Annual Symposium of the Society for the Study of Inborn Errors of Metabolism Birmingham, UK, 4

Home , Aceruloplasminemia, Adenylosuccinate lyase, Glutaric acidemia type 2, Glutaric aciduria type 1, Histidinemia, MOCS2

J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 DOI 10.1007/s10545-012-9512-z

ABSTRACTS

Annual Symposium of the Society for the Study of Inborn Errors of Metabolism Birmingham, UK, 4 – 7 September 2012

This supplement was not sponsored by outside commercial interests. It was funded entirely by the SSIEM.

01. Amino Acids and PKU O-002 NATURAL INHIBITORS OF CARNOSINASE (CN1) O-001 Peters V1 ,AdelmannK2 ,YardB2 , Klingbeil K1 ,SchmittCP1 , Zschocke J3 3-HYDROXYISOBUTYRIC ACID DEHYDROGENASE DEFICIENCY: 1Zentrum für Kinder- und Jugendmedizin de, Heidelberg, Germany IDENTIFICATION OF A NEW INBORN ERROR OF VALINE 2Universitätsklinik, Mannheim, Germany METABOLISM 3Humangenetik, Innsbruck, Austria Wanders RJA1 , Ruiter JPN1 , Loupatty F.1 , Ferdinandusse S.1 , Waterham HR1 , Pasquini E.1 Background: Carnosinase degrades histidine-containing dipeptides 1Div Metab Dis, Univ Child Hosp, Amsterdam, Netherlands such as carnosine and anserine which are known to have several protective functions, especially as antioxidant agents. We recently Background, Objectives: Until now only few patients with an established showed that low carnosinase activities protect from diabetic nephrop- defect in the valine degradation pathway have been identified. Known athy, probably due to higher histidine-dipeptide concentrations. We deficiencies include 3-hydroxyisobutyryl-CoA hydrolase deficiency and now characterized the carnosinase metabolism in children and identi- methylmalonic semialdehyde dehydrogenase (MMSADH) deficiency. On fied natural inhibitors of carnosinase. the other hand many patients with 3-hydroxyisobutyric aciduria have been Results: Whereas serum carnosinase activity and protein concentrations described with a presumed defect in the valine degradation pathway. To correlate in adults, children have lower carnosinase activity although pro- identify the enzymatic and molecular defect in a group of patients with 3- tein concentrations were within the same level as for adults. The difference hydroxyisobutyric aciduria. in activity is caused by different carnosinase isoforms in children and Methods: Case Report: Fibroblasts were collected from several centres adults. Additionally we identified several natural compounds which influ- around the world from patients with 3-hydroxyisobutyric aciduria. The ence carnosinase activity. Carnosine degradation by carnosinase is inhibited patients investigated in this study showed a wide range of clinical signs by the carnosinase substrates anserine and homocarnosine but not by other and symptoms. The only parameter in common was the 3-hydroxyisobuty- histidine-dipeptides (e.g. leucine-histidine). We further measured the influ- ric aciduria. ence of amino acids on carnosinase activity. Whereas glutamine, ornithine, The activity of 3-hydroxyisobutyric acid dehydrogenase was measured as alanine or histidine had no effect on carnosinase activity, homocysteine, described in Loupatty et al. (2006) Mol. Genet. Metab. 87, 243–348). cysteine, methionine, citrulline, lysine and arginine inhibited carnosinase Results: We determined the activity of 3-hydroxyisobutyric acid dehydro- activities significantly, in concentrations found in homocystinuria or urea genase (HIBADH) in a series of patients with 3-hydroxyisobutyric aciduria cycle disorders. and found a full deficiency only in a single patient. Molecular analysis Conclusions: Carnosinase metabolism is highly regulated and controls revealed bona fide mutations in the gene encoding HIBADH. histidinedipeptide concentrations which seem to be important in oxidative Conclusions: We have now identified the first patient with 3-hydroxyisobutyric stress response. The protective function of carnosine and other histidine- acid dehydrogenase deficiency. The defect in the large group of patients with dipeptides and the regulation needs further investigations. 3-hydroxyisobutyric aciduria with normal HIBADH activity remains to establish in the future. S2 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-003 O-005 MELATONIN: A NEW BIOMARKER TO REFLECT BRAIN TETRAHYDROBIOPTERIN RESPONSIVENESS IN PKU: SEROTONIN METABOLISM IN PHENYLKETONURIA PREDICTION WITH THE 48-HOUR LOADING TEST AND Yano S1 , Moseley K1 , Azen C2 GENOTYPE 1Genetics, Pediatrics, USC, Los Angeles, United States Anjema K1 , van Rijn M1 , Hofstede FC2 , Bosch AM3 , Hollak CEM3 , 2Clinical & Translational Sci Inst, USC, Los Angeles, United States Rubio-Gozalbo ME4 , de Vries MC5 , Janssen MCH5 , Boelen CCA6 , Blau N7 , Heiner-Fokkema MR1 , van Spronsen FJ1 Background: Early diagnosis by newborn screening and phenylalanine 1Beatrix Child Hosp, Univ Med Center, Groningen, Netherlands (Phe) restriction has been successful in preventing intellectual disability in 2Wilhelmina Child Hosp, Univ Med Center, Utrecht, Netherlands the majority of PKU patients. However, current studies indicate that PKU 3Academic Medical Center, Amsterdam, Netherlands patients who were early diagnosed and maintained good blood Phe control 4Maastricht University Medical Center, Maastricht, Netherlands have lower executive function and a higher prevalence of depressive and 5Radboud Univ Nijmegen Med Center, Nijmegen, Netherlands anxiety disorders possibly due to deficiencies of neurotransmitters includ- 6Leiden University Medical Center, Leiden, Netherlands ing serotonin and dopamine. 7University Children's Hospital, Zürich, Switzerland Patients and Methods: We studied blood and urine melatonin, a serotonin metabolite in PKU patients, in a randomized double blind crossover study Background: It remains unclear how to efficiently diagnose tetrahydro- consisting of three 3-week phases in 10 adult PKU subjects: Phase 1 biopterin (BH4) responsiveness in patients with PKU. We compared the (washout), Phase 2 (supplementation of large neutral amino acid (LNAA) positive predictive value (PPV) of the 48-hour BH4 loading test and tablets or placebo), and Phase 3 (alternate supplementation). Study subjects genotype. stayed overnight to measure night time blood melatonin and urine 6- Methods: PAH deficient patients with ≥30 % phenylalanine reduction at ≥1 sulfatoxymelatonin and dopamine in first void urine specimens. The Phase time point(s) during the 48-hour BH4 loading test (20 mg/kg/day) were 1 protocol was also conducted in 10 control subjects. regarded potential responders and were invited for the BH4 extension Results and Conclusions: This study showed significantly lower concen- phase. True BH4 responsiveness was defined as long-term ≥30 % reduction trations of these neurotransmitter metabolites in PKU subjects compared to in mean phenylalanine concentration or >4 g/day and/or ≥50 % increase of controls in Phase 1, and significant increases with LNAA supplementation natural protein intake. compared to the washout and placebo phases. Urine 6-sulfatoxymelatonin Results: 177/183 patients successfully completed the 48-hour BH4 loading and dopamine may serve as biomarkers reflecting neurotransmitter metab- test. 80/177 were potential responders. 67/80 completed the BH4 extension olism in the brain, thus optimizing metabolic control. phase. 58/67 showed true BH4 responsiveness (PPV 87 %). Genotype was Conflict of Interest declared. complete in 65 % of 177 patients. 39/41 patients with ≥1 mutation associated with long-term BH4 responsiveness in literature showed potential responsiveness. 31/32 participating in the BH4 extension phase showed true BH4 responsiveness (PPV 97 %). PPV of both methods was not O-004 significantly different (P00.16). Conclusions: Genotyping can be performed first. A genotype with ≥1 INHIBITION OF BRAIN PHENYLALANINE (PHE) TRANSPORT mutation associated with BH4 responsiveness is a good predictor for true IN PAHENU2 MICE WITH METHYL-AMINOISOBUTYRATE BH4 responsiveness. For patients with an unclassified or unknown geno- (MAIB): TREATMENT RAMIFICATIONS FOR type, the 48-hour BH4 loading test is a good alternative to detect BH4 PHENYLKETONURIA responsiveness. Vogel KR1 , Wasek BR2 , Arning EA2 , Bottiglieri T2 , Gibson KM1 Conflict of Interest declared. 1Clin Pharmacol, Wash State Univ, Spokane WA, United States 2Inst Metab Dis, Baylor Univ Med Center, Dallas TX, United States

We documented the capacity of large neutral amino acid (LNAA) transport inhibitors (DL-norleucine (NL); 2-aminonorbornane (NB)) to lower brain phe in Pahenu2 mice [1], and extend this now to examine the effect of the system A transport inhibitor MAIB on brain phe levels in the same system. Pahenu2 mouse cohorts (mixed gender) were formed and fed an 18 % protein diet supplemented with 3 % MAIB (control, 3 % casein) for three weeks. At sacrifice, LNAAs and monoamine neurotransmitters were quantified using UPLC and/or tandem mass spectrometry. MAIB feeding did not alter weight or movement, or enhance morbidity, of subjects. Brain phe (782 +/- 18 (SEM; n05,-MAIB) : 671 +/- 19 (n06, +MAIB; p<0.01, two- tailed t test) and tyrosine (28 +/- 3 (-MAIB) vs. 21 +/- 1.3 (+MAIB; p<0.05) were lowered without alterations of other brain LNAAs. Dopamine and serotonin were unchanged in brain extracts, although turnover and metabolism of both was decreased. These results are the first to document inhibition of LNAA transport in Pahenu2 mice by MAIB. This inhibition, and the milder effects of MAIB in comparison to NL and NB, highlight the potential of MAIB as a therapeutic consideration for PKU. [1] Vogel KR (2012) Molec Genet Metab 105: 359. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S3

O-006 02. Nutrition, Dietetics and Urea Cycle Defects (1) DISCOVERY OF NOVEL PHARMACOLOGICAL CHAPERONES FOR PKU THAT CORRECT PHENYLKETONURIA IN VIVO O-007 Santos-Sierra S1 , Kirchmair J2 , Perna A2 ,ReißD3 ,KemterK3 , Roeschinger KETOGENIC PARENTERAL NUTRITION IN INTESTINAL W4 , Glossmann H1 , Gersting S3 ,MuntauAC5 ,WolberG2 ,LaglerFB6 FAILURE 1Biochem Pharm, Medical University, Innsbruck, Austria ROSS K1 , BISSET WM1 , GROSSI H1 , JOLLANDS A2 2Pharmaceut Chemistry, University of, Innsbruck, Austria 1Royal Aberdeen Children's Hospital, Aberdeen, United Kingdom 3Mol Pediatrics, Ludwig Maxim. University, Munich, Germany 2Ninewells Hospital, Dundee, United Kingdom 4Becker & Olgemoeller Laboratories, Munich, Germany 5Ped I, Med University, Innsbruck, Austria Background: A ketogenic diet (KD) is a high fat, adequate protein, very 6IIEM&Dept Pediatrics, Paracelsus Med Uni, Salzburg, Austria low carbohydrate diet that induces ketosis. It is an effective treatment for children with intractable epilepsy and a specific treatment for Glut -1 Background: Phenylketonuria (PKU) is caused by inherited phenylalanine- deficiency syndrome. Ketogenic enteral feeds are widely used but there is hydroxylase (PAH) deficiency and, in many genotypes, it is associated with very little experience in the use of ketogenic parenteral nutrition. protein misfolding. The natural cofactor of PAH, tetrahydrobiopterin Patient: Our patient was a 6 year old boy with refractory seizures .He (BH4), can act as a pharmacological chaperone (PC) that restores enzyme presented with worsening GI dysmotility and it was impossible to maintain function. However, BH4 shows limited efficacy in some PKU genotypes enteral feeding. There was reluctance to discontinue his KD because of the and its chemical synthesis is very costly. predicted negative effect on seizure control. Primary Objective: Identification of alternative PC for PKU Method: We designed a parenteral nutrition (PN) regimen with a 2.5:1 Methods/Results: We used an integrated drug discovery approach which ratio. (g fat: g CHO+g protein) Conventional PN typically provides 40 % has not been applied to this target before. Shape-focused virtual screening energy from fat; the KD PN has 85 % energy from fat. of the National Cancer Institute's chemical library identified 84 candidate Results: Ketosis was maintained in the range of 2.2-4.6 mmol/l beta molecules with potential to bind to the active site of PAH. An in vitro hydroxy butyrate with no measured hypoglycaemia. Biochemical indices, evaluation of these yielded six compounds that restored the enzymatic including triglycerides remained within normal values. activity of the unstable PAHV106A variant and increased its stability in Conclusion: Short term ketogenic PN was well tolerated and effective in a cell-based assays against proteolytic degradation. During a 3-day treatment child already established on a ketogenic diet. study, two compounds (benzylhydantoin and 6-amino-5-(benzylamino)- uracil) substantially improved the in vivo Phe oxidation and blood Phe concentrations of PKU mice (Pahenu1). Notably, benzylhydantoin was O-008 twice as effective as tetrahydrobiopterin. DAILY PROTEIN TOLERANCE INCREASES WITH AGE AND Conclusion: We identified two PCs with high in vivo efficacy that may be BETTER DIETARY CONTROL IN TYROSINAEMIA TYPE I further developed into a more effective drug treatment of PKU. Daly A1 , Ashmore C1 , McKiernan P1 , MacDonald J1 , MacDonald A1 1Birmingham Children's Hospital, Birmingham, United Kingdom

Introduction: Little is known about the long term natural protein tolerance of patients with Tyrosinaemia Type 1 (HT1) on a low tyrosine diet together with the drug Nitisinone. Aim: To evaluate the natural protein tolerance in all patients with HT1 on diet and drug treatment at a single centre. Methods: 15 HT1 children, median age 7y (range 2y-15y), had their daily natural protein tolerance assessed. Natural protein was increased by 1 g/day when 6 consecutive weekly plasma tyrosine concentrations were maintained within the target range of 200-400 μmol/l. All were taking a tyrosine/phenylalanine-free protein substitute. Results: Median daily natural protein increased with age in 80 % of patients. Mean (range) of natural protein intake (g/d) in the following age groups was: 1-2y (n015), 4 g (3-13); 3-5y (n011), 5 g (3-17); 6- 8y (n011),8g(3-22);9-11y(n04), 12 g (6-18); 12-15y (n02) 21 g (15-28). The median natural protein intake for all age categories was 0.4 g/kg/day. There was a positive correlation between percentage of plasma tyrosine concentrations within target ranges and g/d of natural protein tolerated (r00.72) Conclusion: In HT1 children, natural daily protein tolerance increases with age and better plasma tyrosine control. S4 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-009 O-010 SUGGESTED GUIDELINES FOR THE DIAGNOSIS AND EUROPEAN DIETARY MANAGEMENT OF UREA CYCLE MANAGEMENT OF UREA CYCLE DISORDERS DISORDERS Häberle J1 , Boddaert N2 , Burlina A3 , Chakrapani A4 , Dixon M5 , Huemer Evans S1 , Adams S2 , Assoun M3 , Baruteau J4 , Bigot S5 , Champion H6 , M6 , Karall D7 , Martinelli D8 , Sanjurjo P9 , Santer R10 , Servais A11 , Dassy M7 , Daly A1 , Dawson S8 , Dixon M9 , Dokoupil K10 , Dubois S3 , Valayannopoulos V12 , Lindner M13 , Rubio V14 , Dionisi-Vici C8 Dunlop C2 , Eardley J11 , Eyskens F12 , Faria A13 , Favre E14 ,FergusonC15 , 1Div Metab, Univ Child Hosp & Res Center, Zurich, Switzerland FerreiradeAlmeidaM16 , Goncalves C17 , Heddrich-Ellerbrok M18 ,Jankowski 2Radiol Hôp Necker-Enf Malad, Paris, France C19 , Jassen-Regelink R20 ,JouaultC21 ,LaguerreC4 ,LeVergeS3 ,LowryS22 , 3Dep of Pediatr, Padua, Italy Luyten K12 ,MacDonaldA1 ,MaritzC23 , Meyer U24 , Micciche A11 ,Robert 4Birmingham Childr Hosp, Birmingham, United Kingdom M25 , Robertson L26 , Rocha JC16 , Rohde C27 , Saruggia I28 , Sjoqvist E29 , 5Dietetic Dep, GOSH, London, United Kingdom Stafford J9 , Terry A30 ,ThomR31 , Vande Kerckhove K32 , van Teefelen- 6Dept of Pediatr, LKH, Bregenz, Austria Heithoff A33 ,vanWegbergA20 ,vanWykK11 , Vestergaard H34 , Webster D19 7Univ Childr Hosp, Innsbruck, Austria , White F35 , Wildgoose J36 ,ZweersH20 8Bambino Gesù Childr Hosp, IRCCS, Rome, Italy 1Birmingham Children's Hospital, Birmingham, United Kingdom 2Royal 9Div of Pediatr Metabol, Baracaldo, Spain Hospital for Sick Children, Glasgow, United Kingdom 3Here Dis Metab, 10Univ Childr Hosp, Hamburg, Germany Necker Hosp Sick Child, Paris, France 4Centre Competence Child Hosp, 11Serv Néphr Malad Métab Adulte Hôp Necker, Paris, France Toulouse, France 5Centre Rennes, Rennes, France 6Addenbrookes Hos- 12Hôp Necker-Enf Malad, Paris, France pital, Cambridge, United Kingdom 7Cliniques Universitaires St Luc, 13Univ Childr Hosp, Heidelberg, Germany Brussels, Belgium 8Royal Hospital Sick Children, Edinburgh, United 14Inst Biomed, Valencia, Spain Kingdom 9Great Ormond Street Hospital for Child, London, United Kingdom 10Dr von Hauner Child Hosp, Munich, Germany 11Evelina Background: Urea cycle disorders (UCDs) are inborn errors of ammonia Child Hosp, Guys & St Thomas', London, United Kingdom 12Centre of detoxification that present with hyperammonaemia at any age and can lead IMD, Antwerp, Belgium 13Hospital Paediatr de Coimbra, Coimbra, Por- to death or severe neurological handicap. Despite therapeutic options with tugal 14Ref Center Hereditary Met Dis PR Chabro, Nancy, France 15New- alternative pathway therapy and liver transplantation, outcome remains castle upon Tyne Hospital, Newcastle upon Tyne, United Kingdom poor. This may be in part related to missed and delayed diagnosis due to 16Medical Genetics Centre, INSA, Porto, Portugal 17Servico de Saude da the nonspecific clinical presentation and insufficient awareness of health RAM EPE, Madeira Island, Portugal 18University Children's Hospital, care professionals. Our aim was to develop guidelines based on a trans- Hamburg-Eppendorf, Germany 19United Hospitals, Bristol, United King- European consensus to set standards of care, to help awareness campaigns dom 20Radboud Uni Nijmegen Medical Centre, Nijmegen, Netherlands and to guide practitioners. 21University Hospitals, Angers, France 22Sheffield Children's Hospital, Methods: Experts from seven European countries (clinicians, basic scien- Sheffield, United Kingdom 23Charles Dent Metabolic Unit, London, United tists, radiologists, dietitians) collected all existing evidence, scored it Kingdom 24Hannover Medical School, Hannover, Germany 25Hosp Univ according to the SIGN evidence level system and drew up a series of des enfants Reine Fabiola, Brussels, Belgium 26University Hospitals, statements. The guidelines were revised by external specialists and unrelat- Birmingham, United Kingdom 27Hospital Child & Adoles, Uni Leipzig, ed authorities in the field of UCDs and they were pilot-tested by practicing Leipzig, Germany 28Ref Centre Heredit Met Dis PR Chabrol, Marseille, paediatricians. France 29University Hospital of Skane, Lund, Sweden 30Alder Hey Child- Results: Statements covering all aspects of diagnosis and treatment of UCD ren's Hospital, Liverpool, United Kingdom 31Royal Belfast Hospital for were consented. The guideline also raised unanswered questions that must Sick Children, Belfast, United Kingdom 32Metabolic Dept University be addressed by future research. Hospitals, Leuven, Belgium 33Universitats Kinderklinik, Munster, Germany Conclusion: The proposed UCD guidelines (shortly published in OJRD) 34National University Hospital, Copenhagen, Denmark 35Central Man- will help to harmonize practice, set common standards, spread good prac- chester University Hospitals, Manchester, United Kingdom 36Bradford tices and may improve the overall outcome of UCD patients. Teaching Hospital, Bradford, United Kingdom Introduction: There is no published data comparing dietary management (DM) of urea cycle disorders (UCD) between countries. Aim: To compare DM in UCD centres across Europe. Methods: Cross-sectional data from 37 European IMD centres (16 UK, 6 France, 5 Germany, 4 Belgium, 3 Portugal, 1 Denmark, 1 Netherlands, 1 Sweden) was collected by questionnaire describing management of UCD patients on prescribed protein restricted diets. Results: 412 patients (NAGS, n010; CPS, n027; OTC, n0190; citrullinaemia n096; ASA, n068; arginase deficiency n021 were reported; 71 % [n0292] aged 0-16y; 29 % [n0120] >16y. Prescribed median protein intake decreased with age with little variation between disorders although intakes varied between countries. Compared with UK, other European countries (OEC) gave lower protein amounts in infancy; were more likely to supplement with essential amino acids (EAA) (OEC 43 %; n0101; UK 30 %, n0 53), used a higher % of total protein from EAA, and were less likely to tube feed (OEC 15 %; n035; UK 25 %, n044). Only 3-4 % of all patients used branch chain amino acids. There was wide variation in the composition of emergency feeds although UK practice was consistent. Conclusions: DM for UCD varies significantly throughout Europe. More multi-centre research examining protein and EAA intakes is required. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S5

O-011 O-012 LONG-TERM OUTCOME IN ORNITHINE TRANSCARBAMYLASE SINGLE CENTRE DIETARY EXPERIENCE OF FEMALE DEFICIENCY (OTC): A SERIES OF 90 PATIENTS CARRIERS OF ORNITHINE TRANSCARBAMYLASE DEFICIENCY Brassier A1 ,OttolenghiC2 , Gobin S3 ,ArnouxJB1 , Valayannopoulos V1 , (OTCD) Servais A1 ,TouatiG1 ,RabierD2 ,LesageF4 , Bonnefont JP3 ,deLonlayP1 Gribben J1 , Hallam P1 , Micciche A1 , van Wyk K1 , Walker R1 , Champion 1Inherit Metab Dis, Necker, Paris, France M2 , Mundy H2 , Rahman Y2 , Vara R2 2Biochemistry, Necker, Paris, France 1N & D Dept, St. Thomas' Hosp, London, United Kingdom 3Genetics, Necker, Paris, France 2Ctr Inherited Met Dis, St. Thomas' Hosp, London, United Kingdom 4Réanimation pédiatrique, Necker, paris, France Background: Carriers of Ornithine Transcarbamylase deficiency (OTCD) Background: To investigate the long term outcome of a cohort of patients are well known to have a wide range of phenotypic variability. with Ornithine Trans Carbamylase deficiency (OTC) followed up in the Objectives: To illustrate the variable phenotype and management of OTC same medical centre. carriers. Methods: Between 1971 and 2011, we investigated 90 patients with OTC Methods: Retrospective review of OTC carriers referred to our centre from deficiency, 48 males and 42 females. 1998-2012. Results: 27 patients (18 boys, 3 girls) had neonatal presentation; 52 Results: 33 female carriers of OTC were identified. 9 patients presented patients (21 boys, 31 girls) had late-onset form revealed between clinically at a median age 2 years (1.4-9 years) of which 3 resulted in 1 month and 16 years, and 11 patients (5 boys, 6 girls) revealed their marked neurological deficit. Median protein restriction in paediatric disease in adulthood. Patients with neonatal presentations had higher patients was 1.2 g/kg/day (range 1.1-1.3) and in adult patients 43 g/day mortality (90 % versus 13 %), peak levels of plasma ammonium (range 40 –63). (mean value 960 μmol/L versus 500 μmol/L) and glutamine The remaining 24 female patients (age range 0-60 years) identified follow- (4110 μmol/L versus 1000 μmol/L) at diagnosis. Neonatal forms ing investigation for a family history. 10 had a protein restricted diet, 7 of displayed a greater number of acute decompensations (6.2/patient which were already self-selecting and 3 were prescribed a low protein diet versus 2.5 and 1.4). In the adult group, some patients died during based on biochemistry and symptoms. The remaining 14 patients continued their first episode of coma. Molecular analysis identified a mutation in on normal diets. 59/68 patients investigated (69 % single base substitutions and 12 % All patients were advised on an emergency regimen for use during deletions). Neurological score was not significantly different between illness. the patients alive of the neonatal or late-onset groups, and was not Conclusion: We illustrate the variable age of presentation, protein tolerance correlated to ammonia peak and plasma glutamine concentration at and outcome in female carriers of OTCD. Early recognition of patients diagnosis. presenting clinically is essential to prevent long-term sequelae. The treat- Conclusions: OTC disease remains a severe disease raising a number of ment of asymptomatic carriers remains controversial. diagnostic and therapeutic challenges.

03. Newborn Screening and Urea Cycle Defects (2): Outcomes O-013 CLINICAL AND BIOCHEMICAL CHARACTERISTICS OF PATIENTS WITH ORGANIC ACIDAEMIAS AND UREA CYCLE DISORDERS IDENTIFIED THROUGH NEWBORN SCREENING Barends ME1 , Pitt JJ1 , Tzanakos N1 , Boneh A1 1Metabolic Research, MCRI. RCH, Melbourne, Australia

Newborn Screening (NBS) is aimed to reduce morbidity and mortality associated with inborn errors of metabolism (IEM). NBS detects infants with "classical IEM" with biochemical phenotypes that do not correlate with clinical phenotypes. To elucidate the prevalence and clinical and biochemical spectrum of patients with organic acidaemias (OA) and urea cycle disorders (UCD) diagnosed through NBS in a defined population over 10 years (February 2002 to January 2012), all health records of all patients with the above disorders were reviewed. There were 29 patients with OA and 10 patients with UCD. Diagnosis was confirmed by urine analysis for organic and amino acids (28/29 with OA, 10/ 10 with UCD) or plasma amino acids (7/10 with UCD), enzyme analysis (2/39) or mutation analysis (8/39). No episodes of metabolic decompensation were recorded in 35/39 during the follow-up (up to 95 m). Age-appropriate growth and development were recorded in 33/39 patients. Two symptomatic patients experienced hyperammonaemia only once, as neonates. The spectrum of clinical symptomatology in patients diagnosed through NBS is wide and the prevalence of asymptomatic newborns is high. Elucidating a genotype-phenotype correlation may inform clinicians on the best mode of treatment for these infants. Re- evaluation of the NBS program should be done regularly. S6 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-014 O-016 APPLICATION OF HIGH RESOLUTION MASS SPECTROMETRY NEONATAL ONSET HYPERAMMONAEMIA: FACTORS AFFECTING IN NEWBORN SCREENING SURVIVAL Dénes J1 , Robinette S1 , Hester A2 , Szabó E3 , Szatmári I3 ,Szőnyi L3 , Broomfield AA1 , Venkatesha G2 , Jones A1 , Cleary M1 , Abulhoul L1 , Kreuder J4 , Rauterberg E4 , Takáts Z1 Sharma S2 , Grunewald S1 , Grunewald S1 1Imperial College London, London, United Kingdom 1Dep Met Dis, Great Ormond St Hosp, London, United Kingdom 2Justus Liebig University, Giessen, Germany 2PICU, Great ormond St Hosp, London, United Kingdom 3Semmelweis University, Budapest, Hungary 4University Hospital Giessen and Marburg, Giessen, Germany Objectives: Identification of factors affecting survival in neonatal hyperammonaemia. Background: Metabonomic approach was introduced for newborn meta- Material (Patients) and Methods: Retrospective analysis (Jan 2000 - Dec bolic screening, based on high resolution mass spectrometry data. 2011)ofneonates(<28days),presentingtoourPICUwithplasma Methods: Dried blood spot samples were analyzed by nanoelectrospray- ammonia>250 μmol/l. high resolution single stage mass spectrometry using an Exactive and a Results: 30 patients were identified, 13/30 survived their initial episode of TriVersa NanoMate. This setup provides high-throughput analysis with hyperammonaemia. No differences in age at presentation, sex or birth excellent selectivity and sensitivity. Measurements were performed on weight were noted between survivors (group I) and non-survivors (group identical extracts in both positive and negative ion mode. II). Ammonia (μmol/l) at admission was significantly lower in group I [433 Results: More than 400 metabolic constituents were identified in newborn (SD 187) vs 980 (SD 790) P00.017 as was maximal ammonia [685 (SD DBS samples based on the exact masses and MS/MS measurements. 203) vs 1569 μmol/L (SD 907) P00.02]. No patient with a maximal Concentration values were determined using stable isotope labeled internal ammonia>1000 μmol/l survived. The duration of coma prior to PICU standards. Amino acid and acylcarnitine levels were compared with results admission wasn't a significant factor (1.5 v 2.8 days P00.25). 26 patients of traditional ESI-MS/MS method and additional biomarkers for different received filtration: there were no differences in the time prior or duration of metabolic diseases were determined. Multivariate pattern recognition meth- filtration of group I and II. Those neonates with seizures had significantly ods were used for the comparison of the spectral pattern and for better higher ammonia (702 v 1588 P00.002); however, presence of seizures (13/ understanding of metabolic processes. Preliminary study was performed on 25) was not related to survival. Whereas 4/7 of the organic acidaemias 120 healthy newborn and a set of 50 abnormal samples. PKU and MCADD survived, only 5/19 of the urea cycle survived, (Fischer exact P00.017). were investigated in details and extended biomarker sets were proposed for the Conclusion: The main determinates of survival were degree of hyperam- diagnosis of these disorders. In order to validate the results a large screening monaemia and underlying diagnosis. study has been started with cooperation of the Hesse Child Health Centre. Conclusions: Results indicate significant reduction of false positive-rate compared to other screening approaches. Conflict of Interest declared. O-017 LONG-TERM OUTCOME OF ARGINOSUCCINATE SYNTHETASE DEFICIENCY A CASE FOR EARLY LIVER TRANSPLANTATION? O-015 Vara R1 , Goodman B2 , Singh J3 , Chakrapani A3 , Mundy H1 , Champion NEWBORN SCREENING OUTCOME FOR INBORN ERRORS OF MP1 METABOLISM AT A LARGE FOLLOW UP AND TREATMENT 1Centre for IMD, Evelina Children's Hosp, London, United Kingdom CENTER 2GKT School of Medicine, KCL, London, United Kingdom Jain-Ghai S1 , Ambreen M1 , Tam K1 , Charkraborty P2 , Schulze A1 3Dept of IMD, Birmingham Child Hosp, Birmingham, United Kingdom 1Div Clin Gen & Metab, Hos for Sick Child, Toronto, Canada 2Ontario NBS, CHEO, Ottawa, Canada Background: Citrullinaemia (argininosuccinate synthetase deficiency) dis- plays phenotypic variability however the long-term neurocognitive out- Background: Analytical aspects of expanded NBS have been widely come appears to be poor despite early intervention. reported but reports on clinical outcomes are scanty. Methods: Retrospective review of all cases referred to 2 tertiary metabolic Methods: 817 cases of abnormal NBS for inborn error of metabolism (IEM) units in the UK. were referred to NBS center at the Hospital for Sick Children from August 2007 Results: 14 cases were identified, 8 from consanguineous parents. All patients to December 2010. 174 cases required further investigations. Chart review was survived. Median age at presentation was 2.5 days (range 0 – 3.5years),4had completed for these. Disease specific outcomes were defined and evaluated. a positive family history and 3 cases were treated prospectively. The median Results: Final diagnosis could be reached by 20.5 days (10-120); median plasma ammonia was 700 μmol/L (range 112 – 1581 μmol/L), 10 (71 %) had (interquartile range), with treatment initiated by 12 days (8-19). 123 cases were seizures at presentation and 8 required haemofiltration. All were treated with diagnosed with an IEM. Median observation period was 2.1 years (1.2-3.2). ammonia-lowering agents, arginine and protein restriction. 4 cases had >5 When improvement was compared to the natural course, conditions such as hospital admissions in the first year. 7 cases require supportive feeding. Liver Citrullinemia, MSUD, MMA and Tyrosinemia I demonstrated moderate to transplantation has been performed in 3 (aged 4 months, 3 and 3.5 years) and 2 severe developmental delay and medical disabilities. FAODs, other amino- currently listed. Median age at follow-up is 5.5 years (range 0.3 – 32 years). acidemias and organic acidurias demonstrated normal to mild developmental All children of school age (8) require significant educational support. The issues. Milder phenotypes, detected via NBS remained asymptomatic, but youngest transplanted child has normal motor skills and mild speech delay. posed problems with respect to diagnosis, counseling and management. There The adult case has severe learning and behavioural difficulties. were no deaths in our cohort due to the underlying IEM. Conclusion: Despite prospective management the neurocognitive outcome in Conclusion: NBS certainly alters the natural course of many IEM but this citrullinaemia remains poor. Consideration for early liver transplantation may cannot be generalized to all targeted conditions. Some conditions continue help to improve outcomes. to demonstrate significant developmental delay and disabilities, although mortality is improved in all. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S7

O-018 O-020 THE MANCHESTER EXPERIENCE WITH ASA PATIENTS METHYLATION IMBALANCE LEADS TO OXIDATIVE IMBALANCE Jameson E1 , Breen C1 , Jennions L1 , Vijay S2 , Morris AAM1 AND ENDOTHELIAL ACTIVATION 1Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom Barroso M1 , Tavares de Almeida I2 , Castro R2 , Loscalzo J1 , Handy DE1 2Birmingham Children's Hospital, Birmingham, United Kingdom 1Brigham and Women's Hospital / HMS, Boston, United States 2Metabolism and Genetics Group, iMed.UL, Lisbon, Portugal Introduction: Argininosuccinic aciduria (ASA) is a urea cycle disorder due to a deficiency of argininosuccinate lyase. While ASA presents in the typical manner Homocysteine is a risk factor for vascular disease, but the underlying mecha- of a urea cycle disorder the long-term phenotype appears to differ. There tends to nisms remain incompletely defined. Inhibition of methylation reactions by S- be a predominance of gastrointestinal symptoms with episodes of hyperammo- adenosylhomocysteine (AdoHcy), which accumulates in the setting of hyper- naemia being less frequently seen, as compared to the other urea cycle disorders. homocysteinemia, may contribute to endothelial dysfunction. It was our aim to Objectives: We present a review of the ASA patients followed-up in Man- investigate whether AdoHcy accumulation induces oxidative imbalance in en- chester and Bradford. dothelial cells. Results: There are a total of nineteen patients, of which five are deceased. Intracellular levels of AdoHcy were increased in cultured human endothelial The age range of the patients under follow-up is five months to twenty-two cells. ROS levels were measured by Amplex-Red assay. GPx-1 enzyme activity years. This review discusses in detail the gastrointestinal symptoms found was determined by an indirect assay based on GPx-1-mediated oxidation of in these patients and also includes a case of metastatic hepatocellular glutathione. Adhesion molecule expression was studied using quantitative RT- carcinoma in a four-and–a half-year-old female patient. PCR and immunoblotting. Discussion: The key feature we wish to highlight is the prevalence of chronic Our results show that AdoHcy accumulation increased ROS release from cells. gastrointestinal symptoms, approximately 50 % of our cohort experiencing Furthermore, it led to a significant decrease in GPx-1 activity and protein levels. diarrhoea and/or hypokalaemia. It is unclear whether the latter is secondary to Specifically, the highest concentration of AdoHcy decreased GPx-1 enzymatic the chronic diarrhoea or to the well-described renal tubulopathy. activity and expression by 39 and 44 %, respectively (p<0.05 versus control). Additionally, we observed increased expression of ICAM and VCAM, two cell adhesion molecules, which actively participate in atherogenesis. Moreover, this increase was abrogated either by the presence of antioxidants, or by GPx-1 04. Disorders of Sulphur Amino Acid Metabolism and overexpression. Our study supports the view that the deleterious effect of Miscellaneous Disorders homocysteine on the vasculature can, in part, be a consequence of an oxidative imbalance triggered by cellular hypomethylation. O-019 Supported by FCT-Portugal(PTDC/SAU-ORG/112683/2009;SFRH/BD/ 73021/2010). LETHAL COURSE OF ORNITHINE-TRANSCARBAMYLASE (OTC) DEFICIENCY IN A FEMALE NEWBORN WITH EXTREMELY SKEWED X-INACTIVATION Santer R1 , Vater I2 , Oyen F3 , Lüttgen S4 , Tsiakas K1 , Siebert R2 , Schneppenheim R3 O-021 1 Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany MODULATION OF CYSTATHIONINE ΒETA-SYNTHASE 2 Inst Hum Genet, Univ of Kiel, Kiel, Germany MUTATIONS BY CYSTEAMINE: RESCUING PROTEIN 3 Dept Pediatr Hematol Oncol, Univ Med Cen, Hamburg, Germany MISFOLDING? 4 Inst Hum Genet, Univ Med Cent Eppendorf, Hamburg, Germany Mendes M1 , Smith DEC2 , Colaço H1 , Salomons G2 , Ben-Omran T3 , Vicente J1 , Tavares de Almeida I1 , Rivera I1 , Leandro P1 , Blom H2 A variable course in OTC (Xp21.1)-deficient females due to "Lyonisation" 1Met&Gen, iMed.UL, FFUL, Lisboa, Portugal is well known and might also occur in additional defects of genetic ele- 2Clin Chem, VUMC, Amsterdam, Netherlands ments essential for X-inactivation, including the non-translated XIST gene 3Dep Ped, Haman Med Corp, Doha, Qatar (Xq13.2). Here, we report a female OTC newborn with typical metabolic profile, CBS deficiency is caused by mutations in CBS gene, the majority being positive enzymatic studies (post-mortem LBx), heterozygosity for OTC missense mutations often resulting in protein misfolding. Although the c.67 C>T [p.R23X], and an early lethal course. Analysis of X- world-wide prevalence of CBS deficiency is 1/344000, in Qatar it is approx- inactivation revealed 100 % inactivation of the non-mutated allele in patient imately 1/1800, being the R336C mutation the most prevalent one. It has been blood, liver, and fibroblasts, and in maternal blood. Surprisingly, OTC postulated that Arginine to Cysteine mutations can be rescued by Cysteamine, p.R23X was located to the paternal allele. CGH and SNP array analyses a molecule that binds to Cysteine mimicking the wild-type Arginine structure. revealed heterozygosity for an Xq28 microdeletion in the patient and her We aimed to study the effect of Cysteamine in the rescue of the CBS mother. This deletion comprises ~0.7 Mb including morbid genes like R336C, in E. coli lysates and purified proteins, using the wild-type and RAB39B and genes for histone proteins, H2AFB2 and H2AFB3. They R336H protein and the Cysteine molecule as controls. belong to a region that is repeated three times, once in intron 22 of the F8 Lysates and purified proteins were pre-incubated with Cysteamine or Cysteine gene and twice closer to the Xq telomere. In female cells, H2A proteins followed by evaluation of CBS activity and stability (Western-blot analysis). have not been found in Barr bodies. For the R336C protein, all tested conditions showed an increase in enzy- We speculate that the extremely skewed X-inactivation of our patient was matic activity. A 6-fold increase was observed when samples were incubat- related to the maternal X-chromosomal deletion, either by a negative ed for 7 h with 0.25 mM Cysteamine at 37:C, while no effect on the activity selection mechanism for cells with an active deletion-positive X- or stability was observed for the R336H and wild-type proteins. Cysteine chromosome, or secondary to an unknown dosage effect of the affected was unable to rescue any of the studied proteins. histone proteins. Our results support the hypothesis that Cysteamine is able to specifically rescue mutant proteins presenting an Arginine to Cysteine substitution. Funding: FCT-SFRH/BD/43934/2008 (Mendes M). S8 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-022 O-024 A NOVEL ADJUVANT-TREATMENT STRATEGY FOR CBS SUCCESSFUL TREATMENT IN ISOLATED SULFITE OXIDASE DEFICIENT HOMOCYSTINURIA: TAURINE SUPPLEMENTATION DEFICIENCY PREVENTS OXIDATIVE STRESS MEDIATED IMPAIRMENT OF Burlina AB1 , Del Rizzo M1 , Zanco C1 , Cazzorla C1 , Bordugo A1 , BHMT FUNCTION AND SIGNIFICANTLY IMPROVES THE Manara R2 , Sass JO3 ABILITY OF LONG-TERM BETAINE TREATMENT TO LOWER 1Div Metab Dis, Univ Child Hosp, Padua, Italy HOMOCYSTEINE 2Neuroradiol Div, Univ Hosp, Padua, Italy Maclean KN1 , Jiang H1 , Stabler SP1 , Allen RH1 3Universitätsklinikum Freiburg, Freiburg, Germany 1University of Colorado SOM, Aurora, Colorado, United States Background: Isolated Sulfite Oxidase Deficiency (ISOD) is a very rare and Treatment for pyridoxine non-responsive cystathionine beta-synthase deficient devastating metabolic disorder, characterized by severe encephalopathy and homocystinuria (HCU) typically involves lowering homocysteine levels with a poor prognosis. methionine-restricted diet and betaine supplementation. Compliance with Case report: A 12 month old baby girl presented with acute left limbs palsy methionine-restriction is often poor and increased efficacy of betaine treatment with a history of nystagmus and psychomotor delay. Brain MRI showed mild has the potential to improve clinical outcome. One-week betaine treatment of cerebral atrophy and stroke-like lesions at the basal ganglia. Mental develop- HCU mice on a standard chow diet resulted in plasma homocysteine levels ment was measured by means of GMDS-R (0-2), showing General Develop- around 50 μM and normalization of both pro-inflammatory cytokine expression mental Quotient (GDQ)<50 (100 +/-16). Plasma aminoacids showed high S- and a hypercoagulative phenotype. Treating HCU mice with betaine for six weeks sulphocysteine (28 μmol/l) and low cystine (2 μmol/l;nv16-84). In urine S- revealed that the ability of this compound to lower homocysteine diminishes sulphocysteine (313 mmol/mol.creat) and taurine 722 mmol/mol.creat (12- significantly over time. We observed a 3-fold elevation in plasma homocysteine 159) were elevated and cystine undetectable as well as total plasma homo- compared to the one-week treatment group accompanied by significantly in- cysteine. Genetic testing revealed a novel SUOX gene mutation c.427 C>A in creased pro-inflammatory cytokine levels and reversion to a hypercoagulative a homozygote fashion. A treatment with a low methionine (12 mg/kg/day) and phenotype. This effect was found to be due at least in part, to oxidative stress cystine 6 mg/kg/day)diet was well tolerated. mediated impairment of betaine-homocysteine S-methyltransferase function. Co- After 15 months of therapy, we observed: a) S-sulphocysteine is undetect- administration of the antioxidant taurine with betaine effectively prevented this able; b) complete recovery of left limbs function and gain of motor mile- impairment and restored maximal lowering of homocysteine during a six week stones c) GDQ reached the level of 60; d) no new lesions by brain NMR. trial. Our findings indicate a relatively sharp threshold-effect between severely Conclusions: acute palsy in ISOD has never been reported and dietary elevated plasma homocysteine and thrombotic risk in HCU and indicate that co- treatment has been safe and effective in treating the disorder. administration of taurine has the potential to significantly improve the therapeutic effects of long-term betaine treatment in this disease. 05. Organic Acidaemias and Fatty Acid Oxidation Defects, Pathophysiology and Transplantation O-023 SULFITE OXIDASE DEFICIENCY AN UNUSUAL LATE AND O-025 MILD PRESENTATION A SINGLE U.K. CENTRE EXPERIENCE OF RENAL Rocha S1 , Ferreira AC2 , Vieira JP2 , Silva R1 , Sequeira S2 , Sequeira S2 TRANSPLANTATION IN FOUR METHYLMALONIC ACIDAEMIA 1Neurol Dep, H. D. Estefãnia, Lisbon, Portugal PATIENTS 2Met.Unit, H. D. Estefânia, Lisbon, Portugal Jameson E1 ,HYWU1 , Makin A2 , Bradbury M3 , Jones S1 , Shenoy M3 1Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom Background: Sulfite oxidase deficiency (SOD) is an autosomal recessive 2Manchester Royal Infirmary, Manchester, United Kingdom inherited disease, usually presenting in the neonatal period with severe neuro- 3Royal Manchester Children's Hospital, Manchester, United Kingdom logical symptoms including seizures, often refractory to anticonvulsant therapy, and a rapidly progressive encephalopathy leading to a state resembling that Objective: To describe our experience of renal transplantation in four of neonatal hypoxic ischemia, with premature death. Most patients develop MMA patients. dislocated ocular lenses. Later or milder presentations of SOD are being Results: Two received a living related donor (LRD) transplant and two a reported with increasing frequency. These presentations include neurological deceased donor transplant. The first LRD recipient was a seventeen year old regression with loss of previously acquired milestones or movement disorders. male who had an uneventful course. Within two months he had an episode of Case report: We report a four year old girl presenting with intermittent rejection which correlated with increased plasma MMA levels, both resolved ataxia and uncoordinated limb movements. A similar episode of ataxia had with methylprednisolone. The second LRD recipient was a ten year old male. occurred previously, one year before, with complete neurological recovery He developed haemorrhagic pancreatitis forty-eight hours post-transplant re- and normal developmental milestones. Bilateral lens dislocation was requiring intensive care. He subsequently developed large pseudocysts and cently diagnosed. Cranial MRI demonstrated bilateral globus pallidus en- passed away. The two deceased donor recipients were males aged fourteen hancement. Sulfitest was positive. Further investigation led to the and four respectively. The former had an uneventful course initially but later confirmation of isolated sulfite oxidase deficiency, with no enzyme activity developed complications including endocarditis and intermittently raised cre- detected on skin fibroblast culture. Molecular studies are ongoing. atinine. The latter had a slight rise in amylase associated with feed intolerance Discussion: This case illustrates the clinical variability of SOD and is not in the recovery phase, which resolved with conservative management. All only atypical but also seems to be the mildest form described so far. The patients remained metabolically stable and plasma MMA levels fell. association of ectopia lentis with a movement disorder, even without Discussion: (i) Close collaboration between the renal and metabolic teams psychomotor regression, should prompt us to look for this diagnosis. is vital (ii) Plasma MMA levels may be a potential biomarker for rejection (iii) Amylase should be monitored. In patients with a history of pancreatitis consideration should be given to the use of a modified immunosuppressant regimen with avoidance of steroids. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S9

O-026 O-028 DOMINO LIVER TRANSPLANTATION IN METHYLMALONIC INDUCTION OF APOPTOSIS AND NECROSIS IN MURINE ACIDEMIA HL-1 CARDIOMYOCYTES AFTER STIMULATION WITH Barshop B.A.1 , Nyhan W.L.1 , Khanna A.1 MONOUNSATURATED LONG-CHAIN FATTY ACIDS 1Dept of Pediatrics, UCSD, La Jolla, California, United States Hoffmann L1 , Seibt A1 , Spiekerkoetter U1 1Univ Child Hosp, Duesseldorf, Germany Background: Liver transplantation (LT) has been reported in methylmalonic acidemia (MMA) but long term results are controversial. This raises Inborn errors of long-chain fatty acid oxidation present with disease- ethical concerns using LT for MMA, since other patients with approved specific accumulation of acylcarnitines and lipid droplets in various tissues. indications die before grafts are available. There is increasing evidence, that accumulating fatty acids or their deriva- Objectives: Determine feasibility of using an MMA patient as donor for a tives are crucially involved in the occurrence of severe arrhythmias and patient who requires LT but is unlikely to survive to qualify through cardiomyopathy, two major complications in these metabolic disorders. conventional channels. We measured lipid uptake and intracellular lipid accumulation after stimu- Patients/Methods: A 28 yr-old man with MMA underwent deceased donor lation with C8 – C18 fatty acid species for different time periods. We further LT, and his liver was used as domino graft for a 61 yr-old man with primary assessed apoptosis by analyzing the mitochondrial membrane potential and sclerosing cholangitis but low priority on the LT waiting list. The first transplant TLR-4 expression. was performed in standard fashion and the second in "piggy back" fashion. We found a significant accumulation of various saturated and unsaturated Results: Surgical outcome was successful and nine months post- long-chain fatty acids, but only incubation with monounsaturated C14:1 transplantation both patients have excellent graft function. The patient with and C16:1 fatty acids led to an increase in TLR-4 expression and disruption MMA showed decrease in plasma MMA and eventual plateau to levels of mitochondrial membrane potential, resulting in apoptosis and necrosis. slightly below pretransplant, while dietary protein increased from <0.6 to In contrast, accumulation of saturated C12 and C14 fatty acids had no >1.2 g/kg/d. The recipient developed moderate elevations of plasma MMA adverse effects and medium-chain fatty acids were rapidly metabolized (50-100 μM), but was stable with unrestricted dietary protein (1-1.9 g/kg). without accumulation. Conclusion: LT attenuates MMA, but concerns remain, particularly regard- This study demonstrates that C14:1 and C16:1 fatty acids have a high toxic ing CNS function. Patients with MMA can be domino donors for patients potential on cardiomyocytes in vitro. These results support the observation who would otherwise not survive until LT. of acute cardiac arrhythmias during severe lipolysis and metabolic derangement in those defects, accumulating these fatty acids such as very long- chain acyl-CoA dehydrogenase, carnitine palmitoyl-CoA transferase 2 and O-027 carnitine acylcarnitine translocase. BRAIN DAMAGE IN METHYLMALONIC ACIDURIA: 2-METHYLCITRATE LEADS TO AMMONIA INCREASE AND APOPTOSIS O-029 Jafari P1 , Braissant O2 , Zavadakova P1 ,HenryH2 , Bonafé L1 , ENZYMATIC CHARACTERIZATION AND THERMAL STABILITY Ballhausen D1 ASSAYS ESTABLISH METHYLMALONYL-COA MUTASE 1Mol Pediatrics, Lausanne Univ Hosp, Lausanne, Switzerland MUTATIONS AS A TARGET FOR PHARMACOLOGICAL CHAPERONE 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland SCREENING Forny P1 ,FroeseDS2 , Suormala T3 ,YueWW2 , Baumgartner MR3 A 3D in vitro model of rat organotypic brain cell cultures in aggregates was 1SGC Univ Oxford, Univ Child Hosp, Zurich, Switzerland used to investigate neurotoxicity mechanisms in methylmalonic aciduria. 2SGC Univ Oxford, Oxford, United Kingdom 1 mM methylmalonate (MMA), 2-methylcitrate (2-MCA) or propionate 3Univ Child Hosp, Zurich, Switzerland (PA) were repeatedly added to the culture media at two different time points of the cultures. In cultures treated with 2-MCA, we observed a significant Genetic defects of the mitochondrial enzyme methylmalonyl-CoA mutase increase of lactate in the medium, consistent with a possible inhibition of (MCM) or deficiency of its cofactor, 5-deoxyadenosylcobalamin (AdoCbl), Krebs cycle and respiratory chain, as described earlier in the literature. lead to accumulation of toxic intermediates and cause the severe disorder Interestingly, we further observed that 2-MCA induced an important in- methylmalonic aciduria (MMA). The 80 MCM missense mutations reported crease in ammonia production with concomitant decrease of glutamine to date can be phenotypically classified into whether the mutant enzyme concentrations, which suggests an inhibition of the astrocytic enzyme exhibits residual activity in the presence of high concentrations of AdoCbl glutamine synthetase. These previously unreported findings may uncover (Km variants; mut-), or no detectable activity (mut0). a pathogenic mechanism in this disease with deleterious effects on early To identify the molecular basis of MCM mutations and potentials for small stages of brain development. By immunohistochemistry we could show that molecule therapy, we characterized 15 poorly studied mut-/0 missense 2-MCA substantially increased the number of apoptotic cells. On the mutants. Enzymatic assay in fibroblasts showed 100 to 1300-fold increased cellular level, 2-MCA had a toxic effect (cell swelling and cell death) on Km for AdoCbl for 9 mut- mutants compared to wildtype. Using differen- glial cells, but not on neurons. Surprisingly, MMA seemed to have a growth tial scanning fluorimetry (DSF), we further demonstrated (i) stabilization of stimulating effect on the cultures. We can conclude that 2-MCA was the wildtype MCM by AdoCbl (ΔTm +2.8 °C) and combined with substrate most toxic metabolite in our model for methylmalonic aciduria inducing analogue (ΔTm +13.0 °C); (ii) decreased thermal stability of mutants ammonia accumulation and massive apoptosis in brain cells. compared to wildtype (n09; e.g. P86L, ΔTm -9.1 °C), which can be improved with AdoCbl (n05; e.g. R694W, ΔTm +3.2 °C) and with AdoCbl plus substrate analogue (n05; e.g. G648D, ΔTm +12.7 °C). This work establishes a proof of principle for applying DSF and enzymatic assays to screen small molecules which can stabilize mutant MCM proteins for the future development of pharmacological chaperones to treat MMA. S10 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-030 06. Enzyme Replacement Therapy REDOX IMBALANCE IN LONG-CHAIN FATTY ACID OXIDATION DEFECTS O-031 Pastore A1 , Boenzi S1 , Martinelli D1 , Piemonte F2 , Tozzi G2 ,Di ENZYME REPLACEMENT THERAPY IN CHILDREN AND Giovamberardino G1 , Dionisi-Vici C1 ADOLESCENTS WITH ALPHA-MANNOSIDOSIS 1Div Metab Dis-Bambino Gesù Child Hosp, Rome, Italy Borgwardt L1 , Dali CI1 , Fogh J2 , Månsson JE3 , Olsen KJ4 , Beck HC5 , 2Molec Medicine-Bambino Gesù Child Hosp, Rome, Italy Lund AM1 1Dep of Clin. Gen, Rigshospitalet, Copenhagen, Denmark Long-chain fatty acids represent one of the most important energy source in 2Zymenex A/S, Hilleroed, Denmark humans and their degradation process is mediated by beta-oxidation 3Dep of Psychiatry and Neurochemistry,, Salgrenska, Mölndal, Sweden enzymes in the mitochondrial matrix. It has been suggested that oxidative 4Larix, Ballerup, Denmark damage may contribute to organ damage in patients with FAO defects but 5Danish Techological Institute, Kolding, Denmark in-vivo studies in humans are lacking. An overproduction of reactive oxygen species in the liver of VLCAD mouse was reported as the result Background: Alpha-mannosidosis (AM) is a rare LSD caused by alpha- of tissue fat accumulation (Tucci 2010) and long-chain 3-hydroxy fatty mannosidase deficiency resulting in intellectual disabilities, facial charac- acids accumulation has been shown to elicit oxidative stress in the cerebral teristics and hearing impairment. cortex of young rats (Tonin 2010). A recombinant human alpha-mannosidase enzyme (rhLAMAN) has been Since reduced glutathione plays an important role in the detoxification of developed for intravenous enzyme replacement therapy. We present the oxidative species, we assessed in vivo by HPLC the glutathione status in preliminary data after 12 months of treatment. lymphocyte from 3 patients with VLCAD and 5 with LCHAD/MTP Material and Methods: This phase I-II study aims to evaluate safety and deficiencies. efficacy of the ERT. The results showed in FAO patients an impaired glutathione homeostasis as 10 patients (7-17 y) were treated. The efficacy evaluation of the motor indicated by the significant depletion of reduced glutathione (p<0.001) and function (6MWT, 3MSCT, BOT-2), cognitive function (Leiter-R), measure- by the increase of oxidized/reduced- (p<0.001) and oxidized/total-glutathi- ment of oligosaccharides in serum, urine and CSF, CSF-Tau-protein and one (p<0.0001) ratios. GFA-protein were investigated. Our in-vivo study demonstrate significant redox imbalance in patients with Results: Oligosaccharides: S-, U- and CSF-oligosaccharides decreased, FAO defects that may contribute to organ damage and disease progression respectively by 88.6 % (CI -92.0 -85.2, p<0.001), 54.1 % (CI -69.5- - and suggests that new therapeutic strategies based on antioxidant drugs are 38.7, p<0,001), 25.7 % (CI –44.3- -7.1, p<0.05). needed in combination with standard dietary treatment. Biomarkers: CSF-Tau-protein and GFA-p decreased respectively 15 %, p< 0.009) and 23.1 % (CI -54.4 – 8.2, NS). Motor function: Improvements in 3MSCT (31 steps (CI 6.8-40.5, p<0.01) and in 6MWT (60.4 meters (CI –8.9 -51.1, NS) were achieved. Cognitive function: Improvement in the total Equivalence Age of 4 months (0.34) was achieved CI -0.2-0.8, NS). Conclusion: The data suggests that rhLAMAN is an encouraging new treatment for patients with AM. The study continues for 1 year when final conclusions can be drawn. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S11

O-032 O-033 LONG-TERM SAFETY AND EFFICACYDATAOFTALIGLUCERASE RECOMBINANT HUMAN LYSOSOMAL ACID LIPASE (LAL) ALFA, A PLANT CELL EXPRESSED RECOMBINANT DEMONSTRATES PHARMACODYNAMIC ACTIVITY IN GLUCOCEREBROSIDASE, IN THE TREATMENT OF NAÏVE CHOLESTERYL ESTER STORAGE DISEASE (CESD), THE LATE GAUCHER DISEASE PATIENTS ONSET FORM OF LAL DEFICIENCY Zimran A1 , Mehta A2 , Giraldo P3 , Rosenbaum H4 , Giona F5 , Amato D6 , Jones S1 , Enns G2 , Balwani M3 , Breen C1 , Sharma R4 , Deegan P5 , Petakov M7 , Meillon-Garcia LA8 , Solorio-Meza SE9 , Durán G10 , Elstein Malinova V6 , Honzik T6 , Valayannopoulos V7 , Schneider E8 , Burg J8 , D1 , Paz A11 , Chertkoff R11 , Brill-Almon E11 Quinn AG8 1Shaare Zedek Medical Center, Jerusalem, Israel 1MAHSC, St Mary's Hosp, Univ Manchester, Manchester, United Kingdom 2Royal Free Hospital, London, United Kingdom 2Stanford University School of Medicine, Stanford, California, United States 3Hospital Universitario Miguel Servet, Zaragoza, Spain 3Mount Sinai School of Medicine, New York, New York, United States 4Rambam Medical Center, Haifa, Israel 4Salford Royal NHS Foundation Trust, Salford, United Kingdom 5Universita "La Sapienza", Rome, Italy 5Addenbrooke's Hospital NHS Trust, Cambridge, United Kingdom 6Mount Sinai Hospital, Toronto, Canada 61st Faculty of Med, Charles University, Prague, Czech Republic 7Clinical Center of Serbia, Belgrade, Serbia and Montenegro 7Hôpital Necker-Enfants malades, Paris, France 8Hospital de Especialidades Bernardo Sepú, Mexico City, Mexico 8Synageva BioPharma, Lexington, Massachusetts, United States 9Instituto Mexicano del Seguro Social, Mexico City, Mexico 10Ponfificia Universidad Catolica de Chile, Santiago, Chile We have completed the first study (LAL-CL01) with recombinant human 11Protalix Biotherapeutics, Carmiel, Israel LAL (SBC-102) in patients with CESD. This disease, due to deficiency of LAL, is characterized by liver dysfunction, cirrhosis, splenomegaly, and Background: Taliglucerase alfa is an United States FDA approved enzyme dyslipidaemia. The safety and activity of SBC-102 was investigated in nine replacement therapy for Gaucher disease (GD). patients with LAL Deficiency in 3 sequential cohorts (0.35, 1.0, and Objective: To report 24-month interim results of taliglucerase alfa safety 3.0 mg/kg-1). Subjects received 4 once weekly infusions of SBC-102 with and efficacy from a planned 60 month follow-up assessment. 4 weeks of subsequent follow-up. After completing LAL-CL01, currently 7 Methods: Twenty-six treatment-naïve GD patients from the 9-month tali- subjects have transitioned into the LAL-CL04 extension study. SBC-102 glucerase alfa pivotal study continued double-blind treatment for an addi- was well tolerated at all doses with very infrequent mild infusion reactions tional 15 months at the same dose (30 or 60 U/kg) in an extension study. and no antidrug antibodies detected to date. In LAL-CL01 significant (p≤ Results: At 24 months spleen and liver volumes were reduced from 16.4/ 0.05) decreases in transaminases were seen at Day 28 compared to baseline 16.8 multiples of normal (MN) to 10.0/7.3 MN and from 1.7/1.5 to 1.3/1.2 (ALT: 37±12 versus 76±29) with no clear dose effect. Increases in serum MN for 30 U/kg and 60 U/kg, respectively; chitotriosidase activity was lipids provided additional evidence of activity consistent with tissue lipid reduced by 61 % and 76 %; hemoglobin, platelets and bone disease mobilization. In LAL-CL04, improvements in transaminases from baseline (assessed by quantitative chemical shift imaging) were also improved. All have been sustained with every other week dosing regimens and serum treatment-related adverse events (TEAEs) were mild or moderate in severity lipids have already returned to below baseline in 6/7 patients. In summary, and transient in nature; the most commonly occurring TEAEs were head- SBC-102 demonstrates an encouraging safety profile and clinical activity as ache (n02) and pruritus (n02). evidenced by improvements in transaminases and restoration of more Conclusion: This long-term evaluation demonstrated the ongoing safety normal lipid homeostasis. and efficacy of taliglucerase alfa in the treatment of GD. Conflict of Interest declared. Conflict of Interest declared. S12 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-034 O-035 SEVERE LYSOSOMAL DYSFUNCTIONS LEAD TO LEFT VENTRICULAR FUNCTION IN INFANTILE POMPE NEURODEGENERATION IN A MOUSE MODEL OF MUCOLIPIDOSIS PATIENTS ON ENZYME REPLACEMENT THERAPY (ERT) TYPE II Broomfield AA1 , Crook V1 , Finnegan N1 ,FletcherJ2 ,StewartC3 , Cleary M1 , Kollmann K1 , Damme M2 , Markmann S1 , Morelle W3 , Schweizer M4 , Chakrapani A3 , Malaiya N4 , Jones S2 , Chikermane A5 , Vellodi A1 ,FentonM6 Käkelä R5 , Michalski JC3 , Walkley SU6 , Braulke T1 1Dep Met Dis, Great Ormond St Hosp, London, United Kingdom 1Dept Biochemistry, Univ Child Hosp, UKE, Hamburg, Germany 2Dep Gen+Metab Dis, CMFT, Manchester, United Kingdom 2Dept Biochemistry I, Univ Bielefeld, Bielefeld, Germany 3Dep Met Dis, Birmingham Childrens Hosp, Birmingham, United Kingdom 3Unité de Glycobiologie, Univ Lille, Villeneuve d'Ascq, France 4Cardiology Dep, CMFT, Manchester, United Kingdom 4ZMNH, UKE, Hamburg, Germany 5Cardiology Dep, Birmingham Childrens Hos, Birmingham, United Kingdom 5Dept Biosciences, Univ Helsinki, Helsinki, Finland 6Cardiology Dep, Great Ormond Street Hosp, London, United Kingdom 6Albert EInstein College of Medicine, New York, United States Objective: An examination of the left ventricular structure and function of The majority of newly synthesized soluble lysosomal proteins are modified patients with classical infantile onset Pompe disease on ERT. with mannose 6-phosphate (M6P) residues on N-linked glycans, allowing Results: The echocardiograms of 21 patients from 3 centers in England on their M6P-receptors dependent delivery to endosomes. The first step in the Myozyme® were reviewed .The median follow up was 1 year 4 months (range formation of the M6P-recognition marker on lysosomal hydrolases is cat- 3 months-10 years). The overall mean left ventricular mass ndex (LVMI) on alyzed by the Golgi-resident GlcNAc-1-phosphotransferase. Mutations in baseline echos prior to ERT initiation was 259 mg/M2 (range 83-589). Six had a the genes encoding the GlcNAc-1-phosphotransferase subunits result in left ventricular end diastolic diameter (LVEDD) Z score>2 and 11 had reduced mucolipidosis II (MLII) and III. Biochemically these diseases are charac- fractional shortening. 13/21 patients are still alive. The baseline median LVMI of terized by a hypersecretion and intracellular deficiency of lysosomal survivors (229 mg/m2 (IQR125)) was significantly different from the non hydrolases. survivors (300 mg/m2 (IQR 69.5)), Mann Whitney P00.046. An abnormal We have generated knock-in mice with a common MLII patient mutation baseline fractional shortening was not related to outcome. Of the 13 survivors showing growth retardation, skeletal abnormalities, brain atrophy, elevated 10 have normalized their LVMI; the median observed time taken to do so was lysosomal enzymes in serum, and lysosomal storage in fibroblasts and 386 days (IQR 268). Of these 10 patients, 4/8 whose diastolic parameters were brain, closely mimicking the human MLII disease. To identify lysosomal recorded still had abnormal diastolic dysfunction on their most recent echocar- hydrolases that are strictly dependent on M6P for targeting, we analyzed the diogram. Of the 6 with LV dilatation at presentation 2 have survived, both with lysosomal storage material and found fucosylated and high mannose-type normalization of their LVEDD, though one is still on captopril. N-glycans, GM2 and GM3 gangliosides, cholesterol and bis(monoacylglycero)phosphate accumulating progressively in the brain of MLII mice. Increased LC3-II level and the formation of p62-positive neuronal aggregates indicate an impairment of constitutive autophagy in the MLII brain. O-036 Our findings demonstrate the crucial role of M6P for selected lysosomal ANTIBODY FORMATION TO ENZYME THERAPY IN CLASSIC proteins like alpha-L-fucosidase and alpha-mannosidase to maintain the INFANTILE POMPE DISEASE: IMPLICATIONS OF PATIENT capability for degradation of sequestered components in lysosomes and AGE autophagolysosomes preventing neurodegeneration. van Gelder CM1 , Kroos MA2 , Ozkan L2 , Plug I1 , Reuser AJJ2 , van der Ploeg AT1 1Div Metab Dis, Erasmus MC, Rotterdam, Netherlands 2Div Clin Genet, Erasmus MC, Rotterdam, Netherlands

Objectives: Enzyme-replacement therapy (ERT) with alglucosidase alfa has improved the lifespan of patients with classic infantile Pompe disease. We studied the development of antibodies to ERT and the effect on the clinical outcome. Methods: We determined the endogenous acid alpha-glucosidase expression (CRIM status) and regularly assessed antibody formation and clinical outcome in 11 patients with classic infantile Pompe disease treated with ERT since 1999 for 0.3-12.0 years (median 4.2 years). Results: All infants developed antibodies. The patients who lacked any endogenous acid alpha-glucosidase production (Cross-Reactive Immuno- logical Material: CRIM-negative, n03) did not develop a substantially different antibody titer than those who produced an inactive form of the enzyme (n08). However, the age at start proved to be important; none of the four patients who started ERT before 2 months of age developed titers of more than 1:6,250. Gross motor function and cardiac dimension improved less in patients with high titers and in CRIM-negative patients. The three CRIM-negative patients have died, whereas the eight CRIM-positive patients survived. Conclusion: Antibody formation is common. High antibody titers and a CRIM-negative status are associated with a poorer clinical outcome. Earlier start of ERT may prevent the formation of a severe immune response. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S13

07. Disorders of Energy Metabolism (1) O-039 CLINICAL PHENOTYPE OF 22 SPANISH PATIENTS WITH O-037 MUTATIONS IN NFU-1 WHOLE EXOME SEQUENCING REVEALS THAT SUBUNIT Del Toro M1 , Gonzalez-Gutierrez Solana L2 , Garcia-Cazorla MA3 , MUTATIONS ARE PREVALENT IN COMPLEX I DEFICIENT Campistol J3 , Dorao P4 , Labayru MT5 , Aldamiz L5 , Sanjurjo P5 ,de LEIGH SYNDROME Castro P6 , Labanda J7 , Lara J8 , Martin del Valle F6 , Vaquerizo J9 , Tort Fassone E1 , Taanman JW2 , Sweeney MG3 , Woodward C3 , Hargreaves F10 , Navarro-Sastre A10 , Arranz JA1 , Riudor E1 , Rodriguez-Pombo P11 , IP4 , Hanna MG2 , Taylor RW5 , Duncan AJ1 , Rahman S1 Perez-Cerda C12 , Briones P10 , Ribes A10 , Roig M1 1CMGU, UCL Institute of Child Health, London, United Kingdom 1Hospital Vall d'Hebron, Barcelona, Spain 2UCL Institute of Neurology, London, United Kingdom 2Hospital Niño Jesús, Madrid, Spain 3Neurogenetics Unit, Nat Hosp for Neurol, London, United Kingdom 3Hospital Sant Joan de Deu, Barcelona, Spain 4Neurometabolic Unit, Nat Hosp for Neurol, London, United Kingdom 4Hospital La Paz, Madrid, Spain 5Mitochondrial Research Group, Newcastle upon Tyne, United Kingdom 5Hospital de Cruces, Barakaldo, Spain 6Hospital Gregorio Marañón, Madrid, Spain Background: Leigh syndrome (LS) or subacute necrotising encephalomyelop- 7Hospital Ntra. Señora de Aranzazu, San Sebastian, Spain athy is a progressive neurodegenerative disorder caused by impaired cerebral 8Hospital Puerta de Hierro, Madrid, Spain mitochondrial energy generation. Complex I deficiency is the most frequently 9Hospital Infanta Cristina, Badajoz, Spain identified biochemical defect in LS, accounting for at least 30 % of cases. The 10Hospital Clinic, CIBERER,, Barcelona, Spain genetic diagnosis remains elusive in the majority of cases using conventional 11CEDEM, CBM, CIBERER, Univer. Autonoma, Madrid, Spain diagnostic techniques; we therefore sought to determine whether a whole exome 12CEDEM, CBM, CIBERER, Univer. Autonoma, Madrid, Spain Next Generation Sequencing (NGS) approach could increase the diagnostic rate in a single-centre cohort of 12 paediatric patients with complex I deficient LS. Mutations in NFU1 have been associated with an early fatal progressive Methods: A stepwise diagnostic approach was taken: mitochondrial DNA disease. We report the clinical phenotype of 22 patients in 15 families with sequencing was followed by candidate nuclear gene mutation analysis, and NFU1 mutations. finally whole exome NGS was employed in the remaining unsolved cases. All children were born at term and developed normally throughout neonatal Results: Complex I subunit gene mutations were detected in all patients: 3 period. First symptoms started at age 2–14 months, and death occurred mitochondrially-encoded (MT-ND1, MT-ND5, MT-ND6) and 4 nuclear- between 4-17 months. The initial symptoms were failure to thrive (6), encoded (NDUFV1, NDUFV2, NDUFS1, NDUFS4), including 4 novel pulmonary hypertension (9), neurological regression (6) and shock (2). mutations in nuclear-encoded complex I subunits identified by NGS. Clinical evolution allows classifying them into three groups. A first group Conclusions: We identified the genetic cause of complex I deficient LS in (8 patients) presented failure to thrive and neurological involvement. The all 12 patients. All had mutations in structural subunits of the enzyme, and second group (7 patients) had been previously diagnosed of pulmonary none in any of 9 known complex I assembly factors. This observation will hypertension, and after febrile illness exhibited neurological regression. allow the diagnostic pathway for complex I deficient LS to be rationalised. Brain imaging showed bilateral cavitating white matter lesions. Spongiform degeneration, astrogliosis, and white-matter necrosis with preserved U- fibers were confirmed at autopsy. In the third group (7 patients) pulmonary O-038 hypertension was the main clinical feature accompanied occasionally by failure to thrive. Despite the absence of neurological symptoms, areas of LIPOIC ACID SYNTHETASE DEFICIENCYA COMBINED DEFECT white-matter demyelination, vacuolization and astrogliosis in CNS were OF PYRUVATE OXIDATION AND GLYCINE METABOLISM found. All patients showed hyperglycinemia and lactic acidemia or in- 1 2 2 1 1 Brown RM , Naess K , Wibom R , Stewart B , Brown GK creased Krebs cycle metabolites in urine. 1 Dept Biochem, Univ Oxford, Oxford, United Kingdom Both pulmonary hypertension and neurological white matter involvement 2 Dept Lab Med, Karolinska Institutet, Stockholm, Sweden are highly suggestive of altered NFU1, a protein involved in lipoic acid biosynthesis. Lipoic acid synthetase (LIAS) deficiency, a newly recognised enzyme deficiency, results primarily in impaired function of the pyruvate dehydrogenase complex and the glycine cleavage enzyme. We describe a patient with this condition with a less severe presentation and more prolonged clinical course than in the previously reported case. The patient, a female now aged 6, is the first child of unrelated parents. Pregnancy and delivery were normal. She developed seizures on day 2 which were controlled with phenobarbitone and have not recurred. At age 2 years, she was investigated because of delayed development, hypotonia and ataxia. Urinary lactate excretion was greatly increased (550 mmol/mol creatinine) and glycine concentration was elevated in blood (706-733 μmol/L, normal range 60-320) and cerebrospinal fluid (20 μmol/L, normal range 3-18). Muscle morphology was normal, but ATP formation from pyruvate/malate was impaired and NADH-cytochrome c reductase activity reduced. Pyruvate dehydrogenase activity was 0.24 nmol/mg protein/min (normal 0.7-1.1), but no mutation was found in the genes for any subunit of the complex. Pathogenic mutations were subsequently identified in the LIAS gene. The patient currently has mild learning difficulties, poor co-ordination and attention and hyperactivity. There has been no response to thiamine supplementation or a ketogenic diet. S14 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-040 O-042 MULTIPLE MITOCHONDRIAL DYSFUNCTION SYNDROME PHENYLBUTYRATE THERAPY FOR PYRUVATE DEHYDROGENASE CAUSED BY A MUTATION IN BOLA3 COMPLEX (PDHC) DEFICIENCY Ahting U1 , Haack TB2 , Haberberger B2 ,MayrJA3 , Zimmermann F3 , Ferriero R.1 , Lamantea E.2 , Stacpoole P.3 , Bonafe L.4 , Lee B5 , Zeviani Schum J2 ,StreckerV4 ,GrafE5 ,HoppenT6 , Wittig I4 , Strom T5 , Meitinger M.2 , Brunetti-Pierri N.6 T5 ,ProkischH2 ,SperlW3 ,FreisingerP7 ,RolinskiB8 1TIGEM, Naples, Italy 1Dep Klin Chem, Munich Municipal Hosp, Muenchen, Germany 2Unit of Molecular Neurogenetics Besta, Milan, Italy 2Inst Human Genet, Helmholtz Cent Munich, Neuherberg, Germany 3Dept. Medicine, University of Florida, Gainesville, United States 3Dep Pediat, Paracelsus Med Univ, Salzburg, Austria 4Div Mol Pediatr Universitaire Vaudois, Lausanne, Switzerland 4Mol Bioenergetics, Goethe Univ, Frankfurt, Germany 5Dept Mol & Hum Genet, Baylor Coll Med, Houston, Italy 5Inst Human Genet, Technical Univ Munich, Muenchen, Germany 6Dept. Pediatrics, Federico II Univ, Naples, Italy 6Dep Pediat, Koblenz-Mayen Hosp, Koblenz, Germany 7Dep Pediat, Reutlingen Municipal Hosp, Reutlingen, Germany Deficiency of pyruvate dehydrogenase complex (PDHC) is the most 8Elblab, Elblandkliniken, Riesa, Germany common disorder leading to lactic acidemia. Phosphorylation of specific serine residues of the E1-alpha subunit of the PDHC by pyruvate Inborn errors of mitochondrial energy metabolism often present early in life dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephos- affecting multiple organ systems. Multiple mitochondrial dysfunction syndrome phorylation restores PDHC activity. Phenylbutyrate prevents phosphor- affecting mitochondrial oxidative phosphorylation and pyruvate dehydrogenase ylation of the E1-alpha subunit of the branched-chain ketoacid have been rarely reported so far. We report on two siblings presenting with dehydrogenase complex (BCKDC) and reduces plasma concentrations severe neonatal lactic acidosis, hypotonia, encephalopathy and cardiomyopathy; of neurotoxic branched chain amino acids in patients with maple leading to death within the first months of life. Muscle biopsies showed a syrup urine disease (MSUD), due to the deficiency of BCKDC. We specific combined deficiency of respiratory chain complexes I and II accompa- hypothesized that, similarly to BCKDC, phenylbutyrate enhances nied by a defect of the pyruvate dehydrogenase complex. PDHC enzymatic activity by increasing the portion of unphosphory- Joint exome analysis of both affected siblings uncovered a homozygous mis- lated enzyme. To test this hypothesis, we found that wild-type human sense mutation in BOLA3. The causal role of the mutation was validated in fibroblasts treated with phenylbutyrate have reduced levels of phos- silico and by lentiviral-mediated expression of the mitochondrial isoform of phorylated E1-alpha compared to untreated cells. To investigate the wildtype BOLA3 in fibroblasts of the patient leading to an increase of both effect of phenylbutyrate in vivo, we administered phenylbutyrate to residual enzyme activities and lipoic acid levels. Homology studies suggest that C57B6/L wild-type mice and we detected a significant increase in BOLA3 is involved in the biogenesis of iron-sulfur clusters which are necessary Pdhc enzyme activity and a reduction of phosphorylated E1-alpha for proper function of respiratory chain complexes and pyruvate dehydrogenase. subunit in brains, muscles, and livers compared to saline treated mice. Our results suggest that defects in BOLA3 lead to multiple mitochondrial Next, we showed that phenylbutyrate increases PDHC activity in dysfunction syndrome providing the molecular basis for a new class of mito- fibroblasts from PDHC-deficient patients. Being a drug already ap- chondrial dysfunction syndrome. Moreover exome analysis proved to be a proved for human use, phenylbutyrate has potential for increasing the powerful tool in the diagnostic workup of complex mitochondrial disorders. residual enzymatic activity of PDHC and improving the phenotype of PDHC deficiency.

O-041 A MOUSE MODEL OF MITOCHONDRIAL COMPLEX I DEFICIENCY EXHIBITS PROGRESSIVE CARDIAC DISEASE AND SEIZURES Craigen W.J.1 , Donti T.1 ,LaiY-C.1 ,AtherS.1 ,WehrensX.1 , Graham B.H.1 1Baylor College of Medicine, Houston, United States

Mitochondria are essential for many fundamental cellular processes and dysfunctional mitochondria frequently result in multi-organ disease. Mitochondrial complex I (CI) is composed of approximately 45 subunits and is the most common abnormality in mitochondrial respiratory chain disease. NADH:ubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) is one of the nonenzymatic, nuclear-encoded subunits of CI that is essential for assembly. Defects in NDUFS4 cause CI deficiency and a fatal Leigh-like phenotype in humans. A gene trap allele of Ndufs4 (Ndufs4SAβgeo) in mouse embryonic stem cells has been used to generate transgenic animals. Although Ndufs4 mRNA is reduced to 5 % of wildtype levels, Ndufs4SAβgeo homozygotes are viable and exhibit a significant partial deficiency of CI (20-30 % of control activity), as well as a partial deficiency of Complex III. Unlike other previously reported mutant Ndufs4 mouse strains, Ndufs4SAβgeo mice do not die prematurely, but exhibit increased sensitivity to kainate-induced seizures and defective cardiac function, with conduction abnormalities, bradycardia, and diminished contractile function. Mouse embryonic fibroblasts obtained from mutants exhibit decreased cellular respiration that is corrected by treatment with vitamin E. This mouse model will provide insights into the pathogenesis of mitochondrial complex I diseases as well as facilitate the exploration of novel therapeutic strategies. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S15

08. Disorders of Energy Metabolism (2) O-044 EXOME SEQUENCING IDENTIFIES ACYL GLYCEROL KINASE O-043 (AGK) MUTATIONS AS THE CAUSE OF CARDIOMYOPATHY DIAGNOSING VERY LONG-CHAIN ACYL-COA DEHYDROGENASE AND CATARACTS (SENGERS SYNDROME) DEFICIENCY (VLCADD) Schulze A1 , Siriwardena K1 ,MacKayN2 ,LevandovskiyV3 ,BlaserS2 , Olpin SE1 , Clark S1 , Scott C1 , Croft J1 , Manning NJ1 , Bonham JR1 , Raiman J1 ,KantorP4 ,AckerleyC2 , Robinson BH3 ,CameronJM3 Hind H1 , Andresen BS2 , Khan A3 , Sharma R4 , Kirk R5 , Dalley J1 1Div Clin Met Gen, Hosp Sick Child, Toronto, Canada 1Dept Clin Chem, Sheffield Child Hosp, Sheffield, United Kingdom 2DPLM, Hosp Sick Child, Toronto, Canada 2Biochem Mol Biol, Univ South Denmark, Odense, Denmark 3GGB, Hosp Sick Child, Toronto, Canada 3Med Genet Paed, Alberta Children's Hosp, Alberta, Canada 4Div Card, Hosp Sick Child, Toronto, Canada 4Dept Metab Med, Salford Royal Hosp, Salford, United Kingdom 5Dept Mol Genet, Sheffield Children's Hos, Sheffield, United Kingdom Objectives: Identifying mutations in novel genes causative for Sengers syndrome. Case report: Two siblings with severe neonatal lactic acidosis, cardiomy- VLCADD manifests as severe neonatal disease, often with cardiomyopathy, opathy and cataracts, had abnormal looking mitochondria, markedly high milder infantile disease, primarily with hypoketotic hypoglycaemia or as citrate synthase activity, and citrate synthase crystals in electron micro- late onset myopathic disease. Newborn screening programs suffer from a graphs. High levels of manganese superoxide dismutase protein were con- high false positive rate primarily due to positive screens in carriers. We have sistent with increased free radical damage. compared various methods for discriminating carriers from affected infants Methods: Novel base pair changes or small insertions/ deletions in 34,743 genes and for separating the severe from myopathic phenotypes in fibroblasts found by exome sequencing were reduced to 20 candidate genes. The 99.9 % using fat oxidation flux assays at 26 °C, 37 °C & 41 °C, fibroblast reduction was achieved by selecting only novel, non-synonymous coding sequence acylcarnitines profiling and specific enzyme assay (UV-HPLC detection). changes, and proteins computationally predicted to be targeted to the mitochondria. We have data on 64 VLCADD's including 20 myopathic cases. Mutation Results: We have identified the causative mutations in the Acyl Glyerol data was available for most subjects. The more severe phenotypes are Kinase gene (AGK) in the index patients and subsequently in a second family readily detected by all methods e.g. Myristate/oleate ratio and percentage with Sengers syndrome. The causative mutations were confirmed by compar- activity with oleate for severe infantile (n07) was 0.16 and 12+/-3.1 % at ison with family members and control gDNA. 41 °C (normals 0.98+/-0.13 and 104+/-15 % n010 respectively). Specific Conclusion: AGK deficiency is the underlying cause for Sengers syndrome. AGK VLCAD activity (nmol/mg/min) was 0.4+/-1.6 (n07) in VLCADD, 5.0+/- phosphorylates monoacylglycerol and diacylglycerol to lysophosphatidic acid and 1.6 (n04) in established carriers and 15.1+/-5.4 (n018) in controls. How- phosphatidic acid; these signaling molecules affect the mitochondria's response to ever, all methods showed some overlap between mildly affected phenotypes superoxide radicals. Loss of AGK results in oxidative damage, damaging mitochon- and obligate carriers for a handful of difficult cases. Overall fatty acid flux drial DNA and proteins (respiratory chain enzyme deficiencies and cardiac hyper- at 41 °C appeared as the best discriminator. One adult patient appears to be trophy) and lenses (cataracts), as well as mitochondrial membranes. a symptomatic carrier.

O-045 BEZAFIBRATE AS TREATMENT OPTION IN PATIENTS WITH MITOCHONDRIAL COMPLEX-I DEFICIENCY Haberberger BM1 , Freisinger P2 , Strecker V3 , Ahting U4 , Rolinski B4 , Mayr J5 , Sperl W5 , Wittig I3 , Meitinger T1 , Prokisch H6 1Inst Human Genetics, Tech Univ Munich, Munich, Germany 2Depart Paediatrics, Klinikum Reutlingen, Reutlingen, Germany 3Molekulare Bioenergetik, Univ Frankfurt, Frankfurt, Germany 4Depart Klinische Chemie, Munich, Germany 5Depart Pediatrics, Paracelsus Med Univ, Salzburg, Austria 6Inst Human Genetics, Helmholtz Zentrum, Munich, Germany

Mitochondriopathies are an important cause of metabolic disorders. A majority of them affect the respiratory chain complexes and the cellular ATP production. In spite of rapid progress identifying the molecular cause, curative therapeutic options are barely available. Bastin et al. (2008) and Wenz at al. (2008) provided evidence from in vitro studies and mouse models that activation of the PPAR/ PGC-1-alpha pathway with bezafibrate could be a new therapeutic approach. In order to verify this effect, we collected 32 fibroblast cell lines from patients with isolated complex-I deficiency and a defined molecular diagnosis. The complex-I activity in fibroblasts showed in average a rest activity of 40 % compared to control cell lines. Bezafibrate treatment led to significant improvement of complex-I activity in more than 50 % of the cell lines. The treatment effect was analyzed on genome-wide expression levels, revealing increased expression of genes involved in lipid and fatty acid metabolism as well as transport. In a group of 5 cell lines which responded to bezafibrate treatment with significant increase of complex-I activity, we found increased amounts of complex I assembled in supercomplexes by two-dimensional blue native SDS-PAGE experiments. These results support bezafibrate as a treatment option for a subgroup of patients. S16 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-046 O-047 MUTATIONS IN THE PHOSPHOLIPID REMODELING GENE THE INCIDENCE AND NATURAL COURSE OF GLYCOGEN MEGDEL1 IMPAIR MITOCHONDRIAL FUNCTION AND STORAGE DISEASE TYPE IIIA-RELATED CARDIOMYOPATHY INTRACELLULAR CHOLESTEROL TRAFFICKING AND CAUSE Sentner CP1 , Smit GPA1 DYSTONIA AND DEAFNESS 1Dept Metab Dis, Univ Med Centr, Groningen, Netherlands Wortmann SB1 , Vaz FM2 , Gardeitchik T1 , Kluijtmans LAJ3 , Rodenburg RJ1 , van Hasselt PM4 , Baric I5 , Pronicka E6 , Kalkan Ucar S7 , Naess K8 , Background: The clinical significance of GSDIIIa-related cardiomyopathy Singhal KK9 , Krumina Z10 , Smeitink JAM1 , Lefeber DJ11 , de Brouwer remained unclear due to lack of data. Therefore, we conducted a multi- APM12 , Wevers RA3 , Morava E1 centre retrospective study to investigate the natural course of GSDIII and its 1Dep of Ped/NCMD, RUNMC, Nijmegen, Netherlands cardiac complications. 2LGMD/Dep of Clin Chem & Ped, AMC, Amsterdam, Netherlands Methods: Data on cardiomyopathy included results of clinical-, 3LGEM, Dep of Lab Med, RUNMC, Nijmegen, Netherlands electrocardiographic-, and echocardiographic-evaluation and follow-up. 4Dep of Metab Dis, WKZ, Utrecht, Netherlands Results: In total data was collected on 225 patients; median age 19.9 years 5Dep of Ped, Univ Hosp Centre, Zagreb, Croatia (range: 0.3–78.3); 47.6 % males. 208 patients had received cardiac evaluation; 6Dep of Met Dis, Chil Memorial Health Ins, Warsaw, Poland in 111 patients abnormalities were found. Abnormalities were first found at 7Dep of Ped, Ege Univ Fac of Med, Izmir, Turkey median age 8.0 years (0.3–62); the most frequent finding was left ventricular 8Dep of Ped Neurol, Karolinska Univ Hosp, Stockholm, Sweden hypertrophy in 73 patients. 15 affected patients regressed to normal values 9Dep of Neurol, All India Inst of Med Sci, Delhi, India with age, while 18 patients had progressive cardiomyopathy. Heart failure was 10Med Genet Clinic, Child Univ Hosp, Riga, Latvia diagnosed in 11 patients, in whom pharmacological therapy was required. 11Dep of Neurol, RUNMC, Nijmegen, Netherlands Three patients died due to severe congestive heart failure. 12Dep of Hum Gen, RUNMC, Nijmegen, Netherlands Conclusions: Cardiomyopathy is a frequent complication of GSDIII occurring in 53 % of the evaluated patients, generally first presenting in early We defined MEGDEL syndrome as 3-MethylGlutaconic Aciduria with childhood, and remaing stablein70%oftheaffected patients. Left impaired oxidative phosphorylation, Deafness, Encephalopathy, Leigh- ventricular hypertrophy is the most common finding. Surprisingly, 14 % like MRI, progressive spasticity and dystonia. Whole exome/ Sanger se- of the affected patients regressed to normal values with age, with 16 % quencing identified 17 mutations in MEGDEL1, a gene of unknown func- showing progression. tion in 18 patients from 16 families. Upon the similarity of MEGDEL and Barth syndrome and the presence of a lipase domain in MEGDEL1 we performed phospholipid analysis. This revealed abnor- O-048 mal phosphatidylglycerol acyl chain composition, altered cardiolipin subspecies PHOSPHOGLUCOMUTASE-1 - DEFECTS OF THE GATEKEEPER and low bis(monoacylglycero)phosphate levels in patients fibroblasts. As firmly BETWEEN GLYCOGEN AND GLUCOSE STRONGLY IMPAIR established in Barth syndrome, cardiolipin alterations lead to oxidative phosphor- PROTEIN GLYCOSYLATION WITH DIVERSITY OF PHENOTYPES - ylation dysfunction. Low bis(monoacylglycero)phosphate levels are known to MECHANISM, SCREENING, TREATMENT accumulate intracellular cholesterol, which was confirmed by abnormal filipin Rust S1 , Tegtmeyer LC2 , Fingerhut R3 , Freeze HH4 , Marquardt T2 staining in patients fibroblasts. MEGDEL1 was further shown to be localized at 1Leibniz-Inst. f. Arteriosklerosefoschung, Muenster, Germany the interface between endoplasmatic reticulum and mitochondria, in the mito- 2Uni-Klin. f. Kinder- und Jugendmedizin, Muenster, Germany chondria associated membrane (MAM) fraction, important for the exchange of 3Newborn Screen Lab, Univ Child Hosp, Zuerich, Switzerland phospholipids. 4Sanford-Burnham Medical Res Inst, La Jolla, United States Complementation of patient fibroblast with wild-type human MEGDEL1 by lentiviral infection led to a normalization of the phosphatidylglycerol acyl Background: Congenital disorders of glycosylation (CDG) are due to chain composition, linking MEGDEL1 mutations to the compromised phos- incomplete glycan attachments in glycoproteins. Since about 50 % of all phatidylglycerol remodeling. proteins are glycoproteins that may be affected by CDGs, typically systemic MEGDEL1 is crucial for both mitochondrial function and intracellular effects and a broad phenotypic spectrum are observed. We discovered cholesterol trafficking. Our data identify MEGDEL1 as key player in several patients with unusually mixed type CDG-I/-II, associated with split phosphatidylglycerol remodeling as well as first enzyme in the as yet uvula / cleft palate, hepatopathy, hypoglycemia, rhabdomyolysis, and dila- unknown bis(monoacylglycero)phosphate biosynthetic pathway. tive cardiomyopathy (DCM). Results: By next generation sequencing we identified deficiency of pho- phoglucomutase 1 (PGM1) as underlying cause of mixed type CDG-I/II syndrome. PGM1 catalyzes the bidirectional conversion of Glucose-1P (Glc-1P) to Glc-6P. During low blood glucose liver will try to stabilize blood sugar and switch on glycogenolysis producing Glc-1P. In PGM1- deficiency this can not flow to Glc-6P, instead the GALT-catalyzed reaction with UDP-Gal to UDP-Glc and Gal-1P will reduce UDP-Gal and increase UDP-Glc:UDP-Gal ratio. UDP-Gal is necessary for protein glycosylation, explaining the CDG-phenotype. Feeding with galactose and uridine does reverse this effect almost completely. A simple Guthrie-test was developed to screen pre symptomatic carriers. Conclusion: PGM1-deficieny may present with diverse phenotypes, e.g. milder, resulting in adult diagnosis of rhabdomyolysis on exessive training or eg. life-threatening DCM in children and complications in children necessitating resuscitation. Presymptomatic screening and dietary intervention are suggested. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S17

09. Disorders of Trace Elements and Vitamins (1) O-051 MUTATIONS IN SLC33A1 CAUSE AN AUTOSOMAL RECESSIVE O-049 LETHAL DISORDER WITH CONGENITAL CATARACTS, EFFECTS OF CHELATORS ON COPPER BIODISTRIBUTION IN BILATERAL HEARING LOSS, DEVELOPMENTAL DELAY AND MENKES DISEASE MODEL MICE AS EVALUATED WITH REDUCED COPPER AND CERULOPLASMIN IN SERUM SMALL ANIMAL PET IMAGING Huppke P1 , Brendel C1 , Kalscheuer V2 , Korenke G.C.3 , Freisinger P4 , Nomura S1 , Nozaki S2 , Takeda T1 , Ninomiya E1 , Kudo S1 , Hamazaki T1 , Pitelet G5 , Wilson C6 , Moller LB7 , Kaler SG8 , Gärtner J8 Fujioka H1 , Hayashinaka E2 , Wada Y2 , Hiroki T3 , Fujisawa C3 , Kodama 1Universitätsmedizin Göttingen, Göttingen, Germany H3 , Watanabe Y2 , Shintaku H1 2Max Planck Institute for Mol Genet, Berlin, Germany 1Osaka City University of Medicine, Osaka, Japan 3Dep of Neuroped, Children's Hospital, Oldenburg, Germany 2RIKEN CMIS, Kobe, Japan 4Klinikum am Steinenberg, Reutlingen, Germany 3Teikyo University of Medicine, Tokyo, Japan 5Service de Neuropédiatrie, CHU Nice, Nice, France 6Starship Children's Hospital, Auckland, New Zealand Background: Menkes disease is an inherited disorder of copper metabo- 7Kennedy Center, Glostrup, Denmark lism caused by dysfunctional ATP7A. Copper deficiency causes multisys- 8National Institutes of Health, Bethesda, United States temic disorders with severe neurodegeneration. Although subcutaneous injection of histidine-copper is a mainstay of treatment of Menkes disease, We report that mutations in the SLC33A1 gene coding for AT-1, the acetyl- neurological improvement remains limited. Furthermore, accumulation of CoA transporter for the endoplasmic reticulum, cause a distinct clinical injected copper in the kidney potentially degrades renal function. phenotype with congenital cataracts, hearing loss, severe developmental Objectives: We investigated the effects of two copper chelators on biodis- delay, hypomyelination and cerebellar atrophy. Intriguingly, the disorder is tribution of injected copper by using microPET. also associated with low or non-detectable copper and ceruloplasmin in Materials and Methods: C3H/He mice were used as control groups and plasma, a finding that is so far only known for Menkes disease, Wilson macular mice were used as Menkes disease model. Mice were pretreated disease and aceruloplasminemia. In five patients from four unrelated fam- with chelators (disulfiram and/or penicillamine) for 30 minutes before ilies either homozygous or compound heterozygous mutations in SLC33A1 copper injection. Continuous microPET imaging was performed for 4 hours were found. Biochemical analyses showed that the mutations lead to re- after 64CuCl2 injection. duced or absent AT-1 expression and abnormal intracellular localization of Results: Injected copper barely distributed to the brain, and accumulated in AT-1. To study the role of AT-1 in copper metabolism we performed AT-1 kidney without urinary excretion in macular mice. Pretreatment with the lipo- knockdown in HepG2 cells which led to reduced ceruloplasmin secretion. philic chelator disulfiram markedly increased copper uptake to the brain of These results indicate that the reduced copper levels seen in serum of the macular mice. Pretreatment with the water-soluble chelator penicillamine en- patients are due to reduced ceruloplasmin levels and not a sign of copper hanced urinary excretion and reduced copper accumulation in the kidney. Con- deficiency. Determination of copper and ceruloplasmin should be consid- comitant treatment with disulfiram and penicillamine retained the same effects. ered in patients with congenital cataracts especially when combined with Conclusion: MicroPET imaging was useful to evaluate the effects of neurological symptoms. chelators on copper-biodistribution. Combined use of disulfiram and penicillamine may improve clinical outcomes of Menkes disease.

O-050 A NOVELTHERAPY, COMBINATION THERAPY WITH INJECTIONS OF COPPER AND ORAL ADMINISTRATIONS OF DISULFIRAM, IN PATIENTS WITH MENKES DISEASE ANS OCCIPITAL HORN SYNDROME Kodama H1 , Ogawa E2 , Hiroki T2 1Teikyo Heise Univeristy, Tokyo, Japan 2Teikyo University School of Medicine, Tokyo, Japan

The current therapy for Menkes disease (MD) is a parenteral administration of copper-histidine. However, the treatment is less effective for neurological disturbance and connective tissue disorders. Disulfiram, a lipophilic chelator, has shown beneficial effects in the macular mouse, an animal model of MD. These results led us to treat MD and OHS patients with disulfiram in combination with copper-histidine. Disulfiram was orally administered in two patients with MD and a patient with OHS after approval of the ethical committee of Teikyo University Hospital, with maintenance dosage of 100 mg/day. Supplementation of copper-histidine was unaltered during the study period in patients with MD. Serum levels of copper and ceruloplasmine tended to increase in a MD patient, and he showed enriched emotional expression and behavior more often after disulfiram administration. Bone mineral content was increase by dusilfiram administration in the MD and OHS patients. No Adverse effect was found. These results suggest that copper-disulfiram complex is transported to neurons resulting in neurological improvement. Moreover, the therapy may be effective for the connective tissue disorders in MD and OHS patients. S18 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-052 O-054 INVESTIGATION OF RIBOFLAVIN TRANSPORTER MUTATIONS IN MEASUREMENT OF PLASMA B6 VITAMER PROFILES IN CHILDREN BROWN-VIALETTO-VAN LAERE SYNDROME, A POTENTIALLY WITH INBORN ERRORS OF VITAMIN B6 METABOLISM USING AN TREATABLE METABOLIC DISEASE LC-MS/MS METHOD Pandraud A1 , Clayton P2 , Foley AR3 , Muntoni F3 , Johnson JO4 , Footitt EJ1 , Clayton PT1 , Mills K1 , Heales SJ2 , Neergheen V3 , Singleton AB4 , Reilly MM1 , Houlden H1 Oppenheim M3 , Mills PB1 1MRC Ctr Neuromusc Dis, NHNN, ION, UCL, London, United Kingdom 1CMGU, UCL Institute of Child Health, London, United Kingdom 2Dpt Clin Molec Genetics, ICH, UCL, London, United Kingdom 2Chem Path, Great Ormond Street Hospital, London, United Kingdom 3Dubowitz Neuromusc Ctr, ICH, GOSH, UCL, London, United Kingdom 3National Hospital, Queen Square, London, United Kingdom 4Lab of Neurogen, Nat Inst Aging, NIH, Bethesda, United States Vitamin B6 dependent seizure disorders are an important and treatable cause of Background: Brown-Vialetto-Van Laere (BVVL) syndrome is a rare, gener- childhood epilepsy. In man vitamin B6 ingested from the diet exists as six ally autosomal recessive disease with variable age of onset. Patients may present different vitamers and the breakdown product, 4-pyridoxic acid which is excret- with cranial nerve palsies, sensorineural hearing loss, respiratory insufficiency, ed in urine. We describe an analytical method of liquid chromatography linked lower motor neuron signs and severe sensorimotor neuropathy. Mutations have to tandem mass spectrometry (LC-MS/MS) to measure all vitameric B6 forms in been found in genes encoding riboflavin transporters, SLC52A1, SLC52A2, and plasma and show application of this methodology to a control paediatric SLC52A3 (previously GPR172B, GPR172A and C20orf54, respectively) lead- population and for the investigation of children with vitamin B6 responsive ing to flavin deficiency. Flavin adenine dinucleotide (FAD) and flavin mononu- seizure disorders. We show that patients with inborn errors of B6 metabolism cleotide (FMN), riboflavin metabolites, are involved in redox reactions in such as pyridox(am)ine 5'-phosphate oxidase (PNPO) deficiency have charac- processes of cell energy metabolism, serving as electron acceptors in complexes teristic B6 profiles (elevated pyridoxine, pyridoxine phosphate, pyridoxamine I and II of the mitochondrial respiratory chain and in fatty acid β-oxidation. and pyridoxamine phosphate). These profiles allow them to be differentiated Some BVVL patients have shown clinical and biochemical improvements on a from each other and control populations, even when on treatment with B6. treatment regimen of high-dose oral riboflavin. Children on B6 supplementation show greatly elevated plasma levels of Methods: Coding exons and flanking intronic sequences of SLC52A1, some B6 vitamers (pyridoxal phosphate, pyridoxal and pyridoxic acid) and SLC52A2,andSLC52A3weresequencedin46patientspresentingwithBVVL the consequences of such deviations from normal physiology are unknown. and BVVL-like syndromes including early-onset severe sensorimotor neuropathy, This LC-MS/MS method will be a useful tool for treatment monitoring; it multiple cranial neuropathies, truncal and upper extremity weakness, swallowing may allow optimisation of treatment doses in patient groups and help difficulties and respiratory insufficiency. further our understanding of mechanisms of neurotoxicity which may be Results: Five patients had compound heterozygous mutations in SLC52A2. seen with B6 supplementation. No mutations were identified in SLC52A1 and SLC52A3 in our cohort. Conclusion: SLC52A2 mutations are not a rare cause of BVVL syndrome, a severe neurological disorder potentially treatable with high-dose oral riboflavin.

O-053 VITAMIN B6 METABOLISM IN BRAIN Verhoeven-Duif N.M.1 , Albersen M.1 , Bosma M.1 , De Sain-van der Velden M.G.M.1 , Visser G.2 , Visser W.F.1 , de Koning T.J.3 1Dept. Metabolic Diseases, UMC Utrecht, Utrecht, Netherlands 2dept. Pediatrics, UMC Utrecht, Utrecht, Netherlands 3Dept. Genetics, UMC Groningen, Groningen, Netherlands

Background: Deficiencies of vitamin B6 result in convulsions that can be treated with vitamin B6. Although treatment often is successful in reducing the convulsions, developmental outcome is not always optimal. Methods: We studied the distribution of B6 vitamers in CSF from over 100 children. In addition, we used Neuro2A cells to study B6 metabolism in brain cells under normal and vitamin B6-deficient conditions. Results: The profile of B6 vitamers in CSF was found to be consistently different from the profile in plasma and much higher in preterm than in term neonates. Treatment with oral pyridoxine resulted in an extreme increase of pyridoxine, whereas pyridoxal phosphate only increased to maximal two times normal. The neuro2A cells imported and metabolized all unphosphorylated B6 vitamers. In B6-deficient conditions, both pyridoxal phosphate and pyridoxamine phosphate decreased. Rapid normalization was observed after addition of pyridoxal or pyridoxine. Conclusion: These data raise the question whether pyridoxine is the vitamer of choice to treat B6 deficiency, as it leads not only to normalisation of pyridoxal phosphate, but also to extremely elevated pyridoxine in CSF. These high CSF pyridoxine concentrations may lead to increased intracellular pyridoxine concentrations of which the effect in unknown. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S19

10. Disorders of Neurotransmitters and Glycosylation and O-056 Miscellaneous EXPANSION OF THE GENOTYPIC AND PHENOTYPIC SPECTRUM OF HEREDITARY DOPAMINE TRANSPORTER DEFICIENCY O-055 SYNDROME CLINICAL FEATURES OF PRIMARY DYSTONIAS AND OTHER Ng J1 , Meyer E2 ,LiY3 , Zhen J3 , Rider N4 , Rotstein M5 , Leuzzi V6 , MOVEMENT DISORDERS ASSOCIATED WITH SELECTIVE Reith MEA3 , Kurian MA2 , Kurian MA1 SEROTONIN DEFICIENCY (SSD) 1Neurology, Great Ormond Street Hosp, London, United Kingdom Ng J1 , Carr LJ1 , Devile C2 , Hemingway C1 , Lewis V3 , Mordekar SR4 , 2UCL Institute Child Health, London, United Kingdom Pall H5 , Patel J6 , Smith M7 , Gissen P8 , Clayton P8 , Heales SJR9 , Heales 3Psych & Pharm, New York Univ School Med, New York, United States SJR10 , Kurian MA8 , Kurian MA1 4Clinic for Special Children, Strasburg,PA, United States 1Neurology, Great Ormond Street Hosp, London, United Kingdom 5Tel Aviv Sourasky Medical Center, Tel Aviv, Israel 2Neurology, Great North Children's Hosp, Newcastle, United Kingdom 6Child Neurol & Psych, Univ Rome, Rome, Italy 3Royal Devon and Exeter NHS Trust, Devon, United Kingdom 4Sheffield Children's Hosp, Sheffield, United Kingdom Background: Dopamine transporter deficiency syndrome (DTDS) is a 5University Hospital, Birmingham, United Kingdom hereditary disorder characterised by dystonia-parkinsonism, raised CSF 6Bristol Children's Hosp, Bristol, United Kingdom HVA:HIAA ratio and mutations in SLC6A3, the gene encoding the dopa- 7Birmingham Children's Hosp, Birmingham, United Kingdom mine transporter (DAT). We describe the clinical, genetic and functional 8UCL Institute Child Health, London, United Kingdom features of newly identified DTDS cases. 9Neurometab, National Hosp Queens Square, London, United Kingdom Methods: Patients presenting with a biochemical profile suggestive of DTDS 10Chem Path, Great Ormond Street Hops, London, United Kingdom underwent detailed clinical phenotyping and SLC6A3 mutation analysis. Mutant constructs of human DAT (hDAT) were used for in-vitro functional Background: SSD is increasingly recognized as a neurotransmitter disor- analysis of dopamine uptake and cocaine–analogue binding. der in which the underlying aetiology and pathogenic mechanisms are Results: We report five patients presenting in early infancy (2-9 months) poorly understood, and is defined by isolated low CSF 5- with a progressive movement disorder characterised by hyperkinesia, cho- hydroxyindoleacetic acid (5-HIAA). We aim to delineate the spectrum of rea, dystonia and hypokinetic-rigid features. One patient is still alive at movement disorders in children with SSD. 35 years with features of classical parkinsonism. Patients had markedly Methods: Children with isolated low CSF 5-HIAA with normal neuro- raised CSF HVA: 5 HIAA ratios ranging from 6.8 to 31.9 (normal 1.0-3.7). imaging were identified and clinical records reviewed. Mutations in SLC6A3 were identified in all cases, including novel missense Results: Eleven children were identified with median age at presentation of and splice site mutations. In vitro functional investigation of mutant hDAT 2 years (0–15). Median CSF 5-HIAA level was 21.3 % (1.0–63.8 %) below showed that studied mutations resulted in loss of DAT function. age-dependent lower limit of normal. Conclusion: DTDS is a newly described clinical disorder. Our new case 4/11 children presented with focal dystonia (2 with associated myoclonus). cohort of further DTDS patients expands the clinical phenotype and range 4/11 presented with spastic diplegia, with associated focal dystonia in 1. of mutations identified in DTDS, thereby increasing the clinical and mo- One child presented with chorea and myoclonus. 2 sisters presented with lecular understanding of this disorder. tremor, myoclonus and depression and both had pathogenic DYT11 mutation-positive myoclonus-dystonia syndrome (MDS). All neurometabolic and genetic investigations were negative in the remainder. All had a trial of L-dopa without sustained response. Conclusion: SSD is associated with a spectrum of movement disorders and our findings confirm previous data implicating serotonin in motor control. In addition, low serotonin in DYT11-MDS may contribute to the psychiatric MDS disease manifestations. Further studies are required to elucidate pathogenic mechanisms in the SSD phenotypes identified. S20 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-057 O-059 CENTRAL NERVOUS SYSTEM INVOLVEMENT AND BLEEDING PGM1-DEFICIENCY WITH ABNORMAL PROTEIN GLYCOSYLATION; DIATHESIS IN A NOVEL CMP-SIALIC ACID TRANSPORT DEFECT EASY DIAGNOSIS AND DIETARY INTERVENTION Mohamed M1 , Guillard M2 , Wevers RA2 , de Brouwer A3 , Lefeber DJ2 , van Scherpenzeel M1 , Timal S1 , Raymond K2 ,AdamowiczM3 , Socha P3 , Morava E1 van Spronsen FJ4 , Veltman J1 ,WeversRA1 , Morava E1 ,LefeberDJ1 1Dep Ped, Radb Univ Med Cen, Nijmegen, Netherlands 1Radboud University Nijmegen UMC, Nijmegen, Netherlands 2Lab Gen Metab Endocr Dis,Radb Univ Med C, Nijmegen, Netherlands 2Mayo Clinic College of Medicine, Rochester, United States 3Dep Hum Gen, Radb Univ Med Cen, Nijmegen, Netherlands 3The Children's Memorial Health Institute, Warsaw, Poland 4University Medical Center of Groningen, Groningen, Netherlands Sialic acid transport plays an important role in CNS development. We report on the first patient with intellectual disability, seizures, macrothrombocytopenia, Patients with a Congenital Disorders of Glycosylation (CDG) are grouped in renal and cardiac involvement and a systemic glycosylation defect due to a CDG type I (Endoplasmic Reticulum) or CDG type II (Golgi). Golgi CMP-sialic acid transporter deficiency. We evaluated this patient with Using a CDG-I script for filtering whole-exome sequencing data, we found 4 congenital disorder of glycosylation type IIx by performing mass spectrometry defects in 6 CDG-I individuals1, including two novel subtypes: ALG13-CDG, of serum N- glycans, genetic linkage and mutation analysis, functional analysis an X-linked gene, and PGM1-CDG1. For the two PGM1-CDG patients, a of sialic-acid transport and expression studies. Mass spectrometry demonstrated novel high-resolution glycoprofiling method of intact serum transferrin on a severely abnormal sialylation, linkage analysis revealed homozygosity for the C8-chip-QTOF showed a loss of complete N-glycans, in agreement with CMP-sialic acid transporter gene SLC35A1 and mutation analysis in SLC35A1 CDG-I. In addition, truncated glycans lacking galactose were observed, sug- confirmed a homozygous missense mutation. Parents were carriers. Functional gesting a combined CDG-I/II defect. Via this diagnostic QTOF profile, many analysis in yeast showed a 50 % reduction in sialic acid transport activity. additional PGM1 deficient patients were found in our CDG-II cohort. The Expression studies showed significant gene expression in the CNS and kidneys, clinical phenotype included hepatopathy, cleft palate, dilated cardiomyopathy, comparable with mental retardation and chronic proteinuria in our patient. coagulopathy and exercise intolerance with episodes of rhabdomyolysis. SLC35A1-CDG was previously suspected in a single patient without systemic PGM1 plays a central role in both glycogenolysis and nucleotide sugar glycosylation defect, however one of the compound heterozygous mutations has synthesis. After cellular assays, several patients were treated with a diet, rich been proven to be a common polymorphism. in galactose and complex carbohydrates. Within a few weeks, an improvement We describe the first genetically and biochemically confirmed SLC35A1- of protein glycosylation was observed, while in addition, several of the clinical CDG patient with autosomal recessive inheritance. The presentation is and laboratory abnormalities improved. discriminative from known sialic acid transport defects, and the severe In conclusion, we identified a diagnostic glycosylation profile for a novel CNS involvement demonstrates the importance of sialylation for brain CDG-I/II subtype, PGM1-CDG, that is amenable to dietary intervention. function and development. 1. Timal S, et al. Human Molecular Genetics, accepted.

O-060 O-058 SPINOCEREBELLAR ATAXIAS WITH HYPOGONADISM: IDENTIFICATION OF NOVEL GENES FOR THE MUSCULAR DELINEATING A NEW GROUP OF INHERITED NEUROMETABOLIC DYSTROPHY-DYSTROGLYCANOPATHY SYNDROMES DISORDERS 1 1 1 2 1 Riemersma M1 , Buysse K1 , Roscioli T1 , Willemsen M1 , Kamsteeg EJ1 , Lourenco CM , Simao GN , Sobreira C , Giugliani R , Marques Jr W 1 Wevers RA1 , Morava E1 , van Bokhoven H1 , Lefeber DJ1 University of Sao Paulo, Ribeirao Preto, Brazil 2 1Radboud University Nijmegen UMC, Nijmegen, Netherlands Federal University of Rio Grande do Sul, Porto Alegre, Brazil

In a subgroup of the Congenital Muscular Dystrophies, the dystroglycano- Background: The association between cerebellar ataxia and hypogonadism pathies, the glycosylation of alpha-dystroglycan is affected. The clinical was first described in four sibs by Holmes. Several syndromes with hypo/ spectrum ranges from Walker-Warburg syndrome (WWS) with congenital hypergonadotrophic hypogonadism and ataxia have been published, with re- muscular dystrophy and complex eye and brain abnormalities to milder markable clinical heterogeneity amongst them. Here, we present the clinical data forms with limb-girdle muscular dystrophy and dilated cardiomyopathy. and molecular/biochemical studies of nineteen Brazilian patients with cerebellar Some subtypes can be screened by serum glycosylation screening, like ataxia and hypogonadism. defects in DOLK1 and the DPM2 genes, while other gene defects are Methods: Patients were clinically evaluated in our Neurogenetics clinics by restricted to immunohistochemical diagnostics in muscle tissue. a clinical geneticist. Brain MRI, neurophysiology studies, hormone and A combination of SNP array and whole-exome sequencing lead to the biochemical tests, muscle biopsy with chain respiratory enzyme assays identification of WWS mutations in a novel glycosyltransferase and in and measurement of coenzyme Q10, molecular tests for hereditary ataxias ISPD with unknown role in α-DG O-mannosylation. A novel functional were performed in the course of the investigation. genomics approach was developed using complementation of deficient Results: All patients had cerebellar ataxia as the main reason for referral, alpha-dystroglycan glycosylation in model cells and patient fibroblasts as but the age of the onset was variable. Optic atrophy and retinochoroidal assayed by flow cytometry. In addition, morpholino knockdown of the two degeneration were found in five patients. Cerebellar atrophy with pons or genes in zebrafish showed the characteristic phenotypic and biochemical prominent vermis involvement was a constant feature in 16 patients; 3 abnormalities for the dystroglycanopathies. patients had also leukodystrophy. Coenzyme Q10 deficiency was found in In conclusion, functional studies of new dystroglycanopathy genes will 3 patients. Congenital Disorder of Glycosylation type Ia was identified in allow to elucidate the unique O-linked glycosylation of alpha-dystroglycan. one adult patient. 1. Lefeber DJ, et al, PlosGenetics 2011;7:e1002427 Conclusions: Our study reinforces that the association between cerebellar 2. R Barone, et al. Ann Neurology, accepted. ataxia and hypogonadism comprise hetereogenous entities. Clinical investiga- 3. T Roscioli, et al. Nature Genetics, April 22 2012. tion of further cases with this association surely can enlighten the pathological basis of these fascinating neuroendocrinological syndromes. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S21

11. Pathophysiology, Therapies and Modes of Action in O-062 Lysosomal Storage Diseases LIVER TRANSPLANTATION FOR CHOLESTEROL ESTER STORAGE DISEASE: OUTCOME AT FIVE YEARS O-061 McKiernan PJ1 , Hodges S2 , Sharif K1 , Santra S1 NATURAL HISTORY STUDY OF EARLY ONSET LYSOSOMAL 1Birmingham Children's Hospital, Birmingham, United Kingdom ACID LIPASE (LAL) DEFICIENCY (WOLMAN DISEASE) 2Great Northern Children's Hospital, Newcastle, United Kingdom CONFIRMS A SEVERE AND RAPID DISEASE COURSE Hendriksz C1 , Wraith E2 , Dhawan A3 , Whitley C4 , Banikazemi M5 , Cholesterol ester storage disease (CESD) is a rare disorder due to deficiency Bialer M6 , Guardamagna O7 , Jones S2 , Raiman J8 , Cederbaum S9 , of lysosomal acid lipase. It has a heterogenous clinical phenotype including DiRocco M10 , Domm J11 , Enns G12 , Finegold D13 , Simonds A14 , Eckert abdominal pain, poor growth, hyperlipidaemia and hepatosplenomegaly. S14 , Schneider E14 , Quinn A14 End stage liver disease may occur but there are few reports of successful 1Birmingham Children's Hospital, Birmingham, United Kingdom liver transplantation (LT). There are also concerns that systemic manifes- 2Manchester University Hospital, Manchester, United Kingdom tations of the disease might persist post LT. We report a case with excellent 3Kings College Hospital, London, United Kingdom outcome five years following LT. 4University of Minnesota, Minnesota, United States Case report: A is one of twins who was noted to have asymptomatic 5New York Presbyterian Hospital, New York, United States hepatosplenomegaly during an intercurrent illness and CESD was enzymati- 6North Shore Long Island Jewish Hospital, Manhasset, United States cally and confirmed. He is a compound heterozygote for 2 recognised muta- 7University of Turin, Turin, Italy , 8The Hospital for Sick Children, Toronto, tions. He was treated with fat restricted diet but developed signs of progressive Canada , 9University of California Los Angeles, Los Angeles, United States liver disease from age 5 and subsequently developed hepatopulmonary syn- 10Instituto Giannina Gaslini, Genova, Italy drome. He underwent cadaveric LT aged 9.5 years and recovered rapidly with 11Vanderbilt Children's Hospital, Nashville, United States prompt resolution of hepatopulmonary syndrome. He had temporary fat intol- 12Stanford University Schoold of Medicine, Stanford, United States erance which resolved quickly. 13Children's Hospital of Pittsburgh, Pittsburgh, United States Five years post transplant he has normal growth and no gastrointestinal symp- 14Synageva BioPharma, Lexington, United States toms. He is clinically normal except for splenomegaly. Investigation showed normal blood lipids, liver and renal function tests. Liver histology is normal. LAL Deficiency is caused by mutations in the LIPA gene. Reduced LAL Conclusion: LT results in an excellent functional correction of CESD. activity results in accumulation of lysosomal lipids causing malabsorption, Conflict of Interest declared. hepatosplenomegaly, liver failure, cytopenias, and growth failure. The early onset form (Wolman disease) is progressive and manifests in early infancy leading to death usually within one year. Demographic and clinical infor- O-063 mation on 28 patients (16 males, 12 females) was collected utilizing clinical data abstractions in order to characterize the clinical course, inform evalu- LENTIVIRAL SGSH EXPRESSION FROM THE HUMAN CD11B ation and care of patients, and provide a reference for therapeutic studies. PROMOTER IN TRANSPLANTED HAEMATOPOIETIC STEM This report focuses on 19 patients with growth failure by 6 months of age. CELLS FULLY CORRECTS BEHAVIOUR AND NEUROPATHOLOGY The median age (range) at first symptom, diagnosis, and death were OF MUCOPOLYSACCHARIDOSIS IIIA MICE 1 1 1 1 1.00 month (0.23 – 3.0), 2.17 months (1.05 - 7.07), and 3.44 months Langford-Smith A , Sergijenko A , Wilkinson FL , Langford-Smith KJ , 1 2 2 3 1 (1.45 – 37.37), respectively. Six patients had hematopoietic stem cell (n0 Liao A , Jones SA , Wraith JE , Wynn RF , Bigger BW 1 5) or liver transplants. Excluding these patients, the median age (range) of Stem Cell & Neurotherapies, UoM, Mancehster, United Kingdom 2 death was 2.95 months (1.45 – 5.72). Of the 6 transplanted patients, 5 died Genetic Medicine, St Mary's Hospital, Mancehster, United Kingdom 3 before 9 months of age, while the 6th, the liver transplant recipient, died at Blood and Marrow Transplant Unit, RMCH, Manchester, United Kingdom 37.37 months. In conclusion, the clinical course of early onset LAL Defi- ciency is rapidly progressive leading to near certain death in infancy in spite Mucopolysaccharidosis IIIA (MPSIIIA) is a lysosomal storage disorder of transplantation. caused by mutations in sulfamidase (SGSH), resulting in accumulation of Conflict of Interest declared. heparan sulphate and progressive neurodegeneration. We codon optimised hSGSH to increase expression and compared hSGSH expressing lentiviral vectors under ubiquitous PGK (LV-PGK) or myeloid- specific CD11b (LV-CD11b) promoters. Lineage depleted MPSIIIA bone marrow was transduced with these vectors and transplanted into busulfan conditioned MPSIIIA mice. At 6 months, abnormal behaviour of MPSIIIA mice in the open field was fully corrected by LV-CD11b but not LV-PGK. At 8 months in the brain, LV-PGK increased enzyme activity to 7 % of WT levels whilst LV-CD11b was significantly better at 11 %. Abnormally elevated β-hexaminidase levels were normalised by LV-CD11b and reduced by LV-PGK. Neuro- inflammation measured by microglial infiltration in the cortex was completely normalised by LV-CD11b and reduced by LV-PGK. These data demonstrate that LV-CD11b mediated haematopoietic stem cell gene therapy in autologous MPSIIIA cells fully corrects neuropathology and behaviour of MPSIIIA mice whilst LV-PGK improves neuropathology but did not affect behaviour. This is the first demonstration of efficacy of the autologous stem cell gene therapy approach in MPSIIIA mice using a clinically relevant lentiviral vector and opens the door to clinical application in MPSIIIA patients. S22 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-064 O-065 NEUROPATHOLOGICAL CHANGES ARE MORE PRONOUNCED MIGALASTAT HCL REDUCES GLOBOTRIAOSYLSPHINGOSINE IN MOUSE MODELS OF MUCOPOLYSACCHARIDOSIS (MPS) (LYSO-GB3) IN PLASMA OF FABRY PATIENTS AND IN TISSUES TYPE IIIA AND IIIB OVER MPSI OF FABRY TRANSGENIC MICE Wilkinson FL1 , Holley RF2 , Langford-Smith KJ1 ,BadrinathS1 , Benjamin ER1 , Brignol N1 , Young B2 , Chang HH1 , Khanna R1 , Soska Langford-Smith A1 , Cooper JD3 , Jones SA4 , Wraith JE4 , Wynn RF5 , R1 , Sitaraman SA1 ,GermainDP3 , Guigliani R4 , Hughes DA5 , Mehta A5 , Merry CLR2 , Bigger BW1 Nicholls K6 ,BoudesP1 , Lockhart DJ1 , Valenzano KJ1 1Stem Cell & Neurotherapies, UoM, Mancehster, United Kingdom 1Amicus Therapeutics, Cranbury, United States 2Stem Cell Glycobiology, UoM, Mancehster, United Kingdom 2University of Capetown, Capetown, South Africa 3Ped Stor Dis Lab, King's Coll, London, United Kingdom 3University of Versailles, Garches, France 4Genetic Medicine, St Mary's Hospital, Mancehster, United Kingdom 4Medical Genetics Service, HCPA/UFRGS, Porto Alegre, Brazil 5Blood and Marrow Transplant Unit, RMCH, Manchester, United Kingdom 5Univ College London, Royal Free Campus, London, United Kingdom 6Royal Melbourne Hospital, Parkville, Australia Mucopolysaccharide (MPS) diseases are caused by deficiency of glycosaminoglycan (GAG) degrading enzymes, leading to GAG accumulation. Fabry disease (FD) is caused by mutations in the gene that encodes α- Neurodegenerative MPS diseases exhibit cognitive decline, behavioural galactosidase A (α-Gal A), and involves pathological accumulation of globo- problems and shortened lifespan. We characterised neuropathological triaosylceramide (GL-3) and lyso-Gb3. Migalastat-HCl (AT1001/GR181413A) changes in mouse models of MPSI, IIIA and IIIB at 4 and 9 months of is a pharmacological chaperone that can selectively bind, stabilize, and increase age using biochemical and quantitative immunohistochemical analysis to cellular levels of α-Gal A. Oral administration of migalastat-HCl reduces GL-3 provide a better understanding of these events. in tissues of Fabry transgenic mice (hR301Q Tg/KO), and in urine of FD patients MPSI, IIIA and IIIB mice all showed significantly elevated total heparan with "addressable" mutations (i.e., mutant forms that respond in cell-based sulphate (HS), abnormally N-, 6-O and 2-O sulphated HS, increased lysosomal assays according to pre-defined criteria). The effect on lyso-Gb3 was studied compartment size, GM2 ganglioside storage, astrocytosis, microgliosis, MCP- using liquid chromatography-tandem mass spectrometry. hR301Q Tg/KO mice 1, MIP-1α, IL-1α and decreased and mislocalised synaptic markers (VAMP2 orally administered migalastat-HCl (100 mg/kg) daily or less-frequently (4 days and Homer-1). Overall, MPSIIIA and B showed significantly more pro- on/3 off) for 28 days showed maximal lyso-Gb3 reductions of -64 %, -59 %, and nounced pathology than MPSI in lysosomal storage, astrocytosis, microgliosis -81 % in heart, kidney, and skin, respectively (mean difference from untreated). and the percentage of 2-O sulphation of HS. We also observed significant time Plasma lyso-Gb3 was analyzed in six male subjects orally administered progression of all genotypes from 4-9 months in lysosomal storage, astrocy- migalastat-HCl (150 mg QOD) in Phase 2 studies (NCT00283959/ tosis, microgliosis and synaptic disorganisation but not GM2 gangliosidosis. NCT00283933). Baseline levels were elevated 4- to 58-fold above normal. Transmission electron microscopy of MPS brains revealed dystrophic axons, Three males with addressable mutations showed plasma lyso-Gb3 reductions axonal storage, and extensive lipid and lysosomal storage. ranging from -17 % to -32 % (at 24 weeks compared to baseline); three males These data lend novel insight to MPS neuropathology, suggesting that MPSIIIA with non-addressable mutations showed no reductions. Migalastat-HCl is in and IIIB have more pronounced neuropathology than MPSI, yet all are still Phase 3 studies to further assess safety and efficacy for the treatment of FD progressive, at least in some aspects of neuropathology, from 4-9 months. (NCT00925301/NCT01218659). Conflict of Interest declared.

O-066 PROTEIN DISULFIDE ISOMERASE IS A KEY REGULATOR FOR RESIDUAL FORMYLGLYCINE GENERATING ENZYME (FGE) ACTIVITY IN MULTIPLE SULFATASE DEFICIENCY Schlotawa L1 , Radhakrishnan K2 , Wachs M2 , Dierks T2 , Gaertner J1 , Schmidt B3 1UMG, Dep Ped and Ped Neurology, Goettingen, Germany 2Univ of Bielefeld, Biochemistry I, Bielefeld, Germany 3UMG, Biochemistry II, Goettingen, Germany

Multiple Sulfatase Deficiency (MSD) is a rare inborn error of metabolism. It results from a defect in the post-translational activation of sulfatases due to an impaired function of the Formylglycine Generating Enzyme (FGE) caused by mutations in the SUMF1 gene. Residual enzyme activity as well as stability of FGE variant proteins contributes to the clinical presentation of MSD. Most of the SUMF1 mutations are missense mutations that lead to protein instability. The impact of amino acid exchanges on the protein structure/stability of variant FGE and its interaction with components of the protein folding/degradation machinery in the ER remains unclear. We were able to identify that protein disulfide isomerase (PDI) traps and facilitates degradation of misfolded MSD-causing FGE variants by forming mixed disulfide complexes. Interac- tion with PDI is a key determinant for FGE variants' residual catalytic activity, which is inversely correlated with PDI expression levels. We provide proof of principle that intervening at FGE interaction with the protein folding machinery could lead to therapeutic approaches in MSD. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S23

12. Peroxisomes, Sterol and Bile Acid Synthesis, Potential O-069 New Treatments A NOVEL HUMAN GENETIC DISORDER OF PEROXISOME DIVISION O-067 Ebberink MS1 , Koster J1 , Visser G2 , van Spronsen FJ3 , Stolte-Dijkstra I3 , IDENTIFICATION AND CHARACTERIZATION OFA NEW MODIFIER Smit GPA3 , Fock JM3 , Kemp S1 , Wanders RJW1 , Waterham HR1 GENE FOR X-LINKED ADRENOLEUKODYSTROPHY 1Lab. Genet Metab Dis, Acad Med Center, Amsterdam, Netherlands van Engen CE1 ,BarbierM2 , Dijkstra IM1 , Ofman R1 , Schackmann MJA1 , 2Wilhelmina Children's Hospital, Utrecht, Netherlands Wanders RJ1 ,AubourgP2 ,KempS1 3University Medical Center Groningen, Groningen, Netherlands 1Lab Gen Metab Dis, Academic Med Center, Amsterdam, Netherlands 2INSERM UMR 745, Univ Paris-Descartes, Paris, France Peroxisomes are organelles that proliferate continuously and play an indispensable role in human metabolism as evidenced by the existence of numerous X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the often severe peroxisomal disorders, including the peroxisome biogenesis dis- ABCD1 gene. Patients may develop a severe or a milder phenotype, known orders. Currently, 13 different PEX proteins have been implicated in various as respectively childhood cerebral ALD (CCALD) and adrenomyeloneurop- stages of peroxisome assembly and protein import. Defects in any of these athy (AMN). There is no genotype-phenotype correlation. CYP4F2 is a proteins result in a peroxisome biogenesis disorder. We report a novel genetic candidate modifier gene as it encodes for a key enzyme in the omega- defect specifically affecting the division of peroxisomes. oxidation of very long-chain fatty acids (VLCFA) to very long-chain dicar- The patient presented with congenital cataracts, mild intellectual disability, boxylic acids, a possible escape route for the deficient peroxisomal beta- progressive hearing loss, sensory nerve involvement, gastrointestinal problems oxidation of VLCFA in X-ALD. 151 patients with either CCALD or AMN and recurrent migraine-like episodes. Although microscopical investigations were genotyped for rs2108622, a coding CYP4F2 polymorphism, revealing an of patient fibroblasts indicated a clear defect in peroxisome division, all association of the minor allele with CCALD (P00.004, OR02). We over- biochemical parameters commonly used for diagnosing peroxisomal disorders expressed two CYP4F2 variants in ABCD1 and in PEX13 knock-down HEK were normal. After excluding mutations in all PEX genes previously impli- Flp-In cell lines and assessed the effects on omega-oxidation activity and cated in peroxisome biogenesis disorders, we found that the defect was caused VLCFA levels. Over-expression of CYP4F2 encoded by the minor allele by a homozygous non-sense mutation in the PEX11β gene. The peroxisome was less efficient at reducing VLCFA levels than over-expression of CYP4F2 division defect was exacerbated when the patient's fibroblasts were cultured at encoded by the major allele. However, the encoded amino acid substitution did 40oC, which correlated with a marked decrease in the expression of PEX11γ. not affect CYP4F2 catalytic activity when VLCFA were used as substrates in This novel isolated defect in peroxisome division expands the clinical and in vitro omega-oxidation assays. The impact of rs2108622 on VLCFA levels is genetic spectrum of peroxisomal disorders and indicates that peroxisomal probably related to its effect on CYP4F2 protein levels, which are strongly defects exist, which cannot be diagnosed by standard laboratory investigations. decreased when encoded by the minor allele.

