Abstracts

1

001-P

THE COST-EFFECTIVENESS OF NEONATAL POPULATION SCREENING FOR MEDIUM-CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY BY TANDEM MASS SPECTROMETRY. AN EMPIRICAL, PROSPECTIVE STUDY IN THE NORTH-EASTERN REGION OF THE NETHERLANDS. G.P.A.Smit*, J.G.Loeber*, and D-J. Reijngoud*. *Beatrix Children's Hospital, Department and Laboratory of Metabolic Diseases, University of Groningen, and *Diagnostic Laboratory for Infectious Diseases and Perinatal Screening, Bilthoven, The Netherlands.

Heritable defects in fat oxidation bring about life-threatening periods of very low blood glucose levels which occur suddenly in apparently healthy children. These periods result in high mortality and high risk of chronic disabilities. Prevention is e¡ective by prescribing frequent carbohydrate-enriched meals. Medium chain acyl-CoA dehydrogenase de¢ciency (MCADD) is the most frequent defect with a determined prevalence of 1:12.000 births in The Netherlands. Persons with MCADD can be detected by screening of heel puncture derived blood spots with tandem mass spectrometry. In this project a large scale prospective screening of MCADD in the north-eastern region of The Netherlands is combined with a retrospective study of the hospital ¢les of persons with MCADD. The costs of screening are related to the bene¢ts of prevention of the sequela of the disease. The project will ultimately result in a decision on extending the current screening program of PKU/CHT with MCADD.

002-P

RESOURCE UTILIZATION IN AN ACADEMIC BIOCHEMICAL GENETICS LABORATORY: THE OREGON EXPERIENCE K.M. Gibson, K.A. Wilcox Biochemical Genetics Laboratory, Department of Molecular and Medical Genetics, Oregon Health Sciences University, Portland, OR, USA

The Biochemical Genetics Laboratory (BGL) at Oregon Health Sciences University (OHSU) is a moderate-sized genetics subspecialty laboratory which o¡ers a wide spectrum of clinical diagnostic testing. The BGL currently receives 25% of samples from in-house referrals, the remainder from outlying hospitals/clinics within and outside of Oregon. In 1999, the BGL performed the following analyses: 659 metabolic screens, 1137 amino acids, 823 organic acids, 433 carnitines, 175 peroxisomal, 178 mucopolysaccharide, 96 hexoseaminidase, 53 orotic acid, 47 oligosaccharide and 24 galactose-1-phosphate. Some of these tests are not coste¡ective in terms of volume, yet smaller academic laboratories such as ours o¡er these tests to maintain pro¢tability. This is important since larger regional diagnostic laboratories in the US o¡er many of the same high throughput, small molecule tests at considerably reduced prices. While their pricing represents a distinct market advantage, larger diagnostic laboratories often lack diagnostic acumen, do not o¡er STAT analyses, and lack critical ancillary personnel (metabolic physicians, dieticians, nurse practitioners, etc.) associated with academic institutions which are essential in terms of quality patient care. In addition, many State referral laboratories contract with large diagnostic laboratories based upon pricing mechanisms, precluding specimen referral to smaller academic laboratories unless physicians speci¢cally request this. Smaller academic laboratories must continue to o¡er a broad array of testing, and must develop additional specialized testing, in order to maintain pro¢tability in an expanding and competitive US healthcare market.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

2

Abstracts

003-P

A NEW APPROACH TO THE PROBLEM OF DEMOGRAPHIC ENTRY AND DATA HANDLING IN NEONATAL SCREENING M.J. Henderson and R.G. Jones Department of Chemical Pathology and Immunology, Leeds Teaching Hospital Trust, Leeds, UK A multidisciplinary screening laboratory is being created in Leeds supported by the UK Department of Health Modernisation of Pathology Fund. The project is resulting in the centralisation of analytical work for antenatal and neonatal screening, biochemical and haematological. This is having major bene¢ts of e¤ciency and enables state-of-the-art resources like tandem mass spectrometry to be used to maximum e¡ect. It will be possible to introduce new screening modalities to the programme at marginal cost. A key element to the project is the use of automated sample processing facilities wherever possible. A new version of the UK National Newborn Screening Card that enables the patient demographics to be scanned and recognised by OCR software has been developed. A barcoded number, incorporated into the original screening card, will link the demographic details to the various analytical results and enable the controlling software to create a database. Electronic links are being established with client clinical departments. The database will provide a unique continuous record of results for individual pregnancies. It will also facilitate accurate auditing of screening processes. By bringing together specialist expertise in a variety of ¢elds there is the potential for signi¢cant improvement in the quality of service provided.

004-P

ETHICAL ISSUES IN GENETIC TESTING AND NEWBORN SCREENING Linda L. McCabe1,2 and Edward R.B. McCabe2,3, Departments of 1Human Genetics and 2Pediatrics, UCLA, and 3Mattel Children's Hospital at UCLA, Los Angeles, CA 90095-1752 The information from the Human Genome Project is blurring the boundary between biochemical and molecular genetics. The power of the human genome database must be balanced with a responsibility for ethical use of genetic information and the protection of personal privacy. For these reasons, the United States Secretary of Health and Human Services (HHS), Donna Shalala, established the Secretary's Advisory Committee on Genetic Testing (SACGT) to advise her on all aspects of the development and use of genetic tests. Ethical concerns, particularly regarding privacy, discrimination and informed consent were discussed extensively during the SACGT's outreach to the American public. We will discuss these concerns, as well as the need for education, to ensure the ethical and e¡ective use of genetic testing among informed individuals. Nearly every baby born in the developed world has a genetic test performed in the neonatal period, through newborn screening programs. A recent Task Force on Newborn Screening, co-sponsored by the Health Resources and Services Administrations of the US HHS Department and the American Academy of Pediatrics concluded that newborn screening experiences may vary greatly by state and that a national agenda should be developed to ensure a more uniform ethical foundation for this public health genetic testing program. Ethical issues in genetic testing are complex and evolving. It is critical to be proactive in development of policies regarding genetic testing and newborn screening.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

3

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30 YEARS OF MASS-SCREENING: NEW-BORN VERSUS SELECTIVE. ANALYSIS OF BENEFITS AND COSTS Pa©mpols T1 , Maya A1 , Chaba¨s A1 and Sau J2. (1)Institut de Bioqu|¨ mica Cl|¨ nica and (2)Hospital Cl|¨ nic. Corporacio¨ Sanita©ria Cl|¨ nic. Barcelona. Spain.

The aim is to analyse current costs (in euros) and to compare long term bene¢ts of the screening of 1,139,236 new-borns(N) versus the selective screening of 19,914 symptomatic patients(S). Target population and costs of N: 66,525 new-born/year from Catalonia and Balearic Islands are screened for PKU and CH. Global costs 567,334. Costs per new-born 8.5. Costs for positive case 16,209 (6 hyperphenylalaninemia +29 CH per year). Target population and costs of S: 1,609 symptomatic patients/year from all Spain are investigated for around 130 diseases, mainly disorders of intermediary metabolism, lysosomal and peroxisomal diseases and other related diseases. Global costs 706,775. Costs per patient 439.2. Costs for each diagnosed case 6,929 (102 cases/year, which represents 6.3% of patients studied). N versus S: Global costs and individual cost are respectively 1.2 and 51.6 times higher for S. In contrast, costs of each diagnosed case are 2.3 times higher for N. Savings of early detection of PKU and CH are evident. For S, diagnosis save costs, even for untreatable cases, in the sense that contributes to avoid unnecessary tests or unsuitable therapies, and also reduces the number and length of hospital stages. In terms of primary prevention in the population, the impact of S is clearly higher: with N we have detected 675 cases from which only 205 have a genetic cause (PKU and 10% of CH), whereas with S we have diagnosed 1,429 cases of hereditary diseases. Therefore it can be concluded that not only N, but S, should be considered an important matter of Public Health.

006-P

THE INTRODUCTION OF TANDEM MASS SPECTROMETRY INTO THE SOUTH AUSTRALIAN NEONATAL SCREENING PROGRAMME: BENEFITS AND COSTS E. Ranieri, R Gerace, B Bartlett, K Barnard and JM Fletcher Department of Chemical Pathology Women's and Children's Hospital, Adelaide, South Australia

We introduced tandem mass spectrometry (MSMS) into front-line newborn screening in February 1999 as part of an Expanded South Australian (SA) Neonatal Screening Programme. In the ¢rst 12month period of operation 19,942 neonatal blood-spot screening tests have been analysed using a SCIEX API 365 MSMS instrument, performed on dried blood-spot samples collected from neonates at 48 hours of age. Within this cohort 9 infants with an IEM were detected; one with medium chain acyl-CoA dehydrogenase de¢ciency (an additional case identi¢ed in the pilot phase), an isovaleric acidemia, 3-methyl crotonyl Coenzyme A carboxylase de¢ciency, carnitine de¢ciency, four PKU's and one hyperphenylalaninemia. The collective incidence in the screened cohort was 1 in 2055. In order to make these diagnoses, 118 infants had repeat neonatal screening tests requested; 33 for abnormal amino acid pro¢les and 85 for abnormal acylcarnitine pro¢les (0.43% of all babies screened). Thirteen infants had persistently abnormal results and were recalled for counselling and further metabolic testing, of whom 9 have received a diagnosis of an IEM. A direct request for urine and plasma metabolic testing was made in some infants with grossly abnormal results. Either enzymology or DNA mutational analysis was used to con¢rm the diagnosis of IEM's other than PKU and carnitine de¢ciency. Excluding the initial capital cost of the MSMS instrument the additional cost of incorporating MSMS screening into an existing programme is $US0.60 per infant screened, including reagents and technical sta¡. The incremental cost increase for the additional IEM's detected was $US3000 per diagnosis with a detection rate of 1 in 2,055 infants screened. We note major advantages of earlier collection times to 48 hours after birth and a reduction in the overall recall rate.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

4

Abstracts

007-P

TWO YEARS OF ROUTINE NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY (MSMS) IN NEW SOUTH WALES, AUSTRALIA Bridget Wilcken, Veronica Wiley, Kevin Carpenter. NSW Newborn Screening, New Children's Hospital, Sydney, Australia. Since 1998, electrospray MSMS has been used routinely in the NSW newborn screening programme. We tested samples from 190,000 babies on usually day 3 of life for 21 selected amino acids and acylcarnitines and retrospectively analysed stored newborn screening samples from patients diagnosed clinically over the last 20 years. The detection rate with MSMS was approximately the expected rate, based on previous experience or known mutation frequencies. We detected 24 PKU babies (19 expected), 2 biopterin defects, 4 MCAD babies (6 expected), 1 atypical MCAD, and 2 with other defects of fatty acid oxidation, 3 with organic acid defects, 7 with amino acidopathies and 4 non-inborn errors (neonatal hepatitis and maternal B12 de¢ciency). There were 3 known false negatives, cases of tyrosinaemia type 1, NKHG and cobalamin C defect. Only 162 repeat samples were requested. The incremental cost was A$0.92, including overheads and depreciation, but not including cost of collecting resamples. We have insu¤cient data to draw ¢rm conclusions. Our prospective and retrospective results suggest that some disorders can be detected with con¢dence (including PKU, many organic acidaemias, classic MSUD), and some almost always (eg. MCAD de¢ciency, glutaric acidaemia). Disorders which probably cannot reliably be detected without an unacceptable recall rate include mild MSUD, homocystinuria, tyrosinaemia type I, and probably most fatty acid oxidation defects.There is a problem in setting action levels with so many primary analytes, but no justi¢cation in not seeking a disorder simply because it cannot be detected reliably on every occasion.

008-P

PROSPECTIVE STUDY OF MS-MS NEWBORN SCREENING IN BAVARIA, GERMANY. INTERIM RESULTS A Roscher1, B Liebl2, R Fingerhut3, B OlgemÎller3 1 Children's Hospital, University of Munich, 2Public Health Screening Center, Oberschleissheim, Germany, 3 Newborn Screening Laboratory, Munich, Germany A 3-year prospective TMS-based newborn screening (NBS) study was outlayed in Bavaria, Germany (125,000 births/year) in consensus with legal, ethical, scienti¢c and medical bodies. Features: Voluntary participation, written consent, extensive information policies, early screening (day 3), standardized operation under rigid quality assurance (QA) rules, scienti¢c surveillance. 7 disorders are screened routinely by TMS, an expanded disease range under pilot criteria. Interim results: Participation rate is 4 98 %. Among 166,000 newborns TMS screening identi¢ed 49 con¢rmed cases of IEM (1:3390) that required treatment and/or counselling. The main progress resided in the highly speci¢c (5 0.05% recall) and frequent (1:9000) detection of treatable FAO defects. Besides 13 MCADs, the ¢rst cases of LCHAD, Carnitine-uptake defect and CPT-I were detected in prospective NBS. 3-MCC de¢ciency (of yet unknown signi¢cance) appears to be the most frequent OA-disorder within our population (3 isolated cases, 2 asymptomatic mothers). 2 newborns with severe variants of VLCAD and MAD were seen in NBS, but died (day 3) prior to diagnosis. No false-negative cases among speci¢ed disorders were noted so far. A newly developed data interpretation concept (multianalyte pattern recognition analysis) and rigid QA procedures proved to be essential for save operation and low overall TMS-related recall rates (0.3 %). Intermediate audit and feed-back by questionnaires a¤rms the adequacy of the new screening system and a very high acceptance within the medical community and the public.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

5

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AUDIT OF A MS-MS BASED NEWBORN SCREENING MODEL PROGRAM IN BAVARIA. INTERIM REPORT B Liebl1, U Nennstiel-Ratzel1, R Fingerhut2, B OlgemÎller2, AA Roscher3 1 Public Health Newborn Screening Center, OberschleiÞheim, 2Newborn Screening Laboratory, Munich, 3Children¨s Hospital, University of Munich, Germany

A newborn screening model program based on a coordinated cooperation between private sector (laboratoy), public health services, clinical follow-up network and scienti¢c surveillance was implemented in Bavaria, Germany. Special features are: Shift from written dissent to written consent, early sampling (day 3), expanded disease coverage using tandem mass spectrometry (MS-MS), de¢ned QA-policies and hotlines for clinical intervention and support. A demographic tracking system is matching all screeed children against birth noti¢cations on a regional basis and ensures consequent follow-up and evaluation of recalls. The move towards written consent has had little impact on compliance. Comprehensive written information to parents and health care workers has resulted in 4 98% voluntary coverage. 122,301 newborns were screened in 1999. A second screening due to early discharge (5 48 hr) was requested in 1.8%. Screening was made up for 87 missing children detected by tracking. 75 children with disorders (30 by MS-MS) were identi¢ed. MS-related recall rates were low (0.3%) with no false negative cases known so far. Policies for reporting, urgent intervention and con¢rmation of ``new disorders'' proved to be e¡ective and created very little adverse e¡ects. However, 20% of pending (low urgency) recalls required special contacting of physicians, midwives or parents. 2 children with severe FAO disease variants died prior to diagnosis. In all other positive cases timely treatment was initiated. Feedback by questionnaires is testifying very high acceptability within the medical community and the public.

010-P

ETHICAL IMPLICATIONS OF EXPANDING NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY ^ A GERMAN PERSPECTIVE AA Roscher, B Liebl1, Children's Hospital, University of Munich, 1Public Health Screening Center, Oberschleissheim, Germany The era of TMS technology in newborn genetic screening (NGS) marks the transition to a ``one method many disorders'' procedure and requires reconsideration of screening criteria. However, the widely followed tenets of ethical and public health arguments that justify NGS have been formulated at the realities 30 years ago. From the experiences of the ``Bavarian Screening Study'' we propose that decison making and safeguarding ethical standards surrounding TMS-NGS can better be guided by recent bioethics principles rather than by the old criteria. These would in part ignore the principle of solidarity with children su¡ering from not perfectly treatable IEM. Leading principles followed by legal, ethical and medical bodies were: Recognization that NGS is a medical act in the context of preventive medicine that should lead to medical intervention for the bene¢t of the newborn. This creates a relationship between participants to ensure the respect for the fundamental principles of proper information, written consent, con¢dentiality and of ensuring outcome. NGS as medical act also imposes limits (above those of costs) on the degree of state intervention or on composition of medical and ethical commitees, that decide on screening activities. On the other hand the preposition that NGS should be universally and equitably available and non-discriminatory creates an obligation for state authorities to provide the framework for fully integrated programs from the act of information to validation of testing procedures, quality assurance and the act of follow-up. Application of these principles in a TMS-NGS program (e.g. written consent) proved to be workable and of very high public acceptability.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

6

Abstracts

011-P

A MODEL FOR MEDICAL EVALUATION OF EXPANDED SCREENING WITH MS-MS S Albers, SW Waisbren, LA Finkelson, MS Korson, IV Bailey, HL Levy Children's Hospital, Harvard Medical School, Boston, MA, 02115, USA This study aims at assessing the value of presymptomatic identi¢cation of patients with metabolic disorders diagnosed by newborn screening with tandem mass spectrometry (MS-MS) and at providing a model of integrated health care for this new group of patients. The New England Consortium of Metabolic Centers integrates newborn screening, primary care, metabolic evaluation and investigation to develop a comprehensive and e¤cient system for follow-up of metabolic patients. Infants born in Massachusetts or Maine with a metabolic disorder (except phenylketonuria) identi¢ed by expanded screening with MS-MS are compared with those diagnosed on a clinical basis in the other New England states, in which MS-MS has not been approved. Outcome data include clinical and developmental examination of the child, assessment of parental stress and level of health care required. Since the initiation of MS-MS in Massachusetts in 02/99, 8 infants have been identi¢ed: 1 3-methylcrotonyl-CoA carboxylase de¢ciency (3-MCC), 4 mediumchain-acyl-CoA dehydrogenase de¢cency (MCADD), 2 short-chain-acyl-CoA dehydrogenase de¢ciency (SCADD) and 1 propionic acidemia (PPA). All patients have been treated presymptomatically and have so far maintained normal growth and development. We also diagnosed 5 asymptomatic siblings (4 MCADD, 1 3-MCC). The New England Consortium is likely to be an e¡ective model to assess the bene¢ts and potential problems of expanded newborn screening.

012-P

NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY; RESULTS OF PILOT STUDY AND RETROSPECTIVE ANALYSIS IN JAPAN Y. Shigematsu1, K. Fujisawa1, I. Hata1, M. Mayumi1, Y. Tanaka2, M. Sudo3 Department of Pediatrics, School of Medicine1, Central Research Laboratories2, Fukui Medical University3

Although tandem mass spectrometry has been reported to be a powerful tool for newborn screening, its speci¢city and ability to diagnose a variety of inherited metabolic diseases remain to be clari¢ed. We analyzed patients' blood spots, which had been collected in the newborn period and stored in a freezer, and tested our cut-o¡ values. The ratios of propionylcarnitine to acetylcarnitine concentration in blood spots were markedly higher in three patients wit methylmalonic aciduria (late-onset type) and a patient with propionic acidemia (late-onset type) than our cut-o¡ value (mean + 3SD), while they were lower in a patient with benign methylmalonic aciduria. Two patients with propionic acidemia, both of whom were assumed to be the late-onset type on the basis of residual enzyme activities, were screened using our cut-o¡ values in this pilot study. The acylcarnitine pro¢les of two patients with glutaric aciduria type II were atypical as compared with those reported in the acutely ill patients. Two newborns with severe asphyxia and hypoglycemia showed lower palmitoylcarnitine concentrations than our cut-o¡ value for carnitine-palmitoyltransferase I de¢ciency. A patient with carbamylphosphate synthetase I de¢ciency (late-onset type) and a patient with ornithine transcarbamylase de¢ciency (onset at 2 weeks of life) showed lower citrulline concentrations than our cut-o¡ value of 8 nmol/ml, while a female patient with the latter disease did not. A newborn who died unexpectedly showed an acylcarnitine pro¢le which was not speci¢c to any types of fatty acid oxidation disorders ever reported. We should test our method further by analyzing more samples of patients and newborns.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

7

013-P

A COST^BENEFIT EVALUATION OF NEONATAL SCREENING PROGRAM IN CHINA X.F. Gu, J. Ye, J.J. Wang, X.M. Cheng Xin Hua Hospital, Shanghai Second Medical University and Shanghai Institute for Pediatric Research, China Objective: In order to make the best use of health care resources, to achieve the maximum social and economic bene¢ts and to lay the foundation for the popularization of neonatal screening for phenylketonuria (PKU) and congenital hypothyroidism (CH), a cost^bene¢t analysis of neonatal screening program was conducted. Methods: Cost for and bene¢t gained for screening were calculated according to the average incidence of two diseases and data published in national economics statistics. Results: The cost of neonatal screening, treatment for PKU with low phenylalanine milk powder and follow-up, the total add up to 128 793 Yuan. However, the direct and indirect ¢nancial bene¢t is 481 263 Yuan. Ratio of cost to bene¢t was 1:3.7. The cost of neonatal screening for CH is 129 175 Yuan. However, the ¢nancial bene¢ts including money saved in treatment, nursing care, special education and the loss of income avoided is 468 470 Yuan, the ratio of cost to bene¢t was 1:3.6. Conclusion: The neonatal screening for PKU and CH in this country re£ects a better economic and social bene¢t and merit further popularization.

014-P

A NEW APPROACH IN NEONATAL SCREENING FOR HEREDITARY TYROSINEMIA TYPE I A. Schulze, D. Frommhold, E. Mayatepek, and G.F. Ho¡mann Dept. Gen. Ped., University Children's hospital, INF 150, Heidelberg, Germany

Introduction: Hereditary tyrosinemia type I (McKusick 276700) is a disease with a serious prognosis if untreated leading to liver failure, neurological crises, and hepatocarcinoma. With NTBC a treatment option emerged. Therefore, tyrosinemia type I ful¢ls criteria for neonatal screening. However, elevation of tyrosine can also be caused by several conditions di¡erent from tyrosinemia type I. Methods: The ``second tear'' strategy consists of tyrosine determination by means of electrospray MS/MS followed by the indirect determination of succinylacetone from the same blood spots when the tyrosine level is higher than a given threshold. We have developed a fast spectrophotometric microplate assay based on the inhibition of phorphobilinogen synthase by succinylacetone, which accumulates in tyrosinemia type I. Results: The quantitative phorpho-bilinogen synthase assay clearly discriminates between tyrosinemia and healthy newborns. The assay turned out to be reliable, accurate, and cheap. The ``second tear'' strategy applied in over 70.000 neonates until now led to a strong decline of false positives. Conclusion: Indirect measurement of succinylacetone by the described quantitative assay can be used as con¢rmatory test if the false-positive rate of single tyrosine measurements turns out to be unacceptable. Up to now, our experience is too limited with respect to sensitivity of this strategy because of the low prevalence of tyrosinemia in Germany. However, we recommend inclusion of hereditary tyrosinemia type I in neonatal screening.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

8

Abstracts

015-P

MULTIPLE DISEASE CATEGORIES SCREENING ANALYSIS (MULTISCAN) IN URINE BY TANDEM MASS SPECTROMETRY H. Korall, M. GÎggerle, G. Frauendienst-Egger, F.K. Trefz Zentrum fÏr Sto¡wechseldiagnostik Reutlingen GmbH (Metabolic Unit), WÎrthstraÞe 47, D-72764 Reutlingen, Germany, Tandem mass spectrometry (TMS) is now well established in neonatal screening for aminoacids, free carnitine and acylcarnitins in dried blood spots. Using TMS we developed multiple disease categories screening analysis (MULTISCAN) - an advanced selective screening method in urine for detecting inborn errors of metabolisms. Urine metabolites were separated via a HPLC column and quanti¢ed in the multiple reaction monitoring mode by a Tandem Mass Spectrometer. MULTISCAN is a rapid, sensitive and reliable diagnostic tool in e¡ective screening for inborn errors of metabolism. Urine analysis using TMS may be done either by a single analysis (Table 1A) or an additional analytical run for speci¢c metabolites (Table 1B). ``Conventional'' diseases resp. metabolites detected by conventional newborn screening are not listed. Table 1 A, B: Multiple Disease Categories Screening Analysis in urine by MULTISCAN Category of Disease(s) A. Single Urine Analysis Aminoacidopathias Peroxisomal disorders Purine/Pyrimidine Disorders Sul¢te Oxidase De¢ciency Creatine De¢ciency

Metabolite(s) quanti¢ed

Category of Disease(s)

Metabolite(s) quanti¢ed

Urea Cycle disorders Ureidopropionase De¢ciency

Argininosuccinic acid 3-Ureidopropionic acid

Amino acids Pipecolic Acid Uric Acid B. Additional Analytical Run Sulfocysteine Gluthatione Synthetase Def. Guanidinoacetate, Creatine Canavan Disease

5-Oxoproline N-Acetylaspartic Acid

Applications of the method for diagnosis of various diseases are demonstrated.

016-O (mito)

A NOVEL DISORDER AFFECTING MULTIPLE MITOCHONDRIAL FUNCTIONS, LOCALIZED BY MICROCELL-MEDIATED TRANSFER TO CHROMOSOME 2 (1)Agnieszka Seyda, (2)Suzann Malaney, (3)Andrew Cuthbert, (3)Rob Newbold, (4)Thomas Hudson, (1)Brian H. Robinson (1)The Research Institute, Hospital for Sick Children, 555 University Ave., Toronto, Ontario (2)Garvan Institute of Medical Research, Darlinghurst, Australia (3)Dept. of Biology and Biochemistry, Brunel University, Uxbridge, UK (4)Montreal General Hospital, Montreal, Quebec Mitochondria isolated from skin ¢broblasts of 3 sibs and 1 unrelated individual with combined defects of the a-keto and dehydrogenase complexes and complex III were investigated. Immunoblot analysis of selected complex II subunits (core 1, cyt c1, and ISPs) as well as the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins ruling out the possibility of an import and/or assembly factor being involved. Microcell-mediated chromosome fusion analysis was performed between the studied ¢broblasts (recipients) and a panel of A9 mouse:human hybrids. Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. Two intervals on chromosome 2 have been identi¢ed so far as potentially containing the locus for the unknown factor. To our knowledge this is the ¢rst reported defect with such profound impact on the mitochondrial enzymes involved in the electron transport chain, the Kreb cycle, carbohydrate metabolism and amino acids metabolism which does not involve import and/or assembly mechanism.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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9

EVALUATION OF A NEW KIT FOR PKU SCREENING BY TANDEM MASS SPECTROMETRY Michael E. Jackson, Chet Knopsnider, Susan C. Leonard PerkinElmer Life Sciences 3985 Eastern Road Norton Ohio USA

017-P

Mass spectrometry is an emerging technology for newborn screening for metabolic disorders. PerkinElmer Life Sciences have begun development of a newborn screening system using mass spectrometry including the Wallac MS2, Neogram software and assay kits. The ¢rst kit under development is for screening for Phenylketonuria (PKU). The kit consists of methanolic solutions of PHE-d5 and TYR-d4, the £ow solvent wet acetonitrile, and microplates for the assay. The kit protocol is to punch one 3mm blood spot, extract with 200ul of the standards solution, shake for 15 minutes, and let stand an additional ¢fteen minutes, and then analyse the extract by MRM. The performance of the kit has been examined based on the NCCLS protocol EP-10A. Low, medium and high samples were prepared by adding phenylalanine and tyrosine to blood, and spotting this blood on S&S 903 ¢lter paper cards. These blood spots were analysed for ¢ve consecutive days using the Wallac MS2, and the described protocol. The amount of phenylalanine in the blood spots was also determined by HPLC and the results compared to the MS2 results. The three levels averaged 1.4, 6.1 and 8.7 mg/dl PHE by mass spectrometry. The HPLC measurements averaged 2.3, 6, and 8 mg/dl for the same samples. The HPLC and MS results are linearly related by PHE (MS) = 1.17 PHE (HPLC)-1.17, with r = 0.998. The overall coe¤cients variation of the three levels of PHE by MS were 9.4, 8.1 and 7.2% for the low, medium and high samples respectively. We conclude that this kit with the associated assay protocol and software is suitable for further development to a product for newborn screening for PKU.

018-P

NEONATAL SCREENING FOR INHERITED METABOLIC DISORDERS: EVALUATION OF GC/MS METHOD Takahiro Inokuchi, Ichiro Yoshida, Kumiko Aoki, Kyoko Tashiro, Satomi Tashiro, Misa Jogo and Masatoshi Tanaka Research Institute of Medical Mass Spectrometry, Kurume University, School of Medicine

Inherited metabolic disorders screening is based on the urease treatment on neonatal urine (Shoemaker and Matsumoto). A total of 18563 urine samples from healthy neonates including premature babies were examined during the 4-year-2-month study period (1996^2000). The samples were analyzed by automated GC/MS for organic acids, sugars, nucleioc acids with a 15 minute analysis time/sample. A computer-assisted metabolic pro¢ling was established by cut-o¡ (M + 5SD). We found 1 case of OTC de¢ciency, 1 case of citrullinemia, 1 case of glycerol kinase de¢ciency, 2 cases of 2-aminoadipic aciduria, 2 cases of neuroblastoma and 18 cases of transient hypertyrosinemia. Other transient abnormalities include 34 cases of 3-hydroxybutyric aciduria, 35 cases of lactic aciduria, 9 cases of homovalinic and vanylmandelic acidurias, 16 cases of galactosuria and 8 cases of cystinuria. The results obtained demonstrate that GC/MS method is useful to detect several metabolic disorders which Guthrie method and Tandem Mass Spectrometry can not. However, this method cannot take the place of Guthrie test or Tandem Mass Spectrometry because of time consuming, cost and inability to detect hypothyroidism.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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URINE METABOLIC SCREENING IS USEFUL IN THE ASSESSMENT OF CHILDREN WITH DEVELOPMENTAL DELAY N.K. Poplawski1, J.R. Harrison2, J.M. Fletcher2. Departments of Clinical Genetics1 & Chemical Pathology2, Women's & Children's Hospital, North Adelaide, SA 5006, Australia. Introduction: The role of metabolic screening in the evaluation of children with developmental delay/ intellectual disability (DD/ID) remains the subject of ongoing debate. Aims: To determine 1) the reason for testing, 2) the proportion referred for DD/ID and 3) the diagnostic yield of a urine metabolic screen (UMS) in an unselected paediatric and adult population. Method: Retrospective review of UMS referrals 1/1/92^31/12/98, using gas chromatography/MS for organic acids, ion exchange chromatography for amino acids and a colorimetric assay for orotic acid. Results: 3316 individuals 428 days old were referred for UMS. Most referrals were males (60.8%), with neurological symptoms (66%) and a diagnosis was determined in 1.8% (61/3316). More than 60% of diagnoses had known Mendelian or mitochondrial inheritance and thus signi¢cant recurrence risks. For individuals with DD/ID (43.4% of all referrals) the diagnostic yield was 1.1% (16/1447). A similar diagnostic yield was seen for isolated DD/ID and DD/ID with other features (9/828 versus 7/619, w2=0.006, P=0.56). Speci¢c therapies were available for 10/16 diagnoses made in referrals with DD/ID, and 14/16 had known Mendelian or mitochondrial inheritance. Conclusion: Urine metabolic screening is an important part of the evaluation of children with DD/ ID as it enables them to receive appropriate early intervention and therapy. Early screening of these children also allows parents to choose to avoid a second a¡ected pregnancy.

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URINARY ACYLGLYCINES IN MITOCHONDRIAL ENERGY METABOLISM DEFECTS AND ORGANIC ACIDURIAS: ELECTROSPRAY TANDEM MASS SPECTROMETRIC MEASUREMENT L. Bonafe¨1, H. Troxler1, T. Kuster1, C. W. Heizmann1, N. A. Chamoles2, A. B. Burlina3, N. Blau1 1 Div. of Clinical Chemistry and Biochemistry, Univ. Children's Hospital, Zurich, Switzerland; 2Lab. of Neurochemistry, Buenos Aires, Argentina; 3Depart. of Pediatrics, Univ. of Padua, Italy Urinary acylglycine (AG) excretion was retrospectively analyzed in 26 patients with mitochondrial energy metabolism disorders, 55 patients with organic acidurias, and 54 controls by ESI-MS/MS, by monitoring precursor ions of m/z 90. Urinary concentrations were quanti¢ed using deuterated internal standards. Normal values for the most important AG were established. In MCAD de¢ciency hexanoyl- phenylpropionyl- and suberylglycine were detected in all the samples; in addition, hydroxyoctanoyl- and methylhexanoylglycine were also detected. The main metabolites excreted in MAD de¢ciency (neonatal form) were C4- and C5-, followed by C6-glycine. In isovaleric aciduria, propionic aciduria, and 3-methylcrotonylglycinuria typical glycine conjugates were always found in large amounts. Methylmalonic aciduria (mutase de¢ciency), multiple carboxylase de¢ciency, and 3hydroxy-3-methylglutaric aciduria always revealed pathological acylglycine pro¢les, even if not speci¢c for the disease: propionylglycine was always detectable in methylmalonic aciduria and in holocarboxylase synthetase de¢ciency, and 3-methylcrotonylglycine was constantly present in 3-OH3-methylglutaric aciduria. In all these diseases AG excretion seems to be less in£uenced by the clinical status than the organic acid excretion. ESI-MS/MS is a fast and useful diagnostic tool for these metabolic disorders complementary to organic acids and acylcarnitine pro¢les.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

11

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SCREENING FOR INBORN ERRORS OF METABOLISM: A TEN YEAR EXPERIENCE IN CHILDREN'S HOSPITAL OF RABAT (MOROCCO) L. Chabraoui*, S. Dahri*, H. Talbaoui*, C. Vianey-Saban** and I. Maire** Laboratoires de Biochimie, *Hoªpital d'enfants de Rabat, Maroc, **Hoªpital Debrousse, Lyon, France

The diagnosis of IEM requires expensive methods. Therefore it is necessary to screen them before deciding to employ sophisticated biochemical investigations. So we developed, in the children's hospital of Rabat, a screening program. Patients were selected on conventional clinical criteria. From January 1991 to March 2000 a total of 1432 children aged 1 week to 17 years were screened for IEM by urine and/or plasma analysis using TLC (for amino acids, oligos accharides and sugars studies), colorimetric and electrophoretic determination of urinary glycosaminoglycans and other tests such as ammonia, carnitine, oxalate, lactate and pyruvate determinations. According to the results of these screening methods and to the clinical features, further investigations were performed. Among the 1432 children studied, 134 were detected to have an IEM. Amino acid disorders were found in 74 cases: PKU (38), type I Tyrosinemia (14), Homocystinuria (8), Alcaptonuria (4), MSUD (2), Biopterin biosynthesis de¢ciency (1), Imminodipeptiduria (1) and generalized hyperaminoaciduria (6 cases). 42 patients with MPS were categorized as type I or II (26), type III (8), type IV (6) and type VI (2 cases). We also detected: type III Mucolipidosis (2 sibs), Arylsufatase A de¢ciency (2), type I Glutaric aciduria (1), type I.

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PILOT SCHEME OF SELECTIVE SCREENING ON REGIONAL BASIS TO IMPROVE DETECTION OF INBORN ERRORS OF METABOLISM (IEM) Sykut-Cegielska J., Gradowska W., Gizçewska M.*, Walczak M.*, Korczowski R. **, W|¨ tro¨bska S.**, Nowakowska I.***, Pronicka E. Children's Memorial Health Institute Warsaw, Medical School of Szczecin*, Regional Hospitals of Rzeszo¨w**, Wocawek***, Poland

Only a few cases of GC-MS abnormal pro¢les were identi¢ed in Poland up to 1991 (5200 urine samples examined per year) giving the recognition rate of IEM less than 1:100 000 births. Since then the number of examined samples continuously increased to over 1200 per year, and the IEM detection rate - up to 1:17000 births in 1997. The aim of two years pilot study (1998-1999) was to improve e¡ectiveness of the selective screening for IEM in three regions of the country (covering 5% of births) by programme of medical education, regional net of samples collection and transportation, direct personal contact of three local paediatricians with the CMHI, in which a set of screening methods (including GC-MS) was developed according to ERNDIM standards. The programme was supported by the Ministry of Health and the National Research Committee (PB 346/P05/97/12). Signi¢cant elevation of number of examined samples and detected IEM were achieved in three regions covered by the screening programme in comparison to three regions where the screening was not specially promoted. Birth number (per two years), number of samples examined by GC-MS, and the number of IEM identi¢ed were 40922 and 94321, 1531 and 32, and 7 and 1 in two compared areas respectively. The frequency of the IEM recognised by GC-MS methods in both areas di¡ers markedly being 1:5800 and 1:94000. The results con¢rm the e¡ectiveness of the employed management of selective screening for IEM in the country.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

12

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023-P

DIFFERENCES IN ACYLCARNITINE PROFILE BETWEEN TERM AND PRETERM INFANTS J. Meyburg, A. Schulze, D. KohlmÏller, G.F. Ho¡mann, O. Linderkamp, E. Mayatepek Department of Pediatrics, University of Heidelberg, Germany Background: Tandem mass spectrometry is a powerful technique for detection of inborn errors of fatty acid oxydation. Normal values derived from full-term infants are also used in the screening of preature children. We compared acylcarnitine pro¢les of term and preterm infants to study the neessity for di¡erent normal ranges. Materials: 80 infants were divided in four groups: A (gestaional age (GA) 24 to 27 wks.), B (GA 28 to 31 wks.), C (GA 32 to 36 wks.) and D (GA 37 to 41 wks.). Blood samples were taken on day 5 and spotted on a Guthrie card. Measurements: Small circles of dried blood were excised and extracted with methanol. The exracts were evapoated after addition of internal deuterated standards. The residue was butylated and analysed using a PE Sciex API 365 electrospray tandem mass spectrometer. Carnitine levels were adjusted for haemolobin values. The Student's t-test for unpaired samples was used to test for statistical diferences. Results: Levels of free carnitine, propionyl- (C2), butyryl- (C3), isovaleryl- (C5), hexanoyl- (C6), octanoyl-(C8), decanoyl(C10), decenoyl- (C10:1), lauroyl- (C12), myristoyl- (C14), myristoleyl- (C14:1), hydroxymyristoyl(C14OH), palmitoleyl- (C16:1), hydroypalmioyl- (C16OH), hydroxypalmitoleyl- (C16:1OH), stearoyl- (C18), oleyl- (C18:1) and hyroxyoleylcarnitine (C18:1OH) were signi¢cantly higher (p50.01 or less) in group A compared to the term infants. Less pronounced di¡erences were found in groups B and C compared to group D. Acetyl- (C2) and palmitoylcarnitine (C16) did not vary with gestational age. Conclusions: Leels of free and most acylcarnitines are signi¢cantly higher in preterm compared to term infants. Di¡erent normal ranges should thus be used in the screening of very immature (GA 528 wks.) neonates.

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THE MUTATIONAL SPECTRUM IN THE MCAD GENE OF NEWBORNS IDENTIFIED BY PROSPECTIVE TANDEM MS SCREENING FOR ``DIAGNOSTIC'' ACYL-CARNITINES IN BLOOD SPOTS DIFFERS FROM THAT OBSERVED IN CLINICALLY AFFECTED PATIENTS. B.S. Andresen1,2, S.F. Dobrowolski3, L.. O`Reilly4, P. Engel4, I. Knudsen1, R. Banas3, D. Chace3, E. Naylor3, N. Gregeren1. Research Unit for Molecular Medicine, Aarhus University Hospial1, Institute of Human Geetics, Aarhus University, Denmark2. NeoGen Screening, Pittsburgh, USA3. Department of Biochemistry, Univeristy of Dublin, Ireland4. MCAD de¢ciency (MCADD) is the most common defect of fatty acid b-oxidation, and it is potentially fatal. Because treatment of diagnosed patients may prevent/milden disease presentation, early diagnosis is important. Therefore new-born screening for MCADD has been initiated in several countries as pilot studies using tandem MS analysis for ``diagnostic'' acyl-carnitines in blood spots. Despite this, no validation of the method by genetic analysis has yet been performed. We have investigated 4250 clinically a¡ected patients with MCADD and identi¢ed and characterised 450 di¡erent mutations. Based on this knowledge, we have performed a validation of the tandem MS based new-born screening in several states of the US. So far, we have investigated all samples with an acyl-carnitine pro¢le indicative of MCADD from more than 700.000 individuals tested, and we have identi¢ed both known and unknown mutations. The severity of the mutations was evaluated by overexpression of recombinant protein. By comparing the mutational spectrum observed in clinically a¡ected patients to that observed in new-borns with elevated ``diagnostic'' acyl-carnitines in plasma a determination of the diagnostic speci¢city of the tandem MS method used for new-born screening for MCADD was possible.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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A HIGH RISK SCREENING ANALYSIS OF URINARY ACYLCARNITINES BY GC-MS WITH SOLVENT EXTRACTION AND ACYLOXYLACTONE DERIVATIVES. R.Giguere, D.Cyr, C.Auray-Blais. B.Lemieux University of Sherbrooke, Medical School, Genetic Service, Sherbrooke, Quebec,Canada.

The transesteri¢cation of long-chain fatty acids is essential in transporting molecules into the mitochondria. The carnitine palmitoyltransferase-I converts long-chain fatty acyl-CoA to acylcarnitines which can then enter into the mitochondria. The analysis of acylcarnitines can help us con¢rm the diagnosis of organic acidurias already obtained by organic acid analysis. Several methods have been described for the detection of acylcarnitines. The most successful approach relied on tandem mass spectrometry: fast atomic bombardment and electrospray ionisation. These methods require very expensive instrumentation not available in all laboratories. We have developed a simple and reliable method for the analysis of acylcarnitines by GC-MS o¡ering the advantage of easy implementation in any biochemical genetic laboratory, combining the speed of solvent extraction and the speci¢city of acyloxylactone derivatives. The method is based on a simple butan-1-ol extraction of a random urine sample acidi¢ed to pH 2, followed by derivatization of acylcarnitines into their characteristic acyloxylatones and analysis by GC-MS using selective ion monitoring mode. This simple and rapid solvent extraction method enables modest laboratories to utilize routine instrumentation like a benchtop GC-MS detection system. The proposed GC-MS method is an interesting alternative to the more powerful analysis of blood acylcarnitines by ESI-MS/MS.

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SUPPLEMENTAL NEWBORN SCREENING OF AMINOACIDS (AA) AND ACYLCARNITINES (AC) BY ELECTROSPRAY TANDEM MASS SPECTROMETRY (ESIMS/MS): EXPERIENCE IN ARGENTINA Abdenur JE, Chamoles NA, Schenone A, Guinle A, Fusta M, Gaggioli D. Fundacio¨n para el Estudio de las Enfermedades Neurometabo¨licas (FESEN). Buenos Aires, Argentina.

ESI-MS/MS analysis of AC and AA in blood spots is a rapid and accurate method for the diagnosis of several inborn metabolic diseases (IMD). We present the results of a 4 year (1996-1999) supplemental newborn screening program by ESI-MS/MS in Argentina. Methods: We analyzed 9320 newborn samples collected after the 2nd day of life, in ¢lter paper (S&S 903). Analysis of AC and AA, was performed by ESI-MS/MS (Millington et al), with a triple quadrupole VG QUATTRO instrument (Micromass, UK). Deuterated standards were obtained from Dr. ten Brink (Amsterdam). Results: We detected 5 IMD, one each with: MCAD, PKU, MAD, GA I and MSUD. Four of them had an a¡ected sibling. In the PKU and MSUD patients the diagnosis was clear prior to 24 h of age. All patients remain asymptomatic on treatment. Additionally, in 5 patients we retrospectively analyzed the neonatal samples (which had been obtained for the state newborn screening) after they were diagnosed with: VLCAD de¢ciency (15m, Reye syndrome), propionic acidemia (3m, ketoacidosis), citrullinemia (6d, coma), 3-MCC de¢ciency (4m, hypotonia) and HMG-CoA-lyase (6m, hypoglycemia and acidosis). In all cases the neonatal pro¢les were clearly diagnostic. No missed cases are known. Conclusion: ESI-MS/MS appears to be a reliable method for the diagnosis of several IMD in newborns screening programs. The high sensitivity of this technique will probably lower the impact of early specimen collection

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

14

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EVALUATION OF ELEVATED HYDROXYISOVALERYLCARNITINE (C5-OH) IN THE NEWBORN SCREEN BY TANDEM MASS SPECTROMETRY. W. E. Smith, D. S. Millington, P. S. Kishnani, M. McDonald and D. D. Koeberl. Dept. of Pediatrics/Div. Of Medical Genetics, Duke University Medical Center, Durham, North Carolina, USA. Tandem Mass Spectrometry (TMS) is a powerful diagnostic technique for newborn screening. Although very sensitive, TMS is not entirely speci¢c and the detection of abnormal but nondiagnostic metabolites is common. We evaluated the relevance of elevated C5-OH in the newborn screening TMS. Since the addition of TMS to the North Carolina Newborn Screen in April 1999, ¢ve infants have had persistently abnormal levels of 3-hydroxy-isovalerylcarnitine (C5-OH) on their whole blood spot acylcarnitine pro¢le. A plasma acylcarnitine pro¢le and urine for organic acids was obtained on all ¢ve infants: four infants had variable amounts of 3-hydroxyisovaleric acid in the urine and two of these infants had increased plasma levels of C5-OH. The latter two infants had elevations of both 3-hydroxyisovaleric acid and 3-methylcrotonylglycine in their urine, consistent with 3methylcrotonyl-CoA carboxylase (3-MCC) de¢ciency. Urine organic acid analysis for the mothers of these two patients was normal. One infant has been diagnosed with 3-MCC de¢ciency; her enzyme activity was 43% of control activity in lymphocytes and 2% of control activity in ¢broblasts (UCSD Biochemical Genetics Laboratory). Therefore, of the ¢ve infants with elevated C5-OH identi¢ed by TMS newborn screening in North Carolina, one has been diagnosed with 3-MCC de¢ciency and a second infant has been identi¢ed with probable 3-MCC de¢ciency. Absolute levels of C5-OH on newborn screening were not predictive of a ¢nal diagnosis. Evaluation of newborns with persistently elevated C5-OH levels should include at least urine organic acid analysis and a plasma acylcarnitine pro¢le.

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SPECTRUM OF EXPRESSION OF MCAD AND SCAD DETECTED BY NEWBORN SCREENING. D Marsden, T Zytkovicz, C Larson, V Shih, G Grady New England Newborn Screening Program, Boston, MA, USA

Eleven ``candidate'' cases of MCAD have been detected among the ¢rst 108,000 newborns screened by MS/MS in Massachusetts (*100,000 births) and Maine (*8,000 births). Octanoylcarnitine (C8) above a population-based cuto¡ of 0.5 uM, and accompanied by elevations of C6, C10, or C10:1, was the primary determinant in the initial specimen obtained typically on day 2 of life. Because repeat specimens, typically from 5 to 9 days after birth, showed diminished C8 values, some falling below the cuto¡, the A985G DNA marker was assayed. Two copies of the marker were present in the one baby who had the highest C8 (11 uM), but one copy was present in most others (8/10). One case has been shown now to have a second unique mutation. Urine organic acid analysis in two single-copy babies was consistent with MCAD. Twelve ``candidate'' cases of SCAD with elevated levels of butyrylcarnitine (C4 4 1.9 uM) were identi¢ed. The C4 was diminished on the repeat specimens. Urine organic studies con¢rmed the 4 SCAD cases. The one SCAD with DNA to date, has been shown to be homozygous for the G625A mutation. In vitro oxidation studies on a second case were consistent with SCAD. CONCLUSION: The incidence of SCAD in the Massachusetts population may be higher than previously thought. The optimal time to identify infants with MCAD or SCAD is within the ¢rst few days of life.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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15

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PREVALENCE OF FATTY ACID OXIDATION DISORDERS AND ORGANIC ACIDEMIAS IN NEW ENGLAND NEWBORNS SCREENED BY TANDEM MASS SPECTROMETRY. D Marsden, T Zytkovicz, C Larson, V Shih, G Grady New England Newborn Screening Program, University of Massachusetts, Boston, MA, USA

Newborn screening by tandem mass spectrometry for MCAD and 19 other rare metabolic disorders was introduced in Massachusetts in February 1999 and screening for MCAD in Maine in September 1999, in addition to the traditional screen. 2 MCAD cases were detected among the ¢rst 8000 Maine newborns screened and 2 among 100,000 Massachusetts newborns screened. Two additional MCAD ``candidate cases'' are under investigation. Of the 4 con¢rmed MCAD cases, one was homozygous for the A985G mutation; 3 were heterozygous for this mutation: 1 so far has now been shown to have a second unique variant. Additional disorders in the 100,000 Massachusetts newborns include the folowing: 4 SCAD, 1 LCAD, 2 PA, 1 MCC, and 1 CPT-II. The CPT-II had the severe neonatal form with multiple organ anomalies and expired on the day the newborn specimen was drawn [day four of life]. No tyrosinemia or urea cycle defects were identi¢ed in the population screened. The positive predictive value was about 20% for the fatty acid oxidation defects and about 10% for the organic acidemias. Conclusion: Newborn screening by tandem mass spectrometry in New England for fatty acid oxidation defects and organic acidemias yields a prevalence of approximately 1:10,000.

030-O

DEFECTS IN THE CARNITINE CYCLE DETECTED BY NEWBORN SCREENING USING TANDEM MASS SPECTROMETRY Bridget Wilcken, Veronica Wiley, Keow Giak Sim, Kevin Carpenter. NSW Newborn Screening & Biochemical Genetics Service, New Children's Hospital, Sydney, Australia.

In our ongoing investigation of the uses of tandem mass spectrometry in newborn screening we recorded free carnitine levels in dried blood samples from 149,000 newborns undergoing routine newborn screening., We retrospectively analysed newborn screening samples from three babies with clinically diagnosed carnitine transporter defect (CTD) and one with carnitine palmitoyl transferase I de¢ciency (CPT). Whole blood free carnitine levels did not di¡er markedly between days 2 to 8 (median 26.6 to 27.5 mmol/L). 0.65% of values were below 10 and 0.06% below 7mmol/L. 0.12% were 4 100mmol/L. Low birth weight babies had higher values. Action limits for recall adopted initially were 5 and 120mmol/ L. One baby had free carnitine of 4 mmol/L and was subsequently shown to have carnitine transporter defect. Only one of three patients previously diagnosed clinically had an unequivocally low free carnitine in the newborn sample, which would have been diagnosable by newborn screening. The baby with CPT l de¢ciency had free carnitine of 160 and 154mmol/L on days 1 and 4. Only 1 other newborn sample had a level over 136mmol/L, a baby with sepsis. Action limits for free carnitine could be modi¢ed, but this would increase the recall rate needed to diagnose few cases of very rare diseases. Because there are over 20 diagnostic primary analytes in MSMS screening, recall rates for each must be kept low. Experience of many cases is needed before the cut-points for recall can be optimised.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

16

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NEWBORN SCREENING FOR MCAD DEFICIENCY: THE NEW SOUTH WALES EXPERIENCE Kevin Carpenter, Veronica Wiley, Keow Giak Sim, Judith Hammond, Daphne Heath, Bridget Wilcken. NSW Newborn Screening & Biochemical Genetics, New Children's Hospital, Sydney. Medium chain acyl-CoA dehydrogenase de¢ciency (MCAD) is a good example of a disorder with signi¢cant morbidity and mortality, which may be detected presymptomatically by newborn screening using tandem mass spectrometry. In Australia, the carrier frequency for the common mutation, A985G, is 1:80 implying a birth prevalence of 1:30,000 (95% CI 1:12,500 ^ 1:105,000). Retrospective analysis of newborn screening cards from 13 patients diagnosed clinically with MCAD revealed all but one to have octanoylcarnitine (C8) concentrations in excess of 1mmol/L (2.4 ^ 6.8). Since April 1998, we have prospectively analysed 160,500 consecutive newborns. During this period only 13 babies had C8 levels 41mmol/L. All were analysed for the common mutation, 7 did not carry 985G on either allele, 6 of whom had normal acylcarnitines on repeat testing (one infant died soon after birth). One baby was homozygous and 2 heterozygous for the common mutation with urinary markers and/or ¢broblast fatty acid oxidation studies consistent with MCAD. Three infants were heterozygous for the common mutation. Two had intermittent and mildly abnormal urinary acylglycines and atypical tritium release fatty acid oxidation studies. ETF based assays (Dr Saban, Lyon) show one to have classical MCAD and the other to fall into the heterozygous range. Further molecular studies have so far failed to reveal a second mutation in the MCAD gene. We conclude that newborn screening with tandem mass spectrometry may not reveal all MCAD patients and may detect atypical cases whose risk of developing clinical problems is unknown.

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TWO CASES OF CONGENITAL PORTOSYSTEMIC SHUNT HAVING HYPERGALACTOSEMIA AT THE TIME OF NEONATAL SCREENING A. Ohtake, K. Murayama, A. Kimura, S. Ishii, K. Kawada, H. Niimi, N. Sasaki Department of Pediatrics, Saitama Medical School, Moroyama, Saitama 350-0495, Japan

The manifold etiology exists for the patient whose hypergalactosemia is indicated by neonatal mass screening. With improvements in diagnostic imaging techniques, the number of infants with documented portosystemic shunt (PSS) has increased. We report two cases of PSS (multiple hepatic hemangiomata and patent ductus venosus) discovered by the mass screening program. Patient 1 was a female infant who admitted to our hospital at 21 days of age. At 2 days of age, di¡use cutaneous hemangiomata had been noticed. Hypergalactosemia was indicated by the mass screening at 4 days of age. Liver functional impairment with the lowering of the clotting factors was found out. Elevation of blood ammonia and serum total bile acid levels were also noticed. In the abdominal MRI image, ¢ve hepatic hemangiomata of TI low brightness and T2 high brightness were recognized. Prednisolone was administered. At 1 year and 10 months old, only one collapsed hemangioma remains. Patient 2 was a female infant who admitted to our hospital at 36 days of age. Hypergalactosemia was indicated by the mass screening. Liver functional impairment with the lowering of the clotting factors, elevation of blood ammonia and serum total bile acid levels were also noticed. With imaging techniques, abnormal shunt blood vessel which seemed to be an Arantius duct was found out. Spontaneous closure of the shunt vessel would occur. CONCLUSION: For many patients, neonatal screening for hypergalactosemia provides an important opportunity for diagnosis of PSS.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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EVALUATION OF DHPLC AS A SCREENING TOOL FOR GLYCOGEN STORAGE DISEASE TYPE 1 NON-A Rischewski, J.1, Schneppenheim R.1, Block G.2, Kinner M.2, Wendel U.3, Schaub J.2, Santer R.2 Dpmt of Paediatric Haematology and Oncology, University Childrens`s Hospital, Martinistr. 52, D20246 Hamburg, Germany(1) ; University Children`s Hospitals Kiel (2) and DÏsseldorf (3)

Objective: Mutations in the gene of the microsomal glucose-6-phosphate transporter (G6PT1) have been found in glycogen storage diseases type 1 non-a (GSD1non-a). In the past, we have shown that 17 of 21 german patients (80%) with GSD1non-a carry at least one exon 8 mutation. We now compared speci¢c mutation prediction by denaturating high pressure liquid chromatography (DHPLC) in a blinded fashion to direct sequencing, in order to evaluate DHPLC as a possible routine diagnostic tool for the molecular diagnosis of GSD1non-a. Methods: DNA extraction from 21 unrelated GSD1 non-a patients and 24 parents. PCR ampli¢cation of exons 1 to 6, 8 and 9 of G6PT1. Sequencing of all products. Cloning and sequencing of exon 8 products containing deletions. Optimization of DHPLC (WAVE, Transgenomic) analysis conditions using exon 8 PCR products previously sequenced. PCR and DHPLC analysis of 10 DNA samples in a blinded fashion. Mutation prediction from the retention pattern. Comparison with the results of direct sequencing. Results and conclusion: The retention pattern determined by DHPLC allowed speci¢c mutation prediction in all tested cases. Since DHPLC is less time consuming and cheaper than sequencing, these results favor the application of this method for routine G6PT1 gene mutation screening.

034-P

DHPLC AS A MASS SCREENING TOOL FOR HUMAN FRUCTOSE INTOLERANCE Rischewski J.; Weihe v. M., Schneppenheim R. Department of Paediatric Haematology and Oncology University Childrens`s Hospital, Martinistr. 52, D- 20246 Hamburg, Germany

Objective: Human Fructose Intolerance (HFI) is caused by a de¢ciency of aldolase B in liver, kidney and small intestine. Early diagnosis can avoid dietary or inadvertent iatrogenic fructose ingestion or infusion with possible deleterious consequences. Molecular diagnosis has become the method of choice. We established a partially automatized high throughput screening method to detect the most prevalent mutations in Europe in a Duplex-PCR using Denaturing High Pressure Liquid Chromatography (DHPLC) (WAVE, Transgenomic). We compared mutation prediction to sequencing to evaluate DHPLC as a tool for newborn screening and diagnostic mutation detection. Methods: Duplex-PCR ampli¢cation of exon 5 and 9 of the aldolase B gene from HFI patients carrying mutations in exon 5 (A150P, A175D), exon 9 (N335K), compared to wildtype (WT) DNA. Mixing with WT-PCR products and heteroduplex induction. Optimization of DHPLC analysis conditions to allow for mutation speci¢c detection in two subsequent runs. Comparison with the results of direct sequencing. Results and conclusion: The DHPLC-retention pattern allowed speci¢c mutation prediction in all homo-, hetero- and compound- heterozygous samples. Hands-on-time is only required for PCR, heteroduplex-induction, and evaluation of DHPLC result ¢les. DHPLC proved to be a sensitive and mutation speci¢c screening method for HFI.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

18

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ENZYME-BASED SCREENING FOR BETA-THALASSAEMIA E Escuredo1, JA Duley1, JB CIegg2, DJ Weatherall2, DC Rees2,3 1 Purine Research Laboratory, Guy's and St Thomas' Hospitals NHS Trust, London; 2MRC Molecular Haematology Unit, Institute of Molecular Medicine Oxford; 3Royal Hallamshire Hospital, She¤eld. Beta-thalassaemia as a public health problem and its control relies on ante-natal screening, with fetal diagnosis and selective termination where appropriate. Screening is problematic. Although increased levels of HbA2, are sensitive and speci¢c, the assay is complex and expensive, relying on electrophoresis or chromatography, while osmotic fragility tests are unreliable. Activity of the red cell enzyme pyrimidine 5'-nucleotidase (P5N) has previously been shown to be low in betathalassaemia. We have developed a new test, using the ratio of activities of the two isoenzymes, P5N-1 and P5N-2. Methods: We tested 43 healthy (HbA) individuals, 15 alpha-thalassaemics (1or 2 gene deletions on Southern blotting), 22 beta-thalassaemia traits (with high HbA2), 3 HbE traits, 4 HbE homozygotes, and 15 unspeci¢ed iron-de¢ciencies. Haemolysates were incubated with UMP (P5N-1) and deoxyUMP (P5N-2), generating uridine or deoxy-uridine which were quantitated by HPLC. The ratio of P5N-1/P5N-2 was calculated. Results: All cases of beta-thalassaemia (including HbE) had a ratio of 50.7. Of the iron de¢cient cases, 9/15 had a ratio of 50.7. All cases of HbA and 11/15 alpha-thalassaemics had ratios 40.7. Conclusions: The test was sensitive and speci¢c for beta-thalassaemia. Iron de¢ciency should be excluded ¢rst. The assays generate phosphate and have the potential for use in autoanalysers or in a simple kit for use in developing countries, allowing rapid mass screening. It may also be useful to assess atypical cases of beta-thalassaemia, such as those with normal levels of HbA2.

036-P

A SCREENING ASSAY FOR GLUCOSE-6-PHOSPHATE DEHYDROGENASE DEFICIENCY Than K-A1, Snodgrass M1, Padilla C2. 1 PerkinElmer Wallac, Inc., 3985 Eastern Road, Norton, OH 44203 USA; 2UP-Philippines General Hospital, Taft Avenue, Manila 1000, Philippines. This assay is designed for the determination of Glucose-6-Phosphate Dehydrogenase (G6PD) activity in dried blood spots collected on ¢lter paper. It is a £uorometric assay, which calculates the G6PD activity (U/gHb) from a standard curve. The assay is performed at room temperature, and semiquantitative results are produced in under an hour. The within-run SD (CV) for samples below, near and above normal values were 0.08 (10%), 0.19 (5.8%) and 0.28 (3.9%) respectively. The assay is linear from 0 6 U/gHb. An evaluation of the Wallac assay was conducted on 2397 neonatal samples in the Philippines. There is good correlation between our assay and the Formazan and BoehringerMannheim (B-M) assays. Of 112 samples tested by all three methods, there was agreement on 41 normal and 50 de¢cient samples. Of the 21 unmatched results, 15 samples appear to be in the equivocal zone of both the Wallac and Formazan methods; the Wallac and Formazan results agreed on 6 samples which were not matched by the B-M method. The distribution of the 2397 samples appeared normal. At a cut-o¡ of 1.5 U/gHb for the Wallac assay, 154 samples were determined de¢cient; the Formazan method, at a cut-o¡ of 6.1 mm, determined 116 samples de¢cient. The Wallac results for 38 samples did not match the Formazan method. Of these, 26 were at 1.6 to 3.0 U/gHb by the Wallac assay and 12 were 3.1 U/gHb and above. Simple and fast with a high throughput, this assay is suitable for screening neonates for G6PD de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

19

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DNA-BASED NEWBORN SCREENING FOR HEPATIC CARNITINE PALMITOYL TRANSFERASE 1 (CPT1A) DEFICIENCY IN A TARGETED POPULATION OF MANITOBA, CANADA: A PILOT PROJECT C Prasad1, 2, JP Bonnefont3, 4, LA Dilling1, L Thuillier3, C Prip-Buus5, R Singal2, JRG Thompson6, JC Haworth1 and CR Greenberg1, 2 1 Pediatrics and Child Health & 2Biochemistry and Medical Genetics, University of Manitoba, 6 Cadham Provincial Laboratory, Winnipeg, Canada, 3Genetic Biochemistry Unit and 4Unit INSERM U393, CHU Necker, Paris, 5Ceremod CNRS UPR 1524 MEUDON CPT1A de¢ciency is a rare autosomal recessive disorder of mitochondrial beta-oxidation, which can be fatal if undiagnosed. We hypothesize an increased carrier frequency of *1/15 based on 1/1000 a¡ected infants and a birth rate of *250/year amongst Manitoba Hutterites (a genetic isolate). A novel homozygous mutation G?A transition at nt 2129 predicting a Gly710?Glu (G710E) substitution has been identi¢ed in the Hutterites {Abadi N et al 1999}. Objective: To determine the true carrier frequency of this unique CPT1A mutation among Manitoba Hutterites, a one-year pilotscreening program was begun after obtaining ethics and community approval. Methods: Consecutive Hutterite newborns are identi¢ed by their unique surnames. DNA extracted from a 3mm-diameter disk punched from the newborn screening card (coded) is ampli¢ed by PCR and submitted to restriction digest using BslI, with the G710E mutation suppressing a BslI restriction site. Results: Of 47 samples screened to date (since Feb. 1, 2000), 4 carriers have been identi¢ed. Conclusions: Preliminary results suggest a carrier frequency of *1/12. The ¢ndings support inclusion of CPT 1 de¢ciency in the newborn screening program for the Manitoba Hutterite population. Ongoing education and counselling is provided to the community as part of this newborn screening program.

038-O

RAPID DIAGNOSIS OF LYSOSOMAL STORAGE DISORDERS BY TANDEM MASS SPECTROMETRY P.D. Whit¢eld, P. Sharp, D. Johnson, R. Taylor1, J. Fletcher, J.J. Hopwood and P.J. Meikle Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, South Australia, Australia and Department of Animal Science, University of Sydney, Sydney, New South Wales, Australia1

Lysosomal storage disorders (LSD) are a group of genetic diseases which result from a defect in the activity of speci¢c lysosomal hydrolases. A number of LSD are characterised by the storage of glycosphingolipids. A¡ected individuals su¡er visceral, skeletal and neurological abnormalities and have a reduced life expectancy. We have developed rapid, sensitive and speci¢c methods using electrospray ionisation^tandem mass spectrometry (ESI-MS/MS) for determining glycosphingolipid concentrations in various body tissues from LSD patients. Glycosphingolipids are extracted, isolated using solid-phase extraction procedures and then directly analysed by ESI-MS/MS. Central nervous system (CNS) tissues, skin ¢broblasts, urine, plasma and dried blood spots have been analysed. In comparison to age-matched controls we have demonstrated signi¢cant elevations of glucosylceramide in Gaucher disease, ceramide trihexoside in Fabry disease, sulphatide (galactosylceramide sulphate) in metachromatic leukodystrophy and galatosylsphingosine (psychosine) in Krabbe disease. It is anticipated these methods will prove useful in the diagnosis, screening and therapeutic monitoring of LSD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

20

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039-P

FOLLOW UP EVALUATION OF 50 ADULTS WITH PHEYNYLKETONURIA (PKU) WHO WERE ENROLLED IN THE COLLABORATIVE STUDY OF CHILDREN TREATED FOR PKU (1968^84)* R. Koch1, J. Coldwell2, R. Peterson3, W. Rhead4, B. Rouse5, J. Wol¡6, A. Stern1, F. Guttler7, and C. Azen1 Childrens Hospital Los Angeles and The University of Southern California School of Medicine1, Children's Medical Center, Tulsa, Oklahoma2, San Diego Regional Center, San Diego, California3, Department of Pediatrics, University of Iowa, Iowa City, Iowa4, Department of Pediatrics, University of Texas Medical Branch, Galveston, Texas5, Waisman Center on Mental Retardation & Human Development, University of Wisconsin6, John F. Kennedy Institute, Glostrup, Denmark7 From 1968^84, 211 newborns were enrolled in a 12-year study of children treated for PKU. By the end of the study, only 111 children had completed the protocol outlined for the project. This 16-year follow-up was stimulated by the ongoing controversy of whether to continue a phenylalaninerestricted diet into adulthood or to discontinue or to simply relax the diet by allowing higher blood phenylalanine levels in adulthood, compared to childhood. On follow-up, a medical history, physical examination, career accomplishments, current WAIS-R, blood phenylalanine and mutations of the phenylalanine bydroxylase gene were obtained. While the study is not completed, the following observations appear useful. Only 9 of the 50 have remained on a phe-restricted diet. While WAIS-R data show that IQ is preserved, depression occurred in 8, phobias in 10, panic attacks in 5, hyperactivity in 4, and convulsions in 1. These problems a¡ected employment and social achievement. *Funded by NICHD, Bethesda, MD. Gratitude is expressed to Felix de la Cruz, M.D., Chief of the Mental Retardation and Development Disabilities Branch for support.

040-P

RESULTS OF THE INTERNATIONAL MATERNAL PKU STUDY R. Koch1, W. Hanley2, H. Levy3, R. Matalon4, B. Rouse4, F. Trefz5, K. Matalon4, L. Platt6, F. Guttler7, E.G. Friedman1, and C. Azen1 Children's Hospital Los Angeles and The University of Southern California School of Medicine1, The Hospital for Sick Children, Toronto, Ontario, Canada2, Children's Hospital, Boston, Massachusetts3, Child Development Division, University of Texas, Galveston, Texas4, Universitat Tubingen, Reutlingen, Germany5, Cedar^Sinai Medical Center, Los Angeles, California6, John F. Kennedy Institute, Glostrup, Denmark7 The Maternal PKU Study began in 1984 and enrolled 572 pregnancies in women with hyperphenylalaninemia (HPA) plus 99 controls. 26% of the women with HPA, who delivered live neonates, began a phe-restricted diet before conception, 36% by 8 weeks of pregnancy, and 38% thereafter. The recommended treatment range was 120^360 micromoles/liter. Microcephaly and Congential Heart Disease (CHD) did not occur in the pregnancies that were in control by 8 weeks of pregnancy, but occurred in 27% and 12% respectively, in those not in control by 10 weeks of pregnancy. The mean IQ of the mothers on enrollment measured by the WISC-R was 83. In women with two severe phenylalanine hydroxylase (PAH) mutations, it was 80. The mutational status of mothers was directly correlated to the assigned phe level documented at entry into the study; the more severe the mutations of the PAH gene, the higher the assigned phe level and the lower the mean maternal IQ. Therefore, PKU women with two severe PAH mutations need closer follow-up during the child bearing years and intensive services for family planning. Counseling for the latter should begin with the onset of adolescence. NICHD Contract Numbers: N01-HD-2-3148, N01-HD-2-3149, N01-HD-2-3155, and N01-HD-3156. Grant: National Health Research & Development Program, Ottawa, Number 6606-3265

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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21

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CONGENITAL HEART DISEASE IN MATERNAL PHENYLKETONURIA: REPORT FROM THE MATERNAL PKU COLLABORATIVE STUDY H.L. Levy1, P. Guldberg2, F. Guttler2, W.B. Hanley3, R. Matalon4, B.M. Rouse4, F. Trefz5, C. Azen6, F. de la Cruz7, R. Koch6 Children's Hosp., Boston1; John F. Kennedy Inst., Glostrup, Denmark2; Hospital for Sick Children, Toronto3; Univ. of Texas, Galveston4; Children's Hosp., Reutlingen, Germany5; Children's Hosp. Los Angeles6; National Inst. of Child Health and Human Dev., Bethesda7

Congenital heart disease (CHD) is known to occur in 12^15% of o¡spring from untreated maternal phenylketonuria (PKU) pregnancies. We examined the e¡ect of treatment and the phenylalanine hydroxylase (PAH) genotype on CHD in the Maternal PKU Collaborative Study (MPKUCS). The 416 o¡spring from 412 maternal PKU pregnancies in the MPKUCS that produced live births and the 100 o¡spring from 99 control pregnancies were included in this examination. Metabolic control was de¢ned as a blood phenylalanine (phe) level 5 600 mmol/L (10 mg/dL). Among the 235 pregnancies in women with a basal phe level 4 900 mmol/L and not in metabolic control by the eighth gestational week, 34 had CHD, a frequency of 14% compared to 1% (1/100) among control o¡spring. Patent ductus arteriosus, coarctation of the aorta, tetralogy of Fallot and hypoplastic left heart were overrrepresented as compared to CHD in the population. A basal maternal phe 1500 mmol/L signi¢cantly increased the risk for bearing a child with CHD (P = 0.004). PAH mutations in the mothers and o¡spring did not have an independent relationship to CHD. The data indicate that women with the most severe degree of PKU are at highest risk for bearing a child with CHD, that PAH mutations do not have an independent e¡ect on the occurrence of CHD but act through determining the basal phe level in the mother, and that prevention requires initiation of diet before conception or su¤ciently early in pregnancy for metabolic control by the eighth gestational week.

GROWTH OF PHENYLKETONURIC (PKU) CHILDREN UP TO TWO YEARS L. Fiori, E. Verduci, ML. Giann|© , L. Colombo, E. Riva, S. Scaglioni, M. Giovannini Department of Pediatrics, San Paolo Hospital, I-20142 Milan, Italy.

042-P

Background. The semisynthetic diet for PKU children after diagnosis may be limited in some essential nutrients. Aim. To check the growth indices of treated PKU children in the ¢rst two years of life. Subjects and methods. 25 term PKU infants followed in the 1994-1998 period. Comparison with reference groups of either formula-fed (FF, n=65) or breastfed (BF, n=73) healthy term infants through z-scores of the World Health Organization. Statistical analysis with non-parametric tests. Results. Considering treated PKU infants as an arti¢cially-fed population from very early ages, we have compared them with the FF reference group. PKU showed lower (P50.05) z-scores for weight at 3, 4, 6, 9, 12, 18 and 24 months and for length at 18 and 24 months. We have then compared the growth indices of PKU exclusively breastfed up to diagnosis (n= 11) with those of BF infants to eliminante any environmental bias. PKU subjects (tendencially lighter at birth, P=0.12) showed lower z-scores for weight at 1, 2, 3, 4, 6, 12, 18 and 24 months and lower z-scores for length at 3 months. Conclusions. The dietary treatment of PKU subjects is associated with depressed growth indices in the ¢rst 24 months of life. Even considering those initially breastfed, their growth is limited compared to a reference BF group. Since the nutrient supply and growth rate of healthy BF infants is characteristically limited compared to those of healthy FF counterparts, our ¢ndings show that well-controlled PKU infants have lower growth indices in the ¢rst 24 months of life. The dietary treatment may be a major cause, but the trend of a weight di¡erence already present at birth generates the hypothesis of some negative in£uences on fetal growth in utero

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

22

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043-P

THE SEARCH FOR RANDOMISED CONTROLLED TRIALS IN PHENYLKETONURIA 1 Poustie VJ, 1Beaven OJ, 2Rutherford P, 1Smyth RL. 1 Institute of Child Health & 2Department of Dietetics & Nutrition, Royal Liverpool Children's Hospital, Liverpool, L12 2AP, UK. Introduction: A comprehensive search for randomised controlled trials (RCTs) in phenylketonuria was undertaken for the Cochrane Cystic Fibrosis (CF) & Genetic Disorders Group register of possible RCTs and controlled clinical trials (CCTs). Methods: The Cochrane Controlled Trials Register and Medline were searched to identify relevant trials. The Journal of Inherited Metabolic Disease was hand searched from its inception (1978) to the end of 1998. Manufacturers of dietetic products and experts in the ¢eld of metabolic disease were contacted for information on ongoing or unpublished trials, and the conference proceedings of the SSIEM (1981-2, 1987-1991, 1995, 1998) were also hand searched. Results: A total of 58 references to RCTs and CCTs were identi¢ed, 37 from searching the electronic databases and a further 21 were found during the hand searching process. No unpublished trials were identi¢ed by the manufacturers of dietetic products or by the expert panel. Conclusions: If a review of the literature is restricted to searching for RCTs and CCTs on electronic databases, it is likely that a number of eligible trials will be missed. It is therefore recommended that other searching strategies are employed, for example, hand searching conference proceedings or relevant journals. The Cochrane CF & Genetic Disorders Group register holds a comprehensive collection of RCTs and CCTs identi¢ed using these methods.

044-P

EVIDENCE FOR NEURON APOPTOSIS INDUCED BY PHENYLALANINE X.F. Gu, X.W. Yang, R.G. Chen Xin Hua Hospital, Shanghai Second Medical University and Shanghai Institute for Pediatric Research, China Objective: This study is to investigate the neurotoxicity of hyperphenylalanine and to explore the possible mechanisms. Methods: The primary cultured cortical and hippocampal neurons of embryonic rats were cultured in Neurobasal, exposed to hyperphenylalanine (Phe) and assayed the survival rate. The morphological changes were observed by special staining. Bcl-2, fas and c-fos were tested by immunohistochemistry and RT^PCR. Results: The survival rate of cortical neurons was signi¢cantly decreased compared to normal controls in present Phe at 300 mmol/L, 600 mmol/L, 1200 mmol/L and shown a dose dependent. Positive apoptotic neurons increased with the concentration of Phe by TUNEL staining and observed under electronic microscope. The positive neurons of fas and c-fos increased than normal control by immunohistochemistry with the exception of bcl-2. The mRNA expression of c-fos increased accordingly while that of bcl-2 decreased. Conclusion: Apoptotic neurons were induced by hyperphenylalanine. The abnormal expression of some genes may hasten the neuronal apoptosis, such as the upregulation of fas and c-fos genes and the downregulation of bcl-2.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MAGNETIC RESONANCE IMAGING STUDY OF BRAIN MYELINATION IN PATIENTS WITH UNTREATED PHENYLKETONURIA Z.S. Zhou, W.M. Yu, X.Z. Zhang and W. Wang China^Japan Friendship Hospital, Beijing 100029, China

Objective: To investigate the abnormalities of brain myelination in untreated phenylketonuria (PKU) patients by using magnetic resonance imaging (MRI). Methods: 36 patients with untreated PKU were divided into two groups based on having seizure or not. 15 cases with seizure and 21 without seizure were included in the group one and group two separately. The myelination in ten sections of brain were evaluated by using MRI, T1. WI and T2 WI with Staudt's standard of brain myelination in healthy children. Results: Of 36 patients with PKU, delayed myelination was detected in 28 cases (78%). Delayed myelination on MRI is that the myelin manifesting hyperintense on T1 WI and hypointense on T2 WI had not yet reached the area which normal myelination should reach at certain ages. Delayed myelination was located mainly in the cerebral lobes and corpus callosum. There existed abnormal hyperintense foci in white matter of peritrigonal and periventricular regions in 33 of all 36 patients with untreated PKU. Delayed myelination was more apparent in the group with seizure than in the group without seizure (100% vs 62%). The occurrence frequency of severe mental retardation in the group with seizure was higher than that in group without seizure (53% vs 19%). Conclusion: The patients with untreated PKU had a high occurrence of the delayed brain myelination. The delayed myelination may be one of the reasons responsible for seizure and mental retardation. MRI plays an important role in the diagnosis of delayed myelination in PKU patients.

EXPERIENCE WITH BREASTFEEDING IN PHENYLKETONURIC BABIES Chiesa, A, Keselman A, Fraga C, Prieto L, Pardo ML, Gru·eiro de Papendieck L. Fundacio¨n de Endocrinolog|¨ a Infantil (FEI). Buenos Aires. Repu¨blica Argentina.

046-P

Introduction Breastfeeding can be a successful technique to manage PKU infants, especially in those countries where mothers are encouraged to breastfeed in order to prevent infectious diseases. Objective: To show our experience with breastfeeding in comparison to arti¢cial lactation in PKU babies.Material and methods: PKU according to our ¢ndings in the neonatal screening program has in our country an incidence of 1:15678. Retrospective data are shown from 22 PKU babies with adequate metabolic control. In 12 (G1) diagnosis was con¢rmed at a mean age of 24 days. They were treated with breastfeeding and Analog XP according to Greve et al guidelines till their weaning at a mean age of 6.5 months. The other 10 PKU children (G2) were weaned after diagnosis (29 days) and received phenylalanine free products and formula with known amounts of phe. Time of achievement of metabolic control (phe 5480 mmol/l), height, weight and head circumference (HC) were compared till age 2 in both groups and till age 4 in 6 patients of G1 and 8 of G2. Maturation scores (CD) obtained with Gessell test were compared till age 3 and WIPSI scale (IQ) at age 4. Results: There were no signi¢cant di¡erence between breastfed an bottle-fed babies in the studied variables. CD at 0.5, 1 , 2 and 3 years of age and IQ at four were: G1:100þ8, 102þ7 ,100þ10,104þ9 and 110þ9 ; G2: 100þ7, 101þ11, 97þ8, 98þ6 and 104þ14 without di¡erences between groups. Breastfeeding required more e¡ort in the professional team to support the mother early in lactation giving the family more time to elaborate the arrival of a PKU baby with a better bonding. Conclusion: Breastfeeding was a useful technique in PKU management in our country, being safe, economic and e¤cient for the good control and normal growth and development of these children, improoving these families' quality of life.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

24

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047-P

CARNITINE STATUS IN PHENYLKETONURIC PATIENTS ON DIETARY TREATMENT IN CHILE G.P. Dura¨n, V. Cornejo, A. Valiente, L. Mun¬oz, E. Raimann INTA, Universidad de Chile. Macul 5540, Santiago, Chile Low serum carnitine concentration in phenylketonuric patients on diet has been reported previously, the decrease of level could be produced because low dietary intake, de¢ciency of the precursors of the endogenous synthesis or increase of urinary loss. The Chilean neonatal screening program for PKU started in 1992 and our local reality is not homologabily to developed countries. For this we evaluated the carnitine, methionine, lysine and ferritine level in 27 PKU children (15 female and 12 males) aged 9 months to 7 years (average 3.0) and 25 healthy age and sex matched controls. Their protein intake was estimated by a 24-hour recall of the three previous days. Students' t test was used for the statistical analysis. Results: there was no statistical di¡erence in the total, free and steri¢ed carnitine and esteri¢ed carnitine/free carnitine ratio. Serum methionine and lysine were in the normal range and there was no di¡erence in the ferritine levels. Protein intake of the control group was statistically higher than PKU group, protein from vegetables was above 25% in both groups. The 75% of protein intake in PKU patients came from free-phe amino acid mixture not forti¢ed with carnitine. We conclude that despite low dietary intake of carnitine its levels remain normal in this group, maybe because a good availability of the amino acid and cofactor for the endogenous synthesis. We note that it is necessary to re-evaluate these patients a second time, looking for a long-term e¡ect of the PKU diet.

048-P

RAPID PSYCHOTIC DETERIORATION AFTER COMING OFF THERAPY IN AN ADULT PATIENT WITH PHENYLKETONURIA Ichiro Yoshida, Shigeto Yamada, Kumiko Aoki, Takahiro Inokuchi, Kyoko Tashiro, Satomi Tashiro, Misa Jogo, and Masatoshi Tanaka Research Institute of Medical Mass Spectrometry, Brain Institute, Kurume University, School of Medicine The patient is a 35-year-old female. At 2 years 6 months old, she was referred to hospital because of delayed psychomotor development and had been on diet therapy since that time. She stopped the diet therapy at 15 years old. At 35 years old, she developed auditory hallucination, psychomotor excitement, and paranoic thought. At the time, her serum levels of phenylalanine and its metabolite, phenylacetic acid (PAA) were 24 mg/dl and 1322 ng/ml, respectively which were signi¢cantly higher than the normal levels. The plasma levels of homovanilic acid (HVA), 3-methoxy-4-hydroxyphenylglycol (MHPG), and beta-phenylethylamine (PEA) were lower than the control levels. Reintroduction of low phenylalanine diet caused dramatic improvement of the psychotic symptoms which were associated with the reduction of Phe and PAA level and with the increase in the plasma levels of HVA, MHPG and PEA. These data suggest that the psychotic symptoms were not caused by the hyperdopaminergic mechanism or high level of PEA which structure was similar to methamphetamine. The normalization of the BCAA to Phe ratio might have contributed to improvement of the psychotic symptoms.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BONE STATUS OF PHENYLKETONURIC MICE IS NEGATIVELY AFFECTED BY ELEVATED PLASMA PHE CONCENTRATIONS S. Yannicelli and D.M. Medeiros The Ohio State University, Columbus, Ohio, USA

Children with PKU are at risk of fractures. This study used a phenylketonuric (PKU) murine model (PAHenu-2) to evaluate e¡ects of moderate dietary protein restriction and elevated plasma PHE concentration on bone integrity. Fifty-four male weanling PKU and control mice (BTBR) were assigned to either an elemental PHE-restricted diet (PHR) or PHE-unrestricted diet (PHU) with low or normal protein levels for 56 days. PHU and control mice received equal amounts of dietary PHE; PHR mice consumed prescribed dietary PHE to maintain plasma PHE concentrations between 120^ 480 mmol/L. Plasma PHE, osteocalcin (bone formation marker), and urine deoxypyridinoline (DPD/ creatinine) (bone resorption marker) were obtained. Femur strength, bone mineral density (BMD) and bone mineral content (BMC) were analyzed at day 56. Mean plasma PHE concentrations were signi¢cantly greater in PHU vs PHR and control mice (p50.0001). Total body weight was signi¢cantly less in PHU vs control mice (p50.01). Mean femur weight was reduced in PHU mice vs both PHR and control mice (p50.03). PHU mice had smaller mean femur length than control mice (p50.002). Femur strength was greater in PHR compared to control (p50.01) but not PHU mice. No signi¢cant di¡erence among groups was found in BMD and BMC. At day 56 there was a statistical trend (p50.056) towards higher DPD excretion in PHU mice than PHR mice. Plasma PHE was positively correlated with urine DPD/creatinine but not to total DPD excretion. Moderate protein restriction did not signi¢cantly a¡ect bone status. These data suggest that hyperphenylalaninemia may adversely a¡ect bone status in PKU mice. This study was supported by a grant from the American Dietetic Association and Ross Products Division/ Abbott Laboratories (Columbus, OH).

050-O

GENOTYPE AND PHEYNYLALANINE (PHE) TOLERANCE OF WOMEN WITH PKU DURING THE 1ST TRIMESTER OF PREGNANCY P.B. Acosta1, K. Matalon2, C. Azen3, R. Koch4 Ross Products Division, Columbus, OH1, University of Houston, TX2, Children's Hospital of Los Angeles3, University of Southern California, Los Angeles4

Nineteen treated women in the MPKU Collaborative Study with plasma PHE concentrations 5360 mmol/L by 10 weeks gestation were evaluated to determine if genotype in£uenced PHE tolerance. Two alleles were analyzed in 14 women and 1 allele was analyzed in 5 women. Mean (+ SD) PHE intake was calculated from 3-day diet diaries recorded monthly and compared by genotype. For the 247 women for whom diet diaries were available, 1st trimester Spearman correlation coe¤cients were run to determine if PHE, protein, fat or energy intake was correlated with plasma PHE concentration. Mean (+ SD) PHE intake by 7 women with 2 alleles resulting in PHE hydroxylase (PAH) activity 51% was 518 + 182 mg/d vs 503 + 205 mg/d in 5 women with 1 allele resulting in 1^ 10% activity and 1 allele resulting in 51% activity. The two women who had 1 allele each resulting in 51% and 410% PAH activity ingested means of 417 and 514 mg/d. Spearman correlation coe¤cients indicated that protein (r -0.163, p=0.01) and fat (r -0.143, p=0.02) intakes were signi¢cantly and negatively correlated with plasma PHE concentration. Energy intake was negatively correlated (r -0.076, p=0.232) with plasma PHE concentration but the correlation was not signi¢cant while PHE intake was not correlated with plasma PHE concentration. These data suggest that protein and fat intakes during 1st trimester may have a greater in£uence on plasma PHE concentration than PHE intake or genotype. Acknowledgements. F. Guttler, JFK Institute, Glostrup, Denmark completed mutation analysis. This study was supported by National Institute of Child Health & Development Contracts 1-HD-2-3148, 1-HD-2-3149, 1-HD-2-3155, and 1-HD-3-3156; Project No 6606-3265 of the National Health Research & Development Program, Health & Welfare, Ottawa, Canada; The Danish Health Insurance Foundation Grant 11/224-98; the Danish Medical Research Council Grant 950 212-28807-7001, the Novo Nordisk Foundation; Franz Ho¡mann's Memorial Fund; Else Hjorth's Fund; and Ernst Husman's Fund, Copenhagen, Denmark.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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051-P

LONG CHAIN POLYUNSATURATED FATTY ACID (LC PUFA) SUPPLEMENTATION OF A LOW PHENYLALANINE (PHE) INFANT FORMULA ^ GROWTH, ANTIOXIDANT STATUS AND PHE CONTROL A. Harvie (Co-ordinator), European PKU LC ^ PUFA Supplementation Trial Participants Department of Child Health, Yorkhill Hospital, Glasgow, UK As reported previously (SSIEM 1998), we investigated the e¡ects of LC PUFA supplementation in 40 infants with PKU for one year in a randomised double-blind controlled European study. The infants receiving the LC PUFA supplemented formula (XP Analog LCP), which as a comparable fatty acid content to breast milk, maintained docosahexaenoic acid (DHA) and arachidonic acid (AA) status in red cell membrane closer to that of breast-fed infants compared to controls. Growth, antioxidant status and phe control results are now reported. Weight, length and OFC were measured regularly with Z scores for weight below. Growth patterns were normal in both groups. Table: Z-scores for Weight

Control (Mean (SEM)) Supp (Mean (SEM))

Entry to Study

160 Days

1 Year

-0.66 (0.20) -0.66 (0.20)

-0.24 (0.27) 0.33 (0.19)

0.02 (0.28) 0.56 (0.24)

Vitamins A & E, selenium, glutathione peroxidase and superoxide dismutase measured at study entry, 20 weeks later and 1 year were highly variable between individual infants but no signi¢cant di¡erences between the groups were seen. Phe control was comparable between the two groups. In conclusion, i) the LC PUFA supplemented group grew at least as well as the non-supplemented group, ii) the antioxidant capacity was not compromised by improvement in DHA status, iii) phe control during study period was acceptable for both groups.

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PHENYLKETONURIA: NO SPECIFIC FRONTAL LOBE DEPENDENT DEFICITS OF EARLY TREATED PATIENTS IN COMPARISON TO DIABETIC PATIENTS R. Feldmann, PhD; J. Denecke, MD; M. Pietsch, PhD; M. Grenzebach, PhD; J. Weglage, MD Department of Pediatrics, University of Mu«nster, Albert-Schweitzer-St. 33, D-48129 Mu«nster, Germany, Phone: +49251-8347774, Fax: +49251-8347765, [email protected] Objective: Neuropsychologic studies have shown that even early treated phenylketonurics are su¡ering from phenylalanine related de¢cits. Elevated phenylalanine levels can interfere with the development and function of the CNS. Speci¢c impairments of frontal lobe functions in patients with PKU have been reported. This study was performed to determine whether PKU children show frontal lobe dependent de¢cits if compared to diabetic patients. Methods: The comparative study covered 42 PKU patients 10 to 18 years of age (mean: 14.7 y) and 42 diabetic patients matched for sex, age, and socioeconomic status. Patients were assessed for IQ (Culture Fair Intelligence Test), information processing (Wisconsin Card Sorting Test), and selective (Stroop task) and sustained attention (Test d-2). Results: PKU patients had signi¢cantly poorer results than the diabetic patients. Within all tests, this was due to reduced performance speed but not to de¢cits in speci¢c frontal lobe functions (i.e. no impaired selection abilities, no de¢cits in information processing, insight, and learning). Conclusions: Elevated phenylalanine levels may cause an imbalance in neurotransmitter metabolism. However, this refers to a global neurotoxic e¡ect rather than to speci¢c e¡ects on the dopaminergic system which would a¡ect the activation of the frontal lobes.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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27

MEASURING SERUM SELENIUM IN ADULTS WITH PHENYLKETONURIA Philip Lee and Margaret Lilburn Metabolic Unit, University College London Hospitals, London United Kingdom

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To lower blood phenylalanine concentrations (Phe) in patients with phenylketonuria (PKU) necessitates a reduction in natural protein intake, placing them at risk of de¢ciency of micronutrients, such as selenium (Se). Se is an important determinant of anti-oxidant status, and de¢ciency may impair cardiac, skeletal muscle and immune function. Concentrations of serum Se have been found to be low in children with PKU on Phe-restricted diets, but its clinical signi¢cance is not clear and few older patients have been studied. We evaluated serum Se in adult PKU patients attending our metabolic clinic on a variety of restricted and unrestricted diets. Serum Se was measured in 190 PKU patients aged 13 to 49 years (median 27) between January 1996 and February 2000. At the time, 84 were on an unrestricted diet for at least 6 months. Of those on a Phe-restricted diet (n= 106), 59 were on a nutritionally complete supplement (XP Maxamum, group A), 25 were taking Aminogran metabolic mineral mix (group B), 15 were taking Aminogran plus Forceval (group C), and 7 were taking Paediatric seravit (group D). Serum Se was signi¢cantly lower in group B (mean + SD, 0.55+0.33mmol/l) than those on an unrestricted diet (1.05+0.35mmol/l, p50.001) or in group A (0.97+0.23mmol/l, p50.001). Forceval supplements increased serum Se (0.82+0.30mmol/l), but they remained lower than normal (p=0.011) and compared to group A (p=0.08). There were no strong correlations with Phe tolerance. Despite these di¡erences, no clinical consequences of this relative Se de¢ciency were observed. We conclude that serum Se can be normalised by appropriate supplementation in PKU, but long-term e¡ects of any de¢ciency are not apparent in this young adult population.

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VARIATION IN BLOOD PHENYLALANINE WITH MENSTRUAL CYCLE IN WOMEN WITH PHENYLKETONURIA (PKU) Philip Lee & Margaret Lilburn Metabolic Unit, University College London Hospitals, The Middlesex Hospital, Mortimer Street, London W1N 8AA, United Kingdom

Phenylalanine (Phe) restricted diet must begin before conception in women with PKU to protect fetal brain and cardiac development and so prevent the maternal PKU syndrome in their o¡spring. Based upon recommendations of the Medical Research Council Working Party (1993), our unit aims for Phe concentrations between 100 and 250mmol/l in early morning blood spot samples. To achieve adequate metabolic control, Phe is measured 23 times per week by HPLC. Phe tolerance varies between individuals according to residual phenylalanine hydroxylase, but also depending on intercurrent illness or change in body weight. Here we examine the variation in Phe levels in relation to menstrual cycle, another potential in£uence on metabolic control. Between 1979 and 1999, 69 infants were born to 46 women with PKU. 10 women were on a Pherestricted diet for at least 20 weeks without conceiving (mean 49, range 20-124 weeks). Their blood Phe concentrations were analysed, comparing the week before and the week after the ¢rst day of menses. Two women had signi¢cantly higher Phe levels after menstruation (mean increases of 149 and 179mmol/l). The patterns were not consistent in the remaining eight, varying from a mean fall of 44mmol/l to a mean increase of 43mmol/l. We conclude that sex hormones have variable e¡ects on Phe metabolism in women with PKU, but these can be signi¢cant in certain cases and a¡ect decisions about the dietary advice o¡ered.

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28

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INDIVIDUAL BLOOD^BRAIN BARRIER PHENYLALANINE TRANSPORT IN PHENYLKETONURIA (PKU) J. Weglage, J. Denecke, R. Feldmann, H.G. Kock, K. Ullrich, H.E. Mo«ller Department of Pediatrics, University of Mu«nster, Albert-Schweitzer-Str. 33, D-49129 Mu«nster, Germany, Phone: +49251/8347753, Fax: +49251/8347735, e-mail: [email protected] Objective: Phenylketonuria is the most common treatable enzymopathy of amino acid metabolism in man. Under a strict diet patients' neurologic and intellectual development is close to normal. Duration and strictness of the diet are still under discussion. An individual vulnerability of patients to elevated blood phenylalanine levels is well known. The pathogenesis of di¡erent clinical outcome in spite of comparable blood phenylalanine levels is still unclear. One possible explanation could be interindividual variations in brain concentrations of phenylalanine, which crosses the blood^brain barrier slowly, mediated by a saturable transport system. Methods: In vivo nuclear magnetic resonance spectroscopy was used in 25 patients with classical PKU to measure time courses of intracerebral phenylalanine concentrations before and after an oral phenylalanine load (100 mg/kg). Results: The kinetic investigations revealed transport Michaelis constants and ratios of the brain in£ux, as well as consumption rates of a continuous distribution within a wide range (Kt,app = 0.1 to 1.10 mmol/L; Tmax/Vmax = 2.55 to 14.0). Conclusions: Such variations seem to be major causative factors for the individual vulnerability to PKU.

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PROPER DIETARY TREATMENT ELIMINATES THE INFLUENCE OF PAH GENOTYPE ON COGNITIVE PERFORMANCE IN FEMALES WITH PKU AND RESULTS IN OFFSPRING WITH NORMAL IQ F. GÏttler1, C. Azen2, P. Guldberg1, A. Romstad1, R. Koch2, H.L. Levy3, W.B. Hanley4, R. Matalon5, B.M. Rouse5, F. Trefz6, F. de la Cruz7. 1 John F. Kennedy Inst., Glostrup, Denmark, 2Children's Hosp. Los Angeles, 3Children's Hosp., Boston, 4 Hospital for Sick Children, Toronto, 5Univ. of Texas, Galveston, 6Childrens Hosp., Reutlingen, Germany, 7National Inst. of Child Health and Human Dev., Bethesda. Cognitive development was normal (mean IQ = 108) and independent of phenylalanine hydroxylase (PAH) genotype in 108 Danish adolescents with phenylketonuria (PKU) detected by neonatal screening and still on diet at age 14 years. In the Maternal PKU Collaborative Study cognitive performance was dependent on genotype in 106 females treated for 6 years of life. Mean IQ (WAISR) was 83 in 89 females with a severe PKU genotype versus 96 in 27 females with a mild PKU genotype (P 5 0.001). Females who were treated for 4 6 years showed IQ scores 10 points above average for their group. Cognitive development (WISC-R at age 7 years) in 107 children of these mothers was dependent on maternal IQ, maternal genotype and metabolic control during pregnancy. Mean IQ was 103 in 48 children of mothers with IQ 4 85. Mean IQ was 79 in 50 children of mothers with severe mutations and IQ 5 85. Average phenylalanine exposure was kept below 360 mmol/l in 22% of these pregnancies and IQ of the o¡spring was 102. Our observations indicate that dietary therapy throughout adolescence allows normal cognitive development in females with severe PKU mutations and results in o¡spring with normal IQ.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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29

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BREASTFEEDING IS POSSIBLE EVEN IF MOTHER & BABY HAVE PKU Dorothy E. M. Francis, A. Boneh, M. K. Thong, Victorian Clinical Genetic Services, Royal Children's Hospital, Flemington Rd, Parkville, Victoria, Australia 3052.

Partial breastfeeding is the ideal treatment for babies with PKU, aiming to keep the blood phenylalanine (Phe) level between 200-400umol/L. Following exclusive feeding with a Phe-free formula for 2-3 days, titrated amounts of the formula are given before each breastfeed. The amount of formula prescribed is deduced from the baby's blood Phe levels. A mother with PKU who had excellent biochemical control on diet before and throughout pregnancy was keen to breastfeed her baby. The baby had PKU (blood Phe level of 633 at 2 days & 1049 at 9 days). The mother's blood Phe level rose to 1233 after delivery, and she was encouraged to maintain her blood Phe levels at 5700. The baby was demand-fed *150ml/kg/day of a Phe-free formula (XP Analog) and her blood Phe level dropped to 137 within 48 hours. The volume of XP Analog was reduced to 50ml/kg/day, given in divided doses before each breastfeed. The mother's & baby's blood Phe levels were measured twice then once weekly and the amount of XP Analog adjusted accordingly. The baby's blood Phe level dropped to 10 on day 16 so the XP Analog was further reduced to 30ml/kg/day. Satisfactory biochemical control was subsequently maintained using this regime. Only mild modi¢cation of maternal diet was needed. There was no correlation between the mother's and her baby's blood Phe levels. At 5 months the baby's growth and development are normal. We conclude 1) It is safe for a mother with PKU to partially breastfeed her baby with PKU, provided the baby's blood Phe levels is maintained in the treatment range. 2) Routine screening of partners of individuals with PKU prior to or early in pregnancy.

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PROTEIN INSUFFICIENCY AND IMPAIRED GROWTH IN CHILDREN WITH PKU Arnold GL1, Vladutiu CJ1, Kirby RS2. 1. Univ of Rochester School of Medicine & Dentistry, Rochester, NY, 2. Univ of Wisconsin School of Medicine, Milwaukee, WI, USA.

Children with phenylketonuria (PKU) often exhibit sub-optimal growth. Although protein insu¤ciency has been implicated as an etiology, most studies using traditional measures of protein su¤ciency (e.g. plasma albumin) have been inconclusive. We reviewed the records of 38 children ages 3-20 years with early and continuously treated classic PKU in order to study the relationship between plasma prealbumin and growth. Variables included plasma prealbumin, age, age-adjusted body mass index percentiles (BMI) and height percentile for age (from National Center for Health Statistics growth curves). Growth parameters and plasma chemistries were retrospectively retrieved from routine assessments in the Inherited Metabolic Disorders Clinic. The mean age was 8.9 years, mean height was at the 45.8%ile, and mean plasma prealbumin was 20.6 mg/dl. We identi¢ed a signi¢cant negative correlation between height percentile and prealbumin, such that children with lower plasma prealbumin tended to be shorter (r=0.387, p50.02). This correlation was very strong, and after controlling for age and BMI, having prealbumin less than 200 g/L (20 mg/dl) was associated with a reduction of 43 percentiles in height! There was also a strong correlation between age and prealbumin, with older children having higher prealbumin (p50.01), but there was no correlation between height percentile and age. These data suggest that protein insu¤ciency plays a signi¢cant role in impaired growth in children with PKU.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

30

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FATTY ACID STATUS OF PKU CHILDREN THREE YEARS AFTER LCPUFA SUPPLEMENTATION M. Giovannini, C. Agostoni, L. Fiori, MG. Bruzzese, M. Silano, E.Verduci, E. Riva Department of Pediatrics, San Paolo Hospital, I-20142 Milan, Italy. Background. Long-chain polyunsaturated fatty acid (LCPUFA) have relevant roles in growth and neural development. Dietary treated phenylketonuric (PKU) children are supplied with foods lacking LCPUFA. In a previous trial in 10-year old PKU a balanced supplementation with LCPUFA for one year restored physiological LCPUFA levels in blood. Aim. We have checked the LCPUFA status of all PKU children who completed the previous trial three years after completion of the supplementation. Subjects and methods. 20 dietary treated PKU (mean age 13.5 years), who had been randomized to receive a supplementation with 0.5 g fat caps/4 kg body weight, were sampled after an overnight fasting. Fat caps provided either 26% fatty acids (FA) as LCPUFA (18:3n-6 = 4.6%, 20:4n-6 = 7.4%, 20:5n-3 = 5.5%, 22:6n-3 = 8%) or olive oil (control group). The composition of FA methyl esters (wt%) of plasma total lipids, TL and lipid fractions (phospholipids, PL, cholesterol esters, CE, triglycerides, TG) has been measured with capillary gas-chromatography. Statistics: non-parametric tests. Results. The two groups did not di¡er for the major LCPUFA levels in plasma TL, CE and PL. A trend for higher levels of n-3 LCPUFA (20:5n-3 and 22:6n-3) was present in plasma TG of the supplemented group. In this group the 22:6n-3 levels in plasma PL three years before were associated with the actual 22:6n-3 levels in plasma TG (Rsq=0.41, P=0.03). Conclusions. Since plasma PL re£ect current dietary habits and fasting plasma TG are a partial marker of stored fats, a supplementation with LCPUFA may have a long-term e¡ect in treated PKU.

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DEVELOPMENT OF A SKIN BASED ``PHENYLALANINE SINK'' Christensen R1, Kolvraa S1, Blaese RM2 & Jensen TG1. 1 Institute of Human Genetics, University of Aarhus, Denmark, 2Kimeragen, Inc., Newtown, PA, USA. Phenylketonuria, PKU, is caused by de¢ciency of phenylalanine hydroxylase (PAH) and increased levels of phenylalanine. The required cofactor for PAH, BH4, is normally synthesized in the liver. The rate limiting step in this synthesis is GTP cyclohydrolase (GTPch). We have investigated whether overexpression of two enzymes: PAH as well as GTPch, in primary human keratinocytes, leads to clearance of phenylalanine without BH4 supplementation. Results. Integration of multiple PAH and GTPch transgenes was achieved after optimized retroviral transduction. Phenylalanine clearance was measured in primary human keratinocytes co-transduced with PAH and GTPch (more than 370 nmol/24hrs/106 cells), a level exceeding that of liver cells. Cells expressing either one of the enzymes alone did not clear phenylalanine. Mixing cells transduced separately with PAH and GTPch also resulted in phenylalanine clearance. This ``metabolic cooperation'' was dependent on direct physical contact, since growing the two kind of cells in two separate layers, i.e. without cell-to-cell contact, did not lead to phenylalanine clearance. Phenylalanine clearance continued at a high level when transduced keratinocytes were induced to di¡erentiate into a multilayered tissue (raft cultures). Conclusion. Co-expression of PAH and GTPch in primary human keratinocytes leads to high clearance of phenylalanine without BH4 supplementation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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31

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LONG-TERM RESULTS OF A SIMPLE APPROACH TO BREAST FEEDING THE INFANT WITH PHENYLKETONURIA A.G.F. Davidson, L.T.K. Wong, J. Ternes, C. Hartnett, Y. Lillquist Biochemical Diseases Clinic, BC Children's Hospital, Department of Paediatrics, Vancouver, Canada

Therapy of infants with Phenylketonuria (PKU) involves precise dietary control of phenylalanine intake. Although breast-feeding has signi¢cant advantages for infant and mother, human milk provides more phenylalanine than can be tolerated by the PKU infant. As a consequence, PKU infants are usually bottle-fed with formula. Di¡erent approaches to modify breast feeding and thereby restrict phenylalanine intake to therapeutic levels has been tried, but in general these introduce signi¢cant stress by requiring manual expression, test weighing or supplemental feeding devices. As a result, relatively few PKU infants are breast-fed after diagnosis and even then for a short time. Since 1981, we have used a simple regimen based upon the physiological autoregulation of intake by healthy infants, to allow breast-feeding of PKU infants by mothers who wish to do so. The PKU infant is allowed to breast-feed in the normal way. Test weighing or other calculation of breast milk intake is not required, but the infant is given a speci¢ed amount of low phenylalanine formula before being allowed to breast-feed ad lib. The amount of formula given before each breast-feeding is adjusted according to the blood phenylalanine level. If the blood level is increased, mother is advised to increase the amount of formula (usually 45^75 cc) given before each breast-feed. If the blood phenylalanine level is below therapeutic range, the amount of formula is decreased. From 1981 to 1999, over 63% of 52 newly diagnosed PKU infants attending our clinic have been successfully breast-fed for as long as 15 months. E¤cacy of treatment as measured by Phenylalanine Exposure Index, growth, and psychological testing at 5 and 10-year follow-up is comparable or superior to age-matched formula-fed PKU infants. We conclude that breast-feeding can be satisfactorily used in the management of infants with PKU with excellent outcome.

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ADULTS WITH PKU: 33 YEARS OF THERAPY IN SOUTH AUSTRALIA JMFletcher2,3, M Netting1, DB Ketteridge2, I Chapman3, A Sweeney1, EF Robertson2 Departments of Nutrition and Dietetics1 and Chemical Pathology2 Women's and Children's Hospital, North Adelaide and Department of Medicine, Royal Adelaide Hospital3

Introduction: Neonatally- and late-diagnosed PKU children and adults have been treated from diagnosis, irrespective of age, in South Australia since 1966. We present our current approach to the treatment of adults with PKU. Methods: Following the diagnosis of PKU, never-treated adults are commenced on a phenylalanine-free protein supplement and a low protein diet to provide 0.8 to 1.0 grams per kg per day of protein. Independent of age at diagnosis, we commence therapy immediately rather than introducing the diet in a step-wise fashion. In late-diagnosed patients, skin, re£exes, behaviour, mood swings, anxiety and tremor are scored on and o¡ the diet. Results: In our Clinic, 8 of 17 late-diagnosed adults remain on treatment and show improved clinical status. One woman is ``possibly better'' and remains on diet. Four late-treated adults discontinued diet because they were considered ``no better o¡'' or the diet was ``too hard''. The remainder is intermittently on diet, notably during pregnancies. Twelve of 25 locally born adults treated from birth remain on diet, compared to only 1 of the 8 whose diagnosis and initial treatment was interstate. Conclusions: Late treatment of adults with PKU is feasible and not unduly di¤cult. In most cases PKU treatment results in improved quality of life for both community- and institution-based residents.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PROLACTIN, A MARKER OF CEREBRAL DOPAMINE DEFICIENCY IN PATIENTS SUFFERING FROM PHENYLKETONURIA (PKU)? Denecke J, MÎller H, Schlegel W, Koch HG, Feldmann R, Harms E, Weglage J Department of Pediatrics, University of MÏnster, Albert-Schweitzer-Str. 33, D-49129 MÏnster, Germany. e-mail: [email protected] Objective: Even early treated PKU-patients are su¡ering from reversible neuropsychological de¢cits associated with elevated phenylalanine levels. Reduced concentrations of neurotransmitters especially dopamine- are proposed to be causative. Dopamine regulates prolactin release by inhibitory control. We therefore addressed the question whether prolactin concentrations are correlated with phenylalanine levels and whether prolactin is a suitable marker of cerebral dopamine de¢ciency. Methods: First, we determined blood phenylalanine and prolactin levels repeatedly in 150 PKUpatients visiting our outpatient clinic. Additionally, we simultanously measured cerebral phenylalanine concentrations by magnetic resonance spectroscopy and blood prolactin levels after an oral load with L-phenylalanine (100 mg/kg) in 20 patients. Results: Evaluating 350 blood samples of 150 PKU-patients no signi¢cant correlation could be computed between blood phenylalanine and prolactin levels, neither intra- nor interindividually. After oral load with L-phenylalanine blood and brain phenylalanine increased signi¢cantly within 30 to 60 minutes. Prolactin increased in some patients but there was no signi¢cant correlation between prolactin and blood or brain phenylalanine levels. Conclusion: Prolactin seems to be no suitable marker of cerebral dopamine de¢ciency.

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DIFFICULTIES IN DIETARY COMPLIANCE IN LATE CHiLDHOOD & ADOLESCENT PATIENTS WITH PKU D. E. M. Francis, P. Anderson, S Wood, V Anderson, A. Boneh, Murdoch Children's Research Institute, Royal Children's Hospital, Flemington Rd, Parkville, Victoria, Australia 3052.

Even early treated patients with phenylketonuria (PKU) are found to frequently exhibit white matter abnormalities (WMA) on magnetic resonance imaging (MRI). Previous studies suggested that reversal of cerebral WMA can be achieved by better compliance with diet (Cleary et al J Paediatr. 1995; 127: 251-255). MRI's were performed on 42 children aged between 5-18 years; 76% showed WMA. A cohort of 16 children aged 9-18 (av 13.2) years with severe WMA (Cleary scale 46) was counselled to improve dietary compliance aiming at improving MRI ¢ndings. The cohort included 4 adolescents treated for 47 years but who had subsequently ceased diet. Of these 14 children were motivated to improve their diet and blood Phe levels for 12 weeks with an MRI thereafter. The diet aimed at fasting blood Phe levels of 200-400umol/L in those 510 years & 5700 in those 410 years. Weekly fasting blood tests were obtained from 86% of the children. Compliance and achievement of the target Phe levels were variable, ranging from 0-100% with 58% of the results within range during the 12 week period. Reasons given for not achieving the target Phe levels included: formula refusal; social eating demands; hunger; dietary product costs; time for diet preparation; peer pressure & embarrassment about the diet; infections. The PKU diet is an ongoing ¢nancial and socialpsychological burden to those with PKU and their families even when well motivated. Education of the children & peer support mechanisms for these patients, through PKU camps and similar activities, should be encouraged to help improve compliance.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PHENYLKETONURIA: IS DIET `FOR LIFE' NECESSARY? R. Cerone, M.C. Schia¤no, D. Gianotti, 8E. Veneselli, R. Lorini University Dept. of Pediatrics, 8Neurological Science^Child Neurology, G. Gaslini Institute, Genoa, Italy

In 1986 we reported (1) that the mean IQ scores of PKU patients who discontinued the diet between 9 and 10 y were not signi¢cantly di¡erent 2 y after diet termination. However, we were aware that IQ alone was not an e¤cient control for assessing the e¡ects of high Phe levels. For this reason we investigated in more detail 16 patients (aged 14^19 y) with early treated classic PKU but o¡ diet for 6 y. The evaluation included a detailed neurological examination, IQ, EEG, BAEPs, F-VEPs and SEPs. All subjects had normal neurological and intellectual function; the plasma Phe o¡ diet ranged between 21 and 56 mg/dl. IQ scores of our patients were 106.8+5.5 at age 11 y and 102.8+4.1 after 6 y o¡ diet. Using a non-parametric test, the IQ of the PKU patients o¡ diet is signi¢cantly below that of the same patients on diet (p50.01). We found no abnormality in FVEPs, in SEPs or in BAEPs. Di¡use slow EEG abnormalities of moderate or mild degree were present in 6 patients: these abnormalities were already evident at the last control before diet discontinuation and showed a positive correlation with the presence of a high blood Phe level for a prolonged period. All pts, apart from one, revealed minor neurological signs: intention tremor, unusually brisk tendon re£exes, mild ankle clonus. Two pts exhibited hyperactivity and emotional instability. We conclude that the diet should be continued during adolescence and adult life; higher Phe levels (510 mg/dl) can be accepted when the problems of dietary compliance in these age groups are taken into account. 1) Cerone et al. J. Inherit Metab Dis 9 Suppl 2: 223^225, 1986

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QUALITY OF LIFE IN PKU PATIENTS AFTER DIET INTRODUCTION/ REINTRODUCTION Campistol J, Gassio¨ R, Vilaseca MA, Lambruschini N, Cambra FJ, Gomez L, Fuste¨ E. Grup de Seguiment PKU. Hospital Universitari Sant Joan de De¨u. Barcelona. Spain.

We evaluate the quality of life in adult patients with classical PKU treated with a Phe-restricted diet. We studied 20 patients with classical PKU, whose ages ranged from 17 to 37y (mean 27y). All patients started a diet low in Phe, some of them after a period of at least 1 year of a free diet, others after a late-onset diagnosis of PKU. All patients and/or parents were submitted to a questionnaire of 24 items related to quality of life (state of mind, behavioural or relational problems, general health state). We evaluated the accomplishment of diet with the index of dietary control (good, plasma Phe: 120480 mmol/L, regular: 480-600 mmol/L and bad 4600 mmol/L). Dietary control was bad in 55%, regular in 20% and good in 25% of patients. After beginning/reintroducing a Phe-restricted diet, 45% of patients consider their health good, 40% of patients think they are better with diet than without it, 50% of patients are less nervous and 55% are more quiet and calm, 35% are happier than before and 40% are more concentrated in their work. In general they did not notice special changes in their behaviour, and 55% consider that their quality of life has improved with diet. Conclusions: 1) 55% of patients had bad dietary control after reintroduction of the Phe-restricted diet. 2) It is very di¤cult to reintroduce and specially to achieve a good dietary control after a free-diet period. 3) After introduction/reintroduction of diet, health state, quality of life and behavioural state did not worsen in any case. 4) Only 55% of patients considered that their quality of life improved with diet. 5) 50% of patients were less nervous, more quiet and calm after diet reintroduction.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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INCREASED PHENYLALANINE REQUIREMENTS IN A PRETERM INFANT WITH CLASSICAL PHENYLKETONURIA R. Randall1, J.A. Gick2, S. Bird3, M.P. Champion2 Department of Nutrition and Dietetics1; Department of Paediatric Metabolic Medicine2, Department of Chemical Pathology, Guys Hospital, London, UK3 Cases of classical phenylketonuria (PKU) in the preterm infant are rare in the literature. We report a male infant with classical PKU detected on neonatal screening 19 days post delivery (phenylalanine level of 1926mmol/l. He was born at 30 weeks gestation to non-consanguineous parents, birth weight 2.36kg (490th centile). The neonatal course was otherwise uneventful, and he was discharged home aged 23 days. Phenylalanine levels were rapidly controlled with cessation of natural protein and the introduction of phenylalanine free formula (XP-Analog). Following the reintroduction of phenylalanine (SMAgold), phenylalanine levels remained below 120mmol/l (range 10^73 mmol/l) until an intake of 100mg/ kg/day was achieved, twice the recommended daily requirement1. Weight gain was maintained at 23^ 31g/day. Requirements normalised at term. We conclude that preterm infants with classical PKU may require higher phenylalanine intakes in order to meet the demands of growth. 1

Barnes J, PKU in Clinical Paediatric Dietetics, (Eds Shaw & Lawson) Blackwell Science, London 1994: 177^ 84.

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DETERMINANTS OF ADHERENCE TO A PHENYLALANINE RESTRICTED DIET M.R. Crone1, K. Oudshoorn1, F.J. van Spronsen2, M. van Rijn2, J. Bekhof2, P.H. Verkerk1 1. TNO Prevention and Health. 2. Beatrix Children's Hosptal, University hospital Groningen The objective of this study was to determine the factors that are related to the adherence to the phenylalanine (PHE)-restricted diet. A questionnaire was disseminated among Dutch parents of PKU-patients diagnosed by the screening (after 1 September 1974). The questionnaire was based on the concept that attitudes, social in£uence, personal e¤cacy and knowledge in£uence the adherence to the diet. The adherence to the diet was determined as the average PHE-concentration of the PKUchild between 1994 and 1996. The parents of 169 patients completed the questionnaire (response 71%). Eight percent of the parents was not from Dutch origin and the PHE of their child was in average 128 mmol/l higher than of a child from parents of Dutch origin (p=0.024). The hospital attended by the parents was also related to the PHE: the di¡erence between the hospital with the lowest and the highest average PHE was 207 mmol/l (p=0.005). Parents who had a lower personal e¤cacy had children with higher PHE: especially when it concerned the e¤cacy to make their child eat the amino acid supplement at least tree times a day. This caused di¤culties among 24% of the parents: the di¡erence in the PHE of the children of parents with and without problems was 266 mmol/l (p50,001). It can be concluded that extra attention should be given to the intake of the amino acid supplement three times a day. Special attention should also be given to parents who are not from Dutch origin and to the factors (treatment, information and culture) that may in£uence the di¡erences in PHE between the di¡erent hospitals.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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KNOWLEDGE OF THE DISEASE AND DIETARY TREATMENT IN PKU J Bekhof1, MR Crone2, M van Rijn1, K Oudshoorn2, PH Verkerk2, FJ van Spronsen1 1 Beatrix Children's Hospital, University Hospital Groningen, The Netherlands; 2TNO Health and Prevention, Leiden, The Netherlands

Introduction: The concept that metabolic control may improve when patients/parents are principally responsible for metabolic control in PKU gradually gains ground. In this respect knowledge of PKU and dietary treatment is a prerequisite. Objective: To develop educational programs knowledge about PKU in Dutch PKU patients/parents was evaluated and possible association between knowledge and metabolic control was determined. Methods: Sixty-two PKU patients (12-22 yrs) and parents of 169 PKU patients (0-22 yrs) ¢lled in a questionnaire to evaluate the knowledge. Multivariate analysis, correcting for the patient's age, Phe tolerance, pre-treatment Phe concentrations and parent's educational level, was performed to identify the association of knowledge with metabolic control (p50.10). Results: Disease speci¢c knowledge was better than diet speci¢c knowledge. Univariate analysis showed that when knowledge increased the metabolic control ameliorated. After correction for the other variables this signi¢cant association disappeared and pre-treatment Phe concentrations had the strongest association with metabolic control (parents: b=0.79; patients: b=0.91: p50.001). Conclusion: This study showed that in the present situation, in which the dietician/physician is mainly responsible for dietary adjustments, pre-treatment Phe are stronger predictors of metabolic control than knowledge. Still for a successful introduction of self-treatment in PKU, educational programs aiming at improvement of diet speci¢c knowledge need to be advocated.

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UBIQUINONE DEFICIENCY IN PKU. THE INFLUENCE OF PHENYLALANINE AND DIETARY CONTROL Artuch R, Colome¨ C, Vilaseca MA, Lambruschini N, Campistol J. Hospital Sant Joan de De¨u. Universitat de Barcelona. Spain.

Plasma ubiquinone concentrations are lower in PKU patients than those of a reference population. Our aim was to investigate the factors involved in this de¢ciency: the special diet of PKU patients; the high concentrations of phenylalanine (Phe), that it may down-regulate the mevalonate pathway; the low concentrations of tyrosine (Tyr), which is essential for ubiquinone biosynthesis. We studied plasma ubiquinone, cholesterol and amino acid concentrations in 79 PKU patients, classi¢ed in two groups according to the index of dietary control index (IDC5450 n= 44; IDC4450 n=35). Ubiquinone was analysed by HPLC with UV detection, and amino acids by ion exchange chromatography. Ubiquinone was lower (p5 0.0005) in the PKU patients (median 0.6; range 0.291.0 mmol/L) compared with age-matched controls (median 0.8; range 0.46-1.38 mmol/L). Negative correlations (p50.01) were observed between ubiquinone concentrations and IDC, Phe and Phe/Tyr ratio in well controlled PKU patients (IDC5450). No correlations were observed in the PKU patients with an IDC4450. We applied a multiple linear regression test to avoid the confusing e¡ects of age and cholesterol in ubiquinone concentrations. We observed a negative correlation (P50.01) between ubiquinone and Phe concentrations in the well controlled PKU patients. These results suggest that plasma Phe in£uence ubiquinone concentrations only in PKU patients with a good dietary control. We did not observe this in£uence in PKU patients with IDC4450, probably due to the ubiquinone supply from the diet.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

36

Abstracts

071-P

CEREBRAL MRI/MRS STUDIES IN PHENYLKETONURIA Rex Moats* **, Marvin Nelson*, Alan Stern* and Richard Koch* *The Children's Hospital of Los Angeles and the University of Southern California School of Medicine; **California Institute of Technology MRI/MRS studies of brain phenylalanine (phe) content were carried out for three years on adults with phenylketonuria (PKU). Six control, 10 carriers of the PKU gene and 50 adults with a diagnosis of PKU were studied. Eight individuals with signi¢cantly elevated blood phe levels but with low brain phe levels consistent with a carrier status were identi¢ed. The blood phe and brain phe levels were simultaneously obtained. Unfortunately, the role of large neutral amino acid content in the diet was unknown prior to the study and was not identi¢ed in these 8 persons. Five were not receiving a medical product and exhibited a mean IQ on the WAIS-R of 118. The mean IQ of the 2 on a medical product was 122 on the WAIS-R. All 8 are healthy and do not exhibit any neurological problems, however one person o¡ diet has had an episode of depression and has been seen for counseling. Six are employed and two are college students. Five have severe mutations and are considered classical; two have one severe and one mild PKU gene mutation and one has 2 mild mutations. In conclusion, the occurrence of these individuals in this study of PKU adults is 16% and deserves further study.

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BLOOD^BRAIN PHENYLALANINE RELATIONSHIP IN PHENYLKETONURIA (PKU) SUBJECTS IMPROVED BY INGESTION OF LARGE NEUTRAL AMINO ACIDS Rex Moats, Ph.D., Richard Koch, M.D., Kathryn Moseley, R.D. and Marvin Nelson, M.D. Two subjects with classical PKU were studied with MRI/MRS technology before, during and after ingestion of two doses of large neutral amino acids. The ¢rst subject was a 40-year-old male, originally diagnosed at 11/2 years of age with a developmental quotient (DQ) of 66. Mutation analysis of the phenylalanine hydroxylase gene revealed IVS12ntl/IVS12ntl. He remained on a pherestricted diet with good control (120^900 mmol/liter) until 15 years of age. Thereafter his blood phe levels varied between 1200^1860 mmol/liter. At 27 years of age, he resumed dietary treatment and has maintained it to the present, with a mean of 26 blood phe levels of 900 mmol/liter. His present WAISR is 98 full scale. Arithmetic is his lowest score. He is happily married and has one normal child. He is employed as a professional painter and is purchasing a home. The second subject is a 30-year-old woman who was diagnosed at 3 months of age with a blood phe level of 3000 mmol/liter. She has remained on a phe-restricted diet all her life with a mean of 80 blood phe levels of 720 mmol/liter. She is a college graduate, working full time in an environmental control agency. At present she is unmarried. She has an IQ of 108 on the WAIS-R. Her verbal and performance scores range from 9^18. Both subjects were tested similarly. Diet remained unchanged during the 49 hours of observation. Initially MRI/MRS baseline brain and blood phe levels were obtained and a dose of 24 grams of large neutral amino acids was given at 15 and 30 hours. During the study the brain levels dropped signi¢cantly. This study was undertaken as a result of the work of Pietz et al. in Heidelberg.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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37

073-P

THE DIETARY KNOWLEDGE OF PKU PATIENTS N. Janecke*, P. Burgard University Children's Hospital, Paediatric and Developmental Psychology, Heidelberg, Germany; *Present address: University Hospital of Psychology and Psychotherapy, Innsbruck, Austria

Dietary treatment of phenylketonuria (PKU) in childhood, adolescence and adulthood is impossible without co-operation of the patient who has su¤cient dietary knowledge. We investigated the dietary knowledge of 45 patients of 4 age groups of 8-10, 11-13, 14-16, and 17-30 years. Psychophysical tests were established to test the abilities of seriation and information integration of several nutritional combinations with regard to their phenylalanine contents, as well as in a control task of twodimensional physical stimuli. A control group matched for age and IQ was tested only for the control task. All patients and controls showed the ability of seriation. In both groups only subjects older than 16 years were able to integrate information. All PKU patients gave poor estimates of phenylalanine content and weight of food. Especially, the 14-16 years old PKU patients were unable to combine phenylalanine contents of di¡erent food and overall produced the worst results regarding the knowledge of their diet. Age graded training programs for dietary knowledge especially for adolescents seem to be necessary to improve dietary competence and dietary independence of patients with PKU.

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EXECUTIVE FUNCTION IMPAIRMENT AND NEUROTRANSMITTER DEFICIENCY IN EARLY-TREATED PHENYLKETONURIC (PKU) PATIENTS UNDER DIETARY TREATMENT A.B. Burlina1, L. Bonafe¨2, M. Cendron1, P. Bisiacchi3, A. Suppiej1, N. Blau2, A.P. Burlina4 1 Dept of Pediatrics,3General Psychology,4Neurology, Univ. of Padua, Italy; 2Div. of Clinical Chem. and Biochem., Univ. Children's Hospital, ZÏrich, Switzerland

Long-term prognosis of early-treated PKU and the utility of longlife diet are still controversial. We present a study on 15 early-treated PKU patients (aged 14 + 5 years) who never interrupted the dietary treatment. All patients attended regular school. Some patients presented subtle clinical neurological changes (tremors, brisk tendon re£ex). Plasma Phe and Tyr at the time of the study were 850 umol/L (720-1030) and 32 umol/L (21-54), respectively. Nine patients (group A) showed white matter changes in brain MRI, and signi¢cant decrease of CSF homovanillic acid (HVA; mean 67.3 nmol/L, range 23-136; normal: 109-444) and 5-hydroxyindoleacetic acid (5HIAA; mean 60.2 nmol/ L, range 20-186; normal: 75-214). Six patients (group B) showed normal MRI ¢ndings; three of them underwent CSF examination, showing low levels of 5HIAA and high HVA/5HIAA ratio. I.Q. score was normal in all patients as well as language functions, memory, and visual constructive function. The main impaired cognitive domain was represented by executive functions, such as visual selective attention, action planning, fonemic £uency, sustained attention and shift tasks, working memory tasks, spatial planning and inhibition tasks. In conclusion, despite a continuous dietary control of plasma Phe levels, PKU patients can present subclinical cognitive impairment associated to monoamine neurotransmitter de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

38

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075-P

THE RELATIONSHIP BETWEEN BRAIN WHITE MATTER ABNORMALITIES AND NEUROPSYCHOLOGICAL FUNCTIONING IN PATIENTS WITH EARLY-TREATED PHENYLKETONURIA (PKU) Peter Anderson, V.Anderson, S.Wood, L.Coleman, D.Francis & A.Boneh, Murdoch Children's Research Institute, Flemington Road, Parkville, Victoria, 3052, Australia. Previous Magnetic Resonance Imaging (MRI) studies have identi¢ed white matter abnormalities (WMA) in patients with early-treated PKU, although the clinical relevance of the WMA remains unclear. We examined the relationship between WMA and cognitive functions in patients with earlytreated PKU. Forty-two children aged between 5 years and 17 years received MRI and neuropsychological assessments (WISC-III, TEACh, Contingency Naming Test, Rey Complex Figure, WRAT3). Of this cohort 76% exhibited WMA according to the scale developed by Cleary and associates (1994). Patients were categorised into 3 groups according to the MRI ¢ndings (No WMA, MRI score=0, n=10; Mild WMA, MRI score=1 to 5, n=16; Major WMA, MRI score45, n=16). The WMA groups di¡ered in terms of age (p5.01) and blood phenylalanine levels (p5.01). Full scale IQ did not signi¢cantly di¡erentiate the groups (No WMA, M=96.2, SD=9.8; Mild WMA, M=94.3, SD=10.5; Major WMA, M=90.8, SD=6.4), however the Major WMA group approached the ``Low Average'' range. More speci¢cally, the Major WMA group performed signi¢cantly poorly on tests of speed of processing (p5.05), attention (p5.10) and mathematics (p5.05). These de¢cits are consistent with white matter pathology. In conclusion, Major WMA exhibited in patients with early treated PKU are associated with speci¢c neuropsychological de¢cits. The relationship between these ¢ndings and blood phenylalanine levels is less clear.

076-P

EARLY PHENOTYPING OF PKU BY ORAL PHENYLALANINE LOADING M Spada, L Roasio, S Baglieri, G Battistoni, A Peduto and A Ponzone Department of Pediatrics, University of Torino, Italy The assessment of the metabolic phenotype in PKU patients may be achieved through di¡erent ways, including the measurement of phenylalanine (Phe) turnover after an oral loading. This test has been largely employed after the 6th month of age and at varying basal plasma Phe concentrations, whereas no study was performed in the neonatal period, nor at normal plasma Phe concentration. 25 PKU subjects, later assigned to one of three di¡erent classes of phenylalanine hydroxylase de¢ciency (severe, mild, benign or non-PKU hyperphenylalaninemia) according to their dietary Phe tolerance, had an oral Phe load (100 mg/kg) in the ¢rst month of life, after plasma Phe normalization was obtained by a Phe-free diet. Results are shown in the Table. Table : Time course of plasma Phe concentrations (mean and range) during an oral Phe loading (100 mg/kg) in 25 PKU newborns

Patients classic PKU (n=11) mild PKU (n=4) benign PKU (n=10)

0h

plasma Phe (mmol/L) 3h 7h

43 (10-81) 624 (465-729) 30 ( 6-53) 564 (490-666) 43 ( 4-87) 535 (446-786)

11h

596 (363-717) 561 (376-763) 517 (456-595) 394 (315-542) 487 (346-664) 368 (225-448)

The outcome of the oral Phe loading was di¡erent as a group according to the class of phenylalanine hydroxylase de¢ciency. Consistent overlap, however, among patients belonging to the di¡erent groups precludes the test to be used for early PKU phenotyping.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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39

HETEROLOGOUS GENE THERAPY FOR TREATMENT OF MURINE PHENYLKETONURIA - DEVELOPMENT OF MICE WITH PHENYLALANINE HYDROXYLASE (PAH) EXPRESSION IN BONE MARROW CO Harding, K Wild, MW Ne¡. Oregon Health Sciences University, Portland, OR, USA

077-O

Treatment of many inherited liver enzyme de¢ciencies requires the removal of toxic intermediate metabolites from the blood of a¡ected individuals. We propose that circulating toxins can be adequately cleared and disease phenotype in£uenced by enzyme expressed in tissues other than liver. For this experiment, our speci¢c hypothesis was that bone marrow expression of PAH would lower blood phenylalanine levels in Pahenu2 mice, a model of human PKU. To test this hypothesis, we have used germline modi¢cation to develop mice that constitutively express PAH in erythrogenic bone marrow under the control of a modi¢ed human Þ globin locus control region (mLCR). One line of transgenic mice (designated LCRPAH) carries approximately ten copies of the transgene integrated as a tandem repeat. Transgenic animals have normal hematocrit and peripheral blood smears, are physically healthy, and readily reproduce. PAH speci¢c activity in bone marrow homogenate is approximately one tenth that of liver homogenate. We have bred LCRPAH mice to Pahenu2 mice to generate progeny that are homozygous for the Pahenu2 mutation and therefore exhibit liver PAH de¢ciency but also carry the LCRPAH transgene and have PAH activity in bone marrow. Unfortunately, these animals exhibit hyperphenylalaninemia despite bone marrow PAH expression. Possible explanations for this ¢nding include an insu¤cient level of PAH expression or insu¤cient supply of the required cofactor, tetrahydrobiopterin (BH4). Further experiments to explore these possible limitations are currently ongoing.

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LABORATORY, PSYCHOLOGICAL AND MOLECULAR GENETIC STATUS OF HUNGARIAN PKU PATIENTS A¨. Schuler1, C.S. Somogyi1, I. Va¨radi1, I. To«ro«s1, T. Siska1, E. Kiss1, K. Ne¨meth2, M. Garami2, GY Fekete2 Buda Children's Hospital, PKU Laboratory, Budapest, HUNGARY1, 2nd Department of Pediatrics, Semmelweis University, Budapest2 The aim of our study was to examine the outcome of our classical PKU patients with wide phenylalanine (phe) tolerance by laboratory, psychological and molecular genetical methods. The most common Hungarian PAH mutations (R 408W, R 158Q, IVS12 splice) were examined in 101 patients and 3 further mutations (R252W, R261Q, IVS10nt546) in 4 cases with PCR technic. Phe loading test (100 mg/kg phe; SHS) was performed on 30 patients (16 girls, 14 boys, 1, 5^22 years). Se phe and tyrosin (tyr) levels were measured at 0 and 60 minutes of the loading spectro£uorometrically. Phe2/tyr ratios were calculated at the 60th minute (norm:536). IQs were examined by Brunet^Lesine (53 years), Budapest^Binet (3^14 years) and Wechsler test (414 years). The average blood phe levels of the last year were in the optimal range in most of the patients (n=23). The results of patients homozygote for the most severe R408W mutation (n=7) were better than expected (IQ: 80^122), phe2/tyr: 336^1502. From the 19 patients heterozygotes for R408W mutation on the other allele R158Q in one patient and IVS12 splice mutation in the other one were found, 17 patients had a non-detected allele. The IQs and the phe loading test results of these patients showed a wide range (IQ: 87^136, phe2/tyr: 128^2926) which could be explained with the variations of the non-detected alleles. In 4 cases none of the alleles were found. These 4 patients had high IQs (95^126) and relative lower phe loading test results (phe2/tyr: 175^1082), which indicate mild mutations. Phe loading test, mutation analysis and regular psychological examinations are helpful to reach good results.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GENOTYPE-PHENOTYPE CORRELATIONS IN PORTUGUESE HYPERPHENYLALANINEMIC PATIENTS Isabel Rivera1, Paula Leandro1, Isabel Tavares de Almeida1, Uta Lichter-Konecki2 1 Centro Patoge¨nese Molecular, Faculdade Farma¨cia U.L., Lisboa, Portugal; 2 Center for Medical Genetics, Marsh¢eld Medical Research Foundation, Marsh¢eld, USA A wide spectrum of mutations (4400, PAH Mutation Consortium Database) in the gene encoding phenylalanine hydroxylase is the molecular basis of phenylketonuria (PKU) and related hyperphenylalaninemias (OMIM 261600) in all populations. The mutation heterogeneity results in a high number of patients being compound heterozygotes of two di¡erent mutations. On the other hand, in vitro expression studies show that every mutation a¥icts enzyme activity di¡erently. As a consequence, the hyperphenylalaninemic patients display a remarkable phenotypic heterogeneity. In order to understand the basis for the clinical heterogeneity of phenylalanine hydroxylase de¢ciency among Portuguese hyperphenylalaninemic patients, genotype-phenotype correlations were established . A group of 37 patients, early diagnosed and treated, was fully genotyped and 17 di¡erent mutant alleles were identi¢ed in 22 di¡erent genotypic combinations. The results obtained by this study displayed a strong correlation between the predicted residual enzyme activity, as deduced from the genotype of the patients, and the biochemical phenotype represented by the diagnostic parameters: phenylalanine levels before treatment (r=-0.773, P50.000002, n=37) and the dietary phenylalanine tolerance (r=0.684, P50.002, n=17). It was also demonstrated a stong association between a parameter related to treatment (median of the year medians of phenylalanine levels during the ¢rst 9 years of life) and the IQ values displayed by the patients (r=-0.621, P50.023, n=13), but it was observed that only a judicious follow-up and compliance with the diet could allow the correct assessment of the clinical phenotype of the patients.

