients had enhanced circulating sphingolytic activity compared with controls [31 and 135.5 vs. 12.8(+/-)1.2 nmol/h/mL]. Protein carbonyl content, a marker of cumulative oxidative stress, was increased 8-fold and 4.4-fold, respectively. Conclusions: Protein oxidative modifications can lead to severe biological function failure and cell death. Activation of the sphingomyelinase plasmatic isoform with subsequent increases in the bioactive lipid mediator ceramide may play a critical role in apoptosis induction.

P-573 P-575 Measuring the activity of N-acetylgalactosamine-6-sulfatase in DBS in the diagnosis of MPS IV a: adaptation of incubation time sample

Biochemical parameters of arylsulfatase B in blood samples collected on filter paper

Cé J1, Breier AC1, Mezzalira J1, Daitx VV1, Coelho JC1 Breier AC1, Cé J1, Mezzalira J1, Daitx VV1, Coelho JC1 1

LSD lab -Dept Biochem, UFRGS, Porto Alegre, Brazil 1

LSD lab -Dept Biochem, UFRGS, Porto Alegre, Brazil

Mucopolysaccharidosis IVA is caused by deficiency of Nacetylgalactosamine-6-sulfatase (GALNS). The diagnosis is made by enzymatic assays in leukocytes. The measurement of enzyme activity in dried blood spots on filter paper (DBS) is important because facilitates sending of samples to laboratories, but is currently used only as screening test. The aim of this study was to correlate the activity of GALNS in leukocytes and DBS using the same reagents and incubation time. GALNS activity (Van Diggelen et al., 1990) was measured in leukocytes and DBS 3mm eluted with BSA 0.2% (DBS-BSA) or distilled H2O (DBSH2O), from healthy individuals. DBS was incubated for 17h at 37°C. The mean activity in leukocytes was 115.3±68.23nmol/17h/mg of protein. DBS-BSA was 11.5±5,15nmol/17h/mL and in DBS-H2O was 12.9±8.86nmol/17h/mL. The correlation between the enzyme activity in leukocytes and DBS-BSA or DBS-H2O had an r value of 0.1167 (p>0.05) and r=0.9999 (p<0.0001), respectively.

The blood collected on filter paper is a common alternative for screening in populations at risk in the investigation of inborn errors of metabolism. The Mucopolysaccharidosis VI is a lysosomal storage disorder resulting from deficiency of enzyme arylsulfatase B (ASB). The screening by measurement of enzyme activity on filter paper is well-established technique. The aim of this study was to investigate the biochemical parameters (Km, Vmax and optimum pH) of ASB in blood samples collected on filter paper (DBS) of healthy individuals. The ASB activity was measured according Castillos et al, 2011. To determine the optimum pH tested various pHs in the range of 3.0 to 7.0. The Km and Vmax were determined using substrate concentrations from 2.5 to 15mM. Through this work we can establish an optimum pH of the enzyme in DBS 4.9. The optimum pH was used for the reactions of the curve defining a substrate Km of 8.5mM and a Vmax of 17.29nmol/h/mL.

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These results are important for the characterization of this type of enzyme sample and may be helpful in diagnosis. This possibility has been reported in other studies, as Gaucher disease and Mucopolysaccharidosis I, finding differences for these parameters in normal subjects and patients.

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P-576

Nishida H1, Shimada Y1, Kobayashi H1, Higuchi T1, Eto Y2, Ida H3, Ohashi T1

Proteasome inhibitor improves the function and subcellular localization of mutant lysosomal alpha-glucosidase in fibroblasts from patients with Pompe disease

1 Dept Gen Thera, Jikei Univ Sch Med, Tokyo, Japan; 2Adv Clin Res Cent, Inst Neuro Dis, Kanagawa, Japan; 3Dept Pediat, Jikei Univ Sch Med, Tokyo, Japan

Shimada Y1, Nishida H1, Kobayashi H1, Higuchi T1, Eto Y2, Ida H3, Ohashi T1 1 Dept Gen Thera, Jikei Univ Sch Med, Tokyo, Japan; 2Adv Clin Res Cent, Inst Neuro Dis, Kanagawa, Japan; 3Dept Pediat, Jikei Univ Sch Med, Tokyo, Japan

Pompe disease is an autosomal recessive lysosomal storage disorder caused by a deficiency of acid alpha-glucosidase (GAA). We previously found that the activity of mutant GAA in patient fibroblasts carrying c.546G>T mutation is enhanced by treatment with proteasome inhibitor bortezomib as well as pharmacological chaperon N-butyl-deoxynojirimycin. However, the effect of proteasome inhibitor on other GAA mutations has not been clarified yet. In this study, we investigated whether bortezomib restores the function of mutant GAA in fibroblasts from Pompe disease patients carrying pharmacological chaperon-responsive mutation M519V and -non responsive mutation C647W. Bortezomib promoted the stabilization of patient GAA and processing of them to mature forms in patient fibroblasts. Increased colocalization of GAAwith the lysosomal marker LAMP2 were observed in patient fibroblasts after treatment with bortezomib. Furthermore, bortezomib significantly increased GAA activity in the patient fibroblasts and in transiently transfected HEK293T cells. These results suggest that bortezomib may be effective for a broad range of GAA mutations in Pompe disease patients. Conflict of Interest declared. P-577 Cataplexy in three patients with early-infantile Niemann-Pick disease type C Cak T1, Haliloglu G2, Yuce A3, Topcu M2 Psychiatry, Hacettepe Univ Child Hosp, Ankara, Turkey; 2Ped Neurol, Hacettepe Univ Child Hosp, Ankara, Turkey; 3Gastroenterol, Hacettepe Univ Child Hosp, Ankara, Turkey 1

Cataplexy is a brief episode of bilateral loss of muscle tone with intact consciousness, triggered by a variety of strong emotions. Niemann-Pick Type C is a neurodegenerative lysosomal storage disease, and cataplexy is a relatively specific and common neurologic sign seen in almost 50% of all patients. Three girls with NPC1, between the ages 6-9, mild to moderate mental retardation, on miglustat treatment will be presented. All presented with difficulty in walking between the ages of 14 months-2 years, developmental delay, ataxia, oculomotor apraxia and splenomegaly. They developed cataplexy triggered by laughter or giggling in the last 12 months mostly in antigravity muscles. None of the patients described excessive daytime sleepiness or hypnagogic hallucinations. Awake and sleep electroencephalography (EEG) were normal. Two girls benefitted from imipiramine, one free of cataplexy for the last 6 months and other for the last 2 months. One patient did not respond to imipiramine, fluoxetine or methylphenidate, and currently is on sodium valproate treatment. Early diagnosis of NPC disease in the era of substrate reduction therapy is crucial. Cataplexy is rather a very difficult symptom to treat in NPC population, and despite improvement in neurologic stabilization, the course of the disease may be complicated.

Proteasome inhibitor improves the function of mutant beta-glucuronidase in fibroblasts from Mucopolysaccharidosis type VII patient

Mucopolysaccharidosis type VII (MPS VII) is an autosomal recessive lysosomal storage disease caused by a deficiency of beta-glucuronidase. Pharmacological chaperone therapy using small molecules is an emerging strategy for a potential treatment of lysosomal storage diseases, but specific molecule for mutant beta-glucuronidase has not been identified yet. On the other hands, we recently found that the function of mutant lysosomal beta-glucosidase in fibroblasts from patient with Pompe disease is improved by treatment with proteasome inhibitor bortezomib as well as pharmacological chaperon Nbutyl-deoxynojirimycin. In this study, we investigated whether bortezomib rescues the function of mutant beta-glucuronidase in fibroblasts from patient with MPS VII. Skin fibroblasts from MPS VII patient carrying E540K and D152G mutations were cultured for 24h with or without bortezomib, and analyzed by assay of beta-glucuronidase activity. Treatment with bortezomib significantly increased enzyme activity in patient fibroblasts, and enhanced the activity in a time dependent manner (2.8-fold). Furthermore, the elevated enzyme activity was sustained for more than two days after removal of bortezomib. These results suggest that bortezomib may be effective treatment not only for Pompe disease, but also for MPS VII. Conflict of Interest declared. P-579 Longitudinal outcomes from the international disease registry for Niemann-Pick disease type C (NP-C) Pineda M1, Mengel E2, Wijburg FA3, Muller A4, Schwierin B4, Drevon H5, Vanier MT6, Patterson MC7 Fundació Hospital Sant Joan de Déu, Barcelona, Spain; 2Villa Metabolica, University of Mainz, Mainz, Germany; 3University of Amsterdam, Amsterdam, Netherlands; 4Actelion Pharmaceuticals Ltd, Allschwil, Switzerland; 5Numerus Ltd, Wokingham, United Kingdom; 6INSERM Unit 820, Lyon, France; 7Mayo Clinic, Rochester MN, United States 1

Background: Data from a prospective observational study of patients included in the international NPC Registry who received continuous miglustat therapy are presented. Methods: All patients diagnosed with NP-C were eligible for inclusion. Demographics, disease characteristics, disability status and treatment data were collected between September 2009 and August 2012. Disability scale scores rating ambulation, manipulation, language and swallowing from 0 (normal) to 1 (worst) were assessed. Patients were categorised as 'improved/stable' if ≥3/4 domain scores were lower/unchanged, or as 'worsened' if <3 domain scores were lower/unchanged. Results: 80/190 enrolled patients (mean [SD] age at enrolment 17.8 [11.5] years) received continuous miglustat therapy; mean (SD) exposure between enrolment and last follow-up, 1.19 (0.69) years. Ten of 74 (14%) patients with available data had early-infantile (<2 years), 24 (32%) had late-infantile (2 to <6 years), 24 (32%) had juvenile (6 to <15 years) and 16 (22%) had adolescent/adult (≥15 years) onset of neurological manifestations. Overall mean (95%CI) composite disability scores were 0.39 (0.34,0.45; N=75) at enrolment and 0.45 (0.39,0.51; N=76) at last follow-up, with 52/72 (72%) patients 'improved/stable' and 20/72 (28%) 'worsened'. Conclusion: Disability status was improved or stable in the majority of continuous miglustat-treated patients observed for an average of 1.2 years. Conflict of Interest declared.

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P-580

P-582

Hypopituitarism in Hunter Syndrome

Niemann Pick type C disease: a novel NPC mutation and a hot spots in a Greek island

Nour M. A.1, Stephure D. K.1, Wei X. C.2, Khan A.3 1

Section Ped Endocrin, Alberta Child Hosp, Calgary, Canada; 2 Diagnostic Imaging, Alberta Child Hosp, Calgary, Canada; 3Metab Dis Clin, Alberta Child Hosp, Calgary, Canada Background: Growth failure is a ubiquitous feature in Hunter Syndrome (HS) while hypopituitarism has not been reported previously. We report two cases of HS with incidental discoveries of anatomic pituitary anomalies; one found to have hypopituitarism, the other with intact pituitary function. Methods: Retrospective case review. Results: Patient 1, a 10-year-old boy with HS, presented for evaluation of impaired growth following MRI identification of an ectopic posterior pituitary gland. Comprehensive functional endocrine testing revealed growth hormone deficiency, secondary adrenal insufficiency, and tertiary hypothyroidism. He demonstrated significant improvement in growth with hormone replacement treatment (Pre-GH growth velocity 3.7cm/year, -2.0SD; Post-GH velocity 9.3cm/year, +5.6SD). Patient 2, a 13-year-old male with HS, was evaluated for growth failure following incidental finding of a large empty sella with posteriorly displaced pituitary. Functional endocrine testing revealed intact pituitary functioning. GH was not instituted and growth remained unchanged. Conclusions: We present 2 patients with HS and abnormal pituitary imaging. Patient 1 having anterior panhypopituitarism and a significant response to GH; while patient 2 had intact pituitary function. Panhypopituitarism associated with pituitary anomalies has not been previously reported in HS, but may occur in some patients. Further investigation is required to determine the frequency of this association. P-581 Treatment effect of coenzyme Q10 and an antioxidant cocktail in fibroblasts of patients with Sanfilippo disease Matalonga L1, Arias A2, Coll MJ2, Garcia-Villoria J2, Ribes A2, Gort L2 Hospital Clínic, IDIBAPS, Barcelona, Spain; 2Hospital Clínic,IDIBAPS, U737-CIBERER, Barcelona, Spain 1

CoQ10 plays a key role in the exchange of electrons in the lysosomal membrane, which contributes to protons' translocation into the lumen and to the acidification of the intra-lysosomal medium which is essential for the proteolytic function of hydrolases. In addition, the reduced form of CoQ10 protects the lysosomal membrane from ROS and oxidative stress. Our aim was to evaluate whether treatment with CoQ10 or with an antioxidant cocktail were able to ameliorate the biochemical phenotype in fibroblasts of Sanfilippo patients. Fibroblasts from five patients affected of Sanfilippo.A and B diseases were treated with CoQ10 and an antioxidant cocktail. None of the Sanfilippo.A fibroblasts increased their deficient enzymatic activity, but the two Sanfilippo.B cell lines showed 1.25 and 1.9 fold increase, respectively. Upon CoQ10 treatment, one Sanfilippo.A and the two Sanfilippo.B fibroblasts showed a significant reduction of glycosaminoglycans accumulation (66%, 16% and 10%, respectively compared to basal levels). After treatment with the antioxidant cocktail, the reduction was of 49%, 16% and 35%, respectively. Fibroblasts responsive to treatment enhanced their exocytosis levels. Our results provide a proof-of-concept that some cellular alterations observed in Sanfilippo disease can be partially restored by CoQ10 or other antioxidant treatments. Supported by Fundación StopSanfilippo, Spain.

Mavridou I1, Cozar M2, Douzgou S1, Xaidara A3, Lianou D4, Dimitriou E1, Vanier MT5, Grinberg D2, Vilageliu L2, Michelakakis H1 1

Dept Enz Cel Function,Inst Child Health, ATHENS, Greece; 2Dept Genet, Univ Barcelona, Barcelona, Spain; 3Dept Ped,Univ Athens, ATHENS, Greece; 4Ped Clin,Ag.Sophia Child Hosp, ATHENS, Greece; 5Lab GilletMérieux,Hop Lyon Est, Lyon, France Niemann-Pick type C (NPC) disease, a neurodegenerative autosomal recessive disease,is a cellular lipid trafficking disorder characterized by the accumulation in the lysosomal /late endosomal system of a variety of lipids, especially unesterified cholesterol. So far two genes, NPC1 or NPC2, have been linked to the disorder. It is a panethnic disease for which two isolates have been described. We present a novel NPC1 mutation (p.A1132P; c.3394G>C) identified in homozygosity in two patients originating from the same small town of an Aegean Sea island and the results of the broad screening of their extended families. Overall 153 individuals have so far been investigated and a total of 64 carriers were identified. Moreover a common descent of the individuals tested was revealed and all carriers could be traced back to a common surname, apparently originating from a common ancestor couple six generations back. The mutation was found associated with an uncommon haplotype in the island that is also present in other populations. In conclusion the diagnosis of these two patients has led to the identification of a hot spot of NPC disease on a Greek island and has allowed genetic counselling and carrier detection in the extended family tree. Conflict of Interest declared. P-583 Proteasomal inhibitors reduce cholesterol accumulation in NiemannPick type C cells harbouring missense mutations Macías-Vidal J1, Girós M1, Serratosa J2, Bachs O3, Coll MJ1 1 Sec Err Cong Metab (IBC), Hosp Clínic, Barcelona, Spain; 2Dep Isq Cer i Neur, IIBB-CSIC, Barcelona, Spain; 3Dep Bio Cel, Fac Med, Univ Barcelona, Barcelona, Spain

Background: In Niemann-Pick disease type C (NPC) cell lines harbouring determined missense mutations, NPC1 protein shows a significant reduction of its half-life due to degradation via ubiquitin-proteasome. Objectives: The aim of this study was to demonstrate if the treatment with proteasomal inhibitors is able to reverse the loss of NPC1 protein caused by protein misfolding, and in cases like that, determine the functionality of the NPC1 mutants. Patients: Samples were obtained from 18 unrelated patients previously NPC diagnosed. Methods: 1) Treatment with cycloheximide (protein synthesis inhibitor) and MG132 or ALLN (proteasomal inhibitors) of cultured patients' fibroblasts; 2) Quantification of NPC1 gene expression by real-time PCR; 3) Quantification of NPC1 protein expression by western blot analysis; 4) Cellular colocalization studies by immunofluorescence microscopy; 5) Cholesterol determination by filipin staining, capillary gas-liquid chromatography and a fluorimetric method. Results: The stability of different NPC1 mutant proteins has been examined and there is evidence that the proteasomal inhibitors partially reverse NPC1 loss in some of these mutants. Restored mutant proteins are able to significantly reduce free cholesterol levels, thus recovering their lysosomal localization. Conclusion: These findings can provide support for future trials to treat NPC disease caused by specific mutations.

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P-584

P-586

Glycosaminoglycans (GAGS) differentiation in patients with various types of mucopolysaccharidoses (MPS)

Efficacy of enzyme replacement therapy versus hematopoietic stem cell transplantation on brain involvement in MPS II

Stepien KM1, Petty J1, Thornley M1, Cooper A1, Church HJ1, Tylee KL1

Tanaka A1, Okuyama T2, Suzuki Y3, Sakai N4, Hamazaki T1, Sawada T1, Kosuga M2, Yabe H5, Ishige M6, Mugishima H6, Kato S5

1

1

Willink Biochemical Genetics Unit, CMFT, Manchester, United Kingdom Background: Urine GAGs are measured in the diagnosis of MPS. Our laboratory uses 1,9-dimethylmethylene blue (DMB) quantitative method. Results: for DMB for sulphated GAGs are expressed in terms of urinary creatinine concentration. The range of GAGs excreted by normal children is well established for different age groups. Our aim was to determine any potential differences in GAG levels for different types of MPS patients at diagnosis. Methods: GAG levels were obtained from results achieved over a 30-year period. Numbers of analysed results were different for different MPS types (I, II, IIIA, IIIB, IIIC, IV, VI, VII, multiple sulphatases). Results: GAG results were divided into different types of MPS. All data was separated for individual subgroups respective of age. Types IV, I and II were the most prevalent MPS types in our database, whereas types IIIC and IV had the highest average age. In view of the rarity of multiple sulphatases, MPS IIIC and VII, ranges were calculated for small numbers which may not be valid statistically. Conclusions: GAGs ranges for various MPS types and age groups were established for our population with the aim of detecting potential trends in GAGs between disorders and ultimately to facilitate in interpretation of results. P-585 Novel biomarkers for Fabry disease detected by a mass spectrometry metabolomic approach Auray-Blais C1, Lavoie P1, Boutin M1

Osaka City Univ Graduate School Med, Osaka, Japan; 2National Center Child Health Develop, Tokyo, Japan; 3Gifu University School of Medicine, Gifu, Japan; 4Osaka University Graduate School of Med, Osaka, Japan; 5Tokai University School of Medicine, Isehara, Japan; 6 Nippon University School of Medicine, Tokio, Japan Background: Intravenous enzyme replacement therapy (ERT) does not show efficacies on brain in MPS II patients. Hematopoietic stem cell transplantation has been indicated in patients with MPS II since 1980s before the approval of ERT in Japan. Objective: To evaluate the efficacy of two therapies, ERT and HSCT, on brain function. Patients and Methods: A nationwide survey was conducted in Japan to collect the records of IQ/DQ of patients with MPS II severe form who received HSCT, and compared with those under ERT. Patients were divided into two clinical phenotypes by the developmental delay; Delay was prominent after 2 years of age (Type C) or before 2 years of age (Type D). Numbers of patients in each group were 10 (HSCT, Type C), 9 (HSCT, Type D), 7 (ERT, Type C), and 10 (ERT, Type D). The value of correlation coefficient between chronological age and developmental age was calculated in each group. Results: Positive correlation was shown both in Type C and D with HSCT (r=0.835 and 0.563), while negative correlation both in Type C and D with ERT (r=-0.536 and -0.840). Conclusion: HSCT is beneficial treatment for the patients with MPS II, especially for the patients with brain involvement. P-587 Genotypes and phenotypes in japanese patients with Mucopolysaccharidosis type VII

1

Dept Pediatrics Université de Sherbrooke, Sherbrooke, QC, Canada

Background: Fabry disease is an X-linked lysosomal storage disorder characterized by the accumulation of glycosphingolipids, mainly globotriaosylceramide (Gb3) and globotriaosylphingosine (lyso-Gb3) in biological fluids, and different organs. The objectives were to discover novel urinary/plasma biomarkers related to lyso-Gb3 using a metabolomic approach, elucidate the chemical structure of the targeted biomarkers and quantify them. Materials and Methods: Urine and blood samples from 63 untreated Fabry patients and 59 healthy controls were collected. The analyses were done using a quadrupole-time-of-flight mass spectrometer. Results: Seven novel biomarkers were detected in urine and plasma with the following mass/charge (m/z) ratios: 758, 774, 784, 800, 802, 820, and 836. These biomarkers are analogues of lyso-Gb3 with modifications on the sphingosine moieties. A lyso-Gb3-glycine internal standard was synthesized for relative quantification. Some analogues were excreted in larger quantities than lyso-Gb3. No significant level of lyso-Gb3 analogues was found in controls. Urinary excretion levels of the analogues correlate with patient gender and treatment in men. One male Fabry patient with a cardiac variant mutation (N215S) had a normal urinary Gb3/creatinine level, but abnormal levels of urinary/plasma lyso-Gb3 and the analogues. Conclusion: This metabolomic study is the first to demonstrate the presence of lyso-Gb3 analogues in Fabry patients. Conflict of Interest declared.

Yamashita K1, Hamazaki T1, Takeda Y2, Tomiwa K2, Shintaku H1, Tanaka A1 1

Osaka City Univ Graduate School Med, Osaka, Japan; 2Todaiji Medical Educational Center, Nara, Japan Background: Since mucopolysaccharidosis type VII (MPS VII) is very rare in the world, phenotypes and genotypes have not been well known. In Japan, four patients have received the diagnosis as MPS VII so far. Objective: We report two Japanese patients with MPS VII. Case report: [Case 1]: 30 year-old female. She received the diagnosis as MPS VII at age 7. She showed severe skeletal deformities and a slight elevation of urinary glycosaminoglycan (GAG). She was homozygous for A382C in β-glucuronidase gene. Her height is 95cm. She has normal intelligence, mild corneal opacities, and thickening of mitral and aortic valves without regurgitation. She has no organomegaly. [Case 2]: 21 year-old male. He showed hydrops fetalis at birth. He showed mild dysmorphism and mental retardation at age 5. He received the diagnosis at age 21. He was a compound heterozygote with R110X and IVS3, -3t>g. His height is 156cm and IQ is 60. He showed no elevation of urinary GAG or hepatosplenomegaly. He has slight corneal opacities, and thickening of mitral and aortic valves.

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P-588

P-590

Particular myosonographic examination of the proximal and distal muscles in Pompe disease

Fabry disease screening in end-stage renal disease patients due to unidentified causes within the fifth decade of life: the first Thailand study

Karabul N1, Gokce S1, Beck M1, Mengel KE1 1

Villa Metabolica, Dep. of Ped, UMC Mainz, Mainz, Germany

Introduction: Glycogen storage disease type II (Pompe disease, GSDII) is an autosomal recessive metabolic disorder, which causes damages in muscle cells throughout the body due to deficiency of the lysosomal GAA. There is currently no good biomarker for objective assessment of disease, patient status and muscle involvement. Follow up proceeds with measure of vital capacity (supine/horizontal), Hex4 (urine) and grading of strength using MRC scales for example. Methods: We established the myosonography in addition to MR images and studied in detail proximal and distal muscles. Myosonography detected particular damage of isolated muscle fibres indicating nerverelated muscle changes. During the examination we found out that the muscle however still acts functionally and shows also fasciculation. We developed a scoring system describing status of proximal and distal muscles. Patients younger than 35 years have no or less muscle involvement. Case Report: Image presentation of the myosonography of biceps, triceps, pectoralis, trapezius, quadriceps medial, lateral and autochthonous back muscle groups of a childhood GSDII (6 years). Conclusions: Myosonography is an easily accessible and a favourable alternative or additional tool to MRI. Myosonography is considered in follow up investigations in Pompe Disease. Conflict of Interest declared. P-589

Trachoo O 1 , Jittorntam P 1 , Pibalyart S 1 , Kajanachumphol S 1 , Suvachittanont N1, Sae-chew P1, Pitidhammabhorn D1, Patputthipong S2, Chuengsaman P3, Nongnuch A1 1

Fac Med, Ramathibodi Hosp, Mahidol Univ, Bangkok, Thailand; Uttaradit Hosp, Uttaradit, Thailand; 3Banphaeo Hosp-Prommitr Br, Bangkok, Thailand 2

Background: Previous studies in various ethnic groups show 0-2% amongst dialysis patients carrying Fabry disease (FD). Since FD patients usually reach end-stage renal disease (ESRD) within the fifth decade of life, we aimed to explore FD in ESRD patients with the disease onset less than 60 years. Methods: Patients with known causes of ESRD were excluded. Alphagalactosidase A (AGAL) screening was performed from dried-blood spots using fluorometry. Molecular confirmation was done using DNA sequencing of GLA gene. Results: Total 142 patients of both genders were included in the study. Fifteen of them exhibited significant decrease of AGAL activity. None showed any certain mutations. However, five revealed a novel nucleotide variant in 5'UTR at -10 C>T from the initiation codon. Subsequent genetic association study shows this variant being the susceptible allele for a decrease of AGAL activity in ESRD. Conclusion: Definite FD in this population group remains unclear since certain pathogenic mutation cannot be characterized. However, the discovered nucleotide variant is firstly identified in our series and implied as a susceptible marker for AGAL deficiency. Hence, further functional genomic study is required to prove that this variant may have caused FD in Thai population.

Renal sonography in inborn metabolic disorders P-591 Karabul N1, Arash-Kaps L1, Kalkum G1, Gokce S1, Beck M1, Mengel KE1

Analysis of 18 Niemann-Pick type C patients with visceral involvement: a single center experience

1

Villa Metabolica, Dep. of Ped, UMC Mainz, Mainz, Germany

Introduction: Inborn metabolic diseases (IMD) are rare diseases that are characterized among other things by a great variability in their clinical expression. A central institution is required to cover all aspects of these diseases (diagnosis, follow-up, treatment, research). Villa Metabolica offers the diagnosis, multidisciplinary treatment of metabolic and especially of lysosomal storage diseases (LSD). Different cases in IMDs are presented in this article. Method: This presentation is a little insight into a part of LSDs: increased echogenity in the renal parenchyma (Figure 1), nephrocalcinosis (see Figure 2) and parapelvic cysts (see Figure 3) with proteinuria may provide sonographic evidence of the presence of a storage disease. Adrenal calcifications (see Figure 4) can give an indication of the existence of Wolman disease with deficiency of lysosomal acid lipase, which results in the accumulation of cholesterol esters and triglycerides in various organs. We will discuss examples of Fabry disease, the galacto-nephrosialidosis, the Wolman disease and the hypophosphatasia. Increased renal echogenity may be the first indication of nephrosialidosis or hypophosphatasia. Parapelvic cysts in adults with proteinuria may be an indication for the presence of Fabry disease. Summary: When renal abnormalities are seen in ultrasound, IMD should be added to the differential diagnosis. Conflict of Interest declared.

Yuce A1, Gurakan F1, Haliloglu G1, Demir H1, Saltik-Temizel I N1, Aydemir Y1, Hizal G1, Topcu M1, Ozgul K1, Dursun A1, Runz H2 1

Hacettepe University Children's Hospital, Ankara, Turkey; 2Institute of Human Genetics, Heidelberg, Germany Background: Niemann-Pick type C (NPC) disease is a lysosomal disease due to mutations in two genes (NPC1 and NPC 2). Although the typical presentation is characterized by progressive neurological abnormalities, the patients may have visceral involvement. The diagnosis is difficult because of nonspecific visceral findings. Here we report 18 patients with visceral involvement at diagnosis. PATIENTS: The medical records of 24 patients with NPC disease diagnosed between 1993 and 2012 were reviewed. The patients were diagnosed with Filipin staining and /or mutation analysis. Results: Eighteen patients had visceral involvement at the time of diagnosis. 11 had only visceral involvement, 7 had both visceral and neurological features. Patients with only visceral involvement were younger. All patients had splenomegaly, 15 had hepatomegaly. Twelve patients had neonatal cholestasis or history of neonatal cholestasis, 4 had fetal ascites and 2 had pulmonary involvement at diagnosis. While all of the 11 patients with only visceral involvement died, only one of the patients with neurovisceral manifestations died.

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Conclusion: Manifestations of visceral involvement during early ages are nonspecific and prognosis of the patients with only visceral involvement is poor. New diagnostic and therapeutic studies targeting visceral involvement in children without neurologic involvement should result in better prognosis P-592 Non-competitive pharmacological chaperones as a new strategy for treating a Lysosomal Storage Disease Rodriguez-Pascau L1, Delgado A2, Pizcueta P1, Aymami J3, Barril X2, Vilageliu L4, Grinberg D4, Martinell M1 Minoryx Therapeutics, Mataró (Barcelona), Spain; 2Facul Farmacia; UnivBarcelona, Barcelona, Spain; 3Dep Enyen Química; Univ Polic Barcelona, Barcelona, Spain; 4Dep Gen, Fac Biology;Univ Barcelona, Barcelona, Spain

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The platform applies proprietary technology to identify druggable allosteric sites and candidate binders, which are then experimentally validated through a battery of dedicated assays, such as differential scanning fluorimetry (DSF) and enzyme enhancement based assays on cells. Results: By using SEE-Tx Minoryx has identified novel druggable allosteric sites for three different targets related with three neurometabolic diseases currently incurable. The most advanced project is on Lead Optimization stage for a lysosomal storage disease. The other projects are related with disorders of aminoacid metabolism. Conclusion: SEE-Tx is a very fast & cost-effective solution to target noncompetitive binding sites. This approach is particularly powerful for targets related with metabolic diseases, because their active sites are usually poorly druggable

1

Background: Most pharmacological chaperones (PC's) described up to date are substrate analogues which compete with the natural substrate. Consequently, these compounds inhibit the target protein at higher concentrations and have poor drug-like properties particularly for CNS indications. Objective: By using Minoryx's proprietary technological platform (SEE-Tx; Site-directed Enzyme Enhancement Therapy), to develop novel non-competitive PC's for a Lysosomal Storage Disease (LSD). Material and Methods: Minoryx's platform applies proprietary technology to identify druggable allosteric sites and candidate binders, which are then experimentally validated through a battery of dedicated assays, such as differential scanning fluorimetry (DSF) and enzyme enhancement cell-based assays. Results: Novels series of non-competitive PC's have been identified for a LSD. Previously, only competitive chaperones were described. These series showed excellent ligand efficiency, a comprehensive SAR and enzyme enhancement activity both in cells transfected with wild type protein and clinically relevant mutants. Additionally, enzyme inhibition assays confirmed that these series do not inhibit the target enzyme, in contrast to previously described competitive PC's which exhibited doseresponse inhibitory activity. Conclusions: We have identified a novel series of non-competitive pharmacological chaperones for a LSD. The project is currently in lead optimization and we expect to nominate a candidate by the end of 2014. P-593 Site-directed enzyme enhancement therapy (SEE-Tx): a novel platform for identifying non-competitive pharmacological chaperones

P-594 Gene therapy for MPS VI is effective in cats without pre-existing immunity to AAV8 Ferla R.1, O'Malley T.2, Calcedo R.3, O'Donnell P.2, Wang P.2, Cotugno G.1, Claudiani P.1, Wilson J.M.3, Haskins M.2, Auricchio A.1 1 Telethon Inst of Genetics and Medicine, Naples, Italy; 2 Dept Pathobiol, School of Vet, Upenn, Philadelphai, United States; 3Gen Ther Prog, Dept Path Lab Med, Upenn, Philadelphia, United States

Liver gene transfer with adeno-associated viral (AAV) 2/8 vectors is being considered for therapy of systemic diseases like mucopolysaccharidosis VI (MPS VI), a lysosomal storage disease due to deficiency of arylsulfatase B (ARSB). We have previously reported that liver gene transfer with AAV2/8 results in sustained yet variable expression of ARSB. We hypothesized that the variability we observed could be due to pre-existing immunity to wildtype AAV8. To test this, we compared the levels of AAV2/8-mediated transduction in MPS VI cats with and without preexisting immunity to AAV8. In addition, since levels of lysosomal enzymes as low as 5% of normal are expected to be therapeutic, we evaluated the impact of preexisting immunity on MPS VI phenotypic rescue. AAV2/8 administration to MPS VI cats without pre-existing neutralizing antibodies to AAV8 resulted in consistent and dose-dependent expression of ARSB, urinary glycosaminoglycan (GAG) reduction, and femur length amelioration. Conversely, animals with pre-existing immunity to AAV8 showed low levels of ARSB expression and limited phenotypic improvement. Our data support the use of AAV2/8-mediated gene transfer for MPS VI and other systemic diseases, and highlight that pre-existing immunity to AAV8 should be considered in determining subject eligibility for therapy. P-595

Barril X1, Rodriguez-Pascau L2, Delgado A1, Pizcueta P2, Aymami J3, Grinberg D4, Vilageliu L4, Martinell M2

In vitro and in vivo characterization of an engineered arylsulfatase B for therapy of Mucopolysaccharidosis VI

1

Facul Farmacia; UnivBarcelona, Barcelona, Spain; 2 Minoryx Therapeutics, Mataró (Barcelona), Spain; 3Dep Enyen Química; Univ Polic Barcelona, Barcelona, Spain; 4Dep Gen, Fac Biology; Univ Barcelona, Barcelona, Spain Background: Most pharmacological chaperones (PC's) described up to date are substrate analogues. Consequently, these compounds inhibit the target protein at higher concentrations and have poor drug-like properties particularly for CNS indications. Objective: To use Minoryx's proprietary technological platform (SEETx; Site-directed Enzyme Enhancement Therapy), to identify novel non-competitive PC's for a battery of inborn errors of metabolism. Material and Methods: Minoryx has developed a technological platform which allows identifying non-competitive PC's for selected targets.

Ferla R.1, Claudiani P.1, Cotugno G.1, Petrey A.2, Steet R.2, Auricchio A.1 1 Telethon Inst of Genetics and Medicine, Naples, Italy; 2Compl Carbohyd Res Cent, Univ Georgia, Athens, United States

Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disorder caused by deficiency of the enzyme arylsulfatase B (ARSB). The amelioration of skeletal dysplasia remains one of the major challenges for treating MPS VI. We have reported that liver-directed ARSB gene transfer using adeno-associated viral vectors (AAV) results in significant yet incomplete rescue of the feline MPS VI skeletal phenotype.To improve this, we modified the wild type (wt) hARSB by adding a 6-aspartic acid tag (hARSB.6asp), which has been reported to have high affinity for the

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hydroxyapatite in the bone matrix. In vitro hARSB.6asp has similar enzymatic activity and is secreted two times more than wt hARSB. Similarly serum ARSB activity is higher in MPS VI mice receiving 2x1012 gc/kg of AAV vector encoding hARSB.6asp than encoding wt hARSB. However, the in vivo data did not show increased efficacy of hARSB.6asp compared to wt hARSB. We found that hARSB.6asp has less mannose 6phosphate modification than wt hARSB both in vitro and in vivo and it is less up-taken than wt hARSB by human MPS VI fibroblasts. Our data suggest that modifications that result in more efficient secretion of lysosomal enzymes may impact on their post-translational modifications and therapeutic efficacy. P-596 Engage: a phase 3, randomized, double blind, placebo controlled, multi center study to investigate the efficacy and safety of eliglustat in adults with Gaucher disease type 1: 9 month results Ben Turkia H M-F1, Lukina E2, Amato D3, Baris H4, Dasouki M5, Ghosn M 6 , Mehta A 7, Packman S8 , Pastores G9 , Petakov M 10, Assouline S11, Balwani M12, Danda S13, Hadjiev E14, Mistry PK15, Shankar S16, Marulkar S17, Peterschmitt MJ17 1 Hôpital La Rabta, Tunis, Tunisia; 2Hematology Research Center, Moscow, Russian Federation; 3 Mount Sinai Hospital, Toronto, Canada; 4Rabin Medical Center, Petach Tikvah, Israel; 5University of Kansas Hospital, Kansas City, United States; 6Hôtel-Dieu de France University Hospital, Beirut, Lebanon; 7The Royal Free Hospital, London, United Kingdom; 8UCSF School of Medicine, San Francisco, United States; 9New York University School of Medicine, New York City, United States; 10Clinical Center of Serbia, Belgrade, Serbia and Montenegro; 11 Jewish General Hospital, Montreal, Canada, 12 Mount Sinai Hospital, New York City, United States; 13Christian Medical College, Vellore, India; 14University Hospital Alexandrovska, Sofia, Bulgaria; 15Yale University School of Medicine, New Haven, United States; 16Emory University, Atlanta, United States; 17Genzyme, a Sanofi company, Cambridge, United States

Background/objectives: ENGAGE is a randomized, double-blind, placebo-controlled, Phase 3 trial that investigated the efficacy and safety of eliglustat, an investigational novel oral substrate reduction therapy, in untreated adults with Gaucher disease type 1. Patients/methods: Forty patients (mean age: 31.8 years; 20 males, 20 females) with both splenomegaly and thrombocytopenia and/or anemia were stratified by spleen volume and randomized 1:1 to receive eliglustat (50 or 100 mg BID depending on plasma levels) or placebo for 9 months. The primary efficacy endpoint was percent change in spleen volume (multiples of normal). Other efficacy measures included hemoglobin, liver volume, platelets, disease-related biomarkers, and quality of life. Safety evaluations included spontaneously reported adverse-events, laboratory evaluations, and ECGs. Results: In patients receiving eliglustat compared to placebo, mean spleen volume decreased (-28% vs. +2%, P<0.0001), mean hemoglobin increased (0.69 vs. -0.54 g/dL, P<0.0006), liver volume decreased (-5.20% vs. +1.44%, P<0.0072), and platelet level increased (+32.00% vs. -9.06%, P<0.0001). No patients discontinued due to an AE, all of which were classified as mild to moderate. Only arthralgia and nasopharyngitis occurred in >10% of eliglustat-treated patients. Conclusion: The ENGAGE study met its primary and secondary efficacy endpoints and eliglustat was generally safe and welltolerated. Conflict of Interest declared.

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P-597 Effects of oral eliglustat on bone disease in Gaucher disease type 1: results from the randomized, placebo-controlled engage trial Dasouki M1, Lukina E2, Ben Dridi M-F3, Amato D4, Baris H5, Ghosn M6, Mehta A7, Packman S8, Pastores G9, Petakov M10, Assouline S11, Balwani M12, Danda S13, Hadjiev E14, Mistry PK15, Shankar S16, Marulkar S17, Peterschmitt MJ17 University of Kansas Hospital, Kansas City, United States; 2Hematology Research Center, Moscow, Russian Federation; 3Hôpital La Rabta, Tunis, Tunisia; 4Mount Sinai Hospital, Toronto, Canada; 5Rabin Medical Center, Petach Tikvah, Israel; 6Hôtel-Dieu de France University Hospital, Beirut, Lebanon; 7The Royal Free Hospital, London, United Kingdom; 8UCSF School of Medicine, San Francisco, United States; 9New York University School of Medicine, New York City, United States; 10Clinical Center of Serbia, Belgrade, Serbia and Montenegro; 11 Jewish General Hospital, Montreal, Canada; 12Mount Sinai Hospital, New York City, United States; 13Christian Medical College, Vellore, India; 14University Hospital Alexandrovska, Sofia, Bulgaria; 15Yale University School of Medicine, New Haven, United States; 16Emory University, Atlanta, United States; 17Genzyme, a Sanofi company, Cambridge, United States 1

Background: Eliglustat is an oral substrate reduction therapy in development for treatment of adults with Gaucher disease type 1. We evaluated the effect of eliglustat on bone in the context of the ENGAGE trial. Methods: Randomized (1:1 eliglustat:placebo), double-blind, 39-week Phase 3 trial (primary efficacy endpoint: percent reduction in spleen volume). Bone endpoints included bone marrow burden (BMB) scores (via MRI), bone mineral density changes (DXA), MIP-1β (bone disease biomarker), and others. Results: Forty patients were randomized (20/arm). At baseline, 80% of eliglustat patients and 75% of placebo patients had marked-severe bone marrow burden. After 39 weeks, significant improvements (eliglustat vs. placebo) were observed for total (-1.1 vs. 0.0, p=0.002), spine (-0.6 vs. 0.1, p=0.002), and femur (-0.5 vs. 0.0, p=0.026) BMB scores. Although eligibility criteria excluded patients with symptomatic bone disease, absolute change in total spine DXA z-scores approached significance (LS mean treatment difference=0.2, p=0.06). All patients had elevated MIP-1β at baseline which decreased markedly for all eliglustat patients; response in placebo was variable. Conclusions: Eliglustat, after 9 months, demonstrated significant effects on bone marrow and a trend toward BMD improvement in spine compared with placebo. Conflict of Interest declared. P-598 Encore: a multi-national, randomized, controlled, open-label non-inferiority study comparing eliglustat to imiglucerase in Gaucher Disease type 1 (GD1) patients on Enzyme Replacement Therapy (ERT) who have reached therapeutic goals Cox TM1, Drelichman G2, Cravo R3, Balwani M4, Burrow T5, Martins AM6, Lukina E7, Rosenbloom B8, Ross L9, Angell J9, Puga AC9 1

University of Cambridge,Dept of Medicine, Cambridge, United Kingdom; Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina; 3 HEMORIO, Rio de Janeiro, Brazil; 4Mount Sinai Hospital, New York City, United States; 5Cincinnati Children's Hospital Medical, Cincinnati, United States; 6Universidade Federal de São Paulo, São Paulo, Brazil; 7 Hematology Research Center, Moscow, Russian Federation; 8Tower Hematology Oncology Medical Group, Beverly Hills, United States; 9 Genzyme, a Sanofi company, Cambridge, United States 2

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Background: Eliglustat is a novel oral substrate-reduction therapy in development for adults with Gaucher disease type 1. This Phase 3 trial (ENCORE) compares eliglustat with imiglucerase. Methods: Randomized (2:1 eliglustat:imiglucerase), controlled, open-label non-inferiority trial of 160 patients previously receiving ERT for ≥ 3 years who had met therapeutic goals. The primary efficacy endpoint was percent of patients remaining stable after 52 weeks (using a pre-specified composite of spleen, hemoglobin, platelet, and liver parameters). Results: Eliglustat was statistically non-inferior to imiglucerase: 84% of eliglustat and 94% of imiglucerase patients maintained stability goals for all 4 parameters (lower bound of 95%CI of difference [-18.6%] within pre-specified non-inferiority thresholds [-25%]). Individually, 94% of eliglustat patients maintained stability criteria for spleen, 95% for haemoglobin, 93% for liver, and 96% for platelets. Baseline bone mineral density scores were normal in most patients and remained normal. No serious treatment-related adverse events occurred. Most adverse events in both arms were mild or moderate. Overall, 156/160 patients completed the trial and 2% of patients in each arm discontinued because of an adverse event. Conclusions: Eliglustat was well tolerated and was non-inferior to imiglucerase in maintaining stability for 52 weeks in previously treated GD1 patients. Conflict of Interest declared.

P-599 Difficulties in confirming the diagnosis of Mucolipidosis II / III: case reports Sperb F1, Cury GK1, Velho RV2, Alegra T2, Matte U1, Vairo F2, AeKim C3, van Meel E4, Kornfeld S4, Schwartz IVD2 1

Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil; 2PPG Biol Cel Mol - UFRGS, Porto Alegre, Brazil; 3Genetics Unit- Univ São Paulo, São Paulo, Brazil; 4Dep Int Med- Washington Univ School Med, St Louis, United States Background: Mucolipidoses II and III (ML II and III) are autosomal recessive lysosomal storage disorders (LSD) in which the mannose 6-phosphate recognition marker system is deficient. They are caused by mutations in GNPTAB or GNPTG genes, which encode the N-acetylglucosamine-1-phosphotransferase, although there are other genes involved in this pathway (such as NAGPA gene). Aims: To describe 4 patients, with inconclusive molecular diagnosis. All patients had high levels of lysosomal acid hydrolases in plasma and low levels in fibroblasts. Other LSD were excluded. Methods: Acid hydrolases in plasma of the patients were assessed for the presence of mannose-6-phosphate residues. Genetic analysis was performed by sequencing of GNPTAB, GNPTG and NAGPA. CGH analysis was performed for one patient. Results: Phosphorylation of lysosomal acid hydrolases was severely impaired in all patients. Two patients were found to be heterozygous for a pathogenic mutation in the GNPTAB gene, but no mutations were found in two patients. No mutations were found in GNPTG and NAGPA, and no change was observed in CGH. Conclusions: Mutations may be occurring in a regulatory region. This report demonstrates the difficulties of making a DNA diagnosis in a subset of patients with clinical diagnosis of ML II/III. Support: FIPE/HCPA, FAPERGS, CNPq.

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P-600 Antibody (AB) response to enzyme replacement therapy with idursulfase and clinical outcomes in pediatric and adult Mucopolysaccharidosis II (MPS II, hunter syndrome) patients with an attenuated phenotype Barbier AJ1, Pano A1, Bielefeld B1, Whiteman DAH1, Natarajan M1, Amato DA2 1

Shire Human Genetic Therapies, Lexington, MA, United States; Vertex Pharmaceuticals, Cambridge, MA, United States

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Objectives: We examined the association between Ab status and outcomes in a post-hoc analysis of data from 63 MPSII patients who received 2 years' treatment with 0.5 mg/kg IV weekly idursulfase in the pivotal phase 2/3 trial (1 year duration) and its extension study (2 years duration) Safety: Patients who were antibody positive (Ab+); persistently Ab+ at 3 consecutive visits; or neutralizing Ab+ had no increased risk of serious adverse events, but had a higher infusion-related adverse event (IRAE) rate than did antibody negative patients. The IRAE rate was highest in the time period before antibodies developed. The increased risk appeared to be ameliorated after a first IRAE through standard preventive measures for all genotypes or antibody groups. Patients with nonsense/frameshift mutations appeared more likely to become Ab+ than patients with missense mutations. Efficacy: Positive antibody status of any kind did not significantly lessen the clinical response to idursulfase in the domains of mobility (assessed by the 6-minute walk test), pulmonary function, and liver/spleen volume. Conclusion: Clinical efficacy as assessed does not appear to be affected by antibody status. After the first reports of IRAEs, standard preventive measures largely mitigated later IRAE risk regardless of genotype and anti-drug antibody status. Conflict of Interest declared. P-601 Mutation spectrum of the IDS gene in a sample of Mexican patients with Hunter Syndrome (MPS II) Alcantara-Ortigoza MA1, Gonzalez-del Angel AE1, García-de Teresa B1, Fernandez-Hernandez L1 1

Instituto Nacional de Pediatría, Mexico City, Mexico

Background: Hunter syndrome is a recessive X-linked lysosomal disease, due to deficiency of iduronate-2-sulfatase, an enzyme encoded by the IDS gene. Over 300 mutations have been described and 90% of mothers have been found to be carriers. Mutational spectrum of IDS in Mexican patients is unknown. Objectives: 1) Characterization of IDS mutations 2) Carrier identification of related women 3) Performance of prenatal diagnosis (PD). Patients and methods: Twenty-one unrelated male patients were included; five had a family history of MPSII. The IDS/IDSP1 inversion was looked for using PCR followed by enzymatic restriction, while the 9 exons of IDS were sequenced. Carrier status and PD were determined by a direct molecular strategy. Results: Mutations were recognized in 20 cases: 9 had point mutations, 3 showed the IDS/IDSP1 inversion, 3 displayed a complete deletion of IDS/IDSP1, one presented a complex rearrangement and 4 exhibited microdeletions/duplications. Obligate carrier status was confirmed in 80% of mothers, and a case of gonadal

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mosaicism was found. Three affected fetuses were identified by PD. Discussion: Nine mutations not enlisted in the HGMD database were identified; complex rearrangements represented one third (33%) of mutations. Although uncommon in MPSII, gonadal mosaicism must be considered for appropriate genetic counseling.

P-602 Common mutation analysis of Gaucher's disease: patients from India and Pakistan in resource limited small labs Kudalkar KV1, Jalan AB1, Jalan RA1, Sawant LM1, Shinde DH1, Pawaskar MS1, Khan SA1, Alam MA2 1

NIRMAN, Navi Mumbai, India; 2Bio Care Lab, Islamabad, Pakistan

Background: Gaucher's Disease is the most common LSD in India and Pakistan. Objective: To identify frequency of common mutations in patients of Gaucher's Disease from India and Pakistan with the use of Gaucher's strip assay and evaluate usefulness of strip assays in small labs and genetic clinics with limited resources. Materials and method: 18 patients with Gaucher's disease presenting with hepatosplenomegaly, anemia, generalized osteopenia and Gaucher's cells on bone marrow biopsy were selected. Glucocerebrosidase, Chitotriosidase and CCL18 were used for diagnosis and mutation study was performed by Gaucher Disease Strip Assay®, Vienna Labs. Result: We found a total of 23 L444P and 9 IVS2+1 G>A mutant alleles. 8 patients (44.44%) were homozygous for L444P (c.1448 T>C) and 7 (38.88%) to be compound heterozygous for L444P / IVS2+1G>A. 2 patients (11.11%) were IVS2+1 G>A heterozygotes, where second mutation could not be identified. For identification of these unidentified mutations and one patient where no mutation was identified, gene sequencing is under progress. Conclusion: L444P (23/36) and IVS2+1 (9/36) are the two commonest alleles in our cohort. In countries with limited resources recommended strategy would be to perform 8 common mutation scan as an initial step followed by gene sequencing if required. P-603 Early diagnosis of Niemann-Pick C type 2 (NPC-2) disease by oxysterols measurement Polo G1, Burlina AP2, Giordano L1, Bordugo A1, Guariso G3, Dardis A4, Burlina AB1 Div Metab Dis,Univ Hosp, Padova, Italy; 2Neurological Un, St. Bassiano Hosp, Bassano del Grappa, Italy; 3Pediatric Dept, Univ Hosp, Padova, Italy; 4Centre Rare Diseases, Univ Hosp, Udine, Italy

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red spot. All metabolic investigations were normal, except for increased plasma oxysterols. Results: The oxysterols analysis performed by LC-MS/MS, modified from the method of Jiang X et al. 2011, showed very high levels of cholestane-3β5α6β-triol (515 ng/ml; n.v.<30 ng/ml). Molecular analysis showed a nonsense homozygous mutation (436C>T, already reported in the Arabian population) in the NPC-2 gene. Conclusions: To our knowledge, this is the first report of a NPC2 patient diagnosed by oxysterols analysis. We suggest that in patients with jaundice and hepatosplenomegaly, oxysterols analysis is a fast and reliable marker for the diagnosis of NPC-1 and NPC-2. Conflict of Interest declared. P-604 Changes in chitotriosidase activity with lysosomal storage disease : a four year experience Biberoglu G1, Hasanoglu A1, Ezgu FS1, Tumer L1, Udgu B1 1

Gazi Univ Med fac, div ped metabolism, Ankara, Turkey

Background: Chitotriosidase is a chitinase group enzyme, secreted by active tissue macrophage. Recently it was demonstrated that high plasma chitotriosidase activity is related to the lysosomal storage diseases .The aim of the present study was to evaluate the increase of the plasma chitotriosidase activity is in relation with lysosomal storage diseases in our laboratory. Methods: Plasma chitotriosidase activity was measured according to the method described by Hollak et al. Chitotriosidase activity was determined in 99 patient with lysosomal storage disease.Fourty one of them are Gaucher disease, 54 are Niemann-Pick type A/B and 4 are GMI gangliosidosis. Results: Plasma chitotriosidase levels were found to be significantly higher in patient with 37 Gaucher (339-16437 nmol/h/ml), 54 Niemann-Pick type A/B (165-2307 nmol/h/ml) and 4 GMI gangliosidosis ( 223-424 nmol/h/ml ).Referance range was 0-114 nmol/h/ml. Also plasma chitotriosidase activity were lower in patient with 4 Gaucher disease. Conclusions: Chitotriosidase activity can be used as a biomarker for some lysosomal storage disease such as Gaucher disease, NiemannPick type A/B and GMI gangliosidosis except the patients with chitotriosidase deficiency. P-605 Functional analysis of mutations and effects of a chemical chaperone on various mutations identified in Korean patients with Gaucher disease

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Background: Niemann-Pick C type 2 disease is caused by mutations in the NPC-2 gene which leads to a defect in the lysosomal cholesterol transport protein. Laboratory diagnosis is based on filipin staining results, and mutation analysis sequence. Recently, plasma oxysterols (especially cholestane-3β5α6β-triol) has been reported to rapidly identify patients with NPC-1, but it has never been used to identify NPC-2 form. Case Report: At 20 days of life our child, male, Tunisian origin, was admitted for prolonged neonatal jaundice and significant hepatosplenomegaly. Normal neurological development and respiratory function were reported. Ophthalmologic examination showed a cherry

Cheon CK1, Lee BH2, Heo SH2, Yoo H-W2 1 Pusan National Univ Child Hosp, Yangsan, Korea, Republic of; 2Asan Medical Center Child Hosp, Seoul, Korea, Republic of

Background: Gaucher disease(GD) is lysosomal storage disorder resulting from deficient activity of ß-glucocerebrosidase(GBA). Ambroxol(ABX) has the biochemical characteristics of a effective enzyme enhancement therapy agent for treatment of GD. To date, no research studies on efficacy of PC on common GBA mutations in Korea have been conducted. Objectives: To evaluate the biochemical effects of the ABX on the function of various GD genotypes Methods: Six missense mutations were cloned into mammalian expression vectors. To evaluate the effects of the ABX, analysis of GBA

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activity and protein expression in mutant cultured medium containing ABX was performed. Results: Enhancement of GBA activity by 82.9% was observed in the G46E in the presence of 30 μM ABX. In addition, significant enhancement of enzyme activities of N188S, G46E/R257Q, F213I, R277C, G46E/F213I, R257Q, and G46E/L444P was observed in the presence of ABX. However, enzyme activity of double mutant N188S/R257Q did not show significant enhancement in the presence of ABX. Conclusions: This data suggest that the use of ABX resulted in more effective enhancement of enzyme activity in all GBA mutants, excepting the N188S/R257Q double mutant. This result will provide understanding of effect of ABX on GBA mutants commonly identified in Korean patients with GD.

and identify genotype-phenotype correlations to help inform prognosis; 2) facilitate high quality research assessing the clinical and cost-effectiveness of standard care including high cost drugs and new interventions in a realworld setting; 3) develop educational material for patients and healthcare professionals. Ultimately it is hoped that experience gained from establishing the EUNPD registry will inform the development of other rare disease registries.

P-606

Moreno-Giraldo LJ1, Satizabal-Soto JM2

Biochemical parameters of acid alpha-D-glucosidase in blood samples collected on filter paper

1

Mezzalira J1, Breier AC1, Daitx VV1, Cé J1, Coelho JC1 1

LSD lab - Dept Biochem, UFRGS, Porto Alegre, Brazil

Pompe disease(PD) is a lysosomal storage disorder wherein the enzyme acid alpha-D-glucosidase(GAA) is deficient, generating a glycogen accumulation in the lysosomes of cells. Early diagnosis of PD is an essential step to the treatment to be more effective. For this, we have developed screening methods which measure the activity of lysosomal enzymes directly on dried blood spots(DBS). The aim of this study was to investigate the biochemical parameters (Km, Vmax and optimum pH) of GAA in DBS of healthy individuals. The GAA activity was measured according Castilhos et al (2011). To determine the optimum pH, we tested various pHs in the range of 2.0 to 6.0. The Km and Vmax were estimated using artificial substrate concentrations from 2 to 15mM. Through this work we can establish an optimum pH of the enzyme in 4.5. The optimum pH was used to the assay that determined a Km of 4.85mM and a Vmax of 7.72nmol/20h/mL. These results are important for the characterization of this enzyme in this type of sample and they can be helpful in PD diagnosis. This possibility has been reported in other studies that found differences for these parameters in healthy individuals and patients with lysosomal disorders. P-607 An international registry for Niemann-Pick diseases Hiwot T1

P-608 Diagnosis biochemical, molecular characterization and implementation of enzymatic replacement therapy in a patient adult without mental retardation affected Mucopolysaccharidosis type II (Hunter syndrome)

Dpto Ped,Uni Valle. Genomics IPS, Cali, Colombia; 2Dpto Sci Phisiol, Uni Valle., Cali, Colombia The mucopolysaccharidoses(MPS) are a group of EIM caused by deficiency of lysosomal enzyme needed to break-down glycosaminoglycans (GAG). MPSII(Hunter syndrome) occurs by deficient iduronate sulfatase enzyme(IDS). Structure IDS gene(24Kb) contains 9 exons and 8 introns, located on chromosome X. Clinically there are two forms of MPSII. The severe, with mental retardation progressive; the attenuated with minimally impaired intellect. Objective: To apply confirmatory biochemical, enzymatic and molecular tests IDS deficit, and implement Enzyme Replacement Therapy (ERT) in an adult patient with clinical suspicion of MPSII. Patient: male, 28 years, height 1.33m, weight 35kg. Presents coarse facies, macrocephaly, cataracts, pectum carinatum, hypertrichosis, repeated respiratory infections. Joint mobility angles globally limited, muscle atrophy. Mental and sexual development normal. Physical growth retardation. Methods and Results: Metabolic Screening: Albumin-Acid, CetylPyridium-Chloride, AzurI: Positive. GAGs electrophoresis: Excretion increased chondroitin and heparan-sulfate. Leukocyte Enzymatic Analysis: Alfa-iduronidase: 11.85nmol/mgP/h Normal, Iduronate-Sulfatase 0.12nmol/mgP/h Deficient, ArylsulfataseB 142.96nmol/mgP/h Normal. Molecular analysis: PCR and sequencing of both strands of the entire coding region and the highly conserved region intron-exon splice junction: (c.1122C>T p.G374G) ERT: Idursulfase 0.5mg/kg/week. Conclusions: Confirmed diagnosis of MPS-II.At 18 months of ERT seen increased angles mobility, increased foot stability, increased walking capacity, greater independence in daily activities. No recurrent infections.They tested the benefits of ERT. P-609

1

Endocrinology / IMD, QE Hosp Birmingham, Birmingham, United Kingdom We have secured 1.2m from the European Commission Executive Agency for Health and Consumers to establish a Europe-wide secure open-access web-based registry for Niemann-Pick diseases (NPD) A, B and C. Aims: to establish the largest and most comprehensive single inventory of patients with NPD, to include biochemical and genetic data, treatment history and clinical course; increase awareness of NPD and quality of patient care. Methods: data will be captured in consensus core and extended datasets which will provide the foundation for developing a multifunctional webbased registry. We will achieve high usage of the registry by linking it to laboratory testing. Learning needs of patients and healthcare professionals will be identified through questionnaires and focus groups. Anticipated use of registry data: 1) consolidate knowledge of the natural history of NPD, create a comprehensive mutation database for NPD genes,

A de novo or germline mutation in a family with Mucolipidosis III gamma: implications for molecular diagnostics and genetic counseling Velho RV1, Alegra T1, Sperb F2, Saraiva-Pereira ML1, Matte U2, Schwartz IVD1 1 PPG Biol Cel Mol - UFRGS, Porto Alegre, Brazil; 2Hospital de Clínicas de Porto Alegre, Porto Alegre, Brazil

Introduction: Mucolipidoses II or III are rare autosomal-recessive disorders characterized by the abnormal trafficking and subcellular localization of lysosomal enzymes due to the phosphotransferase deficiency, enzyme which is coded by GNPTAB (ML II or III α/β) and GNTPG (ML III γ) genes.