O-070 O-068 RESVERATROL AND BEZAFIBRATE BOTH DRAMATICALLY THEX-ALDMOUSE2.0:ANEWMODELFORX-LINKED INDUCE TRANSCRIPTION OFASPARTOACYLASE A POTENTIAL ADRENOLEUKODYSTROPHY NEW TREATMENT FOR CANAVAN DISEASE? Engelen M1 , Dijkstra IM2 , Engelen-Lee JY3 , Wanders RJ2 , Kemp S2 Andresen B.S.1 , Doktor T.K.1 , Sass J.O.2 , Bastin J3 , Djouadi F3 , 1Div Ped Neurol, Academic Medical Center, Amsterdam, Netherlands Christensen E4 , Lund A.M.4 , Andersen H.S.1 2Div Metab Dis, Academic Medical Center, Amsterdam, Netherlands 1Dept Biochem & Mol Biol, Univ South DK, Odense, Denmark 3Univ Med Center, Utrecht, Netherlands 2Universitätsklinikum Freiburg, Freiburg, Germany 3INSERM U747, Univ Paris Descartes, Paris, France Background: X-linked adrenoleukodystrophy (X-ALD) is a metabolic 4Dept Clin Genet, Rigshospitalet, Copenhagen, Denmark disorder caused by mutations in the ABCD1 gene, deficient peroxisomal beta-oxidation of very long-chain fatty acids (VLCFA, >22 carbons) and Bezafibrate (BZF) and Resveratrol (RSV) are potent modulators of gene elevated VLCFA levels. Patients may develop a severe or a milder pheno- expression, and are currently being tested as drugs for fatty acid oxidation type, known as respectively childhood cerebral ALD and adrenomyeloneur- defects. The rationale being that BZF or RSV may increase expression opathy. Interestingly, brain VLCFA levels correlate with the severity of the sufficiently to alleviate disease manifestation in patients who have missense clinical presentation. This suggests that VLCFA play an important role in mutations that result in some residual enzyme activity. the onset of cerebral ALD. Unfortunately, this can not be investigated in We employed Next Generation Sequencing of RNA (RNA-seq) from fibro- Abcd1-/- knockout mice as they do not develop cerebral ALD. VLCFA are blasts stimulated with RSV or BZF to shed light on global effects on gene mainly derived from endogenous synthesis through elongation of long- expression. Interestingly, aspartoacylase (ASPA) is dramatically up-regulated chain fatty acids by ELOVL1. by both treatments. We validated this by QPCR including other cell types. The Methods: We developed an Abcd1-/- knockout mouse carrying a Cre- effect of RSV could be partly antagonized by sirtinol, indicating Sirt-1 in- inducible ELOVL1 transgene. Using various Cre driver mice, cell type- volvement. Moreover, RNA-seq and QPCR/PCR validation revealed RSVand specific ELOVL1 activation and increased VLCFA synthesis can be BZF mediated induction of transcription factors with binding sites in the ASPA achieved. promoter and up-regulation of proteins important for myelin synthesis and Results: Data from breeding experiments using a ubiquitous Cre driver function. Aspartoacylase is important for myelin synthesis in the brain and indicates the development of an embryonic phenotype in males. Neuropath- mutations in ASPA cause Canavan Disease (CD). RSV stimulation of fibro- ological analysis of astrocyte-specific ELOVL1 expressing Abcd1-/- mice blasts from CD patients with different missense mutations showed dramatic revealed signs of demyelination accompanied with microglial activation. increase (5-10x) in ASPA transcription. Conclusions: These encouraging initial results indicate that our approach We are currently exploring the stimulatory effects of RSV and BZF on may result in a cerebral ALD mouse model that will allow us to study the ASPA expression further in order to elucidate their potential as drugs for role of VLCFA in the pathophysiology of X-ALD. treatment of Canavan Disease. S24 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

O-071 13. Amino Acids ATYPICAL PARKINSONISM AND CEREBROTENDINOUS XANTHOMATOSIS: REPORT OF A FAMILY WITH CORTICOBASAL P-001 SYNDROME AND LITERATURE REVIEW EVIDENCE THAT ORNITHINE AND HOMOCITRULLINE Rubio-Agusti I1 , Kojovic M2 , Edwards MJ2 , Chandrasehkar HS3 , Bhatia PROVOKE OXIDATIVE DAMAGE IN CEREBELLUM OF YOUNG KP2 ,MurphyE1 ,LachmannRH1 RATS 1Charles Dent Metabolic Unit, London, United Kingdom Zanatta A1 , Viegas CM1 , Tonin AM1 , Moura AP1 , Busanello ENB1 , 2Sobell Department for Movement Disorders, London, United Kingdom Grings M1 , Lobato VGA1 , Leipnitz G1 , Wajner M1 3Lysholm Department of Neuroradiology, London, United Kingdom 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil

Background: Cerebrotendinous xanthomatosis (CTX) is a disorder of Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is cholesterol metabolism, presenting with systemic and neurological symp- biochemically characterized by tissue accumulation of ornithine (Orn), ammonia toms, rarely including parkinsonism. and homocitrulline (Hcit) and clinically by lethargy, cerebellar ataxia, choreoa- Methods: Clinical description of a family with CTX and parkinsonism and thetosis, delayed development and liver dysfunction. Considering that the review of 13 additional cases from the literature, to characterise this clinical pathophysiology of the cerebellar symptoms of this disorder is virtually un- syndrome. known, the aim of the present work was to investigate the in vitro effects of Hcit Results: The index case developed corticobasal syndrome, previously not and Orn (0.1 - 5 mM) on important parameters of oxidative stress in cerebellum reported in CTX. His brother had parkinsonism with cerebellar and cognitive of 30-day-old rats. Orn and Hcit significantly increased thiobarbituric acid- impairment. Review: Median age of onset of parkinsonism was 40 years. All reactive substances (TBA-RS) levels, indicating that lipid peroxidation was patients had walking difficulties. Most patients had other neurological features: induced in rat cerebellum. In addition, these metabolites significantly decreased cognitive (93 %), pyramidal (93 %), cerebellar (53 %). Systemic features were the non-enzymatic antioxidant defenses determined by glutathione (GSH) lev- common: cataracts (93 %), tendon xanthomata (87 %). Frequent MRI abnor- els. Orn also induced sulfhydryl oxidation. In contrast, carbonyl formation was malities included cerebellar atrophy (100 %), cerebral atrophy (80 %) and not altered by Orn or Hcit. Therefore, it is presumed that disruption of redox dentate nuclei signal changes (80 %). Functional dopaminergic imaging often homeostasis by Orn and Hcit may be involved in the pathophysiology of the demonstrated presynaptic denervation. Improvement with levodopa was fre- cerebellar ataxia characteristic of the patients affected by HHH syndrome. quent (91 %), but mild. Progressive neurological decline occurred in 92 % of Financial support: CNPq, PROPESq/UFRGS, FAPERGS, PRONEX, FINEP patients despite treatment with chenodeoxycholic acid. Rede Instituto Brasileiro de Neurociência (IBN-Net) # 01.06.0842-00, Insti- Conclusions: CTX should be considered in the differential diagnosis of tuto Nacional de Ciências e Tecnologia-Excitotoxicidade e Neuroproteção atypical parkinsonism, particularly with early age of onset and with a (INCT-EN). complex neurological phenotype. Tendon xanthomata, cataracts and radiological findings of cerebellar atrophy with lesions of the dentate nuclei are useful clinical clues. Levodopa may help with symptoms, but progressive neurological decline is frequent despite treatment.

O-072 THE METABOLIC BASIS OF PRIMARY HYPEROXALURIA P-002 TYPE 3 GLYCINE INTRACEREBROVENTRICULAR ADMINISTRATION Pitt JJ1 , Belostotsky R2 , Frishberg Y2 COMPROMISES BRAIN ENERGY METABOLISM OF YOUNG 1Murdoch Childrens Research Institute, Melbourne, Australia RATS 2Shaare Zedek Medical Center, Jerusalem, Israel Moura AP1 , Grings M1 , Tonin AM1 , Zanatta A1 , Parmeggiani B1 , Alvorcem LM1 , Fritch L1 , Ribeiro CAJ1 , Wajner M1 , Leipnitz G1 Primary hyperoxaluria type 3 (PH3) is a recently identified type of child- 1UFRGS, Porto Alegre, Brazil hood kidney stone disease caused by mutations in HOGA1. HOGA1 encodes a mitochondrial 4-hydroxy-2-oxoglutarate (HOG) aldolase which Nonketotic hyperglycinemia (NKH) is an inborn error of glycine (Gly) cleaves HOG to pyruvate and glyoxylic acids in the 4-hydroxyproline metabolism caused by a defect in Gly cleavage system, leading to brain catabolic pathway. Paradoxically, glyoxylic is an immediate precursor of accumulation of this amino acid. Clinically, affected patients present severe oxalate. We measured large increases in HOG, its reduced product 2,4- neurological dysfunction, whose pathophysiology is still unclear. In the dihydroxyglutarate and 4-hydroxyglutamate, the immediate precursor of present study, we investigated the ex vivo effects of a single intracerebro- HOG, in the urine of PH3 patients. These data confirm that HOGA1 ventricular Gly administration (5 μmol) on important parameters of energy mutations result in loss of function and that accumulating HOG can exit metabolism in cerebral cortex and striatum of young rats 30 min after mitochondria. In vitro studies using a non-PH3 human hepatocyte cell line injection. Our results demonstrate that Gly significantly reduced CO2 demonstrated significant HOG aldolase activity in mitochondrial and cyto- production from glucose in striatum, but not in cerebral cortex. Further- solic fractions. No HOGA1 protein was detected in the cytosolic fraction more, Gly decreased the activities of complex I-III and complex IV of the indicating that it contains other enzyme(s) with HOG aldolase activity. respiratory chain in the striatum and in the cerebral cortex, respectively. We Aldolase enzymes are known to have broad substrate specificity and several also observed that Gly inhibited the activity of creatine kinase in both brain cytosolic aldolases could fill this role. We propose a model for PH3 structures, without affecting the activity of Na+, K±ATPase. The present whereby accumulating HOG exits mitochondria and is partially metabol- data indicate that Gly impairs brain homeostasis at the level of energy ised to glyoxylate and oxalate in the cytosol. Mutation detection has been transfer and formation. Therefore, it may be presumed that bioenergetic the only means of diagnosing HP3 to date and the finding of a diagnostic dysfunction contributes, at least in part, to the brain injury found in NKH. pattern of urine metabolites is an important advance in screening larger Supported by: CNPq, PROPESq/UFRGS, FAPERGS, FAPESP, PRONEX, cohorts for PH3. FINEP IBN-Net and INCT-EN. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S25

P-003 CHARACTERISATION OF A NOVEL METABOLIC DEFECT IN PROLINE SYNTHESIS AND PATHOMECHANISM IN AUTOSOMAL P-005 RECESSIVE CUTIS LAXA SYNDROME TYPE 2B THERAPEUTIC DOSAGES OF NITISINONE AND HYPERTYROSI- Gardeitchik T1 , Fischer B2 , Kouwenberg D1 , Mohamed M1 , Kornak U2 , NEMIA COULD BE SAFE FOR THE HUMAN FETUS Nijtmans L1 , Morava E1 Garcia Segarra N1 , Roche S1 , Benoist JF1 , Spraul A2 , Ogier de Baulny H1 1Radboud University Nijmegen MC, Nijmegen, Netherlands 1Div Metab Dis, Hôpital Robert Debré, Paris, France 2Charité-Universitätsmedizin Berlin, Berlin, Germany 2Laboratoire de Biochimie, CHU Bicêtre, Paris, France

Mutations in the Pyrroline-5-carboxylate reductase 1 (PYCR1) gene have This 22 year-old woman with tyrosinemia type I was treated with nitisone been recently discovered as underlying aetiology of autosomal recessive during her pregnancy and gave birth to a healthy, tyrosinemic girl. The cutis laxa syndrome type 2B (ARCL 2B). PYCR1 catalyzes the NAD(P)H- mother diagnosed at the age of one year, started nitisone therapy at dependent conversion of pyrroline-5-carboxylate to proline. The disease is 2.5 years. Progressively poor compliance with diet resulted in sustained closely linked to P5CS deficiency, caused by mutations in ALDH18A1, hypertyrosinemia (500- 800 μmol/l) and obesity (100 kg). She married her coding for an enzyme catalyzing an earlier step in proline synthesis. Both cousin, who is heterozygote for the FAH mutation that our patient has in disorders are associated with progeroid features, lax joints, dysmorphic homozygosity. Diet with a theoretical tyrosine intake around 300 mg/day, features, microcephaly and developmental delay. While patients with and nitisone (60 mg/day) were continued throughout pregnancy. Median P5CS deficiency have variable hyperammonaemia and abnormal amino plasma tyrosine levels were 560 μmol/l (range: 375-838, n021) and niti- acid levels, no obvious metabolic abnormalities have been described in sone 51 μmol/l (range: 41-57, n03). Uncomplicated delivery gave birth to a PYCR1 patients. 2615 gr baby with hypertyrosinemia (860 μmol/l blood cord) and nitisone We studied the metabolic and phenotypic characteristics of six PYCR1 levels of 14 μmol/l. In parallel, mother's tyrosine was 501 μmol/l and patients. Mitochondrial function and respiratory complex activity have been nitisone 14 μmol/l. During the first week she had sustained hypertyrosine- evaluated in patient cells and in HeLa cells after knockdown of PYCR1. mia (1128 μmol/l at day 2), normal liver function tests, low plasma alpha- Proline synthesis and associated metabolic markers were studied in differ- fetoprotein levels, urinary excretion of phenolic acids without succinylace- ent body fluids of patients and cell culture media. tate. Specific treatment began at day 16 following molecular confirmation. Our results confirm mitochondrial localisation of PYCR1 and suggest that At that time she had low nitisone levels (2 μmol/l) and detectable urinary mutations cause a unique metabolic defect altering the intracellular endog- succinylacetate. Previously, biological diagnosis wasn't obvious due to the enous proline buffer, changing the balance in mitochondrial NAD(H) mother's treatment. concentration and mitochondrial membrane gradient. Careful evaluation of both clinical and biochemical features of patients implies that this recognizable dysmorphology syndrome is a mitochondrial disorder as well.

P-006 TYROSINEMIA TYPE 1: EARLY EFFECT OF NTBC ON RENAL P-004 TUBULAR FUNCTION TYROSINEMIA TYPE I: MOLECULAR CHARACTERIZATION Maiorana AM1 , Emma FE2 , Deodato FD1 , Boenzi SB1 , Martinelli DM1 , OF SIX TUNISIAN PATIENTS Rizzo CR3 , Dionisi-Vici CDV1 Hammami MB1 , Nasrallah F1 , Hadj Fredj S2 , Hadj Taieb S1 , Omar S1 , 1Div Metab Dis, Bambino Gesù Child Hosp, Rome, Italy Sanhaji H1 , Feki M1 , Messaoud T2 , Kaabachi N1 2Div Nephr, Bambino Gesù Child Hosp, Rome, Italy 1Lab of Biochemistry, Rabta Hospital, Tunis, Tunisia 3Metab Lab, Bambino Gesù Child Hosp, Rome, Italy 2Lab of Biochemistry, Children's Hospital, Tunis, Tunisia Background: Patients with Hereditary Tyrosinemia type1 (HT1) display Background: Tyrosinemia type I (HTI) is an autosomal recessive disease kidney involvement with Fanconi syndrome, characterized by renal tubular caused by a deficiency in fumarylacetoacetate hydrolase (FAH), the last acidosis, phosphaturia, glycosuria, generalized aminoaciduria and rickets. enzyme of the catabolic pathway of tyrosine. At this time, more than 40 Treatment with NTBC improves the outcome of HT1 resulting in the mutations of the gene encoding the FAH protein have been identified with a majority of patients in the recovery of liver dysfunction. However, NTBC prevalent mutation (IVS6-1(G-T)) in the Mediterranean area. We report the effects on renal tubular function has been so far poorly investigated. mutations responsible for FAH deficiency in six Tunisian patients. Methods: We retrospectively explored in 5 HT1 patients the early effect of Patients and methods: Six unrelated Tunisian patients with HTI, aged NTBC on renal tubular function during the first 12 days from the beginning 1 month to 4 years, were tested. Restriction analysis with AluI of PCR of therapy. product of 296 bp, across FAH exon 6 and 7, was performed. AluI cuts the Results: Treatment resulted in the rapid improvement of renal function with normal sequence into 156, 75, 29, 16 and 17 bp fragments. The IVS6-1(G- marked rise of plasma phosphate (2.56±1.3 vs. 4.21±0.8 mg/dl), renal T) mutation removes one restriction site, giving fragments of 156, 104, 16 tubular phosphate reabsorption (65.8±34.6 % vs. 84.1±15.3 %) and tubular and 17 bp. Moreover, the genotypes were confirmed by sequencing using maximum for phosphate corrected for GFR (TmP/GFR, 1.49±0.8 vs. 3.25± the dideoxy-chain termination method. 1.15) along with decrease of UGluc/UCr (12.7±13.1 vs. 2.1±2.5) and UCa/ Results: Clinical manifestations are acute liver failure with renal tubular UCr ratios (0.79±1.2 vs. 0.33±0.2). dysfunction and failure to thrive. The six patients were homozygous for the Conclusions: These preliminary data demonstrate the immediate efficacy IVS6-1(G-T) transversion. of NTBC on renal tubular function. Striking changes in phosphate metab- Conclusion: In agreement with other studies, the IVS6-1(G-T) is the most olism seems to indicate this analyte as the most reliable parameter to predominant mutation in Tunisian patients. monitor renal improvement under NTBC therapy. S26 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-007 P-009 PATIENTS WITH TYROSINEMIATYPE I AND FIRST EXPERIENCE TYROSINEMIA TYPE 1 AND NEUROGENIC CRISIS: A CASE WITH NITISINONE TREATMENT IN MACEDONIA REPORT Kostovski A1 , Zdraveska N1 , Konstantopoulou V2 Bulut FD1 , Kör D1 , Onenli-Mungan N1 , Yükselmiş U1 , İncecik F1 , 1University Children's Hospital Skopje, Skopje, The F.Y.R of Macedonia , Yıldızdaş D1 2Dept Ped and Adolesc med,Med Uni Vienna, Vienna, Austria 1Faculty of Medicine, Univ of Çukurova, Adana, Turkey

Background: Hereditary tyrosinemia type I (HT1) is an autosomal reces- Background: Tyrosinemia 1 is an inherited metabolic disease leading to sive inborn error of tyrosine metabolism. hepatic insufficiency, renal Fanconi Syndrome, peripheral neuropathy, and Objectives: To present patients with HT1 diagnosed in a period of 3 years liver carcinoma without treatment. After the introduction of nitisinone, and our first experience with nitisinone (Orfadine) treatment. neurogenic crisis became an unexpected complication. We report a patient Material and Methods: Retrospective study was carried out analyzing 4 with neurological crisis. children diagnosed with HT1 in the period from 2009 to 2011. Data were Case Report: At 5 days of life regarding to family history the patient analyzed from medical records for phenotype characteristics, therapy and diagnosed as tyrosinemia 1. With diet and nitisinone he had no problem. He outcome. hospitalized for gastroenteritis, hyponatremia, self mutilation, weakness, Results: All patients presented in infancy, mean age was 87 days. One pain and irritability at the age of 11 months, after 15 days interruption of patient had other family members affected, prenatal diagnosis was refused nitisinone. Neurologic crisis including progressive ascending polyneurop- from the parents. The main clinical presentations were acute liver failure, athy, diaphragmatic paralysis and respiratory distress requiring mechanical hepatomegaly, edemas, ascites and coagulation disturbances. The diagnosis ventilation with elevated levels of succinylacetone, δ-aminolevulinic acid, of HT1 was based on positive succinylacetone in urine. Two patients died and AFP was settled down. With the initiation of nitisinone polyneuropathy because of progressive liver failure at age of 34 and 219 days. In one patient clinically got better. At the end of four months in pediatric intensive care treatment with nitisinone was initiated with tyrosine and phenylalanine unit with mechanical ventilation he died due to a very severe nosocomial restriction. One year after therapy there is a complete clinical and biochem- pneumonia and sepsis. ical normalization. Due to limitation of nitisinone therapy in Macedonia Discussion: After nitisinone neurogenic crisis is never being a major cause treatment was not started on time in one patient and now he is liver of mortality and morbidity in tyrosinemia 1. But it is still a problem in our transplantation candidate. country due to the lack of family awareness and social problems. So, when Conclusion: Our first experience with nitisonine treatment showed prom- patients admit with irritability, pain, weakness, and hyponatremia neuro- ising results improving the prognosis for patients with HT1. genic crisis should be inconsideration.

P-008 P-010 CLINICAL, LABORATORY FINDINGS AND OUTCOME OF 34 TYROSINEMIA TYPE I: THE MOROCCAN EXPERIENCE IN PATIENTS WITH TYROSINEMIA TYPE 1 (TT1): EXPERIENCE THE DIAGNOSIS AND MONITORING OF A SINGLE CENTER Dahri S1 , Talbaoui H1 , Meskini T1 , Erreimi N1 , Vianey-Saban C2 , Aktuğlu Zeybek AC1 , Cansever MS1 , Soyucen E1 , Kiykim E1 , Altay S2 , Chabraoui L1 Aydin A1 1Univ Hosp of Rabat, Univ Moha V Souissi, Rabat, Morocco 1Div Ped Nutr Met, Cerr Med Fac, Ist Univ, Istanbul, Turkey 2Hospices Civils de Lyon-Est France, Lyon, France 2Div Diet, Cerr Med Fac, Ist Univ, Istanbul, Turkey Tyrosinemia type I (Tyr-I) is an autosomal recessive inborn error of metab- Background: In countries where TT1 isn't a part of the neonatal screening olism due to fumarylacetoacetase deficiency. If left untreated it affects liver, programme, the diagnosis is based on clinical and laboratory findings. kidney, bone and peripheral nerves. The dietary restriction treatment asso- Methods: A retrospective study was carried on a total of 34 TT1 patients ciated to Nitisinone (NTBC) is effective. In this study, we report clinical followed at Cerrahpasa Medical Faculty, Pediatric Nutrition and Metabo- and biochemical findings in 60 patients diagnosed at age from birth to lism Division, between 1996-2012. 10 years. Increased blood levels of tyrosine and methionine associated with Results: Among 34 patients, 8 were classified as acute, 17 as subacute and the presence of succinylacetone allowed the diagnosis. Hepatic cirrhosis 9 as chronic form according to the age at the onset of symptoms. Most and elevated α-fetoprotein has concerned more than half of patients because common complaints were pallor, poor appetite, irritability, vomiting, ab- of the unavailability of the treatment. Only 3 patients have been treated with dominal distension, failure to thrive, jaundice and an unpleasant urine odor, NTBC. Two siblings aged respectively 6 and 10 years, whose older sister while common physical findings were anemia, hepatosplenomegaly, rachi- had a liver transplant, were screened at birth and immediately treated with tis, failure to thrive and jaundice. The mean time between the onset of NTBC with efficient response. Nevertheless, severe complications leading symptoms and diagnosis was 10,13±15,3 months. Statistically significant to death were noted in the third patient having begun the NTBC treatment differences were found between three forms in PT, a PTT, INR, AST, ALP, late (17 months). The delay of the dietary and NTBC management is due to alpha-fetoprotein, urine deltaaminolevulinicacid-succinylacetone and plas- the lack of these products in Morocco. However, recent procedures were ma succinylacetone levels. Among 31 patients treated with NTBC, two organized to facilitate access to these treatments. underwent hepatic transplantation with hepatocelular carcinoma (HCC) This report emphasizes the importance of early diagnosis and rigorous suspicion, two patients died from HCC, one hepatic insufficiency, one treatment of Tyr-I and shows the success of treatment in cases screened at osephageal variceal bleeding, and one severe porphria-like attack. birth. Conclusion: Although clinical and laboratory findings can differ among 3 forms of TT1, early diagnosis medical treatment can be life saving and prevent or delay hepatic transplantation. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S27

P-011 P-013 THE IMPORTANCE OF HEREDITY IN ORNITHINE AMINO- DISEASE OR NOTA DISEASE: INSIGHTS INTO THE MOLECULAR TRANSFERASE (OAT) DEFICIENCY: TWO FAMILIES CASE BASIS AND CLINICAL HETEROGENEITY OF SARCOSINEMIA REPORT Pode-Shakked B1 , Bar-Joseph I2 , Pras E2 , Reznik-Wolf H2 , Marek-Yagel Noorian Shahab1 , ghavami Mojgan1 , Rabbani Ali1 , Rezaei Nima2 , D2 , Abu-Horwitz A2 , Dushintzky M2 , Goldstein N2 , Reinstein S2 , Dekel Ghassemi Fariba3 ,ShamsHormoz3 , Sotoudeh Aria1 , Nakhaei Maryam1 , M2 , Zlotnik J1 , Benarrosh A3 , Gillery P3 , Hofliger N4 , Auray-Blais C5 , Mosallanezhad Asieh1 Garnotel R3 , Anikster Y1 1Div Metab Dis,Child Med Cen,Univ Tehran, Tehran, Iran, Islamic Republic of 1Metab Dis Unit, Sheba Medical Center, Tel-Hashomer, Israel 2Div Genetic,Child Med Center, Tehran, Iran, Islamic Republic of 2Inst Hum Genet, Sheba Medical Center, Tel-HaShomer, Israel 3Eye Res Cen,Farabi Hosp,Univ Tehran, Tehran, Iran, Islamic Republic of 3Lab Ped Biol Res, American Memorial Hosp, Reims, France 4Nephrology Clinic, Zuger Hospital, AG, Switzerland Mitochondrial enzyme ornithine aminotransferase (OAT) deficiency is a 5Serv Genet, Dep Ped, Univ de Sherbrooke, Quibec, Canada rare congenital metabolic disorder with an autosomal recessive inheritance pattern that is characterized by progressive loss of vision due to gyrate Background: Sarcosinemia is an autosomal recessive metabolic trait man- atrophy of the choroid and retina, high myopia with marked astigmatism, ifested by relatively high concentrations of sarcosine in blood and urine. early cataract formation, and hyperornithinemia. Sarcosine is a key intermediate in 1-carbon metabolism and under normal In this paper, we describe two female related cases (28 and 23 years old circumstances is converted to glycine by the enzyme sarcosine sisters) with OAT deficiency suspicion. Both of them presented with a dehydrogenase. history of myopia and nyctalopia from childhood. The older had a cataract Patients and Methods: We encountered 6 families from 2 different surgery at 18. Amino acid analysis revealed high serum ornithine levels. descents (French and Arab), each with at least one individual with elevated We also describe a 28 years old female with a history of myopia, and a levels of sarcosine in blood and urine. Using the "candidate gene approach" cataract surgery. She and her sister had high serum ornithine levels. Her we sequenced the gene encoding sarcosine dehydrogenase (SARDH), brother had the same problems. Their father's eye disorders resulted in which plays an important role in the conversion of sarcosine to glycine. blindness when he was 50. According to metabolic lab data and early Results: Our analysis revealed 4 different mutations (P287L, V71F, R723X manifestation of eye lesions, the diagnosis of OTA deficiency is suspected ,R514X) in three patients. In an additional patient we found a Uniparental in these patients. To detect the mutations, genetic tests were required. disomy (UPD) in the region of SARDH gene. In two other patients we did Transient hyperammonaemia (due to ornithine deficiency in the urea cycle) not find any mutations in this gene. in early infants of families with other involved members, arouse the OTA Conclusion: We have shown for the first time, that mutations in the deficiency suspicion, and pyridoxine may prevent the gyrate atrophy in SARDH gene are associated with sarcosinemia. In addition, our results them. indicate that other genes are most probably involved in the pathogenesis of this condition.

P-012 P-014 PLASMA AMINO ACIDS PROFILE: ESTABLISHED REFERENCE TAILORED INHIBITION OF CYSTINE STONE FORMATION AS RANGE IN INDIAN CHILDREN A THERAPY FOR CYSTINURIA Pandey Sanjeev Kumar1 , Kapoor Seema1 , Polipalli Sunil Kumar1 , Dubey Sahota A1 ,YangM1 ,ShikhelS1 ,LewisMR2 , Goldfarb DS3 ,WardMD4 , AP1 Tischfield JA1 1Maulana Azad Medical College, New Delhi, India 1Dept Genetics, Rutgers Univ, Piscataway, New Jersey, United States 2Dept Biomolec Med, Imperial College, London, United Kingdom Introduction: The aim was to know the plasma amino acid profiles in 3Dept Med, NYU Langone Med Ctr, New York, New York, United States healthy children to establish a reference range in paediatric population by 4Dept Chemistry, New York Univ, New York, New York, United States reverse phase HPLC. Methods: Plasma proteins were precipitated with SSA. Norvaline served as Background: Cystinuria, caused by mutations in SLC3A1 or SLC7A9, is an IS. OPA used for derivatisation for primary amino acids and FMOC for characterized by excessive excretion of cystine in the urine and cystine secondary amino acids. Seventeen amino acid derivatives were separated in stones in the urinary tract. Cystine stones are difficult to treat surgically and 248 children (168 boys and 80 girls). medical treatments have major side effects. We are using Slc3a1 knockout Results: The boys had significantly higher levels of glutamine and trypto- mice for evaluating new treatment modalities for cystinuria. We recently phan than girls (p<0.05), and the girls had a significantly higher level of showed that cystine analogs such as cystine dimethyl ester (CDME) inhibit alanine than boys (p<0.05). Compared with the 0 to 3 month group, the cystine crystallization in vitro, suggesting that this analog might inhibit 6 month to 1 year group had significantly higher levels of glutamate, serine, cystine stone formation in Slc3a1 knockout mice. threonine and lower levels of glycine, alanine, and methionine (p<0.05). Methods: We administered 200 μg CDME daily by stomach tube to two- Compared with the 1 to 5 years group, the 6 years to 12 year group had month old cystinuria male mice for four weeks. Male mice at this age have slightly higher levels of aspartate, tyrosine and phenylalanine and signifi- cystinuria but no cystine stones in the urinary tract. cantly lower levels of glycine, threonine, arginine, valine and methionine Results: In the water-treated group, there was a wide variation in stone size in (p<0.05). the bladder, ranging from 0.5 to 9 mm (47 stones total). In the CDME-treated Conclusion: The amino acids range in Indian children is different from that group, all stones were in the size range 0.5 to 2 mm (181 stones). The stone for Caucasian children. This reference range will be facilitating in liver number and weight were significantly different between the two groups. diseases, inborn error of metabolism and other clinical diagnosis. Conclusions: CDME-treated mice had a smaller stone size range and mass than water-treated mice. These studies strongly suggest that CDME may be a potential therapeutic agent for cystinuria. S28 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-015 P-017 METHYLMALONATE SEMIALDEHYDE DEHYDROGENASE ADVERSE EFFECT OF RESTRICTED BRANCHED-CHAIN AMINO (MMSADH) DEFICIENCY: IDENTIFICATION OF A NEW CASE ACIDS DIET IN MAPLE SYRUP URINE DISEASES (MSUD) USING A NEWLY DEVELOPED ASSAY Rusli Sjarif D1 , Amir I1 Wanders RJA1 , Ruiter JPN1 , Ferdinandusse S1 , van Kuilenburg ABP1 , 1Dept Pediatrics, Universitas Indonesia, Jakarta, Indonesia Waterham HR1 , Dacremont G2 , Yamaguchi S3 1Div Metab Dis, Univ Child Hosp, Amsterdam, Netherlands Background: Early diagnosis and dietary restriction of BCAAs (leucine, 2University of Ghent, Ghent, Belgium isoleucine,valine) following the regular measurement of plasma BCAAs 3Shimane University School of Medicine, Shimane, Japan could prevent complications in MSUD patients. However, over restriction or imbalance of BCAAs led to anemia, skin desquamation and failure to Background, Objectives: In literature, several patients with a presumed thrive. deficiency of methylmalonate semialdehyde dehydrogenase (MMSADH) Case Report: M , 7 days old girl referred due to coma. She was breastfed have been described. Identification of patients has been hampered by the normally until the 5th day when poor feeding and unusual urine odor were fact that few, if any, reliable methods for enzymatic analysis of MMSADH occurred. Coma and respiratory failure were noticed on the 7th days. have been described in literature. Here we report the development of a new Metabolic acidosis with hyperammonaemia and ketonuria were demonstrat- reliable assay for MMSADH. ed. Protein restriction and glucose infusions of 8 mg/kg/min were instituted. Material and Methods: The activity of MMSADH was measured using a Newborn screening (Adelaide, Australia) showed elevated of BCAAs sug- coupled assay in which the propionyl-CoA was transformed into its gested MSUD. The MSUD-Anamix. and thiamine 100 mg/d were imme- corresponding carnitine ester followed by tandem-MS analysis. Methylmal- diately given and clinical improvement was observed, subsequently. Due to onate semialdehyde was prepared in-house according to published lack of facilities to measure plasma BCAAs, the intake of leucine is procedures. estimated based on age. The acrodermatitis enteropathica-like skin rash Results: We have set up a robust assay for MMSADH using methylmalo- was developed later, suspected due to amino acid imbalance. The skin rash nate semialdehyde which we prepared ourselves as substrate and tandem- was worsening, followed by severe anemia, chronic lung disease and finally MS for quantification of the product after conversion of propionyl-CoA into she died due to sepsis. propionyl-carnitine. The assay was fully validated and then used to measure Conclusion: Plasma amino acids analysis is essential for early diagnosis MMSDAH in fibroblasts of candidate patients. We identified one patient and prompt treatment of aminoacidopathies. Therefore, it's availability in with undetectable MMSADH activity. Molecular analysis revealed bona- the developing countries needs the attention of SSIEM. fide mutations in the gene encoding MMSADH. Conclusions: We have set up a robust assay for MMSADH analysis and used the method to identify a new case of MMSADH deficiency. P-018 CHRONIC ADMINISTRATION OF BRANCHED-CHAIN AMINO ACIDS THAT ACCUMULATE IN MAPLE SYRUP URINE P-016 DISEASE IMPAIRS SPATIAL MEMORY AND INCREASES BRAIN-DERIVED NEUROTROPHIC FACTOR TOTAL AND FREE CHOLINE AND ETHANOLAMINE LEVELS Scaini G1 , Jeremias IC1 , Morais MOS1 , Mina F1 , Comim CM1 , Quevedo IN AMNIOTIC FLUID OF NEURAL TUBE DEFECT CASES J1 , Pasquali MAB2 , Gelain DP2 , Moreira JCF2 , Schuck PF1 , Ferreira Imbard A1 , Barto R2 , Schlemmer D1 , Muller F1 , Blom HJ2 , Benoist JF1 GC1 , Streck EL1 1Biochemistry, Robert Debré Hosp, APHP, Paris, France 1Univ Extr Sul Catarinense, Criciúma, Brazil 2Metab Lab, VUmc, Amsterdam, Netherlands 2Univ Federal do Rio Grande do Sul, Porto Alegre, Brazil

Background: Choline is an essential component of membrane phospholi- Background: Maple Syrup Urine Disease (MSUD) is a neurometabolic pids required for foetal growth, especially for brain development. Low disorder that leads to accumulation of branched chain amino acids (BCAA) maternal intake or plasma levels of choline are related to an increased risk leucine, isoleucine, and valine. Because the neurotoxic mechanisms of factor for neural tube defects (NTDs) in their offspring. We studied amni- MSUD are poorly understood, this study examined the effects of BCAA otic fluid (AF) levels of total and free choline as well as ethanolamine, and chronic administration on spatial memory and levels of brain-derived neu- of choline oxidation products betaine and dimethylglycine in relation to rotrophic factor (BNDF) and pro-BDNF, treated or not with antioxidants. NTD risk. Methods: Wistar rats (7 days) received a pool of BCAA or saline twice a Material and Methods: We collected AFs of 141 NTDs cases (123 spina day for 21 days, and were also supplemented with N-acetylcysteine (20 mg/ bifida and 18 cephalic pole) and 185 controls sampled for fetal karyotyping kg) twice a day and deferoxamine (20 mg/kg) every two days. Twelve hours because of maternal age and/or Down syndrome risk. after the last administration the rats were submitted to open field task and Results: No differences in betaine and dimethylglycine levels were ob- object recognition. Subsequently, rats were killed by decapitation, and served. Total choline levels were significantly higher in AF of NTDs cases cerebral cortex, hippocampus and striatum were isolated. versus controls (p<0.001). Among NTDs, total and free choline and etha- Results: BCAA administration increased BDNF in hippocampus and cere- nolamine levels were higher in the cephalic pole than in the spina bifida (p< bral cortex, but not pro-BDNF, accompanied by memory impairment in 0.001, p00.007, p00.002, and p00.002 respectively). spatial memory tasks; antioxidant treatment prevented both changes. This Conclusion: This is the first study on choline and ethanolamine in AF of increase of BDNF may be related to the impairment of spatial memory. In NTD cases. We obtained no evidence that low choline is a risk factor for addition, we demonstrated that antioxidant treatment prevented the negative NTD. We hypothesize that total and free choline and ethanolamine in AF consequences related to BCAA administration. relates to the severity and/or the size of the NTD lesion. Conclusions: These findings suggest that oxidative stress might be involved in the pathophysiological mechanism(s) underlying the brain damage observed in MSUD. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S29

P-019 P-021 SERINE DEFICIENCY DISORDERS IN TWO TUNISIAN PATIENTS LONG TERM OUTCOME IN HYPERPROLINEMIA TYPE II Nasrallah F1 , Kraoua I2 , Hammami MB1 , Hadj Taieb S1 , Sanhaji H1 , van de Ven S1 , Gardeitchik T1 , Kouwenberg D1 , Kluijtmans L2 , Morava Fontaine M3 , Briand G3 , Feki M1 , Khouja N2 , Kaabachi N1 E1 1Lab of Biochemistry, Rabta Hospital, Tunis, Tunisia 1Dep of Ped, RUNMC, Nijmegen, Netherlands 2Department of Child and Adolescent Neuro, National Institute of Neurology, 2LGEM, RUNMC, Nijmegen, Netherlands Tunis, Tunisia 3Lab of Biochem, Molec Biolo and met dis, Lille, France Although Hyperprolinemia type II has obvious metabolic consequences, and is associated with involvement of the neurological system, the casual relation Background: L-Serine deficiency is a rare, inherited, metabolic disorder of between the metabolic abnormalities and the clinical features, except for those L-serine biosynthesis. The majority of the cases reported show a decrease in of the secondary B6 deficiency, has been frequently debated. We search for 3-phosphoglycerate dehydrogenase activity resulting in low fasting serum disease frequency and evaluated the clinical, neurological, laboratory and and cerebrospinal fluid (CSF) L-serine levels. Detection of this disorder is metabolic features and therapeutic success in metabolically confirmed HPII. essential, as it is potentially treatable. Searching the LGEM database revealed 20991 urinary organic-acid profiles, The aim is to contribute to a better understanding of serine deficiency by the while 16720 samples from these individuals were parallel investigated by presentation of two Tunisian patients serum amino acid analysis between 1992-2010, revealing four patients with Materials and methods: Two patients were oriented with clinical features increased urinary P5C and HPII. Western blot analysis was performed for and were diagnosed by plasma and CSF amino acids levels performed by expression analysis, oxygen consumption was measured to assess ATP pro- ion exchange chromatography. duction in patients fibroblasts and the Mitochondrial Disease Score was used Results: Patients (P1) and (P2) were aged 36 months and 22 months to evaluate clinical mitochondrial dysfunction. The child behaviour checklist respectively, their clinical features included: congenital microcephaly, in- was used to screen for psychopathology. Western blot analysis confirmed tractable seizures, severe psychomotor retardation, epilepsy and spastic mitochondrial co-localisation of P5CDH. All children had low normal B6 tetraparesis. Plasma serine level was: P1, 38 and P2, 42 μmol/L (normal concentration, and three had biochemical markers suggesting mitochondrial range: 90 - 78) and in CSF: 7 and 6 μmol/L (normal range: 25 - 45).The dysfunction. Mitochondrial dysfunction was confirmed in one case. Intellec- CSF glycine level was: 3 μmol/L in two patients (Normal range: 5 – 11). tual disability was found in two young adults. All patients showed seizures and Conclusion: The diagnosis of serine deficiency is relatively easy with amino significant behavioural problems, including agressivity and hallucinations. acids analysis in CSF which is not influenced by dietary amino acids. The clinical course was non-progressive and independent from the B6 concentration and B6 therapy.

P-020 P-022 EXAMINATION OF CSF AMINO ACID ROSTROCAUDAL EARLY AND RAPID PROGRESSION OF RETINAL CHANGES GRADIENTS IN INFANTS AND SHORT-TERM EFFECT OF STEROIDOTHERAPY IN 11- Horman AJ1 , Neergheen V1 , Heales SJR1 , Land JM1 YEAR-OLD PATIENT WITH GYRATE ATROPHY 1Neurometabolic Unit, NHNN (UCLH), LONDON, United Kingdom Sykut-Cegielska J1 , Kocyła-Karczmarewicz B2 , Sredzinska M1 , Hautz W2 , Kowalik A1 , Taybert J1 , Kusmierska K3 Background: Cerebrospinal fluid (CSF) amino acid analysis is an impor- 11Dept Metab Dis, Child Memor Health Inst, Warsaw, Poland tant diagnostic tool in the investigation of metabolic disease particularly in 22Dept Ophtalm, Child Memor Health Inst, Warsaw, Poland infantile seizures. Whilst there is evidence to support the existence of 33Dept Lab Diagn, Child Memor Health Inst, Warsaw, Poland rostrocaudal gradients for some amino acids in adults drawn by comparison between samples taken at different sites, data is sparse regarding such Hyperornithinemia with gyrate atrophy of choroid and retina (HOGA) is a relevance in infants during isolated lumbar sampling. rare autosomal recessive inborn error of ornithine metabolism with the Objectives: Examine possible evidence of rostrocaudal amino acid gra- whole spectrum of ocular symptoms, leading to blindness in the third- dients in infants aged less than 1 year. Assess clinical impact. fourth life decade. We report on the girl now 11-year-old, born from Material (Patients) and Methods: Anonymised CSF was obtained from 9 consanguineous parents (first cousins), diagnosed and treated (together with infants aged less than one year. From each patient, CSF amino acids were her older brother) since the age of 4 years. Treatment has been based on quantified by ion-exchange chromatography with ninhydrin detection in low-protein diet (from 1.0 to 0.5 g/kg/d) with further supplementation of each of three sequential lumbar puncture samples (0.5 ml, 0.5 ml and 1 ml essential amino acids, vitamin B6 and creatine monohydrate, what resulted containing DETAPAC and DTE) previously used as part of a routine CSF in lowering plasma ornithine levels to median value 433 μmol/L. service in our laboratory. In spite of good compliance and careful follow-up, the rapid significant visual Results: No significant differences in amino acid concentrations were loss and cystoid macular oedema (CME) were observed 18 months ago. Oral observed between successive CSF fractions and analysis did not appear to acetazolamide, topical dexamethasone and topical sodium diclofenac reduced be affected by DETAPAC or DTE. CME and improved visual acuity. But after 10 months CME with low vision Conclusion: These data add to the knowledge of pre-analytical and analyt- have recurred. Because of no improvement after above treatment, systemic ical aspects of CSF amino acid interpretation in the context of lumbar steroidotherapy was additionally started - 5 days intravenous infusion of sampling in a population most relevant to the utility of this test. Solumedrol ® followed by oral prednisone – with good functional and morphological results. Such early and rapid clinical deterioration is infrequent in HOGA; cause remains unexplained and management standards - undefined. Long-term effect of steroidotherapy needs further monitoring. S30 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-023 P-025 DANDY-WALKER MALFORMATION AND OBSTRUCTIVE PRENATAL DIAGNOSIS ANALYSING AMNIOTIC FLUID AMINO HYDROCEPHALUS ASSOCIATED WITH 5,10-METHYLENE ACIDS FAILED TO DETECT CLASSICAL MAPLE SYRUP URINE TETRAHYDROFOLATE REDUCTASE DEFICIENCY DISEASE Breen C1 , Baumgartner M2 , West S3 , Josan V4 , Morris AAM1 SIM K1 , Devanapalli B1 , Mowat D2 , Fietz M3 , Christodoulou J4 , 1Genet Med,Manchester Acad Health Sci Cen, Manchester, United Kingdom Carpenter K1 2Div Metab Dis, Univ Child Hosp, Zurich, Switzerland 1Children's Hospital at Westmead, Westmead, Australia 3Paed Neurol, Royal Manchester Child Hosp, Manchester, United Kingdom 2Sydney Children's Hospital, Sydney, Australia 4Neurosurg, Royal Manchester Child Hosp, Manchester, United Kingdom 3Women and Children's Hospital, Adelaide, Australia 4University of Sydney, Sydney, Australia Introduction: Severe 5,10-methylene tetrahydrofolate reductase (MTHFR) deficiency usually presents with progressive encephalopathy in infancy. Maple syrup urine disease (MSUD) is an autosomal recessive aminoacid- Obstructive hydrocephalus has been reported previously in two infants opathy caused by a deficiency of the branched-chain alpha-keto-acid dehy- (Baethmann et al. 2000). drogenase complex. Biochemical markers are elevated concentrations of Objectives: We report a further patient with obstructive hydrocephalus. branched-chain amino acids (BCAA) valine, leucine, isoleucine, allo- Case report: Our patient presented aged 8 weeks with poor feeding, hypotonia, isoleucine (diagnostic), and branched-chain alpha-keto acids in body fluids. nystagmus, stridor and irregular breathing. Plasma homocysteine was raised We report an attempted prenatal diagnosis of a fetus at risk of MSUD (two with low methionine and normal urine organic acids; MTHFR deficiency was previously affected boys who died and termination of an affected pregnan- confirmed by enzymology and mutation analysis. Treatment with betaine, cy), in order to plan for delivery and instigate early treatment to prevent hydroxocobalamin and folic acid led to biochemical improvement but the adverse neurocognitive outcome if found to be affected. Amniocentesis was patient deteriorated with reduced responsiveness, a bulging fontanelle and performed at 32 weeks gestation. Allo-isoleucine was not detected in sun-setting of the eyes. MRI showed periventricular oedema, atrophy of the analysis of amniotic fluid and the other BCAA were considered normal. forebrain and cerebellar hypoplasia; the fourth ventricle communicated with a Cultured amniocyte leucine decarboxylation assay enzyme activity was large posterior fossa cyst. low: 2.1 units/hr/μg protein (reference range 8.4-41) but above observed Results: Insertion of a ventriculoperitoneal shunt at 11 weeks led to rapid range of affected cases (<0.7). improvement. Unfortunately, subsequent head growth and developmental Plasma collected at 24 hours of life showed elevated BCAA, leucine progress has been poor. 396 μmol/L (reference range 35-154), isoleucine 164 μmol/L (reference range Discussion: The hydrocephalus was associated with a Dandy-Walker-like 12-80), valine 370 μmol/ (reference range 58-228), allo-isoleucine 14 μmol/L cyst in our patient and in one of the previous cases. Cerebellar malforma- (reference range 0). By day 3, plasma leucine, allo-isoleucine were 672 and tions have also been reported in a mouse model of MTHFR deficiency. It is 64 μmol/L respectively, confirming classical MSUD in this baby. unclear why this inborn error causes brain malformations but a disturbance We conclude that amniotic fluid amino acids analysis is unreliable for of purine and pyrimidine synthesis in the brain may be involved. prenatal diagnosis of MSUD.

P-024 P-026 DNA DAMAGE IN AN ANIMAL MODEL OF MAPLE SYRUP CONTROL OF HYPERGLYCINEMIA AND SEIZURES WITH A URINE DISEASE MODIFIED LOW-PROTEIN DIETAND GLYCINE FREE FORMULA Streck EL1 , Scaini G1 , Morais MOS1 , Jeremias IC1 , Borges GD1 , IN NONKETOTIC HYPERGLYCINEMIA Munhoz BP1 , Leffa DD1 , Andrade VM1 , Schuck PF1 , Ferreira GC1 Perszyk A1 1Univ Extr Sul Catarinense, Criciúma, Brazil 1Med Genetics, Univ of Florida, Jacksonville, United States

Background: Maple Syrup Urine Disease (MSUD) is caused by a defi- Nonketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism ciency of the branched chain alpha-ketoacid dehydrogenase complex with that presents with seizures, hypotonia, vomiting, and failure to thrive. We accumulation of branched-chain amino acids (BCAA) leucine, isoleucine, recently diagnosed a child with NKH that presented with uncontrolled and and valine. Neurological dysfunction is a common finding in patients with unrelenting seizures. When initial labs showed the high serum glycine a MSUD. Thus, we investigated whether acute or chronic administration of protein restricted diet was started, while awaiting further lab results. Marked BCAA causes transient DNA damage in brain and blood of rats, treated or improvement in seizure frequency and level of alertness in the infant was not with antioxidants. observed immediately after protein restrictive diet was started. After con- Methods: For acute administration, Wistar rats (10 days) received three firmation of the disorder by cerebrospinal fluid (CSF) measured levels of injections (1 h interval) of BCAA pool or saline. For chronic administra- glycine, infant was given a specialized glycine free infant formula. Several tion, Wistar rats (7 days) received a BCAA pool or saline twice a day for simultaneous levels of glycine from blood and CSF were obtained during 21 days; the animals were also supplemented with N-acetylcysteine (20 mg/ the first two weeks of the start of the dietary changes and after one month on kg) twice a day and deferoxamine (20 mg/kg) every two days. One hour the diet. Correlation of the serum glycine and the CSF glycine were (acute) or twelve hours (chronic) after the last injection, rats were killed by recorded. Subsequent serum measurements were obtained and followed decapitation, cerebral cortex, hippocampus and striatum were isolated, and for continued dietary monitoring without the need to measure CSF levels. serum was collected for alkaline comet assay. Over several months, there was good correlation between serum glycine Results: Acute administration of BCAA increased DNA damage frequency level and number and frequency of seizures seen clinically and on EEG. and damage index in hippocampus. Chronic administration of BCAA Patient education of the specialized diet and coordination of care are increased DNA damage frequency and damage index in hippocampus and essential for treatment of this rare metabolic condition. striatum; antioxidants were able to prevent this damage. Conclusions: These findings suggest that oxidative stress may be involved in DNA damage in MSUD. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S31

P-027 P-029 CLINICAL, BIOCHEMICAL, MOLECULAR AND THERAPEUTICAL TWO NEW CASES OF SERINE DEFICIENCY DISORDERS TREATED ASPECTS OF 2-AMINOADIPIC SEMIALDEYHE SYNTHASE WITH L- SERINE DEFICIENCY Brassier A1 , Valayannopoulos V1 , Bahi-Buisson N2 , Wiame E3 , Hubert Artuch R1 , Perez-Cerdá C2 ,SanzP2 , Perez B2 , Arriola G3 ,OrmazabalA1 , L1 , Boddaert N1 ,KaminskaA2 ,RabierD4 , Van Schaftingen E3 , Tondo M1 , Espinos C4 , Perez-Dueñas B1 Ottolenghi C4 , de Lonlay P1 1Hosp Sant Joan de Déu, Esplugues, Spain 1Inherit Metab Dis, Necker, Paris, France 2CEDEM, CBM, Univ Autonoma, Madrid, Spain 2Neuropediatrics, Necker, Paris, France 3Hosp Univ de Guadalajara, Guadalajara, Spain 3de Duve Institute, Université Catholique, Brussels, Belgium 4Hosp La Fe, Valencia, Spain 4Biochemistry, Necker, Paris, France

Objectives: To report 2 new cases of hyperlysinaemia caused by amino- Background: Serine deficiency (SD) is potentially a treatable disease. We adipic semialdehyde synthase (AASS) deficiency. Patients: Case 1 (10-years- report two new cases of SD. old) presented febrile seizures (10-months-old), learning difficulties, hyperki- Patients: Patient 1 had 3-phosphoglycerate dehydrogenase (PHGDH) defi- nesia and clumsiness. Case 2 (1-year-old) showed generalized febrile seizures ciency; Patient 2 had phosphoserine aminotransferase (PSAT1) deficiency. fully controlled with valproate, hyperkinesia and poor attention span. Both presented withmicrocephaly (< 3rd centile at birth), encephalopathy Results: Both cases disclosed hyperlysinaemia (1208 and 1472 μmol/L respec- with spasticity. Patient 1 had intractable seizures. A fetus (Patient 2family) tively: Reference values <164), with hyperlysinuria, no saccharopine excretion presented severe antenatal microcephaly. and increased plasma pipecolic acid concentrations. Urine excretion of cystine, Results: Serine in plasma and CSF was very low, as it was in foetal cord arginine, ornithine, methionine and citrulline was increased. In both cases, het- blood. Patient 1 carried two mutations in PHGDH gene, c.403 C>G erozygous mutations in the AASS gene were detected: c.976-978del (p.Gln326fs) (p.R135W) and c.487 C>T (p.R163W). Patient 2 and the foetus were and c.1925 C>G (p.Ser642term) in case 1 and c.874A>G (p.I292V) and c.2662+ homozygous for mutation c.129 T>G (S43R) in PSAT1 gene. A decreased 1_2662+5 delGTAAGinsTT in case 2. Treatment with lysine restriction and Vmax (0.25 μmol/mn/mg protein versus 1.06) and an increased Km for pyridoxal-phosphate only modified the phenotype in case 2. This child had not phosphoserine (95 μM versus 40 μM) compared to the wild type protein suffered further seizures, and video-EEG recording showed complete resolution were found. Treatment with serine, started at age 4.5 years and 3 months, of the paroxysms previously registered (spike-wave complexes bilaterally in led in patient 1 to a significant reduction of seizures but to moderate central-parietal brain areas). Moreover, this child showed a reduction of plasma improvement of psychomotor development. In patient 2, serine led to an lysine values (971 μmol/L), while no lysine reduction was observed in case 1. improvement of spasticity and head circumference. Conclusions: AASS deficiency is a very rare condition where the main Conclusion: Our data reemphasize the need to measure CSF and plasma challenge is to elucidate the contribution of hyperlysinaemia in the neuro- serine levels in all cases of antenatal microcephaly as serine deficiency can logical dysfunction of the disease. be treated successfully only if the treatment starts very soon.

P-028 CHARACTERIZATION OF MUTATIONS AND PRENATAL DIAGNOSIS IN MAPLE SYRUP URINE DISEASE IN INDIAN PATIENTS: P-030 IDENTIFICATION OF THREE NOVEL MUTATIONS CITRULLINE CONTRIBUTION FOR DIAGNOSIS IN LEIGH 1 1 1 1 1 2 Gupta D , Bijarnia S , Saxena R , Kohli S , Puri R , Shigematsu Y , DISEASE 3 4 1 Yamaguchi S , Deb R , Verma IC Veríssimo C1 , Simues M1 , Pratas J1 , Macário MC2 , Diogo L3 , Grazina 1 Cent of Med Genet, Sir Ganga Ram Hosp, New Delhi, India M4 2 Dept Hlth Sci, Dept of Ped, Univ Fukui, Fukui, Japan 1Center Neurosciences and Cell Biology, Coimbra, Portugal 3 Dept Ped, Shimane Univ Sch med, Izumo, Shimane, Japan 2CHUC - Adult Neurology Department, Coimbra, Portugal 4 AIB, Amity Univ,, Noida, UP, India 3CHUC - Pediatric Hospital, Coimbra, Portugal 4Fac Medicine & CNC/UC, Coimbra, Portugal Maple Syrup Urine Disease is an important metabolic disorder with genetic heterogeneity and adverse consequences, if left untreated. Prenatal diagnosis Leigh syndrome, like other mitochondrial disorders, has no specific and (PND) is essential to prevent recurrence in the families. Molecular studies are a sensitive biomarkers. High levels of lactate are frequently found, but not in pre-requisite for PND. So far, no molecular studies have been reported from India. all cases. Some reports indicate hypocitrullinemia as a possible biomarker We studied eleven Indian patients with MSUD for mutations in the 3 genes, of Leigh disease in cases with m.8993 T>G mutation. BCKDHA, BCKDHB & DBT, for confirmation of diagnosis for enabling In this communication we present five cases of suspected Leigh syndrome. accurate prenatal diagnosis in pregnancies. Our goal is to evaluate the role of citrulline as a possible biomarker in these PCR and SSCP were done followed by gene sequencing where required. cases. Pathogenic variations have been found in 6/ 11 samples studied till date. Three According to our results two patients suspected of Leigh syndrome pre- types of mutations observed were: 5 missense, 1 splice site and 1deletion. sented low citrulline levels, associated with the m.8993 T>G mutation. We Three novel mutations were observed in homozygous condition: c.844 G> suggest that when Leigh disease is suspected, evaluation of citrulline levels C in BCKDHA gene and c.1022 T>A & IVS1-2A>G in BCKDHB gene. should be performed and, if hypocitrullinemia is detected, m.8993 T>G Two patients were homozygous for the reported mutations, c.940 C>T mutation should be immediately investigated in blood. (BCKDHA gene) and c.554 C>T (BCKDHB gene). One patient was Although our findings need confirmation in larger cohorts, we believe that compound heterozygous for c.293 T>G and c.1065delT in BCKDHB gene. hypocitrullinemia could be a helpful biomarker in Leigh disease, particu- Two prenatal diagnoses were performed in two families based on mutations larly related to the presence of m.8993 T>G mutation. The quantification of found, resulting in one affected foetus. this amino acid could also be valuable in other mitochondrial respiratory In conclusion, mutations in Indian population were diverse. Identification chain disorders such as MELAS and Pearson, as previously reported. of the mutations facilitated precise prenatal diagnoses in families at risk, thus leading to primary prevention. S32 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-031 P-033 TWO-GENERATIONAL ALKAPTONURIA IN A MAMMALIAN INSULINOMA CELLS SYNTHESIZE AND NON-CONSANGUINEOUS FAMILY FROM LITHUANIA SECRETE D-ALANINE Cimbalistienė L1 , Rimkus V2 , Songailienė J1 ,Kučinskas V1 Visser WF1 , Albersen M1 , Verhoeven-Duif NM1 , de Koning TJ2 1Dep of Hum and Med Genetic, Vilnius Univ, Vilnius, Lithuania 1Dpt Metab Disease, Univ Med Cntr Utrecht, Utrecht, Netherlands 2Vilnius Univ Emergency Hosp, Vilnius, Lithuania 2Dept Genetics, Univ Med Centre Groningen, Groningen, Netherlands

AKU was first described by A.Garrod as autosomal recessive disorder in D-serine and D-aspartate have recently been discovered as important sig- 1902. A dominant inheritance has been reported, but was attributed to nalling molecules, particularly in the central nervous system. Using a newly extended consanguinity. Whether there are AKU cases with dominant mode developed method, we have revealed the presence of other D-amino acids in of inheritance is still under debate. human plasma, urine and cerebrospinal fluid, raising the question whether Case report: A 74 year old woman was referred due to hip joint ochronosis. some of these D-amino acids may also have functions in human physiology, During the operation an ochronosis was observed in the caput of femori and most probably as signalling molecules. We now show that a rat insulinoma acetabular cartilage. Physical examination revealed bluish-grey pigmentation cell line, widely used as a model for pancreatic beta-cells, synthesize and of pinna, thickened ear cartilage, black spots bilaterally over the sclera. X-ray secrete D-alanine and D-leucine upon stimulation by high levels of glucose. and MRI revealed characteristic features of ochronotic spondyloarthropathy. By utilizing stable isotope labelled precursors and inhibitors of The family was Lithuanian origin and consanguineous relationships were pyridoxal-5-phosphate dependent enzymes, we were able to elucidate denied. Patient's father, grandfather and two brothers were diagnosed with the metabolic steps involved in the synthesis of these molecules. arthropathy and their urine turned dark upon standing. Patient's daughters at These results strongly suggest that D-alanine is synthesized from the moment are healthy and blackening of urine is not observed. GC/MS pyruvate and glutamate in the mitochondrial compartment, presumably analysis of patient's urine revealed high intensity peak of homogentisic acid. via the action of a currently unresolved aminotransferase, whereas D- HGD gene mutation analysis of patient's family is in progress. leucine is synthesized from L-leucine, presumably by an amino acid Conclusions: We have observed a non-consanguineous Lithuanian family racemase. The newly synthesized D-amino acids are then secreted into with AKU presenting in a dominant manner over two generations. It would the extracellular medium. Our results conclusively show that mammals be the first case of autosomal dominant transmission of AKU reported in are able to synthesis D-alanine and D-leucine and suggest that they Lithuania. HGD mutation analysis of consulted AKU family could serve as have a function in glucose homeostasis. evidence.

P-032 A-001 BIOCHEMICAL RESPONSE TO FOLINIC ACID TREATMENT IN A NEW CASE WITH BIOTINIDASE DEFICIENCY AND THE PATIENT WITH FORMIMINOTRANSFERASE- ALKAPTONURIA CYCLODEAMINASE DEFICIENCY 5 MONTHS EXPERIENCE M Kose1 , E Canda1 , H Onay2 , S Kurtgoz3 , M Kagnici1 , SK Ucar1 ,S Songailiene J1 , Matuleviciene A1 , Grigoniene J2 , Enziniene M2 , Habif4 , O Bayindir4 , F Ozkinay2 , M Coker1 Kasnauskiene J1 ,UktveryteI1 , Spaapen L3 ,BakkerJ3 1Pediatric Metabolism Ege University, Izmir, Turkey 1Dept Human&Med Genetics, Vilnius Univ, Vilnius, Lithuania 2Medical Genetics Ege University, Izmir, Turkey 2Lithuanian Univ of Health Sciences, Kaunas, Lithuania 3Medical Genetics Adnan Menderes Univ, Aydın, Turkey 3Lab Bioch Genetics, Univ Hosp Maastricht, Maastricht, Netherlands 4Clinical Biochemistry Ege University, Izmir, Turkey

Glutamate formiminotransferase–cyclodeaminase deficiency (OMIM: Biotidinase deficiency is an autosomal recessive inborn error of biotin 229100) is a rare autosomal recessive disorder of the histidine catabolism. metabolism. Alkaptonuria is a genetic disorder of tyrosine metabolism Biochemically, this disorder is characterized by massive excretion of formi- which is caused by mutations in homogentisate 1,2-dioxygenase (HGD) minoglutamate (FIGLU) and hydantoin-5-propionic acid (H5P). It is not clear gene. We report a case of alcaptonuria who had concomitant biotinidase yet whether reduction of FIGLU excretion has any clinical value. deficiency , which has not been reported hitherto. A 5-year-old girl with mental retardation and autistic features was diagnosed Our patient is a boy in the first month of life and was born from cross- with FTCD deficiency: increased FIGLU in plasma and increased excretion of cousin marriage. He was referred to our department after detected biotini- FIGLU and H5P were found. FTCD genotype: hemizygous for c.403 C>T dase deficiency in newborn screening programme. Biotinidase activity was (p.R135C) with quantitative PCR indicating deletion of the other allele. 18 % ( biotidinase level :1,3 nmol /min/ml ). We observed that his mother Treatment with folinic acid (Leucovorine;15 mg/day) leaded to dramatic also has incomplete biotinidase deficiency. reduction of FIGLU in plasma from 26 to 2 μmol/l and in urine from 523 to Results: of urinary organic asid analyses showed elevated excretion of 31 μmol/l, H5P from 108 to 27U/mmolcreat within 1 month. Steadily reduced homogentisic acid; unexpectedly. In the view of such information we levels of these metabolites were observed during the continuation of folinic reviewed family history again and ascertained that his mother's two uncles acid therapy. Clinical picture did not show significant changes, except for and mother's aunt have alcaptonuria. Sequence analysis of biotinidase gene reduced susceptibility to infectious diseases and improved toilet habits. from genomic DNA revealed that patient and his mother have a compound Long term follow-up of the patient with FTCD deficiency is required to heterozygote with previously described mutations of p.R157H and determine possible beneficial effect of folinate on clinical picture. We p.D444H. Genetic studies of HGD gene was not concluded yet. suggest that clinical picture of FTCD deficiency might be dependent on In conclusion; this paper reports findings in the case of a Turkish boy with impaired FTCD function of structural-functional mediator between cell Biotinidase deficiency and alkaptonuria, which is unique in the literature. organelles and cytoskeleton rather than enzymatic function. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S33

14. Phenylketonuria and BH4 P-036 MATERNAL PHENYLKETONURIA INTERNATIONAL P-034 COLLABORATIVE STUDY REVISITED: DOES GLOBAL DNA NEOPTERIN ANTIOXIDANT EFFECTS IN GLIAL CELLS HYPOMETHYLATION CONTRIBUTE TO CONGENITAL Ghisoni K1 , Olivera S2 , Léon C2 , Quincozes A3 , Gottfried C3 , Barbeito HEART DEFECTS? L2 , Latini A1 Moseley K1 , Azen C2 , Yano S1 1Dept of Biochemistry, UFSC, Florianopolis, Brazil 1Gen. Div, USC/Keck School of Medicine, Los Angeles, United States 2Institut Pasteur, Montevideo, Uruguay 2CTU, CHLA, USC/Keck School of Med, Los Angeles, United States 3Dept of Biochemistry, UFRGS, Porto Alegre, Brazil MPKU is associated with increased risk of CHD in offspring, if diet is not Neopterin (Neo), a biochemical product of the guanosine triphosphate appropriately managed. We hypothesized other biochemical components in addi- pathway, is found at increased levels in cerebrospinal fluid and plasma of tion to Phe might have significant affects on fetal outcome in MPKU pregnancies. patients with immune system activation. However the exact cell type, 416 pregnancies resulting in live births reported in MPKUCS were grouped stimulus that triggers Neo production, and the role of this pteridin are by offspring with CHD or without. The groups were compared on first virtually unknown. Therefore, the effect of Neo was investigated in glial trimester values of data including plasma amino acids, protein and Phe cells. C6 and striatal primary astrocytes were exposed to Neo or in combi- intake, and RBC folate. nation with H2O2 or azide for 1 or 3 hours, in order to assess free radical 28 pregnancies resulted in newborns with CHD. The CHD group had a production, Neo and lactate release, and hemeoxygenase-1 (HO-1) immune significantly higher blood Phe, lower protein intakes, and lower levels of content. Neo pre-treatment significantly prevented free radical generation, RBC folate and plasma amino acids: Proline, Valine, Methionine, Isoleu- when C6 cells were exposed to a pulse of H2O2. Differential results were cine, Leucine, Lysine, and Arginine. After controlling for blood Phe, observed when Neo was co-incubated with H2O2, where the Neo antiox- reduced protein intake and lower levels of amino methionine and RBC idant property displayed a U curve effect. Additionally, we observed that folate remained significant predictors of CHD. the inhibition of mitochondrial activity led to increased Neo production, Data from MPKUCS revealed lower plasma Methionine and RBC folate in the lactate release, and HO-1 content in primary striatal astrocytes. Further- CHD group, indicative of global DNA hypomethylation which has been sug- more, the co-incubation of Neo plus azide reduced free radical production. gested as a contributing factor for CHD in non-PKU pregnancies. Future studies These data indicate that astrocytes may produce and release neopterin when should include measurement of homocysteine, SAM, and SAH in addition to the mitochondrial function is impaired and that this compound might plasma amino acids and RBC folate in MPKU individuals, as abnormal DNA possess antioxidant properties, possibly by Nrf-2-mediated inhibition of methylation is likely one of the contributing factors for fetal CHD. free radical production.

P-035 P-037 PLATELET FUNCTION IN PATIENTS WITH PHENYLKETONURIA EVALUATION OF DIGITAL AND PRINT INFORMATION ABOUT UNDER STRICT DIETARY TREATMENT PKU FOR PATIENTS Mütze U1 , Beblo S1 , Kortz L2 , Brügel M3 , Thiery J4 , Kiess W1 , Burgard P1 , Cay T1 , Garbade S2 , Özkan Ö1 Ceglarek U4 1Centre for Paediatric and Adolescent Med, Heidelberg, Germany 1Univ Child Hosp, University of Leipzig, Leipzig, Germany 2Faculty of Psychology, SRH-University, Heidelberg, Germany 2LIFE – University of Leipzig, Leipzig, Germany 3Div Lab Medicine, LMU, Munich, Germany Background: The internet is a source of data used by many people seeking 4Div Lab Medicine, University of Leipzig, Leipzig, Germany health information. Quality of information on the net about PKU has not been investigated systematically so far. Background: The dietary treatment of phenylketonuria (PKU) results in Objectives: Evaluation of digital and print media by the quality criteria for low dietary intake and reduced concentrations of long-chain polyunsaturat- consumer health information of the DISCERN instrument. ed fatty acids (LC-PUFA) in plasma and cell membranes. Eicosanoids, LC- Material and Methods: 37 print media (26 German, 11 English) and 62 PUFA metabolites, are important for platelet function. Therefore, the die- internet links (19 German, 30 English, 13 Turkish) very evaluated. tary regime in PKU may influence the manifestation of vascular disease Results: German and English print media scored likewise with mean values typically occurring in later adulthood. We investigated platelet function and of 8.13/8.59 (max013) on the DISCERN scale. Correlation across items eicosanoid release in PKU-patients under strict dietary treatment. was 0.78. German and English digital media scored similar with mean Methods: A standardized protocol for platelet activation by collagen and values of 7.89/8.03 but higher than Turkish websites (5.80). Correlations analysis of subsequent eicosanoid release by liquid chromatography/tandem across items were 0.91 (German-English), 0.86 (German-Turkish) and 0.89 mass spectrometry was developed. 12 (6f/6 m) children with PKU under (English-Turkish). Print media scored low in both languages for reference good metabolic control and 8 (5f/3 m) healthy controls were included. to sources of information and areas of uncertainty, and support for shared Results: The established protocol resulted in highly reproducible platelet decision-making. Digital media scored low in all languages for reference to activation. PKU-patients showed lower median (range) LC-PUFA concen- sources of information and areas of uncertainty, description of risks of trations in plasma than controls (eicosapentaenoic acid 63.7 (17-96.2) vs. treatment, effects on quality of life, and support for shared decision-making. 81.9 (40.6-148) ng/ml, p00.08; docosahexaenoic acid 279 (141-526) vs. Conclusion/Discussion: Quality criteria should not only be used to evalu- 406 (223-890) ng/ml, p00.07), but significantly higher platelet counts (329 ate health information but also to improve their development. (257-435) vs. 272 (197-330) Gpt/l, p<0.05). There were no differences in platelet function or eicosanoid release (thromboxane B2, B3, arachidonic acid, prostaglandine D2, E2, 11-S- and 12-S-hydroxyeicosatetraenoic acid). Conclusion: PKU-patients show normal platelet function despite lower LC-PUFA concentrations in plasma but higher platelet counts. S34 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-038 P-040 MOLECULAR CHARACTERIZATION TO PREDICT IDENTIFICATION OF LARGE DELETIONS IN A POPULATION TETRAHYDROBIOPTERIN (BH4) RESPONSIVENESS IN ANALYSIS OF THE PHENYLALANINE HYDROXYLASE GENE PHENYLALANINE HYDROXYLASE (PAH) DEFICIENCY Groselj U1 , Zerjav Tansek M1 , Battelino T1 , Trebusak Podkrajsek K1 Bettocchi I1 , Nicoletti A1 , Baronio F1 , Monti S1 , Rizzello A1 , Baldazzi 1University Children's Hospital Ljubljana, Ljubljana, Slovenia L1 , Cassio A1 , Pession A1 , Bal MO1 1Neon Screen Centre, Dep Pediatrics, Bologna, Italy Background: Over 600 mutations of the phenylalanine hydroxylase (PAH) gene have been identified to date, among them just over 30 large deletions Background: Hyperphenylalaninemia (HPA, OMIM #261600), which (LDs). LDs represented less than 3 % of the mutations in most analyzed includes phenylketonuria (PKU), is an autosomal recessive metabolic dis- phenylketonuria (PKU) populations. ease, caused by mutations in the gene encoding phenylalanine hydroxylase Objective: To identify LDs in PAH gene in Slovene PKU population. (PAH). Traditional treatment is a phenylalanine restricted diet for all life- Methods: In all PKU families (out of 108) with at least one uncharacterized long but the supplementation with BH4 can alleviate the diet burden. PAH allele, MLPA analysis was performed to identify LDs, which were Objective: to study molecular analysis of PAH gene and evaluate the confirmed with long-range PCR. prevalence of BH4-responsive mutations in a group of patients living in Results: Two LDs were found in four unrelated patients and were subse- Emilia Romagna, Italy. quently confirmed with breakpoint analyses. Deletion of 955 bp spanning Patients and Methods: We performed PAH gene analysis using DNA between intron 4 and intron 5 (EX5del955, systematic name extracted from peripheral blood samples in 48 subjects. The prevalence of g.50448_51402del955) was found in three patients; deletion of 4765 bp BH4-responsive mutations was studied by consulting the PAHdb spanning between intron 2 and intron 3 (EX3del4765, systematic name (www.pahdb.mcgill.ca). g.21560_26324del4765) was found in one patient. Cumulatively, LDs were Results: We obtained a full genotypic characterization of 46 patients, 36/46 found in 2 % of all Slovenian PKU alleles. All patients with LDs were on diet: We found 87 mutant alleles and 35 different mutations: the most compound heterozygotes. frequent IVS10-11 G>A (19.3 %), R261Q (9.1 %), R158Q (9.1 %), R408Q Discussion: Both LDs found were first reported in Czech population. (6.8 %), A403V (5.7 %) and two new ones (L287, N223Y) not previously Proportion of LDs in Slovene PKU cohort (2 %) was similar as in some described. 27/36 subjects on diet carried at least one BH4-responsive other European populations (3 % in Czech population; 1.7 % in Italian mutation and 9/36 did not show BH4-responsive mutations. population; 2-3 % in Polish population); however, studies on British and Conclusions: by genotypic characterization we found 75 % of potentially Danish-German cohorts reported proportions below 1 %. BH4-responsive subjects. This treatment, combined with a less restricted diet or in some cases as monotheraphy, can reduce nutritional complications and improve patient's quality of life. P-041 P-039 GENOTYPE-PHENOTYPE CORRELATIONS IN A POPULATION SEVERE AZOTEMIA AND HYPERNATREMIC DEHYDRATION ANALYSIS OF THE PHENYLALANINE HYDROXYLASE GENE 1 1 1 1 IN AN INFANT WITH PHENYLKETONURIA Groselj U ,ZerjavTansekM ,KovacJ , Hovnik T , Trebusak Podkrajsek 1 2 Unal O1 , Duzova A2 , Hismi B1 , Dursun A1 , Tokatli A1 , Coskun T1 , K , Battelino T 1 Sivri HS1 University Children's Hospital Ljubljana, Ljubljana, Slovenia 2 1Hacettepe Univ, Pediatrics, Metabolism, Ankara, Turkey Faculty of Medicine, Univ. of Ljubljana, Ljubljana, Slovenia 2Hacettepe Univ, Pediatrics, Nephrology, Ankara, Turkey Background: Phenylketonuria (PKU) is caused by mutated phenylalanine Introduction: Potential renal solute load (PRSL) refers to solutes of dietary hydroxylase (PAH) gene, resulting in a spectrum of metabolic phenotypes. origin that are needed to be excreted in the urine not used in synthesis of Mostly, good correlation between genotype and metabolic phenotype is new tissue or lost through nonrenal routes. Here we present severe azotemia observed. and hypernatremic dehydration due to acute renal solute load in an infant Objectives: To assess correlation between genotype and metabolic pheno- with phenylketonuria (PKU). type; to predict the functional impact of PAH mutations in functional Case report: A 35 -day- old boy with PKU was admitted to our hospital hemizygotes or homozygotes. with subfebrile fever and watery stool for a week and with dehydration. He Methods: The PAH gene was analyzed in 108 families, whom also meta- had reportedly received six times more protein by mistake due to incorrect bolic phenotypes were determined. Patients were assigned to phenotype preparation of Phe free formula. He had severe azotemia and hypernatremia categories according to their pre-treatment plasma phenylalanine levels and due to exposure of very high protein and sodium content. Following a bolus dietary phenylalanine tolerance. infusion of isotonic saline, maintenance fluid and free water deficit were Results: Spectrum of 37 different mutations and 65 different genotypes was instituted, he recovered well. Renal ultrasonography, blood biochemistry, found. In six of 18 genotypes found in at least two families, inconsistencies urinalysis, arterial blood pressure were normal on six -month- old control with metabolic phenotypes were observed. In three cases p.R158Q mutation visit. His mental and motor development was also normal. was involved; in other three cases one mutation had substantial predicted Conclusion: There is limited clinical information regarding long term residual activity (PRA) (p.E390G, p.A403V, p.415 N) and metabolic phe- clinical effects of high protein intake on renal functions in infants. In the notypes were classified both as mild PKU and MHP. Thirty-two genotypes presented case, short term renal exposure of high protein noted. Therefore, were functionally hemizygous and three homozygous, which enabled func- long term monitorization and follow up is needed in such cases. tional prediction for 17 mutations, including all five novel mutations. Interestingly, the p.L48S and p.R261Q mutations correlated with classic PKU in all nine functionally hemizyguous families despite their substantial PRA. Conclusion: In most patients, good correlation was observed between genotype and metabolic phenotype. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S35

P-042 P-044 ABERRANT PHENYLALANINE METABOLISM IN PKU (PAH) DEMONSTRATION OF A BONE PHENOTYPE AND ITS MITIGATION HETEROZYGOTES WITH A LOW-PHENYLALANINE GLYCOMACROPEPTIDE DIET IN Nardecchia F1 , Carducci C2 , Carducci C2 , D'Agnano D1 , Leuzzi V1 A MOUSE MODEL OF PHENYLKETONURIA 1Dep of Ped and Child Neuro and Psych, Rome, Italy Solverson P1 , Murali SG1 , Litscher SJ1 , Blank RD1 , Ney DM1 2Department of Experimental Medicine, Rome, Italy 1University of Wisconsin-Madison, Madison, United States

Several attempts were made to set up a biochemical test to recognize The etiology of skeletal fragility in phenylketonuria (PKU) is unknown and carriers of phenylalanine (Phe) hydroxylase (PAH) mutations without de- may reflect the genotype and/or the amino acid (AA) dietary treatment. finitive results. Present study was designed to explore in vivo residual Glycomacropeptide (GMP), an intact low-phe dietary protein, provides an enzymatic PAH activity in carriers of PAH gene mutations. We investigated alternative to AA formula. Our objective was to determine the impact of the blood Phe clearance after oral Phe loading test (100 mg/kg bw) in 23 PKU genotype and dietary protein source on growth and bone development. carriers of missense mutations at the PAH gene (PAHH) and 10 PAH Wild type (WT) and PKU (Pahenu2/enu2) mice were fed casein, low-phe AA, normozygote controls (PAHC). Blood Phe and Tyr were determined in and low-phe GMP diets between 3-23 weeks of age. Femoral bone develop- fasting condition and every hour for six hours after Phe loading. Phe/ ment was assessed by size, bone mineral density and biomechanical properties Tyrosine (Tyr) ratio, Phe peak values and the time lag before the basal using the 3-point bending test. PKU mice showed similar plasma phe concen- Phe value was restored were analyzed. Phe/Tyr ratio peak was reached in tration, growth and lean body mass when fed the GMP or AA diets. Regardless most PAHH (20/23 subjects) and controls at the first hour after the loading of diet, PKU femora were more brittle and broke more easily than WT femora, and was significantly higher than in controls from the first to the third hour as manifested by lower post-yield displacement and maximal load tolerated (p<.001) and still at the fourth hour (p<.05). The Phe peak value was before fracture. Regardless of genotype, the AA diet reduced femoral cross- 655,63 μmol/L (SD 152,80) and 454,10 μmol/L (SD 153,20) respectively sectional area with a consequent reduction of maximal load compared with the in PAHH and PAHC (p<.001). Basal blood Phe value was restored in 3/23 GMP diets. Thus, the GMP diet improved bone size and bone strength in both PAHH and in 9/10 controls after six hours (p<.0001). Conclusions: Phe/ WT and PKU mice. Skeletal fragility is an inherent feature of PKU and the Tyr ratio at the first hour after Phe loading can differentiate carriers from bone phenotype is improved with the GMP compared with an AA diet. controls. Conflict of Interest declared.

P-043 P-045 MODULAR PKU TRAINING PROGRAMME WITH A USE OF CANTAB COMPUTERISED NEUROPSYCHOLOGICAL MULTIPROFESSIONAL APPROACH: BENEFIT FOR AFFECTED BATTERY TO ASSESS EXECUTIVE FUNCTIONING IN FAMILIES PHENYLKETONURIA (PKU) Weber L1 , Meyer U1 , Gebert N2 , Wagner P3 , Ernst G2 , Lange K2 , Das Thompson N.J.1 , Abulhoul L.1 , Cleary M.A.1 , Fitzachery C.1 , Renshaw AM1 G.1 , Skeath R.1 , Mumford N.1 , Lucock A.1 , Bond K.1 1Dept Paed, Hann Med School, Hannover, Germany 1Great Ormond Street Hospital, London, United Kingdom 2Dept Med Psyc, Hann Med School, Hannover, Germany 3Dept Pead, Charité, Berlin, Germany Studies have found subtle neuropsychological impairments in executive function for patients with phenylketonuria (PKU) despite early instigation Introduction: Treatment of Phenylketonuria involves regular medical, of dietary control. We present preliminary results from a clinic audit of dietary and psychosocial counselling which is usually done individually CANTAB computerised neuropsychological tests of executive function and for each family. In contrast to other more common chronic diseases (e.g. working memory. asthma, diabetes mellitus) there is no established multiprofessional stand- Results: were analysed with respect to blood phenylalanine concentrations ardised group intervention. The German ministry of health has solicited measured on the day of testing, historical averages and variability since modular group training programmes for chronic paediatric diseases initial diagnosis. A group of 35 treatment-adherent PKU patients aged (MODUS). We have developed such a programme for Phenylketonuria as between 5-18 years were routinely assessed. Neuropsychological results a metabolic model disease. were compared to standardised scores of Wechsler tests of IQ, word reading Methods: The modular training programme consists of 3 generic psycho- and numerical operations to account for variations in individual ability and educational and 3 disease-specific modules. developmental age. Our initial findings show significant differences be- 39 parents of children aged 0-12 years, 5 children aged 8-12 years and 6 tween PKU subjects and controls, non-linearity resulting from patients for children aged 13-17 years participated. Disease-specific knowledge was whom impairments were more severe, and elevated error rates where assessed both in parents and older children using a standardized question- overall performance was normal. We discuss the sensitivity of the CAN- naire 1) directly before the group-training, 2) directly after finishing the TAB in detecting working memory deficiencies; and its suitability in a intervention, 3) six weeks after training. P <0.05 was regarded as clinical setting for detecting traits including impulsivity. We recommend significant. that extra validation of the measures is needed to further our progress Results: Almost all participants were highly motivated and participated refining a protocol for routine screening and monitoring of executive actively. There was a highly significant and sustained gain of specific function in PKU children as an indicator of frontal lobe dysfunction. knowledge both in parents and children with Phenylketonuria. Conclusion: We developed a modular group training programme with a multiprofessional approach for families affected by Phenylketonuria which led to a sustained gain of knowledge, thus hopefully improving metabolic control in the long run. S36 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-046 P-047 COMPARATIVE STUDY OF INTELLECTUAL DEVELOPMENT PKU IN ITALY AMONG PHENYLKETONURIC CHILDREN, BIOLOGICAL Giovannini M1 , Burlina A2 , Spada M3 , Cerone R4 , Bal M5 , Pasquini E6 , SIBLINGS AND CONTROL Stoppioni V7 , Leuzzi V8 , Cotugno G9 , Andria G10 , Correra A11 , Papadia Cornejo V1 , Castro G1 , De la Parra A1 , Cabello JF1 , Arias C1 , Betta K1 , F12 , Concolino D13 ,MeliC14 ,CasertaM15 , Lilliu F16 , Salvatici E1 , Peredo P1 , Colombo M2 Paci S1 1LabGEM, INTA, University Chile, SANTIAGO, Chile 1Paed Dept, San Paolo Hosp, Univ Milan, Milan, Italy 2Van Buren Hospital, Valparaiso, Chile 2University of Padova, Padova, Italy 3University of Torino, Torino, Italy Introduction: Studies show that children with PKU diagnosed and treated 4Gaslini Hospital, Genova, Italy early in their lives have a normal intellectual development, but lower than 5University of Bologna, Bologna, Italy their siblings. 6Meyer Hospital, Firenze, Italy Objective: To determine differences in intelligence quotient (IQ), subtests 7Centro Screening e Cura, Fano (PU), Italy grouped into factors (verbal comprehension, perceptual organization and 8University of Roma (La Sapienza), Roma, Italy distraction) of classical PKU children with good metabolic control, a non- 9Paed Hosp Bambino Gesù, Roma, Italy PKU biological sibling and a control group matched by sex, age, socioeco- 10University of Napoli (Federico II), Napoli, Italy nomic status and nutritional status. 11Santobono-Pausillipon Annunziata Hosp, Napoli, Italy Method: The Wisc-R scale was applied, consisting in 12 subtests to 12Paed Hosp Giovanni XXIII, Bari, Italy determine IQ and factors; nutritional status was determined by BMI and 13University of Catanzaro, Catanzaro, Italy the socioeconomic status by modified Graffar method. The statistical anal- 14University of Catania, Catania, Italy ysis was performed by SPSS (ANOVA). 15Paed Hosp G. di Cristina, Palermo, Italy Results: PKU Group: 16 children (12 men, 9 women); PHE: 5.1±1.7 mg/dl 16Metab Dept, Cagliari, Italy in men and 5.7±2.8 mg/dl in women; 69 % were eutrophic, 25 % overweight; total IQ in PKU0100, siblings0103, control0104. Distraction Neonatal screening for Hyperphenylalaninemia (HPA) is compulsory by factor in PKU patients07.8, siblings09.7, control09.6 (p<0.006 with law in Italy since 05/02/1992. siblings, p<0.009 with controls). Italy's incidence of newborns affected by any form of HPA is equal to Conclusion: We did not find significant differences between IQ of PKU 1:3.494 live births, with 1:8.681 requiring specific treatment (Annual Tech- children in comparison with their siblings nor with a control group. How- nical Report 2010-SIMMESN). ever, PKU children present a significant lower performance in tasks sensi- The following data were obtained by a questionnaire sent to all 16 Italian tive to inattention. There were not significant differences in PKU IQ Centers involved in PKU management. according to socioeconomic status. The total number of patients is 3527 (44 % males, 56 % females) plus 471 late diagnosis, and 2474 of them are actually in follow up. Adult patients (more than 18 years of age) are 904, with 649 females in childbearing age; 83 pregnancy are reported, 54 of them with normal fetal/neonatal outcome. 33 % are classic PKU, while 42,6 % are HPA on free diet. In use strategies for PKU treatment are: 88 % dietary treatment, 7 % BH4 and 5 % Large Neutral Amino Acids (LNAA). 47 patients with a diagnosis of BH4 deficiency are reported. Since 2010 a National PKU Registry has been established, supported by SIMMESN, with 817 patients actually included. In Italy, in the last 40 years, centers' collaboration, experiences' interchange, research and studies collaborative commitment, Italy's engagement in EU project has guaranteed and guarantee the best clinical management for PKU patients, beyond rare disease. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S37

P-048 P-050 THE CHARACTERISTICS OF THE SLOVENE PHENYLKETONURIA GENOTYPE-PHENOTYPE ANALYSIS OF SERBIAN P.L48S PKU POPULATION PATIENTS Groselj U1 ,ZerjavTansekM1 ,KovacJ1 , Hovnik T1 , Trebusak Podkrajsek Stojiljkovic M1 , Djordjevic M2 , Klaassen K1 , Tosic N1 , Sarajlija A2 , K1 , Battelino T1 Zukic B1 , Radmilovic M1 , Spasovski V1 , Kotur N1 , Pavlovic S1 1University Children's Hospital Ljubljana, Ljubljana, Slovenia 1IMGGE, University of Belgrade, Belgrade, Serbia and Montenegro 2Mother and Child Healthcare Institute, Belgrade, Serbia and Montenegro Background: Phenylketonuria (PKU) is the most common inborn error of amino acid metabolism, caused by mutated phenylalanine hydroxylase Phenylketonuria (PKU) is caused by mutations in phenylalanine hydroxy- (PAH) gene. Mandatory neonatal PKU screening in Slovenia has been lase gene (PAH). Mutation p.L48S (39 % residual activity) was found in implemented in 1979. many European populations, but usually with frequency lower than 5 % and Objectives: To identify spectra of the PAH mutations and the metabolic never as the most frequent one. phenotypes in the Slovene PKU population; to estimate the incidence of Over the years, we have genotyped 59 unrelated PKU patients by PCR- PKU in Slovenia. RFLP and DGGE/DNA sequencing analysis of PAH gene. We identified 26 Methods: The PAH gene was analyzed with dHPLC and subsequently disease-causing mutations and p.L48S was the most frequent one (30.5 %). automated sequencing was performed. Patients were assigned to phenotype PKU patients, their siblings and parents (9 p.[L48S];[L48S] and 12 categories according to their pre-treatment plasma phenylalanine levels and p.[L48S];[null]) were assigned to phenotype categories (classical, moder- dietary phenylalanine tolerance. ate, mild and HPA) based on phenylalanine tolerance and/or pretreatment Results: Cumulatively, 108 families were investigated; mutations were iden- phenylalanine blood level. tified on 211 alleles (detection rate 98 %). Thirty-seven different disease- We used Fisher's exact test to compare the effect of p.L48S on phenotype in causing mutations were identified, including five novel (c.43_44insAG, homozygous versus functionally hemizygous patients. We found that dis- c.56_59+1delACAGG, p.V45A, p.L62P and p.R157S). The most frequent tribution of phenotypes significantly varied between two genotype groups mutation was p.R408W in exon 12 (29 % of the alleles). Other common (p00.011). When combined with a null mutation, p.L48S was in 83.3 % mutations were: p.R158Q, p.A403V, p.P281L and p.E390G, accounting for cases associated with the classical PKU. However, p.L48S homozygotes 9 %, 7 %, 7 % and 7 % of the alleles respectively. The metabolic phenotype were associated with all four phenotype categories. In this group, majority was classified as classic PKU in 54 %, as mild PKU in 26 % and as mild HPA was associated with mild PKU (55.5 %). in 20 % families. The incidence of classic PKU was estimated to be 1/10.000. Our findings show that the effect of p.L48S is masked if it is combined with a Conclusions: The characteristics of the Slovene PKU population were null mutation. Moreover, phenotypic inconsistency found in homozygotes comparable as in neighbouring and/or related populations. implies that additional factors play a role in genotype-phenotype correlation.

P-049 P-051 DIETARY MANAGEMENT AND GROWTH OF TETRAHYDRO- STABILITY OF BLOOD PHENYLALANINE (PHE) LEVELS IN BIOPTERIN RESPONSIVE TURKISH PKU PATIENTS PHENYLKETONURIA (PKU) IS NOT INDEPENDENT FROM Gökmen-Özel H1 , Ünal Ö2 , Kalkanoğlu-Sivri HS2 , Köksal G1 , Dursun ABSOLUTE BLOOD PHE LEVELS AND MIGHT BE A MISLEADING A2 , Tokatlı A2 ,Coşkun T2 CONCEPT FOR OUTCOME STUDIES 1Hacettepe Univ, Nutr Diet Dept, Ankara, Turkey Burgard P1 , Lindner M1 , Haege G1 2Hacettepe Univ, Dept Ped, Metab Unit, Ankara, Turkey 1Centre for Paediatric and Adolescent Med, Heidelberg, Germany

Background: Tetrahydrobiopterin reduces blood phenylalanine concentra- Background: In addition to absolute phe levels variability in blood phe- tions in certain patients with phenylketonuria. The effects of tetrahydrobiop- nylalanine (phe) levels have been repeatedly claimed to have an indepen- terin on dietary intake and growth have not been sufficiently studied. This dent negative effect on outcome parameters like intelligence or offspring of study investigated whether tetrahydrobiopterin-responsive phenylketonuric mothers with phenylketonuria. patients could maintain blood phenylalanine control and growth while increas- Objectives: Investigation of theoretical and empirical associations between ing dietary phenylalanine. measures of central tendency and dispersion of blood phe levels. Methods: Forty-nine BH4 responsive children with phenylketonuria (27 Material and Methods: Phe levels measured during the first year of life in boys, 22 girls) aged between 1 and 15.5 yrs (median 5.7) were recruited. 364 patients (234 classical PKU, 56 mild PKU, 74 mild hyperphenylalani- Daily phenylalanine intake was increased by 10 mg/kg on a weekly basis. naemia) were analysed regarding means and SDs, as well as the association Blood phenylalanine level was checked every week. Weight for age (WAZ), between these parameters. height for age (HAZ) and body mass index for age (BMIZ) z scores were Results: Individual means were highly correlated with individual SDs in calculated using WHO Anthro programmes. patients with classical (r00.9) or mild PKU(r00.79) as well as with MHP Results: With tetrahydrobiopterin, 37 patients were on free diet, 8 patients (r00.88). Using a cut-off of 6 mg/dl (360 μM), individual SDs were much still required protein substitutes (PS). Mean dietary phenylalanine tolerance higher correlated with means of values >6 mg/dl than with means of values significantly increased 2.46 folds (p00.000). Median WAZ increased from <0 6 mg/dl. 0.30 to -0.70 (p00.295). Median HAZ improved from -0.51 to -0.28 (p0 Conclusion/Discussion: The standard deviation (SD) of the mean is a 0.081). Median BMIZ score decreased from 0.92 to 0.73 (p00.170). Total misleading concept to model the causal impact of blood phe levels on 748 blood samples were analyzed and 65.2 % of the blood phenylalanine outcome variables. Distributions of phenylalanine levels are bounded at the levels were within the normal ranges. lower tail, and intermittently low levels probably will not have the same Conclusions: Tetrahydrobiopterin-responsive patients experienced in- negative effect (if any) as high levels. creased phenylalanine tolerance and reduced PS. While WAZ and HAZ Conflict of Interest declared. seemed to improve during the period, BMIZ did not change significantly. Conflict of Interest declared. S38 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-052 P-054 PHENYLKETONURIA - THE EFFECT OF LNAA ON AMINO LONG-CHAIN POLYUNSATURATED FATTY ACID STATUS ACID PROFILE IN CHILDREN, ADOLESCENTS AND ADULTS WITH Ahring K.K.1 , Mxller L.B.1 , Andersen J.R.2 PHENYLKETONURIA DOES NOT DIFFER FROM HEALTHY 1Kennedy Centre, Glostrup, Denmark CONTROL SUBJECTS 2State University Hospital, Copenhagen, Denmark Gramer G1 , Haege G1 , Langhans CD1 , Schuhmann V1 , Lindner M1 , Burgard P1 Background: Supplementation of large neutral amino acid (LNAA) and a 1Univ Children's Hosp, Div Metab Dis, Heidelberg, Germany semi-free (SF) diet has been shown to have a positive effect on well-being on adults with PKU. However, finding the optimal dosage in relation to Background: Patients with phenylketonuria have been reported to be defi- stabilising the amino acid profile (AAP) has yet to be determined. The aim cient in long-chain polyunsaturated fatty acids (LCPUFAs). It has been pos- of this study was among others to determine the effect on the total AAP tulated that good compliance with the dietary regimen negatively influences from LNAA in different dosages and combinations. LCPUFA status. Material (Patients) and methods: This was a prospective, double-blind, Patients and Methods: LCPUFA-levels were evaluated in 36 patients with cross-over study consisting of four consecutive 3-week phases. Twelve phenylketonuria and 18 age-matched healthy control subjects. LCPUFAs subjects (6 males, 6 females) with PKU were recruited, 11 completed the were measured in plasma phospholipids, plasma cholesterol esters, eryth- study. Each phase consisted of either LNAA1 or 2, either in low or high rocyte phosphatidylcholine and phosphatidylethanolamine. Docosahexae- dosage. Subjects were instructed to follow their usual SF diet and complete noic acid (DHA) and eicosapentaenoic acid (EPA) levels were compared a 3-day food record at start and at the end of each period. between patients and controls. Results: The AAP analysis includes 19 amino acids (AA), where 9 of the Results: Levels of EPA and DHA did not differ significantly between patients AA were supplemented as LNAA. Three AA were above range level in all and controls in plasma and erythrocyte fractions (p00.304). Correlations phases. There was no correlation between AAP and the amount or brand of between SD (standard deviation) scores of DHA-levels from the respective LNAA. age-matched control group and patients' phenylalanine(phe)-levels were sig- Conclusions: LNAA1 & 2 in different dosages or combinations do not nificant for erythrocyte phosphatidylethanolamine (concurrent phe-level - change the AAP. 0.446; p00.007; long-term phe-level -0.362; p00.032) and erythrocyte phosphatidylcholine (concurrent phe-level -0.350; p00.043; long-term phe-level - 0.350; p00.042). Patients with lower (higher) phe-levels had positive (negative) DHA-SD. DHA-levels did not differ between patients consuming LCPUFA supplemented amino-acid mixtures and patients consuming 0 P-053 amino-acid mixtures without LCPUFA supplementation (p 0.673). Conclusion: In contrast to previous reports we did not find lower LCPUFA-levels PREVALENCE OF VITAMIN B12 DEFICIENCY IN in patients with phenylketonuria compared to healthy control subjects. The PHENYLKETONURIA PATIENTS ON- AND OFF-DIET LCPUFA content of the amino acid mixture did not influence patients' DHA-levels. Liu A1 , Khan A U1 , Walker R2 , Ramachandran R2 , Rahman Y2 1King's College London Medical School, London, United Kingdom 2IMD, Evelina Child Hosp, Guys&Thomas', London, United Kingdom P-055 Background: PKU patients who are off diet are more likely to experience VITAMIN D, B12 AND FOLATE CONCENTRATIONS IN DIET-TREATED neurological signs and symptoms, and these have traditionally been attrib- PHENYLKETONURIA PATIENTS uted to phenylalanine toxicity. Although on an unrestricted diet, animal Khan AU1 , Liu A1 , Walker R2 , Ramachandran R2 , Rahman Y2 protein intake may remain low and these patients are therefore at 1King's College London, Medical School, London, United Kingdom increased risk of vitamin B12 deficiency. Neurological symptoms such 2Cen Inhrt Metab Dis, Evelina Child Hosp, London, United Kingdom as personality changes, blunted affect, and emotional liability have been reported in patients with plasma vitamin B12 levels below Introduction: Dietary protein restriction alongside phenylalanine free pro- 400 ng/L, and this may also account for the neurological signs and teins, vitamins and essential minerals supplementation is the cornerstone of symptoms seen in PKU patients. Phenylketonuria (PKU) treatment. These supplements are often the only Aims: To assess serum vitamin B12 and prevalence of vitamin B12 defi- source of vitamin D, B12 and folate. ciency in patients on and off PKU diet. Aim: To evaluate the efficacy of the current recommended doses of Method: Retrospective analysis of the most recent biochemical records of supplementation. 91 adult PKU patients in a single metabolic centre. Method: The most recent vitamin D, B12 and folate in diet restricted PKU Results: Serum Vitamin B12 levels were significantly lower in patients off patients (n052, mean age 32 years, range 16-65 years, male n019, female n0 diet compared to those on diet. 48 % of patients on diet and 97 % of patients 33) attending a single adult IMD centre, were retrospectively analysed. Vitamin off diet had B12 levels below 400 ng/L. D>40 nmol/L, B12>400 ng/L, and folate>4 ng/L were defined as sufficient. Conclusions: PKU patients are at risk of vitamin B12 deficiency, particu- Results: 36 % of patients (n013, 8 female, 5 male) were vitamin D deficient larly if off diet. Serum vitamin B12 screening and supplementation as best (median 52 nmol/L, range 12-99 nmol/L). 48 % (n025, 18 female, 7 male) were practice may improve PKU management. B12 deficiency must first be deficient in vitamin B12 (median 415, range [64-1254]). 6 % (n03, 2 female, 1 excluded in PKU patients presenting with neurological symptoms. male) were deficient in folate (median 13.5 ng/L, range 2.4->20.0 ng/L). Conclusions: While most patients were folate replete, a significant number of PKU patients are vitamin B12 or D deficient, suggesting that current recommended doses for supplementation of these vitamins in PKU patients may be inadequate. Annual patient monitoring and additional supplementation may be needed. Further longitudinal studies regarding vitamin levels and long-term compliance may be useful. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S39

P-056 P-058 SUPPLEMENTATION OF PKU PATIENTS WITH KEYOMEGA® MAN MADE DISEASE: EFFECTS OF LOW PHENYLALANINE NORMALIZES DHA AND AA LEVELS LEVELS IN AN INADEQUATELY TREATED PHENYLKETONURIA Jans JJ1 , de Sain-van der Velde M.1 , van Hasselt P.M.1 , van den Hurk PATIENT AND MOUSE MODEL D.A.2 , Vaz F.M.3 , Visser G.1 , Verhoeven-Duif N.M.1 Pode-Shakked B1 , Shemer-Meiri L1 , Harmelin A2 , Stettner N2 , Brenner 1Dept. of Metab Dis, Univ Med Center, Utrecht, Netherlands O3 , Schwartz G1 , Anikster Y1 2Dept. of Dietetics and Nutrit, UMC, Utrecht, Netherlands 1Metab Dis Unit, Sheba Medical Center, Tel-Hashomer, Israel 3Dept. of Clin Chem and Ped, Acad Med Cen, Amsterdam, Netherlands 2Dep Vet Res, Weizmann Inst of Science, Rehovot, Israel 3Dep Mol Genet, Weizmann Inst of Science, Rehovot, Israel Objectives: To evaluate the LC-PUFA status of PKU patients supplemented with fish oil or KeyOmega ®. Background: Phenylalanine (Phe) deficiency had been previously de- Patients and Methods: Plasma and erythrocyte docosahexaenoic acid scribed in sporadic case-reports, and associated with listlessness, eczema- (DHA) and arachidonic acid (AA) levels were determined in 54 patients tous eruptions and failure-to-thrive (FTT). (1-18.5 years of age) with confirmed PKU. The influence of supplementa- Case Report: A 9-month old patient with phenylketonuria presented with tion with fish oil versus KeyOmega ® on LC-PUFA status was investigated diarrhoea, severe erythema, desquamation, alopecia, corneal perforation and compared to the status in samples obtained from unsupplemented and FTT. Following an elaborate medical evaluation of her history it was patients. revealed that prior to hospitalization, the patient's diet consisted of the Results: In line with previous reports, unsupplemented PKU patients fre- phenylalanine-free medical-formula alone, without addition of an infant quently showed a deficient LC-PUFA status. Whereas supplementation formula as recommended. with fish oil increased the level of DHA, the AA concentration did not Objective: To investigate the effects of a phenylalanine-free diet in a mouse increase to normal values in these patients. In contrast, both DHA and AA model. levels increased and reached reference values upon supplementation with Materials and methods: Twenty mice were fed either a Phenylalanine- KeyOmega ®. deficient or normal diet. Necropsies were performed either early or late after Conclusion: KeyOmega ® offers additional benefit over fish oil since both diet initiation, or following reintroduction of normal diets. AA and DHA status is normalized in PKU patients supplemented with Results: Gross lesions noted on necropsy in the Phe-deficient mice includ- KeyOmega ®. The clinical relevance of the difference in LC-PUFA status ed scruffy coat, tendency toward weight loss, reduction in thymic mass, and between the two supplementation types, however, remains to be severe gastric dilation, none of which were seen in the controls. Histological established. findings included thymic depletion, hepatocellular vacuolation, and exocrine pancreatic atrophy. No lesions were evident in the brain or eyes. Conclusions: Iatrogenic Phenylalanine deficiency, which manifests in gastrointestinal, dermatological and ocular findings, requires a high index of suspicion. Mice fed a phenylalanine-deficient diet display somewhat similar P-057 organ involvement, although no ocular abnormalities were seen. BIOCHEMICAL MARKERS AND TREATMENT OPTIONS IN PATIENTS WITH DIHYDROPTERIDINE REDUCTASE P-059 DEFICIENCY BREASTFEEDING IN PHENYLKETONURIC (PKU) AND Opladen T1 , Lindner M1 , Hoffmann GF1 , Jaeken J2 , Regal L2 , Blau N1 1 HYPERPHENYLALANINEMIC (HPA) INFANTS: HOW AND WHY Univ Children's Hosp, Div Inborn Met Dis, Heidelberg, Germany 1 1 1 1 1 1 2 Giovannini M ,PaciS ,LopsA , Salvatici E , Verduci E , Banderali G , Univ Hosp Gasthuisberg, Div Metab Dis, Leuven, Belgium 1 Riva E 1Paediatric Department,San Paolo Hospital, Milan, Italy Background: Sapropterin (tetrahydrobiopterin; BH4) is not a standard treatment in dihydropteridine reductase (DHPR)-deficient patients because According to WHO/UNICEF recommendations, breastfeeding should be potentially neurotoxic dihydrobiopterin (BH2) accumulates, leading to in- also encouraged in phenylketonuric infants because, even if practised only hibition of aromatic amino acid hydroxylases and NOS. for a short period (20–40 days), its beneficial effect have been largely Methods: Aim of the study is to investigate biochemical markers of DHPR demonstrated on intelligence quotients, behavioural development and visu- deficiency: BH2 and neurotransmitter metabolites 5HIAA and HVA in al performance, if compared with formula feeding. patients who are in addition to L-dopa/carbidopa/5-OH-Trp on treatment The most important advantages are due to its lower amount of Phe, its with either low-Phe diet or BH4. optimal Phe/Tyr ratio and its content of long-chain polyunsaturated fatty Results: In 63/217 DHPR-deficient patients tabulated in the BIODEF acids (LCPUFAs), which positively influence brain's growth and functional database (www.biopku.org) CSF analysis of biogenic amines is available development, making human breast milk a relatively low-Phe food. before treatment. 96.8 % presented with low 5HIAA and 92.1 % with low Our aim was to investigate by a questionnaire "ad hoc" 100 mothers of PKU HVA in CSF at time of diagnosis. BH2 and/or total biopterin were always and HPA children, diagnosed by neonatal screening and followed up at our elevated in CSF. Data from a single case demonstrate an increase of BH2 Metabolic Department, and to analyse rates of breastfeeding (compared to (from 25 to 43 nmol/L; normal <18 nmol/L), and decrease of 5HIAA (from healthy population in Lombardy-Italy), the role of the diagnosis' commu- 106 to 88 nmol/L; normal 130-362 nmol/L), and HVA (from 205 to nication on lactation, and the neurological-neurophycological outcome of 156 nmol/L; normal 313-824 nmol/L) in CSF while on treatment with patients in primary school-age. sapropterin (5 mg/kg qid), in addition to unchanged L-dopa/carbidopa/5- Breastfeeding duration rates in our patients are notably lower than in OH-Trp treatment. healthy infants. We speculate that the diagnosis of a chronic disorder Conclusion: Preliminary biochemical data suggest a beneficial use of low- requiring a strict clinical and dietary management may negatively affect Phe diet in patients with DHPR deficiency. In DHPR-deficient patients BH2 the maternal attitude towards breastfeeding. After diagnosis, mothers need accumulates after BH4 administration and possibly inhibits neurotransmit- psychological and technical support to maintain breastfeeding in order to ter biosynthesis in CNS. gain the expected advantages on infant. S40 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-060 P-061 MANAGEMENT OF ADULT PATIENTS WITH PHENYLKETONURIA: 48-HOUR BH4 LOADING TEST VERSUS ALGORITHMIC RESULTS FROM AN INTERNATIONAL SURVEY PHENYLALANINE CHALLENGE: INTERIM ANALYSIS OF Trefz F1 , Van Spronsen F2 , MacDonald A3 , Feillet F4 , Muntau A5 , AN OPEN-LABEL MULTICENTRE STUDY Belanger-Quintana A6 , Burlina A7 ,DemirkolM8 , Giovannini M9 , Lagler FB1 , Brunner-Krainz M2 , Moeslinger D3 , Konstantopoulou V3 , Gasteyger C10 Roscher A3 ,ErwaW2 , Sterl L2 , Karall D4 , Scholl-Bürgi S4 , Zschocke J5 , 1School of Medicine Univ Tuebingen, Reutlingen, Germany Sperl W6 , Volkmar B6 ,PleckoB7 2University Medical Center Groningen, Groningen, Netherlands 1IIEM&Dept Pediatrics, Paracelsus Med Uni, Salzburg, Austria 3The Children's Hospital, Birmingham, United Kingdom 2Univ Child Hospital, Graz, Austria 4Hôpital d'enfants Brabois, Vandoeuvre les Nancy, France 3Child Hospital, Med Uni, Vienna, Austria 5Ludwig-Maximilians-University Munich, München, Germany 4Ped I, Med University, Innsbruck, Austria 6Hospital Ramon y Cajal, Madrid, Spain 5Division of Human Genetics, Med Uni, Innsbruck, Austria 7University Hospital, Padova, Italy 6Dept Pediatrics, Paracelsus Med Uni, Salzburg, Austria 8Istanbul University Children's Hospital, Istanbul, Turkey 7Dept Pediatrics, Univ Hospital, Zurich, Switzerland 9San Paolo Hospital, University of Milan, Milan, Italy 10Merck Serono S.A, Geneva, Switzerland Background: The BH4 loading test (BLT) recommended by the European Phenylketonuria Group should be performed at ≥400 μmol/l blood phenyl- Background: Information on the treatment practices of adult phenylketon- alanine. However many patients, who qualify for Kuvan® treatment target uria (PKU) is scarce. lower levels. Furthermore BLT doesn't capture an increase of phenylalanine Objectives: To evaluate the clinical practices and characteristics of centres tolerance (PheTol). Thus we developed a novel algorithm with a BLT for the treatment of adult PKU. followed by a 13 days standardised algorithmic Phe challenge (APC). Material and methods: A questionnaire (25 questions) was sent via email Primary Objective: Feasibility and validity of the APC vs. BLT. (www.surveymonkey.com) in early 2012 to 204 healthcare practitioners Patients/Methods: PKU Patients, aged 4-18 years with potentially BH4 ≥ (HCPs) who treat adults with PKU across the world. responsive genotype were eligible. Responsiveness was defined as a 30 % ≥ Results: HCPs (40 %; 81/204) from 24 countries responded. Most followed decrease in blood Phe levels and/or an increase of PheTol by 10 mg/kg/d ≥ adult patients have classical PKU (67 %); 70 % are aged 18–29 years; 60 % (or 350 mg/d absolute) We report on the first 11 patients. are females. The maximum recommended target blood phenylalanine con- Results: 6/11 responded in both tests. 2/11 could increase their PheTol by only centrations varied between 480 and 1200 μmol/L. HCPs (51 %) reported 5 mg/kg/d, despite a decrease of blood Phe-levels of>80 % on BLT. 3/11 increased being in contact with their adult patients three times a year; contact was lost their PheTol by 350-525 mg despite non-responsiveness in the BLT. No participant with 14.3 % of females and 18.8 % of males. 71 % are treated with a showed harmful hyperphenylalaninemia. Two patients completed the 6-months- phenylalanine-restricted diet only; 22 % receive no treatment at all; 4 % follow-up visit. Both maintained an increased PheTol, 1/2 has had a negative BLT. receive a phenylalanine-restricted diet plus BH,4/sapropterin. Most adult Conclusion/Discussion: Our data indicate feasibility and additional diag- patients (90 %) are still treated in paediatric centres. nostic value of our APC protocol. Conclusion: This study shows that most adult patients with PKU are in Conflict of Interest declared. follow-up, possibly due to a young mean age of the cohort, and are treated. Recommendations to pursue life-long treatment seem to be followed by P-062 most clinics. THE 48-HOUR TETRAHYDOBIOPTERIN (BH4) LOADING TEST Conflict of Interest declared. AND LONG-TERM OUTCOME OF PATIENTS WITH BH4 TREATMENT IN TURKISH PHENYLKETONURIA (PKU) PATIENTS Unal O1 , Gokmen-Ozel H2 ,CoskunT1 ,HismiB1 , Tokatli A1 ,DursunA1 , Ozgul RK1 ,SivriHS1 1Hacettepe Univ, Pediatrics, Metabolism, Ankara, Turkey 2Hacettepe Univ, Dept. Nutrition and Diet, Ankara, Turkey

The 48-hour BH4 loading test (BLT) is used to detect BH4-responsiveness in patients with PKU due to phenylalanine hydroxylase deficiency. For responsive patients, BH4 is a treatment option. BH4-responsiveness was investigated in Turkish PKU patients and long- term outcomes of the treatment were evaluated. Methods: The 48-hour BLT was performed on 121 patients (age range: 0– 16 years). Phe loading was performed with L-Phe powder. Test was started when baseline Phe level was ≥400 μmol/L. A ≥30 % decrease of the Phe level compared to the baseline was interpreted as BH4 responsiveness. BH4 treatment was instituted to responsive patients. In the 121 patients that BLT was performed on (hyperphenylalaninemia: 13, mild PKU: 25, moderate PKU: 55, classical PKU: 28), 62 of them were found as BH4- responsive. BH4 treatment was used in 53 patients (age range: 0–15 years) for 3 months–2 years period. While on BH4, 37 patients were on free diet, 8 patients required protein substitutes. Mean dietary phenylalanine tolerance significantly increased median 2.46 folds. In 4 patients treatment was ceased because of secondary unresponsiveness. The 48-hour BLT is an effective test to detect BH4 responsive patients and BH4 is a safe and effective treatment option for responsive patients. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S41

P-063 P-064 CLINICAL AND BIOCHEMICAL FEATURES, TREATMENT SIGNIFICANCE OF NEONATAL BH4 LOADING FOR DIFFERENTIAL STRATEGIES AND FOLLOW-UP OF 626 PATIENTS WITH DIAGNOSIS OF HYPERPHENYLALANINEMIA TETRAHYDROBIOPTERIN DEFICIENCIES Trefz F1 , Feillet F2 , Demirkol M3 , Muntau A4 , Belanger-Quintana A5 , Opladen T1 , Hoffmann GF1 , Blau N1 Blau N6 1Div Metab Dis, Univ Child Hosp, Heidelberg, Germany 1MVZ Kreisklinik. Reutlingen, Gammertingen, Germany 2Hôpital d'enfants Brabois, Vandoeuvre l, Nancy, France Objectives: Inherited defects in tetrahydrobiopterin (BH4) metabolism lead 3University, Children's Hospital, Instanbul, Turkey to progressive neurological dysfunction. The study summarizes clinical and 4Ludwig-Maximilians-University, München, Germany biochemical findings, current treatment strategies and follow-up. 5Hospital Ramon y Cajal, Madrid, Spain Methods: We analyzed data of 626 patients with BH4 deficiencies [355 6Univ. Children's Hosp. Hd. and Zürich, Zürich, Switzerland with 6-pyruvoyl-tetrahydropterin synthase (PTPS), 217 with dihydropteridine reductase (DHPR), 31 with autosomal recessive GTP cyclohydrolase Background: Neonatal BH4 Loading may be useful for differential diag- (GTPCH), and 23 with pterin-4a-carbinolamine dehydratase (PCD) defi- nosis of BH4 deficiencies (BH4-D) and BH4 responsiveness in phenylala- ciencies] tabulated in the BIODEF Database. nine hydroxylase deficiency (PAH-D). Results: Up to 57 % of neonates with BH4 deficiencies are clinically Objectives: Retrospective evaluation of standardized BH4 loading tests in symptomatic. During infancy, the predominant symptoms are muscular patients found in the PKU screening in 4 different European countries. hypotonia and developmental delay. Children develop age-dependent Method: Measurement of blood phenylalanine (phe) concentration be- movement disorders. After diagnosis of hyperphenylalaninemia in newborn tween 0 and 8 (BH4-D) and 0-24 hours (PAH-D) in newborns under screening, BH4 deficiency is confirmed by urinary or dried blood spot standardized condition. Response to BH4-loading (20 mg/kg BW) in pterins and the measurement of DHPR activity in the blood. Missing PAH-D was defined as a decline of >29 % after 24 hours. hyperphenylalaninemia might delay diagnosis in patients with sepiapterin Results: In 199 patients with BH4-D and 341 with PAH-D half time (t 0 )of reductase and in some with autosomal recessive GTPCH deficiency. Treat- elimination of blood phe was evaluated. Patients with a defect in BH4 ment includes L-dopa, 5-hydroxytryptophan, and BH4 or low- synthesis (n0146) show a rapid decline of blood phe (t01.5 hours) whereas phenylalanine diet. Medication doses vary widely among patients. Experi- patients with BH4-D due to dihydropteridine reductase deficiency (n053) ences with dopamine agonists and monoamine catabolism inhibitors are have a much slower decline (8.5 hours). Patients with PAH-D show different limited. BH4 responsiveness in different countries (27-52 % responsive) (t0range 7-9 Conclusions: BH4 deficiencies are treatable pediatric neurotransmitter dis- hours). They cannot be differentiated from patients with dihydropteridine orders, characterized by motor dysfunction, mental retardation, impaired reductase deficiency. muscle tone, movement disorders and epileptic seizures. Although the out- Conclusion: Neonatal BH4 loading has the advantage of a well standard- comes are highly variable, early diagnosis and treatment are highly bene- ized condition . Rapid information about BH4 responsiveness is available ficial for the good outcome. before introduction of specific treatment, but further biochemical and molecular testing is still essential. S42 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-065 P-066 FOLLOW-UPAND OUTCOME OF PKU PATIENTS ON SAPROPTERIN/ INFLUENCE OF INDIVIDUAL PAH MUTATIONS ON SAPROPTERIN BH4 - PRELIMINARY RESULTS OF A RETROSPECTIVE RESPONSE IN PKU PATIENTS: RESULTS OF A SINGLE CENTER EUROPEAN MULTICENTER STUDY STUDY Keil S1 , Kern I2 , Anjema K3 , Van Spronsen F3 , Bosch A4 , Lambruschini Leuders S1 , Wolfgart E1 , van Teeffelen-Heithoff A1 , Vogelpohl L1 ,OttT1 , N5 , Couce ML6 , Burlina A7 , Cerone R8 , Riva E9 , Giovannini M9 , Meli Weglage J1 , Marquardt T1 , Feldmann R1 ,RutschF1 C10 , Feillet F11 , Lotz-Havla AS12 ,MuntauAC12 , Belanger-Quintana A13 , 1Dep Gen Pediatrics, Univ Child Hosp, Muenster, Germany Blau N1 1Univ Childr Hosp, Zürich, Switzerland It would be desirable to predict sapropterin response based on individual 2Hôpital des Enfants, Geneva, Switzerland phenylalanine hydroxylase (PAH) mutations in PKU patients. 3Univ Med Center, Groningen, Netherlands We investigated sapropterin response during two weeks of sapropterin 4University Hospital, Amsterdam, Netherlands therapy (1st week: 10 mg/kg per day, 2nd week: 20 mg/kg per day) in 5Hospital Sant Joan de Déu, Barcelona, Spain 101 PKU patients (age 4 to 44 years) in an outpatient setting. Response was 6University Hospital, Santiago de Compostela, Spain defined as a reduction in serum phenylalanine levels of more than 30 %. 7University Hospital, Padova, Italy PAH was sequenced conventionally in all patients, and individual mutations 8University of Genoa, Genoa, Italy were correlated with sapropterin response. 9Ospedale San Paolo, Milano, Italy 37 of 101 patients responded to sapropterin. The mutation Y414C was most 10University Hospital, Catania, Italy frequently associated with a response to sapropterin (present on 13 alleles in 11CHU Brabois - Hôpital des Enfants, Vandoeuvre les Nancy, France 37 responders). In the 64 non-responders, the mutation R408W (33 alleles) 12Dr. Von Hauner Children's Hospital, Munich, Germany and the mutation IVS12+1 G>A (17 alleles) were detected most frequently. 13U. Enferm Metab Hosp Ramón y Cajal, Madrid, Spain No mutation could clearly predict sapropterin response in our cohort, since most mutations were found in heterozygous state in non-responders and in Background: Sapropterin/BH4 is an approved drug for treatment of re- responders. Homozygosity for the Y414C genotype, however, was always sponsive PKU patients. The purpose of this study was to collect data on the associated with sapropterin response. Response to sapropterin therapy use of Sapropterin/BH4 and its long-term effect on metabolic control and cannot be predicted based on the presence of a single mutation on one patient-related outcome in five European countries. allele alone. Instead, the complete PAH genotype should be taken into Methods: A questionnaire was developed to assess quality of life (QOL), account. mood changes, adherence to diet, changes in blood Phe levels and Phe S. Leuders and E. Wolfgart contributed equally. tolerance and patients' phenotype and genotype. Conflict of Interest declared. Results: 129 patients from 11 European centers (42.5 % MHP, 49.6 mild PKU, 7.9 % classical PKU) were accessed (62 male, 67 female, age 2- 46 years). No adverse events on Sapropterin/BH4 were reported. Average dose of Sapropterin/BH4 was 10,45 mg/kg/day; range 5,15. Improvement in QOL was reported qualitatively in 52.3 % (31.3 % did not report); improvement in adherence to diet in 52.3 % (in 17.2 % question not applicable); improvement in adherence to treatment in 68.0 % (in 9.4 % question not applicable); improvement in mood was reported in 14.1 % (in 41.4 % question not applicable) of patients. Conclusion: Our data suggest a beneficial effect of sapropterin/BH4 in PKU patients who respond to oral administration of BH4 by lowering their blood Phe levels by >30 %: it increases daily tolerance for dietary Phe intake and improves dietary adherence and QOL. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S43

P-067 P-068 SECOND INTERIM ANALYSIS OF THE KUVAN® ADULT THE SAFETY OF SAPROPTERIN: SECOND INTERIM ANALYSIS MATERNAL PAEDIATRIC EUROPEAN REGISTRY (KAMPER): OF THE KUVAN® ADULT MATERNAL PAEDIATRIC EUROPEAN PATIENT CHARACTERISTICS REGISTRY (KAMPER) Burlina A1 , Trefz FK2 , Bélanger-Quintana A3 , Muntau AC4 , Lagler FB5 , Bélanger-Quintana A1 , Burlina A2 , Alm J3 , Trefz FK4 , Muntau AC5 , Feillet F6 , Vincent C7 , Alm J8 Feillet F6 , Champigneulle A7 , Lagler FB8 1University Hospital, Padova, Italy 1Hospital Ramon y Cajal, Madrid, Spain 2School of Medicine Univ Tuebingen, Reutlingen, Germany 2University Hospital, Padova, Italy 3Hospital Ramon y Cajal, Madrid, Spain 3Karolinska University Hospital, Stockholm, Sweden 4Ludwig-Maximilians-University Munich, München, Germany 4School of Medicine Univ Tuebingen, Reutlingen, Germany 5Paracelsus Medical University, Salzburg, Austria 5Ludwig-Maximilians-University Munich, München, Germany 6Hôpital d'enfants Brabois, Vandoeuvre les Nancy, France 6Hôpital d'enfants Brabois, Vandoeuvre les Nancy, France 7Merck Serono S.A, Geneva, Switzerland 7Merck Serono S.A, Geneva, Switzerland 8Karolinska University Hospital, Stockholm, Sweden 8Paracelsus Medical University, Salzburg, Austria

Objectives: Over 15 years, KAMPER aims to provide information on long- Objectives: KAMPER aims to provide information on the long-term out- term outcomes of approximately 625 sapropterin-treated patients with comes of approximately 625 sapropterin-treated patients with hyperpheny- hyperphenylalaninaemia due to phenylketonuria (PKU) or BH4 deficiency. lalaninaemia due to phenylketonuria (PKU) or BH4 deficiency, over Methods: Observational, multicentre drug registry, including a maternal 15 years. sub-registry. Methods: Observational, multicentre drug registry, including a maternal Results: At the 2-year interim analysis, six countries contributed a total of sub-registry. 203 patients (89 % [n0180] PKU; 11 % [n023] BH4-deficiency; male: Results: The 2-year interim analysis included data from 203 patients (89 % female ratio 50:50). Median(Q1–Q3) age was 10.1(7.0–15.0) years in PKU [n0180] PKU; 11 % [n023] BH4-deficiency). Median sapropterin daily patients and 12.7(6.1–19.1) years in BH4-deficiency patients. Median(Q1– dose was 10.4 and 5.0 mg/kg/day in PKU (n0154) and BH4-deficiency (n0 Q3) phenylalanine concentration (μmol/L) before sapropterin and at 17) patients, respectively. To date, 41 adverse events (AEs) have been 12 months, respectively, was 386(256–533;n0138) and 377(277–578;n0 reported in 26 patients; 34 AEs in 23 PKU patients, and 7 AEs in 3 BH4- 44) in PKU patients, and 91(67–442;n016) and 82(66–165;n04) in BH4- deficiency patients. The most common AEs were headache (5 events), deficient patients. In PKU patients, median(Q1–Q3) actual phenylalanine obesity (3), tonsillitis (2), cough (2) and tonsillectomy (2). Three AEs were intake (mg/day) before sapropterin and at 12 months was 595(310–1514;n0 considered possibly related to sapropterin (PKU: rhinitis, headache; BH4- 92) and 900(650–1739;n028), respectively. In PKU patients, at baseline deficiency: tic). One serious AE (SAE) was recorded in each patient group and 12 months, respectively, mean(SD) height Z-scores were -0.1(1.0;n0 (PKU: nephrolithiasis, unrelated to sapropterin; BH4-deficiency: haema- 153) and 0.1(1.0;n042); mean(SD) BMI Z-scores were 0.4(1.2;n0153) and temesis, unlikely related to sapropterin). All AEs and SAEs were mild to 0.6(1.2;n042). At baseline, osteopenia was detected in 3/41 patients aged moderate in severity. No AEs during three pregnancies and no develop- 12, 13 and 29 years and osteoporosis in 1 patient aged 17 years. mental problems in two newborns (one pregnancy still ongoing) have been Conclusion: Although results should be cautiously interpreted due to reported in the maternal sub-registry to date. limited available data at follow-up, KAMPER provides an opportunity to Conclusion: Consistent with clinical trial evidence, these results continue increase knowledge on the effect of sapropterin treatment including growth. to demonstrate a favourable safety profile for sapropterin. Conflict of Interest declared. Conflict of Interest declared. S44 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-069 A-003 WHAT IS THE OPTIMAL DURATION OF TETRAHYDROBIOPTERIN DIETETIC MANAGEMENT OF PREMATURE INFANT BORN AT 30 LOADING TEST IN PATIENTS WITH MILD PKU AGED OVER GESTATIONAL WEEKS WITH CLASSICAL PHENYLKETONURIA 8 YEARS ? Uudelepp ML1 , Vals MA2 , Kallas E2 , Päärson K3 , Joost K1 , Õunap K1 Gerard M1 , Homedan C2 , Simard G2 , Namour B3 , Jouault C1 , Colin E4 , 1Dpt of Genetics, Tartu Univ Hosp, Tartu, Estonia Bonneau D4 , Barth M4 2Children's Clinic of Tartu Univ Hosp, Tartu, Estonia 1Pediatric department, CHU Angers, Angers, France 3Dpt of Pediatrics, Univ of Tartu, Tartu, Estonia 2Metabolic biochemistry, CHU Angers, Angers, France 3Molecular biochemistry, CHU Nancy, Nancy, France Background: Management of premature phenylketonuria (PKU) patients is 4Genetic department, CHU Angers, Angers, France challenging due to absence of parenteral phenylalanine (Phe)-free solutions and scarce knowledge about Phe requirement, tolerance and safe plasma Background: In 1999, Kure showed that phenylketonuric patients with concentrations related to gestational age. Also the effect of high Phe level to residual phenylalanine hydroxylase activity can respond to tetrahydrobiopterin the premature brain is unknown. Until now only few cases about PKU treatment. Since, to screen responders, different loading test have been pro- treatment of premature infants have been reported in the literature. posed with duration between one day to one month. However, no protocol Case report: 11 days old boy with intrauterine hypotrophy born at 30 consider tetrahydrobiopterin loading test according to phenylketonuria (PKU) gestational weeks was diagnosed with classical PKU. Due to feeding phenotype. difficulties he was on parenteral nutrition until the age of 10 days and blood Objective: Determining the optimal duration of tetrahydrobiopterin loading Phe levels where extremely high on new born screening (1581,7 μmol/L on test in patients with mild PKU aged over 8 years. 4th day and 1799,8 μmol/L on 7th day) compared to average levels of PKU Methods: All patients with mild PKU, aged over 8 years followed in the babies born at term and on enteral feeding. With dietary treatment Phe pediatric departement of the university hospital of Angers, were treated by levels maintained <240 μmol/L after age of 17 days. Phe tolerance at the 20 mg/kg/d of sapropterin dihydrochloride during one month and assayed their corrected age of 40 gestational weeks was 60-70 mg/kg/day. We suggest, phenylalaninemia at day 1, 2 and every week during one month. Patient was that restricted Phe intake may play a role in altering weight gain and growth declared as responder when phenylalaninemia decreased by 30 % from even if caloric, total protein and tyrosine intake is appropriate. Early baseline. psychomotor development of this patient at the age of 5 months was Results: 21 patients have been included, 13 responded: 1 at 24 hours, 6 at normal. 36 hours, and 6 until 11 days. No patients responded after 11 days. The probability of BH4-responsiveness was about 5 % at 24 hours and 66 % at day 11. Conclusion: A tetrahydrobiopterin loading test of at least 11 days should be done before declaring a patient with mild PKU as non-responder. BH4 loading test lenght should be chosen according to PKU phenotype. A-004 INVESTIGATION INTO THE CONCENTRATION OF SERUM PRE-ALBUMIN AND TRACE ELEMENTS TO MONITOR THE A-002 NUTRITIONAL STATUS OF PHENYLKETONURIC AND HYPERPHENYLALANINEMIC PATIENTS HYPERPHENYLALANINAEMIA DUE TO PHENYLALANIN Prochazkova D1 , Kolbova L1 , Jarkovsky J2 , Vinohradska H1 , Konecna P1 , HYDROXYLASE DEFICIENCY: EVALUATION OF Dolezel Z1 TETRAHYDOBIOPTERIN RESPONSIVNESS IN PATIENTS 1Univ Hosp and Med Fac Masaryk Univ, Brno, Czech Republic FROM TURKEY 2Inst Biostat Analys Masaryk Univ, Brno, Czech Republic Kucukcongar A1 , Ezgü FS1 , Tümer L1 , Hasanoğlu A1 1Div Metab Dis, Univ Gazi, Ankara, Turkey Background: Phenylketonuria is an inherited disorder of metabolism of the amino acid phenylalanine caused by a deficit of the enzyme phenylalaninhy- Recent studies showed that patients with hyperphenylalaninaemia by phe- droxylase. It is treated with a low-protein diet containing a low content of nylalanine hydroxylase deficiency (HPA-PAH) can have serum levels re- phenylalanine to prevent mental affection of the patient. The objective of the duced when receiving oral tetrahydrobiopterin (BH4). present study was to assess the compliance of our phenylketonuric (PKU) and The aim of the study was the evaluation of BH4-responsivness in PKU hyperphenylalaninaemic (HPA) patients; to determine the concentration of patients. serum pre-albumin and trace elements to discover the potential correlation Methods: Thirtyfive patients were enrolled in this study (mean age is three between the amount of proteins in food and their metabolic control. years; 1 month-23 years) (Initial Phe levels were; above 20 mg/dl in 22 patients, Material and Methods: The prospective study contained altogether 174 10-20 mg/dl in seven patients, below 10 mg/dl in six patients). Diagnosis of patients, of which 113 were children, 60 with PKU and 53 with HPA and 61 BH4-responsivness was performed by oral BH4 loading test (20 mg/kg/day, in were adults, 51 with PKU and 10 with HPA. two days). According to the National Guidelines We defined BH4 responsive- Results: We did not prove a statistically significant difference in the levels ness as more then 30 % Phe concentration reduction within 24 hours. of serum pre-albumin, zinc and iron among the respective groups. We Results: Seven patients from moderate, two patients from classic, two proved statistically significant difference in the level of serum selenium patients from mild HPA group were responsive to the BH4. All responsive among PKU and HPA patients in adulthood (p00.006, Mann-Whitney U patients were under four years of age except two patients. The ratios of BH4 test). responsiveness are 9,9 % for classic PKU, 99 % for HPA (initial Phe level is Conclusion: Long-lasting dietary restrictions in patients with PKU and below 20 mg/dl) HPA increase the risk of late complications of dietary therapy such as Conclusion: BH4 therapy in patients with BH4 responsive PKU is at main- selenium deficiency. taining serum phenylalanine levels within normal control range. We found the most responsive patients were from the groups with Phe level below 20 mg/dl. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S45

A-005 A-007 CLASSIC PHENYLKETONURIA AND CHANGES IN IMMUNE BRAIN-DERIVED NEUROTROPHIC FACTOR LEVELS ARE RESPONSES: A SYSTEMATIC REVIEW DECREASED BY PHENYLALANINE ADMINISTRATION IN Kazemi sefat GE1 , Kazemi sefat NA2 , Talebi S3 , Talebi S4 BRAIN OF YOUNG RATS 1Genetics Dept,Islamic Azad Univ.(SRBIAU), Tehran, Iran, Islamic Republic of Schuck PF1 , Macan TP1 , Rodrigues LB1 , Scaini G1 ,FerreiraGC1 , Streck EL1 2Kerman University of Medical Sciences, Kerman, Iran, Islamic Republic of 1Universidade do Extremo Sul Catarinense, Criciuma, Brazil 3Avicenna Research Institute (ACECR), Tehran, Iran, Islamic Republic of 4Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic of Background: Accumulation of phenylalanine (Phe) in tissue and body fluids is the hallmark of phenylketonuria (PKU). Brain injury is a clinical Background: There is an increased susceptibility to infections in some characteristic of PKU patients, although the pathophysiology of this dam- patients with Phenylketonuria. Different mechanisms have been suggested age is poorly understood. for changes in humoral and/or cellular immunity in Phenylketonuria. Objective: The aim of this study was to measure brain-derived neuro- Objectives: A systematic review of all published articles was done in order trophic factor (BDNF) and nerve growth factor (NGF) levels in brain of to review the available literature on alterations of immune responses in rats submitted to an experimental model of PKU. Phenylketonuric patients. Material and Methods: Male 30-day-old Wistar rats received a single Methods: Search in PubMed with MeSH headings (such as "Phenylketo- subcutaneous injection of phenylalanine (Phe) (5.2 μmol/g) and/or p- nurias/immunology") and appropriate keywords was performed. The refer- chlorophenylalanine (p-Cl-Phe; 0.9 μmol/g), an inhibitor of phenylalanine ences of all identified relevant studies were searched for further potentially hydroxylase. Control group received saline solution at the same volume. related articles. One hour after the injection, cerebral cortex, striatum and hippocampus were Results: The computerized searches between years 1960-2012 yielded 12 isolated and BDNF and NGF levels were determined. articles of which 83 % were potentially appropriate for inclusion, and Results: The simultaneous administration of Phe and p-Cl-Phe diminished reference checking of these publications added another 8 citations. Of a BDNF levels in cerebral cortex and striatum. In addition, the administration total of 18 potentially suitable articles, four were about the autoimmune of only Phe decreased BDNF levels in all structures. Furthermore, p-Cl-Phe disorders which had no specific immunological assessment. No abstract or decreased these levels in striatum. On the other hand, NGF levels were not full-text were accessible for two of them. In the last 12 publications, 75 % altered in any structure tested. reported at least an alteration in acquired cellular and or humoral immunity. Conclusion: Considering that alterations of BDNF levels may impair In 33 % of publications there were some alterations in both acquired cellular memory, it is tempted to speculate that our results might be related to brain and humoral immunity. damage in PKU patients. Conclusion: Although there are few valid articles, it seems that classic Financial Support: L'Oreal/ABC/UNESCO and CNPq. Phenylketonuria may affect immunity in different ways. More controlled studies are necessary to validate this hypothesis.

