J Inherit Metab Dis (2008) 31 (Suppl 1) DOI: 10.1007/s10545-008-9975-0

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Contents 015-P Sarcosinaemia: a case report with evolutive neurologic symptoms R Garnotel, A Kuvbachieva-Bennarosh, S Bakchine, P Gillery

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016-P N-Acetylamino aciduria: a benign biochemical ¢nding! J Calvin, RA Wevers, U Engelke, W Lissens, J Perrier, T Giardina, JE Smet, S Hogg, L Abulhoul, V Puthi, RN Van Coster

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017-P Development of a succinylacetone method for use as a potential newborn screening test for tyrosinaemia type I S Dowden, R Webster, MA Preece, P Gissen

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018-P Tyrosinemia type I: hepatocellular carcinoma in patient with new liver lesion without increase of AFP FJ van Spronsen, A Gauw, EJ van der Jagt, KP de Jong, HJ Verkade

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019-P Epilepsy and electrophysiological abnormalities in children with hereditary tyrosinaemia type 1 S Santra, P Mckiernan, S Seri, E Wassmer

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020-P Pharmacokinetics of NTBC (nitisinone) following a single dose to 5 children with tyrosinemia type 1 M Pohorecka, M Pohorecka, Z Wawer, M Filipek, J Sykut-Cegielska, E Pronicka

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021-P Determination of NTBC in serum samples from patients with hereditary tyrosinemia type I by capilary electrophoresis MS Cansever, AC Aktuglu-Zeybek, FB Erim

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022-P Unusually severe gyrate atrophy causing unilateral blindness in a 12 year old girl C Barnett, W Lam, A Schulze

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023-P Evidence that homocitrulline and ornithine compromises bioenergetics in cerebral cortex of young rats CM Viegas, AM Tonin, A Zanatta, L Knebel, ATS Wyse, M Wajner

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024-P Evidence for the founder origin of 117delC MSUD mutation in Portuguese Gypsies S Quental, A Gusma¬o, L Vilarinho, A Amorim, MJ Prata

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025-P Comparison of managements of metabolic decompensation in maple syrup urime disease by plasma exchange and nutritional measures in siblings J Okada, M Yoshino, Y Watanabe

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026-P Pancreatic beta cell reserve and insulin sensitivity in maple syrup urine disease A Dursun, YU Sar|kabaday|, ZA Ozon

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01. Amino Acids 001-P Implementation of a new approach for the quantitative analysis of amino acids in human physiological £uids using UPLC S Bekri, B Hecketsweiler, A Paccou, P Hong, TE Wheat 002-P Sensitive and rapid analysis of amino acid concentrations in CSF by stable isotope dilution LC-MSMS NM Verhoeven-Duif, M Van der Ham, BHCMT Prinsen, R Berger, L Klomp, MGM De Sain-Van der Velden 003-P Ion exchange chromatography, an `old' technique for prompt NKH detection: 3 case reports C Ver|¨ ssimo, M Simo¬es, A Estevinho, P Garcia, L Diogo, CR Oliveira, M Grazina 004-P The signi¢cance of amino acids analyzes as an indicator of protein-caloric intake in patients on low protein diet T Honzik, M Magner, S Sulek, P Jesina, E Hruba, P Chrastina, J Zeman 005-P LCPUFA status in patients with di¡erent inborn errors of metabolism MA Vilaseca, O Ayllon, J Moreno, R Artuch, N Lambruschini, L Go¨mez, A Gutierrez, J Campistol

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006-P Methionine induces oxidative stress in liver and brain of young rats FM Stefanello, CD Pederzolli, AG Kurek, CB Mattos, J Kolling, CS Dutra-Filho, CMD Wannmacher, M Wajner, ATS Wyse

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007-P Cystinuria prevalance in an Anatolian city and e¤ciency of sodium nitroprusside test in screening programmes Tunc°, B Hizel, F Tanzer, Albayrak

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008-P Sul¢te oxidase and molybdenum cofactor de¢ciency: EEG, metabolic and neuroradiologic study D Martinelli, P Mariotti, MC Stefanini, D Battaglia, D Antuzzi, C Veredice, A Sacco, F Guzzetta, E Mercuri

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009-P Serine synthesis defects: a treatable cause of microcephaly V Valayannopoulos, F Raqbi, D Rabier, V Abadie, I Desguerre, P de Lonlay

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010-P Diagnosis of glutathione synthetase de¢ciency by newborn screening using tandem mass spectrometry E Thimm, R Fingerhut, E Risto¡, E Mayatepek, U Spiekerko«tter

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011-A Neonatal non-ketotic hyperglycinaemia: a case with a challenging diagnosis E Martins, I Carrilho, P Rocha, M Santos, ML Cardoso, R Chora¬o

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027-O Interference of valproic acid on the branched chain amino acid oxidative metabolism PBM Luis, JPN Ruiter, L IJlst, R Ofman, L Diogo, P Garcia, M Duran, J Vockley, I Tavares de Almeida, RJA Wanders, MFB Silva

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012-P The neonatal case diagnosed with nonketotic hyperglycinemia with a preliminary diagnosis of the chloralhydrate intoxication I Okur, FT Eminoglu, L Tumer, FS Ezgu, G Biberoglu, A Hasanoglu

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013-P Glycine induces lipid oxidative damage and reduces the antioxidant defenses in brain cortex of young rats G Leipnitz, A Solano, B Seminotti, AU Amaral, CG Fernandes, LA Knebel, CMD Wannmacher, CR Vargas, M Wajner

028-P Functional analysis of the nucleotide variation c.288 +9c4t identi¢ed in the BCKDHA gene of a variant MSUD patient P Rodr|¨ guez-Pombo, K Weisiger, S Packman, R Navarrete, M Ugarte

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014-P Lysine inhibits creatine kinase activity and induces oxidative stress in rat brain B Seminotti, AM Tonin, G Leipnitz, PF Schuck, G da Costa Ferreira, AU Amaral, A Solano, M Grings, LA Knebel, CMD Wannmacher, ATS Wyse, M Wajner

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029-O Hepatocyte transplantation (HTx) extends lifespan, increases branched chain alpha-keto acid dehydrogenase (BCKDH) activity, and improves amino acid pro¢les in a murine model of intermediate maple syrup urine disease (iMSUD) KJ Skvorak, HS Paul, K Dorko, F Marongiu, D Chace, C Ferguson, KM Gibson, GE Homanics, S Strom

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J Inherit Metab Dis (2008) 31 (Suppl 1) 044-P Search for pheno-and genotypical conformities in folate cycle defects beyond the usual genetics OY Grechanina, RV Bogatirova, RK Matalon, BB Holmes, YB Grechanina, VA Gusar

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045-O Mutations in cystathionine gamma-lyase gene and their phenotypic consequences V Kozich, B Janosikova, RA Hegele, C Jakobs, SP Stabler, RH Allen, JP Kraus

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046-P Severe neonatal MTHFR de¢ciency, report of a new case with composite mutations C Chery, S Audonnet, T Josse, B Chabrol, F Feillet, JL Gueant

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047-P Venous thrombosis secondary to congenital intrinsic factor de¢cient secretion in a vegetarian young male C Besseau, C Chery, J Devignes, F Namour, F Feillet, JL Gueant

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048-P Homocysteine/folate metabolism in Italian children with Down syndrome I Scala, B Granese, F Sampietro, S Salome©, E Scarpato, C Figliuolo, S Paladino, A Zuppaldi, A D'Angelo, G Andria

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049-P Variation and expression of dihydrofolate reductase (DHFR) in relation to spina bi¢da IJM van der Linden, SH Heil, B Franke, HJ Gellekink, M den Heijer, HJ Blom

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050-P Clinical and molecular manifestations of Taiwanese patients with homocystinuria YH Lu, HC Yu, MY Lo, JH Gao, KW Chong, CH Kao, KH Cheng, TT Liu, NC Lee, DM Niu

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02. Homocysteine 030-O Biochemical and molecular neonatal screening for homocystinuria in the Qatari population T Ben-Omran, H Gan-Schreier, M Kebbewar, J Wilrich, G Abdoh, N Shahbek, C Fischer, B Janssen, H Al Rifai, M Lindner, J Zschocke, G Ho¡mann 031-A Dietary compliance in later diagnoses homocystinurics patients BJ Frangipani, C Micheletti, RB Oliveira, JR Lauandos, CSC Mendes, MH Rand, T Vertemati, V D'Almeida, AM Martins 032-O No longer just an intermediate: cystathionine protects against endoplasmic reticulum stress induced lipid accumulation and apoptotic cell death KN Maclean, H Jiang, S Lhotak, RC Austin, LS Greiner 033-P In vitro e¡ect of S-adenosyl homocysteine on H19 gene expression R Castro, A Gomes, M Rocha, R Esse, N Clode, LM Grac°a, I Rivera, HJ Blom, C Jakobs, I Tavares de Almeida 034-P Behavioral analysis and hippocampal gene expression pro¢ling in a mouse model of CBS de¢ciency reveals epigenetic e¡ects leading to misexpression of homeobox genes KN Maclean, JR Evans, SP Stabler, RH Allen, H Jiang 035-O S-adenosyl homocysteine hydrolase inhibition a¡ects protein and DNA methylation in HUVEC R Castro, R Esse, M Rocha, I Gonc°alves, N Clode, LM Grac°a, I Rivera, T Teerlink, C Jakobs, HJ Blom, I Tavares de Almeida

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036-O Inhibition of methylation and changes in gene expression in relation to neural tube defects IJM van der Linden, SH Heil, M van Egmont Petersen, HW van Straten, M den Heijer, HJ Blom

051-P The high incidence of homocystinuria in the Tao tribe of Taiwan YH Lu, HC Yu, MY Lo, JH Gao, JH Hsu, KW Chong, CH Kao, KH Cheng, TT Liu, PY Hung, DM Niu

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037-P Global DNA methylation ^ comparision of two di¡erent methods M Rocha, R Castro, R Kok, I Rivera, C Jakobs, Y Smulders, I Tavares de Almeida, HJ Blom

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052-O CblD defect of vitamin B12 metabolism: molecular mechanism whereby mutations in the MMADHC gene cause three di¡erent phenotypes D Coelho, M Stucki, T Suormala, MR Baumgartner, B Fowler

038-P Homocysteine and MTHFR polymorphisms in a population of healthy adolescents from Madeira Island H Caldeira Arau¨jo, A Pimenta, R Ornelas, I Rivera, R Castro, I Torres, I Tavares de Almeida 039-P Global DNA methylation measured by liquid chromatography ^ tandem mass spectrometry: analytical technique, normal values and determinants in healthy subjects RM Kok, DEC Smith, HJ Blom, R Barto, AMW Spijkerman, T Teerlink, C Jakobs, YM Smulders

03. Organic Acids 10

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040-P Does C776G polymorphism in the TCN2 gene a¡ect vitamin B12 status? A pilot study in a Portuguese healthy population R Castro, M Barroso, A Pereira, R Ramos, I Rivera, I Tavares de Almeida 10 041-P Endothelial dysfunction in patients with homocystinuria and their parents B Caliskan, H O£az, M Demirkol, T Baykal, I Ozer, N Polat, A Gurdal, G Gokcay 042-P Methionine, homocysteine and methylmalonic acid status in 9 patients with cobalamin or folate de¢ciency M Schi¡, JF Benoist, O Rigal, O Fenneteau, H Ogier de Baulny 043-P Methylentetrahydrofolate reductase de¢ciency presenting with hemolytic uremic syndrome A Ku«ster, C Chery, JM Alberto, S Audonnet, T Josse, G Roussey, E Allain-Launay, I Tea, D Darmaun, JC Roze, F Feillet, JL Gueant

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053-P Urease method for urine organic and amino acid analysis: a powerful extension of an established method V Young, S Lo, WJ Rhead, J Shoemaker, A Thomas 14 054-P Organic acid disorders detected in the Brazilian pediatric population from 1994 to 2007 M Wajner, DM Coelho, ENB Busanello, R Ingrassia, AB Oliveira, AM Carvalho, TM Silva, RF Pires, CF Souza, R Giugliani, CR Vargas

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055-P Organic acidurias in high risk Egyptian patients E Fateen, A Gouda, HJ Boehles

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056-P Life-threatening episodes of hypoglycemia and altered consciousness observed in patients treated with pivalategenerating antibiotics: a new case and review Y Watanabe, T Inokuchi, K Tashiro, K Aoki, J Okada, T Matsuishi, M Yoshino

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057-O Are the current methods of treatment for methylmalonic acidemia adequate? K Michals-Matalon, K Bilger, N Ross, D Freedenberg, R Matalon

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058-P Blood spot methylmalonic acid as a second-tier test for tandem mass newborn screening with propionylcarnitine PW Chen, YH Chien, WL Hwu, LC Chen, SC Tseng

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059-P Methylmalonic academia: report of 78 cases and review of the literature S Farshidi, S Saber, M Hoshmand 15

J Inherit Metab Dis (2008) 31 (Suppl 1)

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060-P Neuroimaging patterns in glutaric aciduria type I: the value of functional techniques in magnetic resonance imaging B Pe¨rez-Duen¬as, A De la Osa, A Navarro-Sastre, A Capdevila, A Leist, A Ribes, A Garc|¨ a-Cazorla, M Pineda, J Campistol

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061-P Two patients with glutaric aciduria type I and infantile spasm A Reims, D Papadopoulou, J Lundgren

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062-P Encephalopatic crisis in glutaric aciduria type I. Are seizures or dystonic atacks a common symptom? J Campistol, A Cerisola, B Pe¨rez-Duen¬as, P Poo, M Pineda, A Garc|¨ a-Cazorla, FX Sanmart|¨ , A Ribes, MA Vilaseca 16 063-P Unreliability of plasma creatinine and the Schwartz formula to identify renal impairment in methylmalonic aciduria B Ko«nig, MA Cleary, PT Clayton, JV Leonard, W van't Ho¡, S Gru«newald

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064-O Methylmalonyl-CoA mutase expression in rat brain suggests autonomous methylmalonate production in CNS D Ballhausen, L Bonafe¨, O Boulat, O Braissant 16 065-P Functional and structural impact on ATP:cob(i)alamin adenosyltransferase of the I96T cblb mutation associated to cobalamin responsiveness B Perez, C Aguado, Lr Desviat, A Jorge-Finnigan, R Banerjee, M Ugarte

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066-P Defect of mitochondrial acetyl-CoA thiolase (MAT): a case report R Garnotel, N Bednarek, P Morville, P Gillery

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067-O Characterization of a weak but speci¢c e¥ux transport of glutaric, 3-hydroxyglutaric, and methylmalonic acid across the blood^brain barrier SW Sauer, JG Okun, A Mahringer, G Fricker, S Koelker, MA Morath 17 068-A E¡ect of ketone bodies on oxidative stress parameters in brain from young rats AP Beskow, CG Gonc°alves, G Leipnitz, B Seminotti, LB da Silva, M Grings, ATS Wyse, M Wajner 069-O 3-Hydroxyisobutyric aciduria due to methylmalonate semialdehyde dehydrogenase de¢ciency JO Sass, M Walter, JPH Shield, S Henger, C Ko«nig, AM Atherton, U Garg, D Scott, CG Woods, LD Smith 070-P Intracerebroventricular administration of isovaleric acid reduces Na+,K+-ATPase activity and induces oxidative damage in cerebral cortex of young rats CAJ Ribeiro, G Leipnitz, B Seminotti, AU Amaral, G de Bortoli, M Wajner

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071-P Greater vulnerability of brain to oxidative damage induced by the metabolites accumulating in 3-hydroxy-3methylglutaric aciduria compared to the liver G Leipnitz, B Seminotti, CG Fernandes, CAJ Ribeiro, CAJ Ribeiro, AU Amaral, AP Beskow, LB da Silva, A Zanatta, CS Dutra-Filho, M Wajner 18 072-O Dynamic changes of striatal and extrastriatal MR abnormalities in glutaric aciduria type I I Harting, E Neumaier-Probst, A Seitz, EM Maier, B Assmann, I Baric, M Troncoso, C Mu«hlhausen, J Zschocke, N Boy, GF Ho¡mann, SF Garbade, S Ko«lker

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073-P Guideline for the diagnosis and management of glutaric aciduria type I S Ko«lker, E Christensen, JV Leonard, CR Greenberg, AB Burlina, AP Burlina, M Dixon, M Duran, SI Goodman, DM Koeller, C Mu«hlhausen, E Mu«ller, ER Naughten, E Neumaier-Probst, JG Okun, M Kyllerman, RA Surtees, B Wilcken, GF Ho¡mann, P Burgard 19

074-P Mutational spectrum and oxidative stress studies in methylmalonic acidemia and homocystinuria, cblC type patients E Richard, A Jorge-Finnigan, LR Desviat, B Merinero, F Leal, M Ugarte, B Pe¨rez

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075-P Blood carnitine concentrations in premature newborns by tandem mass spectometry R Pons, EW Naylor, DH Chace, D McMahon, DC De Vivo

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076-P Postnatal carnitine concentrations in full term newborns R Pons, EW Naylor, DH Chace, D McMahon, DC De Vivo

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077-O Aminoacylase 1 in vivo and in vitro JO Sass, S Meincke, M Walter, H Olbrich, H Omran

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078-P Methylmalonic aciduria with signs of Asperger syndrome J Saligova, L Potocnakova, T Rosenbergerova, J Strnova, D Behulova, L Mrazova, J Zeman 20 079-P Two cases of malonic aciduria detected by newborn screening (NBS): same analyte, di¡erent conditions DK Gavrilov, RH Gavrilova, A McDonald, D Peck, RE Hillman, K Schoonderwoerd, S Tortorell, DB Dawson, KM Gibson, D Matern, P Rinaldo

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080-P a-Ketoisocaproic acid compromises cellular respiration in rat brain AU Amaral, G Leipnitz, CAJ Ribeiro, B Seminotti, CG Fernandes, AP Beskow, LA Knebel, A Zanatta, M Grings, CS Dutra-Filho, M Wajner

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081-P Good outcome of pregnancy in isovaleric aciduria J Sykut-Cegielska, A Kowalik, W Gradowska, K Kusmierska, A Malinowska-Polubiec, MK Kornacka

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082-P Acute decompensation of isovaleric acidemia induced by Graves' disease A Kimmoun, G Abboud, J Straczeck, M Merten, JL Gueant, F Feillet

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083-O Implication of birth cohort, age at onset, enzymatic subgroup and cobalamin responsiveness on long-term outcome in isolated methylmalonic acidurias F Ho«rster, SF Garbade, T Zwickler, HI Ayd|n, MR Baumgartner, AB Burlina, OA Bodamer, AM Das, JBC deKlerk, C Dionisi-Vici, G Go«kcay, S Gru«newald, N Gu¡on, EM Maier, E Morava, S Geb, JH Walter, U Wendel, FA Wijburg, M Lindner, S Ko«lker

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084-P Mutation identi¢cation for Taiwanese patients with isolated methylmalonic acidemia TT Liu, MY Liu, YC Chang, YL Fang, SH Chiang, DM Niu, KJ Hsiao

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085-P 3-Hydroxy-3-methylglutaric aciduria in Portuguese patients: clinical, neuroradiological and genetic characterization: a neurodegenerative disorder? E Lea¬o Teles, E Rodrigues, S Castro, M Ayres-Basto, F Silveira, J Guimara¬es, A Magalha¬es, ML Cardoso

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086-P Quanti¢cation of 3-hydroxyisovalerylcarnitine by high performance liquid chromatography tandem mass spectrometry in two patients with holocarboxylase synthetase de¢ciency Y Nakajima, T Ito, K Yokoi, H Ohmi, M Nagao, Y Maeda, Y Kurono, N Sugiyama, H Togari

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087-P The impact of a 2nd tier blood-spot methylmalonic acid (MMA) using tandem mass spectrometry (MS/MS) on routine newborn screening E Ranieri, R Gerace, B Bartlett, JR Harrison, JM Fletcher

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088-P Methylmalonic aciduria: molecular analysis of the MUT and MMACHC genes in 47 Italian patients C Cavicchi, MA Donati, E Pasquini, R Parini, F Furlan, M Sibilio, G Parenti, C Dionisi-Vici, A Bartuli, F Papadia, E Zammarchi, R Guerrini, A Morrone

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J Inherit Metab Dis (2008) 31 (Suppl 1)

089-P A case of methylmalonic aciduria type B: biochemical and genetic diagnosis E Riva, D Casero, D Minghetti, L Fiori, E Salvatici, C Agostoni 23 090-P Biochemical variability of ¢ndings in patients with glutaryl-CoA dehydrogenase de¢ciency L Varholakova, P Chrastina, E Kostalova, S Stastna

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091-O Glutaric aciduria type 1: synthesis, enzymatic activity, stability, and oligomerisation of mutant glutaryl-CoA dehydrogenase C Mu«hlhausen, B Keyser, A Dickmanns, E Christensen, K Ullrich, T Braulke

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092-P Renal transplant in methylmalonic acidemia: a therapeutic option with or without renal failure V Valayannopoulos, D Rabier, G Guest, Y Aigrain, JF Benoist, P Niaudet, P de Lonlay

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093-P Liver hepatoblastoma and multiple OXPHOS de¢ciency in the follow-up of a patient with methylmalonic aciduria MA Cosson, G Touati, F Lacaille, V Valayannnopoulos, C Guyot, G Guest, V Verkarre, D Chre¨tien, D Rabier, A Munnich, JF Benoist, Y de Keyzer, P Niaudet, P de Lonlay

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096-P X-Inactivation studies through cDNA analyses in two females with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) de¢ciency J Garc|¨ a-Villoria, L Gort, C Fons, P Briones, J Campistol, A Ribes

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097-O Fetal liver cell transplantation for methylmalonic aciduria using mice with an intermediate phenotype H Peters, N Buck, S Pennell, L Wood, J Pitt, K Allen

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098-P Biotinidase de¢ciency: the results of local newborn screening study pioneered nationwide screening I Ozer, T Baykal, G Gokcay, R Kose, S Celik, M Demirkol

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103-P Treatment of hyperammoniaemia with Ncarbamylglutamate in an adult patient with propionic aciduria A Mora¨is, RA Lama, A de la Mano, C Pe¨rez-Cerda¨, MJ Garc|¨ a

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106-O Aspartoacylase entry to the brain of Canavan mouse with hyaluronidase R Matalon, G Bhatia, S Suzucs, K Michals-Matalon, S Tyring, J Grady

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107-O CpG islands around exon 1 in succinyl-CoA:3-ketoacid CoA transferase (SCOT) gene were hypomethylated even in human and mouse hepatic tissues where SCOT gene expression was completely suppressed T Fukao, G Zhang, N Matsuo, N Kondo

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109-A An unusually high propionylcarnitine in a patient without a metabolic disorder CM John, S Poh, SJ Yeo, CE Hart, J Ang, ES Tan, DLM Goh, VS Rajadurai

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110-P Investigation of the `unmeasured' anions in critically ill patients with metabolic acidosis W Ruitenbeek, AM Terpstra, M Moviat, LA Kluijtmans, U Engelke, P Pickers

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111-P Abnormal C5-hydroxy acylcarnitine (C5OH) values detected in Portuguese newborn screening program H Rocha, A Marca¬o, H Fonseca, C Sousa, A Gaspar, L Diogo, E Lea¬o Teles, E Martins, M Noronha, P Garcia, E Rodrigues, I Tavares de Almeida, L Vilarinho

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112-O Multiple OXPHOS de¢ciency and mitochondrial DNA depletion in tissues of patients with methylmalonic and propionic aciduria Y de Keyzer, V Valayannopoulos, JF Benoist, JB Arnoux, F Batteux, D Chretien, AS Lebre, B Chadefeaux-Vekemans, D Rabier, G Touati, A Munnich, P de Lonlay 29

04. Fatty Acid Oxidation

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114-P Growth in 10 Swedish patients with long-chain 3OH-acyl-CoA dehydrogenase (LCHAD) de¢ciency C Bieneck Haglind, S Ask, M Halldin, J Gaîrdman, G Nyberg, J Alm, U von Do«beln, A Nordenstro«m

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115-O Unique organization of the mitochondrial beta-oxidation in insulin-producing beta cells: discrepantly high L-3hydroxyacyl-CoA dehydrogenase expression and its role in glucose sensing GA Martens, K Hellemans, G Stange¨, HV Van Thi, M Van de Casteele, D Pipeleers

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116-P Treating LCHADD in an extremely premature infant LN Carney, J Ganesh

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117-P Primary carnitine de¢ciency due to a novel mutation: report of two cases A Hasanoglu, FT Eminoglu, I Okur, G Biberoglu, L Tumer

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118-P Long-chain ketoacyl-CoA thiolase de¢ciency in the Dutch newborn screening program: a new case BHCMT Prinsen, TJ de Koning, MGM de Sain-van der Velden, M Duran, RJA Wanders, R Berger, NM Verhoeven-Duif

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100-P A novel single-base substitution (c.1124A4G) that activates a 5-base upstream cryptic splice donor site within exon 11 in the human mitochondrial acetoacetyl-CoA thiolase gene T Fukao, A Boneh, N Kondo 26

102-P Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia S Scholl-Bu«rgi, JO Sass, P Heinz-Erian, E Amann, E Haberlandt, U Albrecht, C Ertl, S Baumgartner Sigl, F Lagler, K Rostasy, D Karall

105-P Methylmalonic acidemia: enzyme and molecular study in eight Chilean patients V Cornejo, B Marinero, E Fernandez, G Castro, A Valiente, JF Cabello, M Colombo, E Raimann, B Perez, M Ugarte

113-P LCHAD de¢ciency ^ the most frequent fatty acid oxidation disorder in newborn screening in the Czech Republic P Chrastina, E Kostalova, M Paulova, L Varholakova, S Stastna, M Elleder, J Zeman

099-P Long-term outcome of methylmalonic aciduria: a series of 30 French patients MA Cosson, JF Benoist, G Touati, M Deschaux, N Boddaert, Y de Keyzer, D Rabier, P Niaudet, V Valayannopoulos, P de Lonlay 26

101-P Neuropathology of succinic semialdehyde dehydrogenase (SSADH) de¢ciency (gamma-hydroxybutyric aciduria) I Knerr, KM Gibson, G Murdoch, GS Salomons, L Pope, C Jakobs, GA Catanese, PL Pearl

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108-P Urine screening tests are old but not impractical: an example in diagnosis of a Turkish infant with alkaptonuria I Kurt, A Hasimi, O Ozturk, HI Ayd|n, MK Erbil 28

094-P Renal function in twenty seven children with methylmalonic acidemia (MMA): is there a good marker? HI Ayd|n, A Duzova, HS Kalkanoglu Sivri, A Dursun, T Aksoy, P Kiratli, A Tokatli, A Bakkaloglu, T Coskun, B Fowler 25 095-P Symptomatic patient with partial biotinidase de¢ciency: e¡ect of biotin treatment E Kostalova, P Chrastina, M Paulova, L Varholakova, S Stastna, F McKay

104-P Twenty seven patients a¡ected with propionic academia, Iranian experience T Zaman, SH Parvizi, R Moradian

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J Inherit Metab Dis (2008) 31 (Suppl 1) 119-P Isobutyryl-CoA dehydrogenase de¢ciency ^ case report AC Ferreira, G Mira, S Sequeira

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120-P Abnormal fatty acid metabolism in childhood spinal muscular atrophy (SMA) may predispose to perioperative risks for mitochondrial decompensation and liver failure Z Zolkipli, J Hutchison, M Sherlock, A Gri¤ths, I Tein 31 121-P Preeclampsia and HELLP syndrome: mitochondrial function in human umbilical venous endothelial cells S Illsinger, N Janzen, J Sander, KH Schmidt, J Bednarczyk, L Mallunat, J Bode, F Hagebo«lling, B Vaske, T Lu«cke, AM Das 31 122-P Late-onset very-long-chain acyl-CoA dehydrogenase de¢ciency presented with rhabdomyolysis M Coker, S Kalkan Ucar, S Habif, B Yildiz, RD Goksen Simsek, S Darcan, O Bayindir, RJA Wanders

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123-P Molecular basis of VLCADD in Portugal: three novel mutations H Rocha, M Marques, A Gaspar, E Lea¬o Teles, M Noronha, E Rodrigues, A Marca¬o, C Sousa, H Fonseca, I Tavares de Almeida, L Vilarinho 32 124-P MS/MS screening for VLCAD-de¢ciency: follow-up of positive cases U Spiekerkoetter, F ter Veld, M Mueller, M Stehn, R Santer, Z Lukacs

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125-O Development of dilative cardiomyopathy in carnitinesupplemented very long-chain acyl-CoA dehydrogenasede¢cient mice as studied by in-vivo NMR F ter Veld, C Jacoby, U Floegel, U Spiekerkoetter

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126-A Very long-chain acyl CoA dehydrogenase de¢ciency which was accepted as infanticide FT Eminoglu, L Tumer, I Okur, Z Go«kmen, FS Ezgu, G Biberoglu, A Hasanoglu

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127-P Unusual presentation of carnitine membrane transporter de¢ciency: bicytopenia S Kalkan Ucar, Y Aydinok, S Habif, E Levent, R Ozyurek, O Bayindir, RJA Wanders, M Coker

33

128-P Outcome of an elevated octanoyl carnitine (C8) form newborn screening dried blood spot (DBS) samples obtained during screening for medium chain acyl-CoA dehydrogenase de¢ciency (MCADD) in those not a¡ected with MCADD MJ Sharrard, MJ Downing, NJ Manning, SE Olpin, S Clark, AJ Matthews, JR Bonham 33 129-P Urinary hexanoylglycine in the diagnosis of medium chain acyl CoA dehydrogenase de¢ciency (MCADD) from newborn screening: detection and quanti¢cation M Downing, NJ Manning, BS Andresen, RN Dalton, S Krywawych, J Oerton, SE Olpin, A Patterson, MA Preece, J Till, C Dezateux, UK CSNS-MCADD 33 130-P In vivo glucose and fat metabolism at rest and during moderate-intensity exercise in patients with medium-chain acyl-CoA dehydrogenase de¢ciency HH Huidekoper, MT Ackermans, R Koopman, LJC van Loon, HP Sauerwein, FA Wijburg 34 131-P Molecular analysis of medium-chain acyl-CoA dehydrogenase de¢ciency in Portugal A Luz, S Violante, A Gaspar, M Lobo Antunes, IA Rivera, MFB Silva, A Ramos, H Rocha, C Sousa, A Marca¬o, H Fonseca, FV Ventura, P Leandro, L Vilarinho, I Tavares de Almeida

34

132-P Expanding role of clinical nurse specialists (CNS) ^ nurse led clinics for children with medium chain acyl-CoA dehydrogenase de¢ciency M McSweeney, M Dixon, S Gru«newald, PT Clayton, MA Cleary 34

133-O Studies on the substrate speci¢city of carnitine palmitoyltransferase 2: implications for acylcarnitine pro¢ling S Violante, L IJlst, I Tavares de Almeida, RJ Wanders, FV Ventura

34

134-P CPT 2 de¢ciency detected by newborn screening: 2 novel mutations despite normal follow-up acylcarnitine pro¢le S Illsinger, M Peter, RJ Wanders, M Deschauer, I Handig, T Lu«cke, W Wuyts, AM Das

35

135-P Lethal dilated cardiomyopathy in a girl with SCAD de¢ciency D Ballhausen, N Sekarski, PY Jeannet, O Boulat, N Gregersen, L Bonafe¨

35

136-P Transcriptional analysis of HADHA and HADHB genes encoding the mitochondrial trifunctional protein M Brivet, S Tubiana, A Boutron, C Acquaviva, P de Lonlay, H Ogier de Baulny, N Gu¡on, F Feillet, A Cano, L Faivre, L Burglen, A Labarre-Vila, MT Zabot, C Vianey-Saban, A Legrand 35 137-P Is octanoyl-CoA a novel complex III inhibitor? SW Sauer, JG Okun, GF Ho¡mann, S Koelker, MA Morath

35

138-O Mitochondrial long chain fatty acid oxidation: man versus mouse M Chegary, H te Brinke, JPN Ruiter, FA Wijburg, M Stoll, H Schulz, C Hoppel, RJA Wanders, SM Houten

36

139-P Evidence that cis-4-decenoic acid acts as an uncoupler of oxidative phosphorylation by opening pores in mitochondrial membrane PF Schuck, GC Ferreira, EB Tahara, AJ Kowaltowski, M Wajner

36

140-P Five year follow up of D,L-3-hydroxybutyrate treatment of multiple acyl-CoA dehydrogenase de¢ciency (MADD) S Gru«newald, J Marek, J Dean¢eld, S Olpin, JV Leonard

36

141-O Investigation of the e¡ect of ribo£avin on the steady-state amount of variant electron transfer £avoproteins (ETF: QO) identi¢ed in patients with ribo£avin-responsive multiple acyl-CoA dehydrogenase de¢ciency (RR-MADD) N Cornelius, RKJ Olsen, TJ Corydon, N Gregersen 36 142-P Multiple acyl-CoA-dehydrogenase de¢ciency in a 55-yearold woman P Kaminsky, C Acquaviva-Bourdain, L Pruna, J Jonas, C Vianey-Saban 37 143-O Expression patterns of organic cation/carnitine transporter family in adult murine heart: implications for hypertrophic cardiomyopathy and arrhythmias AM Lamhonwah, J Wong, C Tam, L Mai, I Tein 37 144-P Lipid peroxidative stress in SCAD de¢ciency (SCADD) and response to antioxidants Z Zolkipli, DC Lehotay, BH Robinson, I Tein

37

145-P Short-chain acyl-CoA dehydrogenase (SCAD) de¢ciency: homozygosity for the common c.625G4A variation may contribute to SCAD dysfunction in cultured patient ¢broblasts CB Pedersen, V Stenbroen, RJA Wanders, JPN Ruiter, F Wibrand, SP Young, DS Millington, PP Madsen, BS Andresen, S MÖller-Larsen, M Kjeldsen, N Gregersen 37 146-P Ethylmalonic aciduria: clinical and molecular characterization of short-chain-acyl-CoA deydrogenase de¢ciency in 9 Italian patients S Funghini, A Morrone, E Pasquini, E Procopio, S Gasperini, MA Donati

38

147-P Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) de¢ciency and newborn screening by tandem mass spectrometry Y Shigematsu, I Hata, Y Tanaka, K Shichijo, T Umemoto, T Nakatsu, T Yoshida, E Naito 38

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J Inherit Metab Dis (2008) 31 (Suppl 1)

148-P `White matter disorders' and `inborn errors of metabolism'; bridging clinical phenotypes with biochemical and molecular defects C Prasad, C Campbell, CA Rupar, S Levin, AN Prasad 38 149-P The diagnostic potential of skeletal muscle acylcarnitine pro¢les in identifying defects of fatty acid beta-oxidation G Lynes, IP Hargreaves, JM Land

38

150-P Diagnosis of mitochondrial b-oxidation defects in Russia GV Baydakova, PG Tsygankova

39

05. Carbohydrates 151-P Determination of a galactose-1-phosphate uridyltransferase (GALT) reference range using genotypephenotype from 257 subject values S Minnich, K Raymond, D Matern, D Oglesbee, J O'Brien 39 152-P Long term follow-up of biotinidase activity in 25 patients with hepatic glycogen storage disease. How reliable is a single value? CJ Angaroni, AE Paschini-Capra, AN Giner-Ayala, NB Guelbert, LD Martinez, R Dodelson de Kremer 153-P Mutation in hepatocyte nuclear factor 4a gene in a Saudi family with maturity-onset diabetes of the young S Mohamed, A Hellani, S El-Kholy 154-P Molecular genetics of glycogen storage disease type IX (GSD-IX) in Poland: mutation spectrum and genotype/ phenotype correlation NJ Beauchamp, J Taybert, Z Li, A Dalton, S Tanner, E Pronicka, M Sharrard

39

39

40

155-P The importance of the molecular con¢rmation in suspected glycogen storage disease (GSD) type IX with various clinical presentations M Zerjav Tansek, M Stopar Obreza, N Bratanic, T Battelino

40

156-P Glycerol kinase de¢ciency and partial de¢ciency of mithochondrial respiratory chain complexes: case report E Rodrigues, H Santos, I Azevedo, MM Campos, ML Cardoso, E Lea¬o Teles

40

157-P Two patients with fructose-1,6-diphosphatase defect and late diagnosis C Michot, V Valayannopoulos, M Barth, S Romano, JB Arnoux, C Baussan, M Brivet, P de Lonlay

40

158-P Safety of long term G-CSF administration in two siblings with glycogen storage disease type Ib T Ito, Y Nakajima, K Yokoi, N Sugiyama, H Togari

41

164-P Improvement in the management of patients with glycogen storage disease type I (GSD-I) using uncooked glycosade compared to uncooked cornstarch K Bhattacharya, MF Lilburn, DW Morley, F Maillot, PJ Lee 42 165-P Molecular genetic diagnosis of glycogen storage disease (GSD) type III: experience from a large international cohort R Santer, K Tsiakas, J Bergmann, U Steuerwald, O Sovik, CP Sentner, GPA Smit, B Steinmann, A Gal, K Ullrich

42

166-P Antioxidative defence in pediatric patients with glycogen storage disease type IA and III S Kalkan Ucar, M Coker, E Sozmen, A Anik, RD Goksen Simsek, S Darcan

43

167-P Coagulopathy with severe von Willebrand factor (VWF) de¢ciency in a family with glycogen storage disease Ia (GSD-Ia) not corrected by glucose infusion MJ Sharrard, JH Payne, GT Gillett, AJ Vora

43

168-P A cross-sectional survey of the diets of children and adults with glycogen storage disease type 1 (GSD-I) K Bhattacharya, MF Lilburn, R Orton, M Dixon, PJ Lee 43 169-P Bone metabolism impairment in glycogen storage disease type 1: a case^control study D Melis, G Parenti, R Pivonello, R Della Casa, F Balivo, M Cozzolino, V Gaudieri, L Minichini, F D'Elia, L Di Vuolo, G Lombardi, A Colao, G Andria 43 170-P The GH-IGF axis in glycogen storage disease type 1 (GSD1): evidence of di¡erent growth patterns and IGF levels in patients with GSD1a and GSD1b D Melis, R Pivonello, G Parenti, V Gaudieri, R Della Casa, M Salerno, F D'Elia, P Piccolo, G Lombardi, A Colao, G Andria

44

171-P Glycogenosis Ia in a woman with a mitochondrial disorder and alterations in the protrombin gene JA Arranz, JM Go¨mez-Argu«elles, MA Mart|¨ n, C de Diego, A Bla¨zquez, M del Toro, E Riudor 44 172-P Molecular study of 11 cases of GLUT1 de¢ciency syndrome S Vuillaumier-Barrot, N Bahi-Buisson, S Odent, M Mayer, D Chaigne, A de Saint-Martin, E Flori, S Bekri, V DrouinGarraud, E Ra¡o, B Chabrol, S Julia, C Le Bizec, N Seta

44

173-P Duarte (DG) galactosemia: a study of biochemical and neurodevelopmental assessment in children detected by newborn screening C Ficicioglu, N Thomas, C Yager, PR Gallagher, C Hussa, BJ Forbes

44

174-P Galactosemia patients in Estonia, 15 years of selective screening K Krabbi, K Kall, T-M Laht, K O¬unap, K Joost, R Zordania

45

159-P Bone mineralization in GSD I and III E Riva, S Paci, I Giulini Neri, M Gasparri, G Cagnoli, M Bonza, C Agostoni

41

160-P Absence epilepsy and/or myoclonic epilepsy in children, think of Glut1 de¢ciency syndrome (two case reports) B Chabrol, H Mansour, A Cano, M Milh, I Ticus, N Seta

41

161-P Absence of severe recurrent infections in GSD Ib S Paci, M Gasparri, I Giulini Neri, G Cagnoli, E Salvatici, M Giovannini

41

175-P GALT-activity, galactose metabolites and hormones during pregnancy in a classic galactosemia patient CS Gubbels, CS Gubbels, S Kuppens, JA Bakker, S Konings, P Menheere, LJ Spaapen, MG De Sain, WK Wodzig, ME Rubio-Gozalbo, ME Rubio-Gozalbo 45

42

176-P Twenty ¢ve patients a¡ected with galactosemia, a report from Iran T Zaman, R Moradian, S Mazdarani

42

177-P Spectrum of GALT mutations in Spain and Portugal. Seven new mutations in seventeen new patients E Quintana, L Gort, S Moliner, L Gonzalez-Quereda, T Lopez-Hernandez, I Rivera, M Santos Leite, L Vilarinho, P Briones 45

162-P Case report: GSD type I and pregnancy S Paci, M Gasparri, I Giulini Neri, M Bonza, E Salvatici, M Giovannini 163-O Glycogen storage disease type IX (GSD IX) with an unusual PHKB mutation presenting with developmental delay, deafness, hypotonia and hepatopathy NJ Beauchamp, JH Blackburn, R Kirk, M Sharrard, U Ramaswami

45

178-P Glycogen storage disease type I: Impact of medium chain fatty acids on metabolic control and growth AM Das, T Lu«cke, U Meyer, H Hartmann, S Illsinger 46

J Inherit Metab Dis (2008) 31 (Suppl 1)

vii

179-P Twenty-one additional cases of familial renal glucosuria: absence of genetic heterogeneity, high prevalence of private mutations, and further evidence of volume depletion J Calado, Y Sznajer, D Metzger, A Rita, M Hogan, A Kattamis, M Scharf, V Tasic, J Greil, F Brinkert, M Kemper, R Santer 46 180-P Over 25 years enzymatic diagnosis of glycogen storage diseases. Enhanced activity of debranching enzyme in patients with glycogenosis IX GC Schoonderwoerd, O van Diggelen

46

181-O Paroxysmal exercise-induced dyskinesia and epilepsy due to mutations in SLC2A1, encoding the glucose transporter, GLUT1 D Cassiman, A Suls, P Dedeken, K Go¤n, H Van Esch, P Dupont, J Kemp£e, T V Wuttke, Y Weber, H Lerche, Z Afawi, A D Korczyn, S F Berkovic, D Ekstein, S Kivity, P Ryvlin, LRF Claes, L Deprez, S Maljevic, A Vargas, T Van Dyck, D Goossens, J Del-Favero, K Van Laere, P De Jonghe, W Van Paesschen 46

49

194-O Immunological, developmental and behavioural abnormalities in a mouse model for congenital disorder of glycosylation-IIc D Popovici, J Lu«bbehusen, M Sperandio, D Frommhold, P Gass, C Ko«rner

50

195-P Biochemical tools in the diagnosis of glycosylation related myopathies M Guillard, K Verrijp, N Voermans, M Huizing, E Morava, H Van Bokhoven, B Van Engelen, RA Wevers, M Lammens, DJ Lefeber 50

50

197-P Transferrin IEF patterns and plasma aspartylglucosaminidase activity in CDG, galactosemia and fructosemia M Moraitou, I Mavridou, H Michelakakis

50

198-P Amniotic £uid alpha1-fetoprotein in prenatal diagnostics of CDG E Marklova, Z Albahri

51

47

199-O Cutis laxa syndromes with congenital disorder of glycosylation: clinical, biochemical and genetic review E Morava, M Guillard, R Rodenburg, U Kornak, Z Urban, D Lefeber, RA Wevers

51

47

200-P Congenital defect of N- and O-glycosylation in an infant with cutis laxa syndrome K Tsiakas, E Morava, P Meinecke, GG Gillessen-Kaesbach, R Wevers, R Santer 51

47

183-P Signal symptoms of IEM and inclusions in GSD IV as an example S Stastna, P Chrastina, M Paulova, M Elleder

47

185-P Response to the ketogenic diet in a wide spectrum of GLUT-1 phenotypes B Pe¨rez-Duen¬as, M Pineda, V Gonza¨lez, I Ma¨laga, L Go¨mez, A Gutie¨rrez, JM Pascual, R Artuch

49

193-P A mouse model for congenital disorder of glycosylation Ia (CDG-Ia) A Schneider, C Thiel, HJ Gro«ne, C Ko«rner

196-P Congenital disorders of glycosylation in Southern Africa. A pilot study M Dercksen, LJ Mienie

182-O Pancreatic dopamine metabolism in congenital hyperinsulinism. A [18F]£uoro-L-DOPA PET study MJ Ribeiro, C Bellanne¨-Chantelot, V Valayannopoulos, T Delzescaux, F Jaubert, Y Aigrain, C Nihoul-Fe¨ke¨te¨, F Brunelle, P de Lonlay

184-P Autoimmune endocrine disorders in a patient a¡ected by glycogen storage disease 1b: causal relationship between neutropenia and autoimmunity? D Melis, G Parenti, R Pivonello, V Gaudieri, R Della Casa, M Salerno, F D'Elia, G Lombardi, A De Bellis, A Colao, G Andria

192-P Congenital disorders of glycosylation presenting with neuro-regression K Vijayakumar, P Prabhakar, V Ganesan, V Worthington, M Jackson, P Mills, T Dupre, S Vuillaumier-Barrot, N Seta, S Gru«newald

06. Glycosylation 186-P Pitfalls in interpretation of transferrin isoforms determined by capillary electrophoresis K Carpenter, K Green, C Ellaway, J Pitt

48

187-P Late presentation of cardiomyopathy in CDG type IX EJ Footitt, A Karimova, M Burch, S Grunewald

48

188-P New mutation of the ATP6V0A2 gene in an autosomal recessive Cutis laxa type 2 patient A Bruneel, V Drouin-Garraud, F Habarou, W Morelle, F Foulquier, E Charluteau, C Bouchet, S VuillaumierBarrot, G Durand, X Balguerie, T Frebourg, N Seta

48

189-O A possible role for the Conserved Oligomeric Golgi complex subunit 1 (COG1) in autosomal recessive cerebrocostomandibular syndrome R Zeevaert, R Van Damme-Lombaerts, F Foulquier, E Reynders, W Annaert, G Matthijs, J Jaeken

48

190-P Congenital disorder of glycosylation type Ik (CDG Ik): a frequent form of CDG in France? T Dupre, S Vuillaumier-Barrot, H Sadou-Yaye, C Le Bizec, P de Lonlay, S Moore, N Seta

49

191-P A novel mutation in the COG5 locus a¡ects N- and Oglycosylation causing CDG type II P Paesold-Burda, H Troxler, P Kleinert, C Maag, T Hennet, S Malich, B Steinmann, M Baumgartner

49

201-P Functional analysis of three splicing mutations identi¢ed in the PMM2 gene: towards new therapeutic approaches in congenital disorder of glycosylation type-Ia A Vega, C Pe¨rez-Cerda¨, LR Desviat, G Matthijs, M Adamowicz, M Ugarte, B Pe¨rez 51

07. Mitochondrial Disorders 202-P Recurrent rhabdomyolysis and mitochondrial disease A Bandeira, M Santos, P Rocha, I Carrilho, A Guimara¬es, ML Cardoso, E Martins

52

203-P Mitochondrial cardiomyopathies: biochemical and genetic heterogeneity A Estevinho, S Oliveira, J Pratas, M Simo¬es, C Mendes, MJ Santos, M Oliveira, L Diogo, C Maca¨rio, CR Oliveira, M Grazina 52 204-P Dihydrolipoamide dehydrogenase de¢ciency in a case of Reye syndrome. E¡ect of anaplerotic substrates in vivo and in vitro JB Arnoux, L Le Moyec, L Hubert, D Chretien, M Brivet, V Valayannopoulos, AM Bertrand, A Munnich, Y de Keyzer, P de Lonlay 52 205-P West syndrome and mitochondrial dysfunction S Silva, C Robalo, P Garcia, M Grazina, CR Oliveira, L Diogo

52

206-P Complex I de¢ciency due to a NDUFAF2 defect in a boy with acute fulminant course of Leigh disease and typical brainstem lesions W Sperl, H Prokisch, E Boltshauser, J Koch, C Rauscher, W Radauer, R Forstner, P Freisinger, B Rolinski, RJT Rodenburg, JA Mayr

53

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J Inherit Metab Dis (2008) 31 (Suppl 1)

207-O Defects in ATP synthesis are associated with hypertrophic cardiomyopathy W Sperl, J Koch, P Freisinger, JA Mayr 53 208-P Major depression in adolescent children consecutively diagnosed with mitochondrial disorder S Koene, TL Kozicz, R Rodenburg, B van den Heuvel, C Verhaak, JAM Smeitink, E Morava 209-P Clinical, biochemical and genetic features in 18 children with 3-methyl-glutaconic aciduria type IV SB Wortmann, MC de Vries, RJ Rodenburg, LP van den Heuvel, LA Kluijtmans, U Engelke, K Heldt, JAM Smeitink, R Wevers, E Morava 210-P Third reported case of pyruvate dehydrogenase phosphatase 1 (PDP 1) de¢ciency: promising response to treatment with sodium bicarbonate C Barnett, M Maj, J Raiman, B Robinson, A Schulze

53

224-P Oxidative stress in childhood mitochondrial respiratory disease ^ 20 cases N Neves, P Garcia, T Proenc°a, I Baldeiras, M Grazina, L Vilarinho, CR Oliveira, L Diogo

57

225-P Mitochondrial ND1 involvement in frontotemporal dementia MJ Santos, S Cleto, C Mendes, J Pratas, M Simo¬es, I Santana, CR Oliveira, M Grazina

57

53

226-P The estimation of mtDNA polymorphism in patients with mitochondrial pathology in Ukraine VA Gusar, OYa Grechanina, SI Zhadanov, YuB Grechanina, OP Zdybskaya, NP Fedoseeva, TG Schurr 57

54

227-P Mitochondrial G12300A mutation is associated with a severe mitochondrial multisystemic disorder R Mart|¨ n-Jime¨nez, E Mart|¨ n-Herna¨ndez, B Santiago, L Mendan¬a, A Cabello, EM Fernandez, Y Campos

58

228-P Cerebral palsy (CP) and oxidative phosphorylation (OXPHOS)disorders: clues to a mitochondrial diagnosis A Cozens, J Yaplito-Lee, A Boneh

58

58

211-O Pyruvate dehydrogenase phosphatase (PDP1) de¢ciency. Infantile lactic acidemia and severe phenotype due to a null mutation BH Robinson, N MacKAY, V Levandovskiy, M Maj, J Raiman, A Feigenbaum, A Shulze, JM Cameron

54

213-P Tissue-speci¢c pyruvate dehydrogenase de¢ciency in a boy with novel mutations in the E1 b subunit JA Mayr, J Koch, C Rauscher, G Bernert, W Sperl

54

229-P High throughput mutation screening in patients with combined respiration chain complex defect I Hillier, M Freitag, U Ahting, M Taverna, B Rolinski, P Freisinger, M Tesarova, M Zeviani, T Meitinger, H Prokisch

54

230-P Gender di¡erences in the genotype/phenotype distribution of PDHA1 IC Lee, J Ganesh, B Maranda, LY Tang, W Craigen, F Li, LJC Wong 58

214-P Immunostaining techniques in patients with mtDNA depletion R Van Coster, B De Paepe, J Smet, S Seneca, W Lissens, L De Meirleir, F Roels 215-P Role of BN-PAGE in the diagnosis of mitochondrial DNA depletion R Van Coster, J Smet, B De Paepe, S Seneca, W Lissens, L De Meirleir

55

216-P Heart transplantation in respiratory chain disorders H Mundy, I Hargreaves, M Ashworth, M Burch, S Gru«newald

55

231-P Tissue copper content in children with isolated cytochrome c oxidase de¢ciency H Hansikova, K Vesela, L Stiburek, J Zeman 59 232-P Ocular involvement in mitochondrial disease: biochemical and genetic diversity outline in central Portugal MM Grazina, J Pratas, M Simo¬es, C Mendes, S Oliveira, M Oliveira, C Maca¨rio, L Diogo, P Garcia, CR Oliveira 59

217-P Coenzyme Q10 de¢ciency associated with a mitochondrial DNA depletion syndrome R Montero, P Briones, R Garesse, A Navarro-Sastre, ME Gallardo, J Montoya, R Marti, A Ribes, M Pineda, A Garc|¨ a-Cazorla, P Navas, R Artuch 55

233-P Two novel mutations in thymidine kinase-2 leading to an early onset fatal encephalomyopathy and severe mtDNA depletion K Naess, N Lesko, R Wibom, I Nennesmo, N Solaroli, U von Do«beln, A Karlsson, NG Larsson

59

218-P Segregation study in family with severe variant of the A3302G mutation in the mitochondrial tRNA LEU (UUR) D Ballhausen, F Guerry, D Hahn, L Bonafe¨, S Jacquemont

55

234-P Complex I de¢ciency in Portuguese patients: analysis of the `core' subunits M Ferreira, FM Santorelli, L Vilarinho

59

219-P A novel mutation in pyruvate carboxylase de¢ciency type C TF Lang, D Wang, DC De Vivo, CL Loughrey

56

235-P Contribution of POLG1 mutations to nuclearmitochondrial intergenomic communication disorders M Ferreira, T Evangelista, MJ Rosas, MC Maca¨rio, A Guimara¬es, FM Santorelli, L Vilarinho

60

236-P Analysis of secondary respiratory chain de¢ciency in a hepatoblastoma model of propionic acidemia A Brassier, B Chadefaux-Vekemans, A Munnich, P de Lonlay, Y de Keyzer

60

220-O Expanding knowledge on SUCLG1 mutations causing atypical methylmalonic aciduria R Carrozzo, S Lucioli, C Bruno, AB Burlina, D Haas, F Ho«rster, S Yano, LA Kluijtmans, M Bianchi, MC Meschini, D Verrigni, RA Wevers, C Rizzo, S Boenzi, FM Santorelli, C Dionisi-Vici

56

221-P Mutation studies in patients with pyruvate carboxylase de¢ciency LE Heptinstall, JH Walter, AAM Morris, SA Jones, S Rahman, GTN Besley

56

222-P A comparison of two methods to determine the REE in children diagnosed or suspected of a mitochondrial disease AMJ van den Berg, E Morava, E Rasmussen 56 223-P Nutrition, growth and the mitochondrial function S Koene, S Wortmann, R Rodenburg, A van de Berg, H Zweers, L van den Heuvel, J Smeitink, E Morava

57

237-P Inhibition of mitochondrial complex I in cerebral cortex of immature rats following seizures induced by homocysteic acid (HCA) P Jesina, J Folbergrova, Z Drahota, R Haugvicova, V Lisy, A Pecinova, J Houstek 60 238-P Mitochondrial morphology changes in mutant NDUFA1 and NDUFV1 complex I de¢ciency patients M Mora¨n, H Rivera, D Ferna¨ndez-Moreira, A Bla¨zquez, C Ugalde, J Arenas, MA Mart|¨ n

60

239-P High throughput mutation screening in patients with isolated respiratory chain complex I de¢ciency P Freisinger, M Taverna, F Madignier, U Ahting, B Rolinski, J Mayr, W Sperl, M Tesarova, T Meitinger, H Prokisch 61

J Inherit Metab Dis (2008) 31 (Suppl 1) 240-P Acute necrotizing encephalopathy ^ a mitochondrial disorder? R Castelo, P Garcia, M Vasconcelos, O Rebelo, A Dinis, M Grazina, L Diogo 241-P Corneal dystrophy as a ¢rst clinical symptom in KearnsSayre syndrome K Tsiakas, B Kruse, V Knospe, P Freisinger, R Santer

ix

61

256-P Alpha-methyl CoA racemase (AMACR) de¢ciency in an adult presenting with aphasia NJ Manning, RM Talbot, SE Olpin, A Gibson, GT Gillett, OL Bandmann, S Ferdinandusse, RJA Wanders

65

61

257-O Peroxisomal disorders and newborn screening S Tortorelli, CT Turgeon, MJ Magera, WC Hubbard, AB Moser

65

258-P Two novel mutations in ABCD1 gene ^ one Portuguese and one Serbian F Laranjeira, M Lemos, S Perdiga¬o, L Lacerda

65

259-O Screening small molecules for rescue of peroxisome assembly defects NE Braverman, R Zhang, SJ Steinberg

66

260-P Protein oxidation and lipid peroxidation in plasma of patients with peroxisome biogenesis disorders M Deon, A Sitta, AG Barschak, GB Biancini, AB Oliveira, DM Coelho, R Giugliani, M Wajner, CR Vargas

66

261-P Disturbances in the central nervous system of L-valine metabolism are consistently present in patients with a peroxisomal biogenesis disorder FJ Eyskens

66

262-P VLCFA levels in various approaches to the X-ALD treatment in one 7 years old patient TJ Stradomska, K Drabko, E Moszczyn¬ska, A Tylki-Szyman¬ska

66

263-P X-linked adrenoleukodystrophy: the relative frequency of phenotypic variants in the Portuguese patients R Ferreira, M Lemos, L Lacerda

67

264-P Searching polymorphic variants of methionine metabolism in several clinical phenotypes of X-linked adrenoleukodystrophy in Argentinean patients SE Bender, CL Grosso, NB Guelbert, A Oller- Ramirez, A Becerra, C Amorosi, R Dodelson de Kremer

67

242-O Identi¢cation of novel and recurrent mitochondrial DNA mutations in paediatric onset mitochondrial disease using the MitoChip resequencing array AJ Duncan, MG Sweeney, E Stern, RW Taylor, C Woodward, MB Davis, MG Hanna, S Rahman 61 243-P Diagnosis of respiratory chain defects by enzyme analysis in small biopsies from liver and muscle F Wibrand, E Òstergaard, M DunÖ, AM Lund, E Christensen 62 244-O LRPPRC mutations in French-Canadian Leigh syndrome cause a phenotypically distinct form of cytochrome c oxidase de¢ciency FG Debray, C Morin, J Villeneuve, B Maranda, R Laframboise, BH Robinson, M Lambert, G Mitchell 62 245-P Diagnostic mtDNA analysis in body £uids other than blood RGF Gray, S Ball, R Quinlivan, C Hendriksz

62

246-P Haplogroup determination and frequency of mtDNA LHON associated sequence variations in multiple sclerosis J Pratas, MC Maca¨rio, M Oliveira, MJ Santos, CR Oliveira, M Grazina 62 247-O Myopathy with de¢ciency of succinate dehydrogenase and aconitase together with cardiomyopathy associated with mutations in ISCU G Kollberg, M Tulinius, A Melberg, N Darin, O Andersen, A Oldfors, E Holme 63 248-P Severe axonal polyneuropathy due to dichloroacetate in a patient with pyruvate dehydrogenase de¢ciency A Dursun, A Tokatly, HS Sivri, T Coskun 63 249-P Splicing mutations in the PDHA1 gene CK Ridout, RM Brown, GK Brown 250-P Expression pattern of PDHA1 and PDHA2 genes in human spermatogenesis A Pinheiro, I Faustino, MJ Silva, D Henrique, M Sousa, A Barros, I Tavares de Almeida, I Rivera

63

63

251-P A new mutation in PDHA1 in a girl with severe clinical phenotype but normal activity of pyruvate-dehydrogenase complex in muscle P Freisinger, J Mayr, K Hartmann, B Rolinski, U Ahting, R Woessner, W Sperl 64 252-P Human testis-speci¢c PDHA2 gene: evaluation of the methylation status in promoter region of somatic cells I Faustino, A Pinheiro, MJ Silva, I Tavares de Almeida, I Rivera

64

08. Peroxisomal Disorders 253-P A peroxisomal form of spinocerebellar ataxia caused by mutations in PEX10 L Re¨gal, M Ebberink, R Wanders, B Wuyts, H Waterham, M Van Driessche, R Van Coster, E Achten, L De Meirleir 254-P A peroxisomal biogenesis disorder causing spinocerebellar ataxia in an adult L Re¨gal, M Ebberink, N Goemans, R Wanders, H Waterham, J Jaeken 255-P Expression of the peroxisomal plasmalogen synthesizing enzymes in the CNS AM Bams-Mengerink, R Ofman, D Troost, RJA Wanders, BT Poll-The, P Brites

265-P 7 Tesla proton magnetic resonance spectroscopic imaging in adult X-linked adrenoleukodystrophy FS Eichler, E Ratai, T Kok, C Wiggins, G Wiggins, E Grant, B Gagoski, G O'Neill, E Adalsteinsson 67 266-P 2-pentanone: an indicator of peroxisomal acyl-CoA thioesterase activity? V Walker, JP Begley, EM Stansbridge

67

267-P Batyl alcohol as a therapeutic option in rhizomelic chondrodysplasia punctata AM Bams-Mengerink, P Brites, A Vyth, M Duran, RJA Wanders, HSA Heymans, BT Poll-The

68

268-O Identi¢cation of three PEX16-defective patients with import competent peroxisomes in ¢broblasts MS Ebberink, S Denis, PAW Mooijer, CJM Dekker, PT Clayton, RJA Wanders, HR Waterham, S Ferdinandusse

68

269-P Genetic classi¢cation of peroxisome biogenesis disorders MS Ebberink, PAW Mooijer, J Gootjes, RJA Wanders, HR Waterham

68

270-P A Zellweger syndrome associated with non-immune hydrops fetalis, lung hypoplasia and dermal erythropoiesis A Dursun, Y Do«rtttepe, S Gucer, S Yigit, RJA Wanders 68 64

271-P Toxicity of peroxisomal C27-bile acid intermediates S Ferdinandusse, S Denis, G Dacremont, RJA Wanders

69

09. Creatine 64

65

272-O Brain cells take up guanidinoacetate and convert it to creatine O Braissant, E Be¨ard, C Torrent, H Henry

69

x 273-P Knock-down of AGAT, GAMT and SLC6A8 by RNA interference in brain cells: new experimental models of creatine de¢ciencies in CNS E Be¨ard, O Braissant 274-P Favourable outcome with oral creatine supplementation: clinical and laboratory ¢ndings in ¢ve patients with GAMT de¢ciency G Haliloglu, OK Karli, A Dursun, A Tokatli, O Bodamer, T Coskun, M Topcu 275-P The screening of creatine transporter defect among Estonian families with X-linked mental retardation K O¬unap, H Puusepp, K Kall, I Talvik, M Mannamaa, C Jakobs, GS Salomons 276-P Arginine-guanidinoacetate-creatine pathway in pediatric renal transplants F Andrade, JA Prieto, G Ariceta, M Aguirre, P Sanjurjo, L Alda¨miz-Echevarr|¨ a 277-P Creatine de¢ciency syndromes in Portuguese population C Valongo, L Vilarinho, LS Almeida, GS Salomons, C Jakobs, D Quelhas

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69

280-O Identi¢cation and cloning of a novel splice variant of the creatine transporter gene SLC6A8 C Mart|¨ nez Mun¬oz, EH Rosenberg, C Jakobs, GS Salomons

283-P A rapid method for the determination of creatine and creatinine in urine by electrospray tandem mass spectrometry: a new tool for the diagnosis of creatine trasporter de¢ciency Cl Carducci, S Santagata, Ca Carducci, R Battini, V Leuzzi, I Antonozzi 284-P Simultaneous determination of creatine, creatinine and guanidinoacetate in plasma and urine by stable-isotope dilution UPLC-MS/MS WAH Waterval, JLJM Scheijen, JA Bakker, J Bierau 285-P Urinary stability of creatine and guanidinoacetate under di¡erent storage conditions JA Prieto, F Andrade, S Mart|¨ n, P Sanjurjo, L Alda¨miz-Echevarr|¨ a

288-O Creatine synthesis and transport defects: diagnosis and treatment in a series of 7 patients V Valayannopoulos, N Boddaert, A Chabli, I Desguerre, A Philippe, C Jakobs, A Munnich, G Salomons, P de Lonlay

74

70

292-P Increased spontaneous osteoclastogenesis in phenylketonuria F Porta, I Roato, M Spada, R Ferracini, A Mussa

74

70

293-P Large neutral amino acids and late diagnosed phenylketonuria KD Moseley, CG Azen, R Koch, S Yano

74

294-P Phenylketonuria in adulthood in the Moravian region (Czech Republic) D Prochazkova, P Konecna, E Hrabincova, H Vinohradska, H Hrstkova

74

295-P How many PKU-children can bene¢t from tetrahydrobiopterin? M Bik-Multanowski, B Didycz, JJ Pietrzyk

75

296-O Reduced cerebral £uoro-L-dopa uptake and release in adult PKU patients C Landvogt, E Mengel, P Bartenstein, R Feldmann, J Weglage, P Cumming, R Santer, K Ullrich

75

71

71

71

72

72

72

286-P The transport of creatine (Cr) in the brain: in vitro experiments on rat brain and human cancer cell cultures V Leuzzi, Cl Carducci, Ca Carducci, S Santagata, C Artiola, M Baletsrino, E Adriano, I Antonozzi 72 287-O S-adenosyl-methionine may be bene¢cial for decreasing guanidinoacetate in GAMT de¢ciency DL Renaud

73

291-O Renal NaS1 sulfate transporter defect in autistic patients FG Bowling, H Heussler, P Dawson, J VanDongen, D Markovich

10. Phenylketonuria and BH4

281-P Guanidinoacetoacetate methyltransferase (GAMT) de¢ciency: clinical and biochemical pro¢le in Tunisian patients FN Nasrallah, IK Kraoua, MF Feki, NG-K Gouider-Khouja, GB Briand, NK Kaabachi 71 282-P Long term outcome of AGAT de¢ciency patients during creatine supplementation R Battini, M Casarano, MG Alessandr|© , C Casalini, M Tosetti, MC Bianchi, V Leuzzi, G Cioni

290-O Asperger autism associated with folate receptor autoantibodies and cerebral folate de¢ciency V Ramaekers, J Sequeira, N Blau, E Quadros

70

278-P Creatine transporter defect: results of 6 months' treatment B Wilcken, E Fagan, K Sim, KH Carpenter, GS Salomons 70 279-P Epilepsy spectrum in cerebral creatine transporter de¢ciency. Genotype correlation C Fons, F Sanmart|¨ , A Sempere, P Po¨o, M Pineda, A Arias, A Ribes, R Artuch, MA Vilaseca, J Campistol

289-P A strategy in the treatment of creatine transporter defect: L-arginine supplementation in three Italian CT1 patients R Battini, AM Chilosi, U Caruso, MM Mancardi, V Leuzzi, Italian Group on Creatine Metabolism and Transport (GISMet-Cr) 73

73

73

297-P PAH de¢ciency in Portugal: identi¢cation of potential BH4-responsive patients I Rivera, P Leandro, A Queiro¨s, A Gaspar, M Lobo Antunes, L Vilarinho, I Tavares de Almeida 75 298-P Prevalence of (potentially) BH4 responsive mutations in PKU patients from Ghent, Belgium P Verloo, L De Meirleir

75

299-P Factors in£uencing compliance in PKU during the ¢rst 6 years of life P Burgard, SF Garbade, J Adler

76

300-P The natural history of 6-pyruvoyl-tetrahydropterin synthase (PTPS) de¢ciency (PTPSD). A late diagnosed case V Leuzzi, Cl Carducci, Ca Carducci, ML Di Sabato, C Caforio, T Giannini, S Pozzessere, I Antonozzi

76

301-P Functional analysis and phenotypic outcome of S231F mutation in phenylalanine hydroxylase gene M Stojiljkovic, B Perez, LR Desviat, C Aguado, M Ugarte, S Pavlovic

76

302-P The phenotypic variability in 6-pyruvoyl-tetrahydropterin synthase (PTPS) de¢ciency (PTPSD). Clinical presentation and outcome of the Italian patients V Leuzzi, A Burlina, R Cerone, D Concolino, MA Donati, L Fiori, A Ponzone, F Porta, P Strisciuglio, Ca Carducci, Cl Carducci, C Vagnoni, S Pozzessere, I Antonozzi 76 303-P Studies on the interaction of mutant forms of human phenylalanine hydroxylase with molecular chaperones I Cristo, R Almeida, J Leandro, I Tavares de Almeida, P Leandro

77

304-O Interallelic complementation and phenylketonuria: isolation of hybrid forms of human phenylalanine hydroxylase (hPAH) J Leandro, I Tavares de Almeida, P Leandro, T Flatmark

77

J Inherit Metab Dis (2008) 31 (Suppl 1)

xi

305-P Assembly of hybrid heteroallelic mutant forms of human phenylalanine hydroxylase produced in a prokaryotic dual vector expression system R Almeida, J Leandro, I Cristo, I Tavares de Almeida, P Leandro 77 306-P Phenylketonuria as a protein misfolding disease: mutant pG46S human phenylalanine hydroxylase has a propensity to self-associate and form amyloid ¢brils J Leandro, N Simonsen, I Tavares de Almeida, P Leandro, T Flatmark 77 307-P Determinants of obesity risk in adult patients with phenylketonuria J von Berlepsch, R Feldmann, B Koletzko

78

308-P Treatment of the hyperphenylalaninemia with tetrahydrobiopterin (BH4) and its in£uence in the pattern of amino acids and fatty acids from childhood to adult age I Garc|¨ a-Jime¨nez, A Baldellou-Va¨zquez, H Navarro-Azna¨rez, Mi Salazar, Fj Lo¨pez-Piso¨n

78

309-P Neuropsychological impairment in adult patients with treated phenylketonuria R Feldmann, J von Berlepsch, S Kloska, J Weglage, B Koletzko

78

310-P Preliminary investigation of the mutation spectrum of PKU in Tunisian patients S Khemir, N Tebib, W Cherif, F Nassrallah, N Esseghir, R Jemaa, N Khouja, S Abdelhak, MF Ben Dridi, N Kaabachi

78

311-P Diagnosis, treatment, and incidence of tetrahydrobiopterin (BH4) responsive mild PKU in Japan H Shintaku, H Fujioka, K Aoki, T Yamano 79

320-P Whole body composition analysis by the BodPod airdisplacement plethysmography method in children with phenylketonuria M Albersen, M Bonthuis, NM De Roos, TAM van den Hurk, EC Carbasisus-Weber, MMWD Hendriks, TJ Koning, G Visser 81 321-P L-carnitine de¢ciency contributes to oxidative stress in treated phenylketonuric patients A Sitta, AG Barschak, M Deon, AT Barden, C Vanzin, JF de Mari, CF de Souza, IV Schwartz, C Netto, R Giugliani, M Wajner, CR Vargas

81

322-P Early treatment prevents oxidative damage in phenylketonuric patients A Sitta, AG Barschak, M Deon, JF de Mari, AT Barden, GB Biancini, PR Vargas, CF de Souza, IV Schwartz, C Netto, R Giugliani, M Wajner, CR Vargas

81

323-P Tetrahydrobiopterin (BH4)-responsiveness and long-term treatment with BH4 in hyperphenylalaninemia I Scala, C Ungaro, S Paladino, A Nastasi, A Zuppaldi, M Sibilio, C Figliuolo, E Scarpato, B Capaldo, G Cardillo, A Daniele, R Della Casa, G Parenti, A Andria 82 324-P Signi¢cance of genotype in tetrahydrobiopterin (BH4)responsive phenylketonuria (PKU) FK Trefz, D Scheible, H Go«tz, G Frauendienst-Egger

82

325-P Social adaptation indicators of PKU patients using ready to use form of protein substitute PV Novikov, AS Latypov, ZI Va¢na, EV Denisenkova, LI Kuznetsova, GG Listopad, SA Matulevich, TA Golihina, NV Nikitina, EB Nikolaeva

82

326-P Long-term nutrition and dietary impact of tetrahydrobiopterin therapy in phenylketonuria RHS Singh, TD Douglas

82

312-P Clinical response to Kuvan (sapropterin) in patients with hyperphenylalaninemia/phenylketonuria (PKU) ^ early observations and future directions S Mo¢di, E Lim-Melia, D Kronn

79

313-P Relative frequency of BH4 responsive PAH mutations in Southern Italy G Bonapace, F Ceravolo, S Sestito, M Montesani, A Piccirillo, P Strisciuglio, D Concolino

79

328-P Four apparently unrelated haplotype groups in the phenylalanine hydroxylase gene S Kalb, S Luf, B Janssen, J Zschocke

83

79

329-P Long-term follow-up and outcome of patients with tetrahydrobiopterin de¢ciency L JÌggi, MR Zur£u«h, A Schuler, A Ponzone, F Porta, E Salvatici, M Giovannini, R Santer, GF Ho¡mann, H Ibel, U Wendel, D Ballhausen, MR Baumgartner, N Blau

83

330-P Management of phenylketonuria (PKU) in Europe: a review from 8 countries N Blau, A Be¨langer-Quintana, M Demirkol, F Feillet, M Giovannini, A MacDonald, FK Trefz, FJ van Spronsen

83

331-P Phenylketonuria in an era of neonatal screening MPA Hoeks, M den Heijer, MCH Janssen

84

332-P PKU in Europe ^ what is daily practice? KK Ahring, M Gisewska, F von Spronsen

84

333-O Experience with large neutral amino acids (LNAA) in the treatment of phenylketonuria(PKU) AB Burlina, AP Burlina, K Michals-Matalon, J Coldwell, R Hillman, G Bhatia, P Galvin-Parton, J Grady, SK Tyring, R Matalon

84

334-P Long term tetrahydrobiopterin (BH4) treatment of phenylalanine hydroxylase (PAH) de¢cient patients V Leuzzi, S De Leo, Cl Carducci, Ca Carducci, C Artiola, S Santagata, I Antonozzi

84

335-O Long-term correction of hyperphenylalaninemia in a mouse model for PKU by liver or intramuscular delivery of AAV expressing PAH with various serotypes A Rebu¡at, CO Harding, Z Ding, B Thony

85

314-P Hyperphenylalaninemia and potential renal acid load (PRAL) D Casero, M Borzani, F Sala, G Cagnoli, E Salvatici, S Paci, M Giovannini

315-P Intelligence, cognition and socio-emotional development of children, adolescents and young adults early treated for PKU: the need of school and professional guidance C Carmona, MF Almeida, JC Rocha, L Vilarinho, ML Cardoso, MR Lima 80 316-P Psychosocial distress in PKU female patients with good and bad metabolic control M Giovannini, P Mirazita, S Paci, D Casero, L Fiori, E Riva 317-O Ine¡ectiveness of tetrahydrobiopterin in phenylalanine hydroxylase de¢ciency F Porta, A Mussa, M Spada, A Ponzone

80

80

318-P Brain MRI di¡usion weighted imaging (DWI) is useful to assess white matter abnormalities progression in classical pku patients AB Burlina, R Manara, V Citton, M Ermani, C Carollo, C Zanco, AP Burlina 80 319-P Assessment of tetrahydrobiopterin (BH4) responsiveness in a population of Southern Italy screened for PKU D Concolino, M Rapsomaniki, MG Pascale, MT Moricca, G Bonapace, P Strisciuglio

81

327-O New insights into phenylalanine hydroxylase kinetics and the mutual impact of substrate and cofactor concentrations M Staudigl, SW Gersting, KF Kemter, DD Messing, MK Danecka, AC Muntau 83

xii 336-P Birth of new exons in LINE-2 and in antisense AluSq by intronic mutations in the PTS gene leading to tetrahydrobiopterin (BH4)-cofactor de¢ciency D Meili, J Kralovicova, L Bonafe, L Fiori, N Blau, I Vorechovsky, B Thony 337-P Phe plasma concentration and cognitive performance: preliminary outcomes A Ferreira, B Oliveira, J Gradinayna, A Guerra, A Oliveira 338-P Hypotyrosinemia, a major potential adverse event in the treatment of classical phenylketonuria (PKU) using high doses of tetrahydrobiopterine (BH4) BJM Franc°ois

J Inherit Metab Dis (2008) 31 (Suppl 1)

85

85

85

11. Urea Cycle Disorders 339-P Hyperornithinemia, hyperammonemia and homocitrullinuria (HHH) syndrome: adult presentation JM Hernandez-Perez, J Garcia-Villoria, M Rodes, E Martinez-Hernandez, A Tessa, F Santorelli, A Ribes

86

340-P Rapid detection of 3 common mutations in human OTC gene associated with late-onset OTC de¢ciency in male M Yoshino, S Numata, I Ueki, Y Watanabe, C Fujii, Y Kohda

86

341-P The natural history of ornithine transcarbamylase de¢ciency late-onset form due to prevalent in Poland A208T mutation D Rokicki, E Ciara, E Popowska, J Sykut-Cegielska

86

342-P Using the GaitRite2 walkway system to assess gait patterns in children with arginase de¢ciency M Wood, M Mcsweeney, P Natalia, M Cleary

86

343-P Late onset carbamoylphosphate synthetase de¢ciency AC Ferreira, C Moc°o, R Quental, L Azevedo, S Sequeira

87

344-P Ornithine transcarbamylase de¢ciency: phenotype and genotype characterization in 25 patients ^ experience from a Portuguese tertiary center E Lea¬o Teles, E Rodrigues, S Soares, E Trindade, L Vilarinho, ML Cardoso, L Azevedo

347-P Classical citrullinemia: Possible cluster in a limited geographic area of Argentina LE Laro¨vere, CJ Angaroni, SL Antonozzi, MB Bezard, M Shimohama, R Dodelson de Kremer 348-P Atypical presentation of citrullinemia type I FJ van Spronsen, M Cuppen, MWM Eling, DJ Reijngoud, GPA Smit, M de Groot, FW Verheijen, KE Niezen-Koning, EHHM Rings

89

353-P Inhibition of N-acetyl-glutamate synthase activity in valproate-associated hyperammonemia CCP Aires, A van Cruchten, J Ruiter, L IJlst, I Tavares de Almeida, M Duran, RJA Wanders, MFB Silva

89

354-P Valproate induced hyperammonaemic encephalopathy syndrome. Treatment with carglumic acid C Pedro¨n Giner, L Lo¨pez Mar|¨ n, P Quijada Fraile, J Lara Herguedas, JJ Garc|¨ a Pen¬as, A Duat Rodriguez, MJ Garc|¨ a Mun¬oz

89

355-P E¤cacy of carbaglumic acid for neonatal hyperammonaemia in type I cytrullinaemia S Gasperini, E Pasquini, S Funghini, S Malvagia, MA Donati

90

356-O Hindsight into urea cycle defects a¡ecting regulatory sites: the acetylglutamate site of carbamoyl phosphate synthetase I (CPSI) and the arginine site of acetylglutamate synthase (AGS) S Pekkala, E Sancho-Vaello, L Ferna¨ndez-Murga, I Ye¢menko, V Rubio, J Cervera 90 357-O Citrin de¢ciency: prolonged neonatal jaundice and failure to thrive AP Fernandes, C Nogueira, E Martins, S Soares, M Almeida, D Quelhas, ML Cardoso 90 358-P Argininosuccinic aciduria associated with pancreatitis A Dursun, HS Sivri, A O«zon, Z Akcao«ren, A Tokatl|, HG Koch, T Cos° kun

90

359-P Argininosuccinic aciduria. Report of a case with epileptic encephalopathy D Casero, A Vignoli, F La Briola, S El Oksha, V Colombo, E Riva 91 87

345-P Improved clinical outcome in girls with positive allopurinol loading test following institution of low protein diet DL Renaud, SK Eckert 87 346-P Phenotypic variability among hyperornithinemia^ hyperammonemia^homocitrullinuria (HHH) syndrome patients homozygous for the delF188 mutation in SLC25A15 FG Debray, M Lambert, B Lemieux, R Drouin, B Maranda, R Laframboise, G Mitchell

352-P MRI abnormalities due to neonatal hyperammonemic encephalopathy are not predictive of irreversible brain damage A Ku«ster, T Le Franc°ois, V Valayannopoulos, N Boddaert, JM Dejode, JC Roze, C Vianey-Saban, J HÌberle, P de Lonlay

87

88

88

349-O Analysis of residual enzyme activities in citrullinemia type I by in vitro expression studies C Berning, I Bieger, S Pauli, T Vermeulen, B Rolinski, K Gempel, J HÌberle

88

350-P Transient fulminant hepatitis in a patient with classical citrullinemia H Faghfoury, A Schulze

88

351-P E¡ective treatment of hyperammonaemia in a neonate with citrullinemia type 1 FJ Eyskens, W Peper

89

360-P Argininosuccinic aciduria: a survey of eight Tunisian infants N Esseghir, H Azzouz, M Fontaine, S Khemir, F Nassrallah, N Gandoura, N Tebib, N Ben Jaballah, F Amri, G Briand, MF Ben Dridi, N Porchet, N Kaabachi 91 361-P Liver transplantation for argininosuccinate lyase de¢ciency: clinical, biochemical and neuroimaging follow-up L Re¨gal, R Zeevaert, R Van Damme-Lombaerts, L Salviati, J Jaeken 91 362-P Preliminary data on adult patients with urea cycle disorders (UCD) in an open-label, switch-over, doseescalation study comparing a new ammonia scavenger, glyceryl tri (4-phenylbutyrate) [HPN-100], to Buphenyl (sodium phenylbutyrate [PBA]) B Lee, MR Garovoy, SE Gargosky, SA Berry

91

363-P Sodium phenylbutyrate (Ammonaps) in the treatment of patients with urea cycle disorders ^ European postapproval data DD Dobbelaere

92

364-O Long-term quanti¢cation of enzyme transfer by liver cell transplantation (LCT) for neonatal carbamoylphosphate synthase 1 (CPS1) de¢ciency J Meyburg, JM Nuo¡er, M Lindner, H Bruns, L Grenacher, H Kriegbaum, J Weitz, J Schmidt, GF Ho¡mann 92

J Inherit Metab Dis (2008) 31 (Suppl 1)

xiii 380-O New mutation in M. Anderson-Fabry: e¡ect on lipid composition, lipid raft-associated proteins and enzyme tra¤cking AM Das, J Jia, M Keiser, HY Naim

12. Lipids, Sterols, Lipoproteins 365-P MLPA in di¡erential diagnostics of congenital adrenal hyperplasia ZV Vrzalova, EH Hrabincova, LF Fajkusova, JV Vseticka, LK Kozak 366-P Plasma and thrombocyte levels of coenzyme Q10 in children with Smith-Lemli-Opitz syndrome (SLOS) and the in£uence of HMG-CoA reductase inhibitors D Haas, P Niklowitz, GF Ho¡mann, W Andler, T Menke 367-P Bile acid pro¢les in patients with four di¡erent bile acid biosynthesis defects P Ruiz-Sala, I Ferrer, M Briones, B Merinero, C Pe¨rez-Cerda¨, MJ Garcia, M Ugarte

92

92

382-P Chitotriosidase determination in plasma and in dried blood spots: a comparison using two di¡erent substrates in a microplate assay MDB Rodrigues, AC Oliveira, KB Mu«ller, AM Martins, V D'Almeida

96

93

383-A Diarrhoea and hepatocellular cytolysis pro¢le leading to Pompe disease diagnosis DD Dobbelaere, CA Alexandre, PL Laforeªt, KM Mention

97

384-O Slowly progressive clinical phenotype of San¢lippo A patients exhibiting the mutation p.S298P A Meyer, K Kossow, A Gal, C Mu«hlhausen, K Ullrich, T Braulke, NM Muschol

97

385-O LIMP-2 sorting pathway: a cell type speci¢c de¢ciency of beta-glucocerebrosidase A Balreira, P Gaspar, D Caiola, J Chaves, I Beira¬o, J Lopes Lima, JE Azevedo, MC Sa¨ Miranda

97

386-O Surrogate biochemical markers for lysosomal storage disorders M Fuller, J Hopwood

97

387-P Phase 2 clinical trials of the pharmacological chaperone AT1001 for the treatment of Fabry disease DP Germain on Behalf of AT1001 Study Group, J Castelli, A Shenker, H Do, B Wustman, D Palling, DJ Lockhart

98

388-O Disease severity in multiple sulfatase de¢ciency is determined by stability and residual activity of mutant formyl-glycine-generating enzyme L Schlotawa, T Dierks, B Schmidt, J GÌrtner

98

389-P Chaperone e¡ects on molecular pathology in GM1gangliosidosis Y Suzuki, E Nanba, K Higaki, Y Sakakibara, H Jo, K Yugi, M Iida, S Ogawa

98

390-O Miglustat in patients with Niemann-Pick type C disease (NPC): a multicentre retrospective survey M Pineda, JE Wraith, F Sedel, WL Hwu, M Rohrbach, B Bembi, GC Korenke, C Luzy, P Schieber, MC Patterson

98

391-A Juvenile-onset neuronal ceroid lipofuscinosis (JNCL, Batten): clinical and molecular investigation in a large family in Brazil ER Valadares, RHC Amorim, LR Oliveira, TMM Pinheiro, HH Santos, MX Pizarro, RR Queiroz, GC Lopes, ALB Godard

99

392-O Lentivirus mediated gene therapy for murine model of Pompe disease SO Kyosen, S Iizuka, A Morita, T Kimura, H Kobayashi, Y Eto, H Ida, T Ohashi

99

368-P Diagnosis of chylomicron retention disease by mutation analysis of the SARA2 gene M Busch, K Tsiakas, J Bergmann, M Heiduk, KP Zimmer, R Santer

93

369-P Hypocholesterolemia in the newborn with trisomy 13 V Bzduch, D Behulova, E Veghova

93

370-P Insulin resistance in pediatric patents with familial hyperlipidemia S Terlemez, S Kalcan Ucar, E Bozdemir, RD Goksen Simsek, S Darcan, C Kabaroglu, M Kay|kcioglu, S Habif, O Bayindir, M Coker 93 371-P X-linked steroid sulphatase de¢ciency: review of 8 cases H Santos, I Leite, D Moreira, EO Ferreira, JS Marques

94

372-P Type IIB lipoprotein pattern with elevated liver enzymes as presenting symptom of cholesteryl esters storage disease (CESD) S Decarlis, Cv Agostoni, F Ferrante, S Scarlino, E Riva, M Giovannini 94 373-P Familial lipoprotein lipase de¢ciency in three infants ^ case report E Starostecka, A Lange, ME Funkowicz, M Chmara, J Krzyzçanowska 94 374-P Newborn screening for CAH using MS/MS as a primary screen DC Lehotay, R Thompson, P Van Caeseele, S Taback

94

375-P Abnormal metabolite screening in a patient with Berardinelli-Seip congenital lipodystrophy NM Verhoeven-Duif, TJ de Koning, MAM de Vroede, N Hamers, MGM de Sain-van der Velden, BHCMT Prinsen, EH Jeninga, R Berger, L Klomp, E Kalkhoven 95 376-P Clinical and molecular genetic analysis and treatment outcomes of four patients from three Chinese families with sitosterolemia HC Yu, YH Lu, MY Lo, HJ Gao, JH Hsu, KW Chong, CH Kao, KH Cheng, TT Liu, PY Hung, DM Niu 95

13. Lysosomal Disorders 377-P Autopsy of a case of Gaucher disease type I on enzyme replacement therapy. Comment on dynamics of the persisting storage process H Hulkova, H Poupetova, A Kohout, V Malinova, M Elleder

95

378-P The clinical, genetic and pathological studies of 5 Japanese patients with perinatal lethal Gaucher disease M Kobayashi, T Ohashi, T Fukuda, Y Eto, H Ida 95 379-O Placebo-controlled study of alglucosidase alfa in adults with Pompe disease A van der Ploeg, P R Clemens, D Corzo, D Escolar, J Florence, P Laforet, S Lake, J Mayhew, C Morgan, A Pestronk, B Rosenbloom, A Skrinar, M Wasserstein

96

381-A Chitotriosidase activity in dried blood spots of a Brazilian sample: determining de¢cient and increased activities MDB Rodrigues, KB Mu«ller, AM Martins, V D'Almeida 96

96

393-P Reference limits for components with strong age dependence from laboratory production data ^ exempli¢ed by urinary glycosamino-glycans (U-GAG) L MÖrkrid 99 394-P The neurological manifestations of Gaucher's disease type 1: the French observatoire on Gaucher disease (FROG) P Cherin, Ch Rose, Ch De Roux Serratrice, D Dobbelaere, B Grosbois, E Hachulla, R Jaussaud, RM Javier, E Noel, P Clerson, A Hartmann

99

xiv

J Inherit Metab Dis (2008) 31 (Suppl 1)

395-O Digital micro£uidics: a novel platform for multiplexed detection of lysosomal storage diseases with potential for neonatal screening D Millington, V Srinivasan, A Eckhard, C Cotten, R Goldberg, V Pamula 100 396-P Reduction of left ventricular hypertrophy correlates with a signi¢cant decrease in B-type natriuretic peptide in a patient with infantile Pompe disease treated with recombinant acid alpha-glucosidase I Berkau, C Lilje, A Hahn, N Muschol, R Santer, K Ullrich 100 397-P Mucopolysaccharidosis type II (MPS II): evaluate of food intakes in patients on enzyme replacement therapy RB Oliveira, BJ Frangipani, JR Lauandos, C Micheletti, MT Silveira, T Vertemati, AM Martins 100 398-P Metachromatic leucodystrophy: arylsulfatse A mutations in Russian patients TM Boukina, EYu Voskoboeva, AM Boukina, SV Michailova, IV Tsvetkova 100 399-P Mortality rate of patients with MPS VI during the period of 16 months tracked by patients associations in Brazil AM Martins, C Micheletti, M Kerstenetzky, EM Ribeiro, DG Horovitz, I Gomy, PFV Medeiros, T Amorim, AX Acosta, D Bom¢m, R Boy, M Cipriano, TM Pereira, D Giovannetti 101 400-P Identi¢cation of ambroxol as a potential enzymeenhancement agent for Gaucher disease GHB Maegawa, MB Tropak, J Buttner, G Kornhaber, B Rigat, JTR Clarke, D Mahuran 401-P Impact of ERT on familiar life T Vertemati, C Micheletti, FB Piazzon, S Canossa, RM Okubo, AM Martins

404-P Aneurysm of the left ventricle and cirrhosis associated with mucopolysaccharidosis type II (MPS II) ER Valadares, BEG Quirino, FHC Melo, VHR Leite, P Godoy, RR Arantes, TCA Ferrari, LOS Rocha

104

412-P Anomalies in T and iNKT populations in Fabry mice but not in Fabry patients A Balreira, MF Macedo, C Gira¬o, LG Rodrigues, JP Oliveira, MC Sa¨ Miranda, FA Arosa 104 413-P Echocardiographic and electrocardiographic evolution of 5 paediatric MPS type 1 patients under enzyme replacement therapy ^ 4 year follow-up P Martins, P Garcia, G Ramalheiro, L Diogo, E Castela 104 414-P Clinical presentation of 1 patient with late onset Pompe disease and ¢rst results after 6 months of ERT N Gu¡on, I Forest, F Aubert, F Gormand, A Fouilhoux

104

415-P Animal model of Fabry disease: study of early enzyme replacement therapy (ERT) I Mendes, LG Rodrigues, M Pais-Vieira, MC Sa¨ Miranda

105

416-P Reduction of aldurazyme infusion time for MSP I patients: the Lyon experience N Gu¡on, I Forest, N Reynes, A Fouilhoux 105 417-P Home therapy is feasible for Scheie patients N Gu¡on, I Forest, C Caire, M Chateau, B Renaud, M Magnet, A Fouilhoux

105

418-P Clinical characteristics of patients in the MPS I registry FA Wijburg, D Viskochil

105

101

419-P Treatment trends in MPS I: The MPS I registry N Gu¡on, J Clarke

106

101

420-P Mucolipidosis type IV in a Mapuche patient with cerebral palsy GP Duran, M Hernandez, I Huete, S Gonzalez

106

421-P Neurological complications in type 1 Gaucher disease M Biegstraaten, IN van Schaik, JMFG Aerts, CEM Hollak

106

102

422-P The prevalence of lysosomal storage disorders in the Czech Republic H Poupetova, J Ledvinova, J Hlavata, M Fialova, M Hrebicek, V Kozich, S Stastna, E Hruba, E Kostalova, H Jahnova, V Malinova, B Asfaw, L Kuchar, Z Novotna, J Zeman, M Elleder

106

102

423-P Synthesis of speci¢c sphingolipids isoforms using immobilized sphingolipid ceramide N-deacylase L Kuchar, J Ledvinova, J Lenfeld, D Horak, J Rotkova, Z Bilkova

107

402-P Arylsulfatase A gene: identi¢cation of common mutations by real time PCR H Bock, EA Carpes, GF Rodrigues, MG Burin, U Matte, R Giugliani, ML Saraiva-Pereira 101 403-P Physiotherapeutic evaluation in MPS II patients after twenty six weeks of enzyme replacement therapy (ERT) with idursulfase (Elaprase) E Fraccaro, E Menegatti, A Ricarte, FB Piazzon, T Vertemati, C Micheletti, MH Rand, AM Martins

411-P Danon disease ^ report of two Portuguese patients E Rodrigues, E Martins, A Vieira, H Santos, L Castro, S Carpenter, E Lea¬o Teles

405-P Preliminary data of ERT in MPS VI patients under 3 years old M Kerstenetzky, EM Ribeiro, DG Horovitz, J LLerena, CA Kim, R Honjo, AC Paula, T Moura¬o, D Giovannetti

102

406-P Chitotriosidase levels in cystinosis A Xaidara, E Karavitakis, K Kosma, E Dimitriou, H Michelakakis

102

407-P Lysosomal enzymes in dried blood spots, as a diagnostic tool of lysosomal storage disorders LS Almeida, C Caseiro, E Silva, L Lacerda 103

424-P MS/MS sphingolipid isoform pro¢ling ^ useful diagnostic tool in disorders with Gb3Cer and sulphatide storage L Kuchar, J Hlavata, B Asfaw, J Ledvinova 107 425-P Long-term e¤cacy and safety of agalsidase alfa in women with Fabry disease C Whybra, C Kampmann, E Miebach, A Gal, K Baron, M Beck 107

408-P Eight-year clinical outcomes of long-term enzyme replacement therapy in 884 children with type 1 Gaucher disease H Andersson, P Kaplan, K Kacena, J Yee 103

426-P Molecular analysis of 82 mucopolysaccharidosis type I patients: mutational spectrum in the european population and identi¢cation of 28 novel mutations F Bertola, R Parini, G Casati, A Tylki-Szymanska, I Okur, B Tuysuz, J Dalmau, A Gonzales Meneses, D Antuzzi, R Barone, C Dionisi Vici, A Donati, M Filocamo, O Gabrielli, G Parenti, M Scarpa, G Uziel, A Biondi 108

409-P Substrate reduction therapy with Miglustat in juvenile GM2 gangliosidosis GHB Maegawa, B Banwell, S Blaser, P van Giersbergen, G Sorge, M Tlopak, C Arckerley, C Hawkins, J Hayes, JTR Clarke

103

427-P A phase 2 clinical trial of the pharmacological chaperone AT2101 for the treatment of Gaucher disease N Weinreb on Behalf of AT2101 Study Group, E Schneider, Q Dinh, C Duke, F Insinga, K Scott, H Do, B Wustman, D Palling, DJ Lockhart 108

410-P Experience in Lyon of MPS II patients with enzyme treatment N Gu¡on, I Forest, A Fouilhoux

103

J Inherit Metab Dis (2008) 31 (Suppl 1) 428-P Prenatal diagnosis of Niemman Pick disease type C without family history I Ribeiro, O Amaral, H Ribeiro, C Caseiro, J Dupont, A Couceiro, L Lacerda

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108

429-P Bisphosphonate therapy in osteoporosis in lysosomal storage diseases E Procopio, F Ciani, S Gasperini, E Pasquini, E Zammarchi, MA Donati 109 430-P Niemann-Pick type C: no neurologic imvolvement after three years of treatment with Miglustat A Fiumara, A Dardis, S Federici, R Barone, M Di Rocco

109

431-P Vertebro-medular imaging ¢ndings in mucopolysaccharidosis types II and IV S Castro, M Ayres-Basto, E Rodrigues, MM Campos, J Guimara¬es, E Lea¬o-Teles

109

432-P Behavioural, neurophysiological and neurochemical alterations in male and female (homozygous) Fabry mice LG Rodrigues, MJ Ferraz, M Pais-Vieira, MM Sousa, MC Sa¨ Miranda 109 433-P Cerebral imaging ¢ndings in mucopolysaccharidosis types II and IV S Castro, M Ayres Basto, E Rodrigues, J Guimara¬es, A Magalha¬es, E Lea¬o Teles 110 434-P Experience with enzyme replacement therapy in 6 MPS VI patients V Valayannopoulos, A Chabli, M Lemoine, T Odent, A Goldenberg, V Barbier, C Caillaud, M Lemerrer, P de Lonlay

110

435-P Progressive spinal instability in MPS ^ use of recombinant human bone morphogenetic protein-2 (rhBMP-2) to augment posterior spine fusion G Solanki, S Vijay, A Chakrapani, C Hendriksz 110 436-P Psychological follow-up of children on enzyme replacement therapy for lisosomal storage disorders and their parents F Rodrigues, C Vaz, M Almeida, F Martins, L Diogo, P Garcia 110 437-P Open-¢eld and dark-light tests: behavioural characterization of Fabry knockout mice MJ Ferraz, A Magalha¬es, LG Rodrigues, MC Sa¨ Miranda 111 438-P Chondrogenic di¡erentiation in mesenchymal stem cells: glycosaminoglicans synthesis and GALNS activity ML Gutierrez, LF Malaver, OY Echeverri, LA Barrera 111 439-P Which is the frequency of Fabry disease? P Gaspar, D Rodrigues, J Herrera, MC Sa¨ Miranda 440-P Molecular characterization of Portuguese patients with pathologies related to the lysosomal multienzymatic complex: sialidosis and galactosialidosis MF Coutinho, L Lacerda, MJ Prata, H Ribeiro, S Alves

445-P Assessment of shoulder £exion and abduction in MPS-II Hunter syndrome Polish patients on Genistein A Tylki-Szyman¬ska, J Marucha, G Wegrzyn, 113 J Jako¨bkiewicz-Banecka, E Piotrowska, A Kloska 446-P Functional characterization and chemical chaperones e¡ect on alpha galactosidase gene mutations identi¢ed in Fabry Italian patients C Filoni, A Caciotti, L Carraresi, C Cavicchi, R Parini, S Feriozzi, P Poisetti, S Garman, R Guerrini, E Zammarchi, MA Donati, A Morrone

113

447-P Morquio Syndrome: Gene expression pro¢ling and elastic ¢ber assembly in patients' ¢broblasts A Caciotti, L Carraresi, C Filoni, R Parini, D Antuzzi, R Ricci, M Scarpa, E Procopio, A d'Azzo, E Zammarchi, R Guerrini, MA Donati, A Morrone

113

448-P Enzymatic replacement therapy in 17 patients a¡ected by MPS I, II and VI M Rigoldi, L Tedesco, ML Melzi, A Lastrico, R Parini

113

449-P Annual increase of height in MPS I patients on enzyme replacement therapy with laronidase A Tylki-Szyman¨ska, J Marucha, E Arasimowicz, A Rozçdzçyn¨ska

114

450-P Preliminary results of a phase 2 clinical trial of Genz112638 in patients with type 1 Gaucher disease E Lukina, N Watman, EA Arreguin, M Banikazemi, M Iastrebner, H Rosenbaum, A Zimran, F O'Brien, SE Smith, AC Puga, J Peterschmitt

114

451-P Retinitis pigmentosa and adult-onset neurological deterioration revealing San¢lippo A disease MC Nassogne, A Jeanjean, C Kestens, C Grandin, MF Vincent, W Lissens

114

452-P The cardiac e¡ects of enzyme replacement therapy for japanese Fabry disease: comparison between female and male patients M Fujiwara, T Ohashi, H Ida, Y Eto 114 453-P Determination of stability of total hexosaminidases and beta-galactosidase over time on ¢lter paper dry blood spots in di¡erent conditions of temperature CD Castilhos, FG Werlang, MG Burin, JC Coelho 115 454-P Derangement of mannose-6-phosphate receptor tra¤cking impairs lysosomal enzyme uptake in ¢broblasts from lysosomal storage diseases M Cardone, C Porto, A Tarallo, B Rossi, R Tuzzi, F Donaudy, F Fontana, G Andria, A Ballabio, G Parenti 115

111

455-P E¤ciency of a new visual test for the quick detection of pathological excretion of glycosaminoglycans in urine F Andrade, JA Prieto, P Sanjurjo, M Montejo, L Alda¨miz-Echevarr|¨ a

111

456-P Clinical phenotype of Italian patients with Hunter syndrome: data from HOS ^ the Hunter Outcome Survey R Parini, ML Melzi, M Rigoldi, S Sala, A Rampazzo, O Gabrielli, M DiRocco, C Feliciani, M Castorina, A Cicognani, M Scarpa 115

441-P Time interval between diagnosis of type 1 Gaucher disease and initiation of enzyme therapy and splenectomy are determinants of avascular necrosis PK Mistry, P Deegan, A Vellodi, JA Cole, M Yeh, NJ Weinreb 112 442-A The same novel mutation determined in 2 Hurler-Scheie patients who are the children of di¡erent families A Hasanoglu, I Okur, FT Eminoglu, L Tumer, G Biberoglu, F Bertola, FS Ezgu 112 443-P In vitro e¤cacy of N-octyl-4-epi-beta-valienamine (NOEV) in a patient with infantile GM1 gangliosidosis H Ayd|n, H Kalkanoglu Sivri, I Sinici, A Tokatli, T Coskun, Ha Ozkara, I Kurt, K Higaki, E Nanba, Y Suzuki 112 444-P Fabry disease: a new atypical variant form L Lacerda, O Amaral, I Ribeiro, E Silva, C Ferreira, E Pinto, G Soares, H Antunes 112

457-P Cognitive and neuroradiological improvement in 3 attenuated MPS I patients treated by laronidase V Valayannopoulos, N Boddaert, V Barbier, M Lemerrer, P de Lonlay

115

116

458-P Fabry disease in childhood: renal involvement ^ the value of general evaluation E Lea¬o Teles, E Rodrigues, MJ Baptista, H Jardim, C Moura, A Magalha¬es, L Lacerda, JP Oliveira 116 459-P Enzyme replacement therapy for mucopolysaccharidosis VI in italy M Scarpa, R Barona, A Fiumara, L Astarita, G Parenti, A Rampazzo, S Sala, R Parini 116

xvi 460-P Experience with idursulfase in 11 MPS II patients including 6 patients under the age of ¢ve V Valayannopoulos, A Chabli, C Caillaud, M Lemoine, S Lyonnet, M Lemerrer, V Cormier-Daire, P de Lonlay 461-P Newborn screening for Fabry disease in Japan K Nakamura, K Hattori, S Matsumoto, Y Nakamura, T Mochida, H Tajiri, H Mitsubuchi, F Endo

J Inherit Metab Dis (2008) 31 (Suppl 1)

116

117

462-P An extreme case of Lyonisation in a X-linked disorder: a Turner patient with Fabry disease FJ Eyskens, R Brouns, K de Boeck

117

463-P Pompe disease in Thai infants ^ report of 4 cases P Wasant, N Vatanavicharn, S Liammongkolkul, WL Hwu

117

464-P Risk for carpal tunnel syndrome in patients with mucopolysaccharidosis type I on enzyme replacement therapy QGA Teunissen, CEM Hollak, FA Wijburg

117

465-P Use of high protein diet in infantile Pompe disease V Hopkins, A MacDonald, A Daly, C Bunford, A Chakrapani, C Hendriksz

118

466-P Cholesterol and sphingolipid tra¤cking alterations in CLN6-de¢cient human ¢broblasts CA Teixeira, C Bessa, Y Gong, R Bittman, R Pagano, MG Ribeiro

118

467-P Diagnosis and management of mucopolycaccharidoses in Ukraine NA Pichkur, NV Olkhovich, EA Makushenko, NG Gorovenko 118 468-P Mucopolysaccharidosis type IIID (San¢lippo syndrome D): clinical data on 10 new patients and identi¢cation of 8 novel mutations MJ Valstar, GJG Ruijter, AM Bertoli-Avella, MW Wessels, B de Graaf, BJHM Poorthuis, OP van Diggelen 118 469-P Mucopolysaccharidosis IVA: A severe phenotype in patients homozygous for the c.347G4T (p.Gly116Val) mutation S Vijay, S Ball, C Hendriksz, L Simmons, G Gray, T Hutchin, A Chakrapani 119 470-P Tigroid pattern of leukodystrophy in a case of Krabbe disease AC Ferreira, MC Vale, S Sequeira

119

471-P Enzyme replacement therapy for mucopolysaccharidosis type I in Japan T Okuyama, T Tanaka 119 472-P High incidence of vitamin D de¢ciency in patients with Pompe disease S Waldek, M Roberts

119

473-P Baseline characteristics of patients in the Canadian Fabry disease initiative SM Sirrs, R Casey, JTR Clarke, DG Bichet, K Lemoine, ML West

120

474-P Experience with enzyme replacement therapy for mucopolysaccharidosis VI in 2-year old patient Z Fehervizyova, A Hlavata

478-P Persistent e¡ect of miglustat on four children with Niemann-Pick C disease YH Chien, WL Hwu, NC Lee, LK Tsai, AC Huang, SF Peng

121

479-P Enzyme therapy in early symptomatic adult male patients does not prevent disease progression SM Rombach, GE Linthorst, FA Wijburg, JMFG Aerts, CEM Hollak

121

480-P The correlation between genotype, clinical systemic severity and ocular manifestations in children with Anderson-Fabry disease EC Cosgrave, U Ramaswami, JP Kersey, LE Allen

121

481-P Fabry disease in children and response to enzyme replacement therapy: results from the Fabry outcome survey U Ramaswami, R Parini, G Pintos-Morell, R Hartung, C Kampmann, M Beck

122

482-P ERT using galsulfase for Maroteaux-Lamy syndrome in Japan T Tanaka, M Furujo, T Kubota, T Ohashi, A Tanaka, Y Suzuki, Y Eto, T Orii, T Okuyama

122

483-P Post mortem studies on a patient with mucopolysaccharidosis type 1: histopathologic ¢ndings after one year of enzyme replacement therapy S Yano, K Moseley, Z Pavlova

122

484-P Nonsense-mediated mRNA decay process in nine alleles of Niemann-Pick type C patients from Spain J Macias, L Gort, M Lluch, MJ Coll 122 485-P Computer-assisted teaching of mucopolysaccharidosis by patient management problems F Al-Jasmi, L Moldovan, JTR Clarke

123

486-P E¤cacy of miglustat on dysphagia in four Niemann Pick patients D Bruschini, S Fecarotta, L Astarita, A Romano, G Mansi, M Amitrano, H Dolezalova, R Della Casa, G Parenti, G Andria 123 487-P Phenotype variability in six cases with MPS VI in Lithuania L Cimbalistiene, J Songailiene, B Czartoryska, V Kucíinskas 488-P Metachromatic leukodystrophy is not always a progressive disease A Bley, R Santer, Z Lukacs, A Kohlschuetter

123

489-P Determination of urinary globotriaosylceramide by UPLC-MS/MS: urine dried on ¢lter paper versus liquid urine S Forni, X Fu, L Sweetman, R Schi¡mann

124

490-P Attenuated form of San¢lippo type B: clinical and molecular characterisation of nine Dutch patients S Kalb, S Luf, K Hinderhofer, I van Mierlo, H van Schrojenstein Lantman-de Valk, C Marcelis, J Zschocke, U Moog

124

124

123

120

475-P Dysregulation of intracellular homeostasis in murine model of mucopolysaccharidosis type I VG Pereira, LC Rodrigues, EM Lauro, ML Gazarini, AM Martins, V d'Almeida

491-P Acute lymphoblastic leukemia in a child treated by Elaprase1 for Hunter's disease F Feillet, A Salmon, A Kimmoun, J Straczeck, JL Gueant, P Bordigoni

120

476-P Oxidative stress analysis in patients with Fabry disease KB Muller, MDB Rodrigues, VG Pereira, AM Martins, V d'Almeida

492-P Neuronal ceroid lipofuscinosis from our children's hospital MS Perez Poyato, M Pineda Marfa, V Cussi, I Ferrer, M Mila 124

120

493-P Novel mutation on a Brazilian patient with Fabry disease CFM Souza, C Netto, F Pereira, U Matte, M Burin, R Giugliani 125

477-P Di¡erential stability of lysosomal enzymes activity in dried blood spots on ¢lter paper KB Mu«ller, MDB Rodrigues, VG Pereira, M Miranda, AM Martins, V d'Almeida

121

494-P Adult-onset Niemann-Pick Type C disease: a novel case presenting with seizures MC Maj, T Rupar, JW Callahan, CF Morel

125

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xvii

495-P The pharmacological chaperone AT2220 increases GAA activity and represents a potential new treatment for Pompe disease JJ Flanagan, HV Do, X Wu, AC Powe, R Khanna, W Liang, K Tang, C Pine, H Williams, R Soska, L Pellegrino, S Shao, ER Benjamin, BA Wustman, KJ Valenzano, DJ Lockhart 125 496-P Mutation analysis in Niemann-Pick disease type C: a practical approach SB Knight, LE Heptinstall, C Whitehouse, A Cooper, J Imrie, JE Wraith 497-P Expression analysis in COS-1 cells of missense alleles of the GLB1 (acid b-galactosidase) gene with presumptive in£uence on catalytical enzyme function E Paschke, D Hofer, K Fantur, K Paul, A Morrone, A d'Azzo

125

126

498-P Dietary intake, energy expenditure and body composition of patients with Pompe disease: a pilot study E van der Louw, L van den Berg, P Kramer, N Kruijer, AA Zandbergen, MLC Hagemans, AT van der Ploeg 126 499-P Infantile form of Pompe's disease: two cases with dramatical response to enzymological therapy B Chabrol, A Cano, H Mansour

126

500-P E¡ect of Epstein Barr virus on lysosomal hydrolases activities from lymphocytes: comparison before and after cryopreservation JC Coelho, AS Mello, K Michelin-Tirelli, M Camelier, JF Mari, MG Burin, R Giugliani 126 501-P Enzyme replacement therapy for late-onset acid maltase de¢ciency: clinical experience of fourteen patients treated with alglucosidase alfa for one year A Amado Fondo, A Cousins, PJ Lee, RH Lachmann 127 502-P Dietary intervention in children receiving substrate reduction therapy with miglustat (Zavesca1) H Champion, RH Lachmann, TM Cox, N Wright, U Ramaswami

127

503-P Biochemical screening of Farber disease using a combined approach I Ribeiro, M Alves, O Amaral, MG Ribeiro

127

504-P Dried blood spot assays for Fabry and Pompe disease: how do multiplex tandem mass spectrometry and £uorometry compare to each other? B Wuyts, V Stove

127

505-P Novel clues on the intracellular localization of CLN6, the protein defective in the neurodegenerative LSD-NCL type 6 M Alves, C Bessa, CA Teixeira, RM Boustany, MG Ribeiro 128 506-P Enzymatic method with acarbose used in dried blood spots and leukocytes enables diagnosis of di¡erent types of Pompe disease A Lugowska, R Rola, E Lewandowska, M Rakowicz, T Wierzba-Bobrowicz, A Tylki-Szymanska, H Mierzewska, A Fidzianska 128 507-P A pharmacogenetic approach to predict response to pharmacological chaperone therapy for Fabry disease ER Benjamin, X Wu, R Khanna, SA Sitaraman, DJ Palling, R Schi¡mann, DJ Lockhart, KJ Valenzano

128

508-P Quality of life is signi¢cantly improved following six months of enzyme replacement therapy in late-onset acid maltase disease AL Cole, PJ Lee, AJ Cousins, A Amado Fondo, RH Lachmann 128

509-P Successful `transplantation' of MRI Quantitative Chemical Shift Imaging (QCSI) technology for the detection of bone marrow fat signal fraction in type I Gaucher disease, from Amsterdam to Leuven D Cassiman, R Peeters, J Jaeken, M Maas, EM Akkerman 129 510-P Gb3 analysis for Fabry disease by LC-MS/MS using urine ¢lter paper samples C Auray-Blais, D Cyr, JTR Clarke, R Drouin 129 511-P Morphologically targeted DNA screening of neuronal ceroid lipofuscinoses CLN5 and CLN6 in Argentina IA Cismondi, R Kohan, N Guelbert, V Tapia Anzolini, A Ghio, SE Mole, W Xin, F Santorelli, R Dodelson de Kremer, AM Oller Ram|¨ rez, I Noher de Halac

129

512-P Enzyme replacement therapy in a patient infantile form of Pompe's disease with severe cardiomyopathy F Tanzer, D Buyukkayhan, E Cansu Mutlu, F Kalender Korkmaz

129

513-P Alglucosidase alfa in infants and children with Pompe disease W-L Hwu, B Byrne, E Wraith, N Leslie, H Mandel, M Nicolino, PS Kishnani

130

514-P Development of a disease severity scoring system for patients with Pompe disease EH Giannini, K Berger, A van der Ploeg, L Case, C Dandrea, PS Kishnani, D Marsden 130 515-P Hemiballism-hemichorea associated with mucopolysaccaridosis type IIIA G di Rosa, M Bonsignore, MR Piperata, G Tortorella

130

516-P A clinical trial of idursulfase in Hunter syndrome patients 5 years old and younger A Tylki-Szymanska, R Giugliani, W-L Hwu 130 517-P The Pompe registry: tracking Pompe disease symptoms in a broad patient population B Byrne, P S Kishnani, L Case, L Merlini, W Mu«eller-Felber, A van der Ploeg, J Park, D Marsden 131 518-P Idursufase therapy in Hunter syndrome S Salehpour, M Farahmand Monfared

131

519-P Branched-chain amino acid as supportive therapy for type-A Niemann-Pick patient RS Damayanti, C Tanjung

131

520-P A simple procedure to reduce the number of falsepositive results in mucopolysaccharidosis screening GJG Ruijter, RG van den Berg, JGM Huijmans

131

521-P Neonatal screening for Pompe disease ^ a feasibility study Z Lukacs, P Nieves Cobos, A Keil, R Santer

132

522-P Niemann-Pick type C disease: natural history and clinical course in ten Brazilian patients CM Lourenco, FTS Souza, JC Coelho, R Giugliani, VD Marques, P Toscano, PG Bastos, W Marques Jr

132

523-P The clinical, radiological and histological features of a patient with a novel form of late infantile neuronal ceroid lipofusinosis: CLN7 EE Glamuzina, J Flemming, E Siintola, A Lehesjoki, J MacFarlane, CJ Wilson

132

524-P Quality assessment of enzyme analysis for lysosomal storage disorders (LSD's) three European + QA-pilots with 40^49 participants OP van Diggelen, L Oemardien, I de Graaf, C Weykamp

132

525-P Central nervous system in£ammation in Fabry disease O Lidove, MP Chauveheid, C Caillaud, R Froissart, L Benoist, S Alamowitch, S Doan, R Szalat, N Baumann, JF Alexandra, P Lavallee, I Klein, F Vuillemet, F Sedel, Y Samson, E Roullet, T Papo

133

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14. Vitamins 526-P Screening for pyridoxine dependent epilepsy (PDE): HPLC-MS/MS analysis of lysine degradation metabolites H Korall, S Wallner, N Ermandraut, J Beil, F-K Trefz 133 527-P Neurological outcome in three patients with combined methylmalonic aciduria and homocystinuria (CblC) M Sibilio, R Della Casa, A Romano, G Mansi, A Morrone, MA Donati, F Fontana, L Minichini, C Ungaro, C Cavicchi, D Bruschini, G Andria, G Parenti 133 528-P CblD defect of vitamin B12 metabolism: functional analysis of mutant MMADHC alleles and mitochondrial targeting M Stucki, T Suormala, D Coelho, P Paesold-Burda, B Fowler, MR Baumgartner 133 529-P Biotinidase newborn screening experience in Tuscany S Malvagia, S Funghini, G la Marca, A Morrone, MA Donati, E Pasquini

134

530-P Molecular con¢rmation of cblC patients identi¢ed by expanded newborn screening C Nogueira, C Aiello, C Dionisi-Vici, E Martins, E Lea¬o, L Diogo, R Cerone, U Caruso, S Sequeira, F Kok, F Deodato, FM Santorelli, L Vilarinho 134 531-P Favourable therapeutic outcome for combined methylmalonic acidemia and homocystinuria due to cobalamin defects YS Choy, YS Choy, MY Zabedah

134

532-P Elevated propionylcarnitine on newborn screening and vitamin B12 levels F Al Murshedi, F Al Jasmi, E Crushell, L Kyriakopoulou, A Feigenbaum

134

533-P Folinic acid responsive seizures associated with folate receptor autoimmunity V Ramaekers, N Blau, J Sequeira, E Quadros

135

534-P Vitamin B12 de¢ciency in 50 patients with an inborn error of protein metabolism K Green, K Carpenter, B Wilcken

135

15. Neurotransmitters 535-P Functional tryptophan hydroxylase de¢ciency DA Schott, IMLW Keularts, L Dorland, M Duran, NGGM Abeling, JE de Vries, J Bierau, LMJ Spaapen, J Vles, E Rubio-Gozalbo, J Nicolai, WJM Gerver

135

536-P CSF homocysteine a useful adjunct for the investigation of patients with cerebral folate de¢ciency? G Lynes, J Land, A Briddon, S Heales

135

537-P Developmental delay and cerebral palsy associated with tryptophan hydroxylase de¢ciency. Possibilities for treatment? FAA Langius, N Abeling, M Duran, BT Poll The

540-P Abnormal neurotransmitters: pitfall in the diagnosis of Rett syndrome HM Engbers, J de Ruyter, MGM de Sain-van der Velden, M Wevers, G Visser

137

542-P GTP Cyclohydrolase de¢ciency: report of a family with atypical clinical features S Duarte, E Calado, C Nogueira, P Gaspar, L Azevedo, L Vilarinho

137

543-O Pramipexole in tetrahydrobiopterin de¢ciency F Porta, A Mussa, D Concolino, M Spada, A Ponzone

137

544-P Increased 4-aminobutyrate (GABA) in embryos with succinate semialdehyde dehydrogenase (SSADH) de¢ciency suggest an heightened excitatory state during development EEW Jansen, EA Struys, C Jakobs, SC Guimond, EJ Hager, OC Snead, KM Gibson

137

545-P Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase B Thony, AC Calvo, T Scherer, RM Svebak, J Haavik, N Blau, A Martinez

138

546-O Pyridoxal phosphate availability and aromatic amino acid decarboxylase activity. Implications for AADC de¢ciency and inborn errors of vitamin B6 metabolism G Allen, P Clayton, J Land, K Hyland, S Heales 138 547-O Amino acid and neurotransmitter abnormalities in murine intermediate maple syrup urine disease (iMSUD): signi¢cant improvement of cerebral dopamine and serotonin de¢ciency with hepatocyte transplantation (HTx) KJ Skvorak, GE Homanics, HS Paul, K Dorko, S Strom, Q Sun, E Arning, T Bottiglieri, EEW Jansen, C Jakobs, KM Gibson 138 548-O E¡ect of patient-derived mutations on 3-phosphoglycerate dehydrogenase multimerization and function L Tabatabaie, NJF van den Broek, AB Brenkman, NM Verhoeven-Duif, R Berger, TJ de Koning, LW Klomp 139 549-P Functional studies of a duplication mutation of valine at 235 by a transient expression assay in a boy with MCT8 de¢ciency M Itoh, H Kakinuma 139 550-O Mutations in urocanase gene in a patient with urocanic aciduria, mental retardation and intermittent ataxia R Artuch, C Espinos, A Ormazabal, MA Vilaseca, LJM Spaapen, D Martinez-Rubio, F Palau, M Pineda

139

551-O Dihydrolipoamide acetyltransferase de¢ciency in cases of atypical pantothenate kinase associated neurodegeneration CA McWilliam, RM Brown, RC McWilliam, CK Ridout, J Tolmie, GK Brown 139

16. Dietetics and Nutrition 136

538-P Tyrosine hydroxylase de¢ciency: identical genotype in three apparently unrelated patients. A common ancestor hypothesis M Serrano, A Ormaza¨bal, I Orfanou, S Youroukos, K Drakaki, A Giannakopoulos, J Campistol, A Garcia-Cazorla, R Artuch, B Cormand, R Pons 136 539-P Prevalence of the IVS6+4 A4T mutation in Taiwanese patients with aromatic amino acid decarboxylase de¢ciency NC Lee, WL Hwu, YH Chien, WT Lee, AC Huang, RM Wu

541-P A sibling case of early onset dystonia with MRI abnormality H Fujioka, H Shintaku, T Yokoi, T Yamano

136

136

552-P Growth development of children with classical PKU during their ¢rst year of life C Gibson, M Halldin Stenlid, C Eklund

140

554-P Treatment with ketogenic diet in the pyruvate dehydrogenase de¢ciency L Oliveros, MT Garcia, JM Moreno, E Martin, O Garcia, R Simo¨n, P Briones

140

555-P Malnutrition as an entry point for application of inborn errors of metabolism work-up into health services in the developing countries D Rusli Sjarif

140

556-O Cessation of protein substitute in tyrosinaemia type III A Daly, A Macdonald, V Hopkins, A Chakrapani, C Hendriksz, S Vijay

140

J Inherit Metab Dis (2008) 31 (Suppl 1)

xix

557-P The use of a new essential amino mix in gyrate atrophy A MacDonald, A Daly, V Hopkins, MA Preece, C Hendriksz, S Vijay, A Chakrapani

141

558-P Dietary management of PKU: the experience of CGM MF Almeida, JC Rocha, C Carmona, MR Lima

141

559-P The use of ready to drink liquid protein substitute in nonPKU amino acid disorders A Daly, A Macdonald, V Hopkins, A Chakrapani, C Hendriksz, S Vijay 141 560-O Nutritional management of PKU in Saudi Arabia SA Aljammaz, PT Clayton, TM Hesketh

141

561-P Long term compliance with a novel vitamin and mineral supplement in older people with PKU A MacDonald, P Lee, P Davies, A Daly, V Hopkins, M Lilburn, C Hendriksz, MA Preece, A Chakrapani 142 562-P Ready to use protein substitute for children with PKU A MacDonald, C Ferguson, A Daly, V Hopkins, SK Hall, G Rylance, C Hendriksz, A Chakrapani 563-P A new novel protein substitute for young children with GA1 A MacDonald, A Daly, V Hopkins, C Hendriksz, S Vijay, A Chakrapani 564-P Glucose continuous monitoring in hepatic glycogen storage diseases ^ what dietectic bene¢ts Z Patr|¨ cio, C Figueiredo

142

578-P Treatment of fatty acid oxidation disorders, glycogen storage disease type 1A and hypertriglyceridemia with new medium chain triglycerides sources CE Hanou, LJ Wilkinson, LE Bernstein 146 579-P Assessment of the e¡ectiveness of metabolic university: an entry level program for registered dietitians, nurses and genetic counselors LE Bernstein, C Freehauf, JA Thomas, JM Gessner 146 580-P Propionate enhances resuscitation with D-3hydroxybutyrate in an infant rat model of hypoketotic hypoglycemic encephalopathy PW Schutz, PK Wong, S Innis, E Struys, S Sto«ckler

146

17. General Topics

142

581-P Inherited metabolic disorders, experience from a multidisciplinary team B Oliveira, A Oliveira, J Farinha, A Ferreira, A Guerra

146

142

582-P The impact of inherited metabolic diseases on quality of life: a pilot study L Tumer, FT Eminoglu, AS Soysal, I Okur, A Hasanoglu

147

583-P Metabolomics: a new approach to identi¢cation of metabolic processes in mental retardation HM Engbers, G Visser, M Hendriks, J Gerrits, R Berger

147

584-A Investigation of plasma free carnitine de¢ciency in children with suspicion of inborn errors of metabolism GM Allegri, WMS Cruz, FB Scalco, MLC de Oliveira

147

585-P Approach to IEM in Indian NICU AB Jalan, S Tambde, ND Telawane, V Vedak, R Masand, A Muehl, O Bodamer

147

565-P Importance of information spread on inherited metabolic diseases for dieticians RB Oliveira, T Vertemati, S Araujo, CAF Satiro, C Souza, E Leite, S Raskin, H Pimentel, R Giugliani, E Valadares, L Santana, AM Martins, BJ Frangipani, C Micheletti 143 566-O Three or four protein substitutes per day in pre-conception and maternal phenylketonuria (PKU)? CM Maritz, C McWhinnie, M Lilburn, Cole A, PJ Lee 143 567-O Assessment of body composition of GSD type I patients A Faria, A Mota, S Silva, P Garcia, T Mota-Castelo, L Diogo 143 569-P Dietary intervention in adults with late diagnosed phenylketonuria ^ west of Scotland experience S Adam, B Cochrane, P Galloway

143

570-P The ketogenic diet; more than just fat?! ThAM van den Hurk, EJTM van der Louw

144

571-O Dietary therapy in isovaleric acidaemia EJ Footitt, M Dixon, MA Cleary

144

572-O Improvement in cardiomyopathy in a case of malonyl-CoA decarboxylase de¢ciency on LCT-restricted/MCT supplemented diet EJ Footitt, J Sta¡ord, M Dixon, MA Cleary 144 573-P Responsiveness of Kuvan in PKU patients during the expanded access program and following FDA approval LE Bernstein, S Myers, JA Thomas, C Freehauf, C Hanou

144

574-P The importance of prealbumin concentration in phenylketonuric patients JC Rocha, MF Almeida, C Carmona, ML Cardoso, N Borges, I Soares, G Salcedo, MR Lima, I Azevedo, FJ van Spronsen

145

575-P Service improvement with PKU clinics A MacDonald, S Carver, A Daly, V Hopkins, C Hendriksz, A Chakrapani 145 576-O Practicalities of emergency regimens in IMD A MacDonald, A Daly, V Hopkins, C Hendriksz, S Vijay, A Chakrapani

577-P Results of implementing a new method to vary foods within the phenylalanine-restricted diet of adult PKU patients C Timmer, CF Jonkers, E van der Ploeg, FJ van Spronsen, HW de Valk, NM de Roos 145

145

586-P Managing biobanks with an e¤cient model in biomedical genetics research C Auray-Blais, J Patenaude 148 587-P A pro¢le of inborn errors of metabolism in Rio de Janeiro, Brazil MLC de Oliveira, FB Scalco, RE Simoni, A Bernstein, WMS Cruz, GM Allegri, VF Leal, HJC Bezerra Netto, CPH de Oliveira 148 588-P Survey of IEM diagnosed by the Brazilian information service for inborn errors of metabolim (SIEM) CFM Souza, S Herber, L Giugliani, B Costa, C Netto, MT Sanseverino, L Refosco, C Refaelli, R Giugliani

148

589-P Metabolic investigations in a structured multidisciplinary diagnostic evaluation of developmental delay KT Verbruggen, DJ Reijngoud, RJ Lunsing, OF Brouwer, FJ van Spronsen 148 590-P Inborn errors of metabolism as a cause of liver disease and their genetic structure NB Gusina, ES Budzeika, TV Dzemidovitch, SV Dubovik, TS Zimovina, AV Zinovik, ON Gritsenko 149 591-A Preliminary data of a follow-up campaign in patients with an inherited metabolic disease at the `Instituto Canguru' RB Oliveira, T Vertemati, CAF Satiro, S Araujo, BJ Frangipani, L Santana, C Souza, E Leite, E Valadares, H Pimentel, R Giugliani, S Raskin, AM Martins, C Micheletti 149

xx

J Inherit Metab Dis (2008) 31 (Suppl 1)

592-P Data analysis related to the work carried out by a Brazilian NGO with health professionals and patients with inherited metabolic diseases C Micheletti, T Vertemati, RB Oliveira, S Araujo, E Leite, BJ Frangipani, S Raskin, H Pimentel, CAF Satiro, L Santana, R Giugliani, C Souza, AM Martins 149 593-P Outcome of cardiomyopathy due to inborn errors of metabolism ^ a cohort of 42 patients YS Choy, YS Choy, I Haifa, YS Choy, I Haifa, G Kanthavelu, Y Zabedah

149

594-A Proposal of decentralization of health care for families with inborn errors of metabolism (IEM) in Minas Gerais, Brazil ER Valadares, LR Oliveira, RHC Amorim, TMM Pinheiro, VL Rocha, RR Arantes, HH Santos, CP Souza, ALC Trindade, RR Queiroz, MX Pizarro, GC Lopes, ALB Godard 150

607-A Blood lead and serum selenium, zinc, copper and cobalt levels in primary school children in a risky area Sanli, H Bulbul, D Albayrak, P Kocak

153

608-P Inherited forms of pancreatic dysfunction YB Grechanina, LS Ozerova, OP Zdybskaya, OV Vasylieva, OY Grechanina

153

609-P Crisponi syndrome due to a novel mutation on the cytokine receptor-like factor 1 (CRLF1) gene I Okur, L Tumer, FT Eminoglu, L Crisponi, P Cinaz, I Yenicesu, A Hasanoglu

153

610-P Structure-function relationships of mutations in the porphobilinogen deaminase gene D Ulbrichova, P Martasek

153

611-P ADMA, an established cardiovascular risk factor in adults, in children with phenylketonuria, homocystinuria, or glycogenosis NK Kanzelmeyer, D Tsikas, AJ Fuchs, B Beckmann, S Illsinger, AM Das, T Lu«cke 154

595-P Pattern of metabolic disorders presenting to a metabolic clinic in a developing country over two years S Mohamed, A Khodary, A Elshwaf, S Belushi, A Hellani, S El-Kholy, E El-Melegy

150

596-P Portuguese metabolic neonatal screening performance and major causes of false positive results A Marca¬o, H Rocha, C Sousa, H Fonseca, L Vilarinho

612-P Cyclic pamidronate therapy in children with osteogenesis imperfecta S Salehpour, A Rajaii 154

150

597-P Threshold challenge in India ^ newborn screening for aminoacids, organic acids and fatty acid oxidation disorders M Kaur, S Birla

613-P Optimal sample type for diagnosis and treatment follow up of erythropoietic protoporphyria RBG Bonilla Guerrero, KMK Kloke, KR Raymond, ST Tortorelli 154

150

614-P Renal hypouricemia caused by deletion in human urate transporter 1 gene I Sebesta, K Ichida, M Hosoyamada, B Stiburkova, E Hruba

154

599-P Expansion of newborn screening in Ontario, Canada: lessons learned in the ¢rst 2 years SJ Kennedy, JL Milburn, MT Geraghty MT, L Fisher, C McRoberts, T Chiu, S Zelenietz, T Bouwman, G Mettler, P Chakraborty 151

615-P A novel cause for an abnormally low urinary creatinine concentration UFH Engelke, I Konijnenberg-Kramer, A Bilos, LA Kluijtmans, W Ruitenbeek, E Morava, RA Wevers

155

616-P Higher copper accumulation, a possible cause for hepatocelluar carcinoma in patients with Wilson disease H Kodama, K Shiga, F Kaga, C Fujisawa, YH Gu

155

600-P Hospital based expanded newborn metabolic screening in a developing country: challenges and outcome S Mohamed, M Hamasha, A Shanaa, R Ritmiller, H Khodary

617-P Developmental changes of oxalate excretion in preterm neonates on enteral nutrition S Illsinger, T Lu«cke, B Vaske, KH Schmidt, B Bohnhorst, AM Das

155

598-P The role of the metabolic laboratory in an age of tandem mass spectrometry newborn screening JM Fletcher, E Ranieri, M-U Trinh, J Blake, G Owens, R Velardo R, JR Harrison 151

601-P Newborn mass urine screening experience for amino acids and organic acids C Auray-Blais, JTR Clarke, R Drouin 602-P Selective screening with urine paper samples by tandem mass spectrometry: experience of 5 years MM Rebollido, DE Castin¬eiras, JA Cocho, C Rizzo, MD Bo¨veda, ML Couce, JM Fraga

151

151

152

603-P Cooperation between Hamburg (Germany) and Ecuador for neonatal screening and metabolic diagnosis ^ four years of experience Z Lukacs, R Santer, A Kohlschu«tter, P Nieves Cobos, F Moncayo 152 604-P Newborn screening by tandem mass spectroscopy ^ experiences of two newborn screening centers in Taiwan CH Kao, YH Lu, HC Lo, MY Lo, HJ Gao, JH Hsu, KW Chong, KH Cheng, TT Liu, CC Chien, DM Niu, CC Chiang

619-P Fatal manifestation of Wilson disease in a child after vaccination against hepatitis B D Behu¨lova¨, D Holeova¨, A Vasilenkova¨, Z Laluhova¨-Strieencova¨, L Pevalova¨, J Tuharsky¨, J Ponec 156 620-P APOE4 in Rett syndrome patients: a potential modulation factor? D Zahorakova, M Jachymova, A Puchmajerova, A Baxova, P Martasek 156 621-P Urinary coproporphyrin isomers in an HBsAg-positive patient with DubinJohnson syndrome I Kurt, A Hasimi, O Ozturk, HI Ayd|n, MK Erbil

152

18. Miscellaneous 606-P Antisense RNA therapy in inherited metabolic diseases B Pe¨rez, A Rinco¨n, C Aguado, A Vega, C Pe¨rez-Cerda, B Merinero, L Rodr|¨ guez-Pascau, MJ Coll, L Vilageliu, D Grinberg, LR Desviat, M Ugarte

618-P Clinical, biochemical and genetic ¢ndings in three patients with a b-ureidopropionase de¢ciency ABP van Kuilenburg, J Meijer, K Saito, K Eto, R Meinsma, NGGM Abeling, L Zoetekouw, B Assmann 155

152

156

622-P Mutation screening of ATP7A, ATP7B and ATOX1 genes in patients with Menkes and Wilson diseases in Czech Republic L Kralik, L Pospisilova, E Flachsova, R Bruha, Z Marecek, P Fruhauf, A Puchmajerova, J Zeman, P Martasek 156 623-P Copper and zinc status in the breast milk of mothers with Wilson disease F Kaga, H Kodama, K Siga, C Fujisawa, Y Yanakawa, K Kobayashi, H Koyama

157

J Inherit Metab Dis (2008) 31 (Suppl 1) 624-P HFE genotyping in children with Wilson's disease and iron overload SI Polyakova

xxi

157

625-P Application of the genotyping microarray in the molecular diagnostics of Wilson disease L Gojova, E Jansova, S Pouchla, L Fajkusova 157 626-P Wilson's disease: an experience over a mean period of 15 years D Prochazkova, V Mejzlik, S Pouchla, J Michalek

157

627-P The contribution of GC-MS urinary steroid metabolome in the study of a consanguineous family with 6 members with apparent mineralocorticoid excess (AME) C Knopf, D Magen, A Suheir, K Skorecki 158 628-P Prefrontal cortex oligodendroglia cells from ADSL de¢cient patient produce SAICAR and SAMP L Zidkova, J Krijt, J Sladkova, A Hlobilkova, J Zeman, M Magner, M Elleder, T Adam

158

629-P Screening and diagnosis of purine and pyrimidine metabolism disorders among Malaysian children by rapid resolution liquid chromatography BC Chen, LH Ngu, MY Zabedah, MK Thong, WT Keng 158 630-P Purine and pyrimidine newborn screening by tandem mass spectrometry on urine ¢lter paper samples MM Rebollido, DE Castin¬eiras, JA Cocho, G La Marca, MD Bo¨veda, C Cristobal, ML Couce, JM Fraga

158

631-P Peculiar ¢ndings in intermediate type of adenylosuccinate lyase de¢ciency D Ballhausen, G Lazzarino, B Tavazzi, M Jequier, E Roulet-Perez, S Jacquemont, C Roux, L Bonafe¨

159

Author Index

161

SSIEM

Society for the Study of Inborn Errors of Metabolism The SSIEM was founded in 1963 in the North of England and has developed into the largest international organisation concerned with all aspects of inherited metabolic diseases. The aim of the Society is to promote the exchange of ideas between professional workers in different disciplines who are interested in this group of disorders. This aim is pursued in scientific meetings and publications. The Society holds an annual symposium concentrating on different topics each year with facilities for poster presentations. There is always a clinical aspect as well as a laboratory component. The meeting is organized so that there is ample time for informal discussion; this feature has allowed the formation of a network of contacts throughout the world. The international and multidisciplinary approach is also reflected in the Journal of Inherited Metabolic Disease. If you are interested in joining the SSIEM then contact the Treasurer: Dr. Maureen Cleary, Metabolic Office, 7th floor, Southwood Building, Great Ormond Street NHS Trust, Great Ormond Street, London WC1N 3JH, UK. The subscription includes the 6 issues of the Journal of Inherited Metabolic Disease. The SSIEM web site is on .

J Inherit Metab Dis (2008) 31 (Suppl 1)

001-P

IMPLEMENTATION OF A NEW APPROACH FOR THE QUANTITATIVE ANALYSIS OF AMINO ACIDS IN HUMAN PHYSIOLOGICAL FLUIDS USING UPLC Bekri S1, Hecketsweiler B1, Paccou A2, Hong P3, Wheat TE3 1 Lab Biochimie Meèdicale, CHU de Rouen, Rouen, France, 2Waters EHQ, Guyancourt, France, 3Waters Corporation, Milford, USA A turnkey solution for the quantitative analysis of physiological amino acid analysis has been developed. Amino acid analysis is required for the diagnosis and the follow up of aminoacidopathies and for the identi¢cation of secondary variations of amino acids. The amino acids and their derivatives are commonly analyzed using ion-exchange chromatography. A new method has been developed, combining amino acid derivatization chemistry with the very high resolution and sensitivity allowed by UltraPerformance LC technology (UPLC). Such system was implemented in January 2008 in the `Laboratoire de Biochimie Meèdicale' CHU de Rouen ^ France. This analytical solution ensures accurate and precise qualitative and quantitative analysis of amino acids and their derivatives. The patented derivatization procedure `AcqTag' is very easy to handle and derivatized samples are stable for several days. Importantly, this preparation requires a small amount of sample (20 ml for plasma and 10 ml for urine and CSF) which is a great advantage in presence of pediatric samples. A 150 mm `QC tested Acquity'. C18 column is used. Amino acids are detected by UV (260 nm) which permits to avoid the use of ninhydrine, a toxic reagent commonly used to detect amino acids. The analysis is achieved in a signi¢cantly shorter run time compared to current ion exchange methods. Thus, a complete pro¢le of 42 amino acids and derivatives is obtained in a 45 min cycle. The UPLC analysis of amino acids in biological samples was validated and the obtained data were compared and correlated to those of the Biotronik LC 3000 analyzer.

002-P

1

003-P

ION EXCHANGE CHROMATOGRAPHY, AN `OLD' TECHNIQUE FOR PROMPT NKH DETECTION: 3 CASE REPORTS Ver|èssimo C1, Simo¬es M1, Estevinho A2, Garcia P3, Diogo L3, Oliveira CR2, Grazina M2 1 Centro Neurocieªncias Biologia Celular, Coimbra, Portugal, 2Faculdade de Medicina, Univ Coimbra, Coimbra, Portugal, 3Hospital Pediaètrico de Coimbra, Coimbra, Portugal Background/Objectives: Glycine encephalopathy or nonketotic hyperglycinemia (NKH; OMIM 605899) is a severe autossomal recessive disease, due to glycine cleavage system (GCS) de¢ciency. Ion exchange chromatography (IEC) is an `old' technique for amino acid analysis, which is far from being obsolete. In order to highlight the importance of this method, we present three clinical cases in which amino acid quanti¢cation disclosed the diagnosis. Methods: From 2004 to 2007, we have analysed 1155 samples as part of our screening programme by IEC and classical neonatal NKH was identi¢ed in 3 patients. Case I presented with multifocal, severe seizures and epileptic encephalopathy at 2.5 months of age. Case II was admitted in a neonatal intensive care unit (UCIN) with progressive hypotonia and apnoea followed by epilepsy and severe development delay. Case III had hypotonia and feeding di¤culties since birth; at day 6 of life she was admitted in UCIN with severe hypernatremic dehydration and coma, ensued by epileptic encephalopathy. Results: Diagnosis was done based on hyperglycinemia and CSF/ plasma glycine ratios of 0.07, 0.23 and 0.16 (normal: 0.01^0.02), at 7 months, 7 days and 18 days of age, respectively for cases I, II and III. Conclusions: A glycine ratio above 0.08 is diagnostic for NKH; values of 0.04^0.1 are found in mildly a¡ected patients. In fact, patients II and III presented higher ratios correlated with poorer prognosis. So far, a missence mutation (homozygosity) in AMT gene was found (Prof. MO Rolland) in patient I and con¢rmed diagnosis.

004-P

SENSITIVE AND RAPID ANALYSIS OF AMINO ACID CONCENTRATIONS IN CSF BY STABLE ISOTOPE DILUTION LC-MSMS Verhoeven-Duif NM1, Van der Ham M1, Prinsen BHCMT1, Berger R2, Klomp L2, De Sain-Van der Velden MGM2 1 Dept Metabolism and Endocrine Diseases, UMCU, Utrecht, Netherlands; 2Netherlands Metabolomics Center, Utrecht, Netherlands

THE SIGNIFICANCE OF AMINO ACIDS ANALYZES AS AN INDICATOR OF PROTEIN-CALORIC INTAKE IN PATIENTS ON LOW PROTEIN DIET Honzik T1, Magner M1, Sulek S1, Jesina P1, Hruba E2, Chrastina P2, Zeman J1 1 Dept of Pediatrics, Charles University, Prague, Czech Republic, 2Inst Inherit Met Dis, Charles University, Prague, Czech Republic

Background: For the diagnosis of inborn errors a¡ecting brain amino acid metabolism, quanti¢cation of amino acids in CSF is invaluable. A prerequisite for accurate quanti¢cation is a method with high sensitivity, as both increased and decreased concentrations should be detectable. Methods: We developed a rapid and sensitive LC-MSMS method for quanti¢cation of amino acids in CSF. After addition of fourteen stable isotope labelled internal standards to 50 ml of CSF, samples were deproteinized and analyzed (C18 column equipped with a pre column). Detection was achieved by positive multiple reaction monitoring (MRM). For glycine, a separate sample preparation procedure including butylation was necessary. Analysis of glycine was performed without prior chromatographic separation. Calibration curves were included in every run. Results: Limits of quanti¢cation were below the lower limits of the reference ranges for all amino acids. Between run variation was 520% for all amino acids, except taurine and citrulline, which were therefore excluded from the diagnostic protocol. The applicability of the method was demonstrated by the analysis of samples from patients a¡ected with known disorders in brain amino acid metabolism, such as non-ketotic hyperglycinemia and serine biosynthesis defects. Conclusion: This method is very sensitive and enables rapid (51 h) quanti¢cation of all amino acids of diagnostic interest in less than 100 ml of CSF. In addition, due to the use of MRM, a high speci¢city is achieved. Compared to the conventional ninhydrin-based methods, the LC-MSMS method has many advantages which make implementation of this method in a diagnostic setting useful.

Aims: To evaluate the glycine/BCAA (branched chain amino acids) and alanine/BCAA ratios (Oberholzer, 1990), and methionine level as an indicator of protein-caloric intake and diet compliance in patients with tyrosinemia type I (TYR I) and phenylketonuria (PKU). Study design: 187 aminograms from 8 TYR I patients (aged 1^16 years), 218 aminograms from 16 PKU patients (under 10 years) and 12 aminograms obtained from healthy children were analyzed. The optimal protein intake was characterized by plasma level of tyrosine 250^499 mmol/L in TYR I patients, phenylalanine 200^400 mmol/L in PKU patients and methionine 18^40 mmol/L in both group of patients. Excessive strict protein diet was characterized by low level of methionine, tyrosine or phenylalanine. Conversely elevated tyrosine or phenylalanine level was referred to poor compliance with high protein intake. Results: Signi¢cantly higher alanine/BCAA (1.18+0.4) and glycine/BCAA (0.9+0.3) ratios were observed in patients with low tyrosine, phenylalanine and methionine level in comparison with patients on well-balanced diet and controls (0.94+0.16; 0.67+0.15). Furthermore, higher ratios were found in patients with optimal tyrosine or phenylalanine, but with low methionine level. Level of methionine correlates positively with tyrosine level in PKU patients and may serve as a marker of amino acids formula dosage. No di¡erence was found in ratios when patients with high tyrosine and phenylalanine level were compared with compliant patients. Conclusion: Glycine/BCAA and alanine/BCAA ratiogram, along with measurement of methionine, tyrosine a phenylalanine concentration, is an additional e¡ective tool for assessment of diet compliance.

Supported by MSM 0021620806 and VZ 64165

2

005-P

LCPUFA STATUS IN PATIENTS WITH DIFFERENT INBORN ERRORS OF METABOLISM V|laseca MA, Ayllon O, Moreno J, Artuch R, Lambruschini N, Goèmez L, Gutierrez A, Campistol J Hospital St Joan de Deèu, Barcelona, Spain Background: Patients with inborn errors of metabolism (IEM) treated with protein-restricted diets are a risk population for reduced LCPUFA status. Methods: Patients: cross-sectional study of 204 patients with IEM (PKU/HPA (n = 102), urea cycle (n = 27), galactosemia (n = 10), organic acidemias (n = 10), homocystinuria (n = 11), mitochondrial diseases (n = 11), b-oxidation defects (n = 7), lysosomal diseases (n = 14) and a miscellaneous group (n = 12). Essential fatty acids (EFA) and LCPUFAs were analyzed by gas-chromatography (expressed as % of total fatty acids). Results: EFAs and arachidonic acid values were not signi¢cantly di¡erent compared with 43 control values of similar ages. Docosahexaenoic acid (DHA) values in plasma and erythrocytes were signi¢cantly lower (ANOVA with Bonferroni correction; p50.0001) in PKU, urea cycle and organic acidemia patients when compared with controls and other IEM patients without protein-restricted diet. W6/W3 ratio was signi¢cantly higher only in PKU patients compared with other IEM patients and control values. Considering patients according to natural protein intake, DHA was signi¢cantly lower in patients with good dietary compliance compared with those with poor dietary control or with a free diet (ANOVA with Bonferroni correction; p50.0001). Conclusions: Independently of the IEM, patients under proteinrestricted diet with good compliance present a low DHA status and a high W6/W3 ratio. Our data reinforce the previous observations that DHA supplementation may be important for a good neurological development and physical growth in IEM patients under proteinrestricted diet.

006 -P

METHIONINE INDUCES OXIDATIVE STRESS IN LIVER AND BRAIN OF YOUNG RATS Stefanello FM, Pederzolli CD, Kurek AG, Mattos CB, Kolling J, Dutra-F|lho CS, Wannmacher CMD, Wajner M, Wyse ATS Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil Background: Very little is known about the mechanisms underlying the liver and brain damage present in metabolic diseases that accumulate methionine. Objective and Methods: The objective of the present study was to investigate the e¡ect of chronic administration of methionine on some parameters of oxidative stress, namely chemiluminescence and thiobarbituric acid-reactive substances (TBARS) (lipid peroxidation), total radical-antioxidant potential (TRAP), total thiol content, as well as on the activity of the antioxidant enzyme catalase in liver and hippocampus of hypermethioninemic rats. Animals were treated daily with two subcutaneous injections of methionine (1.34^2.68 mmol/g of body weight) or saline from the 6th to the 28th day of life and were sacri¢ced 12 h later. Results: Methionine signi¢cantly increased chemiluminescence, decreased TRAP and catalase activity in rat liver. Besides, chronic hypermethioninemia enhanced TBARS in hippocampus of rats. The other parameters were not altered in both structures. Conclusions: These ¢ndings suggest that the induction of oxidative stress caused by methionine may be, at least in part, one of the mechanisms related to the brain and liver damage observed in hypermethioninemia. F|nancial support: Research grants from CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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CYSTINURIA PREVALANCE IN AN ANATOLIAN CITY AND EFFICIENCY OF SODIUM NITROPRUSSIDE TEST IN SCREENING PROGRAMMES Tunc°1, Hizel B1, Tanzer F2, Albayrak1 1 K|r|kkale Univ, Dept Pediatrics, K|r|kkale, Turkey, 2Cumhuriyet Univ, Dept Pediatrics, Sivas, Turkey Background: Cystinuria is a common inherited disorder of defective renal reabsorption of cystine, ornithine, lysine and arginine. It may cause nephrolithiasis, recurrent urinary track infections, obstruction and renal insu¤ciency. If recognised early, it is possible to prevent urinary tract calculi and protect the kidney function. Objectives: To determine the reliabilty and e¡ectiveness of the cyanidenitroprusside test (Brand's test) test and to demonstrate cystinuria prevelance in K|r|kkale, a total of 8000 children were screened for cystinuria. Methods: All the subjects were questioned for self and family history of renal stone and urinary tract infection. At ¢rst, sodium nitroprussid test was performed to all collected samples, if the test was positive cystin concentration was measured by Cyanid nitroprusside test and the diagnosis was con¢rmed by paper chromotografy. Results: Cystinuria was determined in six subjects. Among all students, 43% have had renal stone and 2.1% had a family history of renal stone, 412/8000 students have experienced urinary tract infection (UTI) at least one time in life period. There was a signi¢cant correlation between having a renal stone and UTI (p50.05). Conclusions: We conclude that the incidence of subjects at risk for cystine stones in the K|r|kkale Community is 1/1333. As autosomal recessive disorders are frequently seen in countries with high rates of consanguineous marriages, physicians should be careful about cystinuria and consequent complications. Cheap, easy to perform and reliable sodium nitroprusside test, could be performed for public screening to avoide the future complications and high treatment costs of cystinuria.

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SULFITE OXIDASE AND MOLYBDENUM COFACTOR DEFICIENCY: EEG, METABOLIC AND NEURORADIOLOGIC STUDY Martinelli D1, Mariotti P1, Stefanini MC1, Battaglia D1, Antuzzi D2, Veredice C1, Sacco A1, Guzzetta F1, Mercuri E1 1 Ped Neurol and Psich Unit, Pol Gemelli, Rome, Italy, 2Ped Unit, Pol Gemelli, Rome, Italy Background: Isolate sul¢te oxidase de¢ciency (ISOD) is a rare inborn error of the metabolism of sulfated amino acids. Since the neonatal period a¡ected individuals commonly show intractable seizures, dysmorphic features, and severe neurological abnormalities. Most cases of this disorder are associated with de¢ciency of the molybdenum-containing pterin cofactor (MoCo de¢ciency). Clinical features of SOD and MoCo de¢ciency are well described, but EEG and neuroradiologic ¢ndings followup is less known. Moreover, pathophysiology of these disorders is still controversial. O b j e c t i v e s : To d e s c r i b e a n d c o n f r o n t c l i n i c a l , m e t a b o l i c , electroencephalographic and MRI ¢ndings of two children, one with ISOD and one with MoCo de¢ciency. Methods: Both patients underwent a periodic assessment including neurological and ocular examination, metabolic tests, serial long-term video-EEG and MRI. Results Both patients su¡ered from neonatal generalized seizures. Plasma total homocysteine was not detectable. Uric acid was undetectable in MoCo de¢ciency. Urinary sul¢te, thiosulfate, and S-sulfocysteine levels were elevated. Seizures, developmental delay, spastic quadriplegia, and abnormal movements were prominent features in both cases. Ectopia lentis was detected only in MoCo de¢ciency infant, whereas dysmorphic features were present only in ISOD infant. EEG follow-up showed progressive slowing of background activity with intermixed fast rhythms and spike-wave discharges. Progressive cerebral atrophy and cystic encephalomalacia, mimicking hypoxic ischemic encephalopathy, characterized neuroimaging ¢ndings. Conclusions: Our cases suggest that a follow-up as long as possible of EEG and neuroimaging is considerable also for a new insight of the mechanisms of these diseases.

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SERINE SYNTHESIS DEFECTS: A TREATABLE CAUSE OF MICROCEPHALY Valayannopoulos V1, Raqbi F2, Rabier D3, Abadie V4, Desguerre I5, de Lonlay P1 1 Metab Unit, Necker-Enf Malades Hosp, Paris, France, 2Pediatrics Dept, Creil General Hosp, Creil, France, 3Bioch Lab, Necker-Enf Malades Hosp, Paris, France, 4Pediatrics Dept, Necker-Enf Malades Hosp, Paris, France, 5Ped Neurology, Necker-Enf Malades Hosp, Paris, France

NEONATAL NON-KETOTIC HYPERGLYCINAEMIA: A CASE WITH A CHALLENGING DIAGNOSIS Martins E1, Carrilho I2, Rocha P3, Santos M2, Cardoso ML4, Chora¬o R2 1 Metab Unit ^ Child Hosp Maria Pia, Porto, Portugal, 2Neurol Div, Child Hosp Maria Pia, Porto, Portugal, 3Intensive Care Unit, Child Hosp Maria Pia, Porto, Portugal, 4Biology Unit, CGM Jacinto Magalha¬es, Porto, Portugal

Background: Serine biosynthesis defects are due to 3 di¡erent enzyme defects namely phosphoserine phosphatase, phosphoglycerate dehydrogenase and phosphoserine aminotranferase and are caracterized by microcephaly, psychomotor retardation and epilepsy. These defects are treatable by oral serine with various outcomes depending on the defect and on the precocity of treatment onset. Case Report: We present a 8 months old boy born from consanguineous parents of turkish origin presenting at birth with severe microcephaly (head circumference (HC) at birth: 28 cm) who rapidly developed failure to thrive, swallowing di¤culties, central hypotonia, pyramidal spasticity and dystonia. Brain MRI performed at age 2 months showed severely retarded myelination. Serine was extremely low in CSF, very low in plasma. Glycine was low in plasma but normal in CSF. The enzymatic and molecular diagnosis is pending. Results: We treated the child for so far 6 months by oral serine (400 mg/ kg/day then 600 g/kg/day) and folinic acid. He displayed less hypertonia and spasticity and social contact improved. His HC growth progressed by 5 cm. Feeding problems improved. No seizures or EEG changes have been observed. Serine levels normalized in plasma but in CSF remain in the normal-low range. Conclusions: This case report illustrates the importance of CSF chromatography in case of congenital or acquired microcephaly in order to identify a serine biosynthesis defect that may bene¢t from an early treatment by oral serine.

Background: Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive disorder characterized by progressive neurological symptoms in the neonatal period or early infancy (hipotonia, myoclonic seizures, lethargy and coma) and elevation of glycine in urine, plasma and CSF, with an increased CSF to plasma glycine ratio. Case report: Female, 1st child of non-consanguineous parents, born at 36 weeks of gestation with 8/9/10 Apgar index. She started convulsions and episodes apnea by the second day of life. The clinical picture progressed to multifocal myoclonic seizures refractory to antiepileptic drugs, hypotonia and lethargy. Considering investigation, plasma, urine and CSF amino acids were normal, with CSF/plasma glycine 0.029 (N 0.02), on a ¢rst sample (although CSF and blood samples were not simultaneous). Other metabolic investigations (organic acids, lactate, piruvate, respiratory chain, neurotransmitters), cariotype and brain MRI were normal. EEG showed a supression-burst (SB) pattern, with long periods of asymmetrical almost £at tracing that persisted after the neonatal period. Repeated investigation at 3 months show normal glycine plasma levels, CSF glycine of 25.7 mmol/L (N 4.8^8.47 mmol/ L) and CSF/blood glycine 0.11 (N 0.02). The diagnosis was con¢rmed by measurement of glycine cleavage enzyme in liver biopsy. Conclusions: The normal values of glycine in plasma, urine and CSF in the ¢rst sample were not suggestive of NKH. However, both the clinical ¢ndings and a persistent SB pattern on the EEG gave rise to a second investigation that showed an increasing of CSF glycine and CSF/plasma levels, which strongly suggested the diagnosis of NKH.

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DIAGNOSIS OF GLUTATHIONE SYNTHETASE DEFICIENCY BY NEWBORN SCREENING USING TANDEM MASS SPECTROMETRY Thimm E1, F|ngerhut R2, Risto¡ E3, Mayatepek E1, Spiekerko«tter U1 1 Dept General Pediatrics, Univ Child Hosp, Du«sseldorf, Germany, 2 Labor Becker, Olgemo«ller and Kollegen, Munich, Germany, 3Dept Pediatrics, Karolinska Institutet, Stockholm, Sweden Background/Objectives: Glutathione synthetase de¢ciency is an autosomal recessive metabolic defect in the gamma-glutamyl cycle. Main clinical symptoms of the disease are hemolytic anemia and metabolic acidosis. Detection of high excretion of 5-oxoproline in urine leads to the diagnosis that is con¢rmed enzymatically or by mutation analysis. Case report: We report the case of a full term infant born to nonconsanguineous Turkish parents. The newborn presented with icterus precox on the second day of life and chronic severe metabolic acidosis necessitating correction with bicarbonate. Extended newborn screening by tandem mass spectrometry (MS/MS) according to the German newborn screening guidelines was normal. However, selective screening in the dried blood spot obtained for screening at 44 h of life revealed a twentyfold elevation of pyroglutamine suggesting glutathione synthetase de¢ciency. Further analyses showed a massive excretion of 5-oxoproline in urine of 38 700 mmol/mol creatinine (normal: 542.5). Diagnosis was con¢rmed by mutational analysis. Conclusions: In newborn infants with severe metabolic acidosis and icterus precox due to hemolytic anemia, newborn screening by MS/MS including pyroglutamine may already on day 2 of life correctly identify glutathione synthetase de¢ciency. This is of importance as early diagnosis and early initiation of treatment are predictors of survival and long-term outcome. It therefore has to be discussed if glutathione synthetase de¢ciency should be included in extended newborn screening programmes.

THE NEONATAL CASE DIAGNOSED WITH NONKETOTIC HYPERGLYCINEMIA WITH A PRELIMINARY DIAGNOSIS OF THE CHLORALHYDRATE INTOXICATION Okur I, Eminoglu FT, Tumer L, Ezgu FS, Biberoglu G, Hasanoglu A Gazi Univ Hosp, Dept Ped Nutr Metab, Ankara, Turkey Unlike the classic neonatal form of the disorder, the clinical ¢ndings in atypical or mild nonketotic hyperglycinemia are phenotypically heterogeneous and nonspeci¢c. In the neonatal form of atypical NKH the presentation can be similar to classical NKH with hypotonia and apneic episodes that may require assisted ventilation, though seizures are less severe. Chloralhydrate overdosage also produces symptoms like coma, hypotension, respiratory depression and cardiac arrhythmias. We report a neonatal case, who had been hospitalized with a preliminary diagnosis of chloralhydrate intoxication, which turned out to have atypical nonketotic hyperglycinemia. Two month-old girl, who was born at term by normal vaginal delivery, admitted to our hospital due to the di¤culty in sucking, hypotonia and apneic episodes. In history, it was learnt that the spinal MRI due to sacral dimple had been performed in another hospital, for which has been administered cloralhydrate before this process. It was thought that the reason of all clinical ¢ndings could be explained with the cloralhydrate intoxication. The patient is clinical condition was improved and she was discharged from hospital. One month later, she had admitted due to hypotonia and seizures again and the CSF/plasma glycine ratio was determined to be high. The glycine cleavage enzyme system activity in the liver biopsy was very low. The patient, who is currently 10 months-old, does not seizures and has any mental-motor retardation. Inborn metabolic diseases should always be included in the di¡erential diagnosis for the patients present with a clinical picture consistent with intoxication.

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GLYCINE INDUCES LIPID OXIDATIVE DAMAGE AND REDUCES THE ANTIOXIDANT DEFENSES IN BRAIN CORTEX OF YOUNG RATS Leipnitz G1, Solano A1, Seminotti B1, Amaral AU1, Fernandes CG2, Knebel LA1, Wannmacher CMD1, Vargas CR3, Wajner M4 1 Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Universidade Luterana do Brasil, Canoas, Brazil, 3Hospital de Cl|ènicas de Porto Alegre-SGM, Porto Alegre ^ RS, Brazil, 4Hospital de Cl|ènicas de Porto Alegre, Porto Alegre, Brazil Background: Patients a¡ected by nonketotic hyperglycinemia (NKH) usually present severe neurological symptoms and su¡er from acute episodes of intractable seizures, whose pathogenesis is not fully established. Objectives: The present study evaluated the in vitro e¡ects of glycine (GLY) that accumulates in the brain of patients a¡ected by NKH, on important parameters of oxidative stress in cerebral cortex from 30-dayold rats. Methods: Thiobarbituric acid-reactive substances (TBA-RS), chemiluminescence, reduced glutathione (GSH), carbonyl formation and sulfhydryl oxidation were measured. Results: GLY signi¢cantly increased TBA-RS and chemiluminescence values, indicating that this metabolite provokes lipid peroxidation. Furthermore, the addition of the antioxidants melatonin, a-tocopherol and GSH fully prevented GLY-induced increase of lipid peroxidation, indicating that free radicals were involved in this e¡ect. GLY also decreased the concentrations of GSH, the major brain antioxidant, and this e¡ect was prevented by a-tocopherol and melatonin. On the other hand, carbonyl formation and sulfhydryl oxidation, parameters of protein oxidative damage, were not changed by GLY. Conclusions: It is postulated that oxidative stress may be involved in the pathophysiology of NKH. F|nancial support: Research grants from CNPq, PROPESQ/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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LYSINE INHIBITS CREATINE KINASE ACTIVITY AND INDUCES OXIDATIVE STRESS IN RAT BRAIN Seminotti B, Tonin AM, Leipnitz G, Schuck PF, da Costa Ferreira G, Amaral AU, Solano A, Grings M, Knebel LA, Wannmacher CMD, Wyse ATS, Wajner M Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil Background: Familiar hyperlysinemia (FH) is an inherited metabolic disease characterized by tissue accumulation of lysine (Lys). Although many patients are asymptomatic, a considerable number of a¡ected individuals present neurological dysfunction. Objectives: The present work investigated the in vitro e¡ects of Lys on parameters of energy metabolism and oxidative stress in cerebral cortex of young rats. Methods: The activities of creatine kinase and respiratory chain complexes and CO2 production from glucose, as well as lipid and protein oxidative damage and GSH concentrations were determined. Results: Lys inhibited total and cytosolic creatine kinase activity, but did not alter aerobic glycolysis, neither changed the activities of complexes I-V of the respiratory chain. Lys also induced lipid peroxidation, diminished the non-enzymatic antioxidant defenses and provoked protein oxidative damage in rat cerebral cortex. Furthermore, the addition of free radical scavengers fully prevented Lys-induced lipid oxidative damage and creatine kinase inhibition. Conclusions: These deleterious e¡ects may provoke higher vulnerability of the central nervous system to other insults such as systemic infections in symptomatic patients a¡ected by familiar hyperlysinemia. F|nancial support: Research grants from CNPq, PROPESQ/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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SARCOSINAEMIA: A CASE REPORT WITH EVOLUTIVE NEUROLOGIC SYMPTOMS Garnotel R1, Kuvbachieva-Bennarosh A2, Bakchine S2, Gillery P1 1 Pediatric Biochemical Unit, AMH, CHU, REIMS, France, 2Neurologic Unit, HMB, CHU, REIMS, France Sarcosinaemia is an autosomal recessive condition due to the de¢ciency of sarcosine dehydrogenase, a liver mitochondrial matrix £avoenzyme catalysing the conversion of sarcosine (N-methylglycine) to glycine. Generally, sarcosinaemia is considered as a benign condition. But, a great variety of symptoms has been also reported in sarcosinaemia such as mental retardation, growth failure, hepatomegaly, syndactyly, and cardiomyopathy. We present a case of sarcosinaemia with evolutive neurologic symptoms. The patient was the second child of nonconsanguineous marriage and born after a 34-week pregnancy with birthweight of 1200 g. He stayed in the neonatal intensive care unit during 2 months. At the age of 7 months, he presented hypotonia, mental and motor retardation. At 8 years, he developed neurological symptoms, including gait disorder, intentional tremor, as well as cognitive impairment with IQ of 57. At the age of 18 years, gait disorders and dystonia had increased with frequent falls. Magnetic resonance imaging (MRI) of the brain was normal. The analysis of plasma amino acids showed sarcosine to be highly increased to 325 mmol/L and with all other amino acids in normal concentrations. The sarcosine excretion amounted to 1214 mmol/mmol of creatinine. Sarcosine was present in CSF (7 mmol/L). Besides sarcosinaemia, 2 other diseases (folate de¢ciency and glutaric aciduria type II) could also lead to hypersarcosinaemia and had to be ruled out before the diagnosis of sarcosinaemia could be retained. The patient's parents and sister/ brother excreted normal concentrations of sarcosine. The severity of symptoms in patients such as this one could justify pharmacological trials.

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N-ACETYLAMINO ACIDURIA: A BENIGN BIOCHEMICAL FINDING! Calvin J1, Wevers RA2, Engelke U2, Lissens W3, Perrier J4, Giardina T4, Smet JE5, Hogg S1, Abulhoul L6, Puthi V7, Van Coster RN5 1 Biochemical Genetics, Addenbrooke's Hosp, Cambridge, UK, 2 Pediatrics and Neurology, Univ Med Centre, Nijmegen, Netherlands, 3 Center for Medical Genetics, Brussels, Belgium, 4Sciences Moleèculaires, Univ Paul Ceèzanne, Marseille, France, 5Peds, Div Neurol and Metab, Univ Hospital, Ghent, Belgium, 6Paediatrics, Leicester Hosp, Leicester, UK, 7Paediatrics, Peterborough Hosp, Peterborough, UK We present two members of a consanguineous Pakistani family with biochemical evidence of aminoacylase 1 de¢ciency (ACY1D, MIM609924) yet disparate phenotypes. The proband (at 3 years) is profoundly hypotonic with severe developmental delay. Father, brother (8 years) and sister (9 years) are healthy with normal development. His mother has signi¢cant learning di¤culties. The proband and his healthy brother excrete excess Nacetylamino acids in the urine, characteristic of ACY1D. No excess of Nacetylamino acids was detected in plasma or CSF of the proband and ACY1 enzyme activity [EBV lymphoblasts (EBVL), ¢broblasts, skeletal muscle, rectal muscle] gave inconsistent results, re£ecting low expression in these tissues. Western blotting revealed no detectable protein in samples from the two brothers and reduced protein in the sister, mother and father consistent with carrier status. Genomic sequencing of the ACY1 gene (all 14 exons/ exon-intron boundaries) failed to identify a mutation. ACY1 mRNA of normal length and amount was present in EBVL from all family members and in all tissues sampled from the proband. Sequencing of cDNA from a control and the proband showed 100% identity. To date a genetic cause of ACY1D has not been demonstrated in this family. Reports of ACY1 individuals existing as heterocomplex are lacking, although interaction with sphingosine kinase 1 has been described, raising the possibility of defective complex formation. This family adds to the speculation regarding the phenotype of ACY1D. More cases are required to ascertain whether this disorder has direct consequences, confers a risk of neurological sequelae or is simply a benign biochemical variant.

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Tyrosinaemia type I (TTI) is a severe metabolic disease characterised by de¢ciency of fumaryl acetoacetate hydrolase. Untreated TTI leads to liver failure and hepatocellular carcinoma. Early treatment with nitisinone (NTBC) improves outcome. TTI is a candidate for newborn screening but tyrosine has proved unsatisfactory as a screening test. Succinylacetone is a suitable marker and several methods have been published using various derivatives. We have adapted a method described by Johnson et al. (1) for use as a ¢rst line screen. 10 mmol/L Girard-T reagent with 10 mmol/L 13C-succinylacetone IS was added to 3 mm DBS, previously extracted with methanol, and incubated for 20 h at room temperature. Dried extracts were reconstituted in mobile phase and analysed using a Waters Quattro Micro. MRM data was acquired monitoring transitions 2724185 and 2764189 for 12C4 and 13C4 succinylacetone respectively. In-house DBS calibrators (2^50 mmol/L) were used. The method was linear to 100 mmol/L. Interbatch imprecision was 11.5% at 2 mmol/L and 7.8% at 20 mmol/L. 1070 anonymised newborn screening DBS were analysed as described. 1068 samples had concentrations 53 mmol/L (median 0. 35 mmol/L, range 0^ 2.38 mmol/L). Two samples had concentrations of 9.52 and 10. 23 mmol/ L. A DBS collected at 2 days of age from a TTI patient and stored for 2 years at room temperature had a concentration of 5.6 mmol/L. A fresh DBS from a newly diagnosed 2 year old had a concentration of 38 mmol/L. We have developed a method for succinylacetone that could be used for newborn screening of TTI. Further validation work is required. (1) Rapid Communication Mass Spectrometry 2007; 21(1):59^63

Background: Tyrosinaemia type 1 (fumarylacetoacetate hydrolase de¢ciency), causes multisystemic disease characterized by liver and renal tubular dysfunction and neurological crises. We aimed to quantify the incidence of EEG abnormalities and epilepsy in these children. Methods: A retrospective review of case and neurophysiology records of 31 children with tyrosinaemia type 1. EEG recording was for symptoms suggestive of seizures or as part of liver transplant assessment. Results: EEG data was available for 10 children. Three were performed for symptoms suggestive of seizures and 7 were done as part of transplant assessment. All symptomatic and one asymptomatic patient had an abnormal EEG. Patient 1 was asymptomatic and had brief left frontal spikes. Patient 2 had a mixture of complex-partial, tonic-clonic and absence seizures with di¡use 3Hz spike/wave discharges with a frontal lead and was treated with carbamazepine then lamotrigine. Patient 3 presented with episodic eye deviation with frontocentral spikes/sharp waves. Patient 4, who had had previous meningitis, presented with myoclonic attacks associated with multifocal epileptiform discharges and was treated with phenobarbitone. Conclusions: EEG abnormalities were present in 40% of children who had neurophysiological testing and in 13% of all children with tyrosinaemia. Seizures occurred in 10% of children. Although a potential alternative cause for epilepsy was present in one child, these ¢gures are greater than background rates and raise the possibility of under-recognition of subtle seizures in these children. EEG changes varied but frontal abnormalities predominated. We would recommend EEG investigation in any child with tyrosinaemia who presents with possible seizure-like episodes.

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In tyrosinemia type I (HTI), a late start of NTBC, a slow decrease or not normalizing AFP are risk indicators of later hepatocellular carcinoma (HCC) development. HTI patients, however, always showed a clear rise when HCC is present. We present a girl in whom there was a clearly increased risk of HCC development, but in whom timing of liver transplantation was postponed by unclearness of the meaning of abnormalities seen at ultrasound, MRI and CT scanning of the liver, and the lack of a clear increase of the AFP in blood. The patient was diagnosed at 15 months because of delay of motor development with hepatomegaly. At diagnosis AFP was 528.568 mg/L, ultrasound showed one hepatic lesion. NTBC was started immediately. AFP decreased to 180 in 1 year and slowly decreased afterwards, the lesion disappeared. Almost 4 year after the start of NTBC, ultrasound showed a new lesion originally classi¢ed as hemangioma. CT scans and MRI showed one and later two lesions considered uncharacteristic of HCC. AFP stabilized at 23, but when there was a tendency to rise (highest value 28) we agreed to proceed to liver transplantation. Even though the AFP did not rise further within the next 3 months (latest value 26.7) the expanded liver showed HCC at two locations. Conclusions: This patient shows that (a) a new lesion in HTI patients under NTBC is HCC until proved otherwise, and (b) lack of increase of AFP does not exclude HCC.

Background: NTBC (nitisinone) is the ¢rst choice therapy in tyrosinemia type 1. Until now there are scarce data about NTBC pharmacokinetics in the children. Methods: In 5 patients with tyrosinemia type 1 (using HPLC method with UV detection) following NTBC pharmacokinetic properties were settled upon the one-dose strain test with 1 mg of the medicine. Results: There were: mean maximum concentration (Cmax) 38.28 +3.910 mmol/L, mean half-life (t12 ) 12.276+3.91 h and mean area under the NTBC concentration curve in time [AUC (0^24)] 508.848 +170.427 mmol/h/L. The absorption of NTBC lasted from 1 to 3 h at the mean constant absorption value 0.556 h^1 (SD+0.185 h^1). The time after which the concentration reached maximum value (Tmax) was calculated for about 2.5 h. Later NTBC distribution was monitored as well as a elimination phase, in which the geometric mean of NTBC concentration in serum reached 46.6% of the maximum concentration value (40.4%^53.3%; CV% = 11.67). After 8^10 h another peak to the geometric mean of 90.1% Cmax (67.9^143.9%; CV% = 37.1) was observed. Mean b elimination constant was 0.050+0.013 h^1, and MRT (mean residance time) 19.50+5.12 h. Conclusions: In our study the pharmacokinetic properties of NTBC in the children di¡er considerably from the data published so far only once in the adult volunteers. Revealing that the half-life of NTBC is markedly shorter, and the curve graph of blood concentration is diphase, make it possible to try to monitor NTBC dosing by measurement of its serum concentration.

DEVELOPMENT OF A SUCCINYLACETONE METHOD FOR USE AS A POTENTIAL NEWBORN SCREENING TEST FOR TYROSINAEMIA TYPE I Dowden S1, Webster R1, Preece MA1, Gissen P2 1 Clin Chem Birmingham Child Hosp, Birmingham, UK, 2Inherit Metab Dis Birmingham Child Hosp, Birmingham, UK

TYROSINEMIA TYPE I: HEPATOCELLULAR CARCINOMA IN PATIENT WITH NEW LIVER LESION WITHOUT INCREASE OF AFP van Spronsen FJ1, Gauw A2, van der Jagt EJ3, de Jong KP4, Verkade HJ1 1 Beatrix Children's Hospital, UMCG, Groningen, Netherlands, 2Dept Pathol, Univ Med Cent of Groningen, Groningen, Netherlands, 3Dept Radiol, Univ Med Cent Groningen, Groningen, Netherlands, 4Dept Surgery, Univ Med Cent of Groningen, Groningen, Netherlands

EPILEPSY AND ELECTROPHYSIOLOGICAL ABNORMALITIES IN CHILDREN WITH HEREDITARY TYROSINAEMIA TYPE 1 Santra S, Mckiernan P, Seri S, Wassmer E Birmingham Children's Hospital, Birmingham, UK

PHARMACOKINETICS OF NTBC (NITISINONE) FOLLOWING A SINGLE DOSE TO 5 CHILDREN WITH TYROSINEMIA TYPE 1 Pohorecka M, Pohorecka M, Wawer Z, F|lipek M, Sykut-Cegielska J, Pronicka E The Children's Memorial Heath Institute, Warsaw, Poland

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DETERMINATION OF NTBC IN SERUM SAMPLES FROM PATIENTS WITH HEREDITARY TYROSINEMIA TYPE I BY CAPILARY ELECTROPHORESIS Cansever MS1, Aktuglu-Zeybek AC1, Erim FB2 1 Div Metab Dis, Istanbul Univ Cerrah Med Fac, Istanbul, Turkey, 2Dept Chem, Istanbul Techn Univ, Istanbul, Turkey Background: 2-(2-nitro-4-tr i£uoromethylb en zoyl)-1,3cyclohexanedione (NTBC) has been successfully used as a treatment for hereditary tyrosinemia type I since the ¢rst introduction in 1992. Following our ¢rst study on the rapid and easy capillary electrophoresis (CE) method for the determination of succinylacetone in urine samples from patients with hereditary tyrosinemia type I, in the present study, we developed a new CE method for the identi¢cation and quantitative determination of NTBC in serum samples. So far, the only study in the literature for the analysis of NTBC in plasma is a complex column liquid chromatographic method. Method: NTBC was detected in only 4 min by the developed capillary electrophoretic method. Limit of detection of NTBC was obtained as 3.17 mmol/L using UV detection at 278 nm. Results: Repeatability of migration times and peak areas were 0.73% RSD and 2.59% RSD, respectively. The linearity was in the concentration range of NTBC from 25 to 200 mmol/L with 0.99611 regression. Conclusions: The utility of the method was demonstrated by the identi¢cation of NTBC in serum samples from patients with hereditary tyrosinemia type I, using this drug. The developed method is rapid, simple, inexpensive, and suitable for the screening of NTBC levels in clinical serum samples.

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EVIDENCE THAT HOMOCITRULLINE AND ORNITHINE COMPROMISE BIOENERGETICS IN CEREBRAL CORTEX OF YOUNG RATS V|egas CM, Tonin AM, Zanatta A, Knebel L, Wyse ATS, Wajner M Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil Background: Hyperornithinemia^hyperammonemia^homocitrullinuria (HHH) syndrome is biochemically characterized by tissue accumulation of ornithine (Orn), ammonia and homocitrulline (HCit). A¡ected patients present lethargy, vomiting, ataxia, choreoathetosis, delayed development and severe cerebral retardation whose pathogenesis is not yet understood. Objectives: The present work investigated the in vitro e¡ect of Orn and HCit (0.1^5 mM) on bioenergetics in cerebral cortex of 30-day-old rats. Methods: 14CO2 production from glucose or acetate, and the activities of the respiratory chain (RC) complexes I^III, II, II^III, IV, succinate dehydrogenase and creatine kinase (CK) were measured in the presence of various concentrations of Orn or HCit. Results: Orn and HCit signi¢cantly reduced aerobic glycolysis and citric acid cycle activity (reduction of 14CO2 production from glucose and acetate), with no alteration of the activities of the RC complexes. Moreover, HCit, but not Orn, markedly reduced CK activity indicating a disturbance on intracellular ATP transfer. Conclusions: It is presumed that energy metabolism impairment may be involved in the pathophysiology of HHH syndrome. F|nancial support: Research grants from CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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UNUSUALLY SEVERE GYRATE ATROPHY CAUSING UNILATERAL BLINDNESS IN A 12 YEAR OLD GIRL Barnett C1 Lam W2 Schulze A1 1 Div Clin Met Gen, Hosp Sick Children, Toronto, Canada, 2Div Ophtho, Hosp Sick Child, Toronto, Canada

EVIDENCE FOR THE FOUNDER ORIGIN OF 117DELC MSUD MUTATION IN PORTUGUESE GYPSIES Quental S1, Gusma¬o A2, V|larinho L3, Amorim A2, Prata MJ2 1 Fac of Sciences of Univ of Porto, Porto, Portugal, 2IPATIMUP, Porto, Portugal, 3Genetics Medical Center, INSA, Porto, Portugal

Background: Gyrate atrophy is a rare condition of the retina and choroid caused by hyperornithinemia secondary to ornithine aminotransferase (OAT) de¢ciency. Inheritance is autosomal recessive. It usually presents in late childhood and slowly progresses to blindness in the 5th and 6th decades. It is more common in the F|nnish population. We present a 12 year old girl with severe gyrate atrophy causing blindness in the right eye. To the best of the authors' knowledge this is the ¢rst report of a blind eye due to gyrate atrophy in a child of this age. Case report: A 12 year old girl presented with severe deterioration in vision over several months. She was diagnosed with myopia at age 7 and her vision deteriorated gradually from this point until severe deterioration occurred in the months prior to diagnosis. At presentation she had no vision in her right eye. Her general health, growth and development had been normal. Her father was F|nnish. There was no family history of metabolic disease or early blindness. Examination con¢rmed no vision on the right and 20/40 vision on the left. Fundoscopy revealed right sided chronic retinal detachment and severe chorioretinal atrophy consistent with gyrate atrophy. Milder changes were seen on the left. Plasma ornithine was markedly elevated (1220 mmol/L, normal range 26^106). Hyperornithinemia was initially treated with vitamin B6 with little e¡ect. After commencing a low arginine diet, ornithine levels fell dramatically over 3 months. The reason for the advanced stage of disease in our patient is not known.