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Objectives: Report one patient with ML III γ resulting from the inheritance of one paternal mutant allele and one allele that likely resulted from a de novo or maternal germline mutation. Methodology: GNPTG gDNA of patient and their parents were analyzed. For maternity testing, DNA samples were evaluated using 32 markers. Results: The genotype of proband was c.[244_247dupGAGT]+ [328G>T] (p.[F83X]+[E110X]). Paternal sequencing identified the heterozygous mutation p.E110X. Surprisingly, in the mother, no mutation was detected. Evaluation of 32 different DNA markers in patient and their parents confirmed maternity with over 99.9% certainty. Conclusion: We propose that the new mutation c.244_247dupGAGT (p.F83X) in this family originated as a result of germline mosaicism, in the mothers' ova or a de novo mutation in one ovum that took place during cell division. This first report of de novo mutation in ML III γ has significant implications for molecular diagnostics and genetic counseling in recessive disorders. (Support: CNPq, FIPE/HCPA, FAPERGS) P-610 Factors affecting compliance with enzyme replacement therapy with idursulfase in patients with Hunter syndrome: data from the Hunter Outcome Survey (HOS) Solano M1, Morin I2, Lampe C3on behalf of the HOS Investigators 1

Department of Neuropediatrics, Fundación Cardioinfantil, Bogotá, Colombia; 2Shire Human Genetic Therapies, Eysins, Switzerland; 3 Department of Pediatric and Adolescent Medicine, Villa Metabolica, University Medical Center, Johannes Gutenberg University, Mainz, Germany Background: Enzyme replacement therapy with idursulfase (Elaprase®, Shire HGT) is used as a long-term treatment for Hunter syndrome. Treatment consists of weekly intravenous infusions that are generally administered over 3 hours. Patients sometimes miss scheduled infusions. Methods: Data in the Hunter Outcome Survey (HOS; a global, observational registry sponsored by Shire HGT) were used to investigate the frequency of, and reasons for, missed idursulfase infusions and stopping treatment. Results: As of January 2013, data regarding missed infusions and stopping treatment were available for 574 patients followed prospectively in HOS who had received idursulfase. The mean time on idursulfase from HOS entry to last visit was 33.4 months. In total, 862 missed infusions were reported in 141/574 patients (24.6%). The most common reasons given were illness (32.0% of missed infusions), holiday/vacation (17.2%) and caregiver/family issues (8.4%). At last visit, 44/574 patients (7.7%) had stopped treatment; the most common reason given (31.8%) was the patient/their parents deciding to stop treatment. Conclusions: Analysis of data in HOS reveals a variety of factors affect compliance with treatment; the most common reason for missing an infusion was illness. However, over 75% of patients receiving idursulfase did not miss an infusion, and few patients stopped treatment. P-611 Wolman disease: recombinant lysosomal acid lipase replacement therapy prolongs survival and reverses disease manifestations in an affected infant Valayannopoulos V1, Quinn AG2, Brassier A1, Le Quan Sang KH1, Broissand C3, Arnoux JB1, Le Verge S1, Mary S4, Grevent D5, Levade T6, Benlian P4, de Lonlay P1 1 Ref Centre IEM, Necker-Enf Malades Hosp, Paris, France; 2Synageva Biopharma Corp, Lexington, United States; 3Pharmacy Dept, NeckerEnf Malades Hosp, Paris, France; 4U4M, Inst Biochem Mol Biol, Univ Hosp, Lille, France; 5Ped Radiology, Necker-Enf Malades Hosp, Paris, France; 6Metab Biochem Lab, Purpan Hosp, Toulouse, France

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Background: Wolman disease (WD) is a rare disorder of lipid metabolism caused by lysosomal acid lipase (LAL) deficiency. Affected infants display severe malnutrition and liver complications leading to death in the first year of life. We present here results of the first human treatment experience with recombinant LAL (rhLAL) involving a young infant affected with WD. Methods: A 3-month old infant presented with failure to thrive, abdominal distension and hepatosplenomegaly. He was started on low-fat diet and cholestyramine for 1.5 months before commencing rhLAL weekly infusions for 32 weeks on a compassionate basis, then in a clinical trial (LAL-CL05). Primary objective was survival at age 12 months; secondary objectives included long term safety of rhLAL, growth, liver and spleen volume and function, haematological, lipid and nutritional parameters. Results: Weekly ongoing rhLAL infusions (currently n=104) dramatically improved nutritional status and growth, with associated decreases in hepatosplenomegaly and improved liver and lipid biology. Haematological parameters normalized. No adverse events of medical concern were noted. Cognitive development remained normal. Conclusion: rhLAL showed rapid and sustained correction of growth, prolonged survival and improvement of disease markers in the first human subject; rhLAL was well tolerated and demonstrates a promising therapeutic potential for WD. Conflict of Interest declared. P-612 Bone densitometry in 13 Mucopolysaccharidosis (MPS) patients Esposito AC1, Barth AL1, Lima TA1, Boechat MCB1, Llerena Jr JC1, Horovitz DDG1 1

Instituto Fernandes Figueira / Fiocruz, Rio de Janeiro, Brazil

Evaluation of the presence of osteopenia/osteoporosis in a group of 13 MPS patients (8female, 5male, ages 5-13years; 1MPS-I, 1MPS-II, 6MPS-IV and 5MPS-VI), bone mineral density (BMD) was performed by GE Lunar densitometer iDEXA and analyzed with a pediatric software. Bone density for lumbar region L1-L4, right hip and whole body were evaluated; results were compared with age-paired children, without adjustments for height. Except for the MPS-I and MPS-II patients, all had L1-L4 BMD below expectancy for age. BMD in right hip was within normal age-range in 9 patients and above limits in a MPS-II boy. Whole body DMD was within normal limits in 10 patients. Three MPS-IV girls had BMD below normal range in spine, hip and whole body. Some studies show BMD lower than expected in MPS patients when compared with the same age-group. There is some disagreement among published data, as some suggest correction for height. Assuming these patients accumulate GAGs in osteoclasts, osteoblasts and chondrocytes, such abnormalities could alter bone density/resilience. Bone deformities and short stature in MPS could alter BMD results, interfering with the indication of anti-resorptive therapy. Therefore, BMD values should be adjusted for age, but caution is recommended before height adjustment is considered. Conflict of Interest declared. P-613 Somatosensory and motor evoked potentials - neurophysiological evaluation in cervical myelopathy in Mucopolysaccharidosis (MPS) Souza e Silva D1, Esposito AC1, Barth AL1, Magalhães TSPC1, Pena e Costa A1, Llerena Jr JC1, Horovitz DDG1 1

Instituto Fernandes Figueira / Fiocruz, Rio de Janeiro, Brazil

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

Objectives: Cervical myelopathy occurs in MPS leading to different degrees of functional impairment. Somatosensory (SEPs) and motor evoked potentials (MEPs) may offer a look at physiologic anatomy and provide a sensitive tool for assessment of spinal cord and brain stem posterior columns, lemniscal tracts and central motor pathways. Methods: We present 28 patients (5MPS-I, 8MPS-II, 1MPS-III, 5MPSIV and 9MPS-VI) in whom we recorded ulnar and posterior tibial nerve SEPs and calculated central conduction time; we also performed MEPs using magnetic stimulation of roots and brain and calculated central motor conduction time. Results: SEPs were performed 32 times in 25 patients, withabnormal results after upper and lower limbs stimulation in almost all cases (2 exceptions). MEPs were performed 25 times in 17 patients, and abnormal in all. Conclusions: Evoked potentials provides a view of functional anatomy. MPS patients often develop spinal cord compression; the term 'disfunction' seems more adequate, as neurophysiology showed severe conduction impairment in sensory and motor central pathways in all. Evoked potentials allow mapping anatomic specificity, functional sensitivity and have the ability to monitor changes over time; they can be a valuable tool for aiding medical/surgical decision and should be part of the periodical evaluation of MPS patients. Conflict of Interest declared. P-614 Longitudinal natural history and galsulfase treatment in Mucopolysaccharidosis VI (MPSVI, Maroteaux-Lamy syndrome): 10 year follow-up of patients who previously participated in a MPSVI survey study

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P-615 Biochemical characteristics of chitotriosidase enzyme between Gaucher disease patients with and without treatment Garcia CS1, Coelho JC1 1

LSD lab - Dept Biochem, UFRGS, Porto Alegre, Brazil

Chitotriosidase(CT) activity is increased in some lysosomal storage diseases. The aim of this study was to establish and compare biochemical parameters of CT in Gaucher Disease individuals before(G) and after(GT) treatment in order to observe if there was a change in their behavior. CT activity was measured in plasma. Optimum pH: the pH of assay varied from 3.5 to 6.8. Km and Vmax: substrate concentrations varied from 0.005 to 0.02mM for all groups. Thermal stability: samples were preincubated for 1, 3, 5, 10 and 15min at 50°C and 1, 3 and 5min at 60°C in the absence of substrate. The activity was expressed by % of enzyme inactivation compared with the preincubation conducted at 0°C. We were able to differentiate the group C from G according to the Km, Vmax and thermal stability at 50 and 60°C. C group differentiate from GT according to the Km and thermal stability at 60°C and group G differentiate from GT according to the Vmax of the reaction. Thus, unless by measuring the Vmax of the enzyme reaction, it was not possible to distinguish individuals with and without treatment or even observe the effectiveness of this action by biochemical parameters of this enzyme. P-616

Giugliani R1, Lampe C2, Guffon N3, Ketteridge D4, Leão-Teles E5, Wraith JE6, Jones SA6, Piscia-Nichols C7, Quartel A7, Harmatz P8

Vacuolization of peripheral lymphocytes in a complicated patient suffering from NCL7

1

Jahnová HJ1, Hůlková H1, Vlášková H1, Stibůrková B1, Dvořáková L1, Ješina P1

Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil; University Medical Center, Univ of Mainz, Mainz, Germany; 3 Hôpital Femme Mère Enfant, Bron, France; 4 Women's and Children's Hospital, Adelaide, Australia; 5Centro Hospitalar de São João, Porto, Portugal; 6Manchester Children's Hospital, Manchester, United Kingdom; 7BioMarin Pharmaceutical Inc, Novato, CA, United States; 8Children's Hospital and Research Center, Oakland, CA, United States 2

A cross-sectional survey-study of 121 patients was conducted to define the clinical heterogeneity and natural history of MPS VI; 59 patients participated in the resurvey-study (55 ERT exposed; 4 naïve). uGAG level, measured using different methods between the 2 time points, showed greater overall decrease in ERT than in naïve patients. Height Z-score in ERT patients with baseline uGAG ≤200 μg/mg creatinine changed from -2.5 to -2.9; in patients with >200 μg/mg creatinine from -5.1 to -7.2. ERT patients who completed 6-minute walk test and walked <400m at baseline had a mean increase 76.0±104.0m from baseline mean of 266.1±88.6m (p=.0001); all age groups improved. Mean change from surveystudy for respiratory function tests was 0.365±0.5L vs 0.700±0.3L for FVC and 0.211±0.4L vs 0.603±0.2L for FEV1 in the ERT and naive groups respectively. FVC improvements were seen in all age and uGAG groups. In echo-evaluated patients, for most cardiac valves there was no increase in percent of patients with regurgitation or stenosis, except for an increase in aortic regurgitation from 48.5 to 75.8%. Of the 87 patients with available survival data, mortality rate was 20.8 % in the ERT group (n=77) vs 60 % in the naïve group (n=10). Conflict of Interest declared.

1

First Fac Med, Charles Univ Hosp, Prague, Czech Republic

Among neuronal ceroid lipofuscinoses vacuolization of peripheral lymphocytes has been specifically referred in NCL3. We report a male with progressive visual impairment and subsequent neuropsychological deterioration from 3,5 years. Intensive vacuolization of lymphocytes was found. Nevertheless, suspicion of NCL3, supported also by typical ultrastructural findings, was not confirmed by the CLN3 gene analysis. Because of patient's Roma ethnicity the genes for NCL5 (CLN5) and NCL7 (MFSD8) were analysed subsequently. Homozygous mutation p.T294K in the MFSD8 gene and heterozygous polymorphism p.R2C in the CLN5 gene were found. Incidental finding of clinically asymptomatic, but significant hypouricemia, unexplained by common causes, led to analysis of the gene for familiar renal hypouricemia type 1 (SLC22A12). The novel homozygous mutation p.L415_G417del was detected. No vacuolated peripheral lymphocytes were found in another unrelated NCL7 patient and in an unrelated heterozygote for familiar mutation in the SLC22A12 gene. However, vacuolated lymphocytes were proved in two healthy siblings, heterozygous for the both, familiar NCL7 mutation and NCL5 polymorphism, and homozygous for the familiar SLC22A12 gene deletion. Conclusion: Vacuolated peripheral lymphocytes are not specific only for NCL3. Their presence likely reflects disturbance in NCL proteins networking. Relationship with urate transport disorder needs more research. (Grant RVO-VFN64165/2012, MH CR)

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P-617

P-619

Keratan sulfate (KS) analysis in Morquio A patients: LC-MS/MS analysis of KS disaccharides demonstrates that urine is a better source than plasma to monitor dynamic change in KS

A novel CLN8 missense mutation underlies variant late infantile neuronal ceroid lipofuscinosis in South America

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1

1

1

1

1

Miller N , Taniguchi G , Decker C , Zhou H , Shediac R , Quartel A 1

BioMarin Pharmaceutical Inc., Novato, United States

Insufficient activity of N-acetylgalactosamine-6-sulfatase (GALNS), a lysosomal enzyme that catabolizes the glycosaminoglycan (GAG) keratan sulfate (KS), causes Morquio A Syndrome. KS levels are not accurately measured by standard dye-based GAG analysis. In contrast, LC-MS/MS specifically and with high sensitivity quantifies urine or plasma KS in Morquio A patients using mono- or di-sulfated KS disaccharides generated after keratanase II digestion. Urine is a better source than plasma to monitor dynamic changes in KS as measured by LC-MS/MS because: 1) Urine has higher concentrations of KS than plasma and excretion of KS in urine is significantly higher in Morquio A patients than in unaffected individuals; 2) Plasma KS is elevated in Morquio A, but by a smaller factor (approximately 2-fold) compared to urine (approximately 15-fold); 3) Plasma KS concentrations in Morquio A patients overlap those from unaffected persons; and 4) Treatment of 18 Morquio A patients with enzyme replacement therapy in a Phase I/II study resulted in a reduction of urine KS (mean decrease 40.6% from 26.4 μg/mL after 36 weeks) and minimal change in plasma KS (mean decrease 3.6% from 1.5 μg/mL after 36 weeks). Conflict of Interest declared. P-618

Pesaola F1, Cismondi IA2, Guelbert N1, Kohan R1, Carabelos MN1, Alonso G1, Pons P3, Oller-Ramirez AM1, Noher de Halac I4 1

CEMECO, Child Hosp, Nat Univ Cordoba, Córdoba, Argentina; Dentistry Dept, Nat Univ Cordoba, Córdoba, Argentina; 3EM Center, Med Sc Dept, Nat Univ Cordoba, Córdoba, Argentina; 4National Research Council-CONICET, Buenos Aires, Argentina 2

Introduction: The Neuronal Ceroid Lipofuscinoses (NCLs) are inherited neurodegenerative lysosomal diseases appearing in individuals of all ages. The accumulation of autofluorescent lipopigments in the brain and in peripheral tissues is the main characteristic of the NCL group. The mutated CLN8 is known to be associated with Progressive Epilepsy with Mental Retardation (EPMR) and variant Late Infantile (vLI) phenotypes. Aim: To characterize genetically CLN8 in Latin America. Methods: Fifteen individuals with CLN8 suspicion were tested by PCR, DNA sequencing and in silica analysis. Results: The previously registered c.685C>G change, assumed as the disease causing mutation in 2 subjects, is a frequent polymorphism in the Argentinean population. The c.1A>G mutation is a new pathogenic missense mutation, found in heterozygous state in 1/15 vLI individuals. This mutation affects the translation by losing the first methionine, producing downstream a new initiation codon, a frameshift and a premature stop codon. The severity of the vLI phenotype is compatible with the predicted effect of the mutation on the protein. The search of a second mutation in non-coding or intronic regions is in progress. Conclusion: The c.1A>G mutation in CLN8 is the first confirmed mutation associated to a vLI phenotype in Latin America.

Mesenteric Gaucher cell pseudotumor- a rare cause for diarrhea and tetany in a 19 year-old gaucher type 3b patient

P-620

Perez-Colon E1, Sanchez-Valle A1, Patel S1, Murphy M1

Comparison between alpha-galactosidase a activity in dried blood spots, plasma and leukocytes of healthy men and male patients of a hemodialysis screening study

1

University of South Florida, Tampa, United States Daitx VV1, Mezzalira J1, Breier AC1, Cé J1, Moraes V1, Coelho JC1

Background: In Gaucher disease, an autosomal recessive disorder due to glucocerebrosidase deficiency, glucocerebrosides accumulate within macrophage lysosomes. Cumulates of these in tissue are called Gaucher cell pseudotumor or "Gaucheroma". Case Report: A 19-year-old female with Gaucher disease Type 3b presented with a 1.5 month history of diarrhea. The initial physical exam revealed hypotension, tetany, and hepatosplenomegaly. Aggressive fluid replacement was started after lab work showed severe metabolic disturbances. A CT enterography revealed a dense soft tissue mass at the mesentery root, with coarse calcifications occluding the superior mesenteric vein (SMV). In 2004, she had similar symptoms, and a mesenteric mass biopsy confirmed Gaucher cell pseudotumor. Due to concerns for "Gaucheroma" recurrence she was made NPO and started on hyperalimentation, resulting in complete resolution of her diarrhea. Further evaluation determined her "Gaucheroma" was non-resectable due to proximity to the SMV. Discussion: We postulate that: 1. The diarrhea is secondary to bowel wall edema due to obstruction of lymphatic and blood vessels by her pseudotumor, and 2. The diarrhea, in turn, is causing severe metabolic and electrolyte disturbances. Because "Gaucheromas" in the gastrointestinal tract are uncommon, there is limited literature about this potentially serious morbidity in patients with Gaucher disease.

1

LSD lab -Dept Biochem, UFRGS, Porto Alegre, Brazil

Fabry disease is an X-linked lysosomal storage disorder caused by deficiency of Alpha-Galactosidase A (GLA). Males with low GLA activity develop a series of symptoms related to glycosphingolipids accumulation, which includes end-stage renal failure. Screening studies in male hemodialysis patients suggest that up to 1.2% of patients have sub-normal GLA activity. In a screening study using dried blood spots (DBS) with 1647 hemodialysis patients, 111 patients showed low enzyme activity on DBS assay. Confirmatory tests using plasma and leukocyte showed normal activity in all the cases. Considering the false positive DBS results, this study aimed to compare the GLA activity in DBS, plasma and leukocytes of healthy men and male hemodialysis patients. GLA activity was determined by fluorometric assays. For DBS, GLA activity in healthy men (n=16) was significantly different from that in hemodialysis patients (n=111) (3.75±0.2899, and 1.92±0.1268nmol/h/mL, respectively; p<0.0001). The same was observed for leukocyte (healthy men: 51.36 ± 4.367nmol/h/mg; hemodialysis patients: 29.27±1.721nmol/h/mL; p<0.0001). In plasma no significant difference was observed between GLA activity in healthy men (9.869±0.9309nmol/h/mL) and hemodialysis patients (8.032±0.5427nmol/h/mL) (p>0.05). Our results confirm the need to establish specific reference values based on the differences in GLA activity between healthy men and hemodialysis patients.

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P-623

Metabolic disorders represent a significant cause of non-immune hydrops fetalis referred to an australian centre

External quality assesment for enzymatic analysis of lysosomal storage disorders. comparison of enzymatic performance in fibroblasts and dry blood spots

Stark S1, Chin S1, Fong B1, Barnard K1, Pyragius T1, Brion K1, Leo A1, Bayly B1, Gurner M1, Fletcher J1, Fietz M1

Schoonderwoerd K1, de Graaf I2, Weykamp C2 1

1

Dept.Clin.Gen. ErasmusMC, Rotterdam, Netherlands; 2 SKML, Beatrix Hospital, Winterswijk, Netherlands

Aim: To review the diagnosis rate of the 112 samples referred since 1991 from Australasian, Pacific basin and Middle Eastern centres. Methods: Enzyme activities of fibroblast samples cultured from amniocytes or skin were determined for GM1-gangliosidosis, Gaucher disease, Niemann-Pick disease types A and B, mucopolysaccharidosis type VII, acid lipase deficiency, I-cell disease, GM2-gangliosidosis types 1 and 2, metachromatic leucodystrophy, Krabbe disease, fucosidosis, alpha-mannosidosis and ceroid lipofuscinosis neuronal types 1 and 2, mucopolysaccharidosis types I and IVa, galactosialidosis, sialidosis and congenital disorder of glycosylation type 1a (CDG1a). Filipin staining and/or cholesterol esterification studies were performed for NiemannPick disease type C testing and measurement of free N-acetylneuraminic acid for sialic acid storage disorder testing. Results: A positive diagnosis for one of the above disorders was made in 10 cases (8%). MPS VII was the most common diagnosis (n=5). Single cases of Gaucher disease (n=1), Niemann-Pick C (n=1), galactosialidosis (n=1), sialidosis (n=1), and CDG1a (n=1) were identified. Conclusion: IEM represent a significant cause of fetal hydrops in our experience. The autosomal recessive inheritance of these conditions is associated with significant recurrence risk for future pregnancies.

Background: Enzyme diagnostics is acknowledged as a key component in the diagnostics of LSD's. External quality assurance schemes are needed and very important for improvement of the reliability of diagnostics. Since 2010 ERNDIM offers an EQA scheme for lysosomal enzymes. In 2012 the performance of the enzyme diagnostics between fibroblasts and DBS was compared. Methods: Freeze dried samples of fibroblast homogenates were shipped to about 64 participants. Dry blood spots were sent to about 20 participants. Enzyme activities of 6-10 lysosomal enzymes were measured in fibroblasts and DBS. Results: In fibroblasts about 90 - 100 % of the participants accomplished the correct diagnosis on base of biochemical enzyme measurements of all enzymes. In DBS about 90% of the participants accomplished correct diagnosis for Hurler and Fabry, about 60% for MPS VI and Gaucher and about 75% for Hunter. The intra-laboratory and interlaboratory variation are 2 - 3 times higher in DBS compared to fibroblasts. Conclusion: Freeze dried fibroblasts are suitable for an EQA scheme. Enzyme diagnostics in fibroblasts is reliable. In DBS enzyme diagnostics is only reliable for MPS I and Fabry but not for MPS II, VI and Gaucher, implying an increased risk of false diagnostics of these patients.

SA Pathology Women's & Children's Hosp, Adelaide, Australia

P-624 P-622 Molecular oxidative stress in Niemann-Pick disease type C Bone marrow transplant for the treatment of inborn errors of metabolism: a two year retrospective review Nicol R1, Wilson C1, Glamuzina E1, Teague L1

Rodríguez-Sureda V1, Irún P2, Sánchez O1, Pocoví M2, Domínguez C1 1

CIBERER, CIBBIM, Hosp Univ Vall Hebron, Barcelona, Spain; Bioch Biol Mol Cel Dept, CIBERER, UNIZAR, Zaragoza, Spain

2 1

Starship Children's Hospital, Auckland, New Zealand

Hematopoietic stem cell transplantation (HSCT) is a well recognised treatment for certain inborn errors of metabolism (IEM). The New Zealand National Metabolic Service referred seven patients for HSCT between 2012 and 2013. Four had Mucopolysaccharidosis (MPS) Type I – Hurler disease, one had Mucopolysaccharidosis Type II and two had early childhood cerebral X-linked Adrenoleukodystrophy. Age at transplant ranged from 1.5-3 years for MPSI-H, 5 years for MPSII and 10 years for X-ALD. Two of the transplants were from matched sibling donors and similar conditioning regimens were followed. The preparation for HSCT was more complex for those with MPS than X-ALD and included a comprehensive multi-system review. This revealed a requirement for all the children with MPS to undergo adenoidectomy +/- tonsillectomy as well as insertion of a VP shunt in one MPS1-H case. Children with MPS received enzyme replacement therapy following diagnosis until after engraftment was achieved. Complications included non engraftment (requiring a second transplant in three children), graft versus host disease (n=2) and autoimmune thrombocytopaenia, hepatitis and massive hepatosplenomegaly in one. Average length of stay was 60 days (24-105). Average reduction in urine glycosaminoglycan was 100mg.mmol creatinine. MRI changes continued to occur during engraftment in X-ALD. All survived.

Background: Niemann-Pick type C disease (NPC) is an autosomal recessive neurovisceral lipid storage disorder characterised by the lysosomal/late endosomal accumulation of unesterified cholesterol and other lipids. The pathogenic mechanism/s that link intracellular cholesterol accumulation to cell death in NPC are only partially known. This study aimed to further our understanding of the NPC pathogenesis related to oxidative stress pathways. Methods: Fifteen NPC patients were included. In all cases, analysis of genomic DNA confirmed the presence of mutations in the NPC1 gene. Indicative lipid and protein oxidation markers were measured in plasma of NPC patients. Intracellular reactive oxygen species (ROS) generation was assessed by 2',7'-dichlorofluorescein fluorescence in cultured fibroblasts. Results: Our study showed significant elevation of malondialdehyde, the main product of lipid peroxidation (0.85 vs. 0.32 nmol/mL, p< 0.0001) and carbonyl proteins (1.8 vs. 0.95 nmol/mg prot, p=0.007). Plasma levels of oxidizable lipids were not significantly associated with lipid and protein oxidation products. Intracellular ROS production by fibroblasts from NPC patients was significantly increased. Conclusions: Our results demonstrate enhanced intracellular ROS generation in NPC fibroblasts and show systemic oxidative stress to be a common manifestation in NPC patients. Thus, molecular oxidative modifications may be implicated in the pathogenesis of NPC disease.

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P-625 Niemann Pick type C: improved diagnostics by oxysterol measurement Reunert J1, Kannenberg F2, Fobker M2, Marquardt T1 Klinik f Kinder- & Jugendmed, Univ Klin, Münster, Germany; 2Zentr f Laboratoriumsmedizin Univ Klin, Münster, Germany 1

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iPSCs showed massive vacuoles in the lysosomes with TEM and glucosylsphingosine in GD-iPSCs were accumulated more than 4.6-fold, compared with that of Healthy-volunteer-iPSCs. FDiPSCs had many inclusion bodies in the lysosomes using TEM. iPS technology is a powerful new tool for pathological study for genetic disease such as LSDs. Conflict of Interest declared. P-627

Background: The available screening methods for Niemann-PickType-C (NPC) disease are limited to staining of the accumulated cholesterol in fibroblasts and measuring of the chitotriosidase activity in plasma. Both methods are unreliable as they lead to several false positive and false negative results. Methods: Evaluation of a new sensitive and non-invasive biomarker with the possibility to detect the disease by a simple blood test. Due to an increased oxidation of the accumulated cholesterol, NPC patients show an elevated amount of plasma oxysterols, e.g. cholestan-3β,5α,6β-triol, which can be detected by GC/MS analysis. Results: As part of a study with 311 plasma samples, cholestantriol was evaluated as stable biomarker for NPC with a reference value <0.05ng/μl. 23 samples of confirmed NPC patients served as control group and showed elevated plasma oxysterols up to 0.84ng/μl. In 10 out of 288 suspicious samples, the cholestantriol amount was increased (range 0.07-0.22ng/μl) and a following genetic analysis confirmed the NPC diagnosis in 8 cases. In one case, the second sample revealed normal values. In one patient, the genetic result is awaited. Conclusion: These findings suggest that measuring plasma oxysterols is a sensitive and efficient new screening method which leads to a reliable and rapid diagnosis of NPC. Conflict of Interest declared. P-626 Pathological features of induced pluripotent stem cells (iPSCs) generated from skin fibroblasts of various lysosomal storage diseases Higuchi T1, Kawagoe S1, Otsu M2, Shimada Y3, Kobayashi H3, Hirayama R1, Eto K4, Ida H5, Ohashi T3, Nakauchi H6, Eto Y1

A simple enzyme assay in leukocytes distinguishes between metachromatic leukodystrophy patients and pseudodeficient individuals Wibrand F1, Glarborg K1, Dali CI1, Lund AM1, Duno M1 1

Dept Clinical Genetics, Rigshospitalet, Copenhagen, Denmark

Background: Measurement of arylsulfatase A (ASA) activity in leukocytes is often the first laboratory analysis in the diagnosis of metachromatic leukodystrophy (MLD). It is generally accepted, however, that the enzyme assay cannot distinguish between MLD patients and individuals who carry the ASA psedodeficiency (PD) allele, which does not cause disease. We have evaluated the ability of an assay with absolute specificity for ASA to discriminate between MLD patients and pseudodeficient individuals. Methods: ASA activity was determined in leukocytes from >700 individuals. p-Nitrocatechol sulfate was used as substrate and the assay was carried out at 0° C for 18 h. Samples with low ASA activity were subjected to mutation analysis of the ARSA gene. Results: The lowest ASA activity was found in 3 MLD patients (0.2-0.6 nmol/h/mg protein) and in a patient with multiple sulfatase deficiency (0.7). An individual who was heterozygous for a MLD allele and a PD allele had an activity of 0.9. Individuals homozygous for a PD allele, heterozygous for a PD allele plus polymorphisms, or heterozygous for a MLD allele had activities between 1.4 and 2.9. The reference range (2.5-97.5 percentile) was 4.314.9 (median 9.2). Conclusions: Our assay appears to distinguish between MLD patients and pseudodeficient individuals. P-628

Dep Genet Dis Geno Sci, Jikei Univ, Tokyo, Japan; 2Stem Cell Bank, IMS, Univ Tokyo, Tokyo, Japan; 3Dep Gene Thera, Jikei Univ, Tokyo, Japan; 4Koji Eto Lab, CiRA, Kyoto Univ, Kyoto, Japan; 5Dep Pedi, Jikei Univ, Tokyo, Japan, 6Div Stem Cell Thera, IMS, Univ Tokyo, Tokyo, Japan 1

Most lysosomal storage diseases (LSDs) are caused by a deficiency of lysosomal enzyme. Induced pluripotent stem cells (iPSCs) were established by somatic cells. iPSCs can be theoretically differentiated into any cell type in the body. Here, we established LSD patient-specific-iPSCs with Pompe disease (PD), Gaucher disease (GD) and Fabry disease (FD) and tied to understand the pathogenesis of their diseases using LSD-iPSCs with ultrastructural analysis. The iPSCs generated either by retrovirus or Sendai virus vector were identified by undifferentiated stem cell markers and high-level of alkaline phosphatase expression. Infantile-onset and late-onset PD-iPSCs were positive for PAS staining. Infantile-onset PD-iPSCs showed significant accumulation of glycogen in the lysosomes with Transmission Electron Microscopy (TEM) analysis. Although late-onset PD-iPSCs showed minimum accumulation of glycogen in the cells. Infantile-onset PD-iPSCs treated with acid-alpha-glucosidase (GAA) demonstrated less accumulation of glycogen which was depending on the added amounts of GAA concentrations. GD-

Diagnostic approach of Mucopolysaccharidoses Colón C1, Alonso M1, Barros F2, Alvarez JV1, Cocho JA1, Castiñeiras DE1, Fernández-Marmiesse A1, Bóveda MD1, Alonso-Fernández JR1, Couce ML1, Fraga JM1 1 U. de Enf Metabolicas C H U. de Santiago, Santiago de Compostela, Spain; 2 Fundación Pública de Medicina Xenómica, Santiago de Compostela, Spain

Mucopolysaccharidosis are a group of inherited lysosomal metabolic diseases caused by the absence or malfunction of certain enzymes necessary for processing glycosaminoglycans. These substances accumulation causes permanent and progressive cell damage. It has been identified seven main mucopolysaccharidosis types and different clinical subtypes, very different in presentation and in general, hard to diagnose. We report an analytical approach for the identification and classification of these diseases using a single blood-spot sample and a urine-spot impregnated on paper Whatman 903. The first step in our method is to identify the presence of glycosaminoglycans in the eluted urine sample using a colorimetric assay [J Clin Lab Anal. 2010;24(3):149-53]. In a second

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phase we would identify what type of glycosaminoglycan is elevated in an electrophoresis method on the same urine sample. In the third step we would quantify the enzyme activity and we would perform the gene sequencing over the dry blood spot sample. We present results of 37 MPS-I, 6 MPS-VI and 3 MPS-IV, identify by this method.

developmental milestones, severe hypotonia with minimal spontaneous movements, complete ophthalmoplegia and loss of swallowing ability and gag reflex reflecting multiple cranial nerve palsies. MRI showed diffuse cerebral edema and eye examination revealed cherry red spot and marked optic nerve atrophy. HEXB gene sequencing revealed a homozygous novel missence mutation c.272G>C. P-631

P-629 Use of miglustat in four children with infantile-onset Niemann-Pick disease type C

A rare cause of fatal pulmonary alveolar lipoproteinosis: NiemannPick disease type C2 and a novel mutation Eminoglu FT1, Yaman A2, Kendirli T2, Deda G3, Kansu A4, Ince E5

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1

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Hasanoğlu A , Küçükçongar A , Tümer L , Ezgü FS , Kasapkara CS

Dept Pediatr Metab Dis, Ankara Univ Hosp, Ankara, Turkey; 2Dept of PICU, Ankara Univ Hosp, Ankara, Turkey; 3Dept of Pediatr Neurol, Ankara Univ Hosp, Ankata, Turkey; 4Dept Pediatr Gastroenterol, Ankara Univ, Ankara, Turkey; 5Dept Pediatr Hematol, Ankara Univ Hosp, Ankara, Turkey 1

1

Div Metab Dis, Univ Gazi Hosp, Ankara, Turkey

Miglustat is the only therapy options in NPC patients. Here discussed the effect of miglustat therapy in four children with NPC disease and initial symptoms of visceromegaly. Case 1 A 10 days old female infant was diagnosed NPC. Due to her vertical gaze palsy, motor retardation, the miglustat therapy was started at the age of 3 years. She can walk with support outside, start to speak at the first year of the therapy.. Case 2 A 10 months old female was diagnosed NPC. Neurological development was completely normal until the age of five years. The abnormal saccadic eye movement was noted and she was started on miglustat therapy. Case 3 A 3 years old male patient was diagnosed NPC. Because of his walking, speech disabilities, abnormal eye movements, miglustat therapy was started at 4 years old. His walking condition was stable, his speech was improved. Case 4 A 2 months old girl patient was diagnosed NPC. The miglustat therapy was started at the age of one year because of her neurodevelopmental delay. But we had to stop the treatment at the three months of the therapy, because of the intractable diahrrea. The level of chitotriosidase activity was decreased in all patients.

Niemann-Pick diseases are hereditary neurovisceral lysosomal lipid storage disorders, of which the rare type C2 almost uniformly presents with respiratory distress in early infancy. We present herein a patient who died at an early age from pulmonary involvement associated with NPC2. The parents of the patient were consanguineous, the pregnancy was normal, and the female child was born at term. Hepatosplenomegaly, conjugated hyperbilirubinemia, poor motor development and muscle hypotonia, and decreased tendon reflexes were noted between 2 and 3 months of age. The metabolic investigations were normal. Cholestasis was resolved spontaneously at the age of 4 months. The storage cells were not detected in bone marrow smear and liver biopsy. Chitotriosidase activity was normal. She subsequently developed progressive respiratory insufficiency with opacification of the lungs on X-ray examination. Lung lavage, computer tomography, and histology showed typical signs of pulmonary alveolar proteinosis. Genetic analysis revealed a homozygous novel mutation (c.434T>A) in NPC2 gene. BMT therapy was planned, but she died 8 months of age because of respiratory failure. The pathophysiological mechanisms of respiratory distress in Niemann-Pick type C2 disease are still unclear. However, this diagnosis should be added to the list of diseases causing alveolar proteinosis. P-632

P-630 First description of motor neuron disease in an Omani child with infantile-onset Sandhoff disease: a case report 1

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Al Murshedi F , Al Futaisi A , Koul R , Al Azri F , Al Thihli K , Al Kindy A1 1

Sultan Qaboos University Hospital, Muscat, Oman

Sandhoff disease is a progressive neurodegenerative disorder characterized by impaired lysosomal breakdown of GM2 gangliosides. Early onset disease is characterized by early neuroregression and weakness that begins in the first 6 months of life with mean age of death of below 3 years of age. Motor neuron disease (MND) is a recognized presentation in a small subset of adult-onset Sandhoff disease. There is no case report to the best of our knowledge that describes MND in the infantile form of Sandhoff disease. We hereby report a child with Sandhoff disease presenting with features consistent with MND. This first-born male infant to consanguineous parents had uneventful perinatal history except for the finding of right hand complete syndactyly with oligodactyly. He presented at 8 months of age with developmental delay, central hypotonia and brisk deep tendon reflexes. At the age of 18 months, he developed subacute deterioration with loss of all

Bietti crystalline corneoretinal dystrophy with liver involvement: a new inborn error of metabolism Kalkanoğlu-Sivri HS1, Özmert E2, Ünal Ö1, Akçören Z3, Özgül RK1, Kaymaz F4, Tokatlı A1, Dursun A1, Coskun T1 1 Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey; 2Ankara Univ, Dept of Ophtalmology, Ankara, Turkey; 3Hacettepe Univ, Dept Ped Pathology, Ankara, Turkey; 4 Hacettepe Univ, Dept Histol Embriology, Ankara, Turkey

Background: Bietti crystalline corneoretinal dystrophy (BCD) is an autosomal recessively inherited chorioretinal dystrophy, characterized by presence of yellowish glistening crystals in the retina, choroidal sclerosis and crystalline deposits in the peripheral cornea. BCD leads to progressive atrophy and degeneration of the retinal pigment epithelium and results with initially, progressive night blindness, then, reduced visual acuity and total blindness. BCD is associated with the mutations in the CYP4V2 gene. This gene codes for cytochrome P450s involved in fatty acid synthesis. Until now, no systemic involvement or metabolic disorder was found in any of patients with BCD. Case Report: Here we report the first case affected with systemic form of the disease. A 55-year-old man was referred to our clinic with

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findings of retinopathy and cirrhosis. Serum chitotriosidase level was found highly elevated (3040 μmol/l/h, N: 4–120). Liver biopsy showed the same histopathologic findings in the hepatocytes including glystening crystals as in the eyes, with extensive fibrosis. Molecular genetic analysis showed homozygous 1168C>T; p. Arg390Cys mutation in the CYP4V2 gene, and the diagnosis of BCD was confirmed. Conclusion: These findings suggest that BCD might be a new systemic inborn error of metabolism. P-633 Co-formulation of the pharmacological chaperone migalastat HCl with a novel recombinant human α-galactosidase a as a potential next-generation enzyme replacement therapy for Fabry disease Xu S1, Brignol N1, Chang K1, Frascella M1, Yasukawa H2, Shardlow C3, Churchill A3, Feng J1, Soska R1, Garcia A1, Ketkar A4, Robertson N3, Flanagan JJ1, Guillen D1, Miyamoto M2, Mihara K2, Benjamin ER1, Lockhart DJ1, Hirato T2, Fowles S3, Valenzano KJ1, Khanna R1 1 Amicus Therapeutics, Cranbury, NJ, United States; 2JCR Pharmaceuticals, Kobe, Hyogo, Japan; 3GlaxoSmithKline R&D, Ware, Hertfordshire, United Kingdom; 4GlaxoSmithKline R&D, King of Prussia, PA, United States

Fabry disease is an X-linked lysosomal storage disorder caused by a deficiency in α-galactosidase A (α-Gal A) activity, which leads to progressive accumulation of lysosomal globotriaosylceramide (GL-3) in multiple tissues. Previously we demonstrated that oral preadministration of the pharmacological chaperone migalastat HCl (1deoxygalactonojirimycin HCl; AT1001; GR181413A) improves the pharmacological properties of recombinant human α-Gal A (rhα-Gal A) via binding and stabilization, leading to improved enzyme uptake and GL-3 reduction in GLA knock-out (KO) mice. JR-051 is a novel rhα-Gal A that is currently in non-clinical development. When co-formulated, migalastat HCl stabilizes JR-051 in the formulation and in vivo. In human whole blood ex vivo, migalastat HCl increased the stability and minimized denaturation of JR-051 at neutral pH/37°C. In mice, intravenous administration of coformulated migalastat HCl + JR-051 increased the circulating exposure of enzyme up to 4-fold. A single infusion of migalastat HCl + JR-051 in GLA KO mice resulted in up to 2.5-fold greater uptake of α-Gal A and correspondingly greater GL-3 reduction in disease-relevant organs compared to enzyme alone. These data demonstrate the potentially beneficial effects of migalastat HCl + JR-051, thus warranting further investigation of a co-formulated ERT for the treatment of Fabry disease. Conflict of Interest declared. P-634 Immunogenicity of idursulfase and clinical outcomes in very young Mucopolysaccharidosis II (MPS II) patients (16 months to 7.5 years old) Pano A1, Barbier AJ1, Bielefeld B1, Whiteman DAH1, Amato DA2

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(IRAEs). Overall safety and efficacy were associated with the patients' genotype. Complete deletion/large rearrangement (CD/LR) mutation patients had a high probability (>88%) of developing antibodies/neutralizing antibodies. They also had a higher probability of developing IRAEs and showed a muted response (assessed by decreases in urinary glycosaminoglycan (uGAG) levels and liver size) versus those with missense (MS) mutations. Frame-shift or splice site (FS/SSM) patients antibody responses were intermediate between CD/LR and MS patients. All patients experienced decreases in liver size, spleen volume and uGAGs by week 18, but in the CD/LR group liver size approached baseline values by week 53. Conclusions:No association for IRAEs was observed with the type/magnitude of the idursulfase immune response. MS patients showed more benign safety profiles/greater efficacy responses than CD/LR patients, but this should be interpreted with caution due to patient heterogeneity. Conflict of Interest declared. P-635 Translational program on Neuronal Ceroid Lipofuscinoses (NCLS): a model 10-years experience on rare disease studies in latin America Kohan R1, Cismondi IA2, Guelbert N1, Carabelos MN1, Pesaola F1, Pons P3, Alonso G1, Dodelson de Kremer R1, Oller-Ramírez AM1, Noher de Halac I4 1

CEMECO, Child Hosp, Nat Univ Córdoba, Córdoba, Argentina; Dentistry Dept, Nat Univ Córdoba, Córdoba, Argentina; 3EM Center, Med Sc Dept, Nat Univ Córdoba, Córdoba, Argentina; 4National Research Council- CONICET, Buenos Aires, Argentina 2

Rare Diseases (RDs) are more than 6,900 pathologies that affect fewer than 200,000 people in US, or less than 5 people/10,000 in the EU. Disease specific programs at a regional level could benefit the patients, and at the same time stimulate, coordinate and support research, facilitating drug and biologic products development for treatments. A Translational Program for the study of NCLs, a group of inherited neurodegenerative RDs, was implemented along the last 10 years at the Children´s Hospital in Córdoba-Argentina, integrating all key traits of knowledge (diagnoses, phenotypes/genotypes, care/palliative medicine, education/social aspects), and promoting families participation. An important clue was the international collaboration that enabled high technology inputs. The NCL-Program is a model approach on RDs in a middle income country. Forty two individuals were diagnosed and CLN2 was stated as the most frequent form (66.6%). Phenotypes/genotypes were revealed, allowing more effective and less costly molecular diagnostic studies, shortening the time lapse for diagnoses, and allowing families a better quality of life. The under and miss-diagnoses were gradually overcome. Future meetings in our country will provide an excellent opportunity to spread out the scientific knowledge and bring together scientific experts and NCL families from around the world. P-636 Phenotypic subgroups of Neuronal Ceroid Lipofuscinosis type CLN2 in Latin America

Shire Human Genetic Therapies, Inc., Lexington, United States; Vertex Pharmaceuticals, Inc., Cambridge, United States

Kohan R1, Carabelos MN1, Guelbert N1, Cismondi IA2, Pons P3, Alonso G1, Dodelson de Kremer R1, Oller-Ramírez AM1, Noher de Halac I4

Objective: Relationships between genotype, antibody formation, and clinical outcomes were analyzed in a 53 week study of 28 MPS II patients receiving 0.5 mg/kg weekly idursulfase enzyme replacement therapy. Results: 19/28(68%) patients seroconverted by week 9. 14/28(50%) developed persistent neutralizing antibodies (neutralizing antibodies present in three consecutive samples)(PNAb+) by week 36. There was no association between antibody status and risk for infusion-related adverse events

CEMECO, Child Hosp, Nat Univ Córdoba, Córdoba, Argentina; Dentistry Dept, Nat Univ Córdoba, Córdoba, Argentina; 3EM Center, Med Sc Dept, Nat Univ Córdoba, Córdoba, Argentina; 4National Research Council- CONICET, Buenos Aires, Argentina

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Introduction: The Neuronal Ceroid Lipofuscinoses (NCLs) are inherited neurodegenerative lysosomal pathologies frequent in childhood,

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characterized by the pathological accumulation of ceroid-lipofuscin in the brain and extra-cerebral tissues. The CLN2/TPP1 gene encodes the lysosomal enzyme Tripeptidyl-Peptidase1 (TPP1). The high phenotypic variability of CLN2 mutated genotypes was not explained yet. Aims: To establish phenotype/genotype correlations in classic late infantile (LI) and variant juvenile (J) forms. Methods: 1) Clinical studies; 2) Evaluation of TPP1 activity; 3) Transmission electron microscopy; 4) Screening of mutations and polymorphisms; 5) "In silica" mutation studies. Results: 26 CLN2 individuals showed LI (65.4%), or J (34.6%) phenotypes. TPP1 deficiencies: n=8, residual; n=11, null activity; n=7, unknown. Morphology: pure curvilinear bodies (CBs), n=16; mixed CBs, n=2; fingerprints, n=1; not performed, n=7. CLN2 mutations, n=15: n=7 novel; n=8 worldwide known. The mutation c.827A>T (p.Asp276Val-exon7) in homozygosis was associated to the LI phenotype. The intronic mutations c.887−10A>G (intron7) and c.89+5G>C (intron2), in heterozygosis, were associated to the J phenotype. Discussion and Conclusions: The J form, rarely mentioned in the literature, appeared in more than the 30% patients, usually associated with residual TPP1 activity. The most frequent mutations in South America were described, and those frequently associated with each CLN2 phenotype were recognized.

Objectives: We report results from the first-in-human trial (NCT01155778) of IT heparan N-sulfatase (HGT-1410) for the treatment of MPSIIIA and preliminary data from its extension (NCT01299727). Primary objective: HGT-1410 safety/tolerability. Secondary objectives included monitoring CSF heparan sulfate (HS) levels and cognitive status. Methods: HGT-1410 (10mg, 45mg, 90mg) was administered via IT drug delivery device (IDDD) every 4 weeks in patients aged ≥3 years (developmental age ≥1 year). Cognitive status was assessed every 6 months. Results: Twelve patients (median 5.7 years) were enrolled and 11 continue in the ongoing extension study. Patients were heterogeneous in age, disease stage and phenotype. In the initial trial, 10 serious adverse effects were recorded: none were considered HGT-1410-related, 9 were related to IDDD. CSF HS levels declined after treatment. Response for 45mg and 90mg groups appeared superior versus 10mg group. HGT-1410 had no discernible effect on cognitive status, observed over 6-24 months, when compared with natural history study data (NCT01047306). Conclusions:HGT-1410 was biologically active and generally well tolerated. IDDD failures present challenges in this population. These preliminary data do not suggest an effect on cognitive decline. However, this Phase I/II trial was not designed to test efficacy: further clinical studies of this approach are warranted. Conflict of Interest declared.

P-637 P-639 Alpha-mannosidosis: a report of 2 siblings Surgical interventions before mucopolysaccharidoses diagnosis Kasapkara CS1, Tümer L2, Biberoglu G2, Ozbek MN3, Hasanoglu A2 Ribeiro EM1, Bezerra KRF2, da Silva CAB3 1

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Div Metab Dis,Diyarbakır Children's Hosp, Diyarbakır, Turkey; Div Metab Dis, Univ Hosp of Gazi, Ankara, Turkey; 3Div Endoc Dis, Diyarbakır Children's Hosp, Diyarbakır, Turkey Alpha-mannosidosis is a rare lysosomal storage disorder with a heterogeneous clinical presentation that is inherited in an autosomal recessive pattern. Deficient alpha-mannosidase activity leads to lysosomal accumulation of mannose-rich oligosaccharides. It occurs in approximately 1 of 500000 live births and can be caused by 40 different mutations in the gene MAN2B1 located on chromosome 19. We describe a set of siblings with alpha-mannosidosis. They were born at term to first cousin parents. A 12 years old girl and 2,5 years old sister were admitted to our hospital because of coarse facial appearence. Two cases showed frequent respiratory infections, coarse facies, mild dilatation of left ventricle, hearing deficit. Skeletal survey demonstrated dysostosis multiplex. Peripheral blood Alpha mannosidase enzyme levels were 3.12 and 3.01 nmol/h/mgprotein (normal range 231.4±81) respectively. Alpha mannosidosis has been suggested to have three clinical types. The clinical and radiographic findings in those patients suggest type 2 moderate alpha mannosidosis as the phenotype is clinically recognized before 10 years of age.These disorders, although rare, should be considered in the approach to a child with dysmorphism, developmental delay, skeletal deformities, and visceromegaly. P-638 First results of a 6 month, open label, phase I/II clinical trial of intrathecal (IT) enzyme replacement therapy (ERT) and its extension in Mucopolysaccharidosis IIIA (MPSIIIA, Sanfilippo syndrome) patients

Hospital Infantil Albert Sabin, Fortaleza, Brazil; 2 Faculdade UniChristus, Fortaleza, Brazil; 3Federal University Rio Grande do Norte, Natal, Brazil 1

Background: Despite of surgical intervention is often required at a young age in cases of mucopolysaccharidoses (MPS), there are only a few studies about surgical history of these patients, especially when the pre-diagnostic period is considered. Objectives: To characterize surgical histories of patients before MPS diagnosis with thereby broadening understanding of the natural history of these patients and helping physicians recognize the disease. Material and methods: Cross-sectional study. Patients were carried out from 1995 to 2012. Following consent, a clinical assessment form was completed, based on a review of medical records, an interview with patients and/or family and clinical examination. Results: Surgical interventions were performed in 22/51 (43%) of patients before MPS diagnosis. The hernia occurred in 78% and adenoidectomy in 23% of cases.The first surgery was performed at a median age of 3 years when a median age of MPS diagnosis was 10.2 years. Herniorrhaphy was the surgical procedure more frequent, mainly in MPS II patients. Two patients died of anesthetic complications in the second surgical procedure prior to diagnosis of MPS. Conclusions: Surgical procedures before diagnosis MPS are common. If MPS diagnosis was made before surgical procedures we believe that complications as related anesthetics should be avoided Conflict of Interest declared. P-640

Wijburg FA1, de Ruijter J1, Marchal JP1, Breen C2, Heap F3, Rust S3, Baez K4, Nair N4, Haslett P4, Jones SA2

Infusion related reactions to enzyme replacement therapy in patients with mucopolysaccharidoses

Academic Medical Center, Amsterdam, Netherlands; 2St Mary's Hospital, Manchester, United Kingdom; 3 Royal Manchester Children's Hospital, Manchester, United Kingdom; 4Shire Human Genetic Therapies, Inc., Lexington, United States

Ribeiro EM1, Bezerra KRF2, da Silva CAB3 1 Hospital Infantil Albert Sabin, Fortaleza, Brazil; 2 Faculdade UniChristus, Fortaleza, Brazil; 3Federal University Rio Grande do Norte, Natal, Brazil

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Background: Infusion–related reactions (IRR) to enzyme replacement therapy (ERT) in patients with mucopolysaccharidoses (MPS) were common, but there are few reports in the literature about this. Objective: To evaluate the manifestations and management of IRR in patients with ERT for MPS. Material and methods: It was an observational study at a single Brazilian center. Data were obtained from review of medical records, interview with patients/ families and physical examination. Results: IRR were observed in 10 (38%) of 26 patients with MPS I (1/4), MPS II (6/12) and MPS VI (3/10). Patient ages ranges from 5 days old to 19.years old. IRR were mild in 60% of cases. When an IRR occurred, the infusion was suspended until signs and symptoms had subsided. ERT was restarted on the same day except one patient (MPS VI). Intravenous corticosteroids were administered in 5 cases. Patients with MPS II had more IRR (50%) than MPS I and MPS VI. Conclusions: IRR generally were mild and easily managed. The lack of filter was not considered risk factor to IRR. Our clinical experiences in this cohort of patient demonstrated safety of ERT for treatment of MPS I, II and VI. Conflict of Interest declared.

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described in an Argentine region called "Valle de Traslasierra". Mutations c.445+1G>A and p.S261Cfs12X were found in 98,7% and 1,3% of mutant alleles, respectively. In previous population based studies the carrier frequency has been estimated to be 1 in 16-29, all of them with c.445+1G>A. Recently, we detected new mutations in five Argentinian patients: c.1082+5G>A, c.1242+1G>A, c.1451G>A (p.Gly484Glu), c.1597C>T (p.Arg533Cys) and c.1601G>A (p.Cys534Tyr). Objectives: To update the mutation frequencies in SD patients. To study the heterozygote frequency for new mutations in the population at risk. Material and methods: 54 genotyped SD patients. Blood samples from 200 healthy subjects born in the region. Mutation analysis was performed by PCR/Sequencing of specific DNA sequences. Results: Allele frequencies in SD patients: 92.03% for c.445+1G>A, 2.27% for c.1242+1G>A and 1.14% for each other. We found 9 carriers of c.445+1G>A and none of other mutations among healthy subjects (c.1082+5G>A has not yet been investigated). Conclusion: This results suggests that the frequency for new mutations is very low and confirm the role of c.445+1G>A as a founder mutation in the population at risk. P-643

P-641 Orofacial features in patients with mucopolissaccharidoses Ribeiro EM1, Freitas AB2, Fonteles CSR2, Bezerra KRF3, da Silva CAB2 1

Hospital Infantil Albert Sabin, Fortaleza, Brazil; 2Federal University of Ceara, Fortaleza, Brazil; 3Faculdade UniChristus, Fortaleza, Brazil

Small molecule inhibition of glucosylceramide synthase affects bone remodeling in mice Leger AJ1, Luo Z1, Wu I-H1, Yew NS1, Ryan S1, Malley K1, Sweet L1, Schiavi S1, Sampath TK1, Cheng SH1 1

Genzyme, a Sanofi Company, Framingham, United States

Background: Although orofacial findings in Mucopolysaccharidosis (MPS) have received limited attention in previous reports, they comprise very specific characteristics that may assist in the early identification and follow-up of these disorders. Objectives: To describe orofacial features of 26 unrelated Brazilian patients with MPS and verify any possible associations between these findings and specific MPS-types. Material and methods: Patients were diagnosed with MPS and systemically evaluated. Following consent, a clinical assessment form was completed. Facial and intraoral examination was performed, evaluating facial pattern, malocclusions, dental caries, tooth identification. Results: Midface deficiency, increased lower facial third, anterior open bite, convex profile, macroglossia, gingival enlargement and spaced arches were the most frequently observed features. These findings did not allow a differential diagnosis between different types of MPS, except for pitting enamel, which significantly associated with MPS IVA (p=.000). Open bite was statistically related to MPS types I, II, III and VI, whereas only 1 patient with MPS IV expressed this feature (p=.043). Conclusions: Pitted enamel is most likely a feature of MPS IVA. Orofacial features in MPS may help pediatric dentists recognize this disorder and minimize the delay between the initial signs/symptoms and diagnosis of the disease. Conflict of Interest declared.

Patients with type I Gaucher disease (GD1) present not only with hematological and visceral manifestations, but multiple bone abnormalities including osteopenia, Erlenmeyer flask deformity, bone crisis, and avascular osteonecrosis. A mouse model of GD1 generated via conditional knockout of glucocerebrosidase in hematopoietic and mesenchymal cells displayed GD1-like bone pathophysiologies that were attributed to defects in osteoblast proliferation and differentiation. Given the potential relationship between glucosylceramide (GL-1) and osteoblast activity, we investigated the impact of inhibiting GL-1 synthesis upon bone physiology. We administered the glucosylceramide synthase inhibitor Genz-667161 (60 mg/kg/day) in chow to C57BL/6 mice for six weeks and subsequently examined trabecular bone in femurs and lumbar vertebrae via histomorphometric analysis. In treated mice in distal femur we observed an increase in bone volume fraction with a concomitant decrease in trabecular spacing. In L4 lumbar vertebrae we also noted a decrease in trabecular spacing with an associated increase in trabecular number. The effects in lumbar vertebrae would be expected to provide an increase in bone strength. These preliminary data suggest that GL-1 and perhaps other glycosphingolipids may play a role in beneficial bone remodeling and provide the rationale for further studies on the effect of GL-1 upon bone formation and resorption. Conflict of Interest declared.

P-642

Delay diagnosis of nephrophatic cystinosis in Mexico

New mutational spectrum of Sandhoff disease in Argentina and population based study

Belmont ML1, Vela AM1, Ibarra GI2, Fernandez LC1, Guillen LS1, Monroy SS1

Mugnaini J1, Dardis A2, Azar NB1, Becerra AB1, Amorosi CA1, Zampieri S2, Dodelson de Kremer R1, Oller Ramírez AM1

IEM Screen Lab, Nat Inst Pediatr, Mexico City, Mexico; 2Nutr Genet Unit, Biomed Res Int, Mexico City, Mexico

1 CEMECO, Child Hosp, Nat Univ Córdoba, Córdoba, Argentina; 2Reg Coord Ctr for Rare Dis, Univ Hosp, Udine, Italy

Background: Nephropathic cystinosis (NC) is a lysosomal disorder that may represent a challenge to suspect and diagnose because of its low prevalence and similar symptoms with other diseases. Objective: To estimate the time gap between the age at initial clinical manifestations and the age at diagnosis in NC Mexican patients.

Background: Sandhoff Disease (SD) is a lysosomal storage disorder caused by mutations in the HEXB gene. A high incidence of SD has been

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Material and Methods: Longitudinal clinical observations collected from NC patients' historical record. Results: Thirty six NC patients (14 females/22 males) from 25 families were analyzed. Ninety four percent of patients had the infantile form of the disease. The mean ages at onset of symptoms and at diagnosis were 28 months and 5 years respectively. The mean time gap between the onset of symptoms and diagnosis was 37 months; in the worst case, diagnosis was delayed 16 years. All patients had Fanconi syndrome and failure to thrive, 92% had corneal crystals and 83% presented rickets. Four patients were diagnosed after kidney transplant. Conclusions: The time between onset of symptoms and diagnosis was very long in studied patients, suggesting that NC is a difficult disease to suspect. Health professionals should be trained about the main clinical symptoms of NC in order to establish treatment opportunely and ameliorate the course of the disease.

features contained large vacuoles with increase glycogen and acid phosphatase activity. The second case presented at 13 months with proximal muscle weakness. His muscle biopsy showed small vacuoles in few fibres. Vacuolated fibres showed acid phosphatase activity and contained increased glycogen. The third patient presented with mild hypotonia and hyperlaxity of proximal joints at the age of one. At this age muscle biopsy showed acid phosphatase positive/PAS negative inclusions as the main pathological sign in 3% of muscle fibres. The fourth case was asymptomatic apart from elevated CK levels which were detected on a routine analysis. His muscle biopsy did not show abnormalities at the light or ultrastructural levels. These data show that globular inclusions, previously only described in adults, can also be frequently observed in infantile Pompe's patients and that they can be found by their own or in combination with vacuoles. P-647

P-645 How different are Sanfilippo A and B with respect of clinical and neuroradiological findings? 1

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Kalkan Ucar S , Kitiş Ö , Özbaran B , Canda E , Kağnıcı M , Köse M , Coker M1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2Div Neurorad, Ege Univ Hosp, Izmir, Turkey; 3Div Child and Adol Psych, Ege Univ, Izmir, Turkey 1

Mucopolysaccharidosis type III (MPS III) or Sanfilippo syndrome is characterized by progressive nervous system involvement and severe behavioral problems. There are four types III-A, B, C and D, linked to different enzyme defects. The aim of presented study was to investigate MPS IIIA (n=7) and MPS IIIB(n=5) patients with respect of clinical and neuroradiological findings. The age of diagnosis of enrolled patients was (5.3±2.2 years) and (6.1±1.8 years) for types A and B, respectively. Delayed psychomotor development, severe behavioral problems and sleeping problems were observed in all type A patients and 40% of type B patients. Hypertrichosis, coarse face, macrocephaly and deafness were reported in both types. Ventricular enlargement (71.42%-A, 42.85%-B), cortical and callosal atrophy (71.42%-A, 28.50%-B), thickening of the dura (14.28%-A,B ) were the main MRI findings. White matter changes at the parieto-occipital area were detected in all MPS III A patients. However, this area was unaffected in MPS IIIB. Interstingly, there was no involvement of basal ganglia in neighter MPS IIIA nor IIIB. Cribiform changes were reported only in MPS IIIA patients. In conclusion, in spite of the fact that MPS IIIA-B demonstrates a rather homogeneous clinical picture with respect to CNS involvement there are some differences. P-646 Late onset nfantile Pompe's disease, variability in clinical and histopathological spectrum Ortez C1, Jou C1, Nascimento A1, Olivè M2, Cusí V1, Corbera J1, Colomer J1, Jimenez-Mallebrera C1

Fabry patients under enzyme replacement therapy present decreased antioxidant defenses Biancini GB1, Vanzin CS1, Faverzani JL2, Wayhs CAY3, Netto CBO2, Jardim LB2, Giugliani R2, Vargas CR1 1 PPG CB:Bioquímica, UFRGS, Porto Alegre, Brazil; 2Serv Gen Médica, HCPA, Porto Alegre, Brazil; 3PPG Ciências Farmacêuticas, UFRGS, Porto Alegre, Brazil

Background: Antioxidant defenses are potentially important for biological systems because of its capacity on preventing oxidative damage to cells and tissues. The biochemical determination of these defenses is useful to evaluate the susceptibility of oxidative stress, which is involved in various pathological processes. Studies reported the evidence that oxidative stress may be involved in Fabry disease (FD) pathophysiology. Objective: To investigate enzymatic and non-enzymatic antioxidant defenses in Fabry patients under Enzyme Replacement Therapy (ERT). Methods: Heparinized blood samples of treated Fabry patients and controls were used to determine the erythrocyte concentration of reduced glutathione (GSH) and the activity of the antioxidant enzymes glutathione peroxidase(GPx), catalase (CAT) and superoxide dismutase (SOD). Results: Patients presented decreased erythrocyte content of GSH and GPx activity, added to increased SOD/CAT ratio. Conclusions: This in vivo study shows that Fabry patients under ERT present lower levels of antioxidant defenses, including the main intracellular antioxidant – GSH, when compared to healthy controls. Moreover, the increased SOD/CAT ratio indicates that probably hydrogen peroxide is more available to oxidize biomolecules in Fabry patients. Further research and clinical trials are needed to reveal the safety and effectiveness of antioxidant supplementation in combination with ERT in these patients. P-648 Assessment of enzyme replacement therapy in two patients with Hunter disease Cabrera AM1, Fain HE1, Buiras BM1, Bonetto VV1, Fain C1

Hospital Sant Joan de Déu - UB, Barcelona, Spain; 2IDIBELLHospital Univ.de Bellvitge, Barcelona, Spain

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Pompe's disease is a clinically and pathologically heterogeneous glycogen storage disease due to mutations in the a-glucosidase gene.We describe the clinical and patho logical spectrum in 4 patients with infantile Pompe's disease. The first patient presented in the first years of life with hypotonia and respiratory complications. The muscle

Objective: To evaluate the tolerance and response of the enzyme replacement therapy (ERT) in two patients with MPSII. Materials and methods: We evaluated the tolerance and response of ERT in two patients suffering from MPS II, with absence iduronate-2-sulfatase activity and consistent molecular biology.

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Results: When diagnosed, the first patient showed neural development delay, hepatosplenomegaly;obstructive sleep apnea episodes, with desaturations.He had clear joint disorders. He could walk for 20 m alone.Enzyme replacement therapy is started at 5 years, two years after diagnosis and he was evaluated 36 months later.He can grasp small objects. He can walk 100 m in six minutes. He can climb stars if helped. He still suffered obstructive sleep apnea but with saturations of around 95%. Improvement in hepatosplenomegaly. The second patient started TRE five month after diagnosis, at 2 years of age, with a mild expression of the illness. He showed a good tolerance of the therapy and improved his mild hepatosplenomegaly, the only remarkable symptom.He doesn't show signs of progression of the desease. Conclusion: ETR is effective and safe even in young children in mucopolysaccharidosis type II, improving quality of life and comorbidities.Health services must provide treatment despite of early age. P-649

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Objectives: To compare renal function 24m pre- and post-switch in Japanese Fabry disease patients who changed treatment from agalsidase-β (1 mg/kg every other week) to agalsidase-α (0.2 mg/kg every other week). Patients and Methods: The medical records of 18 adult patients (10 women and 8 men) with Fabry disease were used to obtain information concerning patient demographics and renal function pre-and post-switch. Results: There were no significant differences in measures of renal function such as serum urea nitrogen (SUN), serum creatinine (SC), estimated glomerular filtration rate (eGFR) and urinary total protein/creatinine between agalsidase-β and agalsidase-α when comparing values 24m pre- and post-switch. For eGFR, the changes 24m preswitch vs. 24m post-switch were: males -6.58 vs. 1.26 ml/min/1.73m2, females -3.38 vs. -1.91 ml/min/1.73m2. For SC the changes were: males 0.018 vs. -0.013 mg/dl, females 0.038 vs. -0.001 mg/dl. Similar small non-significant changes were recorded for SUN and TP/Cr. Conclusion: Renal function remained stable following the switch from agalsidase-β to agalsidase-α in Japanese patients with Fabry disease. Conflict of Interest declared.