080-O

CHEMICAL CHAPERONES INDUCES THE STABILITY OF PAH MUTANT PROTEINS: GLYCEROL EFFECT AP Leandro1, I Tavares de Almeida1, MC Lechner1 and DS Konecki2 1 Centro de Patoge¨nese Molecular, Faculdade de Farma¨cia de Lisboa, Av. Forc°as Armadas 1600 Lisboa, Portugal; 2Center for Medical Genetics, Marsh¢eld Medical Research Foundation, 1000 North Oak Avenue, Marsh¢eld WI 54449 USA Characterisation of several mutant forms of human phenylalanine hydroxylase enzymes (hPAH) have shown that the majority of the PAH mutations a¡ect protein folding, causing altered oligomerization and accelerated proteolytic degradation. The ultimate e¡ect of these mutations is reduced cellular levels of the PAH mutant proteins. Recently, in vitro studies in transfected cells have revealed that the use of chemical chaperones can correct the defective folding of some mutant proteins. In the present study we produced the wild-type and two mutant forms of the hPAH protein (R270K and V388M) in a prokaryotic expression system. We have investigated the e¡ect of glycerol as a chemical chaperone, when added to the culture medium during induction, on the correct folding, solubility and speci¢c activity of the resulting recombinant enzymes. Our results demonstrate that the use of a chemical chaperone not only improved the yield of the produced soluble hPAH mutant proteins (2 to 3 fold depending on the mutant enzyme), but also increased the speci¢c activity of the puri¢ed recombinant enzymes. These data strongly suggest that further studies are needed to determine the possible e¡ect of chemical chaperones in the treatment of metabolic diseases associated with protein misfolding.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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41

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GENOTYPE-PHENOTYPE CORRELATION IN PATIENTS WITH PHENYLKETONURIA FROM SOUTH BRAZIL Pereira MLS, Silva LCS, Carvalho TS, Silva FB, Morari L, Pires RF, Giugliani R. Medical Genetics Service, Hospital de Cl|¨ nicas de Porto Alegre; Departments of Biochemistry and Genetics, Federal University of Rio Grande do Sul Porto Alegre RS BRAZIL.

Phenylketonuria (PKU) is an autosomal recessive disease caused by the de¢ciency of phenylalanine hydroxylase (PAH), a enzyme that catalises the conversion of phenylalanine (Phe) in tyrosine (Tyr). The locus of the PAH gene is located on chromosome 12 and spans 90 kb of genomic DNA divided into 13 exons. The aim of this study was to characterise mutations presents in PKU patients from South Brazil and correlate these ¢ndings with response to dietary treatment. We have search the whole coding region of the PAH gene in 55 non-related mutant chromosomes. Mutational analysis was performed using a combined approach of PCR and single strand conformational polymorphism (SSCP) analysis followed by digestion with restriction endonuclease and/or direct sequencing. The approach was able to detect approximately 87% of mutant chromosomes, and a total of 24 di¡erent molecular alterations have been identi¢ed, including two novel alterations and few polymorphisms. The most frequent mutations were I65T (18,2%), R408W (9,1%), R261X (9,1%), IVS2nt5g4c (7,3%), R261Q (7,3%) and V388M (7,3%). Results con¢rmed that biochemical outcome is associated to mutations interactions, as well as to polymorphisms presents in the gene. This work brought a new insight on genotype-phenotype correlation in PKU patient from South Brazil (Supported by CNPq, CAPES, FAPERGS, FIPE/HCPA, PROPESQ/UFRGS and MCT/PRONEX).

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GTP CYCLOHYDROLASE DEFICIENCY: NEW HOMOZYGOUS MUTATION IN AN INFANT WITH FAVOURABLE RESPONSE TO DEPRENYL E.H. Touma1, A. Romstad2, A. Sakr3, I. Khneisser1, J. Loiselet1, J.-L. Dhondt4 Laboratoire de Ge¨ne¨tique Me¨dicale, Universite¨ St. Joseph, Beirut, Lebanon1. John F. Kennedy Institute, Glostrup, Denmark2. Analytical Testing Laboratory, Beirut, Lebanon3. Laboratoire de Biochimie, Faculte¨ Libre de Me¨decine, Lille, France

The patient was the fourth child born to ¢rst cousins healthy parents. The ¢rst sibling su¡ered from hyperphenylalaninemia and died at 7 years of age untreated. At 3 months of age plasma phenylalanine (PA) level was 847 mmol/l, tyrosine 62 mmol/l on regular infant formula. The patient was noted to have microcephaly, a dystonic posture with truncal hypotonia, increased limb tone and pronated hands. She su¡ered from di¤culty in swallowing, irritability, episodes of hyperthermia, oculogyric spasms, excessive drooling and vomiting. EEG showed a generalized dysrythmia. Urine organic acid analysis did not show vanillylmandelate and homovanillate. Biopterin and neopterin assayed in dried urine ¢lterpaper were both below control levels, consistent with the diagnosis of GTP cyclohydrolase de¢ciency. Age in months Patient (normal range), PKU

4; 5 0^4

Neopterin mmol/mmol Cr

Biopterin mmol/mmol Cr

0.110; 0.122 (1.02 ^ 4.46), 2.06 - 15.5

0.073; 0.392 (0.46 ^ 1.17), 2.03 ^ 10.3

The patient was started on L-DOPA (10 mg/kg/day progressively) + carbidopa, 5-OH tryptophan 5 mg/kg/day progressively, and BH4 10 mg/day and half of her daily milk was given as PA-free formula Analog XP. Blood PA levels were monitored every two weeks then every month. Deprenyl was started at 2 years of age allowing a better neurologic control while Lamotrigin was discontinued. DNA analysis found a homozygous mutation in exon 6, M213T (ATG-4ACG) not described previously. At 3 years of age her growth and neurodevelopment were appropriate except for a mild delay in speech.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

42

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083-P

HYPERPHENYLALANINEMIA DUE TO PTERIN-4A-CARBINOLAMINE DEHYDRATASE DEFICIENCY (PCD): ANALYSIS OF INTESTINAL BIOPSIES Blau N1, Boerth SR2, Bailey SW2, Giugliani R3, Braegger C1, ThÎny B1, and Ayling JE2 1 Dept. of Pediatrics, Univ. of Zurich, 8032 Zurich, Switzerland, 2Dept. of Pharmacol., College of Med., Univ. of S. Alabama, Mobile, AL 36688, USA, 3Hospital de Cl|¨ nicas, Porto Alegre, Brazil The hyperphenylalaninemia variant primapterinuria is characterized by the excretion of 7-biopterin (primapterin). This disorder is thought to be due to a de¢ciency of PCD, but a lack of tissue activity has not been directly demonstrated. The six mutations so far recognized in patients with primapterinuria are associated with either a single amino acid change or a premature stop codon. Only C81 R has been successfully expressed in soluble form, and was found to have 40% of normal activity. Tissues which could be obtained by minimally invasive procedures were analyzed for dehydratase activity. None was detected in normal human white cells or ¢broblasts. However, activity was found in intestine of rat, dog, pig, and particularly humans where it was only eight times lower than in liver. Distribution along the length and across the wall of small intestine was relatively uniform. Moreover, the dehydratases from human liver and intestinal mucosa have identical kinetic properties. A biopsy of duodenal mucosa from a patient with homozygous E96K dehydratase had activity of 55 nmol x min-1g-1 mucosa compared to 329 + 32 nmol x min-1 g-1 tissue in controls (n=12). The 6-fold lower tissue activity of the E96K mutant alone may not be su¤cient to account for the biochemical symptoms of primapterinuria in this patient. However, accumulation of a 4a-hydroxy-BH4 degradation product (a side-chain cyclic adduct), which has been observed in vitro and appears to be a dehydratase inhibitor, may further exacerbate the problem.

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ENCEPHALOPATHY IN CHILDREN WITH CALCIFICATION OF BASAL GANGLIA CHARACTERIZED BY INTRATHECAL PTERINS EXCESS ¨ N. Blau1, L. Bonafe¨1, B. ThÎny1, V. Ramaekers2 1 Division of Clin. Chem. and Biochem, University Children's Hospital, 8032 ZÏrich, Switzerland; 2 Division of Pediatric Neurology, University Hospital Aachen; Germany. Calci¢cation of basal ganglia may be part of sever encephalopathy described under the heading of ``Fahr syndrome'' or it can be associated with inherited metabolic disorders like dihydropteridine reductase de¢ciency or 5,10-methylenetetrahydrofolate reductase de¢ciency. The clinical pattern is most variable with respect of age and neurological manifestations. In Aicardi-Gouitie©res syndrome, presenting with persistent lymphocytosis and elevated interferon alpha in CSF, so far no metabolic abnormalities were found. We investigated ¢ve infants, aged 10 mo to 3 yr, all from nonconsanguineous parents, with symmetrical calci¢cations of the striatum and periventricular regions, retarded myelinisation, microcephaly, and neurogical abnormalities like extrapyramidal movement disorders (dystonia, athetosis), and metal retardation. Prenatal infectious encephalopaties and calcium-phosphorus imbalance were ruled out. Routine and special metabolic screening as well as interferones and interleukines in CSF were normal. Investigations of CSF revealed normal neurotransmitter metabolites and folates, but extremely elevated levels of neopterin 650 + 133 nmol/l (normal:12-30) and biopterin 137 + 12 nmol/l (normal:15-40). In order to characterize this new syndrome we also investigated cultured skin ¢broblasts from one of the patients for tetrahydrobiopterin metabolizing enzymes, and initiated DNA analysis of the GTP cyclohydrolase feedback regulatory protein (GFRP).

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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43

085-O

DIAGNOSIS OF DOPA-RESPONSIVE DYSTONIA (DRD) AND OTHER TETRAHYDROBIOPTERIN (BH4) DEFECTS BY STUDY OF PTERIN METABOLISM IN CYTOKINE STIMULATED FIBROBLASTS L. Bonafe¨, B. ThÎny, N. Blau. Div. of Clinical Chemistry and Biochemistry, Univ. Children's Hospital, ZÏrich, CH

Autosomal recessive BH4 de¢ciencies (GTP cyclohydrolase I, GTPCH; 6-pyruvoyl-tetrahydropterin synthase, PTPS; dihydropteridine reductase, DHPR) are usually diagnosed by the presence of hyperphenylalaninemia, analysis of urinary and CSF pterins, followed by enzyme assays and mutation analysis. DRD, the autosomal dominantly inherited GTPCH de¢ciency, is diagnosed mainly by clinical criteria and CSF examination. In order to establish a diagnostic tool for these disorders, neopterin (N) and biopterin (B) production in cytokine (IFNg and TNFa)-stimulated ¢broblasts as well as enzyme activities were measured in 22 patients with di¡erent BH4 disorders and 38 controls. Reference ranges for N and B production and enzyme activities were age-dependent. GTPCH-de¢cient cells showed very low N and B levels; enzyme activity was signi¢cantly decreased, being lower in DRD. In PTPS-de¢cient cells very high levels of N and no B were produced. DHPRde¢cient cells exhibited intracellular pterins levels in the normal range, and no detectable enzyme activity. In conclusion, cultured skin ¢broblasts are a useful system for the diagnosis of all forms of BH4 de¢ciency. The same method was used successfully in cultured amniocytes for prenatal diagnosis of PTPS and DHPR de¢ciencies. Fibroblasts from DRD patients showed typical pterin production patterns and low residual enzyme activity; both are essential for the diagnosis of this disease.

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THE STUDY OF TETRAHYDROBIOPTERIN DEFICIENCY IN NORTHERN CHINESE WITH HYPERPHENYLALANINEMIA W.M. Yu1, T.T. Liu2, M. Chang1, Z.S. Zhou1, M. Shen1, and K.J. Hsiao2 China^Japan Friendship Hospital, Beijing, China1, National Yang-Ming University, Taipei, Taiwan2

A screening program for tetrahydrobiopterin (BH4) de¢ciency in hyperphenylalaninemia (HPA) from northern Chinese has been performed at China^Japan Friendship Hospital since 1993. The following tests were done in 280 patients with HPA: (i) analysis of pterins in urine; (ii) measurement of dihydropteridine reductase activity in erythrocytes from Guthrie cards. Of the 280 HPA patients, 10 patients with BH4 de¢ciencies have been recognized as a result of screening carried out. All of these 10 BH4 de¢cient patients su¡ered from 6-pyruvoyl-tetrahydropterin synthase (PTPS) de¢ciency. The frequency of BH4 de¢ciency is 3.6% in northern Chinese HPA. Four single base mutations at nucleotides 166(G4A), 209 (T4A), 259(C4T), and 286(G4A) on PTPS cDNA were detected in northern Chinese PTPS-de¢cient HPA by polymerase chain reaction and solid phase DNA sequencing. By analysis of 18 PTPS mutant alleles from 9 unrelated northern Chinese PTPS-de¢cient HPA families, the allele frequencies of these mutations were determined to be 5.6% (166G4A), 5.6% (209T4A), 61.0% (259C4T) and 27.8% (286G4A), respectively. The latter two mutations are common mutations in northern Chinese PTPS-de¢cient HPA. Eight patients su¡ering from PTPS de¢cient HPA were treated with neurotransmitter precursors as well as with tetrahydrobiopterin, and were followed up by health workers. Five patients were treated when the infants were less than 6 months old, the mental and physical developments of them were normal. Three patients were treated at the age of 1^2 years, although their neurological symptoms were remarkably improved, they still have various degrees of mental retardation. These results again suggest that early di¡erential diagnosis of HPA is essential for the treatment and outcome of HPA patients.

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SCREENING FOR TETRAHYDROBIOPTERIN DEFICIENCY AMONG HYPERPHENYLALANINEMIC SOUTHERN CHINESE Jun Ye MD, Xiao Q. Liu MD PhD, Xie Q. MA, MD*, Rui G. Chen MD, Xue F. Gu MD PhD Xin Hua Hospital, Shanghai Institute for Pediatric Research, Shanghai Second Medical University, Shanghai, China. Maternal and Child Health Care Hospital, Guangzhou, China* Objective: To ¢nd the incidence of tetrahydrobiopterin de¢ciency (BH4D) among patients with hyperphenylalaninemia in Southern Chinese and evaluate the clinical outcome of BH4D) patients. Methods: Analysis of urinary neopterin and biopterin was done in 87 patients with hyperphenylalaninemia by HPLC. Gene mutation analysis was performed for patients with BH4D and their parents and the patients were treated soon after diagnosis. Results: Eleven patients were diagnosed as having 6-Pyruvoyl- tetrahydropterin synthase (PTPS) de¢ciency at age of 21d^6y, with the incidence of BH4 de¢ciency of 10%. PTPS gene mutations (P87S, N52S, D96N and G144R) were detected from 5 PTPS-de¢cient families. The ¢ve PTPSde¢cient patients were treated with synthetic BH4 (1^2mg/kg.d), neurotransmitter precursors L-dopa (5^10mg/kg.d), and 5-hydroxytryptophan (5^7mg/kg/d). They had satisfactory physical and mental development (IQ70^80) after treatment. Conclusions: Our results emphasise that the screening for BH4 de¢ciency should be carried out on all patients with hyperphenylalaninemia.

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DIHYDROPTERIDINE REDUCTASE DEFICIENCY ACCOUNTS FOR ALL PHENYLKETONURIA IN THE MALTESE POPULATION R. Farrugia1, I. Dianzani2, S. Attard Montalto3, A.E. Felice1 1 Laboratory of Molecular Genetics, Dept of Pathology, University of Malta, Msida MSD 06, Malta; 3 Department of Paediatrics, St. Luke's Hospital, G'Mangia, Malta; 2Dipartimento di Scienze Pediatriche e dell'Adoloscenza e Dipartimento di Genetica, Universita' delgi Studi di Torino, Italia Phenylketonuria (PKU) is a rare disorder of phenylalanine metabolism, generally with an occurrence of 1in 1000 live births. Dihydropteridine reductase (QDPR) de¢ciency is a rare form of PKU found in 2% of hyperphenylalaninaemia patients. In the last ¢ve years 3 probands have been identi¢ed. All are under 4 years of age and are not in any way related. The aim of this study was to identify the causative mutation in the probands and their family members, and use the information obtained to investigate the occurrence of QDPR de¢ciency in the Maltese population. Biochemical analysis and enzyme activity tests showed that in all three cases, the de¢cient enzyme was not phenylalanine hydroxylase but dihydropteridine reductase. DNA was extracted from whole blood samples of the 3 probands, their parents and siblings. The QDPR gene was ampli¢ed by the polymerase chain reaction (PCR). The resulting exon 1 fragment was tested by restriction enzyme analysis using HinfI, for the G23D mutation (QDPR exon 1 68G?A). All 3 probands were found to be homozygous for G23D. The presence of the mutation was con¢rmed by restriction enzyme analysis of parent DNA and by dideoxy sequencing of QDPR exon 1. PCR and restriction digest were used to diagnose a newborn brother of one of the probands. A cohort of random cord DNA samples was tested for the presence of the G23D mutation. Our preliminary results on 100 samples show a carrier frequency in the region of 2%. The G23D mutation is the only hyperphenylalaninaemia causing mutation identi¢ed so far in the Maltese population. This study shows that the occurrence of QDPR de¢ciency in the Maltese population is higher than that previously determined for other populations. J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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TETRAHYDROBIOPTERIN-RESPONSIVE PHENYLALANINE HYDROXYLASE DEFICIENCY - A NOVEL CLINICAL ENTITY S. Kure, D.C. Hou, *T. Ohura, **H. Iwamoto, ***S. Suzuki, ****N. Sugiyama, *O. Sakamoto, Y. K. Fujii, Suzuki, Y. Matsubara, K. Narisawa. Depts. of Medical Genetics and *Pediatrics, Tohoku University School of Medicine, Sendai, Japan; **Division of Pediatric Neurology, Kanagawa Children's Medical Center, Japan; ***Dept. of Pediatrics, Osaka Medical Collage, Osaka, Japan; ****Dept. of Pediatrics, Yokkaichi Municipal Hospital, Japan.

Hyperphenylalaninemia (HPA) is caused by a de¢ciency of either phenylalanine hydroxylase (PAH) or its cofactor, tetrahydrobiopterin (BH4). BH4 and combined phenylalanine (Phe) and BH4 loading tests are currently used for di¡erential diagnosis, since the serum Phe concentration decreases after BH4 administration in BH4, but not PAH de¢ciency. Recently we encountered four patients with HPA whose elevated serum Phe concentrations gradually decreased following the oral administration of BH4 (Kure, et al., J Pediatr, 1999;135: 375-378). They showed no abnormalities in urinary pteridine analysis or in dihydropteridine reductase activity, but had mutations in the PAH gene. The observation suggests a novel subtype of PAH de¢ciency that may be treated with cofactor supplementation.

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TETRAHYDROBIOPTERIN-RESPONSIVE HYPERPHENYLALANINEMIA (HPA) IN DUTCH NEONATES L.J.M. Spaapen1, J.A. Bakker1, C. Velter2, W. Loots2, M.E. Rubio3, P.P. Forget3, M. Duran4, L. Dorland4, B.T. Poll-The4, H.K. Ploos van Amstel4, J. Bekhof5, N. Blau6 1 Dept. of Biochem. Genetics and 2Molec. Genetics, Stichting Klinische Genetica ZON, 3Dept. of Pediatrics, Academic Hospital Maastricht, 4University Medical Center Utrecht, 5Dept. of Pediatrics, Academic Hospital Groningen, The Netherlands, 6Dept. of Clin. Chem. and Biochem., University Children's Hospital, Zurich, Switzerland

Four neonates with a positive phenylalanine screening test (Phe concentrations between 258 and 1250 mmol/l) were investigated further in order to di¡erentiate between PAH de¢ciency and variant HPA forms. In patients 1 and 2 a BH4 load caused a signi¢cant decrease of the plasma Phe levels. A combined phenylalanine/BH4 loading test was performed in patients 3 and 4. In the latter patients plasma Phe concentrations completely normalised within 8 hours after the BH4 load (20 mg/kg). Basic urinary pterins were normal in all 4 patients. The activity of DHPR in erythrocytes was normal in patients 2 and 3 and 50% of control values in patient 4. In patient 3 a subsequent phenylalanine loading test with concomitant analyses of plasma biopterins revealed a normal increase of biopterin excluding a BH4 biosynthesis defect. Pterins and neurotransmitters in CSF of patient 3 and 4 were found normal. DNA mutations found in the PAH gene of patients 1-4: V190A; R243X, A313T; 1099 ins C, R241C; A403V, A300S; A403V. The results are suggestive for a mutant PAH enzyme with a decreased a¤nity for the co-factor BH4. These HPA variants will only be found if a BH4 loading test is included in the di¡erential diagnosis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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EFFECT OF EARLY L-DOPA AND TETRAHYDROBIOPTERIN TREATMENT ON PREFRONTAL FUNCTION IN 6-PYRUVOYLTETRAHYDROPTERIN SYNTHASE DEFICIENCY Y. Tanaka1, M. Kato2, F. Saito2, H. Shintaku3, Y. Okano3, K. Tanaka4, H. Kondo4, S. Yamamoto5, T. Nukazawa6, N. Matsuo7 Department of Pediatrics, Tokyo Dental College, Ichikawa General Hospital1, Department of Neuropsychiatry, Tokyo Dental College, Ichikawa General Hospital2, Department of Pediatrics, Osaka City University Medical School3, Department of Neurology, Niigata University4, Department of Pediatrics, Chiba University5, Department of Psychiatry, Kanagawa Rehabilitation Center6, Department of Pediatrics, Keio University School of Medicine, Japan7 Objective: 6-pyruvoyltetrahydropterine synthase (PTPS) de¢ciency is characterized by dopamine de¢ciency in central nervous system which is likely to result in irreversible prefrontal dysfunction. To test the hypothesis that the prefrontal dysfunction may be rescued by early treatment with l-dopa and tetrahydrobiopterin (BH4), we studied six PTPS de¢ciency patients for the relationship between performances on various neuropsychological tests and the age at the start of the therapy. Methods: The age of the patients when the therapy started was 0.1 year (male) in #1, 0.6 year (male) in #2, 1 year (male) in #3, 2.6 years (female) in #4, 27 years (male) in #5 and 41 years (female) in #6. Age of the subjects ranged from 10 to 47 years and IQ of the subjects ranged from 44 to 75 on WAIS-R. They were studied for attention (cancellation test, digit span), verbal ability (word £uency), memory (paired association learning) and prefrontal function (Wisconsin Card Sorting Test [WCST], Tower of Hanoi [TOH]). Results: Attention, memory and verbal ability in each patient was comparable to that of controls. WCST and TOH were comparable to that of IQ matched control in #1, #2, #3 and #4 and were uncomparable in #5, and #6. Conclusion: Prefrontal dysfunction may be minimized in PTPS de¢ciency patients if the treatment is started during early childhood.

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PRENATAL DIAGNOSIS OF DHPR DEFICIENCY BY DIRECT MUTATION ANALYSIS USING DGGE Romstad A.1, Kalkanogïlu H.S.2, Cos° kun T.2, GÏttler F.1 1 The John F. Kennedy Institute, Department of Inherited Metabolic Diseases and Molecular Genetics, DK-2600 Glostrup, Denmark; 2Hacettepe University, Faculty of Medicine, Department of Pediatrics, Division of Nutrition and Metabolism TR-06100 Sihhiye, Ankara, Turkey

Dihydropteridine reductase (DHPR) is an enzyme involved in the recycling of tetrahydrobiopterin (BH4), which is an obligate cofactor of aromatic amino acid hydroxylases. DHPR de¢ciency is a rare, autosomal recessive disorder caused by mutations in the QDPR gene. DHPR de¢cient patients are diagnosed by a lack of response to a low-phenylalanine diet and by severe neurological symptoms. Treatment of DHPR de¢ciency can be di¤cult and the outcome is not always satisfying, even if all treatment strategies are followed. Therefore prenatal diagnosis is of great importance in a¡ected families. Prenatal diagnosis is possible by measuring DHPR activity in di¡erent cell types, but this is time consuming. More than 20 di¡erent mutations have by now been identi¢ed in the QDPR gene and direct identi¢cation of a mutation in a fetus would be easy and fast. We have developed a method based on denaturing gradient gel electrophoresis (DGGE) for the analysis of the QDPR gene. The method is useful for rapid and simultaneous scanning of all exons and £anking intronic sequences of the QDPR gene. We describe the ¢rst prenatal diagnosis using this method.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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LATE DIAGNOSIS OF 6-PYRUVOYL-TETRAHYDROBIOPTERIN SYNTHASE DEFICIENCY IN AN 18-YEAR OLD WOMAN PREVIOUSLY TREATED FOR PHENYLKETONURIA Maria Gizçewska1, Cezary Zçekanowski2, Maria Nowacka3, Wioleta Bich1, Mieczyslaw Walczak1 1 II Department of Paediatrics, Pomeranian Academy of Medicine, Szczecin, Poland; 2Department of Genetics, Nationale Research Institute of Mother and Child, Warsaw, Poland; 3Paediatric Clinic, Nationale Research Institute of Mother and Child, Warsaw, Poland

De¢ciencies of tetrahydrobiopterin (BH4), account for 1-2% of all cases of persistent hyperphenylalaninemia. The procedures for early detection of these disorders, as a part of all newborn screening, allow for early treatment with BH4 and neurotransmitter precursors. However, lack of proper diagnosis may still occurs, especially among the older patients with PKU, who were not previously examined for BH4 de¢ciency, do not response to the diet and demonstrate progressive neurological derangements. We present a case of an 18-year old woman, who has been treated with lowphenylalanine diet for typical PKU. Although the Phe levels were always satisfactory, since she was 15 we observed increasing irritability, learning disabilities, aggression, drowsiness and progressive mental retardation. Performing the di¡erential diagnosis for BH4 de¢ciency, the following results allowed as to detect a mild form of PTPS de¢ciency: positive combined BH4 and Phe loading test, proper DHPR activity, urinary pterins level (mmol/mol cr.) neopterin 2.57, biopterin 0.61 and N72L/N36K mutations in PTPS gene. Since the patient has been introduced to BH4, L-Dopa and 5hydroxytryptophan therapy her neurological as well as psychological status has markedly improved.

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TREATMENT OF MILD PHENYLKETONURIA (PKU) BY TETRAHYDROBIOPTERIN (BH4) Friedrich Trefz1, Nenad Blau2, Christa Aulehla-Scholz3, Herbert Korall4, Georg FrauendienstEgger1 Klinik fu«r Kinder und Jugendmedizin Reutlingen, School of Medicine, University of Tu«bingen, Germany1; Abteilung fu«r Klinische Chemie und Biochemie, Kinderspital Zu«rich, Switzerland2; Institut fr Klinische Genetik Stuttgart, School of Medicine, University of Tu«bingen3; Zentrum fu«r sto¡wechseldiagnostik Reutlingen GmbH, Reutlingen, Germany4 A patient with mild PKU responsive to BH4 supplementation was found in the newborn screening program with blood phenylalanine (phe) of 288 mmol/l increasing to 885 mmol/l at 14 days of age. BH4-loading (60 mg corresponding to 20 mg/kg bodyweight) resulted in a decrease of blood phe to 96 mmol/l 8 hours post load. Under a normal feeding with an adapted formula plasma phe levels went up again to 934 mmol/l. Under a daily supplementation of 45 mg of BH4 blood phe levels dropped down again and remained between 84^222 mmol/l. Surprisingly there was no coenzyme de¢ciency (normal values for neopterin and biopterin in urine, normal dihydropteridinereductase activity in red blood cells, normal neurotransmitters in cerebrospinal £uid). However, mutation analysis of the phenylalanine hydroxylase gene revealed the two mutations IVS10G-11A in intron 10 and E390G in exon 11. The ¢rst one creates a zero activity of the enzyme, the second one is a missense mutation, both together result in a phenotype having mild PKU (1). We speculate that there may be more mutations resulting in a Km-variant of the phenylalanine hydroxylase enzyme in which enhancement of the residual activity can be achieved by supplementation of BH4 as recently also found in HPA patients in Japan (2). Our observation strongly emphasizes the necessity of the BH4 loading test in the newborn period and further DNA analysis in HPA patients responsive to BH4 supplementation. 1) Scriver CR et al.: Hum Mutat 2000; 15(1):99^104. 2) Kure S et al.: J Pediatr 1999; 135(3) 375^8

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RESPIRATORY ALKALOSIS IS NOT PATHOGNOMONIC OF UREA CYCLE DEFECTS A.B. Chakrapani, J.H. Walter Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester, M27 4HA, UK Respiratory alkalosis has been stated to be pathognomonic of urea cycle defects presenting in the neonatal period. In our experience, some of these cases may present wih acidosis. On the other hand, respiratory alkalosis may be initially present in other early-onset metabolic disorders. We reviewed the initial blood gases in all cases of urea cycle defects presenting to our unit in the 1st 4 weeks of life, and of other acute metabolic conditions presenting in the neonatal period for the presence of initial respiratory alkalosis. RESULTS: Out of a total of 12 cases presenting with neonatal-onset urea cycle defects, data on initial blood gases was available for 11. Six patients presented with a respiratory alkalosis, 2 had a normal pH with compensated metabolic acidosis, whereas 3 had uncompensated acidosis. Of the patients with acidosis (pH57.35), two cases (one with citrullinaemia, one with ornithine carbamyltransferase de¢ciency) had a metabolic acidosis on admission and another with citrullinaemia had a mixed acidosis. 3 out of 13 patients with early-onset propionic acidaemia had respiratory alkalosis at presentation. Respiratory alkalosis was not a presenting feature of other metabolic disorders. CONCLUSION: Though respiratory alkalosis is commonly an early feature in neonatal-onset urea cycle disorders, acidosis may be seen at presentation in some cases. Alkalosis may also be an early sign of neurologic distress in other metabolic disorders such as propionic acidaemia. In addition to hyperammonaemia, other abnormal metabolites may contribute to the pathogenesis of respiratory alkalosis in such cases.

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DEVELOPMENTAL OUTCOME IN NEONATAL ONSET UREA CYCLE DEFECTS: EARLY PROGNOSTIC INDICATORS A.B. Chakrapani, J. Sandju, J.H. Walter Willink Biochemical Genetics Unit, Hospital Road, Manchester, M27 4HA, UK There is no consensus regarding early prognostic indicators of the neurodevelopmental outcome of children with neonatal onset urea cycle defects (UCD). A review of all patients presenting to our unit with UCD in the neonatal period was performed. Neurodevelopmental outcome was correlated with the peak plasma ammonia and glutamine levels and the duration of the initial hyperammonaemic coma. RESULTS: 16 patients with neonatal-onset UCD have been seen on our unit, of which 4 have been treated prospectively. The survivors are currently 4 months to 11 years of age. Of the 12 cases presenting with acute hyperammonaemia, 2 patients died during the presenting episode. 5 have severe neurodevelopmental problems (DQ550), 2 have moderate developmental di¤culties (DQ 51^80) and 5 have a relatively good outcome (DQ481) or normal neurological examination). The peak ammonia and glutamine levels did not correlate with developmental outcome. A good developmental outcome was observed if initial ammonia levels were reduced to less than 250mmol/l and/or the initial hyperammonaemic coma resolved within 48 hours of the onset of symptoms, but moderate to severe developmental problems occurred if this duration was 4 days or longer. The 4 patients treated prospectively have all shown a favourable outcome. CONCLUSION: The outcome of neonatal onset UCD appears to correlate with the duration of initial hyperammonaemic coma rather than the peak plasma ammonia or glutamine levels. Very high ammonia levels at the outset maybe compatible with near normal development if the initial hyperammonaemic coma is reversed within 2 days. Neonates with urea cycle defects must be managed aggressively irrespective of the peak plasma ammonia levels.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PLASMA GLUTAMINE/GLUTAMATE, ASSOCIATED WITH HYPERAMMONEMIA OF VARIOUS ORIGIN J.G.M. Huijmans1, J.B.C. de Klerk2, R. Jankie1, M. Williams2 and M. Duran1 Erasmus University, Dept of Clinical Genetics1 and Pediatrics2, Rotterdam, The Netherlands.

Hyperammonemia in children is a serious condition which may lead to acute and/or chronic neurological damage. Various metabolic conditions have been shown to be associated with persistent hyperammonemia, such as congenital defects of the urea cycle, several organic acidemias, dysfunctioning of glutamate dehydrogenase, and the administration of valproate. Ammonia which is highly neurotoxic itself- can be scavenged by various metabolic processes, such as: 1. formation of glutamate from 2-oxoglutarate and 2. formation of glutamine from glutamate. The correlation between the plasma glutamine+glutamate (GLU) level (measured with a Pharmacia Biochrome 20 Analyzer) and the ammonia level was investigated in patients with various types of urea cycle defects and organic acidemias. Results indicate that in general there is only a poor correlation between GLU and ammonia in plasma. Patients with OTC de¢ciency have higher GLU levels (800-1600 mM) than patients with NAGS de¢ciency, citrullinemia or argininosuccinic aciduria (250-850 mM). In these OTC patients the ammonia level on treatment tends to be higher (15-200 mM), compared with the other urea cycle defects (10-100 mM). Patients with various organic acidemias show GLU levels in acute episodes, which are decreased, in spite of sometimes extremely high ammonia levels. These results show, that there is no causal relation-ship between GLU and ammonia, but only a poor correlation. In addition one should be aware of possible toxic e¡ects of glutamine in causing encephalopathy, especially in OTC patients.

098-P

EVALUATION AND MANAGEMENT OF UREA CYCLE DISORDERS USING STABLE ISOTOPE INFUSIONS Fernando Scaglia1, William O'Brien1, Judy Rosenberger2,3, Qiping Zeng1, Peter Reeds2,3 and Brendan Lee1. 1 Dept. of Molecular and Human Genetics, 2Dept. of Pediatrics, and 3Children's Nutritions Research Center, Baylor College of Medicine, Houston TX.

We have used the ratio of isotopic enrichments of 15N urea/15N glutamine (15N-U/G) as a sensitive index of in vivo urea cycle activity. We have now applied this method to the 1) diagnosis of asymptomatic at-risk partial ornithine transcarbamylase de¢ciency (OTCD) females in two OTCD sibships, and 2) comparison of di¡erences in the bioavailability of enteral vs. systemic sources of nitrogen for total body urea production. By determining the 15N U/G ratio in ¢ve at-risk females in two OTCD sibships in which mutations in the OTC gene were not found in the index case, normal and abnormal urea production were measured enabling us to determine carrier status. In one of these two sibships, we used in vivo measurements of urea synthesis to complement enzymatic measurements of fetal liver biopsy specimens to evaluate the carrier status of an at-risk female and her pregnancy. In the second study, we tested the hypothesis that there is more e¤cient utilization of intestinally derived ammonia and alanine as precursors for ureagenesis. By comparing the transfer of 15 N from oral and intravenous [15N]H4Cl and [15N]-alanine to urea, we found individuals who have partial urea cycle activity metabolize excessive dietary protein more e¤ciently supporting previous observations that these patients are susceptible to fasting and stress. These data may help to guide nutritional management of patients with urea cycle disorders especially during situations of illness and catabolism.

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MONITORING THE EFFICACY OF DIETARY TREATMENT IN UREA CYCLE DISORDERS L.T.K. Wong, A.G.F. Davidson, Y.P. Lillquist, C. Hartnett, D.A. Applegarth and H. Vallance Departments of Pediatrics and Pathology, Children's and Women's Health Center of British Columbia, Vancouver, BC, Canada Introduction. Hyperammonemia in urea cycle disorders can be e¡ectively controlled with strict dietary protein restriction, sodium phenylbutyrate/phenylacetate/benzoate and amino acid supplementation to prevent hyperammonemia and consequent brain damage. Care has to be taken not to overzealously restrict the diet and induce dietary de¢ciencies particularly of amino acids. In order to properly manage these patients, regular plasma amino acid monitoring is essential. However there is no consensus as to the optimal timing of the blood collection in relation to meals. Aim. This study examines the dynamic changes of plasma amino acids following a meal in order to determine the most appropriate timing for blood monitoring. Method. 8 patients with urea cycle disorders: Four with Ornithine Transcarbamylase De¢ciency (OTC), two with argininosuccinic lyase de¢ciency (ASA), one with argininosuccinic synthetase (ASS) (Citrullinemia) and one with carbamyl phosphate synthetase de¢ciency (CPS) were studied. Amino acid results were analyzed after (a) `Regular' 6 to 10 hours fast; (b) Fasting and 2 hour post formula; (c) Diet loading. Results. There were signi¢cant changes in blood ammonia and plasma amino acids (glutamine, ornithine, citrulline and arginine) following meals. Maximal changes occurred 1^2 hr post-meal. The 4hr post-meal samples returned to levels more closely re£ecting the steady rate particularly with regard to the amino acids proximal and distal to the metabolic block. Conclusion. 4-hour fasting levels appear to be the most appropriate choice for blood monitoring in urea cycle patients.

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CONFUSING ALLOPURINOL TESTS IN GENOTYPIC OTC HETEROZYGOTES William L. Nyhan, Bruce A. Barshop and Vicente Rubio University of California San Diego, U.S. and Instituto de Biomedicine de Valencia, Spain Allopurinol challenge has been useful in the detection of heterozygous carriers of the gene for ornithine transcarbamylase (OTC) de¢ciency. This inhibitor of orotidine monophosphate decarboxylase detects accumulation of carbamyl phosphate by assay of the urine for orotic acid and/or orotidine. It has been used most commonly in relatives of infants with the classic neonatal onset phenotype. We studied a male with a variant phenotype whose ¢rst episode of hyperammonemia was at 12 years of age and who with treatment has not had a subsequent symptomatic hyperammonemic episode. His asymptomatic mother and sister were tested with allopurinol. Peak excretions of orotic acid were 3 and 10 mmoles of orotic acid per mole creatinine, considerably less than the mean of 83 in 5 heterozygotes. Analysis of the OTC gene in the proband revealed an A to T transversion at position 36 specifying a Leu301 Phe amino acid replacement in the enzyme. Both mother and sister were found to carry the mutation. It is concluded that allopurinol testing may not be adequate for the detection of carriers of variant forms of this enzyme de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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FLUCTUATIONS OF OROTIC ACID AND URACIL LEVELS DURING MILD HYPERAMMONEMIC ATTACKS IN PATIENTS WITH OTC DEFICIENCY Tetsuya Ito 1, Satoshi Sumi1, Kiyoshi Kidouchi2, Kyoko Ban1, Akihito Ueta2, Hajime Togari1 and Yoshiro Wada1 Department of Pediatrics, Nagoya City University Medical School, Nagoya, 467-8601, Japan1, Department of Pediatrics, Nogoya City Higashi General Hospital, Nagoya, 464-0071, Japan2

Ornithine transcarbamy lase (OTC) de¢ciency is an X linked disorder of the urea cycle. High excretory levels of pyrimidines are observed in these patients because accumulated carbamylphosphate is utilized to de novo synthesis of pyrimidines. Pyrimidine excretory patterns in OTC de¢ciency have been reported, however, £uctuations of pyrimidines during a hyperammonemic attack in mild patients are not clear. We report here £uctuations of uracil and orotic acid levels during mild hyperammonemic attacks in a female and a partial de¢cient male patient with OTC de¢ciency. Case one was a 4-year-old female with OTD de¢ciency (OTC activity in liver tissue; 17% of controls) presenting hyperammonemia and liver dysfunction. Case two was a 9-year-old male. He showed acute encephalopathy at 14-months-old and diagnosed by liver biopsy at 28-months-old (OTC activity; about 10% of controls). Orotic acid and uracil levels in urine samples from the patients were analyzed continuously with column switching HPLC method. Mild hyperammonemic attacks (the range of blood ammonia levels; 68^131 mmol/l) were observed during the period. Orotic acid excretion increased dramatically (above 10 times higher than before) and recovered within a few days. Uracil excretion remained at high levels throughout the period. The results suggest that the orotic acid excretory level is a very sensitive parameter for attacks, while the uracil excretion level, which may re£ect a chronic state of urea cycle dysfunction, is suitable for the screening method of partial OTC de¢ciency.

102-O

PASSIVE AVOIDANCE AND SPATIAL LEARNING IN ORNITHINE TRANSCARBAMYLASE-DEFICIENT MICE R. D'Hooge (1), B. Marescau (1), I.A. Qureshi (2), P.P. De Deyn (1) (1) Laboratory of Neurochemistry and Behaviour, Born-Bunge Foundation, University of Antwerp, Belgium; (2) Service de Ge¨ne¨tique Me¨dicale, Hoªpital Ste-Justine, Montre¨al, Canada

Mutant sparse fur (spf) mice are a model for the congenital de¢ciency of ornithine transcarbamylase (OTC), the most common inborn error of urea synthesis in man. In this study, spf and control mice of 8-10 weeks old were behaviourally tested under normal (Arg +) or arginine-free (Arg -) diet conditions. Baseline plasma ammonia concentrations were signi¢cantly higher (202 þ 39 mM, n=7) than in controls (53.1 þ 9.6 mM, n=7). In spf mice, but to a lesser extend also in the control group, plasma ammonia concentration was signi¢cantly increased during Arg(-) diet. Performance of spf mice on Arg(+) diet in the passive avoidance learning test was normal, and they showed no evidence of impaired spatial learning and memory in the Morris water maze test. However, passive avoidance retention was impaired in spf mice which had been on Arg(-) diet during the training period. Mutant mice on Arg(-) diet during the ¢rst 5 trial blocks of hidden-platform water maze acquisition also showed spatial acquisition and retention de¢cits. Learning to ¢nd a visible platform was not impaired in spf mice on Arg(-) diet. The learning and memory de¢cits described here might relate to the cognitive de¢cits seen in patients with OTC de¢ciency, and might be useful outcome measures in therapeutic studies using the spf model. (Supported by Born-Bunge Foundation and MRC Canada)

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LATE-ONSET ORNITHINE TRANSCARBAMYLASE DEFICIENCY IN MALE PATIENTS: PROGNOSTIC FACTORS AND CHARACTERISTICS OF PLASMA AMINO ACID PROFILE Yoshino M, Nishiyori A, Tokunaga Y, Kuriya N and Kato H. Department of Pediatrics and Child Health, Kurume University School of Medicine Background: The occurrence of male patients with ornithine transcarbamylase (OTC) de¢ciency during adolescence or in adulthood has now been recognized. Objectives: To determine the prognostic factors that a¡ect the prognosis of life, to explore a basis of therapeutic strategy. Methods: In ten patients, nine of whom carried the R40H mutation and the other one, the Y55D mutation in the OTC gene, 32 demographic and laboratory data were ¢rst compared between survivors (n=4) and non-survivors (n=6) using the unpaired t test. The factors with signi¢cant di¡erence were then subjected to multiple regression analysis. Results: The factors that exhibited signi¢cant di¡erence were; age at onset, concentration of plasma ammonium, blood pH, and concentrations of eight amino acids in plasma. The multiple regression analysis then revealed concentrations of ammonium, leucine, lysine, isoleucine, phenylalanine, glutamine and proline to be signi¢cant prognostic factors. The amino acid pro¢le in the ten patients showed increase in glutamine, proline, lysine, valine and methionine, and decrease in serine, ornithine and arginine. There was an inverse correlation between the age at onset and the level of the residual hepatic OTC activity. Conclusion: The results implied that 1) in addition to plasma ammonium concentration, the plasma concentration of each of the six (above-mentioned) amino acids was a signi¢cant predictor of prognosis, 2) the plasma amino acid pro¢le was unique, in comparison to other liver diseases, and hence was of diagnostic value.

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DEATH AFTER TRANSPLANTATION OF A LIVER FROM A DONOR WITH UNRECOGNIZED ORNITHINE TRANSCARBAMYLASE DEFICIENCY. A NEED FOR PRETRANSPLANTATION IEM SCREENING ? E. Ploechl1, W. Ploechl2, S. Stoeckler-Ipsiroglu3, B. Wermuth4, A. Roscher5 1 Clinical Genetics, St. Johanns Hospital, Salzburg; Departments of 2Anaesthesiology and General Intensive Care and 3Pediatrics, University of Vienna; 4Central Chemical Laboratory, Inselspital, University of Berne; 5Hauner'sches Children's Hospital, University of Munich Thorough medical evaluation of both donor and recipient is essential for the success of organ transplantation. Standard procedures, including chemical, immunological and microbiological analysis may not be su¤cient in cases of unclear cause of death of the donor. We report on the fatal outcome of a liver transplantation from a previously healthy 26 year old man who had died of cerebral edema due to unrecognized late onset OTC-de¢ciency. OTC de¢ciency was retrospectively suspected when the liver graft recipient developed cerebral edema due to hyperammonemia, and the diagnosis was established by enzyme and molecular analysis in a biopsy of the transplanted liver. This fatal event highlights the fact that asymptomatic or unrecognized patients with late-onset IEM, e.g. urea cycle disorders, organic acidurias and fatty acid oxidation defects , are at high risk of metabolic decompensation or may even die during surgical procedures. With the more widespread availability of fast and e¡ective screening technology (MS-MS) in the future, it might be considered to screen for selected IEM prior to surgeries or during pretransplantation procedures.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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OTC DEFICIENCY AND SUBACUTE NECROTIZING ENCEPHALOPATHY Haas R.H., Barshop B.A., Nyhan W.L. and Billman, G.F. University of California San Diego and San Diego Children's Hospital and Health Center, San Diego, CA, USA.

The brain pathology of ornithine transcarbamylase (OTC) de¢ciency is thought to result from hyperammonemia and cerebral edema. Cavitary change with neuronal loss and gliosis with Azheimer type II astrocytes is seen. We report neuropathologic changes of subacute necrotizing encephalopathy in the case of a 7 year old girl with OTC de¢ciency. The patient was diagnosed at age 3 (assay-proven with demonstrated gene deletion) and had been well for 4 years without neurological disability. She was a strict vegetarian who did not drink milk. Management consisted of a low protein (1g/kg) diet, essential amino acids, multivitamins, citrulline and phenylbutyrate. She was admitted with a 3 day history of lethargy and 2 episodes of vomiting, and maximum ammonia of 232 mM. There was a good response to standard treatment. Four days later, she developed headache, irritability and draging of her leg. She rapidly developed intractable status epilepticus and died 6 weeks after admission with CT/ MR evidence of progressive brain atrophy with basal ganglia necrosis. Investigations found no cause, and lactate remained normal. The brain had extensive subacute necrotic change, cerebral cortex laminar necrosis, necrosis in putamina, tegmentum and basis pontis, medullary pyramids and mamillary bodies. The histology was typical for Leigh's or Wernicke's encephalopathy, with spongiform change, capillary proliferation, gliosis and relative preservation of neurons, a neuropathology compatible with thiamine de¢ciency. A low protein vegan diet limits thiamine intake. This patient's vitamins were administered with antacids. Thiamine is unstable in alkaline solution. We hypothesise that parenteral glucose precipitated a thiamine de¢ciency crisis resulting in CNS injury. Patients on low protein diets may be at risk for this complication and thiamine status should be considered.

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DIFFERENTIAL DIAGNOSIS OF HYPERAMMONAEMIA: ORNITHINE TRANSCARBAMYLASE DEFICIENCY PRESENTING WITH NORMAL URINARY OROTIC ACID EXCRETION Raiman JAJ1,Champion MP2, Baker AJ1, Dalton RN 2. 1 Department of Paediatric Hepatology, Kings College Hospital, London ;2 Department of Paediatric Metabolic Medicine, Guys Hospital, London, UK.

The measurement of urinary orotic acid is an essential component of the di¡erential diagnosis of hyperammonaemia. Classically ornithine transcarbamylase (OTC) de¢ciency (McKusick 311250) is characterised biochemically by increased urinary orotic acid excretion in the presence of hyperammonaemia, increased glutamine, reduced citrulline and arginine, and normal organic acids. We report an eight month old child who presented with an acute encephalopathy and liver dysfunction. Initial results of raised plasma ammonia (198 mmol/l), glutamine (1060 mmol/l) and reduced arginine (22 mmol/l) were indicative of a urea cycle defect. Urinary orotic acid excretion measured at that time was only 3.8 mmol/mol creatinine (normal 55) suggesting a diagnosis of either carbamylphosphate synthetase (CPS) de¢ciency or N-acetylglutamate synthetase de¢ciency. However, subsequent enzymology revealed the true diagnosis to be OTC de¢ciency, activity 4.0 mmol/hr/mg protein (normal 13-43), with normal CPS activity, 5.9 mmol/hr/mg protein (normal 0.65.5). We conclude that the absence of elevated orotic acid excretion does not exclude the diagnosis of OTC.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CENTRAL PONTINE AND EXTRAPONTINE MYELINOLYSIS, A REVERSIBLE CAUSE OF COMA IN PARTIAL OTC DEFICIENCY Grant Mitchell (1), Patrick Cossette (1), Anne Lortie (1), J-Claude De¨carie(2), Franc°ois Proulx (1), Ijaz A Qureshi (1), Marie Lambert (1) [Dept Pediatr (1) and Radiol (2) Ste-Justine Hosp, Montreal, Canada] A 3-year-old girl with partial ornithine transcarbamylase (OTC) de¢ciency (OMIM 311250) developed central pontine and extrapontine myelinolysis (CEPM). There was a 4-mo history of lethargy and irritability. The patient was hospitalised for an episode of somnolence. Plasma ammonia was 491 mM (N, 10-65), glutamine, 1397 mM (475-745), citrulline 12 mM (10-45); plasma amino acids were otherwise not diagnostic; ALT, 635 (5-25); AST 238 (5-60); INR 1.91 (0.9-1.1). Urine orotate was 4287 mM/g creatinine (N534). OTC de¢ciency was subsequently con¢rmed on liver biopsy. Cerebral CT was normal and EEG showed di¡use slow waves without spike activity. After 4 days of clinical and biochemical improvement (ammonia, 117 mM), during which neither hyponatremia nor hypoosmolality were documented, she developed coma and left-sided partial seizures refractory to treatment. She remained comatose for 5 days, then slowly improved. At discharge 6 weeks later she had pseudobulbar palsy, severe visual impairment and ataxia that resolved over 2 months. She is now 6 years old and neurologically normal. Cerebral MRI was initially normal but typical features of CEPM were evident 8 days after the onset of coma. This second report of CEPM in OTC de¢ciency (see also Mattson et al Am J Med Genet 1995, 60: 210-3) establishes the relationship between these two conditions. CEPM is a potentially reversible cause of post-hyperammonemic encephalopathy. Because it may be associated with coma and because typical MRI images only appear after several days of coma, it is important to rule out CEPM in patients who remain comatose after improvement of hyperammonemia, particularly before considering cessation of life support.

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LONG-TERM TREATMENT WITH SODIUM PHENYLBUTYRATE IN ORNITHINE TRANSCARBAMYLASE (OTC) DEFICIENT PATIENTS Burlina A.B.1, Ogier H.2, Korall H. 3 and Trefz F.3 1 Dept. Paediatrics,University of Padova,Italy;2Service Neurope¨diatrie et Maladies me¨taboliques, Hoªpital Debre¨, Paris, France;3Klinik fÏr Kinder und Jugendmedizin, Reutlingen, Germany OTC de¢ciency is a very heterogeneous urea cycle disorder resulting in hyperammonaemia with various presentations from neonatal period through adulthood. We performed a retrospective study in nine patients (4M/5F, age at diagnosis varying from 6 days to 14 years) to evaluate the safety and e¤cacy of sodium phenylbutyrate (Ammonaps1) in long-term treatment. All patients were diagnosed by DNA mutational analysis and/or liver enzyme measurement. They had previously been treated with sodium benzoate (median dose of 248 mg/kg/d,range 106-275) and low protein diet (0.84 g/kg/d) and were switched to sodium phenylbutyrate (median dose of 352 mg/kg/d) at 8.9 and 4.9 years of age (median) in males and females respectively. We analysed clinical and biochemical data in a follow-up trial of 26 months. During that time, there were no hyperammonaemic episodes requiring hospitalisation in 5 patients,only one in 2 patients, and 5 in the last 2 patients. Median plasma ammonia and glutamine levels were 30 and 902 umol/L., respectively. Total protein intake could be increased to 0.95 g/kg/d after 18 months. All patients except for one were attending regular school. No side-e¡ects related to therapy were observed. In conclusion, our results con¢rm the safety and e¤cacy of phenylbutyrate treatment in OTC de¢cient patients. Further prospective studies should be performed to de¢ne the optimal dosage of sodium phenylbutyrate and the requirements for protein diet at di¡erent ages.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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OROTIDINE RESPONSE IN ALLOPURINOL TEST FOR OCT DEFICIENCY IS DOSEDEPENDENT: RE-EVALUATION OF TEST PERFORMANCE WITH DOSE-NORMALIZED RESPONSES Riudor E, Rode¨s M1, Rubio V2, Burlina A3, Pi·ol F, Sent|¨ s M, Arranz JA. Unitat de Malalties Neurometaboliques. Hosp. MaternoInfantil Vall d'Hebron. Barcelona. 1: Institut de Bioqu|¨ mica Cl|¨ nica. Barcelona. 2: Instituto de Biomedicina de Valencia. Spain. 3:Dipartimento di Pediatria. Universita di Padova. Italy.

Allopurinol test is used clasically in the diagnosis of OCT de¢ciency not only in carrier women but in pediatric population. We evaluated the in£uence of allopurinol dose, body weight and age in urine orotate/orotidine (OA/ODN) responses in control population: 15 infants, 20 children and 11 women. OA/ODN were analyzed by anion-exchange HPLC after anion-exchange puri¢cation. ODN response was highest in infants. However multiple regression analysis between ODN response and per Kg dose and age as independent variables was signi¢cative only with per Kg dose (r=0.8192, p=0.0000). So, higher per Kg dose was responsible for increased values in younger controls. Once normalized, ODN response was not dependent of age. Cut-o¡ values of dose-normalized OA/ODN responses per Kg weight to maximize both sensitivity and speci¢city were 1.89 and 0.99 mmol/mol creat./mg allop./Kg respectively (ROC analysis) in our controls plus 9 OCT-de¢cient patients. Again, ODN showed better diagnostic sensitivity than OA: 0.89 vs 0.78 and only slightly lower speci¢city 0.94 vs 0.98. By this way an only control group and cut-o¡ values can be established in order to standardize and making easier test interpretation in all age range, specially in younger patients.

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OBSERVATION OF DOMINANT NEGATIVE EFFECTS FOR ORNITHINE TRANSCARBAMYLASE IN CULTURED CELLS COEXPRESSED WITH WILD-TYPE OTC AND VARIOUS MUTANT OTCS Toshiaki Kogo1, Masaki Kanazawa2, Shigenori Yamamoto2, Masaki Takayanagi3, Akira Ohtake4, Masataka Mori5, Youichi Kohno2 1 Division of Pediatrics, National Shimoshizu Hospital, Chiba, Japan, 2Department of Pediatrics, Chiba University School of Medicine, Chiba, Japan, 3Division of Metabolism, Chiba Children's Hospital, Chiba , Japan, 4Department of Pediatrics, Saitama Medical School, Saitama, Japan 5 Department of Molecular Genetics, Kumamoto University School of Medicine, Kumamoto, Japan Ornithine transcarbamylase (OTC) (EC.2.1.3.3) is the second enzyme in the urea cycle, and its bioactive form is found as a homotrimer in the mitochondrial matrix. Mutation of the OTC gene results in OTC de¢ciency (OTCD), which leads to hyperammonemia. The gene therapy has the potential to be a highly e¡ective therapy, a dominant-negative e¡ect caused by the action of endogenous mutant protein on recombinant wild-type protein activity has been reported. In this study, we analyzed this dominant-negative e¡ect on enzyme activity and protein stability. OTC activity was lower in cells that coexpressed both wild-type OTC and mutant OTCs compared to cells that expressed only wild-type OTC. However, the degree of interference of OTC activity and stability of the coexpressed proteins were di¡erent for each mutant. Our results showed suppression of OTC activity was higher in cells cotransfected with CRM(+) mutant compared with the CRM(-) mutants. We propose that our method is a useful and simple means to analyze the molecular mechanism of possible dominant-negative e¡ects and allows for the evaluation of possible wild-type/mutant protein interactions for use in OTCD gene therapy.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

56

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CITRULLINEMIA: CLINICAL PRESENTATION, EVOLUTION AND MOLECULAR ANALYSIS OF FIVE MEDITERRANEAN CASES Vilaseca MA1, Kobayashi K2, Briones P3, Lambruschini N1, Campistol J1, Tabata A2, Alomar A4, Rode¨s M3, Lluch M3, Saheki T2. Hospital Sant Joan de De¨u1. Institut de Bioqu|¨ mica Cl|¨ nica3. Barcelona. Hospital Son Dureta. Palma de Mallorca4. Spain. Kagoshima University. Japan2. We present the diagnosis, outcome and molecular studies of ¢ve Mediterranean patients, from four unrelated families: four neonatal classical forms and one subacute form presenting with severe hepatic encephalopathy at the age of seven years. Diagnosis was performed by amino acid analysis and con¢rmed by enzymatic studies in post-mortem liver tissue and/or by 14C-citrulline incorporation by ¢broblasts. Mutations were identi¢ed using RT-PCR/sequencing method and genomic DNA. Two neonatal patients died, one in the ¢rst week of life and the other at the age of four months in a severe metabolic decompensation. Three patients survived, two neonatal forms with good neurological outcome and the third patient (late-onset form with Down syndrome) with mental retardation. Mutational analysis revealed three alleles with a common mutation and 5 new mutations. Patients had the following genotypes: two Moroccan siblings are homozygous for the G390R mutation, the subacute form with Down syndrome is compound heterozygous for G390R/G117D (new mutation), and two other neonatal forms are compound heterozygous for four new mutations: V69A/E270Q and T119I (R108L)/E45X?. Phenotype variability and genetic heterogeneity are common in citrullinemia, even in the neonatal form. Combined heterozygosity is a common feature in nonconsanguineous families, and this makes di¤cult phenotype-genotype correlation. Early diagnosis and aggressive treatment are essential to predict good outcome in neonatal citrullinemia.

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MODERATE CITRULLINEMIA WITHOUT HYPERAMMONEMIA IN A CHILD WITH MUTATED AND DEFICIENT ARGININOSUCCINATE SYNTHETASE W. Ruitenbeeck1, T. Saheki2, J.A.M. Smeitink3, A.M.C. Simonis-Bik4, K. Kobayashi2, R.A. Wevers1, H. Kwast1, R.A. De Abreu1, J.G.N. de Jong1, J.M.F. Trijbels1 University Medical Centre Nijmegen (Neth.), Laboratory of Pediatrics and Neurology1, and Inst. of Pediatrics3; Kagoshima University Sakuragaoka (Japan), Dept. of Biochemistry2; Hospital Velp (Neth.), Dept. of Pediatrics4 Feeding problems, restlessness, microcephaly and left-sided hypertonia were the presenting symptoms of a 4-month-old girl from non-consanguineous Turkish parents. Right-sided hemiatrophy and temporoparietal brain wasting were seen with brain MRI. EEG showed hypofunctional and moderate irritative activities. Metabolic screening revealed moderate increase of the citrulline blood concentration (200^350 mmol/l) and urinary excretion (600^900 mmol/mmol creat) without other abnormalities in the amino acid, organic acid and purines/pyrimidines pattern. Blood ammonia content was normal even after a meal and after protein loading. Allopurinol administration did not result in production of orotic acid. In vitro NMR revealed the presence of a substantial amount of N-acetylcitrulline in urine. In ¢broblasts no argininosuccinate synthetase (ASS) activity could be detected. Sequencing of cDNA demonstrated the hitherto unknown G1,085T point mutation in exon 14 of the ASS gene, introducing valine instead of glycine at position 362 of the protein. The patient appeared to be homozygote for this mutation. The mutation was con¢rmed in genomic DNA. The ¢ndings in this patient clearly underline that moderate citrulline increase in blood and urine can form an important indication for ASS de¢ciency, even in the absence (under basal or stressed conditions) of hyper-ammonemia or -glutaminemia or abnormalities in other metabolites being considered as rather characteristic for urea cycle defects. Also the phenotype can be little characteristic. J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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TRANSIENT CITRULLINAEMIA ASSOCIATED WITH GENERALISED LIVER DISEASE C.L. Evans1, M.J. Henderson1, M. Smith1, M.A. Cleary2 and P. McClean3 Departments of Chemical Pathology1 and Paediatrics3, St. James's University Hospital, Leeds, UK. Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester, UK2

We investigated 3 babies with raised plasma and urine citrulline concentrations which returned to normal after 2 months, 7 m and 3 yrs. All had evidence of transient liver disease. In each case citrulline concentrations were su¤ciently abnormal to suspect a urea cycle defect. The most recent case had normal results of citrulline incorporation studies performed on cultured ¢broblasts. He was term, male and had ¢rst cousin Asian parents. Neonatal screening blood spots showed a slightly elevated phenylalanine, but plasma at 14 days old had elevated citrulline (467mmol/L, ref: 536) and threonine (579mmol/L, ref: 5350), with all other amino acids within their reference ranges. There was evidence of liver dysfunction and hepatomegaly. Urine showed elevated citrulline (761mmol/mmol creatinine, ref: 57), threonine (1020 mmol/mmol creat., ref: 5158), arginine (280mmol/mmol creat., ref: 10), lysine (399mmol/mmol creat., ref: 5137) and cystine (59mmol/mmol creat., ref: 540). Urine orotate was normal. Galactose was detected in the urine, but GAL-1-PUT was normal. Urine organic acids showed elevated p-hydroxyphenyl lactate and p-hydroxyphenyl pyruvate and no succinyl acetone. Serial plasma citrulline concentrations (mmol/L) were: 467 (14 days), 720 (28 d), 262 (4 m) and 12 (7 m), and urine citrulline concentrations (mmol/mmol creatinine) were: 761 (14 d), 4930 (28 d), 489 ( 4 m) and 55 (7 m). In conclusion, if moderately elevated plasma citrulline, together with disproportionately raised urine citrulline is observed, transient citrullinaemia should be considered.

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TWO NOVEL MUTATIONS IN A NEONATE WITH LETHAL ``CLASSICAL'' CITRULLINEMIA (CTLN1) P Augoustides-Savvopoulou1 A Andreou2 K Kobayashi3 W Kleijer4 Ayako Tabata3 M Papouli 2 T Saheki3 University 1st Pediatric Biochemistry Lab1 Neonatal ICU, 2 Hippocration Gen.Hospital Thessaloniki Greece, Dept of Biochemistry Faculty of Medicine, Kagoshima University Japan 3 Clinical Genetics, Erasmus University, Rotterdam, The Netherlands 4 Argininosuccinate synthetase (ASS-McKusick 215700) de¢ciency is an autosomal recessive urea cycle defect resulting in hyperammonemia, citrullinemia, citrullinuria, and orotic aciduria. Over 50% of cases are symptomatic a few days after birth with death in the neonatal period (CTLN1) but a late-onset form has also been reported (CTLN2). A Greek infant with lethal CTLN1 in which two novel mutations were found is described. The proband, a female, the ¢rst and only o¡spring of healthy unrelated parents, born full-term by caesarean section, manifested severe respiratory distress and seizures on the 4th day of life , became comatose within two days and succumbed on the 9th day of life. The presence of respiratory alkalosis (pH 7.48), hyperammonemia (760 mmol/l), increased glutamine, massive citrullinuria, citrullinemia and orotic aciduria indicated CTLN1 and the severely de¢cient 14C citrulline incorporation in skin ¢broblasts (17 dpm/Ùg, control 3403) con¢rmed the diagnosis. Prenatal diagnosis of a second pregnancy by ASS analysis in chorionic villi revealed an a¡ected fetus. Mutation analysis performed by sequencing of ampli¢ed ASS cDNA using RT-PCR and total RNA extracted from the patient's ¢broblasts revealed one missense mutation (R363G) due to a change from Arg (CGG) at position 363 to Gly (GGG) by a substitution from C to G of nucleotide at position 1087 in exon 14 of ASS mRNA and one nonsense mutation (Q380X) due to a change from Gln (CAG) at position 380 stop codon (TAG) by a substitution of nucleotide from C to T at position 1138 in exon 15 of ASS mRNA. Our results present further evidence that mutations causing CNTL1 are extremely heterogeneous and that compound heterozygosity is the rule in nonconsanguinity.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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HELPER-DEPENDENT ADENOVIRAL VECTORS IN THE TREATMENT OF CITRULLINEMIA Brendan Lee, Benjamin Mull, Asad Mian, Gabriele Toietta, Olaf Bodamer, Lucio Pastore, William O'Brien and Arthur Beaudet. Dept. Molecular and Human Genetics, Baylor College of Medicine, Houston TX USA. Citrullinemia is a urea cycle disorder characterized by de¢ciency of argininosuccinate synthetase (ASS). Gene therapy correction in both humans and animal models will require high levels of hepatocyte transduction and long-term transgene expression. To achieve these objectives, we have generated helper-dependent adenoviral vectors devoid of viral coding genes which express human ASS (hASS) from a ubiquitously active elongation factor 1 (BOS) promoter (Ad?BOShASS). Evaluation of Ad?BOShASS by Southern analysis, real-time quantitative PCR, and £uorescent in situ hybridization (FISH) after intravenous injection into C57BL/6 mice show helper virus contamination of 51%, stable vector structure, and e¤cient hepatocyte transduction. Northern analysis using a transgene-speci¢c probe showed mRNA expression levels in liver comparable to wild type murine ASS. Our previous data suggested that use of liver-speci¢c and endogenous genomic promoters may increase transgene expression and blunt the host immune response. We analysed mice at 3 days and 8 weeks after injection with ¢rst generation adenoviral vectors expressing hASS from either the ubiquitously expressing CAG promoter or the liver-speci¢c albumin promoter. hASS mRNA expression persisted in mice treated with the albumin promoter vector compared to the CAG promoter virus. These data have prompted us to evaluate helper-dependent vectors containing the endogenous ASS and alpha-1-antitrypsin promoters driving human ASS minigenes for treatment of bovine and human citrullinemia.

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PREDICTION OF PLASMA AMMONIA (NH3) FROM PLASMA AMINO ACIDS IN ARGININOSUCCINATE (ASA) LYASE DEFICIENCY Russell LJ, Barnes RD, Lambert DM. Departments of Genetics and Pediatrics, McGill University, Montreal, Quebec, Canada.

GLN and glutamate (GLU) in the hepatic nitrogen pool favor ammonia formation in the waste nitrogen pathway. We examined the correlations of these and other amino acid levels with plasma NH3 in ¢ve children with ASA lyase de¢ciency. 145 pairs of GLN / NH3 assays showed no signi¢cant linear correlation (r=0.08, p=0.33). 93 pairs of GLU / NH3 and 149 pairs of arginine (ARG) / NH3 assays showed signi¢cant but minor correlations (GLU: r=0.26, p=0.011; ARG: r=-0.26, p=0.0015). Scatterplots suggested against quadratic or logarithmic transformations of GLN, GLU or ARG values for correlation analyses. Ornithine (ORN) levels were more strongly correlated with NH3, especially when logarithmically transformed (n=52, r= -0.71, p=0.0001). However, a receiver operating curve failed to identify a useful cut-o¡ of ORN to predict hyperammonemia with adequate sensitivity and speci¢city. We conclude that plasma GLN, GLU, ARG and ORN do not independently predict hyperammonemia in children with ASA lyase de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GENOTYPE/PHENOTYPE-CORRELATIONS IN PATIENTS WITH ARGININOSUCCINIC ACIDURIA IN FINLAND M Linnebank1 , A Siitonen2, H Willenbring1, R Schultz2, A Homberger1, E Tschiedel1, J Haeberle1, M Salo2, HG Koch1 Departments of Pediatrics, 1University Hospital Muenster, Germany, and 2Tampere University Hospital, Finland

Argininosuccinic aciduria is a rare disorder caused by de¢ciency of the argininosuccinate lyase (ASL) revealing a striking variability of the clinical phenotype. So far, only 8 mutations of the ASL gene have been identi¢ed in 9 patients, however, without genotype/phenotype correlations. In the present study we characterised the genotypes of a¡ected patients of 9 families from Finland which could be compared with the clinical course. We identi¢ed two novel mutations: the transitions 299T4C (exon 4, I100T) and 1153C4T (exon 15, R385C). The mutation 299T4C was found homozygously in two siblings who presented with a late onset of disease (age at onset 11 and 14y) and a mild clinical course with a good outcome. The mutation 1153C4T was found in all other patients in a homozygous state indicating that there is a founder e¡ect for this mutation in the Finnish population. These patients revealed a more severe course, however, there was considerable variability. Manifestation varied from hyperammonemic crisis in the ¢rst week of life up to psychomotor retardation at the age of eleven months. In spite of treatment most of the patients demonstrate an unsatisfying outcome since they present cerebral seizures and/or psychomotor retardation. This study demonstrates for the ¢rst time that the highly variable clinical course of argininosuccinic aciduria is at least partly correlated to the genotype. The identi¢cation of a founder mutation for ASL de¢ciency provides the opportunity to study the impact of modulating factors.

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HYPERLIPIDAEMIA IN ARGININOSUCCINIC ACIDURIA. AN UNDERREPORTED FINDING ? GJ Shortland1 and H Losty2, Dept. of Child Health 1 and Dept of Biochemistry 2, University Hospital of Wales, Heath Park, Cardi¡, Wales, UK. Argininosuccinic lyase (AL) de¢ciency is a common urea cycle defect. Early detection and aggressive management have resulted in long-term survival. This may be complicated by intermittent hyperammonaemia and other biochemical derangement's. To our knowledge there have been no reports of lipid abnormalities associated with this condition and long term management. We report two patients with raised cholesterol and triglyceride levels with AL de¢ciency. These patients presented initially at seven days and ¢ve days of age and are now aged 4 years (male) and 10 years (female). Both patients have received treatment including a reduced protein intake, essential amino acids, overnight feeding, sodium benzoate and sodium phenylbutyrate. Both patients have signi¢cant raised random cholesterol and triglyceride levels Lipaemia was ¢rst evident in the older patient at 12 weeks of age and was a recurrent feature over the ¢rst 5 years of life (peak levels, cholesterol 9.3 mmol/l, triglyceride 13.3 mmol/l). The second patient has had similar but higher levels (peak levels, cholesterol 20.0 mmol/l, triglyceride 25.9 mmol/l). No clear correlation with medication or diet was noted. The e¡ect on the long term outcome of these patients is unknown however prolonged raised cholesterol is a signi¢cant risk factor for coronary heart disease and raised triglyceride levels are linked with pancreatitis. The pathogenesis in our patients, and frequency of these ¢ndings in other patients with AL de¢ciency requires further de¢nition.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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LIVER TRANSPLANTATION IN HYPERARGININEMIA Autors: Santos Silva E, Martins E, Cardoso ML, Barbot C, Vilarinho L, Medina M Institutions: Hospital de Crianc°as Maria Pia and Instituto de Gene¨tica Me¨dica Jacinto de Magalha¬es Porto Hyperargininemia is an autossomal recessive disorder of urea cycle characterized by a clinical picture consisting of progressive spastic quadriplegia and mental retardation presenting in childhood. Liver insult is common but mild and asymptomatic. We present the case of a caucasian girl, ¢rst daughter of a healthy and nonconsanguineous couple in whom diagnosis of hyperargininemia was made at 2 months age after a through investigation for cholestatic jaundice and hepatic insu¤ciency. At the time, plasmatic arginine level was 1756 mmol/l and diagnosis was con¢rmed by the absence of residual activity of arginase in erythrocytes and the presence of homozygote mutation R21X. Clinical outcome was to liver cirrhosis and portal hypertension with marked hypercholesterolemia, xanthomatosis, and mild pruritus. We think a second disease may be involved (a biliary acid synthesis error that unfortounately we could not exclude neither con¢rme). At 7 years old developpment milestones and neurological examination were normal. A liver transplant was performed and plasmatic arginine levels completely normalized and remain normal under a total protein free diet. Althought liver transplantation has been performed in other urea cycle disorders it was never done in a patient with hyperargininemia probably because the disease is extremely rare and the classical clinical picture with progressive neurological disease may be considered a contraindication to the procedure. Our patient is the ¢rst without established neurological features and terminal liver disease compeling to consider liver transplantation.

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A MOUSE MODEL FOR HUMAN ARGINASE DEFICIENCY R. K. Iyer1,3, R. M. Kern3, P. K. Yoo3, N. Rozengurt1, W. E. O'Brien4, A. W. Butch1, K. V. Lu1, W. W. Grody1,2,3 and S. D. Cederbaum2,3. Departments of Pathology and Laboratory Medicine1, Pediatrics2, and the Mental Retardation Research Center3, UCLA School of Medicine, Los Angeles, California, USA; Department of Molecular and Human Genetics4, Baylor College of Medicine, Houston, Texas, USA.

Arginase is the ¢fth and ¢nal enzyme of the urea cycle. Two isoforms of arginase (AI & AII) are known to exist in humans and rodents. De¢ciency of the liver isoform (AI) causes hyperargininemia; a disorder characterised by progressive mental impairment, growth retardation, spasticity, and punctuated, often fatal episodes of hyperammonemia. We constructed a 'knock out' mouse strain carrying a non-functional AI gene, by homologous recombination. These mice completely lack liver arginase (AI) activity, exhibit severe symptoms of hyperammonemia, and die at day p10-14. Unlike human patients where AII activity in kidney is induced 3-34 fold during hyperammonemic episodes, no elevation of AII activity in either kidney or brain was found in these mice, possibly accounting for their more rapid and severe clinical course. During hyperammonemic crises, plasma ammonia levels were increased 410-fold, compared to normal animals. Livers of AI-de¢cient animals, harvested during these episodes showed hepatocyte abnormalities including cell swelling and inclusions. Glucose and electrolyte (Ca++, Mg++, K+) levels were normal. Plasma amino acid analysis is in progress. In summary, the AI-de¢cient mouse duplicates several pathobiological aspects of the human condition, and should prove to be a useful model for further study of the disease mechanism(s), and to explore novel treatment options.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BETAINE INCREASES METHIONINE/HOMOCYSTEINE RATIOS AND DECREASES TOTAL PLASMA HOMOCYSTEINE (tHcy) IN CYSTATHIONINE BETA-SYNTHASE DEFICIENCY. MT Steen, WD Kruger, RH Singh, M Pasquali, LJ Elsas II. Division of Medical Genetics, Department of Pediatrics, Emory University, Atlanta, GA, USA.

Metabolic homeostasis is di¤cult to achieve by diet alone in patients with pyridoxine (B6) nonresponsive, cystathionine beta-synthase de¢ciency (CbS, EC 4.2.1.22). Betaine (N, N, N trimethylglycine) is an alternative methyl-group donor for remethylation of homocysteine to methionine. Twelve patients (8 White, 4 Black) with CbS de¢ciency were genotyped for their CbS mutations. Of 8 mutant alleles in Black patients, 5 were a novel missense mutation (T353M) that is common in Blacks with CbS de¢ciency. Five non-B6 responsive patients (3/9 White, 2/4 Black) strictly managed by methionine restriction were given betaine (50-150 mg/kg/day) to determine its additive e¡ects. This open-label trial began with dietary ``optimization'' using methionine-restriction and cystine-supplementation. Plasma methionine, tHcy and total plasma cysteine were quantitated and compared before initiation and after maximal dosing of betaine. Comparisons were by the paired student's Ttest. Total plasma homocysteine decreased an average of 47.4mM (-21.2 to -104.0mM p=0.05), while total plasma cysteine did not change (34mM, -53 to +146mM, p=0.4). Ratios of plasma methionine/ tHcy increased an average of 5.45 (1.5 to 15.3, p=0.05). The plasma methionine did not change (+33.8mM, -20 to +92mM, p=0.2). We conclude that betaine decreases tHcy and increases ratios of methionine/tHcy in patients who are B6-nonresponsive and optimally treated with methionine restriction.

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IN VIVO STUDIES OF METHIONINE METABOLISM IN HOMOCYSTINURIC PATIENTS Bruce A. Barshop and Robert D. Steiner, Departments of Pediatrics, University of California San Diego, La Jolla, California and Oregon Health Sciences University, Portland, Oregon.

In evaluating homocystinuria, it is important to distinguished those with defective transuluration (TS), as in cystathionine-b-synthase (CbS) de¢ciency, and those with defective reethylation (RM), including methylenetetrahydrofolate reductase (MTHFR) de¢ciency. We have studied methionine (met) metabolism in both forms of homocystinuria, using a standard oral met load (100 mg/kg, 0 and 4 hr time points), and a continuous infusion of the stable isotope [1-13C/sup;C; Me-2H3]-methionine. The isotope method in principle allows direct estimation of RM and TS. The control ratio of RM/TS was 0.96 under these (postabsorptive) conditions. A 19 year old man with B6-responsive CbS de¢ciency (on B6) had a RM/TS ratio of 2.25 when his plasma total homocysteine (THcy) was 92 mM. His mother had a RM/TS ratio of 1.69, and a moderately abnormal met response also, the THcy rising from 12.2 to 34.6 mM. A 25 year old woman with MTHFR de¢ciency (genoype C1081T/ G167A) was studied on 20 g/day betaine and after two days without betaine. O¡ betaine, her RM/TS ratio was low (0.61). The baseline THcy was markedly elevated (158.4 mM) with no further increase following a methionine load. Her father and mother also had abnormal responses to methionine loads (14.4 to 36.3 and 12.3 to 36.9 mM, respectively). The isotope method did not clearly reveal the abnormality in the MTHFR heterozygous mother; her RM/TS ratio was 1.17. However, in the MTHFR de¢cient daughter, the isotope method showed the an increase in RM/TS more than threefold (to 1.92) during treatment with betaine. Stable isotope methods may be useful in the diagnosis and monitoring of homocystinuric and hyperhomocyst(e)inemic individuals.

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FREE AND BOUND CYSTEINE IN HOMOCYSTINURIA Anthony Briddon and Iain P Hargreaves Department of Clinical Biochemistry, National Hospital for Neurology and Neurosurgery, Queen Square, London, WC1N 3BG, UK. The raised concentration of protein bound homocysteine in homocystinuric (HCU) patients displaces protein bound cysteine and increases the free to bound cysteine ratio in plasma. This ratio is independent of the albumin concentration. Results from 39 adult HCU samples were compared to 40 control samples. Free cystine concentrations did not di¡er signi¢cantly (P40.9) but total cysteine results were signi¢cantly lower in patients with HCU (P50.001). This appears to result from an increased free/bound cysteine ratio in HCU patients; (mean+SD for normal, 0.50 + 0.147; and homocystinurics, 1.40 + 1.360). Ex vivo homocysteine-protein binding experiments revealed the free/bound cysteine ratio to be linearly related to the amount of homocysteine bound, (r = 0.907, P50.001). However, the magnitude of this increase was rather less than that found in HCU samples with comparable concentrations of total homocysteine. Total glutathione (mmol/L) was measured in 12 of the HCU samples and did not di¡er signi¢cantly from controls, (mean + SD: controls 5.7 + 1.85, range 3.0 - 8.3; HCU 4.9 + 2.24, range 1.2 - 9.0, P = 0.75). The total glutathione result of 1.2 mmol/L was found in one severely a¡ected HCU patient with a free cystine of 4 mmol/L and total cysteine of 60 mmol/L (normal 200 - 350). We conclude that measurement of free cystine does not adequately re£ect the true cysteine de¢cit in HCU but our preliminary results suggest that the increased free/bound cysteine ratio may help to maintain glutathione concentrations in HCU.

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PREGNANCY IN PYRIDOXINE NON-RESPONSIVE HOMOCYSTINURIA WITH GOOD MATERNAL AND FETAL OUTCOME S Yap1, C Barry-Kinsella2, ER Naughten1. 1 National Center for Inherited Metabolic Disorders, The Children's Hospital, Temple Street and 2The Rotunda Hospital, Dublin, Ireland. The reproductive ¢tness of patients with homocystinuria due to cystathionine b-synthase de¢ciency, particularly pyridoxine non-responsive, has always been guarded due to the maternal risk of thrombosis and spontaneous abortions. We report on the management of a pyridoxine nonresponsive maternal homocystinuria with good maternal and fetal outcome. A 19 year old girl, diagnosed through newborn screening and commenced on a methionine restricted, cystine supplemented diet, became pregnant. Cofactors supplementation included pyridoxine 200mg tds, B12 4mg tds and folate 5 mg daily. Betaine was given at 1.5g tds. Methionine tolerance increased from 225 mg to 575 mg (9g to 23g natural protein) predelivery. Synthetic protein intake was 1 g/kg body weight with a total protein of 1.25 g/kg body weight. Free homocystine mean (SD) = 8.2 (5.5) mmol/L, total homocysteine mean (SD) = 74.8 (20.6) mmol/L, methionine mean (SD) = 61.8 (33.9) mmol/L with normal serum B12, folate and ¢brinogen throughout the pregnancy. Subcutaneous heparin 5000U bd was given from one week prepartum until six weeks postpartum. A male term infant was delivered uneventfully, birth weight was 4kg, length 48cm, head circumference 36.5 cm. An obligate heterozygote, he had a normal physical examination, cranial ultrasound, echocardiogram, total homocysteine and remains developmentally appropriate at 10 months now. We conclude that appropriate treatment with good biochemical control can lead to good maternal and fetal outcome in pyridoxine nonresponsive homocystinuria.

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IDENTIFICATION OF MICROINFARCTS IN PATIENTS WITH TREATED HOMOCYSTINURIA : BRAIN MRI AND NEUROLOGICAL FINDINGS S Yap1, D Annesley2, A Ryan3, S Pittock3, P Brennan2, O Hardiman3,ER Naughten1. 1 National Center for Inherited Metabolic Disorders, The Children's Hospital, Temple Street, and the Departments of 2Radiology and 3Neurology, Beaumont Hospital, Dublin, Ireland.

Untreated patients with homocystinuria (HCU) due to cystathionine b-synthase de¢ciency have a 30% chance of a thromboembolic event before age 20 years. Twenty-two treated HCU patients (B6 nonresponsive; n=19) with a mean (range) age of 22.6 (15-36) years had brain MRIs and neurological examinations. Of these, 15 were detected through newborn screening, the remaining seven were latedetected. Biochemical control was re£ected by the lifetime mean free homocystine (fHcy;mmol/L). Results are expressed as mean (SD): Mode of diagnosis Newborn-good control Newborn-poor control Late-detected

N

Age-yrs

8 7 7

17.3 (1.6) 23.4 (3.8) 27.8 (7.1)

fHcy Abnormal ¢ndings (n) (lifetime) MRI Neurological 15.2 (10.8) 25.3 (14.5) 13.4 (8.1)

0 7 3

0 3 4

In this group of treated HCU patients, only two had cerebrovascular accidents but MRI ¢ndings consistent with microinfarctions were detected in ten. Only seven had abnormal neurological ¢ndings. Poorly compliant HCU patients are at-risk of strokes. This data would suggest that MRI is a useful adjunct to treatment in the early identi¢cation of pre-stroke HCU patients.

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EFFECTS OF TREATMENT ON PSYCHOMETRIC PARAMETERS IN PATIENTS WITH HOMOCYSTINURIA S Yap, H Rushe, PM Howard, ER Naughten. National Center for Inherited Metabolic Disorders, The Children's Hospital, Temple Street, Dublin, Ireland.

Mental retardation is one of the recognised pathological sequelae of untreated homocystinuria due to cystathionine b-synthase de¢ciency. A total of 24 patients (B6 non-responsive, n=23) and 10 nona¡ected sibling controls were assessed using age-appropriate standardised psychometric tests. The results are expressed as mean (SD) as follows: Mode of diagnosis

N

Age-years

FIQ

VIQ

PIQ

Newborn-good control Newborn-poor control Late detected Untreated Controls

16 3 3 2 10

15.6 (6.6) 18.9 (5.4) 19.5 (1.2) 17.0 (7.6) 14.9 (10.1)

104.0 (10.9) 66.3 (24.7) 89.0 (11.5) 52.5 (0.7) 102.0 (12.1)

109.0 (12) 70.7 (24) 90.3 (16) 59.5 (2.1) 107.0 (12.9)

95.8 (13.2) 67.0 (20) 89.3 (6.4) 52.0 (0) 96.6 (12.9)

The Full IQ, Verbal IQ and Performance IQ of patients commenced on early treatment with good control is not signi¢cantly di¡erent to that of the non-a¡ected sibling controls. Although the study group is relatively small, this data would suggest that early treatment with good biochemical control does prevent mental retardation.

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HOMOCYSTEINE PATHWAY: OXIDATIVE STRESS AND ENDOTHELIAL METABOLISM IN WISTAR RATS WITH STREPTOZOTOCIN-DIABETES R. Cerone, G. Minniti, 8D. Boeri, 8M. Maiello, D. Campanella, 8A. Piana, 8U. Armani, R. Lorini Dept. of Pediatrics II, G. Gaslini Institute, 8Thrombosis and Center, DI.M.I. University Genoa, Italy Di¡erent studies have indicated that a mild degree of hyperhomocysteinemia is an independent risk factor for cardiovascular disease and atherosclerosis. In type 2 diabetes increased plasma concentrations of homocystein are signi¢cantly associated with macroangiopathy. Homocysteine is mainly degraded through the pathway of cistathionine B-synthase. However, several issues need to be clari¢ed: a) the speci¢c contribution of the di¡erent tissues to homocysteine catabolic pathways; b) whether the presence of diabetes modi¢es homocysteine metabolism; c) if the possible changes are related to glycemia and to nitric oxide production. The endothelium is the main target of the negative e¡ects of homocysteine but its possible contribution to homocysteine metabolism is not yet understood. In our study, in tissues (heart and kidney) of Wistar rats with and without streptozotocin-induced diabetes, in human endothelial cell from umbilical vein (HUVEC) chronically exposed to 30 mM glucose, to mimic hyperglycemia and to 5 mM as control, cysteine, homocysteine (by HPLC) and Nitric Oxide Synthase (NO-S detect assay, Stratagene) were measured. The activity of cystathionine B-synthase is elevated in the kidney of control rats. In diabetic rats the heart concentrations of homocysteine are slightly increased (Controls: 9.5+-0.90; Diabetic rats: 9.88+2.41 umol/L. A positive correlation between cardiac homocysteine concentrations and renal cysteine concentrations is demonstrated (p50.05), while in diabetics rats the correlation is negative (p50.05). These results suggest that elevated tHcy in diabetic patients is, probably, related to reduced renal function (i.e. diabetic nephropathy).

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EXTENSIVE METABOLITE SURVEY IN HOMOCYSTINURIA 1 V.Kozich, 1J,Krijt, 2J.Svatos, 3S.P.Stabler, 3R.H.Allen, 1J.Zeman, 2J.Zvarova, and 4J.P.Kraus 1 Institute of IEM,2EuroMISE Charles University, Prague; Dept 3Medicine and 4Pediatrics UCHSC, Denver, USA The most common form of homocystinuria results from de¢ciency of cystathionine b-synthase (CBS). We analyzed by LC-MS and HPLC metabolites of the methionine cycle and of the transsulfuration pathway in 13 CBS de¢cient patients. Data were obtained in periods of treatment, and periods of non-treatment (in newly diagnosed and non-compliant patients, or after 2-4 weeks intervals of dietary treatment with no betaine/vitamins). The mean metabolite concentrations for untreated periods (n=9) vs. treated periods (n=8), with laboratory reference ranges given in parentheses, were as follows: plasma methionine 162 vs.42 mmol/l (13-43); blood S-adenosylmethionine (AdoMet), 2352 vs. 1556 nmol/l (1236-2325); blood S-adenosylhomocysteine (AdoHcy), 236 vs. 48 nmol/l (12-99); AdoMet/AdoHcy ratio, 14 vs. 54 (20-118); plasma total homocysteine (tHcy), 172 vs. 34 mmol/l (5.4-13.9); plasma cystathionine, 37 vs. 37 nmol/l (44-342); plasma total cys-teine, 102 vs.188 mmol/l (200-361); blood free glutathione (GSH),1094 vs. 1196 mmol/l (789-1351); plasma Nmethylglycine (MG), 8.0 vs.9.4 mmol/l (0.6-2.7); and plasma serine 80 vs. 92 mmol/l (97-267). The data show that CBS de¢ciency alters AdoMet/AdoHcy ratio thus possibly a¡ecting the methylation reactions, that the excessive AdoMet is channeled to N-methylglycine, and that despite the partially blocked transsulfuration GSH is not depleted (at least not in blood cells). Analysis of variance revealed that tPA and PAI-1 correlate positively with plasma tHcy (p50.0005 and 50.0002, respectively) suggesting a direct role of Hcy in endothelial dysfunction. Although usual treatment normalizes many of the metabolic abnormalities in CBS de¢ciency, pos-sible new targets such as increasing serine levels or AdoMet to MG conversion await exploration.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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ORAL ASCORBIC ACID AMELIORATES HOMOCYSTEINE-MEDIATED ENDOTHELIALDYSFUNCTION IN CLASSICAL HOMOCYSTINURIA, BOTH ACUTELY AND CHRONICALLY 1 JR Bonham,3SJ Moat, 2CH Bones, 2IFW McDowell, 2MJ Lewis, 3HJ Powers 1 Department of Chemical Pathology, She¤eld Children's Hospital, She¤eld, S10 2TH, UK. 3 University Division of Child Health and 2Cardiovascular Sciences Research Group, Wales Heart Research Institute, University of Wales College of Medicine, Cardi¡, UK.

Elevated plasma total homocysteine (tHcy) is associated with an increased risk of vascular disease. The e¡ect may be mediated by oxidative damage to the endothelium. We evaluated the e¡ects of acute (2g) and chronic (1g/day for 6 months) administration of the antioxidant ascorbic acid on endothelial function in 5 subjects with homocystinuria and 5 age and sex-matched controls. Flow-mediated brachial artery dilatation (endothelium-dependent and endothelial-independent (glycerol trinitrate, GTN) was measured by ultrasonic high-resolution wall tracking. Plasma tHcy at baseline was 100.8 (27.5mmol/l and 9.2 (0.8 in the patients and controls respectively (P=0.009) and remained signi¢cantly higher in the patients throughout the study (P=0.0001). Endothelium-dependent responses at baseline were impaired in the patient group (0.02 ( 0.02mm) compared with the controls, (0.12 (0.01mm) (P=0.009) whereas endothelial-independent responses were similar in both groups (P=0.175). 4 hours following ingestion of 2g ascorbic acid endothelial-dependent responses in the patient group had improved signi¢cantly to 0.16 (0.03mm (P=0.009) and were comparable to those of the control group (0.13 (0.02mm, P=0.116). This improvement in endothelial-dependent response was maintained after ingestion of 1g/day of ascorbic acid for 2 weeks (0.17 ( 0.03mm) and 6 months (0.16 ( 0.04mm). Our data suggest that ascorbic acid taken orally ameliorates homocysteine-mediated endothelial dysfunction both acutely and chronically.

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HOMOCYSTEINE METABOLISM IN COMPLICATED PREGNANCIES HW de Valk, AJM Huisjes, N van der Wijk, HW Bruinse, A Franx, GHA Visser Dept. Internal Medicine and Obstetrics, University Medical Center Utrecht, The Netherlands.

Pre-eclampsia (PREC (hypertension occurring after the 20th week of pregnancy with proteinuria)), HELLP syndrome (haemolysis, elevated liver enzymes, low platelet count) and abruptio placentae (AP) are thought to result from vascular placental pathology. Abnormal homocysteine metabolism is implicated in vascular pathology. The aim of this study was to assess the frequency of abnormal homocysteine metabolism in patients with these obstetric abnormalities. Homocysteine metabolism was studied by methionine loading test (MLT). Normal basal homocysteine level was below 15.9 mmol/l, after loading below 49 mmol/l. Abnormal MLT (AMLT) was de¢ned as an elevated basal level and/or elevated level after loading. Plasma levels of the co-factors folic acid, pyridoxine and B12 were measured before loading. MLT was performed at least 3 months after delivery. The group consisted of 118 women with PREC, HELLP or AP occurring before the 34th week; PREC in 46, HELLP in 57, and AP in 15 patients. Mean age: 31.9 years (range: 21-44). AMLT was observed with PREC in 26.1%, with HELLP in 24,6%, and with AP in 13,3%. Plasma folic acid and pyridoxine levels were signi¢cantly lower with AMLT than with normal MLT (p=0.001 and p50.05) with no signi¢cant di¡erence in plasma B12 levels. Time since delivery (3-6 months vs 46 months) did not in£uence percentage AMLT or co-factor levels. In conclusion, there is a high frequency of abnormal homocysteine metabolism after complicated pregnancies with lower plasma levels of pyridoxine and folic acid playing a role, but bene¢cial e¡ects of treating this metabolic disorder on the outcome of subsequent pregnancies needs to be established.

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PHARMACOKINETICS OF ORAL BETAINE IN HEALTHY SUBJECTS B. Schwahn, D. Hafner1, T.Hohlfeld1, M.D. Laryea, U. Wendel. Children's Hospital and 1Institute of Pharmacology and Clinical Pharmacology, Heinrich-HeineUniversity, 40225 Duesseldorf, Germany Large oral doses of betaine proved e¡ective in lowering homocysteine blood levels in severe hyperhomocysteinemia. Pharmacokinetic characteristics and metabolism of betaine in humans have not been assessed. Drug monitoring for betaine therapy is not available. We studied pharmacokinetics of betaine and its metabolite dimethylglycine (DMG) in healthy subjects: 12 male volunteers underwent an open label study. After one single administration of 50mg betaine/kg body weight and during continuous intake of twice daily 50mg/kg bw, serial blood samples and 24 hours urines were collected to determine betaine and DMG plasma levels and urinary excretion, respectively, with a previously developed precise and convenient HPLC method. We found rapid absorption (t50abs 0.28+/- 0.17h) and distribution (t50a 0.59 +/- 0.21h) of betaine. Plasma peak concentrations Cmax of 0.94 +/- 0.19mmol/l were reached after Tmax of 0.90 +/- 0.33h. The elimination half life was calculated as t50b 14.38 +/- 7.17h. After repeated dosage, distribution (t50a 1.29 +/- 0.42h) and elimination half lives (t50b 35.96 +/- 12.32h) increased signi¢cantly, while absorption kinetics remained unchanged. DMG levels increased signi¢cantly after betaine administration, thus providing the ¢rst direct evidence for the catabolism of betaine in healthy subjects. Urinary excretion of betaine was equivalent to 4% of the ingested dose, DMG excretion to only 1%. We conclude that betaine plasma levels change rapidly after application. Betaine is primarily eliminated by catabolism, urinary clearance being negligible. Patient studies are needed to con¢rm the results and to decide whether dosage intervals and monitoring of betaine therapy should be adapted to these kinetic characteristics.

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CYSTATHIONINE b-SYNTHASE: THE GENE, THE PROTEIN, AND THE MUTATIONS Jan P.Kraus, Department of Pediatrics,University of Colorado School of Medicine, Denver,Colorado 80262, U.S.A.

Cystathionine b-synthase (CBS) catalyzes the pyridoxal 5'-phosphate (PLP) dependent condensation of homocysteine and serine to cystathionine, which is then hydrolyzed to cysteine. In addition to PLP, CBS binds two other ligands, S-Adenosylmethionine (AdoMet) and heme. AdoMet acts as an allosteric activator of CBS but the function of the heme group is unknown. De¢ciency of CBS is the most common cause of inherited homocystinuria. We have recently determined the DNA sequence of the entire CBS gene by sequencing a 28 kbp region of chromosome 21 including *5 kbp of 5'£anking sequence that contains at least two functional promoter regions. Enzyme and promoter assays have indicated that both yeast and human CBS are coordinately regulated with proliferation. Mutant recombinant CBS proteins carrying the recently discovered mutations S420L, S466L, and I435T appear to be impaired in their interaction with AdoMet. The mutant I435T CBS protein has been puri¢ed to homogeneity and shown to have normal catalytic activity but *10x lower a¤nity for AdoMet. Recently, we have identi¢ed lysine 119 as the residue in CBS responsible for PLP binding and determined a preliminary three-dimensional structure of human CBS by X-ray crystallography. We have also recently reported the expression, puri¢cation, and characterization of yeast CBS. Yeast CBS is a PLP dependent enzyme that does not contain heme and is not activated by AdoMet. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in human CBS. *

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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ACUTE CEREBRAL EDEMA IN A PATIENT WITH CYSTATHIONINE b-SYNTHASE (CBS) DEFICIENCY R Yaghmai1, MT Geraghty1, RH Allen2, SP Stabler2, A Tangerman3, C Wagner4, SH Mudd5, N Braverman1. 1 Johns Hopkins Univ, 2Univ Of Colorado, 3Univ Hosp Nijmegen, 4Vanderbilt Univ, 5NIMH.

We report an 11 yo with B6 non-responsive CBS de¢ciency who developed cerebral edema on betaine therapy. There was no infarction and we suspected a metabolic etiology. AF is noncompliant and runs plasma tHcy up to 270uM and methionine (met) 1000uM. After starting betaine her tHcy was 260, met 3000. In 3 months she developed headaches and had increased intracranial pressure. MRI showed di¡use hypo-attenuation and increased T2 signal. Ventricles were slit and basal cisterns e¡aced. NMR spectroscopy showed di¡use reductions in Nacetylaspartate and normal choline, lactate and creatine consistent with axonal damage without active demyelination or ischemia. B12, serum and RBC folate levels were normal. Compared to the range seen in therapy, betaine was normal and dimethylglycine markedly elevated. Met transamination metabolites were modestly elevated. Her AdoMet/AdoHcy ratio was reversed. We stopped betaine and met intake. Clinical symptoms and metabolic changes corrected. The edema and abnormal T2 signal resolved. AF is a MTHFR C677T heterozygote. MAT1A was sequenced for the dominant R264H mutation which was not found. These investigations do not provide a clear explanation for the clinical picture although a transient global metabolic insult seems likely. It is possible that the betaine metabolite, dimethylglycine, could have an unknown toxic e¡ect or the high met itself could impair CNS transport of neutral amino acids. Finally, defects in transmethylation might be responsible if AdoMet/AdoHcy was inverted prior to the CNS changes. This case cautions physicians to monitor met levels in their patients on betaine therapy.

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SKELETAL ABNORMALITIES IN LATE DIAGNOSED HOMOCYSTINURIA S.M. Karam, I.V.D. Schwartz, A.C. Puga, C.F.M. Souza and R. Giugliani Medical Genetics Service, Hospital de Cl|¨ nicas de Porto Alegre, and Department of Genetics, UFRGS, Porto Alegre, RS, Brazil The cystathionine B-synthetase de¢ciency leads to the autosomal recessive disorder called homocystinuria which is characterized by mental retardation, dislocation of lens, arterial and venous oclusion and skeletal abnormalities, which includes scoliosis, osteoporosis, platyspondyly, pectus carinatum/excavatus and, less frequently, genu valgum. We report two male patients from di¡erent families who had a late diagnosis (at 4 and 6 years, respectively). Both patients present, besides the classical signs of lens dislocation, myopia, mental retardation and pectus excavatus, a severe valgus deformities of the knees. The skeletal x-rays showed, in addition, osteoporosis and generalized metaphyseal abnormalities. Both were treated with low protein diet and oral administration of folic acid and pyridoxine. The levels of methionine in plasma and of homocystine in urine were reduced. They presented, after treatment, physical and cognitive improvement and a better school performance. The youngest patient is now 8 years old and, despite his radiological studies show increased bone mineralization, a femur's osteotomy was necessary. The other patient is 10 years old, and his skeletal abnormalities became worse in spite of regular treatment. These ¢ndings suggest that early diagnosis and treatment is important to avoid or reduce the impact of skeletal deformities in homocystinuria.

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HYPERMETHIONINEMIA AND HYPERHOMOCYSTEINEMIA IN METHIONINE ADENOSYLTRANSFERASE I/III DEFICIENCY F Lagler, AC Muntau, S Beblo, W RÎschinger, M Linnebank, B Fowler, HG Koch, AA Roscher Dr. von Hauner Children's Hospital, Munich, Germany, University Children's Hospital, Basel, Switzerland, Children's Hospital, University of MÏnster, Germany Methionine adenosyltransferase (MAT) transfers the adenosyl moiety of adenosine triphosphate to methionine in a reaction essential to both the production of S-adenosylmethionine and the synthesis of homocysteine. MAT I/III de¢ciency is caused by mutations in the MAT1A gene and leads to persistent hypermethioninemia. Concurrent hyperhomocysteinemia has not yet been described. A¡ected individuals usually do not show clinical symptoms, however neurological signs, learning disabilities, and demyelination have been observed. We report on two patients who had been detected by newborn screening programs and do not show clinical symptoms by now. Patient 1 is a 20 year old woman revealing plasma methionine values ranging from 850 to 1100 mmol/l (normal 20-40) with total homocysteine concentrations between 30 and 45 mmol/l (normal 5 10). Treatment with hydroxycobalamin, vitamin B6, and folic acid did not a¡ect the biochemical ¢ndings. Patient 2 was detected by MS-MS based newborn screening. Plasma methionine levels ranged from 390 mmol/l at the third day of life to 2069 mmol/l at the age of now 7 months. Plasma total homocysteine was consistently elevated (37 to 59 mmol/l). S-adenosylmethionine in CSF was moderatly decreased (190 nmol/l, normal 260 to 370). Analysis of the MAT1A gene revealed two novel mutations: patient 1 was homozygous for c.65C?T (exon 1, Ser22Leu) and patient 2 was homozygous for c.125T?C (exon 2, Leu42Pro). Our data suggest that MATI/III de¢ciency might cause a complex dysregulation of methionine and homocysteine metabolism rather than isolated hypermethioninemia. A¡ected individuals should be investigated for clinical symptoms associated with hyperhomocysteinemia.

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METHIONINE SYNTHASE DEFICIENCY: COMPLEMENTATION GROUP DIFFERENT FROM CblG AND CblE IN TWO PATIENTS T Suormala1, JE Wraith2, A Burlina3, B Fowler1. 1 Univ. Child. Hosp. Basel, Switzerland; 2Manchester Child. Hosp. UK; 3Dept. Pediatrics, Univ. Padua, Italy. Disturbed conversion of homocysteine to methionine in the absence of methylmalonic aciduria (MMA) due to functional methionine synthase (MS) de¢ciency is associated with defective MS apo-enzyme (complementation group cblG, gene MTR) or MS reductase (cblE, MTRR). Two patients with hyperhomocysteinaemia and hypomethioninaemia without MMA, with clinical and biochemical evidence of functional MS de¢ciency, have been investigated revealing features suggestive of a new MS mutant class. Patient 1 (P1, JIMD 1997:21 Suppl.1,21) presented at 6.5 years with learning di¤culties, ataxia, absent lower limb re£exes, cerebral and cerebellar atrophy, patient 2 (P2) at 3 months with seizures, severe hypotonia, nystagmus, dystonic movements and reduced myelination (brain MRI). Haemoglobin was 11.9 and 8.5 g/dL, MCV 94 and 105 fL and plasma B12 787 and 156 ng/L, in P1 and P2 respectively. Treatment with hydroxocobalamin led to biochemical and clinical improvement in both patients. In intact ¢broblasts, synthesis of methyl cobalamin was de¢cient (P1 4% and P2 6 % of total, controls 40-76%) and formation of methionine from [14C] formate was reduced in normal medium (P1 0.30, P2 0.21 nmol/mg prot/ 16 h, controls 1-4), as observed in cblE and cblG cell lines but reached normal levels in medium supplemented with 1 mg/L hydroxo-cobalamin, in contrast to cblE/G cell lines. Methionine synthase activity, measured under high reducing conditions was lowered, but not markedly de¢cient, as also seen in cblG cell lines. Cell fusion studies using PEG revealed 2-9 fold increases of methionine formation after fusion of either of the two patient cell lines with both cblE and cblG cell lines. The two patient cell lines did not complement with each other. These studies provide evidence for a further mutant class of MS de¢ciency, and emphasise the complexity of the MS reaction.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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TWO COMMON MUTATIONS 677C?T AND 1298A?C IN THE MTHFR GENE: ASSOCIATION WITH PLASMA TOTAL HOMOCYSTEINE AND FOLATE CONCENTRATIONS R Castro 1, I Rivera 1, C Jakobs 2, HJ Blom 3, I Tavares de Almeida 1 1-CPM, FFUL, Lisboa, Portugal; 2- Dep Clinical Chemistry, VU University, Amsterdam, The Netherlands; 3- Dep Pedriatrics, Univ. Hospital Nijmegen, Nijmegen, The Netherlands

Methylenetetrahydrofolate reductase (MTHFR) is one of the main regulatory enzymes of homocysteine (hcys) metabolism. Previous studies revealed that: (1) elevated plasma total hcys (thcys) is a major risk for occlusive vascular pathology; (2) the 677C?T mutation in MTHFR gene leads to decreased enzyme activity and to increased plasma hcys level in association with low plasma folate level; (3) a newly described 1298A?C mutation in the MTHFR gene clearly reduces MTHFR activity but there is no signi¢cant relationship between the homozygous genotype and plasma total hcys or folate concentrations. We determined the prevalence of these two mutations in a Portuguese cohort population (n=88) and related them to thcys and folate concentrations. The 677C?T mutation was checked by PCR and HinfI digestion, and the A1298C allele was determined by PCR followed by MboII restriction analysis. The allele frequencies were 0.32 and 0.26, respectively. Individuals homozygous for the 677C?T mutation had signi¢cantly elevated plasma thcys (p50.01) and signi¢cantly lowered plasma folate (p50.05) concentrations than subjects without the mutation. In the individuals homozygous for the 1298A?C mutation, when compared with the wild-type genotype, neither a signi¢cantly increase on thcys (p40.05) nor a signi¢cantly decrease on folate (p40.05) plasmatic concentrations were observed. Heterozygosity for both mutations was not signi¢cantly (p40.05) associated with increased thcys or reduced folate plasmatic levels. Further studies are required to clearly establish the impact of these mutations and on thcys levels and their association with occlusive vascular disease.

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FIVE YEARS OF NTBC TREATMENT OF TYROSINEMIA (HT1): SUMMARY & PERTINENCE TO NEONATAL SCREENING Que¨bec NTBC Study Group (QNS): G. Mitchell (1), F Alvarez (1), R. Barnes (2), J-F. Bussie©res (1), L. Dallaire (1), J. Dubois (1), C. Dupuis (1), F. Faucher (1), D. Fenyves (3), J-M. Forest (1), P. Goodyer (2), M-F. Goyer (1), A. Grenier (4), E. Holme (5), N. Labbe¨ (4), R. Laframboise (4), D. Lambert (2), M. Lambert (1), R. Lambert (1), J. Larochelle (6), Y. Lefe©vre (1), S. Lindstedt (5), L. Longtin (1), A. Merouani (1), J. Mitchell (2), K. Paradis (1), G. Parizeau (6), L. Pelletier (4), V. Phan (1), M. Quash (1), D. Regimbald (1), P. Rinaldo (7), R. Scott (8) C. Scriver (2), E. Treacy (2). Hoªp Ste-Justine (1), Mtl Child Hosp (2), CHUM Pav St-Luc (3), Montre¨al; CHUL, Que¨bec (4), Sahlgren's Hosp, Gothenberg (5) Cent Hosp Sagamie, Chicoutimi (6), Mayo Clin, Rochester (7), U Wash, Seattle (8) Que¨bec has performed neonatal HT1 screening since 1970. All living nontransplanted HT1 patients participate in the multicentric QNS trial, and are evaluated yearly at Hoªp Ste-Justine. 36 pts have received NTBC (duration, 2 wk-6 yr). Before NTBC, status was: hepatic, acute 4; chronic, 5; neurol, acute 4, chron, 1; chronic renal, 1; asympt 470 d, 7; asympt 5 70 d, 14. No neurologic or hepatic crises occurred after NTBC. Deterioration of glomerular ¢ltration in one pt was stopped. 6 pts with liver disease before NTBC treatment were transplanted; it is unknown whether their disease progressed or simply underwent ¢brotic resolution of preexisting damage. No pts treated rapidly after screening required transplantation. NTBC is an e¡ective short and midterm treatment for earlytreated HT1 patients. Recommendations for its long term use require further research. Two paradigms of HT1 management, (1) neonatal screening and immediate NTBC treatment (+ elective transplantation based upon clinical course) and (2) clinical diagnosis, often as hepatic failure, with palliative medical treatment and emergency or elective transplantation in survivors, should be reevaluated in light of the e¡ectiveness of NTBC.

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ETHNIC DIVERSITY AND MUTATIONS IN THE FUMARYLACETOACETATE LYASE GENE R G F Gray 1, A Harper 1 P Davies1 and P McKiernan 2. 1 Department of Clinical Chemistry, Children's Hospital, Birmingham, UK. 2 Liver Unit, Children's Hospital, Birmingham, UK. Tyrosinaemia type 1 is caused by mutations in the gene coding for fumarylacetoacetate lyase. The gene is mapped to chromosome 15q 23 ^ 25 and contains 14 exons. At least 34 disease-causing mutations have been reported of which four have been reported to account for the majority of the alleles. We have validated PCR/Restriction Digestion methods to detect the four most common mutations and applied them to specimens from 39 patients in 30 separate family groups from the West Midlands Region. In this region 60% of our a¡ected families originate from the Indian Sub-Continent (=Asian). When calculating allele frequencies only one a¡ected member of family group was counted to avoid bias by families with several a¡ected members. The IVS12g+5?a mutation showed a similar allele frequency in our Non-Asian and Asian disease populations (25% and 28%) suggesting it is a very old mutation. The IVS6g-1?t and G1009?A mutations were not detected in the Asian group. However the G192?T mutation allele frequency was 33% in the Asian population and was not detected in our Non-Asian group. This allele has, to date, only been reported in Asian individuals, which would suggest a founder e¡ect in this population. These results show that in devising strategies for screening populations for mutations in the fumarylacetoacetate lyase gene the ethnic diversity of the population needs to be taken into consideration.

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A CASE OF TYROSINEMIA TYPE 2 DETECTED BY NEWBORN SCREENING AND TREATED EARLY VE Shih, T McCormick, U von Schenck*, E Moench*. Massachusetts General Hospital, Pediatrics and Nutrition Services, Boston MA USA; *Children's Hospital, Charite, Humboldt University, Berlin, Germany Tyrosinemia type 2 is an autosomal recessively inherited trait due to tyrosine aminotransferase de¢ciency. Clinical manifestations include mental retardation and lesions of the skin and cornea. We report the ¢rst case to our knowledge of tyrosinemia type 2 detected by newborn screening. The patient was born in Berlin and routine screening showed high plasma tyrosine (Tyr, 20.7 mg/dl) and normal phenylalanine (Phe, 0.6 mg/dl). Urine showed Tyr metabolites without succinylacetone. Evaluation at age 2 mo showed normal development, no ophthalmologic abnormalities, no enlargement of liver or spleen. The infant had been fed breast milk exclusively before changing to a Tyr and Phe free formula with limited breast milk intake. Tyr intake was 29 mg/kg body weight and plasma Tyr levels were kept below 4.7 mg/dl. At age 6 mo mental development was appropriate for age but growth failure and thin hair were evident. Tube feeding was necessary to provide adequate nutrition. At age 10 mo when the family moved to the USA, plasma Tyr was well-controlled (60 mM) and Phe was low (10 mM). Height and weight were below the 3rd percentile; the patient was hypotonic and almost completely dependent on tube feeding. Increased dietary intakes of Tyr and Phe resulted in better growth and a gain in motor skills; tube feeding was discontinued. Plasma Tyr ranged from 300^ 750 mM, Phe between 1550 mM, methionine normal. At age 3 years the patient's intellectual develpment is normal but growth remains less than the 3rd percentile. There are no corneal or skin lesions.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BODY FLUID NMR SPECTROSCOPY OF PATIENTS WITH HAWKINSINURIA Moolenaar, S.H.1; Engelke, U.F.H.1; Lehnert, W.2; van den Berg, G.B.3; Wevers, R.A.1 1 Laboratory of Pediatrics and Neurology, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands; 2University Children's Hospital, Freiburg, Germany; 3Observation Centre De Hondsberg, Oisterwijk, the Netherlands. 1

H-NMR spectroscopy provides an overall view of almost all proton containing metabolites in body £uids. Moreover, the technique can be used for metabolite quanti¢cation. In this study, we measured body £uids from 3 unrelated patients su¡ering from Hawkinsinuria using high resolution NMR spectroscopy. Several unknown resonances were observed in the urine spectra of the patients. These resonances could be assigned to hawkinsin and 4-hydroxycyclohexylacetic acid using two-dimensional NMR techniques (COSY, JRES, TOCSY, HSQC, HMBC). The NMR spectra were used for quanti¢cation of the metabolites. The concentration of hawkinsin varied between 190 and 2100 mmol/mmol creatinine. 4-Hydroxycyclohexylacetic acid may be absent in urine samples of young patients. The concentration varied between 220 and 1100 mmol/mmol creatinine for older patients. Moreover, 5oxoproline could be observed in the urine spectra of 2 of the patients. Our results showed that two-dimensional NMR experiments can be helpful for the identi¢cation of unknown metabolites. Urine 1H-NMR spectra can be used for diagnosing Hawkinsinuria patients due to the ability to identify and quantify relevant metabolites.

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THREE NOVEL MUTATIONS IN THE ORNT1 GENE OF JAPANESE PATIENTS WITH HHH SYNDROME Takeshi Miyamoto,1 Naomi Kanazawa,1,2 Tohya Ohashi,3 Yoshikatsu Eto,3 Toyokazu Saito,4 Junichi Kira,5 Takeshi Yamada,5 Seiichi Tsujino1 1 Department of Inherited Metabolic Disease, National Institute of Neuroscience, NCNP, 2Research Division of Biochemistry, Kitasato University School of Medicine, 3Department of Pediatrics, Institute of DNA Medicine, Jikei University School of Medicine, 4Department of Neurology, Kitasato University East Hospital, 5Depatment of Neurology, Kyushu University Medical School Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome presents with various neurological symptoms; mental retardation, spastic paraparesis with pyramidal signs, cerebellar ataxia and episodic disturbance of consciousness or coma due to hyperammonemia. Analyses of reverse transcription-polymerase chain reaction (RT-PCR) products amplifying the mitochondrial ornithine transporter gene (ORNT1) cDNA from cultured skin ¢broblasts RNA of patients or genomic PCR identi¢ed three novel mutations in the ORNT1 gene of three Japanese patients with HHH syndrome. These include a nonsense mutation (R179X), a missense mutation (G27E), and an insertion of AAC between codons 228 and 229, leading to an insertion of amino acid Asn. Any mutation was only di¡erence in the coding region of the ORNT1 gene of each patient, and was con¢rmed by genomic PCR. All three patients were homozygous for their respective mutations. This study con¢rms that defects in the ORNT1 gene cause the HHH syndrome, and that the genetic basis in Japanese patients is heterogeneous.

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EXON-INTRON STRUCTURE OF THE ORNT1 GENE AND NONSENSE-MEDIATED EXON SKIPPING IN A JAPANESE PATIENT WITH HHH SYNDROME Seiichi Tsujino,1 Takeshi Miyamoto,1 Naomi Kanazawa,1,2 1 Department of Inherited Metabolic Disease, National Institute of Neuroscience, NCNP, 2Research Division of Biochemistry, Kitasato University School of Medicine Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome is characterized by the three biochemical abnormalities stated in the name. Recently, Camacho et al cloned the cDNA of the mitochondrial ornithine transporter gene (ORNT1) and showed that mutations in the ORNT1 gene cause HHH syndrome. We clari¢ed by genomic polymerase chain reaction (PCR) that the ORNT1 gene consists of at least six exons, and all exon-intron junction sequences conform to the GT/AG rule. Approximate lengths of introns 1-5 are 6 kb, 6 kb, 2 kb, 1 kb and 1 kb, respectively. In a Japanese patient with HHH syndrome, we observed skipping of exon 4 in the ORNT1 mRNA. Genomic PCR identi¢ed a nonsense mutation (R179X) almost at the center of exon 4, but no other nucleotide change around any splice junction of the gene. This implied that the exon skipping is mediated by the nonsense mutation. Although this phenomenon has been reported in several genes causing human genetic diseases, the mechanism is not yet clear. Northern blot analysis showed that the amount of ORNT1 mRNA of the patient was not reduced, excluding the possibility that exonskipping mRNA was e¤ciently ampli¢ed by RT-PCR due to reduction of full-length mRNA with socalled nonsense-mediated mRNA decay.

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GENOTYPE AND PHENOTYPE FINDINGS IN THE HYPERORNITHINEMIAHYPERAMMONEMIA-HOMOCITRULLINURIA (HHH) SYNDROME VE Shih and C Ficicioglu Massachusetts General Hospital, Harvard Medical School, Boston MA USA The HHH syndrome is an autosomal recessive disorder caused by defective import of ornithine into the mitochondria. The clinical symptoms are related to hyperammonemia and resemble those of the urea cycle disorder ornithine carbamyltransferase de¢ciency. The basic defect is at the level of the mitochondrial ornithine transporter (ORNT1). The human ORNT1 gene has been mapped to chromosome 13q14. Three di¡erent ORNT1 mutant alleles have been previously reported. We have now performed mutational analysis using cDNA in a total of 11 patients in 10 unrelated families. Primers for PCR ampli¢cation were chosen from the published human cDNA sequence and PCR products were sequenced. We identi¢ed two novel mutations in two unrelated patients. A change of 44C to A resulting in amino acid change A15E was identi¢ed in a Palestinian infant with neonatal hyperammonemia. A 2 bp insertion following bp 96 was identi¢ed in a South American girl with mental retardation. Additionally, of the 9 patients in 8 French Canadian families that we studied, 7 patients were homozygous and 2 patients were heterozygous for the F188 deletion. As previously reported, our data also shows that the F188 deletion is the most common mutation in patients of French Canadian ancestry. However, we have found signi¢cantly di¡erent phenotypic variation in the patients who were homozygous for the F188 deletion mutation. The clinical presentations ranged from the severe neonatal form to a mild adult onset form. Supported in part by PHS grant NS05096.

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AMINO ACID LOADING STUDIES AND L-SERINE SUPPLEMENTATION IN 3PHOSPHOGLYCERATE DEHYDROGENASE (3-PGDH) DEFICIENCY Kathryn J. Swoboda1, Mark S. Korson2, Basil T. Darras2, Marvin R. Natowicz2 1 University of Utah, Salt Lake City, UT, USA 2 Harvard University, Boston, MA, USA

Background: 3-PGDH de¢ciency is a recently described inborn error of serine biosynthesis. Treatment with serine and glycine has been previously associated with reduced seizure activity in two patients. Objective: Minimal data is available regarding serine homeostasis in humans. We performed amino acid loading studies and serine supplementation in two siblings with documented 3-PGDH de¢ciency to evaluate kinetics and guide amino acid dosing. Methods: Loading studies were performed with serine and glycine,100 and 200 mg/kg/dose. Results: Oral serine 100 mg/kg every 4 hours was associated with substantial and sustained reduction in clinical seizure activity within the ¢rst two weeks of treatment, as well as improved interactiveness, visual behavior, growth and reversal of anemia. Conclusions: High dose serine supplementation (300 mg/kg/day in divided doses) normalized CSF and plasma serine and glycine levels and improved seizure control in these patients. The rapid reduction in clinical seizures with serine supplementation supports a primary role for serine de¢ciency in the pathogenesis of epilepsy, and con¢rms prior reports of bene¢t with serine treatment. Oral serine increased glycine levels considerably in CSF and plasma, making supplemental glycine unnecessary. The neurologic bene¢t associated with serine supplementation merits early detection of this disorder in infants with nonsyndromic congenital microcephaly.

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PRENATAL DIAGNOSIS IN 3-PHOSPHOGLYCERATE DEHYDROGENASE DEFICIENCY TJ de Koning, LWJ Klomp, FA Beemer, C van Oppen, M Duran, BT Poll-The, IET van den Berg, JK Ploos-van Amstel, R Berger. Departments of Metabolic Diseases, Pediatric Gastroenterology, Medical Genetics and Obstetrics, University Medical Center Utrecht, KC 03.063.0, PO Box 85090, 3508 AB Utrecht, The Netherlands. [email protected]

3-Phosphoglycerate dehydrogenase (PHGDH) de¢ciency is a severe neurometabolic disorder of serine metabolism. A¡ected patients present with congenital microcephaly, severe psychomotor retardation and intractable seizures. Short term results showed beni¢cial e¡ects of high doses of Lserine and glycine in the treatment of seizures. However, no long term follow-up data are available. The fact that all patients with PHGDH de¢ciency presented with congenital microcephaly, indicates that fetal brain development is disturbed in this disorder. We report the ¢rst prenatal diagnosis in a Turkish family with two a¡ected children. At 10 weeks of pregnancy a villus biopsy was performed for DNA analysis. The fetus was homozygous for a V490M mutation in the PHGDH gene which is associated with the disease phenotype in this family (Klomp et al, submitted). The parents opted not to terminate the pregnancy. At the moment the pregnancy is monitored carefully, the mother will receive L-serine as a possible treatment for the fetus. We will report on the results of this fetal treatment during pregnancy.

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CYSTINURIA TYPE I, GROWTH, MENTAL RETARDATION, AND DYSMORPHISM ASSOCIATED WITH SLC3A1 GENE DELETION - A NEW SYNDROME ? Eli Hershkovitz1, Irena Brodiansky1, Orly Elpeleg2, Ruti Parvari3 1 Pediatric Department and 3 Molecular Genetic Laboratory, Soroka Medical Center, Ben-Gurion University, Beer Sheva, 2Metabolic Unit, Shaare-Zedek Hospital, Jerusalem, Israel Cystinuria is a heritable disorder of cystine and dibasic amino acids transport. We describe six patients with a unique clinical picture and deletion of all exons of the SLC3A1 gene (Solute carrier family 3, member 1) which encodes the carrier transporter of dibasic amino acids and cystine. The patients were diagnosed during the ¢rst year of their life with facial dysmorphysim, neonatal seizures, hypotonia, and severe somatic and developmental delay. Renal stones were found in 4 patients. The major laboratory ¢ndings were: neonatal hypocalcemia and hypoglycemia and lactic acidosis (3 patients). Urinary cystine, lysine,arginine and ornitine, levels were compatible with its level in cystinuric homozygotes patients.The parents and all the apparently healthy siblings had normal urinary excretion pro¢le of amino acids, establishing the diagnosis of cystinuria type I. Muscle biopsy in one patient showed red ragged ¢bers and low activity of complex I,IV, and V, with normal complex II and PDHc activities. Linkage analysis by homozygosity disclosed a linkage to D2S119 and D2S177 close to the locus of the SLC3A1 gene. The 10 SLC3A1 exons could not be ampli¢ed from genomic DNA by PCR. DNA samples from the parents yielded normal ampli¢cations of the SLC3A1 gene's exons. Since cystinuria is probably not associated with mental retardation and as no clinical segregation were found among the rest of the family members, we suggest that this unique clinical syndrome is most likely a result of a deletion that include the SLC3A1 locus. The deletion could also a¡ect the function of additional genes, involved in the synthesis of mitochondrial encoded proteins.

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A PARADOX OF THE NMR URINALYSIS ^ THE ABSENCE OF SIGNALS OF CYSTINE IN THE URINE FROM CYSTINURIA Shuichi Yamaguchi, Tomohiro Matsumoto, Akira Yamaguchi, Hideaki Yamamoto, Masamichi Chiba, Hiroshi Mochizuki, Naoya Koda Division of Endocrinology and Metabolism, Department of Radiological Technology, Department of Laboratory Medicine, Saitama Children's Medical Center, Saitama, Japan Nuclear Magnetic Resonance (NMR) urinalysis has been applied to the diagnosis of some kind of inherited metabolic diseases. Cystinuria is an autosomal recessive disorder of amino acid transport a¡ecting the epithelial cells of renal tubules and gastrointestinal tract. It commonly presents with renal stones that, if untreated, may cause renal damage. The disease is characterized by the higher excretion of cystine, lysine, arginine and ornithine. In our 15 cases with cystinuria, the analysis with NMR did not detect cystine, but detected lysine, arginine and ornithine. If cystine is added to the urine from cystinuria, signals of cystine is visible. The pretreatment of the urine from cystinuria such as the alkalization, acidi¢cation, heating did not a¡ect the visibility of cystine. The patients with lysinuric protein intolerance (LPI) excrete in the urine, lysine, arginine and ornithine, never excrete cystine. The NMR analysis of LPI urine is similar to that of cystinuria. The di¡erential diagnosis of cystinuria and LPI is impossible by NMR urinalysis only. It is probable as the screening of dibasic aminoacidopathies, cystinuria and LPI. We should take care for the interpretation of the NMR urine with respect to t he invisibility of chemical compounds.

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BIOCHEMICAL CLASSIFICATION OF CZECH CYSTINURIA PATIENTS S. Stastna1, K. Peskova1, E. Pospisilova1, Z. Skopkova2 Institute of Inherited Metabolic Disorders, General Faculty Hospital, Prague, Czech Republic1; Dept. of Clinical Biochemistry, University Hospital, Olomouc, Czech Republic2

Background. Cystinuria is a renal tubular and an intestinal absorption disorder of cystine and dibasic aminoacids. Three types (I, II and III) and 9 biochemical phenotypes of cystinuria can be distinguished. Cystine excretion of homozygotes of all types, mixed heterozygotes and heterozygotes II/N and III/N can exceed the threshold of urinary cystine solubility and lead to stone forming. Heterozygotes I/N excrete cystine in the normal range, heterozygotes II/N and III/N excrete cystine in moderate to marked range. Recently, 2 cystinuria loci were found in the chromsomes 2p2.1 (type I) and 19q13.1 (type II and III). Methods. Urinary cystine excretion was studied in a group of 140 Czech patients with cystinuria and their parents. Probands were identi¢ed through our selective screening programme for urolithiasis. Fifty-seven patients were classi¢ed into cystinuria types by their and parental urinary cystine excretion (others were excluded because of incomplete family investigation). Urinary aminoacids were quanti¢ed by ion-exchange chromatography with ninhydrine detection. Results. In the group of 57 patients with cystinuria we distinguished 24 patients with type I/I, but no patients with subtype II/II nor III/III were identi¢ed. We classi¢ed 11 patients as compound heterozygotes with type I/III, 6 patients with type I/II and 2 patients with type II/III, 6 patients are heterozygotes with type III/N and 8 with type II/N. Biochemical ¢ndings are given and statistically evaluated. Conclusions. Classical recessive type I/I is the most frequent subtype of cystinuria in Czech population. Type II/ ? seems to be more frequent in Czech population than in other ones. Precise biochemical determination of the type of cystinuria is the necessary step before molecular genetic studies. Following molecular genetic studies will check correct biochemical determination. Supported by Grant GACR 303/00/0928

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PROLIDASE DEFICIENCY ASSOCIATED TO HYPER IgE Martins E, Silva A, Lopes I, Pena R, Pereira J, Vilarinho L Hospital de Crianc°as Maria Pia Porto, and Instituto de Gene¨tica Me¨dica Jacinto de Magalha¬es Porto

Prolidase de¢ciency (McKusick 170100) is a rare inherited metabolic disorder characterized by dysmorphic phenotype, chronic dermatitis, recurrent infections (respiratory and coetaneous), splenomegaly, anemia, mental retardation and massive urinary excretion of imidodipeptides. The diagnosis is con¢rmed by measures of prolidase activity. The authors present the case report of a 5 years old girl with coarse facies, erythematous dermatitis and respiratory, recurrent infections since the age of 1 month. Diagnosed of prolidase de¢ciency at 4.5 months of age. By this time, she presented total IgE elevated, which has been progressively increasing the actual values higher than 20.000 U/ml. She also presents T linphocytosis with elevation of CD3 and CD4 and impaired neutrofhyls quimiotaxis. All these immunological alterations are associated to prolidase de¢ciency except one importance increase of IgE. The authors reinforce the results with dietary supplements of ascorbic acid, oral MnCl associated to hyperproteic regimen and topic proline.

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RECURRING MUTATIONS IN P- AND T-PROTEINS: A STRATEGY FOR THE MOLECULAR INVESTIGATION OF PATIENTS WITH NONKETOTIC HYPERGLYCINEMIA (NKH) J.R. Toone1,3, D.A. Applegarth1,2,3, M.B. Coulter-Mackie1,2, E.R. James1 Departments of Pediatrics1 and Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia2. Biochemical Diseases Laboratory, B.C.'s Children's Hospital, 4480 Oak St., Vancouver, British Columbia, V6H 3V4, Canada3 NKH is an autosomal recessive disorder of glycine metabolism resulting in severe neurological disease. The defective enzyme complex, glycine cleavage enzyme, is made up of 4 proteins: P, T, H, and L and almost all NKH patients have P or T protein defects. Enzymatic con¢rmation of NKH requires a liver sample and is not done in most patients because of the di¤culty in obtaining this sample: even fewer patients are tested for their speci¢c protein defect. We have devised PCR/restriction enzyme assays for 2 point mutations: R515S in P-protein and R320h in T-protein and tested 100 normal and more than 100 NKH alleles. Neither mutation was found in normal controls. In NKH patient alleles, R515S(P) was detected in 4.5% (6 heterozygotes) and R320H(T) in 4.5% (2 homozygotes, 2 heterozygotes). The second mutation in the R320H heterozygotes was found by sequencing the T-protein gene. DNA screening for the 2 mutations described can detect 9% of NKH alleles and identify the defective protein without the requirement of a liver sample. Enzymatic prenatal diagnosis requires a large CVS biopsy and false negative and false positive results have been reported. Molecular testing would o¡er improved diagnosis and prenatal diagnosis in informative families.

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DEVELOPMENT OF A NOVEL STABLE-ISOTOPE DILUTION ASSAY OF GABATRANSAMINASE (GABA-T) IN ISOLATED WHITE CELLS, AND FURTHER EVIDENCE THAT GABA-T AND BETA-ALANINE TRANSAMINASE (BAT) ARE IDENTICAL E.A. Struys1, D.S.M. Schor1, B.M. Hogema1,2, K.M. Gibson2 and C. Jakobs1 Metabolic Unit, Dept. of Clinical Chem., Free University Hospital Amsterdam, The Netherlands1; Dept. of Molec. and Med. Genet., Oregon Health Sciences University, Portland, USA2 GABA-T de¢ciency is an inborn error of GABA degradation. Two patients with GABA-T de¢ciency have been identi¢ed. It remains uncertain whether or not hyper-b-alaninemia is the same disorder. Several methods have been published to measure GABA-T activity, but these methods are either impracticable due to the use of considerable amounts of radioisotopes, or insu¤ciently sensitive to determine small enzyme activities in white cell extract. We developed a method for the measurement of 15N-glutamic acid which is derived from 15N-GABA and a-ketoglutaric acid (aKG), catalyzed by GABA-T. Method: lysates of lymphocytes or lymphoblasts were incubated at 378C with 15N-GABA and aKG (in the presence of DTT, pyridoxal-5-phosphate, potassium phosphate bu¡er pH=8). The incubation was terminated (HCLO4) and 2 nmol glutamic acid [2,3,3,4,4-d5] was added as internal standard. Glutamic acid was derivatized in the aqueous reaction mixture with methyl chloroformate. The derivative was isolated by solid phase extraction and methylated directly in the acidi¢ed methanolic eluate. Quantitation was accomplished by GC^MS, operated in the electron impact mode. GABA-T de¢cient lymphoblasts showed signi¢cantly diminished enzyme activity (control values (n=6): 44 + 11 pmol min1 (mg protein)-1, range: 23^55; patient: 1.2). Enzyme activity in this patient was also de¢cient with 15N-b-alanine as substrate, further suggesting that GABA-T and BAT are identical when using aKG as nitrogen acceptor. This new assay is sensitive and speci¢c, and obviates the need for use of radioisotopes. The isotope dilution assay should prove bene¢cial for the future analysis of patients with suspected GABA-T de¢ciency and those identi¢ed with hyper-b-alaninemia.

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STUDIES OF THE CORRELATION BETWEEN PHENOTYPE AND GENOTYPE IN HEREDITARY GLUTATHIONE SYNTHETASE DEFICIENCY Runa Njlsson, Ellinor Risto¡, Katarina Carlsson, Andreas Winkler, Camilla Augustsson, Svante Norgren, Agne Larsson Dept. of Pediatrics, Karolinska Institutet, Huddinge Hospital, Sweden

Introduction: Glutathione synthetase (GS) catalyzes the last step in the synthesis of glutathione (GSH): g-glutamylcysteine + glycine + ATP ? g-glutamylcysteinyl glycine (GSH) + ADP + PI. Gs de¢ciency is a heterogeneous disease both clinically and genetically. The mildest forms result in haemolytic anaemia only; whereas the most severe forms also involve 5-oxoprolinuria, metabolic acidosis, CNS damage and possibly early death. We are testing the hypothesis that there is a correlation between phenotype and genotype. Methods: Clinical data was collected from 17 patients in 13 families with GS de¢ciency, and their GS mutations were established. Results: 15 di¡erent deletions, missense and nonsense mutations were identi¢ed in the 34 GS alleles. 10 patients were homozygous and 6 were compound heterozygous. One patient did not have any mutation in the coding sequence of the GS gene. Analysis of recombinant mutant enzyme has revealed decreased stability as well as decreased catalytic activity, revealed by changes in Vmax and Km. Conclusion: No simple correlation between genotype and phenotype has been found so far. It is possible, however, that environmental factors including treatment (acidosis correction, vitamin C and E) in£uence the outcome. Furthermore, the clinical symptoms caused by a single mutation are di¤cult to identify in patients who are compound heterozygous. Analysis of more GS de¢cient patients is required to elucidate the correlation between genotype and phenotype in GS de¢ciency.

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QUANTIFICATION OF S-SULPHOCYSTEINE, A MARKER FOR SULPHITE OXIDASE DEFICIENCY, BY TANDEM MASS SPECTROMETRY D.S. Millington1, R.D. Stevens1, K.V. Rajagapolan2 and J.L. Johnson2. Depts 1Pediatrics and 2Biochemistry, Duke University, Durham, North Carolina, USA.

Molybdenum cofactor de¢ciency or isolated sulphite oxidase de¢ciency should be considered in the evaluation of newborns with seizures. S-sulphocysteine is a disease- speci¢c metabolite that is elevated in urine and blood of patients with either form of the de¢ciency. We have developed a stable isotope dilution tandem mass spectrometric (TMS) method for quantifying S-sulphocysteine in urine. A ¢xed amount of internal standard (13C3, 15N-S-sulphocysteine) is added to an aliquot of urine. The mixture is dried and derivatised to the butyl ester, desalted by solid phase extraction analysed by TMS monitoring a speci¢c product ion. The ratio of the parent ions m/z 256 and 260, corresponding to Ssulphocysteine and internal standard, is determined and S-sulphocysteine concentration calculated using a standard curve. Values are normalised to creatinine. The method o¡ers certain advantages over existing methods such as electrophoresis or chromatographic amino acid analysis as it is speci¢c and rapid. In the present format, analysis of 10 samples can be completed in 1 hour. The method is readily adaptable to a microtitre plate for derivatisation and LC-TMS analysis to increase sample throughput and reduce labour costs . The range of S-sulphocysteine levels found for patients with documented sulphite oxidase de¢ciency was 600-1590 mmoles/g creatinine (n=6) with normal values 5 25 mmoles/g creatinine (mean + 2 SD, n=50). We are currently extending this method to measure levels from urine ¢lter paper spots.