15. Urea Cycle Disorders A-006 EFFECT OF SUPPLEMENTAL TREATMENT WITH SAPROPTERIN P-070 DIHYDROCHLORIDE ON EMOTIONAL AND BEHAVIORAL CASE OF KWASHIORKOR SUGGESTING UREA CYCLE FUNCTIONING IN CHILDREN WITH PHENYLKETONURIA (PKU) DISORDER 1 2 2 3 3 3 Chen D , Greenberg L , Graefe A , Jethva R , Legido A , Grant ML Behulova D1 , Fabriciova K2 ,BuzassyovaD3 , Szokeova A4 ,HolesovaD1 , 1 Dept Psych, Temple Univ, Philadelphia, United States Ostrozlikova M1 ,SebovaC1 , Skodova J1 , Salingova A1 , Pereckova J1 , 2 Dept Psych, Drexel Univ, Philadelphia, United States Syrova D1 3 Drexel Univ College Med, Philadelphia, United States 1Dept Lab Med, Univ Child Hosp, Bratislava, Slovakia 21st Dept Ped, Univ Child Hosp, Bratislava, Slovakia Background: Sapropterin dihydrochloride, synthetic form of BH4 (cofac- 3Dept Ped Anest Intens Med, Un Child Hosp, Bratislava, Slovakia tor for PAH enzyme), was approved in 2007 for supplemental treatment in 4Dept Ped, Univ Hospital, Martin, Slovakia Phe-responsive PKU individuals in USA, but there remains a paucity of published information regarding impact upon psychosocial outcomes. We report the first child of young Caucasian parents. The girl was on breast- Objectives: Comparison of behavioural/emotional and metabolic function- feeding during three months, afterwards milk formula was recommended. No ing in children treated with PKU-Diet or combination of Diet+Sapropterin. signs of failure to thrive or development delay were noticed. At the age of Methods: Retrospective chart review of children treated in PKU centre yielded 7 months, progressive decrease of consciousness and respiratory difficulties fourteen children (ages 6-14) diagnosed at birth and receiving routine care with appeared suddenly. Generalized oedema, hepatomegaly and very low plasma behavioural rating scales (Achenbach CBCL) and Phenylalanine (PHE) levels at protein concentration were found at the hospital. Because of persistent hyper- baseline (Time1) and one-year follow-up (Time2). Eight subjects remained on ammonaemia 65 – 160 μmol/L (CV<60), hepatopathy and unexplained PKU-diet between Times 1 & 2; six subjects on PKU-diet at Time1 responded to clinical course an inherited metabolic disease was also considered. Repeated and maintained supplemental treatment with Sapropterin. investigations revealed extremely high plasma glutamine 3313 and Results: No significant group differences for gender, age, Time1 PHE or 3840 μmol/L, moderately increased plasma glycine, serine , threonine, proline, CBCL Internalizing, Externalizing or Total Problems T-Scores at Times 1 or 2. histidine, orotic aciduria 22.1 μmol/mmol crea (CV<7.8) and decreased serum At Time2, Sapropterin+group had significantly lower PHE levels (p<.05), carnitine. Laboratory results indicated possible urea cycle disorder (UCD), and hierarchical regressions indicated that Sapropterin and PHE at Time2 were however several unusual findings required re-evaluation of medical history. significant predictors of behavioural change scores over time. Since fourth months of age the girl was fed mainly on a commercial rice drink Conclusion: Findings suggest improved metabolic functioning and reduced with 10-times lower amount of proteins than any milk formula contains. The behavioural/emotional symptoms over time in children treated with combina- diagnosis of kwashiorkor was established and treated effectively. We would tion of PKU-diet plus Sapropterin compared to children treated with diet alone. like to emphasize that severe protein malnutrition can develop in Central Conflict of Interest declared. Europe unexpectedly nowadays. Protein aversion might suggest a UCD. S46 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-071 P-074 SUCCESSFUL APPLICATION OF THE BIMDG 'PROSPECTIVE SINGLE CENTRE DIETARY EXPERIENCE OF LATE ONSET MANAGEMENT GUIDELINES' TO PREVENT NEONATAL MALE ORNITHINE TRANSCARBAMYLASE DEFICIENCY HYPERAMMONAEMIA IN INFANTS WITH CITRULLINAEMIA (OTCD) O'Sullivan S1 , Morris A A2 , Chakrapani A3 , Broomfield A1 , Grunewald Gribben J1 , Hallam P1 , Micciche A1 , van Wyk K1 , Walker R1 , Champion S1 , Leonard J V1 , Cleary M1 M2 , Mundy H2 , Rahman Y2 , Vara R2 1Great Ormond Street Hospital, London, United Kingdom 1N & D Dept, St. Thomas' Hosp, London, United Kingdom 2Royal Manchester Children's Hospital, Manchester, United Kingdom 2Ctr Inherited Met Dis, St. Thomas' Hosp, London, United Kingdom 3Birmingham Children's Hospital, Birmingham, United Kingdom Background: Ornithine Transcarbamylase deficiency (OTCD) is well Background: Without prospective management, citrullinaemia is associat- known to present outside the newborn period. ed with severe neonatal hyperammonaemia and a high morbidity and Objectives: To illustrate the variable phenotype and management of late mortality. The BIMDG 'prospective management of UCD' guidelines were onset male OTCD. introduced in 2008, from current best practice, to improve the emergency Methods: Retrospective review of all patients with late onset male OTCD management of patients. referred to our centre from 1998-2012. Case report: One of these guidelines was used to treat an infant at risk of Results: 19 male patients with late onset OTCD were identified. 11 patients citrullinaemia. The affected sibling had died on Day 3 of life with severe presented clinically, the majority with encephalopathy, at a median age hyperammonaemia. The current infant was immediately treated with intra- 6 years (range 0.8-50 years). One paediatric case resulted in marked venous 10 % glucose, sodium benzoate and phenylbutyrate as per guide- neurological deficit and 2 adult males with fatal outcome. Median protein line. Plasma citrulline at 12 hours was 359 μmol/L, and 2527 μmol/L on restriction was 1.3 g/kg/day (range 1.3-2) in paediatric presenting cases; day 7. Orotic acid excretion was markedly raised. He remained asymptom- 60 g/day (range 55 g/day – normal diet) in adults. atic with normal ammonia (consistently <80 μmol/l) and was discharged on The remaining 8 patients (age range 0-56 years) were identified following inves- day 11 taking a diet containing 2.1 g/kg/day protein, with arginine and tigation for a family history. 5 had a protein restricted diet, 2 of which were sodium benzoate. already self-selecting and 3 were prescribed a low protein diet based on bio- We know of two other cases of citrullinaemia in whom hyperammonaemia chemistry and symptoms. The remaining 3 patients continued on normal diets. was avoided in the neonatal period using this guideline. All patients were advised on an emergency regimen for use during illness. Conclusion: It is important to report outcomes from the clinical application Conclusion: We illustrate the variable age of presentation, protein tolerance of guidelines. We recommend further pooling of data to allow auditing the and outcome in late onset male OTCD. Early recognition and treatment is use of these and European guidelines, which are currently being formulated. essential for prevention of long-term sequelae.

P-077 COMPARISON OF TWO SODIUM PHENYLBUTYRATE GRANULE P-073 FORMULATIONS LONG-TERM OUTCOME AND INTERVENTION OF UREA Guffon N1 , Kibleur Y2 ,PickupE3 ,TissenC4 , Breitkreutz J4 , Pretorius A3 CYCLE DISORDERS IN JAPAN 1HFME Hosp, Metabolic Disease Centre, Lyon, France Endo F1 , Kido J1 , Matsumoto S1 , Mitsubuchi H1 , Nakamura K1 2Lucane Pharma, Paris 75011, France 1Dept Pediatrics, Kumamoto Univ, Kumamoto, Japan 3Parexel International, Bloemfontein, South Africa 4Institute of Pharmaceutics and Biopharma, Düsseldorf, Germany Urea cycle disorders (UCDs) are one of the most frequently inherited metabolic diseases in Japan. Here, we investigated the clinical manifesta- Background: Sodium phenylbutyrate (NaPB), a treatment for urea cycle tions, treatment, and prognosis of 177 patients with UCDs who were disorders, has an extremely unpleasant, bitter taste compromising compli- evaluated and treated from January 1999 to March 2009. In 2009, we sent ance and metabolic control. a questionnaire to 928 institutions, including the departments of paediatrics, Objectives: A new, taste-masked, coated-granule formulation is under devel- endocrinology and metabolism, neonatology, genetics, and transplant sur- opment and the taste characteristics, dissolution and bioequivalence, including gery, asking doctors if they had diagnosed or provided medical care to UCD safety and tolerability, of this form was compared to the licensed granule product. patients. The most common UCD was ornithine transcarbamylase deficien- Results: The in vitro taste profile of NaPB indicated a highly stimulant molecule. cy (OTCD), which accounted f or 116 out of 177 patients. This result is The new formulation released NaPB only after a lag-time of ~10s followed by a similar to a previous study performed between 1978 and 1995 in Japan: very slow release during several minutes compared with complete, immediate OTCD accounted for about two-thirds of the total number of UCD cases. release from licensed granules. Pharmacokinetic evaluation demonstrated bioe- We studied the relationship between prognosis and the peak blood ammonia quivalence of a 5g dose of both products in 13 healthy volunteers. No statistical level at the onset in 151 UCD patients. Compared with a previous survey difference was observed for maximal plasma concentration (Cmax) or area under conducted in Japan, we found that a greater number of patients survived the plasma concentration-time (AUC). Confidence intervals for Cmax and without any mental retardation despite their peak blood ammonia levels AUC0-¥ were between 0.80 - 1.25. Both products were well-tolerated. The new being greater than 360 μmol/l. The 5-y ear survival rate of patients with formulation was significantly more acceptable, less bitter and less salty (p<0.01). OTCD improved to 86 % for those with the neonatal-onset type and to 92 % Conclusion: In vitro and in vivo taste profiles indicate that the new formulation f or those with the late-onset type. granules can be swallowed before stimulation of taste receptors by NaPB. Moreover the new formulation is bioequivalent to the licensed product. The availability of the new formulation should improve compliance and efficacy of NaPB treatment. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S47

P-079 P-081 DETERMINATION OF OROTIC ACID IN URINE BY ION TWO CASES OF NEONATAL ONSET TYPE II CITRULLINEMIA CHROMATOGRAPHY DIAGNOSED BY URINE ORGANIC ACIDS BASED NEWBORN Zeydanli D1 , Destanoglu O1 , Cansever MS2 , Gumus Yilmaz G1 SCREENING 1Dep Of Chem, Fac Art&Sci, Ist Tech Univ, Istanbul, Turkey Watanabe Y1 , Tashiro K1 , Aoki K1 , Seki Y2 , Yanagi T2 , Okada J2 , 2Div Ped Nutr & Met, Cerr Med F, Ist Univ, Istanbul, Turkey Mizuochi T2 , Inokuchi T1 , Yoshino M2 , Matsuishi T2 1Research Institute of GC/MS, Kurume Univ, Kurume, Japan Determination of orotic acid in urine is crucial to diagnose some inherited 2Dept Pediatrics, Kurume Univ, Kurume, Japan diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. It is important to develop new methods for the determination of orotic acid Background: Citrullinemia type II caused by citrin deficiency, can manifest in urine samples. The study was aimed at developing chromatographic in newborns as neonatal intrahepatic cholestasis (NICCD). Symptoms associated conditions and pre-treatment method for reliable analysis of orotic acid in with NICCD including liver dysfunction and failure to thrive generally resolve urine. Samples were analyzed by ion chromatography / conductivity detec- within one year with appropriate treatment. Biochemical analytes including tor with a Dionex Ion Pac® AS20 analytical column and a Dionex Ion Pac® citrulline, arginine, methionine, phenylalanine, tyrosine, threonine, threonine/ AG20 guard column using gradient NaOH elution. Interferences from serine ratio, and galactose are often elevated in patients with NICCD. A pilot matrix in urine were removed by strongly cation exchange resin and study of urinary organic acid (UOA) based newborn screening has been solid-phase extraction after centrifugation and dilution. The calibration conducted since 1996 in the southwest area of Japan. We report pre symptom- curves showed good linearity in the ranges of 10-10000 μg/L with the r2 atic diagnoses in two cases of NICCD were made by UOA based NBS. values of 0.999. Under optimized condition, the limit of detection (S/N03) Case reports: Case 1 is a 2784 gram, term male infant. Blood and urine was found to be 0.7 μg/L. Significant results were obtained for various specimens were collected on age 4 days for NBS. A high urine 4- spiked urine samples with % recovery in the range of % 85-95. The hydroxyphenyllactic acid (4OH-PLA) was noted. Blood based NBS was advantages of the method described herein are: (i) it allows determining unremarkable. Case 2 is an 1808 gram, 33 2/7 weeks, preterm male infant. trace amount of orotic acid from human urine samples, (ii) the sample High 4OH-PLA and 4OH-phenylpyruvic acid levels were noted on urine preparation is overly simple, inexpensive and (iii) the test is not time specimens collected on age 18 days. A high blood galactose was noted on demanding and could be employed in routine analysis. age 21 days although blood NBS on age 5 days was unremarkable. Conclusions: Urine 4OH-PLA was elevated before the other biochemical markers were affected. UOA might be useful in early diagnosis of NICCD.

P-080 ABSENCE OF INCREASED URINARY OROTATE EXCRETION IN A PATIENT WITH ORNITHINE TRANSCARBAMYLASE (OTC) P-082 DEFICIENCY IN VIVO MONITORING OF UREA CYCLE WITH ORALLY Prunty H.1 , O'Sullivan S.1 , Krywawych S.1 , Turner C.2 , Abulhoul L.1 , 1 ADMINISTERED 13 C SODIUM ACETATE Grunewald S. 1 1 1 2 3 1 Zangerl K , Opladen T ,LindnerM , Marquardt T ,DasAM , Great Ormond Street Hospital, London, United Kingdom 1 2 Hoffmann GF WellChild Lab, Evelina Children's Hosp, London, United Kingdom 1Div Metab Dis, Univ Child Hosp, Heidelberg, Germany 2Div Metab Dis, Univ Child Hosp, Münster, Germany Background: OTC deficiency is the most common urea cycle disorder: the 3Div Metab Dis, Medical School, Hannover, Germany main biochemical features being hyperammonaemia with raised plasma glutamine, low citrulline and elevated urinary orotate. Background: Urea cycle disorders mostly present neonatally with se- Case: A male infant presented with progressive lethargy around 18 hrs vere hyperammonaemia and a high case fatality rate. Long-term prog- secondary to hyperammonaemia. Around 30 hrs he was commenced on IV nosis depends on the residual activity of the defective enzyme. An easy dextrose, Na-benzoate, Na-phenylbutyrate and arginine. Ammonia peaked and reliable in-vivo method to estimate urea cycle activity does not μ at 619 mol/L around 44 hrs and haemofiltration was required to normalise exist yet. Aim: of the study is the evaluation of oral 13 C-sodium his ammonia. Ultimately, his hyperammonaemia proved refractory to med- acetate loading for assessment of urea cycle function in 20 patients with ical therapy and he died aged 3 weeks. urea cycle disorders, 12 heterozygous mutation carriers and 10 healthy Results: Urinary orotate was 2 μmol/mmol creatinine (normal <5) just prior probands. to treatment, and normal orotate excretion was observed on four subsequent Methods: 13 C-labelled sodium acetate is applied orally in a single dose. occasions. At these times, ammonia and glutamine were not significantly 13 C urea in plasma and urine as well as the 13CO2 exhalation is measured elevated. Retrospective analysis showed significantly elevated plasma orotate for 2-4 hours. (38.6 μmol/L) corresponding with the peak ammonia concentration. The Results: So far, the method and the production of 13 C urea in plasma were genetic diagnosis of OTC deficiency was confirmed on day 8. The proband's examined in 10 patients with urea cycle disorders and 7 asymptomatic brother, who had the same mutation, had presented at day 4 with grossly carriers. μ elevated ammonia and urinary orotate of 2060 mol/mmol creatinine. Conclusion: Preliminary results suggest that in vivo monitoring of the urea Discussion: Urinary orotate measurement failed to confirm the diagnosis, cycle function is possible with 13 C-labelled sodium acetate and the method whereas plasma orotate during hyperammonaemic crisis was clearly abnor- may gain importance for the direction of clinical management in the future. mal. Given the difficulties in obtaining appropriately timed urine speci- Conflict of Interest declared. mens, plasma orotate measurement may be a useful adjunct in the differential diagnosis of hyperammonaemia. S48 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-099 P-101 ARGININOSUCCINATE LYASE DEFICIENCY - TWO CASES VARIABILITY OF ARGININOSUCCINATE LYASE DEFICIENCY PRESENTING AFTER SURGICAL INTERVENTION FOR CAN BE CAUSED BY SPLICE VARIANTS RESULTING IN UNRELATED CONDITIONS HETEROTETRAMER FORMATION Griffith C1 ,CooperJ1 ,GallC2 ,HartC1 , Sharma R2 , Smith G1 ,WalterJ1 , Hu L1 , Eggimann S2 , Pandey A3 , Nuoffer JM2 , Häberle J1 Wu H1 1Div Metab, Univ Child Hosp & Res Center, Zurich, Switzerland 1Central Manchester Foundation Trust, Manchester, United Kingdom 2Inst it Clin Chem, Berne, Switzerland 2Salford Royal Foundation Trust, Manchester, United Kingdom 3Univ Child Hosp, Berne, Switzerland

Argininosuccinate lyase deficiency (ASL) is a urea cycle disorder, causing Background: Argininosuccinate lyase (ASL) is a homotetrameric cytosolic hyperammonaemia and developmental delay. Acute presentations in the urea cycle enzyme cleaving argininosuccinate into arginine and fumarate. neonatal period occur but it frequently presents chronically at a later age. ASL deficiency (ASLD) results in argininosuccinic aciduria with a wide We report two patients presenting at opposite ends of the age spectrum, both clinical variability ranging from asymptomatic or mild to severe life- subsequently diagnosed with ASL after unrelated surgical intervention. threatening. The molecular basis of this variability is poorly understood. Case one is the first child of Pakistani parents, delivered by spontaneous vaginal We investigate whether ASL transcript variants contribute to the known delivery at 34 weeks after a pregnancy complicated by polyhydramnios and variability based on the published frequent occurrence of alternative exon found to have jejunal perforation. A portion of necrotic bowel was removed splicing at the ASL locus. surgically and recovery was not straightforward but relatively unremarkable until Methods: Cloning of the most common ASL transcript variants affected by a clinical deterioration at 12 days of age. Investigations for hypotonia, marked deletion of exon 2 or 7 was performed to express the mutant ASL proteins in hyperammonaemia and encephalopathy gave a diagnosis of ASL. human embryonic kidney cells (293 T) followed by western blot analysis. The Case two is a 59 year old female with a history of mild learning difficulties and residual ASL activities were photometrically determined after expression or co- seizures who underwent elective surgery for rectal carcinoma. Postoperative expression of ASL wild type, exon 2- or 7-deleted mutant proteins, respectively. recovery was poor and abnormal EEGs were noted. Raised ammonia and Results: The exon 2-deleted ASL mutant protein is stable but has no encephalopathy initiated metabolic investigations which diagnosed ASL. The activity. It can form a truncated heterotetramer with ASL wildtype after co- patient subsequently deteriorated and died, despite a peak ammonia concentra- expression showing reduced activity. Exon 7-deleted mutant protein is prob- tion of only 141 μmol/L and aggressive treatment including haemofiltration. ably instable and has, thus, no effect on ASL wild type activity if co-expressed. These unusual presentations highlight the importance of raising awareness Conclusion: ASL transcripts might contribute to the phenotypic variability of metabolic conditions that may be unmasked by surgical intervention for of ASLD through formation of a truncated heterotetramer with wild type unrelated conditions. causing reduced ASL activity.

P-100 EXPRESSION OF NATURALLY OCCURRING ARGININOSUCCINATE LYASE MUTANTS IN 293 T CELLS IMPROVES THE CORRELATION OF RESIDUAL ENZYME ACTIVITY WITH THE CLINICAL P-102 PHENOTYPE Balmer C1 ,HuL1 , Eggimann S2 , Pandey A3 , Wettstein V1 , Nuoffer JM2 , UROLITHIASIS IN TWO INFANTS WITH CITRULLINEMIA 1 2 2 2 Häberle J1 Öztürk Y , Torun Bayram M , Soylu A , Kavukçu S 1 İ 1Div Metab, Univ Child Hosp & Res Center, Zurich, Switzerland Div Metab Dis, Dokuz Eylul Univ, zmir, Turkey 2 İ 2Inst it Clin Chem, Berne, Switzerland Dokuz Eylul Univ. Div Ped. Neph, zmir, Turkey 3Univ Child Hosp, Berne, Switzerland Citrullinemia is a rare inborn error of urea cycle metabolism caused by a Background: Argininosuccinate lyase deficiency (ASLD) caused by muta- deficiency of argininosuccinate syn-thetase. Many anomalies and organ tions in the ASL gene is a rare autosomal recessive urea cycle disorder with disease have been reported in surviving citrullinemia cases. However, only variable biochemical and clinical phenotype. The limited prediction of ASL one neonatal case with urolithiasis has been reported so far. mutants associated with mild ASLD can be partly explained by insufficient Both male patients presented in the newborn period with the symptoms of sensitivity for detection of residual ASL activity. To achieve an improved hypotonia, tachypnea, and difficulty in feeding. Metabolic screen showed eukaryotic expression system with a more sensitive ASL assay, we estab- severely elevated ammonia and citrulline levels in both patients (1387 and μ μ lished the ASL measurement in human embryonic kidney cells (293 T). 934 mol/L; 1292 and 1142 mol/L). The patients were treated by perito- Methods: Construction of 11 ASL mutants associated with mild or asymp- neal dialysis and sodium benzoate, L-arginine and low protein diet. During tomatic ASLD (c.35 G>A, c.91 G>A, c.283 C>T, c.299 T>C, c.532 G>A, follow-up, both patients developed urinary tract infections at 8 and c.566A>G, c.577 C>T, c.1003 G>T, c.1135 C>T, c.1153 C>T, c.1334 G>C) 12 months of age. Urinary system imaging demonstrated radioluscent and of 2 severe ASL mutants (c.857A>G, c.1154 G>T) as well as the benign nephrolithiasis in both cases. Analysis of the stone was consistent with uric polymorphism c.1164 C>T (as positive control) were established to express acid stone. Metabolic evaluation for urolithiasis was normal except low uric ASL mutant proteins in 293 T cells. Their residual activities in whole cell acid levels (1.3 and 1.4 mg/dL) and high fractional excretion of uric acid extracts were photometrically determined and compared to wild type activity. (27.1 % and 30.6 %). Results: 7/11 variant ASL mutants showed significant residual activities The patients have low serum uric acid along with high fractional uric acid (>5 % of wild type) while the remaining four yielded levels≤2 % of wild excretion. In addition, stone analysis showed uric acid stone in one patient. type activity which was also the case for the severe ASL mutants. Thus, we emphasize the association of the disease with urolithiasis, espe- Conclusion: The in-vitro expression of ASL mutants in 293 T cells cially uric acid stones, in cases who survive the neonatal period. improves the correlation of residual ASL enzyme activity with the phenotype in variant ASLD. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S49

P-103 P-105 ARGININEMIA: A RARE TREATABLE CAUSE OF SPASTIC HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS AND LYSINURIC PARAPARESIS PROTEIN INTOLERANCE: CASE REPORT Kendirci Mustafa1 , Kardas Fatih1 , Soylu Pembe1 , Gumus Hakan2 , Per Baronio F1 , Cantarini ME2 , Bal M1 , Martini AL1 , Bettocchi I1 , Pession Huseyin2 , Poyrazoğlu Hatice Gamze2 , Canpolat Mehmet2 A1 , Cassio A1 1Ped Nutr and Met Dept, Erciyes Univ, Kayseri, Turkey 1Neon Screen Centre Dept of Pediatrics, Bologna, Italy 2Ped Neurology Dept, Erciyes Univ, Kayseri, Turkey 2Ped Hematol-Oncol, Dept of Pediatrics, Bologna, Italy

Background: Argininemia is a rare, autosomal recessive, inborn metabolic Background: lysinuric protein intolerance (LPI) is an autosomal recessive disorder due to arginase deficiency. Estimated prevalence of the disorder is hereditary aminoaciduria (SLC7A7 gene) with heterogeneous clinical pic- 1/363.000-2000000 individuals. Clinical manifestations of the disease are ture. It is described the association with hemophagocytic lymphohistiocy- different from other urea cycle defects. Most patients with argininemia tosis (HLH). exhibit insidious onset and progression of neurologic manifestations such Case report: we report the case of a Chinese child, son of non- as spastic paraparesis, mental retardation, failure to thrive and seizures. consanguineous parents referred to us for severe hepatosplenomegaly. Case report: A four years-old male, the second child of consanguineous Biochemical tests show: hypertriglyceridemia (1037 mg /dL), high LDH parents, was admitted to our hospital because of psychomotor retardation, (2357 U / L), high serum levels of Interleukine 2 receptor (2683 IU / mL) difficulty of walking and progressive tiptoeing. Physical examination and hyperferritinemia (486 ng / mL). Bone marrow aspirate shows hemopha- revealed increased tonus of lower extremities, tiptoeing and mental retar- gocytosis with histiocytes and immature granulocytes. Interstitial lung hilar dation. Laboratory investigations revealed mildly elevated hepatic enzymes tumescence was present at chest x-ray. The aminoacidogram shows: increased and plasma arginine concentration (454 micromol/L, normal range 0-100), urinary Citrulline, Ornithine, Lysine, with hypercitrullinemia. Urinary orotic increased blood ammonia level (38.4 micromol /L, normal range 11.2-35.4) acid was markedly elevated. Molecular analysis of the gene SLC7A7 shows and increased urinary excretion of arginine and orotic acid. Analysis of compound heterozygosity for 1673delG, and IVS4 +1 G>A mutations, con- ARG1 gene revealed a c.231 C>A homozygote mutation, but in vivo firming the suspicion of LPI. The child started hypoproteic diet and treatment effects of the mutation have not yet been characterized. with citrulline, lysine, and sodium phenylbutyrate. During follow-up (6 years) Conclusion: Argininemia is a rare treatable cause of spasticity. Low protein the patient did not show hyperammonaemia . Splenomegaly, hypertriglicer- diet, nitrogen scavenging medications decrease arginine and ammonia idemia slight hyperferritinemia and elevated LDH are still present. levels and their unwanted effects.It should be suspected more frequently Conclusion: our case shows the importance to suspect LPI in patients with in the differential diagnosis of slowly progressive neurologic manifestations HLH. Citrulline treatment seems to be sufficient even for the control of HLH. especially progressive spastic paraparesis.

P-104 P-106 MISDIAGNOSED BIPOLAR DISORDER TREATED WITH SODIUM UNUSUAL PHENOTYPIC PRESENTATION IN CITRULLINEMIA DIVALPROATE (DEPAKOTE .) REVEALING A LATE-ONSET TYPE I (CTLN1) ORNITHINE TRANSCARBAMYLASE (OTC) DEFECT Eiroa H.1 , Bay L.1 , Häberle J.2 MESLI S1 , Mousset-Hovaere M2 , Robert S2 , Lamireau D3 , ,Bonnefont JP 1Serv. de Err. Cong. Metab, Htal Garrahan, Buenos Aires, Argentina JP4 , Rabier D5 , Cousin A1 , Balestrat S1 , Bertuletti B1 , Laucher MH1 , 2Division of Metabolism, Univ. Child.Htal, Zurich, Switzerland Redonnet-Vernhet I1 1Lab de Bioch, Pellegrin, CHU Bordeaux, BORDEAUX, France Background: Initial symptoms of CTLN1 are frequently neurologic dete- 2Réa Médico-chirurg, CHG, DAX, Dax, France rioration with mild liver disease.We report a case of recurrent liver failure in 3Pédiatrie, Unité MHM,CHU de Bordeaux, Bordeaux, France a 2 year old patient with CTLN1 without neurological involvement. 4Génétique Moléculaire, Hôp Necker,enf M, Paris, France Case report: 22 month-old first child of a non-consanguineous couple. 5Service de Bioch Métabol, Hôp Necker, Paris, France First symptoms at 18 months were vomiting during infection with signs of liver failure:AST 2200U/L,ALT 2300U/L,Prothrombin Time(PT)17 %, ac- Background: OTC deficiency is the most common inherited defect of the tivated Partial Thromboplastin Time(aPTT)94 sec but no acidosis and urea cycle, transmitted as a partially dominant X-linked trait. The most severe normal glucose.Symptoms improved after a high glucose infusion. He form presents almost exclusively in males as acute neonatal hyperammonemic was referred to our center for evaluation of liver transplantation. Since lab coma. Late-onset OTC deficiency is very rare in male patients. result were improved(PT 53 %, aPTT 59 sec,AST 920U/l,ALT 862U/l, Case report: we describe a 55-year-old man who has been diagnosed as a γGT 135U/l, normal ammonia and no encephalopathy),the patient was bipolar disorder and consequently treated with Depakote® for 1 year. After discharged with no diagnosis with follow up as an outpatient.3 months a protein-rich meal, he presented an unexplained coma. Elevated blood later, vomiting liver dysfunction recurred: TP 13 %, ammonia 63 mg/dl, ammonia in a context of sodium divalproate treatment led us to suspect an plasma citrulline 1711 μmol/l, plasma arginine 13 μmol/l, and high urine urea cycle disorder. An ornithine transcarbamylase deficiency (OTC) was orotic acid.With a suspected diagnosis of citrullinemia type I,treatment was first suspected by plasma amino acid profile and high urinary orotic acid started with diet and drugs and good outcome with normalization of liver excretion. Despite aggressive therapy, the patient rapidly developed fatal function. Diagnosis was confirmed by detection of the ASS1 gene muta- cerebral edema. OTC deficiency was confirmed by a decreased hepatic tions on exon13: c.970 G>A(p.Gly324Ser) and on exon15: c.1168 G>A enzyme activity. Molecular analysis of the OTC gene showed that (p.Gln390Arg) on each one parental allele. Retrospectively, in his neonatal the patient was hemizygous for a new mutation c.214 G>A (p.E72K). screening card, he already had elevated citrulline. Conclusion: we present a case of a late-onset OTC deficiency in a male patient Conclusions: CTLN1 may rarely present with acute hepatocellular dys- receiving valproic acid therapy in a misdiagnosed bipolar disorder. Clinicians function without neurological involvement. and psychiatrists in particular should be aware of atypical psychiatric symptoms and subtle organic signs that are suggestive of an urea cycle defect. S50 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

A-008 16. Homocysteine THE MAJOR METABOLITES ACCUMULATING IN HYPERORNITHINEMIA-HYPERAMMONEMIA- P-075 HOMOCITRULLINURIA SYNDROME COMPROMISE BRAIN HOMOCYSTEINE, FOLATE AND VITAMIN B12 IN PORTUGUESE REDOX HOMEOSTASIS IN VIVO CHILDREN: GENETIC DETERMINANTS AND REFERENCE Viegas CM1 , Tonin AM1 , Busanello ENB1 , Zanatta Â1 , Moura AP1 , INTERVALS Agostini C1 , Leipnitz G1 , Wajner M1 Caldeira Araújo H1 , Pimenta A1 , Barroso M2 , Rivera I2 , Castro R2 , 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil Tavares de Almeida I2 1Unit Medical Sciences, Univ Madeira, Funchal, Portugal Ornithine, ammonia and homocitrulline are the major metabolites accumulating 2iMed.UL, Univ Lisbon, Lisbon, Portugal in hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome, an inherited disorder characterized by neurological dysfunction whose pathogenesis Hyperhomocysteinaemia is a well-established risk factor for vascular disease. is still not understood. We investigated the in vivo effects of intra cerebroven- B vitamins status and polymorphisms in genes coding for key enzymes of tricular administration of ornithine or homocitrulline in young rats submitted to homocysteine metabolism are predictors of total homocysteine (tHcy) plasma hyperammonaemia by intraperitoneal urease treatment on important parameters concentrations. Limited data are available about the influence of these factors of oxidative stress in cerebral cortex. Ornithine increased thiobarbituric acid- on tHcy in pediatric populations. Therefore, we evaluated plasma tHcy, folate reactive substances (TBA-RS) levels and carbonyl formation, without altering and vitamin B12 levels in a healthy population of 9-year-old children (n0195; sulfhydryl content and reduced glutathione (GSH) levels. However, the combi- 107 males and 88 females) from Madeira Island, Portugal, with the aim of nation of hyperammonaemia with ornithine resulted in a significant decrease of proposing reference values for these biomarkers. Furthermore, we investigated sulfhydryl levels and GSH concentrations, highlighting a synergistic effect of the role of genetic determinants on metabolites concentrations. ornithine and ammonia. Furthermore, homocitrulline caused increases of TBA- The studied group revealed a mean plasma tHcy concentration of 4.55 and RS values and carbonyl formation, as well as a decrease of GSH concentrations 4.63 μmol/L for males and females, respectively (95 % CI 4.36-4.75 and without altering sulfhydryl content. Finally, we observed that urease treatment 4.37-4.88) quite low when compared with adolescents and adults, reinforc- per se was able to enhance TBA-RS levels. Our data indicate that the major ing the idea that tHcy plasma levels are age-dependent. Mean folates were metabolites accumulating in HHH syndrome provoke lipid and protein oxidative 22.88 and 22.75 nmol/L (95 % CI 21.00-24.76 and 20.62-24.87 nmol/L) damage and reduce the antioxidant defences in the brain. Therefore, it is and total B12 vitamin 535.73 and 518.73 pmol/L (95 % CI 491.62-579.84 presumed that oxidative stress may represent a pathomechanism involved in and 483.26-554.20) for males and females, respectively. Differences be- the brain damage found in patients affected by this disease. tween gender were not statistically significant (p>0.05). Supported by: CNPq, PROPESq/UFRGS, FAPERGS, FAPESP, PRONEX, MTHFR-677 C>T, MTHFR-1298A>C and MTR-2756A>G genotypes FINEP IBN-Net. did not affect plasma tHcy and folate levels significantly. Moreover, vitamin B12 did not differ between TCN2-776 C>G or TCN2-66 C>G genotypes. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S51

P-083 P-085 PLASMA METABOLITE PROFILES IN ELEVEN CHILDREN HUMAN ENDOTHELIAL CELLS HYPOMETHYLATION, INDUCED DIAGNOSED WITH COMBINED METHYLMALONIC ACIDEMIA BY THE HOMOCYSTEINE PRECURSOR, UP-REGULATES ICAM-1 AND HOMOCYSTINURIA, COBALAMIN C TYPE, ON NEWBORN EXPRESSION SCREENING AND TREATED SINCE BIRTH Florindo C1 , Barroso M1 , Tavares de Almeida I1 , Castro R1 Weisfeld-Adams JD1 , Bottiglieri T2 , Young SP3 , Diaz GA1 1Metabolism and Genetics Group, iMed.UL, Lisbon, Portugal 1Mount Sinai School of Medicine, New York, NY, United States 2Baylor Research Institute, Dallas, TX, United States Hyperhomocysteinemia (HHcy) causes atherosclerosis but the underlying 3Duke University Medical Center, Durham, NC, United States mechanisms are not fully understood. The homocysteine precursor, S- adenosylhomocysteine (AdoHcy), accumulates in the setting of HHcy, and is Combined methylmalonic aciduria and homocystinuria, cblC type, results from a strong inhibitor of most cellular methyltransferases. Inhibition of cellular defective production of methylcobalamin and adenosylcobalamin, essential co- methylation reactions by AdoHcy has been suggested to contribute to factors for methionine synthase, and methylmalonyl-CoA mutase, respectively. homocysteine-induced vascular toxicity. Early-onset cblC is associated with disordered white matter development on ICAM-1 (inter cellular adhesion molecule-1) is a cell adhesion molecule, MRI, and developmental delays and visual difficulties are common, even in which actively participates in atherogenesis by facilitating leukocyte endo- treated patients. The underlying biochemical basis to these abnormalities may thelial transmigration. Here we investigate whether intracellular accumula- be multifactorial and has not been fully elucidated. We measured fasting plasma tion of AdoHcy impacts endothelial ICAM-1 expression. metabolite levels in young children (11 patients, 18 samples) treated from birth Our study was conducted in human umbilical vein endothelial cells, in which for molecularly-confirmed cblC. Mean±standard deviation values for these we supplemented culture media with a methylation inhibitor. This pharmaco- metabolites were homocysteine 43.7±12.7 μmol/L (normal <15), methionine logical approach has been shown by us to increase intracellular AdoHcy 26.1±10.8 μmol/L (normal 14-46), S-adenosylhomocysteine 40.5±21.4 nmol/ accumulation and foster cellular hypomethylation.1 Using flow cytometry, L (normal 13.2-18.2), S-adenosylmethionine 149.4±71.4 nmol/L (normal 33.0- we measured the levels of ICAM-1 expression in our cell model, before and 94.8), methylmalonic acid 2.6±1.4 μmol/L (normal <0.6), guanidinoacetate after 72 h of incubation in control or in supplemented medium. Our results (GAA) 0.9±0.2 μmol/L (normal 0.1-1.7), creatine 51.8±17.9 μmol/L (normal showed that, under hypomethylation, the levels of surface ICAM-1 increased 3-114). Plasma GAA has been previously reported as markedly abnormal in 3-fold, when compared to the control. In addition, this increase was even more cblC, but consistently normal levels on LC-MS/MS testing in our cohort pronounced after cell membrane permeabilization. suggest mechanisms other than elevated GAA are responsible for neurotoxicity In conclusion, our study shows that AdoHcy accumulation induce endothe- and developmental delay in treated cblC patients. We suggest that quantification lial activation trough ICAM-1 expression up-regulation. of these metabolites in cerebrospinal fluid (CSF) may better characterize This work was supported by FCT-Portugal (PTDC/SAU-ORG/112683/2009). neuropathological processes in cblC. 1 - Amino Acids. 2012;42(5):1903-11.

P-086 P-084 DIET-INDUCED HYPERHOMOCYSTEINEMIA IN RATS AFFECTS INVESTIGATION OF OXIDATIVE STRESS IN HOMOCYSTINURIC PROTEIN ARGININE METHYLATION IN A TISSUE-SPECIFIC PATIENTS BEFORE AND DURING THE TREATMENT MANNER 1 1 2 2 1 Vanzin CS , Biancini GB , Sitta A , De Souza CFS , Wyse ATS , Esse R1 , Imbard A2 , Rocha MS3 , Teerlink T3 , Castro R1 , Tavares de 1 1 Wajner M , Vargas CR Almeida I1 , Blom HJ3 1 PPG Bioquímica - UFRGS, Porto Alegre, Brazil 1iMed.UL, Univ Lisbon Fac Pharm, Lisbon, Portugal 2 Serviço de Genética Médica - HCPA, Porto Alegre, Brazil 2Servic Bioch-Hormon, Hôpit Robert Debré, Paris, France 3Dept Clin Chem, VU Univ Medic Center, Amsterdam, Netherlands Homocystinuria is an inherited error of metabolism caused in most cases by β deficiency of cystathionine -synthase. As result of enzymatic deficiency, Elevated homocysteine (Hcy) induces S-adenosylhomocysteine (AdoHcy) occur accumulation of homocysteine and methionine in biological fluids. build-up, which supposedly fosters cellular hypomethylation. We aimed to Recent studies demonstrate that the oxidative stress may be involved in determine whether diet-induced hyperhomocysteinemia (HHcy) elicits tis- pathophysiology of various inherited errors of metabolism. Studies in sue protein arginine hypomethylation. animal models have been shown a relationship between Hcy and oxidative During 2 weeks, 8-10 Wistar rats were fed: standard chow (control); a stress, but scarce studies exist evaluating the oxidative stress in homocys- methionine-enriched diet (HM); a diet deficient in B vitamins (LV); or a high tinuric patients. Therefore, the aim of this study was to evaluate parameters methionine, low B vitamins diet (HMLV). Tissue S-adenosylmethionine (Ado- of oxidative stress in plasma of homocystinuric patients at diagnosis and Met) and AdoHcy levels were measured by LC-MS/MS. Plasma total Hcy was during treatment. We found a significant increase of carbonyl groups and measured by HPLC, as well as protein-incorporated L-arginine and asymmetric malondialdehyde levels, as well as a reduction of sulfhydryl groups in (ADMA) and symmetric (SDMA) dimethylarginines in liver, heart and brain. plasma of homocystinuric patients at diagnosis relative to controls. Patients HHcy was induced in the HM, LVand HMLV groups. Those diets didn't affect under treatment presented a significant reduction of the content of malon- AdoMet levels, but significantly increased AdoHcy concentrations in liver (LV dialdehyde, Hcy and methionine concentrations relative to patients at diag- and HMLV groups) and in heart (HMLV group), but not in brain. Interestingly, nosis. Furthermore, a significant negative correlation between sulfhydryl ADMA levels were significantly decreased in heart (less 7.5±1.0 % and 9.3± group content and Hcy levels was demonstrated and a significant positive 1.0 % in the LVand HMLV groups, respectively) and in brain (less 9.6±0.8 % correlation between malondialdehyde and Hcy levels. Taken together, our and 13.2±1.1 % in the LV and HMLV groups, respectively) but not in liver. data indicate that oxidative stress may play an important role in pathophys- SDMA levels did not change between diets in all tissues. iology of homocystinuria, probably due to high Hcy concentrations found We conclude that diet-induced HHcy disturbs protein arginine asymmetric in these patients. dimethylation in a tissue-specific manner. This study was supported by the FCT (SFRH/BD/48585/2008; PTDC/ SAU-ORG/112683/2009). S52 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-087 P-089 SUCCESSFUL PRENATAL AND POSTNATAL TREATMENT IN IS S-ADENOSYLMETHIONINE S-ADENOSYLHOMOCYSTEINE THE COBALAMIN C (CBLC) DEFECT RATIO IMBALANCE INDUCED BY HYPERHOMOCYSTINEMIA Trefz F1 , Scheible D2 ,HuemerM3 ,SuomalaT3 ,FowlerB4 , Baumgartner M3 COMPENSATED IN BRAIN? 1MVZ Kreisklinik. Reutlingen, Gammertingen, Germany Imbard A1 , Esse R2 , Rocha M2 , Barto R2 , Schlemmer D1 , Benoist JF1 , 2Kinderklinik Kreiskliniken, Reutlingen, Germany Blom HJ2 3University Children's Hospital Zürich, Zürich, Switzerland 1Biochemistry, Robert Debré Hosp, APHP, Paris, France 4Universitäts-Kinderspital beider Basel, Basel, Switzerland 2Metab Lab, VUmc, Amsterdam, Netherlands

Background: Despite early diagnosis treatment success in CblC deficiency Background: S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine is suboptimal. (AdoHcy) are considered to reflect the methyl donor balance in tissues. Meth- Objective: To demonstrate that prenatal together with postnatal treatment ylation is particularly crucial in the brain for myelin, neurotransmitter and may prevent cerebral damage and result in a normal outcome. phospholipids synthesis. The aim of this study was to measure AdoMet and Method: Biochemical and enzymatic prenatal diagnosis was performed in a AdoHcy in brain and liver in models of diet induced hyperhomocystinemia. pregnancy at risk for the CblC defect in a mother with a severely affected Material and methods: Female Wistar rats were fed with different diets child with cblC and multiple developmental problems despite treatment from inducing hyperhomocysteinemia (low vitamin diet or high methionine diet, early infancy. After confirmation of CblC in the fetus the mother was treated or their combination and control diet). Mean plasma homocysteine concen- from 15 weeks of gestation with intramuscular OH-Cbl (3x10 mg/week) and trations were 83.8, 34.8, 22.7 and 4.8 μM respectively. We measured folic acid (5 mg/day). Postnatal treatment consisted of betaine 200 mg/kg BW/ AdoMet and AdoHcy in liver and brain homogenates by LC-MS/MS. day, folic acid 5 mg/day and intramuscular OH-Cbl 1 mg/day. Results: In liver of rats fed with high methionine the AdoMet/AdoHcy ratio Results: Pregnancy and delivery were normal, the infant healthy, without birth did not change. The groups fed with low B-vitamins (with or without high defects. Psychomotor development was in the normal range, the child is now methionine) had significantly lower liver AdoMet/AdoHcy ratios than seven years of age and attends the normal school. Methylmalonic acid excretion controls (p00.001, p<0.001). In brain this ratio was not influenced by all over the years was below 60 mmol/mol creatinine (normal <20) and serum these different diets. homocysteine between 40-60 μmol/l (normal <12). Both affected children have Conclusion: Among tche different diets inducing hyperhomocysteinemia, the same enzymatic defect and the same genotype (p.Arg91Lysfs*/p.Arg153*). only those with low B-vitamins resulted in an impaired methyl donor imbal- Conclusion: Early and adequate treatment with a high dose OH-Cbl during ance in liver. However, the methylation ratio in brain was not influenced pregnancy may prevent intrauterine damage in CblC deficiency. despite a 5-10 fold increased homocysteine. We would like to hypothesise that methylation in brain is very well regulated via compensatory mechanisms.

P-090 P-088 PLASMA CBS ACTIVITY IN VITAMIN B6 RESPONSIVE AND BETAINE DEFICIENCY IS INDUCED BY DIFFERENT FORMS OF NON-RESPONSIVE HOMOCYSTINURIA HYPERHOMOCYSTEINEMIA: AN ARGUMENT FOR BETAINE Kozich V1 ,KrijtJ1 ,VlaskovaH1 , Zeman J1 ,JesinaP1 ,AlcaideP2 ,Ruiz-Sala SUPPLEMENTATION P2 , Merinero B2 ,KormanSH3 Imbard A1 , Barto R2 , Schlemmer D1 , Esse R2 , Benoist JF1 , Blom HJ2 1Inst Inher Metab Dis, General Univ Hosp, Prague, Czech Republic 1Biochemistry, Robert Debré Hosp, APHP, Paris, France 2Centr Diagn Enf Mol, Univ Autonom Madrid, Madrid, Spain 2Metab Lab, VUmc, Amsterdam, Netherlands 3Hadassah-Hebr Univ Med Centr, Jeruslaem, Israel

Background: Betaine is an essential osmolyte and a methyl donor in Background and objectives: We have shown previously that cystathionine homocysteine remethylation. We studied betaine and dimethylglycine lev- beta-synthase (CBS) is released into circulation. In the present study we els in rats with hyperhomocysteinemia induced by various diets. examined whether CBS activity in plasma correlates with pyridoxine re- Material and methods: Hyperhomocystinemia was induced in female Wistar sponsiveness and genotype in patients with CBS deficiency. rats with diet: low B-vitamin diet, high methionine diet, low B-vitamin and Patients and methods: The LC-MS/MS based method of CBS activity high methionine diet; N08 per group compared to standard diet (N020). determination in plasma (Krijt et al, JIMD 34, 2011:49-55) was used in two Mean plasma homocysteine concentrations were 83.8, 34.8, 22.7 and centers with some modification. Center A analyzed 67 controls (median activity 4.8 μM respectively. Betaine and dimethylglycine concentrations in liver 404 nmol/L plasma/hour ), center B analyzed 4 controls and pooled plasma were measured by LC-MS/MS. (median activity 127 nmol/L/hr). Plasma CBS activity was determined in Results: The rats fed with the 3 hyperhomocysteinemia inducing diets had samples from 30 pyridoxine-responsive and 38 pyridoxine non-responsive significantly decreased liver betaine resulting in a lower betaine/dimethyl- CBS deficient patients; to account for different control values the data are shown glycine ratio (p<0.001) compared to reference rats. in percentages of multiples of median of controls in the respective center. Conclusion: Betaine deficiency was found in all the different hyperhomo- Results: Pyridoxine non-responders showed substantially lower plasma cysteinemia rat models, possibly due to increased conversion via homocys- CBS activity (median 0 % of controls, range 0-9 %) than pyridoxine teine remethylation by betaine-homocysteine methyltransferase. We would responders (median 4 %, range 0-190 %). The CBS activity associated with like to hypothesize that betaine insufficiency is present in different forms of homozygosity for the common pyridoxine-responsive p.I278T mutation hyperhomocysteinemia and by itself could be deleterious (eg. via hampered (median 7 % of controls, n018) was higher than for the non-responsive osmotic regulation). As a consequence betaine supplementation may be mutations p.G307S (median 0 %, n05) and p.T191M (median 1 %, n06). beneficial. Betaine insufficiency has also been found in metabolic syn- Conclusion: Plasma CBS activity determination may be useful for diag- drome, lipid disorders, and diabetes. We would like to suggest that betaine nosing pyridoxine non-responsive CBS deficiency. should be monitored in hyperhomocysteinemia irrespective its cause. Acknowledgement: Supported by grant RVO-VFN64165/2012. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S53

A-009 17. Creatine Disorders CLASSICAL HOMOCYSTINURIA AND INSULIN-DEPENDENT DIABETES MELLITUS P-091 Cardoso M T1 , Cardoso M T2 , Chaves P C1 , Chaves P C2 , Fortuna A3 , SIMULTANEOUS DETERMINATION OF CREATINE METABOLITES Vasconcelos C4 , Lacerda L3 , Teles E L4 (CREATINE, CREATININE, ORNITHINE, GUANIDINOACETATE 1Adults Metab Dis Unit, Centro Hospitalar, São João, Porto, Portugal AND ARGININE) IN PLASMA, BLOOD AND DRIED BLOOD 2Int Med Dept, Centro Hospitalar, São João, Porto, Portugal SPOT BY LIQUID CHROMATOGRAPHY-TANDEM MASS 3INSA/ Centro de Genética Médica, Porto, Portugal SPECTROMETRY 1 2 2 2 4Metab Dis Unit, Paed Dept, Centro Hosp, São João, Porto, Portugal Tran CT , Yazdanpanah MY , Levandovskiy VL ,NauferAN , Kyriakopoulo LK2 , Schulze AS1 Classic homocystinuria is an autosomal recessive metabolic disease char- 1Clin Metab Gen, Sickkids Hosp, Toronto, Canada acterized by increased plasma homocysteine and methionine and decreased 2Ped Lab Med, Sickkids Hosp, Toronto, Canada cysteine. The clinical picture is extremely heterogenous, ranging from patients presenting with all of the complications to individuals with no To develop an accurate stable isotope dilution assay for determination of overt clinical involvement. Among its pathological sequelae, which include creatine (CT), creatinine (CTN), ornithine (Orn), guanidinoacetate (GAA) mental retardation, ectopia lentis, osteoporosis and vascular events, insulin- and arginine (Arg) for the purpose of explorative pharmacologic studies on dependent diabetes mellitus is an unusual finding. mouse models of creatine deficiency syndromes. We describe two women, one, 28 years of age and history of ectopia lentis Internal standard solution (D3-CT/D3-CTN/D2-GAA/D6-Orn/D7-Arg) from 3 years old, insulin-dependent diabetes mellitus since age 11, cerebral was added to pooled blood into three different ways: whole blood (WB), venous thrombosis at age 16, epilepsy since 21 years and mental retardation. plasma, and WB then spotted onto filter paper (DBS). After methanol-water She has genu valgum, pes cavus and osteopenia. The other, is 49 years old and extraction, butylated samples were analyzed by LC-MS/MS. with a history of ectopia lentis since 7 years, infertility, insulin-dependent The method was linear up to concentrations of 500/250/250/500/5 μmol/l, and diabetes mellitus since 31 years and family history of stroke. She presents LOD in plasma was 0.8/1.5/0.14/1.0/0.05 μmol/L for Orn/Arg/CT/CTN/ malar flushing and pes cavus. Association with genotypes linked to increased GAA, respectively. Intra-assay CVs for all analytes (except Arg in WB, risk of vascular disease, such as the factor V Leiden H1299R mutation and 22.7 %) were <8 % in WB and plasma and between 13.8 and 16.6 % in A1298C mutation of the MTHFR gene, were reported in this case. DBS. Compared to plasma, in WB, CTN/CT/Orn concentrations were higher In both patients the diagnosis was delayed to 16 and 23 years respectively. by 113 %/445 %/7 %, while in DBS, CTN/CT were higher by 71 %/402 %, Insulin-dependent diabetes mellitus is a rare complication present in each and Orn/GAA/Arg were lower by 10 %/15 %/65 %. One week of storage at - case and probably related to vasculopathy. 20 C led to 15-20 % increase of Arg and Orn in WB but not in plasma. A sensitive stable isotope dilution LC-MS/MS assay was developed for simultaneous determinations of CT/CTN/Orn/GAA/Arg. Absolute analyte concentrations were different in plasma, WB, and DBS. Plasma is the preferred specimen, but WB is reliable if small sample volume, e.g. 10-15 uL is required.

P-092 CREATINE DEFICIENCY AND GUANIDINOACETATE ACCUMULATION IN A NEW MODEL OF GAMT DEFICIENCY BY AAV2-TRANSDUCED RNAI IN 3D ORGANOTYPIC BRAIN CELL CULTURES IN DEVELOPMENT Hanna-El-Daher L1 , Béard E1 , Henry H1 , Loup M1 , Braissant O1 1Biomedicine, University Hospital, Lausanne, Switzerland

Amongst creatine deficiencies, GAMT-deficient patients present the stron- gest neurological alterations due to toxic effects of guanidinoacetate accumulating in CNS. In contrast to GAMT deficiency in humans, the described GAMT-/- KO mouse showed only mild neurological alterations. Our aim was to develop a GAMT deficiency model in organotypic cultures of developing brain cells to better understand the molecular and cellular effects of GAMT deficiency on CNS. 3D rat brain cell cultures in aggregates were transduced by AAV2 viruses expressing a specific GAMT shRNA to knock down GAMT expression by RNAi. The AAV2-transduced GAMT shRNA knocked down GAMT expression in aggregates (-83 % decrease of GAMT protein) with maximal effects between DIV5 and DIV8 of culture. GAMT RNAi induced a specific creatine deficiency paralleled by a stoechiometric accumulation of guanidinoacetate in the cultures. GAMT knock-down altered neuronal differentiation and increased apoptotic pathways in brain cells (cdk5/p35/p25 alteration; caspase 3 activation). Toxicity to brain cells was also caused by AAV2 per se, which altered cell development. While AAV2 was efficient to knock down GAMT expression, thus allowing analysis of some effects of GAMT deficiency in developing brain cells, future work will aim at optimizing AAV vectors to minimize toxicity while improving RNAi efficiency. S54 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-093 P-094 BIOCHEMICAL DIAGNOSTICS OF CREATINE TRANSPORTER FUNCTIONAL AND ELECTROPHYSIOLOGICAL DEFICIENCY IN ESTONIA CHARACTERIZATION OF 4 NON-TRUNCATING MUTATIONS Kall K.1 , Reimand T.2 , Talvik I.3 , Pop A.4 , Salomons G. S.4 , Ounap K.2 RESPONSIBLE FOR CREATINE TRANSPORTER DEFICIENCY 1Central Laboratory of Health Board, Tallinn, Estonia SYNDROME 2United Laboratories, Univ Hosp, Tartu, Estonia Valayannopoulos V1 , Bakouh N2 , Mazzuca M3 , Nonnenmacher L4 , 3Children's Clinic, Tartu University Hosp, Tartu, Estonia Hubert L3 , Makaci FL2 , Chabli A4 , Salomons GS5 , Mellot-Draznieks C6 , 4Metab Unit, Dep of Clin Chem, VU Univ, Amsterdam, Netherlands Brule E7 , de Lonlay P1 , Toulhoat H6 , Munnich A3 , Planelles G2 ,de Keyzer Y3 Creatine Transporter Deficiency is an X-linked disease caused by mutations 1Ref Center for IEM, Necker-Enf Malades, Paris, France in Creatine Transporter gene SLC6A8 in Xq28. The hallmarks of the 2INSERM U845, Necker-Enf Malades, Paris, France disorder are X-linked mental retardation, expressive speech delay, epilepsy, 3INSERM U781, Necker-Enf Malades, Paris, France developmental delay and autistic behavior. 4Metab Biochem Lab, Necker-Enf Malades, Paris, France Five patients from 3 families are diagnosed in Estonia. Three brothers with 5Metab Unit, Dept Clin Chem, VU UMC, Amsterdam, Netherlands a novel hemizygous missense mutation (c.1271 G>A; p.Gly424Asp) have 6IFP Energ Nouv, Rueil-Malmaison, France been published previously. Two other patients are non-related boys who 7Chim Paris Tech, ENSC, CNRS7223, Paris, France have a novel variant c.1529 T>A (p.Met510Lys) and previously reported c.1222_1224delTTC mutation, respectively. The p.Met510Lys is a con- Background: Mental retardation coupled with epilepsy are clinical hall- firmed pathogenic mutation, proven by overexpression of this variant in marks of the creatine (Cr) transporter deficiency syndrome resulting from SLC6A8 deficient fibroblasts using the methods as previously described mutations in the SLC6A8 gene. (Betsalel, Mol Genet Metab 2012). Western blotting confirmed the presence Objectives: to characterize the electrogenic and pharmacological properties of the EGFP-SLC6A8 fusion protein in the transfectants, but the creatine of non-truncating SLC6A8 mutations identified in 4 patients presenting uptake could not be restored. with variable clinical severity. Stable isotope dilution GC-MS method was used for measure Creatine in urine. Material and Methods: wt and mutant mRNA were introduced in X. Samples were treated with hexafluoroacetylacetone+toluene. Toluene layer was laevis oocyte expression system. Electrophysiological and pharmaco- derivatized with N-(tert-Buthyldimethylsilyl)-N-methyl-trifluoroacetamide.The logical properties with Cr and 4 Cr analogs in wt and four mutants product was analysed using GC-MS in Selected Ion Monitoring mode. were studied. [14]C-Cr uptake was assessed in X. laevis and in wt Creatine/Creatinine ratio was elevated in all male patients over 2x above and mutant fibroblasts. normal. The ratio is strongly influenced by patients' age. We show that there Results: Mutations led to a complete loss of both electrogenic and transport is a creatine/creatinine value for every age that only Creatine Transporter activities in X. laevis and Cr uptake in patients' fibroblasts. Among the Cr Defective patients exceed. analogs studied in oocytes injected with wt SLC6A8, only guanidinopro- pionate and PCr-Mg-CPLX induced full and partial electrogenic activities respectively, while no electrogenic activity with any Cr analogs was observed in mutant injected oocytes. Conclusions: Despite mutations affecting various domains of SLC6A8, Cr uptake and electrogenic properties were completely abolished and could not be dissociated. Besides the metabolic functions of Cr, the loss of SLC6A8 electrogenic activity may also play a role in the impaired brain function observed in affected patients. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S55

18. Purine / Pyrimidine Disorders P-078 TREATMENT WITH URIDINE IN HEREDITARY OROTIC P-072 ACIDURIAWITHOUT MEGALOBLASTIC ANAEMIA EXPERIENCE ATTENUATED ADENYLOSUCCINATE LYASE DEFICIENCY: A IN ONE PATIENT REPORT OF ONE CASE AND A REVIEW OF THE LITERATURE Breen C1 , Jameson E1 , Rust S2 , Morris AAM1 Jurecka A1 , Zikanova M2 , Jurkiewicz E1 , Tylki-Szymańska A1 1Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom 1The Children's Memorial Health Institute, Warsaw, Poland 2Royal Manchester Children's Hospital, Manchester, United Kingdom 2Institute for Inherited Metabolic Dis., Prague, Czech Republic Introduction: Hereditary orotic aciduria usually presents with megaloblastic We present a case of adenylosuccinate lyase (ADSL) deficiency type II with anaemia then poor growth and developmental delay, although patients can no obvious signs of disease progression and degradation. Additionally, we rarely present with hereditary orotic aciduria without megaloblastic anaemia. review the previously published cases of attenuated patients. The patient Treatment with uridine is successful for the haematological complications, but presented at the age of 5 months with a history of global developmental it is not known if treatment for learning difficulties associated with hereditary delay with limited eye contact, stereotypic features and myoclonic move- orotic aciduria without megaloblastic anaemia is beneficial. ment. Because of strange contact disturbances and psychomotor retardation, Objectives: To monitor the effect of uridine treatment in a patient with she was suspected of having ADSL deficiency and screening of urinary learning difficulties secondary to hereditary orotic aciduria purine metabolites revealed elevation of SAdo and SAICAr (a ratio of 2.1). Case report: Our patient had confirmed hereditary orotic aciduria, with mild Mutation analysis revealed a compound heterozygosity for missense muta- learning difficulties, normal growth and no haematological complications. tions: p.R426H and p.D268H. She began to walk independently at the age of Materials: Treatment with uridine (at 150 mg/kg/day) was commenced at 3 years. From the age of 4 she improved her communication, presenting less the age of 12 years and continued for one year. Neuropsychological testing autistic features. Currently, at the age of 9, the girl is attending a special was carried out before treatment and after 12 months. kindergarten, is a very cheerful, smiling and curious child. She actively Results: Neuropsychological testing revealed comparable results before participates in the group classes and communicates verbally using more and and after the treatment period. more words. Her statements are short, but begin to form a logical continuity. Discussion: Our patient continued to develop at the same rate and retained She also makes progress in simple manual operations, and can perform simple his position relative to his peer group but improvements in cognition were verbal commands. The patient participates in therapies such as pet therapy, not observed on the measures administered. It may be that treatment at an hippotherapy, speech therapy, gymnastics, hydrotherapy and, music therapy. earlier age would be beneficial.

P-076 P-095 GENETIC DEFECTS OF PURINE AND PYRIMIDINE METABOLISM LEUKOCYTE INOSINE TRIPHOSPHATASE EXPRESSION IS IN ARGENTINA (PERIOD 1996-2012) ALTERED IN HIV-SEROPOSITIVE INDIVIDUALS Laróvere LE1 , Fairbanks L2 , O´Neill JP3 , Arenas M2 , Marinaki A2 , Vroemen WHM1 , Leers MPG2 , Verbon A3 , Peltenburg C4 , Löwe SH5 , Escuredo E2 , Dodelson de Kremer R1 Bakker JA1 , Bierau J1 1CEMECO, Hosp Niños de Córdoba, UNCor, Córdoba, Argentina 1Dep Biochem Genet, Maastricht UMC, Maastricht, Netherlands 2Research Lab, GSTS, St Thomas´ Hospital, London, United Kingdom 2Dep Clin Chem, Atrium MC, Heerlen, Netherlands 3Dept of Pediatrics, Univ of Vermont, Burlington, Vermont, United States 3Dep Int Med, Maastricht UMC & Erasmus MC, Maastricht & Rotterdam, Netherlands , 4Dep Int Med, Maastricht UMC, Maastricht, Netherlands The genetic defects in purine and pyrimidine metabolism affect the nucle- 5Dep Int Med & Med Microb, Maastricht UMC, Maastricht, Netherlands otide metabolism; they are a complex and heterogeneous group of diseases in their clinical, biochemical and molecular characteristics. The lack of Background: The polymorphic ITPA gene encodes for the housekeeping en- previous research in Argentina and Latin America pointed out the necessity zyme inosine triphosphatase (ITPase), which maintains low levels of intracellular to enter upon this wide chapter. The aim of the project has been the inosine nucleotides ([d]ITP) and scavenges potentially cyto- or genotoxic non- development of a protocol in different levels of complexity of the specific canonical purine triphosphates. Recently, our research group demonstrated that the defects: i) phenotype compatibility, ii) determination of metabolites and majority of HIV+patients had decreased erythrocyte ITPase activity. It turned out enzymatic assay by liquid chromatography (hplc), iii) mutation analysis by that this finding was independent of ITPA genotype and/or highly active anti- PCR and sequencing. During the 1996-2012 period we carried out the retroviral therapy. To date, it is unclear whether less ITPase protein was synthe- biochemical and molecular diagnosis of: I) Hypoxanthine-guanine phos- sized or that the stability of the protein was reduced. Therefore, we wanted to phoribosyltransferase deficiency: 9 patients from 8 not related families with determine ITPase expression in the different peripheral leukocyte subpopulations Lesch-Nyhan disease (mutations: c.203 T>C; c.209 G>A; IVS8+6 T>G; of a HIV infected population and additionally determine their ITPA genotype. IVS7+1 G>A; 212dupG; deletion of 4-6 exons; 2 subjects without the Methods: For this purpose, we used our recently developed novel 6-color/ molecular analysis) and 13 patients (5 apparently not related families) with 8-parameter multiparameter flow cytometric assay to determine ITPase the neurological/hyperuricemic variant without self-injury (mutations: expression. ITPA genotype was determined by gene sequencing. c.584A>C; c.143 G>A; IVS6+5 G>A); II) Cofactor molybdenum defi- Results: No significant difference in ITPase expression was observed for ciency (2 patients, different families), genotypes: homozygote for MOCS2 the total leukocyte fraction between the HIV+population and reference c.726-727 AA del and MOCS2 c.413 G>A and III) Dihydropyrimidine values of normal persons. However, within the T-lymphocytes, the CD8+ dehydrogenase deficiency (1 patient), genotype: c.257 C>T7/c.257 C>T. lymphocytes and NK-cells of the HIV+cohort contained less ITPase (P< This experience establishes CEMECO as a reference for the diagnosis, 0.05). Contrarily, a striking increase in ITPase expression was observed in treatment and monitoring of purine and pyrimidine defects in Argentina. the B-lymphocyte subpopulation of HIV+patients (P<0.0001). Conclusion: Leukocytic ITPase expression is altered in HIV-seropositive individuals. S56 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-096 P-098 CLINICAL, BIOCHEMICAL AND GENETIC FINDINGS IN TWO HEREDITARY OROTIC ACIDURIA : IS ANEAMIA ALWAYS PATIENTS WITH DIHYDROPYRIMIDINASE DEFICIENCY PRESENT? Nakajima Y1 , Meijer J1 , Dobritzsch D2 , Roelofsen J1 , Meinsma R1 , Fairbanks LD1 , Arenas M1 , Escuredo E1 , Marinaki A1 Zhang C3 ,WangX4 ,ZhengH4 ,ZhaoJ4 ,ItoT5 ,SaitoS5 ,van 1Purine Res Lab, GSTS , St Thomas' Hosp, London, United Kingdom Kuilenburg ABP1 1Lab Genet Metab Dis, Univ Amsterdam, Amsterdam, Netherlands Introduction: Hereditary orotic aciduria occurs when there is a defect with 2Dept Med Biochem Biophy, Karolinska Inst, Stockholm, Sweden Uridine Monophosphate Synthetase (UMPS). This enzyme is a bifuctional 3MILS International, Kanazawa, Japan enzyme that catalyses 2 reactions Orotate phosphoribosyltransferase 4Beijing Children's Hospital, Beijing, China (OPRT) and Orotidine monophosphate decarboylase (ODC), the 5th and 5Dept Pediatr, Med Sch, Univ Nagoya City, Nagoya, Japan 6th steps in the pyrimidine de-novo pathway. This causes very elevated levels of orotic acid. Classically this disorder also presents with anaemia. Background: Dihydropyrimidinase (DHP) is the second enzyme of the We review 6 cases of UMPS seen in the Purine Research Laboratory. pyrimidine degradation pathway and until now, only 28 patients have been Methods: Enzyme analysis and sequencing of genomic DNA or cDNA was described with complete DHP deficiency. used to define mutations. Patients: The first patient was a Chinese girl experiencing an episode with a Results: Pat: UMPS ((OPRT/ ODC):Mut. anal : Presentation: loss of consciousness at the age of 3 months. When she was 10 years old, she 1 : nd/nd : homo c.275 C>T :OAuria ,anaemia presented with dizziness, unconsciousness and seizures. The second patient 2 : nd/nd : hetero c.245A>C& c.1027 C>A :OAuria. anaemia, seizures was a 22-month-old Chinese girl who presented with seizures, mental and 3 : nd/nd : not done :OAuria, dev. delay motor-developmental delay in addition to bilateral hip subluxation. 4 : 43/28 : not done :OAuria, dev. delay Results: Both patients presented with strongly elevated urinary levels of the 5 : nd/nd : homo c.928 T>G :OAuria, visual problems dihydropyrimidines. Analysis of DPYS showed that both patients were com- dev. delay pound heterozygous for the missense mutation c.1001A>G (p.Q334R) and a 6 : 659/nd: hetero c.867 T>A&c.1351 C>A:OAuria,dev.delay novel splice-site mutation c.1443+5 G>A. Heterologous expression of the nd0not detected mutant enzyme in Escherichia coli showed that the p.Q334R mutation yielded Range :(OPRT/ ODC)[124-497/ 79-1737] activity pmol/h/mgHB mutant DHP proteins with a 10.1 % of wild type residual activity. The crystal Conclusions: Anaemia was present in one third of cases, and neurological structure of human DHP showed that the Q334 might affect two hydrogen problems were present in 4 cases. However, orotic aciduria was the reason for bonds resulting in increased structural instability and impaired catalytic activity. referral in all cases. Conclusion: The finding of mutation Q334R and a novel splice-site mutation in two unrelated Chinese patients may indicate that a DHP deficiency may be more common in China than generally considered.

P-097 19. Organic Acids DETERMINATION OF PYRIMIDINE METABOLITES FOR INBORN ERROR OF METABOLISM IN PYRIMIDINE SYNTHESIS AND P-107 DEGRADATION SYSTEM BY USING UPLC/MS/MS NEUROPATHOPHYSIOLOGICAL MECHANISMS IN GLUTARIC Kato A1 , Maeda Y1 , Nakajima Y2 , Kato S2 , Sugiyama N3 , Saito S2 , ACIDURIA TYPE I: 3-HYDROXYGLUTARIC ACID LEADS TO Kimura K1 , Ito T2 AMMONIA INCREASE AND NON-APOPTOTIC CELL DEATH 1Dept Hosp Pharm, Nagoya City Univ, Nagoya, Japan Jafari P1 , Braissant O2 , Zavadakova P1 ,HenryH2 , Bonafé L1 , 2Dept Pediatr, Ngoya City Univ, Nagoya, Japan Ballhausen D1 3Dept Pediatr, Aichi-Gakuin Univ, Nagoya, Japan 1Mol Pediatrics, Lausanne Univ Hosp, Lausanne, Switzerland 2Biomedicine, Lausanne Univ Hosp, Lausanne, Switzerland Background: Inborn errors of metabolism have been reported in pyrimidine synthesis and degradation system. Orotic acid in pyrimidine synthesis system A 3D in vitro model of rat organotypic brain cell cultures in aggregates was accumulates in orotic aciduria and OTC deficiency. Pyrimidine catabolites used to investigate neurotoxicity mechanisms in glutaric aciduria type I (uracil, thymine, dihydrouracil, dihydrothymine, beta-ureidopropionate and (GA-I). 1 mM glutarate (GA) or 3-hydroxyglutarate (3OHGA) were repeat- beta-ureidoisobutylate) accumulate for each enzyme deficiency in the degrada- edly added to the culture media at two different time points. In cultures tion system. We investigated simultaneous determination method of orotic acid treated with 3OHGA, we observed an increase in lactate in the medium, and pyrimidine catabolites by UPLC/MS/MS for diagnosis of these disorders. pointing to a possible inhibition of Krebs cycle and respiratory chain. We Method: Urine without pretreatment was injected onto Acquity UPLC HSS further observed that 3OHGA and to a lesser extend GA induced an T3 column (2.1 x 150 mm, Waters). Orotic acid was detected in negative increase in ammonia production with concomitant decrease of glutamine ion mode and pyrimidine catabolites were analyzed in positive ion in concentrations, which may suggest an inhibition of the astrocytic enzyme multiple reaction monitoring (MRM) mode of Quattro Premier XE triple glutamine synthetase. These previously unreported findings may uncover a quadrupole mass spectrometer (Waters). pathogenic mechanism in this disease which has deleterious effects on early Results: Orotic acid and pyrimidine catabolites were analyzed in 10 min by using stages of brain development. By immunohistochemistry we showed that UPLC and accumulated in OTC deficiency. Although beta-ureidopropionate and 3OHGA increased non-apoptotic cell death. On the cellular level, 3OHGA beta-ureidoisobutylate accumulated in beta-ureidopropionase deficiency, accumu- and to a lesser extend GA led to cell swelling and loss of astrocytic fibers lation of the upper metabolites was not observed. whereas a loss of oligodendrocytes was only observed for 3OHGA. We Discussion: We developed determination method for orotic acid and py- conclude that 3OHGA was the most toxic metabolite in our model for GA-I. rimidine catabolites by positive and negative ion simultaneous analysis in 3OHGA induced deleterious effects on glial cells, an increase of ammonia MS/MS. The correct diagnosis for inborn errors of metabolism in pyrimi- production, and resulted in accentuated cell death of non-apoptotic origin. dine synthesis and degradation system could be achieved by this method. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S57

P-108 P-110 A MODIFIED MODEL OF LYSINE INDUCED NEUROLOGICAL HYPERCALCAEMIA IN METHYLMALONIC ACADEMIA DISEASE IN GCDH-DEFICIENT MICE (MMA): A CHALLENGING COMPLICATION Opp S1 ,SauerSW1 , Millford D2 ,HeilandS2 , Mittelbronn M3 , O'Sullivan S1 , Abuhoul L1 , Shroff R S1 , van't Hoff W1 , Skeath R1 , Komatsuzaki S1 , Okun JG1 , Kölker S1 Dixon M1 , Grunewald S1 1Div Metab Dis, Univ Child Hosp, Heidelberg, Germany 1Great Ormond Street Hospital, London, United Kingdom 2University Hospital Dep Neuroradiology, Heidelberg, Germany 3Goethe-University, Frankfurt am Main, Germany Objectives: Review of hypercalcaemia, its treatment and complications including pancreatitis in MMA patients. Background: Glutaric aciduria type I is an inherited disorder of lysine Methods: Retrospective review of MMA patients between 2000 and 2012 . metabolism due to deficiency of glutaryl-CoA dehydrogenase (GCDH) Hypercalcaemia was defined as calcium >2.8 mmol/L, ionised calcium causing neurodegeneration of basal ganglia in patients. It was shown that >1.4 mmol/L, needing intervention. Diagnosis of pancreatitis was based lysine-enriched diet induces a similar neurological phenotype in Gcdh-/- on elevation of lipase +/- amylase, abdominal pain and vomiting. mice. Our study reports essential modifications as well as a profound Results: 6/26 patients developed hypercalcaemia, 2 patients had recurrent biochemical, histological, and MRI analyses of this model. episodes. Age of onset ranged from three months to 5 yrs. All patients had Methods: Different amounts of lysine (0.188 mg-0.376 mg lysine/mouse/ chronic kidney disease (CKD). Hypercalcaemia was linked to decompen- day) were applied to Gcdh-/- and control mice. Dietary effects were eval- sations, or excess Vitamin D supplementation. Treatment included reducing uated by MRI and biochemical, enzymatic and histological analyses. calcium intake and discontinuation of Vitamin D, forced alkali diuresis, Results: Our study shows that the published lysine-enriched diet is not pamidronate +/- calcitonin. The aetiology of hypercalcaemia remains elu- sufficient to induce a stable neurological disease. We show a clear threshold sive then in the 2 cases of excess Vit D intake. of applied lysine necessary to induce neuronal damage that is characterized 4/6 patients developed pancreatitis; 3 acute of which one was recurrent and with dramatic increase in cerebral glutaric acid concentrations. Elevating 1 acute on chronic pancreatitis. Age of onset of pancreatitis ranged from daily lysine intake above this level did not further enhance neurological 3 yrs to 4 yrs 10/12. In 3 patients, pancreatitis was observed alongside outcome. MRI analysis of symptomatic mice showed time dependent hypercalcaemia. There was no evidence of calcifying pancreatitis. Contrary decreases of T2 and increases of T1 signals as well as disturbed hemody- to recent reports, 3 of our patients had elevated amylase, elevated lipase was namics. Histological examination showed severe spongiosis and neuronal observed in all 4. pyknosis more prominent in hippocampus than in basal ganglia. Conclusion: Hypercalcaemia in MMA is common but the aetiology needs Conclusions: Our study provides a modified and stable model for lysine- to be better understood to improve management. induced neurological disease in Gcdh-deficient mice described by cytotoxic neuronal damage and disturbed hemodynamics.

P-109 P-111 GENE STUDIES IN PROPIONIC ACIDEMIA IN INDIAN PATIENTS: COULD NEUROLOGICAL SYMPTOMATOLOGY PRECEDE AN REPORT OF SEVEN MUTATIONS, INCLUDING ONE NOVEL ACUTE METABOLIC CRISIS IN PATIENT AFFECTED BY SPLICE SITE MUTATION, AND PRENATAL DIAGNOSIS PROPIONIC ACIDURIA (PA)? A PARTICULAR CASE REPORT Gupta D1 , Bijarnia S1 , Saxena R1 , Kohli S1 , Puri R1 , Shigematsu Y2 , Paci S1 , Vincenti S1 , Selmi R1 , Salvatici E1 , Riva E1 Yamaguchi S3 , Sakamoto O4 , Deb R5 , Verma IC1 1Paed Dept, San Paolo Hosp, Univ Milan, Milan, Italy 1Cent of Med Genet, Sir Ganga Ram Hosp, New Delhi, India 2Dept Hlth Sci, Dept of Ped, Univ Fukui, Fukui, Japan PA is an inborn error of metabolism due to propionyl-coenzymeA carbox- 3Dept Ped, Shimane Univ Sch med, Izumo, Shimane, Japan ylase deficiency, often characterized by episodic metabolic decompensation 4Tohoku UnivSchool of Medicine, Sendai, Japan at risk for neurologic sequelae. We report the case of a 13 years old girl, 5AIB, Amity Univ,, Noida, UP, India diagnosed after a severe metabolic decompensation episode at 7 days of age. From diagnosis, the patient follows a strict dietary and pharmacolog- Propionic Acidemia (PA) is one of the classical organic acidemias causing ical treatment with optimal compliance, showing an isolated decompensa- burden on families of affected children. Two genes, PCCA & PCCB are tion episode during illness at 1 year of age. involved in carboxylation of Propionyl CoA to D-Methylmalonyl CoA. No At 12 years old, in wellness, she started to present progressive (about gene studies have been reported from India so far. 2 weeks) generalized asthenia with marked hyposthenia at legs and inter- The coding sequences of nine patients enrolled were examined by PCR mittent eyelid ptosis. For the worsening progress of symptoms (psychomo- followed by SSCP. The samples showing mobility shift in SSCP were then tor agitation and an episode of vomiting) she was admitted to hospital: subjected to sequencing. In some cases where SSCP was not informative, Astrup, ammonia, lactic acid, complete blood count, hepatic and renal direct sequencing of the coding region was done. function tests and urine parameters were completely normal. After 6 hours We identified mutations in seven out of 9 samples; 2 nonsense: c.123 C>T & she started to present excessive sweating, superficial breath and 2 vomiting, c.1426 C>T, 1 deletion: c.284delA and 1 novel splice site mutation: IVS7+ leading to severe metabolic acidosis with lactic and ketotic component, 1 G>T in PCCA gene. One missense: c.1210 G>A and 2 splice site mutations; marked hyperglycaemia and ketonuria, requiring admission to intensive 1 patient compound heterozygous for IVS3+2 T>C & IVS 1+5 G>A and care unit. another patient homozygous for IVS 1+5 G>Awere found in PCCB gene. We Our case report underline the importance to consider any neurological and performed six prenatal diagnoses in 5 unrelated families, based on mutations neurophycological symptoms and signs, even if not yet associated to identified in affected children. The results showed one affected fetus. metabolic blood and urine alterations, as prelude of a possible acute met- In our study, mutation identification played a pivotal role in PND and prevention abolic decompensation. of burdensome disorder. There were no common mutations amongst the cohort studies, thus suggesting mutational heterogeneity in PA patients in India. S58 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-112 P-114 OPTIC ATROPHY AS A COMPLICATION OF METHYLMALONIC LONG-TERM COMPLICATIONS OF PROPIONIC ACIDURIA IN AND PROPIONIC ACIDAEMIA: A CASE SERIES OF FOUR THE ABSENCE OF METABOLIC ACIDOSIS PATIENTS Komatsuzaki S1 , Sakamoto O1 , Fuse N1 , Uematsu M1 , Matsubara Y1 , Jameson E1 , Chan WH2 , Tolhurst-Cleaver M3 , Thornton W4 , Ashworth Ohura T2 J2 , Walter JH1 1Tohoku University School of Medicine, Sendai, Japan 1Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom 2Sendai City Hospital, Sendai, Japan 2Manchester Royal Eye Hospital, Manchester, United Kingdom 3Stepping Hill Hospital, Stockport, United Kingdom Background: Propionic acidemia (PA) is an autosomal recessive disease 4Royal Manchester Children's Hospital, Manchester, United Kingdom caused by deficiency of propionyl-CoA carboxylase. The clinical manifestations of PA are metabolic acidosis, hyperammonemia and various neuro- Introduction: Our centre currently manages thirty-five patients with meth- logical symptoms. Despite improvement of management, the patients ylmalonic acidaemia (MMA) and eighteen with propionic acidaemia (PA). Over present with various complications including basal ganglia lesions, optic the past two years four of our patients have developed visual impairment. nerve atrophy, and cardiomyopathy. Moreover, long-term complications Objective: To describe our experience of optic atrophy in these four patients. have not been well described. Results: Cases one and two describe male patients with stable MMA who Case: A male patient presented with tachypnea, metabolic acidosis, and developed visual deterioration at six years and ten years of age respectively. hyperammonemia at the age of 6 days, and was treated with exchange Case three is a male MMA patient whose visual problems at twelve years of transfusion and a low protein diet. The diagnosis of PA was confirmed by age coincided with a deterioration in renal function. During an episode of decreased activity of propionyl-CoA carboxylase. After emergency interven- metabolic decompensation there was further deterioration with subsequent tion, the patient was treated with a low protein diet and carnitine supplemen- partial recovery back to the previous visual state. Case four is a female PA tation. Despite nearly normal development up to 4 years of age, his intellectual patient who suffered acute, severe pancreatitis at eleven years of age and ability subsequently deteriorated, without metabolic decompensation (IQ 54 at developed visual loss in the recovery phase of this illness. All four children 15). At the age of 22, ophthalmological examination showed optic nerve have bilateral optic atrophy and are registered as partially sighted. atrophy. At the age of 23, brain MRI showed subcortical lesions, and pro- Discussion: We suggest that ophthalmology review should be part of the gressed over several years in the absence of any metabolic acidosis. standard follow-up for children with an organic acidaemia. Early involve- Conclusion: These findings suggest that conventional management is ment of the visual impairment team can improve quality of life and ensure insufficient to improve the long-term prognosis for patients with PA, thus adequate educational support is in place. revealing a need for novel therapeutic approaches based on a better understanding of the pathophysiology.

P-113 PROPIONIC ACIDEMIA PRESENTING WITH PERSISTENT P-115 PULMONARY HYPERTENSION IN TWO NEONATES GLUTARIC ACIDURIATYPE I AND GLIOMA: THE FIRST REPORT Hişmi B1 ,Tekşam Ö2 , Ünal Ö1 , TakçıŞ3 , Ertuğrul İ4 , Sivri HS1 , Dursun IN A YOUNG ADULT PATIENT A1 , Tokatlı A1 , Coskun T1 Burlina AP1 , Danieli D2 , Malfa F1 , Manara R3 , Del Rizzo M4 , Bordugo 1Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey A4 , Burlina AB4 2Hacettepe Univ Child Hosp, Div Ped Emerg, Ankara, Turkey 1Neurological Unit, St Bassiano Hosp, Bassano del Grappa, Italy 3Hacettepe Univ Child Hosp, Neonatolgy, Ankara, Turkey 2An Pathol & Histol Unit, St Bortolo Hosp, Vicenza, Italy 4Hacettepe Univ Child Hosp, Ped Cardiol, Ankara, Turkey 3Neuroradiology Unit, Padova, Italy 4Div Inherit Metab Dis, University Hosp, Padova, Italy An association of non-ketotic hyperglycinemia and pulmonary hypertension (PH) was previously described and "glycine toxicity" was postulated Case report: We report a 23-years-old man, affected with glutaric aciduria for etiology. Propionic acidaemia (PA), an inborn error of intermediary type I (mutation: R402W), who recently developed a brain tumor. At the age of metabolism caused by propionyl-CoA carboxylase deficiency, also leads 2 years bilateral subdural hematoma was detected. From this age onwards, the to hyperglycinemia caused by secondary inhibition of the glycine cleavage patient started to develop an extrapyramidal movement syndrome. At the age system in liver. Here two neonates with propionic acidemia complicating of 8 years, after a febrile episode with metabolic decompensation, movement with persistent pulmonary hypertension (PPH) are described and possible disorders worsened dramatically. In the last 8 years, trihexyphenidyl, clonaze- roles of hyperglycinemia and/or oxidative stress are discussed. To our pam, and tetrabenazine were used with benefit on dystonia, dyskinesia and knowledge this is the first description of such an association. myoclonic jerks. Brain MRI performed in 2008 showed white matter abnor- Patient-1 presented with grunting and tachypnoea on 20th hours of life. malities in the centrum semiovale and thalami. Echocardiography showed PH and severe tricuspid regurgitation. Despite In January 2012, the patient was transferred to our hospital because of alkalinization and oxygen therapy severe PH persisted and profound met- sudden episodes of vomiting. He became lethargic and a brain CT scan abolic acidosis with ketosis led to further metabolic screening which showed an extensive lesion, with intraparenchymal hemmorrhage, of the revealed PA. Refractory acidosis recovered with peritoneal dialysis, he is right basal ganglia. The patient was immediately transferred to the Neuro- discharged on day 15 with normal echocardiography. surgery Unit. Subsequent histological examination showed that the lesion Patient-2 presented with tachypnoea and severe metabolic acidosis on third was a glioblastoma (G-IV, WHO classification). day of life. An index case in the family prompted PA diagnosis and therapy Conclusion: This is the first report of cerebral glioma in an adult patient was started immediately. Although metabolic acidosis recovered with alkalin- with glutaric aciduria type I. Brain tumors have been reported in patients azation within a day tachypnoea and hypoxemia persisted, echocardiography affected with D/L-2 hydroxyglutaric aciduria. We believe that common showed severe PPH which required vasodilatation therapy (iloprost and silde- pathogenic mechanisms, involving cerebral organic acidurias and cerebral nafil citrate). She is discharged on day 18 with normal echocardiography. neoplasms, should be investigated. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S59

P-116 P-118 CHEMICAL CHAPERONE (PHENYLBUTYRIC ACID) REACTIVE COMPLEMENTARY DIET IN GLUTARIC ACIDURIA TYPE I PROPIONIC ACIDEMIA EXPLOITING THE BLOOD-BRAIN BARRIER FOR TREATMENT Mienie LJ1 , Dielwart P1 , Vorster BC1 Boy SPN1 ,HeringerJ1 ,MüllerE1 , Maier EM2 ,EnsenauerR2 , 1Centr Hum Metab, North-West Univ., Potchefstroom, South Africa Mühlhausen C3 , Schlune A4 , Greenberg CR5 , Koeller DM6 , Hoffmann GF1 , Haege G1 , Burgard P1 , Kölker S1 Background: Propionic acidemia, the most common metabolic disease 1University Children's Hospital, Heidelberg, Germany amongst the Afrikaans speaking South African population, is always fatal 2Reference Centre for Inherit Metab Dis, Munich, Germany and seems to be untreatable. Since 1983 more than 80 patients were 3University Medical Centre, Hamburg, Germany diagnosed who presented with severe metabolic acidosis, ketosis, hyper- 4University Children's Hospital, Düsseldorf, Germany ammonemia, and coma almost immediately after birth and were treated with 5Winnipeg Children's Hospital, Winnipeg, MB R3A 1R9, Canada low branched chain amino acid intake, L-Carnitine, sodium benzoate, 6Doernbecher Children's Hospital, OHSU, Portland, OR 97239, United States alanine and sodium bicarbonate. None of the patients survived the age of 10 years and of the 80 diagnosed patients only 5 are still alive, all of them Background: The cerebral entrapment of neurotoxic dicarboxylic metabo- severely mentally and physically affected. Although the treatment was very lites plays an important role in the neuropathology of glutaric aciduria type I strictly followed, no clear difference on the organic acid profile was ever (GA-I). Low lysine diet in combination with carnitine supplementation and visible (except for hippuric acid). In 2011 phenylbutyrate became available emergency treatment improves the outcome. Cerebral lysine influx via the in SA and a phenylbutyrate/benzoate mixture was used in the prevention of blood-brain barrier can be modulated by arginine. hyperammonemia in two new born babies with propionic acidemia. Methods: The aim of this prospective follow-up study is to evaluate the Results: Surprisingly, not only the ammonia levels decreased, but the effect of lysine-free, arginine-fortified amino acid supplements (AAS) on organic acid profile almost normalized within a week after treatment the outcome. A cohort of 34 neonatally diagnosed GA-I patients (median started. PCC assembly is known to be chaperone depended. age, 7.43 years) who received metabolic treatment according to guideline Conclusion: This is the first case report indicating that phenylbutyrate recommendations were included. Patients received AAS product lines with could be used as a chemical chaperone in the treatment of propionic different arginine content: group 1 (Milupa Metabolics), group 2 (Nutricia). acidemia. Results: During the first year of life, group 2 patients received more arginine from AAS (mean, 145 mg/kg BW) than group 1 patients (mean, 111 mg/kg BW; p<0.001). However, both groups received more arginine than infants not receiving AAS. The frequency of dystonia was low in both groups (group 1: 12.5 %; group 2: 8 %), and patients achieved gross motor milestones in time. Conclusions: The development of complementary dietary strategies P-117 exploiting transport competition between lysine and arginine for treatment EXPERIMENTAL EVIDENCE THAT ACUTE LYSINE of GA-I seems promising. More work is required to understand neuro- ADMINISTRATION PROVOKES MARKED REDUCTION OF protective mechanisms of arginine. NA+, K+-ATPASE AND CREATINE KINASE ACTIVITIES IN GLUTARYL-COA DEHYDROGENASE DEFICIENT MICE Amaral AU1 , Cecatto C1 , Seminotti B1 , Zanatta A1 , Fernandes CG1 , Busanello ENB1 , Braga LM2 , Ribeiro CAJ1 , de Souza DOG1 , Woontner P-119 M3 , Koeller DM4 , Goodman S3 , Wajner M5 MOLECULAR DIAGNOSIS OF BARTH SYNDROME FIRST CASE 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil REPORT FROM INDIA 2FEPPS, Porto Alegre, Brazil Bijarnia - Mahay S1 , Borkar M1 , Kotecha U1 , Saxena R1 , Verma IC1 3School Medicine University of Colorado, Denver, United States 11Center Med Genet, Sir Ganga Ram Hospit, New Delhi, India 4Oregon Health & Science University, Portland, United States 5Hospital de Clinicas Porto Alegre SGM, Porto Alegre, Brazil Barth Syndrome, also known as 3-Methylglutaconic acidurias type II, is an X-linked disorder presenting at variable ages, with dilated We evaluated the effects of a single intra-peritoneal injection of saline (Sal) or cardiomyopathy, growth retardation and neutropenia in 25 % of cases. lysine (Lys - 8 μmol/g) on bioenergetics parameters (the activities of the respi- There is deficiency of a mitochondrial membrane protein (taffazin) ratory chain complexes II, II-III and IV, α-ketoglutarate dehydrogenase (α- affecting phospholipid metabolism. This disorder has never been KGDH), creatine kinase (CK) and synaptic Na+, K±ATPase) in brain, heart reported from India, as its recognition is difficult not only because and skeletal muscle from 15-day-old glutaryl-CoA dehydrogenase deficient mice of the rarity of the condition, but also because of the non-availability (Gcdh-/-), compared to wild type (WT) mice. No differences of all evaluated of diagnostic testing. parameters were detected between Gcdh-/- and WT mice injected at baseline We report a case of Barth syndrome, where the diagnosis was made after the (Sal). Besides mild increases in the activities of some respiratory chain complexes death of child – based on clinical presentation, dilated non-compaction type (II-III and IV) in heart and skeletal muscle of Gcdh-/- and WT mice, marked cardiomyopathy, neutropenia and a positive family history consistent with decreases of the activities of Na+, K±ATPase in brain and CK in brain and X-linked inheritance. Molecular studies were undertaken on the DNA skeletal muscle of Gcdh-/- mice were caused by Lys administration. However, sample stored from the baby at post-mortem sampling. A novel mutation, brain α-KGDH activity was not altered in WT and Gcdh-/- injected with Sal or p.W144G (c.T430G) was found in exon 5 of TAZ gene. This would now Lys. It is presumed that reduction of Na+, K±ATPase and CK activities may play enable prenatal diagnosis in this family and also facilitate carrier testing in an important role in the pathogenesis of the neurodegenerative changes in GA I. all females at risk. Financial support: Research grants from FIPE/HCPA, CNPq, PROPESq/ Thus, a high index of suspicion in cases of dilated cardiomyopathy, UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neuro- especially with a history of X-linked inheritance is very helpful in ciência (IBN-Net) # 01.06.0842-00, INCT - EN. reaching a diagnosis. This report highlights the importance of family history and storage of samples in affected children in any resource poor country. S60 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-120 P-122 IDENTIFICATION OF DEREGULATED PROTEINS IN DEVELOPMENT AND VALIDATION OF A NOVEL LIQUID METHYLMALONIC ACIDEMIA (MMA) CHROMATOGRAPHY TANDEM MASS SPECTROMETRY ASSAY Caterino M1 , Chandler RJ2 , Venditti CP2 , Ruoppolo M3 FOR THE DETERMINATION OF HOMOGENTISIC ACID 1Fondazione SDN, napoli, Italy Carling RS1 , Dowley M1 , Rahman Y2 2Nat. Hum. Gen. Res. Inst., NIH, Bethesda, United States 1Biochemical Sciences, GSTS Pathology, London, United Kingdom 3DBBM, Univ. Naples Federico II, Naples, Italy 2Cen In Metab Dis, Evelina Children's Hos, London, United Kingdom

Background: Methylmalonic acidemia (MMA) is typically caused by Alkaptonuria results in the accumulation of homogentisic acid (HGA) in deficient activity of methylmalonyl-CoA mutase (MUT). The main treat- cartilage, connective tissue, plasma and urine. Patients typically present ment for MMA patients is dietary restriction of propiogenic amino acids with non-specific joint or cardiac symptoms in early adulthood and most and carnitine supplementation. The outcome has historically been poor, diagnoses are made as incidental findings. Until recently treatment options leading to elective liver and/or combined liver/kidney transplantation as a were limited to analgesia and symptomatic measures, with dietary manip- therapy for severely affected patients. ulation and vitamin C being of limited value. However, Nitisinone, a potent Objectives: To identify deregulated proteins and metabolites in liver explants inhibitor of the second enzyme in the tyrosine catabolic pathway, has been from unaffected donors compared to patients with MUT deficiency. suggested as potential therapy. A 3-year follow-up study has been encour- Methods: Whole cell liver extracts were subject to DIGE (Differential Gel aging, showing significant reduction of urinary HGA following treatment. Electrophoresis). Spots with a variation index greater than 1.1 were con- HGA can be quantitated by spectrophotometry and HPLC but no method is sidered significant and were excised, fragmented by trypsin digestion and routinely available in the UK. We have developed a novel liquid chroma- analyzed by mass spectrometry. tography tandem mass spectrometry method, utilising deuterated 3,4-dihy- Results: DIGE showed a high degree of similarity (> 80 % of the spots droxyphenylacetic acid as internal standard. Analysis is performed in superimpose) between the groups. 60 spots were significantly deregulated (p< negative ion mode and chromatographic separation is achieved isocrati- 0.01). Among the set of proteins decreased in the MMA patient livers were cally. Calibration was linear to at least 2.5 mmol/L (R200.9955), inter and enzymes involved in the ETC, branched chain aminoacid oxidation, gluconeo- intra batch CV both<10 % (n010). Ion suppression was negligible, recov- genesis, glycolysis and the urea cycle. Fewer proteins were increased (19 erywas>95%andLODwas7μmol/L. Analysis of samples from proteins), and included proteins involved in Krebs cycle anapleurosis. Western Alkaptonuria patients demonstrated the ability of this method to discrimi- blotting confirmed the differential expression of 5 candidates. nate patients from controls and to monitor the reduction in HGA with Conclusions: Deregulated pathways in MMA affect numerous enzymes Nitisinone therapy involved in intermediary metabolism. Proteomic analyses have provided new insights into disease pathophysiology.

P-121 P-123 4-HYDROXY-6-METHYL-2-PYRONE AND KETONURIA IN DIFFUSION EDITED NMR SPECTROSCOPY OF INTACT BLOOD HMG-COA SYNTHASE DEFICIENCY PLASMA FOR METABOLIC STUDIES HOGG SL1 , PIERRE GM2 , BUCK J3 , THALANGE N4 , CHAMPION Engelke UFH1 , Goudswaard AG1 , Wevers RA1 MP5 , CALVIN J1 1Laboratory Medicine, Radboud UMCN, Nijmegen, Netherlands 1Biochem Gen Unit; Cambridge Uni Hosps, Cambridge, United Kingdom 2Paed Dept; Bristol Child Hosp, Bristol, United Kingdom Background: 1 H-NMR spectroscopy of blood has successfully been 3Paed Dept; Ipswich Hosp, Ipswich, United Kingdom applied to the field of inborn errors of metabolism (IEM). However, the 4Paed Dept; Norfolk & Norwich Uni Hosp, Norwich, United Kingdom presence of macromolecules can hamper NMR analysis and they are there- 5IMD Dept; Evelina Child Hosp, London, United Kingdom fore often removed prior to NMR measurement. Diffusion edited NMR (DE-NMR) spectroscopy applied to intact blood plasma provides an alter- Mitochondrial 3-Hydroxy-3-methylglutaryl-CoA (HMG) synthase (EC native for spectral simplification. 2.3.3.10) catalyses the rate-limiting step in ketogenesis. Deficiency of this Objective: To investigate the use of DE-NMR spectroscopy to measure low enzyme (OMIM #605911) is therefore predicted to be a cause of hypoketotic molecular mass metabolites in intact blood plasma. hypoglycaemia. We report two non-related patients with genetically proven Material and Methods: DE-NMR spectroscopy was performed on blood HMG-CoA synthase deficiency who presented with hypoglycaemia (plasma plasma from controls and patients with IEM (histidinemia (HIS), phenyl- glucose 1.7 and 0.3 mmol/L). Mild ketonuria was demonstrated in both ketonuria (PKU), methylmalonic aciduria (MMA) and isovaleric aciduria patients on at least one occasion by urine organic acid analysis along with (IV)). Sample preparation was limited by adding the chemical shift refer- increased plasma branched chain amino acids, consistent with ketosis. Despite ence (TSP) and a concentration reference (maleic acid) to the blood plasma. this, blood spot acylcarnitines showed normal acetylcarnitine with low/low- Results: DE-NMR spectra of control plasma allowed the identification of 22 normal free carnitine. One patient had a raised free fatty acid:3-hydroxybuty- low molecular mass metabolites and 9 macromolecules (lipoproteins and choles- rate ratio (13.5) at the time of hypoglycaemia. This patient also had hepato- terol). Abnormalities could be demonstrated in the DE-NMR spectra of HIS megaly at presentation and abnormal transaminases (ALT 520 IU/L). (histidine), PKU (phenylalanine) MMA (methylmalonic acid) and IV (3-hydrox- The pattern of organic acid metabolites was non-specific and varied from sample yisovaleric acid) patients. Metabolites were quantified using the maleic acid peak. to sample, but included combinations of ethylmalonic and dicarboxylic, hydrox- Conclusion: DE-NMR spectroscopy can be used to identify low molecular ycarboxylic, 3-hydroxydicarboxylic (including 3-hydroxyglutaric) and 3- mass molecules in intact blood plasma. The identification was possible for oxodicarboxylic acids. 4-hydroxy-6-methyl-2-pyrone previously postulated to be abnormal metabolites of patients with HIS, PKU, MMA and IV. a biochemical marker for this condition, was present in 6 out of 8 urine samples. In our experience 4-hydroxy-6-methyl-2-pyrone is a valuable marker for HMG synthase deficiency, whilst ketonuria does not preclude the diagnosis. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S61

P-124 P-126 NEURODEGENERATIONINAPATIENTWITH INTERACTION OF GLUTARIC ACIDURIA 1-RELATED GCDH METHYLMALONIC ACIDURIA FOLLOWING COMBINED LIVER WITH MITOCHONDRIAL MATRIX PROTEINS AND KIDNEY TRANSPLANTATIONS: THE MECHANISM OF Lamp J1 , Braulke T1 , Mühlhausen C1 NEUROTOXICITY? 1Univ Child Hosp, Hamburg-Eppendorf, Germany Khan M1 , Cole DS1 , Badminton MN1 , Shortland GJ2 , Thomas DM3 , Moat SJ1 Glutaric aciduria type 1 is an inherited neurometabolic disorder caused by 1Dept Medical Biochem, Univ Hosp Wales, Cardiff, United Kingdom mutations in the glutaryl-CoA dehydrogenase (GCDH) gene. The homote- 2Dept Child Health, Univ Hosp Wales, Cardiff, United Kingdom trameric mitochondrial GCDH complex catalyzes the oxidative decarbox- 3Neph Transp Dir, Univ Hosp Wales, Cardiff, United Kingdom ylation of glutaryl-CoA in the degradation pathway of lysine and tryptophan. GCDH deficiency leads to the accumulation of glutaric and 3- The poor neurological outcome of patients with methylmalonic aciduria is well- hydroxyglutaric acid in tissues and body fluids. During catabolic states, recognised, and various mechanisms of neurotoxicity have been proposed. affected patients are prone to encephalopathic crises, characterized by the We describe a patient with methylmalonic aciduria who underwent two destruction of striatal neurons and a subsequent irreversible movement combined liver and kidney transplants early in life. Neurological compli- disorder. Considerable variation in severity of the clinical phenotype is cations appeared later in life, including a haemorrhagic stroke at the age of observed with no correlation to the genotype. 20, and development of a movement disorder. The patient died at the age of Several pathogenic mutations are localized at the surface of the GCDH 31, following complications of acute tubular necrosis, and a second cere- protein and show an impaired capability to form complexes with other brovascular infarct. proteins which might regulate GCDH activity. To identify potential inter- Following neurological deterioration in the last week of life, CSF samples were acting partners, mitochondrial matrix proteins were examined by GCDH- obtained. Organic acid analysis of CSF demonstrated increased levels of lactate and affinity chromatography followed by mass spectrometry of bound proteins. methylmalonic acid (MMA), in the absence of propionate, 3-hydroxypropionate, Four potential binding partners were identified. The interaction of a 50 kDa methylcitrate, and other metabolites of mitochondrial dysfunction. Quantitation protein with GCDH was confirmed by co-precipitation and protein com- revealed grossly increased CSF MMA (499 μmol/L, RI 0.27-0.57), with normal plementation assay experiments. Currently the binding affinities between CSF 5-methyltetrahydrofolate. These findings do not support hypotheses of neu- mutant GCDH and the interaction partner are quantified by microscale rotoxicity previously described, such as disturbance of mitochondrial function and thermophoresis and fluorescence resonance energy transfer. The data may accumulation of multiple toxic dicarboxylic acids in the brain. provide new insight into the regulatory mechanism of GCDH activity. This case illustrates that organ transplantation in methylmalonic aciduria is not curative, and despite long-term aggressive management, neurological complications still occur. The CSF studies suggest MMA to be the principal neurotoxic metabolite; further studies are required to explore the mechanisms of its neurotoxicity. P-127 MITOCHONDRIAL DYSFUNCTION AS A PHENOTYPIC MODIFIER IN ISOLATED METHYLMALONIC ACIDURIA TYPE CBLB P-125 Richard E1 , Brasil S1 , Desviat LR1 , Ugarte M1 , Perez B1 DETECTION OF URINARY ORGANIC ACIDS IN HIGH RISK 1Cent Diag Enf Mol, UAM, Madrid, Spain EGYPTIAN CHILDREN BY ELECTROSPRAY TANDEM MASS SPECTROMETRY In metabolic disorders, several genotype-phenotype inconsistences have 1 1 1 Fateen E.M. , Gouda A , Elbaz A been described. Here we report differences in the disease outcome of 1 Dep Bioch Genet,National Research Centre, Cairo, Egypt siblings with methylmalonic aciduria cblB type and their relation with mitochondrial function. We have analysed several parameters to evaluate a) Objectives: Establishing an approach for early diagnosis and monitoring the mitochondrial dysfunction in two pairs of siblings with p.Ile96Thr/ of disorders associated with accumulation of organic acids in high risk p.Ser174fs (P1 and P2) and p.His183Leu/p.Arg190dup (P3 and P4) geno- Egyptian children using liquid chromatography tandem mass spectrometry types. ROS levels, oxygen consumption, glycolytic parameters and mito- (LC/MS/MS) chondrial morphology are significantly altered in those patients who had a b) Design and Methods: Organic acids were detected in urine using LC/ severe presentation of the disease (P1 and P3) compared to their siblings MS/MS and results were then confirmed by gas chromatography/ mass with a milder presentation and no clinical symptoms. The results suggest spectrometry (GC/MS). For confirmation of the type of organic acidurias that ROS could alter the bioenergetic reserve capacity and might lead to (OA), acylcarnitines and/ or amino acids profiles using LC/MS/MS were fission and a grain-like structure of the mitochondrial reticulum in those determined in all patients showing abnormal organic acids profiles patients more severely affected. Additionally, permeabilized derived-patient c) Results: LC/MS/MS detection of urinary organic acids revealed 31 out fibroblasts compared to healthy control cells revealed that in vitro mito- of 50 subjects (62 %) with 11 different OA. chondria display an enhanced Ca2+ uptake velocity and premature opening d) Conclusion: Organic acids profile must be determined in all neonates of the permeability transition pore which has been described to provoke the admitted to critical care units with unexplained neurological manifestations. release of mitochondrial proapoptotic factors. These studies using cell LC/MS/MS is a fast efficient technique for the detection of many organic models establish that mitochondrial dysfunction could be considered as a acids in urine and can facilitate the future screening for OA among high risk putative disease mechanism probably involved in the clinical phenotype of patients. the disease. S62 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-128 P-131 DIFFERENTIAL CHEMICAL DIAGNOSIS OF PRIMARY EVIDENCE THAT THE METABOLITES ACCUMULATING IN HYPEROXALURIA TYPE II USING URINE GC/MS ANALYSIS 3-HYDROXY-3-METHYLGLUTARYL-COA LYASE DEFICIENCY Inokuchi T1 , Tashiro K1 , Aoki K1 , Inaba M1 , Inoue K1 , Watanabe Y1 , INDUCE OXIDATIVE STRESS IN RAT STRIATUM IN VIVO Tanaka S2 , Matsuishi T2 Fernandes CG1 , da Rosa MS1 , Seminotti B1 , Leipnitz G1 , Wajner M1 1Res Inst of GC/MS,Kurume Univ, Kurume, Japan 1UFRGS, Porto Alegre, Brazil 2Pediatrics, Kurume Univ, Kurume, Japan 3-Hydroxy-3-methylglutaryl-CoA lyase deficiency (HMGLD) is a neuro- Introduction: We have used urine GC/MS analysis since 1996 in neonatal metabolic disorder biochemically characterized by the predominant accu- screening for inherited metabolic disorders. During this screening process, mulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric we encountered a patient with elevated urinary excretion of glyceric acid (MGA) acids. Affected patients usually present neurological dysfunction (GA). GA exists as two optical isomers: a D-form; and an L-form. D- whose pathogenesis is still not established. In this work we investigated the glycerate kinase deficiency is characterized by elevated D-GA levels, while ex vivo effects of an acute intrastriatal injection of HMG and MGA on primary hyperoxaluria type II (PH2) is characterized by elevated L-GA important parameters of oxidative stress, such as thiobarbituric acid- levels. Differentiation of these diseases is critical, as different treatments are reactive substances (TBA-RS) levels, carbonyl formation, reduced glutathi- required. one (GSH) levels, nitric oxide production and the activities of various Patient: The patient had been followed up since hematuria and renal calculi antioxidant enzymes in rat striatum. HMG and MGA increased TBA-RS were identified at 2 years old. The patient's doctor identified proteinuria and carbonyl formation, reflecting an induction of lipid and protein oxida- when the patient was 5 years old and requested urine analysis from our tive damage, respectively. Furthermore, only HMG increased nitric oxide facility. production, suggesting a role for reactive nitrogen species in HMG-induced Methods and Results: D- and L-GA were isolated and identified using GC/ toxic effects. HMG and MGA also decreased GSH levels, the most impor- MS analysis by O-acetyl-(+)-2-butylation of GA according to the methods tant cellular antioxidant in brain and changed the activities of the antioxi- of Inoue et al. GC/MS analysis by this method showed that the GA present dant enzymes, implying that the brain antioxidant defenses were at high levels was L-GA, achieving chemical diagnosis of PH2. compromised by the major metabolites accumulating in HMGLD. The data Conclusions: Our method, which involves butyl esterification of GA, strongly indicate a disruption of redox homeostasis caused by HMG and allows the separation of optical isomers of GA without the need to recon- MGA in striatum in vivo, and this may represent a pathomechanism of brain figure the column or analytical conditions. This appears to be a very useful damage found in HMGLD. method for chemically differentiating between D-glycerate kinase deficien- Supported by: CNPq, PROPESq/UFRGS, FAPERGS, FAPESP, PRONEX, cy and PH2. FINEP IBN-Net and INCT-EN.

P-130 P-132 MITOCHONDRIAL BIOENERGETICS AND CELLULAR ENERGY HIGH DIETARY LYSINE SUPPLEMENTATION DISRUPTS TRANSFER ARE COMPROMISED BY 3- BRAIN REDOX HOMEOSTASIS IN GLUTARYL-COA METHYLCROTONYLGLYCINE IN THE HEART OF YOUNG RATS DEHYDROGENASE DEFICIENT MICE Moura AP1 , Lobato VGA1 , Zanatta A1 , Busanello ENB1 , Grings M1 , Seminotti B1 , Amaral AU1 , da Rosa MS1 , Fernandes CG1 , Leipnitz G1 , Tonin AM1 , Ribeiro CAJ1 , Leipnitz G1 , Wajner M1 de Souza DOG1 , Woontner M2 , Goodman S2 , Koeller DM3 , Wajner M4 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil 2School Medicine University of Colorado, Denver, United States 3-Methylcrotonyl-CoA carboxylase deficiency (3-MCCD) is an inherited disor- 3Oregon Health & Science University, Portland, United States der biochemically characterized by tissue accumulation and high urinary excre- 4Hospital de Clinicas Porto Alegre SGM, Porto Alegre, Brazil tion of 3-methylcrotonylglycine (3-MCG), as well as 3-hydroxyisovalerate, and 3-hydroxyisovaleryl-carnitine to a lesser extent. Clinically, patients usually We investigated important parameters of oxidative stress in brain (cerebral present myocardiopathy and other clinical features whose pathogenesis is still cortex, striatum and hippocampus), liver and heart of 30-day-old GCDH unclear. We investigated the in vitro effects of 3-MCG (0.1 -5 mM) on important deficient knockout (Gcdh-/-) and wild type (WT) mice submitted to a parameters of energy metabolism in heart of 30-day-old Wistar rats. 3MCG did normal (0.9 %), or a high lysine (Lys) diet (2.8 or 4.7 % Lys) for 60 not change CO2 production from acetate but reduced the activity of complex IV hours.The dietary supplementation of 2.8 % or 4.7 % Lys elicited noticeable of the respiratory chain. We also observed that 3MCG inhibited the activity of oxidative stress, as verified by increase of malondialdehyde concentrations mitochondrial creatine kinase (mCK). Furthermore, antioxidants prevented the (lipid oxidative damage) and 2-7-dihydrodichlorofluorescein (DCFH) oxida- effect of 3MCG on the activity of mCK suggesting the involvement of reactive tion (free radical production), as well as decrease of reduced glutathione levels species on this inhibitory effect. Our results indicate that 3-MCG impairs heart and alterations of various antioxidant enzyme activities in cerebral cortex and homeostasis at the level of energy formation and transfer. It is therefore pre- striatum, but not in hippocampus, liver and heart from Gcdh-/- mice, as sumed that these mechanisms may be involved in the pathophysiology of the compared to WT mice receiving the same diets. The results indicate a disrup- cardiomyopathy presented by patients affected by 3-MCCD. tion of redox homeostasis in cerebral cortex and striatum of young Gcdh-/- Financial support: Research grants from CNPq, PROPESq/UFRGS, mice exposed to increased Lys diet. It is presumed that this pathomechanism FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neurociência triggered by Lys overload may contribute to the brain injury observed in (IBN-Net) # 01.06.0842-00, Instituto Nacional de Ciência and Instituto children under metabolic stress in the early stages of glutaric acidemia type I. Nacional de Ciências e Tecnologia – Excitotoxicidade e Neuroproteção. Financial support: Research grants from FIPE/HCPA, CNPq, PROPESq/ UFRGS, FAPERGS, PRONEX, FINEP Rede Instituto Brasileiro de Neuro- ciência (IBN-Net) # 01.06.0842-00, Instituto Nacional de Ciências e Tec- nologia – Excitotoxicidade e Neuroproteção. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S63

P-133 P-135 3-HYDROXY-3-METHYLGLUTARYL-COENZYME A SEVERE NEONATAL PRESENTATION IN A NEW PATIENT (HMG-COA)-LYASE DEFICIENCY: A DISORDER OF KETOGENESIS WITH DIHYDROLIPOAMIDE DEHYDROGENASE DEFICIENCY AND LEUCINE CATABOLISM Merinero B1 , Belanger A2 , Sanz P1 , García-Muñoz F1 , Ecay MJ1 , Ferrer Sass JO1 , Beermann F2 , Spiekerkoetter U3 , Hismi B4 , Coskun T4 , I1 , Navarrete R1 , Rodríguez-Pombo P1 , Martínez-Pardo M2 , Ugarte M1 Wegener VM2 , Bähr L1 1CEDEM, CBM-SO, UAM, Madrid, Spain 1Kinderspital Zürich, Klin Chem Biochem, Zürich, Switzerland 2Serv Pediatr, Hosp Ramón y Cajal, Madrid, Spain 2ZKJ, Lab Klin Biochem Stoffw, Freiburg, Germany 3ZKJ, Päd Stoffwechselstörungen, Düsseldorf, Germany Dihydrolipoamide dehydrogenase (E3) is the common component of the 4Hacettepe Univ Fac Med, Dept Paediatr, Ankara, Turkey three α-keto acid dehydrogenase complexes: pyruvate dehydrogenase, α- ketoglutarate dehydrogenase and branched-chain α-keto acid dehydroge- In HMG-CoA-Lyase (HMGCL) deficiency both ketogenesis from fatty acids nase. Its deficiency is a very rare disorder, and only very few cases have and leucine catabolism are impaired. So far, less 100 patients have been reported. been described with a variable clinical presentation. We present four children of Turkish or Arab origin, who all showed feeding We describe a new case with neonatal, severe metabolic acidosis, hyper- difficulties, tachydyspnoea and metabolic acidosis within the first five days ammonemia and hypoglycemia during E.Coli sepsis, diagnosed at 7 days of of life. In all patients the pattern of urinary organic acids suggested HMGCL age due to the mild increase of branched-chain amino acids in body fluids deficiency, while the acylcarnitine profile pointed in this direction in three of (Leu 490 μmol/L, NV: <120; Alloileu 27, NV nd), an important increase of four cases only. Two of the children receive normal feedings, the other two are lactic acid in plasma (13.8 mmol/L, NV <2) and CSF (7.5 mmol/L; NV on a diet restricted in leucine and supplemented with L-carnitine. <1.9), and an impressive urinary excretion of lactic, 2-hydroxybutyric and Assessment of HMGCL activity in immortalized lymphocytes yielded α-ketoglutaric acids. E3 activity was decreased in cultured fibroblasts (3 % strongly decreased values in all cases. Three mutations were detected in of controls), as well the [1-14 C]Leucine decarboxylation rate (<10 % of the HMGCL gene, which have not been reported previously. In addition to controls). The analysis of the DLD gene identified two novel variations: the stop mutation c.31 C>T (p.Arg11X), a mutation that affects splicing c.259 C>T, causing the missense change p.Pro87Ser predicted as patho- (c.497+1dupG) and a missense mutation (c.272 T>A; p.Val91Asp) were genic; and c.946 C>T causing a premature stop termination codon identified. Following early diagnosis, all four patients have shown normal p.Arg316term. development so far. However, two of them are just one year old. Systematic Under a restricted diet in branched-chain amino acids with riboflavin and assessment of the clinical course including biochemical and genetic character- thiamine, patient presents a very mild central hypotonia without peripheral istics appears prudent. It may contribute to a better understanding of HMGCL hypertonia at 6 months of age. Plasma amino acids have normalized and deficiency and can possibly help to predict the outcome in this disease. only a mild excretion of α-ketoglutaric acid persists. Conflict of Interest declared.