Umbilical cord blood transplantation in Hurler syndrome using busulfan, fludarabine, clofarabine, ratg conditioning regimen

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Boyadjiev SA1, Dvorak CC2, Bivina L1, Wahlstrom J2, Horn B2, Bailey-Olson M2, Cowan MJ2

Spectrum of disease in non-neuronopatic sphingomyelinase deficiency severe form of Niemann-Pick type B in Iranian population

1

Hadipour F1, Shafeghati Y1, Hadipour Z1, Banikazemi M2

Pediatrics / Genetics Univ CA Davis, Sacramento, CA, United States; Ped Allerg Immun BMT, UCSF, San Francisco, CA, United States

2

The mucopolysaccharidoses (MPSs) are a family of metabolic disorders caused by the deficiency of glycosaminoglycan degrading lysosomal enzymes. Although enzyme replacement therapy (ERT) with recombinant forms of the deficient enzymes improves the somatic manifestations of MPS I and MPS II patients, ERT has no effect on the central nervous system (CNS). Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) or bone marrow may slow or halt the progression of MPS I and MPS II by providing sufficient enzyme activity directly to the CNS of patients. A recent cooperative study of 93 patients with Hurler syndrome receiving unrelated donor cord blood transplantation indicates 70% 3-year EFS with 88% durable engraftment rate. majority of patients (89%) in this report received Busulfan and cyclophosphamide-based conditioning regimen. We report our preliminary experience with unrelated donor UCB transplant in 3 patients with MPS I, using Busulfan, Fludarabine, Clofarabine and Thymoglobulin conditioning regimen, designed to increase the engraftment rate while decreasing the rate of transplant-related toxicities. All patients received ERT prior to HSCT and for 4 weeks following engraftment. We will present the details of conditioning regimen, GVHD prophylaxis, HLA match, and the stem cell dose, as well as clinical outcomes of these patients. P-650 The effect of switching treatment from agalsidase-β to agalsidase-α on renal function in 18 adults with Fabry disease Wakabayashi T1, Sakuma M2, Morita A3, Ohashi T3, Eto Y4, Ida H1 1 Dep of Pediatrics, Jikei University, Tokyo, Japan; 2Div Gener Med, Dep Int Med, Hyogo Coll, Hyogo, Japan; 3Dep of Gene Therapy, Jikei University, Tokyo, Japan; 4Adv Clin Res Cent, Inst Neuro Dis, Kanagawa, Japan

Background: Enzyme replacement therapy with agalsidase-α and agalsidase-β has revolutionized the management of Fabry disease. However, in recent years, a supply problem for agalsidase-β has resulted in the need for alternative treatment strategies.

1 Sarem Cell Research Cen Hosp, Med Gen, Tehran, Iran, Islamic Republic of; 2New York Medical College Dept of Pediatr, New York, United States

The non-neuronopatic type of acid sphingomyelinase deficiency designated as Niemann-Pick disease type B is regarded as mild, later-onset, nonneurological form that presents with visceromegaly, and interstitial lung disease. Longitudinal data on Twenty Iranian patients with confirmed enzymatic diagnosis of NPD-B is reviewed. In 50% of patients facial coarseness and severe growth retardation were the striking finding. In these cases motor development was also significantly delayed. However, the mental developments reflected in language and communication skills were progressive in all cases. Hematologic indexes were reduced in 50% of patients; HDL cholesterol was reduced. Viseromegaly and Pulmonary disease were extremely severe in several young patients. There was no evidence of skeletal abnormalities. Consanguinity of parents documented in all of our cases (100%). Among 13 patients with available DNA analysis, homozygous missense mutation in exon 6 showed high prevalence. This is the first report of severe form of NPD-B with high morbidity and mortality seen among Iranian population. In these cases pulmonary disease and viseromegaly are the most debilitating features. As enzyme replacement therapy (ERT) with recombinant sphingomyelinase being developed special focus on this group of patients is warranted. P-652 Reference intervals for lysosomal enzyme activity in a south Australian reference laboratory Mordaunt D1, Metz M1, Stark S1, Fietz M1, Fletcher J1 1

SA Pathology, Adelaide, Australia

Background: Lysosomal enzymology has historically been performed as a white cell enzyme panel in our laboratory, including 15 lysosomal enzymes measured in plasma and leukocytes, and performed by either fluorimetric, spectrometric or radionuclide analysis- galactosidase, total

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beta-hexosaminidase, beta-hexosaminidase A, arylsulphatase A, betaglucuronidase, alpha-mannosidase, alpha-fucosidase, sphingomyelinase, beta-glucocerebrosidase, beta-galactocerebrosidase, acid lipase, palmitoylprotein thioesterase 1, tripeptidyl peptidase-I, alpha-Nac-galactosaminidase (plasma), and alpha-mannosidase (plasma). As with many specialised clinical assays, the origins of our reference intervals have been "lost in the mists of time". Thus, our goal was to establish reference intervals from a clinical reference population, using evidence-based standards. Methods: Entries were extracted from an institutional database, with 3008 requests identified between 25 June 2007 and 23 October 2012. Results: were filtered using a standardised and reproducible approach. Samples were partitioned according to sex, with comparative analysis between partitions performed in addition to analysis of the combined group. Reference intervals and comparative statistics were calculated using non-parametric methods, in compliance with the Clinical and Laboratory Standards Institute, C-28A protocol (CSLI-C28A). Reference limit plots were produced in compliance with CLSI guideline C28-A3 9.4.1 / 9.5.1.

Objective: To clarify relationship between left atrial enlargement, cardiac function, HCM and age by echocardiogram. Patients and a method: 93 patients with Fabry disease are enrolled in this study. Cardiac lesions are evaluated by echocardiogram. Left atrial enlargement is evaluated using z-score, LA z-score. Results: Neither moderate nor severe mitral regurgitation, systolic dysfunction nor DCM was found in all the cases. HCM, AF/Af were seen in 37, 4, respectively. LA z-score<2, 2-3, 3-4, >4 were seen in 34, 24, 26, and 9 patients repectively. 12 patients were under 20 years old, none presented with HCM, but 2 (16.6%) patients had LA z-score>2. One patient presented with HCM of 8 patients with LA z-score>2 among 18 patients who were 21-30 yearsold. LA z-score>2 was found in 25(42.4%) patients without HCM, and in 32(94.1%) patients with HCM. Conclusion: In Fabry disease, mitral valve regurgitation is not severe which affects LA enlargement. LA z-score become >2 with age, which may then progress to HCM. LA z-score is useful for evaluation and in predicting early cardiac lesion in Fabry disease.

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P-655

Molecular testing and genotype-phenotype correlations in Gaucher disease: experience from three southern Indian centres

Value of the biomarker heparin cofactor II-thrombin complex in the early diagnosis of mucopolysaccharidoses I, II and VI

Bhat M1, Ambika KS1, Shetty S1, Sanjeeva GN2, Muranjan M3, Patil S3, Nampoothri S4

Llarena M1, Andrade F1, Sanjurjo P1, Aldámiz-Echevarría L1 1

Cruces University Hospital, Barakaldo, Spain

1

2

Centre for Human Genetics, Bangalore, India; Indira Gandhi Institute of Child Health, Bangalore, India; 3Seth GS Medical College and KEM Hospital, Mumbai, India; 4Amrita Institute Medical Sciences, Kochi, India Background: Gaucher Disease (GD) is the commonest LSD. Little data exists on the frequency of specific mutations and corresponding phenotypes in Indian patients. Materials and methods: 58 GD affected Indians were enrolled. Clinical details in all and mutation studies from 33 patients are described. Mutation screening of the GBA gene included N370S, L444P, c84-85insG and IVS2+1-G>A and Exons 9 and 10 sequencing for the 55bp deletion and for complex recombinant alleles between the active gene and pseudo-gene. Results and discussion: 58 affected individuals (52 families) across three centres were included. 28 were female and 30 male (age at diagnosis: 15mths-26yrs). 26/52 (50%) had parental consanguinity. 51(88%) had GD 1 / III with visceral, skeletal and ocular findings. 4 others had neonatally lethal Type II GD, 2 had classical Type IIIC variant with homozygous D409H point mutations. GBA mutations were negative in three, later diagnosed as Saposin C deficiency. 15 individuals were on ERT (duration: 3mths to 13yrs) with excellent response. Mutation studies completed in 33 showed homozygous L444P (17), homozygous other mutations (8) and compound heterozygous mutations (5). N370S mutation was not detected in this cohort. Regional variations, clinical findings and corresponding mutations will be discussed for each affected individual. Conflict of Interest declared.

The identification and validation of disease biomarkers for the early detection of mucopolysaccharidosis (MPS) is vital. Recently, the levels of the Heparin cofactor II-thrombin (HCII-T) complex have been shown to be highly elevated (25 to 100-fold) in the serum of patients with MPSI, II and VI, which store dermatan sulphate (DS); and less elevated (4-fold) in MPSIII, in which heparan sulphate (HS) is stored. These data indicate that serum HCII-T represents a suitable biomarker to help identify and diagnose patients with MPSI, II and VI. HCII-T has also been demonstrated to be measurable using dried blood spot samples (DBS), though there are not enough reported studies that would confirm the potential value of this biomarker in newborn screening. Here, we have extended these investigations and measured the HCII-T complex in DBS. Our results shown that the combination of several biomarkers: quantitative determination of urinary glycosaminoglycans, HCII-T measurements in serum and DBS (MPSI, II and VI) and a ELISA immunoassay for HS (MPSIII), together with the enzyme activity analysis (MPSI, II, VI and III) would provide us with the tools to distinguish among MPS, using convenient methods for the collection and shipping of samples, and thus facilitate early diagnosis of MPS. P-656 Very high incidence of the classic infantile form of Pompe disease in French Guyana

P-654 Benoist JF1, Ogier de Baulny H1, Poulain Y2, Verloes A3 Left atrial enlargement is progressive before hypertrophic cardiomyopathy in Fabry disease Fujiwara M1, Ohashi T1, Eto Y1, Ida H1 1

The Jikei Unniversity School Of Medicine, Tokyo, Japan

Background: Fabry disease presents cardiac lesion which is progressive with age. Minimal cardiac lesion may be easily overlooked.

Reference Centre Metab Dis, Hop R Debré, Paris, France; 2pediatry, CHOG, St Laurent du Maroni, France; 3Genetic Dep, Hop R Debré, Paris, France 1

Pompe disease, an autosomal recessive disorder due to a deficiency of acid α-glucosidase activity, has a worldwide incidence around 1 in 40 000. In French Guyana we found a very high incidence (1/4000) of the classic infantile form of the disease. All patients are from the same

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Bushinengé ethny issued from the Caribbean maroons slaves originated to western Africa. All the 13 patients diagnosed during the last 10 years shared two mutations most often in coumpond heterozygozity. The first p.Arg854X is a nonsense mutation frequently found in African American population which has been traced to central Africa. The second p.Gly648Ser is a missens mutation responsible for a null enzyme activity. All presented the classic infantile form with severe cardiomyopthy responsible for cardiac insufficiency by the age of 5 months. We developed enzymatic and genetic tests from dried blood spots for a rapid diagnosis and management of the patients. We have estimated the frequency of the heterozygous carrier around 1/30 for each mutation. This high incidence, due to a founder effect, raises the question of a newborn and heterozygote screening in this peculiar population. However, enzyme therapy in such population would be very difficult to follow.

gastrointestinal symptoms. Examination revealed a tense non-tender abdomen, hepatomegaly and reactive BCG scar (5months following inoculation). She had a microcytic anaemia, mildly elevated transaminases and normal hepatic synthetic function. Other significant findings included altered T-cell function and eosinophilia. The finding of adrenal calcification on abdominal CT prompted urgent measurement of LAL activity. Results: Wolman Disease was confirmed with markedly reduced LAL 46μmol/g/h (350-2000) and elevated Chitotriosidase 786μmol/L/h (4-120). Conclusion: Despite the absence of hallmark clinical features of Wolman Disease evidence of adrenal calcification should be sought in all infants presenting with hepatomegaly. Altered T-cell function is previously un-described. The clinical significance is unknown, and warrants further investigation.

P-657

Enzyme replacement therapy enhanced a therapeutic efficacy of bone marrow transplantation in MPS II mouse

Symptomatic carriers of Pompes disease revealed by selective screening in hyperckemia Spada M1, Vercelli L2, Mongini T2, Porta F1 1 Dept of Pediatrics, University of Torino, Torino, Italy; 2 Dept Neuroscience, University of Torino, Torino, Italy

Background: Pompe's disease is an inherited metabolic myopathy caused by acid α-glucosidase deficiency. No symptomatic carriers are have been identified so far. Methods: We screened for Pompe's disease one-hundred-thirtyseven consecutive patients with unexplained hyperCKemia by measuring acid α-glucosidase activity on dried blood spot. Second tier testing in positive patients included the assessment of α-glucosidase activity on lymphocytes or muscle tissue and molecular analysis. Results: Three patients were diagnosed with later-onset Pompe's disease, revealing 2.2% prevalence in paucisymptomatic hyperCKemia. Moreover, three patients positive to the screening revealed abnormal biochemical second tier testing, but were heterozygous for the common c.-32-13T>G mutation at molecular level. Conclusion: The selective screening for later-onset Pompe's disease in paucisymptomatic hyperCKemia allowed the identification of affected patients in a pre-clinical stage. Additionally, the identification of carriers with biochemical alterations related to Pompe's disease extends the spectrum of its manifestations to heterozygous subjects.

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Akiyama K1, Shimada Y1, Higuchi T1, Iizuka S1, Ohtsu M2, Nakauchi H2, Crawford B3, Brown J3, Fukuda T4, Kobayashi H1, Eto Y5, Ida H6, Ohashi T1 1 Dept Gene Ther, Jikei Univ Sch Med, Tokyo, Japan; 2Div Stem Cell Ther, Univ Tokyo, Tokyo, Japan; 3Zacharon pharmaceuticals Inc, San Diego, CA, United States; 4Dept Neuropatho, Jikei Univ Sch Med, Tokyo, Japan; 5Adv Clin Res Cent, Inst Neuro Dis, Kanagawa, Japan; 6 Dept Pediat, Jikei Univ Sch Med, Tokyo, Japan

Atypical wolman disease with evidence of immune dysfunction

Mucopolysaccharidosis type II (MPS II), is characterized by deficient activity of iduronate-2-sulfatase (IDS), resulting in accumulation of glycosaminoglycans (GAGs) in tissues. Enzyme Replacement Therapy (ERT) and bone marrow transplantation (BMT) are available. In this study, we compared the efficacy of BMT, ERT and combination therapy (BMT+ERT) to murine model of MPS II. We assayed total GAG (tGAG), and pathological GAG (pGAG) accumulating due to the deficient of IDS. BMT have therapeutic effect to reduce GAG accumulation in some of tissues except brain and kidney, but ERT reduced GAG more profoundly than BMT. The combination therapy allows promoting reduction of GAG accumulation than only BMT in liver, heart, spleen and lung. In brain, all treatment failed to reduce the GAG and Y-maze test also failed to demonstrate functional recovery. The pGAG assay make a more clearly difference in amount of GAG accumulation between wild type and MPS II. In conclusion, additive effect of ERT to BMT in clearance of GAG was observed. ERT reduced GAG more profoundly than BMT; however we did not detect any antibody formation against enzyme. Thus we overestimated efficacy of ERT in murine system. In addition, pGAG is better biomaker to evaluate efficacy of treatment than tGAG. Conflict of Interest declared.

O'Leary OA1, Hawkes CP1, Cody D1, Hughes JAF2

P-660

1 Our Lady's Children's Hospital, Crumlin, Dublin, Ireland; 2NCIMD, Children's University Hospital, Temple Street, Dublin, Ireland

Pompe disease: new mutations and polymorphisms in GAA gene

P-658

Background: Wolman disease is a rare autosomal recessive inborn error of metabolism due to a deficiency of lysosomal acid lipase (LAL) activity resulting in massive accumulation of cholesteryl esters and triglycerides. It classically presents in the first weeks of life with hepatosplenomegaly, vomiting and steatorrhoea progressing rapidly to severe malabsorption and malnutrition resulting in death before one year of age. Method: We report a 51/2 month old girl presenting with a 3 month history of increasing abdominal distension on a background of normal growth and development. She was well at presentation with no

Catarzi S1, Caciotti A2, Menni F3, Ruggiero L4, Donati MA5, Guerrini R1, Morrone A1 1

Dept NEUROFARBA, AOU Meyer, Un. Florence, Florence, Italy; Mol and Cell Biology Lab, AOU Meyer, Florence, Italy; 3Dept of Ped, De Marchi Hospital, IRCCS, Milan, Italy; 4Dept of Pediatrics, University of Naples, Naples, Italy; 5Metabolic Disorders Unit, AOU Meyer, Florence, Italy 2

Background: Pompe disease (PD) is a disorder of glycogen metabolism caused by deficiency of alpha-glucosidase (GAA). Infants

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typically experience myopathy, hypotonia, hepatomegaly and heart defects, less likely involved in late onset PD. The diagnosis is performed through biochemical and molecular analysis. Objective: GAA pseudodeficiency can complicate the diagnosis of PD. We aim to provide an accurate molecular characterization of GAA gene in patients with suspected PD. Methods: Molecular analyses of GAA gene were carried out in 17 suspected PD patients with reduced enzyme activity. Results: Out of 17 patients GAA clear cut genetic lesions were identified only in 10 patients. Among them we identified four new mutations: two splicing defects and two small deletions. Between the remaining 7 suspected patients one carries only one mutation and the others showed only polymorphisms. Among them two patients carry polymorphisms that are known to cause pseudodeficiency.. Conclusions: Molecular analysis of the GAA gene proved to be essential and confirmed that the biochemical diagnosis of PD is complicated by psedudodeficiency. Supplementary studies on the patients' polymorphism aplotypes are necessary to characterize the effect of the polymorphic nucleotide substitutions.

at the start of ERT 11-21 years, median 15 years) and the effects of ERT cessation (range 2-8 months, median 3 months) on their clinical status. Additionally, we review previously published cases. In our series, a worsening of the patients' clinical status was observed. Symptoms after ERT discontinuation included recurrent respiratory infections (severe pneumonia) with respiratory insufficiency (80%), difficulty with walking/standing (60%), increased joint stiffness (40%), but also decreased hematological parameters (40%), renal insufficiency (40%) and death (20%). The literature review confirms that the beneficial clinical effects of ERT are soon lost if treatment is discontinued in MPS I and Pompe patients. Conclusions: 1. Rapid cessation of ERT results not only in the loss of the beneficial effects, but in a significant worsening of the patient's clinical status. 2. Decisions about the introduction of ERT, especially in patients severely affected, should be made carefully. 3. Once started, it is essential to keep an adequate administration schedule of ERT to maintain the clinical benefits of enzyme therapy.

P-661

Chaperone effects of new aminocyclitol derivatives on mutant glucocerebrosidases causing Gaucher disease

Anthropometric evaluation of a group of patients with Mucopolysaccharidosis VI

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Serra J1, Díaz L2, Gutiérrez de Terán H3, Sánchez-Ollé G1, Bujons J4, Michelakakis H5, Delgado A2, Grinberg D1, Vilageliu L1, Casas J2

Vasconcelos C1, Rodrigues E1, Rola M1, Leao Teles E1 Dpt Genètica, UB; IBUB; CIBERER, Barcelona, Spain; 2RUBAM, Dpt Quim Biomed, IQAC-CSIC, Barcelona, Spain; 3Fund Pub Gal Med Xenom - SERGAS, Santiago de Compostela, Spain; 4Dpt Quim Biol Mod Mol, IQAC-CSIC, Barcelona, Spain; 5Dpt Enzym Cell Funct., Ins Child Health, Athens, Greece 1

1

Unidade Doenças Metabólicas, CHSJ, Porto, Portugal

Background: Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) is a lysosomal storage disease that is characterized by systemic clinical manifestations and significant functional impairment. Nutritional status and anthropometric assessment of these patients is still a gap in the characterization of the disease. Objectives: To assess the anthropometric measures of 5 of the 8 patients with Mucopolysaccharidosis VI, with enzyme-replacement therapy (ERT) ongoing, followed in Metabolic Disease United. Material and Methods: We assessed 5 patients: 2 males and 3 females aged between 5 and 24 years (mean 17,6 + 7,34 years) and ERT time of 3-10years. It was assessed weight, height and 4 skinfolds (bicipital, triceps, subscapular and suprailiac) of these patients. Thereafter, starting from the Durnin/ Womersley formulas for sex and age, body density was determined, and then, using the Siri formula was determined percentage of body fat. Results: Based on NIH/WHO BMI guidelines, just one male presents overweight and all has fat mass within the normal range for age and sex. However, all patients present height lower than normal for the age. Discussion: Despite their particular phenotype, with deviations in height, patients have fat mass and body mass index within the normal upper limits. P-662 Effect of rapid cessation of enzyme replacement therapy: a report of 5 cases and a review of the literature

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency of the lysososmal enzyme glucocerebrosidase (GBA1). It produces the accumulation of glucosylceramide in cells of the reticuloentothelial system and causes multisystemic manifestations. The limited efficacy and high cost of the current treatments has led to the development of new strategies, including the use of pharmacological chaperones. These are small molecules, generally competitive inhibitors of the target enzyme, which at sub-inhibitory concentrations may induce the proper folding and trafficking of the mutated enzyme resulting in a concomitant increase in the residual activity. The aim of this work is to report on the ability of eleven compounds to increase the residual GBA1 activity on fibroblasts from seven Gaucher patients bearing different genotypes. Nine of the products are new aminocyclitols (LD family), and the remaining two are N-(n-nonyl)deoxynojirimycin (NN-DNJ) and isofagomine (IFG). All compounds were highly active as enzyme activity enhancers on fibroblasts from GD patients containing the G202R mutation. Moreover, using the novel tagged sphingolipid ω-azidosphingosine, a reduction in the tagged GlcCer accumulation was also observed for selected aminocyclitols. Additionally, some of the compounds were slightly active on fibroblasts bearing other mutations, including the highly prevalent N370S mutation. P-664

Jurecka A1, Żuber Z2, Opoka-Winiarska V3, Węgrzyn G4, TylkiSzymańska A1 1 The Children's Memorial Health Institute, Warsaw, Poland; 2S. Louis Regional Children's Hospital, Cracow, Poland; 3Medical University of Lublin, Lublin, Poland; 4University of Gdańsk, Gdańsk, Poland

An infant with a diagnosis of Sialidosis type II Kilic M1, Yildiz D2, Dogan Y3, Ozdemir O2, Kocak M2, Gunbey S2, Kandemirli G4, Kilic E5 Kecioren Train-Research Hosp Div Metab, Ankara, Turkey; 2Kecioren Train-Research Hosp Child Unit, Ankara, Turkey; 3Firat Univ, Div Child Gastroent, Elazığ, Turkey; 4Hacettepe Univ, Faculty of Medicine, Ankara, Turkey; 5Hacettepe Univ, Pediatric Genetic Unit, Ankara, Turkey 1

Enzyme replacement therapy (ERT) is a treatment modality available for several of the lysosomal storage diseases including mucopolysaccharidosis type II (MPS II). We report a series of patients with MPS II (n=5, age range

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Background: Sialidosis is a lysosomal storage disease (LSD) due to deficiency of alpha-N -acetyl neuraminidase. Case Report: A 3 months-girl presented with complaints of swelling in the body and respiratory distress. Patient history revealed an episode of one month hospitalization with similar symptoms where she was treated for pulmonary hypertension and heart failure. Physical examination showed coarse facial features, gingiva hypertrophy, cataract in left eye, decreased breathing sounds, hepatosplenomegaly, edema in tibia and dorsum of foot, cutaneous telangiectasia, bilateral hearing loss and developmental delay. Laboratory results showed vacuolated lymphocytes in peripheral blood smear, dysostosis multiplex in X-ray, in abdominal sonography liver parenchymal echogenicity showed mild coarse granular features, echo showed hypertrophic cardiomyopathy and secundum ASD. Urine quantitative sialic acid testing showed free sialic acid levels were normal, bound sialic acid levels were elevated . Neuraminidase level were low but ß-galactosidase were normal in fibroblast culture. Based on her age and symptoms, diagnosis of type II congenital sialidosis was made. At 4 months old, she presented with pneumonia, heart failure and died secondary to respiratory failure. Conclusion: Edema in type II congenital sialidosis is a key finding in differentiating from other LSD. P-665 Outcome in 2 females siblings with MPS VI after 269 weeks of ERT Franco JFS1, Bertola DR1, CRDC Q1, Borlot F1, Honjo R1, Pesquero JB2, Kim CA1 1

Background: MPS VI is an lysosomal storage disorder determined by mutations in the arylsulfatase B (ARSB) gene with progressive multisystem involvement due to deficient activity of the enzyme arylsulfatase B.Treatment with enzyme replacement therapy (ERT) using Naglazyme for MPS VI shows important clinical benefits in affected individuals younger than 5 years. Objectives: To compare clinically two sisters affected with MPS VI after 269 weeks of ERT. Case report: First patient was diagnosed at 8y9mo and presented coarse face with short stature 99 cm, severe multiplex dysostosis, mitral valve replacement and severe multiplex dysostosis. Urine glycosaminoglycans (GAGs) was elevated: 655 mg/mg creatinine and low ASB activity in leucocyte 4 nmol/h/mg (normal range-(nr) 44-106 nmol/h/mg). ASB gene mutation was c.1143−8t>g. Her sib was affected too at 8mo, 87 cm height, presented only mild prominent cheek and thickening of mitral valves. Elevated urine GAGs and low ASB activity confirmed MPS VI. They began to enzyme therapy at 8y9mo and 1y5mo. Results: After 269 weeks ERT, younger sib presents the mild coarse face, absent gibbus, mild multiplex dysostosis, height 112cm(not short stature), normal GAGs, apnea index 4(nr <2), mild mitral envolvement and without stenosis cervical spine. Discussion: Earlier enzyme therapy demonstrates better improvement in clinical parameters P-666 Safety of investigational intrathecal (IT) enzyme replacement therapy (ERT) in mucopolysaccharidosis II (MPSII, Hunter syndrome) children with cognitive impairment and effect on cerebrospinal fluid (CSF) biomarkers Muenzer J1, Shi P2, Pan L2, Wang N2, Fan Z1, Hendriksz CJ3, Vijay S3, Barbier AJ2 1

P-667

2

Unid Genética Inst Criança FMUSP, São Paulo, Brazil; Dep. Biofísica Univ. Fed. São Paulo, São Paulo, Brazil

Univ of North Carolina at Chapel Hill, Chapel Hill, NC, United States; Shire Human Genetic Therapies, Lexington, MA, United States; 3 Birmingham Child Hosp NHS Found Trust, Birmingham, United Kingdom 2

Background: There is no effective therapy for cognitive impairment in MPSII. Methods: A phase 1/2 study (HGT-HIT-045) examined the safety of recombinant iduronate-2-sulfatase formulated for IT administration (idursulfase-IT) and its effect on CSF biomarker (total glycosaminoglycan [GAG]) concentrations. Sixteen cognitively impaired MPSII children previously treated with intravenous idursulfase received either no treatment, or 10 mg, 30 mg, or 1 mg idursulfase-IT monthly for 6 months via intrathecal drug delivery device (IDDD) or lumbar puncture. Results - Safety: Idursulfase-IT was generally well tolerated with normal CSF white cells prior to dosing when the device was intact. Device-related issues necessitated partial or total surgical revision or removal of the IDDD in 6/12 treated patients, leading to lumbar puncture dosing. Results - Pharmacodynamic: All 3 treatment groups showed decreased CSF GAG concentrations, with the major portion of the decline occurring after the first idursulfase-IT dose. The reductions were more pronounced in the 10- and 30-mg dosing groups than in the 1-mg IT dosing group. After 6 months of treatment, the 1-mg IT dosing group started transitioning to 10-mg monthly dosing. Conclusion: Further investigation is warranted to determine whether idursulfase-IT could represent a potential treatment for the cognitive decline associated with the severe form of MPSII. Conflict of Interest declared.

Mutation analysis in IDUA gene in Turkish patients with MPS type I Atceken N1, Karaca M1, Yilmaz-Yucel D2, Dursun A2, Tokatli A2, Sivri S2, Coskun T2, Ozgul RK2 1 Dept. Biol. Aksaray University, Aksaray, Turkey; 2 Hacettepe University, Dep. of Metabolism, Ankara, Turkey

Mucopolysaccharidosis type I (MPS type I) is a rare autosomal recessive disorder resulting from deficiency of the lysosomal enzyme α-Liduronidase. Patients with MPS type I are unable to degrade the glycosaminoglycans dermatan sulfate and heparan sulfate, which causes the progressive storage of glycosaminoglycan within lysosomes. In this present study, mutation screening for IDUA gene in 8 Turkish patients with MPS type I were performed by using direct sequence analysis. A total of 7 different types of disease-causing mutations were identified; including two missense (p.A327P, p.P533L), three nonsense (p.Y64X, p.W402X, p.R628X), one splicing (c.494-1G>A) and one translational initiation site (p.M1L) mutations. Beside mutant alleles, different types of polymorphisms (A8A, A20A, H33Q, R105Q, IVS5-8C>T, A314A, T410T) were also described in the patients. Out of 7, three patients have the same c.4941G>A splicing mutation which was only reported in Turkish patients. The mutational analyses on IDUA gene will identify structure/function relationships of the enzyme, more accurate interpretation of genotype/phenotype correlations and for choosing appropriate treatments. This study was supported by the Scientific and Technology Research Council of Turkey (TÜBİTAK-SBAG-111S217) P-668 Platelet function and coagulation abnormalities in Gaucher disease patients Kose M1, Kagnıcı M2, Canda Ebru2, Balkan Can3, Çoker M1 1 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 3Div Hemat Dis,Ege Univ Child Hosp, İzmir, Turkey

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Background: Bleeding manifestations are common in Gaucher disease (GD) patients.Although usually attributed to thrombocytopenia, some patients with relatively high platelet counts and normal coagulation tests have hemorrhagic phenomena. Materials and Methods: We performed platelet adhesion-aggregation and coagulation tests on 19 Gaucher patients who were not severely thrombocytopenic (platelet counts >50 W 109/L). Results: Ten patients had abnormal partial thromboplastin time. In seven, platelet aggregation was markedly reduced in response to collagen and ADP and in three absent in response to epinephrine.In seven patients, ristocetin- induced aggregation was impaired.We observed no improvement in ristocetin-induced aggregation in patients whom we have of the data before and after enzyme replacement therapy.In contrast to ristocetin we found improvement in aggregation with epinephrin. Interestingly one of our patients who had impaired ristocetin-induced aggregation was GD Type III. Conclusion: Unresponsive phenomenon to ristocetin in our patients might be meaningful for speculating that glycoprotein structure of platelets could be affected in GD patients.In addition to this ; best of our knowledge there is no data about Gaucher types except Type I. Further studies should therefore be performed, not only in GD1.

enzyme replacement therapy (ERT) for Gaucher disease type 3 is discussed. Case: 11-year-old female patient was admitted with complaints of frequent infections, hepatosplenomegaly, psychomotor retardation, pancytopenia at the age of 14 months. The initial diagnosis was Gaucher disease type 3 with glucocerebrosidase activity 10 pmol/min/mgprotein (N:2570) and homozygous L444P mutation. Thrombocytopenia, anemia, hepatosplenomegaly resolved within the first 2 years after ERT but severe psychomotor retardation and neutropenia persisted. Recurrent pyogenic infections with severe neutropenia were treated with antibiotics and granulocyte-colony-stimulating-factor (G-CSF) during 11 hospital admissions. A second bone marrow after 2 years of ERT showed maturational arrest of myelocytes and promyelocytes. G-CSF therapy was reserved to febrile periods. Molecular study for congenital neutropenia showed a heterozygous single-nucleotide insertion (p.W44X) in the HAX1 gene but no mutation in ELA2 gene. Conclusion: In the evaluation of response to ERT for Gaucher disease co-morbid disorders with similar symptoms should also be considered. P-671 Sanfilippo disease: how common is the attenuated form?

P-669 Arash-Kaps L1, Reinke J1, Amraoui Y1, Beck M1, Mengel E1 Adult MPS VI patient diagnosed with cardiac manifestation 1

Villa Metabolica, Univ Child Hosp, Mainz, Germany

Canda E1, Kose M1, Kagnici M1, Simsek E2, Ucar SK1, Hasdemir Can2, Coker M1 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2Div Cardiology, Ege Univ, Izmir, Turkey

Mucopolysaccharidosis type VI (MPS VI) is a rare lysosomal, autosomal recessive storage disorder caused by deficient activity of Nacetylgalactosamine-4-sulfatase (ARSB). Twenty four years old girl with MPS type VI diagnosed after the determination of atrioventricular block. She had visited our emergency department because of heart burn and dyspnea. On investigation complete atrioventricular block was found and a pacemaker was implanted. There was second degree consanguinity. She had cousins with the diagnosis of MPS type VI. On her physical examination she had a coarse face, 2/6 systolic murmur, spastic tetraparesis. Dysostosis multiplex, neurogenic electromyography findings, cerebral atrophy, and cervical cord compression were investigated. ARSB level was 0 nmol/h/mg protein. She had enzyme replacement therapy for 1.5 month. She died at home due to cardiopulmonary complications. Cardiac and pulmonary diseases were the most common causes of the mortality and the morbidity in mucopolysaccharidosis. We reported this case to mention the importance of early diagnosis and treatment of the patients with mucopolysaccharidosis.

Background: Sanfilippo diseases (Mucopolysaccharidosis type III AD) are the most common MPS disorders caused by a deficiency in one of several enzymes involved in the degradation of heparan sulfate. The classical phenotype of MPS III is known as developmental delay, especially speech delay in early infancy. Patients: Here we present six patients with Sanfilippo syndrome III A-C. All six patients had mild to moderate development delay in their early infancy. These patients are between 10-29 years of age with the age of diagnosis between 6-14 years. Two oldest patients with MPS III B are siblings. Different from patients with primary development disturbance, these patients present early dementia with secondary loss of skills resulting in aggressivity, sleep and emotional disturbance. Four patients can still walk. Two present severe spasticity and dystonia. All six patients have no apparent signs of MPS such as hepatosplenomegaly or coarse facial features. Conclusion: All of the patients described here presented early mildmoderate development changes without typical facial or skeletal signs of MPS. All are diagnosed several years later than patients with classical phenotype. Patients with developmental and speech delay even without typical coarse facial features should be screened for MPS III. P-672 Niemann Pick type B patient with arthritis: should we think niemann Pick type B disease for the differential diagnosis of rheumatologic disorders

P-670 Gaucher type 3 with severe neutropenia under enzyme replacement therapy

Canda E1, Kagnici M1, Kose M1, Sozeri B2, Alpman A3, Ucar SK1, Onay H3, Ozkinay F3, Coker M1

Ersoy M1, Balci MC1, Demirkol M1, Devecioglu O2, Gokcay G1 Div Nutr Metab, Child Hosp, Ist Med Fac, Istanbul, Turkey; Div Ped Hemato Onc, Ist Med Fac, ISTANBUL, Turkey

Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2 Div Rheumotology, Ege Univ Child Hosp, Izmir, Turkey; 3Div Genetics, Ege Univ, Izmir, Turkey

Background/Objective: Gaucher disease type 3 is a lysosomal storage disorder presenting with hepatosplenomegaly, pancytopenia, bone and neurologic involvement. Severe congenital neutropenia characterized by maturation arrest of granulopoiesis with neutrophil counts below 0.5x10(9)/L is another disorder mostly due to mutations in ELA2 and HAX1 genes. The management of severe congenital neutropenia during

Niemann-Pick disease (NPD) types A and B are autosomal, recessively inherited, lysosomal storage disorders caused by deficient activity of acid sphingomyelinase (acute neuropathic form type A and non-neuropathic form type B). Most clinical findings were splenomegaly, hepatosplenomegaly, lung disease, hyperlipidemia. We reported a case with NPD type B with symptoms and findings of arthritis.

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Fourteen years old boy referred us due to hepatosplenomegaly and his liver biopsy revealed lipid storage disease. Hemogram and routine biochemical analysis were normal. Sphingomyelinase enzyme level was low; 0.1 μmol/g/h. We found homozygote c.482T>C mutation on SMPD1 gene and he had the diagnose of NPD type B. During his follow up the symptoms of arthritis on his both knees and left hip had been recognized. Hip and knee MR investigation showed articular effusion and right medial menniscus degeneration. Acute phase reactants were normal, Otoimmune markers and viral serology were negative. HLA B27 antigen was also negative. He had salazopyrin and methotrexate treatment. We showed complete remission at the 6. months of methotrexate treatment. Skeletal involvement is common and previously unrecognized manifestation of NPD type B. Differential diagnosis for rheumotolojic disorders Niemann Pick disease should be kept in mind. P-673 Nieman Pick Type-C disease with possibly a novel mutation in intron site: a case report Aydogdu SD1, Yıldırım GK1, Yarar C1, Ceylaner S2, Basmak H3, Yakut A1 Osmangazi Uni., Med. Fac. Dept.of Ped, Eskisehir, Turkey; 2Intergen Genetics Centre, Ankara, Turkey; 3Osmangazi Uni., Med. Fac. Dept.of Ophtal, Eskisehir, Turkey

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Aim: Clinical characterization of GD-I patients, diagnosed in pediatric age, under enzyme replacement therapy (ERT). Methods: Observational study with retrospective analysis of patients diagnosed in a tertiary Centre of North of Portugal, between 1986 and 2012. Results: 5 GD-I patients: 3 females/2 males; follow-up period: 26-3 years. Diagnosis age: 22 months-13 years (median 7 years). At the diagnosis: 3 of five patients had mild complains: asthenia (1), epistaxis (1), prolonged fever (1), abdominal distension (1) and limb pain (2). Two had growth delay; all had hepatosplenomegaly, cytopenias and bone involvement. No patient had pulmonary, cardiac or neurological involvement. Diagnosis always confirmed by enzymatic and mutational studies. All patients received ERT. Time of ERT ranged from: 3-20 years. There was an improvement at subjective and objective parameters in all patients with ERT. There were no side effects. One female had two uneventfully pregnancies. Good correlation with PGS3. Discussion: Gaucher disease is one of the few LSD with available ERT. Severity score system will be essential to classify and monitor disease progression with or without therapy. P-675 Follow-up of Niemann Pick Type C patients: Ege university experience

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Background: Niemann Pick Type-C disease(NPC) is a rare progressive neurodegenerative disorder characterized by accumulation of cholesterol and glycosphingolipids in lysosomes. The disease is caused by autosomal recessive transmission of mutations in NPC1 and NPC2 genes. The patients have mutations mostly in NPC1 gene and mostly in exons. Methods: We presented an eighteen months old girl who was admitted to the hospital because of lung infection. She had splenomegaly, generalized hypotonia, increased deep tendon reflexes and mental retardation. Vertical supranuclear gaze paralysis wasn't present. Bone marrow smear showed foamy cells and sphingomyelinase activity wasn't low enough for the diagnosis of Niemann Pick A and B. Result: Genetic analysis for NPC showed homozygous mutation as "IVS9+5G>A" on NPC1 gene occurred at the localization of 9th intron. cDNA study is also going on. Conclusion: Both of the parents are heterozygotes for this mutation. But we didn't detect in any of 500 healthy persons. The first child of the family who died with the suspicion of NP disease. We accepted that this variation is possible pathogenic mutation due to these findings.

Canda E1, Kagnici M1, Kose M1, Ucar SK1, Coker M2 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2Div Genetics, Ege Univ, Izmir, Turkey

Niemann-Pick type C (NP-C) disease is a neurodegenerative lysosomal storage disorder characterized by accumulation of unesterified cholesterol in the lysosomal/late endosomal system. NP-C is characterized by clinical variability including visceral organ involvement, neurological, psychiatric signs. The aim of this study was to evaluate our patients with NP-C. Seven patients have been followed with diagnosis of NP-C. Mean age of the patients at diagnosis was 6.2 ± 11.4 years (1 month – 32 years). Four patients had consanguinity. Five patients had hepatosplenomegaly, two patients had occulomotor apraxia. Six patients had neurological involvement. Abnormal liver function tests with or without cholestasis found five of them. Bone marrow biopsy and liver biopsy revealed lipid storage. Cranial MR findings of the patients were; cerebral atrophy, hypomyelinisation and thin corpus callosum. Diagnosis was made by mutation analysis in two patients and fibroblast culture – Filipin staining in 5 patients. Two patients had miglustat therapy. Niemann Pick Type C disease was kept in mind when the presence of visceral involvement and progressive neuropathy. Early miglustat treatment has an effect on stabilization of the disease and improves neurological signs. We reported our cases to mention the importance of early diagnosis and treatment of the patients with NP-C

P-674 P-676 Type I gaucher disease in pediatric population clinical characterization and correlation with pediatric gaucher severity scoring system (PGS3) Leão-Teles E1, Rodrigues E1, Campos T1, Vieira A2, Chaves P3, Cardoso T3, Lacerda L4, Sá-Miranda C5 Unidade Doenças Metabólicas, CHSJ, Porto, Portugal; 2Serv Radiologia, CHSJ/ FMUP, Porto, Portugal; 3U Doenças Metabólicas/Dep Medicina, CHSJ, Porto, Portugal; 4Centro Genética Médica Jacinto Magalhães, Porto, Portugal; 5Unilipe, IBMC, Universidade do Porto, Porto, Portugal

Altered expression of MMP-2 and MMP-9 in the serum of patients with lysosomal storage disorders Batzios SP 1 , Zafeiriou DI 1 , Vargiami E 1 , Karakiulakis G 2 , Papakonstantinou E2

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Introduction: Type I Gaucher disease (GD-I) is the most prevalent lysosomal storage disorder. Patients usually present multysistemic involvement with high morbidity even in young ages.

1 1st Paediatric Department, AUTH, Thessaloniki, Greece; 2 2nd Department of Pharmacology, AUTH, Thessaloniki, Greece

Background and Objectives: Lysosomal storage disorders (LSDs) represent a heterogeneous group of hereditary diseases presenting with numerous clinical features. The aim of this study is to elucidate the expression and activity of gelatinases (MMP-2, MMP-9) and the

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expression of their tissue inhibitors (TIMP-1, TIMP-2) in the serum of pediatric patients with LSDs. Patients and Methods: Fifteen patients formed the study group (mean age±SD: 10.3±6.4 years). Serum gelatinase activity was assessed by gelatin zymography and the concentration of circulating MMP-2, MMP-9, and of their tissue inhibitors TIMP-1 and TIMP-2 was measured by ELISA. Results: Serum activity and protein levels of MMP-9 were significantly reduced whereas of MMP-2 were significantly increased in patients with Mucopolysaccharidoses, as compared to controls. The differences were even more pronounced in Sanfilippo patients. Differential expression of gelatinases was found in patients with Krabbe disease, Niemann-Pick C disease, as well as in 2 patients with Pompe disease where an alteration in the expression of studied molecules was noticed after the initiation of enzyme replacement therapy. Conclusions: The reported alterations in the expression of gelatinases suggest that these molecules may be used as potential biomarkers for the diagnosis, follow-up and response to therapy in this group of disorders. P-677 Follow-up of Niemann Pick type B patients: ege university experience Canda E1, Kose M1, Kagnici M1, Aykut A2, Ucar SK1, Onay H2, Ozkinay F2, Coker M1 1 Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey; 2Div Genetics, Ege Univ, Izmir, Turkey

Niemann-Pick disease type B (NP-B) is a lysosomal storage disorder caused by the deficiency of acid sphingomyelinase activity. NP-B is characterized by a clinical variability including visceral organ involvement. The aim of this study was to evaluate our patients with NP-B. Nine patients has been followed with the diagnosis of NP-B. Mean age of the patients at diagnosis was 3.6 ± 3.02 years (4 month - 9 years). All patients had hepatosplenomegaly. We observed elevation of liver function tests in 5 patients. Anemia was observed in four patients, both anemia and thrombocytopenia was observed in 1 patient. Lipid storage was observed in liver biopsies of three patients and bone marrow biopsies of 6 patients. Sphyngomyelinasis enzyme levels were low in all patients (mean 2,14 ± 2,17 nmol/hour/mg protein). We observed clinical and radiological lung involvement in 5 patients. Osteoporosis was observed in 2 patients. One patient had pulmonary hypertension, one patient had 1st degree aortic insufficiency. Atypically, we observed joint involvement in one patient. In one patient, we observed homozygote M694V mutation on MEFV gene. NP-B disease has high morbidity and mortality especially in childhood. We reported our cases to discuss the clinical and laboratory findings of our patient. P-678 Mucopolysaccharidosis VI: long term outcome post BMT (case report) Oates S1, Ketteridge D1, Bratkovic D1, Martin AJ2, Fletcher JM1 SA Pathology (WCH), North Adelaide, Australia; 2Women's and Children's Hosp, Adelaide, Australia 1

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reduction surgery at age 16, and Lefort 1 osteotomy procedure aged 22, respiratory function improvements have been preserved. Serial echocardiograms show long-term stable cardiac disease, and functional improvements in mobility have been sustained. Corneal clouding progressed bilaterally, and left corneal transplant was performed in 2010 Cervical canal stenosis required decompression 1yr post BMT aged 13. Cervical MRI at age 26 shows mild narrowing and physical exam shows early long track signs in the lower limbs. Conclusion: Respiratory function shows improvement post bone marrow transplant in this patient. However, there appears to be slow recurrence of cervical cord compression, and corneal clouding has progressed P-679 Coexistence of Gaucher disease and severe congenital neutropenia in a Turkish patient Kose M1, Kagnıcı M2, Canda E2, Karadaş N3, Okur V4, Alpman A5, Kalkan Uçar S2, Onay H5, Yıldırım Sözmen E6, Karapınar D7, Özkınay F5, Çoker M2 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 3Div Hemat Dis,Ege Univ Child Hosp, İzmir, Turkey; 4Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 5Div Genetic,Ege Univ Med Fac, İzmir, Turkey; 6Div Clinic Chem,Ege Univ Med Fac, İzmir, Turkey; 7Div Hemat Dis,Ege Univ Child Hosp, İzmir, Turkey 1

Gaucher disease (GD) is a lysosomal storage disorder, in which a deficiency of the enzyme glucocerebrosidase. Severe congenital neutropenia (SCN) is a very rare disease which is characterized by arrest of neutrophil maturation at the promyelocyte stage and recurrent fatal infections. In this paper; we report a patient with Gaucher Type III and SCN The patient; 4 months of age; was referred to us because of anemia and neutropenia. The family pedigree revealed that his parents were first degree cousins and had four sibling exitus history with recurrent fatal infections.Initial laboratory revealed severe neutropenia and anemia.Beta glicosidase enzyme level was low.We detected L444P homozygous substitution on GBA gene. Patient was diagnosed with GD.Enzyme replacement therapy (ERT) was initiated. On follow-up severe neutropenia persisted.Bone marrow findings showed maturation arrest of myelopoiesis. We found a homozygous W44X mutation on HAX1 gene and he was diagnosed as Kostmann Syndrome. Best of our knowledge; this is the only report of the combination of GD and Kostmann Syndrome .Through our follow up;he had no febril episode.In comparison with other exitus siblings with Gaucher and probably SCN , the clinical course of the case was much less severe, speculating that ERT had a modifying effect. P-680 Musculoskeletal disease in MPS VI patients: an MRI study Barone R1, Andria G2, Fiumara A3, Palmucci S4, Parini R4, Scarpa M5, Sorge G3 Child Neurol, Univ Child Hosp, Catania, Italy; 2Univ Child Hosp, Napoli, Italy; 3Div Metab Dis, Univ Child Hosp, Catania, Italy; 4 Radiodiagnostic Unit, Univ Hosp, Catania, Italy; 5Div Metab Dis Univ Child Hosp, Padova, Italy 1

Introduction: There is limited data available on long term outcomes of Bone Marrow Transplant (BMT) in MPS VI. Aim: To present serial data over 15 years on a severely affected MPS VI patient following BMT at age 12. Case: MPS VI male diagnosed at aged 3. Pre transplant MPS VI related interventions included VP shunt aged 5, carpal tunnel surgery aged 8 and CPAP commenced from age 10. Post BMT respiratory function showed improvement at 4 years post transplant . In conjunction with a change from CPAP to BiPAP, tongue

Background Mucopolysaccharidosis VI, (ASB deficiency) is a multisystem disorder with a constant musculoskeletal involvement including joint restriction and impaired endurance. The impact of enzyme replacement therapy (ERT) on joint disease in MPS VI has been documented by improving of functional parameters. Unlike X-ray, Magnetic Resonance

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(MR) affords an opportunity to visualize articular cartilage, subarticular bone, ligaments and synovium (synovitis/effusion). Methods This multicenter national study was aimed to evaluate musculoskeletal disease in eight Italian MPS VI patients (2 males and 6 females, aged 4 to 20 years). At the time of the study patients underwent ERT for 20month to 6-year periods. MR imaging of wrist, metacarpophalangeal jointMCP and knee were performed by 1.5T scanners using FSE and gradient echo sequences, according to an ad-hoc protocol and scoring system. Results Musculoskeletal MR findings in MPS VI patients include epiphyseal and metaphyseal remodelling, bone marrow edema and bone erosions and osteonecrosis of the subcondral bone without thickening and/or gadolinium enhancement of the synovial membrane. Subcondral bone MR changes were not obvious in the younger patient after 24- and 36-month ERT period. Conclusions MR evaluation might be considered for the assessment of musculoskeletal disease at baseline and upon ERT in MPS VI. P-681 Clinical effectiveness of idursulfase in patients with Hunter syndrome: 5-year data from the Hunter Outcome Survey (HOS) Muenzer J1, Giugliani R2, Tanaka A3, Tylki-Szymańska A4, Morin I5 and Beck M6: The HOS Global Executive Committee on behalf of the HOS Investigators 1

Department of Pediatrics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 2Medical Genetics Service/HCPA, Department of Genetics/UFRGS and INAGEMP, Porto Alegre, Brazil; 3Department of Pediatrics, Osaka City University Graduate School of Medicine, Abeno-ku, Osaka, Japan; 4Department of Metabolic Diseases, Children’s Memorial Health Institute, Warsaw, Poland; 5Shire Human Genetic Therapies, Eysins, Switzerland; 6Department of Pediatrics, University Medical Center, Johannes Gutenberg University, Mainz, Germany Background: The Hunter Outcome Survey (HOS), a global, observational registry sponsored by Shire HGT, was established in 2005; longterm data are now available. Methods: To investigate the clinical effectiveness of idursulfase (Elaprase®, Shire HGT), clinical measures recorded in HOS at annual time points over 5 years were compared with baseline. As of January 2013, 499/747 patients followed prospectively in HOS had received ≥1 idursulfase infusion; those who had received a bone marrow transplant or were enrolled in the TKT024 clinical trial were excluded. Median age at first treatment was 7.3 years; median treatment duration was 38.7 months. Results: Urinary glycosaminoglycan levels, 6-minute walk test results, left ventricular mass index, pulmonary function, and liver and spleen sizes were improved after 5 years of treatment compared with baseline. However, the number of patients with baseline and post-treatment data available for any given parameter was low, particularly at 5 years (year 1, n=20–111; year 5, n=1–15). Conclusions: These results suggest that clinical effectiveness of idursulfase is maintained over 5 years. Nonetheless, future assessment of long-term disease progression may be aided by a disease severity scoring tool. The ongoing HOS Data Completeness Initiative will be crucial in maximizing the registry’s long-term potential. P-682 Molecular analysis of 15 MPS VI patients from south Turkey and identification of one novel mutation 1

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Önenli-Mungan N , Zanetti A , Kör D , Özbek MN , Lenzini E , Scarpa M2, Tomanin R2 Çukurova University, Adana, Turkey; 2University of Padova, Padova, Italy; 3Diyarbakır Children's Hospital, Diyarbakır, Turkey 1

Background: Mucopolysaccharidosis type VI is an autosomal recessive disorder caused by the deficit of arylsulfatase-B (ARSB) and characterized by growth retardation, dysostosis multiplex, coarse face, stiff joints, cardiac complications, respiratory difficulties, hepatosplenomegaly, hernias, corneal clouding, hearing loss, and hydrocefalus. More than 140 mutations were reported in the ARSB locus. Results: In this study we analyzed 11 unrelated families in which parents are all first degree cousins; we found 8 different mutations, one of which novel: p.H178D. The p.L321P is the most frequently detected mutation. All patients except one were homozygotes. Conclusions: This study evidenced a very high degree of consanguinity in the population examined. A genotype-phenotype correlation was not so unambiguous. Clinical evaluation of the 8 subjects homozygote for the p.L321P mutation showed a continuum of phenotypes from slowly to the rapidly progressing form. Also for c.1036delG mutation, we observed both a mild and a severe phenotype. These findings hypothesize the existence of other genetic or epigenetic factors important for the determination of the clinical phenotype. The number of homozygotes reported in this study reflects the high degree of consanguinity. Carrier identification accompanied with genetic counselling in families at risk may be a solution to minimize the effects of consanguinity. P-683 Idursulfase treatment in six female patients with Hunter syndrome: data from the Hunter Outcome Survey (HOS) Scarpa M1, Morin I2, Giugliani R3, Tylki-Szymańska A4 on behalf of the HOS Investigators 1 Department of Pediatrics, University of Padua, Padua, Italy; 2Shire Human Genetic Therapies, Eysins, Switzerland; 3Medical Genetics Service/HCPA, Department of Genetics/UFRGS and INAGEMP, Porto Alegre, Brazil; 4Department of Metabolic Diseases, Children’s Memorial Health Institute, Warsaw, Poland

Background: Hunter syndrome is rare in females. Little is known about clinical manifestations or the use of idursulfase (Elaprase®) in female patients. Methods: Data in the Hunter Outcome Survey (HOS) were used to investigate disease characteristics and the efficacy and tolerability of idursulfase in females. HOS is a global, observational registry of patients with Hunter syndrome, sponsored by Shire HGT. Results: As of January 2013, 11/896 patients enrolled in HOS were female. Of the nine female patients for whom information was available, neurological, pulmonary and cardiovascular manifestations were present in seven, six, and five individuals, respectively. Eight of the 11 female patients in HOS were followed prospectively; six had received intravenous idursulfase infusions. Eleven non-serious adverse events (AEs) were reported, all in one patient. All AEs were mild in severity and considered not to be infusion-related. In the three individuals for whom more than one urinary glycosaminoglycan (uGAG) measurement was available, uGAGs were elevated initially but dropped to within the normal range following idursulfase treatment. Conclusions: Analysis of data from six females in HOS showed that idursulfase infusions were generally well tolerated. Further analysis in more patients is required to define the best management approaches in females with Hunter syndrome. P-684 Krabbe disease novel mutation with interfamilial variability Shuaib T1 1

Pediatric Dep, King Abdulaziz University, Jeddah, Saudi Arabia

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Background: Krabbe disease is autosomal recessive disorder. It's due to deficiency of galactocerebrosidase enzyme. The infantile type is characterized by progressive neurological deterioration and death before 2years. We are reporting interfamilial variability in two families with similar genotype. Method: 2 families with 2 affected patient's suspected Krabbe disease were included. All had routine metabolic screen, and galactocerebrosidase enzyme assay followed by molecular confirmation. Brain MRI was done. Results: Patient 1, 18m old had early developmental delay, spasticity, and irritability. Had non specific brain MRI changes. He had progressive course with death at 18m. Patient 2, 15m old had mild early developmental delay with regression at 6m. His neurological condition continued to deteriorate. MRI brain was normal. Galactocerebrosidase was low in both patients and in the same range. They had identical novel homozygouse changes in GLAC gene. Conclusion: Krabbe disease is a progressive neurodegenerative disorder. Variable phenotypes, with poor genotype phenotype correlation is known. We are reporting two patients from different families sharing the same enzymatic level and genotype but variable phenotype. That suggests that interfamilial variability also exist. Non genetic factors might contribute to this variation which needs further studies with larger sample size. P-685 Cholesteryl ester storage disease in Greece: longterm follow - up of 24 patients

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Pediatr Neurology, Hosp Vall d'Hebron, Barcelona, Spain; 2Pediatr Orthopedics, Hosp Vall d'Hebron, Barcelona, Spain; 3Neurophysiology, Hosp Vall d'Hebron, Barcelona, Spain; 4Pediatr Haematology, Hosp Vall d'Hebron, Barcelona, Spain; 5CIBBIM Nanomed, Hosp Vall d'Hebron, Barcelona, Spain Background: Carpal tunnel syndrome (CTS) is frequent in patients with mucopolysaccharidosis (MPS) due to glycosaminglycans deposit in connective tissue. It is considered the most common cause of CTS in children. Clinical diagnosis can be difficult when neurological impairment is present. We describe our experience with CTS in MPS patients at our hospital. Patients and method: Retrospective study of 26 patients, (10 MPSI, 9 MPSII, 1 MPSIII, 4 MPSIV, 1MPSVI, 1 MPSVII), followed-up in the last 10 years. Results: 9 patients were screened with electromyography (EMG) and all diagnosed with bilateral severe CTS, showing more involvement in dominant hand. Medium age at diagnosis was 9.9 years (4 to 15 years). MPS distribution was: 6 MPSI, 2 MPSII, 1 MPSVI. All patients but one received therapy, either ERT or BMT. All patients showed claw hands with motor deficit and 44% (4/9) paresthesis. In all cases surgical proceedings consisted in central carpal ligament section and median nerve neurolisis. All patients showed clinical and neurophysiological improvement. Conclusion: Screening for TCS with EMG should be included in protocols of MPS patients specially in young ones or severely affected because of the difficulty in identifying clinical symptoms. Early surgical intervention results in clinical and functional improvement. P-687

Drogari E1, Mollaki V1, Skouma A1, Zellou A1, Kyrkou E2, Manolaki N3, Stamatelopoulos K2 1

Un.Metab.Dis.,1st Dept.Univ.Child.Hosp., Athens, Greece; Lab.Vasc.,Dept.Univ.Intern.Med.,Gen.Hosp, Athens, Greece; 32nd Dept. NHS, Child. Hosp., Athens, Greece 2

Background: CESD is the milder form (adult type) of a monogenic Metabolic Disorder due to the Lysosomal Hepatic Acid Lipase deficiency. This causes accumulation of Cholesteryl Esters mainly in the liver. The patients can suffer from liver cirrhosis and early atherosclerosis. Material:24 patients with CESD (22 families) are described. 76% of them visited the Lipid Clinic at age 3-16 years (4-38 years today) because of high cholesterol and triglyceride levels together with liver disease. The rest were referred because of undiagnosed "hepatitis". Methods: All patients were studied clinically and biochemically. The enzyme deficiency was demonstrated in liver tissue (100%), in lymphocytes and fibroblasts (62%). Five mutations were found although the patients originate from different areas. All members of each family were studied for three continuous generations. Results: Cholesterol ranged between 280-440 mg/dl (homozygotes) and 198-350 mg/dl (heterozygotes). Triglycerides ranged between 432-1100 mg/dl (homozygotes) and 102-280 mg/dl (heterozygotes). HDL-C levels ranged between 21-34 mg/dl (homozygotes) and 32-48 mg/dl (heterozygotes). All patients had fatty liver with mild/moderate cirrhosis. A high frequency of early atherosclerotic disease was found in adult heterozygotes. Conclusion: CESD is a potential dangerous disease. Although hypolipidaemic drugs and special diet are very useful,more radical treatment (ERT) should be considered.

Clinical survey of large cohort of patients with Fabry disease followed at Hospital de Clinicas de Porto Alegre, Brazil Netto CBO1, Dornelles T1, Rocha AC1, Vairo F1, Giugliani R2 Medical Genetic Service-HCPA, Porto Alegre, Brazil; 2Dept Genetics IB- UFRGS, Poto Alegre, Brazil 1

Fabry disease (FD) is an X-linked inherited disorder which results from the build-up of globotriaosylceramide in the lysosomes of endotelial cells of various organs, leading to a multisystemic disorder. It is caused by mutations in the GLA gene which encodes the enzyme alphagalactosidase A, which is deficient in FD. We report here the information obtained from the study of a large cohort (49 patients) of FD patients (26 men and 23 women) followed at the Medical Genetics Service, Hospital de Clinicas, Porto Alegre, Brazil. A large proportion (63.5 %) had impaired renal function, approximately half of the patients (51%) present some cardiac abnormality and about 10% of patients had cerebrovascular events. The average age at onset of symptoms was 15 years and the average age at diagnosis was 32 years. Family history was positive in 87.5% of the cases and half of the patients are on ERT. As FD has specific treatments available, diagnosis allows the introduction of therapies that may prevent or slowdown the damage that usually comes along the time. We emphasize the importance of multidisciplinary care, regular clinical monitoring and genetic counseling for patients with FD and their families. P-688 Cognitive performance in children with Mucopolysaccharidosis II (MPSII, hunter syndrome) receiving investigational intrathecal (IT) enzyme replacement therapy

P-686 Carpal tunnel syndrome in young patients with mucopolysaccharidosis

Muenzer J1, Stein M1, Kearney S2, Fan Z1, Wegner L1, Horton J2, Hendriksz CJ2, Vijay S2, Wang N3, Barbier AJ3 1

Del Toro M1, Ortiz S1, Garcia-Fontecha CG2, Gratacos M3, Elorza I4, Domínguez C5, Aguirre M2

Univ of North Carolina at Chapel Hill, Chapel Hill, NC, United States, Birmingham Child Hosp NHS Found Trust, Birmingham, United Kingdom, 3Shire Human Genetic Therapies, Lexington, MA, United States

2

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Background: There is no effective treatment for cognitive impairment in MPSII. Methods: A phase 1/2 study (HGT-HIT-045) examined the safety of recombinant iduronate-2-sulfatase formulated for IT administration (idursulfase-IT) and the cognitive performance of treated children. Sixteen cognitively impaired MPSII children previously treated with intravenous idursulfase received no treatment or 10 mg, 30 mg, or 1 mg idursulfase-IT monthly for 6 consecutive months via IT drug delivery device or lumbar puncture. Patients were rolled into an extension study (HGT-HIT-046); the longest-treated patient has received 10 mg monthly for 37 months. Results: No serious adverse events related to idursulfase-IT were observed. Most patients had advanced neurodegeneration at baseline, preventing cognitive/functional assessments. Longitudinal General Conceptual Ability scores calculated using the Differential Abilities Test 2nd version were obtained in five patients with 6–24 months follow up. Four, who received the 10- or 30-mg dose, showed stable/improved scores from baseline. One, who received 1-mg dosing, had a decline in function and switched to 10-mg dosing in the extension. Assessments of behavior by the Scales of Independent Behavior-Revised correlated with the cognitive evaluations. Conclusions: Encouraging signs of clinical efficacy were observed, warranting further development of idursulfase-IT as a potential treatment for the severe form of MPSII. Conflict of Interest declared. P-689 HGT-REP-059 treatment protocol: effect and tolerability of openlabel agalsidase alfa in patients with Fabry disease Goker-Alpan O1, Nedd K2, Shankar SP3, Lien YH4, Barshop BA5, Holida M6, Hillman R7, Mardach R8, Rever B9, Forte R10, Desai A11, Wijatyk A12, Chang P12, Martin R12 1

Lysosomal Research & Treatment Unit, Fairfax, United States; Infusion Associates, Grand Rapids, United States; 3 Emory University, Atlanta, United States; 4AKDHC, Tucson, United States; 5 UCSD, San Diego, United States; 6University of Iowa Health Care, Iowa City, United States; 7University of Missouri, Columbia, United States; 8Kaiser Permanente, Los Angeles, United States; 9Central Coast Nephrology, Salinas, United States; 10North Shore University Hospital, Manhasset, United States; 11Stuart Oncology Associates, Stuart, United States; 12Shire Human Genetic Therapies, Lexington, United States

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P-690 Epigenetic GLA regulation in Fabry disease Gervas-Arruga J1, Cebolla JJ2, Giraldo P3, Pocovi M2 Tras Res Unit, Miguel Servet Univ Hosp, Zaragoza, Spain; 2Biochem Mol Cell Dept, Univ Zar, Zaragoza, Spain; 3CIBERER, Zaragoza, Spain 1

Fabry disease (FD), α-galactosidase A deficiency, is an X-linked lysosomal disorder. Heterozygous females can either be asymptomatic or develop the classic phenotype due to random Xchromosomal inactivation. DNA methylation is an important mechanism of X-chromosome inactivation. The aim of this study was to identify the methylation patterns and genotyping of the maternally and paternally derived X-chromosomes inactivation in heterozygous GLA mutated females to perform a correct diagnosis and subsequent genetic counselling. We studied one family, carrier of a point mutation p.G183V. Methylation promoter profile was analysed by pyrosequencing and X-chromosome inactivation was analysed in peripheral blood mononuclear cells and in skin fibroblasts of 2 female carriers by using short tandem repeat fluorescence PCR combined with capillary electrophoresis. Our results showed that methylation profile for female carriers and female patient versus female control was significantly different (p<0.05) in a promoter CpG island region. The skin fibroblast methylation profile was related to the enzyme activity pattern. The enzymatic activity was higher with the increase in the activation of non-mutated chromosome. In conclusion, epigenetic mechanisms could be influenced in the activation on mutated allele. These findings support that the methylation patterns may play a role in the FD phenotype depending on X-chromosome activation P-691

2

Salivary glucose tetrasaccharide (GLC4) as a potential biomarker in infantile pompe disease Prunty H1, Broomfield A2, Vellodi A2, Cleary M2, Harvey K1, Burke D1, Lukovic B1, Heales S1 1

Chemical Pathology, Gt Ormond St Hosp, London, United Kingdom; Metabolic Medicine, Gt Ormond St Hosp, London, United Kingdom

2

Background: The clinical effects and tolerability of agalsidase alfa (agalα) were evaluated in patients with Fabry disease (FD) who were switched from agalsidase beta (switch) or treatment-naïve in a treatment protocol setting. Methods: A multicenter, open-label, treatment protocol (HGT-REP059; NCT01031173; 0.2mg/kg agalα every other week for 24mos) enrolled switch and treatment-naïve patients with FD. Data were collected on change from baseline (CFB) estimated glomerular filtration rate (eGFR), left ventricular mass index (LVMI), midwall fractional shortening (MFS), and adverse events (AEs). Results: 71 switch (median [range] age: 46.6y [5-84]; male:female 40:31) and 29 treatment-naïve (median [range] age 38.7y [12-74]; male:female 14:15) patients were evaluated. No significant CFB in 24 mo were found in either group in LVMI or MFS. Mean±SE annualized eGFR rates of change were -1.92±1.10 (switch) and -1.68±1.98 (naïve). AEs were consistent with the known safety profile of agalα in these patients. Four discontinuations due to AEs (2 switch/2 naïve) and two non-drug-related deaths occurred (1 switch/1 naïve). Conclusions: 24 months of agalα treatment in a heterogeneous population of switch and naïve patients resulted in stable LVMI and MFS; eGFR declined at a mean annual rate of -1.92±1.10 (switch) and -1.68±1.98 (naïve). No new safety concerns emerged. Conflict of Interest declared.