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ISOLATED SULPHITE OXIDASE DEFICIENCY IN A NEWBORN GIRL S.Seneca1, W.Lissens1, E.Gerlo2, D.Hasaerts3, C.Dorche4, I.Liebaers1 and L.De Meirleir3. 1 Center for Medical Genetics, 2Clinical Chemistry, 3Department of Paediatrics, University of Brussels, Belgium; 4Pediatric Biochemistry, Lyon, France Sulphite oxidase de¢ciency is a rare, autosomal recessive inborn error of metabolism. The disease may occur as an isolated de¢ciency of sulphite oxidase or, more often, as a molybdenum cofactor de¢ciency. We report here on a girl, born at term with normal birth weight after an uneventful pregnancy to healthy, unrelated parents. She was admitted to the neonatal care unit with apnoeas, feeding di¤culties and hypothonia. The patient developed symmetrical and asymmetrical clonic seizures with bicycling movements. A MRI and CT scan of the brain revealed di¡use cerebral oedema of the subcortex and white matter. Laboratory studies showed increased S-sulfocysteine and thiosulfate levels, but normal xanthine and hypoxanthine levels in urine. A diagnosis of isolated sulphite oxidase de¢ciency disorder was considered and subsequently con¢rmed by absence of sulphite oxidase enzymatic activity in cultured ¢broblasts. Automatic sequencing analysis of cDNA from ¢broblast mRNA identi¢ed a homozygous G to A point mutation at position 913, resulting in a glycine to serine amino acid substitution (G305S). The mutation was veri¢ed at the genomic level. Both parents were heterozygous carriers of the mutation. The G305S mutation was not found in 25 unrelated controls. A prenatal diagnosis on CVS material was performed for a second pregnancy, and a healthy heterozygous carrier girl was born.

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CHARACTERIZATION OF THE HUMAN HOMOLOGUE OF THE YEAST GENE LYS5, WHICH ENCODES A PHOSPHOPANTETHEINYL TRANSFERASE THAT ACTIVATES aAMINOADIPATE REDUCTASE (LYS2P) Praphanphoj V1, Sacksteder KA2, Geraghty MT1. Depts. Pediatrics1, Biological Chemistry2, Johns Hopkins University School of Medicine, Baltimore, MD, USA In mammals, the early steps of L-lysine degradation are accomplished by its conversion ¢rstly to aaminoadipic semialdehyde by the bifunctional enzyme a-aminoadipate semialdehyde synthase (AASS) and secondly to a-aminoadipate by a-amnioadipate reductase. We recently identi¢ed the gene encoding AASS and reported mutations in this gene in a patient with familial hyperlysinemia. In S cerevisiae the gene LYS2 encodes a-amnioadipate reductase while a second gene LYS5 encodes a phosphopantetheinyl transferase. A recent report demonstrated that LYS5p activates LYS2p by this novel mechanism. We used the LYS5 sequences from S cerevisiae to search the EST database and subsequently cloned a full-length human cDNA. The cDNA contains an ORF of 927 bp predicted to encode 309 amino acids. The human protein is 26% identical to its yeast counterpart. In Northern blot analysis the cDNA crosshybridizes to a single transcript of approximately 3 kb in all tissues except testis, where there is an additional transcript of 1.5kb. Expression is highest in brain followed by heart and skeletal muscle and surprisingly to a lesser extent in liver. Further we have identi¢ed a human genomic DNA BAC clone which contains the structural gene and we have partially de¢ned the intron-exon boundaries. This BAC clone maps to human chromosome 11q22. Complementation studies in the yeast lys5 knockout strain using the human full-length cDNA cloned into a yeast expression vector are underway to further characterize the function of this gene.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

INCREASED NO PRODUCTION IN LPI FIBROBLASTS L.Mannucci,F.Emma,R.Carrozzo,C.Rizzo,C.Dionisi-Vici. Bambino Gesu© Hospital, Rome, Italy.

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LPI is a congenital disorder of dibasic aminoacid transport secondary to mutation of the SLC7A7 gene characterised by postprandial hyperammonemia, renal involvement, alveolar proteinosis and lupus-like autoimmune symptoms. The pathophysiology of systemic involvement remains poorly understood. Generally, plasma arginine (arg) and ornithine are reduced while citrulline levels are increased, despite functional impairment of the urea cycle. Conversely, increased intracellular arg levels have been reported in LPI ¢broblast. We hypothesise that increased intracellular arg promote NO production with secondary induction of in£ammatory pathways. To test this hypothesis, we have studied in vitro NO production in 3 LPI and 3 control ¢broblast cell lines. Inducible and constitutive nitric oxide synthase expression were similar in the two groups of cells, as demonstrated by western blotting and immunocytochemistry. The 15 min 14C-Arg uptake was higher in LPI ¢broblasts (9.9þ0.4 nM/mg of prot.) than in control cells (8.8þ0.3 nM/mg of prot.) but did not reach statistical signi¢cance (p=0.07). However, NO production as measured by NO3 production using the Griess reaction, was signi¢cantly higher in LPI ¢broblasts. NO3 production was 32þ5 nM/mg of prot. in LPI cells and 16þ3 nM/mg of prot. in control cells, corresponding to an average 107% increase (p50.01). These data indicate that impaired arg e¥ux secondary to SLC7A7 protein mutation may induce production of NO. These results are in agreement with our previous clinical studies demonstrating increased plasma NO3 levels in LPI patients (Dionisi-Vici et al., J Inher Metab Dis 19:38;1996). Given the well known activation of various in£ammatory pathways by NO, we speculate that part of the unexplained symptoms presented by LPI patients, may be secondary to increased NO production.

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OUTCOME IN INTERMEDIATE/INTERMITTENT MAPLE SYRUP URINE DISEASE (MSUD) Fanny Lam1, Judith Hammond2, Bridget Wilcken2 1 Clinical Genetic Service, Hong Kong, 2NSW Biochemical Genetics Service, New Children's Hospital, Sydney, Australia

Improved diagnosis and management has improved the outlook in classical MSUD (C-MSUD). Little has been recorded in intermittent/intermediate forms (I-MSUD). In New South Wales we diagnosed clinically 5 C-MSUD and 9 I-MSUD patients from 9 families, and 1 C-MSUD by newborn screening. The birth prevalence was 1:136,000. All patients survived and are now aged 123 years. Only one of the C-MSUD patients has developmental delay needing special school help. Of the I-MSUD cases, 2 treated from birth, and 2 siblings in 1 family, diagnosed at 3 & 15m have normal development. The remaining 5 have signi¢cant delay, 2 with moderate and 3 mild mental retardation. Presenting levels of plasma leucine were 43000 mmol/L for C-MSUD patients, and 1000 - 3000 mmol/L for I-MSUD probands. Age when leucine was ¢rst recorded 51000 mmol/L was 12-15days for C-MSUD, and 3-18months for I-MSUD probands. All I-MSUD patients had leucine levels easy to control within the normal range with minimal protein restriction, except during intercurrent illness. They had mean admissions of 1/3 patient-years (versus 1/2 patient-years for C-MSUD). I-MSUD appears commoner than C-MSUD in NSW, and the intellectual outcome is worse. Detection by newborn screening is vital to ensure good outcome in mildly a¡ected cases.

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CEREBRAL METABOLITE QUANTITATION BY PROTON MAGNETIC RESONANCE SPECTROSCOPY IN PATIENTS WITH MAPLE SYRUP URINE DISEASE B. Schwahn1, U. Wendel1, H. Kugel2 1 Children's Hospital, Heinrich-Heine University Duesseldorf and 2Department of Diagnostic Radiology, University of Cologne, Germany Encephalopathy in maple syrup urine disease (MSUD) is believed to be caused by elevated levels of accumulating branched chain amino acids (BCAA) and branched chain oxo acids (BCOA). Proton magnetic resonance spectroscopy (MRS) is the only tool to gain insight in in vivo tissue concentrations and to determine possible threshold metabolite levels for acute or chronic toxic e¡ects on the brain. We here report on the investigation of three adolescent patients (age 13 - 16y) in good condition but with chronically very high plasma levels due to dietary incompliance (leucine 1.037 +/0.087mmol/l, sum of BCAA/BCOA 2.604 +/- 0.196mmol/l) by means of quantitative MRS. Mean brain tissue BCAA/BCOA sum concentration was estimated as 0.88 +/- 0.29mmol/kg brain water, resulting in a brain/plasma (B/P) ratio of 0.34 +/- 0.13. A previous MRS study of one patient recovering from decompensation showed plasma levels for BCAA/BCOA of 2.536mmol/l and brain tissue levels of 0.9mmol/l using creatine as internal standard, resulting in a B/P ratio of 0.36 (Heindel et al. Pediatr. Radiol 25:296-9, 1995). Sansaricq et al. (Pediatr Res 25:202A, 1989) reported a BCAA CSF/plasma ratio of 0.298 in acutely decompensated infants with MSUD. We conclude that in patients with MSUD brain tissue BCAA/BCOA sum levels can be obtained in vivo and are tolerated up to around 0.88mmol/kg water without symptoms of acute neurotoxicity. B/P ratio and CSF/P ratio seem to be similar. Whether adaptation to these levels due to chronic exposure has to be taken into consideration should be clari¢ed by investigating acutely decompensated patients.

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BONE MINERAL DENSITY (BMD) OF PATIENTS WITH TREATED MAPLE SYRUP URINE DISEASE (MSUD) S Yap1, G Barniville2, M O'Brien2, ER Naughten1. 1 National Center for Inherited Metabolic Disorders, The Children's Hospital, Temple Street, and 2the Osteoporosis Unit, Trinity College, Dublin, Ireland. The treatment of MSUD is dietary restriction of leucine and supplementation with a synthetic amino-acid mix, depleted of leucine, valine and isoleucine. The clinical aims of treatment are to achieve normal intelligence and growth. Synthetic diets can result in de¢ciencies, particularly if there is poor compliance or inadequate provision of nutrients in growing children. BMDs of lumbar spine (L1-4) were assessed in seven MSUD patients with a mean (range) age of 15.9 (8-21) and a total of 111 patient-years of treatment. All had normal serum calcium and phosphate. BMD were expressed as Z-scores (+SD with respect to age and gender matched controls). WHO de¢nitions of osteoporosis was based on T-scores (+SD with respect to peak BMD). The study group had mean (range) Z-scores of -1.29 (-0.19 to -2.41) and T -scores of -2.41 (-0.48 to 5.11). The younger three patients had mean (range) Z-scores of -1.32 (-0.55 to -1.91) and T-scores of -3.67 (-2.83 to -5.11). The remaining older four patients had a mean Z-scores and T-scores of -1.27 and -1.43 respectively. The discrepancies between T- and Z-scores in the younger patients may be due to delayed bone ossi¢cation. The results of this small study suggest that normal BMD in growing children with MSUD on synthetic diets is achievable. Regular biochemical and dietetic monitoring to ensure both good compliance and provision of adequate prescribed products for normal growth are essential. Longitudinal follow-up of this cohort of growing MSUD patients will provide information for further improvement in the treatment and outcome.

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INHIBITION OF GLUTAMATE UPTAKE INTO SYNAPTIC VESICLES OF RAT BRAIN BY THE METABOLITES ACCUMULATING IN MAPLE SYRUP URINE DISEASE CS Dutra-Filho, RG Tavares, CES Santos, CI Tasca, M Wajner, DO Souza. Dept Bioqu|¨ mica, ICBS, Univ Fed do Rio Grande do Sul, Porto Alegre, Brazil

Maple syrup urine disease (MSUD) is caused by severe de¢ciency of the branched-chain L-2-keto acid dehydrogenase complex activity. Neurological dysfunction is common in this disorder. However its pathogenesis is poorly understood. Glutamate, a major excitatory neurotransmitter, is involved in essential functions in the CNS, as well as in excitotoxicity. In this work, we investigated the ``in vitro'' e¡ect of metabolites accumulating in MSUD on [3H]glutamate uptake into synaptic vesicles from the brain of Wistar rats. Synaptic vesicles were isolated (1) and were incubated with L-[3H]glutamate at 358C for 10 min for the uptake studies (2).. We tested amino acids and keto acids at concentrations of 0.25-10 mM. Controls were not supplemented by any acid. Our results showed that L-leucine, L-2keto-isocaproic acid (0.5-10 mM), L-isoleucine and L-2-keto-3-methylvaleric acid (5 and 10 mM) inhibit the glutamate uptake into synaptic vesicles, whereas L-valine and L-2-keto-isovaleric acid showed no e¡ect. These results demonstrate an inhibition of glutamate uptake into synaptic vesicles caused by various metabolites accumulating in MSUD. The consequences of these e¡ects on extracellular glutamate levels leading to excitotoxicity should be considered in the pathophysiology of the neurological dysfunction present in this disorder. Supported by: CNPq, FAPERGS, PRONEX. (1) Fykse EM, Fonnum F. J Neurochem 1988;50:1237-42; (2) Wolosker et al. J Biol Chem 1996;271:11726-31.

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VARIANTS OF MAPLE SYRUP URINE DISEASE (MSUD). CLINICAL PRESENTATION Cabello JF, Cornejo V, Raimann E and Colombo M. Hospital Carlos Van Buren, Instituto de Nutricion y Tecnologia de los Alimentos (INTA), Chile.

Variants of MSUD can be misdiagnosed as they present with variable clinical manifestations, similar to more common pediatric pathologies and because the biochemical alterations can be absent or only be very mild during intercritical periods. Objective: The objective of this study is to show the clinical presentations of this disease and emphasize on the importance of making a metabolic screening in the acute period. Methods: This is a retrospective study analyzing the clinical histories of patients with the diagnosis of variant forms of MSUD done in 2 centers: Hospital Carlos Van Buren, Valparaiso and INTA, Santiago, during the years 1985-1998. The diagnosis was suspected as the result of a positive metabolic screening (ferric chloride and 2-4 dinitrophenylhydrazine tests) and increased amounts of valine, leucine and isoleucine in urine and blood done by paper or thin layer chromatography. Results were con¢rmed by determination of blood aminoacids by Aminoacid Analyzer (Biotronik LC 2000). Results: Nine cases are presented with an average age of diagnosis of 3.3þ1.9 years and an average age of ¢rst symptoms of 1.3þ0.6 years. The main symptomatology was repeated episodes of hyperemesis with a very quick and unexplained dehydration in 7 cases. One had hypotonia and mild developmental delay and one presented as a pseudotumour. The plasma levels of valine, leucine and isoleucine were 547þ315 (averageþSD;range:300-1106), 515þ370 (averageþSD;range:220-1136) and 244þ177 (averageþSD;range:131-574) respectively. All patients were treated with a protein-restricted diet (average 2g/kg/day) and supplemented with thiamin, vitamins and minerals. The evolution was very satisfactory in all children showing normal growth and development. Conclusions: repeated episodes of hyperemesis and dehydration were important symptoms in this group of patients. Early diagnosis and treatment can prevent metabolic decompensations that could be life threatening.

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REDUCTION OF LARGE NEUTRAL AMINO ACID (LNAA) LEVELS IN PLASMA AND BRAIN OF HYPERLEUCINEMIC RATS M. Wajner 1,3, P. Arau¨jo1,3, K. Tallini2, V. Furlanetto3, C.R. Vargas3, C.M.D. Wannmacher1, C.S. Dutra-Filho1, AT.S. Wyse1, G.F. Wassermann2. 1 Departament of Biochemistry, ICBS, UFRGS, 2Departament of Physiology, ICBS, 3Medical Genetics Service, HCPA, Porto Alegre, RS, Brazil The mechanisms underlying the neuropathology of MSUD are poorly known. In the present study we investigated the e¡ect of acute hyperleucinemia on the plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected with 6 mmol Leu per gram body weight subcutaneously. Controls received saline in the same volumes. The animals were sacri¢ced without anesthesia 30 to 120 min after injection, the blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC. The results showed signi¢cant reductions of the large neutral amino acids (LNAA) Phe, Tyr, Iso, Val and Met, as well as Ala, Ser and Hys in plasma and of Phe, Iso, Val and Met in brain, as compared to controls. In vitro experiments to study the in£uence of leucine on amino acid transport and protein synthesis were also carried out using brain slices. Leucine strongly inhibited 14C-Phe transport into brain, as well as the incorporation of 14 C-amino acid mixture, 14C-Phe and 14C-Lys into brain proteins. Considering our previous ¢ndings of reduced levels of the LNAA in plasma and CSF of MSUD patients during crises (1,2), it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmitter synthesis. 1)Arch. Dis. Child. (1999) 80: 579. 2) J. Inher. Metab. Dis (2000) 23:

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HYPERPROLINEMIA REDUCES Na+,K+-ATPase ACTIVITY IN RAT HIPPOCAMPUS ATS Wyse, ZL Pontes, LS Oliveira, CS Bavaresco, R Franzon, M Wajner, CMD Wannmacher. Departamento de Bioqu|¨ mica, ICBS, UFRGS, Porto Alegre, RS, Brazil. Tissue accumulation of proline (Pro) occurs in hyperprolinemia type ll (HP), an inherited metabolic disorder caused by the de¢ciency of delta1 pyrroline-5-carboxilic acid dehydrogenase activity. Epilepsy and mental retardation occurs in approximately 50% of the a¡ected patients. Considering that very little is known about the role of high sustained levels of Pro in the central nervous system and that Na+, K+-ATPase is fundamental to normal brain function, in the present study we investigated the e¡ect of Pro administration to rats on brain Na+,K+-ATPase activity. Pro was injected into rats twice a day from the 6th to the 28th day of age. Control received saline. Synaptic membranes (SM) were prepared from hippocampus and used for the determination of Na+,K+ATPase activity. Our results showed that this enzyme activity was reduced (30%) in the Pro-treated rat group compared to control. In another set of experiments, SM were prepared from hippocampus of non treated rats and incubated with Pro at ¢nal concentrations ranging from 1.0 to 2.0 mM. Na+,K+-ATPase activity was inhibited by 20-30%. Since Pro concentration in plasma of chronically treated rats and of HP children are of the same order of magnitude as those tested in vitro, the results suggest that inhibition of Na+,K+-ATPase may contribute to the neurological dysfunction found in patients a¡ected by HP. Financial Support: FAPERGS, PROPESQ/UFRGS, CNPq and PRONEX ll.

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REDUCTION OF Na+,K+-ATPASE ACTIVITY IN THE MIDBRAIN OF RATS SUBJECTED TO ARGININE ADMINISTRATION IS PREVENTED BY L-NAME ATS Wyse, CS Bavaresco, EL Streck, AI Zugno, C Bandinelli, CMD Wannmacher and M Wajner. Departamento de Bioqu|¨ mica, ICBS, UFRGS, Porto Alegre, RS, Brazil.

We have previously demonstrated that arginine (Arg) administration decreases Na+,K+-ATPase activity in rat midbrain. Considering that nitric oxide (NO) inhibits this enzyme activity, the objective of this study was to investigate whether the inhibitory action of Arg could be through NO formation. We used L-NAME, a nitric oxide synthase inhibitor, together with Arg to test this hypothesis. Sixtyday-old rats received intraperitoneous injections of either Arg (0.8mg/kg), L-NAME (2mg/kg), Arg plus L-NAME or saline (control). The animals were killed 1 h after the injection. Synaptic membranes from midbrain were used for Na+,K+-ATPase activity determination. Our results showed that Na+,K+-ATPase activity was decreased by 40% in the Arg-treated group compared to the control, whereas L-NAME administration did not alter this enzyme activity, but when this compound was coadministered with Arg, it prevented the reduction of Na*,K*-ATPase activity. The results suggest that reduction of Na+,K+-ATPase caused by Arg administration could occurs through NO and free radicals formation. In case this occurs in human hyperargininemia, it may be related to the brain dysfuction observed in this disease. Financial Support: FAPERGS, PROPESQ/UFRGS CNPq and PRONEX ll)

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PHARMACOLOGICAL EVIDENCE THAT a-KETOISOVALERIC ACID INDUCES CONVULSIONS THROUGH GABAergic MECHANISMS M. Wajner 1,3 , A S Coitinho1, T. T. F Lima2, J Bastiani2, M. R. Fighera2, C.F. Mello2 1 Depart. de Bioqu|¨ mica, ICBS, UFRGS, Porto Alegre, RS, Brazil; 2Depart. de Qu|¨ mica, Centro de Cieªncias Naturais e Exatas, UFSM, Santa Maria, RS, Brazil; 3Serviµ o de Gene¨tica Me¨dica, HCPA, Porto Alegre RS, Brazil

MSUD patients predominantly present severe neurologic symptoms whose pathogenesis is not fully understood. Biochemically, the disorder is characterized by tissue accumulation of leucine, isoleucine and valine, as well as their corresponding keto acids a-ketoisocaproic acid (KIC), a-ketoisovaleric acid (KIV) and a-keto-b-methylvaleric acid (KMV). In the present study we investigated the e¡ect of intrastriatal administration of these a-keto acids on the convulsant behavior of rats. After cannula placing, the animals received unilateral intrastriatal injections of KIC, KIV, KMV or NaCl (controls). KIV elicited clonic convulsions, whereas KIC and KMV did not a¡ect this parameter. Furthermore, convulsions provoked by KIV were prevented by intrastriatal preadministration of muscimol, indicating the participation of GABAergic mechanisms in the KIV-induced convulsivant behavior. These results are in line with those reporting an inhibition of glutamate decarboxylase by KIV (1). Therefore, in case the present ¢ndings can be extrapolated to the human condition, it is feasible that KIV may be associated with the seizures characteristic of MSUD. We therefore propose that the levels of KIV should be monitored for adequate management of these patients. 1) Metabolism (1961) 1): 393-402.

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ALPHA-KETOISOCAPROIC ACID INCREASES PHOSPHORYLATION OF INTERMEDIATE FILAMENTS FROM RAT CEREBRAL CORTEX IN VITRO M. Wajner, T. Branco, R. Meirelles, B. B Rocha, A de Mattos-Dutra, R. Pessoa-Pureur Departamento de Bioqu|¨ mica, ICBS, UFRGS, Porto Alegre, Rs, Brazil Maple Syrup Urine Disease (MSUD) is an inherited metabolic disorder clinically characterized by neurological dysfunction. We investigated the e¡ects of a-ketoisocaproic (KIC), a-ketoisovaleric (KIV) and a-keto-b-methylvaleric (KMV) acids, metabolites primarily accumulating in MSUD, on the phosphorylation of intermediate ¢lament proteins of cerebral cortex of rats. Tissue slices were incubated with 32P-orthophosphate in the presence or absence of the acids. The intermediate ¢lament (IF) enriched cytoskeletal fraction was isolated and the radioactivity incorporated into neuro¢lament subunits (NF-M and NF-L), vimentin and glial ¢brillary acid protein (GFAP) was measured. The results demonstrated that KIC strongly increased phosphorylation of the proteins studied in a dosedependent manner. However, this e¡ect was not observed for KIV or KMV. Experiments using the protein kinase inhibitors KN93 and H89 indicated that the e¡ect of KIC was mediated by Ca2+ / calmodulin- and cAMP-dependent protein kinases. This study provides consistent evidence that KIC, a key metabolite accumulating in MSUD, increases phosphorylation of important brain proteins. Therefore, it is possible that an altered brain cytoskeletal metabolism could be related to the CNS damage observed in patients a¡ected by this disorder.

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THE UTILITY OF CORD BLOOD ANALYSIS IN THE DIAGNOSIS OF ORGANIC ACIDAEMIAS A.L. Patterson1, M. Pourfarzam2, M.J. Henderson1 Department of Chemical Pathology, St. James's Hospital, Leeds1; Department of Child Health, Royal Victoria In¢rmary, Newcastle-upon-Tyne2 Rapid diagnosis of organic acidaemias is possible with the advent of the analysis of bloodspot acyl carnitines by tandem mass spectrometry. We describe three cases in which cord blood has been used to make a diagnosis in the immediate post-natal period, one IVA and two MCAD. Appropriate monitoring and therapy was instituted in advance of any metabolic sequelae of the disorders. The ¢rst infant was born to consanguineous parents by elective caesarean section. A previous sibling had died with IVA at seven days of age. Analysis of cord blood from the infant revealed an isovaleryl carnitine level of 8.5 umol/L (ref50.65). 2 hours post-delivery the isovaleryl carnitine level of 13.4 umol/L con¢rmed the diagnosis. Cord blood acyl carnitines were measured post-delivery on six infants with a family history of MCAD. Two of these had elevated octanoyl carnitine levels at 11.35 and 4.5 umol/L (ref50.3). Five days post-delivery octanoyl carnitine concentrations had fallen to 2.04 and 1.05 umol/L respectively. The remaining four babies were una¡ected and had octanoyl carnitine concentrations within the reference range.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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EXPERIENCE IN THE TREATMENT OF ORGANIC ACIDURIAS IN CHILE G.P. Dura¨n, E. Raimann, P. Mabe, V. Cornejo, M. Jimenez, A. De la Parra, A. Valiente INTA, Universidad de Chile, Macul 5540, Santiago, Chile

Propionic acidemia (PA) and methylmalonic acidemia (MMA) have the third frequency of the diagnosed IEM in Chile. They are disorders of the metabolic pathway of the essential amino acid isoleucine, valine, threonine and methionine produced by the de¢ciency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase. In the last years 23 children have been diagnosed, 6 MMA (2 neonatal and 4 late onset) and 17 PA (8 neonatal and 9 late onset). Neonatal form presented between 2 and 9 days of age, some of them without metabolic acidosis may be because very high ammonia levels (average 1319 ug/dl), six needed peritoneal dialysis. Proteins were temporarily stopped, high does of parenteral glucose was given, metabolic imbalance was corrected and L-Carnitine was supplemented. Late onset presented after 5 months of age. Natural proteins are restricted in the long-term according to the severity of the disease, plasma amino acids level, ammonia, metabolic balance and growth. Diet is supplemented with amino acids mixture and L-carnitine. The high caloric intake is met principally by carbohydrate. Four MMA are on vitamin B12 (10 mg/day P.O.) with a good result. Eight children have gastrostomy. Seven children died (1 MMA and 6 PA), 4 of them were neonatal form. At present there are 16 patients in follow-up, average age of 4 years in the neonatal form and 7 years in the late onset group, nutritional status has been normal in most of them. All presented developmental delay, the most deteriorated are those with neonatal onset. We think that despite treatment result is not optimum, e¡orts should be done to detect and treat it early, avoiding the sequelae of metabolic coma, especially in the last onset forms and B12 responsive MMA.

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SPASTICITY AND PARESTHESIAS AS PRESENTING SYMPTOMS IN A TEENAGER WITH MILD METHYLGLUTACONIC ACIDURIA : GENETIC HETEROGENEITY IN MGA TYPE III ? Abramowicz MJ, Mavroudakis N, Vamos E, Gerlo E * Hoªpital Erasme, ULB, and *Academisch Ziekenhuis, VUB, Free University of Brussels, Belgium.

A 19-year-old female from nonconsanguineous parents was evaluated for worsening of weakness and paresthesias in the lower limbs of 5 years duration. Spasticity with very brisk deep tendon re£exes was present in the four limbs, as well as mildly decreased sensitivity in the legs. There were no involuntary movements, ataxia, or extrapyramidal signs. Somatosensory evoked potentials were abnormal and indicative of involvement of the long spinal tracts. The optic fundi were normal, as well as a brain MRI, and a lumbar puncture yielded normal CSF under normal pressure. Urine organic acids showed as sole anomalies a mild but signi¢cant hyperexcretion of 3methylglutaconate (19-36 mmol/mol creatinine, Nl 5 13) and 3-methylglutarate (5-11 mmol/mol creatinine, Nl 5 2 ; 5 observations over a 3 months period). Mild hyperexcretion of 3-methylgutaconate and 3-methylglutarate are the hallmark of methylglutaconic aciduria type III (MGA III), an autosomal recessive disorder prevalent in the Iraqi-Jewish community, associated with extrapyramidal dysfunction and optic atrophy (early and almost constant), spasticity, and ataxia. Our patient presented with a slowly progressive course consistent with an inherited disorder, and signs described in MGA III but not optic atrophy. Although this aciduria has exceptionally been reported in asymptomatic subjects and might be a chance association in our patient, our ¢ndings suggest that MGA III may present as a pyramidal syndrome and sensitive de¢cit without optic atrophy, suggesting perhaps genetic heterogeneity in MGA III.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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UNUSUAL BIOCHEMICAL AND CLINICAL PRESENTATION OF SEVERE BIOTINIDASE DEFICIENCY NGGM Abeling, HD Bakker, AJ Stege*, P Vreken, AH van Gennip and RJA Wanders. Univ Amsterdam, Academic Medical Center, Depts Clinical Chemistry & Pediatrics, Amsterdam and * Pediatric Dept, IJsselmeerziekenhuizen, Emmeloord, The Netherlands. Biotinidase de¢ciency interrupts the biotin cycle, which leads to biotin depletion and multiple carboxylase de¢ciency. This results in accumulation of many carboxylase substrates in the body £uids and a serious clinical picture characterized by neurological symptoms, skin rashes and alopecia. Early diagnosis and biotin treatment normally leads to spectacular recovery and good prognosis. Therefore biotinidase de¢ciency is included in some neonatal screening programs. Here we present a 5-year-old girl of non-consanguinous parents who was admitted for metabolic investigations because of severe autistiform psychomotor retardation, convulsive disorder and choreo-athetosis, spasms and hemiparesis. The child had developed normally until the age of 3.5 years. Metabolite pro¢les were unremarkable, except for a slightly elevated urinary 3-OH-isovaleric acid excretion and a high-normal plasma 3-OH-isovalerylcarnitine. Because of the possibility of partial biotinidase de¢ciency, mainly based on the unexplained convulsive disorder, plasma biotinidase activity was measured and surprisingly revealed a de¢ciency (1.6 % of mean normal activity). The absence of the characteristic biochemical abnormalities in this severe and atypical case is as yet unexplained, but raises doubts about the reliability of metabolite-based neonatal screening programs for biotinidase de¢ciency and stresses the need to include biotinidase activity measurements in general metabolic work-up of patients with neurological disorders.

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MUTATIONS CAUSING PROFOUND BIOTINIDASE DEFICIENCY IN SYMPTOMATIC CHILDREN IN WHOM DIAGNOSIS AND TREATMENT ARE DELAYED M. Demirkol1, T. Baykal1, G. Hu«ner1, G. S°arbat1, N. Kuru1, B. Wolf2 Dept. Pediatric Nutrition and Metabolism, Istanbul Faculty of Medicine, University of Istanbul, Istanbul, Turkey1; Dept. Human Genetics and Pediatrics, Medical College of Virginia, Virginia Commonwealth University, Richmond, USA2 Biotinidase de¢ciency (McKusick 253260) is an autosomal recessive disorder of biotin metabolism which usually leads to neurologic and cutaneous symptoms when untreated. The spectrum of mutations in patients with profound biotinidase de¢ciency (BD) diagnosed by newborn screening is di¡erent from that of symptomatic children. We report six children (2 males, 4 females) with profound BD (mean serum biotinidase activity 0.082 + 0.089 nmol/min/ml; mean normal activity is 6.62 + 1.77 nmol/min/ml) who exhibited early onset of symptoms (3.6 + 2.6 months, range: 1^8 months). The neurologic symptoms presented at a median age of 4.5 months (range: 3^18 months). Clinical ¢ndings included coma (n=3), psychomotor retardation (n=6), seizures (n=5), skin rash (n=5), alopecia (n=4), hypotonia or hypertonia (n=4), inspiratory stridor (n=1) and metabolic acidosis (n=3). Five patients were homozygous for the following mutations: R79C (n=2), a frameshift (n=2) and Q456H (n=1), and one patient was compound heterozygous for the 3' splice site and V4574 mutations. All of the patients had residual neurologic damage following treatment. The worst neurologic outcome was observed in the patients who are homozygous for R79C mutation. These ¢ndings indicate the necessity for newborn screening and early treatment of BD.

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BIOTIN DEFICIENCY EFFECTS ON HCS AND CARBOXYLASES GENETIC EXPRESSION Rodri¨guez-Mel¨ndez, S. Cano-Coli¨n, S. Me¨ndez-Cruz, I. Camacho-Arroyo, A. Leo¨n-del-Ri¨o, A. Vela¨zquez Unidad de Gene¨tica de la Nutricio¨n UNAM-INP, Me¨xico City

Biotin de¢ciency is a nutritional phenocopy of Multiple Carboxylase De¢ciency. Regulation of genetic expression of carboxylases and holocarboxylase synthetase (HCS) is poorly understood, and may have important consequences for their genetic defects. We investigated the regulation of HCS mRNA and of pyruvate (PC), propionyl CoA (PCC) and 3-methylcrotonyl CoA (MCC) carboxylases mRNAs and their protein mass, in rat livers and hepatocyte cultures. Biotin is essential for maintenance of normal (high) levels of HCS mRNA. The amount of PC was minimally a¡ected by biotin de¢ciency, whereas that of PCC and MCC decreased substantially in de¢cient livers. No signi¢cant changes were observed in their mRNAs, implying that biotin regulates carboxylases posttranscriptionally. These results likely explain the rapid normalization of PC activity (1 hour), and the slow one of PCC (24 hours), after biotin addition to de¢cient hepatocyte cultures. This work shows that the biotin-related enzymes are regulated by biotin itself. This is one of the ¢rst-known instances of a water soluble vitamin regulating its related apoenzymes and, in the case of HCS, its mRNA levels. (Sponsored by grants from Programa de Apoyo a Proyectos de Investigacio¨n e Innovacio¨n Tecnolo¨gica UN and Consejo Nacional de Ciencia y Tecnologi¨a)

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EVALUATION OF A SHORTENED BIOTINIDASE DEFICIENCY ASSAY Murray Rosenthal1, John Sherwin2, Richard Greeley2. 1 PerkinElmer Wallac, Inc., Norton, OH 44203 USA; 2Genetic Disease Laboratory, California Department of Health Services, Berkeley, CA 94710 USA.

Because of the limited amount of serum in the blood spot samples, most screening assays for biotinidase de¢ciency require an overnight incubation to generate adequate signal. We evaluated an alternate procedure, which can be completed in one day. In this shorter procedure, 4.5 mm blood spot samples were incubated with the biotinyl-6-amidoquinoline substrate in microplates for 3 hours at 37³C, and then 1 hour at 60³C. After precipitation of the proteins with ethanol and a 30-minute settling period, the £uorescent product was read using the Wallac Victor2D plate reader. Our goal was to compare the shorter procedure to the standard overnight procedure (3 mm blood spots, 17 hours at 37³C) using the Wallac Neonatal Biotinidase Kit. We analyzed 252 screening samples using both methods and found the distribution of results (in enzyme units or e.u.) to be nearly identical for both incubation methods (mean + SD - overnight: 157 + 52 e.u.; short: mean 172 + 42 e.u.). All reps of a de¢cient sample were below the cuto¡ (n = 30, cuto¡ = 86 e.u. overnight: 7.2 + 9.8 e.u.; max = 33.6 short: mean 13.3 + 11.9 e.u., max = 31.3). The total precision of the two methods was also compared using normal (Nor) and abnormal (Abn) controls (mean, %CV): Overnight ^ Nor (245 e.u., 19.5%), Abn (57.6, 24.2%); Short ^ Nor (203, 8.3%), Abn (55.4, 13.7%). In comparison to the overnight method for biotinidase de¢ciency screening, the shortened method o¡ers the advantages of same-day results and improved precision.

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MODIFICATION OF GENE EXPRESSION IN MULTIPLE CARBOXYLASE DEFICIENCY (MCD) BY BIOTIN TREATMENT R. Sergio Solo¨rzano-Vargas, Diana Pacheco-Alvarez, Alfonso Leon-Del-Rio. Deparment of Molecular Biology, Instituto de Investigaciones Biome¨dicas, Universidad Nacional Autonoma de Me¨xico Apartado Postal 70228 Me¨xico D.F. 04510 Holocarboxylase synthetase (HCS) catalyses the biotinylation of three mithocondrial and two cytoplasmic carboxylases in humans. Inherited de¢ciency of HCS causes the disorder known as multiple carboxylase de¢ciency (MCD), characterized by metabolic ketoacidosis, abnormal urine organic metabolites and dermatitis. This disorder is potentially lethal if not treated promptly. . In almost all the cases, symptoms can be reverted by pharmacological doses of biotin. Although biotin is believed to be nontoxic in high doses, it has been shown recently that this vitamin a¡ects the transcription of several genes which may be of relevance in the treatment of MCD.. We studied the e¡ect of biotin deprivation and supplementation on the levels of mRNA of enzymes involved in biotin utilization by semiquantitative RT-PCR and nothern blot analysis. Our results show that biotin upregulates the transcription of HCS and mitochondrial and cytoplasmic carboxylases. These results may explain the fast recovery of MCD patients once biotin supplementation is started.

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THE MOLECULAR BASIS OF HUMAN 3-METHYLCROTONYL-CoA CARBOXYLASE (MCC) DEFICIENCY MR Baumgartner1, 4, S Almashanu2, R Cole3, T Suormala4, ER Baumgartner4, D Valle1,2 Dept. of Pediatrics1, Howard Hughes Medical Institute2, Dept. of Biological Chemistry3, Johns Hopkins University, Baltimore, USA; University Children's Hospital4, Basel, Switzerland Isolated, biotin-resistant MCC-de¢ciency is a rare disorder of leucine catabolism inherited as an autosomal recessive trait. MCC is the last of 4 biotin-dependent human carboxylases to be cloned. MCC is composed of 2 nonidentical subunits, an a subunit which covalently binds biotin and a smaller b subunit. Here, we report molecular cloning of a and b subunits of human MCC. Using A. thaliana MCC a and b sequence we identi¢ed EST candidates and assembled full length mouse and human MCC a and b cDNAs. The human candidates predict an MCC a of 725 amino acids and an MCC b of 563 amino acids with 46 and 56% identity to the MCC a and b of A.thaliana, respectively. MCC b has 17 exons and maps to chromosome 5q12. MCC a maps to chromosome 3. We con¢rmed the identity of MCC b by purifying it from mouse liver using streptavidin dyna beads followed by sequencing tryptic fragments using MALDI and electrospray tandem mass spectrometry. Previous studies in 13 probands with MCC-de¢ciency revealed 2 complementation groups (CG). Biochemical studies suggest that CG2 has a primary defect in MCC a. We ampli¢ed patient MCC a and b cDNA from ¢broblast RNA and sequenced the product directly. So far we identi¢ed 4 mutant MCC a alleles in CG2 patients and 7 mutant MCC b alleles in CG1 patients including missense, frameshift and splice mutations. All mutations were con¢rmed by amplifying and sequencing genomic DNA. Our results con¢rm the identi¢cation of the human MCC a and b genes and the correct assignement of CG2 to a primary defect in MCC a.

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LOCALISATION OF THE 3-HYDROXY-ISOVALERYL CARNITINE WITHIN THE CELLULAR FRACTION OF BLOOD IN A CASE OF 3-METHYL CROTONYL COENZYME A CARBOXYLASE DEFICIENCY E. Ranieri, R L Gerace, M Metz, J R Harrison, P V Nelson , M Fietz and J M Fletcher Department of Chemical Pathology Women's and Children's Hospital, Adelaide, South Australia

In the ¢rst 12 months of routine newborn screening using tandem mass spectrometry (MSMS) we identi¢ed an infant with grossly elevated 3-hydroxy-isovaleryl carnitine (C5-OH); 9.4mmol/L whole blood (wb) (NR; 0.06 ^ 0.46 mmol/L wb) in addition to an elevated C5-OH to propionyl carnitine (C3) ratio of 33.5 (NR; 0.01 ^ 0.28) and a free carnitine level of 6 mmol/L wb (NR; 10 ^ 54 mmol/L wb). However when compared to a plasma sample the C5-OH was only marginally elevated at 1.2mmol/L. Urinary organic acid analysis revealed an elevated 3methycrotonylglycine. Family studies were performed on blood and urine collected from the infant's nonconsanguineous parents and 2 year old sibling (also retrieved the original newborn screening card). The dried blood-spot MSMS analysis clearly showed the mother to be a¡ected with a C5-OH, C5-OH/C3 ratio and a free carnitine respectively of 19.5 mmol/L wb, 162 and 5mmol/L wb. The sibling showed a similar abnormal pro¢le using the original newborn screening sample but a normal pro¢le on a recent sample while the father had levels re£ecting a heterozygote state. A decarboxylation assay using 2-14C-Leucine and measuring liberated 14CO2 in cultured skin ¢broblast from the mother gave a very low activity of 1.5 dpm/hr/mg protein (positive control; 1.8, normal; 4.1 and 4.6 dpm/hr/mg protein) consistent with a diagnosis of 3-methyl crotonyl CoA carboxylase de¢ciency. Heparinised whole blood collected from the mother and proband was partitioned to give plasma and cellular fraction, and analysed by MSMS. The level of C5-OH in plasma of the mother and proband was 2.5mmol/ L and 1.2mmol/L respectively while in the lysed cellular fraction it was 26.6mmol/L and 17.2mmol/L. In conclusion, our ¢ndings show that there is a clear partitioning of the C5-OH into the cellular fraction of blood which is *10 times higher than in plasma. We suggest that this can be potentially used to assess whether mother or child is a¡ected.

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MOLECULAR CHARACTERIZATION OF THE HUMAN GENES ENCODING THE MCCA AND MCCB SUBUNITS OF THE 3-METHYLCROTONYL-COA CARBOXYLASE. IDENTIFICATION OF MUTATIONS IN METHYLCROTONYLGLYCINURIA PATIENTS. Pe·alva, M.A.1; Gallardo, M.E.1; Esparza, J.1; Rodr|¨ guez J.M.1; Desviat L.R.2; Pe¨rez-Cerda¨, C.2; Pe¨rez, B.2; Rodr|¨ guez-Pombo, P.2; Navarrete, R.2; Ribes, A.3; Gibson M.4; Rodr|¨ guez de Co¨rdoba, S.1 and Ugarte M.2 1 CIB, CSIC, Madrid, Spain. 2CBM-SO, UAM, Madrid, Spain.3IBC, Barcelona, Spain. 4OHSU, Oregon, USA.

Methylcrotonylglycinuria (OMIM, 210200) is an inherited human disorder of the leucine catabolism and isoprenoid metabolism characterized by the absence of the biotin enzyme 3-methylcrotonyl-CoA carboxylase (MCC, EC 6.4.1.4). MCC is a heteromeric enzyme composed of biotin-containing (MCCA) and non-biotin-containing (MCCB) subunits. Based upon strategies of conventional and in silico screening of cDNA and genomic libraries and databases, we have isolated the full length cDNAs encoding the human MCCA and MCCB subunits and determined the structure of the human MCCA and MCCB genes. The human MCCA and MCCB polypeptides are encoded by single genes located in chromosomes 3p11.2-p13 and 5p12-13.2, respectively. MCCA and MCCB expression was detected in all human tissues analyzed but it was signi¢cantly increased in heart, skeletal muscle, kidney and liver. Initial mutation screening in patients has resulted in the identi¢cation of two missense and one frameshift mutations in MCCB. Analysis of additional patients and complementation studies in MCC de¢cient human cell lines and A. nidulans strains are in progress.

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GENOTYPE^PHENOTYPE CORRELATION IN A SAMOAN PATIENT WITH BIOTINRESISTANT MULTIPLE CARBOXYLASE DEFICIENCY Peters H1, Zammarchi E2, Malvagia S2, Morrone A2, Craig J1, Kahler S1, Boneh A1 1 VGGS, Melbourne, Australia, 2Neurometabolic Unit, Dept. of Ped., Univ. of Florence, Italy Holocarboxylase synthetase (HLCS) is involved in the biotinidation of four biotin-dependent carboxylases. De¢ciency of this enzyme results in multiple carboxylase de¢ciency (MCD), characterised by psychomotor retardation, seizures, rash, acute metabolic acidosis due to lactic and organic acidemia, and hyperammonemia. We describe a male Samoan infant diagnosed with severeneonatal-onset MCD who is biotin-resistant despite doses of 1000 mg/day. At age 21 months he presents with the following clinical problems: 1) Global developmental delay: assessment at 16 months indicated functioning at an 8^9 month level. 2) Frequent bacterial illnesses with metabolic decompensation. 3) Severe eczematous rash with secondary fungal and bacterial infections. 4) Seizures. Plasma lactate remains persistently elevated at 3^7 mmol/L. Urine organic acid analyses show persistent excretion of 3-hydroxy-isovaleric, 3-methyl-crotonylglycine and methylcitric acids. Molecular analysis of the HCLS gene identi¢ed a homozygous nucleotide substitution T647?G that leads to the L216R amino acid change. Patients homozygous for this mutation have not been reported previously. This mutation is located in the 5' end of HCLS gene, in which no biotin binding domain has been reported. The severe clinical phenotype and biotin-resistance observed in our patient are in accordance with homozygosity for a mutation in this region, which may be a pivotal region for the function of this enzyme.

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DIAGNOSIS OF PROPIONIC ACIDEMIA IN NEONATAL SCREENING BY TANDEM MASS SPECTROMETRY: 1-YEAR FOLLOW UP OF AN EARLY TREATED PATIENT I Knerr1, R Fingerhut2, W Rascher1, R Baumgartner3, B OlgemÎller2, B Liebl4, AA Roscher5. 1 Children¨s Hospital of University of Erlangen, Germany, 3University of Basel, Switzerland, 5University of Munich, Germany, 2Screening Laboratory, Munich, Germany, 4Public Health Screening Centre, Oberschleissheim, Germany. Propionic acidemia (PA) is due to propionyl-CoA carboxylase (PCC) de¢ciency. It leads to lifethreatening episodes which may be prevented by early neonatal screening. In 1999 in the Bavarian Newborn Screening study acylcarnitine and amino acid pro¢les of dried blood samples from 122.300 newborns were investigated by tandem mass spectrometry (MS/MS). We report a case of PA, in whom newborn screening was performed on day 3: Free carnitine was low (10 mmol/l), propionylcarnitine elevated (17,7 mmol/l) with pathological ratios (C3/C4 and C3/C16). A control blood sample con¢rmed the pathological acylcarnitine pro¢le. On admission to hospital at day 10 the child was in good clinical condition with only minor signs of hypotonia. Ammonia was 155 mmol/l, glycine 1139 mmol/l, urinary organic acid analysis revealed methyl-citrate, 3-hydroxypropionate, and tiglylglycine. Treatment was initiated promptly (protein restriction, special formula diet, carnitine supplementation). The diagnosis was con¢rmed by enzymatic analysis in ¢broblasts (PCC activity 9.5 pmol/min/mg protein, reference median and range 740 [207-2150]). The patient is now 1 year old and shows no signs of neurological abnormalities. He never had an acute crisis, but transient anaemia in the ¢rst trimenon (Hb 6,2 g/dl). His psychomotor development is in the lower normal range for children of the same age. Diagnosis of PA by early MS/MS-based neonatal screening and intervention prior to initial metabolic decompensation may improve the chances for good clinical outcome.

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PROPIONIC ACIDEMIA WITH BASAL GANGLIA STROKE: EFFECTIVENESS OF LDOPA TREATMENT A. B. Burlina1, C. Baracchini 3, C. Carollo2, A. P. Burlina 3 1 Dept of Pediatrics, 2Div. of Neuroradiology, 3Dept of Neurological and Psychiatric Sciences, University of Padua, Italy

``Metabolic stroke'' has been reported as a complication of propionic acidemia (PA). We observed a patient with PA who was admitted at the age of 15 years with a 3-day history of upper respiratory tract infection and vomiting. Prominent clinical ¢ndings were a cachectic state (weight below the 3d percentile) with poor muscle masses. Metabolic investigations including amylase and lipase were normal. He was treated with intravenous £uid and L-carnitine with disappearance of the symptoms. In few days, his neurological condition dramatically collapsed. He became dysphagic, amimic and akinetic and stopped to talk; he held his head £exed forward; he presented muscle rigidity of the limbs associated with cogwheel rigidity at the left wrist, and tremor in the extremities (¢ngers and jaw). MRI of the brain showed high T2 signal in caudate nuclei and putamina. At this point. therapy with benserazide-L-DOPA (Madopar dispersible 2 mg/kg/d) and we registered a rapid reduction of muscle rigidity, tremor, and disappearance of the akinetic syndrome. After 5 days of therapy, he suddenly presented euphoria, anxiety, irritability, explosive laughter, and orofacial and lingual dyskinesia. Therefore, L-DOPA was stopped. Dyskinetic phenomena, euphoria and irritability disappeared slowly in the following 10 days. After 5 months from the episode he is neurologically normal. We suggest that L-DOPA therapy may improve neurological condition of patient with PA presenting with an acute extrapyramidal disease.

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THE USE OF INTRAVENOUS SODIUM BENZOATE AND SODIUM PHENYLACETATE IN PROPIONIC ACIDEMIA WITH HYPERAMMONEMIA Praphanphoj V, Brusilow S, Hamosh A, Geraghty MT. Dept. Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA. Hyperammonemia commonly occurs in propionic acidemia. Whereas IV sodium benzoate (BA), sodium phenylacetate (PA) and arginineHCl (ArgHCl) have been used successfully to treat acute hyperammonemia in urea cycle defects, there are theoretical risks to the use of these drugs in propionic acidemia. Sequestration of CoA in propionate metabolites might inhibit activation of both PA and BA. The drugs might exacerbate acid-base balance disturbances. Finally, PA might be less e¡ective in propionic acidemia as plasma glutamine levels tend to be in the low range of normal, independent of plasma ammonium levels. We report the use of the IV drugs on 9 occasions in 5 symptomatic patients. Plasma ammonium levels (range; 129-560 mmol/L) decreased on treatment. Initially, glutamine levels (range; 243-561) were in the low range of normal, despite concurrent hyperammonemia, and decreased further during IV treatment. Glycine levels (range; 247-906) were elevated but lower than levels obtained when the patients were euammonemic and also decreased during treatment. Arginine and ornithine increased during therapy. Where data was available (5 events) the plasma concentrations over time of PA, BA, and metabolites were comparable to those when used in treatment of urea cycle defects. No serious side e¡ects were seen. Nausea and a mild respiratory alkalosis were seen after loading doses. Hypokalemia and an increase in the anion gap were seen in all patients while on the medications. These preliminary data suggest that BA, PA and ArgHCL are e¡ective in the treatment of hyperammonemia in propionic acidemia. Acid-base balance, serum electrolytes and plasma amino acids should be monitored carefully in patients with increased anion gaps or severe hypoglutaminemia.

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NEW MUTATIONS DETECTED IN USA PROPIONIC ACIDEMIA FAMILIES P. Rodr|¨ guez-Pombo, B. Pe¨rez, S. Muro, C. Pe¨rez-Cerda¨, L.R. Desviat, KM. Gibson1 and Ugarte M. CBM. Universidad Auto¨noma Madrid, Spain. 1Dept Mol and Med Genet and Peds. Oregon Health Sci Univ. Portland, USA. Mutations in the PCCA (a subunit) or PCCB (b subunit) gene can cause the inherited metabolic disease propionic acidemia (PA). Here we report the molecular characterisation of three USA patients and their families. Mutational studies of index cases (AB, ChB, BN) were performed on PCR products derived from the ampli¢cation of the 15 di¡erent exons of PCCB gene. Three out of the six PCCB gene mutations identi¢ed were new, one single nucleotide deletion (1204delG) that disrupts the open reading frame creating a stop codon 41 residues downstream the mutation, one mutation a¡ecting a 3' acceptor consensus splice junction (IVS7-2delA) and one missense mutation (G112D). The remaining were mutations previously detected, ins/del described as the most frequent among PCCB Caucasian patients, 1170 insT detected until now only in Spanish and Latin American patients and IVS4+3del4 previously described in a Japanese patient and that a¡ect a short direct repeat region which sustain a higher mutation rate. The absence of immunoreactive b-PCC protein from the ¢broblasts of the three patients studied (as demonstrated by Western blot ) suggests that these mutations severely a¡ect the mRNA or protein stability. On the other hand, several carriers of the disease have been identi¢ed among the family members and a prenatal diagnosis has been performed, showing the carrier status of the fetus. This study illustrates the expanding genotypic spectrum of propionic acidemia and highlight the feasibility of DNA based methods for prenatal diagnosis and detection of carriers for this disease.

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CHARACTERISATION OF STRUCTURAL AND HOMOMERIC ASSEMBLY DEFECTS IN THE PCCB GENE CAUSING PROPIONIC ACIDEMIA S. Muro, B. Pe¨rez, P. Rodr|¨ guez-Pombo, L.R. Desviat, C. Pe¨rez-Cerda¨, and M. Ugarte. Centro de Biolog|¨ a Molecular ``Severo Ochoa''. Universidad Auto¨noma de Madrid. Spain.

Propionyl-CoA carboxylase de¢ciency can be caused by defects a¡ecting either of the genes PCCA or PCCB, which encode the two subunits of this dodecameric enzyme, a- and b-PCC, leading to the inherited disease propionic acidemia (PA). To investigate the e¡ect caused by the PCCB gene mutations S106R, E168K, R512C, L519P, and W531X, two approaches were used, including a protein interaction study through the in vivo two-hybrid assay and the in vitro synthesis and stability analysis using the TnT cell-free system, both of them at di¡erent temperatures. The amino changes S106R and E168K only reduced partially the b-PCC homomeric assembly at 378C but not at 278C, and showed di¡erent temperature dependent patterns of stability. On the other hand, the carboxyterminal changes R512C, L519P and W531X resulted in the total elimination of the b-b association under the two conditions and did not a¡ect signi¢cantly the in vitro stability of these peptides. The results obtained indicate that the mutations S106R and E168K, which show the typical temperature dependent behaviour associated to structural variations, would a¡ect primarily the folding/structure of this subunit, whereas the changes R512C, L519P and W531X would be directly involved in the quaternary assembly of the protein and would not a¡ect signi¢cantly the biogenesis of the b-PCC precursors. These ¢ndings document the pathological mechanism underlying the disease-causing e¡ect of the changes analysed and allow to argue experimentally the potential genotype/phenotype correlation observed in the correspondent PA patients.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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ACYLCARNITINE MEASUREMENT FOR MONITORING TREATED PATIENTS WITH PROPIONIC ACIDEMIA (PPA) AND METHYLMALONIC ACIDEMIA (MMA) B. Klupsch1, M. Go«ggerle2, H. Korall2, F. Trefz1 Klinik fu«r Kinder und Jugendmedizin Reutlingen, School of Medicine, University of Tu«bingen, Germany1, Zentrum fu«r Sto¡wechseldiagnostik Reutlingen, Wo«rthstrae 47, 72764 Reutlingen, Germany2

Dietary treatment and carnitine supplementation has greatly improved long-term outcome of patients with PPA and (vitamin B12 unresponsive) MMA. However, metabolic decompensation may be frequent and ¢nal outcome in most patients show various handicaps. To investigate the usefulness of measuring free carnitine and acylcarnitines in dried blood by tandem mass spectrometry, we investigated 9 patients with PPA and 5 with MMA in a period of 8 months by weekly capillary blood punctures performed by the parents. Ages of the patients were from 0.5 until 18 years. Clinical status at the time of blood drawing was evaluated by regular phone calls. Free carnitine in all patients substituted by oral carnitine treatment (50^100 mg/kg/day BW) was normal. The parameter best re£ecting clinical status was the C3/C16-acylcarnitine quotient. Mean value in MMA and PPA patients showed a range of 11.5^29.4 (normal 1.5 +/- 0.36, n=18), there was no di¡erence between PPA and MMA patients. Individual mean values of the patients signi¢cantly increased when the patient was ranked higher in the clinical score system or during decompensation. Since measurement of acylcarnitines in dried blood by tandem mass spectrometry is easy to perform, this method may be used for home monitoring of patients with MMA and PPA.

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MUTATION SPECTRUM AND HAPLOTYPE ANALYSIS OF THE HOLOCARBOXYLASE SYNTHETASE GENE Y. Suzuki, X. Li, X. Yang, O. Sakamoto, Y. Aoki, M. Hiratsuka, *K. Inui, **K. M. Gibson, ***T. Suormala, ***E. R. Baumgartner, S. Kure, Y. Matsubara, K. Narisawa Dept. of Medical Genetics, Tohoku Univ. School of Medicine, Sendai, Japan, *Dept. of Pediatrics, Osaka Univ. Faculty of Medicine, Osaka, Japan, **Dept. of Molecular and Medical Genetics, Oregon Health Sciences Univ., Portland, OR, U.S.A., ***Metabolic Unit, University Children's Hospital, Basal, Switzerland

Holocarboxylase synthetase (HCS) catalyses biotin incorporation into four carboxylases. Since biotin is essential for their enzymatic activities, defect in HCS results in decreased activity of multiple carboxylases, a¡ecting various metabolic pathways. HCS de¢ciency (McKusick 253270) is therefore called also as biotin-responsive multiple carboxylase de¢ciency. We identi¢ed 24 mutations in 11 Japanese and 10 non-Japanese families with HCS de¢ciency. The results showed that: 1) there was no common mutations; 2) two mutations (508Arg4Trp and 550Val4Met) were found in multiple ethnic groups; 3) none of three mutations (942insA, 237Leu4Pro, and 1067delG) found in multiple Japanese families was found in non-Japanese patients. The data suggest that the spectrum of the mutations in HCS gene is di¡erent among di¡erent ethnic groups. We also determined haplotype of the HCS gene in Japanese patients and control subjects by typing two tetranucleotide repeat markers in the gene. Fourteen haplotypes were found in 100 alleles of the control subjects. All seven 237Leu4Pro and all four 1067delG were associated with 2-2 haplotype. Both 508Arg4Trp and 550Val4Met were associated with either 2-3 or 1-4. The data suggest that the 237Leu4Pro and 1067delG show a founder e¡ect in Japanese population, whereas 508Arg4Trp and 550Val4Met represent hot spot mutations.

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UPDATE ON COMBINED LIVER^KIDNEY TRANSPLANTATION FOR METHYLMALONIC ACIDAEMIA W.G. van't Ho¡1, A. Burroughs, D. Patch, K. Rolles and J.V. Leonard Nephrology Unit, Institute of Child Health, UCLMS, London, WC1N 1EH, UK1 Patients with early-onset methylmalonic acidaemia develop chronic renal failure (CRF) and require either isolated kidney or combined liver^kidney transplantation (LKT). We now update our experience with 3 patients who developed end-stage renal failure (J Pediatr (1998) 132:1043; Eur J Paediatr (1999) 158[S2]:S70). Patient 1 received haemodialysis (HD) for 6 months and, at 13 years, underwent LKT. There were no immediate complications; he had one minor episode of liver rejection (due to non-compliance) but has remained well. At last review (age 18 years), he is in full-time employment, plasma creatinine (Pcr) was 99mmol/l and liver function was normal. Plasma methylmalonate concentration has fallen substantially (but not to normal), from a mean pre-dialysis level of 3.5mmol/l to 0.10mmol/l after LKT. Patient 2, a 19-year-old female, underwent LKT after a period of HD. Post-LKT she developed marked polyuria, had severe liver and kidney graft rejection, seizures and an intracranial haemorrhage. She required a second combined LKT and 3 years later, has made a good neurological recovery, with Pcr 126 and normal liver function. Patient 3, a 15-yearold male, had a catastrophic metabolic decompensation due to a haemodialysis catheter infection, causing severe neurological damage. LKT was undertaken at 16 years but he developed combined severe liver and kidney graft rejection, seizures and had an intracranial haemorrhage. He required further HD and subsequently died. LKT is a potentially successful treatment for CRF in methylmalonic acidaemia but patient selection is important and the long-term outlook is still not clear.

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MOLECULAR CHARACTERISATION OF MUTASE-DEFICIENT METHYLMALONIC ACIDURIA Peters H, Kahler S, Nefedov M, Ioannou P. Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia.

Introduction: Methylmalonic aciduria (MMA) is an autosomal recessive inborn error of organic acid metabolism, which results from a functional defect in the nuclear encoded mitochondrial enzyme methylmalonyl CoA mutase, or its cofactor adenosylcobalamin (AdoCbl). The MUT locus comprises 13 exons spanning greater than 35kb of the genome. Various studies have identi¢ed 31 di¡erent mutations in patients with mut MMA, predominantly missense/nonsense nucleotide substitutions. No common mutations have been identi¢ed and most patients are compound heterozygotes. We are undertaking studies to identify and characterise the speci¢c mutations in our patients with mut0 and mut^ MMA. These mutations will later be used to produce a transgenic mouse model accurate for our MMA patients. Materials and methods: Seven patients were identi¢ed, (from six di¡erent families), who presented over the past 25 years with acute infantile onset classical MMA. Mutations in the probands were sought by direct sequencing of exon-speci¢c PCR products of the mutase locus. Genomic DNA samples from blood and stored ¢broblast lines were used. Results: We have found in one patient, a previously described missense mutation: G to A change at nucleotide position 2087, which results in an amino acid substitution of Val671 for isoleucine (V671I). Further sequencing continues in the remaining patients. Conclusions: This mutation con¢rms previous observations regarding the pathogenesis of de¢cient enzyme activity in mut MMA. Analysis on remaining patients should provide further information on the pattern of mutations in this disorder.

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LIVER TRANSPLANTATION IN CBL B METHYLMALONIC ACIDEMIA S. Packman, P. Rosenthal, K. Weisiger, D. Kostiner, S. Goodman, E. Sherr, C. Ohnstad, C. Mudge, W. Packman, W. Fenton, and J. Roberts University of California San Francisco, University of Colorado, and Yale University

It is becoming apparent that conventional treatment of organic acidemias may not be su¤cient to avert longer-term complications or mortality. Accordingly, organ transplantation is being increasingly considered as a therapeutic option. In this setting, we present our investigations in a now 2-yearold boy with cbl B methylmalonic acidemia (MMA). He presented with acute perinatal metabolic encephalopathy, and recovered after hemodialysis, and initiation and maintenance of IM hydroxocobalamin (OH-B12), ileu and val restriction, and carnitine. A sister had previously died at age 5 days, with intractable metabolic encephalopathy. Complementation analysis identi¢ed ¢broblasts as belonging to the cbl B class. Serum MMA levels in the boy were as high as 316 mM (N 0.09-.28) before treatment, and have since ranged between 100^300 mM. He showed slow acquisition of milestones, with especial delays in ¢ne motor skills and language. Physical growth signi¢cantly declined at age 15 months. Head MRI has shown mild myelination delay, and mild decrease in white matter volume. The child underwent cadaveric orthotopic liver transplant at age 19 months. Posttransplant, he has remained on OH-B12 and carnitine, with relaxation of dietary restrictions. Growth rate rapidly improved and, subjectively, observers have noticed improved alertness and activity. Serum MMA levels are lower, but not normal, ranging from 26^100 mM. CSF MMA is 4.36 higher than simultaneous serum levels. We conclude that liver transplant in cbl B MMA may result in partial biochemical correction, and lead to improvement in parameters of general health, such as growth. CSF MMA levels are signi¢cantly higher than those in serum, either because of concentration or production in the CNS. While liver transplant in MMA and other organic acidemias may have ameliorative and longer-term e¡ectiveness, prognostic counseling must remain guarded.

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METHYLMALONIC ACIDEMIA: IMPROVED RENAL FUNCTION AND REVERSAL OF PANCREATITIS ASSOCIATED WITH IV CARNITINE THERAPY Winter,SC, Valley Children's Hospital, Madera, CA.

Chronic renal failure with tubular dysfunction and acute pancreatitis are life-threatening complications of mut0 methylmalonyl CoA mutase de¢ciency (MMA). Unlike pancreatitis which improves with medical management, renal failure is generally progressive with dialysis and eventual renal transplant as the only long term option. Despite strict dietary control, oral carnitine at 300 mg/kg/day and growth hormone therapy, a 16 yr old male with MMA experienced gradual deterioration in strength, weight loss and bouts of prerenal failure responsive to IV £uids and carnitine over 4 months. He then developed acute pancreatitis and another bout of renal failure with BUN 55mg/dl and creatinine 4.4mg/dl. Profound hypocalcemia, electrolyte disturbances, inability to feed orally, renal failure and acidosis due to MMA created a di¤cult medical management necessitating 3 months of hemodialysis, total parenteral nutrition with specialized amino acid mixture for MMA, and daily IV carnitine 300 mg/ kg/day. After 3 months of this therapy, the pancreatitis resolved and renal failure improved allowing for initiation of oral feeds and a change from hemodialysis to peritoneal dialysis. Initial plans for an emergent combined renal and liver transplant were cancelled due to the excellent response to therapy. Intravenous carnitine continued over the next year and the patient was gradually changed to oral carnitine therapy and peritoneal dialysis was discontinued. Two years later, renal function continues to improve with BUN still elevated (45mg/dl) but creatinine normal at 1.1mg/dl. No further bouts of pancreatitis have occurred.

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A METHOD FOR THE QUANTITATIVE DETERMINATION OF METHYLMALONIC ACID IN PLASMA BY LC-MS/MS STABLE ISOTOPE DILUTION ANALYSIS Mark J. Magera, Dietrich Matern, Piero Rinaldo. Biochemical Genetics Laboratory, Department of Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN 55905 Methylmalonic acid (MMA) can be rapidly (53 min) measured in plasma by LC-MS/MS. Solid phase extraction was applied to 500 mL plasma spiked with 1 nmol d3-MMA. After evaporation of the eluate, the residue is treated with 200 mL 3N HCl in n-Butanol at 658C 15 minutes. MS/MS analysis is preceeded by column separation (Supelcosil LC-18, 33 x 4.6 mm) with acetonitrile: 0.1% aqueous formic acid (60:40, v:v) as mobile phase. The transitions 231-119 m/z and 234-122 m/z were used in the MRM mode for MMA and d3-MMA respectively. Consecutive calibration curves (n= 5) were linear and reproducible (y=0.9320x+ 0.004; r2=0.9996) over the concentration range 0.1 2.5 mmol/L, encompassing the normal and moderately elevated range (40.4 mmol/L). Interference by succinate is avoided by derivatization as butyl-ester, the choice of MRM transitions, and optimization of the chromatographic separation. Recoveries of MMA added to serum at 0.125, 0.5 and 2.0 mM were 595% (n=54). Bland-Altman plot comparisons of 106 plasma specimen values obtained by the LC-MS/MS method agree with a GC-MS SIM method (mean di¡erence = 0.01 mmol/L), 99% of specimen results were within þ2SD of the mean di¡erence. This method is well suited for large scale applications (50-100 samples per day) were a shorter analytical time is highly desirable. Analysis of MMA as butyl-ester could also facilitate the di¡erential diagnosis of elevated propionylcarnitine found by acylcarnitine analysis.

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LIVER TRANSPLANTATION IN METHYLMALONIC ACIDAEMIA R.Parini, D.Codazzi, G.Rossi*, G.Nebbia, C.Corbetta, F.Sala, F.Menni, U.Caruso**, A.Edefonti. Department of Pediatrics and Intensive Care Unit, Azienda Ospedaliera I.C.P.;*Liver transplantation Unit, Ospedale Maggiore IRCCS; Milano, Italy; **Istituto Gaslini, Genova,Italy

The patient, a male child 8 y old, a¡ected by methylmalonic acidaemia unresponsive to B12 (mut³), experienced frequent and severe metabolic decompensations with anorexia, vomiting, metabolic acidosis, decreased urine output with increased serum creatinine ( from 0.7 mg/dl to 1-1.2 mg/dl) and normal or slightly elevated ammonia and lactate, from 6 y on. He also had clinical signs of malnutrition con¢rmed by height 53rd centile ( 112 cm) and delayed bone age (6y). Mental development was normal. Plasma MMA (methylmalonic acid) ranged from 200 to 2000 mmol/l. Urinary MMA from 3 to 20 M/M urinary creatinine. Before liver transplantation (OLT) the child was treated with forced hydration, combined enteral and parenteral nutrition and 3 haemodialytic sessions that reduced plasma MMA concentration of about 30% (mean pre-dialysis value 875-mean post-dialysis 590 mmol/l). A renal biopsy showed moderate tubulo-interstitial nephritis and mild hyalinization of the glomeruli. OLT was performed with a cadaveric liver of an 11 y old donor. Immunosuppression included prednisone and FK 506. The postoperative course was good with normal liver function, without metabolic decompensations and a normal protein intake with diet. Plasma MMA was 500 mmol/l, 1 month after OLT ; urinary MMA was around 10 in the ¢rst month and now, 3 month later, is around 3 M/M urinary creatinine. OLT may have a role in the treatment of severe forms of methylmalonic (and propionic) acidemia.

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RENAL FUNCTION AND FOLLOW-UP IN CHILDREN WITH METHYLMALONIC ACIDAEMIA M De¨chaux, G Touati, R Vargas Poussou, D Rabier, JM Saudubray. Dpts of Physiology, Biochemistry and Pediatric Metabolism Unit, Necker Hospital, Paris, France.

During the last 10 years, earlier recognition of methylmalonic acidaemia (MMA) has led to an earlier beginning of the treatment and to a better evolution. Consequently, renal complications including severe renal failure became obvious leading in some cases to end-stage renal failure. Renal function was assessed in 17 patients aged 1 to 18 yrs and then every year in 11 pts during 2 to 4 yrs. Renal investigation included glomerular ¢ltration rate (inulin clearance, Cin), renal plasma £ow (PAH clearance, CPAH), MMA, urate and urea clearances, calcium and phosphate homeostasis and urine beta2 microglobuline. The ¢rst Cin was normal in 7 pts (1-7 yrs), ranged between 50 and 80 ml/min/ 1.73m2 in 6 others (2-13 yrs), and between 17 and 41 in 4 pts (11 to 18 yrs). Renal failure induced a decrease in MMA excretion rate. Plasma urate was frequently high but could not alone be responsible for the renal failure. Blood pressure was in the normal range. U b2 microglobuline was normal in all cases. Severe renal failure induced in 2 pts a secondary hyperparathyroi«dy. Renal function or evolution was not related to the early or late onset of the disease or the number of metabolic decompensation episodes. Renal follow-up evidenced no change in Cin in the 2 patients with the most severe renal failure (Cin 520 ml/min/1.73m2 with a 4 year follow-up), a decrease in the 3 pts with normal initial Cin values and no change in the others. In conclusion, renal failure is present in 2/3 of MMA. As end-stage renal failure is the likely evolution of the disease, renal follow-up must be performed regularly and renal osteodystrophy must be prevented. A kidney or a combined kidneyliver transplantation will certainly be necessary in these patients.

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PRESYMPTOMATIC DIAGNOSIS & TREATMENT OF CBL C DEFICIENCY DOES NOT PREVENT DISEASE-RELATED RETINOPAHTY C.P. Venditti, A. Laufer-Cahana, G.T. Berry, E.M. Kaye, M. Harrington, M. Miccolis, P. Kaplan University of Pennsylvania School of Medicine, Philadelphia, PA, USA

The introduction of tandem MS has permitted the presymptomatic detection of infants with a variety of biochemical genetic diseases. However, it is not yet known whether early identi¢cation of a¡ected infants will ameliorate complications and/or alter the natural history and progression of disease. Two unrelated neonates with cbl C de¢ciency were identi¢ed because of increased propionyl carnitine detected by tandem MS analysis of newborn blood spots. The pregnancy, labor and delivery were uncomplicated in both cases. Neither infant had medical problems in the neonatal period. Evaluation in the 2nd^3rd week of life demonstrated massively elevated methylmalonic acid and homocystine and low methionine in the CSF and blood. Brain MRI, renal ultrasounds, and dilated eye exams were normal. Treatment with methionine, betaine, IM hydroxycobalamin and protein restriction was initiated with improved biochemical parameters. Both infants developed horizontal nystagmus during the ¢rst 3^4 months of life and have severe retinal pigmentary lesions which will a¡ect vision. One patient shows mild gross motor delays at 8 months. Both have required gastrostomy tube placement for feeding di¤culties.Vigilant nutritional and biochemical monitoring have not prevented some of the complications seen in cbl C disease.

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GROWTH HORMONE DEFICIENCY ASSOCIATED WITH METHYLMALONIC ACIDEMIA J. Thomas, C. Freehauf, L. Bernstein, K. Flugge, C. Greene, and M. Kappy Department of Pediatrics, Univ. of CO School of Medicine, Denver, CO. Despite aggressive dietary management, some patients with methylmalonyl Co-A mutase de¢ciency (MMA) continue to have high levels of methylmalonic acid. Growth hormone (GH) therapy has been considered in such cases to enhance anabolism. We investigated this therapy in a 2 yr. old child with MMA in whom chronically elevated methylmalonic acid levels did not respond to restricted diet, carnitine, and antibiotic therapy. Testing performed prior to initiation of therapy revealed decreased GH secretion in response to provocative glucagon stimulation. A subnormal IGF1 level and the patient's poor growth velocity supported a diagnosis of GH de¢ciency. GH therapy has improved the patient's growth velocity, but methylmalonic acid levels remain high. Evaluation of a second patient with MMA and short stature has also revealed diminished GH secretion in response to provocative stimulation. Poor growth and/or short stature are commonly seen in patients with MMA and are often considered part of the disease process or a consequence of the restricted diet required for management. Our 2 patients, along with previous isolated reports of individuals with organic acidemias and diminished GH secretion, suggest that GH de¢ciency may contribute to the short stature in these patients. The pathophysiology of the de¢ciency has not been explained. Possibilities include alteration of hypothalamic and/or pituitary GH regulation pathways, tissue damage sustained during episodes of metabolic encephalopathy, or others. Conclusion: Our experience and that of others support that provocative testing for GH secretory capacity is indicated in poorly growing children with organic acidemias.

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CBL-C/D DISORDER PRESENTING AS LATE ONSET FAMILIAL CHRONIC THROMBOTIC MICROANGIOPATHIC SYNDROME J. Van Hove1, R. Lombaerts1, S. GrÏnewald1, H. Peters2, B. Van Damme1, J.P. Fryns1, J. Arnout1, R. Wevers3, E. R. Baumgartner4, B. Fowler4. 1 Katholieke Universiteit Leuven, Leuven, Belgium, 2The Murdoch Institute, Melbourne, Australia, 3 Institute of Neurology, University Medical Center, Nijmegen, The Netherlands, 4Univ. Children's Hospital, Basel, Switzerland Two siblings, a boy age 12 and his sister age 4 years, presented with proteinuria and hematuria, hypertension, and chronic hemolytic anemia. At the age of 13 year, the boy developed an episode of severe hypertensive encephalopathy and transient renal failure. Both children are attending normal school, have no neurologic symptoms, and only minimal pigmentary retinal abnormalities. Renal biopsy showed a chronic thrombotic microangiopathic nephropathy. Both patients had hyperhomocysteinemia, and mild methylmalonic aciduria. Fibroblasts had decreased cobalamin uptake, reduced methyl-, and adenosyl-cobalamin formation, and de¢cient formate and propionate incorporation, compatible with the Cbl-C or D complementation group, but milder than usual. Both patients carry a balanced translocation. Parenteral hydroxycobalamin treatment reduced the homocysteine levels, and methylmalonic acid disappeared. Increasing the dosage of hydroxycobalamin from 1 to 2.5, then 5 mg daily, with betaine each further reduced homocysteine levels (boy from 117.9 to 21.8 mM, girl from 59.5 to 14.2 mM). With this treatment, hemolysis has stopped, hematuria has disappeared in both patients, and creatinine clearance has been stable in the boy and normalized in the girl. Investigations for chronic thrombotic microangiopathic syndrome should include this unusual but treatable disorder, regardless of age of presentation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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IMPAIRED FATTY ACID OXIDATION (FAO) IN MALONYL-CoA DECARBOXYLASE (MCD) DEFICIENCY M.J. Bennett, P.A. Harthcock, R.L. Boriack, J.C. Cohen Departments of Pathology and Internal Medicine, University of Texas Southwestern Medical Center, Dallas TX, USA

MCD (EC 4.1.1.9) catalyzes the conversion of malonyl-CoA to acetyl-CoA. It has been postulated that MCD is responsible for regulating the availability of malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase 1, which is rate limiting for mitochondrial FAO. MCD de¢ciency is rare. Since 1984, only 8 cases have been reported. The phenotype includes delayed neurological development, hypoglycemia, hepatic dysfunction, cardiac and skeletal myopathy, many features shared with FAO disorders. It has been proposed, but never demonstrated, that some of the MCD phenotype may be related to secondary inhibition of FAO. To test this hypothesis we have studied FAO £ux as tritiated palmitate (P) and myristate (M) oxidation in cultured ¢broblasts from a patient in whom we recently described the molecular basis for MCD de¢ciency (Gao et al J Lipid Res 40, 178, 1999). The assays were performed under baseline culture conditions with D-MEM containing 10% fetal bovine serum and following supplementation with acetate, a malonyl-CoA precursor, at concentrations of 0,10 and 100mmol/L. At baseline the FAO £ux was reduced in the MCD patient, P was 47% of control, M was 62%. With acetate supplementation, there was a further reduction in the relative £ux such that at 100mmol/l acetate concentration P was 23% of the baseline control, the residual activity for M was 20%. Non-MCD de¢cient ¢broblasts also demonstrated reduced £ux at high acetate concentrations. These results suggest that there is impaired FAO in ¢broblasts in MCD de¢ciency which is further reduced under conditions likely to stimulate high malonyl-CoA production and con¢rms the hypothesis that some of the symptoms of MCD de¢ciency may be related to secondary FAO inhibition.

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PRE-NATAL DIAGNOSIS OF MALONIC ACIDURIA BY A COMBINED METABOLITE, ENZYMOLOGY AND MOLECULAR APPROACH Kevin Carpenter1, Michael Gibson2, Jonathan C Cohen3, Bridget Wilcken1 1 NSW Biochemical Genetic Service, Sydney, Australia, 2Biochemical Genetics Laboratory, Oregon Health Sciences University,Oregon, 3Center for Human Nutrition, Dallas, Texas.

Malonyl CoA decarboxylase (MCD) de¢ciency (McKusick 248360) is a rare disorder with only 7 cases reported in the literature. It is characterised by a combined malonic/methylmalonic aciduria, developmental delay, cardiomyopathy and siezures. We report on the ¢rst successful prenatal diagnosis of an a¡ected foetus using a combined metabolite, enzymology and molecular approach. The index case was an 11 month old girl with Down syndrome who presented with overwhelming cardiogenic shock and metabolic acidosis. She died within 24 hours and a post mortem revealed endocardial ¢broelastosis. Urinary organic acids showed moderate elevation of MMA and gross elevation of malonate. Unfortunately no tissues were available for enzymology or DNA analysis. The mother subsequently presented at 10 weeks gestation requesting prenatal diagnosis. A CVS sample was taken at 11 weeks and MCD activity was markedly reduced, however, propionyl CoA carboxylase, assayed as a control for viability, was also low and the results were felt to be equivocal. Amniocentesis was performed at 14 weeks and stable isotope quantitation revealed malonate 410x the upper limit of normal but MMA within normal limits. Mutational analysis from CVS and parents showed the foetus to be a compound heterozygote carrying a G to C conversion in a highly conserved region of exon 3 from the mother and an A to G transition in exon 1 destroying the initiation codon from the father. The pregnancy was terminated and the diagnosis con¢rmed by enzyme assay in ¢broblasts.

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GLUTARIC ACIDURIA I: CREATINE SUPPLEMENTATION RESTORES CREATINE PHOSPHATE IN MIXED CORTEX CELLS INCUBATED WITH 3-HYDROXY-GLUTARATE A.M. Das, T. Lu«cke and K. Ullrich Department of Paediatrics, University of Hamburg, Germany Introduction: Secondary excitotoxicty due to energy deprivation in neurons is discussed in the pathogenesis of CNS-symptoms in glutaric aciduria I (GA). Recently, we have found reduced concentrations of creatinephosphate (CP) in mixed cortex cultures incubated with 4 mM 3-OHglutarate (OH-glut) while ATP-levels were normal (J. Inher. Metab. Dis. 22, pp 392). Methods: Mixed cortex cultures from neonatal rats were grown for 10 days, then incubated for 24h with 4 mM OH-glut with and without creatine supplementation (1 mM). High-energy phosphate compounds (ATP, CP, ADP) were measured by bioluminescence using a luciferin/luciferase assay. Results: In control cultures (5 preparations) CP-content was 7.3+5.9 nmol/mg prot, after incubation with 4mM OH-glut it dropped to 3.9+2.8 nmol/mg prot. The non-competitive NMDAantagonist MK 801 (20 mM) could prevent this e¡ect showing involvement of NMDA receptorassociated ion channels in CP-depletion. Supplementation of the medium with 1 mM creatine led to restoration of CP. Activity of creatinekinase as well as ATP, ADP were not a¡ected by OH-glut. Discussion: We show that CP-depletion induced by 4 mM OH-glut can be completely prevented by 1 mM creatine. CP-depletion in GA may be due to inhibition of creatine transport across the sarcolemma or the inner mitochondrial membrane by OH-glut. Furthermore, NMDA-associated ion channels seem to contribute to CP-depletion. Creatine-supplementation may be of bene¢t in patients with GA, especially during metabolic crises.

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GLUTAMIC ACIDAEMIA AS A RESULT OF CALCIUM LEVULINATE TOXICITY IN TWO NEW-BORNS WITH HYPOCALCAEMIA M. Williamsa, S. Kamphuisb, M. Duranb, J.B.C. de Klerka, J. Huijmansa, and B.T. Poll-Theb. a Erasmus University Rotterdam, Department of Clinical Genetics and Paediatrics. b University Medical Centre Utrecht, Paediatric department. Two new-born patients receiving calcium levulinate, a common therapeutic drug for hypocalcaemia, developed a very serious lactic acidaemia (blood pH 7.17 and 7.08; serum lactate 7.7 and 7.3 mmol/ L) together with glutamic acidaemia (plasma levels in one of the two patients was 1623 mmol/L) and glutamic aciduria (urine levels were 18170 and 16072 mmol/mol creatinine). One of these patients required ventilatory support. After cessation of calcium levulinate treatment the biochemical abnormalities resolved slowly. Similar toxic e¡ects of calcium levulinate have been described once in the literature1. An observed high lactate/pyruvate ratio in one of the patients suggests that an underlying mitochondrial disorder may have deranged the challenged mitochondrial system. A direct e¡ect of levulinate on glutamate metabolism cannot be ruled out at this point. With respect to the use of calcium levulinate for hypocalcaemia with possible life threatening complications, an advice to use one of the many other calcium containing preparations, seems justi¢able. 1. Teijema H.L., et al. Glutamic acidemia. Monogr. Hum.Genet., vol. 9, pp. 75-79 (1978).

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CREATINE ATTENUATES EXCITOTOXICITY IN AN IN VITRO MODEL OF GLUTARIC ACIDURIA TYPE 1: IMPACT FOR THERAPY? S. Ko«lker1, C. Bachmann2, G.F. Ho¡mann1 University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany1, and Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland2

Objective: It has been previously shown that application of creatine prevents neuronal damage under certain conditions (Wilcken B et al. Ped Res 1998; 43: 8^14). We investigated the e¡ect of creatine on neuronal damage in vitro induced by 3-hydroxyglutaric acid (3-OH-GA) which is the main neurotoxin in glutaric aciduria type I (GA-I). Methods: Primary neuronal cultures from chick embryo telencephalons were preincubated with 1 mmol/L 3-OH-GA for 1 h with or without pre (1^72 h)-, co- or postincubation of creatine (0.01^1 mmol/L). Cell viability was determined by trypan blue exclusion method. Intracellular [Ca2+] as well as reactive oxygen species (ROS) were investigated using Fura-2 AM or dihydrorhodamine 123. Results: Preincubation with creatine decreased neuronal damage, reaching a maximum after a 72 h preincubation at a concentration of 1 mmol/L creatine (18% with vs-40% without creatine, control: 10%). Co- and postincubation with creatine had no e¡ect. Creatine preincubation had no e¡ect on intracellular Ca2+ concentrations, but markedly decreased ROS (16.6 with vs 39.1 £uorescence units [Fl.U.] without a 72 h preincubation with creatine, control: 5 Fl.U.). Conclusions: We conclude from our results that elevation of endogenous creatine concentrations partially protects neurons against neuronal damage in vitro induced by 3-OH-GA. The exact mechanism of protection remains to be elucidated, but decrease in ROS is suggestive of a stabilizing e¡ect of creatine on the mitochondrial energy metabolism.

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NEUROPROTECTIVE POTENCY OF THE RADICAL SCAVENGERS a-TOCOPHEROL AND MELATONIN IN GLUTARIC ACIDURIA TYPE I S. Ko«lker1,2, B. Ahlemeyer2, J. Krieglstein2, G.F. Ho¡mann1 University Children's Hospital, Department of General Pediatrics, Heidelberg1, and Department of Pharmacology and Toxicology, Marburg, Germany2

Objective Reactive oxygen species (ROS) are supposed to play an important role in neurodegeneration as seen in glutaric aciduria type I (GA-I). We investigated the e¡ect of the radical scavengers atocopherol and melatonin on the increase in ROS induced by 3-hydroxyglutaric (3-OH-GA) and glutaric acids (GA). Methods: Primary neuronal cultures from chick embryo telencephalons were maintained in culture for 5 d before they were incubated with 1 mmol/L 3-OH-GA or GA for 1h. Co-, pre- and posttreatments with a-tocopherol (0.1^10 mmol/L) and melatonin (5^500 mmol/L) were performed. ROS was investigated using dihydrorhodamine 123. Cell viability was determined by trypan blue exclusion. Results: Coincubation with a-tocopherol prevented neuronal damage as well as an increase in ROS induced by 3-OH-GA or GA in a concentration-dependent way. Pre- or posttreatment with 10 m M atocopherol were found to be partially neuroprotective but less e¡ective than preincubation. Melatonin (500 mmol/L) only partially decreased neuronal damage and ROS after coincubation, whereas lower concentrations as well as pre- or posttreatment had no e¡ect on ROS levels and neurodegeneration. Conclusion: a-tocopherol is more e¡ective than melatonin in protecting neurons against devastating e¡ects of ROS elicited by 3-OH-GA or GA in vitro. The wide time-frame of neuroprotection make atocopherol a hopeful candidate substance for the prevention of acute neuronal damage in GA-I.

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GLUTARIC ACID: FOE OR FRIEND TO THE DEVELOPING BRAIN? S. Ko«lker1,2, B. Ahlemeyer2, J. Krieglstein2, G.F. Ho¡mann1 University Children's Hospital, Department of General Pediatrics, Heidelberg1, and Department of Pharmacology and Toxicology, Marburg, Germany2 Objective: We have previously reported that long-term preincubation of neuronal cultures with subtoxic concentrations (5100 mmol/L) of glutaric acid led to a downregulation of NMDA receptors. The aim of this study was to investigate, whether preincubation with glutaric acid (GA) protects neurons against subsequent excitotoxic damage. Methods: Primary neuronal cultures from chick embryo telencephalons were preincubated with 10 mmol/L GA for 5 d, and then exposed to 1 mmol/L GA or 0.3 mmol/L NMDA for 1 h. Cell viability was determined by trypan blue exclusion after 24 h. Reactive oxygen species (ROS) were investigated by £uorescence microscopy using dihydrorhodamine 123. Results: Preincubation with 10 mmol/L GA itself had no e¡ect on cell viability (90%, control: 91%) or ROS formation (6 £uorescence units [Fl.U.], control: 4 Fl.U.), whereas it led to a marked increase in cell viability after administration of 0.3 mmol/L NMDA (from 29 to 53%) or 1 mmol/L GA (from 73 to 82%). It also decreased ROS induced by 0.3 mmol/L NMDA (from 59 to 27 Fl.U.). Conclusions: We conclude from our results that GA might have a dual pathogenetic role in glutaric aciduria type I (GA-I), mostly depending on its concentration. At moderately elevated concentrations GA decreases ROS and NMDA receptor expression in vitro, protecting against excitotoxins at high concentrations. On the other hand the chronic decrease in excitatory input may induce a delay in neuronal maturation, which may be responsible for frontotemporal atrophy and reduced opercularization in patients with GA-I.

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ACRODERMATITIS ACIDAEMICA IN GLUTARIC ACIDURIA TYPE I? S. Ko«lker, I. Degen, G.F. Ho¡mann University Children's Hospital, Department of General Pediatrics, Heidelberg, Germany

Acrodermatitis enteropathica (AE)-like cutaneous lesions are rarely found cutaneous lesions of unknown origin in organic acid disorders, also called `acrodermatitis acidaemica' (AA). Previous reports have described AA especially in branched-chain amino acid disorders. Here we report about AE-like lesions in a 5-year-old Turkish girl with glutaric aciduria type I (GA-I). She presented with severe skin lesions that had developed over 6 weeks, starting with moist erythematous lesions in the face, expanding to the extremities including ¢ngers and toes, the axillary intertrigines, the diaper region, and the back. Erythematous lesions changed to an exfoliative dermatitis, later becoming hyperkeratotic and dry in appearance. Following an acute encephalopathic crisis at age 6 m she got a dietary treatment. Severe feeding problems led to an application of a percutaneous gastrostomy at age 21/2 y and of a jejunal tube at age 5 y. Because of compliance problems only an insu¤cient amount of the diet was given to her during 8 weeks before she was presented. Laboratory ¢ndings revealed the picture of hypoproteinaemia, hypoalbuminaemia and an overall de¢ciency of amino acids (including isoleucine). Plasma levels of zinc, selenium and vitamin B6 were also markedly decreased. High calorie, protein-rich diet supplemented with vitamins, zinc and selenium led to a slow recovery over 4 weeks. It seems likely that in our patient AE-like lesions are the result of protein^calorie malnutrition, rather than being primarily associated with GA-I itself. However, one should also consider the possibility of AA in GAI.

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MUTATION ANALYSIS OF GCDH GENE IN ITALIAN AND PORTUGUESE PATIENTS WITH GLUTARIC ACIDURIA TYPE I A. Ribes1,C. Busquets1, M. Soriano1, I. Tavares de Almeida2, B. Garavaglia3, M. Rimoldi3, I. Rivera2,G. Uziel3, M.J. Coll1. 1 Institut de Bioqu|¨ mica Cl|¨ nica, Corporacio¨ Sanita©ria Cl|¨ nic , Barcelona, Spain; 2Centro de Patoge¨nese Molecular, Faculdade de Farmacia da Univ. Lisboa., Portugal; 3Divisione de Biochimica e Genetica and Divisione de Neoropsichiatria Infantile, Istituto Neurologico Carlo Besta, Milano, Italy We report the mutations responsible for GCDH de¢ciency in Italian and Portuguese patients with GAI. Mutational screening by PCR-SSCP of all the coding sequences and the exon-intron boundaries resulted in the characterization of all mutant alleles. Three novel (P248L, G390V and X439W) and 5 already known mutations were identi¢ed in a total of 14 alleles. The substitution X439W is a rare type of mutation, which breaks the stop codon of the GCDH gene. As described in other populations, R402W was the commonest mutation accounting for 5/14 alleles. All the patients, but one, presented high glutarate excretion. Genotype R227P/R402W was found in the patient with low glutarate excretion. This new genotype reinforces the idea that R227P is one of the causative mutations of this biochemical phenotype. Previous results in Spanish GA I patients showed that R402W alleles were always associated with the same intragenic haplotype (Busquets et al. 1999 Human Mutat, Online #291). Results for the Italian and Portuguese patients showed that the R402W bearing chromosomes were also associated with the same haplotype, suggesting a founder e¡ect. However, the possibility of di¡erent founder e¡ects in other populations can not be excluded, as R402W is a CpG mutation. This work was supported by Fondo de Investigaciones Sanitarias (FIS, No. 0049-01).

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3-HYDROXYGLUTARIC AND GLUTARIC ACIDS IMPAIR THE RELEASE OF NERVE GROWTH FACTOR IN CULTURED RAT ASTROCYTES S. Ko«lker1,2, r. Hu«hne2, J. Krieglstein2, and G.F. Ho¡mann1 University Children's Hospital, Department of General Pediatrics, Heidelberg1, and Department of Pharmacology and Toxicology, Marburg, Germany2

Object: We investigated the e¡ect of 3-hydroxyglutaric (3-OH-GA) and glutaric acids (GA) on the release of nerve growth factor (NGF) from cultured rat astrocytes. Methods: Rat cortical astrocytes were maintained for 18^20 d in culture before they were exposed to 160 U/mL IL-1 b, 1 mmol/L Bromo-cAMP, 0.1^1 mmol/L 3-OH-GA or Ga for 48h. NGF release to the culture medium was determined in a commercially available NGF-ELISA (Boehringer Mannheim). Results: Neither GA nor 3-OH-GA itself had a signi¢cant e¡ect on basal NGF (97^127% or 100^ 117% of control). NGF release markedly increased after exposure to IL-1 b (571% of control) or Bromo-cAMP (145% of control). The induction of NGF release by Bromo-cAMP was markedly reduced after coadministration of 0.1^1 mmol/L 3-OH-GA (from 145% to 81^95% of control) or 1 mmol/L GA (from 145% to 84% of control). Only 0.1^1 mmol/L GA but not 3-OH-GA led to a signi¢cant reduction in IL-1b-induced NGF release (from 571% to 417^435% of control). Conclusions: It has been previously shown that NGF is a potent neurotrophin with antiapoptotic potency. But it may also potentiate excitotoxic neuronal damage under certain conditions. A suppression of NGF release could be interpreted in two ways: as an impairment of an important neurotrophic signal or as a protection against excitotoxicity. Future studies may enlighten which role NGF plays in glutaric aciduria type I and by which mechanism NGF release is reduced by 3-OH-GA and GA.

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IONIC DEPENDENCE OF NEURONAL DAMAGE INDUCED BY 3-HYDROXYGLUTARIC ACID IN VITRO S. Ko«lker1,2, B. Ahlemeyer2, J. Krieglstein2, G.F. Ho¡mann1 University Children's Hospital, Department of General Pediatrics, Heidelberg1, and Department of Pharmacology and Toxicology, Marburg, Germany2 Objective: We have previously shown that 3-hydroxyglutaric acid (3-OH-GA) induced neuronal damage via NMDA receptors. We investigated the e¡ect of 3-OH-GA on intracellular [Ca2+] homeostasis and its dependence on extracellular [Ca2+] and [Na+]. Methods: Primary neuronal cultures from chick embryo telencephalons were maintained in culture for 5 d and then exposed to 1 mmol/L 3-OH-GA diluted in Mg2+ -free HEPES-bu¡ered saline containing Na+ (HBS) or N-methyl-D-glucamine (N-HBS) with (HBS) or without Ca2+ (0-HBS). Intracellular [Ca2+] was determined by £uorescence microscopy using Fura-2 AM. Cell viability was investigated by trypan blue exclusion. Results: Exposure to 3-OH-GA in HBS markedly increased intracellular [Ca2+] (232 nmol/L, control: 105 nmol/L), whereas incubation in 0-HBS did not (113 nmol/L). Incubation with 3-OH-GA in N-HBS led to an aggravated increase in intracellular [Ca2+] (342 nmol/L). In parallel to this, cell viability decreased more markedly after exposure to 3-OH-GA in N-HBS (59%; control: 92%) than in HBS (70%), whereas it was not decreased by 0-HBS (84%). Conclusions: 3-OH-GA leads to a disturbance of the intracellular Ca2+ homeostasis by augmented Ca2+ entry from the extracellular space. Replacement of Na+ by N-methyl-D-glucamine, which hyperpolarizes the plasma membrane, further aggravates this e¡ect by enhancing the Ca2+ driving force. Therefore, Na+ may have a regulatory role in Ca2+ entry following NMDA receptor stimulation.

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L-2-HYDROXYGLUTARIC ACIDURIA: REPORT OF 18 TURKISH PATIENTS M. Topc°u1^5, T. C°os° kun2, I. Saatc°i3, K. Yalaz1, Y. Renda1, S. Aysun1, B. Anlar1, H. Topalogïlu1, M. O«zgu«c°4, M. Kesimer2 Hacettepe University Dept of Child Neurology1, Nutrition and Metabolism2, Radiology3, Molecular Biology4, Institute of Neurological Sciences5, Ankara, TURKEY Between 1993^2000, 18 patients from 15 families were diagnosed as having L-2 HO Glutaric aciduria and followed up with a mean duration of 44 months. At the time of the diagnosis the mean age was 9.6 years (between 1^19 years of age). The diagnosis was based on highly elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma and CSF. Megalencephaly was seen in 16/18 cases, two had normal head circumference. The main neurological abnormalities were ataxic gait and intentional tremor. On follow-up abnormal cerebellar tests become more prominent around 12 years of age and no obvious deterioration was noted after 14 years of age. Of note, patients had short attention span and hyperactivity before 10 years of age and become depressed after 15 years of age. IQ scores were between 50^75. None of our patients lost ambulation. 80% of our patients had a history of either febrile or afebrile partial or generalized seizures which were well controlled with anticonvulsant drugs. EEG ¢ndings included background slowing, and focal or generalized spike and wave discharges. VEP and BAEP showed progressive deterioration on serial studies. In addition to MRI ¢ndings described in the literature, MRI studies of our patients revealed that putamen and caudate nuclei were slightly a¡ected and globus pallidus was abnormal in all our patients. The thalami and brain stem were intact. Only two of the patients, both over 15 years of age, had ventricular dilation and cortical atrophy. We are in the process of registering L-2 HO Glutaric aciduria patients for genetic studies.

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CARDIOMYOPATHY IN A PATIENT WITH HMG-CoA LYASE DEFICIENCY F. Eyskens, H. Verhaaren, G. Janssens AZ-Middelheim, Campus Queen Paola Children's Hospital; University Hospital, Antwerp, Belgium

A Moroccan girl, third child of consanguineous parents, presented on the second day of life with convulsions, generalized hypotonia and slight liver enlargement. Laboratory investigations revealed a severe hypoglycemia associated with a metabolic acidosis, hyperammonemia (357 mmol/L) and lactic acidemia (4.5 mmol/L). Organic acid analysis in urine showed the typical pattern of a 3-hydroxy-3methylglutaryl (HMG) coA lyase de¢ciency in decompensation: lactic acid 242 mmol/mol creat, absence of ketone bodies, adipic acid 222 mmol/mol creat, suberic acid 27 mmol/mol, creat, very high excretion of 3-methylglutaconic acid, 3-hydroxy-3-methylglutaric acid, 3-hydroxyisovaleric acid, 3-methylglutaric acid and 3-methylcrotonylglycine. The diagnosis was con¢rmed by enzymatic assay of HMG-coA lyase de¢ciency in leucocytes and ¢broblasts (Amsterdam) and the ¢nding of a nonsense mutation and a deletion in the alleles of the HMG-coA lyase gene (Barcelona). Treatment consists of a relative natural protein restricted high-carbohydrate diet, L-carnitine supplementation and, most important, avoidance of catabolism. The evolution of this patient is very good except for a few decompensations triggered by infectious diseases. At the age of three years a systolic cardiac murmur was found on clinical examination. ECG and uultrasonography of the heart showed a hypertrophy of the interventricular septum. Six years follow-up revealed a very slowly progressive hypertrophic cardiomyopathy of the septum and left ventricular wall without functional implications. Cardiomyopathy is a very rare complication of this metabolic disorder. This case further delineates the appearance and evolution of this complication.

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A NEW MUTATION E96X IN HL GENE LEADS TO A 3-HYDROXY-3-METHYLGLUTARIC ACIDURIA IN AN ITALIAN PATIENT S. Funghini, E. Pasquini, M.A. Donati, A. Morrone and E. Zammarchi; Dept of Pediatrics, Florence, Italy. 3-hydroxy-3-methylglutaric aciduria is a rare A.R. inborn error of metabolism due to the de¢ciency of the mitochondrial enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) lyase that catalyses the cleavage of HMGCoA to acetoacetate and acetyl-CoA. The disease has a neonatal or postnatal onset. Clinical acute episodes are characterised by vomiting, hypotonia, lethargy and coma. Macrocephalia or, less frequently, microcephalia, development delay and dilated cardiomyopathy with arrhytmia may be present. Metabolic acidosis with hypoketotic hypoglycemia on fasting, variable hyperammonaemia and high plasma transaminase levels may also be present. The patients showed a characteristic urinary organic acid pattern: 3-hydroxy-3-methylglutaric acid, 3methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. The HL gene, mapped on 1p36.11, has been cloned. Up to now only few mutations have been reported and most of them were identi¢ed in patients with Arabic origin. We report a girl born at 40th week following an uneventful pregnancy and delivery. Three days after birth she presented metabolic acidosis and a diagnosis of transitory tubular renal acidosis was made. At the age of three months she was admitted to our hospital for lethargy and vomiting. She presented hepatomegaly, and hypotonia. Laboratory investigations revealed a metabolic acidosis with increased anion Gap, hypoglycaemia, hyperlactacidemia, hypertransaminasemia, macrocytic anemia and a low level of L-carnitine. MR of the brain revealed an increased signal of paraventricular white matter. Analysis of urinary organic acid showed the characteristic pattern that leads us to suspect a defect of HMG-CoA lyase activity that was con¢rmed on cultured skin ¢broblasts. In the full length patient's HL cDNA a new transition c286C4T that leads to the stop codon E96X was detected at homozygous level. This mutation was con¢rmed in the patient's and parents genomic DNA. The E96X gives rise to a truncated protein in which a high percentage of the amino acid sequence is lost, including the domain that contains the active catalytic site.

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HMG-CoA-LYASE (HMG-CoA) DEFICIENCY - RESPONSE OF 3-OHISOVALERYLCARNITINE (3-OH-IVC) TO DIETARY TREATMENT AND DIFFERENCES IN ITS WHOLE BLOOD, PLASMA AND RED BLOOD CELL (RBC) LEVELS Abdenur JE, Chamoles NA, Schenone A, Guinle A, Fusta M, Coluccio M, Lavorgna S. Fundacio¨n para el Estudio de las Enfermedades Neurometabo¨licas. Buenos Aires, Argentina. We studied the levels of 3-OH-IVC in a patient with HMG-CoA de¢ciency before and after leucine (L) restriction. Methods: The patient presented at 3m of age with metabolic acidosis and hypoglycemia. Initial treatment consisted of carnitine and a protein restricted diet (PRD) (1.5 gr/ kg, L=155 mg/kg). Later on, a leucine restricted diet (LRD) (L=100 mg/kg) was implemented. Levels of 3-OH-IVC (MS/MS-Micromass, UK) were measured during short fasting tests. Partition of 3OH-IVC was assessed by simultaneous analysis in whole blood, plasma and RBC. Results: Diagnostic sample showed a marked increase of 3-OH-IVC in whole blood (4800%), with only a 37% increase in plasma. Levels of 3-OH-IVC in whole blood remained elevated, without signi¢cant variations throughout the PRD test (range 2.39 to 3.06 uMol, normal 50.6) and decreased by about 25% on LRD (1.76 to 2.15 uMol). Plasma levels were normal or close to normal in all samples. Analysis of 3-OH-IVC partition showed that the metabolite is concentrated in RBC, being the levels in plasma of less than 10 % of whole blood. Conclusion: Measurement of 3-OH-IVC in whole blood is useful for the diagnosis and follow-up of patients with HMG-CoA de¢ciency. Normal levels of 3-OHIVC in plasma should be cautiously interpreted.

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ANAEMIA IN 3-HYDROXY-3-METHYLGLUTARIC ACIDURIA P. Garcia(1), A. Dinis(1) , M. Branco(2), L. Ribeiro(1) M. Lu|¨ s Cardoso(3), L. Vilarinho(3), L. Diogo(1) Hospital Pedia¨trico de Coimbra (1); Maternidade -HUC (2); IGM - Porto(3). Portugal. This girl, born in December 1999, is the second child of a healthy, non consanguineous couple. She was the ¢fth pregnancy with three spontaneous abortions. Pregnancy, delivery and Apgar score were normal. Feeding di¤culties were noted since birth. On her third day of life she had one episode of acute severe hypoglycaemia, easily corrected by glucose administration. Biochemical investigation showed: mild hyperammonemia and metabolic acidosis, positive urinary diphenylhydrazine test with no ketonuria, and raised levels of urinary 3-hydroxy-3-methylglutaric acid, allowing the diagnosis of 3-hydroxy-3-methylglutaric aciduria. Diagnosis was con¢rmed by the ¢nding of undetectable levels of 3-hydroxy-3-methylglutaryl coenzyme A lyase activity in ¢broblasts. Her haemoglobin (Hb) was 19,9 g/dl, on day 3 of life. In spite of apparently good metabolic control, anaemia ensued (Hb was 5,8 g/dl by 2 months of age). At 4 months of age, Hb was 8,3 g/dl, with 0,4% reticulocytes and normal ferritin levels. As far as we know, anaemia as not been described in this disorder, although we are aware of other patients in the north of Portugal in whom anaemia as also been a problem. This disease associates a leucine catabolism defect to an impaired ketone body formation. It is possible that anaemia is related to bone marrow toxicity in this particular case, as no other common cause for it was found, lyase activity is null and anaemia is partially responding to the management of aciduria. (Lyase determinations were done by Dr. M.O. Rolland - service de Biochimie Pe¨diatrique, Hoªpital Debrousse , Lyon, France)

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CLINICAL, BIOCHEMICAL AND MOLECULAR CHARACTERISATION OF 3-HYDROXY3-METHYLGLUTARYL-CoA SYNTHASE DEFICIENCY J Zschocke1, FG Hegardt2, N Casals3, JM Penzien4, R Aledo3, J Pie¨ 2, E Mayatepek1, GF Ho¡mann1 1 Univ. Children's Hospital, Heidelberg, Germany, 2Dept. of Biochemistry, Univ. of Barcelona, Spain, 3 Int. Univ. of Catalonia, Barcelona, Spain; 4Children's Hospital, Augsburg, Germany

Introduction: Mitochondrial 3-hydroxy-3-methylglutaryl-(HMG-)CoA synthase is the key enzyme for the hepatic production of ketone bodies. One child with hypoketotic hypoglycaemia and putative mitochondrial HMG-CoA synthase de¢ciency has been reported, but enzymatic con¢rmation was not possible and molecular studies were not performed. Patients and Results: We report two other children with HMG-CoA de¢ciency. Both presented in infancy with acute hypoketotic hypoglycaemic coma after prolonged fasting during a febrile infection. Urinary organic acids showed a unique pattern of metabolites arising from fatty acid and amino acid metabolism. Acylcarnitines were normal even during hypoglycaemia. Molecular studies in the ¢rst patient revealed two mutations in the HMG-CoA synthase gene, one of which was also identi¢ed in the second patient; further analyses are in progress. With avoidance of prolonged fasting periods the children are developing normally at three resp. two years of age. Discussion: HMG-CoA synthase de¢ciency is the only known disorder that exclusively a¡ects mitochondrial ketogenesis. Unlike beta-oxidation defects or HMG-CoA lyase de¢ciency it does not appear to give rise to toxic acyl-CoA or acylcarnitine metabolites. Clinical presentation appears to be restricted to nonketotic hypoglycaemia coma during fasting, without speci¢c organ dysfunction.

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3-HYDROXY-3-METHYL GLUTARYL-CoA SYNTHASE: DEVELOPMENT OF A NEW ENZYME ASSAY APPLICABLE TO LIVER BIOPSY SPECIMENS L. IJlst, S. Wanrooij, S. Houten, H.R. Waterham, R.J.A. Wanders University of Amsterdam, Academic Medical Centre, Department of Pediatrics (Emma Children's Hospital) and Clinical Chemistry, Amsterdam, The Netherlands

Objectives: In many patients mitochondrial fatty acid oxidation appears to be compromised at least in vivo although subsequent studies in ¢broblasts fail to identify the defect in such patients. 3-Hydroxy3-methylglutaryl-CoA (HMG-CoA) synthase de¢ciency is a good candidate for the enzyme defect in such patients. HMG-CoA synthase, however, is notoriously di¤cult to measure which led us to set up a good, reliable assay. Results: HMG-CoA synthase catalyses the formation of HMG-CoA from acetyl-CoA and acetoacetyl-CoA. Most assays are based on the disappearence of acetoacetyl-CoA which is full of pittfalls. We developed a coupled assay making use of recombinant HMG-CoA reductase which we prepared. In this set-up HMG-CoA synthase can simply be measured by following the absorption at 340 nm since the generation of HMG-CoA is coupled to the conversion of NADPH to NADP+. The assay has been optimized and has turned out to be very reliable. Furthermore, the assay is applicable to small liver biopsy specimens. This assay opens the way to identify more cases of HMG-CoA synthase de¢ciency. Sofar only two cases have been described in literature.

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MITOCHONDRIAL 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE DEFICIENCY: CLINICAL COURSE AND DESCRIPTION OF CAUSAL MUTATIONS IN THE TWO KNOWN PATIENTS Grant A. Mitchell (1), Luigi Bouchard (1), Marie-France Robert (1), Charles A. Stanley (2), Geo¡rey N Thompson (3)*, Andrew Morris (4)*, James V. Leonard (4), Patti Quant (4), Betty Y. L. Hsu (2), Avihu Boneh (3), Youssef Boukaftane (1), Lyudmila Ashmarina (1) [(1) Service de ge¨ne¨tique me¨dicale, Ste-Justine Hospital, Montreal (Que¨bec), Canada, H3T 1C5; (2) The Children's Hosp of Philadelphia, Div of Endocrinol/Diabetes, Philadelphia, PA; (3) The Murdoch Inst, Royal Children's Hosp, Melbourne, Australia; (4) Biochem, Endocrinol and Metabolic Unit (JVL) and Paediatric Surg Unit, (PQ), Inst of Child Health, London, UK] Hereditary de¢ciency of mitochondrial HMG-CoA synthase (mHS, OMIM 600234) is a poorlyde¢ned, treatable, probably underdiagnosed cause of hypoketotic hypoglycemia. Diagnosis is challenging because the symptoms are as yet incompletely de¢ned and the metabolite pattern is not speci¢c and enzyme diagnosis requires sampling of expressing viscera such as liver. We present clinical followup and the ¢rst molecular analysis of the two known mHS-de¢cient patients. Pt 1, a Chinese Australian boy now aged 16 years and Pt 2, a British boy aged 6 years, have normal development and no further decompensations. Using single strand conformational polymorphism (SSCP) screening of amplicons for all 9 coding mHS exons, Pt 1 is a F174L homozygote. A Leu residue was present in this location in HS-like peptides from Arabidopsis and C elegans. We therefore created a bacterial expression system, showing that F174L produced a low level of mHS peptide with no detectable activity. Pt 2 is a compound for a premature termination mutation (R424X) and a second as-yet uncharacterized mutant allele. Molecular analysis is useful in patients suspected of mHS de¢ciency.

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BIOCHEMICAL AND MOLECULAR DIAGNOSIS OF ISOPRENOID BIOSYNTHESIS DEFECTS: MEVALONIC ACIDURIA AND HYPER-IgD AND PERIODIC FEVER SYNDROME S.M. Houten, J. Koster, G.J. Romeijn, R.J.A. Wanders, H.R. Waterham Academic Medical Center, University of Amsterdam, Dept. Of Clinical Chemistry and Div. Emma Children's Hospital, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands

Mevalonic aciduria (MA) and hyperimmunoglobulinaemia D and periodic fever syndrome (HIDS) are two autosomal recessive inherited disorders both caused by a de¢cient activity of the enzyme mevalonate kinase (MK) resulting from mutations in the encoding MVK gene. MA is a severe multisystemic disorder, characterized by psychomotor retardation, failure to thrive, hepatosplenomegaly, anaemia and recurrent febrile crises. HIDS is a relative benign condition, in which patients su¡er, as in MA, from recurrent fever episodes associated with lymphadenopathy, arthralgia, gastrointestinal problems and skin rash. Thus far, disease-causing mutations could only be detected by analysis of MVK cDNA, however, we recently resolved the genomic structure of the MVK gene. This enables mutation analysis at the genome level. We here report the biochemical and molecular characterization of 28 patients with an MK de¢ciency. In total, we identi¢ed 14 di¡erent mutations of which 7 were not reported before. Furthermore, the sequence analysis con¢rmed all previously reported genotypes based on cDNA analysis. Comparison of the genotypes with the corresponding MK enzyme activities provides new insights in the genotype/phenotype relation between HIDS and MA.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MEVALONATE KINASE DEFICIENCY IN A CHILD WITH PERIODIC FEVER WITHOUT HYPERIMMUNOGLOBULIN D N. Di Rocco, U. Caruso**, H.R. Waterham***, P. Picco*, A. Loy*, R.J. Wanders*** *II Pediatric Division `Gaslini' Institute, **DIPE University Genoa, Italy and ***Laboratory of Genetic Metabolic Diseases, Academic Medical Center, Amsterdam

Mevalonic aciduria is the consequence of the de¢ciency of mevalonate kinase key enzyme in the biosynthesis of cholesterol and non sterol isoprenes. Some patients with mevalonic aciduria present with failure to thrive, severe psychomotor retardation, dysmorphic features and cataract and die in the ¢rst year of life, other patients have ataxia, hypotonia, mild psychomotor retardation. Patients from both groups have recurrent crisis of high fever, lymphoadenomegaly, hepatosplenomegaly and diarrhea. Recently a third group of patients with mevalonic aciduria has been reported: they have a genetic disease known as hyperimmunoglobulinemia D and periodic fever (HIDS) (Nat Genet 1999; 22:175^7; Nat Genet 1999; 22:178^ 81). We have studied a 4-year-old boy, only child of healthy unrelated parents, who showed fever spikes from the age of 9 months; these episodes persist for 2^3 days, disappear without treatment with £are-ups every 20 days; the child complains also malaise, myalgias, arthralgias lymphoadenopathy, diarrhea and increase of in£ammatory indexes. IgD were assayed in di¡erent occasions and found to be normal. Slight excretion of urine mevalonic acid was detected by GC-MS during an episode of fever and disappeared when the child was without fever. Mevalonate kinase was assayed on cultured ¢broblasts and found de¢cient (5.3 pmol/min/mg; controls 144 +/67). Our patient with recurrent fever, normal IgD and mevalonate kinase de¢ciency con¢rms that hyperimmunoglobulinemia D is only an epiphenomenon and that mevalonic acid excretion during the episodes of fever is the most reliable biological marker of this disease. Concerning the pathogenesis of autoin£ammatory phenotype in mevalonic aciduria it is probable that it results from abnormal synthesis of isoprenoid, important intermediates in generations of sterols, ubiquinone and prenylated proteins, involved in cell growth and di¡erentiation, glycosylation signal transduction and electron transport.

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2-METHYL-3-HYDROXYBUTYRYL-CoA DEHYDROGENASE (MHBD) DEFICIENCY ^ A NOVEL NEURODEGENERATIVE DISORDER J Zschocke1, L Ijlst2, J Brand3, M Lindner1, E Mayatepek1, R Wanders2, GF Ho¡mann1 1 Dept of General Paediatrics, University Hospital, Heidelberg, Germany; 2 Dept of Paediatrics, University Hospital, Amsterdam, The Netherlands; 3Johanniter Children's Hospital, St. Augustin, Germany

Introduction: We report the ¢rst patient with a genetic de¢ciency of 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD), an isolated defect in the mitochondrial oxidation of 2-methylated fatty acids and isoleucine. Patient: The a¡ected boy was born on term and recovered well from a postnatal episode of metabolic decompensation and lactic acidosis. Psychomotor development in the ¢rst year was moderately delayed. After age 14 months there was progressive loss of mental and motor skills; at age two years he was severely retarded with marked restlessness, choreoathetoid movements, absence of directed hand movements, marked hypotonia and little reaction to external stimuli. Investigations: Notable were marked elevations of urinary 2-methyl-3-hydroxybutyrate and tiglylglycine without elevation of 2-methylacetoacetate, particularly after isoleucine challenge; mild elevations of lactate in CSF and blood; and a slightly abnormal acylcarnitine pro¢le. Enzyme studies showed absent MHBD activity. Treatment: Under dietary isoleucine restriction there was no further deterioration of neurological functions over four months. Discussion: MHBD de¢ciency is a novel neurodegenerative disorders with clinical features that di¡er markedly from other organic acidurias. It can be detected through urinary organic acid analysis but may be mistaken for 3-ketothiolase de¢ciency. Pathogenetic mechanisms are unknown but may be related to the accumulation of short 2-methylated fatty acids.

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PHARMACOLOGIC RESCUE OF LETHAL SEIZURES IN A MURINE KNOCKOUT MODEL OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH) DEFICIENCY K.M. Gibson1, B.M. Hogema1,2, M. Taylor1, R.B.H. Schutgens2, W. Froestl3, O.C. Snead4, M. Grompe1, C. Jakobs2 Molec. and Med. Genet., Oregon Health Sci. University, Portland, OR1; Metab. Unit, Dept. of Clin. Chem., Free Univ. Hosp., Amsterdam2; Novartis, Basel, Switzerland3; Dept. of Neurol., Hosp. for Sick Children, Toronto, Canada4 SSADH de¢ciency is a rare defect of GABA degradation associated with 4-hydroxybutyric (GHB) aciduria in a¡ected patients. To explore pathomechanisms and develop preclinical treatment paradigms, we developed a targeted murine knockout of SSADH de¢ciency in C57/129 mice. In the ¢rst two weeks of life, mutant (-/-) mice showed mildly decreased body weight and ataxia. At postnatal day 17^20 generalized seizures began, leading to rapid death in all -/- animals. SSADH activities in mouse tissue homogenates (nmol/min/mg protein) were: wild-type (+/+) brain (n=16, 22.5 + 5.5 (SD), range 13^34); liver (n=18, 18.6 + 7.0, range 11.3^40.5); and kidney (n=2, 7.6, 9.4); heterozygous (+/-) brain (n=9, 8.2 + 2.5, range 3.3^13.0); and liver (n=9, 7.6 + 2.4, range 4.9^12.4), indicating a gene-dose e¡ect. A¡ected brain (n=6), liver (n=9) and kidney (n=3) manifested nil SSADH activity. Urine GHB and total GABA (umol/L) from +/+ and +/- mice were indistinguishable (GHB, n=12, 5.5^19.1; total GABA, n=5, 111^195), but were increased in two -/- mice (GHB, 1024 and 1350; total GABA, 446 and 707). GHB in +/+ and +/- mice tissue extracts overlapped (brain, 0.07^0.22 umol/g protein, n=11; liver, 0.02^0.19 umol/g, n=12) but were elevated in -/- mice (brain (n=2), 5.3 and 5.4; liver (n=5), 0.9^2.5 umol/g). Intervention in -/- mice with the GABAB receptor antagonist, CGP 35348, signi¢cantly improved survival time. This suggests that GHB acts at the GABAB receptor in -/- mice. The knockout model is valuable for exploring pathomechanisms and developing preclinical treatment strategies applicable to human SSADH de¢ciency.

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PRENATAL DIAGNOSIS (PND) OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH) DEFICIENCY: INCREASED ACCURACY USING DNA, ENZYME AND METABOLITE ANALYSES C. Jakobs1, B.M. Hogema1,2, M. Taylor2, S. Akaboshi2, R.B.H. Schutgens1, B. Wilcken3, S. Worthington3, G. Maropoulos4, M. Grompe2, K.M. Gibson2 Metabolic Unit, Department of Clinical Chemistry, Free University Hospital, Amsterdam1; Department of Molecular and Medical Genetics, Oregon Health Science University, Portland, OR, USA2; Biochem. Genet. Lab., New Children's Hospital, Parramatta, NSW, Australia3; Department of Chemical Pathology, Children's Hospital, Kyriakou, Athens, Greece4 Elucidation of the human SSADH gene sequence should enable DNA-based PND of SSADH de¢ciency (4hydroxybutyric aciduria). We report results of DNA-based PND for three families at-risk from SSADH de¢ciency (Table; horizontal columns representing families 1^3, respectively, top to bottom; CV, chorionic villi; AC, amniocytes; GHB, 4-hydroxybutyric acid in amniotic £uid (control 5 2.2 umol/L, n=30)). Proband Alleles G268E/W204X W204X/R412X G409D/G409D

Fetal SSADH Activity 5 4%, CV and AC Normal, CV 5 2%, CV

Fetal GHB 4.0 not done 1.1

Fetal Mutations both present in CV, AC, fetal DNA neither present, CV heterozygous, CV

Analyses in families 1 and 2 were consistent with an a¡ected and una¡ected fetus, respectively. In family 2, father and fetus shared a polymorphism (1 bp deletion, 75 bp upstream of exon 8) absent in the proband, verifying that proband and fetus inherited di¡erent paternal chromosomes. The CV sample for family 3 was delayed eight days in transit to Portland. Marker enzyme activity (propionyl-CoA carboxylase) was very low in CV extracts, suggesting sample deterioration. Amniocentesis revealed normal GHB concentration; DNA analysis in CV revealed G409D heterozygosity (further AC studies pending). DNA-based analysis represents an additional useful diagnostic tool for the accurate PND of SSADH de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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ON THE PATHOGENESIS OF OCHRONOSIS IN ALKAPTONURIA Beat Steinmann*, Peter R. Huber³ *Div. Metabolic & Molecular Pediatrics, CH-8032 Zurich, ³University Hospital, CH-4031, Basel

Alkaptonuria (MIM 203500) is a rare, autosomal recessive disorder caused by de¢ciency of homogentisic acid oxidase (EC 1.13.11.5), which leads to failure in splitting homogentisic acid (HGA) and increased levels of HGA in body £uids and urine. Deposition of HGA and/or its products lead to darkening of urine upon standing and of cerumen, and to black ochronotic deposits in sclerae, cartilage, heart valves and large arteries. It has been shown in vitro that collagen lysyl hydroxylase (EC 1.14.11.4) is inhibited by HGA in a linear, non-competitive way (Ki 120-180 mM), and that in organ culture biosynthesis of hydroxylysine-derived intermolecular collagen cross-links was inhibited in a dose-dependent manner (Murray et al., J Clin Invest 59:1071-1079, 1977). The authors speculated that their results explain the predilection of alkaptonuric complica-tions for these tissues containing collagens rich in hydroxylysine. To address the question of inhibited hydroxylation of lysyl residues by HGA we measured urinary total lysyl pyridinoline (LP) and hydroxylysyl pyridinoline (HP) by HPLC in three alkaptonurics (age 24 and 35, asymp-tomatic; and 45, symptomatic). LP/HP ratios were normal (0.16-0.18; normal 0.20 + 0.05), in contrast to those in 17 patients with Ehlers-Danlos syndrome type VI (MIM 225400) with docu-mented de¢ciency of lysyl hydroxylase (LP/HP = 6.0 + 1.0) and imply that under in vivo condi-tions the claimed inhibition of lysyl hydroxylase by HGA or its derivatives seem not to be opera-tive, at least not in the organs which are the main source of urinary pyridinolines. Thus, the patho-genesis of the disabeling arthrotic and life-threatening cardiovacular changes remains unexplained.

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QUANTIFICATION OF HOMOGENTISIC ACID IN URINE WITH FT-IR SPECTROSCOPY BS Jakobs1, AP Markus1, DW Swinkels1, RA Wevers2, JM Trijbels2, HL Willems1 Department of Clinical Chemistry/5641 and Laboratory of Paediatrics and Neurology2, UMC St Radboud, PO box 9101, 6500 HB Nijmegen, The Netherlands. Fourier Transform infrared (FT-IR) spectroscopy has been used as a new technique in the investigation of inborn errors of metabolism by studying alkaptonuria. The main advantages of FTIR are the simple, low cost measurements and the short time of analysis. Urine samples from randomly selected hospitalised patients were spiked with homogentisic acid (024 mM). Analysis was performed on a spectrum 2000 FT-IR spectrometer (Perkin Elmer) equipped with a liquid-nitrogen cooled mercury-cadmium-telluride (MCT) detector. Spectra were recorded from 4000-900 cm-1 and co-added from 200 scans (resolution 8 cm-1) using a horizontal ZnSe ATR accessory with 12 internal re£ections. The best chemometric PLS regression model, using full-cross validation, was obtained in the 15001200 cm-1 region, using 5 PLS regression factors, Multiplicative Scatter Correction (MSC) and second derivative calculation. The standard error of prediction (SEP) for homogentisic acid was 0.77 mM. Intra- and inter-assay CV's were 1.4% and 3.2%, respectively, for a urine sample with 29.4 mM homogentisic acid and 5.6% and 16.6%, respectively for a urine sample with 5.4 mM homogentisic acid. Analysis of a urine sample from an alkaptonuria patient using FT-IR and GC-MS resulted in a homogentisic acid concentration of 13.8 mM and 12.8 mM, respectively. This study has demonstrated the potential of FT-IR as an easy and cheap technique for the quantitative analysis of homogentisic acid at pathological concentration levels.

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CLINICAL, RADIOLOGICAL AND MOLECULAR STUDIES IN A GIRL WITH FUMARASE DEFICIENCY V.E. Kimonis1, K Shih2, R. Mandell2, V. Shih2. 1 Southern Illinois University Schl. of Medicine, Spring¢eld, IL, 2Massachusetts General Hospital, Boston, MA.,USA. Fumarase is an enzymatic component of the tricarboxylic acid, and exists as cytosolic and mitochondrial forms. Fumarase de¢ciency is a severe autosomal recessive disorder. Previous documented cases have been associated with early death, microcephaly or severe psychomotor retardation (Zinn et al. 1986, Christensen et al. 1986, Petrova-Benedict et al. 1987, Walker et al 1989, Gellera et al 1990, & Remes et al 1992). We report a 7-year-old Caucasian girl who presented at the age of 5 mo. with seizures. Brain CT/ MRI shows frontal and lateral ventricular dilatation and cerebral atrophy. Urinalysis revealed massive fumaric aciduria, and minimally elevated alpha-ketoglutarate, succinate, and malate. Cultured skin ¢broblast studies con¢rmed severe de¢ciency of fumarase [(0.9 nmol/min/mg protein (control 40.7)]. EEG shows slow background rhythms suggestive of encephalopathy and multifocal epileptogenic activity. She has focal and grand mal seizures treated with phenobarbitone and Dilantin. She has atypical language development, autistic features and an IQ of 35 however continues to make developmental progress. The growth parameters are normal. She has distinct dysmorphic features dolichocephaly, hypotelorism, bitemporal narrowing, prominent ears and a pointed chin. The family is non-consanguineous. Mutation analysis using RNA extracted from cultured lymphoblasts indicated that she is a compound heterozygote for novel mutations: a change of 492C to G resulting in amino acid change P131R (maternal) and a change of 779T to C (paternal) resulting in amino acid change L260S. Clinical, radiological and molecular data on the patient will be presented and fumarase de¢ciency will be reviewed.

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MITOCHONDRIAL ACETOACETYL-CoA THIOLASE DEFICIENCY T Fukao1, CR Scriver2, N Kondo1, and T2 de¢ciency collaboration working group. 1 Dept. of Pediatr., Gifu Univ. Sch. of Med., Gifu, Japan; 2Dept. of Hum. Genet.,McGill Univ., Montreal Children's Hosp. Res. Inst.,Montreal, Canada. Mitochondrial acetoacetyl-CoA thiolase (T2) de¢ciency is a disorder in ketone body metabolism and isoleucine catabolism. T2 de¢ciency is characterized by intermittent ketoacidotic attacks, which usually follow fasting or febrile events such as gastroenteritis. Over 40 patients were reported thusfar. Clinical heterogeneity was noted in the literature. We have analyzed molecular basis of this disorder at the protein and DNA levels and accumulated clinical informations by questionnaire. We identi¢ed 36 gene mutations in 38 T2 de¢cient families. Mutations were not identi¢ed in 7 mutant alleles thusfar (9%). Eleven of the 38 probands had homozygous mutations. Missense mutations were characterized by transient expression assay. This allowed us to evaluate residual activity of mutants. Among 19 missiense mutations characterized, 13 mutations were classi¢ed as null mutations, and the other 6 mutations retained some residual T2 activity. This indicates that 6 of 38 patients have mild mutations at least one of two mutant alleles. Severity of gene mutations could not predict clinical severity, hence, phenotype/genotype correlation is not clear in this disorder. T2 de¢ciency had a favorable outcome in all early diagnosed, early treated patients. We discuss possible factors which also a¡ect clinical severity in T2 de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SUCCINYL-CoA: 3-KETOACID CoA TRANSFERASE DEFICIENCY: PROGRESS IN MUTATION DETECTION AND FUNCTIONAL ANALYSIS Fukao1, G. A. Mitchell2, K.E. Orii1, X-Q. Song1, N. Kondo1 1 Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan; 2Department of Pediatrics, Service de Genetique Medicale, Hopital Ste-Justine, Montreal, Canada

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) is a key enzyme for ketone body utilization in extrahepatic tissues. SCOT de¢ciency is characterized by persistent ketosis and intermittent ketoacidotic crisis. Since there are no speci¢c metabolites in blood and urine enzyme assay is essential to establish the diagnosis. Only 14 SCOT de¢cient families have been reported to date. We have analyzed the molecular basis of this disorder at the protein and DNA levels. We have cloned the human SCOT gene which spans 4 120 kb, and established PCR conditions for all 17 SCOT exons. Mutation analysis at the genomic level is now possible. We have identi¢ed 7 gene mutations in 6 SCOT de¢cient patients thusfar. 5 missense mutations were characterized by transient expression assay. Four of the 5 had no detectable activity but one, V221M, retained some activity. This indicated that only GS05 (V221M homozygote), who interestingly had a mild clinical course reported to date, had detectable residual SCOT activity. In contrast, her ¢broblasts, as well as those of 5 other patients whose mutations produced no detectable SCOT activity, have 20-30 % residual activity in the clinical SCOT assay in whole ¢broblasts. Current ¢broblast assays overestimate SCOT activity, probably because other enzymes compete for substrate. Expression studies of mutant SCOT protein provide a more accurate estimate of true residual activity and will be important for establishing genotypephenotype associations.

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NEONATAL ONSET SUCCINYL-CoA: ACETOACETATE TRANSFERASE DEFICIENCY DIAGNOSED AT THE AGE OF 2 YEARS G. Sarbat1, G. Huner1, T. Baykal1, S. Donmez1, J.P.N. Ruiter2, R.J.A. Wanders2, M. Demirkol1 Dept. Pediatric Nutrition and Metabolism, Instanbul Faculty of Medicine, University of Istanbul, Istanbul, Turkey1. Academic Medical Centre, Univ. of Amsterdam, Dept. of Clinical Chemistry, Amsterdam, The Netherlands2

Succinyl-CoA: acetoacetate transferase de¢ciency, a rare ketolysis defect causes permanent ketosis 7 -year-old girl (MY) born to and episodes of ketoacidosis that may lead to death. We report a 2 12 consanguineous parents after an uncomplicated pregnancy. Two previous siblings were lost with tachypnea and convulsions during neonatal period. This patient presented with ketoacidosis at the second day of her life. She had six hospital admissions with severe episodes of vomiting and keotacidosis. Myoclonic convulsions started at the age of 18 months. At referral to our center, severe dehydration, acidotic breathing, mental retardation, microcephaly and strabismus were observed. Laboratory investigations showed metabolic acidosis (pH: 7.2, HCO3: 5.5 mmol/L) and massive ketonuria. Blood glucose, ammonia, lactate levels were normal with elevated 3-OH-butyrate + acetoacetate (4.18 mmol/L), low free fatty acids (0.13 mmol/L) and increased 3-OH-butyrate (1966 mmol/mol crea) in urine organic acid analysis. Enzymatic analysis in ¢broblasts demonstrated succinyl-CoA acetoacetate transferase de¢ciency (1.3 nmol/min.mg, controls mean:12.11 + 3.60). Moderate restriction of dietary protein and clonazepam therapy prevented ketosis, convulsions and ketoacidotic attacks with improvement in neuromotor development. These ¢ndings indicate that early diagnosis and treatment can prevent mortality and morbidity in succinyl-CoA acetoacetate transferase de¢ciency.

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ENZYMES OF KETONE-BODY UTILIZATION IN HUMAN PLATELETS JÎrn Oliver Sass1, Miranda Klosterhuber1, Walter Nussbaumer2, Wolfgang HÎgler1,2 1 UniversitÌtsklinik fÏr Kinder- und Jugendheilkunde, Sto¡wechsellabor, 2Zentralinstitut fÏr Bluttransfusion und Immunologische Abteilung, Innsbruck, Austria Measurement of activities of ketone-body using enzymes plays a key role in the diagnosis of potentially life-threatening ketolysis defects. Usually enzyme activities are determined in lymphocytes or cultured ¢bro blasts. In patients with the ketolysis defect succinyl-CoA: 3-oxoacyl CoA transferase (SCOT) de¢ciency, enzyme analysis can be impaired by rather high apparent residual activities. Only little is known about the ketone-body metabolism of platelets, altho ugh such cells can be easily obtained from peripheral blood. This has prompted us to compare activities of acetoacetyl-CoA thiolases (with and without added K+ ions) and SCOT in platelet and lymphocyte homogenates of 22 healthy humans. Mean ratios of thiolase activities with K+/without K+ were 1.10 (platelets) and 1.65 (lymphocytes), mean ratios of SCOT activity and thiolase activity with K+ 2.82 (platelets) and 1.27 (lymphocytes). Since mitochondrial methylacetoacetyl-CoA thiolase T2 is the only thiolase activated by K+ , our data indicate that the activity of T2 in platelets is rather low. In contrast, platelets showed much higher SCOT activity than lymphocytes. SCOT activities of di¡erent tissues have been reported to correlate well with their ketolytic ca pacities. Hence our results suggest that ketone bodies can contribute signi¢cantly to the energy supply of platelets, if compared with data from other tissues. It may be assumed that platelets are more suitable for SCOT assessment than lymphocytes, becau se less in£uence of background activities can be expected. This hypothesis could be tested with samples from patients with known SCOT de¢ciency.

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LETHAL NEONATAL PRESENTATION OF CPT I DEFICIENCY Invernizzi F, Donadio A1, Burlina AB2, Allievi S, Rimoldi M, Taroni F and Garavaglia B Div.di Biochimica e Genetica, Istituto Nazionale Neurologico ``C.Besta'' Milano, 1Div. di Pediatria, Arcispedale Santa Maria Nuova, Reggio Emilia, 2Div. di Pediatria, Universita© di Padova, Padova, ITALY Carnitine palmitoyltransferase I (CPT I) catalyzes the transfer of long-chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. CPT I exists as two di¡erent, tissue-speci¢c, isoforms: a liver-type (L-CPT I) and a muscle-type (M-CPT I), which are encoded by two distinct genes. Fibroblasts express only the L-CPT I form, while heart contains both isoforms. CPT I de¢ciency appears to be quite rare, as only 14 patients have been reported thus far. Apparently all of them had a defect of the liver isoform L-CPT I. The disease usually presents in infancy, with recurrent episodes of hypoketotic hypoglycemia triggered by fasting or intercurrent illness. Myopathy or cardiomyopathy have never been observed. We report a patient with CPT I de¢ciency died at 34 hours of life for a severe crisis of bradichardia and cardiorespiratory arrest. T.M. was the fourth child of consanguinous parents of Moroccan origin. He had a sister who died suddenly at 3 days of life. He was born at term after an uneventful pregnancy. He was well until 34 hours of life when he suddenly developed an untreatable episode of bradichardia. Despite pharmacological and mechanical intervention, he died of cardiorespiratory arrest. Postmortem showed microvescicular steatosis of the liver with no abnormality in heart and skeletal muscle. Tandem-mass spectrometry analysis of blood acylcarnitine pro¢le revealed high levels of free carnitine with no long-chain acylcarnitines. In cultured ¢broblasts a marked reduction of [9,103H] Palmitate and [9,103H] Myristate oxidation was found (12% and 15% of controls' mean, respectively). CPT II activity was in the normal range, while CPT I activity was 0.24 nmoli/min mg of protein (normal value 2.3þ0.9). To our knowledge, this is the ¢rst report of CPT I de¢ciency with a lethal neonatal presentation. Cardiac involvement seems to be the cause of death in our patient, although no heart abnormality was found at necroscopy. Heart muscle contains both L- and M-CPT I isoforms. However, while the muscle type is primarily expressed in the adult heart, the liver isoform is highly expressed before birth. It is therefore conceivable that a profound reduction of L-CPT I activity in the neonatal heart could have caused the severe phenotype in our patient.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MOLECULAR ANALYSIS OF HEPATIC CARNITINE PALMITOYLTRANSFERASE I DEFICIENCY (1): cDNA AND GENOMIC DNA ANALYSES OF INFANTS PRESENTING WITH REYE-LIKE ILLNESS Shigenori Yamamoto1,2, Masaki Kanazawa1,2, Atsushi Ogawa1, Masaki Takayanagi3, Akira Ohtake4, Yoichi Kohno1 1 Department of Pediatrics, Chiba University School of Medicine, Chiba, 2National Shimoshizu Hospital, Yotsukaido, 3Chiba Children's Hospital, Chiba and 4Department of Pediatrics Saitama Medical School, Saitama, JAPAN

Carnitine palmitoyltransferase I (CPT I) de¢ciency is a cause of disorders in mitochondrial fatty acid oxidation. Hepatic CPT I de¢ciency is characterized by recurrent episodes of Reye-like illness. CPT IA is the tissue speci¢c isoform of the enzyme present in liver and ¢broblasts. CPT1A has been revealed as the gene responsible for hepatic CPT I de¢ciency. In order to investigate the molecular basis of CPT I de¢ciency, cDNA and genomic DNA of the CPT1A gene were analyzed in three unrelated patients presenting with Reye-like illness. Multiple clones of the reverse-transcriptase polymerase chain reaction (RT-PCR) products of the CPT1A gene in ¢broblasts from each patient were screened and analytically compared to the wild-type. Point mutations were revealed at either 96T4G (Y32X) or 1079A4G (E360G) in patient 1 and at either 1425G4A (W475X) or 1494T4G (Y498X) in patient 2. Genomic DNA analysis con¢rmed all the mutations. The RT-PCR products of patient 3 were truncated and no other mutations were apparent. A four base-pair deletion, 20272028+2delAAGT, in a splicing donor site, was found in the paternal allele of patient 3, which likely caused the splicing error. The e¡ects of the mutations on the enzyme activity were investigated in another paper.