Background: Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched chain alpha-ketoacid dehydrogenase complex. We have previously reported the molecular characterization of 31 Portuguese patients, eleven of them (35.5%) belonging to a Gypsy community from Beja (region in South of the country). As expected, all Gypsy patients were found to be homozygous for the same mutation in BCKDHA gene (117delC). Objective: In order to con¢rm the suspected founder origin of 117delC mutation we have performed a haplotypic study as well as a carrier frequency determination. Methods: Four microsatellite markers £anking BCKDHA gene have been studied in three groups: one corresponded to Gypsy patients and families and, the other two, were control groups of Portuguese Gypsies (137) and non-Gypsies (92) individuals. Results and Conclusions: The same disease-associated haplotype was identi¢ed in all patients carrying the 117delC mutation, thus con¢rming its unique origin. From the 137 control Portuguese Gypsies analysed, one was heterozygous for the 117delC alteration, which was found to be in the same disease-associated STR haplotype. The referred individual was also from a Gypsy community from Beja. This result indicates that MSUD, and particularly the 117delC mutation in BCKDHA, in the Portuguese Gypsy population seems to be restricted to the Beja communities. Knowing that 117delC is a major mutation underling MSUD in Portuguese Gypsies (with an estimated frequency of 0.36%) has a great relevance for public health-care since it is possible to facilitate molecular prenatal diagnosis in families at risk.

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COMPARISON OF MANAGEMENTS OF METABOLIC DECOMPENSATION IN MAPLE SYRUP URINE DISEASE BY PLASMA EXCHANGE AND NUTRITIONAL MEASURES IN SIBLINGS Okada J, Yoshino M, Watanabe Y Kurume University School of Medicine, Kurume, Japan Background: Genetic and hence phenotypic diversity of maple syrup urine disease (MSUD) has made it di¤cult to make comparative study of e¤ciency of therapeutic modalities in acute metabolic decompensation. Objective: To compare therapeutic e¤ciency of plasma exchange and nutritional management in genetically and environmentally identical siblings. Patients and Methods: A boy (patient 1) was found have an elevated leucine concentration on screening test made on 7 days of age. Plasma leucine concentration at 10 days of age was 4.9 mmol/L. The patient was treated by 4 cycles of plasma exchange at ages of 11 days through 14 days. The T1/2 of plasma leucine concentration (T1/2 Leu) during this period was calculated to be 86.4 h. His sister (patient 2), born 4 years later and followed prospectively, also showed an elevated leucine concentration of 0.93 mmol/L at two days of age. The baby was treated by nutritional measure alone that provided 90 to 110 kCal/kg/ day and nutrients free of leucine, isoleucine and valine since 3 days until 9 days of age, when the 3 branched-chain amino acids were added to the formula. T1/2 of leucine concentration during this period was 139.2 h. Conclusions: The e¤ciencies of the 2 therapeutic modalities are very similar. The nutritional measure is the therapy of ¢rst choice in view of its minimum invasiveness and minimum risk of infection.

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PANCREATIC BETA CELL RESERVE AND INSULIN SENSITIVITY IN MAPLE SYRUP URINE DISEASE Dursun A1, Sar|kabaday| YU1, Ozon ZA2 1 Hacettepe Univ Metab and Nutr Unit, Ankara, Turkey, 2Hacettepe Univ Ped Endoc, Ankara, Turkey Acute and chronic pancreatitis have been reported as rare complications of branched chain aminoacid disorders including MSUD, PA, MMA, and isovaleric acidemia. In this study, insulin sensitivity and b-cell reserve are investigated in 14 MSUD patients (8 girls and 6 boys) older than 4 years of age. Sex and age matched patients with PKU (n: 14) were included as negative controls, and 14 healty controls comprised the control group. Insulin sensitivity and b-cell reserve were evaluated using three (fasting insulin level, OUICKI, and HOMA-IR), and two parameters (HOMA B % and insulinogenic index), respectively. Patients with MSUD were studied twice, initially during acute poor metabolic balance, and later during acute good metabolic balance. Additionally, 7 patients with MSUD (48 years) underwent oral glucose tolerance test. Insulin sensitivity and b-cell reserve of patients and controls did not di¡er signi¢cantly. In the literature there are some case reports of abnormal glucose homeostasis during the course of inherited metabolic disorders, although their pathogenesis is not clari¢ed completely yet. Care should be taken on pancreatic endocrine functions and glucose homeostasis during acute and chronic stages of inherited metabolic diesases, especially intoxication types. Support: State Planning Organisation; Prj. No:2006 K 120 640-06-03

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INTERFERENCE OF VALPROIC ACID ON THE BRANCHED CHAIN AMINO ACID OXIDATIVE METABOLISM Luis PBM1, Ruiter JPN2, IJlst L2, Ofman R2, Diogo L3, Garcia P3, Duran M2, Vockley J4, Tavares de Almeida I1, Wanders RJA2, Silva MFB1 1 Met and Gen Group, iMed UL, Fac Farm Univ Lisboa, Lisboa, Portugal, 2 Lab Gen Metab Dis, AMC, Univ Amsterdam, Amsterdam, Netherlands, 3 Hospital Pediatrico de Coimbra, Coimbra, Portugal, 4Dept Ped, School Med, Univ Pittsburgh, Pittsburgh, USA Valproic acid (VPA) is an e¡ective antiepileptic drug that a¡ects several biochemical pathways including the branched-chain amino acids (BCAAs) catabolic pathway. Increased urinary excretion of 3-hydroxyisovaleric and 2methyl-3-hydroxybutyric acids has been detected in patients under VPA therapy suggesting an interference of the drug with the oxidative metabolism of BCAAs, probably at the level of isovaleryl-CoA dehydrogenase (IVD) and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD). Aims: To investigate the potential inhibitory e¡ect of VPA metabolites on speci¢c enzymes from the BCAAs oxidative pathway. Methods: The enzymatic activities of heterologously expressed IVD, isobutyryl-CoA dehydrogenase (IBD), short branched-chain acyl-CoA dehydrogenase (SBCAD) and MHBD were measured using optimized HPLC procedures. Incubations were preformed with valproyl-CoA, valproyl-dephosphoCoA and 3hydroxyvalproyl-CoA. Results: Valproyl-CoA and valproyl-dephosphoCoA inhibited IVD activity signi¢cantly, by a purely competitive mechanism (Ki: 84.7 mM and 216 mM, respectively). IBD activity was not a¡ected by any of the VPA esters. Valproyl-CoA inhibited SBCAD activity by a purely competitive mechanism with a Ki of 280 mM. However, valproyldephosphoCoA inhibited SBCAD activity through a distinct mechanism, which appeared to be of the mixed type. 3-Hydroxyvalproyl-CoA was found to inhibit MHBD by a purely competitive mechanism. Both SBCAD and MHBD participate in the b-oxidation of VPA explaining, at least in part, the inhibitory e¡ect that speci¢c metabolites of VPA exert on these enzymes. Conclusions: VPA metabolism clearly interferes with the leucine and isoleucine oxidative pathways. These data indicate that the use of VPA should be carefully considered in patients with an inborn de¢ciency in the leucine or isoleucine oxidative pathways.

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FUNCTIONAL ANALYSIS OF THE NUCLEOTIDE VARIATION c.288+9c4t IDENTIFIED IN THE BCKDHA GENE OF A VARIANT MSUD PATIENT Rodr|èguez-Pombo P1, Weisiger K2, Packman S2, Navarrete R1, Ugarte M1 1 Centr Biol Mol, Univ Autonoma Madrid, Madrid, Spain, 2Div Med Gen, Dept Ped, UCSF, San Francisco, CA, USA Mutations in any of the BCKDHA, BCKDHB and DBT genes encoding for catalytic components of the branched-chain-alpha ketoacid dehydrogenase can cause maple syrup urine disease (MSUD). The variability of disease expression has stimulated attempts to correlate it with genotypes of patients. Here, we examine the consequences, at the mRNA level of the intronic alteration c.288 +9c4t found in heterozygous fashion in a BCKDHA MSUD patient bearing also the mutation p.Gly249Ser and with clinical and biochemical diagnostic of variant form. Splicing mutations are considered as causative when they a¡ect the conserved canonical sites GT and AG dinucleotides at the 5' and the 3' intron boundaries, but the interpretation of variations at non conserved regions depends on functional studies. Methods: Lymphoblast cells from patient were treated with emetine to prevent potential degradation of unstable mRNAs. All transcripts obtained were cloned, sequenced and quanti¢ed using a Quantitec Primer assay. Results: Quantization of the mRNA transcripts present in patient lymphoblast shown a severely decreased amount of BCKDHA mRNA and a small but measurable quantity of normal full-length BCKDHA mRNA. After emetine treatment we identi¢ed several transcripts, including ones retaining 7 nucleotides of intron 2. We concluded that c.288+9c4t could introduce a new splice site which results in a premature stop codon supporting the possible activation of a nonsense mediated mRNA decay. We also hypothesized that presence of normal full length BCKDHA mRNA could attenuate the phenotypic expression of the disease. So, the enhancement of correct mRNA splicing could be a promising route toward e¡ective therapy.

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HEPATOCYTE TRANSPLANTATION (HTX) EXTENDS LIFESPAN, INCREASES BRANCHED CHAIN ALPHA-KETO ACID DEHYDROGENASE (BCKDH) ACTIVITY, AND IMPROVES AMINO ACID PROFILES IN A MURINE MODEL OF INTERMEDIATE MAPLE SYRUP URINE DISEASE (IMSUD) Skvorak KJ1, Paul HS2, Dorko K3, Marongiu F3, Chace D4, Ferguson C5, Gibson KM3, Homanics GE5, Strom S3 1 Univ Pittsburgh, Dept Mol Gen and Biochem, Pittsburgh, USA, 2 Biomed Research and Technologies, Inc, Wexford, USA, 3Univ Pittsburgh, Dept Path, Pittsburgh, USA, 4Pediatrix Screening, Inc, Bridgeville, USA, 5Univ Pittsburgh, Dept Anesthes, Pittsburgh, USA MSUD (OMIM 248600) is an aminoacidopathy treated primarily by dietary manipulation of branched-chain amino acids (BCAA). Dietary compliance may be di¤cult; conversely, liver transplantation signi¢cantly improves outcomes, but donor organs are scarce. We previously created a murine model of iMSUD (BMC Med. Gen. 2006, 7:33) characterized by elevated blood BCAAs, alloisoleucine, and decreased glutamine. This model was used to investigate HTx as a treatment for MSUD. W|ld-type murine donor hepatocytes were harvested and directly injected (10^6 cells/50 ml) into liver of iMSUD mice (two injections, 1^10 days of age). Neonatal mice were employed to enhance engraftment in the expanding hepatic volume. HTx signi¢cantly extended lifespan [iMSUD-Htx: 70% survival (n = 10) vs untreated iMSUD controls (iMSUD): 25% survival (n = 78) at 37 days (p50.0001)] and improved blood total BCAA/alanine ratio [iMSUD-Htx: 3.9+0.4 (SEM; n = 9) vs iMSUD: 14.7+1.9 (n = 5; p50.0001)]. HTx did not correct total BCAA/alanine ratio in whole brain homogenates, whereas brain alloisoleucine levels were signi¢cantly improved [iMSUD-Htx: 32.9 +7.5 nmol/g tissue (n = 5) vs iMSUD: 80.4+18.5 (n = 6; p50.05)]. F|nally, HTx enhanced liver BCKDH activity [iMSUD-Htx: 175+27 mmol ketoisocaproate oxidized/min/mg (n = 7) vs iMSUD: 82+6 (n = 4; p50.0233)]. Encouraged by our ¢ndings of partial metabolic correction of iMSUD, and the fact that multiple manipulations are possible to further enhance engraftment with HTx, we suggest that HTx represents a promising therapeutic intervention for MSUD. Supported by NIH DK57386 and the MSUD Family Support Group

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BIOCHEMICAL AND MOLECULAR NEONATAL SCREENING FOR HOMOCYSTINURIA IN THE QATARI POPULATION Ben-Omran T1, Gan-Schreier H2, Kebbewar M3, W|lrich J3, Abdoh G1, Shahbek N1, F|scher C3, Janssen B3, Al Rifai H1, Lindner M3, Zschocke J2, Ho¡mann G3 1 Dept of Paed, Hamad Medical Corporation, Doha, Qatar, 2Inst of Human Gene, Ruprecht-Karls-Univ, Heidelberg, Germany, 3Dept of Paed, University Children Hospital, Heidelberg, Germany Background: Homocystinuria is a disorder of methionine metabolism caused by cystathionine-synthase (CBS) de¢ciency. It is characterized by mental retardation, ectopia lentis, a Marfan-like appearance, epilepsy and thromboembolism. Prevalence range from 1:200 000^335 000; however, there is increased incidence of homocystinuria in Qatar caused by founder e¡ects in a highly consanguineous population resulting in homozygosity for the mutations R336C and D234N in the CBS gene. As the best therapeutic results can be achieved in individuals identi¢ed by newborn screening and treated shortly after birth. Methods: We developed a novel biochemical and molecular testing strategy for extended neonatal screening. Total Hcy was determined in dried blood spots by a high performance liquid chromatography method with tandem mass spectrometry detection. In addition the common mutations R336C and D234N in the CBS gene were tested. Results: So far we have analyzed 17 743 newborns from Qatar, 7559 of Qatari origin. A total of 6 neonates with homocystinuria were identi¢ed. All showed highly elevated homocysteine concentrations in dried blood spots whilst methionine was elevated in two neonates only. F|ve children were homozygous for the common mutation R336C; follow-up sequence analysis in the sixth patient revealed homozygosity for mutation G347S, not previously observed in the Qatari population. Conclusions: these results prove the new biochemical method to be well suited for neonatal screening with a higher sensitivity than DNA testing for the detection of homocystinuria in Qatar. Our results support a very high incidence of homocystinuria in Qatar reaching up to 1:900, caused mainly by high consanguinity rate.

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DIETARY COMPLIANCE IN LATER DIAGNOSED HOMOCYSTINURIC PATIENTS Frangipani BJ, Micheletti C, Oliveira RB, Lauandos JR, Mendes CSC, Rand MH, Vertemati T, D'almeida V, Martins AM CREIM /UNIFESP, Sa¬o Paulo, Brazil Background: Homocystinuria due to cystathione beta synthase (CBS) de¢ciency is the most common disorders of sulfur amino acid metabolism that leads to abnormal accumulation of homocysteine (Hcy) in the plasma. In patients who do not respond to pyridoxine, a low methionine diet must be introduced, and supplemented with a methionine-free medical food. In addition pyridoxine, folate and betaine are given orally. For individuals diagnosed late, dietary compliance is di¤cult to achieve. Objective: To present the laboratorial response of nonresponsiveness patients with a low metione diet. Methods: Laboratorial evaluation of 6 pacients. Two patients are adolescents, who were diagnosed when 7 and 8 years old and the others are adults, with were diagnosed when 13 years old. We evaluated the average total Hcy plasma levels during 3 years. All patients had total Hcy plasma level above 300 mmol/L at the diagnoses. Results: We observed an average total Hcy plasma levels such as: patient 1: 16.63 mmol/L, patient 2: 54.8 mmol/L, patient 3: 68.64 mmol/L, patient 4: 73.9 mmol/L, patient 5: 110.88 mmol/L and patient 6: 177.4 mmol/L. Just 2 patients had total Hcy plasma levels under 60 mmol/L, that suggested by good compliance in patients who do not respond to pyridoxine. Conclusions: Dietary compliance in late diagnosed patients are di¤cult and in£uenced by many factors such as, they are used to a normal unrestricted diet, failure to consume prescribed quantity of protein substitute and in Brazil doesn't exist speci¢cally manufactured low protein foods.

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NO LONGER JUST AN INTERMEDIATE: CYSTATHIONINE PROTECTS AGAINST ENDOPLASMIC RETICULUM STRESS INDUCED LIPID ACCUMULATION AND APOPTOTIC CELL DEATH Maclean KN1, Jiang H1, Lhotak S2, Austin RC2, Greiner LS1 1 University of Colorado, Aurora, USA, 2McMasters University, Hamilton, Canada Cystathionine b-synthase (CBS) represents a key regulatory point at the ¢rst committed step in the biosynthesis of cysteine via the transsulfuration pathway. The CBS enzyme catalyzes a pyridoxal 5'phosphate (PLP) dependent b-replacement reaction condensing serine and Hcy (Hcy) to form cystathionine. Cystathionine is a thioethercontaining amino acid currently without a known function other than serving as an intermediate in transsufuration. Cystathionine thus formed, is subsequently converted to cysteine plus ammonia and aketobutyrate by the action of cystathionine g-lyase (CGL). A number of lines of evidence suggest that in neural tissues and at speci¢c periods of development CBS expression is speci¢cally directed for the production of cystathionine independent of cysteine synthesis. Using both whole animal experiments and cell culture models, we have found that cystathionine is capable of blocking the induction of both hepatic and renal lipid accumulation and apoptotic cell death by the endoplasmic reticulum stress inducing agent tunicamycin. Immunohistochemical analysis indicated that this protective e¡ect occurs without modulation of the unfolded protein response. Our experiments indicate that cystathionine is not just an intermediate of transsulfuration but can also function as a cytoprotective agent in neural, renal and hepatic tissues. These results o¡er a possible explanation for the expression of CBS in the absence of CGL during mammalian development and in primate neural tissue.

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IN VITRO EFFECT OF S-ADENOSYL HOMOCYSTEINE ON H19 GENE EXPRESSION Castro R1, Gomes A2, Rocha M1, Esse R1, Clode N3, Grac°a LM3, Rivera I1, Blom HJ4, Jakobs C4, Tavares de Almeida I1 1 Met and Gen Group, iMed UL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal, 2UniMo, IMM, Lisbon, Portugal, 3Dept Obstetrics, Gynec and Reprod Med, Hosp Santa Maria, Lisbon, Portugal, 4Dept Clin Chem, VU Univ Medical Center, Amsterdam, Netherlands

S-ADENOSYL HOMOCYSTEINE HYDROLASE INHIBITION AFFECTS PROTEIN AND DNA METHYLATION IN HUVEC Castro R1, Esse R1, Rocha M1, Gonc°alves I1, Clode N2, Grac°a LM2, Rivera I1, Teerlink T3, Jakobs C3, Blom HJ3, Tavares de Almeida I1 1 Met and Gen Group, iMed UL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal, 2Dept Obstetrics, Gynec and Reprod Med, Hosp Santa Maria, Lisbon, Portugal, 3Dept Clin Chem, VU Univ Medical Center, Amsterdam, Netherlands

The involvement of epigenetic mechanisms in the context of homocysteine (Hcy) and vascular disease is an emergent possibility. Accordingly, evidence has been gained concerning the Hcy ability, via accumulation of its precursor, S-adenosylhomocysteine (AdoHcy), to a¡ect DNA methylation patterns. The present study is aimed at examining whether AdoHcy is able to alter the expression of H19, an imprinted gene. A HUVEC line, heterozygous for a suitable RsaI RFLP in H19 gene, was selected and incubated for 24 h (n = 3) with increasing (0, 2.5 and 5 mmol/L) concentrations of adenosine-2,3-dialdehyde (ADA), an inhibitor of AdoHcy hydrolase. Cytosolic AdoHcy concentrations were measured by LC-MS/MS. Gene-speci¢c methylation of the di¡erentially methylated region (DMR) of H19 promoter was evaluated by bisulphite-sequencing. Allelic expression pattern was analysed by RT-PCR-RFLP with RsaI and H19 transcription level was quanti¢ed by qRT-PCR. The results revealed that ADA induced a highly signi¢cant and dose-dependent increase in intracellular AdoHcy levels (p50.05 and p50.01 for incubations with 2.5 and 5 mmol/L of ADA, respectively, when compared with control cultures), which was already reported to be associated with a decrease in global DNA methylation patterns (1). H19 allelic expression pattern and DMR methylation status remained unchanged. However, HUVEC incubated with the highest ADA concentration displayed signi¢cantly increased transcription levels when compared to control cultures, suggesting that other cellular methylation events involved in H19 expression were a¡ected by AdoHcy accumulation. Further studies are required to elucidate whether/how AdoHcy-induced epigenetic changes are involved in vascular disease. (1) Castro et al. J Mol Med 2005;83:831-836

Background: Evidence has been gained concerning the homocysteine (Hcy) ability, via accumulation of its precursor, S-adenosylhomocysteine (AdoHcy), to a¡ect DNA methylation patterns indicating that Hcy may be regarded as a global demethylating agent. Protein methylation, an emerging regulator of protein function, can also be potentially a¡ected by AdoHcy and may constitute a new mechanism implicated in Hcy-related pathology. The posttranslational methylation of arginine within polypeptides results in di¡erent forms of methylated arginine residues, including asymmetric dimethylarginine (ADMA), which is released upon proteolysis. Thus, ADMA concentration can be regarded as a measure of cellular protein arginine methylation status. Aim: To evaluate the e¡ect of intracellular AdoHcy accumulation on both DNA and protein methylation patterns. Methods: AdoHcy intracellular accumulation was achieved through HUVEC incubation with increasing concentrations of adenosine-2,3dialdehyde (ADA), an AdoHcy hydrolase inhibitor. Extracellular tHcy and ADMA concentrations, after 24, 48 and 72 h of incubation, were quanti¢ed by HPLC. Genomic DNA methylation patterns were evaluated by the cytosine-extension-assay. Results and Discussion: HUVEC exported Hcy and ADMA at a constant rate (1.5+0.2 and 0.21+0.03 mmol/L, respectively). ADA, at any studied concentration, elicited a dose-dependent decrease in the export of both metabolites; the e¡ect on Hcy export was already observed after 24 h of incubation, while the e¡ect on ADMA export was apparent after 48 h. In addition, ADA also decreased global DNA methylation patterns in a dosedependent manner. These results suggest that AdoHcy-mediated inhibition of methyltransferases can be detected at both DNA and protein arginine residues methylation levels.

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BEHAVIORAL ANALYSIS AND HIPPOCAMPAL GENE EXPRESSION PROFILING IN A MOUSE MODEL OF CBS DEFICIENCY REVEALS EPIGENETIC EFFECTS LEADING TO MISEXPRESSION OF HOMEOBOX GENES Maclean KN, Evans JR, Stabler SP, Allen RH, Jiang H University of Colorado, Aurora, USA The pathological mechanisms that underlie mental retardation associated with cystathionine beta-synthase (CBS) de¢cient homocystinuria (CBSDH) are poorly understood. We used two separate tests of spatial memory and learning to examine the HO mouse model of CBSDH for impaired hippocampal function. In a radial maze food retrieval paradigm, HO CBSDH mice showed highly signi¢cant (p50.001) de¢cits in terms of working and reference memory compared to normal controls. Similarly, the HO CBSDH mice were unable to form a conditioned taste aversion. Examination of changes in the hippocampal expression using the A¡ymetrix MOE 430A A¡ymetrix Murine Genome mouse expression chip found a total of 332 genes were signi¢cantly up-regulated compared to controls. We observed a dramatic induction (between 30 and 41000-fold) of 33 homeobox genes and their interacting transcription factors. Although important during development, the normal spatio-temporal activity of homeobox genes is not consistent with expression in the mature hippocampus and they are typically silenced by methylation of their promoter regions. Their expression in the CBSDH mouse hippocampus most likely re£ects an epigenetic e¡ect where elevated Sadenosylhomocysteine (SAH) acts to impair methylation of their promoter regions. This hypothesis is supported by the observation that our microarray analysis detected the induction of another 25 unrelated genes that are known to be rerepressed by promoter methylation. Inappropriate expression of homeobox genes has previously been linked with dysmorphogenesis and impaired organ function. Thus, epigenetic misexpression of homeobox genes is a candidate mechanism for the hippocampal de¢cit and cognitive impairment observed in our animal model of CBSDH.

INHIBITION OF METHYLATION AND CHANGES IN GENE EXPRESSION IN RELATION TO NEURAL TUBE DEFECTS van der Linden IJM1, Heil SH2, van Egmont Petersen M2, van Straten HW3, den Heijer M2, Blom HJ1 1 Metabolic Unit, Clin Chem, VUMC, Amsterdam, Netherlands, 2 Radboud University Nijmegen Medical Cent, Nijmegen, Netherlands, 3 Anatomy and Embryology, Univ Maastricht, Maastricht, Netherlands Background: Impaired DNA methylation has been suggested to underlie the complex etiology of neural tube defects (NTDs). Previously, we have demonstrated that inhibition of methylation by periodate oxidized adenosine (Adox) results in a widening of the anterior neuropore (ANP) in our in vitro chick embryo model. Since DNA methylation is the chief regulator of gene expression, we hypothesize that inhibition of methylation will a¡ect the expression of genes implicated in neurulation. In the present study we therefore examined di¡erential gene expression between Adox treated and control chick embryos. Materials and Methods: Chick embryos of 4/5 somites were cultured in vitro with saline (control) or Adox and cranial parts were excised. Gene expression pro¢ling was determined using the A¡ymetrix Genechip. Chicken Genome Array on RNA isolated from two pools of Adox treated cranial parts (n = 12) and two pools of saline treated cranial parts (n = 12). Microarray data were validated by QPCR analysis. Results: In the Adox treated chick embryos 45 probesets were up regulated (fold 52.0, p50.05) and 32 probesets were down regulated (fold 40.5, p50.05). Among genes selected for QPCR analysis up regulation of phosphoserine phosphatase (PSPH), unc-51-like kinase 1 (ULK1) and chemokine (C-X-C motif) ligand 12/stromal cell-derived factor 1 (CXCL12/SDF-1) was con¢rmed. Conclusions: Inhibition of methylation a¡ects gene expression in our in vitro chick embryo model. Further research will focus on the gene speci¢c methylation patterns of PSPH, ULK1 and CXCL12/SDF-1 and the role of their gene products in neurulation.

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Background: Methylation is one of the most prominent epigenetic modi¢cations of mammalian genomes, being implicated in transcriptional silencing of genes. Disruption of proper DNA methylation patterns is associated with pathogenic mechanisms underlying embryonic development, carcinogenesis and mental retardation and, more recently, vascular disease. The evaluation of global DNA methylation status can thus be envisaged as a potential biomarker for several disease states and, accordingly, methods are needed for simple and reliable measurements. Methods: Global DNA methylation was measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the cytosine extension assay (Cyt-Ext). Genomic DNA was isolated from peripheral blood of healthy subjects (n = 96). Calf thymus and pBR322 DNAs were used as hyper and hypomethylated references. Aim: Compare LC-MS/MS with Cyt-Ext, the currently available methods for determining global DNA methylation. Results: LC-MS/MS versus Cyt-Ext of hypomethylated reference was 0.45+0.02% mCyt/tCyt (CV = 4.9%) versus 9679.7+393.4 Dpm/0.5 mg DNA (CV = 3.7%), and of hypermethylated 6.4+0.09% (CV = 1.3%) mCyt/tCyt versus 4193.4+368.8 Dpm/0.5 mg DNA (CV = 8.4%). Using the 96 healthy subjects the two methods displayed a poor correlation (r = ^0.30; p50.003). Conclusions: LC-MS/MS analysis is extremely simple and very precise, but requires special equipment, not available at all laboratories. Cyt-Ext assay is very laborious and less precise, but needs no special facilities, ensuring its wide-spread use. Both methods were able to discriminate between hyper and hypomethylated reference DNA. The poor correlation of both methods using DNA of healthy controls shows that di¡erent sorts of DNA methylation are determined: LC-MS/MS measures all methylated cytosine, whereas Cyt-Ext only those recognized by the restriction enzyme.

Background: Global DNA methylation monitors epigenetic changes, which relate to various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LCESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA. Methods: DNA was hydrolyzed using formic acid. Cytosine and 5methylcytosine were separated by gradient-elution reversed phase chromatography with a mobile phase containing nona£uoropentanoic acid (NFPA) as ion-pairing reagent and quanti¢ed using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers. Results: Cytosine and 5-methylcytosine showed intense protonated molecular ions under positive electrospray ionization conditions. Good peak shapes and adequate retention times of approximately 3 min and 4 min 15 s were obtained for cytosine and 5-methylcytosine respectively under the described ion-pairing conditions. Linear calibration curves were obtained in the range 0.111^4.422 ng/ml. The intra- and inter-assay CV of the 5-methylcytosine/total cytosine ratio was 1.7% (n = 9) and 3.5% (n = 8) respectively for calf thymus DNA (mean mcyt/cyt ratio 6.5%), and 4.5% (n = 6) and 6.5% (n = 14) respectively for pBR322 DNA (mean mcyt/cyt ratio 0.48%). The limit of detection (S/N = 3) was 2 pg on column for cytosine and 5-methylcytosine. The clinical and biochemical characteristics of the healthy subjects were determined. DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase 677 TT genotype (p = 0.046). No association with B-vitamin status was observed. Conclusions: This LC-ESI-MS/MS method is easy to perform and o¡ers reproducibility, selectivity and sensitivity to study DNA methylation. The method allows a sample throughput of about 200 samples a week.

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Hyperhomocysteinaemia is associated with increased risk for vascular disease. Plasma levels of homocysteine (Hcy) may be a¡ected by nutritional and genetic factors, namely by MTHFR C677T and A1298C polymorphisms. The present work aimed at investigating the genetic and biochemical determinants of homocysteine levels in a population of 123 healthy school adolescents (55 males and 68 females) aged between 16 and 17 years old, from Madeira Island. We found a signi¢cant di¡erence (p50.05) between median plasma total Hcy in males (9.32 mM; 95%CI 9.20^11.28) and in females (7.08 mM; 95%CI 6.99^8.03). In the whole sample, the relative frequency of the 677T allele was 29.3% and the prevalence of the mutant homozygous genotype was 9.8%, consistent with previous results obtained for the Portuguese population. Concerning the A1298C mutation, the relative frequency of the 1298C allele was 34.1% and the prevalence of the mutant homozygous genotype was 15.5% which are both higher than those already reported for the Portuguese population (28.2% and 6.0%, respectively). Individuals homozygous for the 677T allele showed signi¢cantly higher levels of plasma Hcy than those with the CC genotype. On the other hand, plasma Hcy levels were increased in individuals bearing the 1298AA genotype. Genotypic combination of 677TT/1298AA and 677CC/1298CC corresponded to a signi¢cant increase in Hcy levels. Regarding the variables that may modulate plasma Hcy concentration, and in order to exclude folate and vitamin B12 in£uence, these nutritional determinants need to be further evaluated.

Background: Mild hyperhomocysteinemia is considered an independent risk for premature atherosclerosis and may result from a disturbed cobalamin metabolism. After absorption, only vitamin B12 carried by transcobalamin II (holotranscobalamin or active vitamin B12) is available for cellular uptake and is physiologically relevant in the remethylation of homocysteine (Hcy) to methionine through the transfer of a methyl group from folate via vitamin B12. The main focus of this study was to evaluate the e¡ect of the TCN2 C776G polymorphism on vitamin B12 (total and active), Hcy and folate status. Methods: A group of healthy Portuguese subjects (n = 122; 42 M and 80 F), with a mean age of 45.9+12.6 years were genotyped. Plasma Hcy, active and total B12 and folate levels, as well as the erythrocyte folate levels, were measured (Abbott ASSYM). The C776G polymorphism was screened by PCR-RFLP with ScrF1. Results: Allele frequencies for C and G were 0.52 and 0.48, respectively, while genotype relative frequencies were 31.1% (CC), 41.0% (CG) and 27.9% (GG). The C776G polymorphism showed no e¡ect on either tHcy or total B12 levels. But active B12 and erythrocyte folate levels were signi¢cantly (p50.05) lower in GG individuals when compared to CC individuals: 52.2+17.3 versus 64.7+22.8 pmol/L and 139.8+260.1 versus 554.8+266.4 nmol/L, respectively. Regarding plasma folate levels, a trend towards signi¢cance (p50.07) was observed (6.2+5.7 (GG) versus 17.1+9.7 (CC) nmol/L). Conclusions: These results suggest that the common TCN2 C776G polymorphism modulates vitamin B12 and folate cellular availability, which can be predicted by active B12 plasma levels.

GLOBAL DNA METHYLATION ^ COMPARISION OF TWO DIFFERENT METHODS Rocha M1, Castro R1, Kok R2, Rivera I1, Jakobs C2, Smulders Y2, Tavares de Almeida I1, Blom HJ2 1 Met and Gen Group, iMed UL, Fac Pharmacy, University of Lisbon, Portugal, 2Dept Clin Chem, VU Univ Medical Center, Amsterdam, Netherlands

HOMOCYSTEINE AND MTHFR POLYMORPHISMS IN A POPULATION OF HEALTHY ADOLESCENTS FROM MADEIRA ISLAND Caldeira Arauèjo H1, Pimenta A1, Ornelas R2, Rivera I3, Castro R3, Torres I1, Tavares de Almeida I3 1 Dept Health Sciences, Univ Madeira, Funchal, Portugal, 2Dept Phy Ed, Univ Madeira, Funchal, Portugal, 3Med and Gen, iMed UL, Fac Pharm, Univ Lisbon, Lisbon, Portugal

GLOBAL DNA METHYLATION MEASURED BY LIQUID CHROMATOGRAPHY ^ TANDEM MASS SPECTROMETRY: ANALYTICAL TECHNIQUE, NORMAL VALUES AND DETERMINANTS IN HEALTHY SUBJECTS Kok RM, Smith DEC, Blom HJ, Barto R, Spijkerman AMW, Teerlink T, Jakobs C, Smulders YM VU University Medical Center, Amsterdam, Netherlands

DOES C776G POLYMORPHISM IN THE TCN2 GENE AFFECT VITAMIN B12 STATUS? A PILOT STUDY IN A PORTUGUESE HEALTHY POPULATION Castro R, Barroso M, Pereira A, Ramos R, Rivera I, Tavares de Almeida I Met and Gen Group, iMed UL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal

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ENDOTHELIAL DYSFUNCTION IN PATIENTS WITH HOMOCYSTINURIA AND THEIR PARENTS Caliskan B1, O£az H2, Demirkol M1, Baykal T1, Ozer I1, Polat N2, Gurdal A2, Gokcay G1 1 Div Nutr Metab, Istanbul Med Fac, Child Hosp, Istanbul, Turkey, 2Div Cardiol, Istanbul Med Fac, Istanbul, Turkey Background: To investigate endothelial dysfunction, in patients with homocystinuria and their parents, using noninvasive studies. Methods: 17 patients with classical homocystinuria, aged 10^34 years (mean+SD 21.4+7.8), 13 obligate heterozygous parents aged 34^57 years (mean+SD 44.2+8.1) were studied. Study groups were compared with two di¡erent control groups, matched for baseline characteristics. Using high-resolution ultrasound, brachial artery diameter at rest, during reactive hyperemia and carotis intima media thickness (IMT), were measured. Echocardiographic assessment has also been performed. Results: The mean brachial artery £ow mediated dilation (FMD) values of the patient and control group were 5.66+4.70% and 13.79+8.31%, respectively (p = 0.001). The mean carotid IMT of the patient group (0.73 + 0.11 mm) was signi¢cantly higher than the control group (0.46 + 0.10 mm) (p = 0.000). The left ventricle ejection fraction (EF) values of the patient group (71.42+8.47%) were lower than the control group (77.47+4.14%) (p = 0.014). The di¡erence between isovolumetric relaxation time (IVRT) values of the patient (109.46+13.49 msn) and control groups (89.00+7.14 msn) was statistically signi¢cant (p = 0.003). The di¡erence between the mean FMD values of heterozygous parents (5.79+5.14%) and parents control group (10.17+5.03%) was statistically signi¢cant (p = 0.038). The IMT, EF and IVRT values of the two groups were similar. Conclusions: Patients with homocystinuria and their heterozygous parents had impaired endothelial function in the systemic arteries. Patients also had impaired diastolic and systolic functions of heart that were preserved in parents.

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METHIONINE, HOMOCYSTEINE AND METHYLMALONIC ACID STATUS IN 9 PATIENTS WITH COBALAMIN OR FOLATE DEFICIENCY Schi¡ M1, Benoist JF1, Rigal O1, Fenneteau O2, Ogier de Baulny H1 1 Div Metab Dis, Univ Child Hosp R. Debreè, Paris, France, 2Lab Haematol, Univ Child Hosp R. Debreè, Paris, France Background: In childhood, plasma de¢ciency in cobalamin (Cbl) and folate are rare conditions. Cbl de¢ciency is secondary either to insu¤cient intake especially in infants born to vegan mothers, or to congenital malabsorption. In contrast, folate de¢ciency is mainly due to congenital malabsorption. Biologically, these de¢cits share megaloblastic anemia with low level of plasma Cbl or red blood cell folate, low to normal methionine (Meth) plasma level and high plasma homocysteine (HCy) level; abnormal urinary methylmalonic acid (MMA) excretion is only seen in Cbl de¢ciency. Precise values of these metabolic biomarkers are poorly reported. Objective: To document pre treatment levels of plasma Meth, plasma free and/or total HCy in patients with Cbl or folate de¢ciency and urinary MMA in Cbl defective patients. Methods: The data from 7 patients with Cbl de¢ciency (one dietary Cbl de¢ciency, 2 intrinsic factor de¢ciencies and 4 Imerslund-Gra«sbeck diseases) and 2 patients with congenital folate malabsorption were analyzed. Results: Meth levels were low to normal in 6 patients (range 8 to 20 mM, normal values 15^25 mM). Free and/or total HCy levels were elevated in 8 patients (range 22 to 64 mM, normal value 515 mM). Urinary MMA was increased in six Cbl defective patients (range 13 to 3120 mmol/mol creatinine, normal value 55 mmol/mol creatinine) and not determined in one. Conclusion: These data provide precise values of metabolic biomarkers from nine Cbl or folate defective patients. Even if their variations remain moderate, these parameters are crucial elements in the diagnostic workup and follow-up of megaloblastic anemia.

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METHYLENTETRAHYDROFOLATE REDUCTASE DEFICIENCY PRESENTING WITH HEMOLYTIC UREMIC SYNDROME Ku«ster A1, Chery C2, Alberto JM2, Audonnet S2, Josse T2, Roussey G1, Allain-Launay E1, Tea I1, Darmaun D1, Roze JC1, Feillet F2, Gueant JL2 1 INRA,UMR Phan,CIC004,Metab Dis,Univ Hosp, Nantes, France, 2 INSERM U724, Div Metab Dis, Univ Hosp, Nancy, France Remethylation defects of homocysteine account for several rare atypical hemolytic uremic syndromes (HUS) particularly when associated with dyserythropoiesis. Methylentetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of plasma homocysteine levels. We report the ¢rst case of MTHFR de¢ciency with HUS. The 18 month-old girl presented with asthenia, aregenerative anemia (Hb 3.5 g/dl), thrombopenia (16 000/ml) and microangiopathy in a context of HUS. Bone marrow showed dysmyelopoiesis. Serum cobalamin and folate levels were normal. Low plasma methionine levels (16 mmol/L, N: 15^ 29), hyperhomocysteinemia (50 mmol/L) and absence of urinary methylmalonic acid were indicative of a remethylation defect. Response to treatment with OH-cobalamin, folic acid and betaine was slow but complete with transfusions and antihypertensive drugs being stopped after 3 months. Enzymatic phenotyping of ¢broblasts showed MTHFR de¢ciency (1.4 nmol/h/mg; patient versus control ratio: 0.2) while methionine synthase (MTR) activity was normal (ratio 0.93). Genotyping of MTR, MTRR and MMACHC showed absence of mutations in the genes open reading frame. Genotyping of MTHFR revealed a c.1215C4T heterozygous silent mutation (p.L405L) and a heterozygous polymorphism 677CT. The genetic defect of MTHFR leading to de¢cient activity remains to be identi¢ed and may correspond either to abnormal transcription rate or splicing. This ¢rst report of atypical HUS due to MTHFR de¢ciency highlights the importance of considering defective remethylation in di¡erential diagnosis of atypical HUS, as it is a treatable condition.

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SEARCH FOR PHENO- AND GENOTYPICAL CONFORMITIES IN FOLATE CYCLE DEFECTS BEYOND THE USUAL GENETICS Grechanina OY1, Bogatirova RV1, Matalon RK2, Holmes BB2, Grechanina YB1, Gusar VA1 1 Institute of Clinical Genetics, Kharkov, Ukraine, 2UTMB, Texas, Galveston, USA High frequency, neural tube defects and insu¤cient folate intake, incidence and mortality from vascular pathology are characteristic for Ukraine. Investigation of in£uence of polymorphisms in folate cycle genes of Ukrainian population is carried out. 200 Ukrainians specimens, 16 specimens of patients with suspicion for homocystinuria (HCU), 118 specimens with suspicion for folate cycle disorder. Ampli¢cation of MTHFR, MTRR, RFC-1 genes fragments (primers of Bio-Synthesis Inc., Lewisville, Texas), restriction of ampli¢cated fragments; test-systems `Lytech' (Russia). An average allele frequency in the Ukrainian population for: MTHFR (C677T) ^ 27.39%, MTHFR (A1298C) ^ 28.25%, MTHFR (G1793A) ^ 2.31%, MTRR (A66G) ^ 57.00%, RFC-1 ^ 40.00%. The genotypes and allele frequencies of genes investigated are determined in 16 patients: MTHFR (C677T) ^ 34.40%, MTHFR (A1298C) ^ 12.50%, MTHFR (G1793A) ^ 12.50%, MTRR (A66G) ^ 56.60%, RFC-1 ^ 34.40%. Among the 118 patients, frequency of heterozygotes for MTHFR (C677T) ^ 44.10%, for MTRR (A66G) 40.67%; frequency of homozygotes for MTHFR (C677T) ^ 5.93%, for MTRR (A66G) ^ 29.66%. Detailed study of pheno- and genotypic conformities enable us to distinguish two alternative phenotypes and their intermediate forms, as well as to assume that in HCU, epigenetic phenomenon of allelic genes interaction in heterozygous state is clearly observed, which leads to inherited expression change of one of alleles (paramutation). It is possible, donor de¢cit of methyl groups in£uences on genic expression regulation processes and leads to disorder of individual development as a consequence of autoteratogenesis. The ¢ndings obtained proved to be beyond the classic genetic ideas.

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MUTATIONS IN CYSTATHIONINE GAMMA-LYASE GENE AND THEIR PHENOTYPIC CONSEQUENCES Kozich V1, Janosikova B1, Hegele RA2, Jakobs C3, Stabler SP4, Allen RH4, Kraus JP5 1 Inst IMD, Charles Univ, 1st Fac Medicine, Prague, Czech Republic, 2 Blackburn Cv Genet Lab, Robarst Res Inst, London, Canada, 3VU University Medical Center, Amsterdam, Netherlands, 4Dept Med, Univ Colo Health Sci Ctr, Denver, USA, 5Dept Pediatr, Univ Colo Health Sci Ctr, Aurora, USA Cystathioninuria due to cystathionine gamma-lyase (CTH) de¢ciency is a biochemical trait with extremely variable clinical phenotype and largely unknown genetic basis. In this study we examined plasma sulfur amino acids and the CTH gene mutations in three Czech patients and in a cohort of 165 Czech controls. Patients 1 and 2 were ascertained in childhood due to developmental delay while patient 3 (with unimpaired cognitive functions) was diagnosed in adulthood during a workup for mild/moderate hyperhomocysteinemia detected following a crural thrombosis. The plasma cystathionine levels were grossly elevated (4412, 6000 and 22 596 nmol/L, ref. range below 342 nmol/L) while total homocysteine was only slightly elevated or normal (34, 7.1 and 34 mmol/L, ref. range below 15 mmol/L) in patients 1, 2 and 3, respectively. Interestingly, the total cysteine concentrations were only borderline low suggesting possible alternative pathways for cysteine synthesis in CTH de¢ciency. Only two mutant alleles ^ c.344C4T (p.T67I) and a severe 14.5 kbp deletion of exon 2 to 5 (delEx2__5) have been found in these three patients. Patient 1 is a compound heterozygote for these two mutations while patients 2 and 3 are homozygotes for the c.344C4T and the delEx2__5 mutations, respectively. These two mutant alleles are also frequent among Czech controls (population frequencies 0.015 and 0.003, respectively) with a predicted incidence of CTH de¢ciency being about 1:3000. Our data suggest show that CTH de¢ciency may be a common biochemical trait in the Czech Republic and that it should be looked for in the work-up for mild/moderate hyperhomocysteinemia.

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VENOUS THROMBOSIS SECONDARY TO CONGENITAL INTRINSIC FACTOR DEFICIENT SECRETION IN A VEGETARIAN YOUNG MALE Besseau C, Chery C, Devignes J, Namour F, Feillet F, Gueant JL Hematology Lab, CHU Brabois, Vandoeuvre les Nancy, France We report the case of a 30 year old vegan male who presented a penile venous thrombosis. He was treated with omeprazole for 2 years for gastroesophageal re£ux. Biological screening revealed a mild hyperhomocysteinemia (40 mmol/L) and a low serum vitamin B12 level. There was no other haematological abnormality. Thirty years ago, his mother had an episode of anaemia, treated by vitamin B12, during pregnancy. Gastric endoscopy (including biopsies) was normal. Gastric intubation with pentagastrine stimulation (the treatment with omeprazole was stopped for 10 days) showed a de¢ciency in intrinsic factor secretion (6.4 ng/ml in basal and 3.1 ng/ml after stimulation). Under combined treatment with folic acid and vitamin B12, homocysteinemia normalized. Genotyping showed a MTHFR 677 TT polymorphism (also found in the mother) and heterozygous c.435__437delGAA in exon 4 of intrinsic factor gene (GIF). This heterozygous defect was responsible for the dramatic decreased of intrinsic factor stimulated secretion with subsequent de¢ciency in cobalamin absorption. In this case, vitamin B12 de¢ciency is multifactorial: genetic intrinsic factor de¢ciency; inhibition of gastric secretion (omeprazole) and vegan diet. Moreover, the MTFR 677 TT polymorphism adds a risk factor of hyperhomocysteinemia. This observation underlines the possible association between inherited and non inherited thromboembolic factor risks in the same patient.

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SEVERE NEONATAL MTHFR DEFICIENCY, REPORT OF A NEW CASE WITH COMPOSITE MUTATIONS Chery C1, Audonnet S1, Josse T2, Chabrol B3, Feillet F1, Gueant JL1 1 Ref Cent Inb Err Metab INSERM U724, Nancy, France, 2Serv Neonatology, CHU Purpan, Toulouse, France, 3Ref Cent Inb Err Metab, CHU T|mone, Marseille, France

HOMOCYSTEINE/FOLATE METABOLISM IN ITALIAN CHILDREN WITH DOWN SYNDROME Scala I1, Granese B1, Sampietro F2, Salome© S1, Scarpato E1, F|gliuolo C1, Paladino S1, Zuppaldi A1, D'Angelo A1, Andria G1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2Coagul and Thromb Unit, Inst S. Ra¡aele, Milan, Italy

We report the case of a one month-old girl who was addressed in Neonatal intensive care unit for apnoea, hypotonia, hepatomegaly, vomiting and feeding di¤culties. Ultrasound revealed enlarged cerebral ventricles and there was abnormal EEG pattern. Biological data showed pancytopenia (haemoglobin: 8 g/dl; thrombocytopenia: 147 000/mm3 and neutropenia 730/mm3), increased lactate, very low methionine (0 mmol/L) and hyperhomocysteinemia (319 mmol/L). Serum cobalamin and folate were normal. She died two days later from respiratory failure. Enzymologic studies on ¢broblasts showed severe methylenetetrahydrofolate reductase (MTHFR) de¢ciency (patient versus control ratio: 0.16) while methionine synthase (MTR) activity was normal (ratio: 0.80). Genotyping of MTHFR showed composite defects with heterozygous c.523G4A missense mutation (p.A175T) in exon 4, 677 CT polymorphism in exon 5 and c.1240C4T and heterozygous nonsense mutation (p.W389X) in exon 7. The two mutations were in trans since the mother was heterozygous for the mutation in exon 4, and 677 TT homozygous, while the father was heterozygous for exon 7 mutation and 677 CT. This is the ¢rst report for severe neonatal MTHFR de¢ciency with proven composite mutations. The clinical description is similar to the rare reports already described with severe neurological symptomatology.

Background: Down syndrome (DS) is due to trisomy 21. The cystathionine-beta-synthase (CBS) and the reduced-folate-carrier (RFC1) genes map on chromosome 21 and are critical regulators of the homocysteine/folate metabolism. To investigate whether the homocysteine/folate pathway is disrupted in DS, we analysed the CBS844ins68, the RFC1A80G, the methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, the methionine synthase reductase (MTRR) A66G and the methionine synthase (MTR) A2756G polymorphisms as well as plasma homocysteine, methionine, folates, B12 and B6 vitamins. Methods: Genotype analysis was performed by RFLP. Genotype and allele frequencies were evaluated in 134 DS subjects and 93 controls, and the odds ratio was used as a measure of association between polymorphisms and DS. Biochemical data were analyzed by t-test. The relation between homocysteine/folate levels and genotypes was also evaluated. Results: No association was found between the analysed polymorphisms and the risk of DS. A negative interaction was found between RFC80G homozygotes and: MTR 2756AA and AA/AG; MTRR 66AG/AA and AG/GG; MTHFR 1298AA/AC and AA; MTHFR 677CC/CT; CBSins^. Homocysteine and methionine levels were lower in DS subjects (p = 0.004 and p = 0.011, respectively), while folates, B12 and B6 levels were not di¡erent. Low homocysteine levels were associated to the following genotypes: MTHFR677CT (p = 0.012); MTHFR1298AA (p = 0.006); MTRR66AG (p = 0.008); MTRR66GG (p = 0.001); MTR2756AA (p = 0.006); CBSins^/^ (p = 0.022). Among DS subjects, low folate levels were associated with the MTR2756GG genotype. Conclusions: Homocysteine/folate metabolism is impaired in DS with a reduction of homocysteine and methionine, partly depending on the genotype. This may a¡ect cellular methylation reactions.

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VARIATION AND EXPRESSION OF DIHYDROFOLATE REDUCTASE (DHFR) IN RELATION TO SPINA BIFIDA van der Linden IJM1, Heil SH2, Franke B2, Gellekink HJ2, den Heijer M2, Blom HJ1 1 Metabolic Unit, Clin Chem, VUMC, Amsterdam, Netherlands, 2 Radboud University Nijmegen Medical Cent, Nijmegen, Netherlands

THE HIGH INCIDENCE OF HOMOCYSTINURIA IN THE TAO TRIBE OF TAIWAN Lu YH1, Yu HC1, Lo MY1, Gao JH1,Hsu JH2, Chong KW2, Kao CH2, Cheng KH2, Liu TT, Hung PY2, Niu DM2 1 Dept Pediatrics, VGHospital, Taipei, Taiwan, 2Genome Research Cente, Yang-MING Univ, Taipei, Taiwan

Background: The dihydrofolate reductase (DHFR) enzyme regulates folate availability, folate turnover and DNA synthesis. The 19-bp deletion in intron-1 of DHFR has been associated with the risk of having spina bi¢da a¡ected o¡spring, supposedly by changing DHFR gene expression. Recently, a 9-bp repeat in exon 1 of the mutS homolog 3 (MSH3) gene was found in the 5'UTR of DHFR and may possibly a¡ect DHFR gene expression as well. We examined the association between these DHFR variants and spina bi¢da risk and investigated their e¡ect on DHFR expression. Methods: 121 mothers of a spina bi¢da a¡ected child, 109 spina bi¢da patients, 292 control women and 234 pediatric controls were screened for the DHFR 19-bp deletion and the 9-bp repeat. DHFR gene expression was measured in 66 spina bi¢da patients, using real-time PCR analysis. Results: The DHFR 19-bp del/del genotype was not associated with spina bi¢da risk in mothers and children (OR 0.8; 95%CI 0.4^1.5 and OR 1.2; 95%CI 0.6^2.2, respectively) and both the WT/del and the del/ del genotype did not a¡ect DHFR expression relative to the WT/WT genotype (relative expression = 0.89, p = 0.46 and relative expression = 1.26, p = 0.24 respectively). The DHFR 9-bp repeat was not associated with spina bi¢da risk in mothers and children. DHFR expression of the 6/6 allele was 73% increased compared to the 3/3 allele, although not signi¢cantly (relative expression = 1.73, p = 0.09). Conclusion: The DHFR 19-bp deletion or 9-bp repeat do not increase risk on spina bi¢da. An e¡ect of the 6/6 repeat genotype on DHFR expression cannot be ruled out.

Homocystinuria caused by cystathionine beta-synthase de¢ciency a¡ects at least 1 in 200 000 to 335 000 people worldwide. The disorder appears to be more common in some countries, such as Ireland (1 in 65 000), Germany (1 in 17 800), Norway (1 in 6400), and Qatar (1 in 3000). The prevalence of homocystinuria in Taiwan is extremely low (51 in 1 000 000). Recently, we found 8 patients with homocystinuria from Tao tribe at Orchid Island in Taiwan. All of these patients were found to be the homozygous of a novel mutation, D47E. A total of 428 persons have been screened for the D47E mutation and 51 heterozygous carriers and 2 homozygous were found in this screening study. The carrier rate of this D47E mutant is around 1/7.78. The estimated prevalence of homocystinuria caused by this mutation is around 1/240 in this population. This is the highest prevalence of homocystinuria, as we known, in the world. In this report, the clinical manifestations and expression result of these patients will be also presented.

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CLINICAL AND MOLECULAR MANIFESTATIONS OF TAIWANESE PATIENTS WITH HOMOCYSTINURIA Lu YH1, Yu HC1, Lo MY1, Gao JH1, Chong KW1, Kao CH1, Cheng KH1, Liu TT2, Lee NC3, Niu DM1 1 Dept Pediatrics, VGH Hospital, Taipei, Taiwan, 2Genome Research Cente, Yang-MING Univ, Taipei, Taiwan, 3Dept Pediatrics, NTUH Hospital, Taipei, Taiwan Homocystinuria (HCU) is an inherited autosomal recessive defect in methionine metabolism that is caused by a de¢ciency in cystathionine beta-synthase (CBS). Major clinical manifestations involve the eyes and the central nervous, skeletal, and vascular systems. More than 130 pathogenic mutations, mostly in the Caucasian population, have been described. The worldwide prevalence of HCU has been reported to be around 1 in 300 000. The prevalence of homocystinuria in Chinese has never been formally reported. In Taiwan, the newborn screening for HCU started in 1984. Until now, over ¢ve million newborns have been screened and only three babies were found to have HCU. The CBS gene of these three patients has been analyzed and 4 mutations were identi¢ed, including three known mutations (R121L, E176K, V320G) and one novel mutation (G259D). To characterize these mutations, normal or mutated forms of CBS were cloned into pFLAG-CMV2 expression vector followed by transfection into mammalian cells for transient expression. In this report, the clinical manifestations and expression results of these patients will be presented.

CBLD DEFECT OF VITAMIN B12 METABOLISM: MOLECULAR MECHANISM WHEREBY MUTATIONS IN THE MMADHC GENE CAUSE THREE DIFFERENT PHENOTYPES Coelho D1, Stucki M2, Suormala T1, Baumgartner MR2, Fowler B1 1 Metab Unit, Univ Child Hosp, Basel, Switzerland, 2Div Metab Mol Pediatr, Univ Child Hosp, Z u«rich, Switzerland Background: We recently identi¢ed MMADHC as the gene responsible for the cblD defect (Coelho et al, N Engl J Med 2008). This defect is unusual in that mutations in a single gene result in three clinical and biochemical phenotypes: isolated homocystinuria (HC), isolated methylmalonic aciduria (MMA) and both (MMA/HC). We propose that the nature and location of MMADHC mutations explain the three di¡erent phenotypes. Methods: Biotinylated MMADHC protein translated from cDNA in vitro was subjected to SDS-PAGE and stained with streptavidin-alkaline phosphatase or with rabbit anti-MMADHC antibodies. Mutations were analysed in ¢ve patients in addition to the seven previously reported (14 mutations in total). Fluorescence microscopy was performed to localize MMADHC. Results: Each of three cblD-HC patients carries a missense mutation in the C-terminal part of the protein. All ¢ve patients with cblD-MMA carry a stop codon upstream of a potential site for re-initiation of translation and produce a smaller protein than wild-type and missense HC alleles. The estimated size of protein products supports our hypothesis of re-initiation of translation from the Met62 codon. Four patients with cblD-MMA/HC have truncating mutations downstream of the putative re-initiation site, with no detectable protein at all in one cell line and a markedly smaller protein in the other three. Fluorescence microscopy suggests mitochondrial colocalisation and excludes association with lysosomes. Conclusion: Our data indicate that re-initiation of translation downstream of a stop codon explains MMA without HC in cblDMMA patients, missense mutations cause cblD-HC and truncating mutations downstream of potential sites of reinitiation lead to cblDMMA/HC.

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UREASE METHOD FOR URINE ORGANIC AND AMINO ACID ANALYSIS: A POWERFUL EXTENSION OF AN ESTABLISHED METHOD Young V1, Lo S1, Rhead WJ2, Shoemaker J3, Thomas A3 1 Children's Hospital of W|sconsin, Milwaukee, WI, USA, 2Medical College of W|sconsin, Milwaukee, WI, USA, 3Metabolic Screening Laboratory, St. Louis, MO, USA Urine organic acid analysis by GCMS represents the standard for quantitation of acidic amino acid and fatty acid metabolites. Unfortunately, many neutral and positively charged compounds of metabolic and biochemical interest are not detected using these techniques. However, pretreatment with urease permits TMS derivatization of most organic molecules in body £uids. Using published methods (J Chrom Biomed Appl (1991) 562; 125^38), we have expanded the range of metabolites analyzed and quantitated. Our Agilent 5973 GCMS is calibrated with 11 deuterated organic acid, amino acid and carbohydrate internal standards. Individual calibration curves for all metabolites are established and integrated using the Agilent ChemStation Program. Metabolite identities are con¢rmed both by using NIST, St. Louis University Metabolic Screening Laboratory and/or Pitt/Sweetman libraries, and by direct comparison with the pure compound mass spectra analyzed in our instrument. We can currently quantitate 57 organic acid metabolites, 39 amino acids, 10 acylglycines, 9 carbohydrates, 7 neurotransmitters and 6 purines and pyrimidines during a 45 min run. We will soon add an additional 25 to 30 metabolites to our panel, including pentoses, heptoses, Ssulfocysteine, dipeptides, purines, pyrimidines and peroxisomal metabolites. Disadvantages include a more laborious sample preparation than for traditional organic acid GCMS analyses (currently 25^30 samples/week); analysis of these elution pro¢les with many overlapping peaks often requires visual con¢rmation of peak identity and mass spectra. In our view, the utility of the information gained largely outweighs these inconveniences. This technique clearly invites serious consideration by the biochemical genetic and metabolic disease communities.

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ORGANIC ACID DISORDERS DETECTED IN THE BRAZILIAN PEDIATRIC POPULATION FROM 1994 TO 2007 Wajner M1, Coelho DM1, Busanello ENB2, Ingrassia R2, Oliveira AB2, Carvalho AM1, Silva TM2, Pires RF1, Souza CF1, Giugliani R1, Vargas CR1 1 Hospital de Cl|ènicas de Porto Alegre-SGM, Porto Alegre ^ RS, Brazil, 2 Universidade Luterana do Brasil, Canoas, Brazil Background: Organic acidemias (OA) are usually diagnosed by qualitative urinary organic acid analysis, which is a relatively expensive methodology for developing countries. Methods: During 1994^2007 we have analyzed organic acids by gas chromatography/mass spectrometry (GC/MS) in urine samples from 6731 children with suspicion of metabolic disorders. Results: We diagnosed 219 cases of organic acidurias (3.25%). The most frequent diseases were lactic acidemias (54, 24.65%), methylmalonic acidemia (34, 15.52%), glutaric acidemia type I (33, 15.07%), propionic acidemia (18, 8.22%), 3-hydroxy-3methylglutaric acidemia (17, 7.77%), L-2-hydroxyglutaric acidemia (9, 4.11%), multiple carboxylase de¢ciency (9, 4.11%), isovaleric acidemia (7, 3.2 %), glutaric acidemia type II (7, 3.2%) and other less frequent organic acidemias (31, 14.1%). Age at diagnosis was as follows: 16.43% before 1 month of age, 28.57% from 1^12 months, 12.14% from 12^24 months, 37.14% above 2 years of age and 5.71% unknown. The most prominent clinical and laboratory ¢ndings were neurological dysfunction (67.86%), metabolic acidosis (40.71%), hypo/hyper/ dystonia (32.86%), hypoglycemia (24.28%), vomiting (23.57%), feeding di¤culties (16.43%), failure to thrive (15.71%), hepatomegaly (15.0%) and hyperammonemia (11.43%). Conclusions: OA disorders had never being diagnosed in Brazil until GC/MS technology was introduced in our country. The set up of OA analysis in our laboratory provided a diagnostic clue not only to OA but to other IEM including amino acid, urea, carbohydrate and mitochondrial fatty acid oxidation disorders. The diagnosis of these entities allowed us to prompt treatment and better outcome in our patients. F|nancial support: Research grants from FIPE/HCPA and CNPq.

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ORGANIC ACIDURIAS IN HIGH RISK EGYPTIAN PATIENTS Fateen E1, Gouda A1, Boehles HJ2 1 Bioch Genet Dept, National Research Centre, Cairo, Egypt, 2Child Hosp Goethe Univ, Frankfurt, Germany Background: Amino acids, fatty acids and carbohydrates can be metabolized to organic acids. Organic acidurias are inherited metabolic disorders in which organic acids accumulate in tissues and physiologic £uids leading to acute or chronic intoxication. They are considered the most frequent metabolic disorders among severely ill children. Methods: 117 patients, 79 males (67.5%) and 38 females (32.5%) were studied to determine the prevalence and types of organic aciduria in high risk Egyptian children with clinical signs and symptoms suspicious of inherited metabolic diseases. Their age ranged from 3 days to 12 years. Analysis of urine organic acids by gas chromatography/ mass spectrometry was performed to all patients. Results: 22 (18.8%) cases of organic acidurias were diagnosed among the tested individuals. The disease pro¢le showed increased lactate in 12 cases (55%) and was consisted of glutaric aciduria type I (3), PKU (2), MSUD (1), glutaric aciduria type II (1), methylmalonic aciduria (1), canavan disease (1) and non ketotic hyperglycinemia (1). Conclusion: The results demonstrate the importance of high risk screening and diagnosis of organic acidurias in Egypt. The diagnosed organic acids pattern in this study showed that 55% of the patients had a mitochondrial energy defect, while 13.8% su¡ered from glutaric aciduria type I. A larger study is recommended to determine the prevalence of the di¡erent organic acids among our population.

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LIFE-THREATENING EPISODES OF HYPOGLYCEMIA AND ALTERED CONSCIOUSNESS OBSERVED IN PATIENTS TREATED WITH PIVALATE-GENERATING ANTIBIOTICS: A NEW CASE AND REVIEW Watanabe Y1, Inokuchi T2, Tashiro K2, Aoki K2, Okada J1, Matsuishi T1, Yoshino M1 1 Dept Pediat, Kurume Univ, Kurume, Japan, 2Research Inst of Med Mass Spectrometry, Kurume, Japan Background: Pivalate-generating prodrugs have been known to cause hypocarnitinemia and low muscle carnitine content. Metabolic consequences of use of Pivalate-generating prodrugs including secondary systemic carnitine de¢ciency and fatty acid oxidation defects have been raised as a potential adverse e¡ect of the drugs. Objectives and Methods: Clinical and laboratory observations on a 1year-old girl who developed an altered consciousness and convulsions after a chronic use of Pivalate-generating antibiotics are presented. Seven additional cases that have been presented or reported somewhere are also reviewed. Results: All of the eight cases presented with hypoglycemia and prolonged altered consciousness have been reported from Japan over the past 5 years. All of the patients were under age two years except for a four-year-old boy. Levels of serum free carnitine and blood glucose were at 3.6^9.6 mM and 10^26 mg/dl, respectively. The highest value of blood ammonia was 319 mg/dl. All patients were treated with glucose infusion with/without L-carnitine and recovered without any neurological sequella. Use of Pivalate-generating antibiotics ranged from 19 days to more than six months. Conclusions: Secondary carnitine de¢ciency induced by Pivalategenerating antibiotics resulted in life threatening events with hypoglycemia, altered consciousness and convulsions in at least 8 patients. It seems that infants less than age 2 are at higher risk in developing serious symptoms. Caution should be exercised with use of Pivalate-generating prodrugs particularly for infants since the adverse e¡ects can be serious and are considered to be preventable.

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ARE THE CURRENT METHODS OF TREATMENT FOR METHYLMALONIC ACIDEMIA ADEQUATE? Michals-Matalon K1, Bilger K2, Ross N2, Freedenberg D3, Matalon R4 1 University of Houston, Houston, USA, 2Dell Children's Hospital, Austin, USA, 3Vanderbilt University, Nashville, USA, 4University of Texas Medical Branch, Galveston, USA Background: Severe methylmalonic acidemias, mut o (MMAo), are treated with protein restriction, carnitine supplementation, correction of acidosis and antibiotics during acute illness. Two unrelated children with MMAo were seen at the age of 4 years with poor growth, frequent bouts of hospitalizations with acidosis, hair loss and skin rashes. They had low blood concentration of isoleucine, valine, arginine and leucine. Albumin and pre-albumin were low. The treatment protocol that was followed by two institutions include restriction of natural protein 0.5^ 0.7 g/kg and 1.1 g/kg from medical food. Method: Both children showed protein de¢ciency. The diet was changed to include 1.2^1.5 g/kg natural protein and 2.2^2.5 g/kg protein from medical food. Calories were increased from 5100 cal/kg to 130 calories/kg. Carnitine was changed from 100 mg/kg/day levocarnitine to 50 mg/kg/day of levocarnitine and 50 mg/kg/day acetyl-carnitine. Bicitra was increased from 6^7 cc daily to 30^40 cc daily. Results: One patient moved after 1 week of treatment. The other patient showed catch up growth in height and weight and improved energy level and biochemical pro¢le. The loss of hair stopped and for 3 years she did not require hospitalization. Conclusion: Over restriction of protein in MMAo can be deliterious and we suggest that current treatment policies of MMAo should be revised. Increase of citric acid intake (Polycitra), and acetate (acetyl-carntine) need to be considered in treatment of MMAo.

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BLOOD SPOT METHYLMALONIC ACID AS A SECOND-TIER TEST FOR TANDEM MASS NEWBORN SCREENING WITH PROPIONYLCARNITINE Chen PW, Chien YH, Hwu WL, Chen LC, Tseng SC National Taiwan University Hospital, Taipei, Taiwan Background: Propionylcarnitine is the current marker for methylmalonic acidemia and propionic acidemia in newborn screening. However, the speci¢city of propionylcarnitine is low which causes the high false-positive rate and the delay in diagnosis. Therefore we determine dry blood spot methylmalonic acid by LC-MS/MS to serve as a second-tier test for propionylcarnitine screening. Methods: One 1/8-inch punched dry blood disc was extracted with 100 ml of 90% acetonitrile (contains the internal standard) for 20 min. The extract was separated through a 5-cm C18 column and analyzed directly by LC-MS/MS in 5 min. Both newborn samples and samples from methylmalonic acidemia patients were tested. Results: Methylmalonic acid could not be detected in samples from normal newborns. Samples from 8 patients revealed elevated propionylcarnitine level and C3/C2 ratio, and persistently high methylmalonic acid level. In 25 newborns who had elevated propionylcarnitine levels at the ¢rst screening, 17 had elevated C3/C2 ratio. But none of these newborn samples contained methylmalonic acid. A second blood sampling from the 25 babies all revealed normal propionylcarnitine levels. Conclusions: Measurement of dry blood spot methylmalonic acid is rapid and sensitive, and can be served as a secondtier test to reduce the false-positive rate for newborn screening with propionylcarnitine.

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METHYLMALONIC ACADEMIA: REPORT OF 78 CASES AND REVIEW OF THE LITERATURE Farshidi S1, Saber S2, Hoshmand M2 1 Welfare Organization, Prevention O¤ce, Tehran, Islamic Republic of Iran, 2Internatinal Institute of Biotechnology, Tehran, Islamic Republic of Iran Methylmalonic acidemia is a rare autosomal-recessive inborn error of metabolism which is caused by a downstream defect in the propionate metabolic pathway. The metabolic block in these disorders often leads to severe keto- and organic acidosis, hyperammonemia, hyperglycinemia, and hypoglycemia with marked accumulation of methylmalonate in body £uids and tissues of infants and children. The disorder is lethal in its early (neonatal) onset form, and may also manifest as a chronic problem, with episodes of acute decompensation and, in rare cases, the patient may be completely asymptomatic (`benign' form). In this survey the natural histories of 78 patients with these etiologies: 33 mut0, 10 mut^ 24 cblA, and 21 cblB will be described. The most common signs and symptoms were similar in all etiologies. Patients in mut0 class presented earlier than those in other groups. Whereas 80 percent of children in the mut0 class became ill in the ¢rst week of life, less than half the children in the three other groups were ill during this interval. The laboratory ¢ndings in a¡ected patients also show marked similarity between the etiologies. The prevalence of methylmalonic acidemia is di¤cult to de¢ne precisely. A much greater prevalence of between 1:1000 and 1:2000 has been reported in Middle Eastern populations. Conclusion: Methylmalonic acidemias are a group of rare but severe inborn errors of metabolism caused by non-functional methylmalonylCoA mutase. According to increasing consanguinity marriages rate in our country early diagnosis and intervention for this patient is crucial.

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NEUROIMAGING PATTERNS IN GLUTARIC ACIDURIA TYPE I: THE VALUE OF FUNCTIONAL TECHNIQUES IN MAGNETIC RESONANCE IMAGING Peèrez-Duen¬as B1, De la Osa A1, Navarro-Sastre A2, Capdevila A3, Leist A4, Ribes A2, Garc|èa-Cazorla A1, Pineda M1, Campistol J1 1 Neurol Dept St Joan de Deèu Hosp, Barcelona, Spain, 2Division of IEM, Hospital Cl|ènic, Barcelona, Spain, 3Neuroradiol Dept St Joan de Deèu Hosp, Barcelona, Spain, 4Dept Diagnostic Imaging C. M. Teknon, Barcelona, Spain Background: Acute striatal necrosis is a devastating consequence of encephalopathic crisis in patients with glutaric aciduria type I (GA-I), but the mechanisms underlying brain injury are not completely understood. We aimed to approach pathophysiological aspects of brain injury in GA-I by means of functional techniques in magnetic resonance imaging (MRI). Methods: Four patients during an acute encephalopathic crisis and three asymptomatic siblings with GA-I underwent single-voxel hydrogen magnetic resonance spectroscopy (MRS) and brain MRI including gradient echo T1weighted, FLAIR, T2-weighted and di¡usion-weighted imaging. Results: The study was performed between three and eight days after the onset of acute encephalopathic crisis. Isotropic di¡usion images showed high signal changes with corresponding low apparent di¡usion coe¤cient values within the putamen, caudate nuclei and globus pallidus (four patients), and the cerebral peduncles including the substantia nigra (one patient). The study disclosed normal ¢ndings in asymptomatic siblings. MRS showed decreased N-acetyl-aspartate/creatine ratio at the basal ganglia in encephalopathic patients when compared to their asymptomatic siblings. Conclusions: By functional imaging techniques, we could demonstrate the presence of a cytotoxic edema and a reduction of neuronal integrity during acute striatal necrosis. Involvement of the basal ganglia may be asymmetrical in patients with unilateral motor disorder and may extent to the cerebral peduncles and substantia nigra, which may be responsible for the acute onset dystonia in some patients. Functional techniques failed to demonstrated any abnormalities within the basal ganglia in asymptomatic patients that might help to predict which child with GA-I is at risk for developing striatal necrosis.

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TWO PATIENTS WITH GLUTARIC ACIDURIA TYPE I AND INFANTILE SPASM Reims A1, Papadopoulou D2, Lundgren J2 1 Queen Silvia Children's Hospital, Gothenburg, Sweden, 2Dept Paed, Univ Hosp Lund, Lund, Sweden Background: Glutaric aciduria type I (GA I) is a genetic disorder caused by a de¢ciency of glutaryl-CoA dehydrogenase. The defect breakdown of the aminoacids lysine, hydroxylysine and tryptophan leads to accumulation of 3-hydroxyglutaric acid and glutaric acid. Most children are diagnosed between the age 6 and 18 months. Acute neurologic deterioration is usually triggered by febrile illness and dehydration. We describe two children with early onset of the disease. Both children developed infantile spasm a few months after the presentation of the disease. Patients: Both patients were born to nonconsangineous parents. Patient one was under investigation for macrocephali and presented with seizures and hypoglycaemia during gastroenteritis at the age 7 months. Three months later she developed infantile spasm with hypsarrhythmia. Patient two presented with hypotonia from birth and a lifeless attack at the age of 6 days. She showed a large urinary secretion of 3hydroxyglutaric acid. At the age of 7 months she also developed infantile spasm. Patient one responded to an increase of V|gabatrin and adding Topiramat. Patient two responded well to treatment with V|gabatrin. Both patients did also show the typical MR ¢ndings with wide cerebrospinal £uid spaces. Conclusion: GA I can present at any age, even as early as in the newborn period. To our knowledge infantile spasm has not been described earlier in GA I. One explanation could be the early onset of the disease and infantile spasm being the seizures seen in this age group.

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ENCEPHALOPATIC CRISIS IN GLUTARIC ACIDURIA TYPE I. ARE SEIZURES OR DYSTONIC ATACKS A COMMON SYMPTOM? Campistol J1, Cerisola A1, Peèrez-Duen¬as B1, Poo P1, Pineda M1, Garc|èa-Cazorla A1, Sanmart|è FX1, Ribes A2, V|laseca MA1 1 Hospital Sant Joan de Deuè, Barcelona, Spain, 2D Biochem and Mol Gen, Hospital Cl|ènic, Barcelona, Spain Background: Glutaric aciduria type I (GAI) begins with an acute encephalopatic crisis with hypotonia and generalized rigidity, neurologic depression, irritability, seizures and dystonia. Many times, dystonic movements may be confused with seizures. Objective: To describe the clinical features and the initial electroencephalographic ¢ndings in GAI patients. Methods: Descriptive, retrospective study based on the review of clinical histories of GAI patients. Results: We review 13 GAI patients, 9 males and 4 females, mean age 8.7 months (range 3^15 months). 4/13 had consanguineous parents; 7/ 13 had macrocephaly. 12/13 patients (92%) had seizures or pseudoseizures. In 8 of the 13 patients (62%) those episodes were the ¢rst neurologic symptom. In 11/13, the dystonia appeared after the second day. Other clinical features included irritability (12/13), neurologic depression (11/13) and hypotonia (7/13). 35 EEG were analyzed during the ¢rst year (25 EEGs in the ¢rst month). EEG paroxysms in 2 patients; 8/13 slow background activity and 4/13 asymmetries in background activity. In the follow-up of 11 patients, none of them had seizures later. Antiepileptic drugs were discontinued during the ¢rst year. Discussion: Even though seizures are part of the symptoms of the GAI onset, certain paroxysmal movements could be dystonic episodes and not genuine seizures. This hypothesis is supported by the fact that seizures do not continue to occur after dystonic tetraparesis is noticed, EEG paroxysms are infrequent in the acute stage, antiepileptic drugs are not needed on follow-up.

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UNRELIABILITY OF PLASMA CREATININE AND THE SCHWARTZ FORMULA TO IDENTIFY RENAL IMPAIRMENT IN METHYLMALONIC ACIDURIA Ko«nig B, Cleary MA, Clayton PT, Leonard JV, van't Ho¡ W, Gru«newald S Great Ormond Street Hospital, London, UK Background: Chronic renal failure is a common long-term and severe complication of methylmalonic aciduria (MMA). Early detection of renal impairment is essential for optimising treatment. MMA patients are on a low-protein diet and therefore receive little exogenous creatine. In addition muscle mass is often poor. Therefore using the plasma creatinine or Schwartz formula will not give an accurate measure of patient's renal function. Methods: Since 1985 we have used the clearance of chromium EDTA to measure the glomerular ¢ltration rate (GFR) in MMA patients. This method is independent of plasma creatinine concentrations. We have cumulative data of 104 GFRs of 21 MMA patients. The EDTA clearance has been compared with value estimated by the Schwartz formula. We show that there is a major discrepancy between the calculated GFRs and those obtained with the clearance of EDTA chromium, which usually give much lower results. Moreover, the decline of GFR often pre-empts the rise of creatinine or urea. Conclusions: The use of creatinine as a marker, combined with the Schwartz formula, is of limited value in determining the renal impairment in MMA. It hinders the early recognition of chronic renal failure and delays appropriate treatment.

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METHYLMALONYL-CoA MUTASE EXPRESSION IN RAT BRAIN SUGGESTS AUTONOMOUS METHYLMALONATE PRODUCTION IN CNS Ballhausen D1, Bonafeè L1, Boulat O2, Braissant O2 1 IEM, Molecular Pediatrics, CHUV, Lausanne, Switzerland, 2IEM, Clinical Chemistry, CHUV, Lausanne, Switzerland In methylmalonic acidemia, methylmalonate (MMA) and propionate (PA) are mainly produced in the liver from isoleucine, leucine, threonine, and odd-chain fatty acids. Catabolic states (infections, fasting, major surgery) mobilize branched-chain amino acids from muscle and odd-chain fatty acids from adipose tissue, resulting in lifethreatening accumulation of PA and MMA. Liver transplantation is a therapeutic option to `cure' the metabolic disease. The experience of liver transplantation is limited to a small number of cases and shows a high incidence of peri-/post-operative brain damage, despite strong reduction of plasma MMA after transplantation. It was observed that, in contrast to the signi¢cant decrease in plasma, MMA remains high in CSF. This raises the question whether autonomous MMA production exists in the brain and if this mechanism is responsible for neurological degradation despite liver transplantation. The open reading frame cDNA of the rat methylmalonyl-CoA-mutase (MCM) gene was cloned from rat brain mRNA by reverse transcription coupled to PCR, allowing the synthesis of digoxigenin-labelled in situ hybridization (ISH) riboprobes. The MCM mRNA expression was then analyzed by ISH on cryosections of adult rat brain. MCM was found expressed in the whole rat brain. MCM is expressed by neurons but is absent from glial cells. This supports the hypothesis that autonomous MMA production is possible in the brain, which could be the determining prognostic factor in liver transplanted patients. Further studies are needed to provide evidence on the presence of this pathway and to understand the mechanisms leading to neurotoxicity in methylmalonic aciduria.

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FUNCTIONAL AND STRUCTURAL IMPACT ON ATP:COB(I) ALAMIN ADENOSYLTRANSFERASE OF THE I96T CBLB MUTATION ASSOCIATED TO COBALAMIN RESPONSIVENESS Perez B1, Aguado C1, Desviat LR1, Jorge-F|nnigan A1, Banerjee R2, Ugarte M1 1 Centro de Biologia Molecular, UAM. CIBERER, Madrid, Spain, 2Biol Chem Depart, Univ Michigan, Michigan, USA

CHARACTERIZATION OF A WEAK BUT SPECIFIC EFFLUX TRANSPORT OF GLUTARIC, 3-HYDROXYGLUTARIC, AND METHYLMALONIC ACID ACROSS THE BLOOD^BRAIN BARRIER Sauer SW1, Okun JG1, Mahringer A2, Fricker G2, Koelker S1, Morath MA1 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Inst for Pharm and Mol Biotech, Univ Heidelberg, Germany

ATP:cob(I)alamin adenosyltransferase (ATR) is an enzyme of vitamin B12 metabolism that converts reduced cob(I)alamin to the adenosylcobalamin cofactor required for the activity of methylmalonylCoA mutase. Mutations in the human MMAB gene result in a block in adenosylcobalamin synthesis and are responsible for the cblB type methylmalonic acidemia. The goal of this study was the functional analysis of the I96T mutation associated with B12 responsiveness in order to determine the underlying molecular basis of dysfunction. This sequence change was identi¢ed in three Spanish cblB a¡ected patients, in one patient in combination with the missense change R191W, and in two siblings in combination with the splicing change c.584G4A. All three patients exhibited in vitro responsiveness to B12. W|ld type ATR and the mutant I96T protein were stably expressed in a prokaryote expression system. The mutant protein was enzymatically active with KM for ATP and KD for cob(I)alamin similar to wild type enzyme, but exhibited a 40% reduction in speci¢c activity. Size exclusion chromatography revealed that the mutant protein like the wild type enzyme was present predominantly in a trimeric form. T|me degradation assays using the human recombinant ATR expressed in bacterial system revealed that the I96T is less stable than wild-type protein. Structural/functional characterization of mutant ATR will contribute to the understanding of the molecular basis of cobalamin responsiveness and allow the use of transcriptional drugs such as statins or beza¢brate to improve the residual activity.

Background: Glutaric (GA), 3-hydroxyglutaric (3-OH-GA) and methylmalonic acid (MMA) are key metabolites of glutaric aciduria type I and methylmalonic aciduria respectively. Recently, we have shown that the blood^brain barrier (BBB) is only weakly permeable for GA and 3-OH-GA in vivo and in vitro (`trapping hypothesis'). To investigate whether this small £ux across the BBB is carrier-mediated we performed speci¢c transport studies using porcine brain capillary endothelial cells (PBCEC). Because the role of BBB in methylmalonic aciduria is unknown, we included MMA in these studies. Methods: Transport of d3-MMA, d4-GA, and d5-3-OH-GA across PBCEC was measured depending on Na+ gradient and ATP content as well as in the presence of competitively transported substrates. Metabolites were detected by GC/MS analysis. Results: E¥ux (CNS to blood) of d3-MMA, d4-GA, and d5-3-OH-GA was higher than in£ux. Transport was inhibited in the absence of Na+ and by the OAT 1/3 substrate para-aminohippuric acid. In contrast, addition of NaCN did not in£uence the transport. Conclusions: Our results suggest that MMA, GA and 3-OH-GA are transported via OAT1/3. Expression of OAT3 was shown at the apical and basolateral site of BBB. Apical expression is only weak which is in line with small in£ux rates. The sodium dependency of transport suggests an involvement of NaDC1/3, sodium-dependent dicarboxylic acid transporters that were shown to be co-expressed with OAT1/3 in renal proximal tubule and choroid plexus. Stimulation of the e¥ux of toxic dicarboxylic acids at the BBB may represent a therapeutic strategy in glutaric aciduria type I and methylmalonic aciduria.

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DEFECT OF MITOCHONDRIAL ACETYL-CoA THIOLASE (MAT): A CASE REPORT Garnotel R1, Bednarek N2, Morville P2, Gillery P1 1 Pediatric Biochemical Unit, AMH, CHU, REIMS, France, 2Neonatal Intensive Care Unit, CHU, REIMS, France Mitochondrial acetyl-CoA thiolase (MAT, beta-ketothiolase) is responsible for 2-methylacetoacetyl-CoA cleavage in isoleucine metabolism, acetoacetyl-CoA formation in ketogenesis and acetoacetyl-CoA cleavage in ketolysis. Hence ketone body metabolism and isoleucine catabolism are disturbed in MAT de¢ciency. The patient is the ¢rst child of consanguineous marriage. The baby was a full-term female of a normal pregnancy. Neurological development was normal. She presented at 16 months a febrile episode with viral rhinitis and a progressive severe consciousness distress with hyperventilation, and was admitted in intensive care unit. Neuroimagery was normal. After a speci¢c diet (hypercaloric and protein free) alertness rapidly improved and she was discharged at home four days later. She is 2 years old now and neurological exam is normal. Her diet is normocaloric and normoproteic. Blood gas analysis revealed a severe metabolic acidosis with pH = 7.3 and bicarbonate: 2.1 mmol/L. Blood glucose level was more often than not normal, with extreme values 3.2 to 11.1 mmol/L. Hyperuricemia (1070 mmol/L) and hyperammonemia (71 mmol/L) were observed. 3-hydroxy-butyric acid and acetoacetic acid concentrations were respectively 3.9 mmol/L (N: 50.3 mmol/L) and 0.62 mmol/L (N: 50.2 mmol/L). Urinary organic acid analysis showed an excessive excretion of 2-methyl-3-hydroxybutyrate (1660 mmol/mmol creatinine), 3-hydroxy-isovalerate (1220 mmol/mmol creatinine), 2-methyl-3ketobutyrate (1600 mmol/mmol creatinine) and tiglylglycine (1000 mmol/mmol creatinine). Potassium-ion dependent acetoacetyl-CoA thiolase assay in cultured ¢broblasts con¢rmed the defect of mitochondrial acetoacetyl-CoAthiolase. In the MAT defect, prolonged fasting must be avoided and carnitine supplementation should be supplied. The evolution was good with normal neurologic development.

EFFECT OF KETONE BODIES ON OXIDATIVE STRESS PARAMETERS IN BRAIN FROM YOUNG RATS Beskow AP1, Gonc°alves CG1, Leipnitz G2, Seminotti B2, da Silva LB1, Grings M2, Wyse ATS2, Wajner M2 1 Universidade Luterana do Brasil, Canoas, Brazil, 2Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil Background: Ketone bodies (KB) accumulation occurs during fasting, in diabetes and during metabolic decompensation in inherited neurometabolic disorders, especially in acetoacetyl-CoA thiolase and succinyl CoA: 3-oxoacid CoA transferase de¢ciencies. Pro-oxidant and antioxidant activities have been reported for these compounds in peripheral tissues, but virtually nothing is described in the brain. Objectives and Methods: The present study investigated the e¡ect of acetoacetate (AcAc) and b-hydroxybutyrate (BHB) on a large spectrum of oxidative stress parameters in cerebral cortex of young rats. Results: We observed that lipid peroxidation (chemiluminescence and TBA-RS levels), protein oxidative damage (sulfhydryl oxidation and carbonyl formation), as well as the non enzymatic (total antioxidant reactivity ^ TAR and GSH levels) and enzymatic (glutathione peroxidase, superoxide dismutase and catalase activities) antioxidant defenses were not changed by AcAc and BHB at doses as high as 25 mM. We also veri¢ed that lipid peroxidation induced by the Fenton reaction (hydroxyl radical generation), as well as by 3-hydroxy-3methylglutaric and 3-methylglutaconic acids were not altered by BHB and AcAc. F|nally, these KB were not able to oxidize a commercial GSH solution in a cell free system. Conclusion: The data do not support BHB- and AcAc-induced prooxidant or antioxidant e¡ects in the CNS, as previously reported in the liver, heart, erythrocytes and endothelial cells. F|nancial support: Research grants from CNPq, PROPESQ/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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3-Hydroxyisobutyric aciduria (HIBA) is an organic aciduria with a poorly understood biochemical basis. It has been assumed that de¢ciency of 3-hydroxyisobutyrate dehydrogenase (HIBADH) is the underlying enzyme defect, but Loupatty et al. provided evidence that genetic HIBADH de¢ciency is not causative for HIBA (Mol Genet Metab 2006;87:243^8). HIBA may be secondary to respiratory chain defects or can be caused by de¢ciency of methylmalonate semialdehyde dehydrogenase (MMSDH), an enzyme of valine and pyrimidine catabolism. However, reports on mutations in the ALDH6A1 gene coding for MMSDH are sparse. We present two patients with a developmental delay who presented with high urinary concentrations of 3-hydroxyisobutyric acid. Both children were products of consanguineous unions, parents of a boy were of European descent, those of an a¡ected girl from Pakistan. At the age of 2 years the boy developed a febrile illness and subsequently died in a metabolic decompensation. Further studies were initiated and included tests of the enzymes HIBADH and 3-hydroxyisobutyryl-coenzyme A hydrolase in ¢broblast homogenates, which yielded normal activities. Sequencing of the ALDH6A1 gene of the boy revealed a missense mutation in exon 7 which resulted in substitution of an evolutionary highly conserved serine residue by tyrosine, which was not found in 210 control alleles. Mutation analysis of the ALDH6A1 gene of the girl con¢rmed the presence of a di¡erent missense mutation. Mutation analysis in the ALDH6A1 gene can reveal the cause of HIBA, which may present with slight increases in urinary levels of 3-hydroxyisobutyric acid, if a patient is metabolically stable.

Background: 3-Hydroxy-3-methylglutaric aciduria (HMGA) is a disorder biochemically characterized by tissue accumulation of 3hydroxy-3-methylglutarate (HMG), 3-methylglutarate (MGA), 3methylglutaconate (MGT) and 3-hydroxyisovalerate (OHIVA). Patients a¡ected by HMGA usually present neurological dysfunction and a few hepatomegaly with mild hyperammonaemia. Objective: We investigated the in vitro e¡ects of HMG, MGA, MGT and OHIVA on various parameters of oxidative stress in cerebral cortex, striatum and liver from young rats. Methods: The parameters analyzed were lipid and protein oxidation, as well as glutathione (GSH) levels in cerebral cortex, striatum and liver of 30-day-old rats. Results: All metabolites induced similar degree of lipid peroxidation (TBA-RS increase), GSH and sulfhydryl oxidation in cortical and striatal structures. In contrast, only HMG was able to induce lipid peroxidation and oxidize GSH in the liver, but to a lesser degree. Conclusion: The data suggest that cerebral cortex and striatum present higher vulnerability to oxidative stress elicited by the metabolites accumulating in HMGA compared to the liver, which corroborates with the clinical ¢ndings of this disorder. F|nancial support: Research grants from CNPq, PROPESQ/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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3-HYDROXYISOBUTYRIC ACIDURIA DUE TO METHYLMALONATE SEMIALDEHYDE DEHYDROGENASE DEFICIENCY Sass JO1, Walter M1, Shield JPH2, Henger S1, Ko«nig C1, Atherton AM3, Garg U3, Scott D3, Woods CG4, Smith LD3 1 Lab Clin Biochem Metab, Univ Child Hosp, Freiburg, Germany, 2Dept Child Health, Univ, Bristol, UK, 3Med Gen Mol Med, Child Mercy Hosp, Kansas City, USA, 4Dept Med Genet, Univ, Cambridge, UK

INTRACEREBROVENTRICULAR ADMINISTRATION OF ISOVALERIC ACID REDUCES Na+,K+-ATPase ACTIVITY AND INDUCES OXIDATIVE DAMAGE IN CEREBRAL CORTEX OF YOUNG RATS Ribeiro CAJ1, Leipnitz G1, Seminotti B1, Amaral AU1, de Bortoli G2, Wajner M3 1 Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Universidade Luterana do Brasil, Canoas, Brazil, 3Hospital de Cl|ènicas de Porto Alegre, Porto Alegre, Brazil Background: Patients a¡ected by isovaleric acidemia (IVAcidemia) su¡er from acute episodes of encephalopathy, whose underlying mechanisms are not fully established. Objectives: In the present study we investigated the e¡ects of intracerebroventricular (icv) isovaleric acid (IVA) administration, which accumulates in high amounts in IVAcidemia, on important parameters of oxidative stress and on energy metabolism in cerebral cortex of rats. Methods: Thirty-day-old male W|star rats received one icv injection of IVA (5 mmol, 2 ml in each side), 24 h later the animals were sacri¢ced by decapitation, the cerebral cortex dissected, homogenized and used for the biochemical assays. Results: IVA administration provoked a signi¢cant selective inhibition of Na+,K+-ATPase activity from synaptic plasma membranes with no alteration of creatine kinase and the respiratory chain complexes II^IV activities. Furthermore, IVA administration also induced lipid and protein oxidative damage, but did not change GSH content and the activities of the antioxidant enzymes glutathione peroxidase, catalase and superoxide dismutase in cerebral cortex. Conclusions: It is therefore presumed that the inhibitory e¡ect of IVA on Na+,K+-ATPase activity and IVA-induced oxidative damage in the brain may be involved in the pathophysiology of the neurological dysfunction of isovaleric acidemic patients. F|nancial support: Research grants from CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

GREATER VULNERABILITY OF BRAIN TO OXIDATIVE DAMAGE INDUCED BY THE METABOLITES ACCUMULATING IN 3-HYDROXY-3-METHYLGLUTARIC ACIDURIA COMPARED TO THE LIVER Leipnitz G, Seminotti B, Fernandes CG, Ribeiro CAJ, Ribeiro CAJ, Amaral AU, Beskow AP, da Silva LB, Zanatta A, Dutra-F|lho CS, Wajner M Universidade Luterana do Brasil, Canoas, Brazil

DYNAMIC CHANGES OF STRIATAL AND EXTRASTRIATAL MR ABNORMALITIES IN GLUTARIC ACIDURIA TYPE I Harting I1, Neumaier-Probst E2, Seitz A1, Maier EM3, Assmann B4, Baric I5, Troncoso M6, Mu«hlhausen C7, Zschocke J8, Boy N9, Ho¡mann GF9, Garbade SF9, Ko«lker S9 1 Dept of Neuroradiology, Heidelberg, Germany, 2Dept of Neuroradiology, Mannheim, Germany, 3Dept of Biochem Gen and Molec Biology, LMU, Munich, Germany, 4Dept of Gen Pediatrics, Du«sseldorf, Germany, 5Dept of Paediatrics, Zagreb, Croatia, 6Hospital Clinico San Borja Arriaran, Santiago, Chile, 7Dept of Gen Pediatrics, UKE, Hamburg, Germany, 8Institute of Human Genetics, Heidelberg, Germany, 9Dept of Gen Pediatrics, Div of Metab Dis, Heidelberg, Germany Background: Untreated, most patients with glutaric aciduria type I develop acute striatal injury during a ¢nite period of brain development (age 3^36 months) following an acute encephalopathic crisis (AEC). Most studies have focused on this prognostically relevant episode, whereas extrastriatal abnormalities are less well studied. Methods: Sixty-seven MRIs of prospectively followed 38 patients were assessed by three neuroradiologists independently using a standardized protocol. For all patients, detailed information on date of birth, diagnosis, genotype, biochemical and clinical phenotype, neurodevelopment and treatment were reported. Results: In accordance with previous studies, major neuroradiological di¡erences between MRIs from patients with or without AEC were signal changes or atrophy of the putamen (100% vs 0%; p50.001) and caudate (92% vs 11%; p50.001). Interestingly, isolated pallidal abnormalities were frequently found in patients without AEC (53%). In addition to nucleus lentiformis, a wide array of abnormalities were found in other brain regions of both patient groups, such as wide anterior temporal and Sylvian CSF spaces (97%), white matter disease (62%), and signal changes of substantia nigra (19%) and dentate nuclei (13%). Discussion: These results highlight that MR abnormalities of glutaric aciduria type I are more complex and dynamic than previously described. Some MR abnormalities are related to age and appear to manifest following a distinct time pattern. Pathophysiological and therapeutic concepts should not only focus on acute striatal injury and its prevention but should also integrate extrastriatal abnormalities.

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GUIDELINE FOR THE DIAGNOSIS AND MANAGEMENT OF GLUTARIC ACIDURIA TYPE I Ko«lker S1, Christensen E2, Leonard JV3, Greenberg CR4, Burlina AB5, Burlina AP6, Dixon M7, Duran M8, Goodman SI9, Koeller DM10, Mu«hlhausen C11, Mu«ller E1, Naughten ER12, Neumaier-Probst E13, Okun JG1, Kyllerman M14, Surtees RA3, W|lcken B15, Ho¡mann GF1, Burgard P1 1 Dept of General Pediatrics, Division of Metab Disease, Heidelberg, Germany, 2Dept of Clinical Genetics, Copenhagen, Denmark, 3Institute of Child Health, London, UK, 4W|nnipeg Children's Hospital, W|nnipeg, Manitoba, Canada, 5Metabolic Unit, Dept of Pediatrics, Padova, Italy, 6 Dept of Neuroscience, Neurol Clinic, Padova, Italy, 7Dietetic Dept, Great Ormond Street Hospital, London, UK, 8AMC, Lab Gen Metab Dis, Amsterdam, Netherlands, 9Dept of Pediatrics, UCHSU, Denver, CO, USA, 10Dept of Pediatrics, Mol and Med Genet, OHSU, Portland, OH, USA, 11Dept of General Pediatrics, UKE, Hamburg, Germany, 12 National Centre of Inherited Metabolic Disease, Dublin, Ireland, 13 Dept of Neuroradiology, Mannheim, Germany, 14Queen Silvia Children's Hospital, Go«teborg, Sweden, 15The Children's Hospital at Westmead, Sydney, Australia Background: Guidelines for inborn errors of metabolism are still rare. Major arguments against the development of guidelines for orphan disease are that guidelines (1) must always be based on randomized controlled trials (RCTs); (2) automize or restrict clinical decision making; and (3) are expensive and time-consuming. Methods: The guideline developmental process was initiated in October 2003. Four further meetings were performed between 2004 and 2007. The guideline was developed by a group of international experts and external consultants following the methodology of SIGN (www.sign.ac. uk). A systematic literature review and internet searches were carried out. Main searches were selected and evaluated by the group before conclusions were considered as evidence. Results: At this time of rapid expansion of neonatal screening glutaric aciduria type I, the major aim of this guideline was to re-assess the common practice and to formulate recommendations based on the best available evidence. Thirty-one evidence-based statements on neonatal screening, con¢rmation of diagnosis, maintenance and emergency treatment, and therapy monitoring were worked out. Supplementary materials were added such as evidence tables of systematic literature review, diagnostic pitfalls, and dietary recommendations. Thus far, the guideline has been endorsed by the APS (Germany), ESN (The Netherlands), SISMME (Italy), and SPDM (Portugal). Conclusions: The development of scienti¢c guidelines for orphan diseases is manageable ful¢lling all criteria of evidence base and consensus building even in the absence of RCTs.

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MUTATIONAL SPECTRUM AND OXIDATIVE STRESS STUDIES IN METHYLMALONIC ACIDEMIA AND HOMOCYSTINURIA, CBLC TYPE PATIENTS Richard E, Jorge-F|nnigan A, Desviat LR, Merinero B, Leal F, Ugarte M, Peèrez B Centro de Biologia Molecular,UAM, CIBERER, Madrid, Spain Methylmalonic acidemia with homocystinuria, cblC type is the most frequent genetic disorder of vitamin B12 metabolism. In this work, we present the mutational spectrum of 29 patients belonging to the cblC complementation group and intracellular reactive oxygen species (ROS) studies in order to provide insight on the phenotype-genotype correlation. The mutational spectrum included seven previously described mutations (M1L, R73X, R91fs, R132X, R153X, R161X and R189S) and one new splicing change (IVS1nt2T4G). The most frequent change was the known mutation R91fs (c.271dupA) which accounts for 82% of the mutant alleles characterized and 71% of the cblC patients are homozygous. The frequency of c.271dupA is the highest described and in our population allows the genetic diagnosis of the disease by speci¢c mutational analysis using high resolution melting. Most cblC a¡ected patients were biochemically responsive to B12 although they clinically exhibited a fatal outcome with a high frequency of dead patients. In order to provide insight about the severity of the neurological symptoms even in responsive patients, we have evaluated various parameters involved in oxidative stress by £ow cytometry. All patients exhibited increased levels of ROS ranging from 110^300% of control range. Particularly, we have detected higher ROS levels in compound heterozygous of the R91fs mutation although ROS levels are highly heterogeneous in homozygous patients for R91fs. These results indicated that not only the homocysteine and methylmalonic acid but other genetic factors could be responsible for the oxidative stress and for the fatal outcome of the cblC a¡ected patients.

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BLOOD CARNITINE CONCENTRATIONS IN PREMATURE NEWBORNS BY TANDEM MASS SPECTOMETRY Pons R1, Naylor EW2, Chace DH2, McMahon D3, De V|vo DC3 1 Agia So¢a Hospital, Univ Athens, Athens, Greece, 2Pediatrix-Obstetrix Center for Research, Florida, USA, 3Div Child Neurology, Columbia Univ, New York, USA Background: In a previous study of whole blood carnitine concentrations determined by electrospray tandem mass spectrometry in the early neonatal period in 24 644 newborns, we reported normative carnitine data in full-term newborns (37^42 weeks gestation) (Pediatr Res 53:823^829, 2003). In continuing data analysis we have analyzed carnitine concentrations in 2295 premature newborns (34.23+2.45 weeks gestation). Results: Total carnitine (TC) was 73.76+21.42, free carnitine (FC) 48.19+16.40 and acylcarnitine (AC) 25.58+7.83 mmol/L. The AC/FC ratio was 0.57+0.19. Carnitine concentrations (TC, FC) in prematures were statistically higher than in full terms, while AC and AC/FC were statistically lower. The magnitude of these di¡erences, although statistically signi¢cant, was small. On correlation analysis there was no meaningful association between carnitine fractions and gestational age. The AC/FC ratio correlated positively with birth weight, while FC correlated negativelyWhen we divided our cohort in groups of di¡erent gestational age we found that FC was higher in the more immature newborns (24^27 weeks gestation) gradually decreasing up to term (37 weeks gestation), while AC and AC/FC were lowest in immature newborns gradually increasing with fetal maturation. Conclusion: Our ¢ndings suggest a gradual build up of fetal tissue carnitine stores from peripheral blood as the fetus matures and body mass increases. The increase in the proportion of acylated carnitine may be related to the metabolic function of the feto-placental unit. Our ¢ndings support the concept that premature infants are at risk for tissue carnitine de¢ciency.

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POSTNATAL CARNITINE CONCENTRATIONS IN FULL TERM NEWBORNS Pons R1, Naylor EW2, Chace DH2, McMahon D, De V|vo DC 1 Pediatrix-Obstetrix Center for Research, Florida, USA, 2Div Child Neurology, Columbia Univ, New York, USA Introduction: The human fetus adapts from a glycolytic-lipogenic state to a more complex metabolic state shortly after birth. A cluster of metabolic functions emerge, including fatty acid oxidation, ketogenesis and gluconeogenesis. Given the role of carnitine in fatty acid oxidation, we hypothesize that these adaptive changes will be re£ected in the carnitine status of the newborns during the ¢rst days of life. Methods: We performed comparative analysis of carnitine concentrations in full term newborns (n = 19 414) measured by electrospray tandem mass spectrometry during the ¢rst 5 postnatal days of life. In addition, we measured cord blood carnitine in 50 full term newborns (day 0). Results: All carnitine fractions increased within the ¢rst 24 h after birth. Thereafter, all carnitine fractions, including total carnitine (TC), free carnitine (FC) and acylcarnitine (AC) remained constant from postnatal day 2 to day 4. AC decreased signi¢cantly and FC remained constant on post-natal day 5. Conclusion: We speculate that the increase of all carnitine fractions within the ¢rst 24 h of life re£ects the immediate stress response to birth, and it is likely that the rising blood carnitine concentrations re£ects an e¥ux of carnitine from tissue stores. The decrease of AC on the 5th day of life may re£ect a transition from a catabolic state to an anabolic state and a net in£ux of carnitine from blood to tissues. These ¢ndings are important for neonatal screening of defects of the intermediary metabolism associated with secondary carnitine de¢ciency.

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AMINOACYLASE 1 IN VIVO AND IN VITRO Sass JO1, Meincke S1, Walter M1, Olbrich H2, Omran H2 1 Lab Clin Biochem Metab, Univ Child Hosp, Freiburg, Germany, 2Dept Ped Neurol Mus Dis, Univ Child Hosp, Freiburg, Germany Canavan disease (MIM 271900) is a well known neurodegenerative disease with leukodystrophy presenting early in life. It is due to aminoacylase 2 (ACY2, aspartoacylase, EC 3.5.1.15) de¢ciency caused by mutations in the ASPA gene. ACY2 is responsible for the enzymatic cleavage of N-acetyl-L-aspartic acid. In contrast, other acetylated Lamino acids are hydrolyzed by aminoacylase 1 (ACY1, EC 3.5.1.14). Recently, we have reported that recessive mutations in the ACY1 gene result in ACY1 de¢ciency, a novel inborn error of metabolism (Am J Hum Genet 2006;78:401-9; Neurology 2007;68:2151^3). All a¡ected individuals exhibited markedly increased urinary excretion of several N-acetylated amino acids. Most patients with ACY1 de¢ciency (MIM 609924) present with neurologic symptoms. Epileptic seizures have frequently been noted. Due to young age of most ACY1-de¢cient individuals, the clinical course cannot be fully predicted. In order to further elucidate possible pathomechanisms, we have now performed in vitro tests with puri¢ed porcine ACY1, which is closely related to the human enzyme. A wide range of acylated compounds has been tested as potential ACY1 substrates, reaching far beyond typical acetylated derivatives of proteinogenic amino acids and and with a focus on compounds with a role in the nervous system. Our results do not only con¢rm the enantioselectivity of the enzyme as a rule and support that (in addition to N-acetyl-L-aspartic acid) N-acetyl-L-proline is not a substrate of ACY1, but provided also a basis for ongoing mechanistic work on ACY1 de¢ciency.

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METHYLMALONIC ACIDURIA WITH SIGNS OF ASPERGER SYNDROME Saligova J1, Potocnakova L1, Rosenbergerova T2, Strnova J3, Behulova D4, Mrazova L5, Zeman J5 1 Child Fac Hosp, Kosice, Slovakia, 2Fac Hosp, Kosice, Slovakia, 3Med Univ, Bratislava, Slovakia, 4Child Fac Hosp, Bratislava, Slovakia, 5 Charles Univ, Prague, Czech Republic Background: Methylmalonic aciduria (MMA) is pathogenetically and clinically heterogeneous group of disorders of intermediary metabolism. Asperger syndrome is a disorder of social interactions associated with limited stereotypical and repeated spectrum of interests and activities. Case report: Authors present a 20 year old patient with MMA due to a mutation in apoenzyme of methylmalonyl-CoA-mutase. The disease manifested at the age of 4 months with typical signs of organic acidurias and the patient started to keep the standard therapy. F|rst years of his life are characterised by metabolic decompensations during infections. Pronounced regression of attacks of metabolic derangement is characterised for second decade of life, with a normal physical and neurological development. Psychopathologic symptomatology became predominating transitory depression, unequal performance in verbal and non-verbal abilities, ADHD syndrome with speci¢c learning disability especially in mathematics, emotional immaturity and signs of Asperger syndrome. Patient has serious problems with social interactions, special even limited spare-time activity and interest in trains and buses, he spends hours by travelling alone and knows tra¤c schedules by heart. Actually his laboratory results correspond with a compensated state of MMA. Conclusion: Autism belongs to clinical symptoms of MMA, therefore we suppose Asperger syndrome belonging to pervasive disorders is a rare consequence of MMA found in our patient.

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TWO CASES OF MALONIC ACIDURIA DETECTED BY NEWBORN SCREENING (NBS): SAME ANALYTE, DIFFERENT CONDITIONS Gavrilov DK1, Gavrilova RH2, McDonald A1, Peck D3, Hillman RE3, Schoonderwoerd K4, Tortorell S1, Dawson DB5, Gibson KM6, Matern D1, Rinaldo P1 1 Biochem Genet Lab, Mayo Clinic, Rochester, MN, USA, 2Dept Med Genet, Mayo Clinic, Rochester, MN, USA, 3Div Med Genet, Univ Missouri, Columbia, USA, 4Dept Clin Genet, Erasmus MC, Rotterdam, Netherlands, 5Molec Genet Lab, Mayo Clinic, Rochester, MN, USA, 6 Biochem Genet Lab, Univ Pittsburgh, Pittsburgh, PA, USA Background: Malonic aciduria has been reported in less than 30 patients, most of them caused by a de¢ciency of malonyl-CoA decarboxylase (MCD). Few patients have been categorized as combined malonic and methylmalonic aciduria with normal MCD activity (CMAMMA). Both conditions are associated with variable phenotypes ranging from severe neonatal crisis to developmental delays and/or neurologic abnormalities. Case reports: We report two cases of malonic aciduria detected by NBS. Patient 1 remains asymptomatic at age 11 months, his in vitro MCD activity was normal (13 nmol/h/mg protein; reference range: 45.7), no mutations were found in the coding sequence of the MLYCD gene. Patient 2 presented at day three of life with hypoglycemia, seizures and later developed cardiomyopathy. He responded to treatment but has residual developmental delays and microcephaly. MCD activity was 51.0 nmol/h/ mg protein. A large deletion involving exon 1 of the MLYCD gene was detected. Both patients excreted large and comparable amounts of malonic acid (4500 mmol/mol creatinine). Additionally, mild methylmalonic aciduria (21^197 mmol/mol creatinine) was documented in patient 1. Conclusions: To our knowledge, patient 1 is the ¢rst patient with CMAMMA detected by NBS. This ¢nding suggests that, like most other NBS analytes, malonylcarnitine is not a speci¢c marker for a single condition, but requires a di¡erential diagnosis at least between two conditions. The clinical signi¢cance of CMAMMA and its underlying defect remain to be elucidated.

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a-KETOISOCAPROIC ACID COMPROMISES CELLULAR RESPIRATION IN RAT BRAIN Amaral AU1, Leipnitz G1, Ribeiro CAJ1, Seminotti B1, Fernandes CG2, Beskow AP2, Knebel LA1, Zanatta A1, Grings M1, Dutra-F|lho CS1, Wajner M1 1 Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Universidade Luterana do Brasil, Canoas, Brazil Background: Maple syrup urine disease (MSUD) is a neurometabolic inherited disorder caused by the mitochondrial branched-chain a-keto acid dehydrogenase (BCKAD) complex de¢ciency. Leucine (LEU), aketoisocaproic acid (KIC) and a-hydroxyisovaleric acid (HIV) accumulate at the highest concentrations in MSUD. Considering that the neurotoxic mechanisms in MSUD are poorly known, the objective of the present work was to investigate the in vitro e¡ect of LEU, KIC and HIV on respiratory parameters measured by oxygen consumption in puri¢ed mitochondria prepared from brain of young rats. Methods: Mitochondrial preparations were obtained from brain of 30day-old rats and the respiratory parameters state III, state IV and the respiratory control ratio (RCR) were measured in the presence of 1.0^ 5.0 mM LEU, 1.0^5.0 mM KIC or 1.0^5.0 mM HIV by using the Clark electrode. Glutamate/malate (2.5 mM each), succinate (5.0 mM) and aketoglutarate (5 mM) were used as the respiratory substrates. Results: We observed that KIC, at 1.0^5.0 mM concentration, signi¢cantly increased state IV and decreased the RCR when the oxygen consumption was stimulated by glutamate/malate or succinate. State III was also decreased by KIC when a-ketoglutarate was the substrate. In addition, LEU and HIV did not change the tested respiratory parameters. Conclusions: The data indicate that KIC behaves as an uncoupler of oxidative phosphorylation and that cellular respiration is compromised by this metabolite. F|nancial support: Research grants from CNPq, PROPESQ/UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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GOOD OUTCOME OF PREGNANCY IN ISOVALERIC ACIDURIA Sykut-Cegielska J1, Kowalik A1, Gradowska W2, Kusmierska K2, Malinowska-Polubiec A3, Kornacka MK4 1 Dept Metab Dis, Endoc and Diab, CMHI, Warsaw, Poland, 2Dept Lab Diag, CMHI, Warsaw, Poland, 3Dept Obst, Warsaw Med Univ, Warsaw, Poland, 4Neonat Dept, Warsaw Med Univ, Warsaw, Poland Background: Isovaleric aciduria may cause signi¢cant complications during pregnancy and postpartum period. Methods: We report a pregnant 21-year-old woman with chronic form of isovaleric aciduria, history of recurrent vomiting (followed by ketoacidotic coma) until the age of 10 years, when she was diagnosed, already then with slight mental retardation. The low-protein diet and therapy with glycine and carnitine supplementation were introduced, but compliance was poor. No prenatal diagnostics was performed. Results: Since the gestational age of 16 hbd a strict follow-up with recommendation of daily protein content 0.8 g/kg, calorie intake above 2000 kcal and oral carnitine 3^4 g, glycine 9 g, and emergency therapy during perinatal period has been planned. Clinical and biochemical monitoring with detailed evaluation of diet compliance were undertaken at following periods: 16, 25, 28, 33, 38 hbd and 2 days, 6 weeks and 6 months after delivery. During pregnancy the patient remained clinically metabolically stable. Biochemical results revealed acceptable blood total protein and albumin level, constantly low free/total carnitine serum concentration, transient plasma glycine level increase and continuously excreted isovalerylglycine indicating compensation status. Among others biochemical abnormalities decreased serum calcium level was detected. Three days diet record showed in subsequent evaluations daily calorie intake 1690^2590 kcal and protein content 0.7^0.8 g/kg. A healthy boy was born at term after an uneventful labor and delivery. Conclusion: To our knowledge it is the third report of the pregnancy in isovaleric aciduria. Careful monitoring of pregnancy in such case shall allow for good maternal and newborn outcomes.

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ACUTE DECOMPENSATION OF ISOVALERIC ACIDEMIA INDUCED BY GRAVES' DISEASE Kimmoun A1, Abboud G2, Straczeck J1, Merten M1, Gueant JL1, Feillet F1 1 Ref Cent Inb Err Metab INSERM U724, Nancy, France, 2Int Care Unit, Hop Bel Air, Thionville, France We report the case of a 24 year old girl with isovaleric acidaemia who was admitted for vomiting, drowsiness, abdominal pain and weight loss. Two days after admission, her neurological state worsened (Glasgow coma score 11) with dyspnea and paroxysmal atrial ¢brillation. She was subsequently admitted in the intensive care unit. Blood analysis showed ketoacidosis (PH: 7.25, HCO3^: 5.8 mmol/L) without hyperglycaemia (5.2 mmol/L), hyperammonemia (100 mmol/L) and urine organic acids analysis showed huge excretion of isovaleric acid. The metabolic abnormalities normalised within 24 h with IV glucose, lipid and carnitine infusions. However 24 h later, she developed permanent severe atrial ¢brillation, confusional syndrome and visual hallucinations. A Graves' disease was con¢rmed by a suppressed TSH (50.005 mU/L), high LT3 (11.7 pmol/L), LT4 (43 pmol/L) and the presence of circulating TSH receptor antibody. After treatment with b blockers and neomercazole, clinical recovery was achieved within 15 days. The early recognition and treatment of hyperthyroidism allowed a rapid clinical and biological recovery. In our case, the metabolic episode is mainly due to hyperthyroidism. In IVA decompensation, in case of unusual evolution after a classic therapy, hyperthyroidism must be screened as well as other catabolic factors.

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IMPLICATION OF BIRTH COHORT, AGE AT ONSET, ENZYMATIC SUBGROUP AND COBALAMIN RESPONSIVENESS ON LONG-TERM OUTCOME IN ISOLATED METHYLMALONIC ACIDURIAS Ho«rster F1, Garbade SF1, Zwickler T1, Ayd|n HI2, Baumgartner MR3, Burlina AB4, Bodamer OA5, Das AM6, deKlerk JBC7, Dionisi-V|ci C8, Go«kcay G9, Gru«newald S10, Gu¡on N11, Maier EM12, Morava E13, Geb S14, Walter JH15, Wendel U16, W|jburg FA17, Lindner M1, Ko«lker S1 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Dept Metab, Child Hosp Hacettepe Univ, Ankara, Turkey, 3Metab Mol Ped Univ Child Hosp, Zurich, Switzerland, 4Div Metab Dis, Dept Ped, Univ Hosp, Padova, Italy, 5Dept Ped, AKH, V|enna, Austria, 6MHH, Pa«d II, Hannover, Germany, 7Dept Meatb Dis, Sophia Child Hosp, Rotterdam, Netherlands, 8Div Metab Dis, Hosp Ped Bambino Gesu, Rome, Italy, 9 Dept Nutr Metab, Med Fac Child Hosp, Istanbul, Turkey, 10Metab Unit Great Ormond Street Hospital, London, UK, 11Eduard Herriot Hosp, Lyon, France, 12Dr v Hauner Child Hosp, Munich, Germany, 13Radboud Univ Med Center, Nijmegen, Netherlands, 14Klinik Kinderheilk I, Univ Hosp, Frankfurt, Germany, 15W|llink Unit, Royal Child Hosp, Manchester, UK, 16Dept General Ped, Univ Child Hosp, Du«sseldorf, Germany, 17Dept Ped, Acad Med Center, Univ Hosp, Amsterdam, Netherlands Background: Isolated methylmalonic acidurias (MMA) are caused by de¢ciency of methylmalonyl-CoA mutase (mut0 or mut^ disease) or by defects in the synthesis of its cofactor 5'-deoxyadenosylcobalamin (cblA and cblB). The aim of this study was to evaluate the e¡ects of birth cohort, presymptomatic diagnosis, age at onset, cobalamin responsiveness and enzymatic subgroup on long-term outcome as de¢ned by survival, developmental delay, handicap and chronic renal failure (CRF). Methods: Standardised questionnaires were sent to 17 European metabolic centres asking for biochemical and clinical outcome parameters. Data were evaluated using complex statistical models: Cox regression and partitioning, accelerated failure time model, logistic regression and proportional odds model. Results: 244 patients were included. Neonatal onset of the disease was associated with high mortality, high incidence of developmental delay and severe handicap. Cobalamin non-responsive patients with neonatal onset who were born in the 70s and 80s had a particularly poor outcome. A more favourable outcome was found in patients with late onset of symptoms who were cobalamin responsive or were enzymatically classi¢ed as mut^ or cblA/B. Presymptomatic diagnosis was identi¢ed as possible protective factor concerning occurrence of handicap. Surprisingly,estimated cumulative distribution frequency of CRF did not show signi¢cant di¡erences between enzymatic subgroups. However, CRF did manifest earlier in mut0 patients. Conclusions: Outcome in MMA is related to enzymatic subgroup, cobalamin responsiveness, age at onset and birth cohort. It remains unfavourable in patients with neonatal disease onset who are cobalamin-nonresposive, particularly mut0 patients.

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MUTATION IDENTIFICATION FOR TAIWANESE PATIENTS WITH ISOLATED METHYLMALONIC ACIDEMIA Liu T-T1, Liu M-Y2, Chang Y-C1, Fang Y-L1, Chiang S-H3, Niu D-M4, Hsiao K-J3 1 Genome Res Center, Natl Yang-Ming Univ, Taipei, Taiwan, 2Inst of Genome Sci, Natl Yang-Ming Univ, Taipei, Taiwan, 3Dept Med Res and Edu, TPE Veter Gen Hosp, Taipei, Taiwan, 4Dept Pediatr, TPE Veterans General Hosp, Taipei, Taiwan Background: Isolated methylmalonic academia (MMA) is an autosomal recessive disorder of organic acid metabolism caused by dysfunction of methylmalonyl CoA mutase (MCM, E.C.5.4.99.2; gene symbol: MUT) which requires adenosylcobalamin (Adocbl) as a cofactor. Defects in the MCM apoenzyme (mut type) or Adocbl synthesis (cblA type) may cause isolated MMA. Methods: Mutations in the MUT, MMAA and MMAB genes were determined in ten unrelated Taiwanese mut type MMA and one consanguineous B12-responsive MMA by PCR-based sequencing analysis. Results: Eight mutations in the MUT gene, designated c.316A4C (p.T106P), c.682C4T (p.R228X), c.919T4C (p.F307L), c.1280G4A (G427D), c.1630__1631GG4TA (G544X), c.1741C4T (R581X), c.755dupA (p.H252Qfs*6), and c.1561^1G4A, were identi¢ed in ten unrelated mut type patients. None of 100 alleles for 50 unrelated normal individuals were found to have these novel alterations. These data indicated that these alterations identi¢ed in Taiwanese patients might be disease-causing mutations in mut type MMA. The allele frequency of c.1280G4A mutations was 45% (9/20) in Taiwanese mut type MMA. The c.1280G4A mutation was linked to a 190 bp allele of a microsatellite marker D6S269. The allele frequency of 190 bp allele in Taiwnese mut MMA with c.1280G4A mutation was statistically di¡erent from that in the normal population. One c.742C4T (p.Q248X) mutation in the MMAA gene was identi¢ed in a B-12 responsive MMA from a consanguineous family. No alteration was found in the MMAB gene for this patient and thus con¢rm as a cblA type MMA. Conclusions: The c.1280G4A mutation is a common mutation in Taiwanese mut type MMA and might have founder e¡ect.

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3-HYDROXY-3-METHYLGLUTARIC ACIDURIA IN PORTUGUESE PATIENTS: CLINICAL, NEURORADIOLOGICAL AND GENETIC CHARACTERIZATION. A NEURODEGENERATIVE DISORDER? Lea¬o Teles E1, Rodrigues E1, Castro S2, Ayres-Basto M2, Silveira F3, Guimara¬es J1, Magalha¬es A4, Cardoso ML5 1 Metab Dis Un, Ped Dept,Univ/Hosp St Joa¬o, Porto, 2Neuroradiology Dept, Univ/Hosp St Joa¬o, Porto, 3Neurophysiology Serv,Univ/Hosp St Joa¬o, Porto, 4Ophthalmology Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 5 Genet Med Cent Jacinto Magalha¬es, Porto, Portugal Background: 3-Hydroxy-3-methylglutaric aciduria (3HMG, OMIM 246450), is a rare autossomal recessive disorder caused by the de¢ciency of 3-hydroxy-3methylglutaryl-CoA lyase (HL), a¡ecting ¢nal step of leucine catabolic and ketogenic pathways. The HMGCL gene is located on chromosome 1p36.11. HL de¢ciency seems to be rare in Europe. Our experience suggests that is one of the most frequent organic aciduria in the Northern Portugal population. Aim: to present/discuss the evolution of a 3HMG population. Material: 10 patients, were identi¢ed in our unit, with variable clinical presentation; all had organic acid pro¢le characteristic of 3HMG and enzymatic/molecular studies con¢rming diagnosis; two had fast fatal outcome (one late onset/one newborn screening diagnosis); from the remaining, six have a continuous follow-up in our centre. The main symptoms of clinical/ biochemical presentation were the usual. In all patients the same novel nonsense E37X mutation in exon 2 was found. During the follow-up (2^17 years) the patients had an apparent good control with usual approach without metabolic decompensations; all of them developed macrocephaly and in three patients insidious neurological deterioration was registered, with pyramidal/ extrapiramidal signs. The cerebral imaging showed progressive involvement along the years; all patients disclosed di¡use slightly hiperintense fronto-parietal white matter in T2W1 and DW1, sparing U ¢bers, with alterations in MRS, suggesting neuronal loss. Neurophysiological studies registered also alterations. Conclusions: We emphasise a neurodegenerative aspects of HL de¢ciency that is suggested by the clinical follow-up and the evolution of the neurological studies in our patients. Larger population and accurate interpretation is needed.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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QUANTIFICATION OF 3-HYDROXYISOVALERYLCARNITINE BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY IN TWO PATIENTS WITH HOLOCARBOXYLASE SYNTHETASE DEFICIENCY Nakajima Y1, Ito T1, Yokoi K1, Ohmi H2, Nagao M2, Maeda Y2, Kurono Y2, Sugiyama N3, Togari H1 1 Dept Pediatr, Nagoya City Univ Med, Nagoya, Japan, 2Lab Hosp Pharm, Nagoya City Univ Pharm, Nagoya, Japan, 3Dept Pediatr, AichiGakuin Univ, Pharm, Nagoya, Japan Background: 3-Hydroxyisovalerylcarnitine (C5OH-I) is an important metabolites for diagnosis of organic acidemias such as holocarboxylase synthetase de¢ciency (HCSD) 3-methylcrotonyl-CoA dehydrogenase de¢ciency. The quanti¢cation of C5OH-I was di¤cult because the authentic compound was not easily available. We have reported a HPLC-MS/MS method for separation and quanti¢cation of acylcarnitines and its isomers. Here we applied this method to two siblings with MCD using newly synthesized standards of C5OH-I, 3metylcrotonylcarnitine (C5:1-M) and tiglylcarnitine (C5:1-T). Case 1: A 5 year old girl who was genetically diagnosed as HCSD and has been treated with biotin and carnitine. Serum and urine samples were obtained. Case 2: A 1-year-old sister had received prenatal biotin therapy, however metabolic acidosis was observed after birth. She was also diagnosed as HCSD and has been similarly treated. Her cord blood and serum samples before and during therapy were obtained. We analyzed acylcarnitine pro¢les of these samples by HPLC-MS/MS. Result: C5OH-I and C3 concentrations in serum and urine of both patients were much higher than that of controls. Low concentration of C5:1-M appeared in urine but not in serum. In case 2, concentrations of C5OH-I and C3 in cord blood were higher than in controls. At 2 h after birth, higher serum levels of these acylcarnitines were also observed. Although C3 slightly decreased after administration of biotin and carnitine, C5OH-I increased. Conclusion: We developed the determination method of C5OH-I and C5:1 by HPLC-MS/MS. The correct quanti¢cation of acylcarnitine was useful for futher understanding these two patients clinical condition.

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METHYLMALONIC ACIDURIA: MOLECULAR ANALYSIS OF THE MUT AND MMACHC GENES IN 47 ITALIAN PATIENTS Cavicchi C1, Donati MA1, Pasquini E1, Parini R2, Furlan F2, Sibilio M3, Parenti G3, Dionisi-V|ci C4, Bartuli A4, Papadia F5, Zammarchi E6, Guerrini R1, Morrone A1 1 Metabolic Unit, Meyer Children's Hospital, Florence, Italy, 2S Gerardo Hospital, Monza, Italy, 3Dept Pediatrics, University of Naples, Naples, Italy, 4Metabolic Unit, Bambino Gesu© Hospital, Rome, Italy, 5Metabolic Unit, Giovanni XXIII Hospital, Bari, Italy, 6Dept Pediatrics, Florence University, Florence, Italy Background: Mutations in MUT and MMACHC genes, lead to isolated methylmalonic aciduria (MMA) mut type and combined MMA and homocystinuria cblC type, respectively. Methods: Molecular analyses of the MUT and MMACHC genes were performed in 47 unrelated Italian MMA patients, 21 a¡ected by isolated MMA and 26 by combined MMA and homocystinuria. Results: F|broblast studies, carried out in 17 cases, identi¢ed 9 mut0, 2 mut^ and 6 cblC patients. In the remaining cases, diagnosis was con¢rmed by molecular analysis. Four novel (p.Gln30X, p.Tyr110X, c.1092__1114del23, p. Leu358Pro) and 20 known mutations were identi¢ed in the MUT gene. The novel mutations were detected in severe mut patients; two of whom died during an acute metabolic crisis. The known p.Gly215Ser and p.Asn219Tyr were the most frequent mut0 identi¢ed mutations. In the cblC cohort, one new (c.626^627insT) and 12 known mutations were found. The c.626^ 627insT was detected at heterozygous state with the p.Arg73X in a patient who suddenly died at 1 month of life. The known c.271^272dupA represented the most common MMACHC allele (52% total) followed by p. Met1Ile (11%) and both correlated with early onset. Molecular prenatal diagnosis, performed in 3 unrelated at risk pregnancies, identi¢ed one mut heterozygous fetus and two a¡ected (one mut and one cblC) foetuses. Conclusions: This study contributes to existing knowledge about the spectrum of mutations leading to MMA and corroborates the use of molecular analysis as a reliable and rapid tool for con¢rming proband's diagnosis, especially when patients' ¢broblasts are unavailable, for identifying carrier status and performing prenatal diagnosis.

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Background: A 2nd tier blood-spot test for methylmalonic acid (MMA) was added as a speci¢c diagnostic marker for a group of inherited disorders collectively known as methylmalonic acidemias. Methods: A non-derivatised blood-spot LC-MS/MS MMA method using a 56100 mm Phenomenex C6-phenyl column with acetonitrile: water:formic acid at a £ow rate of 150 ml/min directly into an API4000 MS/MS (MDS-SCIEX) operated in negative ion mode. MMA was eluted from a 3 mm blood-spot and determined against a (d3)-MMA using the MRM pairs of 117.1/73.1 and 120.1/76.1 in a 5 min LC run. Results: In over 154 000 blood-spots tested 207 (0.13%) required a repeat sample due to an elevated C3-carnitine in addition to the ratios C3/C2, C3/C16 and C3/methionine. Of the 207, 12 babies with an elevated C3 were recalled for a plasma and urine MMA and B12 determination at an average infant age of 21 days. Of these 9 had an elevated MMA and shown to be signi¢cantly B12 de¢cient. Retrospective analysis of the 207 blood-spot samples using the bloodspot MS/MS MMA method showed an elevated MMA (41.0 mmol/L whole blood) in all 9 con¢rmed B12 de¢cient cases. Since introducing the MS/MS MMA blood-spot method we have identi¢ed a mother, as a result of an elevated blood-spot C3 and MMA in her baby, with pernicious anaemia due to antibodies against intrinsic factor. Conclusion: The incorporation of a 2nd tier MS/MS MMA blood-spot method into a routine newborn screening programme has had a signi¢cant impact on reducing the false positive rate associated with the measurement of C3-carnitine.

Introduction: Methylmalonic acidurias (MMA) are autosomal recessive metabolic disorder due to the de¢ciency of methylmalonyl-CoA mutase activity, a vitamin B12-depent enzyme. Mutations in the gene of MMAB protein (cobalamin adenosyltransferase), co-factor of the methylmalonyl-CoA mutase, are responsible for the cblB complementation group of B12-dependent MMA. Case report: A 3 year-old male patient was admitted to our emergency department for recurrent vomiting, poor growth, progressive muscular hypotonia and prostration (withouth lethargy or coma episodes), starting at nine months of age. F|rst level metabolic analyses showed metabolic acidosis (EGA: pH 7.27, HCO3 11.5 mmol/L, BE-13.7 mmol/L), hyperammoniemia (93 mmol/L) and high plasma levels of lactate (3.8 mmol/L). Acylcarnitine pro¢le in blood showed a reduction of total carnitine, with an increased of propionil-carnitine. Organic acid analisys in urine revealed high levels of methylmalonic acid (3976 mM/ M Creat/ur), 3-hydroxypropionate and methylcitrate. Enzymatic studies on cultured ¢broblasts indicated a defect of AdoCbl synthesis, without increase of propionate incorporation after addition of B12 to culture. F|nally, molecular analysis of MMAB gene revealed homozygosity for the R190H mutation, resulting in the diagnosis of cblB de¢cient MMA. Therapy was based on low-protein high-energy diet, carnitine and bicarbonate supplementation. Only 40 percent of cblB patients are reported to respond to Cbl supplements. Conclusions: This report highlights the importance of routine ¢rst level metabolic analysis and organic acids evaluated in urines of children with recurrent vomiting and poor growth, since organic acidemias should be considered in these patients.

THE IMPACT OF A 2ND TIER BLOOD-SPOT METHYLMALONIC ACID (MMA) USING TANDEM MASS SPECTROMETRY (MS/MS) ON ROUTINE NEWBORN SCREENING Ranieri E, Gerace R, Bartlett B, Harrison JR, Fletcher JM Dept of Genetic Medicine, W and C Hospital, Adelaide, Australia

A CASE OF METHYLMALONIC ACIDURIA TYPE B: BIOCHEMICAL AND GENETIC DIAGNOSIS Riva E, Casero D, Minghetti D, F|ori L, Salvatici E, Agostoni C Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy

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BIOCHEMICAL VARIABILITY OF FINDINGS IN PATIENTS WITH GLUTARYL-COA DEHYDROGENASE DEFICIENCY Varholakova L, Chrastina P, Kostalova E, Stastna S Inst Inh Metab Dis, General Faculty Hosp, Prague, Czech Republic Background: To point out potential di¤culties in di¡erential diagnosis of glutaric aciduria type I (GA I), we present inter- and intra-individual biochemical variability of laboratory ¢ndings in 5 patients with GA I monitored in our department since 2001. Results: Diagnostic di¤culty in infancy is highlighted by the fact that glutaric aciduria may be absent, even at times of acute neurologic decompensation. Some patiens are identi¢ed by the presence of 3hydroxyglutaric acid rather than glutaric acid in the urine. The presence of glutarylcarnitine in blood spot and increased ratio of acylcarnitines to free carnitine in plasma and urine may be diagnostic. Thus when clinical suspicion of this diagnosis is strong and the quanti¢cation of acylcarnitines in blood fails to reveal the diagnosis, the enzyme assay of glutaryl-CoA dehydrogenase and/or mutation analysis in GCDH gene must be undertaken. The methods used for di¡erential diagnosis of GA I include: GC/MS analysis of urinary and serum organic acids, tandem mass spectrometric analysis of acylcarnitines in dried blood spot and determination of conjugated and free carnitines in plasma and urine. Molecular-genetic investigation is available as well. Conclusion: Di¡erential diagnosis of GA I might be complicated since de¢ciency of glutaryl-CoA dehydrogenase is associated with great biochemical variability of biochemical ¢ndings. Thus repeated and multiple urine and blood samples may be necessary to conclude the diagnosis of GA I. De¢nitive diagnosis requires direct enzyme assay or molecular-genetic analysis. The work was supported by the project VZ 64165 of Ministry of Health of Czech Republic.

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GLUTARIC ACIDURIA TYPE 1: SYNTHESIS, ENZYMATIC ACTIVITY, STABILITY, AND OLIGOMERISATION OF MUTANT GLUTARYL-CoA DEHYDROGENASE Mu«hlhausen C1, Keyser B1, Dickmanns A2, Christensen E3, Ullrich K1, Braulke T1 1 Dept Pediatr, Univ Med Center, Hamburg, Germany, 2Dept Mol Struct Biol, Univ Go«ttingen, Go«ttingen, Germany, 3Dept Clin Genet, Rigshospitalet, Copenhagen, Denmark Background: Glutaric aciduria type 1 is caused by mutations in the glutarylCoA dehydrogenase gene (GCDH), leading to accumulation of glutaric and 3-hydroxyglutaric acids. Considerable variation in clinical phenotype is observed. We report on expression studies of the four mutations p. Arg138Gly, p.Met263Val, p.Arg402Trp, and p.Glu414Lys identi¢ed in GA1 patients. Methods: BHK cells were transfected with wildtype and mutant GCDH, and cell extracts used for further analyses. Activity and expression were examined by GCDH activity assay and Western blotting. Metabolic labelling experiments were used to analyze synthesis and stability of mutant proteins, the capability to form oligomeric complexes was tested by chemical cross-linkage. E¡ects of mutations on GCDH structure were evaluated by comparative 3D in silico modelling analyses. Results: Expression of mutant GCDH in BHK cells revealed that all mutants were enzymatically inactive except p.Met263Val which showed 10% activity of wildtype enzyme. Western blot analyses demonstrated that the amount of expressed p.Arg402Trp protein was signi¢cantly reduced compared with wildtype. Pulse-chase experiments and double-label immuno£uorescence-microscopy showed that rapid intramitochondrial degradation rather than decreased synthesis is responsible for low steady state levels of mutant protein. The formation of tetrameric GCDH was strongly impaired in p.Met263Val and p.Arg402Trp mutants. Additionally, GCDH appears to interact with three distinct polypeptides, and comparative 3D in silico modelling suggests that Met263 at the surface of GCDH might be part of the contact interface. Conclusion: These data indicate that both intramitochondrial proteolysis and loss of GCDH interaction with mitochondrial matrix proteins may a¡ect the disease course.

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RENAL TRANSPLANT IN METHYLMALONIC ACIDEMIA: A THERAPEUTIC OPTION WITH OR WITHOUT RENAL FAILURE Valayannopoulos V1, Rabier D2, Guest G3, Aigrain Y4, Benoist JF5, Niaudet P3, de Lonlay P1 1 Metab Unit, Necker-Enfants Malades Hosp, Paris, France, 2Bioch Lab, Necker-Enfants Malades Hosp, Paris, France, 3Ped Nephrology, NeckerEnfants Malades Hosp, Paris, France, 4Ped Surgery, Necker-Enfants Malades Hosp, Paris, France, 5Bioch Lab, Robert-Debreè Hosp, Paris, France Background: In methylmalonic acidemia (MMA), liver, kidney, or combined liver and kidney transplantation is a possible therapeutic option. Case Report: We report on a 5-year-old boy with MMA. The ¢rst symptoms occurred at 4 days of life, namely hyperammoniemic coma and ketoacidosis. After medical treatment his clinical course was uneventful until the 2nd year of life when he displayed several episodes of metabolic decompensation with vomiting and failure to thrive that required continuous enteral feeding. His neurological condition deteriorated (tremor, developmental arrest) while MMA levels remained high. We decided to propose renal transplantation for this patient despite normal renal function because liver transplantation was considered at high risk because of his impaired metabolic condition. Results: The procedure was uneventful and MMA levels in blood and urine fell rapidly to very low levels. Ten months after the transplant the patient is at home, started oral feeding and has normal somatic and cognitive developments and moderate protein restriction. No further metabolic decompensation occurred, except from a single and short episode of acute encephalopathy with low urine and plasma MMA levels, neither hyperammoniemia nor ketoacidosis, but elevated MMA levels in CSF. Conclusion: Although liver is the major site of methylmalonyl-CoA mutase activity, this case and similar ones suggest that the smaller mutase activity present in the transplanted kidney may be su¤cient to ensure partial correction of the metabolic defect. However organ transplantation does not prevent neurological complications where the blood^brain barrier by trapping toxic metabolites may play a major role.

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LIVER HEPATOBLASTOMA AND MULTIPLE OXPHOS DEFICIENCY IN THE FOLLOW-UP OF A PATIENT WITH METHYLMALONIC ACIDURIA Cosson MA1, Touati G1, Lacaille F2, Valayannnopoulos V1, Guyot C3, Guest G4, Verkarre V5, Chreètien D6, Rabier D7, Munnich A6, Benoist JF8, de Keyzer Y6, Niaudet P4, de Lonlay P1 1 Metab unit, Necker-Enfants Malades Hosp, Paris, France, 2Hepatology, Necker-Enfants Malades Hosp, Paris, France, 3Nephrology, Me©re-Enfant Hosp, Nantes, France, 4Nephrology,Necker-Enfants Malades Hosp, Paris, France, 5Pathology, Necker-Enfants Malades Hosp,Paris, France, 6 INSERM-U781, Necker-Enfants Malades Hosp, Paris, France, 7 Biochem unit, Necker-Enfants Malades Hosp, Paris, France, 8 Biochemistry unit, Robert-Debreè Hosp, Paris, France A 10 day old boy was diagnosed with methylmalonic aciduria (MMA). He presented persistent hepatomegaly from age 4 with increased circulating hepatic enzyme levels, Leigh syndrome and renal failure requiring kidney transplantation. A massive hepatoblastoma led to death by the age of 11 years. MCM activity was undetectable on both cultured skin ¢broblasts and kidney biopsy and multiple respiratory chain de¢ciency was demonstrated in the kidney. Mitochondrial dysfunction and/or post-transplant immunosuppressive therapy should be regarded as a possible cause of liver cancer in MMA.

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RENAL FUNCTION IN TWENTY SEVEN CHILDREN WITH METHYLMALONIC ACIDEMIA (MMA): IS THERE A GOOD MARKER? Ayd|n HI1, Duzova A2, Kalkanoglu Sivri HS3, Dursun A3, Aksoy T4, Kiratli P4, Tokatli A3, Bakkaloglu A2, Coskun T3, Fowler B5 1 Div Metab Dis, Dept Ped, Gulhane MMF, Ankara, Turkey, 2Div Ped Neph, Dept Ped, Hacettepe Univ, Ankara, Turkey, 3Div Metab Dis, Dept Ped, Hacettepe Univ, Ankara, Turkey, 4Dept Nuclear Medicine, Hacettepe Univ, Ankara, Turkey, 5University Children's Hospital, Basel, Switzerland Background: Unlike other organic acidemias, renal disturbance and chronic renal failure is a prominent feature in MMA. Aim: To evaluate renal function and to determine urinary N-acetyl-beta-Dglucosaminidase (NAG) activity, serum cystatin C (Cys-C) and beta-2microglobulin (B2M) levels in children with MMA. Patients/Methods: 27 patients with isolated MMA (14 M, 13 F; mean age 6.9 years; 14 mut 0 , 1 mut ^ , 5 CblA/B, 7 unknown) were studied, phenylketonuria (PKU) and propionic acidemia (PA) patients served as controls. Tubular phosphate reabsorption (TPR), NAG/Cre, albumin/Cre ratio (ACR), serum Cys-C and B2M levels were measured under good metabolic control. GFR was estimated based on the Schwartz formula and creatinine clearance measured by collecting 24-h urine. Renal DMSA scintigraphy was performed. Results: Compared to PKU and PA patients serum Cys-C, B2M and urinary NAG/Cre and ACR, but not serum creatinine, were higher (p50.05) and TPR was lower (p50.05) in MMA patients. Age, malnutrition, mild elevation of transaminases or anemia had no e¡ect on Cys-C and B2M. DMSA revealed decreased uptake in 9 out of 18 patients. GFR was below 90 ml/min/L. 73 m2 in 5 patients (19.3%) with MMA. The correlations between NAG/Cre and TPR (r: ^494) and ACR (r: 0.472) were signi¢cant (p50.05). The correlation between di¡erent markers and GFR was highest with serum B2M (r: ^0.687, p50.001). Conclusions: Renal scintigraphy, serum B2M, Cys-C, urinary NAG/Cre ratio, ACR may be valuable tools in the follow up of renal functions in MMA. Longitudinal studies are required to ¢nd out best marker(s) in detecting renal problems in patients with MMA.

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X-INACTIVATION STUDIES THROUGH cDNA ANALYSES IN TWO FEMALES WITH 2-METHYL-3-HYDROXYBUTYRYLCoA DEHYDROGENASE (MHBD) DEFICIENCY Garc|èa-V|lloria J1, Gort L1, Fons C2, Briones P1, Campistol J2, Ribes A1 1 Dept Biochem Mol Genet, Hospital Cl|ènic, Barcelona, Spain, 2Serv Neurology, Hosp Sant Joan de Deèu, Barcelona, Spain Background: MHBD is a mitochondrial enzyme involved in the degradation pathway of isoleucine. MHBD de¢ciency (MIM 300256) is an X-linked defect characterised by progressive neurodegeneration in males. Only two a¡ected females have been described, both with psychomotor and speech delay. The gene that encodes MHBD is HADH2, located in Xp11.2, and has been reported as one of the few genes that escapes X-inactivation. Objective: Molecular studies trying to explain the di¡erent clinical presentation in two females heterozygous for a mutation in HADH2. Methods: cDNA was obtained, ampli¢ed and sequenced by usual methods. Mutated and non-mutated alleles were quanti¢ed by realtime PCR. Results: We present two females with di¡erent clinical picture. One of them carried mutation p.N247S and presented with a severe form of the disease. Like her brother, MHBD activity in ¢broblasts showed a severe de¢ciency (20% of controls), and only the mutated cDNA was detected in both siblings. The other carrier female, is the mother of a hemizygous patient with mutation p.P210S, and only was borderline mentally retarded. MHBD activity was within normal range, and molecular studies demonstrated the presence of both, mutated and non-mutated, cDNAs in her cells, while her son showed a low MHBD activity and only the mutated cDNA was detected. Conclusions: Our results indicate that MHBD gene does not totally escape X-lyonization, as previously reported, and the clinical presentation of the carrier females is directly related to the amount of mutated MHBD mRNA in their tissues.

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SYMPTOMATIC PATIENT WITH PARTIAL BIOTINIDASE DEFICIENCY: EFFECT OF BIOTIN TREATMENT Kostalova E1, Chrastina P1, Paulova M1, Varholakova L1, Stastna S1, McKay F2 1 Inst Inher Metab Dis, Gen Fac Hosp, Prague, Czech Republic, 2Mol Genet, Ninewells Hosp and Med School, Dundee, UK

FETAL LIVER CELL TRANSPLANTATION FOR METHYLMALONIC ACIDURIA USING MICE WITH AN INTERMEDIATE PHENOTYPE Peters H1, Buck N2, Pennell S2, Wood L2, Pitt J1, Allen K2 1 Metabolic unit, MCRI, Parkville, Australia, 2Cell and Gene Therapy Group, MCRI, Parkville, Australia

Background: Biotinidase (BTD) de¢ciency (BTDD, MIM 253260) is an autosomal recessive inherited metabolic disorder of cleaving and recycling biotin. Patients with profound BTDD have 510% of mean normal serum BTD activity while patients with partial BTDD have 10^ 30% of mean normal serum BTD activity. BTDD can be prevented or e¡ectively treated with biotin. Recommendations for treatment of partial BTDD are not uniform. We present the patient with partial BTDD who is treated with biotin. Clinical picture: The patient was diagnosed at the age of 18 years. Clinical features were predominantly ophtalmological and neurological (loss of vision, oculomotor apraxia, ataxia, hemiparesis, mental delay, seizures). W|thin one year of biotin treatment neurological ¢ndings improved more than ophtalmological. Ataxia and hemiparesis resolved. Seizures were sporadic and well controlled. W|thin next ¢ve years of treatment patient's clinical features including IQ remain static. Methods/Results: Serum BTD activity was constantly decreased (0.6^ 1.8 nmol/min/ml, normal activity 45.4 nM/min/ml). Organic aciduria has never been found. Sequencing of DNA showed compound heterozygosity for the mutation for profound de¢ciency (c.643C4T) and for mutation c.1330G4C. The second one results in 50% loss of normal enzyme activity. These mutations in combination are a cause of partial BTDD. Conclusions: (1) The patient with partial BTDD exhibited strong clinical symptoms. (2) Biotin treatment led to the improving of his clinical features. (3) Some symptoms remain irreversible. (4) Our case supports recommendations for biotin treatment in partial BTDD with the same genotype. Supported by the project VZ 64165/2 of Ministry of Health of Czech Republic

We aim to investigate the use of fetal liver cells for the treatment of methylmalonic aciduria (MMA) using a mouse model with an intermediate phenotype of MMA. Fetal liver cells (embryonic day 16^ 18) from congenic enhanced green £uorescent-tagged mice were transplanted into a MMA mouse model and then culled 4 weeks later. Donor cell engraftment was determined using quantitative real time PCR and £ow cytometric analysis. Disease correction in mice was assessed based on urine and plasma MMA concentration and mouse weight change. Initial studies indicated that optimal conditions for fetal liver cell transplantation included pre-transplantation sublethal irradiation followed by transplantation with 5 million cells. Fetal liver cells delivered via the tail vein were able to engraft and repopulate liver, spleen and BM of wildtype and MMA mice at 4 weeks posttransplantation. We anticipated an equivalent level of donor cell engraftment between wildtype and MMA mice in the absence of selective advantages based on underlying pathological damage. Surprisingly, the MMA mouse model demonstrated reduced bone marrow and liver engraftment. Disease correction was not e¡ected within the study time frame. Higher levels of engraftment than those seen in this study or allowing longer experiment times may be required for correction of disease. The potentially inhibitory e¡ect of elevated organic acids on bone marrow and liver engraftment warrants further investigation. In conclusion, liver engraftment of a mouse with mild metabolic liver disease transplanted with donor fetal liver cells is possible; however the methods need to be modi¢ed to a¡ect the disease condition.

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BIOTINIDASE DEFICIENCY: THE RESULTS OF LOCAL NEWBORN SCREENING STUDY PIONEERED NATIONWIDE SCREENING Ozer I1, Baykal T1, Gokcay G1, Kose R2, Celik S1, Demirkol M1 1 Div Nutr Metab, Ist Med Fac, Child Hosp, Istanbul, Turkey, 2Ministry of Health, Ankara, Turkey

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A NOVEL SINGLE-BASE SUBSTITUTION (C. 1124A4G) THAT ACTIVATES A 5-BASE UPSTREAM CRYPTIC SPLICE DONOR SITE WITHIN EXON 11 IN THE HUMAN MITOCHONDRIAL ACETOACETYL-CoA THIOLASE GENE Fukao T1, Boneh A2, Kondo N1 1 Dept Pediatr, Grad Sch of Med, Gifu Univ, Gifu, Japan, 2Murdoch Inst, Royal Child Hosp, Melbourne, Australia

Background: Early diagnosis and treatment of biotinidase de¢ciency (BD) is life saving. In Istanbul, BD screening has been included in the `Newborn Screening Program' since 1991. The worldwide incidence of BD is 1/100 000. In Istanbul, the incidence is 1/11 763 in 1 170 167 newborns. We compared the outcome of patients diagnosed at newborn screening (NS) to late diagnosed (LD) patients to test the bene¢t of early diagnosis. Methods: Patients with profound BD (serum biotinidase activity 510% of mean normal activity) diagnosed at NS (n = 69) and selective screening (n = 28) in Istanbul, since 1991 were studied. The ¢ndings of the two groups were compared. Results: The mean age (+SD) at diagnosis was 1.06+0.9 months in the NS group, 49.6+143.7 months in LD patients. The mean biotinidase activities (+SD) in NS and LD patients were 0.27+0.23 and 0.28+0.24 nm/min/ml (normal: 4.2^8.4), respectively. The ¢ndings at diagnosis in the NS group were mild dermatological ¢ndings (n = 30) and convulsions (n = 1). During follow up period (7.8+3.2 years) normal developmental milestones were attained in all patients, except one. LD patients had dermatological (n = 13), neurological ¢ndings (n = 19), visual (n = 3) and hearing (n = 1) impairment at diagnosis. In the follow up period (7.5+3.9 years) dermatological ¢ndings recovered but neurologic, visual and hearing disturbances persisted. Conclusions: The high incidence of BD and favourable outcome with early treatment necessitates NS. Our results were e¡ective in in£uencing the Ministry of Health to include BD screening in `Newborn Screening Program' in 2009, in Turkey.

Background: Most mutations related to aberrant splicing occur in conserved splice acceptor and donor sites. Some exonic mutations also a¡ect splicing. We have been analyzing molecular basis of mitochondrial acetoacetyl-CoA thiolase (T2) de¢ciency. Case and Methods: GK43 is an Australian T2 de¢cient patient. We searched GK43 mutations at genomic level followed by at the cDNA level. Mini gene splicing experiment was done to con¢rm that c.1124A4G was responsible for aberrant splicing. Results: GK43 is a homozygote of c.1124A4G, which activates a cryptic splice donor site 5 bases upstream from c.1124A4G within exon 11, causing aberrant splicing in most transcripts. The aberrant splicing results in c.1120^1163 (44-base) deletion, causing a frameshift in T2 mRNA. This cryptic splice site has a Shapiro and Senapathy score (70.0) in a normal sequence but if mutated, the score (84.3) becomes higher than the one in the authentic splice donor site of intron 11 (81.4). A mini-gene splicing experiment con¢rmed that the c.1124A4G substitution was responsible for this aberrant splicing. A small amount of transcripts were revealed to use the authentic splice donor site of intron 11 instead of the activated cryptic splice site. In the latter case, the mutation results in N375S missense mutation. Transient expression analysis of N375S mutant cDNA revealed that N375S mutant does not retain any residual activity. Conclusion: This is an example in which a point mutation activates a cryptic splice donor site motif that is used preferentially over a downstream authentic splice site.

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LONG-TERM OUTCOME OF METHYLMALONIC ACIDURIA: A SERIES OF 30 FRENCH PATIENTS Cosson MA1, Benoist JF2, Touati G1, Deschaux M3, Boddaert N4, de Keyzer Y5, Rabier D6, Niaudet P3, Valayannopoulos V1, de Lonlay P1 1 Metab unit, Necker-Enfants Malades Hosp, Paris, France, 2 Biochemistry Unit, Robert-Debreè Hosp, Paris, France, 3Nephrology, Necker-Enfants Malades Hosp, Paris, France, 4Radiology, NeckerEnfants Malades Hosp, Paris, France, 5INSERM-U781, Necker-Enfants Malades Hosp, Paris, France, 6Biochem Unit, Necker-Enfants Malades Hosp, Paris, France In order to detail the natural history of patients with methylmalonic aciduria (MMA). Methods: 30/41 MMA patients with a homogeneous management and surviving more than 18 mths were included. Two subgroups were delineated, mut^ defect and mut0 defect. Results: The median follow-up period was 9.7 years. 5/30 died (7^11 years) during metabolic decompensation. 15/30 had a neonatal onset, and more metabolic decompensations than late forms (p = 0.32). 43% of patients had signi¢cant neurological disease, without correlation with number of decompensations, urinary MMA excretion levels, initial coma if present and plasma ammonium level at diagnosis. Chronic renal failure (CRF) occurred in 46.7%, with a median age of onset at 6.8 years (1.5^18.6). The follow-up of CRF was stable for 9 patients after a mean period of 5.6 years. Two patients reached the step of end-stage renal failure. 14/22 patients were classi¢ed as mut0 and 8 as mut^, with mutations in MUT gene in all mut0 and in 2 mut^ patients. Mortality was higher in mut0 patients (3/14) than in mut^ patients (0), as well as the number of acute decompensations (p = 0.04), levels of urinary MMA excretion (p = 0.002), neurological disease (p = 0.017). The mean age of survey was 6.6 years in mut0 and 12.6 years in mut^ patients (p = 0.01). Concerning the number of patients with CRF, no signi¢cative di¡erence was found. However, the onset of CRF was earlier in mut0 patients, and CRF was more severe. In conclusion, our cohort study shows the importance of the mut0 or mut^ phenotype as a predictor of the outcome in MMA patients.

NEUROPATHOLOGY OF SUCCINIC SEMIALDEHYDE DEHYDROGENASE (SSADH) DEFICIENCY (GAMMAHYDROXYBUTYRIC ACIDURIA) Knerr I1, Gibson KM2, Murdoch G2, Salomons GS3, Pope L3, Jakobs C3, Catanese GA4, Pearl PL5 1 Children's and Adolescents' Univ Hosp, Erlangen, Germany, 2Biochem Genet Lab, Univ Child Hosp, Pittsburgh PA, USA, 3Metab Unit, VU University Medical Center, Amsterdam, Netherlands, 4Nassau County Medical Examiner O¤ce, Nassau County NY, USA, 5Dept of Neurology, CNMC, GW Univ, Washington DC, USA Background: Succinic semialdehyde dehydrogenase (SSADH) de¢ciency is a rare disorder of gamma-aminobutyric acid (GABA) metabolism. Its biochemical hallmark is the accumulation of gamma-hydroxybutyric acid (GHB) in £uids and tissues. The diagnosis can be detected via urinary organic acid analysis for GHB excretion and con¢rmed via enzymatic or mutation testing. Patients present with psychomotor retardation, hypotonia, ataxia, convulsions and other neuropsychiatric symptoms. Post-mortem diagnosis and neuropathological ¢ndings have not yet been reported. Objective: A woman born to consanguineous Greek parents experienced a nocturnal death at age 19 years, six years following onset of generalized tonic-clonic seizures with recent acceleration in convulsive activity. Postmortem neuropathology revealed striking discoloration of the globus pallidi, leptomeningeal congestion, and gliosis in frontal cortex. Conversely, there were no systemic abnormalities. Her younger sister presented later with mental retardation and seizures, and was diagnosed with SSADH de¢ciency based on clinical and urinary organic acid ¢ndings. Molecular analysis identi¢ed the pathogenic mutation p.Gly409Asp in the Aldh5a1 gene. Therefore, SSADH de¢ciency was suspected in the decedent and molecular analysis performed. Conclusions: This is the ¢rst case report on post-mortem suspected SSADH de¢ciency. It highlights (1) the discoloration of the globi pallidi which may occur in SSADH de¢ciency (consistent with the most common magnetic resonance imaging ¢nding of abnormal pallidal signal), (2) the value of evaluating for metabolic diseases such as organic acidurias in patients with sudden unexpected death in epilepsy (SUDEP).

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CHANGES IN PLASMA AMINO ACID CONCENTRATIONS WITH INCREASING AGE IN PATIENTS WITH PROPIONIC ACIDEMIA Scholl-Bu«rgi S1, Sass JO2, Heinz-Erian P1, Amann E1, Haberlandt E1, Albrecht U1, Ertl C1, Baumgartner Sigl S1, Lagler F1, Rostasy K1, Karall D1 1 Innsbruck Medical University, Innsbruck, Austria, 2Clin Biochem Metab, Univ Child Hosp, Freiburg, Germany Introduction: Propionic acidemia (PA) is an inherited disorder of propionyl-CoA degradation caused by de¢ciency of propionyl-CoA carboxylase. Accumulating concentrations of propionyl-CoA and its metabolites may have an in£uence on amino acid concentrations through inhibition of enzymes catalyzing amino acid degradation. The objective of this study was to analyze plasma amino acid concentrations in PA to reveal possible correlations between propionyl-CoA carboxylase de¢ciency and distinct amino acid changes. Methods: Plasma concentrations of 19 amino acids were measured in 240 random samples from eleven patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sample collection between Jan 2001 Dec 2007). Results: Decreased plasma concentrations were observed for glutamine, histidine, threonine, isoleucine, leucine, valine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated. Serine, asparagine, tyrosine, ornithine and glutamate levels were normal. For methionine, proline and lysine a clear association was not possible. Signi¢cant correlations (Pearson) with age were found for 14 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine and arginine; negative correlation: leucine, tyrosine, phenylalanine, ornithine, glutamate and aspartate). Summary: This study gives new insights into long-term changes in plasma amino acid concentrations. The decreased levels of several amino acids and may provide options for future therapies in PA patients.

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TREATMENT OF HYPERAMMONIAEMIA WITH NCARBAMYLGLUTAMATE IN AN ADULT PATIENT WITH PROPIONIC ACIDURIA Moraèis A1, Lama RA1, Mano A de la1, Peèrez-Cerdaè C2, Garc|èa MJ2 1 U de Nutr y Enf Metab, H Univ Inf La Paz, Madrid, Spain, 2Centro Diag Enf Molec, U Autonoma Madrid, Madrid, Spain Introduction: Treatment with N-carbamylglutamate (NCG) has been recently proposed for decreasing ammonia (NH4) levels in patients with propionic aciduria (PA), an inborn disorder of branched-chain aminoacid metabolism, in which accumulation of propionyl-CoA inhibits N-acetylglutamate synthetase activity. The consequent lack of N-acetylglutamate-mediated activation of carbamylphosphate synthetase causes hyperammoniaemia during metabolic crises. Case report: A 17-year-old boy with neonatal-on-set PA presented moderate hyperammoniaemia (200 mmol/L) during an episode of viral respiratory infection. He had been presenting £uctuating, mildly high NH4 levels since the end of pubertal growth. Treatment with NCG, added to intravenous hydration and diet therapy, was able to restore normal NH4 levels in the ¢rst 24 h. Further on, NCG was used in this patient at the beginning of several episodes of mild hyperammoniaemia, in order to prevent severe metabolic crises and reduce the days of hospital treatment. NCG daily dose used was between 4000 and 6000 mg in all cases (2200^3100 mg/m2/day). Lately, he presented an episode of acute diarrhoea which caused high NH4 levels (maximum 180 mmol/ L), restored using NCG. Maintaining drug therapy the following days made possible to keep normal NH4 levels, despite the development of two severe septic episodes, caused by Candida parapsilosis and Klebsiella pneumoniae, which required life-support therapy because of the presence of septic shock. Conclusions: (1) In this patient, treatment with NCG contributed to control mildly high NH4 levels. (2) Early treatment with NCG prevented metabolic crises during severe septic episodes.

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TWENTY SEVEN PATIENTS AFFECTED WITH PROPIONIC ACIDEMIA, IRANIAN EXPERIENCE Zaman T, Parvizi SH, Moradian R Tehran University IEM Department, Tehran, Islamic Republic of Iran Introduction: (PA) is one of the most common inborn errors of metabolism, caused by a de¢ciency of propionyl CoA carboxylase. We present our experience at two main reference centers. Materials and Methods: The diagnosis was made on elevated propionyl carnitine and propionic acid on MS/MS, urine GC/MS respectively. 27 patients were studied (ages: 1 day^14 years, 14 male and 13 female, average age of 51.62 months), 10 cases in neonatal period, 2 neonates by newborn screening, 10 cases: 1 month^2 years and seven cases in childhood. Positive family history in 72%. The chief presentation was attacks of vomiting in 11 cases, 8 of them in neonatal period, convulsion in ¢ve; 3 cases in neonatal period and two at 1 month^2 years. Lethargy in16, poor feeding in 19, failure to thrive, usually after six months in 15, psychomotor delay in 8, hypotonia in 20 and abnormal movements in three, inverted nipple in 3. Hyperammonemia and hyper lactatemia in all, metabolic acidosis in 13 respiratory alkalosis in 1, highly elevated propionyl carnitine, and high C3/C2 ratio in all, high propionic acid in organic acid pro¢le in two of two with normal propionyl carnitine, neurological outcome: 9: very well, 4 diagnosed by newborn screening, 9 whose treatment was started 1 month^2 years after onset: mild mental retardation, the rest, specially those with abnormal movement: moderate retardation. Conclusion: Propionic acidemia should be considered in di¡erential diagnosis of unexplained neurological disorders and failure to thrive. Early diagnosis, timely measures for adequate treatment can contribute a lot to improve prognosis.

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METHYLMALONIC ACIDEMIA: ENZYME AND MOLECULAR STUDY IN EIGHT CHILEAN PATIENTS Cornejo V1, Marinero B2, Fernandez E1, Castro G1, Valiente A1, Cabello JF1, Colombo M3, Raimann E1, Perez B2, Ugarte M2 1 LabGEM, INTA, Universidad de Chile, Santiago, Chile, 2Centro de Biolog|èa Molecular, UAM, Madrid, Spain, 3Hospital Van Buren, Valparaiso, Chile Introduction: Isolated methylmalonic acidemia (MMA) is due to a defect of methylmalonyl CoA mutase (mut) or in at least three of the enzymes implied in the synthesis of active cofactor AdoCbl (complementation groups cblH-D, cblA and cblB). The cblA form is due to a defect of unknown protein MMAA and cblB form is due to the defect of adenosylcobalamine transferase (ATR). Methodology: Enzyme activity (mut) and propionate incorporation (cbl) were measured and mutations in ¢broblasts mutation were analyzed. Results: We presented 8 MMA cases with urine MM acid (gas chromatography) between 0.7 to 2.5 mg/mg (NV 5.00047). Three cases of 8 presented normal mut activity and propionate incorporation, ruling out MMA. The other 5 cases presented normal mut activity, with abnormal cblA. The molecular study of MMAA gene found two cases in homozygosis: G188R/G188R; E199fs/E199fs and 3 cases were compound heterozygosis: Q120X/c.733G4A; Q120X/L272fs; P151fs/ S189fs. All patients even those carrying null mutations are vitamin B12responsive with exception to the patient with the G188R mutation in homozygosis. All cases that are vitamin B12-responsive `in vitro' or `in vivo', maintain C3 value 510 mM/L (4.0 VN 5mM/L), C0 = 40 mM/L, with the exception of G188R mutation in homozygosis (C3 30^40 mM/ L ). Conclusion: the importance of measuring enzyme activity or its cofactor is emphasized, since the presence of urine MMA does not con¢rm the disease. To con¢rm the enzyme defect and mutations can establish the prognosis of the disease.

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ASPARTOACYLASE ENTRY TO THE BRAIN OF CANAVAN MOUSE WITH HYALURONIDASE Matalon R1, Bhatia G1, Suzucs S1, Michals-Matalon K2, Tyring S3, Grady J1 1 University of Texas Medical Branch, Galveston, USA, 2University of Houston, Houston, USA, 3University of Texas Health Sci Center, Houston, USA

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URINE SCREENING TESTS ARE OLD BUT NOT IMPRACTICAL: AN EXAMPLE IN DIAGNOSIS OF A TURKISH INFANT WITH ALKAPTONURIA Kurt I1, Hasimi A1, Ozturk O1, Ayd|n HI2, Erbil MK1 1 Dept Clin Biochem, Gulhane School of Med, Ankara, Turkey, 2Dept Pediatrics, Gulhane School of Med, Ankara, Turkey

Objective: Enzyme replacement therapy (ERT) is an accepted method of treatment, even though there is poor entry to some tissues such as brain. In a previous study we have shown that adding hyaluronidase (Hyase) to alpha-glucosidase, improved signi¢cantly the entry of the de¢cient enzyme to the brain. The purpose of this study is to ¢nd out whether adding Hyase to aspartoacylase (ASPA) improves the penetration of ASPA to the brain. Method: Knock out mice for Canavan disease was used. Two sets of experiments were carried out, with two mice in each group. One pair received ASPA only, another received Hyase only, and the other received Hyase and ASPA. Mouse Hyase (Sigma), 3000 U were injected intraperitoneally. Recombinant mouse ASPA, 120 mg/mouse were given 15 min after the Hyase injection. One set of animals was given two treatments, every other day, and the other set of mice was given three treatments every other day. Animals were sacri¢ced 6 h after the last dose, and the brains were removed to determine ASPA activity. Results: Statistically signi¢cant increase in the activity of ASPA was found in the brains of the knock out mice with ASPA and Hyase, in each set of experiments. The APSA only group had an increase in activity, but ¢fty percent less than ASPA and Hyase. Conclusion: The data suggest that enzyme replacement for Canavan disease (CD) should be tried as therapeutic modality. It is anticipated that mild forms of CD stand to bene¢t most from such treatment.

Background: The simple urine screening tests were utilized for newborn screening purposes commencing at mid 1960s; however have become a part of the ¢rst-line analyses in metabolic screening of newborns lately. After implementation of advanced techniques, bene¢ts of these tests in metabolic screening or re£ex testing have been questioned by some scientists recently. In this study we present a Turkish infant with alkaptonuria who had been diagnosed by odd coloring of urine screening tests. Patient and Results: A 5 month old female infant was referred due to darkening of overnight napkins, beginning at newborn period. Her routine laboratory analyses were within normal limits. While screening with simple urine spot tests, an odd color formation other than yielding the presence of glucose was seen as the qualitative Benedict test result. Addition of ferric chloride to urine sample gave a transient blue-green color. Darkening of standing urine residue at the napkin accompanied by atypical urine test results reminded us the diagnosis of alkaptonuria. The patient was regarded as alkaptonuria ad interim, in accordance with darkening of fresh urine sample after adding sodium hydroxide solution. De¢nitive diagnosis of alkaptonuria was later con¢rmed NMR method. Conclusion: Although non-speci¢c features of simple urine screening tests limit their utilization in large-scaled metabolic centers, they may still successfully be used as a ¢rst-line analyses in small-scaled centers as well as in diagnostic laboratories with limited technical equipment.

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CPG ISLANDS AROUND EXON 1 IN SUCCINYL-CoA: 3-KETOACID CoA TRANSFERASE (SCOT) GENE WERE HYPOMETHYLATED EVEN IN HUMAN AND MOUSE HEPATIC TISSUES WHERE SCOT GENE EXPRESSION WAS COMPLETELY SUPPRESSED Fukao T, Zhang G, Matsuo N, Kondo N Dept Pediatr, Grad Sch of Med, Gifu Univ, Gifu, Japan Background: Ketone bodies are important vectors of energy transfer from the liver to extrahepatic tissues, especially when glucose is in short supply. SCOT catalyzes the rate-determining step of ketone body utilization (ketolysis) in extrahepatic tissues. SCOT mRNA expression is almost completely suppressed in human liver, the site of most ketone body synthesis. We previously analyzed 5' £anking region by Luciferase assay and showed two GC boxes were responsible for basic expression of SCOT gene but failed to identify cis-elements elements responsible for complete suppression of SCOT gene in the hepatic tissues in the 2.2 kb 5' £anking region. Methods: Genomic DNAs from human liver tissues and HepG2, Chang liver cells, and HeLa cells, and those from mouse liver, heart, kidney were analyzed by Sodium bisul¢te treatment and sequencing to analyze methylation status around CpG islands around exon 1. Results: Most CpG dinucleotides in the CpG islands of human SCOT promoter region were not methylated in DNAs from HeLa and Chang cells. Even HepG2 and two hepatic DNAs were hypomethylated in this CpG island. Moreover, in case of mouse CpG islands were almost completely unmethylated in the liver DNA as well as heart and kidney DNAs. Conclusions: CpG islands around promotoer region of SCOT gene were hypomethylated in both human and mouse hepatic tissues. Hence, methylation status is not contributed to the hepatic tissue- speci¢c SCOT gene suppression.

AN UNUSUALLY HIGH PROPIONYLCARNITINE IN A PATIENT WITHOUT A METABOLIC DISORDER John CM1, Poh S1, Yeo SJ1, Hart CE2, Ang J1, Tan ES3, Goh DLM4, Rajadurai VS5 1 Nat Extend Newborn Screen, KK Hosp, Singapore, Singapore, 2BG Lab, KK Hosp, Singapore, Singapore, 3Paed Med, KK Hosp, Singapore, Singapore, 4Dept of Paed, Nat Uni Sing, Nat Uni Hosp, Singapore, Singapore, 5Dept Neonatol, KK Hosp, Singapore, Singapore Patient: F|lipino boy, 4th child of non-consanguineous parents screened through the national screening programme. He was large for gestational age, 4200 g at 39 weeks, born by emergency caesarian section. Throughout the course of investigations he was well. Results: The newborn screening sample showed moderate increase in free carnitine and acetylcarnitine (C2), 135 and 70 mmol/L respectively (99 percentile 74 and 49) and a signi¢cant increase in propionylcarnitine (C3) 13.4 mmol/L (99 percentile 4.9). In the ¢rst repeat sample free and C2 remained elevated to a similar level and C3 had increased to 20 mmol/L. Organic acids were requested on the baby and acylcarnitines on the mother. The organic acids showed no metabolites associated with a raised C3 and the mother's acylcarnitines were normal. At one month all acylcarnitines were within normal range. Discussion: Elevated free, C2 and C3 together are not indicative of a metabolic disorder, although they are often associated with carnitine supplementation and can be a physiological ¢nding in the newborn. Elevated C3, particularly at these levels, is usually associated with either MMA or PA. However, no metabolites were seen on organic acid analysis. We have observed transient elevations of C3 being associated with hyperbilirubinaemia. Our patient had a total bilirubin level of 180 mmol/L which remained elevated for 4/5 days and was treated with phototherapy. This has also been noted by another screening programme, but the mechanism is unclear. However, the very high level of C3 in this patient is particularly remarkable.

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INVESTIGATION OF THE `UNMEASURED' ANIONS IN CRITICALLY ILL PATIENTS WITH METABOLIC ACIDOSIS Ruitenbeek W1, Terpstra AM2, Moviat M2, Kluijtmans LA1, Engelke U1, Pickers P2 1 Lab Pediat Neuro, Radboud UMCN, Nijmegen, Netherlands, 2Dept IC Medicine, Radboud UMCN, Nijmegen, Netherlands Background: The chemical nature of the strong ion gap (SIG), which correlates to the frequently applied anion gap, is for the major part unknown. Contribution of citric acid cycle intermediates and acetate has been suggested. Methods: Blood of 31 intensive care patients with metabolic acidosis was examined as to amino acids, organic acids and uric acid. Two groups of patients were compared: 8 patients with calculated SIG 42.0 and 12 patients with SIG 55.0 mEq/L. Results: Concentrations of the anionic compounds aspartic acid, uric acid, succinic acid, pyroglutamic acid, p-OH-phenyllactic acid and homovannillic acid were statistically signi¢cantly increased in the SIG 55.0 group compared to the SIG 42.0 group. The mean di¡erence between both groups for the contribution to SIG by the total organic acids was 5.6% (430 mEq/L), by uric acid 2.2% (169 mEq/L) and by total amino acids 0.07% (5 mEq/L). Together these compounds accounted for only 7.9% of the SIG in IC patients with metabolic acidosis. Conclusions: This study does not endorse a contribution of citric acid cycle intermediates or acetate to SIG. A largely unclari¢ed portion of the chemical nature of SIG in humans remains to be explored.

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ABNORMAL C5-HYDROXY ACYLCARNITINE (C5OH) V ALUES DETECTED IN PORTUGUESE NEWBORN SCREENING PROGRAM Rocha H1, Marca¬o A1, Fonseca H1, Sousa C1, Gaspar A2, Diogo L3, Lea¬o Teles E4, Martins E5, Noronha M2, Garcia P3, Rodrigues E4, Tavares de Almeida I6, V|larinho L1 1 Portuguese NBS Laboratory ^ CGMJM-INSA, Porto, Portugal, 2Met Dis Unit, HSM, Lisbon, Portugal, 3Hospital Pediaètrico de Coimbra, Coimbra, Portugal, 4Hospital St Joa¬o, Porto, Portugal, 5Centro Hosp Porto ^ Unid Maria Pia, Porto, Portugal, 6Met and Gen Group, iMed UL, Faculdade Farmaècia Univ Lisboa, Lisboa, Portugal Elevations of C5-hydroxy acylcarnitine (C5OH) in expanded newborn screening may be associated with of a broad spectrum of conditions, namely 3methylcrotonyl-CoA carboxylase de¢ciency (3MCCD), 3-methylglutaconic hydratase de¢ciency, 2-methyl-3-hydroxybutyryl-CoA dehydrogenase de¢ciency, 3-hydroxy-3-methylglutaryl-CoA lyase de¢ciency, b-ketothiolase de¢ciency, holocarboxylase synthetase de¢ciency and sometimes biotinidase de¢ciency. Besides these, are common elevations in C5OH due to maternal 3MCCD. The goal of this study is to evaluate the outcome of newborns with elevations of C5OH at newborn screening so we can better de¢ne screening approaches as well as the follow-up of these newborns. To achieve this, a retrospective analysis of the data from 250 000 newborns screened in Portuguese Newborn Screening Program, was done. A total of 22 newborns were screen-positive for elevations of C5OH. Biochemical/molecular follow-up con¢rmed the diagnosis in eleven cases: six 3MCCD (three newborns and three mothers); three HMG-CoA lyase de¢ciency; one holocarboxylase synthetase de¢ciency and one biotinidase de¢ciency. No diagnosis was achieved so far, in the remaining eleven newborns besides the persistent elevations of C5OH in some of them. The review of C5OH values as well as some ratios, namely C5OH/C5, C5OH/C8 and C5OH/C0 couldn't help to di¡erentiate between true and false positive cases. In conclusion, follow-up of positive C5OH screening results, lead to the identi¢cation of a metabolic disorders in 11 out of 22 cases. From the remaining cases some continue with elevations of C5OH, but no diagnosis could be achieved so far. Additional investigation is needed so more accurate screening approaches can be achieved and de¢ned more appropriate follow up testing.

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MULTIPLE OXPHOS DEFICIENCY AND MITOCHONDRIAL DNA DEPLETION IN TISSUES OF PATIENTS WITH METHYLMALONIC AND PROPIONIC ACIDURIA de Keyzer Y1, Valayannopoulos V2, Benoist JF3, Arnoux JB2, Batteux F4, Chretien D1, Lebre AS1, Chadefeaux-Vekemans B5, Rabier D5, Touati G2, Munnich A1, de Lonlay P1 1 INSERM U781, Necker Hosp, Paris, France, 2Metabolic Dept, Necker Hosp, Paris, France, 3Biochemistry, Robert Debre Hosp, Paris, France, 4 EA1833, Cochin Hosp, Paris, France, 5Biochemistry, Necker Hosp, Paris, France Background: Respiratory chain (RC) de¢ciency has been suggested to be responsible for the development of various late complications in organic acidemias (OA). Objective: Determine the extent of OXPHOS and tricarboxylic acid cycle (TCA) dysfunction in OA patients presenting severe late complications. Patients: Six pediatric patients, two with PA presenting severe cardiomyopathy and four with MMA with neurological disease (3/4) and renal failure (2/4), were followed. Methods: We measured RC activity, mtDNA content, TCA cycle enzyme activity in various tissues and assessed oxidative metabolism in ¢broblasts. Results: RC was de¢cient in the liver, heart and muscle in PA patients and in kidney and liver in MMA patients investigated. Residual mtDNA contents were decreased in the liver of PA patients, and in muscle of one MMA patient. TCA enzyme activities and oxidative metabolism were normal. Conclusions: Mitochondrial dysfunction was found in all investigated tissues of 6 patients with OA and severe late complications. Cardiomyopathy quickly reversed after liver transplantation in one PA patient. We hypothesize that despite normal TCA enzymatic activity, reduced £ux through the TCA is likely to impair RC actvity and contribute to late complications development.

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LCHAD DEFICIENCY ^ THE MOST FREQUENT FATTY ACID OXIDATION DISORDER IN NEWBORN SCREENING IN THE CZECH REPUBLIC Chrastina P, Kostalova E, Paulova M, Varholakova L, Stastna S, Elleder M, Zeman J Inst Inher Metab Dis, Gen Facul Hosp, Prague, Czech Republic Background: To prepare conditions for national-wide newborn screening of inherited metabolic disorders (IMDs) in Czech Republic by tandem mass spectrometry (MS/MS) we analysed 83 820 samples from neonates born between 2000 and 2007 from our catchment area. Methods: The metabolites were extracted from blood spots into a methanol solution with deuterium-labeled internal standards and then were derivatized before analysis by MS/MS. Results: We detected 21 patients with IMD (146 PKU/HPA, 36 LCHAD de¢ciency, 16 MCAD de¢ciency, 16 3-methylcrotonyl-CoA carboxylase de¢ciency, 16 propionic acidemia and 16 methylmalonic acidemia). The frequency of screened IMD in our catchment area is 1:3991. The frequency of phenylketonuria and LCHAD de¢ciency are 1:5988 and 1:27 940, respectively. Conclusions: Routine newborn screening for IMD by MS/MS is useful for diagnoses of IMD. Early detection of IMD enables counselling of families, close monitoring of child and the opportunity to provide appropriate medical treatment. LCHAD de¢ciency is the most frequent fatty acid oxidation disorder in newborn screening by tandem mass spectrometry in comparison with high incidence of MCAD de¢ciency in Western Europe. The work was supported by the project VZ 64165 of Ministry of Health of Czech Republic

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GROWTH IN 10 SWEDISH PATIENTS WITH LONG-CHAIN 3OH-ACYL-CoA DEHYDROGENASE (LCHAD) DEFICIENCY Bieneck Haglind C1, Ask S1, Halldin M2, Gaîrdman J1, Nyberg G1, Alm J1, von Do«beln U3, Nordenstro«m A1 1 Childrens Hosp, Karolinska Univ Hosp, Stockholm, Sweden, 2Dept Endocrinology, Univ Childrens Hosp, Uppsala, Sweden, 3CMMS, Karolinska, Univ Hosp, Huddinge, Stockholm, Sweden LCHADD is a recessively inherited disorder caused by a defect in the mitochondrial b-oxidation of long chain fatty acids. Ten children (7 girls, 3 boys) 4^19 years of age, with LCHADD are treated at our centers. Eight patients are homozygous for the common mutation 1528 G4C. Following diagnosis all children were started on dietary treatment with markedly reduced fat intake with 20 E % MCT fat including supplementation of essential fatty acids and docosahexaenoic acid. Nine children had continuous night feeds. During infections with fever a carbohydrate-rich supplement or i.v. glucose were administered. Height, weight and BMI were measured at the age of 4, 8 and 16 years. At 4 years of age (n = 10) the median height SDS was 0 (range ^1.5 ^ 2.0 SDS) and the median BMI SDS 1.25 (range ^2.0 ^ 2.25 SDS), at 8 years (n = 7) the median height SDS was 1.0 (range ^1.0 ^ 1.5 SDS) and the median BMI SDS 1.25 (range 0 ^ 2.25 SDS) and at 16 years (n = 3) of age the median height SDS was ^1.0 (range ^2.0 ^ 0.75 SDS) and the median BMI SDS 2.0 (range 0 ^ 2.0 SDS). The patients' growth pattern unexpectedly showed an accelerated growth, possibly due to the increase in BMI. The BMI SDS indicates a tendency to development of overweight over time as a result of the continuous anabolic situation. However there were no obese patients in this group. It is important to carefully balance the energy intake to avoid fasting as well as excess intake of calories.