Background: Urinary Glc4 has been used as a biomarker in infantile Pompe disease as it is thought to reflect the degree of glycogen storage. However, we have occasionally observed unexpectedly low results which cannot be readily explained by the clinical picture; despite stability studies indicating that urinary Glc4 should be stable for at least 7 days at room temperature. These results may reflect degradation due to bacterial action or excessive urinary enzyme accumulation secondary to enzyme replacement therapy. Aims: An alternative sample medium is to be explored. Glc4 analysis in plasma and bloodspots has proved unsuccessful using the published HPLC-ECD method due to interfering peaks. Saliva presents an attractive alternative, being non-invasive, easy to collect, and requiring minimal preparation. At 666.75Da, Glc4 is small enough to enter the saliva by ultrafiltration between the acinar cells and could therefore be a potential candidate for salivary analysis. Results: Preliminary studies have shown no detectable Glc4 and no interfering peaks in control subjects (n=5). Glc4 was detectable in spiked control saliva down to a concentration of 5μmol/L. Conclusions: We have confirmed that salivary analysis of Glc4 is practically possible and are looking to assess its efficacy as a potential biomarker in Pompe patients.

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P-692 Bicyclic derivatives of L-idonojirimycin as pharmacological chaperones for neuronopathic forms of Gaucher disease

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Conclusions: These results highlight the importance of screening children with MPS for OSA using polysomnography, irrespective of MPS type and previous complaints of snoring. Financial support: IGEIM. P-694

Alfonso P1, Andreu V2, Pino-Angeles A3, Moya-García AA3, GarcíaMoreno MI MI4, Sánchez-Jiménez F1, Pocoví M5, Ortiz-Mellet C4, García-Fernández JM6, Giraldo P1 1 CIBERER, Valencia, Spain; 2UIT, HUMS, Zaragoza, Spain; 3D. of Molecular Biology and Biochemistry, Malaga, Spain; 4Department of Organic Chemistry, Sevilla, Spain; 5IACS, Zaragoza, Spain; 6Institute for Chemical Research (IIQ), Sevilla, Spain

Background: Gaucher disease (GD) is caused by mutations in the gene encoding for lysosomal glucocerebrosidase (GCase). The mutant enzymes exhibit impaired cellular trafficking as a consequence of aberrant folding. Current investigational therapeutic strategies including the development of ligands capable of promoting those conformational changes that are required for efficient folding and restoring trafficking, they are called "pharmacological chaperones". However, most of the pharmacological chaperones under study are iminosugars that behave as broad spectrum inhibitors and are not active for glucocerebrosidase mutations located outside the domain containing the active site and are associated with neurological involvement. Objective: To develop molecules with high binding specificity towards GCase, high ratio of chaperone versus inhibitor activity and capable of producing an increase in the enzymatic levels. Methods: The chaperone potentials of three bicyclic derivatives of Lidonojirimycin were evaluated in vitro using stable transfectants of glucocerebrosidase mutants (N370S and L444P) in COS-7 cells and skin fibroblasts of homozygous GD patients for these above mentioned mutations. Results: Results showed significant increases in GCase activity for COS-7 mutants and slight increases for the L444P homozygous GD fibroblasts at various chaperone concentrations. Conclusion: The use of bicyclic L-idonojirimycin-based compounds could be considered as a therapeutic alternative for GD.

Design of an analysis of long-term effectiveness of agalsidase alfa in the Fabry outcome survey Larroque S1, Gerin R1, Wijatyk A2 1 Shire Human Genetic Therapies, Eysins, Switzerland; 2Shire Human Genetic Therapies, Lexington, United States

Background: The Fabry Outcome Survey (FOS), a Shire-sponsored international database, collects information on patients with Fabry disease (FD) who are untreated, or treated with agalsidase alfa. Methods: FOS data (extracted November 2012) from treated patients will be evaluated for morbidity (age at or time from treatment start to first onset of clinical composite events [renal, cardiac, cerebrovascular, death]) and mortality (age at death). Renal function will be assessed by estimated glomerular filtration rate (eGFR). Progression of cardiac disease will be evaluated using left ventricular mass indexed to height to the 2.7 power (LVMI). Results: Treated FOS patients will be subdivided into an evaluable cohort and further subdivided into renal, cardiac, and overall morbidity cohorts. Planned analyses include Kaplan-Meier survival for morbidity and mortality, average slopes from mixed models of available data for eGFR and echocardiographic LVMI, and supportive sensitivity analyses to confirm robustness of data. This will be compared with published data from FD natural history and placebo control group data. Conclusions: The methodology will include the evaluable FOS sample with frequent measurements over a long follow-up period. Conflict of Interest declared. P-695

P-693

Highly sensitive and specific clinical diagnostics of lysosomal storage diseases using multiple reaction monitoring mass spectrometry, fluorimetry, and affinity-proteomics

Prevalence of obstructive sleep apnea in children with Mucopolysaccharidosis in Sao Paulo, Brazil

Przybylski M1, Cozma C1, Lindner K1, Pierson N2, Iurascu M1, Ion L1, Petre A1, Gross M3, Maeser S4, Clemmer D2

Moreira GA1, Kyosen SO2, Martins AM2

1

1

Instituto do Sono, Sao Paulo, Brazil; 2CREIM, Universidade Federal de Sao Paulo, Sao Paulo, Brazil

Dept. Chemistry, University of Konstanz, Konstanz, Germany; 2Dept. Chemistry, Indiana University, Bloomington, United States; 3Dept. Chemistry, Washington University, St.Louis, United States; 4Centogene AG, Rostock, Rostock, Germany

Background: Prevalence of respiratory problems is still unclear in with mucopolysaccharidosis (MPS) patients, and studies have reported the importance of screening MPS patients for obstructive sleep apnea (OSA). We aimed to verify the OSA prevalence in MPS patients, based on the first polysomnography. Methods: Retrospective study of data from de medical files of MPS patients at the CREIM. Results: Data from 46 patients (17 MPSI, 16 MPSII, and 13 MPSVI), 12F/34M, mean age 4.6 yr (range: 2.7–8.3), median age (months) of disease onset 12 for MPSI, and 18 for MPS II and VI. Diagnosis was established 58.4, 44, and 57 months after the initial manifestation for MPS I, II and VI, respectively. The patients had underwent surgeries (mainly adenoidectomy and/or tonsillectomy) before the diagnosis, 23% of MPSI and 50 and 53.8% of MPSII and VI. Parents reported snoring in 35.3, 31.3 and 30.8% of patients with MPS I, II and VI respectively; however the OSA was prevalent in 76.5, 56.3 and 53.8% in patients with MPS I, II and VI.

Background: Lysosomal storage disorders (LSDs) are a group of approximately 60-70 "rare" metabolic disorders caused by deficient metabolic activitiy of single lysosomal enzymes, or by a defect lysosomal protein. LSDs resulting from inherited genetic defects or biochemical defects are manifesting through a spectrum of life-threatening clinical symptoms including bone, muscle and organ enlargement differing by disease, age of onset and severity. For some LSDs, enzyme replacement therapies (ERT) have been successfully developed; however, ERT is critical to start as soon as possible, thus rendering rapid and specific diagnosis of key importance. Methods and Results: Here we describe highly specific and sensitive diagnostics on dry blood spots (DBS) for (i), molecular determinations of LSDs, particularly muco-polysaccaridoses and muco-lipidoses, by simultaneous fluorimetric and mass spectrometric analysis using identical substrates and standards; (ii), clinical diagnostics of LSDs by multiplex- MSMRM analysis; (iii), the clinical validation of the new methods in patients with established LSDs. Moreover, most recent methods enable the direct

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determination of LSD proteins in vivo by affinity- proteomics and -mass spectrometry, using epitope- specific antibodies. Conclusions: These methods enable a substantial extension and improvement of current diagnostics, for a spectrum of LSDs not hitherto amenable to clinical diagnostics.

variation during treatment were observed in relation with genotype. Therefore, ChT activity in naïve patients depends on the presence/absence of c.1049_1072dup24 and p.G102S polymorphisms in CHIT1. However, the percentage of ChT decrease after a year on ERT is independent of the presence of any of these polymorphisms.

P-696

P-698

Effectiveness of miglustat in the treatment of a patient with Tangier disease, description of a clinical case

Utility of chitotriosidase and CCL18/parc as biomarkers in clinically suspected and confirmed Niemann Pick disease type C

Sechi A1, Dardis A1, Zampieri S1, Calandra S2, Platt F3, Bembi B1

De Castro-Orós I1, Irún P2, Mallén M3, Pueyo MJ1, Cebolla JJ4, Giraldo P5, Pocoví M6

1 1Regional Centre Rare Dis, Univ Hosp, Udine, Italy; 22Dep BioSci, Univ Modena-Reggio Emilia, Modena, Italy; 33Dep Pharm, Univ Oxford, Oxford, United Kingdom

A female patient aged 59 years presenting with pancytopenia, splenomegaly, dysartria, dysphagia, ataxia, neurodermatitis and leg lymphedema was misdiagnosed as Niemann Pick Type C (NPC) and put on substrate reduction therapy (SRT) with miglustat. After 6 months of treatment with 300 mg/day of miglustat the patient showed significant clinical improvement with resolution of the neurological symptoms and reduction of skin lesions and lymphedema. However, the patient was later correctly diagnosed with Tangier disease due to an homozygous deletion of exons 32-34 of the ABCA1gene, which results in abnormal sterol levels and HDL formation. Therefore, miglustat treatment was stopped. Two month after miglustat discontinuation skin lesions, lymphedema and general conditions worsened. Electron microscopic analysis of the skin lesions showed accumulation of lipids in the Schwann cells. Considering the patient's clinical improvement on SRT, we hypothesized that glycosphingolipids may accumulate in Tangier cells. Therefore, the intracellular accumulation of GM2 ganglioside was investigated in Tangier fibroblasts in culture by immunofluorescence. Preliminary data showed an accumulation of this ganglioside within the lysosomal compartment. Conclusions: Our data suggest that SRT with miglustat may be an effective therapy for neurological manifestations of Tangier disease. Conflict of Interest declared.

1

IIS Aragón & Bioquím. Univ de Zaragoza, Zaragoza, Spain; CIBERER; Bioquím. Univ. de Zaragoza, Zaragoza, Spain; 3Bioquímic. Univ de Zaragoza, Zaragoza, Spain; 4FEETEG, Zaragoza, Spain; 5 CIBERER; Hematol, Uni Hosp Miguel Servet, Zaragoza, Spain; 6 CIBERER; FEETEG; Bioq. Univ. de Zaragoza, Zaragoza, Spain 2

Influence of DUP24 and G102S polymorphisms on CHIT1 gene in chitotriosidase activity at diagnosis and its change during enzymatic replacement therapy in Gaucher disease

Background: Niemann Pick type C (NP-C) is a lysosomal storage disease with heterogeneous signs and symptoms. The diagnosis remains difficult both clinically and biologically. Objectives: To assess the utility of two surrogated biomarkers: chitotriosidase activity (ChT) and CCL18/PARC concentration as indicators of NP-C, being the diagnosis confirmed by NPC1 and NPC2 genes sequencing. Material and Methods: Plasma ChT activity and CCL18/PARC concentration were determined in 239 Spanish subjects with clinical suspicion of NP-C. When those levels were increased; NPC1 and NPC2 genes (promoter, exons and exon-intron boundaries) were sequenced. The NP-C Suspicion Index was calculated by ww.npc-si.com. Results: Twelve subjects of 239 with an increase in ChT activity and/or CCL18/PARC were genetically diagnosed for NP-C. Moreover, 7 presented one causal mutation for NP-C in heterozygosis. Only in 5 of 12 (41,6%) cases had the NP-C Suspicion Index > 70. We have identified 18 mutations, 6 new (p.T375A, p.L846P, p.D1097N, p.R1173G, p.R1274W and a microdeletion in 18q11-q12). Family studies and/or bioinformatics (Mutation Taster, PolyPhen2 and SIFT) showed causal association with NP-C. Conclusion: According to our experience, plasmatic determination of chitotriosidase activity and CCL18/PARC concentration in subjects with a NP-C clinical suspicion might be useful biomarkers for Niemann Pick type C diagnosis.

Irún P1, Alfonso P1, Aznarez S2, Giraldo P3, Pocovi M2

P-699

1 CIBERER, Zaragoza, Spain; 2 Dep Bioquimica. Universidad de Zaragoza, Zaragoza, Spain; 3Haematol Serv, Miguel Servet Univ Hosp, Zaragoza, Spain

Metabolic diseases in four babies with non-immune hydrops fetalis

P-697

Plasma chitotriosidase activity (ChT), a biomarker for Gaucher disease (GD), varies in general population due to polymorphisms in CHIT1 gene. Our aim was to analyze the frequency of c.1049_1072dup24 and p.G102S polymorphisms and their influence on ChT activity and its change during enzymatic replacement therapy (ERT). Genotypes were analyzed by PCR, gel electrophoresis and restriction isotyping in 269 type I GD patients. ChT activity was analyzed before and after a year on ERT using 4-methylumbelliferyl-β-D-N, N′,N″-triacetylchitotrioside. We found allelic frequencies for dup24 and p.G102S of 0.22 and 0.27 respectively, 4% of patients were homozygous (HOMOdup) and 37% heterozygous (HETdup) for dup24 and 9% homozygous (HOMO102) and 37% heterozygous (HET102) for p.G102S. Patients were classified in two groups: negative for dup24 (NEGdup) and negative for p.G102S (NEG102). Significative differences were found in ChT before ERT between HETdup versus NEGdup and also for HET102 and HOMO102 versus NEG102. No differences in the percentage of ChT

Moreno CA1, Steiner CE1, Baptista MB1, Gomes CP2, Burin M3, Giugliani R3, Froissart R4 1

Depart Medical Genetics, FCM, Unicamp, Campinas, Brazil; Department of Pathology, FCM, Unicamp, Campinas, Brazil; 3 Medical Genetics Service, HCPA, UFRGS, Porto Alegre, Brazil; 4 Lab des Maladies Héréd du Metabol, Lyon, Brazil 2

Non-immune hydrops fetalis (NIHF) is the presentation of many disorders, including inborn errors of metabolism (IEM). Although controversial, the frequency of IEM among NIHF has been recently reported to reach nearly 6%. The purpose of this presentation is to report four cases of NIHF caused by IEM, emphasizing the relevance of the placental and radiological examinations. Hydrops was identified in the 3rd trimester (27-32 weeks), and firstly the main associated causes were excluded. Subsequent metabolic evaluation detected three cases of lysosomal storage disorder (LSD) - gangliosidosis GM1 [2] and

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infantile sialic acid storage disease (ISSD), and one congenital glycosylation defect (CDG-Ia). Except for this last one, the other IEM were molecularly confirmed. Placental evaluation in two cases (gangliosidosis GM1 and ISSD) identified vacuolization of trophoblast and stromal cells suggestive of LSD. The X-ray was clearly abnormal in ISSD case (osteopenia, coarse trabeculation, metaphyseal fraying, and periosteal cloaking), and more subtle in the others one (abnormal pubic ossification in CDG-Ia and some changes in upper lumbar vertebral bodies in gangliosidosis GM1). These results reinforce the importance to have in mind IEM during NIHF investigation and to look at for indirect (radiological and placental) signs that could suggest a metabolic disease.

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Developmental assessment at 3 years of age using Bailey scales showed a cognitive age of 19 months and a gross motor age of 1 year. At 4 years of age she is able to run and climb onto furniture. She remains neurologically stable and continues to make slow progress with no signs of regression. Discussion: Early treatment with Miglustat significantly altered the disease course in this patient compared with the sibling, highlighting the importance of early diagnosis of the onset of neurological disease in NPC1. P-702 Clinical heterogeneity of NPC patients: a challenge to diagnosis

P-700 Benefits of active screening project of Pompe disease in Slovakia

de Souza CFM1, Donelles T1, Pereira ML1, Brites A1, Timm F1, Koladicz K1, Giugliani R1

Hlavatá K1, palek P2, Mattošová S3, Hlavatá A1

1

1

Niemann-Pick C (NPC) disease is an autosomal recessive condition related to cholesterol trafficking defects. We describe different forms of presentation of NPC disease in 6 patients, and correlate with laboratory findings. Patients were 2 females and 4 males, being 2 with adults form, 2 with juvenile form and 2 with infantile form (one deceased). All patients were referred to investigation due to neurological regression. In addition, the infantile cases presented hepatosplenomegaly. The Filipin test in fibroblasts was positive in 4/6 patients, and inconclusive in 1/6 case. Molecular analysis of the NPC1 gene showed pathogenic mutations in 5/6 patients (2 mutations are not previously described in the literature). Familial recurrence was observed in 3/6 cases and consanguinity was present in 2/6 families. Miglustat was started in the 3/5 patients who are still alive (1 infantile, 1 juvenile and 1 adult), and is being well tolerated. This report illustrates the broad heterogeneity of NPC presentation, even when a small number of patients is analyzed, and stress the need of increasing awareness about this disease and of making available the appropriate tools for the laboratory diagnosis.

2nd Dep of Paediat, Univ Child Hosp, Bratislava, Slovakia; 2Center for Neuromusc Dis, Bratislava, Slovakia; 3Center of Medical Genetics, Univ Hosp, Bratislava, Slovakia Background: Pompe disease is an autosomal recessive, hereditary, metabolic lysosomal storage disorder, that is caused by deficient acid alpha-glucosidase (acid maltase, GAA) activity and results in accumulation of glycogen in lysosomes of all kind of tissues, but predominantly in skeletal and cardiac muscles. Clinical manifestation is variable, from most severe, rapidly progressive new-born and infant form, to late manifestation in adulthood with slower progression of symptoms. Methods: In Slovakia there has been a project of active screening for patients with Pompe disease since January 2009,based on measuring of alpha-glucosidase activity in dry blood spot. Positive findings are confirmed by measuring the activity of GAA in lymphocytes and molecular-genetic analyse of mutations in gene for acid GAA in chromosome 17q25.3. Results: Until the end of year 2012,115 centres have joined the project, 840 Slovak patients have been tested. Most commonly the samples were sent by neurologists from adult patients. Until now 7 patients from Slovakia have been diagnosed with Pompe disease, two of which have infantile form, and five have adult form. Conclusions: Early diagnosis and treatment of Pompe disease could be lifesaving in infantile form and as for juvenile and adult form it can help the patients to achieve a good quality of life. P-701 Effect of miglustat on neurological outcome in early infantile niemann pick C : a case report Sreekantam S1, Simmons L1, Santra S1, Vijay S1, Chakrapani A1 1 Dept of IMD, Birmingham Childrens Hosp, Birmingham, United Kingdom

Introduction: Miglustat, a small iminosugar with its ability to cross the Blood Brain Barrier has proven disease-modifying benefit on neurological manifestations in Niemann Pick type C1 (NPC1). We report the benefit of early use of this drug in infantile onset NPC1. Case Report: A female infant presented with neonatal cholestasis and hepatosplenomegaly and was diagnosed with NPC1. Her previous affected male sibling had early infantile onset NPC1 with similar presentation. He was in a vegetative state by 4years and died aged six. She was started on Miglustat at 5months of age after the cholestasis resolved. At 30months of age, her Niemann Pick disease related disability score was 4 with normal ambulation, manipulation, language and swallowing.

Medical Genetic Service, Porto Alegre, Brazil

P-703 NPC Brazil network: 444 subjects with suspected Niemann-Pick C disease investigated from 2010 to 2012 Timm F1, Santos S2, Silveira B2, Bohn E2, Michelin-Tirelli K2, Burin M2, Bock H2, Polese MG2, Matte MC2, Saraiva-Pereira ML1, Giugliani R3 1

Department of Biochemistry, UFRGS, Porto Alegre, Brazil; 2Medical Genetics Service, HCPA-UFRGS, Porto Alegre, Brazil; 3Department of Genetics, UFRGS, Porto Alegre, Brazil Niemann-Pick type C is an autosomal recessive inborn error of cholesterol trafficking,characterized by the storage of cholesterol inside the lysosomes,which lead to a wide range of clinical manifestations,usually involving the CNS.As there is one specific treatment approved and several therapeutic strategies in development,diagnosis is becoming increasingly important.However, identification of this disease is challenging,as signs and symptoms overlap with many other conditions and as the standard diagnostic method(Filipin test)is performed in growing fibroblasts,which requires the collection of a skin biopsy.To help the identification of affected patients,we built in Brazil a program called "NPC Brazil Network",available for medical doctors across the country.From 2010 to 2012 we received clinical information and biological samples(blood and/or skin biopsy)from 444 suspected patients,and were able to obtain suitable fibroblasts to perform the Filipin test in 296 cases.From these,50(17%) had a positive result,58(20 %)were inconclusive and 188(63%)negative. Patients with inconclusive results in the Filipin test(and also patients with negative results but with strong clinical suspicion)were

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further studied by molecular analysis of NPC1 and NPC2 genes. Along the first 3 years of activity, the NPC Brazil Network proved to be a valuable resource for the identification of NPC patients in Brazil. P-704 Natural history of Japanese patients with Fabry disease: the indication of enzyme replacement therapy for female patients Kobayashi M1, Ohashi T2, Ida H1 Dep Pediatr, Jikei Univ Sch of Med, Tokyo, Japan; 2Dep Gene Therapy, Jikei Univ Sch of Med, Tokyo, Japan 1

Objective: The clinical severity of female patients with Fabry disease is heterogeneous. The aim this study is to clarify the natural history of Japanese female patients with Fabry disease to consider the indication of enzyme replacement therapy (ERT). Material and Method: The study patients were 65 Japanese female patients with Fabry disease who had never received ERT (4-72 years, average ± SD: 40.0 ± 18.6). We studied the incidence of proteinuria, end-stage renal disease (ESRD), left ventricular hypertrophy (LVH) and cerebrovascular disease. Result: The incidence (mean age of onset ± SD) of proteinuria, left ventricular hypertrophy and cerebrovascular disease were 33.8% (47.1±11.8 years), 29.2% (57.2±9.2 years) and 10.8% (61±11.5 years), respectively. Half of the study patients had proteinuria, LVH or cerebrovascular disease by 54 years of age and all of patients had LVH by 72 years. Three (4.6%) patients had ESRD and the duration between age of onset of proteinuria and ESRD was 13.3±1.53 years (mean±SD). Conclusion: Physicians should be cautious of the onset of proteinuria, LVH and cerebrovascular disease in female patient as well as male patients with Fabry disease and start ERT immediately after the onset of these complications. P-705 Survey of the first 1000 patients identified by the MPS Brazil network Giugliani R1, Jesuino K2, Brites A2, Burin MG2, Matte U2, LeistnerSegal S2, Bittar CM2, Rafaelli CL2, Schwartz IVD1, Toralles MB3, Acosta AX3, Ribeiro EM4, Valadares ER5, Silva LCS6, Kim CA7, Lourenço CM8, Horovitz D9, Boy R10, Steiner C11, Ribeiro MG12, Federhen A2 1 Department of Genetics, UFRGS, Porto Alegre, Brazil; 2Medical Genetics Service, HCPA-UFRGS, Porto Alegre, Brazil; 3HUPESUFBA, Salvador, Brazil; 4Hospital Infantil Albert Sabin, Fortaleza, Brazil; 5Medical School-UFMG, Belo Horizonte, Brazil; 6UFPA, Belem, Brazil; 7 IC-HC-USP, Sao Paulo, Brazil; 8 FMRP-USP, Ribeirao Preto, Brazil; 9 IFF-FIOCRUZ, Rio de Janeiro, Brazil; 10 HUPE-UERJ, Rio de Janeiro, Brazil; 11Dep Medical GeneticsUNICAMP, Campinas, Brazil; 12IPPMG-UFRJ, Rio de Janeiro, Brazil

The MPS BRAZIL-NETWORK(MBN)was created to improve diagnosis and management of MPS diseases in Brazil.Since then, physicians from all Brazil have requested support for the investigation of patients with suspected MPS.The contact with MBN has been performed through website(www.mps.ufrgs.br), email([email protected]) or a tollfree helpline(0800-510-2030).Informative materials and instructions for sample collection and shipment,as well as educational material about MPS can be downloaded in the website.Services from all Brazilian regions sent biological samples to MBN headquarters,located at the Medical Genetics Service-HCPA,where the laboratory

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investigation for MPS is performed.From 2004 to 2012 1000 patients with MPS were identified,being 542 new diagnosis(average 5.2/month) .The most frequent type of MPS diagnosed was MPS II,confirmed in 300/ 1000(29.9%)of MPS patients, followed by MPS VI(23.3%),MPS I (19.4%)and MPS IVA(11.5%).Most MPS I patients came from South or Southeast regions(49.6%),while most MPS VI patients came from Northeast region(49.5%).MPS III-B and IV-A are also frequent in South with 42% and 31.5% of patients,respectively coming from this region.Easy access to information and to diagnostic tests provided by MBN helped to identify many patients with MPS in Brazil, transforming MBN in a template to develop similar initiatives for other rare diseases. P-706 Long-term use and follow-up of substrate reduction therapy with miglustat in type 1 Gaucher disease in Spain. Zagal study Giraldo P1, Alfonso P1, Irun P1, Atutxa K2, Barez A3, Franco R4, Fernandez-Galan MA5, Roig I2, Giner V6, Villalon L7, Martinez E8, Luño E9, Loyola I10, Salamero O2, de la Serna J11, Pocovi M12 1 CIBERER, Zaragoza, Spain; 2Spanish Working Group GD, Zaragoza, Spain; 3H Virgen de Sonsoles, Avila, Spain; 4H Punta Europa, Cadiz, Spain; 5H Virgen del Puerto, Plasencia, Spain; 6H Verge dels Lliris, Alcoy, Spain; 7H Alcorcon, Madrid, Spain; 8H Virgen de Valme, Sevilla, Spain; 9H General de Asturias, Oviedo, Spain; 10H Montecelo, Pontevedra, Spain; 11H Doce de Octubre, Madrid, Spain; 12Bioch Mol and Cell Biol Dep. University, Zaragoza, Spain

The ZAGAL study was planned for monitoring the real-life use of miglustat ('Zavesca™') in Spanish adult patients with mild-to moderate disease. The study included GD1 patient's naïve to therapy as well as patients who have previously been treated with ERT. To date a total of 302 GD1 patients have been diagnosed in Spain (Orphanet J Rare Dis. 2012); from 2004 until 2011, 52 of them have been treated with miglustat (17.2%). Currently 42 patients are on miglustat therapy (15 during 7 years). The therapeutic goals (normalized blood counts, reduced liver and spleen volume and absence of bone crisis) have been achieved or maintained. Fourteen patients had transitory gastrointestinal disturbances. Ten patients discontinued therapy: 3 died by non-related causes (2 cancer, 1 heart attack), one to become pregnant and the remaining 6 due to gastrointestinal discomfort or intolerance.60% of patients have a fine tremor. Conclusion: The follow-up of GD patients treated with Miglustat for long term shows that it achieves and/or maintains the therapeutic goals. A high individual variability had been observed in terms of miglustat gastrointestinal intolerance apparently unrelated with GBA genotype, gender and age, but possibly associated with disaccharidasesinhibition and food habits. Conflict of Interest declared. P-707 Induction of oral tolerance to N-acetylgalactosamine 6-sulfate sulfatase used for enzyme replacement therapy in Morquio A syndrome Montano AM1, Sosa AC1, Kariuki B1, Knutsen A1, Tomatsu S1, Barrera LA2, Bellone C3 1 Dept Pediatrics, St. Louis University, St Louis, United States; 2Inst Err Inn Metab, Javeriana University, Bogota, Colombia; 3Dept Mol. Micr. Imm, St Louis University, St Louis, United States

Background: Morquio A syndrome (MPS IVA) is an autosomal recessive disorder caused by N-acetyl-galactosamine-6-sulfate sulfatase (GALNS) deficiency that results in the intralysosomal accumulation of

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keratan sulfate and chondroitin-6-sulfate. MPS IVA is a good candidate for enzyme replacement therapy (ERT). Immune response against the infused protein in ERT has been reported as the main limitation in therapeutic effectiveness. Currently, unspecific immunosuppressive protocols have been evaluated for improving ERT efficacy in which the riskbenefit profile is less clear, due to the associated side effects in the patient. Specific induction of tolerance to proteins used in ERT without associated adverse reactions is crucial for the effectiveness of the treatment. Objective: The main goal of this study is to induce specific tolerance to GALNS in a mouse model by a natural mechanism before its use in ERT. Results: One of the most immunodominant peptides identified previously and GALNS were used for oral tolerance induction to ERT, resulting in decreased humoral and cellular response towards GALNS. In addition, levels of glycosaminoglycans in tissues were dramatically reduced. Conclusion: Induction of oral tolerance showed a significant improvement of ERT. This model could be extrapolated to other LSDs in which the immune response hinders the development of ERT. P-708

include hepatosplenomegaly, hematologic problems, and low bone density. Current treatment of the disease involves a choice among enzyme replacement therapy (ERT), substrate reduction therapy and stem cell transplantation. Miglustat, an inhibitor of glucosylceramide synthase, is administrated orally and has been shown clinically to increase bone mineralization over a shorter time course. However, the mechanism through which bone remineralization occurs with Miglustat is unknown. Objective: Characterize the mechanism of action of Miglustat on bone density and whether those beneficial effects are through: i) decreasing levels of the accumulating substrate or ii) by a novel mechanism acting directly on cells of bone remodeling. Methods: The analysis of expression markers associated with osteoclasts and osteoblasts treated with Miglustat were studied in a low bone density mouse model. Gene candidates were identified based on a minimum two-fold increase or decrease of in vitro expression of a gene between groups. Results: Our findings suggest a possible role for osteoclasts in promoting bone formation via expression and synthesis of BMPs, which then would play an important role in promoting the recruitment, proliferation, and differentiation of osteoblasts at bone resorption sites. Conflict of Interest declared.

Dried-cell filter paper samples: a new tool for the identification of LSDS

P-710

Civallero GE1, Viapiana-Camelier M2, Mari JF de2, Burin MG2, Giugliani R3

Demographic characterization of female Brazilian patients enrolled in the Fabry registry

1

Martins AM1, Garrote J1, Kyosen SO1, Marques F2, Rosa M2

Med Genet Service-HCPA and INAGEMP, Porto Alegre, Brazil; Medical Genetics Service, HCPA-UFRGS, Porto Alegre, Brazil; 3 Dep Genetics-UFRGS and INAGEMP, Porto Alegre, Brazil 2

1

CREIM, Universidade Federal de Sao Paulo, Sao Paulo, Brazil; Hospital Santo Antonio, Tapejara, Brazil

2

Diagnosis of lysosomal storage disorders(LSDs)is largely based in the detection of specific enzyme deficiencies.As therapies are now available for many LSDs,accurate and timely diagnosis became more important.Assays in dried-blood filter paper samples are already performed for some enzymes, allowing fast and easy detection of affected patients,but are not available for all LSDs so far.Additionally, methods have been adapted to evaluate glycosaminoglycans and other metabolites in dried-urine filter paper samples.Following these strategies, we developed an enzyme assay which uses dried cell homogenates impregnated in filter paper, initially for the detection of heparin sulfamidase (MPS IIIA) and beta-galactosidase(GM1 gangliosidosis,MPSIVB,galactosialidosis) deficiencies.We prepared filter paper samples with dried-leukocytes or dried-fibroblast(DLFP,DFFP)from healthy individuals and from patients with confirmed MPS IIIA or GM1 by direct transfer of cell homogenates to filter paper discs.Heparan-sulfaminidase and beta-galactosidase were tested by incubating samples with specific buffers and substrates.For heparin-sulfamidase,a clear discrimination was found between normal controls and MPS IIIA patients both in DLFP and DFFP samples.For beta-galactosidase,distinction between normal controls and GM1 patients was evidenced only in DLFP samples so far.Although the method still needs further development,we conclude that assays for these two enzymes could be performed in dried-cell homogenates,and could an useful tool for the identification of LSDs.

Background: Fabry disease is an X-linked inborn error of metabolism caused by alpha-galactosidase A deficiency. The Fabry Registry (NCT00196742) is an observational database that collects clinical data of patients with Fabry disease. Methods: We characterized the age at symptom onset, age at diagnosis, and presenting symptoms at enrollment in Brazilian Fabry Registry female patients. Only natural history data (untreated patients or prior to treatment with ERT) were included. Results: As of 04 January 2013, 94 female Brazilian patients had been enrolled in the Fabry Registry. The mean age at symptom onset was 15.3 ± 11.5 years (± SD, n = 69). The mean age at Fabry diagnosis was 30.6 ± 15.69 years (± SD, n = 89). The mean time from the onset of symptoms until diagnosis was 16.6 ± 14.69 years (± SD, n = 63). Neurologic pain was the presenting symptom most frequently reported (57.4%). Other symptoms and signs reported were gastrointestinal (9.6%), renal (3.2%), ophthalmologic (4.3%), cerebrovascular (3.2%) and cardiovascular (2.1%). Conclusion: Fabry disease is treatable, and the long delay from symptom onset until diagnosis in Brazilian female patients is very concerning, as irreversible damage may occur during this time. Conflict of Interest declared.

P-709

P-711

Effect of miglustat on bone remodeling in vitro in a low bone density mouse model

Nanoparticles as delivery systems for drugs in lysosomal disorders Andreu V1, Alfonso P2, Santamaría J3, Pocoví M1, Arruebo M3, Giraldo P4

1

2

Montano AM , Ozdemir E , Lee D

2 1

Dept Pediatrics, St. Louis University, St Louis, United States, Sch Med, St Louis University, St Louis, United States

Aragon Health Sciences Institute (I+CS)., Zaragoza, Spain; 2CIBERER, ISCIII., Zaragoza, Spain; 3Institute of Nanoscience of Aragon (INA), Zaragoza, Spain; 4Miguel Servet University Hospital, Zaragoza, Spain

Background: Gaucher Disease is a lysosomal storage disease caused by the deficiency of β-glucocerebrosidase. Major clinical symptoms

The lysosomal disorders are produced by an inborn error of metabolism.The possibility to apply small quantity of drugs with activity

1

2

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in the lysosomal targets can produce more effective effects with less quantity of drug and low cost. The aim of this work was the development of new delivery systems of the drugs allows modifying its release profiles and minimizing its side effects. By controlling the pharmacokinetics, reduced side effects are expected. In this work, two different types of materials have been synthesized: MCM41 type ordered mesoporous silica materials (inorganic materials) and PLGA (poly(lactic-co-glycolic acid)) nanoparticles (organic materials). The porous structure of MCM41 is used to host the drug and to control its release profile ideally following a zero orden. Spherical PLGA nanoparticles are widely studied as carriers because they are biodegradable and biocompatible. The physico-chemical stability under simulated gastric and intestinal fluids of the different carriers has been evaluated. Both carriers showed different erosion profiles being the MCM41 almost unaffected by a harsh environment (gastric fluid). In conclusion, it has been developed two different therapeutic delivery vehicles for the encapsulation of different drugs that are physically stable to the transit through gastrointestinal tract showing different pharmacokinetics.

In our laboratory Leucocytes Enzymes activities were determined by spectrofluorimetric and spectrophotometric method. Since 1998 we investigated 1175 patients suspected to have LSD. During this period 272 patients with LSD's were diagnosed. In MPS group, we diagnosed MPSI (110 cases) , MPSII (10 cases), MPSIII (33 cases: 13 type A, 9 type C, 1 type B, 10 non determined), MPSIV (24 cases)and MPS VI (6 cases). We also diagnosed Gaucher (26 cases), Niemann-Pick A-B (11 cases), Metachromatic leucodystrophy (11 cases), GM1 Gangliosidosis 9 cases), Sandhoff disease (9 cases), Fabry (7 cases), Tays Sacchs (4 cases), Krabbe (2 cases), CLN 2 cases) and Pompe disease (6 cases). The most frequent LSDs in our series are Hurler and Gaucher diseases. The incidence of Hurler Disease is approximately 1/100 000 that of Gaucher disease is estimated at 1/250000. The incidence of LSDs should be higher because of the high frequency of consanguineous marriages. Early diagnosis is important for treatment of LSDs. Currently seven Hurler patients, two Gaucher patients and one Fabry patient are treated by ERT.

P-712

Infantile Sandhoff disease: clinical and biochemical recognition

A neonatal case with I-cell disease and phenylketonuria: a novel mutation in the gnptab gene

Pérez-Dueñas B1, Casado M2, Cuesta L1, Ortigoza JD1, Artuch R2, Campistol J1

Okur I1, Gunduz M1, Alves S2, Koc N1, Biberoglu G3

Child Neurol Dep, Sant Joan de Déu Hosp, Barcelona, Spain; 2Clin Biochem Dep, Sant Joan de Déu Hosp, Barcelona, Spain

Div Ped Metab, Ankara Diskapi Child Hosp, Ankara, Turkey; 2INSA, CGMJM, Porto, Portugal; 3Div Ped Nut Metab, Gazi Univ Hosp, Ankara, Turkey

P-714

1

1

Inherited metabolic diseases are more frequent in countries with high prevalence of consanguineous marriages as Turkey because they are usually autosomal recessive. While I-cell is a rare lysosomal disease as 1/100000-250000 of their global prevalence, phenylketonuria is a aminoacid metabolism disease that Turkey is the country with the highest incidence in the worldwide. The genes responsible for both disorders (PAH and GNPTAB), are on the long arm of chromosome 12. Case Report: The girl patient was single child born from healthy parents who were consanguineous. At 14 days of age, she referred to our department due to hyperphenylalaninemia in national neonatal screening. The blood Phe level was 1962 mmol/L (ND, 24-150) and she was diagnosed classical phenyketonuria. We suspected from mucolipidosis because she had dismorphic findings in physical examination. Enzyme and mutation analysis revealed mucolipidosis type II (I-cell). Genetic testings revealed homozygous IVS10+546G>A mutation in PAH gene and homozygous c1124delG(exon 10) (R375QfsX2)(a novel mutaion) in GNPTAB gene. This novel mutation creates a premature STOP codon leading to the formation of a truncated protein. Both parents are heterozygous for the mutation. Conclusion: Here we presented first case with phenylketonuria and Icell, having a novel mutation in GNPTAB gene.

Background: Sandhoff disease (SD) is a lethal inborn error of βhexosaminidase A and B, characterized by lysosomal storage of GM2 ganglioside and irreversible neuronal injury. Early clinical and biochemical recognition may be increasingly important as specific therapies (i.e.pharmacological chaperones or substrate reduction therapy) become available. Methods: We analyzed clinical and radiological features of two children with SD (age 15 months and 6 years). Selective screening of SD was performed with the determination of oligosaccharides by capillary electrophoresis. Results: Both infants with SD presented with rapid neurological deterioration and progressive visual loss at age 6 and 15 months. They showed a cherry-red macular spots, diffuse cerebral white matter abnormalities and bilateral symmetric thalamic T2 hypo-intensities. An abnormal excretion of oligosaccharides in urine was highly suggestive of SD, which was confirmed by enzyme assay. The older patient was homozygous for the mutation p.G282E in the HEX B gene. Conclusions: Infants with SD presented with rapid deterioration and widespread lesions involving the white matter and thalamic nuclei on MRI. Determination of oligosaccharides by capillary electrophoresis may be useful for early recognition and future therapeutic interventions. P-715

P-713

Lipidomics for Gaucher disease

Epidemiological study of lysosomal storage diseases in Morocco: experience of central laboratory of biochemistry in Rabat

Fuller M1 1

SA Pathology, Adelaide, Australia

Talbaoui H1, Puech JP2, Dahri S1, Kriouile Y1, Meskini T1, Benouchane T1, Otmani S3, Caillaud C2, Chabraoui L1 1 University Hospital of Rabat, Rabat, Morocco; 2Laboratory of Genetic and Inherited Path, Cochin Paris, France; 3University Hospital of Fes, Fes, Morocco

Lysosmal Storage Diseases (LSDs) are characterized by storage of substrates in lysosomes related with defects of lysosmal enzyme activity or defects of proteins of lysosmal membrane receptor.

Gaucher disease is a complex disorder of sphingolipid metabolism caused by a genetic defect resulting in an enzyme deficiency in the catabolic pathway. This block in catabolism alters the overall flow of sphingolipid metabolites. Using high-throughput lipidomics to measure over 200 individual species of lipids simultaneously demonstrated that in addition to the substrate for the deficient enzyme, the levels of other sphingolipids, phospholipids and lyso-derivatives were altered in Gaucher disease. The identification of elevated glucosylsphingosine has led to the development of a rapid assay to measure this metabolite

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in blood spots and 0.01 mL of plasma with 100% sensitivity and specificity from the analysis of more than 500 samples. This metabolite clearly outperforms the current method of chitotriosidase determination as both a diagnostic and therapeutic monitoring biomarker for Gaucher disease. Additionally, the identification of alterations in a lysosomal phospholipid, has allowed its partial normalisation by supplementation with linoleic acid with a flow on effect that reduces primary substrate accumulation. This may prove useful as an adjunct therapy. Highthroughput lipidomics has thus enabled improvements in diagnosis and biochemical monitoring of Gaucher disease as well as identifying new possible treatment strategies. This approach is equally relevant for other disorders of sphingolipid metabolism.

mechanography. The change was not explained by any changes joint mechanics, body mass or respiratory function. This type of improvement is similar to that seen in subjects after exercise training although our patient was not on an exercise program. We hypothesize that the mechanism of the improvement may either be a direct effect on muscle velocity or improved patient mobility leading to gradual increases in muscle use and improved performance. This is the first instance we are aware of where an improvement in muscle power, with minimal changes in joint mobility, has been demonstrated as a mechanism of improved mobility with recombinant enzyme therapy in Hunter syndrome. Conflict of Interest declared.

P-716

16. Enzyme replacement therapy

Abnormal filipin staining unmasking lysosomal acid lipase (LAL) deficiency: not all that shines is Niemann-Pick type C

P-718

Lourenco CM1, Timm F2, Burin M2, Burg J3, Quinn A3, Hamilton J4, Giugliani R2, Marques Jr W1

Sibling study of enzyme replacement therapy for Mucopolysaccharidosis type VI Furujo M1, Kubo T1, Okuyama T2

1

2

University of Sao Paulo, Rineirao Pretto, Brazil; Federal University of Rio Grande do Sul, Porto Alegre, Brazil; 3Synageva BioPharma Corp, Lexington, United States; 4 Yorkhill Hospital, Glasgow, United Kingdom LAL Deficiency is a lysosomal storage disease characterized by cholesteryl ester and triglyceride accumulation. Niemann-Pick type C (NPC) is a complex lysosomal disease caused by defective intracellular transport of cholesterol leading to unesterified cholesterol storage in the late endosome/lysosome system. Unlike LAL Deficiency, NPC cannot be diagnosed by enzyme assay. NPC utilizes filipin staining to show the intralysosomal accumulation of unesterified cholesterol. Here we describe a 5 year old boy, born to non-consanguineous parents, noted to have cholestasis in the first months of life. His cholestasis remitted but a residual degree of hepatomegaly remained. At age 4, he underwent a liver biopsy which revealed features of storage disease. Investigation for Gaucher disease and Niemann-Pick types A/B were negative. Given his high levels of chitotriosidase, a skin biopsy for filipin staining was performed, revealing "classical" NPC pattern in his fibroblasts. Since molecular genetics analysis of NPC1/2 genes failed to identify mutations, further investigation was warranted. Dried blood spot (DBS) enzyme assay for LAL showed decreased activity, compatible with LAL Deficiency. To our knowledge, this is the first report of a patient with LAL Deficiency with positive filipin staining; suggesting that this cytochemical test could help identify patients with LAL Deficiency. P-717 Improvement in muscle power after one year of idursulfase treatment in a 9 year-old boy with Hunter syndrome Khan A1, Ramage B2, Robu I2, McNeil C3, Casey R1 Alberta Child Hosp, Univ Calgary, Calgary, Canada; 2Neurosci, Alberta Child Hosp, Calgary, Canada; 3Met Clinic, Alberta Child Hosp, Calgary, Canada 1

Impairment of mobility is a key factor affecting quality of life in patients with Hunter syndrome. Treatment with recombinant iduronate-2sulfatase (I2S) has been shown to increase the 6-minute walk distance after 1 year of treatment compared to placebo. However, the mechanism by which mobility is improved is not clearly understood. We report the effects in mobility seen in a 9-year old boy treated with I2S for Hunter syndrome. After one year of of biweekly infusions of Idursulfase, we measured a 32% increase in muscle power output using jumping

NHO Okayama Medical Center, Okayama, Japan; 2National Center for Child Health and Dev, Tokyo, Japan 1

Here, we report sibling study of Enzyme Replacement Therapy (ERT) for Mucopolysaccharidosis type VI (MPSVI) . The elder brother (sibling 1) was diagnosed as MPSVI, started ERT at the age of 5.6 years old and he is now 12 years old. The younger sister (sibling 2) was diagnosed as MPSVI at just after delivery, started ERT at 6 weeks old and she is now six years old. She does not show any symptoms typical of MPS VI including short stature, progressive dysmorphic facial features, hepatosplenomegaly, otitis media, hearing difficulty, corneal clouding and joint contracture. On the other hand, her older brother had all typical MPS VI phenotypic features at the age of three years old before treatment. Some of his symptoms were improved by treatment of ERT, but his skeletal deformity and short stature have not yet been improved. Based on these observations, we conclude that that early initiation of ERT is essential for good therapeutic outcome, and newborn screening of MPS is conceivable P-719 Systemic manifestations of early treated type III Gaucher disease in patients homozygous for the p.L444P mutation Hwu WL1, Lee NC1, Chien YH1, Wong SL2, Sheen JM3, Tsai FJ4, Peng SF5, Leung JH6, Chao MC7, Shun CT8 Med Gen, Nat Taiwan Univ Hosp, Taipei, Taiwan; 2Div Ped End, DMF Chia-Yi Chri Hosp, Chia-Yi, Taiwan; 3Ped, Kaohsiung Chang Gung Hosp, Kaohsiung, Taiwan; 4Dep Ped, China Med Univ Hosp, Taichung, Taiwan; 5 Radio, Nat Taiwan Univ Hosp, Taipei, Taiwan; 6Radi, DMF Chia-Yi Christ Hosp, Chia-Yi, Taiwan; 7Div Gen, Kaohsiung Med Univ Hosp, Kaohsiung, Taiwan; 8Path, Nat Taiwan Univ Hosp, Taipei, Taiwan Background: Gaucher disease (GD) is caused by a deficiency in the activity of the lysosomal acid β-glucosidase (GBA). Recombinant human GBA (rhGBA) is a treatment for GD, but the outcome of neuropathic GD is variable. The p.L444P mutation of the GBA gene is commonly associated with neuropathic GD. Objectives: To see the benefits of rhGBA therapy in early treated Gaucher patients. Material and methods Clinical information of 7 children who were homozygous for the p.L444P mutation with rhGBA starting from a median age of 2.1 years (range 1-2.9 years) were retrospectively collected.

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Results: After treatment with rhGBA for 7.6 years (range 2.2-12.0 years), all 7 patients exhibited normal hemoglobin levels and platelet numbers. Neurological symptoms, including slow progressing horizontal gaze palsy, seizures, or mild mental retardation, were observed in only 3 patients. Five patients exhibited kyphosis over the thoracic spines or at the thoracolumbar junctions. Lymphadenopathy in the neck, thorax, and abdomen were observed in 4 of the treated patients. The lymphadenopathy was asymptomatic and non-progressive in these patients. Conclusion: Early treated GD patients that were homozygous for the p.L444P mutation was neurologically stable. However, systemic manifestations, including kyphosis and lymphadenopathy, should need an attention.

to cynomolgus monkeys and Dachshunds was 100- to 1000-fold higher than that in plasma. ICV rhTPP1 distributed widely in the brain resulting in concentrations 2- to 10-fold greater than the endogenous wild-type level. An expected delivery device related inflammation was observed along the catheter track in all animals, including vehicle treated controls. Anaphylactoid reactions observed after repeat dosing in Dachshunds were mitigated by increasing the duration of the infusions and administration of diphenhydramine. The positive nonclinical profile of rhTPP1 offers promise for the planned first in human clinical trial in NCL2 patients. Conflict of Interest declared.

P-720

Immunomodulation and enzyme replacement therapy for infantile onset Pompe disease: Australias first case

Mucopolysaccharidosis type VI (MPS VI) five years treatment with galsulfase: a comparative study Sales Marques J1, Aires Pereira S1, Carriço A1, Varandas R1, Carvalho I1, Romariz J1, Correia S1, Santos H1 1

Centro Hospitalar V. N. Gaia/Espinho, Vila Nova de Gaia, Portugal

Background: The deficient enzyme of MPS VI is Arylsulfatase B. Galsulfase is used for replacement therapy. We evaluate five year treatment with galsulfase a thirteen years old boy with MPS VI. The comparative study included weigh, height, growth velocity, body mass index, ECG, heart ultrasound, respiratory function, vision, endurance test, psychomotor development and Gags. Methods: We diagnosed a 15 month old boy with MPS VI. He has hypotonia, umbilical hernia and hip dysplasia. He started galsulfase at eight years old. Results: The height/weight maintained the same percentile. The growth velocity was same. Body mass increased 10% during the treatment.ECG maintained normal. Heart ultrasound improved from thick mitral and aortic valve to normal structure. FEV1/FEV ratio increased from 85% to 97%. The eyes has the same changes.The 12 minute walk has more 575 meters and he finished the test. The 3 minute stairs climb has more 6 stairs. He showed development quotient- 69 in Griffiths scale and total IQ - 49 in WISC scale. The GAGS reduced during the treatment. Conclusions: Galsulfase improved endurance test and reduced urine Gag, showed benefit in the patient respiratory function, heart structure and avoid so far more severe changes in the eyes. P-721 Nonclinical development of TPP1 enzyme replacement for NCL2 Vuillemenot BR1, Tsuruda LS1, Kennedy D1, Katz ML2, Coates JR2, Lobel P3, Henshaw J1, Musson D1, Keve S1, Cahayag R1, Tiger P1, O'Neill CA1 1 BioMarin Pharmaceutical Inc., Novato, CA, United States; 2University of Missouri, Columbia, MO, United States; 3Univ Medicine and Dentistry New Jersey, Piscataway, NJ, United States

Neuronal Ceroid Lipofuscinosis 2 (NCL2) is a fatal neurodegenerative lysosomal storage disorder caused by lack of tripeptidyl-peptidase I (TPP1) enzyme activity. BioMarin is developing intracerebroventricular (ICV) recombinant human (rh) TPP1 enzyme replacement therapy. The nonclinical pharmacology, pharmacokinetics, and safety of rhTPP1 were assessed in animal models after CNS delivery. In TPP1-null mouse and Dachshund NCL2 disease models, administration of rhTPP1 resulted in reduction of lysosomal storage, attenuation of clinical decline, improvement of function, and lifespan extension. Peak CSF exposure after ICV infusion

P-722

Ellaway CJ1, Owens P1, Wong M1 1

Sydney Children's Hospital Network, Sydney, Australia

Enzyme replacement therapy (ERT) for infantile Pompe disease has improved survival however there is marked variability in clinical outcomes as a result of many factors including CRIM (cross-reactive immunologic material) status. CRIM negative status is associated with a poor response to ERT due to the formation of antibodies to rhGAA. Our patient with CRIM negative Pompe disease started ERT at age 4 months. Initially there was an improvement in his muscle strength. Antibody levels rose steadily, however given the ongoing clinical improvement it was decided not to immunomodulate. At one year he suffered with pneumonia, complicated by an episode of aspiration. Continuous BiPAP and naso-jejunal tube feeds were required. The antibody levels reached a maximum of 819,200, associated with infusion related adverse reactions of escalating severity. He commenced immunomodulation therapy with rituximab age 13 months, followed 2 months later by bortezomib, methotrexate and high dose intravenous immunoglobulin. At 16 months the patient's antibody levels began to fall, and the infusions were better tolerated. His clinical condition initially stabilised however subsequently deteriorated and he died at 19 months. Immunomodulation should be considered in CRIM negative infantile onset Pompe disease in patients with rising antibody titres and declining clinical response to ERT. P-723 Agalsidase alfa in paediatric patients with Fabry disease: a 7-year open-label study Schiffmann R1, Castaneda V2, Lien YH3, Chang P4, Martin R4, Pastores G5, Wijatyk A4 Baylor Research Institute, Dallas, United States; 2Renown Children's Hospital, Reno, United States; 3AKDHC, Tucson, United States; 4Shire Human Genetic Therapies, Lexington, United States; 5New York University School of Medicine, New York, United States 1

Background: Signs and symptoms of Fabry disease (FD) can emerge in early childhood with incidences increasing with age. We evaluated the long-term effect and safety of agalsidase alfa (agalα) in a FD paediatric population. Methods: An open-label, multicenter, extension study (TKT029, NCT00084084) enrolled children with FD. After 4 years' agalα (0.2mg/kg agalα every other week), patients could transition into study phase 2 (updated agalα manufacturing process). Phase 2 results are reported (~7ys' treatment), including safety/tolerability and heart rate variability (HRV), left ventricular mass index (LVMI), estimated glomerular filtration rate (eGFR), and plasma/urine globotriaosylceramide (Gb3).

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Results: 11/17 patients transitioned into phase 2 (baseline median [range] age: 10.8y [8.6-17.3]; 10 males). All patients experienced ≥1 treatmentemergent adverse event (AE; ≥1 drug-related n=8; infusion-related n=6; discontinuations due to AEs n=0; serious AEs n=2 [none drug-related]) and no deaths occurred. Three patients formed anti-agalα antibodies (1 patient IgG [agalα-neutralizing, but no apparent clinical impact], 1 IgM, 1 IgM/IgA). eGFR and LVMI generally remained in the normal range. HRV showed a trend towards improvement. Plasma/urinary Gb3 reductions were maintained. Conclusions: Agalα was generally well tolerated and efficacy endpoints remained generally stable in this clinical trial assessment of ERT in paediatric FD. Conflict of Interest declared. P-724 A multicenter, double-blind, randomized safety and efficacy study of two dose levels of taliglucerase alfa in pediatric patients with Gaucher disease Zimran A1, Gonzalez-Rodriguez DE2, Abrahamov A1, Elstein D1, Paz A3, Brill-Almon E3, Chertkoff R3 1

Gaucher Clinic, Shaare Zedek Medical Ctr, Jerusalem, Israel; I.P.H.I.C., Asuncion, Paraguay; 3Protalix Biotherapeutics, Carmiel, Israel

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Background: LAL Deficiency, an autosomal recessive disorder, results in abnormal cholesteryl esters and triglycerides accumulation within the lysosome. Patients present with dyslipidemia, elevated transaminases and/or hepatosplenomegaly which often progress to cirrhosis and early death. Methods: LAL-CL04, a phase 1/2 extension trial, evaluates the longterm effects of every-other-week infusions of sebelipase alfa (recombinant human lysosomal acid lipase) in LAL deficient adults (n=8). Many patients were on stable background lipid-lowering therapy. Results: At 52 weeks, sebelipase alfa produced mean percent decreases in ALT and AST from baseline of 56% and 40%, respectively (p=0.031 each). The mean percent decreases observed for LDL, total cholesterol and triglyceride were 63%, 42% and 47%, respectively, with a mean increase in HDL of 29% (p=0.031 each). AEs were mainly mild and unrelated. Two SAEs occurred in one subject and were deemed unlikely to be related to sebelipase alfa. Infusion-related reactions (IRRs) were uncommon. One patient had a moderate (Grade 2) allergic-type IRR and has paused treatment pending skin testing. No anti-drug antibodies were detected in any patient to date. Conclusions: These results suggest that long-term dosing with sebelipase alfa produces sustained improvement in patients' transaminases and lipid profile. A phase 3 clinical trial is underway (ARISE: NCT01757184). Conflict of Interest declared. P-726

2

Background: Taliglucerase alfa is a plant cell–expressed betaglucocerebrosidase approved for enzyme replacement therapy in adults with type 1 Gaucher disease (GD). Objectives: To investigate the safety and efficacy of taliglucerase alfa in pediatric GD patients. Methods: Multicenter, double-blind, parallel-dose, 12-month study of safety and efficacy in GD patients aged 2 to <18 years. Results: Eleven patients were randomized to taliglucerase alfa 30 U/kg (n=6) or 60 U/kg (n=5). Median (interquartile range) for the primary endpoint, hemoglobin concentration, increased from baseline to month 12 by 12.2% (20.6) and 14.2% (10.4) for taliglucerase alfa 30 U/kg and 60 U/kg, respectively. By month 12, mean spleen volume decreased from 22.2 to 14.0 multiples of normal (MN) and 29.4 to 12.9 MN; mean liver volume decreased from 1.8 to 1.5 MN and 2.2 to 1.7 MN, platelet counts increased by 30.9% and 73.7%, and chitotriosidase activity was reduced by 58.5% and 66.1% for 30-U/kg and 60-U/kg doses, respectively. Nearly all adverse events were mild or moderate, unrelated to treatment, and transient in nature. No patient discontinued. Conclusion: As previously observed for adult patients, this study suggests that taliglucerase alfa has the potential to be a therapeutic treatment option for children with GD. Conflict of Interest declared. P-725 Long term effect of sebelipase alfa after one year in patients with late onset lysosomal acid lipase deficiency (LAL deficiency) Valayannopoulos V1, Malinova V2, Sharma R3, Bourdon C4, Boyadjiev S5, Kessler B6, Schneider E7, Twelves C8, Whitley C9, Quinn AG7 Ref Centre IEM, Necker-Enf Malades Hosp, Paris, France; 21st Faculty of Medicine Charles Univ, Prague, Czech Republic; 3Salford Royal NHS Foundation Trust, Salford, United Kingdom; 4Health Sciences North, Sudbury, Canada; 5Univ of California Davis Medical Center, Sacramento, United States; 6Eureka Internal Medicine, Eureka, United States; 7Synageva BioPharma Corp, Lexington, United States; 8 St James's University Hospital, Leeds, United Kingdom; 9University of Minnesota, Minneapolis, United States 1

The randomized blind start trial: a new study design to assess clinical outcomes in rare, heterogeneous patient populations Kakkis ED1, Signorovitch J2 Ultragenyx Pharmaceutical Inc., Novato, United States; 2Health Economics and Outcomes Research, Boston, United States 1

The clinical development of therapies for diseases as rare as mucopolysaccharidosis (MPS) 7, with < 100 identified patients worldwide, can benefit from improvements to conventional small-sample, open-label clinical trial designs. The novel Blind Start study design randomizes patients to ≥ 3 groups which initiate double-blind active therapy at different times from baseline, preceded by 0, 1, 2 or more intervals of placebo. Compared to a single-arm design, Blind Start provides more objective assessments of effort-based or patient-reported outcomes, and allows treatment effect estimation from double-blind initiation of active therapy. Since all patients are assessed from last pre-treatment assessment to a set interval post-treatment, Blind Start maximizes treatment outcome collection in limited sample settings, and reduces the impact of baseline randomization imbalance. Placebo treatment data can be used for additional supportive analyses. To evaluate the theoretical and practical utility of the Blind Start, a simulation study was conducted in comparison to single-arm and parallel-group designs. Results: indicate that Blind Start can improve precision for treatment effect estimation vs. parallel-group design and reduce risk of bias vs. single-arm design. Endpoint choice and statistical analysis strategies for the Blind Start design can maximize the assessment of treatment effects on multiple outcomes. Conflict of Interest declared. P-727 Comparison of growth patterns of patients with Mucopolysaccharidosis type II who started enzyme replecement therapy before 6 years of age and patients who were naove to ERT Różdżyńska-Świątkowska A1, Żuber Z2, Jurecka A3, Tylki-Szymańska A1 1 The Children's Memorial Health Institute, Warsaw, Poland; 2St Louis Children's Hospital, Cracow, Poland; 3University of Gdańsk, Gdansk, Poland

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Background: The mucopolysaccharidoses II (MPS II) leads to profound disruption in the normal mechanism of growth and development. For the reliable evaluation effects of treatment, it is necessary to compare them with the natural history of these diseases. Objective: To compare a natural growth pattern in children with MPS II with growth of MPS II patients who started enzyme replacement therapy (ERT) before 6 years of age. Material and Methods: An analysis of longitudinal anthropometric data of 11 patients (65 measurements) who started ERT before 6 years of age was performed and compared with retrospective analysis of data for 60 MPS II patients (280 measurements, naive for ERT). Results: The course of average growth curve for MPS II ERT group was very similar to growth pattern in MPS naïve group. But the average value of body height in subsequent years in MPS II ERT group was a bit greater than in MPS II naïve group. However this differences were not statistically significant. Conclusions: The growth pattern of MPS II patients who started enzyme replacement therapy before 6 years of age was similar to growth pattern of MPS II patients who were naïve to ERT. P-728 Efficacy of long-term velaglucerase alfa on haematological and visceral parameters in patients with type 1 Gaucher disease Zimran A1, Kisinovsky I2, Lukina EA3, Elstein D1, Zahrieh D4, Crombez E4, Giraldo P5 Shaare Zedek Medical Center, Jerusalem, Israel; 2Your Health SA, Buenos Aires, Argentina; 3Hematology Research Center, Moscow, Russian Federation; 4Shire Human Genetic Therapies, Lexington, United States; 5CIBERER, HU Miguel Servet, Zaragoza, Spain 1

Background: We assessed the long-term efficacy of velaglucerase alfa in patients with type 1 Gaucher disease (GD1). Methods: Treatment-naïve GD1 patients aged ≥2 years received velaglucerase alfa (45 or 60 U/kg every other week [EOW]) in two Phase III trials and an extension study. Results: 39 GD1 patients were randomized to 45 or 60 U/kg EOW and enrolled in the extension study. Mean (SD) treatment duration was 54.5 (9.9) months. At baseline: median age, 29 years (range 6, 62); n=8 <18 years; n=21 male; n=9 splenectomized. Longitudinal analysis showed mean (95% CI) changes from baseline to months 24, 36 and 60, respectively, for: haemoglobin (g/dL), 2.68 (2.25, 3.10), 2.68 (2.26, 3.11) and 3.32 (2.72, 3.92); and platelets (109/L), +100.3 (76.1, 124.6), +102.5 (77.9, 127.1) and +88.3 (54.3, 122.4). Longitudinal analysis showed mean (95% CI) changes from baseline to months 24, 39 and 63, respectively, for: splenic volume (n=30) -66% (-71%, -59%), (n=27) -71% (-76%, -66%) and (n=9) -78% (83%, -73%); and hepatic volume, -27% (-31%, -22%), -33% (-37%, -28%) and -39% (-44%, -33%). No new safety concerns were identified. Conclusions: Sustained improvements in haematological and visceral parameters were observed in GD1 patients receiving long-term velaglucerase alfa treatment. Conflict of Interest declared.