P-136 NEPHROTOXICITY OF MALEIC ACID AND METHYLMALONIC P-134 ACID - ON THE DIFFERENCE OF FANCONI SYNDROME AND PROPIONIC ACIDEMIA: IS THERE A DECREASE IN TUBULOINTERSTITIAL NEPHRITIS TRICARBOXYLIC ACID CYCLE ENZYME FUNCTIONS? Ruppert T1 , Tuncel* AT1 , Opp S1 , Suormala T2 , Okun JG1 , Kölker S1 , 1 1 1 Chapman KA , Markowitz-Shulman A , Cunningham G , Cabrera-Luque Morath MA1 , Sauer SW1 1 1 J , Summar M 1University Children's Hospital, Heidelberg, Germany 1 Children's National Medical Center, Washington, United States 2University Children's Hospital, Zürich, Switzerland

Propionic acidemia (PA, OMIM #606054) is an organic acidemia Background: Chronic renal damage is a common finding in inborn defects caused by dysfunction of propionyl CoA carboxylase leading to accu- of energy metabolism. Patients can develop either Fanconi syndrome or mulation of propionic acid and decrease of down-stream intermediates tubulointerstitial nephritis. including the tricarboxylic acid cycle (TCA) intermediate, succinyl CoA. Objectives: Our study aimed to differentiate the pathomechanism underly- Thus, TCA cycle function can be decreased in PA explaining, in part, ing both forms of chronic kidney damage. Therefore, we stressed human the decreased energy phenotype seen in patients. Our studies demon- proximal tubule epithelial cells either with maleic acid, a known inducer of strate that total protein levels do not change under low glucose con- Fanconi syndrome in animal models, or methylmalonic acid, accumulating ditions compared to regular growth conditions for lymphoblastoid cell in methylmalonic aciduria. Patients affected with the latter disease often lines from individuals with PA. In addition the level of the TCA cycle develop tubulointerstitial nephritis. enzyme, oxoglutarate dehydrogenase (OGDH) as measured semi- Material and Methods: Cytotoxic effects of used agents were detected by quantitatively by Western blot when cells are grown in low glucose measuring LDH release as well as ethidium homodimer and calcein AM conditions as compared to typical conditions is unchanged. Unlike staining. Subsequently, rescue experiments were performed with different OGDH, fumarate hydratase (FH) levels are increased in low glucose amino acids and transporter inhibitors. We additionally investigated the conditions compared to typical conditions after one day. Despite effect of both agents on energy homeostasis, i.e. glycolysis, respiratory increases in total protein between days one and three, fumarate hydra- chain, citrate cycle, and tubular transport processes. tase amounts are decreased day 3 compared to day 1. Perhaps, cells Results and Discussion: Methylmalonic acid did not show any acute toxic from PA individuals are programmed, on the short term, to bypass the effects on proximal tubule cells, whereas maleic acid induced cell death, site of their decreased intermediate (succinyl CoA) by increasing protein disturbance of energy metabolism and of cellular calcium homeostasis. These distal to the block (FH), while leaving protein levels proximal to the effects could be blocked by selected amino acids and calcium chelators. Our block (OGDH) less affected. findings indicate that in inborn errors of energy metabolism Fanconi syndrome but not tubulointerstitial nephritis occurs due to disturbed energy supply. S64 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-428 A-011 AN EXONIC SPLICING ENHANCER MUTATION IDENTIFIED IN KIDNEY TRANSPLANTATION IN A 26-YEAR-OLD JAPANESE GERMAN BETA-KETOTHIOLASE DEFICIENT PATIENTS MALE WITH METHYLMALONIC ACIDURIA PRESENTING Fukao T1 , Sass JO2 , Konstantopoulou V3 , Marquardt T4 , Frauendienst- END STAGE RENAL FAILURE (SECOND REPORT) Egger G5 , Kondo N1 Yamamoto S1 ,IchimotoK2 ,KakinumaH3 , Takayanagi M4 , Shigematsu Y5 1Dept Pediatr, Grad Sch Med, Gifu Univ, Gifu, Japan 1Dept Pediat, Shimoshizu Natl Hosp, Yotsukaido, Japan 2Kinderspital Zürich, Clin Chem & Biochem, Zürich, Switzerland 2Dept Pediat, Chiba Univ Hosp, Chiba, Japan 3Univ Child Hosp Heidelberg, Heidelberg, Germany 3Chiba City Sakuragi-en Insti, Chiba, Japan 4Univ Child Hospital Münster, Münster, Germany 4Div Metab, Chiba Children's Hosp, Chiba, Japan 5Klinikum am Steinenberg, Reutlingen, Germany 5Dept Pediat, Univ Fukui Fac Med Sci, Eiheiji, Japan

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inherited Background: Methylmalonic aciduria (MMA) is an inborn error of organic disorder affecting isoleucine catabolism and ketone body metabolism. We acid metabolism. Patients with severe disease develop many complications previously identified c.951 C>T(D317D) in exon 10 which diminish the despite treatment; often, the disease progresses to tubulointerstitial nephritis effect of the exonic splicing enhancer SF2/ASF and causes exon 10 skip- with progressive renal failure or to severe damage of the central nervous ping. We currently analyzed mutations in two German patients (GK59 and system. Liver, kidney, or combined liver and kidney transplantation is advo- GK63). GK59 was a compound heterozygote of IVS8(+1)g>t and D317N. cated when medical treatment is ineffective. Liver or combined liver and GK63 was also a compound heterozygote of N158D and D317N. This c. kidney transplantation is effective to improve the metabolic decompensation; 949 G>A (D317N) mutation occurred at the same codon as c.951 C>T however neurological deterioration has occurred in some individuals after the (D317D). Computer search using ESE finder showed that c. 949 G>A also transplantation. Some individuals have received only renal allografts. affects the SF2/ASF sequence CT949GACGC. In vivo splicing experiment Case Report: We report a Japanese male with MMA MUT0 (R93H, IVS2 clearly showed that exon 10 skipping was induced in the c.949 G>A +5 G>A), who received kidney transplantation because of end stage renal mutant construct as well as c.951 C>T mutant construct. These results failure at age 26 years. The clinical symptoms appeared at age 7 months. He confirmed that both c.951 C>T and c.949 G>A diminished the effect of the progressed into renal dysfunction at age 6 years. Serum levels of creatinine, exonic splicing enhancer and caused exon 10 skipping. It is possible that methylmalonic acid and propionylcarnitine decreased 6.33 to 1.30 mg /dl, some transcripts include exon 10 much less effectively than those skipping 3,360 to 421 nmol / ml, and 90.4 to 25.5 nmol / ml, respectively, a month exon 10. In transient expression analysis of the mutant D317N cDNA, after the transplantation. Neurological deterioration has not occurred mutant protein was unstable and retained no residual activity. 10 months after the transplantation.

A-010 A-012 A REVIEW OF ISOVALERIC ACIDEMIA IN IRANIAN PATIENTS MANAGEMENT OF COMPLICATIONS IN GA I FOLLOWING Zaman TZ1 , Taheri ZT2 , Goyens PG3 , Meulemann AM3 , Moradian RM4 , FORMULA DIET IN AN INDIAN CHILD Ehdaeivand MR1 Lokesh Lingappa1 1Research Unit of Iranian National Societ, Tehran, Iran, Islamic Republic of 1Rainbow Children Hospital, Hyderabad, India 2IEM Unit, Tehran Univ, Tehran, Iran, Islamic Republic of 3Pediatric Lab,Fabiola Hospital,ULB, Brussels, Belgium Introduction: Nutritional deficiency in children on special diets in inborn 4Research Unit of Iranian National Societ, Tehran, Iran, Islamic Republic of errors of metabolism needs to be avoided and recognized early to prevent morbidity and mortality in formula treated children. Background: Isovaleric acidemia (IVA)is an inborn error of leucine me- Case Report: A five month old child with GA I was on tryptophan, lysine tabolism, caused by a deficiency of the enzyme isovaleryl –coA dehydro- free diet since birth presented with diarrhea and skin rashes. Treatment for genase(IVD),which results in the accumulation of isovaleryl-co A and its two days did not resolve the symptoms the child became increasingly derivatives in body fluids. restless requiring admission. Plasma amino acids revealed tryptophan defi- Methods: Retrospective review of all the patients with isovaleric acidemia ciency. Amino infusions were given to raise the tryptophan levels. A diagnosed by the detection of incresed isovaleric acide in urine( GC-MS) fulminate course led to ventilating the child. Child recovered from acute and/or increased level of plasma isovaleryl carnitine(MS/MS), referred to tryptophan deficiency but developed extra pyramidal symptoms of oral our unit between 1999-2011. dyskinesia with no response to Pacitane. Baclofen was started with com- Results: 14 patients were identified, (male;9) age at onset; 1 day – 4 years plete relief in 24 hours and continued till recovery. The child was dis- (mean;17 months). The first symptom was; continuous vomiting; 42.9 %, charged on GA I diet and regular biochemical analysis for tryptophan and seizure; 35.7 %, developmental delay; 21.5 %, sweaty feet odor;14.3 %, lysine. Progressive improvement with no residual neurological damage was failure to thrive; 14.3 %, coma; 7.1 %. High anion gap metabolic acidosis; observed at four years age, the child attends school and has normal mental 64.3 %, hyperammonaemia ; 35.7 %, hypoglycemia;28.6 %, anemia; and motor development. 14.3 %, pancytopenia; 7.1 %, microlithiasis; 14.3 %. With glycine and /or Conclusion: Deficiency of restricted amino acids in the treatment of IEM carnitine, protein restricted diet and emergency treatment of acute hyper- should be watched for and promptly treated to prevent untoward ammonemia and metabolic acidosis, all the cases showed better condition in complications. follow up visits. Mild mental retardation was detected in 28.6 %. Conclusion: With early and effective treatment, morbidity and mortality of this disease can be prevented successfully. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S65

A-013 20. Fatty Acid Oxidation BETA-KETOTHIOLASE (T2) DEFICIENCY DETECTED WITH NEWBORN SCREENING (NBS) P-137 Bettocchi I1 , Baronio F1 , Martini A1 , Bal MO1 , Marsigli A1 , Cassio A1 EFFECTS OF A MEDIUM-CHAIN TRIGLYCERYDE (MCT) DIET 1Neon Screen Centre, Dep Pediatrics, Bologna, Italy ON DILATED CARDIOMYOPATHY IN VLCAD-DEFICIENT MICE Tucci S1 , Flögel U2 , Sturm M1 , Borsch E3 , Spiekerkoetter U1 T2 deficiency is a rare inborn error of metabolism caused by a defect of a 1Div Metab Dis, Univ Child Hosp, Düsseldorf, Germany mitochondrial enzyme involving ketone body and isoleucine catabolism. It 2Dept Mol Cardiol, HHU, Düsseldorf, Germany is associated with recurrent ketoacidotic crises that follow increased dietary 3Dept Gastroent Hepat Infect, HHU, Düsseldorf, Germany protein intake, infections or fasting. These episodes can determine neurological impairments or even death. In Italy NBS programs does not always Hypertrophic cardiomyopathy and arrhythmias are typical manifestations of include metabolic diseases, in particular this defect. In our Centre, we the severe early-onset phenotype of very-long-chain acyl-CoA perform this analysis for about a year and we found one newborn affected. dehydrogenase-deficiency (VLCADD). The recommended treatment She was born at term, pregnancy and delivery were uneventful, breastfed. includes the application of a MCT-modified diet associated with reversal We found at NBS elevated 3-hydroxyisovalerylcarnitine (C5OH) and tiglyl- of cardiomyopathy. However, the impact of MCT as long-term treatment carnitine. At 6 days of age the baby was clinically stable. Routine labora- with respect to the cardiac phenotype is still unknown. tory exams (glucose, electrolytes, blood gas, ammonia) were normal; Here, we study the effects of a long-term MCT supplementation on cardiac plasma acylcarnitine analysis confirmed the elevation of C5OH and tiglyl- morphology and function in VLCAD-deficient (VLCAD-/-) mice. Cardiac carnitine and urinary organic acids analysis revealed elevated tiglylglycine parameters were assessed by in vivo MRI at two different time points. and 2-methyl-3-hydroxybutyrate. We established a mild protein restricted Metabolic adaptations were determined by the expression of genes regulat- diet and we started L-carnitine supplementation. She remained asymptom- ing glucose and lipid metabolism using RT-PCR. These data were subse- atic, she never showed ketonuria but a persistent elevated excretion of quently correlated with cardiac histology. tiglylglycine. Molecular analysis of ACAT1 gene is in progress. Six-months-old VLCAD-/- mice showed under both a control and a MCT- Conclusions: Early diagnosis of T2 deficiency can prevent episodes of modified diet the reactivation of the foetal gene program, indicative of ketoacidosis and lead to a normal development. We suggest to include this metabolic compensatory mechanisms. In one-year-old VLCAD-/- mice analysis in neonatal screening program. the symptoms progressed towards dilated cardiomyopathy with histological evidence of fibrosis and degenerative alterations. These symptoms were more pronounced in MCT fed VLCAD-/- mice. This study shows that in VLCAD-/- mice compensatory mechanisms based on substrate switch in the long-run do not prevent the development of A-014 cardiomyopathy. In VLCAD-/- mice, a continuous long-term MCT diet CHEMICAL DIAGNOSIS OF INBORN ERRORS OF METABOLISM aggravates cardiac alterations. The exact pathogenic mechanisms need to WITH CONCURRENT URINE GC/MS AND BLOOD MS/MS be further studied. ANALYSES Inokuchi T1 , Aoki K1 , Tashiro K1 , Inaba M1 , Inoue K1 , Yanagiuchi C1 , Watanabe Y1 , Matsuishi T2 1Res Inst of GC/MS,Kurume Univ, Kurume, Japan 2Pediatrics, Kurume Univ, Kurume, Japan

At present, we concurrently use two mass spectrometry applications; GC/MS to analyze urease-treated urine and MS/MS to analyze serum. to chemically diagnose inborn errors of metabolism(IEM). Our analysis thus far of about 4,000 primarily high-risk children has revealed 20 diseases in 47 children, including six cases of MMA. Around the world, MS/MS has become an alternative to the Guthrie method as a mass-screening application for MCAD deficiency and other fatty acid oxidation disorders, but the quantitative precision achieved with filter-paper blood samples used is inferior to results from serum samples, because the amino acid and acylcarnitine fraction in filter- paper blood degrades significantly with aging and suboptimal sampling conditions. Urine GC/MS analysis is an established application for definitively diagnosing organic acid metabolism disorders, but sample pretreatment is troublesome and time-consuming. Treating urine with urease followed by ethanol extraction appears simpler and much quicker than the conventional organic solvent extraction process, with the added benefit of also allowing the analysis of both organic acids and more polar substances. Concurrent use of these two mass spectrometry applications should greatly increase the number of diagnosable diseases and diagnostic accuracy, and is the most effective and accurate way to chemically diagnose IEM. S66 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-138 P-139 DISORDERS OF LONG-CHAIN FATTY ACID OXIDATION: METABOLIC CHANGES DURING EXERCISE AND MCT USE IN MANAGEMENT AND OUTCOMES IN A SINGLE CENTRE IN CARNITINE PALMITOYL TRANSFERASE II DEFICIENCY THE UNITED KINGDOM Santra S1 , Whitlock M2 , Webster R2 , Preece M A2 , MacDonald A3 , Glamuzina E1 , Glamuzina E2 , Dixon M2,3 , Prunty H2 , Pitt M2 , Russell- Chakrapani A B1 Eggitt I2 , Abulhoul L2 , Clayton P2,3 , Grunewald S2,3 1Clinical IMD, Birmingham Children's Hosp, Birmingham, United Kingdom 1Starship Children's Hospital, Auckland, New Zealand 2Biochemistry, Birmingham Children's Hosp, Birmingham, United Kingdom 2Great Ormond Street Hospital, London, United Kingdom 3Dietetics, Birmingham Children's Hosp, Birmingham, United Kingdom 3Inst Child Health, Univ Col London, London, United Kingdom Background: Patients with Carnitine-Palmitoyl-Transferase-II deficiency Background: Long-chain fatty acid oxidation disorders (LC-FAOD) result in (CPT2), can develop hypoketotic hypoglycaemia, cardiomyopathy and energy deficiency and accumulation of toxic LC acylcarnitines. Expanded exercise-induced rhabdomyolysis. Little is known about the metabolic newborn screening (ENBS) has broadened the clinical spectrum. Accordingly, changes occurring during exercise or the effects of dietary medium-chain dietary management has been modified particularly for Very long acyl-CoA triglyceride (MCT) in this condition. dehydrogenase deficiency (VLCADD). However the optimal management of Objective: To analyse metabolic changes during and after exercise, with LC-FAOD patients remains to be defined. carbohydrate or MCT, in a CPT2-patient. Objectives: To review outcomes of a 'pre-ENBS' cohort, all of whom were Methods: A 12-year-old male, diagnosed with CPT2 after presenting with a on comparable management. hypoglycaemic seizure at 2 years of age [fibroblast enzyme activity 0.3 nmol/ Methods: Retrospective review of 12 patients: VLCADD (n03), Tri- min/mg protein], was exercised twice, one week apart, on an exercise cycle for functional protein (TFPD) (n01), LC 3-hydroxy acyl-CoA dehydrogenase 30 minutes at 15-20 W resistance and 70 rpm. Blood was taken before, during deficiency (LCHADD) (n08), treated between 1989 and 2012 at Great and for up to 6 hrs after exercise. Before exercising he was given either 0.75 g/ Ormond Street Hospital. kg/dose of glucose/maltodextrin or 0.5 g/kg/dose of MCT. Results: All presented symptomatically (2 days to 8 months) and were Results: Glucose stayed >4.0 mmol/l throughout both tests. There was no treated with minimal LC triglyceride diet, overnight feeding and essential significant change in creatine kinase, alanine/aspartate transaminases or lac- fatty acids. All VLCADD and 6/9 TFPD/LCHADD patients developed tate. 3-hydroxybutyrate rose to 552 micromol/l after exercise with MCT but recurrent rhabdomyolysis. Retinopathy was observed in 5/9 TFPD/ remained <50 micromol/l with carbohydrate. C16-carnitine+C18:1-carnitine/ LCHADD patients; 8/9 had neuropathy (youngest 26 months). Cognitive C2–carnitine ratio increased to 1.0 after exercise with carbohydrate but was impairment was seen in 5/12. Gastrointenstinal symptoms included chronic <0.35 with MCT. There were no clinical symptoms of exercise intolerance. diarrhoea (4/12) with eosinophilic colitis in 3/4. Conclusions: We demonstrated that a CPT2 patient can metabolise MCT Conclusions: Despite intensive dietary treatment the full spectrum of long- safely during exercise. This may be superior to using carbohydrate alone. term complications occurred. The effect of this management on the onset and rate of progression of complications in TFPD/LCHADD is unknown. It does not prevent rhabdomyolysis. Enteropathy is a previously unreported finding.

P-140 IS THERE ANY EFFECT OF GROWTH HORMONE THERAPY ON CARNITINE AND ACLYLCARNITINE STATUS IN CHILDREN? Biberoglu Gursel1 ,HasanogluAlev1 , Eminoglu Fatma Tuba1 ,OkurIlyas1 , Yesilkaya Ediz2 , Tumer Leyla1 ,EzguFatihSuheyl1 , Bideci Aysun2 ,Derin Betul3 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 2Div Ped Endoc, Gazi Univ Hosp, Ankara, Turkey 3Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey

Background: Growth hormone has effects on body fatty acid metabolism. Fatty acids are being moved into the mitochondria by esterification with carnitine. Carnitine is an important mediator for mitochondrial fatty acids oxidation reactions. Objective: The aim of the study was to evaluate the free carnitine and acylcarnitine levels of children under growth hormone therapy. Methods: Free carnitine and acylcarnitine levels were measured by tandem mass spectrometry. Patient group was divided into two groups. First group includes children using growth hormone less than one year and second group includes children under treatment between 1-3 year. Results: Free carnitine, propionyl carnitine and palmitoyl carnitine levels were found to be higher than controls in both of the patient groups. Acetylcarnitine levels were detected higher than controls in the first patient group but lower in the second patient group. Conclusion: It is predicted that the levels of free carnitine and some acyl carnitines are increased by GH treatment. This may be due to the increasing effect of GH on mitochondrial fatty acid oxidation reactions. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S67

P-141 P-143 BEZAFIBRATE IN THE TREATMENT OF VERY LONG-CHAIN TWO ADOLESCENT PATIENTS WITH RIBOFLAVIN ACYL-COA DEHYDROGENASE DEFICIENCY (VLCADD) RESPONSIVE MULTIPLE ACYL-COA DEHYDROGENASE Sturm M1 , Tucci S1 , Herebian D1 , Spiekerkoetter U1 DEFICIENCY (RR-MADD): A RARE DISORDER WITH 1Dept General Pediatrics, Univ Child Hosp, Duesseldorf, Germany HETEROGENEOUS CLINICAL FINDINGS Aktuglu Zeybek AC1 , Kiykim E1 , Cansever MS1 , Ceylaner S2 , Altay S3 , VLCADD is the most common long-chain fatty acid oxidation (FAO) Aydin A1 disorder presenting with hepatic, cardiac or muscular symptoms. Currently, 1Div Ped Nutr Met, Cerr Med Fac, Ist Univ, Istanbul, Turkey dietary modification is the mainstay of therapy. As mitochondrial FAO is 2Intergen Gen Cen, Ankara, Turkey , 3Div Diet, Cerr Med Fac, Ist Univ, regulated via peroxisome proliferator-activated receptors (PPARs), treat- Istanbul, Turkey ment with the PPAR agonist bezafibrate could improve VLCADD. In this study VLCADD and wildtype (WT) mice were fed for two weeks Riboflavin responsive MADD is characterised by intermittent vomiting, with chemise containing 0.3 % bezafibrate or control chemise. Blood, liver, abdominal pain, hypoglycemia, hepatomegaly, metabolic acidosis and cardiac and skeletal muscle were analyzed with respect to bezafibrate may develop progressive proximal myopathy. concentration, mRNA and protein expression of FAO and PPAR genes as A 12 years old male patient was admitted with recurrent episodes of well as C8-CoA and C16-CoA oxidation and carnitine palmitoyl transferase appetite loss, abdominal pain, muscle weakness and pain, headache and 2 activity. joint pain. He was formerly diagnosed as FMF with no mutation and no Although bezafibrate reached sufficient blood and tissue levels, protein and response to colchicine treatment and as acute intermittent porphyria with mRNA expression of PPAR and FAO genes was only significantly in- positive urine porphobilinogen before referred to Pediatric Metabolic Dis- creased in hepatic tissue confirmed by increased enzyme activities. Impor- eases Unit. Another 18 years old male patient was admitted to Pediatric tantly, no effects were observed in cardiac or skeletal muscle in WT and Metabolic Diseases Unit with slowly progressive muscle weakness, fatiga- VLCAD-deficient mice based on other FAO enzymes but VLCAD regulat- bility in neck and lower limbs, exercise intolerance for 2 years which started ed by PPARs. In WT mice VLCAD expression was not stimulated. after a vigorous exercise. EMG was consistent with myopathy, muscle In the mouse model, the expected benefit of bezafibrate in the treatment of biopsy with lipid myopathy. myopathic VLCADD is questionable, despite sufficient muscular drug Both patients were diagnosed as RR-MADD with increase in blood C5-, concentrations. In contrast, beneficial hepatic effects could be observed, C8-, C10-, C12-, methylglutaryl-, C14:2- and C14:1-carnitine levels and however accompanied by signs of hepatopathy. urinary 3-OH-isovaleric, 3-OH-butyric-, glutaric-, adipic-, 2-OH-glutaric-, suberic acid excretion. Mutation analyses are pending. Both patients responded well to riboflavin, carnitine, CoQ10, low-fat diet with uneventful daily life and normalisation of laboratory findings. RR-MADD is a rare disorder and diagnosis is often difficult when symp- P-142 toms are nonspesific. Early diagnosis is essential to achieve the best and HIGHLY EFFICIENT KETONE BODY TREATMENT IN GLUTARIC earliest treatment response. ACIDURIA TYPE II RELATED LEUKODYSTROPHY Gautschi M1 , Nava E2 , Nuoffer JM3 1Metab Dis Unit, Univ Child Hosp, Bern, Switzerland 2Dept Neurology, Univ Hosp, Bern, Switzerland 3Instit Clinical Chem, Univ Hosp, Bern, Switzerland

Background: Glutaric Aciduria Type 2 (GA-II)-associated leukodystrophy may be severe and progressive despite treatment with protein- and fat- restricted diet, carnitine, riboflavin and coenzyme Q10. Administration of ketone bodies (Van Hove et al, 2003) is a promising but rarely documented adjunct. Objectives: To show the clinical efficacy of sodium hydroxy-butyrate (NaHB) therapy in a patient. Case report: A Portuguese boy of consanguineous parents developed progressive muscle weakness, followed by severe metabolic decompensation during a viral infection (hypoglycaemia and coma) at 20years. MR imaging showed leukodystrophy. The diagnosis of GA-II was made by biochemical and molecular analyses. Material and Methods: Enteral NaHB was administered every 4 hours at 300 mg/kgBW/d, progressively increased to 900 mg/kgBW/d and, after 3 months, maintained at 600 mg/kgBW/d (≈3 % caloric need). Clinical monitoring included regular assessments using a motor function score (GMFC-MLD, 00normal, 60maximum impairment), neuropsychological testing (SON-R) and a quality of life questionnaire (Ped QL 2). Results: Follow up was 21 months. The initial GMFC-MLD score of 5-6 gradually improved to 1 after 5 months. Despite significant recovery of speech, global developmental delay persisted (last SON-IQ 66). Social functioning and quality of life considerably improved. Conclusion/Discussion: Sodium hydroxy-butyrate is a highly effective treatment of GA-II-associated leukodystrophy. S68 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-144 P-146 FATAL REFRACTORY STATUS EPILEPTICUS IN A CHILD WITH REVERSIBLE SEVERE CARDIOMYOPATHY INDUCED BY VLCAD DEFICIENCY CALORIC RESTRICTION IN AN ADOLESCENT VLCADD PATIENT O'Callaghan M1 , González V1 , Pineda M1 , Sans O1 , García-Villoria J2 , Diekman E.F.1 , Reimer A.2 , Visser G.1 , de Vries M.C.3 Meavilla S1 , Ormazábal A1 , Jou C1 , Briones P3 , López-Gallardo E4 , 1Dept Metab Dis, Univ Medical Center, Utrecht, Netherlands Montoya J3 , Artuch R3 , García-Cazorla A1 2Dept Ped Cardiology, Radboud Med Center, Nijmegen, Netherlands 1Hospital Sant Joan de Déu, Barcelona, Spain 3Dept Ped Metab Dis, Radboud Med Center, Nijmegen, Netherlands 2Institut Bioquímica Clínica, Hosp Clinic, Barcelona, Spain 3CIBERER, ISCIII, Madrid, Spain Cardiomyopathy is a severe complication of very long-chain acyl-CoA 4Bioch and Molec Biol dept, Univ Zaragoza, Zaragoza, Spain dehydrogenase deficiency (VLCADD). Cardiomyopathy in VLCADD is most often reported in the neonatal period. We describe a presentation of Background: Deficiency of very long-chain acyl-CoA dehydrogenase cardiomyopathy later in life, induced by caloric restriction. (VLCAD) is associated with three phenotypes: the severe early-onset car- A girl, 15 years old, diagnosed with VLCADD shortly after birth diac and multiorgan failure form, the hepatic or hypoketotic hypoglycemic (c.104delC, p.Pro35LeufsX26; <5 % enzyme activity), presented with form in early childhood and the later-onset episodic myopathic form. tachypnea, coughing and wheezing for the last few months. ECG revealed Objectives: to report a case of VLCAD deficiency associated with refrac- disturbed repolarisation. Echocardiography showed impaired left ventricu- tory myoclonic status. lar function (ejection-fraction: 37 %) with a dilated aspect; and mitral-valve Case Report: We report the case of a previously healthy child who insufficiency grade III. Her CK was 250 U/l and NTpro-BNP 21423 pg/mL. presented at 9 months old, with gastroenteritis, hypoketotic hypoglycemia, In contrast, routine cardiac evaluation 9 months earlier was normal. and decreased level of consciousness and myoclonic seizures in extremities With iv Milrinone, iv glucose and diuretic treatment on the intensive care and at facial level. EEG showed multifocal polyspikes. Seizures and EEG unit, she revived within two days. Her history revealed that she had results did not change in spite of glucose infusion and anticonvulsvants. extremely reduced her caloric intake to lose weight. In the last 9 months Organic acids and acylcarnitines suggested VLCAD deficiency. she had lost 11 kg. After three days, enteral feeding (12En% LCT fat, Brain MRI disclosed diffuse cerebral edema, T2 high signal in striatum, pallidus, 20En% MCT fat, 18En% protein, 50En% carbohydrates, 1750 kcal) was corticospinal tracts. During the subsequent 9 weeks the child did not improve restarted and clinical improvement continued. In conclusion, caloric restric- with diet for VLCAD deficiency and antiepileptic drugs, and died in a fatal tion leading to rapid weight loss may induce cardiomyopathy in patients epileptic status. Mutations in ACADVL gene (c.[848 T>C+1748 C>T]; with VLCADD. Timely improvement of caloric intake may resolve this (p.[Val283Ala+Ser583Leu])) confirmed the beta-oxidation defect. No mutations complication (ejection-fraction after 3 months: 57 %). or depletion in muscle mitochondrial DNA were found as well in POLG1 gene. Conclusions: this is the first case of VLCAD deficiency associated with fatal refractory epilepsy. It is difficult to explain this association from a pathophysiological point of view.

P-145 P-147 DIAGNOSIS OF FATTY-ACID β-OXIDATION DEFECTS (FAOD) PSEUDO-DOMINANT INHERITANCE OF GLUTARIC ACIDURIA IN SUDDEN DEATH CASES: BENEFIT OF ACYLCARNTINE TYPE-2 IN A FAMILY: THREE PATIENTS, DIFFERENT CLINICAL ANALYSIS IN HEPATIC TISSUE COURSE MESLI S1 , SAINT-JEAN B1 , COUSIN A1 , CARLES D2 , DUCINT D3 , Tsiakas K1 , Schürmann M2 , Horváth R3 , Ullrich K1 , Santer R1 REDONNET-VERNHET I1 1Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany 1Lab Bioch, hop Pellegrin, CHU Bordeaux, BORDEAUX, France 2Inst Hum Genet, Univ Med Cent Lübeck, Lübeck, Germany 2Lab Anapath, hop Pellegrin, CHU Bordeaux, BORDEAUX, France 3MGZ, Med Genet Cent, Munich, Germany 3Lab Pharmac, hop Pellegrin, CHU Bordeaux, BORDEAUX, France Glutaric aciduria type-2 (GA2) is an autosomal-recessive disorder caused by Mitochondrial fatty acid β -oxidation disorders are a group of clinically and mutations of electron transfer flavoprotein (ETF) or ETF dehydrogenase biochemically heterogenous inherited metabolic defects. About 5 % of all (ETFDH). The clinical spectrum is broad with severe neonatal-onset and milder cases of sudden death are likely caused by an FAOD. Strategies for the late-onset forms. Here, we report a pedigree with dominant inheritance pattern pathological diagnosis of FAOD from post-mortem specimens have been and three affected family members with different severity of their disease. developed. Among these, acylcarnitine profiling in dot blood specimen Case report: A 2-year-old girl, the second child of a non-consanguineous (DBS) as an initial screen appears to be the most convenient and the most family died of severe GA2. Fifteen years later, her mother developed informative test. Unfortunately, in sudden death cases, blood (even DBS from metabolic acidosis and rhabdomyolysis during an acute illness. Acylcarni- neonatal screening) and urine are not always available. However, during tine profile and urine organic acids suggested late-onset GA2. Similar autopsy, systematic pathological investigations, including histological analysis findings confirmed GA2 in the first child of this family, a 16-year-old of the main organs, are executed. In sudden death cases, presence of micro- asymptomatic boy. Molecular genetic analyses revealed that GA2 in this vesicular steatosis at autopsy is suggestive of FAOD. In three sudden death family is caused by three different ETFDH mutations. All affected members cases of FAOD (one Long -chain 3-Hydroxyacyl-CoA dehydrogenase and two were compound heterozygous for two missense mutations, while the non- Very- long -chain Acyl-CoA dehygrogenase deficiencies) presenting with affected father was heterozygous for one. Treatment with riboflavin caused steatosis, acylcarnitine profiles were performed on hepatic specimen. Interest- improvement of symptoms and biochemical parameters of the mother and ingly in these three patients, the hepatic acylcarnitine profiles, compared to her son but had minor impact on metabolites of the daughter. normal post-mortem hepatic tissues, were found in good agreement with their Conclusion: We describe pseudodominant inheritance of GA2. The differ- DBS acylcarnitine profiles. Acylcarnitine profiling on hepatic samples is a ent phenotypes might reflect different severity of the three mutations. Two very simple and no-time consuming method which could be useful in sudden of them are novel and can probably be added to the list of pathogenic death cases in which FAOD are suspected and blood not available. ETFDH variations. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S69

P-148 P-150 CLINICAL OUTCOME, BIOCHEMICAL AND THERAPEUTIC INTRACELLULAR ACYLCARNITINE PROFILING USING IN FOLLOW UP IN AUSTRIAN PATIENTS WITH LONG-CHAIN VITRO PROBE ASSAY AT VARIOUS C0 CONCENTRATIONS 3-HYDROXY ACYL COA DEFICIENCY (LCHADD) SELECTIVELY IDENTIFIES CPT-1 DEFICIENCY AND PRIMARY Karall D1 , Kogelnig K1 , Brunner-Krainz M2 , Plecko B3 , Möslinger D4 , CARNITINE DEFICIENCY Konstantopoulou V4 , Volkmar B5 , Sperl W5 , Scholl-Bürgi S1 Yamaguchi S1 , Purevsuren J1 , Yamada K1 , Takahashi T1 , Mushimoto Y1 , 1Pediatrics I, Medical University, Innsbruck, Austria Kobayashi H1 , Hasegawa Y1 , Takayanagi M2 , Fukuda S1 2Univ Children´s Hospital, Graz, Austria 1Dep Pediatr, Shimane Univ Sch Med, Izumo, Japan 3Univ Children´s Hospital, Zürich, Switzerland 2Chiba Childrens Hospital, Chiba, Japan 4Univ Children´s Hospital, Vienna, Austria 5Univ Children´s Hospital, Salzburg, Austria Background: Neonatal mass screening for mitochondrial fatty acid oxidation (FAO) disorders using MS/MS is becoming popular. In vitro probe Introduction: To evaluate patients with LCHADD in the national setting, acylcarnitine assay (IVP assay) with cultured cells and MS/MS is used for we collected clinical, biochemical and therapeutic data retrospectively. enzymatic diagnosis. However, the conventional IVP assay is not specific Patients: Data of 12 LCHADD patients (median 6, range 1-13 years, 6 male / 6 for CPT-1 deficiency and primary carnitine deficiency (PCD), because a female) were collected. Eight patients were premature, 3 pregnancies compli- large amount of free carnitine (C0) is overloaded in the medium. Herein we cated by HELLP syndrome. Six showed clinical symptoms, the other six were amended IVP assay to detect CPT-1 deficiency and PCD. identified by newborn screening. In 9, biochemical diagnosis was also confirmed Methods: Fibroblasts from patients with CPT-1 deficiency and PCD were enzymatically in fibroblasts. All are homozygous for the common mutation. incubated in a medium containing 0.2 mM palmitate and different concen- Results: All patients have normal psychomotor development. 10/12 present tration of C0 (C0-reduced condition, 10 μM; and C0-excess, 400 μM) for hyperpigmentation of retina, clinically relevant retinopathy only one (age 10 years). 96 h. The AC profiles in both culture medium and cell lysates were None has signs of polyneuropathy. There have been 102 episodes of rhabdomylosis determined using MS/MS. (CK>1000 U/l) (range 1000-95.000 U/l) in 10, 2 have not had CK elevations Results and Discussion: In the C0-excess condition, the ratio of intracel- measured. Therapy consists of fat defined diet in all patients (30-40 % total energy lular C0/C16 was high in CPT-1 deficiency and PCD, but it was difficult to intake, 50 % MCT (8/12) or heptanoate (4/12) based). 2/12 have night feeds, 3/12 distinguish CPT-1 deficiency from PCD because of their extensive overlap. before physical activity. 2/12 are partially or totally fed by naso-gastric tube or PEG. In the C0-reduced condition, however, the ratio of C0/C16 in CPT-1 Conclusion: From the data there is no difference between the patients deficiency was higher than that of PCD and clearly distinguishable. These treated in the centers. Management in all patients is similar to the interna- data demonstrate that intracellular AC profiling using IVP assay at various tional policy. Concluding from single case reports, anaplerotic therapy with C0 concentrations selectively identifies CPT-1 deficiency and PCD. heptanoate could eventually be considered.

P-149 P-151 MCAD DEFICIENT PATIENTS ARE SUBJECTED TO LIPID AND EFFECT OF PROINFLAMMATORY CYTOKINES ON PROTEIN OXIDATIVE STRESS MITOCHONDRIAL FATTY ACID OXIDATION IN DEVELOPMENT Derks TGJ1 , Touw CML1 , Ribas GS2 , Biancini GB2 , Negretto G2 , OF ACUTE ENCEPHALOPATHY Mescka CP2 ,VanzinCS2 , Reijngoud DJ1 ,SmitGPA1 ,WajnerM2 ,VargasCR2 Purevsuren J1 , Hasegawa Y1 , Kobayashi H1 , Yamada K1 , Takahashi T1 , 1Univ of Groningen, UMC Groningen, Groningen, Netherlands Ichiyama T2 , Yamaguchi S1 2Univ Fed do Rio Grande do Sul, Porto Alegre, Brazil 1Shimane University Faculty of Medicine, Izumo, Japan 2Yamaguchi Univ Grad School of Medicine, Yamaguchi, Japan Background: Medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency is the most common disorder of mitochondrial fatty acid oxida- Background: Acute encephalopathy such as influenza associated enceph- tion (mFAO). Animal studies and in vitro studies report an association alopathy (IAE) happens to break out in children. Fatty acid oxidation between defect mFAO and oxidative stress (OS). disorders (FAOD) are also associated with acute encephalopathy. The Objective: To determine whether OS might play a role in asymptomatic underlying mechanism for IAE is still controversial, however increase of MCAD deficient patients. some proinflammatory cytokines is observed in IAE. Herein, the effect of Methods: Study of parameters of oxidative damage and antioxidant de- the proinflammatory cytokines on FAO was determined. fence in MCAD-deficient patients and controls. Patients were subdivided Methods: Fibroblasts from controls and patients with various FAODs were based on therapy: patients without supplementation, with carnitine, and cultured in the presence of cytokines such as IL-1β, IL-6, IL-10 and TNF-α with carnitine+riboflavin. with palmitate loaded condition. FAO capacity was determined by in-vitro Results: Patients presented increased thiobarbituric acid reactive species (TBA- probe acylcarnitine (AC) assay and tandem mass spectrometry. RS) reflecting lipid damage. Protein damage was indicated by decreased concen- Results: The AC profile in the presence of IL-6 and IL-10 was similar to trations of plasma sulfhydryl content and increased urinary di-tyrosine. Patients that of absence of those cytokines in all cases. In the presence of IL-1β and receiving carnitine displayed significantly higher carbonyl content than patient TNF-α, C16 was significantly increased in CPT-2 deficiency, while C14 without supplementation, while TBA-RS were lower. In patients on carnitine+ and C16 were elevated in VLCAD deficiency. Wide ranged ACs (C6-C16) riboflavin, these effects were opposite. Enzymatic antioxidant defences, as deter- were significantly increased in glutaric acidemia type 2, whereas ACs were mined by superoxide dismutase and catalase activities, were increased. barely affected in MCAD deficiency and controls. Conclusions: These are the first indications that MCAD-deficient patients Conclusion: It is suggested that IL-1β and TNF-α inhibit mitochondrial may be subjected to lipid and protein oxidative damage. Increased antiox- FAO, in particular in long chain fatty acids related disorders. Patients with idant enzyme activities suggest involvement of free radicals. Carnitine and long chain fatty acid related deficiencies may be more susceptible to riboflavin might have opposite effects, but the possible mechanisms yet cytokine induced illness. remain speculative. Further studies must determine the clinical relevance, including the place of antioxidant therapy. S70 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-152 P-154 UNCOUPLING EFFECT OF LONG-CHAIN HYDROXYACIDS ON ABNORMAL INFLAMMATORY PROFILES IN PATIENTS WITH OXIDATIVE PHOSPHORYLATION IS DEPENDENT ON CARBON LONG CHAIN FATTY ACID OXIDATION DEFECTS WITH WELL CHAIN LENGTH IN MITOCHONDRIAL PREPARATIONS FROM CONTROLLED GLUCOSE LEVELS RAT HEART Vockley J1 , de Vallejo AN1 Wajner M1 , Tonin AM1 , Busanello ENB1 , Grings M1 , Hickmann F1 , 1Univ of Pittsburgh School of Med, Pittsburgh, United States Ritter L1 , Ribeiro C1 , Leipnitz G1 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil Long chain FAODs have long been treated with medium chain triglyceride oil as an alternative energy source. A clinical trial on treatment of long Long-chain hydroxyacids accumulate in isolated long-chain 3-hydroxyacyl- chain FAODs with triheptanoin has shown that hypoglycemia, but not CoA dehydrogenase (LCHAD) deficiency, a severe inborn error of fatty acid rhabdomyolysis, is essentially resolved with therapy. RNA microarray oxidation. Affected individuals present encephalopathy, hepatic dysfunction expression studies indicate up-regulation of inflammatory pathways in the and cardiomyopathy, whose pathophysiology is virtually unknown, especially VLCAD but not LCAD deficient animals, a finding substantiated by cyto- regarding the cardiac symptoms. We investigated the in vitro effects of 3- kine studies. We hypothesized that these patients might be experiencing hydroxydecanoic (3HDCA), 3-hydroxydodecanoic (3HDDA) and 3- clinically significant inflammation leading to muscle instability. To examine hydroxytetradecanoic (3HTA) acids, which accumulate in LCHAD deficiency, this, anonymous blood samples from 8 patients with VLCAD deficiency on the respiratory parameters state 4, state 3, respiratory control ratio (RCR), as were examined. Affected patients had higher levels of the inflammatory well as on the mitochondrial membrane potential and the matrix NAD(P)H cytokines (expressed either as means or medians) IL1b, IL6, IL17, inter- levels in mitochondrial preparations from heart of young rats. 3HTA markedly feron IFNg, TNFa and the inflammatory monokines MCP1 and MIPb. increased state 4 respiration and diminished the RCR using glutamate plus TNFa is known to be myolitic, while IL6 has been shown to attenuate malate or succinate as substrates, whereas 3 HDDA only diminished the RCR muscle contraction. Co-elevation of TNFa and IL6 has been correlated with with succinate as substrate. 3HDDA and 3HTA also reduced the mitochondrial frailty syndrome and sarcopenia. IL12 and IFNg are the key players of the membrane potential and the matrix NAD(P)H content. The effects of these Th1 pathway, suggesting these two patients may have hyperactive T cells. fatty acids were dependent on the carbon chain length since 3HTA showed the Our results suggest that long-chain FAODs activate inflammation, and that most pronounced effects at lower concentrations as compared to 3HDCA and disease management of VLCAD deficiency may need to include monitoring 3HDDA. These findings indicate that the 3-hydroxy fatty acids that accumu- and possibly therapy of systemic inflammation. late in LCHAD deficiency act as uncouplers of oxidative phosphorylation therefore potentially impairing heart energy homeostasis. Supported by: CNPq, PROPESq/UFRGS, FAPERGS, FAPESP, PRONEX, FINEP IBN-Net and INCT-EN.

P-153 P-155 HOW FAR SHOULD WE GO IN THE INVESTIGATION OF SEARCH FOR VARIATIONS IN THE ETHYLMALONYL-COA VERY-LONG-CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY DECARBOXYLASE GENE IN PATIENTS WITH ETHYLMALONIC (VLCADD)? ACIDURIA Schiff M1 , Karunanidhi A2 , Yeasted R2 , Mohsen A-W1 , Vockley J1 Olsen RKJ1 , Bie A1 , Van Schaftingen E2 , Gregersen N1 1UPMC, Dep Pediatrics, Children's Hosp, Pittsburgh, United States 1Res Unit Mol Med, Aarhus University, Aarhus, Denmark 2Dep Pediatrics, Univ Pittsburgh, Pittsburgh, United States 2Welbio Duve Insti, Univ Catholiq Louvain, Brussels, Belgium

Background: VLCADD is identified in the US through newborn screening Background: Variations in the genes ACADS, encoding short chain (NBS). NBS can unequivocally identify affected individuals, but a growing acyl-CoA dehydrogenase, and ETHE1, a sulfor dioxygenease, are the number of variant patterns may represent mild disease or heterozygote two main causes of ethylmalonic aciduria. Besides rare inactivating carriers. Our aim was to investigate 11 patients suspected of VLCADD variations, the most frequent variations found in the ACADS gene are and review current laboratory tests for VLCADD. c.511 C>T, p.Arg171Trp and c.625 G>A, p.Gly209Ser commonly Methods: Eight of the studied patients had abnormal NBS. Three exhibited found in the population. This suggests that sequence variations in other suggestive clinical findings and blood acylcarnitine profile (two of them genes may contribute to the development of ethylmalonic aciduria. Our had normal NBS and the third one was unscreened). Fibroblasts VLCAD purpose was to check for variations in the ECHDC1 gene encoding activity, immunoblotting/staining for VLCAD on fibroblasts, and sequenc- ethylmalonyl-CoA decarboxylase, a recently described cytosolic enzyme ing of ACADVL were performed. Prokaryotic mutagenesis and expression that metabolizes ethylmalonyl-CoA. studies were performed for all the uncharacterized missense mutations. Methods: All exons of the ECHDC1 gene were PCR-amplified and se- Diagnostic tests including WBC enzyme assay or fibroblast acylcarnitine quenced using DNA from patients (n080) with ethylmalonic aciduria, who profiling for 55 and 84 samples respectively were retrospectively reviewed. had no inactivating variations in the ACADS gene or only one or both of Results: VLCAD activity was abnormal in all of the abnormal NBS the two frequent sequence variations. patients and in the unscreened patient, and normal in the two patients with Results: Twoc variations affecting the coding sequence of ECHDC1 were normal NBS. For these 9 patients, immunoblotting/staining showed vari- found in the heterozygous state in two different patients. able presence of VLCAD; all but one had two mutated alleles. Eight Discussion: As a reduction in ethylmalonyl-CoA decarboxylase activity pathogenic missense mutations were characterized. increases the formation of ethylmalonic acid in cell cultures, these findings Fibroblast acylcarnitine profile was the best predictive screening test. suggest that variations in ECHDC1 may contribute to increased ethylma- Conclusions: These results emphasize the importance of exhaustive inves- lonic aciduria in some rare cases. tigation of abnormal NBS or clinical testing suggestive of VLCADD. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S71

P-156 P-158 FETAL INVOLVMENT IN MULTIPLE ACYL-COA MUTATIONAL SPECTRUM AND GENOTYPE-PHENOTYPE DEHYDROGENASE DEFICIENCY RELATIONS IN 139 PATIENTS SUSPECTED TO SUFFER FROM Martins E1 , Brandão O2 , Bandeira A1 , Rocha H3 , Vilarinho L3 MULTIPLE ACYL-COA DEHYDROGENATION DEFICIENCIES 1Hospital de Crianças Maria Pia - CHP, Porto, Portugal Olsen RKJ1 , Olpin SE2 , Cornelius N1 , Andresen BS3 , Gregersen N1 2Hospital de São João, Porto, Portugal 1Res Unit Mol Med, Aarhus University, Aarhus, Denmark 3Laboratório Nacional de Rastreio, Porto, Portugal 2Dep of Clin Chem, Children's Hospital, Sheffield, United Kingdom 3Dep Biochem Mol Bio, Univ South. Denmark, Odense, Denmark Background: Multiple acyl-CoA dehydrogenase deficiency (MADD) has been described with three main clinical phenotypes: type I neonatal onset Multiple acyl-CoA dehydrogenation deficiencies (MADD) are a heteroge- with congenital anomalies; type II neonatal onset without anomalies; type neous group of disorders characterized by impaired function of several III mild or later onset. mitochondrial flavoprotein dehydrogenases that use FAD – derived from Objectives: Report prenatal and neonatal data (clinical, histological and riboflavin – to oxidise fatty acids and branched-chain amino acids. Based molecular) in two affected siblings with type II MADD. on clinical and biochemical findings patients are divided into severe Case reports: The index case was a first child of non-consanguineous (MADD:S), milder (MADD:M) or riboflavin-responsive (MADD:RR) parents born at 38 weeks. The physical and neurological examinations were forms. Genetic defects of electron transport flavoprotein (ETF) or its dehy- normal. He died suddenly, at the second day of life. In post-mortem drogenase (ETFDH) are the major cause of all three disease forms. Recently investigation, a severe steatosis was identified in liver, as well as in in genetic defects of cellular riboflavin transporters have been identified in a heart, kidney and skeletal muscle cells. The diagnosis was suspected based few patients with MADD:RR. Here we report on the ETF/ETFDH muta- on the acylcarnitine profile and confirmed by molecular analysis (homozy- tional spectrum in 139 patients suspected to suffer from MADD. ETFDH gous for the mutation p.R155G in ETFDH gene) Three years later, a mutations were identified in 53 % of the patients and ETF mutations in termination of second pregnancy was performed at 20th week of gestation, 17 %. In the remaining 30 % patients no disease-causing ETF/ETFDH after confirmation of the homozygous condition of the fetus for the same mutations were identified. Fourteen of these patients were clearly mutation. The fetus presented no significant abnormalities, with only small riboflavin-responsive and may have mutations in genes involved in ribo- droplets of lipids – oil red positives – in some liver cells, flavin metabolism or transport. 77 % of the mutant MADD:S alleles were Comments: Present data support the idea that the disturbance of energy null mutations with devastating effect on enzyme function. At least one of metabolism in type II MADD produces effects in early in fetal life. two mutant alleles in MADD:M and MADD:RR patients were of the missense type giving rise to temperature sensitive fatty acid oxidation as demonstrated in fibroblasts from some of the patients.

P-157 P-159 STUDIES ON THE TRANSLOCATION OF ACYLCARNITINES PHENOTYPING A NEW FAO DEFICIENT MOUSE MODEL ACROSS THE OUTER MITOCHONDRIAL MEMBRANE: ROLE Diekman E.F.1 , van Weeghel M.1 , Visser G.2 , Houten S.M.1 OF CARNITINE PALMITOYLTRANSFERASE 1A 1Dept Gen Metab Dis, Univ Medical Center, Amsterdam, Netherlands Violante S1 , IJlst L2 , te Brinke H2 , Tavares de Almeida I1 , Wanders RJA2 , 2Dept Metab Dis, Univ Medical Center, Utrecht, Netherlands Ventura FV1 , Houten SM2 1Met&Gen, iMed.UL, Fac Pharm, Univ Lisbon, Lisbon, Portugal Background: Very long chain acyl-CoA dehydrogenase deficiency 2Lab Gen Met Dis, AMC, Univ. Amsterdam, Amsterdam, Netherlands (VLCADD) is a disorder of long chain fatty acid beta-oxidation. Patients present with hypoglycaemia, hepatomegaly, cardiomyopathy and myopa- Carnitine palmitoyltransferase 1A (CPT1A), a transmembrane protein of thy. Although the phenotype of the LCAD KO mouse is closely related to the outer mitochondrial membrane (OMM) with the active and regulatory humans, it does not have myopathy. As mice have two enzymes for sites facing the cytosol, is responsible for the conversion of long-chain acyl- degradation of long chain fatty acids (LCAD and VLCAD) we studied a CoAs into the respective acylcarnitines. It remains controversial how these model in which one VLCAD allele was deleted in combination with the acylcarnitine intermediates cross the OMM to be further transported into LCAD KO. LCAD VLCAD double KO mice are not viable. mitochondria for β-oxidation. Previously it has been shown that rat CPT1A Objective: To phenotype the LCAD-/-; VLCAD+/- mouse model. has a hexameric structure potentially arranged as a pore, which may Methods: We cross-bred LCAD+/-; VLCAD+/- mice with LCAD-/-; function as a channel for acylcarnitines in the OMM. We aimed to inves- VLCAD+/+ and characterized the resulting 4 genotypes using metabolic tigate if CPT1A is crucial for the entry of acylcarnitines into the mitochon- cages. dria. We measured oxidation of palmitoylcarnitine in mitochondria isolated Results: Enzymatic activity was significantly decreased and C14:1/C2 from fibroblasts derived from WT and CPT1A null individuals. In addition, acylcarnitine ratio was significantly increased in the LCAD-/-; VLCAD+/- we assessed oxidation of pristanic acid in intact cells, since this pathway mice opposed to all other genotypes. Heat production was decreased in also involves transfer of acylcarnitine intermediates from peroxisomes to LCAD-/-; VLCAD+/+ and LCAD-/-; VLCAD+/- mice, but the RER was mitochondria. We found that the oxidation of both substrates did not differ not significantly changed. Locomotor activity was reduced in the LCAD-/-; between controls and CPT1A deficient cell lines, suggesting that acylcar- VLCAD+/- opposed to all other genotypes. nitines are able to cross the OMM for further oxidation even with severe Discussion: The lower enzymatic activity and deviated acylcarnitine profile impairment of CPT1A function and structure. In conclusion, the hypothet- both indicate decreased FAO flux in LCAD-/-; VLCAD+/-. In addition, ical pore formed by oligomerization of CPT1A does not seem to function as locomotor activity was decreased in LCAD-/-; VLCAD+/- mice suggesting a channel for mitochondrial uptake of acylcarnitines. a phenotype more closely related to humans. S72 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-160 P-161 CARNOSINASE METABOLISM IN VLCAD-DEFICIENT AND IN DUAL EFFECT ELICITED BY ACUTE ETHYLMALONIC ACID DIABETIC MICE ON ACETYLCHOLINESTERASE ACTIVITY AND MRNA LEVELS Tucci S1 , Spiekerkoetter U1 , Forsberg E2 , Zschocke J3 , Schmitt CP4 , IN CEREBRAL CORTEX OF RATS Klingbeil K4 , Peters V4 Ferreira GC1 , Rodrigues LB1 , Furlanetto CB1 , Machado JL1 , Fraga DB1 , 1Div Metab Dis, Univ Child Hosp, Düsseldorf, Germany Ghedim F1 , Pereira TCB2 , Zugno AI1 , Bogo MR2 , Streck EL1 , Schuck 2Dept Mol Med Surg, Karolinska Institute, Stockholm, Sweden PF1 3Div Hum Gen, Medical University, Innsbruck, Austria 1Universidade do Extremo Sul Catarinense, Criciuma, Brazil 4Centre Paed Adol Med, Medical University, Heidelberg, Germany 2Pont Univ Cat do Rio Grande do Sul, Porto Alegre, Brazil

Carnosinase, a N-glycosylated dipeptidase, degrades histidine-containing Background: High concentrations of ethylmalonic acid (EMA) occur in dipeptides, such as carnosine and anserine. Those dipeptides are known to tissues of patients affected by short-chain acyl-CoA dehydrogenase defi- have several protective functions, especially as antioxidant agents. We ciency (SCADD) and ethylmalonic encephalopathy (EE), diseases charac- recently postulated that low carnosinase activities result in high histidine- terized by neurological symptoms. dipeptide concentrations which protect from oxidative stress and (diabetic) Objective: Considering that the pathophysiological mechanisms responsi- nephropathy. ble for brain damage in these diseases are virtually unknown, we investi- We now compared carnosinase metabolism in diabetic mice (db/db), gated the in vitro and in vivo EMA effects on acetylcholinesterase (AchE) VLCAD-deficient (VLCAD-/-) and VLCAD-/- mice supplemented with a activity and its gene expression in rat cerebral cortex. medium-chain triglycerides (MCT) over one year. The application of this Material and Methods: For in vitro studies, cerebral cortex homogenates diet resulted in increased hepatic oxidative stress and a clinical phenotype were incubated in the presence of EMA (0.5, 1 and 2.5 mM) for 1 h. For in similar to that of the metabolic syndrome. vivo experiments, animals received three subcutaneous EMA injections Whereas renal carnosinase activity was clearly elevated in diabetic mice, (6 μmol/g; 90 min interval) and were killed 1 h after the last injection. carnosine and anserine metabolism in non-treated VLCAD-/- mice was Control animals received saline in the same volumes. After that, AChE unaffected. In contrast, mice supplemented with a long-term MCT diet activity and semi-quantitative RT-PCR for AchE gene expression were displayed a significant increase in carnosinase activity. measured. VLCAD deficiency did not influence the renal carnosine and anserine Results: We observed that EMA in vitro and in vivo increased AchE metabolism. However, a long-term MCT-diet clinically resulted in a meta- activity. On the other hand, EMA administration significantly decreased bolic syndrome and in increased carnosinase activities as demonstrated in AchE gene expression when compared to saline group. diabetic mice. Increased oxidative stress might be due to decreased Conclusion: Altogether, the present results suggest that cholinergic alter- histidine-dipeptide concentrations as result of elevated carnosinase activity. ations may be involved in the pathophysiology of brain damage found in It is known, that carnosinase activity is influenced by N-glycosylation. It patients affected by SCADD and EE. needs to be further studied which mechanisms are responsible for its up- Financial Support: CNPq and UNESC regulation in MCT-treated VLCAD-/- mice. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S73

P-162 P-163 RISK STRATIFICATION BY RESIDUAL ENZYME ACTIVITY A NOVEL MUTATION IN SLC22A5 (OCTN2) GENE CAUSING AFTER POSITIVE NEWBORN SCREENING FOR MEDIUM-CHAIN REVERSIBLE DILATED CARDIOMYOPATHYAND HYPOCHROMIC ACYL-COA DEHYDROGENASE DEFICIENCY: DATA FROM A ANEMIA IN A SUDANESE FAMILY COHORT STUDY Choy Y,S,1 , Mazeni A.2 , Kandaveloo G.2 , Zabedah M.Y.3 , Choo K.K.4 , Touw CML1 , Smit GPA1 , de Vries M2 , de Klerk JBC3 , Bosch AM4 , Waerham H.R.5 Visser G5 , Mulder MF6 , Rubio-Gozalbo ME7 , Niezen-Koning KE8 , 1Prince Court Medical Centre,, Kuala Lumpur, Malaysia Elvers B9 , Wanders RJA10 , Waterham HR10 , Reijngoud DJ8 , Derks TGJ1 2National Heart Institute, Kuala Lumpur, Malaysia 1Dept Metab Dis,Univ Med Centre Groningen, Groningen, Netherlands 3Institute of Medical Research, Kuala Lumpur, Malaysia 2Inst Gen Metab Dis,Univ Med Cen Nijmegen, Nijmegen, Netherlands 4Sime Darby Medical Centre, Subang Jaya, Malaysia 3Centre Lys Metab Dis, Erasmus Med Centre, Rotterdam, Netherlands 5University of Amsterdam, Amsterdam, Netherlands 4Dept Metab Dis, Academic Med Centre, Amsterdam, Netherlands 5Dept Metab Endocr Dis, Univ Med Centre, Utrecht, Netherlands Dilated cardiomyopathy usually runs a progressive downhill course despite anti- 6Section Metab Dis, VU Univ Med Centre, Amsterdam, Netherlands failure medications and many patients ended up requiring cardiac transplant. The 7Dept Ped Lab Gen Metab Dis, Univ Med Cen, Maastricht, Netherlands etiologies for dilated cardiomyopathy are diverse and half are familial. We 8Lab Metab Dis, Univ Med Centre Groningen, Groningen, Netherlands followed up a cohort of 65 patients with dilated cardiomyopathy in National 9Lab Infect Dis Perinat Screen, RIVM, Bilthoven, Netherlands Heart Institute. Twenty patients (30 %) were found to have an underlying 10Lab Gen Metab Dis, Academic Med Centre, Amsterdam, Netherlands genetic/metabolic cause. Recently we diagnosed a Sudanese family with systemic primary carnitine deficiency when a 11-year-old boy presented with Introduction: Newborn bloodspot screening (NBS) for medium-chain progressive cardiac failure associated with anemia unresponsive to hematinics acyl-CoA dehydrogenase (MCAD) deficiency identifies subjects with var- for 6 months. Parents were first cousins and his elder brother passed away a year iant ACADM (gene encoding MCAD) genotypes that have never been ago after suffering from similar illness. He had been previously healthy and there observed in clinically ascertained patients. was no muscle weakness. Chest X-ray showed a large globular heart and Objective: To determine whether residual MCAD enzyme activity can echocardiography revealed severely dilated cardiac chambers with very poor contribute in risk stratification after positive NBS. left ventricular contractility and ejection fraction of 18 %. The serum carnitine Methods: A retrospective study was performed of all patients identified upon level was near zero (0.83 μmol/L). Sequencing of the SLC22A5(OTCN2) gene NBS in The Netherlands between 2007-2010. MCAD enzyme analyses were identified a novel homozygous mutation c.740 C>G (p.Pro247Arg) which is performed in leukocytes or lymphocytes, using 3-phenylpropionyl-CoA as a highly conserved amongst species, confirming the diagnosis of primary systemic substrate. carnitine deficiency. Carnitine supplementation (100 mg/kg/day) resulted in Results: Eighty-four patients were identified. Residual MCAD enzyme remarkable improvement. The disease is uncommon in Malaysian population activities were significantly lower in patients with classical ACADM gen- with no case reported for past 15 years since metabolic service was available. otypes (median 0 %, range 0-8 %), when compared to subjects with variant ACADM genotypes (median 25 %, range 0-63 %) (p<0.01). Neonatal presentations were reported in 7 patients, who all displayed residual MCAD P-164 enzyme activities <1 %. After diagnosis, hypoglycaemia, and carnitine FUNCTIONAL ANALYSIS OF MUTANT MCAD PROTEIN FOUND supplementation were only reported in patients with residual MCAD en- IN JAPAN zyme activities <10 %. Hara K1 , Tajima G1 , Okada S1 , Sakura N2 , Takihara Y3 , Kobayashi M1 Conclusion: Residual MCAD enzyme activity can contribute in risk strat- 1Dept.peds, Hiroshima Univ., Hiroshima, Japan ification after positive NBS. All patients with residual MCAD enzyme 2N.H. for S.M.I.D. Suzugamine, Hiroshima, Japan activities <10 % should be considered 'at risk' for clinical symptoms, 3Dept.stemcell biology, Hiroshima Univ, Hiroshima, Japan regardless of ACADM genotype. There are, however, arguments in favor of abandoning the advice on avoidance of prolonged fasting in subjects Background: To date, over 20 Japanese patients with Medium-chain acyl-CoA with residual MCAD enzyme activities ≥10 %. dehydrogenase deficiency (MCADD) have been found. Recently, c.449- 452delCTGA (449del4) mutation had been reported as a common mutation. The other mutations are unique and uncharacterized. To characterize these mutants, we performed analysis of their functions using gene expression system. Methods: Eleven patients with MCADD were studied. Five were diagnosed after metabolic decompensation and 6 were detected from the newborn screening (NBS). We generated eleven mutations found among them by mutagenesis. Plasmid DNAs carrying the WT or each mutants were introduced into HEK293 cells. We evaluated n-octanoyl-CoA dehydrogenase activity of crude cell lysate including mutant MCADD protein by measuring 2-octenoyl-CoA formation at 37 °C. Results: Six mutants showed less than 10 % residual activity. On the other hand, five showed over 50 % activity. We examined residual activities of the latter 5 mutants at higher reaction temperature, and found that one showed significant decline. These 5 mutants had been detected from the NBS and from slight symptom. Conclusions: Our results suggest that some of those newborns detected from the NBS could have high residual enzyme activity. Minute genetic and enzymatic evaluation is essential for appropriate follow-up of cases suspected with MCADD by MS/MS-NBS in Japan. S74 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-165 A-015 RAPID AND UNEQUIVOCAL IDENTIFICATION OF LCHAD/MTP OCTN2 GENE MUTATIONS IN TURKISH PATIENTS WITH DEFICIENCY USING AN IMPROVED U-HPLC BASED ASSAY PRIMARY CARNITINE DEFICIENCY WITH IMPLICATIONS FOR NEONATAL SCREENING Yücel-Yılmaz D1 ,ErsoyM2 , Candan Ş3 ,Balcı M2 ,KılıçM1 ,GökçayG2 , Wanders RJA1 , Ruiter JPN1 , Ferdinandusse S.1 , van Kuilenburg ABP1 , Dursun A1 ,ÖzgülRK4 Waterham HR2 1Dept Pediat, Metab Unit, Hacettepe Univ, Ankara, Turkey 1Div Metab Dis, Univ Child Hosp, Amsterdam, Netherlands 2Div Nutr Metab, Child Hosp, Ist Univ,, İstanbul, Turkey 2University Hospital Amsterdam, Amsterdam, Netherlands 3Bakirkoy Kadin Dogum ve Cocuk Hastanesi, İstanbul, Turkey 4Ins of Child Health, Hacettepe Univ, Ankara, Turkey Background, Objectives: LCHAD/MTP deficiency is one of the more frequent FAO disorders and has been included in neonatal screening programs Primary systemic carnitine deficiency (SCD) is a rare autosomal recessive around the world. Distinction between true and false positives has remained disorder of fatty acid oxidation due to defective carnitine transport. SCD is a problematic. We have used enzymatic analysis of the long-chain 3- potentially lethal, characterized by progressive cardiomyopathy, skeletal my- hydroxyacyl-CoA dehydrogenase (LCHAD) and long-chain 3-ketothiolase opathy, hypoketotic hypoglycaemia, and hyperammonaemia. Primary carni- (LCKT) components of MTP in lymphocytes from neonates to resolve tine deficiency is caused by mutations in OCTN2 gene (SLC22A5), which whether candidate patients are truly LCHAD/MTP deficient or not. encodes a plasma membrane carnitine transporter. In this study, mutation To develop an improved method for the analysis of the LCHAD and LCKT screening of ten exons in OCTN2 gene was performed in 6 Turkish patients components of MTP using U-HPLC rather than spectrophotometric or with SCD. Four different types of pathogenic mutations (two missense, one fluorometric assays. deletion and one duplication) were detected in OCTN2 gene. Mutation anal- Material and Methods: We have transferred our original assay for LCHAD ysis has harboured for two novel mutations including p.Ser190Leu, c. and LCKT to U-HPLC (see Wanders et al. (2010) J.Inherit.Metab.Dis., 33, 63_65delCTT, and known mutations c.254_264dupGGCTCGCCACC and 479-494 for details on assay conditions). p.Leu363Pro. Since oral L-carnitine supplementation makes a significant Results: Using U-HPLC we have now developed a rapid, unequivocal method improvement in clinical symptoms in patients, early diagnosis is of utmost for the analysis of the LCHAD and LCKT activities of MTP which allows importance. Therefore molecular genetic analyses are essential and useful analysis to be completed within a few hours so that results can be reported the diagnostic tool for patients with asymptomatic SCD. It is need to make same day in any case in which the sample has arrived in our lab before noon. genotype –phenotype correlation for severe and mild mutations in OCTN2 Conclusions: We have improved our assay for LCHAD/MTP deficiency gene to understand the clinical outcome in early symptomatic and asymptom- which now allows rapid discrimination between true and false positives atic cases with primary carnitine deficiency. with obvious implications for neonatal screening.

P-166 A-016 NOVEL DIAGNOSTIC RATIO FOR LCHAD DEFICIENCY IN A SHORT CHAIN ACYL- COA DEHYDROGENASE DEFICIENCY, SELECTIVE SCREENING BY TANDEM MASS SPECTROMETRY REPORT OF NINE CASES; IRANIAN EXPERIENCE Baydakova GV1 Zaman TZ1 , Moini HM2 , Mahdizadeh MM3 , Moradian RM4 1Reseach Center for Medical Genetics RAMS, Moscow, Russian Federation 1Research Unit of Iranian National Societ, Tehran, Iran, Islamic Republic of 2IEM Unit, Tehran Univ, Tehran, Iran, Islamic Republic of Severe secondary carnitine deficiency is a frequent finding in fatty acid 3MRI Unit Tehran Univ, Tehran, Iran, Islamic Republic of oxidation disorders and this can lead to a lack of diagnostic acylcarnitines. 4Research Unit of Iranian National Societ, Tehran, Iran, Islamic Republic of In a selective screening by tandem mass spectrometry in 7 patients from 17 (40 %) with long-chain 3-hydroxy-acyl-CoA dehydrogenase deficiency Background: Short chain acyl co A dehydrogenase deficiency (SCADD) is (LCHADD) we observed a decrease of free carnitine concentration and as a an autosomal recessive inborn error of mitochondrial fatty acid beta oxida- consequence - reduce of diagnostic long-chain 3-OH-acylcarnitines concen- tion at the first step of the short chain fatty acid oxidation spiral, which tration. To improve the use of tandem mass spectrometry to selective screening results in accumulation of C4-CoA .SCADD is biochemically characterised for LCHADD we take a new ratio (С18ОН+С18:1ОН+С16ОН)/С0. In all by increased C4-carnitine in blood and ethyl malonic acid in urine. Most patients with LCHADD, this ratio was statistically different from the normal patients affected by this poorly pathophysiologically defined disease suffer and patients with other disorders of fatty acid oxidation. The (С18ОН from neurologic and muscular symptoms instead of attacks of fasting coma. +С18:1ОН+С16ОН)/С0<0,03 cut-off gives 100 % sensitivity, specificity Methods: A retrospective review of all children with SCADD, referred to and positive predictive value of this method. This ratio can be used for our unit between 1999-2011.Cases: 6 male, 3 female; age at referral: 1.25 - improved diagnosis of LCHADD in selective screening by tandem mass 10 years (mean;4.69 years); age at onset:3 days-18 months spectrometry. (mean;9.4 months). Results: Hypotonia and developmental delay; 6/9, vomiting; 4/9, convulsion; 2/9 (one had intractable convulsion despite treatment with four anti- convulsive medication), orthostatic acrocyanosis ;2/9. Mild metabolic acidosis; 3/9, hyperammonaemia; 4/9, high C4 carnitine; 9/9. Undetectable lymphocyte SCAD activity 9/9.Two had normal follow up (one diagnosed by neonatal screening and the other with intractable convulsions); three with severe phenotype and abnormal brain MRI demonstrating high signal in semioval internal capsule and lateral ventricles. Conclusion: SCADD should be considered in the differential diagnosis of patients with developmental delay, hypotonia and seizures. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S75

21. Carbohydrate Disorders P-169 FUNCTIONAL MAGNETIC RESONANCE IMAGING OF LANGUAGE P-167 PRODUCTION IMPAIRMENTS IN CLASSIC GALACTOSEMIA USE OF CONTINUOUS GLUCOSE MONITORS IN CHILDREN Timmers I1 , van den Hurk J2 , Jansma B. M.2 , Rubio-Gozalbo M. E.3 WITH KETOTIC HYPOGLYCAEMIA 1Fac Psych Neurosc, UM; Dept Ped, MUMC, Maastricht, Netherlands , McSweeney M1 , Skeath R1 , Abulhoul L1 , Cleary MA1 2Fac Psych Neurosc, UM, Maastricht, Netherlands 1Dept Metabolic Medicine, GOSH, London, United Kingdom 3Dept Ped, MUMC, Maastricht, Netherlands

Background: Ketotic hypoglycaemia is a common cause of hypoglycaemia Background: Language production impairments are prominent complica- in children. It is a homogeneous condition and the diagnosis is made after tions in classic galactosemia despite dietary treatment. We aim to elucidate exclusion of specific enzyme deficiencies. The prognosis is generally good the neural correlates of these impairments. and many have no further episodes, but a small number continue to be Methods: In an ongoing study, we use magnetic resonance imaging (MRI) problematic with parents reporting hypoglycaemia even without the prov- in 12 patients and 12 controls, obtaining diffusion tensor imaging and ocation of intercurrent illness. functional MRI during rest and language production. At present, 4 patients Objectives: To identify glucose trends and ascertain the true incidence of (age 13.4-17.8y) and 4 healthy controls (age 13.9-18.2y) have been studied. hypoglycaemia in children with ketotic hypoglycaemia. We will present the functional data (3 T head scanner; T2*-weighted Method: The iPro™ 2 professional continuous glucose monitoring system sequence; imaging parameters: TR02000 ms, TE030 ms, 32 slices of (CGMS) was inserted into patients for a period of about 7 days whilst 3.5 mm). During the task, participants either passively watched or overtly children remained at home. Parents kept a concurrent record of daily described a visual animated scene (using minimal or sentence-level syntax). activity and dietary intake. Results: Based on an independent dataset, a region of interest (ROI) was Results: Ten patients were monitored with CGMS. Three profiles were chosen in the left inferior frontal gyrus, a region related to syntactic normal, five showed periods of hypoglycaemia even when well and the planning (LIFG; peak Talairach-coordinates: x0-58, y07, z06). Prelimi- children were commenced on uncooked cornstarch at bedtime. One showed nary ROI analyses revealed that controls showed higher activity during episodes of hypo and hyperglycaemia. One study was invalidated by the language production than during passive watching (p<.001), whereas the child removing the device. Three of the ten patients had repeat monitoring patients did not (p0.92). Further, the controls showed more activity in LIFG following dietary changes which showed that the profile had normalised. during language production (p<.001) compared to the patients. Conclusion: CGMS helps identify extent of hypoglycaemia in children Conclusion: These preliminary results indicate a clearly deviating recruit- whose parents report recurrent episodes and allows individualisation of ment of LIFG (part of Broca's area) during sentence production in the treatments. patients with galactosemia.

P-168 P-170 VITAMIN E SUPPLEMENTATION IMPROVES NEUTROPENIA LIVER TRANSPLANTATION FOR GLYCOGEN STORAGE DISEASE AND REDUCES THE FREQUENCY OF INFECTIONS IN TYPE 1 NON-A: A SINGLE CENTRE EXPERIENCE PATIENTS WITH GLYCOGEN STORAGE DISEASE TYPE 1B Santra S1 , Daly A1 , Salmons E1 , Lloyd C2 , Chakrapani A1 , Sharif K2 , Balivo F1 , Minopoli G1 , Cozzolino M1 , Rossi A1 , Della Casa R1 , Andria McKiernan P2 G1 , Parenti G1 , Melis D1 1Clinical IMD, Birmingham Children's Hosp, Birmingham, United Kingdom 1Depart of Pediatrics "Federico II" Univ, Naples, Italy 2Hepatology, Birmingham Children's Hosp, Birmingham, United Kingdom

Background: Glycogen storage disease type 1b (GSD1b) patients show Background: Glycogen Storage Disease Type 1 Non-A (GSD1nA) is a rare neutropenia and neutrophil dysfunction, which cause recurrent infections disorder of glucose-6-phosphate-translocase deficiency associated with se- and inflammatory bowel disease (IBD). These manifestations are due to verely limited fasting tolerance, hypoglycaemia, hepatomegaly, neutropenia increased intracellular reactive oxygen species of neutrophils. Aim of the and inflammatory bowel disease (IBD). Liver transplantation (LT) has been study was to evaluate the efficacy of antioxidant therapy with vitamin E on indicated for fasting intolerance and reported to improve neutropenia. We clinical and biochemical parameters, neutrophil counts and function, fre- report three cases with metabolic correction at up to 3 years post-cadaveric- quency of infections and IBD in GSD1b patients. LT but with persisting neutropenia and gastrointestinal symptoms. Patients and methods: Eighteen GSD1b patients, median age 12 years, Cases: Patient 1 (c.1105-1106insA-homozygous in SLC37A4) presented 18- were studied over a 2-year-period; only during the second year vitamin E hours-old, hypoglycaemic with lactic acidosis. Prior to LT aged 6, fasting supplementation was prescribed. tolerance was 90 minutes. 3 years post-LT she has normal growth and fasting Results: During vitamin E supplementation frequency (6. ±0.6 vs1.5±0.1, tolerance but remains GCSF-dependent for neutropenia and had perianal p00.003) and severity of infections (score of 3.7±0.4 vs 2.2±0.2,p0 abscesses. 0.0001) significantly decreased. Neutrophils and neutrophil function were Patient 2 (c.1105-1106insA-homozygous in SLC37A4) presented 13-weeks- higher during vitamin E supplementation (1583±668 vs 941±809, p00.03 old, hypoglycaemic with hepatomegaly. Prior to LT aged 8, fasting tolerance and 19.7±5.6 vs 11.7±5.6, respectively) while a significant reduction in the was 2 hours. 2 years post-LT she has normal growth and fasting tolerance but inflammatory activity during vitamin E supplementation (13±1.2 vs 9±1.4, remains GCSF-dependent for neutropenia with chronic diarrhoea. p00.006) was showed. Patient 3 (c.936dupA-homozygous in SLC37A4), presented 6-months-old, Conclusion: Our study suggests a role of vitamin E supplementation at hypoglycaemic with hepatomegaly during bronchiolitis. Prior to LT aged 3, least as an adjunct therapy of GSD1b to reduce G-CSF doses or the fasting tolerance was 2 hours. 5 months post-LT she has normal growth and frequency of G-CSF administration. Vitamin E has evident advantages, as fasting tolerance but remains GCSF-dependent for neutropenia. it can be assumed orally, and it has not been associated with severe side Conclusion: LT results in excellent correction of GSD1nA fasting intoler- effects. ance but in our experience neutropenia and symptoms of IBD persist. S76 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-171 P-173 ASYMMETRIC DIMETHYLARGININE (ADMA) IN CHILDREN PITUITARY DYSFUNCTION IN GLYCOGEN STORAGE DISEASE WITH GLYCOGEN STORAGE DISEASE TYPE I TYPE III Tumer L1 , Kasapkara C S1 , Biberoglu G1 , Kasapkara A2 , Hasanoglu A1 Melis D1 , Rossi A1 , Taurisano R1 , Ginocchio V1 , Minopoli G1 , Della 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey Casa R1 , Andria G1 , Parenti G1 2Div Cardiology, Dışkapı Hosp, Ankara, Turkey 1Depart of Pediatrics "Federico II" Univ, Naples, Italy

Patients with glycogen storage disease type 1 (GSD-1) often have marked Background: Glycogen storage disease type III (GSD III) is a rare disorder hyperlipidaemia with abnormal lipoprotein profiles. This metabolic abnor- caused by a defective activity of glycogen debranching enzyme primarily mality may be at high risk for the development of atherosclerosis. A affecting liver and muscle. Late complications include: growth retardation, recently discussed cardiovascular risk factor, asymmetric dimethylarginine osteopenia/osteoporosis, polycistic ovary disease, hepatocellular carcino- (ADMA), a naturally occurring product of asymmetric methylation of ma. A systematic endocrine assessment has never been reported. proteins, is an endogenous inhibitor of endothelial nitric oxide synthase. Patients and Methods: 9 GSD III (6 males and 3 females) and 18 age and ADMA causes endothelial dysfunction, vasoconstriction, blood pressure sex-matched controls were enrolled. The serum levels of Growth hormone elevation, atherosclerosis, and kidney disease progression. A high preva- (GH), insulin-like growth factor 1 (IGF-1), Gonadotropins, Adrenocortico- lence of elevated plasma ADMA levels is observed in adults with hyper- tropic hormone (ACTH), Cortisol, Dehydroepiandrosterone sulfate cholesterolemia, hypertension, chronic kidney disease, diabetes mellitus, (DEHAS), Androstenedione and Thyroid hormones were evaluated. peripheral arterial disease, coronary artery disease, preeclampsia, heart Results: Pituitary hormone levels were lower in GSD III than in controls: GH failure, liver disease, stroke, and many other clinical disorders. The aim (p00.01), IGF-1 (p00.00), FSH (p00.01), LH (p00.00), ACTH (p00.04). of the study was to evaluate ADMA level in children with GSD type I. Moreover Androstenedione was lower in GSD III than in controls ( p00.00) ADMA level was determined in plasma using HPLC based method. ADMA and fT3 was higher in GSD III than in controls (p00.00). No significant level was similar between children with GSD type I and in the aged difference was observed for TSH, DEHAS, T3, T4, fT4, Cortisol matched healthy control group (0.93±0.23 vs. 1.04±0.41 μmol/l, p0 Conclusion: Our data suggest the presence of a pituitary dysfunction in 0.11). The preservation of normal endothelial function may result from GSD III and further studies are required to be confirmed. A regular endo- diminished platelet aggregation, increased levels of apolipoprotein E, de- crine surveillance is recommended. creased susceptibility of LDL to oxidation-possibly related to the altered lipoprotein fatty acid profile in GSD I- and increased antioxidative defences in plasma protecting against lipid peroxidation.

P-172 P-174 CONTINUOUS GLUCOSE MONITORING IN CHILDREN WITH GSD 1B AND CYSTIC FIBROSIS: MULTIDISCIPLINARY GLYCOGEN STORAGE DISEASE TYPE I MANAGEMENT IN A PATIENT Kasapkara C S1 , Cinasal Demir G1 , Hasanoglu A1 , Tumer L1 Menni F1 , Salera S1 , Bonarrigo F1 , Mirra N1 , Claut L2 , Parini R3 , 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey Colombo C2 1Ped Dep, IRCCS F.Policlinico, Univ Studi, Milan, Italy Glycogen storage disease type I (GSD I) is characterized by impaired 2Center Cystic Fib, IRCCS F Policlinico, Milan, Italy production of glucose from glycogenolysis and gluconeogenesis, results 3Metab Unit, MBBM S. Gerardo Hosp.,, Monza, Italy from defects in the glucose 6-phosphate enzyme system. The clinical manifestations are hepatomegaly, failure to thrive, severe fasting hypogly- We describe the report of 10 years old child suffering from cystic fibrosis cemia within 3-4 h after a meal, hyperlactatemia, hyperuricemia and hy- and glycogen storage disease type Ib. He was born from consanguineous perlipidemia. The aim of the present study was to examine the efficacy of a parents. The diagnosis of cystic fibrosis was performed by neonatal screen- continuous subcutaneous glucose monitoring system, to determine the ing and confirmed by molecular study (homozygous for ΔF508). GSD, magnitude and significance of hypoglycemia in GSD I and to evaluate the suspected from the first week of life (persistent hypoglycaemia<2 mmol/L efficacy of the revised dietary treatment. Sixteen children with GSD I were after fasting of 2 – 3 hours), was confirmed only 9 years later by molecular studied over a 72-h period. All the patients completed the study without any analysis. Enzymatic study for glucose-6-phosphate translocase performed at adverse events. None of the patients experienced irritation or infection at the 10 months of life was normal. Despite treatment with GCSF, neutropenia insertion site. Significant periods of asymptomatic hypoglycemia (below conditioned repeated respiratory infections by multi-resistant germs with 4 mmol/L, 70 mg/dl) were noted, especially during night time. There was a worsening of respiratory function and a bad metabolic control (frequent close correlation between CGM sensor and capillary blood glucose values hypoglycemic attacks during infection in spite of optimal dietetic compli- measured by glucometer. CGM was repeated in all patients 3 months after ance with frequent meals supplemented with uncooked starch and contin- the first monitoring to examine the effects of revised dietary instructions on uous nocturnal gastric drip feeding with sucrose/lactose free formula glycemic control. CGM indicated a considerable reduction in duration of enriched with maltodextrin). The boy had a splenectomy (spleen05 Kg) hypoglycemia, liver size and improvements in secondary metabolic at 7 years of age with improvement of neutropenia. No autoimmune prob- derangements such as hyperlacticacidemia and hyperlipidemia. lems were found until now, but into account his clinical condition a double- lung and liver transplant was considered. Conclusions: A multidisciplinary management involving metabolic, hema- tologic and pulmonary units was mandatory to control the complications related to cystic fibrosis and GSDIb. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S77

P-175 P-177 INSULIN RESISTANCE AND DIABETES IN GLYCOGEN STORAGE MCARDLES DISEASE FEATURING ATYPICAL ISCHAEMIC DISEASE: PRESENTATION OF 5 CLINICAL CASES EXERCISE RESPONSE AND A NOVEL MUTATION Sechi A1 , Ellerton C1 , Peters C2 , Gissen P3 , Mundy H4 , Lachmann RH1 , Petrou P.1 , Pantziaris M.1 , Dionysiou M.1 , Drousiotou A.1 ,KyriakidesTh.1 Murphy E1 1The Cyprus Inst. of Neurol. and Genetics, Nicosia, Cyprus 1C D Metab Unit, Nat Hosp Neurol Neuros, London, United Kingdom 2Paed Endoc, Gr Ormond Str Child Hosp, London, United Kingdom McArdle's disease (glycogen storage disease type V) is an inherited meta- 3In Metab Dis Unit, Gr Ormond Str Ch Hosp, London, United Kingdom bolic myopathy caused by deficiency in the muscle isoform of glycogen 4Dep In Metab Dis, Evelina Child Hosp, London, United Kingdom phosphorylase. Affected individuals are characterized by exercise intolerance due to their inability to mobilize glycogen stores in skeletal muscle. Background: Glycogen Storage Disease (GSD) and its treatment may Typical symptoms include exercise induced myalgias, cramps, premature predispose to the development of insulin resistance and hence type 2 fatigue and recurrent myoglobinuria. diabetes mellitus (DM). We report the first diagnosis of McArdle's disease in Cyprus. The patient, a Case 1: male with GSD Ia, presented with DM aged 23 years. Glycaemia 25 year old male, presented with myalgias, cramps and elevated creatine >7.8 mmol/L; HbA1c 9.7 %. He was overweight (BMI 29.1), and hyper- kinase. Upon forearm ischaemic exercise test evaluation, he exhibited an tensive. After altering dietary intake he lost 10 kg, and HbA1c decreased to exaggerated increase in ammonia (x29) and a normal increase in lactate 5.6 %, without hypoglycaemia. (x3). This represents an atypical response since the usual finding is a lack of Case 2: male with GSD Ia, presented with DM aged 56 years. Post-prandial lactate rise. The diagnosis of McArdle's disease in our patient was con- glucose 18 mmol/L, HbA1c 6.5 %. BMI 26.8. He had hypertension and firmed by the lack of phosphorylase staining and enzymatic activity in dyslipidaemia. After modifying his diet he lost 7 kg, and HbA1c fell to muscle biopsy. 5.9 % without hypoglycaemia. Molecular investigation revealed the presence of a novel homozygous Case 3: male with GSD IX, presented with DM aged 43 years. After 1 year c.1151 C>T transition resulting in the substitution of the highly conserved of treatment with metformin and repaglinide, but poor dietetic compliance, Ala384 by valine. The allele frequency of this mutation in the Cypriot his HbA1c decreased from 9.3 % to 8.0 %. population was estimated to be 0,0097. As appraised by immunoblot Case 4 and 5: brother and sister with GSD Ia, aged 12 and 11 years. They analysis, this mutation does not compromise either the size or the amount had evidence of insulin resistance following 45 g cornstarch load: glucose of phosphorylase synthesized, suggesting that it may directly interfere with rose to 8 mmol/L, insulin 187 mU/L, with hypoglycaemia at 4.5 hours. enzymatic activity. Conclusion: DM is a probably underestimated complication or comorbidity of GSDs. Strict diet optimization, minimizing obesity, and reducing the effects of prolonged exposure to glucose toxicity are important.

P-178 P-176 ABNORMAL CENTRAL TEGMENTAL TRACT SIGNAL IN A PATIENT WITH MILD GLYCOGEN STORAGE DISEASE CLINICAL CHARACTERISTICS IN TWO NEWLY DIAGNOSED TYPE 1A (GSD1A) TRANSALDOLASE DEFICIENT POLISH PATIENTS A 1 2 3 3 1 1 2 2 Renaud DL , Janousek ST , Freese DK , Schwenk WF Tylki-Szymańska A , Stradomska TJ ,JakobsC ,SalomonsGS , 1 1 1 1 2 Department of Neurology, Mayo Clinic, Rochester, MN, United States Taybert J ,Dąbrowska-Leonik N , Rurarz M , Wamelink MMC 2 1 Noran Neurological Clinic, Minneapolis, MN, United States The Children's Memorial Health Institute, Warsaw, Poland 3Department of Pediatrics, Mayo Clinic, Rochester, MN, United States 2VU University Medical Center, Amsterdam, Netherlands Case Report: A 15 month old boy, born to non-consanguineous parents, Transaldolase (TALDO) deficiency (OMIM 606003) is a multisystem se- presented for evaluation of spells. His development and neurological examina- vere disease due to an inborn error of the pentose phosphate pathway. Up to tion were normal. He had hypoglycemia as a newborn. No subsequent episodes now 11 patients from 7 families have been described. Affected patients have of hypoglycemia were documented. His EEG was normal. The MRI revealed abnormal polyol concentrations and seven-carbon chain carbohydrates, abnormal signal in the central tegmental tracts bilaterally. sedoheptulose, mannoheptulose and sedoheptulose-7P in body fluids, most- Evaluation in the Neurometabolic Clinic revealed an elevated uric acid of 11.1 ly in urine. They also present severe symptoms in the neonatal period and (2.4-5.4) and lactic acid at 7.8 (normal <3.2). Hyperlipidemia was present. The some signs already prenatally. The leading symptoms in transaldolase ALT was increased at 479. Neutropenia was absent. Urine organic acids deficient patients are anaemia, bleeding problems with thrombocytopenia, revealed 3-methylglutaconic acid and 3-methylglutaric acid. An abdominal hepatosplenomegaly, nodular, progressive hepatic fibrosis and later on ultrasound demonstrated marked hepatomegaly with normal hepatic echoge- nephropathy. We here present the clinical features of two recently diagnosed nicity. Kidneys were mildly enlarged. The biochemical and clinical profile transaldolase deficient Polish patients from two families. Two boys, cur- were suggestive of GSD1a despite the limited history of hypoglycemia. rently one and two years old presented already prenatally with IUGR, Enzyme analysis of glucose-6-phosphatase activity in liver revealed a ascites and oligoamnion and after birth, anaemia, bleeding diathesis hep- significantly decreased (0.9 micromole/min/gram tissue) but not absent atosplenomegaly, liver involvement. Additionally, both present characteris- enzyme activity (normal 3.50 +/- 0.8). Sequencing revealed a homozygous tic thin, silky skin with visible vessels net and multiple haemangiomas. mutation, c.648 G>T, which results in the creation of a new splice donor Such unusual skin changes made us think straightaway of transaldolase site in exon 5 and premature translation termination. deficiency. Even though transaldolase deficiency has characteristics easily Conclusions: Central tegmental tract abnormalities have been described in identified on the basis of prenatal history, neonatal and postnatal signs and patients with disorders of amino acid, organic acid and tetrahydrobiopterin symptoms it is still an underdiagnosed condition and needs more awareness metabolism. GSD1a should be added to the differential diagnosis of this from clinicians. MRI finding. S78 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-179 P-181 FANCONI-BICKEL SYNDROME : A MILD FORM ASSOCIATED LEPTIN LEVELS IN ADULTS AND CHILDREN WITH CLASSIC WITH GLUT2 MUTATION GALACTOSAEMIA Baronio F1 , Santer R2 , Bettocchi I1 , Graziano C3 , Bergmann J2 , Bal M1 , Knerr I.1 , Coss K.P.2 , Doran P.2 , Hughes J.1 , Treacy E.P.1 Rimondini D1 , Cassio A1 1NCIMD, Univ Child Hosp, Dublin, Ireland 1Neon Screen Centre Dept of Pediatrics, Bologna, Italy 2UCD Clin Res Centre, Mater Univ Hosp, Dublin, Ireland 2Dept of Pediatrics, Univ Medical Center, Hamburg, Germany 3Med Genet Unit, Dept of Pediatrics, Bologna, Italy Background: Classic Galactosaemia is caused by profound deficiency of galactose-1-phosphate uridyltransferase (GALT). The majority of Irish Fanconi-Bickel syndrome (FBS) is caused by mutations in SLC2A2 encod- patients harbour the severe Q188R Galt mutation. Many patients experience ing the glucose-transporter-protein-2 (GLUT2). Recently, two patients with long-term complications such as speech deficits, osteopenia, and premature an unusual mild phenotype were reported (Grünert et al 2012). Here, we ovarian insufficiency with subtle abnormalities of reproductive function describe the similar case of an 8-year-old Italian boy, referred to us because recently reported in males. Leptin is an adipocytokine which affects the of the incidental finding of glucosuria and galactosuria first detected at neuroendocrine reproductive axis. Data on leptin in individuals with Gal- 8 months of age. Due to postprandial hyperglycemia, he was on insulin actosaemia is lacking. glargine for several years in addition to a galactose-restricted diet. In our Methods: We measured serum leptin concentrations in 22 adults (18-37 yrs.) department, oGTT, ivGTT, and abdominal ultrasonography did not show and 19 children (2-17 yrs.) with Galactosaemia. All patients had been treated any severe abnormality, particularly no hepato- and nephromegaly. Urine with lactose-free diet in the Metabolic Centre in either Dublin or Belfast. We examination showed only mild glucosuria but no other signs of tubulopathy. then compared their results with reference data for healthy individuals. Osteopenia was diagnosed by DXA, statural growth and psychomotor Results: In males and in the entire adult cohort, the physiological associ- development were normal. A mild form of FBS was suspected and SLC2A2 ation between log-leptin and body mass index (BMI) was no longer detect- analysis revealed homozygosity for c.589 G>A [p.V197I]. able (r200.1360, and r200.0070). In children with Galactosaemia leptin Conclusion: (i) This is the first FBS case with homozygosity for the well- standard deviation scores (SDS) were lower than the corresponding average characterized variant p.V197I, the first known dysfunctional mutation in a of the controls (mean -0.64 for girls and -0.69 for boys). human facilitative glucose transporter, originally found in a heterozygous Conclusion: Our findings, particularly an absent association between BMI diabetic subject (Tanizawa et al 1994, Mueckler et al 1994). (ii) Mild forms and log-leptin in adults and the reduction in leptin-SDS in children with of FBS should be suspected in patients with glucosuria and only mild Galactosaemia could indicate leptin dysregulation. This may contribute to impairment of glucose metabolism, even without other signs of FBS. long-term complications in patients with Galactosaemia.

P-180 P-182 A NOVEL FAMILY WITH TRANSALDOLASE DEFICIENCY WITH THE INCIDENCE AND NATURAL COURSE OF HEPATIC A HETEROGNEOUS CLINICAL PHENOTYPE RANGING FROM COMPLICATIONS IN GLYCOGEN STORAGE DISEASE TYPE III ASYMPTOMATIC AT 9 YEARS TO LIVER TRANSPLANTATION Sentner CP1 , Smit GPA1 AT 1 YEAR OF AGE 1Dept Metab Dis, Univ Med Centr, Groningen, Netherlands Wamelink MMC1 , Leduc CA2 , Shen Y3 , Sun X3 , Martinez M2 , Jakobs C1 , Salomons GS1 , Chung WK2 Background: Based on multiple small cohorts, hepatic complications such 1Dept of Clin Chem, VU Univ Med Center, Amsterdam, Netherlands as adenomas seem to occur in 10-25 % of the GSDIII patients. The 2Dept of Pediatr, Columbia Univ, New York, NY, United States incidence and nature of GSDIII-related hepatic complications however, 3Dept of Biomed Inform, Columbia Univ, New York, NY, United States remained unclear. Therefore, we conducted a multi-centre retrospective study to investigate the natural course of GSDIII and its hepatic Transaldolase (TALDO) deficiency is a rare inborn error of metabolism in complications. the pentose phosphate pathway (PPP). We report a novel presentation in a Methods: Data on hepatic complications included evaluation by clinicians, family with four boys, of whom the two youngest presented with infantile ultrasound, and MRI. hepatomegaly and cirrhosis. The youngest developed hepatocellular carci- Results: In total data was collected on 225 patients; median age 19.9 years noma (HCC) and received an unrelated cadaveric liver transplant at one (range:0.3–78.3); 47.6 % males. 220 patients had received hepatic evalua- year of age. By RNA sequencing of the tumour a p.Ser171Phe mutation in tion; 24 patients had abnormalities. 16 patients had hepatic cirrhosis (me- TALDO1 was identified and confirmed in the germline in the two affected dian age 18.0 years (0.5–54)), three patients progressed to hepatocellular boys. Sequencing of the parents and two healthy brothers confirmed het- carcinoma (median age 32.0 years (31–35)). Hepatic adenoma was found in erozygosity in the parents and homozygous normal alleles in the oldest 12 patients (median age 24.5 years (1–45); single nodule in five patients; brother. Surprisingly, one of the asymptomatic brothers was found to be multiple nodules in seven patients). Orthotopic liver-transplantation was homozygous for the pathogenic mutation. Metabolite analysis in urine performed in six patients, two patients died of severe liver failure. showed high-normal excretion of erythritol, ribitol and arabitol with clear Conclusions: In the largest GSDIII-cohort to date, cirrhosis is the most elevations of the heptuloses and sedoheptulose-7P in the three affected frequent hepatic complication, while adenomas occur less frequently than children, including the youngest child after his liver transplant. The oldest previously presumed. However, as this cohort is relatively young, the child had a normal metabolite profile. We present here the first asymptom- frequency of hepatic complications may increase with age. atic child (9 years) with TALDO deficiency and the first human with HCC, broadening the clinical phenotype. After liver transplantation, the abnormal urine metabolite profile persists indicating the important role of other tissues the PPP metabolism. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S79

P-183 P-185 CK, ALAT RELATED TO DECREASED BONE MINERAL DENSITY TRANSALDOLASE DEFICIENCY IN 12 PATIENTS WITH NEW IN GLYCOGEN STORAGE DISEASE TYPE III ADULTS PHENOTYPE Sentner CP1 , Minarich L2 , Weinstein DA2 , Smit GPA1 EYAID W1 , Al Harbi T2 , AL SALMAH M2 , Anazi S3 , Alkuraya FS4 , 1Dept Metab Dis, Univ Med Centr, Groningen, Netherlands Alfadhel M2 , Wamelink M5 , Jakobs C5 2Glyc Storag Dis Prog, Univ Florida, Gainesville, FL, United States 1King Abdulaziz Med.City .Ped.Med. Gen., Riyadh, Saudi Arabia 2King Saud bin Abdulaziz University, Riyadh, Saudi Arabia Background: GSDIII patients are known to have decreased bone mineral 3King Faisal Specialist Hospital-Med.Gene, Riyadh, Saudi Arabia density (BMD; Mundy 2008) and an increased risk of fractures. The 4College of Medicine, Alfaisal University, RIYADH, Saudi Arabia underlying mechanism is unknown, and laboratory markers for lowered 5VU medical Center ,Metabolic Unit, Amsterdam, Netherlands BMD have not yet been identified. Therefore, we searched for laboratory values that are associated with BMD in adult GSDIII patients. Transaldolase deficiency is an inborn error of the pentose phosphate path- Methods: DEXA-BMD measurement Z-scores (whole body and lumbar way (PPP) and 19 patients with this disorder have been described. spine) were collected for GSDIII patients (age≥18 years) in combination We report on another 12 children from 6 families who have been followed with cholesterol, triglyceride, ASAT, ALAT, and CK-values. Based on by our genetics clinics. DEXA-BMD-measurements, patients were classified as having normal They were born with intrauterine growth retardation, and dysmorphic BMD, osteopenia, or osteoporosis. Kruskal-Wallis-Test was used to assess features in the form of triangular face with excessive wrinkles on the differences in laboratory values between groups. forehead. Some of them showed generalized cutis laxa. The abdominal Results: 32 patients were included (median age 31.5 yrs [range:19–55]). A exam showed hepatosplenomegaly, and inguinal hernia. They had mild significant difference was found for CK (p00.004) and ALAT (p00.027) hypotonia at birth. Their development appears normal between groups; the osteoporosis group had the highest CK/ALAT values, They have a wide range of cardiac anomalies that range from septal defects to and the normal BMD-group had the lowest. No significant difference was dextrocardia. They had pancytopenia that resolves by 3-4 months of age. Liver, shown for cholesterol/triglycerides/ASAT. skin, and bone marrow biopsies failed to show any unusual inclusions. They start Conclusions: CK and ALAT correlated inversely with BMD in GSDIII showing signs of hepatic fibrosis by 6 yrs of age. Some patients had bleeding adults. Importantly, the highly significant relationship between decreased diathesis that was found to be secondary to platelets aggregation defect in response BMD and elevated CK indicates that muscle involvement is a crucial to Arachodonic acid in the absence of salicylic acid ingestion. Such finding, which component in developing osteoporosis/osteopenia in GSDIII. we confirmed in all subjects, is a novel feature of Transaldolase deficiency. Homozygosity scan highlighted the TALDO gene as a likely candidate. All patients were found to be homozygous for a frame shift mutation NM_006755:c.793del (p.Gln265Argfs*56).

P-184 NEUROMUSCULAR ALTERATIONS IN GLYCOGEN STORAGE DISEASE TYPE I AND IIIA P-186 1 2 2 3 Sentner CP , van der Hoeven JH , Maurits NM , Verbeek RJ , Smit A NEW VARIANT OF CLASSICAL GALACTOSEMIA DUE TO A GPA1 , Sival DA3 1 NOVEL MUTATION IN THE GALT GENE Dept Metab Dis, Univ Med Centr, Groningen, Netherlands 1 2 3 4 2 2 Choy Y.S. , Breda Klobus A , Zabedah M.Y. , Endo F. , Rolfs A. Clin Neurophys, Univ Med Centr, Groningen, Netherlands 1 3 Prince Court Medical Centre,, Kuala Lumpur, Malaysia Dept Child Neurol, Univ Med Centr, Groningen, Netherlands 2Centogene, Rostock, Germany 3Institute of Medical Research, Kuala Lumpur, Malaysia Background: In GSD, altered metabolic control and/or disease progression 4Kumamoto University, Kumamoto, Japan could affect neuromuscular integrity, but details are lacking. In GSDI and GSDIIIa patients, we aimed to evaluate neuromuscular involvement. Classical form of galactosemia is life threatening in the neonatal period with Patients & Methods: We included 12 children (7 GSDI; 5 GSDIIIa); median multiorgan involvement. Long term sequelae include cognitive impairment, – age 7 years (2 17), and 12 adults (8 GSDI; 4 GSDIIIa); median age 31 years speech defects and ovarian failure. It is due to a severe deficiency of the – (18 42). Neuromuscular parameters involved muscle-ultrasound-density enzyme galactose-1-phosphate uridyltransferase(GALT). Milder variants (MUD, measuring water-content; MUD increases by fat-deposition and/or have been described. fibrosis), electromyography and dynamometry (in patients>4 years). MUD We report here a new variant of the disease in an 18- month- old Malaysian and muscle force were expressed as Z-scores (corrected for gender and age). Chinese girl of Fujian, Anhui ancestry. She was fully breast fed since birth. She According to treatment guidelines (Rake,2002; Kishnani,2010) patients with presented with severe hyperbilirubinemia of 587 μmol/L associated with vomit- compromised metabolic control were identified. ing on day 7 of life. There was progressive elevation of liver enzymes and direct Results: Electromyography was normal. In GSDI and IIIa children, MUD bilirubin. The disease culminated with E. coli sepsis,disseminated intravascular was increased (Z>2.0SD and Z>1.5SD, respectively) in association with coagulopathy and psedotumour cerebri on day 14. Dried blood spot galactose 0 compromised metabolic control (p 0.028). In GSDI, however, increased was markedly elevated (9967 μmol/L) and GALT was low 2.2 U/g Hb. pediatric MUD and muscle weakness (Z<-2SD) normalized into adulthood. Galactose- 1-phosphate was elevated 11.2 mg/dL after 2 days of lactose free Exclusively in GSDIIIa adults, we observed an age-related MUD increase, milk. Epimerase assay was normal. GALT gene sequencing revealed a novel, independent of metabolic control. Comparing muscle force, revealed more homozygous mutation in intron 4 (c.377+2dup). So far she has been doing very muscle weakness in GSDIIIa than in GSDI adults (p<0.05). well on lactose-free diet with normal milestones. Long term follow- up and Conclusions: In GSDI and IIIa children, transiently compromised meta- functional study of this genetic variant is needed. Further evaluation of the family bolic control can induce reversible neuromuscular alterations. Thereafter, members and Chinese of Fujian, Anhui ancestry may provide further informa- GSDIIIa adults may show age-related neuromuscular deterioration. tion about this genetic variant. S80 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-187 P-189 MOLECULAR DIAGNOSIS OF HEREDITARY FRUCTOSE LYMPHOPENIA AND ALTERATION OF SPECIFIC LYMPHOCYTE INTOLERANCE SUGGESTION OF A FOUNDER MUTATION SUBPOPULATION AND RISK FOR AUTOIMMUNE DISORDERS IN IN A COMMUNITY FROM INDIA? GSD1B PATIENTS Bijarnia - Mahay S1 , Puri RD1 , Kotecha U1 , Saxena R1 , Treacy R2 , Minopoli G1 , Carbone F2 , Casertano A1 , Della Casa R1 , Andria G1 , Drummond J2 , Verma IC1 Parenti G1 , Matarese G2 , Melis D1 11Center Med Genet, Sir Ganga Ram Hospit, New Delhi, India 1Depart of Pediatrics "Federico II" Univ, Naples, Italy 2E A Med Gen Service, Addenbrooke's hosp, Cambridge, United Kingdom 2Lab Immun, Endo, Exper Oncol, IEOS-CNR, Naples, Italy

Hereditary fructose intolerance is an inborn error of metabolism which is Background: GSD1b patients present autoimmune disorders including difficult to prove, as it requires either invasive liver biopsy based enzyme inflammatory bowel disease (IBD) and Myastenia Gravis. Although these assay or expensive molecular genetic analysis. Therefore, worldwide there manifestations impact significantly on patients' life, their pathophysiology has been a trend towards finding 'common mutations' in distinct ethnic has long remained obscure. groups to simplify the process of diagnosis. The non-specific presentation Patients and methods: Seven GSD1b and fourteen age and sex-matched of the disease often confusing with other metabolic liver disorders like controls were enrolled. The presence of autoimmune disorders, serum levels Glycogenoses, Galactosemia and Tyrosinemia leads to much delay in of autoantibodies, subclasses of lymphocytes, number of T-cells regulating diagnosis and consequent harm to the patient. system (TRegs) were investigated. We report molecular mutations in ALDOB gene, in two Indian patients Results: The serum levels of peripheral lymphocytes were lower in GSD1b from non-consanguineous families but from the same community of than in controls when considering lymphocyte (1539±317 vs 3052±369, Agrawals. Both boys were infants presenting with jaundice, vomiting (with p00.01), CD3 (937±169 vs 1986±192, p00.001), CD4 (540±112 vs 1048 or without diarrhoea), failure to thrive and significant hepato-cellular dys- ±103, p00.005), CD8 (298±51 vs 741±80, p00.001), NK (155±58.6 vs function. The mutation, c.324+1 G>A in ALDOB gene is a novel mutation 320±45, p00.04), CD4CD28 (485.5±104 vs 850±87, p00.01), that was homozygously detected in both children. The community has been CD3CD45RA (523±159 vs 1143±141, p00.01), CD3CD45RO (414±63 known to harbor founder mutations in other genes such as the MLC1 and vs 842±98.9, p00.004), CD4CD45RO (225±44 vs 499±69.6, p00.008). PANK2 genes, thus providing a strong suggestion of a founder effect in the The patients showed reduced TRegs numbers. community for HFI. This may pave the path for a simpler and quicker test Autoimmune diseases correlated with CD8DR cells prevalence (r00.88, p0 that is made available at least for this community in India. 0.019). IBD inversely correlated with NK (r0-0.85, p00.032) and CD4CD8 (r0-0.88, p00.01) prevalence. The serum levels of antibodies anti-acetylcholine inversely correlated with lymphocytes counts (r0-0.97, p00.023), CD4CD8 (r0-0.98, p00.02), CD4CD45RA (r0-0.95, p00.04) prevalence. Conclusion: The data showed a reduction of specific lymphocyte subpo- P-188 pulations and the association between autoimmune disorders and some of CHARACTERIZATION OF LIVER AND MUSCLE PHENOTYPES these abnormalities. The pathophysiology of this association has to be IN A CANINE MODEL OF GLYCOGEN STORAGE DISEASE further investigated. TYPE IIIA Sun B1 ,YiH1 , Thurberg B2 , Austin S1 ,FyfeJ3 , Koeberl D1 ,KishnaniP1 1Dept Peds, Duke Univ, Durham, United States 2Genzyme Corp, Framingham, United States 3Lab Comp Med Genet, Michigan State Univ, East Lansing, United States

Background: Mutations in debranching enzyme cause glycogen accumulation in liver and muscle in glycogen storage disease type IIIa (GSD IIIa). Clinical symptoms vary widely in patients. Objectives: To characterize liver and muscle phenotypes in a canine model of GSD IIIa. Methods: Eight GSD IIIa dogs were studied from age 2 to 16 months. Blood was collected monthly to test the routine chemistry panel. Liver and skeletal muscle biopsies were performed at 4, 12, and 16 months for biochemical and histological analyses. Results: Gradually increasing levels of liver enzymes ALP, ALT and AST were observed in all dogs. CPK activity was elevated after 12 months. Liver glycogen was extremely high at all ages; muscle glycogen gradually increased to high level with age. H&E staining revealed normal hepatic cellular architecture at 4 months but markedly distorted at 16 months due to progressive fibrosis. PAS staining showed scattered cytoplasmic glycogen particles in muscle at 4 months and significantly larger glycogen lakes at 12 and 16 months. Conclusions: Progressive liver fibrosis and muscle damage caused by glycogen accumulation are the main features of affected dogs. This is a very valuable animal model for understanding disease pathogenesis and developing treatment interventions. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S81

P-190 P-192 CARDIAC PATHOLOGYAND CLINICAL FINDINGS COMPARISON THE BITTERNESS OF GLUCOSE/GALACTOSE: NOVEL IN PATIENTS AND DOGS WITH GLYCOGEN STORAGE DISEASE MUTATIONS IN THE SLC5A1 GENE TYPE III Pode-Shakked B1 ,ReishO2 , Aktuglu-Zeybek C3 , Kesselman D4 , Bujanover Austin SL1 , Proia AD2 , Kanter R1 , Drake E1 , Sun B1 , Wechsler SB1 , Y5 ,AniksterY1 Kishnani PS1 1Metab Dis Unit, Sheba Medical Center, Tel-Hashomer, Israel 1Dept of Peds, Duke Univ Med Ctr, Durham, United States 2Genetics Institute, Assaf Harofeh Hosp, Zerifin, Israel 2Dept of Pathology, Duke Univ Med Ctr, Durham, United States 3University of Istanbul, Cerrahpasa Med F, Istanbul, Turkey 4Dep Bio Resources Eng, Univ Maryland, Maryland, United States Glycogen storage disease type IIIa (GSD IIIa) is an autosomal recessive 5Ped Gastr Unit, Sheba Medical Center, Tel-Hashomer, Israel disease caused by deficiency of glycogen debranching enzyme in liver and muscle. Limited information is known about the clinical/pathological car- Glucose Galactose Malabsorption (GGM) is an autosomal-recessive disor- diac findings. A naturally occurring dog model has been described in curly- der characterized by life-threatening osmotic diarrhea at infancy. When the coated retrievers (CCR). We will describe the known cardiac findings in intake of the offending sugars is ceased, the diarrhea promptly stops. GSD III patients and the CCR model. We documented elevated left ven- Mutations in the SLC5A1 gene, encoding the sodium-glucose co- tricular mass and wall thickness in GSD IIIa patients with relatively pre- transporter located on enterocytes, have been showed to cause the disease. served ventricular systolic function. We will describe the gross and Over 300 subjects have been reported worldwide, most of whom a result of microscopic findings of cardiac tissue and the clinical records of three consanguineous unions. GSD IIIa individuals who died/underwent cardiac transplantation. Our We examined eleven patients presenting with symptoms consistent with examination revealed cardiac fibrosis (n01), moderate to severe vacuola- GGM and responsive to the appropriate fructose-based diet. Genomic DNA tion of cardiac myocytes (n03), mild to severe glycogen accumulation in of the patients was PCR-amplified for the 15 exons of SLC5A1 and the atrioventricular node (n03), and glycogen accumulation in smooth analyzed by nucleotide sequencing. muscle cells of intramyocardial arteries associated with smooth muscle Two novel mutations were identified: 1915 del C, a frameshift-mutation hyperplasia and profoundly thickened vascular walls (n01). We employed leading to a premature stop at codon 645; and a substitution missense mutation cardiac MRI to characterize fibrosis in one GSD IIIa patient. Pathological of T to C on nucleotide 947 causing a L316P substitution. In addition, two and 10 electrophysiological studies of the CCR model will be presented. previously described mutations were identified (G426R, C255W). In five of Our findings document diffuse though variable involvement of the heart in the nine families no mutation was found, possibly indicating a condition other these groups with a potential for serious arrhythmia and symptomatic heart than GGM. As most patients did not undergo a full diagnostic algorithm (i.e. failure in some GSD III patients. one that consists of a glucose breath hydrogen test and an attempt of a dietary switch), we suggest that molecular genotyping of such patients should only follow appropriate clinical evaluations.

P-191 P-193 DOES HYPERGLYCAEMIA CONTRIBUTE TO CIRRHOSIS IN GALT AND GALK ACTIVITY REGULATION FROM PRENATAL GLYCOGEN STORAGE DISEASE DUE TO PHKG2 DEFICIENCY? TO ADULT LIFE IN A SHEEP MODEL Bhattacharya K1 , Dennison B1 , Thompson S1 Rubio-Gozalbo M.E.1 , Bakker J.A.2 , Kramer B.W.3 , Bierau J2 1Children's Hospital at Westmead, Sydney, Australia 1Depts Paed & Clin Gen, Biochem Gen, MUMC, Maastricht, Netherlands 2Dept Clin Gen, Biochem Gen, MUMC, Maastricht, Netherlands Background: PHKG2 is a rare cause of glycogen storage disease type IX. 3Dept Paed, MUMC, Maastricht, Netherlands There is a high incidence of liver cirrhosis in reported cases but aetiology is unknown. Galactose-1-phosphate-uridyltransferase (GALT) is deficient in classical Case report: We report a pair of siblings with homozygous mutations in the galactosemia. Galactose-free diet quickly resolves acute symptoms, how- PHKG2 gene. The oldest was referred for assessment by our team after he ever cognitive problems and gonadal dysfunction in females still develop. was identified as having short stature, liver dysfunction and hepatomegaly Galactokinase (GALK) is a potential therapeutic target as it produces with liver biopsy demonstrating portal bridging fibrosis, nodule formation galactose-1-phosphate, substrate of GALT. Knowledge of the regulation and prominent cytoplasmic glycogen. Subsequent fasting studies showed of both enzyme activities from prenatal life into adulthood may provide hypoglycaemia (2.1 mmol/L) and ketosis (5.6 mmol/L) and hyperlipidae- additional insight into the pathophysiology of galactosemia and therapeutic mia. The younger sibling was seen at 30 months with hepatomegaly and modulation. The objective of this study was to measure GALT and GALK liver dysfunction ALT-938(10-50U/L), AST 1434(10-50U/L). He had com- activities in different organs, pre- and postnatally in a sheep model. Tissues menced high energy supplemental feeds for short stature but the LFTs of 5 preterm lambs and 5 adult ewes were used. GALT activity was highest deteriorated ALT1511, AST2369. Substituting with uncooked cornstarch in liver in preterm lamb and ewe (5.68±0.97 and 0.73±0.23 mol/(μg led to improvement in LFTs – AST 736, ALT 604 with concomitant proteinWh), respectively). In preterm lamb liver>small intestine>kidney improvement in lipid profile. He too had a low fasting lactate 0.9 mmols/ showed highest activity levels whereas brain activity was relatively low. L and glucose 2.0mmols/L with ketosis 3.33 mmol/L with post prandial Ewes had higher activity in cerebrum and cerebellum than preterm lambs lactate elevation after a modified glucose tolerance test of 5.5mmols/L. (0.17±0.03 and 0.37±005 respectively vs 0.27±0.04 and 0.52±0.16 mol/ Management with a low glycemic index based diet and cornstarch led to (μg proteinWh)). In sheep, target organs of neonatal involvement in galac- stable biochemistry and improved growth. tosemic humans, have the highest prenatal activity. Conversely, brain and Conclusion: Liver function in some forms of GSD could deteriorate with ovary, target organs for diet-independent long-term complications in excessive simple sugar. humans have a significantly lower GALT activity. Determination of GALK activity is on-going. S82 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-194 P-196 MULTIPLEX LIGATION-DEPENDENT PROBE AMPLIFICATION CLINICAL APPLICATION OF NEXT GENERATION SEQUENCING (MLPA) ANALYSIS IN HEREDITARY FRUCTOSE INTOLERANCE TO MOLECULAR DIAGNOSIS OF GENETICALLY (HFI) HETEROGENEOUS GLYCOGEN STORAGE DISEASE Santer L1 , Steglich C1 , Bergmann J2 , Tsiakas K2 , Schneppenheim R3 , Wang J1 , Cui H1 , Lee NC2 , Craigen WJ1 , Zhang VW1 , Wong LJ1 Santer R2 1Mol Hum Genet, Baylor College Medicine, Houston, Texas, United States 1Inst Hum Genet, Univ Med Cent Eppendorf, Hamburg, Germany 2Pediatr, national Taiwan Univ Hospital, Taipei, Taiwan 2Dept Pediatr, Univ Med Cent Eppendorf, Hamburg, Germany 3Dept Pediatr Hematol Oncol, Univ Med Cen, Hamburg, Germany Background: Glycogen storage diseases (GSDs) are a group of genetic disorders of glycogen metabolism. Clinical features include hypoglycemia, Congenital defects of ALDOB are the molecular basis of HFI. While certain point hepatomegaly, developmental delay and muscle cramps. Untreated patients mutations (p.A150P, p.A175D) are frequent, the role of long-range deletions has may suffer devastating outcome, therefore, prompt and accurate diagnosis is remained unclear. Here, we report our experience with MLPA analysis, a method critical. Here we report the utility of a cost effective next generation which can detect both known mutations but also copy number variations. sequencing (NGS)-based molecular analysis for GSDs. Patients/Methods: A total of 88 HFI patients were enrolled in the study (72 Method: The coding exons and their flanking 20 bp intron regions of 16 previously published, Hum Mutat 2006). Among them were 7 cases suspicious genes; GYS2, GYS1, G6PC, SLC37A4, GAA, AGL, GBE1, PYGM, for homo- or heterozygosity for a long-range deletion. MLPA analysis was PYGL, PFKM, PHKA2, PHKB, PHKG2, PHKA1, PGAM2, and PGM1, performed with the SALSA MLPA P255 ALDOB probemix (MRC Holland) are captured using DNA probes, followed by fragmentation, template and by seperation of exon- and mutation-specific PCR products by capillary library preparation, and massively parallel sequencing (MPS). electrophoresis. Results were confirmed by conventional Sanger sequencing of Results: All bases in the target regions have been unbiasedly enriched with junction fragments. deep coverage. Validation was performed by parallel Sanger sequencing. Results: The two common point mutations were reliably detected. In 5/7 cases, MPS demonstrated 100 % sensitivity and specificity compared to Sanger long-range deletions were found in exons 1, 6, and 2-6, respectively, and in 4/5 sequencing. This method correctly identified all types of mutations, includ- cases breakpoints were determined (del c.540+567_c.651 insATGGGTT [del ing single nucleotide substitutions, small deletions and duplications, and 1315 ins 7 bp], del -2053_c.624+63 [del 6449 bp], del -1747_c.624+41 [del large deletions involving one or more exons. Furthermore, novel mutations 6131 bp]). Furthermore, a novel 2-bp-deletion, c.893_4delTA [p.L298Hfs37X] have been identified in previously undiagnosed patients. was detectable. Detection rates for **two/*°at least one ALDOB mutation in our Conclusion: This report demonstrated the clinical utility of MPS-based tech- cohort were compared: sequencing **92.0/*°97.7, MLPA **75.9/*°92.0, nology in molecular diagnosis of a group of clinically overlapping and geneti- MLPA (extended for p.N335K) **83.9/*°97.7 %. cally heterogeneous disorders such as GSD, in a cost and time efficient manner. Conclusion: MLPA is a reliable first diagnostic step in HFI.

P-197 MOLECULAR SPECTRUM OF GLYCOGEN STORAGE DISEASE TYPE III IN MALAYSIAN PATIENTS P-195 Yap S1 , Abdullah IS2 , Ngu LH3 , Teh SH2 , Ong SY1 , Chong SY4 , Boey COULD GSD TYPE I EXPAND THE SPECTRUM OF DISORDERS CCM4 , Lee WS4 , Mohamed Z2 WITH ELEVATED PLASMA CHITOTRIOSIDASE ACTIVITY? 1Div IMD, Paeds Dept & UMMC, Univ Malaya, Kuala Lumpur, Malaysia Tumer L1 , Kasapkara C S1 , Biberoglu G1 , Hasanoglu A1 2Inst Biol Sc, Univ of Malaya, Kuala Lumpur, Malaysia 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 3Dept Genet & Metab, Hosp Kuala Lumpur, Kuala Lumpur, Malaysia 4Div Hep & GE, Paeds Dept & UMMC, UM, Kuala Lumpur, Malaysia Glycogen storage disease type I (GSDI) is characterized by accumulation of glycogen and fat in the liver and kidneys, resulting in hepatomegaly and Glycogen storage disease type III (GSD III) is a rare disorder of glycogen renomegaly. Human chitotriosidase is a recently described fully active debranching enzyme (GDE) deficiency which is caused by ethnically depen- chitinase expressed by activated macrophages. Marked elevation of chito- dent mutations in the AGL gene. There is biochemical and clinical heteroge- triosidase activity was initially observed in plasma of patients with gaucher neity reflecting the severity of the AGL mutations. Clinically, it affects the disease. Subsequently, elevation was also observed in various lysosomal liver, cardiac and skeletal muscles. GSD IIIa is the most common (85 %) and storage disorders such as fucosidosis, galactosialidosis and glycogen stor- shows both liver and muscle involvement, while GSD IIIb affects the liver age disease type IV. The aim of the present study was to evaluate plasma only. This study reports on the molecular spectrum of the AGL gene, which is chitotriosidase activity in 19 children with Glycogen storage disease type I. as yet unknown, in an ethnically diversed Malaysian population. Nine patients Plasma chitotriosidase levels were found to be significantly higher in (7 males; 2 females) from 8 unrelated families were studied; seven Malays and children with GSD type I than healthy age-matched controls (21.3±16.4 two Chinese. The mean age was 8 years (range: 2.4 -12.5); Mean age of vs. 12.3±8.9 nmol/h/ml, p00.04). All the patients reported here presented diagnosis is at 2.8 years (range: 1.1-7.0). Six had GDE enzymology confir- with hepatomegaly. Irrespective of the mechanisms involved, our report mation. Of the 16 independent alleles studied, 8 different mutations were expands the spectrum of disorders that should be included in the differential found; six novel (c.4490 G>A, c.1423+1 G>T, c.3814_3815del AG, diagnosis of patients with increased plasma chitotriosidase activity. c.2914_2915delAA, c.4333 T>G, c.4531_4534delTGTC) and 2 previously reported (c.2681+1 G>A, c.99 C>T). The commonest mutation c.2681+1 G >A (n07 alleles; 70 %) was found in five Malay patients. Conclusion: The Malaysian AGL gene spectrum shows heterogeneity. The novel c.4531_4534delTGTC mutation is associated with a milder phenotype with relatively good response to carbohydrate and protein supplementation. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S83

P-198 A-017 TYPE I COLLAGEN POST-TRASLATIONAL MODIFICATIONS: EPIMERASE DEFICIENCY GALACTOSEMIA: A CASE REPORT IMPLICATION IN THE PHYSIOPATHOLOGY OF CONGENITAL Kör D1 , Onenli-Mungan N1 , Shin-Podskarbi Y2 , Bulut FD1 GALACTOSEMIA 1Faculty of Medicine, Univ of Çukurova, Adana, Turkey Lacombe C1 , Untereiner V1 , Manfait M1 , Sockalingum G.D1 , Garnotel 2Children's Hospital, Munich, Germany R1 1MEDyC, FRE CNRS/URCA 3481, REIMS, France Background: Galactosemia is caused by inherited deficiencies in one of three enzymes involved in the metabolism: galactose-1-phosphate uridyl- Congenital galactosemia is due to a deficiency of galactose-1-phosphate transferase, galactokinase, and uridine diphosphate galactose-4-epimerase. uridyltransferase, an enzyme involved in the metabolism of galactose. This The rarest and poorly understood form of galactosemia is epimerase defi- enzymatic deficiency causes an accumulation of galactose, galactose-1- ciency. There are three forms of epimerase deficiency; generalized, periph- phosphate, as well as galactitol, a reduction product of galactose. eral, and intermediate. Here we report a patient with epimerase deficieny The aim of our work is to show that galactose induces a post-translational galactosemia. To the best of our knowledge this is the first case reported modification of proteins via the nonenzymatic 'galactation', and in particular from Turkey. type I collagen, thus causing the early and late symptoms. Case report: A 5-month old male patient was referred from an ophthal- To verify this hypothesis, we modified type I collagen with galactose and its mologist because of bilateral cataract detected during the routinely evalua- derived products in a time- and concentration-dependent manner. We evi- tion of prematurity problems. His family history was non-contributory. dence that galactose binds to collagen more quickly than glucose (reference Except cataract physical examination was unremarkable. He did not have hexose) and this in a concentration-dependent manner, showing a structural jaundice or hepatomegaly. Liver function tests were normal. Galactose was modification of collagen. This was confirmed by the analysis of collagen positive in urine. After confirmation of decreased epimerase and normal fibrillation. These modified collagens were able to influence the activation levels of other galactose metabolism enzymes in RBC hemolysate, the of inflammatory cells with a modulation of the release of reactive oxygen patient was put on galactose restricted diet. As the patient had only cataract species, analysed by nitro-blue of tetrazolium (NBT) reduction. In parallel, we thought that it was an intermediate form. On follow-up he had no a spectral analysis of pathological and healthy plasmas by infrared spec- problems. Genetic study is planned. troscopy showed subtle differences that were sufficient to differentiate Discussion: Epimerase deficiency is a very rare and frequently benign form between them. otherwise then cataract. Our patient is the first epimerase deficient galacto- Thus, our findings seem to indicate that 'galactation' is an important phe- semia reported from Turkey. nomenon in galactosemia.

P-199 22. Lysosomal Disorders STUDY OF QUALITY OF LIFE FOR CHILDREN WITH GLYCOGEN STORAGE DISEASE TYPE I AND THEIR PARENTS P-200 Tumer L1 , Kasapkara C S1 , Soysal A S2 , Hasanoglu A1 AN EXTERNAL QUALITY ASSURANCE SCHEME FOR 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey DIAGNOSTIC TESTING OF MUCOPOLYSACCHARIDOSES IN 2Clin Psychology, Gazi Univ Hosp, Ankara, Turkey URINE Ruijter G1 , De Graaf I2 , Van den Berg R1 , Weykamp C2 , Huijmans J1 Glycogen storage disease type I (GSD I) is caused by deficiency of the 1Dep Clin Gen, Erasmus Medical Center, Rotterdam, Netherlands glucose-6-phosphatase catalytic subunit in type Ia or of glucose-6- 2SKML, Hosp Koningin Beatrix, Winterswijk, Netherlands phosphate transporter in type Ib. The presence of GSD type I is associated with reduced quality of life, independent functioning, and elevated levels of Objective: To establish External Quality Assurance (EQA) for Mucopoly- internalizing distress and parental stress. saccharidoses (MPS) testing in urine. Material-Method: The aim of the present study was to evaluate the quality Methods: In 2010 and 2011 we carried out a pilot study and circulated 14 of life (QOL) among 19 individuals with type I GSDs. The Kiddy-KINDL, authentic urine samples (11 MPS, 3 normal controls) to 90 participants. a parent based instrument for assessing health related quality of life in Participants were asked to perform quantitative and qualitative GAG anal- children (age 4-7 years) and the Kid-KINDL, a self-report questionnaire ysis and to give the most likely diagnosis. for children (age 8-16 years) used to measure degrees of health and Results: The MPS pilot scheme had participants from Europe (63), North & adaptability in relation to QOL. Parents also completed questionnaire for South America (9), Asia (9), Australia (7) and Africa (2). The majority (76) measuring parental distress, parenting stress, child adaptive behaviour, and submitted at least 10 out of 14 sample reports. Diagnostic proficiency in this child emotional and behavioral functioning. group was: 51/76: >70 % of the samples interpreted correctly; 18/76: 50- Results: Relative to their children, parents generally reported that their child 70 % correct interpretation; 7/76: less than 50 % correct. Among the was more impaired, which suggests the need for multiple informants during different sample types diagnostic proficiency was good for MPS I, II, assessment and active parental involvement during psychological treatment. classic III, VI and normal controls, while relatively poor results were Conclusion: These points should be kept in mind when assesing and obtained for MPS IV, mild III and VII. treating those patients with this disease and their families as psychological Conclusion: We have successfully established an international EQA interventions that target areas of concern (e.g.adherence, coping with hav- scheme for urine MPS diagnostic testing. The results show that many ing a chronic disease) may be helpful for improving child and family laboratories perform high quality MPS diagnostics, while improvement is outcomes. necessary for others. The MPS scheme has tremendous educational value by making available positive urine samples from these rare disorders and will be a full ERNDIM scheme in 2012. S84 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-201 P-203 HOME SLEEP STUDY CHARACTERISTICS IN PATIENTS WITH ANTI-IDURSULFASE ANTIBODY AND THE EFFICACY OF MUCOPOLYSACCHARIDOSIS ENZYME REPLACEMENT THERAPY IN THE PATIENTS Kasapkara C S1 , Tumer L1 , Hasanoglu A1 , Ezgu F S1 , Arslan A2 , WITH MUCOPOLYSACCHARIDOSIS II Kokturk O3 , Kucujcongar A1 Tanaka A1 , Hamazaki T1 , Sawada T1 , Kadono C1 , Kudo S1 , Shintaku H1 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 1Osaka City University, Osaka, Japan 2Div Ped Chest Dis, Gazi Univ Hosp, Ankara, Turkey 3Div of Chest Dis, Ankara Univ, Ankara, Turkey Background: Among the patients with severe form of MPS II, a few patients showed infusion related reaction with high anti-idursulfase antibody. Mucopolysaccharidoses (MPS) are inherited lysosomal storage disorders Objective: The efficacy of ERT and the development of antibody were caused by the deficiency of hydrolases involved in the degradative pathway studied in the patients with MPS II treated for more than 2 years. of glycosaminoglycans. In MPS, progressive respiratory disease may se- Patients and Methods: Thirteen patients were analyzed at the baseline and verely affect morbidity and mortality. Obstructive sleep apnea (OSA) or every 6 months after the ERT. Patients were divided into four groups; obstructive sleep apnea syndrome is the most common type of sleep apnea type A (normal intelligence), B (normal-borderline intelligence), C (retard- and is caused by obstruction of the upper airway. Material- Method: ed after 2 years of age), and D (retarded before 2 years of age). Measurements of apnea, apnea-hypopnea index, hypopnea index, oxygen Results: The patients included four type A, one type B, three type C, and desaturation index, minimal oxygen saturation were obtained through home five type D. All the patients with type D developed anti-idursulfase anti- sleep study. We evaluated upper airway obstructive disease and related body, while the patients with type A, B, and C did not. Liver volume and clinical findings through home sleep study in 19 patients (11 with MPS urinary GAG were decreased remarkably in all patients except two of type VI, 4 with MPS I, 4 with MPS II) with mucopolysaccharidoses followed at D patients, who developed extremely high titer of antibody and showed Gazi University Pediatric Metabolic Unit. severe urticaria during or after infusion. Results: For 19 children, the disorder was mild (OAHI 1.5-5/h) in 6, Conclusion: Anti-idursurfase antibody was developed in all the patients moderate (OAHI 5-10) in 2, and severe (OAHI>10) in 10. The prevalence with type D, the most severe form of MPS II. The patients with extremely of OSA was 94.7 %(18/19) in patients with MPS. Snoring, witnessed apnea, high titer of antibody showed poor efficacy of ERT, which suggested the pectus carinatum, and macroglossia were the main clinical findings. Echo- anti-idursurfase antibody reduced the enzyme activity. cardiograms showed evidence of pulmonary hypertension in 13 patients. Conclusion: Home sleep study is a very quick, easily available screening test to determine the abnormalities of breathing during sleep and urge the clinicians to take necessary action for patients with severe manifestations.