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UNIQUE ORGANIZATION OF THE MITOCHONDRIAL BETAOXIDATION IN INSULIN-PRODUCING BETA CELLS: DISCREPANTLY HIGH L-3-HYDROXYACYL-CoA DEHYDROGENASE EXPRESSION AND ITS ROLE IN GLUCOSE SENSING Martens GA1, Hellemans K1, Stangeè G1, Van Thi HV2, Van de Casteele M1, Pipeleers D1 1 Diabetes Research Center, VUB, Brussels, Belgium, 2Lab Pediatrics, Brugmann Hosp ULB, Brussels, Belgium To function as the body's glucose-sensors, pancreatic beta cells have a specialized intermediary metabolism that translates the magnitude of the nutrient stimulus into proportionate insulin synthesis and release. Using tissue-comparative mRNA expression pro¢ling, we focused on metabolic enzymes that were abundantly expressed in puri¢ed beta cells, as compared to glucagon-producing alpha cells and liver, adipose, muscle, pituitary and brain tissues. This disclosed a novel unique property of their mitochondrial beta-oxidation (Martens G, J Biol Chem 2007): rodent beta cells expressed the highest levels of L-3hydroxyacyl-coA dehydrogenase (HADH, formerly HADHSC) mRNA and protein of all tissues tested. This contrasted with their low rates of palmitate oxidation and low expression of all other enzymes of this pathway. We have now con¢rmed this discrepantly high HADH expression in human beta cells, indicating that the specialization is evolutionary conserved and potentially crucial for normal beta cell function. This view is strengthened by the hyperinsulinemic hypoglycemia that occurs in the rare humans with loss-of-function HADH mutations (OMIM 609975). Speci¢c HADH knockdown by RNA interference increased the glucose responsiveness of rodent beta cells, causing excessive insulin secretion. HADH suppression was not unequivocally associated with decreased fatty oxidation, as the cells developed compensatory up-regulation of the upstream ACAD enzymes. Combined, our work shows that HADH plays a role in the beta cell's glucose sensing but could do so by ful¢lling hitherto unknown role in their mitochondria, in addition to its classical involvement in boxidation, e.g. by operating as mitochondrial NADH/NAD+ sensor.

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TREATING LCHADD IN AN EXTREMELY PREMATURE INFANT Carney LN, Ganesh J Div Metab Dis, Children's Hosp Philadelphia, Philadelphia, USA Background: Expanded NBS has increased diagnosis of FAOD. Treatment of long-chain 3-hydroxyacyl CoA dehydrogense de¢ciency (LCHADD) includes 30^40% energy from fat, 50% from MCT utilizing medical formulas, which contain cow's milk protein. Provision of essential fatty acids (EFA) is crucial. It is challenging to meet the nutritional needs of premature infants a¡ected by IEM. Parenteral nutrition (PN) and lipids (IL) are often necessary for premature infants; 0.5 g IL/kg/day prevents essential fatty acid (EFA) de¢ciency. Feeding premature infants may be hindered by: GER, delayed gastric emptying, and cow's milk protein allergy. Methods: PN and IL were initiated for infant MY, born at 24 6/7 weeks and diagnosed with LCHADD. MY received 0.5 g IL/kg/day during feed titration. Challenges included: inadequate protein, calorie, vitamin and mineral intake; providing LCFA 520% energy, treating EFA de¢ciency, GER, and cow's milk protein allergy. F|nal formula contained: pregestimil lipil, polycose, complete amino acid mix, £ax seed oil, and MCT oil. Results: Outcomes included growth, CK, EFA pro¢le, and blood glucose. Growth was normal for corrected age. CK levels were 134 to 12 250 U/L. MY was not ill during high CK levels, etiology was catabolism due to inadequate formula intake and emesis. EFA de¢ciency resolved with 20% energy from LCFA, 5.9% from LA, and 1.3% from ALA. Hypoglycemia occurred rarely. Conclusions: W|th prematurity increasing, treating IEM is more complex. Clinicians must modify the plan of care frequently. Collaboration between genetics and neonatology is necessary. Further research is warranted.

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PRIMARY CARNITINE DEFICIENCY DUE TO A NOVEL MUTATION: REPORT OF TWO CASES Hasanoglu A, Eminoglu FT, Okur I, Biberoglu G, Tumer L Dept of Nutr and Metab, Gazi Univ Hosp, Ankara, Turkey In primary carnitine de¢ciency (CTD), sodium-dependent transport of carnitine across the plasma membrane is absent in muscle and kidney. This leads to severe reduction of carnitine and these levels of carnitine are low enough to impair fatty acid oxidation. We present two sibs with CTD due to a novel mutation. Case report: Patient 1, a 7 year old boy, was the second child of nonconsanguineous parents. He applied a hypoketotic hypoglycemic coma that triggered with infection at age of 8 months. The metabolic investigations showed decreased levels of free, C2, C3, C16 carnitines in plasma and increased excretion of carnitine in the urine. The hypertrophic cardiomyopathy was determined by echocardiogram. Carnitine treatment was started. The patient who has ceased the follow up since the age of 2 years, again applied to us with the ¢ndings of cardiac insu¤ciency at age of 6 years. It was learned that the carnitine treatment has not been taken. The diagnosis was con¢rmed by the absence of carnitine transport in ¢broblast cultures. The patient was the homozygous for a novel mutation (c.1519^1526delTTGTTTCT|nsAT). His 12 year old sister had low plasma carnitine levels and hypertrophic cardiomyopathy in echocardiogram. His sister was also homozygous for the same mutation and his parents were heterozygous. Two siblings who are homozygous for the same mutation have shown the di¡erent clinical course from each other. These phenotypic variations of our cases may be the result of factors that may modify the genes or the environmental factors such as infections.

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LONG-CHAIN KETOACYL-CoA THIOLASE DEFICIENCY IN THE DUTCH NEWBORN SCREENING PROGRAM: A NEW CASE Prinsen BHCMT1, de Koning TJ1, de Sain-van der Velden MGM1, Duran M2, Wanders RJA2, Berger R1, Verhoeven-Duif NM1 1 UMC Utrecht, Dept of Metab and Endo Dis, Utrecht, Netherlands, 2 AMC Medical Center, Amsterdam, Netherlands

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ABNORMAL FATTY ACID METABOLISM IN CHILDHOOD SPINAL MUSCULAR ATROPHY (SMA) MAY PREDISPOSE TO PERIOPERATIVE RISKS FOR MITOCHONDRIAL DECOMPENSATION AND LIVER FAILURE Zolkipli Z1, Hutchison J2, Sherlock M3, Gri¤ths A3, Tein I1 1 Div Neurology, Hosp for Sick Children, Toronto, Canada, 2Critical Care Med, Hosp for Sick Children, Toronto, Canada, 3Div Gastroenterol, Hosp for Sick Children, Toronto, Canada

Background: In 2007, the newborn screening program (NBS) in the Netherlands was extended with fourteen treatable inborn errors of metabolism (IEM), including LCHAD de¢ciency (LCHADD). As tandem mass spectrometry (LC-MS/MS) of acylcarnitines does not discriminate between isolated LCHADD and other defects of mitochondrial trifunctional protein (MTP), screening for LCHADD inevitably means screening for MTP-de¢ciency and the very rare condition long-chain 3-ketoacyl-CoA thiolase de¢cieny (LCKATD). Patient: A girl died at the age of 10 days due to a severe necrotizing enterocolitis (NEC) with hypertrophic cardiomyopathy, just before the screening results were available. Subsequently, an elevated C16:OHcarnitine (1.44 mmol/L) was measured. Methods: (Keto)-acylcarnitine concentrations were analysed in plasma by LC-MS/MS as their butylesters derivates. The three activities of MTP: LCHAD, long-chain enoyl-CoA hydratase and LCKAT were assayed in lymphocytes. Results: C14^C18-carnitines concentrations [(un)saturated, hydroxyl] were strongly elevated, while free carnitine concentration was reduced. Analysis of keto-acylcarnitines showed the presence of 3-keto-C18:1carnitine and 3-keto-C18:2-carnitine, suggesting LCKATD. LCHAD activity was low but not de¢cient (12 nmoles/min/mg protein, controls: 23.9+8.9 nmoles/min/mg protein), while LCKAT activity was strongly reduced (0.9 nmoles/min/mg protein; controls 10.8+1.7 nmoles/min/ mg protein) con¢rming LCKATD. Discussion: Here, we present a girl in which NBS results were consistent with LCHADD, but in whom the primary defect was a severe untreatable LCKATD. The results of the NBS enabled genetic counseling. W|thout the NBS screening results, this patient would have remained undiagnosed, suggesting that LCKATD may be an underdiagnosed condition.

Background: SMA children have FAO abnormalities proportional to their phenotypic severity and hypothesized to be related to molecular defects of the SMA-pathogenic SMN gene or to reduced b-oxidation of denervated atrophied muscle. Methods/Results: This mildly obese boy with SMA II underwent uneventful spinal fusion at 13 years for scoliosis and su¡ered catabolic crisis at *4 days postop. He developed lactic acidosis (12.5 mM), hyperammonemia (173 mM), fulminant liver failure (LDH 33854, ALT 2797, AST 412, GGT 97 U/ L, albumin 25), DIC (INR 4.9; platelets 6000), renal failure requiring dialysis, melena, muscle CK elevation (513 U/L), and coma. Serum carnitine was reduced (T/F = 27/16 mM); urine OA revealed moderate lactic acid. Brain MRI showed hemorrhages in supratentorium, thalami, basal ganglia, brainstem. Metabolic abnormalities were reversed with anabolic (TPN with amino acids at 1 gm/kg/d, glucose + insulin) and mitochondrial therapies (L-carnitine, steroids, antioxidant vitamins C, E and CoQ10) with normalization of lactate and NH3 in *12 h. Liver bx: 80% necrosis + micro/macrovesicular steatosis. After stabilization and reversal of DIC, he underwent liver transplant. He made excellent recovery returning to baseline. Post-transplant liver bx's: macrovesicular steatosis. Conclusions: We hypothesize that a combination of surgical stresscate cholamine indu c ed lipolysis, general anaesthesia, postop acetaminophen (no toxicity by urine pyroglutamate) on a background of decreased b-oxidative capacity were potentially factors in this Reye-like syndrome. SMA children undergoing general anesthesia and surgery may be at risk for severe mitochondrial decompensation. Rigorous attention should be paid to perioperative nutrition, medications and prevention of lipolysis.

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ISOBUTYRYL-CoA DEHYDROGENASE DEFICIENCY ^ CASE REPORT Ferreira AC1, Mira G2, Sequeira S1 1 Metabolic Unit ^ Hosp Dona Estefaªnia, Lisbon, Portugal, 2Serv Paediatrics ^ Hosp Esp|èrito Santo, Eèvora, Portugal Background: Isobutyryl-CoA dehydrogenase de¢ciency (IBDD) is a rare defect in the catabolism of valine. Lately, quite a few cases have been identi¢ed through neonatal screening due to increased C4-acylcarnitine and are mostly asymptomatic. The gene for IBDD (ACAD8) has been cloned and mutations have been identi¢ed in several patients. We present a symptomatic case of IBD de¢ciency diagnosed before the generalised implementation of newborn screening in Portugal and report a new mutation in ACAD8 gene. Case: We report a girl, fourth child of non-consanguineous parents, born after a non-followed pregnancy and normal delivery. During the ¢rst months of life she had feeding di¤culties, failure to thrive, axial hypotonia and developmental delay. She also had a systolic murmur and the echocardiogram revealed slight enlargement of the left ventricle. Presently she is four years old and on no treatment has normal psychomotor development and normal echocardiogram. Metabolic investigation showed persistent elevation of plasmatic isobutyryl/ butyrylcarnitine (0.56 to 1.12 mmol/L ^RV 0.08^0.38), total and free carnitine in blood within the normal range and normal organic acids. Mutation analysis of the ACAD8 gene showed homozygosity for the variant S171C (c.512C4G) in exon 5. Comments: It is yet uncertain whether IBDD may cause signi¢cant morbidity and whether treatment (carnitine supplementation and protein restricted diet) is necessary as children who remain asymptomatic have been identi¢ed. In view of the limited experience worldwide, careful monitoring of children a¡ected is recommended.

PREECLAMPSIA AND HELLP SYNDROME: MITOCHONDRIAL FUNCTION IN HUMAN UMBILICAL VENOUS ENDOTHELIAL CELLS Illsinger S1, Janzen N2, Sander J2, Schmidt KH1, Bednarczyk J1, Mallunat L1, Bode J1, Hagebo«lling F1, Vaske B3, Lu«cke T1, Das AM1 1 Dept of Paed II, Med School, Hannover, Germany, 2ScreeningLaboratory, Hannover, Germany, 3Inst of Biomed Statistics, Med School, Hannover, Germany Background: Fetal inborn errors of fatty acid oxidation (FAO) are associated with obstetric complications including preeclampsia (PE) and hemolysis, elevated liver enzymes, low platelets (HELLP) syndrome. It is unclear whether a causal relationship exists. Due to discrepancy between frequencies of these complications and incidence of inborn errors of FAO, we hypothesize that acquired changes of mitochondrial energy metabolism in HUVEC (human umbilical venous endothelial cells as selected part of the fetoplacental unit) may be involved in these obstetric complications. Methods: Analysis of cultured HUVEC from newborns (25^40 weeks of gestation, 35 from PE/HELLP mothers and 41 age-matched controls) for overall FAO, activities of respiratory chain (RC) enzymes, hexokinase and LDH and levels of energy-rich phosphates. Acylcarnitine pro¢les in blood from mothers were investigated. Results: RC-activities of complexes II+III, IV, V, citratesynthase and overall FAO in HUVEC from pregnancies complicated by PE/HELLP syndrome in the mother were all signi¢cantly compromised. Levels of energy-rich phosphates remained constant. Activities of LDH and hexokinase were higher in preterm than in term newborns and LDH in preterm cells from complicated pregnancies was higher than in controls. Activities of RCenzymes and CS decreased, those of FAO increased signi¢cantly with gestational age. Healthy mothers had signi¢cantly lower free carnitine than mothers with complications. Conclusion: Compromised mitochondrial function is associated with PE and HELLP-syndrome and may play a crucial role in the pathophysiology of these obstetric complications. Toxic metabolites resulting from these impaired pathways may contribute to organ dysfunction in the mother.

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LATE-ONSET VERY-LONG-CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY PRESENTED WITH RHABDOMYOLYSIS Coker M1, Kalkan Ucar S1, Habif S2, Y|ldiz B1, Goksen Simsek RD1, Darcan S1, Bayindir O2, Wanders RJA3 1 Div Ped Metab End, Ege Univ Child Hosp, Izmir, Turkey, 2Div Bioch, Ege Univ Hosp, Izmir, Turkey, 3Div Clin Chem Ped, Emma Child Hosp, Univ Amst, Amsterdam, Netherlands Very-long-chain acyl-CoA dehydrogenase (VLCAD) is the initial enzyme in fatty acid oxidation. In the vast majority of cases, the defect manifests itself in early infancy as acute metabolic decompensation dominated by hypoketotic hypoglycemia, coma or cardiac hypertrophy. More rarely patients may present later in life with fasting-induced or exercise-induced recurrent myoglobinuria. An apparently healthy 17year-old female had a history of more than ten episodes of muscle pain and weakness usually brought on by moderate exercise. She denied the intake of any herbs or drugs and had no family history of similar conditions. Her examination was unremarkable apart from generalized muscle weakness. Laboratory investigations during attacks revealed elevated creatine kinase 51228 U/L; lactate dehydrogenase 2381U/L. Urinalysis was negative for ketones. Further investigations revealed normal serum lactate, pyruvate, and ammonia. Muscle biopsy showed only nonspeci¢c myositis changes; there were no ragged red ¢bres, inclusions or mitochondrial abnormalities and myo¢brils were intact. The serum acylcarnitine pro¢le found an increase in very long-chain acylcarnitines (C14 = 0.72 mmol/L(50.5 mmol/L); C14:1 = 1.05 mmol/L (50.5 mmol/L). Urine organic acid pro¢le showed no dicarboxylic acids. The overall biochemical ¢ndings were highly suggestive of a fatty acid b-oxidation defect. The diagnosis of VLCAD de¢ciency was con¢rmed by enzyme assays in cultured ¢broblasts by [13C]-palmitate loading test, which showed a marked reduction in VLCAD activity of 0.21 nmol/min/mg protein (normal range: 2.28+0.92 nmol/min/mg). In conclusion, genetic defects of fatty acid b-oxidation might be considered when an adolescent presents with unexplained episodes of rhabdomyolysis, which is induced by prolonged exercise and fasting.

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MOLECULAR BASIS OF VLCADD IN PORTUGAL: THREE NOVEL MUTATIONS Rocha H1, Marques M1, Gaspar A2, Lea¬o Teles E3, Noronha M2, Rodrigues E3, Marca¬o A1, Sousa C1, Fonseca H1, Tavares de Almeida I4, V|larinho L1 1 Portuguese NBS Laboratory ^ CGMJM-INSA, Porto, Portugal, 2Met Dis Unit, HSM, Lisbon, Portugal, 3Hospital St Joa¬o, Porto, Portugal, 4 Met and Gen Group, iMed UL, Faculdade de Farmaècia Univ Lisboa, Lisboa, Portugal Mitochondrial fatty acid b-oxidation is one of the main energy producing metabolic pathways in eukaryotes. Very long-chain acylCoA dehydrogenase (VLCAD) is one of the four acyl-CoA dehydrogenases involved in the initial step of mitochondrial fatty acid b-oxidation. VLCAD is responsible for the initial step of spiral oxidation of C10 C18 or longer, straight chain fatty acids. VLCAD de¢ciency (VLCADD; MIM 211475) is an autossomal recessive disorder of fatty acid b-oxidation, usually characterised by infantile hypoketotic hypoglycemia, hepatic dysfunction or cardiomyopathy. Since the more severe forms can lead to death, early detection and accurate diagnosis are essential to achieve a favourable outcome, bellowing to the group of disorders that are being rapidly introduced in Newborn Screening programs. The gene coding for VLCAD (HUGO symbol, ACDVL) is located in chromosome 17 (17p13) and is composed by 20 exons. Since the identi¢cation of the ¢rst mutation associated to VLCADD, back in 1995, more than 100 pathogenic mutations have already been reported. The authors will present molecular data from the characterisation of three VLCADD patients detected through newborn screening. Two of the patients are heterozygous (p.G222R/p.G476A and p.R615X/p.G327A) and one homozygous (p.G441A/p.G441A). Mutations p.G327A (p.Gly327Ala; c.980G4C), p.G441A (p.Gly441Ala; c.1322G4C) and p.G476A (p. Gly476Ala; c.1427G4C) are described here for the ¢rst time. These results allowed not only to con¢rm the diagnosis in one of the patients, but also provide valuable information for genetic counselling and to increase the knowledge on the molecular pathology of this disorder.

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MS/MS SCREENING FOR VLCAD-DEFICIENCY: FOLLOW-UP OF POSITIVE CASES Spiekerkoetter U1, ter Veld F1, Mueller M1, Stehn M2, Santer R2, Lukacs Z2 1 Dept Pediatr, Univ Child Hosp, Du«sseldorf, Germany, 2Dept Pediatr, Univ Child Hosp, Hamburg, Germany Background: Screening for very long-chain acyl-CoA dehydrogenase de¢ciency (VLCADD) by tandem mass spectrometry (MS/MS) has only recently been implemented in many European countries. However, the disease-speci¢c acylcarnitine pro¢le often only allows detection of VLCADD during catabolism on days 2^3 of life. Methods: Over a period of 2 years, all newborns identi¢ed with acylcarnitine pro¢les suggestive for VLCADD on days 2^3 (Screening Laboratory Hamburg, Germany) have been investigated by palmitoylCoA oxidation in lymphocytes. Patients with residual enzyme activities 530% have been further characterized by molecular analysis. Results: From a total of 100 000 newborns, 55 presented with an acylcarnitine pro¢le suggestive for VLCADD. In 48/55, palmitoylCoA oxidation was performed. In 2/48, palmitoyl-CoA oxidation was clearly reduced to 510% residual activity (3% and 4%, respectively) and con¢rmed VLCADD (disease prevalence of 1:50 000). In both patients, 2 pathogenic mutations in the VLCAD gene were delineated. In 3/48 newborns, palmitoyl-CoA oxidation was reduced to residual activities of 11 to 20%, in 5/48 to 21 to 30%. In the latter 8 newborns, diagnosis was not con¢rmed by molecular analysis. Conclusions: Diagnosis of VLCADD can well be missed, if MS/MS newborn screening occurs after the age of 3 days. On the contrary, screening on days 2^3 of life identi¢es many false positive cases. Con¢rmatory enzyme and molecular analyses are essential to correctly identify VLCADD. Further evaluation of the respective acylcarnitine pro¢les is necessary to analyze whether a better di¡erentiation between patients and healthy individuals is possible.

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DEVELOPMENT OF DILATIVE CARDIOMYOPATHY IN CARNITINE-SUPPLEMENTED VERY LONG-CHAIN ACYLCoA DEHYDROGENASE-DEFICIENT MICE AS STUDIED BY IN-VIVO NMR ter Veld F, Jacoby C, Floegel U, Spiekerkoetter U Dept Cardiovasc Physiol, University, Du«sseldorf, Germany Background: The long-term e¡ects of carnitine-supplementation, as a therapeutic strategy in very long-chain acyl-CoA dehydrogenase de¢ciency (VLCADD), are unknown. Negative e¡ects due to the increased production of possibly toxic acylcarnitines are proposed. The VLCAD-de¢cient mouse displays similar stress-induced clinical phenotypes to humans, however, cardiomyopathy has not been observed during follow-up. Methods: VLCAD-de¢cient and wild-type (WT) mice received oral carnitine supplementation for 20 months after weaning (in a dose in accordance to clinical use, of 100 mg kg^1 day^1). Using in-vivo NMR, we studied cardiac function of carnitine-supplemented VLCADde¢cient mice. Six to eight contiguous ventricular short-axis highresolution heart images were acquired (slice-thickness = 1 mm) to cover the entire heart. Results: Long-term carnitine-supplemented VLCAD-de¢cient mice displayed clear signs of dilative cardiomyopathy as re£ected by an increase in end-diastolic (EDV) and end-systolic volumes (ESV) (EDV and ESV were 78.5+2.8 and 27.4+1.7 ml, respectively; as compared to WT: 58.4+3.6 and 14.2+0.2 ml, respectively; p40.05; mean+SEM). Compensation of both EDV and ESV resulted in similar stroke volume for carnitine-supplemented VLCAD-de¢cient and WT mice (51.1+2.3 and 44.2+4.0 ml, respectively; n.s., mean+SEM). The ejection-fraction of carnitine-supplemented VLCAD-de¢cient heart was signi¢cantly impaired (65.2+1.9%, as compared to 75.3+2.7% for WT; p40.05; mean + SEM). We observed no signi¢cant changes in left ventricular mass and end-systolic wall diameter. Conclusions: Our ¢ ndings reveal development of dilative cardiomyopathy with carnitine-supplementation in VLCAD-de¢cient mice.

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VERY LONG-CHAIN ACYL CoA DEHYDROGENASE DEFICIENCY WHICH WAS ACCEPTED AS INFANTICIDE Eminoglu FT1, Tumer L1, Okur I1, Go«kmen Z2, Ezgu FS1, Biberoglu G1, Hasanoglu A1 1 Dept of Nutr and Metab, Gazi Univ Hosp, Ankara, Turkey, 2Dept of Neonatology, Konya, Turkey Very long-chain acyl CoA dehydrogenase (VLCAD) is a recently identi¢ed enzyme bound to the inner mitochondrial membrane. It catalyzes the dehydrogenation of acyl CoA esters of 14^20 carbon length. It is a rare cause of sudden infant death syndrome (SIDS), or unexplained death in the neonatal period. Case report: One month old girl patient was referred to our clinic for having 3 siblings who died from unknown cause in newborn period. Our patient was a second live child of his mother 6th pregnancies. At an estimated gestational age of 39 weeks, she was delivered by cesarean section. Birth weight of the neonate was 2600 g. The mother and father were cousins. The parents' ¢rst and second children died suddenly after normal deliveries in 24 h, and a third child died also in 4 days after birth. After the last death, the parents were investigated for infanticide. The fourth child was healthy (male, 13 months old). Patient's physical examination was normal. The metabolic investigations showed increased C 14:1, C14, C18:1 acylcarnitines in plasma. The levels of CKMB, PRO-BNP, Troponin were increased. An echocardiogram revealed hypertrophic of interventricular septum and left ventricle. F|broblast culture was performed in She¤eld Children's Hospital and the results are consistent with VLCAD (myristate = 63%, palmitate = 36%, oleate = 7%). The mutation analysis is in course. As a result, fatty acid oxidation defects show di¡erent clinical course in di¡erent age groups. A family who has multiple SIDS and suspected infanticide, must be investigated for fatty acid oxidation defects.

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UNUSUAL PRESENTATION OF CARNITINE MEMBRANE TRANSPORTER DEFICIENCY: BICYTOPENIA Kalkan Ucar S1, Aydinok Y1, Habif S2, Levent E1, Ozyurek R1, Bayindir O2, Wanders RJA3, Coker M1 1 Div Ped Metab End, Ege Univ Child Hosp, Izmir, Turkey, 2Div Bioch, Ege Univ Hosp, Izmir, Turkey, 3Div Clin Chem Ped, Emma Child Hosp, Univ Amst, Amsterdam, Netherlands Carnitine transporter defect is an autosomal recessive disorder caused by lack of functional OCTN2 carnitine transporter. The disease spectrum includes cardiomyopathy, metabolic crisis of Reye-like syndrome and recurrent hypoglycaemic hypoketotic encephalopathy. Here we describe siblings of a consanguineous marriage with two distinct presentations of disease. Eleven year old boy presented with persistent, non-progressive moderate normocytic anaemia and neutropenia since early childhood with normal platelet count. He su¡ered from generalized weakness. Bone marrow smear revealed erythroid dysplasia in a relatively hypocellular marrow. Bone marrow biopsy con¢rmed hypo cellularity and dyserythropoiesis. Serum B12 and folate levels were normal while serum ferritin decreased. Although, serum ferritin increased to normal with oral iron therapy, haemoglobin achieved only to 10 g/dl while neutropenia persisted. In the history, it was informed that his older brother manifested cardiomyopathy with unknown aetiology. Therefore, metabolic screening was performed. Plasma carnitine was severely reduced (in various samples: free carnitine: 0.74^2.28 mmol/L (N: 7^80 mmol/L)). A carnitine transporter defect was suspected and con¢rmed by the study of L[methyl-3H] carnitine uptake by ¢broblasts 0.01 (patient)/0.03 (his brother) pmol/min.mg (1.01+0.26). Chronic treatment of oral carnitine was initiated for both siblings. Although, carnitine is well known to have a role in red cell metabolism and anemia was noted in patients of primary carnitine transporter de¢ciency presented with cardiomyopathy, there is no report of bicytopenia related with this disorder. Further, it is interesting that the same defect would cause two distinct manifestations of disease (etc; cardiomyopathy in one and bicytopenia in the other sibling).

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OUTCOME OF AN ELEVATED OCTANOYL CARNITINE (C8) FORM NEWBORN SCREENING DRIED BLOOD SPOT (DBS) SAMPLES OBTAINED DURING SCREENING FOR MEDIUM CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY (MCADD) IN THOSE NOT AFFECTED WITH MCADD Sharrard MJ1, Downing MJ2, Manning NJ2, Olpin SE2, Clark S2, Matthews AJ2, Bonham JR2 1 Paediatrics, Children's NHS Trust, She¤eld, UK, 2Clinical Chemistry, Children's NHS Trust, She¤eld, UK Introduction: Elevated C8 in DBS samples obtained on day 5 is used to screen for MCADD. Occasionally C8 remains elevated when MCADD has been excluded. Aim: To investigate the outcome of infants with elevated C8 when MCADD and carrier state had been excluded. Method/Results: As part of the UKCSNS-MCADD study 2004^2008 we have screened 269 030 newborns for MCADD by detecting C8 40.5 mmol/ L. Presumptive positives were investigated with repeat DBS and plasma acylcarnitine analysis, urine organic acids and MCADD genotyping. 3 infants had persistently elevated C8, no mutations in the ACADM gene and biochemistry inconsistent with MCADD, but initial acylcarnitine pro¢le suggestive of multiple acyl-CoA dehydrogenase de¢ciency (MADD). Infant 1 had screening C8 = 0.62 mmol/L, elevated C4^C14 and C5dicarboxylic acylcarnitines and follow-up C8 = 0.68 mmol/L and. Urine contained signi¢cant ethylmalonate, suggesting mild MADD. The mother had similar changes. Abnormalities did not correct with ribo£avin. Mother and infant had normal fat oxidation in ¢broblasts. Infant 2 had initial C8 = 0.54 mmol/L with increased C6^C14 acylcarnitines. Abnormalities have persisted. Organic acids were unremarkable. Fat oxidation in ¢broblasts was signi¢cantly reduced at 378C consistent with MADD, but normalised at 418C. Infant 3 had initial C8 = 0.74 mmol/L with increased C4^C14 acylcarnitines. Organic acids showed multiple acylglycine increases. DBS acylcarnitines normalised on ribo£avin supplementation, but C10:1 increased on withdrawal. These infants have remained asymptomatic. Conclusion: Elevated C8 may be associated with persistent biochemical abnormalities inconsistent with MCADD but suggestive of MADD. These individuals require long-term follow-up for late onset disease.

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URINARY HEXANOYLGLYCINE IN THE DIAGNOSIS OF MEDIUM CHAIN ACYL CoA DEHYDROGENASE DEFICIENCY (MCADD) FROM NEWBORN SCREENING: DETECTION AND QUANTIFICATION Downing M1, Manning NJ1, Andresen BS2, Dalton RN3, Krywawych S4, Oerton J5, Olpin SE1, Patterson A6, Preece MA7, 5 T|ll J8, Dezateux C5, CSNS-MCADD UK 1 2 î She¤eld Children's Hosp, She¤eld, UK, A rhus Univ, Aîrhus, Denmark, 3 Guy's Hosp, London, UK, 4Great Ormond Street Children's Hosp, London, UK, 5UCL Inst of Child Health, London, UK, 6St James' Univ Hosp, Leeds, UK, 7Birmingham Children's Hosp, Birmingham, UK, 8 Manchester Children's Hosp, Manchester, UK Introduction: Qualitative organic acid analysis (UOA) is used to detect hexanoylglycine (HG) in `non crisis' urine samples investigated for MCADD. However, HG was not detected by UOA in *10^30% of ERNDIM EQA samples at HG concentrations below *8.0 mmol/mmol creatinine. We compared HG detected by UOA with quanti¢ed HG (qHG) in presumptive positive (PP) infants (dried blood spot octanoylcarnitine 50.5 mmol/L) identi¢ed in a prospective multicentre UK screening study of *740 k newborns. Methods: HG detection by UOA of trimethylsilyl derivatives; qHG (mmol/mmol creatinine) by GCMS SID of methyl esters. Results: qHG ^ measured in 93/105 PP cases (all asymptomatic) ^ was abnormal in 80 (range 1.2^47.7, median 21.7). HG by UOA ^ available in 91 infants ^ was detected in all 43 homozygous for c.985A4G (qHG 510.8; median 22.5) but in only 1 of 13 with normal qHG (51.1): nine c.985A4G carriers, three c.985A4G heteroallelic uncertain phenotypes (IVS5-9C4T; c.1091T4C; IVS7-21T4G) and one MADD. HG was not detected by UOA in 4 abnormal samples (qHG: 1.2^5.9) comprising one c.985G4A carrier, two heteroallelic (c.985A4G; c.797A4G; c.985A4G; c.253G4T) and one MADD. Conclusion: HG was detected by UOA in all c.985A4G homozygotes but not in 4 of 10 infants with abnormal qHG 56.0, two of whom were diagnosed as MCADD of uncertain phenotype. UK policy is to recommend that diagnostic follow up of PP infants identi¢ed through screening include quanti¢cation of HG by GCMS-SID in all those where UOA does not detect HG to ensure milder MCADD phenotypes are ascertained. (Funder: Department of Health).

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IN VIVO GLUCOSE AND FAT METABOLISM AT REST AND DURING MODERATE-INTENSITY EXERCISE IN PATIENTS WITH MEDIUM-CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY Huidekoper HH1, Ackermans MT1, Koopman R2, van Loon LJC2, Sauerwein HP1, W|jburg FA1 1 Acad Med Centr, Univ Amsterdam, Amsterdam, Netherlands, 2Dept Hum Mov Sci, Univ Maastricht, Maastricht, Netherlands Objective: To study glucose and fat metabolism at rest and during moderate-intensity exercise in patients with medium-chain acyl-CoA dehydrogenase de¢ciency (MCADD). Methods: contemporary stable isotope methodology with [U-13C] palmitate and [6,6-2H2]glucose in combination with deuterated water was applied to compare fat and glucose metabolism between four adult patients with MCADD (age: 27.3+9.3 years, BMI: 24.6+3.8 kg/m2) and four matched control subjects (age: 27+4.6 years, BMI: 23.4+3.5 kg/m2) at rest and during 1.5 h of moderate-intensity cycling exercise (50% Wmax). Results: No signi¢cant di¡erences were detected between patients with MCADD and control subjects in glucose kinetics, either at rest or during exercise, or in palmitate turnover, FFA turnover, whole-body fat oxidation, carbohydrate oxidation, muscular glycogen oxidation or fat oxidation from plasma and muscle derived triglycerides during exercise. Plasma FFA oxidation was signi¢cantly lower in patients at the end of exercise. At rest, plasma FFA turnover was signi¢cantly higher in patients with MCADD, whereas plasma FFA concentrations were not signi¢cantly di¡erent. Norepinephrine at rest was signi¢cantly higher in patients with MCADD, but no signi¢cant di¡erences in regulatory hormones were detected between patients and control subjects at the end of exercise. Conclusions: Whole-body fat oxidation is not impaired and gluconeogenesis is stimulated normally in adult patients with MCADD during moderate-intensity exercise. Plasma FFA turnover at rest is elevated in MCADD patients and might result in ectopic fat accumulation, eventually causing insulin resistance.

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MOLECULAR ANALYSIS OF MEDIUM-CHAIN ACYL-CoA DEHYDROGENASE DEFICIENCY IN PORTUGAL Luz A1, V|olante S1, Gaspar A2, Lobo Antunes M2, Rivera IA1, Silva MFB1, Ramos A3, Rocha H3, Sousa C3, Marca¬o A3, Fonseca H3, Ventura FV1, Leandro P1, V|larinho L3, Tavares de Almeida I1 1 Met and Gen Group, iMed UL, Fac Farmaècia, Univ Lisboa, Lisboa, Portugal, 2Div Metab Dis, Clin Univ Ped HSM, Lisboa, Portugal, 3Natl Newborn Screening/CGM, Porto, Portugal Medium chain acyl-CoA dehydrogenase de¢ciency (MCADD) is a potentially fatal fatty acid oxidation defect, highly prevalent in NorthWestern Europe. About 80% of MCADD patients are homozygous for the MCAD gene mutation 985A-4G in association with haplotype112. While the molecular basis of MCADD has been extensively studied in several populations, the mutational background of the Portuguese MCADD patients is rather unknown. We aimed to genotype the MCADD patients diagnosed in Portugal and determine the 985A-4G allele frequency in a non-consanguineous Portuguese volunteer population (n = 135). From the 56 MCADD patients studied, 48% (27/ 56) were acknowledged since 2004 after inclusion of MCADD in the National Newborn Screening, 44/56 are of gypsy origin, 8/56 are nongypsy Portuguese, 1/56 is Argentinean and 3/56 are Brazilian. Among the gypsy patients, 11 (including 2 mothers) were asymptomatic identi¢ed through familial studies. The G985 allele, haplotype-112 associated, was found in homozygosity in all gypsy patients, in 6 nongypsy Portuguese and in 2 Brazilian patients. Three/56 were heterozygous compound states for G985. During this study the previously reported 218A-4G and 1045C-4T mutations and 3 novel substitutions were identi¢ed (205C-4T, 503A-4T and 1205G-4T) in heterozygosity. Moreover, a high frequency of the G985 allele in the control Portuguese population (7.2) was found, although few MCADD patients with Portuguese origin have been identi¢ed, a picture that may change with the tandem-MS screening implementation. It must be emphasized that MCADD spectrum in Portugal may vary with the new genetic background brought by the immigrant populations especially from western-European countries and Brazil.

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EXPANDING ROLE OF CLINICAL NURSE SPECIALISTS (CNS) NURSE LED CLINICS FOR CHILDREN WITH MEDIUM CHAIN ACYL CoA DEHYDROGENASE DEFICIENCY McSweeney M, Dixon M, Gru«newald S, Clayton PT, Cleary MA Great Ormond Street Hospital, London, UK Background: A CNS for metabolic disorders was introduced to Great Ormond Street Hospital (GOSH) six years ago to bridge the gap between specialists and local/community services. CNSs are highly quali¢ed having received additional training in physical examination, history taking and prescribing according to the GOSH criteria for nurse-led clinics. Methods: Newborn screening for MCADD was introduced to GOSH in 2004 as part of the UK Collaborative study of newborn screening. This resulted in signi¢cant increase in MCADD patients in our metabolic department. CNS involvement begins from the moment a presumptive positive result is identi¢ed to follow up counselling and overall management. The CNS provides a vital role for education of families and professionals and o¡ers support in illness management at home and local hospital. Children diagnosed on screening are followed up in the nurse-led clinic with the family's acceptance. The clinic runs monthly, has dietetic input and support from metabolic consultants. The clinic has expanded to include MCADD patients, identi¢ed pre-screening. Feedback is very positive indicating high levels of satisfaction. Conclusions: Nurse-led clinics are an e¡ective method of delivering patient care. The expertise of the CNS ensures a high quality of patient care similar to that previously delivered by consultants. This model, based on clear clinical management guidelines can be developed for several metabolic disorders. The suitability of nurse led clinics for other metabolic conditions needs to be carefully assessed

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STUDIES ON THE SUBSTRATE SPECIFICITY OF CARNITINE PALMITOYLTRANSFERASE 2: IMPLICATIONS FOR ACYLCARNITINE PROFILING V|olante S1, IJlst L2, Tavares de Almeida I1, Wanders RJ2, Ventura FV1 1 Met and Gen Group, iMed UL, Fac Pharmacy, Univ of Lisbon, Portugal, 2 Lab Gen Metab Dis, AMC, Univ of Amsterdam, Netherlands Over the last years acylcarnitines emerged as important biomarkers for the diagnosis of mitochondrial fatty acid b-oxidation disorders (mFAOD) and branched-chain aminoacid oxidation defects (BCAAOD). Nevertheless, the origin of these intermediates remains rather unidenti¢ed. It is generally assumed that the acylcarnitines' pro¢les as observed in mFAOD re£ect the intramitochondrially accumulating acyl-CoAs, namely via carnitine palmitoyltransferase 2 (CPT2). Yet, this has not been studied in detail for the human enzyme. Aims: Establishment of CPT2 substrate speci¢city pro¢le, allowing the elucidation of the role of CPT2 in the intramitochondrial synthesis of acylcarnitines. Methods: Expressed human CPT2 was incubated with acyl-CoA esters ranging from C2^C26, including FAO intermediates of di¡erent chain lengths and acylCoAs from the BCAAO. The produced acylcarnitines were quanti¢ed by ESIMS/MS. Results: We show that CPT2 is active towards medium (C8-C12) and long-chain acyl-CoA esters (C14^C18), whereas virtually no activity was found with shortand very-long-chain acyl-CoA intermediates as well as with BCAAO intermediates. The trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Conclusions: The results obtained support the hypothesis that the carnitine cycle can operate in the reverse direction for medium and long-chain acyl-CoAs, with exception of trans-2-enoyl-CoAs. This contributes to the abnormal acylcarnitines' pro¢les detected in mFAOD, likely the output of a mitochondrial detoxi¢cation pathway. Poor detoxi¢cation of the enoyl-CoA intermediates by CPT2 can explain the absence of trans-2-enoyl-carnitines in mitochondrial trifunctional protein de¢cient patient's pro¢les. Furthermore, their accumulation may cause the discrepancy between the clinical features of this disorder and very-long-chain acyl-CoA dehydrogenase de¢ciency.

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CPT 2 DEFICIENCY DETECTED BY NEWBORN SCREENING: 2 NOVEL MUTATIONS DESPITE NORMAL FOLLOW-UP ACYLCARNITINE PROFILE Illsinger S1, Peter M2, Wanders RJ3, Deschauer M4, Handig I5, Lu«cke T1, Wuyts W5, Das AM1 1 Dept of Paed II, Med School, Hannover, Germany, 2ScreeningLaboratory, Hannover, Germany, 3Clin Chem Lab, Gen Metab Dis, AMC, Amsterdam, Netherlands, 4Dept of Neurology, University Hospital, Halle W|ttenberg, Germany, 5Dept Med Genetics, University Hospital, Antwerp, Belgium Background: Several variants of CPT2-de¢ciency are known including a lethal neonatal form a¡ecting multiple organ systems, a severe infantile form with hepato- cardiomuscular symptoms and an adult-onset myopathic variant. Studies of milder phenotypes suggest complex interactions with modulators leading to higher residual fatty acid oxidation, a wide range of CPT2 activity but little evidence for genotype-phenotype correlation. Case Report: After an uneventful pregnancy the male newborn presented with a pathological acylcarnitine pro¢le in routine newborn screening (3rd day of life). The spectrum showed elevations of long chain- acylcarnitines while the ratio of C16+C18:1/C2-carnitine was increased, therefore CPT2de¢ciency was suspected. A follow-up acylcarnitine pro¢le (day 9) was normal under breast milk feeding. No clinical symptoms and no excretion of dicarboxylic- acids were observed. Methods and Results: The activities of CPT2 and overall fatty acid oxidation were decreased to 20% and 10% of controls in healthy ¢broblasts, respectively. Sequence analysis showed two mutations in exon 4: a severe truncating c.748-749delAA, and a c.1436A4G (p.Tyr479Cys)- missense mutation. Both have not been described before. Parents are heterozygous for one of the mutations each. Until now, no clinical symptoms occurred despite marked impairment of long-chain fatty acid oxidation in the child. Conclusion: Con¢rmation of suspected mitochondrial fatty acid oxidation defects based on an initial abnormal newborn-screening by MS/MS should include enzyme and if possible molecular genetic analysis despite normal 2nd screening (resulting from anabolism). CPT2 de¢ciency could be missed if only follow-up acylcarnitines in blood and organic acids in urine were analyzed.

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LETHAL DILATED CARDIOMYOPATHY IN A GIRL WITH SCAD DEFICIENCY Ballhausen D1, Sekarski N2, Jeannet PY3, Boulat O4, Gregersen N5, Bonafeè L1 1 IEM, Molecular Pediatrics, CHUV, Lausanne, Switzerland, 2Pediatric Cardiology, CHUV, Lausanne, Switzerland, 3Child Neurology, CHUV, Lausanne, Switzerland, 4IEM, Clinical Chemistry, CHUV, Lausanne, Switzerland, 5Res Unit Mol Med, Aarhus University, Aarhus, Netherlands SCAD de¢ciency (SCADD) is a fatty acid beta-oxidation defect with heterogeneous phenotypic features. Developmental delay, epilepsy, behavioral problems, and hypoglycemia are most often reported. Myopathy is described in Ashkenazi Jews and cardiomyopathy has been described in one Ashkenazi Jewish patient; however, myocardial dysfunction is very unusual in SCADD patients. The clinical course is relatively benign in most symptomatic patients. Frequent SCAD variants (625G4A and 511C4T) cause susceptibility for clinical disease, requiring other genetic/cellular/environmental factors to manifest. We report on a 7 year-old girl of Tamil origin, adopted at 18 months. She had growth retardation, microcephaly, bilateral clubfeet, and moderate developmental delay. She presented acutely ill with fever, vomiting, hepatomegaly, and transient paralysis of the right body side. Brain CT scan revealed hypodensities in the lenticular nuclei compatible with stroke. One month after this episode she presented with cardiac insu¤ciency and dilated cardiomyopathy. No viral infection could be demonstrated by extensive microbiological studies. Despite maximal pharmacological therapy, heart failure occurred rapidly and the child died 5 months later. Biochemical investigations showed increased urinary excretion of ethylmalonate, methylsuccinate and increased plasma C4-acylcarnitine. No hypoglycemia was detected during our clinical observation. Molecular analysis of SCAD gene revealed 2 homozygous changes: the common variant 625G4A (G209S) and the previously undescribed putative pathogenic mutation 730G4A (E244K). This case further expands the phenotypic spectrum of SCADD and underlines that severe dilated cardiomyopathy and rapidly progressive heart failure may be a feature of SCADD.

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TRANSCRIPTIONAL ANALYSIS OF HADHA AND HADHB GENES ENCODING THE MITOCHONDRIAL TRIFUNCTIONAL PROTEIN Brivet M1, Tubiana S1, Boutron A1, Acquaviva C2, de Lonlay P3, Ogier de Baulny H4, Gu¡on N5, Feillet F6, Cano A7, Faivre L8, Burglen L9, Labarre-V|la A10, Zabot MT2, V|aney-Saban C2, Legrand A1 1 Biochemistry, AP-HP Hopital de Bicetre, Le Kremlin Bicetre, France, 2 Centre de Biologie et Pathologie Est, Lyon, France, 3Div Metab Dis, AP-HP Hopital Necker, Paris, France, 4Div Metab Dis, AP-HP Hopital R Debre, Paris, France, 5Div Metab Dis, Hopital E Herriot, Lyon, France, 6Div Metab Dis, Hopital d'Enfants, Vandoeuvre les Nancy, France, 7Div Metab Dis, Hopital de la T|mone, Marseille, France, 8 Genetics, Hopital d'Enfants, Dijon, France, 9Genetics, AP-HP Hopital A. Trousseau, Paris, France, 10Neurology Dept, CHU, Grenoble, France Aim: To assess cDNA sequencing as a screening strategy to identify HADHA and HADHB mutations in patients with defects of the mitochondrial trifunctional protein (MTP) and evaluate the involvement of nonsense mediated mRNA decay (NMD) in this disorder. Methods: Mutations were screened, using genomic DNA and cDNA, in 45 patients having a biochemical hallmark of MTP de¢ciency. Transcripts levels, from emetine-treated (+) and -untreated (^) ¢broblasts, were measured by quantitative real-time RT-PCR and expressed as HADHA relative to HADHB levels (or vice-versa). Results: 38 patients had HADHA mutations (19 being homozygous for the common missense mutation c.1528G4C, also carried on 46/76 alleles) and 7 patients had HADHB mutations, including a large heterozygous deletion (5932 bp) encompassing the translation initiation site. Sixteen mutations resulted in the appearance of a premature termination codon (PTC) (11/18 novel mutations and 5 previously described mutations); 7/16 of these mutations appeared only on the cDNA sequences of emetine (+) ¢broblasts. Patients with two PTCs or one PTC, showed mRNA levels in the range of 0.05^ 0.17 and 0.5^0.63 respectively, compared to values of 0.96^1.19 in controls and patients with missense mutations. Blocking NMD by emetine signi¢cantly restored the amount of HADHA or HADHB mRNAs. Unexpectedly, the large HADHB deletion and one 5'-proximal PTC resulted in a normal transcript level, suggesting a likely mechanism of translation re-initiation. Conclusion: Our results emphasize the value of cDNA analysis in the characterization of MTP defects and highlight the variability of transcript expression levels as a basis of phenotypic heterogeneity.

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IS OCTANOYL-CoA A NOVEL COMPLEX III INHIBITOR? Sauer SW, Okun JG, Ho¡mann GF, Koelker S, Morath MA Div Metab Dis, Univ Child Hosp, Heidelberg, Germany Background: Accumulation of short- and medium-chain acyl-CoAs results from di¡erent metabolic disorders such as beta-oxidation defects, organic acidurias, Reye syndrome, and Jamaican vomiting sickness. We investigated the e¡ect of these acyl-CoAs on respiratory chain. Unexpectedly, octanoyl-CoA revealed to be a complex III inhibitor. To evaluate the underlying mechanism, we performed competition studies (octanoyl-CoA vs decylubihydroquinone and cytochrome c) and spectral shift experiments. Methods: Complex III activity was measured with increasing concentrations of octanoyl-CoA (0.05^3 mmol/L). Further, enzyme activity was recorded in presence of varying concentrations of octanoyl-CoA (125^1000 mmol/L), decylubihydroquinone (1^50 mmol/ L) and cytochrome c (4^60 mmol/L). For spectral shift experiments complex III spectra of oxidized and reduced enzyme as well as reduced enzyme with a saturating amount of octanoyl-CoA were recorded. Results: The inhibition of complex III by octanoyl-CoA was concentration dependent (e.g. 1 mmol/L, 78% inhibition; 0.05 mmol/ L, 25% inh ibition) as well as non- competitive regarding decylubihydroquinone and uncompetitive regarding cytochrome c. In spectral shift experiments octanoyl-CoA caused a blue shift in the gamma band and a minimum at 561 nm in the alpha band referring to cytochrome b. Conclusions: Our results indicate that inhibition of complex III by octanoyl-CoA is based on inhibited reduction of cytochrome b, an integral component in the low-potential chain of this complex. This e¡ect may play a role in the pathogenesis of medium-chain and multiple acyl-CoA dehydrogenase de¢ciency, Reye syndrome, and Jamaican vomiting sickness which are inherited and acquired conditions of intracellular accumulation of octanoyl-CoA.

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MITOCHONDRIAL LONG CHAIN FATTY ACID OXIDATION: MAN VERSUS MOUSE Chegary M1, te Brinke H2, Ruiter JPN2, W|jburg FA3, Stoll M4, Schulz H5, Hoppel C4, Wanders RJA2, Houten SM2 1 Dept of Pediatrics, AMC, Amsterdam, Netherlands, 2Lab GMZ, AMC, Amsterdam, Netherlands, 3Dept of Pediatrics, AMC, Amsterdam, Netherlands, 4Dept of Pharmac, Univ School of Med, Cleveland, USA, 5 Dept of Chem, City Univ of New York, New York, USA Background: Several mouse models have been developed to aid in the study of the pathogenesis of mitochondrial fatty acid oxidation (FAO) defects. The objective of this study was to select a mouse model for human very long chain acyl-CoA dehydrogenase de¢ciency (VLCADD). Methods: We compared FAO of long chain acyl-CoA dehydrogenase (LCAD-KO) and VLCAD knockout (VLCAD-KO) mouse ¢broblasts with ¢broblasts of VLCADD patients, by using a combination of analytical, biochemical and molecular methods. Results: We found signi¢cant di¡erences in the mitochondrial long chain FAO between human and mouse. Incubation with saturated and unsaturated fatty acids caused acylcarnitine accumulation in VLCADD ¢broblasts, whereas no evident accumulation of acylcarnitines was observed in the VLCAD-KO ¢broblasts. The LCAD-KO ¢broblasts showed only signi¢cant acylcarnitine accumulation after incubation with unsaturated fatty acids. The oleic acid oxidation and the dehydrogenation of various long chain acyl-CoAs were less de¢cient in both the VLCAD- and LCAD-KO ¢broblasts. We provide evidence that these di¡erences in FAO between man and mouse are primarily caused by a di¡erence in LCAD expression levels. In human, LCAD is neither detectable on mRNA level, nor on protein level, whereas in mice both LCAD and VLCAD are highly expressed. Moreover, our results indicate that substrate overlap between VLCAD and LCAD in mice may explain the di¡erence in phenotypic severity of FAO defects between man and mouse. Conclusion: We consider the LCAD KO mouse as the best model to study VLCADD, despite the observed di¡erences.

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EVIDENCE THAT CIS-4-DECENOIC ACID ACTS AS AN UNCOUPLER OF OXIDATIVE PHOSPHORYLATION BY OPENING PORES IN MITOCHONDRIAL MEMBRANE Schuck PF1, Ferreira GC1, Tahara EB2, Kowaltowski AJ2, Wajner M1 1 Dept Bioquimica, ICBS, UFRGS, Porto Alegre, Brazil, 2Dept Bioquimica, USP, Sa¬o Paulo, Brazil Background: Patients a¡ected by medium chain acyl-CoA d e hy d ro g e n a s e ( MC A D ) d e ¢ c i e n c y p r e s e n t p r o g r e s s i v e encephalopathy, drowsiness and lethargy, besides high levels of cis-4decenoic acid (cDA) in their tissue and body. Methods: In the present work, we investigated the in vitro e¡ect of cDA on mitochondrial membrane potential, H2O2 production, NAD(P)H content, mitochondrial respiration and swelling in mitochondrial preparations from rat brain, using glutamate/malate or succinate as substrates. Results: cDA diminished membrane potential, H2O2 production and NAD(P)H content in mitochondrial preparations. Moreover, cDA diminished state III and RCR, while increased state IV in the presence of both substrates. Interestingly, when atractyloside was added to the incubation medium, the uncoupler e¡ect of cDA was only mildly prevented, indicating that a mechanism distinct of other fatty acid is probably involved. F|nally, we found that mitochondrial swelling was increased in the presence of cDA. Conclusions: Taken together, these data strongly suggest that cDA may lead to impairment of ATP production acting as an uncoupler of oxidative phosphorylation. In case of our results could be extrapolated to human condition, they could explain, at least in part, the characteristic brain dysfunction observed in MCAD de¢cient patients. F|nancial support: CNPq, FAPERGS, PRONEX and the FINEP research grant Rede Instituto Brasileiro de Neurocieªncia (IBN-Net) # 01.06.0842-00.

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FIVE YEAR FOLLOW UP OF D,L-3-HYDROXYBUTYRATE TREATMENT OF MULTIPLE ACYL-CoA DEHYDROGENASE DEFICIENCY (MADD) Gru«newald S1, Marek J1, Dean¢eld J1, Olpin S2, Leonard JV1 1 Great Ormond Street Hospital, London, UK, 2Children's Hospital, She¤eld, UK Background: In multiple acyl-CoA dehydrogenase de¢ciency (MADD), the transfer of electrons from fatty acids to the electron transport chain is disrupted by genetic defects of the electron transfer chain. MADD has a variable phenotype, the severe forms having a high mortality; cardiomyopathy being just one of the life threatening complications. Case report: This is a follow-up report of two distantly related children that presented at 2 and 5 months with heart failure and have been reported earler [1]. The diagnosis of ribo£avin unresponsive MADD was established biochemically. Four other children in this extended family had died in early infancy. Cardiac ultrasound showed dilated hypertrophic cardiomyopathy with a shortening fraction of 6% and 16% respectively. Despite intensive conventional treatment the children remained critically ill. Only after starting sodium-D,L-3hydroxybutyrate supplementation (initially 400 mg/kg/day, gradually increased to maximum of 800 mg/kg/day), the cardiomyopathy resolved rapidly. Peripheral muscular hypotonia improved more gradually. The children have been on 3-hydroxybutyrate treatment for more than ¢ve years without any complications such as metabolic alkalosis or nephrocalcinosis. There have been no metabolic crises and regular cardiac examinations during follow-up have been consistently normal. The children are also developing normally. Conclusion: In this family, treatment with D,L-3-hydroxybutyrate has proved safe and e¡ective for MADD with a major and rapid e¡ect on the severe cardiomyopathy. [1] Van Hove JL, Gru«newald S, et al D,L-3-hydroxybutyrate treatment of multiple acyl-CoA dehydrogenase de¢ciency (MADD). Lancet. 2003;361:1433^5.

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INVESTIGATION OF THE EFFECT OF RIBOFLAVIN ON THE STEADY-STATE AMOUNT OF VARIANT ELECTRON TRANSFER FLAVOPROTEINS (ETF:QO) IDENTIFIED IN PATIENTS WITH RIBOFLAVIN-RESPONSIVE MULTIPLE ACYL-CoA DEHYDROGENASE DEFICIENCY (RR-MADD) Cornelius N1, Olsen RKJ1, Corydon TJ2, Gregersen N1 1 Res Unit for Mol Med, Aarhus Univ Hosp, Aarhus, Denmark, 2Inst of Hum Gen, Aarhus Univ, Aarhus, Denmark RR-MADD is a metabolic myopathy in which patients show urinary organic acids pro¢les compatible with either glutaric aciduria type 2 or ethylmalonic-adipic aciduria. Biochemically, the disease is characterised by decreased activities of a number of £avin-dependent acyl-CoA dehydrogenases and respiratory chain enzymes. Clinical and biochemical parameters are either totally or partially corrected after treatment with ribo£avin, which is the precursor of £avins. We and others have recently demonstrated that variations of the £avindependent enzyme ETF:QO are associated with RR-MADD. In the current study we have tested our hypothesis that RR-MADD may be caused by ribo£avin-sensitive variant ETF:QO proteins. Three ETF:QO variants that have been identi¢ed in RR-MADD patients were overexpressed in HEK cells cultured in media with di¡erent concentrations of ribo£avin. Surprisingly, the steady-state amounts of wild-type and variant proteins were decreased similarly in response to decreased ribo£avin concentrations indicating that the defects result in ribo£avin-sensitive loss-of-function without a¡ecting protein amount or that the variant ETF:QO proteins themselves are not responsible for the ribo£avin-sensitive nature of the phenotype.

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MULTIPLE ACYL-CoA-DEHYDROGENASE DEFICIENCY IN A 55-YEAR-OLD WOMAN Kaminsky P1, Acquaviva-Bourdain C2, Pruna L1, Jonas J1, V|aney-Saban C2 1 Ctr Ref Her Metab Disorder, Nancy, France, 2Her Metab Dis Dept, Lyon, France Background: Multiple acyl-CoA-dehydrogenase de¢ciency (MADD) is an autosomal recessively disorder resulting from de¢ciency of the electron transfer £avoprotein (ETF) or of its dehydrogenase (ETFQO). Three clinical phenotypes have been described: type I and type II are neonatal-onset forms, type III form has been reported in childhood or in teenagers and exceptionally in young adult patients. We describe the case of a 55 year old woman who presented with the type III form. Case Report: This patient complained from severe myalgia and limb girdle muscular weakness. Blood analysis showed an increased CK level at 8000 Ui/LL. Full blood analysis was normal. She was never hypoglycaemic. Electromyogram showed myopathic patterns. The muscle biopsy found a moderate ¢bre lipid accumulation. Results: Total blood carnitine level, urinary organic acids chromatography and plasma amino acid chromatography were normal. Acylcarnitines, measured using tandem mass spectroscopy, showed increased levels of short-, medium-, and long-chain acylcarnitines. While ETF activity was normal, activity of ETF-QO was dramatically decreased at 0.013 nmol/min/g of protein versus 0.288 +0.109 in controls, as well as the activity ratio ETF-QO/SCHAD at 0.06 (vs 1.75 in controls). This was consistent with the diagnosis of MADD by ETF-QO de¢ciency. The ETFDH genotype was p. His293Asp/?. The patient was successfully treated with be£avine and L carnitine. Discussion: Our case illustrates the diagnostic di¤culties in adult-onset myopathies and the need for thorough investigation including measurement of acylcarnitines. MADD may present as a proximal myopathy even in old adult, and may not be associated with hypoglycaemia.

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EXPRESSION PATTERNS OF ORGANIC CATION/CARNITINE TRANSPORTER FAMILY IN ADULT MURINE HEART: IMPLICATIONS FOR HYPERTROPHIC CARDIOMYOPATHY AND ARRHYTHMIAS Lamhonwah AM, Wong J, Tam C, Mai L, Tein I Div Neurology, Hosp for Sick Children, Toronto, Canada Background: Organic cation/carnitine transporters (OCTN1, OCTN2 and OCTN3) transport carnitine, drugs, and xenobiotics, (e. g. choline, acetylcarnitine, quinidine, verapamil), maintain critical homeostasis in the intracellular acyl CoA:CoA and carnitine pools, support fatty acid oxidation, and are expressed in heart, muscle, blood vessels, etc. hOCTN2 de¢ciency presents with progressive, lethal hypertrophic cardiomyopathy, myopathy and recurrent coma and is reversible with L-carnitine therapy with good long-term outcome. Methods: To characterize the expression patterns and potential roles of mOctn1, -2 and -3 in adult murine heart, we applied our transporterspeci¢c antibodies to mOctn1,-2 and -3, followed by secondary antibody and DAB peroxidase detection to serial adult murine heart sections counterstained with hematoxylin. Results: All 3 transporters showed strong expression in ventricular 4atrial cardiomyocytes, ¢broblasts of the lamina ¢brosa of cardiac valves, the perivascular interstitial spaces and walls of the great arteries and walls of myocardial arterioles, and showed di¡erential expression patterns in the specialized spindly cardiomyocytes and extracellular tissue of the sinoatrial and atrioventricular nodes innervated by the vagal nerves. Conclusions: Octn1, -2 and -3 are expressed in many regions of the murine heart with a pattern suggestive of key roles in modulating myocardial bioenergetics, supporting valvular structure/function and participating in the anapleurotic generation of acetylcholine for the vagal innervation of the cardiac parasympathetic conduction system. This distribution likely plays a role in the pathogenesis of the hypertrophic cardiomyopathy and arrhythmias seen in hOCTN2 de¢ciency and may also in£uence the absorption and/or elimination of organic cationic cardiac drugs.

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LIPID PEROXIDATIVE STRESS IN SCAD DEFICIENCY (SCADD) AND RESPONSE TO ANTIOXIDANTS Zolkipli Z1, Lehotay DC2, Robinson BH, Tein I1 1 Univ of Saskatchewan, Regina, Canada, 2Genetics/Metabol, Hosp for Sick Children, Toronto, Canada Background: Given similarities of our SCADD case to complex I de¢cient phenotype of cataracts and cardiomyopathy with excess superoxide radicals, we hypothesized that free radical production, arising from dysfunction of FAD-linked SCAD, may play a role in the pathogenesis of SCADD. Case: We previously reported a 23 year old female who presented with congenital-onset myopathic facies, progressive severe limb girdle and axial myopathy, contractures, lumbar lordosis, progressive external ophthalmoplegia, ptosis, cataracts, left ventricular dysfunction with pneumonia, minicore myopathy and EMA. SCAD activity was 0% with no detectable protein on Western blot. She was homozygous for c.319 C4T mutation. Independent 1 year trials of high carbohydrate/ low fat diet+uncooked corn starch qhs, L-carnitine, and ribo£avin did not improve strength. Methods/Results: To assess oxidative stress, we measured 20 plasma aldehydes (lipid peroxidation products) in vivo by GC/MS and found a marked increase in our SCADD case of 2997.56 nM compared to normal controls (2041.3+228.3, n = 12), comparable to elevations in Complex I de¢cient patients (3377.8+595.7, n = 14). We incubated our SCADD ¢broblasts in vitro with 25 mM menadione, a free radical ampli¢er, and found 0% cell viability at 4 h in contrast to controls that remained 100% viable at 48 h. We thus instituted a 6 month clinical trial with antioxidants (V|tamin C and E) and documented a 6-fold increase in endurance on sustained deltoid abduction from 15^90 s. Conclusion: Lipid peroxidative stress may contribute to SCADD pathology and warrants a clinical antioxidant trial.

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SHORT-CHAIN ACYL-CoA DEHYDROGENASE (SCAD) DEFICIENCY: HOMOZYGOSITY FOR THE COMMON C. 625G4A VARIATION MAY CONTRIBUTE TO SCAD DYSFUNCTION IN CULTURED PATIENT FIBROBLASTS Pedersen CB1, Stenbroen V1, Wanders RJA2, Ruiter JPN2, W|brand F3, Young SP4, Millington DS4, Madsen PP5, Andresen BS1, MÖllerLarsen S6, Kjeldsen M1, Gregersen N1 1 Res Unit Mol Med, Aarhus Univ Hosp, Skej, Aarhus, Denmark, 2Dept Clin Chem Pediatr, ACM, Univ of Amsterdam, Netherlands, 3Dept Clin Gen, Rigshospitalet, Copenhagen, Denmark, 4Dept Pediatr, Div Med Gen, Duke Univ, Durham, North Carolina, USA, 5Mol Diag Lab, Aarhus Univ Hosp, Skej, Aarhus, Denmark, 6Inst Hum Gen, Aarhus, Denmark Short-chain acyl-CoA dehydrogenase (SCAD) de¢ciency is an inborn error of the mitochondrial fatty acid oxidation associated with elevated urine ethylmalonic acid (EMA) in some individuals homozygous for the common SCAD c.625G4A variation. In addition to be overrepresented among EMA patients, the c.625G4A variation is present in homozygous, 4^10%, and heterozygous, 22^ 43%, form in asymptomatic European and American individuals. In the present study we investigated SCAD de¢ciency in cultured ¢broblasts from 11 patients presenting with clinical symptoms and c.625G4A homozygosity without other variations in the ACADS gene. All patients had elevated excretions of EMA (440 mmol/mol creatinine), but only six demonstrated an SCAD de¢cient phenotype, de¢ned as 520% SCAD enzyme activity, in cultured ¢broblasts. Although SCAD mRNA levels were within the observed control range, these patient cells showed low SCAD enzyme activity due to elimination of soluble SCAD protein or signi¢cant levels of soluble inactive SCAD protein, thus for the ¢rst time demonstrating SCAD protein misfolding in cultured ¢broblasts. In addition to the ¢broblasts with documented SCAD de¢ciency, another ¢ve patient cells showed normal or slightly reduced SCAD enzyme activity when cultured at 378C. These ¢ndings demonstrate that SCAD de¢ciency due to c.625G4A homozygosity is very heterogeneous and that individual genetic/environmental factors may predispose to disease. As a preliminary approach to identify factors predisposing for disease, the patient cells were examined for defects in the ETHE1 gene and in the mitochondrial respiratory chain/ATP production as defects in these components may also contribute to secondary SCAD de¢ciency and elevated EMA.

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ETHYLMALONIC ACIDURIA: CLINICAL AND MOLECULAR CHARACTERIZATION OF SHORT-CHAIN-ACYL-CoA DEYDROGENASE DEFICIENCY IN 9 ITALIAN PATIENTS Funghini S, Morrone A, Pasquini E, Procopio E, Gasperini S, Donati MA Meyer Children's Hospital, Florence, Italy Short-chain-acyl-CoA deydrogenase de¢ciency is an autosomal inherited metabolic disorder a¡ecting mitochondrial b-oxidation of the short chain fatty acids. The clinical phenotype varies from acute episodes like fatal metabolic decompensation in early life to neuromuscular dysfunction or to asymptomatic patients. We report results obtained by a large study on 9 patients: 6 patients presented, at the diagnosis, neurological involvement, one was diagnosed at neonatal screening and 2 were mothers diagnosed during the study of their carried status. All patients showed increased levels of urinary ethylmalonic acid. We performed molecular analysis of ACADS gene. All the patients presented the c.625G4A common allelic variant at heterozygous or homozygous status. The full gene ACADS sequences of all patients were made and we found 4 new genetic lesion: c.826G4A, c.65G4T, c.842G4C, c.1066G4A. The new c.826G4A was found in 5 patients. We suppose that c.826G4A mutation is frequent in the Italian patients. We found that the mother of a patient with severe neurological involvement had the same genotype of her proband but she was completely asymptomatic. In conclusion, it is not possible to make a genotype/phenotype correlation in SCAD patient but in our experience the patients a¡ected by severe neurological involvement carried a complex allele with c.625G4A common allelic variant and c.826G4A genetic lesion. Environmental and other genetic factor might act synergically with generic lesion in ACADS gene to make the clinical phenotype.

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SHORT-CHAIN 3-HYDROXYACYL-CoA DEHYDROGENASE (SCHAD) DEFICIENCY AND NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY Shigematsu Y1, Hata I1, Tanaka Y2, Shichijo K3, Umemoto T3, Nakatsu T3, Yoshida T3, Naito E4 1 Dept Pediatr, Univ Fukui, Fukui, Japan, 2Centers Advanced Res Support, Univ Fukui, Fukui, Japan, 3Div Pediatr, Tokushima Red Cross Hosp, Tokushima, Japan, 4Div Pediatr, Tokushima RC Hosp Hinomine, Tokushima, Japan In a newborn screening by tandem mass spectrometry in Japan, SCHAD de¢ciency was not a target disorder, but was tentatively monitored using markers such as C4-hydroxyacylcarnitine (C4OH) (cut-o¡ value; 0.23 nmol/ml) and a C4OH/acetylcarnitine (C2) ratio (cut-o¡ value; 0.011). A Japanese girl was bone with a birth weight of 1230 g and a gestation age of 31 weeks. Acylcarnitine analysis in the ¢rst dried blood spots (DBS), which was made on day 49 after respirator therapy for respiratory distress, showed increased levels of C4OH (0.72 nmol/ml), C4OH/C2 (0.028), and glutarylcarnitine (C5DC) (0.28 nmol/ml, cut-o¡; 0.25). Because of the decreased level of C5DC (0.22 nmol/ml) in the second DBS on day 63, additional DBSs were not requested, although levels of C4OH (0.48 nmol/ml) and C4OH/C2 (0.026) were still increased. Because of several episodes of convulsion since 5 months of age and hypoglycemia (22 mg/dl) at 7 months of age, the patients had laboratory work-up, which indicated increased insulin levels during hypoglycemia. Serum acylcarnitines analysis revealed increased levels of C4OH, C4OH/C2, and glutarylcarnitine, and urinary organic acid analysis showed increased excretion of 3hydroxyglutaric acid. The patient did not experience hypoglycemia after diazoxide therapy. The diagnosis of SCHAD de¢ciency was con¢rmed by DNA analysis. The present case indicates that the markers mentioned above may be useful for the screening of SCHAD de¢ciency and should be tested further.

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`WHITE MATTER DISORDERS' AND `INBORN ERRORS OF METABOLISM'; BRIDGING CLINICAL PHENOTYPES WITH BIOCHEMICAL AND MOLECULAR DEFECTS Prasad C, Campbell C, Rupar CA, Levin S, Prasad AN LHSC and Univ Western Ontario, London, Canada Background: White matter abnormalities are frequently detected during cranial magnetic resonance imaging studies done in children presenting with neurodevelopmental symptoms. Methods: Neurometabolic clinic (2003^present) list search for patients with white matter abnormalities on MRI, spasticity, and poor head growth. Results: Of 47 patients evaluated, there were 7 patients who met above criteria. The following metabolic disorders were con¢rmed Krabbe (2) and metachromatic leukodystrophy (1), urea cycle defects (2), methylenetetrahydrofolate reductase de¢ciency (1), and the following previously unreported genotype/phenotype associated with isolated ethlymalonic aciduria (1). A 13 year old right handed male, born to consanguineous parents, presented at age 2 years with ataxia, intention tremor, and increasing spasticity (legs 4 arms). Imaging studies (MRI) showed an atypical di¡use pattern of symmetric increase in signal intensity involving the white matter of cerebral hemispheres. Urine organic acid assays showed large and isolated increases in ethylmalonic acid (93.85 mg/mg Cr; normal range 0.5^20.2 mg/mg Cr). No other unusual acylglycines were reported. ETHE1 gene sequence was normal. Fatty acid oxidation probe assay showed butyrylcarnitine (0.337 mmol/g protein; controls 50.128, n = 88) elevation supporting a diagnosis of SCAD de¢ciency. DNA sequencing showed the presence of homozygous 625G4A SCAD gene variant. Conclusion: Children presenting with white matter abnormalities and progressive spasticity need to be investigated for biochemical and molecular defects. In addition to well established metabolic etiologies, SCAD de¢ciency should be considered, whenever isolated ethylmalonic aciduria is detected. A reappraisal of biochemical ¢ndings and clinical phenotypes in the case of SCAD de¢ciency is warranted.

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THE DIAGNOSTIC POTENTIAL OF SKELETAL MUSCLE ACYLCARNITINE PROFILES IN IDENTIFYING DEFECTS OF FATTY ACID BETA-OXIDATION Lynes G, Hargreaves IP, Land JM Nat Hosp for Neurology and Neurosurgery, London, UK Analysis of blood or plasma acyl carnitine pro¢les is commonly used to identify defects in beta-oxidation [1]. Since skeletal muscle homogenates are routinely assayed within our laboratory to determine mitochondrial respiratory chain enzyme activities we investigated whether assessment of skeletal muscle acyl-carnitine pro¢les would o¡er diagnostic potential in identifying patients with disorders of beta-oxidation. Our existing LC-MSMS plasma acylcarnitine method was modi¢ed to assess muscle carnitine/acylcarnitine. Reference intervals for individual acylcarnitine species were established for skeletal muscle (C14:1, C6, C8 and C10). A comparison was then made between the skeletal muscle acylcarnitine pro¢les from 3 con¢rmed VLCAD and 1 MCAD patients and the reference interval. In contrast to plasma, skeletal muscle does not appear to show consistent characteristic acylcarnitine pro¢les for VLCAD or MCAD. This may re£ecteither the resting state of the muscle at the time of the biopsy and the reduced £ux through the fatty acid B-oxidation pathway or the absence of an overnight fast prior to biopsy. In conclusion, this study indicates that assessment of muscle acylcarnitine pro¢les may not be informative in the diagnosis of VLCAD or MCAD. [1]. Adv Exp Med Biol. 1999;466:327^37.

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DIAGNOSIS OF MITOCHONDRIAL b-OXIDATION DEFECTS IN RUSSIA Baydakova GV, Tsygankova PG Lab Inher Metab Dis, Res Center Med Gen, Moscow, Russian Federation Inborn errors of fatty acid oxidation (FAO) are a group of recessively inherited disorders presenting with a wide spectrum of manifestations. During last four years in the laboratory of inherited metabolic diseases (Russian research center for medical genetics, RAMS) we provide the wide-spectrum investigation on the mitochondrial fatty acid boxidation. Using ESI-MS/MS we analysed 917 samples from patients suspected of FAO defects. Positive tests were found in 34 samples. In 9 cases diagnosis of FAO defects were con¢rmed. MCAD de¢ciency was con¢rmed in 3 patients. C8 and C10:1 acylcarnitines were elevated in all cases, C10 acylcarnitine was elevated only in one cases and C6 acylcarnitine was elevated in two cases. Frequent mutation c. [985A4G] revealed in two sibs. LCHAD/MTP de¢ciency (elevated C16OH, C18OH, C18:1OH acylcarnitines) was diagnosed in 4 patients. In one cases free carnitine level was strongly decreased and C18OH, C18:1OH levels were at the upper cut-o¡. Two of them were homozygous for c.[1528G4A] and two another were heterozygous for this mutation. Two patients with VLCAD de¢ciency (elevated C14 and C14:1 acylcarnitines in one cases and C14:1, C14:2 in other cases) were compound heterozygous for the following mutations: c.[266C4T] + c. [562G4A] and c.[1097G4A] + c.[335delT], consequently. The diagnostic rate is 0.98%. It is the ¢rst experience of complex diagnosis of FAO defects in Russia.

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LONG TERM FOLLOW-UP OF BIOTINIDASE ACTIVITY IN 25 PATIENTS WITH HEPATIC GLYCOGEN STORAGE DISEASE. HOW RELIABLE IS A SINGLE VALUE? Angaroni CJ, Paschini-Capra AE, Giner-Ayala AN, Guelbert NB, Martinez LD, Dodelson de Kremer R CEMECO, Child Hosp, Coèrdoba Univ, Coèrdoba, Argentina Background: An elevated serum biotinidase (BTD) activity has been proposed as an important biomarker in the hepatic glycogen storage diseases (GSDs); although reduced sensitivity for GSD-III and GSD-IX has been reported. Objective: To communicate BTD activities obtained during a long-term follow-up in patients with hepatic GSDs. Subjects. 25 GSD patients were included: GSD-Ia (n = 13), GSD-Ib (n = 1), GSD-III (n = 5), GSD-VI (n = 1), GSD-IX (n = 5). Method: BTD activity was assayed by measuring the hydrolysis of nbiotinyl-p-amino benzoate. Results: The enzyme determinations began in 1996 and the number of tests performed on each patient depended on the attendance to their medical check-ups. The mean and range of BTD activity from healthy individuals were 9.08 nmol/min/ml; 4.68-13.48 (n = 60). (A) Patients with persistently elevated BTD activities: GSD-Ia (8/13); GSD-III (1/ 5); GSD-IX (3/5). (B) Patients with oscillating BTD activities (from normal to elevated): GSD-Ia (4/13; patient (P)1: 11.0^24.8; P2: 11.9^ 28.3; P3: 11.7^27.2; P4: 10.9^17.2); GSD-III (2/5; P1: 11.6^15.8; P2: 7.8^18.4); GSD-IX (1/5; P1: 11.9^15.5). (C) Patients with persistently normal BTD activities: GSD-Ia (1/13, P1: 6.7^12.8, follow-up for 9 years), GSD-Ib (n = 1; post liver transplantation), GSD-III (2/5), GSDVI (1/1), GSD-IX (1/5). Conclusion: This work shows that not all GSD-Ia attend with an increase of this biomarker. In addition, oscillating BTD activity was observed in patients with GSD-Ia, GSD-III and GSD-IX. Thus, it is necessary to be cautious with a single BTD determination as a tool in the presumptive hepatic GSD diagnosis.

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Background: GALT de¢ciency is a common cause of galactosemia. The objective of this study was to evaluate our laboratory's galactosemia testing strategy by determining reference ranges for GALT-de¢cient phenotypes based on molecular alterations (GAL6) and enzymatic phenotype (IEF). Methods: Our laboratory measures GALT activity by enzyme-coupled spectrometric assay. Six molecular alterations were included for analysis: Q188R, S135L, L195P, K285N, N314D (Duarte) and L218L (Los Angeles). Data was tabulated into phenotypes based on the IEF banding pattern (Group 1) and by the presence of at least two molecular alterations (Group 2). Reference ranges (mid-90th percentile) were established for the phenotypes in each group. Results: Signi¢cant overlaps between phenotypes within the groups were detected. In addition, distinct di¡erences in the ranges for the GG, DD and LA/G phenotypes were noted (Table1). Literature describes the LA variant to have 75% activity. Interestingly, ¢ve of the six subjects assigned the LA/G genotype by GAL6 were determined by IEF to be phenotypically GG due to undetectable enzyme activity. One of the subjects identi¢ed by GAL6 to be GG was determined by IEF to be NG with signi¢cant enzyme activity.

Introduction: Maturity-onset diabetes of the young (MODY) is characterized by nonketotic diabetes mellitus, an autosomal dominant mode of inheritance, an onset usually before the age of 25 years and frequently in childhood or adolescence. Method and results: We report on a Saudi family with MODY 1. The index patient is LB, a female who presented at 13 years of age with symptoms suggestive of diab etes. Investigations revealed hyperglycemia, glycosuria and ketonuria with no acidosis. She responded well to insulin therapy. Father is diabetic diagnosed at 16 years of age, responded initially to insulin therapy but developed recurrent hypoglycemia and hence shifted to oral hypoglycemic agent. Mother has type 2 diabetes. A 16 year old brother has diabetes for 5 years that treated with insulin. A 24 year old sister has diabetes for 8 years that was treated with insulin. DNA from LB was extracted, ampli¢ed by PCR and sequenced. The analyzed sample carries a heterozygous substitution at position nt5 in intron 1 of Hepatocyte nuclear factor (HNF) 4 gene. The entire family (7 sibling and parents) was screened and showed that 2 brothers, 2 sisters and the father are carriers of the same mutation. Two of the siblings are asymptomatic. Conclusion: We report a mutation in HNF 4a gene in 6 members of a Saudi family with MODY 1.

DETERMINATION OF A GALACTOSE-1-PHOSPHATE URIDYLTRANSFERASE (GALT) REFERENCE RANGE USING GENOTYPE-PHENOTYPE FROM 257 SUBJECT VALUES Minnich S, Raymond K, Matern D, Oglesbee D, O'Brien J Dept Lab Med, Mayo Clinic, Rochester, USA

N Group 1 DD GG Group 2 DD GG LA/G

Table1 GALT (mg/g Hgb) Mean 5th 95th

8 36

12.1 0.3

8.2 0.0

15.8 1.5

10 7 6

9.9 1.5 2.8

5.2 0.0 0.0

15.8 7.0 14.9

Conclusions: Based on the retrospective analysis of our GALT, GAL6 and IEF subject values, sequencing of the GALT gene should be explored before establishing speci¢c genotype references ranges using.

MUTATION IN HEPATOCYTE NUCLEAR FACTOR 4a GENE IN A SAUDI FAMILY WITH MATURITY-ONSET DIABETES OF THE YOUNG Mohamed S, Hellani A, El-Kholy S Saad Specialist Hospital, Alkhobar, Saudi Arabia

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MOLECULAR GENETICS OF GLYCOGEN STORAGE DISEASE TYPE IX (GSD-IX) IN POLAND: MUTATION SPECTRUM AND GENOTYPE/PHENOTYPE CORRELATION Beauchamp NJ1, Taybert J2, Li Z1, Dalton A1, Tanner S1, Pronicka E2, Sharrard M1 1 She¤eld Children's NHS Foundtn Trust, She¤eld, UK, 2Dept Metab Dis, Endocr and Diab, CMHI, Warsaw, Poland Introduction: GSD-IX results from mutations in PHKA2, PHKB or PHKG2. We aimed to investigate genotype/phenotype correlation. Methods: We investigated 12 M and 1 F GSD-IX patients, diagnosed by blood cell and liver enzymology, and/or clinical features including hepatomegaly, hypertransaminasaemia, hyperlipidaemia, tendency to fasting hypoglycaemia, and hyperlactatemia after intravenous glucose intake. All exons of PHKA2 and PHKG2 and/or PHKB where sequenced, where appropriate. Results: Nine patients had reduced erythrocyte phosphorylase kinase (PHK) indicating X-linked glycogenosis type 1 (XLG1). Four novel and three reported PHKA2 mutations were identi¢ed in eight patients: three small deletions and a duplication (p.I826LfsX13, p.R1070del, p.Met1170dup, p. R1217SfsX26), single nonsense (p.R352X), splice site (c.366-2__-1delAG) and missense mutations (p.P1205L). All had normal fasting tolerance. One XLG1 patient was homozygous for the PHKG2 p.R44X mutation while mutations were not identi¢ed in the ninth. The female was homozygous for PHKG2 p.Thr46LeufsX14. The remaining 3 patients had normal erythrocyte PHK. One patient underwent liver biopsy revealing reduced PHK and thus XLG2. Novel PHKA2 missense mutations were identi¢ed in each of the patients (p.C326R, p.E893K and p.E1117K). While the p. E1117K mutation is close to previous XLG2 mutations, p.C326R and p. E893K are distant from the domains associated with XLG2. F|ve of these six patients had reduced fasting tolerance. Conclusions: In our cohort, patients carrying PHKG2 mutations and PHKA2 missense mutation with XLG2 had reduced fasting tolerance in childhood. GSD-IX should be considered in children with hepatopathy without liver failure, especially with mild hypertransaminasaemia, hyperlipidaemia, slight tendency to fasting hypoglycaemia, and hyperlactatemia after intravenous glucose intake.

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THE IMPORTANCE OF THE MOLECULAR CONFIRMATION IN SUSPECTED GLYCOGEN STORAGE DISEASE (GDS) TYPE IX WITH VARIOUS CLINICAL PRESENTATIONS Zerjav Tansek M, Stopar Obreza M, Bratanic N, Battelino T Univ Child Hosp, Dept of Metab Dis, Ljubljana, Slovenia Background: In children with suspected GSD (hepatomegaly and hypoglicaemia) enzymology is not always diagnostic. We present two families with X-linked GDS type IX (XLG-2 variant) and defects in gene PHKA2, encoding the liver isoform (alpha subunit) of enzyme phosphorylase kinase (PHK). Results: Case 1. Extreme glycogen storage was con¢rmed on liver biopsy and in erythrocytes (Er) in 10 month old boy with severe hepatomegaly and fasting hypoglycaemia. Enzymology in blood cells revealed normal PHK activity, GDS type III, IV, VI were highly unlikely. Patient had transient liver failure during primary EBV infection. At 10 years of age his development and stature were normal. Molecular analysis revealed mutation c.977G4A [p.C326Y] of PHKA2 in the evolutionarily conserved residue (She¤eld Molecular Genetics, UK). Patient's 44 year old uncle with the same genotype has mild hepatomegaly and hyperlipidaemia but no other symptoms or secondary consequences of GSD. Case 2. The liver biopsy of 21 months old boy with hepatomegaly was consistent but not diagnostic of GSD, glycogen content of Er was not increased. Enzymology in blood cells con¢rmed normal PHK activity. At 12 years of age he had normal development and growth. X-linked GDS IX was suspected because his cousin had transient hepatomegaly in early childhood. Analysis revealed a small deletion c.3335__3336delAG of PHKA2 gene that leads to a frameshift and premature termination of translation [p. E1112fs]. Conclusions: A diverse spectrum of phenotypes and possibility of genetic counseling advocate molecular analysis to provide a de¢nitive diagnosis of GSD IX without need for liver biopsy.

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GLYCEROL KINASE DEFICIENCY AND PARTIAL DEFICIENCY OF MITHOCHONDRIAL RESPIRATORY CHAIN COMPLEXES: CASE REPORT Rodrigues E1, Santos H1, Azevedo I2, Campos MM3, Cardoso ML4, Lea¬o Teles E1 1 Metab Dis Un, Ped Dept,Univ /Hosp St Joa¬o, Porto, Portugal, 2 Pneumol Un, Ped. Dept, Univ/ Hosp St Joa¬o, Porto, Portugal, 3 Neuroped, Ped Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 4Genet Med Cent Jacinto Magalha¬es, Porto, Portugal Glycerol kinase de¢ciency (GKD) is an X linked recessive disorder characterized by hyperglycerolemia and glyceroluria. Two types had been described: isolated and complex forms, this associated with Duchene muscular dystrophy and congenital adrenal hypoplasia. Aim: Discuss unusual presentation of GKD. Case Report: Male child, ¢rst son of healthy, non consanguineous parents. Eutocic delivery at 41 weeks; Apgar 8/10. An arthrogryposis was seen in the delivery room conducting to a clinical investigation. A normal growth and development were registered. At the age of two months, following an episode of vomit aspiration, he was admitted to pediatric ICU due to a cardio-respiratory arrest. He developed a severe hypoxic-ischemic encephalopathy resulting in a permanent vegetative state. Background: Usual etiological investigation was negative. The echocardiogram showed an hypertrophic cardiomyopathy and cerebral MRI registered a thin corpus callosum and hipoplasic left cerebellum lobe. The metabolic study revealed hypertriglyceridemia, glyceroluria and hiperglycerolemia, suggesting GKD, latter con¢rmed by molecular study; endocrinological studies, CK and muscular structure were normal. Furthermore Respiratory Chain study showed a combined partial de¢ciency of complex I and II. The patient now with 18 months developed hydrocephalus and dysautonomic responses to stress or postural changes. Discussion: GKD can presents with repeated vomiting/hypoglycemia, which may had been responsible by the initial event. To our knowledge the ¢ndings of arthrogryposis, hypertrophic cardiomyopathy and c erebral malformations have not been described in this disorder. The authors suggest that there could be associated X linked other disorders contributing to these ¢ndings and the dismal evolution of our patient.

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TWO PATIENTS WITH FRUCTOSE-1,6-DIPHOSPHATASE DEFECT AND LATE DIAGNOSIS Michot C1, Valayannopoulos V1, Barth M1, Romano S1, Arnoux JB1, Baussan C2, Brivet M1, de Lonlay P1 1 Metabolic Center, Necker Hospital, Paris, France, 2Biochemistry, Biceªtre, France Background: Fructose-1,6-bisphosphatase de¢ciency is a disorder characterized by impaired gluconeogenesis, of excellent prognosis if diagnosed. Here we report on two patients with a late diagnosis. Patient 1. A girl, born to cousin Turkish parents, presented with vomiting and acidosis at age 36 days. She was operated on a duodenal malformation at age 1 mth. At age 14 months, a metabolic acidosis was noted (bicarbonates 1 mmol/L) during gastroenteritis. Glycemia was 2.9 mmol/L, and was associated with hepatic dysfunction and lactate in urine. After that, the patient presented permanent hyperlactatemia (4^5 mmol/L). She displayed several episodes of lactic acidosis without triggering factors, during which glycaemia had not been paid attention to. Patient 2. A boy, born to cousin Marocan parents, had a sib died at age 4 years in Morocco, with a previous notion of hypoglycaemia and hepatomegaly noted at age 2 years. At age 6 months, he presented a hypoglycaemia (1.9 mmol/L) during gastroenteritis, with bicarbonates 15 mmol/L, pH 7.38, plasma lactate 10 mmol/L, high liver enzymes and ketonuria. Recurrent hypoglycaemia occurred at a short fasting with permanent hepatomegaly. Glucagon test was positive at 5 min, then negative (glycaemia 1.4 mmol/L) after 15 min. Results: For both, the diagnosis of fructose-1,6-diphosphatase de¢ciency has been performed and con¢rmed by molecular study. Conclusion: We report here two patients with fructose-1,6diphosphatase de¢ciency; one with permanent hyperlactacidemia for whom hypoglycaemia has not been taken into account, the other with recurrent hypoglycaemia at short fasting mimicking glycogen storage defect.

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SAFETY OF LONG TERM G-CSF ADMINISTRATION IN TWO SIBLINGS WITH GLYCOGEN STORAGE DISEASE TYPE IB Ito T1, Nakajima Y1, Yokoi K1, Sugiyama N2, Togari H1 1 Dept Pediatr, Nagoya City Univ Med, Nagoya, Japan, 2Dept Pediatr, Aichi-Gakuin Univ Pharm, Nagoya, Japan Background: Glycogen storage disease (GSD) type Ib is caused by a defect in the transport of glucose-6-phosphate into the microsome. Patients with GSD type Ib shows the same symptoms of type Ia, in addition neutropenia and impaired neutrophil function. These patients are troubled by recurrent bacterial infection such as stomatitis, otitis media and furuncles. G-CSF is used for the treatment of the neutropenia, but in the long term therapy, speci¢c antibody production and malignant cell induction are concerned. Here we present GSD Ib siblings treated with G-CSF administration for over 10 years and continuously monitored production of antibody against G-CSF and monosomy-7 which is characteristic chromosome aberration of G-CSF related leukemia. Case 1: A 13-year-old boy. In his neonatal period, neutropenia was pointed out. At two months old, hepatomegaly was observed and he was diagnosed as GSD Ib after close investigation. He had been treated with noctournal nasogastric infusion and uncooked cornstarch, and at the age of two years; G-CSF hypodermic injection was started. Case 2: An 11-year-old brpther was pointed out hypoglycemia and neutropenia at the age of two months. The same treatment with his brother was started after the diagnosis of GSD Ib and G-CSF injection was started at 8-month-old. Method: Blood samples were serially taken from the patients to analyze G-CSF antibody and monosomy-7. Result and discussion: Neither G-CSF antibody nor monosomy-7 was detected in the both patients. G-CSF administration was safe and useful therapy for the GSD Ib siblings.

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ABSENCE EPILEPSY AND/OR MYOCLONIC EPILEPSY IN CHILDREN, THINK OF GLUT1 DEFICIENCY SYNDROME (TWO CASE REPORTS) Chabrol B1, Mansour H1, Cano A1, Milh M2, T|cus I2, Seta N3 1 Metab Ref Cent, Univ Child Hosp T|mone, Marseille, France, 2Neuroped Unit, Univ Child Hosp T|mone, Marseille, France, 3Biochemical Lab, Hoªpital Bichat, Paris, France Background: GLUT-1 de¢ciency is a disease that is characterized by acquired microcephaly, spasticity, developmental delay, hypotonia, and ataxia; as well as absence, myoclonic and partial seizures, refractory to anticonvulsive drugs, but responding to ketogenic diet. Methods: We report the cases of two children with Glut-1 de¢ciency. The ¢rst patient, who has an acquired microcephaly started having abnormal eye movements by the age of 4 months, then he started having head lags with myoclonic episodes, and gaze ¢xation followed by an episode of hypotonia of 30 min duration. Dystonic movements and ataxia appeared shortly after. The second patient presented an absence of seizures by the age of two years, at twenty episodes/day, mainly the morning, along with myoclonic movements. At eight years of age the patient started having gait disturbance and ataxia following long walk, and was considered as severe myoclonic epilepsy. Both patients have developmental delay, none showed a response to classical antiepileptic drugs. EEGs showed generalized paroxystic activities. Results: In both of the patients, decreased CSF glucose level was found, as well as a CSF glucose/blood glucose level ratio less than 0.46. The molecular studies found SLC2A1 gene mutation, and ketogenic diet was started. Conclusions: GLUT-1 de¢ciency syndrome remains an under diagnosed entity. This diagnosis should be considered in the cases of early onset epilepsy with microcephaly and developmental delay, and £uctuation of neurological symptoms related to fasting conditions. A ketogenic diet will prompt clinical improvement.

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Patients a¡ected by glycogen storage disease type I (GSD I) show a reduced bone mineral content (BMC) and are at risk of osteopenia and its clinical complications (such as fractures). A recent study evidenced an abnormal bone mineral content also in patients a¡ected by GSD III. The bone mineral density (BMD) in 11 patients with GSD I (5 type Ia, 6 type Ib) and 5 with GSD III were also investigated by dual energy X-ray absorptiometry (DEXA). Measurements were recorded at L2^L4 vertebrae level; Z-scores were calculated by comparing BMD with age (6^16 years) and aged and sex-matched reference values according to the manufacturer's internal reference database. All patients were supplemented with calcium from age of diagnosis. Three patients showed osteoporosis (1 GSD Ia, 2 GSD Ib), 6 osteopenia (3 GSD III, 2 GSD Ia, 1 GSD Ib) while the others had a normal lumbar spine BMD. Literature shows that osteopenia develops in adolescence or adulthood; in our experience also two prepuberal patients showed reduced BMD. According to literature data, patients receiving G-CSF don't signi¢cantly di¡er from the others. Our data con¢rm that osteopenia and osteoporosis are related to metabolic control in patients with GSD: in fact we evidenced poor metabolic control and chronic lactic acidosis in 2 of 3 patients (1 GSD Ia and 1 GSD Ib) with osteoporosis; the third patient is a¡ected by in£ammatory bowel disease which could in£uence calcium and phosphate absorption. Further studies are needed to understand the correlation between dietary/metabolic treatment and bone mineralization.

In addition to the typical signs and symptoms of glycogen storage disease type I (GSD I), patients a¡ected by subtype Ib usually present with neutropenia and neutrophil dysfunction. We here report on two patients with GSD Ib who never required G-CSF (granulocyte colonystimulating factor) therapy. The ¢rst one (M.C.) is a 28-year-old boy with neutropenia and neutrophil dysfunction who never su¡ered of severe recurrent infections. The second one (C.S.) is a 25-year-old boy who never developed neutropenia. Case 1: the diagnosis was performed when he was 21 months old (adopted child) by liver biopsy and genetic analysis showing 350 DelG homo (exon 2) mutation. His neutropenic status started at 6 years, with a leukocyte count ranging between 3000 and 4000/mm3 (neutrophil 8^10%). Except for a Salmonella enteritis at the age of 5, recurrent oral aftosis represents the only remarkable sign of neutropenia/neutrophil dysfunction. Case 2: C.S. was diagnosed by enzymatic analysis when 1 month old (brother a¡ected by GSD Ib), with recognized mutation 1211^1212 DEL CT-HOMO, exon 8. After being diagnosed C.S. has never developed any severe infection. According to our reports further studies are required to identify the real mechanisms involved in the de¢nition of GSD Ib phenotype.

BONE MINERALIZATION IN GSD I AND III Riva E, Paci S, Giulini Neri I, Gasparri M, Cagnoli G, Bonza M, Agostoni C Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy

ABSENCE OF SEVERE RECURRENT INFECTIONS IN GSD IB Paci S, Gasparri M, Giulini Neri I, Cagnoli G, Salvatici E, Giovannini M Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy

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CASE REPORT: GSD TYPE I AND PREGNANCY Paci S, Gasparri M, Giulini Neri I, Bonza M, Salvatici E, Giovannini M Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy Pregnancy in women a¡ected by glycogen storage disease (GSD) type I has been rarely reported. We describe a case of successful pregnancy in a 24-year-old patient with GSD type Ib with presumed partial de¢ciency followed in our department with monthly controls. The metabolic control was poor before pregnancy, but then become optimal. Only two symptomatic hypoglycaemic episodes were recorded at the 10th and 32nd weeks of gestation treated i.v. 10% glucose up to stabilization. Blood liver parameters and lipid pro¢le remained stable, with a persistent status of lactic acidosis. Increased uric acid levels were observed after stopping allopurinol therapy, with further normalization after post-delivery reintroduction. Kidney function worstened, with onset of hyper¢ltration (214 ml/min), then followed by proteinuria (229 mg/L) persisting after delivery. Blood pressure remained within a normal range. Abdominal ultrasounds showed absence of liver adenomas through all pregnancy, with normal intrauterine growth. Labour and delivery progressed uneventfully to the 38th week of gestation, when a male infant weighing 2700 g (AGA) was delivered. The child is healthy with a good psychomotor development at 6 months of age. Liver ultrasounds performed 6 weeks after delivery showed an adenoma at the second hepatic segment (maximum diameter 20 mm) even if signi¢cant increase in size or number of liver adenomas during or after pregnancy are not reported. An optimal strict metabolic control during pregnancy in GSD I female patients is the pre-requisite of a favourable infant's outcome, as suggested by the described case.

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GLYCOGEN STORAGE DISEASE TYPE IX (GSD IX) WITH AN UNUSUAL PHKB MUTATION PRESENTING WITH DEVELOPMENTAL DELAY, DEAFNESS, HYPOTONIA AND HEPATOPATHY Beauchamp NJ1, Blackburn JH1, Kirk R1, Sharrard M1, Ramaswami U2 1 She¤eld Children's NHS Foundtn Trust, She¤eld, UK, 2Addenbrooke's University Hospital, Cambridge, UK Introduction: Mutations in the PHKB gene result in mild GSD IX characterised by mild hepatomegaly, liver dysfunction and occasionally hypotonia. PHKB is alternatively spliced in liver and brain using exon 27 and in muscle using exon 26. Case Study: A female infant was diagnosed with sensory neuronal hearing loss at 7 months and is awaiting cochlear implants. She also has signi¢cant gross motor and speech delay. At 15 months she experienced an apparent life threatening episode, with choking, rigidity and breathing di¤culties. On examination she had 5 cm hepatomegaly and was £oppy with central and peripheral hypotonia. Transaminases, CK, cholesterol, triglycerides and lactate were elevated. Height and weight were on the 50th percentile. Hypoglycaemia was not observed following a 17 h fast. Pre-prandial lactate (1.5 mmol/L) was normal but raised post glucose load (5.5 mmol/L). Transaminase levels continue to be elevated. Enzymology revealed a reduced erythrocyte phosphorylase kinase activity 0.6 mmol/min/g Hb (NR: 10^90). The CK has now normalised. Results: Sequence analysis of PHKG was negative. Analysis of PHKB revealed a single base deletion, c.2088delT, p.Ser697ValfsX28 in exon 23 and a missense mutation, c.2326C4T, p.Arg776Cys in the alternatively spliced exon 27. Conclusions: This is the ¢rst reported missense mutation in the tissue speci¢c, di¡erentially spliced, exon 27 of PHKB. The expected e¡ect would be normal activity in muscle but reduced in liver and brain. The degree of gross motor delay and hypotonia are unusually severe for GSD IX. Deafness has not been previously reported.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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IMPROVEMENT IN THE MANAGEMENT OF PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE I (GSD-I) USING UNCOOKED GLYCOSADE COMPARED TO UNCOOKED CORNSTARCH Bhattacharya K1, Lilburn MF1, Morley DW2, Maillot F3, Lee PJ1 1 Charles Dent Metabolic Unit, Nat Hosp Neu, London, UK, 2Dept Envion Change, Uni Col Lond, London, UK, 3Cent Hosp Reg et Uni de Tours, Tours, France Aim: To assess short-term metabolic control and long-term starch use of Glycosade (GA) compared to uncooked cornstarch (UCCS) in adults with GSD I. Method: Randomised double-blind crossover trial of 10 adults with GSD I, (age 16^38, six male) After an individualised fast, subjects were randomised to take a 50 g starch-load of either GA or UCCS. The starch-load terminated when the blood glucose was 53.0 mmol/L or the subject felt subjectively hypoglycaemic. Glucose and lactate data were assessed by 2 blind investigators and a starch administration schedule recommended. Product was delivered in coded sachets and starch intake was monitored for the following 16 weeks. After a washout period, the protocol was repeated with the alternative product. Results: 4 subjects failed to establish therapy on the cross-over limb (2 GA and 2 UCCS). Of the 7 paired starch load data, median duration of normoglycaemia was longer with GA (6.5 h vs 5 h; p = 0.059), there was a slower decrease in the glucose curve (0.357 mmol/h vs 0.632 mmol/h; p = 0.028), a lower glucose curve in the ¢rst 4 h (p50.02) and lower median insulin (p50.02) and area under insulin curves (p = 0.03). Two of six subjects took 50% or less GA compared to UCCS and one took more GA. Plasma triglycerides, cholesterol and uric acid were unchanged after each phase of starch use. Conclusion: Glycosade leads to a signi¢cant reduction in insulin release and to reduced starch use in some GSD patients.

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MOLECULAR GENETIC DIAGNOSIS OF GLYCOGEN STORAGE DISEASE (GSD) TYPE III: EXPERIENCE FROM A LARGE INTERNATIONAL COHORT Santer R1, Tsiakas K1, Bergmann J1, Steuerwald U2, Sovik O3, Sentner CP4, Smit GPA4, Steinmann B5, Gal A6, Ullrich K1 1 Dept Pediatr, Univ Medical Center, Hamburg-Eppendorf, Germany, 2 Dept Occup Publ Health, Torshavn, Faroe Islands, Denmark, 3Dept Pediatr, Univ Child Hosp, Bergen, Norway, 4Dept Pediatr, Univ Child Hosp, Groningen, Netherlands, 5Dept Pediatr, Univ Child Hosp, Zurich, Switzerland, 6Inst Human Genet, Hamburg, Germany Due to the small size of the genes and the existence of common mutations, genetic diagnosis has become the accepted standard in GSD Ia and I non-a. Here, we report our experience with molecular genetic diagnosis in GSD III (amyloglucosidase de¢ciency). Methods: Automated direct sequencing of the AGL gene in 53 GSD III patients from 46 not obviously related families from all over the world. Results: Two patients carried previously described exon 3 mutations and su¡ered from GSD IIIb; all others were IIIa cases. Among the latter, 7 cases from the Faroe Islands were homozygous for the known c.1222C4T [p.R408X] mutation. Likewise, 3 of 4 patients from the Bergen area, in Norway (a region with a previously reported local cluster of cases [Moe PJ. Glycogen storage disease in Norway. Acta Paediatr Scand 1972]) were also found to be AGL p.R408X homozygous, supporting the notion that this mutation had occurred in Scandinavia at the age of the V|kings who brought it to the Faroe Islands. The next most common mutations in this study were c.1020delA [p.E340DfsX8; in 3 families], c.753-6delCAGA [p. L250fsX23], c.2590C4T [p.R864X], and c.3980G4A [p.W1327X; all 3 in 2 Turkish families]; all others were found in single families. Among the total of 39 mutations detected, 27 were novel. Conclusion: The study shows that AGL mutations are very variable and that most are con¢ned to single families. Nevertheless, also in GSD III molecular genetic diagnosis is feasible and is suggested to replace more invasive techniques in typical cases.

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ANTIOXIDATIVE DEFENCE IN PEDIATRIC PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE IA AND III Kalkan Ucar S1, Coker M1, Sozmen E2, Anik A1, Goksen Simsek RD1, Darcan S1 1 Div Ped Metab End, Ege Univ Child Hosp, Izmir, Turkey, 2Div Bioch, Ege Univ Hosp, Izmir, Turkey Patients with glycogen storage disease type Ia (GSDIa) and III (GSDIII) do not develop premature atherosclerosis despite hyperlipidemia. The aim of the study was to investigate the whole antioxidative status in GSDIa and III patients. We measured lipid pro¢le, lipid peroxidation products, antioxidative parameters: total antioxidant activity, antioxidant enzymes (catalase, superoxide dismutase, paraoxonase, arylesterase), aqueous (vitamin C, uric acid, bilirubin) and lipid-soluble antioxidants (alpha-tocopherol, beta carotene). Alterations in serum copper, zinc, and iron concentrations and their carrier proteins: ceruloplasmin, transferin, albumin were also investigated. The study included 50 individuals: 22 with GSDIa, 9 with GSDIII, and 19 healthy subjects. The patients with GSDIa showed a marked hypertriglyceridaemia, GSDIII patients demonstrated hypercholesterolemia with elevated LDL-cholesterol and decreased HDL-cholesterol levels. Lipid peroxidation levels were increased in both GSDs. The antioxidant activity was elevated in GSDIa. The activities of antioxidant enzymes in GSDs patients were not di¡er from controls. Uric acid and alpha-tocopherol levels were increased, whereas beta-carotene was reduced in both GSDs groups. Serum copper and iron levels were signi¢cantly higher in GSDIa and GSDIII, respectively. However, ceruloplasmin levels were decreased in both groups. Zinc, transferrin and albumin showed no signi¢cant di¡erences between groups. In conclusion, this study supplied the data for antioxidative defense in patients with GSDs. Normal activity of antioxidant enzyme mainly contributed by elevated uric acid levels may protect the dyslipidemic GSD patients from atherosclerosis. Higher levels of copper in GSD Ia, iron in GSDIII, and decreased ceruloplasmin levels in both GSD are other main results of the study.

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A CROSS-SECTIONAL SURVEY OF THE DIETS OF CHILDREN AND ADULTS WITH GLYCOGEN STORAGE DISEASE TYPE 1 (GSD-I) Bhattacharya K1, Lilburn MF1, Orton R2, Dixon M3, Lee PJ1 1 Charles Dent Metabolic Unit, Nat Hos Neu, London, UK, 2Gastro Dept, Great Ormond St Hos, London, UK, 3Dietetic Department, Great Ormond St Hos, London, UK Aim: To document current nutritional intake of patients with GSD-I in one centre with a view to making future recommendations. Method: A cross sectional survey of 20 patients (age 3^42, 8 female, 8 GSD Ib), using a prospective diet diary and nutrition related biochemistry. Analyses of intake were performed on Dietplan 6 software (Forest¢eld, Horsham, UK) Data outputs including energy intake, macronutrient and micronutrient composition were related to UK dietary reference values, and age and sex matched mean UK population intakes. Results: Mean carbohydrate intake was 67.8% of total energy intake across the group, although mean absolute energy intake was 110% of that recommended. Mean absolute carbohydrate intake was 174% of age matched controls and 147% of recommendations for GSD from published literature. Protein intake was broadly adequate but fat intake ranged from 10.2% to 32.5% (mean 21%) of total energy intake. Two subjects, however, also had low protein intake with serum V|tamin B12 concentration below the reference range. Calcium intake failed to meet recommendation in 12 (60%), whereas in 4 (20%) the intake was 176 254% of recommendations. Inadequate selenium intake was predicted from assessment in 60% of individuals and was below the plasma reference range in 30%. Conclusion: The intensive carbohydrate therapy of patients with GSD-I can lead to life-long disorder in eating patterns. Many adults in particular do not require such intensive therapy and are at risk of varied nutritional de¢ciencies and psychosocial disturbance due to their substantial di¡erence from the general population.

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COAGULOPATHY WITH SEVERE VON WILLEBRAND FACTOR (VWF) DEFICIENCY IN A FAMILY WITH GLYCOGEN STORAGE DISEASE IA (GSD-IA) NOT CORRECTED BY GLUCOSE INFUSION Sharrard MJ1, Payne JH2, Gillett GT3, Vora AJ2 1 Paediatrics, Children's NHS Trust, She¤eld, UK, 2Haematology, Children's NHS Trust, She¤eld, UK, 3Clin Bioch, Teaching Hospitals NHS Trust, She¤eld, UK

BONE METABOLISM IMPAIRMENT IN GLYCOGEN STORAGE DISEASE TYPE 1: A CASE^CONTROL STUDY Melis D1, Parenti G1, Pivonello R2, Della Casa R1, Balivo F1, Cozzolino M1, Gaudieri V1, Minichini L1, D'Elia F1, Di Vuolo L3, Lombardi G2, Colao A2, Andria G1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2Dept of Molecular and Clin Endocr Metab, Naples, Italy, 3Physiol. Nutrition Unit, Dept Neuroscienc, Naples, Italy

Introduction: Abnormal bleeding and bruising associated with VWF de¢ciency are complications of GSD-Ia. It is recommended that coagulopathy is corrected with intravenous glucose infusion prior to surgery. Case history: A 13 year old girl with GSD-Ia (genotype c.79delC; p.Gln27fs, c.231G4A; p.Trp77X) presented with severe facial bruising following mild blunt trauma. She had easy bruising previously, but had undergone portacath insertion without signi¢cant bleeding. She had a prolonged APTT (40.0 s, normal 28.7^37.6), with normal PT and ¢brinogen; previously APTT had been normal. Factor VIIIC, vWF(Rag) and FVIIIRistCofactor were low (0.43 U/mL, [normal 0.5^1.50], 0.15 U/mL, [0.46^1.46], 0.05 U/mL, [0.50^1.72] respectively), indicating severe type IIa vWF de¢ciency. The bruising resolved with factor concentrate. Following a 48 h 10% glucose infusion, the APTT decreased slightly (38.1s) whilst vWF (Rag) and FVIIIRistCofactor showed no increase. Her 8 year old sister with GSD-Ia has similar VIIIc, vWF (Rag) and FVIIIRistCofactor values. Although she bruises easily, tonsillectomy and portacath insertion were uneventful. The 20 year old older sister with GSD-Ia has a normal APTT but low FVIIIRistCofactor (0.23 U/mL). She had undergone dental extractions, liver biopsy and multiple central venous catheterisation without abnormal bleeding, and was not menorrhagic. Mother denied abnormal bleeding or bruising, but father was not available. Conclusion: In GSD-Ia there may be severe vWF de¢ciency with normal coagulation times, which is not corrected by glucose infusion. All GSD-Ia patients should be investigated with clotting factor assays and vWF de¢ciency treated with intermediate purity factor concentrate prior to surgery or at times of severe trauma or bleeding.

Background: Impaired bone metabolism is a frequent complication of glycogen storage disease type 1 (GSD1). Material and methods: Twenty-two patients (15 GSD1a; 7 GSD1b) and forty-four controls were enrolled. Biochemical parameters of mineral metabolism, pituitary and gonadic hormones and bone mineral density (by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound of proximal phalanges (QUS)) were evaluated in patients and controls. Results: In GSD1a patients, alkaline phosphatase (p = 0.014) and parathormone (PTH) (p = 0.008) serum levels were lower and calcium excretion (p = 0.0001) higher than in controls. Higher though not signi¢cant magnesium serum levels and lower magnesium excretion were also detected in GSD1a patients than in controls (p = 0.08). In GSD1b patients, calcium serum levels were lower (p = 0.009), while calcitonin (p = 0.019) and osteocalcin (p = 0.009) levels higher than in controls. In all patients hydroxyproline excretion (GSD1a: p = 0.022; GSD1b: p = 0.000) was higher than in controls. DEXA (GSD1a: p = 0.006, GSD1b: p = 0.0001) and QUS Z scores (GSD1a: p = 0.0001, GSD1b: p = 0.033) were lower in patients than in controls. In GSD1a patients DEXA Z score directly correlated with age (p = 0.002), IGF-II (p = 0.035), ALS levels (p = 0.037) and inversely with hydroxyproline excretion (p = 0.007); QUS Z score correlated with PTH (p = 0.025) and V|tamin D levels (p = 0.037). In GSD1b patients DEXA data were correlated to BMI (p = 0.007) and calcitonin levels (p = 0.041). QUS Z score correlated with free IGF serum levels (p = 0.008). Conclusion: GSD1a and GSD1b patients show a di¡erent impairment in the biochemical parameters involved in mineral metabolism. Bone mineral density data are associated to several factors known to be in£uenced by metabolic control.

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THE GH-IGF AXIS IN GLYCOGEN STORAGE DISEASE TYPE 1 (GSD1): EVIDENCE OF DIFFERENT GROWTH PATTERNS AND IGF LEVELS IN PATIENTS WITH GSD1A AND GSD1B Melis D, Pivonello R, Parenti G, Gaudieri V, Della Casa R, Salerno M, D'Elia F, Piccolo P, Lombardi G, Colao A, Andria G Dept of Molecular and Clin Endocr Metab, Naples, Italy Background: GSD1 patients show growth impairment but no de¢nitive data are available on their GH-IGF axis. Aim: The aim of the current study was to investigate the GH-IGF system in GSD1 patients and to compare it in the two forms of the disease, GSD1a and GSD1b. Patients and Methods: Ten GSD1a, seven GSD1b and 34 sex, age and BMI-matched healthy controls entered the study. Baseline GH, IGFI, IGFII, IGFBP1, IGFBP3, ALS, GH response to GHRH plus arginine test and the presence of anti-pituitary (APA) and anti-hypothalamus (AHA) antibodies was investigated. Results: Short stature was detected in 10.0% of GSD1a (chi2 = 1.86, p = 0.35), 42.9% of GSD1b patients (chi2 = 7.00, p = 0.02) and in none of the controls. Only in GSD1b patients, serum total and free IGFI levels were lower than in controls (p = 0.0001). IGFII (p = 0.001) and insulin (p = 0.02) serum levels were higher in patients than in controls. Serum IGFII levels correlated with height SDS (p = 0.01). At dynamic test, a decreased GH response was found in patients compared to controls (p = 0.0001). APA were demonstrated in 42.9% of GSD1b and in no GSD1a patient (chi2 = 6.8, p = 0.02). The presence of APA inversely correlated with GH response to GHRH plus arginine test (r = ^0.87, p = 0.02). Conclusions: GSD1a and GSD1b patients have a di¡erent impairment of GH-IGF pathway. GSD1a patients have a functional GH de¢ciency, whereas GSD1b have an impaired GH secretion probably due to the presence of APA. The increased IGFII serum levels might be responsible of the improved growth pattern observed in patients under strict dietary treatment.

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GLYCOGENOSIS IA IN A WOMAN WITH A MITOCHONDRIAL DISORDER AND ALTERATIONS IN THE PROTROMBIN GENE Arranz JA1, Goèmez-Argu«elles JM2, Mart|èn MA3, de Diego C4, Blaèzquez A3, del Toro M1, Riudor E1 1 Unit Metab, Hosp Univ Vall d'Hebron, Barcelona, Spain, 2Serv Neurol, Hosp Nac Parap, Toledo, Spain, 3Lab Enf Mit, Hosp Univ 12 octubre, Madrid, Spain, 4Serv Genet, Hosp V|rgen de la Salud, Toledo, Spain A 3 month old girl born to consanguineous parents presented with a typical clinical-biochemical picture of GSD Ia, i.e. hypoglycaemia, hepatomegaly, hypertriglyceridemia, lactic acidosis, positive glucagon test. GSD Ia was con¢rmed by a decrease of glucose-6-phosphatase (G6Pase) activity. A novel homozygous missense mutation (p.W70R) in the G6PC gene was identi¢ed in the proband and also in her asymptomatic father and two brothers, but was absent in 120 control alleles. W70 residue is near to the active centre in a highly conserved region where similar mutations (p.W63R, p.W77R) have been reported. Hypertriglyceridemia, hypertension and obesity remained until age of 21 when she presented with a sudden loss of visual acuity, progressive leg weakness, sensorineural deafness and hypercoagulability which was associated with heterozygosis for the genetic susceptibility factor G20210A in prothrombin gene. She died from pneumonia 6 months later. A combined defect of mitochondrial respiratory chain complexes and a mild reduction of mitochondrial DNA copy number were observed. These puzzling ¢ndings points to the in£uence of mitochondrial dysfunction on the clinical expression of GSD Ia otherwise silent in related people- and perhaps in the late-onset problems in coagulation status.

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MOLECULAR STUDY OF 11 CASES OF GLUT1 DEFICIENCY SYNDROME Vuillaumier-Barrot S1, Bahi-Buisson N2, Odent S3, Mayer M4, Chaigne D5, de Saint-Martin A5, Flori E6, Bekri S7, Drouin-Garraud V8, Ra¡o E9, Chabrol B10, Julia S11, Le Bizec C1, Seta N1 1 AP-HP, Biochimie, Hoªp Bichat, Paris, France, 2Neuropeèdiatrie, Hoªp Necker, Paris, France, 3Geèneètique Meèdicale, CHU, Rennes, France, 4 Neuropeèdiatrie, Hoªp Trousseau, Paris, France, 5Peèdiatrie, CHU Hautepierre, Strasbourg, France, 6Cytogeèneètique, Chu Hautepeirre, Strasbourg, France, 7Biochimie, CHU, Rouen, France, 8Geèneètique Meèd, CHU, Rouen, France, 9Meèd Inf1, Hoªp d'Enfants, CHU, Nancy, France, 10 Neurologie Peèd, CHRU, Marseille, France, 11Geèneètique Meèd, Hoªp Purpan, Toulouse, France Background: Autosomal dominant Glut-1 de¢ciency syndrome (GLUT1 DS, OMIM 606777) resulting in impaired glucose transport across the blood^brain barrier is characterized by infantile seizures, developmental delay, acquired microcephaly, ataxia, and hypoglycorrachia (glycorrachia/glycaemia 40.4). Previous data demonstrated that mutations in SCL2A1 gene encoding GLUT1 correlates with hypoglycorrachia and reduced erythrocyte glucose transporter activities, suggesting that the latter is not mandatory for the diagnosis. In the last 3 years, 22 patients were addressed for clinical suspicion of GLUT1 de¢ciency for who direct sequencing of SCL2A1 gene was performed. Methods: We sequenced the 10 exons and £anking introns of SCL2A1 gene and performed quantitative PCR when no mutation was detected. Results: 11 patients out of 22 (median diagnosis age: 10 years) were found to harbor 11 di¡erent de novo heterozygous SCL2A1 mutations con¢rming the diagnosis of GLUT1 DS. Nine were novel mutations including 2 frameshift: c.737__740del AAGA and c.32__33del GC; 1 splicing defect: c.1257C4T (p.Gly419AlafsX28); 3 nonsense: c.338C4A (p. Ser113X), c.143G4A (p.trp48X) and c.121G4T (p.Glu41X); 3 missense: c.68C4T (p.Ser23Phe), c.884C4T (p.Thr292Me) and c.388G4A (p.Gly130Ser) which were predicted to a¡ect protein function by phenotype prediction programs and not to be a common SNP (4100 healthy individuals screened). The two remaining mutations (c.436G4A, p.Glu146Lys and one large deletion from exon 2 to 10) were previously described. Conclusions: Molecular screening of SCL2A1 gene seems e¤cacious in the diagnosis of GLUT1 DS. Implication for clinicians may be important considering the viable treatment option, ketogenic diet.

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DUARTE (DG) GALACTOSEMIA: A STUDY OF BIOCHEMICAL AND NEURODEVELOPMENTAL ASSESSMENT IN CHILDREN DETECTED BY NEWBORN SCREENING F|cicioglu C, Thomas N, Yager C, Gallagher PR, Hussa C, Forbes BJ Children's Hospital of Philadelphia, Phialadelphia, USA The newborn screening for galactosemia has shown a high prevalence of partial galactose uridyl transferase de¢ciencies such as Duarte (D/G) galactosemia. Study objective: To determine whether (a) there is any clinical impact of D/G galactosemia on development (b) there is a relationship between outcome and biochemical parameters in patients who receive no treatment. Study population: 28 children with DG galactosemia. Group I: 17 children were on a lactose-restricted diet in the ¢rst year of life. Group II: 11 children were on regular diet since birth. Methods: Developmental, physical and ophthalmologic assessments were done on both DG children. RBC gal-1-P, and urine galactitol were monitored during the follow-up visits in every child with D/G galactosemia. Gal-1-P, urine galactitol, liver function tests, and FSH were tested at the time of study visit. Results: The groups had statistically signi¢cant di¡erences on RBC Gal1-P and urine galactitol at the 2 week, 1 month, 6 month, and 1 year time points. There was no statistical di¡erence of Gal-1-P or urine galactitol in Group I and Group II at the time of study. The groups had statistically signi¢cant di¡erences on adaptive scores, but not on language or IQ. None of the DG subjects had abnormal liver function at the time of diagnosis or the study visit. The FSH levels were normal . There were no statistically signi¢cant relationships between the ¢rst year metabolic values and developmental outcomes. Conclusions: The data presented here indicate that clinical and developmental outcome in DG galactosemics is good regardless of any diet changes.

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Background: Galactosemia usually presents in infancy. It has high mortality, if untreated. In Estonia selective screening for sugar metabolism defects is o¡ered when the hospitalized infant has suggestive symptoms: jaundice, hypoglycemia, weight loss, ascites. Methods: Sugars are quanti¢ed using HPLC (Aminex-HPX87H column, RI and UV detectors). Urine samples need no pretreatment. Pretreatment for plasma samples is the same as for amino acid (AA) analysis. AA's are determined using classical ion chromatography method. Body £uid samples are ¢xed with sulfosalicylic acid. Urine organic acid (OA) pro¢ling is made by GC/MS, quanti¢cation with standards. Results: In the ¢rst three years of screening we found seven patients with elevated galactose in urine and serum, classical galactosemia was con¢rmed four times. Following years we usually found 2^3 cases per year. In emergency samples blood galactose concentrations were over 20 mg/dl, in urine up to 60 g/L. Lactate in serum was highly elevated but glucose almost undetectable. AA and OA analyses showed changes referring to liver malfunction. GC/MS analyses showed highly elevated p-hydroxyphenyllactate, malate, fumarate and sugar peaks. On lactosefree diet most metabolites normalized in hours. Conclusions: The incidence of galactosemia in Estonia is high, about 1:13 000, but we are still missing older patients with unspeci¢c symptoms. Classical galactosemia is the second most frequent metabolic disease in Estonia after PKU. We also have cases with higly elevated galactose at birth and normal range afterwards, which could be associated with immature hepatic functions. HPLC is quick (30 min) and reliable method for selective galactosemia screening.

Objective: A case series study of 25 patients a¡ected with galactosemia who were diagnosed among 547 high risk neonates and infants, from January 2003^January 2006. Introduction: Galactosemia is a rare inborn metabolic disease caused by enzymatic de¢ciency on galactose pathway, usually GALT. De¢native diagnosis is made by measurement of enzyme activity usually in RBCs. Materials and methods: 25 patients a¡ected with galactosemia were studied. Diagnosis was made by measuring enzyme activity in RBC by DBS-GALT enzyme analysis using BIO RAD kit and total galactose by Quantose kit. Results: 23 patients a¡ected with classic form, one galactokinase and one UDP-galactose; four epimerase (age: 3 day^18 years 17 male; 8 female). Age at onset: 11 cases 51 month, 3 cases 1^2 months, 9 cases 2 months^2 years respectively. Hepatomegaly with liver failure was discovered in 6, prolonged hyperbilirubinemia in 10, cataract in 7, renal involvement in 9, CNS in 13 including psychomotor retardation (11), (2), seizure (2). Two asymptomatic. GALT activity; less than 261 (homozygote): 9 cases, between 261^430 (heterozygote or variant): 9 between 430^800 (suspicious): 5, mutation detection analysis examined in 25% and revealed Q188R mutation in four. Improvement in CNS in 5. renal: 7 out of 9, liver failure 6 out of 6, bilirubin normalization 6 out of 6, cataract 4 out of 4, thyroid 4 out of 4. Conclusion: Though mass screening is still not available in Iran, early detection and early institution of therapy is possible. The outcome of properly treated children to be good except for CNS involvement.

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GALACTOSEMIA PATIENTS IN ESTONIA, 15 YEARS OF SELECTIVE SCREENING Krabbi K1, Kall K2, Laht T-M1, O¬unap K3, Joost K4, Zordania R4 1 Tallinn University of Technology, Tallinn, Estonia, 2Centr Chem Lab of Health Protection Insp, Tallinn, Estonia, 3Tartu University Hospital, Tartu, Estonia, 4Tallinn Children's Hospital, Tallinn, Estonia

GALT-ACTIVITY, GALACTOSE METABOLITES AND HORMONES DURING PREGNANCY IN A CLASSIC GALACTOSEMIA PATIENT Gubbels CS1, Gubbels CS2, Kuppens S3, Bakker JA2, Konings S4, Menheere P5, Spaapen LJ2, De Sain MG6, Wodzig WK5, Rubio-Gozalbo ME1,2 1 Dept of Paediatrics, Univ Med Cent, Maastricht, Netherlands, 2Lab Inherit Metab Dis, Univ Med Cent, Maastricht, Netherlands, 3Dept of Gynaecology, Catharina Hosp, Eindhoven, Netherlands, 4Dept of Internal Med, Catharina Hosp, Eindhoven, Netherlands, 5Dept of Clin Chemistry, Univ Med Cent, Maastricht, Netherlands, 6Metab Dis, W|lhelmina Child Hosp, Utrecht, Netherlands Background/Objectives: Premature ovarian failure (POF) is a common complication in women with classic galactosemia. However, pregnancies occur and may not be as rare as assumed, even when POF is diagnosed. We report a third pregnancy in a 26-year old patient compound heterozygous for the Q188R/ N314D-mutations, despite POF-diagnosis. Research in a rat-model demonstrated an increased galactose-1-phosphate uridyl transferase (GALT) activity during pregnancy, quickly dropping after delivery. Therefore, the patient's GALT activity before, during and after pregnancy is shown. Methods: Measurement of GALT activity in red blood cells (RBC), galactose and galactitol in blood and urine, 17-b-estradiol, follicle stimulating hormone (FSH), luteinizing hormone (LH), anti-mu«llerian hormone (AMH) and progesterone were performed in serum before, during and after the third pregnancy. Furthermore, an exogenous FSH ovarian reserve test (EFORT) was performed between her second and third pregnancy. Results: The performed EFORT showed no estradiol-response. The gonadotropins were in the postmenopausal range before her pregnancy, and hormonal values £uctuated during pregnancy and puerperium. Galactose and galactitol in blood and urine rose during the third trimester and dropped again during breastfeeding. The GALT activity measurements showed £uctuation during pregnancy and puerperium. Conclusions: The negative EFORT indicated the absence of ovarian reserve, which was supported by the pre-pregnancy hormonal levels. However, two months after the test the patient became pregnant, indicating a varying degree of POF. The rise in galactose and galactitol was as expected and might be explained by the £uctuation in GALTactivity, which is a new observation in humans.

TWENTY FIVE PATIENTS AFFECTED WITH GALACTOSEMIA, A REPORT FROM IRAN Zaman T1, Moradian R2, Mazdarani S1 1 Tehran University IEM Department, Tehran, Islamic Republic of Iran, 2 Tehran University IEM Department, Tehran, Tehran, Islamic Republic of Iran

SPECTRUM OF GALT MUTATIONS IN SPAIN AND PORTUGAL. SEVEN NEW MUTATIONS IN SEVENTEEN NEW PATIENTS Quintana E1, Gort L1, Moliner S1, Gonzalez-Quereda L1, LopezHernandez T1, Rivera I2, Santos Leite M2, V|larinho L3, Briones P4 1 Inst Bioq Clin ^ Hosp Cl|ènic, CIBERER, Barcelona, Spain, 2iMed UL, Faculty of Pharmacy, Universityof Lisbon, Lisboa, Portugal, 3Inst Gen Medica Jacinto de Magalhaes, Porto, Portugal, 4Inst Bioq Clin ^ Hosp Cl|ènic and CSIC, CIBERER, Barcelona, Spain Background/Objective: Classical galactosaemia is an autosomal recessive inherited metabolic disorder due to mutations in the galactose-1-phosphate uridyltransferase gene (GALT). We previously reported molecular analysis of 83 Spanish and Portuguese galactosaemic patients. Here we present the molecular results of seventeen additional unreported a¡ected individuals. Results: Twelve patients of Spain were analysed. We detected ¢ve alleles carrying p.Q188R, accounting for 21%. Other six alleles (25%) were identi¢ed with the mutation p.K285N. Remarkably, the two patients that were homozygous for this change were of North African origin. We also identi¢ed six novel mutations: p.Q9X, c.328+2T4C, p.I170N, p.C180F, p. V233L, p.P257L. Taking into account all the Spanish galactosaemic patients, mutation p.Q188R is still the most frequent mutation identi¢ed (44.4%). The second most frequent mutation is p.L195P (13.5%) followed by p.K285N (12.7%). The increase of the immigration experimented in Spain during the last years is clearly responsible for the change in mutation frequencies of some inherited diseases such as galactosaemia. F|ve new Portuguese patients were analysed. In ¢ve alleles p.Q188R was detected, representing 50%. One novel mutation was identi¢ed, p.F171C. Mutations p.L195P and p.K285N still remain undetected in Portuguese patients. Concerning the whole group of 37 Portuguese patients analysed so far, mutation p.Q188R remains the most frequently identi¢ed (56.7%). Conclusion: Our results con¢rm the already published observation that p. Q188R is the most frequent mutation in Iberian Peninsula among galactosaemic patients (48.5%). Moreover, our molecular analyses on these seventeen new galactosaemic patients provide seven novel mutations to the database with more than 200 disease-causing mutations already reported.

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Introduction: Hypoketotic hypoglycaemia and hypertriglyceridaemia are biochemical hallmarks of GSD I. They are caused by increased malonyl-CoA production from accumulating acetyl-CoA. MalonylCoA compromises the mitochondrial b-oxidation of long chain fatty acids via CPT1-inhibition which is part of the carnitine shuttle. Inhibition of the carnitine shuttle impairs energy balance and ketogenesis. As the oxidation of medium chain fatty acids (MFA) does not depend on this shuttle we studied the e¡ect of a MFA-enriched diet on metabolic control and growth in SnGSD SnI. Methods/Patients: An adult female, a twelve year old girl, a 1.8 year old boy with GSD Ia and a 6.5 year old girl with GSD Ib were enrolled. Their `classical' GSD I diet was enriched with up to 20 g MCT (oil and margarine) per day. Serum glucose, lactate, triglycerides, uric acid, acylcarnitine-pro¢les and ketone bodies as well as organic acids in urine were determined. Results: There were no clinical side-e¡ects, acid-base state, amylase and lipase in blood as well as organic acids in urine as biochemical parameters were normal. MFA-feeding resulted in lowering of lactate, triglyceride and uric acid levels in blood of patients with GSD I, reduced the amount of carbohydrates and total calories required to maintain euglycaemia and led to improvement in growth. Capacity of ketogenesis during hypoglycaemia increased. Conclusion: We observed a positive e¡ect of a MFA-enriched diet on metabolic control and growth of patients with GSD I.

Background: To obtain an overview of the incidence of the di¡erent glycogen storage diseases, age at diagnosis etc., we analysed these results over the past 25 years. Methods: Enzyme activities were measured in blood cells (490%), muscle, liver or ¢broblasts. Results: In the past 25 years in about 387 patients glycogenosis was con¢rmed enzymatically. The distribution of the di¡erent glycogenosis were type I 5%, type II 53%, type III 15%, type IV 4%, type V 7%, type VI 1%, type VII 0.3%, type VIII 0% and type IX 17%. The age of onset of the di¡erent GSD's were: type I all in the ¢rst year. Type II 38% infantile and 52% adult. Type III about 80% age below the age of three and about 15% was over 40 years old. Type IV all patients below the age of 3, one patient had adult polyglucosan body disease and was 49 years old. Type V 75% patients adult, 25% juvenile. Type IX most patients below the age of 3. The most remarkable ¢nding was an enhanced activity of debranching enzyme in all of our GSD IX patients, even in blood cells of a GSDIXa2 patient with normal activity of phosphorylase kinase in erythrocytes. Conclusion: Debranching enzyme activity should always be measured by suspicion of glycogen storage disease type IX and can be used as a marker for glycogen storage disease IX

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GLYCOGEN STORAGE DISEASE TYPE I: IMPACT OF MEDIUM CHAIN FATTY ACIDS ON METABOLIC CONTROL AND GROWTH Das AM, Lu«cke T, Meyer U, Hartmann H, Illsinger S Dept Paediatrics, Hannover Med. School, Hannover, Germany

TWENTY-ONE ADDITIONAL CASES OF FAMILIAL RENAL GLUCOSURIA: ABSENCE OF GENETIC HETEROGENEITY, HIGH PREVALENCE OF PRIVATE MUTATIONS, AND FURTHER EVIDENCE OF VOLUME DEPLETION Calado J1, Sznajer Y2, Metzger D3, Rita A4, Hogan M5, Kattamis A6, Scharf M7, Tasic V8, Greil J9, Brinkert F10, Kemper M10, Santer R10 1 Dept Genet, Fac Med Sci, New Univ Lisbon, Lisbon, Portugal, 2Genet Unit, Enfants Fabiola, ULB, Bruxelles, Belgium, 3Pediatr Endocrinol, BC Child Hosp, Vancouver, Canada, 4Dept Nephrol, Hosp Curry Cabral, Lisbon, Portugal, 5Div Nephrol, Mayo Clin, Rochester, USA, 6Dept Pediatr, Univ Athens, Athens, Greece, 7Pediatr Endocrinol Diab Center, Curitiba, Brazil, 8Dept Pediatr Nephrol, Child Hosp, Skopje, The FYR of Macedonia, 9Dept Pediatr, Univ Heidelberg, Heidelberg, Germany, 10 Dept Pediatr, Univ Med Center, Hamburg-Eppendorf, Germany Familial renal glucosuria (FRG) is a rare renal tubular disorder caused by mutations in the SLC5A2 gene. It is characterized by persistent glucosuria in the absence of hyperglycemia and any signs of generalized tubular dysfunction. In small series of patients previously reported, the molecular and phenotypic ¢ndings in FRG have been described. We have now extended this analysis to another 21 consecutive cases from 17 pedigrees, including 10 cases with severe glucosuria. Methods: Mutation analysis was performed by direct sequencing of the genomic SLC5A2 coding region. In 2 cases with severe glucosuria, basal plasma renin activity (BPRA) and serum aldosterone concentration (SAC) were determined. Results: W|thin the 17 pedigrees, we have identi¢ed a total of 20 di¡erent SLC5A2 mutations, with 15 being novel. In all glucosuric individuals tested, at least one SLC5A2 mutation could be identi¢ed. Heterozygous individuals were found to have mild glucose excretion whereas homozygous or compound heterozygous patients had severe glucosuria (10^86.5 g/1.73 m2/24 h). In 2 patients of the latter group, BPRA and SAC were found to be raised to an average of 4.6 fold and 3.1 fold the upper limit of the normal range, respectively. Conclusion: The identi¢cation of at least one mutated allele in every a¡ected individual in this cohort of 17 consecutively investigated families strongly suggests that genetic heterogeneity is not prevalent in FRG; in addition most mutations are private. Our ¢nding of an activation of compensatory mechanisms for salt loss may warrant more detailed studies of long-term hormonal and metabolic imbalances in patients with FRG.

OVER 25 YEARS ENZYMATIC DIAGNOSIS OF GLYCOGEN STORAGE DISEASES. ENHANCED ACTIVITY OF DEBRANCHING ENZYME IN PATIENTS WITH GLYCOGENOSIS IX Schoonderwoerd GC, van Diggelen O Dept Clin Gen, ErasmusMC, Rotterdam, Netherlands

PAROXYSMAL EXERCISE-INDUCED DYSKINESIA AND EPILEPSY DUE TO MUTATIONS IN SLC2A1, ENCODING THE GLUCOSE TRANSPORTER, GLUT1 Cassiman D1, Suls A2, Dedeken P2, Go¤n K3, Van Esch H1, Dupont P4, Kemp£e J4, Wuttke TV4, Weber Y4, Lerche H4, Afawi Z5, Korczyn AD5, Berkovic SF6, Ekstein D7, Kivity S8, Ryvlin P9, Claes LRF2, Deprez L2, Maljevic S4, Vargas A4, Van Dyck T2, Goossens D2, DelFavero J2, Van Laere K1, De Jonghe P2, Van Paesschen W1 1 Metabolic Ctr, Univ Hospitals, Leuven, Belgium, 2Neurol/Neurogenet, Antwerp, Belgium, 3Dept Neurol, Univ Hospitals, Leuven, Belgium, 4 Univ Ulm, Germany, 5Univ Tel Aviv, Tel Aviv, Israel, 6Univ Melbourne, Heidelberg West, Australia, 7Univ Medical Ctr, Jerusalem, Israel, 8 Children's Medical Ctr, Petach T|kvah, Israel, 9INSERM, Lyon, France Background: Paroxysmal exercise-induced dyskinesia (PED) can occur in isolation or in association with epilepsy, but the genetic causes and pathophysiological mechanisms are still poorly understood. Methods: We performed a clinical evaluation and genetic analysis in a ¢ve-generation family (56 members, of which 39 were examined, 22 with symptoms) with co-occurrence of PED and epilepsy in the absence of overt mental retardation, suggesting that this combination represents a clinical entity. PED was characterized by choreoathethosis, dystonia or both, a¡ecting mainly the legs. Predominant epileptic seizure types were primary generalized. Based on a whole genome linkage analysis we ¢nally screened SLC2A1, encoding the glucose transporter of the blood^brain barrier, GLUT1. Results: Heterozygous missense and frameshift mutations in GLUT-1, segregating in this and 3 other nuclear families with a similar phenotype, were found (total patient number 25). Pathogenicity of these mutations was proven by the demonstration of a reduced glucose uptake by the mutated transporters after transfection in Xenopus oocytes. A moderately decreased blood/cerebrospinal £uid glucose ratio (median of 0.52, vs normal 50.60) was found in the patient population. Functional imaging studies implicated alterations in glucose metabolism in the corticostriate pathways in the pathophysiology of PED and in the frontal lobe cortex in the pathophysiology of epileptic seizures. Three patients were successfully treated with ketogenic diet. Conclusion: Co-occurring PED and epilepsy can be due to autosomal dominant heterozygous SLC2A1 mutations, expanding the phenotypic spectrum associated with GLUT1 de¢ciency and providing a potential new treatment option for this clinical syndrome.

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PANCREATIC DOPAMINE METABOLISM IN CONGENITAL HYPERINSULINISM. A [18F]FLUORO-L-DOPA PET STUDY Ribeiro MJ1, Bellanneè-Chantelot C2, Valayannopoulos V3, Delzescaux T1, Jaubert F3, Aigrain Y3, Nihoul-Feèkeèteè C3, Brunelle F3, de Lonlay P3 1 CEA, DSV, I2BM, SHFJ, Orsay, France, 2Deèp Geèn, Hop PitieèSalpeªtrie©re, AP-HP, Paris, France, 3HÂp Necker-Enfants Malades, APHP, Paris, France Background: Neuroendocrine diseases are a heterogeneous group of entities with the ability to uptake amine precursors (as L-DOPA) and to convert them into biogenic amines (as dopamine) by decarboxylation. Among these diseases, congenital hyperinsulinism (HI) is secondary to either focal adenomatous hyperplasia or a di¡use abnormal pancreatic insulin secretion. If focal HI can revert by selective surgical resection, di¡use form requires subtotal pancreatectomy when resistant to medical treatment. We evaluated the use of positron emission tomography (PET) with [18F]£uoroL-DOPA to assess dopamine metabolism and its relationship with insulin production in HI. Methods: 53 children with HI were enrolled in this study. PET acquisition started between 30^60 min after injection of [18F]£uoro-L-DOPA. Three children were studied using a PET-CT scanner. For operated children, immunohistochemical detection of DOPAdecarboxylase was performed. Results: An abnormal focal pancreatic uptake of [18F]£uoro-L-DOPA was observed in 18 patients, whereas a di¡use uptake of the radiotracer was observed in the pancreatic area of the other patients. Patients with focal radiotracer uptake and patients with pancreatic di¡use radiotracer accumulation, unresponsive to medical treatment, were submitted to surgery. In focal HI, proinsulin and DOPAdecarboxylase were strongly enhanced in all of the aggregated abnormal beta-cells. By contrast, in di¡use forms, the intralobular islets had reduced staining for proinsulin and a normal level of DOPAdecarboxylase. The localization accuracy of the abnormal radiotracer uptake is improved with the use of PET-CT scanner. Conclusion: [18F]£uoro-L-DOPA PET is an accurate non-invasive technique to evaluate pancreatic dopamine metabolism allowing di¡erential diagnosis between focal and di¡use HI.

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SIGNAL SYMPTOMS OF IEM AND INCLUSIONS IN GSD IV AS AN EXAMPLE Stastna S, Chrastina P, Paulova M, Elleder M Inst Inherit Met Dis, Gen Teach Hosp, Prague 2, Czech Republic Signal symptoms of IEM are clinical, laboratory or imaging symptoms, rare and speci¢c for one or a small group of IEM. The smaller the group of IEM is, where the symptom could be found, the more speci¢c the symptom is and the easier and the faster is the way to reach the right diagnosis. Glycogen storage disease type IV (GSD IV) is an IEM with mutations in the GBE1 gene. Clinically, GSD IV is heterogeneous with hepatic or neuromuscular for m. Histopatologic and electronmicroscopic investigation reveals the signal symptom of GSD IV, intracytoplasmic inclusions containing accumulated ¢lamentous amylopectin-like polysaccharide called polyglucosan bodies or corpora amylacea. The only disorder with similar pattern is Lafora disease with myoclonic epilepsy and mutations in EPM2A and EPM2B/NHLCR1 gene. The ¢rst Czech patient with GSD IV exhibited signi¢cant hepatopathy with impaired blood clotting. Liver biopsy with polyglucosan bodies and absence of myoclonic epilepsy directly led to the diagnosis of GSD IV. Enzymatic investigation showed no activity of branching enzyme in leucocytes. Conclusion: Signal symptoms provide useful and cost-e¤cient opportunity to improve diagnostics of IEM. In GSD IV, intracytoplasmatic polyglucosan bodies are such a signal symptom, but liver biopsy is necessary. Enzymatic investigation is accessible, fast and simple. Supported by Grant 64165 by Ministry of Health of Czech Republic

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AUTOIMMUNE ENDOCRINE DISORDERS IN A PATIENT AFFECTED BY GLYCOGEN STORAGE DISEASE 1B: CAUSAL RELATIONSHIP BETWEEN NEUTROPENIA AND AUTOIMMUNITY? Melis D1, Parenti G1, Pivonello R2, Gaudieri V1, Della Casa R1, Salerno M1, D'Elia F1, Lombardi G2, De Bellis A3, Colao A2, Andria G1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2Dept of Molecular and Clin Endocr Metab, Naples, Italy, 3Dept Clin Exp Medic Surg, Sec Univ, Naples, Italy Background: Glycogen storage disease type 1b (GSD1b) is an inborn error of glycogen metabolism, caused by mutations in the glucose 6-phosphate translocase (G6TP) gene. GSD1b patients su¡er from chronic neutropenia and functional de¢ciencies of neutrophils, resulting in recurrent infections. It has been recently proposed that the peculiar haematologic ¢ndings of these patients are predisposing factors for autoimmune disorders, such as autoimmune thyroiditis, Myasthenia Gravis and Crohn's disease. Case Report: The patient was born from consanguineous parents. GSD1b diagnosis was performed by both enzyme studies and mutation analysis of the G6PT gene. When he was 6 months old, neutropenia and recurrent infections were detected and granulocyte-colony-stimulating-factor therapy was started. At the age of 7 years, for the presence of stomatitis aphthosa and perianal ulcers, ileocolonoscopy was performed, showing an unspeci¢c chronic in£ammation. When he was 10-year-old, a diagnosis of autoimmune thyroiditis with subclinical hypothyroidism was performed on the basis of the detection of increased both thyroid stimulating hormone and thyroid auto-antibodies levels. At the age of 11 years, thyroid function was normal and no therapy was needed. When he was 14-year-old, short stature with delayed bone maturation was observed. GHRH plus arginine test was performed showing a decreased GH response (peak = 7.8 ng/dl). High levels of anti-pituitary antibodies recognizing growth hormone (GH)producing cells (41:8) were detected and a diagnosis of autoimmune GHde¢ciency was proposed. Pituitary magnetic resonance imaging gave normal results. Discussion: This report provides further support to the hypothesis that GSD1b patients have an increased risk for autoimmune disorders.

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RESPONSE TO THE KETOGENIC DIET IN A WIDE SPECTRUM OF GLUT-1 PHENOTYPES Peèrez-Duen¬as B1, Pineda M1, Gonzaèlez V1, Maèlaga I2, Goèmez L3, Gutieèrrez A3, Pascual JM4, Artuch R5 1 Neurol Depart St Joan de Deèu Hosp, Barcelona, Spain, 2Neurol Dept Hosp Univ Central Asturias, Oviedo, Spain, 3Nutrition Dept Hosp St Joan de Deèu, Barcelona, Spain, 4Neurol Univ Texas Southwestern Med Cen, Texas, USA, 5Biochem Dept Hosp St Joan de Deèu, Barcelona, Spain Background: Glut-1 de¢ciency is a treatable disorder of brain energy metabolism with an increasing spectrum of phenotypes known worldwide. Methods: F|ve patients (mean age 10 years, range 3^19) were diagnosed of Glut-1 de¢ciency based on biochemical and molecular analysis. Results: All patients showed hypoglycorrhachia with CSF:blood glucose ratio 50.4 and heterozygous mutations in the SLC2A1 gene. One patient manifested developmental delay and minor chorea, but she never developed seizures and her head circumpherence was in the 85th percentile for age. MRI disclosed cortical atrophy in the occipital lobes at 5 years. Four patients developed seizures between 3 and 10 months of life. Seizure types at onset were focal (1), absences (1), £exion spasms and tonic (2). EEG abnormalities were: di¡use background slowing (2), frontal predominant paroxysms (1), generalized 3-to 4-Hz polyspikewave discharges (4). Fasting aggravated seizures in all patients. After a mean period of 19 months (range: 8^36) on ketogenic diet, ataxic gait improved in all patients and one wheelchairbound patient recovered deambulation. In the non epileptic patient, choreic movements completely disappeared, but a mild global cognitive impairment persisted after two years on ketogenic diet. Epileptic patients became seizure-free and antiepileptic drugs were discontinued in two patients. Adverse e¡ects of ketogenic diet were carnitine depletion (1) and high plasma cholesterol levels (1). Conclusions: Despite the highly variable severity of the disease and age at diagnosis, ketogenic diet was e¡ective in controlling seizures and improving the motor pattern of all a¡ected children.

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PITFALLS IN INTERPRETATION OF TRANSFERRIN ISOFORMS DETERMINED BY CAPILLARY ELECTROPHORESIS Carpenter K1, Green K2, Ellaway C3, Pitt J4 1 NSW Bio Gen and Disp Gen Med, Univ Sydney, Sydney, Australia, 2 NSW Biochemical Genetics Service, Sydney, Australia, 3Child Hosp at Westmead, Sydney, Australia, 4Murdoch Children's Research Institute, Melbourne, Australia Transferrin isoform analysis is the cornerstone of investigation of Nglycosylation defects. Classical methods involve isoelectric focussing (IEF) followed by immunoprecipitation of transferrin and staining. The technique is labour intensive and with an increasing clinical spectrum to the CDG defects demand for testing is increasing. Many labs have now turned to methods which can be semi-automated including HPLC and capillary electrophoresis (CE). We report on two cases where transferrin isoform analysis by CE gave UV absorbing peaks initially incorrectly identi¢ed as disialotransferrin. The CE method, (CEo¢xTM CDT kit), involves dynamic capillary coating with peak detection at 200 nm. Our cases were investigated for possible CDG defects with presentations of hyperinsulinism (case 1), and prematurity complicated by thyroid dysgenesis (case 2). In the ¢rst case, multiple samples were tested over one month, all of which showed a peak at diasialotransferrin, representing 14 to 18% of total transferrin. Normal phosphomannomutase and phosphomannoisomerase activities were fou n d an d an alys i s of t ran s fe r r i n i s ofo r m s by IE F an d immunoprecipitation revealed a normal result (Prof. Jaeken, Belgium). We performed further analysis by HPLC (using detection of the irontransferrin complex at 470 nm) which also gave a normal pattern. Similar CE results on the second case led straight to HPLC analysis which showed normal results. The identity of the interfering compound is unknown but it is removed by ultra¢ltration suggesting it may be a protein. We suggest atypical abnormal patterns obtained by CE should be con¢rmed by another technique before more complex investigations are performed.

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LATE PRESENTATION OF CARDIOMYOPATHY IN CDG TYPE IX Footitt EJ1, Karimova A2, Burch M2, Grunewald S1 1 Metab Dept, Grea t Ormond Street, London, UK, 2Cardiac Dept, Great Ormond St, London, UK Background: Congenital disorders of glycosylation (CDG) are inherited multisystem disorders characterised by defects in glycosylation of proteins. Cardiomyopathy has been previously described in several CDG cases, presenting within the ¢rst 2 years of life. Acute heart failure at 11 years of age only led to the diagnosis of CDG. Case Study: The11 year old female presented with a short history of lethargy, dyspnoea and ankle oedema. She had a background of learning di¤culties of unknown aetiology. Initial investigations demonstrated a poorly contractile, dilated heart with thrombi present in the left ventricle. Her cardiac function deteriorated and she required mechanical support with V-AECMO and subsequently a Berlin Heart VAD as bridge to transplantation. She had multiple complications related to di¤culties with anticoagulation ^ thrombosis of brachial artery requiring thrombectomy, and signi¢cant clots of mechanical devices. At this point, the diagnosis of CDG type I was made by transferrin isoelectric focussing. Enzyme assay in leucocytes excluded CDG type Ia and b. Secondary causes of abnormal transferrin isoelectric focussing were excluded. Various serum proteins (protein C, S, factor XI, antithrombin) were low, in keeping with the diagnosis. Problems with thrombosis and later haemorrhages continued and she died from overwhelming sepsis. Conclusion: This is the ¢rst reported case of late presenting cardiomyopathy in CDG. It emphasises the need to screen all children with cardiomyopathy for CDG, regardless of age. It also highlights the di¤culties of e¡ective anti-coagulation and the ethical considerations of cardiac transplant and use of mechanical devices in this group of disorders.

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NEW MUTATION OF THE ATP6V0A2 GENE IN AN AUTOSOMAL RECESSIVE CUTIS LAXA TYPE 2 PATIENT Bruneel A1, Drouin-Garraud V2, Habarou F1, Morelle W3, Foulquier F4, Charluteau E1, Bouchet C1, Vuillaumier-Barrot S1, Durand G1, Balguerie X5, Frebourg T2, Seta N1 1 AP-HP, Biochemistry, Bichat Hospital, Paris, France, 2Clinical Genetic, CHU Rouen, Rouen, France, 3UMR CNRS/USTL 8576, Lille ^ V|lleneuve d'Ascq, France, 4Lab Mol Diag, Univ of Leuven, Leuven, Belgium, 5Dermatology, CHU Rouen, France, Rouen, France Background: Subtypes of autosomal recessive cutis laxa (ARCL) are rare inherited diseases presenting with wrinkling skin and systemic involvements in cluding dysmorphism, microc ephaly, joint abnormalities, large fontanels and psychomotor retardation. Mutations in ¢bulin gene have been involved in a few cases of ARCL-I. Very recently, consanguineous ARCL-II patients sharing mutations in the gene encoding the a2 subunit of V-type H+ ATPase (ATP6V0A2) have been reported. We present here the ¢rst French ARCL-II patient harboring a new mutation in ATP6V0A2. Patient: The girl from apparently non consanguineous parents presented at birth intraventricular communication, axial hypotonia and large fontanels in addition to major hypotrophy. She developed psychomotor retardation and anterior fontanel remained large until 8 years-old. At the age of twelve years, she presented microcephaly, low length and body weight and cutis laxa, joint laxity, nasal voice and strabismus were also noted, leading to glycosylation defects screening. Results: Two-dimensional electrophoresis showed abnormalities in serum transferrin and apoC-III, while mass spectrometry further revealed hypogalactosylation and/or hyposialylation of N- and Olinked glycans. Western-blotting of COG subunits showed no abnormality but brefeldin A induced a signi¢cant delay in the vesicular Golgi tra¤cking of patient's cells. Lastly, DNA sequencing of ATP6V0A2 showed an homozygous G deletion leading to a stop codon. Conclusion: Glycomic tools and typical clinical ¢ndings allowed us to diagnose a novel case of ARCL-II-associated CDG sharing one original ATP6V0A2 mutation.

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A POSSIBLE ROLE FOR THE CONSERVED OLIGOMERIC GOLGI COMPLEX SUBUNIT 1 (COG1) IN AUTOSOMAL RECESSIVE CEREBROCOSTOMANDIBULAR SYNDROME Zeevaert R1, Van Damme-Lombaerts R1, Foulquier F2, Reynders E3, Annaert W3, Matthijs G2, Jaeken J1 1 Dept Pediatrics, Univ Hosp Leuven, Leuven, Belgium, 2Centre Human Genetics, Univ Hosp Leuven, Leuven, Belgium, 3VIB, KULeuven, Leuven, Belgium Since 2004, nine patients with a mutation in one of the seven subunits of the conserved oligomeric golgi (COG) complex have been described. One of these is a patient with a truncating COG1 mutation, c.26592660insC, and a mild phenotype (1). We describe a new patient with autosomal recessive cerebrocostomandibular syndrome and a clear reduction of Cog1 protein level on western blot, without a¡ecting the levels of the other 7 subunits of the complex. A homozygous intronic mutation, c.1070+5G4A, disturbing the splice donor and activating alternative splicing, led to the presence of two transcripts: a normal one and one lacking exon 6 with a frameshift and a premature stop codon. The parents and 2 non-a¡ected siblings were heterozygous for this mutation. We used real time RT-PCR to quantify the transcripts. The patient had only 1% of transcript containing exon 6 compared to control. The amount of total transcript was reduced to 15% of control. Puromycin treatment, to prevent nonsense mediated decay, led to higher expression levels with 8% of normal transcript and 41% of total transcript compared to control. Treatment of ¢broblasts with brefeldin A showed a delay in retrograde tra¤cking. This delay has also been shown in other patients with a COG de¢ciency. We identi¢ed a patient with a truncating mutation in COG1 without a¡ecting the interaction between the Cog1 and Cog8 subunit. The clinical presentation, however, is much more severe than the ¢rst patient described with a COG1 mutation. (1) Zeevaert R et al. Mol Genet Metabol 2008; 93:261^278

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CONGENITAL DISORDER OF GLYCOSYLATION TYPE IK (CDG IK): A FREQUENT FORM OF CDG IN FRANCE? Dupre T1, Vuillaumier-Barrot S1, Sadou-Yaye H1, Le Bizec C1, de Lonlay P2, Moore S3, Seta N1 1 Lab Biochimie Hosp Bichat AP-HP, Paris, France, 2Dept Pediatrie, Hosp Necker, APHP, Paris, France, 3Inserm U773 CR3B, Paris, France Background: CDG are a rapidly growing group of inherited errors of metabolism a¡ecting glycan biosynthesis. Type I CDG are caused by defects in the biosynthesis of the lipid-linked oligosaccharide (LLO) required for protein N-glycosylation in the ER. Presently, 14 CDG I subtypes (CDG Ia-n) have been identi¢ed at the biochemical and molecular levels but cannot be di¡erentiated on the basis of clinical presentation alone. Therefore diagnosis must be established at the biological level. Applying a biological diagnosis strategy to untyped CDG I, 4 patients could be identi¢ed as CDG Ik. Methods: Four patients with a CDG I positive screening and exclusion of CDG Ia (normal phosphomannose activity) and Ib (normal phosphomannose isomerase), metabolic labelling of cultured ¢broblasts with 3H-mannose or 3H-glucosamine was performed in order to identify defective glycosylation steps through the characterisation of accumulated oligosaccharide intermediates. Results: We identi¢ed four patients that manifested abnormal accumulations of dolichol-PP-GlcNAc2, suggesting a de¢ciency of ALG1, the enzyme that adds the ¢rst mannose residue to LLO. ALG1 sequences derived from the 4 patients revealed 8 mutations: 3 mutations known to be causal (c.773C4T (Ser258Leu), c.434G4A (p. Gly145Asp), c.450C4G (p.Ser150Arg)) and 5 unknown mutations (c.765G4A (p.Thr255Thr), c.1129A4G (Met377Val), c.740G4T (p. Arg247Leu), c.740+5G4A, c.1263G4A (p.Cys396X)). Allelic inheritance and splicing error studies are underway. Conclusion: CDG Ik could be a frequent type of CDG I in France.

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A NOVEL MUTATION IN THE COG5 LOCUS AFFECTS N- AND O- GLYCOSYLATION CAUSING CDG TYPE II Paesold-Burda P1, Troxler H1, Kleinert P1, Maag C2, Hennet T2, Malich S3, Steinmann B1, Baumgartner M1 1 University Children's Hospital, Zurich, Switzerland, 2Institute of Physiology, Zurich, Switzerland, 3Kantonsspital Aarau, Aarau, Switzerland Congenital disorders of glycosylation (CDG) are a group of autosomal recessive disorders caused by aberrant biogenesis of N-glycans. Here, we describe a patient from healthy and apparently unrelated parents presenting mental retardation, motor developmental delay and cerebral ataxia. Isoelectric focusing (IEF) of serum transferrin showed increased amounts of trisialo and disialo transferrin. Analysis of transferrin by electrospray ionization mass spectrometry revealed increased amounts of an isoform lacking one sialic acid residue. These ¢ndings are in agreement with 2D-PAGE analysis of serum alpha1-acid glycoprotein: the molecular mass remained constant, whereas a shift was observed at di¡erent isoelectric points. Analysis of the O-glycan apolipoprotein CIII by IEF and Western blot showed markedly elevated asialo apoC-III. Congenital defects a¡ecting both N- and O-glycosylation have been described in patients with a de¢ciency in one of the eight subunits of the conserved oligomeric golgi (COG) complex. In addition, mutations in the COG complex alter the distribution of golgi resident proteins. We observed that retrograde transport of the golgi resident GalT glycosyltransferase to the ER via Brefeldin A induced tubules was signi¢cantly slower in the patient ¢broblasts compared to normal cells. Sequencing of the eight Cog subunits revealed a homozygous mutation in the COG5 gene. Further characterization of the mutation will be presented.

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CONGENITAL DISORDERS OF GLYCOSYLATION PRESENTING WITH NEURO-REGRESSION V|jayakumar K1, Prabhakar P1, Ganesan V1, Worthington V2, Jackson M3, Mills P4, Dupre T5, Vuillaumier-Barrot S5, Seta N5, Gru«newald S1 1 Great Ormond Street Hospital, London, UK, 2National Hospital for Neurology, London, UK, 3St Thomas' Hospital, London, UK, 4Institute for Child Health, London, UK, 5Biochimie Metabolique, Hoªpital Bichat, Paris, France Background: The majority of congenital disorders of glycosylation (CDG) present with neurological symptoms, a¡ecting motor and cognitive development. It has been thought, that CDG patients progress with time albeit to a very variable degree. We present two CDG cases with clear neuro-regression. Case reports: Patient 1 initially showed normal development. Laryngomalacia and occasional myocloni were noted at six weeks. At 4 months, the boy showed dystonic movements, central hypotonia and he lost his social smile. Seizure activity increased, evolving to multifocal drug resistant epilepsy, associated with apnoeas. Increasing swallowing di¤culties required tube feeding. MRI showed generalised lack of white matter and bilateral high signal in the thalami. He died at the age of one year. The isoelectric focusing of transferrin revealed a type I CDG pattern. Lipid linked oligosaccharide studies showed an increase of the DolP-Man 9 peak. Mutation analysis of the hALG6 gene detected a missense and a splicing mutation, con¢rming CDG-Ic. Patient 2 was born to unrelated parents. The girl's gross-motor development was initially mildly delayed. Towards the end of second year of life, a regression in di¡erent areas was observed, with loss of speech. Her gait became increasingly unsteady. IEF of transferrin and ApoCIII revealed a combined N- (CDG II pattern) and O-glycosylation defect. Total plasma glycan structure analysis showed abnormal fucosylated `hybrid type' glycans. The underlying enzymatic defect still needs to be determined. Conclusion: The di¡erential diagnosis of CDG should be considered in patients with unexplained neuro-regression.

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A MOUSE MODEL FOR CONGENITAL DISORDER OF GLYCOSYLATION IA (CDG-IA) Schneider A1, Thiel C1, Gro«ne H-J2, Ko«rner C1 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Div Cell Mol Path, Ger Cancer Res Center, Heidelberg, Germany During the last years the group of inherited multisystemic human disorders, called congenital disorders of glycosylation (CDG), notable gets into the focus of medical investigation and diagnostics. Defects within the biosynthesis of glycoproteins describe one thematic priority of metabolic diseases. CDG-Ia, the most frequent type of the CDG (more than 500 patients world wide), is evoked by mutations in the Phosphomannomutase 2 gene (PMM2), thereby leading to a severely reduced conversion of mannose-6-phosphate to mannose-1-phosphate in the cytosol. The symptoms of CDG-Ia patients include psychomotor and mental retardation, peripheral neuropathy, cerebellar atrophy, retinitis pigmentosa, hepatopathy and blood clotting problems. To investigate pathologic aspects of the disease and develop therapeutic approaches, an animal model for CDG-Ia is indispensable. For these purposes we generated mice with either a complete loss of Pmm2 activity or a weak residual enzyme activity as has been observed in all known CDG-Ia patients. Complete leakage of Pmm2 activity in the mouse leads to early embryonic lethality around day 2.5. Moreover, mating of heterozygous Pmm2-de¢cient male and female mice with WT mice revealed that maternal transmission of the Pmm2 null allele is severely impaired. Mouse models, which display residual Pmm2 activity, were generated by introduction of point mutations which are known from CDG-Ia patients. They reveal a broad spectrum of phenotypes which reach from early embryonic death (compound heterozygous mutation) to normal viability (F119L mutation).

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IMMUNOLOGICAL, DEVELOPMENTAL AND BEHAVIOURAL ABNORMALITIES IN A MOUSE MODEL FOR CONGENITAL DISORDER OF GLYCOSYLATION-IIC Popovici D1, Lu«bbehusen J1, Sperandio M2, Frommhold D2, Gass P3, Ko«rner C1 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Div Neonat, Univ Child Hosp, Heidelberg, Germany, 3Inst Mental Health, Univ Hd, Mannheim, Germany Congenital disorder of glycosylation IIc (CDG-IIc) is caused by a defect of GDP-fucose import into the golgi. This leads to a generalized hypofucosylation of glycoproteins. CDG-IIc patients su¡er from persistent leukocytosis, severe infections and retardation of mental and physical development. By inactivation of the murine golgi GDP-fucose transporter we generated a mouse model for CDG-IIc, which resembles the pathological phenotype of CDG-IIc patients in many aspects. Besides a strong reduction of fucosylated glycoconjugates in tissues and isolated cells, transporter-de¢cient mice show severe growth retardation, leukocytosis, dilated lung alveoles and hypocellular lymph nodes. Adhesion and rolling of leukocytes is substantially impaired by interruption of P-, E- and L-selectin ligand function. Most recent investigations led to results concerning the dependency of ER-located protein O-fucosylation on the golgi GDP-fucose transporter and the existence of alternative GDP-fucose transport mechanisms. Characterization of the lung phenotype demonstrated upregulation of matrix metalloproteases, possibly caused by a disturbance of growth factor signaling arising from a generalized fucosylation defect. Additionally, behavioural phenotyping showed several abnormalities of behaviour in transporter-de¢cient mice. The CDG-IIc mouse model allows for detailed analysis of additional so far poorly understood complex fucosylation-dependent mechanisms in mammalians, whose importance is undelined by the signi¢cantly increased mortality rate of GDP-fucose transporter de¢cient mice.

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BIOCHEMICAL TOOLS IN THE DIAGNOSIS OF GLYCOSYLATION RELATED MYOPATHIES Guillard M1, Verrijp K2, Voermans N3, Huizing M4, Morava E5, Van Bokhoven H6, Van Engelen B3, Wevers RA1, Lammens M2, Lefeber DJ1 1 Lab Ped and Neurol, Radboud UMC, Nijmegen, Netherlands, 2Dept Pathology, Radboud UMC, Nijmegen, Netherlands, 3Dept Neurology, Radboud UMC, Nijmegen, Netherlands, 4Medical Genetics Branch, NIH, Bethesda, USA, 5Dept Pediatrics, Radboud UMC, Nijmegen, Netherlands, 6Human Genetics, Radboud UMC, Nijmegen, Netherlands The classical congenital disorders of glycosylation (CDG) are characterized by a highly variable multisystem presentation including liver involvement, eye anomalies, neurological symptoms, coagulopathy, etc. Muscle involvement is rare with the exception of CDG-Ie presenting with elevated CK levels. In contrast, the group of dystroglycanopathies and Nonaka myopathy are glycosylation disorders with a distinct phenotype a¡ecting the skeletal muscles and the CNS. Some patients with an alpha-dystroglycanopathy present with severe congenital muscular dystrophy in combination with structural brain defects and ophthalmological problems, others are only mildly a¡ected as in limb-girdle muscular dystrophy (LGMD) patients. Children diagnosed with hereditary inclusion body myopathy (HIBM/Nonaka) exhibit a late onset, slowly progressive distal myopathy. Due to the variable presentation of the muscle disease many cases remain unresolved and incorporation of biochemical tools in routine diagnostic protocols might help to reveal the underlying etiology. A panel of lectins was chosen for immunohistochemistry on muscle biopsies and conditions were optimized. Subsequently, a series of muscle biopsies was screened by immunohistochemistry with IIH6 alpha-DG speci¢c antibody and a panel of lectins. In 2 out of 6 patients with a clinical phenotype of Nonaka and abnormal lectin staining, GNE mutations could be identi¢ed. Western blotting of muscle homogenates of dystroglycanopathy patients provided additional evidence for a dystroglycanopathy. Besides genetic methods such as mutational analysis of the known genes (POMT1/2, POMGnT, FKRP, fukutin and LARGE) and homozygosity mapping, biochemical tools were investigated to further unravel genetic defects such as alpha-DG western blotting on cell homogenates and immuno£uorescence.

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CONGENITAL DISORDERS OF GLYCOSYLATION IN SOUTHERN AFRICA. A PILOT STUDY Dercksen M, Mienie LJ Lab Inh Metab Def, Biochem Dept, NWU, Potchefstroom, South Africa Background: Congenital disorders of glycosylation (CDG) results in a broad spectrum of clinical manifestations. This fast expanding group of diseases is rapidly growing with an estimated cumulative disease incidence of 1:20 000.International cases reported, consist of 385 CDG type 1 patients (mostly type 1a) and 11 CDG type 2 (Orphanet Report series 2008). Preliminary results indicated an even higher prevalence of these defects in Southern Africa. Methods: In 1998, a pilot study was initiated for the detection of CDG defects in patients with clinical symptoms indicative of CDG-defects. Blood card analysis was used in the study. The analyses were done with isoelectric focusing (IEF) of Transferrin and positive results were con¢rmed by the AMC, Amsterdam, The Netherlands. Since 1998, material of only 210 patients was analyzed. Abnormal transferrin pro¢les were identi¢ed in 20 patients. Result: This study indicates an elevated prevalence of these defects, as well as a unique transferrin pro¢le, uncommon to the European countries. We identi¢ed 20 patients with CDG defects. The group consists of 8 patients with CDG type 1, 4 patients with CDG type 2 (one Cog2 and one Cog8) and 8 uncon¢rmed cases. The oligosaccharide LC-MS-MS pro¢le indicated some unique markers. Conclusion: A high incidence of CDG-defects was found in Southern Africa. Neonatal screening tests must be included, on a routine basis, in the future. IEF tends to be labour intensive and is not amendable for high throughput. The establishment of a blood card CDG screening is a necessity in African Clinics.

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TRANSFERRIN IEF PATTERNS AND PLASMA ASPARTYLGLUCOSAMINIDASE ACTIVITY IN CDG, GALACTOSEMIA AND FRUCTOSEMIA Moraitou M, Mavridou I, Michelakakis H Institute of Child Health, Athens, Greece N-glycosylation defects are in most instances associated with abnormal transferrin IEF patterns (Tf IEF) that di¡erentiate them into types I and II. Elevation of plasma acid aspartylglucosaminidase (AGA) has also been reported in the majority of CDGI patients. Galactosemia and fructosemia are two disorders where abnormal TfIEF patterns can be observed. We report on AGA plasma activity in GDGI, CDGII patients and patients with galactosemia and fructosemia. The patients studied included 4CDGIa, 3CDGIx and 5CDGIIx (all with combined N- and O-glycosylation defect), 5 with classical galactosemia (GALT), 4 with fructose-1,6-diphosphatase de¢ciency and 2 with hereditary fructose intolerance (HFI). Elevated AGA activity was found in all CDGI cases, whereas in CDGII cases it was normal. In galactosemia normal TfIEF patterns and AGA activity were seen in an asymptomatic one day old neonate, a severely ill 12 days old neonate receiving iv-glucose for two days and symptom free patients on treatment. Elevated plasma AGA activity and abnormal TfIEF pattern were observed in 2 GALT and one of the two HFI patients before treatment. Both parameters normalized upon two-months of dietary treatment. Thus increased AGA activity could serve as an indicator of CDGI but not CDGII disorders. Its activity in GALT and HFI seems to correlate with abnormal TfIEF patterns and both, when found abnormal, could be used in monitoring response to therapy. Supported by Euroglycanet LSHM-CT-2005-512131

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AMNIOTIC FLUID ALPHA1-FETOPROTEIN IN PRENATAL DIAGNOSTICS OF CDG Marklova E, Albahri Z Dept Paediatr, Charles Univ Fac ^ Hosp, Hradec Kralove, Czech Republic Background: Congenital disorders of glycosylation (CDG) represent an ever increasing group of defects (22 types known so far) in the common posttranslational process of protein modi¢cation. Reliable prenatal diagnostics is based on the particular mutation analysis. A problem arises when a patient with typical clinical symptoms and highly abnormal pattern of proteins glycosylation dies and identi¢cation of the actual mutation and CDG typing could not be achieved. Considering the partially maternal origin of transferrin (commonly used for postnatal CDG screening) in amniotic £uid, an e¡ort was made to establish a prevalent glycosylation pattern of alpha1fetoprotein, which is synthesized mainly by the foetal yolk sac and liver. Methods: Isoelectric focusing method with immunodetection, silver staining and densitometry has been elaborated and over 100 samples of amniotic £uid from 14^18th week of gestation analysed. The total concentration of alpha1-fetoprotein was determined by rocket immunoelectrophoresis. Results: The separation pattern in samples from physiological pregnancies is regarded as normal, in contrast to the isoforms distribution in the cases with foetal death, open neural tube or ventral wall defects. Conclusions: The system is ready for analysis of samples from CDGsuspicious pregnancies revealed by ultrasonography (oligohydramnion, microcephaly, atresias, hydrops fetalis, etc.) and namely for one family at risk mentioned above. The study was supported by Research Project MZO 00179906

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CUTIS LAXA SYNDROMES WITH CONGENITAL DISORDER OF GLYCOSYLATION: CLINICAL, BIOCHEMICAL AND GENETIC REVIEW Morava E1, Guillard M1, Rodenburg R1, Kornak U2, Urban Z3, Lefeber D1, Wevers RA1 1 Radboud University Medical Center, NIjmegen, Netherlands, 2Institute of Medical Genetics, Charite, Berlin, Germany, 3Department of Genetics, St Louis Univ, St Louis, USA W|th an international consortium we recently described mutations in the ATP6V0A2 gene in several consanguineous families with autosomal recessive cutis laxa (ARCL) syndrome. This genetic defect is associated with a characteristic phenotype and abnormal glycosylation in the Golgi apparatus, leading to the de¢nition of a distinct combined N- and Olinked glycosylation disorder. The clinical spectrum of the various ARCL syndromes is highly heterogeneous, both with respect to organ involvement and severity. In some families mutations have been demonstrated in the ¢bulin 4 and 5 genes, but the underlying biochemical and genetic etiology in the majority of patients is still unknown. We couldn't con¢rm mutations in the ATP6V0A2 gene in a large series of patients diagnosed with various cutis laxa syndromes without an associated glycosylation defect. We detected abnormal Oglycosylation, however, in additional patients with cutis laxa, not carrying an ATP6V0A2 defect. The variable presence of protein glycosylation disorders in the wrinkled skin-cutis laxa syndrome spectrum necessitates routine screening for glycosylation disorders in all patients diagnosed with CL to o¡er adequate prognosis assessment and counselling. In addition, we are developing new biochemical tools for metabolic analysis of the Golgi system including novel functional studies. The present paper aims at describing the spectrum of clinical and biochemical features in various forms of ARCL syndromes. Based on the recently unravelled novel ATP6V0A2disease entity we also review clinical and genetic aspects in 12 patients, including genotypephenotype correlations and suggest a practical diagnostic approach.

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CONGENITAL DEFECT OF N- AND O-GLYCOSYLATION IN AN INFANT WITH CUTIS LAXA SYNDROME Tsiakas K1, Morava E2, Meinecke P1, Gillessen-Kaesbach G3, Wevers R2, Santer R1 1 Dept Pediatr, Univ Medical Center, Hamburg-Eppendorf, Germany, 2 Dept Pediatr, Radboud Univ Med Center, Nijmegen, Netherlands, 3Inst Hum Genet, Univ Med Center SH, Lu«beck, Germany In patients with cutis laxa syndrome and variable central nervous system involvement, a combined defect of N- and O- glycosylation (CDGIIx), due to mutations of ATP6V0A2, the gene coding for the alpha-2 subunit of V-Typ H+ATPase, has been recently reported (Eur J Hum Genet 16:28, 2008). We describe an infant with cutis laxa and con¢rmed CDG IIx diagnosis. Case report: F|rst child of healthy consanguinous parents originating from Kurdistan. Growth and head circumference of the female newborn were below ^2 SD. Generalized wrinkled skin, abnormal fat distribution with lipodystrophy, bilateral hip dislocation, a very large anterior fontanel and dysmorphic features such as hypertelorism, down-slanting palpebral ¢ssures, beaked nose with a broad nasal bridge, a high, narrow palate, and retrognathia were noted. Furthermore, joint laxity but no muscle hypotonia were present. Psychomotor development was only mildly delayed. Analysis of transferrin isofocusing pro¢le (TIEF) was normal, whereas the apolipoprotein CIII pro¢le was abnormal, indicating impairment of mucin type O-glycosylation. Genetic analysis showed mutations of ATP6V0A2. Discussion: This new type of CDG with a combined defect of N- and Oglycosylation is an important di¡erential diagnosis in newborns with cutis laxa. TIEF as a diagnostic tool for defects of N-glycosylation has been reported to be normal in the ¢rst 6 months of age in these cutis laxa patients. Diagnosis can be established through an abnormal ApoCIII pro¢le, compatible with a combined defect of N- and Oglycosylation and can be con¢rmed by genetic analysis of ATP6V0A2.

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FUNCTIONAL ANALYSIS OF THREE SPLICING MUTATIONS IDENTIFIED IN THE PMM2 GENE: TOWARDS NEW THERAPEUTIC APPROACHES IN CONGENITAL DISORDER OF GLYCOSYLATION TYPE-IA Vega A1, Peèrez-Cerdaè C1, Desviat LR1, Matthijs G2, Adamowicz M3, Ugarte M1, Peèrez B1 1 Centro de Biologia Molecular,UAM, CIBERER, Madrid, Spain, 2 Center for Human Genetics, Univ of Leuven, Leuven, Belgium, 3The Children's Memorial Health Institut, Warsaw, Poland Congenital disorders of glycosylation (CDG) are a group of diseases caused by defects in at least 21 di¡erent genes a¡ecting N-glycosylation. In this work we describe the functional analysis by cell based splicing assays and transcriptional pro¢ling in ¢broblast cell lines of three splicing mutations identi¢ed in the PMM2 gene (CDG-Ia). We have also included the use of antisense morpholino oligonucleotide (AMO) and the overexpression of several trans-acting splicing factors to restore the normal splicing. We have studied two new nucleotide changes identi¢ed in 3' splice site intronic sequences (c.256-1G4C and c.6409T4G) and one previously described deep intronic nucleotide change in intron 7 (c.640-15479C4T). Using cell based splicing assays we have demonstrated that all are disease-causing splicing mutations. Overexpression of the cellular splicing factors SC35 and ASF/SF2 promoted the normal splicing of exon 8 in the pre-mRNA transcribed from a minigene carrying the disease causing mutation c.640-9T4G and no e¡ect was detected with the minigene bearing c.256-1G4C. F|nally we have demonstrated that the deep intronic change c.64015479C4T caused the activation of a pseudoexon inclusion sequence from intron 7 as has been previously described in patient's ¢broblasts. Using AMO targeted to the 5'and 3' cryptic splice sites to block access of the spliceosome machinery we have obtained correctly spliced mRNA that was e¤ciently translated into a functional and immunorreactive PMM2 protein. Our results o¡er a possible, novel but mutation-speci¢c therapeutic approach in this genetic disease where other e¡ective treatment is not possible.

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RECURRENT RHABDOMYOLYSIS AND MITOCHONDRIAL DISEASE Bandeira A1, Santos M2, Rocha P3, Carrilho I2, Guimara¬es A4, Cardoso ML5, Martins E1 1 Metabolic Unit, Centro Hosp Porto, Porto, Portugal, 2Neuroped Service, Centro Hosp Porto, Porto, Portugal, 3Inten Care Unit, Centro Hosp Porto, Porto, Portugal, 4Pathologist Unit, Centro Hosp Porto, Porto, Portugal, 5CGM Jacinto Magalha¬es, Porto, Portugal Background: Mitochondrial respiratory chain abnormalities can a¡ect muscle speci¢cally or as a part of a multisystem disease, typically involving the central nervous system. Rhabdomyolysis associated with mitochondrial disease is rare but have been described in sporadic cases with mutations in the cytochrome b and cytochrome c oxidase genes. Clinical reports: A male patient, 8 years old, referred for investigation of muscular pain with miositis and muscular weakness and a female patient, 14 years old, referred for investigation of myoglobinuria. She needed intubation for respiratory insu¤ciency. The muscular enzymes were elevated CK 2.600 U/L in the boy and 40 000 U/L in the female case. In both cases, physical examination was normal without muscular atrophy, pain or muscular de¢cit between episodes. The plasma carnitine, acylcarnitine pro¢l in one of the episodes, lactate and urinary organic acids were normal. A skeletal muscle biopsy was performed: histochemical evaluation revealed increased in lipid droplets and mitochondrial proliferation in the ¢rst case and normal morphology in the second, immunohistochemical study for dystrophine was normal. The respiratory chain enzyme analyses showed reductions in complex I, II and IV activities (in the boy 27%, 34%, 40%; in the female 30%, 23%, 30%, respectably). Conclusions: In these cases, recurrent rhabdomyolysis was the single clinical manifestation of reduced activity in I, II and IV complexes of respiratory chain. The detection of a mitochondrial mutation and the clinical follow-up will permit to establish the true aetiology of these episodes of rhabdomyolysis.