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CARNITINE PALMITOYLTRANSFERASE I DEFICIENCY: ACYLCARNITINE PROFILES IN BLOOD SPOTS ARE HIGHLY SPECIFIC R Fingerhut1, AC Muntau2, W RÎschinger2, E Lainka3, M Baethmann3, A Superti-Furga4, H Troxler4, B Liebl5, B OlgemÎller1, AA Roscher2 1 Newborn Screening Laboratory, Munich, 2Children's Hospital, University of Munich, 3University Children's Hospital, Essen, Germany, 4University Children's Hospital, ZÏrich, Switzerland, 5Public Health Screening Center, Oberschleissheim, Germany

In carnitine palmitoyltransferase-I (CPT-I) de¢ciency, long-chain fatty acids are not transferred from CoA to carnitine to form acylcarnitines (AC) and thus cannot enter the mitochondrion for betaoxidation. The blood acylcarnitine pro¢le in CPT-I has been considered unremarkable (except for elevated free carnitine as secondary e¡ect), as no pathologic acylcarnitine species are detectable. We have analysed newborn and high-risk screening blood spots of 3 CPT-I patients, including those of the ¢rst patient to be detected prospectively in MS-MS newborn screening. Palmitoyl- and stearylcarnitine concentrations were below the 0.5th , and free carnitine above the 99th centile, in all samples analyzed, including those from the neonatal period. Other long-chain AC were below detection limit. The molar ratio of free carnitine to the sum of palmitoyl- and stearylcarnitine was highly elevated. Retrospective analysis of our data base of 4150,000 acylcarnitine pro¢les, including those of patients with other disorders, with or without carnitine supplementation, did not reveal any sample giving an even similar pattern. We conclude that CPT-I can be identi¢ed both in newborn and in later blood spots by virtue of high carnitine, low long-chain AC, and speci¢cally by the free carnitine/palmitoyl- plus stearylcarnitine ratio. Because of the potentially life-threatening features of the disorder, and of the possible circumvention of the metabolic block by MCT feeding with good clinical outcome, we consider CPT-I an excellent candidate for inclusion in MS-MS based metabolic screening programmes.

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MOLECULAR ANALYSIS OF HEPATIC CARNITINE PALMITOYLTRANSFERASE I DEFICIENCY (2): EXPRESSION OF MUTATED CPT1A CDNAS IN COS-7 CELLS Masaki Kanazawa1,2, Shigenori Yamamoto1,2, Atsushi Ogawa1, Masaki Takayanagi3, Akira Ohtake4, Yoichi Kohno1 1 Department of Pediatrics, Chiba University School of Medicine, Chiba, 2National Shimoshizu Hospital, Yotsukaido, 3Chiba Children's Hospital, Chiba and 4Department of Pediatrics Saitama Medical School, Saitama, JAPAN In order to investigate the molecular basis of hepatic carnitine palmitoyltransferase I (CPT I) de¢ciency, immunoblot analysis of ¢broblasts from three unrelated Japanese patients and expression of mutant proteins have been performed. Patient 1 was compound heterozygous for 96A4G (E360G) and 1079T4G (Y32X) in the CPT1A gene; patient 2 was compound heterozygous for 1425G4A (W475X) and 1494T4G (Y498X). Patient 3 carried a 4 base-pair deletion, 20272028+2delAAGT, of a donor splice site on the paternal allele, but mutations on the maternal allele were not identi¢ed. Major RT-PCR products obtained from ¢broblasts from patient 3 were a 153 b a s e - p a i r d e l e t i o n ( 1 8 7 6 - 2 0 2 8 d e l ), a n 1 8 b a s e - p a i r i n s e r t i o n ( 2 0 9 7 - 2 0 9 8 i n sGTCTCTTCCACTTCTTCC) and a 3 base-pair deletion (2026-2028del). Immunoblot showed the CPT1A proteins were markedly reduced in ¢broblasts from patients 1 and 2, but not patient 3. We transiently expressed mutated CPT1A proteins in COS-7 cells with expression vectors containing either the three RT-PCR products from patient 3, or mutated CPT1A cDNA encoding E360G. Each expressed mutant was an unstable protein and/or had remarkably decreased CPT I activity. These results indicate that these mutations are responsible for CPT I de¢ciency.

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EVOLUTION OF PLASMA ACYLCARNITINES PATTERN IN A NEWBORN WITH PRENATAL DIAGNOSIS OF CPT II DEFICIENCY: EVIDENCE OF IMPROVEMENT UPON CARNITINE SUPPLEMENTATION M.C. Nassogne1, C. Jakobs2, M. Brivet3, M. Sharrard1, A. Garcia1, D. Rabier4, G. Touati1, J.P. Bonnefont5, J.M. Saudubray1 Dept of 1Pediatrics, 4Biochemistry and 5Genetic Biochemistry Unit Necker Enfants-Malades Hosp, & 3 Dept of Biochemistry, Biceªtre Hosp, Paris, France; 2Lab of Neurochemistry, Buenos Aires, Argentina There are controversial data about carnitine supplementation in long-chain fatty acids oxidation disorers due to the potential toxic e¡ects of accumulated long-chain acylcarnitines. We report the evolution of plasmatic acylcarnitines in a newborn with prenatal diagnosis of CPT II de¢ciency. The ¢rst child of the family presented with neonatal hypoglycemia, tubulopathy, and hepatomegaly. Diagnosis of CPT II de¢ciency was con¢rmed by a low enzymatic activity on ¢roblasts and an homozygous 1085 A?G transversion in the CPT II gene. This girl died at the age of 2 years from deshydratation. During the following pregnancy, a prenatal diagnosis con¢rmed CPT II de¢ciency. Immediately after birth, intravenous glucose infusion associated with MCT-enriched milk feeding by nasogastric drip was given. FAB-MS/MS on umbilical cord blood showed increased levels of C12C18 acylcarnitines, while organic acids pro¢le was still normal on the ¢rst urines. Levels of acylcarnitines remained high during the following days. At the age of 6 days, carnitine supplementation was given, leading to the decrease of C12-C18 acylcarnitines levels. At the age of 9 months, development is normal. This case illustrates ¢rst that plasmatic acylcarnitines pro¢le is already abnormal in umbilical cord blood allowing early diagnosis and secondly the bene¢t of carnitine supplementation on plasmatic acylcarntines levels.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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IMPAIRED SODIUM STIMULATION OF CARNITINE TRANSPORT IN PRIMARY CARNITINE DEFICIENCY Nicola Longo, Yuhuan Wang, Telly Meadows. Div. Medical Genetics, Dept. Pediatrics, Emory University, Atlanta GA, USA.Division of Medical Genetics, Department of Pediatrics ,EmoryUniversity,AtlantaGA,USA.

Primary carnitine de¢ciency is a recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia, skeletal and cardiac myopathy. It is caused by mutations in the sodiumdependent carnitine cotransporter OCTN2. Most natural mutations identi¢ed in this and other Na+solute symporters introduce premature termination codons or impair insertion of the mutant transporter in the plasma membrane. OCTN2 transporters containing a missense mutation (E452K) identi¢ed in a patient with primary carnitine de¢ciency retained normal Km toward carnitine, suggesting impaired maturation to the plasma membrane. However, E452K-mutant OCTN2 transporters tagged with the green £uorescent protein had normal membrane localization by confocal microscopy. Since OCTN2 is a Na+-solute symporter, we analyzed whether the E452K mutation a¡ected interaction of the transporter with sodium. The Km toward carnitine of the mutant transporter normally decreased to its physiological value at low concentrations of Na+, indicating that the E452K mutation was not close to the carnitine/sodium recognition site(s). By contrast, the E452K mutation increased the concentration of sodium required to half-maximally stimulate carnitine transfer (KNa) from the physiological value of 11.6 mM to 187 mM. Substitution of the E452 residue with Glutamine (E452Q), Aspartate (E452D) and Alanine (E452A) caused intermediate increases in KNa. The Vmax for carnitine transport decreased exponentially with increased KNa in these mutant transporters, con¢rming an essential role of the E452 residue in the transfer of the Na+carnitine complex. The E452K mutation is the ¢rst natural mutation in a mammalian cotransporter a¡ecting sodium stimulation of solute transfer.

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SUDDEN INFANT DEATH FOLLOWING PIVAMPICILLIN TREATMENT IN A PATIENT WITH CARNITINE TRANSPORTER DEFICIENCY Christensen E 1, Holm J 1, Hansen SH 2, SÖrensen N 3, Nezu J 4, Tsuji A 5, Skovby F 1 1) Department of Clinical Genetics, Rigshospitalet, Copenhagen, Denmark 2) Department of Forensic Pathology, University of Copenhagen, Denmark. 3) Department of Medicine, Landssygehuset, Thorshavn, Faroe Islands. 4) Chugai Research Institute for Molecular Medicine, Ibaraki, Japan. 5) Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan. A 14-month-old girl died unexpectedly after one day of illness. Autopsy showed a fatty liver. The urinary organic acid excretion dominated by saturated and unsaturated dicarboxylic acids as well as 3-hydroxydicarboxylic acids without signi¢cant ketone bodies pointed to a mitochondrial fatty acid b-oxidation defect. Pivaloylglycine was found in the urine indicating recent treatment with antibiotics containing pivalic acid. The concentration of carnitine in a blood sample obtained postmortem was very low (0.7 mmol/l). Although carnitine depletion could have been due to treatment with pivaloylesteri¢ed antibiotics a defect of the carnitine transporter was an alternative explanation. This disorder had been diagnosed previously in another patient from the Faroe Islands (population about 50.000). A search for mutations in the carnitine transporter gene SLC22A5 (coding for the transporter protein OCTN2) revealed homozygosity for an asn32ser mutation in both patients. Carnitine transporter de¢ciency has been reported in a case of sudden neonatal death (Rinaldo et al. J Pediatr 1997 131:304-5). Treatment with pivampicillin may be hazardous in patients with this disorder.

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FUNCTIONAL ANALYSIS OF MUTATIONS IN THE OCTN2 TRANSPORTER CAUSING PRIMARY CARNITINE DEFICIENCY: LACK OF GENOTYPE-PHENOTYPE CORRELATION. Nicola Longo1, Yuhuan Wang1, Franco Taroni2, Barbara Garavaglia2,. 1 Div. Medical Genetics, Dept. Pediatrics, Emory University, Atlanta, GA, USA, and 2Istituto Nazionale Neurologico C. Besta, Milano, Italy. Primary carnitine de¢ciency is an autosomal recessive disorder of fatty acid oxidation caused by mutations in the novel organic cation transporter OCTN2. This disease can present abruptly early in life with hypoketotic hypoglycemia or later in life with progressive skeletal myopathy or cardiomyopathy. The objective of this study was to determine whether the variation in phenotypic expression was due to di¡erent degrees of impairment of carnitine transport caused by di¡erent mutations. Fibroblasts from two patients with absent carnitine transport were homozygous or compound heterozygous for mutations producing premature STOP codons (R282X, Y401X, and 458X). Expression in CHO cells of missense mutations identi¢ed in other patients indicated that three of these (R169W, G242V, W351R) completely abolished carnitine transport, while two others (A301D and E452K) retained small, but signi¢cant carnitine transport activity (2-4% of control). Two mutations (G242V and W351R) a¡ected predicted transmembrane domains, while three others (R169W, A301D, and E452K) were located in putative intracellular loops. No correlation could be established between residual carnitine transport and severity of the phenotype or age at presentation. These results suggest that the variability in phenotypic expression in primary carnitine de¢ciency is most likely related to environmental factors, such as the supply of carnitine in the diet or fasting and infections, which increase energy demand and the requirement for fatty acid oxidation.

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3-METHYLGLUTACONIC ACIDURIA: ASSOCIATION WIH CARNITINE TRANSPORTER DEFECT M. Maradin1, K. Fumic¨1, V. Sarnavka2, D. Begovic¨2, Z. Mandic¨3, J. Vrdoljak3, I. Tein4, A.M. Lamhonwah4, I. Baric¨2 Clinical Institute of Laboratory Diagnosis1, Department of Pediatrics, University Hospital Center, Zagreb, Croatia2, Department of Pediatrics, University Hospital Osijek, Croatia3, The Hospital for Sick Children, University of Toronto, Canada4 3-Methylglutaconic aciduria represents a heterogeneous group of metabolic disorders characterized by increased excretion of 3-methylglutaconic acid. The di¡erential diagnosis includes 3-methylglutaconyl-CoA hydratase de¢ciency, Barth syndrome, Coste¡ syndrome, etc. We report the ¢rst case of 3methylglutaconic aciduria in a child with carnitine transporter defect. The girl presented at 6 months of age with vomiting and diarrhea followed by hypotonia, convulsions and hypoketotic hypoglycemic encephalopathy. Aminotransferases and creatine kinase were moderately and ammonia slightly elevated. Organic acids showed increased excretion of 3-methylglutaconic (69 mmol/mol creatinine) and 3-methylglutaric acid. The child was referred to our center where mild hypertrophic cardiomyopathy was noted. Leucine loading test resulted in a mild transient increased excretion of both acids. Total and free plasma carnitine were extremely low (2.08 and 1.15 mmol/L, respectively). Carnitine transport defect was con¢rmed in cultured lymphoblasts. Carnitine uptake was negligible throughout the entire range of carnitine substrate concentration. Following moderate protein restriction, carnitine, folate and thiamine therapy, the child underwent a complete clinical recovery. 3methylglutaconic aciduria decreased from 28^69 mmol/mol creatinine before therapy to 5^22 mmol/mol creatinine. Association of 3-methylglutaconic aciduria with carnitine transporter defect remains unclear but adds another potential etiology to be considered in di¡erential diagnosis of 3methylglutaconic aciduria.

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CLINICAL FOLLOW-UP AND MOLECULAR ETIOLOGY OF THE ORIGINAL CASE OF CARNITINE TRANSPORTER DEFICIENCY S. Cederbaum, K. Dipple, E. Vilain, M. Miller, S. Koo-McCoy, B.Y.L. Hsu, A. Ganguly, C. Stanley. Departments of Psychiatry, Pediatrics and Human Genetics, UCLA, Los Angeles; Division of Endocrinology, Children's Hospital of Philadelphia; Department of Genetics, Univ. of Penna Sch of Med.

A 25-year-old man, ill since age 3 months and having su¡ered 8 cardiac arrests was diagnosed with systemic carnitine de¢ciency at 3.5 years. Oral carnitine substantially repleted his tissue carnitine, eliminated the cardiomyopathy and has allowed an illness-free period of 22 years. A recent echocardiogram was normal. Hip x-ray showed some osteopenia. Carnitine uptake in his lymphoblasts was de¢cient when compared to controls (0.006 vs. 0.33 pmol/min/mg protein). Analysis of his plasma membrane carnitine transporter gene (OCTN2) revealed a homozygous frameshift mutation, 1027delT, in exon 4. The resulting polypeptide terminates after amino acid 262. Both his parents were heterozygous for the mutation. The deletion resulted in predominantly abnormal mRNA splicing with either a 13 or 19 bp insertion from the 5' end of intron 3. The 13/19 nt insertions were found in both parents, predominantly in cis to the deletion, and rarely seen with normal alleles from either parents or controls. Carnitine transporter de¢ciency is con¢rmed in this original patient with systemic carnitine de¢ciency. Long-term followup demonstrates the continued, and apparently complete, e¤cacy of carnitine repletion therapy. The disease causing mutation in the OCTN2 gene is homozygous despite the lack of parental consanguinity and results in an exaggeration of aberrant splicing at a cryptic donor site in the preceding intron.

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GFP-HUMAN HIGH-AFFINITY CARNITINE TRANSPORTER OCTN2 PROTEIN: SUBCELLULAR LOCALIZATION & FUNCTIONAL RESTORATION OF CARNITINE UPTAKE IN MUTANT CELL LINES WITH THE CARNITINE TRANSPORTER DEFECT Anne-Marie Lamhonwah and Ingrid Tein Division of Neurology, Dept. of Pediatrics and Dept. of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada M5G 1X8

Background: Individuals with the plasmalemmal high-a¤nity carnitine transporter defect present with progressive infantile-onset carnitine-responsive cardiomyopathy, lipid storage myopathy, recurrent hypoglycemic hypoketotic encephalopathy and failure to thrive. The carnitine uptake defect (CUD) has been documented in their cultured skin ¢broblasts, lymphoblasts and/or myoblasts. The cDNA encoding the high-a¤nity sodium-dependent human carnitine transporter, OCTN2, has recently been cloned. Objective: To use the green £ourescent protein (GFP) as a living marker for positively transfected cells in our expression studies of the high-a¤nity carnitine transporter OCTN2 cDNA in cell lines with CUD. Methods: Transfection of cell lines from twelve unrelated patients (nine ¢broblast and three lymphoblastoid) with GFP construct harbouring the wild-type full-length OCTN2 cDNA was done using LipoTAXI. Results & Conclusions: Transient and stable expression of the recombinant GFP-human carnitine transporter OCTN2 cDNA was surveyed, and transient transfection of the ¢broblast and stable transfection of the lymphoblastoid cell lines was achieved. There was a functional restoration of carnitine uptake in the transfected mutant cell lines, thereby con¢rming the identity of the transfected cDNA. In addition, we report the ¢rst demonstration of the subcellular localization of an in-frame fusion GFP-human high-a¤nity carnitine transporter OCTN2 protein in the plasma membrane by confocal laser scanning £uorescence microscopy. J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CODING REGION MUTATIONS ASSOCIATED WITH ABERRANT mRNA SPLICING IN PATIENTS WITH CARNITINE / ACYLCARNITINE TRANSLOCASE (CACT) DEFICIENCY B.Y.L. Hsu, V. Iacobazzi, Z. Wang, H. Harvie, R. Chalmers, J.M. Saudubray, F. Palmieri, A. Ganguly, C. A. Stanley. Children's Hospital of Philadelphia; Departments of Pediatrics & Genetics, University of Pennsylvania, School of Medicine, USA; Department of Pharmaco-Biology, University of Bari, Italy. CACT is an inner mitochondrial membrane carrier that transfers fatty acylcarnitines into mitochondria in exchange for free carnitine. CACT de¢ciency was ¢rst identi¢ed in a boy who died at 2 years of age of intractable heart and liver failure; most subsequent cases have also been lethal. This report describes the molecular defects in the ¢rst case and in two other unrelated infants with CACT de¢ciency. Patients #1 and #2 were of European and Chinese origin; #3 was from consanguineous Turkish parents. CACT activity was de¢cient in cultured skin ¢broblasts from all 3 patients as compared with normal ¢broblasts (0.004 þ 0.0002 vs. 0.70 þ 0.06 nmol/mg protein/min). Patient #1 was heterozygous for a paternal frameshift mutation, 120delT in exon 1, and a maternal splice site mutation, 10 T-4G in intron 2. Patient #2 was heterozygous for the ^10T-4G intron 2 mutation and a frameshift mutation, 362 delG in exon 3. Patient #3 was homozygous for a frameshift mutation, 306delC in exon 3. Analysis of cDNA from all three patients revealed aberrant transcripts with skipping of exon 3. Aberrant exon 3 splicing was also noted in the father of #1 with the exon 1 coding region mutation. Transcripts skipping exons 3 & 4 were also prominent in patient #2 and faint, but detectable, in patients #1 and #3. This unusual molecular defect of aberrant exon 3 splicing by distant mutations in CACT exons 1 and 3 de¢nes CACT de¢ciency in the ¢rst reported case and in two other a¡ected infants.

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MITOCHONDRIAL CARNITINE/ACYLCARNITINE TRANSLOCASE (CACT) DEFICIENCY: IDENTIFICATION OF NEW CASES USING A NOVEL ASSAY FOLLOWED BY MOLECULAR ANALYSIS OF THE CACT-GENE L. IJlst1, J.P.N. Ruiter1, W. Oostheim1, H.R. Waterham1, V. Iacobazzi2, F. Palmieri2, R.J.A Wanders1 1 University of Amsterdam, Academic Medical Centre, Depts. Pediatrics, Emma Children's Hospital, Clinical Chemistry and Neurology, Amsterdam, The Netherlands, 2University of Bari, Bari Italy Objectives: Mitochondrial carnitine/acylcarnitine translocase (CACT) de¢ciency has been recognized in an increasing number of patients. Only few centres are capable of performing identi¢cation of patients since determination of CACT-activity is notoriously di¤cult. We therefore developed a simple method based on the use of enzymatically synthesized [14C] acetylcarnitine to identify patients and subsequently performed mutation analysis to identify the molecular defect. Methods: The method involves incubation of selectively permeabilized ¢broblasts with [14C] acetylcarnitine followed by quanti¢cation of the [14C] CO2 produced. Results: The newly developed assay has been optimized in terms of incubation, concentration of acetylcarnitine, a.o. and subsequently validated in known patients. Using this assay new patients were identi¢ed and in the 10 patients we have collected, mutation analysis has been performed. In most patients aberrant cDNA species were found whereas only few point mutations were identi¢ed. The mutant cDNAs were expressed in S. cerevisiae to evaluate the consequences of the aminoacid substitutions identi¢ed: in all cases the activity of the carrier was severely reduced. Conclusions: Identi¢cation of CACT-de¢ciency in candidate patients is greatly helped by the newly developed assay, which can also be used for prenatal diagnostic purposes in cultured chorionic villous cells.

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IDENTIFICATION OF TWO NOVEL MUTATIONS IN THE CARNITINEACYLCARNITINE TRANSLOCASE (CACT) GENE IN A PATIENT WITH CACT DEFICIENCY Atsushi Ogawa(1), Shigenori Yamamoto(1), Masaki Kanazawa(1), Masaki Takayanagi(2), Shuji Hasegawa(3), and Yoichi Kohno(1). (1) Department of Pediatrics, Chiba University School of Medicine, 1-8-1 Inohana, Chuou-ku, Chibashi, Chiba 260-8670, Japan. (2) Division of Metabolism, Chiba Children's Hospital, 579-1 Heta-chou, Midori-ku, Chiba-shi, Chiba 266-0007, Japan. (3) Chiba City Institute of Health and Environment, 13-9 Saiwai-chou, Mihama-ku, Chiba-shi, Chiba 261-0001, Japan.

Carnitine-acylcarnitine translocase (CACT) transports acylcarnitines across the inner mitochondrial membrane in exchange for free carnitine, and is therefore an essential component within the fatty acid oxidation pathway. CACT de¢ciency is an autosomal recessive disease caused by mutations in the CACT gene. We have identi¢ed two novel mutations in the CACT gene in a patient with CACT de¢ciency. The ¢rst, a deletion mutation (146 del T), leads to premature termination and results in a very immature CACT protein. The second, a splicing mutation (261-10T4G), was found in the 3' end of the consensus branchpoint sequence (YNYYRAY: Y, pyrimidine; R, purine; N, any base) in the acceptor splice site of exon 3, and results in either skipping of exon 3 and 4, or of exon 3 alone, leading to truncation of the protein. Analysis of the transcript derived from the fragments ampli¢ed by RT-PCR revealed that this patient is a compound heterozygote for the 146 del T and 261-10T4G mutations. Each of these mutations is hypothesized to destroy the function of the CACT protein. Each of these mutations in the CACT gene is proposed to play a causative role in the disease.

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CLINICAL AND MOLECULAR HETEROGENEITY IN CARNITINE-ACYLCARNITINE TRANSLOCASE DEFICIENCY F. Invernizzi, B. Garavaglia, A. Ribes,* C. Dionisi-Vici,³ R. Parini,½ V. Iacobazzi,} and F. Taroni. Ist. Naz. Neurologico ``C. Besta'', Milan, Italy; *Inst. Bioquim. Clin., Barcelona, Spain; ³Osp. Bambino Gesu©, Rome, Italy; ½Univ. di Milano, Milan, Italy; }Univ. di Bari, Bari, Italy. The enzyme Carnitine-AcylCarnitine Translocase (CACT) is involved in the transport of long-chain fatty acids into mitochondria. We report here the clinical, biochemical and molecular features of 4 CACT-de¢cient patients from Italy and Spain. Pt 1 was an Italian baby who presented at 4 days of age with hypoketotic hypoglycemia, hypotonia and hepatomegalia. Despite a low-fat diet supplemented with carnitine, he died of untreatable cardiac arrhytmias at 6mo. Residual ¢broblast CACT activity was 5%. Molecular investigation showed a maternal novel missense mutation C397T (Arg-4Trp) in exon 4 and a paternal splice site mutation (IVS7+1C-4G) in intron 7. Pt 2 was an Italian boy with an apparently milder clinical picture. At 4mo he had severe generalized hypotonia, important liver enlargement and severe hypertrophic cardiomyopathy. Residual ¢broblast CACT activity was 6%. He is now 5y old with normal physical and psychic development, slight hypotonia and no hepatomegaly. Sequence analysis of genomic DNA demonstrated that the boy was homozygous for a single 47-bp deletion in intron 8 (IVS8del[4-50]) which was carried in heterozygous form by both parents. Pt 3 was a clinically asymptomatic 17-mo Spanish baby. Her elder brother had died suddenly at 2d. Residual ¢broblast CACT activity was 8%. She had low levels of plasma free carnitine and elevated long-chain acylcarnitines. She was a compound heterozygote for two mutations, a maternally-inherited doublet (insT159/delA163) in exon 2 that causes the double amino acid substitution of Gly-Thr with Trp-Ala, and a paternally-inherited point mutation C532T in exon 5 causing frameshift and premature translation termination. Pt 4 was a Spanish baby deceased at 4mo of cardiorespiratory arrest. His elder sister had died at 40 hours. He had severe hypoglycemia and hepatosplenomegaly. Residual ¢broblast CACT activity was 3%. Interestingly, he was found to be homozygous for the same point mutation in exon 2 carried by Pt 3, which might therefore be a relatively common mutation in CACTde¢cient patients of Spanish ancestry. Furthermore, this mutation appears to be associated with both mild and severe phenotypes. In conclusion, our data clearly indicate that CACT de¢ciency is a usually very severe metabolic disorder with clinical and molecular heterogeneity. Available enzyme data in ¢broblasts also show that there is a poor correlation of the clinical picture with the biochemical phenotype, which makes it necessary to carry out in vitro functional analysis of the mutations.

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BIOCHEMICAL, FUNCTIONAL CHARACTERIZATION, AND CLINICAL IMPLICATIONS OF THE COMMON POLYMORPHIC VARIANTS OF SCAD J. Vockley and T. Nguyen, Mayo Clinic, Rochester, MN, USA and M. Corydon and N. Gregersen, Aarhus University Hospital, Skejby Sygehus, Denmark. Short chain acyl-CoA dehydrogenase (SCAD) catalyzes the ¢rst intra-mitochondrial step in the boxidation of fatty acids. Two polymorphisms in this enzyme (Arg147Trp, and Gly185Ser) are associated with increased EMA excretion in urine. To characterize the biochemical consequences of these polymorphisms, the variant enzymes were expressed in E. coli and purifed. The variant enzymes were not stable unless co-expressed with the bacterial chaperonin GroEL/ES. The kcat/Km values of puri¢ed wild type, Arg147Trp, and Gly185Ser enzymes were 2044, 1915 and 599 mM-1 min-1, respectively, as measured with the ETF reduction assay, and 61.8, 11.4 and 12 mM-1 min-1, respectively measured with DCIP as the ¢nal electron acceptor. Spectrophotometric binding studies showed that both of the variant proteins could e¤ciently establish the charge-transfer enzymatic reaction intermediate. In contrast, near-UV circular dichroism (CD) studies revealed signi¢cant alterations in the observed spectrum of the variant enzymes at 292 and 309 nm. Furthermore, characteristic changes of the CD spectrum at these wavelengths induced by substrate binding were impaired in the variant enzymes, indicating a reduced ability to assume conformational changes necessary to release electrons to an electron acceptor. pH- and thermal stability of the Gly185Ser variant enzyme were signi¢cantly reduced compared to wild type, while these parameters were normal for the Arg147Trp variant. These ¢ndings suggest that the common SCAD polymorphisms may lead to clinically relevant alterations in enzyme function, and emphasize the need for additional in vivo studies on these variant enzymes.

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STUDIES OF CHAPERONES AND PROTEASES INVOLVED IN DEGRADATION OF MUTANT SHORT-CHAIN ACYL-COA DEHYDROGENASES Peter Bross, Vibeke Winter, Thomas J. Corydon*, Mette Wilsbech, Brage S. Andresen*, Lars Bolund*, Niels Gregersen. Research Unit for Molecular Medicine, Skejby Sygehus, Brendstrupgaardsvej, 8200 Ðrhus N, Denmark, * Institute for Human Genetics, Aarhus University, 8000 Ðrhus C, Denmark, and Danish Center for Human Genome Research. We have earlier shown that a disease-causing missense mutant (R22W) of short-chain acyl-CoA dehydrogenase (SCAD) is normally imported into mitochondria but rapidly degraded. In the present study we have synthesized wild type and R22W mutant SCAD polypeptides by in vitro transcription/ translation as probes for characterization of the chaperones and proteases involved in handling of proteins in the mitochondrial matrix. The polypeptides were imported into isolated rat liver mitochondria and production of tetrameric enzyme, complex formation with chaperones/proteases and degradation were analysed. At 37³C R22W SCAD is degraded more rapidly and much less tetramer is formed for the mutant protein compared to wild type. In addition, more of the R22W protein forms complexes with the chaperonin Hsp60 and probably with the chaperone Hsp70 and the protease ClpP. These experiments document that impaired tetramer formation and rapid degration of complexed folding intermediates are involved in the disease mechanism. The results furthermore suggests that the residual level of functional enzyme is dependent on the mitochondrial protein quality control system.

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SHORT CHAIN ACYL-CoA DEHYDROGENASE (SCAD) DEFICIENCY WITH COMPLEX NEUROPSYCHIATRIC MANIFESTATIONS AND ETHYLMALONIC ACIDURIA (EMA) SH Korman, A Gutman, N Gregersen1, C Vianey-Saban2, A Lossos. Hadassah University Hospital, Jerusalem, Israel, 1Aarhus University Hospital, Aarhus, Denmark and 2 Hoªpital Debrousse, Lyon, France.

SCAD de¢ciency is a rare disorder which may present as myopathy, seizures, developmental delay or neonatal metabolic acidosis. A 25-year-old man with chronic paranoid psychosis since age six was evaluated for a progressive cerebellar and pyramidal syndrome of 7 years duration. His identical twin had psychosis with pyramidal signs; their non-consanguineous parents were neurologically normal. Both twins had multifocal white matter involvement on brain MRI. The proband's CSF was positive for oligoclonal bands and muscle biopsy revealed lipid accumulation but normal respiratory chain enzyme activity. The patient, his twin, the father and the mother all had marked EMA (38-115, 40125, 42, 34 mmol/mol creatinine respectively, normal 57) with increased methylsuccinic acid excretion and elevated butyrylcarnitine on tandem MS examination of blood acylcarnitines. In the twins only there was a 50%-60% reduction in lymphocyte oxidation of 14C-butyrate in vitro. Speci¢c muscle SCAD activity in the proband was 6% of control. Both twins carried the SCAD 625 G4A EMA-susceptibility change on the allele from the mother (who was 625A homozygous) and the 319C4T disease-causing mutation on the allele from the father, who had the same 319T-625G/625A haplotype as the twins. This report expands the clinical spectrum of SCAD de¢ciency to include neuropsychiatric features and delayed CNS involvement. The phenotypic disparity between the genotypically SCAD-identical father and twins suggests that additional genetic &/or environmental factors are involved in the pathogenesis.

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IN VITRO DIAGNOSIS OF SCAD DEFICIENCY USING AN MCAD INHIBITOR Sarah P. Young1, D Matern1,2,3, J Vockley3, N Gregersen4, H-M Liu1, DS Millington1. 1 Dept. of Pediatrics, Duke University, Durham, NC; Depts of 2Laboratory Medicine & 3Medical Genetics, Mayo Clinic, Rochester, MN; 4Resarch Unit for Molecular Medicine, Aarhus University, Denmark.

Incubation of ¢broblasts with C16:0 and excess L-carnitine (LC) results in a build up of acylcarnitine species (ACs) in the culture medium that can be diagnostic of fatty acid oxidation disorders. The contribution of medium chain acyl-CoA dehydrogenase (MCAD) activity towards the metabolism of butyryl-CoA limits the usefulness of this assay for the diagnosis of short chain acyl-CoA dehydrogenase de¢ciency (SCADD). Studies using spiropentaneacetic acid (SPA; kind gift of Dr. Ch. Hoppel), a speci¢c inhibitor of MCAD, were carried out to determine whether signi¢cant accumulation of C4 -carnitine by SCADD ¢broblasts could be induced. Cells from patients diagnosed with SCADD by biochemical and molecular genetic testing were incubated with 100 mmol/L SPA, 200 mmol/L C16:0, 0 to 50 mmol/L C4:0 and excess LC in serum-free medium for 5 days and ACs in medium were analysed by tandem mass spectrometry. Inhibition of MCAD activity was indicated by a 10-fold accumulation of medium chain ACs over 5 days in all cell lines incubated with SPA as compared to no addition of SPA. Incubation with 50 mmol/L C4:0 caused a higher C4carnitine increase (range: 0.45-0.72 mmol/L; n=3) in the SCADD group compared to controls (0.100.15 mmol/L; n=3). The C4/C2 carnitine ratio was also higher in the SCADD group (range: 1.15-3.25; n=3) vs. controls (0.10-0.30; n = 3) suggesting that this ratio is a useful index to identify SCAD de¢ciency in an in vitro system when MCAD activity is inhibited.

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ISOLATED 2-METHYLBUTYRYLGLYCINURIA DUE TO SHORT-BRANCHED-CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY: IDENTIFICATION OF A NEW ENZYME DEFECT, RESOLUTION OF ITS MOLECULAR BASIS AND EVIDENCE FOR DISTINCT ACADS IN ISOLEUCINE AND VALINE METABOLISM B.S.Andresen 1,2, E. Christensen3, T.J. Corydon2, P.Bross1, B. Pilgaard4, R.J.A. Wanders5, J.N.P. Ruiter5, H. Simonsen6, V. Winter1, I. Knudsen1, L.D. Schroeder1,2, N. Gregersen1, F. Skovby3 Res.Unit f. Molec. Med.1 and Inst. of Hum. Genet.2, Aarhus University, Denmark2. Dept. of Clin. Genet., Rigshosp., Copenhagen, Denmark3. Dept. of Pediatr., Roskilde Amtssygehus, Denmark4. Dept. of Pediat., Univ. Hosp. Amsterdam, The Netherlands5. Statens Serum Inst., Copenhagen, Denmark6. Acyl-CoA dehydrogenase (ACAD) defects in isoleucine and valine catabolism have, so far not been proven enzymatically or genetically, and it is unknown if one or two ACADs are involved. We investigated a patient with isolated 2-methylbutyrylglycinuria and found no mutations in ACAD-8, but a 100 bp deletion in his SBCAD cDNA. Our identi¢cation of the SBCAD gene structure (11 exons, 420 kb.) enabeled analysis of genomic DNA. This showed that the cDNA deletion was caused by skipping of exon 10 due to homozygosity for a 1228G4A mutation in the patient. The mutation was not present in 118 control chromosomes. In vitro transcription/translation experiments and overexpression in COS cells con¢rmed the disease-causing nature of the mutant SBCAD protein, showed that ACAD-8 is an isobutyryl-CoA dehydrogenase and that both wild-type proteins are imported into and form tetramers inside mitochondria. Enzyme assay of patient ¢broblasts, using 2-methylbutyryl-CoA as substrate, con¢rmed the defect. Tandem MS analysis of the newborn blood spot from the patient showed a normal acyl-carnitine pro¢le. This ¢nding, together with our ¢nding of homozygosity for the 1228G4A mutation in the asymptomatic mother of the patient, may indicate that this enzyme defect is underdiagnosed. In conclusion, we report the ¢rst mutation in the SBCAD gene, show that it results in an isolated defect in isoleucine catabolism, and indicate that ACAD-8 functions in valine catabolism.

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BIOCHEMICAL AND GENETIC STUDIES IN A FAMILY WITH SHORT-CHAIN ACYLCOA DEHYDROGENASE (SCAD) DEFICIENCY Vreken P1, Bok LA2 Gregersen N3, Ruiter JPN1, Denis S1 Ijlst L1, Wijburg FA1, Waterham HR1, Wanders RJA1 1 Academic Medical Center, University of Amsterdam, Dept. of Clinical Chemistry and Div. Emma Children's Hospital, Amsterdam, The Netherlands; 2 IJsselmeerziekenhuis, Dept. Pediatrics , Lelystad, the Netherlands; 3 Dept. Molecular Medicine, Aarhus University Hospital, Denmark The index patient was the seventh child of healthy consanguineous parents (¢rst cousins) and born at 29 weeks gestational age after a pregnancy complicated by HELLP-syndrome. At birth the patient su¡ered from bronchopulmonal dysplasia and hepatosplenomegaly. Metabolic screening revealed a high ethylmalonic acid (EMA) excretion (4 100 mmol/mol creatinine) and high plasma butyrylcarnitine (4 3 mmol/L), suggesting SCAD de¢ciency. Screening of ¢ve sisters and the parents revealed EMA-uria and elevated butyrylcarnitine in a 20 year old sister and the father. Enzymatic SCAD analysis in ¢broblasts showed de¢cient values for the patient (0.10) and father (0.12), whereas activity in the mother was in the heterozygous range (0.34 (reference 0.4-0.8 nmol /min/mg protein)). Genetic analysis showed that the patient was homozygous for a 1138C4T mutation. The parents and two sisters were heterozygous for this mutation. In addition the father had a 625A allele, which in combination with the 1138C4T mutation explains the EMA-uria and low SCAD activity. Surprisingly, the sister with EMA-uria was also homozygous for the 1138C4T mutation, but clinically completely normal. At two years of age the patient has delayed growth, but is otherwise normal, illustrating the mild presentation of SCAD de¢ciency in this family .

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LACK OF EFFECT OF CARNITINE SUPPLEMENTATION AND A LATE EVENING MEAL IN SUPPRESSING LIPOLYSIS AND OCTANOATE PRODUCTION IN MCAD DEFICIENCY M.E.J. den Boer1, P. Vreken2, L.van Lint2, R.J.A. Wanders1,2, F.A.Wijburg1, Academical Medical Centre, Departments of 1Paediatrics, 2Clinical Chemistry, Amsterdam, the Netherlands

Most patients with Medium-Chain AcylCoA Dehydrogenase de¢ciency (MCADD) present with metabolic decompensation, consisting of hypoglycemia, hepatic dysfunction and encephalopathy, after a period of prolonged fasting, often in combination with an infectious disease. Therapy aims to suppress the FAO pathway by frequent meals and to increase urinary excretion of accumulating toxic medium-chain acylCoA esters as acylcarnitines by supplementation of carnitine. Especially octanoate can be raised and is probably related to the CNS-involvement. We studied the in£uence of a late evening meal (23.00 hr) and carnitine supplementation (100 mg/ kg/day) under 3 di¡erent conditions: 1.with carnitine and with late evening meal, 2.with carnitine, without late evening meal, 3.without carnitine, without late evening meal, in 4 MCADD patients (2-5 yrs). We measured glucose, FFA's plus octanoate and the acylcarnitine levels from immediately after dinner, until breakfast (19.00-07.00 hr). Glucose-levels remained stable (44 mmol/L) and the increase of FFA-, octanoate- and C8-carnitine-levels did not di¡er under the various testcircumstances. We conclude that in MCADD children, a late evening meal, as well as carnitine supplementation, does not play an important role in suppressing the production of potentially toxic metabolites by the MCAD de¢cient FAO pathway, during an overnight fast. Their role during a period of illness probably remains essential and both should be started at times of an infection.

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TANDEM-MS NEWBORN SCREENING DETECTS MILD MCAD DEFICIENCY WITH NEGATIVE PHENYLPROPIONIC ACID TEST M. Lindner 1, J. Zschocke1, A. Schulze1, S. Fiesel1, K. OlgemÎller1, G.F. Ho¡mann1, R.J.A. Wanders2, and E. Mayatepek1 1 Div. of Metabolic Diseases, University Children's Hospital, Heidelberg, Germany, and 2Lab. Genetic Metabolic Diseases, Dept of Paediatrics, University Hospital, Amsterdam, The Netherlands

Introduction: MCAD-de¢ciency (MCADD) is the most common defect of fatty acid b-oxidation. The characteristic presentation is hypoketotic hypoglycemia following ``metabolic stress'' in early childhood. Presymptomatic diagnosis is possible through tandem-MS newborn screening where patients display a characteristic acylcarnitine pro¢le. Oral loading with phenylpropionic acid (PPA) is widely used in clinical practice to con¢rm or discard the diagnosis. Patients and Results: We report on 4 children detected in newborn screening with abnormal acylcarnitine pro¢les indicative of MCAD. Urinary organic acid analyses were repeatedly normal. PPA loading tests with 25mg PPA/kg body weight resulted in normal excretion of hippuric acid without excretion of phenylpropionylglycine in all subjects. Enzyme studies showed residual MCADactivities between ``classical'' MCADD and heterozygotes (16%-27% of the mean of healthy controls). MCAD gene analysis revealed compound heterozygosity for the common K329E mutation and a novel mutation in three cases, and homozygosity for a novel mutation in the child of a consanguinous Turkish couple. Conclusion: Newborn screening by acylcarnitine pro¢ling identi¢es subjects with mild MCADD who otherwise show normal urinary organic acids and normal results after a PPA-loading test. The clinical relevance of mild MCADD remains to be determined.

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EPISODIC FASTING-RELATED SYMPTOMS IN A TODDLER WITH A HETEROZYGOUS DOUBLE MUTATION [K329E; I233T] IN THE MCAD GENE: COINCIDENCE OR MILD MCAD-DEFICIENCY? M. Lindner1, J. Zschocke1, A. Schulze1, S. Fiesel1, S. Karch2, G.F. Ho¡mann1, R.J.A. Wanders3, and E. Mayatepek1 1 Div. of Metabolic Diseases and 2 Dept of Paediatric Neurology, University Children's Hospital, Heidelberg, Germany, 3Lab. Genetic Metabolic Diseases, Dept of Paediatrics, University Hospital, Amsterdam, The Netherlands Introduction: MCAD-de¢ciency (MCADD) is the most common defect of fatty acid b-oxidation. It is recognised through analysis of organic acids in urine and acylcarnitines in dried blood spots. There is one common MCAD gene mutation (K329E); carriers are thought to be asymptomatic. Case report: We report on a 4 year old boy who presented with recurrent episodes of reduced consciousness after overnight fasting, relieved by food intake. Acylcarnitine pro¢les were clearly abnormal and indicative of MCADD during the acute episodes but were normal in the interval. Urinary organic acids were consistently normal. Mutation analysis in the MCAD gene revealed heterozygosity for a double mutant allele with K329E and a novel mutation, I233T. This allele was inherited from the father; no mutation was identi¢ed on the maternal chromosome. Enzyme studies showed markedly decreased MCAD-activity of 30% of the mean normal (heterozygous carriers: 4060 %) in the patient and in his father. Conclusion: Heterozygosity for [K329E; I233T] in our patient caused a reduction of enzyme activity greater than usually observed in MCADD carriers. This may have contributed to the development of clinical symptoms in our patient, although coincidence cannot be ruled out completely.

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LOW FREQUENCY OF THE G985A MCAD MUTATION IN BRAZIL Roberto Giugliani, Eduardo Lewis, Jurema F. de Mari, Cristina D. Castilhos and Eurico Camargo Neto DNA4 Laboratory and Medical Genetics Service, Hospital de Cl|¨ nicas de Porto Alegre, RS, Brazil email: [email protected] MCAD (medium-chain acyl-CoA dehydrogenase) de¢ciency is reported as one of the most common genetic metabolic disorders, with a frequency of 1:7000 births in some areas. Clinical symptoms include progressive lethargy, which could lead to coma. Death could occur in around 20% of cases. Up to 10% of cases of sudden death infant syndrome were attributed to MCAD de¢ciency by some authors. The point mutation which results in the change of adenine by guanine in exon 11 - position 985 (G985A) is found in around 90 % of the alleles of a¡ected patients, allowing the estimation of the frequency of MCAD de¢ciency by the molecular identi¢cation of this common mutation. We analysed 2,421 dried blood samples referred to the Neonatal Screening Centre (Porto Alegre, Brazil), through PCR of exon 11 of the MCAD gene and cleavage of the ampli¢ed product with the restriction enzyme StyI. This approach allows the detection of A985 bands in the normal subjects and of G985 bands in the a¡ected homozygous. No homozygous for G985 bands were detected, but we identi¢ed 6 heterozygous subjects (presenting A985 and G985 bands), a proportion of 1:402. This low frequency suggests that, if G985A is actually present in 90 % of alleles of patients with MCAD de¢ciency, this disorder would be extremely rare in Brazil (about one case in half million births). However, further studies, including MS/MS screening, are necessary to clarify if the MCAD de¢ciency is actually rare in Brazil or if the proportion of G985A alleles in a¡ected patients is lower than that reported for North America and Europe.

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FULMINANT HEPATIC FAILURE ASSOCIATED WITH MUTATIONS IN THE MEDIUM AND SHORT CHAIN L-3-HYDROXYACYL-CoA DEHYDROGENASE GENE Laurie K. O'Brien1, Piero Rinaldo2, Harold F. Sims1, Estella M., Alonso3, Joel Charrow3, Patricia M. Jones4, Michael J. Bennett4, Joseph J. Barycki5, Leonard J. Banaszak5, and Arnold W. Strauss1 1 Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA, 2Mayo Clinic and Foundation, Rochester, MN, USA, 3Northwestern University, Chicago, IL, USA, 4 University of Texas Southwestern Medical Center, Dallas, TX, USA, 5University of Minnesota, Minneapolis, MN, USA A patient who presented at age 3 with fulminant hepatic failure was considered likely to have mutations in the medium and short chain L-3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) gene based on ¢ndings of elevated medium chain fatty acid metabolites in plasma and urine. A liver biopsy revealed centrilobular necrosis and lipid accumulation. The patient received a living-related liver transplant and recovered. Genomic DNA was isolated from this patient, and each of the 8 exons of M/SCHAD was sequenced. Two mutations were found in this patient. Exon 1 G118A changes alanine-28 to threonine, and exon 2 C171A changes aspartate-45 to glutamate. Both mutations are in the conserved NAD-binding domain of the protein. The mutant proteins were expressed in bacteria and puri¢ed. The kinetic and dissociation constants of native M/SCHAD and both mutants were determined. The D45E mutation decreases the a¤nity of the enzyme for NAD+. These are the ¢rst reported mutations in M/SCHAD.

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A MOUSE MODEL FOR MEDIUM AND SHORT CHAIN L-3-HYDROXYACYL-CoA DEHYDROGENASE DEFICIENCY Laurie K. O'Brien1, Harold F. Sims1, Michael J. Bennett2, and Arnold W. Strauss1 1 Department of Pediatrics, Washington University School of Medicine, St. Louis, MO USA, and 2 Departments of Pediatrics and Pathology, University of Texas Southwestern Medical Center, Dallas, TX USA

Medium and short chain L-3-hydroxyacyl-CoA dehydrogenase (M/SCHAD) catalyzes the third reaction of the mitochondrial aª-oxidation spiral, the oxidation of 3-hydroxyacyl-CoA to 3-ketoacylCoA, for medium and short chain fatty acids. To create a model for M/SCHAD de¢ciency, exon 1 of the mouse gene was ablated by homologous recombination in mouse embryonic stem cells. No M/ SCHAD mRNA or protein is present in the homozygous knockout mice in any tissue examined, based on Northern and Western blotting. The homozygous M/SCHAD knockout mice have no enzymatic activity toward acetoacetyl-CoA and reduced activity toward 3-ketooctanoyl-CoA and 3ketopalmitoyl-CoA in extracts from liver, heart, skeletal muscle, kidney, brain, and ¢broblasts. Under normal conditions, the M/SCHAD knockout mice are healthy and viable. However, they are sensitive to metabolic stress. Upon fasting and exposure to the cold (4³C), wild-type mice 6 weeks of age can survive longer than 24 hours. The homozygous M/SCHAD knockout mice of the same age die within 10 hours, with lipid accumulation in the liver and kidneys. This phenotype is similar to that of a patient with fulminant hepatic failure and mutations in M/SCHAD and to mouse knockout models for other aª-oxidation disorders.

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MITOCHONDRIAL b-OXIDATION OF FATTY ACIDS IN THE RETINAL PIGMENT EPITHELIUM Tyni T, Eaton S, Pourfarzam M, Turnbull DM. Department of Neurology, University of Newcastle upon Tyne; Sir James Spence Institute of Child Health, Newcastle upon Tyne; Unit of Paediatric Surgery, Institute of Child Health, London, UK. Progressive pigmentary retinopathy leading to blindness is an important but unexplained clinical feature of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) de¢ciency. Pathogenic mechanisms of this manifestation remain unknown. Previous studies show that the retinal pigment epithelium (RPE) is a¡ected early in this retinopathy (1). We studied mitochondrial fatty acid oxidation in RPE-cells to clarify pathogenesis of the retinopathy. Mitochondrial b-oxidation was measured from cultured porcine RPE-cells by incubating harvested cells with U-13C hexadecanoate. The intermediates were analysed by tandem mass spectrometry. Acylcarnitine analysis showed normal chain-shortening of the labelled palmitate and the production of acetyl-carnitine. Both aand b-subunits of mitochondrial trifunctional protein (MTP) were identi¢ed in RPE-cells by immunoblot analysis with antibodies to the human MTP. LCHAD and long-chain 3-ketoacyl-CoA thiolase (LCKT) activities were detected in RPE-cells (LCHAD 31.55 mU/mg protein, LCKT 125.8 mU/mg) (2). RPE-cells also showed carnitine uptake capacity. Detection of mitochondrial boxidation in RPE-cells is a new ¢nding, which could partly explain the pathogenesis of LCHAD de¢ciency retinopathy 1. Tyni T et al. (1998) Ophthalmic pathology in long-chain 3-hydroxyacyl-CoA dehydrogenase de¢ciency. Curr Eye Res 17: 551-3. 2. Eaton S et al. (1998) Control of mitochondrial b-oxidation: sensitivity of the trifunctional protein to [NAD+]/[NADH] and [acetyl-CoA]/[CoA]. BBA 1429:230-8

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PLACENTAL FLOOR INFARCTION INDUCED BY FETAL LONG-CHAIN L-3HYDROXYACYL-COA DEHYDROGENASE (LCHAD) DEFICIENCY Dietrich Matern1, Bahig M. Shehata2, Prem Shekhawat3, Arnold W. Strauss3, Michael J. Bennett4, Piero Rinaldo1. 1 Department of Laboratory Medicine, Mayo Clinic, Rochester, MN; 2Children's Hospital of Toledo, OH; 3Department of Pediatrics, Washington University, St. Louis, MO; 4Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA Placental £oor infarction (PFI) is a rare complication of pregnancy of unknown etiology, associated with intrauterine growth retardation (IUGR), prematurity and fetal death. Histology reveals ¢brin deposits, which encase the villi, often compromising intravillous circulation causing villous necrosis. LCHAD is an enzyme of the fatty acid oxidation (FAO) pathway and its de¢ciency in a fetus is considered a cause of IUGR, prematurity, and maternal acute fatty liver of pregnancy. After birth, LCHAD de¢ciency is associated with episodes of hypoketotic hypoglycemia, liver disease, cardiomyopathy, and sudden death triggered by common infections. We report a patient born at 34 weeks gestation by emergent C-section. Examination of the placenta revealed PFI. He developed a hypoxic-ischemic encephalopathy and died unexpectedly at 8 months old in acute cardiovascular failure. Autopsy revealed fatty in¢ltration of the liver, heart, and kidneys. Biochemical analyses on postmortem liver suggested a long-chain FAO disorder, and molecular testing established a diagnosis of LCHAD de¢ciency, the patient being compound heterozygous for two mutations (G1528C, delA1095). The combination of yet another pregnancy complication and fetal LCHAD de¢ciency is unlikely to be a mere coincidence. Neonates born following pregnancies complicated by PFI should be routinely evaluated for LCHAD de¢ciency to prevent severe manifestations and to con¢rm this possible relationship.

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CLINICAL AND BIOCHEMICAL FEATURES OF TRIFUNCTIONAL PROTEIN DEFICIENCY A. Chakrapani1, S. Olpin2, J. Till1, R. Edwards1, L. Heptinstall1, M.A. Cleary1, J.H. Walter1, J.E. Wraith1, G.T.N. Besley1 Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester, M27 4HA, UK1 and She¤eld Children's Hospital, She¤eld, UK2

Very few cases of trifunctional protein (TFP) de¢ciency have so far been reported. We present a review of ¢ve cases recently diagnosed on our unit and con¢rmed by enzyme analysis. The complex clinical and biochemical presentation of this rare disorder can lead to considerable diagnostic di¤culties (table). CASE 1 Age at Presentation 5 days Clinical & biochemical features LA, HG, E, N Age at death 26 days Maternal 3rd trimester complications Liver dysfunction G1528C mutation Absent Urine organic acids Normal Acylcarnitine pro¢le Abnormal

CASE 2

CASE 4

CASE 5

1 day LA, HG, CMP 2 days

CASE 3 3 days LA, CMP, E, LD 5 days

3 months LA, HG, SA, N, M, E, LD 11 months

2 days LA, HG, CMP 2 month

HELLP syndrome Absent Equivocal Abnormal

Liver dysfunction Absent Lactate, no DCA Abnormal

Nil Absent Typical DCA Abnormal

Nil Absent normal Abnormal

LA = lactic acidosis, HG = hypoglycaemia, CMP = cardiomyopathy, E = encephalopathy, LD = liver dysfunction, SA = sideroblastic anaemia, N = neutopaenia, M = myopathy, DCA = dicarboxylic aciduria

CONCLUSION: TFP de¢ciency presenting in infancy is a potentially fatal condition with many features resembling a respiratory chain disorder. Maternal complications (HELLP) can occur in the 3rd trimester similar to isolated LCHAD de¢ciency. Urine organic acids and mutation analyses can be misleading but the plasma acylcarnitine pro¢le is more helpful. Con¢rmation of the diagnosis requires ¢broblast enzyme studies. Suspected cases should be treated until shown to be una¡ected.

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LACK OF MITOCHONDRIAL TRIFUNCTIONAL PROTEIN IN MICE CAUSES NEONATAL HYPOGLYCEMIA AND SUDDEN DEATH Jamal A. Ibdah1, Hyacinth Paul1, Yiwen Zhao1, Scott Binford1, Ken Salleng1, Mark Cline1, Michael J. Bennett2, Piero Rinaldo3 and Arnold W. Strauss4. 1 Wake Forest Univ. School of Medicine, Winston-Salem, NC, USA; 2Univ. of Texas, Dallas, TX, USA; 3 Mayo Clinic, Rochester, MN, USA; and 4Washington Univ., St. Louis, MO, USA.

Mitochondrial TFP is a hetero-octamer of 4 a and 4 b subunits that catalyze the ¢nal 3 steps of long chain fatty acid oxidation. Pediatric TFP de¢ciency is associated with cardiac and muscular abnormalities and may cause sudden unexplained neonatal death. We used gene targeting replacement strategy to generate a-subunit null allele. 87 litters (809 pups) of a cross between +/- mice were studied and compared to 37 litters (342 pups) of a cross between +/+ mice. -/- mice were TFPde¢cient by enzymatic assays with lack of expression of both the a and b subunits by Northern and Western blot analyses. All -/- mice developed hypoglycemia and su¡ered sudden neonatal death 6-36 hours after birth. -/- pups had statistically signi¢cant low birth weight compared to the +/- and +/+ pups. The -/- mice had biochemical features identical to those seen in human cases with TFP de¢ciency, particularly elevated C8-C14 urine dicarboxylic acids and C14-C18 serum acylcarnitines and free fatty acids. -/- mice had signi¢cant micro- and macro- vesicular fatty in¢ltration of the liver with necrosis and acute degenerative changes in the heart and diaphragm. In conclusion, de¢ciency of mitochondrial TFP in mice causes clinical and biochemical features similar to those in humans. Lack of TFP in mice is associated with intrauterine fetal growth retardation, neonatal hypoglycemia and sudden death.

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LIVER TRANSPLANTATION IN A PATIENT WITH THE MITOCHONDRIAL TRIFUNCTIONAL PROTEIN (TFP) DEFICIENCY G.T. Berry,1 M. Bennett,2 D. Millington,3 P. Jones,2 D. Piccoli,1 E. Maller,1 E. Rand,1 K. Oltho¡,1 E. M.Kaye,1 P. Kaplan,1 M. Harrington,1 A. Cahana;1 C. Vendetti,1 A. Strauss.,4 University of Pennsylvania School of Medicine,1 Philadelphia, PA, Children's Medical Center of Dallas,2 TX, Duke University Medical Center,3 Durham, NC and Washington University School of Medicine,4 St. Louis, MO A one year old male with 30% 3-hydroxy long chain acyl-CoA dehydrogenase and 33% long chain ketothiolase residual enzyme activities presented in the newborn period with biliary atresia and underwent a Kasai procedure. Two rare TFP b-subunit gene mutations were detected, arg 84 gly (exon 6) and arg 38 cys (exon 4). The infant developed cirrhosis associated with severe hypotonia due to myopathy and peripheral neuropathy. Acute episodes of obtundation were followed by prolonged periods of recovery. Liver transplantation was performed at 10 months of age. This resulted in a 90% reduction in blood C14, C16 and C18 3- hydroxyacylcarnitine content but no sustained decrease in free and bound plasma C14 and C16 3- hydroxy-long chain fatty acids. Probably largely related to perioperative complications, the patient was severely encephalopathic post-transplantation. The plasma 3-hydroxy C14 and C16 fatty acids have steadily-risen from being just above the normal range (50.5 mM) pre-transplant to at least 4 times normal possibly due to the stress of post-operative complications. We conclude that liver transplantation fails to eradicate the accumulation of circulating 3-hydroxy-long chain metabolites synthesized in extra-hepatic tissues in TFP de¢ciency.

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LONG-CHAIN 3-KETOACYL-COA THIOLASE KNOCKOUT MOUSE MODEL OF MITOCHONDRIAL FATTY ACID BETA-OXIDATION Raphael B. Merriman*, Wenning Qin, Harold F. Sims, Arnold W. Strauss. Division of Pediatric Cardiology, *Division of Gastroenterology, Washington University School of Medicine, St. Louis, MO 63110, USA. Background: Long-chain 3-ketoacyl-CoA thiolase (LKAT) catalyses the ¢nal enzymatic step in the fatty acid beta-oxidation spiral and comprises the beta subunit of the trifunctional protein (TFP) complex. Expression of LKAT is necessary for stable TFP formation. TFP de¢ciency is associated with skeletal myopathy and cardiomyopathy and sudden infant death. Objective: Generation and evaluation of a mouse model de¢cient in LKAT providing insight into mechanisms of regulation of fatty acid oxidation and disease pathogenesis. Methods: Targeted gene deletion of a 5274 base pair segment encompassing 1Kb of the intergenic region and exons 1, 2 and 3 of the beta subunit, and replacement with a neomycin cassette. Results: LKAT de¢cient mice were generated. Matings of heterozygote-deleted mice produced the expected ratios of normal, heterozygote (+/-) and de¢cient (+/-) o¡spring. De¢ciency of LKAT in -/and partial de¢ciency in +/- was con¢rmed by Western blot and Northern blot analyses. LKAT de¢cient mice appear to survive normally up to 5 months and are capable of reproducing. However, de¢cient pups weigh 40% less (p50.002) than +/- and +/+ littermates and the di¡erence persists into maturity. Early data suggest that the -/- mice die rapidly when exposed to cold. Conclusions: De¢ciency of LKAT is compatible with survival and initial phenotypic analysis indicates low body mass and cold exposure susceptibility.

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VALPROATE INDUCES IN VITRO ACCUMULATION OF LONG-CHAIN FATTY ACYLCARNITINES M. F. B. Silva 1,2, C. Jakobs3, M. Duran4, I.Tavares de Almeida1 and R.J.A.Wanders 2 1 C.P.M., Fac. de Farma¨cia da Univ. de Lisboa, Portugal; 2Dept. of Clin. Biochem., Univ. Hosp. Amsterdam; 3Dept. of Clin. Chem., Free Univ. Hospital, Amsterdam; 4Univ. Children's Hospital, Wilhelmina Kinderziekenhuis, Utrecht, The Netherlands.

Objectives: Valproic acid (VPA) can trigger an inhibition of mitochondrial fatty acid b-oxidation (dicarboxylic aciduria and reduced levels of ketone bodies) in humans and to induce secondary carnitine insu¤ciency. To elucidate the putative mechanisms of these VPA e¡ects, the pro¢le of acylcarnitine formation was studied in vitro using human skin ¢broblasts. Methods: Intact cells obtained from control individuals and from patients with previously de¢ned defects of mitochondrial fatty acid b-oxidation were incubated for 96h at 378C with 0.2 mmol/L [U-13C]palmitic acid, 0.4 mmol/L L-carnitine, and VPA (0, 0.5 and 2 mmol/L). After extraction, acylcarnitines were analysed and quanti¢ed by gas chromatography chemical ionisation mass spectrometry (GC-CI-MS). Results: VPA was found to impair the production of acetylcarnitine and to increase the levels of the long-chain acylcarnitines (from C10 to C16), both in control and in mitochondrial fatty acid boxidation de¢cient cell lines (VLCAD, LCHAD, MTP). Conclusions: These results show that VPA inhibits long-chain fatty acid b-oxidation. The observed accumulation of long-chain acylcarnitines may well explain the carnitine de¢ciency in patients given valproate, where the excretion of valproylcarnitine could not per se justify the fact. In addition, these ¢ndings may account for the pathogenic mechanism of hepatic toxicity such as steatosis, often observed upon VPA therapy.

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CLINICAL AND BIOCHEMICAL PHENOTYPES OF LCAD VERSUS VLCAD DEFICIENCY IN THE MOUSE P.A. Wood1, K.B. Cox1, P.S. Berger1, C.A. Pinkert1, J.R. Lindsey1, D. Matern2, J. Vockley2, D.S. Millington3, P. Rinaldo2, W.J. Rhead4 University of Alabama at Birmingham1, Mayo Clinic, Rochester, MN2, Duke University, Durham, NC3, University of Iowa, Iowa City4

Inborn errors of mitochondrial fatty acid oxidation remain an important, yet confusing, group of disorders to diagnose and treat. To address these problems we have created and characterized mouse models with a gene knockout for long-chain acyl-CoA dehydrogenase (LCAD) or very long-chain acyl-CoA dehydrogenase (VLCAD). Each de¢ciency results from a null mutation as demonstrated by immunoblot and enzyme activity analyses. Side-by-side comparisons were done of both enzyme de¢cient models. Both mutant lines had acute characteristics of fatty change in the liver, absence of ketone body generation, and hypoglycemia along with fatty change in the heart after fasting. All of these characteristics were much more severe in the LCAD -/- mice. Gestational losses (50%) occurred in the LCAD mutants, and based on in vitro studies, appeared to be due to failure to develop beyond the morula stage. Gestation was normal in the VLCAD mutants. Estrogen sensitive cardiac hypertrophy (50% enlargement) occurred in LCAD-/- males and a signi¢cant but milder hypertrophy was found in VLCAD-/- males. Young LCAD-/- males and females showed a similar hypertrophy that reduced as they matured on a soy-based diet. A phytoestrogen-free diet markedly exacerbated these changes. Finally, metabolite analyses of bile, blood, and muscle acylcarnitines, along with serum and liver fatty acid analyses demonstrated a clear chain-length speci¢c substrate accumulation of C12^14 substrates in LCAD de¢ciency and C16^18 substrates in VLCAD de¢ciency.

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CLEAR GENOTYPE/ PHENOTYPE CORRELATION OF VERY-LONG-CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY OBSERVED IN FIVE JAPANESE PATIENTS Yuichi Takusa (1, 2), Toshiyuki Fukao (2), Masahiko Kimura (1), Atsushi Uchiyama (1), Yukitoshi Takahashi (2), Kenji E Orii (2), Naomi Kondo (2), and Seiji Yamaguchi (1) 1) Department of Pediatrics, Shimane Medical University, Izumo, Japan; 2) Department of Pediatrics, Gifu University School of Medicine, Gifu, Japan VLCAD de¢ciency has been classi¢ed into 3 clinical phenotypes; a severe childhood form, a milder childhood form, and a myopathic form. Five patients with VLCAD de¢ciency have been identi¢ed in Japan up to now. We analyzed them at the protein and gene levels. Cases 1, 2 and 3 were the myopathic form, case 4 was the milder childhood form, and case 5 was the severe. Immunoblot analysis showed that VLCAD protein was detected with decreased amounts in ¢broblasts from cases 1, 2, and 3, whereas no signal for VLCAD in cases 4 and 5. DNA analysis revealed compound heterozygotes of A416T and R450H (case 1); K264E and M437V (case 2); A416T and c.1798delA (case 3); and P89S and IVS16-3delAA (Case 4). Transient expression analysis showed that the above 5 missense mutations retained some temperature-sensitive residual VLCAD activity (K264E4P89S4A416T4R450H4M437V4Mock). Both mutations in case 5, E130del and K382Q, retained null activity. Our results supported the current hypothesis that there is a clear correlation between clinical phenotype and severity of gene mutations.

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VLCAD DEFICIENCY IN RIBOFLAVIN-RESPONSIVE MULTIPLE ACYL-CoA DEHYDROGENASE DISORDER (RR-MADD) Lamantea E, Invernizzi F, Rimoldi M, Taroni F, and Garavaglia B. Divisione di Biochimica e Genetica, Istituto Nazionale Neurologico ``C.Besta'', Milano, Italy. RR-MADD is a genetic metabolic disorder characterized by impaired function of several FAD-containing dehydrogenases. Mitochondrial b-oxidation of fatty acids is particularly a¡ected. Although RR-MADD is one of the most frequently observed defects of fatty-acid oxidation, its etiology is still obscure. The clinical, morphological, biochemical, and physiological responses to oral ribo£avin supplementation are dramatic. In RR-MADD patients, multiple de¢ciencies of short-chain acyl-CoA dehydrogenase (SCAD), medium-chain acyl-CoA dehydrogenase (MCAD), and long-chain acyl-CoA dehydrogenase (LCAD) are usually reported. However, activity of the very-long-chain acyl-CoA dehydrogenase (VLCAD), a mitochondrial membrane-associated enzyme, has never been documented. Here, we report a biochemical investigation on VLCAD activity performed in muscle tissue of a RR-MADD patient, before and after ribo£avin supplementation. To determine VLCAD activity, 80 mg of frozen skeletal muscle were fractionated into a soluble matrix-containing fraction and a pellet containing mitochondrial membrane. Palmitoyl-CoA dehydrogenation was measured in both the soluble and the membrane fraction. Before therapy, membrane-associated palmitoyl-CoA (C16) dehydrogenation was 3.05 nanomol per min per mg of protein (controls 21.3þ6.8 n=8), while in the soluble matrixcontaining fraction the activity was 0.05 nanomol per min per mg of protein (controls 5.01þ1.02 n=8). Using lignocerylCoA (C24), a substrate with greater a¤nity for VLCAD, the activity in the membrane-associated fraction was 0.47 nanomol per min per mg of protein (controls 6.18þ2.48 n=8), and in the soluble fraction was 1.2 nanomol per min per mg of protein (controls 4.04þ1.25 n=8). The reduction observed in the soluble matrix-containing fraction is likely to be due to both the release of VLCAD during muscle storage and fractionation and LCAD contribution to C16/C24 dehydrogenation. After therapy, in the membrane fraction the activity with C16 raised to 14.7 (69%) nanomol/min/mg of protein, while in the soluble fraction it raised to 4.6 (92%) nanomol per min per mg of protein. Using C24, the activities in the membrane fraction and in the soluble fraction were 3.4 (55%) and 6.6 (100%) nanomol per min per mg of protein, respectively. These data clearly indicate that, similarly to other FAD-dehydrogenases of b-oxidation cycle, RR-MADD causes an impairment of VLCAD activity, which is reverted by ribo£avin supplementation.

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SIDS IN VLCAD DEFICIENCY: ACYLCARNITINES IN BILE VS. PLASMA M. Duran1, T.J. de Koning1, J. Dijksman1, M.M.J. de Barse1, D.P. Knoll,2 R.J.A. Wanders3, J.P.N. Ruiter,3 P. Nikkels1, B.T. Poll-The1, L. Dorland1, R. Berger1 1 Wilhelmina Children's Hospital, Lundlaan 6, 3584 EA Utrecht, The Netherlands, 2 School Chem.Biochem., Potchefstroom Univ., SA, 3AMC Dept. Clinical Chemistry, NL-Amsterdam VLCAD-de¢ciency, an often fatal defect of mitochondrial fatty acid oxidation, is usually diagnosed by acylcarnitine analysis of blood or plasma. We studied a girl who had died suddenly and unexpectedly at the age of ¢ve months. Vomiting and diarrhoea on the preceding day were the only signs. Hypoglycaemia was evident from a vitreous humor glucose of 5 1.1. mM. There were no signs of cardiomyopathy. The liver showed microvesicular steatosis and the lungs were in¢ltrated. Postmortem plasma and bile were analysed for acylcarnitines by FAB-MS and ES-MS-MS. Surprisingly plasma 14:1 acylcarnitine was normal (0.41 mM; controls 5 0.83). Bile acylcarnitines showed abundant 14:1, 14:2, 14:0, 16:1, and 16:0 species. Free carnitine in plasma was high (74 mM); organic acid analysis showed 3-OH butyrate to be 0.43 mM, FFA 1.99 mM with 14:1 fatty acid 159 mM. VLCAD in ¢broblasts was very low. Our results show that bile is a superior £uid for the diagnosis of long-chain fatty acid oxidation disorders. The analysis of plasma organic acids by GC-MS is still a very valuable tool.

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OBESITY AND TUMORS IN THE VERY-LONG-CHAIN ACYL-COA DEHYDROGENASE DEFICIENT MICE Vernat Exil, Harold F. Sims, Arnold W. Strauss. Dept. of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.

The aim of this study is to investigate a role for fatty acid b-oxidation defects in a novel obesity model in mice. Mice lacking the mitochondrial very-long-chain acyl-CoA dehydrogenase (VLCAD), just like humans, survive in the absence of physiologic stresses. The adult non-stressed mice become obese as early as 12 months of age on a diet containing 4 % fat. We compared 5 VLCAD +/+, 6 VLCAD +/-, and 14 VLCAD -/- littermate mice between 12 and 15 months of age. Median body weight of the VLCAD -/- mice was 55.6 gms (SE +/- 8 gms), compared with 46.8 gms (SE +/- 12 gms) for the VLCAD +/- mice and 36 gms (SE +/- 8 gms) for the VLCAD +/+ mice (p50.001). Upon sacri¢ce, histology revealed fatty in¢ltration of the heart and of the liver in the obese mice. Hepatic adenomas were found in 1 out of 6 VLCAD +/- and 4 out of 14 VLCAD -/- mice. Northern blot form VLCAD -/- brown adipose tissue (BAT) revealed dysregulation of the peroxisome proliferator-activated receptors (PPARs). There was 3 fold increase in the expression of PPAR-a, and a 50% decrease in the expression of PPAR-g in the (BAT) of the VLCAD -/- mice compared with VLCAD +/+ littermates. Importantly, heart and BAT expression of PPAR-â coactivator-1 (PGC-1) was increased 2 fold in the VLCAD -/- mice. Additionally these mice are cold sensitive. These results suggest that key components of energy homeostasis, a defense against obesity, are altered. These changes are likely components of compensatory mechanisms that may allow survival in these non-stressed mice but cause the obese phenotype.

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MOLECULAR BASIS OF GLUTARIC ACIDURIA TYPE II: IDENTIFICATION AND CHARACTERIZATION OF KNOWN AND NOVEL MUTATIONS: GENOTYPE^ PHENOTYPE CORRELATION Rikke Katrine Jentoft Olsen1, Brage Storstein Andresen1,2, Peter Bross1, Ernst Christensen3, Niels Gregersen1 Research Unit for Molecular Medicine, Aarhus University Hospital and Faculty of Health Science, Aarhus, Denmark1; Institute of Human Genetics, University of Aarhus, Aarhus, Denmark2; Metabolic Laboratory, Department of Paediatrics, Rigshopitalet, Copenhagen, Denmark3 Mutations in electron transfer £avoprotein (ETF) and ETF ubiquinone oxidoreductase (ETF-QO) are the molecular basis of Glutaric aciduria type II (GAII), and autosomal recessively inherited and clinically heterogeneous disease that can be divided into three disease phenotypes. We investigated the molecular^genetic basis for the disease in six unrelated patients with all three clinical phenotypes by analysis of the entire protein coding region of the ETFa, ETFb and ETF-QO genes using cDNA and genomic DNA. We report six new and one previously described disease-causing mutant alleles. Genotype^phenotype correlation showed that the ETF/ETF-QO genotype itself is determinant of the residual enzyme activity and thereby also for the clinical phenotype. Identi¢cation and characterization of an ETFb-D128N missense mutation in a patient with mild disease indicates that aberrant protein folding is an important mechanism in the molecular pathogenesis of GAII.

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CLINICAL HETEROGENEITY IN MAD - REPORT OF TWO CASES L.Diogo(1,2), P.Garcia(1), O.Rebelo(2), M.Grazina(2), I. Carreira(3), I.T.Almeida(4) (1) Hospital Pedia¨trico de Coimbra; (2) Centro de Neurocieªncias de Coimbra; (3) Faculdade de Medicina de Coimbra; (4) Faculdade Farma¨cia de Lisboa. Portugal.

Multiple acyl CoA dehydrogenase de¢ciency (MAD) is a clinically heterogeneous autossomal recessive disorder, caused by de¢ciency of electron transfer £avoprotein (ETF) or its dehydrogenase: ETF-ubiquinone oxidoreductase (ETF-QO). We report two boys with MAD. The ¢rst, born in 1995, presented generalised hypotonia and muscle hypotrophy, with episodes of adinamia and prostration after prolonged fasting, one with hypoketotic hypoglycaemia. At two years of age, hyperlactacidemia, no acidosis, organic aciduria, hypocarnitinemia and lipid storage myopathy were noticed. Mitochondrial respiratory chain was normal in muscle.The second boy, born in 1998, was £oppy, with no dysmorphic features, and had feeding di¤culties since birth. On admittion at 2 months of age because of respiratory distress, organic aciduria, typical of MAD, was observed. In spite of treatment, he died at 3 months of age after a multisystem illness with neurological, muscular, hepatic, myocardial and renal involvement. A respiratory chain de¢ciency was demonstrated in muscle and liver. Neither hypoglycaemia nor signi¢cant metabolic acidosis were found. ETF de¢ciency in ¢broblasts was proved in the ¢rst case and ETF-QO de¢ciency, in the second. Levels of organic acids and substrate conversion were compatible with mild MAD in both. This points to a complementary pathogenic role of mitochondrial respiratory chain impairment in the second child. (ETF studies were performed by Dr. C. Vianey-Saban Hoªpital Debrousse, Lyon, France).

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RESPIRATORY CHAIN DISORDERS: CAN WE ESTABLISH CONSENSUS DIAGNOSTIC CRITERIA? DR Thorburn1, MA Cleary1, A Boneh1, CW Chow1, X Dennett2, FP Bernier1,3 1 The Murdoch Childrens Research Institute and 2State Neuropathology Service, Melbourne, Australia, 3 Department of Medical Genetics, University of Calgary, Canada Diagnosis of respiratory chain (RC) defects is complicated by heterogeneity of features, and the absence of any de¢nitive abnormality in many patients. Each diagnostic centre has developed its own ``private'' diagnostic criteria that are often not transparent to others, raising concerns that ``other'' centres over- or under-diagnose RC defects. A consensus framework of conservative diagnostic criteria could decrease such concerns and provide guidelines for clinicians and laboratories. Walker et al (Eur Neurol 1996;36:260-267) proposed a detailed set of criteria (based on adult neurology patients) that classify patients into either de¢nite, probable or possible categories based on the presence of major or minor clinical, metabolic, enzymatic, pathological and DNA criteria. We evaluated the Walker criteria in a retrospective review of 139 consecutive paediatric patients seen and investigated for a suspected RC defect in our hospital. Two major di¤culties were identi¢ed: the lack of paediatric speci¢c features and general di¤culty in categorising essentially continuous data into just major or minor criteria. We modi¢ed the Walker criteria by incorporating paediatric features, allowing additivity of enzyme data and including an additional criterion of RC function. The modi¢ed scheme essentially concurs with our own diagnoses. We encourage other centres to consider this scheme for discussion at Workshop 5.

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MITOCHONDRIAL DYSFUNCTION WITH NORMAL RESPIRATORY CHAIN ENZYME ACTIVITY AND WITHOUT EVIDENCE OF MITOCHONDRIAL DNA MUTATION ACCOMPANIED BY REDUCED PDH ACTIVITY AND LOW FATTY ACID OXIDATION S.E. Olpin1, N.J. Manning1, J.R. Bonham1, S. Clark1, D. O'Neill1, M.S. Tanner2, I. Hargreaves3, J. Land3, S. Heales3, G. Brown4 and C.J. Harrison5 Department of Pathology1 and Paediatrics2, The Children's Hospital, She¤eld, UK and Department of Clinical Biochemistry, National Hospital for Neurology, London3, The Department of Biochemistry, University of Oxford4, Department of Paediatrics, Rotherham, DGH5 We present 5 a¡ected members of an extended multiply consanguineous Asian family presenting either as Leigh's disease or as Familial Erythrophagocytic Lymphohistiocytosis FEL. The index case presented at 14 months of age with developmental regression. A CT scan showed cavitation and P.M. ¢ndings con¢rmed Leigh's disease. Muscle respiratory chain complex activity was normal with no evidence of mitochondrial DNA mutation. Fibroblast PDH activity was low at 0.28 nmol/mg (0.7^ 1.1) as was fatty acid oxidation at 40% of controls. No mutation of the E1a subunit of the PDH complex could be found. Three other members of the extended family developed FEL. Bone marrow ¢broblasts of one patient gave PDH of 0.37 with low fatty acid oxidation (40%). The youngest sibling of the index case has low PDH and low fatty acid oxidation but is well at 6 months. One other well child has normal PDH and fatty acid oxidation. This case illustrates that negative mtDNA ¢ndings and normal respiratory enzyme activity in muscle do not necessarily exclude mitochondrial disease. The di¡erent presentations of Leigh's and FEL in association with low PDH and low fatty acid oxidation is noteworthy.

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FIBER TYPE DISPROPORTION IS CAUSED BY MITOCHONDRIAL RESPIRATORY CHAIN DISORDERS G. Enns, C. Hoppel, S. Schelley, N. Bass, N. Chung, K. Weisiger, S. DeArmond, D. Horoupian, M. Golabi, and S. Packman Stanford University, University of California San Francisco, and Case Western Reserve University Variation in size of type 1 and 2 muscle ¢bers occurs in nemaline myopathy and central core disease, and in disorders of heterogeneous etiology, including: congential hypotonia with type 1 ¢ber predominance (T1FP), and congenital ¢ber type disproportion (CFTD). The pathogenesis of T1FD and CFTD is unknown. We present data from 3 patients with variation in muscle ¢ber type size and proportion, to suggest that ¢ber type disproportion may be caused by abnormalities in respiratory chain complexes. Patient 1 is a 5-year-old boy with hypotonia, proximal weakness, and normal cognition. Muscle histology showed mildly increased lipid, and a predominance (95%) of type 1 ¢bers. EM showed increased number of variable-sized mitochondria. Muscle decyclubiquinolcyochrome c reductase and cytochrome c oxidase activities were reduced, indicating partial de¢ciencies of complexes III and IV. Patient 2 had hypotonia and little spontaneous movement from birth, requiring intubation and ventilation. Muscle histology showed predominance (85%) of type 2 ¢bers; EM was normal. Reduced activity of muscle succinate-cytochrome c reductase suggested a defect in the later components of complex III. Patient 3 has neonatal hypotonia and arthrogryposis, consistent with CFTD. Histology showed T1FP and variation in muscle ¢ber diameter. On EM there were multiple elongated branched mitochondria. Muscle rotenone-sensitive NADH cytochrome c reductase activity was reduced, indicating a complex I defect. We conclude that respiratory chain defects represent the etiology of at least a subset of ¢ber-type disproportion. We speculate that proper functioning of the mitochondrial respiratory chain may be required for the development of a normal distribution of type 1 and 2 muscle ¢bers. Respiratory chain analysis is recommended for patients with muscle histology suggestive of T1FP or CFTD, even in the absence of more typical features of disorders of oxidative metabolism.

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DIAGNOSIS AND INCIDENCE OF MITOCHONDRIAL RESPIRATORY CHAIN DISEASE IN ADULTS R.W. Taylor, C.M. Hayes, P.F. Chinnery, M.A. Johnson, R.S. Kler, R. McFarland, A.A.M. Morris and D.M. Turnbull Departments of Neurology and Child Health, University of Newcastle upon Tyne, UK Aim/Methods: In order to assess the important diagnostic criteria and incidence of mitochondrial respiratory chain disease in an adult population, we have reviewed the clinical features and laboratory investigations (histochemistry, biochemical assays and molecular genetic analyses) of the last 50 patients (416 years old) to present for the investigation of mitochondrial disease. Results/Conclusions: The majority of patients had clear neurological symptoms and signs (ophthalmoplegia, optic atrophy, ataxia, deafness etc) as their presenting feature, whilst other clinical ¢ndings such as diabetes were important. Despite Newcastle being the only centre in the North East of England, these features may re£ect the referral pattern. Abnormal muscle histochemistry (cytochrome c oxidase-negative ¢bres, subsarcolemmal accumulation of mitochondria) was a prominent ¢nding in the majority of cases and proved to be a more valuable indicator of mitochondrial disease than the ensuing biochemical investigations (measurement of individual respiratory chain complex activities). We have also been able to calculate, by retrospective analysis of data collected over a ten-year period, the minimum prevalence of mitochondrial DNA (mtDNA) disorders in adults of working age in the North East of England, and ¢nd this ¢gure (6.57/100,000) to be comparable to other neurological disorders such as amyotrophic lateral sclerosis and Huntington's disease.

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BIOCHEMICAL DIAGNOSIS OF RESPIRATORY CHAIN DEFECTS IN SMALL MUSCLE BIOPSIES F Wibrand, U Gaard, N Horn The John F Kennedy Institute, Glostrup, Denmark

Mitochondrial respiratory chain (RC) defects are now recognized as important causes of human disease. The diagnosis is often based on measurements of enzyme activities in isolated mitochondria from fresh muscle biopsies. We have established spectrophotometric assays for RC complexes I-IV that can be performed on small amounts (420 mg) of frozen muscle. The tissue samples were homogenized and centrifuged (5500 g for 40 s) to obtain low-speed supernatants, which contained about 70% of the mitochondria. The CoQ analogue decylubiquinone (and its reduced form), which is commercially available, was used as substrate in the assays for complexes I, II and III. The rotenone sensitivity in the complex I assay was 490%. RC enzyme activities were divided by the citrate synthase (CS) activity to minimize the e¡ects of variability in mitochondrial content. We analysed the activities in muscle samples from 11 patients (0-16 years) suspected of having RC defects. Reference intervals were established, retrospectively, from 9 of these samples. The RC activities in the ``controls'' were (in mU/mU CS): complex I, 0.31-0.57; complex II, 0.24-0.41; complex III, 1.08-2.23; complex IV, 2.26-4.57. The two other patients had reduced complex I activities of 0.05 mU/mU CS (13% of control median) and 0.09 mU/mU CS (28%), respectively, whereas the activities of complexes II-IV were within the reference intervals. Thus, these two patients are most likely su¡ering from isolated complex I de¢ciency. We believe that our methods are useful for the diagnosis of RC disorders.

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THE AUSTRALIAN EASTERN MEDITERRANEAN COMMUNITY HAS A HIGH PREVALENCE AND WIDE VARIETY OF MITOCHONDRIAL RESPIRATORY CHAIN (RC) DISORDERS Daniela Skladal1,4, John Christodoulou2, David Mowat3, Denise M Kirby1, David R Thorburn1 1 The Murdoch Childrens Research Institute, Melbourne, 2New Children's and 3Sydney Children's Hospitals, Sydney, Australia, 4Children's Hospital, Innsbruck, Austria In South Eastern Australia, RC defects are about 5 times more common in patients of Eastern Mediterranean origin than in the general population. We reviewed the onset, clinical features, enzyme defects and outcome in this group by studying the charts of 31 patients in whom a de¢nite diagnosis of an RC defect was established in our centre and whose parents are of Eastern Mediterranean origin. Results: There were 31 patients from 25 families (19 consanguinous), 20 boys and 11 girls. Age at presentation ranged from birth to 11 years, and 15 patients are currently alive. Clinical presentation was Leigh disease or Leigh-like in 9, lethal infantile mitochondrial disease or primary lactic acidosis in 15, one each of Alpers' disease, cardiomyopathy, myopathy, anemia in 2 and hepatopathy/ encephalopathy in 2 patients. The enzyme defect was RC complex I in 5, complex III in 4, complex IV in 15 and a combined RC defect in 7. No mtDNA or SURF1 mutations were identi¢ed. Conclusion: RC disorders can be common inherited disorders in highly consanguineous communities. In the Australian Eastern Mediterranean community the wide range of type of enzyme defects, clinical presentations, outcome and demographic characteristics implies that the high prevalence cannot be readily explained by a single founder e¡ect, and that multiple genetic defects are likely.

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BIRTH PREVALENCE OF MITOCHONDRIAL RESPIRATORY CHAIN DEFECTS IN CHILDREN Daniela Skladal1,2, Francois P Bernier1,3, Jane L Halliday1, David R Thorburn1 1 The Murdoch Childrens Research Institute, Melbourne, Australia, 2Children's Hospital, Innsbruck, Austria, 3Department of Medical Genetics, University of Calgary, Canada Objective: To estimate a minimal birth prevalence of RC defects in the paediatric population. Methods: In a retrospective analysis children born in the three South-Eastern states of Australia between 1987 and 1996, who have been investigated for possible respiratory chain defects, were evaluated. Data from the birth registries in these states were used for birth prevalence estimates. Results: Of 403 potential cases, 81 had a de¢nite diagnosis of RC defect. Age at presentation ranged from 0 to 47 (mean 6.2, median 3.7) months, age at diagnostic biopsy from 0 to 104 (mean 19, median 10.5) months, time between presentation and diagnostic biopsy from 0 to 98 (mean 12.8, median 4.6) months. The total data set predicts a minimum birth prevalence for RC defects in children of 4.7/100,000 (95% CI 3.2-5.0) (81 patients/ 1,706,694 births). Interpretation: Even though awareness of RC disorders and investigation methods have improved since 1987, not all a¡ected children from the more recent years would have been recognised so far. The minimum birth prevalence of 6.1/100,000 (95% CI 4.3-8.1) for the 42 patients born between 1991 and 1994 is therefore a more accurate estimate. Bearing in mind the incomplete ascertainment and inability to establish a de¢nite diagnosis in numerous patients, we regard a birth prevalence of 10/100,000 as a plausible estimate for RC defects presenting in childhood.

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CONGENITAL HYPERLACTACIDAEMIAS: CLINICAL PRESENTATION AND DIAGNOSTIC PROCEDURE OF 273 CASES A.Garcia-Cazorla, P. Delonlay, MC. Nassogne, G. Touati, JM. Saudubray Department of Metabolism. Hoªpital Necker-Enfants Malades. Paris. We studied 273 patients suspected of congenital hyperlactacidaemia referred to our service from 1977 to 1999. Acquired causes of lactic acidosis (LA) were ruled out, exams to detect multisystem involvement and metabolic tests were performed: 1-``Redox cycle'': glucose, lactate, pyruvate, lactate/pyruvate (L/P), ammonia, 3-hydroxybutyrate(3OHB), acetoacetate(AC), 30HB/AC, and free fatty acids, before and after meals.2- Plasmatic aminoacid chromatography. 3-Urinary organic acid chromatography. 4- Enzymatic and genetic studies. Results:We found 100 respiratory-chain disorders (RCD), 67 non-classi¢ed LA , 63 false metabolic LA, 15 Pyruvate dehydrogenase de¢ciencies (PDH), 15 multiple carboxylase (MC), 9 pyruvate carboxylase (PC) and 4 Krebs cycle (KC) de¢ciencies.Only considering true LA, a ¢nal diagnosis was made in 68% of the cases.. Clinical presentation of RCD was multisystemic in neonatal cases(nn) and mono-organic in the majority of non-nn. LA was higher in nn.Alanine and Proline were nearly constantly increased. Complex I de¢ciency was the most frequent diagnosis. PDH,MC,PC,KC presented with hypotonia and tachypnea in nn and acute intermittent neurological symptoms in the other ages. Highest LA were found in PC, hyperpyruvicemia with a normal or low L/P in PDH, increased citrulline, proline and lysine in PC and a particular pro¢le of organic acids (3OHpropionate, methylcitrate, 3-methylcrotonate) in MC. In conclusion LA associated with a clinical and biological characteristical pattern should lead to the suspicion of a particular enzymatic diagnosis.

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SEVERE CARNITINE DEFICIENCY AND SUCCESSFUL TREATMENT IN A PATIENT WITH THE MITOCHONDRIAL T8993C MUTATION E¨va Morava, Gabor Toth, Judit Bene, Viktor Farkas, Ma¨ria Molnar, Be¨la Melegh

We report on a patient with carnitine de¢ciency presenting with severe cerebellar ataxia, Leigh disease, mild mental retardation and muscular myopathy. Detailed laboratory investigations including an acyl-carnitine pro¢le have shown no metabolic alterations in serum and urine. Total, free and esteri¢ed plasma carnitine levels were signi¢cantly decreased. Molecular genetic studies revealed a familial T8993C pointmutation in the mitochondrial DNA (mt ATPase 6 gene). A second mutation in the carnitine transporter gene has been ruled out. The patient at the age of twelve years was unable to walk due to severe ataxia and muscular weakness. Since the introduction of continous carnitine supplementation at 50 mg/kg body-weight the child has been able to walk without support. He has required a carnitine dose adjustment due to sudden body growth manifesting in worsening ataxia and weakness, responding quickly to therapy. No carnitine de¢ciency has been reported so far in patients with the T8993C mitochondrial mutation. Based on the therapeutic e¡ectiveness of oral carnitine supplementation, we recommend a screening for carnitine de¢ciency in patients with the T8993C mtDNA deletion.

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DIAGNOSIS OF NEONATAL UBIQUINONE DEFICIENCY; A BIOCHEMICAL APPROACH S J R Heales1, S Rahman2, I P Hargreaves1 and P T Clayton2. 1 National Hospital & Institute of Neurology, London, UK. 2Great Ormond Street & Institute of Child Health, London, UK. In our laboratory, the analysis of muscle biopsies for suspected mitochondrial respiratory chain defects involves the enzymatic analysis of the components of the electron transport chain. Whilst complex I and IV are assayed separately, our initial investigations assay complex II+III together. The activity of the latter being dependent on the endogenous ubiquinone concentration. Recently, we investigated a 10 month old male child who had a history of seizures, lactic acidosis with evidence of mild cardiac and kidney abnormalities and cerebellar atrophy. The family history revealed that a sibling had died at 1 day and that there had been 5 ¢rst trimester miscarriages. Analysis of the muscle biopsy revealed normal complex I and IV activity but undetectable II+III activity. However, the separate analysis of complex II and III revealed normal activity. In view of these observations, ubiquinone de¢ciency was proposed. HPLC analysis of the muscle con¢rmed this suggestion by revealing a marked de¢ciency of ubiquinone (0.037, reference range 0.14-0.58 nmol/mg). Addition of ubiquinone to the muscle homogenate restored complex II+III activity and hence suggested the possibility of treatment. Consequently, ubiquinone therapy has commenced. Enzymatic analysis is useful for the identi¢cation of defects in one or more of the components of the electron transport chain. The initial assay of complex II+III is also helpful for the identi¢cation of ubiquinone de¢ciency and has enabled us to identify, what we believe to be, the ¢rst neonatal case.

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IMPROVEMENT OF ATP PRODUCTION IN COMPLEX I DEFICIENT FIBROBLASTS Ann Saada, Maskit Bar-Meir, Orly N. Elpeleg. Metabolic Disease Unit, Shaare Zedek Medical Center P.O.B 3235, Jerusalem 91031, Israel Mitochondrial respiratory chain defects are common inborn errors of metabolism. Among these, isolated complex I de¢ciency is the most frequent abnormality detected in Israel. The prognosis is generally grave and various agents, including coenzymes, free radical scavengers and lactate lowering agents have been administered to the patients, however clear bene¢cial e¡ect was not demonstrated in most cases. We investigated the e¡ect of various agents by measuring ATP production from the patient's digitonin permeabilized ¢broblasts using luminometry. This approach alleviated the need for large amounts of cells, enabling the performance of multiple repeats and including controls. Seven complex I de¢cient cell lines were compared; all had lowered ATP production with pyruvate (24-58% of control) while the ATP synthesis rate with succinate was normal. The e¡ect of carnitine, CoQ derivatives, uridine, dichloroacetate, ribo£avin, thiamin, and vitamin E added to the culture medium was studied. The preliminary results show that ribo£avin is markedly bene¢cial by improving ATP synthesis with pyruvate up to 3-fold, up to values approaching normal controls. Incubation with CoQ10 was also bene¢cial while the other agents proved ine¡ective. It is suggested that evidence from ¢broblasts may form a basis for therapy.

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ALPERS SYNDROME WITH TISSUE-SPECIFIC mtDNA DEPLETION IN A BOY FROM `IN VITRO FERTILISATION' J. Zeman, V. Scherhammer2, L. Wenchich, H. Hans|¨ kova¨, M. Elleder, V. Vobruba, H. Mayr2 and W. Sperl2 Departments of Pediatrics, Charles University in Prague1 and University in Salzburg2

Sporadic and familial occurrence of progressive infantile poliodystrophy (Alpers disease) was described. We present a male infant with Alpers disease born from `in vitro fertilisation'. He developed a progressive neuromuscular disease with liver impairment characterised by central hypotonia, visual disturbances, nystagmus, refractory epilepsy and severe psychomotor retardation. Profound cortical and periventricular atrophy developed between the 11th and 16th month of life. Progressive course of the disease, increased levels of aminotransferases (AST 2.3^3.5 mkat/l, ALT 1.6^2.6 mkat/l) and lactate (B-lactate 5^12 mM, controls 52.3; CSF-lactate 6^8 mM, controls 5 2.3; U-lactate 167^296 mmol/mol creat., controls 5100) with increased B-lactate/pyruvate ratio (20^24) suggested a disturbance in the mitochondrial energy generating metabolism. Liver biopsy showed massive multiplication of abnormal mitochondria. The activities of respiratory chain complexes I, I+III, II+III, III and IV in isolated liver mitochondria were low due to low protein amount of respiratory chain complexes I, III, IV, V in comparison with controls. The activities and protein composition of respiratory chain complexes were only mildly decreased in muscle mitochondria and normal in heart mitochondria. Southern blot analyses showed markedly reduced levels of mtDNA in liver and brain cortex (11 and 15% of the mean values in controls) but no mtDNA deletion was found in muscle. Other studies are necessary to answer the question, if mtDNA depletion may arise from `in vitro fertilisation'. Supported by grant GA-CíR 302/0648/99 and Czech^Austrian grant Kontakt ME 226.

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A NEW POINT MUTATION OF MITOCHONDRIAL ATPASE 6 GENE IN LEIGH SYNDROME Motohiro Akagi, MD, 1 Koji Inui, MD, 1 * Hiroko Tsukamoto, MD, 1 Norio Sakai, MD, 1 Takashi Muramatsu, MD, 1 Minoru Yamada, MD,1 Kouzi Matsuzaki, MD,2 Shintaro Okada, MD,1 Yuichi Goto, MD,3 Ikuya Nonaka, MD,4 1 Department of Developmental Medicine (Pediatrics), Osaka University, Graduate School of Medical Science, 2Suita Municipal Hospital, Departments of 3Mental Retardation and Birth Defect Research, and 4Ultrastructural Research, National Institute of Neuroscience, NCNP, Kodaira, Tokyo, JAPAN Leigh syndrome (subacute necrotizing encephalopathy) is a progressive neurodegenerative disorder in early infancy or childhood leading to death within months or years. We surveyed reported mtDNA mutations in ¢broblasts, muscles and peripheral blood leukocytes of 10 patients clinically diagnosed with Leigh syndrome and found a new T9176G mutation in the mtATPase 6 gene in a familial case of Leigh syndrome. A T-to-G transition at nucleotide (nt) 9176 in the mitochondrial adenosine triphosphate (mtATPase) 6 gene was detected in 2 siblings with Leigh syndrome. Heteroplasmy was observed in the leukocytes of their mother. The T9176G mutation changes a highly conserved leucine residue to an arginine in subunit 6 of the mtATPase gene and is maternally inherited. Another mutation in the same codon (T9176C) has been previously reported in Leigh syndrome. This gives strong support to the relevance of ATP-synthetase dysfunction in Leigh syndrome and the importance of leucine at the 9176 position.

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SUDDEN DETERIORATION IN A 5-MONTH-OLD BABY WITH COMPLEX II DEFICIENCY AND HOMOZYGOUS MUTATION IN THE 70-KDA FLAVOPROTEIN GENE (GLY555GLU) R VAN COSTER, J SMET, L DE MEIRLEIR, W LISSENS

Objective An increasing number of patients with de¢ciencies of the complexes of the respiratory chain were reported during last decades, most frequently of complex IV and complex I but rarely of complex II. The latter has 4 subunits all encoded by the nuclear genome: a 70-kDa £avoprotein subunit (Fp), a 27-kDa iron-sulfur protein (Ip) and two smaller membrane anchoring proteins. Only in the two siblings reported by Bourgeron (1995) and one patient reported by Parfait (2000) mutations were found, all located in the Fp subunit. The 3 patients had Leigh syndrome. Results We present here an additional patient with complex II de¢ciency in whom a homozygous mutation in the gene coding for the Fp subunit was found predicting a Gly555Glu substitution.The parents (¢rst cousins) were heterozygous for the mutation which was absent from controls.At 512 months of age, after a short episode of fever and rhinitis, the proband was noticed to have respiratory di¤culties for which reason she was admitted to the hospital. Soon after arrival sudden apnoea necessitated intubation. Maximal supportive measures notwithstanding, the patient died during this ¢rst day of hospitalisation. In skeletal muscle and cultured skin ¢broblasts a de¢cient activity of complex II was detected. Immunoblotting using speci¢c antibodies against the Fp and Ip subunits revealed severely decreased CRM of both subunits and of the whole complex after SDS-PAGE and Blue Native PAGE, respectively. Conclusion Complex II de¢ciency is a cause of sudden clinical deterioration and fatal outcome at young infantile age.

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PROFOUND AND GENERALIZED DEFECT OF CYTOCHROME-C-OXIDASE DUE TO A MUTATION OF SUBUNIT III PRESENTING WITH A MILD PHENOTYPE G. Uziel, N. Milani, E. Lamantea, P. Corona, V. Tiranti, M. Zeviani Istituto Nazionale Neurologico C. Besta , Milan, Italy We report the ¢rst frameshift mutation of the mtDNA gene encoding COX subunit III. The proband is a 11 year old girl her psychomotor development was normal in the ¢rst year but afterwards her motor and language development slowed down: she achieved indipendent gait at 2 years and her cognitive functions were mildly impaired. Since the age of 4 she developed a progressive spastic paraparesis with dystonic postures and ophtalmoparesis. In spite of the absence of recurrent vomiting her growth was below 3 %. The presence of high level of lactate in plasma (2594 mM) and CSF (4269mM) and bilateral lesions of putamina at the MRI prompted us to perform muscle and skin biopsy. In muscle and ¢broblasts a severe and isolated defect of complex IV was found. Sequence analysis of mitochondrial genes encoding for subunit I, II and III revealed a frameshift mutation in COX subunit III. In order to understand the consequence of the mutation on the assembly of the holoenzyme, we performed western blot analysis using anti-COXI antibodies on patient's mitochondrial proteins separated by 2D blue-native electrophoresis. Rusults showed an accumulation of earlyassembly intermediates of COX, while the fully assembled complex was absent. No mutation was found in the mtDNA of her mother's limphocytes. The clinical features of our patient were extremely less severe when compared to those encountered in other patients with similar biochemical defect e.i. patients with Leigh disease due to SURF 1 mutations.

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LEUKODYSTROPHY ASSOCIATED WITH A NOVEL MUTATION IN THE SURF1 GENE S Rahman1, RM Brown3, WK Chong2, CJ Wilson1 and GK Brown3 1 Metabolic Unit and 2Department of Radiology, Great Ormond Street Hospital, London; 3Genetics Unit, Department of Biochemistry, University of Oxford, UK. Mutations in the SURF1 gene have been demonstrated in a signi¢cant proportion of patients with systemic cytochrome oxidase (COX) de¢ciency. Patients with mutations leading to complete de¢ciency of the SURF1 protein have usually presented with typical clinical and pathological features of Leigh syndrome. A 2 year-old girl, the second child of consanguineous Bengali parents, presented with failure to thrive, global neurodevelopmental regression and lactic acidosis (plasma lactate 6.8 mmol/l, CSF lactate 7.65 mmol/l). Brain MRI demonstrated extensive leukodystrophy with corticospinal tract involvement. Muscle histology revealed non-speci¢c changes and no ragged red ¢bres. Muscle respiratory chain (RC) enzyme assays showed severe isolated COX de¢ciency (COX/citrate synthase ratio 0.004, reference range 0.014-0.034) and there was no detectable COX activity in cultured skin ¢broblasts. Sequence analysis of the SURF1 gene revealed a novel homozygous deletion of an AG couplet in exon 9 which is predicted to produce a truncated SURF1 protein. The mutation creates a BsrI restriction site and both parents were demonstrated to be heterozygous for the mutation. Leukodystrophy is increasingly recognised in patients with mitochondrial disease and the phenotype associated with SURF1 mutations should be extended to include this form of neuropathology. Mitochondrial RC enzymes should be assayed in patients with leukodystrophy and lactic acidosis and the SURF1 gene sequenced in those with isolated COX de¢ciency.

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RESPIRATORY CHAIN COMPLEX I TO IV DEFECTS: WHAT ARE REALISTIC REFERENCE RANGES? Denise M Kirby, C W Chow and David R Thorburn Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia

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Diagnosis of respiratory chain (RC) enzyme defects is based on comparison with a ``normal'' range, using either raw activities or enzyme ratios. Di¡erent centres use di¡erent criteria, and it is often not practical to match patients and controls exactly for all parameters such as age, type of anaesthesia, fresh or frozen samples and tissue pathology. In patients investigated for an RC defect, tissue pathology such as myopathy or hepatomegaly is often present either due to the defect or from another cause. We aimed to determine whether such pathology might widen the ``normal'' enzyme ranges such that ``sick'' tissues may be misdiagnosed as having an RC defect. Complexes I, II, II+III, III, IV and CS were measured in post-600g supernatants from frozen tissue samples from controls (6 healthy livers, 17 muscle biopsies from orthopaedic surgery patients), 20 muscle and 9 liver biopsies from children with inborn errors other than complex I to IV (mtDNA nt8993 mutations, PDHC, LCHAD, multiple carboxylase, Zellweger syndrome), and 16 livers from patients with (nonmetabolic) liver failure. These comparisons showed: (i) Reference ranges for 6 healthy livers were narrow (all values and ratios 4 60% of the mean). (ii) Patients with other inborn errors or ``sick'' tissues can have RC enzyme activities as low as *30% of the mean value. (iii) Complexes II+III, III and II are more labile than complexes I, IV and CS, so use of CS ratios alone may misdiagnose complex II and III defects. (iv) Residual RC enzyme activities & ratios of 25-50% may be regarded as supportive but not diagnostic of RC dysfunction.

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THE MOLECULAR BASIS OF RESPIRATORY CHAIN COMPLEX I DEFICIENCY IN CHILDREN Denise M Kirby, Hans-Henrik M Dahl and David R Thorburn Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia

Although complex I de¢ciency is the most commonly reported respiratory chain defect, the molecular basis has been identi¢ed in very few children. Mutations have been reported in only 4 of the 36 nuclear genes encoding complex I subunits so far. Mutations in the 7 mtDNA-encoded complex I subunit genes and transfer RNA genes, particularly the tRNALeu (UUR) gene, can cause a complex I defect. In our group of more than 60 complex I de¢cient children, mtDNA mutations account for the de¢ciency in 12 patients. Six have tRNA Leu(UUR) mutations (A3243G, G3242A, T3250C, T3271C and C3303T), and 6 Leigh syndrome patients from 5 unrelated families have mutations in complex I subunits (G14459A and G13513A). We anticipate that most patients have nDNA mutations, and have undertaken complementation analysis to de¢ne the number of genes responsible for complex I de¢ciency in our patients. Fibroblast cell lines expressing the defect were transformed with plasmids expressing the SV40 T antigen and resistance to either G418 or Hygromycin B. Transformed cell lines with di¡erent antibiotic resistance were fused and hybrids selected in medium containing both antibiotics. Phenotypic rescue was assessed by measuring complex I activity and comparing ATP synthesis with complex I- and complex II-linked substrates. Lack of complementation was observed with only 2 of the 8 most severely a¡ected cell lines. Unlike complex IV de¢ciency, it appears there is not a single major gene causing most cases of severe complex I de¢ciency, but multiple ``uncommon'' genes.

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MERRF/MELAS OVERLAP SYNDROME ASSOCIATED WITH THE MATERNAL A8344G tRNALys MUTATION OF MITOCHONDRIAL DNA. AN ARGENTINEAN CASE S Bacman1, A Paschini-Capra1, G Civallero1, N Guelbert1, A Giner-Ayala1, I Noher de Halac1, C Depetris-Boldini1, C Angaroni1, R Theaux1, C Argara·a2, R De Kremer Dodelson1. 1 CEMECO, Paediatric Department, School of Medicine, Children¨s Hospital. 2CIQUIBIC. National University of Co¨rdoba, Argentina. Most patients with MELAS syndrome harbour a point mutation at nt 3243 in the tRNALeu mtDNA gene, and 85% of patients with MERRF syndrome have point mutation at nt 8344 within the tRNALys gene. Although these syndromes are clinically very distinctive, there can be overlap between them. In this report, we describe a female case with a MERRF/MELAS overlap syndrome. Clinical features, since nearly 9 years, were progressively developed: myopathy, short stature, stroke-like episodes, deafness and lactic acidosis, commonly found in MELAS syndrome. Ataxia with wide base gait, generalized action myoclonus more prominent in the superior left limbs, neuropaythy and sporadic tonic-clonic seizures, characteristic of MERRF, were also observed. Brain MRI disclosed an infarct-like low density lesion on the right putamen. The proband died at age 16 of respiratory arrest. Urinary organic acids showed pro¢les of mitochondrial disease. Respiratory chain enzyme assays in muscle showed de¢ciencies in Complexes I and IV (51,5% and 27,7% of control means, respectively). A ¢rst muscle biopsy, at age 12, indicated normal histochemistry reactivity, while second biopsy two years later, presented few RRFs, succinate dehydrogenase increase, mild COX de¢ciency and abundant lipid depots. EM revealed clusters of giant mitochondria, others with scarce crestae and vacuoles. Molecular analysis by RFLP showed an heteroplasmic A to G transition at nt 8344 on the tRNALys gene (MERRF mutation). Phosphorimage quanti¢cation of PCR products revealed an 80% of mutated mtDNA in blood and 495% in muscle in the proband and 10% in mother¨ s blood. No mutation was detected in a female sibling. This ¢rst Argentinean case broadens the molecular aetiology of the MERRF/MELAS overlap syndrome, adding the MERRF mutation A8344G to the previous list.

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ALLELE FREQUENCY OF THE UNCOUPLING PROTEIN 1 BCL I POLYMORPHISM IN SIDS VICTIMS A. Fatemi +, C. Item*, S. StÎckler-Ipsiroglu*, O. Ipsiroglu*, W. Sperl ½, W. Patsch&, W. Strobl +, Depts. of Medical Chemistry+ and Pediatrics*, Univ. of Vienna, Depts. of Pediatrics½ and Laboratory Medicine&, Landeskrankenanstalten Salzburg, Austria. Disturbances of thermoregulation in brown adipose tissue (BAT) have been postulated as a cause of SIDS. The human uncoupling protein-1 (UCP-1), expressed in BAT, dissipates the transmitochondrial proton gradient as heat and plays a central role in energy homeostasis and thermogenesis. A Bcl I polymorphism at -3826 relative to the transcription start site of the UCP-1 gene is associated with alterations of energy homeostasis and with reduced UCP-1 mRNA (1). To determine whether the UCP-1 Bcl I polymorphism is associated with the occurrence of SIDS, we extracted DNA from the Guthrie cards of 53 Austrian SIDS victims and 54 controls. The clinical characteristics of the majority of these SIDS patients have been reported previously (2). A 350bp fragment of the UCP-1 promoter was ampli¢ed by nested PCR and digested with Bcl I. We found that the allelic frequencies for the Bcl I UCP-1 polymorphism did not di¡er between the SIDS group (0.65/035) and the control group (0.72/0.28, chi 2: 1.26, p: 0.26). In conclusion our data do not support an association of the UCP-1 Bcl I polymorphism with SIDS. Considering the limited number of subjects studied and the heterogeneity of SIDS, however, a role of UCP-1 in the pathogenesis SIDS can not be ruled out. 1) Esterbauer,H. et al. J Lipid Res 39:834-44 (1998) 2) Kohlendorfer, U.et al. Am J Epidemiol 15;147:960-8 (1998)

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GENOTYPICAL AND PHENOTYPICAL VARIABILITY OF MITOCHONDRIAL DNA DELETIONS: REPORT OF SIX CASES M. M. M. Grazina (1,2), L. M. Oliveira (2), L. Diogo (1,2,3), A. I. P. Fernandes (2), M. Godinho (2), I. M. Carreira (1), M. C. Maca¨rio (4), O. Rebelo (4), P. Santos (5); C. R. Oliveira (1,2,4) (1) Faculty of Medicine; (2) Centre for Neurosciences; (3) Paediatric Hospital; (4) University Hospital; (5) Histocompatibility Centre. Coimbra, Portugal.

Mitochondrial DNA (mtDNA) deletions/ insertions have been associated to mitochondrial syndromes, including mitochondrial myopathy (MM) with progressive external ophtalmoplegy (PEO) and Kearns-Sayre syndrome (KSS). We report six cases of mitochondrial disease (PEO: cases 1-4; KSS: case 5; MM: case 6) in whom we have found three types of mtDNA deletions, detected by long PCR, followed by sequencing. In all cases histological studies were compatible with mitochondrial cytopathy except for case 3. Concerning the PEO cases, the multiple deletions were only found in muscle (cases 2 and 3), while the 5kb deletion was found both in blood and muscle (case 1) and the 6kb deletion was found alone in blood and recombined in muscle (case 4). Cases 5 and 6 presented the 5 kb deletion not only in muscle, but also in ¢broblasts and lymphocytes, respectively. Results of biochemical analyses were heterogenous: cases 1 and 2 had no biochemical mitochondrial respiratory chain abnormalities detected, whereas cases 2 had complex IV de¢ciency in ¢broblasts, plus reduced activities of complexes I+III+IV in muscle and case 5 had both tissues a¡ected with complex IV de¢ciency. (Authors are grateful to Dr P. Rustin, Dr. A. RÎtig and Prof. A MÏnnich, from INSERMU393, Paris, France, for the collaboration).

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EPISODES OF HYPERVENTILATION AS A POSSBLE FACTOR INITIATING LEGH-LIKE BRAIN DAMAGE DUE TO SURF-1 MUTATIONS Pronicka E., Piekutowska-Abramczuk D., Popowska E., Karczmarewicz E., Pronicki M., Dembi·ska-Szyma·ska T. Children's Memorial Health Institute, Warsaw, Poland

Experimental data show that elevation of intracellular pH leads to severe lesions of brain cells. Acidi¢cation of intracellular £uid by accumulation of lactate may compensate an e¡ect of respiratory alkalosis. Increased serum pH, low pCO2 and low HCO3 associated with hyperlactataemia (sometimes incorrectly called ``acidosis'') were reported in children with Leigh disease. The aim of the study is to check if initial respiratory alkalosis is characteristic for patients with Leigh disease due to SURF-1 mutations. Up to now all 5 examined DNA samples (out of 15 quali¢ed for molecular study because of Leigh fenotype and generalised COX de¢ciency) were found to have mutations on one or both alleles (772delCA, 859delCT, 387C4G, 770insAG, 718T4C). Analysis of clinical course of 6 a¡ected children from 5 families carrying the SURF-1 mutation con¢rmed that episodes of hyperventilation and hypocapnia were observed at the onset of the disease (pH 47.400, pCO2 530 mmHg, HCO3 518 mmol/l). Respiratory alkalosis were associated with elevation of lactate level in serum and cerebrospinal £uid and alkalisation of urine. In our hypothesis - devastating e¡ect of recurrent elevations of intracellular pH (often provoked by stress) may lead to irreversible brain damage of the patients with COX de¢ciency due to SURF-1 mutations. Any stress should be avoided in these patients.

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A CASE OF SPORADIC INFANTILE HISTIOCYTOID CARDIOMYOPATHY CAUSED BY THE 8344 (MERRF) mtDNA MUTATION HD Vallance1, G Jevon1, MD. Brown2. Dept of Pathology, Children's & Women's Health Center of B.C., Vancouver, Canada1. Center for Molecular Medicine, Emory University, Atlanta, USA2. The 8344 A4G mitochondrial DNA (mtDNA) mutation is best known for the MERRF phenotype (myoclonic epilepsy, myopathy and ragged red ¢bres). We describe an infant with the 8344 A4G mtDNA mutation who presented with failure to thrive and died suddenly and unexpectedly at eleven months of age. The autopsy revealed a histiocytoid cardiomyopathy, di¡use steatosis of the liver and bilateral retinal hypoplasia. Electron micrographs of cardiac myocytes showed striking mitochondrial hyperplasia, dispersing the sarcomeres. Special stains of frozen heart muscle showed an absence of cytochrome C oxidase in many of the myocytes. Both Complex I and IV were reduced in cardiac muscle; Complex I: 16 nmol/min/mg protein (reference range 79 ^ 292), Complex IV: 240 nmol/min/ mg protein (reference range 1372 ^ 3075). DNA was extracted from cardiac muscle and liver tissue. Both tissues were homoplasmic for the 8344 mtDNA mutation. As far as we know, this is the ¢rst description of the 8344 mtDNA mutation presenting as a sporadic case of infantile cardiomyopathy. Unfortunately the mother was not available for testing to determine if this mutation was a de-novo germline event or maternally inherited. There was no relevant family history, the mother was in good health and has subsequently had a healthy child. This favours a spontaneous mutation in the germ line (mosaicism) or in a single oocyte as the etiology in this case.

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NOVEL DISORDERS ASSOCIATED WITH AN INCREASED MUTATION FREQUENCY IN THE mtDNA CONTROL REGION M Ito, T Higashimoto, ST Le, D Chaudhari and RG Boles. Childrens Hospital Los Angeles, CA USA. We used temporal temperature gradient gel electrophoresis (TTGE) to assay the mtDNA control region (mtDNA-CR) in blood for heteroplasmy in 85 children with idiopathic neuromuscular or multi-system disease and lactic acidosis, and in 100 controls. We found heteroplasmic, single nucleotide transitions in the hypervariable regions (HV1&2) of the mtDNA-CR in 11 a¡ected children and in 0 controls. HV1 homoplasmic single nucleotide transitions were 10 fold more frequent in the 11 cases than in 11 haplogroup-matched controls, indicating that the defect was maternally transmitted over at least a few generations. Children with mtDNA-CR heteroplasmy demonstrated a variable phenotype characterized by transient non-neurological tissue dysfunction with resolution by age 3 years, and/or static and variable neuromuscular disease which can be fatal. Cardiomyopathy, cyclic vomiting and common birth defects were often present. The apparent inheritance pattern is maternal or sporadic in cases with clinical improvement, and their mothers and siblings, but not their fathers, have mtDNA-CR heteroplasmy. Two neonatal-onset fatal cases show apparent autosomal recessive inheritance, and, in one, mtDNA-CR heteroplasmy is present in both parents. In the latter case, mtDNA-CR sequences varied among various tissues in the proband. Our cases likely su¡er from at least 2 novel disorders: a maternally-inherited variant with broad phenotypic expression, and a severe, early onset, autosomal recessive variant. Pathology is likely mediated by an increased mtDNA mutation rate, possibly secondary to faulty mtDNA repair.

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A NOVEL MUTATION IN THE MITOCHONDRIAL CYTOCHROME B GENE ASSOCIATED WITH COMPLEX III DEFICIENCY S Haut1,A.Slama1 ,T Billette de Villemeur 2,A Guiochon-Mantel 3,A.Boutron1 ,A.Legrand1, M.Brivet1. 1-Laboratory of Biochemistry;3-Laboratory of molecular Biology AP-HP Hopital de Biceªtre . 2Department of Pediatrics, AP-HP Hopital Armand Trousseau, Paris, France.

We report on a patient with growth retardation, IgF1 de¢ciency and abnormalities in the white matter on cerebral MRI. An isolated mitochondrial respiratory chain complex III de¢ciency was found in blood lymphocytes and skin ¢broblasts. The sequence analysis of cytochrome b which is the only mitochondrial DNA encoded subunit of complex III revealed an homoplasmic novel mutation at the nucleotide 15498. This G to C transition, resulting in the substitution of a highly conserved Gly to Asp (AA 251) was absent in over than 100 controls. This amino-acid change is located in the region of the interaction of ubiquinon, between the transmembranes helix F1 and F2 of the cytochrome b protein. The G15498A mutation was also found to be homoplasmic in the mother's lynphocytes and ¢broblasts and complex III was found to be de¢cient in ¢broblasts. Nevertheless the mother haven't clinical signs. Our data may suggest that this mutation is pathogenic but is not present in all tissues of the mother.

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CLINICAL, BIOCHEMICAL AND GENETIC STUDIES IN TWO PATIENTS WITH AN MNGIE SYNDROME A.Slama1 , C Lacroix 2, V Plante 2, D Crenn 4, S Haut1, M.Brivet1,A.Legrand1. A GuiochonMantel 3 1-Laboratory of Biochemistry;2-Department of neurology, 3-Laboratory of molecular Biology AP-HP Hopital de Biceªtre; 2-Department of gastroenterology, AP-HP Hopital Bichat, Paris, France. The MNGIE syndrome or mitochondrial neurogastointestinal encephalomyopathy is a fatal and rare autosomal recessive disease characterized clinically by gastrointestinal dysmotility, cachexia, ophtalmoparesis, ptosis, peripheral neuropathy and leucoencephalopathy. The genetic defect has been recently located in the thymidine phosphorylase (TP) gene resulting in an impairment of mitochondrial DNA replication or maintenance. Two 23-year-old patients, a man and a woman were referred for investigation of a severe neuropathy, with a Charcot Marie Tooth like presentation. Both patients did not present with ophtalmoplegia; they experienced intestinal malabsorption associated with diarrhoea and vomitting. Muscle biopsy showed a neurogenic atrophy, a di¡use decrease of staining with oxidative enzymes and Gomori's trichrome and many COX-negative ¢bers. Multiple respiratory chain complex de¢ciency was observed for both patients in muscle homogenate and multiple mt DNA deletions were found by long range PCR analysis. Thymidine phosphorylase activity was dramatically reduced in lymphocytes, indicating that a TP gene abnormality was probably the cause of the MNGIE syndrome. Sequence analysis of the TP gene found that both patients were composite heterozygote. The ¢rst patient had two unreported mutations (A1453G and T2306C). The second patient had two other mutations (A3371C already reported and G3868C not yet reported). In both cases the parents were found t o be heterozygote for one mutation.

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MITOCHONDRIAL DISEASE PRESENTING AS LEUKODYSTROPHY E. M. Kaye, M.D. Biochemical Genetics, Children's Hospital of Philadelphia Three children presenting with leukodystrophy within the ¢rst 6 months of life were found to have mitochondrial dysfunction. Case 1 presented at birth with increased tone and poor feeding, developed seizures by one month, and apnea at six months. The child deteriorated with episodes of severe rigidity and hyperthermia and died at 15 months of age. MRI showed abnormal thalamic signal, di¡use leukoencephalopathy, and atrophy. Case 2 was well until four months of age when failure to thrive developed. Hypotonia followed by hypertonia emerged and the child died by 13 months of age. MRI at 5 months of age revealed a markedly abnormal white matter with cavitary lesions in the centrum semiovale. Serum and CSF lactate levels were increased from 3.6-6.8 mmol/L. Case 3 developed a failure to thrive at 4 months and died by 6 months of age. MRI at 4 months showed severe white matter changes consistent with a leukoencephalopathy. Serum lactates were consistently increased and an elevated lactate peak was identi¢ed on brain MRS. None of these children demonstrated the typical MRI ¢ndings of mitochondrial disease such as increased T2 weighted signal within the basal ganglia. All of these children were thought to represent a typical leukodystrophy until the lactate and other biochemical studies con¢rmed a mitochondrial disease. On isolated mitochondrial studies of muscle, Case 1 was found to have Complex I de¢ciency, Case 2 a Complex III de¢ciency, and Case 3 a defect within the PDH and alpha-ketoglutarate complexes. These cases suggest that mitochondrial disease should be considered in children with di¡use, cavitary white matter lesions on MRI and be con¢rmed with serum or CSF lactate levels and brain MRS.

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CLINICAL COURSE OF SAGUENAY-LAC ST-JEAN CYTOCHROME OXIDASE DEFICIENCY (SLSJ-COX), A RETROSPECTIVE STUDY Annie Janvier (1), Charles Morin (2), Brian Robinson (3), Nana Lee (3), Marc De Braekeleer (4), Marie Lambert (1), Jacques Lacroix (1), Grant Mitchell (1). [(1)Dept Pediatr, Ste-Justine Hosp, Montreal, Canada; (2) Clin de Pe¨diatrie, Chicoutimi, Canada; (3) Biochem Genetics, Hosp Sick Children, Toronto, Canada; (4) U Bordeaux, France.] SLSJ-COX (OMIM 220111) is felt to be a genetically-homogeneous autosomal recessive COX de¢ciency with a strong founder e¡ect, a common chr 2 haplotype and a ~5% carrier rate in SLSJ. 53 potential cases were reviewed. The diagnosis was accepted in 32, using a standardized score including: COX assay, SLSJ ancestory, known SLSJ-COX relatives, acidotic or neurologic crises, Leigh disease and elevated blood or CSF lactate. Several had neonatal signs (hypotonia, 14/28; jitteriness 6/27; tachypnea 12/27; septic workup, 9/32) and mean bicarb was 14.5 mM (n=5). Acidotic crises (i.e. bicarb512 mM) occurred at 0-312 mo (n=48; median, 14 mo, mortality of the ¢rst crisis, 13/27, 48%). Some pts survive to adolescence. Acidotic crises may feature tachypnea (38/ 48, 79%) and acute pulmonary edema (26/48 crises), often misdiagnosed as asthma. Hyperglycemia 415 mM and transaminases 4700 are poor prognostic signs. Purely neurologic crises (stroke-like episodes) occur (n=4). Viral prodromes often preceed crises (43/48, 84%). Patients have a typical morphology, hypotonia 28/30 (93%), tremor 14/28 (50%), strabismus 10/29 (34%), nystagmus 5/29 (17%) and psychomotor delay (median ages: sitting, 12 mo (n=12; range 5.5-16), walking, 23 mo (n=23, range 11-36); pronouncing ``mama'' & ``papa'', 31 mo (n=11; range 15-38), all delayed 490th Denver developmental percentiles). Between crises the predominant blood gas pro¢le was partially compensated respiratory alkalosis (median pH, 7.43+0.03, bicarb 18.2+2.0 mM; pCO2 27.3+4.0) with lactate 3.0+1.2 mM, n=153. CSF lactate (4.35+0.99 mM, n=20) was a more sensitive marker than blood lactate.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PHENOTYPIC DIFFERENCES BETWEEN T8993C AND T8993G MUTATIONS OF MITOCHONDRIAL DNA IN LEIGH SYNDROME Vilarinho L1, Pereira C1, Nogueira C1, Lea¬o Teles E2, Calado E3, Santos M4, Barbot C4 1-Unidade Biologia Cl|¨ nica, Instituto Gene¨tica Me¨dica Jacinto de Magalha¬es, Porto; 2 Servic°o de Pediatria, Hospital S. Joa¬o Porto; 3 Servic°o de Neuropediatria, Hospital D. Estefaªnia, Lisboa; 4 Servic°o de Neuropediatria, Hospital E. C. Maria Pia Porto - Portugal

Leigh syndrome (LS) is a genetically determined neurodegenerative disorder of infancy or childhood characterised by developmental delay with psychomotor regression, signs of brainstem dysfunction, and lactic acidosis. Two of the most common mutations in the mitochondrial DNA (mtDNA) of children occur at nucleotide 8993 (nt8993), in the ATPase 6 gene. The base substitutions of T to G (T8993G) (1) and T to C (T8993C) (2) are routinely screened on patients suspected of having a mitochondrial disorder. In our lab we have diagnosed four T8993G and two T8993C patients by PCR-RFLP analyses and con¢rmed by DNA sequencing (ABI PRISM 310 Applied Biosystem). The less frequent T8993C mutation (L156P) has thus far, been reported in less than 15 families. Comparison of clinical and biochemical features on patients with the T8993C or T8993G mutation suggests that there are di¡erences between the two syndromes. We describe six new patients and discuss clinical and molecular correlation. References: (1) Holt IJ, Harding AE, Petty RKH, Morgan-Hughes JA.. Am J Hum Genet 1990;46:428-433. (2)de Vries DD, van Engelen BGM, Gabreels FJM, Ruitenbeek W, van Oost BA. Ann Neurol 1993;34:410-412

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CLINICAL HETEROGENEITY IN MITOCHONDRIAL DNA (mtDNA) DEPLETION J.E. Abdenur1, M.Garc|¨ a Alvarez1, C. Barreiro1, J. Donari1, N. Spe¨cola1, T.H. Vu2, S. Di Mauro2, N. Chamoles1 Fundacio¨n para el Estudio de las Enfermedades Neurometabo¨licas (FESEN), Buenos Aires, Argentina1; H. Houston Merrit Clinical Research Center for Muscular Dystrophy and Related Diseases, Columbia University, New York, USA2

Objective: We report a series of 6 patients (5 males) with mtDNA depletion and variable clinical presentation. Methods: Case Reports: Two patients developed congenital lactic acidosis with central nervous system involvement. One patient presented progressive dilated cardiomyopathy. Two patients developed myopathy, in one case with early onset and renal tubular acidosis. Finally one patient presented a clinical syndrome mimicking spinal muscular atrophy. Biochemistry, histochemistry and Southern blot hybridization: mitochondrial enzyme assays were performed in muscle. For quantitative Southern blots 5 mg of total DNA was used. Results: Biochemical studies showed a severely decreased COX activity and additional defects in other respiratory chain complexes. Ragged red ¢bers were absent only in both patients with early encephalopathy. Quantitative Southern blot con¢rmed decreased amounts of mtDNA in the 6 patients. Conclusions: Clinical and pathological features of patients with mtDNA depletion are heterogeneous with possible multiorgan involvement. Encephalopathy as the main clinical syndrome may not be rare in patients with this disorder.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CEREBRAL LEIGH-LIKE ALTERATIONS IN CHILDREN WITH MULTISYSTEMIC KEARNS-SAYRE SYNDROME (mKSS) ARE ASSOCIATED WITH WIDE-SPREAD DELETION/DUPLICATION mtDNA REARRANGEMENTS E. Wilichowski, A. Borchert, A. Ohlenbusch, C.G. Korenke, F. Hanefeld Abteilung Paediatrie/Neuropaediatrie, Universitaets-Kinderklinik, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany Background: Large-scale mtDNA rearrangements (deletions, duplications) are the genetic hallmark of Kearns-Sayre syndrome (KSS). This mitochondrial encephalomyopathy presents with external ophthalmoplegia, retinopathy, cerebellar dysfunction, deafness and heart block (classical type, cKSS). In some patients, additional manifestations of various systems have been described (multisystemic type, mKSS). Methods: 21 patients with mtDNA rearrangements ranging from 3670 to 9485 bp were studied. Tissue distribution (muscle, ¢broblasts, blood cells, urine cells) and quanti¢cation of the rearranged molecules were determined using PCR screening systems, quantitative Southern bloting and breakpoint sequencing. Results: 7 patients (33%) show the cKSS with heteroplasmic deletions present only in muscle tissue but not in other tissues studied, whereas 14 patients (67%) present with the mKSS. In all of them, complex pleioplasmic deletion/duplication rearrangements are present in all tissues studied. 8 children of this group showed a progressive clinical course with scoliosis, pyramidal and extrapyramidal disturbances and brain stem symptoms. CT/MR studies of the brain demonstrated symmetric lesions of the basal ganglia, thalamus and/or brain stem. HMR spectroscopy of these Leigh-like alterations disclosed marked lactate accumulation and signs of progressive neuronal loss. Conclusion: Leigh-like lesions in children with a progressive clinical course of mKSS are invariably associated with wide-spread pleioplasmic deletion/duplication mtDNA rearrangements.

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ABNORMAL CARDIOLIPIN AND PHOSPHATIDYLGLYCEROL REMODELING IN BARTH SYNDROME Vreken P1, Valianpour F1, Grivell LA2, Nijtmans LG2, Wanders RJA1, Barth PG1 Academic Medical Center, University of Amsterdam, 1Dept. of Clinical Chemistry and Div. Emma Children's Hospital, 2 Section for Molecular Biology, Institute of Molecular Cell Biology, University of Amsterdam, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands Barth syndrome (MIM 302060) is an X-linked disorder characterized by a cardioskeletal myopathy, intermittent neutropenia, 3-methylglutaconic aciduria, mophologically abnormal mitochondia and variable respiratory chain dysfunctions. The gene responsible for Barth syndrome has been cloned (G4.5) and shares homology with a family of acyltransferases, suggesting a role in phospholipid metabolism. Using cultured skin ¢broblasts from six patients with ¢ve di¡erent mutations in the G4.5 gene, we show that the incorporation of linoleic acid into phosphatidylglycerol (PG) and cardiolipin (CL) is dramatically reduced, whereas the incorpation of saturated and monounsaturated fatty acids in these phospholipids is normal. The de¢cient incorporation of linoleic acid into PG was not observed in normal controls and in patients with documented respiratory chain defects or cardiomyopathies of unknown origin, implicating that this abnormality is speci¢c for Barth syndrome and not secondary to mitochondrial dysfunction. Linoleic acid incorporation into PG and CL is the ¢rst speci¢c biochemical test which discriminates Barth syndrome from clinically or biochemically similar defects and suggests a role for the G4.5 geneproduct in PG and CL remodeling. The abnormal acylcomposition of PG and CL might explain the mitochondrial dysfunction observed in Barth syndrome.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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TWO NOVEL MUTATIONS OF SURF1 IN A JAPANESE CASE OF LEIGH SYNDROME WITH CYTOCHROME C OXIDASE DEFICIENCY Yuji Yokoyama, MichioTeraoka, Sumie Yamashita, Chiyo Inoue, Shinsuke Ninomiya, Yoshiki Seino Dept.of Pediat. Okayama University Medical School 2-5-1 Shikata-cho, Okayama 700, JAPAN

Cytochrome c oxidase (COX) de¢ciency is the most common cause of Leigh syndrome (LS). Nowadays, mutations were found in the surfeit 1 (SURF1) gene in LS families with COX de¢ciency. We identi¢ed two novel mutations of SURF1 in a Japanese case of LS with COX. Case: The patient was the ¢rst son of a non-consanguineous, healthy Japanese couple. From the age of 10 months on, this patient developed neurological signs and symptoms. Respiratory failure developed gradually, and he required intermittent assisted ventilation. Magnetic resonance imaging showed bilateral, symmetrical signal increases in basal ganglia, cerebellum dentate nucleus, and around the aqueduct of the midbrain on T2-weighted scans. No mitochondrial alterations were shown on microscopy. Results and Conclusions: Firstly, a 2-bp deletion at nucleotide position 790 (790delAG) in exon 8 was found, which shifts the reading frame such that the mutant protein has a completely di¡erent amino acid sequence from codon 264 to the premature stop codon at 290. Secondly, we found a T-toG transversion at nucleotide 820 resulting in the substitution of tyrosine by aspartic acid at codon number 274 (Y274D). We also studied the parents' genes, and found that the Y274D mutation was in his father and the 790delAG mutation was in his mother, heterozygously. These are the ¢rst pathogenetic SURF1 mutations identi¢ed in a Japanese family.