P-204 MAROTEAUX LAMY SYNDROME (MPS VI): LONG TERM FOLLOW UP OF SIBLING PAIR: ONE TREATED WITH BONE P-202 MARROW TRANSPLANTATION AND THE OTHER RECEIVED ENZYME REPLACEMENT THERAPY TOWARDS A BETTER UNDERSTANDING OF MPSIII THROUGH O'Sullivan S1 , Woods M1 , Finnegan N1 , Cooke V1 , Vellodi A1 PATIENT REPORT OUTCOMES MEASURES (PROMS) 1Great Ormond Street Hospital, London, United Kingdom Ferreira V1 , Kole A1 , Aiach K1 1LYSOGENE, Paris, France Background: Mucopolysaccharidosis VI (MPS VI) is a lysosomal storage disease with progressive multisystem involvement. There is no cure; man- Background: Patient-Reported Outcomes Measures (PROMs) refers to agement consists of supportive care, bone marrow or haematopoietic stem information about a health condition and its treatment that is directly cell transplantation (HSCT), and more recently enzyme replacement thera- obtained from patients. An experimental treatment aimed at treating Muco- py (ERT). polysaccharidosis Type III (MPS III) (Sanfilippo Syndrome) is being in- Method: We report the long term follow-up of two siblings with MPS VI. vestigated by LYSOGENE. The older sibling, a boy, received bone marrow transplantation (BMT) aged Objectives: To evaluate existing PROMs instruments targeted to Mucopo- 3 yrs. He is now aged 20 yrs. His younger sister has been receiving ERT lysaccharidosis Type III (MPS III) and/or similar pathologies focusing in (Naglazyme) since the age of 11. She is now aged 17. particular on the measurement of HRQoL (Health Related Quality of Life). Results: Both siblings have required bilateral corneal transplants. They Methods: A structured review of existing PROMs instruments was have significant skeletal manifestations requiring orthopaedic and neuro- performed. surgical procedures at different stages, and different rates of progression. Results: Several HRQoL instruments targeted to rare diseases were identi- However, the older siblings has a better facial appearance, posture, joint fied. In addition, three questionnaires covering specific symptomatic mobility and exercise tolerance. aspects of MPS III were found. Importantly, any instrument to evaluate Conclusions: It is unusual to find siblings with the same condition treated the overall HRQoL in MPSIII patients was identified. by different methods and so these siblings provide a unique opportunity, Conclusion: Despite significant progress, the study of HRQoL in MPSIII albeit anecdotal, to compare the two treatments. In our view, BMT has remains a nascent field. As the use of new therapies continues to increase in yielded a superior result. Given the striking reduction in morbidity and the Rare Disease field, additional work directed to develop specific disease mortality from BMT in recent years, it could perhaps be considered for PROMs will be needed. Finally, these questionnaires give insights about the particularly severe cases of MPS VI. wide range of clinical features and progression of these diseases. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S85

P-205 P-207 EVALUATION OF HAND FUNCTION IN PATIENTS WITH EVALUATION OF CARDIAC MANIFESTATIONS IN MUCOPOLYSACCHARIDOSIS IVA MUCOPOLYSACCHARIDOSES Aslam R1 , Hendriksz CJ1 , Jester A1 Kagnici M1 , Canda E1 , Kose M1 , Ulger Z2 , Kalkan Ucar S1 , Levent E2 , 1Birmingham Children´s Hospital NHS Found, Birmingham, United Kingdom Ozyurek R2 , Coker M1 1Div Metab Dis, Dep Ped, Univ Ege, Izmir, Turkey Background: With advances in enzyme replacement therapy for Mucopo- 2Div Cardiol, Dep Ped, Univ Ege, Izmir, Turkey lysaccharidosis VI, we may see increased survival rates with the need to improve self caring and functioning in daily live. Background: Cardiac involvement due to progressive deposition of glyco- Aims: The aim of our study is to assess a level of subjective functioning saminoglycans on cardiac valves can occur in mucopolysaccharidoses and objective function in Morquio patients in order to advance understand- (MPS). In this study, we aimed to evaluate our patients who suffer from ing and hence generate discussion on potential future management. MPS in terms of cardiac involvement. Method: We have studied a series of 10 consecutive Morquio patients Patients and Methods: A detailed description of demographic characteristics, currently under enzyme replacement therapy in our unit. We applied the phenotype and duration of enzyme replacement therapy (ERT) were assessed principal of the World Health Organization International Classification of retrospectively. The echocardiograms (Echo) of 35 patients were reviewed. Functioning, Disability and Health (WHO ICF), using a new patient centred Cardiac valve dysfunction and signs of myocardial involvement were recorded. Hand and Upper Limb Questionnire, as well as range of movement and grip Results: Thirty five MPS subjects (MPS I:4, MPS II:5, MPS IIIa:1, MPS performance. IIIb:3, MPS IIIc:2, MPS IVa:13, MPS VI:6, MPS VII:1) have been evalu- Results: Coupling the hypermobility and limited control of the wrist with ated. Eighteen patients were male and seventeen were female. Of this total, the vastly reduced grip strength and the higher difficulty levels for activities thirteen patients had been treated with ERT with a mean duration of 3,04± involving strength, it seems that the wrist may be the cause of major areas 1,5 years. In the first Echo examination; we detected valve disease in of function and functioning deficit. fourteen patients, which half had mitral insufficiency, and both of valve Conclusion: Our findings suggest the wrist itself represents an area that we disease and myocardial involvement in six patients. Fourteen patients were should focus on to be able to improve the patients' functional abilities and free of any cardiovascular manifestation. We detected valve disease through hence their level of independence on day to day basis. follow–up of ERT receiving two subjects who had no cardiovascular find- Conflict of Interest declared. ings in their first Echo examination. Conclusion: We reported our data to emphasise necessity of monitoring cardiac functions in subjects with MPS.

P-206 P-208 RADIOGRAPHIC ANALYSIS OF WRIST AND HAND IN MPS 4A AN IMPROVED RAPID ISOLATION OF URINARY PATIENTS AND ITS SIGNIFICANCE FOR TREATMENT GLYCOSAMINOGLYCANS OF SMALL VOLUME OF URINE Jester A1 , Aslam R1 , Chapman S1 FOR HIGH-RESOLUTION ELECTROPHORESIS FOR ISOTYPING 1Birmingham Children´s Hospital NHS Found, Birmingham, United Kingdom OF MUCOPLOYSACCHARIDOSES: AN EXAMPLE WITH MPS VI -MAROTEAUX-LAMY Background: Unstable wrists and dysfunctional hands have been a well Tanyalcin T1 , Cayir A2 , Wuyts B3 , Vincent MF4 , Orbak Z2 known problem in MPS 4A patients. With increasing life expectancy under 1Tanyalcin Lab Screening and Metab Unit, Izmir, Turkey ERT an independant, selfdetermined life with good upper limb function has 2Ataturk Univ,Pediat Endoc Div Metab Dis, Erzurum, Turkey gained increasing importance. Treatment plans to stabilize wrists and there- 3Labo Metabole Ziekten 3 K5 Klinische Biol, Gent, Belgium fore improve functionality must be based on an understanding of the 4Clin Univ Saint-Luc ,Lab Maladies Métab, Bruxelles, Belgium underlying problems. Aim: Evaluate bone and soft tissue pathologies of hands and wrist using The urine glycosaminoglycan (GAG) measurement for patients with mucopo- xrays and MRI lysaccharidosis (MPS) is considered to be the first step pr in diagnosis of those Methods: 15 Patients (age 2,3 to 16 years) under treatment at the Birming- heterogenous group of lysosomal storage disorders presenting clinical pheno- ham Childrens Hospital were included. type. In this study, dimethylmethylene blue (DMB) dye method was used Annualy performed skeletal survey xrays were used to evaluate forearm, before GAG differentiation. Measurements were carried out on microtiter wrist and hand skeleton. Two patients had an MRI of hands and wrist, plates at double wavelength 580/690 nm using regression equation polynomi- which was used to determine the soft tissue pathologies. nal second order.The urine GAG excretion of the clinically suspected Results: The comparison hand bone vs. chronological age showed delayed Maroteaux-Lamy case (MPS VI) was strongly increased:1387 mg/g creatinine bone age, the comparison carpal bone vs. chronological carpal bone age (reference range 68-188 )and GAG electrophoresis showed marked excretion showed very delayed maturation carpal bones with the scaphoid bone being of dermatan sulphate;strongly suggestive for MPS VI and the dried blood spot extremely delayed, or not present, the ulna shortened and the radius tilted. was confirmed by arylsulphatase B measurement which was found to be very This finding correlated with the clinical picture. low: 0.13 μmol/L/h (reference range 3.33-14.89.The urine was used to adjust Conclusion: We will discuss the unique changes in forearm and wrist the rapid GAG isolation method using a bench top eppendorf centrifuge. imaging, which determine future treatment plans to improve functionality Different volumes of urine containing different GAG concentrations were of hand and upper limb. used to mix with 1 mL cetylpyridinium(CPC)/citrate buffer. 200 microgram of GAG is the most appropriate concentration that gives the best precipitation and clear pattern of high resolution GAG electrophoresis following high speed centrifugation at 12,200 rpm. This procedure allows GAG isolation and high resolution GAG electrophoresis to be easily performed in routine clinical diagnostic laboratories. S86 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-209 P-210 BRONCHIECTASIS IN MPS TYPE I PATIENTS POST-HAEMOPOETIC DIAGNOSIS OF MUCOPOLYSACCHARIDOSIS IVA: A STEM CELL TRANSPLANTATION COMPREHENSIVE ALGORITHM ENCOMPASSING CLINICAL, Sunther M1 , Jameson E2 , Breen C2 ,MercerJ2 ,WynnR1 ,ChanE1 , Jones S2 RADIOGRAPHIC, AND CLINICAL LABORATORY DATA 1Royal Manchester Children's Hospital, Manchester, United Kingdom Wood TC1 , Bainbridge K2 , Beck M3 , Burin MG4 , Chien YH5 , Church 2Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom HJ6 , D'Almeida V7 , van Diggelen OP8 , Fietz M9 , Giugliani R10 , Harmatz P11 , Hwu WL5 , Ketteridge D9 , Lukacs Z12 , Miller N13 , Pasquali M14 , Introduction: Mucopolysaccharidosis type I (MPS I) is associated with a Schenone A15 , Thompson JN16 , Tylee K6 , Hendriksz CJ17 number of respiratory complications including recurrent respiratory tract 1Greenwood Genetic Center, Greenwood, United States infections, airway obstruction and restrictive lung defects. In the past two 2Great Ormond Street Hosp for Children, London, United Kingdom years four of our patients who have undergone haemopoeitic stem cell 3Children's Hosp, University of Mainz, Mainz, Germany transplantation (HSCT) have developed bronchiectasis. 4Hospital de Clinicas de Porto Alegre, Porto Alegre, Brazil Objective: To describe these four patients in terms of presentation, CT 5National Taiwan University Hosp, Taipai, Taiwan chest findings, lung function tests and management. 6Dept of Genetic Medicine, St Mary's Hosp, Manchester, United Kingdom , Results: Case one is a male patient who underwent transplantation in 2001. 7Universidade Federal de São Paulo, São Paulo, Brazil Post-transplantation he developed an oxygen requirement between 2001 8Erasmus University Medical Center, Rotterdam, Netherlands and 2008 which gradually resolved. Bilateral lower lobe bronchiectasis was 9Women and Children's Hosp, North Adelaide, Australia confirmed in 2010. Cases two and three are female patients who underwent 10Fed Univ of Rio Grande do Sul (UFRGS), Porto Alegre, Brazil transplantation in 2004 and 2008 respectively. Each child went on to 11Children's Hosp Oakland, Oakland, United States develop a chronic, productive cough and were both subsequently diagnosed 12Dept of Ped and Institute Clin Chem, Hamburg, Germany with bronchiectasis in 2011. All receive three monthly intravenous anti- 13BioMarin Pharmaceutical Inc., Novato, United States biotics and are under respiratory review. 14ARUP Laboratories, Salt Lake City, United States Discussion: While bronchiectasis has been described in patients following 15Fund Est Enfermedades Neurometabólicas, Buenos Aires, Argentina bone marrow transplantation for malignancy we have found no literature 16Univ of Alabama at Birmingham, Birmingham, United States regarding this complication in MPS I patients. We recommend that at 17Birmingham Children's Hosp, Birmingham, United Kingdom follow-up patients are specifically questioned regarding symptoms of bronchiectasis as hopefully early treatment will maintain good long-term lung Mucopolysaccharidosis IVA (MPS IVA/Morquio A syndrome) is an auto- function. somal recessive lysosomal storage disorder resulting from genetic defects in N-acetylgalactosamine-6-sulfate sulfatase (GALNS). Diagnosis can be challenging and requires agreement of clinical, radiographic, and laboratory findings. A group of biochemical genetics laboratory directors and clinicians involved in the diagnosis of MPS IVA, convened by BioMarin Pharmaceutical Inc., met to develop recommendations for diagnosis. The following conclusions were reached. Due to the wide variation and subtle- ties of radiographic findings, imaging of multiple areas is recommended. Urinary glycosaminoglycan analysis is particularly problematic for MPS IVA and it is strongly recommended to proceed to enzyme activity testing even if urine appears normal when there is clinical suspicion of MPS IVA. Enzyme activity testing of GALNS is essential in diagnosing MPS IVA. Additional analyses to confirm sample integrity and rule out MPS IVB and multiple sulfatase deficiency are recommended as part of enzyme activity testing. Using leukocytes or cultured dermal fibroblasts for repeat enzyme activity testing to confirm diagnosis is recommended. Molecular testing may also be used to confirm the diagnosis in many patients. However, two known or probable causative mutations may not be identified in all cases of MPS IVA. A diagnostic algorithm is presented. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S87

P-211 P-213 GUIDELINES FOR THE RESUSCITATION OF CHILDREN WITH MOLECULAR DIAGNOSIS OF MPS TYPE IVA (MORQUIO A MUCOPOLYSACCHARIDOSES SYNDROME) AND TYPE VI (MAROTEAUX-LAMY SYNDROME) Santra S1 , Reynolds F2 , Stewart C1 , Thomas S3 , Lavery C3 , Hendriksz IN TAIWAN C1 Tsai Fuu-Jen1 , Lin Wei-De1 , Tsai Chang-Hai2 1Clinical IMD, Birmingham Children's Hosp, Birmingham, United Kingdom 1Genetic center ,China Medical Univ Hosp, Taichung, Taiwan 2PICU, Birmingham Children's Hosp, Birmingham, United Kingdom 2Asia University, Taichung, Taiwan 3Society For Mucopolysaccharide Diseases, Amersham, United Kingdom In this study, 12 MPS IVA and 10 MPS VI patients were investigated. To Children with MPS disorders have unique anatomical differences compared identify possible exonic mutations in the target gene leading to MPS IVA or to other children including short necks, narrow airways, cervical spine MPS VI, the entire coding sequence of the DNA fragments, each covering instability, small chests and large abdominal organs. Some aspects of an exon and its flanking regions, was amplified and subjected to direct standard resuscitation guidelines are inapplicable and there is a need for sequencing. Total RNA was isolated from one MPS VI patient's blood clear disease-specific algorithms suitable for use by professionals and carers sample and performed reverse transcription-polymerase chain reactions alike. We devised algorithms for the choking and fallen MPS child as well (RT-PCR) to identify exon deletion. as a modified basic life support algorithm suitable for widespread use. All patients carried one or two nucleotide substitutions or small deletion in These have been evaluated favourably by MPS Clinicians in the UK as their GALNS gene. The p.M318R was found to be a common mutation in well as families and carers of MPS children the panel of MPS IVA patients, accounting for 23.1 % of the total number of mutant alleles. The allele frequency of undetectable was 19.2 %. The analysis of the patients' ARSB gene, a total of six missense, two nonsense and one deletion mutations were identified. The c.1197 C>G (p.Phe399Leu) and c.395 T>C (p.Leu132Pro) mutations were the most common missense mutation in the Taiwanese MPS VI patients. Most mutations found in our patients were not reported in other populations previously. The undetected rate in all patients' allele was 15 %. P-212 ITEM REDUCTION AND ASSESSMENT OF MEASUREMENT PRIORITIES OF THE HUNTER SYNDROME FUNCTIONAL OUTCOMES FOR CLINICAL UNDERSTANDING SCALE (HS-FOCUS) Wiklund I1 , Raluy M1 , Chen WH2 , Muenzer J3 , Pelletier N4 , Fang J4 , Whiteman DAH5 1United BioSource Corporation, London, United Kingdom 2United BioSource Corporation, Bethesda, MD, United States 3 Dept Pedia, Univ N Carolina Chapel Hill, Chapel Hill, NC, United States P-214 4Shire Human Genetic Therapies, Lexington, MA, United States HIGH-RISK POPULATION SCREENING FOR 5Shire AG, Nyon, Switzerland MUCOPOLYSACCHARIDOSES Nieves Cobos P.1 , Gal A2 , Lukacs Z1 Objectives: The HS-FOCUS questionnaire was used to monitor patient 1Institute of Clinical Chemistry, Hamburg, Germany reported outcomes for mucopolysaccharidosis II (MPS II, Hunter Syn- 2Institute of Human Genetics, Hamburg, Germany drome) patients. Preliminary psychometric analysis suggested some items were redundant or not informative. The objective was to shorten HS- Mucopolysaccharidoses (MPS) are a group of progressive disorders which FOCUS to enhance its validity and reduce respondent burden. derive from deficiency of lysosomal enzymes involved in the degradation Methods: HS-FOCUS data collected in a trial were used. Final items were of glycosaminoglycans. Clinical features may include coarse facies, hep- selected based on content validity, per clinicians' input and psychometric atosplenomegaly and dysostosis multiplex. properties, followed by assessing reliability, validity, and responsiveness. We analyzed 211 samples from patients with symptoms compatible with Results: Altogether, 49 patients and 84 parents completed the HS-FOCUS. MPS I. Dried blood samples (DBS) originated from the Middle East, Items were removed owing to ambiguous wordings, floor effect (> 60 %), Mexico and Europe. In total 17 patients (8 %) with MPS I were identified. and high correlations (> 0.90). Both the shortened patient (32 items) and Samples were also analyzed for MPS II (male patients) as well as MPS VI. parent versions (35 items) contain 5 functional domains. The internal In this way, 11 patients with MPS II (5 %) and 10 patients (5 %) with MPS consistency and test-retest reliability were >0.70 for all domains except VI were found. Using the same DBS sample for DNA analysis, the bio- the Breathing domain in patients. Significant correlation (>0.30) with chemical diagnosis of MPS was corroborated by mutation data in 68 % of Childhood Health Assessment Questionnaire (CHAQ) and 6-Minute-Walk cases, despite limitations in DNA quantity and quality. Another 10 samples test demonstrated concurrent validity. Scores were significantly different in (5 %) showed significantly elevated enzyme activities in DBS which is all domains among levels of disability measured by CHAQ. Change scores compatible with mucolipidoses. were responsive to changes in patients' health status. In spite of moderate extra cost, one should consider that at least MPS I, Conclusions: The shortened HS-FOCUS will reduce patient and parent MPS II and MPS VI are always analyzed in parallel from a single specimen burden without compromising the validity. The shortened HS-FOCUS is as otherwise, some patients may be missed. reliable, valid, and responsive for use in clinical trials or registries. Conflict of Interest declared. Conflict of Interest declared. S88 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-215 P-217 A MULTI-CENTER, OPEN-LABEL STUDY EVALUATING SAFETY ENZYME REPLACEMENT THERAPY (ERT) IN MPS II PATIENTS AND CLINICAL OUTCOMES IN YOUNG CHILDREN (1.47.5 AGED 20 YEARS OR OLDER AT TREATMENT INITIATION: DATA YEARS) WITH HUNTER SYNDROME RECEIVING IDURSULFASE FROM THE HUNTER OUTCOME SURVEY (HOS) ENZYME REPLACEMENT THERAPY Heiberg A1 , Lampe C2 , Whiteman DAH3 Giugliani R1 , Wuh-Liang Hwu P2 , Tylki-Szymańska A3 , Whiteman 1Dept Med Gen, Oslo Univ Hosp, Ntnl Hosp, Oslo, Norway DAH4 2Villa Metabolica, Univ Medical Center, Mainz, Germany 1Dept Genetics/UFRGS, HCPA and INAGEMP, Porto Alegre, Brazil 3Shire Human Genetic Therapies, Eysins, Switzerland 2Dept Ped/Med Gen, Nat Taiwan Univ Hosp, Taipei, Taiwan 3Metab Dis Clinic, Child Mem Health Hosp, Warsaw, Poland Much of the published data for ERT focuses on its use in children. Here we 4Shire AG, Eysins, Switzerland report our experiences with adult patients aged >20 years at treatment start, for whom six-minute walk test (6MWT) and percent predicted forced vital Primary objective was to determine the safety of idursulfase in Hunter capacity (%FVC) data from baseline and treatment years 1, 2, or 3 were syndrome patients ≤5 years old. Idursulfase (0.5 mg/kg) was administered available from the HOS database. Of 52 patients in HOS who began ERT at intravenously weekly (total 52 infusions/patient) in an open-label study. >20 years, 6MWT and %FVC data were available for 11. As of the last Safety monitoring included AEs, anti-idursulfase antibodies, vital signs, lab time-point recorded (either year 1, 2, or 3 of treatment depending upon the tests, physical exam, 12-lead ECG, concomitant medications and proce- data entered in HOS), 6MWT and %FVC were both improved in 2 patients. dures. Secondary outcomes (urinary GAG levels; liver/spleen size changes; Percent improvements from baseline were 20 % for both in 6MWT, and 9 % growth indices) were assessed at baseline and weeks 18, 36 and 53. PK and 12 % for %FVC. Four other patients had improved 6MWT (22 %, parameters were also assessed. 28 boys, aged 1.4–7.5 years, were enrolled 11 %, 188 %, 92 %) but decreased %FVC (-32 %, -30 %, -6 %, -16 %). Two (1 discontinued for non-compliance). All patients reported at least 1 AE. patients had improved 6MWT (19 % and 18 %) and stable %FVC. One Only severe AE was sleep apnea (in 2 boys). All others were mild to patient had stable 6MWT and improved %FVC (10 %). One patient had moderate. 16 patients had infusion-related AEs, similar to those previously stable 6MWT and %FVC. None of the patients had both decreased 6MWT reported. 13 patients experienced at least 1 serious AE: bronchopneumonia/ and %FVC. In conclusion, on-treatment improvements in mobility and pyrexia were the most common (3 patients each). No new ADRs or some measurements of pulmonary function were seen for patients aged clinically important changes in lab parameters/vital signs/ECGs, were >20 years at ERT initiation. reported. 19 patients seroconverted to IgG+. Growth rates/development Conflict of Interest declared. were within normal age-related ranges. Organ size and uGAGs levels decreased vs baseline by week 18 and maintained decreases throughout study. Idursulfase was generally well tolerated, and showed similar PD/ ≥ efficacy in these boys to that previously reported in males 5 years. P-218 Study NCT00607386 funded by Shire HGT. Conflict of Interest declared. QUANTIFICATION OF GLYCOSAMINOGLYCANS IN HUMAN URINE AND MOUSE TISSUE BY UPLC-MS/MS Young SP1 , Zhang H1 , Wood T2 ,FuH3 , Auray-Blais C4 , Koeberl DD1 , Millington DS1 1Div Med Genetics, Duke Univ Med Ctr, Durham, United States P-216 2Greenwood Genetics Ctr, Greenwood, United States FOLLOW-UP OF MUCOPOLYSACCHARDOSIS TYPE IVA 3Res Ins, Nationwide Children's Hospital, Columbus, United States PATIENTS: EGE UNIVERSITY EXPERIENCE 4Peds Genetics, Université de Sherbrooke, Sherbrooke, Canada E Canda1 , M Kose1 , M Kagnici1 , SK Ucar1 , M Coker1 1 Pediatric Metabolism, Ege University, Izmir, Turkey Background: Mucopolysaccharidoses (MPSs) are storage disorders characterized by glycosaminoglycans (GAGs) accumulation in tissues. We Mucopolysaccharidosis (MPS) type IVa (Morquio A disease) is an autosomal developed a sensitive pseudo-stable isotope dilution-tandem mass spectro- recessive multisystemic disease characterized by N-acetylgalactosamine-6- metric method to quantify chondroitin sulfate (CS), dermatan sulfate (DS) sulfatase deficiency. The aim of this study was to evaluate our patients with and heparan sulfate (HS). MPS type IVa Method: Urine or tissue extracts (25 μL) were dried, methanolysed and We retrospectively evaluated our patients with MPS type IVa. Demographical mixed with a deuterium-labeled DS and HS methanolysate mixture. Meth- features, clinical findings, laboratory evaluations of patients were analyzed. anolysed dimers derived from the different GAG species were separated on Mean age was10.3±6.9 years (6 months - 25 years), mean age at diagnosis an amide UPLC column and detected by selected reaction monitoring on a was 5.7±3.9 years (5 months – 16 years). Male/female ratio was 9/5. All tandem mass spectrometer. patients (except a girl who had diagnosis during the 6th months of age) had Results: The assay showed good intra and inter-day precision and accuracy, skeletal system involvement. One patient can't walk because of severe bone and was linear over the calibration ranges for CS, DS and HS. GAG species dysplasia and one patient can be able to walk with assisting device. Thirteen were elevated in patient urine corresponding to the expected excretion patients had corneal clouding. 5 patients had adenoid hyperplasia, 4 patients pattern for the particular enzyme deficiency. Mild elevations of HS and had hepatomegaly, and 4 patients had mitral valve disease. None of the DS were detected in urine from patients with MPS IIIB and MPS VI. HS patients had myocardial involvement or left ventricular dysfunction. DEXA was elevated in tissues from an untreated mouse model of MPS IIIA and analysis showed osteoporosis in 4 patients. was reduced one month after treatment with gene therapy vectors (AAV9- Patients with Morquio A disease should still be treated supportively. In the SGSH, AAVRh-SGSH). near future, patients may benefit from the improvements in enzyme replace- Conclusion: This novel method has the required sensitivity and specificity ment treatments so, patients with Morquio A disease should be followed up for quantifying CS, DS and HS accumulation in urine and tissues and is regularly. useful for diagnosing and monitoring patients with MPS. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S89

P-219 P-221 LONG-TERM EXPERIENCE WITH ENZYME REPLACEMENT ANTIBODY TITERS AND BINDING TO GALSULFASE IN 10 MPS THERAPY (ERT) IN MPS II PATIENTS WITH A SEVERE VI PATIENTS (NEURONOPATHIC) PHENOTYPE: AN INTERNATIONAL Brands MMMG1 , Kroos M.A.1 , Hoogeveen-Westerveld M.1 , van der CASE SERIES Ploeg A.T.1 , Reuser A.J.1 Lampe C1 , Bosserhoff AK1 , Burton BK2 , Souza C3 , Bittar C3 , Giugliani 1Div Metab Dis, Div Clin Genet, ErasmusMC, Rotterdam, Netherlands R4 , Muschol N5 , Olson R6 , Mendelsohn NJ6 1Villa Metabolica, Univ Medical Center, Mainz, Germany Antibody formation is frequently found in patients receiving enzyme- 2Lurie Child Hosp and NW Univ School Med, Chicago, IL, United States replacement therapy (ERT) for lysosomal storage disorders and may affect 3Medical Genetics Service, HCPA, Porto Alegre, Brazil clinical outcome. This topic is still underexposed in mucopolysaccharidosis 4Med Genet HCPA/Dpt Genet UFRGS &INAGEMP, Porto Alegre, Brazil type VI. 5Univ Med Center Hamburg-Eppendorf, Hamburg, Germany We measured antibody titers and correlated these with the binding of 6Child Hosp & Clinics of Minnesota, Minneapolis, MN, United States galsulfase (ERT) in vivo. Ten patients were included in this analysis (treatment duration 1.1- Guidelines recommend ERT be used on a trial basis for neuronopathic MPS II. 4.5 years). Blood was drawn at three months intervals from start of ERT. We describe our experiences with 22 such patients on ERT for >2 years. ERT We developed an ELISA procedure to measure antibody titers. Further- initiation was between 18 months and 21 years of age. Time on treatment was more, we investigated in the patient with the highest antibody titer what between 2 and 6 years. Retrospective data on somatic signs/symptoms, in- percentage of galsulfase was antibody-bound during galsulfase infusion. cluding skeletal disease, joint range of motion, liver and/or spleen size, All of our patients showed seroconversion after 26 weeks of ERT with titers respiratory infections, diarrhoea, skin/hair, and hospitalizations, were collect- ranging from 1:250 to 1:31,250. The highest titer ever measured was ed. All patients experienced reduced urinary glycosaminoglycan levels on 1:156,250 in an 11-year old patient receiving ERT for four years. A treatment. Continued neurocognitive decline was seen in 17 patients; stabili- pharmacokinetic analysis in this patient after 4.5 years of therapy showed zation or slight improvement was noted in 5. One of 22 patients experienced that 70 % of the enzyme was antibody-bound during infusion. This patient improvements in 7 somatic signs/symptoms; 15/22 experienced improvements had experienced two mild infusion-associated reactions. Her clinical con- in 5 to 6 somatic signs/symptoms; and 6/22 experienced improvements in 4 dition approved slightly across the years. somatic signs/symptoms. None experienced fewer than 4 improvements. No In conclusion we ascertained that antibody formation occurred early new safety concerns arose. Infusion-related reactions were experienced by 4/ during course of ERT, and that antibodies can potentially interfere 22 patients but were successfully managed by slowing the infusion and/or with effects of ERT. The clinical implications of these findings are premedication with steroids. In conclusion, long-term treatment with idursul- still under investigation. fase was associated with multiple improvements in somatic signs and symptoms in a case series of patients with neuronopathic MPS II. Editorial assistance was funded by Shire HGT. The authors take responsi- bility for the data and Shire HGT does not guarantee their accuracy. P-222 Conflict of Interest declared. BIOCHEMICAL AND MOLECULAR STUDIES IN MOROCCAN PATIENTS WITH HUNTER DISEASE P-220 Talbaoui H1 , Froissart R2 , Puech JP3 , Dahri S1 , Benouachane T1 , 3 1 NEWBORN SCREENING FOR MUCOPOLYSACCHARIDOSIS I Caillaud C , Chabraoui L 1 (MPS I) BY DRIED BLOOD SPOTS IN TAIWAN Univ Hosp of Rabat, Univ Moha V Souissi, Rabat, Morocco 2 Lin S.-P.1 ,LinD.-S.1 , Chang J.-H.2 ,LinH.-Y.1 , Wang T.-J.1 , Hospices Civils de Lyon-Est France, Lyon, France 3 Huang S.-F.1 , Chuang C.-K.1 Institut Cochin Paris France, Paris, France 1Mackay Memorial Hospital, Taipei 10449, Taiwan 2Taipei Medical University, Taipei 10449, Taiwan Hunter disease (MPSII) is an X linked iduronate-2-sulfatase deficiency (IDS) characterized by the accumulation of glycosaminoglycans, dermatan Background: MPS I is caused by deficient α-L-iduronidase activity due to sulfate (DS) and heparan sulfate (HS). The major clinical manifestations are gene defect. Three major phenotypes with wide clinical spectrum are joint contractures and often mental retardation. identified. Treatments for MPS I become possible recently. However, early Among the patients referred to our laboratory during the last 15 years, 83 diagnosis is crucial and positively related to the prognosis. A pilot MPS I had an electrophoretic profile with DS and HS. Using the fluorimetric assay newborn screening project has been applied in Taiwan. of Iduronate-2-sulfatase enzyme activity in peripheral leucocytes we found Material and Method: Two MPS I patients, 4 MPS I carriers and 23,801 only seven male patients with IDS, while the majority of patients had an newborn samples were collected (from October, 2008 to January 2012). MPS I (Iduronidase deficiency). Dry blood sample was taken on the 3rd day postpartum with 3.2 mm The MPS II patients were aged of 1 year to sixteen years and half. The diameter dry spot applied for duplicate analysis. Fluorescence assay using consanguinity rate is of 14 %. All patients presented with coarse facial features 4-methylumbelliferyl-α-L-iduronide as substrate was executed in this study. and dysostosis multiplex. Molecular analysis, performed on DNA extracted Results: The within–run and between-run precisions were 9.35 % and 11.56 %, from blood leukocytes, showed that one patient have the previously reported respectively. The reference value of α-L-iduronidase activities in newborn control mutation p.R88G, two patients presented deletion in exons 4-7 and two is 9.03~69.52 mol/L blood*20 h. The neonatal samples were analyzed and 57 patients had novel mutations (1216_1217 del CT and c.607 G>T p.G203X). infants had an IDU activity <9.03 mol/L blood*20 h. They have received notice for For the last two patients, mutations have not yet been investigated. a recheck. 16 neonates had completed the recheck (recalled rate 028 %). Only one Our study showed that Hunter disease is much less frequent than Hurler infant showed significant reduction of a-Iduronidase activity. disease in Morocco. The management of Hunter disease patients needs Conclusion: The MPS I newborn screening has a role of early diagnosis. The recall multidisciplinary structure and adequate genetic counseling. rate is lower than that expected. More efforts are needed to promote the test. Conflict of Interest declared. S90 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-223 P-225 ATTENUATED PHENOTYPE IN MPS VI (MAROTEAUX-LAMY) NATURAL HISTORY, INCIDENCE AND PREVALENCE RATES PATIENTS CARRYING THE P.R152W MUTATION OF MUCOPOLYSACCHARIDOSIS TYPE VI IN CENTRAL AND Jurecka A1 , Zakharova E2 , Cimbalistiene L3 ,GusinaN4 , Kulpanovich A4 , EASTERN EUROPE Golda A5 , Opoka-Winiarska V6 , Piotrowska E7 , Voskoboeva E2 , Jurecka A1 , Cimbalistiene L2 , Gusina N3 , Malinova V4 ,Różdżyńska- Tylki-Szymańska A1 Świątkowska A1 , Golda A5 , Kulpanovich A3 , Kaldenovna Abdilova G6 , 1The Children's Memorial Health Institute, Warsaw, Poland Opoka-Winiarska V7 , Õunap K8 , Tylki-Szymańska A1 2Research Center for Medical Genetic, Moscow, Russian Federation 1The Children's Memorial Health Institute, Warsaw, Poland 3Vilnius University Hospital, Vilnius, Lithuania 2Vilnius University Hospital, Vilnius, Lithuania 4National Medical Center 'Mother & Child', Minsk, Belarus 3National Medical Center 'Mother & Child', Minsk, Belarus 5Gliwice General Hospital, Gliwice, Poland 4Charles University, Prague, Czech Republic 6Medical University of Lublin, Lublin, Poland 5Gliwice General Hospital, Gliwice, Poland 7University of Gdańsk, Gdańsk, Poland 6Research Center of Pediatrics, Almaty, Kazakhstan 7Medical University of Lublin, Lublin, Poland Maroteaux-Lamy syndrome or mucopolysaccharidosis type VI (MPS VI) is a 8Tartu University Hospital, Tartu, Estonia rare lysosomal, autosomal recessive storage disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase. The aim of the study was to The aim of the study was to describe the natural history, the incidence and describe the natural clinical course in patients homozygous for R152W muta- prevalence rates of mucopolysaccharidosis type VI (MPS VI) in Central and tion from Central and Eastern Europe. From our database of 31 patients with Eastern Europe. Patients (n031) were identified by retrieving the data from MPS VI, we selected those with the p.R152W mutation in the homozygous seven centers for MPS disorders. For patients with severe phenotype (n08; (n010, median age 27.5 years, range 18-38 years). We performed a cross- 26 %) the mean age at the onset of symptoms and at diagnosis was sectional observational study. Patients homozygous for the p.R152W mutation 9.5 months and 5 years, for patients with intermediate phenotype (n014; showed a much more attenuated course of the disease characterized by a 45 %) 3 and 8 years, while for patients with attenuated phenotype (n010; significantly slower disease progression. First signs of the disease, usually 32 %) 10.3 and 24 years, respectively. The incidence rates ranged from slight range of motion limitations and mild skeletal malformations were 0,0363 per 100 000 live births in Poland to 0,64 per 100 000 live births in observed at the median age of 6 years (mean age 8, range 1-21 years). Over Lithuania. The prevalence rates ranged from 0,013 in Poland to 0,235 in 57 % of these patients showed first signs of the disease after the age of 5 years, Lithuania per 100 000 inhabitants. but even as late as 21 years. In these patients height was only slightly decreased Conclusions: 1. A large number of patients in our study presented with an and MPS VI features developed later in the course of the disease. attenuated phenotype. 2. A patient's height and genotype were related to the Conclusion: The p.R152W mutation in the homozygous state is associated severity of the disease, while no clear trend between a patient's height and with a relatively attenuated phenotype of mucopolysaccharidosis type VI. urinary GAG levels was observed. 3. The incidence values for Poland were the lowest from all previously published, while values for Lithuania were among the highest.

P-224 P-226 RESTRICTED JOINT RANGE OF MOTION IN MPS II PATIENTS: OBSTRUCTIVE SLEEP APNEA SYNDROME AND UPPER AIRWAY CORRELATION WITH AGE AND HEIGHT PROBLEMS IN MUCOPOLYSACCHARDOSIS PATIENTS Marucha J1 , Jurecka A1 , Syczewska M1 ,Różdżyńska-Świątkowska A1 , Gönüldaş B1 , Yilmaz T1 , Kalkanoğlu Sivri SH2 Tylki- Szymańska A1 1Ent Head and Neck Sur,Uni Hosp Hacettepe, Ankara, Turkey 1The Children's Memorial Health Institute, Warsaw, Poland 2Div Metab Dis,Uni Hosp Hacettepe, Ankara, Turkey

The aims of the study were to assess joint range of motion (ROM) in Background: Obstructive Sleep Apne Syndrome (OSAS) and breathing prob- patients with mucopolysaccharidosis type II (MPS II) and to correlate joint lems seen in mucopolysaccharidosis (MPS) patients due to the upper airway mobility with patients' age and height. Passive ROM and SDs of height obstruction because of adenoid hypertrophy, tonsillary hypertrophy, macroglos- were followed in 29 patients with MPS II between years 2005-2010. sia, nasal obstruction, neck stiffness, and short neck. Polysomnography is known Results: 1. Restriction in upper extremities ROM in patients with MPS II as the gold standard test for evaluation of OSAS. Adenotonsillectomy is mostly was observed since the second year of life. These limitations were partic- done for treatment of children with OSAS. It's the same for MPS patients. ularly visible in the elbows (reduced extension), the shoulders (limitation of Materials and Methods: 70 MPS patients evaluated in otolarynghology flexion and abduction) and the wrists (restriction of flexion and extension). outpatient clinic. Polisomnography to 22 of patients randomly selected and 2. ROM limitations intensified and became more severe with the patients' the results compared. Patients grouped according to apnea-hypoapnea index age, making patients' self-care more difficult or impossible. A particularly (A-HI), Upper airway obstruction level, MPS type and age. Kuruskal-Vallis strong correlation between joint mobility and patient's age was visible for and Mann-Whitney U test used in statistical analysis. elbow and wrist extension. 3. A strong correlation was observed between Results: The patient with high obstruction level had severe OSAS. There were ROM and patients' height, particularly for elbow and wrist extension. 4. A no statistical relation with MPS subtypes, age and OSAS scores.90 % of patients strong correlation was observed between patients' age and patients' height. had OSAS.Recurrence rate of adenoid hypertrophy is high after operation. Conclusions: Documentation of the potential ROM limitations as early as Conclusion/Discussion: Incidence of OSAS is very high in MPS patients. the second year of life supports the need for early treatment. To achieve Adenotonsillectomy is the first opsion for treatment but anaesthesiology of holistic benefits of the I2S therapy, physical therapy should be added and MPS patients is so difficult and there is high risk of complication.Before adjusted to the patient's efficiency and capabilities. operation PSG should be done and patients that have high A-HI levels must be treated with CPAP or BPAP. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S91

P-227 P-229 THE EFFECT OF GLOBOTRIAOSYLCERAMIDE (GB3) IN PREGNANCY-RELATED OUTCOMES IN FEMALES WITH FABRY PATIENTS UNDER ENZYME REPLACEMENT THERAPY: FABRY DISEASE: FABRY OUTCOME SURVEY AND OTHER CORRELATIONS WITH OXIDATIVE STRESS AND POSTMARKETING SAFETY DATA INFLAMMATION Deegan PB1 , Barba-Romero MA2 , HollaK CE3 , Giugliani R4 , Larroque Biancini GB1 , Vanzin CS1 , Rodrigues DB2 ,DeonM2 , Barschak AG3 ,Ribas S5 , Verma A6 , Hughes DA7 GS2 ,ManfrediniV4 , Netto CBO2 , Jardim LB2 , Giugliani R2 ,VargasCR5 1Addenbrooke's Hospital, Cambridge, United Kingdom 1PPG Ciências Biológicas:Bioquímica,UFRGS, Porto ALegre, Brazil , 2Albacete University Hospital, Albacete, Spain 2Serviço de Genética Médica, HCPA, Porto Alegre, Brazil 3Academic Medical Centre, Amsterdam, Netherlands 3PPG Bioquímica e Bioprospecção, UFPel, Pelotas, Brazil 4Med Genet Serv HCPA/Dep Genet UFRGS and, Porto Alegre, Brazil 4Lab Hematol e Citol Clínica, UNIPAMPA, Uruguaiana, Brazil 5Shire HGT, Nyon, Switzerland , 6Shire HGT, Lexington, MA, United States 5PPG Ciências Farmacêuticas, UFRGS, Porto Alegre, Brazil 7Royal Free Campus, University College, London, United Kingdom

Background: There is some evidence in the literature, by in vitro studies, Background: Females with Fabry disease (FD), an X-linked lysosomal demonstrating that oxidative stress may be involved in Fabry disease (FD) storage disorder, exhibit variable symptomatology, but FD effects on preg- pathophysiology. Studies reported the existence of an inflammatory process nancy/pregnancy-related outcomes are not well documented. in it. Globotriaosylceramide (Gb3) accumulates in FD patients and is Objective: To provide a descriptive overview of pregnancy events in FD associated with disease progression. females, highlighting specific cases. Objective: To investigate oxidative damage to biomolecules and inflam- Methods: Fabry Outcome Survey (FOS, Shire HGT) data were collected mation in Fabry patients under Enzyme Replacement Therapy (ERT) and (clinical reports, questionnaires) on pregnancies, pregnancy-related events (e.g., correlate the findings with Gb3 levels. miscarriages, newborn clinical characteristics), other clinical characteristics, and Patients and Methods: Urine and plasma samples of treated Fabry patients treatment status (agalsidase alfa 0.2 mg/kg every-other-week, untreated). and controls were used for? biochemical and immunological determinations. Results: 1050/1933 females enrolled in FOS; 41 had available pregnancy data Results: Patients presented with increased plasma levels of malondialdehyde (total 64 pregnancies). Most were untreated (n026/64) or not yet treated (n0 (MDA) and carbonyl groups, reflecting lipid and protein damage, respectively. 29/64) while pregnant. Sixty (93.8 %) pregnancies were normal; 4 (6.2 %) had The pro-inflammatory cytokine interleukine-6 (IL-6) was also increased in spontaneous abortions. Eight (12.5 %) babies had reported FD diagnoses. In patients. Patients presented significant higher level of urinary Gb3, which was addition to FOS data, 14 other women had maternal agalsidase alfa exposure positively correlated with plasma levels of IL-6, MDA and carbonyl groups. during pregnancy (Shire Global Safety System, SGSS, as of March 31, 2012). Conclusion/Discussion: This is the first in vivo study linking oxidative No adverse outcomes in these patients were deemed agalsidase alfa-related. stress, inflammation and Gb3 in Fabry patients. Our findings indicate that Conclusions: FD had no apparent adverse impact on the course of preg- Fabry patients under ERT have a pro-inflammatory and pro-oxidant status, nancy. Pregnancy outcomes of women treated with agalsidase alfa during probably induced by Gb3 accumulation. Further research and clinical trials pregnancy will be further explored through examination of SGSS data and are needed to reveal the safety and effectiveness of antioxidant supplemen- FOS clinician's experiences. tation in combination with ERT in these patients. Conflict of Interest declared.

P-228 FABRY DISEASE SCREENING PROGRAM IN HYPERTROPHIC CARDIOMYOPATHY Eyskens FJM1 , Paelinck B2 1Div Metab Dis, UZA, Antwerp, Belgium 2Div Cardiology, UZA, Antwerp, Belgium

Background: Progressive left ventricular hypertrophy (LVH) is a common cardiac manifestation of Fabry disease (FD). It has been estimated that 3 % of males suffering from LVH and 6 % of males with late-onset LVH have Fabry disease. Up to 5 % of females with late-onset LVH may have Fabry disease. The D313Y mutation of the alfa-galactosidase gene (GLA) was reported in 0.45 % of normal X-chromosomes. We found it in 1.1 % of 453 females with stroke, which was confirmed in the Portystroke study. Objective: To screen systematically for FD in patients, both males and females, from the age of 18 – 60 years, with a diagnosis of unexplained LVH with a minimum left ventricular wall thickness of≥13 mm and/or septum≥13 mm. In males an enzymatic assay of alfa-gal A was used in dried blood spots. In females DNA analysis was performed. Preliminary results: The results of 172 screened individuals after 1 year was obtained. In 115 males normal enzymatic activity was found in blood spots. In 57 women the D313Y mutation was found in 2 patients (3.5 %). Conclusion: Based on our findings the D313Y mutation in the GLA gene seems to predispose to develop cardiac and/or vascular disease. S92 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-230 P-232 RATE OF CHANGE OF GFR AND LV MASS IN FABRY DISEASE: CARDIAC OUTCOMES OF AGALSIDASE BETA TREATMENT TARGETS FOR THERAPEUTIC GOALS FROM THE CANADIAN FOR FABRY DISEASE: ANALYSIS OF CARDIOVASCULAR FABRY DISEASE INITIATIVE (CFDI) DISEASE PROGRESSION IN MEN ENROLLED IN THE Sirrs S1 , Lemoine K2 , Bichet DG3 , Casey R4 , Clarke JTR5 , West ML2 FABRY REGISTRY 1University of British Columbia, Vancouver, Canada Germain DP1 , Abiose A2 , Patel MR3 , Weidemann F4 , Benistan K1 , 2Dalhousie University, Halifax, Canada Waldek S5 , Strotmann J6 3University of Montreal, Montreal, Canada 1Univ Versailles, Hôpital R. Poincaré, Garches, France 4University of Calgary, Calgary, Canada 2North Ohio Heart Center, Westlake, OH, United States 5University of Sherbrooke, Sherbrooke, Canada 3Div Cardiovasc Med, Duke Univ Sch Med, Durham, NC, United States 4Dept Internal Medicine, Univ Würzburg, Würzburg, Germany Background: There is wide discrepancy in the literature on the rate of change 5Independent Medical Consultant, Manchester, United Kingdom (Δ) in clinical parameters like GFR and LV mass index (LVMI) in patients 6Medizin Klinik, Städtisches Krankenhaus, Kiel, Germany with Fabry disease (FD). To facilitate the development of "therapeutic goals" for FD, we report ΔeGFR and ΔLVMI in 285 patients from the CFDI. Background: Patients with Fabry disease often develop cardiovascular Methods: Patients from the CFDI on ERT (Cohorts 1a and 1b) are followed complications, including left ventricular hypertrophy (LVH), which every 6 months and patients not currently meeting criteria for ERT every increases risk for cardiovascular events. 12 months. Annual ΔeGFR (ml/min/1.73 m2/year) and annual ΔLVMI by echo Objectives: To determine the progression of LVH in men with Fabry (%) are reported. Data from the ERT treated cohorts are combined for analysis. disease and to identify specific factors associated with left ventricular mass Results: The mean followup of ERT treated patients was 40 (Cohort 1b) to (LVM) stabilization. 46.9 (Cohort 1a) months and 14 months for untreated (Cohort 1c) patients. Methods: LVM was analyzed in adult men who had 2 or more LVM assess- 87 ERT treated males showed an annual ΔeGFR of -2.05 and ΔLVMI ments within 2 or more years, including men treated with agalsidase beta at an +0.73 %/year. 68 ERT treated females showed an annual ΔeGFR of +0.67 average dose of 1 mg/kg/2 weeks (n0115) and untreated men (n048). and ΔLVMI was +0.93 %/year. 16 untreated males had an annual ΔGFR of Results: In men who initiated treatment at age 18 to <30 years, mean LVM -0.8 and ΔLVMI of -0.1 %/year while annual ΔeGFR in 114 untreated slope was –3.6 g/year (n031), compared to +9.5 g/year in untreated men in the females was -1.3 and ΔLVMI was +0.4 %/year. same baseline age category (n015), p<0.0001. Patients were grouped into Conclusions: Long term data from the CFDI provide data to develop quartiles based on individual LVM regression slopes (N0163). Untreated men therapeutic goals of therapy in patients with FD. had a 3.4-fold likelihood of being in quartile 4 (Q4) than treated men (odds Conflict of Interest declared. ratio 3.43, 95 % CI 1.05–11.22, p00.0415). Additionally, multivariate logistic regression identified factors significantly associated with LVH: baseline age 40 or older and baseline LVM greater than the median (238 g). Conclusions: Agalsidase beta treatment improved or stabilized LVM in men with Fabry disease; and was most beneficial in those who began treatment before age 30. P-231 Conflict of Interest declared. CLINICAL PHENOTYPING AND FAMILY SCREENING OF PATIENTS WITH α-GAL A ENZYME DEFICIENCY OR α-GAL P-233 A GENE ABNORMALITIES IDENTIFIED IN A LARGE FABRY PRODUCTION OF PROINFLAMMATORY CYTOKINES BY SCREENING PROGRAM IN STROKE IN THE YOUNG. MONONUCLEAR CELLS FROM FABRY DISEASE PATIENTS. Eyskens FJM1 , De Deyn PP2 1 ROLE OF GLOBOTRIAOSILCERAMIDE Div Metab Dis, UZA, Antwerp, Belgium 1 1 1 1 2 Rozenfeld PA , De Francesco PN , Mucci JM , Ceci R Div Neurology, ZNA Middelheim, Antwerp, Belgium 1LISIN, Universidad Nacional de La Plata, La Plata, Argentina

Introduction: The results of the Belgian Fabry Study I (1000 patients Background: Fabry disease is a lysosomal storage disorder associated to included) suggested that Fabry disease may play a role in up to 1 % of accumulation of globotriaosilceramide (Gb3). Pathological mechanisms of this young female patients with stroke. We report on genetical, biochemical and disorder are poorly understood, chronic inflammation was suggested as one of clinical investigation results in 4 different families. the contributing factors. The aim is to analyze cytokine levels in peripheral α Methods: All subjects were investigated genetically, biochemically ( -Gal A in blood mononuclear cells (PBMC) from Fabry disease patients, as well as to test plasma and/or leukocytes, Gb3 levels in plasma and urine and lyso-Gb3 levels in the effect of Gb3 addition to normal control cells on cytokine secretion. plasma). They were clinically investigated (heart, cerebral MRI, audiogram, Methods: PBMC from Fabry disease patients and normal controls were incu- ophthalmological examination, dermatological examination, skin biopsy (elec- bated with specific antibodies for surface markers (CD56, CD3, LIN1, HLA- tron microscopy) and renal evaluation (GFR, proteinuria, microalbuminuria)). DR, CD14) and the cytokines IL-1β,IL-6,yTNF-α (intracellular staining), and Results: Genetic family screening revealed 18 individuals who carried a mutation. assessed by flow cytometry. Moreover monocytes and dendritic cells obtained Three of them (two females and 1 male) showed significant organ involvement from normal donors were incubated with Gb3 and DGJ (GLA inhibitor), and (renal and/or cardiac). Normal α-Gal A enzyme activity in plasma was detected in secretion of cytokines were determined in culture supernatants. a non-affected male hemizygous for the A143T mutation. Analysis of Gb3/lyso- Results: We found higher levels of TNF-α and IL-1β (p<0,05) in dendritic Gb3 offers no added value in our population in predicting disease activity. cells and IL-6 in monocytes (p<0,05) from Fabry patients as compared to Conclusions: Our findings indicate the importance of screening protocols normal controls. Exposure of dendritic cells and monocytes from normal for the identification of Fabry patients and their affected family members. donors to Gb3 induced the secretion of IL-1β and TNF-α. Clinicians should be aware that males with atypical forms of Fabry disease Conclusions: These results are compatible with the existence of a proin- with residual enzyme activity can be missed by α-Gal A enzyme activity flammatory condition in Fabry disease. Moreover, Gb3 accumulation could screening (blood spots and plasma). be one of the factors implicated in this phenomenon. Conflict of Interest declared. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S93

P-234 P-236 PATHOPHYSIOLOGY OF M. FABRY: ABNORMALITIES OF VITAMIN D LEVELS AMONGST ADULT PATIENTS WITH 'LIPID-RAFTS'WHICHCANBEREVERSEDBYERTAND FABRY DISEASE, A SINGLE CENTRE EXPERIENCE NB-DNJ INCUBATION IN VITRO Whitby JE1 , Jovanovic A1 , Thompson L1 Maalouf K1 , Naim HY2 , Das AM1 1Adult Metab Dis, Salford Hospital, Salford, United Kingdom 1Hannover Medical School, Hannover, Germany 2University of Veterinary Medicine, Hannover, Germany The National Diet and Nutrition Survey demonstrates that up to a quarter of people in the UK have low levels of vitamin D in their Introduction: It is not clear how alpha-galactosidase deficiency translates blood, making them at risk of the clinical consequences of vitamin D into clinical symptoms of M. Fabry. Disturbed glycosphingolipid metabo- deficiency. Low status is defined by the DOH as a plasma concentra- lism may lead to abnormal lipid composition of cell membranes and tion of 25-hydroxyvitamin D (25(OH) D) of below 25 nmol/l (10 alteration of 'lipid rafts'. microgram/l). Methods: We studied trafficking of the raft-associated protein dipeptidyl- The audit was retrospective. We referred to each individual's patient records peptidase IV and the non-raft-associated protein aminopeptidase N using for our data, recording the first measure for vitamin D3. immunofluorescence in cultured fibroblasts from male patients with M. The audit was conducted on 206 patients, of which 163 had their level of Fabry. Furthermore, we measured 'lipid raft'-associated flotillin 2 with vitamin D2 and D3 measured by tandem mass spectrometry. The patients sucrose density gradients. had an age range of 16-74, with a mean age of 42.36. BMI ranged from 16.5 Results: Trafficking of aminopeptidase N was normal, while traffick- – 44.5, with a mean of 22.23. Vitamin D3 concentration had a range of 2.2 – ing of the raft-associated protein dipeptidylpeptidase IV to the plasma 55 microgram/l, with a mean of 16.357. membrane was impaired. Flotillin 2 shifted to the soluble fraction of 68 % of the 163 patients had vitamin D3 levels below 20 microgram/l, 30 % the sucrose gradient. After treating the diseased cells with agalsidase below 10 microgram/l and 6 % below 5 microgram/l. alfa (10 and 20 μg/ml), flotillin 2 shifted back to the floating In conclusion, many of our patients have less than the recommended fraction, indicating partial reversibility of 'lipid- raft' abnormalities levels of vitamin D3 in their blood. These results show a higher than in M. Fabry. average prevalence of vitamin D3 insufficiency in patients with Fabry Discussion: Trafficking of 'lipid-raft'- associated proteins was compromised disease. in fibroblasts from males with M. Fabry. This may indicate abnormal function of 'lipid rafts' in the plasma membrane contributing to pathophysiology. If these findings could be extrapolated to other cells it may explain multiorgan dysfunction in M. Fabry. By incubating the cells in vitro with P-237 NB-DNJ, we showed that abnormalities in 'lipid rafts' are linked to storage STROKE IN FABRY DISEASE: GENOTYPE-PHENOTYPE processes. CORRELATION Ginsberg L1 , Larroque S2 , Burlina A3 , Giugliani R4 , Jardim L4 , Manara R5 , Reinke J6 , Gal A7 1 P-235 Royal Free Hospital, London, United Kingdom 2Shire HGT, Nyon, Switzerland IDENTIFICATION OF THE POTENTIAL URINARY BIOMARKER 3Neurological Unit, San Bassiano Hospital, Bassano del Grappa, Italy PROACTIVATOR POLYPEPTIDE IN FABRY DISEASE 4Medical Genetics Service, HCPA, Porto Alegre, Brazil Manwaring V1 , Heywood W1 , Heales S2 , Mills K1 5Neuroradiologic Unit,University Hospital, Padova, Italy 1UCL Institute of Child Health, London, United Kingdom 6University of Mainz, Mainz, Germany 2Great Ormond Street Hospital, London, United Kingdom 7Institut für Humangenetik, Universitäts, Hamburg, Germany

Background: Using label-free quantitative proteomics we have shown that Background: Stroke is an important complication of Fabry disease, an X- the levels of peptides from proactivator polypeptide (PSAP, prosaposin) in linked lysosomal storage disorder caused by mutations of GLA, the gene the urine of Fabry disease patients decrease following enzyme replacement encoding alpha-galactosidase A. therapy (ERT). Objective: To characterize genotypes (ie, mutations) according to biochem- Objectives: To confirm our findings by developing a rapid MRM-based ical severity in Fabry patients with stroke. peptide assay (UPLC-MS/MS) to quantitate this potential biomarker. Methods: The Fabry Outcome Survey (Shire HGT) is an international Materials and Methods: Control and pre/post treatment urine samples (n> database containing stroke prevalence data (154/1933 patients08.0 %, as 70) were analysed using UPLC-MS/MS and aquapeptide/stable isotope of September 2010). The patient mutations were divided into two groups: peptide standards. (1) null alleles and missense mutations associated with the classical pheno- Results: Levels of saposin A in the urine of patients undergoing ERT were type, and (2) 'mild' missense mutations, known or predicted to have signif- observed to increase after treatment of patients with Fabrazyme. icant residual enzyme activity. Discussion: Following treatment with ERT, the levels of the detergent like Results: Stroke prevalence was higher in group 1 (8.8 %) than group 2 (1.8 %; protein saposin A were found to increase and we postulate that ERT therapy p<0.001), a difference which remained significant when genders were analysed increases the processing of the prosaposin protein into the functioning separately. Of the 87 patients with the oligosymptomatic p.N215S mutation (a subunits saposin A-D. This rapid UPLC-MS/MS assay may be a useful mutation in group 2), only one, a 65-year-old man, had a stroke. Within each adjunct to Gb3 and lyso-Gb3 for monitoring treatment in Fabry disease. mutation category, stroke patients tended to have more severe phenotypes generally, as determined by an age-dependent disease severity score. Conclusion: Fabry patients with more severe mutations are likelier to have strokes than those with mild mutations. Stroke patients in the latter category may have additional cerebrovascular risk factors (age, other diseases). Conflict of Interest declared. S94 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-238 P-240 FABRY DISEASE: CLINICAL SEVERITY CORRELATES WITH OPHTHALMOLOGICAL MANIFESTATIONS IN CHILDREN GENE MUTATION WITH FABRY DISEASE IN THE FABRY OUTCOME SURVEY Gal A1 , Larroque S2 Kalkum G1 , Beck M1 , Larroque S2 , Parini R3 , Pintos-Morell G4 , 1Institut für Humangenetik, Universitätsk, Hamburg, Germany Ramaswami U5 , Rohrbach M6 , Pitz S7 2Shire HGT, Nyon, Switzerland 1University of Mainz Children's Hospital, Mainz, Germany 2Shire HGT, Nyon, Switzerland Fabry disease (FD) is an X-linked lysosomal storage disorder due to deficiency 3University of Milano-Bicocca, Monza, Italy of alpha-galactosidase A. Clinical manifestations of FD are very heterogeneous 4Germans Trias i Pujol University Hospita, Badalona, Spain and become more severe with age. The relationship between genotype and 5Addenbrooke's Hospital, Cambridge, United Kingdom phenotype has repeatedly been analyzed but only a few studies could show 6University Children's Hospital, Zurich, Switzerland positive correlations for selected phenotypic features. Instead of the widely used 7Johannes Gutenberg University, Mainz, Germany descriptive classification of mutations, we used a functional classification based on the putative biological consequence of the mutations. In this approach, an Background: Classic Fabry disease (FD) clinical features usually manifest amino acid change in the enzyme's active center due to a missense mutation or during childhood, including ophthalmological signs that may help diagnosis one interfering with the proper folding of the protein is considered a null allele. (eg, cornea verticillata [CV], increased vessel tortuosity, posterior cataract). We analyzed 486 males between 6-60 years from the Fabry Outcome Survey Objective: To characterize eye findings in pediatric FD patients. (FOS, Shire HGT) and found significant correlation (non-parametric linear Methods: Ophthalmologic signs and symptoms of agalsidase alfa-treated and trend: p:0.014) between mutation types (null alleles – missense mutations – untreated children (<18-year-old) were obtained from Fabry Outcome Survey missense with residual enzyme activity) and disease severity before enzyme (FOS; sponsored by Shire HGT) data, collected until September 2010. replacement therapy. Disease severity was measured as the difference between Results: 261/308 (84.7 %) children underwent ophthalmologic examinations. patients' actual and predicted age-dependent average MSSI scores. The power of 164/261 (62.8 %) children had any eye finding (eg, CV 151 [57.9 %], vessel new surrogate markers can be tested by the correlation found. Our approach tortuosity 68 [26.1 %], cataracts 13 [5.0 %]). Any ocular finding occurred in might also help to predict the course of disease for making therapeutic decisions. 85/136 (62.5 %) girls (CV 83 [61.0 %], vessel tortuosity 32 [23.5 %], cataract Conflict of Interest declared. 5 [3.7 %]) and 79/125 (63.2 %) boys (CV 68 [54.4 %], vessel tortuosity 36 [28.8 %], cataract 8 [6.4 %]). 91/120 (75.8 %) agalsidase alfa-treated and 73/ 141 (51.8 %) untreated children had any eye finding. Older paediatric patients tended to exhibit higher incidences of CV than younger patients (≥3-<6 years P-239 17/45 [37.8 %] through ≥15-<18 years 73/125 [58.4 %]). ANGIOKERATOMA IN FABRY DISEASE: SIGNIFICANT Conclusions: Most (84.7 %) FOS-enrolled children underwent detailed oph- CORRELATION BETWEEN GENOTYPE AND PHENOTYPE thalmologic examinations. No gender effects were observed on eye findings. Orteu CH1 , Larroque S2 , Mehta A1 , Gal A3 Incidences of CV may increase over time in treated and untreated children. 1Royal Free Hospital, London, United Kingdom Conflict of Interest declared. 2Shire HGT, Nyon, Switzerland 3Institut für Humangenetik, Universitätsk, Hamburg, Germany

Angiokeratoma (AK) is the cutaneous hallmark of Fabry disease (FD), an P-241 X-linked, lysosomal storage disorder. Clinical manifestations are heterogeneous and become more severe with age. We have developed a functional ANTIBODIES TO ENZYME REPLACEMENT THERAPY IN classification of GLA (alpha-galactosidase A gene) mutations, based on FABRY DISEASE: URGENT NEED TO STUDY CLINICAL their putative biological consequence, into 3 groups (null alleles, missense IMPACT 1 1 mutations, and mild missense mutations with residual enzyme activity). Linthorst GE , Hollak CEM 1 Using patient data from the Fabry Outcome Survey (sponsored by Shire Intern Med, Acad Med Centr, Amsterdam, Netherlands HGT), most males with null (80 %) or missense (71 %) mutations, but only 50 % of those with mild missense mutations, had AK. Analysis of all Background: Fabry disease (FD), alfa-galactosidase A (aGal A) deficiency, patients showed a significant (p<0.001) linear correlation between age- causes globotriaosylceramide and globotriasylsphingosine (Gb3 and lysoGb3) adjusted relative disease severity scores (aaFOS-MSSI) and putative bio- accumulation. Enzyme replacement therapy (ERT) with exogenous aGal A logical consequences of the mutations, with a gradual decrease of the scores results in antibody formation towards agalsidase in a subset of male. of patients carrying null, missense, and mild missense mutations. Patients Methods: PubMed search on human studies with>1 year ERT and impact of with AK had significantly (p<0.001) higher aaFOS-MSSI scores in all antibody on biochemical and clinical disease parameters. mutation classes than those without AK. This is the first time that a highly Results: Ten studies were identified (2 on agalsidase alfa, 3 on agalsidase beta, significant correlation has been demonstrated between the type of GLA 5 on both). All studies demonstrated impact of antibodies on urinary Gb3, but mutation and severity of the patients' clinical phenotype. In patients carry- outcome on plasma Gb3 was mixed. Three studies demonstrated that a switch ing mutations of the same class, AK may be a predictor of more severe to higher dose resulted in a decrease in Gb3 in some, but not all patients, while disease. switching to a lower dose generally resulted in (lyso)Gb3 increase. Re- Conflict of Interest declared. occurrence of skin tissue Gb3 was demonstrated in males with the highest AB titers. Clinical effects have not been convincingly shown, but analysis is hampered as ERT is of limited effectiveness in males with advanced disease. Conclusion: FD males often develop antibodies to aGal A, depending on preparation or dose. These antibodies exert a negative effect on biomarkers, which can partially be overcome by dose increase. We postulate that these antibodies also have an adverse effect on clinical outcome. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S95

P-242 P-244 MIGALASTAT HCL, AN INVESTIGATIONAL PHARMACOLOGICAL A NOVEL PHASE 2A STUDY DESIGN TO INVESTIGATE CHAPERONE THERAPY: SCREENING RESULTS FROM FACETS, A THE EFFECT OF AT2220 (DUVOGLUSTAT HCL) ON THE PHASE 3 STUDY IN MALE AND FEMALE FABRY PATIENTS PHARMACOKINETICS OF ACID ALPHA-GLUCOSIDASE IN Nicholls K1 , Castelli J2 , Winkler R2 , Sitaraman SA2 , Benjamin ER2 ,Wu SUBJECTS WITH POMPE DISEASE X2 , Duke C2 , Boudes P2 Johnson FK1 , Kishnani P2 , Tarnopolsky M3 , Sivakumar K4 , Byrne B5 , 1Royal Melbourne Hospital, Parkville, Australia , 2Amicus Therapeutics, Goker-Alpan O6 , Guter K7 , Pervaiz M8 , Dasouki M9 , Levine T10 , Cranbury, United States Roberts M11 , Winkler R1 , Valenzano KJ1 , Lockhart DJ1 , Boudes P1 1Amicus Therapeutics, Cranbury, NJ, United States Fabry disease (FD) is characterized by deficient α-galactosidase A (α- 2Duke University Medical Center, Durham, NC, United States Gal A) activity and accumulation of globotriaosylceramide. Over 600 3McMaster University Medical Center, Hamilton, ON, Canada mutations in the gene encoding α-Gal A are associated with FD. 4Neuromuscular Research Center, Scotsdale, AZ, United States Migalastat HCl (AT-1001, GR181413A), an investigational pharmaco- 5University of Florida, Gainesville, FL, United States logical chaperone, is being studied in Phase 3 for FD (FACETS, 6LSD Treatment and Research Center, Springfield, VA, United States AT1001-011, NCT00925301). Screening included sequencing of the 7Great Falls Clinic, LLP, Great Falls, MT, United States GLA gene to identify the FD mutation. The mutation was considered 8Emory University, Decatur, GA, United States "addressable" if the expressed mutant form of α-Gal A is responsive to 9University of Kansas Medical Center, Kansas City, KS, United States migalastat-HCl in cell-based assays according to pre-defined criteria. 10Phoenix Neurological Associates, LLC, Phoenix, AZ, United States As of December 15th, 2011, 60 males and 120 females have been 11Salford Royal Hope HNS Trust Hope Hospit, Salford, England, United screened. The mean (std. dev.) age was 41 (14) years. 154/180 subjects Kingdom carried a missense mutation. 136/154 (88 %) subjects carried an addressable mutation. For 18/154 (12 %), the mutation was not consid- Pompe disease is an inherited lysosomal storage disease that results from a ered addressable. 68 unique missense mutations were confirmed. Of deficiency in acid alpha-glucosidase (GAA) activity, and is characterized by these, 56 (82 %) were considered addressable. 15 mutations were not progressive accumulation of lysosomal glycogen primarily in heart and previously described. Frequent substitutions were N215S (17 subjects), skeletal muscles. AT2220 is a pharmacological chaperone that selectively R342Q (8 subjects), R112H (7 subjects), and R301Q (7 subjects). All and reversibly binds and stabilizes GAA. AT2220-010 is an open-label, four were addressable. In summary, a majority of the FD subjects single ascending dose, fixed-sequence, two-period, dose-finding study screened for FACETS carried addressable missense mutations and were comprised of four cohorts of 4 to 6 subjects with Pompe disease. Study potentially eligible for the study. Screening for FACETS is complete objectives: 1) evaluate the safety of AT2220 co-administered with rhGAA; and 67 subjects have been randomized. 2) assess the effect of AT2220 on rhGAA plasma and muscle activity. Conflict of Interest declared. During Period 1 subjects receive rhGAA alone, and Period 2 they receive a single oral dose of AT2220 1 hour prior to the start of infusion. Safety data from each completed dose cohort is reviewed by a Data Safety Monitoring Board. An innovative muscle biopsy technique, which requires specialized investigator training, is used to sample quadricep muscle either on Day 3 or P-243 7 of each period. This unique fixed-sequence design allows each subject to serve as their own control and reduces variability compared to parallel COMPARISON BETWEEN ALPHA-GALACTOSIDASE A ACTIVITY designs that require larger sample sizes. The design is especially useful IN DRIED BLOOD SPOTS, PLASMA AND LEUKOCYTES OF for the evaluation of safety and pharmacokinetics in rare disease clinical HEALTHY MEN AND WOMEN studies. 1 1 1 1 Daitx VV , Mezzalira J , Goldim MPS , Coelho JC Conflict of Interest declared. 1Biochem Dept, Univ Fed Rio Grande do Sul, Porto Alegre, Brazil

The enzyme alpha-galactosidase A (GLA) is a lysosomal hydrolase that degrades glycosphingolipids. A deficiency of this enzyme characterizes Fabry disease (FD), an X linked recessive genetic disease. Considering the pattern of inheritance and the difficulty to establish a conclusive biochemical diagnosis of FD, the objective of this study was to compare the GLA activity in dried blood spots (DBS), plasma and leukocytes of healthy men and women. We used 9 ml blood for obtaining DBS, leukocytes and plasma samples. GLA activity was determined by fluorometric assays. For DBS, mean GLA activity in healthy women (n010) was significantly different from that in healthy men (n010) (7.67±3.33, and 4.50± 1.01 nmol/h/mL, respectively; p<0.01). The same was observed for plasma (women: 5.98±1.17 nmol/h/mL; men: 7.57±1.84 nmol/h/mL; p<0.03). However, in leukocytes no such significant difference was observed between GLA activity in women (26.27±10.36 nmol/h/mg) and men (34.25 ±10.57 nmol/h/mg) (p>0.05). New reference and cutoff values were established based on the differences in GLA activity between genders. Our results are important to improve the techniques to diagnose FD, since they confirm the need to establish specific reference values based on gender. S96 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-245 P-247 AN ONGOING PHASE 2A STUDY TO INVESTIGATE THE LABORATORY DIAGNOSIS OF POMPE DISEASE IN GREECE EFFECT OF AT2220 (DUVOGLUSTAT HCL) ON THE THE IMPACT OF ASSAYING ACID A-GLUCOSIDASE IN DRIED PHARMACOKINETICS OF ACID ALPHA-GLUCOSIDASE IN BLOOD SPOTS AND WHITE BLOOD CELLS SUBJECTS WITH POMPE DISEASE: PRELIMINARY RESULTS Dimitriou E1 , Mavridou I1 , Michelakakis H1 Johnson FK1 , Kishnani P2 , Tarnopolsky M3 , Sivakumar K4 , Byrne B5 , 1Institute of Child Health, Athens, Greece Goker-Alpan O6 , Guter K7 , Pervaiz M8 , Dasouki M9 , Levine T10 , Roberts M11 , Sitaraman S1 , Winkler R1 , Khanna R1 , Flanagan J1 , Assaying of acid a-glucosidase (GAA) activity in cultured skin fibroblasts Sjoberg ER1 , Valenzano KJ1 , Lockhart DJ1 , Boudes P1 (CSF) has been considered the gold standard method for the diagnosis of 1Amicus Therapeutics, Cranbury, NJ, United States Glycogenosis II or Pompe Disease (PD). Methods allowing the accurate 2Duke University Medical Center, Durham, NC, United States assay of GAA activity in dried blood spots (DBS) and mixed leucocytes 3McMaster University Medical Center, Hamilton, ON, Canada (WBC) have been described. 4Neuromuscular Research Center, Scotsdale, AZ, United States We present the impact of these methods on the diagnosis of PD in Greece. 5University of Florida, Gainesville, FL, United States GAA was assayed in CSF and since 2008, in DBS and WBC extracts using 6LSD Treatment and Research Center, Springfield, VA, United States the 4MU-substrate, including acarbose in the latter two. 7Great Falls Clinic, LLC, Great Falls, MT, United States In the period 1997-2007, there were 76 referrals and 13 cases were diag- 8Emory University, Decatur, GA, United States nosed (30 % infantile, 70 % late onset). From 2008-2012 there were 390 9University of Kansas Medical Center, Kansas City, KS, United States referrals, fibroblast cultures were initiated in 29, and a total of 16 cases were 10Phoenix Neurological Associates, LLC, Phoenix, AZ, United States diagnosed (19 % infantile, 81 % late onset, 5 % presymptomatic). 11Salford Royal Hope HNS Trust Hope Hospit, Salford, England, United Kingdom No discrepancies were observed in the results obtained by the three methods. Pompe disease is an inherited lysosomal storage disease that results from a In conclusion, assaying of GAA in DBS and WBC reliably diagnosed PD, deficiency of acid alpha-glucosidase (GAA) activity, and is characterized by reduced the time of diagnosis, allowed the expansion?? of the investigated progressive accumulation of lysosomal glycogen primarily in heart and skeletal individuals, increased the number and changed the profile of diagnosed muscles. AT2220 is a pharmacological chaperone that selectively and reversibly cases, allowed presymptomatic diagnosis and greatly reduced the cost. binds and stabilizes GAA, facilitating proper folding and trafficking to lysosomes. The study was partly supported by a grant of Genzyme to the Institute of AT2220-010 is an open-label, single ascending dose, fixed-sequence, two-period, Child Health. dose-finding study comprised of four cohorts of 4 to 6 subjects with Pompe Conflict of Interest declared. disease. During the first period, subjects receive their regularly scheduled intravenous infusion of rhGAA 20 mg/kg alone. During the second period they receive a single oral dose of AT2220 1 hour prior to the start of infusion. Preliminary data will be presented for GAA activity, total GAA protein, and AT2220 concentrations in plasma and muscle tissue. Safety data including urine glucose-containing tetrasaccharide concentrations and anti-GAA antibody titers may also be presented. P-248 Conflict of Interest declared. THE PHARMACOLOGICAL CHAPERONE AT2220 INCREASES RECOMBINANT HUMAN ACID α-GLUCOSIDASE TISSUE UPTAKE AND IMPROVES GLYCOGEN REDUCTION IN A P-246 MOUSE MODEL OF POMPE DISEASE 1 1 1 1 1 1 BLOOD CROSS-REACTING IMMUNOLOGICAL MATERIAL Pellegrino L , Feng J , Soska R , Frascella M , Lun Y , Flanagan JJ , 1 1 1 1 1 (CRIM) TESTING IN POMPE DISEASE Guillen D , Benjamin ER , Lockhart DJ , Valenzano KJ , Khanna R 1 Bainbridge K1 , Cullen E1 , Heales S1 Amicus Therapeutics, Cranbury, United States 1Great Ormond Street Hospital, London, United Kingdom Pompe disease is a lysosomal storage disease caused by a deficiency in acid Background: Pompe disease is an inherited disorder of glycogen metabolism alpha-glucosidase (GAA) activity, and is characterized by glycogen accu- caused by a deficiency of lysosomal a-glucosidase. Enzyme replacement therapy mulation, progressive skeletal muscle weakness, reduced cardiac function, (ERT) is available but response to treatment can be variable. Patients who are and respiratory insufficiency. Biweekly infusion of recombinant human cross-reacting immunologic material (CRIM) negative tend to have a poorer GAA (rhGAA, Genzyme Corp.) is the only approved therapy for Pompe response apparently due to the development of inhibitory antibodies. Immuno- disease. While rhGAA provides some clinical benefit, tolerability and modulation prior to commencing ERT has been shown to improve outcome in efficacy may be attenuated by low stability at neutral pH and body temper- CRIM negative patients. CRIM status can be determined using cultured skin ature, modest tissue uptake and glycogen reduction, and immune responses. fibroblasts, however this involves an invasive skin biopsy and the delay culturing AT2220 (1-deoxynojirimycin-HCl; duvoglustat hydrochloride) is a small the fibroblasts means results are rarely available prior to starting ERT. molecule pharmacological chaperone that binds and stabilizes endogenous Objective: To develop and implement CRIM testing in blood to enable the GAA in cells and tissues, increasing lysosomal GAA activity. We hypoth- rapid determination of CRIM status at initial referral for suspected Pompe esized that AT2220 might interact with exogenous rhGAA to improve its disease before commencement of ERT. pharmacological properties. In human plasma, AT2220 co-incubation in- Methods: Blood samples were collected into cell preparation tubes from creased stability of rhGAA at neutral pH/37 °C. In rats, a single intravenous which mononuclear cells lysates were prepared and analysed by Western bolus injection or 60-minute infusion of rhGAA, following a single oral blotting using a-glucosidase specific monoclonal antibodies. Methodology administration of AT2220, resulted in up to a 2-fold increase in the circu- was optimised and sample stability was assessed. Blood CRIM testing on a lating half life of rhGAA. In GAA knockout mice, oral administration of number of ERT naive Pompe patients were tested. AT2220 resulted in up to 2.5-fold greater uptake and glycogen reduction in Conclusion: Blood CRIM testing is a viable alternative to fibroblast testing skeletal muscles compared to rhGAA alone. Hence, a Phase 2a study and can be used to guide appropriate treatment in addition to providing exploring AT2220 co-administration with rhGAA has been initiated. useful prognostic information. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S97

P-249 P-251 RAPID ASSAY OF A CHARACTERISTIC GLYCOGEN-DERIVED MIGLUSTAT THERAPY IN TYPE 1 GAUCHER DISEASE: TETRASACCHARIDE FOR POMPE AND OTHER LONG-TERM TREATMENT EXPERIENCE FROM A MULTICENTER, GLYCOGEN-STORAGE DISEASES BY UPLC-MS/MS RETROSPECTIVE COHORT STUDY Sluiter W1 , Van den Bosch JC1 , Goudriaan DA1 , Van Gelder CM1 ,De Hollak CEM1 , Kuter D2 , Giraldo P3 , Hughes D4 , Belmatoug N5 , Brand Vries JM1 , Huijmans JGM1 , Reuser AJJ1 , Van der Ploeg AT1 , Ruijter M6 , Muller A6 , Schaaf B7 , Giorgino R6 , Zimran A8 GJG1 1Academic Medical Centre, Amsterdam, Netherlands 1Center Lysosmal Metab Dis, Erasmus MC, Rotterdam, Netherlands 2Massachusetts General Hospital, Boston, United States 3CIBERER, Hosp Univ Miguel Servet, Zaragoza, Spain Objective: The tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) is 4Royal Free Hosp & Univ Coll Med School, London, United Kingdom elevated in urine during various clinical conditions associated with increased 5Hôpital Beaujon, Clichy, France turnover or storage of glycogen, making Glc4 a potential biomarker for 6Actelion Pharmaceuticals Ltd, Allschwil, Switzerland Glycogen Storage Diseases (GSD). We developed a simple method to detect 7Factum, Offenbach, Germany Glc4 in urine without interference of Glc4's isomer maltotetraose (M4). 8Shaare Zedek Medical Center, Jerusalem, Israel Methods: Urine samples were diluted in 0.1 % ammonia containing the internal standard acarbose and filtered. The filtrate was analysed by UPLC Background: A retrospective cohort study evaluated clinical outcomes and (BEH Amide)-ESI-MS/MS. safety-relevant information in treatment-naïve and ERT-pretreated adult Results: - Acarbose, M4 and Glc4 were quantified on the basis of their ion GD1 patients receiving commercial miglustat. pairs m/z 644/161, 665/161 and 665/179. Response of Glc4 was linear up to Methods: 115 patients were enrolled; 34 (30 %) were ERT-naïve and 81 1500 μmol/L, LOQ 2.8 μmol/L and inter-assay CV 16-18 %. Mean Glc4 (70 %) were ERT-pretreated. levels in untreated Pompe patients (GSD type II; n066; 39.4 to 10.3 mmol/ Results: Median (range) miglustat exposure was 15 (1–53) months in ERT- mol creatinine in different age groups) were significantly higher (P<0.01) naïve and 15 (0–62) months in ERT-pretreated patients. At miglustat initiation compared to controls (n0116; 8.9 mmol/mol to 1.0 mmol/mol creatinine). the median (range) haemoglobin concentration in ERT-naïve patients was 12.8 In 5 % of the urine samples M4 was present. Sensitivity for Pompe disease (10.2–16.4) g/dl, and in ERT-pretreated patients it was 13.6 (7.3–17.4) g/dl; was 98.5 %, specificity 92 %. Also in GSD-III (8/9), GSD-IV (2/3) and median (range) changes in haemoglobin were 0.3 (-2.5–3.6) and -0.3 (-4–4.6) g/ GSD-IX (6/10) patients, urine Glc4 was increased. dl, respectively. At miglustat initiation the median (range) platelet count in ERT- Conclusions: – ThepresentdirectassayofGlc4inurinewasrapid, naïve patients was 101 (37–730) x10(9)/l, and in ERT-pretreated patients it was discriminative between Glc4 and M4, and confirmed the diagnosis in 173 (43–382) x10(9)/l; median (range) changes in platelet counts in these groups 98.5 % of the 66 GSD-II cases studied. were 8 (-77–145) x10(9)/l and -10 (-144–434) x10(9)/l, respectively. Forty-nine patients (43 %) discontinued miglustat; tolerability issues, primarily gastrointestinal, were the most frequent reason [32 (28 %) patients]. Tremor was reported in 3/115 (3 %) patients before, and in 18/115 (16 %) after miglustat initiation. P-250 Conclusion: In ERT pre-treated patients haemoglobin levels and platelet RESPIRATORY COMPLICATIONS IN INFANTILE ONSET count tended to either deteriorate slightly or remain stable, while treatment- POMPE DISEASE naïve patients tended to improve. Broomfield AA1 , Finnegan N1 , Crook V1 , Wood M2 , Lozano S3 , Cleary Conflict of Interest declared. M1 , Fenton M4 , Kilner D5 , Vellodi A1 1Dep Met Dis, Great Ormond St Hosp, London, United Kingdom 2Dep Physio, Great Ormond St Hosp, London, United Kingdom 3Dep Speech+Lang, Great Ormond Street Hos, London, United Kingdom 4Cardiology Dep, Great Ormond Street Hosp, London, United Kingdom 5Resp Dep, Great Ormond Street Hosp, London, United Kingdom

Background and Methods: A retrospective review of 17 patients treated with ERT at a tertiary centre whose need for ventilation was determined by the use of serial sleep studies. Results: 6/17 patients survived without respiratory support(median length of follow up 12.5 (IQR 55) Months,4 survived with respiratory support follow up 24 (IQR 76) months and 7 died, median age at death 11 (IQR 12) months. The overall median age at initial administration of ERT was 108 (IQR 155) days with no significant difference seen between the subgroups. Initial chest x-rays showed signs of atelectasis in 11/15; of these, 7 had evidence of left lower collapse (LLLC) while 7 had other parenchymal changes. Those with LLLC had increased Left ventricular mass index (LVMI) (mean LVMI 0366 SD 134 v 145 SD 86 gm/m2, P00.04). Eight patients had reflux and six dysphagia at presentation; neither symptom correlated with outcome. Initial sleep studies were performed in 12/17 at a median age of 52 days (IQR 124) post onset of ERT. Patients with a mean transcutaneous Co2 of>45 had a relative risk of 3.75 of requiring noninvasive ventilation. Conclusion: Respiratory disease occurs early and is common in infantile Pompe disease S98 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-252 P-254 GENOTYPIC FEATURES OF 41 PATIENTS WITH GAUCHER ELIGLUSTAT, AN INVESTIGATIONAL ORAL THERAPY FOR DISEASE FROM TURKEY GAUCHER DISEASE TYPE 1 (GD1): PHASE 2 RESULTS AFTER 4 Hasanoglu A1 , Akay G1 , Ezgu F1 , Biberoglu G1 , Tumer L1 , Okur I1 , YEARS Kucukcongar A1 , Kasapkara C1 , Polat M1 , Ciftci B1 Puga AC1 , Lukina E2 , Dragosky M3 , Iastrebner M4 , Pastores G5 , Avila 1Dep Ped Metabolic Dis, Gazi Univ Med Fac, Ankara, Turkey Arreguin E6 , Zimran A7 , Rosenbaum H8 , Watman N9 , Sysoeva E2 , Aguzzi R1 , Ross LH1 , Peterschmitt MJ1 Background: Gaucher disease (GD) is a lysosomal storage disorder which 1Genzyme, a Sanofi company, Cambridge, United States includes clinical spectrum findings from a perinatal lethal disorder to an 2Hematology Research Center, Moscow, Russian Federation asymptomatic type. The diagnosis of GD is mainly made by the demon- 3IMAI-Research, Buenos Aires, Argentina stration of deficient lysosomal glucosylceramidase enzyme activity in leu- 4Inst Argentino Diagnostico y Tratamiento, Buenos Aires, Argentina kocytes. Identification of two disease-causing alleles in GBA, the only gene 5New York University, New York, United States in which mutations are known to cause GD. Although the clinical spectrum 6Inst Mex Seguro Social Hosp Especialidad, Col. La Raza, Mexico and phenotypic features of patients with GD in Turkey have been well 7Sha'are Zedek Medical Center, Jerusalem, Israel documented, there is limited data about the genotype of the patients. 8Rambam Medical Center, Haifa, Israel Methods: This study was of 41 patients with GD all of whom were confirmed 9Hospital Ramos Mejia, Buenos Aires, Argentina with the demonstration of low leukocyte glucosylceramidase activity. Ge- nomic DNA was obtained from EDTA blood samples and DNA sequence Background: Eliglustat is a potent and specific glucosylceramide synthase analysis was carried out after polymerase chain reaction. The primers were inhibitor that is under development as an oral substrate reduction therapy selected specifically to prevent the pseudogene from amplification. for GD1. Updated long-term efficacy and safety results are provided. Results: The results of the sequence analysis revealed 14 patients homo- Methods: A Phase 2 study enrolled 26 adults with GD1; 19 completed 4 years zygous for N370S, 13 homozygous for L444P, 4 homozygous for IVS2+ of eliglustat treatment. Changes in disease parameters and achievement of 1 G>A, 4 homozygous for D409H, 1 homozygous for R463H, 1 homozy- therapeutic goals (Pastores, Semin Hematol, 2004) were assessed periodically. gous for V460M, 3 compound heterozygous as N370S/L444P, 1 compound Results: Mean hemoglobin level and platelet count increased 2.3 g/dL and heterozygous as N370S/D409H. 95 %, respectively; mean spleen and liver volumes decreased 63 % and 28 %, Conclusion: This study showed that the genotype distribution of Gaucher respectively. All patients met ≥3 of 4 long-term therapeutic goals (spleen, 100 % patients from Turkey had been somewhere between Europe and Middle East. of patients; liver, 94 %; hemoglobin, 100 %; and platelets, 50 %). Median chitotriosidase and CCL-18 each decreased by 82 %; plasma GL-1 and GM3 normalized. Mean lumbar spine bone mineral density increased by 0.8 T-score, with the greatest increases occurring in patients with osteoporosis at baseline. Femur dark marrow was reduced or stable in 17/18 patients. There were no bone P-253 crises. Eliglustat was well-tolerated. Most adverse events (AEs) were mild and ASSESSMENT OF CCL18 FOR THE DIAGNOSIS AND MONITORING unrelated to treatment. Ten drug-related AEs, all mild, occurred in 8 patients. OF GAUCHER DISEASE Conclusions: Eliglustat continues to show promising efficacy and safety, Pandey Sanjeev Kumar1 , Kapoor Seema1 ,MishraTarunKumar1 , Dubey A P1 with clinically meaningful improvements across several disease parameters. 1Maulana Azad Medical College, New Delhi, India Three Phase 3 trials completed enrollment. Conflict of Interest declared. Background: Gaucher disease is characterized by storage of glucosylceramide in lysosomes of tissue macrophages as the result of an autosomal recessively inherited deficiency in glucocerebrosidase. Glucosylceramide- laden tissue macrophages in Gaucher patients secrete large quantities of P-255 chemokine CCL18 resulting in markedly elevated plasma levels. TISSUE SPECIFIC DIFFERENCES IN B-GLUCOSIDASE Method: We have comparatively investigated the occurrence of biomarker ACTIVITY- IMPLICATIONS FOR DIAGNOSING GAUCHER CCL18 in plasma as well as in urine samples of 52 controls (4-12 years) and DISEASE 1 1 1 1 16 Gaucher patients (Age 3-10 years). Hutchin T , Chakrapani A , Vijay S , Preece MA 1 Result: The median level of CCL18 in control plasma was 87.5 ng/mL, Birmingham Children's Hospital, Birmingham, United Kingdom (range25-200 ng/mL), and Gaucher plasma was 824 ng/mL (range 510- 1688 ng/mL). The median value of the CCL18/creatinine ratio in urine Gaucher disease, due to a deficiency of b-glucosidase, typically presents samples of normal subjects was 6.6 pg/μmol (range 2.9–8.8) and in those of with hepatosplenomegaly with or without neurological involvement. Here Gaucher patients was 118.2 pg/μmol (range 26–432). we describe two siblings with a classical clinical presentation of neuro- Conclusion: We observed that urinary levels of CCL18 were highly ele- nopathic type II Gaucher disease but confusing enzyme results. vated in sample from Gaucher patients as compared to controls; the result In the first child, presenting at 6 months, plasma chitotriosidase activity was showed that the low-molecular mass chemokine CCL18 in urine and grossly elevated consistent with Gaucher disease. However, b-glucosidase plasma of Gaucher patients and in controls correlated well. It is concluded activity in leucocytes was normal on two occasions. Subsequent analysis in that assessment of urinary CCL18 level of Gaucher patients can also give fibroblasts showed a significant deficiency of b-glucosidase and the diagnosis insight in the diagnosis and monitoring of Gaucher Disease. of Gaucher disease was further confirmed by DNA analysis which revealed the presence of heterozygote mutations c.754 T>A and RecNcil in the GBA gene. A further sibling, with a similar clinical presentation, also had grossly elevated plasma chitotriosidase but unlike her brother, b-glucosidase was grossly deficient in leucocytes. She carried the same two mutations as her brother. The reason for the disparity in enzyme activity in leucocytes and fibroblasts in this case is not clear but it highlights the need to test alternative tissues where clinical indications of a diagnosis are sufficiently strong. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S99

P-256 P-258 VALIDATION OF FLUOROMETRIC SPECIFIC ACTIVITY ON THE SUBCLINICAL NEUROLOGICAL FINDINGS IN TYPE 1 DBS FOR GAUCHER DISEASE DIAGNOSIS GAUCHER DISEASE Goldim MP1 , Garcia CS1 , Coelho JC1 Kucukcongar A1 ,AydınK2 , Ezgü FS1 , Kasapkara ÇS1 , Tümer L1 , 1Biochem Dept, Univ Fed Rio Grande do Sul, Porto Alegre, Brazil Hasanoğlu A1 1Div Metab Dis, Univ Gazi, Ankara, Turkey Gaucher disease (GD) is the most frequent LSD. It's caused by the defi- 2Div Pediatr Neurology, Univ Gazi, Ankara, Turkey ciency of acid beta-glucosidase (GBA), generating glucosylceramide accumulation in the lysosomes. Chitotriosidase (CT) is also related to GD, and The absence of neurological symptoms and signs is traditionally considered activity may be increased up to 1000 times.? Currently the diagnosis is mandatory for a diagnosis of type 1 Gaucher disease (GD1), but in recent based on the specific activity of enzymes in leukocytes or fibroblasts. Dried years many reports have emerged on neurological manifestations in GD1 blood spots (DBS) have been used only as screening, because there is no patients. validated specific activity. This study aimed to validate fluorometric assays Objectives: The current study replicates the eight turkish GD1 patients with of GBA and CT on DBS eluted with sodium phosphate buffer (pH7.0). The at least one N370S mutation which has been assumed to be protective of sample volume was corrected by total protein quantification, establishing neurological involvement. No neurological symptoms have been described the specific activity. New reference values and cut-off points were estab- in these patients. lished using ROC-curve (100 % specificity and sensitivity). The mean Methods: In all patients were performed electroencephalography (EEG), activity of healthy controls (HC) was 3.43 ±2.34 nmol/h/mg protein and brain magnetic resonance (MR), electromyography (EMG). for patients was 0.10±0.17 nmol/h/mg protein, for GBA. For CT, the mean Result: The EEG of an adult GD1 patient showed mild to moderate focal activity was 7.81±7.07 nmol/h/mg protein (HC) and 283.66±176.58 nmol/ slowing and three adult GD1 patients had diffuse slowing on the back- h/mg protein (patients). HC were significantly different from GD patients ground activity of the EEGs. The brain MR images of three subjects were for both enzymes (p<0.05). The specific activity on DBS has been proven suitable with deep white matter abnormalities. EMG revealed were deter- valid and the coefficients of variation were acceptable (<20 %). Thus the mined myophaty pattern in a child patient and peripheral neuropathy pattern specific activity on DBS can be used as a reliable screening method and as in an adult patient in whom the most common reasons of myopathies and diagnosis of GD. neuropathies had been excluded. Conclusion: We conclude that the brain images and neurologic electrophysiological studies should be a part of routine follow-up guidelines in GD1 patients, and these patients should be carefully followed up especially focusing on the neurologic symptoms. P-257 PLASMALOGEN LEVELS AND GAUCHER DISEASE: FURTHER STUDIES 1 1 2 1 MORAITOU M , DIMITRIOU E , DEKKER N , MONOPOLIS I , P-259 AERTS H2 , MICHELAKAKIS H1 FOLLOW-UP OF GAUCHER PATIENTS: EGE UNIVERSITY 1INSTITUTE OF CHILD HEALTH, ATHENS, Greece EXPERIENCE 2AMC,Univ Amsterdam, Amsterdam, Netherlands E Canda1 , M Kagnici1 , M Kose1 , SK Ucar1 , M Coker1 1Pediatric Metabolism, Ege University, Izmir, Turkey In Gaucher disease (GD) reduced red blood cell (RBC) plasmalogen levels, that showed a significant rise following treatment, have been reported. Aim: Gaucher's disease (GD) is a multisystemic disorder characterized by We further investigated plasmalogen levels in relation to glucosylceramide glucoserebrosidase enzyme deficiency. The aim of this study was to present (GlcCer), ceramide (Cer), glucosylsphingosine (GlcSph) and malonyldial- our series of 22 patients (19 Type 1, 1 type 2 and 2 type 3) with clinical dehyde (MDA) levels in 27 GD patients and 13 controls. follow-up. Plasmalogens were measured as their dimethylacetal derivatives (DMA) by Methods: Demographical features, clinical findings, genetical analyses, GC in lipid extracts of RBC membranes and expressed as the ratios laboratory workup, bone mineral densitometry (DEXA), liver and spleen C16:0DMA/C16:0 and C18:0DMA/C18:0. MDA using the TBA? method, volumes, treatments of patients were analyzed. GlcCer and Cer using HPLC, were also quantified in RBC membrane Results: Mean age was 21.3±14.9 years (5 months - 55 years), mean age at extracts. Plasma GlcSph was quantified by LC-ESI-MS/MS. The results diagnosis was 15.2±15.1 years (1 month - 55 years). Most common were analysed by non parametric tests. presenting symptom was abdominal distension. Two patients had severe GlcSph, GlcCer/Cer and MDA were significantly higher in GD compared to anemia and 5 patients splenectomised at diagnosis period. Most com- controls. In both GD patients and controls a negative correlation between mon mutation was heterozygous N370S. DEXA revealed osteoporosis both plasmalogens and GlcCer, Cer, GlcCer/Cer and GlcSph was observed, in 3 patients, osteopenia in 8 patients. 18 patients had enzyme re- which was statistically significant only for C16:0DMA/C16:0 in GD placement treatment (ERT) and 4 patients had miglustat treatment. patients. A negative, not statistically significant correlation, was observed When we compared the pre and post treatment DEXA's of 7 patients between MDA and both plasmalogen species in GD patients and only who had ERT longer than 1 year, we observed improvement in lumbal C16:0DMA/C16:0 in controls. and femur Z scores (p>0.05). Pre and post treatment liver and spleen Our results show a link between the reduced plasmalogen levels observed in volumes decreased after ERT (p>0.05). GD with the lipid abnormalities characterizing the disorder, as well as an Conclusion: In our series, we observed favorable bone densitometry and association with the increased oxidative stress observed in GD patients. volumetric changes after ERT. S100 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-260 P-262 LONG-TERM EFFECTS OF ENZYMATIC REPLACEMENT PLANT CELL EXPRESSED RECOMBINANT THERAPY IN GAUCHER TYPE 3 DISEASE. GLUCOCEREBROSIDASE - TALIGLUCERASE ALFA AS THERAPY Bertin N1 , Deroma L1 , Ciana G1 , Sechi A1 , Dardis A1 , Deganuto M1 , FOR GAUCHER DISEASE IN PATIENTS PREVIOUSLY TREATED Malini E1 , Bembi B1 WITH IMIGLUCERASE 1University Hospital of Udine, Udine, Italy Pastores GM1 , Szer J2 , Petakov M3 , Cox T4 , Giraldo P5 , Rosenbaum H6 , Amato D7 , Mengel E8 , Chertkoff R9 , Almon-Brill E9 , Zimran A10 Background: The effectiveness of enzyme replacement therapy (ERT) in 1NYU School of Medicine, Neurogenetics, New York, United States the chronic neuronopathic form of Gaucher disease (GD3) is still 2Royal Melbourne Hospital, Victoria, Australia controversial. 3Clinical Center of Serbia, Belgrade, Serbia and Montenegro Methods: In our centre, we have followed nine paediatric GD3 patients up 4Addenbrooke's Hospital, Cambridge, United Kingdom to 15 years. They received bi-weekly alglucerase/imiglucerase infusions 5Hospital Universitario Miguel Servet, Zaragoza, Spain (30-60 IU/kg). To evaluate the long-term effects of ERT we analyzed 6Rambam Medical Center, Haifa, Israel clinical conditions, laboratory values, radiological and electrophysiological 7Mount Sinai Hospital, Toronto, Canada features at baseline, and every 6 to 12 months thereafter. 8Gutenberg-University Mainz, MAINZ, Germany Results: During the follow-up, haematologic parameters and organomegaly 9Protalix Biotherapeutics, Carmiel, Israel improved in all patients, bone pain was observed in 5 of them (2 were 10Shaare Zedek Medical Center, Jerusalem, Israel asymptomatic at baseline), while in most patients bone density was normal. Considering neurological involvement, 3 patients showed neurological Background: Taliglucerase alfa is an United States FDA approved enzyme stabilization on ERT, and one of them never reported electrophysiological replacement therapy for Gaucher disease (GD). abnormalities. Three patients developed progressive myoclonic encepha- Objective: To assess the safety and efficacy of taliglucerase alfa in GD lopathy and died (all of them presented F213I/L444P genotype). Intelli- patients previously receiving imiglucerase. gence quotient decreased in one patient while it remained stable in the other Methods: Eligible patients were switched from imiglucerase to taliglucer- 8. One patient developed symptoms of Asperger syndrome (P159T/ ase alfa at the same dose and received a total of 20 infusions, given every g.4641_J03060.1:g.2828 genotype). 2 weeks. Each patient's previous historical clinical and laboratory stability Conclusions: Long-term ERT demonstrated to improve systemic manifesta- measurements served as controls. tions in all our GD3 patients, showing to control neurological deterioration Results: Twenty-six adult patients were recruited; 25 completed the 9-month only in a minority of them, all presenting with L444P homozygosity. Presence treatment period. Spleen and liver volumes as well as platelet, hemoglobin, of myoclonic epilepsy demonstrated to be an unfavourable prognostic factor. and chitotriosidase measurements remained stable or improved following Conflict of Interest declared. switchover to taliglucerase alfa. All treatment-related adverse events were mild or moderate in severity and transient in nature; the most commonly occurring were headache (n02) and infusion-related reaction (n02). Conclusions: Mean efficacy parameters were maintained and taliglucerase alfa P-261 was well-tolerated in patients switched from imiglucerase to taliglucerase alfa. IS IT POSSIBLE TO EVALUATE DOPAMINERGIC ACTIVITY IN Conflict of Interest declared. GAUCHER DISEASE BY MEASURING PROLACTIN CONCENTRATION? M Kose1 , M Kagnici1 , E Canda1 , SK Ucar1 , M Coker1 1Pediatric Metabolism Ege University, Izmir, Turkey

Background: Since discovery of association between glucocerebrosidase mutations and diseases characterized clinically Parkinsonism; many studies have emphasised this relationship. Aim of our study was evaluate dopaminergic activity by performing prolactin assays in individuals carrying glycocerebrosidase mutation. Methods: Fifteen patients; suffering from GD ; all of whom have being followed by our department were included and prolactin levels was analysed. Results: The mean age of patients was 20.4 ±16.5 . 11 of cases have been treated with enzyme replacement therapy (ERT) while 4 ones have been received Miglustat. Prolactin concentration range was obtained 5.6 - 26.8 pg/ml (mean 15.5±7.44 ). Although not statistically significant; it has been demonstrated that prolactin analyses of two patients which had been performed before enzyme replacement therapy ; was found elevated (13.6 and 25.8 pg/ml). Prolactin level of a patient who has tremor was also obtained high. Conclusions: Increased prolactin concentration of two patients whose ERT was not initated and one patient who has tremor supports theory of neurotoxicity via depletion of glucocerebrosidase enzyme resulting dopaminergic alteration. It will be required further studies consisting larger series with additive parameters to identify link between dopaminergic activity and glucocerebrosidase gene mutation by measuring prolactin concentration. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S101

P-263 P-265 HLA AND KIR GENES IN GAUCHER DISEASE NEUROIMAGING FINDINGS IN LATE INFANTILE GM1 Filippo Vairo FV1 , Portela PP2 , Patricia SPH2 , Netto CBN3 , Mittelstadt GANGLIOSIDOSIS SM4 , Jobim LFJ2 , Schwartz IVDS3 Eminoglu FT1 , Aksoy A2 , Tos T3 , Ozkan M2 1Genetics Department, UFRGS, Porto Alegre, Brazil 1Div Metab Dis,Dr. Sami Ulus Child Hosp, Ankara, Turkey 2Immunology Service, HCPA, Porto Alegre, Brazil 2Div Neurol, Dr. Sami Ulus Child Hosp, Ankara, Turkey 3Medical Genetics Service, HCPA, Porto Alegre, Brazil 3Div Genetics, Dr. Sami Ulus Child Hosp, Ankara, Turkey 4BRAIN Exp Research Group, HCPA, Porto Alegre, Brazil Late infantile (Type II) GM1 gangliosidosis is a metabolic disorder with the According to the literature, the activity of the Natural Killer (NK) cells is clinical features of seizure, progressive motor and mental retardation with- impaired in Gaucher disease (GD). Killer immunoglobulin-like receptors out facial dysmorphism, visceral organomegaly. We present the neuroimag- (KIR) regulate the activity of NK cells through an interaction with specific ing findings in a patient with type II GM1 gangliosidosis. human leukocyte antigen (HLA) class I molecules. Case Report: A 3-years-old girl presented with developmental regression Objectives: To analyze the variability of HLA/KIR genes in a Southern and generalised seizure. Physical examination showed a hypotonic infant Brazilian sample of GD patients. and psychomotor retardation. Hyperactive reflexes and augmented startle Methodology: Thirty one GD patients were analyzed and compared to 250 response to noise were present. The diagnosis was based on a deficiency of healthy controls regarding the frequency of HLA and KIR genes. b-galactosidase activity .T2-weighted MR image revealed hypodensity of Results/Discussion: There was no significative difference in the frequencies the thalamus and normal signal intensity of the basal ganglia. The cerebrum of KIR gene variants between the groups. The HLA B37 allele is more showed almost no myelination of the white matter except the splenium of frequent in patients with GD (p00.011). The age of onset of symptoms was the corpus callosum. associated with KIR2DL2 and KIR2DS2 combination with the ligand HLA- Discussion: Demyelination has been reported in GM1 gangliosidosis. Al- C1 (p00.038). Patients who have the HLA-C2 variant appear to have more though it remains controversial, the cause of the T2W hyperintensity in the mono/polyclonal bands (p00.007, OR021.3). An association between the cerebral white matter has been attributed to primary hypoplasia of the DR11 (p00.008) and DR13 (p00.011) alleles and disease severity was found. myelin (hypomyelination) and/or delayed myelination (dysmyelination) The DR11 allele appears to be associated with neurological impairment, while rather than demyelination resulting from neuronal death due to the accu- the DR13 allele to the development of osteonecrosis. mulation of storage materials. Our findings suggested that the metabolic Conclusion: Although exploratory, our data suggest a possible association defect has a primary influence on myelin development, in addition to effects between KIR genes and HLA genes and GD. These genes may be a related to neuronal storage. phenotype modifying factors of GD. Conclusion: GM1 type II should be in the differential diagnosis of hypomyelinating disorders.

P-264 P-266 TRANSIENT NEONATAL HYPERPARATHYROIDISM: A LATE-ONSET TYPE OF KRABBE DISEASE IS DOMINANT IN PRESENTING FEATURE OF SIALIDOSIS TYPE II JAPAN AND THE LATE-ONSET MUTANT PRECURSOR Eminoglu FT1 , Igdoura S2 , Dursun A3 , Okumus N3 , Zencirlioglu A3 SHOWED EFFECTIVE PROCESSING 1Div Metab Dis,Dr. Sami Ulus Child Hosp, Ankara, Turkey Hossain Mohammad Arif1 , Otomo Takanobu1 , Hamada Yusuke1 , Ozono 2Depts. mol med, McMaster Univ,, Ontario, Canada Keiichi1 , Sakai Norio1 3Div Neonatol, Dr. Sami Ulus Child Hosp, Ankara, Turkey 1Osaka University Graduate School of Medi, Suita, Osaka, Japan

Sialidosis is a lysosomal storage disease caused by the deficiency of alpha- Background: Krabbe disease is an autosomal recessive leukodystrophy N-acetyl neuraminidase-1 (NEU1). Sialidosis is classified into two main characterized by demyelination of the central and peripheral nervous sys- clinical variants: type I, the milder form of the disease, and type II, which tems, caused by the deficiency of galactocerebrosidase. Typical infantile can in turn be subdivided into three forms: congenital, infantile and form is known to share more than 90 % of all Krabbe disease patients; juvenile. however we detected more patients as late-onset. We analyzed the molec- Case report: We report herein a girl patient with Type II sialidosis exhibit- ular pathology for late-onset mutations. ing the congenital forms of the disease with thrombocytopenia, pulmonary Methods: 48 Japanese patients were analyzed for the mutations and compared interstitial thickening and transient neonatal hyperparathyroidism. This with clinical phenotypes. Transient expression for the common mutations was infant showed radiological features of osteopenia and 'rickets-like' changes. performedinCOS1cells.Enzymeactivitiesweremeasuredwiththreesub- The activities of serum alkaline phosphatase and parathyroid hormone level strates. Western blotting was performed with transfected COS1 cells. were markedly elevated. Serum phosphorus was decreased. Serum levels of Results: 88 alleles were detected within 48 Japanese patients and found that 25-hydroxyvitamin D and calcium were normal. Secondary hyperparathy- 57 % of all patients are late-onset. 62 % of all patients can be diagnosed by roidism resolved biochemically and radiologically by age 11 months. the seven mutations. We found, late onset mutations showed 5-20 % of Discussion: As far as we know, this case is the first patient with type 2 normal activity. With western blot, we detected the 80 kDa band as precur- sialidosis exhibiting transient neonatal hyperparathyroidism. We suggest sor GALC protein for all the missense mutations; however expression level that the hyperparathyroidism is secondary to impaired transplacental calci- of 30 kDa band as the processed protein for late-onset is higher than the um transport. infantile mutations. Conclusion: Newborns with sialidosis II can present with radiological and Conclusion: Japanese Krabbe disease has different clinical distribution biochemical signs of hyperparathyroidism. Awareness of this phenomenon from Caucasian people. Classification of the mutations according to enzy- may help in avoiding diagnostic pitfalls and establishing a proper diagnosis matic activities and processing profile may bring important information on and therapy. the prognosis for the patients. S102 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-267 P-269 METACHROMATIC LEUKODYSTROPHY IN ASSOCIATION HIGH PREVALENCE AND NOVEL MUTATIONS OF LYSOSOMAL WITH HIRSCHSPRUNG DISEASE, A CASE REPORT STORAGE DISORDERS IN UNITED ARAB EMIRATES Kagnici M1 , Karaca E2 , Canda E1 , Kose M1 , Kalkan Ucar S1 , Serdaroglu Tawfig N1 , Berniah a2 ,Alib3 ,Talebm1 , Hertecant j2 ,BastakiF1 ,Souida3 G3 , Ozkınay F2 , Gokben S3 , Coker M1 1Latifa Hospital, dubai, United Arab Emirates 1Div Metab Dis, Dep Ped, Univ Ege, Izmir, Turkey 2Tawam hospital, Al Ain, United Arab Emirates 2Div Genetics, Univ Ege, Izmir, Turkey 3United Arab Emirates University, Al Ain, United Arab Emirates 3Div Neurol, Dep Ped, Univ Ege, Izmir, Turkey Lysosomal storage disorders (LSD) are rare entities of recessive inheritance. Background: Metachromatic leukodystrophy (MLD) is an autosomal re- Their prevalence is expected to be higher with consanguineous marriages, cessive inherited lysosomal storage disorder caused by a deficiency in the such as in the Middle East North Africa (MENA) region. The primary aim lysosomal enzyme arylsulfatase A (ASA), which results in sulfatide accu- of this study was to estimate prevalence and report mutation spectrum in mulation in various tissues. The disease is characterized by degeneration of UAE. Between 1995 and 2010, 119 patients were diagnosed with LSD (65 myelin in both the central and peripheral nervous systems, and as a result Emiratis and 54 non-Emiratis). Genotyping was performed in 59 (50 %) progressive motor and mental deterioration develops. patients (39 Emiratis from 17 families and 20 non-Emiratis from 17 fam- Hirschsprung's disease (HD) is a congenital condition characterized by the ilies). LSD prevalence among Emiratis was 26.9/100,000 live births. Sphin- absence of parasympathetic ganglion cells in the myenteric and submucosal golipidoses were common (9.8/100,000), with GM1-gangliosidosis being plexuses of the distal bowel leading to functional intestinal obstruction. It can be the most prevalent (4.7/100,000). Compared to Western countries, the associated with various diseases, including Down syndrome, Neurocristopathy prevalence of fucosidosis, Batten disease and α-mannosidosis were 40-, syndromes, Congenital central hypoventilation syndrome and trisomy 18. 7- and 4-fold higher in UAE, respectively. The prevalence of Pompe disease Case Report: Our patient, a four year old boy who was born from second (2.7/100,000) was similar to The Netherlands, but only the infantile subtype degree relative parents, was diagnosed to have HD, and had an intestinal was detected. Sixteen distinct mutations were identified in 39 Emirati surgery at 40 days old. His growth parameters were normal until 1 years old patients. Eight (50 %) mutations were reported only in Emiratis, of which after which loss of acquired developmental milestones followed. After 3 were novel [c.1694 G>T in the NAGLU gene, c.1336 C>T in the GLB1 enzymatic analysis, (ASA: 1.49 nmol/h/mg protein, N: 12 and 15.9) and gene, and homozygous deletions in the CLN3 gene]. Twenty-seven (42 %) molecular genetic testing (homozygous mutation, C.459+1 G>A) he also patients were clustered in 5 of 70 Emirati tribes. These findings highlight received late infantile MLD diagnosis. the need for tribal-based premarital testing and genetic counseling. Conclusion: As far as we know this is the first case where HD is accompanied by MLD in the literature.

P-268 P-270 I-CELL DISEASE: THE EXPERIENCE OF THREE U.K. CENTRES BURDEN OF LYSOSOMAL STORAGE DISORDERS IN INDIA: Jameson E1 , Wilson B1 , Santra S2 , Broomfield A3 , Jones S1 STUDY OF 946 CHILDREN 1Biochem Genetic Unit, St Mary's Hospital, Manchester, United Kingdom , Sheth J1 ,MistryM1 , Sheth F1 ,OzaN1 , R Shah2 , Kamte M3 , Godbole K4 , 2Birmingham Children's Hospital, Birmingham, United Kingdom Bavdekar A5 , Datar C6 3Great Ormond Street Children's Hospital, London, United Kingdom 1FRIGE's Institute of Human Genetics, Ahmedabad, India 2Ankur children Hospital, Ahmedabad, India Introduction: Mucolipidosis type II, commonly known as I-cell disease, 3KLIS Hospital, Belgam, India has a combined incidence with ML III of 1 in 325,000. It is caused by a 4Deenanath Mangeshkar Hospital, Pune, India deficiency in the Golgi apparatus enzyme GlcNAC phosphotransferase. It 5KEM Hospital, Pune, India , 6Sahyadri Hospital, Pune, India classically presents in the first six months of life with any of hepatosplenomegaly, dysmorphism or failure to thrive. It is a rapidly progressive Present study comprises of 946 children with 307 males and 639 females in disease with no available curative treatment, typically proving fatal in the the age range from 1 day to 16 yrs referred from various parts of India. The first decade of life. most prominent clinical phenotypes observed were coarse facial features, Objective: To provide a comprehensive overview of I-cell disease in the developmental delay, respiratory distress, hepatosplenomegaly, skeletal UK population. dysplasia, neuroregression, leucodystrophy, cerebral-cerebellar atrophy, vi- Results: The data is sub-divided into four categories: (i) Demographics(ii) sual impairment or cherry red spot, seizures and mongolian spots. Presentation (iii) Diagnosis including mutation analysis (iv) Outcome in- The three tier screening approach developed at FRIGE was used for the cluding co-morbidities such as the need for non-invasive ventilation. initial diagnosis. That included screening of urine for GAGs, plasma for I- Discussion: This review provides a comprehensive overview of I-cell cell and chitotriosidase followed by confirmatory enzymes study and rest disease in the UK population. It highlights the common mutations, the with specific enzymes depending on the presenting signs and symptoms multi-system nature of the disease and the resultant co-morbidities. The and clinical suspicion. latter provides support for the early use of a multi-disciplinary team ap- Of 946 cases investigated, 341 (36.5 %) subjects were found to be affected proach in the care of these patients. with LSDs. The most common LSD observed was glycolipids storage disorder detected in 16 % followed by mucopolysaccharide disorders (MPS) in 9 %, sulphatide degradation defect in 5.5 %, glycogen storage disorder type II in 2 %, protein degradation defect and lysosomal transporter deficiency in 2 % each, lysosomal trafficking protein defect in 1 %. Present study thus demonstrates higher incidences of glycolipids and mucopolysaccharide storage disorders in Indian children. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S103

P-271 P-273 LYSOSOMAL STORAGE DISEASES IN OUR COUNTRY : NEW METHODS FOR THE MEASUREMENT OF PALMITOYL RESULTS OF LAST SIX YEARS PROTEIN THIOESTERASE AND TRIPEPTIDYL PEPTIDASE IN Hasanoglu Alev1 , Biberoglu Gursel1 , Ezgu Fatih Suheyl1 , Tumer Leyla1 , DRIED BLOOD SPOTS Kasapkara Çigdem Seher1 , Kuçukçongar Aynur1 , Udgu Basak1 McNeilly JD1 , Hamilton J1 , Galloway PJ1 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 1Royal Hospital for Sick Children, Glasgow, United Kingdom

Lysosomal storage disorders (LSDs) are a heterogenous group of more than Background: Neuronal ceroidlipofuscinoses (NCL) constitute a group of at 40 genetically different diseases. There are different methods for diagnosis least 11 fatal hereditary neurodegenerative diseases characterised by of LSD's but enzymatic confirmation is necessary in the appropriate bio- lipofuscin-like inclusions in various tissues. Infantile (CLN1) and late infantile logical materials like leukocyte, fibroblast. (CLN2) forms are caused by deficiencies in the lysosomal enzymes, palmitoyl Material-Methods: We analysed enzyme activity in leukocytes samples for protein thioesterase (PPT1) and tripeptidyl peptidase (TPP1) respectively. six years. Enzyme activities were determined by spectrofluorometric and Objective: To develop methods for measuring PPT1 and TPP1 in dried spectrophotometric method. Enzyme activities were determined in 2746 blood spots (DBS) and validate these techniques using samples from known suspicious patients. patients with CLN1 and CLN2. Results: During this period, 543 LSD's were diagnosed in 2746 suspicious Methods: PPT1 was extracted from DBS using 5 % triton X-100 and patients. 170 mucopolysaccharidoses, 137 Gaucher disease, 109 Niemann- incubated with 4-MU-6-thiopalmitoyl- β-glucoside plus exogenous β- Pick A-B, 48 Fabry disease, 3 ?-Fucosidosis, 17 GM I , 20 Metachromatic glucosidase. TPP1 was extracted with sodium-acetate+5 % triton X-100 leukodystrophy, 7 Sandhoff disease, 19 Mucolipidosis, 3 Wolman disease, buffer and incubated with 7-amino-4-methylcourmarin (4-AMC) and pro- 2 alpha-?-Mannosidosis, 2 Pompe disease and 6Tay Sachs disease. tease inhibitors pepstatin A and E64. Both assays were performed on 96 Conclusion: In recent years progress has been made? : enzyme replace- well plates, incubated for 20 hr and read at 370/460 nm. ment, substrate reduction and transplantation of bone marrow for the Results: PPT1 and TPP1 activities in normal controls were 17.4-60.6 and 22.2- threatment of LSD's. Early diagnosis is very important for treatment of 60.5 pmol/punch/hr respectively. Patients with known CLN1 and CLN2 had LSD's. In our country the incidence of LSD's is considerably high significantly reduced PPT1 and TPP1 activity <2.4 (n04)and<1.3(n03) because of consanguineous marriages are very often. Most common LSD's respectively. for our country are mucopolysaccharidoses, Gaucher, Niemann-Pick A-B, and Conclusion: Current methods for investigating CLN1 and CLN2 require Fabry. isolation of leucocytes. Our simple methods facilitate reliable determination of PPT1 and TPP1 in DBS which clearly differentiates affected cases from normal controls. P-272 EFFECT OF READTHROUGH TREATMENT ON FIBROBLASTS OF PATIENTS AFFECTED WITH LYSOSOMAL DISEASES P-274 CAUSED BY PREMATURE TERMINATION CODONS GM2 GANGLIOSIDOSIS: LATE-ONSET FORM OF β- Matalonga L1 , Gort L1 , Ribes A1 1 HEXOSAMINIDASE A DEFICIENCY IN THE CZECH REPUBLIC Div IEM, Bio Mol Gen, H Clínic, CIBERER, Barcelona, Spain Poupětová H1 , Jahnová H1 , Honzíková J2 , Fialová M1 ,Příhodová I3 , Kaliová J3 , Zumrová A4 , Ledvinová J1 Background: Nonsense-mediated mRNA decay (NMD) is a quality- 1Inst of Inherit Metab Dis, Charles Univ, Prague, Czech Republic control mechanism that selectively degrades mRNAs harbouring premature 2Inst of Inherited Metabolic Disorders, Prague, Czech Republic termination codons (PTC). It has been described that aminoglycoside anti- 3Dept of Neurol, General Univ Hospital, Prague, Czech Republic biotics and other products such as 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3- 4Dept of Child Neurol, Univ Hosp Motol, Prague, Czech Republic yl]benzoic acid may induce PTC readthrough and elude NMD mechanism. Because PTC are frequently involved in lysosomal diseases, these products Background: GM2 gangliosidoses are autosomal recessive lysosomal storage would be useful as potential therapeutic approaches. disorders characterized by GM2 ganglioside accumulation within neurons. Tay- Objective: To find patient's fibroblasts that may promote PTC readthrough Sachs disease (TSD) is caused by mutations in the gene HEXA coding α-subunit under gentamicin and 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic of lysosomal enzyme β-hexosaminidase A. Late-onset chronic form first occurs in acid treatment, in order to be used as positive controls in the future adolescence and adulthood, and takes a slower course with progressive proximal screening of other compounds. limb weakness and lower motor neuron signs in combination with cerebellar and Patients and Methods: Fibroblasts from ten patients affected with five occasionally also extrapyramidal signs. Activity of β-hexosaminidase A is re- different lysosomal diseases carrying PTC were treated with different concen- duced to below 10 % of the normal values. Late-onset TSD may mimic spinal trations of gentamicin and 3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic muscular atrophy or diseases arising from spinocerebellar degeneration. acid. The enzymatic activities of each enzyme were analyzed by fluorimetry. Results: We present findings in six patients with late-onset form of TSD Results: Four out of ten patients showed 1.2 to 3 fold increase in the identified since 1994. One patient was diagnosed post mortem by HPTLC enzymatic activity after gentamicin or 3-[5-(2-fluorophenyl)-1,2,4-oxadia- chromatography of the brain gangliosides. In the other cases deficiency of zol-3-yl]benzoic acid treatment. Treatments in the other six patients did not β-hexosaminidase A was found in leukocytes, plasma and serum. raise the activity or even lowered it, although cell viability was not altered. Conclusions: In the Czech population calculated birth prevalence of late- Conclusion: As not all PTCs are responsive to the mentioned treatment, we onset TSD is 0.35 per 100 000 live births. Late-onset TSD disease seems to conclude that patients fibroblasts may be helpful to elucidate the respon- be underestimated due to patients misplacement in other groups of neuro- siveness before starting the screening of other candidate products. logic disorders. Fast and simple screening for TSD is available by determination of β-hexosaminidase A activity in serum or plasma. Acknowledgement: This work was supported by the grant project of RVO- VFN64165/2012. S104 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-275 P-276 DIAGNOSING LSDS: A SYSTEMATIC REVIEW OF SECONDARY SEQUENCE AND COPY NUMBER ANALYSES OF HEXB GENE SCREENING AND AWARENESS RAISING PROJECTS IN PATIENTS AFFECTED BY SANDHOFF DISEASE: FUNCTIONAL Stroicz P1 , Sperl W2 , Lagler FB3 CHARACTERIZATION OF 9 NOVEL SEQUENCE VARIANTS 1Inst f IEM, Paracelsus Med University, Salzburg, Austria Zampieri S1 , Cattarossi S1 , Oller Ramirez AM2 , Rosano C3 , Lourenco 2Dept Pediatrics, Paracelsus Med Uni, Salzburg, Austria CM4 , Passon N5 , Moroni I6 , Uziel G6 , Pettinari A7 , Stanzial F8 , 3IIEM&Dept Pediatrics, Paracelsus Med Uni, Salzburg, Austria Dodelson de Kremer R2 , Azar NB2 , Filocamo M9 , Bembi B1 , Dardis A1 1Univ. Hosp. S. Maria della Misericordia, Udine, Italy Background: Lysosomal storage diseases (LSD) are chronic, progressive, 2University of Córdoba, Cordoba, Argentina debilitating and life-threatening conditions. For seven LSDs orphan drugs 3IRCSS San Martino, Genova, Italy are available and further products are under development. The diagnosis of 4University of Sao Paulo, Sao Paulo, Brazil LSDs however can be failed or delayed substantially. Initiatives to foster 5Università di Udine, Udine, Italy early diagnosis mainly comprise of awareness raising projects and high-risk 6Fondazione Istituto Neurologico Besta, Milan, Italy population screenings. The validity of these approaches has not been 7Ospedali Riuniti Ancona, Ancona, Italy analysed systematically to our knowledge. Here we present a systematic 8Azienda Sanitaria dell'Alto-Adige, Bolzano, Italy literature review of awareness and secondary screening projects. 9IRCCS G. Gaslini, Genova, Italy Methods: Systematic literature review. Results: Awareness was assessed for Fabry disease (in rheumatologists, cardi- Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the ologists, and nephrologists), Gaucher disease (orthopedics, rheumatologists, and HEXB gene. To date, 43 mutations of HEXB have been described, includ- pediatricians) and Mucopolysaccharidoses (in pediatricians, and rheumatolo- ing 3 large deletions. Here, we have characterized 14 unrelated SD patients gists). These studies identified factors that influence the probability of consider- and developed a Multiplex Ligation-dependent Probe Amplification ing LSD. But the efficacy of awareness raising interventions was not evaluated. (MLPA) assay to investigate the presence of large HEXB deletions. We Secondary screening programs have been performed for Fabry disease (hemo- identified 16 alleles, 9 of which were novel, including 4 sequence variation dialysis, kidney transplanted, and hypertrophic cardiomyopathy patients). The leading to aminoacid changes [c.626 C>T (p.T209I), c.634 C>A prevalence was 64 to 1170 fold elevated in these high-risk populations. (p.H212N), c.926 G>T (p.C309F), c.1451 G>A (p.G484E)] 3 intronic Conclusions: The published screening projects identified a substantial mutations (c.1082+5 G>A, c.1242+1 G>A, c.1169+5 G>A), 1 nonsense number of LSD patients. It may be more targeted and cost efficient to raise mutation c.146 C>A (p.S49X) and 1 small in-frame deletion awareness in the respective specialist groups, however this remains to be c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, proven. 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant amount activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S105

P-277 P-279 GNPTG VARIATION IN ML II/III BRAZILIAN PATIENTS A PROJECT TO IDENTIFY GENES THAT CAUSE RECESSIVE Velho RV1 , Artigalás OA1 , Cury GKC2 , Alegra TA3 , Matte USM4 , INHERITED LIVER DISEASES Braulke TB5 , Schwartz IVDS1 Gray Z1 , McKay K2 , Lloyd C1 , Hartley J1 , MacDonald F2 , Hendriksz 1Genetics Department, UFRGS, Porto Alegre, Brazil CJ3 , Gissen P4 , Kelly DA1 2BRAIN Exp Research Group, HCPA, Porto Alegre, Brazil 1Birmingham Children's Hospital, Birmingham, United Kingdom 3Medical Genetics Service, HCPA, Porto Alegre, Brazil 2Birmingham Women's Hospital, Birmingham, United Kingdom 4Gene Therapy Center, HCPA, Porto Alegre, Brazil 3Salford Royal NHS Foundation Trust, Salford, United Kingdom 5Hamburg University, Hamburg, Germany 4UCL Institute of Child Health, London, United Kingdom

Mucolipidosis II and III (ML II/ III) present heterogeneity of loci (at least two Introduction/Background: The incidence of liver disease due to rare genes, GNPTAB and GNPTG are recognized, when mutated, as causing these inherited metabolic disorders, including Niemann Pick type C (NPC), Citrin diseases). Deficiency and Progressive Familial Intrahepatic Cholestasis (PFIC), is Objective: To characterize GNPTG in 14 unrelated Brazilian patients with unknown. New sequencing methods, including next generation sequencing, ML II/ III, 11 previously characterized as ML α/ β. permit analysis of multiple genes simultaneously, reducing time to molec- Methods: GNPTG gDNA of all patients was analyzed. RT-PCR and qRT- ular diagnosis and cost. Accurate timely diagnosis is essential to optimise PCR were also performed for patient D. clinical management and instigate specific treatments. Results/Discussion: Six different mutations were identified in 4 patients Objectives: To identify the incidence of inherited metabolic disorders, in (MLα/β01/4), including four novel mutations: c.328 G>T (p.E110X), patients with infantile liver disease, and to make this molecular information c.244_247dupGAGT, c.-112 C>G (n in controls: 0/ 200 alleles) and available early on in the diagnostic pathway. c.233+7 G>T (31/200 alleles). Two patients have MLIIIγ (B: Subjects/Methods: A prospective study of 64 infants, from 12 centres c.[244_247dupGAGT]+[-112 C>G,328 G>T], and D: c.[-112 C> worldwide, with cholestasis, acute liver failure or isolated splenomegaly G,328 G>T]+[-112 C>G, 328 G>T]). One patient remains unclassified had DNA sequenced for mutations in 6 genes. (J: c.[-72 G>T, Results: Diagnosis was confirmed (homozygous pathogenic mutation or -122 C>G]+[N] – both mutations present, in cis, in 12/200 control alleles). compound heterozygous pathogenic mutations) in 1 patient with NPC1, 1 Patient D: RT-PCR showed no abnormality in the GNPTG cDNA, and qRT-PCR with PFIC1 (ATP8B1), 2 with PFIC2(ABCB11)and3withPFIC3 showed a low level of expression of this gene, which could be due to the nonsense (ABCB4). 3 other patients had heterozygous pathogenic mutations, and mediated decay (NMD). Moreover, the difference between the gDNA and cDNA 24 variants of unknown significance were detected. sequences may be explained by stop codon misreading, or mRNA editing. Discussion/Conclusion: This unique study has successfully achieved world- Conclusions: Our data showed that GNPTG presents low allelic heteroge- wide collaboration to identify the incidence of rare inherited metabolic dis- neity. Experiments aiming at evaluating the occurrence of NMD, as well as orders presenting with infantile liver disease. Further collaboration will enable the therapeutic effect of aminoglycosides in ML II/III, are being conducted. this to continue, and determine the significance of novel sequence variants.