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Background: We report growth parameters/bone marrow burden (BMB) in children with type 1 Gaucher disease (GD1) after switching from long-term imiglucerase to velaglucerase alfa treatment. Methods: Children on ≥30 consecutive months' imiglucerase treatment at 15–60 U/kg every other week (EOW) switched to the same dose of velaglucerase alfa in 1-year trial TKT034 and continued in extension HGT-GCB-044. Results: 9 children (baseline: 2 male; median age 13 years [range 9, 16]) participated. Baseline: median (range) skeletal age and height Z-scores -0.12 (-2.45, 0.91) and 0.65 (-1.11, 1.31), respectively. Longitudinal analysis showed mean (95% CI) changes from baseline to Years 2 and 3 in skeletal age Z-scores of 0.69 (0.03, 1.35) and 0.64 (-0.18, 1.47) and to Years 2, 3 and 4 in height Z-scores of 0.05 (-0.11, 0.21), 0.05 (-0.15, 0.25) and -0.06 (-0.32, 0.20), respectively. One patient with skeletal age Z-score <-2 normalized by 2 years (Z-score 0.52); all others remained stable throughout velaglucerase alfa treatment for both skeletal age and height Z-scores. Baseline and final median (range) lumbar spine BMB scores: 1 (1, 5) and 3 (1, 5), respectively. Conclusion: Skeletal age, height-for-age and BMB remained stable in these (8) children on velaglucerase alfa after switching from imiglucerase. Conflict of Interest declared. P-730 Exploratory assessment of growth and bone marrow burden in a pooled subgroup of paediatric patients with type 1 Gaucher disease treated with long-term velaglucerase alfa Zimran A1, Kishnani P2, Elstein D1, Gonzalez DE3, Zahrieh D4, Crombez E4, Cohn GM4 1 Shaare Zedek Medical Center, Jerusalem, Israel; 2Duke University Medical Center, Durham, United States; 3Sanatorio Español, Asunción, Paraguay; 4Shire Human Genetic Therapies, Lexington, United States

Background: We report growth parameters and bone marrow burden (BMB) in a pooled subgroup of treatment-naïve children with type 1 Gaucher disease (GD1) treated with long-term velaglucerase alfa. Methods: The children received 45 or 60 U/kg velaglucerase alfa every other week in trials TKT032 or HGT-GCB-039, and extension HGTGCB-044. Longitudinal analysis assessed mean changes from baseline in skeletal age Z-scores, height Z-scores and BMB score. Results: 8 children (6 male; median age at baseline 8 years [range 6, 16]; 1 splenectomized; median [range] treatment duration 53.5 [46.5, 69] months) were included. Median (range) skeletal age and height Z-scores at baseline were -2.39 (-6.60, 0.01) and -1.54 (-2.70, -0.37), respectively. Mean (95% CI) changes from baseline to 24, 39 and 63 months in skeletal age Z-scores were 1.59 (0.64, 2.55), 2.63 (1.68, 3.59) and 3.52 (1.91, 5.12); and to 24, 36 and 60 months in height Z-scores were 0.58 (0.34, 0.81), 0.83 (0.59, 1.08) and 1.21 (0.80, 1.62), respectively. Baseline median lumbar spine BMB score (n=6): 5 (range 4, 8); this score improved by ≥2 points in 5 patients by the last available assessment. Conclusions: Growth parameters and BMB scores continued to improve in children over long-term velaglucerase alfa treatment. Conflict of Interest declared.

P-729

P-731

Exploratory assessment of growth and bone marrow burden in a paediatric subgroup with type 1 Gaucher disease transitioned from imiglucerase to velaglucerase alfa

Clinical outcome of 5 Mexican patients with Mucopolysaccharidosis type I in enzyme replacement therapy

Giraldo P1, Smith L2, Harmatz P3, Zahrieh D4, Crombez E4, Cohn GM4 CIBERER, HU Miguel Servet, Zaragoza, Spain; 2Children's Mercy Hospital, Kansas City, United States; 3Children's Hospital Oakland, Oakland, United States; 4Shire Human Genetic Therapies, Lexington, United States

Ruiz-Cruz ED1, Campos-García FJ1, Carpio-Hernández JC2, VegaRamírez ME 3 , Márquez-Gutiérrez MA 1 , Ricárdez-Marcial EF 1 , Franco-Ornelas SJ3, González-Vite M3

1

1 Div Med Genet, IMSS La Raza, Mexico City, Mexico; 2Div Pediat Cardiol, IMSS La Raza, Mexico City, Mexico; 3Div Pedia, IMSS la Raza, Mexico City, Mexico

J Inherit Metab Dis (2013) 36 (Suppl 2):S91–S342

Background: Mucopolysaccharidosis type I (MPSI) is due to the enzymatic deficiency of alpha-l-iduronidase, causing malfunction of lysosomes and accumulation of glycosaminoglycans in different organs that leads to a intellectual disability, sight and movement impairment. enzyme replacement therapy can improve some of these complications. Objective: Evaluate the integral clinical evolution of five Mexican patients with MPSI in ERT. Patients and Methods: We analyzed five Mexican patients (three males, two females) with MPSI. Diagnosis was made by clinical examination and enzymatic activity analysis. Three patients have received ERT with Laronidase for 6 years, the other two for one year. A complete clinical check-up was performed yearly, following national and international guidelines. Afterwards, data was collected and analyzed. Results: ERT decreases lower respiratory infections and hepatic size, also improves life expectancy, however with significant disability. We observe no differences from natural history of MPSI in size, skeletal phenotype, CNS and cardiovascular system. Conclusion: We observe no regression either arrest of MPSI in ERT. Only respiratory and hepatic phenotypes improve remarkably. Some aspects like ophthalmologic, cardiovascular and skeletal phenotypes show slow progression.

P-732 Long term safety analysis of BMN110 dosed at 2 mg/kg/week in 52 subjects with mucopolysaccharidosis (Morquio A syndrome, MPSIVA) Harmatz P1, Hendriksz CJ2, Giugliani R3, Quartel A4, Farmer P4, Slasor P4, Butine M4, Haller C4 1

Children's Hospital & Research Center, Oakland, United States; Birmingham Children's Hospital NHS Found, Birmingham, United Kingdom; 3Department of Genetics/UFRGS and INAGEMP, Porto Alegre, Brazil; 4BioMarin Pharmaceutical Inc., Novato, United States 2

A clinical development program investigating safety and efficacy of BMN110, an enzyme replacement therapy for treatment of MPSIVA, was conducted. A long-term safety analysis was done on a subset of 52 subjects with >48 weeks (49-100.1 weeks) of BMN110 exposure at 2.0 mg/kg/week. Mean duration of exposure was 75.3(± 17.49) weeks, and mean weekly dose was 1.99 (± 0.039) mg/kg. To account for varying durations of follow-up in ongoing studies, frequencies of adverse events (AEs) are reported as standardized on an annualized basis. Mean subjectyear frequency of all AEs decreased from 33.33 during the 1-12 week interval to 11.68 during the >48 week treatment duration. Subject-year frequency of the most common AEs, including vomiting, pyrexia, and headache, decreased with treatment duration. Infusion Associated Reactions (IARs) were reported for all subjects, and mean subject-year frequencies decreased with treatment duration. Overall, mean annualized frequency was 11.13 IARs per subject-year. The most common IARs by incidence (and annualized frequency) were pyrexia, 51.9% (0.91), vomiting 46.2% (1.13), and headache 38.5% (1.04). Of the 3630 infusions administered, 23 (0.63%) were interrupted/discontinued due to an AE requiring medical intervention. There were no deaths and no AEs resulting in permanent study discontinuation reported in this subset of subjects. Conflict of Interest declared.

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P-733 A multicenter, open-label, extension study to evaluate the long-term efficacy and safety of BMN110 in patients with mucopolysaccharidosis IVA (MPS IVA, Morquio A syndrome) Hendriksz CJ1, Vellodi A2, Jones S3, Hiwot T4, Santra S5, Slasor P6, Decker C6 1 Birmingham Children's Hospital NHS Found, Birmingham, United Kingdom; 2Great Ormond Street Children's Hospital, London, United Kingdom; 3St Mary's Hosp, Manchester Acad Health C, Manchester, United Kingdom; 4New Queen Elizabeth Hospital, Birmingham, United Kingdom; 5Birmingham Children's Hospital, Birmingham, United Kingdom; 6BioMarin Pharmaceutical Inc., Novato, United States

Twenty MPSIVA patients enrolled in a Phase 1 / 2 36 week dose escalation (0.1, 1.0, 2.0 mg/kg/wk) safety and efficacy study of BMN110 enzyme replacement therapy, followed by a 36-48 week continuation phase at 1.0 mg/kg/wk. 17 patients enrolled in the ongoing extension study at 2.0 mg/kg/wk evaluating long-term outcomes. Interim results (as of July,2012) indicate BMN110 was well tolerated, with <1% of infusions interrupted or discontinued due to adverse event requiring medical intervention. Means trend to increase from baseline in 6-minute walk test and 3-minute stair climb by 13.8 (±63.25) meters and 7.8 (± 13.96) stairs/min after 36 weeks, and 27.2 (±62.51) meters and 9.6 (±19.63) stair/min after approximately 120 weeks. Means trend to decrease by 52.7 (±133.78) meters and 3.3 (±21.97) stairs/min after approximately 156 weeks, due to 4 patients with recent orthopedic surgery who had large decreases in endurance measures which reversed at the following assessment. Respiratory function improved by 16.1 (± 21.96)% in FVC and 10.1(± 27.83)% in MVV after approximately 156 weeks. Urinary keratan sulfate levels decreased and remained low over time, with lowest levels occurring during 2.0 mg/kg/wk dosing in both the dose escalation phase and extension study, demonstrating the drug's pharmacodynamic effects. Conflict of Interest declared. P-734 Plant cellexpressed recombinant glucocerebrosidase: taliglucerase alfa as therapy for Gaucher disease in adults patients previously treated with imiglucerase: 24-month results Pastores GM1, Shankar SP2, Petakov M3, Giraldo P4, Rosenbaum H5, Amato DJ6, Chertkoff R7, Almon-Brill E7, Zimran A8 1 New York University School of Medicine, New York, NY, United States; 2Emory University School of Medicine, Decatur, GA, United States; 3Clinical Center of Serbia, Belgrade, Serbia and Montenegro; 4 Hospital Universitario Miguel Servet, Zaragoza, Spain; 5Rambam Medical Center, Haematology, Haifa, Israel; 6Mount Sinai Hospital, Toronto, Ontario, Canada; 7Protalix Biotherapeutics, Carmiel, Israel; 8 Gaucher Clinic, Shaare Zedek Med. Ctr., Jerusalem, Israel

Background: Taliglucerase alfa is a plant cell–expressed betaglucocerebrosidase approved for enzyme replacement therapy in adults with type 1 Gaucher disease (GD). Objectives: To evaluate the safety and efficacy of taliglucerase alfa in patients previously treated for ≥2 years with imiglucerase. Methods: Study PB-06-002 was a 9-month, phase 3, multicenter, open-label, switchover trial. Eligible patients entered a 12-week disease stability evaluation period based on hematologic parameters. Patients with stable disease were switched from imiglucerase to the same dose of taliglucerase alfa. The control was each patient's historical clinical status. At completion of the 9-

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month study, patients were eligible to enter an open-label extension study, PB06-003. Results: This is an interim report of 15 adult patients who completed 24 months of treatment. Efficacy parameters of hemoglobin concentration, platelet counts, chitotriosidase levels, liver volume and spleen volume were maintained or improved in these patients following switchover to taliglucerase alfa. None of the patients met the criteria for clinically relevant deteriorations as specified by the study protocol. All treatment-related adverse events were mild or moderate in severity and transient in nature. Conclusions: This 24-month evaluation demonstrated that taliglucerase alfa is a safe and effective alternative treatment for GD in patients switched from imiglucerase. Conflict of Interest declared.

Results: The age of diagnosis was 53±36,7 (14-130) days (age: 17±14,3 months, min:8, max:43). Initial symptom was cardiomegaly in three patients, cyanosis, hypotonia, supraventricular tachycardia, and positive family history in the others. ERT was started at a dose of 40mg/kg/every other week in the first year of therapy, and then decreased to 30mg/kg. The first positive effect of ERT was decreased frequency and severity of respiratory problems. Reduced hypotonia, and increased ejection fraction are the other significant benefits. Three of them were able to walk. Conclusions: There is not a consensus about the dose of ERT in pompe. An ERT dose of 40mg/kg in the first year and 30mg/kg in the second year provided significant benefits in our patients. But more detailed studies with larger groups are needed in order to evaluate the optimal dose.

P-735

Preliminary findings evaluating safety and efficacy of recombinant human n-acetylgalactosamine-6-sulfatase (rhGALNS) in pediatric patients less than 5 years of age with Mucopolysaccharidosis IVA (Morquio A syndrome, MPSIVA)

Comparative analysis of taliglucerase alfa efficacy with enzyme replacement therapies for Gaucher disease Chertkoff R1, Paz A1, Almon-Brill E1, Fletcher N2, Golembo M1

P-737

Jones S1, Harmatz P2, Bialer M3, Parini R4, Martin K5, Farmer P5, Slasor P5, Haller C5

Protalix Biotherapeutics, Carmiel, Israel; 2Pfizer, Sydney, NSW, Australia

1

Background: Taliglucerase alfa is a plant cell–expressed betaglucocerebrosidase approved for enzyme replacement therapy in adults with type 1 Gaucher disease (GD). Objectives: To assess the efficacy of taliglucerase alfa according to a 3category classification system and compare it with imiglucerase and velaglucerase alfa. Methods: Retrospective analysis wherein responses were categorized with respect to changes from baseline of the following: spleen or liver volume reduction good (≥30%), moderate (≥10% and <30%), no response (<10%); hemoglobin increase good (≥1.5 g/dL), moderate (>0.5 and <1.5 g/dL), no response (≤0.5 g/dL); platelet count increase good (>30x109/L), moderate (>15x109/L and ≤30x109/L), no response (≤15x109/L). Results: Most or all patients receiving taliglucerase alfa 60 U/kg showed good or moderate responses at months 9, 12 and 24 months. Responses to imiglucerase 60 U/kg, velaglucerase alfa 60 U/kg, and taliglucerase alfa 60 U/kg were similar at the common time point of 9 months; responses to velaglucerase alfa and taliglucerase alfa were similar at the common time point of 12 months. Conclusions: Efficacy responses to 60U/kg taliglucerase alfa continued through 24 months of treatment and at this dose, appear to be comparable for taliglucerase alfa, imiglucerase, and velaglucerase alfa in this retrospective analysis using a 3-category classification system. Conflict of Interest declared.

1 St Mary's Hosp, Manchester Acad Health C, Manchester, United Kingdom; 2Children's Hospital & Research Center, Oakland, United States; 3North Shore LIJ Health System, Manhasset, United States; 4Az. Ospedaliera S. Gerardo, Monza, Italy; 5BioMarin Pharmaceutical Inc., Novato, United States

Preliminary results after 26 weeks of treatment from an ongoing study evaluating safety and efficacy of rhGALNS in 15 MPSIVA subjects <5 years of age are reported. The mean(range) age was 3.1(0.8-4.9) years. Standing height/length (n=15) was severely affected in many subjects; 7 (46.7%) at <3rd, 3(20.0%) at >3rd-<10th, 2(13.3%) at >25th-<50th, and 3 (20.0%) at ≥ 50th percentiles. The most commonly reported adverse events (AEs) were vomiting in 12 (80%), pyrexia in 11 (73.3%), and cough in 8 (53.3%) subjects. The majority of AEs were mild-moderate with 1 event of Grade 3 (severe) tonsillar hypertrophy. No subjects discontinued due to an AE. rhGALNS treatment had a similar safety profile as seen in older children and adults. Normalized urine keratan sulfate (uKS) was increased with a mean(range) of 35.9(18.8-56.5) μg/mg creatinine (n=15). In 8 subjects with 26 weeks of data, rhGALNS led to a substantial decrease in mean(±SD) normalized uKS by 30.5(±15.49)% after 2 weeks and sustained at -35.2(±15.57)% at 26 weeks. Mean height/length for age z-scores didn't demonstrate significant change from Baseline (1.8SD) to week 26 (-2.2SD) for these 8 subjects. Anthropometrics will continue to be assessed in all subjects to determine impact of rhGALNS intervention on long term growth. Conflict of Interest declared.

P-736 P-738 Enzyme replacement therapy in 7 Turkish patients with infantile type Pompe disease

A new assay for fast, reliable crim status determination in infantile-onset Pompe disease

Kör D1, Önenli-Mungan N1, Erdem S1, Hergüner Ö1 Keutzer J1 1

Çukurova University, Adana, Turkey 1

Genzyme, a Sanofi Company, Cambridge, United States

Background: Pompe disease is a lysosomal storage disorder caused by the deficiency of acid α-1,4-glucosidase which results within the accumulation of glycogen. The infantile type presents with hypertrophic cardiomyopathy, hypotonia, respiratory problems, and death generally in the first year of life. After the introduction of enzyme replacement therapy (ERT) the prognosis gets better. We report the results of ERT in 7 CRIM(+) infantile Turkish patients.

Pompe disease is caused by a deficiency of acid α-glucosidase (GAA; EC, 3.2.1.20), and the infantile-onset form is rapidly fatal if left untreated. However, recombinant human GAA (rhGAA) enzyme replacement therapy (ERT) extends survival for infantile Pompe patients. Although crossreactive immunologic material (CRIM) negative patients, who lack detectable endogenous GAA, mount an immune response to rhGAA that

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renders the therapy ineffective, timely induction of immune tolerance in these patients may improve clinical outcomes. Previously, CRIM status has been determined by Western blot analysis in cultured skin fibroblasts, a process that can take several weeks. We present a blood-based CRIM assay that can yield results within 48 to 72 hours. Results: from this assay have been confirmed by GAA Western blot analysis in fibroblasts or by GAA sequencing in a small number of Pompe disease patients. Rapid classification of CRIM status will assist in identifying the most effective treatment course and minimizing treatment delays in patients with infantile-onset Pompe disease. Conflict of Interest declared.

Methods: Medical records were audited for demographic and laboratory data and documentation of treatment effects. Results: Mean frequency of blood tests was every 2.5 weeks. All patients had a 12 month mean Phe of <600μmol/L. Amino acid supplements provided a mean daily protein intake of 1.1g/kg of ideal body weight. Positive treatment effects were most commonly observed in skin condition and behaviour. In some cases care needs were reduced and new skills were obtained. Conclusion: Uncounted low protein diets in this cohort resulted in outstanding compliance and Phe levels. Positive treatment effects were observed in all patients. P-741

17. Dietetics and nutrition Adult Refsums disease: micronutrient status with long term low phytanic acid diet therapy

P-739 Satisfaction survey on a new tool for therapeutic education of parents with PKU children: "le phi tout"

Baldwin EJ1, Feher MD1 1

Chelsea & Westminster Hospital, London, United Kingdom

1

Spiewak Ms 1

Children's Hospital Pellegrin, Bordeaux, France

Spiewak A.1, Ducournau E.1, Viguier S.2 and the dieticians from the SFEIM3 1 CHU Pellegrin, Bordeaux France, 2 Merck Serono s.a.s, Lyon France, 3 Société Française des Erreurs Innées du Métabolisme, France Introduction: PKU patients have few practical tools for the management of their diet. A group of French dieticians therefore decided to create an educational tool to allow patients to easily calculate the Phe content of industrial food. Aims: The objective of this survey was to evaluate satisfaction of dieticians and families. Material and Methods: 107 PKU patients and 22 dieticians evaluated the tool with 2 different satisfaction questionnaires, using a 5-point scale. Blood Phe levels were collected to evaluate possible impact. Results: The best rated items for families were the visual appearance, practical aspect and the interest of a consensus tool. Dieticians appreciated the independency of families and the interest of a consensus tool. There was no negative effects on metabolic balance. Conclusions: Families and dieticians were very satisfied with the tool. 88% of families declared that they wish to continue using it. No negative impact on blood Phe levels was observed. P-740

Background: Adult Refsum's Disease is a disorder of peroxisomal fatty acid metabolism. Phytanic acid (3,7,11,15 tetra-methyl-hexadecanoic acid) metabolism is impaired and accumulates. Clinical features include retinitis pigmentosa, anosomia , deafness, icthyosis and cardiomyopathy. Chronic management is a low phytanic acid diet. Ojectives: To determine the nutritional status of individuals adhering to the Westminster low phytanic acid diet. Method: Dietary intake was assessed by either a seven day weighed food intake or four twenty-four hour recalls taken as a series (including one weekend day and calculated using a dietary software analysis package). Anthropometry and blood analysis taken prior to the recording of dietary intake. Results: From 11 (4 women, 7 men) subjects, Body mass index ranged from 19-38 (mean 27, median 27). Fat soluble vitamin deficiencies were confirmed; vitamin D ( n=6); vitamin E ( n=3); vitamin K ( n=10); vitamin A ( n=2): low iron status was a feature (n=7). All subjects had high sodium intakes (mean intakes 1873-4828mg). Low copper (n=6) and selenium intakes (n=2) were also confirmed. Vitamin B12 status was low (n=4). Conclusions: Comprehensive nutritional screening should be a feature of long term clinical management in Adult Refsum's Disease to prevent and treat micronutrient deficiencies. P-742 Free intake of some vegetables and fruits in treatment of phenylketonuria (PKU)

Successful treatment of late diagnosed adults with phenylketonuria using an uncounted approach to the low protein diet

Hamilton V1, Benedetti G1, Castro G1, Arias C1, Raimann E1, Cabello JF1, Valiente A1, Betta K1, Colombo M1, Cornejo V1

Clover EC1, Chapman IM2, Fletcher JM3 1

Inst Nut Food Tech, Univ of Chile, Santiago, Chile

1

2

Dept Clin Dietetics, Royal Adelaide Hosp, Adelaide, Australia; Div of Medicine, Univ of Adelaide, Adelaide, Australia; 3Genetics & Molecular Path, SA Path, Adelaide, Australia Introduction: Numerous patients with late diagnosed phenylketonuria (PKU) have been referred for treatment. We report the outcomes of treatment in this group by an uncounted approach to diet. In our clinic, caregivers (and independent patients) are educated on an uncounted low protein diet and inclusion of low protein food products. A suitable amino acid supplement is prescribed. Patients are reviewed by physician and dietitian 6 monthly. Phenylalanine (Phe) levels are determined at baseline then fortnightly by tandem mass spectrometry on filter paper specimens collected at home, reported by the dietitian. Patients: 12 adults (6 female, mean age 57 years) with late diagnosed PKU.

Introduction: Phenylketonuria treatment consist in a restricted phenylalanine (Phe) intake, mainly based on special formula, vegetables, fruits and few amounts of cereals. The Phe levels must be maintained below 8 mg/dl. Objective: evaluate if free intake of some vegetables and fruits alters Phe level. Methods: 8 PKU patients 3–15 years old, were included in a longitudinal study. Phe intake was maintained and children could consume as free food all fruits and vegetables with less than 75 mg Phe/100g. Phe intake record was done daily and Phe levels were measured once a week (mass spectrometry method). Results: 4 patients were males (6.5±2.4 years old) and 4 females (7.6±4.6 years old). Before the study Phe intake was 453.6±104.6 mg/d, post intervention increased to 555.7±134.8 mg/d (p<0.05). They ate 30% additional Phe intake from cereal, vegetables and fruits with more than

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75 mg Phe/100 g (p<0.05). Had 102 mg extra Phe intake from vegetables and fruits with less 75 mg Phe. Phe levels before study were 4.1±0.8 mg/dl, and increased to 5.3±0.9 mg/dl during the study (p<0.05). Conclusions: Study suggests that liberation of vegetables and fruits containing less than 75 mg Phe does not increase Phe level above normal range.

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protein/kg/day of which a median of 68% was from phe free protein supplement (prescribed for 27 patients). Moderate to high protein foods or low protein food products were not eaten regularly and half the group did not eat vegetables daily. Mothers expressed a range of concerns about PKU management but most commonly were a little or not worried overall (12/22). There was high expectation of BH4 therapy.

P-743 G-tube in inherited metabolic disorders: improving antropometric parameters and hospitalizations Guillén-López S1 , Villegas-Hernández W2, Monroy-Santoyo S1 , Guevara-Martínez YC2, Vela-Amieva MB.1 National Institute of Pediatrics, Mexico City, Mexico; 2Div Biolog Scien, Univ Aut Metrop, Mexico City, Mexico 1

In diet-dependent inborn errors of metabolism (IEM) nutrition becomes the main treatment, failure to ingest oral adequate energy intake is a frequent problem. Objective: To compare anthropometric data and hospitalizations before and after the g-tube placement in a group of diet-dependent patients with IEM from the National Institute of Pediatrics in Mexico City. Materials and Methods: Retrospective review of 12 patients (age 19 to 144 months) with IEM treated at a tertiary care center. Z scores of weight for height (W/H), weight for age (W/A) and lenght/heigh for age (H/A) and hospital admissions, captured form hospital records, were obtained before and after g-tube. Results: Adequate nutrition status improved for W/H parameter in 41.6% of patients with a Z score of >-1 after g-tube placement, W/A change from 8.3% of our patients before g-tube placement to 25% of patients without acute malnutrition, above -1, with a 16.7% difference. Lastly, H/A increased 8.3%, with a total of 16.6% of patients with a normal heigh for their age. Hospital admissions number decreased from 35 to 18 (48%) after the G-tube intervention. Discussion/Conclusion: G-tube reduced 48% the number of hospital admissions, and is also a useful tool for improving weight, height, additionally prevents decompensations. P-744 Towards BH4 therapy in phenylketonuria: a survey of baseline characteristics, nutrition, food variety and impact of management

P-745 The first Belgian patient diagnosed with citrin deficiency presenting with hepatic cholestasis in infancy Eyskens FJM1, Van De Vijver E2 1 Div Metab Dis, Dept of Paediatrics,UZA, Antwerp, Belgium; 2Div Pediatric Gastroenterology,UZA, Antwerp, Belgium

Case presentation: A male infant, non-consanguineous Belgian parents, was sent to our hospital with severe failure to thrive and growth retardation, cholestatic icterus, hepatomegaly, hypoproteinemia, gastrointestinal bleeding due to decreased coagulation factors. Blood ammonia was only slightly elevated (80-100 μmol/L); the serum amino acid pattern was very suggestive for citrin deficiency: elevated concentration of citrulline, arginine, alanine, threonine. Pancreatic secretory trypsin inhibitor (PSTI) was not measured. Identification of biallelic mutations in SLC25A13, the only gene in which mutations are known to cause citrin deficiency, confirmed the diagnosis: the patient is homozygous for a new splice site mutation c.15G>C (p.K5N), both parents are carriers of the same mutation! Management: The patient was put on a high-protein, lactose-free formula containing medium-chain triglycerides.Fat-soluble vitamins were supplemented. L-arginine was given at a dose of 100 mg/kg/day to prevent hyperammonemia. In addition to dietary treatment, administration of sodium pyruvate may improve growth. This was not needed in our patient as he thrived well under the present diet. Conclusion: Until recently, citrin deficiency was thought to be restricted to Japan; citrin deficiency is now recognized to be pan ethnic. In our knowledge this is the first Belgian patient detected with this rare inborn error of metabolism. P-746

Williams E1, Dennison B2, Watson P2, Thompson S M2 Univ Sydney, Sydney, Australia; 2Gen Metab Dis Serv, Child Hosp Westmead, Sydney, Australia 1

Dietary recommendations for adolescents and adults with classical Galactosaemia - a German survey Meyer U1, Das AM1

Sapropterin (BH4) therapy for phenylketonuria (PKU) is not yet routinely available in Australia. This study determined nutritional intake, food variety, clinical measures and psychosocial impacts of dietary management of hyperphenylalaninaemia/PKU in 28 of 40 children, likely to be responsive to BH4 but not currently receiving BH4 therapy. Four were classified as classical PKU, 24 with milder variants. Clinical records were retrospectively reviewed for diet prescription, anthropometry and blood phenylalanine(phe). Dietary intake and food variety was determined using three non-consecutive 24-hour recall phone interviews and a qualitative food frequency questionnaire. A modified version of the PKU Worry Checklist rated concern about current management and expectations of BH4 therapy. Growth was in the normal range and median blood phe in previous year was 295 μmol/l. Reported protein intake ranged from 0.7-2.9g

1

Department of Paediatrics Medical School, Hannover, Germany

Introduction: Based on recent findings showing that the main source of galactose in adolescents/adults is endogenous production, we recommend relaxation of the diet after the age of 14 years. We distributed a modified version of the questionnaire from the UK Galactosaemia Support Group translated into German among dietitians in Germany, Austria and Switzerland (DACH region). Results: 10 centres responded, 6 German and one Swiss metabolic centre reported their dietary recommendations for 40 adults with galactosaemia. All centres stated that they do not restrict vegetables, legumes and fruits. However, milk and lactose-containing foods are forbidden. The

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recommendations vary regarding the selection of cheese, offal and hidden sources of lactose and how to relax the diet for adolescents and adults with galactosaemia. Only 2 centres recommend relaxation of the diet after the age of 14 years, 5 centres recommend strict diet in adults, however 4 of them do not believe that there is a need for a strict diet after childhood. Discussion: In contrast to current scientific evidence, most centres in the DACH region stick to strict dietary recommendations in adolescence and adulthood. Metabolic centres should be encouraged to recommend relaxation of the diet after 14 years of age.

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The "UCD widget" elaborated by metabolic dieticians and metabolic physicians needs a widely usage to confirm its interest and eventually improved it. P-749 Micronutrient intake from phenylalanine-free supplements used as part of the dietary management of phenylketonuria (PKU) in south Australia Sweeney AL1, Hart SJ2

P-747 Controlled diet in phenylketonuria and hyperphenylalaninemia may cause serum vitamin-B12 and folic acid deficiency: the Czech experience Prochazkova D1, Jarkovsky J1, Vinohradska H1, Mikuskova A1, Jagerova J1, Konecna P1, Machacova L1, Dolezel Z1 1

Med Fac of Masaryk Univ, Univ Hosp, Brno, Czech Republic

Background: Phenylketonuria (PKU) is an inherited disorder of metabolism of the amino acid phenylalanine caused by a deficit of the enzyme phenylalanine hydroxylase. PKU patients are at risk for vitamin-B12 deficiency based on their low-protein diet. The aim of this study was to determine the prevalence of vitamin-B12 deficiency in these patients and correlation of parameters of vitamin-B12. Methods: In 51 phenylketonuric (PKU) and hyperphenylalaninemic (HPA) patients aged 3-48 years, were studied concentrations of vitaminB12, folic acid in serum, plasma concentrations of homocysteine, and methylmalonic acid concentrations in urine. The metabolic control of the patient was defined as the median of concentrations of serum phenylalanine and tyrosine over a period of 6 months retrospectively. Results: We confirmed a statistically significant difference in the serum concentrations of vitamin-B12 (p<0.001; Mann-Whitney U test) and folic acid (p=0.004 Mann-Whitney U-test) in adult PKU and HPA patients. Conclusions: These results indicate that controlled low-protein diet for PKU and HPA may cause a serum deficiency of vitamin-B12 and folic acid in adult patients.

1 Dep Nutrition, Women's & Children's Hosp, Adelaide, Australia; 2Nut Diet Stud, Flinders Uni, Adelaide, Australia

Background: With an increasing number of phenylalanine-free supplements, containing varying amounts of micronutrients, it has become difficult to quickly assess adequacy of supplement intake as patients mix and match supplements in form, flavour and type to assist in compliance. Method: Micronutrient intake from supplements for 27 paediatric patients attending the WCH PKU Clinic were analysed and assessed against the Australian Nutrient Reference Values (NRVs) for adequacy and excess. Micronutrients from patients' diets were not included. Results: Inadequate intake was identified for calcium (n=7), phosphorus (n=10), magnesium (n=3) selenium (n=1), potassium (n=26), manganese (n=18) and folate (n=9). Upper limits were exceeded for copper (n=5), iron (n=1), zinc (n=15) magnesium (n=23), folate (n=3) and Vitamin A (n=3). Conclusion: Food consumed as part of a PKU diet provides some micronutrients identified inadequate e.g. fruit is a good source of potassium. Micronutrient intakes which are low in a PKU diet e.g. calcium, need to be addressed. Micronutrient intake exceeding the upper limit needs assessment for long term safety and effect on bioavailability of other micronutrients. These findings reinforce the need for full nutritional assessment of supplement, diet and nutritional blood tests as part of PKU management to ensure against deficiency and toxicity of micronutrients. P-750 The importance of the diet in the treatment of hypertrophic cardiomyopathy with type 1 hyperlipoproteinemia

P-748 The UCD widget : handy software for dietetic management of UCD patients

Gündüz M1, Koc N1, Özaydın E1, Ekici F2, Tarugi P3 1

1

2

Dubois SD , Wenz MJ

1 Metab Unit, Necker Hosp., PARIS, France, 2Pediatric liver unit, Bicêtre Hosp, Le Kremlin Bicêtre, France

Urea cycle disorders (UCD) are chronic diseases which management requires a strict and life-long follow up. Outcome depends on the good metabolic status and the severity of their hyperammonaemic Decompensation. The "UCD widget" is a new and handy software designed for dieticians in their daily practice and is the first tool proposed for the following up of these patients. It includes synopsis of clinical presentations, biological normal values and therapeutic objectives in UCD. Examples of UCD diets exposing various situations (emergency diet, nutriments calculation, from neonates to adulthood, etc.) are available and could be printed. Data from patients are collected and allow the follow up of physical, biological and nutritional evolution through three types of graphics: Decompensation graphics showing the evolution of ammonaemia and glutaminaemia; Weight graphics; Biochemical graphics showing evolution of orotic acids, amino acids and organic acids.

Div Metab Dis,Ankara Dıskapı Child Hosp, Ankara, Turkey; 2Dep of Cardio,Ankara Dıskapı Chil Hosp, Ankara, Turkey; 3 Dep of Biochemistry,Modena Unv, Modena, Italy Background: Type 1 hyperlipoproteinemia is caused by genetic deficiency of lipoprotein lipase (LPL) or its cofactor apo C II with a frequency of 1/1,000,000. We present a case with type 1 hyperlipoproteinemia and hypertrophic cardiomyopathy (HC) of whom the cardiac findings improved with only diet therapy. Case Report: A 1 month old baby was investigated as his brother was followed in our department. The tryglyceride and cholesterol levels were very high. Prominent HC was observed on echocardiography. The baby was started on a fat restricted diet involving formula with medium-chain tryglycerides (MCT). In the patient, a homozygote mutation (c.644G>A) was found in the LPL gene. From the sixth month of his life, complementary feeding was started, the special formula was continued and MCT oil was added to the meals. Skimmed milk and milk-products, low portion meat, chicken were started gradually in the following months. After two year follow-up, lipid levels were between 1500 and 2000 mg/dl. HC had disappeared in the final cardiologic examination. Conclusion: HC is an unexpected finding in the patients with type 1 hyperlipoproteinemia which can be treated successfully with diet.

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P-751 PKU - So what? living with PKU. a movie to encourage patients and their parents Ellerbrok M1, Schultz S1, Tsiakas K1, Mühlhausen C1, Muschol N1, Santer R1 1

University Medical Center, Hamburg, Germany

Introduction: The diagnosis of PKU often resembles a shock, and parents feel unprepared to deal with these complex challenges. Brochures and training materials explain therapy, but cannot convey how everyday life with PKU can be realized. Therefore, we created a movie intended to help fulfill this desire, and to present a positive prognosis, showing the steps that have to be taken to realize this outcome. Content: The movie shows families talking about their experience after diagnosis. Children are shown at different ages and situations. Parents and children speak openly and candidly. Through seeing children in their recreation, we can see them beyond their diagnosis. Parents can see how sharing their experiences with other parents of affected children can be an invaluable source of advice and support. Conclusion: Our film looks at PKU and the special features of living with it in a frank and warm manner. It is designed to be especially helpful to parents of newly diagnosed children, but also for relatives, teachers, kindergarten nurses and friends. It aims to build understanding and acceptance of PKU, while encouraging the viewer to see how they or their loved ones can live a happy and fulfilled life with PKU. P-752 Self-perceived competence of children with a metabolic disease Simons A1, Van Impe S1, Eyskens FJM2 1 UKJA, ZNA Middelheim, Antwerp, Belgium; 2Div Metab Dis, Dept of Paediatrics,UZA, Antwerp, Belgium

Objectives: The self-perceived competence of children with a metabolic disease was examined. Perceived competence is a factor that influences the motivation of a child (Denham, Wyatt, Bassett, Echeverria, and Knox, 2008). Motivation is an important factor in compliance. It is well known that therapy compliance is a great challenge for persons with a metabolic disease and their caregivers. Methods: Thirty-five children between eight and eighteen years completed the Dutch version of the Self-Perception Profile for Children (CBSK) and Adolescents (CBSA). Results: In this sample we found indications for a deviating self-esteem on various domains; school skills(32.3%), social acceptance (41.2%), sporting skills (35.3%), physical appearance (45.7%), behavior attitude (40%) and global self-esteem (37.2%). Discussion: To optimize therapy adherence psychological interventions can be needed. Literature showed that a feeling of competence is a necessary factor for maintaining therapy. When therapy compliance is an issue, it is important to consider all domains, and work on perceived competence and self-esteem of patients and their environment.

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Introduction: Phenylketonuria (PKU) is a metabolic disease characterized by increased plasma levels of phenylalanine (FA) which cause mental retardation. Through the National Neonatal Screening Programme for PKU 197 cases were diagnosed with classical PKU (100 male and 97 female). Methodology: The treatment involves restricted intake of FA, formula without FA, medical and nutritional assessments, psychometric (Weschsler Bayley Scale) and biochemical evaluations (MS/MS). Results: Average age of diagnosis: 17.6±9.5 days, with initial FA values 18.5±8.9 mg/dl and tyrosine 1.2±0.7 mg/dl. Current age range: 1 month to 22 years. Average diet provided: 18.6±14 mg/kg/day, 2.0±0.6 g Prot/kg/day (75% of the formula), 1251 ± 47 kcal/day. 83% of the PKU patients maintained blood FA values under 8.5 mg/dl (good metabolic control), there was a significant correlation (p <0.01) with IQ. 88% of PKU patients have normal IQ, 11% borderline and 1% mild delay. 69% have normal nutritional status, 14.4% are overweight, 12.4% are obese and 4% are underweight. Conclusion: Diagnosed PKU children neonatal growth and development are within normal ranges when FA levels are maintained under 8.5mg/dl during follow-up. P-754 Dramatic effect of high performance activity on phenylalanine levels in an adolescent female with phenylketonuria MacLeod EL1, Cusmano-Ozog K1 1

Div Gen Metab, Child Nat Med Center, Washington, DC, United States

Case Report: Classical phenylketonuria (PKU) is a metabolic disorder that requires tight dietary control and most individuals tolerate a limited quantity of natural protein to provide the essential amino acid phenylalanine. We present here a 12 year pre-menarche old female with PKU with a history of good dietary control and compliance who now engages in high performance swimming. Before swim activities became extremely competitive, blood phenylalanine averaged 300mg/dL. Since beginning high performance swim, phenylalanine levels have fluctuated between 27 and 1309μmol/L following a variety of dietary interventions. These interventions included: increasing and decreasing natural protein intake, increasing frequency and protein of medical food, increasing calories, and providing activity specific diet requirements. When all activity stopped for two weeks, blood phenylalanine levels dropped from 719μmol/L to 280μmol/L with no dietary intervention. While she did not engage in activity, blood phenylalanine levels averaged 168μmol/L. Once high intensity swimming restarted, blood phenylalanine levels spiked from 84μmol/L to 618μmol/L in four days. Conclusion: Individuals with PKU who engage in high performance activities must be monitored closely and their diet adjusted frequently to account for high protein turnover. Moreover, the effect of high performance activities in individuals with inborn errors of metabolism warrants further investigation. P-755 Intestinal graft versus host disease after hematopoietic stem cell transplantation:successful dietary management

P-753

Koc N1, Gündüz M1, Azık F2, Tavil B3, Özaydın E1, Çetinkaya D3

Follow up of children with classic phenylketonuria diagnosed by newborn screnning program in Chile: 20 years of experience

1 Div Metab Dis,Ankara Dıskapı Child Hosp, Ankara, Turkey; 2Dep of Hem,Ankara Dıskapı Child Hosp, Ankara, Turkey; 3Dep of Ped Hem, Hacettepe Unv, Ankara, Turkey

Castro G1, Arias C1, Cabello JF1, Raimann E1, Hamilton V1, Valiente A1, Betta K1, De la Parra A1, Colombo M1, Cornejo V1 1

INTA, Univ de Chile, Santiago, Chile

Background: Graft versus host disease causes organ damage such as intestinal involvement with long-lasting diarrhea, abdominal cramps and pain. There are some difficulties in the feeding of

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the patients orally and they should receive total parenteral nutrition for a long time. We present six patients who responded to modified diet including low lactose, lipid and fiber. Case reports: Six patients with different diseases aged between 2-14 years were enrolled in the study. A modified diet protocol including five steps was used. In the first step, TPN was initiated for all the patients after HSCT, and minimal enteral nutrition and meat soup were given. In the second step, boiled potatoes and banana in small portions were added. Baked bread and boiled macaroni with very low fats were added in the third step. Boiled chicken and ayran were added in the fourth step, and the last step included the cheese, corn-flakes, peach fruit, apple, and rice. Full oral feeding was started in two weeks. In the beginning of the third week, the patients had no bloody diarrhea. Discussion: The optimal nutritional support is very important during acute GVHD period. Initiating the nutrition step by step is very important in the treatment of the intestinal symptoms. P-756 Moving with the times: development of an email database and electronic newsletter to communicate with paediatric PKU patients Walker R1, Gribben J1, Van Wyk K1, Gick J2, Campbell T2, Barnard C2, Vara R2 N&D Dept, St. Thomas' Hosp, London, United Kingdom; 2Ctr Inherited Met Dis, St. Thomas' Hosp, London, United Kingdom

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Background: Dietary emergency regimens (ER) are commonly used as an initial measure for emergency treatment of patients with some inborn errors of metabolism (IEM). These regimens may sometimes reduce the need for acute hospital admissions. Objectives: Primary objective: to assess whether caregivers prepared the ER according to recommendations given by the treating dietitian. Secondary objective: to assess whether implementation of the ER has contributed to reducing acute hospital admissions rates for patients with IEM. Materials and methods: A survey questionnaire was validated and provided to 26 caregivers of 29 patients with IEM at SQUH. The rates of acute hospital admissions were retrospectively reviewed and compared between 2012 (year of implementation of the ER), and the year preceding implementation of the ER. Results: Only 8% of caregivers prepared the age appropriate concentration of the dietary ER. Both years witnessed 71 acute admission episodes. The total number of days of acute admissions decreased from 322 (2011) to 269 (2012). The acute admissions days per patient per year were 11.1 (2011), and 9.3 (2012). Conclusion: Implementation of the dietary ER requires constant interactions between the dietitian and caregivers. Appropriate and timely initiation of the ER may reduce the need for acute hospital admissions. P-758

1

Comprehensive communication in PKU requires regular patient education and updates. Traditionally this comprises of clinic visits and regular telephone calls. Increasing use of electronic means of communication is a potential tool for effective and efficient delivery of group information. Many carers of PKU patients now use email frequently, both on home computers and smartphones. In 2012, an email address database was developed for 84% of caregivers from a total 76 PKU patients on low protein diets (median age = 5.8y, median phenylalanine exchanges = 6). An electronic newsletter was developed and sent quarterly via an email group; four have been issued to date. We include practical messages, educational articles by members of the MDT, low protein recipes and commercially available lower protein supermarket products. Feedback from caregivers is incorporated into subsequent issues. Since publication we have seen a rise in email correspondence from caregivers including reports and reviews of new supermarket products, suggestions for future newsletters and questions around their child's low protein diet. We conclude that electronic messages can be communicated rapidly en masse; reducing dietetic time spent relaying these individually to families. Increased engagement and awareness by families can potentially enhance dietary concordance. P-757 The practice of dietary emergency regimen application among care givers of patients with inborn errors of small molecules metabolism: a survey and a retrospective study S AlBelushi1, AlHabsi A1, Al Murshedi F2, AlShekaili S1, Al-Thihli K1 1 Depart. of Dietitics and Nutrition, SQUH, Al Khod, Oman; 2Genetic and Developmen Med. Clinic, SQUH, Al Khod, Oman

Association of waist circumference with inflammatory markers in phenylketonuria Rocha JC1,2, van Spronsen FJ3, Almeida MF1, Silva N2,4, Ramos E5,6, Borges N7, Guimarães JT2,4,6 1 Center Medical Genetics JM, CHP, Porto, Portugal; 2Depart Biochem (U38-FCT), FMUP, Porto, Portugal; 3Beatrix Children's Hospital, UMCG, Groningen, Netherlands; 4Depart Clin Pathol, Sao João Hosp Centre, Porto, Portugal; 5Depart Epidem, FMUP, Porto, Portugal; 6Instit Public Health, UP, Porto, Portugal; 7Fac Nutrition and Food Sciences, UP, Porto, Portugal

Background: Limited knowledge exists about the prevalence of obesity in phenylketonuria (PKU) and its metabolic consequences. Inflammation is a link between increased waist circumference (central obesity) and metabolic syndrome. Objective: To investigate the association between waist circumference and inflammatory markers in patients with PKU under dietary treatment. Patients and methods: A sample of 84 PKU patients (3-30 y; 14.2 ± 6.6 y) and 74 controls (3-47 y; 16.4 ± 7.8 y) was studied. In the fasted state, anthropometric and analytical parameters (amino acids, high sensitivity C-reactive protein, uric acid and adiponectin) were measured. For participants >10 years, central obesity was defined using waist circumference measurements according to international defined cut-offs. Results: The prevalence of central obesity in patients was similar to controls (36.1% vs. 37.1%; p=0.999). Both in patients and controls, waist circumference was positively associated with high sensitivity Creactive protein and uric acid, and negatively associated with adiponectin. Conclusion: Despite their special diet, patients with PKU seem to manifest the same association between waist circumference and

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inflammatory markers as observed in controls. More studies are needed to understand the long-term metabolic consequences of the special diet used in the treatment of PKU. P-759 The EU 'Foods for Special Medical Purposes' (1999) directive is not suitable to define the vitamin and mineral composition of l-amino acids supplements in PKU MacDonald A1, Ahring K2, Bélanger-Quintana A3, Dokoupil K4, Gokmen Ozel H5, Lammardo A6, Robert M7, Rocha JC8, van Rijn M9 1

Birmingham Children's Hospital, Birmingham, United Kingdom; Center for PKU, Kennedy Institute, Glostrup, Denmark; 3Unidad Enferm Metab, Hosp Ramon y Cajal, Madrid, Spain; 4Dr von Hauner Children's Hospital, Munich, Germany; 5Dept Nutr & Diet, Hacettepe University, Ankara, Turkey; 6Dept Pediatr, San Paolo Hosp, Univ Milan, Milan, Italy; 7Hôpital Univers des Enfant Reine Fabiola, Brussels, Belgium; 8Centro Genética Médica Jacinto Magalhães, Porto, Portugal; 9 Univ of Groningen, Univ Medical Center, Groningen, Netherlands 2

Background: In PKU, many EU phenylalanine-free, L-amino acids supplements have age-specific vitamin and mineral (VM phe-free L-AA'S) profiles to meet individual requirements. Within the EU, the composition of VM phe-free L-amino acids supplements are governed by the Foods for Special Medical Purposes (FSMP) directive (European Commission Directive number 1999/21/EC and amended by Directive 2006/141/EC). This provides a single regulatory framework, for minimum/maximum levels per 100 kcal of micronutrients. Objectives: To examine if the micronutrient composition of VM phefree L-AA's meets the FSMP guidelines. Methods: The micronutrient composition of 20 EU VM phe-free Lamino acids (from 4 manufacturers) was compared with the FSMP guidelines. Results: the micronutrient composition of all 20 VM phe-free L-AA's exceeded the FSMP guidelines by a median of 15 (range 4-21) nutrients. Vitamin D, folic acid, zinc, selenium and manganese exceeded maximum levels in almost all supplements. Discussion: VM phe-free L-AA's supplements failed to remain within FSMP micronutrient maximum limits due to their low energy content. Their adherence may potentially lead to micronutrient deficiency. Currently, compositional exceptions to the FSMP directive have to be granted for phe-free L-AA's but for safety and control, it is more appropriate to have specific regulations based on their protein equivalent content. Conflict of Interest declared. P-760 Tyrosine requirements in a preterm infant with phenylketonuria (PKU) - a case study Skeath RH1, Baruteau J1, Abulhoul LH1, Cleary MA1 1

Great Ormond St Hosp for Children NHS FT, London, United Kingdom

Background: Treatment for PKU is a phenylalanine-(PHE-)restricted diet with PHE-free, tyrosine-(TYR-)enriched amino acid (aa)

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supplements. Treatment of the neonate is well established and includes TYR supplementation of 100-120mg/kg/day. Less is known for the preterm infant. Case: A twin born at 28 weeks gestation; weight 968g. PKU diagnosed age 14 days; PHE 2263μmol/L, TYR 24μmol/L. Dietary treatment: A combination of parenteral nutrition (dextrose, lipid) with enteral PHE-free, TYR-enriched amino acid formula increased as tolerated (102-120kcal/kg, 0.5-2.8g aa/kg, 55-260mg TYR/kg). Blood PHE was controlled by age 23 days; PHE 203μmol/L, TYR 243μmol/L. Full enteral feeding was achieved by day 27. Abdominal distension developed on day 40. Enteral feeds of fortified expressed breast milk and PHE-free formula (124kcal/kg, 0.4g aa/kg, 97mg PHE/kg, 122mg TYR/kg) were stopped. As no specialised intravenous PHE-free preparation is available standard parenteral nutrition was commenced (120kcal/kg, 103mg PHE/kg, 19mg TYR/kg) with trophic PHE-free formula feeds (0.5g aa/kg, 53mg TYR/kg). Blood PHE levels increased (1886μmol/L) whilst TYR dropped (7 μmol/L). Enteral supplementation of 216-447mg/kg TYR normalised blood TYR concentration. Conclusion: It is challenging to meet TYR requirements in a preterm PKU patient on standard Vaminolact® parenteral nutrition. This infant became TYR deficient on 73mg TYR/kg/day. Enteral TYR supplementation was necessary. P-761 Dietary management in phenylketonuria: the nutritional composition of cooked dishes Pimentel FB1, Alves RC1,2, Costa ASG1, Fernandes TJR1, Torres D3,4, Almeida MF5, Oliveira MBPP1 REQUIMTE, FFUP, Porto, Portugal; 2REQUIMTE, ISEP, Porto, Portugal; 3FCNAUP, Porto, Portugal; 4Dep Biochem (U38-FCT), FMUP, Porto, Portugal; 5Center Medical Genetics JM, CHP, Porto, Portugal 1

Background: Nutritional and dietary management is essential for Phenylketonuria (PKU) treatment. The restrictive diet has to be carefully planned to provide the best ingredient combinations, and allow an adequate nutritional status for PKU patients. Thereby, it is crucial to know the detailed composition of natural and/or cooked foods specifically prepared for them. Objective: To evaluate the nutritional composition of dishes specifically prepared for PKU patients the following parameters were determined: moisture, protein, total fat, total carbohydrates, phenylalanine (Phe), tyrosine (Tyr), fatty acids and vitamins E and B12 contents. Material and Methods: Sixteen dishes were analyzed. The nutritional parameters were determined according to methodologies generally used. Discussion/Conclusions: The nutritional composition of the dishes varied between 29.3-92.0 g/100g, for moisture; 1.2-1.9 g/100g, for protein; 0.5-25.0 g/100g, for total fat; and 5.0-42.0 g/100g, for total carbohydrates. The contents of fatty acids and vitamin E reflected the amount and type of fat used. Boiled rice presented the highest Phe and Tyr contents. All dishes were poor in vitamin B12 (0.28-0.75μg/100g). These data allow a more accurate calculation of the diet, according to patients' individual needs and Phe tolerance. Moreover, point to the need to monitor their nutritional status and to resort to supplementation whenever needed.

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P-762 Obesity and overweight in PKU: the results from 9 centers in Europe and Turkey Gokmen-Ozel H1, Ahring K2, Belanger-Quintana A3, Dokoupil K4, Lammardo AM5, MacDonald A6, Robert M7, Rocha JC8, van Rijn M9 1

Dept Nutr Diet Hacettepe University, Ankara, Turkey; 2Dept PKU Kennedy Centre, Copenhagen Univ, Copenhagen, Denmark; 3Univ Enf Metabolicas Hosp Ramon y Cajal, Madrid, Spain; 4Dept Metab Nutr Dr von Hauner Child Hosp, Munich, Germany; 5Dept Pediatr San Paolo Hosp Univ Milan, Milan, Italy; 6Inh Metab Dis Children's Hosp, Birmingham, United Kingdom; 7Dept Nutr Metab Hosp Univ Enf Reine Fabi, Brussels, Belgium; 8Cent Genét Médica Jacinto de Magalhaes, Porto, Portugal; 9Sect Metab Dis Univ Medical Center, Groningen, Netherlands Background: There are suggestions that overweight and obesity are more common in PKU than the general population, potentially associated with a high carbohydrate diet and high non-phenylalanine protein intake in infancy. In a cross-sectional survey, the prevalence of overweight and obesity was compared among 9 European PKU treatment centres (Belgium, Denmark, Germany, Italy, Netherlands, Portugal, Spain, Turkey, and UK). Methods: The percentage of overweight and obesity, according WHO BMI z scores/ WHO BMI classification was calculated (184 adults from 5 centres: Belgium, Germany, Netherlands, Portugal, Spain; and 887 children <19 y from 8 centres: Belgium, Denmark, Italy, Netherlands, Portugal, Spain, Turkey, and UK) were included. Results: In children, % overweight in centres ranged between 14.7% (Denmark) to 44% (Spain) and obesity from 2.3% (Belgium) to 14.5% (Spain). In adults, % overweight ranged 22.7% (Belgium) to 42% (Spain); and obesity from 4.5% (Belgium) to 18.2% (Germany). Girls were more overweight and obese than boys (data obtained from n=4 centres). Conclusions: Overweight and obesity in PKU varies between treatment centres despite a similar treatment approach. More research is needed to identify the main risk factors associated with its occurrence in PKU. P-763 Large neutral aminoacid supplementation in the treatment of phenlyketonuria-hacettepe experience Gokmen-Ozel H1, Öztürk Hişmi B2, Ünal Ö2, Sivri HS2, Dursun A2, Tokatlı A2, Coskun T2

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(p<0.05). The median decrease and increase in blood phenylalanine and tyrosine level was 13% and 28.7% respectively. Conclusions: LNAA is seen as an alternative to conventional dietary treatment. Further longitudinal studies are necessary to investigate the effect of LNAA on blood phenylalanine and tyrosine levels. P-764 Description of the population of patients with inherited metabolic disease (IMD) on dietary treatment in a public pediatric hospital in Argentina Lavorgna S1, Cresta A1, Bay L1, Eiroa H1, Uicich R1 1

Hospital de Pediatria Juan P Garrahan., Buenos Aires, Argentina

In Argentina the diagnosis of IMD has increased dramatically in recent years Our hospital is a public referral center for complex pediatric diseases including IMD. Dietitians are involved in the treatment of many of these metabolic diseases working in collaboration with an interdisciplinary team. The aim of the study was to describe the hospital population of IMD patients requiring dietary treatment since 1997 301 IMD patients were treated requiring "diet" or a combination of "diet and medication". At the time of this study 220 patients are followed-up and 81 discontinued (17 adult age; 47 unknown/referred to other centers; 17 died). The main disorders treated with diet therapy are: PKU 28; Tyrosinemia 6; UCD 25; Organic acidurias 22; MSUD 9 ; Homocystinuria 5; Galactosemia 10; GSD 54; FAO 9; other IMD 52. Most of patients (56%) lived in other provinces, 43% in the capital or suburbs and 1% in neighboring countries. The increased number of patients led to incorporation of more metabolic dietitians, specialized fellows, as well as a need for new treatment strategies and research. Patient care at the moment is concentrated in the capital and ists surroundings, and there is an urgent need to train more dietitians in order to support patient monitoring through our hospital outreach network working in a multidisciplinary team. P-765 Educational activities to support compliance in inherited metabolic disorders (IMD) patients with protein/aminoacids restricted diet Lavorgna S1, Vinuesa MV1, Blasi S1

Dept Nutr Diet Hacettepe University, Ankara, Turkey; 2Hacettepe Univ, Child Host Div Metab, Ankara, Turkey

1

Background: Because some patients are not able to adhere to the phenylketonuria diet, alternative treatment regimens such as large neutral amino acids (LNAAs) have been developed. Possible LNAA treatment targets include reduction of brain and blood phenylalanine concentrations. The aim of this study was to compare blood phenylalanine and tyrosine levels before and during LNAA treatment. Methods: Thirty-five subjects (22 boys, 13 girls) with phenylketonuria using LNAA for more than 6 months were recruited. The median age was 17.3 years (11.6-26.9 years). 1 g/kg LNAA was given three times daily with meals and 1 g/kg/day protein. LNAA comprised 40% of patients' daily protein requirement and natural protein made up remaining 60%. Blood phenylalanine and tyrosine levels were measured before and at the 6th month of the treatment. Results: With LNAA, blood phenylalanine levels significantly decreased (median 22 and 18.6 mg/dL, respectively), and tyrosine levels (median 0.84 and 1.08 mg/dL, respectively) significantly increased

In diets that require the calculation of protein/amino acids from natural food it is a constant demanding task to turn the dietary prescription into a "meal". Difficulties in dietary compliance are frequent and can be solved with educational strategies involving the child and family, to anticipate everyday situations and problems. Participatory multithematic workshops were designed based on the real needs. The aim of the study was to achieve nutritional treatment compliance through the implementation of educational workshops for IMD patients and their families. Seventeen patients (6m-14y) and 26 relatives attended 3 workshops planned according their needs, including educational and culinary activities. The topics discussed were qualitative (understanding IMD and diet, allowed/prohibited foods, use of medical/low-protein foods, handling special situations, self-care, eating outside the home) and quantitative (weigh-measure food, calculations, food records, exchanges). Recipes were designed to encourage participation and consumption of foods allowed, expanding to a variety of menus.

1

Hospital Garrahan. Area de Alimentación, Buenos Aires, Argentina

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Educational workshops encourage dietary compliance in a practical way, improve conventional instruction, and provide the opportunity to share ideas and experiences in the diet management. The continuation of these activities is important to measure and evaluate the impact of changes on adherence and hopefully improve the longterm metabolic control.

Conclusion: The survey points out - the key person is the dietician who must be easily available and well trained - the metabolic centre must have a good infrastructure. Conflict of Interest declared.

P-766

Nutritional assessment of Niemann Pick C Patients in Colombia

Training of dietitians in a network for inherited metabolic disorders (IMD) in Argentina

Ladino L1, Sepúlveda N1, Ochoa E2

Lavorgna S1, Cresta A1, Blasi S1 1

Hospital Garrahan. Area de Alimentación, Buenos Aires, Argentina

P-768

1 Javeriana University, Bogotá, Colombia; 2Monterrey Institute of Technology, México D.F., Mexico

In Argentina care for patients with inherited metabolic disorders (IMD) is concentrated in the capital city although more than half of them live in remote areas where lack of specialized dietitians complicate dietetic treatment. In this setting we decided to create a network for the care of IMD patients linking our center with centers in different provinces. To enhance coordinated care for patients with IMD receiving dietary treatment through the training of specialized dietitians. Dietitians were identified and enrolled in a course on dietary management of IMD during 2010, 2011, 2012 in a mixed modality. A survey was conducted to evaluate previous knowledge and satisfaction with the course, and students did a final online multiple-choice exam. Dietitians were encouraged to join the provincial network for patient referral. Sixty seven dietitians were enrolled in the course. Forty-nine (73%) passed, 9 (13%) failed, and 9 (13%) abandoned the course. Only 28% had previous knowledge and 90% felt the need for further training and joining multidisciplinary teams. General satisfaction with the course was high (94%) Currently 15 provincial health centers have joined the network. The 3- year experience stimulated us to continue training dietitians and strengthen the initial network for the dietary management.