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DEFECTS OF PYRUVATE METABOLISM IN CULTURED LYMPHOBLASTOID CELLS AND TREATMENT IN 52 JAPANESE PATIENTS WITH LEIGH SYNDROME E. Naito, M. Ito, L. Yokota, T. Saijo, Y. Ogawa, Y. Kuroda Dept. of Pediatrics, School of Medicine, University of Tokushima, Tokushima, Japan

We analyzed the biochemical and molecular defects in cultured lymphoblastoid cells obtained from 52 Japanese patients with Leigh syndrome, and these defects were determined in 26 patients (50%). Pyruvate dehydrogenase complex (PDHC) de¢ciency was detected in ¢ve patients, cytochrome c oxidase (complex IV) de¢ciency in four patients, NADH-cytochrome c reductase (complex I) de¢ciency in four patients, and a point mutation of mitochondrial DNA in 13 patients (A8344G in one, T8993C in two, and T8993G in ten). These ¢ndings suggest that cultured lymphoblastoid cells are useful for elucidating the etiology of Leigh syndrome. The most striking ¢nding in our study was the higher incidence of thiamine-responsive PDHC de¢ciency in PDHC de¢ciency with Leigh syndrome. In four of ¢ve patients with PDHC de¢ciency, PDHC showed a reduced a¤nity for thiamine pyrophosphate. Treatment with high doses of thiamine resulted in a reduction in lactate and clinical improvement in these four patients, suggesting that some patients with Leigh syndrome may have a thiamine-responsive PDHC de¢ciency and high doses of thiamine may be useful at an early stage of neurologic abnormalities in patients with this type of PDHC de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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NOVEL COMPOUND HETEROZYGOUS MUTATION IN A BOY WITH DIHYDROLIPOAMIDE DEHYDROGENASE DEFICIENCY H. Hansi¨kova¨, L. Cíerna¨, L. Wenchich, S. Kmoch2, K. Sípicíkova¨2, V. Malinova¨ and J. Zeman Department of Pediatrics1 and Institute for Inherited Metabolic Disorders2, Charles University in Prague, Czech Republic The lipoamide hedydrogenase (E3 de¢ciency was diagnosed in male infant with vomiting, hypotonia, psychomotor retardation, lactic acidosis and elevated branched chain amino acids and branched chain a-ketoacids. The activity of E3 was low in blood lymphocytes (510% of control values), cultured ¢broblasts (5 14%) and isolated muscle mitochondria (5 5%). A novel compound heterozygous mutation in gene for E3 was found in the patient. The A1081G mutation resulting in the substitution of Met-8 for Val was inherited from the mother and grandfather and the mutation G1123A resulting in the substitution of Glu-18 for Thr was inherited from father and grandfather. Western blot analysis in the ¢broblasts showed decreased amount of E3 protein. So far E3 de¢ciency was described in 21 patients from 15 families. The prognosis of a¡ected infants and small children is unfavourable and current therapeutic possibilities are limited. The treatment includes low-carbohydrate, low-branched chain amino acid and high-fat diet, sodium dichloroacetate (DCA) and lipoic acid. DCA e¡ectively decreased the lactate level in our patient, but during respiratory infections he was repeatedly admitted to the intensive care unit with severe metabolic and lactic acidosis, which correction could be achieved only with high intravenous intake of fat emulsions. No bene¢t from administration of lipoic acid was observed. Supported by grant GA-CíR 302/0648/99.

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DETECTION OF A MUTATION IN CATALYTIC SUBUNIT 2 OF PYRUVATE DEHYDROGENASE PHOSPHATASE GENE IN A PATIENT WITH CONGENITAL LACTIC ACIDEMIA M. Ito, I. Ohigashi, K. Sinahara, Y. Ogawa, S. Saijo, I. Yokota, E. Naito, Y. Kuroda Department of Pediatrics, University of Tokushima, Tokushima, Japan A de¢ciency of pyruvate dehydrogenase phosphatase (PDP) has been suggested as a possible cause of congential lactic acidemia and Leigh's disease due to a defect of activation of pyruvate dehydrogenase complex (PDHC). However, as yet, the molecular genetical analysis of PDP in these patients has not been performed. In this study, we isolated two cDNAs for human catalytic subunits of PDP (PDP1 and PDP2) and detected a mutation in a patient with a defect of activation of PDHC. Human PDP1 and PDP2 were highly homologous in nucleotide and protein levels to the previously reported rat PDH1 and PDH2, respectively. In addition, genes of PDP1 and PDP2 was mapped in chromosome 8, in the region q22^23, and in chromosome 3, in the region of q27^28, respectively. With the nucleotide sequence determination, A716T (D239V) in cDNA for PDP2 was detected in a patient with congenital lactic acidemia due to a defect of activation of PDHC. This amino acid was conserved in human and previously reported PDP1 and PDP2. These results suggest that this mutation is the primary defect of activation of PDHC in this patient.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CONCOMITANT ADMINISTRATION OF SODIUM DICHLOROACETATE AND VITAMIN B1 IS THE FIRST CHOICE IN TREATMENT OF MELAS SYNDROME Y. Kuroda, E. Naito, I. Yokota, T. Saijo and M. Ito Department of Pediatrics, University of Tokushima, JAPAN

We investigated the therapeutic e¡ect of oral sodium dichloroacetate (DCA) in combination with vitamin B1 (B1) in patients with the MELAS syndrome in a Japanese collaboration study group. Twenty-one patients (6^21Y) were treated with oral DCA in dosage of 25^50 mg/kg/day for 3^58 months. Plasma and CSF levels of lactate and DCA were monitored during the therapy. No changes were made in any drug regimens, including B1, initiated prior to the start of DCA therapy. DCA^B1 therapy causes signi¢cant increases in the PDH complex and Krebs cycle in various tissues including the brain. DCA^B1 therapy halted or delayed the clinical deterioration and improved their quality of life in all the patients. Especially, the therapy was e¡ective in eliminating or improving stroke-like episodes, episodic headache and vomiting, myoclonus, abdominal pain, and psychiatric symptom. The therapy also improved the disturbed motor function in ¢ve patients. No serious adverse e¡ects were observed in all the patients. It was shown that the concomitant administration of DCA and B1 was reasonable to improve the clinical ¢ndings and the quality of life in patients with MELAS syndrome.

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INHIBITION OF PYRUVATE DEHYDROGENASE BY FATTY ACID CoA ESTERS: A POSSIBLE CAUSE OF LACTIC ACIDEMIA IN LONG-CHAIN FATTY ACID b-OXIDATION DEFECTS Ulrike Piccolruaz, Bernhard Meister, JÎrn Oliver Sass UniversitÌtsklinik fÏr Kinder- u. Jugendheilkunde, Sto¡wechsellabor, Innsbruck, Austria Lactic acidemia is a well known abnormality in metabolically decompensated patients with defects in long-chain fatty acid oxidation. This ¢nding indicates that not only the utilization of fatty acids is decreased, but th at impaired oxidation of pyruvate may also contribute to energy de¢ciency and cardiac involvement in these disorders. A possibly site of inhibition of pyruvate utilization is the enzyme pyruvate dehydrogenase (PDH). Using PDH from porcine heart in a sp ectrophotometric assay (based on the method by Strumilo et al., BBRC 1999; 256: 341-5) we therefore tested in vitro e¡ects of a variety of fatty acids and fatty acid metabolites, including acyl esters of carnitine and coenzyme A (CoA). While most compounds studied did not alter PDH activity, CoA esters of saturated fatty acids were potent inhibitors. Kinetic data indicated uncompetitive inhibition. Longchain acyl-CoA esters showed strongest inhibition of PDH, palmitoyl-CoA with an IC50 of 83 mM at Km (15.3 mM) of pyruvate. Octanoyl-CoA, a medium-chain fatty acid ester, presented a 15-fold higher IC50, but still inhibited PDH , while octanoic acid and the corresponding carnitine ester showed no e¡ect even at much higher concentrations. Our res ults provide evidence for a direct modulation of PDH, a key enzyme of energy metabolism, by long-chain acyl-CoA. This may contribute to energy de¢ciency and lactic acidemia in disorders of long-chain fatty acid oxidation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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IMPAIRED PYRUVATE OXIDATION IN MUSCLE: EVIDENCE OF A BIOCHEMICAL DEFECT WITHOUT AN IDENTIFIABLE CAUSE I P HARGREAVES1, S J R HEALES1, A BRIDDON1, M HANNA2, G BROWN3 and J M LAND1 Neurometabolic unit, National Hospital for Neurology and Neurosurgery, Dept. Clinical Neurology,Institute of Neurology,Queen Square, London. 3Genetics Unit, Dept. of Biochemistry,Oxford University, Oxford. We present a 20 year old male, born to consanguinous parents, who was mentally retarded and had developmental delay, seizures and a generalised dystonia. Plasma alanine, lactate and pyruvate were moderately elevated, with normal lactate/pyruvate ratio. A skeletal muscle biopsy was obtained for polarographic studies. These revealed a 50% reduction in oxygen consumption with pyruvate/malate substrates suggestive of pyruvate dehydrogenase (PDH) de¢ciency. Direct assay of Ca2+/Mg2+ activated PDH complex revealed a reduced activity (7.6 nmol/min/mg; reference interval: 43.8-92.0 nmol/min/mg). Studies on ¢broblasts also revealed markedly reduced PDH activity (0.1 nmol/min/ mg; reference interval: 0.71.1 nmol/min/mg). However, immunochemical studies showed normal levels of all PDH subunits. A de¢ciency of the E3 subunit was excluded by normal plasma levels of branched chain amino acids. Genetic studies failed to detect a mutation in the gene which codes for E1a subunit. In conclusion, we have evidence of a PDH de¢ciency, but so far have been unable to identify a speci¢c cause. This patient could have a PDH phosphatase de¢ciency. Direct assay of this enzyme is required before this can elucidated

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SUSTAINED HYPERPYRUVICEMIA AND HYPERLACTATEMIA DUE TO A DEFECT OF THE MITOCHONDRIAL PYRUVATE CARRIER M.Brivet1,A.Garcia2,M.C.Nassogne2 ,A.Boutron1,A.Slama1,M.Mine1,A.Legrand1,G.Touati2,J.M.Saudubray2. 1-Laboratory of Biochemistry, AP-HP Hopital de Biceªtre ; 2-Department of Pediatrics, AP-HP Hopital Necker-Enfants Malades ; Paris, France. The patient is the ¢rst child of healthy consanguineous parents. She presented at birth with hypotonia, mild facial dysmorphism, periventricular cysts and a marked metabolic acidosis (pH=7.22, bicarbonates 12 mM), hyperlactatemia (14.3 mM), normal glycemia and normal ammonemia. P e rsistant hyperlactatemia (11-12 mM) with normal lactate/pyruvate ratio, increased blood levels of alanine and proline and increased lactaturia were shown at day 3 of life. Treatment with bicarbonate, thiamine (10 mg/d), dichloropropionate (100 mg/kg/d) and a ketogenic diet failed to decrease the hyperlactatemia. Pyruvate dehydrogenase (PDH) activity was found normal in lymphocytes and ¢broblasts. The possibility of a defect in the mitochondrial pyruvate transport was suggested by the major impairment of 14C-acetylcarnitine production from (2-14C) pyruvate (5% of controls) in digitonin-permeabilized ¢broblasts (Pande et al JCI 1993 ; 91 : 1247). By contrast, in frozen-thawed ¢broblasts, 14C-acetylcarnitine production from (2-14C) pyruvate was normal, demonstrating the normality of PDH and carnitine acetyltransferase activities. A defect of carnitine acylcarnitine translocase activity was also ruled out by demonstrating normal oxidation of long chain fatty acids in ¢broblast monolayers. The defect is likely to be restricted to the pyruvate carrier as suggested by normal 14CO2 production from (U-14C) malate and (1,4-14C) succinate versus greatly impaired decarboxylation of (1- 14C) pyruvate in permeabilized ¢broblasts. Supported by a grant of Association Franc°aise contre les Myopathies.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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DIAGNOSTIC PARAMETERS IN COMPLEX I DEFICIENCY Brian H. Robinson, Sandeep Raha, Gillian McEachern, Jessie Cameron, Nevi MacKay The Research Institute, Hospital for Sick Children, 555 University Ave. Toronto, Ontario

Human Complex I de¢ciency has a wide clinical spectrum of presentation and can be di¤cult to diagnose. We have taken the results from cohorts of patients we have and have compared enzymatic diagnosis in frozen muscle homogenate versus freshly isolated mitochondria in muscle biopsies. We have compared muscle and human skin ¢broblast analysis of both enzymatic and redox state measurements. Finally, we have looked at the status with regard to skin ¢broblast levels of MnSOD and superoxide production. The lowest levels of activity, the biggest changes in lactate/pyruvate ratio and the highest levels of MnSOD are seen consistently with patients with fatal infantile lactic acidosis (FILA) and Leigh Disease with Cardiomyopathy (LD + C). Patients with Leigh Disease only have a slower onset disease and less dramatically changed parameters for L/P ratio and MnSOD. Patients with developmental delay +/- cataracts had low MnSOD and produced more superoxide with little change in redox state. We conclude a combination of muscle biopsy with mitochondrial isolation coupled with ¢broblast analysis of redox state +/- galactose sensitivity as the optimal method of diagnosis but propose we look for ¢ner diagnostic algorithms, including immunoblot and sequencing.

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GENETIC HYPOGLYCAEMIA IN INFANCY AND CHILDHOOD: PATHOPHYSIOLOGY AND DIAGNOSIS J.M. Saudubray, P. de Lonlay, M.C. Nassogne, A. Garcia, G. Touati

Knowledge of the homeostatic mechanisms that maintain the blood glucose concentration between the relatively narrow range of 2.5 and 7.5 mmol/l whether the child eating or fasting, is crucial in elucidating the causes and mechanisms of genetic hypoglycaemia. Characteristic clinical features are very important (age at onset, severity and frequency of hypoglycaemic attacks, timing of hypoglycaemia, glucose requirements to maintain normal blood glucose concentrations, ketosis, glucagon responsiveness, hepatomegaly, short stature, dysmorphy). Hypoglycaemia in the absorptive phase is very likely to be due to hyperinsulinism. Diagnosis relies upon high glucose requirements, glucagon responsiveness and inappropriate low ketones. About half neonates with hyperinsulinism may have focal islet cell hyperplasia that can be treated with partial pancreatectomy. The di¡use hyperinsulinism are inherited heterogeneous disorders involving the gene encoding the sulfonylurea receptor, the inward rectifying potassium channel, the glucokinase, and the glutamo-dehydrogenase gene in cases in which hyperammonemia is associated with hyperinsulinism. Hypoglycaemia in the post-absorptive phase and early starvation phase is very likely to be due to disorders of glycogen metabolism (glycogenosis) presenting with hepatomegaly, ketosis and glucagon unresponsiveness. Hypoglycaemia in early intermediate starvation phase (12 to 16 hours of fast) is mostly observed in gluconeogenesis defects (fructose diphosphatase de¢ciency, fatty acid oxidation disorders), endocrine disorders (growth hormone de¢ciency, panhypopituitarism and adrenal insu¤ciencies), and idiopathic ketotic hypoglycaemia. Fasting hypoglycaemia without ketosis is likely to be due to fatty acid oxidation or ketogenesis disorders. It is extremely important to collect appropriate samples at the time of the acute event. This will provide a diagnosis or will at least indicate which group of disorders is likely. Blood should always be taken for glucose, insulin, growth hormone, cortisol, lactate, amino acids, hydroxybutyrate, free fatty acids, a Guthrie card for acylcarnitine pro¢le and liver function test. The ¢rst urine pass must always be tested for ketones allowing an initial separation between ketotic and non ketotic hypoglycaemia and saved for organic acids analysis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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STABLE ISOTOPE TURNOVER STUDIES ON THE ENDOGENOUS PRODUCTION OF DGALACTOSE IN HEALTHY SUBJECTS AND IN PATIENTS WITH CLASSICAL GALACTOSEMIA Peter Schadewaldt, Kamalanathan Loganathan, Hans-Werner Hammen, Uta SpiekerkÎtter, Annette Bodner-Leidecker, and Udo Wendel Deutsches Diabetes Forschungsinstitut and Kinderklinik, Heinrich-Heine-UniverstitÌt DÏsseldorf, D40225 DÏsseldorf, Germany Knowledge of the quantitative role of endogenous galactose is essential for the judgement of the signi¢cance of lifelong dietary restriction in patients with galactose-1-phosphate uridyltransferase de¢ciency galactosemia (McKusick 230400). We therefore performed galactose turnover studies in overnight fasted adults using a primed continuous infusion approach with i.v. application of D[1-13C]galactose (priming, 8 mmol x (kg bw)-1; infusion, 0.8 mmol x (kg bw)-1 x h-1 for 6 h). As analyzed by a recently established gas chromatography-mass spectrometry method, steady state conditions in plasma [13C]galactose were reached within 3 h. Initial plasma concentrations of galactose were about 0.15 mmol x L-1 in healthy subjects and thus about 10-fold higher than in galactosemic patients. The rate of appearance of galactose in plasma amounted to 0.2-0.4 mmol x min-1 x (kg bw)-1 in controls (n = 5) compared to 1.7-3.3 mmol x min-1 x (kg bw)-1 in the galactosemic patients (n = 3). These ¢ndings indicate that production of free galactose from endogenous sources proceeds at a cosiderable rate in vivo and point at signi¢cant di¡erences of galactose turnover in healthy subjects and patients. The lower rate of release of galactose into the plasma compartment in controls is presumably explained by a higher rate of (re)utilization and/or degradation in extrahepatic tissues. Supported in part by grants from the Elterninitative GalactosÌmie and the Deutsche Forschungsgemeinschaft (DFG We 614/10-1).

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A FURTHER PREGNANCY IN A WOMAN WITH CLASSIC GALACTOSEMIA WHO DELIVERED TRIPLETS FOLLOWED BY PREMATURE OVARIAN FAILURE V. Kimonis, SIU School of Medicine, Div. of Gen. and Metab., Dept. of Ped., Spring¢eld, IL, USA Dietary therapy in classic galactosemia does not prevent ovarian failure and infertility. Most of the previous pregnancies have occurred in black women or in the milder variant types. A 23-year-old woman was diagnosed with classic galactosemia at the age of 2 weeks having presented with vomiting, cataracts, FTT, and hepatitis. She has taken a lactose free diet all her life. She has normal intelligence. Since menarche at age 11 y. until age 16 y. she had amenorrhea lasting up to 6 months. Subsequently her periods became regular, monthly, until her pregnancy at the age of 18 years with triplets . Her gravid Gal-1-P level was 4.12 mg % (normal 5 1 mg%) and Gal-1-P uridyl transferase 0, 0.34 mmol/hr/g Hb (classical galactosemia 0-0.7). She is homozygous for the common mutation Q188R. Her pregnancy was complicated by pre-eclampsia and azotemia during the 29th week of pregnancy. She had an emergency caesarian section and delivered healthy triplets weighing 1770 g, 1290 g, and 1450 g respectively. She took oral contraceptives immediately after the birth of her triplets and discontinued them 17 mo. later. She subsequently noted amenorrhea. She had high luteinizing hormone (LH) level 38.82 mIU/ml and follicle stimulating hormone (FSH) level 94.19 mIU/ml attributable to ovarian failure. She subsequently became pregnant again. The pregnancy was uncomplicated and she delivered a healthy male 5 mo ago. The children are all developmentally normal and have no dysmorphic features.

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APOLIPOPROTEIN E POLYMORPHISM AND COGNITIVE DEVELOPMENT IN CLASSICAL GALACTOSEMIA W. Strobl+, J. Berger*, M. Tiefenthaler½, V. Hinteregger½, S. StÎckler-Ipsiroglu½, S. SchweitzerKranz&, M.P.Manns#. Depts. of Medical Chemistry+, Pediatrics½, and Brain Research Institute*, University of Vienna, Austria; Dept of Pediatrics, Evangelisches Krankenhaus, DÏsseldorf& , Dept. Gastroenterology, Univ. of Hannover, Germany#.

In many patients with classical galactosemia cognitive development is impaired despite of dietary treatment. The extent of cognitive impairment is not related to the onset of treatment or to the level of red cell galactose-1-P. Apolipoprotein E (apoE) is expressed in the brain and participates in its response to injury. The polymorphism of apoE accounts for part of the variability in the outcome after brain injury, and in the onset of dementia in Down syndrome and Alzheimer disease. To determine whether cognitive development in classical galactosemia is in£uenced by apoE polymorphism we determined the apoE genotypes in 25 galactosemia patients aged 6-34 years by PCR/restriction analysis. IQs were measured by intelligence tests based on the Wechsler concept . The distribution of of apoE alleles in galactosemia patients (E-3: 64%, E-2: 22%, E-4: 14%) did not di¡er signi¢cantly from that in controls (E-3: 76%, E-2: 13%, E-4: 12%). No di¡erence in mean IQ was found between patients carrying an apoE-4 allele and those without apoE-4 (77 + 13, n=6 vs. 74 + 17, n=19, n.s.). Similarly, the presence or absence an apoE-2 allele did not in£uence the mean IQ score (67 + 19, n=9 vs. 78 + 16, n=16, n.s.). Our preliminary data obtained in a small group of galactosemia patients provide no evidence for a major e¡ect of the apoE genotype on cognitive development in classical galactosemia.

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DELAYED DIAGNOSIS OF GALACTOSEMIA IN A SEVERELY RETARDED BOY L.J.M. Spaapen1, J.A. Bakker1, U. Moog1, M.E. Rubio2, P.P. Forget2 1 Dept. of Biochemical Genetics, Stichting Klinische Genetica ZON, 2 Dept. of Pediatrics, Academic Hospital Maastricht, Maastricht, The Netherlands

The patient was born at term as the 2nd child of non-consanguineous parents. In the neonatal period he showed failure to thrive; a liver disease was diagnosed but could not be speci¢ed. Psychomotor retardation was evident from the beginning. He was seen at the age of 16 years by a clinical geneticist in an institution for mentally handicapped for evaluation of his mental retardation. He was severely retarded, unable to speak, had a happy disposition, epilepsy and spasticity. Angelman syndrome was ruled out. Ophthalmologic examination showed a possibly slight cataract. At the age of 4 years cytogenetic and metabolic investigations were performed elsewhere. A normal karyotype and no indications for a metabolic disorder were found. Recently, investigation of serum sialotransferrins by IEF showed increased bands of a-, mono-, and disialotransferrin, a CDG-1 like pattern lately reported to be present in untreated galactosemic patients (1). In addition, urinary galactose and galactitol were found increased. Subsequently, a de¢cient activity of GALT was found in erythrocytes. In general patients with galactosemia present at neonatal age with severe liver disease. In the selective screening for inborn errors of metabolism of patients with severe psychomotoric retardation and severe speech disability classical galactosemia should be considered. 1. Spaapen LJM, Vulsma T, et al. (1992) SSIEM, 30th Ann. Symp. Leuven, (abstract)

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RISK FACTORS FOR PREMATURE OVARIAN FAILURE IN FEMALES WITH GALACTOSEMIA RH Singh 1, N Guerrero 1, A Manatunga 1, G Berry 2, R Steiner 3, LJ Elsas II 1, 1 Emory University School of Medicine, Atlanta, GA, 2 University of Pennsylvania School of Medicine Children's Hospital of Philadelphia, PA, 3 Oregon Health Sciences University, Portland, OR. The risk for premature ovarian failure (POF) in females with galactosemia can be predicted by analyzing three areas of risk pathology:1) the patient's molecular genotype for galactose-1-phosphate uridyltransferase (GALT); 2) alternate pathways for galactose metabolism; and 3) the patient's environment at diagnosis and during treatment. Retrospective/Cross sectional information was collected on 53 females with classic galactosemia and their ovarian function was analyzed by serum FSH, LH and clinical observation. The associations were analyzed among POF and the mutations in GALT, the highest erythrocyte galactose-1-phosphate (Gal-1-P) at diagnosis, the age at which dietary treatment was initiated, mean erythrocyte Gal-1-P during treatment, and whole body 13C-galactose oxidation to 13CO2. The most prevalent mutation, Q188R, had a signi¢cant e¡ect of Genotype Category (Q188R/Q188R, Q188R/Other, Other/Other) on POF (p = 0.04, Fisher's and an OR of 8.3). Mean erythrocyte Gal-1-P concentration during treatment was a signi¢cant risk factor for POF (p=0.04). Also, all patients studied with less than 5% total body oxidation of galactose to 13CO2 had POF, whereas those with more than 5% did not have POF (p=0.008, Fisher's). The development of premature ovarian failure in females with galactosemia is more likely if: the patient's genotype is Q188R/Q188R; the mean erythrocyte Gal-1-P is above 3.5 mg/dL during therapy; and if the recovery of 13CO2 from whole body 13C-galactose oxidation for 2 hours is reduced below 5% of administered 13C-galactose.

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OVER-EXPRESSION OF HUMAN UDP-GLUCOSE PYROPHOSPHORYLASE RESCUES GALACTOSE-1-PHOSPHATE URIDYL TRANSFERASE DEFICIENT YEAST LJ Elsas and K Lai. Division of Medical Genetics, Department of Pediatrics, Emory University, Atlanta, GA, USA. Studies in model systems may reveal alternate pathways that reduce toxic precursors and increase de¢cient products relevant to inborn errors in humans. We studied galactose-1-phosphate uridyltransferase-de¢cient yeast (GALT knockouts) that could not grow on galactose medium and selected revertants by their ability to grow. Using Ye6100 GeneChipTM, we compared gene expression pro¢les among wild-type, GALT-knockout and revertant yeast strains and found that the autosomal recessive, revertant strains could down-regulate the GAL regulon (equivalent to the Leloir pathway in humans) and up-regulate the gene for UDP-glucose pyrophosphorylase (UGP1). We found decreased concentrations of galactose-1-phosphate in the revertant cells of 0.65 compared to 4.82 mol/g protein in the GALT knockout strain. We found that UDP-glucose pyrophosphorylase could use galactose-1-phosphate as substrate and catalyze the production of UDP-galactose in the presence of UTP. We then transfected and over-expressed the yeast UGP1 and the human homolog (hUGP2) in GALT-knockout yeast. Both UGP1 and hUGP2 enabled the GALT-knockout yeast to grow on galactose. We conclude that revertant yeast down-regulate their GAL regulon, thereby reducing the accumulation of galactose-1-phosphate and up-regulate UDP-glucose pyrophosphorylase. UGP1 can convert galactose-1-phosphate to UDP-galactose, thus bypassing an inherited metabolic block common to yeast and humans with galactosemia.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MOLECULAR CHARACTERIZATION OF GALACTOKINASE DEFICIENCY IN JAPANESE Y. Okano1, M. Asada1, T. Imamura1, A. Ohtake2, K. Murayama3, K. Choeh4, K-J. Hsiao5, JKV. Reichardt6 and T. Yamano1 1 Dept. of Pediatr., Osaka City Univ. Graduate Sch. Med., Osaka 545-8585, Japan, 2Dept. of Pediatr., 3 Dept. of Opthal., Saitama Med. Sch., Japan, 4Dept of Pediatr, Eulji Univ., Korea, 5Veterans General Hosp-Taipei, and Nat'l Yang-Ming Univ., Taiwan, 6Ins. for Genet. Med., Univ. of Southern California Sch. of Med., USA

Galactokinase (GALK) de¢ciency is an autosomal recessive disorder and causes cataract formation. Using DGGE and sequence analysis, we found 13 missense mutations (M1I, S33P, N39S, E43K, G137R, A198V, R256W, R277Q, T288M, M307R, S313L, T344M, G349S), a nonsense mutation (E245X), two deletions (410delG, 509-510delGT) from 16 Japanese patients with GALK de¢ciency. Using the COS cell expression analysis, in vitro GALK activities of missense mutations were remarkably reduced, and corresponded with those in patients' RBC. The immuno-reactivity of mutant constructs indicated qualitative and quantitative abnormalities of GALK proteins. We found the new variant ``Osaka'' in Japanese, which was associated with A198V. The GALK activities in the patients' RBC with null and A198V were 9.0 - 13.9%, consistent with 18.5% in expression analysis. The population genetics study of A198V revealed 24/582 (4.1%) in Japanese, 8/288 (2.8%) in Koreans, 1/264 (0.4%) in Taiwanese, and 0/188 (0%) in Caucasians. The Japanese patients with bilateral cataracts had a relatively high frequency of A198V (15/200, 7.5%, no signi¢cance). This new ``Osaka variant'' is prevalent in Koreans and Japanese populations, and may be possible to be one of the factors for presenile cataracts.

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MEASUREMENT OF BLOOD GALACTOSE ON FILTER PAPER FOLLOWING DERIVATIZATION WITH 8-AMINO-2-NAPHTHALENSULFONIC ACID BY REVERSED PHASE HPLC IN GALACTOSEMIA Seon-Pyo Hong 1*, Hye-Ran Yoon2, Kyoung Ok Lee 2, Mi-Kyoung Kim1, Yoon S Shin 3, Dong Hwan Lee4 1 College of Pharmacy, Kyunghee University, Korea. 2 Special Biochemistry, Seoul Medical Science Institute, Seoul Korea. 3 Children's Hospital University of Munich, Germany. 4 Dept. of Pediatrics, Sooncheonhyang University Hospital.

De¢ciency of galactose -1-phosphate uridyl transferase(GALT) is a polymorphic enzyme and Duarte(D) is the most common enzyme variant. A new reversed-phase HPLC method has been developed for the measurement of galactose in blood (50mL) on Guthrie ¢lter paper using 8-amino-2naphthalenesulfonic acid (8,2-ANS) as derivatization reagent for the diagnosis of Galactosemia. Galactose was extracted from blood spot on ¢lter paper and derivatized with 8,2-ANS to produce Schi¡ bases, and reduced under sodiu m cyanoborohydride. The linear range was between 7.2 m g/dL and 29 mg/dL and the limit of detection (S/N=3) of this method was 90 ng/dL. The mean recovery of galactose was 104.29 % with a S.D. of 3.62 % with correlation coe¤ciency of 0.9999. Control range of blood galactose in Korean newborn was below 6 mg/dL for male and female without any gender di¡erence (n=5). We applied 11 anonymous blood spots in which diagnosis has already been made by enzyme assay as one of the GALT or epimerase de¢ciencies. All the patients blood spots showed abnormal elevation of galactose. These results suggest that the developed method could be a practical tool for the diagnosis of Galctosemia with high sensitivity.

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FRUCTOSE-1,6-BISPHOSPHATASE DEFICIENCY: MOLECULAR ANALYSIS OF 35 CASES INCLUDING 2 WITH CEREBRAL PALSY Y. Kikawa1, Y. Shigematsu1, M. Mayum1, Y.S. Shin2 Fukui Medical University, Japan1, Mu«nchen Children's Hospital, Germany2 Fructose-1,6-bisphosphatase (FBPase) de¢ciency is an autosomal recessive inherited disorder and a potential cause of sudden unexpected infant death. To facilitate carrier detection, genomic DNA was analyzed in 35 patients with FBPase de¢ciency, including two Japanese cases with cerebral palsy. Cultured monocytes were also used as a source alternative to liver samples for analysis of mRNA, in some Caucasian and in most Japanese patients. In Caucasian patients, mutations were identi¢ed only in 20% (6 among 30 alleles), and 4 mutations in 5 among the 6 alleles were new and di¡erent from those already reported in Caucasian by Herzog et al. While, in Japanese patients, mutations were identi¢ed in 75% (24 among 32 alleles), 960/961InsG being the predominant mutation in 34% (11 among 32 alleles). No mutation was found in the remaining 25% (8 among 32 alleles) with analysis of both genomic DNA and cDNA, in spite of complete loss of enzymatic activities in cultured monocytes. Our results show marked heterogeneity of FBPase mutations in Caucasian compared with those reported in Japanese, and suggest a possibility that mutations, not including FBPase gene itself, caused FBPase de¢ciency at least in these 4 Japanese patients.

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LONG TERM OUTCOME AND MUTATIONAL ANALYSIS IN A CASE OF FRUCTOSE 1,6BISPHOSPHATASE DEFICIENCY 1 M.D. Bain., 2 G. Bano., 3 K. Eschrich., & 1R.A. Chalmers. 1 Paediatric Metabolism Unit, Department of Child Health, 2Division of Gastro- enterology, Endocrinology & Metabolism, St. George's Hospital Medical School, London, UK, and 3Institut fur Biochemie, Leipzig, Germany. The patient is now 25 years of age. Fructose 1,6-bisphosphatase (F1,6-bisP) de¢ciency was diagnosed at 6 months of age when she presented with hypoglycaemia, hepatomegaly, and lactic acidosis. Liver biopsy tissue showed absent F1,6-bisP activity. Treatment was initially with oral hourly daytime glucose feeds and via a nasogastric tube at night. Later, nocturnal cornstarch was introduced. The evolving spectrum of complications has been very similar to that of glycogen storage disease type 1 leading some to doubt the validity of the initial diagnosis: fasting hypoglycaemia, but of reducing severity with age, has persisted as has hepatomegaly; hyperuricaemia requiring allopurinol, pancreatitis aggravated by psychosocial problems; ultrasonographic evidence of enlarged kidneys together with a possible liver adenoma; and most recently musculo-skeletal problems with deteriorating osteopenia (Z score spine and neck of femur -2.23 & -0.62 respectively) that stabilized with bisphosphonate therapy. Leucocyte F1,6-bisP activity recently measured is normal (435 nmol/mg. protein/hr, Ref 101-463). Mutational analysis showed no abnormality in the exons but evidence for splice mutations with no normal cDNA detectable.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SIMPLE AND SENSITIVE SCREENING FOR FRUCTOSE-1,6-DISPHOSPHATASE DEFICIENCY USING GC/MS Iga Misako1, Masahiko Kimura1, Xiao-wei Fu1, Yoshiharu Kikawa2, Toshihiro Ohura3, Seiji Yamuguchi1 Department of Pediatrics, Shimane Medical University, Japan1, Department of Pediatrics, Fukui Medical School, Japan2, and Department of Pediatrics, Tohoku University, Japan3

Fructose-1, 6-disphosphatase (FDPase) de¢ciency is a disorder of gluconeogenesis which often causes severe hypoglycemia. Increased excretion of glycerol-3-phosphate (G3P) and glycerol in urine of acute status in this disorder was previously reported. For early diagnosis, we developed a more simpli¢ed and sensitive screening method for detecting FDPase de¢ciency using GC/MS with SIM mode. Urine specimen containing 0.1 mg Cre was treated with urease, then tropic acid was added as an internal standard. Protein was removed using ethanol. After centrifugation, the supernatant was dried under a nitrogen stream. The dry residue was TMS-derivatized and analyzed by GC/MS. Both glycerol and G3P were detectable in all normal controls (n=30) with this method. In FDPase de¢ciency (n=6), both glycerol and G3P were high. Glycerol was also increased in Glycerol kinase (GK) de¢ciency (n=5) and in Glycerol-infused patients (n=10). The glycerol/G3P ratio enabled us to distinguish FDPase de¢ciency from both GK de¢ciency and Glycerol-infused patients. This method, as well as being a simple method, will, we suggest be of help in the detection of FDPase de¢ciency.

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NEXT EUROPEAN CASE OF FRUCTOSE-1,6-BISPHOSPHATASE DEFICIENCY CONFIRMED BY MOLECULAR FINDINGS Taybert J., Keªpka A., Sawnor-Korszynska D., Jin B. Y., Kikawa Y., Mayumi M., Pronicka E. Children's Memorial Health Institute Poland and Dept. of Pediatrics Fukui Medical University Japan

De¢ciency of fructose-1,6-bisphosphatase (FBP), an enzyme of gluconeogenesis, is characterised by failure to thrive, fasting hypoglycaemia, lactic acidosis and hepatomegaly. Marked mortality of a¡ected neonates and severe episodes of metabolic decompensation precipitated by fasting and infections have been reported. Until recently when Herzog et al (J Inher Metab Dis 1999;22:132-138) found FBP gene mutations in 4 new patients (two of German origin) only Japanese patients had been reported to carry FBP gene mutations. We describe a next European FBP de¢cient case con¢rmed by DNA analysis of the gene. Beata K. an adopted child of unknown family history (probably of Polish origin) was apparently healthy up to the age of 9 years. Since then, up to the age of twelve, she presented with subsequent 5 episodes of unconsciousness at the morning with hypoglycaemia (once with convulsions). Her development has been normal and hepatomegaly has been never observed. Fasting test was performed to assess glucose homeostasis. At the end of the test (22 hours) marked increased serum concentration of lactate (7.9 mmol/l), pyruvate (0.25 mmol/l) and alanine (550 mmol/l), hypoglycaemia (2.1 mmol/l), ketonuria and metabolic acidosis were observed suggesting FBP de¢ciency. Liver biopsy was not made. Activity of FBP in leukocytes was absent and it was also decreased in cultured monocytes (0.11 nmol/min/mg prot., controls 2.90 and 5.02). DNA analysis revealed two new mutations of FBP gene(A618del - frame shift from amino acid 207 and point mutation G619C at the phosphorylation site). Expression studies to con¢rm the mutations pathogenicity is undertaken.

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DIETARY MANAGEMENT OF GLYCOGEN STORAGE DISEASE Ia PATIENTS IN PREGNANCY M v Rijn1),S Huitema1),JP Rake2),G Visser2),MP Heringa3),FJ v Spronsen2),GPA Smit2) 1 Dietary Department, 2 Beatrix Children's Hospital, 3Obstetrics/Gynaecology, University Hospital of Groningen, The Netherlands. e-mail [email protected] The primary aim of treatment of glucose-6-phosphatase de¢ciency (GSD Ia) is prevention of hypoglycaemia by administering exogenous glucose either by gastric-drip-feeding (GDF), frequent meals or slow releasing carbohydrates. At present many of these patients reach adulthood, increasing the demand of females for control in pregnancy. Guidelines for the dietary treatment of these patients are rare and not very extensive. We treated two GSD Ia patients during pregnancy. Dietary interventions were frequent and showed large intra- and inter-individual variations in glucose requirements. Pregnancies were uneventful. Both infants had a normal start and weight. The following items were essential to reach optimal dietary treatment: *Initiation of blood glucose measurements at home; the patients capability to interpret blood glucose levels without actually measuring is disturbed during pregnancy. *(Re-) initiation of GDF to meet high dietary demands and to cope with pregnancy related feeding problems, especially for those being on long-term cornstarch therapy for the night. *Frequent dietary monitoring and good compliance before and during pregnancy. Special attention is needed after delivery, the time needed to return to the original prepregnancy diet was di¡erent and determined by the glucose dependency. We would advocate to inform the patient about the optimal dietary protocol before pregnancy.

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PRIMARY PULMONARY HYPERTENSION AND GLYCOGEN STORAGE DISEASE TYPE I: THE SEROTONIN HYPOTHESIS Philippe Labrune*, Marc Humbert**, Philippe Herve¨**, Didier Samuel***, Ludovic Drouet****, Jean-Marie Launay****, Ge¨rald Simonneau**. *Service de Pe¨diatrie and **Service de Pneumologie, Hoªpital Antoine Be¨cle©re, 92141 Clamart cedex, and ***Centre He¨pato-Biliaire, Hoªpital Paul Brousse, Villejuif, and ****Service de Biochimie, Hoªpital Lariboisie©re, Paris, France. Glycogen storage disease type Ia is an autosomal recessive disorder due to a de¢ciency of hepatic glucose-6-phosphatase activity. Among the numerous complications which may occur in the course of the disease, pulmonary hypertension has been reported in a few cases and the role of vasoconstrictive amines, such as serotonin, in its development has been suggested. Patients and methods: Plasma serotonin concentrations were measured in 16 patients with severe pulmonary hypertension, 13 GSD Ia patients without pulmonary hypertension and one GSD Ia patient with severe pulmonary hypertension . Twenty-six normal age-matched healthy controls were also included in the study. Results: Elevated plasma serotonin concentrations were observed in patients with either severe pulmonary hypertension (38.8 + 7.3 nmol/L) or GSD Ia and no evidence of pulmonary hypertension (36.8 + 12.0 nmol/L) , as compared with controls (8.8 + 0.6 nmol/L, p5 0.001). Similarly, plasma serotonin concentration was markedly raised in the GSD Ia patient with pulmonary hypertension. Conclusions: The demonstration of elevated plasma serotonin in primary pulmonary hypertension and in GSD Ia suggests that this mediator could be involved in the development of pulmonary arterial hypertension in susceptible individuals.

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GLYCOGEN STORAGE DISEASE TYPE I : INCREASED LEVELS OF PROTEINS INVOLVED IN HAEMOSTASIS DESPITE NORMAL CONCENTRATIONS OF TNFa and IL6 Martine Wolf*,Catherine Boyer-Neumann*, Anne Marfaing-Koka*, Dominique Meyer*, Michel Odie©vre**, Philippe Labrune**. *Service d'He¨matologie Biologique, and **Service de Pe¨diatrie, Hoªpital Antoine Be¨cle©re, BP 405, 92141 Clamart Cedex, France.

Glycogen storage disease type I (GSD I) is due to a de¢ciency of the glucose-6-phosphatase system. Besides usual biological manifestations, GSD Ia patients often have hyperprotidemia. In order to elucidate the origin of the latter, we studied the proteins of the haemostatic system. The role of TNFa and IL-6, known to upregulate the synthesis of many haemostatic proteins was also investigated. Methods : Twenty-seven GSD I patients were evaluated and compared with 14 patients with GSD of other types (III and VI), and with 30 healthy controls. Haemostatic proteins were divided in 4 groups : 1) proteins exclusively synthesized by hepatocytes, ie FVIIc, protein C activity (PC Act) and C4bBP antigen (C4bBPAg), 2) proteins synthesized by both endothelial cells and hepatocytes, ie ATIII activity (ATIII Act), PS activity (PS Act), 3) a protein exclusively synthesized by endothelial cells, ie von Willebrand Factor Antigen (vWF Ag), 4) FVIIIc whose synthesis is ubiquitous. TNFa and IL-6 were measured by ELISA. Results : In GSD I patients, FVIIc, PC Act, C4bBP Ag concentrations were signi¢cantly increased, with mean values of at least twice the normal range, whereas the levels of these proteins were normal in the other two groups. ATIIIAct and PS Act concentrations had mean values at one and half the normal range in GSD I. Despite increased levels of FVIIIc, vWAg levels were normal. TNFa and IL-6 were normal. Conclusion : In GSD I, increased protein synthesis only involves hepatocytes.

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MUTATIONS IN THE GLUCOSE-6-PHOSPHATASE GENE OF 46 PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE IA Pascale Trioche*, Jeanne Francoual**, Jacqueline Chalas**, Liliane Capel**, Philippe Labrune**. *Laboratoire de Biochimie, and **Service de Pe¨diatrie, Hoªpital Antoine Be¨cle©re, BP 405, 92141 Clamart Cedex, France. Glycogen storage disease type Ia (GSD I) is due to a de¢ciency of the glucose-6-phosphatase system. Besides usual biological manifestations, GSD Ia patients often have hyperprotidemia. In order to elucidate the origin of the latter, we studied the proteins of the haemostatic system. The role of TNFa and IL-6, known to upregulate the synthesis of many haemostatic proteins was also investigated. Methods : Twenty-seven GSD I patients were evaluated and compared with 14 patients with GSD of other types (III and VI), and with 30 healthy controls. Haemostatic proteins were divided in 4 groups : 1) proteins exclusively synthesized by hepatocytes, ie FVIIc, protein C activity (PC Act) and C4bBP antigen (C4bBPAg), 2) proteins synthesized by both endothelial cells and hepatocytes, ie ATIII activity (ATIII Act), PS activity (PS Act), 3) a protein exclusively synthesized by endothelial cells, ie von Willebrand Factor Antigen (vWF Ag), 4) FVIIIc whose synthesis is ubiquitous. TNFa and IL-6 were measured by ELISA. Results : In GSD I patients, FVIIc, PC Act, C4bBP Ag concentrations were signi¢cantly increased, with mean values of at least twice the normal range, whereas the levels of these proteins were normal in the other two groups. ATIIIAct and PS Act concentrations had mean values at one and half the normal range in GSD I. Despite increased levels of FVIIIc, vWAg levels were normal. TNFa and IL-6 were normal. Conclusion : In GSD I, increased protein synthesis only involves hepatocytes.

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SPLENECTOMY IN GLYCOGEN STORAGE DISEASE TYPE 1b H. Peters1, K. Tiedemann2, A. Auldist3, D. Francis1, S. Casanelia1, L. Warwick1, A. Boneh1 VCGS1, Dept. of Haem.2, Dept. of Surg.3, Royal Children's Hospital, Melbourne, Australia Glycogen storage disease type 1b (GSD1b) is characterised by fasting hypoglycaemia, hyperlactaemia, hepato-splenomegaly, growth failure and recurrent infections as a result of neutropenia and phagocytic dysfunction. In£ammatory bowel disease and arthritis have been increasingly observed. Treatment with granuloctye colony-stimulating factor (GCSF) is accepted as part of the treatment protocol of GSD1b to reduce the number and severity of infections. The most signi¢cant adverse e¡ect of GCSF is severe splenomegaly, which may respond to dose reduction1. Two siblings with GSD1b aged 14 and 10 years, were treated with GCSF for 7 and 8 years at 10mg/ kg/day and 4.5mg/kd/day respectively. Severe splenomegaly and feeding di¤culties developed in both children, with some evidence of hypersplenism developing later. Attempts to reduce GCSF dosage resulted in inadequate neutrophil counts manifesting with aggravation of in£ammatory bowel disease and arthritis in the ¢rst sibling and mouth ulceration in the second. Splenectomy was therefore performed at age 12 and 8 years respectively. Post surgery both siblings had improved appetite and mobility. Marked improvement was observed in the enteritis and arthritis in the ¢rst sibling and mouth ulceration in the second. Notably both siblings now require lower maintenance doses of GCSF. We conclude that splenectomy be considered in GSD 1b patients with splenomegaly leading to adverse e¡ects, in whom reduction in GCSF dosage is not feasible. G. Visser et al. The long-term use of granulocyte colony-stimulating factor in Glycogen Storage Disease type 1 non a. Results of the European study on GSD type 1. JIMD 22:41, 19991

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ANTIOXIDATIVE PROTECTION AND OXIDATIVE STRESS IN GLYCOGEN STORAGE DISEASE IA (GSDIA), DIABETES MELLITUS TYPE I (DMI) AND FAMILIAL HYPERCHOLESTEROLEMIA (FH) B. Wittenstein, M. Klein, B. Finckh, K. Ullrich, A. KohlschÏtter Department of Pediatrics, University Hospital Hamburg, Germany* Background: Oxidative modi¢cation of lipoproteins in the subendothelium of the arterial vessel wall plays a central role in atherogenesis. Patients with DM I and FH are at high risk for premature atherosclerosis. Patients with GSDIa do not develop premature atherosclerosis despite severe hyperlipidemia. We analysed the endogenous antioxidative protection and oxidative stress in patients with GSDIa compared to patients with FH and DMI. Methods: The following parameters in serum and plasma of 72 patients (GSDIa n=17, FH n=18, DM I n=17, healthy controls n=20) (age 2-29 years) were analysed: TRAP (total radical-trapping ability), calculated TRAP, SH-groups, malondialdehyd, vitamin E, vitamin C, uric acid, homocystein, lipid pro¢le, oxidized LDL antibodies. Results: Patients with GSDIa show an elevated TRAP (p50,01) compared to the 3 other groups which can mainly be attributed to elevated uric acid levels (p50,05 vs. control). Patients with DMI show decreased SH-groups re£ecting decreased plasma antioxidative protein groups (p50,01 vs. control), in addition malondialdehyd (p50,001 vs. control) and serum oxidized LDL antibodies (p50,05 vs. control) as indicators of endogenous lipid peroxidation are elevated. In patients with FH TRAP and parameters of oxidative stress did not di¡er from healthy control subjects. Conclusions: Patients with GSDIa show an increased endogenous antioxidative capacity, which may protect against lipid peroxidation and thus against premature atherosclerosis. Patients with DM I exhibit increased oxidative mechanisms related to lipid peroxidation already at childhood age probably promoting premature atherosclerosis. In FH more detailed studies are necessary to evaluate correlations between the family history of early coronary heart disease and endogenous antioxidative capacity. J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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DISTINCT FEATURES IN ARGENTINEAN PATIENTS WITH LIVER GLYCOGENOSIS STORAGE DISEASE Paschini-Capra A, Giner-Ayala A, Guelbert N, De Kremer Dodelson R. CEMECO, Paediatric Department, School of Medicine, National University of Co¨rdoba. Children's Hospital, Argentina

Over a period of 18 years, 33 patients (P) were recognised to be a¡ected by liver glycogenosis storage disease (GSD). The series was composed by: GSD Type 0 (2 P); GSD Type I (9 P); GSD Type II (1 P); GSD Type III (8 P); GSD Type VI (6 P); GSD Type IX (7 P). The aim of this report is to point out those unusual or not described clinical and/or biochemical symptoms or signs. The following observations were noticed: a. Microcephaly in 5 P: GSD Type IX (1); GSD Type III (1); GSD Type Ia (3). b. Rickets: GSD Type Ia (1). c. Oligosaccharides in urine (OU) for TLC showed abnormal excretion in all P with GSD Type IX. d. Glycogen concentration in RBC (enzymatic method) was increased only in GSD Type III. e. Partial de¢ciency of liver glycogen synthase in GSD Type 0. The patients who showed more pronounced microcephaly were correlated with the severe forms of GSD Types I, III and IX. At variance with osteopenia and osteoporosis signs, the con¢rmed rickets (Rx and biochemical data) was a new ¢nding associated with GSD Type Ia. With respect to OU, it should be remarked its presence at childhood in GSD Type IX; these data have not been reported in the screening of liver GSD. We found increased glycogen in RBC only in patients with GSD Type III, which became normal in adolescent period. Our description of the Argentinean cases of GSD Type 0 (Medicina, Buenos Aires 50: 299, 1990) showed a distinct phenotype which contrasted with the severe forms described up to now. The data presented herein broaden the phenotypes in di¡erent types of liver GSD. A better understanding of these variations could be possible through a future genotypephenotype correlation.

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MUTATION ANALYSIS IN GLYCOGEN STORAGE DISEASE TYPE 1 NON-A A.R. Janecke1, M. Lindner2, M. Erdel1, E. Mayatepek2, D. MÎslinger3, T. Podskarbi4, F. Fresser1, S. StÎckler-Ipsiroglu3, G. F. Ho¡mann2, G. Utermann1 1 Institute of Medical Biology and Human Genetics, University of Innsbruck, Austria; 2University Children's Hospital Heidelberg, Germany; 3University Children's Hospital Wien, Austria; 4Labor fÏr Sto¡wechselgenetik, MÏnchen, Germany

Glycogen storage disease type 1 (GSD 1) comprises a group of autosomal recessive inherited disorders caused by de¢ciency of the microsomal multicomponent glucose-6-phosphatase system. Of the two known transmembrane proteins of the system, malfunction of the catalytic subunit characterises GSD 1a. GSD 1 non-a is characterised by defective microsomal glucose-6-phosphate or pyrophosphate/phosphate transport. Mutation analysis of G6PT (glucose-6-phosphate-translocase gene) encoding a microsomal transporter protein revealed mutations on both chromosomes in all 13 patients with GSD 1 non-a investigated, four of which were novel. An Austrian patient was compound heterozygous for a large intragenic deletion detected by FISH analysis, and the missense mutation S45R. A German patient was compound heterozygous for W227X and the splice site mutation, 1292+1G4A. The common mutations, 1211/1212delCT and G339C, accounted for 54% of disease alleles. A speci¢c distribution of certain mutations among patients of di¡erent origin was observed. G6PT mutation analysis thus con¢rmed the diagnosis in all cases including two patients suspected with GSD 1 non-a on clinical ¢ndings thus sparing liver biopsy taking. No correlation of the genotype with the severity of the disease was observed. The possibility of the presence of larger intragenic rearrangements should be kept in mind.

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GENE THERAPY WITH ADENO-ASSOCIATED VIRUS (AAV) VECTORS IN CANINE GLYCOGEN STORAGE DISEASE, TYPE IA D. D. Koeberl, R. M. Beaty, M. Jackson, P. Kishnani, E. Faulkner, S. VanCamp, T. Brown, A. Boney, and Y.-T. Chen. Division of Medical Genetics, Duke University Medical Center, Durham, NC 27710 (DDK, RMB, PK, EF, AB, Y-TC) and College of Veterinary Medicine, North Carolina State University, Raleigh, NC (SVC, MJ, TB). Current, nutritional therapy in glycogen storage disease type Ia (GSD Ia) does not appear to adequately prevent long-term sequelae, including renal dysfunction, gout, and hepatic adenomas. Thus, we have developed AAV vectors encoding glucose-6-phosphatase (G6Pase) as an approach to gene therapy in GSD Ia. We recently described a canine model for GSD Ia, and it resembles severe GSD Ia in humans. A¡ected puppies presented with lethargy and weakness, and developed growth delay prior to an early demise. Laboratory evaluation revealed hypoglycemia, lactic acidosis, hyperlipidemia, and elevated uric acid. Postmortem examination revealed hepatomegaly, nephromegaly, hepatic glycogen accumulation, and de¢cient G6Pase in liver. We have constructed an AAV vector encoding G6Pase driven by the mouse albumin promoter/enhancer (AVAlbG6PGH), and administered AVAlbG6PGH to an a¡ected puppy on day 3 of life. The puppy had hypoglycemia with blood glucose 29 mg/dl in response to fasting prior to administration of the AAV vector. Four weeks following vector administration fasting glucose was normal at 77 mg/dl. Increased growth for this puppy relative to other a¡ected puppies was observed. Liver G6Pase expression was equivalent to that for a carrier puppy, approximately 50% of normal levels. Further analysis will include vector DNA analysis in the liver and liver glycogen content. Initial results indicate that safety and e¤cacy for AAV vector-mediated gene therapy in canine GSD Ia could provide a needed, new approach to therapy for this disorder.

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GLYCOGEN STORAGE DISEASE TYPE IA AND TYPE IB: MOLECULAR CHARACTERIZATION OF 62 JAPANESE PATIENTS Y. Matsubara*, S. Kure*, Y. Suzuki*, T. Nishigaki**, K. Inui**, J. Akanuma*, K. Fujii*, DC Hou*, K. Takahashi*, T. Ohura***, S. Miyabayashi***, Y. Suzuki****, S. Okada**, and K. Narisawa*. Dept. of Medical Genetics and **Dept. of Pediatrics, Tohoku University School of Medicine, Sendai, Japan; ***Dept. of Pediatrics, Osaka University Faculty of Medicine, Suita, Japan; ****Dept. of Pediatrics, Gifu University School of Medicine, Gifu, Japan. Glycogen storage disease (GSD) type I is caused by either a de¢ciency of glucose-6-phosphatase (G6Pase)(type Ia) or an impairment of glucose-6-phosphate transporter (G6PT) across the microsomal membrane (type Ib). GSD-Ib is clinically distinguished from GSD-Ia by the presence of neutropenia. We analyzed G6Pase mutations in 51 Japanese patients with GSD-Ia and G6PT mutations in 11 Japanese patients with GSD-Ib. Mutations were identi¢ed in all 124 mutant alleles. The most prevalent mutation in the G6Pase gene was g727t, accounting for 88 of 102 mutant alleles, followed by R170X, R83H, P257L, IVS1-1g4a, G122D and H179P mutations. Aberrant mRNA splicing associated with the g727t and IVS1-1g4a mutations were readily detected in ``ectopically'' transcribed G6Pase mRNA in cultured lymphoblasts. In GSD-Ib, W118R mutation in the G6PT gene appeared to be a prevalent mutation, accounting for 10 in 22 mutant alleles. Interestingly two patients without neutropenia, clinically suspected of GSD-Ia, carried mutations in the G6PT gene, but not in the G6Pase gene. References: 1) Kure, BBRC 248:426, 1998; 2) Hou, AJMG 86:253, 1999; 3) Akanuma, AJMG 91:107, 2000; 4) Kure, J Pediatr (in press); 5) Takahashi, AJMG (in press).

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GLYCOGEN STORAGE DISEASE IB NEUTROPHILS SHOW OPPOSITE FINDINGS COMPARED TO NEUTROPHILS IN AN ACUTE BLOCKING MODEL WITH CHLOROGENIC ACID ANALOGUE S4048 G Visser1, M Huitema2, JP Rake2, AJ Meijer3, P Limburg2, HJ Burger4, AW Herling4, GPA Smit1, KE Niezen-Koning1. 1 Beatrix Children's Hospital; 2 Department of Clinical Immunology, University Hospital Groningen; 3 Department of Biochemistry Amsterdam, Academic Medical Center; The Netherlands, 4 Aventis Pharma Deutschland GmbH, Frankfurt am Main, Germany. Introduction Glycogen Storage Disease Ib (GSD-Ib) is caused by a glucose-6-phosphate (G6P) transporter de¢ciency. Recently synthetic chlorogenic acid derivatives (a.o S 4048) have been proven to be inhibitors of the G6P-transporter in liver and kidney, thereby mimicking GSD-Ib. Previously we described that neutrophils of patients with GSD-Ib show a decreased superoxide production and decreased intracellular G6P concentrations. Objective To study the e¡ect of S4048 on superoxide production and on intracellular G6P concentration in normal neutrophils. Results In the presence of S4048 and glucose, stimulation of the respiratory burst by PMA resulted in an enhanced superoxide production. Furthermore, after stimulation in the presence of S4048 intracellular G6P concentrations increased. Conclusions In neutrophils in the acute model results are opposite compared to those in the chronic disease state, but rational in view of the working model of the G6P transporter. This might indicate that in GSD-Ib neutrophil dysfunction is a secondary phenomenon.

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HYPERTHERMIA-LIKE SYNDROME AS A CAUSE OF DEATH IN INFANTILE GLYCOGENOSIS TYPE 2 B. Bembi, C. Martini, R. Bussani, F. Katouzian, A. Benettoni, G. Severini, E. Arbustini, G. Ciana. I.R.C.C.S. Burlo Garofolo - Trieste, Italy

Background. Glycogenosis type 2 is a lysosomal storage disorder characterised by an inherited recessive autosomal de¢ciency of a-glucosidase. Di¡erent phenotypes are described according to the age of onset and to the severity of the disease. At present the therapeutic experiences are limited to the dietary management of patients with the juvenile or adult form. Methods. Two patients (a ¢ve years old female and a three years old male) with the type 2 late infantile form of glycogenosis were treated with a high protein diet enriched with the branched chain aminoacids. The diet was accompanied by a physiotherapy program. Results. After 12 months of dietary treatment and physiotherapy the patients' clinical condition showed signi¢cant improvement (muscular strength, nutritional state, respiratory and cardiac function). Both the cases presented with an elevated and persistent fever not apparently related to septic episodes and was not responsive to any antipyretic or antibiotic treatment. The fever was accompanied by a dramatic decrease in seric creatin-kinase and an increase of lactic-dehydrogenase. They felt into a comatose state and died. The autopsy performed in one patient,also demonstrated the involvement of the central nervous system with glycogen storage in the cerebral cortex, cerebellum,brainstem and spinal cord. Conclusions. This study shows how the dietary-phisiotherapy approach only temporarily improves the clinical course of infantile type 2 glycogenosis. Nevertheless it could be a useful approach while waiting for enzyme replacement therapy.

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PREVALENCE AND MOLECULAR GENETIC BASIS OF GLYCOGEN STORAGE DISEASE TYPE IIIA (GSD IIIA) IN THE FAROE ISLANDS R Santer 1, M Kinner 1, U Steuerwald 2, F Skovby 3, YT Chen4 , R Schneppenheim 5, J Schaub 1 University Childrens`s Hospital 1Kiel and 5Hamburg, Germany, 4Durham, NC, USA; 2National Hospital, To¨rshavn, Faroe Islands; 3Dpmt of Clinical Genetics, Copenhagen, Denmark GSD IIIA is relatively rare; it is caused by mutations of the amyloglucosidase gene (AGL). In most populations, none of the AGL mutations described to date is particularly frequent, which is also demonstrated by the fact that we found only novel mutations when investigating 6 families with GSD IIIA living in central Europe. In contrast, GSD IIIA is very frequent in the Faroe Islands. Objective: To de¢ne the molecular basis and the prevalence of GSD IIIA in the Faroe Islands. Methods: Molecular genetic analysis of the AGL gene in (i) 6 out of 14 GSD IIIA patients known to us from the Faroe Islands (population 45,000), (ii) numerous family members, and (iii) in 200 randomly chosen individuals each from the Faroe and the German neonatal screening program. Results: A novel homozygous nonsense mutation c.1222C4T (R408X), always associated with the same haplotype (de¢ned by 4 intragenic polymorphisms), was found in all investigated GSD IIIA cases. This was con¢rmed by restriction enzyme digest with NsiI after mismatch PCR. The mutation was not detectable in 200 German newborns, but 6 of 111 Faroe newborns were heterozygous, predicting a prevalence of one GSD IIIA case per *1,400 individuals (95% CI 1: 280 1:3,500). Conclusion: Probably due to a founder e¡ect, GSD IIIA is extraordinarily frequent in the Faroe Islands. The detection of the molecular defect has facilitated the diagnosis and o¡ers the possibility for prenatal diagnosis of GSD IIIA in this patient group.

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NOVEL FOUR BASE PAIR DELETION IN EXON 10 ASSOCIATED WITH EXON 11 SKIPPING IN A PATIENT WITH GLYCOGEN STORAGE DISEASE TYPE IIID a Tokiko Fukuda, aHideo Sugie, a Masataka Ito, a Yoko Sugie, bTohru Kojo a Department of Pediatric Neurology, Hamamatsu City Medical Center for Developmental Medicine, Hamakita, Japan, b Department of Neurology, Jichi Medical College, Tochigi Objective: Glycogen storage disease (GSD) IIId is a rare subtype of GSD III . We report novel exon 11 skipping mutation in a patient with GSD IIId. Patient: A 38-year-old woman presented with easy fatigability. Muscle histohemistry revealed vacuoles with intense periodic acid Schi¡ (PAS) positivities. The debranching enzyme (AGL) activities in biopsied muscle were undetectable by spectrophotometric assay using phosphorylated limit dextrin (PLD) as substrate, whereas the activities were normal by a methods on labeled-glucose incorporation into glycogen. Methods: (i) cDNA analysis. RT-PCR using total RNA extracted from the patient's muscle and the sequencing of the ampli¢ed products (ii) Genomic DNA analysis. PCR of the £anking and encompassing the abnormal cDNA sequences using the patient's muscle and the sequencing of the ampli¢ed products (iii) Restriction analysis. The region encompassing the mutation was ampli¢ed by PCR. The ampli¢ed fragments were digested by Tru9I. Results: cDNA and DNA analysis revealed 1117-1120del and exon 11 skipping. Restriction analysis con¢rmed this mutation was homozygous. Discussion :This is the ¢rst mutation report of a patient with GSD IIId. Several mutations have been reported in the patients with type IIIa and b, however phenotype and genotype correlation is still uncertain.

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GLYCOGEN STORAGE DISEASE (GSD) TYPE IV: HETEROGENEOUS PHENOTYPES FROM FATAL INFANTILE CARDIOMYOPATHY TO ADULT POLYGLUCOSAN BODY DISEASE (APBD) Yoon S. Shin1, T. Podskarbi2, J.M. Schro«der3, M. Vorgerd4 University Children's Hospital, Munich, Germany1 Metabolism and Genetics Laboratory, Munich, Germany2. Dept. of Neuropathology, University of Aachen, Germany3. Dept. of Neurology, Ruhr University, Bochum, Germany4

De¢ciency of glycogen branching enzyme (GBE) is known to cause GSD type IV (Andersen's disease) characterized by marked cirrhotic hepatomegaly. Recently, however, other phenotypes of GBE de¢ciency such as late onset cardiomyopathy and muscular hypotonia or hydrops fetalis have been described. We report here two unique phenotypes of GBE de¢ciency: the ¢rst case concerns a German premature boy of unconsanguineous marriage with muscular hypotonia, hypertrophic cardiomyopathy, hypoglycemia, macrosomia and hepatomegaly, who died at 41/2 months of bradycardia. The second case concerns a 46-year-old German woman who revealed polyglucosan bodies in the sural nerve biopsy and showed clinical ¢ndings of APBD such as gait disturbance, spastic tetraparesis and progressive hearing loss. In both cases the GBE activity was de¢cient in leukocytes as well as in cultured ¢broblasts. In the latter patient we found not only de¢ciency of GBE as described in a subgroup of Jewish APBD patients, but also two novel missense mutations in the GBE gene, Arg515His and Arg542Gln. Even though clinical phenotypes of these cases are indeed di¡erent, the decrease in the GBE activity in leukocytes were comparable and furthermore all heterozygotes showed an intermediate activity. This suggests again the usefulness of the GBE assay in blood cells for an initial diagnosis of which the con¢rmation can be made by molecular genetic analysis. Thus, multiplicity of GBE de¢ciency should be explored further in respect of such patients described here in addition to previously reported various hepatic and muscular forms.

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MOLECULAR GENETIC ANALYSIS OF JAPANESE PATIENTS WITH MYOPHOSPHORYLASE DEFICIENCY (MCARDLE'S DISEASE) Masataka Ito, MD, Hideo Sugie, MD, Tokiko Fukuda, MD, Yoko Sugie, MD Department of Paediatric Neurology, Hamamatsu City Medical Centre for Developmental Medicine, Hamakita, JAPAN.

Objectives: R49X has been reported as the predominant mutation in McArdle's disease in USA and UK, accounting for 75% and 83% of the cases, respectively. We have reported that there is a distinct ethnic di¡erence in genetic mutation in McArdle's disease (CCA 236:81-86, 1995). We studied molecular genetic analyses in 22 Japanese patients with McArdle's disease and report three novel mutations. Patients and Methods: We studied 22 patients diagnosed as having McArdle's disease (7 female, 15 male), ranging in age from 1 to 79 years. In all patients myophosphorylase de¢ciency was documented biochemically by a muscle biopsy. RT-PCR using total RNA extracted from the patients' muscle biopsies and PCR using genomic DNA with restriction analysis were performed for search genetic mutations. And three reported mutations such as R49X, G204S and K542T, frequently observed in Caucasian patients were also analysed. Results: 708/709delTTC was found in 10 cases (45 %). And we identi¢ed three novel mutations such as 74-87del, R138W and exon 18 skipping. R49X, G204S and K542T were not found in our series. 708/709delTTC is still the predominant mutation in Japanese patients with McArdle's disease, however the molecular genetic heterogeneity is also present in the Japanese population.

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LIVER PHOSPHORYLASE DEFICIENCY: A POTENTIALLY LIFE-THREATENING DISEASE DURING INFECTIONS G. Hu«ner1, G. S°arbat1, T. Baykal1, T. Podskarbi2, Y.S. Shin3, M. Demirkol1 Dept. Pediatric Nutrition and Metabolism, Istanbul Faculty of Medicine, University of Istanbul, Istanbul, Turkey1; Molecular Genetics and Metabolism Laboratory, Munich, Germany2; Children's Hospital, University of Munich, Munich, Germany3 Glycogen storage disease due to a defective hepatic phosphorylase system is a heterogenous group of liver glycogenosis consisting of type VI (liver phosphorylase de¢ciency), Via (phosphorylase kinase de¢ciency) and phosphorylase system defect (type VIII, IX or X). In liver phosphorylase de¢ciency hepatomegaly, growth retardation, mild hypoglycemia, hyperlipidemia, hyperketosis and elevated transaminases with benign course are the prominent features. Two patients (9- and 6.5-year-old females) followed from our department with the diagnosis of liver phosphorylase de¢ciency had ¢ve episodes of life-threatening attacks during infections that required hospitalization. Similar clinical and laboratory pathology was recorded during these attacks. Severe metabolic decompensation with hypoglycemia (glucose: 20 mg/dl), metabolic acidosis (pH: 7.0, HCO3: 3.0 mmol/l), ketonuria (+++), elevated transaminases (ALT: 416 IU/L, AST: 132 IU/L), hyperlactemia (lactate: 7.5 mmol/l) and hyperuricemia (uric acid: 14 mg/dl) were the most pathologic ¢ndings recorded during these episodes. The patients received intravenous glucose and bicarbonate treatment during these attacks. Both patients had normal psychomotor development, physical examination and laboratory ¢ndings at their last out-patient follow-up. Although liver phosphorylase de¢ciency is reported as a benign disorder it may be a life-threatening condition during mild infections.

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IDENTIFICATION OF THREE NOVEL MUTATIONS IN THE PHKA2 GENE IN CZECH PATIENTS WITH X-LINKED LIVER GLYCOGEN STORAGE DISEASE TYPE IX Jana Rudolfova¨ 1, Romana Slova¨cíkova¨ 1, Martin Trbusíek1, Sylvie Sít'astna¨ 2, Karol|¨ na Pesíkova¨ 2, Milan Elleder2, Libor Koza¨k1 1 Research Institute of Child Health, Department of Biochemical and Molecular Genetics, Cernopolni 9, CZ-662 62 Brno, Czech Republic; 2 Institute for Inherited Metabolic Disorders, Prague, Czech Republic Mutations in the gene for a-subunit of liver phosphorylase kinase (PHKA2) are responsible for glycogen storage disease type IX (GSD IX). We analyzed molecular defects in the PHKA2 gene by direct sequencing of RT/PCR products in four patients from three Czech unrelated families with Xlinked liver glycogenosis type IX. Consequently we developed PCR/restriction enzyme digestion analysis of natural or ampli¢cation created restriction sites (ACRS) to analyze other family members and to verify the mode of inheritance. We have found three novel mutations. A point mutation G3629A in exon 33 which causes glycine (GGG) to glutamic acid (GAG) substitution at codon 1210 (G1210E) was found in two male patients in hemizygous state and in one female patient in heterozygous state (family 1). A G272A mutation in exon 3 which causes cysteine (TGC) to tyrosine (TAC) substitution (C91Y) was found in two patients from family 2, male patient is hemizygote and female patient is heterozygote for C91Y. A deletion of one nucleotide 3401delC in exon 32 was found in two male patients (family 3) in hemizygous state. This mutation causes a frameshift and leads to a premature termination. Additionally we screened 100 alleles of healthy controls for each mutation to ensure that these were not polymorphisms. This work was supported by grant no. 302/97/0742 from GA CR, Czech Republic.

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GLYCEROL KINASE DEFICIENCY - CLINICAL HETEROGENEITY AND MOLECULAR FINDINGS Adamowicz M., Hellerud Ch.*, Jurkiewicz D., Ciara E., Taybert J., Kubalska J., Popowska E., Lindstedt S.*, Pronicka E. Children's Memorial Health Institute Warsaw, Poland and *Institute of Laboratory Medicine Go«teborg University, Sweden Glycerol kinase de¢ciency (GKD) can be an isolated monogenic disorder or occur as a part of contiguous gene syndrome together with congenital adrenal hypoplasia (AHC) and/or Duchenne muscular dystrophy (DMD). Clinical presentation of the contiguous gene deletion syndrome is dominated by the life-threatening associated abnormalities. Isolated GKD is a heterogenous disorder. Some cases are asymptomatic (benign) and identi¢ed accidentally by detection of pseudohypertriglyceridemia or glyceroluria. Others were reported to present with e.g. hypoglycaemia, ketonuria and metabolic acidosis. The underlying biochemistry is not fully understood. Five cases of hyperglyceroluria were found by us: three came from urinary GC-MS screening and two others - from hyperlipidaemia screening. De¢ciency of glycerol kinase activity was con¢rmed in leukocytes of all ¢ve patients. We identi¢ed a contiguous gene deletion syndrome, involving GK, DMD and AHC genes in one case (3 months old boy with hypotony and hyperkaliemia) and GK and AHC genes in the second case (15 days old boy with adrenal insu¤ciency). The remaining three patients showed isolated GKD, and presented with rather mild symptoms like: neonatal respiratory failure or episodes of abdominal pain, vomitus and ketonuria during childhood. Molecular studies revealed some new mutations (deletion of GK and DAX-1 genes and part of DMD gene, deletion of GK and DAX-1 genes, point mutations in GK gene: ex 16 1507-1508insG and IVS1+4A4G) responsible for the observed clinical symptoms in 4 examined cases. Both mothers of the patients with point mutations were carriers of the mutation.

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HYPERKETONEMIA IN GLYCEROL KINASE DEFICIENCY D.R. Sjarif1,2, L. Dorland1, W. Sperl3, T.J. de Koning1, F.A. Beemer2, B.T. Poll-The1, M. Duran1 Department of Pediatrics/Metabolic Diseases, Wilhemina Children's Hospital1, Department of Medical Genetics, University Medical Center Utrecht, Utrecht, The Netherlands2, St. Johanns-SpitalLandesklinik fu«r Kinder- und Jugendheilkunde, Salzburg, Austria3

It is generally accepted that ketone bodies are excellent alternative fuels to the brain during starvation, when glucose production becomes limited. Hyperketonemia has been demonstrated to induce an increased cerebral blood £ow, but at the same time causes a reduction of the cerebral glucose metabolism. The total e¡ect of hyperketonemia on the brain is slightly detrimental, as positron-emission-tomography studies have shown that the mesencephalon will not oxidize ketone bodies as e¤ciently as the rest of the brain. Isolated glycerol kinase de¢ciency (GKD) has been associated with episodes of metabolic decompensation leading to drowsiness. These attacks were accompanied by hypoglycemia and/or hyperketosis. We investigated ¢ve patients (age 1^4 years) with isolated GKD who were subjected to a prolonged fasting test. None of the tests gave any adverse e¡ect; hypoglycemia occurred in two patients. The data of the fasting tests were compared with those of Bonnefont et al (1990). In general GKD patients had higher ketone body concentrations than the controls; their blood glucose values did not di¡er. Plasma glycerol concentrations did not increase dramatically. It is hypothesized that the observed hyperketonemia is due to overproduction of ketone bodies as a result of enhanced fatty acid oxidation at the expense of triglyceride synthesis.

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``SIMPLE'' MENDELIAN DISORDERS ARE COMPLEX TRAITS: HUMAN GLYCEROL KINASE DEFICIENCY AS A MODEL KM Dipple, YH Zhang, J Havens, K Nagano, BL Huang, and ERB McCabe. UCLA Department of Pediatrics, Los Angeles CA 90095-1752 Isolated glycerol kinase de¢ciency (GKD) is either symptomatic (episodic metabolic decompensation) or asymptomatic (pseudo-hypertriglyceridemia only). To better understand GKD pathogenesis, we identi¢ed patients' missense mutations, measured their GK activities and mapped the mutations to the E. coli GK protein (65% similar to human GK) three-dimensional structure (Hurley et al., Science, 259:673, 1993). We analyzed mutations in one symptomatic (R405Q) and 5 asymptomatic (D198G, N288D, A305V, M428T, Q438R) patients. The GK activity in lymphoblastoid cells from the symptomatic patient (6.7% of normal control) was indistinguishable from the asymptomtatic patients (4.2 8.4%). Mapping of these six missense mutations and two from reported asymptomatic patients (D440V, Walker et al., Am J Hum Genet 58:1205, 1996; W503R, Sjarif et al., J Med Genet 35:650, 1998) showed that the symptomatic patient's mutation was indistinguishable from the asymptomatic patients'. We conclude that genotype does not predict phenotype for GK missense mutations. We speculate that the phenotype of symptomatic GKD is dependent on reduction of the net £ux into one or more critical intermediates from glycerol, and that £ux will be in£uenced by polymorphisms in related enzymes. Observations of intrafamilial phenotypic di¡erences in humans (Walker et al., Am J Hum Genet 58:1205, 1996) and levels of metabolites in Gyk knockout mice (Huq et al., Hum Molec Genet 6:1803, 1997) support the speculation that the GKD phenotype is a complex trait.

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A NEW DISORDER IN THE PENTOSE PHOSPHATE PATHWAY: LIVER CIRRHOSIS CAUSED BY TRANSALDOLASE DEFICIENCY? Huck JH1,2, Verhoeven NM2, Roos B2, Struys EA2, Douwes AC1, van der Knaap MS1, Jakobs C2 1 Department of Pediatrics and 2Department of Clinical Chemistry, Metabolic Unit, Free University Hospital, Amsterdam The Netherlands The Pentose Phosphate Pathway (PPP) is an alternative pathway for the oxidation of glucose and present in all human tissues. It can be divided into an oxidative irreversible stage and a non-oxidative reversible stage. The reactions of the non-oxidative stage are regulated by two enzymes: transketolase and transaldolase. Transketolase catalyses the conversion of pentose-5-phosphates into glyceraldehyde-3-phosphate (Gly-3-P) and sedoheptulose-7-phosphate (Se-7-P). Transaldolase subsequently regulates the conversion of Gly-3-P and Se-7-P into fructose-6-phosphate (F-6-P) and erythrose-4phosphate (Ery-4-P). We present a 10-year-old girl with congenital liver cirrhosis. Analysis of urine and plasma showed highly elevated levels of D-arabitol, ribitol and erythritol. Presumably, the pentose precursors of these pentitols are formed through the PPP. We developed an enzyme assay in lysed erythrocytes and lymphoblasts for transketolase and transaldolase activities. After incubation with pentose-5-P, the formation of products was followed in time by capillary gas chromatography with a nitrogen phosphorous detector (GC-NPD). In cells from the patient, normal amounts of Gly3-P and Se-7-P were formed, indicating a normal activity of transketolase. However, no formation of F-6-P and Ery-4-P was observed, which points to a de¢ciency of transaldolase. The accumulation of the polyols is thought to be a result of the transaldolase de¢ciency, but the exact metabolism of polyols has, so far, not been revealed. Whether the liver cirrhosis is directly related to either the polyol accumulation or the transaldolase de¢ciency remains to be elucidated.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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L-ARABINOSURIA: A NEW DEFECT IN HUMAN PENTOSE METABOLISM W.Onkenhout, J.E.M.Groener, C.Yin and L.A.E.M.Laan Departments of Pediatrics and Neurology, Leiden University Medical Centre (LUMC), Leiden, The Netherlands

The female patient, the ¢rst child of healthy non-consanguineous Egyptian/Dutch parents, presented at the age of 16 months with delayed motor development and (facial) dysmorphism. In addition she had an extreme kyphosis, resulting in spastic tetraparesis. Oto-Palato-Digital syndrome type I was suspected, however this could not be con¢rmed. Biochemical investigation of urine showed the presence of reducing substances, not being glucose. Enzymatic determination of D-galactose showed an excretion of 6610 mmol/mmol creat, however further TLC analysis of mono-/disaccharides and GCMS analysis of polyols showed that the sugar in question was not D-galactose but L-arabinose. Additionally we found large amounts of L-arabitol and arabinoic acid(-lactone). L-arabinose was normal in both plasma and CSF, however L-arabitol was slightly increased in plasma and 4-fold increased in CSF. To our knowledge, L-arabinose is almost exclusively present in fruit. In the period the patient was hospitalized she took about 200 g of fruit formula daily, containing 1.9-3.9 mg/g of L-arabinose. When she was on a fruit-free diet for one week the abnormalities in urine disappeared. Knowledge of the metabolism of L-arabinose in man is very scarce. Hitherto essential pentosuria is the only well characterized inborn error in human pentose metabolism. Based on the results we presume that L-arabitol dehydrogenase is de¢cient in this patient. At this moment the enzyme has only been described in yeast and is neither puri¢ed nor cloned.

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CORRELATION BETWEEN CLINICAL PHENOTYPE AND GENOTYPE IN CONGENITAL HYPERINSULINISM AND HYPERAMMONEMIA Fujioka H, Okano Y, Inada H, Asada M, Hase Y, Yamano T. Dept. of Pediatr. Osaka City Univ. Grad. Sch. of Med. 545-8585 Osaka, Japan

Congenital hyperinsulinism and hyperammonemia (CHH) is caused by uncontrollable regulations of glutamate dehydrogenase (GDH). We report here the molecular characterization of GDH and the relations between phenotypes and genotypes in two Japanese patients with CHH. In the enzymatic analysis, the basal levels of the GDH activities in lymphoblasts of patients 1 and 2 showed three-folds higher than those in the wild-controls. In the inhibition assay for GDH, half-maximal inhibitory concentration of GTP in wild-controls, patient 1, and 2 were 0.2mM 0.7mM, and 2mM, respectively. In the stimulation assay, half-maximal stimulatory concentration of ADP in patients 1 and 2 were the same as those in the wild-controls. In the genetic analysis, patients 1 and 2 were heterozygous for L413V and G446D, respectively, which were de novo mutations. In vitro GDH activities of L413V and G446D in the COS cell expression system were consistent with those in patients' lymphoblasts. The immuno-reactivities of mutant proteins were the same as the wild-controls. L413V outside the GTP binding site showed milder impairment of GDH activity, compared with G446D in the GTP binding site. In the clinical phenotype, patient 1 with L413V had symptomatic hypoglycemia at 4 months of age. On the other hand, patient 2 with G446D showed hypoglycemia on the 5th day of birth, and had subtotal pancreatectomy to control hypoglycemia at 6 months. Finally, we were able to ¢nd the correlation between genotypes and phenotypes in CHH.

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GLUTAMATE DEHYDROGENASE ACTIVITIES IN LYMPHOBLASTS FROM PATIENTS WITH THE HYPERINSULINISM-HYPERAMMONEMIA SYNDROME Toshihiro Ohura, Yuko Miki*, Tomohiko Taki*, Yasuhide Hayashi*, Osamu Sakamoto, Aki Asanuma, Jun-ichiro Aikawa, Yutaka Igarashi and Kazuie Iinuma Department of Pediatrics, Tohoku University School of Medicine, Sendai, JAPAN; *Department of Pediatrics, Faculty of Medicine, the University of Tokyo The hyperinsulinism-hyperammonemia syndrome (HHS) is a recently identi¢ed genetic disorder that is characterized by hypoglycemia and hyperammonemia in infants and children. In the present study, we analysed three di¡erent mutant GDHs in lymphoblasts cultured from three patients with HHS. The sensitivity of GDH with the S445L mutation, located in the GTP-allosteric domain, to inhibition by GTP was reduced as was previously reported. The GDH from patients with the E296A or the R265K mutation, located outside the GTP-allosteric domain, was also less sensitive to inhibition by GTP. Our ¢ndings suggest that mutations outside the GTP-allosteric domain of GDH also a¡ect the sensitivity to inhibition by GTP (Table). Since a child with mild HHS (Patient 2) exhibited a lower GDH enzyme activity in the absence of e¡ectors, determination of basal GDH activity may be useful in predicting the severity of this syndrome. Table: Activity and allosteric responsiveness of Glutamate dehydrogenase (GDH).

GDH activity (nmol/min/mg) IC50 of GTP (nmol/L)

Control (n=7)

Patient 1 (S445L)

Patient 2 (E296A)

Patient 3 (R265K)

14.9 + 3.1 115 + 25.1

18.8 + 5.6 473 + 32.1

8.27 + 2.6 330 + 90.0

15.2 + 4.0 237 + 15.3

IC50: Half-maximal inhibitory concentration

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FACIAL DYSMORPHISM IN PERSISTENT HYPERINSULINEMIC HYPOGLYCEMIA Pascale de Lonlay1, Vale¨rie Cormier-Daire2, Jean-Christophe Fournet3, Francis Brunelle4, Claire Nihoul-Fe¨ke¨te¨ 5, Claudine Junien6, Jean-Jacques Robert1, Jean-Marie Saudubray1 1 De¨partement de Pe¨diatrie, 2De¨partement de Ge¨ne¨tique, 5De¨partement de Chirurgie Infantile, 4Service de Radiologie Pe¨diatrique, 3Service d'Anatomopathologie, 6INSERM-U383, Hoªpital Necker-Enfants Malades, 149 rue de Se©vres, 75743 Paris Cedex 15, France Persistent hyperinsulinism is the most common cause of recurrent hypoglycemia in infancy due to inappropriate oversecretion of insulin by the pancreas. Pancreatic lesions can be either focal or di¡use and they have distinct molecular basis. We report here facial features in 13 unrelated patients presenting with severe neonatal (n=6) and infancy-onset (n=7) hyperinsulinism, related to focal adenomatous hyperplasia (n=7), di¡use hyperinsulinism (n=3) and hyperinsulinism with hyperammonemia (n=3). SUR1 or Kir6.2 mutations were found in 7/7 focal adenomatous hyperplasia and 1/ 3 di¡use hyperinsulinism. Dysmorphic features included high forehead, large and bulbous nose with short columella, smooth philtrum and thin upper lip in all patients. Large birth weight (mean 4 3870g) was consistantly observed (10/13) but protruding tongue or visceromegaly were never observed and the diagnosis of Beckwith Wiedemann was always carefully ruled out. Facial features have never been reported in hyperinsulinism and their pathogenesis is not understood. Their observation among patients with hyperinsulinism of various mechanisms could be related to a fetal intoxication by insulin.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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HYPERINSULINISM IN ASSOCIATION WITH A SYNDROME-LIKE PHENOTYPE T. Meissner1, B. Beinbrech1, K. Mohnike2, W. Rabl3, G.F.Ho¡mann1, E. Mayatepek1 Department of General Pediatrics, University Children's Hospital 1Heidelberg, 2Magdeburg, 3 MÏnchen (TU), Germany;

Object: Identi¢cation of patients with congenital hyperinsulinism with atypical syndrome-like clinical phenotype and description of such variants. Methods: Analysis of a clinical database of a total number of 68 patients from Germany with documented hyperinsulinism. Results: A total number of 5 patients were identi¢ed with hyperinsulinism and major additional clinical problems. Three of them presented with chronic pulmonary disease, congenital heart defects and severe psychomotor retardation suspicious of a carbohydrate de¢cient glycoprotein syndrome; a fourth patient was clinically characterised by an ondine's syndrome. The ¢fth patient with BeckwithWiedemann-syndrome su¡ered from an unusual severe and persistent hyperinsulinism requiring subtotal pancreatectomy. Conclusions: The basis of the hyperinsulinism of the reported patients remains unclear and requires further evaluation. Hyperinsulinism seems to be more common than previously thought in association with further clinical problems and is in this situation probably not caused by the known primary genetic defects in the regulation of the pancreatic b-cell. Transient or mild episodes of hypoglycemia may result in di¤culties to diagnose hyperinsulinism. However, in most cases medical treatment is necessary.

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PULSATILE INSULIN SECRETION IN PERFUSED LANGERHANS ISLETS FROM PATIENTS WITH CONGENITAL HYPERINSULINISM T. Meissner1, M. Hoemme2, R. Zelezny1, G. F. Ho¡mann1, F. Schaefer2, E. Mayatepek1 Department of Pediatrics, Divisions of Metabolic Diseases1 and Nephrology2, University Children's Hospital Heidelberg, Germany

Object: Insulin secretion of isolated Langerhans islets from 2 patients with congenital hyperinsulinism. Both patients su¡ered from severe hyperinsulinism and subtotal pancreatectomies were performed. Histology revealed di¡use b-cell hyperplasia. Methods: A perfusion chamber system was used to measure the insulin secretion under di¡erent glucose concentrations (1-3-11 mM), diazoxide (250mM) or somatostatin (1mM). Results: Mean insulin secretion of 100 islet equivalents was up to 30 mU/min at 11mM glucose concentration. Islets of both patients responded to an increase of the glucose concentrations (3 to 11 mM) with a 1.3- to 2.8- fold increase of insulin concentrations. Insulin secretion was clearly pulsatile in the phase of glucose increase. Somatostatin suppressed insulin release (44-60 % inhibition). Addition of diazoxide resulted in 40 % inhibition of insulin secretion. Conclusions: The perfusion chamber is a useful system to investigate the temporal dynamics of insulin secretion in Langerhans islets of patients with congenital hyperinsulinism. The Langerhans islets were still responsive to glucose changes but the changes in insulin secretion were relatively poor. Somatostatin as well as diazoxide inhibited the insulin secretion in vitro but the minor responses correlate to the inadequate success of medical treatment in vivo.

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176

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MYOCLONIC EPILEPSY AND PECULIAR ABNORMALITY OF CNS IN A PATIENT WITH HYPERINSULINISM AND HYPERAMMONIEMIA ENZYME OF NEUROACTIVE AMINOA M. Di Rocco, U. Caruso*, P. Picco, G. Minniti, A. Loy, P. Fondelli Gaslini Institute and *DIPE University of Genoa, Italy Hyperinsulism and hyperammoniemia syndrome is a genetic disease due to excessive activity of glutamate dehydrogenase, which oxidizes glutamate to alpha-ketoglutarate. This enzyme is a regulator of insulin excretion in pancreatic beta cells and of ureagenesis in the liver (N Engl J Med 1998; 338:1352^7). We studied an 8-year-old girl, ¢rst child of healthy unrelated parents. From ¢rst days of life she presented with episodes of symptomatic hypoglycemia and hyperinsulinism, diazoxide-responsive. Hyperammoniemia was occasionally discovered: plasma ammonia ranged from 213 and 251 mM per deciliter. Plasma aminoacids, urine orotic acid and urine organic acids were normal. Protein loading did not change plasma ammonia levels. No symptoms of hyperammoniemia such as lethargy or coma were evident. At the age of 7 years she ¢rst had seizures. Clinical and electrophysiological ¢ndings were consistent with a myoclonic epilepsy. Further clinical course was severe because epilepsy was not controlled by antiepileptic drugs and the child developed mental regression. Brain MMR showed a very peculiar picture of lateral ventricles, which appeared irregularly shaped, with symmetric cavities separated by septa from lateral ventricles. To our knowledge this is the ¢rst patient with myoclonic epilepsy and hyperinsulinism and hyperammoniemia syndrome; it is possible that this association is not casual because glutamate dehydrogenase has an important role in brain as regulatory acids (Neurochem Res 1999; 24:11:1387^ 95).

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GLUTAMATE DEHYDROGENASE GENE (GLUD1) MUTATIONS: NOVEL MECHANISMS LEADING TO CONGENITAL HYPERINSULINISM (CH) R Santer 1, R Schneppenheim 2, M Kinner 1, M Passarge 1, A Superti-Furga 3, J Schaub 1 University Childrens`s Hospital 1Kiel , 2Hamburg, Germany, and 3Zurich, Switzerland

Glutamate has been shown to be a key regulator of insulin secretion of pancreatic Þ-cells and activating mutations of the glutamate dehydrogenase gene a¡ecting the allosteric domain of the enzyme (encoded by exons 10, 11, and 12) have been found as a cause of the hyperinsulinismhyperammonaemia syndrome (HHS). Objective: To elucidate the role of GLUD1 mutations and their functional e¡ects in CH. Methods: Molecular genetic analysis of GLUD1 in 7 cases from 4 families with late-onset, mild hyperinsulinism who responded to dietary or medical therapy. Characterization of the regulation of GLUD activity by GTP and ADP in isolated lymphocytes from these patients and 8 controls. Results and Conclusion: In all patients, we found 1 of 3 novel heterozygous GLUD1 mutations: c.705G4A (R231Q), c.833C4T (R274C), c.978G4A (R322H) within exons 5, 6, and 7, resp.. The latter two mutations were associated with a decreased inhibition of GLUD activity by GTP despite the fact that both predict an e¡ect not on the allosteric but on the catalytic domain of the enyme. The exon 5 mutation was associated with a diminished stimulation of GLUD activity by ADP, an entirely novel mechanism in patients with hyperinsulinism. Although dominant inheritance (found in 1 of 4 families), hyperammonaemia (3/4), and leucine sensitivity (2/3) seem to be chaaceristic for HHS, they were not conistently found in the 4 families. We therefore conclude that GLUD1 mutations are probably more commonly responsible for CH than previously anticipated.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GLUT1-DEFICIENCY IN BRAIN Klepper J, Herrmann R, Kutzick, C, Guertsen E, FlÎrcken A, Voit T. Department of Pediatrics and Pediatric Neurology, University of Essen, Germany

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GLUT1-de¢ciency is caused by a defect in glucose transport into brain mediated by the facilitative glucose transporter GLUT1. In addition to 25 patients identi¢ed, we have diagnosed 10 patients in Germany within the past year. Clinical features include infantile seizures (100%), developmental delay (100%), acquired microcephaly (75%) and movement disorders (50%). Symptoms are often associated with fasting. The biochemical hallmark is hypoglycorrhachia in the setting of normoglycemia (CSF/blood glucose ratio 50.4) and low to normal CSF lactate. Neuroimaging is uninformative. The GLUT1-defect can be con¢rmed by immunoblotting, impaired glucose uptake into erythrocytes, and molecular analyses of the GLUT1 gene (1p35-31.3). New diagnostic approaches include immuno£uorescence, £ow cytometry, and PET studies. Several novel private heterozygous mutations have been identi¢ed, suggesting a sporadic, autosomal dominant condition resulting in GLUT1-haploinsu¤ciency. E¡ective treatment is available by means of a ketogenic diet as ketone bodies provide an alternative fuel to the developing brain. In some infants the diet was started as early as age six weeks with good seizure control. This treatable condition should be suspected in children with unexplained seizures and a lumbar puncture under fasting conditions and simultaneous determination of blood glucose, CSF glucose, and CSF lactate concentrations should be performed. The presentation will summarize our experience with this novel clinical entity and will discuss current developments.

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MUTATION ANALYSIS OF THE GLUT2 GENE IN PATIENTS WITH FANCONI^BICKEL SYNDROME Osamu Sakamoto, Eishin Ogawa, Toshihiro Ohura, Kazuie Iinuma (Department of Pediatrics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Japan); Yutaka Igarashi (Igarashi Pediatric Clinic, 1-1-234 Takamori, Izumi-ku, Sendai, Japan), Yoichi Matsubara, Kuniaki Narisawa (Department of Medical Genetics, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai, Japan) Fanconi^Bickel syndrome (FBS) is an autosomal recessive disorder manifesting hepatorenal glycogen accumulation, Fanconi nephropathy, and impaired utilization of glucose and galactose. Recently several mutations in a gene encoding a glucose transporter, GLUT2, have been reported in patients with FBS. We have performed molecular analysis on three Japanese patients and found four novel mutations: a splice-site mutation (IVS2-2A4G), a nonsense mutation (Q287X), and two missense mutations (L389P and V423E). Family members, who were heterozygous for L389P or V423E mutation, showed renal glucosuria. These data suggested that GLUT2 gene defects may be a cause of renal glucosuria.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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THE MOLECULAR BASIS OF RENAL GLUCOSURIA: MUTATIONS IN THE GENE FOR A RENAL GLUCOSE TRANSPORTER (SGLT2) R Santer 1, M Kinner 1, R Schneppenheim 2, G Hillebrand 1, M Kemper 2, J Ehrich 3, P Swift 4, F Skovby 5 J Schaub 1 University Childrens`s Hospital 1Kiel , 2Hamburg, 3Hannover, Germany; 4 Leicester, England; 5Dpmt of Clinical Genetics, Copenhagen, Denmark Objective: We have determined the sequence, structure, and tissue-speci¢c expression of the gene for the sodium-dependant glucose transporter of renal tubular cells (SGLT2) in order to investigate the role of this transport protein in renal glucosuria. Methods: SGLT2 gDNA was sequenced by primer walking starting from known cDNA sequences; unknown £anking sequences were determined by a two step PCR method (Dynal1). For expression studies, multiple tissue Northern blots were hybridized to SGLT2 cDNA probes (Master Blot1, Clontech). In 5 patients from 4 families with severe isolated glucosuria (including the original patient with renal glucosuria ``type 0'', de¢ned by virtual absence of tubular glucose reabsorption) exonspeci¢c PCR products of SGLT2 were analyzed by direct sequencing. Results : SGLT2 was strongly expressed in kidney, to a smaller extent in other tissues like liver, intestine, and lung. All investigated patients were homozygous or compound heterozygous carriers of SGLT2 mutations; in some families heterozygous members were documented to have mild glucosuria. A total of ¢ve SGLT2 mutations was detected: c.500delA(166fs...186X), c.814G4A(G272R), IVS8 +5g4a, c.920T4C(L307P), c.973-7delATGTT(324fs...347X). Conclusion: Homozygous SGLT2 mutations cause the severe, recessive type of renal glucosuria. Haploinsu¤ciency for SGLT2 can result in the milder, dominant types of renal glucosuria.

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A METHOD FOR THE RAPID DETERMINATION OF TRANSFERRIN ISOFORMS BY IMMUNOAFFINITY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY Jean M. Lacey, Mark J. Magera, Dietrich Matern, Piero Rinaldo, John F. O'Brien Biochemical Genetics Laboratory, Department of Laboratory Medicine & Pathology, Mayo Clinic and Foundation, Rochester, MN 55905 Carbohydrate De¢cient Glycoprotein Syndrome (CDGS) is an autosomal recessive disorder that results in elevated serum carbohydrate de¢cient transferrin isoforms (TrfI). We have developed an automated column-switching LC-MS method to determine TrfI. Five mL of 1:5 diluted serum is applied to the a¤nity column, which sequesters TrfI, using pH 7.4 PBS bu¡er. TrfI are eluted from the a¤nity column with pH 2.5 100mM-gly/2% acetate bu¡er and concentrated on a C4 column which is then washed with 1% acetate/methanol/acetonitrile (98/1/1). TrfI are eluted from the C4 column and introduced to the MS using a gradient program with 0.5% acetate-0.02% tri£uroacetic acid/methanol/acetonitrile (5/48/48). Calculation of the TrfI amount ratio (mono-oligosaccharide/ di-oligosaccharide) is routinely possible with as little as 1 mL serum, and is reproducible (average intra- and interassay CV=9.3% (n=10) and 10.3% (n=5), respectively). Advantages of the LC-MS method versus current methods include improved sensitivity, minimal sample preparation, and an analytical time of 10 minutes per sample. Variability in separation seen in traditional methods due to iron loading is not encountered since apotransferrin is analysed. The possibility to detect mass changes indicative of more subtle structural defects in glycoprotein assembly allows for the detection of CDGS variants involving Golgi transferase defects.

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A METHOD FOR THE DETERMINATION OF TRANSFERRIN ISOFORMS BY LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY Jean M. Lacey, Mark J. Magera, Dietrich Matern, John F. O'Brien Department of Laboratory Medicine & Pathology, Biochemical Genetics Laboratory, Mayo Clinic and Foundation, Rochester, MN 55905 Carbohydrate De¢cient Glycoprotein Syndrome (CDGS) is a genetic disorder, which results in elevated serum carbohydrate de¢cient transferrin (CDT). A simple column-switching liquid chromatography-mass spectrometry (LC-MS) method to determine transferrin isoforms and resolve them by mass was developed. A 2 x 1mm a¤nity column is synthesized by coupling polyclonal antibody to POROSTM-aldehyde media. Serum is applied to the a¤nity column, which sequesters transferrin isoforms (TrfI), using pH 7.4 phosphate bu¡er saline (PBS). TrfI are eluted from the a¤nity column with pH 2.5 100mM glycine/2% acetic acid bu¡er and concentrated on a C4 LC column which is then washed with 1% acetic acid/methanol/acetonitrile (98/1/1) to remove excess phosphate and other bu¡er components which suppress MS response. TrfI are eluted from the C4 column and introduced to the MS using a gradient program with 0.5% acetic acid-0.02% tri£uroacetic acid/methanol/acetonitrile (5/48/48). Calculation of the TrfI amount ratio (monooligosaccaride/di-oligosaccaride) is possible with as little as 1 mL serum, and is reproducible (intraand interassay CV%= 9.3 and 10.3, respectively). Advantages of the LC-MS method versus current methods include improved sensitivity, speed and accuracy. Moreover, LC-MS provides more useful observations relative to structural defects in glycoprotein assembly in CDGS and will a¡ord the possibility of determining TrfI in neonates where sample size is limiting.

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ISOELECTRIC FOCUSING (IEF) TRANSFERRIN GLYCOFORMS PATTERN AS A METHOD TO DETECT GALACTOSAEMIA AND FRUCTOSAEMIA Pronicka E., Sykut-Cegielska J., Adamowicz M., Radomyska B.*, Rogaszewska M., Zwitkowska E,.Socha P. Children's Memorial Health Institute and *National Research Institute of Mother and Child, Poland

Children with classical galactosaemia and hereditary fructose intolerance (HFI) on non-restricted diets show abnormal (under-glycosylated) patterns of transferrin which disappeared in few weeks after introducing galactose/fructose dietary restrictions. Since 1996 we use the IEF test as a part of selective screening for inborn errors of metabolism to look for galactosaemia and HFI in all cases presenting with hepatopathy and tubulopathy. Out of 120 tests performed in the programme, 18 samples with abnormal transferrin IEF patterns have been found. Two slightly di¡erent abnormal IEF patterns were observed - one corresponding with galactosaemia, and the second one - similar to described in HFI (and CDGS Ia/b). Further studies have con¢rmed the preliminary diagnosis: 13 new cases of galactosaemia and 3 cases of HFI (and only 2 cases of true CDGS Ia and Ib) were identi¢ed by the method. The test was specially useful in galactosaemic patients who had undergone blood transfusion. The age of diagnosis ranged from 8 days to 2 mos. Activity of galactose uridyltransferase was not measureable in all detected cases. In our experience measuring serum IEF transferrin pattern helps to identify a number of new cases of galactosaemia (and HFI). The method may be specially useful as a part of selective screening programme in the countries where the mass neonatal screening for galactosaemia is not instituted.

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CONGENITAL DEFECTS OF GLYCOSYLATION WITH RETARDATION-DYSMORPHY SYNDROME AND MYOPATHIC CHANGES A.B. Burlina1, L. Bonafe¨1, E. Van Schaftingen2, J. Jaeken3. 1 Dept of Pediatrics, Univ. of Padua, Italy; 2Laboratory of Physiological Chemistry, ICP and Univ. Catholique de Louvain, Brussels, 3Center for Metabolic Disease, KU Leuven, Belgium. Congenital defects of glycosylation (CDG) are genetic, multisystemic diseases with, as a rule, important neurological involvement resulting from defective N-linked glycan synthesis. We describe a patient that seems to represent a new type of CDG. This girl, ¢rst child of non-consanguineous parents, was born at term with Apgar score 8/9. Because of frequent vomiting and severe failure to thrive (53³ %ile), she came to our observation at the age of 14 months. She presented with severe hypotonia, gross motor developmental delay, absence of deep tendon re£exes and mild dysmorphic signs (triangular face, antimongolian slant, dysplastic low set ears, spaced inverted nipples). Brain MRI, fundoscopy, ECG and heart ultrasound were normal. EMG was abnormal with a picture of myotonic dystrophy. Laboratory investigations showed increased liver enzymes (ALT 285 U/L, AST 326) and normal cholesterol, PT, antithrombin III, factor XI, CPK and LDH. Isoelectrofocusing of serum sialotransferrin showed a type 1 pattern and isoelectrofocusing of serum hexosaminidase also showed a cathodal shift. Phosphomannomutase, phosphomannose isomerase and GDP mannose synthase activities in ¢broblasts were normal. After a respiratory infection the patient presented cardio-respiratory arrest needing intubation and tracheostomy. Now she is alive but severely mentally retarded. Further biochemical and genetic studies are underway in order to determine the basic glycosylation defect in this patient.

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CONGENITAL DISORDERS OF GLYCOSYLATION (CDG) MAY BE UNDERDIAGNOSED WHEN MIMICKING MITOCHONDRIAL DISEASE PBriones1, MAVilaseca2, MTGarc|¨ a-Silva3, MPineda2, JColomer2, IFerrer2, JArtigas4, GMatthijs5, JJaeken5, AChaba¨s1. 1 Institut de Bioqu|¨ mica Clinica, 2Hosp. Sant Joan de Deu. Barcelona. 3Hosp. 12 de Octubre. Madrid. 4 Hosp. Parc Taul|¨ . Sabadell. 5Center for Human Genetics. Leuven. CDG and mitochondrial diseases are multisystemic disorders whose clinical characteristics may sometimes overlap. We present here four patients with CDG whose clinical phenotypes suggested the diagnosis of a mitochondrial disease. Patients 1 and 2 are siblings with hemiplegic headache, strokelike episodes, lactic acidemia and history of maternal migraine; their initial clinical diagnosis was MELAS syndrome. Case 3 su¡ers from ataxia, neuropathy, ophtalmoplegia and retinitis pigmentosa suggestive of NARP. Case 4 presented with neurological regression mimicking Leigh disease, ptosis myoclonus, ataxia, and brain-stem and cerebellar atrophy. Screening for mitochondrial disease including enzyme and mtDNA investigations on muscle biopsy were performed in cases 1, 2 and 4 with negative results. %CDT was increased and IEF of serum sialotransferrin showed type I pattern in patients 1-3 and a pattern shifted towards the less sialylated bands in case 4 (CDG-x). De¢cient PMM activity con¢rmed the diagnosis of CDG-Ia in siblings 1-2, who were compound heterozygous for mutations R141H and T237M. Enzyme and molecular studies are in course for patients 3 and 4 (PMM and PMI are normal in case 4). As the search for the primary defect in mitochondrial diseases is very often unsuccessful, the pool of mitochondrial patients that remain without de¢nite diagnosis might include CDG cases. Routine screening for CDG disease may avoid preliminary arduous investigations. (Supported in part by grant FIS no. 98/0049 from the Ministry of Health of Spain).

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PARTIAL DEFICIENTY OF PHOSPHOMANNOMUTASE: A POSSIBLE PITFALL IN THE DIAGNOSIS OF CDG-Ia S. Gru«newald, E. Schollen*, G. Berghenouse8, E. Van Schaftingen8, J. Jaeken*, G. Matthijs* Center for Human Genetics or for Metabolic Diseases, University of Leuven, B-3000 Leuven*; Laboratory of Physiological Chemistry, ICP or University of Louvain, B-1200 Brussels, Belgium8

The most frequent type of congenital disorders of glycosylation (CDG) is due to mutations in the phosphomannomutase 2 (PMM2) gene and up to now, we have identi¢ed more than 30 di¡erent mutations in the PMM2 gene in 99 CDG-Ia families. Routinely, PMM activity is measured in ¢broblasts of patients. The majority of CDG-Ia patients have PMM activities below 10% of normal. However, in some CDG patients, we measured residual PMM activities that reached heterozygous and even normal values. This observation urged us to re-evaluate several patients with the typical IEF pattern and a clinical presentation suggestive of (a milder form) of classical CDG-Ia. We now have documented 28 patients ^ in a series of over one hundred ^ with a partial de¢ciency of PMM (values 4 0.94 mU/mg protein [425% of normal, range 3.77+/-0.86 mI/mg protein]) and identi¢ed PMM2 mutations in 24 of them. The highest PMM value (3.29 mU/mg protein) was found in a patient, compound heterozygous for the I120T and V231M mutations. Preliminary data show that in leukocytes of patients with partial de¢ciency in PMM, the residual activity is consistently lower than in ¢broblasts (<7% of the control). An assay of PMM in leukocytes appears thus to be a more reliable test for CDG-Ia. In conclusion, patients with high residual to quasi-normal activity of PMM in ¢broblasts might still harbour mutations in PMM2, and further scrutiny is warranted, especially if the clinical picture strongly suggests CDG-Ia.

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CONGENITAL DISORDER OF GLYCOSYLATION DUE TO PHOSPHOMANNOMUTASE DEFICIENCY (CDG-IA): SKELETAL STATUS ASSESSMENT BY BONE DENSITOMETRY, ULTRASONOGRAPHY AND BIOCHEMICAL MARKERS OF BONE TURNOVER R. Barone1, V. Pavone2, P. Pennisi, C.E. Fiore3 and A. Fiumara1 1Department of Pediatrics, 2Orthopedics and 3Internal Medicine - University of Catania, Italy.

Congenital disorder of glycosylation (CDG) type Ia (phosphomannomutase de¢ciency) is characterized by a defective glycosylation of glycoprotein. In addition to a major nervous system involvement, many other organs, including the skeleton, may be a¡ected in CDG-Ia patients. The aim of our study was to evaluate bone density, ultrasound parameters and biochemical markers of bone turnover in three patients with CDG-Ia, whose age ranged between 14 and 27 years. Bone mineral density (BMD) values for CDG-Ia patients were signi¢cantly lower than in control subjects matched for age and sex. Ultrasound parameters, with particular regard to Broad band Ultrasound Attenuation (BUA) values were reduced suggesting that in such patients not only the bone mass but also bone structure might be impaired. Free Pyridinoline (Pyr) levels in serum and Pyr and Deoxypyridinoline (D-Pyr) urinary excretions were normal, whereas osteoblast markers, particularly serum bone alkaline phosphatase levels, were increased. The present study suggests that in CDGI-a an intrinsic defect in bone mineralization might occur, possibly dependent on the hypoglycosylation of bone matrix proteins. This might lead to a defective control of the remodeling process, with inappropriate balance between resorption, which remains normal, and formation, which is impaired despite increased osteoblast activity.

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CDG Ia, WITHOUT MENTAL RETARDATION, EXTRANEUROLOGICAL AND DYSMORPHIC FEATURES Diana M. Neele1, Leo M.E. Smit2, Cornelis Jakobs1, Marjo S. van der Knaap2 1 Department of Clinical Chemistry, 2Department of Child Neurology, Academic Hospital ``Vrije Universiteit'', De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands Congenital Disorders of Glycosylation (CDGs) are a group of inherited disorders of glycoprotein Nglycosylation that cause multisystemic abnormalities. These disorders can biochemically be diagnosed by analysis of serum transferrin isoforms using immuno-isoelectric focussing (IEF). Bestdescribed is CDG Ia which usually presents with severe psychomotor retardation, typical dysmorphic abnormalities and variable extraneurological features. Here, we report a patient with an atypical, relatively mild, presentation of CDG Ia. He was evaluated at the age of 11 months because of a retarded motor development. CT scan of the brain showed hypoplasia of the cerebellar vermis. At the age of 6 years he showed signs of cerebellar ataxia and dyspraxia. He had high scores on memory tests, low scores on ¢ne motor tasks while his mental development was age-appropriate. From that age on, he followed special education for speech problems and received extra training for gross motor skills as well. At the age of 8 years he was re-evaluated because of increasing cerebellar ataxia. Transferrin IEF pattern showed increased asialo and disialo bands as seen in CDG Ia, Ib and Ic. Enzyme assays showed a de¢ciency of phosphomannomutase. In conclusion, the presentation of our CDG Ia patient is relatively mild: he has no mental retardation, no extraneurologic signs and no dysmorphic features. This patient illustrates the phenotypic heterogeneity of phosphomannomutase de¢ciency. We recommend transferrin IEF testing for all patients having unexplained cerebellar ataxia.

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CDG-Ia: PHENOTYPIC SPECTRUM OF 25 PATIENTS WITH THE R141H/F119L PMM2 GENOTYPE S. Kjaergaard, M. Schwartz and F. Skovby Department of Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark Congenital disorders of glycosylation (CDG) are a group of inherited multisystem disorders resulting from defects in glycosylation of proteins. The predominant CDG type Ia is due to de¢ciency of phosphomannomutase (PMM) encoded by the PMM2 gene. 25 of our 29 CDG-Ia patients are compound heterozygous for the same mutations, R141H/F119L, and we reviewed their phenotypic spectrum. Age range was 0-19 years, implying an incidence at birth of 1:41000. 19/25 were hospitalized as neonates due to cyanosis, respiratory distress, hypoglycemia, dysmorphism and/or feeding problems. Developmental delay was obvious during the ¢rst three months in all cases. All patients had hypotonia, ataxia, muscular atrophy, esotropia, dysmorphic features, feeding di¤culties, growth retardation and abnormal liver function. Pericardial e¡usions, gastrointestinal symptoms, seizures and stroke-like episodes were frequent, but not uniform ¢ndings. CT of the brain in 20 children was normal in 2 cases examined before 4th month of life, while 18 children had cerebellar atrophy. Bleeding diathesis, thromboemboli, cardiac tamponade and ascites were rare occurrences. The motor ability varied greatly, even among sibs. Only two were able to walk with support, but the majority were mobile in a wheel chair or gait trainer and four children did not achieve ambulatory function. Vocabulary ranged from no words to comprehensible speech. The overall mortality was 20%. The considerable variability of both morbidity and mortality among patients with the same PMM2 genotype is relevant to counselling of families a¡ected by CDG-Ia.

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TWO ADULT SISTERS WITH CDG TYPE Ia WITH NORMAL PHOSPHOMANNOMUTASE ACTIVITY IN FIBROBLASTS AND ATYPICAL ISOELECTRIC FOCUSING PATTERN OF a1-ANTITRYPSIN V. Peters1, P. Vieregge2, R. Hacker3, B. Assmann1, S. Bubel2, J. Fang1, V. Vincon1, J.R. Schaefer3 and G.F. Ho¡mann1 Department of Pediatrics1, University of Heidelberg, Department of Neurology2, University of Lu«beck and Department of Internal Medicine3, University of Marburg, GERMANY Congenital Disorders of Glycosylation (CDG, former Carbohydrate de¢cient Glycoprotein) are autosomal recessive multisystemic disorders characterised by glycosylation defects of glycoproteins. We report here on two sisters, aged 33 and 34 years, with clinical features typical for CDG type Ia such as developmental delay, muscular hypotonia, cerebellar signs with cerebellar hypoplasia on imaging studies, unusual distribution of subcutaneous fat and lack of puberty. Laboratory ¢ndings showed a combination of typical and atypical features for CDG type Ia. The isoelectric focusing (IEF) pattern of serum transferrin showed clearly elevated amounts of a- and disalotransferrin, typical for CDG type I patients. Genetic transferrin variants were excluded by neuraminidase treatment. Reduced activity of phosphomannomutase (PMM) in leukocytes con¢rmed the diagnosis of a CDG type Ia in both sisters. However, activity of PMM in ¢broblasts was normal and the IEF band pattern of a1-antitrypsin was clearly di¡erent to that obtained in serum of con¢rmed CDG type Ia patients. This case demonstrates that enzymatic tests should be performed not only in ¢broblasts. The reason for the unusual IEF band pattern of a1-antitrypsin, possibly pointing to a broader heterogenity in CDG type Ia, as well as the contradictory results of enzymatic tests require further investigations.

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CARBOHYDRATE DEFICIENT GLYCOPROTEIN SYNDROME TYPE I - PORTUGUESE EXPERIENCE Quelhas D1, Vilarinho L1, Jaeken J2. 1 Instituto de Gene¨tica Me¨dica, Porto, Portugal, 2 Dpt. of Paediatrics University of Leuven, Belgium

Carbohydrate de¢cient glycoprotein syndromes are a group of diseases due to glycosylation defects of glycoproteins, as reported by Jaeken et al (1). Several types have been described, but the de¢ciency of phosphomannomutase (PMM) type Ia is by far the most common. The diagnosis of CDG syndromes in general and of PMM in particular is usually made measuring carbohydrate de¢cient transferrin (CDT) and isoelectrofocusing of sialotransferrins (2). We report 8 unrelated Portuguese patients whose clinical diagnosis has been con¢rmed by biochemical analysis. In the great majority of patients, the prominent features were neurological manifestations, failure to thrive, variable dysmorphy and hepatomegaly. All patients had abnormal values of CDT, con¢rmed by IEF of serum transferrins that showed a type I pattern. In 6 patients PMM has de¢cient activity in cultured skin ¢broblasts (Prof.Van Schaftingen, Belgium) con¢rmed by molecular analysis in PMM2 gene (Prof. Matthijs, Belgium). The remaining patients are currently being studied.The patients presented here have heterogeneous clinical phenotypes, ranging from relatively mild to extremely severe, without correlation between the biochemical data and the clinical severity, so it should be advisable to perform CDT and/or isoelectrofocusing of sialotransferrins even in mild clinical phenotypes. References: (1) Jaeken J, Stibler H, Hagberg (eds) 1991 Acta Paediatr Scand (supplement) 375: 5-71; (2) Jaeken J, Matthijs G, Carchon H, Van Schaftingen E (1999); In: Scriver C, Beaudet A, Sly W, Valle D (eds). The metabolic and molecular bases of inherited disease. 8th edn Mc Graw-Hill, New York, in press.

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VARIABLE OUTCOME AND THE EFFECT OF MANNOSE IN CONGENITAL DISORDER OF GLYCOSYLATION TYPE Ib (CDG-Ib) S. Kjaergaard1, V. Westphal2, J.A. Davis2, Peterson, S.M.2, H.H. Freeze2 and F. Skovby1 1 Department of Clinical Genetics, University Hospital Rigshospitalet, Copenhagen, Denmark; 2 The Burnham Institute, La Jolla, California, USA. Patients with phosphomannoisomerase (PMI) de¢ciency have hepatic ¢brosis, protein-losing enteropathy and recurrent venous thrombosis, but normal psychomotor development. Several young CDGIb patients have died before diagnosis and mannose therapy became available in 1998. One of two Danish sibs with the hallmarks of CDG-Ib, described in 1980, died at age 5. We located the surviving sib, now aged 33 years. She is completely healthy, eats a normal diet and has three normal children. Her diagnosis of CDG-Ib was substantiated by an abnormal transferrin isoelectric focusing pattern and a reduced PMI activity. Two missense mutations, 419 T?C (I140T) and 636 G?A (R219Q), and a single base substitution in intron 5, 670+9G?A were found in MPI of both sibs. Three months of mannose therapy improved the surviving sib's transferrin IEF pattern and normalised the antithrombin III level, but both returned to the pre-therapy state when mannose was discontinued. These results demonstrate that CDG-Ib, although serious in childhood, may improve with increasing age, even without mannose therapy, and allow for a normal adult life.

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NEW CASE OF PHOSPHOMANNOSE ISOMERASE DEFICIENCY (CDG 1B) Adamowicz M., Matthijs G.*, Van Schaftingen E.**, Jaeken J.*, Rokicki D., Pronicki M., Pronicka E. Children's Memorial Health Institute, Warsaw, *Center of Human Genetics University of Leuven, **Laboratoire de Chimie Physiologique, Brussels

Congenital disorders of glycosylation (CDG) type Ia (phosphomannomutase [PMM] de¢ciency) and type Ib (phosphomannose isomerase [PMI] de¢ciency) show the same abnormal transferrin glycoforms patterns in IEF (isoelectric focusing), but completely di¡erent clinical picture. The latter disorder seems to be milder, presenting mainly with gastrointestinal and hepatic symptoms, without neurological involvement. Oral mannose supplementation was reported to control the clinical and biochemical abnormalities. We describe a new case of this disease similat to ten described cases. Oliwia K. is a ¢rst child of healthy unrelated parents of Polish origin. First symptoms - diarrhoea, dehydratation, anaemia and hypoproteinaemia appeared at age of 7 mos and needed hospitalisation. Because failure to thrive (height below 3 percentile) and marked elevation of transaminases she was referred to our hospital. Clinical status was stabilised but mild tubular acidosis was observed additionally (not reported in CDG 1b till now) and treated with oral bicarbonate. Liver biopsy showed ¢brosis. At age of 13 mos the transferrin IEF was found to be increased (CDT 38.9%). De¢ciency of PMI was con¢rmed in ¢broblasts (4.4% of control values) and two missense mutations (R219Q known and novel G250S). identi¢ed on both alleles of PMI gene. Mannose supplementation (4 x 0.15 g/kg) caused slow normalisation of IEF pattern (after 55 mos - CDT 23.6%). Dehydratation episodes did not recur. Evolution of tubular acidosis under mannose supplementation is being observed to assess its causal connection with phosphomannose isomerase de¢ciency (CDG Ib).

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THE CLINICAL AND MOLECULAR CHARACTERISTICS OF A NOVEL CONGENITAL DISORDER OF GLYCOSYLATION TYPE IC (CDG-IC) Stephanie GrÏnewald 1,2, Timo Imbach 3, Karin Huijben 4, Hans de Klerk 5, Udo Wendel 2, Gert Matthijs 1, Jaak Jaeken 1, Thierry Hennet 3, Ron Wevers 4. 1 University of Leuven, Belgium, 2 Heinrich-Heine UniversitÌt DÏsseldorf, Germany, 3 University of ZÏrich, Switzerland, 4 Institute Neurology, University Medical Centre Nijmegen, The Netherlands (email: [email protected]), 5 Academic Hospital Rotterdam, The Netherlands.

Congenital Disorders of Glycosylation (CDG) are a family of genetic diseases characterized by defects in the synthesis of glycoproteins. Here we describe the characteristics of eleven cases with CDG-Ic. This is the ¢rst described defect of a human ER localized enzyme in glycan biosynthesis. We have identi¢ed the patients by isoelectric focusing of serum transferrin in a broad screening for Nglycosylation defects in a large pediatric population with mild to severe neurological developmental delay. The accumulation of dolichol-linked Man9GlcNAc2 in patient's ¢broblasts intitated a defect in the human ortholog of the Saccharomyces cerevisiae ALG6 gene. Eight patients were shown to be homozygous for a A333V mutation. In another child the A333V mutation was found next to an intron splice site defect (IVS3+5G4A). Two novel ALG6 mutations, namely 911T4C and 1432T4C, were found in two additional cases. As in CDG-Ia patients, mental retardation, speech delay and epilepsy were present in the CDG-Ic cases. They did not show any of the cardinal symptoms of CDG-Ia as inverted nipples or fat pads. Cerebellar hypoplasia was never observed. The neurological impairment of CDG-Ic patients is less severe than in CDG-Ia. In agreement with this clinical observation the hypoglycosylation of the brain derived b-trace protein was less pronounced in CDG-Ic than in CDG-Ia.

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TOWARDS THE IDENTIFICATION OF THE MOLECULAR DEFECT IN A NEW TYPE OF CONGENITAL DISORDERS OF GLYCOSYLATION (CDG-IIx) WITH A DECREASED TRANSPORT OF GDP-FUCOSE INTO THE GOLGI APPARATUS Torben Lu«bke1, Thorsten Maquardt2, Kurt von Figura1 and Christian Ko«rner1 Institut fu«r Biochemie II, Heinrich-Du«ker-Weg 12, D-37073 Go«ttingen, Germany1; Klinik- und Poliklinik fu«r Kinderheilkunde, 48149, Mu«nster, Germany

Congenital Disorders of Glycosylations (CDG) which were formerly termed Carbohydrate De¢cient Glycoprotein Syndromes (CDGS) comprise a group of inherited multisystemic disorders which are clinically and biochemically heterogenous but have in common the synthesis of glycoproteins with an abnormal N-glycosylation. Previously we could show a new type of CDG (CDG-IIx) due to a 5^10 times decreased translocation of GDP-fucose into Golgi derived vesicles from patient's ¢broblasts and lymphoblasts. Clinically the defect is characterized by mental and growth retardation, dysmorphic signs and recurrent infections. The biochemical hallmark is a general hypofucosylation of N- and O-glycosylated proteins. Since a GDP-fucose transporter could not be identi¢ed so far, and we cannot exclude a defective associated protein, we are currently investigating the molecular defect underlying the GDP-fucose import de¢ciency by a complementation cloning approach. Therefore we set up a binding assay using Aleuria aurantia lectin which allows to distinguish between control and patient's ¢broblasts. A human cDNA library packed into retroviral particles will be used for the infection of patient derived ¢broblasts. In case of a complementation event we should expect a positive staining with the lectin binding assay. The ¢broblast concerned will further be investigated by single cell PCR.

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A NEWLY RECOGNIZED GLYCOSYLATION DEFECT WITH PSYCHOMOTOR RETARDATION, ICHTHYOSIS AND DWARFISM J. Jaeken1, T. Imbach2, B. Schenk3, E. Smeets4, H. Carchon1, E. Van Schaftingen5, G. Matthijs4 Center for Metabolic Disease1 and Center for Human Genetics4, KU Leuven, Belgium; Institute of Physiology2, University of Zu«rich and Institute of Microbiology3, ETH Zu«rich, Switzerland; Laboratory of Physiological Chemistry, ICP and Universite¨ Catholique de Louvain, Brussels, Belgium We describe siblings with a novel type of CDG. A girl was seen at the age of 5 months with feeding problems, psychomotor retardation, ichthyosis, growth retardation with partial growth hormone de¢ciency and hypo-b-lipoproteinemia. A sibling died at 3 months with a similar syndrome. She was seen again at the age of 16 years with an impressive scaly and itching skin disease particularly of the face and lower legs, dry hair, night blindness and sight problems changing in parallel with the severity of the skin lesions and worsening during summer. She showed dwar¢sm but normal pubertal development. She could slowly walk without support and speak a few words. She grasped for objects with gross movements. Deep tendon re£exes were normal. Laboratory investigation showed a type 1 serum sialotransferrin pattern and decreased values of serum LDL-cholesterol (52 mg/dl), cholinesterase, progesterone, oestradiol, and of blood factor XI, antithrombin, protein C and protein S. Serum haptoglobin, transaminases, TBG, growth hormone and IGF-I were normal. Vaginoscopy and hysteroscopy were normal as were echocardiography, nerve conduction velocity and MRI of the brain. Electronmicroscopy of a liver biopsy was normal. Activities of phosphomannose isomerase, phosphomannomutase and GDP-mannose synthase in ¢broblasts were normal. Lipid-linked oligosaccharide analysis in ¢broblasts revealed a novel ER-glycosylation defect (CDG-If).

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A NEW DISORDER CAUSED BY DEFECTIVE BIOSYNTHESIS OF N-LINKED OLIGOSACCHARIDES DUE TO GLUCOSIDASE I DEFICIENCY R van Coster, C de Praeter, G Gerwig, E Bause, I Nuytinck, J Vliegenthart, W Breuer, C Voelker, M Espeel, JJ Martin, G Dacremont

Objective Several inborn errors in biosynthesis of N-linked oligosaccharides have already been reported. Defects were located either in the assembly reactions of the precursor oligosaccharide-PPDolichol in the cytoplasm, or in the enzymatic processing of the carbohydrate chains in the Golgi. Genetic defects in one of the trimming enzymes located in the endoplasmic reticulum, a common pathway of all N-linked oligosaccharides, have hitherto not been described in man or in higher organisms. We report here a patient with a severe defect in this pathway. Results We have identi¢ed a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features.The clinical course was progressive, characterized by the occurence of hepatomegaly, hypoventilation, feeding problems, seizures and fatal outcome at age 74 days. The accumulation of the tetrasaccharide Glc(a1-2)Glc(a1-3)Glc(a1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymatic studies on liver tissue and cultured skin ¢broblasts revealed an a-glucosidase I de¢ciency. Molecular analysis of the glucosidase I gene disclosed two base changes in the cDNA sequence. The accumulation of the abnormal tetrasaccharide in the urine, the normal IEF pattern of serum sialotransferrin and of b-trace protein in CSF, and the morphological abnormalities in liver and cerebrum were di¡erent from the ¢ndings detected in other variants of the carbohydrate-de¢cient glycoprotein syndrome. Conclusion This is the ¢rst reported patient with a defect in one of the trimming enzymes of the N-linked oligosaccharides in the endoplasmic reticulum.

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YET ANOTHER CONGENITAL DISORDER OF GLYCOSYLATION! (CDGS) N. Buist, M. Grompe, R. Steiner, Y. Miura, J. O'Brien, H. Freeze. OR Health Science U. Portland, OR, Mayo Clinic, Rochester, MN, The Burnham Institute, La Jolla, CA. USA.

An 18y old female has a novel CDGS. She attends regular school, taking limited courses. At 16 she reads at the 3-4th grade level, uses a computer appropriately and is very articulate. At birth, she had abnormally wrinkled skin and corneal dystrophy (that resolved by one year). She has abnormal fat distribution over the body and thighs, kyphoscoliosis, facial dysmorphism, amelogenesis imperfecta, mild hepatocellular dysfunction, short stature, small hands and feet with dysplastic 4th metacarpals and metatarsals, normal ¢nger nails but dysplastic toe nails. Deep tendon re£exes are normal. She has pigmentary iris defects, mild optic atrophy and retinal pallor but no pigmentary retinopathy; the ERG at one year showed abnormal rod and cone function. Breast development started at 12y, and periods at 15 y. X Rays at 9 mo showed anterior beaking of L1, 2 &3 and minimal hypoplasia of the femoral heads; bone age was normal. At 8 y, the bones were undermineralized, the long bones were thin with large medullary spaces, the vertebrae and ribs were dysplastic. The cerebellum is normal on MRI. Liver bx was normal by light and em. Transferrin electrophoresis showed abnormal reduction of ``tetrasialo'' and increased ``disialo'' forms. Enzyme studies showed normal activity of phosphomannomutase, phosphomannose-isomerase and GlcNAc Transferases I & II. Analysis of transferrin by ESI-MS and by MALDI-TOF showed that some protein molecules lacked an entire sugar side-chain as well as 2-3 monosaccharides from other side chains, with mass sizes consistent with a sialic acid, a hexose and GlcNAc. A precise enzyme defect has not yet been elucidated.

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ESTABLISHMENT OF A EUROPEAN-WIDE RESEARCH & DIAGNOSTIC NETWORK FOR INBORN ERRORS OF PURINE & PYRIMIDINE METABOLISM HA Simmonds1, AM Marinaki1, AH van Gennip2 Guy's Hospital, London GB1 , Academic Medical Center, Amsterdam, NL2 Purine and pyrimidine (PP) disorders are relative newcomers to the ¢eld of inborn errors. The few diagnostic services in existence for these defects in Europe at the end of the 20th Century (comprehensive in only 4 countries) had developed on an ad hoc basis from speci¢c research interests in this area of human metabolism. An EC grant under the orphan diseases section (BMH4-CT98-3079) to `develop a European research and diagnostic network..') involving 19 countries has enabled the ¢rst major step forward for the 21st Century: January 2000 saw the publication and distribution of an Interim Directory of Laboratories Diagnosing Inborn Errors of Purine and Pyrimidine Metabolism in Europe embracing all 19 countries, made possible through their individual e¡orts and assistance in data gathering. This grant has also enabled: & expansion of transnational collaborative research projects involving these potentially lethal or debilitating disorders (eg pathogenesis/molecular basis of Lesch-Nyhan syndrome, familial juvenile gouty nephropathy, hereditary oroticaciduria, dihydropyrimidine dehydrogenase de¢ciency-see posters) & establishment of an internet database on all European patients with PP disorders, to include all defects, and facilitate information presentation, dissemination, and encourage awareness. These disorders jeopardise the lives of children and adults, are tragic to the family and costly to European health services. Collaborative research will be enhanced by the increased patient numbers identi¢ed through this network and improvement in information exchange. Knowledge so derived will be used to devise new diagnostic tests and improve e¤cacy of treatments for PP disorders, as well as to develop improved drugs for more common disorders of immune regulation, neoplasia and heart disease.

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ANALYSIS OF PYRIMIDINES AND THEIR CATABOLITES IN URINE OR URINE SOAKED FILTER PAPER BY HPLC/ESI TANDEM-MS Albert H. van Gennip, Henk van Lenthe, Tetsuya Ito, Arno van Cruchten and Andre¨ B.P. van Kuilenburg. Academic Medical Centre, Emma Children's Hospital Dept of Clinical Chemistry, F0-224, PO Box 227000, 1100 DE Amsterdam, The Netherlands Introduction: The degradation of uracil and thymine is controlled by dihydropyrimidine dehydrogenase, dihydropyrimidinase and ureidopropionase respectively. Patients with a de¢ciency of one of these enzymes present with variable clinical picture. However, they can easily be detected by the abnormal concentrations of the accumulating substrates in urine. Objectives: Until now methods allow analysis of only part of the index metabolites or lack speci¢city and are time-consuming. Therefore we developed a rapid and speci¢c method to measure all index metabolites. Material and Methods: ESI tandem-MS was connected to HPLC to separate analytes with the same fragmentation from each other and from interfering substances. Multiple reaction monitoring was used in electropositive mode and stable isotope labeled reference compounds were used as internal standards. Results: Adequate transitions and instrument settings were established. Urine was only centrifuged, urine soaked ¢lter paper strips were extracted by water/methanol. Recoveries ranged from 89 to 99% (n=13) for spiked (100mM) urine and from 93 to 103% (n=13) for the ¢lter paper strips. Total analysis time is 15 min., which is about 1/64 compared to the conventional procedure by amino acid analysis after acid hydrolysis of the di¡erentially isolated compounds. Analysis of urine samples from patients with established disorders yielded the correct diagnoses and excretion values. Conclusion: HPLC/ESI MSMS of urine allows rapid, speci¢c and quantitative analysis of pyrimidine degradation products.

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CEREBROSPINAL FLUID PURINES AND PYRIMIDINES MAY REFLECT EXCITOTOXICITY IN ORGANIC ACIDURIAS AND LYSOSOMAL DISORDERS L. Laro¨vere, A. Latini, C.E. Coronel*, C. Grosso, R. De Kremer Dodelson CEMECO, Departments of Paediatric and *Biological Chemistry, School of Medicine, National University of Co¨rdoba, Argentina It is known that ATP degradation and oxygen radical generation are associated with glutamate-mediated excitotoxicity. Many evidences suggest that purine and pyrimidine CSF alterations can re£ect this mechanism. Our objective was to study if these CSF metabolites could express excitotoxicity in de¢ned inborn errors of metabolism. The metabolites were analysed by HPLC; the results are expressed as mmol/L: Disorder Glutaric aciduria Type I Medium-chain acyl-CoA dehydrogenase de¢ciency 3-Methylcrotonyl-CoA carboxilase de¢ciency 3-OH-3-methylglutaryl-CoA lyase de¢ciency b-Ketothiolase de¢ciency Mucopolysaccharidoses Type II Mucopolysaccharidoses Type III (n=2) Canavan disease Sandho¡ disease (n=2) Controls (n=15)

Hypoxanthine

Uridine

Uric Acid

Xanthine

4.96 6.90 5.22 5.32 9.13

1.15 2.43 1.51 1.41 2.50

52.4 34.2 35.7 45.6 108

4.15 4.45 19.63 16.52 10.45

2.45 2.6/7.6 2.71 5.1/5

1.41 0.4/3.3 0.98 2.4/1.4

8.2 6.1/12.1 10 14.4/11

1.21 1.3/3 1.71 2.6/2.5

2.6 + 0.6

1.1 + 0.2

22 + 7.4

2.5 + 0.7

The increased hypoxanthine in most of the patients would depict energetic depletion, and in a lesser degree uridine might do it. Otherwise, abnormal xanthine and uric acid levels in organic acidurias seem to be most likely related to free radical generation by xanthine oxidase, which is activated in brain by energy failure and intraneuronal Ca2+. It is interesting to note that both mechanisms, energetic impairment and oxidative stress, could be involved in organic acidurias while ATP depletion would be prominent in lysosomal disorders.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

189

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PREDICTING DRUG TOXICITY AND NON-RESPONSE: AZATHIOPRINE AND THIOPURINE METHYLTRANSFERASE AS A METABOLIC MODEL M Shobowale-Bakre1, C Hassan2, A Ansari2, J Duley1, A Marinaki1, J Meenan2, P Seed3, J Sanderson2. Purine Research Laboratory1 and Departments of Gastroenterology2 and Obstetrics3, Guy's & St. Thomas' Hospitals Trust and GKT School of Medicine, London, UK.

Immunosuppression by azathioprine is commonly used for transplantation, eczema, in£ammatory bowel disease etc, but is discontinued in up to a third of patients because of either clinical nonresponse or toxicity. Azathioprine metabolism is regulated by an allelic polymorphism of thiopurine methyltransferase (TPMT) activity. Homozygous de¢cient individuals experience severe bone marrow depression with azathioprine, while heterozygotes are at increased risk of side e¡ects. Thus TPMT screening is increasingly requested prior to therapy. We retrospectively assessed patient outcome on azathioprine compared with predicted response based on erythrocyte TPMT. Methods: Erythrocyte TPMT activity was assayed in 108 patients with in£ammatory bowel disease on azathioprine. 52 patients including all intermediate TPMTs were genotyped. Clinical response, adverse e¡ects and haematological parameters were correlated to TPMT activity and genotype. Results: 98 patients had normal or high TPMT activity, 10 (9%) had intermediate activity and carried mutant alleles. Complete or partial remission on azathioprine was achieved in 80% of cases, while 18% were intolerant to azathioprine. 14 (13%) of the patients with normal or high TPMT had side e¡ects, compared with 6 (55%) with intermediate activity (p=0.0015). Conversely, high TPMT activity (414nmol/hr/ml.rbc) predicted non-responsiveness to azathioprine (p=0.018). Conclusions: Intermediate TPMT activity is associated with increased risk of azathioprine toxicity. High TPMT predicts non-response and, in theory, a need for higher doses of azathioprine to achieve clinical remission. Metabolic pro¢ling has a role in rational planning of drug therapy.