P-280 P-278 NIEMANN-PICK B DISEASE WITH PSEUDONORMAL M. NIEMANN-PICK TYPE C1: RESTORATION OF LIPID SPHINGOMIELINASE ACTIVITY: CLINICAL FEATURES RAFTS AND OTHER BIOCHEMICAL ANOMALIES BY AND BIOCHEMICAL APPROACH 1 1 1 1 1 N-BUTYL-DEOXINOJIRIMYCIN Pichkur NA , Olkhovich NV , Trofimova NS , Ivanova TP , Gorovenko NG 1 Maalouf K1 , Naim HY2 , Das AM1 Cent Metab Dis, Nal Child Hosp Ohmatdet, Kiev, Ukraine 1Hannover Medical School, Hannover, Germany 2University of Veterinary Medicine, Hannover, Germany Background: The Niemann-Pick A/B (NPA/B) disease is caused by deficiency of acid sphingomyelinase (ASM). There is the phenomenon of Introduction: One potential mechanism leading to clinical symptoms in pseudonormal ASM activity in patients with NPA/B, carrying the Q292K Morbus Niemann –Pick type C (NPC) may be the altered composition of mutation in SMPD1 gene. We present two siblings have been diagnosed cellular membrane lipids with subsequent impairment of lipid raft function with NPB with pseudonormal ASM activity. and protein-trafficking. Miglustat (N-butyl-deoxynojirimycin, NB-DNJ), an Methods: The patients were examined with morphological study of bone N-alkylated imino-sugar which inhibits the ceramide-specific glucosyl- marrow, biochemical activity of ASM in leukocytes and chitotriosidase transferase has been advocated in the treatment of NPC. activity of blood plasma. The molecular analysis of mutations Q292K in Methods and results: We examined the effects of NB-DNJ on membrane SMPD1gene was carried out. trafficking in fibroblasts of NPC1 patients. We found a substantial increase in Results: Two siblings demonstrated the specific clinical features: coarse facial dipeptidyl peptidase IV (DPPIV) levels in the untreated patient's fibroblasts, features, mild hepatosplenomegaly, and mental retardation. The+blue-sea cells which was reversed after treatment with NB-DNJ. Furthermore, NB-DNJ were revealed in bone marrow. The NPB disease was suspected. The biochemical resulted in a partial restoration of cholesterol-and sphingolipid- enriched analysis detected the increased chitotriosidase activity and the normal ASM activity membrane microdomains (lipid rafts) as assessed by increased levels of using pure fluorogenic substrate 6-Hexadecanoylamino-4-methylumbelliferyl flotillin 2 (a protein marker of lipid rafts) in the floating fractions of sucrose phosphorylcholine. The reaction with mixed substrate with lysosphyngomyeline density gradients in Triton X-100 lysates of NB DNJ-treated NPC1 fibroblasts. (van Diggelen et al, 2005) demonstrated the dramatically increased ASM activity. Endocytosis of DPPIV appeared to be slower in NPC1 cells, a phenotype This biochemical phenomenon is characterized ? as pseudonormal ASM activity which was enhanced when the NPC1 cells where incubated with NB-DNJ. and could confirm the diagnosis of NPB in these patients. To confirm the diagnosis Conclusion: Our study demonstrates that NB-DNJ as a potential therapeu- the detection of mutations Q292K in the SMPD1 gene was performed. tic agent for NPC patients reverses membrane lipid abnormalities in NPC Conclusions: The presence of phenomenon of pseudonormal ASM activity fibroblasts. increases the risk of false negative diagnosis of NPA/B disease. S106 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-281 P-282 NIEMANN-PICK DISEASE TYPE C (NP-C) SUSPICION INDEX MOLECULAR GENETIC TESTING FOR NIEMANN-PICK DISEASE (SI) TOOL: FURTHER ANALYSES BY AGE AND ASSOCIATIONS TYPECINBIRMINGHAM BY LEADING SYMPTOMS McKay KE1 , Bruce CK2 , Hunt J1 , Hartley JL3 , Hendriksz CJ4 , Kelly Sedel F1 , Walterfang M2 , Fahey M3 , Hendriksz CJ4 , Pineda M5 , Wijburg DA3 , Macdonald F1 , Gissen P5 FA6 , Patterson MC7 , Chadha-Boreham H8 , Kolb SA8 , Wraith JE9 1Birmingham Women's Hospital, Birmingham, United Kingdom 1Groupe Hospitalier Pitié-Salpêtrière, Paris, France 2Coventry University, Coventry, United Kingdom 2Royal University of Melbourne, Melbourne, Australia 3Birmimgham Children's Hospital, Birmingham, United Kingdom 3Monash University, Victoria, Australia 4Salford Royal Hospital, Salford, United Kingdom 4Salford Royal Hosp NHS Foundation Trust, Manchester, United Kingdom 5University of Birmingham, Birmingham, United Kingdom 5Hospital Sant Joan de Déu, Barcelona, Spain 6Academic Medical Centre, Amsterdam, Netherlands Background: Niemann-Pick disease type C (NPC) is a lysosomal lipid 7Mayo Clinic, Rochester, United States storage disorder. It is a progressive neurological disease, typically present- 8Actelion Pharmaceuticals Ltd., Allschwil, Switzerland ing in childhood and clinical features include ataxia, vertical supra-nuclear 9Royal Manchester Children's Hospital, Manchester, United Kingdom gaze palsy (VSGP), cognitive decline and seizures. It is caused by mutations in the genes NPC1 in 95 % of cases and NPC2 in 4 % of cases, and is Objectives: To examine the effect of age on the discriminatory power and autosomal recessively inherited. symptom association of the NP-C SI screening tool. Methods: Mutations analysis was carried out in 171 patients with suspected Methods: Original retrospective NP-C positive cases (Neurology, 2012) NPC. Initially this was done on a research basis using a custom-designed were split into three age groups: 16 years (n030), 4–16 years (n018), and resequencing microarray (n0118). Testing was then transferred to a diagnostic <4 years (n023). Patients' NP-C Risk Prediction Score (RPS) was analysed laboratory (n053) where the methods used were bi-directional Sanger se- by logistic regression. Symptom co-occurrence within/across domains (vis- quencing and multiplex ligation-dependent probe amplification (MLPA). ceral, neurological, and psychiatric) by leading symptoms (ataxia, cognitive Results: 16 cases resulted in positive molecular diagnoses, due to the decline, psychosis, and splenomegaly) were analysed descriptively. presence of two previously reported pathogenic mutations. A further 17 Results: NP-C positive cases vs. controls (>16 years and 4–16 years) cases had one pathogenic mutation present; however a second mutation was showed strong discriminatory power of RPS compared to infants (<4 years), not identified. Variants of unknown significance (VUS) were identified in a generating Receiver Operating Characteristic area under curve values of further 17 cases and 129 cases had no mutations. 0.964, 0.981, and 0.562 respectively. Patients with RPS <70 displayed a Conclusions: The overall pickup rate for this testing was 20 % (20 % in the lack of psychiatric symptoms and low levels of neurological involvement, research laboratory and 19 % in the diagnostic laboratory). No mutations suggestive of a more visceral phenotype. In infant patients, the main have yet been detected by MLPA. Clinical features of the diagnosed cases symptoms were splenomegaly and delayed developmental milestones. In will be discussed. patients >4 years, ataxia most frequently co-occurred with dystonia, dysarthria/dysphagia, and cognitive decline. Psychotic symptoms co-occurred with dysarthria/dysphagia but also with cognitive decline and treatment- resistant psychiatric symptoms. P-283 Conclusions: The SI tool maintains strong discriminatory power in SLEEP DISORDERS IN CHILDREN WITH NIEMANN-PICK DISEASE patients >4 years and can provide information regarding the associa- TYPE C tion of symptoms in patients with NP-C. Singh J1 , Rajdev S1 , Simmons L1 , Vijay S1 , Wassmer E1 Conflict of Interest declared. 1Birmingham Children's Hospital, Birmingham, United Kingdom

Background: Polysomnographic and biochemical studies have demonstrated disturbed sleep in children with NPC including Narcolepsy-Cataplexy like symptoms. [Kanbayashi et al 2003, Vankova et al 2003]. Methods: Parents of children with childhood NPC filled a Sleep Distur- bance Scale for Children(SDSC)questionnaire based on observation of sleep abnormalities over the previous six months. The SDSC assesses for disorders of 1.Sleep breathing 2.Initiating and maintaining sleep 3.Arousal 4.Sleep-wake transition 5.Excessive somnolence and 6.Sleep hyperhydro- sis. Summing all sleep factor scores derived a total sleep problem score. T scores greater than two standard deviations above the mean (T score >70) were taken as clinically significant and Total T-scores of 50-70 were considered borderline. Results: Five of six children identified, mean age 48 months (range 21 m- 108 m), returned the questionnaire. None of the children were found to have a clinically significant Total Sleep problem score, although 3 children had a borderline score. Only one of the children were found to have a clinically significant specific sleep disorder- Sleep breathing disorder. The two children who were being treated with Miglustat did not differ from the rest of the group. Conclusions: Sleep disorders were not a major feature of the disease phenotype of Niemann-Pick Disease type C in our cohort. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S107

A-018 A-020 THE VALUE OF THE SURROGATE TESTS (SERUM DIPEPTIDYL METACHROMATIC LEUKODYSTROPHY: MORTALITY DATA PEPTIDASE-IV, ADENOSINE DEAMINASE-1, CHITOTRIOSIDASE ) TO SUPPORT COUNSELLING OF PARENTS IN THE DIAGNOSIS OF MUCOPOLYSACCHARIDOSIS Singh J1 , Simmons L1 , Chakrapani A1 , Wassmer E1 Kurt I1 , Hasanoglu A2 ,AydinHI3 ,OkurI2 , Sertoglu E1 , Tapan S1 ,EzguFS2 1Birmingham Children's Hospital, Birmingham, United Kingdom 1Dept of M.Biochem,Gulhane School of Med, Ankara, Turkey 2Dept Ped Met and Nutr,Gazi Uni Fac Med, Ankara, Turkey Background: Leukodystrophies are a group of neurodegenerative diseases 3Dept Pediatrics, Gulhane School of Med, Ankara, Turkey affecting the white matter. Counselling parents of affected children is difficult as clinical characteristics, incidence, disease burden and descrip- Objectives: Urinary glycosaminoglycans (GAG) measurement and measure- tion of outcomes of such disorders are sparse. Gastrostomy as well as ment of the activity of missing enzyme is being used for the clinical diagnosis advanced palliative care may influence the outcome. The purpose of our of mucopolysaccharidoses (MPS). In this study, we aim to investigate the study was to describe common presenting features and the mortality and value of surrogate tests (serum dipeptidyl peptidase-IV, adenosine deaminase- time course of deaths in patients with MLD. 1 and chitotriosidase ) in the diagnosis of MPS. Methods: The present study Methods: Retrospective and concurrent review of medical records of MLD included 21 controls and 22 MPS patients ( 4 MPS II, 4 MPS I, 9 MPS VI, 5 among children younger than 16 years presenting to a regional children's MPS IV) which were previously diagnosed by enzymatic analysis. Serum hospital. DPP-IV, ADA-1 and chitotriosidase activities were measured. Results: A total of 11 children with MLD were identified. 3 children died at Results: There was approximately 3-fold increase in serum DPP-IV (patients: the age of 5.5, 7.2 and 7.7 years and this was a mean of 4.8 years(range 3.5- 122 nmol/min/ml; controls:44), 2-fold increase in ADA-1 (patients: 9 U/L; 6 years) after first symptom. The current age of the rest is a mean of controls:5), while there was no increase in chitotriosidase (patients:27 nmol/ 10.1 years(range 4.9-20.6 years). hour/ml; controls:27). DPP-IV activity in 20/22 of MPS patients and ADA-1 The most common presenting feature was developmental regression at activity in 15/22 of MPS patients were over the upper limit of reference range. approximately 2 years but 2 children manifested symptoms after age 5 years. Conclusion: Increase in serum DPP-IV, compatible with Beesley et al One 10.5 year old child with mild symptoms had a bone marrow transplant (2009), and ADA-1 suggests an inflammation response in MPS. Small at 5 years. numbers in our study limits the reliability but measurement of serum Conclusions: A body of evidence is required to inform discussions with parents DPP-IV activity can potentially be used as first-line screening test comple- regarding the morbidity and mortality in Leukodystrophies. This observational mentary to urinary GAG/creatinine for MPS screening. data will help clinicians and parents caring for children with MLD.

A-019 A-021 REDUCTION IN NUMBER OF OBSTRUCTIVE SLEEP APNOEA A NOVEL MUTATION DESCRIBED İN TWO BROTHERS WITH EVENTS AFTER ENZYME REPLACEMENT THERAPY FOR METACHROMATIC LEUKODYSTROPHY MUCOPOLYSACCHARIDOSIS IVA Kiykim E1 ,Aktuğlu Zeybek AÇ1 , Soyuçen E1 , Cansever MŞ1 , Laçinel S1 , Breen C1 ,EllicottR2 , McBride K2 , Hensman P1 ,EagletonT3 , Decker C3 , Aydin A1 Cunningham S4 , Jones S1 1Div Ped Nutr & Met, Cerr Med F, Ist Uni, İstanbul, Turkey 1Genet Med,Manchester Acad Health Sci Cen, Manchester, United Kingdom 2Wellcome Trust Clin Research Facil, Manchester, United Kingdom MLD is an autosomal recessive disorder. Multiple affected sibs of both 3BioMarin Pharm Inc, Novato, United States sexes and normal parents have often been observed. The age at onset of the 4Resp Med Royal Hosp Sick Child, Edinburgh, United Kingdom juvenile form ranges between 3 and 14 years. Failure in school, behavioural problems of disturbance of cognitive function may precede motor abnor- Background: Patients with MPS IVA commonly have respiratory prob- malities, especially in patients with a later onset. Progressive difficulties in lems, which historically were considered to be mostly restrictive in nature, walking with pyramidal signs and peripheral neuropathy, together with but increasingly are thought to be due to a combination of restrictive and cerebellar ataxia constitute the most common presentation. More than 100 obstructive airway defects. different mutation of the arylsulfatase A ARSA gene (22q13) are known. Objectives: To describe and document the reduction in obstructive sleep Here, we report two siblings having a novel mutation that hasn't been apnoeic events in a MPS IVA patient participating in a clinical trial with reported before. ERT (enzyme replacement therapy). 17 year old boy admitted to the Metabolic Disease Department with the Methods: An eight year old girl with MPS IVAwho previously experienced complaint of difficulty in walking. His complaint was recognized 3 months documented nocturnal hypoxia secondary to significant obstructive sleep ago. On his physical examination Gowers' sign was positive. He had diffuse apnoea (OSA) as measured by polysomnography continued to be assessed white matter involvement on cranial MRI. Electromyography was consis- for OSA following enrolment in an open label Phase 1/2 clinical trial with tenrt with demyelinating polyneuropathy. The Aryl sulfatase A enzyme BMN110 (a recombinant form of N-acetylgalactosamine-6-sulphatase, level was 15,18 NCS/mg pr (N: 50-126). His brother also had the same GALNS), ERT for MPS IVA. complaints with the same cranial MRI and EMG findings. Aryl sulfatase A Results: Per protocol, in 3 successive 12 week periods, the patient received enzyme level was 16.50 NCS/mg pr(N: 50-126). escalating doses of ERT initially of 0.1 mg/kg/week, followed by 1.0 mg/kg/week, Mutation analysis of both patients revealed c.467 G>A(p.C156Y) homo- then up to and remaining on a dose of 2.0 mg/kg/week. After 13 months on ERT zygous mutation that had never been reported before. the patient experienced a reduction in the number of obstructive events. Her height increased by 0.4 cm from baseline to week 120 (from baseline of 89.9 cm). Conclusion: Treatment with ERT may be associated with improvements in respiratory parameters such as OSA. Conflict of Interest declared. S108 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

A-022 A-024 ALPHA-MANNOSIDOSIS: TWO SIBLING CASES DESENSITIZATION OF AN INFANT WITH POMPE DISEASE Kardas Fatih1 , Soylu Pembe1 , Kendirci Mustafa1 AND A HISTORY OF ANAPHYLAXIS TO ALGLUCOSIDASE 1Ped Nutr and Met Dept, Erciyes Univ, Kayseri, Turkey ALFA Kucukcongar A1 ,Ertoğ Karagöl İ2 ,EzgüFS1 ,TümerL1 , Kasapkara ÇS1 , Background: Alpha-mannosidosis is an inherited lysosomal storage disor- Hasanoğlu A1 der characterized by immune deficiency, facial and skeletal abnormalities, 1Div Metab Dis, Univ Gazi, Ankara, Turkey hearing impairment and intellectual disability. The disorder is caused by 2Div Pediatric Allergy, Univ Gazi, Ankara, Turkey lysosomal α-mannosidase deficiency and due to mutations in the MAN2B1 gene located on chromosome 19. It occurs in approximately 1 of 500000 Many medications may cause allergies which may manifest in a wide live births. Main features are immune deficiency, skeletal abnormalities clinical spectrum from light pruritus to anaphylactic shock. Recombinant (mild to moderate dysostosis multiplex, scoliosis and deformation of the alglucosidase alpha (rhAGA) which is used in Pompe disease (PD) is an sternum), hearing impairment (moderate to severe sensorineural hearing immunogenic compound. loss), impairments of mental functions and speech, and periods of We propose a new desensitization protocol, which follows general desen- psychosis. sitization rules, used for the youngest infantile Pompe patient, who devel- Case reports: A 5 10/12 years-old boy and his 7 years-old sister were oped anaphylaxis to rhAGA. admitted to our hospital because of frequent ear inflammation and psycho- Case Report: Rh AGA was started on a patient who was diagnosed as PD motor retardation. Characteristically; two cases showed coarse facies, flat- antenatally with the Standard dose of 20 mg/kg alternate weekly in the 1st tened nasal bridge, macroglossia, short neck, hepatomegaly and sea blue month of the postnatal period. Her growth and physical examination were histiocytosis in bone marrow aspiration. Alpha-mannosidase enzyme levels normal. On echocardiographic examination, hypertrophy in interventricular were 1.66 and 1.52 nmol/h/mg protein (normal range 231.4±81 nmol/h/mg septum was observed. Anaphylaxis developed 15 minutes within start of the protein) respectively. 12th ERT infusion. An intradermal test with rh AGA at 1 /1.000 concen- Conclusion: Alpha-Mannosidosis belongs to a group of disorders de- tration was made and found to be positive. We treated the patient with the scribed as oligosaccharidoses or glycoprotein storage diseases and resem- new desensitization protocol without serious complications. bles Hurler's syndrome and its clinical manifestations. Disease should be The patient is now 16 month-old, shows normal growth, echo and electrocardio- suspected in individuals with mental retardation, skeletal changes, hearing grams and is able to do every activity consistent with her age. She has received loss and recurrent infections. Differential diagnosis should be done from ERT therapy without any further allergic reactions the age of two months. other lysosomal storage disease especially mucopolysaccharidosis. To our knowledge this case is the youngest patient reported with PD desensitized to AGA.

A-023 A-025 EARLY-ONSET RESPIRATORY MANIFESTATIONS IN HUNTER GOOD OUTCOME OF ERT OF TWO INFANTILE POMPE DISEASE DISEASE: CASE REPORT CASES Sarajlija A1 , Minic P2 , Djordjevic M1 , Rodic M2 , Kecman B1 , Savic N2 , Hasanoğlu A1 , Kucukcongar A1 , Ezgü FS1 , Tümer L1 , Reuser AJJ2 Baljosevic I3 1Div Metab Dis, Univ Gazi, Ankara, Turkey 1Metab Unit, Mother and Child Health Inst, Belgrade, Serbia and Montenegro 2Div Clin Genetic, Univ Erasmus, Rotterdam, Netherlands 2Pulmo Unit, Mother and Child Health Inst, Belgrade, Serbia and Montenegro 3ORL Unit, Mother and Child Health Inst, Belgrade, Serbia and Montenegro Some patients with classic infantile Pompe patients don't express any endogenous acid alpha-glucosidase (GAA) whose cross reactive immun Background: Respiratory involvement is present in most types of muco- material (CRIM) status are negative. They are prone to develop a high polysaccharidosis (MPS). According to Hunter Outcome Survey report of antibody titer against the recombinant human GAA, counteracting the effect 2008, significantly enlarged adenoid and tonsils mostly occured after 3 years of enzyme-replacement therapy (ERT). Therefore CRIM status is important of age. Extension of pathological storage beyond level of segmental bronchi for the prognosis. Here described the outcome of our two CRIM positive is rarely encountered. infantil Pompe disease patients. Methods: We evaluated history of boy diagnosed with MPS during inves- Two cases were both diagnosed in the newborn period as infantil Pompe tigations in our Institute. disease and have been started on ERT at one month of age. CRIM was Case report: Fourteen months old boy was referred to our Institute for determined positive for both. prolonged cough, dyspnea and recurrent respiratory infections. Examina- The first case had globally cardiac hypertrophy before ERT. He is now tion showed stridor, wheezing and crackles, hepatomegaly and skeletal 16 months old and is being treated with ERT since 15 months regularly. His changes suggestive of MPS. Urinary glycosaminoglycans turned positive. motor and mental development is normal according to his age and cardiac HRCT scan showed stenotic changes in left main bronchus with solid tissue finding was improved. infiltrations surrounding trachea and bronchial tree and within lung paren- The second case is being treated with ERT currently, she is now 17 months chyme. Flexible bronchoscopic exam revealed severely enlarged tonsils and old, her development is normal and only mild interseptal cardiac hypertro- adenoid, possible storage in tracheal mucosa and stenosis of left main phy was shown by ecocardiography. bronchus. Enzyme assay confirmed Hunter disease. Sleep study showed Conclusion: Early diagnosis and CRIM positive status results in a favorable sleep disturbance with desaturation below 90 % in more then 20 % of outcome in severe infantil Pompe disease patients. sleeping time. Tonsiloadenoidectomy was performed at age of two years and resulted in improvement of respiratory status and overall health. Conclusion: Onset of serious respiratory disturbance before 18 months of age is not frequent but could be encountered in boys with Hunter disease. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S109

A-026 A-028 CERVICAL CORD DECOMPRESSION IN TWO PATIENTS WITH BONE METABOLISM IN PATIENTS AFFECTED BY GAUCHER MUCOPOLYSACCHARIDOSIS DISEASE Hui J1 , Zhu XL1 , Chiang G1 , Chu WCW1 , Tang NL1 Hasanoglu A1 , Tumer L1 , Ezgu F S2 , Kasapkara C S1 , Kucukcongar A1 1The Chinese University of Hong Kong, Hong Kong, Hong Kong, China 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 2Div Metab Dis, Gazi Univ Hosp, Ankara, Turkey Introduction: Spinal cord compression is a known complication of Muco- polysaccharidosis which may occur at the cranio- cervical junction due to Gaucher disease is the most prevelant hereditary lysosomal storage disor- (1) atlanto-axial subluxation +/- foramen magnum, vertebral canal stenosis der, affecting multiple organ systems. It is characterized by a deficiency of (2) meningeal hypertrophy caused by deposition of mucopolysaccharides. the enzyme glucocerebrosidase that leads to accumulation of glucosylcer- We presented 2 patients with this complication who underwent neurosurgi- amide in cells of the monocyte - macrophage lineage. It is classified in three cal intervention with good neurological recovery. types, according to the presence of central nervous system involvement Patient 1: TH was a 7 years old boy with Mucopolyssacharidosis I who (type 2 and 3) or not (type 1). Majority of patients present with hepatos- sustained cervical cord injury with initial tetraplegia after an accidental fall. plenomegaly, anemia, thrombocytopenia, bleeding tendencies, skeletal pa- MRI spine showed C1/C2 subluxation with cervical cord compression. He thologies, growth retardation and in severe cases pulmonary disease. The underwent decompression and internal fixation. He recovered and was able bone manifestations include bone infarcts, avascular bone necrosis, lytic to walk independently 1 year after the operation. lesions osteosclerosis, osteopenia or osteoporosis. This article gives an Patient 2: CH was an 18 years old man with Mucopolysaccharidosis VI. He overview on bone manifestations of 20 Gaucher patients followed at Gazi underwent HLA identical umbilical cord blood transplant at 6 years of age. University Hospital Pediatric Metabolic Unit and review the current litera- Cord compression at the upper cervical region was noted on serial MRI ture. 19/20 patients with Gaucher disease showed low levels of 25(OH)D examination. He underwent decompression surgery with laminectomy and (<30 ng/ml). Considering the essential role of vitamin D in bone homeo- posterior cranial-cervical fusion. Post operatively, he did well but developed stasis and the prevalence of hypovitaminosis D, we suggest to evaluate this obstructive hydrocephalus which required shunting. parameter routinely in all Gaucher patients. – – Conclusion: MPS patients should be routinely screened for clinical and radiological evidence of spinal cord compression. Timely neurosurgical intervention is vital in preventing morbidity and mortality arising from A-029 irreversible cord compression. MULTIPLE SULFATASE DEFICIENCY- REPORT OF A NOVEL MUTATION IN AN INDIAN FAMILY Kotecha U1 , Movva S1 , Verma J1 , Puri R1 , Bijarnia S1 , Verma IC1 1Cent Med Gen,Sir Gangaram Hospital, New Delhi, India A-027 GAUCHER DISEASE TYPE 2: A NOVEL MUTATION IN INFANT Multiple sulfatase deficiency (MSD) is an autosomal recessive disorder OF SLAVIC ORIGIN with an estimated prevalence of 1 in 1.4 million live births. We hereby 1 1 2 3 3 Bzduch V , Fabriciova K , Behulova D , Mattosova S , Chandoga J , describe a case with classical features of MSD wherein mutation analysis 4 5 Jajcaiova-Zednickova N , Majidi A revealed him to be compound heterozygous for two novel mutations. 1 First Dept Pediat Univ Children´s Hosp, Bratislava, Slovakia Case Report: The proband, a four year old boy was the first born of non 2 Centre of Inherited Metabolic Diseases,, Bratislava, Slovakia consanguineous parents of Indian ethnicity. presented with history of neuro- 3 Centre of Medical Genetics Univ Hosp, Bratislava, Slovakia regression. His initial development was normal till 15 months following 4 Dept Hematol, Bratislava, Slovakia which parent's regression of attained milestones. Currently at 4 years he is 5 Inst Pathol, Health Care Surv Authority, Bratislava, Slovakia able to sit with support momentarily and speech is limited to vocalizations. On examination positive findings included a coarse facies with a depressed Background: Gaucher disease type 2 (GD2) is the rarest and most severe nasal bridge, bushy eyebrows, bilateral broad great toes and icthyosis. form of the disease with genotyping heterogeneity and presents pre- or Central nervous system examination showed axial hypotonia with appen- perinatally, or in the first few months of life (classical variant). dicular hypertonia. Skeletal survey was suggestive of dyostosis multiplex. Case report: A female neonate was born to Czech father and Slovak A clinical suspicion of MSD was entertained and further investigations mother. At the age of six months she appeared developmentally delayed advised with hypertonicity, facial dysmorphy and psychomotor regression. At the Biochemical: urine analysis showed mucoploysacchariduria. age of 11 months during respiratory infection marked splenomegaly with Enzyme levels of MPS II, IIIA, IVA and arylsulfatase A were low. anemia and thrombocytopenia was found. Plasma chitotriosidase activity Molecular analysis of SUFM1 gene : – was grossly elevated [10,630 nmol/h/ml (normal range 4 200)]. On a bone Compound heterozygous: exon 3 (c.451A>G) and ins T exon 5 (690- marrow aspirate Gaucher cells were seen. Leukocyte enzyme assay showed 691InsT,p.W231LfsX10). a significant decrease in the levels of ß- glucocerebrosidase (GBA) activity Both the above mutations are novel mutations and have not been reported [0,7 nmol/h/mg protein (normal range 4-12)]. DNA sequence analysis of previously. the GBA gene revealed mutation L444P and a novel mutation C4W in exon 3 at cDNA nucleotide position 129 . End stage disease was characterised by cerebral deterioration. She died before reaching one year of age due to respiratory failure. Conclusion: We found compound heterozygosity L444P/C4W in infant with GD2 of Slavic origin, which had a major impact on residual GBA activity (17,5 % of normal) and resulted in classical variant of disease. S110 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

A-030 A-033 SUCCESSFUL MANAGEMENT OF INFUSION-ASSOCIATED NIEMANN-PICK DISEASE TYPE C: TWO NOVEL MUTATIONS REACTIONS IN A 23 YEARS OLD PATIENT WITH OF FOUR CASES MUCOPOLYSACCHARIDOSIS VI RECEIVING RECOMBINANT Kardas Fatih1 , Soylu Pembe1 , Kendirci Mustafa1 , Gumus Hakan2 , Arslan HUMAN ARYLSULFATASE B Duran3 , Ozturk Adnan4 Hasanoglu A1 , Kasapkara C S1 , Celik G2 , Kucukcongar A1 , Ezgu F S1 , 1Ped Nutr and Met Dept, Erciyes Univ, Kayseri, Turkey Tumer L1 2Ped Neurology Dept, Erciyes Univ, Kayseri, Turkey 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 3Ped Gastroenterology Dept, Erciyes Univ, Kayseri, Turkey 2Div of Chest Dis, Ankara Univ, Ankara, Turkey 4Neonatology Dept, Erciyes Univ, Kayseri, Turkey

Mucopolysaccharıdosıs Type VI (MPS VI), or Maroteaux-Lamy syndrome, is Background: Niemann-Pick disease type C (NP-C) is a lysosomal storage a lysosomal storage disorder that results from a deficiency of the enzyme N- disease in which impaired intracellular lipid trafficking leads to excess storage acetylgalactosamine 4-sulfatase, or arylsulfatase B (ASB). Here we report on a of cholesterol and glycosphingolipids in the brain and other tissues. NP-C has 23 year old male patient with MPS VI who developed an IAR ( a light an extremely heterogeneous clinical presentation characterized by a wide range erythema and generalized itching) within 1 month of initiating weekly infu- of symptoms that are not specific to the disease. NPC1 gene mutations are sions (1 mg/kg) of galsulfase therapy with the typical infusion lasting 4 hours. present in 95 % and NPC2 mutations are present in approximately 4 % of cases. When infusion-related reaction occurred, ERT was immediately suspended Case reports: The first case was 4 10/12 years-old female, presented with until the patient's symptoms had resolved. Antihistamines and antipyretics early infantile form of the disease, the second one was a 5-day female newborn, were administered to treat the acute symptoms of hypersensitivity reactions. pre/perinatal form, and the third one was one-month-old female with neonatal When ERT was restarted; we were able to continue treatment successfully by cholestatic form. The first and the second cases were sisters, third one was their the addition of montelukast 10 mg, acetylsalicylic acid 300 mg and phenir- cousin. The fourth case, 4 years-old male was not a relative of the first three amine (avil) 1 mg/kg to the premedication regimen (1 hour before ERT cases. A homozygote mutation, g.8749 del.A, was identified in NPC2 gene of administration) and by significantly reducing the rate of drug infusion. This these first three cases. c.2000 C>T (p.ser667Leu) and c.3709_3711delGC- case is the oldest patient reported with MPS VI desensitized to galsulfase. We CinsTT mutations were identified in NPC1 gene of the fourth case. have demonstrated that by significantly reducing the rate of infusions and Conclusion: We found two novel mutation (g.8749 del.A and adjusting the premedication regimen, galsulfase infusions can continue with c.3709_3711delGCCinsTT)in four cases with NP-C and the fenotypes of no additional observance of infusion-associated reactions. the disease were different in three cases that the same mutation detected.

A-032 23. Glycosylation Disorders CHOLELITIASIS IN A YOUNG PATIENT WITH TYPE 1 GAUCHER DISEASE, A CASE REPORT P-284 Kagnici M1 , Coker A2 , Kalkan Ucar S1 , Coker M1 A BROAD SPECTRUM OF CONGENITAL DISORDER OF 1Div Metab Dis, Dep Ped, Univ Ege, Izmir, Turkey GLYCOSYLATION SUBTYPES IN ARGENTINA 2Div Surgery, Univ Ege, Izmir, Turkey Asteggiano CG1 , Bistué Millón MB2 , Delgado MA2 ,SarriónP3 ,Martínez Domenech G2 ,GuelbertN2 ,BalcellsS3 ,GrinbergD3 , Dodelson de Kremer R2 Background: Type 1 Gaucher disease (GD1), an autosomal recessive 1CEMECO/UNC/UCC/ CONICET, Córdoba, Argentina lysosomal storage disorder, results from deficient activity of β- 2Centro Estudio Metabolopatía Congénita, Córdoba, Argentina glucocerebrosidase, leading to glucocerebroside accumulation in cells of 3Departament de Genética, Fac. Biol. UB, Barcelona, Spain reticuloendothelial system. Gall stones have been recognized as a manifestation of GD1. In this report, we discuss a case with GD1 having gallstones CDG are due to defects in N-, O-glycoproteins and glycolipids. Their spectrum in a younger age than expected. ranges from multisystem to mono-organ disease. Most CDG are autosomal Case Report: The female patient, was single child born from healthy recessive, except multiple osteochondromatosis (EXT1/EXT2-CDG), charac- parents who were not consanguineous. Medical history revealed that her terized by the formation of multiple cartilage-capped tumors. Two glycosyl- first complaints were abdominal pain and abdominal distension at three transferases involved in heparan sulphate elongation have been identified. years old, and after a short period, she had undergone splenectomy. After The aim is to increase the knowledge on human glycobiology in a broad referral to our clinic, a deficiency in β-glucocerebrosidase activity spectrum of CDG in Argentina. (1.8 nmol/prot/h, N>6.56) were found. Genetic testing revealed compound Using transferrin isoelectric-focusing, mass spectrometry and lipid-Linked- heterozygosity with N370S (N370S/T134l). She was diagnosed to have Oligosaccharides, we identified six patients with abnormal N-glycosylation. GD1 in 2005. ERT and miglustat therapy have been applied since. At the Studies are in progress to elucidate the defect. The initial work-up has been age of 19 years she began complaining about right upper quadrant pain. completed in P1 and the exome was sequenced in collaboration with Prof. Gert Abdominal ultrasonography revealed multiple gall stones and cholecystec- Matthijs. P1 has two mutations in GALT gene, despite normal galactose levels. tomy was performed in 2011. Regarding EXT1/EXT2-CDG, DNA was studied by genotyping and MLPA. Conclusion: The patient was in the second decade of her life when diagnosed Twenty Latin-American patients had a severe phenotype and two patient with gallbladder disease, which is a unique situation in the literature. Splenec- presented malignant transformation to chondrosarcoma. We have found the tomy in early age can result in gallbladder disease, sooner than expected. molecular bases of 85 % patients, 65 % in EXT1 and 20 % in EXT2. By MLPA we found two complete deletions in EXT1 and in EXT2 gene. This interdisciplinary research program in Argentina is the beginning of a "CDGnet Latin America" to promote scientific research, diagnosis and specific management of patients with Congenital Disorders of Glycosylation. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S111

P-285 P-287 GLYCOSYLATION DEFECTS AND EPILEPSY SURVIVING TO ADULTHOOD IN ALG6-CDG; UNIQUE Fiumara A1 , Sturiale L2 , Cocuzza M3 , Castiglione D3 , del Campo G1 , MALFORMATIONS AND A RECOGNIZABLE PHENOTYPE Barone R3 IN A LARGE COHORT OF PATIENTS 1Center for Metabolic Diseases, Catania, Italy Morava E1 , Tiemes V1 , Tiel C2 , Seta N3 , de Lonlay P4 , de Klerk H5 , 2CNR, Catania, Italy Visser G6 , van Hasselt P6 ,MulderM7 ,RubioE8 ,MatthijsG9 ,JaekenJ10 , 3Child Neurology and Psychiatry, Catania, Italy Lefeber D11 , Wevers RA12 1Dep of Ped, RUNMC, Nijmegen, Netherlands Investigation for inherited metabolic diseases are rarely performed in epi- 2Dep Hum Genet, Heidelberg, Germany leptic children. Usually these patients are classified on the basis of the 3Métabolique Hôpital Bichat-Claude Bernar, Paris, France seizures phenotype and drug response. Seizures are described in Congenital 4Ref Cent of Metab Diseases, Necker, Paris, France Disorders of Glycosylation (CDG). 5Dep of Ped, Erasmus Hospital, Rotterdam, Netherlands In the past years (2005-2011) we analyzed more than three thousand 6Dep of Ped, UMC Utrecht, Utrecht, Netherlands samples from Italian patients by transferrin IEF, positive ones were typed 7Dep of Ped, VU, Amasterdam, Netherlands by MALDI-TOF. 8Dep of Ped, UMC Maastricht, Maastricht, Netherlands We identified 21 patients with PMM2-CDG (CDG Ia) and seven with 9Dep Hum Genet, Leuven, Belgium different types of non-PMM2 CDG. Among these, drug resistant epilepsy 10Dep of Pediatrics, Leuven University, Leuven, Belgium in the first year of life was the presenting sign in six, commonly infantile 11Dep of Neur, Nijmegen, Netherlands spasms, myoclonic or tonic-clonic seizures. Dysmorphisms were a constant 12LGEM, RUNMC, Nijmegen, Netherlands finding (7/7), followed by microcephaly (6/7), severe mental retardation (6/ 7), visual impairment (5/7) and only 1/7 had cerebellar atrophy. Serum ALG6-CDG has been described as a syndrome of hypotonia, developmen- transferrin IEF and MALDI-TOF defined a type I underglycoslation profile tal delay and epilepsy with a high frequency of bleeding-anomalies but no in these patients but PMM2 was normal. Definitive diagnosis was ALG6- significant systemic involvement. Based on the EUROGLYCANET data- CDG Ic; ALG3-CDG Id and DPM2 in one patient respectively and is base registration we approached the referring clinicians and collected com- pending in four (CDG Ix). prehensive data, including clinical-metabolic features and mutation analysis We wish to underline that early onset epileptic encephalopathies and drug- in 44 unreported patients. We found hypotonia and developmental delay in resistant epilepsy, in the first months of life, should arouse the suspicion of all cases and epilepsy and ataxia consecutively in 70 % and 50 %. Coag- non PMM2-CDG. This could allow the recognition of a number of undi- ulation anomalies were present in less than 50 % without spontaneous agnosed CDG cases. bleedings. Four patients had cardiomyopathy. The majority of children had failure to thrive and many children had severe protein losing entherop- athy. Nine children deceased. Unique hand anomalies were observed in 7 patients with missing phalanges and brachydactyly. CBCL and quality of P-286 life assessment showed cyclic behavioral changes and depressive behaviour A NOVEL NON-NEUROLOGICAL CONGENITAL DISORDER OF as one of the most significant complaints. Most patients were diagnosed by GLYCOSYLATION CAUSED BY MUTATIONS IN THE PHOSPHO- LLO analysis and only 3 patients underwent direct primary mutation GLUCOMUTASE 1 GENE analysis. The most common mutations were p.A333V and p.I299Del with- Pérez B1 , Medrano C1 , Ecay MJ1 , Ruiz Sala P1 , Martínez-Pardo M2 , out geno-phenotype correlation. The oldest patient is a Dutch lady, current- Ugarte M1 , Pérez-Cerdá C1 ly 36 years old, can make some steps independently, has mild speech delay 1CEDEM,CBM,CIBERER, Univ Autonoma Madrid, Madrid, Spain and a stable encephalopathy. ALG-CDG remains the second most common 2U Enf Metab. S Pediatr. H Ramón y Cajal, Madrid, Spain CDG type with recognizable phenotype and a non-progressive course.

We report a new subtype of non-neurological congenital disorders of glycosylation (CDG) involving the impaired cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. A patient presented with muscle fatigue, elevated creatine kinase, growth hormone deficiency, and first branchial arch syndrome. These findings, together with the abnormal type II plasma transferrin isoform profile detected, was compatible with a CDG. Functional testing and clinical analyses suggested a deficiency in the interconversion of glucose-1-phosphate and glucose-6-phosphate catalysed by phosphoglucomutase (PGM1), a problem previously described as glycogenosis type XIV. PGM1 activity in patient-derived fibroblasts was significantly reduced, as was the quantity of immunoreactive PGM1 protein. Mutation analysis of PGM1 and subsequent functional analysis investigating transient expression of PGM1 in immortalized patient fibroblasts, followed by ex vivo splicing assays using minigenes, allowed the characterization of two novel pathogenic mutations: c.871 G>A (p.Gly291Arg) and c.1144+3A>T. The latter represents a severe splicing mutation leading to the out-of-frame skipping of exon 7 and the formation of a truncated protein (p.Arg343fs). MALDI mass spectra of permethylated protein N- glycans from the patient's serum suggested a marked hypoglycosylation defect. The present findings confirm that, in addition to a rare muscular glycolytic defect, PGM1 deficiency causes a non-neurological disorder of glycosylation. S112 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-288 P-289 COG5-CDG: FIVE NEW PATIENTS EXPANDING THE PHENO- CONGENITAL DISORDERS OF GLYCOSYLATION TYPE IA: TYPICAL SPECTRUM ARE VACUOLATED PERIPHERAL BLOOD CELLS A DIAGNOSTIC Rymen D1 , Rymen D2 , Keldermans L1 , Race V1 , Mallezie A1 , Foulquier CLUE? F3 , Régal L2 , Deconinck N4 , Dionisi-Vici C5 , Fung CW6 , Matthijs G1 , Ersoy M1 ,BalciMC2 , Özgüven A.A1 ,ÜnüvarA1 ,MatthijsG3 , Jaeken J4 , Jaeken J2 Demirkol M1 ,GökçayG1 1Cent Hum Gen, Univ Hosp Gasthuisberg, Leuven, Belgium 1Div Nutr Metab, Child Hosp, Ist Med Fac, Istanbul, Turkey 2Cent Metab Dis, Univ Hosp Gasthuisberg, Leuven, Belgium 2Dep of Dietetics, Ist Medical Fac, Istanbul, Turkey 3Struct Funct Glycob Unit, Univ of Lille, Lille, France 3Centrum of Hum Genetics, Uni of Leuven, Leuven, Belgium 4Univ Child Hosp Queen Fabiola, Brussels, Belgium 4Centrum of Metab Dis, Uni of Leuven, Leuven, Belgium 5Div Metab, Bambino Gesù Hosp, Rome, Italy 6Duch Kent Child Hosp, Univ Hongkong, Hong Kong, Hong Kong, China Background: Several metabolic diseases can result in abnormal accumulation of metabolic by-products, resulting in abnormal cytoplasmic vacuo- Background: The Conserved Oligomeric Golgi (COG) protein complex lation in peripheral blood cells. Phosphomannomutase-2 (PMM2) consists of eight subunits. It is involved in the retrograde trafficking of deficiency (CDGIa) is the most frequent type of congenital disorders of Golgi components, thereby affecting the localization of Golgi glycosyl- glycosylation (CDG), a family of multisystem metabolic disease. PMM-2 transferases. Deficiency of a COG-subunit leads to defective protein gly- catalyzes the transformation of mannose-6-phosphate (M6P) to mannose-1- cosylation, and thus Congenital Disorders of Glycosylation (CDG). phosphate. M6P is also part of a novel mammalian stress-signaling path- Mutations in subunit 1, 4, 5, 6, 7 and 8 have been associated with CDG- way, responding to pathological viral stress and plays an essential role in II. Previously only one COG5-CDG case was described (Paesold-Burda et phagosome formation, membrane trafficking and inflammatory response. al. Hum Mol Genet 2009; 18:4350-6). Contrary to most patients with other Objectives: CDG type Ia could be one of the diseases with vacuolated COG-CDG, the patient presented a mild/moderate phenotype, i.e. moderate peripheral blood cells and the evaluation of peripheral blood smear could be psychomotor retardation with language delay, truncal ataxia, slight hypoto- a discriminating finding. nia but pronounced pontocerebellar hypotrophy. Material/Method: Peripheral blood smears of six patients, 3 with CDGIa, Methods: CDG-IIx patients from our database were screened for mutations 2 with CDGIb, and one with CDG Ix were evaluated. in COG5. Clinical data were compared. Brefeldin A treatment of fibroblasts Results: All three patients with CDGIa had cytoplasmic vacuolation in and Western Blot experiments were performed to support the diagnosis. peripheral blood cells, especially in monocytes and neutrophils, while the Results and conclusion: We identified 5 patients with 6 novel mutations in blood smear of the other patients were unremarkable with respect to COG5. All patients presented hypotonia, microcephaly and psychomotor vacuolation. retardation (PMR). Three presented very severe PMR. Language develop- Discussion: Evaluation of peripheral blood cells of CDG patients could be ment was consistently delayed or absent. Cortical blindness and deafness a discriminating finding. We could speculate that free glycans and unbal- were present in two patients. Variable findings were failure to thrive, anced glycoprotein production released in response to elevated M6P levels important liver involvement, and recurring urinary tract infections due to could result in abnormal cytoplasmic vacuolation in peripheral blood cells. neurogenic bladder. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S113

P-290 P-292 EXPANDING OUR KNOWLEDGE OF CONGENITAL DISORDER EVIDENCE OF RETINAL DYSFUNCTION AT THE ON-BIPOLAR OF GLYCOSYLATION-DPAGT1 TYPE SYNAPSE IN PMM2-CDG Perez B1 ,VegaA1 ,MedranoC1 , Martín-Higueras C1 , Pascual SI2 , Arroyo I3 , Thompson DA1 , Lyons RJ1 , Russell-Eggitt I1 , Liasis A1 , Jägle H2 , Domingo R4 ,ArtuchR5 , Briones P6 ,UgarteM1 , Pérez-Cerdá C1 Grünewald S3 1CEDEM-CBMSO, UAM-CSIC, CIBERER-Madrid,, Madrid, Spain 1Ophthalmology, Great Ormond St Hosp, London, United Kingdom 2Hospital La Paz, Madrid, Spain 2Univ Eye Clinic, Regensburg, Germany 3Hospital San Pedro de Alcantara, Cáceres, Spain 3Metab Dept, Great Ormond St Hosp, London, United Kingdom 4Hospital Virgen de la Arritxaca, Murcia, Spain 5Hosp. San Joan de Deu, CIBERER, Barcelona, Spain Background: Two siblings with PMM2-CDG showed profound dysfunc- 65 Hosp Clinic, IDIBAPS, CIBERER, Barcelona, Spain tion at the retinal cone on-bipolar cell synapse. Objective: We sought confirmatory evidence of retinal on-pathway dys- Congenital disorders of glycosylation (CDG) is a clinically and biochemi- function in a larger series of patients with PMM2-CDG. Patients and cally heterogeneous group of genetic diseases caused by defects in the Methods: The ERGs and clinical reports of 8 genetically confirmed synthesis (CDG-I) or processing (CDG-II) of glycans that form glycocon- PMM2-CDG patients (aged 1-16 years), were reviewed retrospectively. jugates. In this work we described the clinical, biochemical and genetic ERGs were recorded with corneal electrodes to International Society of studies of three type I CDG patients owned to genetic defects in DPAGT1 Clinical Electrophysiology of Vision standards where possible, or in youn- gene (UDP-GlcNAc:dochicol phosphate N-Acetyl glucosamine-1- ger children with peri-orbital skinelectrodesandahandheldflash phosphotrnasferase, EC 2.7.8.15). All three patients presented a transferrin stimulator. type I profile and a broad spectrum of clinical symptoms. Protein analysis Results: The patients exhibited a wide range of clinical and neurological by western blotting and immunofluorescence staining of patient-derived severity; 3/8 patients had experienced acute crises at a young age. We found fibroblasts revealed a moderate reduction of DPAGT1 protein. Genetic ERG features of retinal on-pathway dysfunction, in the form of reduced b- studies allowed the identification of five novel nucleotide changes wave amplitude, in 5/8 patients. The remaining 3/8 patients had severe (p.Leu385Arg, p.Arg301Cys, p.Val264Gly, p.Phe110Ser and p.Leu120- photoreceptor dysfunction, which markedly attenuated both a- and b-wave Met) and one previously described mutation (p. Arg301His). Next, we have amplitudes. studied the endoplasmic reticulum (ER)stress using derived-patients fibro- Conclusions: ERG features of on-pathway dysfunction are evident in early blasts to provide clues to understand the pathophysiology of the disease. stages of PMM2-CDG. In children referred to ophthalmology with yet to Although the protein is the target of the tunicamycin, drug used to achieve establish, presumed, metabolic diagnosis, ERG findings can be helpful to cellular ER stress, these patients only had a slight increase of biochemical streamline investigations. Furthering our understanding of N-glycan inter- markers of stress and apoptosis. This may be explained by a moderate effect actions at the retinal photoreceptor synapse with on-bipolar cells may help of some mutations on the physiological enzymatic activity of DPAGT1 design interventions that ameliorate the progressive visual loss associated even though the patients presented a severe and broad clinical outcome. with PMM2-CDG.

P-291 GENETIC ANALYSIS OF CONGENITAL DISORDERS OF GLYCOSYLATION (CDG) PATIENTS USING CANDIDATE GENE GENOMIC CAPTURE AND NEXT GENERATION SEQUENCING Medrano C1 , Perez-Cerdá C1 ,Gómez-PuertaP2 , Desviat LR1 ,UgarteM1 , Pérez B1 1CEDEM-CBMSO, UAM-CSIC, CIBERER-Madrid,, Madrid, Spain 2CBMSO. Lab. Bioinformática,, Madrid, Spain

The clinical and subsequent biochemical diagnosis allow to classify the affected patients as CDG type I caused by defects in cytoplasmic and endoplasmic reticulum proteins or CDG type II caused by defects in the Golgi apparatus. Genetic diagnosis is required to identify the affected gene using a high-time consuming approach to sequence gene by gene. The aim of this study was to improve molecular diagnosis for CDG by developing a customized array. We present the results obtained by combination of a targeted in solution capture from Agilent and subsequent next generation sequencing using the Solid platform. In this work 16 barcoded patients have been analyzed (seven CDGI and nine CDGII). On average, coverage was 45 to 60 fold and we have detected close to one-hundred SNV per patient. The SNV were filtered excluding common variants and prioritizing in coding variants or consensus splice sites changes either previously reported (HGMD®) or classified as damaging by SIFT and polyphen 2. The initial results have allowed the identification of pathogenic mutations in DPAGT1, RTF1 and COG7 genes. In summary the development of next generation sequencing panels in the genetic diagnostic laboratory allows a most efficient genetic diagnosis compared with the conventional gene-by-gene sequencing. S114 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-294 P-295 DEFICIENCY OF SUBUNIT 6 OF THE CONSERVED OLIGOMERIC PGM1 DEFICIENCY: CLINICAL SPECTRUM OF A NEW GOLGI COMPLEX (COG6-CDG): SECOND PATIENT, DIFFERENT SECONDARY CDG PHENOTYPE Rymen D1 , Rymen D2 , Keldermans L1 , De Meirleir L3 , Van Schaftingen Goyens PJ1 , Huybrechts S2 ,DeLaetC1 , Bontems P3 , Rooze S1 , Souayah E4 , Matthijs G1 , Jaeken J2 H4 , Sznajer Y5 ,SturialeL6 , Garozzo D6 ,MatthijsG7 ,FersterA2 , Jaeken J8 1Cent Hum Gen, Univ Hosp Gasthuisberg, Leuven, Belgium 1Nutr Metab, Univ Child Hosp Queen Fabiol, Brussels, Belgium 2Cent Metab Dis, Univ Hosp Gasthuisberg, Leuven, Belgium 2Hemato-Oncology, Univ Child Hosp Queen F, Brussels, Belgium 3Dep Ped Neur and Metab, Univ of Brussels, Brussels, Belgium 3Gastroenterol, Univ Child Hosp Queen Fab, Brussels, Belgium 4Lab Phys Chem, Duve Inst, Cath Univ Louv, Brussels, Belgium 4Pediatrics, CHU St Pierre, Brussels, Belgium 5Med Genetics, Clin Univ St Luc, Brussels, Belgium Background: Congenital Disorders of Glycosylation (CDG) are caused by 6Ist Chim e Tecnol Polimeri, Catania, Italy deficient protein and lipid glycosylation. Recently, phosphoglucomutase 1 7Human Genetics, Univ Hosp Gasthuisberg, Leuven, Belgium (PGM1) deficiency was reported to have a combined type 1/2 serum 8Metab Diseases, Univ Hosp Gasthuisberg, Leuven, Belgium transferrin pattern on glycan analysis. PGM bidirectionally converts glucose-6-phosphate into glucose-1-phosphate. Its deficiency will lead to CDG-II due to defects in one of the eight subunits of the COG-complex secondary CDG due to a defect not only in glucosylation (type 1 pattern), represent a novel group of protein glycosylation disorders. We describe a but also in galactosylation (type 2 pattern) due to further processing of the 42-month-old girl with COG6 deficiency. First child of healthy consan- monosaccharide into UDP-galactose. Since PGM1 deficiency also could be guineous Moroccan parents, she presented at birth with dysmorphic fea- classified as a glycogen storage disease, one can predict that patients will tures: microcephaly, polydactyly, broad palpebral fissures, retrognathia and present with muscle weakness and hypoglycemia. anal anteposition. The clinical phenotype was further characterised by Methods: Candidates were selected on a clinical basis and screened for multiorgan involvement including mild psychomotor retardation, inflam- mutations in PGM1. Enzymatic activity of PGM1 was measured in leuko- matory bowel disease, micronodular liver cirrhosis, proximal tubulopathy, cytes. Clinical files of patients with confirmed PGM1-CDG were compared. associated with life-threatening and recurrent infections due to combined T- Results: Patients with confirmed PGM1 deficiency presented with various and B-cell dysfunction and neutrophil dysfunction. No bleeding tendency combinations of periodic hypoglycemia, muscle weakness, cardiomyopa- was observed. Mutation analysis showed the patient to be homozygous for thy, cleft palate, cerebral thrombosis, hepatopathy and growth delay. the c.G1646T mutation in the COG6 gene. She is the second reported Discussion: PGM1 deficiency is a novel cause of secondary CDG such as patient with COG6 deficiency and carrier of the same mutation as the index e.g. galactosemia. Patients with a combined type 1/2 pattern on glycan patient (Lübbehusen et al, 2010). Although both patients are homozygous analysis should be screened for mutations in PGM1, especially if they for the same mutation, they present a markedly different clinical picture. present with combinations of the above mentioned symptoms (mainly Indeed immunodeficiency as well as inflammatory bowel disease has not periodic hypoglycemia and muscle weakness). been described previously in patients with any COG-CDG. Mutation c.G1646T produces instability of mRNA rather than a degradation of mutated protein; individual factors might therefore interfere with the degradation speed of mRNA and contribute to the inter-individual phenotypical variation. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S115

P-296 24. Mitochondrial Disorders SRD5A3-CDG: AN IMPORTANT GROUP OF DOLICHOL- P-293 PHOSPHATE SYNTHESIS DEFECTS Rymen D1 ,RymenD2 ,KeldermansL1 ,QuelhasD3 , Snyder FF4 ,AlehanF5 , COMPLEX V DEFICIENCY AND MIS-ASSEMBLY: A CASE FOR Erol I5 , Mübeccel D6 , Gokçay G6 ,MitchellG7 , Matthijs G1 ,JaekenJ2 INCREASED INVESTIGATION UTILISING BLUE NATIVE GEL 1Cent Hum Gen, Univ Hosp Gasthuisberg, Leuven, Belgium ANALYSIS 1 1 2 1 2Cent Metab Dis, Univ Hosp Gasthuisberg, Leuven, Belgium Hargreaves IP , Chasalani A , Heales SJ , Land JM 1 3Dep Clin Biol, Inst Med Gen, Porto, Portugal Neurometabolic Unit, National Hospital, London, United Kingdom 2 4Dep Med Gen Ped, Univ Calgary, Calgary, Canada Div Metab Med, ICH, London, United Kingdom 5Div Ped Neurology, Baskent Univ, Ankara, Turkey The mitochondrial respiratory chain (MRC) is composed of four oxido-reductive 6Div Nutr and Met, Istanbul Univ, Istanbul, Turkey complexes which generate an electrochemical force across the inner mitochondrial 7Med Gen Div, Univ of Montréal, Montréal, Canada membrane. MRC Complex V (F1 FO-ATP synthase) utilises this force to generate ATP. Complex I and IV deficiencies are the most commonly diagnosed MRC Background: Dolichol-phosphate synthesis defects are an upcoming group defects and analyses can be undertaken in either fresh or frozen tissue. Classically of Congenital Disorders of Glycosylation (CDG). Recently defects in three Complex V deficiency was diagnosed polarographically in fresh tissue by observ- consecutive steps of this pathway have been discovered, i.e. DHDDS, ing enhanced substrate oxidation rates in the presence of oligomycin. Complex V SRD5A3 and DK1 respectively. Patients with SRD5A3-CDG are the larg- activity can also be assessed spectrophotometrically but this also requires fresh est group up to now. The clinical spectrum is characterized by psychomotor tissue and is difficult. We have applied Blue Native gel analysis (BN Gel) to frozen retardation, severe ocular involvement and ichthyosiform skin lesions. tissueandfoundthistobeareliablemethod for recognising defects in both Patients and methods: 88 CDG-Ix patients were screened for mutations in Complex V activity and assembly. Complex V deficiency has been noted in a SRD5A3 by Sanger-sequencing. Clinical information was retrieved and compared. range of clinical phenotypes including neonatal hypotonia, lactic acidosis, hyper- Results: The study revealed 10 patients from 8 families with 6 novel mutations trophic cardiomyopathy, developmental delay and peripheral neuropathy. We have in SRD5A3. All patients, except one who presented a homozygous missense become aware that Complex V deficiency may present with minimal phenotypes mutation, were diagnosed with loss of function mutations. Different degrees of such as myalgia, or peripheral neuropathy in the absence of lactic acidosis, in which psychomotor retardation were observed. Convulsions were present in 3 children. histology may only show alterations of mitochondrial morphology. We suggest BN Half of the children presented with important ocular involvement i.e. optic Gel should be applied to any unexplained mitochondrial disease patient with atrophy. Only one child had skin involvement with ichthyosiform lesions. otherwise "normal" investigations. Discussion and conclusion: Patients with SRD5A3-CDG represent a growing group within the CDG. Severe ocular involvement and ichtyosis are indeed more present in patients with defects in the dolichol-phosphate pathway. Though, the phenotype varies considerably, not only between different families, but also within families. P-298 A PROGRESSIVE LEUKOENCEPHALOPTHY DUE TO ALTERED LIPOIC ACID METABOLISM: MUTATIONS IN BOLA3 GENE Roche S1 , Imbard A1 , Boutron A2 , Brivet M2 , Ogier de Baulny H1 , P-297 Boespflug-Tanguy O3 , Benoist JF1 1 THROMBOTIC COMPLICATIONS IN PATIENTS WITH PMM2-CDG Unité Mal. Métaboliques, Hopital R Debré, Paris, France 2 Linssen M1 , Mohamed M1 , Wortmann S1 , Wevers RA2 , Lefeber DJ3 , Biochimie, Hôpital Bicêtre, Le Kremlin Bicêtre, France 3 Morava E1 Unité leucodystrophies, Hôpital R Debré, Paris, France 1Dep of Ped, RUNMC, Nijmegen, Netherlands 2LGEM, RUNMC, Nijmegen, Netherlands These two cousin patients were each born to consanguinous parents and died at 3Dep of Neur, Nijmegen, Netherlands the age of 8 months, after a rapidly progressive encephalopathy. Both patients displayed a diffuse demyelination on cerebral MRI and similar biochemical Many proteins regulating normal coagulation, including factor IX, factor profile. It comprised moderate hyperlactacidemia, hyperlactatorachia with XI, Antithrombin-III, Protein C and Protein S are deficient in CDG. Be- increased lactate/pyruvate ratios. Various intermediates of Krebs'cycle, espe- α– cause of the imbalance in pro- and anticoagulation factor, some patients cially ketoglutarate were increased in urine. In one patient the aminoacid μ develop acute vascular events, such as thrombosis. Identifying patients with analyses disclosed high levels of L-glycine in plasma (600 mol/l, nv: 174- μ μ a high risk for thrombotic events could prevent serious complications, 256), urine (4177 mol/mmol creat, nv: 110-356), and in CSF (43 mol/l, nv: unnecessary deaths or irreversible damage. < 16) while glycine accumulation was less severe in the other patient: plasma μ μ μ We performed a meta-analysis involving patients reported with PMM2-CDG 922 mol/l, urines 3622 mol/mmol creat, CSF 16 mol/l. This later patient between 1990 and 2012 and evaluated our own patient cohort with PMM2- had reduced pyruvate dehydrogenase complex (PDHc) activity (70 %) mea- CDG for vascular events. Out of the total of 176 patients one or more sured in leukocytes contrasting with normal activity in fibroblasts. coagulation factors were evaluated in 91 patients. We compared patients with These biological results associating hyperlactacidemia, hyperglycinemia α– and without a thrombotic event based on the alterations of the following and excretion of ketoglutatrate suggested a defect of lipoic acid metab- α– factors: PT, aPTT, Antithrombin-III, Protein C, Protein S, factor IX and XI. olism a common cofactor for PDHc, glycine cleavage system and Additionally we evaluated the family history and provoking events. ketoglutatrate dehydrogenase. Homozygous nonsense mutation found in From the group of 91 PMM2-CDG patients 11 had a thrombotic event, either BOLA3 (exon 2) confirmed the diagnosis. These cases underlie that BO- venous or arterial. Relatively high factor IX and XI activity were predictive for LA3 mutation is responsible for early progressive and severe encephalop- thrombosis. However low activity of anticoagulation factors did not correlate athy. Unfortunately, premature death did not give us the opportunity to test with thrombotic events. Prolonged PT and aPTT did not protect against lipoic acid therapy. thrombosis in the patients. Evident and easily preventable risk factors included immobility, elective surgical procedures and decreased fluid intake. S116 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-299 P-301 EXOME SEQUENCING AND FUNCTIONAL BIOLOGY REVEAL NOVEL MUTATION IN MTMETHIONYL-TRNA- NOVEL MITOCHONDRIAL DISEASE GENES FORMYLTRANSFERASE (MTFMT): A DEFECT OF Bonnen PE1 , Donti T1 , Besse A1 , Raghavan A1 , Lalani S1 , Scaglia F1 , MITOCHONDRIAL TRANSLATION WITH A MODERATE Craigen WJ1 , Graham BH1 PHENOTYPE 1Baylor College of Medicine, Houston, United States Freisinger PJK1 , Haack T2 , Kopajtich R2 , Mayr JA3 , Ahting U4 , Sperl W3 , Sperl W3 , Plecko B5 , Willichowski E6 , Strom T2 , Meitinger T7 , Mitochondrial disease is a diverse group of disorders that are estimated to Prokisch H2 occur with an incidence of 1/5,000. Approximately ninety percent of pediatric 1Dep. Pediatrics, Klinikum Reutlingen, Reutlingen, Germany onset cases are caused by high penetrance recessive mutations in the nuclear 2Inst. Hum. Genetics, Helmholtz-Zentrum, München, Germany genome, however after exhausting all available diagnostic tests most patients 3Dep. Pediatrics, Paracelsus Medical Univ, Salzburg, Austria remain without a molecular diagnosis. Using a combination of sequencing, 4Dep. Clin. Chemistry . Städt. Klinikum, München, Germany bioinformatics, and cellular and molecular biology we have discovered path- 5Dep.Neurol, Kinderspital, Univ. Zürich, Zürich, Switzerland ogenic mutations. We identified pediatric patients who have been pre-screened 6Dep. Ped. Neurol. , Univ. Medicine, Göttingen, Germany and shown to be negative for mitochondrial and known nuclear gene candi- 7Inst. Hum. Genetics, Techn. Universität, München, Germany dates but have overwhelming evidence supporting a mitochondrial disease diagnosis. A battery of mitochondrial functional assays have been performed Exome sequencing has contributed to the identification of proteins involved on all samples with available tissues including electron transport chain anal- in mitochondrial transcription or translation and their pathogenic role in ysis, FACS based analysis of relative mitochondrial membrane potential and combined respiratory chain complex (RCC) deficiencies. Recently it was mitochondrial DNA copy number. Exome sequencing has been completed on shown, that mutations in MTFMT cause decreased formylation of Met- 20 patients. We have discovered and validated the pathogenic mutation in five tRNAMet, which is crucial for initiation of mitochondrial translation. patients. A causal relationship has been established using cDNA complemen- (Tucker EJ. 2011, Haack T 2012). tation and mitochondrial functional assays. This work has resulted in the We report a 6-year old boy presenting from infancy with microcephaly and discovery of novel disease genes, improved molecular diagnosis, and new slowly progressive muscular hypotonia, mild ataxia and moderate develop- insights into the pathogenetic mechanisms underlying mitochondrial disease. mental delay. Lactic acidemia and Leigh-like MRI-changes were noted at 3 years. Muscle biopsy revealed a reduced RCC I activity (0,09 mU/mU CS, normal 0.14-0,35). P-300 Exome sequencing identified a heterozygous mutation c.[626 C>T], p.[Ser209Leu] in MTFMT. This mutation has been found in all 4 previously SURF1 DEFICIENCY: NATURAL HISTORY STUDY OF A UK COHORT described patients (Tucker EJ 2011; Haack T 2012). Sanger sequencing of Wedatilake Y1 ,BrownRM2 , McFarland R3 , Chakrapani A4 ,MorrisAA5 , the MTFMT gene detected a novel heterozygous nonsense mutation, Champion MP6 , Jardine PE7 ,DobbieA8 ,HanrahanD9 , Simmons L4 , c.[73 C>T], p.[Gln25*] predicting a premature truncation of >90 % of Collins JE10 , Brown GK2 ,RahmanS1 the MTFMT protein. 1UCL Institute of Child Health, London, United Kingdom This patient, like the 4 others shows a relatively mild, slowly progressive, 2Dept of Biochem, University of Oxford, Oxford, United Kingdom pure neurologic phenotype. This might be explained by a mechanism 3Newcastle University, Newcastle upon Tyne, United Kingdom partially compensating for MTFMT-deficiency. Furthermore the presence 4BCHIMD Birmingham Children's Hospital, Birmingham, United Kingdom of a common mutation p.[Ser209Leu] in all patients (homozygous or 5Central Manchester University Hospital, Manchester, United Kingdom compound heterozygous) could indicate a central role of this position in 6Evelina Children's Hospital, London, United Kingdom MTFMT. 7Bristol Royal Hospital for Children, Bristol, United Kingdom 8Yorkshire Regional Genetics Service, Leeds, United Kingdom 9Royal Belfast Hospital fr Sick Children, Belfast, Ireland 10Great Ormond Street Hospital, London, United Kingdom

Background: Mutations of SURF1, encoding an assembly factor for cytochrome c oxidase (COX), are the most frequently identified cause of COX- deficient Leigh syndrome (subacute necrotising encephalomyelopathy). Objectives: To describe the natural history of SURF1 deficiency in a large cohort. Methods: Retrospective multi-centre case notes review of all patients diagnosed in the UK since 1998, when SURF1 mutations were first linked to Leigh syndrome. Results: Fifty-three patients with SURF1 mutations were identified. Clinical data were ascertained for 38 cases. Clinical features included hypotonia (90 %), developmental delay/regression (84 %), recurrent vomiting (79 %), poor growth (74 %), respiratory impairment (63 %), ataxia (47 %), hypertrichosis (32 %), movement disorder (27 %), peripheral neuropathy (18 %) and optic atrophy (18 %). MRI brain revealed basal ganglia and brain stem involvement except for two patients with leukodystrophy. The most prevalent mutation was insAT/delTCTGCCAGCC in exon 4 (14 homozygous, 9 heterozygous). Median age of death was 34 months (range 4 months-14 years) and was frequently due to respiratory failure. Six patients had a milder course and survived >10 years of age Conclusions: SURF1 deficiency usually causes early onset severe neurological disease and mortality before 10 years of age. Early recognition of the clinical featureswouldleadtomorerapidgenetic diagnosis of affected children. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S117

P-302 P-304 A CASE OF PYRUVATE CARBOXYLASE DEFICIENCY WITH DGUOK RELATED MITOCHONDRIAL DEPLETION SYNDROME NEW NEUROIMAGING FEATURES IN A CHILD WITH AN EARLY DIAGNOSIS OF GSDS Kasapkara C S1 ,AydınK2 , Hasanoglu A1 , Kucukcongar A1 , Ezgu F S1 , Kasapkara C S1 , Tumer L2 , Kucukcongar A1 , Hasanoglu A1 , Seneca S3 , Tumer L1 De Meirleir L4 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 2Div Ped Neurology, Gazi Univ Hosp, Ankara, Turkey 2Div Metab Dis, Gazi Univ Hosp, Ankara, Turkey 3Vrije Univ Brussel, Center for Med Gen, Brussels, Belgium Pyruvate carboxylase (PC) is a biotin-containing, nuclear encoded mitochondrial 4Vrije Univ Brussel, Pediatric Neurology, Brussels, Belgium protein that catalyzes the first step of gluconeogenesis by converting pyruvate to oxaloacetate. PC deficiency is a rare autosomal recessively inherited disorder Mitochondrial DNA (mtDNA) depletion syndrome encompasses a clinical- presenting with lactic acidaemia and neurological involvement. We report a ly heterogeneous group of disorders sharing marked reduction of mtDNA 13 months old child diagnosed with neuroradiological aspects and clinical presen- copy number in one or more tissues. DGUOK (deoxyguanosine kinase, tation. She was admitted to our hospital for severe psychomotor retardation. dGK), MPV17, POLG1(polymerase gamma), C100RF2 and RRM2B muta- Biological tests revealed increased levels of alanine 773 (nv: 144-348) and gluta- tions are the major causes of mitochondrial DNA (mtDNA) depletion mine: 124 (nv: 24-68) on plasma amino acid profile and showed persistent hyper- associated with hepatocerebral syndromes and mutations in DGUOK are lactacidaemia worsening on fasting with increased L/P ratio L/P: 18, normal<15) the most commonly reported. Deoxyguanosine kinase catalyzes the first and normal ketonemia. A brain MRI showed ventriculomegaly, cerebral dysgen- step of the mitochondrial deoxypurine salvage pathway, the phosphoryla- esis and corpus callosum agenesis. These MRI findings and biochemical test results tion of purine deoxyribonucleosides. The two forms of DGUOK deficiency prompted us to measure the PC enzyme activity. Pyruvate carboxylase in leuko- are a hepatocerebral mitochondrial DNA depletion syndrome and isolated cytes was found 0.3 nmol/min/mg Protein (nv: 5-15). The pyruvate carboxylase hepatic disease. Herein, we describe a homozygous stop codon mutation in activity in leukocytes was decreased, which was compatible with pyruvate carbox- the DGUOK gene found in a male patient with a severe clinical phenotype ylase deficiency. The reported neuroradiological findings in PC deficiency are who died of liver failure at the age of four months. periventricular leukomalacia, delayed myelination and subcortical leukodystrophy. In conclusion, we present additional MRI findings. Our data suggest that when investigating a developmentally delayed child with such a brain MRI aspect and clinical findings, one should search for PC deficiency. P-305 PROFOUND LACTIC ACIDOSIS, METHYLGLUTACONIC ACIDURIA AND HYPERTROPHIC CARDIOMYOPATHY: THINK P-303 TMEM70 Abbot V1 , Deshpande C2 , Alston CL3 , Simpson J4 , Agalou S5 , Taylor SEVERE EARLY ONSET ETHYLMALONIC ENCEPHALOPATHY RW4 , Champion MP1 WITH WEST SYNDROME 1Department of IMD, Evelina Child Hosp, London, United Kingdom Papetti L1 , Spalice A1 , Nardecchia F1 , Papoff P2 , Nicita F1 , Fabiana U1 , 2Clinical Genetics Dept. Guy's Hospital, London, United Kingdom Leuzzi V1 3Wellcome Trust Mitochondrial research, New Castle upon Tyne, United 1Dep of Pediatr&Child Neur,Sapienza Univ, Rome, Italy Kingdom 2Dep Pediatr, Sapienza University, Rome, Italy 4Cardiology dept. Evelina Child Hosp, London, United Kingdom 5IMD lab, Biochem Scien, St Thomas's Hosp, London, United Kingdom Case report: A 4-months girl presented with bilateral and symmetrical spasms of the trunk and extremities. Interictal-EEG showed hypsarrhyth- Background: TMEM70 mutations are a recognised cause of isolated mito- mia. Brain MRI revealed abnormal signal intensity involving basal ganglia chondrial ATP synthase (complex V) deficiency. The phenotype comprises of and the brainstem. Laboratory study showed increased serum and CSF severe congenital lactic acidosis, 3-methylglutaconic aciduria (MGA), hyper- lactate and ethylmalonic acid (EMA) in blood (6.6 μmol/L) and urine trophic cardiomyopathy (HCM), encephalopathy and dysmorphic facies. Most (247.4 mmol/mol creatinine). Skeletal muscle biopsy showed severe COX cases are homozygous for a c.317-2A>G splice site mutation and share a deficiency. The genetic analysis confirmed ETHE1 mutations1 (p.R163Q4 common Roma ancestry. We report 3 cases, born to parents of Eastern and the two novel p.Q208deletion and p.I114F aminoacid change). She European descent, diagnosed on basis of a highly suggestive phenotype. started carnitine, riboflavin and antiepileptic drugs, and therapy with met- Methods: Case 1- born at 30 weeks to consanguineous parents. HCM was ronidazole (30 mg/Kg/day) and N-acetylsysteine (100 mg/Kg/day)2. By the noted in the neonatal period. She presented at 5 months with profound lactic age of five months petechiae and acrocyanosis became manifest while rare acidosis, hypoglycaemia and hyperammonaemia following a diarrhoeal illness. episodes of diarrhoea were observed. Subsequent EEGs showed progres- Case2- antenatal diagnosis of potential Potter's sequence. Ultrasound sive pseudorhythmic suppression-burst. The girl died at the age of 9 months showed oligohydramnios, growth retardation and renal anomalies. Dysmor- after acute metabolic acidosis triggered by respiratory infection. phic features were noted at birth, developing severe lactic acidosis and Discussion: The present is the first case of EE with West syndrome at onset hyperammonaemia at 24 hours. Echocardiography revealed HCM. while the classical symptoms (recurrent petechiae, acrocyanosis, chronic Case 3- collapsed at 36 hours with severe lactic acidosis, encephalopathy, diarrhoea) appeared later and remained extremely mild. Moreover the hypoglycaemia and mild cardiac dysfunction. severe COX deficiency and lactic acidosis dominated the biochemical Results: Prominent MGA was present in all cases. Sequencing of TMEM70 picture of our patient, while EMA levels remain relatively low. Finally, identified the homozygous founder c.317-2A>G mutation confirmed reces- metronidazole and N-acetylsysteine proved to be ineffective in preventing sive inheritance, facilitating prenatal diagnosis in one family. the progression of the disease. Conclusion: GA, a degree of hypertrophic cardiomyopathy and profound lactic acidosis should prompt TMEM70 screening and may negate requirement for a diagnostic muscle biopsy. S118 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-306 P-308 NOVEL PHENOTYPE ASSOCIATED WITH OPA1 MUTATIONS? THE GENETIC ORIGIN OF LEIGH SYNDROME IN BELARUS Honzík T1 , Tesařová M1 , Stránecký V2 , Kratochvílová H1 , Hájková Z1 , Gusina N.B.1 , Zimovina T.S.1 , Demidovich T.V.1 , Zinovik A.V.1 , Sládková J1 , Spáčilová J1 , Hansíková H1 , Hartmannová H2 , Nosková L2 , Danilenko N.G.2 Piherová L2 , Lalonde E3 , Majewski J3 , Kmoch S2 , Zeman J1 1Centre "Mother and child', Minsk, Belarus 1Dep Pediatrics, Univ Hosp, Charles Univ, Prague, Czech Republic 2National Academy of Sciences, Minsk, Belarus 2Inst Inherit Met Dis, Charles Univ, Prague, Czech Republic 3Dep Hum Gen, McGill Univ Health Center, Montreal, Quebec, Canada Objectives: Leigh syndrome is a neurodegenerative mitochondrial disorder with wide genetic heterogeneity. The knowledge of genetic origin of Leigh Mutations in OPA1 gene were found to be responsible for autosomal phenotype is important for appropriate genetic counselling because some dominant optic atrophy. Moreover OPA1 mutations may be responsible forms are the consequence of nuclear genes defects and some are the results for a continuum of phenotypes. In Caucasian non-consanguineous family, of mtDNA substitutions. It's also the basis for prenatal diagnosis in families 3 boys were born with lactic acidosis, hypotonia and apathy. Patient 1 died at risk. on day 9. In his younger brothers (died aged 10 and 12.5 months), vision Patients and Methods: A group of 41 patients with clinical signs of Leigh/ and hearing impairment, brain atrophy and hypertrophic cardiomyopathy Leigh-like phenotype were examined. The methods included MS-MS, were present from the early infancy. In patient 3, Leigh syndrome was enzyme assays, mtDNA point mutations analysis, fragment assay and direct observed at autopsy. Metabolic examination revealed lactic acidosis, in- sequencing of SURF1 gene. creased excretion of KC intermediates and 3-methylglutaconic acid. Muta- Results: The most common cause of Leigh syndrome was SURF1 gene tions in mtDNA, TMEM70, TAZ, SCO2, SCO1 were excluded. Genome- mutations, detected in 11 patients. The most common mutation was wide copy number analysis was unremarkable. Whole-exome sequencing 845delCT (12 of 22 mutant alleles), allele IVS8-1 G>C was detected in 2 revealed heterozygous mutations: p.E354del (novel mutation) and of 22 mutant alleles of SURF1 gene. In one case pyruvate carboxylase p.R590W in OPA1. Steady-state levels of OPA1 isoforms were significantly deficiency appeared the cause of Leigh phenotype. No one case of mtDNA decreased in muscle, heart and frontal core. Pronounced reduction of short T8993C/G substitution was detected. OPA1 isoforms (S3, S4, S5) was observed in patients fibroblasts compared Conclusions: It looks like in Belarus Leigh syndrome is a rather common to parents. To conclude, despite on going analyses to confirm OPA1 cause of neurodegenerative diseases in children. The direct sequencing of mutations as cause of the disease, symptoms described in our patients mtDNA must be included into the diagnostic algorithm for this disease to may enlarge phenotypic variability associated with OPA1. Supported by increase the diagnostic effectiveness. PRVOUK-P24/LF1/11, NT12165-5/2011 and IGA NT 13114-4/2012.

P-307 P-309 MITOCHONDRIAL CARDIOMYOPATHY IN JAPANESE CHILDREN. MALNUTRITION AND NUTRITION-RELATED PROBLEMS IN Fukuoka Shoji1 , Murayama Kei1 , Fushimi Takuya1 , Kawachi Emi1 , ADULTS WITH MITOCHONDRIAL DISEASES. Ajima Masami1 , Mori Masato2 , Okazaki Yasushi3 , Takayanagi Masaki1 , Zweers H1 , Wanten G1 , Smits B1 , Janssen M1 Ohtake Akira4 1Radboud Univ Nijmegen Med. Centre, Nijmegen, Netherlands 1Div Metab Dis, Child Hosp, Chiba, Japan 2Dep Pediatr,Jichi Med Univ, Tochigi, Japan Background: The treatment of Mitochondrial Diseases (MD) is mainly 3Research Cent Genom Med,Med Univ, Saitama, Japan supportive. By describing the nutritional characteristics of this patient group 4Dep Pediatr, Med Univ, Saitama, Japan we hope to develop better nutritional intervention strategies. Methods: An inventory was made of anthropometric data of 77 adult MD Background: Cardiomyopathy (CM) is a frequent manifestation of mito- patients with the m3243AG mutation or CPEO and their nutrition-related chondrial respiratory chain deficiency (MRCD). We reveal the clinical problems. In 10 patients we performed a Nutritional Assessment and con- manifestation and biochemical, pathological and molecular findings in ducted a nutritional survey. Japanese patients with mitochondrial cardiomyopathy. Results: MD patients were underweight more frequently (26 %) and Subject and Methods: Of 624 candidate patients, 218 patients were diagnosed to overweight less frequently (29 %) when compared with the average popu- have MRCD based on Bernier et al. criteria. We classified seventeen patients lation (2 % underweight, 48 % overweight). Patients had a reduced Fat Free who had CM predominantly as mitochondrial cardiomyopathies and summa- Mass (90 %) and a higher Fat Mass (130 %). Their energy expenditure was rized them in clinical manifestation and biochemical, histological and molecular lower (1679 Kcal SD 318) than recommended (2348 Kcal) and calculated findings. energy requirement (1909 Kcal SD 247). There was a discrepancy between Results: Of the 17 patients with CM, eleven patients had hypertrophic CM, four measured energy expenditure and dietary intake. Gastrointestinal symptoms had dilated CM, one had left ventricular non-compaction and one had restrictive (48 %) and dysphagia (41 %) were the most common diet-related com- CM. Thirteen patients were died, and eleven had died by one year of age. plaints. Patients with dysphagia had a lower BMI (20.4) compared with Enzyme activities of fibroblast, skeletal muscle, liver and myocardium were patients without dysphagia (24.3). each decreasing in 7/9, 6/8, 7/12, and 13/13, respectively. Complex I (47 %) Conclusion: Despite a reduced energy requirement MD patients have an was the most common enzyme defect. increased risk for malnutrition. Frequent gastrointestinal problems, dyspha- Pathological study was performed in 8 patients and found the myofibrillar structural gia and fatigue are probable contributing factors. Discrepant nutrient intake disarray and greatly increased numbers of abnormal mitochondria in their heart. and demand suggests that nutritional intervention is promising. There were no mtDNA mutations in spite of whole sequence of mtDNA. One patient had mutation of TAZ gene. Discussion: CM is common and poor prognosis in MRCD, and has several pathological variation. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S119

P-310 P-311 SPASTIC DIPLEGIA IN A NOVEL FORM OF NON-KETOTIC NOVEL DOMINANT POLG MUTATIONS CAUSE DISTAL HYPERGLCYCINAEMIA DUE TO GLRX5 DEFICIENCY MYOPATHY Bhattacharya K1 , Gupta S1 , Wilcken B1 , Procopis P1 , Spector E2 , Pitceathly RDS1 , Tomlinson SE2 , Hargreaves I3 , Bhardwaj N4 , Holton Creadon-Swindel G2 , Aicher J2 , Thorburn D3 , Scharer G2 , Van Hove J2 JL1 , Evans J5 , Smith C5 , Fratter C5 , Woodward C6 , Sweeney MG6 , 1Children's Hospital at Westmead, Sydney, Australia Hanna MG1 , Rahman S7 2Dept Ped, Clin Gen & Metab Univ Colorado, Denver, United States 1MRC Cen Neur Dis, UCL Inst Neurol, London, United Kingdom 3Murdoch Children's Research Institute, Melbourne, Australia 2Syd Med Scho, Uni of Syd, Sydney, Australia 3Neuromet Unit, Nat Hosp Neur Neurosurg, London, United Kingdom Objective: To describe the phenotype in a novel genetic disorder affecting both 4Dub Neuro Centre, UCL Inst Chil Heal, London, United Kingdom glycine cleavage (GCE) and pyruvate dehydrogenase complexes (PDHC). 5Oxf Reg Molec Gen Lab, Oxf Rad Hosp, Oxford, United Kingdom Case Presentation: Two girls with normal development from apparently unre- 6Neurogen Unit, Nat Hosp Neur Neurosur, London, United Kingdom lated families originating from the same village in North Lebanon, presented in 7Mito Res Group, UCL Inst Chil Heal, London, United Kingdom childhood with progressive spastic diplegia. Plasma and CSF glycine levels were elevated with normal CSF: plasma ratios. Cranial MRI revealed leuko- Background: Mutations in the nuclear-encoded mitochondrial DNA main- dystrophy (Case 1- 2.5 years) and small areas of hyper-intense signal in the tenance gene POLG, encoding the catalytic subunit of DNA polymerase deep frontal white matter for Case 2 (8 years). Spinal MRI in both cases gamma, present a considerable diagnostic challenge since they exhibit showed large linear T2 hyper-intense lesions in the posterior cervical cord. highly variable clinical phenotype and age of onset. Case 1 was confirmed to have hepatic deficiency in the glycine cleavage Objective: To report a novel phenotype, namely distal myopathy, associ- enzyme system. Both cases had deficiency of pyruvate dehydrogenase com- ated with dominant POLG mutations. plex identified on cultured skin fibroblasts A hypothesis of defective lip- Cases reports: Two unrelated patients presented with a distal, predomi- oylation was proposed as causing both deficiencies. nantly upper limb, myopathy. Onset was in early adolescence in the first Case 1, now 11 years old, is able to ambulate with the help of crutches and case, and in the fourth decade in patient 2. Patient 1 had a ragged-red fibre has mild learning difficulties. Case 2, now 10 years old, ambulates inde- myopathy association with multiple mitochondrial DNA deletions. pendently with orthotics and has normal intellect. Results: Sequence analysis of all exons and exon-intron boundaries of POLG Further work presented by Van Hove and colleagues demonstrated defi- revealed novel dominant heterozygous missense POLG mutations (Leu896Arg ciency of GLRX5 in iron-sulur cluster biogenesis leading to combined in patient 1 and Tyr951His in patient 2) within the catalytic polymerase domain PDHC and GCE deficiencies in both cases. of the protein. Multiplex ligation–dependent probe analysis was used in both cases to exclude large-scale rearrangements in POLG on the other allele. Conclusions: These findings highlight the diverse clinical spectrum of POLG- related mitochondrial disorders, which may present with distal myopathy before more typical features of mitochondrial disease become apparent. We suggest that POLG mutations should be excluded prior to muscle biopsy to investigate mitochondrial dysfunction in cases of distal myopathy. S120 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-312 P-314 ATYPICAL MANIFESTATIONS OF A PATIENT WITH EPILEPTIC PHENOTYPES IN CHILDREN WITH PROVEN MITOCHONDRIAL MYOPATHY, ENCEPHALOPATHY, LACTIC MITOCHONDRIAL DISEASE AND EPILEPSY ACIDOSIS AND STROKE-LIKE (MELAS) EPISODES Illingworth MA1 , Boyd S1 , Varadkar S1 , Rahman S2 Castro-Chaves P1 , Nunes A2 ,SantosAraújoC2 , Oliveira A2 ,BecoA2 , 1Neurosciences, GOSH, London, United Kingdom Quelhas D3 , Vasconcelos C4 ,PestanaM2 , Teresa Cardoso M1 , Leão Teles E4 2Mitochondrial Research Group, UCL, ICH, London, United Kingdom 1Adults Metabolic Disease Unit, CH S Joao, Porto, Portugal 2Nephrology Department, CH S Joao, Porto, Portugal Aims: To describe epileptic phenotypes and response to treatment in children 3INSA / Centro de Genética Médica, Porto, Portugal with biochemically/genetically confirmed mitochondrial disease and epilepsy. 4Metab Dis Unit, Paed Depart, CH S Joao, Porto, Portugal Methods: Retrospective case-note review of children attending a tertiary referral centre. Mitochondrial disorders (MD) have protean clinical manifestations. Results: 44 children were identified. 16/44 had genetic confirmation; others We describe a 29 years-old woman with short stature, migraine and normal had deficiency of one/more respiratory chain enzyme complexes (most often psychomotor development until the age of 16 years-old when depressive and Complex IV (COX) (21/44)). Genetic defects included mitochondrial DNA obsessive-compulsive behaviour was noted. At the age of 23, a stage 4 chronic deletions, point mutations, POLG mutations and RARS2 mutations. Seizures kidney disease of unknown aetiology was diagnosed. Five years later, she was were the manifesting complaint in 16/44. Presenting semiology included admitted with diarrhoea, emesis and persistent hyperlactacidaemia. Afterwards, she infantile spasms (7/44), status-epilepticus (SE) (5/44), febrile SE (2/44), en- developed seizures and focal neurological signs. A mutational study in a peripheral cephalopathy (6/44). Subsequent seizures included EPC, GTCs, absences and blood sample was performed and the MELAS-related mutation mt.3243A>G was myoclonic seizures. Initial EEG patterns included, background slowing (11/ identified. Later, brain MRI showed areas of hyper intensity in T2/FLAIR involv- 44), hypsarrhythmia (5/44), high-amplitude semi-rhythmic slowing with runs ing precentral circunvolutions and occipital cortex bilaterally and a muscle biopsy of rhythmic delta (2/44) and discontinuous burst-suppression (2/44). Subse- showed red ragged fibers. Due to renal function worsening and acute respiratory quent patterns included, excess slowing (7/44) with posterior emphasis (2/44), failure, she began peritoneal dialysis, ventilatory support, nutritional care and rhythmic high-amplitude epileptic activity (3/44), ESES (2/44). antioxidant treatment. She recovered well but, two months later persistent head- Treatment included: aches were noted and an extensive cerebral vein thrombosis was identified. A - Levetiracetam (11/44) prothrombotic study was negative and she began anticoagulation. - Sodium valproate (13/44) This clinical case illustrates the challenging difficulties involved in the management - Steroids (11/44) of MD in the adult patient with concomitant end stage kidney disease and draws - Ketogenic diet (KD)(3/44) attention for the role that peritoneal dialysis may have in the treatment of this disease. 7/44 (5/7 with COX deficiency) became seizure free. 13/44 children died, within fourteen years from initial seizure. Conclusions: Seizures are common in mitochondrial disease. COX deficiency was the most common biochemical defect, although seizures infre- quently recurred following the primary episode. Favourable responses to P-313 Levetiracetam and KD were not observed. SEVERE MITOCHONDRIAL ENCEPHALOPATHY CAUSED BY TWO LEBERS HEREDITARY OPTIC NEUROPATHY MUTATIONS 1 1 2 2 Mikhaylova S , Volkova I , Itkis Yu , Zakharova E P-315 1Russian Children hospital, Moscow, Russian Federation EARLY MANIFISTATION OF THE LBSL SYNDROME 2Research Center for Med Genetics RAMS, Moscow, Russian Federation Grechanina OYa1 , Zdybskaya OP1 1Ukrainian Institute of Clinical Genetics, Kharkiv, Ukraine CNS involvement in LHON (Leber's hereditary optic neuropathy) is generally limited to the visual pathway. LHON is mainly associated with three Background: Leukoencephalopathy with brain stem and spinal cord in- common mitochondrial DNA (mtDNA) mutations. We present a 10-year- volvement and lactate elevation (LBSL) is an autosomal recessive disease. old female with normal neurodevelopment until 9 years of age, when appear Mutation in gene DARS2 is associated with LBSL-syndrome. refractory partial and generalized tonic-clonic epileptic seizures, cognitive Case report: A boy of 1 year 5 months old presented with delayed motor decline, visual impairment and epileptic aphasia. Axial hypotonia, ataxia and speech development and excessive body weight (16 kg). Weight at birth and bilateral pyramidal signs became evident. Fundus examination revealed was 3300 g. He had hypotonia , nystagmus and changes on EMG sugges- bilateral optic atrophy. MRI showed lesions in the right thalamus region and tive of neuropathic type disorder or a myopathic syndrome. Brain MRI in in the brainstem. Her mother has optic nerve atrophy since childhood and T1, Т2 and FLAIR-mode showed that cerebral white matter and the cere- no other neurological symptoms. Patient was screened for mtDNA common bellum was affected. The karyotype was 46,XY. The speed of phosphory- mutations using MLPA analysis and sequencing. Unexpectedly we found lation was lowered 111,8 mmol/min. Partial analysis of the gene sequencing two mtDNA mutations in our patient-11778 G>A and 14484 T>C in using the DARS method revealed a heterozygous mutation s492+2 T-S in homoplasmic state. Her mother and grandmother also have both mutations the 5th locus of the gene. . (Research was carried out in the medico-genetic (14484 T>C- homoplasmic, 11778 G>A– 90 % and 50 % respectively). scientific center, Moscow). Several reports about association between typical LHON mtDNA mutation Conclusion: As a rule, the disease presents between ages of 3-15 years. In and other mitochondrial encephalopathies have been reported. Despite of the pre symptomatic phase of the disorder psychomotor and speech devel- strong association of some mtDNA mutations with mitochondrial syn- opment corresponds to the age, later on as it progresses movement impair- dromes, the complex relationship between genotype and phenotype still ment increase, which leads to the disability of patients from the second - remains unclear. Our observations show that typical LHON-associated fourth decade of their life. mutations can lead to severe mitochondrial disease and diagnostic panel for mitochondrial diseases should include all common mtDNA mutations. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S121

P-316 P-318 FUMARASE DEFICIENCY IN TWO PORTUGUESE PATIENTS A COMBINED TREATMENT APPROACH TO COMPLEX I Beleza-Meireles A1 , Garcia P2 , Grazina M3 , Alfaiate C2 , Quelhas D2 , DEFICIENCY DUE TO A NOVEL ACAD9 MUTATION Diogo L2 Burlina AB1 , Bordugo A1 , Del Rizzo M1 , Cazzorla C1 , Cerutti A1 , 1Dep Medical Genetics, Paed Hosp, Coimbra, Portugal Zeviani M2 2Dep Metab Dis, Paed Hosp, Coimbra, Portugal 1Div Metab Dis, Univ Child Hosp, Padua, Italy 3Centre for Neuroscience, Coimbra Univ, Coimbra, Portugal 2Neurological Institute C. Besta, Milan, Italy

Background: Fumaric aciduria is a very rare autosomal recessive disorder of Background: Acyl-CoA dehydrogenase 9 (ACAD9) mutations have been the tricarboxylic acid cycle caused by mitochondrial fumarate hydratase recently reported as an important cause of complex I deficiency and treat- deficiency due to mutations in FH gene. It is characterized by severe neuro- ment with riboflavin has been recently proposed. logical impairment in early childhood. Milder cases have been described. Case report: We report a baby girl who showed after 24 hours sudden Methods: We describe two Caucasian girls with fumarase deficiency, respiratory distress with normal cardiac function. Metabolic evaluation diagnosed after fumaric aciduria detection in the diagnostic workup of showed a severe metabolic acidosis (pH 6.51,BE -31, lactate 19 mEq/l) development delay. and massive ketonuria by organic acids analysis. Despite high bicarbonates, Results/Case-Report: Patient 1, 11yo, presents severe developmental de- carnitine and ubiquinone, she had a persistent acidosis (lactate 10 mmol/l). lay, growth retardation, cerebral atrophy and persistent hyperlactacidaemia At 6 months of age, clinical evaluation showed growth parameters <3rd (≤7,3 mM). Patient 2, 6yo, has mild developmental delay, gait imbalance, percentile, moderate developmental delay and mild left ventricle hypertro- epilepsy, delayed myelination and corpus callosum hypoplasia and inter- phy. Severe reduction in the complex I respiratory chain activity (1.3 nmol/ mitent hyperlactacidaemia (≤2,74 mM). Family history was irrelevant, min mg) on muscle was detected. Molecular analysis revealed a new except for miscarriage in the first pregnancy of patient 2' mother. Fumarase mutation: ACAD9 compound heterozygosis for the missense mutations activity in lymphocytes was decreased in both (7,61 % and 6,71 % of the c.857 T>C/c.1240 C>T. High dosage of riboflavin was administered with- normal medium, respectively). The analysis of the coding sequence of FH out modification of lactate level. Then dicloroacetate (50 mg/kg/day) was gene in the two girls evidenced no mutation. administered, with a consistent lactate levels reduction. After 16 months of Conclusions: It is possible that mutations in non-coding sequences of FH combined treatment, we obtained a stabilization of clinical cardiomyopathy gene could be disclosed or that fumarase deficiency is secondary to other and improvement in weight and mental development. disorders, namely those of the mitochondrial respiratory chain. Although Conclusions: In patients with complex I due to ACAD9 assembly factor there is no specific treatment for either, molecular diagnosis is essential for defect with severe acidosis combined treatment, riboflavin and DCA, adequate genetic counseling. ameliorates the course of the disease.

P-317 P-319 LEUKOENCEPHALOPATHY WITH BRAIN STEM AND SPINAL MITOCHONDRIAL ELECTRON TRANSPORT CHAIN CORD INVOLVEMENT AND LACTATE ELEVATION (LBSL) DYSFUNCTION ASSOCIATED WITH STATIN THERAPY HIGH OUTCOME VARIABILITY AMONG TWO SIBLINGS Hargreaves IP1 , Heales SJR2 , Duncan AJ3 , Land JM1 Tylki-Szymanska A1 , Jurkiewicz E1 , Zakharova Y2 , Bobek-Billewicz B3 1Neurometabolic Unit, National Hospital, London, United Kingdom 1Dept Metab Dis, Child Mem Inst, Warsaw, Poland 2Chem Path, Great Ormond St. Child Hosp, London, United Kingdom 2Nat Res Centre Med Gen, Moscow, Russian Federation 3Inst. Child Health, London, United Kingdom 3Centre Oncol, Gliwice, Poland To date, the patho-physiological mechanisms of statin [3-hydroxy-3-meth- Leukoencephalopathy with brain stem and spinal cord involvement and lactate ylglutaryl coenzyme A (HMG-CoA) reductase inhibitor] induced myotox- elevation is a rare disorder caused by a mutation in the gene encoding a icity are still not clearly understood. In order to investigate the potential mitochondrial aspartyl-tRNA synthetase DARS2. The clinical features include involvement of mitochondrial electron transport chain (ETC) dysfunction in childhood or juvenile onset, slowly progressing ataxia, spasticity, and dorsal the myotoxicity associated with statin therapy, assessment was made of column dysfunction with mild cognitive deficit. MR imaging shows a highly ETC activity and ubiquinone status in two patients experiencing myopathy characteristic leukoencephalopathy with multiple long tract involvement. MR following treatment with simvastatin (40 mg/day) and cyclosporin (patient spectroscopy shows lactate elevation. Different disease outcomes were noted 1) and simvastatin (40 mg/day) and itraconazole (patient 2). Analysis of among two brothers, aged currently 42 and 35 years. In the elder brother skeletal muscle biopsies revealed a decreased ubiquinone status (77 and cognitive abilities were normal, seizures were observed at the age of 6 months 132; reference range: 140-580 pmol/mg) and cytochrome oxidase (complex and first symptoms of ataxia only at the age of 12. From the age of 13 he IV) activity (0.006 and 0.007 reference range: 0.014-0.034). To assess statin became wheelchair dependent. His younger brother presented severe motor treatment in the absence of possible pharmacological interference from retardation form the age of 7 months he was never able to walk alone. He never cyclosporin or itraconazole, primary astrocytes were cultured with lovastat- had seizures. MRI showed typical for LBSL changes. Surprisingly, they are in (100 microM). Lovastatin treatment resulted in a decrease in ubiquinone more pronounced in the brother with milder clinical phenotype. Molecular (97.9 +/- 14.9; control: 202.9 +/- 18.4 pmol/mg; p<0.05), and complex IV analysis revealed compound heterozygosity for two common mutations; activity (0.008 +/- 0.001; control: 0.011 +/- 0.001; p<0.05) relative to CS072 183c.492+2 T>C/CX072638c.228-20_21delTinsC. control. These data, coupled with the patient findings, indicate a possible Heterogeneity in clinical presentation makes the diagnosis challenging, but on association between statin treatment, decreased ubiquinone status, and loss the other hand characteristic MR imaging and lactate elevation in spectroscopy of complex IV activity. strongly suggest LBSL, which should be confirmed by molecular analysis. S122 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-320 P-321 CLINICAL AND LABORATORY MANIFESTATIONS OF DIFFERENT LABORATORY AND MUSCLE BIOPSY FINDINGS THIAMINE-RESPONSIVE AND NON-RESPONSIVE PATIENTS IN FAMILY WITH M.8851 T>C MUTATION IN MITOCHONDRIAL WITH PYRUVATE DEHYDROGENASE COMPELX DEFICIENCY MTATP6 GENE van DONGEN S1 , Brown RM2 , Brown GK2 , Thorburn DR3 , Boneh A1 Jesina P1 , Honzik T2 , Tesarova M2 , Vinsova K2 , Hansikova H2 , Magner 1Metabolic Research, MCRI. RCH, Melbourne, Australia M2 , Zamecnik J3 , Kratochvilova H2 , Zeman J2 2Depart of Biochem, University of Oxford, Oxford, United Kingdom 1Inst Inher Metab Dis, Charles Univ, Prague, Czech Republic 3Mitochondrial Research, MCRI, Melbourne, Australia 2Dept. Pediatr, Charles Univ, Prague, Czech Republic 3Dept. Pathol Mol Medicin, Charles Univ, Prague, Czech Republic Pyruvate dehydrogenase complex (PDHC) deficiency leads to impaired energy metabolism and a range of clinical manifestations. Thiamine pyro- Here we present a family with a very rare m.8851 T>C mutation in mitochon- phosphate is a co-factor of the PDHC. Little is known about the prevalence drial ATP6 gene. A 2 yr 6 m old girl with high mtDNA mutation load (87-97 %) of thiamine responsiveness and the clinical and laboratory differences has normal development till the 8th months of life. Then failure to thrive, between responsive and non-responsive patients. microcephaly, psychomotor retardation, hypotonia, ataxia and MRI finding of To characterise clinical manifestations, neuroimaging, biochemical and mo- Leigh syndrome were documented. Neurological examination revealed central lecular findings in thiamine-responsive and non-responsive patients, we retro- hypotonic syndrome, ataxia, and moderate hearing impairment. MRI of the spectively reviewed medical records of all PDHC-deficient patients (n020), brain demonstrated diffuse symmetric hyperintensity in bilateral basal ganglia suspected thiamine deficient patients (n02) and a patient with PDHC defi- including nuclei caudata. Her 26-year-old mother (level of heteroplasmy in ciency secondary to 3 hydroxyisobutyryl-CoA hydrolase deficiency at our blood 68 %) developed peripheral neuropathy and muscle weakness at the age centre (January 1982 to January 2012). of 22 years. Biochemical investigation found metabolic acidosis, elevated Eight patients were thiamine-responsive (regaining spontaneous breathing, lactate in blood (6.4 mmol/l) and cerebrospinal fluid (5.0 mmol/l) and increased mobility, developmental improvement and a decline in seizures). Age at alanine in blood (649 μmol/l). Elevated ethylmalonate was detected in profile of presentation was 0-6 m and 0-27 m in the non-responsive and responsive organic acids in urine. Muscle biopsy revealed focal sub sarcolemmal accumu- patients, respectively. Seizures were noted in 3 and 6 responsive and non lation of SDH reaction product. But spectrophotometric enzyme measurements responsive patients, respectively; ataxia in 5 and 2; corpus callosum abnor- showed normal activity of respiratory chain complexes. Electrophoretic analy- malities in 1 and 4; and basal ganglia lesions in 3 and 0 patients. No sis showed decreased ATP synthase content in muscle. differences were found in biochemical parameters. Conclusion: - We report a second case of very rare mtDNA 8851 T>C Thiamine-responsiveness cannot be predicted from clinical, biochemical mutation in ATP6 gene. Our findings extend clinical and laboratory phe- and neuroimaging manifestations. High dose thiamine treatment should be notype associated with the m.8851 T>C mutation. initiated in all patients. Mutation analysis should be performed to avoid This work was supported by the grant RVO-VFN64165/2012. missing thiamine-responsive patients and misdiagnosing patients with secondary PDHC deficiency.

P-322 COMPREHENSIVE SEQUENCING OF MITOCHONDRIAL DNA IN PATIENTS WITH SUSPECTED MITOCHONDRIAL DISEASE: IS THERE A NEED FOR A REVISED MOLECULAR DIAGNOSTIC ALGORITHM? Ezgu F1 , Kucukcongar A1 , Ciftci B1 , Kasapkara C1 , Hasanoglu A1 , Tumer L1 , Okur I1 , Gunduz M1 , Polat M1 , Bahceci S1 1Dep Ped Metabolic Dis, Gazi Univ Med Fac, Ankara, Turkey

Background: Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutations of nuclear or mitochondrial DNA (mtDNA). Several clinical and molecular diagnostic guidelines have been suggested, and mtDNA sequencing obtained from peripheral blood has been proposed as the initial molecular diagnostic assay in many of these guidelines. Methods: In a total of 60 patients aged 1 month-37 years, who were clinically suspected to have mitochondrial disorder according to the Nijme- gen Criteria, mtDNA was analysed with DNA sequence analysis. Results: A total of 31 novel changes, in addition to one unpublished and many previously published polymorphisms were found. The occurrence of novel mutations was more frequent in the protein coding regions than the tRNA genes. The possible effect of some of the novel changes was investigated by analyzing the asymptomatic family members. Conclusion: Considering the high frequency of polymorphisms and novel changes with clinically unknown significance in mtDNA and the significant proportion of nuclear DNA encoded mitochondrial disorders, gene panels designed according to the major clinical presentations in the era of next generation sequencing could be considered as the initial diagnostic assay. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S123

P-323 P-324 CLINICAL UTILITY OF MASSIVELY PARALLEL SEQUENCING THE NHS SPECIALISED SERVICES BARTH SYNDROME (MPS) IN COMPREHENSIVE ANALYSES OF MITOCHONDRIAL SERVICE - A CLINICAL AND LABORATORY CASE AUDIT DNA: DETECTION AND QUANTIFICATION OF HETEROPLASMIC Honeychurch J1 , Pennock M1 , Bowron A2 , Steward C3 , Newbury-Ecob MUTATIONS AND LARGE DELETIONS WITH BREAK POINTS R4 , Tsai-Goodman B5 , Kinning E6 , Williams MM1 PRECISELY MAPPED 1Bristol Genetics Laboratory NB NHS Trust, Bristol, United Kingdom Wong LJ1 , Cui H1 , Zhang VW1 2Dept Clin Biochem, Bristol Royal Infirm, Bristol, United Kingdom 1Mol Hum Genet, Baylor College Medicine, Houston, Texas, United States 3Dept Stem Cell Trans Bris Roy Hosp Child, Bristol, United Kingdom 4Clin Gen Dept, St Michaels Hospital, Bristol, United Kingdom Background: Mitochondrial disorders are a group of complex dual genome 5Dept Card, Bris Royal Hosp Child, Bristol, United Kingdom diseases. Current molecular diagnosis requires multiple different methods, 6Clin Gen, Royal Hospital for Sick Child, Glasgow, United Kingdom including sequencing, qPCR, Southern blot or array CGH, for the detection and quantification of mutations. In addition, the highly polymorphic nature Barth Syndrome (OMIM 302060) is a rare recessive metabolic disease of mtDNA makes multiplex PCR-based analyses problematic. characterised by dilated cardiomyopathy ( mainly in infancy ), skeletal Methods: A unique Next Generation Sequencing NGS-based approach by myopathy, neutropenia, growth retardation and 3-methylglutaconic acidu- a single long-range (LR)-PCR has been developed and validated, that ria. It may be relatively under diagnosed due to clinical variability. Barth would eliminate the interference of nuclear mtDNA homologs (NUMT) can present in females as a history of multiple male stillbirths and fetal and allow a comprehensive diagnosis. cardiomyopathy. (PrenatDiagn.2010Oct;30(10):970-6). Results: With one-piece LR-PCR enrichment of the entire mitochondrial Barth syndrome is caused by mutations in the TAZ gene (Xq28.12) encod- genome and proper qualitative and quantitative controls, this unique method ing the protein tafazzin, an acyltransferase which remodels cardiolipin in was able to sequence the "true mtDNA", detect low heteroplasmy (to 1.5 % the inner mitochondrial membrane. without the interference of the NUMT) and large single or multiple deletions Bristol Genetics Laboratory forms part of a multidisciplinary Barth team with breakpoints precisely mapped. This is particularly important in assessing providing the UKNSS specialist service. Males with positive cardiolipin the carrier status of family members or in various types of tissues from the assays proceed to TAZ gene sequencing (11 exons) for formal disease proband in order to determine if a mutation is inherited, de novo, or somatic. confirmation. Genetic testing is also undertaken where samples are not This information has a dramatic impact in genetic counselling. available for cardiolipin analysis e.g. neonatal death. Conclusion: Our unique deep sequencing, one-step approach provides Genetic testing has confirmed a diagnosis in 27 Barth families, facilitated 70 comprehensive molecular analysis for patients with suspicion of mtDNA familial tests and 3 prenatal diagnoses. Cardiolipin triageing of tests imple- diseases in a timely, accurate, and cost-effective manner. mented in April 2010 has improved targeting of genetic testing. We present clinical/laboratory data from our most interesting cases, including one patient with dilated cardiomyopathy who has a MHY7 variant p.Met982Thr and is also hemizygous for the novel TAZ intron 3 splice variant c.284+3 G>T where bioinformatics and RNA functional studies have proven pathogenicity. S124 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-325 P-326 BARTH SYNDROME IN TWO BROTHERS WITH NORMAL DEVELOPMENT OF A MASS SPECTROMETRY METHOD LEUKOCYTE CARDIOLIPIN CONCENTRATIONS AND WITH (MS/MS) FOR QUANTIFICATION OF COENZYME Q10 IN CSF NO NEUTROPENIA Duberley KEC1 , Hargreaves IP2 , Chaiwatanasirikul K3 , Heales SJ4 , Bowron A1 , Honeychurch J2 ,PennockM2 , Williams MM2 , Tsai-Goodman Rahman S5 , Mills K6 , Eaton S3 B3 , Shortland G4 , Steward CG5 1Dept Mol Neuro, Inst Neurol-UCL, London, United Kingdom 1Clin Biochem, Bristol Royal Infirmary, Bristol, United Kingdom 2Neurometab unit, NHNN-UCLH, London, United Kingdom 2Bristol Genetics Lab, Southmead Hosp, Bristol, United Kingdom 3Unit Paed Surg, Inst Child Health-UCL, lONDON, United Kingdom 3Dept Cardiology, Bristol Children's Hosp, Bristol, United Kingdom 4Dept Clin Path, GOSH, London, United Kingdom 4Dept Metab Dis, Univ Hosp Wales, Cardiff, United Kingdom 5Mito Res Group, Inst Child Health-UCL, London, United Kingdom 5Dept Haem, Onc, BMT, Bristol Child Hos, Bristol, United Kingdom 6Biochem Res Group, Inst Child Health-UCL, London, United Kingdom

Background: Barth syndrome (BTHS) is an X-linked disorder character- Background: Neurological dysfunction is common in primary Coenzyme Q10 ised by cardiomyopathy, skeletal myopathy, growth retardation, neutrope- (CoQ10) deficiencies; the most readily treatable subgroup of mitochondrial nia, 3-methylglutaconic aciduria (3-MGCA), tetra-linoleoyl cardiolipin disorders. Therapeutic benefit from CoQ10 supplementation is also noted in (CL4) deficiency and TAZ gene mutations. We describe two brothers with other neurodegenerative diseases, particularly Friedreich's ataxia. Whilst CoQ10 genetically confirmed BTHS but normal CL4 and no neutropenia. can be measured by HPLC in plasma, muscle or leucocytes, there is currently no Cases: Boy A presented at 2 years with global motor delay, failure to thrive, reliable method to determine CoQ10 levels in cerebrospinal fluid (CSF). dilated cardiomyopathy and 3-MGCA; he was diagnosed with BTHS. He is Methods: CoQ10 was measured by tandem mass spectrometry using a now on ACE inhibitors and diuretics, has poor growth and skeletal myop- novel d6-CoQ10 internal standard and 5 mM methylamine as an ion-pair athy but no neutropenia (neutrophil counts 3.8-7.2x109/l). Boy B presented reagent. Chromatography was performed using a Hypsersil GOLD C4 at 13 months with bronchiolitis, dilated cardiomyopathy and 3-MGCA column (150 x 3 mm, 3 μ). resulting in a diagnosis of BTHS. He is now well, is on no medication, Results: CoQ10 levels were linear over a concentration range of 2 to 200nM and has had no recorded neutropenia (neutrophil counts 2.4-5x109/l). (R200.9995). The lower limit of detection was 2nM. The inter-assay CV was Results: A previously unreported exon 2 TAZ mutation, c.118A>G 3.6 % (low spike010nM) and 4.3 % (high spike020nM), and intra-assay CV (p.Asn40Asp), was found in both patients. Leukocyte CL4 concentrations 3.4 % (low spike010nM)and3.6%(highspike020nM). CoQ10 was mea- were 171 and 120pmol/mg protein (controls >61). Monolysocardiolipin sured in CSF of 17 controls (no evidence of CSF monoamine metabolite (MLCL) was increased, resulting in an elevated MLCL/CL ratio (0.10 and disorder): mean concentration07.7nM, interquartile range05.7-9.0 nM. 0.14, controls <0.01). This was lower than other BTHS patients (range >3.0). Conclusion: We have developed a method to determine CoQ10 levels in Conclusion: We found normal CL4 and no recorded neutropenia in CSF, which could potentially be used diagnostically to identify patients brothers with genetically confirmed BTHS. Further investigation of this with neurological CoQ10 deficiency. This could also be useful for moni- mutation may help elucidate the pathogenesis of this complex disorder. toring CSF CoQ10 levels following supplementation in mitochondrial and other neurodegenerative diseases. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S125

P-327 P-329 IS PLASMA CREATINE CONCENTRATION A REAL BIOMARKER A PATIENT WITH PYRUVATE CARBOXYLASE DEFICIENCY OF MITOCHONDRIAL DISEASES? AND NEMALINE RODS ON MUSCLE BIOPSY Arias A1 , Pajares S1 , Garcia-Villoria J2 , Briones P3 , Ribes A2 Unal O1 , Hismi B1 , Dursun A1 , Tokatli A1 , Coskun T1 , Wibrand F1 , 1BMG, H Clinic. CIBERER. Equal contribut, Barcelona, Spain Sivri HS1 2Div IEM, Dpt BMG, H Clinic. CIBERER, Barcelona, Spain 1Hacettepe Univ, Pediatrics, Metabolism, Ankara, Turkey 3Div IEM,Dpt BMG, H Clinic. CIBERER. CSIC, Barcelona, Spain Introduction: Nemaline rods are the pathological hallmark of nemaline Background: Recent investigations have reported increased extracellular myopathy, but they have also been described as a secondary phenomenon in creatine (Cr) levels in patients with mitochondrial respiratory chain defi- a variety of other disorders including mitochondrial disorders. Nemaline ciencies, suggesting Cr as biomarker of mitochondrial diseases. rods have not been reported in pyruvate carboxylase (PC) deficiency before. Objective: To corroborate the previous findings. Case report: A -two- day old patient was referred to hospital because of Materials and Methods: We analysed plasma Cr in 36 patients with different refractory lactic acidosis and hypotonia. A mitochondrial disorder was mitochondrial diseases. Control values were obtained from 196 healthy chil- suspected because of the multisystem involvement (brain, liver and muscle) dren and adults. Quantification of the buthylated Cr, using 2 H3-Cr as internal combined with lactic acidosis, and therefore thiamine, biotine, carnitine, standard, was performed by HPLC-MS/MS equipped with a 2.1x50 mm coenzyme Q10 and dichloroacetic acid treatments were commenced. Mus- Symmetry C18 column, 3.5 μm particle size, and operating in the electrospray cle biopsy and skin biopsy for fibroblast culture were taken for investiga- positive ion mode using multiple reaction monitoring mode. tion of mitochondrial disorders. Muscle histology showed mild variation in Results: The mean of Cr concentrations was significantly higher in the fibre size. On modified Gomori trichrome stain, subsarcolemmal rods were cohort of 36 patients with mitochondrial diseases compared with the found in about 25 % of the fibres. Measurement of pyruvate carboxylase corresponding age-matched control group. However, individually, only 10 (PC) enzyme activity in cultured fibroblasts revealed that the patient suf- patients showed a clear increase. This group of positive patients includes 5 fered from PC deficiency. Screening for mutations in the PC gene revealed patients with respiratory chain deficiencies, 4 patients with mitochondrial a homozygous missense mutation, c.2606 G>A (p.Gly869Asp), affectect- DNA depletion and 1 patient with mutations in NFU1. ing a highly conserved glycine residue. Conclusion: Only 28 % of the total patients with mitochondrial diseases Conclusion: The nemaline rods may be due to cellular energy shortage and showed an increase of plasma Cr, suggesting that Cr is not a specific altered energy metabolism. The mechanism of nemaline rod formation may biomarker of mitochondrial diseases but, if increased, it could provide be associated with the role of PC in cellular energy pathways. additional information to help the diagnosis.

P-328 P-330 MLPA APPROACH TO SEARCH OF MTDNA MUTATIONS HOW TO IMPROVE DIAGNOSTIC OF EARLY FORMS OF Itkis Yu1 , Tsygankova P1 , Zakharova E1 MITOCHONDRIAL OXPHOS DISEASES? 1Research Center for Med Genetics RAMS, Moscow, Russian Federation Tsygankova PG1 , Itkis YS1 , Mikhailova SV2 , Zakharova EYu1 1Research centre for medical genetics, Moscow, Russian Federation Mitochondrial diseases it's just developing area of medicine, but very 2Russian child hospital, Moscow, Russian Federation important, because nowadays it's highly difficult to diagnose these disorders due to extremely heterogeneous clinical manifestations, especially in cases Inherited mitochondrial disorders is a group of metabolic diseases affected of diseases caused by mutations in mitochondrial DNA (mtDNA). Similar children and adults. They are characterized by high severity of clinical phenotypes could be caused by different mutations of mtDNA and vice symptoms, genetic heterogeneity and few or even absence of biochemical versa. Earlier if a patient with MELAS (MERRF/ Leigh) phenotype did not markers. We provided DNA tests in about 250 patients from different reveal m.3243A>G (m.8344A>G/ m.8993 T>G/C) mutation, we per- groups of OXPHOS in disorders (Leigh syndrome, Alpers disease, mito- formed mtDNA sequence analysis. Later on a base of our results and our chondrial hepatopathy, Pearson syndrome). We confirmed the diagnosis in experience we've developed two specific sets of primers for MLPA (Mul- 40 % of patients. We also developed fast test for frequent mutations and tiplex Ligation-dependent Probe Amplification) analysis which allows us to made a kind of selective screening with wider clinical criteria: from more hasten a search of mutations in mtDNA. These sets include both frequent than 200 patients suspected with one of mito disorder in 5 we found mutations of mtDNA such as m.3243A>G (MELAS), m.8344A>G mutations typical for another form of mito disorder. So the panel for (MERRF), m.3460 G>A, m.11778 G>A, m.14484 T>C (LHON), frequent mutations in mtDNA and in nuclear genes gives an opportunity m.8993 T>G/C (NARP) and rare mutations also found in our patients to reveal patients with a lack information about clinical symptoms. Our (e.g. m.3697 G>A, m.8363 G>A, m.13094 T>C, m.13513 G>A, algorithm of molecular diagnostic has efficiency not more than 40 %. Our m.14459 G>A). In retrospective analysis of 200 patients with proposed next steps to make it more efficient will be 1. Oxymetry assay in small mitochondrial pathology we've received interesting results using MLPA amounts of muscle biopsy will allow reveal biochemical defect and may approach. E.g. in two patients with MELAS phenotype we've found LHON give hints for DNA analysis. 2. PGM (Personal Genome Machine) sequenc- mutation (m.11778 G>A), in two patients with Leigh syndrome mutations ing is able to check thousands of mito genes simultaneously in a group of m.13094 T>C and m.13513 G>A have been revealed respectively. Thus, patients with revealed biochemical defect. our results show the efficiency of MLPA approach in mitochondrial diseases diagnosis. S126 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-331 P-332 URINARY ORGANIC ACID PROFILES IN MITOCHONDRIAL ANALYSIS OF COQ SYNTHESIS USING LABELED NON DISEASES RADIOACTIVE SUBSTRATES. APPLICATION FOR THE Pajdowska M1 , Gradowska W1 , Piekutowska-Abramczuk D1 , Baczyńska DIAGNOSIS OF PRIMARY COQ10 DEFICIENCIES A1 , Iwanicka-Pronicka K2 , Sykut-Cegielska J1 , Pronicka E1 Buján N1 , Arias A1 , Montero R2 , García-Villoria J3 , Lissens W4 , Seneca 1The Children's Memorial Health Institut, Warsaw, Poland S4 , Espinós C5 , De Meirleir L4 , Artuch R2 , Briones P6 , Ribes A3 2The Institute of Physiology and Patholog, Warsaw, Poland 1BMG, H Clinic. CIBERER. Equal contribut., Barcelona, Spain 2Serv Bioquímica, H Sant Joan de Déu, Barcelona, Spain Background: Urinary organic acid profiles (OA) in mitochondrial diseases 3Div IEM, Dpt BMG,H Clinic. CIBERER., Barcelona, Spain (MD) are neither specific nor sensitive, and are rarely reported in detail. 4UZ Brussel, Vrije Universiteit, Brussel, Belgium Objective: The aim is to characterize OA in a group of MD with known molecular 5CIBERER, Valencia, Spain background and assess in which extent GC-MS analysis may contribute to 6Div IEM, Dpt BMG,H Clinic. CIBERER. CSIC, Barcelona, Spain detection of MD in comparison to the age and the type of mitochondrial defect. Material and Methods: Retrospective analysis of OA was performed in a Background: CoQ10 deficiencies are associated to mutations in genes of group of 143 patients with MD with mutations in mtDNA, SURF1, SCO2, and CoQ biosynthesis and respond to CoQ10 supplementation. Early treatment mtDNA depletion. Age of the patients ranged from one day to adulthood. allows better outcome. Therefore, early diagnosis is desirable. Results: Various urinary OA changes were present in 76.2 % of the MD group. Objectives: Developing a non-radioactive methodology for fibroblast CoQ10 Increased excretion of lactate (45.5 %), Krebs cycle metabolites (30.8 %), fatty biosynthesis quantification that enables discriminating primary deficiencies. oxidation (24.5 %), liver (19.1 %) and intestinal motility (16.8 %) dysfunction Methods: Fibroblasts were incubated 72 h with 28 μM 2 H3-mevalonate or parameters, 3-metaglutaconic acid (11.1 %) and others were observed. Krebs 1.65 mM 13 C6-p-hydroxybenzoate. The labeled CoQ10 synthesized was cycle metabolites the most frequently occurred in SURF1 subgroup (59 %) analyzed by HPLC-MS/MS. and the liver dysfunction in mtDNA depletion (62 %). Patients: A) To validate the method: one patient with confirmed primary OA selective screening was a first metabolic measure in the mtDNA depletion deficiency (COQ2 mutation) and 6 patients with CoQ10 deficiency second- (87.5 %; median age 6 weeks), more frequently than in SCO2 and SURF1. ary to other inborn errors of metabolism (ETF, ETFDH, VLCAD, MELAS Conclusion: Urinary OA characteristics may help in MD mutation search, or Nieman-Pick Type C). especially in unexplained pathology of young infants. B) 3 groups of CoQ10 deficiencies: 1) 3 Patients heterozygous for one COQ Supported by NCN-NN403130636,407118939,407285739. gene; 2) 5 without mutations in the genes studied but responsive to CoQ10, 3) 9 clearly deficient, without further genetic or therapeutical studies. Results and discussion: A) We have demonstrated that our method is suitable for discriminating between primary and secondary deficiencies. B) 10/17 patients' fibroblasts investigated showed significantly decreased CoQ10 synthesis (40-88 % of the lower control) and mutational study of COQ genes should be performed. In patients with normal rates the deficiency is probably secondary. Conclusions: Our method is a good tool for the diagnosis of these treatable diseases. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S127

P-333 P-335 ISOLATED DEFICIENCY OF ATP SYNTHASE POINTS TO GLUCOSE KINETICS IN SUBJECTS WITH MELAS SYNDROME: MTATP6 AND MTATP8 GENES SEQUENCING INTERIM RESULTS Mendes C1 , Simues M1 , Santos MJ1 , Pratas J1 , Ferreira C1 , Diogo L2 , El-Hattab AW1 , Emrick L2 , Hsu J2 , Jahoor F2 , Scaglia F2 , Craigen W2 Garcia P2 , Macário MC3 , Gaspar A4 , Moreno T4 , Grazina M5 1University of Missouri Health Care, Columbia, United States 1Center Neurosciences and Cell Biology, Coimbra, Portugal 2Baylor College of Medicine, Houston, United States 2CHUC - Pediatric Hospital, Coimbra, Portugal 3CHUC - Adult Neurology Department, Coimbra, Portugal Background: The mitochondrial encephalomyopathy, lactic acidosis, and 4Centro Hospitalar Lisboa Norte, Lisboa, Portugal stroke-like episodes (MELAS) syndrome is a common mitochondrial dis- 5Fac Medicine & CNC/UC, Coimbra, Portugal order in which diabetes mellitus (DM) occurs in one third of affected individuals. We hypothesize that DM develops in MELAS due to multiple Mitochondrial respiratory chain (MRC) disorders are clinically heterogeneous, defects in glucose metabolism. but certain clinical and biochemical laboratory features can point to molecular Methods: We assess glucose metabolism using stable isotope infusion in defects in specific genes. subjects with MELAS who have DM, subjects with MELAS who do not From 2003 to 2011, we have performed the evaluation of MRC complexes have DM, and in healthy controls. activity and we have found 16 patients (median age 2.50) with isolated Results: To date, 6 controls, 4 subjects with MELAS and DM, and 4 non- complex V deficiency (activity below 40 % of the reference mean value diabetic subjects with MELAS have completed the study. Both groups of normalized to CS), according to Grazina 2012. subjects with MELAS (with and without diabetes) show increased glucose These results suggested the sequencing of mtDNA complex V subunit production and gluconeogenesis rates. Additionally, diabetic subjects with genes, MTATP6 and MTATP8. Total DNA was extracted from biopsies MELAS exhibit higher insulin resistance. by standard methods and the analysis of MTATP6 and MTATP8 sequences Conclusions: Subjects with MELAS and diabetes showed both increased were performed by automatic sequencing. It was found 9 different sequence glucose production and higher insulin resistance, suggesting that DM variations (8 in MTATP6 and 1 in MTATP8 sequences), one of which is develops due to multiple defects in glucose metabolism in MELAS. Sub- pathogenic (8993 T>C), 7 are described as polymorphisms, and the remain- jects with MELAS who do not have DM have higher rates of glucose ing is a reported mutation (9055 G>A) described as Parkinson disease production that can predispose them to develop diabetes. The completion protective factor. The pathogenic mutation was identified in the liver of a of this study will result in a better understanding of the pathophysiological Leigh syndrome diagnosed child with a benign phenotype of Ornithine mechanisms of DM in MELAS and may provide further insights into the transcarbamylase deficiency (Henriques et al., 2012). pathogenesis of DM in mitochondrial diseases in general. Our results show the importance of evaluation of MRC complexes activity to guide the molecular approach in MRC disorders, pointing to representa- tive genes to be sequenced.