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MITOCHONDRIAL CARDIOMYOPATHIES: BIOCHEMICAL AND GENETIC HETEROGENEITY Estevinho A1, Oliveira S2, Pratas J2, Simo¬es M2, Mendes C2, Santos MJ2, Oliveira M2, Diogo L3, Macaèrio C4, Oliveira CR1, Grazina M1 1 Faculdade de Medicina, Univ Coimbra, Coimbra, Portugal, 2Centro Neurociencias Biol Cel, Univ Coim, Coimbra, Portugal, 3Hosp Pediaètrico de Coimbra, Coimbra, Portugal, 4Hosp da Univ Coimbra, Coimbra, Portugal Background: Cardiomyopathy is a severe condition associated to impairment of heart contractile function, with progressive congestive heart failure. Genetic defects disturbing the maintenance of mitochondrial respiratory chain (MRC) system will reduce ATP levels and may underlie heart dysfunction. Decrease in MRC complexes I and III activities are the most typical ¢ndings; mtDNA defects plus mutations in nuclear genes encoding mitochondrial proteins have been described. Our aim is to analyse a group of patients presenting cardiomyopathy, concerning mtDNA and MRC abnormalities. Methods: We have analysed 86 samples (lymphocytes, myocardium, liver, muscle) from 48 patients (aged 2^43 years) suspected of cardiomyopathy. Total DNA was extracted by standard methods. Analysis of mtDNA was performed using standard techniques (PCR, PCR-RFLP, sequencing). MRC assays were performed as described (Rustin et al., 1994), normalized to CS. De¢ciency was considered if value540% of mean reference value. Results: Complexes I, III and IV were the most a¡ected (68%, 65% and 60%, respectively) in the samples analysed. The results show decreased activity of all complexes in myocardium, complex III in muscle, complexes IV, I and V in liver, and complexes IV and III in lymphocytes. Abnormalities of mtDNA found were heterogeneous and included deletions, insertions and sequence variations. Conclusions: We con¢rmed that complexes I, III and IV are the most a¡ected in cardiomyopathies, which is in agreement with literature. No correlation has been found between biochemical and genetic data. The majority of cases presented multiple MRC de¢ciencies, suggesting complex genetic origin, possibly involving both genomes.

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DIHYDROLIPOAMIDE DEHYDROGENASE DEFICIENCY IN A CASE OF REYE SYNDROME. EFFECT OF ANAPLEROTIC SUBSTRATES IN VIVO AND IN VITRO Arnoux JB1, Le Moyec L2, Hubert L3, Chretien D3, Brivet M4, Valayannopoulos V1, Bertrand AM1, Munnich A3, de Keyzer Y3, de Lonlay P1 1 Metabolic Dept, Necker Hosp, Paris, France, 2CNRS UMR7033, Bobigny, France, 3INSERM U781, Necker Hosp, Paris, France, 4 Biochemistry, Kremlin-Bicetre Hosp, Paris, France Background: The molecular origin of Reye syndrome and its consequences on respiratory chain (RC) activity remain unknown. Objective: Study OXPHOS activity in Reye syndrome and evaluate the therapeutic potential of anaplerotic treatments. Patients and methods: A consanguineous family with three children presenting episodes of Reye syndrome. We performed molecular genetic analysis, and measured RC activity in liver and in ¢broblasts treated with lipoate or an anaplerotic substrate. We measured urinary Krebs cycle intermediates by MNR after aspartate treatment. Results: The patients carried a homozygous G4T mutation in the dihydrolipoamide dehydrogenase (DLD) gene encoding the E3 subunit of pyruvate (PDH) and alpha-cetoglutarate dehydrogenases changing glycine 229 into cysteine. DLD activity was reduced by 75% and PDH activity by 30%. RC de¢ciency was demonstrated in the liver and in skin ¢broblasts. Treatment of ¢broblast with lipoate or octanoate increased RC activity after 4 days. Aspartate treatment increased urinary oxaloacetate and citrate but not pyruvate or lactate. Conclusions: DLD mutations are a cause of Reye syndrome and induce secondary RC de¢ciency. Our results suggest that aspartate improves Krebs cycle activity in vivo and that selected anaplerotic substrates may present a therapeutic interest.

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WEST SYNDROME AND MITOCHONDRIAL DYSFUNCTION Silva S1, Robalo C2, Garcia P1, Grazina M3, Oliveira CR3, Diogo L1 1 Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal, 2 Neuropediatria, CDC, Hosp Ped, Coimbra, Portugal, 3Inst Bioq Faculdade Med Univ Coimbra, Coimbra, Portugal Background: Mitochondrial respiratory chain diseases (MRCD) often involve the central nervous system, being a possible aetiology of West syndrome (WS). Objective: To report children with WS in whom MRCD was investigated. Methods: Seven children of a cohort of 62 with epilepsy studied for MRCD, ful¢lled criteria for West syndrome. Investigation included mitochondrial enzymatic studies and mtDNA abnormalities screening in muscle biopsies. Other causes of WS were excluded. Patients were classi¢ed according to Bernier's criteria for MRCD. Results: Three females and four males, aged 8 months to 5 years (median: 2 years) at the time of the study, presented between the neonatal period and 9 months-old (median: 3 months) with myoclonic (5 cases) or multifocal partial clonic and tonic (2) seizures. Brain cortical and/or subcortical atrophy and delayed myelination were identi¢ed by MRI in ¢ve and three children, respectively. Hyperlactacidemia and/or elevated cerebrospinal £uid lactate (6 patients), hyperalaninemia (3) and lactate (1) or Krebs cycle intermediates (1) in urine were the most relevant biochemical abnormalities. Major mitochondrial respiratory enzyme activity de¢ciencies (cytochrome oxidase, isolated or in combination) were detected in three patients. No mtDNA abnormalities were found. Patients were classi¢ed as de¢nite (3), probable (3) and possible (1) MRCD. Seizures were refractory to treatment in three patients and six survived, although with severe handicap. Conclusions: MRCD should be considered in the diagnosis workup of infantile spasms, especially when encephalopathy and/or elevated lactate are present. However, the demonstration of a DNA mutation is mandatory to con¢rm that enzyme de¢ciencies are not secondary events.

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COMPLEX I DEFICIENCY DUE TO A NDUFAF2 DEFECT IN A BOY WITH ACUTE FULMINANT COURSE OF LEIGH DISEASE AND TYPICAL BRAINSTEM LESIONS Sperl W1, Prokisch H2, Boltshauser E3, Koch J1, Rauscher C1, Radauer W1, Forstner R4, Freisinger P5, Rolinski B5, Rodenburg RJT6, Mayr JA1 1 Dept Pediatr, Paracelsus Med Univ, Salzburg, Austria, 2Inst Hum Genet, Techn Univ, Munich, Germany, 3Univ Children's Hosp, Zurich, Switzerland, 4Dept Radiol, Paracelsus Med Univ, Salzburg, Austria, 5 Children's Hosp Schwabing,Techn Univ, Munich, Germany, 6Radboud Univ, Nijmegen Med Center, Nijmegen, Netherlands In a boy with a fulminant and lethal autopsy proven Leigh disease complex I de¢ciency was diagnosed in muscle tissue and cultivated ¢broblasts. The clinical course of the disease was dramatic with respiratory failure at an age of 24 months and death within 6 weeks. He had so far developed nearly normally, only a mild nystagmus was noticed. After an upper respiratory tract infection the child was admitted to our intensive care unit with recurrent severe apnoic crises. After a ¢rst recovery within a few days, symptoms relapsed and he died due to severe respiratory failure despite assisted ventilation. MRI revealed bilateral symmetric brainstem lesions involving pons, substantia nigra/lemniscus medialis, reticular formation with a relative sparing of the basal ganglia. Lactate was repeatedly normal and only in the ¢nal state slightly elevated. W|th a high throughput protocol for genetic screening of complex I patients using high resolution melting point analysis (Idao-Light Scan) and by direct sequencing a homozygous nonsense mutation (c.9G4A p.Trp3X) in the NDUFAF2 gene was detected. This gene encodes a molecular chaperone for complex I assembly. In all 3 patients so far described with NDUFAF2 mutations acute episodes of encephalopathy and a similar pattern of brainstem involvement was described, indicating together with our ¢ndings a similar clinical phenotype of this genetic disorder. We recommend in all patients with Leigh disease and predominant brainstem involvement to investigate complex I activity and to screen for mutation in the NDUFAF2 gene.

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DEFECTS IN ATP SYNTHESIS ARE ASSOCIATED WITH HYPERTROPHIC CARDIOMYOPATHY Sperl W, Koch J, Freisinger P, Mayr JA Children's Hosp Schwabing, Techn Univ, Munich, Germany A disorder of ATP synthesis can be caused by defects in the F1F0-ATP synthase, the adenine nucleotide translocator (ANT) or phosphate carrier (PiC). These three enzyme defects result in a reduced ATP production and can manifest as systemic mitochondrial diseases with di¡erent organ involvement, frequently with cardiac a¡ection. 13/14 patients with an isolated ATP synthase defect of nuclear genetic origin presented with a hypertrophic cardiomyopathy, in all of them an elevated excretion of 3-methylglutaconic acid in urine was detected. Until now two children have been identi¢ed with a PiC-defect, both with a severe lactic acidosis and hypertrophic cardiomyopathy and early death within the ¢rst year of life. A de¢ciency of the ANT can cause Sengers syndrome, which is characterized by cataracts and hypertrophic cardiomyopathy. In defects of the mitochondrial genes ATP6 and ATP8 as well as ANT1 gene mutations with accompanying mtDNA deletions cardiac a¡ection is not a predominant symptom, maybe due to ability of tissue-speci¢c compensation by varying rates of heteroplasmy of mtDNA mutations. In conclusion ATP synthesis disorders should be considered in patients with hypertrophic cardiomyopathy, especially in combination with cataracts or 3-methylglutaconic aciduria. In all disorders of ATP synthesis functional investigation of mitochondrial respiration from unfrozen tissue is crucial to make the diagnosis.

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MAJOR DEPRESSION IN ADOLESCENT CHILDREN CONSECUTIVELY DIAGNOSED WITH MITOCHONDRIAL DISORDER Koene S1, Kozicz TL2, Rodenburg R1, van den Heuvel B1, Verhaak C1, Smeitink JAM1, Morava E1 1 Radboud University Medical Center, Nijmegen, Netherlands, 2Catholic University of Nijmegen, Nijmegen, Netherlands A higher incidence of major depression has been described in adults with a primary oxidative phosphorylation disease. Intriguingly however, not all patients carrying the same mutation develop symptoms of major depression, pointing out the signi¢cance of the interplay of genetic and non-genetic factors in the etiology. In a series of paediatric patients evaluated for mitochondrial dysfunction, out of 35 children with a biochemically and genetically con¢rmed mitochondrial disorder, we discovered ¢ve cases presenting with major depression prior to the diagnosis. The patients were diagnosed with di¡erent molecular genetic defects, including the common ¢ve kb mtDNA deletion and mutations in MTTK, MTND1, POLG1, or PDHA1. The children showed biochemical defects in the mitochondrial energy generating system in muscle, compatible with their molecular genetic defects. Elevated lactate levels were demonstrated in the cerebral spinal £uid, while protein and glucose concentrations, immunoglobins, anti-gangliosides and neurotransmitters were normal. No signi¢cant di¡erence could be con¢rmed in the disease progression or the quality of life, compared to the other, genetically con¢rmed mitochondrial patients. In three out of our ¢ve patients a signi¢cant stress life event was con¢rmed in the medical history. We propose the abnormal central nervous system energy metabolism as the underlying cause of the mood disorder in our paediatric patients. Certain medications in children with major depression might cause an additional deteriorating e¡ect on mitochondrial function. Exploring the genetic etiology in children with mitochondrial dysfunction and depression is essential both for optimal therapy and adequate counselling.

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CLINICAL, BIOCHEMICAL AND GENETIC FEATURES IN 18 CHILDREN WITH 3-METHYL-GLUTACONIC ACIDURIA TYPE IV Wortmann SB1, de Vries MC1, Rodenburg RJ2, van den Heuvel LP2, Kluijtmans LA2, Engelke U2, Heldt K3, Smeitink JAM1, Wevers R2, Morava E1 1 Dept of Ped, Radboud Univ Med Centre, Nijmegen, Netherlands, 2Lab of Ped/Neu, Radboud Univ Med Centre, Nijmegen, Netherlands, 3Dept of Gen Ped, Univ Child Hosp, Du«sseldorf, Germany The heterogeneous group of 3-methylglutaconic-aciduria (3-MGA) includes the classic organic aciduria 3-MGA type I, Barth (type II) as well as Coste¡ syndrome (type III). In addition, the quite unspeci¢ed 3-MGA type IV consists of patients with 3-MGA and OXPHOS-dysfunctioning. Previously within this group we de¢ned a distinct clinical phenotype with sensorineural deafness, encephalopathy, and Leigh-like syndrome (MEGDELassociation). Typ e V was re c ently descr ibed with ataxia and cardiomyopathy. Here we describe the clinical and biochemical phenotype in 18 children with 3-MGA type IV. In addition to the phenotype with MEGDEL-association we de¢ne 4 clinical subgroups. The hepatocerebral phenotype presents mostly with complex-I-de¢ciency and mtDNAdepletion, a third of the children carrying POLG1-mutations. The cardiomyopathic type demonstrates encephalomyopathy and an overlapping phenotype with that described by Sperl with isolated ATP-asede¢ciency. One male with a myopathic form, severe combined OXPHOSdisorder and RYR1-mutations displays the pathognomic histology of central core disease. In the rather nonspeci¢c clinical group of children with an encephalomyopathic phenotype without liver involvement, SUCLA2-mutations were detected in 3 patients with MMA-uria, Leighlike encephalomyopathy, dystonia and deafness. Biochemical analysis and NMR spectroscopy in urine showed concentrations of 22^196 mmol/mmol creat and 1:1 ratio of `cis' and `trans' forms of 3-MGA in all patients. Interestingly this ratio di¡ers from the 2:1 ratio of patients with 3-MGA type I. By delineating patient-groups we elucidated the genetic defect in 7 out of 18 children. We suggest POLG1, SUCLA2 and RYR1 sequence analysis and mtDNA-depletion studies in children with 3-MGA type IV.

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THIRD REPORTED CASE OF PYRUVATE DEHYDROGENASE PHOSPHATASE 1 (PDP 1) DEFICIENCY: PROMISING RESPONSE TO TREATMENT WITH SODIUM BICARBONATE Barnett C1, Maj M2, Raiman J1, Robinson B2, Schulze A1 1 Div Clin Met Gen, Hosp Sick Children, Toronto, Canada, 2Met Res Prog, Dept Biochem, HSC, Toronto, Canada

TISSUE-SPECIFIC PYRUVATE DEHYDROGENASE DEFICIENCY IN A BOY WITH NOVEL MUTATIONS IN THE E1 b SUBUNIT Mayr JA1, Koch J1, Rauscher C1, Bernert G2 Sperl W1 1 Dept Pediatr, Paracelsus Med Univ, Salzburg, Austria, 2Preyer Kinderspital, V|enna, Austria

Background: The pyruvate dehydrogenase complex (PDHc) is a mitochondrial matrix complex whose primary function is to catalyse the oxidative decarboxylation of pyruvate to acetyl CoA. PDHc function is regulated by phosphatases (PDP 1 and 2) and kinases (PDH kinases 1-4) and an inheritable de¢ciency of PDP1 has been recently shown to cause PDHc de¢ciency. PDHc de¢ciency causes pyruvate accumulation and resultant lactic acidosis, which can be lethal in the neonatal/infantile period. Here we describe the third case of PDP1 de¢ciency in the literature. Case report: A 2 month old infant was seen following lactic acidosis in the neonatal period. She was born to a G5P1 mother in a consanguineous union. Following delivery at 38 weeks she was admitted to the NICU with e¡ortless tachypnoea. Hypoglycemia (BSL 1.8), metabolic acidosis and elevated lactate (8.9 mmol/L) were noted. Biventricular hypertrophy was seen on echocardiography. After a period of apparent stabilisation, lactic acidosis was again noted at 4 weeks of age and the patient was re-admitted. Both the metabolic acidosis and elevated lactate responded well to treatment with sodium bicarbonate during hospitalization. Using the equation for required HCO3 dose for full correction (0.36wt6BE), the improvement in base de¢cit was consistently greater than expected. Lactate consistently fell after bicarbonate administration and after stopping regular bicarbonate treatment, lactate rose from a baseline of 2.4 mmol/L to 14.3 mmol/L within 48 h. The patient was discharged on maintenance bicarbonate therapy but died after a presumed acute decompensation secondary to a viral infection at 4 months.

Pyruvate dehydrogenase complex (PDHC) de¢ciency can lead to the clinical picture of Leigh syndrome and is caused mostly by mutations in the E1 a subunit, rarely in other subunits or the pyruvate dehydrogenase phosphatase. We report on a patient with novel E1 b mutations and a delayed onset of the disease and a relatively mild clinical course with Leigh-like MRI ¢ndings. F|rst symptoms were noticed at an age of 16 months with recurrent episodes of infection triggered metabolic crises with somnolence, transient tetraparesis, ptosis, strabism and lactic acidosis. In muscle tissue biochemical investigations showed a reduced oxidation of pyruvate substrates and a diminished PDHC activity. However in ¢broblasts PDHC activity was normal. Western blot analysis of a 600 g supernatant revealed a clear reduction of E1 b but also E1 a protein in skeletal muscle while these proteins were only mildly reduced in ¢broblasts. Two novel compound heterozygous mutations p. [Met101Val]+[Arg105Gln] were identi¢ed in a conserved region of the E1 b subunit. In conclusion we present a new patient with E1 b de¢ciency with Leigh-like symptoms which was detected by enzyme analysis in muscle tissue but would have been missed by PDHC measurement in ¢broblasts.

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PYRUVATE DEHYDROGENASE PHOSPHATASE (PDP1) DEFICIENCY. INFANTILE LACTIC ACIDEMIA AND SEVERE PHENOTYPE DUE TO A NULL MUTATION Robinson BH1, MacKay N1, Levandovskiy V1, Maj M1, Raiman J1, Feigenbaum A1, Shulze A1, Cameron JM1 1 Div Genetic Metab Dis, Hosp Sick Child, Toronto, Canada While the existance of an entity related to poor activation of PDH has been postulated, PDP1 de¢ciency has only been de¢ned in one family at the molecular level. This was in two brothers with a presentation of exercise intolerance and mild develomental delay (Maj, M et al., J. Endicrinol. Metab 90, 4101^4107, 2005). A female patient, born of consnaguinous parents, developed lactic acidemia soon after birth which £uctuated between 2.5 and 17 mM until her death at 6 months of age. CSF lactate was found to be elevated by functional MRS. There was a suspicion of cardiac conduction abnormalities in the neonatal period which seemed to resolve. The child exhibited hypotonia and the MRI showed bilateral parietal oedema. The native pyruvate dehydrogenase complex activity was 35% of the fully activated complex compared to the normal range of 58^91% in cultured skin ¢broblasts. The percentage of active native PDH also declined with passage number in a reproducible fashion. Sequencing of PDP1 demonstrated a homozygous G277T (E93X) nonsense mutation in both cDNA and genomic DNA. Immunoblotting showed a complete absence of PDP1 protein in ¢broblast mitochondria. PDHC activity could be restored by addition of recombinant PDP1. This is the second family reported with a molecular diagnosis of PDP1 de¢ciency. The severity indicates that, compared to exercise intolerance seen in the ¢rst case, there is ample room for a spectrum of severity associated with this inborn error depending on the molecular defect.

IMMUNOSTAINING TECHNIQUES IN PATIENTS WITH mtDNA DEPLETION Van Coster R1, De Paepe B1, Smet J1, Seneca S2, Lissens W2, De Meirleir L2, Roels F3 1 Ped Neurol and Metabol, Univ Hosp Ghent, Ghent, Belgium, 2Cent of Med Genet, Univ Hosp Brussels, Brussels, Belgium, 3Dept of Pathol, Univ Hosp Ghent, Ghent, Belgium Background: mtDNA depletion is a prevalent cause of multiple OXPHOS de¢ciency. The depletion, however, is tissue speci¢c and the clinical phenotype is heterogeneous. Therefore, the diagnosis can be di¤cult. We investigate the role of immuno-staining techniques in the diagnosis and evaluated for common and speci¢c features. Methods: Cultured skin ¢broblasts and liver tissue from one patient with DGUOK (P1) and ¢ve patients with POLG (P2^6) mutation were retrospectively evaluated for their immuno-cytochemical and immunohistochemical staining patterns for OXPHOS complexes I to V. Results: Spectrophotometric analysis showed normal OXPHOS activities in the ¢broblasts of all patients. However, in the cell cultures from three patients a mosaic of immuno-negative and immuno-positive ¢broblasts was found: complex I and complex IV mosaicism in P4 and P6, complex I mosaicism in P5. F|broblasts from three patients (P1^P3) displayed normal immuno-staining patterns. Spectrophotometric analysis of liver tissue showed combined OXPHOS de¢ciencies and preserved complex II activity. Immuno-histochemical staining was carried out in liver from P2, P3 and P5. All three displayed a mosaic staining pattern for complex I and IV, with positive and negative hepatocytes found alongside, in contrast to homogeneous complex II staining. In these patients, histochemical staining con¢rmed the mosaicism for COX activity in hepatocytes. Conclusions: Although mtDNA depletion is a frequent cause of multiple OXPHOS de¢ciency, diagnosis can be di¤cult as spectrophotometric analysis of OXPHOS complex activities can be normal. Our data show that the implementation of immuno-detection of OXPHOS proteins can be an additional tool to successfully identify patients with mtDNA depletion.

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ROLE OF BN-PAGE IN THE DIAGNOSIS OF MITOCHONDRIAL DNA DEPLETION Van Coster R, Smet J, De Paepe B, Seneca S, Lissens W, De Meirleir L Cent of Med Gent, Univ Hosp Brussels, Brussels, Belgium Background: The group of mtDNA depletion (MIM 251880), a prevalent cause of mitochondrial disease, is a clinically and genetically heterogeneous group of autosomal recessive diseases characterized by reduced mtDNA copy number. Responsible genes are involved either in mtDNA replication or in recycling of the nucleotides in the mitochondria. The aim was to study the role of BN-PAGE in the diagnosis. Methods: T|ssue samples from 434 clinically suspected patients were analyzed using spectrophotometrical analysis and Blue Native PolyAcrylamide Gel Electrophoresis (BN-PAGE) followed by activity staining in the gel. The amount of mtDNA was measured using realtime PCR. In the patients in whom mtDNA depletion was found, DNA analysis of thymidine kinase 2 (TK2), deoxyguanosine kinase (DGUOK) and DNA polymerase gamma (POLG) was performed. Results: In 91 patients a decreased enzyme activity in one or more OXPHOS complexes was detected using spectrophotometrical analysis. In 44 patients the presence of V subcomplexes was detected following BN-PAGE evaluation. Extensive testing of the mtDNA revealed 25 patients in whom a mtDNA defect was identi¢ed. In nine of them mtDNA depletion was found. Mutations were detected in the POLG (7), DGUOK (1) and TK2 gene (1). Conclusions: Our study shows that mtDNA depletion is a prevalent cause of OXPHOS dysfunction. Application of BN-PAGE proves to be a powerful tool in selecting the patients for genetic evaluation of the mtDNA.

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HEART TRANSPLANTATION IN RESPIRATORY CHAIN DISORDERS Mundy H1, Hargreaves I2, Ashworth M3, Burch M3, Gru«newald S3 1 St Thomas' Hospital, London, UK, 2National Hospital for Neurology, London, UK, 3Great Ormond Street Hospital, London, UK Objectives: We report on two patients with complex IV de¢ciency presenting with hypertrophic cardiomyopathy in end-stage heart failure. Both were accepted for heart transplantation. Case reports: The ¢rst patient was diagnosed with complex IV de¢ciency in h i s ¢ rst months of life. He had undergone postnatal echocardiography, as his older sister had died in cardiac failure. His initial mild cardiomyopathy improved on conservative management. He was monitored carefully and remained well until the age of 17 months, when he unexpectedly rapidly deteriorated, requiring ECMO. After 2 weeks, he was changed to a Berlin heart as a bridging procedure to heart transplantation. The multidisciplinary decision was reached on the background of his age appropriate development and lack of any other organ system involvement. He was transplanted 5 months later. At his last assessment at the age of 28 months, he remains very well. The second patient was heart transplanted at the age of 12 years, shortly after been diagnosed with complex IV de¢ciency. His clinical history included failure to thrive, hearing and renal impairment. His development was normal. Both children sailed through the heart transplantation surgery, but had considerable complications post surgery including stroke and infarction, resulting only in transient neurological sequelae. The older patient however, developed acute on chronic renal failure, his hearing deteriorated and there is progressive retinal dystrophy. Conclusions: The suitability for patients with mitochondrial disorders for heart transplantation remains a subject to debate and is a very complex decision.

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COENZYME Q10 DEFICIENCY ASSOCIATED WITH A MITOCHONDRIAL DNA DEPLETION SYNDROME Montero R1, Briones P2, Garesse R2, Navarro-Sastre A2, Gallardo ME2, Montoya J2, Marti R2, Ribes A2, Pineda M2, Garc|èa-Cazorla A2, Navas P2, Artuch R1 1 Biochem Dept, Hosp St Joan de Deu, Barcelona, Spain, 2CIBER de Enfermedades Raras (CIBERER), ISCIII, Spain Background: Mitochondrial DNA depletion syndromes are a common cause of mitochondrial diseases in infancy. In spite that mutations in several nuclear genes have been associated with this syndrome, there are still mtDNA depletion cases from unknown origin. Methods: We report on a one-day-old consanguineous girl. The child was born prematurely (33 weeks). Generalized hydrops, poor activity, hypotonia and hyperlactacidaemia. Other biochemical tests for screening of inborn errors of metabolism were normal. The child died at ten days of life. CoQ concentration and mitochondrial respiratory chain enzyme activities were analyzed in muscle and skin biopsies by reverse-phase HPLC with electrochemical detection and by spectrofotometric analysis respectively. mtDNA depletion was investigated by quantitative real-time-PCR. Results: Muscle biopsy revealed a decrease in citrate synthase, complex II + III activities of the mitochondrial respiratory chain and in CoQ content (46 nmol/gram of protein: reference values: 125^450) and cultured skin ¢broblasts (19 nmol/gram of protein: reference values: 51^119). Mitochondrial DNA revealed a 78% of depletion in muscle. Mutational study of DGUOK, POLG, TK2, MPV17, SUCLA2 and RRM2B genes sequencing of all exons and intron boundaries failed to reveal any pathogenic mutation. Conclusions: This is the ¢rst time that mtDNA depletion syndrome has been associated with CoQ de¢ciency. Our results support the hypothesis that CoQ de¢ciency might be involved in the pathophysiology of this case.

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SEGREGATION STUDY IN FAMILY WITH SEVERE VARIANT OF THE A3302G MUTATION IN THE MITOCHONDRIAL tRNA LEU (UUR) Ballhausen D1, Guerry F2, Hahn D3, Bonafeè L1, Jacquemont S2 1 IEM, Molecular Pediatrics, CHUV, Lausanne, Switzerland, 2Medical Genetics, CHUV, Lausanne, Switzerland, 3Clinical Chemistry, Inselspital, Bern, Switzerland The mutation A3302G in mitochondrial tRNA LEU(UUR) is associated with respiratory chain complex I de¢ciency and has been described in 11 individuals of 5 families so far. Disease manifestation mainly occurs in young adults with slowly progressive myopathy and exercise intolerance. Early death (10^35 years) due to cardiorespiratory failure has been reported in 5 patients (4 of the same family). Genetic counseling is very di¤cult in these families due to variable age of onset and lack of reliable data on correlation between mutation load in di¡erent tissues and clinical manifestation. We report on a 7 year-old girl with a rapidly progressive myopathy since the age of 2 years. A severe complex I de¢ciency and a mutation load of 498% for A3302G was found in muscle. At present, she is wheel-chair bound, su¡ers from frequent bronchitis with respiratory decompensation and undergoes a therapeutical trial with ketogenic diet. We performed a segregation study for A3302G mutation load in several tissues in three generations of this family. Muscle biopsies were performed in the mother and her 2 siblings enabling OXPHOS assays. All participants were healthy and did not show any myopathic traits. We found a mutation load of up to 45% in muscle with normal results of OXPHOS assays in clinically not a¡ected adults. The variation of mutation load between di¡erent tissues (blood, ¢broblasts, buccal swab, hair and muscle) was relatively small. These results indicate that relatively high mutation loads are necessary to alter complex I activity leading to clinical manifestation.

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A NOVEL MUTATION IN PYRUVATE CARBOXYLASE DEFICIENCY TYPE CLang TF1, Wang D2, De V|vo DC2, Loughrey CL1 1 Clin Biochemistry, Belfast HSC Trust, Belfast, UK, 2Dept Neurology, Columbia UMC, New York, USA Background: Pyruvate carboxylase (PC) de¢ciency typically presents with overwhelming neonatal lactic acidosis or an intermittent acidosis with severe developmental delay in infancy. The rarest form (type C) is benign, presenting as episodic acidosis. We present a case of type C PC de¢ciency with a novel mutation in the PC coding region, resulting in a premature stop codon. Case History: A 12-month-old boy presented acutely with lethargy and vomiting. Investigations revealed a raised anion gap metabolic acidosis (HCO3 7 mmol/L) and raised lactate (10.2 mmol/L). He was managed initially with IV £uids including sodium bicarbonate. Peritoneal dialysis was instituted after 24 h for persistent acidosis. The lactic acidaemia was not exacerbated by fasting. Lactate: pyruvate ratio was 12.8. Cultured ¢broblasts were found to exhibit 51% of normal PC activity. Mutation analysis identi¢ed compound heterozygous mutations: c.[1705A4G,=] +[=,3499-3500delCT] in DNA level; p.[T569A,=]+[=, L1137VfsX1170] in protein level. The child is now age 3.5 years, and has been admitted on several occasions with minor intercurrent illnesses which are managed conservatively. His baseline lactate is approximately 5.0 mmol/L and he does not require bicarbonate on an ongoing basis to maintain normal acid-base status. Psychomotor development is thus far normal. Discussion: We present a case of PC de¢ciency which is typical of the benign form, with a previously unreported mutation. The complete enzyme de¢ciency exhibited by this child is typical of this phenotype. This case represents the third reported case of type C PC de¢ciency.

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EXPANDING KNOWLEDGE ON SUCLG1 MUTATIONS CAUSING ATYPICAL METHYLMALONIC ACIDURIA Carrozzo R1, Lucioli S1, Bruno C2, Burlina AB3, Haas D4, Ho«rster F4, Yano S5, Kluijtmans LA6, Bianchi M1, Meschini MC1, Verrigni D1, Wevers RA6, Rizzo C1, Boenzi S1, Santorelli FM1, Dionisi-V|ci C1 1 Div Metab and Mol Med, Bambino Gesu© Hosp, Rome, Italy, 2Div Neuromusc Dis, Gaslini Hosp, Genoa, Italy, 3Div Metab Dis, Univ Child Hosp, Padua, Italy, 4Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 5Med Genet, Child Hosp, Los Angeles, USA, 6Lab Pediatr Neurol, Radboud Univ AMC, Nijmegen, Netherlands Background: Atypical forms of MMA has been recently observed in patients with mtDNA depletion carrying mutations in succinyl-CoA synthetase (SCS), the enzyme complex that catalyzes the conversion of succinyl-CoA to succinate in TCA cycle. SCS is composed by an alfa-subunit (coded by SUCLG1) and by beta-subunits (coded by SUCLA2 and SUCLG2). SUCLA2 mutations have been reported in several patients all presenting Leigh-like encephalomyopathy, dystonia and deafness, whereas SUCLG1 mutations has been found only in three related patients. Their phenotype was extremely severe with antenatal manifestation of the disorder (IUGR, microcephaly, dimorphisms, polydactyly), neurological and liver signs, neonatal onset and early death. Patients/results: We report six patients from 4 families presenting an atypical MMA. F|ve are alive (age 1.8^17 years) and one died at 2 years. Onset of symptoms ranged from 1 day to 5 months. All presented hypotonia, dystonia, and developmental delay. Additional signs include deafness (1/6), optic atrophy (2/6), peripheral neuropathy (2/6), and seizures (1/5). MRI showed Leigh-like lesions (5/6) and frontal lobe atrophy (1/6). All had lactic acidosis. Urine organic acid showed elevated MMA (range 76^2400 mmol/mol creat), methylcitrate, lactate, and Kreb's cycle intermediates. Blood acylcarnitines showed elevation of C4-dicarboxylyl-carnitine (i.e. succinyl-carnitine). Multiple OXPHOS defects and mtDNA depletion were detectable in muscle and/or ¢broblasts. Sequence analysis of candidate genes (SUCLA2, SUCLG1 and SUCLG2) showed the presence of 6 new mutations in SUCLG1. Conclusions: This report enlarges the spectrum of the phenotypic expression of SUCLG1 mutations and con¢rms that mild MMA and succinyl-carnitine esters constitute the biomarker of SCS defects.

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MUTATION STUDIES IN PATIENTS WITH PYRUVATE CARBOXYLASE DEFICIENCY Heptinstall LE1, Walter JH1, Morris AAM1, Jones SA, Rahman S2, Besley GTN1 1 W|llink Biochem Genet Unit, Manchester, UK, 2Institute of Child Health, London, UK Pyruvate carboxylase (PC) is an important gluconeogenic and anapleurotic enzyme. Its de¢ciency may lead to severe (Type B) or mild (Type C) presentations of lactic acidosis. The condition is rare and apart from studies in the intermediate/North American (Type A) variant, little has been published on the underlying mutations. We have studied mutations in 9 families diagnosed with PC de¢ciency. Our patients ranged from severe to mild; 5 were British Caucasians, 3 were of Asian origin and one from South Africa. F|broblast DNA was extracted and sequenced on an ABI 3130 sequencer following PCR ampli¢cation. Mutations were identi¢ed in 16/18 alleles. Twelve novel mutations were identi¢ed in 9/20 exons. The 3 unrelated and severe Asian patients were homozygous for 3 di¡erent mutations: c.3381__3399del, p.G169D and c.1154__1155delGC. These were from consanguineous families; each led to a severe PC de¢ciency (51%). All other patients were compound heterozygotes. Three clinically mild patients had signi¢cant residual PC activity (2, 6 and 24%) and 2/3 patients had the same R451H mutation in exon 9 and on the same codon as a previously reported mutation. From our studies and those previously reported, it would appear that there were `hot spots' on exons 15 (cation-binding site), 20 (biotinbinding site) and codon 451.

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A COMPARISON OF TWO METHODS TO DETERMINE THE REE IN CHILDREN DIAGNOSED OR SUSPECTED OF A MITOCHONDRIAL DISEASE van den Berg AMJ, Morava E, Rasmussen E UMC St Radboud, Peadiatric Department, Nijmegen, Netherlands Background: Children, diagnosed with a mitochondrial disease, are often known with malnutrition and eatingproblems. The aim of this prospective study is to compare two methods to determine the resting energy expenditure (REE) in children: the standard method of Scho¢eld (S) and the indirect calorimetry (IC). Method: Until now 26 children (0^20 years) were included. The IC was planned in the out-patient clinic. The circumstances were as optimal as possible: relaxed, parents accompanying their children, according the standard procedure in measurements. Results: The IC failed 6 times (23%); 5 children (19.2%) were too stressed. The measurement by IC was performed 20 times (76.9%) successfully. The comparison of the results measured by IC and the results calculated by S showed the following di¡erences: the REE of 2 children measured by IC was equal to the REE calculated by S, the REE of 4 children measured by IC was higher (mean 58 kcal) than calculated by S, but the REE of 14 children measured by IC was lower (mean 134 kcal) than calculated by S. Conclusion: The REE measured by IC in 18 children (90%) showed remarkable di¡erences in comparison with the standard calculation method by S. These di¡erences should be known in order to optimize the nutritional support as adequate as possible in treatment of children diagnosed of a mitochondrial disease.

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NUTRITION, GROWTH AND THE MITOCHONDRIAL FUNCTION Koene S, Wortmann S, Rodenburg R, van de Berg A, Zweers H, van den Heuvel L, Smeitink J, Morava E Radboud University Medical Center, Nijmegen, Netherlands Children with mitochondrial dysfunction often present with feeding problems, gastrooesophageale re£ux and failure to thrive. Secondary deterioration of the mitochondrial function has been reported in various chronic disorders in combination with anorexia. Previously we reported on an improvement of the mitochondrial energy production in children following nutritional intervention. We prospectively evaluated 172 children undergoing muscle biopsy for a suspected OXPHOS-disorder and evaluated the correlation between growth, the metabolic and clinical condition, nutritional state, resting energy expenditure and mitochondrial energy production. Dietary evaluation was performed and nutritional intervention was initiated when needed prior to muscle biopsy. Additional measurement of the resting energy expenditure (indirect calorimetry) was requested in 28 consecutively evaluated children. Mitochondrial dysfunction was con¢rmed by detection of enzyme complex de¢ciencies and/or by mutations in 83 children, in 14 patients no biochemical abnormalities were found. In our prospective study group, as well as in the subgroup with enzyme complex de¢ciency, a signi¢cant correlation was found between the ATP production and the age related BMI, but not with respect to the weight and height for age. Indirect calorimetry in the patient group with abnormal mitochondrial function demonstrated highly variable levels of resting expenditure, resulting in individualized nutritional intervention. In summary we found a positive correlation between the mitochondrial energy production and the age related BMI in patients with the clinical diagnosis of OXPHOS disease. An individualized diet and optimal dietary intake in children with a mitochondrial disease improve the energy generating capacity, the clinical condition and the quality of life in patients.

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MITOCHONDRIAL ND1 INVOLVEMENT IN FRONTOTEMPORAL DEMENTIA Santos MJ1, Cleto S2, Mendes C1, Pratas J1, Simêes M1, Santana I3, Oliveira CR2, Grazina M2 1 Centro Neurocieªncias e Biologia Celular, Coimbra, Portugal, 2 Faculdade Medicina, Universidade Coimbra, Coimbra, Portugal, 3 Hospitais da Universidade de Coimbra, Coimbra, Portugal Background/Objectives: Frontotemporal dementia (FTD) is the second most common type of primary degenerative dementia, frequently presenting neuropathological and clinical overlaping with Alzheimer's disease (AD). Following the report of mtDNA alterations found in a FTD patient, correlated with mitochondrial respiratory chain (MRC) de¢ciency of complex I, we aim to determine the involvement of mtDNA ND1 in FTD. Methods: We studied 27 patients recruited at the Neurology Unit of HUC with probable diagnosis of FTD, aged between 43 and 81 years old (mean+sd: 58+11). Total DNA was extracted from peripheral blood (in EDTA) by standard methods. Analysis of ND1 complete sequence was performed using the Kit mitoSEQr Resequencing System. Peripheral leukocytes were isolated from 50 ml of blood (in EDTA) and MRC complex I (NADH-cytochrome c oxidoreductase; EC 1.6.5.3) activity was evaluated as described (Rustin et al., 1994). The results were expressed normalized to CS. De¢ciency was considered if 540% of mean reference value. Results: We have found 11 mtDNA ND1 sequence variations, 6 polymorphic, 3 pathogenic and 2 unreported, in 12 patients (44%). Decreased complex I activity was detected in 15% of cases. Conclusions: The results suggest the involvement of mtDNA and respiratory chain in FTD, similarly to AD, reinforcing their role in dementia. Phenotype^genotype correlation shows heterogeneity of patterns, but normal MRC is preferentially associated to the absence of mtDNA variations (7% higher). The exact role of mtDNA needs further examination, but our data support mitochondrial cascade hypothesis in FTD etiopathogeny. F|nancial support: Projecto GAPI n: 19/07

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OXIDATIVE STRESS IN CHILDHOOD MITOCHONDRIAL RESPIRATORY DISEASE ^ 20 CASES Neves N1, Garcia P1, Proenc°a T2, Baldeiras I2, Grazina M3, V|larinho L4, Oliveira CR3, Diogo L1 1 Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal, 2Lab Neuroq, Dept Neur, HUC, Coimbra, Portugal, 3Inst Bioq Faculdade Med Univ Coimbra, Coimbra, Portugal, 4Laboratoèrio Nacional Rastreios, IGMJM, Porto, Portugal

THE ESTIMATION OF mtDNA POLYMORPHISM IN PATIENTS WITH MITOCHONDRIAL PATHOLOGY IN UKRAINE Gusar VA1, Grechanina OYa2, Zhadanov SI3, Grechanina YuB2, Zdybskaya OP2, Fedoseeva NP1, Schurr TG4 1 Specialized Medical Genetic Center, Kharkiv, Ukraine, 2Institute of Clinical Genetics KhNMU, Kharkov, Ukraine, 3Institute Cytology and Genetics, SB RAS, Novosibirsk, Russian Federation, 4University of Pennsylvania, Philadelphia, USA

Background: Increased generation of reactive oxygen species and enhanced oxidative stress underlie the pathophysiology of respiratory chain mitochondrial disorders (MRCD). Objectives: Evaluate blood antioxidant status in a group of 20 children classi¢ed as de¢nite MRCD, according to Bernier's diagnostic criteria. Methods: Uric acid, a-tocoferol/cholesterol ratio, reduced glutathione (GSH), malondialdehyde (MDA) and total antioxidant capacity in plasma and atocoferol, GSH, superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes were measured by standard methods in 20 MRCD and 17 healthy children (control group-CG). Median ages were 5.3 (SD+4.7) and 8.4 (SD+4.7) years, respectively in MRCD group and CG. Student T test was used for statistical analysis. MRCD patients presented as unspeci¢ed encephalopathy in 11 cases, Leigh syndrome (four: one 8993T4G; two SURF1 gene mutations; a Q8L mutation and an insertion in exon 1), multi-system disease (three), myopathy with cardiomyopathy (3302 A4G) and migraine with alternate hemiplegia (one each). Hyperlactacidemia and a major respiratory chain enzyme complex de¢ciency in muscle were detected in 17 and 18 patients, respectively. None was treated with anti-oxidants at the time of the study. Results: No statistically signi¢cant di¡erences in plasma values between MRCD and the CG groups were found. In erythrocytes, the results were signi¢cantly di¡erent for a-tocoferol: 9.7+7.7 v 2.2+1.6 nmol/gHb (p50.01), GSH: 1.55 +0.73 v 9.95+5.37 mmol/gHb (p50.01) and SOD: 873.0+305.6 v 582.0+158.4 U/gHb (p50.001). Conclusions: Changes in antioxidant defense system in our group of MRCD patients was demonstrated only in erythrocytes. Erythrocyte might undergo adaptive changes, (enzyme overexpression and accumulation of GSH) over time, plasma changes being transient.

Background: One of the important and challenging aspects of mitochondrial genetic studies is to understand the role of population genetic background in penetration of pathogenic mutations and expression of mitochondrial diseases. To further investigate their relationship we have analyzed 50 samples of patients with mitochondrial pathology from Ukraine. Methods: Sequencing of hypervariable segment HVS I of mtDNA control region. Restriction analysis of mtDNA coding region. Phylogenetic analysis. Statistical analysis (http://cmpg. unibe. ch/software/ arlequin3). Results: We found polymorphism in 55 sites of the mtDNA. 51 nucleotide substitutions were transitions, with prevalence pyrimidine under purine as follows: C ^ 40.73%, T ^ 27.90%, A ^ 23.05%, G ^ 8.32%. Transversions were found at 4 sites. Overall, 36 haplotypes were found; unique from them were 60.0%. The most common haplotype (14.0%) belonged to CRS. Indexes of genetic diversity for selection of patients were D = 0.990+0.007 and yp = 6.13, accordingly. Screening for polymorphic sites established the presence of mtDNA haplogroups (86.0%) with Pan-European distribution: H^24.0%, pre-V2.0%, V-2.0%, J^12.0%, T^16.0%, U (U3, U5)^18. 0%, I^2.0%, W^2.0%, X^8.0%. There were Eastern Eurasian variants (A, C) at low frequency, 4.0%. An additional polymorphisms in haplogroups U5, T and é were found in combination with their basic motif. Conclusion: Further researches of mtDNA haplotypes of patients will allow to estimate certain role of genetic background in the expression of mitochondrial diseases.

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MITOCHONDRIAL G12300A MUTATION IS ASSOCIATED WITH A SEVERE MITOCHONDRIAL MULTISYSTEMIC DISORDER Mart|èn-Jimeènez R1, Mart|èn-Hernaèndez E2, Santiago B2, Mendan¬a L1, Cabello A2, Fernandez EM1, Campos Y1 1 Instituto de Salud Carlos III, Majadahonda, Spain, 2Hospital 12 de Octubre, Madrid, Spain Mitochondrial G12300A transition, located in the anticodon of the tRNA Leu (CUN) gene, has been described as a suppressor mutation of the mitochondrial A3243G point mutation in the tRNA Leu (UUR) gene. We report a patient with mitochondrial encephalomyopathy and high levels of the G12300A mutation. Methods: We studied an 8 year-old patient with sensoryneural hearing loss, cerebellar syndrome, myoclonus epilepsy, pigmentary retinopathy, lactic acidosis and bilateral calci¢cations of basal ganglia. Her mother su¡ered partial sensoryneural hearing loss. A muscle and skin biopsies were taken from the patient and muscle histochemistry and mitochondrial respiratory chain complex activities were analysed as described. Mitochondrial DNA (mtDNA) deletion and depletion were excluded by southern blot; mitochondrial tRNA genes were screened by direct sequencing and single-¢ber PCR-RFLP analysis was done. Mitochondrial membrane potential (Cm) was measured in ¢broblasts by £ow cytometry and JC-1 and mitochondrial network was observed by confocal microscopy and MitoTracker red 580. Results: Muscle biopsy showed 12.5% cytochrome oxidase negative ragged-red ¢bers (RRF) and combined defects of the respiratory chain complexes I, III and IV in muscle and ¢broblasts. The mutation was heteroplasmic, very abundant in both tissues from the patient and less abundant from her mother blood. Single muscle ¢ber PCR showed higher levels of mutant genomes in RRF than in normal ¢bers. F|broblasts with 89% of mutated mtDNA had a signi¢cant Cm reduction and a disturbed mitochondrial network was observed. Conclusions: mitochondrial G12300A point mutation ful¢lls accepted criteria for pathogenicity and is associated with a severe multisystemic disorder.

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CEREBRAL PALSY (CP) AND OXIDATIVE PHOSPHORYLATION (OXPHOS) DISORDERS: CLUES TO A MITOCHONDRIAL DIAGNOSIS Cozens A, Yaplito-Lee J, Boneh A Metabolic Service, GHSV, Melbourne, Australia Background: CP describes a group of motor disorders caused by ¢xed lesions of the developing brain. OXPHOS disorders are progressive, neurodegenerative conditions, but there may be long periods of stability, when CP may be wrongly diagnosed. Di¡erentiating these conditions provides important prognostic and genetic information, and may a¡ect treatment decisions. Method: Case note review. Results: We found 7 patients with all three types of CP movement disorder: spastic (2), dyskinetic (1) and ataxic (3). We describe the clinical features of three representative patients with established CP, stable for years, subsequently diagnosed with OXPHOS disorder. Patient 1. A boy born preterm with right hemiparesis (`spastic CP') and delay. He had seizures age 3, and 12 years. At age 14 years he had cluster seizures, encephalopathy and metabolic acidosis. Diagnosis: MELAS. Patient 2. A girl aged 2 years with `ataxic CP'. At age 5 years she had focal seizures hemiparesis and progressive myoclonic epilepsy. Diagnosis: Alpers syndrome (POLG). Patient 3. A girl aged 9 months with `dyskinetic CP', profound delay, seizures at 14 months and a new squint at 2 years. Seizures worsened, with rapid loss of vision aged 18 years. Diagnosis: Complex I de¢ciency. Conclusions: None had good evidence to support the diagnosis of CP. Only one had adverse perinatal history; all had normal early MRI scans. Unusual clinical features included: unexpected patterns of motor disability, new motor signs; new onset squint and intermittent nystagmus; explosive onset of seizures. Recommendation: Children with CP of no clear etiology, and/or new onset clinical symptoms, should be investigated for OXPHOS disorders.

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HIGH THROUGHPUT MUTATION SCREENING IN PATIENTS WITH COMBINED RESPIRATION CHAIN COMPLEX DEFECT Hillier I1, Freitag M1, Ahting U2, Taverna M1, Rolinski B2, Freisinger P3, Tesarova M4, Zeviani M5, Meitinger T6, Prokisch H1 1 HelmholtzZentrum, German Res Cent Health, Mu«nchen, Germany, 2 Dept Clin Chem, Klinikum Schwabing, Mu«nchen, Germany, 3Dept of Pediatrics, Technical University, Mu«nchen, Germany, 4Dept of Pediatrics, Charles-University, Prague, Czech Republic, 5Institute of Neurology C. Besta, Milano, Italy, 6Inst Human Genet, Technical University, Mu«nchen, Germany Introduction: Mitochondria are the main energy-producing organelles of the cell. F|ve complexes embedded in the mitochondrial inner membrane, together constituting the oxidative phosphorylation (OXPHOS) system, comprise the ¢nal steps in cellular energy production. About one third of patients with a mitochondrial defect su¡er from a so-called combined de¢ciency with decreased enzymatic activities of two or more complexes of the OXPHOS system. The genetic cause for most of the diseases is not known. Here we describe a highthroughput mutation screening in candidate genes. Method and Patients: We perform a genetic screen by high-resolution melting point analysis of PCR-ampli¢ed DNA fragments, covering the coding region of the genes of interest and the neighboring non-coding regions. The investigated group of patients showed combined OXPHOS complex defect in the muscle biopsy. The majority of the patients were pediatric. Results: So far we have screened 10 genes coding mitochondrial translation factors (initiation, elongation and termination) in 96 DNA samples. We found 63 rare variations in 119 analysed exons. Most of them are not described in the literature yet. Discussion: By using the melting point screen we have found several not yet known rare variants in candidate genes of patients with combined OXPHOS complex defect so far. Additional patients and candidate genes will be included in the screen. Based on the screening results the correlation between genotype and clinical symptoms has to be investigated.

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GENDER DIFFERENCES IN THE GENOTYPE/PHENOTYPE DISTRIBUTION OF PDHA1 Lee I-C1, Ganesh J2, Maranda B3, Tang L-Y4, Craigen W4, Li F5, Wong L-JC5 1 Dept of Pediatrics Neurology, CSMU, Taichung, Taiwan, 2Sec of Bio Gene, Child Hosp of Phila, Philadelphia, USA, 3Deèpt de peèdiatrie, CHUL-CHUQ, Quebec, Canada, 4Molecular and Human Genetics, BCM, Houston, TX, USA, 5Molecular and Human Genetics, BCM, Houston, TX, USA Background: The PDHA1 gene, encoding the E1a subunit of the pyruvate dehydrogenase complex (PDHc), is the most frequently mutated gene in PDHc de¢ciency. The gender di¡erence in the phenotype and genotype of PDHc defect is noteworthy. Methods: The PDHA1 gene of approximately 100 patients suspected of PDHc de¢ciency were sequenced. The clinical features and molecular genetics of reported cases were reviewed. Results: We identi¢ed PDHA1 mutations in 21 unrelated families. The average age at the time of a molecular diagnosis is 58-month-old. The majority (64%) of manifestating females exhibited brain atrophy or malformations. Ten novel mutations were identi¢ed in a total of 17 di¡erent mutations. Among them, 11 (65%) are missense mutations and the remaining are splice site and out of frame insertion/deletions. Together with previously reported cases, 77% of male patients carry missense mutations, whereas less than 50% of female patients have missense mutations (p50.001). The most common mutation; p.R302C, has been observed in 13/84 female patients but in none of the 90 males whom have been studied. Conclusions: We add 10 novel mutations to the growing list of reported PDHA1 mutations. Mutations in exons 10 and 11 account for half of the total 110 mutations. Gender speci¢c mutations are observed. The p. R302C mutation is found only in female, while more than 90% of patients carrying p.R263G mutation are male. The majority of manifestating females have brain atrophy or brain malformations resulting in developmental delay, microcephaly, and seizures, whereas the majority of male patients present with Leigh disease.

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TISSUE COPPER CONTENT IN CHILDREN WITH ISOLATED CYTOCHROME C OXIDASE DEFICIENCY Hansikova H, Vesela K, Stiburek L, Zeman J Department of Pediatric, Charles Univ, Prague 2, Czech Republic Human cytochrome c oxidase (COX) is composed of 13 structural subunits and contains two copper centers CuA and CuB that are essential for enzyme catalytic activity. Aim: of our study was to analyze copper content and COX activity and amount in muscle, liver, heart and brain from 10 children with isolated COX de¢ciency due to mutations in nuclear encoded genes for various COX assembly factors. Material and methods: The samples of muscle, liver, heart and brain tissues were obtained at autopsy in 5 children with mutations in SCO2 gene (36 1541G4A/1541G4A, one 1280C4T/1541G4A and 1518delA/1541G4A), 4 children with mutations in SURF1 gene (c.790___792delCT/c. 845__846delCT, c.312insATdel10/c. 821del18, c.668C4T/c. 668C4T and c.469C4T/c. 845__846delCT) and child with mutation in SCO1 gene (c.394G4A/c. 394G4A). Copper content was analyzed by atomic absorption spectrophotometry. COX activity and amount were analyzed by spectrophotometry, 2D-BlueNative electrophoresis and western blotting. Results: The Sco1 de¢cient muscle sample showed profoundly decreased copper content to 20% of age related controls. Similarly, also the various Sco2 de¢cient tissue samples showed very low copper content, with the heart samples being the most a¡ected (33% of controls). This low copper content in heart from SCO2 patients corresponded well with the most decreased COX activity. Surprisingly, the copper content of Surf1 de¢cient tissues was also signi¢cantly decreased. Conclusion: While the copper de¢cient phenotype of SCO1/SCO2 tissues appears consistent with the function of both gene products in regulation of cellular copper homeostasis, the severe copper de¢ciency of Surf1 de¢cient tissues remains elusive. Supported by GACR 303/07/0781

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OCULAR INVOLVEMENT IN MITOCHONDRIAL DISEASE: BIOCHEMICAL AND GENETIC DIVERSITY OUTLINE IN CENTRE PORTUGAL Grazina MM1, Pratas J2, Simo¬es M2, Mendes C2, Oliveira S2, Oliveira M2, Macaèrio C3, Diogo L4, Garcia P4, Oliveira CR1 1 Faculdade de Medicina de Coimbra, Coimbra, Portugal, 2Centro de Neurocieªncias Biologia Celular, Coimbra, Portugal, 3Hospitais da Universidade de Coimbra, Coimbra, Portugal, 4Hospital Pediaètrico de Coimbra, Coimbra, Portugal Background: Ocular manifestations in mitochondrial disease include chronic progressive external ophthalmoplegia, retinopathy and optic nerve atrophy. Abnormalities of mitochondrial DNA (mtDNA) presenting with the disease may include deletion or point mutations. Optic nerve atrophy with bilateral loss of central vision, one of the most common manifestations, is typical of Leber's hereditary optic neuropathy (LHON) phenotype, maternally inherited, a¡ecting predominantly young men. Mutations 11778G4A, 3460G4A and 14484T4C (PPM), in complex I subunits genes of mitochondrial respiratory chain (MRC), are frequently found. Methods: We evaluated 28 patients, followed at Neurology Unit, HUC and Paediatric Hospital of Coimbra (aged 0.5^70 years), initially suspected of LHON. Total DNA was extracted by standard methods. Analysis of mtDNA was performed using standard techniques (PCR, PCR-RFLP, sequencing). MRC assays were performed as described (Rustin et al., 1994), normalized to CS. De¢ciency was considered if value 540% of mean reference value. Results: In follow up, clinical ¢ndings were compatible with other optic neuropathy in 7 patients, multiple sclerosis in 1 case and LHON in 20 cases, 14 of whom carrying mtDNA alterations, classical mutations (40%, frequency 1:384 215), deletions (20%) and other sequence variations (26.7%), including a novel transition 9165C4T. Complex I was a¡ected in 5 of 8 muscle samples analyzed; other de¢ciencies were also found. Conclusions: Our results reinforce the pattern heterogeneity of mitochondrial diseases and represent an additional contribution for characterizing mtDNA abnormalities in LHON. However, disease speci¢city is di¤cult to explain and nuclear genes probably play an important role, namely X-linked modi¢ers, as recently reported.

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TWO NOVEL MUTATIONS IN THYMIDINE KINASE-2 LEADING TO AN EARLY ONSET FATAL ENCEPHALOMYOPATHY AND SEVERE DNA DEPLETION Naess K1, Lesko N1, W|bom R1, Nennesmo I1, Solaroli N2, von Do«beln U1, Karlsson A2, Larsson NG1 1 Centre of Inherited Metabolic Diseases, Stockholm, Sweden, 2Inst Laboratory Medicine, Karolinska, Stockholm, Sweden De¢ciency of thymidine kinase-2 (TK2) has been described in children with early onset fatal skeletal myopathy. We describe two novel mutations in the TK2 gene in a patient with severe encephalomyopathic mitochondrial depletion syndrome. The patient had a very early onset of disease with intractable seizures from six weeks of age. She had signs of myopathy with a markedly increased level of creatine kinase. Her disease was rapidly progressive and she died at the age of three months. A muscle biopsy showed a high amount of ¢bres with a prominent reduction of cytochrome c oxidase activity and electron microscopy revealed abnormal mitochondria. Biochemical studies of respiratory chain function showed decreased ATP production rate and very low enzyme activity in complex I, III and IV. Southern blot analysis of muscle revealed profound mtDNA depletion. On clinical and biochemical grounds we suspected a TK2 de¢ciency and upon sequencing of the gene two novel mutations were detected; a CG insertion at position 219 in exon 3 (creating a stop codon at position 230) and R130W in exon 6. In vitro activity of recombinant TK2R130W was 53% compared to wild-type recombinant TK2. Our ¢ndings show that these novel mutations cause an extremely severe phenotype with overwhelming cerebral symptoms, not commonly seen in patients with TK2-de¢ciency. We conclude that the severity of the clinical presentation was due to a virtual lack of TK2 activity in the mitochondria of this patient.

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COMPLEX I DEFICIENCY IN PORTUGUESE PATIENTS: ANALYSIS OF THE `CORE' SUBUNITS Ferreira M1, Santorelli FM2, V|larinho L1 1 Centro de Geneètica Meèdica, INSA, Porto, Portugal, 2Ospedale Bambino Gesu, Med Molecolare, Roma, Italy Background/Objectives: De¢cient complex I activity represents the most frequent form of OXPHOS defect in children and young adults. Mutations in both mtDNA and nDNA-encoded subunits have been associated with an ample array of clinical phenotypes, including leukodystrophy and Leigh syndrome. In complex I, 14 subunits are essential for catalysis of electron transfer from NADH to ubiquinone and for generation of the membrane potential (`core' subunits). Half of them are encoded by the nuclear genome (NDUFS13, NDUFS78, NDUFV12) and the remaining by mtDNA (ND16, ND4L). Nonetheless, there are patients in whom no molecular defect can be identi¢ed in any of the known subunits. W|th the ultimate goal of studying the molecular defect in 11 Portuguese patients with complex I defect, we analysed the genes encoding the `core' subunits of complex I. Methods: Eleven patients with isolated complex I de¢ciency (2 men and 9 woman; age at diagnosis 3.65+8.23 years; mean residual complex I activity 31+7.87%) were screened by direct sequencing for the sequences of the mitochondrial (ND16, ND4L) and nuclear `core' genes (NDUFS1-2, NDUFS4, NDUFS7-8 and NDUFV1-2). The most common point mutations of mtDNA as well as large-scale rearrangements had been previously excluded. Results: None of our patients showed variants of pathological signi¢cance. A number of sequence changes were deemed as neutral polymorphisms using generally accepted criteria. Conclusions: It seems likely that the molecular defects of Portuguese patients with complex I de¢ciency reside in the accessory subunits or in assembly factors, or in boths.

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CONTRIBUTION OF POLG1 MUTATIONS TO NUCLEARMITOCHONDRIAL INTERGENOMIC COMMUNICATION DISORDERS Ferreira M1, Evangelista T2, Rosas MJ3, Macaèrio MC4, Guimara¬es A5, Santorelli FM, V|larinho L1, 1 Hospital Sta. Maria, Neurologia, Lisboa, Portugal, 2Hospital S. Joa¬o, Neurologia, Porto, Portugal, 3HUC, Neurologia, Coimbra, Portugal, 4 HGSA, Anatomia Patoloègica, Porto, Portugal, 5Ospedale Bambino Gesu, Med Molecolare, Roma, Italy Background/Objectives: Diseases caused by nuclear genes that a¡ect mtDNA stability are termed disorders of nuclear-mitochondrial intergenomic communication. In these conditions, a primary nuclear gene defect in SLC25A4/ANT1, Twinkle, POLG1 and POLG2, have been associated with multiple mtDNA deletions, a qualitative abnormality of mitochondrial genomes. The aim of this study was to identify the spectrum of mutations in a cohort of patients having multiple mtDNA deletions, by direct sequencing of the aforementioned genes. Methods: Twenty-one Portuguese and Italian patients were screened by direct sequencing for mutations in SLC25A4, Twinkle, POLG1 and POLG2. The patients were selected according to their clinic phenotype, and the presence of multiple deletions and RRFs (ragged red ¢bres). Results: Eight di¡erent mutations were identi¢ed in POLG1, ¢ve of which have already been described and three are novel (p.W918R; p.A862T and p. R1081Q). We also found two new sequence variations (c.1080G4T and c.3480G4A) and a number of already described single nucleotide polymorphisms. No mutations were identi¢ed in SLC25A4, Twinkle and POLG2. Conclusions: Nine out of the 21 patients with multiple mtDNA deletions tested in this study showed a con¢rmatory molecular diagnosis. POLG1 mutations account for the vast majority of cases with but other genes are believed to contribute to quantitative intergenomic disorders. Our ¢ndings expand the array of mutations associated with multiple rearranged mitochondrial genomes and con¢rmed that the range of the associated phenotypes is ample and includes among others ocular and skeletal myopathy, the clinical triad seen in the Alpers syndrome, and sensory ataxia and neuropathy.

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INHIBITION OF MITOCHONDRIAL COMPLEX I IN CEREBRAL CORTEX OF IMMATURE RATS FOLLOWING SEIZURES INDUCED BY HOMOCYSTEIC ACID (HCA) Jesina P1, Folbergrova J2, Drahota Z2, Haugvicova R2, Lisy V2, Pecinova A2, Houstek J2 1 Pediatric Dept,1st Med Fac,Charles Univ, Prague, Czech Republic, 2 Inst Physiol, Acad Sciences, Prague, Czech Republic The present study demonstrates the marked decrease (*60%) of respiratory chain complex I (CI) activity in cerebral cortex of immature rats following seizures induced by HCA. The decrease was already evident during the acute phase of seizures and persisted during long periods of survival (6 days, 3 and 5 weeks), i.e. period of the occurrence of spontaneous seizures (epileptogenesis) in this model. Activities of complex II, IV and citrate synthase remained una¡ected. Inhibition of CI was not associated with changes in its content. Based on enhanced lipoperoxidation and decreased aconitase activity, it is assumed that the cause of inhibition could be oxidative modi¢cation of CI. Mitochondrial respiration and cortical ATP levels remained in the control range, apparently due to excess capacity of CI documented by energy thresholds. However, in mitochondria from HCA-treated animals the enhanced production of reactive oxygen species was observed. Decrease of CI activity was attenuated by substances providing an anticonvulsant e¡ect and also with selected free radical scavengers. We can assume that CI inhibition may elicit enhanced formation of reactive oxygen species and thus contribute to neuronal injury in this model. We also suggest that inhibited CI may play a role in the process of epileptogenesis. Supported by Grant Agency of the Czech Republic, No. 309/08/0292

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Background/Objectives: Secondary respiratory chain (RC) de¢ciency associated with mitochondrial DNA (mtDNA) depletion was suggested to be the cause of late complications observed in propionic acidemia (PA). Propionate-derived metabolites have been shown to inhibit various enzymes of the tricarboxylic acid (TCA) cycle and the RC but some complications are observed in treated patients despite apparent satisfactory metabolic control, suggesting additional mechanisms. Our study aims to determine the potential of anaplerotic substrates to prevent secondary respiratory de¢ciency. Methods: Inhibit the activity of propionyl-CoA carboxilase (PCC) by expression of miRNA targeting the PCCA mRNA encoding its alpha subunit. Measure RC activity, mtDNA and TCA cycle intermediates levels with or without anaplerotic treatment. Results and conclusions: We generated stables clones in the HepG2 human hepatoblastoma cell line expressing two di¡erent PCCA miRNA sequences under the control of a doxycycline inducible promoter. PCC activity is inhibited by 85^90% after 5 days induction and by 595% after 10 days. Cells were treated from days 5^10 of doxycyclin induction with nonanoate to activate the propionate pathway and/or aspartate. Cell mortality was observed under nonanoate treatment, alone or combined, suggesting that PCC inhibition is strongly detrimental when the propionate pathway is activated. These cells represent a new model of PA and should help study the mechanisms of the secondary RC de¢ciency.

Background: Mutations in genes coding for respiratory chain proteins have been associated with mitochondrial network disturbances and reactive oxygen species (ROS) production. We have assessed mitochondrial morphology, ROS production and mitochondrial membrane potential in 3 patients with isolated complex I de¢ciency harboring mutations in the nuclear genes NDUFV1 (patient 1, leukodystrophy and epilepsy) and NDUFA1 (patient 2, Leigh syndrome; patient 3, myoclonic epilepsy and developmental delay). Methods: Control and patients skin ¢broblasts were cultured in DMEM supplemented with foetal calf serum. Mitochondrial morphology and complex II content was studied by immuno£uorescence. Hydrogen peroxide production was determined by £ow cytometry and by confocal microscopy with dicloro£uorescein diacetate. Mitochondrial membrane potential was estimated by £ow cytometry using the JC-1 probe. Results: A slight increase in length, and mitochondrial density was observed in patients 2 and 3, whereas in mutant NDFUV1 patient entangled and fragmented mitochondria were present. Mitochondrial membrane potential was decreased in all patients. ROS production was similar in patients and control cells. Conclusions: The raising in mitochondrial density without ROS overproduction observed in patients 2 and 3 might constitute part of an adaptive response of the cell which in turn would increase electron in£ux through complex II to compensate for the reduced complex I activity.

ANALYSIS OF SECONDARY RESPIRATORY CHAIN DEFICIENCY IN A HEPATOBLASTOMA MODEL OF PROPIONIC ACIDEMIA Brassier A, Chadefaux-Vekemans B, Munnich A, de Lonlay P, de Keyzer Y INSERM U781, Necker Hosp, Paris, France

MITOCHONDRIAL MORPHOLOGY CHANGES IN MUTANT NDUFA1 AND NDUFV1 COMPLEX I DEFICIENCY PATIENTS Moraèn M, Rivera H, Fernaèndez-Moreira D, Blaèzquez A, Ugalde C, Arenas J, Mart|èn MA Mitoch Dis Lab, Res Cent Hosp 12 Octubre, Madrid, Spain

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HIGH THROUGHPUT MUTATION SCREENING IN PATIENTS WITH ISOLATED RESPIRATORY CHAIN COMPLEX I DEFICIENCY Freisinger P1, Taverna M2, Madignier F2, Ahting U3, Rolinski B3, Mayr J4, Sperl W4, Tesarova M5, Meitinger T2, Prokisch H2 1 Child Hosp Univ Technology, Mu«nchen, Germany, 2Human Genet Helmholtz Inst, Mu«nchen, Germany, 3Dept Clin Chem, Klinikum Mu«nchen, Mu«nchen, Germany, 4Childrens Hosp Univ Salzburg, Salzburg, Austria, 5Dept Pediatr Karls University, Prag, Czech Republic

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CORNEAL DYSTROPHY AS A FIRST CLINICAL SYMPTOM IN KEARNS-SAYRE SYNDROME Tsiakas K1, Kruse B1, Knospe V2, Freisinger P3, Santer R1 1 Univ Med Center, Hamburg-Eppendorf, Germany, 2Dept Ophthalmol, Univ Med Center, Hamburg-Eppendorf, Germany, 3Dept Pediatr, Tech Univ, Munich-Schwabing, Germany

Congenital disorders of mitochondrial respiratory chain present a heterogeneous phenotype ranging from pure myopathy to multi-system involvement. Isolated Complex I de¢ciency, the most common defect (*20%) also shows various phenotypes as e.g. neonatal lactic acidosis, Leigh syndrome, cardiomyopathy-encephalophathy. Complex I consists of 45 proteins, 38 encoded by nuclear DNA, 7 by mtDNA. MtDNA mutations are detected in only 20% of the patients with isolated Complex I de¢ciency, suggesting that most patients harbour mutations in nuclear genes that encoded structural subunits or assembly factors. We performed a genetic screen by high-resolution melting point analysis of PCR-ampli¢ed DNA fragments, covering the coding region of the genes of interest and the neighboring non-coding region. 95% of the 267 amplicons coding for the subunits and known assembly factors of Complex I were screened. After exclusion of the most frequent mtDNA mutations 90 samples have been analyzed. Additional 94 DNA samples are in progress. The sensitivity of the method was over 90%. Most likely causative mutations in the structural subunits of complex I have been identi¢ed in 20% of patients. Single variants were identi¢ed in an additional 30% of samples. In 50% of samples, no mutations could be detected suggesting defects in unknown genes. A diagnostic platform for the systematic, low-cost and e¡ective screening for mitochondriopathies caused by complex I de¢ciency is now available. Based on the screening results we already provide molecular diagnosis for several families, including prenatal diagnosis.

Kearns-Sayre syndrome (KSS) is a clinically heterogeneous multisystemic disorder caused by large-scale deletions of mitochondrial DNA or point mutations of tRNAs. Corneal dystrophy has been reported in association with many other genetic defects, but according to our knowledge, has only been described in two cases with KKS. We report on a patient who presented with corneal dystrophy as a ¢rst manifestation of KSS. Case report: We describe a 19-year old female KSS patient whose ¢rst symptoms at the age of 5 years were a reduction of visual acuity due to both macular degeneration and corneal dystrophy. Around the same time, sensorineural hearing loss was ¢rst detected. Short stature and ptosis without ophthalmoplegia developed later. All symptoms were slowly progressive. Laboratory examinations showed renal Fanconi syndrome and an increased protein content in cerebrospinal £uid. She has had no seizures but EEG showed sharp waves. Brain-MRI revealed symmetrical lesions of thalamus and globus pallidus with a lactate peak on spectroscopy. Muscle biopsy was morphologically and histochemically unremarkable. Biochemical examination of respiratory chain enzymes was normal in muscle, but a reduced activity of cytochrome c oxidase was found in ¢broblasts. Molecular analysis of mtDNA in muscle showed a 7 kb deletion with a degree of heteroplasmy of 40%. Discussion: KSS should be considered as a di¡erential diagnosis of corneal dystrophy in children, even if other signs or symptoms are not (yet) present. Although a causal therapy of KSS does not exist, early evaluation and recognition of multisystemic involvement may be important.

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ACUTE NECROTIZING ENCEPHALOPATHY ^ A MITOCHONDRIAL DISORDER? Castelo R1, Garcia P1, Vasconcelos M2, Rebelo O3, Dinis A4, Grazina M3, Diogo L1 1 Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal, 2 Neuropediatria, CDC, Hosp Ped, Coimbra, Portugal, 3C Neurocieªncias Biol Cel Univ Coimbra, Coimbra, Portugal, 4UCI, Hosp Ped, Coimbra, Portugal Background: Acute necrotizing encephalopathy (ANE) is a rapidly progressive neurologic depression following 2^4 days of a viral respiratory febrile illness. The diagnosis relies upon exclusion of other diseases. CT, MRI and neuropathology demonstrate symmetric brain lesions a¡ecting the thalami and brainstem. CSF reveals elevated protein and absence of in£ammatory cells or infectious agents. Metabolic studies are normal, except for possible mitochondrial dysfunction. Case Report: A previously healthy 2 year-old girl with febrile rhinopharingitis evolved to coma in 24 h. Despite vigorous hemodynamic and ventilatory support she died 3 days later with cerebral oedema. Admission CT scan revealed symmetrical bilateral thalamic lesions and brain swelling. Normal values for lactate, ammonia, acylcarnitine pro¢le, l ive r an d re n al fu n c ti o n, h a e moglobi n, l eu ko cy te, p l atel ets, immunoglobulins, lymphocytary population and complement were found. A nonspeci¢c abnormality of lymphoblastic transformation test was disclosed. Liver mitochondrial functional study showed a minor de¢cit of complexes II and V. CSF revealed increased protein, normal glucose and cell count. Cultural and PCR methods for virus and bacteria in CSF, blood and urine were negative except for in£uenza B virus. Autopsy didn't suggest other organ involvement apart from brain. Her older sister, at 13 months of age, developed a similar clinical, biochemical and imagiologic presentation with fatal outcome. Liver evaluation disclosed major de¢cit of mitochondrial complexes I and III. Conclusions: Clinical evolution, imagiology and pathology ¢ndings strongly suggest ANE. Although a de¢nite diagnosis of mitochondrial disease could not be established, liver involvement in both may not be coincidental and further molecular investigation needed.

IDENTIFICATION OF NOVEL AND RECURRENT MITOCHONDRIAL DNA MUTATIONS IN PAEDIATRIC ONSET MITOCHONDRIAL DISEASE USING THE MITOCHIP RESEQUENCING ARRAY Duncan AJ1, Sweeney MG2, Stern E1, Taylor RW3, Woodward C2, Davis MB2, Hanna MG4, Rahman S1 1 Mito Res Group, UCL Inst Child Health, London, UK, 2Neurogenet Unit, Nat Hosp for Neurology, London, UK, 3Mito Res Group, Newcastle Univ, Newcastle, UK, 4Centre for Neuromusc Dis, NHNN, London, UK Background: Disorders of the mitochondrial respiratory chain a¡ect 1 in 5000 children and may arise from mutations in the mitochondrial or nuclear genomes. Identifying the correct genome responsible for the disease is essential for accurate genetic counselling. We sought to evaluate the feasibility of using the A¡ymetrix MitoChip v2 to sequence the entire mitochondrial genome in routine diagnostic practice, by comparing it to conventional cycle sequencing in 20 previously undiagnosed patients. Methods: Complete mitochondrial genome sequence analysis was performed in DNA extracted from muscle, ¢broblasts or blood from 20 patients with suspected mitochondrial disease referred to the National Commissioning Group mitochondrial genetics laboratories in London and Newcastle. Identical samples from each patient were analysed using (i) the MitoChip v2 array and (ii) cycle sequencing (ABI BigDye Terminator Chemistry v1.1). Results: Six known pathogenic mutations were detected: 4 in genes encoding complex I subunits and 2 in the MTTL1 gene. In addition, four novel mutations were identi¢ed, two of which are probably pathogenic. All changes were detected by both methods, but the overall call rate was lower for the MitoChip. Conclusions: Sequence analysis of the entire mitochondrial genome yielded a higher rate of mutations (40% of this highly selected cohort) than expected, suggesting that routine analysis of the mitochondrial genome might be worthwhile in all patients with a strong clinical suspicion of mitochondrial disease. The MitoChip v2 promises to be cheaper and less labour-intensive and time-consuming than conventional sequencing, but the method needs further optimization for use in routine diagnostic practice.

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DIAGNOSIS OF RESPIRATORY CHAIN DEFECTS BY ENZYME ANALYSIS IN SMALL BIOPSIES FROM LIVER AND MUSCLE W|brand F, Òstergaard E, DunÖ M, Lund AM, Christensen E Dept Clin Genet, Natl Univ Hosp, Copenhagen, Denmark

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DIAGNOSTIC mtDNA ANALYSIS IN BODY FLUIDS OTHER THAN BLOOD Gray RGF1, Ball S1, Quinlivan R2, Hendriksz C3 1 Clinical Chemistry, Childrens Hospital, Birmingham, UK, 2Dept Neurology, Childrens Hospital, Birmingham, UK, 3Clinical IMD, Childrens Hospital, Birmingham, UK

Objective: Respiratory chain (RC) defects can be tissue-speci¢c and it is important to be able to measure RC enzyme activity in liver as a suppl ement to muscle. The aim was to develop a set of spectrophotometric methods for measuring complex I-IV activities in small biopsies from both tissues. Methods: Supernatants prepared from frozen tissues were stored at 808C until use. Di¡erent detergents were added to the reaction mixtures to maximize the individual enzyme activities. No additional pretreatment of samples was required. Results: Using these methods we analysed 9 liver biopsies (6^17 mg) and 460 muscle biopsies from patients suspected of having a RC defect. Analysis of the liver biopsies revealed 2 patients with combined RC de¢ciency (complexes I, III and IV). The ¢rst patient was subsequently found to be homozygous for a N46S mutation in the DGUOK gene. DGUOK de¢cient patients are known to have markedly reduced RC activity in liver, but not in muscle. The second patient presented with a Sengers-like syndrome and he also had combined RC de¢ciency in muscle. The other liver biopsies were normal based on their similar complex II-corrected activities of complexes I, III and IV. In muscle, we identi¢ed two patients with complex I de¢ciency, one patient with complex III de¢ciency and two additional patients with combined RC de¢ciency. C o n c l u s i o n s : We h av e e s t a b l i s h e d s i m p l e a n d r e l i a b l e spectrophotometric methods that are suitable for detection of RC defects in both liver and muscle.

Background: In mitochondrial DNA encoded disorders the proportion of mutant DNA varies from tissue to tissue usually being expressed at its highest level in the most severely a¡ected tissue. Readily available cells such as leucocytes are only occasionally a¡ected in these disorders so it is often di¤cult to exclude the disease in a relative of a patient with a known mutation without invasive sampling. We have looked at the use of readily available body £uids in diagnosis. Methods: Samples of blood, saliva and urine were collected with full consent from patients with known mtDNA disorders and their relatives. DNA was extracted from them and tested for the presence of the known mutation by RFLP/PCR methods. Results: Samples from urinary epithelial cells and saliva from patients with the MELAS m. 3243A4G, NARP m. 8993T4G or C, MERRF m. 8344A4G and FSBN m. 9176T4C mutations showed comparable and in many cases higher levels of heteroplasmy for the mutation. The analysis was used to con¢rm the mutation in family members and was of particular bene¢t in a needle phobic parent. Conclusions: mtDNA analysis of alternative, readily accessible, cells and body £uids may obviate the need for invasive biopsies in patients with mtDNA disorders and provide alternative tests in needle phobic patients.

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LRPPRC MUTATIONS IN FRENCH-CANADIAN LEIGH SYNDROME CAUSE A PHENOTYPICALLY DISTINCT FORM OF CYTOCHROME C OXIDASE DEFICIENCY Debray FG1, Morin C2, V|lleneuve J2, Maranda B3, Laframboise R3, Robinson BH4, Lambert M5, Mitchell G5 1 Dept Human Genet, CHU of Lie©ge, Lie©ge, Belgium, 2Dept Pediatrics, CHU Sagamie, Chicoutimi, Canada, 3Dept Med Genet, CHU of Queèbec, Queèbec, Canada, 4Sick Children Hospital, Toronto, Canada, 5Div Med Genet, CHU Sainte-Justine, Montreal, Canada Objective: To determine the clinical spectrum, phenotypic speci¢cities and outcome in a homogeneous cohort of patients with cytochrome c oxidase (COX) de¢ciency. Methods: We determined the clinical and molecular characteristics of patients with Saguenay-Lac-Saint-Jean (SLSJ)-COX de¢ciency identi¢ed in Queèbec. Results: F|fty-six French-Canadian patients were retrospectively identi¢ed having COX de¢ciency due to mutations in LRPPRC, one of the assembly factors required for biogenesis of COX. Due to a founder e¡ect, all alleles except one showed the A345V mutation. One patient was compound heterozygote for this mutation and C1277Xdel8. Chronic clinical features included developmental delay, distinctive dysmorphic features and failure to thrive. More speci¢cally, 91% of patients experienced single or recurrent acute metabolic or neurological crises, characterized by profound and fulminant lactic acidosis, and Leigh syndrome or stroke-like episodes, respectively. Global mortality rate, which was caused by these crises, was 46/56 (82%) and median age at death, 1.6 years. Among 71 crises recorded at a median age of 1.4 years, mortality rate was 52%. Fatal outcome was caused by multiple organ failure and/or Leigh disease. We identi¢ed hyperglycemia, hepatic cytolysis and alteration of consciousness at admission as major predictors of mortality (p50.005). Compared to reported SURF1-de¢cient patients, SLSJ-COX de¢ciency has earlier and higher mortality rate (Hazard ratio: 2.29 [95%CI 1.34^3.92]; p50.001). Metabolic crises appear to be speci¢c for SLSJ-COX de¢ciency. Conclusions: The SLSJ-COX phenotype caused by LRPPRC mutation is distinct from that of other COX de¢ciencies. Occurrence of LRPPRC-linked COX de¢ciencies in other ethnic background remains to be demonstrated.

HAPLOGROUP DETERMINATION AND FREQUENCY OF mtDNA LHON ASSOCIATED SEQUENCE VARIATIONS IN MULTIPLE SCLEROSIS Pratas J1, Macaèrio MC2, Oliveira M1, Santos MJ1, Oliveira CR3, Grazina M3 1 Centro Neurocieªncias e Biologia Celular, Coimbra, Portugal, 2Hospitais da Universidade de Coimbra, Coimbra, Portugal, 3Faculdade de Medicina da Univ Coimbra, Coimbra, Portugal Background: Multiple sclerosis (MS) is the most frequent neurological autoimmune disease and the main cause of severe neurological damage among young adults. Several reports have shown positive association of mtDNA LHON sequence variations with MS cases. Our aim is to evaluate the signi¢cance of mtDNA LHON sequence and phylogenetic variations in Portuguese MS patients. Methods: We have studied 87 MS patients and 52 healthy controls using PCR, PCR-RFLP and sequencing analysis techniques, followed by Chi-square test statistical analysis. We evaluated the frequency of primary (3460G4A, 11778G4A, 14459G4A, 14484T4C), secondary LHON mutations (4216T4C, 4917A4G, 13708G4A, 15257G4A) and haplogroups. Results: No primary mutations have been detected. The frequency distribution of LHON secondary mutations T4216C, A4917G, G13708A and G15257A is signi¢cantly di¡erent (p50.0001) in patient and control groups: 0.23, 0.11, 0.17, 0.05 and 0.06, 0.04, 0.02, 0.02, respectively. Considering the presence or absence of sequence variations, frequencies distribution (0.86 and 0.14, 0.44 and 0.66, respectively for control and MS patients groups) is signi¢cantly di¡erent (p50.0001). So far, haplogroup frequency analysis has shown signi¢cant (p = 0.0181) increased JT and decreased H haplogroups in MS patients (n = 50). No signi¢cant correlation has been found with age of onset, disease duration or EDSS severity scale, in either case. Conclusions: The results reinforce the association of LHON sequence variations with MS and show evidence of mitochondrial genome abnormalities contributing for MS phenotype. Furthermore, our data suggest that haplogroup variation may in£uence the presentation of the disease, possibly dependent on nuclear genome factors.

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MYOPATHY WITH DEFICIENCY OF SUCCINATE DEHYDROGENASE AND ACONITASE TOGETHER WITH CARDIOMYOPATHY ASSOCIATED WITH MUTATIONS IN ISCU Kollberg G1, Tulinius M2, Melberg A3, Darin N2, Andersen O4, Oldfors A5, Holme E1 1 Sahlgrenska Univ Hosp, Dept Clin Chem, Go«teborg, Sweden, 2Queen Silvia Child Hosp, Dept Paediat, Go«teborg, Sweden, 3Uppsala Univ Hosp, Dept Neurosc, Neurol, Uppsala, Sweden, 4Sahlgrenska Univ Hosp, Dept Neurol, Go«teborg, Sweden, 5Sahlgrenska Univ Hosp, Dept Pathol, Go«teborg, Sweden Background: ISCU encodes the iron-sulphur (Fe-S) cluster assembly protein IscU. Fe-S cluster containing proteins are essential for e.g. iron homeostasis and the respirartory chain function. In a genome-wide search in patients with `Myopathy with de¢ciency of succinate dehydrogenase (SDH) and aconitase' we had localized a shared homozygous segment spanning *405 kb on chromosome 12 encompassing ISCU which was an obvious candidate gene. A deep intronic IVS5+382G4C homozygous mutation a¡ecting mRNA splicing associated with the disease was identi¢ed in 8 individuals originating from northern Sweden. Our aim was to investigate patients with a di¡erent clinical phenotype where the laboratory results were consistent with IscU de¢ciency. Results: Two a¡ected brothers were of half F|nnish descent. Like all patients, who were homozygogous for IVS5+382G4C, they su¡ered from severe exercise intolerance and had mitochondrial myopathy with SDH, partial cytochrome c oxidase, aconitase and complex I de¢ciency. No one except the two brothers had cardiomyopathy. The two brothers were compound heterozygous for the IVS5+382G4C mutation and a c.149G4A missense mutation in exon 3 changing a glycine to glutamate. The consequence of the intronic mutation was a pseudoexon insertion matching an intronic sequence in intron 5. Expression of the pseudoexon will lead to frame-shift and premature truncation. Western blot revealed a severe reduction of IscU in mitochondrial protein extracts from patients and visualized a band that might represent a truncated protein in patients. Conclusions: Defects in IscU may cause either pure myopathy or a combined myopathy and cardiomyopathy depending on the nature of the ISCU mutation.

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SEVERE AXONAL POLYNEUROPATHY DUE TO DICHLOROACETATE IN A PATIENT WITH PYRUVATE DEHYDROGENASE DEFICIENCY Dursun A, Tokatly A, Sivri HS, Coskun T Hacettepe Univ Metab and Nutr Unit, Ankara, Turkey De¢ciency of pyruvate dehydrogenase enzyme complex (PDH) present with neonatal lactic acidosis, Leigh's encephalopathy and intermittant ataxia. Minor dysmorphic anomalies including microcephaly, frontal bossing, broad nasal bridge, upturned nose, long philtrum, low set ears, short ¢ngers, Simian creases, and £ared nostrils are also reported. The patient presented here is an 11 year-old male diagnosed in the neonatal period due to respiratory di¤culty for severe lactic acidosis. He was on thiamine (100 mg/d), dichloroacetate (DCA 50 mg/kg/d), and ketogenic diet until 10 years old. At 4 years of age, the diagnosis was con¢rmed by enzyme analysis showing reduced actvitiy of PDH (0.04 mU/mg protein; contols: 0.76^1.70). He complained of di¤culty in walking 1.5 years ago. At that time, electromyography showed severe axonal polyneuropathy involving motor and sensory nerves. DCA was stoped and thiamine dose was increased to 250 mg/d. Six months later, slightl improvement in gait was observed. This was con¢rmed by EMG which showed increased nerve conduction time in both motor and sensory nerves. Polyneuropathy is a well known complication of DCA treatment, however there is no report about polyneuropathy as a complication of DCA during treatment of PDH. We want to stress that potential complications of DCA should be followed closely in patients with PDH de¢ciency on DCA treatment. Support: State Planning Organisation; Prj. No:2006 K 120 640-06-03

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SPLICING MUTATIONS IN THE PDHA1 GENE Ridout CK, Brown RM, Brown GK Genetics Unit, Biochem Dept, Oxford Univ, Oxford, UK Background: Pyruvate dehydrogenase (PDH) de¢ciency is mostly caused by mutations in PDHA1, the X-linked gene for the E1a subunit. In other human genes, *30% of pathogenic mutations a¡ect splicing and usually involve the highly conserved dinucleotides in the consensus sequences at exon/intron boundaries. However, this is not the case for the few splicing mutations found in PDHA1. We describe ¢ve PDHA1 splicing mutations, 3 of which are novel, all with unusual consequences. Methods: Clones of ¢broblasts expressing the mutant PDHA1 allele were isolated from female patients and splicing intermediates analysed after blocking transcription. In vitro studies of splicing constructs containing exons 4, 5, 6 and 7 of the PDHA1 gene con¢rmed the consequences of these mutations. Results: These mutations are not distributed randomly. One mutation, in intron 5, results in exonisation, a rare outcome in which a central portion of an intron is included in the mRNA. The remaining four mutations, in intron 4 and exon 5, lead to skipping of exon 5 in some transcripts and, unexpectedly, skipping of both exons 5 and 6 in others. Analysis of splicing intermediates shows that the removal of exons in PDHA1 is not sequential and can occur by several simultaneous pathways. Conclusions: The order of PDHA1 exon splicing is unexpectedly complex. In addition, several of the splice junctions have poor homology with the consensus sequences. Identi¢cation of these factors provides signi¢cant insight into the splicing mechanism and emphasises that extensive investigations, beyond standard sequence analysis, may be required for the diagnosis of PDH de¢ciency.

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EXPRESSION PATTERN OF PDHA1 AND PDHA2 GENES IN HUMAN SPERMATOGENESIS Pinheiro A1, Faustino I1, Silva MJ1, Henrique D2, Sousa M3, Barros A4, Tavares de Almeida I1, Rivera I1 1 Met and Gen Group, iMed UL, Fac Pharmacy, University of Lisbon, Portugal, 2IMM, Fac Medicine, University Lisbon, Lisbon, Portugal, 3 Lab Cell Biol, ICBAS, University Porto, Poto, Portugal, 4Dept Genet, Fac Medicine, Univ Porto, Porto, Portugal Background: An essential step in aerobic energy metabolism is the reaction catalyzed by the pyruvate dehydrogenase complex. Its rate-limiting component is E1a subunit encoded by two di¡erent genes: the somatic PDHA1 (X-linked) and the testis PDHA2 (autosomal). The presence of this testis-speci¢c isoform is important in spermatogenesis, due to the absence or inactivation of X-chromosome, allowing sperm cells to generate nearly all their energy. During spermatogenesis, germ cells undergo a complex process of cell di¡erentiation and morphological restructuring, resulting in the formation of mature spermatozoa. All this process depends on the coordinated expression of di¡erent genes in a stage-speci¢c manner. Given the scarce available information regarding the mechanisms controlling gene expression in human spermatogenesis, PDHA genes constitute a reliable model for studying and understanding this complex process. Objective: Elucidation of the temporal- and cell-speci¢c transcription and tran sl ati o n p atte r n s of PDH A 1 an d PDH A 2 du r i ng hu m an spermatogenesis. Material and Methods: Normal adult testicular biopsies were analyzed by immunostaining with monoclonal antibody against E1a protein and by in situ hybridization with RNA probes either for PDHA1 or PDHA2 transcript. Slides were analyzed by photo microscopy after speci¢c counterstaining. Results and Conclusion: The results revealed a stage-speci¢c expression pattern of PDHA1 and PDHA2 genes during human spermatogenesis. These ¢ndings show that the testis-speci¢c and somatic form of PDHA transcripts and the synthesis of E1a protein are developmentally regulated in male germ cell di¡erentiation. Work supported by FCT (POCI/SAU-MMO/57052/2004 and SFRH/BD/ 31264/2006)

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A NEW MUTATION IN PDHA1 IN A GIRL WITH SEVERE CLINICAL PHENOTYPE BUT NORMAL ACTIVITY OF PYRUVATE-DEHYDROGENASE COMPLEX IN MUSCLE Freisinger P1, Mayr J2, Hartmann K1, Rolinski B3, Ahting U3, Woessner R4, Sperl W2 1 Child Hosp Univ Technology, Mu«nchen, Germany, 2Childrens Hosp Univ Salzburg, Salzburg, Austria, 3Dept Clin Chem, Klinikum Mu«nchen, Mu«nchen, Germany, 4Univ Child Hosp, Wuerzburg, Germany

A PEROXISOMAL FORM OF SPINOCEREBELLAR ATAXIA CAUSED BY MUTATIONS IN PEX10 Reègal L1, Ebberink M2, Wanders R2, Wuyts B3, Waterham H2, Van Driessche M3, Van Coster R3, Achten E4, De Meirleir L3 1 Metab Center, Dept Pediatr, Univ Hosp, Leuven, Belgium, 2Lab Genet Metab Dis, Acad Med Center, Amsterdam, Netherlands, 3Div Child Neurol Metab Dis, Univ Hosp, Ghent, Belgium, 4Dept Radiol, Univ Hosp, Ghent, Belgium

De¢ciency of pyruvate-dehydrogenase-complex (PDHc), an X-linked inherited disorder, is an important cause of neonatal lactic acidosis with lethal outcome if not treated adequately. A baby girl with normal family history presented at birth with severe lactic acidosis (up to 15 mmol/L), muscle hypotonia, hypoplasia of the corpus callosum and pulmonary valve stenosis. Muscle biopsy was performed and showed activity of the PDH-complex at the lower level of the control in two di¡erent laboratories. On the basis of the typical clinical picture and in spite of the biochemical result we analyzed the PDHA1-Gene and found a mutation c.707C4A leading to an amino acid exchange p.Ala236Glu. Sequencing of genomic DNA showed that the mutation was heterozygous however in cDNA from muscle the mutation was expressed higher than the normal variant. This mutation, which has not been described so far, is most probably pathogenetic as the amino acid at this position is phylogenetically highly conserved and the described amino acid exchange most probably a¡ects the protein structure. We suspect that the contrast of normal activity of PDHC in muscle and the severe clinical phenotype is the result of variable Xinactivation in di¡erent tissues. Treatment with thiamine and ketogenic diet (80% fat) was started 10 days after birth. The patient, now 4 months old, responded well to diet and shows lactate levels up to 4 mmol/L and developmental progress. This case illustrates the importance of PDHA1 mutation-screening in patients with typical clinic but normal enzyme activity in muscle especially in female patients.

Background: Mutations in 12 di¡erent PEX genes, resulting in generalized defects of peroxisomal biogenesis (PBD), are the cause of the Zellweger spectrum. It is characterized by severe global neurologic involvement and variable systemic features, including death in infancy or early childhood. We report an 11-year-old boy of normal intelligence with spinocerebellar ataxia caused by a PBD. Case report: The patient presented with balance problems, worsening from age 5. He had ataxia, absent ankle re£exes, and reduced vibration sense. Paraclinical examinations showed cerebellar atrophy, axonal motor neuropathy and posterior column dysfunction. Very-long-chain fatty acid levels and plasmalogens were within the normal range, but phytanic acid, pristanic acid levels, bile acid intermediates and pipecolic acid were clearly increased. Dietary restriction of phytanic acid lowered plasma levels of phytanic and pristanic acid and stabilized ataxia. Methods: Di¡erent peroxisomal enzymes were evaluated in cultured skin ¢broblasts. Immuno£uorescence studies for catalase were performed in cultured skin ¢broblasts. Candidate PEX genes were sequenced. Cultured ¢broblasts were transfected with PEX10. Results: Analysis of peroxisomal enzymes revealed no clear abnormalities. Immuno£uorescence studies showed peroxisomal mozaicism. Two mutations in PEX10 were found, c.764__765insA and R331Q. Transfection with PEX10 resulted in normal peroxisome formation, con¢rming that defective PEX10 caused the PBD. Discussion: The mild clinical and biochemical involvement in our patient is unusual for a PBD. Spinocerebellar ataxia without mental retardation caused by mutations in PEX10 has not been reported before. The evolution after dietary measures suggests this peroxisomal form of spinocerebellar ataxia may be partially treatable.

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HUMAN TESTIS-SPECIFIC PDHA2 GENE: EVALUATION OF THE METHYLATION STATUS IN PROMOTER REGION OF SOMATIC CELLS Faustino I, Pinheiro A, Silva MJ, Tavares de Almeida I, Rivera I Met and Gen Group, iMed UL, Fac Pharmacy, University of Lisbon, Portugal

A PEROXISOMAL BIOGENESIS DISORDER CAUSING SPINOCEREBELLAR ATAXIA IN AN ADULT Reègal L1, Ebberink M2, Goemans N, Wanders R2, Waterham H2, Jaeken J1 1 Lab Genet Metab Dis, Acad Med Center, Amsterdam, Netherlands, 2 Div Child Neurol, Dept Pediatr, Univ Hosp, Leuven, Belgium

Background: Pyruvate dehydrogenase complex is essential in the control of aerobic energy metabolism and its pivotal subunit, E1a, exists as two isoforms encoded by two di¡erent genes: PDHA1 located on Xp22. 1 and selectively expressed in somatic tissues, and the intronless PDHA2, located on chromosome 4 and only expressed in testis after the onset of spermatogenesis. Several authors postulated that the activation of testisspeci¢c genes, in particular those which are temporally expressed during spermatogenesis, is likely to require several levels of regulation, including DNA hypomethylation. Recently, we have reported the case of a Portuguese girl displaying the expression of the PDHA2 gene in circulating lymphocytes and now we are aiming to elucidate the mechanisms underlying the somatic expression of this testis speci¢c gene. Objective: Investigate the potential role of DNA hypomethylation upon the de-repression of PDHA2 gene in somatic cells of this individual. Methods: Genomic DNA was isolated from circulating lymphocytes and the PDHA2 promoter region was PCR ampli¢ed in four overlapping fragments and characterized by direct sequence analysis. An aliquot of DNA was subjected to bisulphite treatment and the inner CpG island was analysed by methylation speci¢c PCR. Results and Conclusion: The results revealed distinct methylation patterns at speci¢c CpG sites in the promoter region between index case and ten controls, which may contribute to explain the expression of PDHA2 gene in lymphocytes. However, further studies are running to clarify the involvement of DNA methylation status upon the tissue speci¢c expression of PDHA2 gene, namely the study of testicular tissues.

Background: The Zellweger spectrum is a group of peroxisomal biogenesis disorders (PBD) characterized by generalized neurologic and multisystem involvement of variable severity, typically leading to death in infancy or early childhood. We present a 21-year-old patient with spinocerebellar ataxia without cognitive impairment, caused by a PBD. Case report: The patient presented at the age of 6 years with slowly progressive gait problems. He had cerebellar ataxia, are£exia, pes cavus, and decreased vibration sense. Neurophysiologic studies showed an axonal motor neuropathy and posterior column dysfunction. On brain MRI there was severe cerebellar atrophy. Cholesterol was low and there was mild elevation of liver and muscle enzymes in blood. Levels of verylong-chain fatty acids (VLCFA) were not elevated, but phytanic acid, pristanic acid, bile acid intermediates and pipecolic acid were elevated, indicating a PBD. Dietary restriction of phytanic acid and bile acid supplementation was started. Methods: On cultured skin ¢broblasts, immuno£uorescence studies for catalase were performed. Results: There was absent visualization of peroxisomes in 20% of cells, con¢rming the diagnosis of a PBD. Complementation studies and molecular characterization are underway. Discussion: Our patient expands the clinical spectrum of PBD with spinocerebellar ataxia in adults. Remarkably, VLCFA levels, the classic screening method for PBD, did not indicate a PBD. We suggest that peroxisomal metabolites, including pipecolic acid, should be measured in adult patients with the combination of axonal motor neuropathy, posterior column involvement and cerebellar ataxia.

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EXPRESSION OF THE PEROXISOMAL PLASMALOGEN SYNTHESIZING ENZYMES IN THE CNS Bams-Mengerink AM1, Ofman R2, Troost D3, Wanders RJA2, Poll-The BT1, Brites P2 1 Emma Child Hosp, AMC, Amsterdam, Netherlands, 2Lab Gen Metab Dis, AMC, Amsterdam, Netherlands, 3Dept Neuropath, AMC, Amsterdam, Netherlands Background: Plasmalogens are a special group of ether bond containing phospholipids. They are claimed to function as scavengers of reactive oxygen species (ROS), as storage depot for polyunsaturated fatty acids and to be involved in cellular signalling and of membrane dynamics. Plasmalogens are especially abundant in the myelin sheets of the central nervous system. So far little attention has been paid to where plasmalogens are formed. Since patients with a severe de¢ciency in plasmalogens, as in rhizomelic chondrodysplasia punctata (RCDP), already show major neurological problems early in life, we are interested in the expression of the plasmalogen synthesizing enzymes DHAPAT and AGPS in the central nervous system of mice and humans during the embryonal and (early) postnatal period. Methods: Immunohistochemistry with antibodies against AGPS and DHAPAT was performed on brain tissue of wild type mice aged E14 to P180 and human fetuses with a gestational age of 20 weeks to postnatal age 1 year. Comparison was made between wild type and PEX7 knock out mice. Results: In both human and murine tissue the plasmalogen synthesizing enzymes are expressed in a time dependent manner. In murine cortex, medulla and cerebellum there seems to be a decreased expression around P10. In human cerebellum peak expression is around 35 weeks gestational age. The expression in cerebellum of the Pex7 knockout mice is less than in wild type mice, which is in agreement with the pathological ¢ndings in RCDP patients.

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ALPHA-METHYL COA RACEMASE (AMACR) DEFICIENCY IN AN ADULT PRESENTING WITH APHASIA Manning NJ1, Talbot RM1, Olpin SE1, Gibson A2, Gillett GT2, Bandmann OL3, Ferdinandusse S4, Wanders RJA4 1 She¤eld Children's Hospital, She¤eld, UK, 2She¤eld Teaching Hospitals, She¤eld, UK, 3Doncaster Royal In¢rmary, Doncaster, UK, 4 AMC, Amsterdam, Netherlands Background: Previous reports of AMACR de¢ciency include symptoms such as tremor, epilepsy, motor neuropathy and spastic paraparesis. We describe a patient presenting with novel clinical features including aphasia. Case: A 37 year old female experienced several weeks of headache and progressive confusion culminating in loss of speech, epilepsy and incontinence. There was facial twitching, vacancy with a right sided loss of vision and paresis with sti¡ness and weakness in both legs. MRI scan showed extensive abnormalities of the left hemisphere cortex (with lesser changes a¡ecting the right), the thalamus bilaterally, the peritrigeminal area on the left and sub-cortically in the frontal lobes. EEG indicated bilateral cerebral dysfunction more prominent in the left anterior quadrant. Results: 3 weeks after admission, plasma pristanic acid was markedly increased at 342 mmol/L (ref 51.8) with normal phytanic acid at 3.0 mmol/L (ref. 519). Analysis of bile salts showed increased taurotrihydroxycholestanoic acid indicating AMACR de¢ciency. No AMACR activity was detected on ¢broblast enzyme assay. A lowphytanate diet reduced the pristanate to 235 mmol/L within 2 weeks. Clinical improvement was noted soon after dietary restriction, with partial resolution of the profound aphasia and hemianopia. Subsequently plasma pristanate decreased and 14 months after diagnosis the concentration was 38 mmol/L. The patient continues to improve: she is able to walk unaided, but with impaired ¢ne coordination; some nominal dysphasia persists. Conclusion: AMACR de¢ciency should be considered for patients presenting with aphasia as a primary symptom.

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PEROXISOMAL DISORDERS AND NEWBORN SCREENING Tortorelli S1, Turgeon CT1, Magera MJ1, Hubbard WC2, Moser AB3 1 BGL, Mayo Clinic College of Medicine, Rochester, MN, USA, 2Dept Clin Pharmacol Johns Hopkins Univ, Baltimore, MD, USA, 3Kennedy Krieger Institute, Baltimore, MD, USA Objective: To develop a MS/MS method suitable for neonatal screening for X-linked adrenoleukodystrophy (X-ALD) and other peroxisomal disorders. X-ALD is the most common peroxisomal disorder a¡ecting approximately one in 21 000 males. It is a progressive and fatal disorder that a¡ects the nervous system, the adrenal cortex and the testis. Adrenal insu¤ciency can be totally prevented by presymptomatic therapy, while the cerebral phenotype, a¡ecting 40% of all male patients, can be prevented by dietary therapy and hematopoietic transplant. However, those interventions have a narrow window of opportunity to be e¡ective. Lysophosphatidylcholine species (lyso-PC) on blood spots have been demonstrated to be abnormally elevated in newborns with peroxisomal disorders. Methods: Using a £ow injection analysis MS/MS (FIA-MS/MS) method, we have measured lyso-PC species (C26:0, C24:0, C22:0, C20:0) on blood spots from 6 male patients with X-ALD, and 6 patients a¡ected by a peroxisomal biogenesis disorder (PBD). Data were compared with 89 controls (NBS blood spots). Results: Concentrations of lyso-PC fraction containing hexacosanoic acid (C26:0) were distributed as follows (range, median): X-ALD 0.39^ 1.05 ng/ml, 0.64 ng/ml; PBD 0.88^3.12 ng/ml, 1.05 ng/ml; control 0.13^0.39 ng/ml, 0.21 ng/ml. To better discriminate X-ALD and controls we used the sum of the lyso-PC species. The range for X-ALD was 0.99^1.77, with 1.50 as median, while the same parameters were distributed as follow in controls: 0.38^0.72, 0.54. Conclusions: The data of the present study strongly indicate that our method can be used as a screening tool for X-ALD and other peroxisomal disorders.

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TWO NOVEL MUTATIONS IN ABCD1 GENE ^ ONE PORTUGUESE AND ONE SERBIAN Laranjeira F1, Lemos M1, Perdiga¬o S2, Lacerda L1 1 Unid Enzimologia, C Genet Meèdica, INSA, Porto, Portugal, 2Ser Neurol, Unid Local Sauède Matosinhos, Matosinhos, Portugal Background: X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder presented in various forms as far as phenotype goes, and it can di¡er even within the same family. X-ALD is due to a peroxisomal failure in b-oxidation of VLCFA, thus making its measurement the primary diagnostic test, which is e¡ective for male patient detection, but not for all female carriers. However, the ultimate diagnostic test is the study of the ABCD1 gene. Objective: Screen for mutations in the ABCD1 gene. Methods: The DNA testing was performed according to Boehm et al (1999). Results: We report two novel mutations in the ABCD1 gene: c.1244A4G (p.Y415C) and c.2036G4A (p.W664X). The c.1244A4G mutation was found in a Portuguese family whose index case initiated symptoms at the age of one, presented atypical clinical features and borderline VLCFA in plasma, but patient-like in cultured ¢broblasts. However, all the family heterozygous females studied, showed typical carrier levels of VLCFA in plasma. The second mutation, c.2036G4A, was found in a 34 year old male of Serbian origin, with adrenomyeloneuropathy (AMN), whose ¢rst symptoms occurred at the age of 31, and led to a walking inability within 18 months. VLCFA were typical of a male patient. Conclusion: The genetic study is a valuable diagnostic tool. Based on the ¢ndings (clinical and analytical), along with comparison with other mutations already described we can assume that these nucleotide changes are causal to X-ALD.

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SCREENING SMALL MOLECULES FOR RESCUE OF PEROXISOME ASSEMBLY DEFECTS Braverman NE1, Zhang R2, Steinberg SJ3 1 Montreal Child Res Inst, McGill Univ, Montreal, Canada, 2Inst Genet Med, Johns Hopkins, Baltimore, USA, 3Neurogenet, Kennedy Krieger Inst, Baltimore, USA Background: Zellweger spectrum disorder (ZSD) is a heterogenous group of autosomal recessive disorders with high morbidity and mortality caused by a failure to assemble normal peroxisomes. There are no therapies for ZSD. Nevertheless about half of the patients have a phenotype milder than Zellweger syndrome and exhibit a progressive disease course. Thus patients would bene¢t if therapies become available and are instituted early. Recent observations suggest that improving the conformation of a defective PEX protein, increasing its expression, or increasing peroxisome numbers results in partial recovery of peroxisome function in ZSD ¢broblasts. Methods: We engineered a cell line homozygous for the common missense allele, PEX1-G843D, and over-expressing a GFP-tagged PTS1 matrix protein that remains cytosolic at baseline. The 2000 compound MSSP Spectrum library was screened ¢rst. The cells were cultured in each compound in duplicate for two days, ¢xed and imaged using epi£uorescent microscopy on a high content imaging platform. Redistribution of the GFP tagged matrix protein from the cytosol to the peroxisome membranes was scored visually and compared to both positive and negative controls. Results: Currently we have identi¢ed four compounds that recover peroxisome matrix protein import and con¢rmatory studies are in progress. Initial studies indicate that PEX1-G843D homozygous primary ¢broblasts attain similar functional improvement when cultured in these compounds. Conclusions: We have shown that this assay is e¤cient and reliable for high throughput analysis. F|nally, it is a robust platform for drug identi¢cation because analysis is based on recovery of downstream function, and is not biased toward mechanism.

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PROTEIN OXIDATION AND LIPID PEROXIDATION IN PLASMA OF PATIENTS WITH PEROXISOME BIOGENESIS DISORDERS Deon M1, Sitta A1, Barschak AG2, Biancini GB2, Oliveira AB2, Coelho DM2, Giugliani R2, Wajner M2, Vargas CR3 1 Prog Poès-Grad Bioqu|èmica, ICBS, UFRGS, Porto Alegre, Brazil, 2 Servic°o de Geneètica Meèdica, HCPA, Porto Alegre, Brazil, 3Dept Anaèlises, Fac Farmaècia, UFRGS, Porto Alegre, Brazil Background/Objectives: Peroxisomes are ubiquitous cell organelles with di¡erent metabolic functions. The importance of the peroxisome became more evident by the existence of various severe genetic disorders associated by the failure of the functions of peroxisomes. Defects in peroxisomal functions are associated with major, and often fatal, changes at the neurological level during human development. The peroxisomal disorders are subdivided into two major categories including: the single peroxisomal enzyme (transporter) de¢ciencies (PEDs) and the peroxisomal biogenesis disorders (PBDs). Neurological symptoms and brain abnormalities are characteristic of patients with PBDs. However, very little is known about the pathomechanisms involved in the tissue damage of these disorders. Considering that peroxisome is involved in oxidative reaction and that in a previous study we showed evidence that oxidative stress is probably involved in pathophysiology of other p eroxisomal disease ^ X-linked adrenoleukodystrophy, in the present study we evaluated two oxidative stress parameters in plasma from patients with PBDs. Methods: Protein thiol content and thiobarbituric acid-reactive species (TBA-RS) were measured in plasma of patients with PBDs and in controls. Results: It was observed a signi¢cant decrease of protein thiol content, indicating a possible protein oxidation, and a signi¢cant increase of plasma TBA-RS measurement, indicating a stimulation of lipid peroxidation. Conclusions: It is therefore proposed that oxidative stress may be involved in the pathophysiology of the disorders of peroxisome biogenesis. F|nancial support: CNPq, CAPES, PROPESQ/UFRGS, PROREXT/ UFRGS, FIPE/HCPA, FAPERGS.

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DISTURBANCES IN THE CENTRAL NERVOUS SYSTEM OF LVALINE METABOLISM ARE CONSISTENTLY PRESENT IN PATIENTS WITH A PEROXISOMAL BIOGENESIS DISORDER Eyskens FJ CEMA Antwerp, Antwerp, Belgium We describe 6 infants, who presented from birth with severe hypotonia and convulsions; 4 of them had concomitant liver dysfunction in a varying degree of severity; dysmorphic features were present in het majority of these infants. In the cerebrospinal £uid (CSF) of all of these patients abnormalities in the organic acid pattern were found with an elevated 2-hydroxyisovaleric acid concentratrion as the most prominent abnormality: concentration range from 41^104 mmol/L (reference value: Undet. ^ 18 mmol/L). These abnormalities were not present in plasma and urine. Further diagnostic investigations revealed that all patients were a¡ected by a peroxisomal biogenesis defect. A Dutch girl, second child of consanguineous parents, showed slightly and transiently elevated plasma VLCFA concentration in the neonatal period. Despite the elevated 2-hydroxyisovaleric acid concentration found in the CSF (84 mmol/L) and in contrast to all other patients, all biochemical parameters linked to a peroxisomal disorder (VLCFA, pipecolic acid, C27 bile acids) were normal. Enzymatic assays performed in ¢broblasts were consistent with a peroxisomal biogenesis defect; molecular genetic analysis con¢rmed the diagnosis of a peroxisomal biogenesis defect in ¢nding homozygosity for two splice site mutations (IVS12+5G4A; IVS12-85G4C) in the PEX5-gene of this patient. Conclusion: Abnormalities in L-valine metabolism in the central nervous system, whatever the underlying defect or pathogenesis may be, are consistent and very speci¢c in patients a¡ected by a peroxisomal biogenesis disorder. These abnormalities are even present in patients with normal to near-normal results of biochemical parameters linked to a peroxisomal disorder as is demonstrated by the case presented.

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VLCFA LEVELS IN VARIOUS APPROACHES TO THE X-ALD TREATMENT IN ONE 7 YEARS OLD PATIENT Stradomska TJ1, Drabko K2, Moszczynèska E1, Tylki-Szymanèska A1 1 Children's Memorial Health Institute, Warsaw, Poland, 2Univ Child Hosp, Lublin, Poland Background: X-linked adrenoleukodystrophy is a neurodegenerative peroxisomal disorder connected with mutation in the ABCD1gene encodes, an ABC half transporter, ALDP adrenoleukodystrophy protein. Impairment of peroxisomal b-oxidation VLCFA (very long chain fatty acids) process resulting from defect membrane transport caused their accumulation in blood and tissues. Major biochemical biomarker for diagnosis X-ALD is detected VLCFA levels in serum measurement by chromatographic methods. Methods: Patient (GP) was diagnosed at the age of 6 month through the family screening, his elder brother presented CCALD. The patient was management with Lorenzo oil (LO) and bone marrow transplantation. Serum VLCFA levels was detected by GC method. Analysis of chimerism and MRI was performed. Results: The treatment with LO was started at the age of 2, after 2 months of LO administration serum VLCFA levels decreased to the normal level. Bone marrow transplantation from family donor was performed twice, at the age of 4.5 and after rejection at the age of 5.8 years again. Serum VLCFA level soon after the engraftments decreased but later increased to the levels characteristic for X-ALD hemizygotes. At the age of 7 he is asymptomatic although the signs of adrenal insu¤ciency were noted. Conclusion: Despite of the analyzed therapeutic methods C24:0 level decreases earlier, than C26:0. Chimerism correlates with VLCFA serum levels. Monitoring of serum VLCFA levels is useful in the treatment e¡ectiveness.

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X-LINKED ADRENOLEUKODYSTROPHY: THE RELATIVE FREQUENCY OF PHENOTYPIC VARIANTS IN THE PORTUGUESE PATIENTS Ferreira R, Lemos M, Lacerda L UE- C. Genet Med Jacinto Magalha¬es, INSA, Porto, Portugal Background: X-linked adrenoleukodystrophy (X-ALD) is the most common inherited disorder of peroxisomal metabolism. Clinical presentation involves mainly the white matter of the central nervous system and the adrenal cortex. Biochemically it is characterized by an increase of unbranched saturated very long chain fatty acids in tissues and body £uids. According to the age of onset and the organs principally a¡ected, at least six phenotypic variants can be distinguished, even within the same kindred, being the childhood cerebral ALD (CCALD) and the adrenomyeloneuropathy (AMN) the two most common phenotypes. Methods: Patient's phenotype assignments were made either at diagnosis, at present or at death, using medical history and ¢ndings of neurological examination, from laboratory records collected over a 19year period. Results: Of 54 a¡ected males studied from 34 kindreds, the phenotypic distribution was 20% CCALD, 4% adolescent cerebral ALD, 2% adult cerebral ALD, 48% AMN, 20% Addison only and 6% remained asymptomatic when last examined. Adrenocortical dysfunction was diagnosed in 40 of 54 patients (74%) in whom 85% showed clinical signs before ¢fteen years of age. Conclusions: The AMN is clearly the most common phenotypic variant in this Portuguese survey of X-ALD patients. This study also points out that the ¢rst neurological symptoms start in adulthood for half of the patients and the ¢rst Addison symptoms show high prevalence in childhood and adolescence.

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SEARCHING POLYMORPHIC VARIANTS OF METHIONINE METABOLISM IN SEVERAL CLINICAL PHENOTYPES OF XLINKED ADRENOLEUKODYSTROPHY IN ARGENTINEAN PATIENTS Bender SE, Grosso CL, Guelbert NB, Oller- Ramirez A, Becerra A, Amorosi C, Dodelson de Kremer R CEMECO, Child Hosp, Coèrdoba Univ, Coèrdoba, Argentina The X-linked adrenoleukodystrophy (X-ALD) ranges from severe childhood cerebral (CC) form to milder phenotypes as adrenomyeloneuropathy (AMN), Isolated Addison (IA) and Asymptomatic type. At present, no correlation has been found among ABCD1 gene mutations/X-ALD phenotypes. Objective: To study the inter-intrafamilial phenotypic variability of Argentinean X-ALD patients through two functional methionine polymorphisms: 5,10methylenetetrahydrofolate reductase (MTHFR) c.677C4T and cystathionine beta-synthase variant c.844ins68 (CBS). The MTHFR 677T/T and CBS wt/Ins genotypes were proposed to be risk and protective factors for CNS demyelination, respectively (Linnebank et al., 2006). Material/Methods: Molecular analysis was performed by PCR and restrictionenzyme assays in 95 controls and 20 X-ALD patients (9 hemizygotes and 11 heterozygotes). Results: In our control population the frequencies of the studied polymorphisms were: MTHFR 677T/T 11/95, 677C/T 45/95, 677C/C 39/95 and CBS Ins/Ins 0/ 95, wt/Ins 10/95, wt/wt 85/95. In the pathological group (including carriers) the 677T/T risk genotype was absent; 677C/T was present in 8/20 X-ADL distributed in all clinical phenotypes: 1 CC, 1 AMN, 2 IA and 4 carriers (2 symptomatic and 2 asymptomatic). Notably, this genotype was absent in the asymptomatic hemizygote. The CBS wt/Ins genotype was only found in 2 asymptomatic carriers. Discussion: The high 677C/T frequency in controls, near 50%, is a remarkable ¢nding that deserves additional studies. The small size of the investigated X-ALD group hinders the estimation of the putative risk and protective e¡ect associated to these polymorphisms. Thus, these genetic conditions should be considered in each hemi/heterozygote X-ALD individual to attempt novel therapeutic approaches based on nutritional modulation.

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7 TESLA PROTON MAGNETIC RESONANCE SPECTROSCOPIC IMAGING IN ADULT X-LINKED ADRENOLEUKODYSTROPHY Eichler FS1, Ratai E1, Kok T2, W|ggins C1, W|ggins G1, Grant E1, Gagoski B2, O'Neill G1, Adalsteinsson E2 1 Massachusetts General Hospital, Boston, USA, 2Massachusetts Institute of Technology, Cambridge, USA Background: Adult patients with X-linked adrenoleukodystrophy (XALD) remain at risk for progressive neurological deterioration. Phenotypes vary in their pathology, ranging from axonal degeneration to in£ammatory demyelination. The severity of symptoms is poorly explained by conventional imaging. We set out to test the hypothesis that neurochemistry in normal appearing brain di¡ers among adult phenotypes of X-ALD, and that neurochemical changes correlate with the severity of symptoms. Patients and Methods: Using a 7 Tesla scanner we performed structural and proton MRSI in 13 adult patients with X-ALD, including 4 patients with adult cerebral ALD (ACALD), 5 with adrenomyeloneuropathy (AMN) and 4 female heterozygotes. Studies were also performed in nine healthy controls. Results: Among adult X-ALD phenotypes, MI/Cr was 46% higher and Cho/Cr 21% higher in normal appearing white matter of ACALD compared to AMN (p50.05). Both NAA/Cr and Glu/Cr ratios were lower in AMN patients (p = 0.028 and p = 0.036, respectively) than in controls. There were no signi¢cant di¡erences between AMN and female heterozygotes. In cortex, ACALD patients had lower values of NAA/Cr compared to female heterozygotes and controls (p = 0.022). The global MI/Cr ratio demonstrated a signi¢cant association with the EDSS (Spearman rho = 0.66, p = 0.039). Conclusion: 7 Tesla proton MRSI reveals di¡erences in the neurochemistry of ACALD but is unable to distinguish AMN from female heterozygotes. MI/Cr correlates with the severity of the symptoms and may be a meaningful biomarker in adult X-ALD.

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2-PENTANONE: AN INDICATOR OF PEROXISOMAL ACYLCoA THIOESTERASE ACTIVITY? Walker V1, Begley JP2, Stansbridge EM1 1 Clin Biochem, Southampton Gen Hosp, Southampton, UK, 2Clin Biochem Poole Hosp, Poole, UK Background: The methylketone, 2-pentanone, is a volatile constituent of urine of unknown source. Using solid- phase microextraction with GCMS, we commonly ¢nd a small amount. Some patients have a large transient increase when sick. We looked for an explanation. Method: The pro¢les of urine volatiles analysed since 1999 were reviewed and patients with high excretion identi¢ed. Results and Discussion: 113 patients had detectable 2-pentanone. 23 of them had increases: age 0.5^ 28 years; diagnoses: recurrent vomiting (8); fasting hypoglycaemia (6); inborn errors (5) (MCADD (1); MADD (2); VLCAD (1); methylmalonic aciduria with pancreatitis 1); miscellaneous (4) (recurrent seizures (1); malodour (1); encephalopathy (1); ethylene glycol ingestion (1). In one case, a 28 year old man with possible glycogen synthetase defect, 75 g glucose decreased excretion considerably: urine 2-pentanone 2.258; 2.075; 0.328 mmol/mmol creatinine, fasting, 1 and 2 h post-load; plasma 3-hydroxybutyrate: 0.708; 0.145; 0.061 mmol/L. 2-Pentanone production appears to increase with abnormal fasting stress. Serrendipitously, we found that the mould Penicillium roquefortii produced methylketones when growing on margarine: 2-heptanone, 2-nonanone and 2-undecanone unsupplemented, plus 2-pentanone with hexanoic acid or hexanoylCoA addition. This, and other moulds and fungi, produce methylketones from medium chain fatty acids, supposedly by incomplete b-oxidation when free CoASH is depleted. Oxidation proceeds to formation of 3-oxoacyl-CoA which, in lieu of cleavage by the CoASH-consuming thiolase reaction, is hydrolysed by a thioesterase, liberating CoASH. The oxo-acid is decarboxylated to a methylketone. This pathway might explain increased 2-pentanone production in man, perhaps via ACOT8, a highly conserved acyl-CoA thioesterase regulated by free CoASH and induced by fasting via PPARa.

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BATYL ALCOHOL AS A THERAPEUTIC OPTION IN RHIZOMELIC CHONDRODYSPLASIA PUNCTATA Bams-Mengerink AM1, Brites P2, Vyth A3, Duran M2, Wanders RJA2, Heymans HSA1, Poll-The BT1 1 Emma Child, Hosp, AMC, Amsterdam, Netherlands, 2Lab Gen Metab Dis, AMC, Amsterdam, Netherlands, 3Dept Pharm, AMC, Amsterdam, Netherlands Background: Rhizomelic chondrodysplasia punctata is an autosomal recessive disorder of peroxisomal metabolism. The key biochemical defect is a de¢ciency in plasmalogens. There is a strong relation between plasmalogen levels in erythrocytes and severity of the disease. Plasmalogens can be made from batyl alcohol, bypassing peroxisomal enzymes. In patients with Zellweger syndrome and in the mouse model for RCDP (PEX7KO) plasmalogens can be restored by adding batyl alcohol to the diet. Objectives: Can plasmalogen levels in RCDP patients be restored by adding batyl alcohol to their daily diet? What are the clinical e¡ects of batyl alcohol supplementation? Methods: Dutch RCDP patients were included in an open cohort study. 5^50 mg/kg/day Batyl alcohol was administred orally for one year. Primary endpoint was the plasmalogen level in erythrocytes. Secondary endpoints were growth and bone development. Results: 8 patients with RCDP participated in the study. C18:0DMA/ C18:0 levels in erythrocytes in patients with the milder phenotype increased to normal values at 50 mg/kg/day. C18:0DMA/C18:0 levels in erythrocytes in patients with the severe phenotype increased to 35^ 60% of lower normal values. One patient dropped out, because of loss of appetite and subsequent weight loss. No other side e¡ects were noticed. Growth and bone density increased in 75% of the patients. Conclusion: Plasmalogen levels in patients with RCDP can be increased by supplementation of batyl alcohol in a dose dependent manner. 50 mg/kg/day is su¤cient in patients with the milder phenotype of RCDP. In patients with the severe form of RCDP higher doses are recommended.

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GENETIC CLASSIFICATION OF PEROXISOME BIOGENESIS DISORDERS Ebberink MS, Mooijer PAW, Gootjes J, Wanders RJA, Waterham HR Lab Gen Metab Dis, AMC, Amsterdam, Netherlands Background: The peroxisome biogenesis disorders (PBDs) comprise a group of severe, often lethal, inherited multi-systemic disorders that can be caused by a defect in any of at least 12 di¡erent PEX genes. The large genetic heterogeneity underlying PBDs has made geneti c complementation testing an important ¢rst step towards the identi¢cation of the a¡ected PEX gene in the diagnostic workup of patients with a PBD. We have assigned more than 550 PBD cell lines to di¡erent genetic complementation groups (CGs) by means of complementation testing. Method: Skin ¢broblasts from PBD patients were assigned to CGs using the traditional polyethylene glycerol (PEG) fusion complementation assay and a recently developed PEX cDNA transfection assay. To establish the PEX cDNA transfection assay, all known human PEX cDNAs were subcloned in the mammalian vector pcDNA3. Using the AMAXA nucleofector technology, the pcDNA3-PEX expression vectors were co-transfected with the peroxisomal reporter protein GFP-SKL into PBD cells with an unknown PEX gene defect. Two days after transfection, cells are examined by £uorescent microscopy for the localization of GFP-SKL. Results and Conclusions: The PEX cDNA transfection assay allows for rapid identi¢cation of PEX genes defective in PBD patients. The more than 550 di¡erent PBD cell lines could all be assigned to the 12 known CGs, providing a representative overview of the frequency of the di¡erent PEX gene defects. No novel CG was identi¢ed. Our results suggest that all the PEX gene defects that may give rise to a Zellweger syndrome-like PBD are currently known.

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IDENTIFICATION OF THREE PEX16-DEFECTIVE PATIENTS WITH IMPORT COMPETENT PEROXISOMES IN FIBROBLASTS Ebberink MS1, Denis S1, Mooijer PAW1, Dekker CJM1, Clayton PT2, Wanders RJA1, Waterham HR1, Ferdinandusse S1 1 Lab Gen Metab Dis, AMC, Amsterdam, Netherlands, 2UCL Inst of Child Health, London, UK

A ZELLWEGER SYNDROME ASSOCIATED WITH NONIMMUNE HYDROPS FETALIS, LUNG HYPOPLASIA AND DERMAL ERYTHROPOIESIS Dursun A1, Do«rtttepe Y1, Gucer S2, Y|git S3 Wanders RJA4 1 Hacettepe Univ Metab and Nutr Unit, Ankara, Turkey, 2Hacettepe Univ Ped Pat, Ankara, Turkey, 3Hacettepe Univ Neonatoloji Unit, Ankara, Turkey, 4Amsterdam Univ, Amsterdam, Netherlands

Background: Peroxisome biogenesis disorders (PBDs) can be classi¢ed into 12 genetic complementation groups. Patients belonging to group D have a defect in PEX16 that encodes a 336 amino acid integral peroxisomal membrane protein which is involved in peroxisomal membrane assembly. Previously, PEX16-defective ¢broblasts were shown to have a complete defect in import of both matrix and membrane proteins, resulting in a total absence of peroxisomal remnants. Here, we report 3 unrelated patients, who have homozygous mutations in the PEX16 gene but catalase-positive peroxisomes in ¢broblasts. Methods: Biochemical parameters were studied in plasma and cultured ¢broblasts, and molecular analysis of all known PEX genes was performed. Results: Plasma analysis revealed biochemical abnormalities suggesting a peroxisomal disorder, including elevated levels of very long-chain fatty acids, phytanic acid and C27-bile acid intermediates. Biochemical parameters in ¢broblasts were only mildly abnormal or even within normal range, like phytanic acid alpha-oxidation, pristanic acid beta-oxidation and DHAPAT activity. Immuno£uorescence with antibodies against catalase and ALDP revealed the presence of peroxisomes, which were increased in size. Subsequent sequencing of all known PEX genes revealed three novel homozygous mutations in PEX16, including a missense mutation and 2 small deletions, all located at the carboxy-terminal end of PEX16. Conclusions: Our results show that in case of only very mild biochemical peroxisomal abnormalities in ¢broblasts a PBD cannot be excluded. Moreover, although PEX16 is involved in peroxisomal membrane assembly, PEX16 defects can present with import-competent peroxisomes in ¢broblasts.

At six hours of life, physical examination of the patient, a male infant born at 38th week of gestation, revealed di¡use edema, hepatomegaly, ascites, wide anterior fontanel, hypertelorism, bilateral hypoplastic supraorbital notches, epicanthus, low set ears, short neck and hypoplastic toenails, bulish colored areas on the right lower abdomen and the scrotum. Laboratory investigation showed hypoplastic lung in chest X-ray, severe acidosis, and hypoxia, mild-moderate anemia (Hb: 10.7 g/dl), hemolysis, mild elevated liver enzymes, hypoproteinemia, hypoalbuminemia, and abnormal coagulation tests, and normal karyotype (46, XY). Blood types of mother and infant were O Rh negative and B Rh positive, respectively. Direct antiglobulin (Coombs) test was negative. Bone marrow examination showed marked erythroid hyperplasia. VLCFA levels of plasma were compatible with Zellweger syndrome. Peroxisomal biogenesis disorders (PBD) was proven with the absence of dihidroxy-acetonephosphate acyltransferase (DHAPAT) enzyme in ¢broblast culture and showing the absence of peroxisomes by immuno£uorescence study. Postmortem investigation showed hyaline membranes and meconium aspirates in the lungs, 2+ siderosis with minimal portal ¢brosis in the liver, glomerular cysts in renal cortex, dermal erythropoeisis, microgyri and neuronal heterotopy in CNS. To the best of our knowledge, hydrops fetalis, lung hypoplasia and dermal erythropoiesis have not been reported in patents with Zellweger syndrome and further investigations are needed to elucidate this association. Support: State Planning Organisation; Prj. No:2006 K 120 640-06-03

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Background: Peroxisomes play an important role in the synthesis of bile acids because the last steps of the biosynthesis pathway are performed by the beta-oxidation system located inside peroxisomes. As a consequence, C27-bile acid intermediates accumulate in several peroxisomal disorders. It has been suggested that the C27-bile acids are especially toxic and contribute to the liver disease associated with peroxisomal disorders. For this reason, we studied the toxicity of the C27-bile acids and the underlying mechanisms. Methods: The e¡ects of conjugated and unconjugated C27-bile acid intermediates (t-THCA, g-THCA, THCA and DHCA) on cell viability, mitochondrial respiratory chain function and the production of oxygen radicals in the rat hepatoma cell line McA-RH7777 were studied and compared with the e¡ects of the mature C24-bile acids (CDCA, gCDCA, CA, t-CA and g-CA). Results: Cell viability decreased progressively after incubation with increasing concentrations of DHCA, THCA, t-THCA, g-THCA, CDCA and g-CDCA. DHCA was clearly the most cytotoxic bile acid studied. In addition, the di¡erent bile acids produced a dose-dependent decrease in ATP synthesis by isolated mitochondria oxidizing malate, which enters the respiratory chain at complex 1. The order of potency of i n h i b i t i n g t h e AT P s y n t h e s i s w a s a s fo l l o w s : DHCA4THCA4CDCA4g-THCA4g-CDCA4t-THCA4CA. F|nally, there was a dose-dependent stimulation of ROS generation in the presence of t-THCA, g-CDCA, THCA and g-THCA (in order of potency). Conclusions: Our studies showed that C27-bile acids are more cytotoxic than the mature C24-bile acids. C27-bile acids are potent inhibitors of oxidative phosphorylation and enhance mitochondrial ROS production by inhibiting the respiratory chain.

While it is known that AGAT, GAMT and SLC6A8 are expressed in CNS, many questions remain on the speci¢c e¡ects of AGAT, GAMT and SLC6A8 de¢ciencies on brain cells. Our aim was to develop new experimental models of creatine de¢ciencies by knock-down of AGAT, GAMT and SLC6A8 genes by RNAi in brain cells. Speci¢c siRNA sequences for each of the rat AGAT, GAMT and SLC6A8 mRNAs were cloned as shRNA-producing inserts into an expression plasmid vector co-expressing GFP for the follow-up of the siRNA-producing cells (pRNAT-CMV3.2/Neo). These shRNA-producing vectors were stably transfected into ROC cells (hybridoma between C6 astroglioma and oligodendrocytes), which normally express AGAT, GAMT and SLC6A8. We show that RNAi against AGAT, GAMT and SLC6A8 genes induces the knock-down of their expression in ROC cells. Using a test allowing the quantitative measure of their respective mRNA decrease through RNAi (co-transfection with a psiCHECK plasmid expressing the mRNA sequences of AGAT, GAMT or SLC6A8 downstream of the open reading frame of Renilla luciferase gene), we demonstrate that AGAT, GAMT and SLC6A8 knock-down is at least of 70% in ROC cells. Gene knock-down by RNAi of AGAT, GAMT and SLC6A8 in brain cells appears as a powerful experimental tool to analyze the speci¢c alterations induced, in creatine de¢ciency syndromes, on CNS development and the interactions and functions of speci¢c brain cells.

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TOXICITY OF PEROXISOMAL C27-BILE ACID INTERMEDIATES Ferdinandusse S1, Denis S1, Dacremont G2, Wanders RJA1 1 Lab GMZ, AMC, Amsterdam, Netherlands, 2Dept Pediatrics, Univ Ghent, Ghent, Belgium

BRAIN CELLS TAKE UP GUANIDINOACETATE AND CONVERT IT TO CREATINE Braissant O, Beèard E, Torrent C, Henry H IEM, Clinical Chemistry, CHUV, Lausanne, Switzerland While CNS expresses AGAT, GAMT and SLC6A8, the lack of SLC6A8 in astrocytes around blood^brain barrier limits the brain capacity to import creatine, and suggests that CNS has to rely on endogenous creatine synthesis. This seems contradictory with SLC6A8 de¢ciency, which, despite AGAT and GAMT expression, leads to creatine de¢ciency in CNS. We have shown that in the rat cortex, AGAT and GAMT are expressed in a dissociated way, only a few cells co-expressing both genes (Braissant and Henry, 2008, J Inherit Metab Dis, 31:230^ 239). This suggested that to allow creatine synthesis within CNS, guanidinoacetate must be transported from AGAT- to GAMTexpressing cells, possibly through SLC6A8. 3D primary reaggregating brain cell cultures, issued from fetal rat telencephalon, were used to analyze the guanidinoacetate uptake by brain cells and their synthesis of creatine, by tandem mass spectrometry using stable isotopes. These cultures are made of all cell types found in telencephalon, and express AGAT, GAMT and SLC6A8 as the in vivo brain (Braissant et al., 2008, Eur J. Neurosci., 27:1673^1685). We show here that guanidinoacetate is transported into brain cells, and that this uptake is competed by creatine, suggesting that in CNS, guanidinoacetate, as creatine, uses the SLC6A8 transporter. We further demonstrate that the brain cellimported guanidinoacetate is e¤ciently converted into creatine. Our work demonstrates that guanidinoacetate can be e¤ciently imported by brain cells, which then convert it to creatine. This ¢ts with our hypothesis of guanidinoacetate transport, by SLC6A8, from AGAT- to GAMT-expressing cells, which may explain why SLC6A8-de¢cient patients lack creatine in CNS.

KNOCK-DOWN OF AGAT, GAMT AND SLC6A8 BY RNA INTERFERENCE IN BRAIN CELLS: NEW EXPERIMENTAL MODELS OF CREATINE DEFICIENCIES IN CNS Beèard E, Braissant O IEM, Clinical Chemistry, CHUV, Lausanne, Switzerland

FAVOURABLE OUTCOME WITH ORAL CREATINE SUPPLEMENTATION: CLINICAL AND LABORATORY FINDINGS IN FIVE PATIENTS WITH GAMT DEFICIENCY Haliloglu G1, Karli OK2, Dursun A3, Tokatli A3, Bodamer O4, Coskun T3, Topcu M1 1 Hacettepe University, Pediatr Neurol, Ankara, Turkey, 2Hacettepe University, Radiology, Ankara, Turkey, 3Hacettepe University, Metab Dis, Ankara, Turkey, 4Univ Child Hosp, General Pediatr, V|enna, Austria Cerebral creatine de¢ciency syndromes (CCDS) are neurometabolic diseases that include two autosomal recessive creatine biosynthesis defects (arginine-glycine amidinotransferase and guanidinoacetate methyl transferase and an X-linked creatine transporter defect. Clinical phenotype is characterized by intellectual disability, delay in language, autistic behavior, epileptic seizures and movement disorder. We present ¢ve patients (4 boys, 1 girl) from four di¡erent families with GAMT de¢ciency. There is ¢rst degree consanguinity in all. There is a family history of mental and motor retardation in two families. The age at referral to our hospital was 11 months, 14 months, 3, 10 and 11 years respectively. Clinical symptoms were developmental and speech delay (n = 5), autistic behavior (n = 5), seizures (n = 3), movement disorder (n = 1). Onset of symptoms was within one year of age in all patients. MRI showed bilateral inreased intensity in the globus pallidi (n = 4). Diagnosis was done by biochemical features and absence of cerebral creatine levels by MRS. Interval between onset of symptoms and diagnosis varies between 6 months to 10 years. All of the patients are on creatine substitution treatment. Mean follow-up period is 6 months (3^14 months). Mutational analysis revealed homozygous c.327G4A mutation on exon 2 in the GAMT gene. In our patients, oral creatine supplementation leads to improvement in motor milestones, epilepsy, autistic features and behavioral disturbances, normalization of creatine pool with a decrease in guanidinoacetate levels and basal ganglia signal intensity. Except the ones with a late-onset diagnosis, they all improved concerning language development.

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THE SCREENING OF CREATINE TRANSPORTER DEFECT AMONG ESTONIAN FAMILIES WITH X-LINKED MENTAL RETARDATION O¬unap K1, Puusepp H2, Kall K3, Talvik I4, Mannamaa M4, Jakobs C5, Salomons GS5 1 Dept of Genet, Tartu Univ Hosp, Tartu, Estonia, 2Dept of Ped, Univ of Tartu, Tartu, Estonia, 3Cent Lab of Health Protection Inspect, Tallinn, Estonia, 4Children's Clinic, Tartu Univ Hosp, Tartu, Estonia, 5Dept of Clin Chem, VU Univ Med Center, Amsterdam, Netherlands The aim of this study was to estimate the frequency of creatine transporter (SLC6A8) defect among Estonian X-linked mental retardation (XLMR) families. A large study of unspeci¢c MR is going on in Estonia. From the total investigated group, we selected 49 families who had a suspicion of XLMR by pedigree data. In 11 out of 49 families we detected an increased urinary creatine/creatinine ratio and normal guanidinoacetate levels by GC/MS. In the repeated sample three brothers had still an increased ratio of 0.75, 0.95, and 1.92 respectively, and in another unrelated boy this ratio was 0.9 (normal 50.24). DNA sequence analysis of the 13 exons and adjacent splice sites of the SLC6A8 gene was performed for all patients in 9 families. One novel presumably pathogenic missense mutation was found in the family with three male probands and their mother. The elder son has mild mental retardation (IQ 68), the second son has moderate mental retardation, expressive speech and language delay, autistic behavior and epilepsy and the third son has presently attention de¢cit disorder (IQ 88). Their mother showed reduced creatine ratio in MR spectroscopy (IQ 81). Functional studies to con¢rm the pathogenic nature of the DNA variation are initiated. Our results show similarly to Rosenberg et al. (2004) that the frequency of SLC6A8 de¢ciency among XLMR patients is approximately 2%. This XLMR disease is not easily recognized by clinical characteristics and thus this population needs to be screened by metabolite (urinary creatine/creatinine ratio, MRS of brain), and or molecular tests.

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ARGININE-GUANIDINOACETATE-CREATINE PATHWAY IN PEDIATRIC RENAL TRANSPLANTS Andrade F1, Prieto JA1, Ariceta G2, Aguirre M2 Sanjurjo P1, Aldaèmiz-Echevarr|èa L1 1 Div Metab, Cruces Hospital, Barakaldo-Bilbao, Spain, 2Div Pediatr Nephrol, Cruces Hospital, Barakaldo-Bilbao, Spain Introduction and Objectives: Cardiovascular disease is an important cause of morbidity in recipients of renal transplants due to their hyperhomocysteinemia and other traditional factors of cardiovascular risk, such as hypertension, dyslipidemia, obesity, insulin resistance, immunosuppressive therapy, etc. The aim of the study is to know the status of the arginine-guanidinoacetate-creatine pathway in these patients, given the relation between arginine metabolism, renal function, methionine-homocysteine cycle, and cardiovascular disease. Methods: Patients (median age 13, range 6^18 years) with a renal allograft of at least six months of evolution were enrolled into the study. The levels of homocysteine, methionine, arginine, ornithine and glycine, and urinary creatine and guanidinoacete were analysed. Results: Median plasma concentrations of homocysteine (9.0 mmol/L, p50.001) and glycine (246.5 mmol/L, p = 0.083) were signi¢cantly higher, while urinary excretions of guanidinoacetate and creatine were signi¢cantly lower (18.5 mmol/mol creatinine, p = 0.006, and 89.2 mmol/mol creatinine, p50.001 respectively) than in healthy children. In addition, urinary excretion of guanidinoacetate and creatine correlated positively with creatinine clearance (r = 0.559, p = 0.002 and r = 0.636, p50.001). Urinary excretion of creatine was negatively correlated with plasmatic homocysteine. Conclusions: The demonstration of disturbances in the arginine-creatine pathway in patients with well-functioning renal transplants in absence of chronic renal failure represents a novel ¢nding. We speculate that the low urinary excretion of guanidinoacetate and creatine is probably related to an enzymatic de¢cit in this pathway, and to the defective methylation associated with the presence of hyperhomocysteinemia.

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CREATINE DEFICIENCY SYNDROMES IN PORTUGUESE POPULATION Valongo C1, V|larinho L1, Almeida LS1, Salomons GS2, Jakobs C2, Quelhas D1 1 CGMJM, INSA, Porto, Portugal, 2Dept Clin Chem, Metab U, VU Univ Med Cen, Amsterdam, Netherlands Background: Creatine de¢ciency syndromes are a group of disorders caused by endogenous creatine biosynthesis defects (GAMT and AGAT de¢ciencies), or by a defective creatine transport into the central nervous system (SLC6A8 de¢ciency). The common clinical denominator of disorders of creatine biosynthesis and transport is mental retardation with prominent speech delay. Since 2002 we are screening children and young adults for defects of creatine metabolism or transport. Methods: Simultaneous determination of creatine and guanidinoacetate in urine were performed by a GC-MS-SIM method. Molecular genetic analysis, GAMT enzyme activity and creatine uptake in ¢broblasts were performed in order to con¢rm the biochemical data. Results: 12 patients with GAMT de¢ciency (7 of them identi¢ed in our lab) and 5 patients with X-linked SLC6A8 de¢ciency were diagnosed. Up to now no AGAT de¢cient patients were diagnosed. Furthermore we found low levels of urinary creatine excretion in other metabolic conditions (ornithine carbamyl-transferase de¢ciency, cobalamin C de¢ciency, phenylketonuria and 3-hydroxy-3-methylglutaryl-CoA lyase de¢ciency) under treatment. Conclusions: So far, 17 patients with creatine de¢ciency syndromes were identi¢ed, with GAMT de¢ciency the most frequent in our population. We believe that there are a signi¢cant number of creatine biosynthesis and transport defects still undiagnosed. Other inborn errors of metabolism, under low protein diet, may need to be supplemented with creatine monohydrate in order to reestablish creatine pool, increase muscle mass and prevent neurological deterioration.

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CREATINE TRANSPORTER DEFECT: RESULTS OF 6 MONTHS' TREATMENT W|lcken B1, Fagan E1, Sim K1, Carpenter KH1, Salomons GS2 1 Child Hosp Westmead, and Univ of Sydney, Sydney, Australia, 2VU Univ Med Centre, Amsterdam, Netherlands Background: Creatine transporter de¢ciency, one of the three described cerebral creatine de¢ciencies, is caused by mutations in the SLC6A8 gene, is X-linked, and associated with mental retardation, speech and language delay, and seizures. Creatine medication has been somewhat successful in treating the creatine synthesis defects, but so far no e¡ective treatment has been described for the transporter defect. Case report: A boy with neonatal feeding di¤culties, constipation and hypotonia presented at 6 months with global delay. An MRI/MRS at 11m showed virtual absence of a creatine peak. Plasma creatine and guanidinoacetate levels were normal, but the urinary creatine:creatinine ratio was signi¢cantly elevated, with virtually normal guanidinoacetate levels. Mutation analysis of SLC6A8 showed the presence of a de novo hemizygous mutation in exon 1 which predicts erroneous splicing as it disrupts the splice donor site (c.262__262+1delinsTT). At one year he had global delay, with gross language delay and hand hyperkinesias. Treatment: Treatment was started with oral creatine, arginine and glycine, the latter being precursors of creatine. Clinically he progressed well, sitting unsupported by 16 months, attempting a pull-to-stand by 19 months. Hand hyperkinesias had disappeared. However, language was not improved. A repeat MRI/MRS at 20 months showed an increased creatine peak. The N-acetylaspartate:creatine ratio had decreased by 20% and the choline:creatine ratio decrease was 28%. Conclusions: While it seems that the brain creatine level had increased in the basal ganglia (where measurements were made) it is not clear how much his developmental progress is related to this.

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EPILEPSY SPECTRUM IN CEREBRAL CREATINE TRANSPORTER DEFICIENCY. GENOTHYPE CORRELATION Fons C1, Sanmart|è F2, Sempere A1, Poèo P2, Pineda M1, Arias A3, Ribes A3, Artuch R1, V|laseca MA1, Campistol J1 1 Div Metab Dis, Child Hosp Sant Joan Deèu, Barcelona, Spain, 2Neurol Dept, Child Hosp Sant Joan Deèu, Barcelona, Spain, 3Clin Biochem, Biochem Clin Inst, Barcelona, Spain Background/Objectives: Creatine transporter de¢ciency (CRTR-D) comprises mental retardation, autistic behaviour, severe language delay and mild epilepsy. Seizures are usually well-controlled with common antiepileptic drugs. We describe the epilepsy spectrum in a series of 6 patients with CRTR-D, and try to correlate the SLC6A8 gene mutation with seizure severity. Patients and Methods: We review the charts of six males with genetically con¢rmed CRTR-D (mean age: 19.8 years), followed-up in neurology department. Four cases unrelated and two are brothers. Clinical data (history of febrile seizures, age at seizure onset, seizure types, response to treatment, development of status epilepticus, and outcome), videoEEG, metabolic exams in blood and urine, Cr signal in brain H-MRS, Cr uptake in ¢broblasts, and SLC6A8 mutational analysis are reported. Results: F|ve out six patients presented seizures. Mean age at seizure onset was 5 years+5.1. One case presented family history of generalized epilepsy. Four patients presented febrile seizures with a mean age at onset of 26 months+9.8. V|deo-EEG (3) showed no speci¢c abnormalities, only a slow background activity for age. No speci¢c seizure type was detected. Four cases presented status epilepticus, before treatment with AED and in one case after AED was discontinued. Seizures were well controlled with monotherapy and classical AED (n:4), and politheraphy (n:1). Treatment with Cr monohydrate and L-arginine were both ine¡ective. Conclusions: Guanidine compounds have epileptogenic potential, acting as a partial GABA A receptor agonist contributing interfering in inhibitory GABAergic circuits. Patients with status epilepticus and severe phenotype present deletions in SLC6A8.

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IDENTIFICATION AND CLONING OF A NOVEL SPLICE VARIANT OF THE CREATINE TRANSPORTER GENE SLC6A8 Mart|ènez Mun¬oz C, Rosenberg EH, Jakobs C, Salomons GS VU MC, Dept Clin Chem, Metab Unit, Amsterdam, Netherlands Background: SLC6A8 de¢ciency is caused by mutations in the X-linked creatine transporter gene (SLC6A8), which leads to cerebral creatine de¢ciency, mental retardation, speech and language delay, autistic-like behaviour and epilepsy. Insight in the mechanism of how the transporter is regulated is largely unknown. This may be of great importance for the development of successful treatment strategies of cerebral creatine de¢cient syndromes especially of the biosynthesis defects. Our goal was to characterize CRT2 (SLC6A8B), a published splice variant of the creatine transporter. Methods/Results: Surprisingly, using RT-PCR we found a novel splice variant, SLC6A8C, which is predominantly found in human tissues with a high energy requirement such as brain, kidney, heart, small intestines and skeletal muscle, where SLC6A8 transporter is most required. The 5' untranslated region (UTR) of the SLC6A8C mRNA was identi¢ed using the Smart TM Race cDNA ampli¢cation kit. The SLC6A8C mRNA contains intron 4 and exons 5 through 13 of SLC6A8, including part of the 3' UTR. An open reading frame was found, which predicts a truncated protein identical to the SLC6A8 transporter, comprising the ¢ve last C-terminal transmembrane domains of the SLC6A8 transporter. SLC6A8C-EGFP (enhanced green £uorescence protein) fusion protein was detected by Western Blot analysis. Conclusions: This study reveals the presence of a novel SLC6A8 splice variant, SLC6A8C. The biological relevance is illustrated by the presence of this splice variant in mouse, chimpanzee and African green monkey and humans. The function of SLC6A8C is currently being investigated.

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GUANIDINOACETOACETATE METHYLTRANSFERASE (GAMT) DEFICIENCY: CLINICAL AND BIOCHEMICAL PROFILE IN TUNISIAN PATIENTS Nasrallah FN1, Kraoua IK2, Feki MF1, Gouider-Khouja NG-K2, Briand GB3, Kaabachi NK1 1 Laboratory of Biochemistry. Rabta Hosp, Tunis, Tunisia, 2Infantile neurology unit, Tunis, Tunisia, 3Laboratory of Biochemistry, CHRU Lille, Lille, France Background: Guanidinoacetoacetate methyltransferase (GAMT) de¢ciency; the ¢rst identi¢ed error of the creatine metabolic pathway, is an autosomal recessive disorder of creatine biosynthesis causing increased concentration of guanidinoacetate (GAA) with depletion of creatine (Cr) in muscle and brain. The authors analyzed clinical and biochemical ¢ndings in two ¢rst Tunisian patients with GAMT de¢ciency. Methods: Two patients have been referred to our metabolic laboratory, with signs and symptoms suggestive inborn errors of metabolism. After the ¢rst exploration, urine organic acids and plasma aminoacids was normal and no history of aminoacidemia and organic aciduria. GAA and Cr were quanti¢ed in urine, ¢rstly by gas chromatography-mass spectrometry and secondly by high pressure liquid chromatographytandem mass spectrometry for con¢rmation. Results: Two patients, 12 (P1) and 14 years (P2) of age, exhibited an intellectual disability, epilepsy, signi¢cant movement's disorders, severe abnormalities in expression and cognitive speech, motor handicap and hypotonia. In both patients, biochemical analysis showed very high urinary GAA levels [6431 mmol/L for P1 and 4503 mmol/L for P2 (controls: 9^1142 mmol/L)] and low urinary Cr levels [42 mmol/L for P1 (controls: 36^4964 mmol/L) and 50 mmol/L for P2 (controls: 31^2588 mmol/L)]. Conclusion: In presence of these neurological features, physicians should suspected inborn errors of creatine metabolism and prescribe Cr and GAA assessment. GAMT de¢cit merit rapid diagnosis because the disease may be treated with oral creatine supplementation.

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LONG TERM OUTCOME OF AGAT DEFICIENCY PATIENTS DURING CREATINE SUPPLEMENTATION Battini R1, Casarano M1, Alessandrm MG1, Casalini C1, Tosetti M1, Bianchi MC2, Leuzzi V3, Cioni G1 1 Dept Dev Neurosciences, Univ Pisa, Pisa, Italy, 2Unit of Neuroradiology, Hosp S Chiara, Pisa, Italy, 3Dept Child Neur Psych, Univ La Sapienza, Roma, Italy Arginine:glycine amidinotransferase (AGAT, OMIM 602360) de¢ciency (AGAT-d) is a rare inborn error of creatine (Cr) synthesis. The clinical presentation of AGAT-d is characterized by psychomotor delay, mild to moderate mental retardation and severe language delay. Until now only four AGAT patients are described in literature. In AGAT-d Cr and guanidinoacetate (GAA) are very low in plasma and urine and proton spectroscopy (1H-MRS) shows the lack of peak of Cr. Oral Cr monohydrate therapy restores brain Cr concentration and improves clinical picture. AGAT-d patients were four members of the same Italian family, two sisters (aged 16 and 14 years), one male cousin (12 years) and one presymptomatic child (3 years) in which AGAT gene analysis showed the W149X mutation. We performed consecutive measures of total Cr and phosphocreatine (PCr) in the brain of children during a long period of therapy. The patients' treatment was monitored by means of 1H- and phosphorus ( 31P)-MRS and by biochemical evaluations. The three AGAT-d patients were ¢rstly supplied with 400 mg/kg bw/day of Cr and in the following years we decreased the dose (300^200^100 mg/kg bw/day) to identify the optimal lowest e¡ective dosage. Clinical improvement was remarkable since patients learned to speak and their autistic-like behaviour faded during the ¢rst year of Cr supplementation, although they still show a moderate or mild mental retardation. No side e¡ects were observed and a lower Cr intake is possible. The ¢rst newborn a¡ected by AGAT-d was early treated (100 mg/ bw/day) and at 24 months his psychomotor development was completely normal.

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A RAPID METHOD FOR THE DETERMINATION OF CREATINE AND CREATININE IN URINE BY ELECTROSPRAY TANDEM MASS SPECTROMETRY: A NEW TOOL FOR THE DIAGNOSIS OF CREATINE TRASPORTER DEFICIENCY Carducci Cl1, Santagata S1, Carducci Ca1, Battini R2, Leuzzi V1, Antonozzi I1 1 Sapienza University of Rome, Rome, Italy, 2IRCCS Stella Maris, Pisa, Italy Background: The increase of creatine/creatinine (Cr/Crn) ratio in urine is a peripheral marker of creatine trasporter (CT) defect. To date, the MS/MS methods developed for the determination of Cr in urine using a derivatization step (3 mol/L HCl in n-butanol) have the disadvantage of a long sample preparation procedure. Our aim was to set up a rapid MS/MS method for the simultaneous determination of Cr and Crn in urine without derivatization. Methods: Urine proteins were precipitated with methanol. After a dilution with methanol/water solution containing D3-Cr and D3-Crn, 20 ml were directly injected in MS/MS PerkinElmer spectrometer equipped with electrospray source used in positive ionization. Multiple reaction monitoring was used for the following transitions: 132?90, 135?93 for Cr and D3-Cr, 114?86, 117?89 m/z for Crn and D3-Crn, respectively. Analysis time was 2.5 min. Results: The range of method's linearity was 0.004^4 mmol/L for Cr and 0.05^20 mmol/L for Crn. The between and within run precision was 5.1% for Cr and 4.0% for Crn, 2.5% for Cr and 1.4% for Crn respectively. The detection limits were 0.9 mmol/L for Cr and 11 mmol/L for Crn. The mean of recoveries was 102+7% for Cr and 98+4% for Crn. The method showed to be able to discriminate the urine of a CT1 de¢cient patient among those of 580 patients with di¡erent neurological disorders. Conclusions: This method is suitable for selective screening with the advantage of high-throughput quantitative simultaneous measurement of Cr and Crn

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SIMULTANEOUS DETERMINATION OF CREATINE, CREATININE AND GUANIDINOACETATE IN PLASMA AND URINE BY STABLE-ISOTOPE DILUTION UPLC-MS/MS Waterval WAH, Scheijen JLJM, Bakker JA, Bierau J Lab Biochem Genet, Maastricht Univ Hosp, Maastricht, Netherlands Background/Objectives: The measurement of creatine and guanidinoacetate is a routine procedure in many laboratories involved in the diagnosis of inborn errors of metabolism. A potential pitfall in the diagnosis of creatine disorders may the fact that creatinine is often measured using a di¡erent method than that is used to measure creatine and guanidinoacetate. We have developed and validated the simultaneous quantitative determination of creatine, creatinine and guanidinoacetate in plasma and urine using ultra-performance liquid chromatography coupled with ESI-MS/MS detection. Methods: Plasma and urine samples were mixed with stable isotopelabelled internal standards and deproteinized using acetonitrile. After centrifugation and subsequent evaporation to dryness, creatine and guanidinoacetate were butylated. Creatinine was not derivatized during the procedure. After evaporation, the mixture was reconstituted in acetonitrile and analysed using a Waters Quattro Micro MS/MS system equipped with an Acquity UPLC system. Creatine and guanidinoacetate were detected in the multiple reaction mode and creatinine was detected using the daughter-ion scan mode. The values for creatinine measured in urine were compared to the results obtained with the Ja¡eè-method using Bland-Altman analysis. Results and Conclusion: Calibration curves in plasma and urine were linear over wide concentration range and typical recovery was 100 +10%. Inter assay variation was 410% for all compounds in urine and 47% in plasma. The correlation between the present method and the Ja¡eè method was acceptable. This method allows reliable simultaneous analysis of all relevant creatine metabolites.

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URINARY STABILITY OF CREATINE AND GUANIDINOACETATE UNDER DIFFERENT STORAGE CONDITIONS Prieto JA, Andrade F, Mart|èn S, Sanjurjo P, Aldaèmiz-Echevarr|èa L Div Metab, Cruces Hospital, Barakaldo-Bilbao, Spain Background and Objectives: Creatine is a natural compound that supplies energy to the muscle and brain. Three inborn errors of its metabolism have been described. Diagnosis of these diseases relies on the analysis of creatine and guanidinoacetate levels in urine. Therefore, obtaining accurate control levels and ensuring a correct storage of the sample is crucial in order to make the correct diagnosis based on concentrations. Results: Reference values were obtained from a population of 179 volunteers of both sexes aged 6^18 years. Results were 24.2^1135 and 20.2^104 mmol/mol creatinine for creatine and guanidinoacetate, respectively, for children aged 6^12 years; and 14.3^695 and 13.4^104 mmol/mol creatinine for subjects aged 13^18. A comprehensive study of the stability of creatine and guanidinoacetate in the urine of ten volunteers at three di¡erent temperatures (RT, 48C and ^358C) was carried out, using GC-MS as analytical technique. Creatine concentration increases at room temperature (326% on average after 15 days), and more slowly at 48C (75% on average). However, its concentration decreases after freezing (^37% on average). Guanidinoacetate does not undergo degradation in liquid urine after 15 days, but its concentration decreases when freezing (^36% on average after 15 days). Big individual di¡erences on the stability of creatine have been observed. Conclusions: sample freezing and sonication before the analysis of the urine are required in order to dissolve precipitates and obtain repetitive concentrations. These results will help obtain accurate control values and will increase the reliability of the procedure to make a correct diagnosis.

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THE TRANSPORT OF CREATINE (CR) IN THE BRAIN: IN VITRO EXPERIMENTS ON RAT BRAIN AND HUMAN CANCER CELL CULTURES Leuzzi V1, Carducci Cl1, Carducci Ca1, Santagata S1, Artiola C1, Baletsrino M2, Adriano E2, Antonozzi I1 1 Sapienza University of Rome, Rome, Italy, 2Dept Neurol and V|sion Sci, Univ of Genoa, Genova, Italy The discovery of Cr transporter (CT1) de¢ciency revealed the importance of exogenous Cr for normal brain functioning. However the role of CT1 in Cr tra¤c inside CNS is not completely known. Aim of the present study was to explore the transport of Cr by CT1 in di¡erent kind of CNS cells. Methods: Cr uptake was studied in primary cultures of type I astrocytes and cerebellar granule neurons obtained from rat brains and in human astrocytoma cell lines. Cells were incubated for 24 h using three di¡erent media: (1) Basal medium eagle (BME); (2) BME added with 1 mM d3Cr; (3) BME added with 1 mM d3-Cr and 1mM 3-guanidinopropionic acid (GPA, a competitive inhibitor of CT1). Intracellular D3-Cr concentrations were determined by ESI-MS/MS after derivatization to butyl esters. 13C2-GAA was used as internal standard. Multiple reaction monitoring of the transitions 191?93 m/z for d3-Cr and 176?103 m/z for internal standard were applied. Results: D3-Cr signal detected in lysates of cell incubated with d3-Cr was 200 times higher then basal signal as detected in control cells. Mean concentrations of d3-Cr were: 52 and 508 nmol/mg proteins, in rat neurons and astrocytes, respectively, and 2.17 nmol/106 cells in human astrocytoma cell line. The presence of GPA produced a marked decrease of D3-Cr up-take in all cell types (85%, 68%, 78%, respectively in rat neurones, in rat astrocytes, and in human astrocytoma cells. Conclusions: CT1 is present in astrocytes and neurons and acts as the most important transporter of Cr inside CNS.

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S-ADENOSYL-METHIONINE MAY BE BENEFICIAL FOR DECREASING GUANIDINOACETATE IN GAMT DEFICIENCY Renaud DL Mayo Clinic Foundation, Rochester, MN, USA Background: Guanidinoacetate methyltransferase (GAMT) de¢ciency is an inherited disorder of creatine synthesis. This autosomal recessive disorder results in a decrease in creatine in the brain and an accumulation of guanidinoacetate (GAA). S-adenosyl-methionine is a cofactor for GAMT. Method: A twelve-month-old boy presented with mild global developmental delay and hypotonia. Magnetic resonance spectroscopy (MRS) revealed absence of the creatine peak. Creatine levels were undetectable in plasma and signi¢cantly decreased in urine. Guanidinoacetate levels were signi¢cantly increased in plasma at 12.6 mmol/L (normal 0.4^3.7), suggestive of GAMT de¢ciency. Creatine supplementation was provided orally with near normalization of the MRS creatine peak size. S-adenosyl-methionine supplementation was also provided, initially at a dose of 100 mg once daily then increased to 200 mg once daily. The patient was followed with annual MRS, plasma and urine creatine and GAA levels. Results: At the most recent visit, at age 3 years 9 months, his gross and ¢ne motor skills were age appropriate. He receives speech therapy for articulation but has appropriate expressive and receptive language skills for age. There was no history of abnormal involuntary movements although he has had 2 brief generalized tonic-clonic seizures with febrile illnesses. MRS demonstrated a slightly decreased creatine peak, resulting in an increased dose of creatine supplementation. Supplementation with creatine and S-adenosyl-methionine resulted in a signi¢cant decrease in GAA levels in both blood and urine. The plasma GAA level was 5 mmol/L. Conclusions: S-adenosyl-methionine supplementation in combination with creatine may be bene¢cial for lowering GAA levels in patients with GAMT de¢ciency.

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CREATINE SYNTHESIS AND TRANSPORT DEFECTS: DIAGNOSIS AND TREATMENT IN A SERIES OF 7 PATIENTS Valayannopoulos V1, Boddaert N2, Chabli A3, Desguerre I2, Philippe A4, Jakobs C5, Munnich A4, Salomons G5, de Lonlay P1 1 Metab Unit, Necker-Enf Malades Hosp, Paris, France, 2Ped Neurology, Necker-Enf Malades Hosp, Paris, France, 3Bioch Lab, Necker-Enf Malades Hosp, Paris, France, 4Genet Dept, Necker-Enf Malades Hosp, Paris, France, 5Dept Clin Chem, VU Univ Med Center, Amsterdam, Netherlands Background: Creatine metabolism disorders include biosynthesis defects (AGAT and GAMT de¢ciencies of recessive autosomal inheritance) and of cerebral transport (SLC6A8 gene, X-linked). Patients: We present 2 siblings with GAMT de¢ciency and 5 patients (3 males and 2 females) with creatine transporter (CRTR) de¢ciency. The diagnosis has been suspected upon guanidinoacetate, creatine and creatinine measurements in urine and undetectable or very low creatine on brain magnetic resonance spectroscopy (MRS). The clinical presentation included severe mental retardation for the 2 siblings, behaviour disturbances and intractable seizures. The patients with CRTR de¢ciency presented with mental retardation, speech delay and psychiatric manifestations. Two of the male patients displayed pseudomyopathic symptoms. Methods and Results: The siblings with GAMT de¢ciency have been treated by oral creatine for 3 years with dramatic improvement in seizures, normalisation of creatine in brain MRS but persistence of abnormal behaviour. The patients with CRTR de¢ciency have been treated so far for 2 years with oral creatine combined or not to its natural precursors L-Arginine and L-Glycine. No improvement of their clinical signs (except from myopathic signs) or brain creatine in MRS has been observed so far. Conclusion: the metabolic screening of patients with non-speci¢c mental retardation associated to autistic or other psychiatric symptoms, seizures or dystonia should include brain MRS in order to diagnose patients with creatine metabolic defects. However treatment has been proven of bene¢t only in creatine biosynthesis defects so far.

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A STRATEGY IN THE TREATMENT OF CREATINE TRANSPORTER DEFECT: L-ARGININE SUPPLEMENTATION IN THREE ITALIAN CT1 PATIENTS Battini R1, Chilosi AM1, Caruso U2, Mancardi MM2, Leuzzi V3, Italian Group on Cr metabolism and transport GISMet-Cr4 1 Dept Dev Neurosciences, Univ Pisa, Pisa, Italy, 2Dept Pediatrics and NPI, Gaslini Inst, Genova, Italy, 3Dept Child Neur Psych, Univ La Sapienza, Roma, Italy, 4GISMet-creatina Italian Group, Pisa, Genova, Roma, Italy Creatine transporter de¢cit (CT1) is a inherited metabolic defect that causes mental retardation, epilepsy, speech, language and behavioral de¢cits. No treatment is proved to be successful in this condition until now. Conversely, creatine (Cr) synthesis defects have shown favourable outcome under oral Cr supplementation and dietary. Given that the Cr precursors (arginine and glycine) have their own transporters at level of blood^brain barrier, the supplementation of one or both of these precursors appears an attractive therapeutic option aimed to stimulate the endogenous synthesis of Cr and, in such a way, to overcome the block of Cr transport in the brain. In order to test in CT1 patients the capability of Cr precursors to restore brain Cr levels and to improve symptoms, a pilot study has recently started on three Italian CT1 patients under L-Arginine (Arg) supplementation at the dosage of 300 mg/kg/day. The age of diagnosis was 8 years and 6 months, 5 years and 18 years respectively. All patients showed moderate or severe mental retardation, hyperactivity and impulsiveness and severe language/ speech delay, with oral-motor dyspraxia in one of them. The second one had also a severe epileptic phenotype. Oral supplementation with Arg was started and in two of them we completed one year of follow up, describing a noticeable clinical improvement. In addition, an increase of brain Cr, although well below the normal values, was documented at 1H-MRS. The results suggest that children with CT1 disorder show some residual adaptive plasticity for certain functions even at a quite advanced age.

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ASPERGER AUTISM ASSOCIATED WITH FOLATE RECEPTOR AUTOANTIBODIES AND CEREBRAL FOLATE DEFICIENCY Ramaekers V1, Sequeira J2, Blau N3, Quadros E2 1 Dept Ped Neurol, CHU Lie©ge, Lie©ge, Belgium, 2Dept Cell Biology, State Univ New York, Brooklyn, USA, 3Div Clinical Chemistry, Univ Child Hosp, Zu«rich, Switzerland Background: A boy, now 15 years old, was diagnosed with Asperger (high-functioning) autism. His younger brother was known to su¡er from the infantile-onset cerebral folate de¢ciency (CFD) syndrome associated with folate receptor (FR) autoantibodies, which block membrane-attached folate receptors at choroid plexus epithelial cells and impair folate transport to the spinal £uid compartment and CNS. Methods: After informed consent serum and spinal £uid samples were investigated for the presence of FR autoantibodies of the blocking type in serum and measurement of biogenic monoamine metabolites, pterins and 5-methyltetrahydrofolate in CSF. Results: Investigations in the boy with Asperger syndrome identi¢ed the presence of serum FR autoantibodies of the blocking type and low CSF values of 5-methyltetrahydrofolate (5MTHF) and the serotonin endmetabolite 5-hydroxy- indoleacetic acid. Oral supplements with L-5methyltetrahydrofolate corrected the CSF 5MTHF levels and cured his autism. Conclusion: This report identi¢ed FR autoimmunity and CFD in two brothers who manifested marked phenotypic heterogeneity, being the infantile CFD syndrome and Asperger autism as a new emerging phenotype. Asperger syndrome due to FR autoimmunity can be cured after oral treatment with L-5-methyltetrahydrofolate. Further investigations have been started to de¢ne the role of FR autoimmunity in Asperger autism and to ¢nd explanations for the intrafamilial heterogeneity of the clinical phenotype.

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RENAL NAS1 SULFATE TRANSPORTER DEFECT IN AUTISTIC PATIENTS Bowling FG1, Heussler H1, Dawson P2, VanDongen J1, Markovich D2 1 Dept of Paediatrics, Mater Childrens Hospital, Brisbane, Australia, 2 University of Queensland, St Lucia, Australia

LARGE NEUTRAL AMINO ACIDS AND LATE DIAGNOSED PHENYLKETONURIA Moseley KD1, Azen CG2, Koch R1, Yano S1 1 Gen Div, USC Keck School of Medicine, Los Angeles, USA, 2USC Keck School of Medicine, CHLA, Los Angeles, USA

Background: Studies of autism show 7q31^32 as a consistent susceptibility locus. The locus includes the sulfate transporter gene, NaS1 which is proposed to play a major role in maintaining human sulfate levels. Intracellular sulfate is maintained by transporters and intermediary metabolism. Despite the importance of sulfate, plasma levels are rarely measured clinically and little is known about the physiological consequences of hyposulfataemia. Xenobiotics and endogenous substrates including steroids, bile acids, thyroid hormones and neurotransmitters are sulfonated by cytosolic sulfotransferases. Autistic patients have a reduced sulfation capacity and the aetiology is yet unknown. We propose that the NaS1 sulfate transporter gene is associated with hyposulfataemia and hyposulfonation in autistic patients. Methods: We have cloned the human and mouse NaS1 genes, and generated a NaS1 null mouse. The Nas1^/^ mice exhibit increased urinary sulfate excretion, hyposulfataemia, gastrointestinal disturbances, seizures and behavioural abnormalities, which represent a striking similarity to some of the features in autistic patients. We developed a highly speci¢c novel assay of sulfate, to calculate the fractional excretion index for sulfate. Results: Testing a selected cohort of children with autism (n = 78) meeting ADOS criteria, demonstrated very high FEI SO4-2 (40.8) in 3 children, and elevated FEI SO4-2 (0.6^0.8) (normal range (0.32^0.47)) in 8 children, indicating abolished and reduced renal sulfate reabsorption, respectively. Sulfonation of acetaminophen, peptides, and cholesterol was reduced. Sequence analysis of the NaS1 gene revealed loss of function mutations (R12X and N174S). Conclusion: Loss of function in the Nas1 gene is associated with autism in humans.

Background: Many individuals with phenylketonuria (PKU) born before newborn screening are severely a¡ected and remain untreated since it was surmised that the damage was already done and it was too late to treat. However, based on our recent experience in treating these individuals proper treatment can reduce negative behaviors, improve overall health and reduce costs of care. Objectives: The aim of this study is to assess health outcomes, behaviors and cost bene¢t analyses in PKU adults for one year who are being treated with large neutral amino acid therapy (LNAA). Methods: 10 late diagnosed, severely a¡ected individuals with PKU were evaluated. Baseline and end of study tests include laboratory tests and psychological evaluations. Blood phenylalanine/tyrosine and behavior were evaluated on a monthly basis. Cost bene¢t analysis compared the cost of care in state institutions versus community placement as well as any decrease in medications. Results: There was a signi¢cant correlation between increased tyrosine blood levels and less aggression towards others and improvement on the V|neland daily living scale and, also, signi¢cant declines in phe/tyr ratio. Cost comparisons of psychotropic medications obtained before and after the study revealed a cost savings of 50%. Annual costs for an individual living in a state institution versus living in a community setting decreased approximately 52%. Conclusion: Using LNAA to treat individuals with PKU who previously were untreated resulted in a cost savings as well as improvement in behavior.

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INCREASED SPONTANEOUS OSTEOCLASTOGENESIS IN PHENYLKETONURIA Porta F1, Roato I2, Spada M1, Ferracini R2, Mussa A1 1 Dept of Pediatrics, University of Turin, Torino, Italy, 2CERMS, Torino, Italy Background/Objectives: Phenylketonuria (PKU) is commonly complicated by a progressive bone impairment of uncertain etiology. The protein-restricted diet and the possible noxious role of high plasma phenylalanine (Phe) concentrations on bone have been previously suggested as possible determinant factors. Since osteoclasts (OCs) are involved in bone reabsorbing, they could play a role in determining bone damage in PKU. The previously reported high excretion of bone resorption markers in PKU patients is consistent with this hypothesis. Although di¡erent diseases characterized by bone loss have been related to an increased spontaneous osteoclastogenesis from peripheral blood mononuclear cells (PBMC), to date there is no evidence of increased OCs formation in PKU. Methods: We compared the spontaneous osteoclastogenesis from PBMC in 13 patients (8 males and 5 females, mean age 11.8+6.3 years) a¡ected by PKU with that observed in age- and sex-matched healthy subjects. The PBMC were obtained according to the F|coll method, and the cell incubations performed in triplicate in 96-well/ plates. After 13 days of culture, cells were stained for tartrate-resistant acid phosphatase and the number of OCs was detected in blind by the same operator. Results: Phenylketonuric patients showed a number of OCs almost double with respect to that observed in controls (163.9+87.5 and 84.3 +54.9, respectively; p = 0.013). Conclusions: Possibly, an imbalance between bone formation and bone resorption could explain, at least in part, the pathogenesis of bone loss in this disease. These ¢ndings add new insights in the comprehension of the biological mechanisms underlying bone damage in PKU.

PHENYLKETONURIA IN ADULTHOOD IN THE MORAVIAN REGION (CZECH REPUBLIC) Prochazkova D1, Konecna P1, Hrabincova E2, V|nohradska H3, Hrstkova H1 1 Dept Pediat, Univ Hosp Masaryk Univ, Brno, Czech Republic, 2Center Mol Biol Gene Ther Univ Hosp, Brno, Czech Republic, 3Dept Biochem Univ Hosp, Brno, Czech Republic Background: The objective of this presentation is to give a survey about phenylketonuria (PKU) in adulthood in the Moravian region. Methods: One hundred adult PKU patients aged from 18 to 43 years were examined. Results: 55% of patients with PKU were not on a low-protein diet. 45% of them kept the diet but 22.2% had the phenylalanine blood level higher than 20 mg/dl, i.e. 1200 mmol/l. The educational attainment of patients: 66.2% of patients were apprenticed, 21.7% of them ¢nished their secondary school, 1.2% ¢nished college and 1.2% ¢nished university studies. 6% of patients attended special-needs school and 3.6% of them were unable to educate. The body mass index (BMI) of female patients was 23.6+0.05 (16.8^43.6). BMI of male patients was 24.0+0.8 (16.7^ 38.9). In the case of interruption or extenuation of the diet eleven patients (aged from 18 to 38 years, eight women and three men) restarted a low-protein diet because of following problems: body wasting, angst, breathlessness, anxious disorder and insomnia requiring psychiatric medication, activity disorder, migraine. One female patient was treated for mycosis fungoides. The most frequently observed mutations in this group were p.R408W (41.7% of patients), p. R158Q (16.7%) and p.R261Q (8.3%). Conclusions: The results demonstrate that a controlled relaxation of the low protein diet in PKU adults is allowed, keeping the phenylalanine blood level in some cases below 15 mg/dl. The educational attainment is lower than in the Czech population.

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HOW MANY PKU CHILDREN CAN BENEFIT FROM TETRAHYDROBIOPTERIN? Bik-Multanowski M, Didycz B, Pietrzyk JJ Chair of Pediatrics Jagiellonian Univ, Krakow, Poland Background: Tetrahydrobiopterin (BH4) decreases blood phenylalanine concentration in some persons with hyperphenylalaninemia and phenylketonuria (PKU) allowing for relaxation of dietary treatment regimen. As BH4 is currently not recommended for PKU/ hyperphenylalaninemia treatment in Poland, we aimed to assess the number of our patients who could bene¢t from BH4 use. We also intended to analyze molecular background of BH4-responsiveness in our patients. Methods: We retrospectively analyzed blood phenylalanine concentrations at diagnosis, blood phenylalanine decrease after BH4 loading and mutations of the phenylalanine hydroxylase gene in a group of 94 consecutive newborns with PKU/hyperphenylalaninemia diagnosed in our center. BH4 loading test (10 mg/kg) was performed in the children to exclude BH4 de¢ciency. If initial blood phenylalanine concentration was 5360 mmol/L, oral phenylalanine loading (100 mg/ kg) preceded the BH4 test. Results: Decrease by 430% of the initial blood phenylalanine concentration within 24 h post BH4 loading, which indicates BH4responsiveness, was observed in 25 (27%) of children. However, in 15 of them the basic blood phenylalanine concentration did not exceed 360 mmol/L (no dietary treatment necessary). In the remaining 10 patients, who required dietary treatment at least in early childhood, mild hyperphenylalaninemia (6 cases) or mild PKU (4 cases) were diagnosed. Typical BH4-responsive mutations of the phenylalanine hydroxylase gene were found in this group: (p.Y414C, p.L348V, p. A300S, p.E390G) as well as probably BH4-resposive mutations (p. P211L, p.I306V, p.R297H, IVS10nt-11g4a). Co n c l u s i o n: Approx i m ately 1 0 % o f Pol i sh p ati e n ts w i th hyperphenylalaninemia/PKU could potentially bene¢t from use of BH4. Study supported by government grant N40708032/3085.