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HEMATOLOGICAL TOXICITY DURING THIOPURINE TREATMENT: ASSOCIATION WITH THIOPURINE ENZYME DEFICIENCY RA De Abreu, J. J. Keizer-Garritsen,C. Brouwer, K. Ahment and L.H.J. Lambooy. University Medical Center Nijmegen (NL), Department of Pediatrics, Laboratory of Pediatrics and Neurology.

Azathioprine (AZA) and 6-mercaptopurine (6MP) are thiopurine drugs which are regularly used in the treatment of various diseases; e.g. AZA is used as an immune suppressive drug in rheumatoid arthritis, during post-treatment of renal transplantation, in a variety of dermatological conditions and in gastroenterology. 6MP is used as chemotherapeutic agent during maintenance treatment of childhood leukemia. After intake, AZA is rapidly incorporated into 6MP and the latter is metabolized by multi-step conversion into active metabolites like 6-thio-GTP and 6-methylthio-IMP. The incorporation of 6thio-GTP appears to be the main cause for cellular toxicity of 6MP and AZA. Methylation of 6MP in bone marrow cells and peripheral blood cells, by thiopurine methyltransferase (TPMT), leads to detoxi¢cation of 6MP in these cells. Purine-5'-nucleotidase (5'NT) is the enzyme involved in the ¢rst step in nucleotide breakdown, another detoxi¢cation pathway of thiopurines. In patients with de¢cient activity of either TPMT or 5'NT, treatment with AZA or 6MP may develop severe systemic toxicity of the peripheral blood system due to increased intracellular synthesis of 6-thioGTP. We have diagnosed 9 patients with TPMT de¢ciency and 5 patients with low 5'NT activity, who have developed hematological toxicity during treatment with AZA or 6MP, respectively. From 5 of the patients with TPMT de¢ciency the mutation was established.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

190

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FAMILIAL JUVENILE HYPERURICEMIC NEPHROPATHY (FJHN) : LOCALIZATION OF THE GENE ON CHROMOSOME 16P11.2 AND EVIDENCE FOR GENETIC HETEROGENEITY Stibu©rkova¨ B.1, Majewski J.2, Síebesta I.1,3, Zhang W.2, Ott J.2 and Kmoch S.1 1 Institute for Inherited Metabolic Disorders and 3Department of Clinical Biochemistry, 1st School of Medicine and General Faculty Hospital Prague; 2Laboratory of Statistical Genetics, Rockefeller University, New York Familial juvenile hyperuricaemic nephropathy (FJHN), is an autosomal dominant renal disease characterized by juvenile onset of hyperuricemia, gouty arthritis and progressive renal failure at an early age. Using a genomewide linkage analysis in three Czech a¡ected families we have identi¢ed a locus for FJHN on chromosome 16p11.2 and found evidence for genetic heterogeneity and reduced penetrance of the disease. Maximum two point HLOD score of 4.70 was obtained at a recombination fraction yq = 0 with marker D16S3036; multipoint linkage analysis yielded a maximum HLOD score of 4.76 at the same location. Haplotype analysis de¢ned a 10 cM candidate region between £anking markers D16S501 and D16S3113 exhibiting crossover events with the disease locus. Recent publication on localization of the FJHN gene(1) and our results narow the 1,7 cM candidate region, between markers D16S403 and D16S3113. This region contains several genes expressed in the kidney, one of which, NADP-regulated thyroid-hormone binding protein is currently being analysed. 1. Kamatani et al. (2000) Arthritis Rheum 43: 925-9

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SUCCESSFUL CARRIER ERYTHROCYTE ENTRAPPED ADENOSINE DEAMINASE THERAPY IN A PATIENT M.D. Bain1, B.E. Bax 1 L.D. Fairbanks 2, H.A. Simmonds 2, A.D.B. Webster 3 and R.A. Chalmers1. 1 Paediatric Metabolism Unit, Department of Child Health, St George's Hospital Medical School, 2 Purine Research Laboratories, Guy's Hospital Medical School, and 3 MRC Immunode¢ciency Research Group, Royal Free Hospital Medical School; London, UK. Adenosine deaminase (ADA) de¢ciency, a rare disorder of purine metabolism, causes severe combined immunode¢ciency in which elevated levels of deoxyadenosine triphosphate (dATP) disrupt lymphocyte di¡erentiation and proliferation. Polyethylene glycol-conjugated adenosine deaminase (Pegademase) is available for enzyme replacement, but therapy requires twice weekly parenteral administration, and is exceedingly costly. We have entrapped native and unmodi¢ed pharmaceutical grade ADA within erythrocytes (carrier erythrocytes) as an alternative therapy in a patient with ADA de¢ciency already established on Pegademase. Hypo-osmotic dialysis with therapeutic grade ADA was used to prepare autologous enzymeloaded carrier erythrocytes. These have been used therapeutically over 35 months, and as sole therapy for 24 months. Plasma ADA activity has been 0.4 mmol/hr/ml, demonstrating minimal intravascular haemolysis.. Erythrocyte ADA activity has increased from 57 nmol/hr/mg Hb pre-therapy to a trough level of 250 in the most recent treatment cycles. Erythrocyte [dATP] has ranged between 50 and 97 mmol/l packed erythrocytes over the most recent 9 months of therapy compared to 203 mmol/l on diagnosis. Half-life of entrapped enzyme varies around 16 days. The only side e¡ect noted so far has been iron de¢ciency that responds to supplemental iron. Clinical well-being has been maintained with carrier erythrocyte therapy alone.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts 1

191

H-NMR SPECTROSCOPY MONITORING OF RIBOSE TREATMENT IN ADSL DEFICIENCY Bowling FG, McGill JJ, Moxon-Lester L, Broe D, Cowley DM. Mater Hospital, South Brisbane, Queensland, AUSTRALIA.

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Adenosuccinate lyase (EC 4.3.2.2) de¢ciency is a disorder of purine metabolism in which the conversion of SAICAR to AICAR and S-AMP to AMP are blocked. The intermediates in the purine pathway are di¤cult to characterise and quantitate and 1H-NMR Spectroscopy o¡ers advantages with these metabolites. We report the monitoring of treatment with ribose using 1H-NMR Spectroscopy. Our case is an 8year old boy with mild-moderate disability, lissencephaly, behavioural problems, and autism. Neurological examination, behavioural assessment were characterised before commencement of treatment with Ribose (30gm/daily in 3 divided doses) and reassessed one month after. Urine metabolites were characterised daily before and after. Treatment resulted in a improvement in behaviour. Night terrors have ceased, autistic features are less evident and receptive language skills have improved. Before treatment, SAICA-riboside and succinyladenosine were elevated above controls. Treatment resulted in no signi¢cant change in urinary succinyladenosine excretion, but a decrease in SIACAR. Compounds such as 5-aminoimidazole ribonucleotide, 5-amino-4-carboxy-amino-imidazole ribonucleotide (that precede SAICAR in the pathway) increased. Ribose metabolites also became evident. These data support the role of ribose as increasing pathway £ux at SAICAR. Ribose kindly supplied by SHS, AUSTRALIA

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A MUTATION IN A POTENTIAL NRF-2 BINDING SITE IN THE 5'UTR OF THE ADSL GENE IN A PATIENT WITH ADENYLOSUCCINASE DEFICIENCY S Marie, V Race, MC Nassogne, MF Vincent, G Van den Berghe Laboratory of Physiological Chemistry, Christian de Duve Institute of Cellular Pathology and Universite¨ Catholique de Louvain, B-1200 Brussels, Belgium

Adenylosuccinase (adenylosuccinate lyase, ADSL) de¢ciency has been diagnosed in approx. 50 patients up to now. In accordance with the heterogeneity of the clinical picture, particularly with respect to the degree of mental retardation, 22 di¡erent mutations have been identi¢ed to date in 25 unrelated families. The majority are missense mutations, with the exception of a nonsense mutation, and a splicing error (Marie et al., Hum Mutat 13: 197-202, 1999). Most frequent is a R426H mutation, found in homozygous form in 9, and in compound heterozygote form in 4 families. In an Italian-American girl with ADSL de¢ciency, a c.-49 T-C change was found in the 5' untranslated region (5'UTR), in compound heterozygote form with mutation R426H. To analyze the in£uence of the 5'UTR mutation, we performed functional studies of the ADSL 5' £anking region with luciferase as a reporter gene. We could show that the mutation causes a 4-fold reduction of promoter activity. The mutation was also found to be located within a potential binding site for transcription factor NRF-2. Site directed mutagenesis showed that introduction of another mutation within the core sequence had the same e¡ect as c.-49 T-C. Northern blots showed that the luciferase mRNA level was also reduced 3- to 4-fold with the mutated constructs. The results suggest that the 5'UTR mutation a¡ects the NRF-2 binding site, causing a reduction of either transcription level or stability of ADSL mRNA, rather than a direct e¡ect on translation only.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

192

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DIHYDROPYRIMIDINE DEHYDROGENASE (DPD) DEFICIENCY. IDENTIFICATION OF NOVEL MUTATIONS IN THE DPD GENE Albert H. van Gennip, Janet Haasjes, Rutger Meinsma, Hans R. Waterham, Peter Vreken and Andre¨ B.P. van Kuilenburg Academic Medical Center, University of Amsterdam, The Netherlands. Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases. In children, a de¢ciency of DPD is often accompanied by a neurological disorder but a considerable variation in the clinical presentation among these patients has been reported. So far, only a few mutations have been reported in the DPD gene of patients with a complete DPD de¢ciency. The DPD gene was analysed by PCR ampli¢cation and subsequent sequencing of the genomic regions containing the 23 exons. In a group of 8 patients we could identify 9 novel mutations including 8 missense mutations D949V (exon 22), I370V (exon 10), P86L (exon 4), S201R (exon 6), H978R (exon 23), I560S (exon 13), S492L (exon 12) and Y211C (exon 6) and 1 deletion of two nucleotides (1039-142delTG). The latter mutation causes a frameshift leading to a premature stop codon shortly thereafter. So far, 16 di¡erent mutations have been identi¢ed in 26 families presenting 32 patients with the IVS14+1G4A splice-site mutation being the most common one (allele frequency of 25/64). The identi¢cation of novel mutations will provide an important and reliable tool for carrier detection. The recent cloning of the cDNA coding for human DPD and the sequence of the entire human DPD gene3 (DPYD) has allowed the detection of the defects at the molecular level. Identi¢cation of disease-causing mutations in the DPD gene will allow rapid pre-screening of patients at risk.

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DIHYDROPYRIMIDINE DEHYDROGENASE DEFICIENCY: FURTHER EVIDENCE OF ACUTE NEUROLOGICAL ONSET Fiumara A*, Caruso U³, Marzullo E*, Barone R*, van Kuilenburg ABP½, van Gennip AH½ *Dept of Paediatrics, University of Catania, Italy, Dept of Paediatrics, University of Genova, Italy, ½ Lab. Genetic Metabolic Diseases, Academic Medical Center, University of Amsterdam, The Netherlands. Dihydropyrimidine Dehydrogenase (DPD) acts in the initial step of pyrimidine bases metabolism, catalysing the reduction of uracil and thymine to b-alanine and b-aminoisobutirate. Patients with DPD de¢ciency may remain asymptomatic, developing 5-£uorouracil intolerance if treated for a cancer, or present severe neurological problems since infancy. Psychomotor delay and seizures are present and, less frequently, microcephaly, autism and ocular abnormalities.Acutely developed lethargy and cerebral palsy, are also described. Di¡erent mutations on 1p22 have been identi¢ed. We report a female child, born to Tunisian ¢rst cousins, who was normal up to the age of 1 year, when seizures appeared with fever. In a few days, she deteriorated and presented hypertonia, neck rigidity and hypere£exia, weak cry and absent photopic reaction. Serologic investigations failed to detect any bacterial or viral agent. Organic acids in urine revealed increased excretion of uracil, thymine and 5OH-methyluracil pointing to a possible defect of DPD. This was con¢rmed in ¢bs. Blood and CSF analysis performed some months later, when the patient stabilised, con¢rmed the elevated concentrations of uracil and thymine and 5-OH-methyluracil, but surprisingly, CSF showed normal levels of balanine, probably deriving from diet. This ¢nding might explain the non progressive course of this disorder despite the complete enzyme defect.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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193

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PARTIAL HPRT DEFICIENCY AS THE CAUSE OF RENAL DISEASE IN A LARGE KINDRED P Augoustides-Savvopoulou1 F Papachristou2 LD Fairbanks3 K Dimitrakopoulos 4 I Magoula4 HA Simmonds3 1st Pediatric Biochemistry Lab1 3rd Pediatric Dept2 and 2nd Internal Medicine Dept4 Aristotole University of Thessaloniki, Purine Research Unit Guy's Hospital London UK3

Hypoxanthine-guanine phosphoribosyltransferase (HPRT, EC 2.4.3.8 McKusick 308000, Xq26q27.2)de¢ciency is an X-linked defect of purine metabolism resulting in gross overproduction of uric acid. There is considerable genetic heterogeneity and clinical manifestations of HPRT de¢ciency are usually related to the degree of enzyme de¢ciency. Complete HPRT de¢ciency (Lesch-Nyhan syndrome) is characterized by severe neurological and renal problems, whereas partial de¢ciency usually manifests as a gout-urolithiasis syndrome. A three generation kindred with partial HPRT de¢ciency in which the diagnosis in two siblings led to the diagnosis in two maternal uncles with renal failure is described, the objective being to stress the importance of early diagnosis and initiation of appropriate therapy . The index patients were two male siblings aged 14 and 15 years old respectively who were referred to our department because of repeated episodes of urolithiasis. The family history revealed urolithiasis and renal failure in a maternal brother and uncle. The laboratory work-up revealed hyperuricemia, hyperuricosuria and uric acid urolithiasis in both male siblings and the low activities of HPRT in the RBC of the two index patients and their uncles established the diagnosis of partial HPRT de¢ciency (3.7-5.3 nmol/mg Hb/h, control range 80-132. Intact red cells took up 7090% of 14C hypoxanthine, controls 100%). The siblings responded well to a therapeutic regimen consisting of a high £uid intake, low purine diet and allopurinol 5-7.5mg/kg/d. The favourable response to therapy in our patients in contrast to the unfavourable outcome in their two uncles is a reminder of the importance of early diagnosis and therapy for prognosis of this disorder.

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PARTIAL HPRT DEFICIENCY IN A GIRL I.Sebesta 1,2, M.Kumsta1,2, B.Stiburkova2, S.Kmoch2, J.A.Duley3, A.H. Simmonds3. Inst.Inher.Dis.1, Inst.Clin. Biochem.2, 1st Faculty of Medicine, Charles University, Prague, Czech Republic, Purine Research Unit3, UMDS, Guy's Hospital, London,UK.

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) de¢ciency is an X-linked recessive genetic defect associated with two clinical syndromes. The complete de¢ciency presenting in childhood as Lesch-Nyhan syndrome, characterised by self-mutilation, choreoathetosis, spasticity, developmental retardation, gout or urolithiasis, contrasts with partial de¢ciency (Kelley-Seegmiller syndrome), generally presenting later in adolescence with gout and/or urolitiasis. In partial de¢ciency the neurological symptoms may range from absent to severe but do not impose an inability of the Lesch-Nyhan syndrome. Female carriers are healthy. We recently ascertained a girl with partial HPRT de¢ciency su¡ering from gout and renal colic with no neurological involvement. 7 years ago gouty arthritis with hyperuricaemia was found. At the age of 16 years she was re¡ered for detailed purine metabolic investigation. Activity of the phosphoribosylpyrophosphate syntetase (PRPPs) in erythrocytes was normal. The absence of HPRT activity in lysed erythrocytes con¢rmed partial de¢ciency. The loss of HPRT activity was found also in her father, who had renal colics and gout since the age of 18 years. His brother has the same clinical picture. Molecular biological studies of the family started. This ¢rst description of partial HPRT de¢ciency in a female underlines the need of purine metabolic work up in girls with hyperuricaemia and/or gout. The cause of this phenotype could be not only familial juvenile hyperuricaemic nephropathy or overactivity of PRPPs but also HPRT de¢ciency. Supported by EC project BMH4-CT98-3079 and grant MSM-111100005 Czech Ministry of Education

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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EXCLUSION OF FIVE CANDIDATE KIDNEY DISEASE LOCI BY LINKAGE ANALYSIS IN FAMILIAL JUVENILE HYPERURICAEMIC NEPHROPATHY AM Marinaki1, M Greener2, S Fisher2, C Lewis2, MM Town2, F Moro3, H A Simmonds1, JS Cameron1, JA Duley1, C Mathew2. 1 Purine Research Laboratories & 2Dept. of Medical & Molecular Genetics, GKT, Guy's Hospital, London, 3Renal Unit St Mary's Hospital Portsmouth, UK Familial juvenile hyperuricaemic nephropathy (FJHN) is an autosomal dominant disorder, presenting in both males and females in childhood or early adulthood, with gout and/or hyperuricaemia and renal failure. If untreated, FJHN can progress to end stage renal failure. The molecular nature of the defect underlying the hyperuricaemia is unknown. Previous studies excluded genetic disorders of purine metabolism associated with hyperuricaemia or gout by enzyme and metabolic studies; FJHN patients are normoproducers and hypoexcretors of urate, not overproducers. Hereditary renal diseases associated with hyperuricaemia such as re£ux nephropathy, Alports syndrome and Bartters syndrome were also excluded during the clinical workup. However, there remain several candidate inherited renal diseases associated with hyperuricaemia. These include two loci for autosomal dominant adult polycystic kidney disease (PKD1; chromosome 16p13.3-p13.12 and PKD2; Chr 4q21-q23), medullary cystic kidney disease Type 1 (MCKD1; Chr 1 q21) and autosomal recessive familial juvenile nephronophthisis Type 1 (NPHP1; Chr 2 q13). In addition, linkage to a 9cM region of 16p12 has recently been reported in a Japanese FJHN family. Linkage to PKD1, PKD2, MCKD1 and NPHP1 were excluded using a combination of RFLP and microsatellite markers. No linkage to the candidate FJHN locus on 16p12 was found, suggesting underlying genetic heterogeneity of the FJHN phenotype. A genome wide search for linkage on other chromosomes is in progress.

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HEREDITARY OROTIC ACIDURIA WITHOUT MEGALOBLASTIC ANAEMIA G.T.N. Besley1, J.H. Walter1, L.D. Fairbanks2, H.A. Simmonds2, A.M. Marinaki2, A.H. Van Gennip3 Willink Biochemical Genetics Unit, Royal Manchester Children's Hospital, Manchester, M29 4HA, UK1, Purine Research Unit, Guy's Hospital, London, SE1 9RT2, Lab. Genetic Metabolic Diseases, AMC, 1100 DE Amsterdam, The Netherlands3 Hereditary orotic aciduria is a rare disorder of de novo pyrimidine biosynthesis usually associated with macrocytic hypochromic megaloblastic anaemia. We report a case recently diagnosed who did not have the characteristic haematological ¢ndings. The patient is a male infant born to consanguineous Asian parents. He was normal at birth but at 5 months was investigated for visual problems and found to have congenital ocular motor dyspraxia. Walking was delayed at 21 months and at 2 years he only spoke single words. He was investigated at 9 months for developmental delay, and a urine sample showed a marked increase in orotic acid on routine organic acid investigation. This was con¢rmed on HPLC and levels of orotic acid and orotidine were increased to 598 and 465 mmol/ mmol creatinine (normals 51); uracil was only slightly raised. The pattern was consistent with a defect of UMP synthase activity and a defect in the ODC component of this bifunctional protein is likely. De¢ciency of UMP synthase activity was con¢rmed in erythrocytes (0.019nmol/h per mg protein; control 3.66). Mutation studies have identi¢ed a point mutation (928T4G) leading to an amino acid substitution F310V. This is in a highly conserved region of the ODC domain. A bone marrow aspirate showed normal erythropoiesis with no evidence of megaloblastic anaemia.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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195

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NEUTRAL LIPID STORAGE DISEASE IN 3 TURKISH CHILDREN Ali Dursun*, Serap Kalkanogïlu*, Figen Gu«rakan*, Turgay Cos° kun*, Ays° egu«l Tokath*, Nurten Koc°ak* Hacettepe University, Department of Pediatrics, Ankara, TURKEY

Neutral Lipid Storage Disease (NLSD) (Chanarin^Dorfman disease), inherited autosomal recessively, is characterized by variable degrees of skin, liver, muscle, eye, and nervous system involvement. Although ichthyosis and vacuolisation of leucocytes due to triglyceride accumulation were universal ¢ndings, symptoms related to other systems were detected in older patients. We described here the clinical spectrum of 3 Turkish children with NLSD (Table). So far 27 patients have been reported, two of whom are of Turkish origin. Vacuoles in cytoplasm of the eosinophils were noted in some parents, which was reported especially in the heterozygotes of Middle Eastern origin. In one family, we detected that the mother had vacuoles in eosinophils, but the father had not. This patient is most probably compound heterozygote. The interval between onset of clinical manifestation and the ¢nal diagnosis was 1.5, 3.5 and 8 years in our patients respectively. This data suggested that peripheral blood smears should be examined with special attention to the morphology of the leucocytes in every case with a previsional diagnosis of ichthyosis. Table: Clinical presentation of three patients with NLSD Age/sex

Ichthyosis

Myopathy

Hepatomegaly with high transaminases

Growth retardation

Eosinophils vacuolisation of parents

2y/F 5.5y / F 10 y / M

+ + +

+ +

+ +

+ +

-/+/-/-

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ONE YEAR SCREENING OF INBORN ERRORS OF STEROL METABOLISM IN SPANISH PATIENTS M. Giro¨s1, N. Clusellas1, F. Borja1, T. Vendrell2, E. Gean3 and T. Pampols1 1 Institut de Bioqu|¨ mica Cl|¨ nica, 2Genetic Service Hospital Vall d'Hebro¨n and 3Genetic Service Hospital Sant Joan de Deu Barcelona. Spain. Over a period of one year, we have received 36 requests for biochemical diagnosis of Smith-LemliOpitz (SLO) and 5 for Cerebrotendinous Xanthomatosis (CTX). Sterol determination was carried out in serum following the procedure of saponi¢cation and sterol extraction with hexane. Sterols were quanti¢ed /identi¢ed by gas liquid chromatography as the trimethylsilyl derivatives. 14/36 patients suspected of su¡ering from SLO presented increased levels of 7- and 8-dehydrocholesterol (7DHC/ 8DHC), but only 12 /14 were de¢cient in cholesterol levels. Although the clinical symptoms were neonatal in 11/14, the biochemical diagnosis was done from 6 months to 16 years. All of them are still alive. One patient who had dysmorphic features and ichthyosis in the neonatal period, showed low levels of cholesterol and the presence of a peak co-eluted with 8DHC and another not yet identi¢ed. 3/5 patients suspected of su¡ering from CTX with xantomas presented increased levels of b-cholestanol. In the other two, only a high level of cholesterol was found. Twin patients with spastic paraparesis, seizures and cataracts , but without xanthomas were also diagnosed as CTX due to the elevation of b-cholestanol. In summary, the sterol determination has allowed the diagnosis of 20 cases:14 SLO , 5 CTX and a patient with a still unidenti¢ed problem of cholesterol synthesis. The introduction of sterol determination allows a rapid and cost e¤cient diagnosis of the inborn errors of sterol metabolism (Grant from Spanish Health Ministry: FIS99/0282).

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SMITH-LEMLI-OPITZ SYNDROME: THE MILD END OF THE CLINICAL AND BIOCHEMICAL SPECTRUM F.A.A. Langius,1 H.R. Waterham,2 J. Koster,2 L. Dorland,1 M. Duran,1 T.J. de Koning,1 F.A. Beemer,3 R.J.A. Wanders,2 B.T. Poll The1 1 Department of Metabolic Diseases and 3Medical Genetics, University Children's Hospital, Utrecht, 2 Departments of Clinical Chemistry and Pediatrics, Academic Medical Centre, Amsterdam, The Netherlands Smith-Lemli-Opitz Syndrome (SLOS) is an autosomal recessive malformation syndrome characterised by mental retardation, congenital anomalies and growth de¢ciency. The cause of SLOS is a block in cholesterol biosynthesis at the level of 7-dehydrocholesterol reductase (7DHCR), which results in elevated levels of the cholesterol precursor 7-dehydrocholesterol and its isomers. We studied three patients from two families, who had a very mild form of SLOS. Clinically, they only show syndactyly of toes II / III and some minor dysmorphic features. MRIs of the cerebrum were normal. They enjoy normal primary and secondary education. Biochemically, they only have slightly elevated serum levels of 7- and 8-dehydrocholesterol (7DHC: 2.3-8.6 mmol/L, n: 50.8 mmol/L), but normal cholesterol values. Furthermore, cells of the patients displayed signi¢cant residual 7DHCR activity. Two brothers were heterozygotes for the frequent IVS8-1G4C mutation and the third patient for an E448K mutation in the 7DHCR gene. In addition, all three patients were heterozygotes for a novel mutation a¡ecting the translation initiation (M1I). Our results reveal a clear genotype-phenotype correlation and have important implications for the clinical identi¢cation and diagnosis of patients with very mild forms of SLOS.

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CHOLESTEROL SUPPLEMENTATION DOES NOT ALTER DEVELOPMENTAL PROGRESS IN SMITH-LEMLI-OPITZ SYNDROME M Ruggiero, K Pettit-Kekel, D Sikora, L Linck, WE Connor, RD Steiner. Oregon Health Sciences University, Portland, OR, USA

Smith-Lemli-Opitz syndrome (SLOS) is a multiple malformation/mental retardation syndrome caused by de¢ciency in the ¢nal enzyme in cholesterol synthesis, 7-dehydrocholesterol-a-reductase. It is unclear whether the resulting cholesterol de¢ciency or 7-DHC excess, or both, is responsible for the phenotype. We hypothesized that cholesterol supplementation would improve developmental progress in SLOS by ameliorating cholesterol de¢ciency and inhibiting 7-DHC synthesis. Eight children with SLOS are enrolled in a prospective study assessing developmental progress during cholesterol supplementation of 1/2 to 2 hard-boiled egg yolks per day. At least two developmental evaluations, using the Bayley, Peabody, Vineland, and Stanford Binet tests, were performed during continuous cholesterol supplementation for all but one subject. Average ages were: at ¢rst testing27months (mo.), at most recent testing-49 mo. Duration of cholesterol supplementation at the time of the ¢rst evaluation: 0-29 mo.(ave.-10 mo.), at the most recent evaluation: 9-57 mo.(ave.-32 mo.), and between evaluations: 7-36 mo.(ave.-24 mo.). The average DQ (Developmental age/Chronological age x 100) of those supplemented was 53 at ¢rst testing, and 42 at the most recent testing. The loss of DQ despite continuous cholesterol supplementation would indicate lack of e¤cacy of cholesterol supplementation in preventing mental retardation. These ¢ndings suggest that cholesterol supplementation does not improve developmental progress in SLOS patients, and that suggest placebo controlled trials are indicated.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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WHOLE BODY CHOLESTEROL SYNTHESIS IS REDUCED IN SMITH-LEMLI-OPITZ SYNDROME Steiner RD, Flavell D, Lin D, Connor WE. Oregon Health Sciences University, Portland, OR, USA

Smith-Lemli-Opitz syndrome (SLOS) is caused by de¢ciency of 7-dehydrocholesterol-D7-reductase. This enzyme converts 7-dehydrocholesterol (7-DHC) to cholesterol in the ¢nal step in cholesterol synthesis. The pathogenesis of SLOS may be related to cholesterol de¢ciency or accumulation of noncholesterol sterols. To date there has been no data on whole body cholesterol synthesis in SLOS. We measured whole body synthesis of cholesterol, 7-DHC, and bile acids in 8 subjects and 6 controls. The diets were essentially cholesterol-free and precisely controlled. Stools were collected for one week. Sterols and bile acids in the stools were assayed by gas-liquid chromatography. Cholesterol, total sterol, 7-DHC, and bile acid synthesis were calculated by sterol balance. Cholesterol synthesis in SLOS subjects was signi¢cantly reduced compared with controls (9 vs. 20 mg/kg/d, P50.002). 7DHC was synthesized in SLOS subjects (1.7 þ 1.2 mg/kg/d), but not in controls. Total sterol synthesis was also reduced in SLOS subjects (12 vs. 20 mg/kg/d, P50.022). Bile acid synthesis (3.5 mg/kg/d) did not di¡er signi¢cantly from controls (4.6 mg/kg/d). This study provides direct evidence that whole body cholesterol synthesis is reduced in SLOS and synthesis of 7-DHC is profoundly increased. It is the ¢rst measure of daily bile acid synthesis in SLOS and provides evidence that bile acid supplementation may not be necessary in treatment. These results strengthen the rationale for use of dietary cholesterol in therapy and provide data on which to base dosing estimates.

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EFFECT OF CHOLESTEROL SUPPLEMENTATION ON SERUM LIPID AND APOLIPOPROTEIN LEVELS IN CHILDREN WITH SMITH-LEMLI-OPITZ SYNDROME Behu¨lova¨,D1., Bzdu¨ch,V2., Síkodova¨,J1., Dello Russo,A3., Corso,G3., Ponec,J1., Barosíova¨,J4., Nebeson©í a¨kova¨,E5. Dept. Clinical biochemistry1 and First Dept. Pediatrics2 University Children¨s Hospital, Bratislava, Slovakia, Department of Biochemistry and Medical Biotechnology3, Federico II University, Naples, Italy, Dep.Med.Genetics Nitra4 and Kosíice5, Slovakia.

The characteristic serum lipid and apolipoprotein pattern in children with Smith-Lemli-Opitz syndrome (SLOS) consists of decreased concentration of total cholesterol (C), HDL C, LDL C, apolipoprotein (apo) A-I and normal levels of VLDL C and apo B (Behu¨lova¨ D et al. (2000) Serum lipids and apolipoproteins in children with the Smith-Lemli-Opitz syndrome. J.Inher.Metab.Dis (In press). We compared changes in serum lipids and apolipoproteins (measured by enzymatic and imunoturbidimetric methods) in two SLOS children treated with C (50 mg/kg/day) and ursodeoxycholic acid (15 mg/kg/day) with an untreated SLOS patient. In Patient 1 treatment was initiated at the age of 7 months and continued for 24 months. Biochemical studies before and after therapy revealed the following: total C of 1.65 and 2.65 mmol/l, HDL C of 0.33 and 0.96 mmol/l, LDL C of 0.54 and 1.10 mmol/l, apo A-I of 0.46 and 1.11g/l, apo B of 0.62 and 0.60 g/l. Biochemical analysis in Patient 2 aged 5 years before and after therapy giving during 4 months showed the following: total C of 2.10 and 2.45 mmol/l, HDL C of 0.59 and 0.77 mmol/l, LDL C of 0.92 and 1.20mmol/l, apo A-I of 0.76 and 0.91 g/l, apo B of 0.63 and 0.65 g/l. On the contrary, investigation in the untreated 5 year-old Patient 3 at the time of diagnosis and after period of 24 months revealed the following: total C of 1.33 and 1.22 mmol/l, HDL C of 0.13 and 0.18 mmol/l, LDL C of 0.43 and 0.20 mmol/l, apo A-I of 0.15 and less than 0.11 g/l, apo B of 0.68 and 0.71 g/l. We conclude that long-term treatment with exogenous cholesterol and bile acid in SLOS results in a marked increase or normalization in serum total cholesterol, its HDL and LDL fractions and mainly serum apo A-I levels. This therapy has no signi¢cant e¡ect on serum apo B concentrations.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SIMVASTATIN: A NEW THERAPEUTICAL APPROACH FOR SMITH-LEMLI-OPITZ SYNDROME (SLO): INITIATION OF AN INTERNATIONAL TRIAL R.A. Wevers1, P.E. Jira2, F.K. Trefz3, F.S.M. Janssen-Zijlstra1, J.A.M. Smeiink2. 1 Laboratory of Pediatrics and Neurology, 2Department of Metabolic Diseases, Institute of Pediatrics, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmeen, the Netherlands (e-mail: [email protected]), 3Department of Pediatrics, Reutlingen, Germany. Accumulation of the cholesterol precursors 7DHC and 8DHC may contribute to the poor outcome in SLO. Statins may improve the precursor to cholesterol-ratio and the clinical outcome. Simvastatin was given (0.6-1.0 mg/kg/day) for a median period of 20 months to three SLO patients (1, 3 and 10 months of age). This treatment gave a lasting improvement of the precuror to cholesterol-ratio in plasma, erythrocyte membranes and CSF. Plasma precursors concentrations decreased to 24% (1033%) of the initial level, whereas the cholesterol concentration unexpectedly normalized (42.5 mmol/L) by a more than twofold increase in all. The plasma precursor to cholesterol-ratio improved from 0.28, 0.47 and 0.32 before, to 0.01, 0.06 and 0.04 after treatment, respectively. During the follow-up all morphometric parameters and neuromotor development improved. The therapy was well tolerated without clinical side e¡ects. This is the ¢rst study where the blood cholesterol level in SLO patients normalized with a simultaneous signi¢cant decrease in precursor levels with lasting biochemical and encouraging clinical improvement. Statin therapy is a promising novel approach in SLO. A collaborative international trial will be started in the course of 2000. Details on the trial are available via the Nijmegen metabolic group.

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A GENETIC MOUSE MODEL OF SMITH-LEMLI-OPITZ SYNDROME N.A. Nwokoro1, C.A. Wassif1, P.A. Krakowiak1, L.E. Kratz2, K.P. Battaile3, R. D. Steiner3, F.D. Porter1. 1 Heritable Disorders Branch, NICHD, NIH, Bethesda, Maryland; 2Kennedy Krieger Institute and JHU, Baltimore, Maryland; 3Molecular and Medical Genetics, Oregon Health Sciences University, Portland, Oregon, USA OBJECTIVE: To further our understanding of Smith-Lemli-Opitz syndrome (SLOS) at the biochemical and developmental level by producing and studying a genetic mouse model. BACKGROUND: SLOS is an autosomal recessive multiple congenital malformations syndrome due to mutations in the sterol _7-reductase gene. This enzyme catalyzes the conversion of 7-dehydrocholesterol (7-DHC) to cholesterol. The human enzyme has been cloned and to date, 54 di¡erent mutant alleles have been reported. METHODS: A genetic mouse model was produced by disrupting the sterol _7-reductase gene in mouse embryonic stem cells. RESULTS: Heterozygous mice are viable, fertile, and phenotypically normal. The homozygous mutant mice, similar to many SLOS infants, show decreased movement, are small at birth (77% control size, p 5 0.0001) and have feeding di¤culties. Craniofacial anomalies include cleft palate (9/111) and nasal occlusion (26/67). The mutant pups fail to feed and die within 24 hours. Aspiration limits the ability to hand feed mutant pups. Sterol D7reductase activity, as measured by the conversion of ergosterol to brassicasterol, was 0.3% of control level (p 5 0.0001). GC/MS analysis of serum, brain, liver, kidney and skeletal muscle in the mutant mice showed mildly reduced total sterol, elevated 7-DHC, and decreased cholesterol levels. In mutant brains from E12.5 to birth, a steady increase in both 7-DHC and 7-dehydrodesmosterol levels paralleled the normal increase of cholesterol and desmosterol. CONCLUSION: The development of a genetic mouse model for SLOS a¡ords us an opportunity to characterize further this disorder and design experiments to test therapeutic interventions including gene therapy.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PRENATAL DIAGNOSIS OF SMITH-LEMLI-OPITZ SYNDROME BY MUTATION ANALYSIS Bzdu¨ch,V1.,Koza¨k,L2., Francova¨,H2., Behu¨lova¨,D3. First Department of Pediatrics1 and Clinical Biochemistry3 University Children¨s Hospital, Bratislava, Slovakia ,Research Institute of Child Health, Brno, Czech republic2 Prenatal diagnosis for Smith-Lemli-Opitz syndrome (SLOS, McKusick 270 400) has previously been performed by determination of abnormally elevated amniotic £uid dehydrocholesterol concentration in the amniotic £uid. Recently the gene for 7-dehydrocholesterol reductase (DHCR7) has been cloned and mutation causing SLOS have been identi¢ed. We report the molecular prenatal diagnosis of SLOS in two families with known genotype. In the ¢rst family the the mother carried G410S mutation and the father carried W151X mutation. In the second family either parents were found to be carriers for W151X mutation. Results of the sterol analysis of amniotic £uid by gas chromatography/mass spectrometry indicated that neither foetus should a¡ected by DHCR7 de¢ciency. To con¢rm this assumption molecular analysis was performed. DNA isolated from amniocytes was analysed for the mutation known in corresponding families. Two techniques, polymerase chain reaction together with restriction analysis (PCR/RFLP) and sequencing were used for mutation analysis. It was found that both fetuses are healthy heterozygote carriers, ¢rst for the maternal mutation G410S and second for the mutation W151X. We conclude, that reliable prenatal diagnosis of SLOS by molecular analysis in families with known genotype become now possible.

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BIOCHEMICAL AND MOLECULAR DIAGNOSIS OF CHOLESTEROL SYNTHESIS DEFECTS: SMITH-LEMLI-OPITZ SYNDROME HR Waterham,1 P Vreken,2 GJ Romeijn,2 W Oostheim,2 W Smit,2 J Koster,2 RJA Wanders1,2 Departments of Pediatrics1 and Clinical Chemistry2, Academic Medical Center, Amsterdam

Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive developmental disorder characterized by facial dysmorphism, mental retardation and multiple congenital anomalies. The disorder is caused by a de¢cient activity of 7-dehydrocholesterol reductase (7-DHCR), the enzyme catalyzing the ¢nal step in cholesterol synthesis, which is the conversion of 7-dehydrocholesterol (7-DHC) into cholesterol. As a consequence, most patients have low serum cholesterol and elevated 7-DHC (and 8DHC) levels. Our laboratory has developed a complete package for post- and prenatal diagnosis of SLOS both at the biochemical and the molecular level, including serum analysis of sterol and bile acids by GC-MS, analysis of de novo cholesterol synthesis in ¢broblasts of patients by 14Cmevalonate incorporation, measurement of speci¢c 7-DHCR enzyme activity in cell lysates by ergosterol conversion and mutation analysis of the DHCR7 gene to detect the disease-causing mutations. Finally, we have generated 7-DHCR-speci¢c antibodies to study the e¡ect of the various mutations on the protein by immunoblot analysis. Currently, we have performed post-natal analysis in 32 patients clinically diagnosed with SLOS and performed 4 pre-natal analyses. So far, 27 di¡erent mutations have been identi¢ed which when compared with the corresponding 7-DHCR enzyme activities provide insight into the genotype-phenotype relationship of SLOS.

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DESMOSTEROLOSIS PRESENTING WITH MULTIPLE CONGENITAL ANOMALIES AND PROFOUND DEVELOPMENTAL DELAY HC Andersson, LE Kratz and RI Kelley. Tulane University School of Medicine, New Orleans, LA and Kennedy Krieger Institute, Baltimore, MD. Desmosterol is a sterol intermediate in the cholesterol biosynthetic pathway. We describe the ¢rst living patient with desmosterolosis, an infant with dysmorphic facial features, profound microcephaly, limb anomalies, 2-vessel cord and persistent patent ductus arteriosus. Normal studies included prometaphase chromosome analysis, normal renal and cardiac sonography (other than a PDA), and TORCH titers. Plasma sterol quanti¢cation (GCMS)showed normal cholesterol and 7-dehydrocholesterol levels but a 100-fold increased desmosterol (60mg/ml; nl 0.5 +/- 0.3 (SD)mg/ml) suggestive of steroid-24-reductase de¢ciency. Additional biochemical studies found that both parents had moderately elevated plasma desmosterol levels (mother: 1.4mg/ml; father: 1.8mg/ml), suggestive of heterozygosity for steroid-24-reductase de¢ciency. Analysis of sterol synthesis in transformed lymphoblasts documented de¢cient conversion of desmosterol to cholesterol and low cholesterol levels in the patient's cells: Cholesterol (mg/mg prot) Patient (n=3) Mother (n=1) Control (n=3)

7.0 (SD=0.7) 15.1 10.6 (SD=0.5)

Desmosterol (mg/mg prot) Desm/Sterol Ratio 5.2 (SD=0.96) 0.24 0.02 (SD=0.01)

42.4% 1.6 0.2

At age 3.5y, the patient stands, uses 5 words, and has no major medical problems. This unique patient broadens the spectrum of inborn errors of cholesterol biosynthesis o¡ering important contrasts to the SLO syndrome. Photographs and video of the patient will be presented.

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ABNORMALTIES OF LATERALITY DEVELOPMENT IN X-LINKED DISORDERS OF CHOLESTEROL BIOSYNTHESIS D.K. Grange1, G.E. Herman2, K.J. Kopacz2, L.E. Kratz3 and R. I. Kelley3. 1 St. Louis Univ., St. Louis, MO, 2Children's Hosp. Research Fdn., Columbus, OH and 3Johns Hopkins Univ., Baltimore, MD CHILD syndrome is an X-linked dominant disorder with congenital hemidysplasia, ichthyosis, and limb defects, most often a¡ecting the right side. Large areas of skin not following lines of Blaschko are almost always involved (trunk) or always spared (face). We have studied a total of 4 patients with CHILD syndrome, two with X-linked sterol D8-isomerase def. and two with sterol abnormalities in cultured cells indicating sterol-4-demethylase (NSDHL) def. The latter two patients were heterozygous for the same splicing mutation in NSDHL (IVS7-2 A to C). All 9 published or unpublished patients with CHILD syndrome caused by X-linked defects of sterol metabolism have predominantly right-sided disease. The embryological mechanism by which these X-linked mutations cause unilateral disease is unknown. Unilateral involvement cannot be explained by random X inactivation alone. In the mouse, studies of laterality determination at Henson's node have delineated a cascade of signaling proteins including FGF8 (left-determining) and Sonic Hedgehog (SHH) (right-determining). Because SHH signaling is impaired in another defect of sterol biosynthesis, SLOS, we speculate that in CHILD syndrome abnormal function of SHH and/or related signaling proteins (e.g. Indian HH), plays a role in early embryonic survival of cellular clones expressing normal or mutant alleles. Laterality determining proteins such as SHH and FGF8 expressed by an ``organizer clone'' (Happle) of cells at the midline of the embryo may interact with mutant gene expressing cells, leading to the asymmetric defects in CHILD syndrome.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BIOCHEMICAL AND MOLECULAR DIAGNOSIS OF CHOLESTEROL SYNTHESIS DEFECTS: X-LINKED DOMINANT CHONDRODYSPLASIA PUNCTATA (CDPX2) HR Waterham,1 J Koster,1 W Smit,2 RJA Wanders,1,2 P Vreken 2 Departments of Pediatrics1 and Clinical Chemistry2, Emma Children's Hospital, Academic Medical Center, University of Amsterdam

X-linked dominant chondrodysplasia punctata (CDPX2), also known as Conradi-HunermannHapple syndrome, is a male-lethal disorder characterized by asymmetric rhizomesomelia (shortening of limbs), hyperkeratosis and ichthyosis. Recently, it was demonstrated that CDPX2 is caused by a de¢cient activity of sterol D8-D7 isomerase, one of the enzymes involved in cholesterol biosynthesis. As a consequence, abnormal amounts of the sterol intermediates cholest-8(9)-en-3b-ol and cholesta5,8-dien-3b-ol (8-DHC) can be found in cultured ¢broblasts of patients. Currently, our laboratory has con¢rmed the diagnosis of CDPX2 in 10 suspected patients both at the biochemical (sterol analysis by GC-MS) and the molecular level (mutation analysis). Biochemically, serum of most patients contained readily detectable elevated levels of cholest-8(9)-en-3b-ol and, occasionally, 8-DHC. Both sterols were also clearly detected in ¢broblast cultures of patients grown in sterol-free medium. Mutation analysis of the EBP gene, which codes for the sterol D8-D7 isomerase, revealed di¡erent mutations. In a few patients the mutations were inherited from their mothers but most mutations were de novo in agreement to the sporadic nature of CDPX2.

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CLINICAL AND MOLECULAR GENETIC CHARACTERISTICS OF PATIENTS WITH CEREBROTENDINOUS XANTHOMATOSIS R.A. Wevers 1, A.Verrips 2, E.H. Hoefsloot 3, G.C.H. Steenbergen 1, J.P. Theelen 3, F.J.M. GabreÍls 2 , B.G.M. van Engelen 2, L.P.W.J. van den Heuvel 1. 1 Laboratory of Pediatrics and Neurology, Departments of 2Neurology, and 3Human Genetics, University Medical Centre Nijmegen, P.O. Box 9101, 6500 HB Nijmeen, the Netherlands.

Cerebrotendinous xanthomatosis is a lipid storage disease caused by a de¢ciency of the mitochondrial enzyme 27-sterol hydroxylase, due to mutations in its gene. In this study we report on mutations in 58 patients with cerebrotendinous xanthomatosis out of 32 unrelated families. Eight of these were novel mutations, two of which were found together with two already known pathogenic mutations. Twelve mutations found in this patient group have been described in the literature. In the patients from 31 families, mutations were found in both alleles. In one family, two mutations were found together in one allele. In the literature, 28 mutations in 67 patients with cerebrotendinous xanthomatosis out of 44 families have been described. Pooling our patient group and the patients from the literature together, 37 di¡erent mutations in 125 patients out of 74 families were obtained. Identical mutations have been found in families from di¡erent ethnic background. In 41% of all the patients, 27-sterol hydroxylase gene mutations are found in the region of exons 6 - 8. This region encodes for adrenodoxin and heme binding sites of the protein. A genotype-phenotype analysis was done for 79 homozygous patients out of 45 families, harbouring 23 di¡erent mutations. The patients with compound heterozygous mutations were left out of the genotype-phenotype analysis. There was no genotype-phenotype correlation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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HORMONE-SENSITIVE LIPASE (HSL) DEFICIENCY IN MICE: IMPLICATIONS FOR HUMAN INBORN ERRORS Grant A. Mitchell(1), Shu Pei Wang (1), Nancy Laurin (1), Jean Himms-Hagen (2), Shari Chung (3), Michael A. Rudnicki (3), Emile Levy (1), Marie-France Robert (1), Linghe Pan (1), Luc Oligny(1), Louis Hermo (3), Jacquetta Trasler (3). [(1)Depts of Pediatr (SPW, NL, EL, MFR, LP, GAM) and Pathol (LO), Research Center, SteJustine Hosp, Montreal, CAN; (2) Fac Medicine, U Ottawa, Ottawa, CAN; (3) Dept Anat & Montreal Child Hosp Res Inst, McGill U, Montreal, CAN; (4) Inst Molec Biol & Biotech, McMaster U, Hamilton, CAN] Hormone-sensitive lipase (HSL, E.C.3.1.1.3) is a 84 kDa multifunctional hydrolase of adipocytes, in which it cleaves triacylglycerols in an intricately-controlled fashion. HSL can also hydrolyse fatty acyl esters of cholesterol, retinoic acid and steroid hormones, and is expressed in many tissues including male germ cells. To directly ascertain its physiological roles, we performed constitutive gene targeting in mice. The anticipated HSL-/- phenotype was obesity, fasting intolerance and hypoketonemia. Surprisingly, HSL -/- mice have normal total body weight and tend to reduced abdominal fat mass. They tolerate overnight fasting and prolonged cold exposure. Histologically, both white and brown adipose tissues show striking heterogeneity in cell size. In isolated HSL-/- adipocytes, lipolysis is not increased by beta-adrenergic stimulation, but the basal rate of lipolysis is at least as high as in normal adipocytes. Interestingly, HSL-/- males are infertile. These observations show a previouslyunexpected redundancy at the ¢rst step of lipid energy metabolism and agree with those obtained independently by Osuga et al (PNAS 2000, 97, 787-92). Complete human HSL de¢ciency has not been reported. If similar to the mouse phenotype (male infertility with subtle fat changes at the macroscopic level), it may have been overlooked.

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LEUKOENCEPHALOPATHY WITH VANISHING WHITE MATTER M. Pineda1, E. Gelpi1, A. Aracil1, J. Pineda2, R. Artuch2, C. Colome2, V. Cusi3, M. Baquero4, A. Capdevila4, MA Vilaseca2. S. Neuropediatria1, Bioqu|¨ mica2, Anatomia Patolo©gica3. Unitat Integrada. H. Sant Joan de De¨uCl|¨ nic. C. Diagno©stic Pedralbes de Resona©ncia Nuclear Magne©tica4. Barcelona. Spain.

We reviewed ten patients who ful¢lled all clinical inclusion criteria for diagnosis of vanishing white matter disease. Consanguinity was referred in one family, and two pairs of siblings were present in our series. Initially they showed normal psychomotor development and head circumference, neurologic deterioration started on early-childhood, between 17-30 months (7/10 cases) and on late childhood 3.5-6 years of age (3/10). Infection (5/10) and minor trauma (3/10) preceded episodes of rapid deterioration. Neurologic examination showed predominantly chronic-progressive spasticity (9/10) and cerebella ataxia (3/10). Optic atrophy was present only on (2/10) and epileptic seizures (4/10). Peribucal and action myoclonus was present in 2 patients. Death happened between 4 and 19 years of age. Clinical follow-up has been between 1.8-17 years. Three cases with a milder disease course are still alive. CTscan, MRI and MR-S showed di¡use cerebral hemispheric leukoencephalopathy, in the follow-up progressive increasing areas of abnormal white matter that vanished and were replaced by CSF. Lisosomal and peroxisomal disorders were excluded. Respiratory chain activities in muscle biopsy did not show any mitochondrial disorder in 4 cases. The CSF to plasma glycine ratio was normal in the seven cases studied; glycine loading test showed delayed degradation of glycine in 1/3 patients assayed. Glycine in body £uids analysed during progressive evolution and also during episodes of deterioration has not revealed abnormalities. In our experience, glycine does not seem a good marker for vanishing white matter disease. Supported by a grant for Scienti¢c research (FIS nú 98/0049-01) from the Ministry of Health of Spain.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CYSTIC LEUKOENCEPHALOPATHY - A NEW GROUP OF UNDETERMINED LEUKODYSTROPHIES (UL) Jutta GÌrtner, Fuat Aksu, Christoph HÏbner H. Heine-UniversitÌt DÏsseldorf, Vestische Kinderklinik Datteln and Charite¨ Berlin, Germany

Leukodystrophies are an increasing group of disorders that predominantly a¡ect the white matter of the central nervous system. They have been de¢ned according to their causes, either inherited or acquired. In at least 1/3 of cases the aetiology is unknown. We and others have started to categorise patients with UL into major groups by using clinical and magnetic resonance imaging (MRI) criteria. Examples of already established groups are myelinopathia centralis di¡usa (MCD) and leukoencephalopathy with swelling and mild clinical course. Here we describe one German and three Turkish patients who represent a further major group of UL. Their neurological abnormalities were noted within the ¢rst months of life and include severe intellectual impairment, motor retardation, spasticity, and epilepsy. MRI revealed a unique pattern, the presence of bilateral anterior temporal lobe cysts and multifocal lobar white matter lesions with characteristic sparing of central white matter structures; the signal intensities of the cysts' content were identical to those of cerebrospinal £uid. The MRI spectroscopy pro¢les were only indicative of neuronal dysfunction or loss and did not show a unique diagnostic feature. In all patients, extensive laboratory investigations including those for inborn errors of metabolism and infectious diseases did not reveal any abnormalities. The MRI pattern and identical clinical picture of these patients are distinct from other described UL. They de¢ne a new group of UL and probably a new inherited disease entity.

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AUTOSOMAL DOMINANT FEVER-ASSOCIATED ENCEPHALOPATHY Derek Neilson, Robert Eiben, Charles Hoppel, Matthew Warman, Douglas Kerr Depts. of Pediatrics, Genetics, and Medicine, Case Western Reserve Univ., Cleveland, OH, USA

Objective: To present clinical, radiographic, biochemical, and familial data of a large kindred with acute encephalopathy following fever. Method: Case presentation, pedigree, and metabolic analysis. Results: Three y.o. developmentally normal identical twins developed acute encephalo-pathies following a fever. Ammonia, blood lactate and pyruvate, urine organic acids, and plasma amino acids were normal. CSF lactate, pyruvate, and cell counts were normal. MRI of the brain demonstrated symmetric decreased T1 and increased T2 and FLAIR weighted signal along the pons, midbrain, thalami, external capsule, and subinsular white matter in one case and included the medial temporal lobes and cerebellar hemispheres in the other. Oxidative phosphorylation analysis of intact mitochondria isolated from a muscle biopsy demonstrated an increase in state 4 oxidation rates and low respiratory control, indicating uncoupling. Both twins made remarkable recoveries within two months. A six-generation pedigree reveals 16 individuals with similar acute encephalopathies. Some relatives recovered completely while others did not. Male-to-male transmission in a dominant pedigree demonstrates autosomal dominant inheritance. Una¡ected obligate carriers prove incomplete penetrance. Conclusion: This family represents a previously undescribed, incompletely penetrant, autosomal dominant disorder with features of mitochondrial encephalopathy, but without the characteristic degenerative course.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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FAMILIAL CYSTIC LEUKOENCEPHALOPATHY WITH NEMALINE BODY MYOPATHY IN TWO SIBLINGS GA Mitchell (1), A Lortie(2), Y Robitaille(3), J-C De¨carie(4), M Lambert(1). [Services de (1) ge¨ne¨tique me¨dicale and (2)Neurologie, De¨partement de Pe¨diatrie, and De¨partements de (3) Pathologie and (4) Imagerie me¨dicale, Hoªpital Ste-Justine, Montre¨al, Canada] We report a sister and brother of Haitian origin who developed progressive coma, refractory epilepsy, decerebration and optic atrophy with progressively increased head circumference. Their pregnancies and birth were normal. They developed normally until 7 and 15 months respectively. In each case, they deteriorated over several days following a gastroenteritis-like illness. In the younger patient, di¡use white matter hypodensity was demonstrated on cerebral CT prior to his decompensation at age 15 months. Partial recuperation occurred brie£y after the decompensation of the girl, with subsequent deterioration. Other organ systems were not clinically a¡ected initially. The following were normal: plasma lactate, pyruvate, ammonia, VLCFA, phytanic acid, folate, vitamin B12, lead, cholesterol and triglycerides, urine organic acids and N-acetylaspartate and CSF protein, glucose and cell counts. Fibroblast hexosaminidase, galactocerebrosidase and aspartoacylase were also normal. Autopsy was permitted only for the sister, who died at age 6 years. It revealed a di¡use cystic sudanophilic leukoencephalopathy with evidence of structurally abnormal mitochondria, severe optic atrophy, nemaline body myopathy and tomacular peripheral neuropathy. Mild biventricular cardiac hypertrophy and hepatomegaly with macrovesicular steatosis were also present. The association of signs found in these patients is unusual; the underlying cause is still under investigation.

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ENCEPHALOMYOPATHY IN MICE LACKING THE MITOCHONDRIAL OUTER MEMBRANE VOLTAGE DEPENDENT ANION CHANNELS William J. Craigen, Keltoum An£ous, J. David Sweatt, Edwin Weeber Department of Molecular and Human Genetics and Division of Neuroscience, Baylor College of Medicine, Houston Texas, USA Encephalomyopathy is a frequent consequence of mitochondrial disorders, yet the mechanisms underlying brain dysfunction in the face of mitochondrial abnormalities is poorly understand. We have developed mouse models of mitochondrial dysfunction by disrupting the genes encoding the mitochondrial outer membrane Voltage-dependent Anion Channels (VDACs or porins). Skinned ¢ber studies of cardiac and skeletal muscle of VDAC1 de¢cient mice reveal reduced permeability for ADP but no e¡ect on creatine coupling, while electron micrographs demonstrate structurally abnormal mitochondria. To examine neurologic function, behavioral testing and electrophyiologic studies of the hippocampus were performed. Fearing conditioning paradigms of associative learning are abnormal in all mice, with VDAC1, VDAC3, and VDAC1/VDAC3 de¢cient mice exhibiting a de¢cit in cued conditioning. VDAC3 and VDAC1/VDAC3 de¢cient mice also demonstrate defective contextual fear conditioning. Spatial learning, as tested by the Morris water maze, is also impaired in all mice. Synaptic plasticity of hippocampal neurons is abnormal in all three strains, yet di¡er in the speci¢c types of defects. VDAC1 and double mutant mice demonstrate an almost complete block in tetanus-induced long term potentiation, while VDAC3 animals exhibit a pronounced abnormality in paired pulse facilitation, implying that each channel plays a di¡erent role in synaptic plasticity. These results provide insights into the roles mitochondria normally play in learning and memory formation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SIGNAL TRANSDUCTION DEFECTS: A NOVEL FAMILY OF NEUROMETABLIC DISEASES J. Jaeken1, K. Freson2, M. Hoylaerts2, M. Eyssen1, J. Arnout2, J. Vermylen2, C. Van Geet1,2 Department of Pediatrics1 and Center for Molecular and Vascular Biology2, University Hospital Gasthuisberg, University of Leuven, Leuven, Belgium

Signal transduction disorders are defects in receptors, G proteins or downstream signal transductors. Mutations in the alpha subunit of the Gs proteins (encoded by GNAS 1 located in an imprinting cluster on chromosome 20q13.12-13), cause rare endocrine disorders such as Albright hereditary osteodystrophy (heterozygous inactivating mutations) and McCune^Albright syndrome (activating mosaic mutations). The GNAS1 cluster encodes the XL-GNAS1 exon 1 which is paternally expressed and maternally methylated. Using a modi¢ed platelet aggregation test (C. Van Geet et al. submitted) to examine the Gsa pathway we identi¢ed 5 patients (from 3 families) with an extra repeat and a mutation in the internal repeat region of the XL-GNAS1 exon 1 on the paternal allele. These patients had mainly a neurological syndrome (psychomotor retardation, hypotonia, behaviour disturbances, movement disorders) as well as variable dysmorphy (mainly of face and hands) and, only in one, a bleeding tendency. This defect was associated with a loss of paternal expression of XL-GNAS1, a switch to expression of the maternal allele and an overexpression of GNAS1 with increased cAMP levels in platelets and ¢broblasts upon stimulation. In 2 patients this defect was combined with a polymorphism in b3 and a more severe clinical syndrome. (Jaeken et al. J Inher Metab Dis 1999; 22 (suppl I): 10). Conclusion: G protein defects can be associated with neurological syndromes. Platelet function analysis is a key to the identi¢cation of these and other signal transduction defects.

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ATYPICAL TYROSINE HYDROXYLASE DEFICIENCY Diana M. Neele1, Nanda M. Verhoeven1, Cornelis Jakobs1 , Marjo S. van der Knaap2 1 Department of Clinical Chemistry, 2Department of Child Neurology, Academic Hospital ``Vrije Universiteit'', De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands

Tyrosine hydroxylase is the rate limiting enzyme in the biosynthesis of catecholamines. Tyrosine hydroxylase de¢ciency (THD) is an autosomal recessive inborn error which is L-dopa-responsive and usually presents with i.a. dystonia, rigidity and hypokinesia. Here, we present a case with an atypical presentation of THD. The development of the boy was normal untill the ¢rst vaccination at the age of 3 months; after that he developed tremors of arms and legs and became hypotonic. Subsequent development was grossly retarded, the tremors gradually disappeared but he remained hypotonic. There was no evidence of paresis, but a striking lack of spontaneous movements was observed. Re£exes were normal. MRI of the brain revealed a mild delay in myelination, but no other abnormalities. At the age of 2 years he developed an RS virus infection with high fever. The tremors recurred and a¡ected his arm, legs, perioral muscles and tongue. He became hypertonic, but rigid rather than spastic. Without evidence of an infection, he continued to have episodes of fever since that time and would often sweat profusely. The only abnormal biochemical ¢nding was an extremely low HVA concentration in CSF of 70 nmol/l and 49 nmol/l at two di¡erent occasions (normal values 2001000 nmol/l). Pterines in CSF were normal. These ¢ndings suggested THD. Treatment was started with levodopa and benserazide which gave clinical and biochemical improvement. His CSF HVA concentration has increased to 101 nmol/l. DNA-analysis showed homozygosity for the ``common'' mutation G698A (Dr R. Wevers, Nijmegen). In conclusion, the common G698A mutation can lead to di¡erent phenotypic presentations of THD. In contrast with the typical presentation of THD, our patient was hypotonic and only under the stress of fever he developed the rigidity that is so typical of the known patients with THD.

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DETECTION OF A SEVERE CASE OF TYROSINE HYDROXYLASE DEFICIENCY BY A DIAGNOSTIC URINARY NEUROTRANSMITTER METABOLITE PATTERN NGGM Abeling, *P Delonlay, *MC Nassogne, ** T Billette de Villemeur, ***M Cretz, AH van Gennip, AC van Cruchten, ****RA Wevers and *JM Saudubray Academic Medical Center, Depts Clinical Chemistry & Pediatrics, Amsterdam, The Netherlands, *Dept of Metabolism, Hoªpital Necker-Enfants Malades, **Dept of Pediatric Neurology, Hoªpital Trousseau, Paris, ***Pediatric Dept, Hoªpital de Colmar, Colmar, France and Dept of Neurology, Univ Hospital Nijmegen, Nijmegen, The Netherlands Tyrosine hydroxylase (TH) de¢ciency is an inherited defect in the synthesis of the catecholamines.The enzyme is expressed mainly in the brain and in the adrenal medulla. Diagnosis relies on a characteristic pattern of biogenic amine metabolites in the CSF, while urinary metabolite patterns are usually normal or at least not typically abnormal. Based on experience with the ¢rst patients with this relatively `new' disorder, clinical response on low-dose L-dopa is good and sometimes even spectacular. Here we describe a severe and atypical case of TH de¢ciency, which was detected by a severely abnormal and characteristic biogenic amine metabolite pattern in the urine. The diagnosis was con¢rmed by CSF analysis and establishment of a new homozygous mutation in the TH gene, but the patient appeared to be unresponsive to L-dopa treatment. This case shows the importance of metabolite analyses in urine of TH de¢cient patients, not only as a diagnostic tool, but also to provide further insight in the pathophysiology of this still poorly understood disorder.

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NEUROTRANSMITTER PROFILES IN CSF AND URINE AS A DIAGNOSTIC AID IN CONDITIONS WITH SECONDARY DOPAMINE BETA-HYDROXYLASE DEFICIENCY NGGM Abeling, *SB van der Meer, **IFM de Coo, **CE Catsman-Berrevoets and AH van Gennip. Univ Amsterdam, Academic Medical Center, Depts Clinical Chemistry & Pediatrics Amsterdam, * Pediatric Dept, Atrium Medical Center Heerlen and ** Neurological Dept, Sophia Children's Hospital Rotterdam, The Netherlands. Menkes disease (MD) is a neurodegenerative disorder of intracellular copper transport leading to secondary de¢ciencies of copper enzymes, a.o. dopamine beta-hydroxylase (DBH). Though the contribution of this secondary DBH de¢ciency in the clinical symptoms is still unclear and presumably minor, it can be used as an additional parameter in diagnosis and monitoring of (e.g. copper) treatment. Measurement of serum DBH enzyme activity however is unsatisfactory because of the wide normal range with a lower level of almost zero, particularly for young children. Neurotransmitter (NT) metabolite pro¢les in CSF and to a lesser degree in urine, much better re£ect the functional in-vivo DBH activity. A comparable situation exists for Riley Day Familial Dysautonomia and other hereditary sensory and autonomic neuropathies (HSAN). Here we describe examples of the role of NT pro¢le analysis in diagnosis and therapy monitoring of cases of MD and a case of HSAN type IV.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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LOW CSF 5-HYDROXYINDOLE ACETIC ACID LEVELS IN COCKAYNE SYNDROME CJ Ellaway1,2, A Duggins3, Fung VS3, JW Earl4, R Kamath5, PG Parsons6, JA Antony7, KN North2,8 1 Western Sydney Genetics Program, Royal Alexandra Hospital for Children 2Department of Paediatrics and Child Health, University of Sydney 3Department of Neurology, Westmead Hospital 4 Department of Biochemistry, RAHC 5Department of Gastroenterology, RAHC 6Queensland Institute of Medical Research 7Department of Neurology, RAHC 8Neurogenetics Research Unit, RAHC Cockayne syndrome is a rare, clinically heterogeneous neurodegenerative disorder, characterised by severe growth failure, cognitive impairment, characteristic facies and photosensitivity. Both the central and peripheral nervous systems are involved with pigmentary retinopathy, delayed nerve conduction velocities, sensorineural hearing loss, progressive spasticity and cerebellar involvement with dysarthria, tremor and ataxia. The cerebral histopathological changes most commonly seen are patchy demyelination of the subcortical white matter and microscopic calci¢cations throughout the central nervous system. Calci¢cation of the basal ganglia may be visible on CT scan. The diagnosis is made on clinical grounds in association with the failure of RNA synthesis in cultured ¢broblasts or lymphoblastoid cells to recover to normal rates after UV-C irradiation. We report a patient with Cockayne syndrome in whom cerebrospinal £uid 5-hydroxyindole acetic acid was markedly reduced. To date there are no reports of abnormalities of the cerebrospinal £uid neurotransmitters in association with this disorder. This ¢nding may provide insight into the pathogenesis of the central nervous system abnormalities in Cockayne syndrome.

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TRYPTOPHAN HYDROXYLASE DEFICIENCY V. Th. Ramaekers1, M. Ha«usler1, N. Blau2 Division of Paediatric Neurology, University Hospital Aachen, Germany1, Division of Clinical Chemistry and Biochemistry, University Children's Hospital, Zu«rich, Switzerland2

Serotonin modulates cognitive function, mood, behavior, vegetative functions and spinal motor control. Four children presented with a £oppy infant syndrome, followed by motor developmental delay, £uctuating gait disturbance with ataxia and lower limb weakness, as well as moderate cognitive de¢cits and decreased alertness. Cerebrospinal £uid was analyzed for biogenic amine metabolites, amino acids, pterines and 5methyl-tetrahydrofolate. Urinary excretion of 5-hydroxyindoleacetic acid (5-HIAA), the end-metabolite of serotonin, was also measured. The results of CSF analysis showed lowered 5-HIAA levels in the presence of normal concentrations of homovanillic acid, L-Dopa, 3-O-methyl-Dopa, neopterin, biopterin, 5-methyl-tetrahydrofolate and amino acids. Daily urinary 5-HIAA excretion was diminished in all four patients and did not change after oral loading with a single dose of 50 mg/kg L-tryptophan. However, urinary 5-HIAA excretion increased above the normal reference range after a single oral load with 5-hydroxytryptophan (1.0 mg/kg). Replacement therapy with 5-hydroxytryptophan alone (one patient) or combined with Cabidopa (three patients), resulted in marked clinical amelioration and normalization of 5HIAA concentrations in CSF and 24-hour urinary samples. Our results suggest a putative novel defect of serotonin synthesis attributed to de¢cient activity of the ¢rst and rate-limiting enzyme tryptophan hydroxylase, resulting in reduced conversion of the serotonin precursor tryptophan to 5-hydroxytryptophan. Oral substitution with 5-hydroxytryptophan and Carbidopa appears to be an e¡ective treatment of this new neurometabolic disorder.

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6 YEARS EXPERIENCE WITH THE ANALYSIS OF CSF DOPAMINE AND SEROTONIN METABOLITES, TETRAHYDROBIOPTERIN, NEOPTERIN AND 5METHYLTETRAHYDROFOLATE Lauren A. Arnold and Keith Hyland. Institute of Metabolic Disease, Baylor University Medical Center, 3812 Elm Street, Dallas, TX. 75226, USA. Biogenic amine and folate metabolism were investigated in CSF from 932 neurological patients (520 years of age). 862 were tested for homovanillic acid (HVA), 5-hydroxyindoleacetic acid and 3O-methyldopa, 694 for tetrahydrobiopterin (BH4) and neopterin (N) and 341 for 5-methyltetrahydrofolate (5MTHF). 167 samples (18%) yielded abnormal results. Diagnoses were as follows: 11 aromatic L-amino acid decarboxylase de¢ciencies; 11 6-pyruvoyltetrahydropterin synthase de¢ciencies; 2 dihydropteridine reductase (DHPR) de¢ciencies with hyperphenylalaninemia (HPA); 3 ``central'' DHPR de¢ciencies with no HPA; 1 homozygote GTP cyclohydrolase (GTPCH) de¢ciency with HPA; 2 compound heterozygote GTPCH de¢ciencies with no HPA; 6 heterozygote GTPCH de¢ciencies (dopa responsive dystonia) with no HPA; 11 infants with hypoxic-ischemic encephalopathy with secondary alterations of amine and BH4 metabolism; 6 folinic acid responsive seizures; 1 5-10-methylenetetrahydrofolate reductase de¢ciency and 1 folate binding protein de¢ciency with low 5MTHF; 1 methionine synthetase de¢ciency and 1 putative serine synthesis defect with raised 5MTHF; 1 putative dopamine transporter defect and 11 possible tyrosine hydroxylase de¢ciencies with low HVA. We also found 11 samples with unexplained low 5MTHF, 25 with raised N and 54 samples with unexplained alterations in amine metabolites or BH4. These results emphasise the utility of these analyses in the investigation of paediatric neurological disease.

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CSF POLYOL PROFILES IN INHERITED METABOLIC DISORDERS Latini A, Laro¨vere L, Guelbert N and De Kremer Dodelson R. CEMECO, Paediatric Department, School of Medicine, National University of Co¨rdoba. Children's Hospital, Argentina. Polyols are ubiquitous metabolites formed by the reduction of aldoses and ketoses. They are abundant within the CNS but their brain metabolism is not fully understood yet. The objective of this work was to investigate the polyol pro¢les in CSF from de¢ned Inherited Metabolic Disorders (IMD) and from apparently healthy controls. Material: CSF series was composed by a) 30 controls and b) patients (P) a¡ected by Glucogenosis type I and VI (P1,2); Lowe's Syndrome (P3); Mucopolysaccharidoses type II (P4) and III (P5-6); Metachromatic leukodystrophy (P7); 3-OH-3methylglutaryl-CoA lyase de¢ciency (P8; HMGLD); Glutaric Aciduria type I (P9), Partial Hypoxanthine phosphoribosyl transferase de¢ciency (P10; PHPRTD); Methylenetetrahydrofolate reductase de¢ciency (P11; MTHFRD; heterozygous for the C667T mutation). Polyols were analysed by CG as their trimethylsilyl derivatives. Results: They are expressed as mmol/L. P1 to P11: decreased myo-inosytol (more than 2SD; NV:769þ201); P8: increased ribulose (33; NV:4þ3) and ribose (18; NV:3þ2); P10: increased galactose (189; NV:27þ13); P11: increased threitol (23; NV:3þ2), erythritol (116; NV:31þ20), xylitol (36; NV:4þ5) and xylulose (14; NV:1þ1). Discussion: The most striking ¢ndings were: I. A constant decreased myo-inositol in all patients. It could re£ect the calcium-mediated breakdown of membrane phospholipids leading to cell death by an excitotoxic mechanism. II. Both patients, HMGLD and MTHFRD, showed a clear impairment of brain pentose phosphate pathway, that correlates with the severe CNS involvement. III. The increase of galactose in PHPRTD could be an interesting clue for future physyopatogenic studies on this entity. To our knowledge, this is the ¢rst report of CSF polyol pro¢les in de¢ned IMD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PIPECOLIC ACID ELEVATION IN PLASMA AND CSF OF TWO PATIENTS WITH PYRIDOXINE-DEPENDENT EPILEPSY Barbara Plecko1.2, Sylvia Sto«ckler-Ipsiroglu2, Wolfgang Erwa1, Eduard Struys3, Cornelis Jakobs3 Univ. Klinik fu«r Kinder-und Jugendheilkunde Graz1, Univ. Klinik fu«r Kinder-und Jugendheilkunde Wien2, Free University Hospital Amsterdam, Dep. of Clinical Chemistry3

The biochemical defect of pyridoxine-dependent epilepsy is still unclear. We report on two patients with pyridoxine-dependent epilepsy and isolated elevation of pipecolic acid in plasma and CSF. Peroxisomal disorders were excluded by normal plasmalevels of VLCFA and phytanic acid. In both patients pipecolic acid in plasma was markedly elevated before treatment with pyridoxine (17,6 and 49, normal 0,6^4, 1 mmol/l), decreased while on treatment and increased again after a prolonged, uncontrolled withdrawal of pyridoxine in one patient (16.4 mmol/l). Pipecolic acid was also markedly elevated in CSF of our two patients while on pyridoxine (0,52 and 1,93; normal 0,009^0,12 mmol/l) and further increased over the 72 h controlled withdrawal of pyridoxine in one of them. In contrast pipecolic acid was normal in plasma and CSF of 15 patients with non-pyridoxine-dependent epilepsy (some with recent seizure activity). A Km-variant at the level of the pyridoxine-dependent a-amino adipic transaminase was assumed but normal concentrations of a-amino adipic acid and piperidines were found in CSF of both patients. Though explanation for these ¢ndings is pending we believe, that if proven in further patients, pipecolic acid might be a speci¢c diagnostic marker of pyridoxinedependent epilepsy, and might prevent patients from dangerous pyridoxine withdrawals, which until now have been claimed to prove the diagnosis.

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THE DETERMINATION OF S-ADENOSYLMETHIONINE AND SADENOSYLHOMOCYSTEINE IN CSF USING STABLE ISOTOPE DILUTION TANDEM MASS SPECTROMETRY E.A. Struys, E.E.W. Jansen and C. Jakobs Metabolic Unit, Dept. of Clinical Chemistry, Free University Hospital Amsterdam, The Netherlands

S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are exclusive intermediates in the metabolic pathways of methionine/homocysteine. Low CSF SAM levels have been found in patients su¡ering from cobalamine defects, methylenetetrahydrofolate reductase de¢ciency and aromatic Lamino acid decarboxylase. Additionally there is evidence that SAM is required for myelin maintenance. Up until now CSF SAH levels have been poorly documented most likely due to its low nanomolar concentration which complicates the analysis. We have developed an electrospray tandem mass-spectrometric method for the combined analysis of SAM and SAH in CSF using stable isotope labeled internal standards. The procedure comprises a weak anion exchange solid phase extraction (SPE) procedure to obtain clean extracts which were injected on a C18 analytical column. Detection was accomplished by multiple reaction monitoring (MRM). Total LC-MS/MS runtime was 3 minutes, which is an enormous time reduction compared to other analytical methods. Detection limits for SAM and SAH in real samples were 7.5 and 2.5 nmol/L respectively (S/N 45). Reference values obtained by the analyses of 10 CSF samples from children, in whom no disturbance of the methyl transfer pathway was suspected, were: SAM 248 + 97 nmol/L and SAH 11.3 + 2.8 nmol/L. The total analysis time for a set of 20 samples was 2.5 hours. Their rapid and accurate determination by stable isotope dilution tandem mass spectrometry opens the possibility for large-scale CSF screening to gain more insight into the neurological importance of SAM and SAH.

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ANALYSIS OF PHOSPHATIDYLCHOLINES AND SPHINGOMYELINS IN CSF USING ELECTROSPRAY TANDEM-MASS SPECTRO-METRY Valianpour F1, Overmars H1, de Koning T J2, Poll-The¨ BT2, Barth PG1, Vreken P1 1 Academic Medical Center, University of Amsterdam, Dept. of Clinical Chemistry and Div. Emma Children's Hospital, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands, 2Wilhelmina Children's Hospital, Dept. of Metabolic Diseases,Utrecht, The Netherlands We developed a sensitive semi-quantitative analysis of individual phosphatidylcholines (PC) and sphingomyelins (SM) using electrospray tandem-mass spectrometry. In normal CSF samples over 30 individual molecular species could be identi¢ed, including PC's with C22:6n-3 or C20:4n-6 attached to their sn-2 position and SM's conjugated with very long chain fatty acids. The stuctures of these lipids were veri¢ed using daughter-ion analysis. Quanti¢cation of 13 PC species and 7 SM species correlated well with total lipid phosphorus concentrations, implicating that the species quanti¢ed represent 4 98 % of the phospholipids present in CSF. Analysis of CSF samples from patients with SLO syndrome revealed very low concentrations of PC and SM . Analysis of a sample from a patient with Krabbe's disease revealed a high concentration of C16:0-SM, and a very low concentrations of C18:0-SM. Finally, samples from two patients su¡ering from a serine biosynthesis defect (3phosphoglycerate dehydrogenase de¢ciency) showed reduced concentrations of SM species and lownormal concentrations of PC species. The phosphatidylserine concentration was to low to be measured in the amount of sample available. The method developed can be extented by measuring ceramides, cerebrosides and gangliosides and might be useful in the screening or therapy control of patients with neurological disorders.

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MOLECULAR ANALYSIS OF A NEW CASE OF GAMT DEFICIENCY Carducci Ca., Mercuri L., Prudente S., Leuzzi V., Carducci Cl., Antonozzi I. University ``La Sapienza'' of Rome. Italy

Notwithstanding the relevant importance of creatine (Cr) homeostasis for the energetic metabolism, no primary metabolic disorder of Cr had been reported until 1994, when the ¢rst patient a¡ected by guanidinoacetate methyltransferase (GAMT) de¢ciency was described. The disorder causes accumulation of guanidinoacetic acid (GAA) and depletion of Cr. We report molecular studies of a new patient a¡ected by GAMT de¢ciency, who presented neurological regression, movement disorders and epilepsy during the ¢rst year of life. Brain MRI revealed pallidal and periacqueductal alterations. In vivo 1H MRS and GAA dosage by HPLC in urine plasma e CFS con¢rmed the diagnosis. Full length cDNA sequencing analysis showed the presence of a unique species of transcript di¡ering from wild type sequence only for a G insertion at position of Gly 164 causing a frameshift. To con¢rm the mutation, proband's genomic DNA sequencing analysis was performed showing, surprisingly, the Gins in exon 5 borne in heterozygous way. The only further alteration found in the genomic sequence was a heterozygous G?C transversion in intron 5 at position nt 3, interesting the consensus sequence of the acceptor splice site. The ¢rst mutation, con¢rmed in the father, produced a frameshift and a premature stop codon was originated. The splicing mutation was con¢rmed in the mother and the RNA molecule derived from maternal allele is not detectable under the condition used. A strict coupling between proper 3'-end formation and transcription process has been previously reported which argues for defect occurring also in the transcription of the maternal molecule.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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A NEW DISORDER OF CREATINE METABOLISM; A PATIENT WITH POSSIBLE CREATINE TRANSPORT DEFECT Ton deGrauw, Kim Cecil, William Ball, Brenda Wong, Cornelis Jakobs, and Nanda Verhoeven, Divisions of Neurology and Radiology, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA and Metabolic Laboratory, Division of Clinical Chemistry, Free University Hospital, Amsterdam, The Netherlands.

Proton magnetic resonance spectroscopy (MRS) provides a non-invasive method for assessing metabolic information in vivo. It makes it possible to study the concentration of creatine in the central nervous system (CNS). Several patients have been reported who lack a creatine peak on proton MRS. All of these patients had a de¢ciency of guanidinoacetate-methyltransferase. We present another patient with an absent creatine peak on proton MRS in the CNS. Case: A Caucasian boy was followed for central hypotonia, developmental delay and autistiform features. MRI at the age of 6 years showed small focus of gliosis which was unchanged compared to a previous MRI. Proton MRS of the frontal white matter and basal ganglia showed almost complete loss of creatine signal. Urine creatine: 189 mgs/24 hours (nl:0-40 mgs/24 hrs), serum creatine: 1.2 mg/dL (nl:51.0 mg/dL), serum creatinine: 0.4 mg/dL (nl:0.2-1.2 mg/dL), creatine kinase: 100 U/L (nl:75-215 U/L), guanidinoacetate in urine: 74 mMol/Mol creatine (nl:10.3-98.8 mMol/Mol), guanidinoacetate in plasma: 1.05 muMol (nl:0.65-1.44 muMol). The patient was treated with creatine monohydrate (340 mg/kg/day). Repeat proton MRS after 3 months of treatment did not show any change. It did not e¡ect his clinical course. Conclusion: This patient may have a defect in the CNS creatine transporter.

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IMPROVED TREATMENT OF GUANIDINOACETATE METHYLTRANSFERASE (GAMT) DEFICIENCY A. Schulze1, E. Mayatepek1, and D. Rating2 1 Dept. Gen. Ped., 2Dept. Neuroped., University Children's Hospital, INF 150, Heidelberg, Germany

Introduction: GAMT de¢ciency (McKusick 601240) is characterised not only by lack of creatine and its derivatives but also by accumulation of guanidinoacetic acid, the precursor of creatine. Creatine replacement, the only therapeutic strategy so far, improves creatine concentrations in brain. However, neurotoxic accumulation of guanidinoacetic acid remains unchanged. Therapeutic trial in our patient therefore aims at lowering of guanidinoacetic acid. Methods: Principles of the therapy are arginine restriction (15 mg/kgBW/day) achieved by low protein diet with additional synthetic amino acid mixture on the one hand and ornithine supplementation (100 mg/kgBW/day) on the other hand. Creatine supplementation (1140 mg/kgBW/day) remained unchanged. Guanidino compounds in blood, CSF, and urine were measured with cation-exchange chromatography and electrospray MS/ MS. Results: Reduction of arginine in plasma and urine led to the stepwise and sustained drop down of GAA. The biochemical correction was accompanied by a marked clinical improvement. For the ¢rst time the EEG was free of slow spike waves. The activity and frequency of seizures declined obviously. The girl expressed an increasing interest in her surrounding. Conclusion: Combined arginine restriction and ornithine supplementation is able to decrease elevated GAA concentration permanently. The clinical improvement after the decrease of GAA supports the hypothesis of their neurotoxic action. Therefore, we recommend the combination of creatine replacement and GAA reduction in the therapy of GAMT de¢ciency.

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PRESENCE OF MUSCLE CREATINE IN A PATIENT WITH GUANIDINOACETATE METHYLTRANSFERASE (GAMT) DEFICIENCY R. Ensenauer, T. Thiel#, K.O. Schwab, W. Lehnert University Children's Hospital, Mathildenstr. 1, 79106 Freiburg/Germany; #Section of Medical Physics, University of Freiburg, Hugstetter Str. 55, 79106 Freiburg/Germany Introduction: GAMT de¢ciency, a rare inborn error of creatine biosynthesis, leads to severe disturbances of human brain energy metabolism most probably due to shortage of creatine and creatine phosphate in the central nervous system. So far it is not yet clear whether other tissues like muscles are a¡ected in a similar manner. Material and methods: Diagnosis of GAMT de¢ciency in a 5-year-old boy with mental retardation, severe speech impairment and seizures was suspected by a positive Sakaguchi reaction performed routinely during selective screening for inborn errors of metabolism. Quantitation of guanidinoacetate (GAA) by a new isotope dilution assay con¢rmed the diagnosis. More evidence was obtained by 1H and 31P magnetic resonance spectroscopy (MRS). We determined metabolite concentrations in parietal and occipital regions of the brain and in muscle. Results: 1H and 31P MRS demonstrated an extreme depletion of creatine and creatine phosphate in the brain (5 0.3 mmol/kg compared to 6-7 mmol/kg in age-matched controls). Abnormal high intensities of GAA in the 1H spectra and of GAA phosphate in the 31P spectra were obtained. However, a considerable amount of creatine (8.8 mmol/kg; normal about 19-21 mmol/kg) was measured in the right gastrocnemius muscle. Conclusions: GAMT de¢ciency was con¢rmed in a new patient by MRS revealing complete absence of creatine and creatine phosphate in the brain. For the ¢rst time the presence of muscle creatine in an untreated patient with GAMT de¢ciency was demonstrated.

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GENERATION OF A KNOCKOUT MOUSE MODEL FOR GUANIDINO-ACETATE METHYLTRANSFERASE (GAMT) DEFICIENCY D. Isbrandt1, A. Schmidt1, A. Neu3, J. RÎper3, R. Steinfeld2, K. Ullrich2 1 ZMNH, Institut fÏr Neurale Signalverarbeitung and 2 UniversitÌts-kinderklinik, Martinistrasse 52, 20246 Hamburg, Germany; 3 Anatomical Neuropharmacology Unit, Oxford University, Mans¢eld Rd, Oxford OX1 3TH, UK GAMT de¢ciency is a disease of creatine biosynthesis and manifests as developmental delay or arrest and as heterogeneous neurological symptoms. A de¢ciency of high-energy phosphates in cells with high energy demand or the neurotoxic action of accumulated guanidinoacetate (GAA) might be involved in the pathophysiology of this disease. In order to investigate the pathophysiology of GAMT de¢ciency using a mouse model, the murine GAMT gene was disrupted in exon 1 by homologous recombination in embryonic stem cells. Northern and Western blot experiments with liver preparations of GAMT-de¢cient animals proved the absence of GAMT-speci¢c mRNA as well as of GAMT protein. GAMT-knockout mice are viable and fertile. Till the age of four months, they do not display an apparent neurological phenotype. The analysis of possible biochemical alterations is presently being performed. To study the role of GAA accumulation in GAMT de¢ciency, pathophysiological concentrations of GAA were applied to acute murine brain slices. GAA hyperpolarized globus pallidus neurons and thus reduced their spike frequency. This GAA-induced e¡ect could be blocked by selective GABAA-receptor antagonists. In contrast, creatine and creatinine had no e¡ect on the neuronal activity. The GABA-mimetic action of GAA in the basal ganglia may be a candidate mechanism for the extrapyramidal symptoms in patients with GAMT de¢ciency and will be further characterised in the knockout mice.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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213

OVINE NEURON CULTURES FOR STUDYING BATTEN DISEASE D N Palmer, G W Kay, S M Hughes and M J Oswald Animal and Food Sciences Division, Lincoln University, New Zealand

425-O

The neuronal ceroid lipofuscinoses (NCL, Batten disease) are inherited neurodegenerative storage diseases in children, mutations in di¡erent genes underlying di¡erent forms. Subunit c of mitochondrial ATP synthase is speci¢cally stored in lysosomes in most forms, including one in sheep orthologous with the human CLN6 late infantile disease. Most cell types accumulate subunit c but only a subpopulation of neurons are functionally a¡ected. Cultures of sheep neurons have been established to test possible therapies and study the relationship between protein storage and neurodegeration. An embryo transfer breeding program, the recovery of 200-600 million viable cells from each foetus, and culturing neurons from frozen stocks allows year-round experimentation. Neurons derived from 60-120 day-old foetuses begin to form single processes within one day of plating and create a network by day ¢ve. They persist in culture for up to 60 days and immuno-stain positively for microtubule-associated protein (MAP2). Accumulation of subunit c and of £uorescent storage bodies was observed in cells from a¡ected 90 day foetuses by immunocytochemistry, Western blotting and confocal microscopy. These bodies were not seen in control cultures. Organelle speci¢c dyes demonstrated colocalisation of auto£uorescent bodies with lysosomes, not with mitochondria. Immunocytochemical techniques have been developed for subtyping sheep neurons in terms of neurotransmitter activity and comparing the cell type distribution in control and a¡ected cultures with the development of pathology and neuronal di¡erentiation in foetal and post natal sheep in vivo.

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INCREASED LEVELS OF N-ACETYLASPARTYLGLUTAMATE IN CSF AND URINE OF PATIENTS WITH PELIZAEUS-MERZBACHER DISEASE A.P. Burlina1, V. Ferrari2, A.B. Burlina3, E. Bertini4 , O. Boesp£ug-Tanguy5. 1 Dept. of Neurol. & Psych. Sciences, 2Veterinary Medicine, 3Pediatrics, Univ. of Padua; 4Div. of Neuropediatrics, Hosp. Bambino Gesu©, Rome; 5INSERM U384, Clermont-Ferrand, France

Pelizaeus-Merzbacher disease (PMD) is a rare X-linked dysmyelinating disease. PMD is caused by abnormalities in the proteolipid protein (PLP) gene. Despite tremendous progress in understanding the molecular basis of PMD, knowledge about the biochemical mechanisms underlying dysmyelination process and axonal dysfunction are not yet well understood. Moreover, no biochemical markers are available for diagnostic purpose. N-acetylaspartylglutamate (NAAG) is a dipeptide present in the brain and localized to glutamatergic pathways. It exhibits mixed agonist/antagonist properties at the N-methyl-D-aspartate receptor. We analysed 16 CSF samples and 19 urine samples (random collection) from 20 patients with clinically de¢ned diagnosis of PMD and PLP alterations (mutation or duplication). Control CSF samples were collected from 30 hospitalized patients, age 1- 19 years, free from neurodegenerative or metabolic disorders. Control urines were obtained from 70 subjects without symptoms of neurological, hepatic or renal disease. The content of NAAG in CSF and urine was measured by capillary electrophoresis according to Burlina et al. (Eur. J. Pediatr. 1999;158:406). High concentrations of NAAG were measured in all PMD's CSF samples and in 13 PMD's urine samples. Our results show a high sensitivity (100%) and speci¢city (88%) of CSF NAAG in PMD. These data indicate that NAAG may be a useful biochemical marker for the diagnosis of PMD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MECP2 MUTATIONS IN 50 PATIENTS WITH RETT SYNDROME K. Ho¡buhr1, N. Siranni1, J. Devaney2, J. Giron1, B. LaFleur1, C. Scacheri1, J. Schuette3, J. Inns3, M. Marino2, M. Philippart4, V. Narayanans5, R. Umansky6, E. Ho¡man1, S Naidu7 1 Research Center for Genetic Medicine, Children's Research Institute, Washington DC. 2 Transgenomics Inc., Gaithersburg, MD 3Pediatric Genetics, University of Michigan, Ann Arbor, MI 4 Mental Retardation Research Center, UCLA, Los Angeles, CA 5Children's Hospital, Pittsburgh, PA 6 Child Development Center, Children's Hospital, Oakland, CA 7Kennedy Krieger Institute, Johns Hopkins University. The MeCP2 gene was screened in 83 patients with classical and atypical Rett syndrome. We identi¢ed mutations in 50/83 patients (60%), including 14 novel mutations (2 missense mutations [P152R,K305R], 4 nonsense mutations [S204X, Q244X, R270X, R294X], a A9963G splice site mutation and 7 novel deletions [5 clustered in a region 3' of the conserved methyl binding domain (MBD) and the transcriptional repression domain (TRD)]. 8 mutations were found with multiple recurrences. The most common mutations were R106W (7X), T158M (8X) and R255X (5X). To identify genotype/phenotype correlations, 42 patients with MeCP2 mutations were scored on 5 clinical parameters; deceleration of head growth, frequency and manageability of seizures, respiratory irregularities, scoliosis and ability to walk. Patients were also separated into 5 categories according to type of mutation and location of mutation within the gene. Our studies indicated that mutations within the TRD and deletions near the carboxyl terminus of the MeCP2 gene may be associated with a milder Rett phenotype. X-inactivation studies of 18 patients revealed highly skewed X-inactivation in two girls with mild to no clinical symptoms, suggesting that non-random Xinactivation may also be an important determinant of phenotype.

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LYSOSOMAL PROTEOMICS USING 2D-GELS AND MASS SPECTROMETRY Richard Bagshaw, John W. Callahan, Don J. Mahuran, Research Institute, The Hospital for Sick Children, Dept. Pathobiology and Laboratory Medicine, University of Toronto, Toronto, Ontario, Canada, M5G 1X8. Lysosomal enzymes and membrane transporter proteins play pivotal roles in digestion of macromolecules and recycling of products for re-use. Transporters and membrane proteins are poorly characterized and represent few of the over 35 known storage diseases known. To ¢ll in this gap we are developing proteomic maps of lysosomal subfractions from rat liver and human skin ¢broblasts. Liver lysosomes (enriched 34 fold using marker enzymes) were obtained from rats given a single intra-peritoneal injection of Triton WR-1339 and separated into the soluble (luminal), membrane-associated (Na2CO3 extractable) and detergent extractable, integral membrane fractions. Proteins were separated by two-dimensional-IEF-SDS-polyacrylamide gel electrophoresis and detected by silver or Coomassie blue staining. Lysosomes isolated from iron-Dextran-loaded, metabolically-labeled skin ¢broblasts showed high speci¢c activities of marker enzymes and 2D gel patterns have also been developed for control and San¢lippo C disease. Using rat liver preparations, we have developed reproducible in-gel tryptic digestion, extraction, and desalting techniques that provide excellent signal-to-noise ratio when the resulting peptides are mass ¢ngerprinted using MALDI-TOF and/or de novo microsequenced on the Q-TOF mass spectrometer. To date, we have identi¢ed 22 proteins (including lysosomal protective protein, b-hexosaminidase a-subunit, bglucuronidase, H+-pump ATPase A and B subunits, dipeptidylpeptidase IV) in subfractions from the lysosomal membrane. Five of these appear to be unique. While plasma membrane proteins and their associated complexes have been well characterized, few similar complexes have been de¢ned in the lysosome. Our proteomics approach o¡ers an e¤cient method to de¢ne the composition of lysosomes, the organization of its components into macromolecular complexes and will assist in uncovering the lysosome's role in a variety of human pathologies and in developing new prognostic indicators. (Supported by the Research Institute, HSC).

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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POSTTRANSLATIONAL MODIFICATION OF LYSOSOMAL ENZYMES: LESSONS FROM LYSOSOMAL STORAGE DISORDERS Kurt von Figura, Lioudmila Borissenko, Thomas Dierks, Jens Fey, and Bernhard Schmidt UniversitÌt GÎttingen, Abt. Biochemie II, Heinrich-DÏker-Weg 12, D-37073 GÎttingen Lysosomal enzymes carry private protein modi¢cations and such shared with non-lysosomal proteins. The group of congenital defects of glycosylation (CDG) are representatives for disorders a¡ecting the latter, while in I-cell disease and in multiple sulfatase de¢ciency protein-modi¢cations restricted to lysosomal enzymes are a¡ected. In I-cell disease the addition of the mannose 6phosphate recognition marker is de¢cient. The mannose 6-phosphate residues serve as a lysosomal tra¤cking signal for soluble lysosomal proteins. The biochemical phenotype of I-cell disease and mannose 6-phosphate receptor de¢ciency (in mice) illustrates the importance of this transport mechanism, but points also to the existence of alternative transport pathways. Multiple sulfatase de¢ciency is characterized by the synthesis of inactive sulfatases, in which the modi¢cation of a cysteine to Ca-formylglycine is missing. This Ca-formylglycine is located in the active site of all known sulfatases. Transient esteri¢cation during sulfate ester cleavage of the hydrated Ca-formylglycine explains its essential role for sulfatase activity. The Ca-formylglycine is generated from a cysteine (pro- and eukaryotes) or from a serine (prokaryotes). In eukaryotes the modi¢cation is catalyzed by matrix components of the endoplasmic reticulum prior to folding of sulfatases and directed by a short linear sequence surrounding the cysteine. Sulfatases are the only known targets of this novel posttranslational protein modi¢cation.

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SCREENING FOR POMPE DISEASE WITH ACID a-GLUCOSIDASE Meikle PJ, Umapathysivam K, Whittle AM, Ranieri E1, Bindloss C, Ravenscroft EM, van Diggelen OP2, Hopwood JJ. Lysosomal Diseases Research Unit and State Screening Services1, Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, AUSTRALIA and Department of Clinical Genetics, Erasmus University, Rotterdam, THE NETHERLANDS2.

Pompe disease or glycogen storage disease type II is an autosomal recessive disorder of glycogen metabolism caused by a de¢ciency of lysosomal acid a-glucosidase (EC 3.2.1.3). Pompe disease can present as either infantile, juvenile or adult onset forms. The infantile onset form is characterised by massive cardiomegaly, macroglossia, progressive muscle weakness (including respiratory muscles) and marked hypotonia, with death occurring within the ¢rst 2 years of life. The juvenile and adult onset forms manifest as slower progressive muscular disorders which are limited to skeletal muscle with death usually occurring from respiratory failure. With the development of enzyme replacement therapy for Pompe disease, methods for the rapid, early diagnosis of this disorder will be required. To this end we have developed immune-quanti¢cation assays for the acid a-glucosidase protein and immune-capture assays for enzyme activity. These assays can be performed on a variety of tissues and body £uids including whole blood or dried blood spots and show good speci¢city and sensitivity in the detection of Pompe disease a¡ected patients. The assays have application as a rapid diagnostic screen of patients with a high clinical index of suspicion and also have potential in the area of newborn screening for Pompe disease.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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REDUCTION OF LYSOSOMAL STORAGE IN SLY MOUSE BY TRANSPLANTATION OF GENETICALLY MODIFIED CD18/CD11b POSITIVE CELLS T. Ohashi, T. Yokoo, H. Kobayashi, W.S. Sly and Y. Eto1, Department of Gene Therapy, Institute of DNA Medicine, The Jikei University School of Medicine1, Japan, The Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University2, USA The de¢ciency of human b-glucuronidase(HBG) results in MPS type VII(Sly syndrome). In this study, we tested the ability to target CD18/CD11b positive cells with gene therapy for murine MPS VII. We harvested bone marrow cells from syngeneic normal mice and cultivated in CSF-1 containing medium for 2 weeks. More than 95 % of the cells were double positive for CD18/CD11b by £owcytometry. We gave 2x106 cells to the non-myeloablated Sly mouse. One week post-transplantation, donor cells populated liver and spleen. The HBG activity increased from 0.9þ0.7 to 28.4þ12.5 u/mg and 0.7þ0.4 to 29.7þ23.1 u/mg in liver and spleen respectively. However, the pathology was unchanged. The HBG activity in liver and spleen decreased by 5 weeks to 3.7þ1.5 and 2.3þ0.5 respectively, but the pathological improvements were signi¢cant and glycosaminoglycan storage was largely cleared. Next, the CD18/CD11b cells were collected from Sly mouse by the method above, transduced by HBG expressing retrovirus(MFG-HBG), and given to non-myeloablated Sly mouse. By 5 weeks post-transplantation, HBG activity was only marginally above the control level. However, many HBG positive cells were observed and pathological improvement was signi¢cant. These data suggest that CD18/CD11b cells transplantation is promising for treatment of murine MPS VII without myeloablation, and CD18/CD11b cells may be a good targets for gene therapy for Sly syndrome.

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N-BUTYLDEOXYGALACTONOJIRIMYCIN IS A MORE SELECTIVE COMPOUND FOR THE THERAPY OF GLYCOSPHINGOLIPIDOSES Ulrika Andersson, David Smith, Terry D. Butters, Raymond A. Dwek and Frances M. Platt Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford 0X1 3QU, U.K. The imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits the ceramide speci¢c glucosyltransferase which catalyses the ¢rst step in glycosphingolipid (GSL) biosynthesis. It has the potential to be used for the treatment of the GSL lysosomal storage diseases and has recently been shown to be e¡ective in type I Gaucher patients. However, NB-DNJ is also a potent inhibitor of other enzymes including a-glucosidase I and II, which could cause side e¡ects in patients receiving life-long therapy. A potentially more selective GSL biosynthesis inhibitor, N-butyl-deoxygalactonojirimycin (NB-DGJ) was therefore evaluated in vitro and in vivo. The tissue distribution of the compound and degree of GSL depletion in the liver of mice treated with NB-DGJ was equivalent to NB-DNJ. Mice treated with NB-DGJ had normal body weights and lymphoid organ sizes, whereas NB-DNJ treated mice exhibited weight loss and partial lymphoid organ shrinkage. NB-DNJ partially inhibited glycogen catabolism in the liver, whereas NB-DGJ did not. In vitro experiments also showed that NB-DNJ was a potent inhibitor of sucrase and maltase but not of lactase, while NB-DGJ inhibited lactase but not sucrase or maltase. The therapeutic potential of NB-DGJ was evaluated in a mouse model of Sandho¡ disease (GM2 storage disorder). Preliminary results indicated that NB-DGJ treated Sandho¡ disease mice performed better in behavioural tests and had prolonged life expectancy compared to NB-DNJ treated mice. The study therefore suggests that NB-DGJ is more selective than NB-DNJ, has improved therapeutic potential, and may be the compound of choice for long-term human therapy.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SUBSTRATE REDUCTION USING N-BUTYLDEOXYNOJIRIMYCIN (OGT 918) : CLINICAL TRIAL OF A NOVEL ORAL THERAPY FOR GAUCHER'S DISEASE R.H. Lachmann, C.E.M. Hollak, J.M.F.G. Aerts, S. van Weely, M. Hreb|¨ cek, F.M. Platt, T.D. Butters, R.A. Dwek, C. Moyses, I. R. Gow, D. Elstein, A. Zimran & T.M. Cox.

N-butyldeoxynojirimycin (NB-DNJ/OGT 918) is an inhibitor of glucosyltransferase, the ¢rst committed step in the biosynthesis of glycolipids. Attenuating the synthesis of glucocerebroside by oral administration of NB-DNJ would be expected to reduce lysosomal storage in Gaucher's disease. In this clinical study we have investigated the therapeutic potential of NB-DNJ in 28 adult patients. We have con¢rmed that oral administration of NB-DNJ leads to a reduction in cell surface GM1 expression in leucocytes. The treatment was well tolerated by most patients with mild diarrhoea being the commonest side e¡ect. We have also observed the development of peripheral neuropathy in some patients who have been treated with NB-DNJ for a year or more. At the end of the initial 12 month protocol, mean liver and spleen volumes were signi¢cantly reduced from baseline (by 12% and 19% respectively; both p values less than 0.001), although haematological parameters showed only a small improvement; further improvements have been observed in patients in an extended use protocol. Overall, our experience suggests that substrate reduction is a promising approach and merits further investigation, both in combination with enzyme replacement therapy in Gaucher's and as a sole agent in other glycosphingolipid storage disorders.

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ENZYME ACTIVATION IN LYSOSOMAL STORAGE DISEASES Yoshiyuki Suzuki1, Junichiro Matsuda2, Akihiro Oshima2, Eiji Nanba3, Kousaku Ohno3, Satoshi Ishii4, Jian-Qiang Fan5 International University of Health and Welfare, Otawara, Japan1; National Institute of Infectious Diseases, Tokyo, Japan2; Tottori University, Yonago, Japan3; Usuki Bio Research Center, Usuki, Japan4; Mount Sinai School of Medicine, New York, U.S.A.5

1-Deoxy-galactonojirimycin, an inhibitor of lysosomal a-galactosidase, was found to be an activator of some mutant enzymes (R301Q, Q279E) expressed in somatic cells from patients with Fabry disease. This low molecular compound accelerates transport and maturation of the enzyme molecule in cultured ¢broblasts. Oral administration to transgenic mice overexpressing a mutant a-galactosidase A (Q279E) signi¢cantly elevated the enzyme activity in all tissues examined. Its intralysosomal concentration was lower than that required for inhibition of the enzyme activity. Then we tried this compound to ¢broblasts from patients with b-galactosidase de¢ciency disorders; infantile, juvenile, and adult form of GM1-gangliosidosis. No activation was observed for an infantile patient but signi¢cant amounts of increase were observed for juvenile and adult form patients. However, the concentration required for this e¡ect was higher than that for a-galactosidase de¢ciency in Fabry disease. Recently we have established transgenic mice overexpressing mutant enzymes responsible for each clinical phenotype of b-galactosidase de¢ciency. They will serve as a model system for developing a new therapeutic approach to this disease. We propose a novel molecular therapeutic strategy toward genetic metabolic diseases by administration of competitive inhibitors as `chemical chaperons' at sub-inhibitory intracellular concentrations.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SECRETION OF LYSOSOMAL ENZYMES BY MACROPHAGES AND THEIR ENDOCYTOSIS BY VARIOUS CELL TYPES N. Muschol, R. Steinfeld, V. Gieselmann*, K. Ullrich, T. Braulke Dept. of Pediatrics and Biochemistry, University of Hamburg, Bonn, Germany With rare exceptions BMT did not prevent the progressive neurodegeneration in patients with di¡erent lysosomal storage disorders. We recently described that di¡erent brain cells (astrocytes, oligodendrocytes, neurons) endocytose arylsulfatase A (ASA) only be receptors speci¢c for mannose6-phosphate (J. Inher. Metab.Dis. 21, 313, 1998). As up to 30% of the perivascular glia is derived from blood monocytes, we tested the secretion of lysosomal enzymes by human macrophages as well as their endocytosis by ¢broblasts. The secretion rate of di¡erent lysosomal enzymes by macrophages varied. High relative secretion rates were achieved for b-hexosaminidase, a-mannosidase lower rates for ASA and bgalactosidase whereas nearly no secretion of b-glucosidase was found. Incubation of macrophages in the presence of ammoniumchlorid enhanced 1.3^3.0 fold the rate of enzyme secretion. Pulse chase experiments revealed that preferentially newly synthesized lysosomal enzymes are secreted. In competition experiments using human recombinant I125-ASA for endocytosis in primary mixed rat cortex cultures, glial C6-, neuronal SY5Y-cells as well as ASA-de¢cient ¢broblasts, lysosomal enzymes secreted by stimulated macrophages had only low mannose-6-phosphate inhibition capacity indicating insu¤cient glycosylation/phosphorylation of the secreted enzymes. These results indicate that BMT might not be a proper therapy for di¡erent lysosomal storage disorders like arylsulfatase A de¢ciency.

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STUDIES ON THE MECHANISM OF ACTION OF SAPOSINS A.M. Vaccaro, F. Cia¡oni, M. Tatti and R. Salvioli Laboratotio Metabolismo e Biochimica Patologica, Istituto Superiore Sanita', Roma, Italy The saposins (Sap) are a family of four proteins, Sap A, B, C and D, generated by a common precursor, called prosaposin. They are localized in the late endosomes and lysosomes and exert their function in the acidic environment of these organelles. The saposins are involved in the degradation of sphingolipids by sysosomal hydrolases. In fact, variant forms of sphingolipidoses, such as metachromatic leukodystrophy and Gaucher disease, are due to a de¢cit of speci¢c saposins. For a long time it has been thought that the mechanism of action of Sap A, C and D was based on their interactions with lysosomal hydrolases. Conversely, we have recently demonstrated that Sap C and D preferentially interact with phospholipids, especially anionic phospholipids. Upon interaction, Sap C, whose physiological role is that of favouring the degradation of glucosylceramide by glucosylceramidase, promotes the binding of glucosylceramidase to the lipid surface and reconstitutes the enzyme activity. In order to shed light on the mechanism of action of another saposin, Sap D, we have investigated the behaviour of this saposin towards membranes. The e¡ects of pH and anionic phospholipids on the properties of Sap D have been anayzed and compared with those of Sap C. The changes in the lipid organization of membranes upon interaction with Sap C and D have also been examined. The results highlight marked di¡erences in the mode of interaction of the two saposins with phospholipid membranes. These di¡erences most likely are related to the di¡erent function of the two saposins and suggest distinct mechanisms of action.

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PROSAPOSIN DEFICIENCY DUE TO 1BP DELETION IN SAP B DOMAIN: THE EVIDENCE FOR FUNCTIONAL SAP A DEFICIENCY DUE TO NONSENSE-MEDIATED mRNA DECAY Hríeb|¨ e©ek M, E©ervenkova¨ M, Hu©lkova¨ H, Tocha¨e©kova¨ M, Ledvinova¨ J, Poup|© tova¨ H, Elleder M Institute of Inherited Metabolic Disorders, 1.st Faculty of Medicine, Prague

The ¢rst child of unrelated parents presented at birth with hepatosplenomegaly and generalised seizures and died at 4 months of age due to progressive generalised storage disorder. Storage macrophages were found in multiple organs at post-mortem. Gross elevation of glucosylceramide, globotriaosylceramide and lactosylceramide and moderate elevation of sulfatides and ceramide was found in liver, spleen and kidney. Cholesterol and sphingomyelin concentrations were not elevated. Immunohistochemistry with anti sap- A, D and anti sap C antibodies showed the absence of immunoreactive material in all studied tissues. The patient was homozygous for a single base deletion in sap B domain (c803delG) , leading to a frameshift and premature stop codon. In the heterozygous parents direct sequencing of RT-PCR products revealed only the wild-type cDNA sequence. Mutant cDNA was detected by ARMS only in RNA isolated from nuclear fraction of parents ¢broblasts, but it was not detectable in the cytoplasmic RNA fraction. This con¢rms that the mutant mRNA is in the cytoplasm essentially absent or at least signi¢cantly less abundant - a ¢nding compatible with the nonsensemediated decay of mutant mRNA in the nucleus. These ¢ndings together with the lack of immunoreactive saposins A-D in the patient's tissues allow to postulate that the c803delG in addition to de¢cit of saposins B,C, and D leads also to a functional de¢cit of saposin A due to nonsense-mediated mRNA decay. All eukaryotic cells eliminate mRNAs that encode prematurely terminated products. Nonsense-mediated mRNA decay (NMD) presumably protects the cell from potentially toxic mutant proteins resulting from frameshifts. mRNAs that contain nonsense mutations more than 50 nucleotides upstream of the 3'-most exonexon junction are selectively degraded by nonsense mediated decay(NMD).

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A HUMAN HEXOSAMINIDASE A RESIDUE THAT DETERMINES SUBSTRATE SPECIFICITY F. Kaplan1,2,4, B. Boulay4, P. Cordeiro3 and P. Hechtman1,2,3,4 Human Genetics1, Pediatrics2, Biology3;and the Montreal Children's Hosp Research Institute4, McGill Univ.

Human hexosaminidase (Hex) isozymes share 460% homology yet di¡er in substrate speci¢city. Hex B(bb homodimer) hydrolyzes neutral substrates. Hex A (ab heterodimer) hydrolyzes neutral and charged substrates; including GM2 ganglioside, which accumulates in neurons of patients with TaySachs (TSD) or Sandho¡'s Disease. We are investigating the molecular basis of substrate speci¢city of Hex isozymes. We hypothesized that a basic residue which neutralizes the negative charge of anionic substrates may contribute to substrate speci¢city of Hex A. We identi¢ed 7 lys / arg residues in the asubunit that lack identical / similar residues at corresponding positions in the b-subunit. We used sitedirected mutagenesis to introduce into the HEXA cDNA, the codon for the corresponding amino acid to each of these lys / arg in the b-subunit. Mutant HEXA cDNAs were expressed in a TSD neuroglial cell line, Hex fractions separated by chromatofocusing, and hydrolysis of the synthetic substrates 4MUGS (charged) and 4MUG (neutral) measured for Hex fractions. Only HEXA R424L produced a normal amount of a-subunit with activity only toward 4MUG. We then mutated bsubunit L453, corresponding to a-subunit R424, to R and expressed the mutant Hex B. Ratios of 4MUGS / 4MUG hydrolysis were: Hex B, 0.4%; Hex B L453R, 1.5%. Hex S (aa, unstable) ratio is 40%. Our results indicate that a-subunit R424 is a necessary component of the substrate speci¢city of Hex A, but is insu¤cient to account for all of Hex A activity toward charged substrates. Homology modelling of Hex A R424L showed that R424 projects into the same hydrophilic cavity as do the active site amino acids R178, D258 and E323.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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STUDIES ON ARGENTINEAN HETEROZYGOTES OF SANDHOFF DISEASE. A PRONOUNCED VARIABILITY OF b-HEXOSAMINIDASE ISOENZYMES IN INDIVIDUALS WITH DIFFERENT GENOTYPES A.M. Oller Ram|¨ rez1,2 R. De Kremer Dodelson1, F.E. Kleiman2 and C.E. Argara·a2. CEMECO1, Paediatric Department, School of Medicine, National University of Co¨rdoba, Children's Hospital. CIQUIBIC2, National University of Co¨rdoba, Argentina. We investigated the marked di¡erence in the b-N-acetil hexosaminidase (Hex) activity of plasma and leukocytes from heterozygotes of Sandho¡ disease. All of them, belonging to the high risk native population of the Valle of Traslasierra, Province of Co¨rdoba (Argentina). They have a de¢ned bhexosaminidase gene (HEXB) mutation, a IVS-2+1 G?A (G?A) substitution in the splice site of intron 2 which produce a null allele and a TG deletion (DTG) in the 3' untranslated region (3' UTR) of the HEXB. A group of 55 heterozygotes were analyzed, 50 of them with the predominant genotype (WT/G?A) and 5 double heterozygotes (DTG/G?A); the last group showed Hex B activity values as low as 10 % of those from normal controls. In other 8 subjects with genotype WT/DTG, an undescribed variability in the Hex activity in di¡erent blood samples of the same individual was found. To analyze further the Hex activity di¡erences among those groups, the Hex isoenzymes were separated by DEAE cellulose chromatography. The elution pro¢les and the quanti¢cation of plasmatic Hex B, Hex I and Hex A showed distinguishing patterns in the di¡erent HEXB genotypes: the heterozygotes WT/G?A and WT/DTG genotype had similar behaviour, while those with DTG/ G?A were markedly decreased in all the fractions. Heat sensitivity studies showed in all heterozygotes a residual Hex A activity 2 to 3 times more stable than those of the normal controls. Although double heterozygotes DTG/G?A individuals showed the lowest enzymatic activity, they did not present clinical manifestations of any variant of Sandho¡ disease at least until fourth decade of live. So far, the medical signi¢cance of the DTG in homozygous state is unknown.

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FIRST DESCRIPTION OF b HEXOSAMINIDASE S IN NORMAL INDIVIDUALS AND HETEROZYGOTES OF SANDHOFF DISEASE WITH DIFFERENT GENOTYPES A.M. Oller Ram|¨ rez1,2, R. De Kremer Dodelson1, F.E. Kleiman2 and C.E. Argaran¬a2 CEMECO, School of Medicine, Children's Hospital1. CIQUIBIC, National University of Co¨rdoba, Argentina2 The analysis of b-N-acetil hexosaminidase (Hex) isozymes was carried out in heterozygotes and Sandho¡ patients from a high-risk zone of Co¨rdoba, Argentina. The series was composed by: a) Heterozygotes with IVS-2+1 G?A splice mutation (G?A) and TG deletion (TG) in 3' unstranslated region of the HEXB gene (3' UTR), genotype TG/G?A (n=4) b) Heterozygotes with genotype WT/ G?A (n=4) c) Carriers with genotype WT/TG (n=4) d) Homozygotes patients with genotype G?A/ G?A (n=5) e) Normal control group, genotype WT/WT (n=6). The isoenzymatic analysis of each group was carried out by chromatofocusing. Hex was separated into three peaks, Hex B, Hex A and Hex S. The quanti¢cation of each isozymes of Hex and their respective pI from plasmatic and leukocytes samples was determined. The plasmatic Hex S was detected in controls (9% of the Total Hex). The plasmatic Hex S activity in relation to Total Hex was 29% in heterozygotes TG/G?A, 19% in WT/G?A and 14% in WT/TG. The Hex S activity from leukocytes in relation to the Total Hex was 17% in normal, 55% in TG/G?A, 44% in WT/G?A and 35% in WT/TG. Another ¢nding was the similarity found between Sandho¡ patients (G?A/G?A) and TG/G?A heterozygotes in relation to: a) a high percentage of Hex S (59 and 55%, respectively), b) the presence of two Hex S peaks of activity with pI 4.3 and 3.6, c) same heat lability of Hex S and d) same pI of Hex B. These similarities between both groups allow us to speculate that the genotype TG/G?A could not be excluded to produce a variant of the disease with late onset expression. The demonstration of Hex S in normal individuals, opens interesting prospective research for a better understanding of the role of this isoenzyme, as yet unknown.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GENE DELIVERY TO THE NERVOUS SYSTEM IN MURINE GM2-GANGLIOSIDOSIS B. Cachon-Gonzalez, C. Scarpini, R.H. Lachmann, S. Efstathiou, & T.M. Cox.

We have used murine models of GM2 gangliosidosis as a target for transgene expression mediated by Herpes Simplex virus type 1 vectors. The development of HSV1 gene therapy depends on noncytotoxic vectors that mediate stable transgene expression from the latent vector genome. We have cloned the b-subunit of human hexosaminidase A and after linkage to an IRES, the transgene was inserted 1.5 kb downstream of the latency-activated promoter transcriptional start site of a replication-defective HSV vector lacking thymidine kinase and the essential glycoprotein H gene. After footpad inoculation of 5 x106 pfu into hex-b-subunit knockout mice, tissues were removed for in situ assay for hexosaminidase A and B. The spinal cord and ganglia ¢ve weeks post-inoculation showed abundant perinuclear expression of active hexosaminidase in sensory and motor neurones con¢rming long-term expression of the therapeutic virus in vivo. Stereotactic injections of the recombinant virus into de¢ned areas of the brain of knockout and control mice at speci¢c time points are underway. These show that gene therapy employing HSV1 vectors merits therapeutic development for humans a¥icted by neuronopathic storage disorders. This work is supported by Cantab Pharmaceuticals, BBSRC and the Medical Research Council (DTI-LINK Grant).

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rAAV VECTORS ENABLE TRANSDUCTION OF GLIAL CELLS AND EXPRESSION OF TRANSGENE ARYLSULFATASE A IN BRAIN OF A METACHROMATIC LEUKODYSTROPHY KNOCK-OUT MOUSE Makoto Migita1,2, Kumi Takahashi1, Yukihiko Hirai1, Volkmar Gieselmann3, Takashi Shimada1 Department of Biochemistry and Molecular Biology1, Department of Pediatrics, Nippon Medical School, Tokyo, Japan2, University of Kiel, Germany3

Metachromatic leukodystrophy (MLD) is an autosomal recessive, inherited, lysosomal storage disease caused by a de¢ciency of arylsulfatase A (ASA). As this disease is characterized by progressive demyelination leading to increasingly more severe neurological symptoms, direct gene transfer to oligodendrocytes is an important strategy for the treatment of MLD. A recombinant adeno associated virus (rAAV) vector plasmid containing both ASAcDNA as a therapeutic gene and EGFP cDNA as a marker gene was constructed. To prepare su¤cient amounts of high titer AAV vectors, cell lysate from HeLa cells which were co-transfected with vector plasmid and AAV/ AD in presence of adeno viruses was concentrated up to 1010 to 1011 viral particles / ml. Approximately 5 X 107 particles of rAAV vectors were stereotactically injected into rat brain in vivo. Oligodendrocytes in rat brain showed double positive for both EGFP and a speci¢c antigen, carbonic anhydrase II using a confocal microscope in three months after a single injection. Skin ¢broblasts froma MLD patient were transduced and ASA activity in the transduced cells increased signi¢cantly with rAAV vector. Finally, expression of ASA was observed in the brain of MLD knock-out mouse after stereotactical injection with rAAV of ASA in vivo. These results suggest that rAAV vector could be a potentially useful component of a gene therapy for MLD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

222

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AGE-RELATED COGNITIVE AND NEUROMOTOR DEFECTS IN ARYLSULFATASE ADEFICIENT MICE R. D'Hooge (1), D. Van Dam (1), V. Gieselmann (2), P.P. De Deyn (1) (1) Laboratory of Neurochemistry and Behaviour, Born-Bunge Foundation, University of Antwerp, Belgium; (2) Institut fÏr Physiologische Chemie, Rheinische Friedrich-Wilhelms-UniversitÌt, Bonn, Germany Transgenic mice de¢cient in arylsulfatase A (ASA) were generated as a putative model for the neurodegenerative lipidosis metachromatic leukodystrophy. ASA(-/-) and wildtype control mice were tested between 3 months and 2 years of age using a battery of behavioural tasks. It was found that ASA(-/-) mice display age-related impairments on several tests of neuromotor function and activity as well as on tests of learning and memory. One-year-old ASA(-/-) mice showed impaired rotarod performance and walking with a shorter pace, later evolving into more severe ataxia with tremor in 2year-old animals. These late defects might be related to selective neurodegeneration within the cerebellum of these ageing animals. However, performance of ASA(-/-) mice on tests of learning and memory also showed de¢cits which could not be reduced to neuromotor impairment. Whereas passive avoidance learning was not impaired in 3- and 6-month-old ASA(-/-) mice, 12-month-old animals did show signi¢cantly impaired passive avoidance retention. Acquisition and probe (retention) trial performance in the Morris water maze test of spatial learning and memory was normal in 3-month-old ASA(-/-) mice; slight impairments were seen in 6-month-old animals; whereas 12-month-old animals displayed markedly impaired acquisition and retention performance indicative of more profound cognitive impairment in these older animals.

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ENZYME REPLACEMENT THERAPY IN FABRY DISEASE: Results of a Phase I/II Clinical Trial Eng, C.1; Phelps, R.1; Kim, L.1; Goldman, M.1; Gordon, R.1; Gass, A.1; Winston, J.1; Brodie, S.1; Mehta, D.1; Dikman, S.1; Fallon, J.1; Parsons, R.1; Stacy, C.1; Shinnar, M.1; Rosenberg, M.2; O'Callaghan, M.2; Fitzpatrick, M.2; Huertas, P.2; Desnick, R.1 1 Mount Sinai School of Medicine, New York, NY; 2Genzyme Corporation, Cambridge, MA Fabry disease (a-galactosidase A (a-GAL) de¢ciency) results in the progressive lysosomal accumulation of the glycosphingolipid GL-3, primarily in vascular endothelium. Treatment is currently palliative and a¡ected patients generally die in their forties to ¢fties. Preclinical studies of recombinant human a-GAL (r-haGAL) treatment in knockout mice demonstrated reduction of GL-3 in tissues and provided the rationale for a Phase I/II clinical trial. A single center, open-label, dose ranging study of r-haGAL was conducted in 15 patients, each receiving ¢ve infusions of one of ¢ve di¡erent dose regimens. Infusions were well-tolerated; four patients experienced mild to moderate reactions, suggestive of hypersensitivity, that were managed by conservative measures. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and ultrastructurally. Mean GL-3 content decreased 84% in liver (SD = 22; n = 13) and was markedly reduced in kidney in 4/5 patients with pre- and post-treatment biopsies. GL-3 deposits were reduced in vascular endothelium of kidney, heart, skin and liver by light and electron microscopic evaluation. In addition, trends toward improvements of pain, quality of life, and autonomic function were observed. These results provide the rationale for further investigation of enzyme replacement therapy for the treatment of Fabry disease.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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223

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EXPRESSION OF THE GLYCOSPHINGOLIPID SUBSTRATES IN ARYLSULFATASE A DEFICIENT MICE- A POTENTIAL ANIMAL MODEL FOR METACHROMATIC LEUKODYSTROPHY (MLD)

J.-E. Maînsson1, M. Molander1, Z. Pernber1, P. Fredman1, U. Matzner2, F. Schestag2, V. Gieselmann2. 1 Institite of Clinical Neuroscience, Experimental Neuroscience Section, GÎteborg University, Sahlgrenska University Hospital, S-431 80 MÎlndal, Sweden. 2Biochemisches institut, Christian-Albrechts-UniversitÌt Kiel, Olshausenstr. 40, D-24098 Kiel, Germany. Metachromatic leukodystrophy (MLD) is caused by de¢ciency of the enzyme Arylsulfatase A (ASA) and the substrates for this enzyme are the acidic glycosphingolipids sulfatide, lactosylceramide sulfate and seminolipid. In patients a¡ected by MLD, these lipids are accumulated and stored intralysosomally in the brain and in other organs. The dominating substance stored in MLD is sulfatide, present in both the central and peripheral nervous system and a major myelin component. Early signs of the disease are lack of coordination, intellectual loss and weakness caused to some extent by demyelination. In the present study, we have investigated the developmental changes of sulfatide, lactosylceramide sulfate and semino-lipid in an ASA knockout mouse model (ASA ^/^) (Hess et al. 1996). Biochemical analyses of the brain tissue and immunohistochemical analyses on the brain region cerebellum, mainly responsible for motor control was performed. In the ASA ^/^ mice there was a steadily increasing accumulation of sulfatide (ten-fold) and of the much less abundant ASA substrate seminolipid (two-fold) as compared to the wildtype control mice (ASA +/+), in which sulfatide remained at a steady state after 7 months while seminolipid decreased. We could not detect lactosylceramide sulfate in the mice. With immunohistochemistry and confocal microscopy we found that the ASA ^/^ mice displayed an increased anti-sulfatide staining mainly of the molecular cell layer of the cerebellum as compared to the wildtype mice. The results demonstrate that the accumulation of ASA glycosphingolipid substrates in the animals is in agreement with the biochemical ¢ndings in MLD patients. This animal model should thus represent a useful system to study metabolic disturbances associated with the ASA enzyme de¢ciency and of the MLD disease. Reference: Hess B, Saftig P, Hartmann D, et al (1996) Phenotype of arylsulfatase A-de¢cient mice: Relationship to human metachromatic leukodystrophy. Proc Natl Acad Sci USA 93:14821-14826.

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GENETIC MODIFCATION OF THE LIVER AND LUNG AS PORTALS FOR DELIVERY OF a-GALACTOSIDASE INTO CIRCULATION FOR FABRY DISEASE S.H. Cheng1, C. Li1, R. Ziegler1, R.J. Desnick2, Y.A. Ioannou2 and N.S. Yew1 Genzyme Corporation, Framingham, MA1, Mount Sinai School of Medicine, New York, NY2

Fabry disease is caused by a de¢ciency of the lysosomal enzyme a-galactosidase A resulting in deposition of globotriaosylceramide (GL-3) in vascular lysosomes. Genetic modi¢cation of a depot organ such as the liver or lung may su¤ce to produce corrective circulating levels of the de¢cient enzyme. We have demonstrated the e¤cacy of systemic administration of an adenovirus vector encoding human a-galactosidase A in Fabry mice. Injection of 1011 particles of Ad2/CMVHIagal resulted in high level transduction of the liver and subsequent secretion and uptake of a-galactosidase A by all organs examined. Concomitant with these high levels of enzyme activity was a reduction of GL-3 to near normal levels for up to 6 months. An alternative portal for secretion of the enzyme is through vector administration into the lung. Pulmonary instillation of 1010 particles of Ad2/ CMVHIagal into BALB/c mice resulted in high level transduction and expression of a-galactosidase A in the lung. Enzymatic activity was also detected in the liver, spleen, kidney and plasma. The observation of enzymatic activity outside of thelung along with detection of viral DNA that was limited only to the lung, suggests that the enzyme crossed the air^blood barrier, entered systemic circulation and was internalized by distal tissues. The levels of a-galactosidase A detected in these tissues, although lower than observed following systemic delivery of the virus, were nevertheless within the therapeutic range. Measurement of GL-3 levels at day 28 showed reduction to basal levels in the lung and liver and signi¢cant reductions in the spleen and heart. These studies demonstrate the feasibility of the liver and lung for use as depot organs.

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SECRETION OF RECOMBINANT HUMAN aGALACTOSIDASE A FROM GENETICALLY MODIFIED HEMATOPOIETIC CELLS AND SKELETAL MYOBLASTS, AND ITS UPTAKE BY FABRY PATIENT FIBROBLASTS Nobuaki Takiyama1,2,3, Ryoichi Kase1, John A. Barranger4, Hitoshi Sakuraba1 Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo Metropolitan Organization for Medical Research, Tokyo1, Department of Pediatrics, Saitama Social Insurance Hospital, Urawa2, Department of Pediatrics, Keio University, Tokyo3, Department of Human Genetics, University of Pittsburgh, Pittsburgh4 Fabry disease is an X-linked inherited metabolic disorder caused by a de¢ciency of lysosomal enzyme a-galactosidase A. Enzyme replacement therapy for Fabry disease is currently being evaluated. An alternative therapeutic approach is to develop an enzyme delivery system which could supply agalactosidase A into the circulation continuously. In this study, human CD34+ hematopoietic cells and murine C2 skeletal myoblasts were transduced with a retroviral vector, MGF-a-galactosidase A. The enzyme was secreted into the medium from transduced cells and taken up by Fabry patient ¢broblasts through mannose 6-receptors. These ¢ndings suggest that genetically corrected hematopoietic cells or skeletal myoblasts can be an enzymatic source for neighboring enzyme-de¢cient cells, and can potentially be useful for gene therapy of Fabry disease.

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EFFICIENT ENZYMATIC CROSS-CORRECTION OF FABRY PATIENTS FIBROBLASTS BY ADENO-ASSOCIATED VIRUS VECTOR MEDIATED TRANSFER OF THE aGALACTOSIDASE A GENE T. Shimada1, H. Takahashi1, Y. Hirai1, K. Takahashi1, M. Migita1, R. Kase2, and H. Sakuraba2 Department of Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan1, and Department of Clinical Genetics, The Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan2 Fabry disease is an X-linked inherited metabolic disorder due to a de¢ciency of the lysosomal enzyme, a-galactosidase A (a-gal A). The enzyme defect leads to the systematic accumulation of neutral glycosphingolipids predominantly ceramide trihexoside. No e¡ective treatment is available at this time. Clinical trials of enzyme replacement therapy has just started. Another possible approach is gene therapy using the normal a-gal A gene. Enzymatic correction of patients' ¢broblasts using retroviral and adenoviral vectors has been reported. In this study, we explore the feasibility of replacement gene therapy using adeno-associated virus (AAV) vectors. We generated the high-titer AAV vector containing the a-gal A gene driven by the CAG promoter. High levels of the a-gal A activities were detected in both inside and outside HeLa and C2C12 (mouse myoblast) cells transduced with the AAV vector. Stably transduced HeLa cells continued to secrete the enzyme for at least 6 weeks. The a-gal A activity of skin ¢broblasts from Fabry patients was increased more than 30-fold of the non-transduced control activity. Furthermore, the secreted enzyme from the HeLa cell clone was e¤ciently taken up by patients's skin ¢broblasts in a mannose-6-phosphate receptordependent manner. These studies demonstrated the e¡ectiveness of AAV vectors for delivery and expression of the agal A gene. AAV vector mediated gene transfer into skeletal muscle may provide a continuous source of a-gal A for replacement therapy of Fabry disease.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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A COMPARISON OF MOLECULAR DEFECTS, a-N-ACETYLGALACTOSAMINIDASE (aNAGA) ACTIVITIES AND URINARY PRODUCTS IN TWO PATIENTS WITH KANZAKI DISEASE N. Yoshii*, S. Yotsumoto*, T. Fukushige*, K. Kodama**, Y. Hirabayashi***, and T. Kanzaki* Depts. Of Dermatol, Kagoshima Univ., Faculty of Medicine*, Kagoshima Hokkaido Univ. Sch. Of Medicine**, Sapporo, and Lab. for Cell. Glycobiol., Riken, Wako, Japan***

We investigated a second Japanese case of a-NAGA de¢ciency with angiokeratoma corporis di¡usum and compared this new patient to the previous one. The results are: Case 1st 2nd

Age / Sex

AKCD

I.Q.

59F 47F

+++ ++

70 112

Mol defect C985T G986A

AA in a-NAGA Arg 329 Try Arg 329Gln

a-NAGA activity Ratio of AA in (n mol / h / ml plasma) urinary glyco-AA (normal : 0.940^4.190) (Ser / Thr) 0.005 0.014

8 / 10 2 / 10

Thus, the 2nd case had one base mutation in the same codon as in the 1st case for a-NAGA resulting in Try to Gln and subsequent change in conformation (random coil to b-sheet), then aNAGA activity and ¢nally relative reduction of GalNac-O-Ser compared to GalNac-O-Thr. This suggests that the defected enzyme in the 2nd case had more a¤nity to GalNac-O-Ser than in the 1st case with higher activity to GalNac-O-Ser / Thr.

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BONE MARKER ALTERATIONS IN PATIENTS WITH TYPE I GAUCHER DISEASE Giovanni Ciana, Cristina Martini, Agnese Leopaldi, Giorgio Tamaro, Faraugh Katouzian, and Bruno Bembi Istituto perl'Infanzia ``Burlo Garfolo'', via dell'Istria 65/1, 34137 Trieste, Italy

Background. Bone involvement is one of the most disabling aspect of Type I Gaucher disease and its pathophysiology is still not well understood.The recent development of sensitive tests for bone resorption allows us to study the bone metabolism in a non invasive manner in a group of Type I Gaucher's patients. Methods. We measured the markers of bone formation (alkaline phosphatase and isoenzymes,carboxyterminal propeptide of type I procollagen,osteocalcin) and of bone resorption (serum carboxyterminal telopeptide of type I collagen,urinary hydroxyproline,free-deoxypyridinoline and calcium) in ten Type I Gaucher adult patients with a mild to severe bone disease.The same tests were performed on ten healthy adult subjects matched for sex and age. Results. In Gaucher patients carboxyterminal propeptide of type I procollagen was shown to be signi¢cantly decreased when compared to normal subjects (mean 101.17 ng/ml vs 140.75 ng/ml, p=0.038).In the bone resorption indeces serum carboxyterminal telopeptide of type I collagen showed to be signi¢cantly increased (mean 4.24 ng/ml vs 2.87 ng/ml, p= 0.012).No signi¢cant di¡erences were observed in other bone turnover indexes normally used to monitor bone metabolism. Conclusions. The authors retain that carboxyterminal propeptide of type I procollagen and carboxyterminal telopeptide of type I collagen could be proposed as an e¡ective, sensitive and non invasive method for the study of bone metabolism and a method for monitoring the e¡ectiveness of enzyme therapy in Gaucher disease bone involvement.

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ASSESSMENT OF IN VIVO GENE THERAPY APPROACHES FOR GAUCHER DISEASE J. Marshall, J. Cavanagh, T. Budzinski, R. Ziegler, J. Sullivan, A. Scaria and S.H. Cheng Cheng. Genzyme Corporation, Framingham, MA 01701, USA Gaucher disease (GD) is caused by mutations in the gene for the lysosomal enzyme, glucocerebrosidase (GC) resulting in accumulation of the substrate glucosylceramide in the macrophages of the a¡ected livers and spleens. Current treatment relies on regular infusions of recombinant GC that has been modi¢ed to enhance uptake by macrophages. Gene therapy of GD to date has focused on the use of ex vivo approaches such as retrovirus-mediated delivery of the normal gene into myeloid stem cells. Gene transfer has been demonstrated but at levels that are unlikely to be therapeutic. As an alternative gene therapy approach, we are exploring the use of vectors that would achieve direct in vivo gene transfer. Recombinant adenoviral, adeno-associated viral (AAV), and synthetic vectors encoding GC have been constructed. The objective is to administer these vectors either systemically, intranasally or directly into muscle to transduce a tissue that could act as a depot for the secretion of therapeutic levels of GC into circulation. We have shown that transduction of Gaucher ¢broblasts with these vectors results in the secretion of active enzyme into the media. Furthermore, the secreted GC is competent for internalization by macrophages. The latter is an important consideration since the in vivo synthesized GC, unlike the recombinant GC used in enzyme replacement therapy, will not be modi¢ed for improved uptake by macrophages. Preliminary studies using an AAV vector yielded activity of GC410 fold endogenous levels in the lungs of mice. Taken togethr, these in vitro and in vivo data suggest that gene therapy is a viable strategy for the treatment of Gaucher disease.

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THE IMPACT OF GENOTYPE ON CLINICAL EXPRESSION OF GAUCHER DISEASE: DATA FROM 733 PATIENTS CR Scott, H Andersson, J Charrow, P Kaplan, E Kolodny, P Mistry, G Pastores, B Rosenbloom, R Wappner, N Weinreb. The International Collaborative Gaucher Group (ICGG), Cambridge, MA.