Background: Severe gastrointestinal complications have been described as adverse effects of Miglustat therapy in Niemann-Pick C (NP-C), affecting nutritional status. The aim of this study was to describe the anthropometrical status of NPC patients as well as their dietary intakes. Methods: NPC patients with substrate inhibition therapy were recruited in a nutritional private practice. A thorough nutritional assessment was conducted, including anthropometrical and dietary assessment. Results: Fifteen patients with NP-C on Miglustat therapy were recruited, nine were male. Average age was 11.4±9.4 years. Five out of 13 pediatric patients had adequate nutritional status, 15.4% suffered emaciation and 46.2% were stunted, 33.3% of these patients suffered from emaciation as well. Emaciation might be even higher in NP-C considering that weight appear in the normal range due to hepatomegaly and splenomegaly; thus, body composition assessment is more reliable. Average energy and protein intake was 73.8±45.3 Kcal/Kg/day and 3.3±1.9 g/Kg/day, respectively. When compared to FAO/WHO energy requirements, intake was adequate in 53.3%, excessive in 33.3%, and deficient in 13.3%. Conclusions: Neither stunting nor emaciation was associated with adequacy of diet, demonstrating that the nutritional risks are also independent of neuropathological features that may affect dietary intake. Thus, every NP-C patient should undergo a thorough nutritional assessment.

P-767

P-769

Which are the most important factors influencing adherence of PKU patients to the phenylalanine restricted diet?

Nutritional assessment of mucopolysaccharidosis patients in Colombia Ladino L1, Sepúlveda N1, Ochoa E2

Kiss E1, Simon E1, Bókay J1, Reismann P2, Szőnyi L1 1

1st Dep of Pediatrics, Semmelweis Univ, Budapest, Hungary; 22nd Dep of Medicine, Semmelweis Univ, Budapest, Hungary Introduction: In Hungary the countrywide newborn screening for phenylketonuria (PKU) started in 1975. The incidence of PKU is 1:8500 newborn. The success of PKU treatment depends on the adherence to the low phenylalanine diet. The reasons for non-adherence can be different in different countries. Aims: The objective was to assess the most important factors influencing long-range adherence of PKU patients to the phenylalanine restricted diet in the Hungarian population. Patients and methods: We mailed a questionnaire with 56 questions to 213 PKU patients/parents followed in the Metabolic Centre, Budapest. Results: The number of responders was 131, the main patient age was 16,5 years (0,2 – 48 years). Eight of the responders stopped the diet before receiving the survey. The three most important factors for the patients/parents were: (1) to get the results and the assessment very soon (95,4%), (2) to keep up-to-date with informational materials of the food products (75,5%), (3) the knowledge and the skill of the dietician.

1 Javeriana University, Bogotá, Colombia; 2Monterrey Institute of Technology, México D.F., Mexico

Background: The skeletal dysplasia severely affects linear growth in Mucopolysaccharidosis (MPS) patients. The aim of this study was to describe nutritional status of MPS patients in Colombia. Methods: MPS patients were recruited in a nutritional private practice. A thorough nutritional assessment was conducted, including anthropometrical and dietary assessment. Results: Twenty-three patients with MPS were recruited; 56.5% were male. The average age was 12.2±4.7-years-old. Average height was 101.5±7.9cm, (range 83-121cm). Every patient suffered growth retardation (height for age z-score -6.5±2.1), thus the anthropometrical assessment was done using the age according to their height instead of chronological age. 8.7% had adequate weight; 91.3% suffered overweight or obesity. Body composition analysis demonstrated that overweight is due to higher fat-free mass, 65.2% had arm muscle area z-score above +1 SD while only 4.5% had tricipital skinfold z-score above +1 SD. Energy intake was 74.1±26.1 Kcal/Kg/day, being adequate for 30.4% and excessive for 47.8%; however, it was not associated with a specific nutritional status (χ2 test, p=0.446). Conclusion: Longitudinal and multicenter studies should be promoted in order to evaluate the development of specific growth reference charts

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was 1:1000 live neonates (CH is not included). Early intervention of cases reduces infant morbidity and mortality. These results show the factibility of develop and expanded screening programs and highlight the importance of including more markers in México.

P-770

P-772

Newborn population screening for classic homocystinuria in Qatar "7 years of success"

Performance of the South Australian (SA) expanded neonatal screening programme using tandem mass spectrometry (MSMS)

Ben-Omran T1, Shabeck N1, Ali R1, Abdoh G1, Chandra P1, Al Rifai H1, Elbashir H1, Hoffmann GF2

Ranieri E1, Gerace R1, Bartlett B1, Johnson D1, Fletcher J1

as well as the determination of energy requirements, considering the peculiar body composition in MPS patients.

1

Dir Genetics & Molecular Pathology, Adelaide, Australia

1

2

Dep of Pediatrics, Hamad Medical Corp, Doha, Qatar; Univ Child Hosp, Heidelberg, Germany Introduction: Classic homocystinuria caused CBS deficiency is a rare IE of methionine metabolism with prevalence 1/200,000 births. In Qatar, its prevalence is the highest in the world, of 1:1800 births. If untreated patients present with MR and devastating multi-system complications and premature death. The goal of NBS is the presymptomatic detection of infants so that treatment will be commenced as early as possible to prevent, its complications. Methods: 111,713 neonates (Qatari= 40496, Non-Qatari= 71217) were screened by measuring total homocysteine in DBS. Results: 29 infants with mean age 3.2 years ranging from 3 months to 7 years (15 females, 14 males) with classic homocystinuria were identified in 40,496 native Qatari infants, yielding an incidence of 1:1500. The parents of 29 infants were all consanguineous. Mean homocysteine and methionine levels at diagnosis were 144.9±38.2 and 516.1±287.4 micromol/l respectively. At last follow-up in the Metabolic Clinic at HMC, all are growing and developing normal. Conclusions: Our data support that the incidence of classic homocystinuria in Qatari population is the highest in the world and even higher than previously estimated. In addition, NBS is effective in the prevention of complications in patients with classic homocystinuria when coupled with early dietary and pharmacological treatment. P-771 Incidence of inborn errors of metabolism detected by expand newborn screening in Nuevo León, Mexico: experience of ten years Torres MR1, Martinez LE1, Ruiz MC1, Lopez GA1, Sanchez A1, Gonzalez R1, Castillo ER1, Arrendondo PC2, Villarreal JZ2 Genetics Dept, Medicine School, Uanl, Monterrey, Mexico; 2Servicios de Salud de Nuevo León, Monterrey, Mexico 1

Background: Until 2010, only CH was detected in newborn screening in Mexico then, PKU, adrenal hyperplasia and galactosemia were included. In Nuevo León, (state located at Northeast Mexico), a pilot expand neonatal screening program was initiated ten years ago. Objectives: to known the incidence of IEM and to obtain experience for the implementation of the program in other states. Material and Methods: Blood spots were collect from newborns at 24 Hr life and used to quantify: 17 OHP, IRT (AutoDELFIA) Biotinidase, G-6PD, total galactose (DELFIA), acylcarnitines and amino acids (Ms/Ms). Positive results underwent confirmatory tests and positive cases were follow-up. Results: from December 2002 to November 2012 there were 111.287 newborns screened. A total of 115 cases were confirmed: 51 G-6PD deficiency, 22 organic acidurias, 19 amino acidurias, 11 FAO disorders, 5 cases of CF, 4 adrenal hyperplasia and 3 galactosemias. Conclusion: Incidence of IEM depends on the number of markers included in neonatal programs. The incidence found in the present study

The South Australian (SA) expanded Neonatal Screening Programme using tandem mass spectrometry (MSMS) commenced in February 1999. Over 283,000 dried blood-spot specimens collected at 48 hours of age from infants were analysed to 2012. Of the total, re-sampling was requested on 990 (0.35%), with 290 (0.10%) and 700 (0.25%) for AA & AC profiles. respectively. Forty two (15.1%) had an abnormal AA and were recalled for urine and plasma amino acids. We identified PKU (18), hyperphenylalanemia (9), tyrosinaemia type II & III (1), MSUD (1), OTC (1), pyruvate carboxylase deficiency (1), neonatal haemochromatosis (2), argininosuccinate lyase deficiency (1) and citrullinaemia I (1). Seventy five (10.1%) of the 700 with an abnormal AC were recalled for urine organic acids and plasma carnitine testing. We identified MCAD (22), IVA (2), systemic carnitine deficiency (2), GA1 (2), GAII (4), vLCAD (3), SBCAD (1), cobalmin A defect (cblA) (1) and vitamin B12 deficiency (24). The remainder had normal metabolic screening results. A case of malonyl-CoA decarboxylase deficiency and cobalamin metabolism defect were confirmed retrospectively. To date 74 infants with a metabolic disorder have been identified, with a positive predictive value of 60% and an incidence rate of 1 in 3,840 in the SA population. P-773 The use of a 2nd tier dried blood-spot methylmalonic acid (MMA) into routine newborn screening for inborn errors of metabolism Ranieri E1, Gerace R1, Barlett B1, Johnson D1, Fletcher J1 1

Dir Genetics & Molecular Pathology, Adelaide, Australia

A 2nd tier Methylmalonic acid (MMA) blood-spot assay was implemented into the South Australian neonatal screening in June 2007. MMA was performed in the top 1% of C3-carnitine values. The determination of MMA reduced the recall for further testing due to an elevated propionyl- (C3)-carnitine/ratio. A single 3mm blood-spot was extracted with 100μL methanol containing d3-MMA and was dried and reconstituted in 100μL 50% acetontrile. An LC-MSMS was performed using a Phenomenex Gemini 3μ C6-phenyl column (100 x 2mm) run isocratically with 50% acetonitrile at a flow rate of 100μL/min. To end of 2012 only 85 neonates were recalled for a repeat blood spot or clinical assessment, representing a recall rate of 0.05% compared to 0.14%. A case of cobalamin A deficiency, (cblA, p.R145X/p.D292V, diagnosis confirmed by Dr Brian Fowler, Basel) was identified with a blood spot MMA of 18.3μmol/L whole blood (NR<2.2μmol/L whole blood). Twenty-four of the 85 recalled neonates had significant vitamin B12 deficiency in mother or infant or both. There was also elevations in plasma homocysteine (HcY) and vitamin D deficiency in a high percentage of mothers. This high frequency of Vitamin B12 deficiency (1 in 5,000) may be symptomatic of a wider public health issue.

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P-774

P-776

Confirming the diagnosis by reduced very long-chain acyl-CoA dehydrogenase activity in patients identified by newborn screening

Newborn screening for lysosomal storage disorders: the Children's Mercy Hospital experience

Eyskens FJM1

Atherton AM1, Strenk ME1, Lawson CE1, Heese BA1, Smith LD1

1

1

Introduction: The implementation of screening for medium chain acylCoA dehydrogenase deficiency (MCADD), the most common mitochondrial fatty acid oxidation defect FAO, revealed a great number of apparently healthy individuals who had mild mutations in the ACADM gene. This is also true for a more uncommon FAO defect, namely very long-chain acyl-CoA dehydrogenase deficiency (VLCADD). Methods: MS/MS Xevo (Waters); Neobase kits (nonderivatized method; PerkinElmer). Screening parameter C14:1 acylcarnitine (cutoff: <0.39 μmol/L) and C14:1/C2 ratio (cutoff: <0.023). Results: Blood sampling takes place between 3-4 days postpartum. The recall rate is 0.07%. Specificity: 99.93%. Two newborn girls were found with a C14:1 acylcarnitine concentration of 1.33 and 3.59 μmol/L, resp. The C14:1/C2 ratio was elevated in both patients. The diagnosis was confirmed by enzyme activity measurement in lymphocytes (AMC, Amsterdam, The Netherlands). The residual enzyme activity of VLCADD was 0.61 and 0.24 nmol/min/mg protein, resp (controls: 1.84-4.80 nmol/min/mg protein; 10% enzyme activity = 0.66). Conclusions: A functional enzyme activtity assay is the only reliable method to predict the clinical course in patients with VLCADD detected by newborn screening: patients showing a <10% residual enzyme activity are at risk to develop clinical disease. Early dietary intervention improves the outcome of severely affected patients.

Expanded newborn screening is an important public health initiative to screen for conditions for which interventions are available to significantly reduce morbidity and mortality and is considered standard of care. There is a general international consensus regarding the majority of conditions that are recommended for newborn screening, however, there is variability between countries on what is included on newborn screening panels. In the United States, a universal screening panel exists and states decide which disorders to include on their newborn screening panel. Recently, there has been extensive discussion about the inclusion of lysosomal storage diseases (LSDs) on newborn screening panels and such testing has been mandated in the state of Missouri. A pilot study of newborn screening for five LSDs (Krabbe disease, Hurler syndrome, Gaucher disease, Fabry disease and Pompe disease) is currently being pursued utilizing a rapid microfluidic assay, with plans to add several more disorders in the future. Screening for Krabbe disease began in September 2012 while screening for the remaining disorders began in January 2013. We report on the current status and experience of this pilot study at Children's Mercy Hospital, one of four referral sites for newborn screening in the state of Missouri.

Div Metab Dis, Dept of Paediatrics,UZA, Antwerp, Belgium

P-775 Clinical, biochemical, molecular findings and outcomes of 8 children with cobalamin C disease detected through newborn screening: 13 years experience Clarke C1, Li M1, Payan I1, Weiss B1, Ganesh J1, Kaplan P1, Ficicioglu C1 1

The Children's Hospital of Philadelphia, Philadelphia, United States

Background: Cobalamin C (Cbl C) is the most common inborn error of intracellular Cobalamin metabolism. Patients/Methods: This is a retrospective analysis of clinical, biochemical, molecular and treatment data on eight children with Cbl C disease detected through newborn screening in the past 13 years. Results: Three carried homozygous mutations; one of whom is homozygous for the common 271_272dupA mutation. We identified three nonsense mutations, three missense mutations (1 novel mutation), and six frameshift mutations. Two children had IUGR noted at birth. At the time of diagnosis, five required hospital admission because of poor feeding. Microcephaly was observed in 50% of the children; 50% had mild to moderate developmental delay. Two had nystagmus without other changes, one had atrophic macular changes, three had pigmented retinopathy, retinal dystrophy, macular changes. All eight were treated with protein restriction and OH-Cbl injections with varying frequency. Some children required valine, betaine, carnitine, folate, and/or methionine supplementation. Average lifetime plasma MMA levels varied from 4,437 to 88,772 nmol/L. Average lifetime total plasma homocysteine levels were between 20 and 76 μmol/L. Conclusion: Despite early treatment children continue to develop ocular abnormalities, elevated MMA and homocysteine, developmental delay, and microcephaly.

Children's Mercy Hospitals and Clinics, Kansas City, United States

P-777 Newborn screening and second tier testing for methylmalonic (MMA) and propionic (PA) aciduria Laeremans H1, Van Thi HV1, De Meirleir L2, De Laet C3, Goyens Ph1 1 Lab of Paediatrics Univ of Brussels, Brussels, Belgium; 2Dep of Neurology, UZBrussels, Brussels, Belgium; 3Dep of Metabolics, HUDERF, Brussels, Belgium

With the introduction of mass spectrometry, the screening of lifethreatening metabolic disorders came widely available. MMA and PA are the most frequent forms of branched-chain organic acidurias. However, screening for these diseases is controverial. In Belgium, the level of C3 is used as single parameter. A pilot study, started several years ago in our lab, indicated that specificity and sensitivity is low when only C3 levels are used. To improve the screening, C3/C2 levels were included as well as quantification of MMA and PA with LCMSMS as second tier test. These test results indicated that C3 levels alone gave rise to both false positive and false negatives. Inclusion of C3/C2 and second tier increased specificity and sensitivty and moreover, no false negatives were reported. However, since MMA and PA quantification are expensive and time consuming, we looked into other parameters. Based on a study of 25 known patients and more than 50000 newborn DBS, we can conclude that C3, C3/C2 in combination with PA/MMA gives rise to a very high specificity and sensitivity. This can further be increased to almost 100% by inclusion of C3/C0, C3/C16, C16OH:1/C2 and C16OH:1/C4DC. P-778 Newborn screening: a system to assure universal access Marcadier JL1, Milburn J1, Rodgers I1, Dougan S2, Davies C1, Higgins L1, Sayer M1, Zelenietz S1, Geraghty MT1,3, Chakraborty P1,2,3 1 Newborn Screening Ontario, CHEO, Ottawa, Canada; 2 Better Outcomes Registry & Network, CHEO, Ottawa, Canada; 3Department of Pediatrics, CHEO, Ottawa, Canada

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In Ontario, newborn screening (NBS) is not mandatory, but considered standard of care. A crucial question for all NBS programs is, how do we assure all babies are offered testing? The Better Outcomes Registry and Network (BORN) collects data on prenatal and neonatal care throughout the province from several different databases. This facilitated an alert process between BORN and Newborn Screening Ontario (NSO) for infants without matching birth and NBS records by 14 days of age. Between February 2012-13, 593 potential missed screen alerts (~1 in 243 births) were received. Data entry and informatic errors accounted for 293 (49%) alerts. Delayed samples, received ≥14 days of age accounted for 128 (22%) alerts (4 of these had false positive results). Parents declined screening in 78 (13%) cases. Finally, system error resulted in 194 (33%) samples not being taken. Through follow up 165 (85%) of these true missed cases were received. Midwifery care and hospital transfer (inter and intra) were identified to be major reasons for missed screens with a measurable risk for a false negative result. This system allows prompt follow-up, enumeration and categorization of missed screens as well as documentation if screening is declined.

by a full-time pediatric nurse practitioner with oversight from a physician-director. All referrals are seen by the coordinator with one of 14 Medical Genetcists. We accept all referrals from our catchment and provide comprehensive genetic/metabolic care and social support until a diagnosis is made. Since 2012 we have had 245 referrals. Of these, 100 (41%) were made directly by the Departments of Health, 89 (36%) were made by primary pediatricians. 71 (29%) were for a disorder of fatty acid oxidation, 43 (18%) for organic acidemias, 35 (14%) for an aminoacidopathy, 21 (9%) for a urea cycle disorder, 67 (27%) for galactosemia and 10 (4%) for biotinidase deficiency. Diagnostic testing confirmed 18 fatty acid oxidation disorders (PPV = 25%), 8 organic acidemias (PPV = 19%), 7 aminoacidopathies (PPV = 20%), 1 urea cycle disorder (PPV = 5%), 19 disorders of galactose metabolism (PPV = 28%) and 5 biotinidase deficiencies (PPV = 50%). P-781 First year of expanded newborn screening in western Sicily: preliminary results

P-779 Scaturro G.M.1, Sanfilippo C.1, Giuffrè M.1, Castana C.2, Corsello G.1 Expanded newborn screening: the New South Wales experience Dip Promoz Sal e Mat Infant, Palermo, Italy; 2 Div. Malattie Metaboliche, Palermo, Italy 1

1

2

1

1

Estrella J , Tchan MC , Bhattarcharya K , Ellaway C , Christodoulou J1, Carpenter K1, Lipke M3, Mowat D3, Kirk EC3, Freckman ML3, Wiley V1, Wilcken B1 1 West Syd Gen Program, Child Hosp West, Westmead, Australia; 2Dept Gen Med, Westmead Hosp, Westmead, Australia; 3Syd Chil Hosp Net, Syd Chil Hosp Rndwick, Sydney, Australia

Tandem mass spectrometry (MS/MS) was introduced in New South Wales, Australia in 1998, to screen for selected disorders of amino acid, organic acid, and fatty acid metabolism. Of 1,500,000 babies screened, one in 2,700 was diagnosed with a target disorder. Maternal disorders ascertained through infant screening included 3- methylcrotonyl glycinuria, and deficiencies of the carnitine transporter, citrin, holocarboxylase synthase and the riboflavin transporter. Thirteen affected babies were missed and presented clinically. In five cases (cobalamin-C deficiency (3), VLCAD, and glutaric aciduria, missed on repeat testing) this resulted in modification of analyte cut-off values or protocols prior to 2001. Patients with intermittent MSUD (2), beta-ketothiolase deficiency (2) and argininosuccinic aciduria (2, siblings) had analyte values and ratios below the action limits. These could not have been detected without unacceptable false-positive rates. Cutoffs, regularly reviewed, were not altered. Two infants with citrin deficiency were undetected and presented clinically. Whilst marker amino acids may be increased in citrin deficiency, we do not specifically screen for this disorder. Most of the children are doing well. Analyte ratio and cut-off value optimisation are important, but for some disorders missed cases may have to be accepted to maintain acceptable specificity.

Background: The introduction of the tandem mass spectrometric methods (MS/MS) and electro-spray ionization for metabolite analysis changed our ability to detect intermediates of metabolism on dried blood spot and provides the means to detect a large number of metabolic disorders (MD) in a single analytical run. Objectives: to evaluate expanded newborn screening preliminary results of the first year activity in western Sicily. Cases expected: 12/year in reference areas. Materials and Methods: 18111 neonates were screened by tandem MS/MS for MD from November 2011 to November 2012. Blood spots were collected between 2nd-7th days after birth. Special protocols were used for low birth weight newborns and for neonates receiving Total Parental Nutrition. Results: Cut-offs for the Sicilian Population were established. 1300 newborns (7%) were re-tested. Further diagnostic tests were performed on 53 newborns with elevated screening results by metabolic disease centers and our laboratory, with exclusion of false-positive. We detected 3 patients with MD (primary carnitine deficiency, methylmalonic acidemia, isovaleric acidemia). Conclusions: False positive rate was higher than the desirable value of 3% according to National Guidelines (2008). These results will be optimized with experience and with a more efficient network with birthing centers. P-782 Feasibility of newborn screening for guanidinoacetate methyltransferase (GAMT) deficiency

P-780 Pasquali MP1,2, Schwarz ES3, Yuzyuk T4, De Biase I2,4, Longo N2,5 Design and results of a nurse practitioner-coordinated newborn screening referral program in Washington, D.C. Viall S1, Summar M1, Kirmse B1

1 Dept Path, Univ of Utah, Salt Lake City, United States; 2ARUP Laboratories, Salt Lake City, United States; 3ARUP Inst Clin Exp Path, Salt Lake City, United States; 4Dept Path, Univ of Utah, Salt Lake City, United States; 5Dept Peds, Univ of Utah, Salt Lake City, United States

1

Children's National Medical Center, Washington, DC, United States

In an effort to improve outcomes, optimize linkage to specialty care, and decrease familial anxiety, Children's National (CNMC) supports a dedicated NBS follow-up program (CNMC NBS) while engaging in continuous quality improvement. Here we present a description of the program and referrals since January, 2012. CNMC NBS is coordinated

Background: Guanidinoacetate methyltransferase (GAMT) deficiency) causes brain creatine deficiency characterized by developmental delays, hypotonia, autism and seizures. Therapy initiated at birth for positive family history can prevent mental retardation. Objective: To identify guanidinoacetate methyltransferase (GAMT) deficiency in newborns.

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Methods: Creatine and guanidinoacetate were extracted from 10,000 deidentified blood spots using the same protocol routinely used for newborn screening and quantified by stable isotope dilution using deuterated creatine and guanidinoacetate as Internal Standards. Residual dried blood spots from 3 infants with GAMT deficiency were used to evaluate the sensitivity of the method. A second tier test using UPLC-MS/MS was performed to analyze samples with a concentration of guanidinoacetate >2.44 μmol/L (99.5% of the normal population). Results: 53 blood spots required second tier testing in addition to 7 blood spots from 3 patients with GAMT deficiency retrospectively analyzed. Using our second tier test, only the samples from GAMT deficiency patients showed elevated concentration of guanidinoacetate. Conclusions: GAMT deficiency can be identified in newborns using routine extraction methods. The cost of this additional screening is minimal, as it does not require additional instrumentation or procedure. The use of a second tier test can reduce to a minimum the false positive rate.

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tyrosinemia type I and disorders of methionine and propionate metabolism); first-step markers arginine, citrulline, methionine, propionylcarnitine, succinylacetone and biotinidase activity were added. Between October 2012 and March 2013 we analyzed samples from 14,532 newborns of which 30 samples were subjected to second-tier LC-MS/MS analysis of homocysteine, cystathionine, methylmalonate and methylcitrate, while LC-MS/MS steroid profiling was performed in 167 samples. The FPR was 0.04% for biotinidase deficiency and 0.00% for other IMDs; so far we did not detect any new patient. The FPR for congenital adrenal hyperplasia was reduced by 91% compared to immunochemical method for 17-hydroxyprogesterone. Conclusions: Preliminary data demonstrate a reasonable performance of this panel of markers and the evaluation of pilot phase involving about 40,000 newborn will facilitate decisions on expansion and optimization of neonatal screening program in the Czech Republic. Supported by MH CZ - DRO-VFN64165/2012 and NT/12213–3 IGA by Ministry of Health of the Czech Republic.

P-783 P-785 Difficulties in newborn screening of carnitine palmitoyltransferase-2 deficiency scheduled on 5th day after birth Hara K1, Tsumura M2, Kagawa R2, Okada S2, Tajima G2, Sakura N3, Hata I4, Shigematsu Y4, Kobayashi M2 1 Dept. Pediatrics, Kure Medical Center, Kure, Japan; 2Dept. Pediatr, Hiroshima Univ Gradu Scho, Hiroshima, Japan; 3Nursing House Suzugamine, Hiroshima, Japan; 4Dept. Pediatr, University of Fukui, Fukui, Japan

Background: In 2010, an infant presenting with acute encephalopathy was diagnosed with carnitine palmitoyltransferase-2 (CPT2) deficiency. In newborn screening (NBS), the acylcarnitine (AC) profile of his dried blood spots (DBS) collected on 5th day after birth was normal; C16-AC 3.45 nmol/mL (cutoff 6.3) and C18:1-AC 1.68 nmol/mL (cutoff 3.0). Objectives: To reduce the risk of false negative results we lowered the cutoff for C16-AC to 3.0, and replaced C18:1-AC with (C16+C18:1)/C2 ratio, with cutoff set at 0.62 (mean+6SD), which was 0.75 in DBS of the index patient. Methods: The new criteria were applied to newborns in Hiroshima area of Japan since 2011. Positive results were further evaluated by fatty acid oxidation study using cultured lymphocytes and genetic analysis. Results: By the end of 2012, five out of 43,794 newborns were positive. Mildly impaired capacity of fatty acid oxidation was observed in one subject, but no mutation was detected. The other four were concluded to be normal. Discussion: The high false positive rate arisen from our attempt indicates great difficulties in NBS of CPT2 deficiency scheduled on the 5th day of life. To prevent catastrophic results of false negative decision in NBS of fatty acid disorders, the first DBS specimen must be collected earlier.

Incidence and spectrum of IEMs in critically ill infants (0-90 days) in India and Pakistan Jalan R A1, Jalan A B1, Kudalkar K V1, Sawant L M1, Shinde D H1, Pawaskar M S1, Khan S A1 1

NIRMAN, Navi Mumbai, India

Background: Incidence of IEM in India and Pakistan are not known as both do not have any nationwide screening program despite having very large populations and high rate of consanguineous marriages. Objective: To investigate the spectrum of IEM's in critically Ill infants (0 – 90 days) and recommend screening tests to be performed for critical NICU babies. Materials and method: We investigated 1,226 critically ill infants admitted from various NICU's with a clinical suspicion of IEM for a period of Jan 2000 to April 2013. All these babies were subjected to a standard panel of tests to detect IEMs. Result: We diagnosed 978 cases with IEMs. We herewith present the data for 50 most common disorders in our cohort. NKHG-48 (4.90%), MMA-35 (3.57%), Galactosemia-34 (3.47%) MSUD-31 (3.16%) and Citrullinemia Type I-28 (2.86 %) were the 5 most common disorders. Conclusion: NKHG, Galactosemia, MMA, MSUD and Citrullinemia type I are the 5 commonest IEMs in critically ill infants in our cohort from India and Pakistan. Only 34.35% of IEMs were detectable by standard Expanded Newborn Screening. Therefore besides expanded newborn screening detailed metabolic investigations are recommended for all critically ill babies with suspected IEM's P-786

P-784 Optimization of laboratory newborn screening in the Czech Republic

Twelve patients with MAT1A mutations detected through newborn screening: 13 years experience

Chrastina P1, Bartl J1, Hodik J1, Svacinova R1, Hornik P1, Pinkasova R1, Jezova R1, Pekarkova Matousova V1, Krijt J1, Jesina P1, Peskova K1, Votava F2, Kozich V1

Chadwick S1, Fitzgerald K1, Weiss B1, Ficicioglu C1

1

Background: Methionine Adenosyltransferase (MAT I/III) deficiency is the most common genetic cause of persistent isolated hypermethioninemia. Pateints/Methods: This is a retrospective data analysis of 61 newborns with elevated methionine detected by newborn screen between January 2000 and March 2013. Results: 12 were identified as having classical homocystinuria; 37 were false-positives; and 12 were found to have isolated persistent

Inst Inher Metab Dis, Gen Univ Hosp, Prague, Czech Republic; Pediat, Univ Hosp Kral Vinohrady, Prague, Czech Republic

2

Objectives: The aim of our project is the optimization of the newborn screening program for inherited metabolic diseases (IMD) and steroid synthesis disorders in the Czech Republic. Results: We have expanded the present screening panel (10 IMD) by including 20 additional IMDs (urea cycle disorders, biotinidase deficiency,

1

The Children's Hospital of Philadelphia, Philadelphia, United States

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hypermethioninemia. These 12 individuals underwent MAT1A gene sequencing. 3 were found to have the mild autosomal dominant R264H mutation, 1 was found to be a compound heterozygote for two novel pathogenic mutations, and 3 were found to be heterozygotes for previously reported mutations shown to cause autosomal recessive MATI/III deficiency when present in homozygous or a compound heterozygous state, and the remaining 5 were found to be heterozygotes for novel variants of unknown clinical significance. For the majority of individuals, methionine persisted above the normal reference range but trended downwards over time. No one was started on a low-methionine diet, and all individuals have age-appropriate growth and development. Conclusion: Individuals with even single changes in the MAT1A gene may have elevated methionine detected by newborn screen that may persist for months after birth without an observable impact on overall health or development.

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We present results of an 18 month study carried out between July 2011–December 2012. Out of 220,570 samples received in this period, 101 triggered screen positive values for PA or MMA. Diagnostic evaluation in 93 patients revealed 8 true positives (PA, MMA or cobalamin defects) and 85 false positives including 19 maternal B12 deficiencies, yielding a positive predictive value (PPV) of 8.6%. No false negatives were reported during this period signifying 100% sensitivity. MCA was determined retrospectively in the screen positive specimens. Twenty two samples including all confirmed positives exceeded the established cut-off of 0.7 micromol/l yielding a PPV of 36% and 100% sensitivity. Median MCA concentration in confirmed patients was 8.5 micromol/l (range 0.8-89.4 micromol/l). We anticipate the implementation of MCA as a marker will improve newborn screening for this group of disorders. P-789

P-787 Developmental milestone comparision between the index patients and siblings diagnosed by neonatal screeningin in the same families

Positive newborn screening identifying mothers with biochemical differences: a single institution's experience

Rahbeeni ZA1, Al-Hahshem A1, Shoukri M1

Chapman KA1, Viall S1, Leon E1, Smpokou P1, Lanpher B1, CusmanoOzog K1, Kirmse B1

1

1

Aim: To conclude if early detection of IMDs by neonatal screening can help in preventing serious complications Method: Retrospective study reviewing the data of families who are followed at KFSH (Riyadh, KSA) with two or more affected children with same condition. The subsequent siblings were diagnosed by neonatal or selective screening. Data collected include age, sex,age at diagnosis, compliance with diet, and developmental assessment of index patient and the siblings who were screened and diagnosed early. Disorders included PKU, MSUD, MMA, HMG-CoA layase deficiency, 3-MCC deficiency, homocystinurea and tyrosinemia I (7 diseases). Data were stored in excel form and analyzed with SPSS program. Families with only one affected child were excluded. Chi square test of independence was used to test the association between categorical variables. Results: We included 42 patients from 19 families. The mean age of diagnosis in the index cases was 16 months while the mean was one month in the screened group. Three patients of the index group with late diagnosis had normal developments (3/17, 18%) while 12 of the early diagnosed siblings had normal developments (12/25, 48%); P value was found to be significant (less than 0.05).

Background: Newborn screening is designed to identify patients with inborn errors prior to their presentation. However, it can occasionally identify mothers with biochemical differences. Objectives: To identify the frequency of false positive newborn screens due to low carnitine or 3-methylcrotonyl CoA carboxylase (3MCC) in which we identify biochemically different mothers. Case Reports: We present at least 8 women identified by a newborn screen to have low carnitine levels and potential carnitine uptake deficiency out of 245 infants referred for abnormal newborn screening (from January 2012-April 2013). During this time no infants were identified to have carnitine uptake deficiency. We identified at least 2 women with probable 3-methylcrotonyl CoA carboxylase deficiency, as well as 2 infants with 3-methylcrotonyl CoA carboxylase deficiency over the same time period. Discussions/Conclusions: Newborn screening has increased the number of women with differences in their metabolism and there are few recommendations concerning their follow up. Abnormal screening identifying women with biochemical differences is a relatively frequent (4%) occurrence in our cohort and we are establishing recommendations for their follow-up.

P-788

P-790

Methylcitrate in DBS improves newborn screening for propionic and methylmalonic acidemia

Tandem mass spectrometry screening for inborn errors of metabolism in high risk Ukrainian patients

Al-Dirbashi OY1, McIntosh N1, McRoberts C1, Geraghty MT2, Santa T3, Chakraborty P1

Pichkur NA 1 , Olkhovych NV 1 , Mytsyk NY 1 , Trofimova NS 1 , Samonenko NV1, Shkurko TA1, Ivanova TP1, Gorovenko NG2

1 Newborn Screening Ontario, Ottawa, Canada; 2Dept of Pediatrics, University of Ottawa, Ottawa, Canada; 3The University of Tokyo, Tokyo, Japan

1 Nat Child Hosp "OHMATDET", Kyiv, Ukraine; 2Nat Med Acad of Postdip Educ, Kyiv, Ukraine

KFSH and RC, Riyadh, Saudi Arabia

Newborn screening for propionic (PA) and methylmalonic acidemia (MMA) using C3 and C3/C2 ratio is not specific, producing high false positive rates. Methylcitrate (MCA), a hallmark for propionate metabolic defects, is not detectable using standard newborn screening methodology. Recently, we developed an LC-MS/MS method which involves on-card derivatization to determine MCA in DBS. This method was validated using controls (n=370) and patient samples (n=18).

CNMC, Genetics and Metabolism, Washington, United States

Introduction: The application of tandem mass spectrometry (MS/MS) to screening for inborn errors of metabolism offers the potential of substantially altering the natural history of these diseases by reducing morbidity and mortality. Methods: Specimens of dried blood spots were collected from patients from the all region of Ukraine between 2011 to 2013. Acylcarnitines and amino acids were analyzed as butyl esters on an AB Sciex 2000 MS/MS with electrospray ionization.

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Results: We investigated 2250 patients referred to our center with suspicion for inborn errors of metabolism. On the basis of specific changes in the amino acids and acylcarnitines profiles we have identified 56 patients with IEM (2,5%), including 16 with aminoacidemias (29%), 6 with organic acidemias (11%), 3 with fatty acid oxidation disorders (5%), 3 with carnitine transport defects (5%), 3 with biotinidase defitiency (5%), and 26 with secondary carnitine defitiency (46%). We have also identified 33 cases with unspecific aminoacids and acylcarnitines profiles (1,5%). More detailed biochemical and molecular analysis in unclassified cases should be performed in future. Conclusions: The selective screening using MS/MS is high informative on the detection of inborn errors of metabolism, but requires mandatory use of confirmatory methods for definitive diagnosis.

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We report the eight years experience with MS/MS in Portugal and give an overview of the incidence of diseases, organization, updates on methods and current development, and future aspects. A total of 737,902 newborns were screened by MS/MS for more than 20 diseases between October 2004 and December 2012. Dried blood spot samples were collected (between the 3rd and the 6st day of life) and sent to the National Laboratory for Newborn Screening. The resulting overall incidence of inherited metabolic disorder identified by MS/MS was 1:2,335, including 118 newborns with amino acidemias (1:6,253), 16 with urea cycle disorders (1:46,119), 61 with organic acidurias (1:12,097), and 121 with fatty acid oxidation disorders (1:6,098). The introduction of MS/MS technology, significantly increased the detection of IEM that were previously not covered.

P-791 P-793 Cross-border newborn screening in Greifswald (Germany) and Szczecin (Poland) in the euroregion pomeraniaas a model of an international cooperation in the field of newborn screening Müller C1, Winter Th1, Giżewska M2, Ołtarzewski M3, Seiberling St4, Krzywińska-Zdeb E2, Romanowska H2, Bartkowiak E2, Nauck M1

Detection of epimerase deficiency galactosemia on supplemental newborn screening Lawson CE1, Heese BA1, Smith LD1 1

Div Clin Genetics, Children's Mercy Hosp, Kansas City, United States

1

University Medicine, Inst Clin Chem, Greifswald, Germany; 2 Pomeranian Medical University, Szczecin, Poland; 3 Institut for Mother and Child, Warsaw, Poland; 4Ernst Moritz Arndt University, Greifwald, Germany The aim of this project is a cross-border cooperation for the early detection of selected inborn errors of the metabolic and endocrine system funded by the framework of the European Union (INTERREG IVa). The project is carried out by the screening centres of Greifswald (northeast Germany) and Szczecin (northwest Poland) in partnership with the University of Greifswald and the Institute for Mother and Child in Warsaw. Cooperated planning, financing, organization and realisation lead to improved screening quality, a wider spectrum of detected disorders as well as to intensified and improved care of affected children. The spectrum of detected disorders in Szczecin was increased from 3 (phenylketonuria, hypothyroidism, cystic fibrosis (CF)) to 14 by means of tandem-mass-spectrometry analysis of Polish samples in Greifswald and the establishment of CAH- and galactosaemia screening in Szczecin. For the project time, Greifswald offers CF screening using a two-stage strategy measuring Immunoreactive Trypsin (IRT) and PancreasAssociated Protein (PAP). During the first 7 months of cooperation 14.648 newborns were screened and 6 affected children were detected. Additionally, unique meetings of families with children affected by inborn errors of metabolism contribute to better intercultural understanding on both sides of the border.

Epimerase deficiency galactosemia, a variant form of galactosemia, results from deficiency of the UDP-galactose 4'-epimerase enzyme (GALE). We report on a Caucasian infant who had a normal Kansas state newborn screen but, through an elective supplemental screen by PerkinElmer Genetics, Inc., was found to have an elevated total galactose level of 16.3 mg/dL (ref <15.0). At age three weeks his Gal-1-P level was 394 mcg/g. This child's GALE activity level was 17.8 μmol/hr/gm Hb (ref 17.1-40.1), suggesting heterozygote status. Molecular sequencing revealed no mutations in GALE. An ophthalmology exam at age two months was normal. The severity of epimerase deficiency galactosemia ranges from serious complications, similar to those seen in classical galactosemia, to a relatively benign condition only affecting the red blood cells. Newborn screening programs that only analyze GALT activity will fail to detect GALE deficiency with potentially serious adverse consequences, as the most severely affected children require prompt dietary intervention to avoid liver dysfunction, infection and/or cataracts. Children with the intermediate phenotype may also benefit from intervention. This case suggests further research is necessary to determine the benefits of using total galactose to identify children with GALE deficiency in programs that rely exclusively on GALT activity. P-794 Experience and development of a national program for newborn screening in Uruguay

P-792 Changed incidence of IEM in Portugal after expanded NBS Vilarinho L1, Sousa C1, Fonseca H1, Lopes L1, Carvalho I1, Rocha H1, Marcão A1 1

Newborn Screening Unit, Nat Inst Health, Porto, Portugal

The National Portuguese Newborn Screening Program was introduced in 1979 for phenylketonuria and in 1981 for congenital hypothyroidism. In 2004, the introduction of tandem mass spectrometry (MS/MS) substantially increased the number of detectable inborn errors of metabolism (IEM) and now includes disorders of fatty acid oxidation, organic acidurias and various disorders of amino acid metabolism and urea cycle.

Queijo C1, Garlo G1, Machado M1, Gonzalez F1, Corbo L1, Franca K1, Segovia B1, Tadeo L1, Queiruga G1, Lemes A1 1

Newborn Screening Laboratory, Montevideo, Uruguay

In Uruguay, NBS is mandatory and financed by the government. We have a National NBS Program with a unique NBS Laboratory that process almost all samples. Our National Program began in 1994 with CH. During 2007 we started with CAH and PKU and in 2010 with CF. Since 2009 we have a pilot program for expanded newborn screening by MS/MS. The average number of newborns screened per year is approximately 49,000, reflecting 99% of newborns of the country. The recommended time of sampling is 40 hours of life.

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The methodologies used are ELISA (CH, CAH, CF) and tandem mass spectrometry (PKU and others aminoacidopathies, FAO, organic aciduria). Since November 2007 to December 2012 we processed 246350 samples finding a global incidence of 1/4399 without CH cases. Our results confirm the high frequency of selected congenital diseases, similar to other worldwide National Programs. They also illustrate the advantage of a centralized laboratory relying on a clinical network able to provide fast treatment necessary for a good outcome in the positive cases. At present we are starting with confirmatory assays (GC-MS and HPLC for amino acids) that will provide a complete diagnosis locally.

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Conclusions: It appears that children with type 1 diabetes have a unique metabolic profile at birth that may put them at risk for early development of type 1 diabetes provided additional genetic susceptibility factors are present. This unique profile may enable early detection through newborn screening for type 1 diabetes. P-797 Optimization of newborn screening for homocystinurias Kožich V1, Bártl J1, Chrastina P1, Hodík J1, Krijt J1, Ješina P1, Pešková K1

P-795 1

Inst Inherit Metab Dis, Gen Univ Hosp, Prague, Czech Republic

Prevalence of glucose-6-phosphate dehydrogenase deficiency in newborns in Argentina Suldrup N1, Cesari N1 1

IACA Laboratorios, Depto. Metabolopatías, Bahía Blanca, Argentina

In Argentina newborn screening is mandatory by law for certain conditions but not for Glucose-6-Phosphate Dehydrogenase (G6PDH) deficiency. G6PDH deficiency is an X-linked disorder which causes in most cases neonatal jaundice and even kernicterus and may cause acute intravascular hemolysis in association with intake of certain drugs, foods or oxidative stress. It is one of the most common enzymopathies throughout the world and one of the causes of Chronic Non-Spherocytic Hemolytic Anemia. The aim of this study was to determine the prevalence of G6PDH deficiency in Argentina. We tested 4,500 males from newborn dried blood spots. Enzyme activity was quantitatively determined by a fluorometric method, measuring the rate of formation of NADPH. We found that 0,2% of the total of the samples showed total deficiency, while 0,7 % demonstrated intermediate deficiency; thus, the combine prevalence for both was 0,9 % among the male newborn population. This finding is important as such patients must receive a preventive and educational care. Screening for G6PDH deficiency is feasible and not only would it take early preventive measures against severe hemolysis in the neonatal period, but also other preventive measures in later life.

Introduction: The presently used marker methionine (Met) has a low sensitivity and specificity for detecting newborns with homocystinurias while total homocysteine (tHcy) determination is too expensive for mass screening. We aimed at optimizing a two-tier system for more efficient screening of homocystinurias. Methods: Primary markers were determined by MS/MS after butylation with comercial kit, total homocysteine in dry blood spots was extracted by acetonitrile/water and determined by LC-MS/MS. Results: In a cohort of 2,000 newborns the 1st and 99th centile of primary markers Met, Met/Phe and Met/Xle were 4-50 micromol/L, 0.28-1.10 and 0.10-0.37, respectively. The tHcy determination in dry blood spots has a limit of detection 0.5 micromol/L, reproducibility of 12.8% at 5 micromol/L, recovery 82% and reference range in newborns (n=100, weight>2500g, 5th-95th percentile) between 4.3 and 10.2 micromol/L. Our target is to analyze in the second-tier 1% of samples with the highest and lowest Met, respectively; using all three primary markers, the cut-off for analyzing tHcy will be for Met concentrations <5 and >35 micromol/L. Conclusions: An optimized system using three markers and a second tier-method for tHcy is likely to increase the sensitivity and specificity of screening for homocystinurias. Supported by grants MH-CZ-DRO-VFN64165/2012 and IGA MZNT/12213–3. P-798 Mutation spectrum of BTD gene in east Turkey

P-796

Chang YC1, Tanyalcin I2, Chiu YH3, Liu TT1, Tanyalcin I4, Hsiao KJ5

The potential for newborn screening for the early detection of type 1 diabetes

1 VYM Genome Research Center, NYMU, Taipei, Taiwan; 2VUB Center for Medical Genetics, Uz Brussel, Belgium; 3Dept of Educ & Research, City Hospital, Taipei, Taiwan; 4Tanyalcin Medical Laboratory, Izmir, Turkey; 5Preventive Medicine Foundations, Taipei, Taiwan

Johnson BA1, Waldhör T2, Rami B3, Bodamer OA1 Dept Human Genetics, Univ of Miami, Miami, United States; 2Centre Pub Health, Dept Epidem, Med Univ, Vienna, Austria; 3Div Paed Endo, Univ Child Hosp, Vienna, Austria 1

Background: The incidence of type 1 diabetes, an autoimmune disease, continues to increase albeit the complex nature of its multifactorial pathophysiology is poorly understood. It is yet unknown whether nutritional or metabolic factors in addition to genetic factors may confer an additional risk for type 1 diabetes. Methods: We retrospectively compared the metabolite profiles that have been obtained for neonatal screening by tandem mass spectrometry in 35 day old infants (n=60; 31 female; 3522g + 500g; 39.6 weeks + 1.4 weeks) that later developed type 1 diabetes with a reference population. Results: Concentrations of free carnitine, total carnitine, acetylcarnitine (C2), long-chain acylcarnitine species, guanidinoacetate, and several amino acids were significantly decreased (p< 0.000001 to p< 0.00027) in infants who later developed type 1 diabetes, while concentrations of methionine and other carnitine species were significantly elevated (p< 0.000001 to p< 0.000016).

Background: Biotinidase deficiency (BD, MIM 253260) is an autosomal recessively inherited disorder that affects the endogenous recycling of biotin. The intracellular depletion of biotin leads to low activities of biotin dependent carboxylases and a characteristic clinical symptom including seizures, muscular hypotonia, skin eczema and metabolic acidosis which may result in coma and early death. Human biotinidase was encoded by BTD gene consisting of 4 exons with a total length of 1629 bp. Newborn screening has enabled the identification of children with profound (<10% of residual activity) and partial (10-30% of residual activity) biotinidase deficiency using colorimetric enzyme assay on dried blood spots. Material and Methods: Sixty-two patients were identified by newborn screening or clinical observation. The coding regions and the flanking areas of the BTD gene were analysed by Sanger sequencing. Results: Mutations were identified in 93.2% of 107 mutant alleles. The mutations 470G>A (R157H), 98_104delinsTCC (C33Ffs*36), c.1330G>C (D444H), and 1595C>T (T532M) accounted for 44.7%, 20.4%, 10.7% and 4.9% of BTD mutant alleles, respectively. The

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mutations 470G>A, 98_104delinsTCC and 1595C>T were the most common cause of profound biotinidase deficiency whereas the c.1330G>C was associated to partial biotinidase deficiency. Conclusion: This study demonstrated a different mutation spectrum of BTD gene in East Turkey. P-799 Newborn screening for inborn errors of metabolism at Sultan Qaboos University Hospital (Oman): a pilot study Al-Riyami S1, Al-Maney M1, Bayoumi R1, Krishnan L2, Joshi SN2, Al Murshedi F3, Al-Thihli K3 Department of Biochemistry, SQUH, Al Khod, Oman; 2Department of Child Health, SQUH, Al Khod, Oman; 3Genetic and Developmen Med. Clinic, SQUH, Al Khod, Oman 1

Background: Inborn Errors of Metabolism (IEM) constitute a major burden in developing countries; with relatively high incidence largely reflecting increased rates of consanguinity. Objectives: to estimate the incidence of IEM detectable by tandem mass spectrometry (MS/MS) at Sultan Qaboos University Hospital (SQUH). Methods: all newborns delivered at SQUH were screened for 24 treatable IEM via MS/MS using dried blood spots samples collected between 24-72 hrs of life. Positively screened cases were then evaluated using quantitative plasma amino acids analysis (HPLC) and/or urine organic acids analysis (GC-MS). Results: 3 out of 1780 screened newborns tested positive for an underlying IEM: one with very long chain acyl-CoA dehydrogenase deficiency, one with maple syrup urine disease, and one with propionic acidemia. The incidence of IEM detected by MS/MS at SQUH during the study period was 1 in 600. Discussion: The incidence of IEM in this study is among the highest reported incidences worldwide. This may be partly due to referral bias; the true incidence may not significantly be different from that observed in neighboring countries. Conclusion: The high incidence of IEM shown in this study calls for a larger study and argues for a national newborn screening program in Oman. P-800 Evolution of patients with methionine adenosyltransferase (MAT) I/III deficiency detected by neonatal screening in Spain

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course was monitored, recording mean and range of methionine level over time. Psychomotor development was evaluated. The diagnosis was confirmed by MAT1A molecular testing: 15 patients were heterozygous for the dominant R264H mutation, one patient for the R199C(c.595C>T) mutation, 2 patients were homozygous for the novel mutations p.T288A(c.862A>G) and p.L355K(c.1064T), respectively. Patients with the R264H mutation have only moderately elevated methionine levels and it is believed that their neurological outcome will be similar to their affected parents. The prognosis and outcome in the homozygous individuals remains uncertain because they maintain methionine levels >400 μM. P-801 Pilot newborn-screening in Guatemala - first experience with the NeoPlex4. assay from Luminex Silva Polanco L1, Velasquez O1, Sandoval MA1, Bustamente YY1, Granados L1, Fingerhut R2, Silva Arevalo G1 1

INVEGEM, Newborn Scr & Metab, Santa Lucia Milpas Altas, Sacatepéquez, Guatemala; 2Swiss Newborn Screening, Univ Child Hosp, Zurich, Switzerland Background: Newborn Screening (NBS) for inborn errors of metabolism and endocrinopathies is a worldwide highly accepted tool of preventive medicine with an extremely positive cost/benefit ratio. However, in Guatemala there is so far no nationwide NBS program installed. Outline: In summer 2013 a pilot program for NBS in Guatemala will start in selected hospitals and regions. Proposed coverage is 20,000 samples in the first year, increasing to 60,000 – 100,000 over the next 2 years and leading to nationwide newborn screening in Guatemala. Methods: NBS will be performed with the NeoPlex4® Assay from Luminex, with TSH, and T4 for CH, 17-OHP for CAH, and IRT for CF. The system is validated against samples from external quality control and positive samples from the Swiss Newborn Screening. During the pilot phase of NBS in Guatemala, all results of positive cases will be cross-checked in Zurich. Results: First tests with QC-material showed reasonable CVs, with recoveries between 97 and 120% for all analytes at 3 different concentrations. Out of 183 samples from the Swiss NBS, all positive samples were also found positive with the NeoPlex4® Assay. In addition, we will show results of the first months of our screening pilot program. P-802

Bóveda MD1, Castiñeiras DE1, García-Jimémez C2, Balmaseda E3, Vives I4, Iglesias AJ1, Fraga JM1, Corrales FJ5, Couce ML1

3-Methylcrotonyl-CoA carboxylase deficiency: experience with selective screening in a single center vs newborn screening

1

Hospital Clínico Universitario, Santiago de Compostela, Spain; Hospital Miguel Servet, Zaragoza, Spain; 3Complejo Hospitalario Universitario, Albacete, Spain; 4Hospital Universitario Virgen Arrixaca, Murcia, Spain; 5CIMA. Universidad de Navarra, Pamplona, Spain 2

Methionine adenosyltransferase (MAT) I/III deficiency is an inborn error of methionine metabolism. Thirty seven different MAT1A mutations have been reported in hypermethioninemic humans. Although it can also be symptomatic, most cases are clinically benign and detected by newborn screening (NBS) for hypermethioninemia. The optimal management of MAT I/III deficiency remains to be defined; the long-term prognoses of patients detected by NBS is unknown. We describe the long-term evolution of 18 patients diagnosed by screening in four regions of Spain: 13, 1, 3 and 1, from Galicia, Murcia, Aragón and Castilla-La Mancha respectively. Age, presence/absence of symptoms at diagnosis, methionine levels at diagnosis and at confirmation were recorded for each case. Clinical

Pokora P1, Gradowska W1, Oltarzewski M2, Jablonska E2, Taybert J1, Pajdowska M3, Bogdanska A3, Kowalik A1, Sykut-Cegielska J1 1 Dept Metab Dis, Child Mem Health Inst, Warsaw, Poland; 2Screening Dep, Inst of Mother and Child, Warsaw, Poland; 3Laboratory Diagn, Child Mem Health Inst, Warsaw, Poland

3-methylcrotonyl-CoA carboxylase (MCC) deficiency presents with variable phenotypes from neonatal onset with severe neurological symptoms to asymptomatic adults. It is the most frequent organic aciduria detected by MS/MS newborn screening (NBS). The majority of patients remains asymptomatic even without any treatment. In our center selective screening revealed 11 patients (current age: 4.5-19 years) identified due to sudden hypoglycaemia, metabolic acidosis with vomiting, or recurrent metabolic decompensation during infections, and 5 asymptomatic children diagnosed through family screening (affected

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siblings). NBS detected 19 affected newborns (current age: 3 month11years) and 7 cases of maternal MCC deficiency (with clearly elevated C5OH-carnitine). Biochemical results are comparable in patients diagnosed symptomatically and by NBS. In the selective screening and NBS groups, C5OH-carnitine in dry blood spots was significantly increased in 45% and 68% of cases, respectively. At the time of diagnosis urinary excretion of 3-HIVA and 3-MCG was detected in all but 2 cases (in one has remained undetectable during 11 months of follow-up). Massive elevation of 3-HIVA and 3-MCG was noted in above half of the patients identified symptomatically and by NBS, as well. Conclusion: The biochemical phenotype of MCC deficiency does not allow to predict clinical course or whom to treat.

was taking antibiotic therapy, therefore mild IVA with low excretion of IVG or the presence of another isobaric compound was considered. Further investigations using reverse phase chromatography, fragmentation of the molecule using tandem mass spectrometry and gas chromatography allowed us to identify the C5-carnitine as 2 methyl butiryl carnitine. This metabolite is associated with short branched chain acylCoA dehydrogenase (SBCAD). This is the first case identified in the UK through the screening programme. P-805 Broadening of neonatal screening in the federal district of Brazil through the implementation of tandem mass spectrometry

P-803 Methylmalonic acid (MMA) as a primary marker of neonatal vitamin B12 deficiency in expanded newborn screening

Cardoso MT1, Pogue R1, Oliveira RN1, Oliveira RN1, Viegas MS1, Oliveira IS1, Freitas RS1, Pogue HB1, Pogue HB1, Avelino LMC1, Thomas JV1, Raniere E2 1

Bettocchi I1, Baronio F1, Righetti F1, Rizzello A1, Balsamo A1, Pession A1, Cassio A1

NUGEN,Univ.Catolica Brasilia, Distrito Federal, Brazil; 23SouthAustralian Neonatal Screening Cent, Adelaide, Australia

1

Until 2008, neonatal screening in Brasilia included: phenylketonuria, hypothyroidism and hemoglobinopathies. In compliance with Districtal Law 4190/2008 that instituted a broadened neonatal screening protocol in the public network of the Federal District, tandem mass spectrometry was incorporated for screening of amino acidopathies, defects of beta oxidation, and organic acidemias. Our objective is to describe the implementation of mass spectrometry in the screening protocols of the Federal District. The definition of cut-offs for diseases included in the broadened program for this population was carried out by a sampling process in three steps: with 3,500 samples (provided through collaboration with Dr. Enzo Ranieri); five months later with 9,700 samples; seven months after that with 23,500 samples. The establishment of these values was done through calculation percentiles for upper limits, and for lower limits. The confirmatory tests for children with positive results were performed in partner laboratories . With the paradigm change in analysis methods, after two years of experience and 90,000 samples analyzed, we have achieved a population coverage of 95.6% of children up to seven days of age. Currently, we are the only Brazilian public service that performs neonatal screening for 22 diseases, together with the National Neonatal Screening Program.

Neon.Screen.Center, Ped.Endocr.Prog., Bologna, Italy

Introduction: Vitamin (vit.) B12 deficiency, a treatable condition most commonly due to maternal vit.B12 deficiency (caused by vegetarian diet, pernicious anemia, gastric bypass), is not a primary target of NBS programs. Elevation of propionylcarnitine (C3) may incidentally identify this condition but C3 may not be sufficiently high during the first days of life when NBS samples are collected. We decided to apply second tier testing of MMA for levels of C3 below our usual cut-off (>6.8 micromol/L to 3 micromol/L). Methods: we examined 37.296 infants in our Regional Screening Program between 31st May 2012 and 5th May 2013. NBS samples with C3>3micromol/L had second tier MMA tests. All newborns positive (13) and some of their mother underwent further laboratory testing. Results: We found 5 patients with vit.B12 deficiency secondary to maternal vit.B12 deficiency. All were asymptomatic when the treatment with hydroxycobalamin was started and subsequently all biochemical markers normalized. Conclusion: When undiagnosed, vit.B12 deficiency in infants can result in anemia, failure to thrive, developmental delay, and neurological deficits. The most common cause is maternal vit.B12 deficiency. Without lowering the C3 cut-off none of the 5 infants would have been identified. MMA is a more sensitive marker of vit.B12 status.

P-806 Reduction in the overall incidence of inborn errors of metabolism in the eastern province of Saudi Arabia

P-804 False positive for isovaleric acidaemia in neonatal screening

AlRatrout R1, Tamimi W2 1

1

1

2

Herrera DJ , Henderson MJ , Williams T , Walter JH

1 1

2

Biochem Genet, LTHT, Leeds, United Kingdom; Willink Biochem Genet Unit, Manchester, United Kingdom An English national newborn screening pilot study is in process to evaluate the inclusion of five additional disorders. These are: maple syrup urine disease, homocystinuria (pyridoxine unresponsive), isovaleric acidaemia, glutaric aciduria type 1 and long chain hydroxyl acyl CoA dehydrogenase deficiency. Screening for isovaleric acidaemia (IVA) has proven to be the most challenging with many confounding factors such as distinguishing classical IVA from "mild IVA", false positives associated with antibiotic therapy (Pivmecillinam) and other potential isobaric compounds. We describe a positive screen for IVA with a C5 concentration in the screening sample of 1.1 mmol/L. Diagnostic samples confirmed the initial elevation of C5 with concentrations between 0.9 and 1.2 mmol/L but no isovaleryl glycine was found in the urine. Neither baby nor mother

Newborn Screening Dammam Regional Lab, Dammam, Saudi Arabia, King Fahad National Guard Hospital, Riyadh, Saudi Arabia

2

Background: Screening services for inborn errors of metabolism (IEM) are provided in the Eastern Province of Saudi Arabia. We report the preliminary incidence of IEM in newborns in this area and compare the data to other national and international figures. Patients and Methods: Blood filter papers of 13459 newborns were collected during the 4 months period from January 2013 to April 2013. The samples were examined by tandem mass spectroscopy. Results: Nine cases were confirmed to have an IEM. The overall incidence was found to be 1 in 1495 corresponding to a cumulative incidence of 66 cases per 100,000 live births. This was 2-folds lower than what has been reported previously (1:667). Congenital hypothyroidism was diagnosed in 2 patients (22%). Phenylketonuria (PKU) was found in 3 cases (33%). Two cases of galactosemia (22%) were

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detected. One case of each of biotinidase and 3-Methylcrotonyl-CoA carboxylase deficiency was reported. Conclusion: Incidence of IEM has decreased which may be due to implementation of newly sophisticated technology and improvement of public awareness of the risk factor of consanguineous marriage. Compared with other regions, the incidence of congenital hypothyroidism was lower. However, the incidence of PKU was higher.

mothers with lower vitamin B12 levels presented lower birthweight and higher C3 levels, with higher C3/C2 and C3/C16 ratios. Conclusions: Smoking seems to have a negative effect, and meat consumption a positive effect on maternal vitamin B12 levels. Newborn screening levels of C3, C3/C2 and C/C16 are correlated with maternal vitamin B12 status. P-809

P-807 MS/MS newborn screening for inborn errors of metabolism in Brazil: a 2-year-challenging-experience of Apae de São Paulo

Dihydropterine reductase (DHPR) deficiency detected by newborn screening: a case report Bal MO1, Bettocchi I1, Zazzetta E1, Cantasano A1, Th ny B2, Cassio A1

1

1

1

1

Piazzon FB , Garcia LRRG , Iskandar MAM , Belarmino TMA , Martins HH1, Silva EN1, Bueno C2, Kok F2, Hadachi SM1

Neon Screen Center, Ped Endo Progr, Bologna, Italy; 2Div Clin Chem Biochem, Univ Child Hosp, Zürich, Switzerland 1

1

Apae de São Paulo, São Paulo, Brazil; 2FMUSP/FMUSP, Sao Paulo, Brazil

Electrospray ionization-tandem mass spectrometry (MS/MS) permits efficient identification of several metabolic disorders with acceptable laboratory operating costs and changes in patient mortality. The aim is to describe the positive profile of the newborn screening center, to determine the impact of expanded screening by MS/MS on the local overall detection rate of inborn errors of metabolism (IEM), and to assess patient outcome. From 2010 to 2013, 29553 neonates were investigated for 38 inborn errors of metabolism by MS/MS. The overall value of the screening program was estimated by the sensitivity (94%), specificity (99%) and accuracy (99%). Online follow-up of all detected patients went until confirmation of the diagnosis. Confirmed patients are evaluated and treated at the neurometabolic unit of Children´s Hospital. 15 newborns had a confirmed IEM. Other 6 IEM were confirmed in older patients (not NB screening). 391 tests (1.5%) were false-positive. The screening by MS/MS for more than 30 disorders has drastically increased the detection rate when compared to the conventional techniques. Yet of poor availability in Brazil, this methodological strategy represents unequivocal preventive medicine by enabling diagnosis and treatment before the onset of symptoms, what might be a model for the governmental programs.