P-334 P-336 EXPRESSION OF GENES INVOLVED IN MTDNA TRANSCRIP- DEFECTIVE MITOCHONDRIAL SUPER-COMPLEX ASSEMBLY TION AND MAINTENANCE IN LIVER AND MUSCLE TISSUE IN NOONAN-LIKE SYNDROME WITH LOOSE ANAGEN HAIR DURING HUMAN FETAL DEVELOPMENT AND SHOC2 MUTATIONS Hansikova H1 , Hulkova M1 , Tesarova M1 , Spacilova J1 , Hajkova Z1 , Parikh S1 , Moran R1 Honzik T1 , Zeman J1 1Cleveland Clinic, Cleveland, United States 1Dep Ped, Charles Univ, Gen Univ Hosp, Prague, Czech Republic Background: The rasopathies represent a spectrum of disorders due to muta- The mitochondrial biogenesis and adequate energy production are impor- tions in one of several genes in the Ras-Mitogen-Activated Protein Kinase tant for fetal growth and early postnatal adaptation. (MAPK) pathway leading to Noonan syndrome and related disorders includ- The aim of our study was to analyze mRNA expression profiles of NRF1, ing Cardio-Facio-Cutaneous (CFC), LEOPARD and Costello Syndromes. TFAM, PGC1A and MTCO2 and COX4, mitochondrial DNA (mtDNA) An association has been made between rasopathies and mitochondrial content, activities of cytochrome c oxidase (COX) and protein levels of dysfunction by Kleefstra et al (EJHG 2011), Lee et al (BBA 2010) and COX subunits in human fetal tissues. Aeby et al (JIMD 2007) 26 pairs of liver and muscle tissue samples collected from foetuses aborted Case report: We report the first case of a 6 year old girl with a SHOC2 spontaneously or after genetic indication between 13th and 28th week of mutation leading to Noonan-Like Syndrome with Loose Anagen hair and gestation were investigated. The mtDNA amount and gene expression defective mitochondrial super-complex assembly as seen on proteomic levels were analyzed by the RT-PCR. Activities and amount of COX were analysis via Blue-Native Gel electrophoresis along with mitochondrial analyzed by spectrophotometry and western blot. dysfunction as seen in biochemical analysis of blood, urine, skin and mtDNA content, NRF1, PGC1A, TFAM, COX2 and COX4 expression muscle. She has developmental delays, pulmonic stenosis, hyperpigmenta- levels were increasing during development in fetal liver tissue. In muscle tion, atypical cyclic vomiting responding to carnitine, illness-related ano- tissue, only mtDNA content and COX2 expression level increased between rexia and lethargy and short stature. 13th and 28th week of gestation. Activity and content of COX in fetal Conclusion: This is the first rasopathy patient with defective mitochondrial muscle and liver tissue were significantly lower in comparison with controls supercomplex assembly described and expands the types of rasopathies but no significant changes were detected during gestation. where mitochondrial dysfunction has been identified. Treatment of the Our results suggest that fetal developing liver and muscle differ in the mitochondrial dysfunction helped improve cyclical vomiting, food aversion control of the mitochondrial biogenesis depending on their energy demand when ill and hypotonia and may prevent unexpected deterioration in neuro- and the phase of development as well. logic or medical health. Further study of mitochondrial function in rasopa- Supported by PRVOUKP24/LF1/3, GAUK667612 thies is needed. S128 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-337 P-339 ANALYZING FUNCTIONAL PHENOTYPES IN FIBROBLASTS ANALYSIS OF OPA1 GENE IN A FAMILY CARRYING THE FROM PATIENTS WITH CLINICALLY-DIAGNOSED MITO- M.11778 G>A MUTATION WITH INCOMPLETE PENETRANCE CHONDRIAL DISEASE ASSOCIATED WITH A CASE OF LHON-PLUS Graham B. H.1 , Donti T.1 , Besse A.1 , Craigen W. J.1 , Bonnen P.1 Sousa T1 , Silva F2 , Ribeiro C1 ,PratasJ1 ,SantosMJ1 , Silva E3 ,DiogoL4, 1Dept Hum Mol Genet, Baylor Coll Med, Houston, United States Grazina M5 1Center Neurosciences and Cell Biology, Coimbra, Portugal Mitochondria are intracellular organelles that are essential for many cellular 2IBILI, University of Coimbra, Coimbra, Portugal functions including oxidative phosphorylation and energy metabolism. The 3CHUC- Serviço de Oftalmologia, Coimbra, Portugal mitochondrial proteome is derived from two genomes: the nuclear genome 4CHUC - Pediatric Hospital, Coimbra, Portugal and the mitochondrial DNA (mtDNA). Mutations in either mtDNA 5Fac Medicine & CNC/UC, Coimbra, Portugal encoded or nuclear encoded genes cause mitochondrial disease. Fibroblast cell lines derived from patients with clinically-suspected mitochondrial Leber's hereditary optic neuropathy (LHON) is a mitochondrial genetic disease provide a convenient resource for research studies, but the per- disease characterized by loss of central vision. In some cases there are also ceived low sensitivity of this cell type for displaying abnormal mitochon- neurologic defects. The majority of patients have one of the three muta- drial phenotypes has limited their diagnostic use. For this study, a collection tions: m.3460 G>A, m.11778 G>A, m.14484 T>C. These are character- of primary fibroblast cell lines from 16 unrelated patients with clinically- ized by incomplete penetrance, suggesting the role of other genetic factors diagnosed mitochondrial disease but no molecular diagnosis was subjected in the disease's aetiology. to a battery of assays for mitochondrial function. Tests include assays for Here we report the analysis of OPA1 gene in a family, including a proband with cellular respiration, electron transport chain (ETC) activities, relative the LHON-plus phenotype, harbouring the m.11778 G>A mutation (Grazina et changes in mitochondrial membrane potential (MMP), increased reactive al, 2007). The aim of this study was to find genetic sequence variations in OPA1 oxygen species (ROS), changes in mtDNA copy number, and abnormal that could explain the difference in the expression of the disease. mitochondrial morphology. To date, 7 out of 16 (44 %) patient cell lines We identified 17 genetic variations, two of which probably leading to amino exhibit abnormalities in ETC activities and/or mtDNA content compared to acid alteration in protein sequence. The proband does not have a different controls with other assays pending. A battery of functional assays in genotype from the other relatives carrying the m.11778 G>A mutation. fibroblasts offers the promise of increased diagnostic sensitivity and can We conclude that variations in OPA1 gene sequence are not responsible for ultimately be used to functionally screen candidate disease genes identified the manifestation of the disease in the studied family. Nevertheless, we from whole exome studies. cannot exclude the possibility that these variations contribute to the atypical phenotype of the proband.

P-338 A-033 SPECIFIC CORRELATION BETWEEN THE WOBBLE MODIFI- GASTROINTESTINAL MANIFESTATIONS IN MITOCHON- CATION DEFICIENCY IN MUTANT TRNAS AND THE CLINICAL DRIAL DISEASE: A CLINICAL CASE AND A NEW MUTATION FEATURES OF MELAS Castro-Chaves P1 , Teresa Cardoso M1 ,AlmeidaL2 ,AlvelosM3 , Eyskens FJM1 , Van Goethem G2 Vasconcelos C4 , Vilarinho L2 , Leão Teles E4 1Div Metab Dis, UZA, Antwerp, Belgium 1Adults Metabolic Disease Unit, CH S Joao, Porto, Portugal 2Div Neurology, UZA, Antwerp, Belgium 2INSA / Centro de Genética Médica, Porto, Portugal 3Internal Medicine Department, CH S Joao, Porto, Portugal Background: The deficient wobble modification , lacking the normal 4Metab Dis Unit, Paed Depart, CH S Joao, Porto, Portugal taurine-containing modification (5-taurinomethyluridine) at the anticodon wobble position, is probably the key molecular factor responsible for the Mitochondrial disorders (MD) have a highly variable clinical expression phenotypic features of Myopathy-Encephalopathy, Lactic acidosis-Stroke- making their diagnosis difficult. like episodes (MELAS). We herein describe the case of a 54 year old man with long-standing bilateral Clinical picture of our patient: We report a girl who at the age of palpebral ptosis, ophtalmoplegia and neurosensorial deafness. The patient also 14 year developed an encephalopathy including seizures, periods of had muscle cramps, proximal tetraparesis and axonal sensorimotor polyneur- confusion, recurrent migraine-like headaches and episodes of vomit- opathy. He had left ventricular hypertrophy and no conduction defects. ing, anorexia, visual hallucinations and an episode resembling a stroke Increased lactate and pyruvate serum levels were detected and, in muscle with on MRI an area of cortico-subcortical necrosis in the right biopsy, red ragged fibers. Brain MRI showed elevated lactate in spectroscopy. occipital region. She also complains of muscle weakness and intoler- Respiratory chain study revealed a complex IV partial defect. However, the ance to exercise. Plasma lactic acid is elevated (4.2 mmol/L). The study of the most common MD related mutations was negative. During clinical picture was very suggestive for MELAS and the very rare follow-up, the patient developed progressive weight loss, cachexia, nausea, mtDNA mutation in the tRNAleu(UUR) at nucleotide pair 3291 was emesis, early satiety and episodes of post-prandial abdominal pain. Due to the found in a muscle biopsy. This mutation is in our knowledge only appearance of these gastrointestinal manifestations, a mutational study of described in a Japanese family. The patient we report is a Belgian girl Mitochondrial Neurogastrointestinal Encephalopathy Disease (MNGIE) asso- with a negative family history. ciated Tymp gene was performed. The following mutations were identified: Conclusion: MELAS has different genetic underlying causes (primary c.221 T>A / 478 T>C related to proteins p.M74K e S160P, respectively. defects of mtDNA, POLG mutations). Our patient adds prove to the This case highlights the multisystemic nature of MD and how patient hypothesis that the deficient wobble modification in tRNAleu(UUR) caused follow-up made it possible to reach a final diagnosis. The novel p.M74K by different MELAS-associated mutations result in indistinguishable mutation involves the substitution of a methionine residue by a lysine and, clinical features. due to the nature of this substitution, is considered pathological. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S129

A-034 25. Peroxisomal Disorders SEVERE NEONATAL LACTIC ACIDOSIS WITH MILD METHYL- MALONIC ACIDURIA: CASE REPORT P-340 MESLI S1 , Jouvencel Ph2 , Naudion S3 , Castella C4 , Espil-Taris C5 , VERY LONG-CHAIN FATTY ACIDS IN NEUROLOGICAL Bessou P6 , Lamireau D7 , Cousin A1 , Colombies B1 , Rous I1 , de Verneuil PATIENTS WITH EPILEPSY ON A KETOGENIC DIET H1 , Lacombe D3 , Redonnet-Vernhet I1 Stradomska T.J.1 , Bachański M.1 , Syczewska M.1 , Tylki-Szymańska A.1 1Lab de Bioch, Pellegrin, CHU Bordeaux, BORDEAUX, France 1The Children's Memorial Health Institute, Warsaw, Poland 2Ser. Pédiatrie, CHG, Bayonne, Bayonne, France 3Ser. Génétique.Médicale, CHU de Bordeaux, Bordeaux, France The oxidations of fatty acids are the most important biochemical reactions 4Pédiatrie,Néonat et Réanimation, CHU, Bordeaux, France placed in peroxisomal apparatus. Very long-chain fatty acids (VLCFAs) are 5Ser,Pédiatrie, Neurologie, CHU, Bordeaux, France catabolized exclusively via the peroxisomal beta-oxidation process. Blood 6Imagerie Médicale, Hôpital Pell, CHU, Bordeaux, France levels of VLCFAs are endogenous derivatives (synthesis by a microsomal 7Pédiatrie, Unité MHM,CHU de Bordeaux, Bordeaux, France elongation system), and exogenous (from the diet). Determination of VLCFA levels in body fluids exists as the basic stage of the diagnostic procedure for Background: Recently, mutations in SUCLA2 and SUCLG1 genes coding peroxisomal diseases. respectively for the β and α subunits of the Succinyl-CoA ligase (SUCL) have The objective of these studies was examination of the serum VLCFA levels been reported in neonates with severe lactic acidosis associated with a mild in neurological patients with epilepsy unresponsive to anticonvulsants elevated urinary excretion of methylmalonic acid (MMA) and mitochondrial whilst being managed on a ketogenic diet. DNA depletion. Case report: On the first day of life, N. was lethargic and VLCFA levels were examined in 25 patients (12 males and 13 females, age hypotonic. Feeding problems and vomiting were noted. Increased of plasma range 3 - 16 years) on a ketogenic diet (with LCT oil) with a fat/non-fat ratio of lactate level (23 mmol/L) and hypoglycemia were observed. Urinary organic 4:1. VLCFA levels were detected as methyl esters by gas chromatography acids analysis showed a huge peak of lactate, methylmalonate and tricarbox- technique and 5 parameters were evaluated: C22:0, C24:0, C26:0; C24:0/ ylic acids cycle intermediates (TCA). Plasma homocysteine was normal. Brain C22:0, C26:0/C22:0. MRI showed T2-signal abnormalities in periventricular white matter and in The absolute mean values of individual fatty acids increased in comparison basal ganglia. Muscle respiratory chain enzyme activities showed a combined with the controls levels. The mean C22:0 increased about 15 % more than defect of complexes I and IV. C24:0 and C26:0 concentration. The C24:0/C22:0 and C26:0/C22:0 ratios and Conclusion: Our patient presented a neonatal lactic acidosis with severe C26:0 concentrations were remained below the cut-off limits. The application hypotonia and muscle atrophy associated to a respiratory chain deficiency. of ketogenic diet with LCT does not lead to false-positive VLCFA results. Mild methylmalonic aciduria combined to mitochondrial encephalomyopathy is a useful marker of decreased succinyl CoA ligase activity. Quanti- fication of mitochondrial DNA depletion and mutation analysis of SUCLA2 and SUCLG1 should be considered in patients with severe lactic acidosis with elevated MMA. These investigations are in progress for our patient.

A-035 P-341 EFFICIACY OF DICHLOROACETATE USAGE İN A TERM AND A ARRESTED LEUKODYSTROPHY IN A YOUNG MAN WITH PRETERM NEWBORN ADRENOMYELONEUROPATHY(AMN) KIYKIM E1 , AKTUĞLU ZEYBEK AÇ1 , SOYUÇEN E1 , ERENER Renaud DL1 , Port JD2 , Welker KM2 , Trenerry MR3 ERCAN T2 , CANSEVER MŞ1 , ULUDAĞ D2 , AYDIN A1 1Department of Neurology, Mayo Clinic, Rochester, MN, United States 1Div Ped Nutr & Met, Cerr Med F, Ist Uni, İstanbul, Turkey 2Department of Radiology, Mayo Clinic, Rochester, MN, United States 2Div New Cerr Med F, Ist Uni,, İstanbul, Turkey 3Department of Psychology,Mayo Clinic, Rochester, MN, United States

Dichloroacetate has been administered for several years to patients with Background: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal genetic mitochondrial diseases. The primary rationale for its use is its ability disorder which presents with progressive leukodystrophy in boys or as to stimulate the activity of the pyruvate dehydrogenase complex, progressive spasticity and neuropathy in men (AMN). thereby facilitating aerobic glucose and lactate oxidation and ener- Case Report: A 30 year old male presented to the Neurometabolic Clinic with getics. Most published information on the safety and efficacy of worsening stiff gait and paresthesias, suggestive of AMN, and a recent diagnosis of dichloroacetate in this population is derived from open-label studies X-ALD. He was diagnosed with Addison's Disease at age 4. He was a gifted in 1 or a few individuals. student until high school when he had a change in personality, decreased attention A 7 days old girl was refered to our clinic with lactic acidosis which started and executive functioning. This was progressive during high school and then in the first 24 hours after the delivery. At 7th day of admission she was stabilized. IQ testing at age 16 years revealed a verbal IQ of 115 and performance markedly hypotonic and in the need of mechanical ventilation. Serum IQ of 92. lactate was 18 mmol/L which didn't respond to any other treatment. At At the time of his evaluation, neuropsychological testing revealed a verbal IQ of the 8th hour of administration of the first dose of 50 mg/kg dichloroacetate 115 and an improved performance IQ of 110. MRI of the spine revealed general- the serum lactate level 2,2 mmol/L, without any further increase. No side ized atrophy of the spinal cord and the EMG revealed sensorimotor neuropathy. effect was detected. The MRI scan revealed abnormal signal in the white matter of the parieto- A girl who was born at 30th gestational week weighing 900 g developed occipital, temporal and cerebellar white matter bilaterally which has lactic acidosis at 24th day. Serum lactate level was 16 mmol/L. After remained stable. There was no enhancement or restricted diffusion to excluding the causes of type 2 lactic acidosis 50 mg/kg dichloroacetate suggest active demyelination. MR spectroscopy was normal. was administrated. She is now 8 month old, still using dichloroacetate with Conclusion: This case demonstates a rare arrested form of leukodystrophy no side effect and normal neuromotor development. in a young man with AMN. S130 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-342 P-344 THE CLINICAL PHENOTYPE OF X-LINKED ADRENOLEUKO- PROTEIN AND LIPID OXIDATIVE DAMAGE AND BONE MARROW DYSTROPHY IN WOMEN: A PROSPECTIVE CROSS- TRANSPLANTATION IN X-LINKED ADRENOLEUKODYSTROPHY. SECTIONAL CLINICAL & BIOCHEMICAL STUDY Deon M1 , Rockenbach FJ1 , Marchese DP2 , Manfredini V3 , Mescka C4 ,Ribas Engelen M1 , Barbier M2 , Schur R1 , Dijkstra I.M.E.3 , Aubourg P2 , GS2 , Habekost CT2 ,CastroJrCG5 ,JardimLB2 , Giugliani R2 ,VargasCR1 Wanders R.J.A.3 , de Visser M4 , van Geel B.M.5 , Kemp S3 , Poll-The B.T.1 1PPGCF, UFRGS, Porto Alegre, Brazil 1Academic Medical Center, Div Ped Neurol, Amsterdam, Netherlands 2Medical Genetics Service, HCPA, Porto Alegre, Brazil 2INSERM UMR 745 Univ Paris-Descartes, Paris, France 3UNIPAMPA, Uruguaiana, Brazil 3Academic Medical Center, Lab Gen Met Dis, Amsterdam, Netherlands 4PPGCB:Biochemistry, UFRGS, Porto Alegre, Brazil 4Academic Medical Center, Div Neurol, Amsterdam, Netherlands 5Pediatric Oncology Service, HCPA, Porto Alegre, Brazil 5Medical Center Alkmaar, Div Neurol, Amsterdam, Netherlands Background: Current treatment options for X-linked adrenoleukodystrophy Background and Objectives: X-linked adrenoleukodystrophy (X-ALD) is a (X-ALD) are limited and bone marrow transplant (BMT) has been an effective peroxisomal disorder caused by mutations in the ABCD1 gene. The clinical method to halt cerebral demyelination. Oxidative stress plays an important role phenotype is well described in men, but not in female carriers. This information in the pathophysiology of neurodegenerative diseases, including X-ALD. is important to better inform women diagnosed with X-ALD about the prognosis Objectives: In the present work, we evaluated protein and lipid oxidative of their condition. Primary objective: to describe the phenotype of women with damage parameters in X-ALD patients before and after BMT, in order to X-ALD. Secondary objective: to explain the phenotypic heterogeneity. verify if this treatment is capable to alter these oxidative parameters studied. Methods: Adult women with confirmed X-ALD were eligible to participate Methods: We evaluated sulfhydryl content and malondialdehyde content and visited the hospital once for: history and physical examination, quality (MDA) in plasma of X-ALD patients before and after BMT and in controls. of life assessment (SF-36), disability assessment (ALDS), blood sampling, Results: We found a significant increase of MDA as well as a reduction of skin biopsy, evoked potentials and EMG. sulfhydryl content in plasma of these patients before BMT compared to Results: A total of 46 women were examined. For analyses women were controls. After BMT, there were a significant reduction in levels of MDA divided in age groups: 18 – 39 (I), 40 – 59 (II) and 60 – 79 years (III). History and a significant increment of sulfhydryl content in plasma from X-ALD and neurologic examination was abnormal in 36 % (I), 85 % (II) and 100 % (III). patients compared to before BMT. Biochemical analysis was performed and will be correlated to the clinical data. Conclusions: The results reinforce that X-ALD patients are subject to lipid Conclusion: A large proportion of women with X-ALD have signs and symp- and protein oxidative damage, suggesting a possible involvement of free toms attributable to X-ALD and signs of myelopathy are most prominent. There radicals in the pathophysiology of this disease. Also, the results may suggest is a strong correlation between age and symptomatology. A correlation between that BMT therapy seems to protect against lipid peroxidation and protein biochemical abnormalities and clinical phenotype is being investigated. damage in these patients. Financial support: CNPq, CAPES, FIPE/HCPA.

P-343 P-345 α-METHYL-COA RACEMASE DEFICIENCY: REPORT OF A DISRUPTION OF BIOENERGETICS HOMEOSTASIS BY PHY- NEW MUTATION AND RESPONSE TO TREATMENT IN A TANIC ACID IN CEREBELLUM OF YOUNG RATS PATIENT WITH NEONATAL CHOLESTATIC LIVER DISEASE Busanello ENB1 , Amaral AU1 , Zanatta A1 , Tonin AM1 , Viegas CM1 , AND ATTENTION DEFICIT HYPERACTIVITY DISORDER Agostini C1 , Leipnitz G1 , Wajner M1 Ersoy M1 , Çakir N1 , Balci MC1 , Demirkol M1 , Gökçay G1 1Univ. Fed. Rio Grande do Sul, Dep Bioq, Porto Alegre, Brazil 1Div Nutr Metab, Child Hosp, Ist Med Fac, Istanbul, Turkey Elevated phytanic acid (Phyt) tissue concentrations are found in Refsum Background: α-Methyl-acyl-CoA-racemase (AMACR) deficiency disease, an inherited metabolic disorder clinically characterized by cerebel- (OMIM 604489), a rare peroxisomal disorder, may present with cholestatic lar ataxia whose pathogenesis is poorly known. The in vitro effects of Phyt liver disease in the first months of life and with neuropsychiatric disorders (10-100 μM) on important parameters of energy homeostasis were therefore such as development delay, encephalopathy, demyelinating polyneuropathy, investigated in cerebellum from 30-day-old rats. Phyt decreased state 3 schizophrenia from childhood to late adult life. respiration, as well as complexes I-III, II and II-III activities of the respira- Objectives: AMACR could be in the aetiology of attention deficit hyperactivity tory chain, reflecting a metabolic inhibitory role for this fatty acid. In disorder (ADHD) and can benefit from diet therapy. Case report: 10-month-old addition, Phyt markedly increased state 4 respiration and reduced RCR, girlpresentedwithbleedingtendencywithelevatedprothrombintime(PT), NAD(P)H levels and membrane potential, indicating an uncoupling effect. international normalized ratio (INR) and 5-fold raised transaminases. Liver Phyt also significantly decreased synaptic Na±K±ATPase activity that is biopsy showed mild hepatocellular degeneration. Vitamin-K treatment normal- important for neurotransmission. The data indicate that Phyt acts as a ised PT, INR, transaminases within 6 months. Elevation of C27-bile acids and metabolic inhibitor and as an uncoupler of oxidative phosphorylation, pristanic acid raised the suspect of peroxisomal dysfunction. The absence of the besides affecting neurotransmission. It is presumed that these patho- S-isomers and accumulation of the R-isomers of bile acid intermediates led to the mechanisms may contribute to the cerebellar abnormalities presented diagnosis of AMACR deficiency. Sequence analysis of the AMACR gene by patients affected by Refsum disease in which tissue Phyt concen- showed a new homozygous mutation, c.59 G>A. On follow up although trations are very high. neurocognitive development was normal ADHD was diagnosed at the age of Supported by: CNPq, PROPESq/UFRGS, FAPERGS, FAPESP, PRONEX, three years. After phytanic acid restricted diet pristanic acid levels decreased FINEP IBN-Net and Instituto Nacional de Ciência e Tecnologia – Excitotox- half-fold and patient showed improvement on ADHD symptoms. icidade e Neuroproteção. Conclusion: AMACR is a rare disorder that should be considered in the differential diagnosis of ADHD and implementation of dietary treatment should be evaluated. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S131

P-346 P-348 MITOCHONDRIAL FUNCTION IS DISRUPTED BY PHYTANIC OXALATE CRYSTALS IN PH1 ACTIVATE THE NLRP3 ACID IN RAT HEART INFLAMMASOME Leipnitz G1 , Grings M1 , Tonin AM1 , Zanatta A1 , Knebel LA1 , Moura Ebberink MS1 , IJlst L1 , Turkenburg M1 , Waterham HR1 AP1 , Wajner M1 1Lab Genet Metab Dis, Academic med Center, Amsterdam, Netherlands 1UFRGS, Porto Alegre, Brazil Primary hyperoxaluria type 1 (PH1) is caused by a deficiency of the liver- Refsum disease is a peroxisomal disorder biochemically characterized by specific peroxisomal enzyme alanine:glyoxylate aminotransferase, resulting accumulation of phytanic acid (Phyt). Affected patients present predomi- in excess oxalate production and subsequent deposition. The consequences nantly neurological symptoms, and cardiomyopathy, whose pathogenesis is of this systemic oxalosis range from bone fractures and loss of visual acuity, not yet established. In the present work we investigated the in vitro effects to advanced neural damage, cardiac conductivity blocks and arterial vessel of Phyt on important parameters of oxidative stress and mitochondrial obstruction. In addition, patients often suffer from recurrent urinary tract function in heart of young rats. Our results demonstrate that Phyt induced infections, fever and end-stage-renal-disease, although little is known about lipid peroxidation (increased thiobarbituric acid-reactive substances) and the cause of this inflammatory response. Recently, monosodium urate and protein oxidative damage (increased carbonyl formation), and decreased the calcium pyrophosphate dehydrate crystals have been reported to induce concentrations of reduced glutathione, a crucial non-enzymatic antioxidant inflammation through activation of NLRP3 inflammasomes. We have defense. We also observed that Phyt increased 2',7'-dichlorofluorescin oxi- studied whether calcium-oxalate is also capable of activating the NLRP3 dation, reflecting an increase in reactive species generation. In addition, the inflammasome. Phyt-induced toxic actions were prevented or attenuated by free radical We stimulated human monocytic Thp-1 cells and mouse peritoneal macro- scavengers, reinforcing the involvement of reactive oxygen species in these phages with calcium-oxalate crystals in the absence and presence of the effects. Moreover, Phyt decreased membrane potential and the NAD(P)H caspase-1 inhibitor z-YVAD. The calcium-oxalate crystals caused a strong pool in heart mitochondria and inhibited NADH-cytochrome c oxidoreduc- release of the pro-inflammatory cytokine IL-1β in a caspase-1 dependent tase (complex I-III) activity in heart supernatants, indicating that this fatty manner. Moreover, when peritoneal macrophages from mice deficient in acid acts as an uncoupler of the electron transport chain and as a metabolic inflammasome components ASC or NLRP3 were stimulated with calcium- inhibitor. The data suggest that Phyt-induced oxidative stress and disruption oxalate crystals, the secretion of IL-1β was not induced. of mitochondrial homeostasis may contribute to the cardiomyopathy found These findings show that calcium-oxalate crystals engage the caspase-1 in patients affected by Refsum disease. activating NLRP3 inflammasome, resulting in the production of active IL- Supported by: CNPq, PRONEX, FINEP, IBN-Net # 01.06.0842-00, INCT-EN. 1β, which most probably results in a systemic inflammation.

P-347 A-036 PEX26 ESTABLISHES A CENTRAL HUB IN THE PEROXISOMAL LORENZO'S OIL THERAPY. FOLLOW UP OF THREE PATIENTS. INTERACTOME AND INTERSECTS WITH CELLULAR LIPID Espinosa E1 ,OrtizT2 ,PulidoN.F3 , Ardila Y.A3 ,GuevaraJ.M3 , METABOLISM Echeverri O.Y.3 , Barrera L.A.3 Lotz-Havla AS1 , Woidy M1 , Guder P1 , Erdmann R2 , Muntau AC1 , 1Neuropediatría. Hosp. Militar Central, Bogotá, Colombia Gersting SW1 2Endocrinologa Pediatra, Bogotá, Colombia 1Dr. von Hauner Child Hosp, LMU, Munich, Germany 3I.E.I.M- HUSI. U. Javeriana, Bogotá, Colombia 2Instit Physiol Chem, Med Ruhr Univ, Bochum, Germany X-Linked Adrenoleukodistrophy is a peroxisomal disorder affecting very Numerous metabolic pathways are shared by different organelles and long chain fatty acids (VLCFA) oxidation, biochemically characterized by increasing insight into peroxisomal biochemistry allowed for the recogni- accumulation of principally C26:0 and C24:0. It is a clinically heteroge- tion of a central position of peroxisomes in cellular metabolism. However, neous disease involving adrenal function and central nervous system (CNS) the specific function of many peroxisomal proteins remains unclear and with various degrees of severity. The classical form usually presents during restricted knowledge of the peroxisomal protein interaction network limits childhood, as a rapidly progressive neurodegenerative disorder. Lorenzo's our comprehension of how the organelle is embedded into key cellular oil is the most accepted therapy. There is evidence that its use combined processes. We performed a whole-organelle protein interaction screen for with nutritional management reduces plasma VLCFA levels and can benefit PEX26 associated with peroxisomal disorders of varying severity and patients before any CNS compromise. identified 16 novel binding partners. A network medicine approach We present a three year treatment follow-up of three patients from two revealed a hub position inside the peroxisomal interactome expanding families with Lorenzo's oil. Patients did not manifest any CNS involvement PEX26 function to proliferation, membrane assembly, and fatty acid metab- as evidenced clinically or by images, but were suspected because of their olism. Fine mapping of mutation-specific perturbations allowed for robust family history. All were biochemically tested and presented adrenal func- genotype phenotype correlations. Moreover, the PEX26 interaction network tion alterations that required replacement therapy at initiation of treatment, intersects with cellular lipid metabolism at different steps and genetic yet no CNS compromise was evident. perturbations of the peroxisomal interactome provide a mechanistic link To date all patients present a normal neurological exam, without patholog- to multigenic acquired disorders such as Alzheimer's disease, obesity, and ical changes in their neuroimages, and unchanged adrenal function. diabetes. Although, according to the literature, there has been a good clinical response, there is need for a longer follow-up to determine the treatment's effect based on the patients family history. S132 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

26. Lipids, Sterols, Lipoproteins P-351 THE MOLECULAR BASIS OF FAMILIAL HYPERCHOLESTER- P-349 OLEMIA IN THE CZECH REPUBLIC: SPECTRUM OF LDLR MACROCEPHALY, AN UNUSUAL PRESENTATION OF A MUTATIONS AND GENOTYPE-PHENOTYPE CORRELATIONS PATIENT WITH SMITH-LEMLI-OPITZ SYNDROME (SLOS) Tichy L1 , Freiberger T2 , Zapletalova P1 , Soska V3 , Ravcukova B2 , Zeevaert R1 , Peeters H2 , Vinckx J3 , Régal L1 , Jaeken J1 Fajkusova L4 1Center Metab Dis, Univ Hosp, Leuven, Belgium 1Center Mol Biol Gen Ther, Univ Hosp Brno, Brno, Czech Republic 2Center Hum Genet, Univ Hosp, Leuven, Belgium 2Cent Cardiovasc Surg and Transpl, Brno, Czech Republic 3Dep Pediatrics, Univ Hosp, Leuven, Belgium 32nd Clin Int Med, Masaryk Univ, Brno, Czech Republic 4CEITEC, Brno, Czech Republic Background: SLOS is a genetic defect in cholesterol synthesis associated with dysmorphy and encephalopathy including microcephaly. Background: Familial hypercholesterolemia (FH), a major risk for coro- Patient report: This girl with prenatally diagnosed macrocephaly was born nary heart disease, is predominantly associated with mutations in the genes at the gestational age of 34 weeks with weight 3700 g, length 52 cm and encoding the low-density lipoprotein receptor (LDLR) and its ligand apo- head circumference 37 cm. She presented with dysmorphism including lipoprotein B (APOB). syndactyly of the 3rd and 4th fingers and toes, and postaxial polydactyly Methods: Sequencing, RFLP, DHPLC. of the right hand. She was hypotonic and had an excess of adipose tissue, Results: We characterize the spectrum of mutations causing FH in 2239 Czech kyphosis and joint hyperlaxity. At the age of 2.5 years, her psychomotor probands suspected to have FH. We found 265 patients (11.8 %) with the APOB development was 17 months. Brain MRI showed a Chiari 1 malformation mutation p.(Arg3527Gln) and 535 patients (23.9 %) with a LDLR mutation. In with compression, right parietal en mild temporal polymicrogyria and 535 probands carrying the LDLR mutation, 127 unique allelic variants were extensive bilateral leukomalacia. Now at 6.5 years, her head circumference detected. Fifty five variants were novel, not described in other FH populations. is on 7.9 SDS, whereas weight and height are normal for age. Conclusions: The statistical analysis of lipid profiles was performed in Results: No mutations were identified in the PTEN gene, but the diagnosis of 1722 probands adjusted for age in which biochemical data were obtained SLOS was biochemically and genetically confirmed. Two missense mutations without FH treatment. Significant gradients in i) total cholesterol (LDLR+ were identified in the 7-DHCR gene: c.1348 C>T and IVS8-1 G>C. patients>APOB+patients0LDLR-/APOB- patients) ii) LDL-cholesterol Conclusion: Macrocephaly has never been described in SLOS, but two (LDLR+patients>APOB+patients0LDLR-/APOB- patients in men and patients with another defect in cholesterol synthesis, desmosterolosis, pre- LDLR+patients>APOB+patients>LDLR-/APOB- patients in women), sented with macrocephaly. It remains uncertain whether the macrocephaly iii) triglycerides (LDLR-/APOB- patients>LDLR+patients>APOB+ is part of the SLOS or a feature of a different syndrome. patients), and iv) HDL-cholesterol (APOB+patients>LDLR-/APOB- patients0LDLR+patients) were shown.

P-350 P-352 CLINICAL UTILITY OF THE UKGTN DHCR7 MUTATION ASSOCIATION BETWEEN SOLUBLE CD40 LIGAND AND SERVICE FOR SMITH-LEMLI-OPITZ SYNDROME. PROTHROMBOTIC STATE IN CHILDREN WITH Sawyer H1 , Gable MJ1 , Honeychurch J1 , Greenslade M1 , Burton-Jones HYPERCHOLESTEROLEMIA S1 , Brown AY2 , Williams M1 Kucukcongar A1 , Eminoğlu FT1 , Okur İ1 , Aral A2 , Hasanoğlu A1 , 1Bristol Genetics Laboratory, NBT, Bristol, United Kingdom Tümer L1 2Biochemical Genetics Unit, NBT, Bristol, United Kingdom 1Div Metab Dis, Univ Gazi, Ankara, Turkey 2Div Immunology, Univ Gazi, Ankara, Turkey Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive congenital multiple malformation disorder caused by a deficiency of 7- Hypercholesterolemia is associated with the pathogenesis of atherosclerosis. dehydrocholesterol reductase (DHCR7). The phenotype ranges from intra- This may be related to enhanced thrombotic risk. In this study, we propose to uterine lethality through to mild dysmorphism/mental impairment with a show that the association between soluble CD40 ligand (sCD40L), some carrier frequency of ~3 % in Caucasians. Local diagnostic biochemical clotting and thrombotic factors and cholesterol in childhood period. services have been offered since 1995, however, results may be equivocal, Methods and results: Plasma levels of sCD40L, sP-selectin, 8-hydroxy-2- carrier testing is challenging and not readily available in the UK. deoxyguanosine (8-OHdG), prothrombin fragment 1+2 were determined using A UKGTN DHCR7 mutation service by sequencing (ABI3730) was intro- commercial ELISA kits in 27 hypercholesterolemic patients and 36 healthy duced in 2009 for: 1) diagnosis in biochemically/clinically equivocal/nor- normocholesterolemic subjects. Significant differences were measured between mal cases with a SLOS phenotype, 2) retrospective carrier status of parents two groups in respect of total cholesterol, LDL cholesterol, VLDL cholesterol where no proband material remains, 3) cascade carrier studies. and triglycerides, sCD40L (p<0.05). The values of patients were higher than Two pathogenic mutations have been detected in 22 clinically/biochemi- controls. Although P-selectin values were higher in patients group, but this result cally affected cases with subsequent confirmation of carrier status for 10 was not significant as statistically between the two groups (p00,51, r00,247). couples. 6 deceased and 3 live patients where biochemical testing was not In the Spearman correlation analysis, a positive correlation was observed possible had no mutations. 7 parents with a fetus or deceased child with ? between sCD40L and total cholesterol, LDL- cholesterol, VLDL- SLOS, 3 population risk partners and 2 patients with family history have cholesterol, P-Selectin (p<0.05). But there were no significant correlation also been tested. between sCD40 and triglycerides, 8-OHdG, F1+2. (p>0.05). We present the clinical and mutation data (including one novel variant) of Conclusion: Future prospective studies with larger sample size are needed patients tested to demonstrate the utility of the DHCR7 mutation service in to confirm detection of increased CD40 ligand and P-selectin levels in the facilitating diagnosis, identification of carriers, broad phenotypic range of setting of the pediatric population with familial dyslipidemias may be affected patients and possible genotype-phenotype correlations. helpful to predict an increased risk of incident cardiovascular events. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S133

P-353 P-355 GENOTYPIC AND PHENOTYPIC CHARACTERISATION OF 15 ASSOCIATION BETWEEN INFLAMMATORY MARKER OF SLOVAK PATIENTS WITH SMITH-LEMLI-OPITZ SYNDROME MACROPHAGE ACTIVATION AND ENDOTHELIAL DYSFUNC- Skokňová M1 , Behúlová D2 , Fajkusová L3 , Dolníková D1 , Bzdúch V1 TION IN FAMILIAL HYPERCHOLESTEROLEMIC CHILDREN 11st Dep Ped, Child Univ Hosp, Bratislava, Slovakia Kologlu T1 , Kalkan Ucar S2 , Levent E3 , Delen Akcay Y1 ,Yıldırım 2Dep Lab Med, Child Univ Hosp, Bratislava, Slovakia Sozmen E1 , Coker M2 3Dep Bioch Mol Gen, Res Inst Child Heal, Brno, Czech Republic 1DivMedBioch,EgeUnivChildHosp,Izmir,Turkey 2Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey Objective: Estimated incidence of Smith-Lemli-Opitz syndrome (SLOS) in Slovak 3Div Ped Card, Ege Univ Child Hosp, Izmir, Turkey population is 1/15 000-20 000 live births. The purpose of this study was to determine the characteristics of SLOS patients from a typical middle European country. Background: It has been clearly known that there is a correlation between Methods: Clinical suspicion of SLOS was confirmed by a characteristic inflammation markers and subclinical atherosclerosis markers, in early stages of sterol pattern in serum determined by gas chromatography-mass spectrom- atherogenesis in subjects with familial hypercholesterolemia (FH). Chitotriosidase etry. Rapid PCR/RFLP technique and sequencing was used to detect muta- (ChT) enzyme, as an inflammatory marker, was found as elevated in extracts of tions in the 7-dehydrocholesterol reductase (DHCR7) gene. atherosclerotic tissue. The aim of this study was to investigate potential relationship Results: The most common clinical features were microcephaly (14/15), 2- between the carotid intima media thickness (cIMT), flow-mediated dilation (FMD) 3 toe syndactyly (14/15), oral cleft defects (13/15), micrognathia (13/15), and the inflammation markers of familial hypercholesterolemic (FH) children. cardiac defects (10/15) and ophtalmological abnormalities (8/15) including Methods: 48 FH children and 24 healthy age matched control subjects who have cataract. Four 46,XY individuals had total sex reversal. been managed by Metabolic Disorders outpatient clinic, were taken into study. There were found seven different mutations (W151X, V326L, G410S, Inflammation and macrophage activation markers, serum oxidation as well as lipid R352Q, IVS8-1 G>C, S397L, L109P). Mutations W151X (15/30) and parameters of all patients were measured. cIMT, FMD levels also were determined. V326L (6/30) accounted together for 70 % of all mutant alleles. One of Results and conclusion: Our data suggested that clinically evidenced (by cIMT the most common genotype W151X/W151X was connected with severe and FMD levels) atherosclerosis starts in early ages of children with hypercholes- phenotype and very low plasma cholesterol (0.017 mmol/l). terolemia and LDL oxidation seems as first finding for these patients. Higher Conclusion: The spectrum of DHCR7 mutations in Slovak patients is different cholesterol and oxidatively modified LDL levels strongly correlated with macro- from commonly published data but it is closed to that observed in Poland. High phage activation markers (ChT, YKL-40 and myeloperoxidase). ChT and YKL-40 incidence of cleft palate, cataract and congenital cardiac defects is supposed to be seems more predictable markers of atherosclerosis even in early ages (under 6 years influenced by the prevalent nonsense W151X mutation in the Slovak population. old) than other classical inflammation markers such as hs-CRP, IL-6, TNFα,MPO.

P-354 A-037 EFFECTS OF LOW DENSITY LIPOPROTEIN APHERESIS ON LIPOPROTEIN ASSOCIATED PHOSPHOLIPASE A2 AS AN EARLY ENDOTHELIAL FUNCTION AND PLATELET ACTIVATION IN ATHEROSCLEROTIC MARKER IN HIPERCHOLESTEROLEMIC HYPERCHOLESTEROLEMIC CHILDREN CHILDREN Kologlu T1 , Kalkan Ucar S2 , Levent E3 , Delen Akcay Y1 ,Yıldırım Öngen B1 , Kalkan Ucar S2 , Levent E3 , AzarsızE1 , Koloğlu T1 ,Yıldırım Sozmen E1 , Coker M2 Sözmen E1 ,SağınFG1 , Coker M2 1Div Med Bioch, Ege Univ Child Hosp, Izmir, Turkey 1Div Med Bioch, Ege Univ Child Hosp, Izmir, Turkey 2Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey 2Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey 3Div Ped Card, Ege Univ Child Hosp, Izmir, Turkey 3Div Ped Card, Ege Univ Child Hosp, Izmir, Turkey

Background: The aim of this study was to determine the parameters in Background: Lipoprotein associated phospholipase A2 (Lp-PLA2) is an enzyme relation with endothelial function and platelet activation in children with that hidrolysis oxidized phosphatidylcholine producing potent pro-inflammatory familial hypercholesterolemia by measuring serum homocysteine, assime- agents like oxidized free fatty acids and lysophosphatidylcholine. The purpose of tric dimethyl argininin (ADMA), nitrotyrosine and p-selectin. thisstudyistodetermineLp-PLA2massin hipercholesterolemic children and Methods: 35 heterozygous familial hypercholesterolemic patients, 10 homozygous investigate the relationship between Lp-PLA2andknownmarkers (carotid intima familial hypercholesterolemic patients treating by LDL apheresis, and 25 healthy media thickness-IMT and flow mediated dilatation-FMD) of atherosclerosis. children, all aged between 2 to 16, were enrolled in this study. Echocardiography Methods: Our study included 43 hypercholesterolemic children and 24 healthy was performed and intima-media thickness (IMT), endothelium-dependent vaso- children. Demographic information and family history were obtained from all dilation parameters were evaluated. LDL apheresis was performed by adsorbtion cases. Routine parameters (total cholesterol, LDL, HDL, triglycerides, glucose) method by using double membran filtration technique. Plasma nitrotyrosine, were analyzed in serum samples. Lp-PLA2 mass were determined with a homocysteine, p-selectin and ADMA levels were determined with an enzyme- turbidimetric immunoassay method. Carotid IMT and FMD measures of all cases linked immunosorbent assay (ELISA) using a commercial kit. were conducted by a blind method with a high resolution B-mode ultrasound. Results: Plasma homocysteine (p00,000), ADMA (p00,005), nitrotyrosine Results: In dyslipidemic group total cholesterol, HDL, LDL, TG and (p00,808), p-selectine (p00,466) were lowest in the LDL apheresis group. Lp-PLA2 (p00.000) values were higher than the controls. IMT values were Positive correlation was detected between homocysteine and intima/media thick- higher (p00.001) and FMD values were lower (p00.001) in patient group. ness (r00,334, p00,043). Our data showed that LDL apheresis therapy might Positive correlations were established between Lp-PLA2 and TK decrease serum levels of homocysteine, ADMA, and nitrotyrosine, and eventu- (R00.41, p00.001), LDL (R00.36, p00,004), IMT (R00.44, p00.019) and ally plays an important role in the improvement of endothelial dysfunction and a poor negative correlation was established between Lp-PLA2 and FMD platelet activity. (R00.15, p00.446). Conclusion: This current study has great importance also with the aspect of Conclusions: Lp-PLA2 mass can be used as a marker in hypercholester- being the preliminary study investigating the effects of LDL apheresis in olemic children to determine early atherosclerotic changes. hypercholesterolemic children. S134 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

27. Vitamins and Trace Minerals P-358 THE LYSINE RESTRICTED DIET AS NOVEL ADD-ON THERAPY P-356 FOR PYRIDOXINE DEPENDENT EPILEPSY: PILOT STUDY MOLECULAR CHARACTERIZATION OF FOLATE RECEPTOR 1 RESULTS MUTATIONS REVEAL PHENOTYPIC VARIATIONS OF van Karnebeek CD1 , Hartmann H2 , Mahmutoglu S.3 ,J.S.4 , Das A2 , CEREBRAL FOLATE TRANSPORT DEFICIENCY Cheng B1 , Giezen A1 , Meyer U2 , Struys E5 , Jakobs C5 , van der Lee JH6 , Steinfeld R1 , Grapp M1 , Gärtner J1 Paschke E7 , Plecko B8 , Stockler S1 1Dept of Pediatrics, Univ of Goettingen, Goettingen, Germany 1Div Biochem Diseases,BC Children's Hosp, VANCOUVER, Canada 2Hannover Univ Med School, Hannover, Germany Cerebral folate transport deficiency is an inherited brain-specific folate transport 3Dept Metab Dis, Hosp Sick Kids, Toronto, Canada defect that is caused by mutations in the folate receptor 1 gene coding for folate 4Dept Pediatrics, BC Childrens Hosp, Vancouver, Canada receptor alpha. This genetic defect gives rise to a childhood-onset neurodegener- 5Metab Lab, Free Univ Med Centre, Amsterdam, Netherlands ative disorder. We screened 72 children with low 5-methyltetrahydrofolate 6Dept Pediatr, Acad Med Centre, Amsterdam, Netherlands concentrations in the CSF and neurological symptoms. We identified nucleotide 7Dept Ped, Med Univ Hosp Graz, Graz, Austria alterations in the folate receptor 1 gene in 10 individuals that shared develop- 8Dept Neurol,Zurich Univ Hosp, Zurich, Switzerland mental regression, ataxia, profound cerebral hypomyelination and cerebellar atrophy. We found four novel pathogenic alleles, one splice mutation and three Background: Pyridoxine Dependent Epilepsy classically presents as a neo- missense mutations. Heterologous expression of the missense mutations natal onset epileptic encephalopathy. This cerebral organic aciduria is caused including previously described mutants revealed minor decrease in by deficiency of antiquitin (ATQ) (α-aminoadipic semialdehyde dehydrogen- protein expression but loss of cell surface localization, mistargeting ase, ALDH7A1), an enzyme facilitating lysine catabolism. Despite adequate to intracellular compartments and thus absent cellular binding of folic seizure control with pyridoxine in most cases, neurodevelopmental outcome is acid. These results explain the functional loss of folate receptor alpha poor with 80 % of patients suffering developmental / cognitive deficits. for all detected folate receptor 1 mutations. Noteworthy, three indi- Objective: To evaluate effects of dietary lysine restriction as add-on ther- viduals presenting a milder clinical phenotype revealed very similar apy on biochemical parameters, seizure control and neurodevelopmental biochemical and brain imaging data but partially shared pathogenic outcomes in patients with molecularly confirmed ATQ deficiency. alleles with more severely affected patients. Our studies suggest that Methods: A pilot study with observational n-of-1 cohort design was performed; different clinical severities do not necessarily correlate with residual 4 patients were started at mean age 6 yrs on lysine restriction 50-60 mg/kg/day function of folate receptor alpha mutants and indicate that additional and regular monitoring and outcome assessments during a 12-24mos period. factors contribute to the clinical phenotype in cerebral folate transport Results: The diet was well tolerated; good compliance, no reports of adverse- deficiency. events. Observed reduction of plasma pipecolic acid on diet was 59-84 % of pre-treatment level; for urine α-AASA 57-86 %. (Subjective) Improvements of neurodevelopment, behaviour and seizure control are reported. Conclusions: Our pilot study suggests a positive effect of lysine restriction on relevant outcomes in ATQ deficiency (evidence level 4). The PDE P-357 consortium has initiated further research using novel trial methodologies EXTREME VITAMIN D DEFICIENCY AFTER DISCONTINUATION to generate natural history data and more solid therapeutic evidence, before OF A PROTEIN RESTRICED DIET IN 3-METHYL-CROTONYL- burdensome dietary therapy becomes mainstay treatment. COA-CARBOXYLASE DEFICIENCY Pontes C1 , Mueller E1 , Doering JH2 , Haas D1 1Center Metab Dis, Univ Child Hosp, Heidelberg, Germany 2Dept Ped Neur, Univ Child Hosp, Heidelberg, Germany

3-methyl-crotonyl-CoA carboxylase (3-MCC) deficiency is an organic aciduria, a defect of leucine catabolism. Affected patients show a variable clinical course. When extended newborn screening was implemented, it turned out that most patients remain asymptomatic. Thus, the approach to the disease was revised. A diet or pharmacotherapy is no longer considered appropriate whereas a decade ago patients were treated with leucine restriction including vitamin and amino acid supplementation, carnitine and glycine. We report on a 12-year-old female with 3-MCC deficiency with a massive isolated vitamin D deficiency detected by a routine examination three years after termination of the leucine restricted diet. 25-OH-vitamin D was not detectable, resulting in pronounced hypocalcaemia, secondary hyperparathyroidism, elongated QTc-time and marked elevation of CK. Enteral malabsorption could be ruled out. There were no signs for osteopenia. The patient did not complain about any discomfort. From the nutritional history it became obvious that she still did not consume meat or dairy products, thereby continuing the protein restricted diet, but without amino acid or vitamin supplementation. Conclusion: Due to possible habituation effects, the termination of a protein restricted diet is not trivial. It has to be assured that there is no alimentary deficiency developing even years later. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S135

P-359 P-361 SEVERE SELF-INJURIOUS BEHAVIORS AS MAIN CLINICAL EARLY DIAGNOSIS OF PYRIDOXAL-5'-PHOSPHATE OXIDASE MANIFESTATION OF A NEW FORM OF CEREBRAL FOLATE (PNPO) DEFICIENCY AND OUTCOME AT 8 MONTHS DEFICIENCY. Kern I1 , Plecko B2 , Paschke E3 , Korff C4 , Fluss J4 Leuzzi Vincenzo1 , Mastrangelo Mario1 , Celato Andrea1 , Carducci Carla2 , 1Ped Nephrol & Metabol, Univ Child Hosp, Geneva, Switzerland Carducci Claudia2 2Ped Neurology, Univ Child Hosp, Zurich, Switzerland 1Div Child Neurol Sapienza Univ Rome, Rome, Italy 3Lab Metab Dis, Dep Pediatr, Univ Graz, Graz, Austria 2Dep Exp Med Sapienza Univ Rome, Rome, Italy 4Ped Neurology, Univ Child Hosp, Geneva, Switzerland

Introduction: Cerebral Folate Deficiency (CFD) is a severe neurometabolic PNPO-deficiency abolishes pyridox(am)ine oxidation to pyridoxal-5'- encephalopathy associated with low levels of 5-methyltetrahydrofolate phosphate(PLP), resulting in intractable neonatal seizures. Most of the <20 (5MTHF) in cerebrospinal fluid (CSF) and normal systemic folate metabo- published cases suffered severe/lethal brain damage as treatment was delayed. lism. We report on a new clinical variant of CFD. A 35 week premature newborn presented intractable seizures in the delivery Case report: A 6 years old male with intellectual disability, hypotonia, room (interictal suppression-burst intermixed with continuous electroclin- acquired microcephaly and without epilepsy and autistic features came to ical erratic myoclonic seizures), refractory to phenobarbital, phenytoin and our attention because of severe recurrent self-injuring behaviors (SIBs) pyridoxine. At 24 h, he was sedated and intubated. Upon PLP introduction (hitting his head by hands or feet with a rapid, stereotyped, compulsive at 72 h, he improved progressively, despite initially marked hypotonia and limb movements), which were triggered by selective environmental minimal response to stimulation. changes or frustrating novelties. MRI was normal. He had a low cerebro- Elevated vanillactate with normal pipecolate and α-aminoadipic semialdehyde spinal fluid (CSF) concentration of 5-methyltetrahydrofolate (5MTHF), pointed towards PNPO-deficiency. A new homozygous intronic mutation was increased CSF biopterin and normal values of peripheral blood folate. found, predicted to alter a donor splice-site, resulting in PNPO-truncation. Mutations in Folate receptor 1 gene and the production of folate receptor From the age of 8 weeks, riboflavin(8-10 mg/kg/d), a PNPO-cofactor, was blocking/binding autoantibodies were excluded. MTHFR gene sequencing added to PLP(30-40 mg/kg/d), in an attempt to enhance residual enzymatic and DHPR enzyme activity were normal. Lesch Nyhann, Rett and Angel- activity. At 8 months, he exhibits moderately delayed gross motor mile- man syndromes were excluded. Folate supplementation restored almost stones, mild truncal hypotonia, but close to normal fine motor and early completely CSF folate without influencing either biopterin and biogenic social and communicative skills. No seizures recurred, aside from an amine concentrations or SIBs. accidental 11 h-PLP delay causing a 45' seizure and 2 further seizures after Conclusions: Developmental delay, mental disability, acquired microce- 8 h-intervals, subsiding 15' post-PLP. Current EEG revealed no epileptic phaly, severe SIBs and undetectable 5MTHF in CSF with remarkable focus and age-appropriate background activity. increase of biopterin are all hallmarks of this new condition associated to Severe PNPO-deficiency is compatible with encouraging psychomotor devel- CFD. Epilepsy and autism spectrum disorder are notably absent. opment if treated early. Riboflavine may enhance residual PNPO-activity.

P-360 P-362 BROWN-VIALETTO-VAN LAERE AND FAZIO LONDE SYNDROME VITAMIN D REPLACEMENT IN METABOLIC PATIENTS A REVIEW OF THE LITERATURE Newton R1 , Sharma R1 , Smith E1 Stroek K1 , Bosch AM1 1Dept of Metab Dis, Salford Royal Hosp, Salford, United Kingdom 1Dept Pediatrics AMC, Amsterdam, Netherlands Background: Daily replacements with combined calcium and Vitamin D Background: Brown-Vialetto-Van Laere (BVVL) and Fazio Londe (FL) (VitD) preparation for the patients who are VitD deficient are poorly syndrome is a neurological disorder presenting from early childhood to tolerated and have reduced compliance. There are also concerns regarding adulthood, with symptoms of neurological deterioration, deafness, ponto- increased cardiovascular risks due to calcium supplements. bulbar palsy and respiratory insufficiency. Recently the gene has been This audit was designed to explore whether measuring dietary calcium recovered and the involvement of a riboflavin transporter defect recognized. intake will help in appropriate prescribing of only VitD or a combination Objectives: Gain insight into the course of disease of patients presenting in with calcium in adult patients with metabolic diseases. childhood. Method: Dietary calcium was calculated using validated dietary calcium Methods: A search in Pubmed for BVVL and FL syndrome revealed 29 calculator questionnaire when patients attended adult IMD clinics. Patients retrievable English publications reporting on 57 cases. serum VitD level was measured. The results were reviewed by the con- Results: Patients presenting before age 3 years (mean 12 months) had a sultants prior to prescribing/changing replacement therapy. severe disease with respiratory problems (14/16), bulbar palsy (13/16) and Results: Out of 50 patients who were audited, 19 were on combined hearing loss (9/16). Survival is known for 15 patients of which 10 died prescription. 9(18 %) were changed to VitD only. Two patients required (mean age 1.8 years). 4/5 surviving patients were treated with riboflavin. dietary advice to increase calcium. Additional 24 patients were identified as Patients presenting at age 3-10 years (mean 7.8) demonstrated deafness (16/ low in dietary calcium/VitD or both and were treated accordingly. 16), bulbar palsy (16/16) and respiratory problems (10/16). 8/16 died (mean Conclusion: Dietary calcium calculator helped in appropriate prescribing age 17.2, range 10.5-42 years). Patients presenting at age 11-17 years and improved compliance. The process helped patients in understanding the demonstrated deafness (22/25), bulbar palsy (22/25), and respiratory prob- advice/prescription they were given. This audit mainly included patients lems (11/25). 6/25 died (mean age 18.5, range 12-25 years). with lysosomal storage disorder. It is now standard practice in our depart- Conclusion: Severity of BVVL and FL syndrome is negatively correlated ment for patients to complete a calcium questionnaire prior to prescribing with age and many patients die. All four patients presenting before age three decision. treated with riboflavin survived. S136 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-363 P-365 CALLOSAL AND WHITE MATTER FEATURES IN PYRIDOXINE- MOLYBDENUM COFACTOR (MOCO) DEFICIENCY TYPE B; DEPENDENT EPILEPSY (PDE) CLINICAL, BIOCHEMICAL AND NEUROIMAGING FEATURES Ishak GE1 , Friedman SD1 , Poliakov AV1 , Poliachik SL1 , Shaw DWW1 , OF FIVE PATIENTS WTH TWO NOVEL MUTATIONS Gospe SM2 Hişmi B1 , Ünal Ö1 , Sass JO2 , Beermann F3 , Ichida K4 , Dursun A1 , Sivri 1Rad, U Washington & Seattle Child Hosp, Seattle, United States HS1 , Tokatlı A1 , Coskun T1 2Neuro, U Washington & Seattle Child Hosp, Seattle, United States 1Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey 2Kinderspital Zürich, Clin Chem & Biochem, Zürich, Switzerland Background: Brain white matter abnormalities, ventriculomegaly and dif- 3Lab Klin Biochem Stoffw, ZKJ, Univ.Klin, Freiburg, Germany fuse thinning of the corpus callosum (CC) have been described in patients 4Tokyo Univ, Depart Pathophsiology, Tokyo, Japan with PDE, while some report thinning limited to the posterior CC. Objectives: Evaluate the generalizability of atypical CC in a larger PDE MoCo deficiency is a devastating disease leading to sulfite oxidase (SUOX) cohort including using diffusion tensor imaging (DTI). deficiency with prenatal onset brain disruption. A therapeutic approach Patients/Methods: 11 pyridoxine-dependent (PD) patients, 5 F/6 M, 17.91 using a precursor of MoCo is available for type A, the most frequent form ±11.20 years, and age/gender matched controls (5 F/6 M, 17.27± of this disesase, but only symptomatic treatment exists for type B, caused 10.27 years) underwent brain MRI. The CC was divided in 5 segments by mutations in the MOCS2 gene. Five Turkish type B patients from four (A-E from anterior to posterior). We evaluated 2 ratios; CCsegment:CCtotal familes with severe neonatal presentation are described. Cranial ultrasound (area) and CCsegment:Cerebrum cross-sectional area (same mid-sagittal revealed subependymal germinal-matrix hemorrhage in two children. Cer- slice). DTI (4 patients) was also analyzed. ebral atrophy, multiple subcortical cavities, posterior fossa abnormalites, Results: Analyses showed differences in anterior (PDE>Control) and two poste- cerebellar hypoplasia, corpus callosum dysgensis, mega cisterna magna rior segments (PDEA features suggesting an altered interhemispheric connection pattern. FA and c.G68C>G) have not been reported previously. Our findings extend the findings and literature evaluating other developmental diseases (e.g. CC information on a disease, which can well be detected using simple labo- agenesis) support further evaluation of patterns related to callosal fiber- ratory markers, if it is included in the differential diagnosis. tracking and interhemispheric connectivity. Conflict of Interest declared.

P-364 P-366 EVALUATION OF VITAMIN B12 LEVELS DURING THE FIRST PYRIDOXINE DEPENDENT EPILEPSY: CLINICAL FEATURES TRIMESTER OF PREGNANCY AND PROGRESSIVE SERIAL BRAIN MRI ABNORMALITIES Dayaldasani A1 ,YahyaouiR1 , Ruiz-Escalera JF1 ,Rodríguez-EspinosaM1 , Niermeijer JMF1 , Abeling N2 , Koelman HJ3 , Poll-The BT1 Casero MC1 ,RuedaI1 ,Pérez-ValeroV1 1Dept of Paed Neurol, Ac Med Centre, Amsterdam, Netherlands 1Clin Lab, Carlos Haya Hospital, Málaga, Spain 2Dept of Metabol Dis, Ac Med Centre, Amsterdam, Netherlands 3Dept of Clin Neurophys, Ac Med Centre, Amsterdam, Netherlands Background: During pregnancy, vitamin B12 concentrations may drop physiologically, and concentrations below reference values may not alter Background: Pyridoxine dependent epilepsy (PDE) is a treatable meta- certain parameters. The use of non-pregnant values may not be appropriate bolic autosomal recessive epileptic encephalopathy that is characterised by for assessing vitamin B12 status during pregnancy. a therapeutic response to vitamin B6. It is diagnosed by a pyridoxine trial Method: Vitamin B12 concentrations were evaluated in 204 pregnant and confirmed by analysis of concentrations of pipecolic acid in CSF and women during the first trimester to calculate the reference interval. Possible blood and α-AASA (alfa-aminoadipic semialdehyde) in urine and further confounding factors were analyzed using stepwise multiple linear regres- confirmed by genetic testing. PDE is caused by a mutation in the antiquitin sion models. Other factors including the newborns' birthweight, and pro- gene (ALDH7A1) Brain MRI abnormalities consist of generalised cerebral pionylcarnitine levels, as well as C3/C2 and C3/C16 ratios were considered. atrophy, corpus callosum agenesis, and nonspecific white matter abnormal- Results: The reference interval for vitamin B12 obtained was 272.7– ities. However, little is known about the evolution of the radiological 837.8 pg/mL. Vitamin B12 concentrations were significantly lower in abnormalities in time. smokers, and in women with low meat consumption. Other factors did Methods: We here describe the brain MRI abnormalities and clinical not have any significant effect. Newborns of mothers with lower vitamin features in 4 patients with PDE aged 3 months – 6 years. B12 levels presented lower birth weight and higher propionylcarnitine Results: MRI showed vasogenic oedema, hydrocephalus and aqueduct levels, with higher C3/C2 and C3/C16 ratios. stenosis, and delay of myelination. In three patients there were serial radio- Conclusions: The reference interval for serum vitamin B12 concentrations logical exams, that showed progressive lesions in time. obtained is narrower than the one currently used for our general population. Conclusions: Theoretically the progressive MRI abnormalities could be Smoking seems to have a negative effect, and meat consumption a positive caused by the severity of the continuous shortage of pyridoxine that causes effect on vitamin B12 levels. Mothers with lower vitamin B12 concentra- a depletion of GABA, or by damage secondary to epileptic seizures. As tions during the first trimester of pregnancy seem to have children with literature on this specific aspect of PDE is scarce, the presented case series lower birth weight and higher propionylcarnitine levels. may help in understanding the progressive course of PDE. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S137

P-372 P-374 EFFECT OF DIETHYLDITHIOCARBAMATE (DEDTC) ON A EUROPEAN PATIENT SURVEY IN WILSON DISEASE TO LYSYL OXIDASE (LOX) ACTIVITY IN THE CULTURED IMPROVE THE MANAGEMENT OF THIS RARE DISEASE SKIN FIBROBLASTS FROM PATIENTS WITH MENKES RUANO E1 , WOIMANT F2 , TROCELLO JM1 DISEASE 1EuroWilson, European network for WD, Paris, France Hiroki T1 , Fujisawa C1 , Miyazawa M1 , Ogawa E1 , Kodama H1 2French Nal Ref Centre for Wilson disease, Paris, France 1Dep Pediat, Teikyo Univ School of Med, Tokyo, Japan Background: EuroWilson is a European network for professionals and Menkes disease (MD) is an X-linked disorder characterized by progressive patients' groups from 20 countries dedicated to Wilson Disease (WD). Its neurodegeneration and connective tissue abnormalities due to the defect of mission is to improve the quality of care and access to multidisciplinary a copper-transporting ATPase (ATP7A). In affected cells, copper transport expertise for European patients with WD. by ATP7A from cytosol to Golgi apparatus is disturbed, resulting in Objective: This survey will allow us to better define patients priorities and reduction of the activities of copper-dependent enzyme such as lysyl oxi- expectations from a European network. dase (LOX). Connective tissue disorders are caused by the reduced LOX Method: 23 questions have been elaborated with European patient organ- activity. The current subcutaneous administration of copper histidine does isations and EuroWilson members, available online from September to not improve connective tissue disorder, because the administered copper is December 2011 in English, French, German, Italian and Spanish. not transported into Golgi apparatus due to ATP7A defects. In this study, we Results: 269 patients or relatives of a patient replied to the survey. The investigated the effect of Diethyldithiocarbamate (DEDTC), a lipophilic mean age was 34 years old (3 to 83). 83 % were members of patient's chelater on LOX activity using the cultured skin fibroblasts from MD organisation. patients. The skin fibroblasts were cultured with or without 0.2 μM DEDTC Time to diagnosis was less than one year for 48 % of patients but over 3 years for 48 hours and LOX activities in culture media were assayed. Without for 15 % . This period was not significantly different between countries. DEDTC, LOX activities in the culture media of MD fibroblasts were The access to an expert centre varied between countries (90 % in France, significantly lower than those of control fibroblasts. With DEDTC, LOX 53 % in Spain) which shows real inequalities. Collaboration between local/ activities in the culture media of MD fibroblasts were remarkably increased national organisations needs to be improved. and there was no significant difference in LOX activities between MD and Conclusion: Time to diagnosis must be shortened, facilitating the access for control fibroblasts, suggesting that DEDTC can improve copper transport physicians to detailed information on WD. This survey underlines inequal- from cytosol to Golgi apparatus and consequently the activity of copper- ities in Europe. EuroWilson must take into account these conclusions to dependent LOX. better answer to patient's expectations.

P-373 P-375 HYDROXYCOBALAMIN INJECTIONS IN CHILDREN WITH IMPROVEMENT OF NEUROLOGICAL STATUS BY INTRAVE- COBALAMIN C DEFECT NOUS ADMINISTRATION OF HIGH DOSAGES OF FOLINIC McSweeney M1 , Abulhoul L1 , Cleary MA1 ACID IN TWO GIRLS WITH FOLATE TRANSPORTER DEFI- 1Dept Metabolic Medicine, GOSH, London, United Kingdom CIENCY DUE TO MUTATIONS IN THE FOLR-1 GENE. Nassogne MC1 , Häberle J2 , Thöny B2 , Lindner M3 , Clapuyt P1 , Vincent Background: Cobalamin C defect requires intramuscular (im) hydroxyco- MF1 , Collins C1 , Blaud N3 balamin (OHCbl) injections to achieve satisfactory metabolic control. IM 1Univ cathol Louvain, Clin univ St-Luc, Bruxelles, Belgium injections can be traumatic resulting in needle phobias and aberrant behav- 2University Children's Hospital Zurich, Zurich, Switzerland iours especially in children. There is little data on alternative parenteral 3Univ Children's Hospital Heidelberg, Heidelberg, Germany routes. Objectives: To investigate whether subcutaneous administration of OHCbl A previously healthy infant developed an ataxic gait in the 2nd year of life. At can achieve satisfactory biochemical control in children with cblC and the age of 3, MRI showed a diffuse abnormal signal of the white matter. Clinical reduce associated pain and anxiety. evolution was characterized by severe psychomotor regression and myoclonic Method: Three children with cblC and needle phobia were switched over to epilepsy. Her young sister developed the same clinical picture. A puncture using a subcutaneous pen device for the administration of OHCbl. Dose and revealed a profound deficiency of 5MTHF in CSF (<1 nmol/l; normal : frequency of drug was unchanged. Metabolic control was monitored by 53–182). Molecular investigations confirmed the presence of two mutations tHcy and MMA (plasma and urine). c.332 G>T (p.Glu108X) and c.373 C>T (p.Arg125Cys) in the the FOLR1 Results: Parents reported sc route was easier and faster; the child was more gene. Treatment with folinic acid (5 mg/kg/day po) was started with some cooperative and less anxious. Children still reported injection-associated clinical improvement, but with the persistence of severe epilepsy. CSF analysis pain but preferred sc to IM as the procedure was completed faster. Satis- revealed a normal level of 5MTHF (60.1 nmol/l ; normal : 63-111). Epilepsy factory biochemical control was maintained. remained difficult to treat and the young girl presented with psychomotor Conclusion: These data suggest that sc administration of OHCbl can regression. Due to this evolution, high doses of folinic acid were given by maintain metabolic control in CblC. Since needle phobias are common intravenous administration during 3 days (12.5 mg/kg/day) with a significant when giving regular IM injections, sc route should be explored in this and improvement of the clinical status and the epilepsy. CSF analysis revealed very other metabolic disorders responsive to IM OHCbl. high level of 5MTHF after intravenous infusion ci (–>180 nmol/l ; normal : 31- 130). Folate receptor defect is a newly identified potentially treatable neurometabolic disorder, but dosages of folinic acid and long-term outcome remain unknown. S138 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-381 P-383 PATIENTS WITH BIOTINIDASE DEFICIENCY DETECTED CEREBRAL FOLATE DEFICIENCY ASSOCIATED WITH A THROUGH NEWBORN SCREENING IN EGE REGION OF NOVEL HOMOZYGOUS NONSENSE MUTATION IN FOLR1 TURKEY Aylett S-B1 , Varadkar S2 ,FassoneE1 ,PopeS3 , Neergheen V3 , E Canda1 , H Onay2 , M Kagnici1 , M Kose1 , SK Ucar1 , S Habif3 ,F Hargreaves IP3 , Land JM3 , Heales SJ4 , Rahman S1 Ozkinay2 , O Bayindir3 , M Coker1 1CMGU, UCL Institute of Child Health, London, United Kingdom 1Pediatric Metabolism, Ege University, Izmir, Turkey 2Neurology Unit, Great Ormond St Hospital, London, United Kingdom 2Medical Genetics, Ege University, Izmir, Turkey 3Neurometabolic Unit, Nat Hosp for Neurol, London, United Kingdom 3Clinical Biochemistry,Ege University, Izmir, Turkey 4Chemical Pathology, Great Ormond St Hosp, London, United Kingdom

The aim of the study is to describe our experience in 66 biotinidase Background: Inborn errors of 5-Methyltetrahydrofolate (5MTHF) trans- deficiency (BD) patients. Mean age at diagnosis was 45±21 days. 30 port across the blood-cerebrospinal fluid (CSF) barrier result in decreased patients had profound deficiency. Two patients had skin manifestation and 5MTHF availability in the central nervous system, leading to cerebral folate one patient had hearing loss at the time of diagnosis. All other patients were deficiency (CFD) causing neurological dysfunction including seizures. asymptomatic. Two patients had double mutation (p.R157H; p.D444H), 1 Patient and Methods: The patient presented with global developmental delay at patient had D444H mutation in one allele, c.407-408 Ins A mutation 4 years and subsequently developed an intractable epileptic encephalopathy in the other allel, 1 patient had homozygote R157H, 1 patient had the Lennox-Gastaut spectrum resistant to multiple antiepileptic drugs (AEDs). homozygote F128S, 1 patient had heterozygote R157H, and 1 patient Muscle biopsy revealed cytochrome oxidase deficiency (50 % of lowest control had homozygote p.E64K, 1 patient had c.98,-104delinsTCC mutation activity) whilst CSF analysis demonstrated undetectable 5MTHF. There was no in one allele and D444H in the other allele, 1 patient had c.98,- evidence of systemic folate deficiency. Exon and exon-intron boundaries of 104delinsTCC in one allele and R157H in the other allele. We inves- FOLR1, encoding folate receptor alpha (the major folate transporter across the tigated BD in asymptomatic relatives. Seven relatives had profound blood-CSF barrier), were sequenced in genomic DNA from the patient. deficiency. Double mutation (p.R157H; p.D444H), homozygote Results: Sequence analysis of FOLR1 revealed a novel homozygous nonsense F128S, D444H mutation in one allele, c.407-408 Ins A mutation the mutation (c.610 C>T; p.R204X) in the patient; both parents were heterozygous. other allelehave been detected. Six patients were not detected through Conclusions: We report a case of severe CFD caused by a homozygous nonsense neonatal screening. Mean age was 47.2±23 months. 4 patients had mutation in FOLR1 associated with defective 5MTHF transport across the blood- profound deficiency. Two patients had motor mental retardation CSF barrier. Supplementation with oral folinic acid restored CSF 5MTHF levels (homozygote c.98,-104delinsTCC mutations), the others were asymp- to within the reference range, although seizures continued. FOLR1 mutations are a tomatic. Biotinidase deficiency must be included in the newborn potentially treatable cause of AED-resistant seizures, and CSF 5MTHF analysis screening programs in order to initiate early treatment. should be considered in all children with intractable epilepsy.

P-382 P-384 MOLECULAR CHARACTERISATION OF BIOTINIDASE GENE THIAMINE DEFICIENCY (BERIBERI) IN THE MODERN LIFE MUTATIONS IN TURKISH PATIENTS; AN UPDATE OF THE STYLE OF JAPANEASE CHILDREN: DETECTION BY URINARY RESULTS ORGANIC ACIDS ANALYSIS M Karaca1 , D Yucel2 , O Unal2 , A Guzel2 , A Tokatli2 , T Coskun2 ,A Hasegawa-Omura Y1 , Kobayashi H1 , Takahashi T1 , Yamada K1 , Dursun2 , HS Sivri2 , RK Ozgul2 Purevsuren J1 , Yamaguchi S1 1Dept of Biology, Aksaray University, Aksaray, Turkey 1Shimane University School of Medicine, Izumo, Japan 2Pediatr Metab Dis, Hacettepe University, Ankara, Turkey Background: Beriberi is caused by dietary deficit of the essential vitamin Biotinidase deficiency (BTD) is inherited as an autosomal recessive B1, thiamine and manifests hypotonia, cardiac failure and encephalopathy. manner, which caused by complete or partial absence of biotinidase Although beriberi is considered as a historical disease, we recently identi- enzyme activity. The purpose of this study was to determine the fied 10 Japanese beriberi infants using urinary organic acids analysis. spectrum of mutations of BTD gene in Turkish patients. dHPLC Patients and Methods: During 2004 through 2011, children with organic analysis and direct DNA sequencing were used for screening of 154 acids profiles that suggest vitamin B1-deficiency in GC/MS analysis were BTD patients from 128 unrelated families. We would like to highlight investigated. Their clinical symptoms, laboratory data and dietary histories that the data presented here is an extension of our previous study. were collected. Twenty one different mutations were described which of five were Results: Ten Japanese infants with beriberi were identified. Their urinary novel (c.1212-1222del11, c.1320delG, p.C143F, p.T244I and organic acids profiles demonstrated simultaneous elevation of branched chain p.G480R) and three (p.R209H, p.C418S and p.H447Y) have not been 2-ketoacids, 2-ketoglutaric acid and lactic acid, metabolites of the pathways already reported in Turkish patients. Among mutations, p.R157H, for which thiamine are required as a cofactor. Nine patients had cardiac failure c.98-104del7ins3 and p.D444H were common and accounting for following recurrent vomiting and walking difficulty. One patient had been 68 % of all BTD mutant alleles; with frequencies 38.3 %, 14.8 % taking white polished rice as dietary therapy for atopic dermatitis, and six had and 14.8 % respectively. Measurement of enzyme activity is important kept drinking 1-3.5 L isotonic drink every day for several months. in the diagnosis of BTD, however, mutation screening is crucial in Conclusion: As similar cases have been increasingly reported in Japan, it is confirmation of biochemical measurements and genotype-phenotype important to raise the public awareness of the potential risk of vitamin B1- correlation. It allows accurate and precise diagnosis of disease, early deficiency that could result from excessive intake of isotonic drink or over- treatment and prevention of damage via identifying asymptomatic restricted diets. In this regard, organic acids analysis using GC/MS is useful cases. to identify infantile beriberi. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S139

A-038 P-368 ZINC/COPPER METABOLISM IN AUTISM CLINICAL AND BIOCHEMICAL DELINEATION OF TH GENE- KIYKIM E1 , SOYUÇEN E1 , AKTUĞLU ZEYBEK AÇ1 , CANSEVER NEGATIVE SEVERE INFANTILE DOPAMINE DEFICIENCY MŞ1 , AYDIN A1 Ng J1 , Tuschl K2 , Carr LJ1 , Cleary M3 , Devlin A4 , Manzur A1 , Gissen 1Div Ped Nutr & Met, Cerr Med F, Ist Uni, İstanbul, Turkey P2 , Heales SJ5 , Heales SJR6 , Clayton P2 , Kurian MA2 , Kurian MA1 1Neurology, Great Ormond Street Hosp, London, United Kingdom Zinc is a component of more than 80 enzymes. High concentrations have 2UCL Institute Child Health, London, United Kingdom been found in brain hippocampus, and many researchers believe that zinc is 3Metab Dis, Great Ormond Street Hosp, London, United Kingdom a neurotransmitter. Low zinc levels at these sites could reduce the inhibition 4Neurology, Great North Children's Hosp, Newcastle, United Kingdom of neuron activity leading to abnormal behaviour. 5Neurometab, National Hosp Queens Square, London, United Kingdom Their role in behaviour is not yet clear, but could be involved in the 6Chem Path, Great Ormond Street Hops, London, United Kingdom transport or availability of zinc. Low levels of zinc are associated with behaviour disorders. Copper/zinc ratio appears to be more deci- Background: We report a cohort of children presenting with a severe sively important than either of the individual metals alone. Normal dopamine deficiency syndrome in early infancy. Their clinical features ratio is determined as ≥1. Zinc deficiency often results in elevated mimic infantile onset (Type B) Tyrosine hydroxylase (TH) deficiency. blood levels of copper. Elevated blood copper has been associated with Case Cohort: Ten infants were identified to have low HVA levels (range episodic violence, hyperactivity, learning disabilities, and depression. 60-224 nmol/L,34.7-82.1 %) below age-matched reference range) with The mean blood copper level of 142 patients who were followed with the normal 5-hydroxyindolacetic acid (5-HIAA) and pterins. The HVA/5-HIAA diagnosis of autism was 80.8 mg/L (43-125 mg/L). 76 of these patients' ratios were all low (range 0.17- 0.91, normal 1-3.7) with variable prolactin blood copper level were above the lowest normal level of the laboratory, (range 86-2608mu/L). TH gene screening did not detect any disease- 75 mg/L, and 66 were below the normal levels. More importantly, the causing mutations in this patient cohort. average zinc-copper ratios of these patients was 0.64 and only 13 patients All had features of axial hypotonia, limb hypertonia and extrapyramidal had this ratio ≥1. movements. Other features included ptosis, marked orobulbar dysfunction, In this study, the importance of the ratio of copper-zinc and zinc levels in encephalopathy and contractures. 8/10 infants required ventilatory support autistic patients was determined. for profound apnoea and respiratory insufficiency. 5/8 infants trialled on L-Dopa had a partial improvement of their movement disorder. However, all remained symptomatic on treatment and five have died in early infancy. Conclusion: This group of infants present with a life-threatening severe dopamine deficiency syndrome of undetermined aetiology. The possible pathogenic mechanisms for this neurotransmitter disorder would include 28. Neurotransmitter Disorders impaired differentiation of dopaminergic neurons, reduced dopamine syn- P-367 thesis, receptor defects and dopaminergic tract degeneration. Future work is likely to identify the underlying genetic defect in this cohort. SUCCESSFUL PREGNANCY IN A NEW PATIENT WITH EARLY ONSET DOPA-RESPONSIVE DYSTONIA DUE TO AUTOSOMAL RECESSIVE GTP CYCLOHYDROLASE (AR GTP-CH) DEFICIENCY WITHOUT HYPERPHENYLALANINEMIA. D'Agnano D1 , Carducci C2 , Carducci C2 , Leuzzi V1 1Dept Ped, Child Neur, Psyc,Sapienza Univ, roma, Italy , 2Dept Exper Med, Sapienza Univ, roma, Italy

Background: The effect of pregnancy as well as treatment in genetically confirmed AR GTP-CH deficiency has never been reported. Case report: We report the first case of pregnancy in a 27-year-old woman with this disorder. Since the age of 2 she presented progressive generalized rigidity, limb and cervical dystonia with diurnal fluctuation. At 7 years-old CSF examination showed: neopterin 4 nmol/L (r.v. 9-20), biopterin 13 nmol/L (r.v 10-30), normal homovanillic acid and 5- hydroxyindoleacetic acid. Neopterin and biopterin (mmol/mol Creatinine) were low in urine (0.1 r.v. 0.3-4.0, and 0.24 r.v. 0.5-3.0, respectively). Blood phenylalanine (Phe) was persistently normal, whereas oral Phe loading test revealed a severely impaired Phe clearance. GCH1 gene examination detected two novel missense mutations involving exon 3 (c.508A>G; R170G) and exon 6 (c.745A>G; R249G), which were transmitted by the proband father and mother, respectively. L-Dopa/Carbidopa treatment (10 mg/kg/day) started promptly, normalised neurological status. She became pregnant when 25. L-Dopa/Carbidopa dosage was reduced to 2 mg/kg/day. She delivered a healthy male infant, who was heterozygous for exon 3 mutation and showed a mild transient trunk dystonia during the first 2 years of life. Conclusion: Normal pregnancy and delivery as well as levodopa-carbidopa therapy are compatible with AR GTP-CH deficiency condition. S140 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-369 P-376 EARLY ONSET PARKINSONISM: A NEW CLINICAL AND THERAPEUTIC EFFICACY OF MAGNESIUM VALPROATE IN BIOCHEMICAL PHENOTYPE SUCCINIC SEMIALDEHYDE DEHYDROGENASE DEFICIENCY Galosi Serena1 , Celato Andrea1 , Mastrangelo Mario1 , Carducci Claudia2 , Vanadia E.1 , Gibson K.M.2 , Pearl P.L.3 , Trapolino E.4 , Mangano S.1 , Carducci Carla2 , Bertini Enrico3 , Leuzzi Vincenzo1 Vanadia F.4 1Div Child Neurol Sapienza Univ Rome, Rome, Italy 1Univ Dept Child and Adol Neuropsy, Palermo, Italy 2Dep Exp Med Sapienza Univ Rome, Rome, Italy 2Section Clin Pharmac, Washington, DC, United States 3Bambin Gesù Hosp, Rome, Italy 3Univ Dept Child Neurol, Washington, DC, United States 4Hospital Dept Child and Adol Neuropsy, Palermo, Italy A growing number of metabolic and degenerative diseases results in infantile parkinsonism. We report on a new clinical and biochemical phenotype Succinic semialdehyde dehydrogenase deficiency (SSADHD or gamma- so far not associated to any known aetiology. hydroxybutyric aciduria), a disorder of γ-aminobutyric acid (GABA) This 5-year-old child was born from non-consanguineous Italian parents after metabolism, manifests as a slowly progressive or static encephalopathy. uneventful pregnancy and normal delivery. Psychomotor development was nor- The latter encompasses prominent cognitive dysfunction, neuropsychiatric mal up to 4 months, when minimal tremor, upper limb dystonia and rigidity were morbidity and epilepsy. We report safe and effective treatment with MgVPA detected. At 8 months neurological deterioration occurred associated with limb in an adolescent female with SSADHD and seizures refractory to a broad jerks, generalised hypokinesia/dystonia, and akinetic mutism. CSF examination spectrum of antiepileptics. MgVPA therapy (20 mg/Kg/day) was introduced disclosed low homovanillic acid (HVA) (185.5 nmol/L, r.v. 295-932), neopterin at 7 years based upon behavioural difficulties and EEG alterations without (7.8 nmol/L, r.v. 12-30), and biopterin (5.5 nmol/L, r.v. 15-40). TH and of GCH1 adverse effects. Therapy was halted at age 13 years, and reintroduced at genes were both normal. L-Dopa/Carbidopa treatment resulted in a partial clinical 14 years, due to new onset complex partial seizures. EEG demonstrated improvement. From the age of 4, diurnal fluctuations of symptoms and severe on- improvement in epileptiform activity, associated with behavioural improve- off phenomena required a progressive increase of L-Dopa/Carbidopa posology. ment in disinhibition, aggression and coprolalia. Though typically avoided Social interaction and language comprehension remained relatively spared. A in SSADHD due to inhibitory effects on any residual enzymatic activity, further decline of HVA (77 nmol/L; r.v. 211-871) was found in CSF. SR, PTPS, valproate was effective and safe in our patient. Sodium valproate has GFRP genes sequences, and Array-CGH did not detect any alterations. Repeated previously demonstrated therapeutic utility in SSADHD, but the use of Brain MRI and 1 H-MRS as well as an extensive neurogenetic and neuro- the magnesium conjugate has not been reported. Epilepsy remains well metabolic work-up were all normal. Dopamino-mimetic/synergic drugs (Selegi- controlled in our patient, with concomitant improvements in behavioural lina, Tolcapone, Rotigotina, Pramipexole) proved to be ineffective. symptoms . Our results suggest that MgVPA intervention may have utility in selected cases of SSADHD.

P-370 P-377 PHYSIOLOGICAL MARKERS OF TAURINE THERAPY IN SUCCINIC TRANSDERMAL ROTIGOTINE AS A COMEDICATION IN THE SEMIALDEHYDE DEHYDROGENASE (SSADH) DEFICIENCY TREATMENT OF CONGENITAL DISORDERS OF BIOGENIC Schreiber J1 , Theodore WH1 , Pearl PL2 , Gibson KM3 AMINE 1Clin Epilepsy Section, NINDS/NIH, Bethesda MD, United States Mastrangelo Mario1 , Galosi Serena1 , Celato Andrea1 , D'Agnano Daniela1 , 2Childrens Natl Med Ctr, Washington DC, United States Giannini Maria Teresa1 , Bertino Sara1 , Leuzzi Vincenzo2 3Clin Pharmacol, Wash State Univ, Spokane WA, United States 1Div Child Neurol Sapienza Univ Rome, Rome, Italy 2Dep Exp Med Sapienza Univ Rome, Rome, Italy SSADH deficiency is a disorder of GABA metabolism for which targeted pharmacotherapy remains elusive. Taurine (2-aminoethanesulfonic acid) Background: Rotigotine is a novel non-ergot dopamine agonist, with effects interacts with both GABAA and GABAB receptors in different brain on the biogenic amine neurotransmissions. subsections, and pharmacological doses have shown therapeutic benefit in For this reason we have used transdermal rotigotine in three congenital the corresponding murine model and a single SSADH-deficient patient disorders of these neurotransmitters: Aromatic L-Aminoacid decarboxylase (Saronwala et al 2008; meeting abstracts, www.acmg.net). To build on (AADC) deficiency (OMIM # 608643), Tyrosine hydroxilase (TH) defi- these initial results, we have implemented an open label trial of taurine that ciency (OMIM # 191290) and Dopamine transporter (DT) deficiency will evaluate key SSADH biomarkers and neurocognitive performance, on/ (OMIM # 126455). off taurine (crossover design). Study evaluations include neurological and Case Report: In a 11 years old boy with AADC the replacement of neuropsychological examinations, positron emission tomography (PET) pergolide with transdermal rotigotine resulted in an improvement of trunkal with 11 C-flumazenil (FMZ), magnetic resonance spectroscopy and CSF stability, head control and motor planning. A faster and more autonomous collection to measure GABA levels, and transcranial magnetic stimulation walking without support, a faster execution of motor tasks and an increase (TMS) to measure cortical excitation and inhibition, in patients receiving of muscular strength were observed. taurine. Our goal is to define and characterize biomarkers in SSADH- A similar improvement was also observed in a 15 years old male with TH deficient patients that can be measured during therapeutic intervention. deficiency. In this case the benefits were transitory and after some weeks a Data acquired should enhance our ability to understand inborn metabolic severe bradykinesia was observed. The discontinuation of rotigotine and its errors that present with prevalent phenotypes such as epilepsy, intellectual replacement with levo-dopa removed this last side effect. disability, or autism spectrum disorder, and potentially other GABAergic In a 4 years, 7 months old boy with a DT deficiency no clinical effects were disorders such as GABA-transaminase deficiency. This trial is NIH- obtained while bradykinesia , somnolence and asthenia. approved (PTMS T-N-1976) and openly enrolling. For additional details, Conclusions: our experience suggests that transdermal rotigotine is a please contact Drs. J. Schreiber or P. Pearl ([email protected]; promising treatment in the therapeutic management of AADC deficiency. [email protected]) No results have been obtained in TH and DT deficiency. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S141

P-385 P-387 AROMATIC L-AMINO ACID DECARBOXYLASE DEFICIENCY: FACTORS AFFECTING 5-METHYLTETRAHYDROFOLATE (5MTHF) A NEW CASE FROM TURKEY WITH A NOVEL MUTATION STABILITY: IMPLICATIONS FOR CEREBRAL 5MTHF DEFICIENCY Kasapkara C S1 , Gucuyener K2 , Tumer L1 , Verbeek M3 Aylett S-B1 , Neergheen V2 , Eaton S1 , Land JM2 , Rahman S3 , Heales SJ4 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 1UCL Institute of Child Health, London, United Kingdom 2Div Ped Neurology, Gazi Univ Hosp, Ankara, Turkey 2Neurometabolic Unit, NHNN, London, United Kingdom 3Dep of Neurology,Alzheimer Center Nijmeg, Netherlands, Netherlands 3Metabolic Unit, GOSH, London, United Kingdom 4Chem Path, GOSH, London, United Kingdom Aromatic L-amino acid decarboxylase (AADC), a vitamin B6-requiring enzyme that converts L-dopa to dopamine and 5-hydroxytryptophan to Background: 5-Methyltetrahydrofolate (5MTHF) deficiency has been docu- serotonin. Deficiency of this enzyme results in developmental delay, mus- mented in a range of inherited metabolic disorders, including mitochondrial cular hypotonia, dystonia, involuntary movements, autonomic dysfunction respiratory chain diseases. Identification of factors affecting 5MTHF stability and oculogyric crises. We now report a two years old Turkish boy with are important in the correct diagnosis of cerebral 5MTHF deficiency. AADC deficiency confirmed by greatly reduced AADC activity in the Methods: 5MTHF stability was determined in phosphate buffer (pH 7.4), plasma and by genetic studies. Mutation analysis revealed a homozygous pooled human CSF with/without (+/-) hydroxyl radicals, and in buffer in mutation c.208 C>T(p.His70Tyr) in exon 3 of the AADC gene which has the presence of ascorbate. The relationship between 5MTHF and ascorbate not been described to date. Cerebrospinal fluid neurotransmitter metabolite in CSF was also examined. profile remains a critical tool in identifying patients with suspected neuro- Results: 5MTHF (150 nM) was found to be relatively unstable in buffer compared transmitter deficiency state. Treatment options are limited, in many cases to CSF. In the presence of hydroxyl radicals, the calculated rateconstant(k)for not beneficial, and prognosis is uncertain. This current case showed no or 5MTHF was 0.066±0.005 min-1 for buffer and 0.018±0.002 min-1 for CSF (p< poor response despite different protocols and a combination of different 0.0001). 5MTHF degradation was retarded in buffer by ascorbate. A positive drugs. correlation between CSF 5MTHF and ascorbate was observed (r00.2841, p<0.01). Conclusions: The relative stability of 5MTHF in CSF +/- hydroxyl radicals suggests the presence of protective antioxidant type molecules in CSF. A candidate for the protective antioxidant in CSF is ascorbate, and the positive correlation observed between CSF 5MTHF and ascorbate supports this hypothesis. Current work is investigating CSF ascorbate levels in patients with documented CSF 5MTHF deficiency. P-386 P-388 ESTABLISHMENT OF SERUM PROLACTIN REFERENCE CSFDHAINCREASEDINSEPIAPTERINE REDUCTASE INTERVALS AND THEIR USE AS A 'SCREENING' TOOL FOR DEFICIENCY AS A CONSEQUENCE OF NEUROTRANSMITTER CENTRAL DOPAMINE DEFICIENCY DEFICIENCY Aitkenhead H1 , Heales SJ1 Mazzuca M1 , Maubert MA2 ,DamajL3 ,KaminskaA4 ,ClotF5 ,OdentS3 , 1Chem Path, Gt Ormond St Hosp, London, United Kingdom Bahi-Buisson N6 ,WolfC2 , Cadoudal M7 ,RabierD7 , Benoist JF8 ,De Lonlay P1 , Christa L7 Background: The time between presentation and diagnosis of inborn errors 1Ref Cent for Inh Metab Dis, Necker Hosp, Paris, France of dopamine biosynthesis/dopamine transport can be prolonged. Serum 2Bioch St Antoine Hosp, P et M Curie Univ, Paris, France prolactin has been proposed as a screening tool for selecting patients 3Div Genet, Div Paediatric Neurology, Rennes, France presenting with clinical features of dopamine deficiency, prior to diagnostic 4Div Neurological Explor, Necker Hosp, Paris, France testing which requires a lumbar puncture. However, the interpretation of 5Div Neuro Genet, Pitié Salpétrière Hosp, Paris, France prolactin results is difficult because of a lack of paediatric reference inter- 6Div Paediatric Neuro Metab, Necker Hosp, Paris, France vals especially in infants. 7Div Metab, Necker Hosp, Paris Desc Univ, Paris, France Objectives: To develop age appropriate reference ranges for serum 8Div Biochem, Robert Debré Hosp, Paris, France prolactin. Methods: Data from our laboratory database was used to generate age and We report a patient born from consanguineous parents with two homoallelic sex related prolactin reference intervals (n02369). homozygous nonsense mutations in two genes distant from 1.8 Mb on chromo- Results: During the first year of life serum prolactin sharply declines with some 2p14-p12, ie the malonyl CoA epimerase (MCEE) gene (c.139 C>T; age and continues to decline until 5 years. The sex differences are small p.Arg47X) and the Sepiapterin reductase (SR) gene (c.751A>T; p.lys251X). under 13 years. Using these ranges, we have been able to demonstrate that a Moderate but constant urinary excretion of methylmalonic acid associated with number of patients with diagnoses of inborn errors of dopamine biosyn- mental retardation led to diagnose MCEE deficiency. Furthermore, progressive thesis/ dopamine transport have elevated serum prolactin e.g. 11 year old dystonia and cataplexy narcolepsy led to diagnose SR deficiency. L-dopa/ female with confirmed dopamine transporter defect had a serum prolactin of carbidopa and 5-HTP treatment dramatically improved the clinical features, 688 mU/L and the age appropriate reference interval is 44–548 mU/L. but supplementation with BH4 did not induce additional benefits. In order to Conclusion: Assessment of serum prolactin may be a useful adjunct with investigate the neurological consequences of SR deficiency, the levels of Poly regards to the investigation of patients suspected of having a disorder of Unsaturated Fatty Acid (PUFAs) were analyzed in the CSF during the time dopamine metabolism/transport. course of the treatment (n04 lumbar punctures) and compared to CSF controls (n011). After lipid extraction, the fatty acids (n017) were analysed by gas- chromatography coupled to mass spectrometry. Docahexaenoic acid (DHA), but not the other PUFAs, was higher in the CSF of the patient with SR deficiency than in controls (p00.004). Since DHA also increased in CSF of two patients with Aromatic Amino Acid Decarboxylase deficiency, the results suggested that the level of DHA could be modulated by the biogenic monoamines. S142 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-389 29. Molecular Mechanisms ELEVATED CSF TOTAL NEOPTERIN PLUS/MINUS BIOPTERIN SPECIES. USEFUL INSIGHTS INTO INFLAMMATORY P-371 MECHANISMS ? AN EXTREMELY RARE CASE: OSTEOSCLEROTIC METAPHYSEAL Pope SAS1 , Neergheen V1 , Land JM1 , Heales SJR1 DYSPLASIA 1Nat. Hosp. Neurology and Neurosurgery, London, United Kingdom Kasapkara C S1 , Kucukcongar A1 , Boyunaga O2 , Bedir T1 , Oncu F2 , Hasanoglu A1 , Tumer L1 Background: Assessment of CSF total neopterin (Neo) is a useful marker 1Div Metab Dis, Gazi Univ Hosp, Ankara, Turkey of central nervous system immune activation. It is produced by activated 2Radiology Dep, Gazi Univ Hosp, Ankara, Turkey macrophages and astrocytes in response to cytokines. In these cell types the pterin metabolic pathway is thought to be restricted and does not progress to Osteosclerotic metaphyseal dysplasia(OMD) is a very rare sclerosing bone tetrahydrobiopterin (BH4). disorder, first described by G. Nishimura in two Japenese siblings at 1993. Objectives: To determine if in CSF samples with a documented elevation We report the 4th case of OMD described in the literature of a 12-month-old of Neo, there is any evidence for increased total biopterin (BH4 plus male with hypotonia, developmental delay and sclerosis of the metaphyses dihydrobiopterin, BH2) availability. and epiphyses of specific bones. This 36-week gestation boy was born to a Methods: Neo, BH4 and BH2 were measured in CSF by HPLC with 26 year old Turkish mother and a 27 year old nonconsanguineous father. electrochemical/fluorescence detection. Data from 299 individual Radiographic findings included bilateral symmetrical osteosclerosis of the patients, with no evidence of a primary disorder of pterin metabolism, metaphyseal portions of the long bones in the upper and lower extremities were analysed. with osteopenic shafts. Narrow bands of metaphyseal osteosclerosis were Results: 56 patients had an elevated Neo concentration. Of these, 18 had detected in the short tubular bones of the hands and feet. Growing parts of only a high Neo, 12 had a high Neo plus BH4, 15 had high Neo plus BH2, bilateral scapula, iliac, pubic and ischial bones show sclerotic bands. In and 11 had all three pterins elevated. addition superior and inferior plates of vertebras, transverse processes of Conclusion: For a proportion of CSF samples with an elevated Neo, sacral vertebras, all visible epiphyses, carpal and tarsal bones also show BH4 and/or BH2 are also increased in concentration. These data may sclerotic changes. The skalp was unaffected. Based on the clinical, radio- suggest that, under certain conditions following immune response graphic, and laboratory findings, a diagnosis of OMD was made. We do not activation, cell types where the BH4 biosynthetic pathway is intact know any of the osteosclerotic bone disorder with changes including can also be activated. hypotonia, mental and motor developmental delay and metaphyseal sclerosis of the bones with a unique distribution except OMD.

P-390 SECONDARY ALTERATIONS IN BIOGENIC AMINES AND PTERINES IN CEREBRAL FOLATE DEFICIENCY PATIENTS Ormazabal A1 ,MoleroM1 , Sierra C1 , García-Cazorla A1 , O'Callaghan MM1 , Perez-Dueñas B1 ,SerranoM1 ,ArtuchR1 1Hospital Sant Joan de Deu and CIBERER, Barcelona, Spain

Background: Cerebral folate deficiency (CFD) has been described as a neurological syndrome associated with low 5-methyltetrahydrofolate (5- MTHF) values in cerebrospinal fluid (CSF). Objectives: To report on CSF biochemical data of 8 patients with profound CSF 5-MTHF deficiency (below 10 nmol/L) associated with different diseases. Patients and methods: 8 patients diagnosed of severe Methylentetra- hydrofolate reductase deficiency (MTHFR; n03), Kearns-Sayre syndrome (KSS; n04) and Folate receptor Alpha defect (FOLR1; n01). Biogenic amine metabolites (5-hydroxyindolacetic acid (5HIAA) and homovanilic acid (HVA)), pterins (neopterin, biopterin) and 5-MTHF were analysed by HPLC with electrochemical and fluorescence detection. Results: Only one patient with MTHFR presented low values of HVA, 5-HIAA and pterins, while the others patients with MTHFR and FOLR1 showed normal values of these biomarkers. All patients with KSS presented a clear increment of HVA over the reference values. No correlation was observed among 5-MTHF values and the other parameters studied. Conclusion: Despite the similar values of 5MTHF in both transport and metabolic defects, biogenic amines and pterines profiles were different. MTHFR and FOLR1 showed no or slight abnormalities regarding these metabolites, while KSS showed increment in HVA probably due to an impaired transport through choroid plexus. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S143

P-391 A-039 SOMATIC MUTATIONS IN THE KREBS CYCLE ENZYME ISOCI- ANALYSIS OF SNP - MUTATION OF SOME CYTOKINES GENES TRATE DEHYDROGENASE 1 (IDH1) CAUSE METAPHYSEAL AND DETOXIFICATION XENOBIOTICS ENZYME IN MULTI- ENCHONDROMATOSIS WITH D-2-HYDROXYGLUTARIC ACIDURIA. FACTORIAL DISEASES Bonafé L1 , Vissers LELM2 , Dionisi-Vici C3 , Martinelli D3 , Barbuti D4 , Bezrodnaya AI1 Fano V5 , Sass JO6 , Ballhausen D1 , Suarez P1 , Campos-Xavier AB1 , 1Khark Nat Med Univ, Kharkiv, Ukraine Unger S7 , Veltman JA2 , Brunner HG2 , Superti-Furga A8 1Div Molecular Ped, Lausanne Univ Hosp, Lausanne, Switzerland Background: There are huge number of common candidate genes, or 2Dept Hum Genet, Nijmegen Mol Life Scien, Nijmegen, Netherlands syntropic genes, in genome for many multifactorial diseases causes the 3Div Metab, Bambino Gesù Child Hosp, Rome, Italy development of common biochemical and physiological disturbances for 4Div Radiol, Bambino Gesù Child Hosp, Rome, Italy them (EY Grechanina, JB Grechanina, 2010). 5Hosp Pediatria J.P. Garrahan, Buenos Aires, Argentina Objective: To study the frequency of the genotypes IL4 C-589 T, 6Div Clin Chem Biochem, Univ Child Hosp, Zurich, Switzerland IL17A G-197A, IL17F His-161 Arg, CYP1A1 Ile462Val in pro- 7Dept Med Genet, Lausanne Univ Hosp, Lausanne, Switzerland bands with bronchial asthma (BA), atopic dermatitis (AD), psoriasis 8Dept Pediatrics, Lausanne Univ Hosp, Lausanne, Switzerland (PS). Results: 1) IL4: The genotype of 589 TT is most common in probands with Metaphyseal chondromatosis with hydroxyglutaric aciduria (MC-HGA) is a AD, 3 times more frequently than among probands with BA (p <0,05) and generalized skeletal dysplasia, accompanied by urinary excretion of D-2- almost 2 times - than among PS (p <0,05). hydroxyglutarate (HGA), and variable cerebral involvement. By whole- 2) CYP1A1: Among the representatives with different nosologies does not exome sequencing 2 unrelated patients with MC-HGA, we have found muta- appear genotype 462 Val Val(p <0,05). tions in isocitrate dehydrogenase 1 (IDH1) at codon 132, as apparent somatic 3) IL17A: 197 AA genotype found among representatives of the Kharkov mosaicism. IDH1 is a key enzyme of the Krebs cycle, which converts isocitrate population of patients with PS 1.1 times more frequently than genotype 197 into alpha-ketoglutarate (a-KG). Mutations at IDH1 Arg132 residue have GG. originally been identified in different tumour types (isolated gliomas, leuke- 4) IL17F: 161 His His genotype is found in 29.6 times more likely among mias, and chondrosarcomas). These mutations trans-specify the enzyme activity representatives from 161 asthma than the genotype Arg Arg. resulting in HGA accumulation and a-KG depletion. This induces activation of Conclusions: The frequency of polymorphism 589 CT IL4 highest for hypoxia-inducible factor 1-alpha (HIF-1a), an important regulator of chondro- PS and 40 %. It can be assumed that the 462 Val Val polymorphism cyte proliferation at the growth plate. Differently from Arg132 somatic muta- of the CYP1A1 gene is lethal, because not found among all the tions found in isolated tumours, the mutation in our patients must have occurred pathologies. very early in embryogenesis to cause a generalized dysplasia with involvement Conflict of Interest declared. of all long bones metaphyses and mutation detectability in blood. Identical mutations have subsequently been identified in chondromas excised from patients with multiple chondromatosis (Ollier disease). Tissue distribution of the mutation may explain variable cerebral involvement and the susceptibility to develop tumours in other organs. The postulated pathophysiology of MC-HGA points out the link between Krebs cycle, hypoxia sensing and bone growth.

P-392 MOLECULAR BASIS OF TWO EXON SKIPPING (EXONS 12 AND 13) BY C.1248+5 G>A IN FIBROBLASTS FROM A SCOT DEFICIENT PATIENT. STUDY OF SPLICING ORDER IN SCOT TRANSCRIPTS IN FIBROBLASTS FROM CONTROLS AND THE PATIENT Hori T1 , Fukao T1 , Harding C2 , Kondo N1 1Dept Pediatr, Grad Sch Med, Gifu Univ, Gifu, Japan 2Dept Mol Med Genet, OHSU, Portland, OR, United States

The molecular basis of simultaneous two exon skipping induced by a splice site mutation has yet to be completely explained. The splice donor site mutation c.1248+5 g>a (IVS13) resulted predominantly in skipping of exons 12 and 13 in OXCT1 gene in fibroblasts from a patient (GS23) with succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency. We compared splicing order between controls' and GS23's fibroblasts. Our strategy was to detect the presence or absence of large fused "exon"s comprising successive exons by RT-PCR, using heteronu- clear RNA. Our initial hypothesis was that an exon fusion comprising exons 12 and 13 was formed first followed by skipping of this exon fusion in GS23 cells. However, the exon fusion was clearly identified in two control cell lines but was hardly detected in GS23's fibroblasts. Hence, we compared the splicing order of full-length SCOT transcript between controls and GS23. In controls, intron 11 was the last intron to be spliced. However, the mutation in GS23 cells resulted in a retention of intron 12 and splicing out of a large "intron" comprising intron 11- exon 12-intron 12-exon 13-mutated intron 13. Our result indicates that a mutation at a splice donor site can affect the splicing order of non-adjacent introns. S144 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

30. Proteins, Function and Structure 31. Gene Therapy P-378 P-379 PHARMACOLOGICAL CHAPERONE THERAPY IN MCAD DEFI- GENE THERAPY FOR HEPATIC DISEASES USING NON-VIRAL CIENCY: RATIONALE FOR MUTANT SPECIFIC TARGET SITES. MINICIRCLE-DNA VECTOR Gersting SW1 ,JankJ1 ,ReißDD1 , Sommerhoff CP2 , Maier EM3 ,MuntauAC1 Viecelli HM1 , Harbottle RP2 , Chuah M3 , VandenDriessch T3 , Harding 1Mol Pediatrics, Hauner Child Hosp, LMU, Munich, Germany CO4 , Thony B1 2Clin Chem & Biochem, Surgical Clin, LMU, Munich, Germany 1Div Metab, Dept Ped, Univ Zurich, Zurich, Switzerland 3Dept Pediatrics, Paracelsus Med Univ, Salzburg, Austria 2Nat Heart & Lung Int, Imp College London, London, United Kingdom 3Div Gen Ther&Reg Med, Free Uni Brussels, Brussels, Belgium Background: Medium-chain acyl-CoA dehydrogenase (MCAD) mutation 4Dept Mol Med Gen Pedi, OHSU, Portland, United States K304E is associated with high risk of disease manifestation whereas the pathogenic effect of a second prevalent mutation Y42H is considered to be less severe. Both We have reported long-term correction of hyperphenylalaninemia and hypopig- mutations are associated with protein misfolding, however they map to different mentation of the PKU mouse model, C57Bl/6-Pahenu2, after liver-directed gene MCAD domains and have distinct structural relation to substrate and cofactor transfer with recombinant adeno-associated viral vectors. However, questions of binding sites. The impact of mutations identified in newborn screening on MCAD expression stability, treatment toxicity, potential for insertional mutagenesis, and protein structure and their clinical significance is not well understood. safety required for targeting newborn and paediatric patients for potential life-long Results: Based on protein conformation, stability, and unfolding thermody- treatment remain a risk for viral vector-dependent approaches. To overcome these namics molecular phenotypes linked to mutations identified in newborn drawbacks, we are developing non-viral gene transfer methods for liver targeting. screening were classified as either less severe than Y42H or more severe than The major challenge of using non-viral vectors for the treatment of human diseases K304E with most severe effects for mutations mapping to the β-domain. is transcriptional silencing of the DNA vector. Here we report the successful use of Variant MCAD proteins were stabilized against thermal stress in the presence minicircle (MC) technology to treat murine PKU. MC-DNA vectors containing a of both octanoyl-CoA substrate and FAD cofactor with varying efficacy for de novo designed liver-specific hybrid promoter-enhancer fragment, the murine individual mutations. In 3D structural analyses amino acid side chains of N- phenylalanine hydroxylase gene (mPah), and the bovine growth hormone polyA and C-terminal α-domains and the β-domain displayed diverse interaction signal were constructed and delivered to the liver by hydrodynamic tail vein patterns to substrate and cofactor molecules, respectively. injection. MC-vector titration studies showed that the blood phenylalanine levels Conclusion: Structural relation of amino acid side chain replacements to were normalized concomitant with reversion of hypopigmentation at optimal MC- MCAD domains or binding sites governs the protein-misfolding molecular DNA concentrations for several months. Upon scarifying treated PKU mice, phenotype and opens the way to target site specific personalized pharmaco- sustained transgene expression was confirmed by the presence of hepatic PAH logical chaperon strategies. enzyme activity. In conclusion, MC technology offers a better safety profile and has the potential for gene-therapeutic treatment of liver diseases.

P-380 CLASSICAL GALACTOSEMIA: CHARACTERIZATION OF THE FIRST INTRONIC VARIATION AND ITS CORRECTION BY ANTISENSE THERAPY Lourenço SP1 , Coelho AI1 , SIlva MJ1 , Tavares de Almeida I1 , Vicente JB1 , Rivera I1 1Met&Gen Group-iMed, FacPharm, UnivLisbon, Lisbon, Portugal

Classical Galactosemia is caused by deficient activity of galactose-1- phosphate uridylyltransferase (GALT) and results from mutations in the GALT gene, where more than 200 variations have been described thus far. Mutational analysis of 27 Portuguese patients confirmed the Q188R as the prevalent molecular defect (≈60 %), and revealed the intronic variation c.820+13a>g (IVS8+13a>g) as the second most frequent mutation, accounting for 12.5 % of the Portuguese mutant alleles. This intronic variation was believed to affect pre-mRNA splicing, and therefore its pathogenic mechanism was investigated using a minigene approach. The pSPL3 vector containing either the genomic wild-type or the mutant fragments was transfected into COS-7 cell line. 24 h after transfection, RNA was purified and amplified by RT-PCR. Direct sequencing of the reaction products clearly showed that c.820+13a> g favors the next GT dinucleotide (c.820+14_15) to be used as a new 5'splice donor site, leading to the inclusion of the first 13 nucleotides of intron 8 in the coding sequence, thus inducing a frame shift. Accordingly, c.820+13a>g is indeed a disease-causing mutation, being the first intronic variation in GALT gene whose molecular mechanism was elucidated. In order to revert this alternative splicing, an antisense LNA (locked nucleic acid) oligonucleotide was successfully used, opening new therapeutic perspectives. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S145

32. Enzyme Replacement Therapy P-395 ENZYME REPLACEMENT THERAPY (ERT)RESULTS OF P-393 PATIENTS WITH MUCOPOLYSACCHARIDOSIS TYPE II AUDIOLOGICAL OUTCOMES OF MPS II: BEFORE AND AFTER (HUNTER SYNDROME) ENZYME REPLACEMENT THERAPY Unal O1 , Dogru-Ersoz D2 , Alehan D3 , Saglam M4 ,HismiB1 ,DursunA1 , Yigit-Duran O1 , Unal O2 , Genc A1 , Hismi B2 , Dursun A2 , Tokatli A2 , Tokatli A1 ,CoskunT1 , Sivri HS1 Coskun T2 , Sivri HS2 1Hacettepe Univ, Pediatrics, Metabolism, Ankara, Turkey 1Hacettepe Univ, Audiology Unit, Ankara, Turkey 2Hacettepe Univ, Pediatrics, Pulmonology, Ankara, Turkey 2Hacettepe Univ, Pediatrics, Metabolism, Ankara, Turkey 3Hacettepe Univ, Pediatrics, Cardiology, Ankara, Turkey 4Hacettepe Univ, Dept. Physiotherapy, Ankara, Turkey Introduction: Hearing loss is one of the most common co-existing disorders of MPS. In this study, eight patients who were diagnosed with MPS II Background: Mucopolysaccharidosis (MPS) type II is an X-linked reces- were evaluated audiologically. sively inherited disorder caused by deficiency of lysosomal iduronate- 2- Method: Six patients' audiological evaluation was conducted before and sulphatase involved in the catabolism of glycosaminoglycans. The age of after enzyme replacement therapy. Two patients were evaluated with supra- onset and clinical course are variable. Intravenous idursulphase infusion is aural headphones, while the other four patients were evaluated in sound used as enzyme replacement therapy. field. Except one patient who had ventilation tubes, immitansmetric evalu- Method: ERT (Elaprase®) was given 0.5 mg/kg/dose weekly to eight patients ation made. Patient's audiological findings before and after enzyme replace- (age range: 2.5 to 22 years) with MPS type II for 4 months to 4 years period. ment therapy were compared with Paired Samples T-test. Age range was 2.5 to 22 years. Efficacy of the therapy on growth parameters, Results: No statistically significant difference was found between the six minutes walking test, cardiac and pulmonary function tests were evaluated. evaluations. Although, no significant differences, two patients's hearing Results: Pulmonary functions were slightly improved in a patient who had thresholds were approximately 15-20 dB better after replacement therapy. severe symptoms before the therapy. In two patients who had normal or The other two patients' hearing thresholds had not changed, but patients' mildly impaired pulmonary functions before, test results were slightly family members mentioned improvement in their hearing related behaviors. deteriorated after therapy. Cardiac findings deteriorated in two patients Discussion: Audiological evaluation of the MPS patients is very difficult, under therapy and remained stable in the other patients. Height standard both audiological follow up and management process are crucial. In audio- deviation scores were deteriorated in two patients and improved in only one logic follow-up and management processes, receiving information from patient. Six minute walking tests improved in four of eight patients. family and sharing their experiences play an important role. Audiological Conclusion: Efficacy of enzyme replacement therapy on walking tests, follow up is important either understanding the affect of treatment in their pulmonary functions and visceromegaly were reported previously. In this hearing or supporting their quality of life in monitoring their general health. study, most important positive effect was detected on walking distance.

P-394 P-396 EFFECT OF ENZYME REPLACEMENT THERAPY ON FATIGUE IMMUNE TOLERANCE INDUCTION TO ENZYME REPLACEMENT IN ADULTS WITH POMPE DISEASE THERAPY IN CRIM NEGATIVE INFANTILE POMPE DISEASE Güngör D1 , de Vries JM2 , Brusse EB2 , HOP WCJ3 ,ReuserAJJ4 , Hagemans Tardieu M1 , Soulé N1 , Blasco H1 , Watier H1 , Piraud M2 , Chantepie A1 , MLC1 , van Doorn PA2 ,PlugI1 ,KruijshaarM1 ,vanderPloegAT1 Ogier de Baulny H3 , Kishnani PS4 , Labarthe F1 1Dept. Ped, Erasmus MC, Rotterdam, Netherlands 1CHRU Tours, TOURS, France 2Dept. of Neurology, Erasmus MC, Rotterdam, Netherlands 2Hospices Civils de Lyon, Bron, France 3Dept. Biostat, Erasmus MC, Rotterdam, Netherlands 3Hôpital R Debré, APHP, Paris, France 4Dept of Clin Gen, Erasmus MC, Rotterdam, Netherlands 4Duke University Medical Center, Durham, NC, United States

Background: Fatigue is a common disabling symptom of adult Pompe Background: Infantile Pompe disease results in progressive cardiac, motor, and patients. We assessed whether enzyme replacement therapy (ERT) reduces respiratory failure and early fatal outcome. Enzyme replacement therapy (ERT) fatigue, and whether this could be explained by the effect ERT has on with recombinant human acid-alpha-glucosidase (rhGAA) prolongs survival but muscle strength, pulmonary function and depression. may be less effective in cross-reactive immune material (CRIM) negative patients. Methods: Fatigue was measured in an international longitudinal survey We report successful tolerance induction to ERT in a CRIM-negative patient. using the Fatigue Severity Scale (FSS). Repeated measurements ANOVA Case report: Infantile Pompe disease was diagnosed in a 2-m-old girl was used to describe the data over time. admitted for cardiomyopathy, hypotonia and severe respiratory failure. An Results: 163 patients were followed for a median of 4 years before and homozygous 236_246del mutation was identified and the rapid determination 3 years during ERT. At start of ERT, 85 % were fatigued (FSS≥4), of CRIM status was negative. The patient was treated prophylactically with a compared to 78 % at the last follow-up. FSS scores decreased significantly short course of rituximab, methotrexate, and gammaglobulins, prior to ERT. during ERT compared to the period prior to treatment (difference in slope - Results: Immune therapy induced a transitory generalized B-cell suppression that 0.14 FSS score-points per year, p <0.01). The effect of ERT on fatigue was recovered after 14 weeks. The patient remained immune tolerant to rhGAA after correlated with the effect of ERT on muscle strength (ρ -0.55; p <0.001) and 14 months of ERT, with no detectable rhGAA-antibody. She showed a successful depression (ρ 0.34; p00.01), but not to pulmonary function. clinical response to ERT, with respiratory and cardiac function recovery after Conclusions: Our study confirmed that fatigue is prominent in adult Pompe 3.5-months of ERT. Muscle tone also gradually improved and the patient was able patients. During ERT fatigue decreased, although this effect was small. To to sit up unassisted and to maintain a standing position after 12 months of ERT. optimally treat patients, the exact role of muscle strength, depression and Conclusion: Short course immune therapy is able to induce immune other possible associated factors on fatigue needs to be investigated further. tolerance and successful clinical response to ERT in CRIM negative infan- Conflict of Interest declared. tile Pompe patients. S146 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-397 P-399 IMPACT OF SWITCHING ENZYME REPLACEMENT THERAPY MUCOPOLYSACCHARIDOSIS TYPE II IN FEMALES AND ON PATIENTS AFFECTED BY FABRY DISEASE. RESPONSE TO ENZYME REPLACEMENT THERAPY Jovanovic A1 ,SharmaR1 ,KempshallS1 , Thompson L1 , Bleakley Jurecka A1 ,KruminaZ2 , Żuber Z3 ,Różdżyńska-Świątkowska A1 , C1 ,BellL1 ,TaylorR1 ,WhitbyJ1 , Smith E1 ,MeehanM1 , Hill Kloska A4 , Czartoryska B5 , Tylki-Szymańska A1 A1 , Hendriksz CJ2 1The Children's Memorial Health Institute, Warsaw, Poland 1Salford Royal NHS Foundation Trust, Salford, United Kingdom 2Children's University Hospital, Riga, Latvia 2Manchester Acad Health Science Centre, Manchester, United Kingdom 3S. Louis Regional Children's Hospital, Cracow, Poland 4University of Gdańsk, Gdańsk, Poland Data from 77 Fabry patients managed at a single site that switched 5Institute of Psychiatry and Neurology, Warsaw, Poland their enzyme replacement therapy from agalsidae beta to agalsidase alpha was reviewed. The switch was due to multitude of reasons Mucopolysaccharidosis type II (Hunter syndrome) is an X-linked lysosomal including ERT supply problems but no patient was treated with a storage disease caused by a deficiency of iduronate-2-sulfatase. Two affected reduced dosage although frequency of dosing was reduced in some girls with intermediate and severe forms of MPS II with normal karyotypes for a short period. The mean duration on treatment before switch was and increased urinary dermatan sulphate and heparin sulphate excretion and 46.24 months (median 42 range 4-125 months) and the mean on new marked deficiencies of IDS activity are reported. Molecular studies showed therapy after switch was 20.67 month (median 24 range 4-30 months). that case 1 has a heterozygous mutation c.1568A>G (p.Y523C) associated The calculated GFR seem to be unaffected by the switch and many with almost totally skewed X-inactivation of the normal maternal X chromo- off the other collected parameters like left ventricular mass index, PR some, and case 2 has a heterozygous deletion that includes exons 1-4 of the time and urine creatinine protein ratio seemed to be unaffected by the IDS gene (minimal deletion range c.1-103_184del). The multi-exon deletion switch. The lack of an approved prospective protocol leads to incon- correlated with early onset of the disease and severe phenotype with psycho- sistency of data recordings and hamper the interpretation but all motor retardation, whereas the missense mutation was associated with mod- experience gained from switch patients are important especially the erate developmental delay. Although genotype-phenotype correlation in MPS patient experience of the switch. The relatively short follow up mean II is difficult, gene deletions seem to correlate with more severe clinical 20.67 months on the background of stabilized patients also need to be manifestation of the disease. Enzyme replacement therapy in these 2 females considered and compared to natural history for correct interpretation. resulted in disease stabilization in both. Conflict of Interest declared.

P-398 P-400 RENAL FAILURE IN LATE DIAGNOSIS OF FABRY DISEASE: INCIDENCE AND IMPACT OF NEUTRALIZING ALLO- IRREVERSIBILITY DESPITE ENZYME REPLACEMENT ANTIBODIES AFTER ENZYME REPLACEMENT THERAPY IN THERAPY (ERT) MPSI PATIENTS Menni F1 , Testa S2 , Tel F2 , Cerutti M1 , Bonarrigo F1 , Chiarelli G1 , Saif MA1 , Brookes KE1 , Mercer J2 , Tylee KL2 , Church HJ2 , Bonney Paglialonga F2 DK3 , Jones SA2 , Wraith JE2 , Wynn RF3 , Bigger BW1 1Ped Dep, IRCCS F.Policlinico, Univ Studi, Milan, Italy 1Stem Cell & Neurotherapies, UoM, Mancehster, United Kingdom 2Ped Neph Dial Unit, IRCCS Osp Policlinic, Milan, Italy 2Genetic Medicine, St Mary's Hospital, Mancehster, United Kingdom 3Blood and Marrow Transplant Unit, RMCH, Manchester, United Kingdom We describe a patient with Fabry disease diagnosed at 11 years of age as a result of the discovery of renal failure. In 2006 the child was Mucopolysaccharidosis type I (MPSI) is caused by a deficiency of α-L- hospitalized for palmar and plantar burning sensation. Investigations iduronidase (IDUA). Recombinant human enzyme replacement therapy revealed chronic renal failure, cardiomyopathy and an hemodyalysis (ERT) is currently in clinical use for MPSI, however 91 % of MPSI patients program was started. A renal biopsy was performed with evidence of on ERT raise IgG antibodies to enzyme, but the incidence and impact of storage in glomerules and blood vessel. He was underfed (BMI 13,8 functionally active antibodies is unknown. Wt -4SD, Ht -3,76SD). A Fabry disease was suspected and confirmed In longitudinal patient samples (n08), high titres of IDUA reactive by enzyme dosage: low levels of alpha galactosidase (1,68 nmol/h/mg antibodies were seen in 87.5 % of patients on ERT. The majority protein-n.v.100-800). GLA mutation confirmed the disease and the (62.5 %) of patients demonstrated catalytic inhibition ranging from mother was identified as a carrier. 10-75 % and inhibition of enzyme uptake by MPSI fibroblasts was ERT was started in Romania in 2007 with beta agalsidase, every two seen in 75 % of patients. The antibody titres correlated with bio- weeks. After one year he showed an improvement of general status, markers of disease progression. Haematopoietic stem cell transplant of cardiomyopathy, but persistence of renal failure. He arrived in (HSCT) completely abrogated antibody responses. A cross sectional Italy at 17 years of age after stop of therapy of 3 months with group measured over 1 year after HSCT showed that 85 % (n017) inability to work due to acroparestesia, cardiomyopathy and hypo- patients had no detectable antibodies following HSCT. Of 10 patients hydrosis. We started ERT with agalsidase alfa 50 mg/Kg every two receiving ERT alone 70 % still had functionally active antibodies six week. After 3 months of treatment amelioration of acroparestesia and months or longer after commencing the ERT. cardiomyopathy was noticed. He continued hemodialysis 3 times per The majority of MPSI patients on ERT raise high titres of IDUA reactive week. and functionally inhibitory antibodies which remain high for more than Due to his renal condition, he was nominated for a renal transplant. 6 months after commencement of treatment and appear to neutralise the infused enzyme. Allogeneic HSCT can ameliorate allo-immune responses by inducing immune tolerance in transplant recipients. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S147

P-401 P-403 CARDIOVASCULAR OUTCOME AFTER FIVE YEARS OF MUCOPOLYSACCHARIDOSIS: EFFECTS OF ENZYME- ENZYME REPLACEMENT THERAPY IN THREE MEXICAN REPLACEMENT THERAPY IN 27 CHILDREN WITH MPS I, II PATIENTS WITH HURLER SYNDROME AND VI Carpio JC1 , Ruiz ED2 , Campos FJ2 , González M3 Hasanoglu A1 , Tumer L1 , Ezgu F S1 , Okur I1 , Eminoglu F T1 , Kasapkara 1Div Pediat Cardiol, IMSS La Raza, Mexico City, Mexico CS1 , Kucukcongar A1 2Div Med Genet, IMSS La Raza, Mexico City, Mexico 1Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 3Div Pediat, IMSS La Raza, Mexico City, Mexico The mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal Background: Cardiovascular abnormalities are common and may be storage disorders that are clinically characterized by abnormalities in multiple caused early death in Hurler syndrome, without enzyme replacement ther- organ systems and reduced life expectancy. Patients typically appear normal at apy (ERT), approximately half of all patients die from cause cardiac, with birth, but during early childhood they experience the onset of clinical disease, ERT the cardiovascular symptoms could improve. including skeletal, joint, airway and cardiac involvement, hearing and vision Objective: Evaluate the evolution of cardiovascular development and symp- impairment, and mental retardation. MPS I is an autosomal recessive disorder toms after fiver years of ERT in Mexican patients with Hurler Syndrome. caused by deficient activity of the lysosomal enzyme alpha-L-iduronidase Patients and Methods: We analyzed three patients with Hurler syndrome, leading to accumulation of heparan sulfate and dermatan sulfate resulting in two initiated ERT with Laronidase before five years of age and one at multisystem disease. MPS II is a X-linked lysosomal storage disorder causing 7 years old. Cardiac function was investigated with electrocardiography and a progressive multisystemic accumulation of GAGs. Enzyme replacement two-dimensional echocardiography including Doppler imaging. therapy with idursulfase (0.5 mg/kg/week) has been introduced recently. Results: The three patients remain without cardiovascular symptomatology MPS VI results in accumulation of dermatan sulfate. Enzyme replacement and with normal left ventricle ejection fraction ( >75 %). All patients therapy has been successfully applied in MPS I, II, VI. developed valvular heart disease and have any degree of pulmonary arterial Material-Method: In our study, clinical, biochemical and radiological (PA) hypertension. In two patients the PA pressure remained within similar findings and response to ERT for at least six months were analysed in 27 values than before initializing ERT. Surprisingly, the other patient who patients with Mucopolysaccharidoses (17 MPS VI, 6 MPS I, 4 MPS II) started therapy at 3 years old with a normal PA pressure, developed severe followed at Gazi University Pediatric Metabolic Unit. PA hypertension and diastolic function changes. Results: All three groups demonstrated reductions in urinary glycosamino- Conclusion: Two of our patients on ERT decreased the progression of glycans and hepatosplenomegaly. Improvement in the outcome of the 12- cardiovascular disease, nevertheless without regression or arrest of the min walk test and 3-min stair-climbing test were also noted after ERT. disease. In contrast, one of our patients showed a fast progression of cardiovascular signs despite of therapy.