Gaucher disease is the most common lysosomal storage disease, caused by a de¢ciency of glucocerebrosidase. It is typically considered an adult disorder, manifest with hematologic changes and bone fractures. Data from 733 patients (from 2000+ in the ICGG Registry) with complete genotypes was strati¢ed to correlate with clinical symptoms. Surprisingly, 50% of all patients are less than 10 years when diagnosed. The most common genotype in the registry is N370S/N370S (30%), followed by N370S/L444P (15%), N370S/84GG (12%), L444P/L444P (6%), and N370S/IVS2+1 (3%). Uncharacterized genotypes (N370S/?) or rare alleles are not included in this summary. The age at diagnosis was 28 years for N370S/N370S, 18 years for N370S/L444P, 10 years for N370S/84GG, 11 years for N370S/IVS2+1, and 2.4 years for L444P/L444P. All patients with the N370S allele were classi¢ed as type I and were free of neurologic symptoms. 70% of children with the L444P/L444P genotype had neurologic symptoms and were classi¢ed as type III. Radiologic evidence of bone disease was present in 85-100% of each genotype. The largest spleen volumes were in L444P/L444P (42 x norm), followed by N370S/84GG (20 x), N370S/IVS2+1 (18 x), N370S/L444P (16 x), and N370S/N370S (11 x). Some rare alleles present with unique clinical features. Patients homozygous for D409H have aortic valve calci¢cation and cataracts. Newborns with rare recombinant alleles may have ichthyosis and die at birth from neurological disease.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CLINICAL PRESENTATION AND RESULTS OF TREATMENT OF TWO NEW GAUCHERPATIENTS HOMOZYGOUS FOR THE L 444P MUTATION F.J.M. Eyskens, M. Bertrand, G. Janssens A.Z. Middelheim, Campus Queen Paola Children's Hospital, Antwerp, Belgium1

A Turkish girl, second child of consanguineous parents, was sent to our hospital at the age of 9.5 years with a severe splenomegaly, growth retardation (length 10 cm below the 3d centile of age), chronic fatigue, epistaxis, pain in the legs and sweating during the night. There was a hepatomegaly {volume (CT): 1134cc} and splenomegaly {volume (CT): 1218cc}. Laboratory investigations revealed a slight anemia in a leucopenia and trombocytopenia (43,000/mm3). Bone marrow showed the presence of Gaucher cells. The enzymatic activity of b-glucocerebrosidase was very low in leucocytes and ¢broblasts (1,9mmol/g/hr) (norm.: 5.2^15.8). The chitotriosidase activity in plasma was 25,916 nmol/ml/hr. The genotype of this girl who presented with severe systemic manifestations but with no overt clinical signs of neuronopathic involvement was L444 P/L444P. She showed a fast improvement of the clinical symptoms, the visceromegaly and the hematologic disturbances under enzymereplacement therapy (120 U/kg/4 weeks) given as biweekly infusions. Her baby sister has the same genotype and was treated presymptomatically. Neurophysiologic examinations (BAEPS; eye movements studies), performed at Great Ormond Street, London, revealed brain stem involvement. The enzyme-replacement therapy (ERT) was doubled as a consequence of the ¢ndings of neuronopathic disease. This case indicated that detailed neurological evaluation is warranted in every patient with mutations leading to neuronopathic Gaucher's disease. Follow-up of this patient and her baby sister will probably give outstanding information on the e¡ect of ERT on neurological disease progression in type 3 Gaucher's disease.

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MAINTENANCE DOSAGE EFFICACY OF ENZYME REPLACEMENT THERAPY IN 13 JAPANESE PEDIATRIC PATIENTS WITH GAUCHER DISEASE Masahisa Kobayashi, Hiroyuki Ida, Yoshikatsu Eto Department of Pediatrics, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, JAPAN

To obtain insights into optimizing maintenance dosage of enzyme replacement therapy (ERT) in the unique Japanese pediatric patients population with Gaucher disease (GD) we retrospectively examined e¡ects of three ERT dose reduction schedules from a starting regimen of 60 U/kg of body weight every 2 weeks. Early and marked ERT reduction was associated with development of bone involvement, insu¤cient improvement mean hemoglobin level (Hb) and relative height, and with insu¤cient improvement or worsening platelet count (Plt). Only subgroup given 60 U/kg of ERT every 2 weeks for 36 months had signi¢cant improvement in mean Hb, Plt, angiotensin converting enzyme values, acid phosphatase values and relative height at 36 months. These data suggested that long-term high dose ERT may be required to obtain su¤cient improvement to maintain health in pediatric patients with severe GD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

228

Abstracts

455-P

CHARACTERISTICS OF MUTATION PREVALENCE AND PHENOTYPE IN ASIAN PATIENTS WITH GAUCHER DISEASE Hiroyuki Ida, Takeru Ito, Yoshikatsu Eto Department of Pediatrics, Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, JAPAN To characterize the phenotype/genotype correlation in Asian patients with Gaucher disease we examined unrelated 93 Japanese (type 1:47 cases, type 2:20 cases, type 3:26 cases), 19 Korean (type 1:9 cases, type 2:3 cases, type 3:7 cases) and 6 Taiwanese (type 1:3 cases, type 2:2 cases, type 3:1 cases) patients with Gaucher disease. By 7 common mutation screening (84GG, IVS2+1, F213I, N370S, D409H, L444P, R463C) the L444P mutation was the most common mutation. Its incidence was 32%, 19% and 50% of total mutated alleles in Japanese, Korean and Taiwanese population, respectively. The F213I was the second-common mutation in Japanese and Korean patients accounting for 16% each. Neither the N370S nor 84GG mutations have been identi¢ed in Japanese, Korean and Taiwanese population. These data may explain that type 1 Gaucher disease tends to be severe and progressive, and the high incidence of neuronopathic form in Oriental population since the N370S mutation is associated with more mild disease and non-neuronopathic form.

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GAUCHER DISEASE TYPE 2 PRESENTING AS RECURRENT HYDROPS R.S. Colby, H.A. Taylor, and L.H. Seaver Greenwood Genetic Center, 1 Gregor Mendel Circle, Greenwood, SC 29646 Non-immune hydrops (NIH) was diagnosed by ultrasonography in three pregnancies of a nonconsanguineous Caucasian couple at 23, 31, and 20 weeks gestation. All pregnancies ended in IUFD. Autopsies showed generalized edema, hepatosplenomegaly, enlarged lymph nodes, ascites, joint contractures, and macrophage storage material. The diagnosis of Gaucher disease was made in the third fetus based on very low beta-glucosidase activity in ¢broblasts. Lysosomal storage diseases (LSD) causing NIH are underdiagnosed. Ten LSD have been reported presenting as NIH. Recent prospective studies suggest the incidence is much higher than thought (5.9^14.8%) (Piraud, 1996; Groener, 1999). Gaucher disease has presented as ichthyosis, collodion babies, and NIH, with death at a few hours to several months (Sidransky, 1992). Our cases and others with very low beta-glucosidase activity present with a severe phenotype. The mouse model has a severe phenotype and has no enzyme activity (Tybulewicz, 1991). Tayebi reported a case in 1997 with no activity that presented with NIH and IUFD at 20 weeks. Hypokinesia was a prominent feature in our cases. Evidence of the hypokinesia included ultrasound detection, maternal report, and ¢ndings of joint contractures, absent £exion creases, and muscle atrophy. A possible mechanism of hypokinesia is elevation of glucosylsphingosine in the brain, which is a neurotoxin, and is elevated in the brains of the mouse model and type 2 Gaucher patients. These cases expand the phenotypic spectrum of LSD, and should lead to increased recognition and diagnosis of these disorders.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

229

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BIOCHEMICAL AND MUTATIONAL ANALYSES OF PATIENTS WITH GAUCHER DISEASE Pereira MLS, Michelin K, Goulart L, Morari L, Wajner A, Pires RF, Giugliani R and Coelho JC. Dept. of Biochemistry and Genetics and Medical Genetics Service, HCPA, UFRGS, Porto Alegre, RS, Brazil

Gaucher Disease (GD) is a common sphingolipid storage disorder caused by b-glucocerebrosidase de¢ciency. The aim of present study was to apply a protocol combining biochemical and mutational analysis to detect and characterise patients with GD. The biochemical approach combines the measurement of b-glucocerebrosidase activity, the de¢cient enzyme, plus activity of chitotriosidase, an additional biochemical marker that shows increased activity in GD patients. Mutational analysis uses PCR and digestion with speci¢c restriction endonucleases to detect both N370S and L444P mutations. The biochemical protocol was applied to individuals at risk for GD and 37 patients were observed to show low activity of b-glucocerebrosidase, ranging from 0.1 to 5.7 nmol/h/mg protein (normal range = 10 to 45 nmol/h/mg protein). Chitotriosidase activity in these patients varied from 1336 to 68394 nmol/h/mL (normal range = 8.85 to 132). Molecular analysis performed in 17 unrelated patients with GD and 2 heterozygotes (36 aleles) allowed to estimate the overall frequencies of the N307S and L444P mutations to be 27.8% and 44.4%, respectively. In general, the approach presented here was very e¤cient to detect new cases of GD and protocol can be improved with the addition of a speci¢c search for other common mutations in the gene (Supported by Genzyme do Brasil, FAPERGS, CNPq, MCT/PRONEX).

458-P

THE FACILE DETECTION OF R463Q (IVS10-1 MUTATION IN GREEK GAUCHER DISEASE PATIENTS H. Michelakakis*, M. Moraitou*, S. Van Weely**, M. Verhoek**, H. Aerts*** *Dept. of Enzymology and Cellular Function, Institute of Child Health, Athens, Greece; **Dept. of Biochemistry, University of Amsterdam, The Netherlands

More than 100 disease-causing mutations have been identi¢ed in Gaucher patients. The accuracy of PCR techniques followed by restriction fragmentation analysis, often used in the identi¢cation of mutations, is limited by the fact that often they cannot distinguish between di¡erent point mutations occurring within the recognition site of the restriction enzyme used and in some cases between point mutations and deletions. R463C and R463Q(IVS10-1) are two point mutations occurring at the same codon of the bglucocerebrosidase gene. We investigated the reliability of the identi¢cation of the R463C mutation based upon the commonly employed PCR-restriction fragmentation method using MspI as the restriction enzyme. Four Greek Gaucher patients were studied (3 type I and 1 type II) and a simple modi¢cation of the above method, using HphI as the restriction enzyme, was employed. Our results showed that the patients were in fact carrying the R463Q(IVS10-1) mutation and not the previously assigned F463C mutation. Sequencing of the appropriate fragment con¢rmed our ¢nding. Thus the easily applicable PCR-restriction fragmentation analysis method employed in our study can di¡erentiate between the R463C and the R463Q R463Q(IVS10-1) mutations in Gaucher disease patients. In our type I patients, in which di¡erences in the severity of the clinical expression of the disorder were noted, the R463Q(IVS10-1) was found together with the N370S mutation, whereas in the type II patient it was found together with the D409H mutation.

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230

Abstracts

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PRECLINICAL STUDIES OF ENZYME REPLACEMENT THERAPY FOR TYPE B NIEMANN-PICK DISEASE Edward H. Schuchman, Mount Sinai School of Medicine, New York, NY USA Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders due to the de¢cient activity of acid sphingomyelinase (ASM). In contrast to Type A NPD, a neurodegenerative disorder of infancy, Type B NPD is characterized by the lack of neurological involvement and a phenotypic spectrum ranging from severe monocyte/macrophage disease and early demise, to a milder condition of adulthood. A mouse model of Types A and B NPD has been constructed by targeted disruption of the murine ASM gene (ASMKO mice). Recombinant human ASM (rhASM) was puri¢ed from the media of overexpressing Chinese hamster ovary (CHO) cells and intravenously injected into 16 ¢ve month old ASMKO mice every other day for 14 days. The sphingomyelin content and histopathological lesions were markedly reduced in the hearts, livers, and spleens of these animals by this shortterm enzyme treatment. A group of 10 additional ASMKO mice were then intravenously injected with rhASM for 15 weeks, starting at 3 weeks of age. Although anti-rhASM antibodies were produced in these mice, the antibodies were not neutralizing and no adverse e¡ects were observed. Weight gain and rota-rod performance were slightly improved in the treated animals, but signi¢cant neurological de¢cits were still observed and their life-span was not extended by the enzyme treatment. In contrast to these CNS ¢ndings, striking histological and biochemical improvements were found in the reticuloendothelial system of the treated animals. These studies indicate that enzyme replacement therapy should be an e¡ective therapeutic approach for Type B NPD.

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A STUDY FOR NIEMANN^PICK DISEASE: DISTRIBUTION OF ACID SPHINGOMYELINASE IN HUMAN VARIOUS BODY FLUIDS Ikuko Takahashi, Tsutomu Takahashi, Wataru Watanabe, Goro Takada Department of Pediatrics, Akita University School of Medicine Objective: Recent research demonstrated that acid sphingomyelinase (ASM) is secreted from the various cultured cells. To understand the pathophysiology of Niemann^Pick Disease (NPD), we determined the distribution of ASM in various human cell-free body £uids and attempted to know the function of extracellular ASM. Methods: ASM enzyme activities, cation-independent ASM and Zn2+-dependent, were determined in serum, cerebrospinal £uids (CSF), urines, salivary £uids, tear £uids and synovial £uids by radioenzyme assay. Results: The enzyme activities were determined in all the £uids but CSF and those were intensi¢ed by the addition of Zn2+. Tear and salivary £uids demonstrated much higher activities than those of serum from normal controls (tear £uids: 8 times, salivary £uids: 4 times of the serum). In the synovial £uids from patients with rheumatoid arthritis and osteoarthritis, ASM activities were 2 times higher than those of serum from normal control. In urine, the activity was less active than that of serum. ASM activities were not detected in the CSF. Conclusions: ASM enzymes are broadly distributed in various body £uids. The presence of ASM in the salivary £uids suggests that may have a role in the digestion of sphingomyelin in a normal diet.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SIGNALING PATHWAY FOR RADIATION-INDUCED APOPTOSIS IS PRESERVED IN TYPE B NPD T. Abe1, T. Takahashi1, I. Takahasi1, M. Komatsu1, M. Enoki2, G. Takada1 Department of Pediatrics, Akita University School of Medicine1; Hiraka General Hospital2

Objective: A de¢ciency of acid sphingomyelinase (ASM) is known to cause the defective response of apoptosis induction by x-ray irradiation in lymphoblasts. To know the genetic heterogeneity in the disturbed apoptosis induction, we investigated the radiation-induced apoptosis of lymphoblast cells from the patients with type B, mild non-neurodegenerative, Niemann^Pick disease (NPD). Methods: EBV-transformed lymphoblasts established from the two patients with type B NPD (Genotype: S436R/S436R) and a normal control were irradiated with 20 Gy and incubated for 24 hours. The cells were harvested and the morphological features of apoptosis were observed with DNA-speci¢c £uorochrome bis-benzimide and DNA fragmentation study. Results: Expression study of the causing mutation (S436R) showed that the mutation produced a defective enzyme with little residual catalytic activity. However, in the apoptosis induction by the irradiation, the ratio of apoptosis induction in the NPD lymphoblasts was the same as in the normal lymphoblasts. Conclusions: In the patients with type B NPD, the signaling pathway for radiation-induced apoptosis is preserved in lymphoblast cells. This result suggests that the disturbance of cell signaling system caused by the ASM de¢ciency may be related to phenotypical heterogeneity in type A and B NPD.

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STRESS-INDUCED APOPTOSIS IN LYMPHOBLAST CELLS FROM TYPE A NPD 1 Masali Komatsu, 1Tsutomu Takahashi, 1Tamaki Abe, 2Hiroyuki Ida, 1Goro Takada Department of Pediatrics, 1Akita University School of Medicine, 2Jikei Medical School

Objective: To know the cellular pathology of type A Niemann-Pick disease (NPD), we investigated whether a de¢ciency of acid sphingomyelinase (ASM) have some in£uence on the apoptosis induced by oxidative stress (H2O2) and ultraviolet-C (UV-C). Methods: Epstein-Barr virus (EBV) transformed lymphoblast cells established from the patient with type A NPD and a normal control were treated with H2O2 containing medium or exposure to UV-C. After the incubation for 12 or 24 hours, the cells were harvested and the morphological features of apoptosis were observed. Results: In the H2O2 stimulation, apoptosis was detected in 2.84+/-0.06% and 1.30+/-0.35% of NPD cells by 12 hours and 24 hours, respectively, while it was detected in 8.44+/-0.49% and 4.24+/0.16% of normal cells by 12 hours and 24 hours, respectively. In the UV-C stimulation, apoptosis was detected in 8.85+/-0.52% and 5.85+/-0.43% of NPD cells by 12 hours and 24 hours, respectively, while it was detected in 20.13+/-1.07% and 10.85+/-2.04% of normal cells by 12 hours and 24 hours, respectively. Conclusions: NPD lymphoblast cells were defective in H2O2- and UV-C-inducd apoptosis and our results indicated that ASM plays a important role in the signaling pathway of stress-induced apoptosis. We suggest that some of the pathological mechanisms are to the sphingomyelin cell signaling system in type A NPD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

232

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NIEMANN-PICK C DISEASE: INSIGHTS FROM STUDIES ON MUTATED NPC1 GENE AND PROTEIN Marie T. Vanier and Gilles Millat, INSERM U 189, Lyon-Sud Medical School, 69921 Oullins, and Fondation Gillet-Me¨rieux, Centre Hospitalier Lyon-Sud, 69310 Pierre-Be¨nite, France In order to obtain more information on the functional domains of the NPC1 protein, the mutational spectrum and the presence of immunoreactive protein were investigated in 25 unrelated NiemannPick C1 patients. A homozygote status of the mutation in 13 patients facilitated establishment of genotype/phenotypes correlations. The liver disease was independent of mutations. Its perinatal timing was possibly correlated with a developmental pattern of NPC1 transcripts showing lower liver and higher brain expression in normal adult human tissues, but the triggering factor remains unknown. Five frameshift mutations (3 in the N-terminal loop), a nonsense mutation in the cystinerich luminal loop, but also 3 missense mutations in the sterol-sensing domain (SSD) all corresponded to patients with a severe infantile neurological onset and pronounced alterations of cellular cholesterol tra¤cking. No NPC1 protein was detectable by Western blot in ¢broblasts with SSD missense mutations. Our results thus enhance the functional signi¢cance of the SSD. A signi¢cant (9/ 28) number of mutations were located in a conserved NPC1-speci¢c cystein-rich domain (luminal loop between TM-8 and TM-9). They resulted in a wide variation of clinical and biochemical phenotypes. In the missense mutations, NPC1 protein was present in varying but signi¢cant amounts, with one exception in an infantile neurological case. All 3 mutant alleles which we found de¢nitively correlated with mild alterations of cellular cholesterol transport (''variant'' phenotype) were located on this loop, as were most of the previously reported ``variant'' alleles. Our results indicate a hitherto neglected functional complexity of this domain and the need for further investigations on this region of the protein.

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WOLMAN DISEASE PRESENTING AS PRESUMED ABDOMINAL MALIGNANCY Fiona Bamforth, Carolyn O'Hara, Robert Power, David Rayner. Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada

Wolman disease (acid lipase de¢ciency) is a lipid storage disease presenting in the ¢rst few weeks of life. The disorder is characterised by extensive and widespread lysosomal storage of cholesterol esters and triglycerides. The rapid progression of abdominal distension, anorexia and anaemia may point to an abdominal malignancy. A 3-month-old female fraternal twin presented with a three week history of decreased appetite, failure to thrive, fever and increasing abdominal distension. Ultrasound showed hepatosplenomegaly and a midline abdominal mass. The infant was anaemic (haemoglobin 72 g/L, ref 95-135) and had thrombocytopenia (platelets 125 109/L, ref 140-450). Liver transaminases were elevated but alkaline phosphatase and bilirubin were normal. A tentative diagnosis of abdominal malignancy was made, likely lymphoma or neuroblastoma. On this basis, the abdominal mass was biopsied but histology results were inconclusive. A second laparotomy was performed and an abnormal pale liver, ¢rm spleen and a multilobulated mass at the root of the mesentery were noted. Histology suggested lipid storage rather than malignancy and Wolman disease was con¢rmed by leukocyte enzyme analysis (acid lipase 4 pmol/min/mg protein, ref 55-600). Subsequent CT scan con¢rmed adrenal calci¢cation. The infant died shortly after diagnosis from pulmonary haemorrhage. At autopsy the abdominal mass was found to be enlarged ¢brotic mesenteric lymph nodes distended with lipid. There was extensive lipid storage in liver, spleen and adrenal glands.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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A SINGLE BASE PAIR DELETION IN THE HUMAN LYSOSOMAL NEURAMINIDASE GENE LEADING TO CONGENITAL SIALIDOSIS TYPE II IN PATIENTS OF ANATOLIAN ORIGIN Willenbring H, Krasemann T, Linnebank M, Homberger A, Harms E, Koch HG Kinderklinik der UniversitÌt MÏnster, 48149 MÏnster

Objective: Sequence aberrations in the neuraminidase gene cause the lysosomal storage disorder sialidosis. A heterozygous missense mutation was described in late-onset sialidosis type I characterized by macular cherry-red spots and myoclonus. A homozygous insertion frameshift and two compound heterozygous (missense/missense and deletion/missense) mutations were reported in early-onset sialidosis type II. The clinical heterogeneity of sialidosis type II comprising hydrops fetalis, hepatosplenomegaly and mental retardation with congenital, infantile or juvenile onset requires further distinction by genotype-phenotype correlation. Methods: Mutation analysis was performed on complete neuraminidase cDNA after cloning in E. coli and con¢rmed by direct sequencing of an appropriate fragment of genomic DNA in two unrelated patients of Anatolian origin with severe hydrops fetalis. Results: Both patients harboured a homozygous frameshift deletion (679 del G) which introduced a premature termination codon at amino acid 302. The clinical course exhibited recurrent ascites, progressive hepatosplenomegaly, increasing renal involvement, cardiac and respiratory failure leading to death at 3 and 4 months of age, respectively. Conclusions: The full-blown phenotype and complete enzyme de¢ciency in patient's ¢broblasts is consistent with termination of protein synthesis due to the frameshift mutation.

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MOLECULAR MECHANISM OF SIALIDASE DEFICIENCY IN SIALIDOSIS INVOLVES DISRUPTION OF THE LYSOSOMAL MULTIENZYME COMPLEX A. V. Pshezhetsky, K. Lukong, K. Laundry, M.-A. Elsliger, S. Lefrancois, C. Morales; Service de Ge¨ne¨tique Me¨dicale, Hoªpital St-Justine, Universite¨ de Montre¨al, Montre¨al H3T1C5, Canada and Department of Anatimy and Cell Biology, McGill University, Montre¨al H3A2B2, Canada.

Sialidosis is an autosomal recessive disease caused by the genetic de¢ciency of lysosomal sialidase, which catalyzes the hydrolysis of sialoglycoconjugates. The disease is associated with progressive impaired vision, macular cherry-red spots, and myoclonus (type I) or with skeletal dysplasia, Hurlerlike phenotype, dysostosis multiplex, mental retardation, and hepatosplenomegaly (type II). We have identi¢ed 6 new missense mutations in sialidosis patients, located them on three-dimensional model of sialidase structure and estimated their potential e¡ect on the enzyme. This analysis showed that ¢ve mutations (Gly227Arg, Ala298Val, Leu270Phe, and Gly328Ser) and clustered in one region on the surface of the sialidase molecule. These mutations dramatically reduce the enzyme activity and cause a rapid intralysosomal degradation of the expressed protein. We suggested that this region is involved in the sialidase binding with lysosomal cathepsin A and/or b-galactosidase in their high molecular weight complex required for the expression of sialidase activity in the lysosome. Transgenic expression of mutants followed by density gradient centrifugation of cellular extracts con¢rmed this hypothesis and showed that molecular mechanism of sialidase de¢ciency results from disruption of the lysosomal multienzyme complex.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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ABNORMAL BRAINSTEM AND SOMATOSENSORY EVOKED RESPONSES IN aMANNOSIDOSIS GUINEA-PIGS Crawley A.C.1, Weston P.F.2 and Hopwood J.J.1 Department of Chemical Pathology1 and Neurology2, Women's and Children's Hospital, North Adelaide, South Australia. Alpha-mannosidosis, an inherited lysosomal storage disorder resulting in progressive neurological disease and deafness, is recognised in humans, cattle, cats and more recently also in guinea-pigs. We performed sequential ABER and SER under light anaesthesia in 8 a¡ected and 17 normal guineapigs after clinical neurological examination. ABER thresholds were elevated in a-mannosidosis animals as early as 1 month of age, and increased in severity with age to variable degrees. Delayed SER P1 latencies persisted in all a-mannosidosis animals from 1 month of age. Mild changes in posture, slight hindlimb proprioceptive de¢cits and mild hyperaesthesia could be detected as early as 1 month of age. Neurological disease increased in severity with age, with end-stage disease usually between 10 to 14 months of age. Evoked responses provide objective baseline measures of disease in a-mannosidosis guinea-pigs, with onset of abnormalities prior to signi¢cant clinical disease. Future studies will use this model to evaluate e¤cacy of new therapies targeted across the blood-brain barrier to the CNS.

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COST-EFFECTIVENESS OF BONE MARROW TRANSPLANTATION FOR CHILDREN WITH HURLER SYNDROME LN Venditti1, SJ Lee2, CP Venditti3, KM Kuntz4, EM Kaye3, C Peters5. 1 University of Pennsylvania, Philadelphia, PA, USA; 2Dana-Farber Cancer Institute and Brigham and Women's Hospital, Boston, MA, USA; 3Departments of Clinical and Biochemical Genetics, Children's Hospital of Philadelphia, Philadelphia, PA, USA; 4Harvard School of Public Health, Boston, MA, USA; 5Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA. While untreated patients with Hurler syndrome experience rapid neurologic deterioration and early death, bone marrow transplantation (BMT) has been shown to signi¢cantly ameliorate or halt this course. To address the concerns regarding the considerable costs, risks, and variable outcomes of this treatment, we compared the cost-e¡ectiveness of early BMT with no transplantation on the basis of cost per quality-adjusted life year. Data from 94 patients from the Storage Disease Collaborative Study Group were used to populate a Markov model; this model was developed to estimate e¡ectiveness for a 10-year period beyond the decision to undergo transplantation in a hypothetical cohort of 1 year-old children. Cost data were derived from 30 patients treated at the University of Minnesota. The 10-year incremental cost-e¡ectiveness ratio for BMT using related donor marrow is within the acceptable zone (5100,000 US dollars per quality-adjusted life year gained) while that for BMT using unrelated donor marrow is not. However, these results are sensitive to the time frame of analysis, since the latter seems cost-e¡ective when extrapolating over the patient's lifetime. Results are also in£uenced by the value placed on the patient's quality of life post-BMT, a quality of life characterized by signi¢cant resolution of disease but potentially, years of multiple corrective surgeries and possible unfavorable neurological development.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MR IMAGES IN 23 PATIENTS WITH MUCOPOLYSACCHARIDOSES AND THE EFFECT OF BONE MARROW TRANSPLANTATION Akemi Tanaka1, Toshiyuki Seto1, Kinuko Kono2, Kyoko Morimoto1, Yusuke Inoue2, Horuo Shintaku1, Hideji Hattori1, Osamu Matsuoka1, Tsunekazu Yamano1 Department of Pediatrics1 and Department of Radiology2, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan

A longitudinal study of cranial MR imaging was carried out in twenty-three patients with mucopolysaccharidoses (MPS); one each of types IH, VI and VII, two type IS, ten type II and four each of types IIIB and IVA. Six types of distinct images noted in the MRI of these patients were 1) cribriform changes or spotty changes, 2) high intensity signal in the white matter on T2-weirgted images, 3) ventriculomegary, 4) di¡use cerebral cortical atrophy, 5) spinal cord compression and 6) megacisterna magna. The cribriform changes that corresponded to dilated perivascular space were found in the patients with IS, II, and VI. The patchy or di¡use intensity changes were found in the patient with II and IIIB respectively. Type IH and severe type of II showed marked ventriculomegaly and megacisterbna magna was very frequently found in the type II patients. Spinal cord compression was a feature usually observed in MPS IH, IVA, VI and VII. Marked cerebral atrophy was noted in all MPS IIIB patients and in the severe type of II patients. The generalized damage of central nervous system resulted in the brain atrophy in the patients at progressed stage of types II or IIIB. Megacysterna magna was frequent in the patient with type II (6/10). In two patients out of ¢ve patients the therapeutic e¡ect of bone marrow transplantation was remarkable.

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a-L-IDURONIDASE GENOTYPE/PROTEIN FUNCTION RELATIONSHIPS IN MPS I FIBROBLASTS PR Clements, G Yogalingam, V Muller, DA Brooks and JJ Hopwood, Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia, 5006 Australia.

Catalytic capacity determined in cultured cells derived from patients a¡ected with mucopolysaccharidosis (MPS) is a measure of functional enzyme available to turn over substrate. It is derived from the product of the catalytic e¤ciency (V/K) and the level of immunoquanti¢ed enzyme protein in the same cells [1]. We have previously shown that this parameter correlates well with clinical severity in MPS [1- 3] patients. We propose that catalytic capacity can be applied as a prognostic tool for MPS patients that may present presymptomatically from neonatal screening programs. In this study we have expanded the number of MPS-I patients tested and con¢rmed a correlation between genotype and a-L-iduronidase catalytic capacity determined in cultured skin ¢broblasts from these patients. Patients with attenuated clinical phenotypes, often have missense mutations, and catalytic capacities in the range 0.005-0.3 ng.min-1, whereas ¢broblasts from clinically severe patients, often have nonsense and/or missense mutations, and a-L-iduronidase protein that is either not detectable or has unmeasurable kinetics. Normal controls have catalytic capacities of 270-290 ng.min-1. Therefore catalytic capacity is of value as a predictor of clinical phenotype. 1. Ashton LJ, et al., (1992) Am J Hum Genet 50: 787-794; 2. Brooks DA, et al., (1991) Am J Hum Genet 48: 710-719; 3. Bunge S, et al., (1998). Biochimica Biophysica Acta 1407: 249-256.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BIOCHEMICAL CHARACTERIZATION OF a-L-IDURONIDASE IN PLASMA AND LEUKOCYTES OF NORMAL INDIVIDUALS AND CARRIERS OF A COMMON MUTATION FOR HURLER'S DISEASE JC Coelho, J Mandelli, A Wajner, RF Pires, IV Schwartz, S Leistner, U Matte and R Giugliani Dept. of Biochemistry and Genetics and Medical Genetics Service, HCPA, UFRGS, Porto Alegre, RS, Brazil Mucopolysaccharidosis type I (MPS I), is a metabolic disease caused by de¢ciency of the enzyme a-Liduronidase (IDUA), a hydrolase required for degradation of dermatan and heparan sulfate. With the aim of characterizing IDUA in leukocytes and plasma from normal individuals and from heterozygotes for the most frequent and severe form of the MPS I (Hurler disease) who carry the common mutation W402X, we determined the optimum pH, Km and Vmax of the enzyme. In leukocytes we observed that the highest activity of IDUA was at pH 2.7 for both groups. In plasma, controls had an optimum pH at 2.6 and heterozygotes at 2.7. The Km of the enzyme for W402X heterozygotes was 0.43mM in leukocytes and 0.68mM in plasma. Values obtained in leukocytes and in plasma di¡ered signi¢cantly from the normal group, which had a Km of 0.60mM and 0.21mM in leukocytes and plasma, respectively. Vmax was determined as 60.98 nmol/h/mg of protein in normal leukocytes and it is higher than Vmax for W402X heterozygotes (36.91 nmol/h/mg of protein). In plasma, the enzyme for controls had a Vmax of 7.26 nmol/h/ml which was lower than the Vmax for W402X heterozygotes (13.31 nmol/h/ml). We conclude that Km and Vmax of the enzyme in leukocytes and plasma can be used to discriminate the two groups. The combination of these parameters could represent an e¡ective method for the biochemical identi¢cation of MPS IH heterozygotes families who carry the mutation W402X.

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COMMON MUTATIONS OF MUCOPOLYSACCHARIDOSES TYPE I AND IIIA IN AUSTRIA E.Paschke K.Paul and, I. Milos Department of Pediatrics, University of Graz, Austria

During the past 17 years we performed 104 postnatal and 57 prenatal enzymatic diagnoses on heteroglycanoses in patients ethnically originating from Austria, Hungary, the area of former Yugoslavia and Turkey. Some rare disorders were found in speci¢c regions, as for example infantile sialic acid storage disease and MPS type IVB in Austrian patients or Mucolipidosis II/III in Hungarians. Similar to other European countries however, the majority of cases were MPS I (21), II (12) and IIIA (21). Restriction analysis for mutations reported to be common was performed in PCR products from DNA of ¢broblasts from patients with MPS I (19), II (15) and IIIA (16): In MPS I 18 of 38 alleles (47%) carried the mutation Q70X, W402X and A327P, respectively. 6 of 19 genotypes (33%) could be de¢ned. Similar results were obtained for MPS IIIA (sulfamidase de¢ciency), where 10 of 32 alleles (31%) carried the mutations R74C or R245H, with 2 patients homozygous for R245H. (6,2%). Mutation S66W, common in Italian patients, was not found at all. In MPS II, however, only one of 16 patients carried a common mutation, R88H. Therefore, we suggest that for MPS I and IIIA but not for MPS II simple PCR-based restriction analysis could be helpful to be done routinely in addition to enzymatic diagnosis prior to complete genotype analysis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GENE THERAPY FOR MUCOPOLYSACCHARIDOSES TYPES I, IIIA AND IIIB: HERPES SIMPLEX VIRUS-MEDIATED GENE TRANSFER AND EXPRESSION Bryan Winchester1 Sarah Burton2, Elaine Estruch1, and Robert Co¤n2 1 Institute of Child Health, University College London, 30 Guilford Street London WC1N 1EH, UK and 2Neurovex, Windeyer Institute, University College London, Cleveland Street, London W1P 6DB, UK (FAX: 44 20 7404 6191; e-mail: [email protected])

The mucopolysaccharidoses (MPS) result from de¢ciencies in lysosomal enzymes involved in the catabolism of glycosaminoglycans. The di¤culties in treating symptoms of the central nervous system, have stimulated e¡orts to develop gene therapy for this group of diseases. Herpes simplex virus (HSV) vectors are being developed for use in gene therapy because they target the nervous system naturally. We have used these viruses to supply a- Liduronidase (IDUA), heparan- N- sulphamidase (HNS) and N-acetyl- glucosaminidase (NAGLU) enzymes to cultured ¢broblasts from patients with MPS I (Hurler, Hurler-Scheie and Scheie syndromes), MPS III A (San¢lippo type A) and MPS III B (San¢lippo type B), respectively.HSV vectors containing the IDUA, HNS or NAGLU genes were constructed, puri¢ed, grown to high titre, and then used to transduce ¢broblasts from patients with MPS I, III A and III B. The activities of the de¢cient enzymes in patient ¢broblasts transduced with a control viral vector remained essentially zero. Fibroblasts from patients with MPS I, III A and III B transduced with viruses encoding IDUA, HNS and NAGLU genes, respectively acquired activity comparable to that in normal cells. In addition activity was secreted into the medium to a level up to 300 times greater than that secreted from nontransduced normal cells. The uptake of the secreted enzyme into non-transduced mutant cells has been investigated. Knockout mouse models are being used to assess the e¡ectiveness of the vectors in the central nervous system in vivo. The authors thank Dr. Elizabeth Neufeld and colleagues for the knockout mice and the Society for Mucopolysaccharide Diseases for ¢nancial support.

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CHARACTERISATION OF GANGLIOSIDE STORAGE IN MUCOPOLYSACCHARIDOSIS IIIA BY TANDEM MASS SPECTROMETRY P.D. Whit¢eld, P. Sharp, P.J. Meikle and J.J. Hopwood Lysosomal Diseases Research Unit, Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, South Australia, Australia

Mucopolysaccharidosis (MPS) type IIIA (San¢lippo syndrome) is an inherited disorder caused by the de¢ciency of the lysosomal enzyme heparan-N-sulphatase and is characterised by severe progressive mental retardation and relatively mild somatic features. The primary result of the enzyme de¢ciency is the abnormal storage and excretion of heparan sulphate, however, the accumulation of gangliosides in the central nervous system (CNS) is a secondary e¡ect. We have developed a rapid, sensitive and speci¢c method using electrospray ionisation^tandem mass spectrometry (ESI-MS/MS) to demonstrate the nature of ganglioside storage in brain tissues of patients with MPS IIIA. Gangliosides are extracted from the brain, desalted on C18 solid-phase extraction cartridges and then directly analysed by ESI-MS/MS. A non-physiological monosialoganglioside 1 (GM1 internal standard has been used to quantify neural ganglioside concentrations. We have identi¢ed signi¢cant elevations of GM2 and GM3 gangliosides in the CNS of MPS IIIA patients compared to agematched controls. It has been speculated that the storage of gangliosides may contribute to the progressive CNS dysfunction observed in many of the MPS disorders. It is anticipated that this ESIMS/MS method will prove a valuable tool in monitoring ganglioside concentrations in CNS tissues and £uids of MPS animal models used to evaluate di¡erent therapy options.

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LATE-ONSET VISCERAL PRESENTATION WITH CARDIOMYOPATHY AND WITHOUT NEUROLOGICAL SYMPTOMS OF ADULT SANFILIPPO A SYNDROME J. Van Hove1, R. A. Wevers2, 1G. Matthijs, 1E. Schollen, J. Van Cleemput1, P. Moerman1, J.G.N. de Jong2, W.F. Carey3, V. Muller3, C. Nicholls, K. Perkins3, J. J. Hopwood3. 1 Katholieke Universiteit Leuven, Leuven, Belgium; 2Institute of Neurology, University Medical Center, Nijmegen, The Netherlands; 3Women's and Children's Hospital, Adelaide, Australia. San¢lippo A syndrome usually presents in early childhood with developmental delay, beha-vioral disturbances, and progressive neurodegeneration leading to death in teenage years. Visceral symptoms are limited to mild coarsening and diarrhea. Previously described rare adults have similarly presented. We describe a patient who presented with isolated cardiomyopathy. At age 45 years she had hypertension, and the next year developed a progressively worsening cardiomyopa-thy with prominent apical hypertrophy, and atrial ¢brillation. At age 53, she had severe concentric hypertrophic non-obstructive cardiomyopathy in both ventricles. Percutaneous endomyocardial biopsy showed ballooned cardiomyocytes with storage vacuoles, containing acid mucopolysaccharides on Alcian blue staining. There was no coarsening of features. Neurologic function, skeleton, cornea, liver and spleen were normal. Storage material was absent in a brain biopsy taken during removal of a meningioma. There was a slight increase in urine mucopolysaccharides, with an increased proportion of heparan sulfates. Heparan sulfamidase activity, measured with 3 substrates, was de¢cient in leucocytes, and heparan sulfamidase protein and speci¢c activity were reduced in cultured ¢broblasts. Mutations have not yet been identi¢ed. This new clinical case expands San¢lippo A syndrome to include a primary visceral presentation of cardiomyopathy in adults.

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MOLECULAR ANALYSIS OF a-N-ACETYLGLUCOSAMINIDASE IN THREE JAPANESE PATIENTS WITH MUCOPOLYSACCHARIDOSIS TYPE IIIB (MPS IIIB) Natsuko Takaura1, Masatsugu Kimura2, Tsunekazu Yamano1, Akemi Tanaka1 Department of Pediatrics1 and Department of Biophysics2, Osaka city University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka, 545-8585, Japan Molecular analysis of 3 Japanese patients with MPS IIIB from 3 unrelated families was carried out. Patient 1 was a six-year-old male. He was noted speech delay at the age of 3 years, and the diagnosis of MPS IIIB was done when he was 4 years old. He showed coarse facial features, joint sti¡ness, and hepatomegaly. He deteriorated rapidly. He spoke no words when he was 5 years old. He could not walk at the age of 6 years. Patient 2 was a twenty-four-year old male. He was noted hyperactivity and speech delay at the age of 3 years. He was diagnosed as MPS IIIB at the age of 4 years. He showed coarse facial features and joint sti¡ness. He could not talk at the age of 9 years. Seizures appeared when he was 13 years old. He became bedridden at the age of 15 years and nasal tube feeding at the age of 19 years. Patient 3 is a sixteen-year-old female. She was noted hyperactivity at the age of 6 years, and the diagnosis of MPS IIIB was done. She could not talk when she was 14 years old, and sleep disorders appeared at the age of 16 years. Patient 1 and 2 were homozygoutes of R482W and R565W, respectively. Patient 3 was a compound heterozygote of F314L and R565P. R482W, R565W, and R565P have been reported previously in severe type of IIIB in other ethnic groups. F314L was a novel mutation and was suggested to cause a mild phenotype in patient 3.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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NATURE OF GAGS STORED IN MUCOPOLYSACCHARIDOSIS TYPE VI E. Ranieri, S Ramsay, S Byers, J Turnbull1 and JJ Hopwood. Department of Chemical Pathology Women's and Children's Hospital, Adelaide, South Australia; 1 Department of Biochemistry, University of Birmingham, Birmingham, UK.

Mucopolysaccharidosis type VI (MPS VI) results from a de¢ciency of N-acetylgalactosamine-4-sulphatase, lysosomal accumulation of glycosaminoglycans (gags) and smaller oligosaccharides (OS) of chondroitin sulphate (CS) and dermatan sulphate (DS) to cause signi¢cant skeletal and soft tissue pathology. These gags and OS have repeating disaccharides, with non-reducing end N-acetylgalactosamine-4-sulphate (GalNAc[4-OSO3]) and a uronic acid (glucuronic; GlcA or iduronate; IdoA) that may also be sulphated. We have used gradient polyacrylamide gel electrophroesis (GPAGE) to sequence reducing-end DS/CS OS coupled to a coumarin £uorophore. Speci¢c exoenzymes were used to cleave end-terminal saccharide moieties and give GPAGE gelshifts to provide a precise sequence. Positive and negative ion mode electrospray tandem mass spectrometry (MSMS) of OS derivatives with 1-phenyl-3-methyl-5-pyrazolone was used to con¢rm the sequence. GPAGE analysis of gags and OS in tissues from feline MPS VI revealed a tissue-speci¢c pattern of accumulating gag sizes. Urine, chondrocytes and osteoblasts accumulate signi¢cant amounts of DS/CS gags, with size distribution of gag/ OS fragments di¡ering between cell types. Octasaccharide or lower OS represented 60 % of total stored gags in cultured MPS-VI chondrocytes, whereas urine, kidney tissue or cultured osteoblasts contained between 10 to 15% of this sized OS (Byers S, et al., 1998: Molec Genet. Metab 65:282-290). A similar OS size distribution was observed in urine from human MPS VI with the sequence of the penultimate disaccharide determined to be GalNAc(4-OSO3)b1,4IdoAa1,3. This structure was con¢rmed by MSMS. The spectrum of gags excreted in the urine is therefore most likely a combination of gags/OS from various tissues. This provides an important insight into gag degradation pathway and to the understanding of the pathophysiology of the MPS disorders.

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LONG-TERM MORPHOLOGICAL NORMALIZATION IN THE WHOLE BRAIN OF MICE WITH MUCOPOLYSACCHARIDOSIS VII (MPSVII) BY LOCAL INTRA-CEREBRAL ENGRAFTMENT OF GENE-TRANSDUCED AMNIOTIC EPITHELIAL CELLS Motomichi Kosuga1,2, Torayuki Okuyama1,2, Hidenori Ohkawa3, Ikuko Ogino3, Hajime Arai3, Nobutake Matsuo2, and Norio Sakuragawa4 Department of Genetics, National Children's Medical Research Center1, Department of Pediatrics, Keio University School of Medicine2, Department of Neurosurgery, Juntendo University School of Medicine3, Department of Inherited and Metabolic Diseases, National Institute of Neuroscience3

Cell-mediated therapy for visceral lesions of lysosomal storage diseases is promising, however, treatment of the CNS lesions is still challenging. In this study, we ¢rst showed that amniotic epithelial (AE) cells infected with E1-deleted adenoviral vectors survived for more than six months at the transplanted corpus striatum of the mice brains. This long survival tendency was utilized for the cellmediated therapy for CNS lesions of the mice with mucopolysaccharidosis VII. The cells were transduced with an adenoviral vector expressing human b-glucoronidase (GUSB) and generated AE cells over-expressing and secreting the enzyme. After con¢rming that GUSB from the AE cells was taken up via mannose-6-phosphate receptors in primary cultured neurons, the cells were transplanted into adult MPSVII mice brains. Histochemical study showed extensive distribution of GUSB-positive cells throughout the ipsilateral hemisphere of the recipient brain, and morphological normalization was observed even at the region far away from the site of injection. These results indicate that intracerebral transplantation of gene-transduced AE cells have therapeutic potential for the treatment of CNS lesions in lysosomal storage disorders.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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AN AUSTRALASIAN DIAGNOSTIC SERVICE FOR THE NEURONAL CEROID LIPOFUSCINOSES MJ Fietz, VJ Muller and BC Paton National Referral Laboratory for Lysosomal, Peroxisomal & Related Genetic Disorders, Dept of Chemical Pathology, Women's and Children's Hospital, Adelaide, South Australia The neuronal ceroid lipofuscinoses (NCL) are a family of related genetic disorders that together are believed to a¡ect one child in every 12,500 births in the USA. As part of our laboratory's focus on disorders of the lysosome, we have recently begun to develop diagnostic assays for the NCL in order to o¡er a de¢nitive diagnostic service for the Australasian population. We now o¡er the diagnosis of late-infantile neuronal ceroid lipofuscinosis (LINCL) by assay of tripeptidyl peptidase I (TPP I) activity, followed by a screen for three `common' mutations present in the CLN2 gene. In addition, we have also begun to o¡er a limited diagnostic service for the juvenile (JNCL) and infantile (INCL) forms of the disease on the basis of mutation analysis only. Retrospective analysis of Australasian patients with suspected NCL has revealed that six are a¡ected by LINCL, six by JNCL and, to date, two by INCL. We are yet to complete our analysis of patients suspected of having INCL. Since we began o¡ering a diagnostic service for LINCL in August, 1999, we have diagnosed a further three cases. Mutation analysis of our nine LINCL patients has shown that three mutations constitute 83% of alleles; the mutations include the splice mutations 3556g4c (39% of alleles) and 3556g4a (11%), and the nonsense mutation R208X (33%). Work is ongoing to develop further diagnostic services for the NCL.

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TRIPEPTIDYL PEPTIDASE ACTIVITY IN PATIENTS WITH LATE INFANTILE NEURONAL CEROID LIPOFUSCINOSIS R Steinfeld, K Meyer, J Ait Bya Alla, T Braulke, K Ullrich and A KohlschÏtter Department of Paediatrics, University Hospital Eppendorf, Hamburg, Germany Neuronal ceroid lipofuscinoses (NCL) are lysosomal storage diseases that are characterised by proressive neurodegeneration and onset in childhood. We have recently performed a mutational analysis of the CLN2 gene in 20 families of patients with late infantile NCL. We found two common mutations, a nonsense mutation in exon 6 (c.622C4T or R208X) and a splice-mutation in intron 5 immediately preceding exon 6 (IVS5 -1G4C). At least three previously unknown mutations were identi¢ed. One patient compound heterozygous for a new missense mutation (R127Q) and for the R208X mutation showed a protracted clinical course and was originally diagnosed to have atypical juvenile NCL. Other patients slightly di¡ered in their clinical features from the classical late infantile NCL cases. The CLN2 gene encodes a lysosomal tripeptidyl peptidase (TPP I), and CLN2 mutations have been shown to result in a loss of enzymatic activity. To explain the clinical variations associated with deined CLN2 mutations, we measured the TPP I activity in skin ¢broblasts or lymphocytes from seected patients and their relatives. TPP I activity was calculated from the measured £uorescence, resulting from release of the coumarin residue from Ala-Ala-Phe-7-amido-4-methylcoumarin. All patients investigated so far showed a residual activity smaller than 5 % (skin ¢broblasts) or 10 % (lymphocytes) as compared to controls. In heterozygous persons we could measure around 50-60 % TPP I activity. No signi¢cant di¡erences in residual TPP I activity could be found in patients with a protracted course when compared to patients with a classical clinical course.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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STUDY OF URINARY ORGANIC ACIDS OF PATIENTS WITH PEROXISOMAL DISORDERS Seiji Yamaguchi1, Misako Iga1, Masahiko Kimura1, Toshiyuki Fukao2, Nobuyuki Shimozawa2, Yasuyuki Suzuki2, Naomi Kondo2, Tadao Orii3 Department of Pediatrics, Shimane Medical University, Izumo, Shimane 693-8501, Japan1, Department of Pediatrics, Gifu University School of Medicine2, Chubu Gakuin College, Japan3

We studied urinary organic acids in peroxisomal disorders using GC/MS. Nineteen patients with peroxisomal disorders, including Zellweger syndrome or neonatal adrenoleukodystrophy, were examined. Non-ketotic dicarboxylic aciduria (NKDIC) was observed in 18 of the 19 patients, and the sebacate/adipate (C10/C6) molar ratio was higher in 17 of the 18 patients. Elevation in 2hydroxysebacate (2HS) excretion was seen in 11 of the 19 patients. Tyrosyluria was striking in all of the patients. Hyperexcretion of C12- and/or C14-epoxydicarboxylic acids (EDAs) was observed in 17 of 18 patients tested. In all of the 19 patients, serum very-long-chain fatty acid (VLCFA) was high. Conclusions: In patients clinically suspected of peroxisomal disorders, the following ¢ndings in urinary organic acid analysis by GC/MS as well as high serum VLCFA should be a good diagnostic indicator: (1) NKDIC with high molar ratio of C10/C6, (2) elevation of 2HS, (3) tyrosyluria, and (4) hyperexcretion of EDAs.

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URINARY ORGANIC ACIDS IN PEROXISOMAL DISORDERS C. Rizzo, P. Bertucci, G. Federici, ½R.J.A.Wanders, G. Sabetta, C. Dionisi-Vici. *Depts. Clin Biochem & Metabolism Bambino Gesu© Hospital, Rome, Italy; ½University Hospital Amsterdam, The Netherlands

Peroxisomal disorders (PD) are classi¢ed in two major categories: a) disorders with abnormal peroxisome assembly with multiple defective peroxisomal function (Zellweger syndrome, NALD, infantile Refsum's disease, RCDP); b) disorders with a single peroxisomal protein de¢ciency (i.e. XALD). We evaluated the urinary organic acids in three patients with di¡erent PD: Zellweger syndrome (ZS), NALD, and an isolated b-oxidation defect. All patients showed increased excretion of a) epoxydicarboxylic acids (C10; C12; C13; C14), b) odd-chain C7^C15 dicarboxylic acids (mainly pimelic and azelaic), c) 2-hydroxy-sebacic acid, d) saturated and unsaturated C6-C10 dicarboxylic acids. Epoxydicarboxylic aciduria, which could derive from the abnormal oxidation of rinocileic acid (12-hydroxy-cis-9-octadecenoic acid), was prominent in ZS compared to NALD and the isolated boxidation defect. Raised odd-chain dicarboxylic acids and 2-hydroxy-sebacic acid re£ect increased production and oxidation of 2-hydroxy-VLCFA, which usually accumulate in some PD. Di¡ering from mitochondrial beta-oxidation defects, the sebacic/adipic and the suberic/adipic ratios were found to be 41 in ZS and NALD. Increased 4-hydroxy-phenyllactic and 4-hydroxy-phenylacetic acids were detectable only in ZS patient as a sign of liver dysfunction. We conclude that careful evaluation of urinary organic acids could be a useful tool for the di¡erential diagnosis of PD and dicarboxylic acidurias of mitochondrial origin.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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483-O

PEROXISOMAL DISEASES: MICROSCOPY OF 154 LIVER SAMPLES F. Roels, B. De Prest, M. Espeel & I. Kerckaert Human Anat, Embryol, Hist, Ghent University, Godshuizenln 4, 9000 Ghent, Belgium Cytochemistry of liver by LM and EM contribute to the diagnosis of most peroxisomal disorders, and in addition have led to the discovery of novel variants unpredicted by biochemical and genetic analyses. We have now studied 154 liver biopsy- and autopsy specimens from proven peroxisomal patients. 63 patients showed peroxisomes (Px) containing catalase and/or AGT, 68 did not; 10 livers showed Px mosaicism. Livers without Px have additional pathological features such as cytoplasmic catalase and/or AGT (39), trilamellar 6^14 nm or polarizing inclusions (48), lipid droplets insoluble in acetone. `Empty' membrane ghosts were detected in some livers but were small and very few. Livers which do possess Px often show evidence of peroxisomal pathology: in primary hyperoxaluria (17), AGT was either absent or mislocalised in mitochondria (2); in 6/7 typical and 1 atypical RCDP, some Px were abnormally large (41 mm) and few in some cells; in b-oxidation defects the liver again revealed enlarged Px in 14 (4 oxidase- and 6 bifunctional enzyme de¢ciencies), trilamellar inclusions or insoluble lipid in 9, including the thiolase-de¢cient girl; Px with a nucleoid, marginal plate, or membrane invaginations; in 2 patients elongated Px (min axial ratio 5.35) were the only abnormality. In 2 b-oxidation defects, no Px pathology could be detected by microscopy. In DHAPAT de¢ciency and in X-ALD, liver tissue is not informative, but we are now localizing ALDP in blood monocytes. Unexpected ¢ndings were, besides Px mosaics in liver (age of these patients: 13 m, 4 y, 9, 9, 10, 11, 16, 15, 23, and 41 y): atypical Refsum (enlarged Px without catalase); changes with time revealed by successive samples (2 pts); thiolase localisation in Px in 1 typical and 1 atypical RCDP (in agreement with Hughes); normal AGT localisation and activity in a novel type of PH I (2); presence of all 3 b-oxidation proteins in Px concomitant with a functional defect (2); appearance of symptoms at 11 y in a patient with a mosaic of Px de¢cient in several enzymes; almost normal Px in 6 isolated HPA, distinguishing HPA from PBD; discrepancies between liver and cultured ¢broblasts (5), reported also by others. We thank parents and colleagues in Europe, Asia and America. Supp. by Eur. Commission.

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SCREENING FOR PEROXISOMAL DISORDERS: DETERMINATION OF VLCFA AND PHYTANIC ACID BY LC/APCI-MS S. Schrade, M. GÎggerle, H. Korall Zentrum fÏr Sto¡wechseldiagnostik Reutlingen GmbH (Metabolic Unit), WÎrthstraÞe 47, D-72764 Reutlingen, Germany In peroxisomal disorders degradation of very-long-chain fatty acids (VLCFA, unbranched, 522 carbon atoms) and branched-chain fatty acids is disturbed, resulting in an accumulation of these metabolites in tissues and body £uids. We established a new screening method for peroxisomal disorders by determination of VLCFA (cerotic acid (C26:0), lignoceric acid (C24:0) and behenic acid (C22:0)) and phytanic acid in plasma without derivatisation. Free fatty acids were obtained by hydrolysis of 200 mL (or less) plasma with methanolic KOH followed by extraction with heptane. Heptane-phase is evaporated and the residue is dissolved in methanol. Phytanic acid and VLCFA are separated by a reversed phase column. and detected as their [M-H].-peaks in negative-ion mode by LC/APCI-MS using a PE-SCIEX Tandem Mass Spectrometer. Sample preparation is easily performed within 212 hours. The total run time per sample is 10 minutes. Detection levels for phytanic acid and VLCFA are 4 nmol/l and 15 nmol/l respetively. Testing more than 250 samples concentrations of phytanic acid, C26:0 and ratios of VLCFA from patients with peroxisomal disorders stand out from normal values. These data con¢rm that LC/APCI-MS together with a rapid and simple sample preparation is a highly speci¢c method for diagnosis of peroxisomal disorders by quanti¢cation of phytanic acid, C26:0 and calculation of the ratios of C24:0/C22:0 and C26:0/C22:0.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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TEMPERATURE-SENSITIVE FORMATION OF PEROXISOMES IN PEROXISOME BIOGENESIS DISORDERS (PBDs): IMPLICATION FOR CLINICAL PHENOTYPE Atsushi Imamura1,2, Nobuyuki Shimozawa2, Yasuyuki Suzuki2, Zhongyi Zhang2, Toshiro Tsukamoto3, Tadao Orii4, Yukio Fujiki5, Takashi Osumi3 and Naomi Kondo2 Department of Pediatrics, Ogaki Municipal Hospital1; Department of Pediatrics, Gifu University School of Medicine2; Department of Life Science, Himeji Institute of Technology3; Chubu Gakuin University4; Department of Biology, Kyushu University Graduate School of Science, Japan5

PBDs are clinically classi¢ed into 3 phenotypes: Zellweger syndrome (ZS), the most severe; neonatal adrenoleukodystrophy, intermediate; and infantile Refsum disease, the mildest form. Cell fusion studies revealed that there are at least 12 complementation groups (CGs) in PBDs. Although responsible genes (PEX) have been isolated for many CGs in PBDs, it remains clear about the metabolic and molecular bases of clinical phenotypes. We elucidated the temperature-sensitive (ts) formation of peroxisomes in the ¢broblasts of milder forms of PBDs. Peroxisomal enzymes were normally localized into peroxisomes and biochemical functions of peroxisomes, including VLCFA oxidation and DHAP-AT activity, were restored after incubation for 72 h at 308C. The ts phenomenon was caused by the ts mutations in each PEX genes, including PEX1, PEX2, PEX6 and PEX13. We also identi¢ed the characteristic ts import of acyl-CoA oxidase, the ¢rst enzyme of peroxisome beta-oxidation, in the ¢broblasts from PBDs including ZS belonging to CG-A.

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DEFECTIVE PEROXISOME MEMBRANE SYNTHESIS DUE TO MUTATIONS IN HUMAN PEX3 Ania C. Muntau, Peter Mayerhofer, Barbara C. Paton*, Stefan Kammerer, Adelbert A. Roscher Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University Munich, Germany; Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006* Proteins required for the process of peroxisomal assembly are termed peroxins and are encoded by PEX genes. In most cases defects of PEX genes lead to a disruption of peroxisomal matrix protein import while various peroxisomal membrane remnants (``ghosts'') are synthesized. PEX3 encodes a peroxisomal membrane protein and a human phenotype de¢cient in PEX3 has not yet been described. Here we show that inactivating mutations in the PEX3 gene are associated with a severe type of human hepato-cerebro-renal syndrome of Zellweger. We investigated two patients su¡ering from severe forms of peroxisomal biogenesis disorders (PBD) leading to early death. Fibroblasts of the patients had previously been assigned to complementation group G (Gifu University nomenclature) and were described to lack peroxisomal ghosts. PEX3 mutation analysis revealed that patient 1 (PBDG-01) was homozygous for a frameshift mutation in exon 7 and that patient 2 (PBDG-02) carried a homozygous point mutation in the 3' consensus region of intron 10 leading to skipping of exon 11. Both mutations result in a truncation of the C-terminal part of the protein. Peroxisomal biogenesis in patients' ¢broblasts was restored by transfection with wild type PEX3 cDNA. In vitro binding assays showed that wild type but not the truncated Pex3 proteins bind to immobilized GSTPex19 protein demonstrating that mutations in the PEX3 gene lead to disruption of the crucial interaction between Pex3p and Pex19p shown to be important for the very early steps of de novo peroxisomal biogenesis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

244

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THE N TERMINAL SEQUENCE OF PMP70 IS SUFFICIENT FOR TARGETING TO PEROXISOMAL MEMBRANE IN CHO CELLS BUT NOT IN S. cerevisiae. Shlomo Almashanu and David Valle Howard Hughes Medical Institute, Johns Hopkins University School of Medicine The pathways involved in the targeting, assembly and function of the four (PMP70, P70R, ALDP, ALDR) half ATP binding cassette (ABC) transporters localized to human peroxisomal membranes is poorly understood, as well as their disease associations and their role in the pathophysiology of ALD. To address those questions in a more malleable system, we expressed the four human peroxisomal membrane half ABC transporters in S. cerevisiae with high levels of the corresponding mRNA but no detectable protein. We attempted to reconstitute the human targeting pathway in S. cerevisiae by introducing all human genes potentially involved in this pathway (PEX19, PEX16, PEX3) together with PMP70. The human PEX3 and PEX19 proteins were detectable by western blot but not PEX16 nor PMP70. These results suggest some di¡erence in the targeting pathway of PMP`s between humans and yeast. To identify targeting motifs, we generated a series of N-terminal and C-terminal deletions of PMP70 tagged with a C-terminal green £uorescent protein (GFP), and expressed them in Chinese hamster ovary (CHO) cells. We localized a peroxisomal membrane targeting signal to the 80 N-terminal amino acids, preceding the ¢rst transmembrane domain. A chimeric protein with the Nterminal 183 residues of PMP70 followed by the C-terminal 428 residues of an E. coli homolog (YDDA) tagged with GFP localizes to peroxisomes. Truncated PMP70 GFP fusions that localized to peroxisomes in CHO cells, showed a di¡use cytosolic GFP pattern in yeast.

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PHENOTYPIC HETEROGENEITY VERSUS PEROXISOMAL DYSFUNCTION IN DISORDERS OF PEROXISOME BIOGENESIS: IDENTIFICATION OF A GOOD MARKER REFLECTING THE EXTENT OF PEROXISOME DYSFUNCTION IN FIBROBLASTS J.Gootjes1, P.G. Barth1, P.A.W. Mooijer1, S. Denis1, P. Vreken, R.J.A. Wanders1 1 University of Amsterdam, Academic Medical Center, Depts. Pediatrics, Emma Children's Hospital, Clinical Chemistry and Neurology, Amsterdam, The Netherlands Objectives: Zellweger syndrome is generally regarded as the prototype of the group of peroxisome biogenesis disorders (PBD) which also includes milder phenotypes such as neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). A major problem in the management of such PBD-patients is that sofar the relationship between the extent of peroxisomal dysfunction and the phenotypic presentation and survival of patients has remained elusive. We have now performed a full study in a series of patients with de¢ned phenotypes by performing studies in plasma (very-longchain fatty acids, bile acid intermediates, phytanic, pristanic and pipecolic acid), erythrocytes (plasmalogen) and ¢broblasts (de novo plasmalogen synthesis, C26:0 and pristanic acid b-oxidation, phytanic acid a-oxidation, DHAPAT activity measurements, catalase immuno£uorescence in order to search for the best parameter. Methods: Standard methods were used for measurement of peroxisomal metabolites in plasma and peroxisomal functions in ¢broblasts (see Wanders (1999) Neurochem. Res. 24, 565-580) Results: For each parameter plots were made with age of death on the X-axis and metabolite level or enzyme activity on the Y-axis. For most parameters a poor correlation was found between age of death and biochemical abnormality with the exception of pristanic acid b-oxidation, which therefore proves to the best parameter to determine the extent of peroxisomal dysfunction. These results may have important implications for patient management.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MOLECULAR BASIS OF THE PEROXISOME BIOGENESIS DISORDERS: IDENTIFICATION OF THE MUTANT PEX-GENE VIA MEANS OF COMPLEMENTATION ANALYSIS IN 207 PATIENTS FOLLOWED BY MUTATION ANALYSIS J. Gootjes1, P.A.W. Mooijer1, C. Dekker1, P.G. Barth1, B.T. Poll The1, Y Suzuki2, T Shimozawa2, S. Gould3, H.R. Waterham1, R.J.A. Wanders1 1 University of Amsterdam, Academic Medical Center, Amsterdam, The Netherlands, 2Gifu University, Gifu Japan, 3Johns Hopkins Medical School, Baltimore, USA

The disorders of peroxisome biogenesis (PBDs) include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) and a number of phenotypic variants in between ZS, NALD and IRD. Earlier studies have shown that there is profound genetic heterogeneity within the PBD-group. We have now extended these studies and have performed complementation analysis in 207 PBD-patients. In all patient' cell lines detailed studies were done including global assays like de novo plasmalogen biosynthesis, C26:0 and pristanic acid b-oxidation and phytanic acid a-oxidation followed by individual enzyme activity measurements, and immunoblot analysis plus immuno£uorescence microscopy using antibodies against catalase and the ALDprotein. Complementation was evaluated by means of catalase immuno£uorescence which revealed the existence of 11 groups. Our results further show strong overrepresentation of one particular complementation group representing PEX1-de¢ciency. We show that 136 of the 207 (66%) patients we analyzed belong to this group. The second most frequent group to which 12 of our patients belong (6%) represents PEX6-de¢ciency. Molecular analysis of PEX1 and PEX6 reveals a large variety of di¡erent mutations with only few more frequent mutations.These studies now allow identi¢cation of the true molecular defect in most PBD-patients.

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MOLECULAR BASIS OF ZELLWEGER SYNDROME AND OTHER PEROXISOME BIOGENESIS DISORDERS: MUTATION ANALYSIS OF HUMAN PEX 1 AND PHENOTYPE/GENOTYPE CORRELATION J. Gootjes1, E. Hogenhout1, P. Barth1, B.T. Poll The¨1, C. Walter2, G. Dodt2, R.J.A. Wanders1 University of Amsterdam Academic Medical Center, Depts. Clinical Chemistry and Pediatrics, The Netherlands1; Physiologische Chemie, Ruhr-Universita«t, Bochum, Germany2

Objectives: The peroxisome biogenesis disorders include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD), infantile Refsum disease (IRD) and other phenotypes not easily assignable to either category. Complementation analysis has shown the existence of at least 11 distinct genetic groups with overrepresentation of one particular group (CG1) due to mutations in the PEX 1-gene. We have now studied 110 patients with either ZS, NALD or IRD and performed mutation analysis to establish whether there is a correlation between phenotype and genotype. Methods: Standard methods were used to sequence the PEX1-gene at either the cDNA and/or genomic level. Results: The results can be summarized as followed. We ¢rst checked each patient for the occurrence of the frequent G843D mutation in our population of patients. An overall frequency of 37% was found for the G843D allele. All 23 patients homozygous for the G843D allele showed a mild clinical presentation corresponding to NALD and especially IRD. Patients heterozygous for the G843 allele, showed a much more variable presentation ranging from ZS to IRD. In addition to the frequent G843D mutation a variety of other, mostly private mutations were found. Conclusions: The results obtained show a clear genotype/phenotype correlation where the G483D allele is concerned. Furthermore, resolution of the molecular defect in PBD-patients is very important for diagnostic purposes including prenatal diagnosis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

246

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LATE-ONSET LEUKODYSTROPHY AND NEUROPATHY IN A 6-YEAR-OLD GIRL REVEALING A PEROXISOMAL BIOGENESIS DEFECT M.C. Nassogne1, C. Jakobs2, A. Garcia1, F. Roels3, D. Rabier4, M.O. Rolland5, L. Hertz-Pannier6, G. Touati1, P. Aubourg7, J.M. Saudubray1 Dept of 1Pediatrics, 4Biochemistry and 6Radiology, Necker Enfants-Malades Hosp, & 7INSERM U342, St-Vincent de Paul Hosp, Paris; 2Dept. of Clinical Chemistry, Free University Hosp, Amsterdam; 3 Anatomy-Embryology, Univ Gent; 5Dept. of Biochemistry, Debrousse Hosp, Lyon Late-onset peroxisomal diseases include Refsum disease and X-linked adrenoleukodystrophy. We report a girl a¡ected by a peroxisomal biogenesis defect revealed by a late-onset neuropathy associated with leukodystrophy. Psychomotor development was normal. At the age of 6 years, she presented with gait di¤culties. Neurological exam revealed ataxia, dysmetria and are£exia. NCV displayed demyelinating sensorimotor polyneuropathy. Brain MRI showed severe white matter changes in cerebellum and periventricular areas. In plasma, there are elevated VLCFAs (C24/C22 ratio at 1.66, C26/C22 at 0.077), phytanic (44.94 mmoles/l), pristanic (21.27 mmoles/l) and pipecolic acids, while DHA was slightly reduced. b-oxidation of lignoceric and phytanic acids and DHAP-AT activity were normal on ¢broblasts. In ¢broblasts, immunolocalization of Pex 14 and ALDP revealed a reduced number of peroxisomes, sometimes of enlarged size. Liver microscopy showed absence of peroxisomes, cytoplasmic localisation of catalase and trilamellar inclusions. These results strongly suggested a peroxisomal biogenesis defect. This is the second girl described with late-onset neurological symptoms secondary to a peroxisomal biogenesis defect. This case emphasizes the need to investigate peroxisomal functions in patients with atypical neuropathy or leukodystrophy, even with late-onset symptomatology.

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PEROXISOMAL BIOGENESIS DISORDER (PBD) WITH SEVERE PHENOTYPE IN AN INFANT vs MILD PHENOTYPE IN THE AFFECTED PARENTS RESEMBLING USHER SYNDROME - A UNIQUE FINDING SH Korman, G Eshel1, RJA Wanders2, PAW Mooijer2, J. Gootjes2, HR Waterham2, A Gutman, A Raas-Rothschild. Hadassah Univ Hospital, Jerusalem, 1Assaf Harofeh Medical Center, Zeri¢n, Israel and 2Academic Medical Center, Univ Amsterdam, The Netherlands

Sensorineural deafness (SD) and retinitis pigmentosa (RP) are the hallmarks of Usher syndrome and are prominent features in PBD. The ¢rstborn son of unrelated parents, both of whom had severe SD associated with RP diagnosed as Usher syndrome, presented with SD, RP, marked dysmorphism, severe developmental delay, hepatomegaly and hypsarrhythmia and died from aspiration pneumonia aged 17 mo. A PBD was suspected and supported by ¢nding elevated very-long and branched-chain fatty acids (VLCFA and BCFA) in plasma. Fibroblasts studies revealed a complete loss of peroxisomal functions (plasmalogen synthesis, peroxisomal fatty acid a- and b-oxidation) and catalase immuno£uorescence microscopy demonstrated absence of peroxisomes in both ¢broblasts and liver. Surprisingly, VLCFA and BCFA were elevated in plasma of both mother and father and ¢broblast studies as above con¢rmed that both were a¡ected by a PBD, leading to revision of the initial diagnosis of Usher syndrome. Mosaicism demonstrated in the parents' ¢broblasts might account for their milder clinical and biochemical phenotypes. This is the ¢rst reported case of a PBD where the parents are actually a¡ected rather than carriers. In view of the considerable overlap between Usher syndrome and milder PBD phenotypes, PBD should be excluded by VLCFA screening in all cases of suspected Usher syndrome.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PEROXISOME MOSAICISM IN TWO PBD PATIENTS WITH MILD CLINICAL COURSE AND IDENTICAL PEX1 MUTATIONS S. Weller, N. PreuÞ, U. Brosius, W. Schmitz, E. Conzelmann, J. GÌrtner Heinrich Heine-UniversitÌt DÏsseldorf and Biozentrum UniversitÌt WÏrzburg, Germany

The peroxisome biogenesis disorders (PBD) are a group of lethal autosomal recessive diseases with overlapping clinical phenotypes. Their defects are caused by mutations in di¡erent PEX genes. The cellular hallmark are the failure to assemble normal peroxisomes and the concomitant loss of multiple peroxisomal enzyme activities. We describe two male, 10 and 23 year old patients with the characteristic clinical, biochemical and molecular features of PBD. However, the peroxisome defect of these patients is not expressed in all cells. Catalase immuno£uorescence stained skin ¢broblasts show a peroxisomal as well as a cytoplasmic pattern indicating mosaicism. Electron microscopy studies of skin ¢broblasts and liver are ongoing. Both patients have two compound heterozygous mutations in the PEX1 gene: G843D and c.1960-1961insCAGTGTGGA. Mutations in the PEX1 gene are responsible for more than 50% of all PBD cases. In patients homozygous for the G843D allele a mild clinical course can be expected. In contrast, insertions and deletions in the PEX1 gene are known to cause more severe forms of disease. The origin of the mild clinical courses in these two mosaic PBD patients might be residual activity of the mutant PEX1 alleles, complementation of the di¡erent mutant alleles or the peroxisomal mosaicism. The latter is most likely and the mosaicism might be due to reversion of the PEX1 mutation in one allele or somatic loss of heterozygosity in foetal development.

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D-BIFUNCTIONAL PROTEIN DEFICIENCY WITH A MISSENSE MUTATION IN THE DEHYDROGENASE DOMAIN Yasuyuki Suzuki1, Zhongyi Zhang1, Nobuyuki Shimozawa1, Kazutoshi Nakano2, Makiko Osawa2, Masayuki Itoh3, and Naomi Kondo1 Department of Pediatrics, Gifu University School of Medicine1; Department of Pediatrics, Tokyo Women's Medical University2; National Center of Neurology and Psychiatry, Japan3

D-3-hyroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (DBP) de¢ciency is a fatal inborn error of peroxisomal beta-oxidation charaterized by severe hypotonia, psychomotor retardation, liver dysfunction, accumulation of very-long-chain fatty acids and intermediates of bile acid synthesis. Here we report an infant with D-BP de¢ciency who manifested atypical clinical presentation of fetal chylous ascites and polyhydramnios, and died at 7 months of age. Polymicrogyria, heterotopia, dysplasia of the olivary nucleus, liver ¢brosis, renal microcysts and lamellar inclusions in the adrenal cortex were identi¢ed. Bile acid analysis revealed a presence of characteristic stereoisomer of 24-hydroxy-trihydroxycholestanoic acid which suggests a defect in the dehydrogenase activity of D-BP. Immunoreactive material of D-BP was detected, and a missense mutation R106P in the dehydrogenase domain and a 52bp deletion (1210^1261 del) in the dehydratase domain were identi¢ed. Transfection of normal cDNA restored the peroxisomal lignoceric acid oxidation activity, whereas the mutant cDNA with R106P missense mutation did not restore the activity. Attention should be paid for atypical fetal manifestations of D-BP de¢ciency.

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ANALYSIS OF FREE AND CONJUGATED VARANIC ACID DIASTEREOMERS USING HPLC ELECTROSPRAY TANDEM-MASS SPECTROMETRY IN PATIENTS WITH DBIFUNCTIONAL PROTEIN DEFICIENCY Vreken P1, Overmars H1, van Lint L1, van Grunsven EG1, Hunnemann D2, Wanders RJA1 1 Academic Medical Center, University of Amsterdam, Dept. of Clinical Chemistry and Div. Emma Children's Hospital, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands 2Dept. of Pediatrics, GÎttingen, Germany D-bifunctional protein de¢ciency is the most common peroxisomal beta-oxidation defect and can be divided into three subgroups: isolated enoyl-CoA hydratase de¢ciency, isolated 3-OH-acyl-CoA dehydrogenase de¢ciency, and complete D-BP de¢ciency. In a previous report we showed that a patient with dehydrogenase de¢ciency accumulated mainly 24R,25R-varanic acid, whereas a patient with complete de¢ciency accumulated mainly 24S,25S-varanic acid (Vreken et al J.lipid Res. 39:245258,1998). We now tested patients with dehydrogenase (n=6), hydratase (n=2) and complete de¢ciency (n=2), using HPLC tandem-mass spectrometry instead of GC/MS analysis. Two patients with hydratase de¢ciency showed normal bile acid concentrations and did not accumulate THCA, DHCA or varanic acid. The patients with complete de¢ciency accumulated mainly unconjugated 24S,25S varanic acid and the patients with dehydrogenase de¢ciency accumulated mainly unconjugated 24R,25R varanic acid, but also a substantial amount of the 24S,25S-isomer. 24-THCA also accumulated in these patients, but was not only present in the unconjugated form, but also for a substantial part as taurine conjugate.

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D-BIFUNCTIONAL PROTEIN DEFICIENCY: IDENTIFICATION OF THREE SUBGROUPS AND RESOLUTION OF THE UNDERLYING ENZYMATIC AND MOLECULAR BASIS E.G. van Grunsven, W. Oostheim, S. Denis, P. Vreken, H.R. Waterham, R.J.A. Wanders University of Amsterdam, Academic Medical Center, Depts. Pediatrics, Emma Children's Hospital, Clinical Chemistry and Neurology, Amsterdam, The Netherlands Objectives: Following the recent discovery of D-bifunctional protein (D-BP) de¢ciency by ourselves and Suzuki and coworkers we have identi¢ed many additional cases of D-bifunctional protein de¢ciency. We now report the identi¢cation of three subgroups and the resolution of the underlying enzymatic and molecular basis using newly developed enzymatic, immunological and molecular methods. Results: The results can be summarized as follows. First, we performed complementation analysis to classify patients with a defect in peroxisomal b-oxidation of unknown etiology which led to the identi¢cation of ¢ve groups with most patients in the D-bifunctional protein de¢ciency group. Second, subsequent complementation studies revealed the existence of three subgroups within the D-BP de¢ciency group. Third, using a newly developed direct assay for D-BP followed by immunoblot analysis and molecular studies, we have now resolved the underlying basis of the three subgroups: subgroup A represents complete D-BP de¢ciency, whereas in subgroups B and C there is a selective de¢ciency of either the enoyl-CoA hydratase (B) or 3-hydroxyacyl-CoA dehydrogenase (C) component of D-BP. Conclusions: These results resolve many of the mysteries around D-BP de¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PEROXISOMAL D-BIFUNCTIONAL PROTEIN DEFICIENCY: CLINICAL, BIOCHEMICAL, MORPHOLOGICAL AND MOLECULAR STUDIES OF EIGHT PATIENTS H Mandel1, EG van Grunsven2,W Osteim2, L Zach1,F Roels3, M Espeel3,I Kerckaert3,C Knopf1,C Sheiman1, HR Waterham2,RJA Wanders2. 1 Pediatr Metab Unit, Rambam Med Ctr, Haifa,Israel; 3Hum Anat, Embr,Histol,Ghent Univ,Belgium; 2 Lab Genet Metab Dis, AMC, Netherlands. In the past few years we investigated eight patients whose defect in the peroxisomal b-oxidation pathway was unidenti¢ed. We here report the clinical, biochemical, morpho- logical and molecular ¢ndings of these patients, in whom D-bifunctional protein (D-BP) de¢ciency was recently diagnosed. The patients originated from 5 Arab kindreds in northern Israel. All presented at birth with facial dysmorphism, severe hypotonia and intractable seizures, and died before 1 year of age. Plasma levels of very-long-chain fatty acids, bile-acid intermediates di- and trihydroxycholestanoic acids and pristanic were elevated, whereas phytanic acid was normal. Liver microscopy in 4 kindreds revealed enlarged (41 micron) peroxisomes (PXS), and a peculiar distribution of PXS in contact with lipid droplets. The diagnosis of D-BP de¢ciency was based on: 1 de¢cient activity of D-BP enzyme in ¢broblasts; 2. absence of D-BP as shown by immuno£uorescence and immunoblot analysis; 3. the identi¢cation of 2 clear-cut mutations: 6 patients were homozygous for a 13-bp deletion that caused a frame shift resulting in truncated protein, and one patient had a 342-bp deletion leading to an inframe deletion of 114 amino acids.

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ZELLWEGER-LIKE SYNDROME DUE TO D-BIFUNCTIONAL PROTEIN DEFICIENCY; A DIAGNOSTIC CHALLENGE WITH MAJOR IMPLICATIONS C. Rizzo, C. Dionisi-Vici, E.G. van Grunsven, G. Sabetta, R.J.A. Wanders. Depts. Clin Biochem & Metabolism, Bambino Gesu© Hospital, Rome, Italy; ½University Hospital, Amsterdam, The Netherlands

Isolated peroxisomal b-oxidation enzymatic defects have been rarely reported. We describe a 1 year-old Libyan boy, born from consanguineous parents, showing severe psychomotor retardation, axial hypotonia, distal hypertonia, seizures, macrocephaly with enlarged fontanel, nistagmus, optic atrophy, neurosensorial deafness without liver dysfunction who died at 14 months. MRI showed abnormal myelination and hypolastic corpus callosum. Metabolite analyses showed elevated plasma VLCFA, increased urinary excretion of odd- and even-chain dicarboxylic-, epoxy-dicarboxylic-acids and pipecolic acid. Subsequent studies in ¢broblasts revealed a selective defect in the peroxisomal b-oxidation machinery as concluded from the ¢nding of de¢cient C26:0 and pristanic acid b-oxidation in the presence of normal albeit enlarged peroxisomes. Complementation studies followed by D-bifunctional protein activity measurements indicated D-bifunctional protein de¢ciency. Our results stress the importance of detailed studies in ¢broblasts in all patients with a Zellweger-like presentation, which is mostly due to a defect in peroxisome biogenesis but may also represent an isolated defect in peroxisomal b-oxidation.

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MRI TRANSMISSION THROUGH INTERNET-2, A NOVEL TOOL FOR REMOTE EVALUATION OF PATIENTS WITH X-LINKED ADRENOLEUKODYSTROPHY (X-ALD) G. Jimenez-Sanchez1,3, B.A. Levine2, F. Eichler3, and H.W. Moser3 Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University, Baltimore MD, USA1. Georgetown University Hosptial, ISIS Center, Washington DC, USA2, Kennedy Krieger Institute, Baltimore MD, USA3 In order to evaluate novel therapeutic intervention of X-ALD we have developed a multicenter therapeutic trial in which patients from over 20 countries will participate. Magnetic resonance images (MRI) will be key to assess disease severity and treatment e¡ect. Initial results from an Internet-1 based network between Baltimore and Minneapolis has shown the important bene¢ts of electronically transmitted MRI in the evaluation of patients with X-ALD (See abstract by F. Eichler, et al.). The next generation of the Internet (NGI), also called Internet-2 is currently being developed. The NGI o¡ers signi¢cant advantages over the commercial Internet which is in place today. Besides providing improved reliability of image transfer, increased security options to guarantee patient con¢dentiality, and the ability to send high resolution images as opposed to compressed images, the NGI provides higher transmission speeds of complex MRI and MR spectroscopy images thereby reducing transmission time to seconds rather than hours. This latter feature makes possible a `realtime' interaction between site of origin and a central reading site, so that image acquisition can be monitored and modi¢ed as the image is being produced. The United States Library of Medicine has funded this project towards the use of Internet-2 in X-ALD, as an initial example of the tremendous impact that this emerging technology will have for a wide range of biomedical applications.

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FIBER RECONSTRUCTION AND NEUROPSYCHOLOGICAL TESTING IN X-LINKED ADRENOLEUKODYSTROPHY Florian Eichler, Susumu Mori, Christiane Cox, Hugo Moser, Gerald Raymond Kennedy Krieger Institute, 707 N. Broadway, Baltimore, MD The newly developed Di¡usion Tensor Imaging (DTI) technique permits 3 dimensional axonal tracking (Mori et al, Ann Neurol 1999) and the novel study of connections between brain functional centers. We report a 17 year old patient with X-ALD who is asymptomatic and has an overall IQ of 117. T-2 weighted T-2 MRI images were normal except for a small lesion in the genu of the corpus callosum. However, application of the DTI technique showed strikingly abnormal anisotropy in the tracks extending from the corpus callosum to the frontal cortex. More detailed neuropsychological tests revealed statistically signi¢cant de¢cits in semantic and phonemic £uency (-1.16 and -2.08 SD respectively) and verbal encoding strategies (-3.00SD). Performance on these tasks requires integrity of the connections to and from Broca's area and the dorsolateral prefrontal cortex, corresponding to the tracts implicated by DTI. Detection of these abnormalities has alerted us to the need for careful followup to determine whether he may become a candidate for bone marrow transplant. In addition to this clinical relevance, the DTI technique permits greater understanding of brain structurefunction relationships. Supported by Stipendium Metabolicum (Milupa GmbH & Co)

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SUCCESFUL BONE MARROW TRANSPLANTATION IN A CASE OF PROGRESSIVE JUVENILE ALD L. De Meirleir1, J. De Schepper2, H. Schots3, H.Katzira4, B. Desprechins5, C. Demanet3,S. Seneca6, K. Hofmans1, W. Lissens6. Dep. of Paediatric Neurology1, Pediatrics2, Hematology3, Radiology5, Medical Genetics6 AZ-VUB Brussels, Belgium and Inst. Fur Humangenetik4, Hamburg, Germany.

A 10 year old boy presented with recent onset learning di¤culties. Neuropsychological testing indicated a decline of IQ from 120 to 80. Neurological examination showed poor concentration and hyperactivity, perseveration and desinhibition, suggesting a frontal lesion. On MRI of the brain there was severe demyelination in fronto-parietal regions with Gadolinium enhancement, anterior part of capsula interna, and anterior thalamic nuclei. VLCFA were elevated and an ACTH test showed surrenal insu¤ciency. A mutation in the ALD gene was identi¢ed 1801del AG in exon 5 . The mutation was also found in his mother and grandmother who developed clinically an AMN. HLA typing of family revealed that the father was compatible in HLA class I and II. Further slow regression of his IQ was seen over the next 6 months. In July 1998 he underwent a bone marrow transplantation. Follow-up over the next 18months demonstrated stabilisation in his neuropsychological pro¢le but with a persistent attention de¢cit. He is currently under treatment with lovastatin 20mg bid. A recent MRI did not reveal any further changes. Although frontal demyelination in ALD patients tends to have a more chronic course the severe decline within the ¢rst six months and the Gadolinium enhancement indicated progressive disease but which nevertheless stabilised completely following bone marrow transplantation.

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X-LINKED ADRENOLEUKODYSTROPHY (X-ALD): ELECTRONICALLY TRANSMITTED MAGNETIC RESONANCE IMAGES (MRI) AID EVALUATION OF THERAPY F. Eichler, MD1, D.J. Loes, MD2, E.R. Melhem, MD3, B.A. Levine, MS4, H.W. Moser, MD1, G.V. Raymond, MD1 1) Kennedy Krieger Inst., Baltimore, MD, 2) Fairview Southdale Hospital, Radiology Dept., Edina, MN, 3) Johns Hopkins Univ., Radiology Dept., Baltimore, MD, 4) Georgetown Univ. Hospital, ISIS Center, Washington, DC MRI studies are of key importance to the assessment of disease severity and evaluation of therapy in X-ALD. Loes et al (Am J Neuroradiol 15:761, 1994) have developed a 34 points scale for scoring brain MRI abnormalities in X-ALD. However, the utility of this scoring system has been hampered by decentralized assessment, unfamiliarity with the system and resultant interrater variability. In order to overcome this limitation a network is being designed that allows for the transfer of MR images from any institution to 2 locations, one in Minneapolis, MN (Dr. Loes) the other in Baltimore, MD (Drs. Melhem and Eichler). This network utilizes the Internet and a Digital Imaging standard called DICOM and is being used by the Imaging Science and Information Systems Center (ISIS) at Georgetown University in Washington, DC. MRI's of 37 X-ALD patients were scored independently in Minneapolis and Baltimore. The interrater correlation coe¤cient was 98%. A world-wide internet-1 transmission network is being developed. The data presented suggest that this will be a valuable aid for the evaluation of disease severity and of the e¡ects of therapeutic interventions in X-ALD and may have application in other disease states. Supported by Stipendium Metabolicum (Milupa GmbH & Co)

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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EXTENDED FAMILY SCREENING FOR X-LINKED ADRENOLEUKODYSTROPHY (XALD) Lena Bezman, Ann B. Moser, Hugo W. Moser Kennedy Krieger Institute, Baltimore, MD, USA X-ALD can be diagnosed accurately and non-invasively by assays of very long chain fatty acids (VLCFA) in plasma, combined, when appropriate, with mutation analysis and immuno£uorescence assays. Mass newborn screening is not yet technically feasible and there are ethical concerns about its use for disorders in which there is no consistently e¡ective therapy. Our studies indicate that 56% of X-ALD probands have new mutations. Screening of extended families thus has the potential for identifying a high proportion of patients. We have conducted such a screening program in 617 kindreds with X-ALD. Pedigree analysis identi¢ed 12,787 at-risk members. The rationale for screening was presented to the probands or their immediate family, and they contacted members of the extended family. 4,169 (33%) of the known at-risk members were screened with the plasma VLCFA assay, combined with 238 immuno£uorescence and 170 mutation analyses. Family screens identi¢ed 594 hemizygotes, 250 of whom were asymptomatic at the time of the test, and 1,270 heterozygotes. We recommend that extended family screens be o¡ered whenever a new patient with X-ALD is identi¢ed, since this procedure permits identi¢cation of hemizygotes at a stage where therapy has the greatest chance of success, and identi¢cation of heterozygotes increases the potential for disease prevention.

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PHARMACOLOGICAL THERAPY OF X-ALD: THYROID-HORMONE INDUCTION OF THE ADRENOLEUKODYSTROPHY-RELATED GENE M. Bugaut1, S. Fourcade1, S. Savary1, D. Gauthe¨ 1, F. Cadepond2, C. Gondcaille1, M.R. Lecca1, J. Berger3. 1 Laboratory of Molecular and Cellular Biology, University of Burgundy, France; 2Inserm U488, Kremlin-Biceªtre, France; 3Brain Research Institute, University of Vienna, Vienna. X-linked adrenoleukodystrophy (X-ALD) is a severe genetic disorder due to mutations in the ALD gene encoding a peroxisomal ABC transporter. Overexpression of the highly homologous gene ALDR in X-ALD ¢broblasts can restore the biochemical defect (accumulation of very long chain fatty acids). That suggests that pharmacological stimulation of ALDR gene expression could complement the defective ALD gene. The promoter of the rat ALDR gene was cloned and revealed a putative response element to thyroid hormone (TRE), well conserved in mouse and human. Rats were treated with thyroid hormone (T4), and induction of the ALDR gene was observed in Northern blotting. Functional analysis of the TRE using gel shift with nuclear extracts or in vitro translated proteins showed that the TRE speci¢cally bound thyroid hormone receptor T3Rb and cis-9 retinoic acid receptor RXRa. In presence of 50 nM T3, cotransfected COS cells with T3Rb and a construct containing the TRE upstream from the luciferase gene raised the reporter gene expression by a 7-fold factor. Thyroid hormone treatment of X-ALD ¢broblasts will show whether the defective b-oxidation of very long chain fatty acids can be restored. The present results lead the way for a hormonal treatment of XALD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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BOTH CONSTITUTIVE AND FENOFIBRATE-INDUCIBLE MOUSE ALDR mRNA EXPRESSION IS REGULATED BY PEROXISOME PROLIFERATOR- ACTIVATED RECEPTOR ALPHA J. Berger1, T. Korosec1, I. Weinhofer1, M. Bugaut2, S. Forss-Petter1, A. Netik1 1 Brain Research Institute, University of Vienna, Austria; 2Laboratoire de Biologie Mole¨culaire et Cellulaire, Universite¨ de Bourgogne, Dijon, France

Mutations in the peroxisomal adrenoleukodystrophy protein (ALDP) lead to the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). As it was demonstrated that the ALDPdefect can be overcome by overexpression of the highly homologous ALDR, a novel therapeutic strategy seems to be possible based on stimulation of the gene expression of the ALDR gene. In order to identify cis-acting regulatory elements we have isolated and sequenced 3.5 kb of 5¨ £anking DNA of the mouse ALDR gene. We identi¢ed several candidate regulatory elements, including peroxisome proliferator-responsive elements (PPREs). Northern blot analysis of liver from feno¢brate-treated wild type and PPARa-de¢cient mice showed that both constitutive and induced expression of ALDR mRNA is PPARa-dependent. Electrophoretic mobility shift assay showed that a putative PPRE located 3.2 kb upstream from the transcription start site speci¢cally binds proteins in liver nuclear extracts from feno¢brate-treated mice, which was also con¢rmed by DNase I footprinting, whereas no binding was observed at this site with extracts from control mice. An element of similar sequence 1.7 kb upstream from the transcription start site in the human ALDR promoter also binds to feno¢brate induced proteins in the mouse liver extract suggesting that this element might also have a regulatory function in human cells.

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X-LINKED ADRENOLEUKODYSTROPHY (ALD) IN CHINESE-A MUTATION ANALYSIS Nelson LS Tang, Joannie Hui*, LK Law, KL Cheung*, WL Yeung*, CK Li*, TF Fok* Departments of Chemical Pathology & Pediatrics*, Prince of Wales Hospital, The Chinese University of Hong Kong, Hong Kong. Email: [email protected]

ALD is a disorder caused by mutations in the ALD gene, which encodes a ATP-binding cassette transporter protein located on the peroxisomal membrane. Many mutations have been reported in other ethnic groups and some of them are ethnic group speci¢c mutations. We studied the mutations in ¢ve local Chinese ALD patients with an aim to identify a common Chinese mutation if it exist. Genomic DNA was extracted from peripheral leukocytes. All 10 exons were ampli¢ed into 11 PCR products. The region with paralogous copies (exon 7 to exon 10) were ampli¢ed by ALD speci¢c primers (Boehm CD et al 1999). Sequence variants in the ampli¢ed products were identi¢ed by single stranded conformation polymorphism and then by sequencing. All patients presented with a phenotype of childhood cerebral ALD. Three novel mutations (V251E, R591P & a 20bp deletion in exon1 from nt.1234 to 1253) were found. Both V251E and the 20 bp deletion were located in the membrane domain of ALD. V251 is a highly conserved residue across species and it was found among carriers in the family. So this mutation is predicted to be deleterious. The 20bp deletion leads to a frameshift in the coding sequence and it was a de-novo mutation, which was not inherited from the mother. R591P of exon 7 is located inside the ATP binding cassette, in which R591 is a highly conserved residues across species. Although R591P is a novel mutation, R591Q & R591W have been reported in ALD patients. We also identi¢ed two polymorphisms, A123V and a delC in intron 6. There is no single common mutation in Chinese patients. This ¢nding is in keeping with the observation in Caucasian patients, in which mutations are heterogeneous. One out of three mutation occurred in sporadic case, which indicated the potential for de novo mutations for this Xchromosome gene. We are still investigating for the mutations in the remaining two families. J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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THE FIRST POLYMORPHISM CAUSING AN AMINO ACID EXCHANGE IN THE ALDPGENE. EIGHT NOVEL MUTATIONS AND THREE POLYMORPHISMS IN PATIENTS WITH X-LINKED ADRENOLEUKODYSTROPHY L. DvoÖa¨kova¨ 1, G. Sítorka¨nova¨ 1, M.HÖeb|¨ e©ek1, J.Zeman1, J.Hujova¨1, G.Unterrainer2 and J.Berger2. 1 Institute of IMD, 1st Faculty of Medicine and General Faculty Hospital, Praha, Czech Republic, 2 Brain Research Institute, University of Vienna, Austria X-ALD is characterized by impaired peroxisomal very long chain fatty acids bb-oxidation. The associated gene, located on Xq28, encodes a peroxisomal membrane transporter protein (ALDP). We examined the ALDP mutations in probands from 11 unrelated Czech and Slovak families by direct sequencing of RT/PCR products. One large deletion (229 bp), 3 microdeletions (1bp, 2bp, 4 bp) and 7 point mutations were found, 8 of them being novel. We have identi¢ed 8 heterozygotes out of 22 family members which were screened by PCR/RFLP methods. In one patient we have detected two novel one-base substitutions in exon 1 (c.38 A4?}C and c.649 A4?}G), which cause the amino acid change (N13T and K217E). Transfection studies revealed that only K217E is a deleterious mutation as plasmid carrying N13T was able to restore defective b?}-oxidation in X-ALD ¢broblasts. Thus, N/ T13 represents the ¢rst polymorphism causing an amino acid exchange in the ALDP-gene. As this polymorphism was not observed in 300 X-ALD patients sequenced so far world wide it seams to be very rare or unique. We have found another two novel polymorphisms: c.-59 C4?}T in 5' untranslated region and c.2019 C4?}T (F673F) in exon 10. The incidence of these two polymorphisms at 150 control alleles was 7% and 1%, respectively. Supported by a IGA MH grant NE 5770-3-99) and by the Austrian Science Foundation, project no. P12073MED.

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DIAGNOSIS OF ALD HETEROZYGOTES BY ANTIBODIES AGAINST ADRENOLEUKODYSTROHY PROTEIN (ALDP) Brunhilde Molzer1, Marta Zobel1, Thomas Korosec2 and Johannes Berger2 1 Inst. of Neurology & 2Inst. of Brain Research, Medical Faculty, Univ. Vienna, Vienna, Austria. X-linked adrenoleukodystrophy (X-ALD), a severe genetic disease presents with di¡erent phenotypes even in the same family. Nearly all patients show a defect in the gene encoding the peroxisomal ALDP. In 70% of X-ALD patients no antibody (AB) reaction was detected in peroxisomes e.g. of ¢broblasts, if so, female heterozygotes display a mosaic pattern of normal and defective cells, useful for carrier detection. We investgated 25 X-ALD patients (14 families), with highly elevated very long chain fatty acids (VLCFA), by indirect immun£uoroscence (IF) mainly with the monoclonal AB 1D6 (Mosser et al.1994). In 22 of them ALD-P was not be detectable by AB. Fibroblasts of 24 female relatives of patients (12 families) were investigated for mosaicism. 1). 11 females with elevated and 6 (9 families) with normal VLCFA displayed the typical mosaicism. Thus 6 more heterozygotes could be detected with IF than by VLCFA results alone. 2). The remaining 7 females showed normal VLCFA values and normal IF of ALD-P as well. In all of them (3 families) the normal IF pattern was con¢rmed by lack of the patient(s) speci¢c mutation. 2 are mother and sister of an ALD patient lacking ALD-P. From our IF results a de novo mutation was presumed. Mutational and sequence analyses in the patient¨s ALD gene disclosed indeed a de novo mutation, namely S636I.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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CEREBRAL ADRENOLEUKODYSTROPHY IN A GIRL WITH AN EXTREME SKEWED INACTIVATION OF THE X-CHROMOSOME DUE TO Xq DELETION Eli Hershkovitz1, Zamir Shorer1, Esther Manor2. 1 Pediatric Department and 2Genetic Institute, Soroka Medical Centre, Ben-Gurion University, Beer Sheva, Israel

Adrenoleukodystrophy (ALD), the most common leukodystrophy is a disease linked to the X chromosome. Unlike boys with ALD, who su¡er from the grave consequences of the CNS myelin destruction, the vast majority of carrier girls are asymptomatic. A nine years old girl was refereed to the clinic because of impaired school performance, inattentiveness and poor concentration. Two of her maternal uncles had ALD (juvenile cerebral ALD and AMN). Complete physical and neurologic examination was normal. Brain MRI revealed Severe di¡use demyelination, although visual evoked potential was normal. Formal psychological assessment (WISC-R) found total I.Q of 83 with verbal I.Q 87 and performance I.Q 83. She was easily distracted during the tests. Cortisol response to ACTH test was within the normal range. VLCFA plasma levels were high. Fibroblast analysis of the patient (done by Dr. Ann Moser's lab) found that the amount of C26:0 and the ratio of C26/C22 were higher than normal and consistent with the biochemical defect for X-linked ALD. All cells were CRIM negative with the antibody to the ALD protein. Genetic analysis showed a 46 XX karyotype but a deletion at Xq26-27 region was found . Neither her parents carried the deletion. This de novo deletion in one of her X chromosomes may explain the its unusual highly skewed inactivation leading to a fully expression of the mutated ALD gene carried on the structurally intact X chromosome.

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FEMALE CARRIERS OF ADRENOLEUKODYSTROPHY SHOW SKEWED PATTERNS OF X-INACTIVATION BUT NO FAVORISATION OF THE MUTANT ALLELE Esther M. Maier, Stefan Kammerer, Ania C. Muntau, Adelbert A. Roscher Dr. von Hauner Children's Hospital, Ludwig-Maximilians-University Munich, Germany

X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ALD gene encoding a peroxisomal membrane ABC transporter (ALDP). Highly variable clinical manifestations are known. The majority of female carriers of X-ALD develop neurological abnormalities at higher ages that may be caused by skewed X inactivation. We analyzed X chromosome inactivation in blood of carriers and noncarriers from 30 ALD families as well as of healthy controls, using a PCR-based assay at the human androgen receptor locus (HUMARA). Highly skewed patterns (480:20) were found in 7 of 22 carriers, but not in any of 7 related noncarriers or 35 unrelated controls. Distribution of skewing in X-ALD carriers was signi¢cantly deviant from the Gaussian pattern in controls and could not be explained by other ALD-independent genetic factors or age-related phenomena. Skewing of X inactivation in peripheral blood did not correlate with plasma VLCFA levels. Among the X-ALD carriers with a skewed pattern we found both association with either favoring the mutant or the wildtype allele. No clear favorisation of the mutant ALD allele as the preferentially active one can thus be delineated. The high proportion of skewing of X inactivation of X-ALD carriers might re£ect complex postinactivation selection mechanisms.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

256

Abstracts

511-O

DEFECTIVE PHYTANIC AND PRISTANIC ACIDS METABOLISM IN 70kDa PEROXISOMAL MEMBRANE PROTEIN (PMP70) DEFICIENT MICE RESULTS IN DEFECTIVE NONSHIVERING THERMOGENESIS AND DICARBOXYLIC ACIDURIA G. Jiminez-Sanchez1, I. Silva-Zolezzi1, K.J. Hebron1, S. Mihaelik2, P. Watkins2, A. Moser2, G. Thomas2, P.A. Wood3 and D. Valle1 Howard Hughes Medical Institute and Institute of Genetic Medicine, Johns Hopkins University, Baltimore MD, USA1. Kennedy Krieger Institute, Baltimore MD, USA2, University of Alabama, Birmingham AL, USA3 ATP-binding cassette (ABC) transporters are membrane proteins involved in the transport of a variety of molecules across biological membranes. There are four known human peroxisome half ABC transporters: PMP70, ALDP, ALDP-related (ALDR) and PMP70-related (P70R). Mutations in ALD are responsible for X-ALD and result in accumulation of very long chain fatty acids due to impaired peroxisomal b-oxidation. The function of the other 3 peroxisomal ABC transporters is unknown. To better understand PMP70, we targeted the gene in mice by standard methods. Analysis of PMP70-/- ¢broblasts and liver peroxisome fractions showed a defect in phytanic and pristanic acids oxidation. Moreover, PMP70-/- mice fed with a phytol supplemented diet have a dramatic plasma accumulation of phytanic and pristanic acids. Urinary organic acid analysis in the PMP70-/mice showed a striking medium chain dicarboxylic aciduria (DCA) that increases during the fasted state. In addition, PMP70-/- mice fail to maintain body temperature when exposed to cold stress (48C) suggesting a defect in nonshivering thermogenesis (DNST). These results suggest impaired mitochondrial b-oxidation. PMP70-/- mice also have an increased expression of peroxisomal acyl CoA oxidase and mitochondrial MCAD. We propose that PMP70 is essential for peroxisomal branched chain fatty acid metabolism; lack of PMP70 causes accumulation of these metabolites which inhibit mitochondrial b-oxidation and activate PPARa, leading to DNST and DCA.

512-P

PLASMA ANALYSIS OF DI- AND TRIHYDROXYCHOLESTANOIC ACID (DHCA AND THCA) STEREOISOMERS USING HPLC TANDEM MASS SPECTROMETRY AND ITS APPLICATION IN PEROXISOMAL ALPHA-METHYLACYL-CoA RACEMASE DEFICIENCY S Ferdinandusse, H Overmars, S Denis, HR Waterham, RJA Wanders, P Vreken Academic Medical Center, University of Amsterdam, Dept. of Clinical Chemistry and Div. Emma Children's Hospital, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands It has been demonstrated that the peroxisomal beta-oxidation system is stereospeci¢c, because the ¢rst enzyme, branched-chain acyl-CoA oxidase, can only handle (2S)-substrates. For this reason, another enzyme called alpha-methylacyl-CoA racemase is also involved in the beta-oxidation of branched-chain fatty acids. This enzyme is able to convert (2R)-pristanoyl-CoA, (25R)-DHC-CoA and (25R)-THC-CoA into their (S)-stereoisomers. This conversion is essential for degradation of these substrates, because in case of DHCA and THCA only the (25R)-stereoisomers are produced from cholesterol. We analyzed the C27-bile acid intermediates accumulating in plasma from patients with racemase de¢ciency (n=3), Zellweger syndrome (n=4), and adults with cholestatic liver disease (n=6). In racemase de¢ciency we detected an exclusive accumulation of free and taurine conjugated (25R)-THCA and free (25R)-DHCA, whereas in Zellweger syndrome and cholestatic liver disease the S/R-ratio was between 0.3-0.5 for free THCA, taurine conjugated THCA and free DHCA. Based on these results we describe an easy and reliable method to diagnose alpha-methylacyl-CoA racemase de¢cient patients by plasma analysis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

257

513-P

ANALYSIS OF SERUM PRISTANIC AND PHYTANIC ACID STEREOISOMERS IN ZELLWEGER SYNDROME, REFSUM DISEASE, D-BIFUNCTIONAL PROTEIN DEFICIENCY AND ALPHA-METHYLACYL-CoA RACEMASE DEFICIENCY Vreken P, Rusch H, Ferdinandusse S, van Lint L, Wanders RJA Academic Medical Center, University of Amsterdam, Dept. of Clinical Chemistry and Div. Emma Children's Hospital, F0-224, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands

Naturally occuring phytanic acid is a mixture of (3R,7R,11R)- and (3S,7R,11R)-phytanic acid. After peroxisomal alpha-oxidation, which is not stereoselective, both (2R,6R,10R)- and (2S,6R,10R)pristanic acid are formed, of which only the latter is a substrate for peroxisomal beta-oxidation. GC/ MS analysis of phytanic and pristanic acid stereoisomers as their S-1-phenylethylamine derivatives in serum samples of patients with Refsum's disease (n=7) showed that the ratio 3R/3S-phytanic acid was 7/3, similarly to what was observed in Zellweger syndrome (n=5) and D-BP de¢ciency (n=6), most likely re£ecting the ratio of these isomers in a normal diet. The ratio 2R/2S- pristanic acid ratio was 4/6 in Zellweger patients and 6.5/3.5 in D-BP de¢ciency. In patients with alpha-methylacyl-CoA racemase de¢ciency (n=3) the 2R/2S- pristanic acid ratio was 7.5/3.5. This implicates that in contrast to the exclusive accumulation of the bile acid biosynthesis intermediates 25R-THCA and 25R-DHCA, no exclusive pristanic acid diastereomer accumulates in racemase de¢ciency, probably due to the dietary origin of racemic pristanic and phytanic acid, whereas THCA is produced endogeneously only.

514-O

ALPHA-METHYLACYL-CoA RACEMASE DEFICIENCY:AMACR (MASSION-VERNIORY SYNDROME?) JM Land1, PK Thomas1, M Reilly1, H Manji1, I Hargreaves1, and RJA Wanders2 Clinical Neurology and Biochemistry, National Hospital for Neurology and Neurosurgery Queen Square London1 and Department of Clinical Chemistry and Pediatrics, Academic Medical Center Univ of Amsterdam The Netherlands.2

AMACR is a newly described defect of pristanic acid catabolism. We describe a patient who was well until the age of 28. At that time she developed progressive bilateral painless visual loss, an inability to use her hands and increasing, although mild exercise intolerance. She was registered blind at the age of 32. When seen at the age of 52 examination revealed bony speckled pigmentation of the peripheral retinas and marked optic atrophy. The remainder of the cranial nerve examination was unremarkable. In the limbs she had pes cavus and a ¢ne postural tremor. In the limbs there was wasting of the ¢ne distal muscles of the hands and feet with weakness of movements. Re£exes were preserved but there was a glove and stocking sensory loss to light touch and pin prick in the ¢ngers and toes. Romberg was negative and cognition normal. Investigations revealed normal serum vitamin A and E, very long chain fatty acids but a mildly raised phytanic acid (28 mmol/L RR 0-15) and a grossly raised pristanic acid level (174 mmol/L RR 0-2.0 ). Serum Ubiquinone levels were also reduced at 4.8 mmol/L (RR17105). In cultured ¢broblasts no activity of a-methylacyl-CoA racemase could be detected. This case represents the fourth case described and the ¢rst in which a secondary ubiquinone de¢ciency, presumably due to inhibition of ubiquinone synthesis by pristanic acid, has been noted.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

258

Abstracts

515-O

2-METHYLACYL-CoA RACEMASE DEFICIENCY: DISCOVERY OF A NEW PEROXISOMAL DISORDER ASSOCIATED WITH ADULT-ONSET MOTOR NEUROPATHY S. Ferdinandusse1, S. Denis1, P.T. Clayton3, A. Graham4, J.E. Rees4, J.T.Allen5, B.N. Mc Lean6, A.Y. Brown5, P. Vreken1, H.R. Waterham1, R.J.A. Wanders1 1 University of Amsterdam, Academic Medical Centre, The Netherlands. 3Institute of Child Health, London, UK. 4The Princess Royal Hospital, West Sussex, UK. 5Southmead Hospital, Bristol, UK. 6 Royal Cornwall Hospital, Truro, UK. We present a new peroxisomal disorder due to 2-methylacyl-CoA racemase de¢ciency in three patients, two of whom su¡ered from adult-onset sensory motor neuropathy. In addition, one (male) patient had pigmentary retinopathy suggesting Refsum disease whereas the other (female) patient had upper motor neuron signs in the legs suggesting X-ALD. Full analysis of peroxisomal metabolites in plasma revealed a similar pattern including elevated pristanic acid and di- and trihydroxycholestanoic levels but normal very-long-chain fatty acids pointing to a defect in the peroxisomal b-oxidation of 2methyl fatty acids. Studies in ¢broblasts revealed reduced pristanic acid b-oxidation but normal activities for Branched-chain acyl-CoA oxidase, D-bifunctional enzyme and branched-chain thiolase. These puzzling results prompted us to focus on the enzyme 2-methylacyl-CoA racemase which converts (2R)-pristanoyl-CoA, (2R)-DHCA- and (2R) THCA-CoA into their (2S)-isomers which are the true substrates for b-oxidation. We ¢rst set up a reliable assay for racemase in ¢broblasts and found a full de¢ciency in all patients. We subsequently cloned the human racemase cDNA and identi¢ed clear-cut mutations. Our ¢ndings may have major implications for the diagnosis and subsequent treatment of adult-onset neuropathies of unknown origin.

516-O

UNNATURAL COSUBSTRATE-BASED RESCUE OF ENZYME ACTIVITY IN REFSUM'S DISEASE RESULTING FROM A PAHX MUTATION M.D. Lloyd1, M. Mukherji1, N. Kershaw1, C.H. MacKinnon1, C.J. Scho¢eld1, A.S. Wierzbicki2 Oxford Centre for Molecular Sciences, The Dyson Perrins Laboratory, Oxford, OX1 3QY, United Kingdom1, Department of Chemical Pathology, St. Thomas' Hospital, London, SE1 7EH, United Kingdom2 Phytanoyl-CoA hydroxylase (PAHX) is an iron(II), 2-oxoglutarate (2-OG)-dependent dioxygenase involved in the metabolism of the isoprenoid lipid, phytanic acid. Mutations in PAHX cause Refsum's disease, symptoms of which include irreversible blindness. Highly puri¢ed recombinant PAHX was shown unambiguously to convert 2-OG to succinate and CO2 and to hydroxylate phytanoyl-CoA. The conditions for measurement of PAHX activity were optimised and cofactor requirements clari¢ed. The e¡ects of mutations of Arg-275, the putative binding site for the 5carboxylate of 2-OG, on activity were investigated. The R275W mutant present in some Refsum's disease patients retains 55% activity in vivo when 2-OG is used as cosubstrate. Other 2-oxoacids were assessed for their ability to `rescue' the activity R275W PAHX to 40^60% with respect to the wildtype enzyme with 2-OG; other aliphatic 2-oxoacids were also able to rescue the R275A mutant. Modi¢ed cosubstrate-based rescue of enzyme activity may o¡er a novel method of treatment for inherited metabolic disorders involving enzymes with a requirement for cosubstrates.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

259

517-O

HUMAN PHYTANOYL-CoA HYDROXYLASE: RESOLUTION OF THE GENE STRUCTURE AND THE MOLECULAR BASIS OF REFSUM's DISEASE Gerbert A. Jansen1, Eveline M. Hogenhout2, Sacha Ferdinandusse2, Hans R. Waterham1, Rob Ofman2, Cornelis Jakobs3, Ola H. Skjeldal4 and Ronald J.A. Wanders1,2,+ University of Amsterdam, Academic Medical Centre, Departments of 1Pediatrics (Emma Children's Hospital) and 2Clinical Chemistry, Amsterdam, The Netherlands, 3Free University Hospital, Department of Clinical Chemistry, Amsterdam, The Netherlands, and 4Department of Pediatrics, Institute of Clincal Biochemistry, Rikshospitalet, Oslo, Norway

Refsum's disease (RD) is an inherited neurological syndrome biochemically characterized by the accumulation of phytanic acid in plasma and tissues. Patients with RD are unable to degrade phytanic acid due to a de¢cient activity of phytanoyl-CoA hydroxylase (PhyH), a peroxisomal enzyme catalysing the ¢rst step of phytanic acid a-oxidation. To enable mutation analysis of RD at the genome level, we have elucidated the genomic organization of the PHYH gene. The gene is *21 kb and contains nine exons and eight introns. Mutation analysis of PHYH cDNA from 22 patients with RD revealed 14 di¡erent missense mutations, a 3 bp insertion, and a 1 bp deletion, which were all con¢rmed at the genome level. A 111 bp deletion identi¢ed in the PHYH cDNA of several patients with RD was due to either one of two di¡erent mutations in the same splice acceptor site, which results in skipping of exon 3. Six mutations, including a large in-frame deletion and ¢ve missense mutations, were expressed in the yeast Saccharomyces cerevisiae to study their e¡ect on PhyH activity. The results showed that all these mutations lead to an enzymatically inactive PhyH protein.

518-O

IDENTIFICATION OF A SECOND LOCUS FOR REFSUM'S DISEASE AT CHROMOSOME 6P22-24 Anthony S. Wierzbicki1, John Mitchell2, Michelle Lambert-Hammill1, Margaret Sidey3, Michael D. Feher3, Jacqueline de Belleroche2, F. Brian Gibberd3 1 Department of Chemical Pathology, St. Thomas Hospital, London SE1 7EH, 2Department of Neuromuscular Disease, Charing Cross Hospital, 3Department of Therapeutics, Chelsea & Wetminster Hospital, London SW10 9NH

Refsum's disease is generally assumed to be caused by mutations in the peroxisomal enzyme phytanoyl-CoA hydroxylase. We have previously shown that 55% of patients with Refsum's disease in the U.K. do not show linkage to the phytanoyl-CoA hydroxylase locus on chromosome 10p13 in a study of 8 families comprising 48 living individuals, though they are phenotypically identical to patients with Refsum's disease linked to chromosome 10. In the 4 families not linked to chromosome 10 comprising 27 members with 9 a¡ected individuals, further linkage analysis was conducted using a panel of 5 markers on chromosome 6p between D6S262 and D6S290 Linkage analysis indicated that a homogeneous cohort was present. A maximal LOD score of 1.72 was obtained in the region D6S314 to D6S308 corresponding to 6p22-24. Candidate genes in this region for Refsum's disease include the Peroxin 7 (Pex 7): PTS-2 transporter protein, mutations in which are associated with rhizomelic chondrodysplasia. This data suggests that the classical Refsum's disease phenotype, in common with other peroxisomal `diseases', is actually caused by a genetically heterogeneous group of conditions.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

260

Abstracts

519-P

EPIDEMIOLOGY OF PRIMARY HYPEROXALURIA TYPE 1 IN THE NETHERLANDS: THE DUTCH COHORT OF OXALURIA (DUCO) C.S. van Woerden, J.W. Grootho¡, F.A.Wijburg, R.J.A. Wanders, Academic Medical Centre, Emma Children's Hospital, Amsterdam, The Netherlands Objectives: Descriptive epidemiologic survey on primary hyperoxaluria type 1 (PH1) in The Netherlands. Background: PH1 is an inborn error of glyoxylate metabolism due to AGT de¢ciency. Methods: Questionnaire to Dutch nephrologists and pediatric nephrologists. Results: 49 Patients with PH were identi¢ed. All patients had symptoms of the urinary tract. 22% Was diagnosed at adult age; 44% of these patients had end stage renal disease (ESRD) at time of diagnosis vs. 22% in the pediatric group. Time interval between age at ¢rst symptoms and age at diagnosis was much longer in the adult group (median 5.0, range 0-29.0 years) than in the pediatric group (median 0.25 range 0-10.7 years). Symptoms were present before the age of 18 in 59%, but time period between age at initial symptoms and age at decline of renal function varied widely in this group. In 3 out of 7 neonatal PH1 patients, renal function remained within normal range (median (þ SD) follow-up 12.5 (þ 3.5) years). Signi¢cant decrease of hyperoxaluria was seen in 24 out of 39 patients treated with pyridoxine; 7 of these 24 patients developed nevertheless ESRD. No correlation was found between AGT activity and outcome. Conclusion: The diagnosis of PH1 is easily missed in adult patients. The age of ¢rst presentation is not correlated with long term outcome. Prognosis of PH1 in neonates can be favourable. Prognosis is correlated with pyridoxine responsiveness.

520-P

CHARACTERISATION OF HUMAN GLYOXYLATE REDUCTASE AND THE IMPLICATIONS FOR DIAGNOSIS OF PRIMARY HYPEROXALURIA TYPE 2 David Cregeen and Gill Rumsby University College London, Department of Molecular Pathology Primary hyperoxaluria type 2 (PH2) is a rare autosomal recessive disorder characterised by hyperoxaluria and usually the presence of L-glycerate in the urine. The underlying defect is a reduction in cytosolic glyoxylate reductase (GR) and hydroxypyruvate reductase (HPR) activities. We have previously described a human cDNA encoding a protein with both GR and HPR activities (GRHPR) and are now further characterising the gene product and its role in glyoxylate metabolism. His tagged human GRHPR puri¢ed from a bacterial expression system was used to raise polyclonal antibodies in rabbits. These antibodies detect a protein of 40kDa in human liver which was absent in liver samples from patients (n=6) with PH2. GRHPR crosslinked with BS3 forms a dimer and small amounts of high molecular weight complexes. The puri¢ed recombinant protein when applied to a chromatofocusing column eluted in 2 peaks with pI between 6 and 5 and a further small peak with a pI55. These peaks may therefore represent monomeric and multimeric forms of GRHPR. In contrast, HPR activity in partially puri¢ed liver showed an additional, non-retained peak of pI47.2. This fraction did not, however, have GR activity. We propose that the latter may be partly or wholly due to LDH which is not retained by the column. Further work is in progress to determine the substrate kinetics of recombinant GRHPR.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

261

521-P

PRIMARY HYPEROXALURIA TYPE II (PH2): CLINICAL, BIOCHEMICAL AND MOLECULAR FINDINGS Fiona Bamforth*, Manjula Gowrishankar*, Gill Rumsby**, David Cregreen**. *University of Alberta, Edmonton, Alberta, Canada;**University College Hospital, London, U.K.

Primary hyperoxaluria II (PH2) is a rare autosomal condition characterised by excess oxalate synthesis and deposition of oxalate in the kidney leading to nephrocalcinosis and impaired renal function. The condition is less common and less severe than primary hyperoxaluria I (PH1), alanine:glyoxalate aminotransferase (AGT) de¢ciency, and may be either missed or misdiagnosed as PH1. The de¢cient enzyme is glyoxalate reductase (GRHPR) which catalyses the reduction of glyoxalate to glycolate and hydroxypyruvate to D-glycerate. In PH2, excess oxalate and L-glycerate are produced. The presence of L-glycerate in urine is unique to PH2 and is not found in normal individuals or PH1. The patient presented at age three with recurrent urinary tract infection. Calculi present in the renal parenchyma were initially attributed to chronic infection. Urinary oxalate excretion was increased (482 mmol/24 hours, ref 100-450). Serum creatinine and plasma oxalate were normal (51.0 mmol/L, ref 0.4-3.0). A small amount of L-glycerate was detected in urine. PH2 was con¢rmed by enzyme analysis in liver (Dr. G. Rumsby). Glyoxalate reductase activity was 16 nmol/ min/mg protein (ref 49-213) and hydroxypyruvate reductase activity was 143 nmol/min/mg protein (ref 322-1002). AGT activity was normal. Western blot analysis (Dr. D. Cregreen) demonstrated normal AGT protein but undetectable GRHPR protein. DNA analysis showed the patient to be heterozygous for a previously described single nucleotide (G) deletion at codon 35 in exon 2, inherited from her father, causing a frame shift resulting in a premature stop codon at codon 45. The maternal mutation has not yet been determined.

522-P

MUTATION ANALYSIS IN KOREAN PATIENTS WITH MENKES DISEASE Sihoun Hahn, Kyounam Cho, Jongsoo Kim, Ookjoon Yoo* Department of Pediatrics, Ajou University School of Medicine, Suwon, Korea, Biomedical Center, Korean Advanced Institute of Science and Technology, Taejon, Korea*

Mekes disease is an X-linked recessive disorder of the copper metabolism and a¡ected males su¡er a systemic copper de¢ciency due to malabsorption and defective distribution of dietary copper. Clinical presentations include severe neurologic problems, connective tissue defects and hypothermia which can be ascribed to the reduced activities of copper dependent enzymes. It is caused by a defect in the MNK (ATP7A) gene which encodes a transmembrane copper-transporting P-type ATPase. A variety of mutations were reported, however, only a few mutations in Asian patients were reported. Six Korean patients with Menkes disease diagnosed by their clinical ¢ndings and laboratory ¢ndings such as low serum copper and ceruloplasmin level were studied. By RT-PCR and direct sequencing analysis, we identi¢ed three novel mutations and one known mutation which were con¢rmed at the genomic DNA level later on. Arg645Ter in exon 8, a known mutation was identi¢ed in one patient. Glu646Ter in exon 8, a novel mutation was identi¢ed in one patient and was transmitted from his carrier mother. Gly1118Asp in exon 17 and Gly1255Arg in exon 19 were also novel mutations not previously reported. All the mutations found in Korean patients were clustered in the biologically important region of ATP7A gene. Although the number of cases in this study is limited, genetic heterogeneity appears the rule in Korean patients with Menkes disease as in Caucasian patients.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

262

Abstracts

523-O

SUCCESSFUL EARLY COPPER HISTIDINE THERAPY IN A PATIENT WITH MENKES DISEASE D.Mochizuki, H.Kodama, M.Kobayashi, H.Murata, M.Gondo, N.Nakamoto, M.Miyao, Y.Yanagawa Department of Pediatrics, Teikyo University School of Medicine We report a two and half years-old boy (case TN) with Menkes disease (MD) who has been treated successfully with subcutaneous administration of copper histidine since the age of 22 days. Because this patient has an elder brother with classical MD (case KN), TN was diagnosed as su¡ering from MD prenatally at the 30th gestational week on the basis of a high copper levels in the amniocytes. Expected cesarean section was performed at the 36th gestational week with parents' agreements. Since TN showed a poor response against the oral administration of copper sulfate, subcutaneous copper histidine therapy was initiated at the age of 22 days. Case KN: This classical MD case was diagnosed at the age of 8 months. He had a typical clinical appearance of MD. Radiographic examination revealed brain atrophy with subdural hematoma, typical bone changes, multiple diverticula of the bladder, and tortuous arteries. Genetic analysis has been so far demonstrated no obvious mutation of the ATP7A gene and promoter region. Case TN: Early copper histidine therapy has brought TN almost normal development so far. However, radiographic examination has reveals a small diverticulum of the bladder, spurs on the metaphyses of long bones, and subperiosteal calci¢cations of knee joints. Conclusion: Early copper histidine therapy in this patient appears to be quite e¡ective in neurological development. However, the therapy seems to be less e¡ective in preventing connective tissue disorders.

524-P

POSSIBLE MARKERS FOR CONNECTIVE TISSUE ABNORMALITIES IN PATIENTS WITH MENKES DISEASE M.Kobayashi, H.Kodama, D.Mochizuki, K.Shiga, M.Gondo, N.Nakamoto, M.Miyao, Y.Yanagawa Department of Pediatrics, Teikyo University School of Medicine

Objective: Menkes disease (MD) is a genetic disorder of copper metabolism characterized by copper de¢ciency, resulting in connective tissue abnormalities by reduced activity of lysyl oxidase. To ¢nd suitable clinical markers of connective tissue changes, we investigated some markers for bone turnover in six patients. Materials & Methods: The serum and urine were collected before and during the treatment with copper-histidine injections in six patients with MD. Pyridinoline cross-linked telopeptide domain of type I collagen(ICTP) and carboxytermainal propeptide of type I procollagen (PICP) levels in the serum and telopeptide (NTX) and deoxypyridinoline (D-Pyr) levels in the urine were examined. Thirty one healthy children were enrolled as normal controls. Results & Discussion: The serum ICTP and PICP levels were normal in the patients with MD. Urinary NTX levels were also normal in all patients. However, urinary D-Pyr levels were signi¢cantly lower in MD patients than in normal controls (p50.05). The low levels were observed in the urine not only before the treatment, but also during the treatment. These results suggest that the urinary D-Pyr level may be a useful clinical marker for evaluating of the connective tissue abnormalities in the patients with MD. Our results also support that fact that the connective tissue abnormalities in the patients is not improved by the treatment.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

263

525-P

MUTATION SCANNING OF THE MENKES GENE ATP7A USING THE PROTEIN TRUNCATION TEST MÖller L.B. The John F. Kennedy Institute, Department of Inherited Metabolic Disease and Molecular Genetics, DK-2600 Glostrup, Denmark

Menkes disease is an X-linked recessive disturbance of copper metabolism, caused by a mutation in the ATP7A gene encoding a P-type ATPase. The ATP7A gene contains 23 exons covering a coding sequence of 4503 bp. Mutation analysis of this gene is hampered by the large size of the gene, and the high rate of new mutations. However all mutations, except one out of 58 identi¢ed so far, in the ¢rst 1869 bp of the coding region (exon 2-7), cause premature translation termination. To take advantage of this we have applied a protein truncation test method to screen RNA corresponding to exon 2-7 obtained from Menkes patients. Using in vitro transcription followed by translation from ampli¢ed RNA mutations leading to premature termination codons can easily bee detected due to the shortened protein product. We present the evaluation of this method by analyzing RNA from patients with known mutations, and we have applied the method for the detection of mutations in patients diagnosed as having Menkes disease.

526-P

GERMLINE MOSAICISM IN X-LINKED MENKES DISEASE 1 Poulsen L., 1MÖller L.B., 2TÏmer Z., 1Horn N. 1 The John F. Kennedy Institute, Department of Inherited metabolic Disease and Molecular Genetics, DK-2600 Glostrup, Denmark 2Department of Medical Genetics, The Panum Institute University of Copenhagen, Denmark.

X-linked Menkes disease is a recessive lethal disorder of copper metabolism characterized by progressive neurodegeneration and connective-tissue disturbances. The disease is caused by mutations in the ATP7A gene, which contains 23 exons. We have identiti¢ed a pedigree where the disorder is caused by a deletion of exon 11-23. The mutation was ¢rst identi¢ed in the proband, an hardly a¡ected boy. We have identi¢ed the haplotypes of family members by using Southern blot, marker for the androgen receptor and polymor¢c markers in the ATP7A gene. The deletion was present in the DNA from the mother of the proband, but absent in a sister and a brother of her although both were carrying the at-risk haplotype.The deletion was also absent in the mother¨ s somatic cells (lymphocytes), therefore she probably has a germline mosaicism. The results indicate the presence of germline mosaicism in the mother although its not known whether the mosaicism is restricted to the germinal cells (pure germline mosaicism) or also present in other tissue (somatic/germline mosaicism). This is the ¢rst observation of germline mosaicism in Menkes disease.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

264

Abstracts

527-P

TWO CASES OF MILD WILSON DISEASE Y. Yamaguchi, N. Shimizu, K. Yamaguchi, K. Takahashi*, K. Shibuya*, T. Aoki Second Department of Pediatrics & Department of Pathology*. Toho University School of Medicine, Ohashi Hospital, 2-17-6 Ohashi, Meguro-Ku, Tokyo 153-8515, Japan Wilson disease (WD) is an inherited disease of copper metabolism. In our country, the incidence of this disorder is 1/35000. Most of the patients with WD already have mild liver injuries during teenage period, and liver injuries are easily detected by abdominal ultrasound, CT and MRI. Here we have shown two rare cases of WD who have very mild liver damage at the age of 15 and 31. Case 1: 15-year-old boy who was diagnosed at the age of 9. After oral D-penicillamine had been administered for 4 years, he stopped taking medicine for 2 years. The urinary basal copper excretion was between 45^90 mcg/day. The abdominal images such as ultrasound, CT and MRI revealed normal. Although liver biopsy showed very mild ¢brosis, copper concentration of the liver was 441 mcg/g wet tissue. The analysis of ATP-7B gene revealed compound heterozygotes of 2662delG/ N1270S. Case 2: 31-year-old man who was diagnosed with WD at the age of 25. Oral D-penicillamine have been administered for 6 years. At the period of admission, the abdominal images revealed only fatty change. His serum ceruloplasmin was 12 mg/dl, and urinary basal copper excretion was between 50^95 mcg/day. The liver biopsy revealed moderate fatty change due to alcohol and very mild ¢brosis. The copper concentration of his liver was 191 mcg/g wet tissue. The analysis of ATP-7B is being performed. In both cases, their home doctors wondered to make the correct diagnosis and referred them to our clinic. We expected that they already had moderate liver damage or mild cirrhosis of the liver on the abdominal imaging because of their long duration of non-treatment period. We ¢nally diagnosed measuring liver copper concentration. Although gene analysis of WD is widely performed, it is still important and useful to make the correct diagnosis measuring copper concentration of the liver in such cases.

528-P

ASSESSMENT OF FREE OXYGEN RADICALS IN WILSON'S DISEASE V. Kupcíova¨,1 P. Blazí|¨ cek,3 D. Syrova¨,3 L. Procha¨zkova¨,1L. Turecky¨,2 3rd Department of Medicine1, Institute of Medical Chemistry and Biochemistry2, Medical School of Comenius University, Bratislava, Hospital of Ministry of Defense 3, Bratislava Wilson's disease (WD) is an autosomal recessive inborn error of metabolism causing a continual increase in tissue copper concentrations that become toxic to the liver, brain, kidney, eye, skeletal system and several other tissues and organs. Copper transporting P-type ATPase ATP7B plays a pivotal role in the biliary excretion of copper. The liver is unique among these in being both the site of the aetiologic biochemical abnormality and the organ, that is always a¡ected by copper toxicosis. There are three types of disease presentation, an asymptomatic, a hepatic and a neurologic one. Excess copper in tissues leads to the production of free radicals and to DNA-cleavage. Probably one of the source of damage is through the production of free radicals. To assess the contribution of free radicals reactions catalyzed by nonceruloplasmin copper to the development of complication in this disease, we have vestigated oxidative stress parameters (superoxide dismutase, glutathioneperoxidase, malondialdehyde, lipofuscin, total antioxidant status) in a group of patients with WD with di¡erent degree of hepatic dysfunction and complications and in healthy control group. In patients with WD, series of diagnostic steps were undertaken which include: urine and blood copper, blood ceruloplasmin and slit lamp examination for Kayser-Fleischer rings, liver biopsy, neurological examination including EEG, MR images of brain and molecular genetic analysis. We conclude, that free radical reactions may play a role in pathogenesis of hepatic dysfunction and in clinical complications of WD.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

265

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LIVING RELATED LIVER TRANSPLANTATION FOR WILSON DISEASE PRESENTING ACUTE HEPATIC FAILURE FROM HETEROZYGOTE GENETIC CARRIER Emi Ogawa(1), Atsushi Ogawa(1), Masaki Kanazawa(1), Shigenori Yamamoto(1), Takao Suzuki(2), Seiji Hori(2), Susumu Kobayasi(2), Takenori Ochiai(2), Kohichi Tanaka(3), Yoichi Kohno(1) (1)Department of Pediatrics, Chiba University School of Medicine, Japan. (2)Department of Surgery (II), Chiba University School of Medicine, Japan. (3)Department of Transplantation and Immunology, Faculty of Medicine, Kyoto University, Japan. Liver transplantation is a life-saving procedure in patients with Wilson disease presenting fulminant hepatic failure or end-stage hepatic insu¤ciency, and is associated with excellent long-term survival. But this procedure is di¤cult to perform in Japan because of insu¤cient organ donation. Instead, living related liver transplantation has been applied to the treatment of Wilson disease with increasing frequency in Japan. In that case, a parent who is heterozygous state for the disease would be as a donor. But there is a possibility of a potential risk of recurrence associated with the use of donors who are heterozygous for the Wilson disease genetic defect. We will show a clinical presentation of a 13year-old male patient with acute hepatic failure as a ¢rst manifestation of Wilson disease, who was undergoing a living related liver transplantation. And we show a serum copper and ceruloplasmin level and urinary copper excretion before and after liver transplantation. We conclude that living related liver transplantation is e¡ective treatment of Wilson disease, and grafts chosen from heterozygote carriers of the condition do not appear to confer any risk of recurrence in the recipients.

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DNA HAPLOTYPE ANALYSIS FOR THE DIAGNOSIS OF WILSON DISEASE IN SIBLINGS A. Yu«ce, M. Demirtas° , N. Koc°ak, H. O«zen, F. Gu«rakan, M. O«zgu«c° Division of Pediatric Gastroenterology, Department of Medical Biology and TBIç TAK DNA/Cell Bank, Hacettepe University Medical Faculty, Ankara/RRKIç YE Wilson disease is an autosomal recessive disorder of copper metabolism. The diagnosis is usually based on clinical and biochemical ¢ndings. Sometimes these criteria are insu¤cient for the diagnosis of presymptomatic patients. In this study, we performed DAN haplotype analysis using four microsatellite markers spanning the Wilson disease locus in 16 Wilson patients and in their 20 asymptomatic siblings to compare the results of standard tests and molecular diagnosis. Haplotype analysis indicated that seven siblings diagnosed clinically as presymptomatic patients were homozygous for the disease-carrying alleles. Six of the nine siblings diagnosed as normal individuals were actually shown to have normal alleles while the remaining three were heterozygotes. Four siblings with low serum ceruloplasmin levels, who were suspected of being presymptomatic, were found to be heterozygotes with haplotype analysis. Our study showed that the results of clinical diagnostic criteria and haplotype analysis were almost compatible with each other. We suggest that standard tests are still reliable in the diagnosis and screening of Wilson disease. However, haplotype analysis can aid in di¡erentiation between heterozygotes and presymptomatic patients, if standard tests do not provide a de¢nitive diagnosis.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

266

Abstracts

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GENE ANALYSIS AND COPPER METABOLISM OF PATIENTS WITH MENKES DISEASE AND OCCIPITAL HORN SYNDROME H.Kodama, Y.H.Gu, T.Mochizuki, Y. Mori, N, Nakamoto, M. Kobayashi, Y. Yanagawa Department of Pediatrics, Teikyo University School of Medicine Menkes disease (MNKD) and occipital horn syndrome (OHS), a milder form of MNKD, are caused by mutations in a copper transporting ATPase (ATP7A) gene. Such mutations have been reported to show great variety in patients. We report here the mutations identi¢ed in 16 unrelated Japanese patients with classical MNKD and one OHS patient by SSCP and direct sequencing of the exon PCR products. RT-PCR was also examined. The clinical features of the patients and copper levels in cultured ¢broblasts were also investigated. Each MNKD patient had a di¡erent mutation. The mutations consisted of 6 deletions, 2 insertions, 6 nonsense, 2 misssense mutations. In the OHS patient, a splice-site mutation occurs, leading to the skipping of exon 6. RT-PCR of the OHS patient revealed two transcripts, one associated with a fragment of normal size and one with a smaller fragment. These results suggest that the milder phenotype of the OHS patient is due to the presence of barely detectable amounts of normal ATP7A transcript. The serum copper and ceruloplasmin levels were signi¢cantly low in the MNKD patients, while these levels were normal in the OHS patient. These results also suggest that the OHS patient have a partial defect in copper absorption. However, the copper levels were signi¢cantly high not only in the MNKD patients, but also in the OHS patient, suggesting that the copper levels in cultured ¢broblasts do not re£ect the residual activity of ATP7A.