Background: DHPR deficiency is an autosomal recessive disorder of tetrahydrobiopterine (BH4) regeneration and results in decresed synthesis of dopamine and serotonin. It could be detected by newborn screening if phenyalanine (PHE) levels are high and by measurement of enzyme activity . Subjects with DHPR deficiency may present microcephaly, hypotonia, mental retardation and convulsion. Treatment for DHPR deficiency consists in low PHE diet, L-dopa, 5-hydroxytriptofan and folinic acid supplementation. Despite therapy many patients have significant developmental delays and develop brain abnormalities. Case-report: African female born at term from consaguineous parents. At Newborn screening 1st PHE spot 228.96 micromol/L (n.v. <120), 2nd spot 345.76, DHPR activity (DBS) 0 mU/mg Hb (n.v. 1.8-3.8). CSF analysis showed normal biopterin but low 5-Hydroxyindolacetic and Homovanillic acid. MRI and electroencefalogram were normal and she was asymptomatic. She started low PHE diet at 15 days of life and then also treatment with neurotransmitter precursors. The analysis of the gene QDPR is still ongoing. Conclusions: DHPR deficiency is a rare and often undiagnosed disease because PHE levels can be either normal at birth. All patient with elevated PHE levels at the newborn screening should be investigated for DHPR activity, in order to prevent neurological complications.

P-808 P-810 Correlation of maternal vitamin B12 levels during the first trimester of pregnancy with newborn screening results Yahyaoui R1, Dayaldasani A2, Ruiz-Escalera JF2, Rodríguez-Espinosa M2, Rueda-Fernández I1, Casero MC2, Pérez-Valero V2 1 Newborn Screening. Carlos Haya Univ Hosp, MALAGA, Spain; 2Clin Lab. Carlos Haya Univ Hosp, MALAGA, Spain

Background: Expanded newborn screening programs can detect maternal vitamin B12 deficiency. The correlation of maternal vitamin B12 levels with newborn screening related markers (C3, C3/C2 and C3/C16) is unknown. Methods: Serum vitamin B12 concentrations were determined in 204 pregnant women during the first trimester. To assess the effects of the variables BMI, vitamin supplements (including type and dosage), dietary intake, parity, and smoking habits, on vitamin B12 serum concentrations, stepwise multiple linear regression models using backward elimination were used. Other factors including the newborns' birthweight and expanded newborn screening results (C3 levels, C3/C2 and C3/C16 ratios) were considered. Results: The age range of women was 15-46 years, (mean 30.0, SD 6.11). The mean serum concentration of vitamin B12 was 502.4 pg/mL (SD 142.81). Vitamin B12 concentrations were significantly lower in smokers and in women with low meat consumption. Newborns of

A pilot newborn screening program for mucopolysaccharidosis type I in Taiwan Lin SP1, Lin HY2, Wang TJ3, Huang SF3, Chang CY1, Lin DS1, Tsai CC4, Keutzer J5, Chuang CK6 Dept Pediatr, Mackay Memorial Hosp, Taipei, Taiwan; 2Mackay Medical College, New Taipei City, Taiwan; 3Dept Lab Med, Mackay Memorial Hosp, Taipei, Taiwan; 4 Dept Med Research, Mackay Memorial Hosp, Taipei, Taiwan; 5Genzyme, A Sanofi Company, Cambridge, MA, United States; 6 College of Medicine, Fu-Jen Catholic Uni, New Taipei City, Taiwan 1

Background: Mucopolysaccharidosis type I (MPS I) is a genetic disease caused by deficiency of the enzyme activity of α-L-iduronidase (IDUA). MPS I is classified into 3 clinical phenotypes called Hurler, Scheie, and Hurler-Scheie disease according to the clinical severity. Treatments for MPS I are available. Better outcomes are associated with early diagnosis and early treatment, which suggests a need for newborn screening for the disorder. The goal of this study was to determine whether measuring IDUA activity in dried blood on filter paper is effective in newborn screening for MPS I. Methods: We conducted a newborn screening pilot program for MPS I from October 01, 2008 to December 31, 2012. Screening involved

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measuring IDUA activity in dried blood spots from 32,756 newborns using a fluorometric assay. Results: Of the 32,756 newborns screened, three were recalled for leukocyte IDUA enzyme activity testing. Two of the three had deficient leukocyte IDUA activity. DNA analyses confirmed the diagnosis of MPS I in these two newborns. Conclusion: It is feasible to use the IDUA enzyme assay for newborn screening. The incidence of MPS I in Taiwan estimated from this study is about 1/16,500. Conflict of Interest declared. P-811

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acidurias with 10 different disorders: BKT (21 cases), MMA (13 cases), 5-oxoprolinuria (14 cases), PPA (10 cases), GA2 (6 cases), 3methylglutaconic aciduria, congenital lactic acidemia, MCD, GA1, and 3-methylcrotonyl-CoA carboxylase deficiency. 21.8% of cases were aminoacidopathies (22 case with MSUD & 7 cases with PKU), 13.5% were fatty acid oxidation disorders (MCAD, SCAD VLCAD, CPT2, or CPT1), & 10 cases (7.5%) were urea cycle defects (6 cases with OTC, citrulinemia type 1 or arginosuccinic aciduria). Management saved lives without complication in 23.3%, with complication in 20.3%, and death in 56.4% of cases. Conclusions: Organic acidurias are the most common IEMs at the NHP; beta-ketothiolase deficiency is the most common entity. Early detection of these disorders is essential for effective treatment.

Expanded newborn screening for inherited metabolic diseases in eastern Sicily

P-813

Messina M1, Muccilli V1, Barone R2, Fiumara A1, Meli C1, Pittalà A1, Sorge G1

Measurement of psychosine in dried blood spots: a potential improvement of newborn screening for Krabbe disease

1 Div Metab Dis,, Univ Child Hosp, Catania, Italy; 2Child Neurol, Univ Child Hosp, Catania, Italy

Matern D1, Turgeon C1, Orsini JJ2, Sanders K1, Wasserstein M3, Oglesbee D1, Gavrilov D1, Escolar M4, Tortorelli S1, Rinaldo P1, Raymond K1

Expanded newborn screening in Sicily began as a pilot project in 2011, supported by the Sicilian Region in Eastern and Western Sicily, with the aim of screening an estimated 50,000 newborns per year. Newborns are screened for fatty acid oxidation, organic acid, urea cycle and amino acid disorders. Blood collection is performed between 48 and 72 h of life, and dried blood spots are sent by courier to laboratory. Analyte measurement are extracted from dried blood spots with a solution containing stable-isotope labeled internal standard and analyzed by tandem mass spectrometry. In Eastern Sicily (Catania city and surroundings) during the pilot project, cut-off values for all analytes were established after evaluation of 5000 healthy newborns. Up to now, blood spots from up to 19100 infants have been analyzed and 35 recalls have been made with 9 affected patients identified (maternal Methylcrotonyl-CoA carboxylase, Phenylketonuria, Hyperphenylalaninemia, Hypermetioninemie, Methylmalonic aciduria with homocystinuria, Citrullinemie I). Screening results show sensitivity: 100%; specificity: 99,9%; predictive positive value: 25,7%; and predictive negative value: 100%. Diagnosed newborns are currently followed at the regional referral center and laboratory of inherited metabolic disease. Continuance of expanded newborn screening in Sicily enhances prevention and care of IMD in south Mediterranean countries. P-812 Spectrum of inborn errors of metabolism in the referal center of north Vietnam Vu D1, Nguyen K1, Khu D1, Bich N1, Bui T1, Nguyen H1, Nguyen L1, Fukao T2, Kondo N3, Yamaguchi S3 National Hospital of Pediatrics, Hanoi, Viet Nam; 2Gifu University School of Medicine, Gifu, Japan; 3Shimane University School of Medicine, Izumo, Japan 1

1

Biochemical Genetics Lab, Mayo Clinic, Rochester, MN, United States; New York State Dept of Health, Albany, NY, United States; 3The Mount Sinai Medical Center, New York, NY, United States; 4Children's Hospital of Pittsburgh, Pittsburgh, PA, United States 2

Newborn screening (NBS) for Krabbe disease (KD) is conducted in New York and Missouri by measurement of galactocerebrosidase (GALC) activity using tandem mass spectrometry (MS/MS) with the primary goal to identify and treat early-onset KD (EOKD) cases. So far, the incidence of EOKD is unexpectedly low (1:500,000) while many individuals (ca. 1:7,000) with reduced GALC activity and genotypes of uncertain significance are detected and subjected to costly and repeated comprehensive follow up testing. Psychosine (PSY) is a substrate of GALC and has been suggested as a marker of KD progression. Orsini and colleagues recently described a method to measure PSY in DBS by liquid chromatography MS/MS (Clin Chim Acta. 2013; 419: 73-6) which we now apply as a 2nd tier test in an ongoing NBS study into KD and other conditions. In addition, we have begun to correlate PSY concentrations in DBS of symptomatic (range: 50-98 nM; n=6) and asymptomatic KD cases (range: 2-6 nM, n=4; NBS controls: 99th%ile: 6 nM; n=120). Preliminary results indicate that PSY measurement in DBS could serve as both a 2nd tier assay in NBS for EOKD but also for the follow up and determination of disease progression in cases with later-onset or unspecified KD. P-814 Results of expanded newborn screening with electrospray tandem mass spectrometry (ESI-MS/MS) in 118,389 babies: a ten-year summary (2002-2012) of a single center from Turkey Alsancak S1, Aktuglu Zeybek AC2, Caglayan N1, Akbulut H1, Laleli Y1

Objective: To study the spectrum of IEMs and the survival rate of patients with IEMs at the National Hospital of Pediatrics (NHP) in Hanoi, Vietnam. Patients and Methods: 1229 cases with high risk of IEMs were studied at NHP from 2005 to 2012. Blood amino acid, acylcarnitine, and urinary organic profile were analyzed using Tandem MS & GC/MS. Results: IEMs were identified in 133 cases. Age of onset < 12 months accounted for 80%. 76 cases (57.2%) were organic

Duzen Laboratories Group, Istanbul, Turkey; 2Div Ped Metab Dis, Cerrahpasa Medical Fac, Istanbul, Turkey 1

Background: This study aims to determine the impact of expanded newborn screening by ESI-MS/MS on the overall detection rate of selected inborn errors of metabolism (IEM) in Turkey. Methods: Throughout a ten-year period 118,389 dried blood samples from both asymptomatic and symptomatic term newborns were

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analyzed with ESI-MS/MS for selected aminoacid, organic acid and fatty acid oxidation disorders. Positive screening results were referred to pediatric metabolic disease centers for definite diagnosis. Results: Among 128 babies with confirmed IEM, 76 were 2-4 and 52 were 5-7 days of age while 59 were symptomatic and 69 were asymptomatic when blood samples were taken. The overall incidence of IEMs was 1:925. Aminoacidopathies were the most common disorders detected (1:1784), followed by organic acidemias (1:2819) and fatty acid oxidation disorders (1:5919). 10 children had died before the report of the screening. Conclusions: Screening with MS/MS has increased the detection rate of IEM in Turkey where only phenylketonuria, biotinidase deficiency and hypothyroidism are included in nationwide screening programs (NSP). Using ESI-MS/MS as a NSP would not only be an opportunity in preventive medicine by enabling early treatment but would also enable prenatal diagnosis in future pregnancies of the parents.

nocturnal feeding with nasogastric tube to assure adequate energy intake but we observed worsening of feeding difficulties (vomiting and food refusal). Metabolic balance was improved: mild lactic acidosis persisted (2,7 mmol/L). Enteral nutrition was stopped at 9 months without any improvement. Food allergy and endocrine problems were excluded. Actual diet (2y) is mildly hypoproteic but caloric intake is still low; her growth is worsening (weigh < 3rd centile – height 3rd centile) and feeding difficulties are not improved. Are these symptoms results of overtreatment? Expanded newborn screening can identify asymptomatic patients. In our family negative impact of diagnosis in asymptomatic phase may be related with feeding difficulties. Each patient needs individual evaluation to establish if dietary treatment is necessary but a diagnosis is important to monitor metabolic assessment also in asymptomatic patients when exposed to metabolic stressors.

P-817

P-819

Diagnosis of inborn errors of metabolism by a tandem mass spectrometry method using urine filter paper samples

Methods to improve the accuracy and efficiency of newborn screening for congenital adrenal hyperplasia (CAH)

Rebollido MM1, Castiñeiras DE1, Cocho JA1, Rizzo C2

Rowe AD1, Matern D2, Rinaldo P2, Currier R3, Lorey F3, Lindner M4, Mxrkrid L1

1 Unit Diag and Treat Metab Dis, Ped. HCU, Santiago de Compostela, Spain; 2Repart Malattie Metaboliche.Bambino Gesù, Rome, Italy

1

Sect IEM Dep Med Biochem, Oslo Univ Hosp, Oslo, Norway; Biochem Gen Lab, Mayo Clinic, Rochester MN, United States; 3 California Dept Public Health, Richmond CA, United States; 4Univ Children's Hospital, Heidelberg, Germany Newborn screening (NBS) for CAH uses birth weight or gestational age dependent thresholds for 17-hydroxyprogesterone (17-OHP) to determine whether a sample is normal. Nevertheless, NBS for CAH is challenged by the large variability in 17-OHP levels particularly in premature newborns. Using large datasets from California (n>130,000) and Heidelberg (n>270,000), we have performed a multiple regression analysis of 17OHP dependence upon birth weight, gender, and age at collection, and demonstrated that current cutoffs may result in false negative events that might be avoidable. This statistical method provides a covariate-independent means to assess whether CAH levels are abnormal in any given newborn. 17-OHP levels fall with age at collection and birth weight, and are gender dependent. We provide a formula to correct for all of these parameters simultaneously, allowing us to set a universal cutoff value. 2

Galician's newborn screening program includes blood and urine filter paper strips collected at the third day of live. In 2003 ESI-MS/MS methods were developed to detect aminoacids, acylcarnitines, acylglicines and organic acids in urine filter paper samples. ESI-MS/MS was carried out with an API 4000 (Sciex Applied Biosystems). Data were acquired and processed using Analyst 1.4 and Chemoview software. We used MRM experiments to detect aminoacids and organic acids and precursor scans for acylcarnitines and acylglycines. More than 3000 newborn urine samples were analyzed to calculate the cut-offs as percentiles. To prove the diagnostic efficiency of the developed method we have analyzed 328 samples from 221 patients with 37 confirmed IEM. Moreover samples from 9 IEM detected on neonatal period were also included. All samples were analyzed on filter paper strips after elution. The pathological samples analyzed allowed the determination of the sensibility and the utility of the methods. Only 1 patient with mevalonic aciduria, 2 patients with 4-OH-butyric aciduria and 3 patients with MCAD deficiency were missed by these analytical methods, based on the lower concentration levels of the metabolic markers present. In LCHAD and VLCAD deficiency samples we did not found any characteristic profile in urine.

P-820 Medium chain acyl-CoA dehydrogenase deficiency detected by polish newborn screening

P-818

Poławski T1, Głąb-Jabłońska E1, Ołtarzewski M1

Persistent feedings difficulties in a patient with a mild form of UCD: result of overtreatment following presymptomatic diagnosis on DBS?

1

Salera S1 , Cerutti M 1 , Manzoni F1 , Bonarrigo F1 , Sabatini C 1 , Ravazzani V2, Corbetta C2, Menni F1 1 Ped Dep, IRCCS F.Policlinico, Univ Stud, Milan, Italy; 2Lab. Inborn Errors Metab, AO ICP, Milan, Italy

A diagnosis of citrullinemia was made at 3 months of age with expanded newborn screening performed on request of parents for preservation of umbilical cord blood (plasma citrulline 800 μmol/L, NH3 86 μmol/L, lactate 4,7 mmol/L). Feeding problems (lack of appetite, vomiting, low caloric intake) were the only highlighted features (weight at 3rd centile - length at10th centile). We started hypoproteic diet and

Institute of Mother and Child, Warsaw, Poland

Background: Medium acyl-CoA dehydrogenase deficiency (MCADD) is one of the most common metabolic block of the mitochondrial βoxidation pathway. The clinical symptoms like hypoketotic hypoglycemia and Reye-like syndrome can be observed. Undiagnosed MCADD can lead also to sudden death or irreversible damage to the nervous system in the case of an acute metabolic decompensation. Material and Methods: There were three groups: 1) patients with MCADD, 2) parents of the children with MCADD, 3) control group. MCADD was confirmed by enzyme activity assay in lymphocytes based on oxidation of octanoyl-CoA and reduction of ferrocenium hexafluorophosphate. Results: MCAD activity was compared to acylcarnitines profile analyzed by LC/MS/MS. MCADD was diagnosed when enzyme activity was 0.3% - 25% of controls. Enzyme activity in parents of children with MCADD was 30% - 61% of normal.

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Conclusion: Decreased MCAD activity and elevated levels of octanoylcarnitine was observed but there was no correlation between the enzyme activity and acylcarnitine levels. The quantified concentration of suberylglycine negatively correlated with enzyme activity. In 26% of patients with MCADD, activity was in the range of 17.5% - 26.5% of healthy individuals, which may be related to a lack of genetic homogeneity. 19. Next generation sequencing and other technologies P-822 The use of whole exome sequencing to unravel disease genes causing autosomal recessive disorders in Qatari population

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We have identified 18 mutations in ZNF469 and 4 mutations in PRDM5. In one patient no mutation in either ZNF469 or PRDM5 was found, thus suggesting further genetic heterogeneity in BCS. Comparable and significant downregulation of glypican 6, clusterin, thrombospondin I and procollagen Cendopeptidase enhancer was found in both ZNF469 and PRDM5 mutant fibroblasts. These findings further support our hypothesis that PRDM5 and ZNF469 might be part of the same pathway regulating the transcription of extracellular matrix components. Further elucidation of the ZNF469 and PRDM5 pathway will provide significant insights into corneal development and maintenance. P-824

Ben-Omran T1, Almuriekhi M1, Fahiminiya S2, Ali R1, Nawaz Z3, Abu Khadija K3, Majewski J2 1 Dep of Pediatrics, Hamad Medical Corp, Doha, Qatar, 2Dep of Human Gene, McGill Univ, Montreal, Canada, 3Cytogenetic lab, Hamad Medical Corp, Doha, Qatar

Background: Whole exome sequencing (WES) in consanguineous families with autosomal recessive (AR) disorders is the most rapid and powerful approach for novel gene discovery. Consanguinity is common in Qatar resulting in a high AR disorders. The goal of this study is to identify novel genes causing AR diseases in Qatari population using WES. Methods: whole exome capture was carried out on 3 μg of genomic DNA using the SureSelect Human Exome Kit version 4 (Agilent Technologies, Inc., Santa Clara, CA). All potential candidate genes were manually examined using IGV. Results: out of 32 consanguineous families, WES was performed on the affected individuals from 9 families. The WES was performed on patients and their parents in order to identify de novo as well as homozygous mutations. The WES analysis revealed novel mutations in known genes in 6 families (PHKG2, ARSA, PHEX , IDUA, NSUN2, GJA3& CRYGD), mutation in one novel gene in one family (APC2), and few candidate genes in 2 families. Conclusions: our study highlights the utility of using WES in populations of consanguineous families to identify a host of genes in autosomal recessive disorders. We expect WES to be in widespread clinical use in the near future P-823 ZNF469 frequently mutated in the brittle cornea syndrome (BCS) is a single exon gene possibly regulating the expression of several extracellular matrix components Rohrbach M1, Spencer H2, Porter L2, Burkitt-Wright E2, Bürer C1, Janecke A3, Bakshi M4, Sillence D5, Al-Hussain H6, Baumgartner M1, Steinmann B5, Black G2, Manson F2, Giunta C1 1 Div Metab & CRZ, Univ Child Hosp, Zurich, Switzerland; 2Genet Med Res, Univ of Manch, Manchester, United Kingdom; 3Div Hum Genet, Med Univ, Innsbruck, Austria; 4Dep Clin Genet, Children's Hosp, Sydney, Australia; 5Div Metab Dis, Univ Child Hosp, Zurich, Switzerland; 6Div Oculoplastics and Orbit, Riyadh, Saudi Arabia BCS is a rare generalized connective tissue disorder caused by mutations in ZNF469 and/or PRDM5 and is characterized by extreme thinning and fragility of the cornea that may rupture spontaneously, keratoconus/keratoglobus, high myopia, blue sclerae, hyperelasticity of the skin without excessive fragility, and joint hypermobility. In a cohort of 23 BCS affected patients we performed molecular analysis on both ZNF469 and PRDM5, and compared the expression profile of ZNF469 and PRDM5 mutant fibroblasts for Extra-cellular-matrix-associated components of interest. In addition, we show evidence that ZNF469 is a single exon rather than a two exon gene.

Untargeted mass spectrometry-based metabolomics for the diagnosis of inborn errors of metabolism in in individual patients Engelke UFH1, Kluijtmans LAJ1, van der Heeft E1, de Boer S1, Engel J2, Wevers RA1 1 Lab Medicine, Radboud UMC, Nijmegen, Netherlands; 2IMM, Radboud Univ, Nijmegen, Netherlands

Background: In a proof-of-concept study, untargeted liquid chromatography - mass spectrometry (LC-MS) metabolomics was applied to the diseases phenylketonuria (PKU), 3-methylcrotonylglycinuria (3MCC) and medium chain acyl Coenzyme-A dehydrogenase deficiency (MCAD). Goal: To find and identify, in an untargeted approach, the biomarkers in plasma from individual patients with PKU, 3MCC and MCAD. Method: Control plasma (n=20) and plasma from patients (PKU (n=7), 3MCC (n=5) and MCAD (n=5)) were measured using Agilent-UHPLCQTOF-MS (reverse phase liquid chromatography, electrospray ionization, positive ion mode). Accurate masses and retention times (features) were processed using alignment software (XCMS) and were statistically analyzed (t-test, PCA). The Human Metabolome Database (HMDB) was used to find and identify the diagnostic metabolites. Results: Of the approximately 10000 features measured, all diagnostic metabolites were identified for the 3 diseases (PKU, phenylalanine; 3MCC, 3-hydroxyisovalerylcarnitine; MCAD, octanoylcarnitine, 9decenoylcarnitine and hexanoylcarnitine). Additional biomarkers were found for PKU (glutamylphenylalanine), 3MCC (3-methylcrotonylglycine) and MCAD (capryloylglycine, 2-trans,4-cis-decadienoylcarnitine, nonanoylcarnitine, suberylglycine, hexanoylglycine and heptanoylcarnitine). Furthermore, several unidentified metabolites varied significantly between control, PKU, 3MCC and MCAD. Conclusions: Untargeted MS-based metabolomics in plasma can be used to find abnormal metabolites in patients with an inborn error of metabolism. This approach can be used to examine patients for possible new metabolic disorders. P-825 Next generation sequencing as diagnosis tool in inherited metabolic disorders Vega AI1, Trujillano D2, Medrano C1, Merinero B1, Perez-Cerda C1, Rodriguez-Pombo MP1, Desviat LR1, Estivill X2, Ugarte M1, Pérez B1 1

CEDEM, CBMSO (CSIC-UAM), CIBERER-Madrid,, Madrid, Spain; Genetic Causes of Disease Group, CRG, Madrid, Spain

2

To date the diagnosis of inherited metabolic diseases (IMD) involves biochemical and enzymatic studies, and further time consuming Sanger

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sequencing of genes. Furthermore in heterogeneous disorders such as glycogen storage (GSD) or peroxisomal (PD) disorders among others, the genetic analysis involves the analysis of several genes. To that end, next generation sequencing is changing the gold standard of the diagnosis allowing the one-step analysis of many genes easily by designing diagnostic panels. Here, we report the assessment of clinical analytical sensitivity and specificity of targeted exome sequencing (TES) to detect pathogenic mutations in 111 genes related to aminoacidopathies (21), organic acidurias (26), fatty acid oxidation disorders (9), GSD (22) and PD (17) among others using Haloplex technology. We have analyzed previously Sanger-sequenced samples with known mutations in IMD genes and samples belonging to GSD and PD for which no mutations were known. The results indicate that NGS data were concordant with Sanger sequencing. We have detected a new change in the HSD17B4 gene in a PD patient and mutations in PHKB and ALDOB in patients sent for GSD diagnosis. In summary, we can conclude that gene panels are ready to be used in genetic diagnosis of inherited metabolic diseases. P-826 A composite method with the potential of unifying polar biochemical genetics analyses into a single-column

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Serious adverse myopathic reactions to statin therapy are considered to be rare, however, 200,000 people in the U.S. alone (0.5% of 40 million taking statins) are at risk for life-threatening myopathy and 4 – 6 million (10-15%) develop muscle pain +/- weakness that may not be reversible. We hypothesized that a higher proportion of individuals with statin myopathy will have underlying inborn errors of muscle metabolism than expected in the general population. We used MALDI-TOF microarray technology (Sequenom) to interrogate 334 mutations in 12 muscle disease genes. Of 626 Caucasians screened, 346 had severe statininduced myopathy, 112 were statin-tolerant controls, and 168 had exertional rhabdomyolysis (ER). 16.3% of statin myopathy patients had mutations in 8 genes and 17.4% of ER patients had mutations in 10 genes vs. 4.4% of statin-tolerant controls. Mutant alleles in the RYR1 gene associated with malignant hyperthermia (MH) were found in 1 of 35 and 1 of 42, statin myopathy and ER cases, respectively. Mutations in CACNA1S were found in 1 of 70 statin myopathy cases only. Statins may act as a pharmacologic trigger for myopathy in patients with mutations in muscle disease genes and result in new phenotypes for certain of these disorders. P-828 CSF metabolomics studies in 10 pediatric patients

Gangoiti JA1, Gertsman I1, Waeghe TJ2, Nyhan WL1, Barshop BA1 Horvath GA1, Stockler-Ipsiroglu S1, Mandal R2, Wishart D2 1

UCSD Biochemical Genetics Lab, La Jolla, California., United States; Mac-Mod Analytical, Inc., Chadds Ford, PA, United States

2

Background: Biochemical genetics analyses have been separated into several classes (amino acids, organic acids, acylcarnitines, purines/ pyrimidines) requiring multiple instrumentation, because of the challenges imposed by the different chemical properties of the metabolites. A single platform would consolidate, and therefore, simplify the analysis, allowing for easier inter-laboratory transferability and homogeneity of results. Methods: Urine volumes based on their specific gravity are extracted in ice-cold methanol containing 89 stable-isotope labeled internal standards, evaporated to dryness and reconstituted in water. Underivatized samples are analyzed by LC-ESI-MS/MS using both positive- and negativemode-specific MRM transitions, using a 3 μm alkylpentafluorophenyl reversed-phase HPLC column with a simple binary acetonitrile/water gradient containing 0.1% formic acid, for chromatographic separation. Results are normalized by the co-assayed creatinine concentration. Results: We have developed a composite method that can analyze 42 amino acids (resolves 1-/3-methylhystidine and leucine/isoleucine), 74 organic acids (lactic/oxalic; citric/isocitric; all isomers C4OH, C5OH and C6OH; C4:1DC, C4DC, C5DC, C6DC, C7DC, C5OHDC, C6OHDC, and 2-hydroxy-/3-hydroxyphenylacetic acids) and acylglycines (C4, C5:1 and C5), 15 acylcarnitines (C4 and C5), 43 purines and pyrimidines (3-/5-methylcytidine) and 2 sugars (galactose/glucose+fructose). Validated performance characteristics (selectivity/specificity, precision, accuracy, linearity, recovery, analytical range and bias by comparison with reference methods) are reported along with examples. Conflict of Interest declared.

Biochem Dis, BC Children's Hospital, Vancouver, Canada; 2TMIC, Univ Alberta, Edmonton, Canada 1

Background: Metabolomics has shown significant promise for discovery of new biomarkers for detection complex clinical disorders. With improvements to instrumentation sensitivity, dozens of studies have been conducted that have led to identification and quantification of many previously undetectable CSF metabolites. Objectives: Application of multi-platform metabolomics techniques to characterize CSF samples in pediatric patients, which could be used to identify specific metabolic disorders with "one in all" approach. Identification of specific "biomarkers" would allow easier diagnosis of individual diseases. Material/methods: Three methods (NMR, DI/LC-MS/MS, and GCMS) were employed in a complementary manner and CSF metabolome in 10 patients was characterized. Application of bioinformatics and statistical tools were used to study metabolomics data. Results: Of 10 patients: 5 had known metabolic disease, 2 had fatal neonatal epileptic encephalopathy, 1 had status epilepticus at time of collection, 1 had a movement disorder, and 1 had a suspected organic aciduria. The results are significant although more data needs to be collected. Conclusions: Here we intend to show the potential that multiplatform, quantitative metabolomics holds in contributing to our understanding of human disease. This clinical/biochemical database could be used for future complex genomic testing to identify new metabolomic patterns in known or yet undescribed disorders. P-829

P-827

Transition of biochemical genetics to metabolomics

Inborn errors of muscle metabolism implicated in risk for statin-induced myopathy

Barshop BA1, Gertsman I1, Gangoiti JA1 1

UCSD Biochemical Genetics, La Jolla, California, United States

Vladutiu GD1, Isackson PJ1, Tarnopolsky M2, Baker S2, Peltier W3, Weisman M4 1 Univ at Buffalo, Buffalo, United States; 2McMaster Univ, Hamilton, Ontario, Canada; 3 Med Coll Wis, Milwaukee, United States; 4 Cedars-Sinai Med Ctr, Los Angeles, United States

Background: Biochemical Genetics(BG) methods are essentially "targeted metabolomics," i.e.(semi-)quantitative profiling. We will discuss approaches to transition the BG lab to metabolomics. Methods/Instrumentation: Multiple instruments can be used (e.g.GCMS[OAs], HPLC-photodetector[AAs], LC-MS/MS[acylcarnitines]); single

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platform methods are preferable. Paramount considerations are unambiguous identification, acceptable throughput, and linear dynamic range(LDR) permitting biologically relevant analytical measurement ranges(AMR). Stable isotope standards, as many as feasible, should be included. Most useful instruments are LC-QqQ(targeted), or LC-Q/TOF(untargeted analysis). By definition, AMR cannot be validated for untargeted analytes, and LC-Q/TOFs have had lower LDR, though newer instruments are better suited for quantification. We will discuss extraction and chromatography schemes developed in parallel on LC-QqQ and LC-Q/TOF. Reference ranges: BG reference ranges are generally designed to distinguish normal from gross elevations, to diagnose near-total enzyme deficiencies, and special attention is needed to reliably distinguish values within the normal range, needed for metabolomic studies in populations without primary IEMs. Most analytes' distributions are non-Gaussian; we estimate log-normal distribution z-scores. Analysis/Visualization: Comparison across populations uses multivariate and principal component analysis. Graphical visualization of deviations and co-dependencies is useful. We developed a system of metabolic maps from computer databases, rendered with web-based software. Conclusions: This approach also may improve general BG processing/presentation.

In Mendelian disorders, mutations in single genes are the main contributor to phenotype; thus, they are excellent substrates for gene discovery using whole exome or whole genome sequencing. As of May 2013, the Online Mendelian Inheritance in Man (OMIM) database lists 1800 Mendelian disorders without an identified gene. The RaDiCAL (Rare Disease Collaboration for Autosomal Loci) project starts with the analysis of a single proband in an attempt to identify the causative genetic variants (Rosenblatt DS. A RaDiCAL approach to gene discovery. J Med Genet 48(9):577-578, 2011). The long term aim of RaDiCAL is to collect one proband for each disease in OMIM for which the gene is not known. We have developed an ethics protocol and information and consent forms which can be used for genomic studies of any Mendelian disease. They can be used as templates to study patients worldwide. Results from RaDiCAL are returned to the patient only if disease causing variants are identified. In contrast with the recent ACMG recommendations, a deliberate search for incidental findings is not performed as part of the analysis. Since 2010 we have published on the identification of genes responsible for 7 disorders using this approach. P-833 Mass spectrometry analysis of mutant proteins to study mechanisms of inherited disorders

P-830 First experience with an automated on-line extraction system for MS/MS newborn screening

Palmfeldt J1, Gregersen N1, Bross P1 1

Res Unit Mol Med, Univ Hospital, Aarhus, Denmark

1

2

2

Fingerhut R , Silva Polanco ML , Silva Arevalo G , Swiderska M

3

1

Swiss Newborn Screening, Univ Child Hosp, Zurich, Switzerland; 2 INVEGEM, Newborn Scr & Metab, Santa Lucia Milpas Altas, Sacatepéquez, Guatemala; 3CAMAG, Mutenz, Switzerland Background: Dried blood spots (DBS) have been the standard specimen for Newborn Screening programs for decades. In recent years DBS have attracted the attention of pharmaceutical companies. However, the classical NBS workflow, with punching/extraction, did not totally fulfil the needs for their studies. On-line extraction systems have already been tested and proved to be suitable for these pharmaceutical applications. Methods: The suitability of the automated CAMAG DBS-500 MSinterface has been tested together with a Waters TQD tandem mass spectrometer and MassChrom stable isotope labelled internal standards from Chromsystems (Munich, Germany). Internal standards are added to the extraction solution (methanol/water (9:1 v/v) + 0.1% formic acid). Results: First experiments showed intra assay CVs between 1.5–8.9% for acylcarnitins, and 3.3-5.6% for amino acids. Interassay CVs were 5.5-8.4% for uncharged unpolar amino acids, 8.7-13% for uncharged polar amino acids, 7.8-10.6% for short chain, and 8.5-15.0% for medium chain acylcarnitines. Recovery was between 90-120%. For acidic and basic amino acids, as well as long chain and dicarboxylic acylcarnitins, extraction conditions need further improvement. NBS samples from 3 patients with PKU, MCAD deficiency, and PA, respectively, could clearly be detected with the on-line extraction method. Conflict of Interest declared. P-831

Introduction: Protein mass spectrometry (MS) has developed strongly in the last decade and is a promising tool for analysis of protein sequence variants caused by missense mutations. We describe MSbased analyses of protein variants, and give examples of measurements of disease-causing mitochondrial proteins. Methods: Protein extracts from cultivated cells were trypsin digested to generate peptides for nanoLC-MS/MS analysis. Extracted ion chromatograms (XIC) from protein-specific peptides, with criterion <3 ppm mass deviation, was used to measure amounts of variant and wild type peptides. Results: We describe proof-of-principle studies for characterization of protein variants involved in inherited disorders. The ETHE1 protein responsible for ethylmalonic acid encephalopathy was successfully quantified, as well as an active site variant of Hsp60 chaperone. Moreover, a putative dominant negative effect of heterozygous mutation in glutaryl-CoA dehydrogenase (GCDH) causing glutaric aciduria type 1 (GA-1) was also studied by the methodology. Conclusions: High mass accuracy MS is a suitable tool to prove presence of known disease causing protein variants in samples originating from patient cells, and thus to elucidate whether disease is caused by lack of protein or by gain-of-function effects. The output data might aid design of new treatment opportunities such as chemical chaperones or gene knock-down therapies. P-834 Metabolic gene expression network on Down syndrome critical region in normal human brain Satizabal-Soto JM1, Montoya-Villegas JC2, Sanchez-Gomez A1, Fajardo D1, Moreno-Giraldo LJ3, Garcia-Vallejo JF1

Rare disease collaboration for autosomal loci (RaDiCAL) Pupavac M1, Zawati MH2, Nguyen MT2, Joly Y2, Majewski J3, Rosenblatt DS1

1 Dpto Sci Phisiol,Uni Valle., Cali, Colombia; 2Dpto Sci Phisiol Uni Valle, Uni Aut Occ, Cali, Colombia; 3Dpto Ped,Uni Valle. Genomics IPS, Cali, Colombia

1 Human Genetics Dept, McGill University, Montreal, Canada; 2Centre of Genomics and Policy, Montreal, Canada; 3McGill Univ Genome Quebec Innov Centre, Montreal, Canada

Bioinformatic studies provide the ability to extract and analyze information in databases regarding the level of gene expression along the different brain structures that brings us closer to define these molecular

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events, while at the same time determine the transcendence of its implication both for brain hosmeostasis and for some neuropathologies. Objective: To correlate the transcription levels of 19 genes located in the critical region of chromosome-21, associated with Down syndrome, with brain location and its role in the correct functioning of different brain substructures. Methodology: Based upon gene expression data from experiments of DNA microaarays available at the database human brain project from the Brain Atlas of Allen-Institute for Brain Sciences, the expression profiles of these genes along 24 substructures of the Cerebral-Nuclei and 18 of the Limbic-Lobe were constructed. Results: we found a differential expression of these genes along the structures analyzed, besides registering higher levels of global transcription in certain areas of the brain, which seem to be associated to some learning and memory processes. Discussion: Our results lead to propose the hypothesis about the complexity of gene transcription networks to explain not only the functioning of normal brain but also the Down Syndrome and Alzheimer disease. P-835

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Background: We describe two patients carrying novel mutations in MTP gene and two siblings carrying the same mutation previously reported from Turkey and discuss the clinical and biochemical phenotype. Case reports: All patients were diagnosed during the infancy period. Failure to thrive, acanthocytosis in peripheral smear, low cholesterol and tryglyceride levels were common findings in all patients.The neurologic findings were not observed. The first patient with Asp169Val mutation had hypogammaglobulinemia. The sibling of the second patient with the same mutation had better prognosis. In the follow-up, the growth percentils improved from less than 3 percentil reaching normal percentils in all the patients with high calori, low lipid diet including high medium chain triglycerides. High doses of vitamin E (200 U/kg/day) were initiated in order to prevent neurologic complications but in the follow-up low levels were found in some patients. Discussion: The genotype-phenotype correlation is complex. The three patients with the same mutations may have different clinical phenotypes. We also wanted to emphasize the importance of early diagnosis and treatment in the prevention of mortality and morbidity. With recent knowledge, immune deficiency is another important factor in the poor prognosis and the careful evaluation of the immune system is mandatory.

Molecular defects identified in challenging diagnostic cases by whole exome sequencing

P-837

Suchy SF1, Abdenur JE2, Leydiker K2, Chung WK3, Bale SJ1, Daly A1, Haverfield EV1

Cardiovascular disease (CVD) risk factors in a population of hypercholesterolemic children with genetic diagnosis of heterozgous familial hypercholesterolemia (HEFH)

1 GeneDx, Gaithersburg, MD, United States; 2 CHOC Children's Hospital, Orange, CA, United States; 3Columbia University Medical Center, New York, NY, United States

Giovannini M1, Pederiva C1, Capra ME1, Poli PC1, Barberi S1, Salvini F1, Riva E1, Salvatici E1 1

Accurate diagnosis is essential to the appropriate management of inborn errors of metabolism (IEM). We present two challenging cases involving suspected IEM, in which the molecular cause was identified by whole exome sequencing (WES). Case 1: A one year-old child presented with hypotonia, developmental delay, encephalopathy, seizures, intermittent strabismus and dystonia. There were two similarly affected siblings. Extensive work-up in blood / urine, and imaging studies were non-diagnostic. CSF studies were declined by the parents. WES was performed with the Agilent SureSelect XT2 All Exon V4 kit and an Illumina HiSeq 2000, on the proband and parents. A homozygous p.Phe375Leu mutation in the tyrosine hydroxylase gene (TH) was identified. Case 2: A two year-old male presented with congenital ichthyosis, developmental delay, white matter changes, mild hip dysplasia and constipation. Studies included: normal metabolic work-up (organic acids, very long chain fatty acids, amino acids, acylcarnitine profile) and a normal microarray. WES revealed two mutations in cis in the ALDH3A2 gene (Sjogren-Larsson syndrome): a frame-shift (p.Asn255IlefsX11) and a deletion of exons 1-3. Diagnosis of many IEM remains a challenge and disorders with a broad phenotypic spectrum or an atypical presentation can confound the diagnoses. WES offers another option for these diagnostic dilemmas. Conflict of Interest declared. 20. Disorders of lipid metabolism P-836 The clinical and biochemical characteristics of patients with abetalipoproteinemia and two novel mutations Gündüz M1, Özaydın E1, Büyüktaşlı M1, Koç N1, Kırsaçlıoğlu C2, Köse G3, Cefalu A4, Tarugi P5 Div Metab Dis,Ankara Dıskapı Child Hosp, Ankara, Turkey; 2Dep of Gastro,Ankara Dıskapı Child Hosp, Ankara, Turkey; 3Dep of Neuro, Şişli Etfal Hospital, İstanbul, Turkey; 4Dep of Int Med,Palermo Unv, Palermo, Italy; 5Dep of Biochemistry,Modena Unv, Modena, Italy

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy

Background: It is important to detect severely hypercholesterolemic children to start early CVD prevention. Objectives: Estimate the prevalence of genetically-confirmed heFH in children followed at our Lipid Clinic and determine their CVD risk profile. Patients and Methods: 278 severely hypercholesterolemic children (median age 8.6 y, 134 male/144 female), at their first access to our Lipid Clinic, with positive hypercholesterolemia family history and/or premature CVD, no ongoing pharmacological treatment or secondary causes of hypercholesterolemia, were evaluated for: anthropometric measures, twelve-hour-fasting blood sample for total cholesterol (TC), LDL cholesterol (LDL-C), HDL cholesterol (HDL-C) and triglycerides (TGC) by enzymatic method and genetic analysis for heFH by polymerase-chainreaction. Student's t test or Mann-Whitney test for independent samples. Results: Patients were divided in two groups: 137 had a mutation of the LDL-receptor (FH-group), 141 did not (non-FH-group). The prevalence of heFH in our population is 49%. Lipid profile (mg/dl, mean±ds) in the FH-group and non-FH-group was, respectively: TC 268.4±53 vs 213±46 (p<0.0001), LDL-C 194.4 ±55 vs 136.3±34. (p<0.0001), HDL-C 56.7±11.8 vs 59.2±13.6 (p=0.157), TGC 78±38.1 vs 74.9±33.7 (p=0.628), non-HDL-C 212.3 ±57.5 vs 153.5±46.1 (p<0.0001). Conclusions: In a population of severely-hypercholesterolemic children the prevalence of heFH is very high (49%). Genetically-confirmed heFH children have a worse CVD risk profile. P-838 Cerebral vasospasm and stroke due to acute intermittent porphyria Hemelsoet D1, Van Melkebeke D1, Wuyts B2, Poppe B3, Defreyne L4

1

1 Dpt. Neurol, Ghent Univ Hosp, Ghent, Belgium; 2Dpt. Clin Biol, Ghent Univ Hosp, Ghent, Belgium; 3Dpt. Med Genet, Ghent Univ Hosp, Ghent, Belgium; 4Dpt. Vasc Interv Neuroradiol, Ghent, Belgium

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Background: Besides the clinical picture of systemic symptoms comprising gastro-intestinal, cardiovascular, hematological and skin problems, acute porphyria may be complicated by neurological symptoms like polyneuropathy, seizures, focal deficits, and cognitive problems. Cerebrovascular complications are rare Case Report: A 43-year-old woman with a medical history of suspected endometriosis with severe abdominal pain crises was referred to our centre because of acute blindness, confusion and suspected ischaemic stroke. Two weeks earlier she underwent laparoscopic evaluation for an acute abdominal pain crisis. Upon admission brain MRI showed acute bilateral occipital cytotoxic edema and ischaemic lesions. Classical angiography showed diffuse intracerebral vasospasm. A treatment with 3H-therapy and nimodipine for vasospasm was started, but additional vasopression was needed to avoid hypotension. Urinary analysis was positive for porphobilinogen, uro- and coproporphyrins and delta-aminolevulinic acid. A diagnosis of acute intermittent porphyria was made and a treatment with IV human hemin was started. Neurological symptoms improved quickly with partial recovery of the visual deficit with tunnel vision. Cerebral vasospasm recovered slowly within weeks. Because of fluctuating hypotension with transient clinical worsening, alphafludrocortisone was started with beneficial result. Conclusion: Cerebral vasospasm with stroke is a serious neurological complication of acute porphyria with a need for specific appropriate therapy.

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Background: Lipoprotein(a) (Lp(a)) is a low density lipoprotein-like particle in which apolipoprotein B100 is covalently linked to the unique apolipoprotein(a). There is a mounting body of evidence suggesting a role of Lp(a) in the development of several vascular diseases. In the present study we rewieved clinical and laboratory findings of our pediatric patients who have elevated Lp(a). Objectives: 15 patients with elevated levels of Lp(a) were evaluated by their demographically, clinically, laboratory properties.In addition; we made echocardiographic assessment of patients for carotid intima media thickness (CIMT). Results: Median age of patients was 14.1 ± 3.8 years, median age of diagnosis was 12.1 ± 2.87, the male/female ratio was 8/7. Median initial lipoprotein(a) level was 101± 42.3. Electrocardiograpy was normal in all patients. Median CIMT was 0.42 ± 0.21 mm.When we compared the patients with Lp(a) greater or smaller than 100 mg/dl; median CIMT of the Lp(a) >100 mg/dl group was significantly different.We started nicotinic acid treatment in 4 patients. Conclusion: In consideration of the lack of well-controlled studies of Lp(a) in childhood-onset and given also that has been received little attention in this group, we no report our patients' data to emphasise the importance and frequency of lipoprotein(a) excess in pediatric population. P-841 Follow-up of infants with hypertriglyceridemia

P-839 An atypical case of A/hypobetalipoproteinemia Sequeira S1, Ferreira AC1, Bourbon M2

Kagnici M1, Altinok Y2, Canda E1, Kose M1, Karaca E2, Kalkan Ucar S1, Onay H2, Ozkinay F2, Coker M1 Div Metab Dis,Ege Univ Child Hosp, Izmir, Turkey, 2Div Genetics,Ege Univ Child Hosp, Izmir, Turkey 1

1

Metab Unit, Hosp D Estefânia, Lisbon, Portugal, 2Inst S Dr Ric Jorge, Lisbon, Portugal Introduction: Abetalipoproteinemia (ABL), a very rare disorder, is usually characterized by growth delay, diarrhea with steatorrhea and fat malabsorption, hepatomegaly, and neurological/neuromuscular symptoms. Patients show persistently low LDL cholesterol, triglycerides and apolipoproteins and acanthocytosis. It is inherited in a recessive manner (MTTP gene), whereas hypobetalipoproteinemia (HBL), a similar disorder, is a dominant disorder (APOB gene). Other genes have also been involved. Case report: We describe a 14 year-old-girl who presented at the age of 16 months with failure to thrive, no history of chronic diarrhea, acantocythosis and mildly elevated transaminases and alkaline phosphatase. She also had very low levels of LDL- and HDL-cholesterol, triglygerides, apolipoproteins A1 and B. An endoscopy showed a white mucosa and the intestinal biopsies show enterocytes filled with numerous lipid droplets. She has very low vitamin E levels despite therapy with fat soluble vitamins. Molecular studies were negative for the MTP gene but others are pending. Comments: The diagnosis of ABL/HBL should be considered in patients with low levels of cholesterol and triglycerides. The prognosis in this case seems better than described as the girl shows no clear worsening of symptoms over the time, no history of diarrhea or steatorrhea or retinitis pigmentosa on fundoscopy.

Background: The two classic disorders of exogenous lipoprotein metabolism are caused by defective or missing lipoprotein lipase (LPL) and defective apoC-II, the co-factor for LPL. Patients with classic LPL deficiency present in the first months of life with striking hypertriglyceridemia. Treatment is a diet with very low fat (10–15 % of calories) and special formula enriched by medium chain triglycerides (MCT). Objectives: 32 patients with hypertriglyceridemia were evaluated who received MCT treatment. Results: Median age of diagnosis was 3±3 months. Median triglyceride level was 3.892±4.694 mg/dL initially, was 904±808 mg/dl after first month, was 975±981 mg/dl after second month, and was 1.064±1.286 mg/dL after six months. Consanguinity between parents (14 patients), dyslipidemia in siblings (17 patients), and early onset myocardial infarction in family members were detected in the patient history. LPL gene mutations were identified in nine patients (42.9 %); they were S477X, S447X, R192X, C557G>A, G273R, IVS8.15, I194T and G188E. Conclusion: Addition of MCT to the diet of hypertriglyceridemia patients effectively lowered blood triglyceride levels in the first month. The recovery may halt after six months of therapy because of difficulties to obtain MCT, loss of family motivation, and issues of taste and smell. 21. Networks and metabolic services

P-840

P-842

Follow-up of our pediatric patients with lipoprotein (a) excess

Nurse practitioners are made not born: the journey from metabolic clinical nurse consultant to first Australian metabolic nurse practitioner

Kose M1, Canda E1, Kagnıcı M1, Kalkan Uçar S2, Levent E3, Çoker M4 1 Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey; 2Div Metab Dis, Ege Univ Child Hosp, İzmir, Turkey; 3Div Card Dis,Ege Univ Child Hosp, İzmir, Turkey; 4Div Metab Dis,Ege Univ Child Hosp, İzmir, Turkey

Robinson J1 1

Sydney Children's Hospital, Randwick, Australia

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The Metabolic Service at The Sydney Children's Hospital was established in 1998 and has grown rapidly over the last 10 years.The Metabolic Servcie is responsible for the diagnosis and/or ongoing management of children with inborn errors of metabolism and their families. In 2009, a review of the service was carried out, indicating that a substantial number of children were being missed for follow-up monitoring and appointments and that the cause of this shortfall was an excessive workload on the available staff. The review concluded that the gap in the service could best be filled by a Nurse Practitioner (NP), and the current Metabolic Clinical Nurse Consultant (CNC) agreed to undertake the training required to fill that position. This poster documents the journey from CNC to NP including: / Completing a Master of Nursing with a Nurse Practitioner major. / Applying for endorsement as an NP through the Australian Health Practitioner Regulation Agency and Nursing and Midwifery Board. / Convincing the Sydney Children's Hospital to regrade the position. / Documenting the first scope of practice for an Australian Metabolic NP. P-843 WWW.GUIAMETABOLICA.ORG: beyond disease, towards patients Serrano M1, Gómez-Zuñiga B2, Pousada M2, Cutillas J3, Armayones M2, Fernández JJ3, Fabrega J3, Egea N1, García M4, Pérez-Payarols J3, Vilaseca MA1 1

Neurometab Unit, CIBERER HospSantJoanDéu, Barcelona, Spain; Psychology Sciences IN3, Univ Obert Cat, Barcelona, Spain; 3 Innovation&Research Dpt, HospSantJoanDéu, Barcelona, Spain; 4 Catalonian Assoc PKU and other IMD, Barcelona, Spain 2

Background: www.guiametabolica.org is a 2.0 website whose aim is to provide individuals with an interest in Inherited Metabolic Diseases (IMD) access to clinical and daily life information, facilitating contact with health professionals and other patients/families. We use data from its users to understand the impact of 2.0 webs on e-Health Literacy and Patient Empowerment. Objectives: We aimed to characterize users and to assess their health literacy and empowerment. Methods: An online questionnaire was completed by 88 users of www.guiametabolica.org. The questionnaire covered characteristics of the users, and their assessment of the website, and contained the eHealth Literacy Scale and an empowerment questionnaire. Results: www.guiametabolica.org usage implies some changes, among them: / feeling more informed as a patient; / perceiving an understanding of IMD that allows a better management of it; / feeling more prepared for the doctor's appointment; / perceiving less loneliness. Conclusion: www.guiametabolica.org has previously shown a positive impact regarding solitude and self-efficacy. This time it is demonstrated that it also improves the ability of participants to use information and communications technologies to manage their IMD. Users feel more empowered for the self-management of their disease. www.guiametabolica.org has become a meaningful tool for everyday life of IMD affected people. P-844 Results of a families-researchers collaborative project in Lowe syndrome: ten patients with a wide neurological phenotype Serrano M 1 , Perez-Dueñas B 1 , Camino-León R 2 , Pascual SI 3 , Maldonado E4, Cantarín V5, Ojea T6, Vidal-Valls J7, de la Fuente J8,

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Armayones M9, Pousada M9, Gómez-Zuñiga B9, Nafria B9, Families Spanish10 1 Neurometab Unit, CIBERER HospSantJoanDéu, Barcelona, Spain; 2 Pediatr Dpt, Hosp Univ Reina Sofía, Córdoba, Spain; 3Pediatr Dept. Hosp Univ La Paz, Madrid, Spain; 4Pediatr Dept, Hosp Río Carrión, Palencia, Spain; 5Neuropediatr. Dpt. Hosp Niño Jesús, Madrid, Spain; 6 Clínica ASISA, Málaga, Spain; 7Centre Pilot Arcàngel Sant Gabriel, Barcelona, Spain; 8Facultad de Psicología, Univ Barcelona, Barcelona, Spain; 9Psychology Sciences IN3, Univ Obert Cat, Barcelona, Spain; 10 #LoweResearchProject Families, Barcelona, Spain Background: As ultra-rare disease, there is scarce information about Lowe syndrome's natural history, genotype/phenotype correlations Parents of children with rare diseases develop considerable expertise and are advocating for formal collaboration in research. We aimed to increase biomedical knowledge on Lowe syndrome collaborating families and doctors. Methods: 10 Spanish families participated in an online-community receiving periodical information about the disease and completing medical surveys thereafter. Results: Three patients presented de novo mutations. Achievement of developmental milestones expressed in months were (mean age(range): head control 7,2(3-17); sitting 11,7(6-17); independent walk (8 patients) 35,5(1372); social smile 4,4(3-6); ocular pursuit 5,2(3-9); pincer grasping 14,4(8-24) and four patients say/understand more than 50 words (46(24-72)). Vineland scale gave heterogeneous results. Three patients showed traits of autism disorder. Two patients suffer refractory epilepsy causing neurodevelopmental regression. Neuroimaging was normal in two patients and showed white matter cysts in another. Conclusions: Enabling parents to reflect on and share with researchers their personal knowledge in ultra-rare diseases is an effective strategy to advance research. This collaboration could reduce their suffering. Lowe syndrome shows great heterogeneity of neurological symptoms. Probably, Dent type 2 (also caused by OCRL1 mutations) and Lowe Syndrome are comprised in a continuum spectrum of OCRL1-related disorders. P-845 Should haemophagocytic lymphohistiocytosis (HLH) be screened for in all critically ill children with inborn errors of metabolism having unexplained hepato-splenomegaly? Jalan AB1, Kudalkar KV1, Jalan A1, Alam MA2, Sawant L1, Shinde D1, Pawaskar M1, Khan S1, Rao S3 1

NIRMAN, Navi Mumbai, India, 2Bio Care Lab, Islamabad, Pakistan, Wadia Children Hospital, Mumbai, India

3

Background: HLH is a life threatening disease in which an exaggerated but ineffective immune response leads to severe hyper inflammation. There are two forms of this disorder – primary and secondary Objective and design: To screen for HLH in children presenting with fever, hepatosplenomegaly with suspected IEM. Materials and method: 23 subjects were selected for analysis from Jan2011 – April2013. Children 0 – 18 years who were referred to rule out IEM with unexplained significant spleno-hepatomegaly, fever, anemia, low platelets and failure to thrive. Further analysis with ferritin, LDH, triglycerides and sCD25 (IL2 receptor) was performed. Two subjects with HCC associated with tyrosinemia type 1 were excluded from the study. Result: Out of 21, finally 2 satisfied 5 out of 8 HLH (2004) criteria. One child had familial HLH and another one had lysinuric protein intolerance. sCD assay showed a reference range of 0–85 IU/ml in healthy individuals, 123–380 IU/ml in critically ill children and 3,101 IU/ml in one of the HLH patient. Conclusion: All critically ill children with unexplained hepatosplenomegaly, bicytopenia or pancytopenia, should be screened for

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HLH by triglycerides, ferritin and soluble CD25 since presence of HLH demands special treatment. HLH can be associated with IEM. P-846 Screening of high risk Egyptian children with suspected inborn errors of metabolism using tandem mass spectrometry: a 5 year report Selim L1, Abdelhady S2, Salem F2, Hassan F3, El Mogy F3, Abdel Atty S3, Mehaney D3, Mandour I3, Abdelmonem M3, Fathy M3, Orabi A1, Gamal Eldin I4, El Ayat A4, El Badawy A4, Yousry M1 Neurology dep,Cairo Uni Children's Hosp, Cairo, Egypt; 2Genetic Dep, Cairo Uni Children's Hosp, Cairo, Egypt; 3Chem Path dep,Faculty Med, Univ Cairo, Cairo, Egypt; 4Metab Unit, Cairo Uni Children's Hosp, Cairo, Egypt Background: Inborn errors of metabolism (IEMs) are important causes of morbidity and mortality in pediatric patients. Tandem Mass Spectrometry (MS/MS) allows for expanded metabolic screen of fatty acid oxidation defects, aminoacidopathies and organic acid disorders through simultaneous quantitation of amino acids and acylcarnities in dried blood spots. Objective: To report the results of expanded metabolic screening in high risk Egyptian Pediatric patients over a period of 5 years (2008- 2013). Patients and Methods: 3380 children presented with clinical features of IEMs such as neurodevelopmental delay or with biochemical abnormalities such as metabolic acidosis or hyperammonemia. Amino acids and acylcarnitines were quantified in dried blood spots using MS/MS. Positive cases were confirmed by gas chromatography/mass spectrometry. Results: IEM were confirmed in 200 patients (5.9%), including 124 (62%) with amino acids disorders, 72 patients (36%) with organic aciduriass and 4 cases (2%) with fatty acid oxidation defects. The commonest conditions encountered were phenylketonuria, maple syrup urine disease and methylmalonic acidurias. Conclusion: The prevalence of IEMs (5.9%) is relatively high among high risk Egyptian children, mainly due to a high rate of consanguinity. Fatty acid oxidation disorders are relatively rare among Egyptian pediatric patients. The development of a nationwide screening program is warranted. 1

P-847 The prevalence of inborn errors of metabolism among Egyptian pediatric patients admitted to the intensive care unit using tandem mass spectrometry

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Results: Forty patients (52.2%) were born to consanguineous parents. 9/90 (10%) were confirmed to have IEM (tyrosinemia type I, maple syrup urine disease, propionic acidemia, carnitine palmitoyl transferase I deficiency, glutaric acidemia type II). Hyperammonemia was found in 37 cases (41.1%), hypoglycemia in 13 cases (14.4%) and lactate was increased in 16 cases (17.7%). Conclusion: Inherited metabolic diseases are not uncommon at the PICU. Confirmatory tests such as urinary organic acid profiling are mandatory. P-848 Six-years experience of neurometabolic disease unit Massano A1, Gouveia A1, Domingues J1, Garcia P2, Diogo L2, Grazina M3, Macário MC1 1

Neurology Department, Coimbra Univ Hosp, Coimbra, Portugal; Pediatric Hosp, Coimbra, Portugal; 3Center for Neuroscience and Cell Biology, Coimbra, Portugal 2

Background: The nervous system is often attained in metabolic diseases. Our hospital's neurometabolic unit was created in 2003, aiming to follow the patients previously studied in the Pediatric Hospital who reach adulthood and to study those with late onset-forms of disease. Objectives: To characterize the population of patients seen in our neurometabolic unit. Methods: All patients seen in our neurometabolic unit from January 2007 until February 2013 were included. We reviewed their diagnosis, age of disease onset, age of diagnosis and clinical presentations. Results: We analyzed 160 patients. In 26 patients, metabolic diseases were ruled out after extensive workup. 49 patients (31%) remain without diagnosis. The main group of diseases was energy metabolism disorders and the most common diagnosis were progressive external ophtalmoplegia (19 patients) and Leigh's disease (5 patients). 53 patients (40%) had an adult-onset form of disease; 22 patients (16%) had their first symptoms in childhood and were diagnosed during adulthood and 27 patients (20%) were diagnosed during childhood. Conclusion: These results reveal a high number of patients without diagnosis, which is obviously a major concern. Due to the complexity and rarity of these diseases, it would be extremely useful to create diagnostic algorithms to guide the clinician. P-849

Mehaney D1, Hassan F1, Selim L2, Abdelmonem M1, Gayar D1, Sheta M1, Halawa E3, Gamal Eldin I4, Kaddah A3, Elkaffas R1, Elkady N3 Chem Path dep,Faculty Med,Univ Cairo, Cairo, Egypt; 2Neurology dep,Cairo Uni Children's Hosp, Cairo, Egypt; 3Pediatr ICU,Cairo Uni Children's Hosp, Cairo, Egypt; 4Metab Unit, Cairo Uni Children's Hosp, Cairo, Egypt

Heart disease and inborn errors of metabolism: prevalence, manifestations and correlation with the specific ongoing treatment in 42 patients followed at San Paolo Hospital, Milan

1

Paci S1, Scelsa V1, Paramithiotti C1, Vincenti S1, Poli PC1, Rovelli V1, Salvatici E1 1

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy

Background: Inborn errors of metabolism (IEMs) are not an uncommon entity presenting to the Pediatric Intensive Care Unit (PICU). Tandem Mass Spectrometry (MS/MS) is the state of art technology for the diagnosis of IEMs by simultaneous analysis of amino acids (AAs) and acylcarnitines (ACs) in dried blood spots (DBS). Objective: To assess the prevalence of IEMs detectable by MS/MS among critically ill patients admitted to the PICU at Cairo University Children's Hospital (May 2009 - October 2010). Patients and Methods: 90 patients were clinically or biochemically suggestive of having IEMs because of intractable metabolic acidosis, intractable seizures, electrolyte disturbances, and/or high anion gap. AAs and ACs were quantified in DBS using MS/MS. Positive results were confirmed by urinary organic acid profiling using gas chromatography/mass spectrometry.