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REDUCED CEREBRAL FLUORO-L-DOPA UPTAKE AND RELEASE IN ADULT PKU PATIENTS Landvogt C1, Mengel E2, Bartenstein P3, Feldmann R4, Weglage J4, Cumming P5, Santer R6, Ullrich K6 1 Dept Nucl Med, Uni Mainz, Mainz, Germany, 2Dept Pediatr, Univ Mainz, Mainz, Germany, 3Dept Nucl Med, Univ Munich, Munich, Germany, 4Dept Pediatr, Univ Mu«nster, Mu«nster, Germany, 5Inst Stereolog Res, Bispbjerg Hosp, Copenhagen, Denmark, 6Dept Pediatr, Univ Medical Center, Hamburg-Eppendorf, Germany Positron emission tomography was used to measure the utilisation of 6[18]£uoro-L-dopa (FDOPA) in the brain of seven adult PKU patients as compared to seven age matched healthy volunteers. FDOPA utilisation in striatum was calculated by linear geographic analysis, with cerebellum serving as non-binding region. As suspected, we found a delayed and attenuated in£ux of FDOPA into the brain in PKU patients. Surprisingly, the e¥ux of FDOPA from brain was also delayed. Linear geographic analysis in addition showed a signi¢cant reduction of striatal dopa-decarboxylase activity, a ¢nding described earlier in the brain of PKU-enu2 mice. Conclusion: The results give evidence that the clinical variability of untreated PKU patients may be the consequence of mutations with e¡ects on the function of brain amino acid carriers for Phe in£ux and e¥ux. In addition, the results show for the ¢rst time that the activity of dopa-decarboxylase is also reduced in the brain of PKU patients.

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PAH DEFICIENCY IN PORTUGAL: IDENTIFICATION OF POTENTIAL BH4-RESPONSIVE PATIENTS Rivera I1, Leandro P1, Queiroès A2, Gaspar A3, Lobo Antunes M3, V|larinho L2, Tavares de Almeida I1 1 Met and Gen Group, iMed UL, Fac Pharmacy, University of Lisbon, Portugal, 2Instituto Geneètica Meèdica, Porto, Portugal, 3Unid Doenc°as Metaboèlicas, Hosp Sta Maria, Lisbon, Portugal Phenylketonuria (PKU) and related hyperphenylalaninemias (HPA) are caused by mutations in phenylalanine hydroxylase (PAH) gene and for more than 50 years a low diet in phenylalanine has been the main therapy. Recently, a new form of PAH de¢ciency has been described, which responds to tetrahydrobiopterin (BH4) treatment and it is apparently dependent on speci¢c mutations. The aim of this work is to genotype the Portuguese PKU patients in order to evaluate the average frequency of BH4-responsive patients in Portugal and to identify potential candidates to BH4 therapy. We have characterised 255 independent PKU alleles displaying 34 di¡erent mutations, four of which account for more than 62% of mutant alleles (IVS10, R261Q, V388M and R270K). Among the mutational spectrum, we have identi¢ed 13 mutations with residual PAH activity or associated with BH4-responsiveness (I65T, R68S, R176L, V230I, R241H, R243Q, R261Q, A300S, V388M, E390G, A403V, Y414C and D415N), two of which are among the prevalent ones. This study allowed the full genotyping of 117 patients, 68 of whom harbour BH4-responsive, or potentially responsive, mutations; the calculated frequency of BH4responsiveness in our PKU population is 58%, quite close to the predicted average value for European populations. Most of these patients (43/68) carry the R261Q and V388M mutations, either in heterozigosity with another BH4-responsive mutation or in hemizigosity, and display a moderate PKU phenotype. The results of this study will contribute to the appropriate selection of candidates to integrate the pilot study aiming the implementation of the new treatment with BH4 in Portugal.

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PREVALENCE OF (POTENTIALLY) BH4 RESPONSIVE MUTATIONS IN PKU PATIENTS FROM GHENT, BELGIUM Verloo P, De Meirleir L Dept Child Neur and Metab, Univ Hosp, Ghent, Belgium As a preliminary study to set out the e¡ectiveness of BH4 therapy in our population of PKU patients, we studied their mutations and compared this with reported BH4 responsive mutations in the literature. From 41 of 52 (78%) followed PKU patients detailed genetic information was available. We detected 27 di¡erent mutations in the 82 alleles of these patients. All of the mutations found were previously reported in PKU patients. The most common mutation is the splicing mutation IVS12nt1, the most common mutation in Caucasians (10 out of 82 mutations, 12%). This is followed by R158Q (7/82, 9%), the IVS10nt546 splicing mutation (8/82, 7%) and the R261Q (8/82, 7%) mutation. The G272X, R243Q and L48S mutations share a common fourth place (4/82, 5%). Interestingly, all mutations present in this top four are potentially BH4 responsive. 26 patients (64%) had two mutations potentially associated with BH4 responsiveness. Moreover, 33 from the 41 patients (80%) have at least one mutation potentially associated with BH4 responsiveness. Furthermore our cohort has four patients homozygous for the same PAH mutation, one P225T, one R261Q and two R243Q. R243Q and R261Q are potential BH4 responsive mutations, and these three patients can help to further asses the BH4 responsiveness of this mutation. Conclusion: a very high percentage from our PKU patients is potentially BH4 responsive. We will start to asses BH4 responsiveness with a BH4 loading test in the four homozygous patients, and in the 26 patients with two potential BH4 responsive mutations.

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FACTORS INFLUENCING COMPLIANCE IN PKU DURING THE FIRST 6 YEARS OF LIFE Burgard P, Garbade SF, Adler J University of Heidelberg, Heidelberg, Germany Background: Compliance with PKU recommendations is important in infancy/childhood. About 25% of blood phenylalanine levels during the ¢rst 6 years of life are above the recommendation. We investigated possible factors in£uencing compliance in PKU children. Methods: In an explorative study we interviewed 16 mothers of children with PKU about compliance related behaviour during the ¢rst 6 years of life. We ¢rst divided the sample in two n = 8 groups of `good' (phe 5 recommendations) and `poor' (phe median 4 recommendations) compliance to identify di¡erences in the following variables: thoughts/ feelings after con¢rmation of diagnosis, knowledge/acceptance of disease, parenting strategies, support systems/barriers, rules/ regulations, problem solving, emotions related with PKU. The study was explorative and aimed at the identi¢cation of trends; signi¢cance level has been set at 0.15. Results: Compliance self-rating corresponded group assignment, indicating internal design validity. For the initial period both groups reported equal intensity of negative emotions; the good compliance group reported more positive emotions, indicating net balance to be more important than single emotions. The good compliance group reported general strategies of parenting to be su¤cient, the poor compliance group reported education to be more demanding requiring special education methods. Both groups reported equally positive emotional attitudes. The poor compliance group reported more intensive feelings of guilt, anger, and embarrassment. In both groups 75% of mothers reported situations they have been unable to cope alone, all participants stressed the importance of reliable treatment teams. Conclusion: Interview studies can help to understand patients' perspectives and to develop strategies for improving compliance.

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THE NATURAL HISTORY OF 6-PYRUVOYLTETRAHYDROPTERIN SYNTHASE (PTPS) DEFICIENCY (PTPSD). A LATE DIAGNOSED CASE Leuzzi V, Carducci Cl, Carducci Ca, Di Sabato ML, Caforio C, Giannini T, Pozzessere S, Antonozzi I Sapienza University of Rome, Rome, Italy PTPSd, the most frequent disorder of BH4 metabolism, results in a severe encephalopathy if the brain biogenic amine depletion is not precociously corrected. We report on the clinical history of a patient diagnosed in adulthood. This subject presented during the infancy with p s y c h o m o t o r d e l ay a n d r e c u r r e n t ¢ t s o f o p i s t h o t o n u s. Hyperphenylalaninemia (Phe 5600 mM) was detected when he was 13 months old and a Phe restricted diet was commenced (blood Phe 5300 mM) and continued until the age of 13 years. Rigidity, hypokinesia, and trunk dystonia (opisthotonus), with diurnal £uctuation of these symptoms, occurred after diet discontinuation and vanished once it was reintroduced. At the age of 30, he complained progressive deterioration of neurological conditions. On examination he showed: mild mental retardation (WAIS IQ 47), bradypsichia, parkinsonism, bradylalia and palilalia, and anxious-depressive status. Brain MRI revealed a slight cortical atrophy and T2-weighted parieto-occipital white matter hyperintensity. Blood Phe was 5300 mM and prolactin was normal. The result of Phe/BH4 loading test and the pattern of urinary pterine excretion con¢rmed the diagnosis. CSF analysis disclosed: biopterin 5.64 r.v. 2.4^7.1 mg/L; neopterin 16.98 mg/L, r.v. 2.3^5.1; HVA 30 nM r.v. 98^450; 5HIAA 69 nM, r.v. 45^135. PTPS gene analysis detected a condition of compound heterozygous for R9C/ P87L mutations. BH4 (6.5 mg/kg bw/day) and low dosage L-DOPA/ Carbidopa (100 mg/25 mg) therapy resulted in a dramatic improvement of neurological condition. Comments: low CSF HVA is not always re£ected by blood prolactin; PTPSd should be included in the di¡erential diagnosis of patients presenting with juvenile parkinsonism.

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FUNCTIONAL ANALYSIS AND PHENOTYPIC OUTCOME OF S231F MUTATION IN PHENYLALANINE HYDROXYLASE GENE Stojiljkovic M1, Perez B2, Desviat LR2, Aguado C2, Ugarte M2, Pavlovic S1 1 IMGGE, Belgrade, Serbia and Montenegro, 2Centro de Biologia Molecular, Madrid, Spain We report two siblings, a¡ected with classic phenylketonuria (pretreatment serum phenylalanine level 41200 mmol/L). Molecular genetic study showed that they were compound L48S/S231F heterozygotes. L48S is the most frequent PAH mutation in Serbia. Since S231F is a rare, uncharacterized mutation and L48S/S231F genotype has not been reported before, we performed following genetic and in vitro expression studies to explain the severe phenotype observed in our patients. We performed PCR-RFLP analysis on six polymorphic sites (BglII, PvuIIa, PvuIIb, EcoRI, MspI and XmnI). Due to high heterozygosity of the EcoRI, MspI and XmnI polymorphic sites, haplotype associated with S231F remained uncertain. Nevertheless, we showed association of S231F with 8 VNTRs. We characterized S231F PAH protein in prokaryotic and eukaryotic expression systems. In both systems the mutant enzyme was unstable. Its residual enzyme activity was very low (*1%) showing that S231F is a severe mutation. We have found no GroESL chaperone e¡ect and slightly positive e¡ect of the BH4 on the stabilization of the protein structure. However, due to instability of the enzyme, signi¢cant physiological impact of BH4 is not expected. The previous phenotype-genotype correlation study revealed that L48S was exclusively associated with classic PKU in Serbia. Functional analysis showed that S231F was a severe mutation. These ¢ndings elucidate severe phenotype of the patients with L48S/S231F genotype.

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THE PHENOTYPIC VARIABILITY IN 6-PYRUVOYLTETRAHYDROPTERIN SYNTHASE (PTPS) DEFICIENCY (PTPSD). CLINICAL PRESENTATION AND OUTCOME OF THE ITALIAN PATIENTS Leuzzi V1, Burlina A2, Cerone R3, Concolino D4, Donati MA5, F|ori L6, Ponzone A7, Porta F7, Strisciuglio P4, Carducci Ca1, Carducci Cl1, Vagnoni C1, Pozzessere S1, Antonozzi I1 1 Sapienza University of Rome, Rome, Italy, 2Div Metab Dis, Dept Ped, Un Hosp, Padova, Italy, 3University Dept of Pediatrics-G. Gaslini, Genova, Italy, 4Dept of Ped, University Magna Grecia, Catanzaro, Italy, 5 Meyer Children's Hospital, Met Unit, F|renze, Italy, 6Dept of Ped, S Paolo Hosp Un of Milan, Milano, Italy, 7Dept of Ped, University of Torino, Torino, Italy The phenotypic spectrum and the factors a¡ecting the outcome of PTPSd are not completely known. To contribute to this topic we performed a retrospective study of the Italian patients. Methods: Patients: 19 subjects (13 M/6 F) aged 2 months^33 years. Diagnostic tests: Phe/BH4 loading (19), PTPS genotype (19), CSF pterins and neurotransmitters (16), urinary pterins (10), enzyme activity (8). Results and Conclusions: Hyperphenilalaninemia (160^2500 mM) was detected in all patients at neonatal screening. PTPSd was diagnosed at age 8 days to 13 months (18/19 patients). Pretreatment clinical examination showed: normal neurological status (5), trunk hypotonia and weak suction (10), psychomotor delay (7), failure to thrive (6), microcephaly (5), limb rigidity (3), squint (1), epileptic seizures (1), hyperthermia (1). A patient was diagnosed at age of 32 years, when he developed hypokinetic-rigid syndrome and depression. Treatments: BH4 (5 patients), BH4 plus neurotransmitter precursors (14). Outcome: Except for 8 subjects, who remained normal (4) or normalized (4) with the treatment, all others were symptomatic: psychomotor/mental retardation (7), paroxysmal movements (3), psychiatric (2) language (2) and sleep (2) disorders, dysarthria (2), attention de¢cit (1), epilepsy (1), athetosis (1), parkinsonism (1). PTPS gene analysis revealed a high allelic heterogeneity (17 di¡erent mutations in 32 alleles): 5/18 patients were homozygous, 9/18 compound heterozygous; in 4/18 only 1 mutation was found. Factors conditioning the prognosis were the pretreatment clinical status and the degree of neurotransmitters restoration in CSF. Prolactin at the diagnosis was high in 5/ 7 subjects and decreased on neurotransmitter treatment.

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Molecular chaperones play crucial roles either in the folding of newlysynthesized proteins or in the refolding of non-native proteins and therefore represent important pieces in the pathogenesis of conformational diseases recognizing misfolded and partially folded proteins and deciding whether they should be maintained or degraded. Although it has been recognized that several mutant forms of the cytosolic human phenylalanine hydroxylase (hPAH), the de¢cient enzyme in phenylketonuria (PKU), increase their expression level and enzymatic activity when co-expressed with the GroESL system (Pey et al, Hum Mutat, 2003, 21:370^8) the folding pathway of the newlysynthesized mutant and wild-type forms has not yet been characterized. Using a prokaryotic system recombinant wild-type and hPAH mutant forms were co-expressed with several molecular chaperones (GroES, GroEL, DnaK, DnaJ and GrpE) in order to identify those interacting with wild-type and mutant hPAH proteins and further characterize the observed associations. Recombinant hPAH proteins were expressed using the pTrcHis system (Leandro et al, 2000, Mol Genet Metab, 69:204^12). The pOFX-tac1 vectors expressing the studied molecular chaperones were kindly provided by Olivier Fayet (Castanie et al, Anal Biochem, 1997, 254:150^2). Cross-linking experiments and coimmunoprecipitation assays were preformed after cell lysis and following protein puri¢cation. The enzymatic activity and oligomeric pro¢le of puri¢ed proteins were also analysed. Our results indicate that when co-expressed with molecular chaperones an increase in the amount of recombinant protein in the soluble fraction and in their enzymatic activity was achieved. However, these e¡ects were mutantdependent and molecular chaperone-speci¢c. In addition molecular chaperones/recombinant enzymes associations were demonstrated.

Human phenylalanine hydroxylase (hPAH) is a homo-tetrameric protein associated with phenylketonuria (PKU). Each sub-unit presents an N-terminal regulatory domain, a catalytic domain and a C-terminal domain responsible for tetramerization. Since the majority of PKU patients are compound heterozygous expressing two di¡erent mutant subunits, interactions between the mutant subunits (negative interallelic complementation) have been postulated as the molecular mechanism underlying the observed genotype/phenotype inconsistencies reported in these patients (Okano et al, N Engl J Med, 1991, 324:1232^8). Although the production of hybrid mutant hPAH species has been demonstrated (Leandro et al, Biochim Biophys Acta, 2006, 1762:544^ 50), the impact of the di¡erent subunits on the hybrid assembly had never been addressed. Using a dual vector expression system di¡erent hPAH subunits (mutant and wild-type) were produced as fusion proteins each presenting a puri¢cation tag (6xHis) and a di¡erent epitope (XPress or Myc). Cross-linking and co-immunoprecipation assays were performed before and after protein puri¢cation by IMAC. In addition to I65T, R261Q, R270K and V388M, hPAH N-terminal truncated forms, which are produced mainly as tetramers, and a Cterminal truncated protein, which only assemble into dimers, were also studied. The obtained enzymatic activities, lower than the calculated by the predicted residual activity, suggest that negative interactions occur which are strongly depending on the nature of the mutant protein present in the hybrid modulating the oligomeric assortment. Among the studied mutant sub-units the R270K presented a higher negative e¡ect, particularly over the V388M subunit. Joa¬o Leandro is supported by a FCT grant (SFRH/BD/19024/2004).

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STUDIES ON THE INTERACTION OF MUTANT FORMS OF HUMAN PHENYLALANINE HYDROXYLASE WITH MOLECULAR CHAPERONES Cristo I, Almeida R, Leandro J, Tavares de Almeida I, Leandro P Met and Gen Group, iMed UL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal

INTERALLELIC COMPLEMENTATION AND PHENYLKETONURIA: ISOLATION OF HYBRID FORMS OF HUMAN PHENYLALANINE HYDROXYLASE (hPAH) Leandro J1, Tavares de Almeida I1, Leandro P1, Flatmark T2 1 Met and Gen Group, iMed UL, Univ Lisbon, Lisbon, Portugal, 2 Department of Biomedicine, Univ Bergen, Bergen, Norway Negative interallelic complementation occurs when a hybrid protein, expressed from two di¡erent mutated alleles of a gene, present a catalytic activity lower than the predicted residual activity (PRA; mean of the monoallelic in vitro enzyme activities for each mutation comprising the genotype). This issue is of particular interest in genetic disorders associated with mutant hetero or homo-multimeric proteins as for example phenylketonuria (PKU). PKU is an autossomal recessive disorder caused by the presence of a mutant phenylalanine hydroxylase protein (hPAH), a homotetrameric enzyme expressed mainly in the liver. Although in PKU, genotype-phenotype correlations have been established, some compound heterozygous patients present a more severe phenotype than the determined by the PRA, anticipating the presence of a negative interallelic complementation phenomenon. Using a dual vector expression system we were able to produce two di¡erent hPAH subunits mimicking the natural heteroallelic state occurring in heterozygous individuals or in compound heterozygous patients, proving that interactions occur between the di¡erent subunits. However, in order to isolate and biochemically characterize these hybrid forms, high yields are necessary. In this work we report the development of a bicistronic expression system, using as fusion partner the putative molecular chaperone maltose binding protein (MBP) and di¡erent puri¢cation tags (calmodulin binding peptide, hexahistidyl peptide and Strep II peptide) which allowed the isolation of the di¡erent hybrid forms. The characterization of the interactions between wild-type and mutant subunits (carriers) and between di¡erent mutant subunits (compound heterozygous) will contribute to a better understanding of the metabolic phenotype in the HPA/PKU patients.

ASSEMBLY OF HYBRID HETEROALLELIC MUTANT FORMS OF HUMAN PHENYLALANINE HYDROXYLASE PRODUCED IN A PROKARYOTIC DUAL VECTOR EXPRESSION SYSTEM Almeida R, Leandro J, Cristo I, Tavares de Almeida I, Leandro P Met and Gen Group, iMed UL, Fac Pharmacy, Univ Lisbon, Lisbon, Portugal

PHENYLKETONURIA AS A PROTEIN MISFOLDING DISEASE: MUTANT PG46S HUMAN PHENYLALANINE HYDROXYLASE HAS A PROPENSITY TO SELF-ASSOCIATE AND FORM AMYLOID FIBRILS Leandro J1, Simonsen N2, Tavares de Almeida I1, Leandro P1, Flatmark T2 1 Met and Gen Group, iMed UL, Univ Lisbon, Lisbon, Portugal, 2 Department of Biomedicine, Univ Bergen, Bergen, Norway The human inborn error of metabolism phenylketonuria (PKU) is caused by a loss-of-function of the liver enzyme phenylalanine hydroxylase (hPAH). More than 500 di¡erent mutations have been identi¢ed in the human pah gene, most of them associated with PKU and a smaller number with non-PKU hyperphenylalaninemia (HPA). The current spectrum of PKU/HPA mutations consists of *60% missense (single site) mutations of which *100 have been expressed in several complementary in vitro systems. At least three groups of biochemical/enzymatic phenotypes of PKU/HPA have been identi¢ed, i.e. (i) structurally stable mutant proteins with altered steady-state kinetic properties, (ii) mutant enzymes with normal or almost normal steady-state kinetics, but reduced stability in vitro and in vivo; and (iii) mutations a¡ecting both kinetic and stability properties. Since the majority of hPAH mutations results in enzyme forms with reduced solubility when expressed as recombinant proteins, PKU has often been categorized as a protein misfolding disease. In the present work we have studied the mutant pG46S, a misfolded protein that when expressed in E. coli as MBP fusion protein (MBP-pep(Xa)-G46S) is recovered mainly as tetramers (in addition to a fraction of dimers and soluble aggregates) which rapidly self-associate when cleaved by factor Xa. In order to elucidate the self-association process of G46S tetramers we: (i) examined the multiphasic G46S self-association; (ii) identi¢ed the solvent conditions a¡ecting the process; (iii) studied the e¡ect of substrates, phosphorylation and chaperones on the self-association propensity, and (iv) demonstrated that the self-association results in the formation of amyloid like ¢brils.

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DETERMINANTS OF OBESITY RISK IN ADULT PATIENTS WITH PHENYLKETONURIA von Berlepsch J1, Feldmann R2, Koletzko B1 1 Div Metab Dis, Hauner Child Hosp, Munich, Germany, 2Dept Pediatr, Univ Child Hosp, Mu«nster, Germany Background: The diet of adult patients with phenylketonuria (PKU) might be associated with an increased risk for overweight. The objective was to assess body weight and physical activity in adult PKU patients and controls. Methods: We measured body weight and height, BMI, energy expenditure and physical activity (Sensewear Armband) in 33 patients with PKU and 33 healthy controls. Results: PKU patients had higher BMI (26.5 vs 22.5 kg/m2, p50.001), more obesity (6/33 vs 2/33 subjects, p = 0.011), and lower physical activity level (p = 0.009). Their BMI was inversely correlated to active energy expenditure (r = ^0.473, p50.001) and physical activity (r = ^0.583, p50.001). The time spent with active sports correlated to activity energy expenditure (r = 0.349, p = 0.005) and physical activity level (r = 0.299, p = 0.017), whereas the time spent TV watching was inversely correlated to physical activity (r = ^0.0346, p = 0.006) and active energy expenditure (r = ^0.355, p = 0.004). Conclusions: Adult PKU patients have a higher obesity risk than healthy adults. Their BMI is predicted by physical activity level, which is lower than in healthy controls. Measures to enhance physical activity in subjects a¡ected by PKU should be explored.

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TREATMENT OF THE HYPERPHENYLALANINEMIA WITH TETRAHYDROBIOPTERIN (BH4) AND ITS INFLUENCE IN THE PATTERN OF AMINO ACIDS AND FATTY ACIDS FROM CHILDHOOD TO ADULT AGE Garc|èa-Jimeènez I1, Baldellou-Vaèzquez A1, Navarro-Aznaèrez H2, Salazar Mi3, Loèpez-Pisoèn Fj4 1 Div Metab Dis, Univ Child Hosp M Servet, Zaragoza, Spain, 2Div Pharm, Univ Hosp Miguel Servet, Zaragoza, Spain, 3Bioch U, Univ Hosp M Servet, Zaragoza, Spain, 4Div Neuroped, Univ Child Hosp M Servet, Zaragoza, Spain B a c k g r o u n d : A s i g n i ¢ c a n t p e r c e n t a g e o f p at i e n t s w i t h hyperphenylalaninemia (HPA) respond to doses of BH4. In these patients, treatment with BH4 may substitute for diet therapy. Objective: To compare the answer to the dietetic and pharmacologic treatment with BH4 in HPA, by the analysis of biochemical parameters like quality indicators. It is considered as the null hypothesis that in the responsiveness BH4 patients, some biochemical control parameters (phe, tyrosine, total and essential amino acids (AAt, AAe), and fatty acids) are kept equal or better than only with dietetic treatment. Methods: Quasi-experimental trial in HPA patients, period 2004^08. All of them were o¡ered to be treated with BH4. Two groups were established: the BH4 responders, and unresponders. The BH4 responders were monitoring for the previous and the next year after initiating treatment. Biochemical data were collected (Aat, Aae, and essential fatty acid levels). We performed a retrospective and prospective analysis in the BH4 responders by means of SPSS 15.0.The statistical study includes the descriptive and inferential statistic for comparison of averages by means of the T test for twin samples or test of W|lcoxon for nonparametric samples and the con¢dence interval. Results: There are no statistically signi¢cant di¡erences in any of the analyzed parameters before and after the BH4 treatment. Conclusions: The treatment with BH4 maintains a biochemical pattern of amino acids as good as the classic diet. The treatment with BH4 is a good therapeutic alternative for many patients.

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NEUROPSYCHOLOGICAL IMPAIRMENT IN ADULT PATIENTS WITH TREATED PHENYLKETONURIA Feldmann R1, von Berlepsch J2, Kloska S3, Weglage J1, Koletzko B2 1 Mu«nster Univ, Dept of Pediatrics, Mu«nster, Germany, 2Munich Univ, Dept of Pediatrics, Munich, Germany, 3Mu«nster Univ, Dept of Radiology, Mu«nster, Germany Background: The long-term prognosis of treated phenylketonuria (PKU) and the need for a life-long diet are still under discussion. A controlled study was to assess the impact of elevated phenylalanine (PHE) blood levels on neurological and neuropsychological performance in adult phenylketonuric patients. Methods: We investigated 60 well characterized patients with early treated classical phenylketonuria with an age range from 20 to 45 years (30 patients were older than 33 years) and 60 healthy controls, matched for age and socioeconomic status. Patients and controls were assessed for their clinical-neurological outcome, IQ and information processing abilities. In addition, MRI of cerebral white matter was performed in patients. Results: Clinical-neurological examination revealed a higher incidence of tremor, brisk deep tendon re£exes and clumsy motor coordination in PKU patients. The full scale IQ was signi¢cantly lower in patients compared to controls. Information processing was normal in young patients and in all controls. Patients older than age 33, however, showed a dramatic slowing in their information processing. V|rtually all patients showed cerebral white matter abnormalities in MRI. The extension of abnormalities was not correlated to neurological and neuropsychological test results. Conclusions: Neurological and neuropsychological investigation in adult patients with early treated PKU revealed subtle symptoms of brain damage. This seems to refer to a global neurotoxic e¡ect of elevated PHE levels in adolescence and adulthood. Additionally, our results show a dramatic slow down of information processing speed in older patients. Results indicate a life long dietary treatment in PKU.

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PRELIMINARY INVESTIGATION OF THE MUTATION SPECTRUM OF PKU IN TUNISIAN PATIENTS Khemir S1, Tebib N2, Cherif W3, Nassrallah F1, Esseghir N1, Jemaa R1, Khouja N4, Abdelhak S3, Ben Dridi MF1, Kaabachi N1 1 Biochem Lab, La Rabta Hosp, Tunis, Tunisia, 2Ped Dept La Rabta Hosp, Tunis, Tunisia, 3Institut Pasteur, Tunis, Tunisia, 4Neurology Institute, Tunis, Tunisia Background/Objective: Phenylalanine hydroxylase (PAH) de¢ciency is caused by mutations in the PAH gene (12q22-q24) resulting in a primary de¢ciency of the PAH enzyme activity. More than 500 PAH mutations have been identi¢ed, and their distribution among di¡erent populations varies signi¢cantly. IVS10nt546 (IVS10nt-11g?a) is the most common molecular defect of the PAH gene causing PKU in Mediterranean populations. This mutation was reported to as being associated with some cases of autism. To date there have been no reports on the molecular analysis of PKU in Tunisian population. The objective of this study is to screen Tunisian PKU patients for the presence of the IVS10nt546 (IVS10nt-11g?a) mutation. Patients and Methods: Eighteen PKU unrelated families (23 cases), that originated from various geographic locations in Tunisia were investigated. Diagnosis of PKU was based on plasma phenylalanine level Patients were classi¢ed into phenotypes based on pre-treatment plasma phenylalanine level: classic PKU, moderate PKU and mild hyperphenylalaninemia. F|ve cases have autistic behaviours. After informed consent blood samples were collected and DNA extracted by salting out. Exon 11 was ampli¢ed using polymerase chain reaction (PCR) and screening for the mutation of interest performed by restriction enzyme assay followed by direct sequencing con¢rmation. Results: Among the 18 unrelated PKU patients studied, the IVS10nt546 mutation was found only in 1 female patient. Interestingly, the Tunisian autistic patients do not have this mutation. Conclusion: This mutation has a relatively low frequency (2/36 mutant alleles, 5%) compared to other populations and no association with autism was identi¢ed in the studied population.

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DIAGNOSIS, TREATMENT, AND INCIDENCE OF TETRAHYDROBIOPTERIN (BH4) RESPONSIVE MILD PKU IN JAPAN Shintaku H1, Fujioka H1, Aoki K2, Yamano T1 1 Dept Pediatrics, Osaka City Univ, Osaka, Japan, 2Dept Res Dev, Aiiku Maternal and Child Hea, Tokyo, Japan

RELATIVE FREQUENCY OF BH4 RESPONSIVE PAH MUTATIONS IN SOUTHERN ITALY Bonapace G1, Ceravolo F1, Sestito S1, Montesani M1, Piccirillo A1, Strisciuglio P2, Concolino D1 1 Dept of Pediatrics Univ Magna Graecia, Catanzaro, Italy, 2Current address: Univ of Naples, Napoli, Italy

Background/Objectives: Twelve patients with mild PKU responsive to BH4 were identi¢ed by neonatal PKU screening and investigatorinitiated study of tetrahydrobiopterin (BH4) treatment for mild PKU had been performed in Japan. Half of these patients were treated with BH4 alone without diet therapy. To evaluate the long-term treatment of BH4 we performed an extension study and followed up these patients. To diagnose patients with BH4-responsive mild PKU, we evaluated the responses of BH4 loading in patients with HPA detected by neonatal PKU screening. Methods: In an extension study, 5 patients were treated with BH4 alone and their plasma phenylalanine levels were measured between 2002 and 2006. In all of the 100 patients with HPA detected by neonatal PKU screening, we examined biopterin metabolism and single-dose BH4 loading test. Four-dose and 1-week BH4 loading tests were conducted in patients who had normal BH4 metabolism and decreases in plasma phenylalanine concentrations by over 20% in the single-dose test. Results: All 5 patients were controlled well in plasma phenylalanine levels under normal diet and showed no side e¡ect more than 5 years. Among 100 patients, 19 patients had normal biopterin metabolism, and their mean values of percentage decline in serum phenylalanine from initial values were 40, 43, 52 after single-dose, four-dose, and 1-week BH4 loading tests, respectively. Conclusions: Diagnosis should be done by 1-week BH4 loading tests, and the treatment of BH4 should be performed for all responsive patients. The incidence was about 25% in PAH de¢ciency found in neonatal PKU screening in Japan.

Background and Objectives: Tetrahydrobiopterin (BH4) responsiveness in patients with mutations in the PAH gene, is a recently recognized subtype of hyperphenylalaninemia characterized by positive BH4 loading test. According to recent estimates this phenotype may be quite common, suggesting that a large group of individuals may bene¢t from BH4 therapy. Owing to recent studies associating the BH4 responsive phenotype to many di¡erent mutations (www.bb4.org/ biopku.html) we performed a molecular survey on a group of calabrian patients to select potential BH4 responsive genotypes for BH4 substitution treatment. Methods: By using polymerase-chain reaction and Dna sequencing we screened the 5' UTR, all the exons and polyA region of the PAH gene in 33 calabrian patients with 3 di¡erent PKU phenotypes (classic 15 patients, mild 6 patients and benign hyperphenylalaninemia 12 patients). Results: In 24/33 (72%) of the studied patients a BH4 sensitive mutation has been identi¢ed; the relative allelic frequencies were: L48S 11/66 (16.6%), P281L 6/66 (9%), R261X 5/66 (7.5%), R261Q 4/66 (6%), R158Q 3/66 (4.5%), A300S 2/66 (3%), f55Lfs 2/66 (3%). In 9/33 patients (27%) the BH4 sensitive mutation was in homozygous state with the L48S/L48S genotype as the most frequent. In other 15 patients (45%) the BH4 sensitive mutation was associated to missense or splicing mutations with the IVS10-11G4A/L48S as more frequent. Conclusion: The BH4 responsive phenotype could be quite common in Calabria and about 27% of the patients, according to their homozygous state for a BH4 responsive mutation, could have a bene¢t from a BH4 therapy.

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CLINICAL RESPONSE TO KUVAN (SAPROPTERIN) IN PATIENTS WITH HYPERPHENYLALANINEMIA/ PHENYLKETONURIA (PKU) ^ EARLY OBSERVATIONS AND FUTURE DIRECTIONS Mo¢di S, Lim-Melia E, Kronn D Dept of Pediatrics, NY Medical College, Valhalla, USA The potential to lower and maintain phenylalanine (PHE) levels on a m o r e l ib e ra l i z e d d i et i s p ro m i s i ng to al l p at i e nt s w i t h hyperphenylalaninemia/PKU. Our objective was to evaluate the response in PHE levels with high dose Kuvan therapy. Nine con¢rmed patients over 8 years of age from our clinic population were enrolled in Sapropterin Expanded Access Program and started on Kuvan (20 mg/ kg/day). Levels were obtained at baseline, after 48^72 h, and weekly for the remainder of the study. F|ve patients' levels declined 430% in 48^72 h and another by day 9. Two PKU patients levels dropped by 24 and 27%, respectively at 48 h, and in another by 24% by day 9. No adverse events were reported. All patients reported a general sense of well-being, and have continued on their pre-Kuvan medical foods. An additional 200 mg Phe per day was the minimum increase in our patients' diets. This initial experience with the use of Kuvan reveals the promise and also some concerns. Not all patients responded with a 30% decline immediately; some patients may require extra time on Kuvan without any diet changes. Hyper-phenylalaninemic patients responded the best; however some have managed to eat their way to higher levels. PKU patients may experience a modest diet liberalization, although a small increase in Phe intake may be su¤cient to enhance one's quality of life. Dietary liberalization on Kuvan has to be carefully monitored and patients/families need to be counselled on realistic expectations from treatment to maintain levels in therapeutic control.

HYPERPHENYLALANINEMIA AND POTENTIAL RENAL ACID LOAD (PRAL) Casero D, Borzani M, Sala F, Cagnoli G, Salvatici E, Paci S, Giovannini M Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy Background: Phenylketonuria (PKU) is an inborn error of metabolism characterized by the accumulation of phenylalanine (Phe) requiring a Phe restricted diet when Phe plasma levels at neonatal screening are above 360 mmol/L (1). If phe plasma levels at neonatal screening ranging between 120 and 360 mmol/L (2) diet is not usually not required. In spite of the dietary compliance, some PKU patient may develop ostheoporosis. PRAL is the net amount of acid deriving from diet, calculable with an algorithm including protein, phosphorus, magnesium, calcium and potassium intake and could represent a marker of bone re-arrangement. Objective: To evaluate PRAL in PKU patients and either diet-therapy (1) or free-diet (2). Methods: In 29 PKU patients [18 (1); 11 (2)] a three days diet diary was ¢lled out after performing blood examinations during their annual metabolic controls. PH and SG on urine samples, mean Phe plasma levels of the previous six months, blood amino-acid and nutritional parameters were also evaluated. Results: A signi¢cant (p50.05) correlation is present between PRAL and pre-albumine, blood urea nitrogen, urine pH, non essential/ essential blood amino-acid ratio and dietary intakes of phosphorus, calcium, protein and total calories in both groups. In group (2) a PRAL increase and a pH urine decrease parallel increases of protein intakes is present, as already reported. In group (1) a PRAL decrease and pH urine increments were observed, probably because of the lower protein and higher vegetable intakes. Group (1) shows a poorer calcium intake when complete formulas are not in the diet. Conclusions: PRAL, as an indicator of chronic acidosis, maybe helpful in monitoring metabolic bone status in PKU patients.

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INTELLIGENCE, COGNITION AND SOCIO-EMOTIONAL DEVELOPMENT OF CHILDREN, ADOLESCENTS AND YOUNG ADULTS EARLY TREATED FOR PKU: THE NEED OF SCHOOL AND PROFESSIONAL GUIDANCE Carmona C, Almeida MF, Rocha JC, V|larinho L, Cardoso ML, Lima MR Cent Gen Med Jac Mag, INSA, Porto, Portugal Background/Objectives: The studies on patients who started diet after newborn screening showed that the level of intellectual development seems to be preserved if a correct dietary control in childhood was achieved. However, we found di¡erences between individuals with PKU early treated and still on diet, in the levels of intellectual development, speci¢c cognitive domains and school performance. The main purpose of this study was to evaluate the PKU patients early treated and in follow-up at CGM in terms of intellectual, cognitive and socioemotional development, trying to understand the way they adapt to the chronic condition and plan their school and professional guidance. Methods: We studied 126 patients. We considered the diagnostic and annual medians levels of Phe as independent variables. The treatment outcome was evaluated considering the developmental and intellectual global quotients (DQ/IQ), their cognitive pro¢le and the school performance and adaptation in speci¢c domains. Results: We observed signi¢cant negative correlations between global intellectual performance and the annual median of Phe values till the age of 18. The same results were found when we considered the diagnostic levels of Phe. School failure and a performance signi¢cantly bellow the age expectations in speci¢c cognitive domains were found in patients with screening values 420 mg/dl and poor dietetic control. Conclusion: The di¤culties found in speci¢c domains of intellectual and cognitive performance could interfere with their school progress and professional success. Curriculum adaptation and school and professional guidance have been a successful approach in our PKU population.

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PSYCHOSOCIAL DISTRESS IN PKU FEMALE PATIENTS WITH GOOD AND BAD METABOLIC CONTROL Giovannini M, Mirazita P, Paci S, Casero D, F|ori L, Riva E Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy More severe forms of hyperphenylalaninemia (mild, moderate and severe PKU) due phenylanine hydroxylase de¢ciency are treated with diet through all the life-span. Psychological and social problems with behavioural abnormalities have been described in these patients as secondary consequence of the chronic treatment more than disease itself. Objective of the Study, Patients and Methods: Twenty-one moderate and mild PKU female patients (age 12^33 years) on diet, with normal IQ (WISC-R and WAIS-R scale), followed at San Paolo Hospital, Dept of Paediatrics, University of Milan, Italy, were tested for psychosocial distress related to their disease and treatment with two di¡erent approach, The Eating Disorder Inventory 2 or EDI 2 and a global judgement rating expressed by the paediatrician. Every patient underwent 11 di¡erent evaluation scales (total: 231 scales/21 patients). Metabolic control was evaluated and considered good if plasma Phe was 410 mg/dl. Results: 44% of 231 scales completed by PKU patients were out of normal range, mostly showing problems such as bulimia, social insecurity, interpersonal distrust. Psychosocial impairments were equally distributed within patients either in bad or good metabolic control. This result demonstrates that even patients with a good metabolic control and a good compliance to the diet and to the follow up, even referring good emotional state, may have social and emotional distress. Our results con¢rm that the life-long dietary treatment of PKU may add further reasons of psychosocial distress, as already shown for conditions requiring a chronic dietary control such as type-1 diabetes and coeliac disease.

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INEFFECTIVENESS OF TETRAHYDROBIOPTERIN IN PHENYLALANINE HYDROXYLASE DEFICIENCY Porta F, Mussa A, Spada M, Ponzone A Dept of Pediatrics, University of Turin, Torino, Italy Background/Objectives: In order to evaluate the e¡ect of tetrahydrobiopterin (BH4), the cofactor of phenylalanine hydroxylase (PAH), in reducing plasma phenylalanine (Phe) concentration in phenylketonuria (PKU), we elected to perform this quantitative, dynamic, and self-controlled study. Methods: Seven PKU patients, fully characterized and previously responsive to a cofactor challenge, were enrolled to receive a simple Phe (100 mg/kg) and two combined Phe (100 mg/kg) and BH4 (20 mg/ kg) oral loading tests. Cofactor was administered either before or after the amino acid. The concentrations of Phe, tyrosine (Tyr), and biopterin were measured during 24 h after loading. Results: Analysis of plasma Phe and Tyr variations showed no di¡erences among the three types of loading. Plasma Phe peak levels were reduced 24 h after Phe load by 50% in benign, 20% in mild, and 15% in severe phenylalanine hydroxylase (PAH) de¢ciency, respectively, regardless of BH4 administration. A consistent or moderate increase of plasma Tyr, again independent of BH4 challenge, was seen only in the less severe forms. Mean blood biopterin concentration increased 6 times after simple Phe and 34^39 times after combined loading tests. Conclusions: The administration of BH4 does not alter Phe and Tyr metabolism in PKU patients. The clearance of plasma Phe after oral loading, as well as the Tyr production, is not related to cofactor challenge but to patient's phenotype. The assessment of BH4responsiveness by the methods so far employed is not reliable, and the occurrence of BH4-responsive forms of PKU is still unproven.

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BRAIN MRI DIFFUSION WEIGHTED IMAGING (DWI) IS USEFUL TO ASSESS WHITE MATTER ABNORMALITIES PROGRESSION IN CLASSICAL PKU PATIENTS Burlina AB1, Manara R2, Citton V2, Ermani M2, Carollo C2, Zanco C1, Burlina AP3 1 Div Metab Dis,Univ Hosp, Padova, Italy, 2Neuroradiological Unit,Univ Hosp, Padova, Italy, 3Neurology Unit, S Bassiano Hosp, Bassano, Italy Objective: Emotional and behavioural dysfunctions, neurological complications (upper limb distal tremor, pyramidal signs), brain MRI white matter abnormalities (WMAs) have been reported in early-treated PKU patients at di¡erent age after infancy. No clear clinical value has been assigned to WMAs in PKU. Furthermore, a de¢nitive relationship among WMAs, blood Phe and Tyr, and neurological complications has not yet been determined. In this study we used a quantitative score for WMAs to correlate WMAs, blood Phe and Tyr levels in PKU patients on diet. Methods: 21 early-treated PKU patients on diet (mean-age 21.2 years) and 26 controls (mean-age 25.1 years) were enrolled. T2 and DWI of WMAs were semiquantitatively measured according to Thompson Score (TS). Moreover, a regional MRI score (mTS) was developed, considering extension and intensity of WMAs. Phe and Tyr plasmatic concentrations prior MRI and their mean levels in the year before MRI (Pheyear, Tyryear) were measured. Results: No PKU patients younger than 14 years showed WMAs. In older patients, TS and mTS were signi¢cantly higher on DWI than on T2 images. DWI (mTS) showed a fronto-temporal, occipital and parietal WM progressive involvement. TS and mTS, on both T2-weighted images and DWI, showed no correlation with Tyr levels, a strong correlation with Phe levels and a good correlation with Pheyear levels. Conclusions: This study shows that DWI shows is the best sequence to detect and measure white matter abnormalities in PKU patients. The semiquantitative MRI score may help to monitor the involvement of white matter in di¡erent brain regions.

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ASSESSMENT OF TETRAHYDROBIOPTERIN (BH4) RESPONSIVENESS IN A POPULATION OF SOUTHERN ITALY SCREENED FOR PKU Concolino D1, Rapsomaniki M1, Pascale MG1, Moricca MT1, Bonapace G1, Strisciuglio P2 1 Dept Ped, Univ `Magna Graecia', Catanzaro, Italy, 2Present address: Dept Ped, Univ `Fed II', Naples, Italy Background/Objectives: BH4 loading test useful the management of neonates screened for hyperphenylalaninemia. We evaluated retrospectively in subjects with PKU, detected by neonatal screening program in Calabria, a region of Southern Italy, their responsiveness to BH4 by BH4 loading test (20 mg/kg of body weight). Patients and Methods: A total of 123 neonates with hyperphenylalaninemia were detected from 1991 to 2007 and a BH4 loading test (20 mg/kg) was performed in 34 patients with basal serum phenylalanine 4 of 10 mg/dl; blood phenylalanine (Phe) and tyrosine were determined at 0, 4, and 8 h. BH4 de¢ciency was con¢rmed by the measurement of urinary pterins and enzymatic activities. Results: Four of 34 patients had a more than 90% reduction in their plasma phe levels within 8 h after the load; 3 of them had a ¢nal diagnosis of DHPR de¢ciency and 1 of PTPS de¢ciency. Thirty newborns had a mild PKU (7 patients with blood Phe between 10 to 20 mg/dl) or classic PKU (23 patients with blood Phe 420 mg/dl). Four individuals (13.3%) responded with marked decrease in blood Phe (430%) at 8 h after the load (3 with mild PKU and 1 with classic PKU). Conclusion: Our data show: (1) a high prevalence of BH4 de¢ciency, at least for DHPR de¢ciency, in Southern Italy; (2) lower BH4 responsiveness in our population with PKU, probably due to the 8 h modus of challenge.

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WHOLE BODY COMPOSITION ANALYSIS BY THE BodPod AIR-DISPLACEMENT PLETHYSMOGRAPHY METHOD IN CHILDREN WITH PHENYLKETONURIA Albersen M1, Bonthuis M2, De Roos NM2, van den Hurk TAM3, Carbasisus-Weber EC3, Hendriks MMWD1, Koning TJ1, V|sser G1 1 Dept Metab Endocr Dis, UMCU, Utrecht, Netherlands, 2Div Hum Nutr, Univ Research Center, Wageningen, Netherlands, 3Dept Dietetics, Julius Centre, UMCU, Utrecht, Netherlands Background: Phenylketonuria (PKU), an inborn error of phenylalanine (PHE) metabolism, causes irreversible central nervous system damage unless a PHE restricted diet is maintained. This diet consists of restriction of natural protein and supplementary PHE free amino acids. To prevent growth retardation, a protein/amino acid intake beyond the recommended dietary protein allowance is mandatory. However, data regarding disease and/or diet related changes in body composition are still inconclusive and often retarded growth and or adipositas is reported. The relatively new BodPod whole body air-displacement plethysmography method is a quick, safe and accurate technique to measure body composition, especially in children above 6 years. Aim: to gain insight in the body composition of children with PKU treated with diet from birth. Methods: 24 patients treated in our centre born between 1991 and 2001, were included. Four patients could not be measured due to anxiety or hyperactivity. Patients were matched for gender and age with healthy control subjects (40). Body composition was measured with BodPod (Life Measurement Incorporation)). Independent samples t-tests were used to analyze the data. Results: The mean body fat percentage was signi¢cantly higher in PKU patients compared to healthy controls (25.20 vs 18.42; p = 0.002). When taking apart gender and age, this di¡erence remained statistically signi¢cant in girls older than eleven years (30.10 vs 21.48; p = 0.027). Conclusions: These results indicate a tendency towards an increased body fat percentage in older girls with PKU compared to healthy controls. Further research is needed to determine the exact aetiology of this phenomenon.

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L-CARNITINE DEFICIENCY CONTRIBUTES TO OXIDATIVE STRESS IN TREATED PHENYLKETONURIC PATIENTS Sitta A1, Barschak AG1, Deon M1, Barden AT2, Vanzin C2, de Mari JF2, de Souza CF2, Schwartz IV2, Netto C2, Giugliani R2, Wajner M1, Vargas CR3 1 PPG Cieªncias Bioloègicas Bioqu|èmica UFRGS, Porto Alegre, Brazil, 2 Servic°o de Geneètica Meèdica, HCPA, Porto Alegre, Brazil, 3Depto Anaèlises, Fac Farmaècia, UFRGS, Porto Alegre, Brazil Background: L-Carnitine (LC) plays an important role in the mitochondrial transport of fatty acids, but is also a scavenger of free radicals, protecting cells from peroxidative stress. Phenylketonuria (PKU), an inborn error of phenylalanine (Phe) metabolism, is currently treated with a special diet consisting of restriction of protein enriched foods, therefore potentially presenting reduction of LC levels. Objectives: Determine LC levels and oxidative stress parameters in blood of two groups of PKU patients, one with adherence to treatment and the other with high phenylalanine blood levels. Methods: Treatment consisted in a low protein diet supplemented with a synthetic amino acids formula that does not contain LC. LC and the oxidative stress parameters thiobarbituric acid reactive species (TBARS) and total antioxidant reactivity (TAR) were measured in blood of two groups of treated PKU patients and controls. Results: We veri¢ed a signi¢cant decrease of serum LC in patients who strictly adhered to the diet (Phe 340.6+79.7 mmol/L), as compared to controls and patients who did not comply with the diet (Phe 1055.6+233.7 mmol/L). TBARS measurement was signi¢cantly increased and TAR was signi¢cantly reduced in both groups of patients relatively to controls. We also found a signi¢cant negative correlation between TBARS and LC levels and a signi¢cant positive correlation between TAR and LC levels in welltreated PKU patients. Conclusions: Our results suggest that L-carnitine de¢ciency is involved al least partially in oxidative stress in PKU and that supplementation with Lcarnitine should be considered as an adjuvant therapy for PKU patients. F|nancial support: FIPE/HCPA-CNPq-FAPERGS-CAPES-PROPESQ/ UFRGS

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EARLY TREATMENT PREVENTS OXIDATIVE DAMAGE IN PHENYLKETONURIC PATIENTS Sitta A1, Barschak AG1, Deon M1, de Mari JF2, Barden AT2, Biancini GB2, Vargas PR2, de Souza CF2, Schwartz IV2, Netto C2, Giugliani R2, Wajner M2, Vargas CR 1 Servic°o de Geneètica Meèdica, HCPA, Porto Alegre, Brazil, 2Depto Anaèlises, Fac Farmaècia, UFRGS, Porto Alegre, Brazil Background: Phenylketonuria (PKU) is the most frequent disturbance of amino acid metabolism. Treatment of PKU patients consists of phenylalanine intake restriction. Neonatal screening programmes for phenylketonuria enable early treatment, preventing therefore mental retardation. However, there are still a number of patients who do not adhere to treatment or are not submitted to neonatal screening. These individuals are more prone to brain damage due to long-lasting toxic e¡ects of high levels of phenylalanine or/and its metabolites. In previous studies we veri¢ed that oxidative stress occurs in late-diagnosed PKU patients, probably contributing to the neurological damage in this disorder. Objectives: We aimed to compare oxidative stress parameters in blood of PKU patients with early and late diagnosis. Methods: We evaluated a large spectrum of oxidative stress parameters in plasma and erythrocytes of treated PKU patients with early and late diagnosis and of age-matched healthy controls. Results: The antioxidant defenses glutathione peroxidase activity, total antioxidant reactivity and glutathione levels were signi¢cantly reduced in both groups of PKU patients when compared to the control group. Furthermore, protein oxidative damage, measured by carbonyl formation and sulphydryl oxidation, and lipid peroxidation, determined by malondialdehyde levels, were signi¢cantly increased only in patients diagnosed late, compared to early diagnosed patients and controls. Conclusions: Early dietary treatment prevents the oxidative damage in proteins and lipids probably caused by an increased production of free radicals in PKU patients.

F|nancial support: FIPE/HCPA-CNPq-FAPERGS-CAPESPROPESQ/UFRGS

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TETRAHYDROBIOPTERIN(BH4)-RESPONSIVENESS AND LONG-TERM TREATMENT WITH BH4 IN HYPERPHENYLALANINEMIA Scala I1, Ungaro C1, Paladino S1, Nastasi A2, Zuppaldi A1, Sibilio M1, F|gliuolo C1, Scarpato E1, Capaldo B1, Cardillo G3, Daniele A3, Della Casa R1, Parenti G1, Andria A1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2Physiol Nutrition Unit, Dept Neuroscience, Naples, Italy, 3Ceinge-Adv- Biotechnol, Univ Federico II, Naples, Italy Background: Hyperphenylalaninemia (HPA), due to phenylalanine hydroxylase (PAH) de¢ciency, can be classi¢ed as classic phenylketonuria (cPKU), mild PKU (mPKU) and mild HPA (mHPA) on the basis of tolerance and pre-treatment phenylalanine (Phe) levels. Recent reports show the e¡ectiveness of tetrahydrobiopterin (BH4) in PAH de¢ciency. Patients and methods: BH4-responsiveness was tested in 17 HPA patients (6 cPKU, 5 mPKU and 6 mHPA) by BH4 loading test. Two weeks before and during the testing period Phe intake was distributed throughout the day. BH4 loading test was performed with BH4 tablets (Schircks Laboratories, Switzerland) in two doses of 20 mg/kg at T0 and T24 h. Plasma Phe was analysed at 0, 4, 8, 12, 24, 32 and 48 h. Results: Ten patients (2 cPKU, 2 mPKU and 6 mHPA) were BH4-responsive with a decrease greater than 30% in plasma Phe from T0; 40% of patients responded to BH4 after T24. All BH4-responsive patients were compound heterozygous with at least one partially active allele (p.R261Q, p.L48S, p. P281L, p.R158Q, p.D338Y). Long-term BH4 treatment (10 mg/kg/day) was started in BH4-responsive patients with weekly increases of 200 mg Phe/day. The follow-up period ranged from 2 to 11 months. Phe tolerance increased from 2 to 9.7-fold; one patient achieved a liberalized diet. One patient reported headache, two reported epigastric abdominal pain, one developed appendicitis. Conclusion: The most important predictor of BH4-responiveness is the genotype. Long-term BH4 treatment is an e¡ective therapy in selected patients and it can partially or completely replace a restrictedphenylalanine diet. The study is supported by Agenzia Italiana del Farmaco (Roma, Italy)

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SOCIAL ADAPTATION INDICATORS OF PKU PATIENTS USING READY TO USE FORM OF PROTEIN SUBSTITUTE Novikov PV1, Latypov AS2, Va¢na ZI2, Denisenkova EV3, Kuznetsova LI3, Listopad GG4, Matulevich SA5, Golihina TA5, Nikitina NV6, Nikolaeva EB6 1 Inst Ped and Child Surgery, Moscow, Russian Federation, 2Genetic Centre of Republic Tartarstan, Kazan, Russian Federation, 3Medical Genetic Centre, Moscow, Russian Federation, 4Genetic Centre, Volgograd, Russian Federation, 5Genetic Centre, Krasnodar, Russian Federation, 6Genetic Centre, Ekaterinburg, Russian Federation Background: Classic phenylketonuria (PKU) is a hereditary disease which is caused by phenylalanine hydroxilase gene mutations. Until now PKU treatment has been a restrictive diet when about 80% of protein intake comes with specialized medical substitutes. New substitutes development purpose of the ready to use drinks is to make it possible for the patients to have an active way of life and to get rid of psychological problems. Objective: We estimated the role of separate factors to social adapting the PKU children and persons caring about them due to using the new liquid form of protein substitute. Methods: 49 families with classic PKU children from 8 to 20 years (the average age was 11.7) after starting to use the new ready to use protein substitute (`EASIPHEN', SHS-Int.,UK) were examined. We used the originally designed questionnaire, including 19 questions (8 covering identi¢cations, treatment, education and household environment of a child and 11 some aspects a child's and caregiver's social adaption during 1 week of Easiphen using). Results: Analysis included 71 questionnaires. 43 (88%) children were at secondary school, 2 (4%) at higher education institutions, 2 (4%) at colleges, 2 (4%) on-the-house teaching. The estimation of the general health state and life quality with EASIPHEN intake: `very well' ^ 4 (13%), `well' ^ 17 (55%), `satisfactory' ^ 7 (22.5%), `bad' ^ 0 (0%); without EASIPHEN ^ 3 (8.3%), 14 (38.8%), 17 (47%) and 3 (8.3%) accordingly. Conclusion: The new £uid phenylalanine-free protein substitute doesn't e¡ect negatively upon the general health and improves the quality of life of PKU patients and their families.

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Background: The value of genotyping to identify tetrahydrobiopterinresponsive (BH4-responsive) patients with phenylalanine hydroxylase (PAH) de¢ciency is under debate. Methods: We reviewed 250 patient cases with PAH de¢ciency (198 published cases and 52 cases from our clinic) in which patients underwent BH4-loading tests and mutational analyses. Partial and full BH4 responses were de¢ned as a 10^29% and a 30% decrease in blood phenylalanine levels from baseline, respectively. BH4-responsive alleles were identi¢ed from BH4-responsive patients as either homozygous for a speci¢c allele or compound heterozygous for that allele with a null mutation. Results: Most inconsistencies between observed genotype and BH4 response were associated with mutations in the regulatory domain of PAH (R68S, I65T, R48S and F39C), where 20 of 62 alleles (32.2%) were non-responsive. In the catalytic domain, only 8 of 125 alleles (6.4%) were non-responsive (Y414C, R261Q, E390G, A300S, R241C, A403V and V388M). Eight patients had two alleles that met the criteria for BH4 responsiveness and did not or partially respond. 10 patients (including three from our own clinic) had identical genotypes but inconsistent responses on BH4 loading. Conclusions: These results show that BH4 non-responsiveness is associated with genotype however, especially patients with mutations in the regulatory domain show inconsistent results. In patients with two responsive alleles non responsiveness may be due to negative inter allelic complementation. In patients with the same genotype and inconsistent results to a BH4 load other genetic factors may be responsible. Further in vitro studies are necessary to elucidate genotype-phenotype correlation in BH4 responsive PKU.

Objective: The impact of BH4 on Phe tolerance, long-term dietary patterns, formula/medical food (MF) continuation and nutritional status was investigated. Methods: Six well-controlled PKU children who responded to a dose of 20 mg/kg/day of BH4 with 530% decrease in plasma Phe concentrations after 8 days (p = 0.014) were enrolled in a 12-month study to evaluate the impact of BH4 on Phe tolerance and nutritional status. Maximum dietary Phe tolerance was determined by progressively increasing milk or egg powder over six-weeks while maintaining plasma Phe concentrations between 120 and 360 mmol/L. MF was decreased by 25% each week provided that plasma Phe concentrations and nutrition status markers remained within therapeutic range and the average protein intake met US Dietary Reference Intakes (DRIs). Results: Six weeks: Dietary Phe tolerance increased to a mean+SD of 1380+395 mg/d (baseline 575 mg/d+215) (p = 0.001). Twelve months: Mean plasma Phe concentrations persisted within 120-360 mmol/L throughout the 12-month follow-up period while the mean dietary Phe tolerance was 1545+348 mg/d. Three of the six patients were able to eliminate MF, while the remaining three took MF below baseline. Average protein prescription continued to exceed DRIs. V|tamin and mineral supplementation was required for those who discontinued MF. There was no signi¢cant change in mean energy intake; weight percentiles; but concentrations of prealbumin, hemoglobin, hematocrit and cholesterol improved signi¢cantly (p50.01). Conclusions: Results indicate the need to systematically reduce MF, maintain nutrient adequacy, personalize diet recommendations and provide V|tamin and mineral supplements particularly if medical food has been discontinued. Education for new food choices will also be necessary.

SIGNIFICANCE OF GENOTYPE IN TETRAHYDROBIOPTERIN (BH4)-RESPONSIVE PHENYLKETONURIA (PKU) Trefz FK1, Scheible D1, Go«tz H2, Frauendienst-Egger G1 1 Klinik fu«r Kinder und Jugendmedizin, Kr, Reutlingen, Germany, 2TDB Software GmbH, Schwabach, Germany

LONG TERM NUTRITION AND DIETARY IMPACT OF TETRAHYDROBIOPTERIN THERAPY IN PHENYLKETONURIA Singh RHS, Douglas TD Emory University Dept of Human Genetics, Decatur, USA

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NEW INSIGHTS INTO PHENYLALANINE HYDROXYLASE KINETICS AND THE MUTUAL IMPACT OF SUBSTRATE AND COFACTOR CONCENTRATIONS Staudigl M1, Gersting SW1, Kemter KF1, Messing DD1, Danecka MK1, Muntau AC1 1 Molec Pediatr, Hauner Child Hosp, LMU, Munich, Germany Background: Activity of phenylalanine hydroxylase (PAH) is known to be regulated by its substrate, its cofactor 6R-(L-erythro)-5,6,7,8tetrahydrobiopterin (BH4) and by phosphorylation. Mutations in the PAH gene often result in altered enzyme kinetic parameters. Yet little is understood about the mutual impact of various substrate and cofactor concentrations on PAH kinetics. In light of high variations of both Lphenylalanine and BH4 concentrations among healthy individuals, untreated or treated patients with BH4-responsive PAH de¢ciency, this is a key element in need of further evaluation. Results: Here we present a new automated 96-well £uorescence-based assay allowing for new insights into PAH kinetics. This method enables real-time enzyme activity measurements at a wide range of substrate and cofactor concentrations in one single analysis run. For wild-type PAH, we revealed a shift in peak catalytic activity towards higher BH4 concentrations than previously described. In addition to known substrate inhibition, a cofactor inhibition was found. Some of the variant proteins analyzed show a severe shift in the optimal range of activity based upon varying L-phenylalanine and BH4 concentrations, a ¢nding missed when using the standard enzyme kinetic assay. Conclusion: Taken together, we provide new insights in optimal PAH activity-range. The new 96-well enzyme kinetic assay allows for a better understanding of PAH function, showing the mutual impact of substrate and cofactor concentrations on enzyme activity. Furthermore, these results may have implications for the treatment of PAH de¢cient patients responsive to BH4 and could play an important role towards establishing individual tailored medicine.

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FOUR APPARENTLY UNRELATED HAPLOTYPE GROUPS IN THE PHENYLALANINE HYDROXYLASE GENE Kalb S, Luf S, Janssen B, Zschocke J Inst Hum Gen, Univ Hosp, Heidelberg, Germany Phenylketonuria is caused by mutations in the phenylalanine hydroxylase (PAH) gene which contains a variety of polymorphic markers. In order to establish an expanded haplotype system for population genetic studies we selected 3 diallelic RFLPs, an STR and VNTR markers, and combined them with variants in or adjacent to exons that were not in the original haplotype system. We determined modi¢ed haplotypes in 272 independent families with PAH de¢ciency from Germany and found a small number of distinct haplotype groups linked to speci¢c mutations. For a more detailed analysis we subsequently developed a method for molecular haplotyping of all 22 common SNPs in the 3' half of the PAH gene (introns 5^12) based on allele-speci¢c long-range PCR. So far, 34 individuals with PAH de¢ciency living in Germany and 20 non-a¡ected parents were investigated. 86 independent chromosomes associated with wild type (19 chromosomes) or PKU (21 mutations re£ecting at least 23 di¡erent mutation events) revealed four distinct haplotype constellations that di¡ered from each other in 8 to 16 markers. Our combined data indicate that there are four major haplotype groups in the PAH gene that are di¤cult to relate to each other. The haplotype groups are found in association with various di¡erent mutations in chromosomes of di¡ering ethnic origin. The exclusive association of ancient PKU mutations with speci¢c haplotypes and the apparent rarity of recombination over 100 kb of the PAH gene indicate a haplotype block of remarkable stability throughout the history of Europe.

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LONG-TERM FOLLOW-UP AND OUTCOME OF PATIENTS WITH TETRAHYDROBIOPTERIN DEFICIENCY Ja«ggi L1, Zur£u«h MR1, Schuler A2, Ponzone A3, Porta F3, Salvatici E4, Giovannini M4, Santer R5, Ho¡mann GF6, Ibel H7, Wendel U8, Ballhausen D9, Baumgartner MR1, Blau N1 1 Univ Child Hosp Zu«rich, Zu«rich, Switzerland, 2Buda Child Hosp, Budapest, Hungary, 3Pediatrics, Univ of Torino, Turin, Italy, 4Pediatrics, Univ of Milan, Milan, Italy, 5Pediatrics, Univ Medical Ctr, Hamburg, Germany, 6Pediatrics, Univ of Heidelberg, Heidelberg, Germany, 7 Werneck, Werneck, Germany, 8Pediatrics, Univ of Du«sseldorf, Du«sseldorf, Germany, 9Clinique Infantile, CHUV, Lausanne, Switzerland Background/Objectives: To study the treatment, clinical, and biochemical ¢ndings and the outcome of patients with 6-pyruvoyl-tetrahydropterin synthase (PTPS) de¢ciency (n = 26) and dihydropteridine reductase (DHPR) de¢ciency (n = 10). These are the two most common forms of the autosomal-recessively inherited tetrahydrobiopterin (BH4) de¢ciency. Methods: T|me of diagnosis, dosage of BH4 and neurotransmitter precursors, folinic acid substitution, and levels of 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in cerebrospinal £uid (CSF) are used as parameters in the follow-up of patients. These data as well as the clinical information were collected from the BIODEF database (www.bh4. org). Results: 17 patients with PTPS de¢ciency and 4 patients with DHPR de¢ciency were diagnosed within 2 months after birth. In 14 patients with PTPS de¢ciency (54%; 9 early and 5 late diagnosed) and 2 patients with DHPR de¢ciency (20%; all early diagnosed) no developmental delay is observed, while in 10 patients with PTPS de¢ciency (38%; 6 early and 4 late diagnosed) and 8 patients with DHPR de¢ciency (80%; 2 early and 6 late diagnosed) development was delayed. Two PTPS-de¢cient patients died in the newborn period. DHPR de¢ciency seems to be more severe than PTPS de¢ciency and it is clearly the onset of treatment that determines the outcome. Conclusions: Our data suggest that diagnosis within the ¢rst month of life is essential for a good outcome and that low CSF 5HIAA and HVA values in CSF could be an indicator for the ongoing developmental impairment, even in the absence of neurological symptoms.

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MANAGEMENT OF PHENYLKETONURIA (PKU) IN EUROPE: A REVIEW FROM 8 COUNTRIES Blau N1, Beèlanger-Quintana A2, Demirkol M3, Feillet F4, Giovannini M5, MacDonald A6, Trefz FK7, van Spronsen FJ8 1 Univ Children's Hospital Zu«rich, Zu«rich, Switzerland 2Unidad Enfermendades Metabolices, Madrid, Spain, 3Pediatrics, Istanbul Univ, Istanbul, Turkey, 4Hoªpital d'Enfants, CHU Brabois, Nancy, France, 5 Pediatrics, Univ of Milan, Milan, Italy, 6The Children's Hospital, Birmingham, UK, 7Klinik fu«r Kinder und Jugendmedizin, Reutlingen, Germany, 8Pediatrics, Univ of Groningen, Groningen, Netherlands Objectives and Methods: Current practice for PKU diagnosis and treatment was surveyed in 8 European countries. Written questionnaires were sent to PKU centres, and information was collected regarding newborn screening, the use of tetrahydrobiopterin (BH4) loading tests, and treatment. Results: The number of PKU centres included was: France (22), Germany (29), Italy (14), Spain (16), Switzerland (5), The Netherlands (8), Turkey (9), and UK (16). Newborn screening is performed in all European countries; however no standards exist for the use of the BH4 loading test. It is not performed routinely in the UK or The Netherlands, in France it is not performed at all centres and in Turkey it is performed only in Istanbul. It is well established in Germany, Italy, Spain, and Switzerland. Phe levels for both treatment initiation and therapeutic target di¡er. Treatment consists of a Phe-restricted diet with or without BH4 in all countries, with the exception of UK, The Netherlands, and Turkey, where BH4 in PKU is not routinely used. Although Phe levels have been established as a surrogate for treatment response, the clinical outcome for patients with PKU is still variable. Conclusions: This survey highlights the variability in diagnostic and treatment practices for PKU across Europe. A broader understanding of the clinical outcomes for patients with PKU and optimal clinical management is needed in order to inform on future European recommendations, through consensus conferences and the analysis of pooled long term data in registries.

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PHENYLKETONURIA IN AN ERA OF NEONATAL SCREENING Hoeks MPA1, den Heijer M2, Janssen MCH1 1 Dept Gen Int Med, Radboud University, Nijmegen, Netherlands, 2Dept of Endocrinology, Radboud University, Nijmegen, Netherlands Background: Phenylketonuria (PKU) is a classical example of an inherited metabolic disease, in which mental retardation can be prevented successfully by using a diet. However, in adult PKU new problems occur as a result of the restricted diet or the disease itself, like vitamin de¢ciencies, osteoporosis and the maternal PKU syndrome (MPKUS). Our aim was to describe dietary complications and the occurrence and amount of MPKUS. Methods: We retrospectively studied 47 PKU and 9 hyperphenylalaninaemia (HPA) patients. Results: 69% of the PKU patients had good to intermediate blood phenylalanine (Phe) levels (5900) as well as 100% of the HPA patients. 13.3% of the PKU patients and 22% of the HPA patients had low serum vitamin B12 concentrations (5160 pmol/L). Mean T-score of the bone mineral density di¡ered ^1.10SD from the normal population (95%CI ^1.4 to ^0.7). No associations had been found between bone mineral density and calcium intake, blood Phe levels or age. One of the 12 children born from PKU or HPA mothers had intrauterine growth retardation. No other complications had been seen. Compared with 60 matched controls we found a decreased mean birth weight (^410 g, 95% CI ^746.9 to ^72.2) in our series. Conclusion: In The Netherlands relatively good metabolic control of PKU is achieved, on the other hand signi¢cant vitamin B12 de¢ciencies and osteoporosis occur. Dutch PKU and HPA o¡spring appear to have lower birth weights as well.

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PKU IN EUROPE ^ WHAT IS DAILY PRACTISE? Ahring KK1, Gisewska M2, von Spronsen F3 1 Kennedy Centre, Glostrup, Denmark, 2Pomeranien Hospital, Szczecin, Poland, 3Beatrix Childrens Hospital, Groningen, Netherlands Background: Since the start of the European Society of PKU (ESPKU) 1987, parental organizations of 27 member countries joined in order to share knowledge. There are huge di¡erences in treatment within Europe. A survey among professionals was done to determine goals and practice. However, not all countries were really active at ESPKU level. Method: A questionnaire was sent out to professionals of all member countries, addressing the following issues: diagnostic and treatment procedures, numbers necessary for a PKU centre, guidelines followed, numbers of patients treated and professionals involved in care, target phenylalanine concentrations, availability of products, amount of protein prescribed, frequency of monitoring and clinical visits, need for follow up of various clinical/biochemical data, the importance of various abnormalities and de¢nition of poor compliance. Results: At that time 11 countries (14 centres) answered, and 14 countries could not be reached or did not respond. Di¡erences in care do not only apply to the target phenylalanine concentration. Only at few points there is general consensus. Conclusion: PKU treatment varies a lot between the European countries. Knowing these di¡erences and the lack of clearness about goals (apart from phenylalanine concentrations), the ESPKU started to organize meetings for professionals besides the already existing program for parents/patients. Discussion with all kind of professionals taking care of PKU patients may lead to a European guideline, which de¢nitely needs to address more than target plasma phenylalanine concentrations.

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EXPERIENCE WITH LARGE NEUTRAL AMINO ACIDS (LNAA) IN THE TREATMENT OF PHENYLKETONURIA (PKU) Burlina AB1, Burlina AP1, Michals-Matalon K2, Coldwell J3, Hillman R4, Bhatia G5, Galvin-Parton P6, Grady J5, Tyring SK7, Matalon R5 1 University of Padua, Padova, Italy, 2University of Houston, Houston, USA, 3Universiy of Oklahoma, Tulsa, USA, 4Univesity of Missouri, Colombia, USA, 5University of Texas Medical Branch, Galveston, USA, 6 Stony Brook Medical Center, New York, USA, 7University of Texas Health Sci Center, Houston, USA Background: Treatment for PKU requires diet for life. The restrictive nature of PKU formulas, make compliance di¤cult. The addition of tetrahydrobiopterin (BH4)to the treatment of PKU o¡ers new treatment modality, but the majority of patients still require diet therapy. The discovery that LNAA compete with the absorption of Phe, is another method for treating PKU. The purpose of this report is to present data from independent invetigators using LNAA in PKU. Methods: Twenty two patients, ages 11-54 years were enrolled. All patients had classical PKU, some tried BH4 and failed to respond. Baseline blood Phe, tyrosine, and Phe intake were determined. Patients were given 0.5 g/kg (1 pill) LNAA daily divided with meals. Behaviour, cognitive skills, learning and hyperactvity were reported by parents, teachers and professional sta¡. Results: All patients showed decline of blood Phe when taking LNAA. The range of decline varied, depending on compliance. It was possible to determine compliance based on blood Phe and tyrosine concentrations. Three subjects who did not comply had high blood Phe and sharp increase in tyrosine indicating they had just taken the LNAA before the clinic visit. The compliant patients had statistically signi¢cant drop in blood Phe, averaging 40^50% (p50.0003). School performance, cognitive function, social interaction, and hyperactive behaviour improved while on LNAA. Conclusion: The decline of blood Phe concentration is reproducible with LNAA, when taken in the proper dose, and can be included in the management of all PKU patients. Hyperactive behaviour improved also with LNAA treatment.

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LONG TERM TETRAHYDROBIOPTERIN (BH4) TREATMENT OF PHENYLALANINE HYDROXYLASE (PAH) DEFICIENT PATIENTS Leuzzi V, De Leo S, Carducci Cl, Carducci Ca, Artiola C, Santagata S, Antonozzi I Sapienza University of Rome, Roma, Italy Aim: of the study was to assess the long term e¤cacy of chronic BH4 treatment in patients with PAH de¢ciency. Methods and patients: Ten BH4 responsive PKU patients ^ 9 MPKU, 1 CPKU; 7 M/3 F, aged 5^19 years ^ were enrolled for the study. BH4 treatment was started at the age 1.4^15 years and carried on for 3^4 years. BH4 dosage ranged 5^16 mg/kg/bw/day. To assess the e¡ect of BH4, the following aspects were considered: clinical status, Phe tolerance, blood Phe, Tyr and other amino acids, Phe/Tyr ratio. Results: No clinical variations or side e¡ects were detected with BH4 treatment. Dietary Phe was progressively increased and became compatible with a free diet in 8 out of 10 cases. The median values of blood Phe was 281 to 541 mM and 275 to 757 mM, respectively under Phe restricted diet (as unique treatment) and BH4. However, contrarily to what expected, blood Tyr concentration signi¢cantly declined (96.5 +10.7 vs 86.4+10.3, respectively before and after BH4, p50.03) ^ even if it remained in the normal range and Phe/Tyr ratio increased (4.4+1.3 vs 6.2+2.5, respectively, p50.05). The e¡ect was con¢rmed once the variations of other amino acids were controlled. It was independent from: BH4 dosage, patients' genotype, biochemical phenotype, dietary Phe. Conclusions: BH4 supplementation remains an e¡ective treatment for BH4 responsive PKU subjects. The slight decline of Tyr requires to be further con¢rmed. An increase of Tyr uptake by the cells or the activation of other BH4-dependent hydroxylases can be suggested.

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LONG-TERM CORRECTION OF HYPERPHENYLALANINEMIA IN A MOUSE MODEL FOR PKU BY LIVER OR INTRAMUSCULAR DELIVERY OF AAV EXPRESSING PAH WITH VARIOUS SEROTYPES Rebu¡at A1, Harding CO2, Ding Z3, Thony B1 1 Dept Pediatr, Univ Zurich, Zurich, Switzerland, 2Dept Pediatr, Oregon Health Sci Univ, Portland, OR, USA, 3Inst Bioengineering Nanotechnol, Singapore, Singapore Phenylketonuria (PKU) is a frequent inherited disorder of amino acid metabolism caused de¢ciency of hepatic phenylalanine hydroxylase (PAH) resulting in accumulation of Phe and its metabolites in blood and other tissues. Treatment consists of Phe intake restriction, which prevents severe neurological damage in PKU patients, although mild neuropsychological ¢ndings, such as poor school performance, a slight reduction in intelligence quotient, and the presence of tremor may arise, especially when careful dietary compliance is not achieved. Therefore, investigations of alternative or complementary therapeutic approaches are highly encouraged, including gene therapy. We have recently demonstrated life-long therapeutic correction of PKU in a PKU mouse model by using a recombinant AAV2 pseudotype-8-mediated transfer of the murine PAH gene to liver after portal vein or tail vein administration. E¡ective long-term correction for PKU was successfully obtained also following intramuscular delivery of recombinant triple-cistronic AAV2 serotype 1 expressing ectopically in m u s c l e t i s s u e PA H a l o n g w i t h t wo e s s e n t i a l g e n e s fo r tetrahydrobiopterin biosynthesis. Subsequently, we showed in the same mouse model that long-term clearance of blood Phe and therapeutic treatment of the disease including complete phenotypic reversion can also be achieved by targeting the liver upon a single injection into each of the gastrocnemius muscles of the hind legs using AAV2 expressing the murine PAH gene pseudotyped with either capsid 1, 2 or 8. This non-invasive approach completes our previous studies and allows us to compare di¡erent but complementary strategies for the development of an e¤cient and safe gene therapy for PKU.

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BIRTH OF NEW EXONS IN LINE-2 AND IN ANTISENSE AluSq BY INTRONIC MUTATIONS IN THE PTS GENE LEADING TO TETRAHYDROBIOPTERIN (BH4)-COFACTOR DEFICIENCY Meili D1, Kralovicova J2, Bonafe L3, F|ori L4, Blau N1, Vorechovsky I2, Thony B1 1 Univ Southampton, School Med, Southampton, UK, 2Div Pediatr Molec CHUV, Lausanne, Switzerland, 3Dept Pediatr, Univ Milan, Milan, Italy Tetrahydrobiopterin (BH4)-cofactor de¢ciency leads to an autosomal recessive inherited form of hyperphenylalaninemia in mild cases, but is mostly accompanied by a de¢ciency of the neurotransmitters dopamine and serotonin, termed severe cases. Thus far, over 250 patients with di¡erent recessive mutations in the BH4-biosynthetic gene PTS were described with various degrees of phenotypic severity. Here we report on two unrelated patients with BH4 de¢ciency due to inherited intronic mutations causing aberrant splicing of the PTS pre-mRNA and recruitment of pseudoexons. Cryptic exons or pseudoexons usually arise from mutations that create GT or AG dinucleotides of new 5' or 3' splice sites in introns, often in repetitive elements such as Alus. The PTS gene contains in intron 1 a LINE-2 sequence and in intron 2 an antisense Alu sequence. In one case an optimized branch point sequence was brought to proximity of the pseudoexon 3' splice site by a genomic deletion of 57 bp in intron 2, removing the poly(T)-tail of antisense AluSq and an upstream AG that repressed 3' splice site utilization on normal chromosomes. New Alu exons can thus arise in the absence of poly(T)-tails that served as polypyrimidine tracts for most exonized transposed elements, illustrating extraordinary £exibility of Alus in exaptation. In the other case, pseudoexon was activated by an A4T substitution in intron 1 9 bp upstream of the pseudoexon 3' splice site in the LINE-2 sequence, providing the ¢rst example of a disease-causing exonization of this ancient interspersed repeat.

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PHE PLASMA CONCENTRATION AND COGNITIVE PERFORMANCE: PRELIMINARY OUTCOMES Ferreira A1, Oliveira B1, Gradinayna J1, Guerra A2, Oliveira A1 1 Serv Intern Med, Santa Maria Hosp, Lisbon, Portugal, 2Serv Dieteètica e Nutric°a¬o, Hosp St Maria, Lisbon, Portugal Several psychological studies have shown that even phenylketonuric patients treated early and continuously su¡er from phenylalaninerelated de¢cits in all age periods. The knowledge of the cognitive development in adults who grew up with phenylketonuria (PKU) it's still unknown. This study was designed to determine the impact of the level of plasma phenylalanine concentration in ¢ve di¡erent cognitive tasks, in young adults with PKU. Factorial Primary Mental Aptitude Test (PMA) was used to evaluate the cognitive function of the patients. A total of 13 adult patients with PKU were evaluated according to gender (male and female) and level of Phe (Phe under 6 mg/dl; Phe over 10 mg/dl). At the time of the assessment, there were four women and nine men; the mean age was 29 years; six had a level of Phe under 6 mg/ dl and seven had a level of Phe over 10 mg/dl. Word £uency subtest was found to have the highest score independently of gender or Phe level. In the Reasoning subtest the lowest scores were found in the group of higher level of Phe, independently of gender. In the group that had the lowest level of phe, di¡erences were noticed between genders. The women subgroup scored the lowest in spatial visualization and the men subgroup in the reasoning subtest. Phe levels were inversely related to cognitive £exibility and performance. In spite of the small number of patient with PKU, the results suggest that the lowest level of plasma Phe concentration, the better results on cognitive performance.

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HYPOTYROSINEMIA, A MAJOR POTENTIAL ADVERSE EVENT IN THE TREATMENT OF CLASSICAL PHENYLKETONURIA (PKU) USING HIGH DOSES OF TETRAHYDROBIOPTERINE (BH4) Franc°ois BJM Pinocchio Centre, CHC, LIEGE, Belgium BH4 is the cofactor of the 3 aromatic amino acids hydroxylases: phenylalanine- (PAH) tyrosine- (TH) and tryptophane- (TrpH) hydroxylases. Recent data drawn the attention of a possible BH4 sensitive form of hyperphenylalaninemia (HPA) due to the PAH de¢ciency, as found in classical PKU. We could expect that BH4 during treatment for PKU will increase the metabolic £ux through the 2 other enzymes pathways as well. In order to investigate potential interactions between BH4 and TH activity during BH4 treatment for HPA, we examine the Phe and Tyrosine (Tyr) blood values before and during treatment in 4 individuals (aged 9 to 15 years), de¢ned as responsive by a BH4 loading test (20 mg bw^1) showing a reduction of more than 30% in the Phe blood values; the mutations E390G ^ R291Q ^ P281L ^ A309V were found in the PAH gene of these individuals. Tyr blood levels are signi¢cantly (p50.001) lower (51+16 mmol/L) compared to the pretreatment values (83+27 mmol/L). The Phe/Tyr molar ratio in blood is signi¢cantly higher after treatment. All patients presented a perifollicular hyperkeratosis, after 6 months of treatment, suggesting a Tyr de¢ciency at the skin level. These data may suggest an increase £ux into the TH pathway. Further Tyr supplementation should be considered together with the BH4 treatment for HPA in PKU.

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HYPERORNITHINEMIA, HYPERAMMONEMIA AND HOMOCITRULLINURIA (HHH) SYNDROME: ADULT PRESENTATION Hernandez-Perez JM1, Garcia-V|lloria J1, Rodes M1, MartinezHernandez E2, Tessa A3, Santorelli F3, Ribes A1 1 Inborn Errors (IBC) Hospital Clinic, Barcelona, Spain, 2Neurology Unit, Hospital Sant Pau, Barcelona, Spain, 3Mol Med Lab, Bambino Gesu© Children's Hosp, Roma, Italy A 50-year-old woman was admitted to the hospital because of altered mental status and poor feeding during 2 days. She had been well until 2 days before admission. On examination by a neurologist, the patient had episodes of rasping breath, with incomprehensible sounds, ocular supraversion, she did not follow commands and progressed to decreased consciousness. Laboratory tests showed hyperammoniemia (306 mmol/L, CV550). For that reason proteins were withdrawed and she was kept in absolute diet. Further studies showed hyperornithinemia (370 mmol/L, CV 46^142) with generalised hypoaminoacidemia. Urinary aminoacids showed an increased homocitrulline (40 mmol/mol creat, CV undetectable) and orotic acid (5.49 mmol/mol creat, CV 0.46^ 5.09). Therefore the diagnosis of HHH was suspected and treatment was started accordingly, and clinical improvement was evident. Molecular studies showed that she was homozygous for the mutation p.A70L and the diagnosis of HHH was established. Before an episode of loss of conscience with hyperamoniemia in the adulthood, the study of amino acids is very advisable to reject a disease related to the urea cycle.

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RAPID DETECTION OF 3 COMMON MUTATIONS IN HUMAN OTC GENE ASSOCIATED WITH LATE-ONSET OTC DEFICIENCY IN MALE Yoshino M, Numata S, Ueki I, Watanabe Y, Fujii C, Kohda Y Kurume University, Kurume, Japan Background: Prognosis of late-onset OTC de¢ciency in male (LODEM) is excellent if early, adequate treatment is provided, otherwise it is poor. Three kinds of mutations in the OTC gene, g.119G4A (p.R40H); g.163T4G (p.Y55D) and g.829C4T (p.R277W), have been found in 15 of 17 LODEM patients, all inhabitants in a particular area with a radius of 120 km. Objective: To develop simple and timesaving screening method for these mutant alleles to prevent hyperammonemic crisis. Materials and Methods: DNA was obtained from male patients carrying each of the 3 mutant alleles and volunteers with informed consent. To detect these 3 mutations, we employed ampli¢cation refractory mutation system (ARMS) combined with real-time PCR, using Sybr Green I on LightCycler. Two sets of PCR to detect each of the 3 mutations were designed: one allows ampli¢cation exclusively on wildtype DNA (W-PCR) and the other, exclusively on each mutant DNA (M-PCR). Results: Each W-PCR could produce amplicon on wild-type DNA, but not on mutant DNA from patients, whereas each M-PCR could produce amplicon on each corresponding mutant DNA, but not on wild-type DNA. One patient, a 12 year-old boy, was determined to carry the p.R40H allele on this system. Conclusion: The present technique is useful in this particular area to detect males who carry these mutant alleles, and hence for prevention and early intervention, if they have developed, of hyperammonemic episode.

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THE NATURAL HISTORY OF ORNITHINE TRANSCARBAMYLASE DEFICIENCY LATE-ONSET FORM DUE TO PREVALENT IN POLAND A208T MUTATION Rokicki D1, Ciara E2, Popowska E2, Sykut-Cegielska J1 1 Dept Metab Dis, Endo, Diabeto, CMHI, Warsaw, Poland, 2Dept Genetics, CMHI, Warsaw, Poland Congenital hyperammonemia due to ornithine transcarbamylase de¢ciency (OTCD) is transmitted in an X-linked manner. The age of onset and course of the disease are variable in female patients due to skewed random X-chromosome inactivation, but male patients develop hyperammonemic crisis mainly in the neonatal period or in early infancy. According to the literature in some exceptional male patients a ¢rst episode of hyperammonemic crisis could be delayed as late as during adolescence or adulthood (late-onset OTCD). We present clinical description of 24 Polish cases of OTC de¢ciency due to A208T mutation. We have diagnosed 63 cases of OTCD diagnosed so far. Late onset was noticed in 73% of cases. In 24 of them A208T mutation was detected (13 males, 11 females in 3 separate families). Before establishing the diagnosis of OTCD in 8 cases di¡erent tentative diagnoses were suspected: encephalitis, intoxication, Reye syndrome, cerebral tumor, cholelithiasis, adrenal insu¤ciency, hepatitis, liver insu¤ciency. None of the females had clinical symptoms. Before diagnosis 11 males died suddenly in similar circumstances. Death occurred at mean age of 15.3 years. Clinical picture was dominated by disturbances of consciousness and behaviour, cerebellar signs and coma. Before fatal episode all males were apparently healthy (two of them were sportsmen). Despite pedigree analysis indicates X-linked pattern of inheritance, the proper diagnosis was delayed; in one family it took 28 years. Conclusions: (1) The OTCD late-onset type occurs at any age. (2) Assessment of ammonia concentrations in patients with unexplained neurological and psychiatric symptoms should be routine procedure.

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USING THE GaitRite2 WALKWAY SYSTEM TO ASSESS GAIT PATTERNS IN CHILDREN WITH ARGINASE DEFICIENCY Wood M, Mcsweeney M, Natalia P, Cleary M Great Ormond Street Hospital, London, UK Background: Arginase de¢ciency presents with spastic diplegia. Treatment with a low arginine diet and nitrogen scavenging drugs can halt progression and improve neurological function. Robust assessment tools are important to assess and monitor the response to treatment. The aim of this study is to investigate the value of the GaitRite system (electronic pressure sensitive walkway) in identifying changes in gait pattern. Method: F|ve children (from two families) with a diagnosis ofaArginase de¢ciency are currently on treatment. They were assessed at baseline and at intervals following commencement of treatment. Each patient walked barefoot at preferred speed and data was collected from 4 consecutive passes on the GaitRite mat. Speci¢c parameters of gait velocity, cadence, step length, single support and base of support-were measured. Changes for each individual are analysed and compared with suitable controls. Results: All patients had slower and less e¤cient gait patterns than their peers. The most notable response to treatment is in cadence, velocity and base of support indicating a more stable and e¤cient gait pattern. These changes were not apparent on routine clinical examination. Conclusion: The GaitRite was found to identify changes in gait parameters in arginase de¢ciency. It is a robust and accessible way of providing objective quantitative analysis of gait. It highlights alteration in neurological function prior to overt clinical signs and is a useful monitoring tool in arginase de¢ciency.

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LATE ONSET CARBAMOYLPHOSPHATE SYNTHETASE DEFICIENCY Ferreira AC1, Moc°o C2, Quental R3, Azevedo L3, Sequeira S1 1 Metabolic Unit ^ Hosp Dona Estefaªnia, Lisbon, Portugal, 2Paediatric Serv ^ Districtal Hosp Faro, Faro, Portugal, 3IPATIMUP^Inst Mol Path Immun Univ Oporto, Oporto, Portugal Background: Ornithine transcarbamoylase de¢ciency (OTC) is a rather common metabolic disease that can be diagnosed in teenage girls or women. Carbamoylphosphate synthetase de¢ciency (CPS) is a rarer disorder and not usually thought as a cause of sudden neurological symptoms in patients considered previously healthy. Case: 12-year-old girl, with normal development and growth presented with two episodes, a year apart following febrile episodes, of drowsiness, agitation, incoherent speech, midriasis and ataxia. Ammonia was initially normal but on the third day of the second hospitalisation she presented hyperammoniemia (168 mmol/L). Amino acid pro¢le revealed high levels of glutamine and alanine. Allopurinol test showed moderate increase of orotic acid. This data was compatible with OTC de¢ciency, although molecular studies failed to identify the disease-associated mutation in the coding region of the gene. An alternative diagnosis would embrace CPS de¢ciency, which was con¢rmed with the identi¢cation of two mutations: c.411 T4A (p.Ser137Arg) in exon 4 and c.2885 A4G (p.Tyr962Cys) in exon 23 of the gene. No functional assays were performed to establish the deleterious e¡ect of such substitutions, but considering the clinical and biochemical data, the phenotype^genotype association is dreadfully likely. Comments: We remember that in urea cycle disorders ammonia may not be high immediately after patients present with neurological symptoms. When OTC is excluded, CPS de¢ciency should be investigated in patients whose phenotype suggests late onset urea cycle disorder. Molecular studies of the enormous CPS gene, although expensive and time consuming may alleviate the need of an invasive technique like a liver biopsy.

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IMPROVED CLINICAL OUTCOME IN GIRLS WITH POSITIVE ALLOPURINOL LOADING TEST FOLLOWING INSTITUTION OF LOW PROTEIN DIET Renaud DL, Eckert SK Mayo Clinic Foundation, Rochester, MN, USA Background: X-linked ornithine transcarbamalase (OTC) de¢ciency is the most common disorder of urea cycle metabolism. Methods: F|ve girls, aged 6 months to 27 months, were evaluated for global developmental delay and hypotonia. Two of the ¢ve girls also had seizures. All were found to have persistently elevated orotic acid in the urine. An allopurinol challenge resulted in a signi¢cant rise in the orotic acid in the urine, suggestive of probable OTC de¢ciency. Uracil was normal in the urine for all patients. Molecular studies did not detect deleterious mutations in the OTC gene. All patients were started on a low protein diet, consisting of protein and calories at or near the recommended daily allowance with essential amino acids comprising one third to one half of total protein intake. Results: Institution of this diet resulted in a decrease in the urine orotic acid to the normal range, a decrease in liver function studies where elevated and a lowering of the ammonia levels during periods of wellness. Two patients required sodium benzoate for mild elevations of ammonia during illnesses. Both patients were self restricting protein prior to the institution of the diet. All patients had an acceleration of their development on the diet as well as an improvement in their hypotonia and seizure control. Conclusions: Low protein diet is an e¡ective treatment in girls with biochemical evidence of probable OTC de¢ciency in the absence of a detected deleterious mutation, resulting in improved development, tone and seizure control.

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ORNITHINE TRANSCARBAMYLASE DEFICIENCY: PHENOTYPE AND GENOTYPE CHARACTERIZATION IN 25 PATIENTS ^ EXPERIENCE FROM A PORTUGUESE TERTIARY CENTER Lea¬o Teles E1, Rodrigues E1, Soares S1, Trindade E2, V|larinho L3, Cardoso ML3, Azevedo L4 1 Metab Dis Unit, Ped Dep, Univ/Hosp St Joa¬o, Porto, 2Gastroent Unit, Ped Dept Univ/Hosp St Joa¬o, Porto, 3Genet Med Cent Jacinto Magalha¬es, Porto, 4IPATIMUP, Inst Pathol Mol Immun Univ P, Porto, Portugal

PHENOTYPIC VARIABILITY AMONG HYPERORNITHINEMIA^HYPERAMMONEMIA^ HOMOCITRULLINURIA (HHH) SYNDROME PATIENTS HOMOZYGOUS FOR THE DELF188 MUTATION IN SLC25A15 Debray FG1, Lambert M2, Lemieux B3, Drouin R3, Maranda B4, Laframboise R4, Mitchell G2 1 Dept Human Genet, CHU of Lie©ge, Lie©ge, Belgium, 2Div Med Genet, CHU Sainte-Justine, Montreal, Canada, 3Div Med Genet, CHU Sherbrooke, Sherbrooke, Canada, 4Dept Med Genet, CHU of Queèbec, Queèbec, Canada

Background: Ornithine transcarbamylase de¢ciency (OTCD; OMIM 311250) is the most frequent urea cycle disorder. The OCT gene is located in the short arm of the X-chromosome, with numerous mutations described. The clinical diagnosis, although suggestive in a male newborn with encephalopathy and hyperammoniemia, may be more intricate in late forms or female heterozygote. We present the population followed at our unit with biochemical, enzymatic and/or mutational diagnosis of OTCD. Methods: Retrospective analysis of clinical records. Results: 25 patients (3<), 10 paediatric, from 16 families. The age of presentation varied from 0 to 10 years-old; age at diagnosis between 0 and 68 years-old. From the male patients only one had neonatal presentation; from the female heterozygous patients, presentation occurred in all age groups. The evocative manifestations included repeated vomiting, migraine, disturbed consciousness, including post-partum coma, seizures, acute hepatic insu¤ciency, mental retardation and poor weight gain. All patients had episodes of hyperammoniemia and increased orotic acid excretion during the course of disease. Provocation tests with allopurinol were positive in 9 patients. Enzymatic study con¢rmed diagnosis in 2 patients. Mutational anlysis was accomplished in 24 with both familiar and sporadic cases. Functional signi¢cance is being further studied in 4 cases. Conclusion: Clinical heterogeneity in OTCD is an important obstacle to early diagnosis and treatment. The ability of genetic studies to identify mutations in a¡ected patients has allowed a more accurate detection of carriers and prenatal diagnosis. Our data suggest that patients with late forms of the disease and heterozigotes are problably underdiagnosed

Background: Hyperornithinemia^hyperammonemia^homocitrullinuria (HHH) syndrome (MIM 238970) is caused by impaired ornithine transport across the inner mitochondrial membrane due to mutations in SLC25A15. So far, 22 di¡erent mutations of the SLC25A15 gene have been described in 49 patients belonging to 31 independent families. Methods: To further delineate the phenotypic spectrum of HHH syndrome from description of a genetically homogeneous cohort of patients and identify prognostic factors based on long-term follow-up, sixteen French-Canadian patients were retrospectively and prospectively clinically assessed. Results: Due to a founder e¡ect, 15/16 were homozygous for the F188del mutation in the SLC25A15 gene. The main clinical features at presentation were liver dysfunction (6/16) and neurological disease (9/16), including chronic neurological symptoms (6/9) and acute encephalopathy (3/9). Hyperammonemia was inconstant, usually mild, and uncommon after start of treatment. We identi¢ed a paradoxical decrease of plasma ammonia levels after meal in 30% of observations, especially in HHH patients with mild hyperornithinemia. Long-term follow-up showed that variable intellectual impairment and lower limb spasticity occur frequently, together or separately, with no obvious relationship to age at diagnosis and compliance with treatment. Conclusion: We report the largest known cohort of patients with HHH syndrome. A similar range of severity occurred in clinical course and outcome of delF188 homozygotes and in the 49 other reported patients as compiled from the literature. The poor clinical outcome of HHH patients despite early therapy and repeatedly normal plasma ammonia levels emphasizes the need to better understand the pathophysiology and to reconsider the therapeutic goals for HHH.

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CLASSICAL CITRULLINEMIA: POSSIBLE CLUSTER IN A LIMITED GEOGRAPHIC AREA OF ARGENTINA Laroèvere LE1, Angaroni CJ1, Antonozzi SL1, Bezard MB1, Shimohama M2, Dodelson de Kremer R1 1 CEMECO, Child Hosp, Coèrdoba Univ, Coèrdoba, Argentina, 2Pediatrics Department, Kitasato Univ, Kanagawa, Japan Background/Objective: Classical citrullinemia (CTLN1) is caused by mutations at argininosuccinate synthetase gene. F|fty seven mutations were described; one of the most frequent being p.G390R associated with early-onset/severe phenotype. We report the recognition of 8 patients with CTLN1 and its association with a possible geographic cluster. Material/Methods: The included patients were three male and ¢ve female newborns with hypotonia, neurological impairment, lethargy, coma and death in neonatal period. All biological samples were submitted post-mortem from the same geographic area to our centre. Biochemical determinations (ammonia, amino acids, pyrimidines) were performed by standard methods. Molecular analysis included PCR, sequencing and restriction-enzyme assays. Results: The biochemical diagnosis was based on: hyperammonemia, high citrulline levels in biological £uids and orotic acid and uracil urine excretion. The molecular analysis was performed in four families showing the same mutation. The parents of the index case were heterozygous for the p.G390R substitution. Additionally, this mutation was detected in another patient (homozygous) and in two mothers (heterozygous) of other probands. Conclusions: This study reports the recognition of patients a¡ected by CTLN1 with the same genetic defect in a limited geographic area. This region, £anked by two mountain chains, is located in San Luis Province (Argentina). All families have Spanish ancestry and have lived in the area for several generations. Thus, suggesting the existence of a cluster which may have arisen by a founder e¡ect of that ethnic origin. A population study is in progress in order to perform carrier identi¢cations and genetic counselling as a preventive aim.

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ATYPICAL PRESENTATION OF CITRULLINEMIA TYPE I van Spronsen FJ1, Cuppen M2, Eling MWM2, Reijngoud DJ3, Smit GPA2, de Groot M1, Verheijen FW4, Niezen-Koning KE3, Rings EHHM1 1 Beatrix Children's Hosp, UMCG, Groningen, Netherlands, 2Slingeland Hosp, Doetichem, Netherlands, 3Lab Metab Dis, Univ Med Cent Groningen, Groningen, Netherlands, 4Clinical Genetics, Erasmus Univ, Rotterdam, Netherlands Background: Argininosuccinate synthase de¢ciency (ASS) is especially known to cause severe developmental arrest at neonatal age. Some patients with ASS develop liver cirrhosis. We present two patients of Dutch non-consanguineous parents who were referred to our hospital for liver transplantation. Both patients were diagnosed with ASS and have positive development although feeding problems persist. Patients: Patient A (weight 2190 g) had asphyxia due to insu¤cient progress of labour. She did not develop seizures, but was apathic. Metabolic investigations were not performed. MRI showed intracranial bleeding without clear cause. After 16 months of abnormal, but positive, development without catch-up of growth, she became inactive. Feeding problems and severe liver problems developed. She was referred for liver transplantation (NH3 110 mmol/L). Metabolic investigations showed citrulline 1442, arginine 23 mmol/l, without increased argininosuccinate excretion. Although citrin de¢ciency was considered, treatment aiming at ASS resulted in clear improvement. MRI showed typical ASS abnormalities. Citrulline incorporation studies in ¢broblasts showed 0.4 (normal 25.8) nmol/24 h/mg protein. Patient B developed feeding problems after a normal development and growth until 18 months. He developed delay in development and growth. He was transferred for liver transplantation evaluation because of liver failure, but ASS de¢ciency was diagnosed (NH3 210 citrulline 3000 arginine 20 mmol/L with increased arginiosuccinate excretion) and later con¢rmed (citrulline incorporation 0.8 nmol/24 h/mg protein). Conclusion: Both patients showed that liver failure not only may develop within the course of ASS de¢ciency but even may be presenting symptoms resulting in referral for liver transplantation.

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ANALYSIS OF RESIDUAL ENZYME ACTIVITIES IN CITRULLINEMIA TYPE I BY IN-VITRO EXPRESSION STUDIES Berning C1, Bieger I2, Pauli S1, Vermeulen T1, Rolinski B2, Gempel K3, Ha«berle J1 1 Univ Klinikum Mu«nster, Pediatric Dept, Mu«nster, Germany, 2KH Schwabing, Inst Klin Chemie, Mol Diag, Mu«nchen, Germany, 3Labor Bamberg, Bamberg, Germany Background: Citrullinemia type I, synonymous: argininosuccinate synthetase (ASS) de¢ciency, can present with a broad and continuous spectrum ranging from neonatal fatal hyperammonemia to only a biochemical phenotype. There is no reliable prognostic marker allowing conclusions regarding the type of manifestation, the course of the disorder, and the need for follow-up and treatment. We investigated whether varying levels of ASS residual activities can be used for the severity of the disease at the time of diagnosis. Methods: A bacterial in-vitro-expression system was established allowing the enzymatic analysis of puri¢ed wildtype and mutant ASS proteins. In detail, mutations (W179R, V263M, G362V) identi¢ed in asymptomatic patients detected through newborn screening, mutations (R265C, G324S, G390R) associated with a severe phenotype, the mutation A118T found in a patient with late-onset who died postpartum, and the mutation M302V detected in the clinically and biochemically asymptomatic mother of a patient were analyzed. Results: Mutations associated with a severe phenotype do not show any signi¢cant enzymatic activity. Mutations associated with a mild course yield signi¢cant levels of ASS activity. The mutation A118T results, despite a high residual activity of 62%, in a severe reduction of a¤nity towards the substrates citrulline and aspartate. Conclusions: Even a high level of residual ASS activity is no reliable prognostic marker for an uneventful clinical course. Thus, determination of ASS residual activities can not help in anticipating the risk of a metabolic derangement. This information should guide clinicians as well as patients with mild citrullinemia towards a life-long awareness of the disorder.

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TRANSIENT FULMINANT HEPATITIS IN A PATIENT WITH CLASSICAL CITRULLINEMIA Faghfoury H, Schulze A The Hospital for Sick Children, Toronto, Canada Background: Classical citrullinemia, or CTLN1, is a urea cycle disorder caused by a de¢ciency of argininosuccinate synthetase (ASS) which functions to convert citrulline to argininosuccinate. The condition typically presents with hyperammonemia and encephalopathy in the neonatal period leading to death or signi¢cant morbidity in survivors. There have also been rare cases of pregnant or postpartum patients with CTLN1 presenting in hepatic failure or with fatty liver of pregnancy. We report the only case of infantile onset CTLN1 presenting with transient fulminant hepatic failure. Case Report: A previously well 15-month-old infant was seen following a 10 day history of behaviour consistent with gradually progressive encephalopathy. Initial laboratory testing revealed hyperammonemia, high plasma citrulline levels and normal plasma arginine levels, consistent with a diagnosis of classical citrullinemia. Despite treatment which resulted in improvements in her neurological status and ammonia levels, the patient developed fulminant hepatitis with aminotransferase levels increasing 400-fold above normal values. Investigations to rule out an infectious etiology for the hepatitis were normal. Consideration was given to liver transplant but hepatic function normalized within weeks and the patient had no permanent hepatic or neurological sequelae associated with her presentation. There are reports of patients with urea cycle defects presenting with transient hepatitis during metabolic decompensations, for which the etiology is unclear. The hepatotoxic e¡ect of the accumulated substrates may be a possible explanation. The present case suggests that in some cases of citrullinemia with acute liver failure, emergency intervention such as transplantation is not warranted despite evidence of severe hepatotoxicity.

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EFFECTIVE TREATMENT OF HYPERAMMONAEMIA IN A NEONATE WITH CITRULLINEMIA TYPE 1 Eyskens FJ1, Peper W2 1 CEMA Antwerp, Antwerp, Belgium, 2ZNA Queen Paola Children's Hospital, Antwerp, Belgium Case report: A female neonate, ¢rst child of non-consanguineous Moroccan parents, presented with hypothermia, bradycardia and dyspnoea on the second day of life. The initial working diagnosis was sepsis. She deteriorated rapidly with convulsions and apnoea with need for assisted ventilation. A ¢rst blood ammonia gave an immeasurable high value of 41000 mmol/L; plasma lactate was normal; there was a slight metabolic acidosis. Treatment with high glucose infusions, Larginine PO, phenyl acetate PO and benzoate PO, associated with an exchange transfusion and peritoneal dialysis was started immediately. A dose of Carbaglu1 100 mg/kg (Orphan Europe) was administered IV with no e¡ect on the blood ammonia. The following day amino acid analysis in serum and urine, organic acid analysis in urine and orotic acid and orotidine in urine revealed the diagnosis of citrullinemia. Benzoate and phenyl acetate were given IV as a 10%/10% sterile aqueous solution under the form of Ammonul1 (Swedish Orphan) diluted in glucose 10% at an infusion rate of 25 ml/kg given over 24 h administering 250 mg/kg sodium benzoate and 250 mg/kg sodium phenyl acetate resp. Blood ammonia levels decreased within 4 h after initiating the Ammonul infusion and normalized within 24 h. The infusion of Ammonul was well tolerated. The diagnosis of citrullinemia type 1 was con¢rmed by enzymatic analysis in skin ¢broblasts. Conclusions: Ammonul is very e¡ective in excreting nitrogen by alternative pathways and can o¡er better outcome in urea cycle defects in terms of survival in neonates with very high blood ammonia levels at presentation.

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MRI ABNORMALITIES DUE TO NEONATAL HYPERAMMONEMIC ENCEPHALOPATHY ARE NOT PREDICTIVE OF IRREVERSIBLE BRAIN DAMAGE Ku«ster A1, Le Franc°ois T2, Valayannopoulos V3, Boddaert N4, Dejode JM5, Roze JC1, V|aney-Saban C6, Ha«berle J7, de Lonlay P3 1 INRA,UMR Phan,CIC004,Metab Dis,Univ Hosp, Nantes, France, 2Ped Radiol, Univ Child Hosp, Nantes, France, 3Service Mal Metab, Hoªpital Necker, Paris, France, 4Ped Radiol, Hoªpital Necker, Paris, France, 5 PICU, Univ Child Hosp, Nantes, France, 6Service de Biochimie, Hoªp. Debrousse, Lyon, France, 7Div Metab Dis, Univ Child Hosp, Mu«nster, Germany Background: Urea cycle disorders often present with neonatal-onset hyperammonemia due de¢cient nitrogenous waste elimination. Encephalopathy has been reported to be due to cerebral toxicity of high glutamine and ammonia levels. Prompt recognition of urea-cycle disorders and emergency treatment result in survival in the majority of patients. Evaluation of neurologic sequelae of this acute decompensation is challenging. Besides the severity and duration of the hyperammonemic coma, MR imaging may contribute as an early prognostic indicator. Only few descriptions of brain MR images are currently available. Methods: We present brain MR images performed in two neonates with citrullinemia and one with CPS1 de¢ciency and report follow-up MR imaging at 6 months. Results: Early brain MR images showed similar ¢ndings including axial T2weighted (1) di¡use bilateral symmetrical signal abnormalities of the cortex and subcortical white matter predominantly regarding insular lobes (3 neonates) and (2) high signal intensity of basal ganglia (lenticular nuclei, globi pallidi) (2 neonates). Control imaging revealed complete recovery of the initial cortical abnormalities. Persistance of white matter changes and basal ganglia abnormalities could be observed. Conclusion: Neuroradiological changes in patients exposed to high ammonia were not predictive of neurodevelopmental outcome and did not result necessarily in irreversible brain damage as-even though 2 neonates have mild psychomotor delay-the oldest child, now 16 months old, has an excellent psychomotor development.

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INHIBITION OF N-ACETYL-GLUTAMATE SYNTHASE ACTIVITY IN VALPROATE-ASSOCIATED HYPERAMMONEMIA Aires CCP1, van Cruchten A2, Ruiter J2, IJlst L2, Tavares de Almeida I1, Duran M2, Wanders RJA2, Silva MFB1 1 iMed UL, CPM, Fac Farmaècia Univ Lisboa, Lisboa, Portugal, 2Lab Genet Metab Dis, AMC, Univ Amsterdam, Amsterdam, Netherlands Hyperammonemia is a frequent side-e¡ect of valproic acid (VPA) treatment which can potentially evolve to encephalopathy. Aims: To elucidate the pathogenic mechanisms of hyperammonemia, underlying the interference of VPA and valproyl-CoA with the urea cycle, at two levels: (a) the e¡ect on the synthesis of citrulline and (b) the e¡ect on the synthesis of N-acetylglutamate (NAG). Methods: Citrulline formation was studied in intact mouse liver mitochondria (MLM), incubated in a reaction medium containing succinate, rotenone, ornithine, KHCO3, NH4Cl and VPA (0^10 mM). The synthesis of NAG was quanti¢ed in liver of VPA-treated rats (100 mg/kg for 2 weeks), and after incubation of MLM with VPA (0^10 mM) or valproyl-CoA (0^1 mM), glutamate and acetyl-CoA. Both citrulline and NAG were identi¢ed and quanti¢ed using LC-MS/MS. Results: Citrulline synthesis in MLM was only slightly (13%) inhibited by VPA (2 mM), suggesting that the interference of the drug with the urea cycle is not primarily at the CPS or OTC level. Considering that the formation of NAG controls £ux through this cycle, the level of this metabolite was quanti¢ed in liver tissues of VPA-treated rats, and was found to be decreased as compared with controls. Furthermore, valproyl-CoA was found to be a stronger inhibitor of NAG synthesis in MLM than the parent drug. Di scus sion: the results clearly suggest that VPA-indu c ed hyperammonemia may result from the inhibition of NAG synthase activity by valproyl-CoA. The reduced bioavailability of NAG would account to a reduced £ux through the urea cycle and to a subsequent accumulation of ammonia.

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VALPROATE INDUCED HYPERAMMONAEMIC ENCEPHALOPATHY SYNDROME. TREATMENT WITH CARGLUMIC ACID Pedroèn Giner C1, Loèpez Mar|èn L2, Quijada Fraile P1, Lara Herguedas J2, Garc|èa Pen¬as JJ2, Duat Rodriguez A2, Garc|èa Mun¬oz MJ3 1 Div Nutrition, Univ Child Hosp NJesus, Madrid, Spain, 2Div Neurology, Univ Child Hosp NJesus, Madrid, Spain, 3CEDEM, Universidad Autoènoma, Madrid, Spain Background: The carglumic acid (CA) is an analogue of Nacetilglutamate (NAG), activator of carbamoyl phosphate synthetase (CPS). In valproate-induced hyperammonemic encephalopathy (VIHE) it seems that a valproate metabolite produces a decrease of hepatic NAG, with inhibition of the CPS. Methods: Description of two cases with VIHE and the response to treatment with CA. Results: (1) A 15-year-old girl with chromosomopathy-15, refractory epilepsy in treatment with several drugs. She become to a convulsive status and VPA is added. Quickly, she begins with impaired consciousness. A hyperammonemia (154 mmol/L) is detected with normal VPA. VPA is withdrawn and CA is administered (2 doses 70 mg/kg, 24 h). Ammonia levels decreases rapidly (108 mmol/L, 2 h after the ¢rst dose; 70 mmol/L after the second). The analysis of the cerebrospinal £uid (CSF) before the second dose, samples glutamate and glutamine levels very raised. (2) A 14-year-old girl with dna depletion mitochondrial, refractory epilepsy in treatment with VPA and others. She became with continuous partial epilepsy and she begins acute encephalopathy, with hyperammonaemia (102 mmol/L) and normal VPA levels. VPA is withdrawn and CA is added (¢rst dose, 100 mg/kg and 25 mg/kg/6h for 48 h) with decrease of the ammonemia (60 mmol/L 2 h after the ¢rst dose) and disappearance of symptoms in 24 h. Conclusions: The CA was e¡ective to decrease blood ammonia levels in VIHE. After ¢rst dose, CSF glutamine levels remains raised for what we believe that treatment should be extended a few days in spite of normal ammonia levels are achieved.

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EFFICACY OF CARBAGLUMIC ACID FOR NEONATAL HYPERAMMONAEMIA IN TYPE I CYTRULLINAEMIA Gasperini S, Pasquini E, Funghini S, Malvagia S, Donati Ma Metabolic Unit-Meyer Children Hospital, Florence, Italy Common treatment protocols in neonatal onset citrullinaemia suggest high energy intake intravenously, arginine chloridrate, sodium benzoate and sodium phenylbutyrate. We present two cases of neonatal hyperammonaemia due to type I citrullinaemia. Case 1 showed respiratory alkalosis and hyperammonaemia (715 mg/dl) at 4 days. Orotic acid was 146 mmol/mol creat, plasma glutamine and citrulline were respectively 1822 and 1768 mcmol/L. After 2 h of emergency treatment ammonia decreased to 500 mg/dl, so we started therapy with carbaglumic acid (150 mg/kg) with rapid reduction of ammonia until 190 in about 6 h. Glutamine levels decreased to 274 within 2 days. Case 2 presented hyperammonaemia (460 mg/dl) at 36 h, when the parents reported their ¢rst son died in neonatal period, a¡ected by type I citrullinaemia (they had refused prenatal diagnosis). Orotic acid was 362 mmol/mol creat, glutamine and citrulline were 1860 and 1967 mmol/L. After about 24 h since starting common treatments, ammonia was 370 mg/dl. Thus we started therapy with carbaglumic acid (150 mg/ kg) with dramatic decrease of ammonia until 150 in a few hours. Glutamine levels decreased to 466 within 2 days. Carbaglumic acid is an orphan drug for treatment of hyperammonaemia due to NAGS de¢ciency and not only. In acute crisis of hyperammonaemia (also in Type I cytrullinaemia) we suggest it could be helpful to associate carbaglumic acid to common emergency treatments so as to speed up detoxi¢cation and to prevent peritoneal dyalisis, until ammonia and glutamine levels stabilize.

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HINDSIGHT INTO UREA CYCLE DEFECTS AFFECTING REGULATORY SITES: THE ACETYLGLUTAMATE SITE OF CARBAMOYL PHOSPHATE SYNTHETASE I (CPSI) AND THE ARGININE SITE OF ACETYLGLUTAMATE SYNTHASE (AGS) Pekkala S1, Sancho-Vaello E2, Fernaèndez-Murga L3, Ye¢menko I3, Rubio V3, Cervera J1 1 Centro Investig Pr|èncipe Felipe (CIPF), Valencia, Spain, 2Centro Investig Red Enf Raras (CIBERER), Valencia, Spain, 3Inst Biomedicina Valencia (IBV-CSIC), Valencia, Spain Background/Objectives: CPSI de¢ciency can result from decreased CPSI activation by its essential activator, acetylglutamate (AG). Patients with mutations a¡ecting the CPSI AG site might bene¢t from massive carbamoylglutamate administration. In the enzyme that makes acetylglutamate, AGS, mutations may a¡ect the site for the AGS activator arginine, possibly causing AGS de¢ciency. We aim at identifying and delineating the AG and arginine sites in CPSI and AGS, respectively. Methods: Missense mutations in CPSI C-terminal domain (which hosts the AG site), including mutations found in CPSI de¢ciency, were introduced and their e¡ects on the functionalities and AG activation of puri¢ed CPSI were assessed. The residues having an e¡ect on AG activation were localized on the reported structure of the CPSI C-domain. Missense mutations were introduced in the AGS putative arginine site proposed from sequence comparisons with AG kinase. Their e¡ects on the functionalities and arginine regulation of AGS were assessed. Results: CPSI clinical C-terminal domain mutations mostly hampered AG activation. Key residues for high-a¤nity binding of AG or arginine to CPSI or AGS, respectively, have been identi¢ed, and have been located in 3-D structures, allowing the modelling of the corresponding site in the structure of each enzyme. Since the human AGS structure remains undetermined, the arginine site structure has been inferred from the 3-D structures of bacterial AG kinase and AGS. Conclusions: In CPSI and AGS de¢ciencies, deleterious mutations a¡ecting the AG and arginine regulatory sites can now be assigned unambiguously. In the case of CPSI de¢ciency, these assignations may in£uence therapeutic decisions.

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CITRIN DEFICIENCY: PROLONGED NEONATAL JAUNDICE AND FAILURE TO THRIVE Fernandes AP1, Nogueira C2, Martins E3, Soares S1, Almeida M2, Quelhas D2, Cardoso ML2 1 Centro Hospitalar do Alto Ave, Guimarmes, Portugal, 2Centro Geneètica Meèdica Jacinto Magalha¬es, Porto, Portugal, 3Hospital de Crianc°as Maria Pia, Porto, Portugal Neonatal intrahepatic cholestasis caused by citrin de¢ciency (NICCD) # OMIM 605814 is an autosomal-recessive disease caused by a de¢ciency of citrin, a calcium-binding mitochondrial protein, encoded by SLC25A13 gene. Citrin is a liver-type mitochondrial aspartate-glutamate carrier that plays an important role in the malate-aspartate NADH shuttle, urea synthesis, aerobic glycolysis, gluconeogenesis and protein and nucleotide syntheses. Objectives: The authors report the ¢rst patient with Mediterranean geographic origin with citrin de¢ciency. Methods: He was the second live born term child of non consanguineous white parents. He presented jaundice since neonatal period until four months of age and failure to thrive since the ¢rst month of life. He had slight elevated aspartate aminotransferase with non cholestatic hyperbilirrubinaemia, hyperammonaemia, hypercitrullinaemia and hypermethioninaemia. Urinary analysis showed sustained increased phydroxiphenyllactate, p-hydroxiphenylpiruvate. Resolution of jaundice at ¢ve months of age, although persistence of hepatic biochemical anomalies and of abnormal organic acid pro¢le. He showed no developmental delay, but failure to thrive was maintained. Citrin de¢ciency was suspected. Results: Analysis of the SLC25A13 gene was performed. The patient was a compound heterozygous with c.1056__1060delA and c.1231-1G4A novel mutations on exan 11 and intran 12 of this gene, respectively. The parents were heterozygous carriers. After molecular diagnosis of citrin de¢ciency began hyperproteic diet and carbon hydrate restriction with a very good weight gain. Conclusion: This is the ¢rst report of citrin de¢ciency in an originally European child with prolonged neonatal non cholestatic jaundice and failure to thrive.

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ARGININOSUCCINIC ACIDURIA ASSOCIATED WITH PANCREATITIS Dursun A1, Sivri HS1, O«zon A2, Akcao«ren Z3, Tokatly A1, Koch HG4, Cos° kun T1 1 Hacettepe Univ Metab and Nutr Unit, Ankara, Turkey, 2Hacettepe Univ Ped Endoc, Ankara, Turkey, 3Hacettepe Univ Ped Pat, Ankara, Turkey, 4 Univ Kinderklinik Mu«nster, Mu«nster, Germany Until now, acute and chronic pancratitis has been reported in 24 cases with 10 hereditary metabolic diseases including tyrosinemia type I, GA type I, Citrullinemia (type II), homocystinuria, MSUD, MMA, Isovaleric acidemia, OTC de¢ciency, 3-hydroxy3-methylglutaryl-CoA de¢ciency and, 3-methylglutaconic aciduria. As to the authors knowledge there is not any report on argininosuccinic aciduria (ASA) associated with pancreatitis. Clinical features of ASA, caused by argininosuccinate lyase de¢ciency, include mental retardation, hepatomegaly, skin lesions, trichorrhexis nodosa, and convulsions. We present here a patient with ASA in whom pancreatitis has been detected in postmortem studies. Diagnosis of the patient was made by biochemical investigation at 2.5 years age, and was also con¢rmed by mutation analysis showing R445P (1334 G4C in exon 16), homozygously. In the latest admission, the patient, 5.5 years of age, presented with encephalopathy, convulsions, and severe hyperglycemia attributed to stress and cortisone administration. Unfortunately, neither biochemical nor imaging studies of pancreas could be performed before his death. Post mortem examination of the pancreas showed interstitial lymphocytic in¢ltration and ¢brosis, as well as necrosis of the surrounding fat tissue. These ¢ndings were consistent with chronic pancreatitis. In conclusion we want to stress that evaluation of pancreatic functions and health should be considered during acute and chronic stages of the inherited metabolic diesases, especially intoxication types. Support: State Planning Organisation; Prj. No:2006 K 120 640-06-03

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ARGININOSUCCINIC ACIDURIA. REPORT OF A CASE WITH EPILEPTIC ENCEPHALOPATHY Casero D1, V|gnoli A2, La Briola F2, El Oksha S1, Colombo V1, Riva E1 1 Dept Paedia, San Paolo Hosp, Univ Milan, Milan, Italy, 2Epilepsy Cent, S Paolo Hosp, Univ Milan, Milan, Italy Introduction: Argininosuccinic aciduria is an urea-cycle disorder caused by the autosomal-recessive argininosuccinate-lyase de¢ciency, and characterized by the accumulation of argininosuccinic acid in body £uids and severe hyperammonemia. Nervous system, kidney and liver are the organs more often damaged. Case report: a 6 months male infant was admitted for hepatomegaly, cyclic vomiting and lethargy. Hyperammonemia (94 mg/dl), high plasma liver enzymes, citrulline and glutamine and decreased arginine levels, with high urine arginin-succinic acid, led to the diagnosis of argininosuccinic aciduria. Analysis of the ASL gene identi¢ed a heterozygous genetic condition (V178M; G280fs__X283, novel mutation). A treatment with restricted protein intake and arginine supplementation was started. In spite of a good metabolic control and normal psychomotor development, at 17 months of age the child presented recurrent atonic seizures characterized by sudden falls of head and trunk. The seizures became very frequent (440 episodes/ day) and the child showed a progressive psychomotor regression. Electroencephalography (EEG) showed an epileptiform activity in wakefulness and sleep, consistent with the de¢nition of epileptic encephalopathy according to ILAE classi¢cation. Brain magnetic resonance imaging was normal. Therapy with vigabatrin was immediately started with progressive decrease in seizures and successive normalization of the EEG. Conclusion: our case suggests that seizures arising in patients with argininosuccinic aciduria could respond to vigabatrin treatment together with a good metabolic control including protein restriction and arginine supplementation.

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LIVER TRANSPLANTATION FOR ARGININOSUCCINATE LYASE DEFICIENCY: CLINICAL, BIOCHEMICAL AND NEUROIMAGING FOLLOW UP Reègal L1, Zeevaert R1, Van Damme-Lombaerts R2, Salviati L3, Jaeken J1 1 Metab Center, Dept Pediatr, Univ Hosp, Leuven, Belgium, 2Pediatr Transpl, Dept Pediatr, Univ Hosp, Leuven, Belgium, 3Lab EmOnc Ped, Dip Ped, Univ Hosp, Padova, Italy Background: Argininosuccinic aciduria is an autosomal recessive urea cycle disorder. In the liver, argininosuccinate lyase (ASL) is involved in the urea cycle, while in the proximal tubule it is involved in the biosynthesis of arginine from citrulline generated in the small intestine. It is also involved in the NO cycle. Liver transplantation repairs only the urea cycle. Only 3 ASL de¢cient patients who underwent liver transplantation have been reported previously. Case report: A 2-month-old boy presented with hyperammonemic encephalopathy and hepatomegaly. Blood levels of citrulline and argininosuccinate were elevated. Standard treatment quickly corrected ammonia levels. He was compound heterozygous for R236Q and G206V in the ASL gene. Brain MRI at 5 months showed cerebral atrophy and delayed myelination. Developmental delay gradually deteriorated and levels of argininosuccinate gradually rose, despite excellent control of ammonemia with classical treatment. Orthotopic liver transplantation was performed at the age of 2 years, resulting in clear improvement of development. Blood levels of argininosuccinic acid normalized but citrulline remained elevated (130^229 mmol/L, normal range 10^50). Brain MRI at the age of 5.5 years showed minimal white matter changes but no signs of atrophy. Conclusions: The clinical course of our patient illustrates the potential of liver transplantation in ASL de¢ciency. Remarkably, signs of cerebral atrophy disappeared after transplantation. The persisting elevation of citrulline levels was apparently not associated with deleterious e¡ects, and is best explained by the role of ASL in renal citrulline metabolism.

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Background/Objectives: Argininosuccinic aciduria (ASAuria; MIM# 207900) is a rare autosomal recessive disorder of the urea cycle, caused by de¢ciency of argininosuccinate lyase (ASL) which leads to accumulation of argininosuccinic acid (ASA) in body £uids and severe hyperammonemia. This disorder seems to be frequent in our country; in fact, we report the clinical and biochemical pro¢le of eight cases of ASAuria. Results: Eight infants (3 boys and 5 girls) from unrelated consanguineous families were diagnosed with ASA. The age of diagnosis varied from 2 days to 12 years. The patterns of clinical presentation include poor developmental progress, behavioural problems, vomiting, convulsions, hepatomegaly and three have brittle hair. Biochemical abnor malities were charac te r ized with hyperammonia (up to 222 mmol/L) and marked urinary excretion of orotic acid varied from 21.4 to 201.2 mmol/mmol of creatinine. Plasmatic and urinary amino acid analysis quanti¢cation performed on ions exchange chromatography analyzer, provided su¤cient information to make a con¢dent diagnosis of ASAuria by the presence of the higher plasmatic concentration of ASA varied from 213 to 6423 mmol/L and urinary from 28554 to 158313 mmol/gram of creatinine. Conclusion: The incidence of ASA appears to have been increasing over recent years in many centers around the world, in fact, it is important to establish the prenatal diagnosis in future pregnancies of Tunisian families.

Background: HPN-100 is a novel molecule that delivers PBA in an odorless, tasteless, salt-free liquid. Preliminary ¢ndings in three adult UCD patients are presented from a phase 2, open-label, switch-over, dose-escalation study of the safety and tolerability of HPN-100 compared to PBA in UCD patients. Methods: UCD patients maintained on PBA were switched to a moleequivalent HPN-100 dose. Full safety and pharmacokinetic pro¢les were assessed. Results: Plasma ammonia values showed good control and AUC 0^12 h were lower with HPN-100 treatment than PBA. Plasma metabolites PBA Cmax were lower and PBA Tmax was longer following HPN-100, while urinary excretion of nitrogen (assessed by phenylacetylglutamine, AUC 24 h) was higher. Conclusion: Thus HPN-100 is a new compound and these preliminary results support further investigation of the compound for the treatment of UCD.

ARGININOSUCCINIC ACIDURIA: A SURVEY OF EIGHT TUNISIAN INFANTS Esseghir N1, Azzouz H2, Fontaine M3, Khemir S1, Nassrallah F1, Gandoura N4, Tebib N2, Ben Jaballah N5, Amri F6, Briand Gi3, Ben Dridi MF2, Porchet N3, Kaabachi N1 1 Biochem Lab Rabat Uni Central Hosp, Tunis, Tunisia, 2Dept Ped, Rabat Uni Central Hosp, Tunis, Tunisia, 3Lab Bioch Mol Bio Horm Nut Met Onc CHRU, Lille, France, 4Dept of Ped, Hospital, Bizerte, Tunisia, 5 Dept Ped Reanim, Children Hospital, Tunis, Tunisia, 6Dept Ped Ibn El Jazzar Hospital, Kairouan, Tunisia

PRELIMINARY DATA ON ADULT PATIENTS WITH UREA CYCLE DISORDERS (UCD) IN AN OPEN-LABEL, SWITCHOVER, DOSE-ESCALATION STUDY COMPARING A NEW AMMONIA SCAVENGER, GLYCERYL TRI (4PHENYLBUTYRATE) (HPN-100), TO BUPHENYL (SODIUM PHENYLBUTYRATE (PBA)) Lee B1, Garovoy MR2, Gargosky SE2, Berry SA3 1 Dept Mol and Human Gen, Baylor Coll Med, Houston, Texas, USA, 2 Hyperion Therapeutics Inc, South San Francisco, California, USA, 3 Dept Ped, Division of Gen and Metab, Minneapolis, Minnesota, USA

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SODIUM PHENYLBUTYRATE (AMMONAPS) IN THE TREATMENT OF PATIENTS WITH UREA CYCLE DISORDERS ^ EUROPEAN POST-APPROVAL DATA Dobbelaere DD Ref Center Metab Dis, Univ Hosp, Lille, France Objectives: To assess the e¤cacy (measured by hyperammonaemic crisis and neurological assessments) and safety (adverse events) of sodium phenylbutyrate in urea cycle disorder (UCD) patients. Methods: An analysis was performed of post-marketing case report forms (CRFs) from 187 patients with a UCD receiving sodium phenylbutyrate between 1999 and 2001 in 8 European countries. Results: Data show that 55% of patients were diagnosed at 528 days of age. Eighty-nine of 123 evaluable treated patients (72.4%) had no hyperammonaemic episodes requiring hospitalization within the ¢nal 12 months of follow up. The median plasma ammonia levels for patients who had an episode of decompensation were 2^6 times the median levels of plasma ammonia in corresponding groups with no episode of decompensation. Patients with early-onset disorders were statistically more likely to have neurological (p = 0.003) and cognitive problems (p = 0.025) than those with late-onset disorders. A bene¢t of early treatment (within 30 days) was demonstrated for neurological and cognitive problems in the early-onset group. Treatment-related adverse events were reported in only 9 of 187 patients. Conclusions: Notwithstanding the limitations of data collected from post-marketing CRFs, these results con¢rm the preponderance of patients with late-onset disorders (430 days), ¢rst described by Enns et al in a 2007 NEJM paper. Knowing that elevated plasma ammonia levels that result from metabolic decompensation may lead to detrimental neurological and cognitive changes, it was surprising that few hyperammonaemic crises during the treatment period were documented despite lower average dose of sodium phenylbutyrate in the EU than that approved by the EMEA.

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LONG-TERM QUANTIFICATION OF ENZYME TRANSFER BY LIVER CELL TRANSPLANTATION (LCT) FOR NEONATAL CARBAMOYLPHOSPHATE SYNTHASE 1 (CPS1) DEFICIENCY Meyburg J1, Nuo¡er JM2, Lindner M1, Bruns H3, Grenacher L4, Kriegbaum H5, Weitz J3, Schmidt J3, Ho¡mann GF1 1 Dept Gen Pediatr, Univ Child Hosp, Heidelberg, Germany, 2Dept Pediatr Metab, Univ Child Hosp, Berne, Switzerland, 3Dept Gen V|sc Transp Surgery, Heidelberg, Germany, 4Dept Diagn Intervent Radiol, Heidelberg, Germany, 5Cytonet GmbH and Co KG, Weinheim, Germany Background: Since the ¢rst use of LCT in inborn errors of metabolism 1997, several children have been treated with promising results. However, quanti¢cation of transplant success has been a major problem so far, especially in multifactorial diseases. Methods: Orthotopic liver transplantation (OLT) was performed 15 months after clinically successful LCT in a boy with neonatal CPS1 de¢ciency. A 3D reconstruction of the liver based on a preoperative MRI was used to plan multiple tissue samples of the explanted organ. During liver explantation, the in- and out£ow vessels were preserved as long as possible to achieve a minimal ischemia time. A total of 40 samples (1^1.5 ml) were obtained and immediately frozen. CPS1 activity was determined after thawing by citrullin formation. Results: Calculated enzyme activity in the explanted liver averaged 5.4% of healthy controls (range 0^30.8%). Enzyme activities were irregularly distributed in the liver with lowest ¢gures around the hilus in segment IV. There was no signi¢cant di¡erence between central and peripheral tissue samples. Discussion: We found a mosaic of original hepatocytes with no CPS1 activity and healthy transplanted cells that varied through the liver re£ecting the expected irregular pattern of engraftment. The total enzyme activity was 4.3 fold higher than expected from the original number of transplanted cells. Thus, it seems likely that the transplanted liver cells have proliferated, upregulated their urea cycle activity or both. Since the increase in enzyme activity correlates with clinical improvement, this is the ¢rst unequivocal proof of long-term success in human LCT.

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MLPA IN DIFFERENTIAL DIAGNOSTICS OF CONGENITAL ADRENAL HYPERPLASIA Vrzalova ZV1, Hrabincova EH1, Fajkusova LF1, Vseticka JV2, Kozak LK1 1 Center Molec Biol Gen Therapy, Univ Hosp, Brno, Czech Republic, 2 Dept Med Genet Ostrava, Ostrava, Czech Republic Background: Congenital adrenal hyperplasia (CAH) is a group of inherited diseases, in which cortisol secretion is impaired. 95% of CAH is associated with mutations in the CYP21 gene encoding the steroid 21hydroxylase. 90% of CYP21 mutations result from intragenic recombinations between CYP21 gene and CYP21P pseudogene (nonfunctional chimeric CYP21P/CYP21 genes and point mutations present in CYP21P and transferred to CYP21 are present). DNA diagnosis of CAH could not be closed at 25% of our CAH families. On this account, MLPA (Multiplex Ligation-Dependent Probe Ampli¢cation) has been introduced and subsequently tested in our patients. Methods: Long-range PCR, restriction analysis, sequencing and the MLPA analysis were performed. Results: We present an example of di¤culty during DNA analysis in one CAH family. DNA of a family member was sent us for determination of CAH carrying status. Despite that the woman had no clinical symptoms, we found that she carries the mutation I172N in the homozygous state. Consequently, other family members (normal and a¡ected) were examined but equivocal results were obtained. Using MLPA and DNA sequencing, we identi¢ed two types of nucleotide changes in the annealing position of the 3'-primer. Due to these changes, we could detect full, partial or no ampli¢cation of CYP21 alleles. Conclusions: A total of 197 unrelated CAH patients were examined. In 53 patients MLPA allowed us to ¢nish molecular-genetic diagnostics either by ¢nding polymorphic sites in annealing positions of primers (43) or by detection deletions (5) and duplications (5) of the CYP21 gene.

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PLASMA AND THROMBOCYTE LEVELS OF COENZYME Q10 IN CHILDREN WITH SMITH-LEMLI-OPITZ SYNDROME (SLOS) AND THE INFLUENCE OF HMG-CoA REDUCTASE INHIBITORS Haas D1, Niklowitz P2, Ho¡mann GF1, Andler W2, Menke T2 1 Div Metab Dis, Univ Child Hosp, Heidelberg, Germany, 2Child Hosp Datteln, Univ W|tten, Datteln, Germany Background: SLOS is caused by a defect of cholesterol synthesis. HMGCoA reductase inhibitors have been shown to improve biochemical parameters in this condition, but they have also been associated with CoQ10 de¢ciency in patients with hypercholesterolemia. The aim of this study was to analyse plasma and intracellular CoQ10 levels in SLOS patients and to determine the in£uence of HMG-CoA reductase inhibitors. Methods: Plasma concentrations of CoQ10 and vitamin E were measured in 14 patients; intracellular CoQ10 levels were determined in platelets of 10 patients with SLOS and compared to age-matched controls. Results: Plasma CoQ10 and vitamin E levels were signi¢cantly lower in SLOS patients. This di¡erence equalised after adjustment to cholesterol concentrations. Treatment with simvastatin did not in£uence CoQ10 levels and redox status. Platelet CoQ10 concentrations were similar between patients and controls but there were striking di¡erences in the CoQ10 redox status with reduction of oxidised CoQ10 Conclusion: Reduced concentrations of plasma CoQ10 and vitamin E in SLOS patients are due to a decreased carrier capacity. A redox shift in favour of the reduced part of CoQ10 in platelets points to an upregulation of mitochondrial protection mechanisms. Further studies are needed to evaluate a possible bene¢t of CoQ10 supplementation in SLOS patients.

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BILE ACID PROFILES IN PATIENTS WITH FOUR DIFFERENT BILE ACID BIOSYNTHESIS DEFECTS Ruiz-Sala P, Ferrer I, Briones M, Merinero B, Peèrez-Cerdaè C, Garcia MJ, Ugarte M CEDEM, Universidad Autonoma Madrid, Madrid, Spain Bile acid synthesis defects represent a speci¢c category of metabolic diseases. By the application of tandem mass spectrometry, it is possible to perform rapid diagnosis of inborn errors in bile acid synthesis from urinary bile acid analysis. We present four cases all showing almost absence of primary bile acids. Patient 1: (20 years) presented steatorrhea and xerophtalmia. Urinary bile acid analysis showed high excretion of di- and trihydroxycholenoic acids glycine and/or sulphated conjugated (m/z 462, 469, 485, 526, 542). These ¢ndings point to a 3b-hydroxy-D5C27-steroid dehydrogenase de¢ciency, later con¢rmed by molecular analysis. Patient 2: (4 months) was admitted with cholestasis and giant cells in liver. Urinary bile acid analysis showed high excretion of monoand dihydroxy-3-oxocholenoic acids glycine/taurine conjugated (m/z 444, 460, 494, 510). These results are consistent with a 3-oxo-D4steroid 5b-reductase de¢ciency. Patient 3: (30 years) presented spastic paraparesia and diarrhoea. High excretion of cholestanetetrol, -pentol, hexol and unsaturated glucuronides (m/z 611, 625, 627, 641, 643) were found in the bile acid analysis. Plasma sterol analyses by GC/MS showed high levels of cholestanol, 7-dehydrocholesterol, lathosterol and cholest-8-en-3-ol. These ¢ndings are consistent with a cerebrotendinous xanthomatosis. Patient 4: (27 days) was admitted with liver disease. Bile acid analyses showed increase of taurot e t r a hy d ro x y c h o l e s t a n o i c a c i d (m/z 5 7 2 ) i n u r i n e a n d trihydroxycholestanoic acid (m/z 449) in plasma. In addition, plasmatic VLCFA and pipecolic acid, and urinary 2-hydroxysebacic acid and epoxyacids were elevated. Plasmalogen analysis in erythrocytes was normal. These results are compatible with a biogenesis peroxisomal defect.

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DIAGNOSIS OF CHYLOMICRON RETENTION DISEASE BY MUTATION ANALYSIS OF THE SARA2 GENE Busch M1, Tsiakas K2, Bergmann J2, Heiduk M3, Zimmer KP4, Santer R2 1 Dept Int Med, Med Univ, Hannover, Germany, 2Dept Pediatr, Univ Medical Center, Hamburg-Eppendorf, Germany, 3Dept Pediatr, Univ Child Hosp, Magdeburg, Germany, 4Dept Pediatr, Univ Child Hosp, Giessen, Germany Congenital disorders of intestinal lipid transport are very rare and only few of the underlying molecular defects are known. Abetalipoproteinemia is caused by the de¢ciency of the 97 kDa subunit of the microsomal tr iglyc er i de transfer protei n ( MTTP). Hypobetalipoproteinemia is caused by a defect of apolipoprotein B. Chylomicron retention disease (CMRD, Anderson disease) has only recently been shown to be caused by a defect of Sar1b, a small GTPase involved in intracellular tra¤cking of protein-coated vesicles. Only 8 mutations of its gene, SARA2, have been reported so far. We follow 5 patients with intestinal lipid transport defects who all presented with steatorrhea, failure to thrive, and hypocholesterolemia. Three of them have abetalipoproteinemia ultimately diagnosed by detection of homoor compound heterozygosity for MTTP mutations. The other two were classi¢ed to have CMRD. Methods: Clinical characterization of the defect, electron microscopic and immunohistologic study of intestinal biopsies, automated direct sequencing of SARA2, the gene encoding the Sar1b protein. Results: We identi¢ed two novel mutations of the SARA2 gene: patient 1 was found to be compound heterozygous for c.49__50delCA [p. Q17VfsX8] (together with the previously published mutation c.409G4A [p. D137N]); patient 2 was homozygous for a long range deletion (ex4__7del). Conclusion: Mutation analysis of SARA2 facilitates di¡erential diagnosis of intestinal lipid transport disorders and allows to obviate invasive procedures such as an intestinal biopsy.

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HYPOCHOLESTEROLEMIA IN THE NEWBORN WITH TRISOMY 13 Bzduch V1, Behulova D2, Veghova E3 1 F|rst Dept Ped,Comenius Univ Child Hosp, Bratislava, Slovakia, 2Dept Lab Med, University Children's Hosp, Bratislava, Slovakia, 3Dept Clin Genet, Fac Hosp, Bratislava, Slovakia A male newborn with multiple anomalies was admitted to intensive care unit because of respiratory insu¤ciency 3 h after delivery. He had cheilognathopalatoschisis and small mandibule and attempt of intubation was not successful, so urgent tracheostomy was performed. Another dominant anomalies were microphthalmia and polydactyly of both hands. In di¡erential diagnosis, we speculated about Smith-LemliOpitz syndrome (SLOS), but although cholesterol was relatively low 0.76 mmol/L, its precursor 7-OH cholesterol was undetectable. We concentrated also on lipoprotein system, which is responsible for cholesterol transport in the blood and found evident abnormalities in serum apolipoproteins [Apo A^I 0.22 g/L, (range 1.04^2.02) and Apo B 0.19 g/L, (range 0.6^1.4)]. Analysis of peripheral blood revealed trisomy 13 (47 XY + 13). Patient died at the age of 15 days. Alkuraya et al. (Birth Defects Research Part A, 2005, 73: 569^571) described one unique patient with trisomy 13 with a biochemical pro¢le of SLOS. In series of neonates and foetuses with another multi-system malformation syndrome, trisomy 18, Lam et al. (Eur J Medical Genet 2006, 49:195^ 199) described also abnormally low cholesterol and the lack of increases in other sterol precursors. Our data suggest that abnormal low levels of serum apolipoproteins with sequential hypocholesterolemia and disturbed hedgehog signalling protein may be another hypothetic explanation for multiple malformations in both trisomies.

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INSULIN RESISTANCE IN PEDIATRIC PATENTS WITH FAMILIAL HYPERLIPIDEMIA Terlemez S1, Kalkan Ucar S1, Bozdemir E2, Goksen Simsek RD1, Darcan S1, Kabaroglu C2, Kayykcioglu M3, Habif S2, Bayindir O2, Coker M1 1 Div Ped Metab End, Ege Univ Child Hosp, Izmir, Turkey, 2Div Bioch, Ege Univ Hosp, Izmir, Turkey, 3Div Card, Ege Univ Hosp, Izmir, Turkey Familial hyperlipidemia (FH) is considered as a most common form of primary dyslipidemia and atherosclerosis, frequently associated with insulin resistance (IR), but there are few data on direct relation between FH and IR. The objective of this study was to evaluate IR, lipid pro¢le, free fatty acids and carnitine levels in pediatric patients with FH children of adult patients with FH. We studied 77 (41 female, 36 male) patients with age range between 4 and 18 years, selected by consecutive sampling at the pediatric or adult lipids Unit of our centre and all subjects were divided to hyperlipidemic (n = 52) and normolipidemic (n = 25) groups. Blood samples were obtained following an overnight fast of 12 h. Total cholesterol, triglyceride, HDL-cholesterol, Apo-B, glucose and insulin were measured by standard methods as previously described. The IR was calculated by HOMA-IR. There were no di¡erences in age, gender, BMI, abdominal circumference or systolic and diastolic blood pressure between groups. Plasma fasting glucose concentration was no di¡er between groups, however fasting insulin and HOMA-IR levels were signi¢cantly higher in hyperlipidemic group compared to normolipidemic group (p = 0.000, p = 0.000, respectively). No di¡erences were found in the levels of free fatty acids between the groups. We conclude that FH in pediatric patients is related to IR. So we suggest that the metabolic disturbances observed in adults with FH might begin in early childhood. However, further prospective studies are indicated in order to evaluate the impact of childhood IR in the pathogenesis of adult insulin resistant hyperlipidemic patients.

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X-LINKED STEROID SULPHATASE DEFICIENCY: REVIEW OF 8 CASES Santos H1, Leite I2, Moreira D1, Ferreira EO2, Marques JS1 1 Pediatr Dept, V|la Nova Gaia Hosp Cent, V|la Nova de Gaia, Portugal, 2 Dermatol Dept, V|laNova Gaia Hosp Cent, V|la Nova de Gaia, Portugal Background: X-linked ichthyosis is a generalized queratinization disorder, caused by steroid sulphatase (STS) de¢ciency, encoded by a gene located on the distal short arm of the X chromosome (Xp22.3). Methods: We reviewed cases ¢les of our STS patients, to characterize their presentation, age of onset and diagnosis, associated pathologies, and treatment response. Results: We studied 8 male patients, with current age between 18 months and 18 years, 3 followed in genetics outpatient department and the remaining 5 in dermatology consultation. Most were born by caesarean section. Ichthyosis showed between 1 week and 6 months of age and the diagnosis was ¢rst suspected at a median age of 1.6 years (0.4^12 years). Just two patients had not a clear family history of this disease. Diagnosis was made by measuring steroid sulphatase activity in skin biopsy, at a median age of 3 years. All except one had normal growth and development; one had language delay and 3 had atopic disease. No evidence of contiguous gene syndromes (as Kallmann syndrome) was found. All were treated with topical emollients and 2 with urea cream, with very discrete improvement. Conclusions: There was a considerable delay between ¢rst manifestations, clinical suspicion and con¢rmatory diagnosis. The presence of family history of this pathology should have contributed to an earlier diagnosis. The e¤cacy of available therapy is still disappointing. Atopic dermatitis can delay the diagnosis, but it can be an associated with this disorder. No cases of hypogonadism were detected in our patients.

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TYPE IIB LIPOPROTEIN PATTERN WITH ELEVATED LIVER ENZYMES AS PRESENTING SYMPTOM OF CHOLESTERYL ESTERS STORAGE DISEASE (CESD) Decarlis S, Agostoni Cv, Ferrante F, Scarlino S, Riva E, Giovannini M Dept of Ped, Univ Milan, S. Paolo Hosp, Milan, Italy Background: Lysosomal acid lipase (LAL) de¢ciency results in Wolman disease and cholesteryl esters storage disease (CESD), a more benign form. They're both manifested primarily by hepatomegaly and hypercholesterolemia. CESD has a variable phenotype and hepatomegaly is usually evident at birth or in early childhood. Case Report: An 11 year old girl was referred to our clinic for type IIb hyperlipoproteinemia (total cholesterol 323 mg/dl, triglycerides 259 mg/dl). All family members had normal lipid pro¢le and liver function tests. At 8 years she was admitted for acute Epstein^Barr virus infection, with hepatosplenomegaly and elevation of liver enzymes. Liver-spleen enlargement resolved, but serum alanine and aspartate aminotransferase were persistently twofolds the upper limit, with other liver function tests always within the normal range. Ultrasonography showed normal liver and spleen size and minimal hepatic steatosis. Infectious, autoimmune and metabolic causes of elevated liver enzymes were ruled out, including glycogen storage disease. Dysbetalipoproteinemia was also ruled out (ApoE phenotype: E3E3). In the following 2 years the girl was symptom-free, BMI percentile 50^758 percentile for age and lipid pro¢le unchanged despite a low-fat diet. At 13 years of age low acid lipase activity was demonstrated in leukocytes (10 nmol/h/ mgpt, nv 140^380) and cultured skin ¢broblasts (181 nmol/h/mgpt, nv 100^2400) leading to CESD diagnosis. Conclusion: All CESD cases usually progress to hepatic ¢brosis, with high risk of premature atherosclerosis. This diagnosis may be considered in all subjects with atypical typeIIb hyperlipoproteinemia (usually dominant in transmission and rarely evident in pediatric age) and atypical `fatty liver disease', in absence of overweight.

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FAMILIAL LIPOPROTEIN LIPASE DEFICIENCY IN THREE INFANTS ^ CASE REPORT Starostecka E1, Lange A1, Funkowicz ME2, Chmara M3, Krzyzçanowska J1 1 PolishMother's Health Memorial Institute, Lodz, Poland, 2Medical University, Lodz, Poland, 3Genetic Department, Medical University, Gdansk, Poland Familial lipoprotein lipase de¢ciency (MIM ^ 238600.0010) is a rare autosomal recessive disorder of triglyceride breakdown from chylomicrons and VLDL which usually presents in childhood. The incidence of LPL de¢ciency is 1 in 1 000 000 live births. LPL gene was identi¢ed on chromosome 8p22. Clinically it is characterized by chylomicronemia syndrome. We report the case of lipoprotein lipase de¢ciency in three children twin boys recognized at the age of 17 months (M.W, L.W), and a 2-month-old female infant (M.W). The disease was suspected in infancy because of lipemic plasma appearance. Lipid pro¢le revealed markedly elevated serum triglycerides (6000 mg/dl in twin boys) and 4700 mg/dl in a girl, fasting chylomicronemia. Clinically the boys presented episodes of acute abdominal pain, especially in the course of pancreatitis in one patient. M.W. had a history of failure to thrive and abdominal pain. Genetic testing was performed and the mutations in LPL gene were established: siblings p.R270H (exon 6) and (M.W) ^ homozygotic mutations (exon 5) ^ p.G215E. The nutritional treatment was introduced based on high fat restriction and supplementation MCT oil for cooking in older boys. The infant girl is fed with MCT formula. The e¡ect of dietary restriction was positive with decreasing triglyceride blood levels below 2000 mg/dl. But in follow up observation abdominal pain episodes were observed in siblings who are 13 years old now because of their poor dietary compliance. Conclusion: In every case of recurrent abdominal pain and acute pancreatitis chylomicronemia syndrome should be excluded.

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NEWBORN SCREENING FOR CAH USING MS/MS AS A PRIMARY SCREEN Lehotay DC1, Thompson R2, Van Caeseele P2, Taback S3 1 Saskatchewan Disease Control Laboratory, Regina, SK, Canada, 2 Cadham Provincial Laboratory, W|nnipeg, MB, Canada, 3University of Manitoba, W|nnipeg, MB, Canada Background: A method for newborn screening (NBS) for congenitala Adrenal hyperplasia (CAH) using MS/MS was developed for the Province of Manitoba, Canada. Method: 17-OH-progesterone (17-OHP), androstenedione, and cortisol were eluted from 3/16'' DBS using a TLX-2 Turbulent Flow System (Thermo F|sher), and analyzed by an API-4000 MS/MS (Applied Biosystems) as the primary screen for CAH, not a second tier test for con¢rmation. Results were compared to the DELFIA immunoassay screen for 17-OHP to evaluate performance. Results: 1500 sequentially collected samples were analyzed both by immunoassay for 17-OHP, and for three steroids by MS/MS. 2.3% of samples analyzed by immunoassay were screen positive and require further investigation. In contrast, 18/1500 samples by MS/MS had 17OHP concentrations above the cuto¡ of 25 nmol/L, a total of 1.2%. When the steroid ratio was also calculated for these 18 samples, only 2 had a ratio of 42.5. These were con¢rmed true positive CAH cases. The referral rate for further investigation of the 1500 samples was 0.13% when samples were analyzed by MS/MS, a *95% reduction in the follow up rate compared to the immunoassay. The positive predictive value (PPV) of the three steroid NBS assay by MS/MS was 100% in the ¢rst 1500 samples. Conclusions: The three steroid NBS assay for CAH using MS/MS as a primary screen is rapid, sensitive, and performs signi¢cantly better than the DELFIA immunoassay. Measuring 3 steroids by MS/MS as a primary screen for CAH eliminated the need for a second tier test to rule out CAH.

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ABNORMAL METABOLITE SCREENING IN A PATIENT WITH BERARDINELLI-SEIP CONGENITAL LIPODYSTROPHY Verhoeven-Duif NM1, de Koning TJ2, de Vroede MAM2, Hamers N1, de Sain-van der Velden MGM3, Prinsen BHCMT3, Jeninga EH1, Berger R3, Klomp L3, Kalkhoven E4 1 Dept Metab 38; Endocr Disases, UMCU, Utrecht, Netherlands, 2Dept Pediatrics, UMCU, Utrecht, Netherlands, 3Netherlands Metabolomics Center, Utrecht, Netherlands, 4Dept Pediatr Immunol, UMCU, Utrecht, Netherlands

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AUTOPSY OF A CASE OF GAUCHER DISEASE TYPE I ON ENZYME REPLACEMENT THERAPY. COMMENT ON DYNAMICS OF THE PERSISTING STORAGE PROCESS Hulkova H1, Poupetova H1, Kohout A2, Malinova V3, Elleder M1 1 Inst Metab Disord, F|rst Med Fac, ChUniv, Praha, Czech Republic, 2 Dept Pathol, Fac Med, ChUniv, Hradec Kralove, Czech Republic, 3Dept Pediatr Adol Med, Fac Med, ChUniv, Praha, Czech Republic

Background: Congenital lipodystrophies are characterized by alterations in body fat development and/or distribution, and insulin resistance. Berardinelli-Seip congenital lipodystrophy presents soon after birth with generalized absence or near-absence of adipose tissue and prominent musculature. Metabolic complications include diabetes, hypertriglyceridemia, and markedly depressed leptin and adiponectin. Results: We present a patient born after uncomplicated pregnancy and birth. At the age of one week he was admitted for weight loss and feeding problems. A progressive hepatomegaly was found. Laboratory investigations showed hyperglycemia and hypertriglyceridemia (415 mmol/L). A dexa scan revealed a markedly decreased percentage of body fat (1.4%). Furthermore, a left ventricular hypertrophy was noted. Histological analysis of liver biopsy showed massive micro- and macrovesicular steatosis, suggestive for a metabolic disorder. Metabolic investigations showed (highly) increased concentrations of cholesterol intermediates but a normal cholesterol concentration in plasma. Urinary organic acids at the ages of 3^8 weeks showed consistently increased ethylmalonic acid (60^120 mmol/mol creatinine, n57 mmol/mol creatinine) and mild elevations of fumaric and ketoglutaric acids. In combination with increased lactic acid in blood (3.4^6.3 mmol/L) these ¢ndings are suggestive for impaired mitochondrial function. An undetectable leptin concentration suggested a lipodystrophy, which was con¢rmed by the characterization of a novel splice site mutation in the SEIPIN/BSCL2 gene. Conclusion: In our patient a¡ected with Berardinelli-Seip congenital lipodystrophy, abnormal organic acids and cholesterol biosynthesis intermediates were found. Whether these ¢ndings are associated with the lipodystrophy should be investigated in additional patients.

Objectives: Evaluation of the storage status in autopsy specimens from adult female patient with Gaucher disease (GD) type I (mutation N370S/N370S) treated by imiglucerase for three years with signi¢cant clinical and laboratory improvement. After a high mountain tour she was a¡ected by pulmonary edema followed by sepsis, disseminated intravascular coagulation and multiorgan failure. She succumbed to cerebral hemorrhage aged 59. Autopsy revealed liver cholestatic cirrhosis and liver malignancy. Methods: Analysis of the storage process by histology, electron microscopy and histochemistry. Results: Analysis of the storage revealed broad range with its absence or very low levels in the bulk of the liver and spleen macrophages. Full blown storage was present only occasionally in several spleen nodules composed of typical Gaucher cells (GCs) most probably related to intrasplenic hemorrhage in anamnesis. Typical GCs were further seen in the leptomeninx especially of the cerebellum and as infrequent perivascular aggregates in both the grey and white cerebral matters. The bone marrow was heavily in¢ltrated by GCs especially in its adipocyte rich parts. Gaucher cells in this location displayed besides the typical striations also varied degree of vacuolation caused by cytosolic droplets of triglyceride interpreted as due to phagocytosis of necrotic white adipocytes. Conclusions: The observations point to relative e¤ciency of ERT in covering the standard substrate load which should not be exceeded as it would lead to evolution of the mature GCs known to display increased resistance to the therapy (reviewed by Elleder M. J Inherit Metab Dis 29:707^715, 2006).

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CLINICAL AND MOLECULAR GENETIC ANALYSIS AND TREATMENT OUTCOMES OF FOUR PATIENTS FROM THREE CHINESE FAMILIES WITH SITOSTEROLEMIA Yu HC1, Lu YH2, Lo MY1, Gao HJ1, Hsu JH1, Chong KW1, Kao CH1, Cheng KH1, Liu TT3, Hung PY1, Niu DM1 1 Dept of Pediatrics, VGH hospital, Taipei, Taiwan, 2Dept of Pediatrics, NTUH hospital, Taipei, Taiwan, 3Genome Research Center, Yang-MING Univ, Taipei, Taiwan

THE CLINICAL, GENETIC AND PATHOLOGICAL STUDIES OF 5 JAPANESE PATIENTS WITH PERINATAL LETHAL GAUCHER DISEASE Kobayashi M1, Ohashi T2, Fukuda T3, Eto Y4, Ida H1 1 Dept Pediatr, Jikei Univ Sch Med, Tokyo, Japan, 2Dept Gene Therapy, Jikei Univ Sch Med, Tokyo, Japan, 3Div Neuropathology, Jikei Univ Sch Med, Tokyo, Japan, 4Lysosomal Dis Res Cen, Jikei Univ Sch Med, Tokyo, Japan

Sitosterolemia, also known as phytosterolemia, is a rare autosomal recessive inherited disorder of lipid metabolism characterized by increased plant sterol absorption coupled with reduced biliary excretion, leading to signi¢cantly elevated plasma levels of plant sterols (sitosterol, sitostanol, campesterol, sigmasterol, etc) and sometimes shell¢sh sterol with normal to mildly elevated plasma levels of cholesterol. Patients with sitosterolemia mainly present with tendon and tub erous xanthomas, premature coronary and aortic atherosclerosis, arthralgia and arthritis. Abnormal liver function tests and hemolysis and/or thrombocytopenia are seen occasionally. In this study, we describe the clinical, biochemical, molecular genetic features and treatment outcomes of four patients with sitosterolemia, in three Chinese families. Four mutations, including one novel mutation (Y329X) and three known mutations (R446X, N437K, R389H) were identi¢ed in these three families. R389H is the most common mutation (50% of studied alleles) in our patients. This ¢nding is similar to Japanese sitosterolemia patients, in that R389H is also the most common mutation seen. All patients had a good response to one of the following treatments: diet control, ezetimide or cholestyramine therapy. Interestingly, in our study, two patients in the same family had di¡erent response to ezetimide; one had good response, whereas the other had no response.

Objective: G au cher di se ase i s c ause d by a de¢ ci e n cy of glucocerebrosidase, resulting in storage of glucocerebroside in reticuloendothelial system. Recently several cases with perinatal lethal form of Gaucher disease have been reported. The aim of this study is to clarify the clinical, genetic and pathological features of perinatal lethal Gaucher disease. Material and Method: The study patients were 5 Japanese patients with perinatal lethal Gaucher disease. Their diagnoses of Gaucher disease were made by demonstration of accumulated glucocerebroside in autopsied tissue in 4 cases and by enzyme analysis before death in 1 case. The mutation analysis was carried out by SSCP, PCR and direct sequence. Re sult: All of them had hydrop s, collodion skin and hepatosplenomegaly and died within 52 days after birth. The mutation analysis was carried out in 4 cases, and 1447^1466 del 20 ins TG mutation and L444P mutation were identi¢ed in 3 and 1 alleles out of total 8 alleles respectively. We also identi¢ed IVS2+1 mutation in 1 allele and other 3 alleles were unknown mutation. The massive accumulation of Gaucher cells was found not only in liver and spleen but in adrenal grand, thymus and brain. The brain pathological ¢ndings were characterized by destruction of architecture in addition to accumulation of perivascular Gaucher cells. Conclusion: Severe phenotype mutations such as 1447^1466 del 20 ins TG and L444P mutation were identi¢ed in perinatal lethal Gaucher patients. The profound de¢ciency of glucocerebrosidase activity may cause developing their severe clinical course and pathological ¢ndings.

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PLACEBO-CONTROLLED STUDY OF ALGLUCOSIDASE ALFA IN ADULTS WITH POMPE DISEASE van der Ploeg A1, Clemens PR2, Corzo D3, Escolar D4, Florence J5, Laforet P6, Lake S3, Mayhew J4, Morgan C3, Pestronk A5, Rosenbloom B7, Skrinar A3, Wasserstein M8 1 Erasmus Medical Ctr, Rotterdam, Netherlands, 2Univ of Pittsburgh, Dept of Neurology, Pittsburgh, USA, 3Genzyme Corp, Cambridge, USA, 4 Children's National Medical Ctr, Washington, DC, USA, 5Washington Univ School of Medicine, St. Louis, USA, 6Institut de Myologie, PitieSalpetriere, Paris, France, 7Tower Hematology Oncology, Beverly Hills, USA, 8Mount Sinai School of Medicine, New York, USA Background: Pompe disease (also known as acid maltase de¢ciency) is a rare, metabolic myopathy caused by a de¢ciency of acid a-glucosidase (GAA). In juveniles and adults, the disease is relentlessly progressive, leading to wheelchair dependency and respiratory failure. Methods: Patients were 48 years old, ambulatory, free of invasive ventilation, and had quanti¢able respiratory and lower extremity muscle weakness. Patients (randomized 2:1) received biweekly alglucosidase alfa (Myozyme. ) 20 mg/ kg IV or placebo for 78 weeks in the US and Europe. Distance walked in the 6 min walk test (6MWT) and % predicted forced vital capacity (FVC) were co-primary endpoints. Results: 90 patients (45 male, 45 female; 93% Caucasian; age range 10^70 years) were randomized. Baseline mean 6MWT distance was 327.4+128.0 meters (50.1% predicted) and mean FVC was 54.6+14.8% predicted, indicating considerable disease burden at baseline. By last evaluation, estimated mean absolute di¡erences of 28.1 +13.1 meters in 6MWT distance (p = 0.03) and 3.4+1.2% in % predicted FVC (p = 0.003) were observed in favor of Myozyme vs placebo. The frequency of adverse events and infusion associated reactions were comparable between groups. Three patients in the Myozyme group experienced hypersensitivity reactions and discontinued treatment. One patient in the Myozyme group died from causes unrelated to treatment. All evaluable patients in the Myozyme group (n = 59) developed IgG antibodies to rhGAA. A trend toward decreasing IgG titers was observed. Conclusions: In this ¢rst placebo-controlled study of rhGAA in juveniles and adults with Pompe disease, Myozyme was shown to improve walking and pulmonary outcomes compared to placebo.

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NEW MUTATION IN M. ANDERSON-FABRY: EFFECT ON LIPID COMPOSITION, LIPID RAFT-ASSOCIATED PROTEINS AND ENZYME TRAFFICKING Das AM1, Jia J2, Keiser M2, Naim HY2 1 Dept Paediatrics, Hannover Med School, Hannover, Germany, 2Dept Phys Chem, Univ Veterin Medicine, Hannover, Germany Background: The pathogenesis of clinical symptoms in M. Fabry based on AGAL-de¢ciency is still poorly understood. Previously, we have shown compromised function of respiratory chain proteins in mitochondria, now we looked at proteins in the plasma membrane. We describe here the novel mutation c.658 C4T in the coding region of AGAL in a patient with typical symptoms of M. Fabry Methods and Results: In cultured ¢broblasts from this patient the glycolipid content assayed by HPLC was 3.1% compared to 1.3% in wild type cells, phosphatidyl-inositol increased to 12.5% versus 8.9% in wild type. Confocal laser microscopy showed reduced amounts of labelled AGAL in the lysosome assessed by colocalization using LAMP II as a lysosomal marker. Enzyme stability was not a¡ected but tra¤cking was impaired. The e¡ect of an altered membrane lipid composition on protein association with lipid rafts and protein tra¤cking was investigated by transfection of patient's ¢broblasts with di¡erent membrane proteins. An altered tra¤cking pattern of the raftassociated protein dipeptidylpeptidase IV was demonstrated while the transport of aminopeptidase N which is not raft-associated showed normal transport to the cell surface. Conclusion: We describe a new mutation in M. Fabry leading to a change in cellular membrane-lipid composition in the cell and abnormal tra¤cking of AGAL to lysosomes. Moreover, the transport of raft-associated proteins is impaired compatible with dysfunction of the plasma membrane probably due to altered membrane composition.

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CHITOTRIOSIDASE ACTIVITY IN DRIED BLOOD SPOTS OF A BRAZILIAN SAMPLE: DETERMINING DEFICIENT AND INCREASED ACTIVITIES Rodrigues MDB1, Mu«ller KB1, Martins AM1, D'Almeida V2 1 CREIM-UNIFESP, Sa¬o Paulo, Brazil, 2D. Biosciences and CREIM ^ UNIFESP, Sa¬o Paulo, Brazil Background: Chitotriosidase (CT) is a macrophage glycosylhydrolase referred as a biochemical marker on diagnosis and prognosis for Gaucher disease (GD). Interestingly, about 6% of Caucasian population presents a de¢ciency in CT activity and it seems to be harmless considering that is not related to any disease. The aim of this work is to establish the percentage of CT de¢ciency in healthy volunteers (HV) and in a heterogeneous sample of Brazilian individuals and to evaluate CT activity in con¢rmed patients of the following diseases: GD, Fabry disease (FD) and Pompe disease (PD). Methods: We analyzed samples from 92 HV and from 21 patients, being 12 FD, 6 PD and 3 GD patients. Blood was collected to prepare dried blood spots on ¢lter paper (DBFP). CT activity was measured by a £uorimetric assay using punches of 1.5mm of the DBFP samples. Results: F|ve of our 92 HV presented a de¢cient activity of CT, establishing a percentage of 5.4% of our sample. The mean of activity was 24.95+9.51 mmol/L/h in not CT de¢cient HV, 343.39+115.08 mmol/L/h in GD patients, 16.95+8.92 mmol/l/h in FD patients and 9.62+7.29 mmol/l/h in PD patients. Conclusion: The percentage of CT de¢ciency found in our sample was similar to other populations and only GD patients presented increased CT activity in DBFP. Supported by: FAPESP, CNPq, AFIP and Genzyme do Brasil

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CHITOTRIOSIDASE DETERMINATION IN PLASMA AND IN DRIED BLOOD SPOTS: A COMPARISON USING TWO DIFFERENT SUBSTRATES IN A MICROPLATE ASSAY Rodrigues MDB, Oliveira AC, Mu«ller KB, Martins AM, D'Almeida V D. Biosciences and CREIM ^ UNIFESP, Sa¬o Paulo, Brazil Backgrounds: Chitotriosidase (CT) is a macrophage glycosylhydrolase considered a biochemical marker on diagnosis and prognosis for Gaucher disease (GD). The aim of the study was to validate a microplate assay for plasma and dried blood spots on ¢lter paper (DBFP) for CT activity determination and to compare CT activity using 4-methyl-umbelliferyl-N-N'-N''- triacetilchitotrioside (4MU-C3) and 4-methyl-umbelliferyl-deoxychitobiose (4MU-dC2) as substrates. Methods: Heparinized blood was collected from 12 healthy volunteers (HV) and 14 GD patients. Total blood was used to prepare DBFP and plasma was obtained by centrifugation. Plasma was stored at ^808C and DBFP at 48C until analysis. CT activity measurements were done on tube and microplate by a £uorimetric assay using 10 ml and 5 ml plasma and 3.0 and 1.5 mm punches of the DBFP samples, respectively. Results: Microplate assays for plasma and DBFP were validated (Pearson = 0.98). The use of 4-MU-C3 presented a good correlation across the entire group (Pearson = 0.76), but weaker for GD subjects (Pearson = 0.67). The use of 4MU-dC2 increased the correlation for this group of individuals (Pearson = 0.89). Conclusion: The possibility of DBFP use for CT activity determination on a microplate assay improves laboratory routine. The use of 4MUdC2 may be more appropriate for GD patients' evaluation. Supported by: FAPESP, CNPq, AFIP and Genzyme do Brasil

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DIARRHOEA AND HEPATOCELLULAR CYTOLYSIS PROFILE LEADING TO POMPE DISEASE DIAGNOSIS Dobbelaere DD1, Alexandre CA1, Laforeªt PL2, Mention KM1 1 Univ Metab Referral Center, Lille, France, 2Univ Neuromusc Referral Center, Paris, France Objective and Background: The objective was to determine whether digestive symptoms can predict Pompe disease. Pompe disease is an autosomal recessive type 2 glycogenosis caused by de¢ciency of acid alpha glucosidase (GAA). The infantile-onset form usually starts with rapidly progressive hypotonia, generalized muscle weakness, and hypertrophic cardiomyopathy. The juvenile- or late-onset patients experience as ¢rst symptoms a more slowly progressive dysfunction of limb girdle and respiratory muscles. The diagnosis is supported by frequent total and myocardial-band creatine kinase elevations and inconsistent transaminases with ASAT4ALAT and LDH elevations. Low GAA activity con¢rms the diagnosis. Swallowing di¤culties, and gastro-oesophageal re£ux, are much less frequent except macroglossia and hepatomegaly which occur in more than 60% of the infantile-onset form. Methods: We report here the medical history of a 2.5-year-old girl, and analyse the French registry data on liver and gastro-intestinal symptoms. Results: The medical history of the young girl started with abdominal pain and diarrhoea. Hepatomegaly was moderate with a liver cytolysis pro¢le. Liver function and structure exploration led to histological examination which showed glycogen overload. The GAA activity was low. The French registry data on juvenile and adult-onset patients (n = 62) list the following liver and gastro-intestinal symptoms: dysphagia (12.9%), gastro-oesophageal re£ux (12.9%), diarrhoea (19.35%), and hepatomegaly (8.06%). Conclusions: In this case study, diarrhoea was predictive of Pompe disease. Although rarely described in the literature, the French registry data con¢rm that digestive symptoms should be considered in the diagnosis of this disease.

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SLOWLY PROGRESSIVE CLINICAL PHENOTYPE OF SANFILIPPO A PATIENTS EXHIBITING THE MUTATION p.S298P Meyer A1, Kossow K2, Gal A3, Mu«hlhausen C1, Ullrich K1, Braulke T1, Muschol NM1 1 Univ Med Center Hamburg, Dept of Ped, Hamburg, Germany, 2Univ Med Center Hamburg, Inst Med Psych, Hamburg, Germany, 3Univ Med Center Hamburg, Inst of Genet, Hamburg, Germany Introduction: Mucopolysaccharidosis type IIIA (MPS IIIA, San¢lippo syndrome) is caused by lysosomal N-sulfoglucosamine sulfohydrolase de¢ciency and leads to a defective degradation of heparan sulfate. The onset and progression of the disease is highly variable. At present seventy-¢ve mutations distributed over the SGSH gene have been described. Any relationship between the gene mutation and the onset of symptoms and clinical course of the disease is unknown. Methods: The natural course of the disease was assessed in 71 MPS IIIA patients using a detailed questionnaire and a `Four-Point Scoring System' (FPSS). In 54 of these patients an analysis of the underlying mutations was conducted. Results: By assessing the degree of developmental regression over time a group of seven patients with a slowly progressive course of the disease were identi¢ed. In these seven patients and in 3 other mildly a¡ected patients the missense mutation p.Ser298Pro was found on one allele. These patients showed a lower frequency and later onset of the typical symptoms of the disease. The onset of regression in speech abilities and cognitive functions were delayed by 0.7 and 0.8 years, respectively, and the onset of regression of motor functions occurred 6.1 years later than in all other MPS IIIA patients. Severe regressions in speech, cognitive and motor functions were delayed by 5, 5.9 and 11.2 years, respectively. Conclusion: These data suggest that in MPS IIIA patients carrying the mutation p.Ser298Pro a slowly progressive phenotype can be predicted and this may have an important impact on parental counselling and therapeutic interventions.

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LIMP-2 SORTING PATHWAY: A CELL TYPE SPECIFIC DEFICIENCY OF BETA-GLUCOCEREBROSIDASE Balreira A1, Gaspar P1, Caiola D2, Chaves J3, Beira¬o I4, Lopes Lima J3, Azevedo JE5, Saè Miranda MC2 1 UniLiPe, IBMC, Porto, Portugal, 2UniLiPe, IBMC, Porto, Portugal, 3 Servic°o de Neurologia, Hosp St Antoènio, Porto, Portugal, 4Servic°o de Nefrologia, Hosp St Antoènio, Porto, Portugal, 5OBF, IBMC, Porto, Portugal Background: LIMP-2 is a transmembrane protein mainly found in lysosomes and late endosomes. Besides being required for the biogenesis and maintenance of the lysosomal/endosomal system it was recently shown that LIMP-2 is the mannose-6-phosphate independent receptor for betaglucocerebrosidase, the lysosomal enzyme de¢cient in most cases of Gaucher disease. It was recently proposed, that LIMP-2 binds betaglucocerebrosidase in the endoplasmic reticulum and escorts the enzyme to the late endosomes/lysosomes. Methods: A patient presenting progressive myoclonic epilepsy without intellectual impairment and a nephrotic syndrome with a strong accumulation of C1q in capillary loops and mesangium of kidney was studied. Enzymatic activities of several lysosomal enzymes were determined in ¢broblasts, leukocytes and plasma. The LIMP-2 gene was analysed. LIMP-2 and beta-glucocerebrosidase were analysed by western blotting and Endo H assay. Results: Biochemical analysis revealed a normal beta-glucocerebrosidase activity in leukocytes but a severe enzymatic de¢ciency in cultured skin ¢broblasts. The patient did not present activated macrophages or increased levels of chitotriosidase, the hallmark of Gaucher disease. Molecular studies of the LIMP-2 gene, the sorting receptor for beta-glucocerebrosidase, revealed a homozygous nonsense mutation in codon 178. Besides lacking immunodetectable LIMP-2, patient ¢broblasts presented also alterations in isoforms and amounts of beta-glucocerebrosidase which presented to be Endo H sensitive. Conclusions: Enzymatic results suggested a defect in the intracellular sorting pathway of beta-glucocerebrosidase, con¢rmed by the LIMP-2 de¢ciency, which resulted in an endoplasmic reticulum location of betaglucocerebrosidase.

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SURROGATE BIOCHEMICAL MARKERS FOR LYSOSOMAL STORAGE DISORDERS Fuller M, Hopwood J LDRU Women's and Children's Hospital, Adelaide, Australia The wide phenotypic variability associated with lysosomal storage disorders (LSD) poses many caveats to the e¡ective clinical management of these patients. LSD are characterized by the accumulation of undegraded material in lysosomes of a¡ected cells. The primary storage material is usually the substrate for the de¢cient enzyme in each LSD, and may consist of glycosphingolipids such as glucosylceramide or trihexosylceramide in Gaucher and Fabry diseases respectively; sphingomyelin or cholesterol in Niemann-Pick A/B or C; GAG (glycosaminoglycans) in the MPS (mucopolysaccharidoses); or glycogen in Pompe disease. For many LSD, the primary storage has been shown to result in impaired lysosomal function leading to the accumulation of secondary metabolites that are not substrates for the de¢cient enzyme. We have used electrospray ionization-tandem mass spectrometry (ESI-MS/MS) to identify and quantify primary substrates as well as secondary altered metabolites in urine, blood and skin ¢broblasts in a variety of LSD. These metabolites include low molecular weight GAG fragments isolated and identi¢ed in urine of MPS patients as well as over 100 individual species of sphingolipids and phospholipids. We demonstrate that either individual metabolites or pro¢les of metabolites can be diagnostic for each LSD, can also yield information on prognosis, and further show promise as surrogate biochemical markers of therapeutic strategies. The opportunity to pro¢le metabolites is likely to provide a more detailed picture of disease burden in LSD patients, leading to improved methods for diagnosis, prediction of clinical course and biochemical monitoring of therapy.

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PHASE 2 CLINICAL TRIALS OF THE PHARMACOLOGICAL CHAPERONE AT1001 FOR THE TREATMENT OF FABRY DISEASE Germain DP1on Behalf of AT1001 Study Group, Castelli J2, Shenker A2, Do H2, Wustman B2, Palling D2, Lockhart DJ2 1 Universiteè de Versailles, Garches, France, 2Amicus Therapeutics, Cranbury, New Jersey, USA

CHAPERONE EFFECTS ON MOLECULAR PATHOLOGY IN GM1-GANGLIOSIDOSIS Suzuki Y1, Nanba E2, Higaki K2, Sakakibara Y3, Jo H3, Yugi K3, Iida M4, Ogawa S3 1 International Univ Health and Welfare, Otawara, Japan, 2Tottori University, Yonago, Japan, 3Keio University, Yokohama, Japan, 4 Seikagaku Corporation, Higashi Yamato, Japan

Background: AT1001 (migalastat hydrochloride) is an orally administered, small molecule pharmacological chaperone designed to selectively bind the lysosomal enzyme alpha-Galactosidase A (alphaGal A), thereby increasing alpha-Gal A stability, tra¤cking to the lysosome, and cellular activity. Pre-clinical studies revealed that AT1001 treatment of Fabry transgenic mice expressing a mutant form (R301Q) of alpha-Gal A found in some Fabry patients can increase alpha-Gal A activity and signi¢cantly reduce levels of the substrate globotriaosylceramide (GL-3) in heart, kidney, skin and plasma. In previous Phase 1 trials, AT1001 was shown to be safe and welltolerated and to increase alpha-Gal A levels in leukocytes in healthy human volunteers. Methods: A set of Phase 2 clinical trials were recently conducted to evaluate the e¡ects of AT1001 given at di¡erent doses and dose regimens. Results: AT1001 was generally safe and well-tolerated at all doses evaluated. Twenty-four out of 26 patients demonstrated an increase in alpha-Gal A as measured in leukocytes, kidney, and skin. Kidney GL-3 levels as measured in urine or biopsies were decreased in patients who demonstrated greater increases in levels of alpha-Gal A. Renal and cardiac function results were encouraging, including those in patients treated for nearly two years. Patient alpha-Gal A and GL-3 responses were consistent with the results of in vitro testing of Fabry mutations, suggesting that a pharmacogenetic approach may be used to select likely responders for future studies. Conclusion: These data suggest AT1001 merits further investigation as a potential therapy for Fabry disease.

Background: The concept of chemical chaperone therapy has been con¢rmed by animal laboratory experiments for biochemical, pathological and clinical e¡ects mainly in GM1-gangliosidosis. We assessed molecular and metabolic e¡ects of a chaperone compound N-octyl-4-epi-betavalienamine (NOEV) on cells and tissues expressing mutant betagalactosidase in GM1-gangliosidosis. Methods: Assays of in vitro enzyme inhibition and in situ chaperone e¡ect by valienamine derivatives; computational modeling of the human betagalactosidase structure and theoretical calculation of NOEV-enzyme interaction; NOEV e¡ect on intracellular signal transduction, autophagy, and gene expression. Results: NOEV was the most e¡ective inhibitor and enhancer among the valienamine derivatives tested. Computational prediction of human betagalactosidase illustrated the TIM barrel structure commonly known for glycosidases. The NOEV-enzyme binding was less strong in the lysosomal environment, resulting in dissociation of the complex. DNA microarray revealed an abnormal pattern of gene expression in enzyme-de¢cient human and mouse ¢broblasts, and mouse brain. Phosphorylated Trk and Bip/GRP78 abnormally increased at the late clinical stage of GM1-gangliosidosis in the mouse brain. These pathological changes were corrected by NOEV treatment. Conclusions: Computational modeling of wild-type and mutant betagalactosidase molecules is useful for analysis of molecular pathology in GM1-gangliosidosis. In addition we found various intracellular abnormalities associated with a single enzyme de¢ciency. Correction of these pathological phenomena will be necessary for treatment of phenotypic expression in GM1-gangliosidosis. These new approaches are important for understanding chemical pathology and developing new therapeutic trials in lysosomal diseases.