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EARLY TREATMENT OF MOLYBDENUM COFACTOR DEFECT DOES NOT IMPROVE OUTCOME Blakely EM1, Arnold GL1, Seashore GR2, 1 . University of Rochester School of Medicine and Dentistry, Rochester, NY, 2. Yale University School of Medicine, New Haven, CT, USA. A newborn was diagnosed with Molybdenum Cofactor De¢ciency after presenting with seizures at four hours of life. Early diagnosis was aided by family history and presence of s-sulfocysteine on amino acid analysis. EEG showed markedly abnormal background and burst suppression spikes. Treatment was initiated on day 2 of life using betaine (250 mg/kg/day) and a methionine/cysteine restricted diet (prescribed to deliver 125 kcal/kg, 1.9 g pro/kg and 58 mg methionine plus cyteine). However, the child fed poorly, resulting in inadequate calorie and protein intake and poor growth. A feeding gastrostomy and Nissen fundoplication were performed at 10 months of age, after which growth rate improved but without signi¢cant catch-up growth. At age 18 months he smiles responsively but has few other developmental milestones. He remains on betaine (900 mg/day) and diet (prescribed to provide 1.2g/kg/day protein, 10 mg/kg cysteine and 25-35 mg/kg/day methionine), but continues to have vomiting that interferes with nutrition. Early diagnosis and treatment with betaine and cysteine/methionine restriction did not appear to ameliorate the devastating e¡ects of this disorder on the central nervous system. The failure of early treatment in this child may re£ect pre-existing intrauterine damage, suggesting that post-natal treatment may be ine¡ective in severe cases.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

267

DISADVANTAGES OF GENE ANALYSIS FOR PRIMARY DIAGNOSIS R.J. Pollitt Neonatal Screening Laboratory, Children's Hospital, She¤eld, S10 2TH, UK

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For most inborn errors of metabolism, heterogeneity limits the usefulness of gene analysis in primary diagnosis. However, two fairly common diseases, cystic ¢brosis (CF) and medium-chain acyl-CoA dehydrogenase (MCAD) de¢ciency, are moderately homogeneous in populations derived from Northern Europe. Many neonatal screening programmes for CF use gene analysis as the second stage in a two or three-tier screen. Analysis for the 985A4G mutation is increasingly being used as a ¢rst line test where there is clinical suspicion of MCAD de¢ciency and will probably also have a place when screening by tandem mass spectrometry is introduced. In both these diseases, homozygosity for the commonest mutation is associated with severe disease and is thus a useful predictive ¢nding. However, the practice of routinely genotyping parents to con¢rm such homozygosity is regrettable as it may intrude delicate matters of paternity/maternity into what should be entirely a diagnostic process. Greater problems arise when heterozygosity is found. Una¡ected carriers or borderline compound heterozygotes will outnumber clearly a¡ected cases and it is di¤cult to exclude potentially signi¢cant disease. Even in CF, where much detailed information is available, genotype^phenotype correlation in individual cases is poor. Albeit the disorder in question is absent, the parents of a heterozygous child will need genetic counselling for the risk in future pregnancies, again an inappropriate outcome of a diagnostic investigation.

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MOLECULAR CHARACTERIZATION OF INBORN ERRORS OF METABOLISM Aida I Al Aqeel, MD, DCH, FRCPLond FRCPEdin FACMG, Consultant Paediatrician Inborn Errors of Metabolism, Genetics and Endocrinology, Riyadh Armed Forces Hospital, Riyadh, Saudi Arabia

Inherited genetic disorders are quite common in our Saudi community because of the high degree of consanguineous marriages. Biopterin dependent phenylketonuria is a special form of PKU which is quite common in Saudi Arabia. We studied 25 patients with this disorder and we found various mutations leading to variable phenotypes. Vitamin D dependent Rickets type II is another unique disorder. The c-DNAs from two families were sequenced, it was found that each cell line contained a nucleotide substitution resulting in a stop condon, causing truncated receptor protein of 148 and 291 amino acids which accounts for the severe resistant phenotype. Wilson's disease is another common disease, DNA haplotypes of dinucleotide repeat polymorphism (CA) repeat in the Wilson disease gene have helped us in con¢rming the diagnosis and in ¢nding heterozygote carriers in one family. We found a unique syndrome, with unique dysmorphic feaures, bone and joint abnormalities. We mapped the gene for this disorder to 16q11.2-21. In conclusion our Saudi community is quite unique. Molecular genetic studies of these diseases have helped us in further understanding of these diseases, in their clinical management and in ¢nding a unique molecular genetic defect at the DNA and protein (enzyme) level.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

268

Abstracts

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ANALYSIS OF NUCLEIC ACIDS USING DNA MICROARRAY TECHNOLOGY Ondrova¨ L.1, E¨ervenkova¨ M.1, Síulc V.2, Zicha J.2, Housít|¨ k J.3,HÖeb|¨ e©ek M.1, 4, Kmoch S.1, 4 ; 1 Institute for Inherited Metabolic Disorders; 1st faculty of Medicine; 2Division of Precision Mechanics and Optics, CTU; 3Physiological Institute, ASCR; 4GeneAge Technologies; Prague, The DNA microarray technology appears to be one of the most promising and dynamically evolving approaches ful¢lling all requirements of functional genomics and high throughput DNA diagnostics. The method (which can be seen as a miniaturised version of classic dot-blot analysis) is based on detection and analysis of hybridisation events of tested nucleic acids with an array of single stranded DNA fragments (PCR products, oligonucleotides) immobilised on solid surface. Recent advances in robotics, immobilisation chemistries, sensitivity of DNA labelling, £uorescence detection systems and bioinformatics 1)make technique amenable to miniaturisation and automation, so thousands of various nucleic acid sequences can be analysed in a single experiment and 2) make technology approachable by academic research. We have successfully set up DNA microarray technology platform consisting of preparation and characterisation of cDNA libraries, preparation of aminomodi¢ed DNA probes (PCR products, oligonucleotides), arraying and immobilisation of probes at various densities (up to 4900 probes/cm2; GeneSurfer arrayer, GeneAge Technologies), on poly-Nlysine or epoxysilane modi¢ed microscope slides. The hybridisation of £uorescently labeled DNAs is monitored using laser microarray scanner GeneTac IVL (Genomic Solutions). Current pilot experiments are focused on detection of 19 glucocerebrosidase gene mutations encompassing whole mutational spectrum of Czech Gaucher patients and on expression of 16 ATPase subunits under normal and pathological states in di¡erent tissues and in vitro cellular models (mitochondrial disorders).

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COMPLEX PHENOTYPES AND GENOTYPES AMONG THE TRIGGERABLE MYOPATHIES GD Vladutiu1, D Smail1, MJ Bennett2, 1) Dept. of Pediatrics, SUNY at Bu¡alo, NY and 2) Dept. of Pathology, Univ. of Texas Southwestern Medical Center, Dallas, TX Single-gene disorders have evolved into complex phenotypes with increasing numbers of unexpected genotype/phenotype relationships. The triggerable metabolic myopathies provide a biologically relevant model for studying contributors to complex phenotypes. Symptoms range from mild exercise intolerance in adults to multisystem lethal disease in infants. We hypothesize that it is not uncommon for individuals to have 2 or more co-existing mutations causative for di¡erent myopathies and that the threshold for susceptibility to triggering events will be consequently lowered. 45 patients, including manifesting carriers, were diagnosed with 1 of 3 common triggerable myopathies based on biochemical and molecular evidence: carnitine palm- itoyltransferase (CPT) II de¢ciency (25), McArdle's disease (9), and myoadenylate deaminase (AMPD) de¢ciency (11). Each patient was screened for 5 mutations: S113L and Q413fs (CPT2), R49X and G204S (PYGM), and Q12X (AMPD1). Mutant alleles identi¢ed were: 38/46 (83%) in CPT2, 13/17 (76%) in PYGM, and 15/18 (83%) in AMPD1. 5 seriously a¡ected patients were carriers for a second disorder: 3 with CPT II de¢ciency carried Q12X; of 2 with McArdle's disease, 1 carried Q12X and 1 carried S113L. The ¢nding of the Q12X mutation in 4 of 34 non-AMPD de¢cient patients was approximately one-half the general population frequency. The double carrier for the R49X and S113L mutations was unexpected. An increased prevalence of double carriers for certain disease mutations may indicate a lowered threshhold for the manifestation of symptoms.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

269

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DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) SCREENING FOR MUTATIONS IN mtDNA FROM A FAMILY WITH A CLINICAL PRESENTATION CONSISTENT WITH MERRF SYNDROME K.E. Niezen-Koning1, A. ter Harmsel1, A.E.J. de Jager2, P. Terpstra3, M.H.J. Ruiters3, R.M.W. Hofstra4, W.M. Molenaar5, J.M.F. Trijbels6, G.P.A. Smit7 Dept. Pediatrics, Groningen1; Laboratory1 and Metabolic Diseases7; Dept. Neurology, Groningen2; Medical Biology, Groningen3; Medical Genetics, Groningen4; Pathology & Laboratory Medicine, Groningen5; Laboratory Pediatrics & Neurology, Nijmegen6, The Netherlands

Mitochondrial disorders comprise a heterogeneous group of clinical phenotypes which can be caused by mutations of either mitochondrial (mt) or nuclear DNA, or both. A growing number of pathogenic mtDNA mutations have been published in databases. Clinical presentation in some patients of our investigated family was consistent with MERRF syndrome. Other members of this family had a less pronounced presentation with fatigue, unsteady gait and elevated serum CK level. Muscle biopsy showed myopathy with Ragged Red Fibers and, in one patient, enlarged mitochondria. DGGE and direct-sequencing of the aberrant fragments revealed the variation 8344A4G which was mutated for more than 50% (MERRFphenotype). Apart from this, the variations 4216T4C and 13708G4A, which were mutated for more than 80% (LHON 28 phenotype) and 10398A4G (polymorphism) were found. At this moment the clinical consequence of the LHON 28 mutation in this family is not known. In conclusion, in this family, DGGE has proven to be a useful tool in mutation analysis of mtDNA. E-mail: [email protected]

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MUTATION DETECTION BY TAQMAN-ALLELE SPECIFIC AMPLIFICATION: APPLICATION TO MOLECULAR DIAGNOSIS OF GLYCOGEN STORAGE DISEASE TYPE IA AND MEDIUM-CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY Y. Matsubara*, K. Fujii*, S. Kure*, Y. Suzuki*, P. Rinaldo**, and K. Narisawa*. *Department of Medical Genetics, Tohoku University School of Medicine, Sendai, Japan; **Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester MN, U.S.A. We have devised an allele-speci¢c ampli¢cation method with a TaqMan £uorogenic probe (TaqManASA) for the detection of point mutations (Fujii, et al., Hum. Mutat. 15: 189-196, 2000). Pairwise polymerase chain reaction (PCR) ampli¢cation using two sets of allele-speci¢c primers in the presence of a TaqMan probe, which is doubly labeled with a reporter dye and a quenching dye, was monitored in real-time with a £uorescence detector. Di¡erence in ampli¢cation e¤ciency between the two PCR reactions was determined by ``threshold'' cycles, at which the £uorescence exceeds a baseline, to di¡erentiate mutant and normal alleles. The method measured the e¤ciency of ampli¢cation rather than the presence or absence of end-point PCR products, therefore allowing greater £exibility in designing allele-speci¢c primers and an ample technical margin for allelic discrimination. We successfully applied the TaqMan-ASA method to detect a prevalent g727t mutation in Japanese patients with glycogen storage disease type Ia and a common a985g mutation in Caucasian patients with medium-chain acyl-CoA dehydrogenase de¢ciency. The method does not require post-PCR processing, can be easily automated and may be applicable to the DNA diagnosis of various genetic diseases.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

270

Abstracts

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A RAPID SCREENING METHOD TO DETECT MUTATIONS IN CYP21 Chiyo Inoue, Sumie Yamashita, Shinsuke Ninomiya, Michio Teraoka, Yuji Yokoyama, Yoshiki Seino Dept. of Pediat. Okayama University Medical School 2-5-1 Shikata-cho,Okayama 700,JAPAN OBJECTIVE: To facilitate a rapid and practical molecular diagnosis of 21-hydroxylase (21-OH) de¢ciency, we developed a polymerase chain reaction (PCR) test in which only the gene for 21-OH (CYP21) is ampli¢ed. METHODS: We applied this test to diagnose 23 patients having a salt-wasting type of 21-OHD. The upstream and downstream sequences of CYP21 have been speci¢cally ampli¢ed using a primer containing the 8-bp deletion sequence of exon 3, which is the main di¡erence between CYP21 and its pseudogene, CYP21P. The ampli¢ed PCR products were further subjected to mutation detection by restriction analysis: E1PL by AciI, I2g by PstI, E63a by DraIII, E7VL by ApaLI, E8non by PstI and E8RW by AciI. To detect deletions occuring on exon 3 and gene conversions of exon 3, we desighed primer pairs originally and perfomed £uorescent gene-dosage PCR studies, using Gene Scan Model 373 (Perkin Elmer). Our method is able to elucidate eight common CYP21 mutations using only three primer pairs and six restriction enzymes. RESULTS and CONCLUSIONS: The overall detection ratio of abnormal haplotypes by this method was over 95%. Analysis of the 23 patients revealed 44 mutant alleles. The most common mutation found was I2g (37.0%), followed by a deletion including exon 3 or a gene conversion of exon 3 (34.8%) and E1PL (10.9%). We were sure that this ampli¢cation technique is practical and useful particularly for carrier detection.

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ANALYSIS OF MUTATIONS IN THE HUMAN BUTYRYLCHOLINESTERASE (BCHE) GENE FOLLOWING COMPLICATIONS DURING ANAESTHESIA C. Barta1, P. Enyedi2, A. Devai3, M. Sasvari-Szekely1 and M. Staub1 1 Dept. of Medical Chemistry, Molecular Biology and Pathobiochemistry, 2Dept. of Physiology, Semmelweis University, 3Bethesda Children's Hospital, Budapest, Hungary Objective: The aim of our study was to identify the underlying genetic lesions resulting in decreased activity of the plasma cholinesterase in patients with a history of prolonged neuromuscular block and their families. Methods: DNA was extracted and PCR was performed to amplify all 4 exons of the BCHE gene followed by direct sequencing of the samples. Enzyme activity and dibucaine numbers were also determined. Results: We have identi¢ed a number of mutations in the BCHE gene (including the atypical and K-variant). These would account for the impaired activity of the enzyme and thus explain the prolonged apnoea experienced after general anaesthesia using neuromuscular blockers such as mivacurium and succinylcholine. Conclusions: Genotyping patients and their pedigrees for mutations in the BCHE gene allows precise identi¢cations of plasma cholinesterase variants, some of which are di¤cult to di¡erentiate using the standard in vitro enzymatic test. Analysis of other members in the family may help to prevent the occurrence of unanticipated complications during future anaesthesia. (EC Project: BMH4-CT98-3079, Co-ordinator: H.A. Simmonds)

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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271

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DERIVATIZATION STRATEGIES FOR FATTY ACIDS AND ALCOHOLS IN THE DIAGNOSIS OF INHERITED DISEASES BY ELECTROSPRAY TANDEM MASS SPECTROMETRY D.W. Johnson Women's and Children's Hospital, North Adelaide, South Australia 5006, Australia

The rapid analysis of key metabolites in newborn blood spots for the diagnosis of inherited diseases by electrospray tandem mass spectrometry has been limited to charged analytes (aminoacids, acylcarnitines and conjugated bile acids). Carbohydrates have been analysed only after derivatization by condensation with alkoxylamines, hydrazines, pyrazolones and nitriles or reductive amination. This paper outlines novel derivatization strategies suitable for the many metabolites with carboxylic acid and hydroxyl groups which are poorly ionized by electrospray. Derivatization has been used to couple an electrospray ionizable functional group (e.g. di- and trimethylaminoethyl and dimethylglycine esters), enhance sensitivity, protect labile functional groups and resolve interference. Examples include 1) Quanti¢cation of very long chain fatty acids and branced chain fatty acids for the diagnosis of peroxisomal disorders in 5mL plasma samples and 3mm blood spots (stored for up to 17 yrs). 2) Quanti¢cation of unconjugated C27 and C24 bile acids for subclassi¢cation of peroxisomal disorders and rapid screening for jaundice in plasma and 3mm blood spots. 3) The trace, rapid analysis of other fatty acids, hydroxyacids and dicarboxylic acids.

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ENZYME HYDROLYSIS OF OCTANOYL CARNITINE IN HUMAN PLASMA: METHODOLOGICAL AND PHYSIOLOGICAL SIGNIFICANCE Charles Turner & R. Neil Dalton Department of Paediatrics, Guy's Kings & St Thomas's School of Medicine and Dentistry, King's College London & Guy's & St Thomas's NHS Trust, Guy's Hospital, London UK

Quantitative tandem mass spectrometric (TMS) assay of octanoyl carnitine (OC) is used for acute diagnosis and neonatal screening for MCADD. Surprisingly, during preparation of blood/plasma standards and quality controls, we noted in vitro hydrolysis of OC and release of free carnitine by a heat & organic solvent sensitive mechanism. The rate and speci¢city of this activity in blood and plasma from normal subjects and patients with MCADD was investigated. Analysis used underivatised isotope dilution TMS (MRM mode, intra-assay CV for OC: 14% at 0.05 and 46.5% above 0.45mmol/l). Fresh whole blood was incubated at 25oC with or without addition of OC, acetyl or palmitoyl carnitine. Data in parentheses follow addition of 100mmol/l OC. Incubation Time Normal Subjects MCADD (n=3)

Octanoyl Carnitine (mmol/l) 0h 4h 24h 0.05(90) 1.3

0.05(41) 1.4(50)

0.05(39) 1.4(51)

Free Carnitine (mmol/l) 0h 4h 24h 40(39) 7

38(86) 7(55)

41(90) 7(54)

Plasma or dried blood spots gave similar results. There was no detectable hydrolysis of acetyl or palmitoyl carnitine. Patients with MCADD are at equilibrium (no in vitro fall in endogenous OC) but hydrolyse exogenous OC at the same rate as normal individuals. The activity may enable recycling of signi¢cant amounts of free carnitine in vivo.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

272

Abstracts

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ANALYSIS OF FREE AMINO ACIDS IN HUMAN PLASMA BY CAPILLARY ELECTROPHORESIS: POTENTIAL FOR EMERGENCY DIAGNOSIS OF INBORN ERRORS OF METABOLISM Olivier Boulat1, David G. McLaren2, David D.Y. Chen2 Clinical Chemistry Laboratory, CHUV, CH-1011 Lausanne1, Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z12 Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced £uorescence (LIF) detection (£uorescence was induced with the 488-nm line of a 4-mW argon ion laser, emission was monitored at 520 nm). Twenty-nine AAs (including 2 internal standards) are separated reproducibly in less than 70 minutes. Variations in the migration times of the di¡erent AA derivatives (CBQCA-AAs) were observed between individual CE runs, but this was not found to signi¢cantly a¡ect the general migration pro¢le obtained for the free AAs in plasma. The free AAs present in both normal donor plasma samples and plasma samples of patients with phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied. Despite the fact that quanti¢cation of the CBQCA-AAs was not performed, it can be seen that a clear relationship exists between the signals in the di¡erent aminograms and abnormal plasma concentrations, allowing the pattern of diseases to be identi¢ed. The potential of CE AA analysis of real patient samples in the context of IEM neonatal emergencies is demonstrated.

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THE USE OF ELECTROSPRAY IONIZATION MASS SPECTROSCOPY (ES/MS) TO ASSAY FOR ENZYME DEFICIENCIES CR Scott, S Gerber, X Zhou, F Turecek, M Gelb. Depts. of Chemistry and Pediatrics, Univ. of Washington, Seattle, WA, USA.

We have developed a sensitive approach for measuring enzyme activity in crude cell lysates using ES/ MS as a common instrument platform. The strategy consists of synthesizing substrates consisting of biotin-sarcosine-polyethylene glycol-glycoside. Biotin is linked to sarcosine to provide an a¤nity handle for selective capture and simple puri¢cation by streptavidin-agarose. The PEG diamine linker provides water solubility, contains a basic residue for protonation by electrospray (high sensitivity), allows for conjugate di¡erentiation of coincident mass by insertion or deletion of a PEG unit, and provides a location for introduction of stable isotopes for synthesis of internal standards. Following incubation with cell lysates, the substrate and product are captured by streptavidin-agarose, then separated and quantitated by ES/MS. We have applied the method for assay of enzymes de¢cient in the San¢lippo syndrome (A, B, C, D), Krabbe, Niemann-Pick A, and GM1 gangliosidosis. The assays have excellent reproducibility and are linear with protein and time. Sensitivity is similar to that obtained using radionucleotides, but simpler and safer. The method can measure more than one enzyme reaction simultaneously (multiplexing) and can be developed to identify genetic disorders that have a common phenotype but are caused by di¡erent enzyme de¢ciencies.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Abstracts

273

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EXCELLENT FEASIBILITY OF THE ROUTINE URINE ORGANIC ACID TECHNIQUE WHEN APPLIED TO PLASMA FATTY ACID ANALYSIS Arranz JA, Campos F, Pi·ol F, Sent|¨ s M, Riudor E. Unitat de Malalties Neurometaboliques. Hospital Maternoinfantil Vall d'Hebron. Barcelona. Spain.

Cis-4-decenoate (C10:1n-6) and cis-5-tetradecenoate (C14:1n-9) are diagnostic clues in the diagnosis of medium- and long chain FAO disorders. We have applied the current chromatographic method used in urine organic acid analysis to extracted plasma for FFA analysis. Controls ranging from newborn to adult and MCAD and LCHAD de¢cient patients were evaluated. Excellent resolution permits a clear identi¢cation of C10:1n-6 and C14:1n-9. Increases of both acids must be con¢rmed by MS. Analytical performance compares is similar to other FFA methods: detection limit: 0.2 mM ; CV inter: 12-20%; recovery: 69 (C8)-104% (C18). Normal and pathologic results (mM ) were :

C10:1n-6 C14:1n-9 C14:1n-9/C14:0

Newborns n=7

Rest of populat. n=39

MCAD n=1

LCHAD 1 with MCT

LCHAD 2 without MCT

0.8-8.5 50.2-3.9 50.120

50.2 2.5 50.2 1.8 50.075

71.4; 21.8 50.2 50.017

50.2 1.0 0.33

1.4 ; 0.2 14.1 ; 1.3 0.33 ; 0.35

C14:1n-9/C14:0 ratio has higher semiologic value than absolute results as it is shown in LCHAD patient 2. Values were signi¢catively higher in newborns than in older controls. Since other markers as suberyl- and hexanoylglycine may be absent in newborns and diagnosis may be only supported by FFA analysis, the use of an appropriate age-matched control group is necessary. The technique is reliable for the screening of FAO disorders simplifying routine laboratory work.

RAPID ANALYSIS OF AMINO ACID ISOMERS BY LC-MS/MS Michael Morris and Donald Cooper Micromass UK Ltd, Floats Road, Wythenshawe, Manchester M23 9LZ, UK.

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The use of tandem mass spectrometry for the analysis of acylcarnitines and amino acids from blood samples is well established, and is being used in a number of centres around the world for routine neonatal screening. One of the limitations of mass spectrometry, however, is the inability to di¡erentiate between structural isomers. In the determination of maple syrup urine disease, for example, it is necessary to establish the relative concentrations of the di¡erent isomers of leucine (leucine, isoleucine and alloisoleucine) and to di¡erentiate these from any isobaric interferences (hydroxyproline, propionyl glycine). Analysis of the butylated forms of these compounds shows that there is di¡erential fragmentation between the leucines and some of the interferences, but the leucine iosmer fragmentations are not su¤ciently di¡erent to be rationalised by an untrained operator. We have established a rapid LC separation that allows for the separation and quanti¢cation the leucine isomers, using the same sample that had previously been used for routine screening. Data supporting the use of an LC-MS/MS system as a routine amino acid analyser will also be presented.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

274

Abstracts

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ANALYSIS OF METHYLMALONIC ACID IN URINE WITH SEQUENTIAL INJECTION ANALYSIS (SIA)-FTIR SPECTROSCOPY B Jakobs1, P Markus1, D Swinkels1, R Wevers2, J Trijbels2, B Lendl3 , H Willems1 Department of Clinical Chemistry/5641 and Laboratory of Paediatrics and Neurology2, UMC St Radboud, PO box 9101, 6500 HB Nijmegen, The Netherlands, Institute of Analytical Chemistry3, Vienna University of Technology, Getreidemarkt 9/151, A-1060 Vienna, Austria. The main advantages of FTIR are simple, low cost measurements and reduced analysis time compared to the present analysis performed with GC-MS. Here, we measured methylmalonic acid in urine with SIA-FTIR spectroscopy using anion exchanging polymer beads in a transmission cell according to the method described by [Lendl B and Schindler R, 1999, Vib Spectrosc, 19, 1-10]. Urine from 5 healthy persons was collected, divided into 37 samples, pH adjusted and spiked with methylmalonic acid (10-1000 ÙM). Samples were injected using a SIA £ow system and spectra were collected in the mid-IR region (1600-1000 cm-1) on a spectrum 2000 FT-IR spectrometer with a MCT detector. The best chemometric PLS regression model with full cross validation was obtained in the spectral region 1550-1505 cm-1 using 9 principal components and ¢rst derivative calculation. Standard error of cross validation (SECV) in the IR-predicted methylmalonic acid concentration was 89 ÙM with an R2-value of 99.1 %. Repetitive injections of the same sample were in good agreement These ¢rst results show an increase in sensitivity by a factor of 10 compared to our previous study in which we used a horizontal ZnSe Attenuated Total Re£ectance (ATR) accessory. This study shows that SIA-FTIR spectroscopy can be a valuable quantitative tool for the analysis of methylmalonic acid in urine.

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HPLC METHOD FOR THE DETERMINATION OF GUANIDINO COMPOUDS IN BIOLOGICAL LIQUIDS AND DRIED BLOOD SPOT Carducci Cl., Birarelli M., Leuzzi V., Carducci Ca., Antonozzi I. University ``La Sapienza'' Rome, Italy Guanidinoacetic acid (GAA) is synthesized in human liver, renal cortex and pancreas by arginine:glycine amidinotransferase (AGA T) and is converted into creatine by guanidinoacetate methyltransferase (GAMT). The defect of GAMT, recently described, results in creatine de¢ciency and increase of GAA concentration in biological £uids. For this reason GAA can be considered the bioch e mical marker of the disease. We developed a new automated HPLC method for the assay of guanidino compounds in urine, plasma, CSF and dried blood spot (d.b.s.) using RP-HPLC, precolumn derivatization with benzoin and £uorescence detection. We set up two d i¡erent separation procedures. The ¢rst, suitable for large scale application, achieves a rapid determination (12 min) of creatine plus creatinine and GAA using a TSKgel Super ODS 2 mm column. The second procedure provides a more detailed metabolic information, with the separation of 11 guanidino compounds in 52 min using a Kromasil C18 5 mm column. The within-run and between-run C.V.s were all less than 8 %. The linearity of dose-response curve w as tested and the regression coe¤cients were all greater than 0.998. The lower detection limits were all 5 1 pmol. The validity of the method was tested analysing biological £uids of a patient a¡ected by GAMT de¢ciency. W e found a marked increase of GAA values (nearly 10, 20 and 100-fold higher than mean control values resp. in plasma, urine and CSF). We propose this method, especially with the use of d.b.s., as a useful and not expensive tool for the diagnosis of GAMT de ¢ciency.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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SIMULTANEOUS ANALYSIS OF PLASMA HYDROXY AND FREE FATTY ACIDS IN FATTY ACID b-OXIDATION DISORDERS USING GC/NPD Hye-Ran Yoon1* , Man Jeong Paik1, 2, Kyoung Rae Kim2, Seiji Yamaguchi 3, Piero Rinaldo4 1 Special Biochemistry, Seoul Medical Science Institute, Seoul 140-230, Korea. 2 College of Pharmacy, SungKyunKwan University, Suwon, Korea. 3Dept. Pediatrics S himane Medical University, Shimane, Japan 4Laboratory Medicine & Pathology, Mayo Clinic, Rochester, MN. Analysis of free fatty acid (FFA) is useful for the diagnosis of mitochondrial fatty acid oxidation disorder (FAOD). We have developed a method for the simultaneous analysis of C8-C18 free fatty acids and C6-C12 3-hydroxy fatty acids as cyanomethyl derivatives by GC/NPD. Two hundreds microliter of plasma was extracted with ethyl acetate following deproteinization, and then evaporated and derivatized to cyanomethylation for carboxyl group and to trimethylsilylation (TMS) for hydroxyl group, consecutively. The optimum reaction time determined was 30 min and 20 min at 608C for cyanomethylation and TMS, respectively. In twenty plasma specimens from control subjects and ¢ve patients with known FAOD, FFA and hydroxy FFA were successfully analyzed. The reproducibility of the method was within 10 % (RSD). These results suggest a potential application of this method as a cost-e¡ective alternative to provide biochemical screening of patients with a clinical suspicion of a FA OD where GC/MS and MS/MS methods are not readily available.

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DETERMINATION OF D-GALACTOSE IN HUMAN PLASMA Udo Wendel, Kamalanathan Loganathan, Hans-Werner Hammen, Annette Bodner-Leidecker, and Peter Schadewaldt Kinderklinik and Deutsches Diabetes Forschungsinstitut, Heinrich-Heine-UniversitÌt DÏsseldorf, DÏsseldorf, Germany

A stable isotope dilution method allowing sensitive determination of D-galactose in human plasma was established. Plasma was spiked with D-[13C/sup;C]galactose, D-glucose removed by treatment with D-glucose oxidase and the sample puri¢ed by ion exchange chromatography. For gas chromatographic-mass spectrometric analysis, the aldononitril pentaacetate derivatives were prepared. Using positive chemical ionization, monitoring of the [MH-60]+ ion intensities at m/z 328, m/ z 329, and m/z 334 allowed the assessment of 1-12C-, 1-13C/sup;C-, and U-13C/sup;C6-labelled Dgalactose, respectively. D-galactose concentration was quantitated on the basis of the 13C/sup;Clabelled internal standard. The method was linear (range examined: 0.1 ^ 5 mmol x L-1) and of good repeatability in the low and high concentration range (CV's within- and between runs 5 15%). The limit of quantitation of plasma D-galactose was 5 0.02 mmol x L-1. Measurements in plasma of postabsorptive subjects yielded D-galactose concentrations (means þ SD) of 0.12 þ 0.03 (n = 16), 0.11 þ 0.04 (n = 15), 1.44 þ 0.54 (n = 10), and 0.17 þ 0.07 (n = 5) mmol x L-1 in healthy adults, diabetic patients, patients with classical galactosemia, and obligate heterozygous parents thereof, respectively. These data were considerably lower (3-18-fold) than the values obtained by a conventional enzymatic assay. The procedure was also applied successfully in a stable isotope turnover study aiming at the evaluation of endogenous D-galactose formation. The present ¢ndings now ¢rmly establish that Dgalactose from endogenous sources is feasible in human plasma and show that erroneously high concentrations may be indicated by enzymatic methods.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MEASUREMENT OF BLOOD GALACTOSE ON FILTER PAPER FOLLOWING DERIVATIZATION WITH 8-AMINO-2-NAPHTHALENSULFONIC ACID BY REVERSED PHASE HPLC IN GALACTOSEMIA Seon-Pyo Hong1*, Hye-Ran Yoon2, Kyoung O. Lee2, Mi-Kyoung Kim1, Yoon S. Shin3, Dong H. Lee4 College of Pharmacy, Kyunghee University, Korea1; Special Biochemistry, Seoul Medical Science Institute, Seoul, Korea2; Children's Hospital University of Munich, Germany3; Dept. of Pediatrics, Sooncheonhyang University Hospital4 De¢ciency of galactose-1-phosphate uridyl transferase (GALT) is a polymorphic enzyme and Duarte (D) is the most common enzyme variant. A new reversed-phase HPLC method has been developed for the measurement of galactose in blood (50L) on Guthrie ¢lter paper using 8-amino-2naphthalenesulfonic acid (8,2-ANS) as derivatization reagent for the diagnosis of Galactosemia. Galactose was extracted from blood spot on ¢lter paper and derivatized with 8,2-ANS to produce Schi¡ bases, and reduced under sodium cyanoborohydride. The linear range was between 80 nmol and 20 pmol and the limit of detection (S/N=3) of this method was 0.9 ng/mL. The mean recovery of galactose was 104.29% with a S.D. of 3.62% with correlation coe¤ciency of 0.9999. Control range of blood galactose in Korean newborn was below 6 mg/dL for male and female without any gender di¡erence (n=5). We applied 11 anonymous blood spots in which diagnosis has already been made by enzyme assay as one of the GALT or epimerase de¢ciencies. All the patients' blood spots showed abnormal elevation of galactose. These results suggest that the developed method could be a practical tool for the diagnosis of Galactosemia with high sensitivity.

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THE DIAGNOSIS OF THE KYPHOSCOLIOTIC TYPE OF THE EHLERS-DANLOS SYNDROME (EDS VI) Beat Steinmann*, Cecilia Giunta*, and Peter R. Huber³ *Div. Metabolic & Molecular Pediatrics, CH-8032 Zurich, ³University Hospital, CH-4031, Basel The kyphoscoliotic type of the Ehlers-Danlos syndrome (EDS VI; MIM 225400) is an autosomal recessive disease characterised by muscular hypotonia, progressive severe kyphoscoliosis since birth, skin hyperelasticity, joint hypermobility, tissue fragility, Marfanoid habitus, microcornea and fragility of the eye ball, and considerable osteopenia. Life expectancy is markedly reduced by sponaneous arterial ruptures, pulmonary insu¤ciency and cor pulmonale. It is due to de¢ciency of collagen lysyl hydroxylase (EC 1.14.11.4), encoded by PLOD1 on chromosome 1, which normally catalyses the ¢rst step in collagen cross-linking. Here we present an easy diagnostic test which con¢rms and extends previous work (Steinmann et al. Am J Hum Genet 57:1505-1508, 1995). Total urinary lysyl (LP) and hydroxylysyl pyridinolines (HP) (trivalent collagen cross-link products consisting of 2 lysyl and 1 hydroxylysyl residues and 3 hydroxylysyl residues, respecively) were measured by HPLC. The ratio of LP/HP in 17 patients with documented enzyme de¢ciency was markedly increased (5.97+0.99, range 4.3-8.1; controls 0.20 + 0.05, range 0.1-0.4), age-independent and without overlap with controls. Intraindividual variation of the ratio of 14 uriay portions collected over a time span of 7 weeks was small (5.64+0.31, range 5.21-6.22) and reained una¡ected by ascorbic acid, the cofactor of lysyl hydroxylase. Thus, LP/HP is a relible, quick and inexpensive diagnostic test in contrast to enzyme measurements, collagen and mutation analyses to rule in or out the disease which is still rarely considered, especially in infancy.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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277

METABOLIC ALTERATIONS IN CLEIDOCRANIAL DYSPLASIA Eva Morava, Judit Karteszi, Mehes Karoly University School of Pe¨cs, Dep. Medical Genetics, Pe¨cs, Hungary

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Cleidocranial dysplasia (CCD) is a skeletal dysplasia inherited in an autosomal dominant fashion, characterized by hypoplastic clavicules, patent fontanelles, short stature, teeth anomalies and variable skeletal changes. Di¡erent mutations of the CBFA1 gene have been detected in patients with CCD. The gene is a member of the runt family of transcription factors, playing a major role in osteoblast di¡erentiation and bone development. In mouse experiments the heterozygous mutation of the Cbfa1 gene locus leads to a phenotype similar to human and the homozygous mutation causes a complete lack of bone development. In spite of the essential role of the CBFA1 gene in membranous and endochondral bone formation, no systematic metabolic studies have been reported in CCD patients so far. We investigated a mother-daughter pair with typical features of CCD presenting with low serum phosphorus and calcium levels, hypophosphatasia, and excreting a signi¢cant amount of phosphoetanolamin in urine. Renal function was normal, no calcium-, carbohydrate-, or aminoaciduria was detected. Urine electrolytes and pH were within normal limits. No endocrine abnormality was found. A DNA sequence analysis in our patients showed no mutation in the hypophosphatasia gene. Surprisingly metabolic alterations have gradually normalized in both mother and daughter. However, the alkaline phosphatase derived from bone is still low (10%) compared to other fractions. At present only mild, transient phosphoetanolaminuria can be detected. Besides the typical skeletal changes both mother and daughter had a normal psychomotor and mental development. We suggest that the observed metabolic alterations are due to the CCD gene mutation a¡ecting normal early bone-development and turnover. A recent animal study with the Cbfa1 gene deletion carrier mouse detected decreased serum alkaline phosphatase. This observation of hypophosphatasia in our patient may re£ect a gene dosage e¡ect.

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A NOVEL MUTATION (296 DEL G) OF THE SOX9 GENE IN A PATIENT WITH CAMPOMELIC SYNDROME AND SEX REVERSAL Shinsuke Ninomiya, Yuji Yokoyama, Michio Teraoka, Rintaro Mori, Chiyo Inoue, Sumie Yamashita, Hiroshi Tamai, Masahisa Funato and Yoshiki Seino Dept. of Pediat. Okayama University Medical School 2-5-1 Shikata-Cho, Okayama 700, JAPAN

The campomelic syndrome (CMPS) is a rare and usually lethal congenital chondrodysplasia and 75% of XY patients with CMPS show sex reversal (SR). The human SOX9 gene is responsible for CMPS and SR. In the present study we revealed a novel single base (guanine) deletion upstream of the SOX9 high mobility group (HMG) domain. Patient: The patient has typical CMPS ¢ndings and SR showing a normal 46, XY karyotpe and is still alive at 5 years and 3 months of age although in need of mechanical ventilation support. Mutation analysis: PCR ampli¢cation of the SOX9 gene containing the open reading frame and exon/intron boundaries and sequencing have been carried out using 6 overlapping primer pairs. Results: The patient's DNA sequence analysis of the SOX9 gene revealed a 1 bp guanine deletion, positioned in one allele at nt 296, resulting in a stop codon at 30 bp downstream by frameshift in the HMG domain. The patient's parents did not have mutations. Conclusions: The mutation caused a removal of nearly 80% of the SOX9 protein, lacking most parts of the HMG domain and the complete C-terminal transactivation domain. Most cases with CMPS reported previously died within the neonatal period. Our ¢ndings that the patient has survived for 5 years and 3 months despite a severe impaired SOX9 protein, do not support a linear relationship between the type of mutation and severity of the clinical outcome.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

278

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ANALYSIS OF BILE BILIRUBIN PIGMENT IN DISORDERS OF BILIRUBIN METABOLISM IN EARLY INFANCY: WHEN IS EARLY TOO EARLY? W.S. Lee, P.J. McKiernan, S.V. Beath, M.A. Preece*, D.J. Clarke**, D.A. Kelly, B. Burchell** Liver and Biochemistry* Units, Birmingham Children's Hospital, Birmingham; and Department of Molecular and Cellular Pathology, Ninewells Hospital and Medical School, Dundee Aim: To determine whether early bile bilirubin (BB) analysis in infants with prolonged unconjugated hyperbilirubinaemia (UCH) provides useful diagnostic information. Material and methods: Retrospective review of infants with prolonged UCH referred to the Liver Unit, Birmingham Children's Hospital for the diagnosis of Crigler^Najjar syndrome (CNS), between 1992 to 1999. Bile was collected during upper gastrointestinal endoscopy, frozen and shielded from light and its composition determined by high-performance liquid chromatography. Initial diagnoses were made based on BB pigment analysis. Final diagnoses were made after reviewing the clinical course, response to phenobarbitone, repeat BB composition and genetic studies. Results: During the study period, nine infants 53 months of age (range 10 to 58 days, median 25 days), with UCH underwent BB analysis. Median peak serum bilirubin (SB) was 396mmol/L (range 300^650 mmol/L). Based on BB analyses, two cases of CNS type 1, six CNS type 2, and one Gilbert's syndrome were diagnosed. Four cases, whose initial diagnoses were CNS type 2, had resolution of jaundice and normalization of SB after discontinuing phenobarbitone. One of these cases had a negative genetic study of the mutation of CNS type 2. The diagnoses were revised and thought to be normal. One of the initial CNS type 1 responded to phenobarbitone with an 80% reduction of SB. A repeat BB analysis at seven months of age showed a predominant monoglucuronide, consistent with CNS type 2. Phototherapy was discontinued at one year of age. Overall in ¢ve of the nine cases, the initial diagnosis was reviewed. Conclusion: In suspected disorder of UCH, early bile pigment analysis, before three months of age, did not provide useful diagnostic information.

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CRIGLER-NAJJAR SYNDROME TYPE 1 IN TUNISIA: CONFIRMATION OF A FOUNDER EFFECT Armelle Rivie©re*, Jeanne Francoual*, Jacqueline Chalas*, Liliane Capel*, Anne Myara***, Pascale Trioche**, Philippe Labrune**. *Laboratoire de Biochimie, and **Service de Pe¨diatrie, Hoªpital Antoine Be¨cle©re, BP 405, 92141 Clamart Cedex, and ***Laboratoire de Biochimie, Hoªpital Saint Joseph, 75014 Paris, France. Crigler-Najjar syndrome type I (CN1) is a rare disorder of bilirubin metabolism due to a de¢ciency of bilirubin glucuronosyltransferase hepatic activity (BGT). It is transmitted as an autosomal recessive trait and is a heterogeneous genetic condition due to several di¡erent mutations in the coding sequence of the gene encoding BGT. Previous studies had suggested the existence of founder e¡ects in countries such as Tunisia and Sardinia. Here, we con¢rm the founder e¡ect in three CN1 patients of Tunisian origin. Patients and methods: CN1 syndrome was suspected in three jaundiced neonates born to related parents. Jaundice did not respond to phenobarbital and the diagnosis of CN1 was con¢rmed by enzymatic assay on liver tissue at three months, showing that BGT activity was nil. Genomic DNA was extracted from peripheral leukocytes and the 5 exons (including the exon-intron junctions) were PCR-ampli¢ed and directly sequenced according to previously described techniques. Results: The three patients were homozygous for a A-4G substitution at nucleotide 1070 (exon 3, codon 357)) leading to the replacement of a glutamine by an arginine in the protein (mutation Q357R). No other nucleotide variation was detected within the coding sequence and both parents of each infant were heterozygous for the mutation. Conclusion: The Q357R mutation had previously been identi¢ed, at the homozygous state in two Tunisian children. Thus, this report con¢rms that CN1 is, in Tunisia, due to this mutation since, to date, 10 mutant alleles out of 10 have been found to bear this particular mutation.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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`BURGUNDY' RED URINE IN A YOUNG CHILD WITH ACUTE ABDOMINAL PAIN F. Eyskens, M. Docx, G. Janssens AZ-Middelheim, Campus Queen Paola Children's Hospital, Antwerp, Belgium

A 3.5 year old Turkish boy, the youngest child of a family of 8 children, presented with acute intermittent severe abdominal pain, associated with vomiting, pain in the extremities, muscle weakness and mental disturbances. These symptoms started after an anaesthetic procedure. The family history was negative and there was no consanguinity. Physical examination on admission revealed an agitated boy with low weight for age, a hepatosplenomegaly and icteric sclerae. Laboratory investigations: Hb 6.5mg/dl (normal:10.2^14.9); ASAT 67U/L (normal 5^35); total bilirubin 4.4mg/dl (normal 0.2^1.2) with an elevated indirect fraction. High serum ferritin, LDH and low haptoglobin indicated hemolysis. Hemoglobin electrophoresis was normal. The fact that a urine sample turned a dark reddish color on standing let us decide to perform quantitative porphyrin analysis in urine: uroporphyrins: 117.85 mg/g creat (normal 4.40^21.26), coproporphyrins: 487.31 mg/ g creat (normal 22.53^145); protoporphyrin level in erythrocytes was normal. Porphobilinogen deaminase activity in erythrocytes was reduced to 50% of normal. The diagnosis of acute intermittent porphyria was made and treatment started with high carbohydrate feeding, intravenous glucose, hematin and pain relief medication. His further evolution is good with minor crises during episodes of infection. To our knowledge we present the youngest patient with acute intermittent porphyria (de¢cient PBG deaminase) with manifest symptoms and signs at a very early age precipitated by an anaesthetic procedure. Mutation analysis is planned.

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CONGENITAL ERYTHROPOEITIC PORPHYRIA (CEP): DECREASED ESSENTIAL FATTY ACIDS (EFA) IN ERYTHROCYTES C. Depetris-Boldini, G. Civallero, M.P. Videla, S. Antonozzi, R. De Kremer-Dodelson CEMECO, Paediatric Department, School of Medicine, Children's Hospital of Co¨rdoba, National University of Co¨rdoba, Agentina

CEP is an autosomal recessive disorder resulting from a decreased activity in URO III cosynthase, which leads to the accumulation and hyperexcretion of predominantly type I porphyrins. Clinically, it is expressed by skin photosensitivity and haemolytic crises. Haemolysis pathogenesis in CEP is unknown and remains a contentious issue. It is known that animals fed on EFA de¢cient diet have abnormal fatty acid (FA) composition in erythrocytes, particularly EFA's linoleic and arachidonic acid de¢ciencies. These changes are accompanied by some haemolysis and when cellular EFA levels are increased, a reduced haemolysis rate is noted. These observations led us to look into FA of the erythrocytes of a patient with CEP, which were sampled at di¡erent ages. Eleven normal controls were used. FA were analysed by CG as their methyl ester derivatives and expressed as mg/ml. From 35 FA characterised, 18 were polyunsaturated FA and, only EFA had a decrease of more than 2SD in erythrocytes from the patient (see table). A decreased EFA level observed in our patient might be one of the causes for haemolysis. Therefore we hypothesise that type I porphyrins could interfere with EFA metabolism and alter erythrocyte's membrane architecture. This ¢nding allows us to suggest that a diet rich in EFA could be bene¢cial for patients with CEP. EFA Linoleic (18:2) Linolenic (18:3) Arachidonic (20:4) (20:3) (22:4)

Normal Controls (n=11) 136 + 27 2+1 201 + 43 28 + 5 45 + 13

Patient (range) 67^77 0^0 32^63 8^9 7^16

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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HEREDITARY COPROPORPHYRIA IN A CZECH FAMILY CAUSED BY DELETION OF 390GLY OF COPROPORPHYRINOGEN OXIDASE Malonova¨ E., Rosipal R., Zeman J., Deybach J.C.*, Lamoril J.*, Marta¨sek P. Department of Pediatrics, 1st Faculty of Medicine, Charles University, Prague, Czech Republic and *Centre Franc°ais des Porphyries, Colombes, France Hereditary coproporphyria (HC) is classi¢ed in the group of acute hepatic porphyrias because attacks are acute and the principal site of accumulation of coproporphyrinogen (copro-gen) is the liver. HC is an autosomal dominant disorder that results from partial de¢ciency of copro-gen III oxidase (CPO), a mitochochondrial enzyme that catalyzes the sixth step in heme biosynthesis. Systemic analysis of the CPO gene by DGGE and sequencing in four HC Czech families led to the description of four novel mutations. In a large family, a deletion of 3 bp (GGA) at nucleotide 1168 in exon 5 was found, causing a 390Gly deletion without altering the coding frame. 390Gly occurs within the amino acid sequence ^ RRGRYVEFNL- that is absolutely conserved in all known CPO sequences, from tobacco and E. coli to mammals. The expression of normal and del390Gly mutated proteins showed a residual (51%) activity as compared to normal one. The investigation of the family of proband in four generations led to the ¢nding of the mutation in most family members. Analysis of the CPO gene helps to eliminate, by preventive means, the precipitating factors of life threatening acute porphyric attacks. Studies of the naturally occurring mutated CPO will help to understand structure/function relationships of CPO. (Supported by Granting Agency of Czech Republic GACR 302/99/0651)

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A BOY WITH HEPATIC PORPHYRIA HAVING BEEN FOLLOWED UP AS RECCURENT FEVER OF UNKNOWN ORIGIN SINCE 4 YEARS OF AGE K. Murayama, A. Ohtake, K. Baba, K. Kobayashi, H. Niimi, N. Sasaki Department of Pediatrics, Saitama Medical School, Moroyama, Saitama 350-0495, Japan

The porphyrias are inherited and acquired disorders in which activities of enzymes of the heme biosynthetic pathway are de¢cient. They are classi¢ed as either hepatic or erythropoietic, depending on the principal site of expression of the speci¢c enzymatic defect. We report one family diagnosed as a certain hepatic porphyria. The propositus was 13-year-old boy. Since 4 years of age, he has been su¡ering from recurrent fever, with increasing serum CRP level and granulocytosis. He has also had abdominal pain, headache, £ush cheek, and mild jaundice, gradually. His father and one of his uncle also su¡ered from similar symptoms. Laboratory data showed that serum CRP level rose from 1 mg/ dl to 12 mg/dl during episodes. Speci¢c laboratory ¢ndings were as follows: increased excretion of coproporphyrin, harderoporphyrin, and protoporphyrin in feces; increased excretion of coproporphyrin in urine; and decreased activity of erythrocyte porphobilinogen deaminase. He was given a diagnosis of hepatic porphyria (acute intermittent porphyria, hereditary coproporphyria, or variegate porphyria). Supportive therapy was started, and the frequency of his episodes has been decreased. Determination of gene abnormality for speci¢c diagnosis is now undertaken.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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281

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VARIATION IN THE FLAVIN CONTAINING MONOOXYGENASE 3 (FMO3) GENE AS A RISK FACTOR FOR ESSENTIAL HYPERTENSION Lambert DM1, Hamet P2, Gaudet D3, Akerman BR1, Yang H1, Platt R1, Ryan S1, Genest J2, Treacy EP1,4. 1 Montreal Children's Hospital Research Institute, McGill University, Montreal, 2Hospital Centre of the University of Montreal (CHUM), 3Hospital Complex of Sagamie, Quebec, Canada and 4Temple Street Children's Hospital, Dublin, Ireland.

Homozygosity for rare mutations of the FMO3 gene cause the inborn error of metabolism Trimethylaminuria, and may predispose individuals to essential hypertension (HBP) and abnormal catecholamine metabolism (Treacy et al, 1998). Furthermore characterization of three FMO3 polymorphisms (E158K (c.488 G-A), E308G (c.923 A-G) and V257M (c.769 G-A)) in vivo and in vitro suggests that these polymorphisms in particular combinations (haplotypes) have functional pharmacogenetic e¡ects. We report a case control study of the association between variant FMO3 haplotypes and early onset HBP (5 55 years). Haplotypes were determined and their distribution noted between (1) HBP propositi (n = 60), (2) HBP relatives (n = 229), (3) non-HBP control relatives (n = 145) and (4) unrelated non-HBP French Canadian controls (n = 133). Of 8 possible haplotypes, 6 were observed. There was a signi¢cant di¡erence noted between the distribution of haplotypes between groups (1) and (4) (p = 0.026). Individuals with one variant (and one wild type) haplotype had a relative risk of HBP of 1.22 (95% CI: 1.0 ^ 1.5), individuals with two variant haplotypes: a RR of 1.97 (95% CI: 1.0 ^ 3.9). These data suggest that while rare null FMO3 mutations represent an insigni¢cant population burden of HBP, common polymorphisms of FMO3 with adverse environmental exposures may contribute signi¢cantly to HBP prevalence.

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CHARACTERIZATION AND ENZYME EXPRESSION ANALYSIS OF FMO3 MUTATIONS IN TRIMETHYLAMINURIA USING PROTON NMR SPECTROSCOPY Iles, R.A., Murphy, H.C., Dolphin, C.T., Janmohamed, A., Holmes, H.C., Michelakakis, H., Shephard, E.A., Chalmers, R.A., Phillips, I.R. Depts. of Diabetes and Metabolic Medicine and Biochemistry, Queen Mary and West¢eld College; Dept. of Child Health, St. George's Medical School; and Dept. of Biochemistry, University College; University of London, UK.

Proton NMR spectroscopy (1H-NMR) has been used to detect a variety of metabolic disorders including trimethylaminuria (TMAuria). However, as far as we are aware these cases were not de¢ned genetically as £avin monooxygenase 3 (FMO3) de¢ciency. We describe here the use of 1HNMR both to measure urinary TMA excretion and carry out expression analysis of FMO3 activity. The advantage of NMR for the enzyme assay is that all reactants can be monitored simultaneously. In one case, after detecting raised TMA, subsequent genetic analysis identi¢ed a homozygote for a novel mutation in FMO3, a T?C change in exon 3, which, by substituting a methionine with a threonine at residue 82, abolishes catalytic activity of FMO3, as determined by 1H-NMR. However, despite the presence of a loss-of-function mutation of FMO3, the patient was able to convert some dietary-derived TMA into TMA oxide. The combination of 1H-NMR with gene sequencing and expression technology provides a powerful means of determining genotype - phenotype relationships in trimethylaminuria.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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MEASUREMENT OF TRIMETHYLAMINE AND TRIMETHYLAMINE N-OXIDE RATIOS AS A MARKER OF VARIANT FMO3 MEDIATED METABOLISM Treacy EP1,2, Lambert DM,1 Gaudet D3, Akerman BR1, Choiniere L4, Hamet P5, Mamer OA4. 1 Montreal Childrens Hospital, 4Mass Spectrometry Unit, McGill University, Montreal, 2The Children's Hospital, Temple St, Dublin, Ireland, 3Hospital Complex of Sagamie, Quebec, 5 Hospital Center of the University of Montreal (CHUM), Montreal, Canada. Trimethylaminuria (TMAuria) is an inborn error of metabolism resulting in de¢cient oxidation of TMA to TMAOx. We have previously shown that 9 mutations of the FMO3 gene encoding (FMO3) (EC 1.14.13.8), a phase 1 drug metabolising enzyme cause TMAuria. Our in vitro and in vivo data also suggest that three prevalent FMO3 polymorphisms: (E158K, E308G and V257M) in particular haplotypes function as pharmacogenetic polymorphisms. Using a FABMS stable isotope dilution assay with 15N TMA we measured TMA, TMAOx and TMAOx/TMA ratios in urines from individuals with severe TMAuria (n=7) and 20 adult controls. The a¡ected individuals showed a marked reversal of TMAOx/TMA ratios: (mean 0.91+0.58) vs controls (mean 86+59), (p50.0001). We then measured this ratio in 56 FMO3 genotyped individuals from Quebec. We noted di¡erences between the mean ratios of individuals homozygous for the wild type haplotype (group 1, n=13) with three other subgroups with variant haplotypes. When the values were expressed as %TMA oxidized, a threshold cut o¡ of 5 96.5% di¡erentiated the individuals in group 1 from the groups with variant genotypes (p=0.03). We thus propose that polymorphic variants of the FMO3 gene result in variable TMA oxidation, applicable to other FMO3 substrates.

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CARRIER ASSESSMENT IN FAMILIES WITH LOWE OCULO-CEREBRORENAL SYNDROME: NOVEL MUTATIONS IN THE OCRL1 GENE AND CORRELATION OF DIRECT DNA DIAGNOSIS WITH OCULAR EXAMINATION Wulf RÎschinger, Ania C. Muntau, GÏnther Rudolph,* Adelbert A. Roscher, Stefan Kammerer Depts. of Pediatrics and *Ophthalmology, Ludwig-Maximilians-University Munich, Germany Lowe oculocerebrorenal syndrome (OCRL) is a rare X-linked multisystem disorder characterized by congenital cataracts, mental retardation, and renal tubular dysfunction. The underlying gene OCRL1 encodes a 105-kD phosphatidylinositol (4,5) bisphosphate (PtdIns[4,5]P2) 5-phosphatase that is localized to the Golgi complex. To con¢rm the clinical diagnosis and to assess the carrier state of female relatives for genetic counseling we examined 6 independent patients and their families (a total of 23 individuals) by sequencing of large PCR amplicons. 4 novel and 2 known mutations were identi¢ed: 3 premature terminations caused by either frameshift mutations (1899insT in exon 17 and 2104-2105delGT in exon 18) or a nonsense mutation (1399C4T in exon 12), two missense mutations (1676G4A and 1754C4T in exon 15), and a 6-bp deletion (1609-1614delAAGTAT in exon 14). An ophthalmological examination was performed in all patients and 14 female relatives. All genotypically proven carrier females showed characteristic lenticular opacities, while all proven noncarriers were lacking this phenotypic ¢nding. The results con¢rm, that ophthalmological evaluation is a reliable ¢rst-line method to ascertain the carrier state in OCRL. The high expressivity of lenticular symptoms in OCRL1 gene carriers is consistent with the hypothesis, that (PtdIns[4,5]P2) 5phosphatase activity has low functional reserve capacity for maintaining a balanced homeostasis of lenticular metabolism.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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LONG TERM FOLLOW UP OF HYPNOTHERAPY IN THE METABOLIC CLINIC JM Fletcher1, M Netting2, A Sweeney2, G Wicks3 Departments of Chemical Pathology1 Nutrition and Dietetics2 and Psychological Medicine3, Women's and Children's Hospital, 72 King William Road, North Adelaide, Australia.

Introduction: We have previously described a short-term bene¢t using hypnotherapy to improve dietary variety in children on low protein diets (PKU, MSUD) who are extremely fussy eaters, and adults and children with needle phobia. To evaluate the longer-term e¡ect, we reviewed the diet diaries of 5 children 2 years after the initial hypnotherapy, mindful that it was parents who perceived the child had a problem and the therapy was directed at the child. Methods: Three-day food records were analysed for number of di¡erent foods consumed and families were questioned about their use of the hypnotherapy tapes. The current ``fear'' status of adults and children with needle phobias was also surveyed. Results: Diet diary analysis revealed sustained improvement in dietary variety. The mean number of foods per day (n =5 patients, averaged over 3 days) improved from 4.6 prior to the intervention to 6.4 after hypnotherapy, which persisted to 6.9 foods two years later. No child or adult had listened to their hypnotherapy tape in the 3 months prior to their most recent clinical review. All patients treated for needle phobia reported continued e¡ect. Conclusions: Hypnotherapy is an e¡ective and long-lasting treatment for needle phobia and improves dietary variety in very fussy eaters. Despite parental perception that there was no e¡ect, the bene¢ts of hypnotherapy for poor dietary variety were measurable and sustained.

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DEVELOPMENT OF A NEW INHERITED METABOLIC DISEASES CENTER (IMDC):THE CHALLENGE OF INTEGRATING COMPREHENSIVE SERVICES IN THE CURRENT HEALTH CARE ENVIRONMENT D. Kronn*, L. Ross*, M. Sexton*, L. Stern*, V. Marrero-Stein*, S. Mo¢di*, F. Hommes*, A. Bacon*. New York Medical College* and Westchester Institute for Human Development*, Valhalla, NY.

Background: The Lower Hudson Valley Region of New York State has a growing population of 2.2 million people with over 30,000 births per year. The problems were a lack of metabolic services in the Region and a lack of familiarity of metabolic disorders among healthcare professionals. The goal of this project was to develop a comprehensive IMDC program using existing Medical Center resources and was organised to address the barriers to healthcare for this patient population. Methods: 1) A core team of health professionals already at the Medical Center were organised to develop the program for submission to the State. 2) The program design included an interdisciplinary approach which has a strong parent to parent family support component. 3) The application addressed the barriers to care which included appropriate healthcare insurance, cultural and language issues in a diverse population and the need for education of healthcare professionals. Results: 1) In the ¢rst year of the clinic there were 303 patient visits. The referrals came from physicians (67%), hospital consultation (12%), State Newborn Screening Program (8%), and other community care agencies. 2) In the second (current) year to meet growing demand an additional weekly clinic was added and an outreach site was established. 3) To overcome barriers we have obtained supplementary insurance for families, we have co-ordinated with community agencies to address ethnocultural needs and we have held educational seminars for healthcare professional. Conclusions: An unmet need for an IMDC in this Region has been ful¢lled by this program. This is one of the few state recognised IMDCs that has been established in the USA in the last 5 years.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

284

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RAISED IMMUNOREACTIVE TRYPSIN IN METABOLIC DISEASE OTHER THAN CYSTIC FIBROSIS J. Calvin*, K.H. Poyser++, M. Heeley+ and A.F. Heeley+ Dept of Clinical Biochemistry, Addenbrooke's Hospital, Cambridge, UK*, Dept of Clinical Biochemistry, Kings College Hospital, London, UK++, Biochemical Genetics Unit, Peterborough District Hospital, Peterborough, UK+ Measurement of immunoreactive trypsin (IRT) is used in neonatal screening for cystic ¢brosis (CF) and in the di¡erential diagnosis of infants with prolonged jaundice. Using an age-related reference range (with cut-o¡ values of 60 mg/L for bloodspot samples and 120 mg/L for serum samples in infants of 6 weeks or less) we have encountered raised IRT results in twelve out of sixteen infants diagnosed with galactosaemia. Hitherto involvement of the pancreas in this condition has not been reported. Where measured, the IRT values decreased with the commencement of a galactose-free diet. Raised IRT values have also been noted in a child with a defect in energy production (intermittent lactic acidaemia, raised CSF lactate, Leigh syndrome). The common link in these diverse conditions may be de¢cient activity of the cystic ¢brosis transmembrane conductance regulator (CFTR), either as a result of mutation in CF or secondary to energy de¢cit within the pancreatic cells in other metabolic diseases. Activation of the CFTR chloride channel is thought to be sensitive to cell energy levels, requiring phosphorylation of the regulator domain and allosteric binding of ATP to the nucleotide binding domains1. Quinton PM, Reddy PM (1992) Control of CFTR chloride conductance by ATP levels through non-hydrolytic binding. Nature 360: 79-81.

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HERMANSKY-PUDLAK SYNDROME (HPS): A MODEL FOR INTRACELLULAR VESICLE FORMATION AND TRAFFICKING. M Huizing1, RE Boissy2, WA Gahl1. 1 Heritable Disorders Branch, NICHD, NIH, Bethesda, MD 20892, USA, 2Department of Dermatology, University of Cincinnati, OH 45267, USA. HPS is a rare autosomal recessive disorder a¡ecting formation and tra¤cking of intracellular vesicles such as melanosomes, platelet dense bodies and lysosomes. HPS patients exhibit oculocutaneous albinism, platelet storage pool de¢ciency, and intracellular accumulation of ceroid lipofuscin. HPS HPS occurs worldwide, but especially in northwest Puerto Rico, and is caused by mutations in dif- ferent genes. HPS-1 encodes a 79kD protein of unknown function which is primarily cytoplasmic. ADTB3A, the gene for HPS-2, encodes the B3A subunit of a heterotetrameric adaptor complex, AP-3. AP-3 mediates new vesicle formation from the trans-Golgi network and selects cargo for the nascent vesicles. We now present evidence that, in normal cultured melanocytes, tyrosinase and tyrosinase-related protein (TRP-1) migrate with melanosomes peripherally to the dendrites, while in HPS-2 melanocytes, only TRP-1 migrates normally. The retention of tyrosinase in the perinuclear region indicates that tyrosinase, but not TRP-1, is recognized by AP-3. This is the ¢rst demonstration that tyrosinase and TRP-1 tra¤ck within melanocytes by di¡erent mechanisms. We have also searched for other genes causing HPS. 14 independent mouse models of HPS provide candidate genes. For 5 of these (pale ear, mocha, pearl, pallid,, and gunmetal), the causative genes (HPS-1, delta-3, B3A, pallidin, and RABGGTA) have been cloned. Other candidate genes are those involved in vesicle formation and transport, e.g., rab27A, rab27B, ARF1, and VPS41. We performed mutation analysis of 15 genes on 20 HPS patients lacking HPS-1 or ADTB3A defects, and report the results. Any new genetic causes of HPS will better elucidate the mechanism by which melanosomes, dense bodies, and lysosomes are created.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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PNO METAB LISTSERV - INSTANTANEOUS COMMUNICATION WORLD WIDE FOR REGISTERED DIETIcIANS WORKING WITH PATIENTS WITH INBORN ERRORS OF METABOLISM. RH Singh, K Crawford, P Graham, K Ellsworth, E Strumlauf, LJ Elsas II, Division of Medical Genetics, Department of Pediatrics, Emory University, Atlanta, GA.

Originally conceived as the Pediatric Nutrition On Line bulletin board, the PNO-metab Listserv evolved into a separate group due to the less cumbersome, instantaneous nature of e-mail. Survey input from member dieticians dealing with inborn errors of metabolism (IEM) revealed many needs to be addressed by the listserv. Of primary importance was instantaneous peer communication and feedback, followed by access to a forum to obtain treatment and monitoring advice for optimal nutrition therapy, new clinically useful case report ¢ndings, and announcements of meetings, new nutrition products and medical foods. Currently 137 registered dietitians representing 10 countries are enrolled and applications continue to be received. Members communicate daily by e-mail on a variety of topics. This open forum approach not only facilitates communication between metabolic dietitians and fosters professional development, but also serves as an invaluable resource tool for dietitians seeking advice regarding the medical nutrition therapy of IEM patients. Currently, study is in progress to evaluate additional membership needs. Future expansion plans will incorporate these needs. To our knowledge, this is the only site available to dieticians working with IEM which enables subscribers to communicate, discuss common patient management problems in providing medical nutrition therapy, and share solutions which result in improved research and care of patients with these diseases.

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HYPOXANTHINE IN A FOREARM EXERCISE TEST IN HEALTHY ADULTS AND ADOLESCENTS WITH METABOLIC MYOPATHIES Martina Heumer, Guenter Bernert, Christian Huemer, Dorothea Moeslinger, Alexandra Auterith, Erwin Hauser, Sylvia Stoeckler-Ipsiroglu Department of Pediatrics and Department of Clinical and Medical Statistics, University of Vienna, 1090 Vienna, Austria

A non-ischemic forearm exercise test was performed in 31 healthy volunteers in order to establish normal values of the exercise-induced increase of plasma hypoxanthine and lactate. Hypoxanthine directly re£ects intracellular consumption of high energy phosphates within the muscle cell. Hypoxanthine is measured from a venous blood sample taken proximal from the exercising muscle. No correlation was found between hypoxanthine and sex, height, weight, percentage of lean body mass and lactate. Application of the test to patients with metabolic myopathies revealed characteristic hypoxanthine patterns in patients with glycogen storage diseases type II and V, mitochondrial fatty acid oxidation defects (systemic carnitine de¢ciency and VLCAD) compared to a patient with central core disease and healthy volunteers. Repeated tests in a patient with VLCAD showed the improvement of muscular energy metabolism during treatment with creatine monophosphate. The measurement of hypoxanthine during a standardized forearm exercise test may provide a valuable tool for the identi¢cation of speci¢c defects in muscle energy metabolism and to monitor therapeutic interventions.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

286

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REFOLDING OF GLUTATHIONE-BOUND RIBONUCLEASE A Toshihiko Ubuka Department of Clinical Nutrition, Kawasaki University of Medical Welfare, Kurashiki, Okayama 7010193 and Department of Biochemistry, Okayama University Medical School, Okayama 700-8558, Japan In order to study the folding mechanism of nascent and denatured proteins, e¡ect of GSH bound to proteins was investigated using ribonuclease A-glutathione mixed disul¢de (Rase-SG) and protein disul¢ed isomerase (PDI). Eight di¡erent species of GSH-bound RNase were prepared by treating reduced RNase with GSSG. Each species was isolated by chromatofocusing and it was found that molar ratio of RNase : GSH in these 8 species were 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7 and 1:8. These species were designated as RNase-SG1, RNase-SG2, RNase-SG3, and so on. These species lacked ribonuclease activity and thus they are in the state of denatured proteins. Refolding of these modi¢ed RNase species was followed by the recovery of enzyme activity in the presence of PDI, 1.5 mM GSH and 0.3 mM GSSG. Enzyme activity was determined by hydrolysis of cytidine-2',3'-phosphate, and the rate of recovery was compared with those of reduced and scrambled RNase. The recovery rate of ribonuclease activity, which indicates the refolding rate of RNase, was in the following order: RNase-SG8, RNase-SG7, RNase-SG6, reduced RNase and scrambled RNase at the ratio of 2.8, 2.3, 1.1, 1.0 and 0.8. Results show that refolding of GSH-bound RNase was much faster than those of reduced RNase and scrambed RNase, indicating the importance of GSH in the refolding of denatured proteins, and might suggest that S-thiolation of nascent proteins with GSH occurs during protein folding process.

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BACULOVIRUS MEDIATED GENE TRANSFER INTO THE RAT NEWBORN MIXED GLIAL PRIMARY CULTURE H. Kobayashi1, T. Ohashi1, W.Sly2 and Y. Eto1 Department of Gene Therapy, Institute of DNA Medicine, The Jikei University School of Medicine1, Japan, Department of Biochemistry and Molecular Biology, St. Louis University2, USA Gene therapy of neuronal disease including Sly disease requires e¤cient gene delivery system. Baculovirus vectors have been shown to be capable of e¤cient transduction of human hepatoma cells and primary hepatocytes in culture by mammarian promoters, but not to be cappable of transduction of primary culture of neuronalcells nor in vivo. To assess the possibility of recombinant baculovirus to use in gene therapy for neuronal disease including Sly disease, we investigated of three type recombinant baculovirus to express HBG gene in mammarian liver cell line, and lacZ gene in mammarian neuronal cell. As a ¢rst step, we generated recombinant baculovirus to express HBG gene under controlled by CAG promoter, and infected this virus human liver cell line, Hep G2. Recently, the modi¢ed ``pseudotype''baculovirus was prepared that carried lacZ under control of the CMV promoter, and VSV G under control of the polyhedrin promorter. As a second step, This virus was then used to infect primary cultures of rat new-born mixed glial cells, they were stained by a-gal and double stained by GFAP. Three days after infection, they were stained by a-gal and double stained by GFAP, and there were some double positive cells (gene-transfered astrocytes), by 4.7% of all GFAP positive cells(n=500). This is suggestive that baculovirus mediated gene transfer have a potential possibility in gene therapy for neuronal disease. We would like to thank Dr.James Barsoum (Biogen,Inc.,Cambridge) for providing us the recombinant baculovirus vector.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

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GROWTH OF A CLINIC FOR ADULTS WITH METABOLIC DISEASES: IMPLICATIONS FOR RESOURCE ALLOCATION Sirrs SM, Canning C, Toms M and Abraham J. Vancouver General Hospital, Vancouver, British Columbia, Canada

Objectives: To review the growth and case distribution of patients seen at a clinic for adults with inborn errors of metabolism in the ¢rst year of clinic operation. Methods: All cases seen at the adult clinic for diagnosis, source of referral, requirement for inpatient care, and use of metabolic formulae. Results: The clinic opened with 111 active patients transitioned from the pediatric center. The one year growth rate was 68%, consisting of 56 new referrals, 1 patient transitioned and 18 patients who had been previously lost to follow-up from the pediatric center. The most frequent new referrals were disorders of homocysteine metabolism (18%), metabolic myopathy (14%), and adults with PKU returning to diet (12%). Only 8 patients required hospital admission. Outpatient care was intensive with 15 indirect patient contacts for every clinic visit. Formula costs increased 90% in the ¢rst year to Can$277765.44, largely due to increased use of formula by patients already on diet (58% of cost increase) and adult patients with PKU returning to diet (36% of cost increase). Conclusions: The population of adults with metabolic disorders is growing rapidly. Care for the adult population is largely restricted to the outpatient setting, which requires intensive social work support. Budget planning must consider increased costs for laboratory services (such as for the investigation of myopathies) and metabolic formulae.

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NEWBORN SCREENING FOR LYSOSOMAL STORAGE DISORDERS Meikle PJ, Ranieri E1, Ramsey SL, Whit¢eld PD, Rosaklis T, Gerace RL1, Ravenscroft EM, Chang MHY, Brooks DA and Hopwood JJ. Lysosomal Diseases Research Unit and State Screening Services1, Department of Chemical Pathology, Women's and Children's Hospital, Adelaide, AUSTRALIA. Lysosomal storage disorders (LSD) represent a group of over 40 distinct genetic diseases with a total incidence of approximately 1:5,000 births. Bone marrow transplantation and enzyme replacement therapy are currently in use for the treatment of some disorders and new forms of enzyme and gene replacement therapy are actively being researched. The e¤cacy of these therapies will rely heavily upon the early diagnosis and treatment of the disorder, before the onset of irreversible pathology. The only practical way to detect these disorders pre-symptomatically will be by a newborn screening program. We have developed a two tier screening strategy consisting of a ¢rst tier screen utilising the lysosomal protein markers LAMP-1 and saposin C, followed by a second tier screen quantifying lysosomal substrates by tandem mass spectrometry. The ¢rst tier protein markers have been determined in blood spots taken from Guthrie cards and in plasma samples from over 300 LSD a¡ected individuals representing 25 disorders. The results indicate that these two markers will enable the selection of 80-90% of LSD a¡ected patients. We have developed methodology for the determination of both oligosaccharides and glycolipids, stored in LSD, utilising tandem mass spectrometry. These methods enable the quanti¢cation of selected lysosomal storage substrates in blood spots taken from Guthrie cards and will be used in the second tier of the screening program.

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ACUTE MYOPATHY AND CARDIOMYOPATHY IN AN ADULT WITH EMA/ ADIPICACIDURIA. Margretta Reed Seashore, MD and Cheryl Garganta, MD, PhD. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA This 28 year old woman with EMA/adipic aciduria, presented with muscle weakness and edema. She had been diagnosed at age 7 years after recurrent episodes of non-ketotic hypoglycemia when urine organic acid analysis revealed EMA/adipic aciduria. She had been well on a high carbohydrate diet, modest protein and fat restriction and avoidance of fasting. On evaluation, the patient described adhering to a regimen of 2-3 hours daily of vigorous exercise for several months. Diet during this period was high in carbohydrate, but she exercised in the morning after an overnight fast, before eating breakfast. She did not report myoglobinuria. Examination revealed weakness in all muscle groups, pitting edema of the legs, and a just palpable liver. Serum CK was 259 U/L, CKMB 23.6 ng/ ml (nl55); glucose 74 mg/dL; AST and ALT elevated. Urine organic acid analysis showed signi¢cant elevations of EMA, methylsuccinate, 2-OH glutaryl lactone, glutarate, and C6-C10 saturated and unsaturated dicarboxylic acids. Urine acylglycine analysis by stable isotope dilution showed signi¢cantly elevated hexanoylglycine, suberylglycine, isovaleryl glycine and n-butyryl glycine. The presumptive diagnosis was exacerbation of her disease, with cardiac failure and myopathy. Treatment included restriction of exercise with prescription of cornstarch at bedtime, breakfast in the morning, and avoidance of fasting. Within 4 weeks, the edema had subsided, muscle strength had increased, and laboratory tests improved. This case shows that myopathy and cardiomyopathy can occur in EMA/adipic aciduria in adulthood during a period of metabolic stress such as exercise and fasting and is reversible with caloric support and reduction of exercise.

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

# SSIEM and Kluwer Academic Publishers. Printed in the Netherlands

Author index Abdenur JE, 13, 106, 149 Abe T, 231, 231 Abeling NGGM, 86, 206, 206 Abraham J, 287 Abramowicz MJ, 85 Acosta PB, 25 Adamowicz M, 171, 179, 184 Aerts H, 229 Aerts JMFG, 217 Agostoni C, 30 Ahlemeyer B, 101, 102, 104 Ahment K, 189 Aikawa J-i, 174 Akaboshi S, 110 Akagi M, 141 Akanuma J, 166 Akerman BR, 281, 282 Aksu F, 203 Al Aqeel AI, 267 Albers S, 6 Aledo R, 107 Alla JAB, 240 Allen JT, 258 Allen RH, 64, 67 Allievi S, 114 Almashanu S, 88, 244 Almeida IT, 134 Alomar A, 56 Alonso EM, 127 Alvarez F, 69 Alvarez MG, 149 Anderson P, 32, 38 Anderson V, 32, 38 Andersson H, 226 Andersson HC, 200 Andersson U, 216 Andreou A, 57 Andresen BS, 12, 122, 124, 134 An£ous K, 204 Angaroni C, 144 Anlar B, 104 Annesley D, 63 Ansari A, 189 Antonozzi I, 210, 274 Antonozzi S, 279 Antony JA, 207 Aoki K, 9, 24 Aoki T, 264 Aoki Y, 93 Applegarth DA, 50, 76 Aracil A, 202 Arai H, 239 Arau¨jo P, 82 Arbustini E, 167 Argaran¬a CE, 144, 220, 220 Armani U, 64 Arnold GL, 29, 266

Arnold LA, 208 Arnout J, 98, 205 Arranz JA, 55, 273 Artigas J, 180 Artuch R, 35, 202 Asada M, 159, 173 Asanuma A, 174 Ashmarina L, 108 Assmann B, 183 Aubourg P, 246 Augoustides-Savvopoulou P, 57, 193 Augustsson C, 77 Auldist A, 164 Aulehla-Scholz C, 47 Auray-Blais C, 13 Auterith A, 285 Ayling JE, 42 Aysun S, 104 Azen C, 20, 20, 21, 25, 28 Baba K, 280 Bachmann C, 101 Bacman S, 144 Bacon A, 283 Baethmann M, 115 Baglieri S, 38 Bagshaw R, 214 Bailey IV, 6 Bailey SW, 42 Bain MD, 160, 190 Baker AJ, 53 Bakker HD, 86 Bakker JA, 45, 157 Ball W, 211 Bamforth F, 232, 261 Ban K, 51 Banas R, 12 Banaszak LJ, 127 Bandinelli C, 83 Bano G, 160 Baquero M, 202 Bar-Meir M, 140 Baracchini C, 91 Barbot C, 60, 149 Baric¨ I, 118 Barnard K, 3 Barnes LJ, 58 Barnes R, 69 Barnes RD, 58 Barniville G, 80 Barone R, 181, 192 Barranger JA, 224 Barreiro C, 149 Barry-Kinsella C, 62 Barshop BA, 50, 53, 61 Barta C, 270

289

Barth P, 245 Barth PG, 150, 210, 244, 245 Bartlett B, 3 Barycki JJ, 127 Bass N, 136 Bastiani J, 83 Battaile KP, 198 Battistoni G, 38 Baumgartner ER, 88, 93, 98 Baumgartner MR, 88 Baumgartner R, 90 Bause E, 186 Bavaresco CS, 82 Bax BE, 190 Baykal T, 86, 113, 170 Beath SV, 278 Beaty RM, 166 Beaudet A, 58 Beaven OJ, 22 Beblo S, 68 Beemer FA, 73, 171, 196 Begovic¨ D, 118 Behu¨lova¨ D, 197, 199 Beinbrech B, 175 Bekhof J, 34, 35, 45 Bembi B, 167, 225 Bene J, 139 Benettoni A, 167 Bennett MJ, 99, 127, 128, 129, 130, 268 Berger J, 157, 252, 253, 254, 254 Berger PS, 131 Berger R, 73, 133 Berghenouse G, 181 Berner FP, 135 Bernert G, 285 Bernier FP, 138 Bernstein L, 98 Berry GT, 97, 130, 158 Bertini E, 213 Bertrand M, 227 Bertucci P, 241 Besley GTN, 129, 194 Bezman L, 252 Bich W, 47 Billette de Villemeur T, 147, 206 Billman GF, 53 Bindloss C, 215 Binford S, 129 Birarelli M, 274 Bird S, 34 Bisiacchi P, 37 Blaese RM, 30 Blakely EM, 266 Blau N, 10, 37, 42, 42, 43, 45, 47, 207 Blazï|¨ cek P, 264

290 Block G, 17 Blom HJ, 69 Bodamer O, 58 Bodner-Leidecker A, 156, 275 Boeri D, 64 Boerth SR, 42 Boesp£ug-Tanguy O, 213 Boissy RE, 284 Bok LA, 124 Boles RG, 146 Bolund L, 122 Bonafe¨ L, 10, 37, 42, 43, 180 Boneh A, 29, 32, 38, 90, 108, 135, 164 Bones CH, 65 Boney A, 166 Bonham JR, 65, 135 Bonnefont JP, 19, 116 Borchert A, 150 Boriack RL, 99 Borissenko L, 215 Borja F, 195 Bouchard L, 108 Boukaftane Y, 108 Boulat O, 272 Boulay B, 219 Boutron A, 147, 154 Bowling FG, 191 Boyer-Neumann C, 163 Braegger C, 42 Branco M, 106 Branco T, 84 Brand J, 109 Braulke T, 218, 240 Bravaresco CS, 83 Braverman N, 67 Brennan P, 63 Breuer W, 186 Briddon A, 62, 154 Briones P, 56, 180 Brivet M, 116, 147, 147, 154 Brodiansky I, 74 Brodie S, 222 Broe D, 191 Brooks DA, 235, 287 Brosius U, 247 Bross P, 122, 124, 134 Brouwer C, 189 Brown AY, 258 Brown G, 135, 154 Brown GK, 142 Brown MD, 146 Brown RM, 142 Brown T, 166 Bruinse HW, 65 Brunelle F, 174 Brusilow S, 91 Bruzzese MG, 30 Bubel S, 183 Budzinski T, 226 Bugaut M, 252, 253 Buist N, 187 Burchell B, 278

Burgard P, 37 Burger HJ, 167 Burlina AB, 10, 37, 54, 55, 68, 91, 114, 180, 213 Burlina AP, 37, 91, 213 Burroughs A, 94 Burton S, 237 Busquets C, 103 Bussani R, 167 Bussie©res J-F, 69 Butch AW, 60 Butters TD, 216, 217 Byers S, 239 Bzdu¨ch V, 197, 199 Cabello JF, 81 Cachon-Gonzalez B, 221 Cadepond F, 252 Cahana A, 130 Calado E, 149 Callahan JW, 214 Calvin J, 284 Camacho-Arroyo A, 87 Cambra FJ, 33 Cameron J, 155 Cameron JS, 194 Campanella D, 64 Campistol J, 33, 35, 56 Campos F, 273 Canning C, 287 Cano-Col|¨ n S, 87 Capdevila A, 202 Capel L, 163, 278 Carchon H, 186 Cardoso ML, 60, 106 Carducci Ca, 210, 274 Carducci Cl, 210, 274 Carey WF, 238 Carlsson K, 77 Carollo C, 91 Carpenter K, 4, 15, 16, 99 Carreira I, 134 Carreira IM, 145 Carrozzo R, 79 Caruso U, 96, 109, 176, 192 Carvalho TS, 41 Casals N, 107 Casanelia S, 164 Castilhos CD, 126 Castro R, 69 Catsman-Berrevoets CE, 206 Cavanagh J, 226 Cecil K, 211 Cederbaum SD, 60, 119 Cendron M, 37 Cíerna¨ L, 152 Cerone R, 33, 64 Chaba¨s A, 3, 180 Chabraoui L, 11 Chace D, 12 Chakrapani AB, 48, 48, 129 Chalas J, 163, 278 Chalmers RA, 120, 160, 190, 281

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Chamoles NA, 10, 13, 106, 149 Champion MP, 34, 53 Chang M, 43 Chang MHY, 287 Chapman I, 31 Charrow J, 127, 226 Chaudhari D, 146 Chen DDY, 272 Chen RG, 22 Chen Rui G, 44 Chen Y-T, 166, 168 Cheng SH, 223, 226 Cheng XM, 7 Cheung KL, 253 Chiba M, 74 Chiesa A, 23 Chinnery PF, 136 Cho K, 261 Choeh K, 159 Choiniere L, 282 Chong WK, 142 Chow CW, 135, 143 Christensen E, 117, 124, 134 Christensen R, 30 Christodoulou J, 137 Chung N, 136 Chung S, 202 Cia¡oni F, 218 Ciana G, 167, 225 Ciara E, 171 Civallero G, 144, 279 Clark S, 135 Clarke DJ, 278 Clayton PT, 139, 258 Cleary MA, 57, 129, 135 Clegg JB, 18 Clements PR, 235 Cline M, 129 Clusellas N, 195 Codazzi D, 96 Coelho JC, 229, 236 Co¤n R, 237 Cohen JC, 99, 99 Coitinho AS, 83 Colby RS, 228 Coldwell J, 20 Cole R, 88 Coleman L, 38 Coll MJ, 103 Colombo L, 21 Colombo M, 81 Colome¨ C, 35, 202 Colomer J, 180 Coluccio M, 106 Connor WE, 196, 197 Conzelmann E, 247 Cooper D, 273 Corbetta C, 96 Cordeiro P, 219 Cormier-Daire V, 174 Cornejo V, 24, 81, 85 Corona P, 142 Coronel CE, 188

291 Corydon M, 122 Corydon TJ, 122, 124 Cos° kun T, 46, 104, 195 Cossette P, 54 Coulter-Mackie MB, 76 Cowley DM, 191 Cox C, 250 Cox KB, 131 Cox TM, 217, 221 Craig J, 90 Craigen WJ, 204 Crawford K, 285 Crawley AC, 234 Cregeen D, 260, 261 Crenn D, 147 Cretz M, 206 Crone MR, 34, 35 Cusi V, 202 Cuthbert A, 8 Cyr D, 13 D'Hooge R, 51, 222 Dacremont G, 186 Dahl H-HM, 143 Dahri S, 11 Dallaire L, 69 Dalton RN, 53, 271 Darras BT, 73 Das AM, 100 Davidson AGF, 31, 50 Davies P, 70 Davis JA, 184 de Abreu RA, 56, 189 de Barse MMJ, 133 de Belleroche J, 259 de Braekeleer M, 148 de Coo IFM, 206 de Deyn PP, 51, 222 de Jager AEJ, 269 de Jong JGN, 56, 238 de Klerk H, 185 de Klerk JBC, 49, 100 de Koning TJ, 73, 133, 171, 196, 210 de Kremer Dodelson R, 144, 165, 188, 208, 220, 220, 279 de la Cruz F, 21, 28 de la Parra A, 85 de Lonlay P, 155, 174 de Mari JF, 126 de Mattos-Dutra A, 84 de Meirleir L, 78, 141, 251 de Praeter C, 186 de Prest B, 242 de Schepper J, 251 de Valk HW, 65 DeArmond S, 136 De¨carie J-C, 54, 204 De¨chaux M, 97 Degen I, 102 deGrauw T, 211 Dekker C, 245 Delonlay P, 138, 206

Demanet C, 251 Demirkol M, 86, 113, 170 Demirtas° M, 265 den Boer MEJ, 125 Denecke J, 26, 28, 32 Denis S, 124, 244, 248, 256, 258 Dennett X, 135 Depetris-Boldini C, 144, 279 Desnick RJ, 222, 223 Desprechins B, 251 Desviat LR, 89, 92, 92 Devai A, 270 Devaney J, 214 Deybach JC, 280 Dhondt J-L, 41 Di Mauro S, 149 Di Rocco M, 176 Di Rocco N, 109 Dianzani I, 44 Dierks T, 215 Dijksman J, 133 Dikman S, 222 Dilling LA, 19 Dimitrakopoulos K, 193 Dinis A, 106 Diogo L, 106, 134, 145 Dionisi-Vici C, 79, 121, 241, 249 Dipple KM, 119, 172 Dobrowolski SF, 12 Docx M, 279 Dodt G, 245 Donadio A, 114 Donari J, 149 Donati MA, 105 Donmez S, 113 Dophin CT, 281 Dorche C, 78 Dorland L, 45, 133, 171, 196 Douwes AC, 172 Drouet L, 162 Dubois J, 69 Duggins A, 207 Duley J, 189 Duley JA, 18, 193, 194 Dupuis C, 69 Dura¨n GP, 24, 85 Duran M, 45, 49, 73, 100, 131, 133, 171, 196 Dursun A, 195 Dutra-Filho CS, 81, 82 Dvorïa¨kova¨ L, 254 Dwek RA, 216, 217 Earl JW, 207 Eaton S, 128 Edefonti A, 96 Edwards R, 129 E¨ervenkova¨ M, 219, 268 Efstathiou S, 221 Ehrich J, 178 Eiben R, 203 Eichler F, 250, 250, 251 Ellaway CJ, 207

Elleder M, 140, 170, 219 Ellsworth K, 285 Elpeleg ON, 74, 140 Elsas LJ, 158, 158 Elsas II LJ, 61, 285 Elsliger M-A, 233 Elstein D, 217 Emma F, 79 Eng C, 222 Engel P, 12 Engelke UFH, 71 Enns G, 136 Enoki M, 231 Ensenauer R, 212 Enyedi P, 270 Erdel M, 165 Erwa W, 209 Eschrich K, 160 Escuredo E, 18 Eshel G, 246 Esparza J, 89 Espeel M, 186, 242, 249 Estruch E, 237 Eto Y, 71, 216, 227, 228, 286 Evans CL, 57 Exil V, 133 Eyskens F, 105, 227, 279 Eyssen M, 205 Fairbanks LD, 190, 193, 194 Fallon J, 222 Fan J-Q, 217 Fang J, 183 Farkas V, 139 Farrugia R, 44 Fatemi A, 144 Faucher F, 69 Faulkner E, 166 Federici G, 241 Feher MD, 259 Fekete GY, 39 Feldmann R, 26, 28, 32 Felice AE, 44 Fenton W, 95 Fenyves D, 69 Ferdinandusse S, 256, 257, 258, 259 Fernandes AIP, 145 Ferrari V, 213 Ferrer I, 180 Fey J, 215 Ficicioglu C, 72 Fiesel S, 125, 126 Fietz MJ, 89, 240 Fighera MR, 83 Finckh B, 164 Fingerhut R, 5, 90, 115 Finkelson LA, 6 Fiore CE, 181 Fiori L, 21, 30 Fisher S, 194 Fitzpatrick M, 222 Fiumara A, 181, 192

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

292 Flavell D, 197 Fletcher JM, 3, 10, 19, 31, 89, 283 Flo«rcken A, 177 Flugge K, 98 Fok TF, 253 Fondelli P, 176 Forest J-M, 69 Forget PP, 45, 157 Fors-Petter S, 253 Fourcade S, 252 Fournet J-C, 174 Fowler B, 68, 68, 98 Fraga C, 23 Francis D, 38, 164 Francis DEM, 29, 32 Francoual J, 163, 278 Francova¨ H, 199 Franx A, 65 Franzon R, 82 Frauendienst-Egger G, 8, 47 Fredman P, 223 Freehauf C, 98 Freeze HH, 184, 187 Freson K, 205 Fresser F, 165 Friedman EG, 20 Froestl W, 110 Frommhold D, 7 Fryns JP, 98 Fu X-w, 161 Fujii K, 166, 269 Fujii YK, 45 Fujiki Y, 243 Fujioka H, 173 Fujisawa K, 6 Fukao G, 113 Fukao T, 112, 132, 241 Fukuda T, 168, 169 Fukushige T, 225 Fumic¨ K, 118 Funato M, 277 Fung VS, 207 Funghini S, 105 Furlanetto V, 82 Fusta M, 13, 106 Fuste¨ E, 33 Gaard U, 137 Gabreols FJM, 201 Gaggioli D, 13 Gahl WA, 284 Gallardo ME, 89 Ganguly A, 119, 120 Garami M, 39 Garavaglia B, 103, 114, 118, 121, 132 Garc|¨ a-Silva MT, 180 Garcia A, 116, 154, 155, 246 Garcia P, 106, 134 Garcia-Cazorla A, 138 Garganta C, 288 Ga«rtner J, 203, 247 Gass A, 222

Gass|¨ o R, 33 Gaudet D, 281, 282 Gauthe¨ D, 252 Gean E, 195 Gelb M, 272 Gelpi E, 202 Genest J, 281 Gerace RL, 3, 89, 287 Geraghty MT, 67, 78, 91 Gerber S, 272 Gerlo E, 78, 85 Gerwig G, 186 Giann|© ML, 21 Gianotti D, 33 Gibberd FB, 259 Gibson KM, 1, 76, 92, 93, 110, 110 Gibson M, 89, 99 Gick JA, 34 Gieselmann V, 218, 221, 222, 223 Giguere R, 13 Giner-Ayala A, 144, 165 Giovannini M, 21, 30 Giro¨s M, 195 Giron J, 214 Giugliani R, 41, 42, 67, 126, 229, 236 Giunta C, 276 Gizçewska M, 11, 47 Godinho M, 145 Golabi M, 136 Go«ggerle M, 8, 93, 242 Goldman M, 222 Gomez L, 33 Gondcaille C, 252 Gondo M, 262, 262 Goodman S, 95 Goodyer P, 69 Gootjes J, 244, 245, 245, 246 Gordon R, 222 Goto Y-i, 141 Goulart L, 229 Gould S, 245 Gow IR, 217 Gowrishankar M, 261 Goyer M-F, 69 Gradowska W, 11 Grady G, 14, 15 Graham A, 258 Graham P, 285 Grange DK, 200 Gray RGF, 70 Grazina MMM, 134, 145 Greeley R, 87 Greenberg CR, 19 Greene C, 98 Greener M, 194 Gregersen N, 12, 122, 122, 123, 123, 124, 124, 134 Grenier A, 69 Grenzebach M, 26 Grivell LA, 150 Grody WW, 60

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Groener JEM, 173 Grompe M, 110, 110, 187 Grootho¡ JW, 260 Grosso C, 188 Gru«newald S, 98, 181, 185 Grun¬eiro de Papendieck L, 23 Gu XF, 7, 22 Gu Xue F, 44 Gu YH, 266 Gu«rakan F, 195, 265 Gu«ttler F, 28, 46 Guelbert N, 144, 165, 208 Guerrero N, 158 Guertsen E, 177 Guinle A, 13, 106 Guiochon-Mantel A, 147, 147 Guldberg P, 21, 28 Gutman A, 123, 246 Guttler F, 20, 20, 21 Haas RH, 53 Haasjes J, 192 Hacker R, 183 Haeberle J, 59 Hafner D, 66 Hahn S, 261 Halliday JL, 138 Hamet P, 281, 282 Hammen H-W, 156, 275 Hammond J, 16, 79 Hamosh A, 91 Hanefeld F, 150 Hanley W, 20 Hanley WB, 21, 28 Hanna M, 154 Hansen SH, 117 Hans|¨ kova¨ H, 140, 152 Hardiman O, 63 Harding CO, 39 Hargreaves I, 135, 257 Hargreaves IP, 62, 139, 154 Harms E, 32, 233 Harper A, 70 Harrington M, 97, 130 Harrison CJ, 135 Harrison JR, 10, 89 Harthcock PA, 99 Hartnett C, 31, 50 Harvie A, 26 Harvie H, 120 Hasaerts D, 78 Hase Y, 173 Hasegawa S, 121 Hassan C, 189 Hata I, 6 Hattori H, 235 Hauser E, 285 Ha«usler M, 207 Haut S, 147, 147 Havens J, 172 Haworth JC, 19 Hayashi Y, 174 Hayes CM, 136

293 Heales SJR, 135, 139, 154 Heath D, 16 Hebron KJ, 256 Hechtman P, 219 Heeley AF, 284 Heeley M, 284 Hegardt FG, 107 Heizmann CW, 10 Hellerud Ch, 171 Henderson MJ, 2, 57, 84 Hennet T, 185 Heptinstall L, 129 Heringa MP, 162 Herling AW, 167 Herman GE, 200 Hermo L, 202 Herrmann R, 177 Hershkovitz E, 74, 255 Hertz-Pannier L, 246 Herve¨ P, 162 Heumer M, 285 Higashimoto T, 146 Hillebrand G, 178 Himms-Hagen J, 202 Hinteregger V, 157 Hirabayashi Y, 225 Hirai Y, 221, 224 Hiratsuka M, 93 Hoefsloot EH, 201 Hoemme M, 175 Ho¡buhr K, 214 Ho¡man E, 214 Ho¡mann GF, 7, 12, 101, 101, 102, 102, 103, 104, 107, 109, 125, 126, 165, 175, 175, 183 Hofmans K, 251 Hofstra RMW, 269 Hogema BM, 76, 110, 110 Hogenhout EM, 245, 259 Ho«gler W, 114 Hohlfeld T, 66 Hollak CEM, 217 Holm J, 117 Holme E, 69 Holmes HC, 281 Homberger A, 59, 233 Hommes F, 283 Hong S-P, 159, 276 Hoppel C, 136, 203 Hopwood JJ, 19, 215, 234, 235, 237, 238, 239, 287 Hori S, 265 Horn N, 137, 263 Horoupian D, 136 Hou DC, 45, 166 Housít|¨ k J, 268 Houten S, 107 Houten SM, 108 Howard PM, 63 Hoylaerts M, 205 Hrïeb|¨ e©ek M, 217, 219, 254, 268 Hsiao K-J, 43, 159 Hsu BYL, 108, 119, 120

Huang BL, 172 Huber PR, 111, 276 Hu«bner C, 203 Huck JH, 172 Hudson T, 8 Huemer C, 285 Huertas P, 222 Hughes SM, 213 Hu«hne R, 103 Hui J, 253 Huijben K, 185 Huijmans J, 100 Huijmans JGM, 49 Huisjes AJM, 65 Huitema M, 167 Huitema S, 162 Huizing M, 284 Hujova¨ J, 254 Hu©lkova¨ H, 219 Humbert M, 162 Hu«ner G, 86, 113, 170 Hunnemann D, 248 Hyland K, 208 Iacobazzi V, 120, 120, 121 Ibdah JA, 129 Ida H, 227, 228, 231 Iga M, 241 Igarashi Y, 174 Iinuma K, 174, 177 IJlst L, 107, 109, 120, 124 Iles RA, 281 Imamura A, 243 Imamura T, 159 Imbach T, 185, 186 Inada H, 173 Inns J, 214 Inokuchi T, 9, 24 Inoue C, 151, 270, 277 Inoue Y, 235 Inui K, 93, 141, 166 Invernizzi F, 114, 121, 132 Ioannou P, 94 Ioannou YA, 223 Ipsiroglu O, 144 Isbrandt D, 212 Ishii S, 16, 217 Item C, 144 Ito M, 146, 151, 152, 153, 168, 169 Ito T, 51, 188, 228 Itoh M, 247 Iwamoto H, 45 Iyer RK, 60 Jackson M, 166 Jackson ME, 9 Jaeken J, 180, 180, 181, 183, 184, 185, 186, 205 Jakobs B, 274 Jakobs BS, 111 Jakobs C, 69, 76, 110, 110, 116, 131, 172, 182, 205, 209, 209, 211, 246, 259

James ER, 76 Janecke AR, 165 Janecke N, 37 Jankie R, 49 Janmohamed A, 281 Jansen EEW, 209 Jansen GA, 259 Janssen-Zijlstra FSM, 198 Janssens G, 105, 227, 279 Janvier A, 148 Jensen TG, 30 Jevon G, 146 Jimenez M, 85 Jimenez-Sanchez G, 250 Jiminez-Sanchez G, 256 Jin BY, 161 Jira PE, 198 Jogo M, 9, 24 Johnson DW, 19, 271 Johnson JL, 77 Johnson MA, 136 Jones P, 130 Jones PM, 127 Jones RG, 2 Junien C, 174 Jurkiewicz D, 171 Kahler S, 90, 94 Kalkanogïlu HS, 46 Kalkanogïlu S, 195 Kamath R, 207 Kammerer S, 243, 255, 282 Kamphuis S, 100 Kanazawa M, 55, 115, 116, 121, 265 Kanazawa N, 71, 72 Kanzaki T, 225 Kaplan F, 219 Kaplan P, 97, 130, 226 Kappy M, 98 Karam SM, 67 Karch S, 126 Karczmarewicz E, 145 Karoly M, 277 Karteszi J, 277 Kase R, 224, 224 Kato H, 52 Kato M, 46 Katouzian F, 167, 225 Katzira H, 251 Kawada K, 16 Kaye EM, 97, 130, 148, 234 Kays GW, 213 Keizer-Garritsen JJ, 189 Kelley RI, 200, 200 Kelly DA, 278 Kemper M, 178 Keªpka A, 161 Kerckaert I, 242, 249 Kern RM, 60 Kerr D, 203 Kershaw N, 258 Keselman A, 23

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

294 Kesimer M, 104 Ketteridge DB, 31 Khneisser I, 41 Kidouchi K, 51 Kikawa Y, 160, 161, 161 Kim J, 261 Kim KR, 275 Kim L, 222 Kim M-K, 159, 276 Kimonis VE, 112, 156 Kimura A, 16 Kimura M, 132, 161, 238, 241 Kinner M, 17, 168, 176, 178 Kira J-i, 71 Kirby DM, 137, 143, 143 Kirby RS, 29 Kishnani PS, 14, 166 Kiss E, 39 Kjaergaard S, 182, 184 Kleijer W, 57 Kleiman FE, 220, 220 Klein M, 164 Klepper J, 177 Kler RS, 136 Klomp LWJ, 73 Klosterhuber M, 114 Klupsch B, 93 Kmoch S, 152, 190, 193, 268 Knerr I, 90 Knoll DP, 133 Knopf C, 249 Knopsnider 9 C, Knudsen I, 12, 124 Kobayashi H, 216, 286 Kobayashi K, 56, 56, 57, 280 Kobayashi M, 227, 262, 262, 266 Kobayashi S, 265 Koc°ak N, 195, 265 Koch HG, 32, 59, 68, 233 Koch R, 20, 20, 21, 25, 28, 36, 36 Kock HG, 28 Koda N, 74 Kodama H, 262, 262, 266 Kodama K, 225 Koeberl DD, 14, 166 Kogo T, 55 Kohlmuller D, 12 Kohlschu«tter A, 164, 240 Kohno Y, 55, 115, 116, 121, 265 Kojo T, 168 Ko«lker S, 101, 101, 102, 102, 103, 104 Kolodny E, 226 Kolvraa S, 30 Komatsu M, 231, 231 Kondo H, 46 Kondo N, 112, 113, 132, 241, 243, 247 Konecki DS, 40 Kono K, 235 Koo-McCoy S, 119 Kopacz KJ, 200 Korall H, 8, 47, 54, 93, 242

Korczowski R, 11 Korenke CG, 150 Korman SH, 123, 246 Ko«rner C, 185 Korosec T, 253, 254 Korson MS, 6, 73 Koster J, 108, 196, 199, 201 Kostiner D, 95 Kosuga M, 239 Koza¨k L, 170, 199 Kozich V, 64 Krakowiak PA, 198 Krasemann T, 233 Kratz LE, 198, 200, 200 Kraus JP, 64, 66 Krieglstein J, 101, 102, 103, 104 Krijt J, 64 Kronn D, 283 Kruger WD, 61 Kubalska J, 171 Kugel H, 80 Kumsta M, 193 Kuntz KM, 234 Kupcíova¨ V, 264 Kure S, 45, 93, 166, 269 Kuriya N, 52 Kuroda Y, 151, 152, 153 Kuru N, 86 Kuster T, 10 Kutzick C, 177 Kwast H, 56 Laan LAEM, 173 Labbe¨ N, 69 Labrune P, 162, 163, 163, 278 Lacey JM, 178, 179 Lachmann RH, 217, 221 Lacroix C, 147 Lacroix J, 148 LaFleur B, 214 Laframboise R, 69 Lagler F, 68 Lai K, 158 Lainka E, 115 Lam F, 79 Lamantea E, 132, 142 Lambert DM, 58, 69, 281, 282 Lambert M, 54, 69, 148, 204 Lambert R, 69 Lambert-Hammill M, 259 Lambooy LHJ, 189 Lambruschini N, 33, 35, 56 Lamhonwah A-M, 118, 119 Lamoril J, 280 Land J, 135 Land JM, 154, 257 Langius FAA, 196 Laro¨vere L, 188, 208 Larochelle J, 69 Larson C, 14, 15 Larsson A, 77 Laryea MD, 66 Latini A, 188, 208

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Laufer-Cahana A, 97 Launay J-M, 162 Laundry K, 233 Laurin N, 202 Lavorgna S, 106 Law LK, 253 Le ST, 146 Leandro AP, 40 Leandro P, 40 Leao Teles E, 149 Lecca MR, 252 Lechner MC, 40 Ledvinova¨ J, 219 Lee B, 49, 58 Lee DH, 159, 276 Lee KO, 159, 276 Lee N, 148 Lee P, 27, 27 Lee SJ, 234 Lee WS, 278 Lefe©vre Y, 69 Lefrancois S, 233 Legrand A, 147, 147, 154 Lehnert W, 71, 212 Leistner S, 236 Lemieux B, 13 Lendl B, 274 Leo¨n-del-R|¨ o A, 87 Leon-Del-Rio A, 88 Leonard JV, 94, 108 Leonard SC, 9 Leopaldi A, 225 Leuzzi V, 210, 274 Levine BA, 250, 251 Levy E, 202 Levy H, 20 Levy HL, 6, 21, 28 Lewis C, 194 Lewis E, 126 Lewis MJ, 65 Li C, 223 Li CK, 253 Li X, 93 Lichter-Konecki U, 40 Liebaers I, 78 Liebl B, 5, 5, 90, 115 Lilburn M, 27, 27 Lillquist YP, 31, 50 Lima TTF, 83 Limburg P, 167 Lin D, 197 Linck L, 196 Linderkamp O, 12 Lindner M, 109, 125, 126, 165 Lindsey JR, 131 Lindstedt S, 69, 171 Linnebank M, 59, 68, 233 Lissens W, 78, 141, 251 Liu H-M, 123 Liu TT, 43 Liu Xiao Q, 44 Lloyd MD, 258 Lluch M, 56

295 Loeber JG, 1 Loes DJ, 251 Loganathan K, 156, 275 Loiselet J, 41 Lombaerts R, 98 Longo N, 117, 118 Longtin L, 69 Loots W, 45 Lopes I, 75 Lorini R, 33, 64 Lortie A, 54, 204 Lossos A, 123 Losty H, 59 Loy A, 109, 176 Lu KV, 60 Lu«bke T, 185 Lu«cke T, 100 Lukong K, 233 Ma Xie Q, 44 Mabe P, 85 Maca¨rio MC, 145 MacKay N, 155 MacKinnon CH, 258 Magera MJ, 96, 178, 179 Magoula I, 193 Mahuran DJ, 214 Maiello M, 64 Maier EM, 255 Maire I, 11 Majewski J, 190 Malaney S, 8 Malinova¨ V, 152 Maller E, 130 Malonova¨ E, 280 Malvagia S, 90 Mamer OA, 282 Manatunga A, 158 Mandel H, 249 Mandell R, 112 Mandelli J, 236 Mandic¨ Z, 118 Manji H, 257 Manning NJ, 135 Manns MP, 157 Mannucci L, 79 Manor E, 255 Maînsson J-E, 223 Maquardt T, 185 Maradin M, 118 Marescau B, 51 Marfaing-Koka A, 163 Marie S, 191 Marinaki AM, 187, 189, 194, 194 Marino M, 214 Markus AP, 111 Markus P, 274 Maropoulos G, 110 Marrero-Stein V, 283 Marsden D, 14, 15 Marshall J, 226 Marta¨sek P, 280 Martin JJ, 186

Martini C, 167, 225 Martins E, 60, 75 Marzullo E, 192 Matalon K, 20, 25 Matalon R, 20, 21, 28 Matern D, 96, 123, 128, 131, 178, 179 Mathew C, 194 Matsubara Y, 45, 93, 166, 269 Matsuda J, 217 Matsumoto T, 74 Matsuo N, 46, 239 Matsuoka O, 235 Matsuzaki K, 141 Matte U, 236 Matthijs G, 180, 181, 184, 185, 186, 238 Matzner U, 223 Mavroudakis N, 85 Maya A, 3 Mayatepek E, 7, 12, 107, 109, 125, 126, 165, 175, 175, 211 Mayerhofer P, 243 Mayr H, 140 Mayum M, 160 Mayumi M, 6, 161 McCabe ERB, 2, 172 McCabe LL, 2 McClean P, 57 McCormack T, 70 McDonald M, 14 McDowell IFW, 65 McEachern G, 155 McFarland R, 136 McGill JJ, 191 McKiernan PJ, 70, 278 McLaren DG, 272 McLean BN, 258 Me¨ndez-Cruz I, 87 Meadows T, 117 Medeiros DM, 25 Medina M, 60 Meenan J, 189 Mehta D, 222 Meijer AJ, 167 Meikle PJ, 19, 215, 237, 287 Meinsma R, 192 Meirelles R, 84 Meissner T, 175, 175 Meister B, 153 Melegh B, 139 Melhem ER, 251 Mello CF, 83 Menni F, 96 Mercuri L, 210 Merouani A, 69 Merriman RB, 130 Metz M, 89 Meyburg J, 12 Meyer D, 163 Meyer K, 240 Mian A, 58 Miccolis M, 97

Michelakakis H, 229, 281 Michelin K, 229 Migita M, 221, 224 Mihaelik S, 256 Miki Y, 174 Milani N, 142 Millat G, 232 Miller M, 119 Millington DS, 14, 77, 123, 130, 131 Milos I, 236 Mine M, 154 Minniti G, 64, 176 Misako I, 161 Mistry P, 226 Mitchell A, 113 Mitchell G, 54, 69, 148 Mitchell GA, 108, 202, 204 Mitchell J, 69, 259 Miura Y, 187 Miyabayashi S, 166 Miyamoto T, 71, 72 Miyao M, 262, 262 Moat SJ, 65 Moats R, 36, 36 Mochizuki D, 262, 262 Mochizuki H, 74 Mochizuki T, 266 Moench E, 70 Moerman P, 238 Moeslinger D, 285 Mo¢di S, 283 Mohnike K, 175 Molander M, 223 Molenaar WM, 269 Moller LB, 263, 263 Mo«ller HE, 28, 32 Molnar M, 139 Molzer B, 254 Montalto SA, 44 Moog U, 157 Mooijer PAW, 244, 245, 246 Moolenaar SH, 71 Moraitou M, 229 Morales C, 233 Morari L, 41, 229 Morava E¨, 139, 277 Mori M, 55 Mori R, 277 Mori S, 250 Mori Y, 266 Morimoto K, 235 Morin C, 148 Moro F, 194 Morris A, 108 Morris AAM, 136 Morris M, 273 Morrone A, 90, 105 Moseley K, 36 Moser AB, 252, 256 Moser HW, 250, 251, 252 Mo«slinger D, 165 Mowat D, 137

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

296 Moxon-Lester L, 191 Moyses C, 217 Mudd SH, 67 Mudge C, 95 Mukherji M, 258 Mull B, 58 Muller V, 235, 238 Muller VJ, 240 Mun¬oz L, 24 Muntau AC, 68, 115, 243, 255, 282 Muramatsu T, 141 Murata H, 262 Murayama K, 16, 159, 280 Muro S, 92, 92 Murphy HC, 281 Muschol N, 218 Myara A, 278 Nagano K, 172 Naidu S, 214 Naito E, 151, 152, 153 Nakamoto N, 262, 262, 266 Nakano K, 247 Nanba E, 217 Narayanans V, 214 Narisawa K, 45, 93, 166, 269 Nassogne MC, 116, 138, 154, 155, 191, 206, 246 Natowicz MR, 73 Naughten ER, 62, 63, 63, 80 Navarrete R, 89 Naylor E, 12 Ne¨meth K, 39 Nebbia G, 96 Nebesonïa¨kova¨ E, 197 Neele DM, 182, 205 Nefedov M, 94 Ne¡ MW, 39 Neilson D, 203 Nelson M, 36, 36 Nelson PV, 89 Nennstiel-Ratzel U, 5 Netik A, 253 Neto EC, 126 Netting M, 31, 283 Neu A, 212 Newbold R, 8 Nezu J, 117 Nguyen T, 122 Nicholls C, 238 Niezen-Koning KE, 167, 269 Nihoul-Fe¨ke¨te¨ C, 174 Niimi H, 16, 280 Nijtmans LG, 150 Nikkels P, 133 Ninomiya S, 151, 270, 277 Nishigaki T, 166 Nishiyori A, 52 Njlsson R, 77 Nogueira C, 149 Noher de Halac I, 144 Nonaka I, 141

Norgren S, 77 North KN, 207 Nowacka M, 47 Nowakowska I, 11 Nukazawa T, 46 Nussbaumer W, 114 Nuytinck L, 186 Nwokoro NA, 198 Nyhan WL, 50, 53 O'Brien J, 187 O'Brien JF, 178, 179 O'Brien LK, 127 O'Brien M, 80 O'Brien W, 49, 58 O'Brien WE, 60 O'Callaghan M, 222 O'Hara C, 232 O'Neill D, 135 O'Reilly L, 12 Ochiai T, 265 Odie©vre M, 163 Ofman R, 259 Ogawa A, 115, 116, 121, 265 Ogawa E, 177, 265 Ogawa Y, 151, 152 Ogier H, 54 Ogino I, 239 Ohashi T, 71, 216, 286 Ohigashi I, 152 Ohkawa H, 239 Ohlenbusch A, 150 Ohno K, 217 Ohnstad C, 95 Ohtake A, 16, 55, 115, 116, 159, 280 Ohura T, 45, 161, 166, 174,177 Okada S, 141, 166 Okano Y, 46, 159, 173 Okuyama T, 239 Olgemo«ller B, 5, 90, 115 Olgemo«ller K, 125 Oligny L, 202 Oliveira CR, 145 Oliveira LM, 145 Oliveira LS, 82 Oller Ram|¨ rez AM, 219, 220 Olpin SE, 129, 135 Olsen RKJ, 134 Oltho¡ K, 130 Ondrova¨ L, 268 Onkenhout W, 173 Oostheim W, 120, 199, 248 Orii KE, 113, 132 Orii T, 241, 243 Osawa M, 247 Oshima A, 217 Osteim W, 249 Osumi T, 243 Oswald MJ, 213 Ott J, 190 Oudshoorn K, 34, 35 Overmars H, 210, 248, 256

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

O«zen H, 265 O«zgu«c° M, 104, 265 Pa©mpols T, 3 Pacheco-Alvarez D, 88 Packman S, 95, 136 Packman W, 95 Padilla 18 C, Paik MJ, 275 Palmer DN, 213 Palmieri F, 120, 120 Pampols T, 195 Pan L, 202 Papachristou F, 193 Papouli M, 57 Paradis K, 69 Pardo ML, 23 Parini R, 96, 121 Parizeau G, 69 Parsons PG, 207 Parsons R, 222 Parvari R, 74 Paschini-Capra A, 144, 165 Paschke E, 236 Pasquali M, 61 Pasquini E, 105 Passarge M, 176 Pastore L, 58 Pastores G, 226 Patch D, 94 Paton BC, 240, 243 Patsch W, 144 Patterson AL, 84 Paul H, 129 Paul K, 236 Pavone V, 181 Pe¨rez B, 89, 92, 92 Pe¨rez-Cerda¨ C, 89, 92, 92 Peduto A, 38 Pelletier L, 69 Pen¬alva MA, 89 Pena R, 75 Pennisi P, 181 Penzien JM, 107 Pereira C, 149 Pereira J, 75 Pereira MLS, 41, 229 Perkins K, 238 Pernber Z, 223 Pesíkova¨ K, 75, 170 Pessoa-Pureur R, 84 Peters C, 234 Peters H, 90, 94, 98, 164 Peters V, 183 Peterson R, 20 Peterson SM, 184 Pettit-Kekel K, 196 Phan V, 69 Phelps R, 222 Philippart M, 214 Phillips IR, 281 Piana A, 64 Picco P, 109, 176

297 Piccoli D, 130 Piccolruaz U, 153 Pie¨ J, 107 Piekutowska-Abramczuk D, 145 Pietsch M, 26 Pilgaard B, 124 Pineda J, 202 Pineda M, 180, 202 Pinkert CA, 131 Pin¬ol F, 55, 273 Pires RF, 41, 229, 236 Pittock S, 63 Plante V, 147 Platt FM, 216, 217 Platt L, 20 Platt R, 281 Plecko B, 209 Ploechl E, 52 Ploechl W, 52 Ploos van Amstel HK, 45 Ploos-van Amstel JK, 73 Podskarbi T, 165, 169, 170 Poll-The BT, 45, 73, 100, 133, 171, 196, 210, 245, 245 Pollitt RJ, 267 Pontes ZL, 82 Ponzone A, 38 Poplawski NK, 10 Popowska E, 145, 171 Porter FD, 198 Pospisilova E, 75 Poulsen L, 263 Poup|© tova¨ H, 219 Pourfarzam M, 84, 128 Poustie VJ, 22 Power R, 232 Powers HJ, 65 Poyser KH, 284 Praphanphoj V, 78, 91 Prasad C, 19 Preece MA, 278 Preu¨ N, 247 Prieto L, 23 Prip-Buus C, 19 Procha¨zkova¨ L, 264 Pronicka E, 11, 145, 161, 171, 179, 184 Pronicki M, 145, 184 Proulx F, 54 Prudente S, 210 Pshezhetsky AV, 233 Puga AC, 67 Qin W, 130 Quant P, 108 Quash M, 69 Quelhas D, 183 Qureshi IA, 51, 54 Raas-Rothschild A, 246 Rabier D, 97, 116, 246 Rabl W, 175 Race V, 191

Radomyska B, 179 Raha S, 155 Rahman S, 139, 142 Raiman JAJ, 53 Raimann E, 24, 81, 85 Rajagapolan KV, 77 Rake JP, 162, 167 Ramaekers VTh, 42, 207 Ramsay S, 239 Ramsey SL, 287 Rand E, 130 Randall R, 34 Ranieri E, 3, 89, 215, 239, 287 Rascher W, 90 Rating D, 211 Ravenscroft EM, 215, 287 Raymond GV, 250, 251 Rayner D, 232 Rebelo O, 134, 145 Reedsand P, 49 Rees DC, 18 Rees JE, 258 Regimbald D, 69 Reichardt JKV, 159 Reijngoud D-J, 1 Reilly M, 257 Renda Y, 104 Rhead WJ, 20, 131 Ribeiro L, 106 Ribes A, 89, 103, 121 Rimoldi M, 103, 114, 132 Rinaldo P, 69, 96, 127, 128, 129, 131, 178, 269, 275 Rischewski J, 17, 17 Risto¡ E, 77 Riudor E, 55, 273 Riva E, 21, 30 Rivera I, 40, 69, 103 Rivie©re A, 278 Rizzo C, 79, 241, 249 Roasio L, 38 Robert J-J, 174 Robert M-F, 108, 202 Roberts J, 95 Robertson EF, 31 Robinson BH, 8, 148, 155 Robitaille Y, 204 Rocha BB, 84 Rode¨s M, 55, 56 Rodr|¨ guez JM, 89 Rodr|¨ guez de Co¨rdoba S, 89 Rodr|¨ guez-Mel¨ndez S, 87 Rodr|¨ guez-Pombo P, 89, 92, 92 Roels F, 242, 246, 249 Rogaszewska M, 179 Rokicki D, 184 Rolland MO, 246 Rolles K, 94 Romeijn GJ, 108, 199 Romstad A, 28, 41, 46 Roos B, 172 Ro«per J, 212 Rosaklis T, 287

Roscher AA, 4, 5, 5, 52, 68, 90, 115, 243, 255, 282 Ro«schinger W, 68, 115, 282 Rosenberg M, 222 Rosenberger J, 49 Rosenbloom B, 226 Rosenthal M, 87 Rosenthal P, 95 Rosipal R, 280 Ross L, 283 Rossi G, 96 Rouse B, 20, 20 Rouse BM, 21, 28 Rozengurt N, 60 Rubio ME, 45, 157 Rubio V, 50, 55 Rudnicki MA, 202 Rudolfova¨ J, 170 Rudolph G, 282 Ruggiero M, 196 Ruitenbeeck W, 56 Ruiter JPN, 113, 120, 124, 124, 133 Ruiters MHJ, 269 Rumsby G, 260, 261 Rusch H, 257 Rushe H, 63 Rutherford P, 22 Ryan A, 63 Ryan S, 281 Saada A, 140 Saatc°i I, 104 Sabetta G, 241, 249 Sacksteder KA, 78 Saheki T, 56, 56, 57 Saijo S, 152 Saijo T, 151, 153 Saito F, 46 Saito T, 71 Sakai N, 141 Sakamoto O, 45, 93, 174, 177 Sakr A, 41 Sakuraba H, 224, 224 Sakuragawa N, 239 Sala F, 96 Salleng K, 129 Salo M, 59 Salvioli R, 218 Samuel D, 162 Sanderson J, 189 Sandju J, 48 Santer R, 17, 168, 176, 178 Santos CES, 81 Santos M, 149 Santos P, 145 Santos Silva E, 60 S°arbat G, 86, 113, 170 Sarnavka V, 118 Sasaki N, 16, 280 Sass JO, 114, 153 Sasvari-Szekely M, 270 Sau J, 3

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

298 Saudubray JM, 97, 116, 120, 138, 154, 155, 174, 206, 246 Savary S, 252 Sawnor-Korszynska D, 161 Scacheri C, 214 Scaglia F, 49 Scaglioni S, 21 Scaria A, 226 Scarpini C, 221 Schadewaldt P, 156, 275 Schaefer F, 175 Schaefer JR, 183 Schaub J, 17, 168, 176, 178 Schelley S, 136 Schenk B, 186 Schenone A, 13, 106 Scherhammer V, 140 Schestag F, 223 Schia¤no MC, 33 Schlegel W, 32 Schmidt A, 212 Schmidt B, 215 Schmitz W, 247 Schneppenheim R, 17, 17, 168, 176, 178 Scho¢eld CJ, 258 Schollen E, 181, 238 Schor DSM, 76 Schots H, 251 Schrade S, 242 Schro«der JM, 169 Schroeder LD, 124 Schuchman EH, 230 Schuette J, 214 Schuler A¨, 39 Schultz R, 59 Schulze A, 7, 12, 125, 126, 211 Schutgens RBH, 110, 110 Schwab KO, 212 Schwahn B, 66, 80 Schwartz IVD, 67, 236 Schwartz M, 182 Schweitzer-Kranz S, 157 Scott CR, 226, 272 Scott R, 69 Scriver C, 69, 112 Seashore GR, 266 Seashore MR, 288 Seaver LH, 228 Síebesta I, 190, 193 Seed P, 189 Seino Y, 151, 270, 277 Seneca S, 78, 251 Sent|¨ s M, 55, 273 Seto T, 235 Severini G, 167 Sexton M, 283 Seyda A, 8 Sharp P, 19, 237 Sharrard M, 116 Shehata BM, 128 Sheiman C, 249 Shekhawat P, 128

Shen M, 43 Shephard EA, 281 Sherr E, 95 Sherwin J, 87 Shibuya K, 264 Shiga K, 262 Shigematsu Y, 6, 160 Shih K, 112 Shih V, 14, 15, 70, 72 Shimada T, 221, 224 Shimizu N, 264 Shimozawa N, 241, 243, 247 Shimozawa T, 245 Shin V, 112 Shin YS, 159, 160, 169, 170, 276 Shinnar M, 222 Shintaku H, 46, 235 Shobowale-Bakre M, 189 Shorer Z, 255 Shortland GJ, 59 Sidey M, 259 Siitonen A, 59 Sikora D, 196 Silano M, 30 Silva A, 75 Silva FB, 41 Silva LCS, 41 Silva MFB, 131 Silva-Zolezzi I, 256 Sim KG, 15, 16 Simmonds AH, 193 Simmonds HA, 187, 190, 193, 194, 194 Simonis-Bik AMC, 56 Simonneau G, 162 Simonsen H, 124 Sims HF, 127, 130, 133 Sinahara K, 152 Singal R, 19 Singh RH, 61, 158, 285 Siranni N, 214 Sirrs SM, 287 Siska T, 39 Sjarif DR, 171 Skjedal OH, 259 Skladal D, 137, 138 Síkodova¨ J, 197 Skopkova Z, 75 Skovby F, 117, 124, 168, 178, 182, 184 Slama A, 147, 147, 154 Slova¨cíkova¨ R, 170 Sly WS, 216, 286 Smail D, 268 Smeets E, 186 Smeitink JAM, 56, 198 Smet J, 141 Smit GPA, 1, 162, 167, 269 Smit LME, 182 Smit W, 199, 201 Smith D, 216 Smith M, 57 Smith WE, 14

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Smyth RL, 22 Snead OC, 110 Snodgrass M, 18 Socha P, 179 Solo¨rzano-Vargas RS, 88 Somogyi CS, 39 Song X-Q, 113 Sorensen N, 117 Soriano M, 103 Souza CFM, 67 Souza DO, 81 Spaapen LJM, 45, 157 Spada M, 38 Spe¨cola N, 149 Sperl W, 140, 144, 171 Sípicíkova¨ K, 152 Spiekerko«tter U, 156 Stabler SP, 64, 67 Stacy C, 222 Stanley C, 119 Stanley CA, 108, 120 Sít'astna¨ S, 75, 170 Staub M, 270 Steen MT, 61 Steenbergen GCH, 201 Stege AJ, 86 Steiner R, 158, 187 Steiner RD, 61, 196, 197, 198 Steinfeld R, 212, 218, 240 Steinmann B, 111, 276 Stern A, 20, 36 Stern L, 283 Steuerwald U, 168 Stevens RD, 77 Stibu©rkova¨ B, 190, 193 Sto«ckler-Ipsiroglu S, 52, 144, 157, 165, 209, 285 Sítorka¨nova¨ G, 254 Strauss AW, 127, 128, 129, 130, 130, 133 Streck EL, 83 Strobl W, 144, 157 Strumlauf E, 285 Struys EA, 76, 172, 209, 209 Sudo M, 6 Sugie H, 168, 169 Sugie Y, 168, 169 Sugiyama N, 45 Síulc V, 268 Sullivan J, 226 Sumi S, 51 Suormala T, 68, 88, 93 Superti-Furga A, 115, 176 Suppiej A, 37 Suzuki, 45 Suzuki S, 45 Suzuki T, 243, 265 Suzuki Y, 93, 166, 217, 241, 245, 247, 269 Svatos J, 64 Sweatt JD, 204 Sweeney A, 31, 283 Swift P, 178

299 Swinkels D, 111, 274 Swoboda KJ, 73 Sykut-Cegielska J, 11, 179 Syrova¨ D, 264 Tabata A, 56, 57 Takada G, 230, 231, 231 Takahashi H, 224 Takahashi I, 230, 231 Takahashi K, 166, 221, 224, 264 Takahashi T, 230, 231, 231 Takahashi Y, 132 Takaura N, 238 Takayanagi M, 55, 115, 116, 121 Taki T, 174 Takiyama N, 224 Takusa Y, 132 Talbaoui H, 11 Tallini K, 82 Tamai H, 277 Tamaro G, 225 Tanaka A, 235, 238 Tanaka K, 46, 265 Tanaka M, 9, 24 Tanaka Y, 6, 46 Tang NLS, 253 Tangerman A, 67 Tanner MS, 135 Taroni F, 114, 118, 121, 132 Tasca CI, 81 Tashiro K, 9, 24 Tashiro S, 9, 24 Tatti M, 218 Tavares RG, 81 Tavares de Almeida I, 40, 40, 69, 103, 131 Taybert J, 161, 171 Taylor HA, 228 Taylor M, 110, 110 Taylor R, 19, 136 Tein I, 118, 119 ter Harmsel A, 269 Teraoka M, 151, 270, 277 Ternes J, 31 Terpstra P, 269 Than K-A, 18 Theaux R, 144 Theelen JP, 201 Thiel T, 212 Tho«ny B, 42, 42, 43 Thomas G, 256 Thomas J, 98 Thomas PK, 257 Thompson GN, 108 Thompson JRG, 19 Thong MK, 29 Thorburn DR, 135, 137, 138, 143, 143 Thuillier L, 19 Tiedemann K, 164 Tiefenthaler M, 157 Till J, 129 Tiranti V, 142

Tocha©e©kova¨ M, 219 Togari H, 51 Toietta G, 58 Tokath A, 195 Tokunaga Y, 52 Toms M, 287 Toone JR, 76 Topalogïlu H, 104 Topc°u M, 104 To«ro«s I, 39 Toth G, 139 Touati G, 97, 116, 138, 154, 155, 246 Touma EH, 41 Town MM, 194 Trasler J, 202 Trbusíek M, 170 Treacy E, 69 Treacy EP, 281, 282 Trefz F, 20, 21, 28, 47, 54, 93 Trefz FK, 8, 198 Trijbels JMF, 56, 111, 269, 274 Trioche P, 163, 278 Troxler H, 10, 115 Tschiedel E, 59 Tsuji A, 117 Tsujino S, 71, 72 Tsukamoto H, 141 Tsukamoto T, 243 Tu«mer Z, 263 Turecek F, 272 Turecky¨ L, 264 Turnbull DM, 128, 136 Turnbull J, 239 Turner C, 271 Tyni T, 128 Ubuka T, 286 Uchiyama A, 132 Ueta A, 51 Ugarte M, 89, 92, 92 Ullrich K, 28, 100, 164, 212, 218, 240 Umansky R, 214 Umapathysivam K, 215 Unterrainer G, 254 Utermann G, 165 Uziel G, 103, 142 v Rijn M, 162 v Spronsen FJ, 162 v Weihe M, 17 Va¨radi I, 39 Vaccaro AM, 218 Valianpour F, 150, 210 Valiente A, 24, 85 Vallance H, 50, 146 Valle D, 88, 244, 256 Vamos E, 85 Van Cleemput J, 238 van Coster R, 141, 186 van Cruchten A, 188, 206 Van Dam D, 222

Van Damme B, 98 van den Berg GB, 71 van den Berg IET, 73 Van den Berghe G, 191 van den Heuvel LPWJ, 201 van der Knaap MS, 172, 182, 205 van der Meer SB, 206 van der Wijk N, 65 van Diggelen OP, 215 van Engelen BGM, 201 Van Geet C, 205 van Gennip AH, 86, 187, 188, 192, 192, 194, 206, 206 van Grunsven EG, 248, 248, 249, 249 Van Hove J, 98, 238 van Kuilenburg ABP, 188, 192, 192 van Lenthe H, 188 van Lint L, 125, 248, 257 van Oppen C, 73 van Rijn M, 34, 35 Van Schaftingen E, 180, 181, 184, 186 van Spronsen FJ, 34, 35 van Weely S, 217, 229 van Woerden CS, 260 van't Ho¡ WG, 94 VanCamp S, 166 Vanier MT, 232 Vargas CR, 82 Vargas Poussou R, 97 Vela¨zquez A, 87 Velter C, 45 Vendetti C, 130 Venditti CP, 97, 234 Venditti LN, 234 Vendrell T, 195 Veneselli E, 33 Verduci E, 21, 30 Verhaaren H, 105 Verhoek M, 229 Verhoeven NM, 172, 205, 211 Verkerk PH, 34, 35 Vermylen J, 205 Verrips A, 201 Vianey-Saban C, 11, 123 Videla MP, 279 Vieregge P, 183 Vilain E, 119 Vilarinho L, 60, 75, 106, 149, 183 Vilaseca MA, 33, 35, 56, 180, 202 Vincent MF, 191 Vincon V, 183 Visser G, 162, 167 Visser GHA, 65 Vladutiu CJ, 29 Vladutiu GD, 268 Vliegenthart J, 186 Vobruba V, 140 Vockley J, 122, 123, 131 Voelker C, 186 Voit T, 177

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

300 von Figura K, 185, 215 von Schenck U, 70 Vorgerd M, 169 Vrdoljak J, 118 Vreken P, 86, 124, 125, 150, 192, 199, 201, 210, 244, 248, 248, 256, 257, 258 Vu TH, 149 Wada Y, 51 Wagner C, 67 Waisbren SW, 6 Wajner A, 229, 236 Wajner M, 81, 82, 82, 83, 83, 84 Walczak M, 11, 47 Walter C, 245 Walter JH, 48, 48, 129, 194 Wanders RJA, 86, 107, 108, 109, 109, 113, 120, 124, 124, 125, 125, 126, 131, 133, 150, 196, 199, 201, 241, 244, 245, 245, 246, 248, 248, 249, 249, 256, 257, 257, 258, 259, 260 Wang JJ, 7 Wang SP, 202 Wang W, 23 Wang Y, 117, 118 Wang Z, 120 Wannmacher CMD, 82, 82, 83 Wanrooij S, 107 Wappner R, 226 Warman M, 203 Warwick L, 164 Wassermann GF, 82 Wassif CA, 198 Watanabe W, 230 Waterham HR, 107, 108, 109, 120, 124, 192, 196, 199, 201, 245, 246, 248, 249, 256, 258, 259 Watkins P, 256 Weatherall DJ, 18 Webster ADB, 190 Weeber E, 204 Weglage J, 26, 28, 32 Weinhofer I, 253 Weinreb N, 226 Weisiger K, 95, 136 Weller S, 247 Wenchich L, 140, 152 Wendel U, 17, 66, 80, 156, 185, 275 Wermuth B, 52 Weston PF, 234

Westphal V, 184 Wevers R, 98, 185, 274 Wevers RA, 56, 71, 111, 198, 201, 206, 238 Whit¢eld PD, 19, 237, 287 Whittle AM, 215 Wibrand F, 137 Wicks G, 283 Wierzbicki AS, 258, 259 Wijburg FA, 124, 125, 260 Wilcken B, 4, 15, 16, 79, 99, 110 Wilcox KA, 1 Wild K, 39 Wiley V, 4, 15, 16 Wilichowski E, 150 Willems HL, 111, 274 Willenbring H, 59, 233 Williams M, 49, 100 Wilsbech M, 122 Wilson CJ, 142 Winchester B, 237 Winkler A, 77 Winston J, 222 Winter SC, 95 Winter V, 122, 124 W|¨ tro¨bska S, 11 Wittenstein B, 164 Wolf B, 86 Wolf M, 163 Wol¡ J, 20 Wong B, 211 Wong LTK, 31, 50 Wood PA, 131, 256 Wood S, 32, 38 Worthington S, 110 Wraith JE, 68, 129 Wyse ATS, 82, 82, 83 Yaghmai R, 67 Yalaz K, 104 Yamada M, 141 Yamada S, 24 Yamada T, 71 Yamaguchi A, 74 Yamaguchi K, 264 Yamaguchi S, 74, 132, 241, 275 Yamaguchi Y, 264 Yamamoto H, 74 Yamamoto S, 46, 55, 115, 116, 121, 265 Yamano T, 159, 173, 235, 238 Yamashita S, 151, 270, 277 Yamuguchi S, 161

J. Inherit. Metab. Dis. 23 (2000) Suppl. 1

Yanagawa Y, 262, 262, 266 Yang H, 281 Yang X, 93 Yang XW, 22 Yannicelli S, 25 Yap S, 62, 63, 63, 80 Ye J, 7 Ye Jun, 44 Yeung WL, 253 Yew NS, 223 Yin C, 173 Yogalingam G, 235 Yokoo T, 216 Yokota I, 152, 153 Yokota L, 151 Yokoyama Y, 151, 270, 277 Yoo O, 261 Yoo PK, 60 Yoon H-R, 159, 275, 276 Yoshida I, 9, 24 Yoshii N, 225 Yoshino M, 52 Yotsumoto S, 225 Young SP, 123 Yu WM, 23, 43 Yu«ce A, 265 Zach L, 249 Zammarchi E, 90, 105 Zçekanowski C, 47 Zelezny R, 175 Zeman J, 64, 152, 254, 280 Zemen J, 140 Zeng Q, 49 Zeviani M, 142 Zhang W, 190 Zhang XZ, 23 Zhang YH, 172 Zhang Z, 243, 247 Zhao Y, 129 Zhou X, 272 Zhou ZS, 23, 43 Zicha J, 268 Ziegler R, 223, 226 Zimran A, 217 Zobel M, 254 Zschocke J, 107, 109, 125, 126 Zugno ZI, 83 Zvarova J, 64 Zwitkowska E, 179 Zytkovicz T, 14, 15