Background: inborn errors of metabolism (IEM) account for up to 5% of all pediatric heart defects (HD) but few data are available estimating HD's prevalence in this population. Objectives: to evaluate HD's frequency and manifestations (cardiomyopathy, valvular disease, arrhythmia, atherotrombotic event and hypertension) in IEM affected patients referred to Metabolic Unit – Paediatric Department - San Paolo Hospital - Milan, thus verifying whether they can modify their natural course when underwenting the specific disease's treatment. Patients and methods: 42 patients (18 months to 35 years old) affected by lysosomal storage disorders, glycogen storage diseases, fatty acid oxidation defects, organic acidemias, urea cycle defects, aminoacidopathies, disorders of glycosylation, other carbohydrates'

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metabolism defects and Wilson disease were enrolled. IEM's diagnosis and treatment's start mean age were 23 months. All patients underwent electrocardiogram, echocardiography and blood pressure monitoring. Results: 60% of the cases had HDs: valvular disease and cardiomyopathy were more represented, 43% and 24% respectively. During disease's specific treatment HDs improved in 36% of cases, worsened in 12% and resulted unchanged in 52%. Conclusions: HDs show high prevalence in IEM, thus a cardiac assessment should always be considered. Early IEM's diagnosis and treatment can likely change HD's prevalence and natural course. P-850 Mutation spectrum and prevalence of inborn errors of metabolism in United Arab Emirates

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Results/Conclusions: The Adult Metabolic Unit (AMU) currently serves 119 patients with the following diseases: mitochondrial diseases (32), urea cycle abnormalities (3) protein glycosylation disorders (3), homocystinuria (1), phenylketonuria (40), citrulinemia (1), galactosemia (7), organic acidurias (5), glycogen storage diseases (4) and metabolic myopathies (23). The diagnostic process is performed at clinical, biochemical, histological and functional levels. Other actions will take place: 1) adapting clinical guidelines for adult patients; 2) strengthen relationships with patient organizations; 3) development of research projects; 4) consolidate a scientific annual educational program; 5) collaboration in Rare Diseases with the Department of Health; 6) training of young professionals; 7) collaboration with other national and international AMU; 8) collaborate in national and international registries of IEM. Grants DO_2013/CELLEX and FIS/01199/12. P-852

Al Shamsi A1, Hertecant J1, Souid A2, Al Jasmi F2 Biochemical network for IEM detection 1

Tawam hospital, Al Ain, United Arab Emirates; 2United Arab Emirates University, Al Ain, United Arab Emirates

Uicich R1, Rodriguez R1, Otegui I1, Blasi S2 1

This study was designed to describe the mutation spectrum and the birth prevalence of inborn errors of metabolism (IEM) among Emiratis. Fifty-four distinct IEM were diagnosed in 169 patients since 1995. Twenty (37%) entities were previously reported lysosomal storage diseases (LSD, 65 patients with 39 mutations), with a birth prevalence of 26.87 per 100,000. The remaining 34 (63%) entities involved other metabolic disorders (104 patients with 59 mutations), which are the subject of this report. Due to consanguinity, all mutations were homozygous; 24 (71%) mutations were confined to Emiratis. Two (3%) mutations were found in 3 separate tribes [biotinidase deficiency (BTD, c.1330G>C) and phenylketonuria (PAH, c.168+5G>C)]. Five mutations (8%) were found in 2 separate tribes [lysinuric protein intolerance (SLC7A7, c.499+1G>C, c.999G>C, and c.1005C>A), isovaleric aciduria (IVD, c.1184G>A), and propionic aciduria (PCCB, c.990dupT)]. The remaining 52 (89%) mutations occurred in single tribe. The most prevalent diseases (2.2 to 4.9 per 100,000) were biotinidase deficiency, tyrosinemia (type 1), phenylketonuria, propionic aciduria, glutaric aciduria, glycogen storage type Ia, mitochondrial DNA depletion. The overall estimated birth prevalence for IEM is 65.89 per 100,000. These results justify intense prevention programs that include genetic counseling and screening. P-851 Development of an adult metabolic unit for patients with inborn errors of metabolism Cardellach F1, Grau JM1, Visiedo C2, Forga M3, Garrabou G4, Morén C4, Ribes A5

Lab Nutr y Metab Hospital "Garrahan", Buenos Aires, Argentina; Area Alimentacion, Htal Garrahan, Buenos Aires, Argentina

2

The diagnosis of metabolic diseases is usually difficult in countries with few resources. In Argentina there is only one public laboratory situated at Garrahan's Pediatric Hospital in Buenos Aires, and a few private ones, one of which is important. Garrahan´s Hospital has a system of interdepartmental for these diseases and the suspected patients' samples must be sent sometimes from thousands of kilometres. Sometimes, the hospitals located in the povinces do not have enough resources, and samples usually arrive in very bad conditions. We have just created a biochemical network. Professionals are trained by a distant course wich lasts for four months and includes,nine virtual classes and a face class which is held in each province. We take with us the software of the equipment and samples of true cases. In addition, there is a discussion forum and a final exam. The first course has been taught to 23 participants from 7 provinces. In response to a question that requires the shipping of samples, doctors can contact the biochemists who will guide them whith the preparation and sending of samples for organic acids, aminoacids, acylcarnitines, enzymes, etc. We consider this biochemical network to be essential to facilitate the identification of IEM. The next course is in 2013. P-853 The euro-WABB registry: differences in molecular genetic confirmation between monogenic Wolfram, Alstrvm, and Bardet-Biedl syndromes

1 Internal Med. Hosp. Clínic, CIBERER, Barcelona, Spain, 2Internal Medicine Dep. Hospital Clínic, Barcelona, Spain, 3Endocrinology Dep, Hospital Clínic, Barcelona, Spain, 4CIBERER, Barcelona, Spain, 5IEM Unit, Hosp. Clinic, CIBERER, Barcelona, Spain

López de Heredia M1, Farmer A2, Ayme S3, Maffei P4, Mccafferty S5, Mlynarski W6, Paquis V7, Rohayem J8, Sinnott R5, Parkinson K9, Tillmann V10, Tranebjaerg L11, Nunes V1, Barrett T12

Background: Adult metabolic units (AMU) for patients with inborn errors of metabolism (IEM) already exist in many European countries, but not in Spain. Methods: We developed a multidisciplinary team (Internal Medicine, Neurology, Gynecology, Endocrinology, Nutrition/Dietetics and Medical Genetics) to assist adult patients with IEM. Nurses, social services personnel, pharmacy staff and patient organization representatives will join the unit. The aim is to develop clinical practice, research and teaching activities, with the result of improving quality in clinical safety and treatment of patients with IEM.

LGM-IDIBELL, U-730-CIBERER, L'Hospitalet de Llobregat, Spain; Birmingham Child. Hosp., WTCRF, Birmingham, United Kingdom; 3 SC11-INSERM, Paris, France; 4Med. Sci. and Chir. Dept., Univ; Padova, Padova, Italy; 5NeSC, Glasgow University, Glasgow, United Kingdom; 6Paediatrics, Lodz Medical University, Lodz, Poland, 7CNRS, Délégation Côte d'Azur, Valbonne, France; 8Universitätsklinikum Münster, Münster, Germany; 9Alstrom Syndrome UK, Paignton, United Kingdom; 10 Paediatrics, Tartu University, Tartu, Estonia; 11Bispebjerg Hosp./Univ. Copenhagen, Copenhagen, Denmark; 12Sch. Clin Exp. Med., Birminghan Univ., Birmingham, United Kingdom

1

2

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Wolfram (WS; 1:700,000), Alström (AS; <1:1,000,000) and BardetBiedl (BBS; 1:350,000) syndromes are ultra-rare monogenic diseases with overlapping features of diabetes, blindness, and deafness. We developed an international registry in order to: collect clinical, genetic, and outcome data; establish natural history of the diseases; assess genotype phenotype relations; develop patient information and management guidelines; improve access to genetic information and develop cohorts for future clinical trials. We summarise data from 221 records and describe the mutation database. Patients with a confirmed genetic or clinic diagnosis were recruited in Europe and surroundings by their clinicians. 42 basic fields defined by consensus in order to differentiate between the three syndromes are collected. In order to collect detailed and longitudinal information up to 400 fields can be used that will accurately characterize patients phenotypes. A mutation database was generated including 723 mutations from the 22 genes associated to the WABB diseases and available at http://lovd.euro-wabb.org. The mutation database will enable cross-referencing between genotype and phenotype. Regular updating, including end-user submission of new mutations, means that the mutation database is also a reference source for clinicians caring for patients. Acknowledgement The Euro-WABB project is funded from the EU, in the framework of the Health Programme. P-854 Spanish registry for Wolfram, Alström and Bardet-Biedl syndromes (REWBA) López de Heredia M1,2, Valverde D3, Corrochano V4, Nunes V1,2,5 1

Fundació Institut Investigació Biomèdica de Bellvitge, Laboratorio de Genética Molecular, Barcelona; 2CIBER de Enfermedades Raras, Barcelona; 3 Universidad de Vigo, Vigo; 4 CIBERER-Biobank, Valencia; 5Universidad de Barcelona, Barcelona Main objective is to develop a national registry for the ultra-rare genetic syndromes Wolfram (WS), Alström (AS), Bardet-Biedl (BBS) and other diabetic syndromes that include clinical and genetic data and its consequences. Purposes are to increase visibility for these rare diseases in Spain, to establish natural history and characterize patient cohorts for future clinical trials, to establish genotype-phenotype correlations and generate a sample bio-bank. Main characteristics are that: Patients either themselves or recruited by the clinicians will register via a web application (https:// rewba.idibell.cat). Detailed clinical (161 different symptoms) and genetic data is provided by patients clinician. Patients can select the clinician that has access to their data and consent to share data with international registries and provide samples to the associated bio-bank. Clinical data is accordingly with that collected at EUROWABB (http://www.euro-wabb.org) to facilitate data sharing. Acknowledgments Funded by CIBER de Enfermedades Raras (INTRA10/730.1) P-855 The Canadian Inherited Metabolic Diseases Research Network (CIMDRN): a national, practice-based research network for inborn errors of metabolism Kronick JB1, Chakraborty P 2, Coyle D3, Wilson K4, Dyack S5 , Feigenbaum A1, Hernandez M6, Khan A7, Khangura SD3, Mitchell JJ8, Potter M9, Prasad C10, Siriwardena K1, Sparkes R7, Speechley KN10, Potter BK3, behalf of on11

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Hospital for Sick Children, Toronto, Canada; 2Children's Hospital of Eastern Ontario, Ottawa, Canada; 3University of Ottawa, Ottawa, Canada; 4Ottawa Hospital Research Institute, Ottawa, Canada; 5IWK Health Centre, Halifax, Canada; 6Newborn Screening Ontario, Ottawa, Canada; 7Alberta Children's Hospital, Calgary, Canada; 8Montreal Children's Hospital, Montreal, Canada; 9 McMaster Children's Hospital, Hamilton, Canada; 10 London Health Sciences Centre, London, Canada, 11CIMDRN, Ottawa, Canada All health care decision-makers pursue what Berwick et al call the "triple aim": improving patient experiences, improving health outcomes, and managing health system impacts. Challenges in generating evidence to meet these aims for rare diseases are exemplified by inborn errors of metabolism (IEM): limited and evolving understanding of natural history, limited resources for developing and accessing interventions, and difficulties conducting robust evaluative research. To address these challenges, we have established a national, practice-based research network to improve care and outcomes for Canadian children with IEM. CIMDRN is a multidisciplinary group of approximately 40 investigators, representing all Canadian metabolic treatment centres. Given regionalized care in Canada, near-complete case ascertainment is our objective with an expected enrollment of about 1,000 children with 28 selected IEM. The study emphasizes longitudinal data analysis describing interventions and outcomes from a consent-based research platform that relies on multiple data sources, including clinical and health system data, and self-reported patient/family experiences. Guided by a novel research framework, our analysis focuses on generalizable themes including clinical heterogeneity and the comparative effectiveness of emerging and established interventions. CIMDRN's unique methodological approach promises to support evidence-based care, improve outcomes for IEM, and generate insights relevant to other rare diseases. P-856 Liver transplantation in inborn metabolic disorders the Portuguese experience Gonçalves I1, Garcia P2, Diogo L2, Ferreira S1, Furtado E1 U Transpl Hepático, Hospital Pediátrico, Coimbra, Portugal; 2Centro Desenvolv LB. Hospital Pediátrico, Coimbra, Portugal 1

Liver transplantation (LT) is a therapeutic option for several inborn errors of metabolism (IEM). Long-term survival rates in experienced centers are good. Main indications for LT in IEM are: inadequate metabolic control, frequent metabolic crisis and suspected or confirmed liver malignancy. Medical records of all patients with liver-based IEM submitted to LT in the last twenty years were reviewed. Twenty-seven LT were performed in 23 children at a median age of 83 months (4 to 209 months). One child was submitted to combined liver-kidney transplantation. Most frequent metabolic disorders were glycogen storage disease type I (n = 6), tyrosinemia type I (n =6) and argininemia (n = 3). In children undergoing LT by failure to thrive, a significant increase in the z-score of both weight and height was noticed. In two children with argininemia, the progression of neurological impairment was stopped. The great majority has reversed their metabolic disturbances and quality of life was improved. Three children received a second transplant and one underwent a third. There was a predominance of vascular complications (20%) and long-term survival was 87%. We conclude that LT is a safe and powerful procedure in the management of liver based IMD. Early referral is desirable.

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P-857

P-859

Prenatal diagnosis for inborn errors of metabolism in India experience from one centre

The national institutes of health undiagnosed diseases program: the first four years

Bijarnia-Mahay S1, Verma J1, Saxena R1, Kohli S1, Puri RD1, Kotecha U1, Movva S1, Dubey S1, Verma I C1

Adams DR1, Boerkoel CF1, Godfrey R1, Golas G 1, Groden C1, Gropman A2, Landis D1, Markello TC1, Nihrebecky M1, Pierson T3, Sincan M1, Tifft CJ1, Toro C1, Wahl C1, Wolfe L1, Gahl WA1

1

Center Med Genetics, Sir Ganga Ram Hosp, New Delhi, India 1

Prenatal diagnosis (PND) of inborn errors of metabolism (IEM) assumes great importance in India. Its demand is increasing as awareness is improving. Despite their rarity, many disorders are serious, often causing death or severe disability in a child, affecting the family as well. Treatment eludes most patients owing to lack of specific treatment or availability of drugs / diet or financial constraints. In the absence of newborn screening, all patients are picked up late – after symptoms have occurred, thus reducing the overall good outcome despite treatment. We present 10 years data of PND via enzyme assays for lysosomal storage disorders (LSDs) (246) and 5 years of PND using gene studies for IEMs (128) at our center. Amongst LSDs, MPS (60) and GM1/GM2 gangliosidosis (61) are the major groups followed by Gaucher (36), MLD (28), Krabbe (18), NPD (18), Pompe (15) and NCL (10). Of 128 PNDs via gene studies, LSDs were the largest group (49) followed by congenital adrenal hyperplasia (43), organic acidemias (12), urea cycle defects (9), and other IEMs (15). In addition, 12 amniotic fluid metabolite screens were done in unconfirmed cases. To conclude, in India, prenatal diagnosis is still widely utilised as preventive measure until optimal treatment is available.

Undiagnosed Diseases Program, NIH, Bethesda, United States; Children's National Medical Center, Washingon DC, United States; 3 Depts Peds and Neurology, Cedars-Sinai, Los Angeles, United States 2

Background: The NIH UDP works with persons who remain undiagnosed despite an extensive, standard-of-care medical workup. The UDP strives to make diagnoses where possible and to advance medical knowledge. Methods: Since 2008, approximately 2800 medical records have been reviewed. Of those, 640 were selected for extensive, in-person workup and phenotyping. Evaluations include medical testing, specialist consultation, biomaterial collection, and/or genome-scale sequencing where appropriate. Results: Some level of diagnosis was offered in 24% of the evaluated cohort. Undiagnosed cases are undergoing continuing clinical and research screening to generate new research hypotheses. Conclusions: There is a substantial population of persons with undiagnosed, severe medical conditions. New methods are needed to transition such persons into basic, clinical and translational research. P-860

P-858

Inborn errors of metabolism information service (SIEM): results of 11 years of a pioneer freecall service in Brazil (0800-5102858)

Limitations in the approach of health care providers in end-of-life care decisions

de Souza CFM1, da Rocha ACM1, Longoni N1, Pellini TV1, Refosco LF1, Sanseverino MT1, Netto CB1, Rafaelli C1, Vairo F1, Giugliani R1

Sürücü Dr1, Bülbül P1, Karavaizoğlu D1

1

1

The SIEM is a pioneer freecall service in Brazil which has a team specialized in IEMs,ready to remotely help health professionals to diagnose, to provide immediate care to suspected patients, and to guide the specific management in confirmed cases. Between October 2001 and May 2013, 2,338 cases were recorded. The majority (88.1%) called SIEM to request diagnostic support and early management guidance; 6.9% called to obtain information concerning IEM, and 4.3% to have support in the follow-up. From the total of 2,136 records, 1,361(63.7%) had their investigation for IEM concluded, and in 223 (16.4%) of these cases a diagnosis of IEM was reached; 529 (38.8%) were non-metabolic cases; in 326 (24%) it was not possible to reach a conclusion; and in 293 (21.5%) the follow-up was lost. Out of the 223 cases diagnosed with IEM, 18.4% had organic acidemias, 20.8% amino acid disorders, 15.7% LSDs, 17.6% mitochondrial diseases; 7,5% carbohydrate disorders, and 20% other classes of IEM. Among the IEM cases we observed high levels of consanguinity (19.0%), early death (17.5%) and familial recurrence (27,35%). We believe the SIEM is an extremely important resource in a country were IEM are often unrecognized, aiding different health professionals to efficiently diagnose and treat, in addition of being a source of useful data about relative frequency, distribution and burden of these diseases (acknowledgements: CMW, Comida- Med, UFRGS, HCPA).

Div Metab Dis, Univ Kırıkkale Med School, Kırıkkale, Turkey

Background: At terminal stages of neurometabolic diseases, a sense of loss may start to be experienced even though the child is still alive, and the parents may accept the coming death. Objectives: The present study examines the acceptance of death by families with children in the process of dying, their attitudes during treatment, and their expectations from health professionals. Affected patients had terminal Sandhoff or Krabbe disease. Case Report: A 2.5 year old female Sandhoff patient (diagnosed at 9 months of age), and a 17 month old male Krabbe patient (diagnosed at 5 months of age) were admitted to hospital with hypernatremic dehydration and bronchopneumonia, respectively, within 10 days. Both patients developed respiratory arrest shortly after admission and were supported with mechanical ventilation. Both families gave written consent to end life support but their desires could not be accepted according to Turkish laws. Conclusion: Specialist are expected to have good communication with the families and to give continuous care respecting the opinions of the patients' family on time to end life maintained by mechanical support. However, ethical and legal regulations on how health care professionals should act with regard to such conditions are unclear in Turkey and should be structured rapidly. Conflict of Interest declared.

Medical Genetic Service, Porto Alegre, Brazil

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P-861

22. Miscellaneous disorders

Socio-economic and QOL outcomes in adult patients with inherited metabolic diseases

P-863

Lateef A1, Ramachandran R1, Rahman Y1

Drug treatment of inborn errors of metabolism: a systematic review

1

Evelina at Guys and St Thomas' Hospital, London, United Kingdom We present findings of a one year audit of socioeconomic status and quality of life (QOL) of adults with IMDs attending outpatient clinics in a single centre for adults with IMD. Methods: Patients were invited to voluntarily complete sociodemographic and QOL questionnaires. Results: 117 patients participated (53% female, age: 16 – 78 years). Despite 96 (82%) patients having at least secondary level education [66 (44%) secondary education and 30 (26%) postgraduate qualification] only 52 (44%) are in employment. Although only 13 (11%) patients are known to have learning difficulties, 42 (36%) live with family or reside in institutions. Sixty nine percent of patients receive social support [39 (33%) benefits; 15 (13%) community healthcare support and 14 (12%) other social-care input]. QOL on scale of 10: 64 (55%) rated 8-10, 34 (29%) ranked 5-7, 12 (10%) scored 0-4, 6 (5%) and 1 (no response). Conclusion: These results suggest > 50% report an excellent QOL and > 80% of patients have at least completed secondary education. However, < 50 % of patients are in employment and 36% of patients continue to live with family/in institutions. This reiterates the need for further work towards ensuring integration into society and promoting socio-economic independence. P-862 Etiology, clinical spectrum, treatment and outcome of metabolic liver diseases in Macedonia

Alfadhel M1, Al-Thihli K2, Moubayed H1, Eyaid W1, Al-Jeraisy M1 King Abdulaziz Medical City, Riyadh, Saudi Arabia; 2Sultan Qaboos University Hospital, muscat, Oman 1

Objective: The Dosages of medications used for the treatment of various IEM can be obtained from various sources, and they tend to vary among these sources. Moreover, the published dosages are not usually supported by the level of existing evidence, and they are commonly based on personal experiences. The aim of this study was to summarize the dosages of medications used in the treatment of IEM as supported by the best level of evidence that currently exists. Methods: A systematic literature search was conducted. The medications found and their respective dosages were graded according to their level of evidence, using the grading system Oxford Centre for Evidence-Based Medicine (OCEBM). Finally, we compiled it in one table and we presented this table as WebAPP to translate this knowledge into an information portal that will be an easily accessible reference. Results: 83 medications identified. The dosages of 17 medications (21%) achieved grade 1 while 61 (74%) resided in grade 4 level, 2 medications were in level 2 and 3 respectively and dosages of 3 were graded as grade 5. Conclusion: The authors hope that it will serve as a quickly accessible reference for medications used in this important clinical field. P-864

Kostovski A1, Zdraveska N1 1

University Children's Hospital, Skopje, The F.Y.R of Macedonia

Background: Inherited metabolic disorders (IMD) affecting the liver represent a heterogeneous group of genetic conditions. The diagnosis is often very difficult to make and the treatment remains a big problem in many cases. Objective: To determine the etiology, clinical spectrum, treatment, and outcome, of metabolic liver diseases in a single pediatric tertiary centre in Macedonia. Material: A retrospective study was carried out at Department for Gastroenterohepatology from 2007 to 2012. Patients admitted for a liver disorder and diagnosed with IMD were included. Results: Eleven children with mean age 5.40 ± 4.87 years (range 2 days – 12 years) were studied. The etiologies were: tyrosinemia (HT) type I; Wilson's disease; glycogen storage disease type IX; Niemann-Pick type C; Gaucher disease and bile acid syntheses defect. The main clinical presentations were: hepatomegaly (63,6%); jaundice (45,4%), liver failure (18,2%). Two patients with HT died at age 34 and 119 days. Treatment with nitisinone was provided in only one patient, one was transplanted. There was no available treatment for Gaucher disease. Molecular analysis was performed in 6 patients (54%). Conclusion: The diagnosis of metabolic disorders is a challenge. High index of suspicion, early diagnosing and prompt management are necessary to avoid unfavorable outcomes.

Hyaline fibromatosis syndrome resulting from a new homozygous missense mutation, p.Gly116Val, in ANTXR2 Tümer L1, Kasapkara CS1, Fong K2, Serdaroglu A3, McGrath JA2 Div Metab Dis, Univ Hosp of Gazi, Ankara, Turkey; 2 Div Dermatology, King's College London, London, United Kingdom; 3 Div Neurology, Univ Hosp of Gazi, Ankara, Turkey 1

Infantile systemic hyalinosis (ISH) is a rare autosomal recessive genetic disorder characterized by the development of progressive dermal and subcutaneous fibromatosis, gingival hypertrophy, joint contractures and bone deformities. ISH results from mutations in the ANTXR2 gene that encodes capillary morphogenic protein 2 which has a role in extracellular matrix homeostasis, although there is clinicopathological and molecular overlap of ISH with juvenile systemic hyalinosis. Here we report a Turkish child with ISH who presented because of multiple joint contractures and gingival hypertrophy. Sanger sequencing of genomic DNA identified a novel homozygous transversion, c.347G>T, in ANTXR2 that leads to the amino acid substitution, p.Gly116Val; both parents were asymptomatic heterozygous carriers. Identification of this new mutation improves genetic counselling and makes DNA-based prenatal diagnosis feasible for this family, although effective therapy for ISH remains elusive and the clinical prognosis is poor.

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P-865 Trimethylaminuria (TMAU): the apparent prevalence of the secondary / non-genetic form presenting in the UK population Manning NJ1, Allen KE2, Bonham JR1, Croft JM1, Edwards LA1, Gillett GT3, Kirk RJ2, Olpin SE1, Sharrard MJ4, Smith EJ1, Watkinson JM1, Yap S4, Lachmann R5 1

Clin Chem, Sheffield Children's Hospital, Sheffield, United Kingdom; Sheffield Diagnostic Genetics Service, Sheffield, United Kingdom; 3 Clin Chem, Sheffield Teaching Hospitals, Sheffield, United Kingdom; 4Div Med, Sheffield Children's Hospital, Sheffield, United Kingdom; 5 National Hosp for Neurol and Neurosurg, London, United Kingdom 2

Background: From 1997 to 2012 trimethylamine (TMA) was measured in urine from 1,731 individuals of all ages with reported body odour and under investigation for Fish Odour Syndrome. Methods: Free TMA was measured by headspace GCMS with stable isotope dilution. Total TMA (Free TMA + TMA-N-oxide) was measured as TMA after reduction with titanium chloride. Mutation analysis was performed by PCR. Results: 875/1731 presented with increased TMA excretion (TMAU)(> 11 mmol/mol creatinine). 313 TMAU cases with Free/Total of < 21% were designated Secondary (non-genetic) TMAU (TMAU2). 562 showed increased Free/Total TMA (> 21%), potentially Primary TMAU (TMAU1). 78/562 were tested for mutations of the gene coding for the TMA oxidising enzyme FMO3. Only 21/78 were found to have mutant FMO3 (TMAU1) (TMA: 16 – 766). 57/78 with no mutation were therefore also designated TMAU2 (TMA: 14 – 149). Re-estimated TMAU2 (with Free/Total TMA > 21%): 410/562. Conclusions:TMAU2 may represent 723 (313+410) of the 875 TMAU cases. This apparent prevalence of TMAU2 may be due to changes in intestinal microbiota leading to chronic and intermittent body odour. Dietary restriction of TMA precursors in red meat, offal, seafood and pulses combined with antibiotic and probiotics can reduce excessive enteric TMA production. P-866 Prevalence of inherited metabolic disorders in epilepsy patients: a large, single center study Imhof E 1, Zak M 2 , Hewson S1 , Feigenbaum A 3, Kobayashi J 2 , Minassian B2, Raiman J1, Siriwardena K1, Tein I2, Clarke J1, MercimekMahmutoglu S1 1

Div Clin Gen Metab, Dp Ped, Univ Toronto, Toronto, Canada; 2Div Neurol, Dp Ped, Univ Toronto, Toronto, Canada; 3Biochem Gen Dp Ped Univ California, San Diego, United States Background: Epilepsy affects 2-3% of general population. More than 50% of patients have no identifiable underlying cause. Methods: All epilepsy patients followed in the Neurology and Metabolic Genetics Clinics were included. Electronic patient charts were reviewed and information was entered into an excel database. Results: 1291 epilepsy patients were included. 50% had an identifiable cause e.g. anatomical brain malformation, genetic causes or primary underlying disease. 44 patients had an inherited metabolic disorder (IMD), including 10 neuronal ceroid lipofuscinosis (type 2, 3, 6, 8, unidentified), 2 Sanfilippo disease, one Gaucher disease type 2, one

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juvenile Tay-Sachs disease, 2 Niemann-Pick type C, 11 mitochondrial disorders (complex I and IV deficiencies, Leigh's disease, MELAS, MELAS-like, POLG), 3 non-ketotic hyperglycinemia, 2 fumarase deficiency, one GLUT1 deficiency, one X-linked adrenoleukodystrophy, one vanishing white matter disease, one cobalamin C and one cobalamin G deficiency, 4 pyridoxine-dependent epilepsy, one Menkes disease, one CDG type 1d and one type 1f. Conclusion: Lysosomal storage disorders were the most common subgroup followed by mitochondrial disorders. Prevalence of IMD was 3.5% (44/1291) in epilepsy patients. There was no identifiable underlying etiology in 50% of the patients. Certain of these patients might have an underlying IMD. P-867 Severe psychomotor impairment, thyroid alterations and MRI abnormalities: the key to MCT8 deficiency Colomer J1, Nascimento A1, Ortez C1, Rodrigues F2, Amstrong J3, Morte B4, Bernal J4, Artuch R5, Campistol J6 1 Neromusc Unit, Ped Neur, S.Joan Déu Hosp, Barcelona, Spain; 2Ped Dep, Baixo Vouga Hosp Center, Aveiro, Portugal; 3Genetic Unit, S. Joan Déu Hosp, Barcelona, Spain; 4Biomedic Investig Instit, CIBERER, Madrid, Spain; 5Biochemistry Dep, S. Joan Déu Hosp, Barcelona, Spain; 6Ped Neurology Dep, S.Joan Déu Hosp, Barcelona, Spain

Introduction: Thyroid hormone (TH) plays a major role in the development of different organs, in particular the brain. Recently, MCT8 was identified as a specific transcellular transporter of TH. Mutations in SLC16A2/MCT8 gen cause an X-linked disorder named AllanHerndon-Dudley Syndrome (AHDS), characterized by severe psychomotor retardation. Typically high levels of serum T3 are found and brain MRI features fit with Pelizaeus-Merzbacher Syndrome (PMS). We present a 24-month old boy suffering from severe congenital hypotonia and cognitive impairment, affected by AHDS. Clinical Case: The patient was born from non-consanguineous healthy parents. The mother twin-sister has a son with severe neurological disability. During pregnancy, abnormalities of thyroid function were detected in the mother. Physical examination showed cognitive impairment; global hypotonia and weakness, being unable to hold the head upright and to seat without support. Mild pyramidal signs were observed. Deep tendon reflexes were not elicited on Aquiles's tendon. Recently, epileptic seizures were developed. Serum analysis showed T3 4,84 nmol/L (0,63-3,90) and T4 7,7 pmol/L (9,1-25,0). MRI was compatible with PMS. Genetic studies are in progress. Comments:This clinical phenotype, associated with MRI findings of Pelizaeus-Merzbacher Syndrome, should prompt us to look for specific disturbances in T3 and T4 in order to suspect this diagnosis. P-868 Novel ferrochelatase gene mutation in a Slovak patient with erythropoietic protoporphyria and the prevelance of common single-nucleotide polymorphism that contribute to the genetic predisposition in Slavic and Jewish populations Farrag M S1, Martasek P1 1

1st faculty of medicine,Charles univ, Prague, Czech Republic

Erythropoietic protoporphyria (EPP), a chronic subtype of porphyira that results from the decrease of the ferrochelatase (FECH) activity below threshold of ca. 30%. This leads to accumulation of protoporphyrin, mainly in the erythroid cells. EPP patients ussually suffer from cutaneous photosensitvity and in 1-10% of patients could

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develop hepatic failure. EPP inheritance is complex, always associated with two molecular defects, a private FECH mutation in trans to a common hypomorphic FECH*IVS3-48C allele. In this study, we report a novel mutation in the FECH gene leading to a stop codon compound heterozygous with the hypomorphic allele in a Slovak patient with EPP. Moreover, we screened the frequency of the IVS-48C allele in the Czech population in 312 individuals. We found the IVS-48C allele in 35 individuals out of all the screened subjects. The prevalence was determined to be 11% in the Czech population, 10% among males and 12% among females. We also screened the frequency of the IVS-48C allele in the Jewish population (Ashkenazi, Sephardic and Yemenite Jews). The frequency of the IVS-48C allele was determined to be 15% in Yemenite Jews, 17% in Sephardic Jews and 16% in Ashkenazi Jews. P-869 Platelet function and L-arginine/no pathway in Duchenne muscular dystrophy Weigt-Usinger K1, Hoerster I1, Carmann C1, Köhler C1, ChobanyanJürgens K2, Tsikas D2, Schara U3, Luecke T1 Ruhr-University Bochum, Bochum, Germany; 2Hannover Medical School, Hannover, Germany, 3University Hospital of Essen, Essen, Germany 1

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gene encodes a transmembrane protein called Wolframin, which recent evidence suggests may serve as a novel endoplasmic reticulum calcium channel in pancreatic beta cells and neurons. The clinical features include early-onset bilateral optic atrophy, diabetes mellitus, diabetes insipidus, hearing impairment, urinary tract abnormalities and psychiatric illness. The patient was kept under observation from birth for Peterson's anomaly Type III, congenital glaucoma. At the age of 4 months his hearing was examined and severe hearing impairment to deafness was diagnosed. He was under observation from infant age for severe psychomotor retardation, brain MRI showed atrophy of the cerebellum and extensive arachnoidal cyst. From the age of 2.5 years treated for hypothyreosis. At the age of 3 years the patient was examined for growth retardation and failure to thrive. Insulin-treated diabetes mellitus was diagnosed. The Wolfram Syndrome diagnosis was confirmed by DNA sequence analysis and an as yet undefined mutation c.2425G>A (p.(Glu809lys) was discovered. More than 200 variants were described in the WFS1 gene of patients with the Wolfram Syndrome what complicates determination of a clear genotype-phenotype correlation. P-871 Clinical, analytic and therapeutic evaluation of 17 children with osteogenesis imperfecta Correia J1, Ferreira I1, Martins R1, Martins E1, Bandeira A1 1

Centro Hospitalar do Porto, Porto, Portugal

Blood loss during spinal surgery is higher in Duchenne muscular dystrophy (DMD) than in non-DMD patients. Disturbed platelet aggregation and impaired vessel reactivity have been discussed. The Larginine/NO pathway plays a key role in both platelet aggregation and vessel reactivity. Aim: To evaluate platelet aggregation/adhesion and the L-arginine/NO pathway in 56 DMD patients (11.7±4.8 y) and 52 healthy controls (11.4 ±3.96 y). Methods: Induced-platelet aggregation and platelet adhesion were measured by the Born´s and Hellem´s method, respectively. Parameters of the L-arginine/NO pathway were measured by mass spectrometry. Results: Platelet aggregation induced by all agonists (collagen, p=0.52; adrenaline, p=0.77; ADP, p=0.91; ristocetin, p=0.45) and platelet adhesion (p=0.53) did not differ between DMD patients and controls. Plasma ADMA (632±118 nM vs 543±112 nM, p<0.001), urine ADMA (20.20±10.11 vs 7.12±3.38 μmol/mmol creatinine, p<0.001), urine DMA (79.58±29.40 vs 56.48±61.77 μmol/mmol creatinine, p=0.014), urine nitrite (0.94±1.09 vs 0.37±0.38 μmol/mmol creatinine, p=0.001) and urine nitrate (224.87±127.41 vs 160.61±148.11 μmol/mmol creatinine, p=0.018) were higher in DMD boys than in controls. Plasma L-arginine (75.5±15.6 vs 74.1±14.7 μM) did not differ (p=0.623). Data are mean±SD, n=56 vs 52). Discussion: The L-arginine/NO pathway is upregulated in DMD, without influencing platelet function. ADMA´s role in DMD beyond NO synthesis inhibition is elusive.

Background: Osteogenesis imperfecta (OI) is a hereditary disorder characterized by increased bone fragility and progressive bone deformity. Objectives: Characterize the OI children followed in our center. Methods: Anthropometric data, bone turnover markers and bone mineral density were evaluated. Results: We enrolled 17 children, from 22 months and 18 years (median 12 years), 58.8% males. OI type 1 was present in 64.7%, 58.8% had a family history and 58.8% were diagnosed at birth. 58.8% showed blue sclerae, 23.5% dentinogenesis imperfecta and 58.8% long bone deformations. Fourteen children were treated with intravenous pamidronate, which started between 1.5 to 14 years, median 5.5 years. The number of perfusions/child varied from 1 to 17. After two years of treatment, the number of fractures/year decreased from a median of 0.45 to 0.140 (p=0.037), mean height z-score improved from -1.4 to -1.2 and Z-score of bone mineral density of the lumbar spine improved from -3.0 to -1.0 (p=0.013). Serum alkaline phosphatase decreased from a median of 266U/L to 235 U/L and urinary hydroxyproline decreased from a median of 140μmol/24 hours to 89μmol/24 hours. Conclusion: Patients with OI present a wide spectrum of clinical manifestations. Cyclic intravenous treatment

P-870

G6PD-deficiency in the north of Mexico and a new variant description

Wolfram syndrome: new mutation case report

Garcia N1, Luque F2, Galaviz C3, Rangel H4, Aguilar EM2, Ramos R2, Romero JG2, Arámbula E2, Garcia N2

P-873

Konecna P1, Prochazkova D1, Dolezel Z1, Skotakova J2, Hoeflsloot EH3 1

Ped Dep, Univ Hosp Brno, Brno, Czech Republic; 2Rad Dep, Univ Hosp Brno, Brno, Czech Republic; 3Univ Nijm Med Cent, Nijmegen, Netherlands Wolfram Syndrome is a rare autosomal recessive neurodegenerative disorder (OMIM 222300) caused by mutation of the WFS1 gene. The WFS1

1 Universidad Politécnica de Sinaloa, Mazatlan, Mexico; 2Doctorado Regional en Biotecnología, Culiacan, Mexico; 3CIIDIR, Durango, Mexico; 4Instituto de Investigación en Genética, Guadalajara, Mexico

Background: Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme-pathology; it is X-linked inherited and causes neonatal hyperbilirubinemia, chronic non-spherocytic hemolytic anemia

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and drug-induced acute hemolytic anemia. The aim of this study was to analyze the prevalence of G6PD-deficiency in the North of Mexico. Methods: Population samples from the North of Mexico were biochemically screened for G6PD-deficiency; PCR-RFLP's and sequencing were used to identify mutations. Results: The frequency of G6PD-deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A202A/376G, G6PD A-376G/968C, and G6PD Santamaria376G/542T. Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (p= 0.48336), and the unique exception with high frequency of G6PDdeficiency does not involve a coastal population (Chihuahua: 2.4%). We identified in one individual a new G6PD mutation called Magallanes 193A>G. Analysis of eight polymorphic sites showed only 10 haplotypes. Conclusion: The G6PD deficiency in Mexican population is in agreement with historical records that involve both European and - principally - African origins for the presence of G6PD variants in this country.

causes of ASD can be identified in about 10-20% of cases. The contribution of rare inherited metabolic disorders remains poorly evaluated. The aim of the present study was thus to retrospectively explore the benefits of systematic screening for IMD in ASD patients. Study design and population Data were retrospectively collected for all children who were referred to Cerrahpaşa Medical Faculty Pediatric Metabolic Disorders Unit between January 2009 and September 2012, who subsequently received the diagnosis of ASD, and who underwent the appropriate investigations. In all cases, the diagnosis of ASD was made or confirmed by a child psychiatrist. In all cases metabolic investigations included blood gases, amino acids, homocysteine, ammonia, lactate, acylcarnitine profile in plasma, and organic acids in urine. In selected cases urinary mucopolysaccharides were analyzed. Results: Among 244 ASD patients enrolled in the study, metabolic disorders were found in 8 patients: two with mucopolysaccharidosis type III, two with L-2- hydoxyglutaric aciduria, one with partial biotinidase deficiency, one with short chain acyl-CoA deficiency, one with classical homocystinuria and one with late diagnosed phenylketonuria.

P-874 P-876 A girl with dysmorphic features, aortic coarctation, lymphopenia and regression

Barriers to and facilitators for treatment adherence among patients with HT1: a qualitative study of health care professionals perspectives

Houben ML1, Giltay J2, van Montfrans J1, ter Heide M1, van Hasselt PM3 Malik SM1, Weinman JW2 1

2

Div Pediatrics, UMCU, Utrecht, Netherlands; Div Clin Genet, UMCU, Utrecht, Netherlands; 3Div Metab Dis, UMCU, Utrecht, Netherlands Exome sequemcing is a powerful to tool to detect de novo mutations in single patients, e.g. with mental retardation. Proving pathogenecity of de novo changes in genes not previously associated with a disease depends on finding mutations in this gene in similar patients. Proband presented with aortic coarctation and ventricular septal defect. She had deepset eyes, biparietal narrowing, a metopic ridge, mandibular enlargement, and a tent-shaped mouth. She had severe feeding difficulties during infancy and –initially - mild developmental delay. Age 12 months she acquired severe adenovirus pneumonia requiring high frequency oscillation ventilation. Thereafter, regression and epileptic encephalopathy were evident. Subsequent development was severely attenuated. Recurrent viral respiratory tract infections prompted immunologic testing, demonstrating variably severe lymphopenia. Extensive metabolic investigations in plasma, urine, cerebrospinal fluid, muscle biopsy, and lysosomal enzymes, cerebral imaging, ophthalmologic and ENT examinations, and genetic assessment including karyotype, array CGH, array of known immunodeficiency genes, specific genes (e.g. ADA and PNP, Rett, POLG, myotonic dystrophia)) were all negative. Exome sequencing data are pending. We present a girl suspected to have a new syndrome. Finding similar cases is crucial but cumbersome. There is a clear need for novel strategies to ease this search process.

Atlantis Healthcare, London, United Kingdom; 2Kings College London, London, United Kingdom 1

Background: The appropriate use of Nitisinone medication and adhering to specialist diet is central to the successful management of HT1. This study aimed to explore the views of health care professionals on the key drivers and barriers to medication and dietary adherence among their patients. Methods: Eight qualitative interviews were conducted with health care professionals in the UK and France, who were involved in the care of patients with HT1. The sample included physicians, nurses, dieticians and a pharmacist. Data were analysed using thematic analysis. Results: Adherence to the recommended diet was identified as one of the greatest challenges for HT1 patients and their families. Health care professionals' discussed several barriers to treatment adherence, including: a lack of forward planning, limited understanding of the condition and the consequences of non-adherence, language barriers, the role of extended family, challenging behaviour during adolescence, feeling stigmatised and making the transition from home to school and from child to adult care. Conclusions: These findings highlight that even though patients in France and the UK are surrounded by a supporting network of health care professionals, there are several areas in which HT1 patients and their families could benefit from the provision of additional support/ information. Conflict of Interest declared.

P-875 P-877 Autism or metabolic disorder? Kiykim E1, Aktuglu Zeybek AC1, Soyuçen E1, Cansever M1, Aydin A1 Cerrahpaşa Med Faculty, Div Metab Dis, İstanbul, Turkey

Evolution of magnetic resonance imaging (MRI) findings in a girl with megalencephalic leukoencephalopathy with subcortical cysts (MLC)

1

Autism spectrum disorders (ASD) are neurodevelopmental disabilities characterized by severe and pervasive impairment in reciprocal socialization, restricted interests and repetitive behaviors. Currently, genetic

Renaud DL1, Hobson GM2, Manolakos N2 Mayo Clinic, Rochester, MN, United States; 2 Alfred I. duPont Hospital for Children, Wilmington, DE, United States 1

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Background: MLC is characterized by macrocephaly and mild gross motor delay with characteristic MRI findings including diffuse white matter abnormalities and subcortical cysts in the bilateral anterior temporal regions. Mutations in the MLC1 and HEPACAM genes have been described. Objective: To describe the evolution of MRI findings in MLC. Case Report: A 7 month old girl presented with macrocephaly (with enlarged anterior fontanelle) and mild gross motor delay. Her MRI revealed diffuse white matter abnormalities with some anterior predominance. The white matter of the brainstem and cerebellum appeared normal. MR spectroscopy was normal. Normal evaluation included thyroid function, lysosomal enzymes, peroxisomal panel, organic acids, lactate, GFAP gene analysis and eye exam. At 10 months, the MRI was unchanged with no new myelin formation suggesting hypomyelination. Lumbar puncture for metabolic studies was normal. At 17 months, the MRI revealed unchanged white matter abnormalities but the new finding of subcortical cysts in the anterior temporal lobes. Molecular analysis revealed two heterozygous mutations in the MLC1 gene and one heterozygous mutation in the HEPACAM gene. At 2 years of age, she continues to have normal intelligence with mild gross motor delay. Conclusion: Subcortical cysts may not be present initially in patients with MLC. P-878

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recessive mutations in the GJA12/GJC2 gene encoding the gap junction protein connexin47 (CW47) cause a form of PMLD with hypomyelination, nystagmus, impaired psychomotor development and progressive spasticity. In this study, we have investigated the molecular pathology in a Turkish patient who has been previously excluded for mutations in the PLP1 gene. Deletion/duplication of one or more entire PLP1 exons was excluded by MLPA (Multiplex Ligation-dependent Probe Amplification). After excluding PMD in the patient, mutation screening of the seven exons and the promoter region in GJA12/GJC2 gene was performed to detect disease causing pathologic allele. This analysis revealed a disease causing homozygous p.Met286Thr (c.857C>T) mutation. The Met286Thr mutation which was found to be associated with PMLD, located at the third cytoplasmic domain, showed aggregation of mutated connexins in the endoplasmic reticulum and loss of CW47 function. This study was supported by TÜBİTAK-SBAG-111S217. P-880 Molecular genetic analysis and prenatal diagnosis of pantothenate kinase associated neurodegeneration (PANK2 gene) in India Bijarnia-Mahay S1, Setia N1, Puri R D1, Kohli S1, Saxena R1, Verma I C1

Diagnostic approach in congenital cutis laxa

1

Mohamed M1, Gardeitchik T1, Lefeber DJ1, Wevers RA1, Morava E2

Pantothenate kinase-associated neurodegeneration (PKAN, previously known as Hallervorden-Spatz disease), is a neurodegenerative disorder associated with iron accumulation in brain and a characteristic radiographic appearance (eye of tiger sign). Mutations in the pantothenate kinase (PANK2) gene account for most of the PKAN cases. In the last two years we carried out sequence analysis of the PANK2 gene in 9 families including the parents of a deceased affected child. Prenatal diagnosis was performed in three families. Consanguinity was noted in two, and the same community background (Agrawal) in 5 families. Affected children in all five Agrawal families were homozygous for the same reported mutation, 215_216insA. In the consanguineous couple each parent was found to be heterozygous for the known mutation p. R341X. Each of the four non-Agrawal families showed homozygous mutations, including the known mutations, c.828_829delTG, p. R341X and p. L457X, and the novel mutation p.A490V. Prenatal diagnosis (PND) in three families, including one Agrawal showed unaffected carrier fetuses. Molecular analysis of the PANK2 gene is useful for confirmation of PKAN and differentiation of PKAN from other iron accumulation disorders. Mutation analysis can subsequently be used for carrier testing in other family members and for PND.

1

Radboud Univ Nijmegen Medical Centre, Nijmegen, Netherlands; Tulane Univ New Orleans, New Orleans, United States

2

Cutis laxa, or old looking, sagging skin is a symptom of normal aging in healthy individuals. Interestingly, cutis laxa also occurs as a genetic condition of premature and generalized connective tissue aging, affecting various elastic components of the extracellular matrix. The inherited forms of cutis laxa lead not only to wrinkling of the skin, but aging of other essential tissue structures, such as the elastic fibers in the connective tissue of the vascular system, lungs and bones, causing aneurysms, pulmonary emphysema, skeletal abnormalities, like osteoporosis or joint abnormalities and are frequently associated with intellectual disability. Surprisingly, several types of cutis laxa are inborn errors of metabolism. We evaluated 32 patients with congenital cutis laxa and neurologic involvement, 16 of these showed metabolic abnormalities and were diagnosed with either a glycosylation disorder, a mitochondrial disease or showed other metabolic abnormalities. The extreme clinical and biochemical heterogeneity in cutis laxa syndromes makes diagnosing these patients quite challenging. Based on the inheritance pattern, clinical and biochemical phenotype and brain imaging we offer a diagnostic approach in suspected metabolic cutis laxa leading to guided genetic analysis.

Center Med Genetics, Sir Ganga Ram Hosp, New Delhi, India

P-881 P-879 Identification of a GJA12/GJC2 gene mutation in a Turkish patient with Pelizaeus-Merzbacher-like disease Yilmaz-Yucel D1, Ozgul RK1, Hismi B1, Sivri S1, Coskun T1, Tokatli A1, Dursun A1

Chronic manifestations and incidence of acute attacks in Spanish patients with acute intermittent porphyria Barreda M1, Buendía J2, Ballesta-Martínez MJ1, López-González V1, Glóver-López G3, Guillén-Navarro E1 Med Genet,Virgen de la Arrixaca Hosp, Murcia, Spain; 2Neurol Dept, Los Arcos del Mar Menor Hosp, Murcia, Spain; 3Mol Genet, Biochem and Clin Genet Centre, Murcia, Spain 1

1

Hacettepe University, Dep. of Metabolism, Ankara, Turkey

Pelizaeus–Merzbacher disease (PMD), the prototype of hypomyelinating disorders, is due to X-linked recessive rearrangements or mutations of the proteolipid protein 1 (PLP1) gene. Pelizaeus–Merzbacher-like disease (PMLD) shows clinical and neuroradiologic features that are similar to classic PMD but is not associated with PLP1 mutations. Autosomal

Acute intermittent porphyria (AIP) is a hereditary disorder resulting from a deficiency of the enzyme HMBS (Hydroxymethylbilane synthase). Most AIP patients remain asymptomatic and the remainder present episodic acute neurovisceral attacks.

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The purpose of the study was to determine the clinical manifestation patterns in a Spanish adult AIP cohort. We studied 26 adults, average age 43 years [20-71], 14 women (53.8%), 12 men (46.2 %), 22 of them with the mutation 669_698del (84.6%), and 4 with R26C (15.4%) in the HMBS gene. 46.2% had history of acute attacks, and the risk of suffering them was significantly higher in women (P<0.05). Hypertension was detected in 30.8%, elevated transaminases in 15.4% (none with hepatocellular carcinoma) and dyslipidemia in 30.8%. All these frequencies were similar to those reported for the Spanish general population. 38.5% of patients presented with chronic abdominal pain or dyspepsia. 23.1% suffered chronic renal insufficiency (CRI); this is a higher proportion than that reported in the general Spanish population. In addition we found a significant relation between having a history of acute attacks and the risk of suffering CRI. CRI was the main clinical problem detected. Estimated glomerular filtration rate and proteinuria should be incorporated as part of the annual structured review of adult AIP patients, specially in those with acute attacks.

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creating anxiety in these families. Lumbar puncture to check for neurotransmitters is an invasive test that requires sedation in a newborn that usually looks healthy. Prolactin levels have been shown to be a good biomarker for biopterin disorders. Prolactin is usually elevated and can be used to adjust the therapy dosage if needed. BH4 trial in patients who are already in a protein restricted diet is more complicated because the child needs to go back to a liberalized diet. PAH gene sequencing can be done in two weeks; same turnaround time for two urine pterins tests. I propose that additional testing including prolactin level and PAH gene sequencing should be considered in the first tier of tests for patients with high phenylalanine levels initially detected by NBS. 03. Sulphur aminoacid disorders A-002 Psychiatric manifestation in homocysteinuria type I-case report

P-882 Arash-Kaps L1, Karabul N1, Mengel E1 Outcome of hepatic Wilson's disease at 6 months from diagnosis: response to combination of D-Pencillamine and zinc therapy in a tertiary care centre in India Alam S1, Khanna R1, Sood V1 1

Institute of Liver and Biliary Sciences, New Delhi, India

Thirty three prospectively enrolled cases of Hepatic Wilson's disease were given D-Pencillamine (10-20mg/kg/day) and zinc therapy. Combination therapy (administered at intervals to avoid interference) was shifted to zinc only therapy after the normalization of transaminases. Normal transaminases and negative copper balance at 12 months on therapy were considered successful therapy. Time of clinical and biochemical response (AST & ALT <60 IU/L) were calculated. Thirty three cases (median age of 127.21± 42.27 months) were enrolled. Liver transplantation (LT) was offered to 5 cases with fulminant hepatic failure. Of the rest 28 cases with chronic liver disease (CLD): 4 of the 5 presenting as acute on chronic liver failure (ACLF) and 3 of the 17 decompensated CLD required LT. All 6 compensated CLD, 1 of 5 ACLF and 14 of 17 with decompensated CLD were successfully treated with medical therapy. Twenty one (75%) of the 28 cases successfully treated on medical therapy. Twenty cases showed clinical response within 6 months and 1 case at 12 months. Fifteen cases showed biochemical response within 6 months and remaining 6 cases by 12 months. Conclusions: 75% cases improved on combination therapy with response within 6 months.

1

Villa Metabolica, Univ Child Hosp, Mainz, Germany

Background: Homocystinuria is a rare disorder caused by a cystathionine beta synthase deficiency. It leads to accumulation of the amino acid methionine and homocysteine. Presenting signs are eye involvements such as dislocated lens, intellectual impairment and some of the features of Marfan's syndrome. About half of the known cases show psychosis. Case Report: We report about an obese 24 years old male patient with standard growth. After normal development at the age of 16 years he presented symptoms of paranoid psychosis such as acustic hallucination, delusion, circular thinking and obscessive anxious thoughts. After 6 months he developed recurrent venous thrombosis. When he was 22 years he had a pulmonary embolism. A hyperhomocystinaemia (332 μmol/l), elevated methionine (163,15 μmol/l), homocystine (55,60 μmol/l) and cystine (7,13 μmol/l ) levels in serum were detected that lead us to the diagnosis homocystinuria type I. After treatment with Pyridoxine we could measure immediate decrease of homocysteine (52 μmol/l) and methionine (38 μmol/l) in serum. Conclusion: Several patients with homocystinuria show psychiatric symptoms (about 51 %). Interesting in this case are the early symptoms of paranoid psychosis that was the first sign he presented in the age of 16 years. 06. Organic acidurias A-003

01. Phenylketonuria and BH4 A-001

Candida albicans endocarditis in a patient with methylmalonic acidemia: case report

PAH gene sequencing and prolactin level as part of the diagnostic work-up for PTPS deficiency in the USA

Kitkim E1, Aktuglu Zeybek AC1, Cansever M1, Lacinel 1, Sezen M1, Aydin A1

Leon EL1

1

1

Methylmalonic acidemia is an autosomal recessive disorder of amino acid metabolism, involving a defect in the conversion of methylmalonyl-coenzyme A (CoA) to succinyl-CoA. Clinical findings may occur in any period of life from newborn period to adulthood. 1 year old girl, after an infection she had started to vomit and she had metabolic decompensation. On admission to the ICU she was having anion gap metabolic acidosis, ketonuria, hyperammonemia and she was diagnosed as MMA. On the 12th day of hospitalization she had resistant high fever. There was no physical examination findings that

Children's National Medical Center, Washington, United States

High phenylalanine level can be secondary to many metabolic disorders including PTPS deficiency; the most common cause being PKU. Patients with high phenylalanine levels are placed on phe-restricted diet after they are detected by NBS even though the diagnosis of PKU has not been confirmed. The current diagnostic guideline for PTPS deficiency indicates that urine pterins should be obtained. However, this test has a high false positive rate due to inappropriate handling

Cerrahpaşa Med Faculty, Div Metab Dis, İstanbul, Turkey

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would explain the fever origin. Patient had leukocytosis, CRP was high. Urine, blood, throat, and stool culture samples were taken and broad spectrum antibiotics were started. On the 3rd day of treatment fever was persistent so antifungal therapy was started to the patient who had been for long time at the hospital as a possibility of fungal infections. Bacterial endocarditis echocardiographic imaging was applied to the patient. On the surface of the superior vena cava (VCS) and inferior (VCI) echodens structures were detected. 6x4mm echodens structures were detected at the top of the right atrium. Candida albicans species were detected on hemocultures which were taken during fever periods of patient. 10. Carbohydrate disorders

showed pallor, doll-like face and hepatomegaly. Laboratory investigations showed hypoglycemia, neutropenia, anemia, thrombocytosis, hyperlipidemia, lactic acidosis, hypercalcemia, hypercalciuria and ketonuria. Homozygous c.1211-1212delCT mutation in G6PT gene was found in the patient. In follow up, she was closely fed with breast milk and lactose free formula, received antibiotic prophylaxis, GMCSF (Neupogen®) based on her absolute neutrophil count. Drip infusion during the night and 1.5 gr/kg/dose of corn starch after 6 months of age was planned. It was noticed that recurrent infections attacks were developed together with thrombocytosis, hypercalcemia and elevated C-reactive protein. Conclusion: Thrombocytosis and hypercalcemia may be used as markers for indication of infections and bad metabolic control in GSD1b.

A-004 15. Lysosomal disorders Fluctuation in blood lactate levels and L/P ratios in patients with fructose-1,6-bisphosphatase deficiency; comparison between a severe and a mild case Awaya R1, Ito T2, Kato S2, Nakajima Y3, Hata I4, Shigematsu Y4, Sugiyama N5, Saitoh S2 2

1

Dept Pediatr, Holy Spirit Hosp, Nagoya, Japan; Dept Pediatr, Nagoya City Univ, Nagoya, Japan; 3Lab Genet Metab Dis, AMC, Univ Amsterdam, Amsterdam, Netherlands; 4Dept Pediatr, Fukui Univ, Fukui, Japan; 5Dept Pharmacol, Aichi-Gakuin Univ, Nagoya, Japan Background: Fructose-1,6-bisphosphatase (FBPase) is a key enzyme in gluconeogenesis and its defect causes hypoglycemia during fasting. Hyperlactacidemia is a useful finding in hypoglycemic attack in patients with FBPase deficiency and high L/P ratio can be detected in this condition. We present here a severe and a mild case with FBPase deficiency and compare the fluctuations in lactate levels and L/P ratios to discriminate FBPase deficiency from mitochondrial disorder or other hyperlactacidemia. Method: Two cases of FBPase deficiency, which was confirmed by enzyme and gene analyses, were compared. The severe case had hypoglycemic and lactic acidosis on one day after birth. She had normal develop until one-year-old, but after that, she showed severe attack in every 3 months. The mild case developed his symptom in his age of 4 years and 6 months. After the period, he had lethargy in every 2 to 3 months, but hypoglycemia was not so severe. Results: In the severe case, lactic acidosis with high L/P ratio was observed in her first attack, but was not always observed in other attacks. In the mild case, high L/P ratio was not significant. L/P ratio elevation tends to be observed in prolonged metabolic decompensation.

A-006 Genzyme enzymatic screening card for Pompe, Fabry, Gaucher and mucopolysacharidosis type 1 (MPS 1): one year experience Sales Marques J1, Santos H1, Vila Real M1, Carvalho I1, Rodrigues L1, Varandas R1, Santos F1 1

Centro Hospitalar V. N. Gaia/Espinho, Vila Nova de Gaia, Portugal

Background: Pompe, Gaucher, Fabry and MPS 1 are a group of lysosomal disorders. All have enzyme replacement therapy. Methods: Use Genzyme screening card for Pompe, Fabry, Gaucher and MPS 1 in our outpatient department during one year. All patients with two criteria of one of these diseases were screened. Results: A total of 34 patients were screened in different consultations: Metabolics-26, Neuropediatrics-5, Pneμmology-2, Rheumatology-1. The metabolic disorders that were screened included: Pompe-21, Fabry0, Gaucher-4, MPS 1-9. In Pompe disease, the main clinic reasons for the screening were: hypotonia, scoliosis, scapula alatta, hypertrophic cardiomyopathy, weak muscle, mental retardation, diaphragmatic paresis and high creatine kinase. In Gaucher disorder, the reasons for the test were spontaneous fracture, multiple fractures and dystonia. In MPS 1, the reasons for the spot test examination were corneal clouding, coarse face, hypotonia and scoliosis. No screening was done for Fabry disease. Conclusions: Pompe, Fabry, Gaucher and MPS 1, are disorders that can be under diagnosed because of the multisystem involvement of many organs. The Genzyme test is useful for the diagnosis. Although in our first year experience we did not confirmed any of those diseases by the screening card, we will maintain the test using the same criteria. A-007

A-005 A case with sialidosis presenting with ascites in the neonatal period An infant with a diagnosis of glycogen storage disease type 1b 1

2

2

2

3

Kilic M , Onat E , Gültekin-Soylu A , Saylam E , Kandemirli G , Andiran N2

Ünal Ö1, Hişmi B1, Dursun A1, Kalkanoğlu-Sivri HS1, Tokatlı A1, Coskun T1 1

Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey

Kecioren Train-Research Hosp Div Metab, Ankara, Turkey; 2Kecioren Train-Research Hosp Child Unit, Ankara, Turkey; 3Hacettepe Univ, Faculty of Medicine, Ankara, Turkey 1

Background: Glycogen storage disease type 1b (GSD1b) is an autosomal recessive inherited disease due to deficiency of glucose 6 phosphate translocase (G6PT). Case Report: A 5 months old girl presented firsly with cough and diarrhea when she was 2.5 months old. During newborn period she had hypoglycemia without any definitive diagnosis. Physical examination

Sialidosis is an autosomal recessively inherited lysosomal storage disorder due to neurominidase deficiency in the oligosaccharidoses group. Sialidosis type II has congenital, infantile and juvenile forms. Congenital form may present with intrauterin hydrops fetalis, fetal ascites, edema and hepatosplenomegaly. Dysmorphic features accompany the clinical picture. Three weeks old boy was referred to our center with abdominal distension and inguinal hernia. On his physical examination, trigonocephaly, coarse face, flat nasal bridge, hypertelorism, eyelid, extremity edema, and ascites were noted. On his laboratory

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findings, elevated liver enzymes and bicytopenia were found. Abdominal ultrasound showed hepatosplenomegaly, internal echoes and intra-abdominal free fluid with thick septa appearance. Bone marrow examination revealed increased number of histiocytes, megaloblastic changes and severe bone marrow steatosis. In the hospitalization period, pneumonia and severe sepsis occured and he died on third month of admission. Sialidosis/galactosialidosis or sialic acid storage diseases were suspected in the patient. On urine analysis bound sialic acid level was found 12-folds elevated (809,3 μmol/mmol cre, NV: 41,2-94,6). Enzyme assay showed very low levels of neuraminidase (0,35 μmol/g/h, NV>18), and the patient diagnosed with congenital form of sialidosis type II with enzyme analysis and clinical findings.

Conclusion: Despite being a very rare complication in Pompe disease, hydrocephalus can affect the course of the disease. Besides detailed neurological and cardiorespitory evaluations, repetative examinations of head circumference have importance in diagnosis and follow up.

A-008

Paci S1, Scelsa V1, Gasparri M1, Salvatici E1

Pompe disease in association with hydrocephalus, a case report

1

Kagnici M1, Altinok Y1, Canda E1, Kose M1, Karaca E2, Kalkan Ucar S1, Onay H2, Ozkinay F2, Coker M1

Pompe's disease non-classic infantile form usually leads to death within the first year of life mostly due to cardiomyopathy. We here report our six-month's follow up experience with enzyme replacement therapy (ERT) in a 2-year-old Pompe disease's affected female. Baseline examination: lower limbs' muscle weakness and axial hypotonia with delayed motor skills' acquisition and lack of oral skills, hypomimia and macroglossia. Lab tests: very high values of CPK (789 U/L, n.v. 30-135), LDH (2463 U/L, n.v. 313-618), ALP (591 U/L, n.v. 38-126) and transaminases. Biventricular hypetrophy at the echocardiography. Genetical analysis demonstrated two Pompe's disease classical mutations, with absolutely absent alfa-glucosidase activity on skin biopsy and positive CRIM-testing both on blood and fibroblasts. Once made the diagnosis, ERT was started at 18 months of life. Six months after, therapy is well tolerated, without any adverse reactions up to now. Motors skills parameters clearly improved with achieved steady state locomotion. Echocardiography: reduced cardiac walls' thickness with absolute left ventricular function's improvement (end-diastolic diameters from 26 mm to 34 mm; fractional shortening from 38% to 53%). Biochemical parameters and speech delay did not yet improved to date, suggesting that maybe more time is needed to obtain ERT's efficacy on such issues.

Div Metab Dis,Ege Univ Child Hosp, Izmir, Turkey; 2Div Genetics, Ege Univ Child Hosp, Izmir, Turkey 1

Background: Pompe disease is an autosomal-recessive lysosomal storage disorder caused by a deficiency of acid alpha-glucosidase (acid maltase). Accumulation of glycogen in various tissues leads to a progressive skeletal muscle weakness which results in a decline of locomotive and respiratory functions. Classic infantile patients show first symptoms within the first months of life with heart failure and a profound muscle weakness. Without therapy, most of classic infantile patients die within the first year of life. Case Report: The female patient, born from healthy parents who were consanguineous, was diagnosed with pompe disease while being searched for hypotonicity and hypertropic cardiomyopathy at 7 months old. Progressive increase at head circumference was encountered while she was receiving enzyme replacement therapy. Cerebral magnetic resonance imaging revealed biventricular hydrocephalus. She was planned to be performed ventriculoperitoneal shunting, but she died.

16. Enzyme replacement therapy A-009 Efficacy of enzyme replacement therapy over six-months in a non-classic infantile Pompe disease form: our experience in a 2-year-old affected child

Cl Dep Ped, San Paolo Hosp,Univ of Milan, Milan, Italy