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DISEASE SEVERITY IN MULTIPLE SULFATASE DEFICIENCY IS DETERMINED BY STABILITY AND RESIDUAL ACTIVITY OF MUTANT FORMYL-GLYCINE-GENERATING ENZYME Schlotawa L1, Dierks T2, Schmidt B3, Ga«rtner J1 1 Dept Ped and Ped Neurol, Univ of, Go«ttingen, Germany, 2Dept Chem, Biochem I, Univ, Bielefeld, Germany, 3Biochemistry II, Univ of Go«ttingen, Go«ttingen, Germany Multiple sulfatase de¢ciency (MSD) is a rare inborn error of metabolism, which combines clinical and biochemical features of single sulfatase de¢ciencies due to a combined loss of cellular sulfatase activities. MSD is caused by hypomorphic mutations in the SUMF-1 gene encoding the formylglycine-generating-enzyme (FGE). FGE posttranslationally generates formylglycine in the active site of newly synthesized sulfatases necessary for activity. The clinical course displays neonatal severe to mild juvenile cases. We analysed 11 missense and nonsense FGE mutations and referred them to the patients' clinical phenotype. All mutations were expressed and did not a¡ect localization of FGE in the endoplasmic reticulum. They severely a¡ected FGE activity with residual activities between 1% and 23%. We found two mutations leading to stable proteins (p.A177P, p.W179S), ¢ve to instable proteins still partially passing intracellular quality control mechanisms (p.G247R, p.G263V, p.A279V, p.R345C, p.R349W) and four mutations leading to complete protein instability (p.S155P, p.R224W, p.del149173, p.R327X). Mutations leading to instable proteins with high residual activity as well as stable proteins with low activities were found in patients with mild clinical phenotype, mutations leading to instable protein with low residual activity result in a severe phenotype. Nonsense mutations are present in very severe neonatal form and cause instable protein and very low residual FGE activity. Our results underline a consistent dependence of clinical phenotypes from residual FGE activity as well as from protein stability.

MIGLUSTAT IN PATIENTS WITH NIEMANN-PICK TYPE C DISEASE (NPC): A MULTICENTRE RETROSPECTIVE SURVEY Pineda M1, Wraith JE2, Sedel F3, Hwu WL4, Rohrbach M5, Bembi B6, Korenke GC7, Luzy C8, Schieber P8, Patterson MC9 1 Hosp St Joan de Deu, Barcelona, Spain, 2Royal Manchester Children's Hosp, Manchester, UK, 3Hoªpital Pitieè Salpeªtrie©re, Paris, France, 4 National Taiwan University Hosp, Taipei, Taiwan, 5Zu«rich Kinderspital, Zu«rich, Switzerland, 6Udine University Hosp, Udine, Italy, 7Elisabeth Kinderkrankenhaus, Oldenburg, Germany, 8Actelion Pharmaceuticals Ltd, Allschwil, Switzerland, 9Mayo Clinic, Rochester, USA Background: A clinical trial has suggested that miglustat slows disease progression in patients with NPC [1]. Here we present the interim results from an ongoing international survey retrospectively assessing neurological disease progression in NPC patients treated with miglustat. Methods: This survey includes patients with NPC receiving or having received miglustat. Treating physicians are asked to complete a questionnaire on patient demographics, treatment history, disease progression and general health. A disease-speci¢c disability scale [2] is used to assess dysphagia, dystonia, ataxia and dysarthria severity at diagnosis, treatment initiation and last visit. Results: As of 17 December 2007, 44 patients were included in 17 centers. Median (range) miglustat exposure was 17.4 (0.6^40.2) months. Of 43 evaluable patients (mean+SD age: 9.9+7.2 years at diagnosis, 14.2+9.5 years at miglustat initiation), most remained at least stable after treatment with miglustat as regards dysphagia (n = 36, 84%), dystonia (n = 33, 77%), ataxia (n = 31, 72%), and dysarthria (n = 31, 72%). Thirty-two (74%) patients were classi¢ed as good responders, with at least 3 out of the 4 parameters rated as stable or improved. According to physicians' global assessments, 16/41 (39%) patients improved in general health, and 30/38 (79%) patients experienced good/fair bene¢t. Intention to continue therapy was recorded in 31/33 (94%) patients. Conclusion: These data suggest that miglustat may provide clinically relevant bene¢ts on neurological disease progression in patients with NPC, and are consistent with results from a previous clinical trial [1] in the same patient population. [1] Patterson et al. Lancet Neurol 2007;6:765^72. [2] Iturriaga et al. J Neurol Sci 2006;249:1^6.

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JUVENILE-ONSET NEURONAL CEROID LIPOFUSCINOSIS (JNCL, BATTEN): CLINICAL AND MOLECULAR INVESTIGATION IN A LARGE FAMILY IN BRAZIL Valadares ER1, Amorim RHC1, Oliveira LR1, Pinheiro TMM1, Santos HH1, Pizarro MX2, Queiroz RR2, Lopes GC2, Godard ALB2 1 Faculdade de Medicina da UFMG, Belo Horizonte, Brazil, 2 Departamento de Biologia Geral da UFMG, Belo Horizonte, Brazil Background: JNCL is the most common neurodegenerative disorder of the childhood. Symptoms are blindness, progressive psychomotor deterioration, dementia and seizures, starting around 5 years of age (Munroe et al., 1997). It is an autosomal recessive disease and can be caused by several mutations in the CLN3 gene. The CLN3's protein loss of function leads to the lysosomal accumulation of auto£uorescent material in patient`s tissues (Kwon et al., 2005), leading to neocortical neurons death (Peltonen et al., 2000). Methods: Clinical and molecular analysis of members of a large Brazilian family with JNCL composed of 2 consanguineous couples (brother and sister who got married with their ¢rst-cousins, also brothers) and their kindred of 30 children. Molecular study was performed by allele-speci¢c PCR ampli¢cation. Results: Six children died with neurological regression without diagnosis, 3 have classical symptoms of JNCL and 1 that is six-yearsold has only abnormal retinal pigmentation. Besides the parents, 10 children were examined (the 4 symptomatic and 6 asymptomatic). The molecular analysis detected the 1.02 kb deletion in both of the mutated alleles in all symptomatic children. All the others analyzed were heterozygote for this mutation. Conclusions: The studied family presented the 1.02 kb deletion in the CLN3 gene, which is the most common mutation in JNCL. This mutation extinguishes exons 7 and 8, resulting in protein truncation. The genetic counseling was provided to this family.

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LENTIVIRUS MEDIATED GENE THERAPY FOR MURINE MODEL OF POMPE DISEASE Kyosen SO1, Iizuka S1, Morita A1, Kimura T2, Kobayashi H1, Eto Y3, Ida H4, Ohashi T1 1 Dept Gene Ther, Jikei Univ Sch of Med, Tokyo, Japan, 2Dept Urology, Jikei Univ Sch of Medicine, Tokyo, Japan, 3Res Cent Lys Dis, Jikei Univ Sch of Med, Tokyo, Japan, 4Dept Ped, Jikei Univ Sch of Med, Tokyo, Japan Background: Pompe disease results from mutations in the gene encoding the glycogen-degrading lysosomal enzyme acid a-glucosidase (GAA). Methods: Full-length GAA cDNA was excised with EcoRI from the pJW59 plasmid, and ligated into the lentiviral plasmid pRRL-sin. hCMV-MCS(R)Deco-cppt. The pRRL-hCMV-cppt-GAA vector plasmid was transfected into 293T cells, puri¢ed and concentrated by ultracentrifugation. We performed the intravenous injection of 7.56107 infectant units in newborn Gaa^/^ mice. Mice were sacri¢ced 4 weeks and 8 weeks after injection and biochemical analysis was performed in tissue homogenates. Results: After 4 weeks of injection in newborn mice, the GAA activity (nmol/h/mg protein) was 428.19+264.8, 181.68+54.97 and 90.01+24.94 in heart, liver and quadriceps respectively, while in the untreated group, it was 332+3.12, 108.93+0.34 and 39.09+0.29 in heart, liver and quadriceps respectively, and this di¡erence had a p50.05 in two tailed Student's test in all of tissues. After 4 weeks of treatment, the glycogen concentration in treated mice was 314.89+206.87, 1146.76+427.08 and 676.31+326.04 in heart, liver and quadriceps respectively, while in untreated mice it was 1373.07+133.39, 2578.81+275.49 and 938.82+99.75 in heart, liver and quadriceps respectively, and this di¡erence had a p50.05 in two tailed Student's test in heart and liver. After 8 weeks of treatment, there wasn't any statistically signi¢cant di¡erence between treated and untreated group regarding GAA activity and Glycogen concentration. Conclusion: Our results showed that recombinant lentiviral vector system was e¤cient in the samples analysed. Acknowledgements: Dr YT Chen (Duke University-USA) for providing us the GAA cDNA, Dr N Kasahara (UCLA-USA) for providing the lentivirus generating system.

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REFERENCE LIMITS FOR COMPONENTS WITH STRONG AGE DEPENDENCE FROM LABORATORY PRODUCTION DATA EXEMPLIFIED BY URINARY GLYCOSAMINOGLYCANS (U-GAG) MÖrkrid L Dept Med Biochem Rikshospital Med Center, Oslo, Norway Background: Components with a strong age-dependence may require collection of samples from a large number of healthy individuals, evenly distributed within narrow age intervals (bins), to establish reliable reference limits. This can be avoided by describing the central tendency (mean or median) in each bin by a function, e.g. f (age, p1, p2, pk), and using regression analysis to ¢nd a unique dependence between the central tendency and age. Method: Transformation of age and component values may simplify this function and make the dispersion around the regression curve approximately symmetric and independent of age. A uniform standard deviation (s) around the regression line can be obtained from the residuals and facilitates the calculation of continuous age-dependent reference limits. W|th low-prevalence disorders like inborn errors of metabolism, adequate exclusion of outliers in a laboratory production may render the tedious collection of samples from healthy individuals unnecessary. Material: The regression method outlined above was applied on routine urine samples analyzed with respect to GAG-levels (mg/mmol creatinine) from 824 girls and 1233 boys, aged 1 month^14 years. Results: There was no sex di¡erence. Lg GAG = p1 + p2/age60.25 + e, where e represented the residuals, described the age relationship. F|ftyfour regression outliers were excluded. s = 0.102. Reference limits for lg GAG were 1.9731^0.57388/age60.25+1.96/0.102. Corresponding limits for GAG were found by retransformation. Other examples will be given. Conclusion: Appropriate regression models may simplify the calculation of reference limits when there is a strong age-dependence.

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THE NEUROLOGICAL MANIFESTATIONS OF GAUCHER'S DISEASE TYPE 1: THE FRENCH OBSERVATOIRE ON GAUCHER DISEASE (FROG) Cherin P1, Rose Ch2, De Roux Serratrice Ch3, Dobbelaere D4, Grosbois B5, Hachulla E6, Jaussaud R7, Javier RM8, Noel E9, Clerson P10, Hartmann A1 1 Hoªpital de la Pitie-Salpetriere, Paris, France, 2Hoªpital St V|ncent de Paul, Lille, France, 3Hoªpital Saint Joseph, Marseille, France, 4Hoªpital Jeanne de Flandre, Lille, France, 5Hoªpital Sud, Rennes, France, 6Hoªpital Claude Huriez, Lille, France, 7Hoªpital Robert Debre, Reims, France, 8 Hoªpital de Hautepierre, Strasbourg, France, 9Hoªpital Civil, Strasbourg, France, 10Orgametrie, Roubaix, France Background: Type 1 Gaucher disease (GD1), the most common variant, is classically considered non-neuronopathic. This French national prospective study was implemented for describing the clinical aspects of an adult GD cohort. The patients were systematically evaluated for the presence of neurological signs and symptoms. Methods: Clinical data with complete neurological assessment were collected during a routine visit. No additional tests were performed. The CRF was designed for guidance of the physician as an educational tool. Results: From May 2005 to September 2006, 105 GD1 patients were included (mean age 45.6+13.7 (m+SD) years). Thirty-¢ve percent of patients had at least one sibling with GD and 19% had a family history of Parkinson's disease. Out of the thirty-eight patients with genotyping, 71% had a N370S mutation and 29% had at least one L444P or D409H mutation. F|fty-one patients presented with at least one neurological symptom. Four patients (aged 63, 67, 70 and 74 years old) su¡ered from Parkinson's disease and 22 had Parkinson symptoms or signs frequently associated with Parkinson. These patients were older at diagnosis of GD1, respectively 52+4 and 30+16 years old versus 24+12 years old for the patients without Parkinson's signsMyoclonia occurred in 6 patients, peripheral neuropathies in 5 and epilepsy in 2 patients. Conclusions: These data challenge the current classi¢cation of Gaucher's disease suggesting either a continuum between the three forms or three di¡erent neurological phenotypes. Evaluation of therapeutic options for the neurological dysfunction of GD1 is also needed.

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DIGITAL MICROFLUIDICS: A NOVEL PLATFORM FOR MULTIPLEXED DETECTION OF LYSOSOMAL STORAGE DISEASES WITH POTENTIAL FOR NEONATAL SCREENING Millington D1, Srinivasan V2, Eckhard A2, Cotten C1, Goldberg R1, Pamula V2 1 Dept Pediatr, Duke Univ, Durham, NC, USA, 2Advanced Liquid Logic, RTP, NC, Canada Digital micro£uidics is a new technology that allows reliable dispensing, high-speed transport, rapid mixing, dilution, and disposal of submicroliter sized droplets through software control. This technology could reduce assay costs and specimen requirements with an inexpensive instrument and enzyme-speci¢c disposable chips by multiplexing enzymatic and other types of assay including immunoassays on the same chip. Objective: Demonstrate feasibility of performing multiplexed enzymatic assays for Pompe disease (acid alpha-glucosidase (GAA) de¢ciency), and other LSDs using extracts from dried blood spot (DBS) samples on the digital micro£uidic platform. Methods: Extracts from a single 3 mm DBS punch and enzyme substrates for Pompe, Fabry and Hurler disease plus beta-Dglucuronidase (control) were loaded into reservoirs on a chip programmed to dispense, transport, mix, and incubate 0.3 ml droplets of DBS extract plus ennzyme substrates in separate regions of the same chip. Enzyme activities in the sample cleave £uorogenic substrates and form 4-methylumbelliferone (4-MU) which was measured o¡ the chip using a custom-built micro-£uorometer. Results: A kinetic assay was developed on-chip which reduced time to result from 18 h to 2 h. Normal control specimens and disease positives were assayed and gave results consistent with those measured on the bench by conventional methods. Up to eight fully automated assays performed simultaneously consumed 1.2 ml from a total extract of 150 ml. The results indicate that digital micro£uidics o¡ers an e¤cient means to assess activity of multiple LSD enzymes from the same DBS extract. The technology is well suited to high throughput applications such as neonatal screening.

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REDUCTION OF LEFT VENTRICULAR HYPERTROPHY CORRELATES WITH A SIGNIFICANT DECREASE IN B-TYPE NATRIURETIC PEPTIDE IN A PATIENT WITH INFANTILE POMPE DISEASE TREATED WITH RECOMBINANT ACID ALPHA-GLUCOSIDASE Berkau I1, Lilje C2, Hahn A3, Muschol N1, Santer R1, Ullrich K1 1 Dept Pediatr, Univ Medical Center, Hamburg-Eppendorf, Germany, 2 Dept Pediatr Cardiol, Univ Med Center, Hamburg-Eppendorf, Germany, 3Dept Pediatr Neurol, Univ Giessen, Giessen, Germany Introduction: Pompe disease is a rare autosomal recessive lysosomal glycogen storage disorder caused by a de¢ciency of lysosomal acid alpha-glucosidase (GAA). Infantile Pompe disease usually results in massive left ventricular hypertrophy (LVH) and heart failure. Untreated it is fatal within the ¢rst year of life. To date, no reliable marker of improvement of ventricular function during enzyme replacement therapy (ERT) has been described. Several authors have suggested that serum concentration of B-type natriuretic peptide (BNP) correlates with the severity of ventricular dysfunction (Hahn et al., 2006; Mir et al., 2006) and that BNP therefore might be used as a parameter for assessing the improvement of heart function under ERT in Pompe disease. Patient: We report on a ¢ve month old female patient with infantile onset Pompe disease. Echocardiography revealed severe biventricular hypertrophy. During the ¢rst 12 weeks of ERT left ventricular hypertrophy decreased remarkably. Serum concentration of BNP were determined every fortnight and decreased over this period from 3021 ng/L to 331 ng/L. Conclusion: ERT with recombinant human acid alpha-glucosidase reduces LVH in Pompe disease. BNP seems to be a parameter to monitor cardiac function during ERT.

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MUCOPOLYSACCHARIDOSIS TYPE II (MPS II): EVALUATE OF FOOD INTAKES IN PATIENTS ON ENZYME REPLACEMENT THERAPY Oliveira RB, Frangipani BJ, Lauandos JR, Micheletti C, Silveira MT, Vertemati T, Martins AM CREIM/UNIFESP, Sa¬o Paulo, Brazil Introduction: MPS II is a rare X-linked recessive disease caused by de¢ciency of the lysosomal enzyme iduronate-2-sulphatase, leading to a variable, progressive, multisystem disorder. Clinical manifestations include airway obstruction, skeletal deformities, hepatomegaly, macrocephaly and development delay. Objective: To evaluate the patients feed intakes with MPS II in enzyme replacement therapy (ERT). Methods: It was evaluated 5 patients with MPS II in ERT. It was requested to those whom are responsible for the patients to make the food's records of 3 consecutives days and in theirs returns it was applied a questionnaire of food qualitative frequency. The food records were calculated by Nutwin software and evaluated based on DRIs. The frequency questionnaire was evaluated considering the weekly intakes of food groups. Results: Considering the 5 evaluated patients, 3 of them had adequacy of caloric intakes of macronutrients and inadequacy of some intakes micronutrients assessed. The other patient had caloric intakes above recommended for his age and the last one the caloric intakes were below the recommendation. The analysis of frequency questionnaire showed for 4 patients the exclusion of some food group and it was observed in 1 patient the proper inclusion of all groups. Conclusion: The MPS II patients frequently complaint of diarrhea and di¤culty in swallowing, leading to an important dietetics changes. The food consumption evaluation showed inadequacy in these patients' intakes, suggesting the need of appropriate dietetics orientation.

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METACHROMATIC LEUCODYSTROPHY: ARYLSULFATSE A MUTATIONS IN RUSSIAN PATIENTS Boukina TM1, Voskoboeva EYu1, Boukina AM1, Michailova SV2, Tsvetkova IV3 1 Research Centre for Medical Genetics RAM, Moscow, Russian Federation, 2Russian Federation Hospital for Children, Moscow, Russian Federation, 3Institute of Biolmedical Chemistry RAMS, Moscow, Russian Federation Background: Metachromatic leukodystrophy (MLD) is an autosomal recessive demielinating disorder due to the de¢ciency of the lysosomal hydrolase arylsulfatase A. MLD comprises late infantile, juvenile and adult forms. Two mutations are frequent in European population: c.459+1A4G that associated with the late infantile phenotype and p.Pro426Leu that associated with the adult or juvenile phenotype. In the ARSA gene, single nucleotide polymorphisms are presented wich reduce the enzyme activity. Two most frequent are p.Asn350Ser and *96A4G, known as `ARSA pseudode¢ciency alleles'. Objectives: We present results of molecular genetic investigation of 15 patients with the late infantile and juvenile forms of MLD. Methods: 15 patients were diagnosed as having MLD. 10 patients have the late infantile form disease and 5 ones juvenile form. The diagnosis of MLD was con¢rmed by biochemical analysis. Genes of ARSA was analysed by direct non-radioactive sequencing. Results: Altogether 27 mutation alleles and 17 di¡erent mutations were founded. Among Russian patients c.459+1A4G and p.Ile179Ser were frequent representing 13.3% of mutation alleles everyone. 10 novel mutations were founded: c.109__126dup18bp; c.411__412insC (p. His138ProfsX173); IVS2-2A4G; Val187Met; Tyr201His; Gln215Stop; Gly248Arg; Arg299Trp; Thr304Met; Glu307Lys. The polymorphisms p. Asn350Ser and *96A4G to be described as common in our patients were founded in 6 of 30 alleles and 1 of 30 alleles, respectively. Conclusions: Allele frequency of c.459+1A4G in Russian patients is 13%. Allele frequency p.N350S is 20% and *96+1A4G is 3%. It is speculated that patients with splace site mutations, frame shift mutation, duplication and nonsense mutation show clinically severe phenotype.

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MORTALITY RATE OF PATIENTS WITH MPS VI DURING THE PERIOD OF 16 MONTHS TRACKED BY PATIENTS ASSOCIATIONS IN BRAZIL Martins AM1, Micheletti C1, Kerstenetzky M2, Ribeiro EM3, Horovitz DG4, Gomy I5, Medeiros PFV6, Amorim T7, Acosta AX7, Bom¢m D8, Boy R9, Cipriano M10, Pereira TM11, Giovannetti D12 1 CREIM /UNIFESP, Sa¬o Paulo, Brazil, 2Hosp Bara¬o de Lucena, Recife, Brazil, 3Hosp Inf Albert Sabin, Fortaleza, Brazil, 4FIOCRUZ/ Ins Fernandes F|gueira, Rio De Janeiro, Brazil, 5Univ Sa¬o Paulo-Ribeira¬o Preto, Ribeira¬o Preto, Brazil, 6Univ Fed Campina Grande, Campina Grande, Brazil, 7Univ Federal Bahia, Salvador, Brazil, 8Hosp Universitario de Brasilia, Brasilia, Brazil, 9HUPE ^ UERJ, Rio de Janeiro, Brazil, 10Inst Saude p todos/ ISAD, Campinas, Brazil, 11 Biomarim Pharmaceutical Inc. MSL Latin A, Sa¬o Paulo, Brazil, 12 Biomarim Pharmaceutical Inc. MDLatin A, Sa¬o Paulo, Brazil Background: Mucopolysaccharidosis type VI (MPS VI) or Maroteaux-Lamy syndrome is a lysosomal storage disease that is characterized by systemic clinical manifestations and signi¢cant functional impairment, not typically associated with progressive impairment of mental status. Death usually results from respiratory infection or cardiac disease. Endotracheal intubation is extreme di¤culty and for this reason it happen complications during general anesthesia. Objectives: This study aim to assess risk factors and mortality incidence and related cause of MPS VI. Methods: Selection of the patients identi¢ed and followed by Patients Associations in Brazil in the period of November 2006 to March 2008 (16 months). These patients diagnostic were con¢rmed by enzyme assay, and were they currently been followed by Regional Medical Services. We identi¢ed a number of deaths in this period and theirs causes. Results: In this period were selected 121 patients' whit MPS VI. And 14 patients died (11.6%). Four deaths were related to cardiac failure, eight of them due to pulmonary infection associated with sepsis in three of them, and two patients died in an anesthetic accident. None of these patients were being treating with a speci¢c therapy. Conclusion: This mortality rate never published before call attention for the morbidity related to the disease. As we know the MPS VI is a severe disease and potential fatal. Nowadays is currently available enzyme replacement therapy for MPS VI and provides recombinant arylsulfatase B (galsulfase) which to early start can to avoid and prevent the severe consequence of the disease.

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IDENTIFICATION OF AMBROXOL AS A POTENTIAL ENZYME-ENHANCEMENT AGENT FOR GAUCHER DISEASE Maegawa GHB1, Tropak MB1, Buttner J1, Kornhaber G2, Rigat B1, Clarke JTR1, Mahuran D1 1 Hospital for Sick Children, Univ Toronto, Toronto, Canada, 2ExSar Inc, Monmouth Junction, NJ, USA Gaucher disease, currently treated by enzyme replacement therapy (ERT), is caused by a de¢ciency of lysosomal b-glucosidase (GCase). The disadvantages of ERT include its high cost, its ine¡ectiveness in treating the CNS, and its failure to prevent the unfolded protein response in cells. Small molecule-based enzyme enhancement therapy (EET) is a promising approach that can potentially be used alone or in combination with ERT to address these de¢ciencies. Clinical trials of isofagomine, an inhibitor of GCase (IC50 *0.04 mM), as an EET-agent are being initiated. In order to accelerate the process of obtaining INDstatus for new EET-agents we have screened the NINDS library of FDA-approved drugs for compounds that inhibit and/or stabilize the target enzyme towards heat-denaturation. Using GCase as the target we identi¢ed ambroxol, an expectorant, as a candidate EET-agent. Despite ambroxol being only a weak inhibitor of GCase, IC50 *27 mM, at higher concentrations it compared favorably with isofagomine in its ability to rescue mutant N370S GCase in patients' ¢broblast and lymphoblast cell lines. However, it was not as e¡ective at rescuing F213I mutant GCase. Cells with wild-type GCase also show a mild enhancement in activity. Hydrogen-deuterium exchange mass spectrometry (H/D-Ex) was used to compare the regions in GCase that were stabilized by these compounds. Isofagomine has been already shown through co-crystallization to stabilize a loop structure at GCase311^319. Both compounds were equally e¡ective in stabilizing two additional loop regions at the mouth of the active site, GCase243^ 249 and 386^400, suggesting its importance as a target for EET-agents.

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IMPACT OF ERT ON FAMILIAR LIFE Vertemati T, Micheletti C, Piazzon FB, Canossa S, Okubo RM, Martins AM CREIM/UNIFESP, Sa¬o Paulo, Brazil Background: Brazilian patients with some lysosomal storage disease can be treated in hospitals or specialized centers by replacement enzymatic therapy (ERT). Objectives: Impact's ERT analysis on familiar life regarding economic, emotional and social view points. Methods: An inquiry was formulated by multidisciplinary group and applied in 50 patients in ERT: 11 mucopolysaccharidosis I, 6 mucopolysaccharidosis II, 6 mucopolysaccharidosis VI, 17 Gaucher disease, 7 Fabry disease and 3 Pompe disease. Results: The mean age observed is 14.6 years old, 22 females and 28 males. The average period of ERT's treatment is 2.5 years. The average prescribed time for the infusion is 3.1 h, but the time spent between going and returning home in the day of infusion is around 7.4 h: 1.6 h spent from home to CREIM, 1.8 h return, 0.6 h for reception and admission and 0.3 h in physiotherapy. Of the students 23% feel disturbed in their school activity on ERT consequence. Among goes whom works none lost their jobs. Forty four come accompanied for infusion, and among these companions, 31% work, 16% had problems on theirs job and two lost it. The average month spent with transportation, feeding and medication related to ERT is 54.48. Only ten patients get around 166.69 monthly as bene¢t. Twenty one complain some kind of damage for patient and/or family. Conclusions: Even spending 21.5 h/monthly (2.29 times the infusion session), disposing secondary spent and observing personal and familiar disturbance, all of then consider ERT's bene¢ts compensation for sacri¢ces.

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ARYLSULFATASE A GENE: IDENTIFICATION OF COMMON MUTATIONS BY REAL TIME PCR Bock H1, Carpes EA1, Rodrigues GF1, Burin MG2, Matte U3, Giugliani R2, Saraiva-Pereira ML1 1 Genetics Identi¢cation Lab ^ HCPA, Porto Alegre, Brazil, 2Medical Genet Serv ^ HCPA, Porto Alegre, Brazil, 3Genetic Teraphy Center ^ HCPA, Porto Alegre, Brazil Background: Metachromatic leukodistrophy (MLD) is an autosomal recessive disorder characterized by progressive demyelination of the CNS, caused by arylsulfatase A (ARSA) de¢ciency. ARSA gene is located on chromosome 22 and spans about 3.2 kb genomic DNA, divided into 8 exons. Two mutations, 459+1G4A and P426L, were identi¢ed and described to be common among MLD patients. In addition, two other sequence alterations, N350S and 1524+95A4G, were associated to ARSA pseudode¢ciency (ARSA-PD), a condition characterized by low ARSA activity in healthy individuals. The aim of this work was to introduce and validate a real time PCR based methodology to identify common mutations in the ARSA gene. Methods: In this work we have evaluated a group of 26 MLD patients. DNA samples were isolated and samples were quanti¢ed and diluted to 2 ng/ml. Primers and probes were designed by Primer Express software. All four mutations were analyzed by TaqMan. in the 7500 PCR System. Results: We have observed the following mutation distribution within MLD patients: 19 alleles (36.5%) carrying 459+1G4A mutation, 7 alleles (13.5%) carrying N350S mutation, and 4 alleles (7.7%) carrying 1524+95A4G mutation. No alleles were found to carry P426L mutation. Conclusions: Results presented here indicate that this protocol is e¤cient to detect common mutations in MLD patients, and is also able to identify individuals with ARSA-PD, which is not possible to be distinguished by biochemical analysis. F|nally, mutation detecting using real time PCR is faster and easier to be performed in large scale studies. (F|nancial support: CNPq, FAPERGS and Pela V|da ONG)

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PHYSIOTHERAPEUTIC EVALUATION IN MPS II PATIENTS AFTER TWENTY SIX WEEKS OF ENZYME REPLACEMENT THERAPY (ERT) WITH IDURSULFASE (ELAPRASE) Fraccaro E, Menegatti E, Ricarte A, Piazzon FB, Vertemati T, Micheletti C, Rand MH, Martins AM CREIM/UNIFESP, Sa¬o Paulo, Brazil B a c k g r o u n d /O b j e c t i v e s: T h e c l i n i c a l m a n i f e s t a t i o n s o f mucopolysaccharidosis type II (MPS II) include severe airway obstruction, neurological decline and joint restriction. Weekly intravenous infusions of idursulfase have shown to improve many of the signs and symptoms of the disease, and consequently also improve patients' outcome. The physiotherapeutic evaluation of MPS II patients after twenty six weeks of ERT has been analyzed and emphasis was given to evaluation items like motor ability and joint restriction. Methods: An observational study of four patients between ¢ve and thirteen years old was performed after twenty six weeks of ERT. Parents' reports on topics like motor abilities and daily life activities with family support were taken into account. Goniometer was the instrument used to measure the amplitude of joint mobility. Results: Shoulder mobility improvement was reported in all patients, which enabled them to get dressed. The goniometric evaluation showed shoulder £exion improvement of 5 degrees in 3 patients and of 3 degrees in 1 patient and shoulder extension improvement of 3 degrees in 1 patient and of 4 degrees in 2 patients. Conclusion: the preliminary result was observed in the improvement of joint mobility, especially in shoulder movements in all patients with MPS II, which made it easier for them to perform daily activities and gave the family members a positive impact.

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ANEURYSM OF THE LEFT VENTRICLE AND CIRRHOSIS ASSOCIATED WITH MUCOPOLYSACCHARIDOSIS TYPE II (MPS II) Valadares ER, Quirino BEG, Melo FHC, Leite VHR, Godoy P, Arantes RR, Ferrari TCA, Rocha LOS Faculdade de Medicina da UFMG, Belo Horizonte, Brazil Background: The MPS II is caused by de¢ciency of iduronate sulfatase (IDS). Gargoilism, hepatosplenomegaly, valve and myocardial in¢ltration are classical symptoms. Liver dysfunction and ventricular aneurysm have rarely been associated with MPSs. Our aim is to report unusual ¢ndings in adults with MPS II. Methods: Clinical and anatomopathological evaluation of brothers and review of literature. Result: A 33-year-old man admitted to the hospital with intense dyspnea, short stature, grotesque facial features and thoracolumbar gibbus. His echocardiogram showed di¡use thickening of the wall of the left ventricle (LV), thickening of lea£ets of mitral and aortic valves and moderate aortic insu¤ciency. He died and his autopsy showed LV apical aneurysm. His brother had also gargoilism and IDS de¢ciency was detected. Recently he presented with dyspnea, bulky ascites, hepatosplenomegaly, hard liver and esofagial varicose veins, and died at age 32. His liver biopsy revealed cirrhosis and deposit. Conclusion: Apical aneurysm was reported in 2 patients with MPS II [Oliver, 1982; Kettles, 2002] and 1 with MPS VI [Oudit, 2007]. The accumulation of GAGs in the myocardial interstitium may alter the extracellular matrix and the myocardial response to increased wall stress. The apical aneurysm observed in our case was considered by many as pathognomonic of chronic chagasic cardiopathy. One case report correlates cirrhosis to MPS II [Yoshimoto, 2006].

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PRELIMINARY DATA OF ERT IN MPS VI PATIENTS UNDER 3 YEARS OLD Kerstenetzky M1, Ribeiro EM2, Horovitz DG3, LLerena J3, Kim CA4, Honjo R4, Paula AC4, Moura¬o T5, Giovannetti D5 1 Hosp Bara¬o de Lucena Ref Center of IEM, Recife PE, Brazil, 2Infantil Hosp AlbertSabin and Ceèsar Hosp, Fortaleza, Brazil, 3Fernandes F|gueira Inst/ FIOCRUZ, Rio de Janeiro, Brazil, 4Genetic Unit ^ Children's Inst USP, Sao Paulo, Brazil, 5BioMarin Brazil, Sao Paulo, Brazil Background: Mucopolysaccharidosis VI is a lysosomal storage disease resulting from a de¢ciency in the enzyme arylsulfatase B. When the enzyme activity is insu¤cient, causes a progressive disorder with multiple organ involvement. Enzyme replacement therapy (ERT) for MPS VI is available and has shown positive results in e¤cacy and security in 3 clinical trials, and extention studies. Objectives: Evaluate safety and e¤cacy of (ERT) with galsulfase in patients under 3 years old with mucopolysaccharidose type VI (MPS VI). Methods: Describe 4 patient's evolution before and under ERT for MPS VI through family history and weekly clinical assessment in the infusion procedures. Results: We included 4 patients, 1 M/3 F, followed by 4 di¡erent Services in Brazil. They start therapy among 17 and 35 weeks of age. At the baseline all patients presented in¢ltrated faces, joint limitation, hepatosplenomegaly and dysostosis multiplex, their neurological evaluation was normal. The echocardiogram showed alterations in 3/4 patients. They are now under weekly EV infusion of galsulfase from 7 to 40 weeks, without report of adverse event. They have greater agility and mobility through observation of the daily living activities. The 2 patients under therapy for more time (34 and 40 weeks), presented an decrease of airway infections and hepatosplenomegaly, better sleep pattern and decrease in snoring, demonstrated by sleep study in one of them. Conclusion: The ERT with galsulfase seems to be safe and improve the clinical outcome of the disease. The continuous follow-up of the MPS VI under 3 patients with subsidiary exams will reassure this statement.

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CHITOTRIOSIDASE LEVELS IN CYSTINOSIS Xaidara A1, Karavitakis E1, Kosma K1, Dimitriou E2, Michelakakis H2 1 Aghia Sophia Child Hosp, Athens Univ, Athens, Greece, 2Inst Child Health, Athens, Greece Background: Cystinosis is a rare autosomal recessive disease characterized by intralysosomal accumulation of cystine due to its impaired transport across the lysosomal membrane. The initial symptoms of the classical infantile form of nephropathic cystinosis result from the failure of renal tubules to reabsorb small molecules, causing Fanconi's syndrome. Chitotriosidase is a protein secreted by activated macrophages and its enzymatic activity is elevated in serum of patients with Gaucher's disease and some other lysosomal storage disorders. Methods: We report chitotriosidase levels in a 6 year old boy with cystinosis. The child was diagnosed at the age of 6 months with proximal renal tubule dysfunction. In the routine evaluation at the age of 5 years renal failure stage 4 was found using the Schwartz formula (GFR: 24 ml/min/1.73 m2) and slit-lamp examination revealed corneal crystals suggesting cystinosis. Increased leukocyte cystine content (2.15 nmoles/mg protein, normal50.25 units) established the diagnosis. Oral cysteamine therapy (15 mg/kg/day in four doses, increased to 30 mg/ kg/day 10 days later) and cysteamine eyedrops were initiated. Results: Before treatment, serum chitotriosidase activity was 481 nmol/ ml/h (normal: 0^150 units). After one month of treatment leukocyte cystine content was 2.9 nmols/mg protein and serum chitotriosidase activity had also increased (827 nmol/ml/h). Oral cysteamine was further increased to 35 mg/kg/day in four doses and 3 months later serum chitotriosidase activity decreased to 490 nmol/ml/h. Conclusions: To our knowledge this is the ¢rst report of increased chitotriosidase activity in cystinosis. We suggest that it might represent a marker of response to treatment with cysteamine.

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LYSOSOMAL ENZYMES IN DRIED BLOOD SPOTS, AS A DIAGNOSTIC TOOL OF LYSOSOMAL STORAGE DISORDERS Almeida LS, Caseiro C, Silva E, Lacerda L Unidade de Enzimologia, CGMJM-INSA, Porto, Portugal Background: Lysosomal storage diseases (LSD) are severe and rare disorders and its diagnosis is generally based on speci¢c enzymatic assays performed on plasma, leukocytes and ¢broblasts. Being a certi¢ed state laboratory (member of the European Study Group on Lysosomal Diseases) we receive a large number of samples from all country. Therefore a fast diagnosis requiring a small volume of sample would allow a better service to the community. Moreover, a simple and fast screening method would increase detection rate, especially for screening purposes of at risk groups of undiagnosed patients. Methods: We have set-up a multiplex screening assay for measuring the enzymatic activity of chitotriosidase, glucocerebrosidase, agalactosidase A, a-iduronidase, arylsulfatase B, b-hexosaminidase A, b-galactosidase and a-glucosidase in dried blood spots (DBS), using £uorescent substrates. We have analyzed 100 healthy blood donors and about 200 cases referred to the lab due to a clinical suspicion of LSD, as well as, patients under treatment follow-up. Results: This pilot study in DBS samples (total peripheral blood), allowed establishing reference values in a control population. The reliability of this pilot study was achieved by a parallel determination of the enzyme activities by already established standard methods, performed either in plasma or total leukocytes (biological fractions obtained from total peripheral blood). Conclusions: Since the lab is a member of the National Center for Coordination of Diagnosis and Treatment of LSD we will bene¢t by the DBS lysosomal enzyme activity determination, a fast and reliable method for LSD primary screening and treatment follow-up.

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EIGHT-YEAR CLINICAL OUTCOMES OF LONG-TERM ENZYME REPLACEMENT THERAPY IN 884 CHILDREN WITH TYPE 1 GAUCHER DISEASE Andersson H1, Kaplan P2, Kacena K3, Yee J3 1 Hayward Genetic Cntr, Tulane Univ Med Sch, New Orleans, USA, 2 Children Hosp Phil, Univ Penn, Philadelphia, USA, 3Genzyme Corp, Cambridge, USA Objective: To analyze the clinical responses to enzyme replacement therapy (ERT) in a large international cohort of children with type 1 Gaucher disease (GD1). Methods: Anonymized data from 884 children in the Gaucher Registry were analyzed for the e¡ect of long-term ERT on hematological and visceral manifestations, linear growth and skeletal disease. Data analysis used mixed e¡ects models and Kaplan-Meier curves (bone pain and bone crises). Mean ERT dose was 79 m/kg/4 wks. Results: The median height Z-score for the study population was ^1.4 at baseline and 34% of patients were less than 5th percentile. After 8 years ERT, the median height and growth parameters approximated that of the normal population. Anemia was present in over 50% at baseline and resolved in all patients after 8 years of treatment. More than 50% had platelet counts below 100 000/mm3 at baseline but over 95% had platelets above this level after 8 years of treatment. Liver and spleen volumes decreased over 8 years of treatment. Mean bone density Zscore was ^0.34 at baseline, and normalized within 6.6 years of treatment. In patients reporting bone crisis before treatment (17%), no bone crises were reported after 2 years of ERT. Few (2.5%) patients without bone crises pre-ERT at baseline had a crisis after the start of treatment. Conclusions: W|thin 8 years of ERT most clinical parameters became normal or near normal. The data provide pediatricians a measure of expected response in GD1 patients for individual clinical parameters relative to baseline and o¡er the ¢rst long-term outcomes for a large worldwide pediatric cohort.

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SUBSTRATE REDUCTION THERAPY WITH MIGLUSTAT IN JUVENILE GM2 GANGLIOSIDOSIS Maegawa G HB1, Banwell B1, Blaser S1, van Giersbergen P2, Sorge G3, Tropak M3, Arckerley C1, Hawkins C1, Hayes J1, Clarke JTR1 1 Hosp for Sick Children, Univ Toronto, Toronto, Canada, 2ExSar Inc, Monmouth Junction, NJ, USA, 3Psychology Dept, York Univ, York, Canada Background: Juvenile GM2 gangliosidosis (jGM2g) is an inherited neurodegenerative disease caused by de¢ciency of lysosomal bhexosaminidase A. Substrate reduction therapy (SRT) is considered a potential therapy. Objective: To investigate pharmacokinetics (PK), safety and e¤cacy of SRT using miglustat in jGM2g. Methods: Open-label, singlearm phase I/II clinical trial was designed to evaluate 5 patients, who received miglustat orally at 100^200 mg t.i.d. PK and clinical assessments, safety tests, nerve conduction studies and brain MRI/MRS were performed. Results: Similar PK pro¢les were observed from the single- and multipledose assessments. The major adverse events were weight loss and diarrhea. Most mild laboratory abnormalities were either transient or attributable to concomitant medications. In terms of e¤cacy, most composites of di¡erent neurological outcomes showed evidence of disease progression. Of 3 subjects with psychiatric past history, one patient had an acute psychosis needing hospitalization. One patient showed severe aggravation of her seizure pattern. Three less severely impaired patients showed an overall trend towards stabilization, involving performance on speci¢c cognitive tests. Brain MRI of one patient showed signs of mild progression of cerebellar loss and supratentorium alterations. Electron microscopy analysis showed reduction of blood mononuclear cells containing inclusion-bodies. Conclusion: Miglustat was demonstrated to be safe in jGM2 children, and to have PK parameters similar to those observed in adults. SRT showed limited evidence of e¤cacy towards major neurological dysfunctions, with evidence of some stabilization of cognitive function tests. The results must be interpreted with care owing to the small sample and the lack of a controlarm.

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EXPERIENCE IN LYON OF MPS II PATIENTS WITH ENZYME TREATMENT Gu¡on N, Forest I, Fouilhoux A Metabolic Disease, CERLYMM, HCL, Lyon, France Background: 50 patients have been diagnosed in Lyon. 31 MPSII patients coming from di¡erents areas in France have started treatment with elaprase. The youngest patient on enzyme replacement therapy (ERT) was 5 months of age and 17 patients have severe central nervous system involvement. The duration of infusion is initially 3 h and progressively increased to obtain after about 2 months of treatment 2 h of infusion if no infusion associated reaction occurred. Local infusion sites were trained and most of the patients is transferred. Method: After a few infusions, an in-dwelling intravenous catheter is been inserted for 19 patients during multiple surgical procedures. Only 4 patients had infusion associated reaction with tremor, hyperthermia, anxious, vomitis and one with laryngitis and voice loss. For these patients, we initiated pre medication with paracetamol and/or corticosteroid. Result: On the 31 patients with treatment, one patient after 6 months of ERT has stopped the treatment after discussion with parents. One patient is dead in UK during phase 2/3 clinical study after acute respiratory distress, not related with study drug. For the 29 patients, we do not observe any adverse reaction until now. The patients continue to come to Lyon during the multidisciplinary visits. Elaprase is well tolerated and adverse reactions are managed by slowing or interrupting the infusion and by initiation of premedication. It seems evident that early ERT may slow the progression of the disease and need further observations with MPSII severe patients to assess the bene¢t.

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DANON DISEASE ^ REPORT OF TWO PORTUGUESE PATIENTS Rodrigues E1, Martins E2, V|eira A3, Santos H1, Castro L4, Carpenter S4, Lea¬o Teles E1 1 Metab Dis Unit, Ped Dept Univ/Hosp St Joa¬o, Porto, Portugal, 2Cardiol Ser Univ/Hosp St Joa¬o, Porto, Portugal, 3Cardiol Ser Ped Dept Univ/ Hosp St Joa¬o, Porto, Portugal, 4Anat Path Dept, Univ/Hosp St Joa¬o, Porto, Portugal Background: Danon disease is an X-linked cardioskeletal myopathy caused by primary de¢ciency of lysosome-associated membrane protein-2 (LAMP-2). Clinically is characterized by hypertrophic cardiomyopathy (HCM), myopathy, and variable mental retardation. The pathological hallmark of the disease is the appearance of intracytoplasmic vacuoles containing autophagic material and the absence of LAMP-2 activity in the muscle. De¢nitive diagnosis is made by protein/molecular study. We describe two cases of Danon disease with di¡erent clinical presentation. Case Reports: The ¢rst patient was a 27-year-old female, with previous history of `idiopathic HCM'. After 25 years of age, she was submitted to heart transplantation due to heart failure. After cardiac transplant the morphologic and ultra-structural study of the myocardial biopsy revealed the presence of autophagic vacuoles and glycogen deposits, strongly suggestive of Danon disease. The patient had a major clinical improvement, without any other signi¢cant abnormalities. Molecular characterization is ongoing. The second patient, an 11-year-old boy, was referred for investigation of HCM. Familiar history revealed cardiac symptoms on maternal grandmother. Moderate mental retardation, di¡use muscle weakness and wasting were noted, and a muscular biopsy was performed. Histology and ultra-structural study showed suggestive ¢ndings of Danon disease, con¢rmed by the identi¢cation of a novel mutation in exon 3 of the Lamp-2 gene (G76X:c. 226G4T). Discussion: The HCM are genetically heterogeneous diseases, mainly related with mutations in sarcomeric genes. However in about 1/3 of the cases the aetiology is diverse, including lysosomal storage diseases. Histology could be a guide for the diagnosis of Danon disease.

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ECHOCARDIOGRAPHIC AND ELECTROCARDIOGRAPHIC EVOLUTION OF 5 PAEDIATRIC MPS TYPE 1 PATIENTS UNDER ENZYME REPLACEMENT THERAPY ^ 4 YEAR FOLLOW-UP Martins P1, Garcia P2, Ramalheiro G1, Diogo L2, Castela E1 1 Serv Cardiologia Ped, Hosp Ped, Coimbra, Portugal, 2Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal Background/Objectives: Mucopolysacharidosis type 1 (MPS1) is a lysosomal storage disease that frequently involves the heart. Valvular dysplasia, myocardial hypertrophy, pulmonary hypertension, contractile dysfunction, signs of ventricular hypertrophy with QRS axis deviation and rhythm disturbances have been the most frequent electrocardiographic and echocardiographic changes. Our aim was to analyze the e¡ects of enzyme replacement therapy (ERT) with Aldurazyme1 in the heart of MPS1 children. Methods: F|ve MPS1 patients (2 males, 3 females; aged between 6 and 14 years-old) under ERT were studied prospectively during a 4-year period. The electrocardiograms, echocardiograms, and symptomatic therapy before and after 4 years of ERT were analyzed. Results: Overall, there were no signi¢cant electrocardiographic changes during the period analyzed, except for one patient in whom signs of left ventricular hypertrophy regressed to septal hypertrophy. The major echocardiographic ¢ndings were unchanged over the study period: mitral (4) or aortic (3) dysplasia/regurgitation, diastolic dysfunction (2), high estimated pulmonary systolic arterial pressure (2), ventricular hypertrophy (4) and dilated myocardiopathy (1). Three children were under diuretics and/or ACE inhibitors. Conclusions: In general, our patients were stable during the ERT period. Considering that all but one child have Hurler phenotype, we can postulate that ERT had a positive e¡ect on the natural history of the disease. Nevertheless, these results should be interpreted with caution since vasoactive drugs were used simultaneously.

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ANOMALIES IN T AND iNKT POPULATIONS IN FABRY MICE BUT NOT IN FABRY PATIENTS Balreira A1, Macedo MF2, Gira¬o C1, Rodrigues LG1, Oliveira JP3, Saè Miranda MC1, Arosa FA1 1 IBMC, Porto, Portugal, 2SACS, Universidade de Aveiro, Aveiro, Portugal, 3Servic°o de Geneètica, Hosp St Joa¬o, Porto, Portugal

CLINICAL PRESENTATION OF 1 PATIENT WITH LATE ONSET POMPE DISEASE AND FIRST RESULTS AFTER 6 MONTHS OF ERT Gu¡on N1, Forest I1, Aubert F1, Gormand F2, Fouilhoux A1 1 Metabolic Disease, CERLYMM, HCL, Lyon, France, 2CHLS, Lyon, France

Background: Fabry disease is an X-linked sphingolipidosis, that results from the defective activity of alpha-galactosidase A, leading to lysosomal accu mulation of globotriaosylc eramide and digalactosylceramide. Endogenous glycosphingolipids are thought to bind to CD1 molecules, namely CD1d, and be presented to a subset of NKT cells carrying a conserved TCRValpha chain. Methods: Peripheral blood leukocytes were isolated from Fabry patients under ERT and healthy controls. Hepatic and splenic lymphocytes were isolated from Fabry and background mice. T cells populations were analysed by FACS. Results: Liver CD3+CD4+ T cells were signi¢cantly decreased while CD3+CD8+ T cells were increased in Fabry mice, resulting in a twofold decrease in the CD4+/CD8+ T cell ratios. Imbalances in these populations were also observed in spleen of Fabry mice. Hepatic CD3 +Valpha14+ T cells were decreased in Fabry mice. A similar analysis performed in Fabry patients revealed an absence of anomalies, being the only anomaly found in Fabry patients an upregulation MHC-II on monocytes. A marked reduction in liver CD4+CD3int T cells that resulted in a two-fold decrease in the CD3int/CD3high T cell ratio between Fabry and control mice at 12 weeks was also observed. Discussion: In this study we show that Fabry mice display overt anomalies in liver T and iNKT populations, while no anomalies in T and iNKT cells were observed in Fabry patients. These di¡erences between humans and mice may result from the marked di¡erences between the innate and adaptive arms of the immunological system of these two species.

Pompe disease is an autosomal recessive lysosomal storage caused by de¢ciency of acid-alpha glucosidase which converts glycogen to glucose. This neuromuscular disorder can be classi¢ed into two di¡erent forms: early onset and late onset (LO) forms. Our objective was to characterize the clinical presentation (including muscle function, forced vital capacity, cardiologic exploration and biological parameters) and ¢rst results of ERT in one LO patient. F|rst symptoms were slimming and di¤culties to practice sport at 13^14 years. The time lag between onset and ¢rst symptoms is about 5 years. The patient was diagnosed because of cardiac arrest after valium administration for gastroscopy. No cardiomyopathy or arrhythmia was observed. Initiation of mechanical ventilation became necessary before loss of deambulation. ERT was initiated and results after 6 months of treatment will be discussed. Myozyme is well tolerated, improvement of restrictive respiratory insu¤ciency and total disappearance of alveolar hypoventilation were observed. However, no improvement of muscular weakness has been shown by MFM scale. Further evaluation with longer treatment period will be necessary but early diagnosis of Pompe disease is essential to consider ERT before irreversible muscular damage

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Background: Fabry disease is an inherited de¢ciency of the lysosomal agalactosidase A (a-Gal A) due to mutations in the Gal gene at Xq22. The e¡ects of this de¢ciency are traduced by intralysosomal accumulations of globotriaosylceramide (Gb3). The main objective of the present work was to study the e¡ect of ERT in the phenotype onset of Fabry knockout mice. Methods: ERT was provided by weekly perfusion of placebo or agalsidase beta (3 mg/kg or 1.5 mg/kg) (Fabrazyme) given by Genzyme Corporation. Fabry animals with 4 or 8 weeks of age were perfused during a period of 8 and 4 weeks respectively and the animals were slaughtered 1 week after the last injection. The e¤cacy of the therapy was evaluated by the determination of a-Gal A activity and Gb3 accumulation in several organs as well by the study of the thermosensibility. Results: In this study it was shown that a-Gal A activity was increased and Gb3 accumulation was decreased in the tissues of animals treated with enzyme. However, no di¡erences in thermosensibility were observed between the animals treated with placebo or enzyme. Conclusion: This study demonstrates that the ERT corrects the biochemical phenotype of Fabry knockout mice by normalizing the aGal A activity and decreasing the Gb3 accumulation. This correction seems to be dose dependent.

Background: Two patients diagnosed with Scheie syndrome and treated with weekly infusions of Aldurazyme 100 U/kg for 2 h since the age of 22 and 34 years, switched to home therapy after a total of 222 and 144 infusions in a hospital setting. Based on a good clinical condition and patients' request, it has been decided to transfer the infusion to patients' home. A speci¢c department called HAD organizes infusions at home with a maximum of safety. To consider such a transfer, the patient should have been treated with Aldurazyme for at least 2 years and the patient should not have had any infusion associated reactions for 2 years. The homecare nurses have been specially trained by the multidisciplinary team to prepare Aldurazyme to initiate the infusion, to monitor during the 2-h period of the infusion and to perform the safety assessments. Aldurazyme is delivered by the HAD pharmacy. Every month, the hospital nurses and the home care nurses exchange information on the patient's status. On a monthly basis, a report is sent to the physician. Patients attend yearly multidisciplinary follow up visits at the hospital. Besides, at any time, the infusion can be performed in the hospital if needed. Results: Compliance was optimal and patient's satisfaction high. No adverse events occurred within the follow-up (36 and 35 weeks). Conclusion: Under the responsibility of the prescriber and with very strict conditions, home-therapy with Aldurazyme can be considered. Further follow-up is required to con¢rm the safety of this approach.

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ANIMAL MODEL OF FABRY DISEASE: STUDY OF EARLY ENZYME REPLACEMENT THERAPY (ERT) Mendes I1, Rodrigues LG1, Pais-V|eira M2, Saè Miranda MC1 1 UniLiPe, IBMC, Porto, Portugal, 2Morphophysiology, IBMC, Porto, Portugal

REDUCTION OF ALDURAZYME INFUSION TIME FOR MPS I PATIENTS: THE LYON EXPERIENCE Gu¡on N, Forest I, Reynes N, Fouilhoux A Metabolic Disease, CERLYMM, HCL, Lyon, France Background: Aldurazyme is weekly administered in 3 to 4 h intravenous infusions at hospital, at maximum infusion time of 43 U/kg/h. Method: To improve comfort and reduce time spent by the patient each week at the hospital, we adapted the recommended Aldurazyme infusion scheme for administration in 2 h. A reduction time infusion scheme has been designed. The total volume of Aldurazyme to be infused is 250 ml. Result: The initial £ow rate is 50 ml/h and is maintained during the ¢rst hour, the remaining volume is infused at a rate of 80 ml/h. All subsequent infusions have started at a similar 50 ml/h rate, but from the 3rd infusion, only if the treatment is well tolerated, the rate is accelerated by incremental steps of 10 ml/h at each infusion with a maximum of 200 ml/h at the 14th infusion. Then, after a minimum of 14 weeks treatment, the infusion can be completed in 2 h. V|tal signs are monitored during the infusion. Urinary GAG, leukocyte enzyme activity and antibodies to Aldurazyme are measured at regular intervals.

HOME THERAPY IS FEASIBLE FOR SCHEIE PATIENTS Gu¡on N, Forest I, Caire C, Chateau M, Renaud B, Magnet M, Fouilhoux A Soins et Santeè ^ HAD, Caluire, France

CLINICAL CHARACTERISTICS OF PATIENTS IN THE MPS I REGISTRY W|jburg FA1, V|skochil D2 1 Emma Child Hosp Amsterdam, Dept Ped, Amsterdam, Netherlands, 2 Univ of Utah, Div Med Genetics, Salt Lake City, USA On behalf of the MPS I Registry Board of Advisors Background/Methods: Patient demographics, symptom history, and treatment data for MPS I Registry patients were evaluated to better understand the natural history and phenotype spectrum of MPS I. Results: As of September 2007, the Registry included 659 patients. Patients were classi¢ed as Hurler (H: 55%), Hurler-Scheie (H-S: 22%) and Scheie (S: 11%); phenotype was unknown/unreported for 11% of patients. Median ages at diagnosis were 0.8 years H, 3.7 years H-S and 8.8 years S. Overall, 547/659 patients (83%) are reported to have been treated with enzyme replacement, stem cell transplant, or both. Symptom history data were available for 318 H, 137 H-S and 63 S patients. Symptoms of MPS I reported for at least 70% of patients per phenotype, with median age at onset in years, are: coarse facial features (H: 96%, 0.8 years; H-S: 88%, 3.5 years); corneal clouding (H: 91%, 1.1 years; H-S: 91%, 4.3 years; S: 91%, 10.3 years); hepatomegaly (H: 89%, 1.1 years, H-S: 90%) kyphosis/gibbus (H: 88%, 0.9 years); hernia (H: 81%, 0.9 years; H-S: 82%, 3.3 years; S: 75%, 2.6 years); cardiac valve abnormality (H: 71%, 1.6 years; H-S: 88%, 5.7 years; S: 94%, 10.9 years); joint contractures (H-S: 83%, 3.9 years; S: 88%, 7.2 years); and carpal tunnel syndrome (S: 75%, 12.5 years). Conclusions: Coarse facial features and hepatomegaly are prominent symptoms in severe phenotypes; joint contractures are more frequent in attenuated disease. Corneal clouding and hernia are common in all phenotypes. Median age at onset for corneal clouding is similar to age at diagnosis. Hernia is an early symptom across phenotypes.

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Background: The MPS I Registry collects data on disease progression, treatment, and outcomes of patients with MPS I. We sought to explore how treatment approaches to MPS I evolve over time. Description/Results: As of September 2007, 659 patients were enrolled in the Registry: phenotype distribution was 55% Hurler, 23% HurlerScheie, 11% Scheie, and 11% unknown/unreported. 70% of patients were diagnosed before 2003, when enzyme replacement therapy (ERT) became available in North America and Europe; 30% were diagnosed in 2003^2007. 83% of patients have received treatment with ERT and/or hematopoietic stem cell transplant (HSCT). F|fty-seven of 530 (11%) living Registry patients had not received HSCT or ERT. Age at ¢rst HSCT did not vary over time but time between symptom onset and ERT declined since 2003: the interval between ¢rst recorded symptoms and ¢rst ERT was 4.8 years for patients diagnosed before 2003 and 0.9 years for patients diagnosed in 2003^2007. Median age at ¢rst treatment for patients receiving only ERT decreased, from 6.9 years for patients ¢rst treated in 2004 to 5.2 years in 2006. Patients diagnosed in 2003^ 2007 may be slightly more likely to receive treatment than those diagnosed before 2003 (84.5% vs 82.4%) and are more likely to receive ERT (75.5% vs 51.6%), particularly along with HSCT (23.5% vs 4.2%). Conclusion: In MPS I Registry patients, median age at HSCT has not changed appreciably since 2003; however, the age at initiation of ERT has decreased since commercial availability of enzyme.

Background: Type 1 Gaucher disease is di¡erentiated from type 2 and 3 disease by the absence of nervous system involvement. However, an increasing number of reports have emerged on neurological manifestations in GD1 patients. Whether a strict division in three di¡erent phenotypes is still valid has been the subject of debate. Methods: We reviewed the available literature on this subject. In addition, we investigated retrospectively a large Dutch cohort of GD1 patients for the prevalence of neurological manifestations. At the same time, we are involved in a large prospective study on the prevalence and incidence of polyneuropathies in GD1. Results: The literature search revealed four groups of central nervous system disease: late-onset type 3 disease, Parkinsonian manifestations, myelum compression and a group of sporadic disorders. Peripheral nervous system disease was reported as peripheral neuropathy, dorsal root compression and cranial nerve palsies. In our Dutch cohort (n = 75), a diagnosis of a neurological disease was made 34 times during a median follow-up time of eleven years. In addition, the baseline data from the prospective cohort study (n = 103) showed a prevalence of polyneuropathy of 10.7% which is higher than in the general population (0.12^3.6%). Conclusions: In conclusion, the term non-neuronopathic Gaucher disease does not seem to be appropriate. However, the neurological signs and symptoms in type 1 Gaucher disease are of a di¡erent kind and of less severity in comparison with GD2 and GD3. Therefore, type 1 disease should be classi¢ed as a separate phenotype.

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TREATMENT TRENDS IN MPS I: THE MPS I REGISTRY Gu¡on N1, Clarke J2 1 Metabolic Disease, CERLYMM, HCL, Lyon, France, 2Hosp for Sick Child, Div Clin and Metab Gen, Toronto, Canada On behalf of the MPS I Registry Board of Advisors

MUCOLIPIDOSIS TYPE IV IN A MAPUCHE PATIENT WITH CEREBRAL PALSY Duran GP, Hernandez M, Huete I, Gonzalez S Ponti¢cia Universidad Catolica de Chile, Santiago, Chile Introduction: Mucolipidosis IV (ML-IV), is a lysosomal storage disease of autosomic recessive inheritance, described for the most part in patients Ashkenazi Jewish, caused by mutations of the gene MCOLN1 located on chromosome 19p 13.2^13.3. It characterized for severe developmental delay, corneal opacities, retina degeneration, optic atrophy, corpus callosum hypoplasia, abnormal signal of white matter and basal ganglia. Late in the clinical evolution has been observed cerebral and cerebella atrophy. Ultra microscopy shows characteristic storage of lamellar membranous structures and amorphous cytoplasmic inclusions in the lysosomas of skin and conjunctiva. Purpose: To report a case of ML-IV in a girl of Mapuche heritage, previously treated as a cerebral palsy. Clinical Case: A 9 year old girl of Mapuche's origin, with a diagnosis of cerebral palsy since infancy. At 5 years of age she started slowly loss of motor and cognitive skills associated to visual impairment. Optic atrophy was added to the neurological features which motivated the reevaluation of the diagnosis. The review of neuroimagings showed slow and progressive atrophy of intracerebral structures and ultra microscopy of skin and conjunctiva revealed intracytoplasmic inclusions; consistent with the diagnosis of mucolipidosis IV. Conclusion: ML-IV must be included in the di¡erential diagnosis of cerebral palsy associated to ophthalmologic impairment or course slowly regressive. Electronic microscopy of skin or conjunctiva is a useful diagnostic test in this disease. This case permit to rea¤rm that suspicion of ML-IV must not be restricted to Ashkenazi Jewish population.

NEUROLOGICAL COMPLICATIONS IN TYPE 1 GAUCHER DISEASE Biegstraaten M, van Schaik IN, Aerts JMFG, Hollak CEM Academic Medical Centre, Amsterdam, Netherlands

THE PREVALENCE OF LYSOSOMAL STORAGE DISORDERS IN THE CZECH REPUBLIC Poupetova H1, Ledvinova J1, Hlavata J1, F|alova M1, Hrebicek M1, Kozich V1, Stastna S1, Hruba E1, Kostalova E1, Jahnova H1, Malinova V2, Asfaw B1, Kuchar L1, Novotna Z1, Zeman J2, Elleder M1 1 Inst Inherit Metab Dis, Charles Univ, Prague, Czech Republic, 2Dept Pediatrics, Univ Hosp, Prague, Czech Republic Background: Retrospective study from 1975 to 2007 to determine the prevalence of lysosomal storage disorders (LSD) in the Czech population. Methods: The prevalence of an LSD was calculated according to the method by Poorthuis et al. (1999) Hum Genet 105:151^156: The total number of diagnosed cases since 1975 to 2007 (post- and prenatal diagnoses) with a particular LSD born within a certain period of time was divided by the total number of live births within the same period. Results: 34 di¡erent LSD were diagnosed in 535 individuals in the de¢ned period. The numbers of patients in the various groups of LSD are: 331 lipidoses, 108 MPSs, 73 NCLs, 11 glycogenoses type II, 7 glycoproteinoses and 5 mucolipidoses. Lipidoses as a group are the most frequent LSD with combined prevalence of 6.26 per 100 000 (1:15 974) live births. The most frequent are: Fabry disease (48 hemizygotes and 76 heterozygotes), Niemann-Pick disease C (52 patients) and Gaucher disease (48 patients). The prevalence of all MPSs as a group is 3.4 per 100 000 (1:29 412). The most frequent are MPS III (22 patients) and MPS II (21 patients). LSD not diagnosed in the Czech population: Farber disease, galactosialidosis, fucosidosis, aspartylglucosaminuria, Schindler disease, prosaposin and saposin's de¢ciences. Conclusions: Overall prevalence of LSD in the Czech population is 1:7692 (13 per 100 000) live births. Supported by grant project VZ 111100003 and MSM 0021620806 of the Ministry of Education and Youth of the Czech Republic

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SYNTHESIS OF SPECIFIC SPHINGOLIPIDS ISOFORMS USING IMMOBILIZED SPHINGOLIPID CERAMIDE NDEACYLASE Kuchar L1, Ledvinova J1, Lenfeld J2, Horak D2, Rotkova J3, Bilkova Z3 1 Inst Inherit Metab Dis, Charles Univ, Prague, Czech Republic, 2Inst Macromol Chem, ASCR, Prague, Czech Republic, 3Dept Biol Biochem Sci, Univ Pardubice, Pardubice, Czech Republic Background: Sphingolipid ceramid N-deacylase (E.C. 3.5.1.69) is the hydrolytic enzyme isolated from marine bacterium Pseudomonas sp. TK 4. Besides its primary deacylation function, this enzyme is able to reacylate the lyso-sphingolipids under speci¢c conditions. Methods: We immobilized the enzyme on microporous magnetic bead cellulose and use the reverse acylation reaction for semi-synthesis of speci¢c sphingolipid isoforms. Results: Immobilization provided us with easily operating enzyme system long time stable and available for multiple uses. Using this system, two rarely occuring sphingolipid species with margaric acid, (C17:0) glucosylceramide and (C17:0) sulphatide, were synthesized and used as internal standards for tandem mass spectrometry. High rate of conversion (490% for C17:0 glucosylceramide) was achieved. Both lipids were ready to use without further puri¢cation in contrast with reaction products synthesized by soluble enzyme form where palmitic and stearic fatty acid contaminants were present. This pointed to existence of fatty acids in crude preparation of soluble enzyme. Conclusions: In general, our method has a good potential to prepare speci¢cally labeled sphingolipids of good purity available for di¡erent biochemical applications. This opens the way to further applications in di¡erent ¢elds of sphingolipid biochemistry and pathobiochemistry including MS of stored sphingolipids in lysosomal storage disorders. This work was supported by the grant projects of Ministry of Education MSM 0021620806 and MSM 0021627502

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MS/MS SPHINGOLIPID ISOFORM PROFILING ^ USEFUL DIAGNOSTIC TOOL IN DISORDERS WITH Gb3Cer AND SULPHATIDE STORAGE Kuchar L, Hlavata J, Asfaw B, Ledvinova J Inst Inherit Metab Dis, Charles Univ, Prague, Czech Republic Background: Sphingolipidoses are characterized by accumulation of nondegraded sphingolipids due to mutations in genes of corresponding hydrolases or protein activators. The sphingolipid activator proteins (saposins, SapA, B, C and D) are important cofactors for the lysosomal degradation of sphingolipids with short hydrophilic head groups. De¢ciencies of individual saposins or their precursor prosaposin (pSap) result in the blockade of catabolism of corresponding sphingolipids similarly to related enzymopathies caused by defects of enzyme protein. In SapB de¢ciency, major accumulated lipid is Gb3Cer (also accumulated in Fabry disease, X-linked a-galactosidase a de¢ciency) and sulphatide (also in MLD, arylsulphatase A de¢ciency). In pSap de¢ciency, the storage includes the whole array of sphingolipids. Methods: In our study AB/MDS SCIEX API 3200 tandem mass spectrometer was used for lipid quanti¢cation and pro¢ling from urinary and cell extracts. Spectrum of molecular species (isoforms) based on di¡erent chain lengths of fatty acids in ceramide moiety were scanned for each sphingolipid. Results: Beside increased quantity, changes in pro¢les of accumulated sulfatides and Gb3Cer speci¢c to pathology were detected. The shift in isoform pro¢le toward molecular species with longer fatty acid was clearly evident in all urinary samples with defects of degradation of both Gb3Cer and sulphatides. Conclusion: Based on these ¢ndings, we proposed calculation of the ratio of C24 family species to C18:0 fatty acids as a new simple prediagnostic marker useful in the ¢rst stage of screening. This work was supported by the grant project of Ministry of Education MSM 0021620806

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LONG-TERM EFFICACY AND SAFETY OF AGALSIDASE ALFA IN WOMEN WITH FABRY DISEASE Whybra C1, Kampmann C1, Miebach E1, Gal A2, Baron K1, Beck M1 1 Univ Child Hosp, Mainz, Germany, 2Institute of Human Genetics, Hamburg, Germany Background: Women heterozygous for Fabry disease experience all of the signs and symptoms of Fabry disease as male patients, but with more varied onset and expression. Enzyme replacement therapy (ERT) should bene¢t female Fabry disease patients. Methods: In this open-label, single-centre study, 40 women (average age 47 years) with a con¢rmed Fabry mutation were treated with agalsidase a (Replagal., Shire Human Genetic Therapies, Inc., Cambridge, MA, USA, 0.2 mg/kg iv, every other week). Clinical endpoints (Mainz Severity Score Index (MSSI), estimated glomerular ¢ltration rate (e G F R), p ro t e i n u r i a , a n d l e ft v e n t r i c u l a r m a s s ( LV M, echocardiography) were evaluated at baseline and at yearly intervals. Results: MSSI at baseline was 26.3+1.9 (mean+SE). MSSI was signi¢cantly reduced after 1 year of ERT and decreased to 19.7+1.6 (p50.001) after 4 years. eGFR was signi¢cantly improved in patients with Stage 2 chronic kidney disease at baseline, with improvement seen as soon as 1 year of treatment (mean = 77 ml/min/1.73 m2.7 at baseline, 87 ml/min/1.73 m2.7 after 1 year). T|me of treatment was signi¢cantly associated with a reduction in proteinuria in the 19 patients with baseline protein excretion exceeding 200 mg/day. LVM was reduced from 86.5+7.7 g/m2.7 at baseline to 66.5+5.5 g/m2.7 after 1 year in the 28 patients with baseline LV hypertrophy, and remained reduced through 4 years of treatment. Three patients experienced mild infusion reactions during the study. No anti-agalsidase alfa antibodies were detected. Conclusion: The results of this study suggest that women with Fabry disease demonstrate important clinical bene¢ts while being treated with agalsidase alfa.

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MOLECULAR ANALYSIS OF 82 MUCOPOLYSACCHARIDOSIS TYPE I PATIENTS: MUTATIONAL SPECTRUM IN THE EUROPEAN POPULATION AND IDENTIFICATION OF 28 NOVEL MUTATIONS Bertola F1, Parini R2, Casati G1, Tylki-Szymanska A3, Okur I4, Tuysuz B5, Dalmau J6, Gonzales Meneses A7, Antuzzi D8, Barone R9, Dionisi V|ci C10, Donati A11, F|locamo M12, Gabrielli O13, Parenti G14, Scarpa M15, Uziel G16, Biondi A17 1 Consorzio Genet Molec e Um, Univ Bicocca, Monza, Italy, 2Center Metab Dis, Univ Hosp `S. Gerardo', Monza, Italy, 3Inst Pomnik Centrum Zdrowia Dziecka, Warsaw, Poland, 4Gazi University Hosp, Ankara, Turkey, 5Univ Cerrahpasa Medical Faculty, Istanbul, Turkey, 6Hosp Infantil La Feè, Valencia, Spain, 7Hosp V|rgen del Roc|èo, Seville, Spain, 8 Pediatr Inst, Policlinico `A. Gemelli', Roma, Italy, 9Dept Pediatrics, Policlinico, Catania, Italy, 10Metab Unit, Pediatr Hosp, Roma, Italy, 11 Dept Pediatr, `Meyer' Inst, F|renze, Italy, 12Pre-Postnatal lab, Inst `G. Gaslini', Genova, Italy, 13Dept Pediatr,Univ Hosp `G. Salesi', Ancona, Italy, 14Dept Pediatr, Univ `Federico II', Napoli, Italy, 15Dept Pediatr, Univ Hosp, Padova, Italy, 16Neurologic Inst `Carlo Besta', Milano, Italy, 17 Fond `M Tettamanti' Univ Milano Bicocca, Monza, Italy Background: The phenotype of mucopolysaccharidosis type I (MPSI) ranges from severe (Hurler) to attenuated (Scheie). Information on genotype/phenotype correlation is useful for therapeutic decisions in the ¢rst two years of life and genetic counselling. Methods: Mutational analysis of alfa-L-iduronidase gene was carried out by ampli¢cation and direct sequencing of the coding and splicing regions in 82 MPSI European patients from Italy (32), Poland (20), Spain (16), Turkey (13) and Greece (1) (56.1% severe, 24.4% intermediate, 15.8% attenuated phenotype, 3.7% not available). Results: Mutations were found in 155 of 164 alleles (94.5%), reconstructing the complete genotype in 75/82 patients. 47 di¡erent mutations were identi¢ed: 5 nonsense, 20 missense, 11 insertion/ deletion and 11 splice site mutations. 28 of them were novel mutations: they were considered severe if found in homozygosity in patients with severe phenotype or associated to a known severe mutation and mild if found in homozygosity in patients with attenuated phenotype or associated to a known severe mutation. F|ve recurrent mutations were identi¢ed in 49.4% of alleles; their prevalence varied widely among countries: Q70X Turkey 3.8% 44 Poland 42.5%; W402X Turkey 7.7% 44 Spain 34.4%; P533R Poland 0% 44 Turkey 7.7%; G51D Turkey and Poland 0% 44 Italy 14.1%; P496R, was a common mutation only in Italy (12.5%). Conclusions: Our study allowed to de¢ne the genotypes of most of our patients. Genotype/phenotype correlation was possible in many cases of MPS I, although the wide spectrum of mutations found in the European population makes it lacking for some patients.

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A PHASE 2 CLINICAL TRIAL OF THE PHARMACOLOGICAL CHAPERONE AT2101 FOR THE TREATMENT OF GAUCHER DISEASE Weinreb N1 on Behalf of AT2101 Study Group, Schneider E2, Dinh Q2, Duke C2, Insinga F2, Scott K2, Do H2, Wustman B2, Palling D2, Lockhart DJ2 1 Research Foundation for LSDs Inc, Coral Springs, Florida, USA, 2 Amicus Therapeutics, Inc, Cranbury, New Jersey, USA Background: AT2101 (isofagomine tartrate) is an orally administered small molecule pharmacological chaperone designed to selectively bind and stabilize glucocerebrosidase (GCase), thereby increasing GCase tra¤cking to the lysosome and cellular enzyme activity. Methods: A randomized, open-label trial of AT2101 in patients with Type I Gaucher disease (GD1) was conducted at 11 U.S. sites with the primary objective of evaluating safety and tolerability of di¡erent doses and dose regimens. The secondary objective was to evaluate pharmacodynamic measures of response, including e¡ects on GCase levels in leukocytes, plasma levels of glucocerebroside, and others. Thirty clinically stable subjects with GD1 receiving imiglucerase enzyme replacement therapy (ERT) were enrolled. ERT was interrupted for a 7-week period during which subjects received AT2101 for 4 consecutive weeks. Results: AT2101 was generally well-tolerated at all doses evaluated. No serious adverse events were reported. One subject withdrew because of an adverse event deemed unrelated to AT2101. Leukocyte GCase activity increased in 20 of the 26 evaluable subjects who had a range of di¡erent genotypes; higher doses of AT2101 produced more consistent elevations of GCase. There were no changes in hematological parameters, plasma glucocerebroside, or other markers. Conclusion: 4-week administration of oral AT2101 in patients with GD1 who temporarily suspended ERT was safe and well tolerated and associated with variable increases in leukocyte GCase activity across genotypes and dose regimens. A 6-month AT2101 safety and clinical e¤cacy trial in ERT-naive patients with GD1 is underway.

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PRENATAL DIAGNOSIS OF NIEMMAN-PICK DISEASE TYPE C WITHOUT FAMILY HISTORY Ribeiro I1, Amaral O1, Ribeiro H1, Caseiro C1, Dupont J2, Couceiro A3, Lacerda L1 1 U Enzimol, C Genet Med J Magalhaes-INSA, Porto, Portugal, 2U DPN, Hospital Santa Maria, EPE, Lisbon, Portugal, 3CDPN, Maternidade Bissaya Barreto, Coimbra, Portugal Background: Niemman-Pick disease type C (NPC) is a rare autosomal recessive disease characterised by intralysosomal accumulation of cholesterol due to defective cholesterol sorting. NPC1 gene mutations or, less frequently, NPC2 mutations may lead to indistinguishable phenotypes. A wide spectrum of phenotypes has been associated with NPC. Methods: NPC can be diagnosed by demonstration of intralysosomal accumulation of unesteri¢ed cholesterol by ¢lipin staining in cultured ¢broblasts or amniocytes; diagnostic con¢rmation requires cholesterol esteri¢cation studies or mutation analyses. Results: The recent ¢nding of two cases of nonimmune hydrops fetalis (NIHF), in two pregnancies of young women without family history of NPC but with exuberant ¢lipin staining in cultured amniocytes prompted the mutation analyses of the foetuses samples. The detection of previously identi¢ed causal mutations in the NPC1 gene con¢rmed that both foetuses were a¡ected with NPC disease. One foetus was homozygous for c.351__352delAG and the other one was a compound heterozygote for p.I935K and p.A1035V. Study of parental samples con¢rmed the segregation of the mutant alleles in the respective families. Conclusions: Lysosomal storage disorders are one of the known causes of NIHF, these ¢ndings illustrate how NPC should be considered as a relatively frequent cause in such cases. The ¢nding of three di¡erent mutations, in two a¡ected foetuses from the centre of Portugal, might suggest an increased risk for NPC in that region. Furthermore, the fact that homozygosity for those mutations was previously found to be associated to severe infantile forms, with classic biochemical features, is indicative of a negative prognosis.

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BISPHOSPHONATE THERAPY IN OSTEOPOROSIS IN LYSOSOMAL STORAGE DISEASES Procopio E, Ciani F, Gasperini S, Pasquini E, Zammarchi E, Donati MA Metab and Muscular Unit, AOU Meyer, Florence, Italy Enzyme replacement therapy as treatment for lysosomal storage diseases (LSD) is now available and the safety and e¡ectiviness of ERT for Fabry disease, mucopolysaccharidoses (MPS) I, MPS II and MPS VI, as well as for Pompe's disease have been demonstrated in well designed clinical trials and the treatments are now commercially available. So, life expectancy and quality of life of these patients are now improved. Bone involvement in these diseases can represent a major source of morbidity because of pain and/or limitation of function. A signi¢cant proportion of patients have osteopenia/ osteoporosis. Dual energy X-ray absorptiometry scan (DXA) is recognized the reference method to misure bone mineral density. We investigated the bone mineral status in patients with Morquio A (4 patients), MPS II (2 patients), GM1 gangliosidosis type 3 (1 patient) and Multiple sulfatase de¢ciency MSD (1 patient). Lumbar spine DXA and bone turnover markers are performed. All patients, except for one patient female who had osteopenia, had osteoporosis. We treated ¢ve patients with intravenous biphosphonates infusions: three patients a¡ected by Morquio syndrome, one patient a¡ected by GM1 gangliosidosis type 3 and one patient a¡ected by MSD. The infusions have well been tollerated and all patients experienced bene¢cial e¡ects on everyday activities, back pain and so, improvement of quality of life. Very few data there are in literature about osteoporosis in lysosomal storage diseases. Osteoporosis is a common, undiagnosed complication of LSD. Bisphosphonate IV infusions are an e¡ective and well tolerated treatment for LSD osteoporosis.

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NIEMANN-PICK TYPE C: NO NEUROLOGIC IMVOLVEMENT AFTER THREE YEARS OF TREATMENT WITH MIGLUSTAT F|umara A1, Dardis A2, Federici S3, Barone R1, Di Rocco M3 1 Dept Pediatrics, Catania Univ, Catania, Italy, 2Metabolic Unit, IRCCS Burlo Garofolo, Trieste, Italy, 3Pediatrics, Gaslini Institute, Genova, Italy Background: NPC is a recessive lipid storage disease with a wide clinical spectrum. Newborns usually present cholestasis and progressive hepatosplenomegaly. Hypotonia and psychomotor delay are evident by the age of 12^18 months, followed by loss of acquired skills and spasticity. Zervas (2002) reported delayed neurological signs, increased life span and reduced ganglioside storage in murine and feline NPC models treated with Miglustat, a glicosphingolipid synthesis inhibitor. Case report: A 3 month old girl with persistent severe cholestasis, yet detected at birth, underwent liver biopsy revealing cirrhosis and storage cells. At the age of 6 months, she showed mild axial hypotonia, a 5 month psychomotor development and grade II esophageal varices. MRI and electrophysiological studies were normal. Diagnosis of NPC was made by philipin staining on skin ¢broblasts and NPC1 gene analysis (homozygousity for Y1019C). Miglustat was started at the age of 7 months at the dosage of 25 mg/kg/day in three doses. Results: Today, age 3.7 years, her psychomotor development is normal and no clinical or instrumental sign of neurological impairment is evident. Liver is palpable at the iliac fossa and spleen is 15 cm below the costal margin. Conclusions: Miglustat inhibits glucosylceramide synthase which catalyses the ¢rst step of glycosphingolipid synthesis. Our patient is the youngest NPC case treated for 36 months without any adverse event. She had normal psychomotor achievement without clinical, electrophysiological or neuroradiological signs. This experience, although limited to one case, suggests that miglustat can prevent the neurological disease but not visceromegaly when started early.

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VERTEBRO-MEDULAR IMAGING FINDINGS IN MUCOPOLYSACCHARIDOSIS TYPES II AND IV Castro S1, Ayres-Basto M1, Rodrigues E2, Campos MM3, Guimara¬es J2, Lea¬o-Teles E2 1 Neuroradiology Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 2Metab Dis Unit, Ped Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 3Neuroped, Ped Dept, Univ/Hosp St Joa¬o, Porto, Portugal Introduction: Mucopolysaccharidosis (MPS) evolution leads to progressive accumulation of glycosaminoglycans in several organs, including the spine. Speci¢c bony and ligament changes were described. Our aim is to present MRI ¢ndings in a group of MPS. Methods: We performed cervico-toracic spinal MRI to nine patients (mean age: 13.3 years; seven MPS VI and two MPS II) and toracolombar spinal MRI to six patients (mean age: 13.6 years; four MPS VI and two MPS II) with MPS. Odontoid hipoplasia, peri-odontoid soft tissue mass, posterior ligament hypertrophy, toraco-lombar beaked vertebra, kyphosis and notching of the posterior wall were evaluated. Spinal cord, dural sac and cauda equina involvement were also evaluated. Results: At cervico-occipital junction, peri-odontoid soft tissue mass was present in seven patients, associated to odontoid hypoplasia in three cases. Posterior ligament hypertrophy was positive in only 1 patient. Two patients showed C1/C2 subluxation. Other two went chirurgical decompression prior to MRI. In this region eight patients showed intraspinal repercussion, with severe cord compression in three patients. At toraco-lombar region, all four MPS type VI patients studied presented beaked vertebra and consequent kyphosis with cauda equina stretching in one case. No beaked vertebra was present in type II patients. Notching of the posterior wall was present in all patients. Discussion: Our series presented all spinal imagiological features described to date in MPS. As previously described, type VI patients have severe vertebral changes while type II present milder alterations. Knowledge about these ¢ndings is important to prognosis, monitoring and therapeutic options in this diseases.

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BEHAVIOURAL, NEUROPHYSIOLOGICAL AND NEUROCHEMICAL ALTERATIONS IN MALE AND FEMALE (HOMOZYGOUS) FABRY MICE Rodrigues LG1, Ferraz MJ1, Pais-V|eira M2, Sousa MM3, Saè Miranda MC1 1 UniLiPe, IBMC, Porto, Portugal, 2Morphophysiology, IBMC, Porto, Portugal, 3Nerve Regeneration, IBMC, Porto, Portugal Background: Fabry disease is an X-linked inherited disorder of glycolipid metabolism resulting from de¢cient activity of the lysosomal enzyme, a-galactosidase A. Glycosphingolipids, predominantly globotriaosylceramide (Gb3), accumulates in several tissues. To the date the male hemizygous and the female homozygous were not used to study the peripheral nervous system (PNS) and/or behaviour. The main aim of the present study was to use a sensorimotor screen, the SHIRPA procedure, for assessment of the evolution of the Fabry mice phenotype. Methods: From the SHIRPA we assessed primary behavioural alteration, motor nerve conduction (MNCV) and the hot-plate. The sciatic nerve was studied in order to assess if Fabry mice display alterations in the PNS. Results: When screening for the SHIRPA protocol it was found several parameters altered and a few di¡er between groups and genders. Fabry mice displayed hypoalgesia and no alteration in MVCV for both genders and ages studied. Also accumulation of Gb3 in the PNS was found for the ¢rst time in the Fabry mice. Conclusions: We found slightly di¡erences in the behaviour phenotype between females homozygous and male Fabry mice. In summary our data shows that the peripheral nerves of Fabry mice displays similar features as the ones described for human patients, i.e. accumulation of Gb3, reduced number of unmyelinated axons and small myelinated axons and preserved large diameter myelinated axons. The Gb3 accumulation found in the PNS of the Fabry mice can be a possible explanation for the alterations observed in thermal sensibility and impairment in the sensorimotor function.

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CEREBRAL IMAGING FINDINGS IN MUCOPOLYSACCHARIDOSIS TYPES II AND IV Castro S1, Ayres Basto M1, Rodrigues E2, Guimara¬es J2, Magalha¬es A3, Lea¬o Teles E2 1 Neuroradiology Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 2Metab Dis Unit, Ped Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 3Ophthalmology Dept, Univ/Hosp St Joa¬o, Porto, Portugal

PROGRESSIVE SPINAL INSTABILITY IN MPS ^ USE OF RECOMBINANT HUMAN BONE MORPHOGENETIC PROTEIN-2 (RHBMP-2) TO AUGMENT POSTERIOR SPINE FUSION Solanki G1, V|jay S2, Chakrapani A2, Hendriksz C2 1 Department of Paediatric Neurosurgery, Birmingham, UK, 2Clinical Inherited Metabolic Dsorders, Birmingham, UK

Background/Objectives: Mucopolysaccharidosis (MPS) evolution leads to progressive accumulation of glycosaminoglycans, including at the central nervous system (CNS). Magnetic resonance imaging (MRI) has an important role to assist in the diagnosis and to monitor the treatment e¡ects. Regarding neuroimaging, cortical atrophy, white matter (WM) lesions, dilated perivascular spaces (PVS) and hydrocephalus are commonly observed in MPS patients. Our aim is to present MRI ¢ndings in a group of MPS II and VI. Methods: We performed brain and orbital MRI in nine patients (mean age: 13.3 years): seven MPS II and two VI, all with severe forms besides one MPS VI patient. Lesions in WM, cortical atrophy, ventricular and PVS enlargement, enlarged PVS, optic sheath and sella turcica enlargement was evaluated. Results: Cortical atrophy, hydrocephalus and PVS were the most common ¢ndings in our patients. Cortical atrophy was present in all patients in grade 3, excluding one patient with grade 1. Grade 3 hydrocephalus was the most common ¢nding in type VI patients. PVS were di¡usely present in 8 patients with di¡erent grade of involvement; WM seems to be the most severely involved area; type II patients had always grade 2 or 3. Conclusion: Our series presented all cerebral imagiological features described to date in MPS. Severity of cerebral changes was higher in type VI patients. We found severe cerebral atrophy and hydrocephalus in almost all patients, despite current literature present type VI as one of the less aggressive to brain. PVS seems to have more severe dilation in MPS II patients.

Aims: We consider the use of bone morphogenetic protein (BMP) to enhance spinal fusion. Patient and Methods: 2 children with a diagnosis of mucopolysaccharridoses (MPS) not amenable to enzyme replacement therapy had progressive disease a¡ecting the cervical spine with neurological deterioration. They underwent occipito-cervical ¢xation and fusion. RhBMP-2 applied to an absorbable collagen sponge (ACS) matrix was used in all cases. Results: There was evidence of disease stabilization, good spinal alignment and construct integrity. None of the children deteriorated neurologically. The quality of bone at fusion sites appeared improved. New bone formation was evident and it appeared to incorporate with existing bone. Bony fusion began at 4-weeks and at 3 months very good incorporation and fusion was seen. There was no evidence of spinal canal encroachment and no adverse e¡ects related to the rhBMP-2/ ACS-carrier matrix. Conclusion: To our knowledge rhBMP-2 has not been used in the treatment of MPS related instability. We report our early experience with rhBMP-2 in augmenting posterior spinal fusion in MPS. Its e¡ect on bone of enzyme-treated patients is not known. We discuss the rationale and detail our encouraging results both to e¡ectiveness and speed. However with all new developments we highlight the need for further experience and longer follow-up in children. Disclosure: The use of rhBMP-2 for posterior approaches is o¡ label.

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EXPERIENCE WITH ENZYME REPLACEMENT THERAPY IN 6 MPS VI PATIENTS Valayannopoulos V1, Chabli A2, Lemoine M3, Odent T4, Goldenberg A5, Barbier V1, Caillaud C6, Lemerrer M7, de Lonlay P1 1 Metab Unit, Necker-Enf Malades Hosp, Paris, France, 2Bioch Lab, Necker-Enf Malades Hosp, Paris, France, 3Phys Med, Necker-Enf Malades Hosp, Paris, France, 4Orthoped Surg Dep-Necker Enf-Malades Hosp, Paris, France, 5Genet Dept, Rouen Univ Hosp, Rouen, France, 6 Bioch Lab, Cochin Hosp, Paris, France, 7Genet Dept, Necker-Enf Malades Hosp, Paris, France Introduction: Mucopolysaccharidosis VI (MPS VI), is a progressive multisystemic lysosomal disease, due to de¢ciency of arylsulfatase B. Clinical trials in patients with MPS VI have shown e¤cacy of enzyme replacement therapy (ERT) in the form of galsulfase (Naglazyme. ). Patients: We present 6 MPS VI patients treated by galsulfase for 12 months so far. Patients 1 (18 year old male) and 6 (3 year old male) presented with the severe, early onset form of the disease. Patients 2 (12 year old female) and 3 (4.5 year old female) presented with a mildest form, responsible for small stature and osteoarticular involvement, with hip dysplasia and bone pain. Patient 4 (14 year old) and 5 (12.5 year old), presented with an intermediate form of the disease associating dysmorphic features, reduced joint mobility, hepatosplenomegaly but no severe complications. Methods: All patients received weekly 1 mg/kg galsulfase infusions that were uneventful. Results: Patient 1 displayed reduction of upper-airways obstruction and hepatosplenomegaly but developed cardiomyopathy and was deceased. Patients 2 and 3 displayed better joint mobility and less osteoarticular pain. Growth remained normal for patient 3, whereas it remained impaired for patient 2. No other complication occurred. Patients 4 and 5 displayed better general condition and joint mobility. Patient 6 improved in growth, ENT infections, joint mobility and size of hepatosplenomegaly. Urinary glycosaminoglycan decreased for all patients close to the normal range. Conclusion: The ¢rst results of ERT in our 6 MPS VI patients are encouraging; the bene¢cial e¡ects seem more signi¢cant in younger patients and highlight the importance of early diagnosis and treatment.

PSYCHOLOGICAL FOLLOW-UP OF CHILDREN ON ENZYME REPLACEMENT THERAPY FOR LISOSOMAL STORAGE DISORDERS AND THEIR PARENTS Rodrigues F, Vaz C, Almeida M, Martins F, Diogo L, Garcia P Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal Background/Objectives: Lysosomal storage disorders (LSD) are progressive diseases that a¡ect the quality of life of patients and families. Our aim was to evaluate the cognition, development and adaptative behavior in children with LSD on enzyme replacement therapy (ERT) and their parents. Methods: We evaluated parents and 13 patients (5 males, aged 1 to 21 years): 8 mucopolysaccharidosis (MPS), 3 Pompe, 2 Gaucher. Children were rated with Ruth Gri¤ths Development Evaluation Scale, V|neland Adaptive Behavior Scale and Wechsler Intelligence Scale for Children. Parents answered Beck Depression Inventory (BDI) and a questionnaire about perceptions on LSD and ERT. Results: F|ve MPS-I patients have moderate to severe developmental delay. In the last two years, the adaptative behavior and cognition stabilized. The three MPS-VI patients have normal evaluations except for autonomy and motricity scales, two of them are stable and one worsened his visual skills. The Pompe juvenile patients have severe autonomy delay which didn't progress. Gaucher patients have normal evaluations. Parents tested by BDI revealed some degree of depression in 7/16. ERT expectations in the beginning of the treatment were high to medium in 13/16 parents, and declined after four years of ERT. Two parents have considered stopping treatment. Quality of life and level of familial stress didn't seem to change with therapy. Conclusions: ERT seems to stabilize or slow the progression of these diseases, even in severe forms. Assessing parents expectations about the ERT favors the families well being.

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OPEN-FIELD AND DARK-LIGHT TESTS: BEHAVIOURAL CHARACTERIZATION OF FABRY KNOCKOUT MICE Ferraz MJ1, Magalha¬es A2, Rodrigues LG1, Saè Miranda MC1 1 UniLiPe, IBMC, Porto, Portugal, 2Neuroprotection Group, IBMC, Porto, Portugal Background: Fabry disease (FD) is an X-linked lysosomal storage disorder characterized by de¢cient a-galactosidase A activity and intracellular accumulation of neutral glycosphingolipids, mainly globotriaosylceramide. Among other symptoms, FD present painfull peripheral neuropathy and neurological manifestations that may include CNS dysfunction, such hemiparesis, ataxia and psychosocial and pshychiatric disorders (especially depression, anxiety, demencia and suicide). The animal model of FD provides us with an opportunity to unveil the disease mechanisms. The aim of this study was to asses the behavioural pro¢le of the Fabry knockout mice at two di¡erent ages in the dark^light and open-¢eld tests in order to study possible behavioural alterations of the disorder. Methods: Male Fabry mice at 24 and 48 weeks old and their respective background group (C57BL/6J) were placed in the open-¢eld or dark^ light, and a 5 min session was recorded. The frequency and time course of several behaviours (i.e. exploration, rear, return, sleep, stretch attend, groom, jump, immobility and total locomotor activity) were analysed. Results: Regarding the dark^light test, Fabry mice at both ages displayed a reduced activity and a decrease in the frequency of exploration. In the open-¢eld, Fabry mice showed reduced exploratory activity in the ¢rst minute of the session, and evidenced a signi¢cantly decreased in the exploratory and rearing behaviours concerning the all session. Conclusions: Our observations suggest that, upon exposure to an adverse situation, Fabry mice evidence decreased exploratory activity and signs of anxiety-like behaviour, at 24 and 48 weeks.

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CHONDROGENIC DIFFERENTIATION IN MESENCHYMAL STEM CELLS: GLYCOSAMINOGLICANS SYNTHESIS AND GALNS ACTIVITY Gutierrez ML, Malaver LF, Echeverri OY, Barrera LA Inst Errores Innatos, Univ Javeriana, Bogotaè, Colombia Background: Mucopolysaccharidosis IVA (MPS IVA) is caused by the lysosomal enzyme N-acetyl galactosamine-6-sulfate sulfatase (GALNS) de¢ciency. Although therapies for MPS IVA are under development they have limitations of clinical e¡ect in cartilage lesions, thus other options have been considered. Cellular therapy is one potential alternative due to putative stem cell utilization. Mesenchymal stem cells or stromal cells (MSCs) are believed to be common precursors to di¡erentiated cell lineages found in adult tissues. Because MSCs are multipotent and easily expanded in culture, there has been much interest in their clinical application In order to evaluate their therapeutic potential for MPS IVA disease, MSCs from adipose tissue cells must be ¢rst characterized. Methods: MSCs from processed lipoaspirate and lipectomy were isolated from control individuals and processed as described by Godhart et al. Puri¢ed preparation of MSCs were cultured in DMEM supplemented with 10% FCS. Results: Ex vivo expanded populations were used between passages 2 and 3 for immunophenotypic analysis. Multipotential was con¢rmed by di¡erentiation to adipogenic, osteogenic and chondrogenic di¡erentiation. GALNS activity was quanti¢ed. Before di¡erentiation values ranged from 0.0209^0.0510 nmol/h/mg and decreased drastically as a function of time during the process of chondrogenesis, days 0, 7, 14 and 21. In addition, glycosaminoglycan synthesis increased. Conclusions: To our knowledge this is the ¢rst report for GALNS activity in MSCs from adipose tissue in an in vitro model for chondrogenesis and suggests a decrease in GALNS expression during this process.

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WHICH IS THE FREQUENCY OF FABRY DISEASE? Gaspar P1, Rodrigues D1, Herrera J2, Saè Miranda MC1 1 UniLiPe, IBMC, Porto, Portugal, 2Ser Nefrologia, Hosp Central Astuèrias, Oviedo, Spain Background: Fabry disease (FD) is an X-linked lysosomal storage disorder due to the de¢cient activity of alpha-galactosidase A. The enzyme de¢ciency caused by mutations in the GBA gene leads to progressive accumulation of globotriaosylceramide (GL-3) and related glycosphingolipids, particularly in the vascular endothelium leading to renal failure, cardiac and cerebrovascular disease. The incidence of FD, based on the number of patients identi¢ed after a clinical diagnosis is estimated to be 1 in 117 000 births and 1 in 40 000 males. However, the really prevalence of FD is unknown. Here we report the screening of FD in a haemodialysis population. Methods: Alpha-galactosidase A activity and genotype analysis were performed in dried blood spots (DBS) from 857 Spanish individuals under haemodialysis. Fluorimetric methods, DHPLC and sequencing were used to identify FD in males and females, according to previously de¢ned criteria. Results: GLA mutations were identi¢ed in 7 individuals. According to these results the frequency of FD among this heamodyalisis group is 1/ 122, 1/167 in males and 1/86 in females. Conclusion: These ¢ndings provide for the ¢rst time an estimation of the incidence of alpha-galactosidase A de¢ciency in a Spanish group of individuals under haemodialysis. Further studies are required in order to con¢rm the high incidence of the FD later-onset phenotypes, since disease frequency variations may occur in di¡erent populations.

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MOLECULAR CHARACTERIZATION OF PORTUGUESE PATIENTS WITH PATHOLOGIES RELATED TO THE LYSOSOMAL MULTIENZYMATIC COMPLEX: SIALIDOSIS AND GALACTOSIALIDOSIS Coutinho MF1, Lacerda L1, Prata MJ2, Ribeiro H1, Alves S1 1 Centro de Geneètica Meèdica, INSA IP, Porto, Portugal, 2IPATIMUP, Porto, Portugal Background/Objectives: The functional activity of lysosomal enzymes sialidase, beta-galactosidase and N-acetylaminogalacto-6-sulfate in the cell depends on their association in a multienzyme complex with the lysosomal carboxipeptidase, cathepsin A. Genetic mutations in any of these complex components results in their functional de¢ciency causing severe lysosomal storage disorders. Here we report the molecular defects underlying sialidosis (mutations in sialidase; gene NEU1) and galactosialidosis (mutations in cathepsin A; gene PPGB) in the Portuguese population. Methods: Using gDNA extracted from patient's ¢broblasts, we performed a molecular study of the PPGB and NEU1 genes in the biochemically diagnosed Portuguese patients with galactosialidosis and sialidosis, respectively. The expression of both genes was determined by qRT-PCR. The e¡ect of each mutation was evaluated at protein levels using bioinformatic tools. Results and Conclusions: In the PPGB gene, we identi¢ed two missense mutations, one novel (p.G85V) and one previously reported (p.V132M) as well as two new deletions (c.228-229delC and c.1075-1076delT) both giving origin to transcripts that lead to the synthesis of truncated nonfunctional proteins. In the NEU1 gene, we found two novel missense mutations (p.P200L and p.D234N). At protein levels, these mutations result in the substitution of two aminoacids localized in a surface region of the molecule, already proposed to be involved in the interface s i al i d a s e/c at h e p s i n A. K n owl e dg e of th e s i al i do s i s an d galactosialidosis mutational spectrum is important to allow carrier detection in a¡ected families. The existence of a molecular diagnosis is a particularly important strategy since it helps to overcome the di¤culties associated to the neuraminidase enzymatic assay.

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TIME INTERVAL BETWEEN DIAGNOSIS OF TYPE 1 GAUCHER DISEASE AND INITIATION OF ENZYME THERAPY AND SPLENECTOMY ARE DETERMINANTS OF AVASCULAR NECROSIS Mistry PK1, Deegan P2, Vellodi A3, Cole JA4, Yeh M4, Weinreb NJ5 1 Yale University School of Medicine, New Haven, USA, 2Addenbrooke's Hospital, Cambridge, UK, 3Great Ormond St Childrens Hosp NHS Trust, London, UK, 4Genzyme Corporation, Cambridge, USA, 5Univ Res Found Lysosomal Storage Dis, Coral Springs, USA Objective: Treatment of type 1 Gaucher disease (GD1) is initiated at varying intervals following diagnosis. This study assessed the e¡ect of elapsed time from diagnosis to initiation of enzyme therapy with alglucerase/imiglucerase on the subsequent risk of developing avascular necrosis (AVN), the principal bone manifestation of GD1. The secondary aim was to identify other determinants of AVN. Methods: All alglucerase/imiglucerase-treated patients with GD1 enrolled in the ICGG Gaucher Registry without documented AVN prior to initiation of therapy were included. Incidence rate for the ¢rst occurrence of AVN after starting therapy was calculated according to time intervals from diagnosis to initiation of therapy. Other risk factors investigated included spleen status, GBA genotype, age at therapy initiation and enzyme dose. Results: 2700 patients met the inclusion criteria. Among GD1 patients who began alglucerase/imiglucerase 2 or more years after diagnosis, the incidence rate of AVN was 16.6 per 1000 person-years. Patients with an interval to start of treatment less than 2 years had a lower rate of AVN (incidence rate 8.1 per 1000 person-years; adjusted incidence rate ratio 52 years vs 42 years 0.58, 95% con¢dence interval 0.37^0.91). Patients with antecedent splenectomy (total or partial) had a higher incidence rate of AVN, regardless of the timing of treatment initiation (incidence rate 26.8 per 1000 person-years; adjusted incidence rate ratio vs nonsplenectomized patients 2.35, 95% con¢dence interval 1.73^3.18). Conclusion: W|th an interval of more than 2 years between GD1 diagnosis and initiation of therapy, patients have increased risk of posttreatment osteonecrosis.

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THE SAME NOVEL MUTATION DETERMINED IN 2 HURLERSCHEIE PATIENTS WHO ARE THE CHILDREN OF DIFFERENT FAMILIES Hasanoglu A1, Okur I1, Eminoglu FT1, Tumer L1, Biberoglu G1, Bertola F2, Ezgu FS1 1 Gazi Univ Hosp, Dept Ped Nutr Metab, Ankara, Turkey, 2Cons Gen Molecol Umana, Univ Mil-Bicoc, Monza, Italy Mucopolysaccharidosis type I (MPS I) is a lysosomal disease due to mutations in the gene encoding alpha-l-iduronidase (IDUA) leading to variable clinical phenotypes with progressive severe organomegaly, bone and neurological involvement in the most severe forms. De¢ciency of alpha-L-iduronidase can result in a wide range of phenotypic involvement with 3 major recognized clinical entities: Hurler (MPS IH), Scheie (MPS IS), and Hurler-Scheie (MPS IH/S) syndromes. We reported 2 Hurler-Scheie patients carried the same novel mutation (IVS4+1 G4A) and discussed the relationship between IDUA genotype, alpha-L-iduronidase biochemical parameters and clinical phenotype. F|rst patient is a 7 year old girl and other patient is a 4 year old boy. The clinical ¢ndings of patients were delayed walking, umbilical hernia, corneal clouding, coarse facies, mild hepatomegaly, hypertrichosis, dysostosis multiplex, thorakolomber kyphosis and scoliosis, mild mental-motor retardation. They are the children of di¡erent families. The mutation (IVS4+1 G4A) has not been described in the literature, yet.

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IN VITRO EFFICACY OF N-OCTYL-4-EPI-BETAVALIENAMINE (NOEV) IN A PATIENT WITH INFANTILE GM1 GANGLIOSIDOSIS Ayd|n Hi1, Kalkanoglu SHs2, Sinici I3, Tokatli A2, Coskun T2, Ozkara Ha3, Kurt I4, Higaki K5, Nanba E5, Suzuki Y6 1 Div Metab Dis, Dept Ped, Gulhane MMF, Ankara, Turkey, 2Div Metab Dis, Dept Ped, Hacettepe Univ, Ankara, Turkey, 3Dept Clin Biochemistry, Hacettepe Univ, Ankara, Turkey, 4Dept Clin Biochemistry, Gulhane MMF, Ankara, Turkey, 5Div Func Genomics, Tottori Univ, Yunago, Japan, 6Grad School, Int Univ Health and Welfare, Otawara, Japan Background: GM1 gangliosidosis is an autosomal recessive lysosomal storage disorder caused by a de¢ciency of b-galactosidase. It is mainly characterized by progressive neurodegeneration, hypotonia, dysmorphic facial features, feeding di¤culties, hepatosplenomegaly, bone deformities, and macular cherry red spots, and in its most severe infantile form it leads to death before the age of two. Currently, no e¡ective medical treatment is available for the underlying disorder. It was shown that a new synthetic chaperone compound, N-octyl-4-epi-b-valienamine (NOEV), given orally in GM1-gangliosidosis model mice entered the brain through the blood^brain barrier, enhanced b-galactosidase activity, reduced the substrate storage, and clinically improved neurological deterioration. Case Report: A 1-month-old boy was diagnosed as having GM1 gangliosidosis, on the basis of history of previous brother with GM1 gangliosidosis, and the clinical symptoms of hepatosplenomegaly, dermal melanosis, extensive Mongolian spot, and enzymatic analysis on peripheral cells revealed de¢ciency of lysosomal b-galactosidase (enzyme activity: 4 nmol/mg protein/h). The patient was found to have a novel homozygous G190D mutation in exon 6 of GLB1 gene. The enzyme activity in cultured ¢broblasts was found 18 nmol/mg protein/h (control: 534 nmol/mg protein/ h). Administration of NOEV to the culture medium at 0.2 and 2.0 mM concentrations resulted in elevation of intracellular enzyme activity to respectively 43-74 nmol/mg protein/h of NOEV. Conclusion: NOEV may be a promising treatment for patients with GM1gangliosidosis. Beta-galactosidase activities in patients with novel mutations should be experimented for the e¡ect of NOEV in vitro.

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FABRY DISEASE: A NEW ATYPICAL VARIANT FORM Lacerda L1, Amaral O1, Ribeiro I1 Silva E1, Ferreira C1, Pinto E1, Soares G2, Antunes H3 1 U Enzimol,C Genet Med J Magalhaes-INSA, Porto, Portugal, 2U Consulta,C. Genet Med J Magalhaes-INSA, Porto, Portugal, 3Ser Pediatria, Hospital S Marcos, Braga, Portugal Background: Fabry disease (FD), an X-linked glycosphingolipid catabolism disorder is due to defects in the alpha-galactosidase gene (GLA) and defective activity of the lysosomal alpha-galactosidase (alpha-GAL) with accumulation of glycosphingolipids, mostly globotriaosylceramide (GL-3) in vascular endothelium and other tissues. This multi-systemic disease may present classic symptoms at the renal, ocular, gastrointestinal, dermatologic, cerebrovascular or neurologic level, cardiac involvement can be found in atypical forms. Since the severity and complexity of the clinical symptoms are frequently age-related, the diagnosis of this disease may often be delayed and missed in early childhood. Methods: In this work the authors describe clinical, biochemical and molecular features of two hemizygous FD paediatric patients, belonging to Portuguese and Spanish families, presenting a variant phenotype. Results: Both patients presented the ¢rst symptoms in early childhood, they had low in vitro residual activity of alpha-GAL accompanied by increased GL-3 excretion in urine and they were both hemizygous for a missense mutation: p.M290I. Although the patients di¡er in age by thirteen years, in both cases, the ¢rst symptoms were at the ophthalmic level appearing within a few months after birth, then followed by a neurodegenerative process leading to loss of motor and intellectual capacities. As expected in a degenerative disorder, the younger patient does not yet present the severe degeneration observed in the older patient. Conclusions: These two cases may point to the existence of an overlooked childhood variant of Fabry disease. The atypical symptoms present in hemizygotes with the same mutation may suggest the existence of a genotype/phenotype correlation.

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ASSESSMENT OF SHOULDER FLEXION AND ABDUCTION IN MPS-II HUNTER SYNDROME POLISH PATIENTS ON GENISTEIN Tylki-Szymanèska A1, Marucha J1, Wegrzyn G2, JakoèbkiewiczBanecka J2, Piotrowska E2, Kloska A2 1 The Child Health Inst, Warsaw, Poland, 2Univ Dept of Mol Biology, Gdanèsk, Poland Background: Mucopolysaccharidosis II (Hunter syndrome) is an Xlinked disorder caused by a de¢ciency of lysosomal enzyme iduronate2-sulfatase, which results in accumulation of undegraded glycosaminoglycans (GAGs) in various tissues, contributing to the signs and symptoms of the disease. Common presenting feature is joint sti¡ness and restriction of the range of motion increasing with age. It was demonstrated previously that genistein-rich iso£avone extract inhibits synthesis of GAGs. Recently, a pilot clinical study indicated that oral administration of genistein-rich iso£avone extract, at the dose of 5 mg per 1 kg/bm daily, for 12 months, resulted in improvement of several parameters in MPS patients, including those connected with functions of connective tissue. Methods: The aim of the study was to assess the e¡ect of a 6 months administration of genistein rich soy iso£avone extract on the range of motion of shoulder passive and active £exion and abduction in three MPS II adult patients and its in£uence on activities of daily living. The active/passive shoulder £exion/abduction was measured using a doublearmed goniometer. Results: Six months of oral administration of genisteine rich soy iso£avone extract resulted in these three patients in improvement in active and passive shoulder range of motion from 10 to 30 degrees. The improvement of shoulders' motion facilitates patients' performance of the activities of daily living and makes them independent in some simple everyday activities. Conclusions: treatment with genistein may be considered as a promising therapy in MPS II patients improving joints range of motion and patients' performance in daily living.

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FUNCTIONAL CHARACTERIZATION AND CHEMICAL CHAPERONES EFFECT ON ALPHA GALACTOSIDASE GENE MUTATIONS IDENTIFIED IN FABRY ITALIAN PATIENTS F|loni C1, Caciotti A1, Carraresi L1, Cavicchi C1, Parini R2, Feriozzi S3, Poisetti P4, Garman S5, Guerrini R1, Zammarchi E1, Donati MA1, Morrone A1 1 Metabolic Unit Meyer Hosp, Florence, Italy, 2S. Gerardo Hosp, Monza, Italy, 3Belcolle Hosp, V|terbo, Italy, 4Renal Unit, Hosp Piacenza, Piacenza, Italy, 5Biochem and Mol Biol,Univ Massachusetts, Rockville, USA Background: Fabry disease is caused by de¢ciency of the alpha galactosidase A (GALA) enzyme. In order to clarify missense mutations' in£uences and to test therapeutic e¡ect of molecular chaperones, in vitro expression studies with administration of galactose and deoxygalactonojirimycin (DGJ) were performed. Methods: four mutant constructs, carrying the GLA missense mutations, p. C52Y p.L191P p.Y216C identi¢ed in male patients and p.G183A in a female proband, were generated. GALA enzyme activity, Western Blot analyses against GLA protein and addition of galactose and DGJ in culture media were carried out in transfected COS-1 cells. Results: Three-dimensional structural analyses of the enzyme helped to predict the e¡ect of the novel missense mutations. In vitro transient overexpression revealed that for all mutations there was a reduced GALA activity. The p.C52Y and p.L191P mutations caused a severely reduced GALA activity and the p.Y216C and p.G183A reduced GALA activity to 12% and 30% of wild type activity respectively. Western blot analysis showed the presence of all overexpressed mutant proteins and DGJ treatment showed an increase in residual enzyme activity in mutant transfected cells. Conclusions: In vitro expression studies, western blot analysis and mutant enzyme structural analysis help to clarify the nature of the missense genetic lesions detected in Fabry patients. All the mutations here reported are disease causing mutations and can be correlated with clinical phenotypes. Data show the important in identifying misfolding mutants for testing active-site-speci¢c chemical chaperones which are potentially useful in the treatment of Fabry. Acknowledgement: this work was partially ¢nanced by Genzyme Corporation

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MORQUIO SYNDROME: GENE EXPRESSION PROFILING AND ELASTIC FIBER ASSEMBLY IN PATIENTS' FIBROBLASTS Caciotti A1, Carraresi L1, F|loni C1, Parini R2, Antuzzi D3, Ricci R3, Scarpa M4, Procopio E1, d'Azzo A, Zammarchi E1, Guerrini R1, Donati MA1, Morrone A1 1 S Gerardo Hospital, Monza, Italy, 2Catholic Univ, Rome, Italy, 3 Metabolic Unit, Pediatrics Dept, Padua, Italy, 4Genetics Dept, St Jude, Memphis, USA Background: Morquio A and B syndromes are caused by de¢ciency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS) and beta galactosidase (GLB1). Impaired elastogenesis due to primary and secondary de¢ciencies of EBP (alternative spliced GLB1 gene product) was described. The latter, caused by the accumulation of condroitin sulfate and keratan sulfate, was reported in Costello syndrome and GM1gangliosidosis, respectively. Methods: Mutation analysis, absolute Real-T|me RT-PCR assays and elastic ¢ber analysis of ¢broblasts from two Morquio A (Pt1; Pt2) and four Moquio B (Pt3; Pt4; Pt5; Pt6) patients, are reported. Results: The new p.K129X and c.8991G4C in Pt1, and the known c.120 +1G4A at a homozygous level in Pt2 were found. Among Morquio B patients the common p.W273L was identi¢ed at a homozygous level in two brothers (Pt3; Pt4); Pt5 and Pt6 were compound heterozygotes for p. W273L/ the new p.D441N and p.W273L/the known c.1480-2A4G respectively. Normal amounts of GLB1 and EBP mRNAs were detected in Morquio B and A specimens. In Pt1, aberrant mRNAs introducing premature stop codons were detected, but real time assay showed normal GALNS mRNA level in contrast with low GALNS mRNA in Pt2. Immunohystochemistry analysis of ¢broblasts showed an absence of tropoelastin deposition in Pt1 and Pt2. Conclusions: Data on mRNAs quantitation indicate that p.129X and c.8991G4C mutations produce mRNAs able to escape the NMD pathway making them candidates for new therapies. Impaired elastogenesis, due to secondary EBP inactivation, detected in Morquio A patients' ¢broblasts, can contribute to the disease's severity.

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ENZYMATIC REPLACEMENT THERAPY IN 17 PATIENTS AFFECTED BY MPS I, II AND VI Rigoldi M1, Tedesco L2, Melzi ML1, Lastrico A1, Parini R1 1 Metab Centre Ped Dept, `S Gerardo' Hosp, Monza, Italy 2Rehabilitation Dept `S Gerardo' Hosp, Monza, Italy B a c k g r o u n d : E n z y m at i c r e p l a c e m e n t t h e r a p y ( E RT ) i n mucopolysaccharidoses (MPS) is available for few years and information is needed about its e¤cacy. Methods: We show biochemical and mobility data of patients a¡ected by MPSI (6 patients; mean age 27.6 years; range 14.5^41 years), MPSII (9 patients; mean 13.7 years; 14.4^41 years) and MPSVI (2 patients; mean 6 years; 5.7^6.3 years) on ERT for 33^66 months (MPS I), 12^18 months (MPSII) and 24 months (MPSVI). Results: In MPSI patients, urinary glycosaminoglycans (GAGs) decreased from 74 to 98% (p = 0.0063); the best changes in the range of motion (ROM) were in shoulders abduction (mean +288; from 08 to +558; p50.05); the 6 min walking test (6MWT) showed a mean improvement of +54 meters (from ^24 to +174). In MPSII patients urinary GAGs decreased from 69 to 84% (p = 0.0023); the best ROM improvement was in shoulders abduction (mean +208; from ^158 to +658; p50.05); the 6MWT got worse (mean ^15 meters, from ^83 to +88 meters). In MPSVI patients, GAGs mean reduction was 71%; ROM showed a slight improvement in knees extension (mean +48), whereas the 6MWT had a mean improvement of +129 meters (from +94 to +163). Conclusion: All MPS types show a biochemical improvement particularly in the ¢rst 3 months of ERT. About ROM, statistically signi¢cant improvements are seen in shoulders abduction for MPSI and MPSII patients. Regarding 6MWT, only MPSI and MPSVI had an improvement. The bad results in 6MWT of MPSII patients could also be due to mental retardation leading to poor compliance.

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ANNUAL INCREASE OF HEIGHT IN MPS I PATIENTS ON ENZYME REPLACEMENT THERAPY WITH LARONIDASE Tylki-Szymanèska A, Marucha J, Arasimowicz E, Rozçdzçynèska A The Child Health Inst, Warsaw, Poland Background: Mucopolysaccharidosis type I (MPS I) results from de¢ciency of the activity of lysosomal enzyme a-L-iduronidase (IDUA). Glycosaminoglycans (GAGs) accumulate in multiple organs. Patients with MPS type I present growth de¢ciency. Patients with Hurler phenotype present severe growth de¢ciency comparing to patients with Scheie phenotype. The aim of the study was to analyze the dynamic of growth in MPS I patients without treatment (retrospectively) and during the enzyme replacement therapy with laronidase in dose 100 mg/kg/ bm/every week. Methods: Annual increase of height and standard deviation were analyzed in 14 patients with MPS I, 9 presented Hurler, 2 Hurler/ Scheie and 3 Scheie phenotypes with age range from 1 to 15 years at the baseline of treatment. Seven patients from this cohort started the treatment with laronidase at the age of 1 year. The observation period was from 1 to 4.5 year. Stadiometer was used for the height assessment. Results: In patients who started ERT at the age of 1 year the growth dynamics was comparable to the growth dynamics of untreated. The slower growth dynamics in patients with Hurler phenotype was noted from the age of 2, despite the treatment. In patients with Scheie phenotype the growth dynamics was variable, but the slower growth gain was noted from the age of 5. Conclusions: ERT with laronidase in patients with MPS I does not improve height gain. Growth pattern is similar in treated and nontreated patients.

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PRELIMINARY RESULTS OF A PHASE 2 CLINICAL TRIAL OF GENZ-112638 IN PATIENTS WITH TYPE 1 GAUCHER DISEASE Lukina E1, Watman N2, Arreguin EA3, Banikazemi M4, Iastrebner M5, Rosenbaum H6, Zimran A7, O'Brien F8, Smith SE8, Puga AC8, Peterschmitt J8 1 Hematol Res Ctr Russian Acad Med Sci, Moscow, Russian Federation, 2 Hospital Ramos Mejia, Buenos Aires, Argentina, 3Inst Mex del Seguro Soc Hosp de Especial, Col. La Raza, Mexico, 4NYU, New York, USA, 5 Inst Argentino de Diagnost y Tratamiento, Buenos Aires, Argentina, 6 Ramban Medical Center, Haifa, Israel, 7Sha'are Zedek Medical Center, Jerusalem, Israel, 8Genzyme Corporation, Cambridge, USA Introduction: Genz-112638, an investigational compound, is a novel oral small molecule inhibitor of glucosylceramide synthase being developed for the treatment of Gaucher disease type 1 (GD1). Objective: To assess the e¤cacy, safety, and pharmacokinetics of Genz112638 in patients with GD1. Methods: An ongoing open-label Phase 2 clinical trial of Genz-112638 (50 or 100 mg bid orally) enrolled patients with GD1 in Israel, North America, Russia, and South America. The main e¤cacy endpoints of the study were changes in hemoglobin level, platelet count, and spleen volume after 52 weeks. An extension study will follow. Results: Data were available for up to 21 and 13 patients receiving Genz112638 at 26 and 52 weeks, respectively. All 9 males and 12 females (age range: 18^55 years) were Caucasian. Four were of Ashkenazi Jewish and 1 was of Hispanic descent. At 26 and 52 weeks mean hemoglobin increased by 0.9 g/dl and 1.3 g/dl, respectively; mean platelet count increased by 18% and 34%; mean spleen volume decreased by 27% and 40%; mean liver volume decreased by 7% and 14%; mean chitotriosidase activity decreased by 30% and 50%. Plasma glucosylceramide levels normalized in all patients. Seven related adverse events were reported in 6 patients and all were mild and transient in nature. Conclusions: Initial observations suggest that Genz-112638 may represent a safe, e¡ective, and convenient oral therapy for patients with GD1. Clinical development of Genz-112638 will proceed in ongoing and additional clinical trials.

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RETINITIS PIGMENTOSA AND ADULT-ONSET NEUROLOGICAL DETERIORATION REVEALING SANFILIPPO A DISEASE Nassogne MC1, Jeanjean A1, Kestens C2, Grandin C1, V|ncent MF1, Lissens W3 1 Cliniques universitaires Saint-Luc, Bruxelles, Belgium, 2Clinique SaintPierre, Ottignies, Belgium, 3Vrije Universiteit Brussel, Brussel, Belgium Background: San¢lippo disease is a lysosomal storage disorder characterised by progressive mental deterioration during infancy. Lateonset forms of this disease are rare and not well described. Methods and Results: we report the clinical history of this 40 year-old Belgian woman referred to our clinic for evaluation of pigmentosa retinitis associated with behavioural problems. The parents were not consangui neous and the family h istory was negative for neurodegenerative disease or pigmentosa retinitis. Her history was unremarkable during the ¢rst years of life and attention de¢cit was described at the age of 8. She had attended a secondary education in a specialised school. She had never worked. At the age of 36, pigmentosa retinitis (rod cone dystrophy) was discovered. Behavioural problems increased during the last 5 years with aggressive attitude followed by apathy. Clinical examination showed no dysmorphic features like coarse facies or organ enlargement. Neurological signs consisted of moderate mental slowing; some rigidity in both arms, and di¡use hyperre£exia without Babinski signs. Brain MRI showed a cerebral atrophy with leukoaraiosis. In view of the combination of retinitis pigmentosa and progressive neurological deterioration, a metabolic screening was organised and revealed an increased in total urinary glycosaminoglycans. Enzyme analysis revealed a profound decreased of a-N-sulphoglucosaminidase leading to the diagnosis of San¢lippo type A disease. Conclusions: our patient shows that, when young adult patients present with progressive dementia and retinitis pigmentosa, San¢lippo A disease should be considered as a possible cause, even in the absence of other organ involvement.

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THE CARDIAC EFFECTS OF ENZYME REPLACEMENT THERAPY FOR JAPANESE FABRY DISEASE: COMPARISON BETWEEN FEMALE AND MALE PATIENTS Fujiwara M1, Ohashi T2, Ida H1, Eto Y3 1 Dept of Pediatr, Jikei Univ, Tokyo, Japan, 2Dept of Gene Therapy, Jikei Univ, Tokyo, Japan, 3Lysosomal Dis Research C, Jikei Univ, Tokyo, Japan Background: Fabry disease is an X-linked recessive lysosomal storage disease caused by de¢cient activity of the lysosomal enzyme alphagalactosidase. The cardiac involvement of Fabry disease signi¢cantly in£uences the prognosis of patients. Enzyme replacement therapy was reported to prevent clinical event of Fabry disease. Objective: The object of this study is comparing the e¤cacy of enzyme replacement therapy for cardiac involvement of Fabry disease between female and male. Methods: Forty-two patients with Fabry disease (14 females and 28 males) were enrolled in this study. All patients received intravenous infusion of agalsidase beta (1 mg/kg, every two weeks). The primary end point was the time to ¢rst cardiac event (worsening of mitral regurgitation and aortic regurgitation, thickening of ventricular wall judged by echocardiography. The cumulative incidence of cardiac events was analyzed retrospectively with the Kaplan^Meier method. The statistical signi¢cance was evaluated with the Log-rank test. Results: The cumulative incidence of newly developed or increment of myocardial hypertrophy (p = 0.9860), worsening of mitral regurgitation (p = 0.6071) and worsening aortic regurgitation (p = 0.2990) shown no di¡erences between female and male. Conclusions: Our study strongly suggested that there are no di¡erence of the e¤cacy of enzyme replacement therapy for cardiac lesion between male and female. From this observation, we recommend that enzyme replacement therapy should be initiated for female Fabry patients as same timing for male. The limitation of this study is the existence of several confounding factor such as base line eGFR and LV mass index. We are currently analyzing the data including confounding factors as stated above.

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Introduction: The lysosomal diseases have been highlighted by recent advances in the ¢eld of laboratory diagnosis, through the analysis of ¢lter paper dry blood spots (DBS) and the possibility of treatment that is being gradually introduced to several of these pathological conditions. Objective: To determine the best condition of storage for total hexosaminidases and beta-galactosidase over a period of 6 months in various conditions of temperature in DBS. Methods: We used 15 samples of DBS from normal individuals divided into 4 groups. Each group was placed in a separate environment: ^208C, 88C, 258C and 378C. For the determination of enzyme activity were used speci¢c substrates 4-metilumbeliferil and the assays being conducted in duplicates at the time of incubation, after 3, 10, 17 and 180 days. Results: For the total hexosaminidases there was no signi¢cant di¡erence between the results over time in environments ^208C and 88C, similar to beta-galactosidase at ^208C, 88C and 258C. The betagalactosidase activity showed stable at ^208C, 88C and 258C and a signi¢cant lost of activity in the temperature of 378C. Already the total hexosaminidases remained stable at ^208C and 88C with signi¢cant increase in activity at 258C and 378C. The results were compared by ANOVA. Conclusion: W|th these results we can conclude that the best conditions of storage, for studied enzymes in DBS are in refrigerator or freezer, with stability in the activities over at least 6 months.

Introduction: Mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders characterized by the de¢ciency/absence of one of the enzymes involved in the intralysosomal degradation of glycosaminoglycans. As a result, large amounts of glycosaminoglycans (GAGs) are excreted in urine in some kinds of MPS. The quantization of these metabolites is not routinely performed, thus in case of suspicion, a quick method is needed. Methods: The visual test (GAG-test) is based on the reaction of dimethylmethylene blue (DMB) with GAGs in acidic medium, producing a pink colour. The reagent is stored in a 2 ml glass vial, to which 50 ml of urine is added. The test was assayed with urine of 169 healthy volunteers comprising neonates and adults, and 52 MPS patients with Hunter, Hurler, Morquio, Maroteaux-Lamy and San F|lippo diseases. Results: The test was positive or dubious for all patients, except one with Morquio disease. The other patients with Morquio disease (8) produced a dubious response. This is not surprising, since this kind of MPS only produces a slightly increased excretion of GAGs. Of 169 healthy volunteers, only two urines gave rise to a dubious response, but it was due to the high creatinine clearance. The numerical results are as follows: sensitivity 96.0%, speci¢city 99.4%, positive predictive value 98.0% and negative predictive value 98.8%. Conclusions: The GAGs-test has proven to be suitable for the quick detection of those kinds of mucopolysaccharidoses that undergo with high excretion of GAGs in urine. Con¢rmation will be given by enzymatic assay or mutation analysis.

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DETERMINATION OF STABILITY OF TOTAL HEXOSAMINIDASES AND BETA-GALACTOSIDASE OVER TIME ON FILTER PAPER DRY BLOOD SPOTS IN DIFFERENT CONDITIONS OF TEMPERATURE Castilhos CD1, Werlang FG1, Burin MG2, Coelho JC1 1 Dept Biochem, UFRGS, Med Gen Serv, HCPA, Porto Alegre, Brazil, 2 Med Gen Service, Hosp Clin Porto Alegre, Porto Alegre, Brazil

DERANGEMENT OF MANNOSE-6-PHOSPHATE RECEPTOR TRAFFICKING IMPAIRS LYSOSOMAL ENZYME UPTAKE IN FIBROBLASTS FROM LYSOSOMAL STORAGE DISEASES Cardone M1, Porto C2, Tarallo A2, Rossi B1, Tuzzi R3, Donaudy F1, Fontana F3, Andria G2, Ballabio A1, Parenti G2 1 TIGEM (Telethon Inst of Gen and Med), Napoli, Italy, 2TIGEM and Dept of Ped Federico II Univ, Napoli, Italy, 3Dept of Ped, Federico II Univ, Napoli, Italy Background: Pompe disease (PD) is a metabolic myopathy due to defective activity of the lysosomal hydrolase a-glucosidase (GAA) and resulting in generalized intra-lysosomal storage of glycogen, most prominent in skeletal muscle and heart. The phenotype of PD is a continuum ranging from a severe classic infantile form, to intermediate and late-onset forms. At present enzyme replacement therapy (ERT) with recombinant human GAA (rhGAA) is the only therapeutic approach available to PD, but the response to ERT is variable among di¡erent PD patients. Methods: Aim of this study is to investigate in vitro the pathogenetic mechanisms underlying PD and to assess the possible correlations between these mechanisms and the response to ERT in di¡erent patients. Results: PD ¢broblasts show abnormalities of cellular compartments and of tra¤cking of membrane-bound proteins, including abnormalities of the autophagic pathway; expansion and enlargement of the Golgi and ER compartments; abnormal intracellular distribution and tra¤cking of the cation-independent mannose-6-phosphate receptor (CI-MPR). The degree of cellular abnormalities correlates with disease severity, as the most striking abnormalities are observed in cells from severe and intermediate PD patients. Abnormalities in CI-MPR distribution and tra¤cking, with reduced CI-MPR at the plasma membrane, result in impaired rhGAA uptake. Conclusions: These results have important implications for the e¤cacy and optimization of ERT, and may indicate the need for therapies targeted to these cellular abnormalities, likely through multi-drug therapeutic approach tailored to individual patients. Impaired CI-MPR tra¤cking may also have implications for the e¤cacy of other therapeutic approaches, including gene therapy.

EFFICIENCY OF A NEW VISUAL TEST FOR THE QUICK DETECTION OF PATHOLOGICAL EXCRETION OF GLYCOSAMINOGLYCANS IN URINE Andrade F, Prieto JA, Sanjurjo P, Montejo M, Aldaèmiz-Echevarr|èa L Div Metab, Cruces Hospital, Barakaldo-Bilbao, Spain

CLINICAL PHENOTYPE OF ITALIAN PATIENTS WITH HUNTER SYNDROME: DATA FROM HOS ^ THE HUNTER OUTCOME SURVEY Parini R1, Melzi ML1, Rigoldi M1, Sala S1, Rampazzo A2, Gabrielli O3, DiRocco M4, Feliciani C5, Castorina M5, Cicognani A6, Scarpa M2 1 Center Metab Dis, Univ Hosp `S Gerardo', Monza, Italy, 2Dept Pediatr, Univ Hosp, Padova, Italy, 3Dept Pediatr,Univ Hosp `G Salesi', Ancona, Italy, 4Pediatria II, Pediatr Hosp, `G Gaslini', Genova, Italy, 5Pediatr Instit, Policlinico `A Gemelli', Roma, Italy, 6Pediatr Dept, S OrsolaMalpighi Hosp, Bologna, Italy Background: The Hunter Outcome Survey (HOS) is a worldwide database for the natural history of Hunter syndrome (HS) and the e¡ects of enzyme replacement therapy (ERT). 544 patients were enrolled in HOS as per April 21, 2008. Method: Demographic information from 26 alive Italian HS patients (25 M and 1 F) enrolled in HOS, age 2.8^28.6 years (median 10.9) are reported. Results: Median age at onset of symptoms was 1.2 years and median age at diagnosis 3.3 years with median delay of diagnosis of 1.1 years. Signs and symptoms reported in more than 50% of patients were: typical facial features (87%); enlarged tongue (56.5%), hypertrophy of tonsils and adenoids (64%); otitis (64%); hepatosplenomegaly (88%); hernia (84%); claw hands (79%); kyphoscoliosis (54%); joint sti¡ness (75%); valve disease (67%); cognitive and/or behavioural problems (77%); hypoacusia with need of hearing aids (72%). 60 surgical interventions were reported, the most frequent being hernioplasty (17), adenoidectomy (11) and tonsillectomy (9). Other interventions were: ventriculoperitoneal shunt (4), carpal tunnel syndrome (5), dental surgery (4), ear tubes (4), spine decompression (2), tracheostomy (1) and valve replacement (1). Conclusion: awareness of early symptoms of HS is better than in the past, but has to be further improved to reach the diagnosis at ¢rst symptoms and allow a prompt access to treatment. The burden of illness in all our patients is severe with signi¢cant multiple impairment of organs and systems and frequent need for surgical interventions. The e¤cacy of ERT in preventing at least the non-neurological symptoms of the disease has to be proved in the next years.

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COGNITIVE AND NEURORADIOLOGICAL IMPROVEMENT IN 3 ATTENUATED MPS I PATIENTS TREATED BY LARONIDASE Valayannopoulos V1, Boddaert N2, Barbier V1, Lemerrer M3, de Lonlay P1 1 Metab Unit, Necker-Enf Malades Hosp, Paris, France, 2Ped Radiology, Necker-Enf Malades Hosp, Paris, France, 3Genet Dept, Necker-Enf Malades Hosp, Paris, France Background: Mucopolysaccharidosis type I (MPS I) or Hurler's disease is a severe multi-organ and neurodegenerative lysosomal disease. Laronidase, a recombinant human a-L-iduronidase, e¡ectively alleviates many systemic manifestations of this disease but does not in£uence the neurological impairment. Attenuated forms of the disease (Scheie and Hurler-Scheie syndromes) are characterized by mild or no cognitive impairment and better outcome. Patients: We show data from 3 patients presenting with attenuated MPS I treated with enzyme replacement therapy (ERT) by laronidase for 4 years: 2 female patients aged 14 and 17 with Scheie syndrome and 1 male patient aged 6, with Hurler-Scheie syndrome. One of the Scheie patients and the Hurler-Scheie patient had mild cognitive impairment. All patients had MRI abnormalities. Results: the 2 patients with mild cognitive impairment displayed signi¢cant improvement of their intellectual performances (from 13 to 30 points of IQ on age-matched cognitive scales) within the ¢rst 2 years of ERT. One of these 2 patients displayed improvement on MRI, namely reduction of storage signs of the periventricular white matter. The 3rd patient who had a normal IQ also showed improvement on MRI. Conclusion: We report here e¤cacy of laronidase on cognitive impairment and MRI white matter changes. These data suggest that ERT may be an alternative therapeutic option to hematopoietic stem-cell transplantation for Hurler-Scheie and Scheie patients presenting with mild cognitive impairment. However, as the intravenously infused enzyme is not expected to cross the blood^brain barrier the precise mechanisms accounting for these ¢ndings need further investigation potentially using animal models.

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FABRY DISEASE IN CHILDHOOD: RENAL INVOLVEMENT ^ THE VALUE OF GENERAL EVALUATION Lea¬o Teles E1, Rodrigues E1, Baptista MJ2, Jardim H3, Moura C4, Magalha¬es A5, Lacerda L6, Oliveira JP7 1 Metab Dis Unit, Ped Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 2 Cardiol Ser Ped Dep, Univ/Hosp St Joa¬o, Porto, Portugal, 3Nephrol Unit, Ped Dep, Univ/Hosp St Joa¬o, Porto, Portugal, 4Othorrino Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 5Ophthalmology Dept, Univ/Hosp St Joa¬o, Porto, Portugal, 6Genet Med Cent Jacinto Magalha¬es, Porto, Portugal, 7Genet Dept, Medicine Faculty, Porto, Portugal Fabry disease is a lysosomal storage disorder with X-linked inheritance, caused by de¢ciency of a-galactosidase A, leading to a progressive accumulation of neutral glycosphingolipids. Usually this rare disease is detected in adult patients, when progression to severe and irreversible forms of the a¡ection already occurs. Enzyme replacement therapy (ERT) suggests bene¢cial e¡ects but it's essential early diagnosis and correct disease characterization. Case Report: Seven year old boy with familiar story of Fabry disease. Identi¢cation of involved mutation in this family was performed and prenatal diagnosis of Fabry disease was made. At the age of ¢ve the child was refereed to our unit: he had complains of painful episodic crises in the extremities and recurrent abdominal pain only triggered by fever. Additionally, he presented a cognitive de¢cit and disturbance of concentration with no other symptoms or signs. Cerebral ressonance, neurophysological study and cardiac assessment, (echocardiogram and cardiac resonance) were normal. Nevertheless, it was registered a slight hearing disfunction and the biopsy of the kidney established the signi¢cative presence of the podocytes and tubular cells involvement. The patient started ERT with algasidase alfa in November 2007, without adverse events until now. Discussion: In this case we demonstrated that although only mildly symptomatic, this child presented signi¢cant kidney involvement. We strengthen the importance of increasing awareness of the disease and its manifestations and the relevance of the baseline evaluation that must be informative enough, to establish the clinical picture and to allow the long term follow-up, including the bene¢t from judicious speci¢c therapy.

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ENZYME REPLACEMENT THERAPY FOR MUCOPOLYSACCHARIDOSIS VI IN ITALY Scarpa M1, Barona R2, F|umara A2, Astarita L3, Parenti G3, Rampazzo A1, Sala S4, Parini R4 1 Dept of Pediatrics, Univ of Padova, Padova, Italy, 2Dept of Pediatrics, Univ of Catania, Catania, Italy, 3Dept of Pediatrics, Univ Naples, Naples, Italy, 4Clinica Pediatrica, Ospedale San Gerardo, Monza, Italy Objectives: To review the management of MPS VI patients in Italy. Methods: Data were collected through the Naglazyme phase III clinical trials (four patients) and the Clinical Surveillance Program (nine patients). These, combined with data from the patients' clinical ¢les were reviewed. Results: All nine known patients currently diagnosed with MPS VI in Italy are receiving ERT. Average age is 9.5 years (min 5.6; max 14.6). Heritage was Italian for seven patients, Arabic for one and Hispanic for another one. Average age at diagnosis was 2.3 years (min 1.1, max 5.2). At diagnosis height was below the 3rd percentile for all patients, all patients had dysmorphic facial features, all had gibbus or other orthopaedic disturbances, six had recurrent upper respiratory tract infections and ¢ve had heart valve disease. All patients were treated once ERT became available or within two months after diagnosis. Urinary glycosaminoglycans, shoulder range of motion, eye exam, audiological testing, ECG and Echocardiography were the most frequently performed tests (51/year). Some patients showed an improvement of growth curve although none of the patients reached the 3rd percentile. Overall, all patients showed improvements in clinical manifestations of some of the parameters measured. No relevant safety concerns in regards to ERT were observed. Conclusions: Patients in Italy are diagnosed quite early and treatment is made available rapidly after diagnosis. Main tests performed for follow up are similar in all centres. Endurance tests are not routinely performed. Patients appear to bene¢t from ERT.

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EXPERIENCE WITH IDURSULFASE IN 11 MPS II PATIENTS INCLUDING 6 PATIENTS UNDER THE AGE OF FIVE Valayannopoulos V1, Chabli A2, Caillaud C3, Lemoine M4, Lyonnet S5, Lemerrer M5, Cormier-Daire V5, de Lonlay P1 1 Metab Unit, Necker-Enf Malades Hosp, Paris, France, 2Bioch Lab, Necker-Enf Malades Hosp, Paris, France, 3Bioch Lab, Cochin Hosp, Paris, France, 4Phys Med, Necker-Enf Malades Hosp, Paris, France, 5 Genet Dept, Necker-Enf Malades Hosp, Paris, France Background: Mucopolysaccharidosis II (MPS II or Hunter syndrome) is an X-linked lysosomal disorder caused by de¢ciency of iduronate-2sulfatase. Recently, enzyme replacement therapy (ERT) with recombinant human I2S (idursulfase (Elaprase)) has been approved for the treatment of these patients. Patients and methods: 11 MPS II patients aged from 2 months to 10.5 years old were treated from December 2006, by idursulfase weekly infusions. Among them, 6 patients were less than 5 years old at the onset of treatment. All but the youngest patient presented various clinical manifestations; 4/6 youngest patients and 4/5 older patients presented mild to severe psychomotor retardation with behavior disturbance. Results: at this stage of treatment (12 to 64 weeks) 5 patients presented repeated mild (rash, fever. . . ) or rarely more severe (hypotension) adverse e¡ects. Important reduction in liver and spleen volume has been observed in all patients even though less signi¢cant for the youngest patients. Joint range motion, endurance tests and overall quality of life scores (CHAQ questionnaire) improved in the older patients. Urinary glycosaminoglycan decreased rapidly and signi¢cantly in the older patients but remained above normal range in the youngest patients. No cognitive improvement was observed. Conclusion: our data show e¤cacy of idursulfase in MPS II patients comparable to the data from the clinical trials, including for the patients under 5 years. However in these patients with slower decrease in urinary GAG, liver and spleen volume we suggest using higher doses of enzyme as it has been proposed for the young Hurler patients treated by ERT.

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NEWBORN SCREENING FOR FABRY DISEASE IN JAPAN Nakamura K, Hattori K, Matsumoto S, Nakamura Y, Mochida T, Tajiri H, Mitsubuchi H, Endo F Dept Pediatrics, Kumamoto Univ, Kumamoto, Japan

POMPE DISEASE IN THAI INFANTS ^ REPORT OF 4 CASES Wasant P1, Vatanavicharn N1, Liammongkolkul S1, Hwu WL2 1 Div Med Gen Dept Peds, Fac Med Siriraj H, Bangkok, Thailand, 2Dept Med Gen Nat Taiwan Univ Hosp, Taipei, Taiwan

Fabry disease is an X-linked disorder of alpha-galactocidase A which causes the accumulation of glycolipids in lysosomes. The incidence of the classical type of the disease is approximately1 in 40 000 males. Recent studies have revealed the late-onset type of the disease to have a higher frequency than previously known. To determine the disease incidence in Japan, we screened newborns to measure alphagalactosidase A activity in dried blood spots from 55 945 consecutive Japanese neonates. Enzyme-de¢cient infants were retested, and infants who were double-screening positive were diagnostically con¢rmed by enzymatic activity and mutation analyses. Ten neonates had a de¢ciency in alpha-galactosidase A activities and speci¢c mutations, including two neonates with novel missense mutations (L180V and S238R), and eight neonates with known missense mutations (E66Q, R112H and P146S) identi¢ed previously in late-onset patients. Based on our newborn screening in Japan, the incidence of alpha-galactosidase A de¢ciency was 1 in 5600 (1 in 5600 male). Based on enzymatic activities, the incidence was 1 in 4000 (1 in 3100 male). These results suggest that the late-onset phenotype of Fabry disease is underdiagnosed among both males and females in Japan. The recognition of the existence of these patients suggests the need for both early diagnosis and therapeutic intervention. However, ethical issues need to be taken into consideration in terms of when and whom the screening should be performed.

Background: Pompe disease or glycogen storage disease II or acidalpha-glucosidase de¢ciency, an autosomal recessive disorder, is a prototypic lysosomal storage disease. It is caused by mutation in the gene encoding acid alpha-1,4-glucosidase (GAA; 606800), which has been mapped to chromosome 17. In the classic infantile form (Pompe disease), cardiomyopathy and muscular hypotonia are the cardinal features. Materials and Methods: We herein report 4 cases of Pompe disease in Thai infants. Case 1: 4 months old female (born 1993) with generalized hypotonia and cardiomyopathy since age 3 months. Case 2: 6 months old male (born 1994) with onset of cardiomyopathy since birth, hepatomegaly and failure to thrive. Case 3: 7 month old female (born 1995) with cardiomyopathy and delayed development. Liver biopsies were done and con¢rmed diagnosis of Pompe disease; however, enzyme assay was not available. All 3 cases died before one year of age. Case 4: 8 month old female (born 2007) with cardiomyopathy since age 2 months and liver biopsy demonstrated glycogen accumulation in lysosomes; diagnosis was con¢rmed by enzyme assay. She is now receiving enzyme replacement therapy (ERT)-Myozyme at age 8 months while under ventilator-support. Mutation analysis is being performed. Results: F|rst 3 out of 4 cases of Pompe disease were diagnosed on pathological ground. The diagnosis of case 4 was con¢rmed by enzyme assay. The ERT is being given and patient is still alive. Conclusion: The con¢rmation and treatment of Pompe disease is quite limited in developing country such as Thailand.

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AN EXTREME CASE OF LYONISATION IN A X-LINKED DISORDER: A TURNER PATIENT WITH FABRY DISEASE Eyskens FJ1, Brouns R2, de Boeck K3 1 ZNA Queen Paola Children's Hospital, Antwerp, Belgium, 2ZNA Middelheim, Div Neurology, Antwerp, Belgium, 3ZNA Middelheim, Div Nephrology, Antwerp, Belgium

RISK FOR CARPAL TUNNEL SYNDROME IN PATIENTS WITH MUCOPOLYSACCHARIDOSIS TYPE I ON ENZYME REPLACEMENT THERAPY Teunissen QGA1, Hollak CEM2, W|jburg FA1 1 Dept Pediatr, Div Metab Dis, AMC, Amsterdam, Netherlands, 2Dept Int Med, Div Metab Dis, AMC, Amsterdam, Netherlands

Case report: A female patient aged 20 years, whose brother recently was diagnosed with Fabry disease (FD), was evaluated as part of a family screening. The patient's history was relevant for Turner syndrome. Anamnesis was also positive for acroparesthesia, pain crises and hypohidrosis. On physiological examination, short stature (length: 1.47 m), high-arched palate, webbed neck, hyperconvex nails and short fourth metacarpal were apparent. The diagnosis of Turner syndrome was con¢rmed by karyotyping: 45,X. Sequencing of the GAL gene revealed a pathogenic mutation (Pro259Arg), con¢rming the diagnosis of FD. Additional investigation showed a total de¢ciency of plasma agalactosidase A activity and accumulation of Gb3 in plasma and urine. Evaluation of organ involvement by FD demonstrated cornea verticillata and increased tortuosity of the retinal vessels on ophthalmic examination, normal renal function but marked vacuolation of glomerular epithelial cells on kidney biopsy, repolarisation abnormalities on ECG, but normal ¢ndings on echocardiography and cardiac magnetic resonance imaging. Angiokeratoma were not identi¢ed, but hearing loss with de¢cit for high tones was present on audiogram. Magnetic resonance imaging of the brain showed bilateral periventricular white matter lesions. Conclusion: The X-monosomic condition in this patient can be regarded as an extremely skewed Lyonisation, resulting in loss of all expression of the GAL gene and a phenotype that is more severe compared to the male hemizygotes with FD in the same family. These ¢ndings underline the importance of X-inactivation on phenotypic expression in female patients with X-linked disorders, in cases of Fabry disease.

Background: Despite enzyme replacement therapy (ERT) in MPS I, patients may still develop carpal tunnel syndrome (CTS). We studied the incidence of CTS in MPS I patients in a single center (AMC, Amsterdam) cohort of MPS I patients on ERT. Methods: Eleven patients, who were treated with ERT as sole therapy for a period of at least 12 months (range 12^67 months, median 44 months), were included in this study. F|ve (45.5%) were classi¢ed as severe (Hurler, MPS IH), one (9%) as intermediate (Hurler-Scheie, MPS IHS) and ¢ve (45.5%) as attenuated (Scheie, MPS IS) phenotypes. Electromyogram (EMG) recordings were studied in all patients before and during treatment. Incidence of CTS during follow up was studied. Results: In the group of 11 patients, 2 (18%) had CTS before start of treatment. During ERT 8 (73%) patients developed CTS, with a mean interval of 14.1 months between start of treatment and detection of CTS. All patients with MPS IH and IHS developed CTS in contrast to 2 of the 5 (40%) patients with MPS IS during follow up. Conclusions: Despite ERT, 73% of the MPS I patients still developed CTS on EMG. The incidence of CTS was higher in patients with more severe phenotypes (MPS IH and MPS IHS). Regular monitoring, preferably by EMG, should be performed in all MPS I patients, especially in the severe phenotypes were clinical symptoms may be easily missed.

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USE OF HIGH PROTEIN DIET IN INFANTILE POMPE DISEASE Hopkins V, MacDonald A, Daly A, Bunford C, Chakrapani A, Hendriksz C Birmingham Children's Hospital, Birmingham, UK Background: There is evidence that high protein diets (up to 30% protein/kcal) improve muscle strength in adults with Pompe disease (PD). We followed 2 patients with Infantile PD on high protein diet (HPD) and enzyme replacement therapy (ERT). Method: Case study 1 diagnosed aged 10 months. After 8 infusions of alglucosidase alpha (Myozyme), HPD commenced. Pre-treatment: energy 88 kcal/kg/day (8% protein/kcal); protein 1.8 g/kg/day. Standard paediatric formula (Nutrini Energy Multi¢bre: Nutricia) was forti¢ed with concentrated milk protein (Protifar; Nutricia) to provide 24% protein/kcal (protein: 5.8 g/kg/day) and administered via nasogastric tube. Case study 2 diagnosed aged 28 months. ERT and HPD were started simultaneously. Pre-treatment intake: energy 94 kcal/ kg/day (11% protein/kcal); protein: 3.6 g/kg/day). Intake increased to 24% protein/kcal (8 g/kg/day) using high protein foods and milk protein (Protifar; Nutricia). Results: Case study 1 after 5 months, weight z score improved from ^0.5 to +1.16; height z score improved from +0.16 to +0.59. BMI increased from 16.62 to 18.22. MAMC increased from 12.9 mm to 13.3 mm. Muscle strength improved and she was able to sit unaided and walk. Urea peaked at 11 mmol/L re£ecting increased protein intake. Case study 2 after 4 months, weight z score improved from ^1.34 to ^0.75; height decreased from ^1.22 to ^1.48. BMI increased from 15.22 to 16.42. MAMC increased from 11.4 to 11.9. Posture, energy and language improved. Urea peaked at 9.1 mmol/L. Conclusions: Although motor ability and anthropometric nutritional status improved, it is unclear the speci¢c contribution of the HPD.

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DIAGNOSIS AND MANAGEMENT OF MUCOPOLYCACCHARIDOSES IN UKRAINE Pichkur NA1, Olkhovich NV2, Makushenko EA3, Gorovenko NG1 1 Med Acad of Postdiploma Educations, Kyiv, Ukraine, 2Cent Med Gen, Child Hosp `OKHMATDET', Kyiv, Ukraine, 3Regional Children Hospital, Donetsk, Ukraine Background: We report our experience of the diagnosis of mucopolysaccharidoses in Ukraine. During the period 1996^2007 mucopoly-saccharidosis was indicated in 58 patients from 54 families originating from all regions of Ukraine. Initial work-up was based on clinical and radiological evaluation of the proband. Methods: All patients were observed by selective screening tests (quantitative and qualitative GAG analysis). Lysosomal enzyme assays in peripheral leukocytes were performed according to standard techniques. Results: The database of MPS patients contains the clinical and laboratory data of investigation these patients. Our database includes: 12 patients with MPS I, 15 MPS II, 16 MPS III (6 MPS IIIA, 4 MPS IIIB, 1 MPS IIIC, 5 unidenti¢ed), 8 MPS IV, 7 MPS VI. We have not found any patient with MPS VII. Since 2004 three patients with MPS I (2 Hurler and 1 Sheie) have treated with enzyme replacement therapy (ERT). As the results of ERT, we noted the positive change in clinical picture such as the facilitation of the breathing, increase in amplitude of movement in grand joints (shoulders and knees), improvement of the daily life activities. The visceromegaly was reduced after 3 4 month of ERT. The improvement of cognitive functions was most signi¢cant in younger patient (2 years old). We have not noted essential in£uence of ERT on the heart valves degeneration in our patients. Conclusions: Enzyme replacement therapy may not improve all a¡ected organs and systems but improves the quality of life of MPS patients.

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CHOLESTEROL AND SPHINGOLIPID TRAFFICKING ALTERATIONS IN CLN6-DEFICIENT HUMAN FIBROBLASTS Teixeira CA1, Bessa C1, Gong Y2, Bittman R2, Pagano R3, Ribeiro MG1 1 Med Genet Centre, Health National Inst, Porto, Portugal, 2Dept Chem Biochem, Queens College CUNY, Flushing, NY, USA, 3Mayo Clinic and Foundation, Rochester, MN, USA

MUCOPOLYSACCHARIDOSIS TYPE IIID (SANFILIPPO SYNDROME D): CLINICAL DATA ON 10 NEW PATIENTS AND IDENTIFICATION OF 8 NOVEL MUTATIONS Valstar MJ1, Ruijter GJG1, Bertoli-Avella AM1, Wessels MW1, de Graaf B1, Poorthuis BJHM2, van Diggelen OP1 1 Dept Clin Genet, Erasmus MC, Rotterdam, Netherlands, 2Dept Med Biochem, AMC, Amsterdam, Netherlands

Background/Objective: Neuronal ceroid lipofuscinosis (NCL) comprises a group of at least 8 genetically inherited neurodegenerative disorders characterized by the accumulation of undigested material in lysosomes. Clinical symptoms include progressive loss of vision, epilepsy, motor and mental impairment, with premature death. However, it is not known how mutations in these distinct genes, some of which code for non-lysosomal proteins (CLN6 and CLN8), lead to diseases with clinicopathological similarities. One possible explanation could reside in blocking of intracellular transport to or from lysosomes at several related cellular points with subsequent endosomal/lysosomal jam. In the present study, the authors investigated if intracellular tra¤cking of speci¢c lipid molecules is blocked in the CLN6 variant. Methods: The intracellular distribution of cholesterol was studied in control and CLN6-de¢cient human ¢broblasts by ¢lipin staining, while endocytic tra¤cking in these cell types was examined using BODIPY-LacCer, a £uorescent glycosphingolipid analog. Results: Increased ¢lipin staining, indicative of high levels of intracellular unesteri¢ed cholesterol, was observed in CLN6-de¢cient cells and found to co-localize with the lysosomal marker, Lamp-1. BODIPY-LacCer was endocytosed from the plasma membrane to endosomes/lysosomes in CLN6-de¢cient cells, in contrast to normal control cells where the lipid analog was transported to the Golgi apparatus. Conclusion: Our results suggest that the equilibrium between cholesterol and SLs is disrupted in CLN6-de¢cient cells. Future studies are needed to better understand if these abnormalities are a common theme in NCL pathogenesis. The work was supported in part by FCT, POCI, and FEDER (grant FCT/ POCI/SAU-MMO/55374/2004); CB: PhD fellow (FCT/SFRH/BD/17560/ 2004); CAT: Postdoctoral fellow (FCT/SFRH/BPD/20710/2004)

Background: Mucopolysaccharidosis IIID (MPS IIID or San¢lippo D syndrome) is an autosomal recessive lysosomal storage disorder previously described in 20 patients only. MPS IIID is caused by de¢ciency of N-acetylglucosamine-6-sulfate sulfatase (GNS), involved in the degradation of heparan sulfate. So far only seven mutations in gene encoding GNS(GNS) are known. Methods: The clinical phenotype of 10 new MPS IIID patients from 8 families was studied and mutational analysis of the GNS gene was performed in 13 patients from 9 sibships. Results: Clinical signs and symptoms in MPS IIID patients appear to be similar to previously described patients with MPS III. Early development was normal in most patients with onset of behavioural problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication from the age of 6^8 years and subsequent deterioration of motor functions. Patients showed extensive behavioural problems and mild somatic disease. Although all patients showed the same general pattern of regression, great variation in the clinical course was found among patients. Mutational analysis of the coding regions of GNS resulted in the identi¢cation of 8 novel mutations: 4 missense mutations (p.S94I, p.K366R, p.K340R, p. G418E), 1 nonsense mutations (p.K388X), 2 splice site mutations (c.1098+1G4T, c.1420-2A4G), and a 9 bp deletion (c.911-919del; p.304EKLdel). Conclusions: We clinically describe the largest cohort of MPS IIID patients and report 8 novel mutations in the GNS gene. Clinical course was highly variable even between sibs. A clear phenotype-genotype correlation could not be established.

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MUCOPOLYSACCHARIDOSIS IVA: A SEVERE PHENOTYPE IN PATIENTS HOMOZYGOUS FOR THE c.347G4T (p.Gly116Val) MUTATION V|jay S, Ball S, Hendriksz C, Simmons L, Gray G, Hutchin T, Chakrapani A IMD Dept, Birmingham Childrens Hosp, Birmingham, UK Aims: Mucopolysaccharidosis IVA ( MPS IVA; Morquio A disease) is an autosomal recessive disorder caused by the de¢ciency of lysosomal N-acetylgalactosamine-6-sulfate sulfatase (GALNS). In our study we describe the unusually severe presentation of this cohort of patients with MPS IVA from the West-Midlands region of the UK who are homozygous for the c.347G4T (p.Gly116Val) mutation. Methods: We retrospectively reviewed the clinical, biochemical and genetic data from 14 patients (age range 2 to 27 years) with this mutation who have been managed in our metabolic department. Results: In a highly consanguineous immigrant subpopulation, this aggressive variant of MPSIV leads to signi¢cant morbidity and mortality at an unusually early age. All patients with this mutation originated from the Saleh Khana area of Northern Pakistan. The rapid onset of complications necessitates invasive interventions at an early age in an attempt to prolong life. Even with early cervical stabilisation surgery, mobility and quality of life remains poor. Cervical myelopathy leads to loss of mobility before 10 years of age. Four other patients with this mutation are known to have died early including 2 children died before their ¢fth birthday. Conclusions: The overall frequency of Morquio syndrome in the West Midlands is about 1:63 000, among the highest in the world. We believe that this cohort of patients exhibits a more severe phenotype to that described in other MPSIVA populations. This study enhances our understanding of this condition, enabling timely intervention and appropriate counselling.

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ENZYME REPLACEMENT THERAPY FOR MUCOPOLYSACCHARIDOSIS TYPE I IN JAPAN Okuyama T, Tanaka T National Center for Child Health, Tokyo, Japan Mucopolysaccharidosis I (MPS I) is a lysosomal storage disorder caused by the de¢ciency of a-L-iduronidase (IDUA). MPS I has historically been classi¢ed into three clinical symptoms; Hurler, Hurler-Scheie, and Scheie. MPS I is rare in Japan, we only identify about 30 patients. Laronidase has been approved to use in Japan at Oct 2006. We evaluated safety and e¤cacy of enzyme replacement therapy in three Japanese patients with MPS I; ¢ve year old boy, case 1 and ¢ve year old girl, case 2 of Hurler syndrome and ten year old girl, case 3 of Hurler-Scheie syndrome. All patients received laronidase, 100 units (0.58 mg)/kg body weight every week. Case 1 and 2 have been administered for three years and case 3 for two years. Urinary glycosaminoglycans level reduced after fourth administration (case 1: 430 to 94.4, case 2: 551 to 96.5, case 3: 178 to 73.9 mg/g creatinine, respectively). Liver and spleen volumes are normalized after 24 weeks. By the combination with ERT and otolaryngological or rehabilitation symptomatic therapy, growth and development was facilitated signi¢cantly. Case 1 showed IARs such as rash and mild wheezing. But there was no apparent correlation between laronidase IgG antibody titer and the incidence of IARs. We conclude that enzyme replacement therapy using laronidase is e¡ective for developmental delay as well as physical symptoms in patients with Hurler syndrome, although the improvement in mental retardation might be indirect and transient.

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TIGROID PATTERN OF LEUKODYSTROPHY IN A CASE OF KRABBE DISEASE Ferreira AC1, Vale MC2, Sequeira S1 1 Metabolic Unit ^ Hosp Dona Estefaªnia, Lisbon, Portugal, 2 Developmental Unit -Hosp Dona Estefaªnia, Lisbon, Portugal

HIGH INCIDENCE OF VITAMIN D DEFICIENCY IN PATIENTS WITH POMPE DISEASE Waldek S1, Roberts M2 1 Dept Metab Med Salford Royal Hosp, Salford, UK, 2Dept Neurol Salford Royal Hosp, Salford, UK

Background: Symmetrical periventricular radial stripes of white matter on MRI, described as tigroide pattern is typical of metachromatic leukodystrophy (MLD). In Krabbe disease, a de¢ciency of the lysosomal galactosylceramide beta-galactosidase (GALC), MRI shows di¡use hypodensity of white matter particularly in the parieto-occipital region. Case: We report a 10-month-old female patient, apparently normal till the ¢fth month of age when she presented with irritability, sti¡ness, clenched ¢sts, developmental delay and feeding di¤culties that progressed rapidly to failure to thrive, apathy, psychomotor deterioration, few spontaneous movements and spasticity. She had no dysmorphia, hepatosplenomegaly or macular `cherry red spot'. Tendon re£exes were depressed. Cerebral MRI showed extensive symmetrical cerebral white matter abnormalities, relatively sparing the U-¢bers, with a pattern of radiating stripes with low signal intensity on T2-weighted images that could suggest MLD. However the involvement of the posterior limb of the internal capsule, the pyramidal tracts in the brain stem and the hilus of the dentate nucleus were also a¡ected and arylsuphatase A level in leukocytes was normal. Galactocerebrosidase activity in leukocyte and ¢broblasts was decreased and molecular studies con¢rmed the diagnosis. Comments: MRI suggesting symmetrical periventricular demyelination is recognized in certain lysossomal storage disorders including MLD, Krabbe disease, and X-linked adrenoleukodystrophy. Some clinical features and a careful evaluation of the pattern of the white matter disease and the involvement of other CNS structures are important in the di¡erential diagnosis of leukodystrophies.

Pompe disease (acid maltase de¢ciency, glycogen storage disease type II) caused by a de¢ciency of acid 1,4,glucosidase presents clinically in adults with a progressive proximal myopathy, usually with the lower limbs more a¡ected than the upper, with or without diaphragmatic involvement. In some patients the respiratory symptoms can be more prominent than those attributable to those from the skeletal muscle involvement. In addition, a few patients also can have di¤culty in swallowing. As part of our protocol for assessing patients prior to starting enzyme replacement therapy we performed several investigations including vitamin D status. Levels of 25(OH)2 vitamin D2 and 3 were assayed using standard tandem mass spectrometry techniques. Levels below 15 ngs/ml were taken as low with those between 15 and 19 ngs/ml were deemed borderline. Of 23 patients tested 9 had low levels and two had borderline levels. Subsequent review did not reveal any relationship to severity of disease, ethnicity, gender, or dietary preferences. The cause of these ¢ndings is unclear. However, poor dietary intake and immobility leading to decreased exposure to sunlight may be two factors. As vitamin D de¢ciency can cause muscle weakness and bone disease we suggest that measurement of vitamin D with replacement therapy when low levels are found should become standard management for all Pompe patients.

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BASELINE CHARACTERISTICS OF PATIENTS IN THE CANADIAN FABRY DISEASE INITIATIVE Sirrs SM1, Casey R2, Clarke JTR3, Bichet DG4, Lemoine K5, West ML5 1 University of British Columbia, Vancouver, Canada, 2University of Calgary, Calgary, Canada, 3University of Toronto, Toronto, Canada, 4 University of Montreal, Montreal, Canada, 5Dalhousie University, Halifax, Canada

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DYSREGULATION OF INTRACELLULAR HOMEOSTASIS IN MURINE MODEL OF MUCOPOLYSACCHARIDOSIS TYPE I Pereira VG1, Rodrigues LC1, Lauro EM1, Gazarini ML2, Martins AM1, d'Almeida V3 1 CREIM-UNIFESP, Sa¬o Paulo, Brazil, 2D Biosciences ^ UNIFESP, Sa¬o Paulo, Brazil, 3D Biosciences and CREIM-UNIFESP, Sa¬o Paulo, Brazil

Background: The Canadian Fabry Disease Initiative (CFDI) is an ongoing study enrolling all Canadians with Fabry disease (FD), regardless of treatment status. We present demographic data of the CFDI cohort. Methods: Enrolment began January, 2007 with 3 cohorts: Cohort 1a: patients previously on ERT and maintained on treatment; Cohort 1b: ERT-naive patients, eligible for ERT under CFDI guidelines, and randomized to treatment with either agalsidase a or agalsidase b; Cohort 1c: patients not currently meeting CFDI ERT guidelines. Results: There are currently 195 patients enrolled: Cohort 1a: 76, Cohort 1b: 28 and Cohort 1c: 91. 104 patients (males 63, females 41) are on ERT: 58 on agalsidase a and 46 on agalsidase b. The mean age of all subjects is 41.3 (+14.2) years with a range of 1.9^76.3 years. Indications for ERT treatment are: cardiac: 41%, renal: 41%, stroke: 21%, GI: 19% and pain: 22%. Discussion: The CFDI currently enrols 78% of the estimated 250 Canadian patients with FD and enrolment is ongoing. The CFDI can provide a more complete picture of the spectrum of FD then industrysponsored registries due to lack of enrolment bias. The CFDI also will allow for ongoing randomized comparison of treatment e¡ectiveness for the two commercially available ERT products. Conclusions: The CFDI will provide information on a broad spectrum of patients with FD.

Background: Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disorder caused by a de¢ciency of alpha-Liduronidase (IDUA), which leads to the accumulation of two glycosaminoglycans (GAGs): dermatan and heparan sulfates. The accumulation of undegraded GAGs in the lysosomes may interfere in some metabolic and cell signaling pathways, contributing to the physiopathology of the disease. The aim of this study was to evaluate some oxidative stress markers and intracellular calcium mobilization in the MPS I mouse model. Methods: Total glutathione levels, superoxide dismutase and catalase activities were analyzed in various tissues (brain, liver and erythrocytes) of Idua+/+ and Idua^/^ C57BL/6 mice at the age of 6 months by colorimetric and spectrophotometric methods, respectively. Splenic lymphocytes were isolated for Ca2+ mobilization assays, which were performed with selective inhibitors of Ca2+ and H+ proteins transporters, followed by £uorescence detection approaches. Results: There was no di¡erence in the oxidative stress markers analyzed. However, lower lysosomal concentrations of Ca2+ and H+ and higher concentrations of Ca2+ in the endoplasmic reticulum were observed in the Idua^/^ mice, when compared to the wild-type Idua+/+. Conclusion: Our results suggest that intracellular Ca2+ homeostasis is impaired in the Idua ^/^ mice, which could contribute to the physiopathology of MPS I. F|nancial support: FAPESP, CNPq, CAPES, AFIP and Genzyme from Brazil.

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EXPERIENCE WITH ENZYME REPLACEMENT THERAPY FOR MUCOPOLYSACCHARIDOSIS VI IN 2-YEAR OLD PATIENT Fehervizyova Z, Hlavata A 2nd Dept Univ Child Hosp, Bratislava, Slovakia Background: Mucopolysaccharidosis VI (MPS VI; Maroteaux-Lamy syndrome) is a lysosomal storage disease caused by the de¢ciency of the lysosomal enzyme N-acetylgalactosamine 4-sulfatase (arylsulfatase B, ASB). This inherited disease leads to a progressive disorder with multiple organ involvement. Intelligence is usually normal. Recently, galsulfase (Naglazyme [BioMarin]), a speci¢c recombinant human ASB (rhASB) became commercially available as long-term enzyme replacement therapy (ERT). Methods: Male patient with coarse face, skeletal deformity of the chest, sti¡ness of shoulder was diagnosed with MPS VI at the age of 1 year and 11 months. At the age of 2 years and 11 months we initiated ERT with galsulfase at a dose of 1 mg/kg of body weight given intravenously once weekly for 73 weeks. To minimize the risk of infusion-related reactions premedication with antihistamines and antipyretics was used. Results: From baseline to week 73 we observed 77 percent reduction in urinary GAG excretion. Range of motion of the small joints is still normal. No improvement in large joint sti¡ness was observed, although the patient had only mild large joint sti¡ness at baseline. The treatment is well tolerated, there have been no adverse events, no anaphylactoid reactions during infusion during the whole period of the therapy. Conclusion: In our patient early initiation of ERT reduced lysosomal storage of GAG and prevented the appearance of non-reversible clinical manifestations of the disease. A key issue is to treat young patients, even though patients 55 years were not included in the phase 3 study.

OXIDATIVE STRESS ANALYSIS IN PATIENTS WITH FABRY DISEASE Muller KB1, Rodrigues MDB1, Pereira VG1, Martins AM1, d'Almeida V2 1 CREIM ^ UNIFESP, Sao Paulo, Brazil, 2D Biosciences and CREIM ^ UNIFESP, Sao Paulo, Brazil Background: Fabry disease (FD) is an X-linked recessive lysosomal storage disease caused by the de¢ciency of a-galactosidase A, which results in glycolipid accumulation, particularly globotriaosylceramide (Gb3) in numerous cell types including vascular endothelial cells. The clinical symptoms have been attributed to progressive deposition of Gb3 in vascular endothelium, leading to end-stage renal disease, cardiac and cerebrovascular disease. Oxidative stress is observed in some inborn errors of metabolism due to the accumulation of toxic metabolites leading to excessive free radical production. We propose the involvement of alterations in redox pathway in the physiopathology of FD. Thus, the aim of this study was to evaluate some oxidative stress markers in FD patients. Methods: We evaluated glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) levels, as well as antioxidant enzymes activities by spectrophotometric assays in 16 FD patients (eleven hemizygous and ¢ve heterozygous) and in control subjects matched by age and sex. Results: Heterozygous FD patients presented higher GSH levels (6.37 +0.94 vs 4.77+0.89 mmol/gHb) and catalase activity (73.04+22. 66 vs 97.02+17.47 U/mgHb) compared to controls. No di¡erences were observed between heterozygous FD patients and controls. Conclusions: The alterations observed suggest that oxidative stress may be an important event in FD, mainly among hemizygous patients. Supported by: FAPESP, CNPq, AFIP and Genzyme from Brazil

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DIFFERENTIAL STABILITY OF LYSOSOMAL ENZYMES ACTIVITY IN DRIED BLOOD SPOTS ON FILTER PAPER Mu«ller KB1, Rodrigues MDB1, Pereira VG1, Miranda M1, Martins AM1, d'Almeida V2 1 CREIM ^ UNIFESP, Sa¬o Paulo, Brazil, 2D Biosciences and CREIM ^ UNIFESP, Sa¬o Paulo, Brazil Background: Lysosomal storage disorders (LSDs) are a group of inherited diseases, which have a combined incidence of about 1:7700 live births. LSDs are caused by dysfunction of either a lysosomal enzyme or a lysosome-associated protein involved in enzyme activation or targeting. As consequence undegraded substances accumulation occurs in multiple cell types throughout the body, resulting in a wide range of clinical involvement. Minimally invasive, dried blood spots on ¢lter paper samples (DBFP) are relatively easy to collect and have been widely used in screenings. In the present study we evaluated enzyme activity in DBFP of the same person collected at two di¡erent times. Methods: Eight volunteers collected two heparinizated blood samples one in 2005/2006 and other in 2008 ^ and DBFP was prepared. We evaluated the enzymes alpha-iduronidase, neutral alpha-glucosidase and alpha-galactosidase A. Both samples of each volunteer were analyzed in 2008 using £uorimetric methods (Chamoles et al., 2001 and 2004). Results: We identi¢ed activity loss of alpha-iduronidase and neutral alpha-glucosidase enzymes. The average decrease was about 40% and 20%, respectively. No alteration was observed in alpha-galactosidase A activity. Conclusion: These results suggest that the storage of this kind of material for a long time can interfere in sample quality, compromising enzyme activity measurements, but each enzyme should be considered individually. Supported by: FAPESP, CNPq, AFIP and Genzyme from Brazil

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PERSISTENT EFFECT OF MIGLUSTAT ON FOUR CHILDREN WITH NIEMANN-PICK C DISEASE Chien YH, Hwu WL, Lee NC, Tsai LK, Huang AC, Peng SF National Taiwan University Hospital, Taipei, Taiwan Background: Niemann-Pick C disease (NP-C) is a lipid storage disorder characterized by the accumulation of unesteri¢ed cholesterol and glycolipids in the lysosomes of cells in the nervous system and visceral organs. Clinical symptoms include progressive neurological deterioration and visceral organomegaly. Miglustat, an iminosugar reversibly inhibits glucosylceramide synthase, has been approved for the treatment of Gaucher disease. Initial evidences revealed that miglustat, which can pass through the blood^brain barrier, could be an e¡ective treatment to NP-C. Methods: We treated ¢ve children with NP-C disease. The median age for the ¢rst neurological symptom onset was 7.5 years (range 5^9 years), and the median age for the diagnosis was 8.5 years (range 5.5^10). They were treated by miglustat 100 mg/m2/day since the median age of 12.3 years (range 9^14) for 1^3 years. Currently the median age was 13.4 years (range 12^17 years). Results: Before treatment, all cases had mental impairment, 4 cases had motor impairment, and 4 cases had di¤culties in swallowing. After treatment, improvement in swallowing, as shown by video£uoroscopy, was the most prominent and immediate ¢nding. Other domains that improvement could be found in individual cases included motor function (according to ambulation index), cognitive function (Mini Mental-State Examination, MMSE), and borderline horizontal eye pursuit movement. Acute deterioration was also noted during drug interruption. Conclusion: This study suggests that miglustat can provide therapeutic bene¢ts in neurological symptoms and stabilize the disease in children a¡ected by NP-C.

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ENZYME THERAPY IN EARLY SYMPTOMATIC ADULT MALE PATIENTS DOES NOT PREVENT DISEASE PROGRESSION Rombach SM1, Linthorst GE1, W|jburg FA2, Aerts JMFG3, Hollak CEM1 1 Dept End and Metab, AMC, Amsterdam, Netherlands, 2Dept Pediatrics Div Metab Dis, AMC, Amsterdam, Netherlands, 3Dept Med Biochemistry, AMC, Amsterdam, Netherlands Background: Fabry disease is an X-linked lysosomal storage disorder due to de¢ciency of a-galactosidase A. Although enzyme therapy with recombinant a-galactosidase A can reduce cardiac mass, stabilize renal function and reduce acroparesthesias, progressive disease can occur in patients with advanced disease. Early intervention trials to prevent disease progression are currently employed. Objectives: To study the follow-up of a subset of male Fabry patients with minimal organ damage who started enzyme therapy (either agalsidase-a (n = 4) or agalsidase-b (n = 3)). Methods: Male patients were included when they had normal renal function as measured by true GFR, no cardiac hypertrophy, no cerebral white matter lesions (WMLs) and either no microalbuminuria (group 1) or microalbuminuria (group 2). Mild acroparesthesias and/or hearing loss were allowed. Results: Three patients (age 18, 19 and 32) met the group 1 criteria. Patient 1, 18 years old, was treated for two years and remained stable. The other two patients developed cardiac hypertrophy and hearing loss during 4 and 6 years of treatment. One of them had a decline in GFR from 106 ml/min to 90 ml/min. Four patients (age 16^32 years) met the group 2 criteria. Progression of disease was demonstrated by detection of WMLs in one patient, cardiac hypertrophy in two patients, decreased renal function from 100 ml/min to 74 ml/min in one patient and progression of hearing loss in all. Conclusions: Early intervention in minimally a¡ected adult male patients may not prevent disease progression. We suggest that intervention at an earlier stage/age should be explored.

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THE CORRELATION BETWEEN GENOTYPE, CLINICAL SYSTEMIC SEVERITY AND OCULAR MANIFESTATIONS IN CHILDREN WITH ANDERSON-FABRY DISEASE Cosgrave EC, Ramaswami U, Kersey JP, Allen LE Addenbrooke's University Hospital, Cambridge, UK Background: Anderson-Fabry disease (AFD, OMIM 301500), an Xlinked lysosomal storage disorder due to a de¢ciency of alfa galactosidase A, presents in childhood, with life threatening renal, cardiac or neurological complications in early adulthood. Aims/Objectives: To assess if ocular involvement: (a) is associated with more severe systemic phenotype in a¡ected children; (b) correlates with genotype. Methods: The ophthalmic and systemic records of children with AFD at Addenbrookes Hospital were reviewed and the ¢rst record of ophthalmic examination and clinical ¢ndings for 8 di¡erent systems analysed. Ocular and systemic phenotypes were correlated with genotype, gender, age, systemic manifestations of the disease and commencement of enzyme replacement therapy (ERT). Results: Data represents 18 children, 7 hemizygous males (median age 5.9 years) and 11 heterozygous females (median age 10.9 years) with 9 di¡erent GAL gene mutations. Corneal verticillata was seen in 13/18 children. All 6 children with retinal tortuosity had abnormal neurological examination and 4 had been started on ERT. The most frequent systemic abnormalities were neurological (72%), gastrointestinal (67%) and cardiac (61%). All except one child had multisystem disease with 4^8 organs involved. Children with no ophthalmic abnormality had fewer systemic abnormalities (1^5 systems involved) and were less likely to be on ERT. Conclusions: Ocular features such as corneal verticillata and retinal vascular tortuosity are common in children. Retinal vascular tortuosity appears to be associated with neurological abnormalities such as acroparaesthesiae and abnormal sweating. In the N215S genotype (cardiac variant) systemic abnormalities are detected in childhood while ocular features are uncommon.

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FABRY DISEASE IN CHILDREN AND RESPONSE TO ENZYME REPLACEMENT THERAPY: RESULTS FROM THE FABRY OUTCOME SURVEY Ramaswami U1, Parini R2, Pintos-Morell G3, Hartung R, Kampmann C, Beck M 1 Universita© Milano, Monza, Italy, 2German Trias I Pujol Hospital, Badalona, Spain, 3University Children's Hospital, Mainz, Germany

POST MORTEM STUDIES ON A PATIENT WITH MUCOPOLYSACCHARIDOSIS TYPE 1: HISTOPATHOLOGIC FINDINGS AFTER ONE YEAR OF ENZYME REPLACEMENT THERAPY Yano S1, Moseley K1, Pavlova Z2 1 Genet Pediatr, Univ Southen California, Los Angeles, USA, 2Pathology CHLA Univ Southern California, Los Angeles, USA

Background: Fabry disease, a progressive, X-linked lysosomal storage disorder, leads to major organ failure and premature death. Although signs and symptoms are apparent in early childhood, limited information exists on the e¡ects of enzyme replacement therapy (ERT) in children. Methods: The Fabry Outcome Survey (FOS) is a global database of patients with Fabry disease. Data were analysed on children in FOS who received ERT with agalsidase alfa (Replagal; Shire HGT) when aged under 18 years and who had baseline, 12 and 24 month data. Results: Of 257 children (120 boys, 137 girls), 141 (78 boys, 63 girls) were receiving ERT. Follow-up data were available at 12 months (51 boys, 27 girls) and 24 months (33 boys, 21 girls). For girls, onset of symptoms, diagnosis and treatment initiation occurred 2 years later than for boys. After 12 and 24 months of treatment, vomiting, heat intolerance, abdominal pain and pain attacks were reduced and renal function remained stable (both sexes). Additional sign/symptom reductions included diarrhoea, chronic pain, left ventricular mass (boys), fatigue, cold intolerance and tinnitus (girls). Adverse events (AEs) were reported in 34 patients (25 boys, 9 girls), the most common being mild, infusion-related reactions (n = 50). Conclusions: Data from FOS con¢rm the signi¢cant burden of Fabry disease in a large cohort of boys and girls. Over 1224 months, treatment with agalsidase alfa reduced symptoms with few AEs.

Background: De¢ciency of lysosomal alpha-L-iduronidase results in systemic accumulation of glycosaminoglycans (GAGs). Cardiac lesions due to accumulation of GAGs include hypertrophic cardiomyopathy, valvular insu¤ciency/stenosis, and coronary artery stenosis due to intimal proliferation. Cardiac dysfunction is one of the most common causes of death in patients with MPS-1. Enzyme replacement therapy (ERT) with laronidase has shown clear e¡ects in reduction of hepatomegaly although it is unclear if ERT can improve or prevent the cardiac lesions in patients with mucopolysaccharidosis type I. Methods: Post mortem studies including electron microscopic histopathologic studies were performed. The subject was a 3-year-old boy who was diagnosed with MPS-I at age two years. He developed cardiomyopathy as his initial symptom at age 8 months. He received ERT with laronidase at 100 U/kg/week for one year. He suddenly developed cardiorespiratory failure and died on the same day of C2-3 spinal surgery for instability. Results: Cardiac ¢ndings included endocardial ¢broelastosis, hypertrophic cardiomyopathy, increased interstitial connective tissue, severe mitral valve thickening with shortened chordae, and aortic valve thickening. Histology of the cardiac tissue revealed increased perivascular and interstitial connective tissue in the myocardium, intimal thickening causing stenosis in the myocardial and epicardial vessels, and non-focal hypoxic changes in myocardium. Electron microscopic studies on the papillary muscle revealed enlarged lysosomes. Histology of the liver tissue revealed no accumulation of GAGs. Conclusions: ERT with laronidase seems to have clear e¡ects in removing GAGs from the liver. However, it did not show clear e¡ects on the cardiac tissues.

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Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome) is a lysosomal storage disorder caused by the de¢ciency of N-Acetylgalactosamine 4sulfatase (arylsulfatae B). In Japan, we have identi¢ed only six survived MPSVI patients, and three of them have been already received hematopoietic stem cell transplantation (HSCT). We have recently started enzyme replacement therapy (ERT) using charitably supplied galsulfase for the other three untreated patients: 7-year-old boy (case 1), one-year-old girl, a sister of case 1 (case 2), and 17-year-old girl (case 3). In Case 1 and 3, all typical symptoms for MPSVI were identi¢ed such as coarse facial features, enlarged tongue, corneal clouding, chronic otitis media, hearing loss, cardiac valve abnormalities, enlarged tonsils, hepatosplenomegaly, dysostosis multiplex, and joint contracture. On the other hand, Case 2 had no typical symptoms, because the diagnosis was carried out at neonatal presymptomatic period. They were administered 1 mg/kg body weight galsufase every week for 19 months (case 1) and 12 months (case 2, 3). Urinary GAG excretion is rapidly decreased and CT scan showed normalization of the size of liver and spleen within 6 months. Improvement of hirsutism, dry skin, bustling with activity, and advancing appetite was also observed. Infusion associated reaction was seen only one patient. Although case 2 had mild urticaria, it was possible to keep infusion. Although short-term results described above is acceptable, long-term observation should be necessary to know whether ERT for MPSVI is ultimate prophylaxis. Especially, follow-up study of case 2 is important.

Background: Mutations in NPC1 or NPC2 genes are responsible of Niemann-Pick type C disease (OMIM #257220), an autosomal recessive neurodegenerative lysosomal storage disorder caused by a non-regulation of intracellular lipid tra¤cking. Over 250 di¡erent mutations have been described worldwide in NPC1 gene. Most of them are missense, but other alterations such as nonsense or frameshift mutations generate a premature termination codon (PTC). In these cases anomalous mRNA is created and it is known that could be degraded by the natural cellular mechanism known as nonsensemediated decay (NMD). It has been described that NMD process takes place when premature translation termination occurs more than 50^55 nucleotides upstream of the 3'-most exon-exon junction. Objectives: Analysis of 9 NPC1 mutations which generate a PTC (p. R116X, p.Q119fsX7, p.W260X, p.S425X, p.A558fsX11, p.Q775X, p. G993fsX3, p.R1059X and p.I1061fsX3) in order to determine if their mRNA su¡er NMD process. Methods: We compared ¢broblasts of patients carrying these alleles with and without cycloheximide (CHX) treatment using conventional PCR and real-time PCR. Results: Reverse transcription-PCR of untreated ¢broblasts showed a reduction of the amount of NPC1 mRNA compared to controls in all cases except in three alleles (p.R116X, p.S425X and p.Q775X). After CHX treatment, a recovery of mRNA was detected in di¡erent degrees. However, when real-time PCR was used, the recovery was observed in all the alleles, including those that ¢rst showed no apparent reduction of the mRNA. Conclusions: We demonstrate in our NPC patients the existence of NMD in all mutations that follow NMD-rules suggested for this process.

ERT USING GALSULFASE FOR MAROTEAUX-LAMY SYNDROME IN JAPAN Tanaka T, Furujo M, Kubota T, Ohashi T, Tanaka A, Suzuki Y, Eto Y, Orii T, Okuyama T Japan MPS VI study group, Tokyo, Japan

NONSENSE-MEDIATED mRNA DECAY PROCESS IN NINE ALLELES OF NIEMANN-PICK TYPE C PATIENTS FROM SPAIN Macias J, Gort L, Lluch M, Coll MJ Inst Bioq Clin ^ Hosp Cl|ènic, CIBERER, Barcelona, Spain

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COMPUTER-ASSISTED TEACHING OF MUCOPOLYSACCHARIDOSIS BY PATIENT MANAGEMENT PROBLEMS Al-Jasmi F, Moldovan L, Clarke JTR Hospital for Sick Children, Toronto, Canada

PHENOTYPE VARIABILITY IN SIX CASES WITH MPS VI IN LITHUANIA Cimbalistiene L1, Songailiene J1, Czartoryska B2, Kucíinskas V1 1 Dept of Hum and Med Genet V|lnius Univ, V|lnius, Lithuania, 2Inst Psychiat Neurol, Warsaw, Poland

Background: Computer-assisted patient management problems (PMP) are well-known educational device and it has been used extensively in teaching in other areas, such as the management of chronic illness in adults and psychosocial aspects of practice. However; it has never been applied systematically to the teaching of inborn error of metabolism. Aim: To develop interactive teaching software for independent learning about the management of mucopolysaccharidoses, targeted to be used as a self-training by the general physicians. Method and Result: The software consists of two main parts: the e-book and the clinic. The e.book is visual illustrative with basic and clinical knowledge that is delivered to the trainee in simple and attractive way. All references are hyperlinked to PubMed and websites, for those needing further information or clari¢cation. The e. clinic is the interactive portion of the software. W|thin an environment resembling a real clinic, the trainee will be instructed to carry out a standard history, examine the patient, and order appropriate tests. The software provides real patient data, such as urine MPS, radiograph, and TLC plate. The trainee will be then questioned and provided with a ¢nal score, along with performance feedback. While a trainee is working on a case, he/she can access the e-book through the book icon. In conclusion, our software is an educational tool for independent learning. It adopts the PMP through the eclinic as well as it provides the trainee with comprehensive and thorough information through the illustrative ebook.

Background: MPS VI is clinically heterogeneous ^ onset and rate of progression is variable. We present ¢rst six cases of MPS VI with great variety of clinical manifestations in Lithuania, con¢rmed biochemical and by enzymatic assays. Objective: to evaluate and compare clinical phenotypes of six Lithuanian cases with MPS VI. Methods: determination of urinary GAG's, lysosomal enzymes activities in serum, leucocytes. Results: Patients were from 6 to 31 years old, 2 male, 4 female. There were 3 siblings from one family (6, 8 and 12 years old). The most frequently reported clinical manifestations were short stature, mildly coarse face, reduced shoulder £exion, clow hand deformity and abnormalities in cardiac valves. 2/6 su¡ered from hearing impairment, 5/6 from visual impairment (hypermetropia and astigmatism), 2/6 showed corneal clouding. All patients were with normal intelligence. Hepatomegaly was observed in 2 out of 6 patients. In 2/6 a medullar stenosis of cervical spine was diagnosed. The oldest patient has signs of cerebral atrophy. All of studied patients have elevated urinary GAG's and show the presence of DS and CS. Mean arylsulfatase B activity in leucocytes was 23.2 nmoles/h/mg protein (reference values: 73+38). Conclusion: this rare disease present in our patients with di¡erent clinical phenotypes from mild through the severe forms. Three siblings presented with severe clinical symptoms.

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EFFICACY OF MIGLUSTAT ON DYSPHAGIA IN FOUR NIEMANN PICK PATIENTS Bruschini D1, Fecarotta S1, Astarita L1, Romano A1, Mansi G1, Amitrano M2, Dolezalova H2, Della Casa R1, Parenti G1, Andria G1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2Dept Diagn Imag and Radioth, Fed II Univ, Naples, Italy Background: Niemann-Pick disease type C (NPC) is a rare disorder characterized by defective intracellular lipid tra¤cking, with secondary storage of free cholesterol and glycosphingolipids. Clinical manifestations include hepatosplenomegaly and progressive neurological involvement. Progressive dysphagia, due to neurological involvement and causing secondary malnutrition, can be considered a marker of disease progression. Oral feeding eventually becomes impossible and gastrostomia is required. No speci¢c treatment for NPC is presently available, but promising results have been obtained with miglustat, an inhibitor of glicosphingolipid synthesis, able to cross the blood^brain barrier. Methods: We studied the e¡ects of miglustat treatment on dysphagia in 4 NPC patients (3 NPC1 and 1 NPC2). All patients received miglustat treatment for 2 years at a dose adjusted for body surface and were subjected, every 6 months, to clinical and video£uorographic evaluation of swallowing. Results: At baseline patient 1 (aged 10 years) was presymptomatic, and had no problem in swallowing; patient 2 (12 years) showed a mild de¢cit; patient 3 (9 years) and 4 (11 months) showed severe swallowing di¤culties. Patient 4 also showed severe growth retardation. After 24 months of therapy all patients showed a normal ability to swallow, except patient 3, who showed a mild de¢ciency in the oral phase of swallowing on video£uoroscopy. Patient 4 showed normalized growth parameters. Conclusions: Miglustat appears e¡ective in improving swallowing abilitity in NPC patients. This may indicate e¤cacy of miglustat in improving or delaying neurological deterioration in NPC. This study is part of a national trial supported by AIFA (Rome, Italy).

METACHROMATIC LEUKODYSTROPHY IS NOT ALWAYS A PROGRESSIVE DISEASE Bley A1, Santer R1, Lukacs Z2, Kohlschuetter A1 1 Dept Pediatr Univ Med Center, Hamburg-Eppendorf, Germany, 2Lab Metab Dis, Univ Med Center, Hamburg-Eppendorf, Germany Metachromatic leukodystrophy (MLD) is considered to be a neurodegenerative disease with relentless progression, the speed of which is depending on the severity of the underlying genetic metabolic defect. The variable progression in late infantile, juvenile and adult MLD is thought to re£ect di¡erent residual activities of the defective enzyme, arylsulfatase A. We report on a 21-year-old woman who su¡ered from a progressive loss of mainly cognitive abilities and minor motor dysfunctions between the age of 9 and 13 years. Between the age of 13 and 21 years her clinical situation stabilized and she regained some of her cognitive and motor abilities. Her only treatment during that time was homeopathic medications and intensive mental and motor stimulation. MLD was diagnosed on the basis of typical white matter MRI changes, low arylsulfatase A activity in leukocytes, elevated urinary sulfatide excretion, and molecular genetic analysis of the MLD gene demonstrating compound heterozygosity for the mutation c.251G4A (associated with juvenile/adult MLD) and IVS2+1G4A (associated with variable clinical courses). If the patient had received an early experimental intervention such as bone marrow transplantation or enzyme replacement, her course would have been regarded as success. Conclusion: The natural course of late-manifesting MLD may be stable over a long time. This will make the evaluation of new treatments particularly di¤cult in such `mild' cases that otherwise may be good candidates for enzyme replacement.

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DETERMINATION OF URINARY GLOBOTRIAOSYLCERAMIDE BY UPLC-MS/MS: URINE DRIED ON FILTER PAPER VERSUS LIQUID URINE Forni S, Fu X, Sweetman L, Schi¡mann R Institute of Metabolic Disease, Dallas, TX, USA Objectives: Develop and compare two fast and simple UPLC-MS/MS methods for globotriaosylceramide (Gb3) and creatinine in urine for detection of Fabry disease. Methods: One ml of urine is dried on a 5 cm6 5 cm square of newborn screening ¢lter paper and sent at room temperature. Liquid urine is sent frozen. Internal standards (C17-Gb3 and d3-creatinine) are added to the dried urine card or 0.25 of liquid urine, and extracted with 4 ml methanol. The urine ¢lter paper is shaken 1 h, sonicated 30 s and the liquid urine is ¢ltered. Ten ml are injected into a BEH C8 1.0650 mm column for UPLC-MS/MS by ESI and MRM for a 3-min analysis. Results: Both methods are linear and reproducible up to 25 mg of Gb3. Accuracy is within 10% of the expected value and intra-day relative SD is less than 10% in both matrices. Recoveries averaged 111% for dried urine and 71% for liquid urine. The upper limit of normal (n = 20) is 23 mg Gb3/mmol creatinine (9.0+4.8 SD) for dried urine on cards and 24 mg Gb3/mmol creatinine (7.1+5.5 SD) for liquid urine. F|ve Fabry heterozygotes had elevated levels of 41^81 in dried urine cards and 56^ 91 in liquid urine. Conclusion: Determination of Gb3 from urine dried on ¢lter paper or liquid urine is suitable for Fabry disease diagnosis and may be useful as a biomarker for monitoring treatment.

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ATTENUATED FORM OF SANFILIPPO TYPE B: CLINICAL AND MOLECULAR CHARACTERISATION OF NINE DUTCH PATIENTS Kalb S1, Luf S1, Hinderhofer K1, van Mierlo I2, van Schrojenstein Lantman-de Valk H3, Marcelis C4, Zschocke J1, Moog U1 1 Inst Hum Gen, Univ Hosp, Heidelberg, Germany, 2Dept Clin Gen, Univ Hosp, Maastricht, Netherlands, 3Dept Gen Pract, Univ, Maastricht, Netherlands, 4Dept Hum Gen, Radbound Univ, Nijmegen, Netherlands San¢lippo type B (MPSIIIB) is an autosomal recessive lysosomal storage disorder. The de¢ciency of a-N-acetylglucosaminidase (NAGLU) leads to accumulation of heparan sulphate in lysosomes and to a severe progressive encephalopathy. Some patients show an attenuated form of MPSIIIB with milder clinical features and a more protracted disease course. We present clinical and molecular data of nine Dutch patients with attenuated MPSIIIB. Natural history was characterised by onset in the ¢rst decade with non-speci¢c developmental delay, marked behavioural problems, slow mental decline and survival into (late) adulthood. Additional complications like loss of speech or ambulation occurred in the 4th^6th decade. Sequence analysis of the NAGLU gene was performed and mutations were identi¢ed on 17 of the 18 mutant alleles. There were ten di¡erent missense mutations, six of which were novel. Combining our results with those of previously published patients, mutation analyses have been carried out in a total of 25 patients with an attenuated form of MPSIIIB. 22 di¡erent mutations were found on 39 of 50 mutant alleles. In two published patients no mutation was found. Available data are compatible with the concept that attenuated MPSIIIB is caused by mutations with residual enzyme function on at least one allele. So far, nine known mutations predict an attenuated phenotype whereas for seven mutations the associated phenotype is unclear. In conclusion, MPSIIIB should be considered in patients of all ages with mental retardation and behavioural problems; molecular studies may be useful to predict or con¢rm an attenuated phenotype in a¡ected patients.

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ACUTE LYMPHOBLASTIC LEUKEMIA IN A CHILD TREATED BY ELAPRASE1 FOR HUNTER'S DISEASE Feillet F1, Salmon A2, Kimmoun A1, Straczeck J1, Gueant JL1, Bordigoni P2 1 Ref Cent Inb Err Metab INSERM U724, Nancy, France, 2Ped Haematol Department, Vandoeuvre les Nancy, France We report the case of a 6 year old boy who was diagnosed for MPS II (Iduronate sulfatase activity: (0.2 nmol/h/mg prot [Nl 5^10]); IDS mutation: S333L). He was diagnosed on a typical pattern of early onset MPS II with typical dysmorphism, hepatosplenomegaly (HSMG), dysostosis, bilateral carpal tunnel syndrome and moderate mental retardation. ERT (Elaprase: 0.5 mg/kg weekly) was started in December 2006. Six months after the beginning of the treatment, we observed a decrease in HSMG, the urinary GAG excretion normalized and there was a clinical improvement in respiratory function and in mobility. In June 2007, he presented a chronic respiratory distress related to a mediastinal compression. The diagnosis of acute lymphoblastic T leukemia (with CNS involvement) was done and he was treated by the FRALLE 2000-BT T group protocol. The child was initially corticoid non responder and obtained complete remission at day 42. As planned in the leukemia protocol, a BMT was performed in November 2007. ERT was continued until 2 months after BMT and was stopped when the iduronate sulfatase activity normalized. Clinically, he presented GVH grade IV, and a resolutive adenovirus infection. One month after BMT he presented a severe neurological degradation with mainly mutism. 3 months after BMT the child began again to speak (some words) but he presented a severe respiratory failure and died. This is the ¢rst case of leukemia in a child treated by ERT for Hunter's disease.

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NEURONAL CEROID LIPOFUSCINOSIS FROM OUR CHILDREN'S HOSPITAL Perez Poyato MS1, Pineda Marfa M1, Cussi V1, Ferrer I2, Mila M3 1 Div Metab Dis Hospital Sant Joan de Deu, Barcelona, Spain, 2Bellvitge Hospital, Barcelona, Spain, 3Clinic Hospital, Barcelona, Spain Background: Neuronal ceroid lipofuscinosis (NCL) is an inherited autosomal recessive disease and is one of the most common progressive neurodegenerative diseases of lisosome storage seen in childhood. Depending on the age of onset, the clinical features and the ultrastructure of the storage material, the NCLs can be divided into three subtypes: infantile neuronal ceroid lipofuscinosis (INCL), late infantile neuronal ceroid lipofuscinosis (LINCL) and Juvenile neuronal ceroid lipofuscinosis (JNCL). Objective: (1) Describe the di¡erent clinical phenotypes and their development in the long term in our series; (2) Analyse the anatomopathological, electrophysiological, biochemical and molecular characteristics. Methods: Retrospective review of clinical histories of 22 children (females: 11, males: 11). Ages ranged from 2 ^13 years old) with con¢rmed diagnosis of NCL, gathered over the last 30 years in our hospital. During these years di¡erent biopsies material for electronic microscopy have been used. Results: Our 22 patients, 4 correspond to infantile form, 7 to late infantile form and 11 juvenile (4 variants). We describe the clinical p hen otyp e n eu rophysi ologi cal, n eu roradi ologi cal an d anatomopathological ¢ndings of the di¡erent subtypes and the response to the di¡erent palliative treatments. Conclusions: (1) A correct clinical evaluation, as well as the anatomopathological, biochemical and neurophysiological studies lead us to the di¡erent clinical phenotypes; (2) Juvenile NCL is the most prevalent form in our reference hospital; (3) Research and recent developments in molecular genetics have allowed a better characterization, diagnosis and classi¢cation of these disorders; (4) Precise diagnosis allows us a prognosis, appropriate genetic counselling and the possibility of prenatal diagnosis.

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NOVEL MUTATION ON A BRAZILIAN PATIENT WITH FABRY DISEASE Souza CFM1, Netto C1, Pereira F2, Matte U2, Burin M1, Giugliani R1 1 Medical Genetic Service ^ HCPA, Porto Alegre, Brazil, 2Gene Therapy Center ^ HCPA, Porto Alegre, Brazil Fabry disease (FD) is an X-linked inborn error of glycosphingolipid metabolism due to the de¢ciency of a-galactosidase A. The progressive accumulation of globotriaosylceramide (Gb3), particularly in the vascular endothelium, leads to renal, cardiac and cerebrovascular manifestations, and early death. Detection of female carriers based solely on enzyme assays is often inconclusive. Therefore, mutation analysis is a valuable tool for diagnosis and genetic counseling. The human alfa-galactosidase A gene (GLA) is located in position Xq21.33^ 22, spans 12 kb divided in seven exons. There is high genetic heterogeneity and most mutations are private. We present an 18 yearold boy who, since childhood presented with skin angiokeratomas. At the age of 15 years he started with hypertension and at 17 years the diagnosis of Fabry was stablished (alfa-gal activity of 1.6 nmoles/h/mg prot normal range 26^53). At this moment he presents mild proteinuria, corneal opacity, no cardiological or neurological involvement. The genomic sequence of the seven exons of GLA gene was ampli¢ed by PCR. These fragments were analyzed by automated capillary sequencing and compared to the reference sequence NM000169 (www. ncbi.nlm.nih.gov). Automated sequencing of the seven exons of GLA gene revealed the presence of the mutation 570del16 in the exon 4. The deletion of sixteen bases results in a frameshift from amino acid 191 on with the constitution of a premature stop codon in residue 239. Family investigation using molecular tools is now in progress.

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ADULT-ONSET NIEMANN-PICK TYPE C DISEASE: A NOVEL CASE PRESENTING WITH SEIZURES Maj MC1, Rupar T2, Callahan JW1, Morel CF3 1 The Hospital for Sick Children, Toronto, Canada, 2Dept Biochem and Ped, Univ Western Ont, London, Canada, 3UHN, University of Toronto, Toronto, Canada Background: Niemann-Pick disease type C (NPC) is a rare autosomal recessive, neurodegenerative disorder, arising from the storage of cholesterol by lysosomes and/or late endosomes. The inability to translocate intralysosomal cholesterol into the cytosol is manifested by a failure to esterify cholesterol in-vivo. Onset is usually in infancy or childhood with liver, neurological or pulmonary involvement however an adult form of the disease with dementia or psychiatric symptoms as presenting features has been described. We report a novel case of adultonset NPC where the ¢rst clinical sign was onset of seizures at age 21 years. Twenty years later, the patient developed leukodystrophy on brain MRI and a slowly progressive dementia which prompted investigations for NPC. Methods: Cholesterol esteri¢cation studies and ¢lipin staining were performed on cultured ¢broblasts. Western blot analysis of the NPC1 protein and sequencing of NPC1 and NPC2 genes were undertaken. Results: The diagnosis of NPC has been con¢rmed by a number of methods. Cholesterol esteri¢cation studies indicated enzyme activity 15% of controls accompanied by a visible increase in the cholesterol storage, viewed by ¢lipin staining. Western blot analysis for protein levels of NPC1 showed levels 15^20% of control. Mutational analysis of the NPC1 gene revealed heterozygosity for the previously described W942C mutation and polymorphisms I642M, I858V and N931N. NPC2 gene was normal. Conclusions: Although NPC usually presents in infancy or childhood, adult-onset has been documented. This case illustrates that seizures may be the presenting sign of NPC in adults, prior to the onset of dementia or psychiatric symptoms.

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THE PHARMACOLOGICAL CHAPERONE AT2220 INCREASES GAA ACTIVITY AND REPRESENTS A POTENTIAL NEW TREATMENT FOR POMPE DISEASE Flanagan J J, Do HV, Wu X, Powe AC, Khanna R, Liang W, Tang K, Pine C, W|lliams H, Soska R, Pellegrino L, Shao S, Benjamin ER, Wustman BA, Valenzano KJ, Lockhart DJ Amicus Therapeutics, Inc., Cranbury, New Jersey, USA Background: Pompe disease is caused by de¢cient acid alpha glucosidase (GAA) activity resulting in impaired lysosomal glycogen metabolism. GAA is synthesized in the endoplasmic reticulum (ER) and tra¤cks to the lysosome to degrade glycogen. Certain genetic mutations in GAA produce mutant forms of the protein with decreased physical stability leading to reduced lysosomal levels and enzymatic activity. Methods: We are developing a new therapeutic approach for the treatment of genetic diseases by using small molecule pharmacological chaperones that selectively bind and stabilize proteins to restore protein tra¤cking and increase cellular levels. Results: In this study, we show that the pharmacological chaperone AT2220 (1-deoxynojirimycin-HCl) binds to GAA and increases its stability. Additionally, AT2220 signi¢cantly increases GAA speci¢c activity in patient-derived ¢broblasts and in transiently transfected COS-7 cells expressing certain GAA missense mutations. AT2220 was also evaluated in wild-type mice, rats and monkeys and was shown to increase wild-type GAA levels in disease-relevant tissues. AT2220induced enhancement of wild-type GAA suggests that this pharmacological chaperone may be appropriate for Pompe patients with the relatively common IVS 1 (^13 T4G) splicing defect because this mutation leads to the expression of low levels of wild-type GAA. AT2220 was subsequently shown to increase GAA levels in cell lines derived from late-onset Pompe patients with this splicing mutation. Conclusion: These results indicate AT2220 merits further evaluation as a potential treatment for Pompe disease.

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MUTATION ANALYSIS IN NIEMANN-PICK DISEASE TYPE C: A PRACTICAL APPROACH Knight SB, Heptinstall LE, Whitehouse C, Cooper A, Imrie J, Wraith JE W|llink Biochem Genet Unit, Manchester, UK Niemann-Pick disease type C (NPC) results from mutations in one of two genes (NPC1 or NPC2). Most patients (95%) have defects in NPC1, a relatively large gene (25 exons) with many di¡erent mutations (250 reported). Mutations in NPC1 or NPC2 give rise to defects in cholesterol esteri¢cation and this currently forms the basis for diagnosis. This test on cultured cells is however, complex and timeconsuming but bene¢ts from con¢rmatory DNA mutation analysis. We have studied 24 NPC patients and present our results obtained using a practical 3-tier screening approach. All patients were diagnosed on the basis of clinical and cholesterol esteri¢cation (¢lipin staining) studies. Our initial screen (level 1) covered 6 exons (18^23) which included the `hot spot' region for known mutations including the `common' p.I1061T mutation. 11 patients were fully characterised at this level and mutations in 27/46 alleles (59%) identi¢ed. The next level (level 2) of sequencing covered 9 exons considered likely to yield mutations. This resulted in a further 6 patients being characterised and a total of 83% of alleles with identi¢ed mutations. A further 3 patients were characterised when the ¢nal 10 NPC1 exons were sequenced (level 3). In parallel with level 2 screening, mutations in the smaller (5 exons) NPC2 gene were sought and one patient was identi¢ed with this variant. A total of 25 di¡erent NPC1 mutations (6 novel) were identi¢ed in 20/23 NPC1 patients studied, representing 91% alleles. This approach has proved a practical way to identify mutations in a reasonable time scale.

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EXPRESSION ANALYSIS IN COS-1 CELLS OF MISSENSE ALLELES OF THE GLB1 (ACID b-GALACTOSIDASE) GENE WITH PRESUMPTIVE INFLUENCE ON CATALYTICAL ENZYME FUNCTION Paschke E1, Hofer D1, Fantur K1, Paul K1, Morrone A2, d'Azzo A3 1 Dept Pediatrics, Med Univ Graz, Graz, Austria, 2Dept Ped, Azienda Ospedaliero-Univ Meyer, Florence, Italy, 3Dept Genet and Cell Biol, St Jude Res Hosp, Memphis, USA Background: Mutations in the b-D-galactosidase (b-gal) gene (GLB1) can result in GM1 gangliosidosis (GM1) a neurodegenerative disease of varying onset and a¡ection of connective tissues, or Morquio B disease (MBD) with normal CNS function. In most infantile GM1 genotypes, gene products are absent while in MBD normal synthesis and transport, but impaired catalytic function is suggested. In juvenile or adult GM1, their stability may be reduced and thus sensitive to chaperone treatment. Mutations and gene products of the GLB1 gene have therefore gained particular importance. Methods: Eight novel and several known missense mutations with presumptive in£uence on the catalytical b-gal activity were expressed in COS-1 cells, the increase of b-gal activities measured and the amount of material monitored by Western blots and immuno£uorescence in CHO-cells and ¢broblasts. Results and Conclusions: In COS-1 cells transfected with wild-type cDNA and the mutations M132T, G190D, D198Y, T329I, Q255H, P397A and P597S, the 85 kDa-precursor of b-gal was abundantly present, lacked with Y333H and was signi¢cantly reduced with Q408P. In contrast to catalytic codon D332, the exchange of Tyr333 to His may rather alter stability than catalytic function. Q408P was detected in a rare MBD phenotype, T500A/Q408P. The ¢nding of reduced expression of Q408P further con¢rms the suggested in£uence of T500A on the phenotype. D198Y and P397A yielded b-gal activities 14 and 23% above COS-1. As both of them were found in MBD patients carrying the common W273L allele, their relation to the phenotype remains to be determined in CHO-cells and ¢broblasts.

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DIETARY INTAKE, ENERGY EXPENDITURE AND BODY COMPOSITION OF PATIENTS WITH POMPE DISEASE: A PILOT STUDY van der Louw E1, van den Berg L2, Kramer P1, Kruijer N1, Zandbergen AA3, Hagemans MLC2, van der Ploeg AT2 1 Erasmus MC, Dept of Dietetics, Rotterdam, Netherlands, 2Erasmus MC-Sophia, Div Metab Dis and Gen, Rotterdam, Netherlands, 3Erasmus MC, Dept of Internal Medicine, Rotterdam, Netherlands Background: In Pompe disease de¢ciency of acid alpha-glucosidase leads to accumulation of glycogen in lysosomes. It has been suggested that protein turnover is increased and that speci¢c diets/supplements might have bene¢cial e¡ects. However, much is still unknown about dietary intake, energy expenditure and body composition of patients with Pompe disease. Methods: Thirteen patients, varying in age between 2 months and 70 years and all receiving enzyme replacement therapy, participated in this pilot study. Resting energy expenditure (REE) was measured by indirect calorimetry (DeltaTrac), body composition by skinfold thickness measurement, and dietary intake by 3-day food diary. Results: REE was within 10% of predicted values (Scho¢eld/HarrisBenedict) for 7 patients, 410% lower than predicted for 4 patients, and 410% higher for 2. Respiratory quotient (RQ) was within normal limits for all patients (0.8^1.1). Energy intake was lower than recommended for 9 out of 13 patients. In all patients, protein intake was higher than recommended, but comparable to the general Dutch population. For most patients skinfold thicknesses and percentage body fat were markedly above reference values, but weight-for-height and/or BMI were at the upper limit of normal. Conclusions: For most patients with Pompe disease REE was comparable to predicted values. Dietary intake was relatively low, but adequate based on RQ, probably due to limited physical activity. Body composition di¡ered from the general population with results suggesting a shift from muscle towards fat tissue. Future studies with a larger sample size and including both treated and untreated patients are needed to con¢rm these results.

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INFANTILE FORM OF POMPE'S DISEASE: TWO CASES WITH DRAMATICAL RESPONSE TO ENZYMOLOGICAL THERAPY Chabrol B, Cano A, Mansour H Metab Ref Cent, Univ Child Hosp T|mone, Marseille, France Background: Pompe's disease, or glycogen-storage disease type II, is an autosomal recessive disorder resulting from the de¢ciency of a lysosomal hydrolase (acid alpha-glucosidase). Normal glycogen accumulates in lysosomes and cytoplasm, resulting in deposits interrupting normal functioning of other organelles and leading to cellular injury, with enlargement and dysfunction of the involved organs. Three forms are recognized: infantile, juvenile, and adult-onset. Methods: we report the cases of two children, who presented with hypertrophic cardiomyopathy, along with muscular and respiratory symptoms. The ¢rst child presented hypotonia in the ¢rst months of life and repeated upper respiratory tract infections, with fatigue and a mild motor developpemetal delay, she also presented a mild form of cardiomyopathy, the diagnosis was made at one year of age and the treatment was started at 17 months of age. At birth the second child presented with meconium aspiration, respiratory distress syndrome and chest X-ray showed a global hypertrophic cardiomyopathy. The diagnosis was made at 24 days of age and the treatment was started at 28 days of age. Results: Both of the children bene¢ciated of an early treatment with recombinant human enzyme, and as a result they both showed dramatic improvement of respiratory, muscular and cardiac symptoms. Conclusions: W|th the present treatment, as once every other week infusion, the children lead a normal life, with a favorable 3 years follow up, the younger child still presents a better clinical condition, but the long-term outcomes of enzyme reconstitution therapy remains to be assessed.

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EFFECT OF EPSTEIN BARR VIRUS ON LYSOSOMAL HYDROLASES ACTIVITIES FROM LYMPHOCYTES: COMPARISON BEFORE AND AFTER CRYOPRESERVATION Coelho JC1, Mello AS1, Michelin-T|relli K2, Camelier M2, Mari JF2, Burin MG2, Giugliani R2 1 Dept Biochem, UFRGS, Med Gen Serv, HCPA, Porto Alegre, Brazil, 2 Med Gen Service, Hosp Clin Porto Alegre, Porto Alegre, Brazil Background: Epstein-Barr virus (EBV) infection in vitro causes transformation of B-cells and generates B lymphoblastoid cell lines (LCLs). LCLs have been used for the diagnostic of genetic metabolic disorders and we have already demonstrated that some enzymes do not change their activities when frozen in liquid nitrogen for 30 days. In this study, analyses were performed in transformed B-cell to measure human lysosomal acid hydrolases associated with GM1-gangliosidosis, Gaucher, Pompe and Fabry diseases and Mucopolysaccharidosis type I. Methods: Peripheral blood mononuclear cells were isolated from 6 normal subjects and LCLs were produced by cell culture with EBV for 12 days. The activities of the enzymes b-galactosidase, b-glucosidase, aglucosidase, a-galactosidase and a-iduronidase were measured before and after cryopreservation in liquid nitrogen for 180 days. Results: Before cryopreservation we determined the normal range of enzymes' activities: b-galactosidase (92.6+14.9), b-glucosidase (26.8 +3.7), a-glucosidase (4.5+1.0), a-galactosidase (48.0+12.4) and aiduronidase (18.6+8.9). After cryopreservation all enzymatic activities were within the normal range. When we compared the enzymatic activities before and after freezing we observed that a-glucosidase (3.2 +9.0) and b-glucosidase (21.3+6.1) lower signi¢cativelly while bgalactosidase (94.0+14.7), a-galactosidase (38.8+5.8) and aiduronidase (11.8+3.1) kept their original activities. Conclusion: These data indicate that long-term frozen LCLs could probably a¡ect a-glucosidase and b-glucosidase activities. However, further work with a¡ected subjects has to be carried out to evaluate this di¡erence.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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ENZYME REPLACEMENT THERAPY FOR LATE-ONSET ACID MALTASE DEFICIENCY: CLINICAL EXPERIENCE OF FOURTEEN PATIENTS TREATED WITH ALGLUCOSIDASE ALFA FOR ONE YEAR Amado Fondo A, Cousins A, Lee PJ, Lachmann RH Metabolic Unit, Nat Hosp Neurol Neurosrg, London, UK Background: In adults, acid maltase de¢ciency (AMD) usually presents as a progressive limb-girdle dystrophy with variable diaphragmatic involvement. We present our experience from 14 adult patients who have completed 12 months of ERT. Methods: Assessments were performed at baseline and 12 months. Patients received alglucosidase alfa infusions at a dose of 20 mg/kg every 2 weeks. Results: We have treated 9 male and 5 female patients (aged 28^76 years). At baseline, eight patients required overnight ventilatory support and two also needed intermittent day-time support. Four patients were wheelchair dependent. Ten patients performed 10m walks. The mean time taken decreased from 11.0+4.9 s to 10.0+3.9 s (p = 0.25). Nine patients completed a 6 min walk test. The mean distance walked increased from 250+131 metres to 313+114 metres (p = 0.01). A compound manual muscle testing score was performed in 11 patients and the mean increased from 231+48 at baseline to 266+40 after one year (p50.005). Mean plasma creatine kinase (n = 12) fell from 536 +332 IU/L to 377+196 IU/L (p50.05). Although there were no signi¢cant changes in spirometry, many patients reported a reduction in their use of non-invasive ventilation. Although ten out of the 14 patients have been shown to have detectable antibodies against alglucosidase alpha, only one patient experienced mild infusion associated reactions and there have been no other signi¢cant adverse events. Conclusion: Although the baseline characteristics of our patients were variable, one year of treatment with alglucosidase alpha has resulted in signi¢cant improvements in mobility and muscle strength.

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DIETARY INTERVENTION IN CHILDREN RECEIVING SUBSTRATE REDUCTION THERAPY WITH MIGLUSTAT (ZAVESCA1) Champion H1, Lachmann RH2, Cox TM3, Wright N1, Ramaswami U1 1 Cambridge Univ Teaching Hosp, Cambridge, UK, 2Charles Dent Met Unit, UCL, London, UK, 3Dept of Med, Univ of Cambridge, Cambridge, UK Background: The iminosugar, miglustat, inhibits glucosylceramide synthase, the initial enzyme in the formation of glycosphingolipids, o¡ering an alternative therapeutic approach in Type 1 Gaucher disease and investigation into related sphingolipidoses. Unwanted side e¡ects include watery diarrhoea, £atulence, abdominal cramps, nausea, weight loss and poor appetite. Miglustat inhibits sucrase-isomaltase and other disaccharidases in the small intestinal mucosa, to which its unwanted abdominal e¡ects are attributed. Methods: We evaluated the e¡ect of diet on 9 patients treated with miglustat for a 6 month period (age range 1.1^20.1 years, mean age 11.4 years). Four were supported with a standard unmodi¢ed diet with milkbased supplements containing lactose. F|ve received a modi¢ed diet, low in disaccharides but containing complex carbohydrate and maltodextrins. Results: Patients on the standard diet plus conventional milk-based supplements experienced signi¢cant weight loss, (average ^6.2% body weight and loss of weight gain potential of ^10.0%). Frequent loose stools resulted in missed schooling, but was controlled with loperamide. Patients on diet low in disaccharides had an average weight gain of 8.8% and a weight gain potential of 2.1%. Diarrhoea was not generally experienced in this group. Conclusion: Retrospective analysis of the data from 9 paediatric receiving therapy with miglustat at our centre in Addenbrooke's Hospital shows that with dietary modi¢cation, weight gain during treatment can be maintained in line with weight gain potential and episodes of gastrointestinal disturbances can be much reduced. Adequate provision of energy from fats, maltodextrins and proteins can be successfully used to maintain predicted weight gain.

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BIOCHEMICAL SCREENING OF FARBER DISEASE USING A COMBINED APPROACH Ribeiro I, Alves M, Amaral O, Ribeiro MG Med Genet Centre, Health National Inst, Porto, Portugal Background/Objective: Farber disease (FD) is a rare autosomal recessive neurodegenerative lysosomal disorder caused by mutations in the ASAH1 gene leading to the de¢ciency of the acid ceramidase (AC). As result, ceramide accumulates in lysosomes of several cell types. FD diagnosis is usually established upon the observation of abnormally high ceramide levels in cultured cells, and subsequent demonstration of reduced AC activity. However, the radioactive assay used to assess the AC activity is not easily reproducible. In the current study the authors examined the possibility to establish the diagnosis of FD using speci¢c polyclonal antibodies against each subunit of the heterodimeric enzyme. Methods: Cultured human ¢broblasts from control and FD patients were used in western blotting and immuno£uorescence studies. Results: In total cell lysates, an absence or marked reduction of immunoreactivity was observed in all 3 patients examined so far. Furthermore, in control cells, AC partially co-localized with lamp-1 whereas no detectable signal was observed in FD cells. Conclusion: The data reported indicate the usefulness of this diagnostic method based on the changes in AC immunoreactivity. This relatively simple, speci¢c, and cost-e¡ective method is a promising diagnostic tool for this disease. If complemented by the mutation analysis of the ASAH1 gene, it will provide a more comprehensive approach to the study of Farber patients. The work was supported in part by FCT, POCI, and FEDER (grant FCT/POCI/SAU-MMO/55374/2004). IR: MSc student, ICBAS/ Faculdade de Cieªncias, Porto University; the work programme was developed at the Enzimology Unit. MQA: FCT Research fellow

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DRIED BLOOD SPOT ASSAYS FOR FABRY AND POMPE DISEASE: HOW DO MULTIPLEX TANDEM MASS SPECTROMETRY AND FLUOROMETRY COMPARE TO EACH OTHER? Wuyts B, Stove V Lab Metab Dis, University Hospital Gent, Gent, Belgium Background: After establishment of dried blood spot (DBS) assays, the diagnosis of lysosomal storage disorders is further innovated introducing a multiplex tandem mass spectrometry (TMS) assay. We correlated the diagnostic performances of £uorometric and TMS DBS assays for Fabry and Pompe. Methods: In 7 Fabry and 8 Pompe patients DBS samples were measured £uorometrically and by multiplex TMS assay on a QuattroMicro instrument, monitoring neutral-loss of 100 m/z. Furthermore, analyses were performed in 47 control DBS samples. Results: Results are shown as mean (95% con¢dence interval). Alphagalactosidase activity in Fabry patients di¡ered signi¢cantly (KruskalWallis 50.0001) from controles using £uorometry 0.04 (0.04^0.12) versus 1.86 (1.38^2.24) mmol/L/h and using the TMS assay 0.25 (0.13^ 0.38) versus 0.85 (0.67^0.99) mmol/L/h. Alpha-glucosidase activity in Pompe patients di¡ered signi¢cantly (Kruskal-Wallis 50.0001) from controls using £uorometry with acarbose 0.27 (0.11^0.40) versus 2.49 (2.04^3.15) mmol/L/h; using £uorometry ratio (acarbose/total activity without acarbose) 0.12 (0.06^0.17) versus 0.51 (0.42^0.54) and using the TMS assay 0.12 (0.12^0.14) versus 1.17 (1.04^1.39 mmol/L/h. Spearman correlation coe¤cient between the £uorometric and TMS assay was 0.89 in the Fabry test and 0.85 in the Pompe test. Discussion: All Fabry and Pompe patients that were diagnoses using DBS £uorometry could be identi¢ed using the multiplex TMS assay. Both methods correlate well with each other. The multiplex TMS assay has the advantage of higher speci¢city. Fluorometry can give rise to false negatives due to £uorophore contamination. TMS can be multiplexed, yet is more labour intensive due to the need of liquid-liquid and solidphase extraction.

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NOVEL CLUES ON THE INTRACELLULAR LOCALIZATION OF CLN6, THE PROTEIN DEFECTIVE IN THE NEURODEGENERATIVE LSD-NCL TYPE 6 Alves M1, Bessa C1, Teixeira CA1, Boustany RM2, Ribeiro MG1 1 Med Genet Centre, Health National Inst, Porto, Portugal, 2Dept Ped Biochem, American Univ of Beirut, Beirut, Lebanon Background/Objective: The CLN6 gene codes for a conserved polytopic protein of 311 amino acids which was recently reported as an ERresident dimeric protein. The CLN6 de¢ciency is associated with the worldwide distributed variant late-infantile form of the disease, with 25 mutations identi¢ed to date. However, the protein function remains elusive. Furthermore, it is not completely understood how the de¢ciency of this non-lysosomal protein lead to various materials being stored in the lysosome. Thus, the aim of the present study was to get insights on the subcellular location of the CLN6 protein. Methods: Anti-peptide antibodies were used in subcellular localization studies of the protein from control and CLN6-de¢cient human ¢broblasts. Results: The antibodies were able to detect the endogenous protein with appropriate speci¢city. In control ¢broblast cell lysates, a band of about 40-kDa was observed in Western blotting. In immuno£uorescent studies, the CLN6 protein was observed in di¡erent cell compartments and found to co-localize with markers for the actin and ER. The p. I154del mutation predicted to occur in the luminal side of the protein had no e¡ect on CLN6 cell tra¤cking. Conclusion: The immunodetection of CLN6 in distinct cell compartments suggests novel unpredicted functions for the protein. Future studies aiming to identify CLN6 interacting partners will be important to unravel protein function and to better understand the disease pathophysiology. The work was supported in part by FCT, POCI, and FEDER (grant FCT/SAU-MMO/55374/2004); AM: Research fellow from the Portuguese Science and Technology Foundation; BC: PhD fellow (FCT/SFRH/BD/17560/2004); TCA, Postdoctoral fellow (FCT/ SFRH/BPD/20710/2004)

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ENZYMATIC METHOD WITH ACARBOSE USED IN DRIED BLOOD SPOTS AND LEUKOCYTES ENABLES DIAGNOSIS OF DIFFERENT TYPES OF POMPE DISEASE Lugowska A1, Rola R2, Lewandowska E3, Rakowicz M4, W|erzbaBobrowicz T3, Tylki-Szymanska A5, Mierzewska H6, F|dzianska A7 1 Inst Psychiatry Neurology, Dept Genetics, Warsaw, Poland, 2Inst Psychiatry Neurol, Dept Neurology 1, Warsaw, Poland, 3Inst Psychiatry Neurol, Dept Neuropathol, Warsaw, Poland, 4Inst Psych Neurol, Dept Clin Neurophysio, Warsaw, Poland, 5Child Memo Health Inst, Dept Metab Dis, Warsaw, Poland, 6Inst Mother Child, Dept Child Neurol, Warsaw, Poland, 7Med Res Center PAS, Neuromusc Unit, Warsaw, Poland Background: Recently, enzymatic method has been described measuring acid alpha-glucosidase (GAA) activity in dried blood spots (DBS) or leukocytes and eliminates maltase-glucoamylase activity by the use of speci¢c inhibitor acarbose. Methods: DBS or leukocytes were taken of control individuals and GAA activity was measured with two concentrations of acarbose: 30 mM and 3 mM. In the next step, DBS and/or leukocytes taken of patients suspected of Pompe disease were examined. Enzyme reactions were performed at pH 3.8, 378C, with 70 mM 4-MUG as substrate for 20 h (DBS) or 1 h (leukocytes). Results were expressed as ratios of GAA activity with and without acarbose. Results: Pompe disease was con¢rmed in eight patients: 2 with infantile, 2 with juvenile, 2 with late juvenile, and 2 with adult type of the disease. GAA ratios in DBS of patients ranged from 0 to 0.12 (n = 8) for 30 mM acarbose (normal range 0.280.55, n = 83) and from 0 to 0.32 (n = 5) for 3 mM acarbose (normal range 0.510.87, n = 47). In leukocytes of patients, GAA ratios range was 0.070.09 (n = 6) for 30 mM acarbose (normal range 0.290.52, n = 16) and 0.11, 0.16 for 3 mM acarbose (normal range 0.460.80, n = 7). Conclusion: The enzymatic method with acarbose is a valuable diagnostic tool for the detection of Pompe patients. Both concentrations of acarbose: 30 and 3 mM, enabled distinct division of results obtained for a¡ected and healthy individuals.

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A PHARMACOGENETIC APPROACH TO PREDICT RESPONSE TO PHARMACOLOGICAL CHAPERONE THERAPY FOR FABRY DISEASE Benjamin ER1, Wu X1, Khanna R1, Sitaraman SA1, Palling DJ1, Schi¡mann R2, Lockhart DJ1, Valenzano KJ1 1 Amicus Therapeutics, Cranbury, NJ, USA, 2Baylor Res Inst, Inst Metab Dis, Dallas, TX, USA Background: Fabry disease is an X-linked lysosomal storage disorder caused by inherited mutations in the gene that encodes alphagalactosidase A (a-Gal A), with consequent pathological accumulation of globotriaosylceramide (GL-3). More than 400 mutations have been reported of which more than 60% are missense. The iminosugar AT1001 (migalastat hydrochloride) is a pharmacological chaperone that selectively binds a-Gal A, increasing physical stability, lysosomal tra¤cking, and total cellular amount and activity. Methods: To identify AT1001-responsive a-Gal A mutant forms, each of the 346 known Fabry disease-causing missense mutations and small inframe insertion and deletion mutations were recombinantly-engineered and expressed in HEK-293 cells. Results: Incubation with AT1001 caused a concentration-dependent increase in a-Gal A levels in approximately 60% of the mutant forms. The results obtained in HEK-293 cell assays were comparable to those obtained in both an ex vivo Fabry patient-derived T-cell assay used during screening in Phase 2 clinical trials of AT1001 and the preliminary in vivo white blood cell response dataset from the Phase 2 trials. Importantly, a-Gal A-de¢cient mice that express a responsive human mutant form (R301Q) of the enzyme showed signi¢cant increases in aGal A and decreases in GL-3 levels in multiple tissues and plasma after oral administration of AT1001 for 28 days. Conclusions: Collectively, these results indicate that genotype is predictive of response to AT1001, and thus may be used to identify patients for further clinical evaluation of AT1001 as a treatment for Fabry disease.

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QUALITY OF LIFE IS SIGNIFICANTLY IMPROVED FOLLOWING SIX MONTHS OF ENZYME REPLACEMENT THERAPY IN LATE-ONSET ACID MALTASE DISEASE Cole AL, Lee PJ, Cousins AJ, Amado Fondo A, Lachmann RH Nat Hosp for Neurology and Neurosurgery, London, UK Background: Acid maltase de¢ciency (AMD) is a rare inherited disorder resulting from reduced activity of lysosomal a-glucosidase. Late onset AMD causes progressive proximal myopathy and respiratory impairment. In 2006, recombinant human acid a-glucosidase was licensed for enzyme replacement therapy (ERT) in AMD. Variable improvements in mobility and ventilator use have been reported, as well as functional improvements in activities of daily life, fatigue and energy. Changes in quality of life (QoL) following ERT have not yet been reported. Methods: QoL was measured using the SF-36 questionnaire at baseline and following six months of ERT. All 18 late-onset AMD patients at our centre were assessed. Responses were entered into SF-36 analysis software and analysed using statistics package SPSS. Results: Group baseline characteristics were remarkably similar to the study of Hagemans et al (2004) showing signi¢cantly poorer QoL, particularly `role physical' and `physical function', in AMD when compared to the normal population and those with other long standing illness. Following six months of ERT paired t-tests showed statistically signi¢cant improvements in role physical (p = 0.017), vitality (p = 0.004), role emotional (p = 0.035) and mental health (p = 0.009) components of the SF-36. Conclusion: QoL is in£uenced by individual baseline health, treatment expectations and lifestyle adjustments. Determining, from a patient's perspective, the value and success of treatment is an important component of clinical care and service evaluation. Here patients report a physical and mental health improvement in 4 of the 8 SF-36 scales with ERT. Longer term follow-up is required.

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SUCCESSFUL `TRANSPLANTATION' OF MRI QUANTITATIVE CHEMICAL SHIFT IMAGING (QCSI) TECHNOLOGY FOR THE DETECTION OF BONE MARROW FAT SIGNAL FRACTION IN TYPE I GAUCHER DISEASE, FROM AMSTERDAM TO LEUVEN Cassiman D1, Peeters R2, Jaeken J1, Maas M3, Akkerman EM3 1 Metabolic Center, University Hospitals, Leuven, Belgium, 2Dept of Radiology, University Hospitals, Leuven, Belgium, 3Dept of Radiology, AMC, Amsterdam, Netherlands

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MORPHOLOGICALLY TARGETED DNA SCREENING OF NEURONAL CEROID LIPOFUSCINOSES CLN5 AND CLN6 IN ARGENTINA Cismondi IA1, Kohan R1, Guelbert N2, Tapia Anzolini V2, Ghio A2, Mole SE3, Xin W4, Santorelli F5, Dodelson de Kremer R2, Oller Ram|èrez AM2, Noher de Halac I6 1 Fac Odont, Univ Nac Cord, Coèrdoba, Argentina, 2CEMECO, Hosp Nin¬os, Fac Cs Meèdicas, Coèrdoba, Argentina, 3MRC LMCB and Institute Child Health, UCL, London, UK, 4Mass Gen Hosp, Boston, USA, 5 IRCCS Bambino Gesu© Hosp, Rome, Italy, 6CONICET-RA, Coèrdoba, Argentina

Background: Lumbar spine bone marrow fat signal fraction, determined with MRI (Dixon Quantitative Chemical Shift Imaging (QCSI)) is considered the `gold standard' to determine and follow up bone involvement in type I Gaucher disease, but is currently only available in 1 European center (AMC, Amsterdam, The Netherlands). This precludes a more detailed study of the e¡ect of therapies on bone involvement in Gaucher clinical trials, while bone involvement is one of the most debilitating aspects of the disease. Methods: We transferred Amsterdam QCSI to a university referral center, experienced in MRI techniques (University Hospital Gasthuisberg, Leuven, Belgium), and tested it in the local patient population. Results: Installation required the transfer of the Amsterdam MRI physicist (EMA) for one day, a 1.5 Tesla MRI, Siemens or General Electric, access to sequence programming and a dedicated local physicist. A total of 14 scans were performed in 4 controls and 8 patients over the last year. An untreated patient showed the lowest fat fraction, a patient that had been treated for 12 years showed the highest value, the other patients (shorter treatment duration) showed intermediate values. The planned follow-up study will include scanning of 20 patients and controls in both centers, to prove reproducibility of the scanning results. Conclusion: our preliminary study shows that QCSI is easily transferable from one center to another. This o¡ers the potential for spreading this technique to Gaucher MRI expert centers throughout the world.

Neuronal ceroid lipofuscinoses (NCLs) are common neurodegenerative disorders of childhood accumulating ceroid lipofuscin-like material in brain and extracerebral tissues. Clarifying the morphology of the intra-lysosomal deposits is helpful in targeting CLN5 and CLN6 genetic screening in atypical late infantile phenotypes, allowing predictions at protein level. Twenty-¢ve patients were studied with clinically consistent phenotype and excluded de¢ciencies of palmitoyl-thioesterase1/tripeptidyl- peptidase-I. Blood smears were screened for vacuolated lymphocytes, electron microscopy detection of ceroid lipofuscin-like materials in skin biopsies, DNA-changes in CLN5 and CLN6 analyzed by PCR and full gene sequencing for compilation and predictions. Vacuolated lymphocytes: 0/25 patients. F|ngerprint (FP) and other morphological pro¢les: 25/25 patients. Gen CLN6, 5 changes in 3/25 unrelated patients. Patient 1 showing FPs was compound heterozygous: exon 6c.552dupC = p. Phe185LeufsX17and exon 4c.307C4T = p.Arg103Trp. Patient 2 exhibiting mixed FP/curvilinear bodies was compound heterozygous: exon 4c.486+8C4T and exon 7c.755 G4A=p.Arg252His. Exon/intron 2c.1984C T+104 polymorphism was present in patients 1 and 2, and as the only change in patient 3. Gen CLN5, 3 changes in 7/25 patients. Patient 4 with FP pro¢les; homozygous: exon 4c.1002-1006delAACA. Patient 5 with FP pro¢les; homozygous: exon 1c.291insC. In other ¢ve patients with FPs exon 1c.4C4TArg4Cys polymorphism: 2/5 homozygous; 3/5 heterozygous. Changes in the structure of both proteins were predicted. Ceroid lipofuscin-like bodies with FP pro¢les guided the genetic studies towards CLN6 in 3/25 patients, and towards CLN5 in 7/25. The genetic spectrum of these genes in Argentina showed 8 changes: 4 in gene CLN6 and 2 in gene CLN5 plus one possible polymorphism in CLN6 and one in CLN5.

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Gb3 ANALYSIS FOR FABRY DISEASE BY LC-MS/MS USING URINE FILTER PAPER SAMPLES Auray-Blais C, Cyr D, Clarke JTR, Drouin R Serv Genetics, Universiteè de Sherbrooke, Sherbrooke, Qc, Canada Background: Fabry disease (FD, OMIM 301500) is a complex, multisystemic and clinically heterogeneous disease, associated with increased urinary excretion of globotriaosylceramide (Gb3), the primary substrate of alpha-galactosidase A enzyme de¢ciency. The ¢rst objective was to develop and validate a rapid, e¤cient multiplex Gb3/ creatinine methodology using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of urine samples collected on ¢lter paper. The second objective was to study correlations between urinary Gb3/creatinine excretion and the genotype in FD patients. Method: Gb3/creatinine analysis was developed and validated in a multiplex LC-MS/MS run of 2.6 min using a LC gradient. We studied correlations between urinary levels of Gb3/creatinine excretion and four types of mutations in the GLA gene (missense, nonsense, frameshift, splice-site defects) in 32 children and 78 adult patients with Fabry disease. Results: The mean recoveries for Gb3 and creatinine from urine ¢lter paper standards were 91% and 97%, respectively, with good precision, reproducibility, and linearity. The variance analysis using independent variables of sex, age, types of mutations and treatment showed that the mutation factor is statistically signi¢cant (p = 0.0007). The same correlation was found for sex (p50.0001) and treatment status (p = 0.0011). Conclusions: We found a highly signi¢cant correlation between urinary excretion levels of Gb3 and the type of mutation in FD patients. The results also indicate that the urinary excretion of this speci¢c glycosphingolipid biomarker is directly related to sex and treatment status, but not age.

ENZYME REPLACEMENT THERAPY IN A PATIENT INFANTILE FORM OF POMPE'S DISEASE WITH SEVERE CARDIOMYOPATHY Tanzer F1, Buyukkayhan D2, Cansu Mutlu E3, Kalender Korkmaz F3 1 Div Metab Dis, Cum Univ Child Hosp, Sivas, Turkey, 2Div Neonatolgy, Cum Univ Child Hosp, Sivas, Turkey, 3Childhood Dept, Univ Child Hosp, Sivas, Turkey Background: Pompe disease is a rare autosomal recessive lysosomal storage disease caused by de¢ciency of acid-alpha-glucosidase (GAA) resulting in intra-lysosomal accumulation of in terms of onset, involvement of organs and life expectancy. Infantile onset is the most severe form presenting with prominent cardiomyopathy, hypotonia, hepatomegaly and death before 12 months of life. Methods: We present a female patient with severe hypotonicity, macroglosia, hypore£exia, respiratory infection and cardiomegaly at the age of 4 months. Huge cardiac enlargement was found on thoracic radiography. Her parents were ¢rst cousin. She was a fourth child her family. F|rst child was stilbirth, in the second child was suspected muscle disease, another hospital. But she died at 5 months. In our patient, diagnosis of Pompe disease was made at 5 months of age in a muscle biopsy specimen. This was co¢rmed by zero GAA activity. Enzyme replacement therapy with myozyme at the rate of 20 mg/kg every 2 weeks administered to the patient for a period of six months. Results: The patient being severely hypotonic and in cardiac failure when initiating ERT, showed improvement of both the cardiac and skeletal muscle functions. She subsequently showed signs of decline and her parents discharged her from hospital. After a few days rehospitalyzed and died at 11 months following a severe respiratory infection and cardiac failure. Conclusion: Prognosis is poor because of heart involvement. Enzyme replacement therapy with acid alpha glucosidase o¡er hope for patients su¡ering from this lethal disease.

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ALGLUCOSIDASE ALFA IN INFANTS AND CHILDREN WITH POMPE DISEASE Hwu W-L1, Byrne B2, Wraith E3, Leslie N4, Mandel H5, Nicolino M6, Kishnani PS7 1 National Taiwan University Hospital, Taipei, Taiwan, 2Shands Hospital at the Univ of Florida, Gainesville, USA, 3Royal Manchester Children's Hospital, Manchester, UK, 4Cincinnati Children's Hospital, Cincinnati, USA, 5Rambam Medical Center, Haifa, Israel, 6Hoªpital Debrousse, Lyon, France, 7Duke University Medical Center, Durham, USA Background: Pompe disease is caused by a de¢ciency of acid alphaglucosidase (GAA). Severe GAA de¢ciency manifests during infancy with rapidly progressing muscle weakness, and cardiomyopathy that typically results in death by 1 year. Aim/Methods: Two open-label studies were conducted in patients 56 months (S1, n = 18) or 46^36 months (S2, n = 21) of age with rapidly progressing disease. S1 patients received alglucosidase alfa at 20 or 40 mg/kg qow; S2 patients started at 20 mg/kg qow. Results: Mean age at treatment and median duration of treatment were: 5.1 months and 121 weeks (S1), 15.7 months and 120 weeks (S2), respectively. Cox regression analyses comparing study patients to historical controls; (S1 n = 61; S2 n = 84) indicated that in patients treated at 56 months, treatment reduced risk of death by 95%, death or invasive ventilation by 91%, and death or any ventilation by 87% (all p50.0001). In patients 46^36 months, treatment reduced risk of death by 79% (p = 0.0009) and death or invasive ventilation by 58% (p = 0.02). Sustained decrease in LVM occurred in 94% (S1) and 81% (S2) of patients. Normal growth occurred in 480% of patients, clinically signi¢cant motor gains in 61%. Infusion-associated reactions occurred in 56%; IgG antibodies developed in 92%. Low IgG titres or trending towards decreasing titres occurred with continued treatment in 74% of those seroconverting. Patients with two null GAA mutations and high, sustained IgG titres more often had poor long-term clinical outcomes. Conclusion: These ¢ndings demonstrate clinical bene¢t of alglucosidase alfa in this population and emphasize the need for early treatment.

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DEVELOPMENT OF A DISEASE SEVERITY SCORING SYSTEM FOR PATIENTS WITH POMPE DISEASE Giannini EH1, Berger K2, van der Ploeg A3, Case L4, Dandrea C5, Kishnani PS4, Marsden D 1 NYU School of Medicine, New York, USA, 2Erasmus Medical Center, Rotterdam, Netherlands, 3Duke University Medical Center, Durham, USA, 4Genzyme Corporation, Cambridge, USA Introduction: A Disease Severity Scoring System (DS3) measures disease burden in patients. It consists of critical health domains, described by relevant clinical assessment(s) quanti¢ed via reliable, valid and feasible methods. DS3s are useful in rare, heterogeneous diseases in which evaluating severity and prognosis is di¤cult. Properly con¢gured, a DS3 provides inter- and intra-patient comparisons through time across critical organ systems. A DS3 is being developed for Pompe disease, a rare, autosomal recessive, heterogeneous, neuromuscular disorder. Description: Pompe experts were assembled to identify critical Pompe disease health domains. A broader `Delphi' physician group captured standard medical practice(s) for severity measurement within each critical domain. Selected domains were: cardiac, respiratory, proximal muscle, physician reported outcomes and patient reported outcomes. W|thin each domain, 1^2 clinical assessments were identi¢ed. To test this preliminary model, 9 cases from the Pompe Registry representing a severity spectrum were scored. Results: Results were compared to results from a blinded small expert group assessment of the cases using a scale similar to the Clinical Global Impression (CGI) severity scale, yielding a 0.93 coe¤cient of correlation, indicating preliminary DS3 consistency with expert opinion, suggesting preliminary DS3 validity, reliability and relevance. Validity and reliability testing are being completed using standardized methods. Conclusion: Preliminary results indicate the Pompe DS3 model will help standardize disease terminology and highlight key clinical assessments to quantify disease severity. This tool can evolve into a universal disease `staging' system that permits more exact prediction of important disease outcomes and identify the need for speci¢c medical interventions.

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HEMIBALLISM-HEMICHOREA ASSOCIATED WITH MUCOPOLYSACCARIDOSIS TYPE IIIA di Rosa G, Bonsignore M, Piperata MR, Tortorella G Unit of Inf Neurops-Univ Hosp Messina, Messina, Italy Mucopolysaccaridosis type IIIA (MPS IIIA) ( MIM# 252900) is a rare, autosomal recessive, lysosomal disorder caused by a reduced activity of N-sulfoglucosamine sulfohydrolase (SGSH), tissues accumulation of heparan sulfate and high urine excretion. Several SGSH gene mutations, on 17q25.3, were reported. Neuropsychiatric features prevail on somatic ones and mainly consist of behavioural disturbances, psychomotor and speech delay. Brain MRI shows cystic changes in the corpus callosum, basal ganglia and white matter, and di¡use atrophy. We describe a 13-year-old boy with MPS IIIA from an Italian pedigree with consanguineous healthy parents and a younger a¡ected brother, presenting attacks of hemiballism-hemichorea. These features were not reported in MPS type IIIA. He was severely impaired in social, verbal and motor skills, generalized hypotonia was evident. Di¡use cortical-subcortical atrophy and basal ganglia lesions were detected at MRI. The attacks were unprovoked. His right arm was twitched or jerked with forearm pronated and supinated. His right leg was shaking. His eyes twitched conjugately, bilaterally. The movements were mainly limited to the right side, but could shift from one side to the other during each attack or one series. One series of the attacks lasted about 1 to 3 h and partially remitted by 10 mg endorectal diazepam administration. An ictal video-EEG recording failed to reveal correlated epileptic discharges. Treatment with pimozide stopped the attacks, with 10-months of follow-up. Hemichorea-hemiballism may be considered in patients with MPS IIIA. The involvement of the basal ganglia could explain this ¢nding.

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A CLINICAL TRIAL OF IDURSULFASE IN HUNTER SYNDROME PATIENTS 5 YEARS OLD AND YOUNGER Tylki-Szymanska A1, Giugliani R2, Hwu W-L3 1 Klinika Chorob Metabolicsynch, Warsaw, Poland, 2Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil, 3National Taiwan University Hospital, Taipei, Taiwan Background: Hunter syndrome (mucopolysaccharidosis II, MPS II) is an X-linked disorder of glycosaminoglycan (GAG) metabolism caused by a de¢ciency in the activity of the lysosomal enzyme, iduronate-2sulfatase. The safety and e¤cacy of enzyme replacement therapy (ERT) with weekly infusions of 0.5 mg/kg idursulfase (Elaprase1, Shire Human Genetic Therapies, Inc., Cambridge, MA, USA) has been demonstrated in a pivotal Phase II/III trial in 96 patients aged 5 years and older. However, younger patients, who also exhibit signs and symptoms of Hunter syndrome, may also bene¢t from ERT. Methods: This open-label, multi-center, single-arm clinical trial will enroll up to 30 patients who will be treated for 1 year with weekly doses of idursulfase (0.5 mg/kg infused intravenously over about 3 h). The primary objective of the study is to determine the safety of idursulfase, which will be assessed by the incidence of adverse events (including infusion-related reactions), changes in electrocardiogram, vital signs, standard laboratory parameters, and anti-idursulfase antibodies. Secondary objectives include an evaluation of the pharmacodynamic e¡ects of idursulfase as measured by urinary GAG excretio n and th e a ss e ss m ent of s ing le and re p e at do s e pharmacokinetics. Additional exploratory measurements will include the assessment of liver and spleen volumes, developmental milestones, growth velocity, and signi¢cant clinical events that re£ect disease progression. Results: The study has enrolled 2 patients as of March 2008. Conclusion: When completed, the results of this study will assess the safety of idursulfase in very young Hunter syndrome patients.

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THE POMPE REGISTRY: TRACKING POMPE DISEASE SYMPTOMS IN A BROAD PATIENT POPULATION Byrne B1, Kishnani PS2, Case L3, Merlini L4, Mu«eller-Felber W5, van der Ploeg A6, Park J7, Marsden D7 1 Congenital Heart Ctr Univ of Florida, Gainesville, USA, 2Duke Univ Medical Ctr, Durham, USA, 3Duke Univ Medical Ctr, Durham, USA, 4 Dept Medical Genetics, Univ of Ferrara, Ferrara, Italy, 5Dept of Neurology, Univ of Munich, Munich, Germany, 6Erasmus Medical Ctr, Rotterdam, Netherlands, 7Genzyme Corporation, Cambridge, USA Introduction: Pompe disease is a rare, progressive, often fatal metabolic myopathy, which manifests as a clinical spectrum that varies with respect to age at onset, rate of disease progression, and extent of organ involvement. The underlying pathology is de¢ciency of acid alpha-glucosidase (GAA). To gain a better understanding of Pompe disease, a global, voluntary, observational Registry was developed to collect anonymous, longitudinal data. Preliminary data overview: as of March 2008, 494 patients from 23 countries were enrolled; the majority (72%) was Caucasian. Europe and North America enroll 87% of patients. Median age of infants at symptom onset was 2.0 months (n = 94) and at diagnosis was 4.0 months (n = 93). Median age of adults at symptom onset was 27.7 years (n = 293) and at diagnosis was 35.3 years (n = 289). Symptoms most frequently reported by patients 518 years old (n = 321) include: muscle weakness [lower extremities (80%), upper extremities (69%), trunk (53%)]; shortness of breath after exercise (61%) and at rest (31%); dependence on respiratory support (38%); sleep disturbance/apnea (35%); orthopnea (32%); and scapular winging (31%). Approximately half of patients genotyped expressed the IVS113T44G mutation. Summary: These results show signi¢cant delay from symptom onset to diagnosis in adult patients, highlighting the need for greater disease awareness. Registry data on prevalence and age at onset of symptoms may allow earlier patient identi¢cation, enabling intervention before irreversible muscle damage occurs. Analysis of registry data over time may increase understanding of the evolution of, and interaction between, impairments and function under varying conditions and interventions, allowing improved clinical management.

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BRANCHED-CHAIN AMINO ACID AS SUPPORTIVE THERAPY FOR TYPE-A NIEMANN-PICK PATIENT Damayanti RS, Tanjung C Div Metab Dis, Dept Pediatr,Univ Indonesia, Jakarta, Indonesia Background: Niemann-Pick is lysosomal storage disorders that result from de¢cient acid sphyngomyelinase (ASM) activity and lead to the accumulation of sphyngomyelin. Until now no e¡ective treatment for children a¡ected by Niemann-Pick type A. Usually the patient will die in the ¢rst two years of life. Nutrition and physical therapy are additional supportive treatment options to assist with feeding di¤culties and the decline of motor skills in those children. Methods: We report a case of 2 year 8 month-old boy from nonconsanguineous parents. He came with the diagnosis of tyrosinemia. He was born spontaneously, at term with birth weight 3200 g, length 50 cm. His brother died at 1.5 years with hepatosplenomegaly. When he ¢rst came, his body weight was 10.4 kg, length 84 cm, head circumference was 47 cm (microcephaly). The clinical manifestations are failure to thrive, hepatosplenomegaly, interstitial lung disease and delayed development. The blood examinations showed pancytopenia, hypertrigliseridemia, increasing liver function test and primary hypothyroid. The blood gas analysis showed metabolic acidosis with increased anion gap. Because the clinical manifestation did not in accordance with thyrosinemia and bone marrow puncture showed foam cell appearance, we checked for the ASM activity. The result of ASM activity was 0.97. Results: We managed the failure to thrive by giving calory according to his ideal body weight. Based on the condition of hypothyroid and liver insu¤ciency, we gave branched-chain amino acid besides carbohydrate as the source of energy. Conclusions: The patient died at the age of 3.5 years because of the worsening of lung function

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The main objective of this study was to determine the e¤cacy and safety of recombinant human iduronidate-2-sulfatase (idursulfase) in treatment of muccopolysaccharidosis II. Material and Methods: In this before^after clinical trial study, intravenous idursulfase was administered to 16 children (from 3 to 11 years old) with muccopolysaccharidosis II (Hunter syndrome, in a 0.5 mg/kg single weekly dose for a 14 months duration. Clinical and laboratory status characteristics including cardiac performance, functional class and percentage of predicted forced vital capacity were evaluated and compared with those before the begining of therapy. Results: The patients showed signi¢cant improvement not only in cardiac performance (p50.005) and functional class (p = 0.005, but also in the percentage of predicted forced vital capacity (p = 0.004)after only 10 months of therapy. The drug was well tolerated and signi¢cant infusion reaction was seen in only 4 children, in which i.v. hydrocortisone could completely subside the problems. Conclusion: Intravenous idursulfase in a weekly 0.5 mg/kg dose is e¡ective to improve mucoplysaccharidosis II.

Background: Biochemical diagnosis of mucopolysaccharidoses (MPS) starts with quantitation of GAG excretion in urine. Several methods have been described for GAG quantitation, but the current method of choice is the dimethylmethyleneblue (DMB) test. False-positive results are common (10^20% of the samples analysed), requiring a repeat sample or electrophoretic analysis of GAG. Objective: To developed a simple method to reduce the number of falsepositives. Methods: GAG concentration in urine samples was determined according to De Jong et al. (Clin Chem 1992, 38:803^7). In addition to measuring absorbance at 520 nm for GAG quantitation, the spectrum (400^700 nm) was recorded and the ratio of A595 nm/A650 nm was calculated. Results: In 2006^2007, 13% of the samples analysed (n = 1053) had GAG values above age-matched reference values, including 27 cases of MPS, which were identi¢ed by GAG electrophoresis and con¢rmed by enzyme analysis. Urines of MPS patients had a distinctive spectrum with an A595/650 ratio of 0.14+0.20 (n = 68), compared to 1.11+0.38 (n = 40) in urine samples with normal GAG concentration and 0.85 +0.35 (n = 109) in false-positives. Using a cut-o¡ value of 0.5 for A595/ A650, the number of false-positives was reduced from 109 to 16 increasing speci¢city of the DMB test from 89% to 98%. Two MPS IV urines had A595/A650 values exceeding 0.5, consistent with previous observations that diagnosis of MPS IV by GAG quantitation is challenging. Conclusion: Our procedure reduces the number of false-positive results in MPS screening using the DMB test with 85%, decreasing the need for electrophoretic analysis of GAG and/or repeat samples.

IDURSUFASE THERAPY IN HUNTER SYNDROME Salehpour S1, Farahmand Monfared M2 1 Div Metab Dis, Mo¢d Child Hosp, SBMU, Tehran, Islamic Republic of Iran, 2Div Metab Dis, Genomic Research Center, Tehran, Islamic Republic of Iran

A SIMPLE PROCEDURE TO REDUCE THE NUMBER OF FALSE-POSITIVE RESULTS IN MUCOPOLYSACCHARIDOSIS SCREENING Ruijter GJG, van den Berg RG, Huijmans JGM Dept Clinical Genetics, Erasmus MC, Rotterdam, Netherlands

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NEONATAL SCREENING FOR POMPE DISEASE ^ A FEASIBILITY STUDY Lukacs Z1, Nieves Cobos P1, Keil A1, Santer R2 1 Dept Clin Chem, Univ Med Center, Hamburg-Eppendorf, Germany, 2 Dept Pediatr, Univ Med Center, Hamburg-Eppendorf, Germany Pompe disease is an autosomal recessive disorder which is caused by de¢ciency of acid alpha-glucosidase. As enzyme replacement therapy has been shown to be e¡ective, neonatal screening for this disorder may signi¢cantly ameliorate outcome. Enzymatic diagnosis in blood is hampered by the presence of the neutrophil enzyme maltaseglucoamylase (MGA) which shows a signi¢cant overlap in activity within the acidic pH range. Recently, Chamoles et al. described a novel method for the selective inhibition of MGA in dried blood spots (DBS). We have modi¢ed the assay, so that it can be used for high-throughput screening. In the ¢rst tier, the activity of alpha-glucosidase is measured in the presence of acarbose at pH 3.8. Samples with ambiguous results are repeatedly tested with and without acarbose at pH 3.8, and at pH 7.0 in order to exclude artefacts. For positive samples, molecular genetic testing of the GAA gene would be available in our laboratory. In total, we have screened approx 3500 newborn samples which, as expected from the relatively small number, all showed results within normal limits. Known positive samples (430) admixed to this series were all detected. The assay was robust and less than 0.5% of samples had to be repeated in the laboratory from the same specimen. Handling of the modi¢ed assay protocol provided no problems to skilled laboratory sta¡. In summary, we were able to demonstrate that the novel £uorometric DBS assay using acarbose as a speci¢c inhibitor can be successfully used for newborn screening in a routine laboratory environment.

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THE CLINICAL, RADIOLOGICAL AND HISTOLOGICAL FEATURES OF A PATIENT WITH A NOVEL FORM OF LATE INFANTILE NEURONAL CEROID LIPOFUSINOSIS: CLN7 Glamuzina EE1, Flemming J2, Siintola E3, Lehesjoki A4, MacFarlane J5, W|lson CJ1 1 Starship Children's Hosp, Auckland, New Zealand, 2Paed Dept, Tauranga Hosp, Tauranga, New Zealand, 3Folkha«lsan Inst of Genetics, Helsinki, F|nland, 4Neuroscience Centre, Univ of Helsinki, Helsinki, F|nland, 5Lab Plus, Auckland, New Zealand Background: The neuronal ceroid lipofuscinoses (NCLs) are a group of lysosomal storage disorders characterised by developmental regression, visual impairment and epilepsy. Recently the molecular aetiology of a novel late infantile form of NCL, NCL7 was reported (Am J Hum Genet 2007;81:136^46) in ¢ve Turkish and one Indian family. These children had mutations in the MFSD8 gene at 4q28.1. The MFSD8 gene is thought to encode a predominantly lysosomal protein that belongs to the major facilitator superfamily of transporter proteins. The clinical details of a child with CLN7 have not previously been reported. Methods: The clinical, radiological, neurophysiological and histological details of a child with NCL7 are presented. Results: A F|jian Indian girl presented at age four years with myoclonic epilepsy and a recent deterioration in her gross motor skills. Her clinical progress over the following four years was typical of late-infantile neuronal ceroid lipofuscinosis (LINCL) with marked developmental regression, visual impairment, treatment resistant seizures and eventually death. Investigations, including lysosomal white cell enzymes (including PPT1 and TTP1) and molecular sequencing of the CLN3 and CLN6 genes, were normal. Rectal biopsy showed mixed-type inclusions with neuronal curvilinear bodies and granular osmophilic deposits. The MRI showed a generalised leukodystrophy and mild cerebral atrophy while the EEG late in the disease revealed widespread generalised background abnormalities and slow rhythms. Conclusions: In children presenting with features of LINCL who have normal PPT1 and TTP1 levels and normal molecular studies of CLN 3, 5 and 6, CLN7 should be considered.

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NIEMANN-PICK TYPE C DISEASE: NATURAL HISTORY AND CLINICAL COURSE IN TEN BRAZILIAN PATIENTS Lourenco CM1, Souza FTS2, Coelho JC2, Giugliani R2, Marques VD1, Toscano P1, Bastos PG1, Marques W Jr,1 1 University of Sao Paulo, Ribeirao Preto, Brazil, 2Rio Grande do Sul Federal University, Rio Grande do Sul, Brazil

QUALITY ASSESSMENT OF ENZYME ANALYSIS FOR LYSOSOMAL STORAGE DISORDERS (LSD'S) THREE EUROPEAN + QA-PILOTS WITH 40^49 PARTICIPANTS van Diggelen OP1, Oemardien L1, de Graaf I2, Weykamp C2 1 Dept Clinical Genetics, Erasmus MC, Rotterdam, Netherlands, 2SKML, Beatrix Ziekenhuis, W|nterswijk, Netherlands

Background: In Niemann-Pick disease types A/B, lipid, mainly sphingomyelin, accumulates; in Niemann-Pick type C (NPC), there is, instead, a defect in cholesterol esteri¢cation causing lipid storage. Methods: A clinical retrospective study and review of exams were carried out of patients diagnosed with NPC. Results: Seven were con¢rmed to have NPC by F|lipin test (three patients required molecular analysis). Regarding clinical form, 4 were perinatal and 6 were infantile. All patients had prolonged jaundice and ¢ve of them were diagnosed with `neonatal hepatitis'. The ¢rst symptom of neurological disease was developmental delay in all but one patient whose ¢rst symptom was seizures; dystonia was present in four patients; three had supranuclear gaze paralysis. At the time of the study, seven patiens were deceased ^ the mean age of them was 4.7 years. Two patients have started on an inhibitor of glycosphingolipid storage (one with 18 months and the other with 10 years) and showed regression of hepatosplenomegaly and stabilization of mental deterioration in the older patient. The youngest patient had delayed milestones before the treatment and presented with seizures with 14 months. After one year with the treatment, she does not have seizures anymore and began to walk with 2 years. Electrophysiological studies did not demonstrate any peripheral neuropathy. Conclusions: The onset of neurological presentation of the disease varies among the patients. Substrate reduction therapy seems to be a reasonable approach to treat lysosomal storage disorders, specially those disorders with brain involvement.

Background: Enduring quality assessment (QA) for enzyme diagnostics is needed, but non-existing. In 2006, 40 ESGLD laboratories participated in the 1st QA-pilot for lysosomal enzymes. In 2007 and 2008 16 non-European laboratories joined. We aim at a regular ERNDIM QA-programme for the LSD's in 2010. Methods: (1) Freeze-dried samples were shipped in winter to avoid costly dry-ice shipments; (2) enzyme activities of 10 `easy' lysosomal enzymes were determined (both nmol/h/mg protein and % of the meanenzyme activity of the participants' lab, to normalise); (3) samples were analysed 2^4 times (blind, identical samples) to determine the reproducibility; (4) to diagnose patients, EBV-lymphoblasts from LSDpatients were used; (5) data entry through existing ERNDIM website (www.erndimqa.nl). Results: The 2006, 2007 results (35; 46 labs) showed large variation in speci¢c enzyme activities (grouping di¡erent methods was not done). After normalisation, the inter-laboratory variation decreased but stayed high (ratio lowest/highest activity for most enzymes: 1/6). All laboratories diagnosed Gaucher, Sandho¡ and GM1-gangliosidosis, but two laboratories missed Tay-Sachs. Reproducibility of the best performers had variation coe¤cients 520% for all enzymes. These 6 labs performed equally well in 2006 and 2007. About one third of the labs had all VCs 540%, but several 420%. In 2007, about half the labs had one or more enzymes with a VC in the range 40^130%. The 2008 results will be presented (54 labs). Conclusions: Freeze-dried samples are suitable for QA-programmes for LSD's. Enzyme analysis programmes under supervision of ERNDIM appear realistic.

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CENTRAL NERVOUS SYSTEM INFLAMMATION IN FABRY DISEASE Lidove O1, Chauveheid MP1, Caillaud C2, Froissart R3, Benoist L4, Alamowitch S4, Doan S5, Szalat R1, Baumann N6, Alexandra JF1, Lavallee P7, Klein I8, Vuillemet F9, Sedel F10, Samson Y10, Roullet E4, Papo T1 1 Dept of Int Med, Bichat Hosp, Paris, France, 2Genetics, Cochin Hosp, Paris, France, 3Biochem, Lyon, France, 4Neurology, Tenon, Paris, France, 5 Ophthalmology, Bichat, Paris, France, 6Neurochemistry, Pitieè, Paris, France, 7Neurology, Bichat, Paris, France, 8Radiology, Bichat, Paris, France, 9Neurology, Colmar, France, 10Neurology, Pitieè, Paris, France Background: Neurological phenotype in Fabry disease (FD, OMIM 301 500) include stroke, acroparesthesia, cranial nerve palsies and autonomic dysfunction. Methods/Patients: We report on three patients with chronic meningitis which led to FD diagnosis. F|ve previously reported cases of meningitis related to FD were also studied (6 male, 2 female, in all). Results: Mean-age at onset of meningitis was 33.3 years (18^54). A familial history of FD was present in 6 cases. Non neurological FD symptoms included: cornea verticillata (n = 7), acroparesthesia (n = 6), heart involvement (n = 5), kidney involvement (n = 5), hypohidrosis, angiokeratoma, abdominal symptoms, telangiectasia, and lymphoedema. Central nervous system involvement included stroke (n = 7), sensorineural hearing loss (n = 6), and vertigo (n = 6). Fever was present in 3 cases. Cerebrospinal £uid was abnormal in all cases, showing pleiocytosis (mean, 28 cells/ml) and elevated protein level (mean, 79 mg/dl). Diagnosis ¢rst considered were tuberculous meningitis, central nervous system vasculitis, neurobrucellosis, Whipple disease, multiple sclerosis, CADASIL, neurovestibulitis. FD diagnosis was assessed by low a-galactosidase A dosage and/or gene mutation analysis in all cases. F|ve patients were treated with enzyme replacement therapy and there was no improvement of cerebrospinal £uid analysis. Six patients were treated with corticosteroids, with improvement or normalization of cerebrospinal £uid in 3 cases. Intracranial hypertension occurred in two cases, and responded to corticosteroids and azathioprine. Conclusion: FD may cause steroid-responsive central nervous system in£ammation and should be considered as a cause of chronic aseptic meningitis.

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SCREENING FOR PYRIDOXINE DEPENDENT EPILEPSY (PDE): HPLC-MS/MS ANALYSIS OF LYSINE DEGRADATION METABOLITES Korall H1, Wallner S1, Ermandraut N1, Beil J1, Trefz F-K2 1 Zentrum fuer Sto¡wechseldiagnostik zfs, Reutlingen, Germany, 2 Children's Hospital Reutlingen, Reutlingen, Germany Introduction: Pyridoxine dependent epilepsy PDE (MIM 266100) is a rare, autosomal-recessive inborn error of metabolism. Seizures are caused by a secondary vitamin B6 de¢ciency due to a lack of 2aminoadipic acid semialdehyde (2-AASA) dehydrogease in lysine degradation. Besides seizures mental retardation, in some cases microcephaly and disturbed brain development may occur. Method: Using HPLC and tandem mass spectrometry we established a method for quantitative determination of pipecolic acid, 2-AASA and lysine in urine, plasma and cerebrospinal £uid. Components were eluted with a mixture of water (70%) and methanol (30%) each containing 0.05% tri£uoracetic acid. The eluate was analysed by an Applied Biosystems API 3000 tandem mass spectrometer. Turbo ion spray ionization is used, MS/MS spectra were taken by multiple reaction monitoring of characteristic ions. Results: Limit of quanti¢cation is 1 mmol/L for 2-AASA and pipecolic acid. Patients with seizures who respond to vitamin B6 initially show elevated 2-AASA levels in all body £uids. This proofs pyridoxine dependent seizures. According to these results pipecolic acid was also found in all body £uids. Excretion of 2-AASA seems to be a reliable marker for diagnosis and therapy monitoring of pyridoxine dependent seizures. Patient cases are shown. Discussion: The presented method o¡ers a non-invasive, quick and reliable tool for diagnosis and therapy monitoring of PDE.

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NEUROLOGICAL OUTCOME IN THREE PATIENTS WITH COMBINED METHYLMALONIC ACIDURIA AND HOMOCYSTINURIA (CblC) Sibilio M1, Della Casa R1, Romano A1, Mansi G1, Morrone A2, Donati MA2, Fontana F1, Minichini L1, Ungaro C1, Cavicchi C2, Bruschini D1, Andria G1, Parenti G1 1 Dept Pediatr, Federico II Univ, Naples, Italy, 2AOU Meyer Metab and Muscular Unit, Florence, Italy Background: Methylmalonic aciduria with homocystinuria, cblC type is a disorder of intracellular cobalamin (Cbl) metabolism, due to mutations of the MMACHC gene. CblC is characterized by psychomotor delay, failure to thrive, microcephaly, megaloblastic anemia, ophthalmic abnormalities. Long-term outcome in most patients is characterized by moderate/severe cognitive impairment. Therapy includes hydroxycobalamin and betaine. Objectives: To describe the clinical course in 3 Italian cblC patients. Patients and Methods: We studied 3 cblC patients (2 girls, LN, CE, ¢rst cousins; 1 boy, CG). All patients originated from the same town. Mutational analysis of the MMACHC gene showed compound heterozygosity (c.3G4A and c.271dupA) in LN and CG and homozygosity for the c.3G4A mutation in CE. Follow-up (clinical, biochemical, developmental tests, brain MRI) were performed in all patients since the age of diagnosis. Results: LN was diagnosed early (1 month), and OH-Cbl treatment was started soon. She presently (8 years old) shows normal mental development (DQ 91). Brain MRI and EEG are normal. CE, presently 20 years old, was diagnosed at 9 months and OH-Cbl treatment was started. She shows moderate psychomotor delay (DQ = 46). MRI shows di¡use white-matter loss, cerebral and cerebellar atrophy. CG (15 years old) was diagnosed at 6 years and started OH-Cbl therapy. He shows severe developmental delay (DQ = 20), microcephaly, hypotonia, seizures. Brain MRI shows cerebral atrophy. Conclusions: Clinical variability was observed in patients with the same genotype. Phenotypic severity apparently correlates with age at start of therapy, and suggests that timely treatment may improve the neurological outcome of patients.

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CblD DEFECT OF VITAMIN B12 METABOLISM: FUNCTIONAL ANALYSIS OF MUTANT MMADHC ALLELES AND MITOCHONDRIAL TARGETING Stucki M1, Suormala T2, Coelho D2, Paesold-Burda P1, Fowler B2, Baumgartner MR1 1 Div Metab and Mol Ped, Univ Child Hosp, Zuerich, Switzerland, 2Div Metab, Univ Child Hosp, Basel, Switzerland Background: The cblD defect causes isolated or combined de¢ciency of m i t o c h o n d r i a l a d e n o s y l c o b a l a m i n (A d o C b l ) a n d c y t o s o l i c methylcobalamin (MeCbl) synthesis leading to either isolated methylmalonic aciduria (cblD-MMA), isolated homocystinuria (cblD-HC) or both (cblD-MMA/HC). We show expression of mutations detected in the recently discovered MMADHC gene (Coelho et al. NEJM 2008). Methods: mRNA levels were studied using real-time PCR in ¢broblasts of 10 patients. The e¡ect of 9 mutations on coenzyme synthesis was investigated by transient transfection of cblD ¢broblasts using electroporation. Mitochondrial targeting was studied by fusing the mitochondrial targeting sequence (MLS) of aldehyde dehydrogenase 2 (ALDH2) to MMADHC. Results: mRNA levels were low in only one patient (p.Y140X/p.Y140X), suggesting nonsense mediated mRNA decay. All other patients, even one with a premature stop codon in the N-terminal part (p.C19fsX20/p. c19fsX20) had normal mRNA levels. MeCbl synthesis was rescued in both cblD-HC and cblD-MMA/HC ¢broblasts by the wildtype cDNA and by alleles with N-terminal mutations associated with the cblD-MMA phenotype (p.C19fsX20, p.L20fsX21, p.R54X). Clear correction of AdoCbl synthesis was only achieved with C-terminally located missense mutations associated with cblD-HC (p.D246G, p.Y249W, p.L259P) or a wildtype construct with modi¢ed C-terminus (V5-tag). Replacing the putative MLS with that of the well established ALDH2 resulted in a signi¢cant shift from MeCbl to AdoCbl synthesis. Conclusion: Our results show a correlation between the nature and location of MMADHC mutations and the phenotype as well as a delicate balance between cytosolic MeCbl and mitochondrial AdoCbl synthesis con¢rming its role as a branching point in intracellular cobalamin tra¤cking.

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BIOTINIDASE NEWBORN SCREENING EXPERIENCE IN TUSCANY Malvagia S, Funghini S, La Marca Gla Marca G, Morrone A, Donati MA, Pasquini E Metabolic Unit, Meyer Children's Hospital, Florence, Italy The biotinidase enzyme is essential for recycling the vitamin biotin. Biotinidase de¢ciency (BD) is inherited as an autosomal recessive condition. W|th reference to residual enzyme activity this condition can be profound or partial. Oral biotin supplementation prevents symptoms and the disorder meets the criteria for inclusion in newborn screening programs. In January 2007, qualitative colorimetric assay for BD was introduced in Tuscany as a pilot screening program and molecular analyses of the BTD gene were performed in all positive newborns. Out of a total 46 000 screened newborns, we identi¢ed 7 patients: 2 with profound and 5 with partial de¢ciencies. All showed normal acylcarnitine pro¢les in LC-MS/MS. The ¢ve patients with a partial de¢ciency were found to be compound heterozygotes for the p.D444H mutation in one allele coupled with a mutation causing a profound BD in the other allele. Although this pilot study involved relatively few newborns, our data suggest that the Tuscan population has one of the highest incidence rates worldwide: 1 case per 23 000 for profound BD and 1 per 9200 for partial de¢ciency. These data are very di¡erent from those so far reported worldwide (1:60 000) but close to a ratio reported in Brazil as one of highest known. The di¡erent incidence observed between retrospective clinical studies and our prospective newborn screening study suggests that the defect may be under-diagnosed. We would like underline the importance of screening for BD as early diagnosis prevents possible neurological damage and early death.

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MOLECULAR CONFIRMATION OF cblC PATIENTS IDENTIFIED BY EXPANDED NEWBORN SCREENING Nogueira C1, Aiello C2, Dionisi-V|ci C2, Martins E3, Lea¬o E4, Diogo L5, Cerone R6, Caruso U6, Sequeira S7, Kok F8, Deodato F2, Santorelli FM2, V|larinho L1 1 Med Gen Center Jacinto de Magalha¬es-INSA, Oporto, Portugal, 2 Bambino Gesu Hospital, Roma, Italy, 3Maria Pia Hospital, Oporto, Portugal, 4St Joa¬o Hospital, Oporto, Portugal, 5Children's Hospital, Coimbra, Portugal, 6IRCCS Gaslini, Genova, Italy, 7D Estefaªnia Hospital, Lisbon, Portugal, 8Clin Neurol Univ S Paulo Sch of Med, S Paulo, Brazil Background/Objectives: Combined methylmalonic aciduria (MMA) and homocystinuria, cblC type (MIM 277400), is the most frequent inborn error of vitamin B12 (cobalamin, cbl). The recent identi¢cation of the disease gene, MMACHC, on chromosome region 1p, has allowed preliminary genotype-phenotype correlations. Based on the age of onset, two distinct clinical forms have been recognized: early-onset and late-onset forms. We present our recent experience in 41 new cblC cases from a combined Portuguese and Italian study, and the molecular data of two cblC patients detected by expanded newborn screening. Methods: Sequence analysis of genomic DNA from these patients was performed to identify disease-causing mutations in the MMACHC gene. Results: We found that the c.271dupA (accounting for 55% of the MMACHC alleles in our cohort) followed by c.394C4T (16%) and c.331C4T (9%) were the most frequent mutations. One of the suspected cblC patients was homozygous for c.271dupA and the other was compound heterozygous for c.271dupA and c.565C4A mutations. Conclusions: This study shows that mutation screening for the most common MMACHC mutations occurring in early-onset forms (c.271dupA and c.331C4T) seems to have a high diagnostic yield in a southern European population with cblC defect. Our data corroborate the importance of a molecular testing to con¢rm suspected cblC patients detected by expanded newborn screening, reducing the need of complex, costly, laborious and time-consuming biochemical testing in cultured skin ¢broblasts. This strategy will be important to identify cblC infants presymptomatically or early in the course of the disease.

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FAVOURABLE THERAPEUTIC OUTCOME FOR COMBINED METHYLMALONIC ACIDEMIA AND HOMOCYSTINURIA DUE TO COBALAMIN DEFECTS Choy YS1, Choy YS2, Zabedah MY3 1 Genetic Unit, Prince Court Med Ctr, Kuala Lumpur, Malaysia, 2Genetic Div, Subang Jaya Med Ctr, Petaling Jaya, Malaysia, 3Metab Lab Institute of Med Research, Kuala Lumpur, Malaysia The outcome of treatment for cobalamin defects with combined methylmalonic acidemia and homocystinuria (cobalamin C, D and F) had largely been unsatisfactory. Survivors had been signi¢cantly handicapped despite treatment. Intramuscular hydroxocobalamin had been shown to improve the urinary methylmalonate excretion. Betaine and folinic acid were found to reduce homocysteine level. We reported here the favourable therapeutic outcome for two cases of combined methylmalonic acidemia and homocystinuria due to cobalamine defects. Hydroxocobalamin was given 1 mg i.m. daily for a week on diagnosis followed by biweekly injection and oral hydroxocobalamin 1 mg daily. Oral folinic acid, betaine and L-carnitine were also given. Plasma homocysteine and urinary methylmalonate level were closely monitored. Natural protein intake was moderately restricted to 1.5 mg/ kg/day. The ¢rst patient was a Chinese girl who presented at 2nd month with lethargy, hypotonia, vomiting, chronic diarrhea and failure to thrive. MRI brain showed fronto-temporal atrophy and she had dystonia when cobalamin C defect was diagnosed at 4 months-old. She was found to have compound heterozygous mutations of the MMACHC gene. Intensive rehabilitative therapy was carried out along with the medical therapy. She had catch-up growth and her development almost normalized. She was walking and talking at the age of 14 months. The second patient was a Pakistani boy who presented with learning di¤culties, diplegia, weight loss and recurrent acrodermatitis enteropathica-like rashes at 8 years-old. He improved tremendously in all aspects with the treatment. Patients with these disorders can have favourable outcome in the absence of prenatal brain malformation.

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ELEVATED PROPIONYLCARNITINE ON NEWBORN SCREENING AND VITAMIN B12 LEVELS Al Murshedi F1, Al Jasmi F1, Crushell E1, Kyriakopoulou L2, Feigenbaum A1 1 SickKids Hospital/ Univ of Toronto, Toronto, Canada, 2DPLM/ SickKids Hospital, Toronto, Canada Background: Screening for elevated blood propionylcarnitine (C3) level was incorporated into the expanded newborn screening (NBS) program of Ontario in August 2006. An elevated C3 level may suggest methylmalonic acidemia (MMA), propionic acidemia (PA) or disturbances of cobalamin metabolism. Hypothesis: A signi¢cant number of neonates with elevated C3 on NBS in our population have low vitamin B12 levels. Methods: The records of all babies reported to our centre as having elevated C3 48 mmol/L on NBS by the central screening laboratory for our province from September 1st 2006 October 31st 2007 were reviewed. The following parameters are examined on the recall visit: C3, plasma vitamin B12 level, MCV, urine MMA and plasma total homocysteine (Hcy). The newborns were classi¢ed according to their vitamin B12 levels into low vitamin B12 and normal vitamin B12 groups. We used vitamin B12 cuto¡ level of 200 pmol/L. Mean C3 on the NBS report, MCV and Hcy and standard deviation were calculated for each group. Results: 21 out of 54 babies (39%) who had a false positive NBS with elevated C3 had vitamin B12 5200 pmol/L. Repeat plasma C3 levels were normal on all cases. Urine MMA was positive in one baby. Mean MCV was normal in both groups and di¡erence of mean Hcy is not clinically signi¢cant. Conclusion: This study shows signi¢cantly increased prevalence of low vitamin B 12 level in babies with elevated C3 on the NBS. Follow up is indicated to study the real vitamin B12 status in these babies.

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FOLINIC ACID RESPONSIVE SEIZURES ASSOCIATED WITH FOLATE RECEPTOR AUTOIMMUNITY Ramaekers V1, Blau N2, Sequeira J3, Quadros E3 1 Dept Ped Neurol, IDCHU Lie©ge CHU Lie©geCHU Lie©ge, Lie©ge, Belgium, 2Div Clinical Chemistry, Univ Child Hosp, Zu«rich, Switzerland, 3 Dept Cell Biology, State Univ New York, Brooklyn, USA Background: Two infants manifested intractable seizures from the age of day one and three months respectively, which did not respond to pyridoxine and anticonvulsant drugs, but arrested completely following high dose folinic acid therapy. Neurological follow-up showed normal outcome for both children. The aetiology of folinic acid responsive seizures remains unknown. Methods and Results: CSF analysis showed normal levels for monoamine metabolites, pterins and methyltetrahydrofolate while the previously reported abnormal substance on the neurotransmitter chromatogram was not present. Before folinic acid treatment serum contained a low titer of folate receptor (FR) autoantibodies of the blocking type, which was followed by increasing titres on follow-up. Maternal serum tested negative for FR autoantibodies. Conclusion: Folinic acid-responsive seizures in the two reported infants are characterized by postnatal development of serum FR autoantibodies of the blocking type. High dose folinic acid treatment can circumvent the blockade at the choroid plexus imposed by FR auto antibodies, arrest intractable seizures and lead to normal outcome. Many questions concerning these preliminary observations still have to be addressed, being the absence of spinal £uid depletion of 5methyltetrahydrofolate and the possibility of intrathecal production of FR autoantibodies with an additional blockade of folate transfer across the subependymal-neural tissue barriers.

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FUNCTIONAL TRYPTOPHAN HYDROXYLASE DEFICIENCY Schott DA1, Keularts IMLW2, Dorland L2, Duran M3, Abeling NGGM3, Vries JE de4, Bierau J2, Spaapen LMJ2, Vles J5, Rubio-Gozalbo E2, Nicolai J5, Gerver WJM1 1 Dept Paed, MUMC+, Maastricht, Netherlands, 2Lab Biochem Gen, Dept Clin Gen, MUMC+, Maastricht, Netherlands, 3Lab Gen Metab Dis, AMC, Amsterdam, Netherlands, 4Lab Clin Chem, St. Laurentius Hospital, Roermond, Netherlands, 5Dept Neurology, MUMC+, Maastricht, Netherlands Objective: A 2-year old girl presented with obesity. At the age of 3 years, behavioral problems manifested as well as sleeping disturbances, hypothermia and neurologic problems. Her symptoms were summarized by hypothalamic syndrome. Metabolic screening revealed abnormalities with respect to neurotransmitters. Methods: Endocrinological measurements were performed in addition to metabolic screening. Furthermore, neurotransmitters and pterins were measured; sequence analysis of the tryptophan hydroxylase (TPH2) gene (Prof. Bader, Berlin, Germany) was performed. Results: Thyroid parameters were within normal ranges as well as cortisol. The patient presented with precocious puberty as was con¢rmed by an abnormal response to GnRH (LH increase 410 IU/L). Selective metabolic screening in urine and plasma did not reveal any signi¢cant abnormalities. In CSF however, 5-HIAA was low (29 and 46 nmol/L; ref 89^341 nmol/L) while HVA was normal. In urine, both 5-HIAA and HVA were normal. In order to exclude a defect in the pterin metabolism, we measured CSF neopterin and biopterin and found no signi¢cant abnormalities. CSF 5OH-tryptophan (52 nmol/L; ref 47 nmol/L) was low. On supplementation with 5-OH-tryptophan, carbidopa and seroxat (serotonin re-uptake inhibitor), 5-HIAA levels in CSF normalized and the patient clinically improved. Sequence analysis of the TPH2 gene was performed and no abnormalities were detected. Conclusions: These data suggest a (functional) tryptophan hydroxylase defect, partially explaining the clinical presentation of the patient. Diagnosis of this condition is important because of the promising results of treatment.

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Background: Cobalamin (B12) is available from meat, ¢sh and dairy products. Patients with inborn errors of protein metabolism (IPM) on long term protein restriction may be susceptible to cobalamin de¢ciency. De¢ciency may result in permanent neurological damage. Methods: We de¢ned cobalamin de¢ciency in patients with normal plasma creatinine as plasma or serum cobalamin 41.96 SD below the mean of an age-related reference population (MR). Alternatively cobalamin at marginal levels (5200 pmol/L) with plasma MMA 43SD above the MR or total homocysteine 43SD above the MR with normal red cell or serum folate. We studied 35 adults and 15 children each with one of 9 disorders. There were 25 with PKU, and 8 with MSUD. Most IPM patients received essential amino acids from liquid supplements which may contain cobalamin. Results: None of the children were cobalamin de¢cient but two received less cobalamin than the National Academy of Sciences recommended daily allowance (RDA). F|ve of 27 (18.5%) adults prescribed liquid amino acid supplements and four of eight (50%) adults prescribed low protein diets with vitamins demonstrated clear-cut tissue de¢ciency of cobalamin. F|ve of the 9 cobalamin de¢cient adults had an estimated cobalamin intake less than the adult RDA. Conclusion: Adult patients with inborn errors of metabolism managed with low protein diets are frequently cobalamin de¢cient. They should be carefully monitored to assess cobalamin status and diets should be monitored to ensure su¤cient intake.

Cerebral folate de¢ciency (CFD) can be de¢ned as any neurological syndrome associated with a low CSF 5-methyltetrahydrofolate (5MTHF) in the presence of normal peripheral folate metabolism. A number of causes of CFD have been suggested including development of auto-antibodies against membrane bound folate receptors present on the choroid plexus and impaired energy metabolism leading to diminished active transport of 5MTHF into the central nervous system. Patients with a CFD are identi¢ed, in our laboratory, by the HPLC (£uorescence detection) analysis of CSF and for such patients the magnitude of the 5MTHF de¢ciency varies considerably. Thus, in common with other groups, we have reported patients with undetectable 5MTHF and others where the concentration is just below the reference interval. To date, the e¡ect that varying degrees of CSF 5MTHF de¢ciency have upon central one carbon metabolism are unknown. Consequently, in view of the 5MTHF requirement of methionine synthase, we have developed an assay for CSF homocysteine, with a view to relating 5MTHF status with homocysteine (substrate for methionine synthase) concentration. Using LC-MS, we have produced a provisional reference range for CSF total homocysteine; 6^180 nmol/L. Applying this method to patients with low CSF 5MTHF concentrations, we have so far recorded a CSF homocysteine of 47000 nmol/L in a patient with an undetectable CSF 5MTHF. This study will now be extended to determine the relationship between CSF 5MTHF and homocysteine concentrations and to establish if a metabolic threshold exists for eliciting perturbations of one carbon metabolism.

VITAMIN B12 DEFICIENCY IN 50 PATIENTS WITH AN INBORN ERROR OF PROTEIN METABOLISM Green K1, Carpenter K1, W|lcken B2 1 NSW Biochemical Genetics Service, Sydney, Australia, 2Discp Paed Ch Health Gen Med, Univ Syd, Sydney, Australia

CSF HOMOCYSTEINE A USEFUL ADJUNCT FOR THE INVESTIGATION OF PATIENTS WITH CEREBRAL FOLATE DEFICIENCY? Lynes G, Land J, Briddon A, Heales S Neurometabolic Unit, National Hospital, London, UK

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DEVELOPMENTAL DELAY AND CEREBRAL PALSY ASSOCIATED WITH TRYPTOPHAN HYDROXYLASE DEFICIENCY: POSSIBILITIES FOR TREATMENT? Langius FAA1, Abeling N2, Duran M2, Poll The BT2 1 St Lucas Andreas Hospital, Amsterdam, Netherlands, 2Academic Medical Centre, Amsterdam, Netherlands We present a 12-year-old boy of unrelated parents. Delivery was complicated by umbilical cord strangulation. He developed cerebral palsy and his psychomotor development was strikingly retarded. A cerebral MRI was not indicative for perinatal asphyxia as the causative factor for the clinical problems. At the age of seven, he developed epilepsy and anticonvulsant therapy was started. From the age of 10 a progressive motor restlessness and uncontrolled yelling resembling Tourette syndrome was noticed. This prompted investigation of neurotransmitters in the CSF, after all previous metabolic investigations were negative. Low levels of 5-hydroxyindoleacetic acid (36 nmol/L; ref. 68^115) as well as its precursor 5-hydroxytryptophan (4 nmol/L; ref. 5.2^7.8) were found. As the other aromatic neurotransmitter metabolites were normal, the possibility of isolated tryptophan hydroxylase (TPH) de¢ciency was raised. Mutation analysis will be performed. Subsequent treatment with 5-hydroxytryptophan resulted in a marked biochemical and some clinical improvement as yet. The clinical condition of our patient clearly di¡ers from previously reported patients with presumed TPH de¢ciency, but nevertheless the possibly bene¢cial e¡ect of treatment is intriguing and warrants further investigations, particularly in view of the scarce knowledge about the clinical phenotype of this neurotransmitter defect.

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TYROSINE HYDROXYLASE DEFICIENCY: IDENTICAL GENOTYPE IN THREE APPARENTLY UNRELATED PATIENTS. A COMMON ANCESTOR HYPOTHESIS Serrano M1, Ormazaèbal A1, Orfanou I2, Youroukos S2, Drakaki K2, Giannakopoulos A2, Campistol J1, Garcia-Cazorla A1, Artuch R1, Cormand B3, Pons R2 1 Neurometab Unit, HSJD and CIBER-ER, Barcelona, Spain, 2Ped Dept, Agia So¢a Hosp, Med School, UA, Athens, Greece, 3Dept Genet, UB, Biomed Inst and CIBER-ER, Barcelona, Spain Background: Tyrosine hydroxylase (TH) is the rate limiting step in the biosynthesis of dopamine. There are near 40 cases in the literature reporting mutations in the codi¢cant and promoter regions of TH gene, and being the cause of di¡erent phenotypes. Methods: We describe the clinics, molecular ¢ndings, and response to therapy, in three apparently unrelated girls of Greek origin. Results: Severe motor delay or loss of milestones, intermittent limb dystonia, ptosis, and abnormal eye movements during the ¢rst months of life were the main clinical signs found in all the patients. Tremor and hyperhydrosis were evident in two cases. Cerebrospinal £uid analysis revealed an isolated markedly decreased homovanillic acid and 4hydroxy-3-methoxiphenilglycol. DNA sequencing revealed a T-to-C transition in exon 6 (L236P). This missense point mutation has previously been reported in a Greek patient (Lu«decke et al. 1996) and studied in E. coli model. In our patients nine studied polymorphisms inside the TH gene limits were coincident, supporting the possibility of a common ancestor. After L-dopa treatment increase in spontaneous movements and some developmental progress was noted in all patients. Facial expression, ptosis and oculogyric crises improved in all patients. Hyperhydorsis did not improve. Two patients developed L-dopa induced dyskinesias. Conclusions: Despite the same genotype, biochemical ¢ndings, clinical presentation and response to L-dopa can be di¡erent. The study of polymorphisms in the TH gene enhances the possibility of a common ancestor for patients presenting the same mutation.

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PREVALENCE OF THE IVS6+4 A4T MUTATION IN TAIWANESE PATIENTS WITH AROMATIC AMINO ACID DECARBOXYLASE DEFICIENCY Lee NC, Hwu WL, Chien YH, Lee WT, Huang AC, Wu RM National Taiwan University Hospital, Taipei, Taiwan Background: The aromatic amino acid decarboxylase (AADC) is responsible for the conversion of L-dopa to dopamine, and 5-hydroxytryptophan to serotonin. Typical symptoms of AADC de¢ciency include hypotonia, oculogyric crisis and dystonia, starting from infancy. Most patients die in their early childhood. Previous clinical observation revealed an excess of patients with AADC de¢ciency in the Taiwanese population. Methods: The diagnosis of AADC de¢ciency was made by the typical symptoms or CSF neurotransmitter measurement. Genomic DNA was extracted from blood. All exons of the AADC gene were ampli¢ed and sequenced. Results: Twelve cases of AADC de¢ciency were collected. Among the 24 mutated alleles, 18 were belonged to the IVS6+4 A4T mutation, which then represented 75% of all mutated alleles. None the families had consanguinity or were relatives to each other. There was another mutation that repeated 3 times, besides another 3 sporadic mutations. Conclusion: The high prevalence of the splicing mutation in Taiwanese patients with AADC de¢ciency suggest a founder e¡ect and is likely to explain the excess of this disease in Taiwan.

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ABNORMAL NEUROTRANSMITTERS: PITFALL IN THE DIAGNOSIS OF RETT SYNDROME Engbers HM1, de Ruyter J1, de Sain-van der Velden MGM1, Wevers M2, V|sser G1 1 Div Metab Dis, W|lhelmina Child Hosp, Utrecht, Netherlands, 2Div of Genetics, W|lhelmina Child Hosp, Utrecht, Netherlands Introduction: Suspicion on Rett syndrome (RS), a neurodevelopmental disorder mostly a¡ecting females, is based on clinical criteria. However, patients may lack essential characteristics which may hamper the diagnostic process. We present a patient who presented with mental retardation and persistent abnormal neurotransmitters in who later RS was diagnosed. Casus The female patient, born after a normal pregnancy from nonconsanguineous parents, had a delayed development from the age of 6 months. At the age of 2 years a work-up for developmental delay was performed. Karyotyping (46, XX), DNA examination of Fragile X, deletion of 22q11 and MRI of the brain were all normal, but CSF showed a persistent, isolated decreased 5-HIAA, 75 nmol/L and 53 nmol/L, respectively (normal values 100^400). On suspicion of a tryptophan hydroxylase de¢ciency treatment with tryptophan was started and later Seroxat was added. CSF 5-HIAA levels normalized, but no clinical improvement was seen. At the age of 5.5 years she presented with seizures, laughing spells, and stereotypic hand movements all typical signs of RS. This was con¢rmed by DNA examination: c.880C 4 T (p.Arg 294X) mutation in the MECP2-gen. Conclusion: The diagnosis RS was delayed, because of suspected tryptophan hydroxylase de¢ciency. In retrospect, given the relatively high prevalence of RS (1:10 000) and the fact that many persistent metabolic abnormalities have been described in RS, especially in CSF, RS should have been considered sooner. We suggest that in girls with mental retardation and abnormal neurotransmitters RS should always be excluded even when epilepsy is not present.

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A SIBLING CASE OF EARLY ONSET DYSTONIA WITH MRI ABNORMALITY Fujioka H, Shintaku H, Yokoi T, Yamano T Dept of Pediatr, Osaka Univ, Osaka, Japan

PRAMIPEXOLE IN TETRAHYDROBIOPTERIN DEFICIENCY Porta F1, Mussa A1, Concolino D2, Spada M1, Ponzone A1 1 Dept of Pediatrics, University of Turin, Torino, Italy, 2Dept of Pediatrics, Univ Magna Grecia, Catanzaro, Italy

We report a sibling case of early onset dystonia. Patient 1: She was 12 year old. Her younger brother (patient 2) showed dystonia-suspected posture. No consanguineous marriage was reported. Around 4 years old, she often tumbled down when she was running. She had odd posture around 7 years old. At 8 years old, she was diagnosed as torsion dystonia. No diurnal £uctuation was observed. At 9 years old, she was admitted to our hospital. Mild improvement was observed by administration of L-dopa. In T2-weightened and FLAIR images, high signal was appeared in bilateral basal ganglia 6 months after admission. The dystonic symptom was improved after the administration of highdose benzodiazepine. The symptom was recurred after a few months. No abnormality was detected in cornea and liver. Both serum copper and ceruloplasmin concentrations were also normal. Muscle biopsy negated mitochondrial diseases. No abnormality was detected in DYT1. The high signal in brain MRI was diminished 3 years after admission. Patient 2: He was 10 years old. His standing posture was always stooping. It was suspected to be dystonic. His behavior was impulsive and hyperkinetic. He trotted when he was walking. It seemed that he understood some simple words, although he did not speak any word. No MRI abnormality has been reported until now. Brain MRI image in basal ganglia of patient 1 was similar to W|lson's disease. However no other abnormality was detected in copper metabolism. It was possible that it was a new hereditary dystonia, which was inherited in autosomal recessive manner.

Background/Objectives: Substitutive L-Dopa therapy represents the most critical aspect in the treatment of tetrahydrobiopterin (BH4) de¢ciency. Despite di¡erent adjustments, such as drug fractionation and administration of peripheral decarboxylase (PD), monoamino oxidase (MAO), and catechol-O-methyl transferase (COMT) inhibitors, adverse e¡ects, such as diurnal £uctuations and on-o¡ phenomena, are commonly observed. Though L-Dopa therapy is the physiological cure of dopaminergic dysfunction, new long-acting dopamine agonists may provide continuous, rather than pulsatile, neuroreceptor stimulation. Methods: The recent nonergoline dopamine agonist pramipexole, provided of speci¢c activity at both D2 and D3 post-synaptic receptors, was orally administered at the daily dosage of 0.0041^0.033 mg/kg to 5 patients (4 males, 1 female) a¡ected by 6-pyruvoyltetrahydrobiopterin synthase (PTPS) de¢ciency. The patients were all on treatment with L-Dopa, and PD, MAO, and COMT inhibitors, with adequate results. Cognitive and motor performances were evaluated by the UDPRS questionnaire. Blood prolactin pro¢le and plasma and urine catecholamine were evaluated before and during the treatment. Results: Pramipexole administration allowed to reduce by 20^40% the daily dosage of L-Dopa and the number of daily L-Dopa administrations from 3^4 to 2. Moreover, the prolactin pro¢le was stabilized and catecholamine excretion was reduced. Improvements in the UDPRS scale were obtained in all patients, with a signi¢cant reduction of on-o¡, wearing-o¡ and end-of-dose phenomena. After one year follow up, all the patients experienced improvement in the quality of life, and the stabilization of the prolactin pro¢le was maintained. Conclusions: Dopamine agonists may well result in facilitated clinical response to L-Dopa therapy in BH4 de¢ciency.

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GTP CYCLOHYDROLASE DEFICIENCY: REPORT OF A FAMILY WITH ATYPICAL CLINICAL FEATURES Duarte S1, Calado E1, Nogueira C2, Gaspar P2, Azevedo L3, V|larinho L2 1 Neurology Dept, D Estefania Hosp CHLC, Lisbon, Portugal, 2Centro de Genetica Medica, INSA, Porto, Portugal, 3IPATIMUP, Porto, Portugal Background/Objectives: Segawa syndrome, due to GTP cyclohydrolase de¢ciency is an autosomal dominant disorder with variable expression. We report clinical and laboratorial data from an a¡ected family. Methods: Neurological and psychiatric examination was performed in index case and other family members. CSF study was done in two patients. Molecular study was obtained in nine family members. Results: The ¢rst family member diagnosed, at the age of 14, exhibited a marked bradykinesia, with facial hypomimia, hypofonia, limb hypertonia with dystonic features. She had depression symptoms, motor and cognitive development delay, labelled as dystonic cerebral palsy. CSF study showed reduced 5-HIAA (34 nmol/L N: 95^173), HVA(86 nmol/L N: 211^540), neopterin (2.61 nmol/L N: 9^20) and biopterin (7.8 nmol/L N: 10^30). Tetrahydrobiopterin metabolism in ¢broblasts disclosed very low neopterin and biopterin production and reduced GTPCH activity suggestive of adGTPCH de¢ciency. L-Dopa/ carbidopa treatment resulted in signi¢cant motor improvement. Molecular study did not reveal the causal mutation within the GCH1 coding region. We evaluated possible departures from normal quantitative expression of the gene by real-time PCR. In all a¡ected patients, a decay of mRNA was observed, suggesting a mutation within the promotor or regulatory region of the gene. Four other family members had con¢rmed molecular study for GTPCH de¢ciency: three siblings and the father. Bradykinesia and facial hypomimia were universal features. Father and a¡ected brother had psychiatric manifestations, interpreted as alcoholic disturbance in the father and psychotic disorder in the brother. Conclusions: The clinical spectrum of this disease is still in expansion and family members with atypical psychiatric features should be considered for diagnostic screening.

INCREASED 4-AMINOBUTYRATE (GABA) IN EMBRYOS WITH SUCCINATE SEMIALDEHYDE DEHYDROGENASE (SSADH) DEFICIENCY SUGGEST AN HEIGHTENED EXCITATORY STATE DURING DEVELOPMENT Jansen EEW1, Struys EA1, Jakobs C1, Guimond SC2, Hager EJ2, Snead OC3, Gibson KM2 1 Metab Unit, Dept Clin Chem, VU Med Ctr, Amsterdam, Netherlands, 2 Med Genet, Dept Peds, Child Hosp, Pittsburgh, USA, 3Ped Neurol, Hosp Sick Children, Toronto, Canada SSADH (aldehyde dehydrogenase 5a1 (Aldh5a1); gamma-hydroxybutyric (GHB) aciduria) de¢ciency is a defect of GABA degradation in which the neuromodulators GABA and GHB accumulate. The human phenotype is that of nonprogressive encephalopathy with variable seizures, the latter displayed prominently in Aldh5a1 ^/^ mice with lethal convulsions. Metabolic studies in murine neural tissue have revealed elevated GABA (and its derivatives succinate semialdehyde (SSA), homocarnosine (HC), 4,5dihydroxyhexanoic acid (DHHA) and guanidinobutyrate (GB)) and GHB (and its analogue D-2-hydroxyglutarate (D-2-HG)) at birth. We now address metabolite features during Aldh5a1^/^ embryo development. Embryos were obtained from pregnant dams sacri¢ced at E (embryo day of life) 10^13, 14^ 15, 16^17, 18^19 and newborn mice. Intact embryos were extracted and metabolites quanti¢ed by isotope dilution mass spectrometry (n = 5^15 subjects (Aldh5a1+/+ and Aldh5a1^/^) for each gestational age group). GABA and DHHA were signi¢cantly elevated at all gestational ages in Aldh5a1^/^ mice, while GB was increased only late in gestation, and SSA was not elevated at any time. GHB and D-2-HG increased linearly with gestation. Correlative studies in human amniotic £uid from SSADHde¢cient pregnancies (n = 5) also revealed signi¢cantly increased GABA. During development, GABA in£uences cell migration and is excitatory via depolarization of neurons with high chloride concentrations; GABA inhibition begins *E15 via modi¢cation of chloride potentials regulated by monovalent ion cotransporters. These data suggest early GABAergic alterations in Aldh5a1^/^ mice, possibly exacerbated by other metabolites, which result in an heightened excitatory state that may predispose neural networks in these animals to epilepsy. Supported by NIH NS40270

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TETRAHYDROBIOPTERIN SHOWS CHAPERONE ACTIVITY FOR TYROSINE HYDROXYLASE Thony B1, Calvo AC2, Scherer T1, Svebak RM2, Haavik J2, Blau N1, Martinez A2 1 Dept Pediatr, Univ Zurich, Zurich, Switzerland, 2Dept Biomedicine, Univ Bergen, Bergen, Norway Tyrosine hydroxylase (TH) is the rate limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with L-DOPA responsive and nonresponsive dystonia and infantile parkinsonism. Here we show that both the cofactor tetrahydrobiopterin (BH4) and the feed-back inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 (hTH-1) in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription-translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH4 supplementation to mice increased TH activity and protein levels in brain extracts, while the ThmRNA level was not a¡ected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding-aid e¡ect of BH4 and a secondary e¡ect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH4 crosses the blood^brain barrier and therapeutic regimes based on BH4 supplementation should thus consider the e¡ect on TH. Furthermore, BH4 supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the hTH-1 mutation L205P.

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PYRIDOXAL PHOSPHATE AVAILABILITY AND AROMATIC AMINO ACID DECARBOXYLASE ACTIVITY. IMPLICATIONS FOR AADC DEFICIENCY AND INBORN ERRORS OF VITAMIN B6 METABOLISM Allen G1, Clayton P2, Land J3, Hyland K4, Heales S3 1 Institute of Neurology (UCL), London, UK, 2Institute of Child Health (UCL), London, UK, 3Neurometabolic Unit, National Hospital, London, UK, 4Medical Neurogenetics, Atlanta, USA Pyridoxal 5'-phosphate (PLP) is an essential cofactor for aromatic amino acid decarboxylase (AADC). In addition to facilitating substrate decarboxylation, PLP may have another role by acting as a chaperone to prevent enzyme degradation. To investigate this possibility, we determined plasma AADC activity (in the presence of saturating substrate and PLP concentrations) in two patients with pyridox(am) ine-5' phosphate oxidase (PNPO) de¢ciency, a disorder where plasma PLP concentrations are low. In one child AADC activity was clearly decreased: 14 pmol/min/ml (ref range: 36^129). In the second child, AADC activity was just below the lower limit of the reference range; 35 pmol/min/ml. For this second patient, additional kinetic analysis revealed an apparent Km for PLP of 15 mM. The Km for control plasma was 6 mM. These ¢ndings suggest that PLP availability may have a direct e¡ect upon measured AADC activity, possibly by in£uencing enzyme degradation and kinetic properties. Furthermore, the marked di¡erences in AADC activity observed here may provide an explanation for the variable CSF dopamine and serotonin metabolite levels reported in PNPO de¢ciency. W|th regards to AADC de¢ciency, our data would support the concept that PLP administration may be of bene¢t, i.e. by stimulating both residual AADC activity and limiting its degradation. We are grateful to the AADC research Trust, UK (www.aadcresearch. org) for supporting our work.

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AMINO ACID AND NEUROTRANSMITTER ABNORMALITIES IN MURINE INTERMEDIATE MAPLE SYRUP URINE DISEASE (iMSUD): SIGNIFICANT IMPROVEMENT OF CEREBRAL DOPAMINE AND SEROTONIN DEFICIENCY WITH HEPATOCYTE TRANSPLANTATION (HTx) Skvorak KJ1, Homanics GE2, Paul HS3, Dorko K4, Strom S4, Sun Q5, Arning E6, Bottiglieri T6, Jansen EEW7, Jakobs C7, Gibson KM8 1 Molec Genet Biochem, Univ of Pittsburgh, Pittsburgh, USA, 2 Anesthesiology, Univ of Pittsburgh, Pittsburgh, USA, 3Biomed Res Tech, Wexford, USA, 4Pathology, Univ of Pittsburgh, Pittsburgh, USA, 5 Baylor Coll Med, Houston, USA, 6Inst Metab Dis, Baylor Univ Med Center, Dallas, USA, 7Metab Unit, Dept Clin Chem, VU Med Center, Amsterdam, Netherlands, 8Div Med Genet, Dept Peds, Pittsburgh, USA MSUD is a disorder of branched chain amino acid (BCAA; leucine, isoleucine, valine) catabolism. To explore preclinical treatment paradigms, a viable transgenic murine model of iMSUD (*6% residual branched-chain ketoacid dehydrogenase activity) was developed. We examined amino acid, neurotransmitter and monamine levels in blood and brain, and the e¡ect of HTx on these species. Animals were wild-type (WT; n = 5, age 35 days), iMSUD (n = 6, age 18) and iMSUD-HTx (n = 5, age 35) mice. Metabolites were chromatographically quanti¢ed and hepatocytes administered via transabdominal injection (K Skvorak, SSIEM 2008). In blood, BCAAs, alloisoleucine and glycine were increased whereas glutamine was decreased (p50.05). Total brain extracts revealed extensive alterations, including increased BCAAs, alloisoleucine, taurine, phosphoethanolamine, and ornithine with decreased glutamate, glutamine, aspartate, serine, and alanine (p50.05). A parallel decrease in GABA (WT, 1942+85 (SEM) nmol/g tissue; iMSUD, 1531+100; p50.02) was detected. Monoamine analysis revealed dopamine (DA; WT, 6.92+0.30 nmol/g tissue; iMSUD, 4.30+0.24) and serotonin (5hydroxytryptamine (5-HT); WT, 4.92+0.12; iMSUD, 3.20+0.26) depletion, both of which signi¢cantly improved with HTx. Systemic BCAA/alloisoleucine elevations suggest that iMSUD mice represent a relevant phenocopy of human MSUD, and cumulative amino acid data suggest global disruption of brain bioenergetics. Neurotransmitter alterations (Glu, Gln, Asp, GABA, DA, 5-HT) in brain infer comparable imbalances in the human disorder. Improvements in neural DA and 5-HT de¢ciencies via HTx provide impetus for further examination of this treatment approach. Supported by NIH DK57386 and the MSUD Family Support Group

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EFFECT OF PATIENT-DERIVED MUTATIONS ON 3PHOSPHOGLYCERATE DEHYDROGENASE MULTIMERIZATION AND FUNCTION Tabatabaie L1, van den Broek NJF1, Brenkman AB1, Verhoeven-Duif NM1, Berger R1, de Koning TJ2, Klomp LW1 1 Dept Metab Endoc Dis and Metabolom UMC, Utrecht, Netherlands, 2 Dept Metab Dis, Univ Med Centre, Utrecht, Netherlands Background: 3-Phosphoglycerate dehydrogenase (3-PGDH) plays a crucial role in the biosynthesis of L-serine. 3-PGDH de¢ciency is an autosomal recessive disorder characterized by congenital microcephaly, psychomotor retardation, intractable seizures and low concentrations of L-serine and glycine in CSF. Seven distinct disease-causing mutations have been detected in the human 3-PGDH gene; six missense mutations and one frame-shift mutation. The missense mutations result in stable proteins that display a signi¢cant reduction in maximal enzyme activities. Here we investigated the composition of human 3-PGDH and the e¡ect of mutations on its biochemical properties. Methods and Results: GST pull-down assays in transfected HEK293T cells indicated that 3-PGDH interacts with itself. Subsequent crosslinking experiments and Q-TOF mass spectrometry analysis of WT-3PGDH-Flag established that it forms homo-tetramers. Mutant 3PGDH-Flag retains the ability to form homo-tetramers. GST pulldown assays con¢rmed that mutant 3-PGDH proteins could interact with both WT and mutant 3-PGDH. These results suggested that inactive mutant 3-PGDH inhibits the function of WT-3-PGDH in a dominant negative fashion. To test this hypothesis, mutant and WT 3PGDH were simultaneously overexpressed. Our initial ¢ndings suggest that additional expression of mutant 3-PGDH did not appreciably a¡ect the enzyme activity of WT-3-PGDH. Conclusion: Human 3-PGDH forms homo-tetramers and this process is not a¡ected by the disease-causing missense mutations. The presence of one or multiple mutant isoenzymes in a 3-PGDH tetramer does not preclude enzymatic activity, which explains the normal enzyme activity observed in obligate heterozygous parents.

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FUNCTIONAL STUDIES OF A DUPLICATION MUTATION OF VALINE AT 235 BY A TRANSIENT EXPRESSION ASSAY IN A BOY WITH MCT8 DEFICIENCY Itoh M, Kakinuma H Dept Pediatr, Kanazawa Medical Univ, Uchinada-Machi, Kahoku-Gun, Ishikawa-Ken, Japan Objective: The objective of this study was to determine the functional consequences of MCT8 gene mutation (235dupVal) detected in a boy with monocarboxylate transporter 8 (MCT8) de¢ciency. Materials and Methods: His parents and two brothers, and 101 healthy individuals were investigated for the mutation. The mutation was transiently expressed in CHO-K1 cells or FTC-238 cells in order to analyze T3 uptake or subcellular localization of the MCT8 protein, respectively. Results: The mutation was not detected both in either the family members or healthy individuals, suggesting that the patient has a de novo mutation. The uptake of triiodothyronine (T3) in CHO-K1cells harboring the vectors of the mutation of MCT8 cDNA was lower by half than that in the cells harboring the vectors of the wild type of MCT8 cDNA. In a subcellular localization study, FTC238 cells expressing both the wild-type and mutated MCT8 protein showed strong cell surface expression in contrast to the cells expressing no MCT8 protein. Conclusion: The roles of the MCT8 protein and thyroid hormones remain to be elucidated in the developing fetal brain. The boy showed signi¢cant neurological symptoms despite residual transport activity of T3. If insu¤ciency of thyroid hormones in the fetal neurons causes these developmental impairments, not only the timing and regions of the fetal brain expressing the MCT8 protein, but also the levels of thyroid hormones in the same timing and regions need to be clari¢ed.

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MUTATIONS IN UROCANASE GENE IN A PATIENT WITH UROCANIC ACIDURIA, MENTAL RETARDATION AND INTERMITTENT ATAXIA Artuch R1, Espinos C2, Ormazabal A1, V|laseca MA1, Spaapen LJM3, Martinez-Rubio D2, Palau F2, Pineda M1 1 Hospital Sant Joan de Deèu-CIBERER, Esplugues, Barcelona, Spain, 2 Instituto de Biomedicina-CIBERER, Valencia, Spain, 3University Hospital Maastricht, Maastricht, Netherlands Background: Urocanase is an enzyme in the histidine pathway encoded by the UROC1 gene. Our aim was to report the ¢rst pathogenic mutations in the coding region of the UROC1 gene in a girl who su¡ers from urocanic aciduria presenting with mental retardation and intermittent ataxia. Methods: Urocanic acid in urine was analyzed by HPLC with ultraviolet and diode array detection, pterins and folate by HPLC with £uoresecence detection and neurotransmitters with electrochemical detection. Mutation analysis of the UROC1 gene was performed by direct sequencing of PCR products using primers designed according to the reference sequence NM__144639 that amplify their twenty exons and intronic £anking sequences in an ABI Prism 3130xl autoanalyser. Conservation of residues was analyzed by alignment of related sequences using the program BLAST. Secondary structure predictions were calculated with four di¡erent software tools (GOR, Jpred3, APSSP, and nnPredict). Results: The patient presented p.L70P and p.R450C mutations in the coding region of the UROC1 gene. In silico studies suggest that p.L70P would imply the disruption of an a-helix in N-terminus and that p. R450C would render impossible any interaction between urocanase and its substrate. Patient presented a slight cerebral folate de¢ciency (36 nmol/L; reference values: 42^81) associated with partial de¢ciency of biopterin (10 nmol/L: reference values: 14^36) and homovanillic acid (90 nmol/L: reference values: 124^362). Conclusions: Our data suggest that both mutations could alter the correct activity of urocanase, which would explain the clinical and biochemical ¢ndings described in the patient.

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DIHYDROLIPOAMIDE ACETYLTRANSFERASE DEFICIENCY IN CASES OF ATYPICAL PANTOTHENATE KINASE ASSOCIATED NEURODEGENERATION McW|lliam CA1, Brown RM2, McW|lliam RC1, Ridout CK2, Tolmie J1, Brown GK2 1 Ferguson Smith Centre Clinical Genetics, Glasgow, UK, 2Genetics Unit, Dept Biochem, Univ Oxford, Oxford, UK Background: The clinical signs of acquired episodic dystonia associated with distinctive neuroradiological ¢ndings suggest a diagnosis of pantothenate kinase associated neurodegeneration (PKAN). However, dihydrolipoamide acetyltransferase (DLAT) de¢ciency [1], for which treatment may be bene¢cial, has similar clinical and neurological signs. We describe two sisters with childhood-onset episodic dystonia and pyruvate dehydrogenase de¢ciency caused by defects within the E2 subunit. Both patients had neuroradiological features reminiscent of PKAN, but were found to be compound heterozygotes for mutations in the DLAT gene. Results: Pyruvate dehydrogenase activity in cultured ¢broblasts was 0.49 nmol/mg protein/min (NR 0.7^1.1). Sequencing of the DLAT gene identi¢ed a T4G substitution in exon 3, changing a glutamic acid to a stop codon. A second intronic change was also identi¢ed which is thought to disrupt splicing. The a¡ected younger sister is also a compound heterozygote for the above two mutations, the mother is heterozygous for the exon 3 mutation and father is heterozygous for the intronic mutation. Functional studies are being undertaken at the time of writing. Conclusion: DLAT de¢ciency is a rare and possibly underdiagnosed cause of `atypical PKAN'. The two major clinical clues are dystonia with globus pallidus hyperintensity on MRI. This case suggests that consideration should be given to the diagnosis of DLAT de¢ciency in cases of atypical PKAN and describes the disorder in the heterozygous form. [1] Head RA, Brown RM, Zolkipli Z, Shahdadpuri R, King M, Clayton PT, Brown GK (2005). Clinical and genetic spectrum of pyruvate dehdrogenase de¢ciency: dihydrolipoamide (E2) de¢ciency. Annal Neurol 58: 234^41.

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GROWTH DEVELOPMENT OF CHILDREN WITH CLASSICAL PKU DURING THEIR FIRST YEAR OF LIFE Gibson C, Halldin Stenlid M, Eklund C Univ Child Hosp, Div Endocrinology, Uppsala, Sweden Objectives/Methods: Infants with PKU are given a highly controlled diet during their ¢rst year of life; dietary composition and energy intake has a major in£uence on growth. The main purpose of this study was to investigate if 22 children with classical PKU treated at the Uppsala University Children's hospital had had a more rapid growth in height and/or weight during their ¢rst year of life compared to the Swedish national growth charts. Additionally, it was aimed at correlating growth rate with blood phenylalanine concentrations. The study was a retrospective cohort study of the selected children's journals. Results: Signi¢cant di¡erences between the mean weight curve on the growth charts and the mean weight of the children in the study were shown at all time periods with the exception of the six months measurement. In contrast, the mean height was signi¢cantly di¡erent from the normal growth chart only at the three months measurement. Furthermore, there was a signi¢cant correlation between higher blood phenylalanine concentration and a more rapid weight gain. Conclusion: Swedish infants with PKU show marginal di¡erences in growth development during the ¢rst year of life as compared to the mean curve on the national growth charts. Thus, there is no reason to revise the recommendations concerning protein and energy intake, for growth reasons. Instead, since the identi¢ed accelerated weight gain correlated with increased phenylalanine levels, it shows the importance of following the recommendations for blood phenylalanine concentrations in order to optimize growth.

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TREATMENT WITH KETOGENIC DIET IN THE PYRUVATE DEHYDROGENASE DEFICIENCY Oliveros L1, Garcia MT2, Moreno JM1, Martin E2, Garcia O2, Simoèn R3, Briones P4 1 Clin Nutr Unit, Hosp Univ 12 Octu, Madrid, Spain, 2Inb Err of Metab Unit, Hosp Univ 12 Octu, Madrid, Spain, 3Neuroped Unit, Hosp Univ 12 Octu, Madrid, Spain, 4Instituto Bioquimica Clinica, Barcelona, Spain Introduction: A defect in the complex pyruvate dehydrogenase (PDH) caused a blockage in the path of aerobic oxidation of glucose, the main energy source for cells of the CNS. Treatment with ketogenic diet (KD) in PDH de¢ciency provides an alternative energy substrate to the blocked metabolic pathway, which in some studies has been correlated with increased longevity and better mental development. Objective: Presenting the clinical course of three pediatric patients su¡ering from PDH de¢ciency after introduction of treatment with KD. Methods: After diagnostic con¢rmation in skin biopsy, was established treatment with KD in a ratio fat/protein+carbohydrate, 4:1. Results: In the 3 patients there was a gradual normalization of the analytical parameters as shown in the tables below. Two patients, who presented seizures treated with antiepileptic drugs, improved after ketogenic diet was started. In all cases achieved improvement of hypotonia and better links with the medium.

Patient 1: Lactate (mmol/L) Pyruvate (mmol/L) B-hydroxybutyrate (mmol/L) Patient 2: Lactate (mmol/L) Pyruvate (mmol/L) B-hydroxybutyrate (mmol/L) Patient 3: Lactate (mmol/L) Pyruvate (mmol/L) B-hydroxybutyrate (mmol/L)

Diagnosis

Few days after initiation KD

Last review

7.91 0.598 0.065

1.84 0.097 0.480

1.52 0.097 0.330

2.00 0.190 0.070

1.57 0.112 0.953

6.53 0.591 0.002

1.95 0.146 3.104

2.06 0.113 1.948

Conclusion: Although there is no treatment to correct the PDH de¢ciency, KD seems to have a positive role avoiding hyperlactataemia, seizures, thereby improving the clinical condition and quality of life of these patients.

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MALNUTRITION AS AN ENTRY POINT FOR APPLICATION OF INBORN ERRORS OF METABOLISM WORK-UP INTO HEALTH SERVICES IN THE DEVELOPING COUNTRIES Rusli Sjarif D Dept of Child Health, Univ of Indonesia, Jakarta, Indonesia Malnutrition is still a major public health problem in Indonesia, approximately 25 to 30% of children have mild or moderate malnutrition and 1^3% are severe. The mortality rate from severe malnutrition without hospitalization is about 50%, but with hospital treatment it can be reduced by 10^30% depending on the severity of the case and quality of the hospital facilities. Although all of them were treated nutritionally according to WHO Guidance 1999, unfortunately several of them do not responded adequately. Since malnutrition is one of the clinical ¢ndings suggesting metabolic disorders, laboratory study was performed to look for the possibility of IEM. Twelve among 27 children with severe malnutrition who were relatively unresponsive to the intervention (weight gain 55 g/kg BW/day) recruited. Blood gases analysis, electrolytes, ureum and creatinine were measured, unfortunately lactate could not examined. Sixth of them showed recurrent acidosis metabolic with increased anion gap. Sodium bicarbonate was administrated four hourly to correct acidosis metabolic. Four children who had good compliances showed increased in appetite, energy intake and weight gain (renal failure was excluded, because of lack of facilitation other causes of increased anion gap have not been examined). Based on clinical appearances all of these four patients possible had mitochondrial diseases. Conclusion: malnutrition which is nonresponding to standard nutritional intervention could be used as an entry for application IEM work-up. into health service in the developing countries.

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CESSATION OF PROTEIN SUBSTITUTE IN TYROSINAEMIA TYPE III Daly A, Macdonald A, Hopkins V, Chakrapani A, Hendriksz C, V|jay S Birmingham Childrens Hospital, Birmingham, UK There is no consensus about the long term bene¢t of a low tyrosine (tyr) diet in tyrosinaemia III. In our centre, it is practice to start a low tyr diet if plasma tyr is 5600 mmol/L. Two Asian girls, one who demonstrated clinical improvement with diet, have required minimal dietary treatment (Subject 1: took protein substitute [PS] without tyr and phenylalanine (phen) only [1 g/kg/day]; subject 2: required 1.5 g/kg/day natural protein with 1 g/kg/day PS) to achieve plasma tyr less than 200 mmol/ L. In latter years, PS dose was not increased in either subject. Subject 2 relaxed natural protein intake without loss of tyr control. Dietary treatment was stopped in both girls. Aim: To evaluate diet cessation in tyrosinaemia III patients. Methods: Subject 1 diagnosed 16 months (tyr 1820 mmol; phen 85 mmol) stopped PS at 13 years. Subject 2 diagnosed 18 months (tyr 1400 mmol and phen 100 mmol) stopped diet and PS at 13 years. Twice daily blood samples (fasting early a.m. and p.m.) were collected over 3 weeks and at monthly intervals post diet cessation. Results: Plasma blood levels for tyr and phen remain well controlled o¡ dietary treatment for 6 months in both subjects. Median fasting morning and evening tyr concentrations remained below 230 mmol/L (morning: subject 1: 180 [130-280]; subject 2: 120 [100^170]; evening; subject 1: 230 [150^320]; subject 2 160 [110^170]). Conclusion: Dietary treatment for some patients with tyrosinaemia III may only be required temporarily to achieve plasma tyr levels closer to normal reference levels.

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Low protein diets (with essential amino acids replacing some natural protein) are di¤cult to establish in Gyrate Atrophy (ornithine aminotransferase OMIM 258870 or GA in short). Dietary treatment (DT) is started after diagnosis, usually in the ¢rst or second decade of life. There are no dietary products speci¢cally for GA. Aim: To evaluate a new pre-measured essential amino acid mix (5 g protein equivalent sachets: EAA: V|ta£o), with vitamins and minerals in a 9 year old girl of Pakistani origin with GA. Method: Diagnosis followed a 4 year history of visual problems. A low protein diet (1 g/kg/day: 25 g protein/day); with 10 g natural protein and 15 g protein equivalent from EAA was started. DT was monitored for 6 months; with 2 weekly assessment of quantitative plasma amino acids and growth, with nutritional biochemistry pre-diet and 6 month DT. Results: Plasma ornithine decreased from 896 to 79 mmol/L by week 8 of DT and has remained mainly 5350 mmol/L. Plasma ammonia was normal with DT. Essential amino acids were at the lower end of reference range at baseline and throughout the ¢rst 6m of DT. Nutritional biochemistry was normal with the exception of plasma zinc (low at baseline and at 6 month DT). On DT, height and weight slightly deteriorated (z score height baseline: 0.5; 6 month DT: 0.34; weight baseline: ^0.76; 6 month DT: ^1.03). Compliance with EAA supplement and diet was acceptable but tenuous. Conclusions: Successful DT of GA is possible, but requires meticulous monitoring and follow-up. The new EAA supplement aided compliance and dietary independence.

Protein substitute (PS) is essential in the dietary management of tyrosinaemia (HT) and homocystinuria (HCU). There are few, suitable PS for older patients and poor compliance is common. Two new, (Tyr Cooler for HT; HCU Cooler for HCU [V|ta£o]) low carbohydrate, ready to drink (RTD) PS's (protein equivalent: 15 g/130 ml) are available for aged 8 years plus. There only di¡erence is amino acid pro¢le; Tyr Cooler is without tyrosine and phenylalanine; HCU Cooler without methionine. Aim: To evaluate acceptability and e¤cacy of both liquid PS in older patients with HT and HCU. Methods: 7 subjects (median age 12 years, range 9^16 years) (HT I: n = 3, HT II: n = 1, HT III: n = 3) and 6 subjects (median age 14 years; range 8^34 years) with HCU were recruited. 1 subject with HT and 3 with HCU refused PS pre-trial. Compliant subjects took their usual PS for 1 week, followed by 1 week of liquid PS. Plasma amino acids were estimated and subjects completed an acceptability questionnaire on day 7 of each week. Results: Median protein equivalent intake from the liquid PS was 60 g (range 45^60 g) Only one subject with HCU refused the liquid PS. Compliant patients pre-trial (HT = 6; HCU = 2) continued to have acceptable plasma amino acid control. 4 non-compliant subjects pre trial started taking the liquid PS and one subject with HT had a 9-fold decrease in plasma tyrosine levels. All found the RTD easier and discreet to take. Conclusions: This RTD PS was e¤cacious, and improved dietary compliance in patients with HT and HCU.

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THE USE OF A NEW ESSENTIAL AMINO MIX IN GYRATE ATROPHY MacDonald A, Daly A, Hopkins V, Preece MA, Hendriksz C, V|jay S, Chakrapani A Birmingham Childrens Hospital, Birmingham, UK

DIETARY MANAGEMENT OF PKU: THE EXPERIENCE OF CGM Almeida MF, Rocha JC, Carmona C, Lima MR Cent Gen Med Jac Mag ^ INSA, Porto, Portugal Neonatal screening for PKU in Portugal began in 1979 and was coordinated by IGM. The Guthrie microbiological method was used between 1979 and 1990. Then it was replaced by a quantitative enzymatic method. The program presently covers almost 99.8% of the Portuguese newborns. Until 2007, 2 797 913 newborns have been screened, and 257 PKU patients were diagnosed, with a calculated incidence of 1:10 876. Actually, we follow-up 145 of 257 screened patients. The cut-o¡ blood phenylalanine concentration value is 3 mg/ dl. We implement treatment for all the newborns presenting a value greater than that. The newborns with values between 3 and 6 mg/dl, stay without treatment and are maintain under periodical control. Patients with phenylalanine concentration in blood higher than 6 mg/dl start dietary treatment. A wide variation in the levels of hyperphenylalaninaemia is found in neonates so the diet should be carefully individualized. It is important maintain as longer as possible the maternal milk, or an infant formula, as a source of natural protein. For the satisfation of the total energy and nutritional needs, we provide measured portions of low-phenylalanine containing foods using exchange lists and a milk substitute without phenylalanine content. The compliance of the treatment is monitored by the blood phenylalanine concentrations, which are done with a variable frequency depending on the age of the patient. The authors will present the follow-up of these patients. These data were very helpful on the elaboration of the Portuguese consensus for the nutritional treatment of PKU.

THE USE OF READY TO DRINK LIQUID PROTEIN SUBSTITUTE IN NON-PKU AMINO ACID DISORDERS Daly A, MacDonald A, Hopkins V, Chakrapani A, Hendriksz C, V|jay S Birmingham Childrens Hospital, UK, Birmingham, UK

NUTRITIONAL MANAGEMENT OF PKU IN SAUDI ARABIA Aljammaz SA, Clayton PT, Hesketh TM Institute of Child Health, UCL, London, UK Background: Inborn errors of metabolism are common in Saudi Arabia (incidence estimated at 5 times that in the US). Adherence to a special diet is essential to prevent developmental disability in phenylketonuria (PKU). Our aim is to identify risk factors for poor outcome in PKU patients at King Faisal Specialist Hospital and Research Center in Saudi Arabia and to improve nutritional management. Methods: Qualitative study: Assessed nutritional knowledge, attitudes and practices through interviews (n = 5) and focus groups (2) with health care providers; interviews with patients (6); and families (17). Quantitative study: Took anthropometric measurements, dietary intake, phenylalanine blood levels, developmental assessments, and questionnaires with 40 PKU patients and their families. Results: Major qualitative themes: Lack of su¤cient services; inadequate dietary knowledge; limited resources; social and emotional attitudes towards diet and compliance. Quantitative initial results: Factors contributing to poor outcome (IQ 580) include: (1) Delayed diagnoses. Mean IQ's (+1 SD) according to age at diagnosis were: diagnosed 51 month: IQ 90 (+12.3); diagnosed 1 month^1 year: IQ 74 (+21.8); diagnosed 41 year: IQ 55 (+28.5). Signi¢cantly di¡erent (p50.005). (2) Inadequate dietary phenylalanine control. Patients with mean phenylalanine above 480 mmol/L were 3.8 times more likely to have developmental disability. Conclusion: In Saudi Arabia targeted nutrition management programs need to be developed to capitalize on the expanding neonatal screening program. Education and metabolic care services need major improvements to be able to deliver optimal care for patients and their families.

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LONG TERM COMPLIANCE WITH A NOVEL VITAMIN AND MINERAL SUPPLEMENT IN OLDER PEOPLE WITH PKU MacDonald A1, Lee P2, Davies P1, Daly A1, Hopkins V1, Lilburn M2, Hendriksz C1, Preece MA1, Chakrapani A1 1 Birmingham Childrens Hospital, Birmingham, UK, 2National Hospital for Neurology and Neurosurgery, London, UK Long term e¤cacy of vitamin and mineral preparations in adult patients with phenylketonuria (PKU) is unreported. Aim: in an open, intervention trial, the acceptability, safety and impact on biochemical and haematological micronutrient status of a new vitamin and mineral tablet (Phlexy V|ts: SHS International) was investigated. Methods: 15 subjects with PKU (median age 21 years: range 8^33 years) on low phenylalanine diet from two PKU centres were recruited. No vitamins or minerals were added to their protein substitute and for 12 months they took their daily requirement of vitamin and minerals from Phlexy V|ts (5 tablets/daily). All but two subjects took vitamin and mineral supplements pre-trial. Fasting bloods were taken at baseline (week ^2 and at week 0), 4 and 12 months for a range of biochemical and nutritional measurements. Results: By 4 months, serum vitamin B12 (p = 0.003); serum manganese (p = 0.03); and plasma (p = 0.03) and red blood cell (p = 0.004) glutathionine peroxidase all signi¢cantly increased, but remained within normal reference ranges. By 12 months, serum vitamin B12 (p50.05); and plasma glutathionine peroxidase (p50.05) remained increased. The Phlexy V|ts tablets scored better than conventional vitamin and mineral supplements for overall acceptability (p50.05), and ease of swallowing (p = 0.1) at 4 months, although swallowing score deteriorated by 12 months (p50.05). There was a small but signi¢cant deterioration in compliance with taking the vitamin and mineral supplements between 4 and 12 months (p50.05). Conclusion: These comprehensive vitamin and mineral tablets appeared acceptable, and improved biochemical nutritional status although there were long-term compliance and swallowing issues.

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A NEW NOVEL PROTEIN SUBSTITUTE FOR YOUNG CHILDREN WITH GA1 MacDonald A, Daly A, Hopkins V, Hendriksz C, V|jay S, Chakrapani A Birmingham Childrens Hospital, Birmingham, UK Lysine-free protein substitutes (PS) have an important role in limiting brain lysine uptake in young children with glutaric aciduria type 1 (OMIM 231670, GA1), particularly during illness. Choice of PS is limited. A new PS (42 g/100 g protein equivalent), free of lysine, low in tryptophan (0.27 g/100 g), with vitamins and minerals has been developed (GA Gel; V|ta£o) for children aged 6 months to 10 years. Aim: To investigate the e¤cacy of GA Gel in young children. Methods: Three children (S1, S2, S3) with GA1 (now aged 8 months; 16 months and 3 years; all pre-encephalopathic crisis but one with multiple dystonic movements without history of decompensation) were given GA Gel. Two were white Caucasian; one Asian origin. S1 and S2 started PS at diagnosis (at 2 days; 4 months), S3 at 2 years. 1.5 g/kg/day PS was given less than 1 year of age; and 1^1.3 g/kg/day post 1 year. Natural protein was restricted to 1 g/kg/day from 1 year. Results: GA Gel was introduced pre-meals as a paste in S1 and S2 at 6 months of age; and at 2 years in S3. All three children took PS orally; and it was tolerated throughout illness. V|tamin and mineral intake achieved requirements. Growth was satisfactory in all 3 children (current height z score for subject (S) S1: +1.4; and S2: +2.05; weight z score S1: +1; S2: ^0.5; and S3: +0.12). Nutritional biochemistry and quantitative plasma amino acids were normal. Conclusions: GA Gel was tolerated and accepted by children with GA1. Further amino acid pro¢le modi¢cation may minimise brain lysine uptake.

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READY TO USE PROTEIN SUBSTITUTE FOR CHILDREN WITH PKU MacDonald A1, Ferguson C2, Daly A1, Hopkins V1, Hall SK1, Rylance G, Hendriksz C1, Chakrapani A1 1 Newcastle General Hospital, Newcastle, UK, 2Royal V|ctoria In¢rmary, Newcastle, UK

GLUCOSE CONTINUOUS MONITORING IN HEPATIC GLYCOGEN STORAGE DISEASES ^ WHAT DIETECTIC BENEFITS Patr|ècio Z1, F|gueiredo C2 1 Hosp St Maria -Serv Dietetica e Nutric°a¬o, Lisboa, Portugal, 2Uni D Metab, Serv Pediatria H St Maria, Lisboa, Portugal

In children with PKU, high carbohydrate, powdered protein substitutes (PS) require preparation, hinder independence and may inhibit appetite. Ready to drink (RTD) may overcome these di¤culties. Aim: A 5 week, 3-part randomized controlled trial to evaluate a low carbohydrate, RTD PS in children aged 3^10 years with PKU. Methods: 14 subjects (12 boys), aged 3^9 years (median 6 years) were recruited from 2 PKU centres. In part 1 (one week), subjects took their usual higher carbohydrate powdered PS (PPS). In parts 2 and 3, subjects were randomized to either a RTD PS or their usual PPS for 2 weeks each. During the last 3 days of each study part, daily blood phenylalanine's (phe) were estimated and children were weighed on the ¢nal day of each study part. Each pouch of RTD PS (Cooler 10: V|ta£o) contained 10 g protein equivalent and 5 g carbohydrate. Results: The RTD resulted in increased independence when taking PS (RTD: 100% subjects drank themselves: PPS: 50% (n = 7) were spoon fed by their parents). Almost all (n = 10) rated the PPS as inconvenient compared with none in the RTD group. Median blood phe did not deteriorate with the lower carbohydrate RTD PS (median blood phe: RTD 220 mmol/L (range 81^717); PPS 288 mmol/L (range 157^573)). Weight remained static on both PS's (median change: RTH 0 kg (range ^1 to 0.7); PPS 0 kg (range ^0.6 to 1)). Conclusions: The RTD led to increased independence without deterioration of blood phe control in children with PKU. It was particularly popular in boys.

Introduction: Glycogen storage diseases (GSDs) are a wide group of diseases characterized by normal or abnormal glycogen accumulation in cells of di¡erent tissues. Usually hepatic GSDs are associated with hypoglycaemia, being the risk particularly high in type I. Glucose continuous monitoring is a new technique that seems important in this disease. Dietary therapies have been devised to use the available alternative metabolic pathways to compensate for disturbed glycogenolysis in GSD I. Objectives: The aim of the study was to determine the feasibility of glucose continuous monitoring in GSD type I patients and the dietetic corrections. Methods: Two children with GSD Ia were studied with two measurements, one after and another before the implementation of diet corrections. Results: The results showed that levels of glucose detected by the sensor helped to observe several episodes of hypoglycaemia, allowing doing a dietetic correction more proper, normalizing the glucose values. Conclusion: This technique minimally invasive and without signi¢cant risks, seems useful for management for dietary therapies and education of GSD Ia patients.

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IMPORTANCE OF INFORMATION SPREAD ON INHERITED METABOLIC DISEASES FOR DIETICIANS Oliveira RB, Vertemati T, Araujo S, Satiro CAF, Souza C, Leite E, Raskin S, Pimentel H, Giugliani R, Valadares E, Santana L, Martins AM, Frangipani BJ, Micheletti C Insituto Canguru, Sa¬o Paulo, Brazil Background: `Instituto Canguru' is a Brazilian NGO, whose mission is disseminating knowledge related to Inherited Metabolic Diseases (IMD) among health professionals and society in general, making easier diagnosis and appropriate treatment. IMD are individually rare and little known by dieticians, emphasizing the importance of dietetic intervention to avoid severe consequences of these diseases. Objectives: Identify doubts presented by dieticians who contacted `Instituto Canguru'. Methods: To accomplish its mission, `Instituto Canguru' has been developing its activities highlighting the attendance to health care professionals through a call free and email services. At ¢rst contact, professionals are registered and directed to the technical team (geneticists and dieticians). Data were obtained in our own databases from May 2005 to April 2008. Results: During the evaluation time 422 dieticians were registered: 51% of then were accompanying diagnosed patients in their respective services. Among then, 90% got in touch due unknowing on dietetic conduct to be implemented and 10% were searching for information about metabolic formulas and laboratory analysis. 49% didn't refer being accompanying IMD patients. 39% asked for information and bibliographic references and 61% were registered in congresses and other events. Conclusions: The results demonstrate an elevated lack of knowledge on IMD dietetic conducts, however, was observed interest of dieticians on information and appropriate conduct on dietetic intervention. Thus, we believe in the importance of organizations in dissemination of IMD knowledge, since precocious and appropriate introduction of convenient diet can avoid serious sequel in patients.

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THREE OR FOUR PROTEIN SUBSTITUTES PER DAY IN PRECONCEPTION AND MATERNAL PHENYLKETONURIA (PKU)? Maritz CM, McWhinnie C, Lilburn M, A Cole, Lee PJ Nat Hosp for Neurology and Neurosurgery, London, UK Background: It has been well documented that recommended phenylalanine (phe) control during pregnancies of PKU women can prevent fetal abnormalities including congenital heart disease, mental retardation, microcephaly and low birth weight. We recently demonstrated £uctuations of phe, even within target ranges, has a negative impact on o¡spring outcome (Maillot et al. 2008). A study in children (MacDonald et al. 2006) has suggested even distribution of protein substitute throughout 24 h may produce more stable phe control and contribute to minimising phe £uctuations. No published adult study has assessed this. Methods: We assessed whether frequency of protein substitute in£uenced phe £uctuation over a 24 h period in adult pre-conception or pregnant patients. By retrospective analysis 20 patients (aged 20^41 years) were classi¢ed according to 3 (n = 14) or 4 protein substitutes/day (n = 6). Patients collected ¢ve ¢nger-prick blood spots over a 24 h period, taken before meals and at bedtime. Results: All patients were on diet with phe 5500 mmol/L. Four protein substitutes/day resulted in a consistently higher, although not statistically signi¢cant, mean compared to 3/day (245.47 and 187.05 respectively). A smaller £uctuation was observed in those that took 4 substitutes per day compared to 3 per day (48 and 55.9 respectively). Conclusion: This small study has not shown any di¡erences in stability of phe when taking 3 or 4 protein substitutes/day, contrary to the one previous report. Indeed taking protein substitute more frequently was associated with higher average phe concentrations. A larger, more controlled study would be useful to examine these issues further.

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ASSESSMENT OF BODY COMPOSITION OF GSD TYPE I PATIENTS Faria A1, Mota A2, Silva S1, Garcia P1, Mota-Castelo T1, Diogo L1 1 Unidade Doenc°as Metab, CDC, Hosp Ped, Coimbra, Portugal, 2 Nutrition Unit, Hosp Ped, Coimbra, Portugal Background: Glycogen storage disease type I (GSD I) is an inherited metabolic disorder, caused by a defect in glucose-6-phosphatase, leading to short fast hypoglycaemia. Due to particular food patterns and associated morbidity of GSD I, these patients are more susceptible to malnutrition status. The aim of this study was to assess body composition (total body water, fat and free fat body mass), basal metabolism and body mass index (BMI) in GSD I children followed as outpatients. Methods: F|ve patients (one female), aged 2 to 14 years (median 10 years) with GSD Ia (n = 3) and GSD Ib (n = 2) were studied. Control group included 13 healthy subjects, matched for age. Body composition was assessed using a tetrapolar bioelectrical impedance analyser Akern. Mann-Whitney test was used for statistical analysis. Results: Comparing with controls, our patients had statistically signi¢cant higher percentage of body fat and BMI. Patients less than 10 years-old showed lower total body water (p50.05). There was no signi¢cant di¡erence between GSD Ia and GSD Ib patients in all parameters. All patients presented low total body water and a medium basal metabolism of 36.6 kcal/kg. Conclusions: GSD I patients are submitted to frequent meals with high carbohydrate content, leading to fat accumulation and higher BMI values than healthy individuals. When managing GSD I, attention should be paid not only to glucose requirements but also to nutritional status. Bioimpedance is a useful, non invasive method for assessing body composition of GSD I patients in outpatient clinics.

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DIETARY INTERVENTION IN ADULTS WITH LATE DIAGNOSED PHENYLKETONURIA ^ WEST OF SCOTLAND EXPERIENCE Adam S, Cochrane B, Galloway P Met Serv, Glasgow Royal Inf, RHSC, Glasgow, UK There is increasing anecdotal evidence that implementing dietary treatment for phenylketonuria in late diagnosed adults is improving their health and quality of life. Since 2002 we have managed 14 late diagnosed and previously untreated adult patients with phenylketonuria in the West of Scotland. Patients were referred by a variety of health care professionals including Clinical Psychology, General Practioners and Community Dietitians. Dietary intervention was recommended to help improve: behavioural di¤culties including aggression and agitation; muscle pain, tingling in limbs and di¤culty walking. To support the implementation and continuation of the diet home visits are a mainstay of successful management. In addition, to aid acceptance of the low protein food products and encourage varied diets, frequent educational cookery workshops are held for patients and carers. Carers report that they ¢nd the sessions very bene¢cial and are useful for sharing ideas with other carers. 10 patients remain on the diet. Some of the reported bene¢ts which prompted the carers and family to continue with the diet include: reductions in anti-psychotic medications; improved socialisation, mobility and concentration; markedly less anxiety and agitation. Di¤culties in managing these adults included various types of care and settings, di¡ering views on the e¡ectiveness of the diet and the commitment of the carers. However, some carers have reported improved job satisfaction with seeing a bene¢t to the patient which may lead to a lower rate of sta¡ turnover.

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The ketogenic diet (KD) is a normocaloric, high-fat, low carbohydrate diet stimulating the generation of ketonbodies which are used as alternative fuel for the brain. The KD has been used for decades to treat children with drug-resistant epilepsy. Now KD is also an accepted treatment of diseases of brain energy metabolism: GLUT1 de¢ciency syndrome (GLUT-1) and pyruvate dehydrogenase de¢ciency (PDHC). Children with mitochondrial disease (Complex 1 de¢ciency) can also bene¢t from KD. The principles and use of KD doesn't really di¡er depending on the indication of use. In the Netherlands, every year, 45^ 50 patients with intractable epilepsy and/or metabolic disease start with KD and are monitored by a team of caregivers. In cooperation with the Dutch Multidisciplinary Ketogenic Diet Group, the evidence-based dietary guideline Ketogenic Diet was published in 2007. The Guideline contains theoretical and practical information for caregivers about both the classical- and MCT-version of KD. The amount of prospective scienti¢c studies of KD and epilepsy is international growing but the experience and research of its use in metabolic disease is still scarce. In 2005 the Dutch national database KD was realized and data of 90 patients are registered and currently analyzed. Thus, all conditions are met for future scienti¢c research. The authors want to share experiences with colleagues about the pitfalls during the process of writing, implementation and use of the guideline, the creation and use of the nationwide database and discuss the experience of the treatment with KD in patients with metabolic diseases.

Introduction: Malonyl CoA decarboxylase (EC 4.1.1.9, MCD) de¢ciency is a rare inborn error of metabolism causing developmental delay, seizures, cardiomyopathy and acidosis. MCD is important in both lipid and carbohydrate function, although its precise role is unclear. Most children have been treated with a low fat/high carbohydrate diet but there is little evidence to support such a diet. This case describes dietary intervention with a precise low fat/high carbohydrate diet with subsequent improvement in cardiac function. Case Study: A term Pakistani infant was diagnosed with malonic aciduria following screening due to positive family history. At age 13 weeks echocardiography showed impaired LV function with ejection fraction of 18%. She was commenced on carnitine, an ACE inhibitor and a nutritionally complete infant formula, providing 1.5% energy from long chain triglyceride (LCT), 20% medium chain triglyceride (MCT) and 65% carbohydrate. Cardiac function was optimal at 23 months of age with a fractional shortening of 25% and good systolic function. Thereafter cardiac function deteriorated following a signi¢cant reduction in MCT intake. This reversed following improved intake of MCT. Conclusions: Since the role of MCD is unclear it is di¤cult to know how best to in£uence disease progression. This case of malonic aciduria with cardiomyopathy demonstrates improvement in cardiac function attributable to LCT restricted/MCT supplemented diet.

THE KETOGENIC DIET: MORE THAN JUST FAT?! van den Hurk ThAM1, van der Louw EJTM2 1 Dept of Dietetics, Univ Child Hosp, Utrecht, Netherlands, 2Dept of Dietetics, Univ Child Hosp, Rotterdam, Netherlands

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DIETARY THERAPY IN ISOVALERIC ACIDAEMIA Footitt EJ1, Dixon M2, Cleary MA1 1 Metab Dept, Grt Ormond Street, London, UK, 2Dietetic Dept, Grt Ormond St, London, USA Introduction: Long-term treatment of isovaleric aciduria (IVA) follows the principles of other organic acidurias: carnitine supplementation and protein or leu- restricted diet. Glycine is also used. IVA appears less severe than PA or MMA and there is little evidence that diet confers any extra bene¢t over medication alone. Many extended newborn screening programmes include IVA making it important to understand the necessity of diet in this condition. This study in an unscreened population details the neurological outcome in IVA children correlating outcome with diet restriction. Methods and Results: Seven patients (2 male), age range 4 months^15 years were reviewed. Six presented neonatally and one aged 5 years with psychomotor retardation. Acute management was cessation of protein and IV carnitine. F|ve had oral glycine. Low protein diet (minimal requirements) was commenced in 5 of 6 neonatal cases. One breast-fed infant was weaned onto low protein foods. Diet was relaxed in 3 of 6 cases at 212 ^ 8 years. Only 1 of 7 cases had a further decompensation at age 1 year; she was diet treated. Neurodevelopment is normal in 3 of 7 cases. Two have moderate psychomotor retardation. Two siblings have severe retardation with autism. The neurological outcome was unrelated to the degree and compliance with protein restriction. Conclusions: The degree of protein restriction does not correlate with neurological outcome or number of decompensations in IVA. Medication alone may be su¤cient to manage this group once stabilised; further studies including those from screened populations would help address this issue.

IMPROVEMENT IN CARDIOMYOPATHY IN A CASE OF MALONYL-CoA DECARBOXYLASE DEFICIENCY ON LCTRESTRICTED/MCT SUPPLEMENTED DIET Footitt EJ, Sta¡ord J, Dixon M, Cleary MA Dietetic Dept, Grt Ormond St, London, USA

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RESPONSIVENESS OF KUVAN IN PKU PATIENTS DURING THE EXPANDED ACCESS PROGRAM AND FOLLOWING FDA APPROVAL Bernstein LE1, Myers S2, Thomas JA1, Freehauf C1, Hanou C2 1 Univ CO Denver, Aurora, USA, 2Cl Gen and Metab, The Children's Hospital, Aurora, USA Kuvan (saproterin dihydrochloride) is a FDA approved drug used in the treatment of PKU. Prior to FDA approval our clinic was a test site for the expanded access program (EAP). Data includes patients from both the EAP and since FDA approval. A baseline Phe level was obtained prior to starting the drug with follow-up levels at 7, 14 and 28 days. All patients started on a dose of 20 mg/kg/day taken at meals as a pill or crushed in apple juice. No change to Phe and formula intake occurred. A responder was determined if a decline in Phe level was greater than 30% within the ¢rst 30 days. This was not enforced following FDA approval. 20 patients were trialed on Kuvan (ages 3^42 years). No one discontinued due to adverse e¡ects. 12 patients (60%) were responsive, based on greater than 30% decline in Phe levels after 7 days. There was a negative change for 2 patients the second week, with their levels returning within 10% of baseline. 7 of the 20 (35%) discontinued the drug at 28 days. Presently, 13 patients from the original 20 continue on Kuvan; In 1 case, responsiveness was reported as increased concentration, without a change to Phe levels or diet. 9 patients (69%) have had an increase in Phe from food and a decrease in medical food. The remaining 31% are still in transition.

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THE IMPORTANCE OF PREALBUMIN CONCENTRATION IN PHENYLKETONURIC PATIENTS Rocha JC1, Almeida MF1, Carmona C1, Cardoso ML1, Borges N2, Soares I3, Salcedo G3, Lima MR1, Azevedo I4, van Spronsen FJ5 1 Cent Genet Med Jac Mag, Porto, Portugal, 2FCNAUP, Porto, Portugal, 3 Endoclab, Porto, Portugal, 4Dept Bioq FMUP, Porto, Portugal, 5Beat Child Hosp, Groningen, Netherlands Background/Objectives: Dietary treatment in PKU has a high risk of inadequate nutritional status. The aim of this work was to study the prevalence of protein insu¤ciency de¢ned by low prealbumin concentrations, and the relation with intake of protein substitute, growth and metabolic control. Methods: We collected data in 69 PKU patients on growth and food intake, as well as blood prealbumin and phenylalanine concentrations. Prealbumin concentrations were expressed as z-scores. Protein insu¤ciency was de¢ned as prealbumin z-scores below the 5th percentile of reference population. Results: Nine patients (13%; 1 classical PKU, 5 mild PKU, 3 hyperphenylalaninemia) showed signs of protein insu¤ciency. No correlation was found between prealbumin z-score and height z-score, the amount of prescribed amino acid mixture or the median of blood Phe (metabolic control). Conclusion: A signi¢cant group of patients presented biochemical signs of protein insu¤ciency. Beyond the more common analysis, measurement of prealbumin concentration in PKU patients may help to detect insu¤cient nutritional status. The prealbumin concentration is not clearly explained by amino acid intake or metabolic control nor does it predict growth. The higher prevalence of protein insu¤ciency in milder compared to more severe form of PKU suggests that beyond metabolic control, optimising protein nutritional status remains of importance in these patients as well.

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SERVICE IMPROVEMENT WITH PKU CLINICS MacDonald A1, Carver S2, Daly A1, Hopkins V1, Hendriksz C1, Chakrapani A1 1 Birmingham Childrens Hospital, Birmingham, UK, 2MSA Consulting, Herefordshire, UK Dietary education and practical teaching in traditional PKU clinics (TC) is hampered by time limits, space and personnel. In weekday clinics, children and carers take time away from school or work. In order to improve education and clinic time suitability, clinic day has been changed to Saturday (SC) for school-age children and teenagers. This has enabled the entire clinic waiting room to be transformed into a `PKU show;' with food displays, cookery demonstrations, PKU education, and entertainment. The size/length of clinics have doubled to maximise the use of the educational resources. Aim: To investigate customer satisfaction with SC's and TC's. Methods: An independent researcher developed a satisfaction survey questionnaire using validated criteria important to PKU families. This survey was completed by 40 parent carers and young people (19 in SC; 21 TC), together with an independent researcher. Each criterion was rated on a 5 point scale and respondents ranked criteria for importance. Results: Better customer satisfaction scores were associated with the SC on: availability and range of suitable low protein foods and drinks (special and natural), `take-home' samples; low protein cooking advice, new protein substitutes available, home delivery information and educational materials. The top three factors important to people attending the SC were displays of suitable PKU foods (1), and drinks (3), and session with dietitian (2). At the SC 70% of people learnt something new compared with 57% at TC. Conclusions: Overall satisfaction and e¡ectiveness were higher in a SC developed around the needs of the families.

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PRACTICALITIES OF EMERGENCY REGIMENS IN IMD MacDonald A, Daly A, Hopkins V, Hendriksz C, V|jay S, Chakrapani A Birmingham Childrens Hospital, Birmingham, UK Emergency feeds are used to prevent metabolic decompensation and encephalopathy during illness in many inherited metabolic disorders (IMD). They are commonly based on glucose polymer, although other high sugar drinks may be used. Methods: In order to ascertain patient/carer understanding and practical application of emergency feeds, 15 multiple choice questions were administered to 111 patients' carers with IMD requiring an emergency regimen. They had a median age of 5 years (range 4 months to 28 years). Forty ¢ve (41%) had fatty acid oxidation and 66 (59%) protein metabolism disorders. 21% (n = 23) had non-English speaking mothers. Results: Almost all (87%; n = 97) understood that emergency feeds were high sugar drinks, their function (87%; n = 97) and when to use them. Most used (n = 85; 77%) glucose polymer; the rest used commercial high sugar drinks. 12% (n = 13) used emergency feeds at least monthly, and only 28% (n = 31) used them less than annually. Although 62% (n = 69) had their emergency feeds updated (carbohydrate and/or volume adjusted) in the last 2 years; 25% (n = 28) of carers could not remember when they received an update or if they had ever had a written regimen. In 38% (n = 42), preparing the emergency feeds was the speci¢c responsibility of one parent only; 19% of carers kept no written emergency feed recipe; and 6% (n = 7) used inappropriate drinks or measuring equipment. Conclusions: It is important health professionals check emergency feeds are updated annually, and verify carers understanding of their appropriate preparation and administration.

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RESULTS OF IMPLEMENTING A NEW METHOD TO VARY FOODS WITHIN THE PHENYLALANINE-RESTRICTED DIET OF ADULT PKU PATIENTS T|mmer C1, Jonkers CF2, van der Ploeg E3, van Spronsen FJ4, de Valk HW1, de Roos NM1 1 University Medical Center, Utrecht, Netherlands, 2Academic Medical Center, Amsterdam, Netherlands, 3University Medical Center, Maastricht, Netherlands, 4University Medical Center, Groningen, Netherlands Background: A new method to vary the phenylalanine (Phe) intake within the Phe-restricted diet was developed at the University Medical Centre Utrecht. This method is based on the exchange system, used in the UK. The Phe-content for each type of food is given in easy-to-count points, using 1 point for each 50 mg Phe. Di¡erent from the UK system, points are given to individual items of food (for example: slice of bread). A 3 colour system: green (freely allowed foods), orange (use within limits) and red (not allowed) simpli¢es the right food-choices. This system of colours and points o¡ers PKU patients an easy way to calculate their daily intake of Phe, without the need to weigh the food. Method: Ten adult PKU patients were enrolled in three University Medical Centers to use the new method. Phe-levels of participants before, and 6 months after starting were compared. Satisfaction with the diet and the ability of patients to choose from a variety of food and to calculate their daily Phe-intake were tested before and 6 months after starting this method. Preliminary results: No signi¢cant increase (410%) of mean Phe-levels were observed (776 versus 842 mmol phe/ L, n = 5). Patients were more satis¢ed with the new method to vary within the PKU diet and wished to continue using this method. Conclusions: This method provides an easy and user-friendly way to vary within the Phe-restricted diet. Calculation of approximate Phecontent seems a less accurate method but does not signi¢cantly elevated Phe-levels.

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Medium chain triglycerides (MCT) are a treatment for long chain fatty acid oxidation disorders (FAOD). MCT supplementation may also aid in lowering triglyceride levels in disorders such as GSD1a and hypertriglyceridemia. Six patients were reviewed following the introduction of a new MCT source. Two patients with FAODs previously on MCT oil were changed to MCT Pro-Cal, a neutral tasting modular powder. Including MCT in the diet has been shown to keep acylcarnitine levels lower. W|th transition from oil to MCT Pro-Cal we saw greater compliance with the product. In 1 patient with VLCAD, C14:1 levels improved from 1.16 mmol/L to 0.20 mmol/L with the change to MCT Pro-Cal. Patient 2 with CACT, did not have improved C:16 or C:18 levels, but has had several interim illnesses. Two siblings with GSD 1a showed possible improvement in triglyceride levels with the addition of MCT Pro-Cal. The older sibling had triglycerides of 1527 mg/dl decreasing to 1001 mg/dl. The younger had triglycerides of 861 mg/dl decreasing to 448 mg/dl. Further, two patients with hypertriglyceridemia also had improvement in triglyceride levels when the majority of long chain fat was replaced with MCT Pro-Cal and/or Lipistart, a nutritionally complete powdered formula. The ¢rst patient had an improvement of triglycerides from over 11 000 mg/dl to 280 mg/ dl on MCT Pro-Cal. The second patient had an improvement from over 100 000 mg/dl to 786 mg/dl on Lipistart. This early data shows a potentially broader treatment use and possible improved compliance with new MCT sources.

D-3-hydroxybutyrate (3OHB) is an alternative energy substrate for brain metabolism with potential applications in the management of recurrent or chronic neuroglycopenia in infancy. In contrast to glucose, 3OHB cannot serve as substrate for cerebral anaplerosis. Combination of 3OHB with an anaplerotic substrate, such as propionate, could improve its ability to sustain brain acitivity. We compared the e¡ectiveness of resuscitation with a single dose 3OHB alone or combined with propionate (3OHB+Prop) in a model of insulin-induced hypoglycemic encephalopathy in 13-day-old rats. Plasma glucose and D-3-hydroxybutyrate levels, EEG, respiratory rate, and alertness score were recorded. Animals were resuscitated when their EEG showed severe suppression. 3OHB injections raised plasma 3OHB concentrations from 0.6+0.1 mM before resuscitation to 3.6+0.7 mM during a 40 min period. Glucose concentrations remained below 1 mM. The proportion of clinically resuscitated animals (alertness score 49) was signi¢cantly larger with propionate (4 of 6 vs 0 of 5, p50.05). Respiratory rates improved from 11+2 min^1 before resuscitation to 110+25 min^1 (n = 5) in 3OHB+Prop vs 65+22 min^1 (n = 4) in 3OHB (p50.05). EEG tracings reverted from severe burst suppression before resuscitation to continuous acitivity after resuscitation in both groups. Recovery of EEG mean amplitude after resuscitation was signi¢cantly better in 3OHB+Prop animals (81+10% (n = 5) vs 62+5% (n = 5) of pre-insulin baseline, p50.05). Values are mean+1SD. Propionate enhances the e¡ectiveness of resuscitation with 3OHB in hypoglycemic infant rats at plasma concentrations of 3^4 mM. Supporting brain function with alternative substrates may require a balance of oxidative and anaplerotic input.

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TREATMENT OF FATTY ACID OXIDATION DISORDERS, GLYCOGEN STORAGE DISEASE TYPE 1A AND HYPERTRIGLYCERIDEMIA WITH NEW MEDIUM CHAIN TRIGLYCERIDES SOURCES Hanou CE1, W|lkinson LJ1, Bernstein LE2 1 Cl Gen and Metab, The Children's Hospital, Aurora, USA, 2Univ CO Denver, Aurora, USA

ASSESSMENT OF THE EFFECTIVENESS OF METABOLIC UNIVERSITY: AN ENTRY LEVEL PROGRAM FOR REGISTERED DIETITIANS, NURSES AND GENETIC COUNSELORS Bernstein LE, Freehauf C, Thomas JA, Gessner JM Univ CO Denver, Aurora, USA Metabolic University was designed to meet a need within the ¢eld of inborn errors of metabolism. W|th the implementation of expanded newborn screening and other advances in clinical genetics, the need for healthcare professionals trained in inborn errors of metabolism has increased. Diagnosis and management of these diseases requires an interdisciplinary approach. Metabolic University is targeted to these professionals newly entering the ¢eld. The program was designed to provide information regarding the natural history, biochemistry, genetics and pathophysiology of common disorders of amino acid, organic acid, urea cycle carbohydrate and fatty acid metabolism. The goals were to enhance knowledge and critical thinking skills. Educational methods utilized at Metabolic University included lectures, group teaching and observations. Thirty-¢ve registered dietitians and nurses participated over two sessions of the program. Participant knowledge was measure by a pre-test, given at the onset of the program, and a post-test given at the end. The program itself was evaluated by participants using questionnaires that addressed program content and presentation. Participants had a 20% average increase in correct answers from the pre-test to the post-test (p50.001) and therefore increased their knowledge regarding inborn errors of metabolism. Participant comments regarding the clarity, organization, expertise and enthusiasm. Overall, participants rated Metabolic University a 9.85 on a scale of 1 to 10. Based on these results we concluded that Metabolic University is an e¡ective way to educate entry-level and practicing clinicians in the ¢eld of inborn errors of metabolism.

PROPIONATE ENHANCES RESUSCITATION WITH D-3HYDROXYBUTYRATE IN AN INFANT RAT MODEL OF HYPOKETOTIC HYPOGLYCEMIC ENCEPHALOPATHY Schutz PW1, Wong PK2, Innis S3, Struys E1, Sto«ckler S1 1 Div Biochem Dis, Dept Paediatr, Univ BC, Vancouver, BC, Canada, 2 Diagn N'physiol, Dept Paediatr, Univ BC, Vancouver, BC, Canada, 3 Nutr R Prgr, Child Fam Res Inst, Univ BC, Vancouver, BC, Canada

INHERITED METABOLIC DISORDERS, EXPERIENCE FROM A MULTIDISCIPLINARY TEAM Oliveira B, Oliveira A, Farinha J, Ferreira A, Guerra A Hospital Santa Maria, Internal Medicine, Lisbon, Portugal Background: Inherited metabolic disorders (IMD) are congenital diseases that a¡ect children and that have been usually associated with a dim prognosis, with a very few number of patients reaching adulthood. Earlier diagnosis and more sophisticated nutritional support allow for better metabolic control and with an increasing number of patients reaching young adult age. Methods: The growing number of patients with IMD that are now young adults have determined the creation of a multidisciplinary team involving internists, nutritionists and psychologists. Results: This team have is now following ¢fthy-six patients with diverse diagnosis ranging from disorders of carbohydrate metabolism (2), disorders of amino acid metabolism (44), disorders of fatty acid oxidation metabolism (4), disorders of porphyrin metabolism (2) and disorders of copper metabolism (4). A protocol has been speci¢cally created to each one of the IMD, that in general comprises a regular clinico-laboratorial and nutritional evaluation, emphasizing such aspects as cardiovascular risk factors, bone mineral density studies, strict adherence to nutritional programs and psychological evaluation and support. Some results are emerging from the application of these protocols that will deserve analysis in the scienti¢c community, namely lower bone density in phenylketonuria patients. Conclusion: Because of the innovative character of this project, with a clear leadership assumed by the Internal Medicine Department, we think that is a scienti¢c imperative to share our protocols, experiences and results with other groups working in this ¢eld.

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THE IMPACT OF INHERITED METABOLIC DISEASES ON QUALITY OF LIFE: A PILOT STUDY Tumer L1, Eminoglu FT1, Soysal AS2, Okur I1, Hasanoglu A1 1 Dept of Nutr and Metab, Gazi Univ Hosp, Ankara, Turkey, 2Div General Pediatrics, Gazi Univ Hosp, Ankara, Turkey Objective: In this pilot study, we aimed to examine the impact of metabolic diseases and their treatment modalities on the patients' everyday emotions, schooling, work, friendships, communication, physical activities, self-esteem and body image. Method: We interviewed 68 patients with metabolic disease and their parents who attended our clinic over 2007^2008. The questionnaire used was based on an adaptation of the HRQoL. Life quality scale for metabolic disease group which was developed for the patients with organic acidemias (OA), disorders of carbohydrate metabolism (CMD), and of amino acid metabolism (AMD) was adjusted. Result: The questionnaires were composed of 33 patients with CMD (% 48.5), 14 patients with OA (20.6%), 21 patients with AMD (30.9%). The 88.2% of 60 patients were followed up regularly. According to the degree of compliance with diet program; 48.5% of patients were good, 44.1% of patients were moderate and 7.4% of patients were bad. The scores of HRQoL that performed in the parents of patients with disorders of CMD were lower than parents with OA. The patients with bad diet compliance had the lower scores of school labeling and perceptiveness of disease in both parents' and child's questionnaire form than patients with good diet compliance (p50.05). Conclusion: This report, the ¢rst study which was made in Turkey, describe that the early and regular evaluation and support of possible cognitive problems should be a major part of the protocol for the followup of patients with metabolic disease.

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METABOLOMICS: A NEW APPROACH TO IDENTIFICATION OF METABOLIC PROCESSES IN MENTAL RETARDATION Engbers HM, V|sser G, Hendriks M, Gerrits J, Berger R Div Metab Dis, W|lhelmina Child Hosp, Utrecht, Netherlands Background: The etiology of MR is very heterogeneous and in up to 50% of the patients no cause is found. Metabolomics is a new technique which aims at holistic pro¢ling of all metabolites with advanced methods, e. g. gas chromatography mass spectrometry (GC-MS) and the quadrupole time of £ight mass spectrometer (Q-TOF). We explored whether with metabolomics new metabolic pathways associated with MR could be found. Method: This study included 104 patients with MR of unknown etiology (<50, ,54, median age 5 years). The metabolomics approach with the energy platform consisted of four main stages: (1) plasma sample and clinical data collection of MR patients, (2) sample analysis with GC-MS, (3) data preprocessing and data analysis, (4) biological interpretation of results. Complex GC-MS pro¢les of the samples needed specialized data preprocessing (a.o. deconvolution) to obtain datasets for subsequent analysis. Di¡erences between pro¢les were sought using multivariate data analysis techniques. Results: We developed (1) a digital clinical database to collect relevant clinical data in MR patients for correlation with metabolic pro¢les, (2) a GC-MS plasma analysis protocol for metabolomics global pro¢ling based on an energy platform, (3) a toolbox for data preprocessing and data-analysis to extract information from GC-MS pro¢les. W|th the energy platform thus far no clear abnormalities were found. Conclusion: Special items to perform steps in metabolomics were developed, but di¡erences in metabolic pro¢les have not been found yet. Further exploration of these data with endocrine and immune metabolomics platforms should be performed.

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INVESTIGATION OF PLASMA FREE CARNITINE DEFICIENCY IN CHILDREN WITH SUSPICION OF INBORN ERRORS OF METABOLISM Allegri GM1, Cruz WMS2, Scalco FB1, de Oliveira MLC1 1 Lab EIM (LABEIM), Univ Fed RJ (UFRJ), Rio de Janeiro, Brazil, 2 Dept Nutr Diet, Univ Fed Fluminense (UFF), Rio de Janeiro, Brazil Inborn errors of metabolism (IEM) are, among the genetic diseases, a group of disorders with possibility of treatment. Achieving diagnosis is often di¤cult, owing to the severe clinical picture, early presentation and lack of speci¢c symptoms. Carnitine, the metabolic functions of which include fatty acid transport for energy generation and removal of toxic metabolism products, is an essential nutrient. Carnitine de¢ciency may occur owing to inadequate ingestion and biosynthesis, defect in cellular transport, increase of requirement or loss. The carnitine de¢cient patient may be asymptomatic or present with various symptoms, such as hypotonia, cardiomyopathy, hypoglycemia, convulsions, acidosis, failure to thrive and proteinuria. When de¢ciency occurs, the clinical presentation is severe and may result in death. This work aims to investigate free carnitine de¢ciency in plasma of children with clinical suspicion of IEM. Analysis of 277 samples was performed by enzymatic spectrophotometric method. A total of 135 samples (48.7%) showed de¢ciency values in relation to the reference range of free carnitine (18.9^71.1 nM) and 22% (n = 61) presented insu¤cient levels. Among the clinical manifestations observed in this selection, the most frequent were convulsions (55.5%; n = 75); acidosis (30.4%; n = 41); hypotonia (30.4%; n = 41); hepatomegalia (28.9%; n = 39); anemia (27.4%; n = 37). The result suggests the necessity of carnitine investigation simultaneously with the speci¢c investigation for IEM, since the de¢ciency picture is varied and may be present in di¡erent clinical situations.

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APPROACH TO IEM IN INDIAN NICU Jalan AB1, Tambde S1, Telawane ND2, Vedak V1, Masand R1, Muehl A2, Bodamer O2 1 Biochemical Genetics Div, NIRMAN, Navi Mumbai, India, 2Div of Newborn Screening, AKH Kindeklinik, V|enna, Austria Background: IEM is quite common in sick newborns and correct diagnosis is very important for proper treatment and prognostication. Unfortunately in India, we don't have any authentic data on incidence of IEM in general population or CINB. Because of this there is no standard protocol or SOP in NICUs. Methods: In this paper we have analyzed data of last 8 years (1999^ 2007) from our center. Of the 601 referred babies for evaluation of IEM in CINB, we could complete analysis in 463 children. Here we shall also compare di¡erent approaches used in NICUs and their pick-up rates. At our Centre we use detailed metabolic workup including ammonia, lactate, ABG and electrolytes, TMS of blood, GC-MS of urine, GALT, galactose, biotinidase enzyme, 17OHP, HPLC-orotic acid, VLCFA analysis etc. Results: We could identify IEMs in 335 (72.35%) babies with this approach. Some other approaches would have pick-up rates much lower than comprehensive pro¢le viz. Only GC-MS (33.26%), only TMS (35.42%), both TMS and GC-MS (52.91%) and expanded NBS (56.37%). Conclusion: Detailed symptomatologic approach along with a clinical geneticist's opinion is far better than other approaches for sick neonates. This way we will pickup almost all (*100%) cases. The cost di¡erence is not much.

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MANAGING BIOBANKS WITH AN EFFICIENT MODEL IN BIOMEDICAL GENETICS RESEARCH Auray-Blais C1, Patenaude J2 1 Serv Genetics, Universiteè de Sherbrooke, Sherbrooke, Qc, Canada, 2 Dept Surgery, Universiteè de Sherbrooke, Sherbrooke, Qc, Canada Background: How can the integrity of participants be protected and the security and con¢dentiality of data be guaranteed in research projects using biobanks? How can Research Ethics Boards (REBs) assure the protection of participants and control the use of biological samples and related genetics data when coded samples are requested for research? Methods: We have devised a model for the management of biobanks using a data-protection o¤cer or medical archivist playing a pivotal role. Medical archivists are employees of medical centers and subject to applicable laws of con¢dentiality. W|th this model, we have reduced the burden placed on REBs responsible for evaluating genetics projects, without jeopardizing the protection of research participants. Results: The proposed model includes a means of protecting the information in biobanks; it o¡ers ways to provide follow-up information requested about the participants; it protects the con¢dentiality of participants and deals e¡ectively with the ethical issues at stake in biobanking. This model respects the informed consent of research participants, while having a health-care professional, such as an archivist, in charge of the biobank and to whom they have entrusted their biological samples, related information and data. The use of two non-related keycodes favors the highest level of con¢dentiality and security of the biobank data. Conclusions: The proposed model is simple, e¤cient and o¡ers an attractive solution on a day-to-day basis for REBs, as well as for researchers, assuring protection of participants who donate their samples for biomedical research.

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A PROFILE OF INBORN ERRORS OF METABOLISM IN RIO DE JANEIRO, BRAZIL De Oliveira MLC, Scalco FB, Simoni RE, Bernstein A, Cruz WMS, Allegri GM, Leal VF, Bezerra Netto HJC, de Oliveira CPH Lab EIM (LABEIM), Univ Fed RJ (UFRJ), Rio de Janeiro, Brazil LABEIM, the Laboratory of Inborn Errors of Metabolism (IEM) in Rio de Janeiro, Brazil, completed twenty years of experience in analysis of IEM. During this period, a total of 7500 samples of biological £uids from 6000 patients (90% children) were analysed. Patients came from di¡erent districts of the state of Rio de Janeiro, in which lives a population of approximately 15.5 million inhabitants. LABEIM is the only reference laboratory that serves gratuitously this population. The purpose of this study is to delineate a pro¢le of IEM in this state. During the study period, a total of 361 cases (6%) of IEM were diagnosed. The most frequent disorders were lysosomal storage disease (LSD) (44.5%), followed by de¢ciencies in metabolism of amino acids ( 2 6 % ). A m o ng th e LSD s, th e m o s t fre qu e nt we re mucopolysaccharidosis (n = 113), followed by infantile Pompe disease (n = 14) and infantile gangliosidosis GM1 (n = 10). In relation to aminoacidopathies, the most common were hyperphenylalaninemia (n = 32) and homocystinuria (n = 13). Other important cases of rare IEM included a-mannosidosis (3 cases), aspartylglycosaminuria (1 case), infantile galactosialidosis (1 case), argininemia (1 case), fumaric aciduria (1 case) and combined de¢ciency of methylcobalamine and adenosylcobalamine (1 case), besides other 9 types of IEM. A ¢nding that attracted attention was the elevated number of patients with infantile Pompe disease. This high frequency may be explained by the elevated afro-american miscigenation in the state of Rio de Janeiro.

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SURVEY OF IEM DIAGNOSED BY THE BRAZILIAN INFORMATION SERVICE FOR INBORN ERRORS OF METABOLIM (SIEM) Souza CFM, Herber S, Giugliani L, Costa B, Netto C, Sanseverino MT, Refosco L, Refaelli C, Giugliani R Medical Genetic Service ^ HCPA, Porto Alegre, Brazil IEM are known to be prevalent disorders, but systematic data about this group of diseases are scarce at our country. Accurate diagnosis and prompt treatment are important to bring a better prognosis for the a¡ected patients. SIEM is a toll-free information service which goal is to provide support to health professionals for diagnosis and management of IEM. Between October 2001 and April 2008, 1250 cases were registered at SIEM database. 722 (58%) had their investigation for IEM completed with 16% (118 cases) being metabolic diseases, 39% non-metabolic, 27% inconclusive and in 18% the follow-up was lost. Organic acidemias (21%) and amino acid disorders (18%) were the most frequent, followed by LSDs (17%) and others defects (12%). In 65% of the metabolic cases patients presented the ¢rst symptoms within the ¢rst year, being the most frequent development delay (49%); seizures (36%) and hypotonia (38%). Consanguinity was present in 21% of the metabolic cases and 10% of the non-metabolic. Recurrence was reported in 23% of metabolic cases and 15% of non-matabolic. The mortality rate in the metabolic cases was 17% and 2.7% in the nonmetabolic. The data obtained con¢rm the severity of the metabolic diseases and the need for a more precise and earlier diagnosis, in order to improve survival rate and help to choose of the best therapeutic strategy. SIEM provides support also to the professionals who are far from reference centers, contributing to the lower the mortality rate even on the most remote areas Brazil. (FAPERGS/UFRGS/ Support/Fundac°a¬o Meèdica)

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METABOLIC INVESTIGATIONS IN A STRUCTURED MULTIDISCIPLINARY DIAGNOSTIC EVALUATION OF DEVELOPMENTAL DELAY Verbruggen KT1, Reijngoud DJ2, Lunsing RJ3, Brouwer OF3, van Spronsen FJ1 1 Beatrix Children's Hospital, UMCG, Groningen, Netherlands, 2Lab of Paediatrics, UMCG, Groningen, Netherlands, 3Paediatric Neurology, UMCG, Groninngen, Netherlands Background: Metabolic investigations in the diagnostic evaluation of developmental delay (DD) are of therapeutical importance, but evidence about need and extent of tests is lacking. Objectives: To investigate the diagnostic yield of a broad metabolic screening program in DD; to describe the results of this program in eventually diagnosed cases; to determine whether metabolic screening can be restricted to selected cases. Methods: 349 patients with DD (2002^2006) received structured multidisciplinary diagnostic evaluation in our center, including screening laboratory investigations: urine portion screening of intermediary, lysosomal (water soluble metabolites) and Creatine metabolism, and plasma aminoacids, random lactate, 7-dehydrocholesterol, screening of Nglycosylation, peroxisomal, Creatine metabolism. Results: Abnormal results were noted in 181 patients, mostly of unclear relevance. A metabolic diagnosis was made in 11 patients (thyroid hormone transport, guanidinoacetate methyltransferase, 96 MRCD). In all cases the initial diagnostice clue derived from clinical features. However, among the patients diagnosed with a metabolic disease in the same period from the northern part of the Netherlands, but not included in this study, a few had been diagnosed primarily as a result of metabolic screening because of DD (San¢lippo A, alpha mannosidosis, aspartylglucosaminuria). Conclusion: Metabolic screening in developmental delay has a low diagnostic yield, but has shown to be diagnostic in several cases presenting with aspeci¢c DD. In the majority of diagnosed patients the diagnosis was not indicated by the screening program. Critical clinical reasoning before or after performing screening investigations remains the ticket to diagnose inborn errors of metabolism in patients with developmental delay.

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INBORN ERRORS OF METABOLISM AS A CAUSE OF LIVER DISEASE AND THEIR GENETIC STRUCTURE Gusina NB, Budzeika ES, Dzemidovitch TV, Dubovik SV, Zimovina TS, Zinovik AV, Gritsenko ON National Centre `Mother and Child', Minsk, Belarus Objectives: To investigate the role of inherited metabolic diseases (IMD) as a cause of liver disease in a cohort of Byelorussian patients. Patients and Methods: A group of 1246 patients with liver disease, hepatomegalia/hepatosplenomegalia, or jaundice of unknown etiology was investigated. The diagnosis of IMD was based on the results of biochemical investigation and DNA assays. Results and Conclusions: The IMD appeared the cause of liver disease in 52% (653/1246) of investigated patients. The following IMD were detected: galactosemia ^ 14 cases (mutant alleles Q188R and K285N 62.5%), inherited fructose intolerance ^ 2, glycogen storage disease type Ia 2 (R43C ^ 50%), Ib ^ 3 and III ^ 1, alfa-1-antitrypsin de¢ciency ^ 25 (PiZZ ^ 7, PiMZ ^ 15, PiMS ^ 3) W|lson disease ^ 33 (å1069G, 2299insC, I1102T and 3400delC ^ 66%), Nieman-Pick type C ^ 2, cholesterol ether storage disease ^ 3, hereditary hemochromatosis ^ 2 (C282Y 1, C282Y/H63D ^ 1), Gaucher disease type I ^ 5 and III ^ 2 (N370S, L444P, RecNciI ^ 77%), methylmalonic aciduria ^ 2, LCHAD de¢ciency ^ 1, syndrome HHH ^ 3, CDG-syndrome ^ 1, tyrosinemia ^ 2, Gilbert's syndrome ^ 550. Among Byelorussians the most common mutation associated with Gilbert's syndrome is insertion of an extra TA into the promoter region of the UGT1A1 gene, 77% of patients with unconjugated hyperbilirubinemia appeared to be homozygous for additional TA repeats. IMD are the frequent cause of liver disease and should be kept in mind working with such groups of patients.

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PRELIMINARY DATA OF A FOLLOW-UP CAMPAIGN IN PATIENTS WITH AN INHERITED METABOLIC DISEASE AT THE `INSTITUTO CANGURU' Oliveira RB, Vertemati T, Satiro CAF, Araujo S, Frangipani BJ, Santana L, Souza C, Leite E, Valadares E, Pimentel H, Giugliani R, Raskin S, Martins AM, Micheletti C Insituto Canguru, Sa¬o Paulo, Brazil Introduction: The Instituto Canguru (IC) is a NGO established in Brazil; its main mission is to disseminate knowledge of inherited metabolic diseases among health professionals and society in general, proposing initiatives to facilitate patient access to information and the necessary treatments. Objective: Describe the preliminary data to follow up the patients assisted by IC. Methods: F|les were used to collect all information previously elaborated and tested. Information was collected by telephone. In this ¢rst round a group of diseases was selected according to the complexity of its treatment. Patients with MSUD were selected. Results: Of twenty two registered patients, 3 died, 9 could not be located by the Institute and 10 of them gave us the necessary information. Among these, 2 responsible people for these patients related their di¤culties in dealing with the diet. But all of them followed the diet without breaking it and they visited the Institute on the scheduled day and time. All their answers were according to the expected for the treatment of MSUD. Conclusion: The importance of this study is to follow-up the compliance keeping the IC informed about data of patients treatment who had received our care, avoiding wasting of products that most of times were supplied by government or through donations, all with high cost. Also the patients with regular support of health professionals have lesser di¤culties in following their diet. These patients showed us the importance of maintaining the campaign to update the information.

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DATA ANALYSIS RELATED TO THE WORK CARRIED OUT BY A BRAZILIAN NGO WITH HEALTH PROFESSIONALS AND PATIENTS WITH INHERITED METABOLIC DISEASES Micheletti C, Vertemati T, Oliveira RB, Araujo S, Leite E, Frangipani BJ, Raskin S, Pimentel H, Satiro CAF, Santana L, Giugliani R, Souza C, Martins AM Insituto Canguru, Sa¬o Paulo, Brazil Background: The Brazilian NGO, Instituto Canguru, disseminates knowledge on Inherited Metabolic Diseases (IMD) among health professionals and society in general. Objectives: Data analysis regarding the work carried out by Instituto Canguru with health professionals and IMD patients and families. Methods: The attendance to health professionals and patients' families was carried out through a call free and email services. A registration was done on patients, parents, relatives, health professionals and associations. Instituto Canguru has implemented a Brazilian Nationwide Mapping Campaign on IMD, whose objective was collecting data on patients, health services and a diagnosis campaign with partners in charge of laboratory diagnostic analyses. Results: Data regarding contacts until April 2008: 1127 health p rofe s s i o n als, b e i ng: 5 5 7 p hys i c i an s, 4 2 2 d i eti c i an s, 1 0 phonoaudiologists, 22 physiotherapists and 116 other professionals. 2249 patients and their families registered, being: 1263 parents (733 mothers and 530 fathers), 24 relatives and 962 patients, who were registered by other persons. Aside these, 267 made contact for IMD suspicion and entered to the diagnosis campaign; 35 other diseases' bearers also did contact and were directed to other associations for necessary assistance. Some ignorance about IMD was observed in the most of the attended cases. Conclusions: The analyzed data demonstrate the necessity and importance of organizations to provide information and support to IMD diagnosis and treatment, due the lack of knowledge observed among health professionals and society in general.

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OUTCOME OF CARDIOMYOPATHY DUE TO INBORN ERRORS OF METABOLISM ^ A COHORT OF 42 PATIENTS Choy YS1, Choy YS2, Haifa I3, Choy YS3, Haifa I3, Kanthavelu G3, Zabedah Y4 1 Genetic Div, Subang Jaya Med Ctr, Petaling Jaya, Malaysia, 2Genetic Unit, Prince Court Med Ctr, Kuala Lumpur, Malaysia, 3National Heart Institute, Malaysia, Kuala Lumpur, Malaysia, 4Metab Lab Institute of Med Research, Kuala Lumpur, Malaysia Inbor n errors of metabolism are important etiologies of cardiomyopathy as many of them are treatable and preventable. A retrospective and prospective study was carried out on a cohort of 82 patients diagnosed with cardiomyopathy in National Heart Institute for the past 5 years. Genetic and metabolic evaluation was carried out for all of them. Inborn errors of metabolism were diagnosed in 42 patients (51%) with age ranging from neonate to 33 years old. Half of them had mitochondrial disorders (50%). The rest include 4 Freidreich Ataxia (10%), 4 fatty acid oxidation defects (FAOD, 10%), 3 Pompe disease (7%), 3 mucopolysaccharidosis (MPS, 7%), 3 malonic acidemia (7%), 2 Barth syndrome (5%) and 2 cardiac glycogenosis (5%). All were given the relevant therapy available when diagnosis was con¢rmed. A third of them (28.5%) had passed away. All patients (100%) with malonic acidemia had passed away due to dilated cardiomyopathy within a year of diagnosis. Most of the patients with mitochondrial disease (75%) were still alive and stable on various therapies including coenzyme and vitamin cocktail. Two out of the three Pompe patients (atypical variant) with non-obstructive hypertrophic cardiomyopathy showed remarkable myocardial improvement on enzyme replacement therapy. The two patients with Barth syndrome are still alive and well. The two cardiac glycogenosis patient are waiting for cardiac transplant in chronic decompensation. Most of the patients with FAOD (75%) and MPS are chronically stable. In conclusion, 5 year-survival for many of the cardiomyopathy patients with inborn error of metabolism are fair with positive response to treatment.

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PROPOSAL OF DECENTRALIZATION OF HEALTH CARE FOR FAMILIES WITH INBORN ERRORS OF METABOLISM (IEM) IN MINAS GERAIS, BRAZIL Valadares ER1, Oliveira LR1, Amorim RHC1, Pinheiro TMM1, Rocha VL1, Arantes RR1, Santos HH1, Souza CP2, Trindade ALC2, Queiroz RR2, Pizarro MX2, Lopes GC2, Godard ALB2 1 Faculdade de Medicina da UFMG, Belo Horizonte, Brazil, 2 Departamento de Biologia Geral da UFMG, Belo Horizonte, Brazil Background: The state of Minas Gerais (MG) is the second most populous in Brazil, with almost 21 million inhabitants, with only 2 centers of reference for diagnosis of IEM, insu¤cient to achieve the demand. Most of the population is descendant of Portuguese colonists from northern Portugal and African slaves. Our aim is to improve health care for large families with IEM in MG through decentralization. Methods: Large families with Segawa syndrome, adrenomyeloneuropathy and juvenile neuronal ceroid lipofuscinose (JNCL) were initially evaluated. Proposal of decentralization through: (1) Providing clinical assessment by specialists locally and biochemical and molecular testing in the centers of reference; (2) Training the local health team to recognize and treat patients properly. Results: Segawa syndrome family: 19 individuals of 55 studied have the mutation IVS5+3insT in the gene GCH1. JNCL family: 4 homozygous and 10 heterozygotes for the deletion 1.02 kb in CLN3 gene. Adrenomyeloneuropathy family: 7 patients, ABCD1 gene in sequencing. Adequate treatment was indicated for patients and genetic counseling has been done individually. The family and the local health team were supplied with lectures and informational material. Conclusion: In general, the study of families a¡ected with IEM enables detection of a large number of patients that can bene¢t from a speci¢c treatment and genetic counseling. The involvement of the local health team improves its expertise in the diagnosis and treatment of regional genetic disorders. F|nancial support: FAPEMIG / SUS

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PATTERN OF METABOLIC DISORDERS PRESENTING TO A METABOLIC CLINIC IN A DEVELOPING COUNTRY OVER TWO YEARS Mohamed S, Khodary A, Elshwaf A, Belushi S, Hellani A, El-Kholy S, El-Melegy E Saad Specialist Hospital, Alkhobar, Saudi Arabia Introduction: Metabolic disorders are genetically inherited diseases with a demonstrable biochemical abnormality associated with enzyme defects. Though individually rare, together they constitute a signi¢cant percentage of children presenting with acute problems. Methods and Results: All cases presenting to the metabolic and neurology clinics at Saad Specialist Hospital, Saudi Arabia, from January 2006 to December 2007, with history and clinical picture suggestive of metabolic disorders were included. Patients were subjected to a metabolic work up including biochemical, enzymatic and genetic testing depending on the provisional diagnosis. The diagnosis was con¢rmed in 52 patients including: non ketotic hyperglycenemia (1), Menkes disease (1), phenylketonuria (2) biotidinase de¢ciency (1), peroxisomal disorders (5), neuronal ceriod lipofucinosis (5), glutaric aciduria type 1 (1), isovaleric aciduria (4), congenital disorder of glycosylation type 1a (1), mitochondrial disorder (2), SCAD de¢ciency (2), MCAD (2), VLCAD (1), long chain hydroxyacyl coA dehydrogenase de¢ciency (1), multiple acyl CoA dehydrogenase de¢ciency (3), carnitine acylcarnitine translocase de¢ciency (1), carnitine palmitoyl transferase type 2 (1), glycogen storage disease type 3 (1), Sandho¡ disease (1), citrulinemia (1), methylmalonic aciduria (3), methylcrotonylglycinuria (1), Neiman Pick syndrome (2), Hurler syndrome (1), San¢lippo syndrome (1), MarteuxLammy syndrome (1), galactosemia (1), Wolman disease (1) and OTC (3). Conclusion: A variety of metabolic disorders were diagnosed in patients presented with symptoms and signs suggestive of metabolic disorders.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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PORTUGUESE METABOLIC NEONATAL SCREENING PERFORMANCE AND MAJOR CAUSES OF FALSE POSITIVE RESULTS Marca¬o A, Rocha H, Sousa C, Fonseca H, V|larinho L National Newborn Screening, CGM, INSA, Porto, Portugal The Portuguese Neonatal Screening Program started in 1979 with the screening for PKU and two years later, congenital hypothyroidism (CH) screening was included. In 2005, started the implementation of the expanded neonatal screening, by tandem mass spectrometry (MS/MS), and with this technique it was possible for the last three years to screen 263 612 babies for PKU and 24 more treatable metabolic diseases. During this time, the screening criteria, including the markers, ratios and cut-o¡ values used for metabolic diseases identi¢cation, were reviewed several times in order to use our increasing experience to reach 100% sensitivity, but keeping the false positive rate as low as possible. One hundred and four patients were identi¢ed (approximate positive detection rate: 1:2500) and no false negative cases are known until now (100% sensitivity). False positive results were obtained in 316 tests (false positive rate: 0.12%). In spite of the analysis of a panel of more than 40 markers and 20 ratios for each baby, altered values in only four of these markers (C0, C3, C5, C5DC) are responsible for the majority of the false positives. Maternal metabolic disorders, maternal pernicious anemia, medication, hyperbilirrubinemia, renal problems or insu¤cient milk intake are some of the reasons in the origin of these alterations. Prematurity and a non-identi¢ed contamination that originates two contaminating products (m/z 332 and m/z 388) are also causes of second tests demands.

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THRESHOLD CHALLENGE IN INDIA- NEWBORN SCREENING FOR AMINOACIDS, ORGANIC ACIDS AND FATTY ACID OXIDATION DISORDERS Kaur M, Birla S Dept Genetics, Dr Lal PathLabs, PBagh, N Delhi-110026, India India is the country with greatest contribution to birth rate in the world. Consanguinity rate is also very high in most states on India. Literature frequently states that 1 in every 1000 Indian newborns is born with neonatal disorder, but still newborn screening programme remains at low priority. We have facility for extended newborn screening. Since year 2005, we are participating in Newborn Screening Quality Assurance Programme of CDC. We started doing in an organized way newborn screening including for aminoacids, organic acids, fattyacid oxidation disorders by tandem mass spectrometry. Most of the newborns tested by tandem mass spectrometry were either already sick or adoption cases. Subjects included from all parts of India and from good socio economic background. Our study revealed 0.5% cases positive for CAH; 0.16% for CH (TSH); 4.3% for G6PD de¢ciency; 1% for cystic ¢brosis (IRT); 1% for galactosemia; 4.5% for biotinidase de¢ciency. We observed that disorders of aminoacids, organic acids and fattyacid oxidation disorders are much more common and important candidate for newborn screening in India. 47% of tested sicknewborns and infants were found a¡ected with such disorders-most common being urea cycle disorder, MSUD,methylmalonic acidemia, propionic acidemia, multiple carboxylase de¢ciency, glutaric acidemia type 1, isovaleric acidemia, carnitine transport defect, CPT1, MADD. It is crucial that importance of newborn screening is spread among public in India.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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THE ROLE OF THE METABOLIC LABORATORY IN AN AGE OF TANDEM MASS SPECTROMETRY NEWBORN SCREENING Fletcher JM1, Ranieri E1, Trinh M-U1, Blake J1, Owens G1, Velardo R2, Harrison JR1 1 Dept Genetic Med WCH, Adelaide, Australia, 2Lab Div WCH, Adelaide, Australia Introduction: The e¡ect on the metabolic laboratory workload and diagnosis rate over 3 years was reviewed 6 years after the introduction of tandem mass spectrometry newborn screening. Methods: Laboratory records for samples referred from within South Australia from 01/01/2005 to 31/12/2007 were reviewed for clinical information. Positive diagnoses were obtained from laboratory records and matched to the sample requests. Results: In this 3 year period, 4081 samples were received for metabolic testing (3191 urines, 847 plasma, 43 CSF). Around 1/3 (1047) of the urine samples were for investigation of developmental delay/intellectual handicap. Newborn screening follow up was the reason for the investigation in 45 plasma and 37 urine samples, often from mother as well as baby. Of these NBS positive samples, a diagnosis was con¢rmed in 29 of 48 patients (60%). In addition, a diagnosis was made in 25 nonnewborn screening-related samples on the basis of urine and/or plasma testing. The diagnosis rate in this group was 0.6% or 0.5% excluding the 4 drug-related conditions from analysis. Conclusion: Plasma and urine analyses generated by the newborn screening program were only 2% of the laboratory workload, with a high positive predictive value. Our metabolic laboratory continues to con¢rm these diagnoses as well as detect conditions such as late-onset urea cycle defects, cystinuria and MPS disorders.

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EXPANSION OF NEWBORN SCREENING IN ONTARIO, CANADA: LESSONS LEARNED IN THE FIRST 2 YEARS Kennedy SJ1, Milburn JL1, Geraghty MT1, F|sher L1, McRoberts C1, Chiu T2, Zelenietz S1, Bouwman T1, Mettler G2, Chakraborty P1 1 Ontario Newborn Screening Program, CHEO, Ottawa, Canada, 2 Genetics Department, CHEO, Ottawa, Canada In 2006 Ontario expanded the newborn screening panel from 2 conditions, phenylketonuria (PKU) and congenital hypothyroidism (CH), to 28 as recommended by the ACMG. The laboratory moved from Public Health to an acute care hospital. Expansion necessitated the rapid acquisition of sta¡, equipment and software, a new transportation system and a provincial infrastructure to facilitate the retrieval of screen positive babies, forming the Ontario Newborn Screening Program (ONSP). From April 2006 to March 2008, the ONSP has screened 284 989 babies. Of these, 1418 have screened positive, and 212 infants have been con¢rmed to be true positives. Follow-up data has been received on 817 (58%) of the screen positive cases, in part due to one of the treatment centres not providing followup data citing privacy concerns. Key ¢ndings include: a higher incidence of some disorders than previously estimated (e.g. biotinidase de¢ciency); identi¢cation of a¡ected infants that would have been missed by pre-existing DNA based neonatal screening for targeted subpopulations, (e.g. glutaric acidemia type 1 in the Ojibway-Cree); and improved enumeration and surveillance of the Ontario newborn population (e.g. evidence of a higher birth rate than previously estimated by the provincial birth registry). Ongoing considerations include: provision of timely screening and diagnostic services to remote communities; strati¢cation of turn around times based upon disease acuity; further delineation of diagnostic classi¢cation for targeted inborn errors of metabolism; development of second tier testing; re¢nement of our transportation system to ensure receipt of quality samples and ensuring compliance with privacy and other governing legislation.

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HOSPITAL BASED EXPANDED NEWBORN METABOLIC SCREENING IN A DEVELOPING COUNTRY: CHALLENGES AND OUTCOME Mohamed S, Hamasha M, Shanaa A, Ritmiller R, Khodary H Saad Specialist Hospital, Alkhobar, Saudi Arabia Introduction: Newborn screening is one of the most successful and coste¡ective programs in the history of medicine. It decreases childhood morbidity and mortality. Objectives: The objectives of the newborn screening program are to identify a¡ected infants with genetic diseases before presenting clinically. Methods and Results: We established a newborn screening at our facility in 2005. We faced a lot of problems both ¢nancial, administrative and logistics. Early home discharge and community awareness are also challenges. We started with a panel of 11 disorders and expanded to 33 disorders including aminoacidopathy, fatty acid oxidation defects, G6PD, hypothyroidism and congenital adrenal hyperplasia. Over 30 months we screened 5054 babies. We used Del¢a and MSMS. We identi¢ed the following disorders: 547 cases of G6PD (incidence 10.8%), 2 congenital hypothyroidism, 2 SCAD, 2 VLCAD, 2 glutaric aciduria type 2, 1 MCAD, 1 PKU, 1 galactosemia, 1 nonketotic hyperglycinemia and 1 case of isovaleric aciduria. Conclusion: Establishing a hospital based newborn screening program in a developing country is a real challenge. The incidence of G6PD in our cohort is very high. Other metabolic disorders seem to be prevalent in our area probably due to high rate of consanguinity.

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NEWBORN MASS URINE SCREENING EXPERIENCE FOR AMINO ACIDS AND ORGANIC ACIDS Auray-Blais C, Clarke JTR, Drouin R Serv Genetics, Universiteè de Sherbrooke, Sherbrooke, Qc, Canada Background: Mass urine screening for inborn errors of metabolism in the neonatal period was instigated 36 years ago in the province of Quebec. Nearly 2 550 000 babies have been screened for amino acids and organic acids with samples collected on ¢lter paper by the parents at 21 days of age. Method: Samples are analysed by a multiplex th in layer chromatographic technique using a sequential-four reagent staining methodology. Two unidimensional ascending migrations are performed for higher resolution. We analyse 500 samples/day totalizing 74 000 samples/year. Results: Parents voluntary compliance is good at 90%. We screen 25 disorders divided into two groups: those causing severe clinical problems which necessitate immediate therapeutic intervention, such as urea cycle disorders and organic acidurias (incidence: 1:24 700) and those necessitating surveillance and follow-up of metabolic (1:5900) and transport disorders (1:1900). The overall incidence is 1:1400. We have con¢rmed 63 cases of methylmalonic aciduria (screening started in 1975), 4 methylcrotonylglycinurias and 1 glutaric aciduria type 1 (for the latter 2 disorders, screening started in 2000), 18 argininosuccinic acidurias, 5 citrullinemia type 1, 3 citrullinemia type 2, 4 hyperargininemias, and one triple H syndrome. Conclusions: Our urine screening program is a dynamic model that has evolved throughout the years to screen as many treatable micromolecule disorders as possible and it is also an important means of genetic education in the population. The methodology used is simple, rapid, reproducible, favouring low cost (4.50 $/sample).

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SELECTIVE SCREENING WITH URINE PAPER SAMPLES BY TANDEM MASS SPECTROMETRY: EXPERIENCE OF 5 YEARS Rebollido MM1, Castin¬eiras DE1, Cocho JA1, Rizzo C2, Boèveda MD1, Couce ML1, Fraga JM1 1 Unid Alt Metab, Hosp Clin Univ Santiago, Santiago de Compostela, Spain, 2Div Metab, Bambino Gesu Hosp, Rome, Italy Urine samples are useful to diagnose a wide range of inborn errors of metabolism, but classical methods of analysis are not suitable to be applied in screening programs. Galician's newborn screening program includes blood and urine ¢lter paper strips collected at the third day of live and since 2000 ESI-MS/MS is performed to analyze aminoacids and acylcarnitines in blood. In 2003 there were developed and applied ESI-MS/MS methods to detect aminoacids, acylcarnitines, acylglicines and organic acids in urine ¢lter paper samples letting us to complement our present newborn screening program. We used MRM experiments to detect aminoacids and organic acids and precursor scans for acylcarnitines and acylglicines. We have analyzed more than 3000 urine samples from newborn to calculate the cut-o¡s as percentiles, and a group of more than 300 pathological samples to contrast the sensibility and the utility of the methods. We present ¢ve years experience in urine analysis with the developed methods. During these years the methods were used like a second tier test to reduce false positive and to increase the e¤ciency. The methods were e¡ective in neonatal period for the detection of the following pathologies: MCAD, propionic acidemia, MSUD, homocystinuria, 3-methylcrotonyl-CoA carboxylase de¢ciency, 3-hydroxy-3-methylglutaric acidemia, tyrosinemia, phenilketonuria, alcaptonuria, glutaric aciduria I, methylmalonic acidemia, argininosuccinic aciduria, SCAD, holocarboxylase synthetase de¢ciency, hypermethioninemia and cystinuria. The combination of the data obtained with blood and urine samples let us to speed up the accurate diagnosis and to include a new set of diseases in the newborn screening program.

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COOPERATION BETWEEN HAMBURG (GERMANY) AND ECUADOR FOR NEONATAL SCREENING AND METABOLIC DIAGNOSIS ^ FOUR YEARS OF EXPERIENCE Lukacs Z1, Santer R1, Kohlschu«tter A1, Nieves Cobos P1, Moncayo F2 1 Dept Pediatr, Univ Med Center, Hamburg-Eppendorf, Germany, 2Inst Andino Enfermedades Metab, Quito, Ecuador For four years, cooperation between the Instituto Andino de Enfermedades Metabolicas (IAEM) and the Hamburg University Medical Center (HUMC) has been build for neonatal screening and metabolic diagnosis. The IAEM coordinates local education and logistics while analysis and clinical advice are provided through the HUMC. In 2008 we expect approx. 5000 screening samples, a growth of 500% since the year 2004. Raised awareness of the doctors in Ecuador also yields a growing number of positive diagnostic specimens. Since the beginning of 2007 Swisslab (Frey Computer Systems, Berlin, Germany) is used in Quito for data processing and direct communication with the HUMC via a VPN connection. In the ¢rst three years of neonatal screening (6000 samples) we have found: 1 MCAD, 3 CAH, 1 CH, 1 MCC-de¢ciency, 2 HPA and 1 galactosemia (Duarte variant). In the same period of time 110 clinical samples have been submitted. We have identi¢ed 2 isovaleric acidurias, 1 MCC de¢ciency, 1 propionic aciduria, 1 methylmalonic aciduria, 1 HMG-CoA-lyase de¢ciency, 1 biotinidase def., 1 CF, 1 MLD, 1 ALD, 1 late infantile neuronal ceroid lipofuscinoses, 1 peroxisomal disorder and 1 GM2-gangliosidoses. In summary, the cooperation has proven e¡ective in identifying patients and raising awareness locally for metabolic disorders. It may serve as a potential role model for other developing countries to improve health care services on a grass roots level.

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NEWBORN SCREENING BY TANDEM MASS SPECTROSCOPY ^ EXPERIENCES OF TWO NEWBORN SCREENING CENTERS IN TAIWAN Kao CH1, Lu YH1, Lo HC1, Lo MY1, Gao HJ1, Hsu JH1, Chong KW1, Cheng KH1, Liu TT2, Chien CC1, Niu DM1, Chiang CC3 1 Department of Pediatrics, NTUH Hospital, Taipei, Taiwan, 2 Department of Pediatrics, VGH Hospital, Taipei, Taiwan, 3Chinese Foundation of Health, Taipei, Taiwan Newborn screening by tandem mass spectroscopy is a technology to detect inborn errors of metabolism which include amino acid disorders, organic acid disorders, urea cycle disorders, and fatty acid oxidation disorders. There were 433 874 newborns who received screening at two centers, the Chinese foundation Health and Taipei Insitute of Pathology, Newborn Screening Laboratory, in Taiwan from 2003 to 2008. There were 36 cases of hyperphenylalaninemias which include 4 cases of 6pyruvol-tetrahydropterin de¢ciency, 4 cases of classical PKU, 7 cases of mild PKU, 21 cases of benign hyperphenylalaninemias, 3 cases of maple syrup urine disease, and 1 case of tyrosinemia type II were found. Of the organic acid disorders: 4 cases of methylmalonic acidemia, 4 cases of 3methylcrotonyl-CoA carboxylase de¢ciency (3-MCC), 2 cases of glutaric acidemia type I, and 1 case of 2- methylbutyryl-CoA dehydrogenase de¢ciency (2-MBCD de¢ciency) were found. Two cases of short chain acyl-CoA dehydrogenase (SCAD) de¢ciency were found. In addition, 4 cases of citrullinemia which include 1 case of type I and 3 cases of type II were found. Newborn screening by tandem mass spectroscopy provides a rapid, accurate technology to detect more disorders of inborn metabolic error than ever before. Because some diseases, such as 3-MCC, 2-MBCD de¢ciency, SCAD de¢ciency, may have a benign course, the management of asymptomatic babies with persistent biochemical disturbances is still controversial. A long-term observation is necessary to predict the prognosis of these inborn metabolic diseases.

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ANTISENSE RNA THERAPY IN INHERITED METABOLIC DISEASES Peèrez B1, Rincoèn A1, Aguado C1, Vega A1, Peèrez-Cerda C1, Merinero B1, Rodr|èguez-Pascau L2, Coll MJ3, V|lageliu L2, Grinberg D2, Desviat LR1, Ugarte M1 1 Centro de Biologia Molecular,UAM. CIBERER, Madrid, Spain, 2 Departament de Gene©tica, UB. CIBERER, Barcelona, Spain, 3Institut de Bioqu|èmica Cl|ènica, CIBERER, Barcelona, Spain Development of pseudoexon-skipping therapies by antisense RNA modi¢cation of pre-mRNA splicing represents a type of personalized genetic medicine. Here we discuss the use of modi¢ed antisense oligonucleotides to block the aberrant splicing caused by deep intronic point mutations causing exonization of in frame and out of frame intronic sequences in four inherited metabolic diseases. The detailed genomic and also the cDNA mutation analysis has resulted in increasing detection of this type of mutations and although each disease represents a rare defect the approaches to detect and to treat this type of change are quite similar. We present the results of the functional analysis by cell based splicing models and antisense morpholino oligonucleotide (AMO) transfection of 6 intronic point mutations identi¢ed in 5 genes (MUT, PCCA, PCCB, PMM2, and NPC1) causing methylmalonic acidemia, propionic acidemia, congenital disorder of glycosylation type Ia and Niemann-Pick Type C, respectivly. The AMO targeted to 5' and/or 3' cryptic splice site was transfected in all cases in a disease cell-based model and the e¡ect was visualized by RT-PCR analysis, en zymatic assays and/or immunodetection of the corresponding a¡ected protein. In all cases the AMO provoked a dose and sequence correction of the aberrant splicing resulting in variable rescue of enzymatic activity. For the mutation causing Niemann-Pick C, an AMO-dependent correction was clearly observed at the RNA level and immuno-detection analyses are underway. Our results suggest that the therapeutical approach described in this work could represent a promising therapy in inherited metabolic disease.

J Inherit Metab Dis (2008) 31 (Suppl 1)

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BLOOD LEAD AND SERUM SELENIUM, ZINC, COPPER AND COBALT LEVELS IN PRIMARY SCHOOL CHILDREN IN A RISKY AREA Sanli, H Bulbul, D Albayrak, P Kocak K|r|kkale Univ, Dept Pediatrics, K|r|kkale, Turkey Background: Trace elements play important roles in many metabolic events. Functional and structural defects occur in their de¢ciency. There are few studies on trace elements in healty subjects in Turkey. Aim: The aims of this study were to determine the blood lead level and serum selenium, zinc, copper, cobalt levels in elementary school students in K|r|kkale. Methods: In this cross-sectional study demographic data and blood samples of 533 students, between the ages 7 and 16 years, from four schools within di¡erential distances to factories where lead is used, were collected. Results: Mean blood lead level was 2.54+1.44 mg/dl (min 0^max 6.5 mg/ dl), serum selenium, zinc, copper and cobalt levels were 21.80+6.43 mg/ dl, 53.79+12.33 mg/dl, 84.38+23.10 mg/dl, 0.01+0.04 mg/dl respectively. There was a signi¢cant di¡erence between the high and low risk school students according to the distances of the schools to the lead using factories. Blood lead, serum zinc and copper levels in children whom malnutrition was detected were found to be lower than the well-nourished children (p50.05). Conclusion: In K|r|kkale, blood lead and serum trace element levels of the children were found to be within the, safe ranges. However, it is known that desired lead level is `0'. Therefore, since the children who live near industrial areas are more exposed to lead and people who live in these areas should be controlled regularly in terms of the long-term hazards of lead.

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CRISPONI SYNDROME DUE TO A NOVEL MUTATION ON THE CYTOKINE RECEPTOR-LIKE FACTOR 1 (CRLF1) GENE Okur I1, Tumer L1, Eminoglu FT1, Crisponi L2, Cinaz P3, Yenicesu I4, Hasanoglu A1 1 Gazi Univ Hosp, Dept Ped Nutr Metab, Ankara, Turkey, 2Ist di Neurogen Neurofarm Cit Univ Mons, Monserato, Italy, 3Gazi Univ Hosp, Dept Ped Endocrin, Ankara, Turkey, 4Gazi Univ Hosp, Dept Ped Hemato, Ankara, Turkey Crisponi syndrome is characterized by contractions of the facial muscles in response to tactile stimuli or during crying, with trismus, abundant salivation simulating a tetanic spasm, intermittent hyperthermia, characteristic facial anomalies and camptodactyly. It is caused by mutations in the cytokine receptor-like factor 1 (CRLF1) and is allelic to cold-induced sweating syndrome type 1 (CISS1) Typical facial features in all patients are large face, chubby cheeks, broad nose with anteverted nostrils and long ¢ltrum. We report here a 9 month-old boy with Crisponi syndrome who presents, besides the typical features, some additional features not described until now. The patient was the third child after fourth pregnancy of a healthy consanguineous couple of Turkish descent. He had hypotonia, marked feeding di¤culties, abundant salivation with inability to swallow, malnutrition and persistent hyperthermia. At his physical examination, atypical facial features such as low-set ears, broad nose with anteverted nostrils, a long and somewhat smoothly formed philtrum, thin and contracted lips, incomplete cleft palate, micrognathia, trismus and bilateral camptodactyly were noted. The thin corpus callosum (hypoplasia?) showed in the magnetic resonance imaging (MRI) of the brain and the velopharyngeal insu¤ciency showed in pharyngoesophagography has not been reported until now. Furthermore, the mutation determined on the cytokine receptor-like factor 1 (CRLF1) gene has not been described in literature, yet.

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Inherited pathology (IP) is characterised by a¡ecting many organs, one of them is pancreas. The development of pancreatitis (P) with exocrinic de¢ciency (ED) can be one expression of gastrointestinal manifestation of IP, which is accompanied by sickness, vomiting, stomach ache, malabsorbtion and maldigestion. The aim of investigation was to reveal the spectrum of IP which is accompanied by P symptoms and ED. 830 families (archive materials from 1997 to 2007), which are placed in KSMGC with preliminary diagnosis: inherited inborn of metabolism, there were 68 patients with manifestation of chronic P and ED. The ¢rst stage of investigations includes classic genetic methods: clinical genealogical analysis and somatogenetic investigation with syndromological analysis; the second stage includes modern biochemical, ultrasound and molecular methods of investigations. There are nosological forms which are revealed from the selected group: cystic ¢brosis with pancreatic de¢ciency 35 patients (51.5%); Kearns-Sayre syndrome 4 (5.9%); MELAS 4 (5.9%); MNGIE 3 (4.4%); fatty acids oxidation defects 5 (7,4%); methylmalonic aciduria 2 (3%); glutaric aciduria 3 (9.1%); propionic aciduria 1 (1.5%); leucinosis 2 (3%); homocystinuria 5 (7.4%); connective-tissue dysplasia with second mitochondriopathia 4 (5.9%). W|de spectrum of IP which is accompanied by P syndromes and ED is revealed. Determination of leading link of ethiopathogenesis of inherited forms of pancreatic dysfunction is necessary for design of investigation with the aim of prevention of reccurences of disease.

Background: Acute intermittent porphyria (AIP) is an autosomal dominantly inherited disorder, classi¢ed as acute hepatic porphyria. It is characterized by a de¢ciency of porphobilinogen deaminase (PBGD, EC 4.3.1.8), the third enzyme in heme biosynthesis. Clinical features include autonomous, central, motor or sensory symptoms, but the most common clinical presentation is abdominal pain caused by neurovisceral crises. Diagnosis of AIP heterozygotes is crucial to prevent life-threatening acute attacks among both symptomatic and asymptomatic carriers. The most powerful diagnostic tool in recent years allows detection of DNA variations by molecular techniques, since biochemical diagnosis is problematic in some cases. Methods and Results: To establish the e¡ects of mutations found in the protein structure of PBGD, we expressed mutant constructs with the described mutations in E. coli, and we analyzed their biochemical and enzymatic properties. All puri¢ed enzymes carrying causative mutations exhibited dramatically decreased activities relative to the average level expressed by the normal allele. Conclusion: E. coli and human PBGD amino acid sequences have 35% homology and more than 70% similarity and, considering this fact, it is possible to extrapolate structure/function relationships for human mutations leading to simple amino acid substitution based on comparative E. coli/human analyses. These analyses together with the kinetic studies of existing mutations can help predict the impact on the enzyme function in living organism and further improve our understanding in this ¢eld. (Supported by Grants MSM0021620806, 1M6837805002, GAUK 257540 54007)

INHERITED FORMS OF PANCREATIC DYSFUNCTION Grechanina YB, Ozerova LS, Zdybskaya OP, Vasylieva OV, Grechanina OY National Medical University, Kharkiv, Ukraine

STRUCTURE-FUNCTION RELATIONSHIPS OF MUTATIONS IN THE PORPHOBILINOGEN DEAMINASE GENE Ulbrichova D, Martasek P Dept of Pediatrics, Charles Univ, Prague, Czech Republic

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Background/Objectives: ADMA, an endogenous inhibitor of nitric oxide (NO) synthase, is an established new cardiovascular risk factor in adults. However, only little is known about the signi¢cance of ADMA in the childhood. We studied ADMA and other members of L-Arg/NO pathway in children su¡ering from (1) homocysteinuria, a disease associated with cardiovascular risk; (2) phenylketonuria (PKU) with suspicious but not proven cardiovascular risk; and (3) glycogen storage disease type I with an atherogenetic lipoprotein pro¢le but without evidence of cardiovascular risks. Age-matched healthy children served as control groups. Methods: ADMA in plasma was determined by GC-MS/MS. Results: As expected, in 6 children with homocystinuria ADMA plasma levels were signi¢cantly higher than in age-matched-healthy controls (660+158 vs 475+77 nmol/L, p = 0.035). By contrast, ADMA plasma levels in 51 patients with PKU were signi¢cantly lower compared to healthy controls (512+136 vs 585+125 nmol/L, p = 0.009). ADMA plasma levels were not altered in 6 patients with glycogen storage disease. Conclusions: In children su¡ering from homocysteinuria or PKU, ADMA homeostas is is al tered. ADMA may repre sent a cardiovascular risk factor in these diseases. However, the precise role of the L-Arg/NO pathway, notably of ADMA, in childhood diseases is unexplored and demands thorough evaluation.

Background: Erythropoietic protoporphyria (EPP) is caused by a partial de¢ciency of ferrochelatase, the eighth enzyme in the heme biosynthetic pathway. Patients with EPP typically present with light-sensitive dermatitis during childhood. Symptoms upon exposure to sun-light include itching, burning, erythema and edema. The reduced ferrochelatase activity leads to accumulation of protoporphyrin in erythrocytes, plasma and feces of EPP patients. Diagnosis is straightforward and is based on free protoporphyrin levels in plasma and erythrocytes. Due to the risk to develop liver failure, levels of protoporphyrin need to be monitored in these patients. Objective: To determine the optimal specimen type to measure protoporphyrin levels. Methods: We compared plasma and erythrocyte protoporphyrin levels on initial and 20 follow-up samples from two patients with EPP. Both specimen types were obtained at the same time. Plasma porphyrins were extracted and analyzed via high performance liquid chromatography (HPLC). Erythrocyte porphyrins were extracted and protoporphyrin was fractionated into the zinc-complexed and non-complexed (free) forms via HPLC. Results: Both, total plasma (73.2, controls 51 mg/dl) and erythrocyte protoporphyrin (9589, controls 520 mg/dl) levels yielded a diagnostic result for EPP. However, after treatment, only the erythrocyte free protoporphyrin levels were consistently elevated (1134^6855 mg/dl) while the total plasma protoporphyrin levels ranged from normal to abnormal (51^1099 mg/dl). Conclusions: These data suggest that erythrocyte protoporphyrin levels yield a more consistent result for monitoring patients with EPP. Protoporphyrin in plasma is relatively unstable and therefore, unreliable in tracking the degree of success treatment for EPP patients.

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ADMA, AN ESTABLISHED CARDIOVASCULAR RISK FACTOR IN ADULTS, IN CHILDREN WITH PHENYLKETONURIA, HOMOCYSTINURIA, OR GLYCOGENOSIS Kanzelmeyer NK1, Tsikas D2, Fuchs AJ1, Beckmann B2, Illsinger S1, Das AM1, Lu«cke T1 1 Dept of Ped, Medical School Hanover, Hanover, Germany, 2Inst of Clinc Pharm, Med School Hanover, Hanover, Germany

OPTIMAL SAMPLE TYPE FOR DIAGNOSIS AND TREATMENT FOLLOW UP OF ERYTHROPOIETIC PROTOPORPHYRIA Bonilla Guerrero RBG, Kloke KMK, Raymond KR, Tortorelli ST Mayo Clinic, Rochester, USA

CYCLIC PAMIDRONATE THERAPY IN CHILDREN WITH OSTEOGENESIS IMPERFECTA Salehpour S1, Rajaii A2 1 Div Metab Dis, Mo¢d Child Hosp, SBMU, Tehran, Islamic Republic of Iran, 2Div Metab Dis, Genomic Research Center, Tehran, Islamic Republic of Iran

RENAL HYPOURICEMIA CAUSED BY DELETION IN HUMAN URATE TRANSPORTER 1 GENE Sebesta I1, Ichida K2, Hosoyamada M3, Stiburkova B1, Hruba E1 1 Inst Inher Metab Dis, F|rst Fac Med, Prague, Czech Republic, 2Dept Pathophysiol, Tokyo Univ Pharmacy, Tokyo, Japan, 3Dept Pharm Toxicol, Kyorin Univ School, Tokyo, Japan

Background: Osteogenesis imperfecta is a disorder characterized by osteopenia, frequent fractures, progressive deformity, loss of mobility, and chronic bone pain. The main objective of this study was to determine the e¤cacy and safety of pamidronate in improving bone mineralization and reducing fracture incidence in osteogenesis imperfecta. Methods: Intravenous pamidronate was administered to 64 children (from 21 months to 10 years old) with severe OI, in a 1 mg/kg single daily dose for 3 sequentional days in 4 months' intervals, over 24^48 months duration. Clinical status, bone turnover markers, the bone mineral density of the lumbar spine and femoral neck, and radiologic changes were assessed regularly during treatment. Results: The number of fractures decreased from median of 8 (range 4^ 11) to 0 fractures/year (range 0^4) (p50.05). After 16 months of treatment, there was signi¢cant improvement in bone mineral density (BMD-DEXA) z-scores of the lumbar spine from median of ^5.90 (range ^7.01 to ^4.76) to ^2.70 (range ^4.46 to ^1.98) (p50.001). Serum alkaline phosphatase decreased from a median of 731.0 U/L (range 438^998 U/L) to 183 U/L (range 95^286 U/L) (p50.001), implying a signi¢cant reduction in bone turnover and its resorption and increase in bone mineralization. There was no improvement either in their height standard deviation scores. Mobility and ambulation improved in all but 5 children, (all of ¢ve taken the drug less than 2.5 years). Conclusion: cyclic pamidronate administration is e¡ective in improving bone mineralization and reducing fracture incidence in childhood osteogenesis imperfecta.

Background: Renal hypouricemia (MIM: 220150) is an inborn error of renal transport of uric acid. The known major cause of this disorder is a defect in the SLC22A12 gene which encodes the human urate transporter 1 (hURAT1). This genetic defect is characterized by two biochemical markers: hypouricemia (less then 119 mmol/L) and increased fractional excretion of uric acid (normal range 6^10%). Most patients are asymptomatic, but some may form urolithiasis and /or exercise-induced acute renal failure. To date, the cases with mutations in hURAT1 have been reported in East Asia only. Over one hundred cases were identi¢ed in Japan and seven patients in Korea. Methods: Uric acid was quanti¢ed by enzymic method. The analysis of hURAT1 was performed as previously described (Ichida K et al., J Am Soc Nephrol. 2004;15:164^73). Results: 3400 blood and urine samples of suspicion of purine disorders were investigated during last three years. Based on our diagnostic approach to unexplained hypouricemia we have found a Czech 5-yearold boy su¡ering from urolithiasis with repeated hypouricemia (low then 108 mmol/L) and increased fractional excretion of uric acid (24.5^ 84.2%). No other secondary causes of hyperuricosuric hypouricemia such as Fanconi syndrome or drug-induced tubulopathy were found. Subsequent mutational analysis revealed novel heterozygous 9 bp deletion in hURAT1 gene. Conclusions: F|rst non-Asian patient with defect in hURAT1 was detected. Hereditary renal hypouricemia is still unrecognized condition and probably not widespread in Japan and Korea only. Detailed purine investigations in patients with unexplained hypouricemia are needed.

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A NOVEL CAUSE FOR AN ABNORMALLY LOW URINARY CREATININE CONCENTRATION Engelke UFH1, Konijnenberg-Kramer I1, Bilos A1, Kluijtmans LA1, Ruitenbeek W1, Morava E2, Wevers RA1 1 Lab Ped Neur, Univ Med Hosp Radboud, Nijmegen, Netherlands, 2Dept Pediatrics, Univ Med Hosp Radboud, Nijmegen, Netherlands Introduction: NMR spectroscopy was performed on urine samples from four di¡erent patients with low or very low excretion of creatinine, revealing two unknown resonances at 2.92 and 4.08 ppm. The patients were adults and all urine samples showed signs of a bacterial contamination (pH 49). Methods: NMR spectroscopy was used to con¢rm the low urinary creatinine found with the routine Ja¡eè and enzymatic method and MS/ MS analysis. Furthermore, NMR spectroscopy was used to identify the unknown resonances. Results: The low concentration of creatinine could be con¢rmed in all urine samples. In one case, the creatinine resonances were completely absent. Normally, they are always present as two resonances at 3.13 and 4.29 ppm, with an integral ratio of 3:2. Remarkably, all spectra showed two unusually high resonances at 2.92 and 4.08 ppm with the same integral ratio of creatinine. We could assign these peaks as the CH3 and CH2 protons of N-methylhydantoin. This compound is the product of creatinine degradation by speci¢c bacterial species (The Human Metabolome Database: HMDB 03646). Several lines of evidence were used to con¢rm the bacterial etiology of this phenomenon. Conclusions: The CH3 and CH2 protons of N-methylhydantoin cause the unknown resonances in the NMR spectrum at 2.92 and 4.08 ppm. Correct quanti¢cation of creatinine in urine is important in metabolic screening. It is important to realize that bacterial contamination of urine samples may lead to falsely very low urinary creatinine levels.

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HIGHER COPPER ACCUMULATION, A POSSIBLE CAUSE FOR HEPATOCELLUAR CARCINOMA IN PATIENTS WITH WILSON DISEASE Kodama H1, Shiga K1, Kaga F1, Fujisawa C1, Gu YH2 1 Dept Pediatr, Teikyo Univ School Med, Tokyo, Japan, 2Dept Health Policy Nation Res Inst Child, Tokyo, Japan W|lson disease (WD) is a hereditary disorder showing higher copper accumulation in the liver and other organs. Although the various types of hepatic disorders have been reported in the patients with WD, little is known about hepatocellular carcinoma (HCC). On the other hand, the LEC rat, an animal model of WD, often exhibits HCC, suggesting that copper accumulation in the liver associates with carcinoma. Thus, the characteristics of 26 patients with both WD and HCC reported in 19592007 from various countries were compared with those of HCC patients in the list of national wide HCC survey in Japan. In addition, copper distribution in the liver was examined in our 12 patients with WD. The copper concentration in the hepatic right lobes of patients with WD was much higher than that in the left lobes. In the 26 patients with both WD and HCC reported during 1959^2007, HCC lesions are more apparent in the right lobe than in the left lobes. National wide HCC data show that onset age of HCC in the patients with WD and HCC is signi¢cantly earlier than that in the patients with only HCC, and that the number of male is signi¢cantly higher than that of female. These results suggest that higher copper accumulation in the liver of patients with WD is one of the causes for the oncogenesis of HCC, indicating that patients with WD should regularly undergo the examinations for HCC.

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DEVELOPMENTAL CHANGES OF OXALATE EXCRETION IN PRETERM NEONATES ON ENTERAL NUTRITION Illsinger S1, Lu«cke T1, Vaske B2, Schmidt KH1, Bohnhorst B3, Das AM1 1 Dept of Paed II, Med School, Hannover, Germany, 2Inst of Biomed Statistics, Med School, Hannover, Germany, 3Dept of Paed I, Med School, Hannover, Germany Background: Data on oxalate excretion in preterm neonates are sparse, especially in neonates born 527 weeks of gestation. To further substantiate gestational age-related development of oxalate excretion [Ox/Cr], we studied urinary Ox/Cr in 67 preterm infants born at 23^35 weeks of gestation. Methods: Spot urine of 67 preterm neonates was analysed by ion chromatography. All neonates were orally fed with enriched human milk and/or special preterm milk formula. Infants with any evidence for renal, gastrointestinal, muscular or metabolic diseases were not included; newborns on parenteral nutrition were excluded. Results: An inverse relationship between Ox/Cr excretion and gestational age was observed. 3 age groups (I: 23rd^27th; II: 28th^31st and III: 32nd^35th week of gestation) showed substantial di¡erences in oxalate excretion: The mean Ox/Cr ratio was highest in group I (421.2 mmol/mol+156.8). Ox/Cr excretion correlated inversely with gestational age (r = ^0.45, p50.01) and positively with postnatal age (r = 0.43, p50.01); this phenomenon seems to be most obvious in group I. Ox/Cr correlated inversely with birth weight as well as actual weight at sample collection (r = ^0.45 and ^0.43; p50.01). Ox/Cr ratios were signi¢cantly linked to energy and carbohydrate intake (r = 0.3 and 0.45; p50.05 and 50.01). These results were independent of gender. Conclusion: Ox/Cr excretion inversely correlates with gestational age in preterm neonates. Age-matched data should be used when assessing hyperoxaluria in preterm neonates/infants.

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CLINICAL, BIOCHEMICAL AND GENETIC FINDINGS IN THREE PATIENTS WITH A b-UREIDOPROPIONASE DEFICIENCY van Kuilenburg ABP1, Meijer J1, Saito K2, Eto K3, Meinsma R1, Abeling NGGM1, Zoetekouw L1, Assmann B4 1 AMC, Genet Metab Dis, Clin Chem, Amsterdam, Netherlands, 2Tokyo Women's Med Univ, Inst Med Genet, Tokyo, Japan, 3Tokyo Women's Med Univ, Dept Pediatrics, Tokyo, Japan, 4Univ Child Hosp, Dept General Pediatrics, Du«sseldorf, Germany Background: b-Ureidopropionase de¢ciency is an inborn error of the pyrimidine degradation pathway a¡ecting the cleavage of the Ncarbamyl-b-amino acids. Up to now, only 5 patients have been reported with a b-ureidopropionase de¢ciency. Here, we present three new patients with b-ureidopropionase de¢ciency with a variable clinical presentation. Methods: The concentration of the N-carbamyl-b-amino acids was determined using HPLC-MS/MS. The genomic sequences of UPB1 were sequenced and mutant b-ureidopropionase was expressed in E. coli. Results: Patient 1 is a 5 month-old Japanese girl who presented at the age of 2 months with nictitating spasms for several min. One week later, she had infantile spasms with asymmetrical tonic neck re£exes, 3^4 times per day, lasting 10^50 min. In addition, her mood gradually worsened. Patient 2 is a Turkish boy who presented with one episode of £oppiness, staring and mild cyanosis ate the age of 10 months. His mother seemed to be asymptomatic. Analysis of urine samples from both patients and the mother of patient 2 showed N-carbamyl-b-amino aciduria. Sequence analysis revealed that patient 1 was homozygous for a 977G4A (R326Q) mutation and patient 2 and his mother were homozygous for a 1076C4T (T359M) mutation in UPB1. Expression of the mutant enzymes in E. coli showed that both mutations resulted in a mutant b-ureidopropionase without residual activity. Conclusion: The transient neurological abnormalities and the ¢nding of an individual with a complete b-ureidopropionase de¢ciency without any clinical abnormalities suggest that a b-ureidopropionase de¢ciency is not a sole prerequisite for the onset of a clinical phenotype.

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FATAL MANIFESTATION OF WILSON DISEASE IN A CHILD AFTER VACCINATION AGAINST HEPATITIS B Behuèlovaè D1, Holeovaè D1, Vasilenkovaè A1, Laluhovaè-Strie-Encovaè Z1, Pevalovaè L2, Tuharskyè J2, Ponec J1 1 Dept Lab Med, Univ Child Hosp, Bratislava, Slovakia, 2ICU, Univ Child Hosp, Bratislava, Slovakia Background: W|lson disease (WD) is an autosomal recessive disorder in ATP7B gene characterized by excessive accumulation of copper in the liver with progressive hepatic damage and copper redistribution to extrahepatic tissues. It is stressed to avoid additional insults to the liver. Severe decompensations were reported in WD with systemic viral disease, after liver biopsy, in a WD woman after delivery. Case report: A girl was born to unrelated parents. She was treated because of pollen fever. At the age of 12 years, in January and February, ¢rst two doses of vaccine against hepatitis B were administrated. In March, symptoms such as intermittent abdominal pain, vomiting, anorexia, weakness, apathy appeared. At the beginning of April, she was admitted with severe intravascular hemolysis and fulminant liver failure. Metabolic investigation disclosed mildly decreased ceruloplasmin, normal concentration of total copper in serum and excessively increased excretion of urinary copper 385 mmol/day. Prevalent mutation H1065Q was found, another mutation has been not identi¢ed yet. Despite agressive treatment the patient died on the 13th day of hospitalization. Conclusion: Recently, obligatory vaccination against hepatitis B has been implemented into childhood immunisation programmes in many countries, including Slovakia. Despite several authors have been recommended this vaccination as liver protection in WD, we propose to evaluate its savity in these patients.

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APOE4 IN RETT SYNDROME PATIENTS: A POTENTIAL MODULATION FACTOR? Zahorakova D1, Jachymova M2, Puchmajerova A1, Baxova A3, Martasek P1 1 Dept of Pediatrics, Charles University, Prague, Czech Republic, 2Inst Clin Bioch Lab Dg, Gen Univ Hosp, Prague, Czech Republic, 3Dept Biol Clin Genet, Charles University, Prague, Czech Republic Background: Rett syndrome (RTT) is a severe neurodevelopmental disorder predominantly a¡ecting females. Most of RTT cases are caused by mutations in the MECP2 gene. The phenotypic spectrum associated with the same MECP2 mutation is widely variable. We hypothesize that there might be other genetic factors modulating the RTT phenotype. Apolipoprotein E, the major apolipoprotein in brain, is considered an important cofactor in neuronal metabolism. e4 allele is the known susceptibility factor for earlier onset of cognitive decline in several neurological disorders. We performed the analysis of APOE in RTT patients to evaluate the possible association of e4 allele with the various RTT phenotypes. Methods: We analyzed the frequencies of APOE alleles in a group of 75 RTT patients with MECP2 mutation and 100 unrelated female controls. Molecular genetic analysis of APOE was performed by PCR/RFLP. Results were statistically evaluated by the test of binomial distribution. Results: Our ¢ndings revealed a signi¢cant di¡erence in the frequency of e4 allele between the two groups (p = 0.001). The association between e4 allele and severity of phenotype was studied in 11 patients with the same MECP2 mutation. The most distinctive di¡erence between two groups was the earlier onset of the psychomotor regression and worse motor functions among e4 carriers. Conclusions: To our knowledge this is the ¢rst such study in RTT patients. Understanding the inherited factors that in£uence patients' susceptibility for developing various Rett phenotypes may lead to the development of better and more comprehensive therapies. The study was supported by grants GAUK25792792707, NR9215, MSM0021620849

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URINARY COPROPORPHYRIN ISOMERS IN AN HBsAgPOSITIVE PATIENT WITH DUBIN-JOHNSON SYNDROME Kurt I1, Hasimi A1, Ozturk O1, Ayd|n HI2, Erbil MK1 1 Dept Clin Biochem, Gulhane School of Med, Ankara, Turkey, 2Dept Pediatrics, Gulhane School of Med, Ankara, Turkey Introduction: Dubin-Johnson syndrome (DJS) is a rare autosomal recessive liver disorder characterized by chronic, often intermittent predominantly conjugated, mild hyperbilirubinemia, liver with brown pigmentation and is caused by de¢ciency of multidrug resistance protein (MRP2). DJS is often an occult entity which until various factors and situations accentuate hyperbilirubinemia, resulting in clinically apparent disease. We herein present an HBsAg-positive patient with DJS, who has been diagnosed based on elevated urinary coproporphyrin I isomer. Case and Results: The case was a 21-year-old male who complained about vertigo and dizziness at military conscription examinations. His laboratory data revealed a chronic HBsAg sero-positivity and a mild conjugated/unconjugated hyperbilirubinemia (0.75 mg/dl and 1.2 mg/ dl; respectively); remaining analytical results were otherwise normal. Any pathological changes of liver and biliary tree were not observed on ultrasonography. Due to granular, dark-brownish pigmentation demonstrated on liver biopsy reminded the DJS. Total porphyrin concentration in spot urine sample was within the reference range; nevertheless, diagnosis of DJS was con¢rmed by increased urine coproporphyrin I/total ratio (%97) on porphyrin fractionation by HPLC. Conclusion: Previously delayed rise in the sulfobromophthalein sodium test and evidence for the presence of intracellular brown granular pigments in the liver were used in diagnosis of DJS; however, former approach is discarded yet, and the latter may also be seen in disorders other than DJS. Although de¢nitive diagnosis of this disorder mostly relies on MRP2 mutation analysis, determination of urinary isomers of coproporphyrin may relatively be an easily accessible and strong biochemical indicator for diagnosis of DJS.

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MUTATION SCREENING OF ATP7A, ATP7B AND ATOX1 GENES IN PATIENTS WITH MENKES AND WILSON DISEASES IN CZECH REPUBLIC Kralik L1, Pospisilova L1, Flachsova E1, Bruha R2, Marecek Z2, Fruhauf P3, Puchmajerova A1, Zeman J1, Martasek P1 1 Dept Pediatr, 1st Sch Med, Charles Univ, Prague, Czech Republic, 2 Dept Intern Medicine 4, Charles Univ, Prague, Czech Republic, 3Dept Pediatr, Fac Gen Hosp, Prague, Czech Republic Copper plays an essential role as a cofactor for many enzymes. There are two copper-transferring P-ATPases in human: ATP7A and ATP7B, and a chaperone ATOX1 which delivers copper to them. De¢ciency of ATP7A causes X-linked Menkes disease (MD). A defect in ATP7B causes autosomally recessive inherited W|lson disease (WD). ATP7A and ATP7B share 60% sequence identity; their genes are located on chromosomes Xq13.3 and 13q14.21, respectively. Here we report the mutational analysis of the ATP7A and ATP7B genes of 4 patients with MD and 120 patients with WD from the Czech Republic. Genomic DNA was used to amplify 23 exons of the ATP7A gene and 21 exons of the ATP7B gene. PCR products were examined by RFLP and sequenced. We performed mutational analysis of the ATOX1 gene in patients whose clinical and biochemical phenotypes suggested impaired copper transport, but no mutations were found within the ATP7A and ATP7B genes. Molecular analysis revealed 4 mutations in the ATP7A gene, two of which have not been previously published (Q724X and E1249X). 13 mutations were found in the ATP7B gene, and no mutations were found in the ATOX1 gene. Molecular analysis of the ATP7A gene allows for genetic counselling in families a¡ected by MD. Screening for the prevalent H1069Q mutation in the ATP7B gene shows that the frequency 45.4% of analyzed alleles is in accordance with its occurrence in Central Europe. Supported by Grants IGA MZ NR9406, NR9215, MSMT 1M0520 and GAUK 109/06

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Female patients with W|lson disease (WD) can become pregnant, and most of the mothers want to breastfeed their infants while continuing their treatment for WD. However, the copper concentration has not been investigated in the breast milk of mothers with WD. We report copper and zinc status in the breast milk of mothers with WD who are being treated with a chelating agent or zinc. Materials and Methods: The breast milks were taken several times from ¢ve mothers with WD. Two patients have been treated with trientine, two patients with penicillamine, and another patient was sequestially with zinc and trientine. The copper and zinc concentrations in the breast milk were analyzed with an atomic absorption spectrometry. The copper distribution was also analyzed by HPLC/ICP-MS. Results and Discussion: The mean concentrations of copper and zinc in the control breast mild were 28 and 123 mg/dl, respectively. The copper and zinc concentrations were within normal levels in the breast milks of mothers treated with zinc. The copper and zinc concentrations in the breast milk were lower than controls in one and two patients, respectively, of three patients treated with trientine. In patients treated with penicillamine, the both copper and zinc concentrations were low. HPLC/ICP-MS analysis suggests that the copper in the control breast milk is bound with ceruloplasmin and albumin. In the breast milk of the patients treated with trientine, however, a copper peak bound with trientine was also detected, suggesting that trientine is secreted into the breast milk.

Background: W|lson disease (WD) is a serious autosomal recessive disorder of copper metabolism caused by mutations in ATP7B gene. In our laboratory, a molecular diagnostics of WD consisted in detection of 5 prevalent mutations in ATP7B gene in Czech population by PCR/ RFLP method, followed by direct sequencing of the whole coding region. Methods: To improve the molecular diagnostics of WD, we have developed a genotyping microarray for simultaneous detection of 87 mutations and 17 polymorphisms in ATP7B gene. The principle of analysis is APEX (Arrayed Primer Extension) reaction. APEX consists in incorporation of £uorescently labeled ddNTPs to the 3' end of sense/ antisense probes spotted on chip which hybridize with complementary fragments of analyzed DNA sample. The reliability of chip to detect all selected mutations/polymorphisms correctly has been validated by DNA samples of WD patients with known genotypes and by samples prepared by mutagenesis. Results: At present, we have analyzed 23 WD suspect patients with unknown genotypes by W|lson chip. F|rstly, we have used PCR/RFLP method for detection of 5 prevalent mutations and we have found a mutation p.H1069Q in 22% of analyzed alleles. By the W|lson chip, we have detected the other 26% of mutated alleles. We did not ¢nd any mutation in the rest of 52% alleles by direct sequencing. Conclusion: We have introduced the W|lson chip as a new ¢rst line screening method for 87 mutations and 17 polymorphisms in ATP7B gene and thus facilitated the molecular diagnostics of WD. F|nancial support: MSMT LC06023 and 2B08060

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COPPER AND ZINC STATUS IN THE BREAST MILK OF MOTHERS WITH WILSON DISEASE Kaga F1, Kodama H1, Siga K1, Fujisawa C1, Yanakawa Y1, Kobayashi K2, Koyama H2 1 Teikyo University School of Medicine, Tokyo, Japan, 2Gunma University Graduate School of Medi, Gunma, Japan

HFE GENOTYPING IN CHILDREN WITH WILSON'S DISEASE AND IRON OVERLOAD Polyakova SI Center of Children Health, Moscow, Russian Federation Heterozygous HFE mutation l eads to chronic sideremia, hyperferritinemia and complicate liver injury in adult pts with di¡use liver diseases. Previous investigation of iron status of WD young patients indicated a high frequency of abnormal transferrin saturation (445%), hyperferritinemia and feed-back decreased transferin level (less 200 mg/dl). These data were interpretive as early stage of iron overload (IO) syndrome. Frequency of HFE mutations in WD children is unknown. Methods and Materials: Genotyping of major hereditary haemochromatosis (C282Y, H63D, S65C) mutations was done by PCR in 18 children with WD (mean age 14.8+0.7 years): 10 ^ without IO (male 6/female 4) and 8 with IO (male 4/female 4). Results: Family anamnesis was de¢ned for IO-associated diseases in 18 patients with WD: death in young ages. We compared the frequency of these disorders. Liver cirrhosis, diabetes mellitus, oncology had a place signi¢cantly more in IO group (p50.01). PCR of three major mutations show: in group without IO 3 heterozygote (1-C282Y, 2-H63D) from 10 patients were found, in IO-group only 1 of 8 patients had H63D heterozygous mutation. In 3 IO patients iron concentration in liver biopsy was done. For example, 8 year old WD boy had 560 mg Fe/g of liver, he had heterozyg. H63D, but normal parameters of iron status, and probably he is in group with high risk of IO. Conclusion: Reason of iron overload in WD children is not link to HFE mutations. Investigation of iron status, diet and medicine correction of IO in WD patients would prevent progress of cirrhotic transformation and other complication.

APPLICATION OF THE GENOTYPING MICROARRAY IN THE MOLECULAR DIAGNOSTICS OF WILSON DISEASE Gojova L, Jansova E, Pouchla S, Fajkusova L Cent Mol Biol, Univ Hosp Brno, Brno, Czech Republic

WILSON'S DISEASE: AN EXPERIENCE OVER A MEAN PERIOD OF 15 YEARS Prochazkova D1, Mejzlik V2, Pouchla S3, Michalek J1 1 Dept Pediat, Univ Hosp Masaryk Univ, Brno, Czech Republic, 2Center Cardiovas Transpl Surg, Brno, Czech Republic, 3Center Mol Biol Gene Ther Univ Hosp, Brno, Czech Republic Background: W|lson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by mutations in ATP7B gene (13q14.3q21.1). Methods: We studied twenty one WD patients and fourteen heterozygote carriers aged 2^43 years. The diagnosis was based on clinical symptoms, laboratory tests (ceruloplasmin ^ CP, urinary, and hepatic copper content) and DNA analysis. Results: Eighteen WD patients presented with liver disease, three had mixed neurologic and hepatic involvement with positive KayserFl ei scher r ing. Ni ne patients under went orthotopic liver transplantation (OLT), of which six women developed hemolytic anemia. The level of CP in blood decreased below 0.2 g/L in 35.7% WD carriers (0.27+0.09 g/L) and 58.3% children with WD (0.21+0.13 g/L). The copper level in the 24-h urine collection in WD children was 1.85+1.0 mmol/day. A statistically signi¢cant di¡erence was discovered in the group with OLT (p50.05) for haemoglobin, CP, ALT, ALP and albumine in the blood. In all patients liver transplantation was succesful with a mean survival after the transplantation of 75.9+42.2 month. The most frequent mutations of WD patients were p.H1069Q (52.4%), p.W779X, p.R778G and c.1340del4 (in all 4.8%). In adults p.H1069Q carriers we discovered: tremor, dysarthria, dystonia, drooling and poor motor coordination. Conclusions: Penicillamine and zinc can e¡ectively treat W|lson's disease. Liver transplantation remains the treatment of choice for the end stage liver disease. DNA genetic tests help to rule out WD in family members, especially when diagnostic uncertainty occurs in a heterozygote carrier.

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The ¢rst patient (A, 13 years old) was clinically diagnosed when hypertension (190/125) was accidentally diagnosed after a bone fracture. He was an o¡spring of consanguineous parents from Arab descent. His family history was remarkable for hypertension, and premature death from `kidney problems' in three of his siblings. A 23 year old. sister (B) su¡ered from hypertension and hypokalemia since the age of two. After kidney transplantation, her blood pressure and kalemia normalized. Other brother, (C), had his blood pressure checked twice, one time normal and the other slightly elevated. The most noteworthy of A analysis was hypokalemic metabolic alkalosis (K = 2.3) and low PRA (1.08 ng/ml/h, normal: 1.33^ 3.95), and aldosterone (41 pg/ml, normal: 70^350). The GC-MS urinary pro¢les of all the family members were done (parents and 5 children). The results are expressed as ratios of cortisone metabolites (THE, aCL, bCL) to cortisol metabolites (THF, aTHF, aC, bC). The lack of inactivation of cortisol to cortisone in this disease leads to its reaction with the mineralocorticoid receptor, which normally is triggered by aldosterone. Two ratios were calculated: ratio1=THE/THF+aTHF; ratio 2 = aCL +bCL/aC+bC. The ratios for the a¡ected members were: A: 1 = 0.01, 2 = 0.06; B: 1 = 0.02, 2 = 0.18; C: 1 = 0.26, 2 = 1.04 (reference values: ratio 1 = 0.4^1.8, ratio 2 = 1.5^5.0. The other members had normal ratios. Genetic studies con¢rmed the diagnosis (communicated separately). Conclusions: there is a good correlation between the ratios and the severity of the disease. Although hypertension and hypokalemia normalized after renal transplant in B, and the minimal symptomatology in C, the ratios are pathologic, showing the sensitivity of this diagnostic tool.

Objective: The aim of this study is to screen for disorders of purine and pyrimidine metabolism among patients with suggestive clinical features. Methods: 300 consecutive urine samples that were collected over a 2year period were analysed using reversed phase RRLC with diode array detector. The urinary uric acid / creatinine ratio was also determined as a ¢rst line screening for purine metabolic defects. Those patients showing speci¢c patterns or markers suggestive of purine and pyrimidine disorders were further subjected to enzymatic assay or mutation study for con¢rmation. Results: Our study showed that 9 of the 300 samples (3%) analysed have purine and pyrimidine disorders. The 9 cases identi¢ed were hypoxanthine guanine phosphoribosyltransferase (HPRT) de¢ciency (4), molybdenum cofactor de¢ciency (MOD) (1), familial juvenile hy p e r u r i c a e m i c n e p h ro p at hy ( 1 ), t r y p t o p h a n a e m i a ( 1 ), Adenylosuccinate lyase (ADSL) de¢ciency (1) and urea cycle defect (UCD) (1). Four HPRT de¢cient patients had increased urine uric/ creatinine ratio. They also have typical urinary pro¢le revealing large peak of hypoxanthine, xanthine and uric acid. Elevated xanthine and hypoxanthine with low uric acid was identi¢ed in patient with MOD. The ¢rst Malaysian ADSL case was detected in a severely retarded child who showed the characteristic marker of Succinyladenosine. A large peak of uracil was detected in a patient with UCD. Conclusion: We conclude that there is a signi¢cant incidence of purine and pyrimidine disorders among Malaysian children. Urinary testing of purine and pyrimidine should be considered for patients with suggestive signs and symptoms.

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THE CONTRIBUTION OF GC-MS URINARY STEROID METABOLOME IN THE STUDY OF A CONSANGUINEOUS FAMILY WITH 6 MEMBERS WITH APPARENT MINERALOCORTICOID EXCESS (AME) Knopf C1, Magen D2, Suheir A3, Skorecki K3 1 Metab Unit, Biochem Dept, Rambam Med Cent, Haifa, Israel, 2 Pediatric Nephrology Dept, Haifa, Israel, 3Nephrology Dept, Rambam Med Cent, Haifa, Israel

SCREENING AND DIAGNOSIS OF PURINE AND PYRIMIDINE METABOLISM DISORDERS AMONG MALAYSIAN CHILDREN BY RAPID RESOLUTION LIQUID CHROMATOGRAPHY Chen BC1, Ngu LH2, Zabedah MY3, Thong MK4, Keng WT2 1 Dept Pathology, Hospital Kuala Lumpur, Kuala Lumpur, Malaysia, 2 Div Genetic and Metabolic, Ins. Paed, HKL, Kuala Lumpur, Malaysia, 3 Div Biochemistry, Inst Medical Research, Kuala Lumpur, Malaysia, 4 Dept of Paediatrics, University Malaya, Kuala Lumpur, Malaysia

PREFRONTAL CORTEX OLIGODENDROGLIA CELLS FROM ADSL DEFICIENT PATIENT PRODUCE SAICAR AND SAMP Zidkova L1, Krijt J2, Sladkova J2, Hlobilkova A3, Zeman J2, Magner M2, Elleder M2, Adam T1 1 Lab Inher Metab Dis, Palacky University, Olomouc, Czech Republic, 2 Dept Paed Inst Inher Met D, Charles Uni, Prague, Czech Republic, 3 Dept Pathol, Univ Hosp and Palacky Univ, Olomouc, Czech Republic

PURINE AND PYRIMIDINE NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY ON URINE FILTER PAPER SAMPLES Rebollido MM1, Castin¬eiras DE1, Cocho JA1, La Marca G2, Boèveda MD1, Cristobal C1, Couce ML1, Fraga JM1 1 Unid Alt Metab Hosp Clin Univ Santiago, Santiago de Compostela, Spain, 2Div Metab Dis, Univ-Meyer Child Hosp, Florence, Italy

Background: Dephosphorylated substrates 5-amino-4-imidazole-Nsuccinocarboxamide (SAICA) riboside and succinyladenosine (SAdo) accumulate in body £uids of patients with adenylosuccinate lyase (ADSL) de¢ciency. Content of succinylpurines in cerebrospinal £uid of patients exceeds 100 mmol/L (Marie S. et al., Am J Hum Genet. 2004, 1276) but the source remains obscure. In this study we tested production of the compounds in cultured oligodendroglia obtained at autopsy from a patient with ADSL de¢ciency (compound heterozygote for Y114H and R396H mutations in ADSL gene). Methods: Oligodendroglia were grown in DMEM medium, supplemented with 10% of fetal bovine serum and antibiotics. After preincubation in purine-free medium for 24 h cells were labeled with 13C-glycine or 14C-glycine (glycine enters the purine de novo synthesis in the 2nd step) for 4 h. Purine products labeled with 13C-glycine were measured by LC-MS/MS. Compounds labeled with 14C-glycine were analyzed by 2D-TLC and detected by autoradiography. Results: Intracellular concentrations (`steady state') were 43, 81, 100 and 65 mmol/L for su c cinyladenosi ne monophosphate, SAdo, SAICAribotide and SAICAriboside, respectively. The contribution of compounds newly synthesized during 4-h incubation (labeled) was 7^ 25%. Conclusions: Both methods demonstrated the production of succinylpurines in oligodendroglia from patient with ADSL de¢ciency in vitro and so these cells can be source of the compounds in vivo. Supported by the grants MSM 6198959205 and MSM 0021620806

Galician's newborn screening program includes blood and urine ¢lter paper strips collected at the third day of live. In 2003 we have developed electrospray tandem mass spectrometry (MS/MS) methods in positive (aminoacids and acylcarnitines) and negative (organic acids, acylglycine) ionization modes. In 2006 it were included in the negative ionization method speci¢c MRM assays to detect purine and pyrimidine metabolites without chromatographic separation. Run time was 2.6 min, allowing the daily analysis of a high number of samples. There were studied 17 metabolites and internal standards for they quanti¢cation covering a great range of metabolites: guanosine, deoxiuridine, thymidine, deoxiinosine, orotidine, uracil, thymine, adenine, hypoxanthine, xanthine, orotic acid, uric acid, uridine, deoxiadenosine, adenosine, deoxiguanosine, inosine. We have analyzed more than 400 urine samples from newborn to calculate the cut-o¡s, and a group of 17 pathological samples representing 7 di¡erent pathologies to contrast the sensibility, the utility and the diagnosis capability of the method. Dihydropyrimidine dehydrogenase de¢ciency (DPD), xantinuria (XDH), phosphoribosyl pyrophosphate synthetase superactivity (PRPS), Lesch-Nyhan syndrome, nucleoside phosphorylase de¢ciency (NP), adenosine deaminase de¢ciency (ADA), XDH-molibdenum cofactor de¢ciency. MS/MS technique allows the simultaneous detection of a great number of metabolites in a complex biological sample with a minimum preparation sample time and without the necessity for previous chromatographic separation, doing the procedure faster and less labor-intensively than conventional methods of testing for IEM. In addition the ability to include a greater range of metabolites o¡ers the potential of a more comprehensive screening procedure.

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PECULIAR FINDINGS IN INTERMEDIATE TYPE OF ADENYLOSUCCINATE LYASE DEFICIENCY Ballhausen D1, Lazzarino G2, Tavazzi B3, Jequier M4, Roulet-Perez E4, Jacquemont S5, Roux C6, Bonafeè L1 1 IEM, Molecular Pediatrics, CHUV, Lausanne, Switzerland, 2Dept Chem Sci, University of Catania, Catania, Italy, 3Inst Biochem, Catholic University Rome, Rome, Italy, 4Child Neurology, CHUV, Lausanne, Switzerland, 5Medical Genetics, CHUV, Lausanne, Switzerland, 6IEM, Clinical Chemistry, CHUV, Lausanne, Switzerland Adenylosuccinate lyase (ADSL) de¢ciency (MIM 103050) is a rare autosomal recessive inborn error of purine synthesis characterized by accumulation of succinylaminoimidazolecarboxamide riboside (SAICAr) and succinyladenosine (S-Ado) in body £uids. Clinical signs are psychomotor retardation of varying degree, epilepsy often resistant to medication, muscular hypotonia, autistic features, small stature and microcephaly. A mild, severe and intermediate form has been described. A three year-old girl, ¢rst child of consanguineous Turkish parents was diagnosed with ADSL de¢ciency. S-Ado/SAICAr ratio was 1.43 in urine and 1.26 in CSF. Succinyladenine and 2,8-dihydroxysuccinyladenine (387.57 and 3.17 mmol/mmol creat, respectively) were detected in urine. Enzyme activity in erythrocytes was 59%. Molecular analysis revealed homozygosity for the known mutation D430N. Motor development was slightly retarded. She has ataxia and mental retardation without any language development. Surprisingly, her weight and height are above P97, with relative macrocephaly (P90-97,) although her parents have normal measurements for all three parameters. Frequent agitation is the major behavioral problem. Brain MRI at the age of 2 years showed hypomyelination and enlarged subarachnoid spaces, but no cerebellar hypoplasia. Suspicion of myoclonic seizures during sleep lead to EEG investigation, that revealed fronto-sagittal epileptic potentials when falling asleep. Our patient presents an intermediate type of ADSL de¢ciency with biochemical parameters typical of the severe form, but a rather mild phenotype. In contrast to other reported ADSL patients, growth is not impaired, there is no hypotonia, no cerebellar hypoplasia, but an ataxic movement disorder. This case further broadens the phenotypic spectrum of ADSL de¢ciency.

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Author Index Abadie, V 9-P Abboud, G 82-P Abdelhak, S 310-P Abdoh, Ghassan 30-O Abeling, N 537-P, 535-P, 618-P Abulhoul, L 16-P Achten, E 253-P Ackermans, MT 130-P Acosta, AX 399-P Acquaviva, C 136-P, 142-P Adalsteinsson, E 265-P Adam, S 569-P Adam, T 628-P Adamowicz, M 201-P Adler, J 299-P Adriano, E 286-P Aerts, JMFG 421-P, 479-P Afawi, Z 181-O Agostoni, C 89-P, 159-P, 372-P Aguado, C 65-P, 301-P, 606-P Aguirre, M 276-P Ahring, KK 332-P Ahting, U 229-P, 239-P, 251-P Aiello, C 530-P Aigrain, Y 92-P, 182-O Aires, CCP 353-P Akcao«ren, Z 358-P Akkerman, EM 509-P Aksoy, T 94-P Aktuglu-Zeybek, AC 21-P Al Jasmi, F 532-P Al Murshedi, F 532-P Al Rifai, H 30-O Alamowitch, S 525-P Albahri, Z 198-P Albayrak, D 7-P, 607-A Albersen, M 320-P Alberto, JM 43-P Albrecht, U 102-P Alda¨miz-Echevarr|¨ a, L 276-P, 285-P, 455-P Alessandr|© , MG 282-P Alexandra, JF 525-P Alexandre, CA 383-A Aljammaz, SA 560-O Al-Jasmi, F 485-P Allain-Launay, E 43-P Allegri, GM 584-A, 587-P Allen, G 546-O Allen, K 97-O Allen, LE 480-P Allen, RH 34-P, 45-O Alm, J 114-P Almeida, LS 407-P, 277-P Almeida, M 357-O, 436-P Almeida, MF 315-P, 558-P, 574-P Almeida, R 303-P, 305-P Alves, M 505-P, 503-P Alves, S 440-P Amado Fondo, A 501-P, 508-P Amann, E 102-P Amaral, AU 13-P, 14-P, 70-P, 71-P, 80-P Amaral, O 503-P, 444-P, 428-P Amitrano, M 486-P Amorim, A 24-P Amorim, RHC 391-A, 594-A Amorim, T 399-P Amorosi, C 264-P Amri, F 360-P

Andersen, O 247-O Andersson, H 408-P Andler, W 366-P Andrade, F 276-P, 285-P, 455-P Andresen, BS 129-P, 145-P Andria, A 323-P Andria, G 48-P, 169-P, 170-P, 184-P, 454-P, 486-P, 527-P Ang, J 109-A Angaroni, CJ 152-P, 347-P Anik, A 166-P Annaert, W 189-O Antonozzi, I 283-P, 286-P, 334-P, 302-P, 300-P Antonozzi, SL 347-P Antunes, H 444-P Antuzzi, D 8-P, 426-P, 447-P Aoki, K 56-P, 311-P Arantes, RR 594-A, 404-P Arasimowicz, E 449-P Arau¨jo, HC 38-P Araujo, S 592-P, 565-P, 591-A Arckerley, C 409-P Arenas, J 238-P Arias, A 279-P Ariceta, G 276-P Arning, E 547-O Arnoux, JB 157-P, 112-O, 204-P Arosa, FA 412-P Arranz, JA 171-P Arreguin, EA 450-P Artiola, C 334-P, 286-P Artuch, R 185-P, 217-P, 5-P, 538-P, 550-O, 279-P Asfaw, B 422-P, 424-P Ashworth, M 216-P Ask, S 114-P Assmann, B 72-O, 618-P Astarita, L 486-P, 459-P Atherton, AM 69-O Aubert, F 414-P Audonnet, S 43-P, 46-P Auray-Blais, C 586-P, 601-P, 510-P Austin, RC 32-O Ayd|n, HI 83-O, 94-P, 108-P, 443-P, 621-P Aydinok, Y 127-P Ayllon, O 5-P Ayres-Basto, M 85-P, 431-P, 433-P Azen, CG 293-P Azevedo, I 156-P, 574-P Azevedo, JE 385-O Azevedo, L 343-P, 344-P, 542-P Azzouz, H 360-P Bahi-Buisson, N 172-P Bakchine, S 15-P Bakkaloglu, A 94-P Bakker, JA 175-P, 284-P Baldeiras, I 224-P Baldellou-Va¨zquez, A 308-P Baletsrino, M 286-P Balguerie, X 188-P Balivo, F 169-P Ball, S 469-P, 245-P Ballabio, A 454-P Ballhausen, D 64-O, 218-P, 135-P, 329-P, 631-P Balreira, A 385-O, 412-P

Bams-Mengerink, AM 255-P, 267-P Bandeira, A 202-P Bandmann, OL 256-P Banerjee, R 65-P Banikazemi, M 450-P Banwell, B 409-P Baptista, MJ 458-P Barbier, V 457-P, 434-P Barden, AT 321-P, 322-P Baric, I 72-O Barnett, C 210-P, 22-P Baron, K 425-P Barona, R 459-P Barone, R 426-P, 430-P Barrera, LA 438-P Barros, A 250-P Barroso, M 40-P Barschak, A G 260-P, 322-P, 321-P Bartenstein, P 296-O Barth, M 157-P Bartlett, B 87-P Barto, R 39-P Bartuli, A 88-P Bastos, PG 522-P Battaglia, D 8-P Battelino, T 155-P Batteux, F 112-O Battini, R 283-P, 289-P, 282-P Baumann, N 525-P Baumgartner, M 52-O, 83-O, 191-P, 528-P, 329-P Baumgartner Sigl, S 102-P Baussan, C 157-P Baxova, A 620-P Baydakova, GV 150-P Bayindir, O 127-P, 122-P, 370-P Baykal, T 98-P, 41-P Be¨ard, E 273-P, 272-O Beauchamp, NJ 163-O, 154-P Becerra, A 264-P Beck, M 425-P, 481-P Beckmann, B 611-P Bednarczyk, J 121-P Bednarek, N 66-P Begley, JP 266-P Behu¨lova¨, D 78-P, 369-P, 619-P Beil, J 526-P Beira¬o, I 385-O Bekri, S 1-P, 172-P Be¨langer-Quintana, A 330-P Bellanne¨-Chantelot, C 182-O Belushi, S 595-P Bembi, B 390-O Ben Jaballah, N 360-P Ben Dridi, MF 310-P, 360-P Bender, SE 264-P Benjamin, ER 495-P, 507-P Benoist, JF 42-P, 93-P, 92-P, 99-P, 112-O Benoist, L 525-P Ben-Omran, T 30-O Berger, K 514-P Berger, R 2-P, 118-P, 375-P, 583-P, 548-O Bergmann, J 165-P, 368-P Berkau, I 396-P Berkovic, SF 181-O Bernert, G 213-P Berning, C 349-O

Bernstein, A 587-P Bernstein, LE 573-P, 579-P, 578-P Berry, SA 362-P Bertola, F 426-P, 442-A Bertoli-Avella, AM 468-P Bertrand, AM 204-P Beskow, AP 80-P, 71-P, 68-A Besley, GTN 221-P Bessa, C 505-P, 466-P Besseau, C 47-P Bezard, MB 347-P Bezerra Netto, HJC 587-P Bhatia, G 106-O, 333-O Bhattacharya, K 168-P, 164-P Bianchi, M 220-O Bianchi, MC 282-P Biancini, GB 260-P, 322-P Biberoglu, G 117-P, 126-A, 442-A, 12-P Bichet, DG 473-P Bieger, I 349-O Biegstraaten, M 421-P Bieneck Haglind, C 114-P Bierau, J 284-P, 535-P Bik-Multanowski, M 295-P Bilger, K 57-O Bilkova, Z 423-P Bilos, A 615-P Biondi, A 426-P Birla, S 597-P Bittman, R 466-P Blackburn, JH 163-O Blake, J 598-P Blaser, S 409-P Blau, N 290-O, 330-P, 336-P, 329-P, 533-P, 545-P, Bla¨zquez, A 171-P, 238-P Bley, A 488-P Blom, HJ 33-P, 35-O, 36-O, 37-P, 39-P, 49-P Bock, H 402-P Bodamer, O 83-O, 274-P, 585-P Boddaert, N 457-P, 99-P, 352-P, 288-O Bode, J 121-P Boehles, HJ 55-P Boenzi, S 220-O Bogatirova, RV 44-P Bohnhorst, B 617-P Boltshauser, E 206-P Bom¢m, D 399-P Bonafe¨, L 64-O, 135-P, 218-P, 336-P, 631-P Bonapace, G 319-P, 313-P Boneh, A 100-P, 228-P Bonham, JR 128-P Bonilla Guerrero, RBG 613-P Bonsignore, M 515-P Bonthuis, M 320-P Bonza, M 162-P, 159-P Bordigoni, P 491-P Borges, N 574-P Borzani, M 314-P Bottiglieri, T 547-O Bouchet, C 188-P Boukina, AM 398-P Boukina, TM 398-P Boulat, O 135-P, 64-O Boustany, RM 505-P Boutron, A 136-P

162 Bouwman, T 599-P Bo¨veda, MD 630-P, 602-P Bowling, FG 291-O Boy, N 72-O Boy, R 399-P Bozdemir, E 370-P Braissant, O 64-O, 272-O, 273-P Brassier, A 236-P Bratanic, N 155-P Braulke, T 91-O, 384-O Braverman, NE 259-O Brenkman, AB 548-O Briand, GB 281-P, 360-P Briddon, A 536-P Brinkert, F 179-P Briones, M 367-P Briones, P 177-P, 217-P, 554-P, 96-P Brites, P 255-P, 267-P Brivet, M 136-P, 204-P, 157-P Brouns, R 462-P Brouwer, OF 589-P Brown, GK 249-P, 551-O Brown, RM 551-O, 249-P Bruha, R 622-P Bruneel, A 188-P Brunelle, F 182-O Bruno, C 220-O Bruns, H 364-O Bruschini, D 486-P, 527-P Buck, N 97-O Budzeika, ES 590-P Bulbul, H 7-P, 607-A Bunford, C 465-P Burch, M 216-P, 187-P Burgard, P 299-P, 73-P Burglen, L 136-P Burin, M 402-P, 453-P, 493-P, 500-P Burlina, AB 333-O, 220-O, 73-P, 83-O, 318-P, 302-P Burlina, AP 318-P, 73-P, 333-O Busanello, ENB 54-P Busch, M 368-P Buttner, J 400-P Buyukkayhan, D 512-P Byrne, B 517-P, 513-P Bzduch, V 369-P Cabello, A 227-P Cabello, JF 105-P Caciotti, A 447-P, 446-P Caforio, C 300-P Cagnoli, G 159-P, 161-P, 314-P Caillaud, C 434-P, 525-P, 460-P Caiola, D 385-O Caire, C 417-P Calado, E 542-P Calado, J 179-P Caliskan, B 41-P Callahan, JW 494-P Calvin, J 16-P Calvo, AC 545-P Camelier, M 500-P Cameron, JM 211-O Campbell, C 148-P Campistol, J 5-P, 60-P, 62-P, 96-P, 538-P, 279-P Campos, MM 431-P, 156-P Campos, Y 227-P Cano, A 136-P, 160-P, 499-P Canossa, S 401-P Cansever, MS 2 1-P Cansu Mutlu, E 512-P Capaldo, B 323-P Capdevila, A 60-P Carbasisus-Weber, EC 320-P

J Inherit Metab Dis (2008) 31 (Suppl 1) Cardillo, G 323-P Cardone, M 454-P Cardoso, ML 202-P, 357-O, 344-P, 574-P, 85-P, 11-A, 315-P, 156-P Carducci, Ca 286-P, 334-P, 283-P, 302-P, 300-P Carducci, Cl 300-P, 302-P, 283-P, 334-P, 286-P Carmona, C 558-P, 315-P, 574-P Carney, LN 116-P Carollo, C 318-P Carpenter, K 534-P, 186-P Carpenter, KH 278-P Carpenter, S 411-P Carpes, EA 402-P Carraresi, L 446-P, 447-P Carrilho, I 11-A, 202-P Carrozzo, R 220-O Caruso, U 289-P, 530-P Carvalho, AM 54-P Carver, S 575-P Casalini, C 282-P Casarano, M 282-P Casati, G 426-P Case, L 514-P, 517-P Caseiro, C 428-P, 407-P Casero, D 314-P, 316-P, 89-P, 359-P Casey, R 473-P Cassiman, D 181-O, 509-P Castela, E 413-P Castelli, J 387-P Castelo, R 240-P Castilhos, CD 453-P Casti·eiras, DE 630-P, 602-P Castorina, M 456-P Castro, G 105-P Castro, L 411-P Castro, R 35-O, 33-P, 40-P, 38-P, 37-P Castro, S 85-P, 431-P, 433-P Catanese, GA 101-P Cavicchi, C 88-P, 446-P, 527-P Celik, S 98-P Ceravolo, F 313-P Cerisola, A 62-P Cerone, R 302-P, 530-P Cervera, J 356-O Chabli, A 434-P, 288-O, 460-P Chabrol, B 46-P, 160-P, 172-P, 499-P Chace, D 29-O Chace, DH 75-P, 76-P Chadefeaux-Vekemans, B 112-O, 236-P Chaigne, D 172-P Chakraborty, P 599-P Chakrapani, A 557-P, 435-P, 556-O, 559-P, 465-P, 469-P, 576-O, 561-P, 575-P, 562-P, 563-P Champion, H 502-P Chang, Y-C 84-P Charluteau, E 188-P Chateau, M 417-P Chauveheid, MP 525-P Chaves, J 385-O Chegary, M 138-O Chen, BC 629-P Chen, LC 58-P Chen, PW 58-P Cheng, K-H 50-P, 376-P, 51-P, 604-P Cherif, W 310-P Cherin, P 394-P Chery, C 46-P, 47-P, 43-P

Chiang, CC 604-P Chiang, S-H 84-P Chien, C-C 604-P Chien, YH 478-P, 58-P, 539-P Chilosi, AM 289-P Chiu, T 599-P Chmara, M 373-P Chong, K-W 50-P, 51-P, 376-P, 604-P Chora¬o, R 11-A Choy, YS 593-P, 593-P, 593-P, 531-P, 531-P Chrastina, P 4-P, 113-P, 90-P, 95-P, 183-P Chre¨tien, D 93-P, 204-P, 112-O Christensen, E 91-O, 243-P, 73-P Ciani, F 429-P Ciara, E 341-P Cicognani, A 456-P Cimbalistiene, L 487-P Cinaz, P 609-P Cioni, G 282-P Cipriano, M 399-P Cismondi, IA 511-P Citton, V 318-P Claes, LRF 181-O Clark, S 128-P Clarke, J 419-P Clarke, JTR 400-P, 409-P, 473-P, 485-P, 510-P, 601-P Clayton, P 546-O Clayton, PT 268-O, 63-P, 132-P, 560-O Cleary, MA 63-P, 132-P, 342-P, 571-O, 572-O Clemens, PR 379-O Clerson, P 394-P Cleto, S 225-P Clode, N 35-O, 33-P Cocho, JA 602-P, 630-P Cochrane, B 569-P Coelho, D 528-P, 52-O Coelho, DM 54-P, 260-P Coelho, JC 453-P, 500-P, 522-P Coker, M 122-P, 370-P, 166-P, 127-P Colao, A 184-P, 170-P, 169-P Coldwell, J 333-O Cole, A 508-P, 566-O Cole, JA 441-P Coll, MJ 484-P, 606-P Colombo, M 105-P Colombo, V 359-P Concolino, D 543-O, 302-P, 319-P, 313-P Cooper, A 496-P Cormand, B 538-P Cormier-Daire, V 460-P Cornejo, V 105-P Cornelius, N 141-O Corydon, TJ 141-O Corzo, D 379-O Cosgrave, EC 480-P Coskun, T 94-P, 248-P, 274-P, 358-P, 443-P Cosson, MA 93-P, 99-P Costa, B 588-P Cotten, C 395-O Couce, ML 630-P, 602-P Couceiro, A 428-P Cousins, A 501-P Cousins, AJ 508-P Coutinho, MF 440-P Cox, TM 502-P Cozens, A 228-P Cozzolino, M 169-P Craigen, W 230-P

Crisponi, L 609-P Cristo, I 305-P, 303-P Cristobal, C 630-P Crushell, E 532-P Cruz, WMS 587-P, 584-A Cumming, P 296-O Cuppen, M 348-P Cussi, V 492-P Cyr, D 510-P Czartoryska, B 487-P da Costa Ferreira, G 14-P da Silva, LB 68-A, 71-P Dacremont, G 271-P Dalmau, J 426-P D'Almeida, V 31-A, 475-P, 382-P, 381-A, 477-P, 476-P Dalton, A 154-P Dalton, RN 129-P Daly, A 563-P, 562-P, 561-P, 575-P, 576-O, 465-P, 559-P, 557-P, 556-O Damayanti, RS 519-P Dandrea, C 514-P Danecka, MK 327-O D'Angelo, A 48-P Daniele, A 323-P Darcan, S 166-P, 370-P, 122-P Dardis, A 430-P Darin, N 247-O Darmaun, D 43-P Das, AM 83-O, 121-P, 134-P, 178-P, 380-O, 611-P, 617-P Dau-Ming, N 50-P Davies, P 561-P Davis, MB 242-O Dawson, DB 79-P Dawson, P 291-O D'Azzo, A 447-P, 497-P de Bellis, A 184-P de Boeck, K 462-P de Bortoli, G 70-P de Diego, C 171-P de Graaf, B 468-P de Graaf, I 524-P de Groot, M 348-P de Jong, KP 18-P de Jonghe, P 181-O de Keyzer, Y 236-P, 93-P, 204-P, 99-P, 112-O de Koning, TJ 118-P, 375-P, 548-O de la Osa, A 60-P de Leo, S 334-P de Lonlay, P 9-P, 92-P, 93-P, 99-P, 112-O, 136-P, 157-P, 182-O, 190-P, 204-P, 236-P, 288-O, 352-P, 434-P, 457-P, 460-P, de Mari, J F 321-P, 322-P de Meirleir, L 214-P, 215-P, 253-P, 298-P de Oliveira, CPH 587-P de Oliveira, MLC 587-P, 584-A de Paepe, B 215-P, 214-P de Roos, NM 320-P, 577-P de Roux Serratrice, C 394-P de Ruyter, J 540-P de Sain, MG 175-P de Saint-Martin, A 172-P de Sain-Van der Velden, MGM 2-P, 118-P, 375-P, 540-P de Souza, CF 321-P, 322-P de Valk, HW 577-P de Vivo, DC 76-P, 75-P, 219-P de Vries, JE 535-P

J Inherit Metab Dis (2008) 31 (Suppl 1) de Vries, MC 209-P de Vroede, MAM 375-P Dean¢eld, J 140-P Debray, FG 346-P, 244-O Decarlis, S 372-P Dedeken, P 181-O Deegan, P 441-P Dejode, JM 352-P Dekker, CJM 268-O deKlerk, JBC 83-O del Toro, M 171-P Del-Favero, J 181-O D'Elia, F 184-P, 169-P, 170-P Della Casa, R 486-P, 527-P, 170-P, 169-P, 184-P, 323-P Delzescaux, T 182-O Demirkol, M 41-P, 98-P, 330-P den Heijer, M 331-P, 49-P, 36-O Denis, S 268-O, 271-P Denisenkova, EV 325-P Deodato, F 530-P Deon, M 260-P, 321-P, 322-P Deprez, L 181-O Dercksen, M 196-P Deschauer, M 99-P, 134-P Desguerre, I 288-O, 9-P Desviat, LR 65-P, 74-P, 201-P, 301-P, 606-P Devignes, J 47-P Dezateux, C 129-P Di Rocco, M 430-P Di Rosa, G 515-P Di Sabato, ML 300-P Di Vuolo, L 169-P Dickmanns, A 91-O Didycz, B 295-P Dierks, T 388-O Dimitriou, E 406-P Ding, Z 335-O Dinh, Q 427-P Dinis, A 240-P Diogo, L 3-P, 27-O, 530-P, 436-P, 224-P, 203-P, 111-P, 240-P, 413-P, 567-O, 232-P, 205-P Dionisi-Vici, C 83-O, 88-P, 220-O, 426-P, 530-P DiRocco, M 456-P Dixon, M 132-P, 572-O, 571-O, 73-P, 168-P Do, H 387-P, 427-P Do, HV 495-P Doan, S 525-P Dobbelaere, D 394-P Dobbelaere, DD 363-P, 383-A Dodelson de Kremer, R 511-P, 152-P, 347-P, 264-P Dolezalova, H 486-P Donati, A 426-P Donati, MA 355-P, 529-P, 429-P, 446-P, 447-P, 88-P, 146-P, 302-P, 527-P Donaudy, F 454-P Dorko, K 29-O, 547-O Dorland, L 535-P Do«rtttepe, Y 270-P Douglas, TD 326-P Dowden, S 17-P Downing, M 129-P Downing, MJ 128-P Drabko, K 262-P Drahota, Z 237-P Drakaki, K 538-P Drouin, R 510-P, 601-P, 346-P Drouin-Garraud, V 172-P, 188-P Duarte, S 542-P Duat Rodriguez, A 354-P

Dubovik, SV 590-P Duke, C 427-P Duncan, AJ 242-O DunÖ, M 243-P Dupont, J 428-P Dupont, P 181-O Dupre, T 190-P, 192-P Duran, GP 420-P Duran, M 27-O, 353-P, 73-P, 267-P, 118-P, 537-P, 535-P Durand, G 188-P Dursun, A 94-P, 274-P, 26-P, 270-P, 248-P, 358-P Dutra-Filho, CS 71-P, 80-P, 6-P Duzova, A 94-P Dzemidovitch, TV 590-P Ebberink, M 253-P, 254-P, 269-P, 268-O Echeverri, OY 438-P Eckert, SK 345-P Eckhard, A 395-O Eichler, FS 265-P Eklund, C 552-P Ekstein, D 181-O El Oksha, S 359-P Eling, MWM 348-P El-Kholy, S 595-P, 153-P Ellaway, C 186-P Elleder, M 113-P, 183-P, 422-P, 628-P, 377-P El-Melegy, E 595-P Elshwaf, A 595-P Eminoglu, FT 126-A, 609-P, 442-A, 12-P, 582-P, 117-P Endo, F 461-P Engbers, HM 540-P, 583-P Engelke, U 16-P, 110-P, 209-P Engelke, UFH 615-P Erbil, MK 108-P, 621-P Erim, FB 21-P Ermandraut, N 526-P Ermani, M 318-P Ertl, C 102-P Escolar, D 379-O Espinos, C 550-O Esse, R 33-P, 35-O Esseghir, N 310-P, 360-P Estevinho, A 203-P, 3-P Eto, K 618-P Eto, Y 392-O, 378-P, 452-P, 482-P Evangelista, T 235-P Evans, JR 34-P Eyskens, FJ 351-P, 462-P, 261-P Ezgu, FS 126-A, 442-A, 12-P Fagan, E 278-P Faghfoury, H 350-P Faivre, L 136-P Fajkusova, L 365-P, 625-P Fang, Y-L 84-P Fantur, K 497-P Farahmand Monfared, M 518-P Faria, A 567-O Farinha, J 581-P Farshidi, S 59-P Fateen, E 55-P Faustino, I 250-P, 252-P Fecarotta, S 486-P Federici, S 430-P Fedoseeva, NP 226-P Fehervizyova, Z 474-P Feigenbaum, A 532-P, 211-O Feillet, F 136-P, 46-P, 47-P, 491-P, 82-P, 43-P, 330-P Feki, MF 281-P

163 Feldmann, R 309-P, 307-P, 296-O Feliciani, C 456-P Fenneteau, O 42-P Ferdinandusse, S 268-O, 256-P, 271-P Ferguson, C 29-O, 562-P Feriozzi, S 446-P Fernandes, AP 357-O Fernandes, CG 71-P, 13-P, 80-P Fernandez, E 105-P, 227-P Ferna¨ndez-Moreira, D 238-P Ferna¨ndez-Murga, L 356-O Ferracini, R 292-P Ferrante, F 372-P Ferrari, TCA 404-P Ferraz, MJ 437-P, 432-P Ferreira, A 581-P, 337-P Ferreira, AC 470-P, 119-P, 343-P Ferreira, C 444-P Ferreira, EO 371-P Ferreira, GC 139-P Ferreira, M 235-P, 234-P Ferreira, R 263-P Ferrer, I 367-P, 492-P Fialova, M 422-P Ficicioglu, C 173-P Fidzianska, A 506-P Figliuolo, C 48-P, 323-P Figueiredo, C 564-P Filipek, M 20-P Filocamo, M 426-P Filoni, C 446-P, 447-P Fingerhut, R 10-P Fiori, L 89-P, 316-P, 302-P, 336-P Fischer, C 30-O Fisher, L 599-P Fiumara, A 430-P, 459-P Flachsova, E 622-P Flanagan, JJ 495-P Flatmark, T 304-O, 306-P Flemming, J 523-P Fletcher, JM 598-P, 87-P Floegel, U 125-O Florence, J 379-O Flori, E 172-P Folbergrova, J 237-P Fons, C 279-P, 96-P Fonseca, H 596-P, 123-P, 131-P, 111-P Fontaine, M 360-P Fontana, F 454-P, 527-P Footitt, EJ 571-O, 187-P, 572-O Forbes, BJ 173-P Forest, I 417-P, 416-P, 414-P, 410-P Forni, S 489-P Forstner, R 206-P Fouilhoux, A 410-P, 414-P, 416-P, 417-P Foulquier, F 189-O, 188-P Fowler, B 94-P, 528-P, 52-O Fraccaro, E 403-P Fraga, JM 630-P, 602-P Franc°ois, BJM 338-P Frangipani, BJ 31-A, 592-P, 591-A, 565-P, 397-P Franke, B 49-P Frauendienst-Egger, G 324-P Frebourg, T 188-P Freedenberg, D 57-O Freehauf, C 579-P, 573-P Freisinger, P 241-P, 251-P, 239-P, 206-P, 207-O, 229-P Freitag, M 229-P Fricker, G 67-O

Froissart, R 525-P Frommhold, D 194-O Fruhauf, P 622-P Fu, X 489-P Fuchs, AJ 611-P Fujii, C 340-P Fujioka, H 311-P, 541-P Fujisawa, C 616-P, 623-P Fujiwara, M 452-P Fukao, T 100-P, 107-O Fukuda, T 378-P Fuller, M 386-O Funghini, S 146-P, 355-P, 529-P Funkowicz, ME 373-P Furlan, F 88-P Furujo, M 482-P Gillessen-Kaesbach, G 200-P Gabrielli, O 456-P, 426-P Gagoski, B 265-P Gal, A 384-O, 165-P, 425-P Gallagher, PR 173-P Gallardo, ME 217-P Galloway, P 569-P Galvin-Parton, P 333-O Gandoura, N 360-P Ganesan, V 192-P Ganesh, J 116-P, 230-P Gan-Schreier, H 30-O Gao, He-Jin 604-P Garbade, SF 83-O, 72-O, 299-P Garcia, MJ 367-P Garcia, MT 554-P Garcia, O 554-P Garcia, P 27-O, 111-P, 232-P, 205-P, 567-O, 413-P, 240-P, 3-P, 224-P, 436-P Garc|¨ a, MJ 103-P Garc|¨ a Mu·oz, MJ 354-P Garc|¨ a Pe·as, JJ 354-P Garc|¨ a-Cazorla, A 60-P, 217-P, 62-P, 538-P Garc|¨ a-Jime¨nez, I 308-P Garcia-Villoria, J 96-P, 339-P Gaîrdman, J 114-P Garesse, R 217-P Garg, U 69-O Gargosky, SE 362-P Garman, S 446-P Garnotel, R 66-P, 15-P Garovoy, MR 362-P Ga«rtner, J 388-O Gaspar, A 131-P, 111-P, 297-P, 123-P Gaspar, P 439-P, 385-O, 524-P Gasparri, M 159-P, 161-P, 162-P Gasperini, S 146-P, 355-P, 429-P Gass, P 194-O Gaudieri, V 184-P, 170-P, 169-P Gauw, A 18-P Gavrilov, DK 79-P Gavrilova, RH 79-P Gazarini, ML 475-P Geb, S 83-O Gellekink, HJ 49-P Gempel, K 349-O Gerace, R 87-P Geraghty, MT 599-P Gerrits, J 583-P Gersting, SW 327-O Gerver, WJM 535-P Gessner, JM 579-P Ghio, A 511-P Giannakopoulos, A 538-P Giannini, E H 514-P Giannini, T 300-P Giardina, T 16-P

164 Gibson, A 256-P Gibson, C 552-P Gibson, KM 79-P, 101-P, 544-P, 29-O, 547-O Gillery, P 15-P, 66-P Gillett, GT 256-P, 167-P Giner-Ayala, AN 152-P Giovannetti, D 399-P, 405-P Giovannini, M 161-P, 162-P, 314-P, 316-P, 330-P, 329-P, 372-P Gira¬o, C 412-P Gisewska, M 332-P Gissen, P 17-P Giugliani, L 588-P Giugliani, R 588-P, 493-P, 522-P, 500-P, 402-P, 260-P, 321-P, 322-P, 516-P, 591-A, 565-P, 592-P, 54-P Giulini N, I 159-P, 161-P, 162-P Glamuzina, EE 523-P Godard, ALB 391-A, 594-A Godoy, P 404-P Goemans, N 254-P Go¤n, K 181-O Goh, DLM 109-A Gojova, L 625-P Go«kcay, G 83-O, 41-P, 98-P Go«kmen, Z 126-A Goksen Simsek, RD 122-P, 166-P, 370-P Goldberg, R 395-O Goldenberg, A 434-P Golihina, TA 325-P Gomes, A 33-P Go¨mez, L 5-P, 185-P Go¨mez-Argu«elles, JM 171-P Gomy, I 399-P Gonc°alves, CG 68-A Gonc°alves, I 35-O Gong, Y 466-P Gonzales Meneses, A 426-P Gonzalez, S 420-P Gonza¨lez, V 185-P Gonzalez-Quereda, L 177-P Goodman, SI 73-P Goossens, D 181-O Gootjes, J 269-P Gormand, F 414-P Gorovenko, NG 467-P Gort, L 484-P, 177-P, 96-P Go«tz, H 324-P Gouda, A 55-P Gouider-Khouja, NG-K 281-P Grac°a, LM 35-O, 33-P Gradinayna, J 337-P Gradowska, W 81-P Grady, J 333-O, 106-O Grandin, C 451-P Granese, B 48-P Grant, E 265-P Gray, G 469-P Gray, RGF 245-P Grazina, M 225-P, 224-P, 232-P, 3-P, 246-P, 240-P, 205-P, 203-P Grechanina, OY 608-P, 226-P, 44-P Grechanina, YB 44-P, 226-P, 608-P Green, K 534-P, 186-P Greenberg, CR 73-P Gregersen, N 135-P, 145-P, 141-O Greil, J 179-P Greiner, LS 32-O Grenacher, L 364-O

J Inherit Metab Dis (2008) 31 (Suppl 1) Gri¤ths, A 120-P Grinberg, D 606-P Grings, M 68-A, 80-P, 14-P Gritsenko, ON 590-P Gro«ne, H-J 193-P Grosbois, B 394-P Grosso, CL 264-P Gru«newald, S 132-P, 140-P, 192-P, 63-P, 216-P, 83-O, 187-P Gu, YH 616-P Gubbels, CS 175-P, 175-P Gucer, S 270-P Gueant, JL 82-P, 46-P, 47-P, 491-P, 43-P Guelbert, N 511-P Guelbert, NB 152-P, 264-P Guerra, A 581-P, 337-P Guerrini, R 447-P, 446-P, 88-P Guerry, F 218-P Guest, G 92-P, 93-P Gu¡on, N 410-P, 83-O, 136-P, 414-P, 419-P, 416-P, 417-P Guillard, M 195-P, 199-O Guimara¬es, A 202-P, 235-P Guimara¬es, J 431-P, 85-P, 433-P Guimond, SC 544-P Gurdal, A 41-P Gusar, VA 226-P, 44-P Gusina, NB 590-P Gusma¬o, A 24-P Gutierrez, ML 438-P Gutie¨rrez, A 5-P, 185-P Guyot, C 93-P Guzzetta, F 8-P Haas, D 220-O, 366-P Haavik, J 545-P Habarou, F 188-P Haberlandt, E 102-P Ha«berle, J 349-O, 352-P Habif, S 370-P, 122-P, 127-P Hachulla, E 394-P Hagebo«lling, F 121-P Hagemans, MLC 498-P Hager, EJ 544-P Hahn, A 396-P Hahn, D 218-P Haifa, I 593-P, 593-P, 593-P Haliloglu, G 274-P Hall, SK 562-P Halldin, M 114-P Halldin Stenlid, M 552-P Hamasha, M 600-P Hamers, N 375-P Handig, I 134-P Hanna, MG 242-O Hanou, C 573-P Hanou, CE 578-P Hansikova, H 231-P Harding, CO 335-O Hargreaves, I 216-P Hargreaves, IP 149-P Harrison, JR 87-P, 598-P Hart, CE 109-A Harting, I 72-O Hartmann, A 394-P Hartmann, H 178-P Hartmann, K 251-P Hartung, R 481-P Hasanoglu, A 117-P, 582-P, 126-A, 609-P, 442-A, 12-P Hasimi, A 621-P, 108-P Hata, I 147-P Hattori, K 461-P Haugvicova, R 237-P Hawkins, C 409-P

Hayes, J 409-P Heales, S 546-O, 536-P Hecketsweiler, B 1-P Hegele, RA 45-O Heiduk, M 368-P Heil, SH 49-P, 36-O Heinz-Erian, P 102-P He-Jin, G 50-P, 51-P, 376-P Heldt, K 209-P Hellani, A 595-P, 153-P Hellemans, K 115-O Hendriks, M 583-P Hendriks, MMWD 320-P Hendriksz, C 465-P, 469-P, 563-P, 562-P, 576-O, 561-P, 575-P, 435-P, 556-O, 557-P, 559-P, 245-P Henger, S 69-O Hennet, T 191-P Henrique, D 250-P Henry, H 272-O Heptinstall, LE 496-P, 221-P Herber, S 588-P Hernandez, M 420-P Hernandez-Perez, JM 339-P Herrera, J 439-P Hesketh, TM 560-O Heussler, H 291-O Heymans, HSA 267-P Higaki, K 389-P, 443-P Hillier, I 229-P Hillman, R 333-O Hillman, RE 79-P Hinderhofer, K 490-P Hlavata, A 474-P Hlavata, J 422-P, 424-P Hlobilkova, A 628-P Hoeks, MPA 331-P Hofer, D 497-P Ho¡mann, GF 137-P, 73-P, 72-O, 329-P, 366-P, 364-O Ho¡mann, G 30-O Hogan, M 179-P Hogg, S 16-P Holeova¨, D 619-P Hollak, CEM 479-P, 464-P, 421-P Holme, E 247-O Holmes, BB 44-P Homanics, GE 29-O, 547-O Hong, P 1-P Honjo, R 405-P Honzik, T 4-P Hopkins, V 465-P, 556-O, 563-P, 562-P, 575-P, 561-P, 576-O, 559-P, 557-P Hoppel, C 138-O Hopwood, J 386-O Horak, D 423-P Horovitz, DG 399-P, 405-P Ho«rster, F 83-O, 220-O Hoshmand, M 59-P Hosoyamada, M 614-P Houstek, J 237-P Houten, SM 138-O Hrabincova, E 294-P, 365-P Hrebicek, M 422-P Hrstkova, H 294-P Hruba, E 4-P, 422-P, 614-P Hsiao, K-J 84-P Hsiao-Chi, Y 376-P, 50-P, 51-P Hsu, J-H 51-P, 376-P, 604-P Huang, AC 478-P, 539-P Hubbard, WC 257-O Hubert, L 204-P Huete, I 420-P Huidekoper, HH 130-P

Huijmans, JGM 520-P Huizing, M 195-P Hulkova, H 377-P Hussa, C 173-P Hutchin, T 469-P Hutchison, J 120-P Hwu, W-L 516-P, 463-P, 58-P, 478-P, 539-P, 390-O, 513-P Hyland, K 546-O Iastrebner, M 450-P Ibel, H 329-P Ichida, Kimiyoshi 614-P Ida, H 378-P, 392-O, 452-P Iida, M 389-P Iizuka, S 392-O IJlst, L 27-O, 133-O, 353-P Illsinger, S 611-P, 178-P, 134-P, 617-P, 121-P Imrie, J 496-P Ingrassia, R 54-P Innis, S 580-P Inokuchi, T 56-P Insinga, F 427-P Ito, T 86-P, 158-P Itoh, M 549-P Jachymova, M 620-P Jackson, M 192-P Jacoby, C 125-O Jacquemont, S 631-P, 218-P Jaeken, J 189-O, 509-P, 361-P, 254-P Ja«ggi, L 329-P Jahnova, H 422-P Jako¨bkiewicz-Banecka, J 445-P Jakobs, C 288-O, 277-P, 35-O, 33-P, 39-P, 547-O, 544-P, 37-P, 275-P, 280-O, 101-P, 45-O Jalan, A B 585-P Janosikova, B 45-O Jansen, EEW 544-P, 547-O Jansova, E 625-P Janssen, B 328-P, 30-O Janssen, MCH 331-P Janzen, N 121-P Jardim, H 458-P Jaubert, F 182-O Jaussaud, R 394-P Javier, RM 394-P Jeanjean, A 451-P Jeannet, PY 135-P Jemaa, R 310-P Jeninga, EH 375-P Jequier, M 631-P Jesina, P 4-P, 237-P Jia, J 380-O Jiang, H 32-O, 34-P Jo, H 389-P John, CM 109-A Jonas, J 142-P Jones, SA 221-P Jonkers, CF 577-P Joost, K 174-P Jorge-Finnigan, A 74-P, 65-P Josse, T 46-P, 43-P Julia, S 172-P Kaabachi, N 310-P, 360-P, 281-P Kabaroglu, C 370-P Kacena, K 408-P Kaga, F 616-P, 623-P Kakinuma, H 549-P Kalb, S 328-P, 490-P Kalender Korkmaz, F 512-P Kalkan Ucar, S 370-P, 166-P, 122-P, 127-P

J Inherit Metab Dis (2008) 31 (Suppl 1) Kalkanoglu Sivri, HS 443-P, 94-P Kalkhoven, E 375-P Kall, K 275-P, 174-P Kaminsky, P 142-P Kampmann, C 481-P, 425-P Kanthavelu, G 593-P Kanzelmeyer, NK 611-P Kao, C-H 50-P, 51-P, 376-P, 604-P Kaplan, P 408-P Karall, D 102-P Karavitakis, E 406-P Karimova, A 187-P Karli, OK 274-P Karlsson, A 233-P Kattamis, A 179-P Kaur, Manjit 597-P Kayykcioglu, M 370-P Kebbewar, M 30-O Keil, A 521-P Keiser, M 380-O Kemper, M 179-P Kemp£e, J 181-O Kemter, KF 327-O Kennedy, SJ 599-P Kersey, JP 480-P Kerstenetzky, M 405-P, 399-P Kestens, C 451-P Keularts, IMLW 535-P Keyser, B 91-O Khanna, R 507-P, 495-P Khemir, S 310-P, 360-P Khodary, A 595-P Khodary, H 600-P Khouja, N 310-P Kim, CA 405-P Kimmoun, A 82-P, 491-P Kimura, T 392-O Kiratli, P 94-P Kirk, R 163-O Kishnani, P S 517-P, 513-P, 514-P Kivity, S 181-O Kjeldsen, M 145-P Klein, I 525-P Kleinert, P 191-P Kloke, KMK 613-P Klomp, L 375-P, 2-P Klomp, LW 548-O Kloska, A 445-P Kloska, S 309-P Kluijtmans, LA 615-P, 220-O, 110-P, 209-P Knebel, L 23-P Knebel, LA 13-P, 80-P, 14-P Knerr, I 101-P Knight, SB 496-P Knopf, C 627-P Knospe, V 241-P Kobayashi, H 392-O Kobayashi, K 623-P Kobayashi, M 378-P Kocak, P 607-A Koch, HG 358-P Koch, J 207-O, 213-P, 206-P Koch, R 293-P Kodama, H 616-P, 623-P Koelker, S 137-P, 67-O Koeller, DM 73-P Koene, S 223-P, 208-P Kohan, R 511-P Kohda, Y 340-P Kohlschu«tter, A 488-P, 603-P Kohout, A 377-P Kok, F 530-P Kok, R 37-P

Kok, RM 39-P Kok, T 265-P Koletzko, B 309-P, 307-P Ko«lker, S 83-O, 73-P, 72-O Kollberg, G 247-O Kolling, J 6-P Kondo, N 107-O, 100-P Konecna, P 294-P Ko«nig, B 63-P Ko«nig, C 69-O Konijnenberg-Kramer, I 615-P Koning, TJ 320-P Konings, S 175-P Koopman, R 130-P Korall, H 526-P Korczyn, A D 181-O Korenke, GC 390-O Kornacka, MK 81-P Kornak, U 199-O Ko«rner, C 194-O, 193-P Kornhaber, G 400-P Kose, R 98-P Kosma, Konstantina 406-P Kossow, K 384-O Kostalova, E 113-P, 90-P, 422-P, 95-P Kowalik, A 81-P Kowaltowski, AJ 139-P Koyama, H 623-P Kozak, LK 365-P Kozich, V 422-P, 45-O Kozicz, TL 208-P Krabbi, K 174-P Kralik, L 622-P Kralovicova, J 336-P Kramer, P 498-P Kraoua, IK 281-P Kraus, JP 45-O Kriegbaum, H 364-O Krijt, J 628-P Kronn, D 312-P Kruijer, N 498-P Kruse, B 241-P Krywawych, S 129-P Krzyzçanowska, J 373-P Kue©inskas, V 487-P Kubota, T 482-P Kuchar, L 422-P, 423-P, 424-P Kuppens, S 175-P Kurek, AG 6-P Kurono, Y 86-P Kurt, I 443-P, 621-P, 108-P Kusmierska, K 81-P Ku«ster, A 352-P, 43-P Kuvbachieva-Bennarosh, A 15-P Kuznetsova, LI 325-P Kyllerman, M 73-P Kyosen, SO 392-O Kyriakopoulou, L 532-P La Briola, F 359-P La Marca, G 529-P, 630-P Labarre-Vila, A 136-P Lacaille, F 93-P Lacerda, L 258-P, 263-P, 407-P 428-P, 444-P, 440-P, , 458-P Lachmann, RH 501-P, 502-P, 508-P Laforet, P 379-O Laforeªt, PL 383-A Laframboise, R 244-O, 346-P Lagler, F 102-P Laht, TM 174-P Lake, S 379-O Laluhova¨-Strie-Encova¨, Z 619-P Lam, W 22-P Lama, RA 103-P

165 Lambert, M 244-O, 346-P Lambruschini, N 5-P Lamhonwah, AM 143-O Lammens, M 195-P Land, JM 149-P, 536-P, 546-O Landvogt, C 296-O Lang, TF 219-P Lange, A 373-P Langius, FAA 537-P Lara Herguedas, J 354-P Laranjeira, F 258-P Laro¨vere, LE 347-P Larsson, NG 233-P Lastrico, A 448-P Latypov, AS 325-P Lauandos, JR 397-P, 31-A Lauro, EM 475-P Lavallee, P 525-P Lazzarino, G 631-P Le Bizec, C 172-P, 190-P Le Franc°ois, T 352-P Le Moyec, L 204-P Leal, F 74-P Leal, VF 587-P Leandro, J 306-P, 304-O, 305-P, 303-P Leandro, P 303-P, 305-P, 131-P, 304-O, 306-P, 297-P Lea¬o Teles, E 85-P, 111-P, 123-P, 156-P, 344-P, 411-P, 431-P, 433-P, 458-P, 530-P Lebre, AS 112-O Ledvinova, J 422-P, 423-P, 424-P Lee, B 362-P Lee, IC 230-P Lee, NC 539-P, 478-P, 50-P Lee, PJ 501-P, 561-P, 566-O, 508-P, 164-P, 168-P Lee, WT 539-P Lefeber, DJ 195-P, 199-O Legrand, A 136-P Lehesjoki, A 523-P Lehotay, DC 144-P, 374-P Leipnitz, G 13-P, 68-A, 70-P, 71-P, 80-P, 14-P Leist, A 60-P Leite, E 592-P, 565-P, 591-A Leite, I 371-P Leite, VHR 404-P Lemerrer, M 434-P, 460-P, 457-P Lemieux, B 346-P Lemoine, K 473-P Lemoine, M 460-P, 434-P Lemos, M 258-P, 263-P Lenfeld, J 423-P Leonard, JV 73-P, 140-P, 63-P Lerche, H 181-O Lesko, N 233-P Leslie, N 513-P Leuzzi, V 302-P, 300-P, 286-P, 334-P, 283-P, 289-P, 282-P Levandovskiy, V 211-O Levent, E 127-P Levin, S 148-P Lewandowska, E 506-P Lhotak, S 32-O Li, F 230-P Li, Z 154-P Liammongkolkul, S 463-P Liang, W 495-P Lidove, O 525-P Lilburn, MF 168-P, 164-P, 566-O, 561-P Lilje, C 396-P Lima, MR 315-P, 574-P, 558-P Lim-Melia, E 312-P Lindner, M 30-O, 83-O, 364-O

Linthorst, GE 479-P Lissens, W 16-P, 215-P, 214-P, 451-P Listopad, GG 325-P Lisy, V 237-P Liu, MY 84-P Liu, TT 84-P, 604-P, 51-P, 376-P, 50-P LLerena, J 405-P Lluch, M 484-P Lo, HC 604-P Lo, MY 604-P, 376-P, 51-P, 50-P Lo, S 53-P Lobo Antunes, M 297-P, 131-P Lock Hock, N 629-P Lockhart, DJ 387-P, 427-P, 495-P, 507-P Lombardi, G 170-P, 184-P, 169-P Lopes, GC 594-A, 391-A Lopes Lima, J 385-O Lo¨pez Mar|¨ n, L 354-P Lopez-Hernandez, T 177-P Lo¨pez-Piso¨n, FJ 308-P Loughrey, CL 219-P Lourenco, CM 522-P Lu, YH 50-P 51-P, 376-P, 604-P Lu«bbehusen, J 194-O Lucioli, S 220-O Lu«cke, T 617-P, 121-P, 134-P, 611-P, 178-P Luf S 328-P, 490-P Lugowska, A 506-P Luis, PBM 27-O Lukacs, Z 124-P, 603-P, 521-P, 488-P Lukina, E 450-P Lund, AM 243-P Lundgren, J 61-P Lunsing, RJ 589-P Luz, A 131-P Luzy, C 390-O Lynes, G 536-P, 149-P Lyonnet, S 460-P Maag, C 191-P Maas, M 509-P Maca¨rio, MC 235-P, 246-P, 232-P, 203-P MacDonald, A 556-O, 557-P, 559-P, 465-P, 576-O, 575-P, 562-P, 561-P, 563-P, 330-P Macedo, MF 412-P MacFarlane, J 523-P Macias, J 484-P MacKay, N 211-O Maclean, KN 34-P, 32-O Madignier, F 239-P Madsen, PP 145-P Maeda, Y 86-P Maegawa, GHB 400-P, 409-P Magalha¬es, A 85-P, 458-P, 433-P, 437-P Magen, D 627-P Magera, MJ 257-O Magner, M 4-P, 628-P Magnet, M 417-P Mahringer, A 67-O Mahuran, D 400-P Mai, L 143-O Maier, EM 83-O, 72-O Maillot, F 164-P Maj, MC 494-P, 210-P, 211-O Makushenko, EA 467-P Ma¨laga, I 185-P Malaver, LF 438-P Malich, S 191-P

166 Malinova, V 377-P, 422-P Malinowska-Polubiec, A 81-P Maljevic, S 181-O Mallunat, L 121-P Malvagia, S 355-P, 529-P Manara, R 318-P Mancardi, MM 289-P Mandel, H 513-P Mannamaa, M 275-P Manning, NJ 128-P, 129-P, 256-P Mano, A de la 103-P Mansi, G 486-P, 527-P Mansour, H 160-P, 499-P Maranda, B 244-O, 346-P, 230-P Marca¬o, A 596-P, 111-P, 131-P, 123-P Marcelis, C 490-P Marecek, Z 622-P Marek, J 140-P Mari, JF 500-P Marinero, B 105-P Mariotti, P 8-P Maritz, CM 566-O Marklova, E 198-P Markovich, D 291-O Marongiu, F 29-O Marques, JS 371-P Marques, M 123-P Marques, VD 522-P Marques, Jr W 522-P Marsden, D 514-P, 517-P Martasek, P 620-P, 610-P, 622-P Martens, GA 115-O Marti, R 217-P Martin, E 554-P Mart|¨ n, MA 171-P, 238-P Mart|¨ n, S 285-P Martinelli, D 8-P Martinez, A 545-P Martinez, LD 152-P Mart|¨ nez Mu·oz, C 280-O Martinez-Hernandez, E 339-P Martinez-Rubio, D 550-O Mart|¨ n-Herna¨ndez, E 227-P Mart|¨ n-Jime¨nez, R 227-P Martins, AM 382-P, 381-A, 477-P, 476-P, 475-P, 31-A, 401-P, 399-P, 397-P, 592-P, 591-A, 565-P, 403-P Martins, E 357-O, 111-P, 11-A, 530-P, 411-P, 202-P Martins, F 436-P Martins, P 413-P Marucha, J 445-P, 449-P Masand, R 585-P Matalon, R 333-O Matalon, RK 44-P, 106-O, 57-O Matern, D 79-P, 151-P Matsuishi, T 56-P Matsumoto, S 461-P Matsuo, N 107-O Matte, U 493-P, 402-P Matthews, AJ 128-P Matthijs, G 189-O, 201-P Mattos, CB 6-P Matulevich, SA 325-P Mavridou, I 197-P Mayatepek, E 10-P Mayer, M 172-P Mayhew, J 379-O Mayr, J Mayr, JA 206-P, 207-O, 213-P, 239-P, 251-P Mazdarani, Sh 176-P McDonald, A 79-P McKay, F 95-P McKiernan, P 19-P

J Inherit Metab Dis (2008) 31 (Suppl 1) McMahon, D 76-P, 75-P McRoberts, C 599-P McSweeney, M 132-P, 342-P McWhinnie, C 566-O McWilliam, CA 551-O McWilliam, RC 551-O Medeiros, PFV 399-P Meijer, J 618-P Meili, D 336-P Meincke, S 77-O Meinecke, P 200-P Meinsma, R 618-P Meitinger, T 229-P, 239-P Mejzlik, V 626-P Melberg, A 247-O Melis, D 169-P, 184-P, 170-P Mello, AS 500-P Melo, FHC 404-P Melzi, ML 448-P, 456-P Menda·a, L 227-P Mendes, C 225-P, 203-P, 232-P Mendes, CSC 31-A Mendes, I 415-P Menegatti, E 403-P Mengel, E 296-O Menheere, P 175-P Menke, T 366-P Mention, KM 383-A Meow Keong, T 629-P Mercuri, E 8-P Merinero, B 74-P, 606-P, 367-P Merlini, L 517-P Merten, M 82-P Meschini, MC 220-O Messing, DD 327-O Mettler, G 599-P Metzger, D 179-P Meyburg, J 364-O Meyer, A 384-O Meyer, U 178-P Michailova, SV 398-P Michalek, J 626-P Michals-Matalon, K 57-O, 106-O, 333-O Michelakakis, H 197-P, 406-P Micheletti, C 31-A, 401-P, 403-P, 592-P, 399-P, 591-A, 565-P, 397-P Michelin-Tirelli, K 500-P Michot, C 157-P Miebach, E 425-P Mienie, LJ 196-P Mierzewska, H 506-P Mila, M 492-P Milburn, JL 599-P Milh, M 160-P Millington, DS 145-P, 395-O Mills, P 192-P Minghetti, D 89-P Minichini, L 169-P, 527-P Minnich, S 151-P Mira, G 119-P Miranda, M 477-P Mirazita, P 316-P Mistry, PK 441-P Mitchell, G 346-P, 244-O Mitsubuchi, H 461-P Mochida, T 461-P Moc°o, C 343-P Mo¢di, S 312-P Mohamed, S 595-P, 600-P, 153-P Moldovan, L 485-P Mole, SE 511-P Moliner, S 177-P MÖller-Larsen, S 145-P Moncayo, F 603-P Montejo, M 455-P

Montero, R 217-P Montesani, M 313-P Montoya, J 217-P Moog, U 490-P Mooijer, PAW 269-P, 268-O Moore, S 190-P Moradian, R 176-P, 104-P Mora¨is, A 103-P Moraitou, M 197-P Mora¨n, M 238-P Morath, MA 67-O, 137-P Morava, E 195-P, 199-O, 209-P, 208-P, 83-O, 223-P, 222-P, 615-P, 200-P Moreira, D 371-P Morel, CF 494-P Morelle, W 188-P Moreno, JM 5-P, 554-P Morgan, C 37 9-O Moricca, MT 319-P Morin, C 244-O Morita, A 392-O MÖrkrid, L 393-P Morley, DW 164-P Morris, AAM 221-P Morrone, A 146-P, 497-P, 527-P, 447-P, 446-P, 88-P, 529-P Morville, P 66-P Moseley, KD 293-P, 483-P Moser, AB 257-O Moszczy·ska, E 262-P Mota, A 567-O Mota-Castelo, T 567-O Moura, C 458-P Moura¬o, T 405-P Moviat, M 110-P Mrazova, L 78-P Muehl, A 585-P Mueller, M 124-P Mu«eller-Felber, W 517-P Mu«hlhausen, C 72-O, 73-P, 384-O, 91-O Muller, KB 476-P Mu«ller, E 73-P Mu«ller, KB 477-P, 382-P, 381-A Mundy, H 216-P Munnich, A 93-P, 288-O, 236-P, 204-P, 112-O Muntau, AC 327-O Murdoch, G 101-P Muschol, NM 384-O, 396-P Mussa, A 292-P, 317-O, 543-O Myers, S 573-P Naess, K 233-P Nagao, M 86-P Naim, HY 380-O Naito, E 147-P Nakajima, Y 86-P, 158-P Nakamura, K 461-P Nakamura, Y 461-P Nakatsu, T 147-P Namour, F 47-P Nanba, E 389-P, 443-P Nasrallah, FN 281-P Nassogne, MC 451-P Nassrallah, F 310-P, 360-P Nastasi, A 323-P Natalia, P 342-P Naughten, ER 73-P Navarrete, Rosa 28-P Navarro-Azna¨rez, H 308-P Navarro-Sastre, A 60-P, 217-P Navas, P 217-P Naylor, EW 75-P, 76-P Nennesmo, I 233-P

Netto, C 493-P, 588-P, 321-P, 322-P Neumaier-Probst, E 73-P, 72-O Neves, N 224-P Niaudet, P 93-P, 92-P, 99-P Nicolai, J 535-P Nicolino, M 513-P Nieves Cobos, P 521-P, 603-P Niezen-Koning, KE 348-P Nihoul-Fe¨ke¨te¨, C 182-O Nikitina, NV 325-P Niklowitz, P 366-P Nikolaeva, EB 325-P Niu, DM 604-P, 376-P, 51-P, 84-P Noel, E 394-P Nogueira, C 357-O, 542-P, 530-P Noher de Halac, I 511-P Nordenstro«m, A 114-P Noronha, M 111-P, 123-P Novikov, PV 325-P Novotna, Z 422-P Numata, S 340-P Nuo¡er, JM 364-O Nyberg, G 114-P O'Brien, F 450-P O'Brien, J 151-P Odent, S 172-P Odent, T 434-P Oemardien, L 524-P Oerton, J 129-P O£az, H 41-P Ofman, R 255-P, 27-O Ogawa, S 389-P Ogier de Baulny, H 136-P, 42-P Oglesbee, D 151-P Ohashi, T 392-O, 378-P, 482-P Ohashi, T 452-P Ohmi, H 86-P Okada, J 56-P, 25-P Okubo, RM 401-P Okun, JG 137-P, 67-O, 73-P Okur, I 582-P, 609-P, 117-P, 126-A, 442-A, 12-P, 426-P Okuyama, T 471-P, 482-P Olbrich, H 77-O Oldfors, A 247-O Oliveira, A 581-P, 337-P Oliveira, AB 260-P, 54-P Oliveira, AC 382-P Oliveira, B 337-P, 581-P Oliveira, CR 232-P, 203-P, 225-P, 3-P, 246-P, 224-P, 205-P Oliveira, JP 458-P, 412-P Oliveira, LR 594-A, 391-A Oliveira, M 203-P, 232-P, 246-P Oliveira, RB 31-A, 397-P, 591-A, 565-P, 592-P Oliveira, S 203-P, 232-P Oliveros, L 554-P Olkhovich, NV 467-P Oller Ram|¨ rez, AM 511-P, 264-P Olpin, SE 256-P, 128-P, 129-P, 140-P Olsen, RKJ 141-O Omran, H 77-O O'Neill, G 265-P Orfanou, I 538-P Orii, T 482-P Ormaza¨bal, A 538-P, 550-O Ornelas, R 38-P Orton, R 168-P Òstergaard, E 243-P O¬unap, K 174-P, 275-P Owens, G 598-P Ozer, I 98-P, 41-P

J Inherit Metab Dis (2008) 31 (Suppl 1) Ozerova, LS 608-P Ozkara, HA 443-P Ozon, ZA 26-P, 358-P Ozturk, O 108-P, 621-P Ozyurek, R 127-P Paccou, A 1-P Paci, S 316-P, 159-P, 161-P, 314-P, 162-P Packman, S 28-P Paesold-Burda, P 528-P, 191-P Pagano, R 466-P Pais-Vieira, M 415-P, 432-P Paladino, S 323-P, 48-P Palau, F 550-O Palling, DJ 507-P, 427-P, 387-P Pamula, V 395-O Papadia, F 88-P Papadopoulou, D 61-P Papo, T 525-P Parenti, G 454-P, 426-P, 527-P, 88-P, 170-P, 184-P, 169-P, 323-P, 459-P, 486-P Parini, R 481-P, 459-P, 456-P, 448-P, 426-P, 446-P, 88-P, 447-P Park, J 517-P ParvizI, SH 104-P Pascale, MG 319-P Paschini-Capra, AE 152-P Paschke, E 497-P Pascual, JM 185-P Pasquini, E 146-P, 355-P, 429-P, 529-P, 88-P Patenaude, J 586-P Patr|¨ cio, Z 564-P Patterson, MC 390-O, 129-P Paul, HS 547-O, 29-O Paul, K 497-P Paula, AC 405-P Pauli, S 349-O Paulova, M 113-P, 183-P, 95-P Pavlova, Z 483-P Pavlovic, S 301-P Payne, JH 167-P Pearl, PL 101-P Pecinova, A 237-P Peck, D 79-P Pedersen, CB 145-P Pederzolli, CD 6-P Pedro¨n Giner, C 354-P Peeters, R 509-P Pekkala, S 356-O Pellegrino, L 495-P Peng, SF 478-P Pennell, S 97-O Peper, W 351-P Perdiga¬o, S 258-P Pereira, A 40-P Pereira, F 493-P Pereira, TM 399-P Pereira, VG 476-P, 477-P, 475-P Perez, B 105-P, 301-P, 65-P, 74-P, 606-P, 201-P Perez Poyato, MS 492-P Pe¨rez-Cerda¨, C 201-P, 367-P, 606-P, 103-P Pe¨rez-Due·as, B 62-P, 185-P, 60-P Perrier, J 16-P Pestronk, A 379-O Peter, M 134-P Peters, H 97-O Peterschmitt, J 450-P Pevalova¨, L 619-P Philippe, A 288-O Piazzon, FB 401-P, 403-P

Piccirillo, A 313-P Piccolo, P 170-P Pichkur, NA 467-P Pickers, P 110-P Pietrzyk, JJ 295-P Pimenta, A 38-P Pimentel, H 592-P, 565-P, 591-A Pine, C 495-P Pineda, M 217-P, 62-P, 390-O, 550-O, 279-P, 60-P, 185-P Pineda Marfa, M 492-P Ping-Yao, H 51-P, 376-P Pinheiro, A 250-P, 252-P Pinheiro, TMM 594-A, 391-A Pinto, E 444-P Pintos-Morell, G 481-P Piotrowska, E 445-P Pipeleers, D 115-O Piperata, MR 515-P Pires, RF 54-P Pitt, J 186-P, 97-O Pivonello, R 169-P, 184-P, 170-P Pizarro, MX 391-A, 594-A Poh, S 109-A Pohorecka, M 20-P, 20-P Poisetti, P 446-P Polat, N 41-P Poll The, BT 537-P, 255-P, 267-P Polyakova, SI 624-P Ponec, J 619-P Pons, R 538-P, 75-P, 76-P Ponzone, A 543-O, 317-O, 302-P, 329-P Po¨o, P 279-P, 62-P Poorthuis, BJHM 468-P Pope, L 101-P Popovici, D 194-O Popowska, E 341-P Porchet, N 360-P Porta, F 302-P, 543-O, 317-O, 292-P, 329-P Porto, C 454-P Pospisilova, L 622-P Potocnakova, L 78-P Pouchla, S 625-P, 626-P Poupetova, H 377-P, 422-P Powe, AC 495-P Pozzessere, S 302-P, 300-P Prabhakar, P 192-P Prasad, AN 148-P Prasad, C 148-P Prata, MJ 24-P, 440-P Pratas, J 225-P, 246-P, 232-P, 203-P Preece, MA 557-P, 561-P, 17-P, 129-P Prieto, JA 276-P, 285-P, 455-P Prinsen, BHCMT 118-P, 375-P, 2-P Prochazkova, D 626-P, 294-P Procopio, E 447-P, 429-P, 146-P Proenc°a, T 224-P Prokisch, H 239-P, 206-P, 229-P Pronicka, E 20-P, 154-P Pruna, L 142-P Puchmajerova, A 622-P, 620-P Puga, AC 450-P Puthi, V 16-P Puusepp, H 275-P Quadros, E 533-P, 290-O Queiro¨s, A 297-P Queiroz, RR 594-A, 391-A Quelhas, D 357-O, 277-P Quental, R 343-P Quental, S 24-P Quijada Fraile, P 354-P

167 Quinlivan, R 245-P Quintana, E 177-P Quirino, BEG 404-P Rabier, D 112-O, 99-P, 92-P, 93-P, 9-P Radauer, W 206-P Ra¡o, E 172-P Rahman, S 221-P, 242-O Raiman, J 211-O, 210-P Raimann, E 105-P Rajadurai, VS 109-A Rajaii, A 612-P Rakowicz, M 506-P Ramaekers, V 533-P, 290-O Ramalheiro, G 413-P Ramaswami, U 163-O, 481-P, 480-P, 502-P Ramos, A 131-P Ramos, R 40-P Rampazzo, A 456-P, 459-P Rand, MH 403-P, 31-A Ranieri, E 87-P, 598-P Rapsomaniki, M 319-P Raqbi, F 9-P Raskin, S 592-P, 565-P, 591-A Rasmussen, E 222-P Ratai, E 265-P Rauscher, C 206-P, 213-P Raymond, KR 613-P, 151-P Rebelo, O 240-P Rebollido, MM 630-P, 602-P Rebu¡at, A 335-O Refaelli, C 588-P Refosco, L 588-P Re¨gal, L 361-P, 254-P, 253-P Reijngoud, DJ 348-P, 589-P Reims, A 61-P Renaud, B 417-P Renaud, DL 345-P, 287-O Reynders, E 189-O Reynes, N 416-P Rhead, WJ 53-P Ribeiro, CAJ 70-P, 80-P, 71-P, 71-P Ribeiro, EM 399-P, 405-P Ribeiro, H 428-P, 440-P Ribeiro, I 444-P, 428-P, 503-P Ribeiro, MG 503-P, 505-P, 466-P Ribeiro, MJ 182-O Ribes, A 96-P, 279-P, 217-P, 62-P, 339-P, 60-P Ricarte, A 403-P Ricci, R 447-P Richard, E 74-P Ridout, CK 249-P, 551-O Rigal, O 42-P Rigat, B 400-P Rigoldi, M 456-P, 448-P Rinaldo, P 79-P Rinco¨n, A 606-P Rings, EHHM 348-P Risto¡, E 10-P Rita, A 179-P Ritmiller, R 600-P Riudor, E 171-P Riva, E 316-P, 89-P, 359-P, 159-P, 372-P Rivera, H 238-P Rivera, I 252-P, 250-P, 40-P, 33-P, 35-O, 297-P, 38-P, 37-P, 131-P, 177-P Rizzo, C 602-P, 220-O Rozçdzçyn¨ska, A 449-P Roato, I 292-P Robalo, C 205-P Roberts, M 472-P

Robinson, BH 144-P, 211-O, 244-O, 210-P Rocha, H 596-P, 131-P, 123-P, 111-P Rocha, JC 574-P, 315-P, 558-P Rocha, LOS 404-P Rocha, M 35-O, 33-P, 37-P Rocha, P 11-A, 202-P Rocha, VL 594-A Rodenburg, RJ 209-P, 223-P, 208-P, 199-O, 206-P Rodes, M 339-P Rodrigues, D 439-P Rodrigues, E 123-P, 111-P, 411-P, 344-P, 85-P, 431-P, 156-P, 433-P, 458-P Rodrigues, F 436-P Rodrigues, GF 402-P Rodrigues, LC 475-P Rodrigues, LG 412-P, 437-P, 432-P, 415-P Rodrigues, MDB 382-P, 477-P, 476-P, 381-A Rodr|¨ guez-Pascau, L 606-P Rodr|¨ guez-Pombo, P 28-P Roels, F 214-P Rohrbach, M 390-O Rokicki, D 341-P Rola, R 506-P Rolinski, B 229-P, 349-O, 251-P, 239-P, 206-P Romano, A 486-P, 527-P Romano, S 157-P Rombach, SM 479-P Rosas, MJ 235-P Rose, C 394-P Rosenbaum, H 450-P Rosenberg, EH 280-O Rosenbergerova, T 78-P Rosenbloom, B 379-O Ross, N 57-O Rossi, B 454-P Rostasy, K 102-P Rotkova, J 423-P Roulet-Perez, E 631-P Roullet, E 525-P Roussey, G 43-P Roux, C 631-P Roze, JC 352-P, 43-P Rubio, V 356-O Rubio-Gozalbo, E 535-P Rubio-Gozalbo, ME 175-P, 175-P Ruijter, GJG 520-P, 468-P Ruitenbeek, W 110-P, 615-P Ruiter, JPN 145-P, 353-P, 27-O, 138-O Ruiz-Sala, P 367-P Rupar, CA 148-P Rupar, T 494-P Rusli Sjarif, D 555-P Rylance, G 562-P Ryvlin, P 181-O Saber, S 59-P Sacco, A 8-P Sadou-Yaye, H 190-P Saito, K 618-P Sakakibara, Y 389-P Sala, F 314-P Sala, S 456-P, 459-P Salazar, MI 308-P Salcedo, G 574-P Salehpour, S 518-P, 612-P Salerno, M 170-P, 184-P Saligova, J 78-P Salmon, A 491-P

168 Salome©, S 48-P Salomons, GS 278-P, 288-O, 101-P, 280-O, 275-P, 277-P Salvatici, E 314-P, 161-P, 162-P, 89-P, 329-P Salviati, L 361-P Sa¨ Miranda, MC 415-P, 432-P, 437-P, 412-P, 439-P, 385-O Sampietro, F 48-P Samson, Y 525-P Sancho-Vaello, E 356-O Sander, J 121-P Sanjurjo, P 276-P, 285-P, 455-P Sanli, 607-A Sanmart|¨ , FX 62-P, 279-P Sanseverino, MT 588-P Santagata, S 283-P, 286-P, 334-P Santana, I 225-P Santana, L 565-P, 592-P, 591-A Santer, R 396-P, 296-O, 200-P, 124-P, 241-P, 165-P, 368-P, 488-P, 179-P, 329-P, 521-P, 603-P Santiago, B 227-P Santorelli, FM 220-O, 530-P, 234-P, 235-P, 339-P, 511-P Santos, H 156-P, 371-P, 411-P Santos, HH 594-A, 391-A Santos, M 11-A, 202-P Santos, MJ 203-P, 225-P, 246-P Santos Leite M 177-P Santra, S 19-P Sarykabadayy, YU 26-P Saraiva-Pereira, ML 402-P Sass, JO 102-P, 69-O, 77-O Satiro, CAF 591-A, 592-P, 565-P Sauer, SW 67-O, 137-P Sauerwein, HP 130-P Scala, I 48-P, 323-P Scalco, FB 587-P, 584-A Scarlino, S 372-P Scarpa, M 456-P, 426-P, 459-P, 447-P Scarpato, E 323-P, 48-P Scharf, M 179-P Scheible, D 324-P Scheijen, JLJM 284-P Scherer, T 545-P Schieber, P 390-O Schi¡, M 42-P Schi¡mann, R 507-P, 489-P Schlotawa, L 388-O Schmidt, B 388-O Schmidt, J 364-O Schmidt, KH 121-P, 617-P Schneider, A 193-P Schneider, E 427-P Scholl-Bu«rgi, S 102-P Schoonderwoerd, GC 180-P Schoonderwoerd, K 79-P Schott, DA 535-P Schuck, PF 39-P, 14-P Schuler, A 329-P Schulz, H 138-O Schulze, A 22-P, 350-P, 210-P Schurr, TG 226-P Schutz, PW 580-P Schwartz, IV 322-P, 321-P Scott, D 69-O Scott, K 427-P Sebesta, I 614-P Sedel, F 390-O, 525-P Seitz, A 72-O Sekarski, N 135-P Seminotti, B 70-P, 13-P, 68-A, 71-P, 14-P, 80-P Sempere, A 279-P

J Inherit Metab Dis (2008) 31 (Suppl 1) Seneca, S 214-P, 215-P Sentner, CP 165-P Sequeira, J 533-P, 290-O Sequeira, S 343-P, 119-P, 470-P, 530-P Seri, S 19-P Serrano, M 538-P Sestito, S 313-P Seta, N 188-P, 190-P, 192-P, 172-P, 160-P Shahbek, N 30-O Shanaa, A 600-P Shao, S 495-P Sharrard, MJ 128-P, 167-P, 163-O, 154-P Shenker, A 387-P Sherlock, M 120-P Shichijo, K 147-P Shield, JPH 69-O Shiga, K 616-P Shigematsu, Y 147-P Shimohama, M 347-P Shintaku, H 541-P, 311-P Shoemaker, J 53-P Shulze, A 211-O Sibilio, M 88-P, 323-P, 527-P Siga, K 623-P Siintola, E 523-P Silva, E 444-P, 407-P Silva, MFB 353-P, 131-P, 27-O Silva, MJ 250-P, 252-P Silva, S 567-O, 205-P Silva, TM 54-P Silveira, F 85-P Silveira, MT 397-P Sim, K 278-P Simmons, L 469-P Simo¬es, M 232-P, 203-P, 225-P, 3-P Simo¨n, R 554-P Simoni, RE 587-P Simonsen, N 306-P Singh, RHS 326-P Sinici, I 443-P Sirrs, SM 473-P Sitaraman, SA 507-P Sitta, A 322-P, 321-P, 260-P Sivri Serap, H 358-P, 248-P Skorecki, K 627-P Skrinar, A 3 79-O Skvorak, KJ 29-O, 547-O Sladkova, J 628-P Smeitink, JAM 208-P, 209-P, 223-P Smet, JE 16-P, 215-P, 214-P Smit, GPA 165-P, 348-P Smith, DEC 39-P Smith, LD 69-O Smith, SE 450-P Smulders, YM 39-P, 37-P Snead, OC 544-P Soares, G 444-P Soares, I 574-P Soares, S 357-O, 344-P Solanki, G 435-P Solano, A 13-P, 14-P Solaroli, N 233-P Songailiene, J 487-P Sorge, G 409-P Soska, R 495-P Sousa, C 123-P, 131-P, 111-P, 596-P Sousa, M 250-P Sousa, MM 432-P Souza, C 565-P Souza, CFM 493-P, 588-P, 592-P, 591-A, 54-P

Souza, CP 594-A Souza, FTS 522-P Sovik, O 165-P Soysal, AS 582-P Sozmen, E 166-P Spaapen, LJM 550-O, 175-P, 535-P Spada, M 292-P, 543-O, 317-O Sperandio, M 194-O Sperl, W 213-P, 207-O, 239-P, 251-P, 206-P Spiekerko«tter, U 10-P, 125-O, 124-P Spijkerman, AMW 39-P Srinivasan, V 395-O Stabler, SP 34-P, 45-O Sta¡ord, J 572-O Stange¨, G 115-O Stansbridge, EM 266-P Starostecka, E 373-P Stastna, S 90-P, 113-P, 422-P, 95-P, 183-P Staudigl, M 327-O Stefanello, FM 6-P Stefanini, MC 8-P Stehn, M 124-P Steinberg, SJ 259-O Steinmann, B 165-P, 191-P Stenbroen, V 145-P Stern, E 242-O Steuerwald, U 165-P Stiburek, L 231-P Stiburkova, B 614-P Sto«ckler, S 580-P Stojiljkovic, M 301-P Stoll, M 138-O Stopar Obreza, M 155-P Stove, V 504-P Straczeck, J 82-P, 491-P Stradomska, TJ 262-P Strisciuglio, P 302-P, 319-P, 313-P Strnova, J 78-P Strom, S 547-O, 29-O Struys, EA 544-P, 580-P Stucki, M 528-P, 52-O Sugiyama, N 158-P, 86-P Suheir, A 627-P Sulek, S 4-P Suls, A 181-O Sun, Q 547-O Suormala, T 52-O, 528-P Surtees, RA 73-P Suzucs, S 106-O Suzuki, Y 389-P, 482-P, 443-P Svebak, RM 545-P Sweeney, MG 242-O Sweetman, L 489-P Sykut-Cegielska, J 20-P, 81-P, 341-P Szalat, R 525-P Sznajer, Y 179-P Taback, S 374-P Tabatabaie, L 548-O Tahara, EB 139-P Tajiri, H 461-P Talbot, RM 256-P Talvik, I 275-P Tam, C 143-O Tambde, S 585-P Tan, ES 109-A Tanaka, A 482-P Tanaka, T 471-P, 482-P Tanaka, Y 147-P Tang, K 495-P Tang, LY 230-P

Tanjung, C 519-P Tanner, S 154-P Tanzer, F 512-P, 7-P Tapia Anzolini, V 511-P Tarallo, A 454-P Tashiro, K 56-P Tasic, V 179-P Tavares de Almeida, I 133-O, 37-P, 250-P, 252-P, 303-P, 305-P, 131-P, 111-P, 38-P, 297-P, 306-P, 304-O, 353-P, 40-P, 33-P, 35-O, 123-P, 27-O Tavazzi, B 631-P Taverna, M 229-P, 239-P Taybert, J 154-P Taylor, RW 242-O te Brinke, H 138-O Tea, I 43-P Tebib, N 310-P, 360-P Tedesco, L 448-P Teerlink, T 35-O, 39-P Tein, I 143-O, 144-P, 120-P Teixeira, CA 466-P, 505-P Telawane, ND 585-P ter Veld, F 125-O, 124-P Terlemez, S 370-P Terpstra, AM 110-P Tesarova, M 229-P, 239-P Tessa, A 339-P Teunissen, QGA 464-P Thiel, C 193-P Thimm, E 10-P Thomas, A 53-P Thomas, JA 579-P, 573-P Thomas, N 173-P Thompson, R 374-P Thony, B 336-P, 335-O, 545-P Ticus, I 160-P Till, J 129-P Timmer, C 577-P Togari, H 86-P, 158-P Tokatly, A 358-P, 248-P, 443-P, 94-P, 274-P Tolmie, J 551-O Tonin, AM 23-P, 14-P Topcu, M 274-P Torrent, C 272-O Torres, I 38-P Tortorell, S 79-P Tortorella, G 515-P Tortorelli, ST 613-P, 257-O Toscano, P 522-P Tosetti, M 282-P Touati, G 93-P, 112-O, 99-P Trefz, FK 324-P, 330-P, 526-P Trindade, ALC 594-A Trindade, E 344-P Trinh, MU 598-P Troncoso, M 72-O Troost, D 255-P Tropak, MB 400-P, 409-P Troxler, H 191-P Tsai, LK 478-P Tseng, SC 58-P Tsiakas, K 368-P, 165-P, 200-P, 241-P Tsikas, D 611-P Tsvetkova, IV 398-P Tsygankova, PG 150-P Tubiana, S 136-P Tuharsky, J 619-P Tulinius, M 247-O Tumer, L 117-P, 126-A, 609-P, 582-P, 442-A, 12-P Tunc°, 7-P Turgeon, CT 257-O Tuysuz, B 426-P

J Inherit Metab Dis (2008) 31 (Suppl 1) Tuzzi, R 454-P Tylki-Szyma·ska, A 262-P, 445-P, 449-P, 426-P, 516-P, 506-P Tyring, SK 333-O, 106-O Ueki, I 340-P Ugalde, C 238-P Ugarte, M 301-P, 201-P, 74-P, 606-P, 367-P, 105-P, 65-P, 28-P Ulbrichova, D 610-P Ullrich, K 165-P, 91-O, 384-O, 296-O, 396-P Umemoto, T 147-P Ungaro, C 323-P, 527-P Urban, Z 199-O Uziel, G 426-P Va¢na, ZI 325-P Vagnoni, C 302-P Valadares, ER 391-A, 404-P, 594-A, 591-A, 565-P Valayannopoulos, V 157-P, 92-P, 9-P, 288-O, 182-O, 352-P, 434-P, 204-P, 112-O, 99-P, 460-P, 457-P, 93-P Vale, MC 470-P Valenzano, KJ 507-P, 495-P Valiente, A 105-P Valongo, C 277-P Valstar, MJ 468-P Van Bokhoven, H 195-P Van Caeseele, P 374-P Van Coster, RN 16-P, 253-P, 214-P, 215-P van Cruchten, A 353-P Van Damme-Lombaerts, R 361-P, 189-O van de Berg, A 223-P Van de Casteele, M 115-O van den Berg, AMJ 222-P van den Berg, L 498-P van den Berg, RG 520-P van den Broek, NJF 548-O van den Heuvel, B 208-P van den Heuvel, LP 209-P, 223-P van den Hurk, TAM 320-P, 570-P Van der Ham, M 2-P van der Jagt, EJ 18-P van der Linden, IJM 36-O, 49-P van der Louw, EJTM 570-P, 498-P van der Ploeg, AT 498-P, 514-P, 517-P, 379-O van der Ploeg, E 577-P van Diggelen, OP 524-P, 468-P, 180-P Van Driessche, M 253-P Van Dyck, T 181-O van Egmont Petersen, M 36-O Van Engelen, B 195-P Van Esch, H 181-O van Giersbergen, P 409-P van Kuilenburg, ABP 618-P Van Laere, K 181-O van Loon, LJC 130-P van Mierlo, I 490-P Van Paesschen, W 181-O van Schaik, IN 421-P van Schrojenstein Lantman-de Valk, H 490-P

van Spronsen, FJ 574-P, 18-P, 348-P, 589-P, 330-P, 577-P van Straten, HW 36-O Van Thi, HV 115-O VanDongen, J 291-O van't Ho¡, W 63-P Vanzin, C 321-P Vargas, A 181-O Vargas, CR 321-P, 322-P, 54-P, 13-P, 260-P Vargas, PR 322-P Varholakova, L 95-P, 90-P, 113-P Vasconcelos, M 240-P Vasilenkova¨, A 619-P Vaske, B 121-P, 617-P Vasylieva, OV 608-P Vatanavicharn, N 463-P Vaz, C 436-P Vedak, V 585-P Vega, A 201-P, 606-P Veghova, E 369-P Velardo, R 598-P Vellodi, A 441-P Ventura, FV 131-P, 133-O Verbruggen, KT 589-P Veredice, C 8-P Verhaak, C 208-P Verheijen, FW 348-P Verhoeven-Duif, NM 118-P, 548-O, 375-P, 2-P Ver|¨ ssimo, C 3-P Verkade, HJ 18-P Verkarre, V 93-P Verloo, P 298-P Vermeulen, T 349-O Verrigni, D 220-O Verrijp, K 195-P Vertemati, T 401-P, 31-A, 403-P, 592-P, 591-A, 397-P, 565-P Vesela, K 231-P Vianey-Saban, C 352-P, 142-P, 136-P Viegas, CM 23-P Vieira, A 411-P Vignoli, A 359-P Vijay, S 559-P, 435-P, 556-O, 557-P, 469-P, 563-P, 576-O Vijayakumar, K 192-P Vilageliu, L 606-P Vilarinho, L 177-P, 596-P, 24-P, 131-P, 224-P, 315-P, 234-P, 235-P, 277-P, 123-P, 297-P, 111-P, 344-P, 542-P, 530-P Vilaseca, MA 62-P, 5-P, 550-O, 279-P Villeneuve, J 244-O Vincent, MF 451-P Vinohradska, H 294-P Violante, S 133-O, 131-P Viskochil, D 418-P Visser, G 583-P, 540-P, 320-P Vles, J 535-P Vockley, J 27-O Voermans, N 195-P von Berlepsch, J 309-P, 307-P von Do«beln, U 233-P, 114-P von Spronsen, F 332-P Vora, AJ 167-P Vorechovsky, I 336-P Voskoboeva, EY 398-P Vrzalova, ZV 365-P

169 Vseticka, JV 365-P Vuillaumier-Barrot, S 192-P, 172-P, 188-P, 190-P Vuillemet, F 525-P Vyth, A 267-P Wegrzyn, G 445-P Wajner, M 260-P, 23-P, 321-P, 322-P, 139-P, 54-P, 70-P, 13-P, 68-A, 14-P, 80-P, 6-P, 71-P Waldek, S 472-P Walker, V 266-P Wallner, S 526-P Walter, JH 83-O, 221-P Walter, M 69-O, 77-O Wanders, RJA 267-P, 118-P, 145-P, 256-P, 268-O, 269-P, 353-P, 254-P, 253-P, 133-O, 138-O, 270-P, 271-P, 255-P, 27-O, 134-P, 127-P, 122-P Wang, D 219-P Wannmacher, CMD 6-P, 14-P, 13-P Wasant, P 463-P Wasserstein, M 379-O Wassmer, E 19-P Watanabe, Y 340-P, 56-P, 25-P Waterham, HR 269-P, 268-O, 253-P, 254-P Waterval, WAH 284-P Watman, N 450-P Wawer, Z 20-P Weber, Y 181-O Webster, R 17-P Wee Teik, K 629-P Weglage, J 309-P, 296-O Weinreb, NJ 441-P, 427-P Weisiger, K 28-P Weitz, J 364-O Wendel, U 83-O, 329-P Werlang, FG 453-P Wessels, MW 468-P West, ML 473-P Wevers, M 540-P Wevers, RA 199-O, 195-P, 16-P, 615-P, 220-O, 209-P, 200-P Weykamp, C 524-P Wheat, TE 1-P Whitehouse, C 496-P Whybra, C 425-P Wibom, R 233-P Wibrand, F 243-P, 145-P Wierzba-Bobrowicz, T 506-P Wiggins, C 265-P Wiggins, G 265-P Wijburg, FA 83-O, 418-P, 130-P, 138-O, 464-P, 479-P Wilcken, B 73-P, 278-P, 534-P Wilkinson, LJ 578-P Williams, H 495-P Wilrich, J 30-O Wilson, CJ 523-P Wodzig, WK 175-P Woessner, R 251-P Wong, J 143-O Wong, LJC 230-P Wong, PK 580-P Wood, L 97-O Wood, M 342-P Woods, CG 69-O Woodward, C 242-O Worthington, V 192-P

Wortmann, SB 209-P, 223-P Wraith, E 513-P Wraith, JE 390-O, 496-P Wright, N 502-P Wu, RM 539-P Wu, X 507-P, 495-P Wustman, BA 495-P, 427-P, 387-P Wuttke, TV 181-O Wuyts, B 504-P, 253-P Wuyts, W 134-P Wyse, ATS 14-P, 68-A, 6-P, 23-P Xaidara, A 406-P Xin, W 511-P Yager, C 173-P Yamano, T 311-P, 541-P Yanakawa, Y 623-P Yano, S 220-O, 483-P, 293-P Yaplito-Lee, J 228-P Yee, J 408-P Ye¢menko, I 356-O Yeh, M 441-P Yenicesu, I 609-P Yeo, SJ 109-A Yigit, Sule 270-P Yildiz, B 122-P Yokoi, K 158-P, 86-P Yokoi, T 541-P Yoshida, T 147-P Yoshino, M 25-P, 56-P, 340-P Young, SP 145-P Young, V 53-P Youroukos, S 538-P Yugi, K 389-P Zabedah, MY 531-P, 629-P, 593-P Zabot, MT 136-P Zahorakova, D 620-P Zaman, T 104-P, 176-P Zammarchi, E 447-P, 88-P, 446-P, 429-P Zanatta, A 23-P, 71-P, 80-P Zanco, C 318-P Zandbergen, AA 498-P Zdybskaya, OP 608-P, 226-P Zeevaert, R 361-P, 189-O Zelenietz, S 599-P Zeman, J 422-P, 113-P, 78-P, 622-P, 628-P, 4-P, 231-P Zerjav Tansek, M 155-P Zeviani, M 229-P Zhadanov, SI 226-P Zhang, G 107-O Zhang, R 259-O Zidkova, L 628-P Zimmer, KP 368-P Zimovina, TS 590-P Zimran, A 450-P Zinovik, AV 590-P Zoetekouw, L 618-P Zolkipli, Z 144-P, 120-P Zordania, R 174-P Zschocke, J 72-O, 490-P, 328-P, 30-O Zuppaldi, A 323-P, 48-P Zur£u«h, MR 329-P Zweers, H 223-P