P-402 P-404 ENZYME REPLACEMENT THERAPY AND ITS EFFECTS ON EFFECTS OF ENZYME REPLACEMENT THERAPY FOR PROGRESSION OF FABRY DISEASE, A SINGLE CENTRE MUCOPOLYSACCHARIDOSIS TYPE II INITIATED IN EARLY EXPERIENCE INFANCY Oliver M1 , Sharma R1 , Hendriksz CJ1 , Jovanovic A1 Tajima G1 , Hara K1 , Sakura N1 , Okuyama T2 , Kobayashi M1 1Dept of adult inherited metab dis, Salford, United Kingdom 1Dept Pediatr, Hiroshima Univ Hosp, Hiroshima, Japan 2Dept Clin Lab, Nat Ctr Child Health Dev, Tokyo, Japan Background: Fabry Disease (FD) is an X-linked deficiency in the activity of alphagalactosidase A . Cardio-renal manifestations of FD account for a Background: It has been shown that enzyme replacement therapy (ERT) large burden of morbidity. Enzyme replacement therapy (ERT) involves by recombinant idursulfase can improve several visceral symptoms of replacement of the deficient alpha gal A enzyme. Mucopolysaccharidosis type II (MPS-II). Some lesions seem to be irrever- Objectives and Method: We analysed our local data from the Fabry sible after they are developed, but little is known about preventive effects Registry including eGFR, creatinine, and echocardiogram measure- for pre-symptomatic patients. ments of interventricular septal diameter and ventricular posterior wall Methods: We diagnosed an asymptomatic infant with MPS-II according to thickness to assess changes in cardiac and renal function over time in his family history. His elder brother was diagnosed after typical symptoms patients on ERT. and was introduced into ERT at the age of 3 years. We could start ERT for Results: 179 Fabry patients were assessed. (N0179). Age range 13.6- the younger patient when he was 4 months old. Recombinant idursulfase 71.2 years. Mean age 41 with a mean time on ERT of 6.2 years. 66 was administered weekly at the dosage of 0.5 mg/kg. The duration of ERT patients had baseline cardiac wall measurements prior to ERT initia- was 26 and 24 months, respectively. tion. 40 % had improvement or stabilisation of cardiac indices since Results: During ERT, the younger patients stayed free from most of the ERT initiation (P00.7 & 0.35). Average eGFR at initiation of ERT symptoms that had already appeared in his brother at the same age. His was 87.3 ml/min with 53.6%patients showing an improvement or developmental quotations were still kept around lower limit of normal stabilisation in eGFR over time (P <0.05). Those patients with normal range. Symptoms in elder patient also improved or remained stable in or near normal baseline echocardiogram measurements and higher general, but his mental development seemed deteriorating gradually. baseline eGFRs responded best to ERT Conclusion: These findings suggest that pre-symptomatic initiation of ERT Conclusions: Markers of cardio-renal disease can be stabilised or improved can prevent or attenuate progression of visceral lesions of MPS-II. Further with most improvement seen in those patients with better baseline observation is required to clarify long-term outcome of preventive applica- measurements. tion of ERT for MPS-II. S148 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-405 P-406 EFFECT AND TOLERABILITY OF AGALSIDASE ALFA IN MINIMAL ANTI-DRUG ANTIBODY FINDINGS OF AGALSIDASE PATIENTS WITH FABRY DISEASE ALFA FOR FABRY DISEASE: TREATMENT-NAOVE OR Goker-Alpan O1 , Nedd K2 , Shankar S3 , Lien YH4 , Barshop BA5 , Holida FORMERLY TREATED WITH AGALSIDASE BETA M6 , Hillman R7 , Ibrahim J8 , Mardach R9 , Weinreb N10 , Rever B11 , Forte Goker-Alpan O1 , Nedd K2 , Shankar S3 , Lien YH4 , Barshop BA5 , Holida R12 , Desai a13 , Wijatyk A14 , Chang P14 , Martin R14 M6 , Hillman R7 , Ibrahim J8 , Mardach R9 , Weinreb N10 , Rever B11 , Forte 1Lysosomal Research & Treatment Unit, Fairfax, VA, United States R12 , Desai A13 , Wijatyk A14 , Chang P14 , Martin R14 2Infusion Associates, Grand Rapids, MI, United States 1Lysosomal Research & Treatment Unit, Fairfax, VA, United States 3Emory U, Atlanta,GA, United States 2Infusion Associates, Grand Rapids, MI, United States 4AKDHC, Tucson,AZ, United States 3Emory U, Atlanta,GA, United States 5UCSD, San Diego,CA, United States 4AKDHC, Tucson,AZ, United States 6U Iowa Health Care, Iowa City, IA, United States 5UCSD, San Diego,CA, United States 7U Missouri, Columbia, MO, United States 6U Iowa Health Care, Iowa City, IA, United States 8St Joseph's Children's Hospital, Paterson,NJ, United States 7U Missouri, Columbia, MO, United States 9Kaiser Permanente, Los Angeles,CA, United States 8St Joseph's Children's Hospital, Paterson,NJ, United States 10Univ Res Foundation for LSD's, Coral Springs, United States 9Kaiser Permanente, Los Angeles,CA, United States 11Central Coast Nephrology, Salinas,CA, United States 10Univ Res Foundation for LSD's, Coral Springs, United States 12North Shore University Hospital, Manhasset, NY, United States 11Central Coast Nephrology, Salinas,CA, United States 13Stuart Oncology Associates, Stuart, FL, United States 12North Shore University Hospital, Manhasset, NY, United States 14Shire HGT, Lexington, MA, United States 13Stuart Oncology Associates, Stuart, FL, United States 14Shire HGT, Lexington, MA, United States Background: A multicenter, open-label, agalsidase alfa (agalα) treatment protocol (HGT-REP-059; NCT01031173) included patients with Fabry Background: Potential anti-drug antibody (ADA) formation against agal- disease (FD) who were treatment-naïve or previously treated with agalsi- sidase alfa (agalα) was evaluated in patients with Fabry disease (FD) who dase beta (switch). were treatment-naïve or formerly treated with agalsidase beta (agalβ; Objective: To evaluate tolerability and effect of agalα in patients with FD. switch) over 12-months' agalα treatment. Methods: Adverse events (AEs), estimated glomerular filtration rate Methods: Antibody profiles were measured by enzyme-linked immuno- (eGFR), left ventricular mass index (LVMI), and other clinical endpoints sorbent assay (Baseline, 12-month) in agalα-treated (0.2 mg/kg every- were evaluated in agalα-treated patients (1 yr; 0.2-mg/kg every other other-week) patients with FD. week), controlling for potentially confounding factors. Results: 29 naïve (median age [range] 38.7 yr [12-74]; 15 female) and 62 Results: 29 naïve (median age [range] 38.7 yr [12-74]; 15 female) and 62 switch patients (47.0 yr [5-84]; 29 female) were assessed. One (3.4 %) switch patients (47.0 yr [5-84]; 29 female) were assessed. Most patients naïve and 23 (37.1 %) switch patients were baseline agalα ADA-positive; demonstrated no significant LVMI (naïve/ switch) or eGFR (naïve) changes these switch patients were baseline agalβ ADA-positive, suggesting agalα from Baseline. Switch patients showed statistically significant mean±SE cross-reactivity, since they had no previous agalα exposure. After eGFR (mL/min/1.73 m^2) declines overall (-3.17±1.69, p00.009), but not 12 months, 3 (10.3 %) naïve and 19 (30.6 %) switch patients tested agalα with hyperfiltrators removed (n054; -1.2±1.6; p00.081). AEs were con- ADA-positive; 1 (3.4 %) naïve and 10 (16.1 %) switch patients had agalα sistent with the known safety profile of agalα (most common AEs [naïve/ neutralizing ADA's (NAbs); 4 switch patients became agalα ADA-negative switch]: nasopharyngitis [20.7 %/8.1 %], dizziness [17.2 %/12.9 %], head- and 5 became NAb-negative. No new switch patients developed agalα IgG ache [27.6 %/8.1 %], nausea [20.7 %/11.3 %]). or NAbs during treatment, although 6 developed agalα IgM. Conclusions: After 1 year of agalα for FD, no statistically significant Conclusion: After 12-months' agalα, the number of switch patients with changes were found in LVMI (all naïve or switch) or eGFR (all naïve or agalα ADA's did not increase and some became ADA-negative. Few naïve switch patients minus hyperfiltrators), and no new safety concerns emerged patients developed agalα ADA's, consistent with agalα's known antigenic- (naïve or switch). ity profile. Conflict of Interest declared. Conflict of Interest declared. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S149

P-407 P-408 BONE MINERAL DENSITY RESPONSE TO ENZYME REPLACEMENT PATHOLOGICAL EXAMINATION OF THE LUNG IN A FABRY THERAPY OVER 2 YEARS IN ADULTS WITH TYPE 1 GAUCHER PATIENT ON ENZYME REPLACEMENT THERAPY DISEASE Raiman JAJ1 , Cameron BH2 , Mackrell M1 , Clarke JTR1 , Terry J3 Zimran A1 , Gonzalez DE2 ,KabraM3 , Lukina EA4 ,GiraldoP5 , 1Div Metab Gen, Hosp for Sick Children, Toronto, Canada Kisinovsky I6 ,BavdekarA7 ,BenDridiM-F8 ,GuptaN3 , Kishnani PS9 , 2Dept of Surg, McMaster Children's Hosp, Hamilton, Canada Mehta A10 ,ElsteinD11 ,WangN12 , Hangartner TN13 , Crombez E12 ,Ben 3Dept of Pathol, McMaster Children's Hosp, Hamilton, Canada Turkia H8 1Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel The pathological features of Fabry Disease [FD] have been widely docu- 2Sanatorio Español, Asunción, Paraguay mented; however, lung involvement is not well described especially in the 3All India Institute of Medical Sciences, New Delhi, India setting of long-term enzyme replacement [ERT]. 4Hematology Research Center, Moscow, Russian Federation We report the findings in a teenage male who presented with a spontaneous 5CIBERER & Hosp Univ Miguel Servet, Zaragoza, Spain pneumothorax. He was diagnosed in childhood following confirmation by 6Your Health SA, Buenos Aires, Argentina decreased alfa galactosidase activity of 1 nmol/h/mg protein [13-38] and a 7KEM Hospital Research Centre, Pune, India deletion in the GLA gene [744_745delAC]. He has been treated with ERT 8La Rabta Hospital, Tunis, Tunisia [agalactosidase alpha] for 9 years. 9Duke University Medical Center, Durham, NC, United States He presented with pleuritic pain and dyspnea. The xray showed a right 10Royal Free Campus, University College, London, United Kingdom pneumothorax that was treated by chest drain without resolution. CT scan 11Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel showed right apical blebbing. At operation there were multiple apical 12Shire HGT, Lexington, MA, United States pleural adhesions and a wedge excision of the upper lobe apex was done. 13Wright State University, Dayton,OH, United States Microscopy revealed subpleural simplification of airspaces and inflammatory changes consistent with spontaneous pneumothorax. Also present was Objective: To assess bone mineral density (BMD) response to velaglucer- prominent vacuolization of the respiratory epithelial cytoplasm and appa- ase alfa in adults with type 1 Gaucher disease (GD1). rent thickening of bronchiolar smooth muscle compared to age-matched Methods: BMD was measured by dual-energy x-ray absorptiometry in control tissue. Histological and ultra structural features typically associated GD1 patients, receiving 60 U/kg every other week (EOW) of velaglucerase with untreated Fabry's disease, including the characteristic lysosomal inclu- alfa (velaglucerase/continuous arm) or imiglucerase (imiglucerase/switch sions on EM, were not seen. arm) in HGT-GCB-039 (9-month trial), followed by 15 months' velaglucer- We believe that the impact of ERT may be responsible for lack of findings ase alfa 60 U/kg EOW (both arms) in HGT-GCB-044 (extension study). of Fabry's disease in the lung. BMD Z-scores (age- and sex-matched) were calculated. Conflict of Interest declared. Results: Excluding patients on concomitant bisphosphonates, 11 velaglucerase alfa-arm patients (19-58 years) and 8 imiglucerase-arm patients (20- 44 years) were studied (median [range] lumbar spine [LS] Z-scores at Baseline −1.46 [−3.50, 0.98] and −0.86 [−2.17, 2.02], respectively). Mean LS Z-score changes from Baseline (95 % CIs) in the velaglucerase/contin- P-409 uous and imiglucerase/switch arms were 0.33 (0.10, 0.55) and 0.06 (−0.22, VALUE IN DISEASE REGISTRIES - THE HUNTER OUTCOME 0.34) at 9 months and 0.64 (0.22, 1.06) and 0.54 (0.21, 0.87) at 24 months, SURVEY (HOS) respectively (similar results when patients on bisphosphonates2 continuous Paabxl Larsen M1 , Whiteman D1 [18 and 60 years], 3 switch [35, 48 and 58 years]included). All femoral 1Shire AG, Eysins, Switzerland neck (FN) changes from Baseline (both arms, ±bisphosphonates) were non- significant (P>0.05). Hunter syndrome (MPSII) is an X-linked lysosomal storage disease caused Conclusion: In GD1 adults, LS BMD Z-scores increased significantly (P< by iduronate-2-sulfatase (I2S) deficiency , with progressive accumulation of 0.05) following 9 months' velaglucerase alfa, but not imiglucerase, glycosaminoglycans in organs and body tissues. MPSII is a multi- exposure. symptomatic disease, primarily affecting males (Incidence - 1:162,000). Conflict of Interest declared. The Hunter Outcome Survey (HOS: 2005) is a global, longitudinal disease registry, sponsored by Shire HGT gathering real-world data on MPSII natural history, and monitoring long-term safety and effectiveness data on idursulfase. Patients with confirmed MPSII are eligible for enrolment after informed consent. Participating sites require ethics committee and regulatory approval in accordance with applicable laws. HOS is overseen by MPSII physi- cian advisory boards As of January 2012, 902 patients from 116 clinics across 24 countries were enrolled. Most patients were below 18 years of age at entry. The median ages (10th – 90th percentiles) at symptom onset and diagnosis were 1.5 (0.3 - 4.0) years and 3.3 (1.2 - 7.2) years, respectively. Neurological manifestations were reported in 592/702 (84 %) prospective patients; chest and lung symptoms in 600/703 (85 %) prospective patients. HOS provides real-world data on MPSII. More than 12 papers have been published from HOS data. Ongoing collaboration with participating physicians allows HOS to expand the evidence base. Conflict of Interest declared. S150 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-410 P-411 BONE MINERAL DENSITY IN ADULTS WITH TYPE 1 GAUCHER ERYTHROCYTE ENCAPSULATED THYMIDINE PHOSPHOS- DISEASE RECEIVING VELAGLUCERASE ALFA 60 U/KG EVERY PHORYLASE (EE-TP) FORTHE TREATMENT OF MITOCHONDRIAL OTHER WEEK: 2-YEAR RESULTS NEUROGASTROINTESTINAL ENCEPHALOMYOPATHY (MNGIE) Elstein D1 , Ben Turkia H2 , Gonzalez DE3 , Kabra M4 , Lukina EA5 , Levene M1 , Bain MD1 , Fairbanks LD2 , Moran NF2 , Bax BE1 Giraldo P6 , Kisinovsky I7 , Bavdekar A8 , Ben Dridi M-F2 , Gupta N4 , 1Clin Sci, St George's Univ of London, London, United Kingdom Kishnani PS9 , Mehta A10 , Zahrieh D11 , Hangartner TN12 , Crombez E11 , 2Kings College, London, United Kingdom Zimran A1 1Shaare Zedek Med Cent & Hadassah Med Sch, Jerusalem, Israel MNGIE is a rare and fatal autosomal recessive disorder caused by a 2La Rabta Hospital, Tunis, Tunisia deficiency in thymidine phosphorylase leading to an accumulation of thy- 3Sanatorio Español, Asunción, Paraguay midine and deoxyuridine. EE-TP is under development as an orphan 4All India Institute of Medical Sciences, New Delhi, India designated therapy for MNGIE. In this approach the deficient enzyme is 5Hematology Research Center, Moscow, Russian Federation encapsulated within the patient's own erythrocytes in vitro which are 6CIBERER & Hosp Univ Miguel Servet, Zaragoza, Spain returned to the patient where the enzyme metabolises the pathological 7Your Health SA, Buenos Aires, Argentina plasma metabolites which permeate the erythrocyte by passive diffusion. 8KEM Hospital Research Centre, Pune, India Two patients with confirmed diagnoses of MNGIE were recruited into a 9Duke University Medical Center, Durham, NC, United States compassionate clinical evaluation of EE-TP. Patient 1 received 31 treatment 10Royal Free Hospital, London, United Kingdom cycles over 24 months and Patient 2 received 17 cycles over 17 months. 11Shire HGT, Lexington, MA, United States AdministrationofEE-TP(6-50IU/kg)everytwotofourweekswas 12Wright State University, Dayton,OH, United States effective in reducing the elevated plasma and urine thymidine and deoxyuridine concentrations. Patient 1 after three months reported a reduction in Objective: Bone mineral density (BMD) was evaluated in type 1 Gaucher the number of vomiting attacks, gaining 4 kg in weight. Patient 2 reported disease (GD1) patients receiving velaglucerase alfa 60 U/kg every other after two months an improvement in distal sensation in his hands and week over 2 years. fingers and after 12 months showed improvements in sensory ataxia and Methods: BMD was assessed by dual-energy x-ray absorptiometry of the fine finger functioning allowing the resumption of previously lost physical lumbar spine (LS) and femoral neck (FN) in two Phase III trials (TKT032, activities. Both patients reported an increased quality of life. HGT-GCB-039) and an extension study (HGT-GCB-044). BMD Z- (age- EE-TP offers a promising new treatment for MNGIE. and sex-matched) and T-scores were calculated. Results: 19 adults (19–58 years) completed 2 years' velaglucerase alfa. Median (range) LS and FN Z-scores at Baseline were -1.59 (-4.20–0.98) and -1.2 (-3.53–2.84), respectively. Mean (95 % CI) LS and FN Z-score changes from Baseline were 0.27 (0.10, 0.44) and -0.02 (-0.25, 0.21) at 1 year and 0.64 (0.35, 0.93) and 0.1 (-0.12, 0.32) at 2 years, respectively. The results remained similar when 2 additional patients on concomitant P-412 bisphosphonates were included. By 2 years, 5/19 patients changed WHO ENZYME REPLACEMENT THERAPY IN A GIRL AFTER 15 → classification from osteopenia normal and 1 patient changed from osteo- YEARS OF HEMATOPOIETIC STEM CELL TRANSPLANTATION → → porosis osteopenia in LS; 1/19 patients changed from osteopenia nor- Lee Ni-Chung1 , Chien Yin-Hsiu1 , Yang Chih-Chao2 , Hwu Wuh-Liang1 , → mal, 1 changed from osteoporosis osteopenia, 2 changed from Tsseng Sheng-Hong3 → → osteopenia osteoporosis and 1 changed from normal osteopenia in FN. 1Dep Ped, Nat Taiwan Univ Hosp, Taipei, Taiwan Conclusion: Beginning at 1 year and continuing through Year 2, LS BMD 2Dep Neur, Nat Taiwan Univ Hosp, Taipei, Taiwan improved significantly (P<0.05) among GD1 velaglucerase alfa 60 U/kg- 3Dep NeuroSur, Nat Taiwan Univ Hosp, Taipei, Taiwan treated patients. Conflict of Interest declared. Mucopolysaccharidosis type VI (MPS VI; MIM#253200) or Maroteaux-Lamy syndrome is a rare autosomal recessive disorder due to arylsulfatase B (ARSB) deficiency. The accumulation of dermatan sulfate and chondroitin sulfate within lysosomes in tissues results in hepatosplenomegaly, corneal opacity, skeletal malformation, and growth retardation. In the past two decades, bone-marrow transplantation (BMT) has been considered as the primary treatment option for MPS VI patients. Since 2005, enzyme replacement therapy (ERT) using recombinant human ARSB was approved by the US FDA for clinical utilization. Here we present a girl received bone marrow from her HLA-matched brother at 10 years old with normalization of arylsulfatase activity of the recipient's leukocytes and urinary excretion of glycosaminoglycans (GAGs). However, she developed progressive C-spine stenosis, corneal clouding, malocclusion, and exercise intolerance gradually. She received corneal transplantation and Suboccipital craniectomy and C1-C5 laminectomy at 25 years old, 15 years after BMT. ERTwas given one year later. After ERT, her urine GAG decreased further and her exercise intolerance was improved. For MPS VI patients after BMT, C- spine stenosis, exercise intolerance, corneal clouding, or valvular heart disease may still occur, and an adjunctive ERT could be considered. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S151

P-413 P-414 IMPACT OF IDURSULFASE ON GROWTH RATE IN PATIENTS GLYCOMACROPEPTIDE BASED PROTEIN SUBSTITUTES: ARE WITH HUNTER SYNDROME: DATA FROM THE HUNTER THEY THE NEXT GENERATION FOR PKU ? OUTCOME SURVEY (HOS) Daly A1 , Ashmore C1 , Chahal S1 , MacDonald A1 Jones SA1 , Parini R2 , Harmatz P3 , Giugliani R4 , Mendelsohn NJ5 1Birmingham Children's Hospital, Birmingham, United Kingdom 1MAHSC, St Mary's Hosp, Univ Manchester, Manchester, United Kingdom 2Univ Milano Bicocca, San Gerardo Hosp, Monza, Italy Glycomacropeptide (GMP), casein derived whey peptide, contains no 3Children's Hosp & Research Cntr Oakland, Oakland, CA, United States aromatic amino acids (AA) and only a trace amount of phenylalanine 4Dept Genet/UFRGS and INAGEMP, Porto Alegre, Brazil (phe) rendering it a suitable low-phe protein substitute (PS) in phenyl- 5Children's Hosps & Clinics of Minnesota, Minnesota, MN, United States ketonuria (PKU). AA supplemented GMP has been developed for PKU and initial studies indicate benefit over conventional phe-free We assessed the effects of idursulfase on rate of growth in patients with AA's. Hunter syndrome in HOS, a global, multicentre, longitudinal, observational Aim: An evaluation of the acceptability and tolerance of GMP PS in PKU. database, sponsored by Shire Human Genetic Therapies. Methods: 10 children with PKU, median age 13.5y (range 11-17y; 5 males) As of July 2011, height data before treatment were available for 544 of 713 males took 2 different presentations of GMP PS (Camino Pro Restore sports drink, followed prospectively after HOS entry. Age-corrected standardized height scores 10 g protein equivalent/500 ml; Camino Better Milk, 15 g protein equiv- (Z-scores) before and after treatment start (TS) in 133 patients (aged 8–15 years at alent/49 g powder, Cambrook Foods USA), each for 7 days. All completed TS; data available on ≥1 occasion, +/–2 years of TS; growth hormone-treated an acceptability and tolerance questionnaire. All but 2 took liquid AA pre- patients excluded) were analysed using a piecewise regression model. study. Results: showed that the slope of regression after treatment was significantly Results: All children took at least 500 ml of GMP liquid daily for 7 days; it improved compared with before treatment (estimated slopes before and after was well accepted by 50 % of subjects and 100 % liked its 'normality' and treatment were –0.040 and –0.011, respectively; difference in Z-score 0.029, p< trendy presentation. Its large volume was negatively rated by 30 %. The 0.001). Analysis of covariates indicated a significant influence of mutation type GMP powdered product was not accepted by the majority, its mixing was (height deficit in terms of Z-scores most pronounced in patients with deletions/ difficult, influencing its final texture. large rearrangements, p00.0014), age (most pronounced in 12–15-year group, p< Conclusion: GMP PS offers theoretical benefit over phe-free AA's but it is 0.0001), and an age by time before treatment interaction (growth deficit gets worse essential its presentation and volume equal existing phe-free AA's. at a faster rate in the 12–15-year versus 8–11-year group before TS, p00.023). Conflict of Interest declared. Idursulfase treatment improved growth rate in a large group of patients in HOS, aged 8–15 years at TS. Conflict of Interest declared.

33. Dietetics and Nutrition P-415 LONG TERM GROWTH STATUS OF TURKISH CHILDREN P-129 WITH PHENYLKETONURIA: A RETROSPECTIVE STUDY HANDS-ON SEMINARS AS EDUCATIONAL TOOL USED IN Gökmen-Özel H1 , Gedikli C1 , Köksal G1 ,Coşkun T2 DIETARY MANAGEMENT OF PHENYLKETONURIA (PKU) 1Hacettepe Univ, Nutr Diet Dept, Ankara, Turkey Link RM1 , Lang F2 2Hacettepe Univ, Dept Ped, Metab Unit, Ankara, Turkey 1Chair SSIEM-DG,, Wiesbaden, Germany 2University Childrens Hospital, Mainz, Germany Background: Early experience with a low-phenlyalanine diet in phenylketonuric patients showed a positive effect on mental development but a negative A commonly problem in PKU is poor compliance with diet. Education is a effect on physical growth. Some studies employed longitudinal measurements very important tool to achieve compliance. Mainly one-on-one counseling of parameters related to growth, but no study has reported the growth of and printed material are used for all age groups. Hands-on seminars are phenlyketonuric patients from infancy throughout adolescence. In this single offered rarely, an important tool to acquire basic skills and independence for centre, retrospective study, the aim was to examine changing growth param- it`s own PKU diet. eters in phenylketonuria from infancy to 18 yrs of age. We invited PKU children (6-12 y) with their parents and siblings to prepare Methods: Twenty nine children (16 boys, 13 girls) aged between 18.1 and a tasty "funny-face" sandwich. Different kind of foods as bread, spreads and 31.7 yrs with phenylketonuria were recruited. Weight for height (WHZ), various vegetables were offered. 15 children attended the event for 90 weight for age (WAZ), height for age (HAZ) and body mass index (BMIZ) minutes. The variety of foods and how to prepare them was not known to z scores were calculated using WHO Anthro programmes. Blood phenyl- many children and family members. All enjoyed the sandwiches and alanine concentrations from diagnosis to 18 yrs compared to the local experienced that PKU tastes good, can be eaten without weighing and guidelines. calculating and brings healthy food to the whole family. Practical knowl- Results: Median WHZ scores significantly increased (p00.001), median edge helped caregivers to become positive role models. The seminar was HAZ scores significantly decreased (p00.001) and median WAZ and BMIZ easy to organize and perform, but was not easy to convince the scores did not change significantly with age. Collectively, the percentage of families to participate. Practical experience is important for all age all plasma phenylalanine concentrations >6 mg/dL was 73.8 % in the first groups and should start in preschool age when taste and nutritional 10 years of life and >12 mg/dL was 58.2 % between 10.1 to 18 years of age. behaviour are shaped. The seminar helps to promote practical skills, Conclusions: No abnormalities in physical development were noticed to taste a big range of natural food and to become independent for except poor height gain during low phenylalanine diet. later life. Conflict of Interest declared. S152 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-416 P-418 NUTRITIONAL OUTCOME POST LIVER TRANSPLANTATION HELPING ADHERENCE ON LOW PROTEIN DIETS: AN EVALUATION IN IMD - A CASE REVIEW OF NOVEL LOW PROTEIN, INSTANT SNACK POTS Daly A1 , Ashmore C1 , McKiernan P1 , MacDonald A1 Chahal S1 , Ashmore C1 , Daly A1 , MacDonald A1 1Birmingham Children's Hospital, Birmingham, United Kingdom 1Birmingham Childrens Hospital, Birmingham, United Kingdom

Introduction: Liver transplantation (OLT) eliminates the risk of metabolic Many older patients with inherited metabolic disorders (IEM) on very low decompensation, and allows a normal diet in Urea Cycle (UCD) and natural protein diets (commonly <10 g/daily) have adherence issues Glycogen Storage(GSD) disorders. We report the nutritional outcome of 8 because there are few snack meals which can be prepared quickly in order subjects with successful OLT in GSD and UCD. to satisfy hunger and provide variety. A series of instant low protein (LP) Case studies: Three girls of Pakistani origin with GSD type 1b were instant snack pots based on maize and potato starch have been specifically transplanted at 6y (range 3-9y). All had limited fasting tolerance (maximum developed for patients at their request. 2 h) pre-transplant and were completely dependent on enteral feeding. Aim: To evaluate the acceptability and taste of 2 types of LP snack pots in Mean weight and height z scores pre-transplant were 1.1 and -0.8; post patients with IEM on LP diets. transplant (up to 3y) were 0.1 and -1.1. One child remains on tube feeds, but Methods: LP pasta pots and LP potato pots were given to 2 groups of 22 all have normal fasting tolerance. and 23 patients respectively (aged 1-18y with a median age of 7y). They Five children (3 girls) with UCD (3 Citrullinaemia; 2 OCD) were trans- completed evaluation questionnaires about their taste, appearance, texture, planted at 3y 9 m (1.2 -6.6y). Four children received tube feeds pre- and packaging. transplant; all were on low natural protein diets±essential amino acids. Results: 92 % enjoyed the low protein pasta pots, with 68 % appreciating Mean weight and height z scores pre-transplant were 0.4 and 0.04; post the snack pot presentation. Potato pots were enjoyed by 100 % (n011) of transplant (up to 3y) were -0.24 and -0.9. Two children remain on tube patients ≥11y and 50 % <11y with 50 % overall valuing their presentation. feeds, but all tolerated normal protein intakes. The snack pots were thought to be particularly convenient for school, Conclusions: OLT facilitates dietary relaxation but growth still requires holiday and eating away from home. careful monitoring. Poor growth may be associated with drug therapy or Conclusion: It is essential LP special foods are trendy and ready to use and reflect less intensive nutritional support. follow 'normal' market trends to ensure they are consumed by patients.

P-417 P-419 GROWTH AND NUTRITIONAL SUPPORT IN PROPIONIC MAPLE SYRUP URINE DISEASE: USE OF LEUCINE FREE ACIDAEMIA MEDICAL FOODS AND AMINO ACID SUPPLEMENTS IN Daly A1 , Ashmore C1 , Chakrapani A1 , Vijay S1 , Santra S1 , MacDonald A1 DIETARY TREATMENT 1Birmingham Children's Hospital, Birmingham, United Kingdom Çakir N1 , Tuncer S2 , Balci MC2 , Ersoy M2 , Demirkol M2 , Gökçay G2 1Dep of Dietetics, Istanbul Medical Fac, Istanbul, Turkey Introduction: There are no international guidelines for the dietary manage- 2Div of Nutr and Metab, Istanbul Med Fac, Istanbul, Turkey ment of propionic acidaemia (PA). We present our experience in a group of patients with PA who presented in the neonatal period and all received their Background: Maple syrup urine disease (MSUD) (OMIM 248600) is nutrition via tube feeds. caused by a deficiency of branched-chain 2-ketoacid dehydrogenase Methods: All 7 subjects were Pakistani origin (4 male); with annual, enzyme complex. The management of patients with MSUD consists of retrospective nutritional intake and anthropometric data presented up until limiting the intake of branched chain amino acids (BCAAs) which are 9 years of age. The median age of subjects was 4y, range 1-9y. elevated during critical illness. On treatment plasma concentrations of Results: Total protein intake (natural and PA precursor-free amino acids isoleucine (ILE) and valine (VAL) decrease more rapidly than leucine (AA)) in g/kg/day at 1 m was 2 g; 1-3 y, 1.4 g; 4-6y, 1.3 g; and 7-9y, 1.3 g. (LEU) and they have to be supplemented to prevent deficiency. PA precursor-free AA were given to all patients and provided a mean of Objectives: We compared the response to treatment with LEU-free medical 24 % (range 8-50 %) of total protein intake for all age categories. Their foods and single amino acid supplements when both plasma ILE and VAL mean energy intake in kcal/kg/day at 1 m was 162; 1-3 y, 79; 4-6y, 57; and concentrations were low and LEU elevated. 7-9y, 51. Weight z-score at 1 m was -2.1; 1-3y, -0.1; 4-6y, 1.6; 7-9y, 1.6; Material and Results: Plasma BCAA's were monitored in 8 patients and height/length z-score at 1 m was -2.3; 1-3y, -1.2; 4-6y, -1.2; and during 9 episodes. According to our observation, LEU-free formula is better 7-9y, -1.8. tolerated, caused a rapid decrease in plasma LEU concentrations and a more Conclusions: Achieving optimal growth in PA was challenging, even rapid increase in ILE and VAL concentrations when compared to similar though total protein met safe levels (WHO/UNU/FAO 2007) of intake. amounts of ILE and VAL supplements. We speculate that ILE and VAL Weight gain was difficult to control despite low energy intakes when from LEU-free formula can be absorbed more efficiently than when given patients were well. as single amino acid supplements. Conclusion: In the treatment of patients with MSUD when both ILE and VAL have to be supplemented, LEU-free formula can be used more efficiently than amino acid supplements. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S153

P-420 P-422 FOOD INTAKE OF ADULT DUTCH PATIENTS WITH NUTRITIONAL BLOOD MONITORING IN UK ADULT PHENYL- MITOCHONDRIAL DISEASES KETONURIA AN AUDIT OF PRACTICE Zweers H1 , Janssen M1 , Engelen v. B1 , Wanten G1 Boocock S1 , Robertson LV1 , Weetch E2 , Rogozinski H3 , Wildgoose J3 , 1Radboud Univ Nijmegen Med. Centre, Nijmegen, Netherlands Maritz C4 ,ChanH4 ,EllertonC4 , Danaher S5 , Donald S6 ,McStravickN7 , Terry A8 , MacDonald A9 Background: Mitochondrial Diseases (MD) are among the most fre- 1Univ Hosp Birmingham NHS Found Trust, Birmingham, United Kingdom quent metabolic diseases and their treatment is exclusively supportive. 2Sheff Teach Hosp NHS Found Trust, Sheffield, United Kingdom Micronutrients play a role in MD and it has been reported that 3Brad Teach Hosp NHS Found Trust, Bradford, United Kingdom inadequate energy intake can lead to secondary mitochondrial impair- 4UCLH NHS Found Trust, London, United Kingdom ment. MD patients have low energy requirement leading to a need for 5Guys and St Thoms NHS Found Trust, London, United Kingdom high nutrient density. Since many adult MD patients suffer from 6Cambs Uni NHS Found Trust, Cambridge, United Kingdom malnutrition or obesity this raises the question whether these patients 7Belfast Health & Social Care Trust, Belfast, United Kingdom have an adequate diet. 8Alder Hey NHS Found Trust, Liverpool, United Kingdom Methods: Food records of 20 adult MD patients were collected and com- 9Birmingham Child Hosp NHS Found Trust, Birmingham, United Kingdom pared with Recommended Daily Allowance (RDA) and with the Dutch National Food Survey (DNFS) 2004-2011. There is little published evidence regarding which nutritional bloods should Results: While no patient had an adequate diet, the use of milk be routinely measured in adults with Phenylketonuria (PKU). products was below normal when compared with RDA and with the A questionnaire was distributed to eleven UK metabolic centres to deter- DNFS, possibly due to gastrointestinal problems. 4 patients had an mine which nutritional blood tests are routinely tested, including patients on inadequate protein intake and 5 patients used prescribed diet prod- and off diet and during pregnancy. Parameters included vitamin B12, folate, ucts.12 patients had a high sugar intake and 2/3 of the patients took a ferritin, zinc, selenium and copper. The centres were also asked if they micronutrient supplement. confirm a deficiency by measuring another parameter. E.g. confirm a Conclusion/discussion: MD patients seem not able to meet their nutrient vitamin B12 deficiency by measuring total homocysteine levels. demands. Patients try to compensate their lack of energy by eating con- Eight centres responded: venience foods with high sugar content which makes meeting nutrient / 100 % of centres routinely measure vitamin B12 and folate in all PKU demands even harder. Nutritional intervention may be beneficial for these patients. No centres confirm deficiency by measuring another parameter. patients. / 75 % of centres routinely measure ferritin, increasing to 88 % in maternal PKU. / 13 % and 25 % of centres routinely measure copper and both zinc and selenium respectively for patients on diet. For maternal PKU and those off diet, this increases to 25 % and 50 %. / One centre confirms selenium deficiency by checking other parameters P-421 such as glutathione peroxidase concentrations. TRACE ELEMENT STATUS IN ADULTS WITH PHENYLKETONURIA The results illustrate variable practice across UK centres. Further longitu- 1 1 1 2 Boocock S , Robertson LV , Geberhiwot T , MacDonald A dinal study is needed to determine which nutritional parameters should be 1 Univ Hosp Birmingham NHS Found Trust, Birmingham, United Kingdom measured routinely and a standard UK protocol devised. 2Birmingham Child Hosp NHS Found Trust, Birmingham, United Kingdom

Little has been reported about the trace element status of adults with Phenylketonuria (PKU). The non-fasting plasma zinc and selenium status of adult patients (>16 years) with PKU were reviewed. One zinc and selenium result was recorded for each patient (n049), excluding maternal PKU and patients taking Sapropterin. The patients were divided into 3 groups. The median zinc and selenium concentrations and percentage of results outside the reference ranges were compared (selenium: 0.9-1.7 μmol/l, zinc: 11-24 μmol/l). 1) Patients on dietary treatment with approximately 70 % of blood phenylalanine concentrations<700 μmol/l (median selenium 0.83 μmol/l, median zinc 10.7 μmol/l and 71 % of selenium results below the reference range and 55 % of zinc). 2) Patients on dietary treatment with approximately 70 % of blood phenylalanine concentrations>700 μmol/l (median selenium 0.94 μmol/l, median zinc 10.7 μmol/l and 40 % of selenium results below the reference range and 67 % of zinc). 3) Patients not on dietary treatment (median selenium 1.04 μmol/l, median zinc 10.1 μmol/l and 30 % of selenium results below the reference range and 89 % of zinc). Low zinc and selenium concentrations are common in adult patients with and without dietary treatment. Further work is needed to assess longitudinal trace element status, clinical deficiency and devise practical solutions in adults with PKU. S154 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-423 P-425 PUFA AND LCPUFA PROFILES IN CHILDREN WITH LCHAD CONTINUOUS BLOOD SUGAR MONITORING IN GSD: A DEFICIENCY HELPFUL MONITORING TOOL FOR DIETARY MANAGEMENT Sykut-Cegielska J1 , Kowalik A1 , Wierzbicka A2 , Syczewska M3 Daly A1 ,AshmoreC1 ,SantraS1 , Mckiernan P1 , Salmons E1 , 1Dept Metab Dis, Child Memor Health Inst, Warsaw, Poland MacDonald A1 2Dept Lab Diagn, Child Memor Health Inst, Warsaw, Poland 1Birmingham Children's Hospital, Birmingham, United Kingdom 3Dept Ped Rehab, Child Memor Health Inst, Warsaw, Poland Introduction: Continuous blood glucose monitors (CBGM) have been Background: Children with LCHAD deficiency (LCHADD) are treated introduced to monitor the efficacy of dietary management (DM) in glyco- with low fat diet. Excluding sources of linoleic acid (LA) and α-linolenic gen storage disease (GSD). The aim of DM is to minimise secondary acid (ALA) from diet increases the risk of polyunsaturated- and long-chain metabolic abnormalities by maintaining normoglycaemia. polyunsaturated fatty acids (PUFA and LCPUFA) deficiency. Aim: To report the value of CBGM in GSD from one treatment centre. Objective: Are PUFA and LCPUFA deficient in children with LCHADD? Methods: CBGM (Medtronic) was attempted on 23 occasions for 2-5 days Subjects and in 17 subjects (10 girls, median age 7.4y; (range 3 m-16y). GSD types were: Methods: Plasma phospholipid fatty acid profiles from 17 patients (age 1a, n03; 1b, n04; III, n07; IX, n02. Uncooked cornstarch (UCCS) was 15 months-20 years) with LCHADD were determined by gas chromatog- used in 10 children from all GSD group types; overnights tube feeds (OTF) raphy and expressed as % wt/wt of all fatty acids. The control group was 59 in 13 subjects. healthy children (age 6–19 years). Results: GSD 1a and 1b subjects had significant variation in 24 hour Results: ALA, docosahexaenoic acid (DHA), arachidonic acid (AA) did not blood glucose (BG) control, with both low and high BG throughout differ in the patients and control group. LA, N3, N6 and PUFAs were signifi- day and night. In 58 % (n010) their DM was modified as a result of cantly lower (t-student test, p<0,001), but N3/N6 ratio and eicosapentaenoic CBGM. GSD III and IX consistently achieved normoglycaemia. With acid (EPA) were significantly higher in the patients (t-student test, p00,028 and CBGM, four children (GSD1b 01, GSD III 03) have safely stopped Mann-Whitney's test, p00.016, respectively). Discussion: This study does not OTF using UCCS instead. confirm previous reports about DHA deficiency in the patients on the recom- Conclusion: The CBGM was well tolerated and facilitated DM changes in mended diet with approximate content of 10 % of energy from long-chain fatty the management of GSD. Use of the monitor has allowed an improved and acids, but without supplementation of PUFAs from vegetable oils. Endogenous individual approach to DM. LCPUFA synthesis seems to be sufficient, but further studies are needed. Conclusion: PUFA and LCPUFA status is acceptable in the patients with LCHADD.

P-424 P-427 UNRESTRICTED FRUITS AND VEGETABLES IN THE PKU DIET: ASSESSING NUTRITIONAL STATUS IN PATIENTS WITH A ONE YEAR FOLLOW-UP INBORN ERRORS OF METABOLISM A MULTICENTER Rohde C1 , Mütze U1 , Arelin M1 , Weigel JFW1 , Schulz S2 , Schulz S2 , SURVEY Ceglrek U3 , Thiery J3 , Kiess W1 , Beblo S1 Coelho M1 , Faria A1 , Garcia P1 , Diogo L1 1Child Hosp ot the Univ. of Leipzig, Leipzig, Germany 1Unid Doenças Metab, Hosp Pediátrico, Coimbra, Portugal 2University Children's Hospital, Hamburg-Eppendorf, Germany 3Instit Labor. Med, Univ Leipzig, Leipzig, Germany Background: Nutritional status has been shown to highly influence the outcome in many diseases. Patients with inherited metabolic disorders Objective: Phenylketonuria (PKU) therapy demands protein-calculation submitted to restrictive diets are susceptible to develop nutritional deficits. from natural foods, including small amounts from fruits and vegetables. Although nutritional status assessment of these patients is extremely impor- We investigated whether or not free access to fruits and vegetables impairs tant, no consensus guidelines have been published yet. metabolic control over a one year period. Objectives: Determine how dietitians assess nutritional status of patients Patients/Methods: 19 PKU children (2-10 years) with good metabolic with inborn errors of metabolism. control had free access to fruits and vegetables over one year. Dried blood Methods: An online anonymous survey was created and web link was sent phenylalanine (Phe) concentration was measured three times/week over the to dietitians of several European centers. first two weeks, thereafter bi- to four-weekly. Fruit and vegetable consump- Results: 23 dietitians answered the survey. The great majority assesses nutrition and nutrient intake was analysed from diet records, performed initially, tional status at least once a year using anthropometry (n022), and laboratorial after two weeks, six and 12 months. tests (n020). Calcium, hemoglobin, total protein, albumin, cobalamin and iron Results: Phe intake increased significantly by 58 mg/day (week 2, p< are the most frequently analyzed parameters, whereas other, like selenium and 0.001), 70 mg/day (month 6, p<0.001), 61 mg (month 12, p<0.001). Dried aminoacids, were less mentioned (n010 and n02, respectively). Subjective blood Phe concentrations remained stable (195 +/-108 μmol/l before study global assessment and body composition evaluation (mainly using bioelectri- vs. 178+/-108 μmol/l, 178+/-108 μmol/l, 167 +/-95 μmol/l; p00.894). The cal impedance) are less commonly used (n09 and n08, respectively). When frequency of Phe concentrations above the recommended range remained nutritional deficits are detected, individual dietary counseling and supplemen- unchanged (24+/-33 % initially, vs. 22+/-35 %, 18+/-38 % and 19+/-32 %, tation are regularly used (n022 and n018, respectively). p00.592). Total fruit and vegetable intake was unchanged (initially 345+/- Conclusion: Nutritional status of patients with inborn errors of metabolism 133 g/d; vs 367+/-178 g/d, 379+/-184 g/d, 318+/-102 g/d; p00.692) is usually assessed by anthropometry and laboratorial tests. Establishing Conclusion: PKU diet liberalisation for fruits and vegetables seems unpro- and validating guidelines, based on clinical experience of those who care blematic. Interestingly,despite increased diet flexibility, basic eating habits for these patients, is essential. remained unchanged after one year. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S155

P-429 P-431 SMS TEXT MESSAGE REMINDER SERVICE FOR PHENYLKE- ANALYSIS OF GALACTOSE CONTENT OF CASEINATES AND TONURIA PATIENTS TO MONITOR PHENYLALANINE AGED CHEESES FROM NORTH AMERICA: IMPLICATIONS CONCENTRATIONS FOR NUTRITION MANAGEMENT OF CLASSICAL Robertson LV1 , Boocock S1 , MacDonald A2 , Geberhiwot T1 GALACTOSEMIA 1Univ Hosp Birmingham NHS Found Turst, BIRMINGHAM, United Van Calcar SC1 , Bernstein L2 , Rohr F3 , Scaman CH4 Kingdom 1University of Wisconsin-Madison, Madison, Wisconsin, United States 2Birmingham Child Hosp NHS Found Trust, BIRMINGHAM, United 2Children's Hospital of Colorado, Aurora, Colorado, United States Kingdom 3Children's Hospital Boston, Boston, Massachusetts, United States 4University of British Columbia, Vancouver, British Columbia, Canada It is advocated that adults with Phenylketonuria (PKU) monitor their own blood phenylalanine (phe) concentrations monthly (MRC 1993). The aim of Background: European studies have found that some aged cheeses have this service was to improve adherence with blood taking. negligible galactose and may be appropriate to include in the diet for individuals A text message reminder was set up via the hospital data base. Adult PKU with classical galactosemia. However, analysis of cheeses produced in North patients (>16y) were invited to join the project. An automatic monthly text America has not been completed. Evaluation of caseinates is also needed. message was sent reminding patients to take a blood spot sample. Methods: Various types of cheese, sodium and calcium caseinate were pur- 25 patients were audited. There was a 65 % increase in samples returned chased in different locations in North America. Total galactose content was over 12 a month period (total 117; p00.03). determined from both lactose and free galactose quantitated by HPAEC-PAD. The patients were split into 4 groups: a) patients that increased the number Results: Galactose content of caseinates averaged 36 mg/100 g. U.S manu- of blood spots by 2 or more (44 %, median phe over 2 years 595 μmol/l), b) factured Gruyere and Emmentaler cheese contained galactose below quantifi- patients that performed the same amount, one more or less (32 %, median cation limits. Galactose content of cheddar cheeses aged>9 months ranged phe 890 μmol/l), c) patients that decreased the number of blood spots by 2 from<5 to 104 mg/100 g. Mild and medium cheddar cheeses averaged 5 mg/ or more (12 %, median phe 700 μmol/l), and d) patients that performed 0 100 g. Hard parmesan cheese aged>10 months contained galactose below blood spots before and 0 afterwards (12 %). quantification limits and grated parmesan cheese contained 7 mg/100 g. The text message reminder increased the amount of blood spot sent in. The Conclusion: Caseinates contain minimal amounts of galactose and can reminder service appeared to help patients who were more adherent with be allowed in a galactose-restricted diet. North American Gruyere, their diet therapy. Emmentaler and Parmesan cheeses contain negligible galactose and are also appropriate. In contrast to European studies, aged cheddar cheeses varied in galactose content suggesting moderate consumption needs to be recommended until further analyses are completed.

P-430 OBESITY A CONCERN IN GLYCOGEN STORAGE DISEASE P-432 (GSD) TYPES I AND III? ATTITUDE AND USE OF LOW LACTOSE/GALACTOSE CHEESE Skeath R1 , Stafford J1 , Grunewald S1 , Cleary M1 , Dixon M1 BY CAREGIVERS AND PATIENTS WITH GALACTOSAEMIA 1Great Ormond Street Hosp for Child, London, United Kingdom Ford S1 , Bevington S1 , Bevington M1 , Portnoi P1 , Ford K1 , Rapps H1 , Rapps G1 , Sagal S1 , MacDonald A2 Background: Children with GSDI and III are dependent on frequent 1Galactosaemia Support Group, Birmingham, United Kingdom feedings of carbohydrate to improve growth and help correct secondary 2Birmingham Childrens Hospital, Birmingham, United Kingdom biochemical abnormalities. Objectives: To review growth, dietary intake and prevalence of obesity in Treatment of galactosaemia requires a low lactose/galactose diet and in the UK, until our GSD cohort. 2001, all milk-derived cheeses (MDC) were excluded. Following analysis, some Methods: Review of longitudinal growth and dietary management of GSD cheeses (Emmental, Jarlsberg, Gruyere, Italian Parmesan (Parmigiano Reggiano patients: type Ia n011, 1b n02, III n06. Z scores for weight, height, BMI and Grana Padano) and mature Cheddar cheese produced in one UK area to a were calculated using LMSGrowth®. guaranteed standard) were found to contain minimal lactose/galactose amounts and Results: Age at presentation ranged from 3 days to 14mths GSDI and are now permitted. Usage and attitudes to milk-derived cheeses appear variable. 2mths to 3y GSDIII. Current age range between 2 to 15y GSDI and 2 to Aim: To examine parents and patients attitudes towards suitable MDC in 14y GSDIII. All treated with 2 to 3 hourly daytime feeds or uncooked galactosaemia. cornstarch plus overnight tube feeds (18/19). Height Z score from age 2y Methods: A questionnaire (9 questions) was promoted online (surveymon- was maintained or increased in 11/13 GSDI, (1/13 is 2y) and 4/6 GSDIII, key.com) through the UK Galactosaemia Support Group website. (2/6 are 2y). BMI: 8/13 GSD I are overweight, 5/13 obese and 2/6 GSD III Results: The questionnaire was completed by 131 (81 % caregivers; 19 % are overweight, 3/6 obese, 1/6 normal. patients). They represented 48 % of children (n063) aged <10 y and 23 % Conclusions: Overweight and obesity was prevalent from a young age (n031) 17y and over. 76 % (n099) ate MDC with 64 % (n084) taking this despite maintenance of height growth. Although uncooked cornstarch at least once weekly. The most preferred were West Country Farmhouse enabled less frequent feeding, BMI's did not improve. Possible explanations Cheddar (70 %), Emmental (31 %), Italian Parmesan (24 %), Gruyere include, excess carbohydrate intake, inadequate exercise and endocrine (18 %) and Jarlsberg (14 %). Reasons for non-consumption of MDC disturbances. Early obesity may increase the risk of 'metabolic syndrome' included dislike taste or smell (12 % n014); disbelief/uncertainty that some and type II diabetes. MDC may be suitable (4 %) and non-awareness (2 %). Conclusions: There was wide acceptance and use of suitable MDC in patients/caregivers with galactosaemia, with many requests to extend the lactose/galactose testing to other foods. S156 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

A-040 P-435 LONG TERM FOLLOW UP OF A PATIENT WITH GLUTARIC DETERMINATION OF PHOSPHOGLUCOMUTASE (PGM)- AND ACIDURIA (GA) TYPE 1 GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PDH) ACTIVITY ALTAY I.Suheyla1 , AKTUGLU-ZEYBEK A.C.2 , CANSEVER MS2 , FROM DRIED BLOOD SPOTS (DBS) BY A SIMPLE MODIFICATION KIYKIM E2 , AYDIN A2 OF THE CLASSICAL BEUTLER TEST 1Dep Dietetics, Cerr Med Fac, Ist Univ, Istanbul, Turkey Fingerhut R1 , Tegtmeyer L2 , Rust S3 , Schmugge M1 , Marquardt T2 2Dep Ped Nutr Metab, Cer Med Fac Ist Univ, Istanbul, Turkey 1University Children`s Hospital, Zurich, Switzerland 2University Children`s Hospital, Muenster, Germany Glutaric aciduria is an inborn metabolic disease with predominantly 3Leibniz-Inst. for Arteriosclerosis Res., Muenster, Germany neurological presentation due to deficiency of glutaryl coA dehydrogenase on the catabolic pathway of lysine, hydroxylysine and trypto- Background: The classical "Beutler-Test" is worldwide one of the most phan. The presence of glutaric acid with 3-hydroxyglutaric acid in often used test for newborn screening for classical galactosaemia. However, urine is diagnostic. The patients usually develop normal till initial this test suffers from low specificity. (i) The beutler-test is not a specific acute neurologic episode characterised by acute loss of motor func- assay for galactose-1-phosphate uridyltransferase (GALT), but an assay tions leaving severe dystonia-dyskinesia with mental retardation. cascade which measures consecutively 4 enzymes: GALT, PGM, G6PDH, Chronic treatment consists of riboflavin and carnitine with a low and 6-Phosphogluconate Dehydrogenase (6-PGDH). (ii) Non disease caus- protein diet with low tyriptophan and lysine. There is no concensus ing compound heterozygocity of classical galactosaemia/Duarte variants. on dietary management whether prtoein restriction is needed in Objectives: We describe two simple variations of the classical beutler-test, for patients after an acute attact the determination of PGM-, and G6PDH-activity from DBS respectively. A 14 years old female patient was diagnosed as glutaric aciduria type Methods: The beutler-test was modified by exchanging the substrates for the 1 at the age of 6 months with an acute, progressive neurological GALT (galactose-1-phosphate and UDP-glucose) with the substrate for PGM disorientation after an intercurrent infection. She was put on a low (glucose-1-phosphate), or the substrate for G6PDH (glucose-6-phosphate). Addi- protein diet, riboflavin (100 mg/kg/day) and carnitine (100 mg/kg/d). tionally, the incubation time for the G6PDH assay could be reduced to 15 minutes. A gastrostomy tube was placed because of severe swallowing diffu- Results: Reference ranges (median (range) in U/dL) for PGM and G6PDH were culties and dystonia at the age of 6 years. She has also been followed determined from 1316,and 1048 normal, not pre-selected samples from the by home ventilatory assistance due to respiratory insufficiency. from newborn screening respectively.: PGM 48.1 (17.5-96.2); G6PDH 291.7 (56.4- time to time for nearly 6 years 559.5). Intraassay CV was 4.5 and 3.7 U/dL; interassay CV was 12.2 and 8.1 U/ This case is demonstrative in emphasizing long term follow-up of a patient dL at 14.3 and 39.3 U/dL for PGM. ). Intraassay CV was 3.2 and 2.2 U/dL; with glutaric aciduria interassay CV was 12.1 and 9.2 U/dL at 16.8 and 40.3 U/dL for G6PDH.

34. Newborn Screening P-436 A NOVEL METHOD FOR THE DETERMINATION OFACYLGLYCINES P-434 IN DRIED BLOOD SPOTS (DBS) BY UPLC-TANDEM MASS A RAPID AND SENSITIVE UPLC-MS/MS-METHOD FOR THE SPECTROMETRY SEPARATION AND QUANTIFICATION OF BRANCHED-CHAIN Fisher LJ1 ,Al-DirbashiOY1 , McRoberts C1 , Milburn JL1 , Geraghty MT1 , AMINO ACIDS FROM DRIED BLOOD SAMPLES OF MAPLE Chakraborty P1 SYRUP URINE DISEASE (MSUD) PATIENTS 1Children's Hospital of Eastern Ontario, Ottawa, Canada Fingerhut R1 , Röschinger W2 , Heck M3 , Heck M1 1University Children`s Hospital, Zurich, Switzerland Several acylcarnitines (such as C3, C8 and C5OH acylcarnitines) used as 2Laboratory Becker, Olgemöller&Colleagues, Munich, Germany primary markers for newborn screening lack specificity and thus contribute 3Waters, Baden-Dätwill, Switzerland to a higher false positive rate. The analysis of acylglycines is known to be useful in the diagnosis of inborn errors of metabolism (IEM) including Background: Newborn screening for MSUD is a special challenge since medium chain acyl-CoA dehydrogenase (MCAD) deficiency, isovaleric patients with MSUD can metabolically decompensate rapidly without acidemia, and beta-ketothiolase deficiency, among others. We report a adequate treatment within the first two weeks of life. However, the screen- novel dried blood spot (DBS) acylglycine profile assay highlighting its ing method does not detect the actual marker metabolite (allo-isoleucine) potential application as a second tier screening test. specifically, but only as part of the group of the other isobaric amino acids Acylglycines are extracted from two 3.2 mm DBS punches using methanol: leucine, isoleucine and hydroxyproline. water containing stable isotope internal standards and butylated using Objectives: We describe a sensitive and rapid second-tier UPLC-MS/MS Butanol-HCl at 65 °C for 30 min. Ultra Performance Liquid Chromatog- method to determine branched-chain amino acids from the initial extraction raphy (UPLC) MS/MS with a run time of 10 min permits resolution and of the screening sample. quantitation of 13 acylglycines in DBS, including several that are isobaric. Methods: Quantification is based on a 7-point calibration curve. The Calibration curves are linear from 0.05 to 25.0 μM and reference intervals method can also be used for therapy monitoring in patients with MSUD. (n0150) have been established. DBS samples from patients with a con- Results: Reference ranges (mean±SD in μmol/L) were determined from 179 firmed IEM, as well as other causes for false positive screens (e.g. maternal normal, not pre-selected samples from the newborn screening: Leucine: 72±27; B12 deficiency, MCAD mutation carriers, etc.) have been analyzed. isoleucine: 37±19; valine: 98±46; hydroxyprolin: 23±13. The concentration of We anticipate this method will reduce the false positive rate for a number of allo-isoleucine was in about 55 % of the cases below the detection limit, the IEM targeted by newborn screening. highest concentration was 1.9 μmol/L. In all 30 retrospectively studied screening samples from patients with confirmed MSUD the concentration of allo- isoleucine was significantly increased. In 10 samples with false-positive newborn screening due to significantly increased concentration of leucine+isoleucine (400 to >4000 μmol/L) allo-isoleucine could not be detected. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S157

P-437 P-439 QUANTITATIVE AMINO ACID AND ACYLCARNITINE ANALYSIS A PILOT STUDY OF NEONATAL SCREENING BY ELECTROSPRAY IN PLASMA USING HILIC COUPLED TO SECOND GENERATION IONIZATION TANDEM MASS SPECTROMETRY AND EXACTIVE HIGH RESOLUTION MASS SPECTROMETER FLUOROIMMNOASSAY IN SAUDI ARABIA Bruce SJ1 , Rochat B2 , Roux C1 , Robinson S3 , Bromirski M4 , Henry H1 Al-Dehaimi AW1 , Asmari A1 1Clinical Chemistry Laboratory, CHUV, Lausanne, Switzerland 1Dep Path Clin Lab Med,Ki Fahad Med Ci, Riyadh, Saudi Arabia 2qMSF, University of Lausanne, Lausanne, Switzerland 3Thermo Fisher Scientific, Hemel Hempstead, United Kingdom A Saudi newborn screening program is important to the half million 4Thermo Fisher Scientific, Bremen, Germany Saudi children born each year in the Kingdom of Saudi Arabia.In this pilot study, we introduced electrospray tandem mass spectrometry Background: Our goal was to merge the analysis of amino acids (AAs) and (ESI/MS/MS) and a sensitive fluoroimmunoassay(FIA),to screen acylcarnitines (ACs) in a single run. In addition, we compared two tech- 5046 newborns in King Fahad Medical City to establish the normal nologies for AAs: a Biochrom 30 analyzer, and a high resolution Orbitrap amino acid , acylcarnitine, TSH, biotidinase, 17-hydroxyprogesterone spectrometer (HR-MS), Exactive (Thermo Fisher Scientific) for the deter- and galctose-phosphate uridyl trasferase (GALT) levels. Based on the mination of AAs and their metabolites. upper cut-off levels (98 percentile). 138 (66 ESI/MS/MS and 72 FIA) Methods: The LC method uses an acetonitrile/water gradient (HILIC). The samples studied were considered as questionable. 37 samples were acquisition involved full HR scans and extracted ion chromatograms of from preterm babies. After follow-up samples and urine GC/MS each analytes m/z with a 5 ppm mass tolerance. The Biochrom 30 was used analysis, only 10 were confirmed as true inborn errors. One was as a reference method for AA analysis. identified as propionic aciduria, two identified as argininosuccinic Results: We have evaluated the quantitative performance of the LC-HR- aciduria, three identified as congenital adrenal hyperplasia, one iden- MS method for both ACs and AAs. A linear curve with R2 values ranging tified as Congenital Hypothyroidism, and three biotidinase deficiency. from 0.98 to 1.00 were achieved for the metabolite standards including 25 The positive rate of true inborn metabolic error using ESI/MS/MS was AAs (with the exception of aspartic acid, taurine and pipecolic acid with R2 0.059 % and using FIA was 0.14 %.The false positive rate using ESI/ values between 0.95 and 0.98) and 12 ACs. Quantitative measurements of MS/MS was 1.2 % and using FIA was 1.3 %. We found that ESI/ the AAs obtained with the Biochrom 30 analyzer and with LC-HR-MS MS/MS and FIA neonatal screening were valuable in the early diag- gave acceptable correlations with the exception of aspartic acid. nosis of severe and treatable inborn errors of metabolism. The results Conclusion: The LC-HR-MS method described has great potential for indicated the importance of newborn screening using this technology quantitation of AAs, AA metabolites and ACs in a single run without the in Saudi Arabia. need of derivatization.

P-440 P-438 NEWBORN SCREENING FOR HOMOCYSTINURIA: MACHINE DETECTION OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE LEARNING VS CLINICIANS 1 2 1 3 4 5 DEFICIENCY USING FLUORESCENT SPOT TEST AND MOLECULAR Chen WH , Chen HP , Tseng YJ , Hsu KP , Hsieh SL , Chien YH , 5 1 METHOD FOR PREDICTION OF SEVERE NEONATAL Hwu WL , Lai F 1 HYPERBILIRUBINAEMIA IN NORTH INDIA Grad Ins of Bio Eng & Bio Info, NTU, Taipei, Taiwan 2 Polipalli SK1 ,PandeySK1 ,VyasV1 , Puppala M1 ,LomashA1 , Kumar S1 , Dept CSIE, NTU, Taipei, Taiwan 3 Kapoor S1 Comp & Info Net Center, NTU, Taipei, Taiwan 4 5 1Maulana Azad Medical College & Associate, New Delhi, India NCTU, Hsinchu, Taiwan , Dept Med Gen, NTU Hosp, Taipei, Taiwan

Background & Aim: Our study aimed to determine the occurrence of The National Taiwan University Hospital (NTUH) initiated the newborn G6PD deficiency in neonatal hyperbilirubinemia and comparing the detec- screening research in 1981. At present, NTUH newborn screening laboratory tion rates of glucose-6-phosphate dehydrogenase (G6PD) deficiency in provides medical service to approximately 1/3 of the nation's newborns for neonates by fluorescent spot test (FST) and using molecular methods. inborn errors of metabolism. The objective of the paper is to introduce an Materials & Methods: A total of 1600 subjects were screened for G6PD optimal support vector machine based algorithm in place of cut-off value deficiency, in that only 205 babies presented with neonatal jaundice were decision to evaluate the analyte elevation associated with Homocystinuria included in the study. FST was performed for G6PD deficiencyDNA extrac- (HCU). The experimental datasets of the neonatal screening MS/MS were tion was done using DBS filter paper as well as venous blood leucocytes for collected over the period of 2006-2011. Each set of data consists of 35 confirmed G6PD subjects. Molecular analysis was performed using PCR- analytes. Between the years of 2006-2010, the total 303 suspected cases RFLP procedure for three deficient molecular variants; G6PD Mediterranean including 37 positive ones are partitioned into 50-50 % training-test. The (563 C>T), G6PD Kerala-kalyan (494 G>A) and G6PD odissa. methodologies encompass a support vector machine classifier applying the Result: In our study out of 205 babies only 29 babies showed G6PD total combinations of the analytes ranked by Fisher Score. The optimal trained deficiency( 10.3 % were females while 89.7 % were males) by FST, and model and 8 significant features: ARG, GLY, MET, PHE, VAL, C0, C10, the G6PD mutation could be identified in 20(68.96 %) babies, in all these C14:1 are generated. The model and the features apply to: (1) 2006-2010 babies the mutation identified was G6PD Mediterranean(563 C>T). testing data classification; (2) 2011 predicting data classification. Conclusion: Our findings show low prevalence of G6PD deficiency in The results indicate that both specificity and predicting accuracy are improved north Indian region. G6PD Mediterranean mutation was the most common comparing with cut-off value approaches. The numbers of false positive cases and it has been reported to have significantly lower red cell enzyme activity. and suspected cases are reduced 1/3~1/2 approximately. Therefore, the approach can handle medical resources effectively and efficiently. S158 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-441 P-443 DETAIL ANALYSIS OF ACYLCARNITINES AND AMINO ACIDS SPECTRUM OF METABOLIC DISORDERS IN HIGH RISK FOR INBORN ERROR OF METABOLISM USING UPLC/MS/MS GROUP OF CHILDREN FROM WESTERN INDIA (GUJARAT) Maeda Y1 , Oda H1 , Nakajima Y2 , Kato S2 , Sugiyama N3 , Saito S2 , Sheth Jayesh1 , Mehul Mistry1 , Shah Raju2 , Kachhy Vishal3 , Sheth Kimura K1 , Ito T2 Frenny1 1Dept Hosp Pharmacy, Nagoya City Univ, Nagoya, Japan 1FRIGE's Institute of Human Genetics, Ahmedabad, India 2Dep Pediatr, Nagoya City Univ, Nagoya, Japan 2Ankur children Hospital, Ahmedabad, India 3Dep Pediatr, Aichi-Gakuin Univ, Nagoya, Japan 3Synergy Neonatal & Pediatric Hospital, Ahmedabad, India

Background: Although tandem mass spectrometry (MS/MS) analysis of acyl- Our study comprises of 283 high risk groups of neonates in the age range of carnitines and amino acids has been used to screen newborns, this method 3 months to 3.5 year presented with failure to thrive, encephalopathy, severe cannot separate acylcarnitine and amino acid isomers and the quantification acidosis, seizures, persistent vomiting & jaundice with hepatomegaly. method is semiquantitative analysis by comparing peak sensitivity of analyte 112 children were investigated by GCMS study from urine and 171 children with that of internal standard. We investigated the detail determination method of by tandem mass spectrophotometry from dried blood spot. This has iden- 28 acylcarnitines and 9 amino acids without derivatization by UPLC/MS/MS. tified 46 children (16.25 %) with different metabolic disorders. The most Method: Analytes were extracted from serum of patients using an OASIS common one was fatty acid oxidation defect detected in 32.5 % followed by MCX solid phase extraction cartridge (Waters). The extract was injected organic acid disorder in 30 %, tyrosinemia and urea cycle disorder 10 % onto Acquity UPLC BEH C18 column. Acylcarnitines and amino acids each. MSUD, multiple carboxylase deficiency (MCD) separately 4 % and were analyzed in positive ion MRM mode of Quattro Premier XE triple 2 % each with PKU, Hawkinsinuria and canavan disease. Of these, prenatal quadrupole mass spectrometer (Waters). diagnosis was offered to one family having tyrosinemia and identified novel Results: All analytes were analyzed in 35 min using UPLC. The precision mutations in FAH gene in exon 8 c.648 C>G (p.Ile216Met) and exon 13 data showed that intra-day coefficients of variance (CVs) at three concentra- c.1159>A (p.Gly387Arg) and the same information was supportive in tions were less than 15 %. The detail acylcarnitine and amino acid profiles of prenatal diagnosis in two subsequent pregancncies. the serum samples of patients with inborn error of metabolism were obtained. This study demonstrate that nearly 16 per cent of high risk group of Conclusions: The peak separation by using column avoided variation in the children have underlying cause of metabolic disorders and could have peak intensity by ion suppression. Detail diagnosis was enabled by these been given early treatment in presence of NBS program. Novel profiles. It has the potential to decrease the false positive rate by exact mutation detection in FAH gene has helped the family for precise determination of the near cut-off value. genetic counseling.

P-442 P-444 PILOT STUDY OF NEONATAL URINE SCREENING WITH GC/MS EVALUATION OF SUCCINYLACETONE ANALYSIS AS A FOR INHERITED METABOLIC DISORDERS IN JAPAN NEWBORN SCREENING TEST FOR TYROSINAEMIA TYPE 1 Inokuchi T1 , Aoki K1 , Tashiro K1 , Inaba M1 , Inoue K1 , Yanagiuchi C1 , Dowden S1 , Webster R1 , Johnston C1 , Stokes G1 , Preece MA1 Watanabe Y1 , Matsuishi T2 1NBS&Biochem Genetics B'ham Child Hosp, Birmingham, United 1Res Inst of GC/MS,Kurume Univ, Kurume, Japan Kingdom 2Pediatrics, Kurume Univ, Kurume, Japan Tyrosinaemia type I (TTI) is characterised by a deficiency of fumaryl We have been performing a pilot study for neonatal mass screening using acetoacetate hydrolase. Early treatment improves clinical outcome support- GC/MS methods since 1996. Screening of urine samples from 111,050 ing the introduction of newborn screening (NBS). Succinylacetone has been neonates to date has identified 98 cases of a total of 20 diseases, including identified as a suitable screening test. 14 cases of methylmalonic acidemia. Also identified were cases of neonatal We routinely measure tyrosine as part of the NBS test for phenylkentonuria ketosis, which has been considered rare, several transient pathologies (e.g., at 5-8 days. We previously reported a method for measurement of succiny- tyrosinemia, hyperlactacidemia, orotic aciduria/uraciluria), unanticipated lacetone in dried blood spots (DBS) using mass spectrometry (1). We exposures to chemical substances such as drugs perinatally used by the determined a cut off of 3.5 μmol/L to use as a second line screening test mother or administered to the neonate, and other new findings. The high following a raised tyrosine concentration. overall rate of disease identification of 1 in 1,100 validates the high 80,470 samples were received for NBS during the study period. 1799 of these sensitivity and diagnostic accuracy of our GC/MS method and confirms samples had tyrosine concentrations >200 μmol/L. 1797 samples had succiny- the efficacy of screening. This application for concurrently analyzing meta- lacetone concentrations <3.5 μmol/L (median 1.33 μmol/L range 0-3.38 μmol/ bolic products in urine, although considered a screening tool, is also useful L). 2 samples had succinylacetone concentrations of 3.7 and 4.9 μmol/L for chemically diagnosing diseases, detecting diseases not specifically (tyrosine results 249 and 282 μmol/L respectively). Repeat samples were screened for, and characterizing diseases with high accuracy. This applica- requested and gave a normal result; these babies were not followed up. tion has great potential to uncover more diseases and new findings and is 3 neonates diagnosed because of family history had succinylacetone con- proving to be a most effective and indispensable tool in screening for centrations of 32.4, 51.0 and 19.5 μmol/L inherited metabolic disorders in neonates. Further work to review the cut off is needed to minimise the number of false positive results. 1) Journal of Inherited Metabolic Disease, 31(Suppl. 1), 5. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S159

P-445 P-447 DRIED BLOOD SPOT ANALYSIS OF BRANCHED CHAIN AMINO SELECTIVE SCREENING BY TANDEM MASS SPECTROMETRY: ACIDS BY UHPLC A FOLLOW ON TEST FOR THE UK EXPANDED 5 YEARS EXPERIENCE IN BELARUS NEWBORN SCREENING PILOT Zinovik A. V.1 , Gusina A. A.1 , Miasnikov S. O.1 , Gusina N. B.1 Baggot M1 ,GhansahN1 , Krywawych S1 , Prunty H1 ,PatelH1 , 1National Centre "Mother and Child", Minsk, Belarus Bainbridge K1 , Ifederu A1 , Heales S1 1Chem Path, Great Ormond Street Hospital, London, United Kingdom Introduction: From September 2007 a selective screening by tandem mass spectrometry (MS/MS) has been initiated in Belarus. Our goal was to Background: A 12 month pilot expanded newborn screening programme determine approximate prevalence of metabolic disorders and to make has commenced in the UK. Amongst the conditions now screened is maple recommendations for the future development of neonatal screening syrup urine disease (MSUD). Tandem mass spectrometry will initially be program. utilized to assess the total leucines concentration in dried blood spots Methods: Over a period of five years (2007-2012) 4500 patients were (DBS). If elevated, this will trigger the need for a confirmatory test that evaluated. Blood spots were collected from children suspected of will include measurement of branched chain amino acids (BCAA's). inborn errors of metabolism (IMDs) and also from those died from Objectives: To establish rapid DBS analysis of BCAA's by UHPLC. sudden infant death syndrome within this period. Samples with abnor- Methods: BCAA's were extracted from DBS with a 70 % methanol mal results were repeated and the patients were recalled to confirm solution containing norleucine internal standard. The sample was deriva- with follow-up testing. tised with phenylisothiocyanate and excess reagent removed under vacuum. Results: Totally 16 patients were detected. The most common IMDs were Derivatised BCAA´s were separated by UHPLC (Agilent's 1290 infinity glutaric aciduria type 1 (4 cases) and long-chain 3-hydroxyacyl-CoA dehy- UHPLC system) using a Poroshell EC-C18 2.7 μm 2.1 x 150 mm column drogenase deficiency (3 cases). We also found methylmalonic acidaemia (2 and detected by UV absorbance (254 nm). cases), maple syrup urine disease (1 case), very long-chain acyl-CoA Results: Full chromatography of BCAA's was achieved within 10 minutes. dehydrogenase deficiency (1 case), homocystinuria (1 case), citrullinemia Within batch imprecision for allo isoleucine, leucine and valine ranged from (1 case), formiminoglutamic aciduria (1 case), Zellweger syndrome (2 0.3 to 2.7 %, between batch from 5.0 % to 9.5 % and recovery ranged from cases). 80 % to 106 %. The method was linear up to 5000 mmol/l for isoleucine, Conclusions: MS/MS is useful for diagnosis of selected IMDs. Early leucine and valine and 1250 mmol/l for alloisoleucine. detection of IMDs enables appropriate medical treatment, close monitoring Conclusion: UHPLC analysis of DBS provides a rapid confirmatory test of metabolic status of a child and genetic counseling in families. We for MSUD newborn screening. consider national-wide neonatal expanded screening program by MS/MS may be rational for Belarus.

P-446 P-448 COMPARISON OF TWO LC/MS/MS QUANTITATION METHODS: OUTCOME OF NEWBORNS PRESENTING CLINICALLY IDMS VS 6-POINT CALIBRATION BEFORE RESULTS OF NEWBORN SCREENING ARE Ifederu A1 , Bainbridge K1 , Heales S1 AVAILABLE 1Great Ormond Street Hospital, London, United Kingdom Tal G1 , Tzanakos N1 , Francis I1 , Pitt JJ1 , Boneh A1 1Metabolic Research, MCRI. RCH, Melbourne, Australia Background: In the UK newborn screening for phenylketonuria and medium chain acylCoA dehydrogenase deficiency is carried out using In Victoria, dried blood spots for Newborn Screening using Tandem tandem mass spectrometry (TMS). A 12 month pilot extended screening Mass Spectrometry (NBS-TMS) are collected at 48-72 hours of age programme has expanded the use of TMS to include screening for a further and results are usually available at 7 days (4.8-11.0; 2.5-97.5 percen- five metabolic disorders. Most screening laboratories utilise an isotope tiles). We wished to explore the effectiveness of NBS-TMS in the dilution TMS (IDMS) technique to calculate the concentration of each management and outcome of patients with fatty acid oxidation defects analyte which relies on using defined concentrations of internal standards. (FAOD) and disorders of protein metabolism who present clinically However, at Great Ormond Street Hospital a 6-point calibration curve per before the results are available. plate is used to accurately quantitate each analyte with internal standards We conducted a retrospective review of all clinical and laboratory data from used to correct for recovery. health records of all neonates with FAOD and disorders of protein metab- Objective: To directly compare the reliability, accuracy and limitations of olism who presented clinically prior to their newborn screening results, using IDMS and 6-point calibration to quantitate newborn screening analytes. between February-2002 and January-2012. Methods: IDMS and 6-point quantitation techniques are used simultane- Four patients with MSUD, one with MMA, one with Citrullinaemia Type I ously to calculate the concentration of analytes used in expanded newborn and 4 with MCADD, 1 with VLCADD, 1 with CPT II and 2 with CACT screening on a triple quadrupole tandem LC/MS/MS. The comparability of (deceased) presented clinically. In retrospect, the maximum time between the data is assessed with particular reference to the screening analytical and presentation and availability of results was 6 days. Specific treatment of clinical cut-offs. patients with disorders of protein metabolism was initiated within hours of Conclusion: The concentration of analytes used in newborn screening to notification. Patients with FAOD benefited from glucose infusion as per detect metabolic diseases can differ depending on the technique used to usual practice for sick neonates. quantitate the results. This could have implications for cut-offs and has Newborn screening results for FAOD and disorders of protein metabolism particular relevance to the pilot for extended screening. are valuable in the management of newborns presenting clinically during the first days of life. S160 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-449 A-041 NEWBORN SCREENING IMPORTANCE IN DETECTION OF EVALUATION OF A NOVEL, COMMERCIALLY AVAILABLE ASYMPTOMATIC FORMS OF EPIMERASE DEFICIENCY MASS SPECTROMETRY KIT FOR NEWBORN SCREENING GALACTOSEMIA (EDG) INCLUDING SUCCINYLACETONE WITHOUT HYDRAZINE Bal MO1 , Zanotti M1 , Zazzetta E1 , Bettocchi I1 , Baronio F1 , Rimondini Kasper DC1 ,MetzTF1 , Mechtler TP2 , Herkner KR2 ,MerkM3 , D1 , Balsamo A1 , Pession A1 , Cassio A1 Gottschalk A3 , Lukacin R3 1Neon Screen Centre, Dep Pediatrics, Bologna, Italy 1Dep Pediatrics&Adolescent Med, Med Univ, Vienna, Austria 2Res Core Pediatric Biochem&Analytics, Vienna, Austria Background: EDG is an autosomic recessive disease caused by reduced 3Chromsystems Instrument & Chemicals GmbH, Gräfelfing, Germany activity of UDP-galactose 4-epimerase (GALE) and is a continuum comprising three forms: generalized (precociously symptomatic), intermediate Newborn screening for hereditary tyrosinemia type I (HT 1) is mandatory to and peripheral (usually identified by biochemical testing). identify infants at risk before life-threatening symptoms occur. The analysis Because of the risk of short and long-term complications and the unclear of tyrosine alone is limited. Consequently, the analysis of succinylacetone long-term outcomes, dietary treatment is recommended. (SUAC) is needed. Current protocols are time-consuming, and include Case Report: An African male born at term from nonconsaguineous parents, hazardous reagents such hydrazine. We evaluated a novel, commercial kit with regular neonatal adaptation, only one day of phototherapy for jaundice. to analyze amino acids, acylcarnitines and SUAC with a significantly less Newborn screening showed galactose spot 20,88 mg/dL (nv <15 mg/dl). Traces harmful hydrazine derivative in a newborn screening laboratory. Dried of galactose were present in urine, GALT activity was 2,7 U/g Hb (nv>2,3), blood spot specimens from 4,683 newborns and samples from known RBC gal-1P was 10,95 mg/dl RBC (n.v. ≤ 5 mg/dl RBC). Clinical and patients with inborn errors of metabolism (IEM) were analyzed by a novel ophthalmologic evaluation and abdominal ultrasound were negative. No muta- protocol and compared to an in-house screening assay. All samples were tions were found in GALT gene. He started galactose free diet at 10 days of life. derivatized with butanol-HCl after extraction from 1/8-inch DBS punches. The analysis of GALE gene evidenced the homozygous known K275R For the novel protocol, the residual blood spots were extracted separately for mutation. SUAC, converted into hydrazone, combined with amino acids and acylcarni- Actually he's ten months old, he continues diet and shows normal growth. tines, and analyzed by mass spectrometry using internal isotope-labeled stand- Conclusions: Neonatal screening is important in order to prevent possible ards. All newborns were successfully tested, and 74 patients with IEMs long term complications in asymptomatic newborns with non classical including three with HT 1 were detected accurately. The mean SUAC level galactosemia. As information is sparse regarding the natural history of all in non-affected newborns was 0.68 μmol/l (cut-off 1.29 μmol/l). The novel EDG's, knowledge of forms and options for treatment is limited. assay was demonstrated to be accurate in the detection of newborns with IEM, robust, and without the risk of the exposure to toxic reagents.

P-450 DETECTION OF VITAMIN B12 DEFICIENCY USING EXPANDED 35. Adults and IEM NEWBORN SCREENING IN EASTERN ANDALUCIA, SPAIN Yahyaoui R1 , Dayaldasani A1 , Rueda I1 , Blasco J2 , Serrano J2 , Navas P-451 2 2 3 3 1 VM , Sierra C , Merinero B , Pérez-Cerdá C , Pérez-Valero V DISTURBED BONE METABOLISM IN ADULT GLYCOGEN 1 Clin Lab, Carlos Haya Hospital, Málaga, Spain STORAGE DISORDER (GSD) TYPE 1A A NEGLECTED 2 Pediatrics, Carlos Haya Hospital, Málaga, Spain COMPLICATION? 3 CEDEM, Univ Autonoma, Madrid, Spain Hochuli M1 , Spinas GA1 , Baumgartner MR2 1Div Endo Diab Clin Nutr, Univ Hosp, Zürich, Switzerland Introduction: Although vitamin B12 deficiencies in newborns are not a 2Div Metab Dis, Univ Child Hosp, Zürich, Switzerland primary goal of expanded newborn screening programs, markers for methylmalonic and propionic acidemias (C3 and C3/C2) may identify them. Background: Bone disease in adults with GSD1 is incompletely charac- Methods: Amino acid and acylcarnitine levels were determined from single terized and clear strategies for diagnosis and treatment are not available. dried blood-spot samples from 77,733 newborns, between April 2010 and Patients: Three adult GSD1 cases are presented. Patient 1 (22y, m) has poor December 2011, using MS/MS.A new sample was requested if there was an metabolic control and persistent low testosterone levels after delayed pub- increase in C3 and/or C3/C2.All cases with persistently high levels were erty (5.6 nmol/l, n8.6-29); patient 2 (35y, f) achieves only intermediate studied further, both mother and child, evaluating acylcarnitines, homocys- metabolic control despite a strict diet and has normal sex hormone status teine and vitamin B12 levels in plasma, and organic acids in urine samples. (i.e. regular menses); patient 3 (29y, m) has very stable metabolic control Results: Increased C3 and/or C3/C2 levels were found in 310 newborns; and testosterone levels in the lower third of normal. these markers were persistently elevated in second samples in 24 cases. Results: All patients have osteopenia or osteoporosis, patient 1 is most Biochemical studies showed: 2 methylmalonic acidurias, 1 propionic acid- severely affected (lowest Z-score -3.7, lumbar spine). Intermittent hypercalce- emia, 16 vitamin B12 deficiencies, and 5 false positive cases. mia with PTH-independent calcium release from bone occurred in patients 1 The ratio C3/C2 (14/16) was a more sensitive marker than C3 (7/16)for the and 2. Urinary markers of bone resorption (deoxypyridinoline) were elevated detection of vitamin B12 deficiency. Most newborns were exclusively in patients 1 and 3 and normal in patient 2. Deoxypyridinoline levels normal- breastfed at diagnosis (12/16).One of the mothers had previously undergone ized after initiation of testosterone substitution in patient 1. a partial gastrectomy. Four cases of probable maternal pernicious anemia Discussion: Bad metabolic control and/or severe disease may be risk were detected. factors for disturbed bone metabolism in GSD1. Patients with persistent Conclusions: Identification of vitamin B12 deficiencies in newborns (with low testosterone levels after delayed puberty may profit from hormone a high frequency in our population of 1:4,608), is an added benefit of substitution. Further studies are needed to study pathophysiology, treatment expanded newborn screening programs. and outcome of bone disease in GSD1. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S161

P-452 P-453 LATE-ONSET HOLOCRABOXYLASE SYNTHETASE DEFICIENCY: PORPHYRINS PROFILE BY HIGH PERFORMANCE LIQUID CLINICAL PRESENTATION IN SEVEN FAROESE ADULTS CHROMATOGRAPHY/ELECTROSPRAY IONIZATION TANDEM Steuerwald U1 , Sander S2 , Olsen K3 , Steuerwald U2 MASS SPECTROMETRY FOR THE DIAGNOSIS OF PORPHYRIA 1Dep. Occup. Med. and Public Health, Tórshavn (Faroe Islands), Denmark Gouda A1 , Fateen E1 , Nazim W1 2Screening-Laboratory, Hannover, Germany 1national research center, Giza, Egypt 3National Hospital, Tórshavn (Faroe Islands), Denmark Background: Porphyrias are a group of inherited or acquired disorders of Introduction: Mutations in the holocarboxylase synthetase gene (OMIM certain enzymes in the heme bio-synthetic pathway. Most symptoms are 609018) can result in holocarboxylase synthetase (HLCS)-deficiency. Var- nonspecific and occur intermittently; resulting in missed diagnosis since the iants with higher residual activity might lead to milder course and often go disease itself is a rare one. undiagnosed for many years. Objectives: Establishing a new accurate laboratory method for quantitation Methods: In seven adults from the Faroe Islands, analysis of dried blood of urinary porphyrins by liquid chromatography tandem mass spectrometry spots revealed elevated concentrations of 3-hydroxy-isovaleryl-carnitine. (LC/MS/MS) and the diagnosis of different types of porphyria for the first All were found to be homozygous for IVS10+5 G>A or c.1519+5 G>A, time in Egypt. the predominant mutation in Europe. Patients and methods: Fifty clinically suspected cases were subjected to Results: Typical symptoms at first consultation and examination included screening by plasma fluorescence. The diagnosis is confirmed by quantita- dermatological complains (perioral eczema, "fish-skin", hyperkeratosis of tive determination of porphyrins by (ESI-MS/MS) that was modified further feet, recurrent mycosis), frequent / chronic infections esp. of respiratory by the application of another newly established technique known as Ion tract; periods of ravenousness resulting in obesity; reduced endurance / Mapping without using HPLC- separation and only using the mass detector stamina and behavioural troubles (social phobia, autistic traits and others). for quantitative determination of urinary porphyrins. Discussion: Optimal diet containing several fish meals per week (fish is Result: One case (2 % from total 50) and (12.5 % out of eight patients rich in biotin) and sparse use of antibiotics might have enabled individuals presented with neurological and skin manifestations) was diagnosed with with late-onset HLCS-deficiency in this inbred population to survive into variegate porphyria, while five cases (10 % from total 50) and (31.2 % from adulthood, thus resulting in a very high carrier rate and incidence of the sixteen patients presented with chronic liver disease) diagnosed with por- disorder (1:1.600). Symptoms vanished or at least improved after treatment phyria cutanea tarda. with biotin. Conclusion: Quantitatitation of porphyrins using HPLC/ESI/MS/MS and ion Conclusion: Coincidence of otherwise unexplained dermatological trou- mapping techniques are applicable for the differential diagnosis of porphyria bles; recurrent infections and mental / behavioural abnormalities might be types, since each type has a characterestic porphyrins profile. caused by untreated late-onset holocarboxylase-synthetase deficiency.

P-454 IMPROVED MUSCLE STRENGTH AND ENDURANCE IN MCARDLE'S DISEASE USING CARNITINE, POTASSIUM AND LOW-PROTEIN DIET TO PROMOTE INCREASED USE OF ALTERNATIVE ENERGY PATHWAYS Perszyk A1 1Med Genetics, Univ of Florida, Jacksonville, United States

Limited access to stored muscle glycogen is the basis of McArdle's Disease. Vitamin and nutrient support to the intracellular machinery will allow more efficient switch to fatty acid oxidation intermediates as the main energy source. We have designed a patient education, exercise, vitamin and diet program to promote use of alternative energy pathways for this condition. Illustrated in this report, is an adult patient with previously confirmed McArdle's disease with frequent episodes of painful muscle cramping, uncontrolled muscle twitching, tachycardia episodes, hand weakness, rhabdomyolysis and myoglobinuria with reliance on benzodiazepines. Patients are given a low protein diet with increased water-soluble B vitamin complexes and potassium. Carnitine and other nutrients were utilized before and after exercise. In one month, this patient built muscle bulk, increased work capacity, improved muscle strength, while lowering resting heart rate. Patient could move immediately into sustained aerobic exercise for 60 minute work-outs. Complete cessation of tachycardia/arrhythmia events. Hand weakness resolved. Rhabdomyolysis controlled. Patient also experienced increased self-confidence, attention span and improved memory. Involvement of patient in food selection and exercise goals are key to the success of this intervention. S162 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-455 P-457 BARRIERS TO TRANSPLANTATION IN ADULTS WITH INBORN CACT DEFICENCY: LONG -TERM FOLLOW -UP FROM INFANCY ERRORS OF METABOLISM TO ADULTHOOD Sirrs SM1 , Faghfoury H2 , Hiwot T3 Gasperini S1 , Furlan F1 , Santus F1 , Rigoldi M1 , Corbetta C2 , Parini R1 1University of British Columbia, Vancouver, Canada 1Metab Dis Unit, MBBM Found, S Gerardo H, Monza, Italy 2University Health Network, Toronto, Canada 2Reg Lab Neonatal Screening ICP Hosp, Milano, Italy 3University Hospitals Birmingham, Birmingham, United Kingdom Carnitine-Acylcarnitine Translocase (CACT) deficiency is a rare Background: Transplantation in patients with inborn errors of metabolism inborn error of the carnitine cycle. Patients presenting in the neonatal (IEM) is used as rescue therapy for acute decompensation, organ replace- period show rapidly progressive deterioration and high mortality rate. ment, or disease modifying therapy. We sought to quantify the use of In 1999 (J Inher Metab Dis 22:733-739) we described a male patient transplantation in adults with IEM. with neonatal onset and favourable outcome thanks to a strict dietary Methods: 10 question on-line survey sent out through Metab-L and posted management. We showed that only 50 % of introduced Medium Chain on the SIMD website. Tryglycerides was metabolized to ketone bodies: this prompted us to Results: 13 centers from 5 continents responded. These centers, ranging in reduce fat intake and increase carbohydrates and proteins. The patient, size from. <50 adults (3) to >500 (2), reported 57 adult patients undergoing now 18 years old, has normal growth, mild generalized hypotonia, transplantation. 29/57 (51 %) came from the two largest centers and 27/57 intentional tremors, Gower's sign, lumbar lordosis, is cognitively and (47 %) were renal transplants for Fabry disease (FD). 7 transplants were socially adequate, no cardiomyopathy or arrythmias. He also has done for indications which may have been intended to modify disease bilateral neurosensory auditory deficit. CK plasma range: 800 to course. 8/13 centers had not had patients with IEM bumped from the 8000 UI/L. transplant list but 4 of these 8 had not referred a patient for transplantation. Diet: every 3 hours meals+cornstarch during the day. Nocturnal con- 4/13 centers had patients bumped from the transplant list citing: a. trans- tinuous nasogastric feeding (CNGF) was substituted few months ago, plant team not familiar with IEM b. cognitive impairment raised concerns by every 4 hours meals plus cornstarch during the night. Diet has about compliance c. multi-system involvement d. not at enough risk of life- always been monitored and modified to maintain low plasma free threatening decompensation. fatty acids and avoid acyilcarnitines toxicity. Carnitine therapy (now Conclusions: Excluding renal transplantation for FD, there is low use of 40 mg/Kg/die) was always maintained and assessed on the carnitine transplantation in adults with IEM. Some barriers to transplantation plasma values. We believe that the favourable outcome of this patient reported by adult centers could be improved with education of transplant emphasizes the outmost importance of strict diet and good family programs. compliance.

P-456 P-458 INVESTIGATING ADULT PATIENTS IN A MOVEMENT ADULT REFSUM DISEASE PRESENTING WITH DEMENTIA DISORDER CLINICS: WHEN TO LOOK FOR NIEMANN-PICK AND LEUKODYSTROPHY IN A 72-YEAR-OLD WOMAN TYPE C DISEASE? bompaire F1 , marcaud V2 , Le Trionnaire E3 , Levade T3 , Sedel F1 Lourenco CM1 , Coelho JC2 , Rodrigues GG1 , Tumas V1 , Souza FTS2 , 1Pitié-salpêtrière Hospital, Paris, France Giugliani R1 , Marques Jr W1 2CHR de gonesse, Gonesse, France 1University of Sao Paulo, Ribeirao Preto, Brazil 3CHU de Toulouse, Toulouse, France 2Federal University of Rio Grande do Sul, Porto Alegre, Brazil Background: Adult Refsum disease (ARD) is an autosomal recessive Background: Niemann-Pick type C disease (NP-C) is an inborn error of peroxisomal disorder caused by mutations in the PAHX gene and charac- metabolism caused by defective intracellular transport of cholesterol. terized by the accumulation of phytanic acid in blood and tissues. Clinical Although, early childhood and late infantile presentations are the classical features usually include retinitis pigmentosa, anosmia, sensorineural hear- presentation of the disease, more adult patients with NP-C have been ing loss, chronic sensorimotor neuropathy, cerebellar ataxia, ichthyosis, described, expanding the neurologic spectrum of the disease. hands and feet deformities and cardiomyopathy. Diagnosis confirmation Methods: Retrospective study and review of neuroimaging, neurophysio- relies on the demonstration of isolated elevation of plasma phytanic acid logical, biochemical and molecular studies carried out in the course of 30 and genetic tests. adult patients with NPC suspicion. Case report: We report the very unusual case of a 72 year-old Results: 14 patients were referred because of generalized dystonia with pro- woman who presented with progressive dementia and ataxic gait gressive cognitive decline/dementia; 11 had cerebellar ataxia with vertical oph- lasting for one year. Brain MRI disclosed diffuse leukoencephalop- talmoparesis; 3 patients had dystonia without an apparent cognitive decline, but athy but nerve conduction studies and ophthalmological examination with eye ophtalmoparesis; 2 patients had a complex neurological picture with were normal. In addition, the patient presented none of the classical cerebellar syndrome, generalized dystonia, ophtalmoplegia, cognitive decline features of ARD. Biological tests disclosed elevated plasma phytanic and pyramidal syndrome. Three out of 30 patients were confirmed to have NPC acid (704 μmol/l, N<12), with normal values of pristanic acid, by filipin staining ( 1 patient required molecular analysis). Regarding clinical VLCFA and Plasmalogens. PAHX gene sequencing revealed a new presentation, all positive NPC cases had VSGP with typically downward gaze homozygote mutation. palsy. None of the patients had previous history of prolonged jaundice. Conclusion: This case expands the phenotype of ARD. To our Conclusions: Neurological manifestations in NPC patients are extremely knowledge, this is the first case of a patient presenting with a pure variable. Better understanging of the natural history of the disease is crucial leukodystrophy without any peripheral nervous system and eye for evaluation of potential therapeutic approaches in such devastating disorder. involvement. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S163

P-459 A-042 ATYPICAL PRESENTATION OF A NOVEL SERINE DEFICIENCY ACUTE INTERMITTENT PORPHYRIA: STILL UNCALLED BY SYNDROME IN AN ADULT PHYSICIANS Bompaire F1 , Wiame E2 , Bolgert F1 , Von Schaftingen E2 , Sedel F1 Hişmi B1 , Tanrıöver MD2 , Ünal Ö1 , Şener E2 , Temuçin Ç3 , Sivri HS1 , 1Pitié-salpêtrière Hospital, Paris, France Dursun A1 , Coskun T1 2De Duve Institute, Brussels, Belgium 1Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey 2Hacettepe Univ, Intern Med, Ankara, Turkey Background: Serine synthesis defects caused by deficiencies in 3- 3Hacettepe Univ, Depart Neurol, Ankara, Turkey phosphoglycerate dehydrogenase (3-PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP) have been reported in Here we present a 23-year-old young woman who presented with severe children with severe encephalopathy and microcephaly. In addition, milder abdominal and extremity pain, hyponatremic convulsions, progrssive forms presenting with mild developmental delay/seizures or with prominent muscle weekness,in whome extended laboratory tests, imaging studies polyneuropathy have been recently published. and procedures performed by multiple physicians at multiple visits in a Case report: A 61 year-old man presented at the age of 49 with relapsing university hospital setting fail to give any diagnosis. But a "get-well visit" episodes of lethargy, speech difficulties and ataxic gait. The episodes lasted performed by a relative recalls a similar case in the family, then the patient for several hours and occurred at a mean frequency of 1 crisis per week. diognosis herself as "Acute İntermittent Porphyria". Diognosis was con- Episodes were always observed in the evening, often after prolonged firmed by urine porphyrine analysis, Human hemin was administered for exercise. Plasma amino acids chromatography disclosed very low serine four consecutive days and hyponatremia is aggresively treated in this values (33 to 48 μmol/l, N0139+/-38) as well as low glycine values (113 to patient. Severe demyelinating sensoryneuropathy being the etiology of her 133 μmol/l, N0237+/-56). The patient was treated with oral serine (6 gr per pain, resolved within 6 months. day) which resulted in normalization of serine and glycine concentrations. After treatment onset, the patient noticed a marked improvement of the 36. Miscellaneous 2 lethargic crises with only one episode observed in one year. No mutation was found in the 3PGDH, PSAT and PSP genes. P-461 Conclusion: Our observation suggests the existence of a novel serine syn- THE DIAGNOSTIC CHALLENGE OF HYPOMYELINATING thesis defect not linked to 3PGDH, PSAT or PSP deficiencies. DISORDERS Wassmer E1 , MacPherson L1 , Foster K1 , Santra S1 , Davison JE1 1Birmingham Children's Hospital, Birmingham, United Kingdom P-460 FEVER OF UNKNOWN ORIGIN IN A YOUNG ADULT WITH Objective: Hypomyelinating disorders present a diagnostic challenge. This END-STAGE RENAL DISEASE, PREMATURE CORONARY study describes population incidence and presenting features of hypomye- ARTERY DISEASE AND POLYNEUROPATHY lination disorders in a tertiary Children's Hospital Hişmi B1 ,Yaşar Y2 , Ünal Ö1 ,KılıçL3 , Türkmen E4 , Erdem Y4 , Dursun Method: A retrospective clinical and neuroimaging review of hypomyelination A1 , Sivri HS1 , Coskun T1 disorders among children under 16 years presenting in a 7 year period was 1Hacettepe Univ Child Hosp, Div Metab Dis, Ankara, Turkey conducted. Neurodegenerative disorders were excluded (hypomyelination with early 2Hacettepe Univ, Intern Med, Ankara, Turkey severe cerebral atrophy or basal ganglia / thalamic abnormalities, e.g. infantile 3Hacettepe Univ, Intern Med, Div Rheum, Ankara, Turkey neuronal ceroid lipofuscinosis). Incidence based on county birth rates was estimated. 4Hacettepe Univ, Intern Med, Div Nephrol, Ankara, Turkey Results: 40 children with neuroimaging-confirmed hypomyelination were identified: Pelizaeus-Merzbacher disease (n03, PLP/ GJA12); trichothiodystrophy A 35-year-old man with end-stage nephrolithiatic renal disease admitted to (n01); Cockayne syndrome (n04); hypomyelination with congenital cataract hospital with progressive muscle weakness. He was on hemodialysis for (n02); hypomyelination with hypodontia (4 H syndrome, n03); 18q−syndrome 1.5 years ,waiting for renal transplant and had gone through bypass surgery (n02); Aicardi-Goutiere syndrome (without calcification, n02); fucosidosis (n0 for premature coronary artery disease. ENMG showed chronic iflammatory 1); and SOX10 mutation (n01). No final diagnosis was reached in 22 children. demyelinating polyneuropathy and IVIG was administered. Mild hepatosple- Presenting features included: hypotonia, ataxia, tremor and nystagmus with nomegaly and dilated cardimyopathy. His fever started just after IVIG therapy mild to moderative cognitive impairment (60 %), progressive spastic para- and has become prolonged for 4 months. All diagnostic work-up for FUO paresis (20 %), early onset severe global developmental delay (20 %). 5 revealed nothing, so bilateral nephrectomy was performed for possible foci of children died during follow up (Cockayne (1); Aicardi-Goutiere (1); hypo- infection. Renal biopsy was compatible with chronic pyelonephritis and fever myeliantion with congenital cataracts (1) and unknown (2)). Population was controlled only for a short time. Autoantibodies, FMF gene analysis, incidence was estimated as 1 in 12000 live births. muscle biopsy and CT angiographies showed no sign of vasculitis. Bone Conclusion: The cause of hypomyelination in many children remains marrow biopsy performed for FUO showed birefringent oxalate cyrstals on unknown. A combined clinical and imaging algorithm could aid diagnosis. polarized light microscopy and when previous renal and muscle biopsies were reevaluated they were also full of oxalate cyrstals which made the diagnosis as Primary hyperoxaluria type I (PH1). PH1 is the most common and severe form of primary hyperoxalurias caused by deficiency/mistargeting of alanine:glyoxylate aminotransferase (AGT) in liver peroxisomes. This is the first case of PH1 complicating with FUO. Fever resolved spontanously, he is still waiting for combined renal and liver transplantation. S164 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-462 P-464 INHERITED LEUKODYSTROPHIES:AS SEEN IN CLINIC USING THE NMR SPECTROSCOPY IN DIAGNOSIS OF INBORN Wassmer E1 , Simmons L1 , Hutchin T1 , Santra S1 ERRORS OF METABOLISM 1Birmingham Children's Hospital, Birmingham, United Kingdom Usurelu N1 , Nicolescu A2 , Garaeva S3 , Redcozubova G3 , Postolache G3 , Szönyi L4 , Deleanu C5 Objective: Leukodystrophies, inherited white matter disorders, remain a 1National Cent Repr Health&Med Genetics, Chisinau, Moldova, Republic of diagnostic challenge. Data concerning incidence, clinical characteristics and 2"P.Poni" Inst Macromol Chem Rouman Acad, Iasi, Romania outcomes are sparse. The purpose of our study was to describe the inherited 3Inst Phys Sanocreatol Acad Scie Moldova, Chisinau, Moldova, Republic of leukodystrophies as seen in a clinic population: incidence, clinical features 41st Dept Pediatr Semmelweis Univ, Budapest, Hungary and outcome. 5"C.D.Nenitescu" Inst Org Chem Roum Acad, Bucharest, Romania Method: We retrospectively reviewed the medical records of children (<16 years) presenting with an inherited leukodystrophies to a tertiary Methods: We report on several children (n015, age<1 year) suspected for IEM children's hospital from April 2005 through March 31, 2012.The incidence according to clinical manifestations, that were investigated through liquid chro- was estimated using county birth rates (~70000/year in West Midlands). matography for amino acids in blood and urine, NMR spectroscopy of urine, Results: A total of 114 children with an inherited leukodystrophy were biochemical analyses. All patients were additionally tested by tandem MS. identified; Population incidence was 1 in 5000 live births. Hypomyelination Results: through NMR spectroscopy there were identified highly elevated con- was seen in 40 and dysmyelination in 74. The most common diagnoses centrations in urine for: methylmalonic acid [1631.4 mmol/molCrn](n01); glutaric were metachromatic leukodystrophy (11), Krabbe (6), Mitochondrial (5, acid [4400 mmol/molCrn](n01); lactate(n04); 2-oxo-glutaric acid and lactate confirmed molecular diagnosis), Vanishing White Matter (4) and Pelizaeus- (n02); glucose, 2-oxo-glutaric acid, lactate(n01); 1,2 propandiol(n01). The ele- Merzbacher disease (3). No final diagnosis was reported in 51 patients. A vated blood Homocystheine[17.8 μmol/l] was appreciated by amino acids analyses mitochondrial diagnosis was suspected in 17 of 29 of the undiagnosed at the patient with methylmalonic acid in urine. The tandem MS has been dysmyelination cases based on biochemistry. Mortality was 32 % during confirmed the Methylmalonic Aciduria and Glutaric Aciduria type1 for respec- the 7 year follow up. tively two suspected by NMR spectroscopy patients, other patients had negative Conclusions: Inherited leukodystrophies remain a diagnostic challenge. results. One patient with high 2-oxo-glutaric acid and lactate in urine was suspected These disorders are associated with significant morbidity and mortality. for GSD type1a according to clinic data and has been confirmed through molecular Overall population incidence is higher than expected in this population. investigations. Other patients (n07) having high lactate and 1,2 propandiol in urine were suspected for energy metabolism disturbances according to their clinic manifestations and biochemical findings (high Ala and/or postprandial lactate, Ala/Lys>3). Undiagnosed patients should be additionally examined. Conclusion: the NMR spectroscopy of urine provides high analytic infor- P-463 mation improving the orientation in diagnosis of IEM in unclear patients. PLASMA AMINO ACID FINGERPRINTING OF METABOLIC DISORDERS Ottolenghi C1 , Chadefaux-Vekemans B1 , de Lonlay P2 , Barouki R1 , Rabier D2 1Descartes Univ / Inserm U747, Paris, France 2Ref Ctr Inh Met Dis, Necker Hosp, AP/HP, Paris, France

Background: It is well known that in addition to disorders involving amino acid metabolism, apparently unrelated conditions can show specific plasma amino acid patterns. How many metabolic disorders show subtle yet specific amino acid profiles that could be of clinical relevance? We looked for potential plasma amino acid fingerprints of 30 candidate metabolic disorders from a large hospital population. Methods: Plasma amino acid levels from 8380 patients with no known aminoacidopathies were age-adjusted and transformed into population- normalized scores via binary combinations of linear contrasts. We tested which profiles were most strongly associated with a range of disorders including glycogen storage disorders and mitochondrial beta oxidation defects. Results: We found specific amino acid profiles for 14 of 30 disorders tested with 1-30 % diagnostic positive predictive value. In 6/14 disorders, no therapy could be held responsible for the variation. Variation of many informative amino acids lied within reference intervals, possibly explaining why the profiles had not been uncovered previously. Conclusions: We propose that plasma amino acid profiling may help to study the mechanisms of pathology of a wide range of metabolic disorders, while also possibly providing novel multivariate biomarkers for prediag- nostic screening or follow-up. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S165

P-465 P-467 SYSTEMATIC EVALUATION OF PATIENTS WITH GLOBAL DIAGNOSTIC VALUE OF PROTON MR SPECTROSCOPY IN DEVELOPMENTAL DELAY / INTELLECTUAL DISABILITY CHILDHOOD INHERITED NEUROMETABOLIC DISEASES: FOR TREATABLE INBORN ERRORS OF METABOLISM: FIRST LITERATURE REVIEW CLINICAL STUDY RESULTS Schulze AS1 , Al-Hertani WAH1 , Shin JS1 van Karnebeek CD1 , Armstrong LL2 ,ConnollyM3 , Demos M3 , Giannasi 1Sick Kids Hospital - Univ of Toronto, Toronto, Canada W1 , Horvath G1 ,HoubenR1 ,LafekM1 ,LehmanA2 , Lillquist Y1 , Murphy T3 , Patel M2 , Salvarinova R1 , Sinclair G4 , Vallance H1 , Stockler S1 Background: The proton magnetic resonance spectroscopy (MRS) is a tool 1Div Biochem Diseases,BC Children's Hosp, VANCOUVER, Canada for noninvasive monitoring of brain biochemistry. The metabolic assess- 2Dept Med Genet, BC Children's Hospital, vancouver, Canada ment of metabolic brain disorders offers valued insight for the diagnosis, 3Div Ped Neurol, BC Children's Hospital, Vancouver, Canada staging, and response to treatment. 4Dept Metab Dis, Hosp Sick Kids, Toronto, Canada The purpose of this study is to review the published literature for MRS findings in diagnosed inborn errors of metabolism, and create a metabolite Background: Current recommendations to investigate genetic causes of pattern-based algorithm. intellectual disability (ID) are based on frequencies of single conditions and Method: By performing a literature review via Pubmed, 60+ studies that yield of tests rather than availability of causal therapy. Early recognition of used MRS to study diagnosed neurometabolic conditions was analyzed. treatable inborn errors of metabolism (IEM) is crucial for minimizing brain The metabolite patterns per disease was transferred to a excel spreadsheet damage and reducing of health-care costs. and organized based on class of inborn error of metabolism. Methods&Results: Our literature review identified 81 IEM, presenting with ID Results: Increased NAA strongly indicates Canavan disease but can also be and amenable to causal therapy. Although evidence is limited, therapies are often found in Pelizaeus Merzbacher disease. Lactate is a useful discriminator, effective, safe, accessible. We translated this knowledge into a diagnostic proto- and was present in not only mitochondrial disorders, but also in some col: The 1st tier comprises metabolic screening tests in blood (plasma amino- organic acidurias and vitamin related disorders. Other discriminators acids, homocysteine) and urine (creatine metabolites, glycosaminoglycans, included spectra in the lipid-lactate complex, carbohydrate, and pathogno- oligosaccharides, organic acids, purines/pyrimidines), with potential to identify monic spectras involving glycine, GABA, and Creatine. 62 % of treatable IDs. The second tier focuses on remaining disorders, requiring Conclusion: Magnetic resonance spectroscopy can be a valuable tool for 'single test per disease' approach. A freely available App (www.treatable-id.org) the diagnosis of neurometabolic conditions, and an algorithmic approach supports the protocol. During first 6 months 130 patients were enrolled in a based on metabolite pattern can be of high diagnostic yield. It is important funded study implementing the protocol; confirmed diagnosis in 25 % (i6 to consider the metabolic state and MRS technique when interpreting treatable IEM), pending confirmation in 17 % (5 treatable IEM). results. Conclusions: Our preliminary results indicate that treatable IEM constitute etiology in 3-7 % of ID patients in tertiary care centres. Our novel approach may well improve outcomes and convince colleagues/policymakers to P-468 change practice and care for individuals with ID. ASSOCIATION OF THE SEROTONIN TRANSPORTER PROMOTER GENE POLYMORPHISM (5-HTTLPR/RS25531) P-466 WITH RESPIRATORY SINUS ARRHYTHMIA: THE TRIALLELIC APPROACH MAKES A DIFFERENCE IDENTIFICATION OF NOVEL METABOLITE PATTERNS ON IN Vulturar R.1 , Chis A.1 , Ungureanu L.2 , Miu A.3 VIVO 1 H-MAGNETIC RESONANCE SPECTROSCOPY (MRS) IN 1Dept Cell Mol Biol, Univ. Med and Pharm, Cluj-Napoca, Romania CHILDREN WITH METABOLIC DISEASES 2Dept of Derm, Univ Med Pharm, Cluj-Napoca, Romania Al-Hertani W1 , Mason E1 , Tam T1 , Imran M1 , Schmitt B2 , Blaser S3 , 3Dept of Psychology, Univ Babes-Bolyai, Cluj-Napoca, Romania Branson H3 , Schulze A1 1Div Metab Gen, Hosp for Sick Kids, Toronto, Canada The disturbances in serotonin metabolism have been associated with 2Dept of Radiology, Med Univers Vienna, Vienna, Austria autism, depression, myoclonus related conditions, etc, and a functional 3Div Diagnost Imaging, Hosp for Sick Kids, Toronto, Canada serotonin transporter (5-HTTLPR) polymorphism has been associated with anxiety, migraine, irritable bowel syndrome. 5-HTTLPR is extremely rele- Introduction: The use of MRS for identifying metabolite patterns as a vant for anxiety disorders, mainly because this polymorphism may influ- diagnostic measure of metabolic diseases is a rapidly evolving field, and one ence the brain availability of 5-HTT, which is targeted by the most effective that will be gaining significant attention in the coming years in the medical anxiolytic drugs (SSRIs). Respiratory sinus arrhythmia (RSA) is concep- communities of inborn errors of metabolism and diagnostic imaging. tualized as an index of physiological flexibility related to emotion regula- Methods: We analyzed brain 1 H-MRS data from >2200 children from the basal tory capacity. Although behavioral genetics research indicates that RSA is ganglia (BG) and periventricular white matter (PVW). Measurements from two partly heritable, we examined whether 5-HTTLPR was associated with voxel localizations (BG and PVW) and two echo times (35 ms and 144 ms) were resting RSA among healthy adults. In light of the involvement of reduced collected. Post-processing of MRS data and analysis was performed using LC RSA in anxiety disorders and emotion dysregulation, the finding of a Model software. Retrospective assessment of the MRS data from children with genetic influence that may explain the variance of RSA may have important >20 different diagnoses of IEM was performed, including: Organic Acidurias, implications for the pathogenesis and treatment of these conditions. It has Remethylation defects, Urea Cycle defects, Storage disorders etc. been reported that carriers of the low-functioning allele (s) of the 5- Results: We identified a number of abnormal metabolite patterns including low HTTLPR may display decreased resting RSA. This study investigated total choline (tCho) in MMA, Arginase deficiency, MTHFR deficiency and resting RSA in an ethnically homogenous sample (N0143) of participants SSADH deficiency, low total creatine (tCr) in Citrullinemia, Arginase deficiency genotyped for the insertion/deletion and another functionally connected and Krabbe and high Inositol (Ins) in Citrullinemia and NCL, to name a few. SNP (rs25531) in 5-HTTLPR. Trait/state anxiety and respiration rate were Conclusions: Our continued data exploration will identify a number of controlled. The triallelic 5-HTTLPR/rs25531 genotypes showed no associ- novel metabolite patterns that are diagnostic for IEM and will aid in the ation with resting RSA. elucidation of the underlying neurometabolic mechanisms. S166 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-469 P-471 ABNORMAL RESPONSES TO VISUAL CORTEX ACTIVATION IN GENOTYPE-BASED LEIDEN OPEN VARIATION DATABASE FOR EARLY STAGE HUNTINGTON DISEASE PATIENTS USING MUTATIONS CAUSING INHERITED METABOLIC DISEASES: 31P-NMR SPECTROSCOPY CHALLENGES AND SOLUTIONS Mochel F1 , Nguyen TM1 , Deelchand D2 , Rinaldi D1 , Wary C2 , Carlier Lanthaler B1 , Kalb S2 , Deutschmann A1 , Schossig A1 , Zschocke J1 , PG2 , Durr A1 , Henry PG3 Witsch-Baumgartner M1 1INSERM UMR S975, La Salpêtrière hospital, Paris, France 1Medical University Innsbruck, Innsbruck, Austria 2Institut of Myology, La Salpêtrière hosp, Paris, France 2Paracelsus Medical University, Salzburg, Austria 3CMRR, University of Minnesota, Minneapolis, United States Most inborn errors of metabolism are inherited at autosomal recessive traits, Background: HD is an autosomal dominant neurodegenerative disease and the disease-causing mutations are mostly known. The functional effects caused by a CAG repeat expansion in the HTT gene. We wished to test result from the combined action mutations in both copies of the gene, and the hypothesis that brain energy metabolism is abnormal in patients at an depending on genotype the phenotype often varies strikingly within the early stage of Huntington disease (HD). same disease. Additional effects may be contributed by so-called modifiers Methods: We coupled noninvasive 31P-NMR spectroscopy with activation which could be nucleotide polymorphisms. We report the development of of the occipital cortex in order to measure the levels of ATP, phosphocrea- genotype (mutation) variation databases for IEM which connect genotypes tine (PCr) and inorganic phosphate (Pi) before, during and after a visual to clinical phenotypes and include data about genetic modifiers. stimulus. We studied 15 HD patients at an early stage of the disease and 15 We used the open source software tool LOVD v2.0 which allows custom age- and sex-matched controls. designed columns with regard to specific content for each gene. For Results: In controls, we observed an 11 % increase in Pi/PCr ratio (p0 nomenclature of genes, transcripts, mutations and phenotype we used 0.024) during brain activation, reflecting increased ATP synthesis and ADP international standards and guidelines (as HGNC, HGVS). levels. Subsequently, controls had a return to baseline levels during recov- Databases have been created for ACAD8, ACADSB, AUH, HMGCS2, ery (p00.012). In HD patients, Pi/PCr ratio was unchanged during and after HSD17B10, DHCR7 and FKBP14. For submission of data registration is visual stimulation, reflecting altered mitochondrial bioenergetics. In addi- necessary. Identical genotypes may be listed multiple times if several tion, in HD patients the ratio of Pi/PCr ratio correlated with the UHDRS patients carry the same. Phenotypic descriptions and biochemical data are score during recovery (p00.016), reflecting a correlation between brain included as detailed as possible. For DHCR7 genetic modifier data (the energy metabolism and disease severity in HD. maternal ApoE and ABCA1 genotypes) are added. Conclusions: We have identified a noninvasive functional biomarker of The devised database structure is particularly well suited to the special brain energy deficit to assess therapeutic efficacy in HD. requirements for IEMs and can be easily adapted to other genes.

P-470 EVALUATION OF MTDNA COPY NUMBER AS BIOMARKER IN LUNG CANCER P-472 Bonifácio C1 , Carvalho L2 , Santos MJ1 , Calvinho P3 , Alarcão A2 , Silva SHWACHMAN DIAMOND SYNDROME MIMICS DISORDER OF MR2 , Dias JA2 , Cabete A1 , Wong LJ4 , Grazina M5 GALACTOSE METABOLISM 1Center Neurosciences and Cell Biology, Coimbra, Portugal Kreile M.1 ,DanebergaZ.2 , Ziemele I.1 , Eglite I.2 , Grinfelde I.2 , 2Faculty Medicine, University of Coimbra, Coimbra, Portugal Sviridova D.1 , Krumina Z.2 3CHUC, Coimbra, Portugal 1Riga Stradins university, Riga, Latvia 4Baylor College of Medicine, Houston, United States 2Childrens University Hospital, Riga, Latvia 5Fac Medicine & CNC/UC, Coimbra, Portugal Background: Shwachman Diamond syndrome (SDS) is a rare autosomal Mitochondrial genome (mtDNA) is more susceptible to damage and recessive disorder characterized by exocrine pancreatic insufficiency, bone acquires mutations at higher rate than nuclear DNA. Some types of cancer marrow dysfunction and skeletal abnormalities. Massive galactosuria usu- present increased mtDNA content, while others have been associated to ally occurs in disorders of galactose metabolism. decrease, compared to non-cancerous tissue from the same patient. Case report: Male patient born from G7P6 at 37 weeks, birth weight We have evaluated mtDNA copy number in blood and lung tissue, derived 2290 g, height 47 cm, Apgar score 8/10. Family history: first child – severe from patients with lung cancer, to determine its role as a possible biomarker, mental retardation, fifth - Down syndrome. Child presented soon after birth according to smoking status. with failure to thrive, anemia, visually big abdomen, liver +1 cm, S. aureus It was observed that 66.7 % of blood samples show a significant decrease in sepsis. Urinary screening for IEM showed massive galactosuria. Breast mtDNA copy number when compared with control, while 67.6 % show a feeding was stopped immediately. Within one month on galactose restricted significant increase in mtDNA copy number in cancer lung tissue, com- diet, there was no remarkable improvement - still failure to thrive, anemia, pared to normal. Correlation analysis between clinical parameters and although no signs of steatorrhea. GALT and galactose metabolites analysis mtDNA copy number in normal tissue vs. tumor tissue shows increased were negative. Remarkably decreased serum elastase level was detected, of mtDNA content in normal tissue of active smokers, compared to passive due to pancreatic insufficiency and pancytopenia SDS was suspected, smokers. Analysis of mtDNA copy number has shown statistically signifi- diagnosis later confirmed in Italy by finding two mutations in gene SBDS cant difference between blood and lung tissue. 183-184TA>CT/258+2 T>C which cause SDS. This study suggests that mtDNA content in blood cannot be used as Conclusions: According to the literature galactosuria is characteristic sign biomarker of lung cancer, but mtDNA content is elevated in tumor tissue of SDS, but usually not as remarkable as in our case, so SDS must be compared with normal. considered as differential diagnosis. This case demonstrates clinical overlap To our knowledge, this is the first study comparing mtDNA content in between symptoms of diseases of completely different origin. blood, tumor and normal lung tissue samples of same patient. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S167

P-473 P-475 DEVELOPMENT OF A LIQUID CHROMATOGRAPHY QUADRUPOLE INBORN ERRORS OF METABOLISM (IEM) IN LITHUANIA TIME OF FLIGHT MASS SPECTROMETRY (LC-QTOF) METHOD ACCORDING TO THE DATA OF CENTER FOR MEDICAL FOR THE INVESTIGATION OF THE WHOLE URINE METABOLOME GENETICS Lxvoll SM1 , Xsteby A1 , Bliksrud YT1 , Woldseth B1 , Mxrkrid L1 , Cimbalistienė L1 , Burnytė B1 , Songailienė J1 , Liaugaudienė O2 ,Kučinskas V1 Elgstxen KBP1 1Dep of Hum and Med Genetic, Vilnius Univ, Vilnius, Lithuania 1Dept Med Biochem, Oslo Univ Hosp, Oslo, Norway 2Center for Medical Genetics, Vilnius, Lithuania

The systematic study of all the metabolites in urine, untargeted urine Background: In Lithuania, no information is available (except PKU and metabolomics, has several advantages over targeted urine metabolomics CH) on the prevalence of the IEM. in which specific metabolites are analyzed and quantified. The untargeted Aims: To pinpoint the profile of IEM and to estimate the most frequent approach has the potential to cast new insights into the biochemical alter- disorders prevalence in Lithuania. ations following an inborn error of metabolism, and eventually also dis- Methods: We analyzed data from patients who had been diagnosed with cover new diagnostic metabolites. IEM between 2001 and 2011. During this period 5015 samples of patients The number of metabolites found in an untargeted approach will with symptoms of IEM were subjected for laboratory investigations. HPLC depend on the analytical parameters used. Thus, we have performed a was performed on plasma amino acids, urinary organic acids' profiles were systematic study to develop a LC-QTOF method capable of separating determined by GC/MS. Screening for oligosaccharides has been performed as many metabolites as possible in urine. A mix of urinary metabolites using TLC, quantitative and qualitative analysis of urinary glycosamino- with different chemical properties, and a urine sample from a healthy glycans was studied by the DMB and 1DEF. In case where laboratory control, was used to compare the performance of reversed phase col- findings suggested IEM, confirmatory tests were performed in different umns from Waters, Varian, ACE, Phenomenex and Agilent. A 64 laboratories worldwide. minutes water/methanol gradient was used for chromatography, and Results: The most frequent diagnosed IEM (except PKU) were lysosomal the QTOF were run in MS1 mode (only parent ion – no fragmentation) storage disorders 38 (42.2 %), disorders of amino acids 15 (16.7 %) and in both positive and negative mode. The number of unique molecules lipoprotein metabolism 12 (13.3 %). The most frequent LSD were MPS 17 found by the QTOF was evaluated. The ACE PFP and Agilent (18.9 %), mucolipidoses 8 (8.9 %) and sphingolipidoses 7 (7.8 %). The diphenyl columns gave the highest score with about 790 and 270 prevalence of LSD in Lithuania was calculated 2.00 in 100 000 live births, metabolites in positive and negative mode respectively, and will there- while the prevalence of MPS was 1.08 in 100 000 live births. fore be used in the proceeding studies. Conclusion: According to our data, the most frequent IEMs in Lithuania were lysosomal storage disorders.

P-474 P-476 LONG-TERM TREATMENT WITH LONG ACTING RELEASE RELATIONSHIPS BETWEEN SYMTOMS OF ATTENTION DEFI- OCTREOTIDE IN CONGENITAL HYPERINSULINISM CIT AND HYPERACTIVITY DISORDER AND BLOOD LEVELS Arnoux JB1 , Mamoune A1 ,BrassierA1 , Barbier V1 , Valayannopoulos V1 , OF LEAD AND PHENYLALANINE IN SCHOOL CHILDREN Broissand C1 ,ThalabardJC2 ,LeQuanSangKH1 ,deLonlayP2 Kırlı Dr.1 , Bulbul Prof.Dr.1 , Kurt Dr.1 1Ref Center Metab Dis, Hôp Necker, APHP, Paris, France 1Div Metab Dis, Kirikkale Univ Hosp, Kirikkale, Turkey 2Paris Descartes University, Paris, France Background: Attention Deficit and Hyperactivity Disorder (ADHD) can Background: The medical treatment of diazoxide-unresponsive congenital occur by the interactions of genetic factors, diet and social/physical environ- hyperinsulinism (HI) is challenging, based on a somatostatin analogue. ment where lead plays an important role among all. This study was planned to We evaluated the long-term efficiency and tolerance of long-acting release determine association between ADHD symptoms and blood lead level and to (LAR) octreotide (Sandostatine LAR®, Novartis) administered as an intra- examine whether serum phenylalanine level effects this assosication. muscular injection every 4 to 6 weeks in HI patients. Methods: The study group was consisted of 1413 primary school children. Method: Twenty-five diazoxide-unresponsive HI patients treated with sub- 55 students with mean age of 8.86±1.60 years were selected by using the cutaneous octreotide given in 3 daily injections were changed to LAR DSM-IV Based child and adolescent behavior rating scale. 55 matched octreotide with a mean follow-up of 24 months. The data collected before controls for age, gender and socioeconomic strata were enrolled. the first LAR octreotide injection and at the latest visit were compared using Results: Serum lead, phenylalanine, tyrosine levels, and phenylalanine/ the Wilcoxon test. tyrosine ratio (Phe/tyr)were measured. Mean blood lead level (4.00± Results: (mean +/- SD before LAR treatment vs. most recent visit): 1.93 μg/dL) and Phe/tyr(0.91±0.37) of the case group were significantly Glycemia were maintained in their usual range and HbA1c remained stable higher than the control group [blood lead level (3.11±1.99 μg/dL) and Phe/ (5.34 % +/- 0.57 vs. 5.35 % +/- 0.53, p00.22). The equivalent daily dose of tyr 0.75±0.32)], (p00.019 and p00.018, respectively). There were weak octreotide was decreased by an average of 17.2 % +/- 14, p00.0003. positive correlations between both blood lead level and Phe/tyr with the Patients' growth followed their usual curves (variation from their initial scores of attention deficit subscales(r00.431 and r00.384 respectively, curve: -0,04 SD +/- 1.05, p00.94). IGF1 levels improved, p00,01. No p<0.001)and hyperactivity/impulsivity subscale (r00.267, p00.005). severe side effect was noted and parents' questionnaire of satisfaction for Conclusion: Because symptoms of ADHD is associated with phenylala- LAR octreotide was highly positive. nine/tyrosine ratio which is used for follow-up of individuals diagnosed Conclusion: LAR octreotide is efficient and well tolerated in diazoxide- with hyperphenylalaninemia, it is suggested that phenylalanine metabolism unresponsive HI patients and contributes to a clear simplification of their may have a role in relationship between lead exposure and ADHD. medical care. S168 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-477 P-478 A CHILD WITH MEGALENCEPHALIC LEUKOENCEPHALOPATHY FACILITATING DECISION MAKING FOR DIAGNOSIS IN WITH SUBCORTICAL CYSTS WITH AN EARLY DIAGNOSIS PATIENTS SUSPECTED OF INHERITED METABOLIC DISORDERS OF CANAVAN DISEASE USING BAYESIAN METHODS AydınK1 , Kasapkara C S2 , Tumer L2 , Hasanoglu A2 , Abbink G E M3 , Talebi S1 , Talebi S2 , Samavat A3 , Taghizadeh M2 , Keramatipour M2 Van der Knaap M S3 1Avicenna Research Institute, ACECR, Tehran, Iran, Islamic Republic of 1Div Ped Neurology, Gazi Univ Hosp, Ankara, Turkey 2Tehran University of Medical Sciences, Tehran, Iran, Islamic Republic of 2Div Ped Metab Dis, Gazi Univ Hosp, Ankara, Turkey 3Ministry of Health and Medical Education, Tehran, Iran, Islamic Republic of 3Div Child neurology, VU Univ Med Center, Amsterdam, Netherlands Introduction: Generally, inherited Metabolic Disorders present with differ- Megalencephalic leucoencephalopathy with subcortical cysts is a ent clinical and laboratory findings. It makes diagnosis not an easy task. recessively inherited genetic brain disorder with onset in early child- Differential diagnosis tables as a screening diagnostic tool can potentially hood. Affected infants develop macrocephaly within the first year of be useful for physicians. Complicated and numerous clinical and para- life followed by slowly progressive, incapacitating cerebellar ataxia clinical variables, however, make effective judgment difficult. and spasticity. MRI shows diffuse signal abnormality and swelling of This study introduces a new methodology to ease judgment for physicians, the cerebral white matter, with evidence of highly increased white when using differential diagnosis tables. matter water content. In most patients, the disease is caused by Methods: A Bayesian model was developed in order to estimate the mutations in the gene MLC1. probability of each metabolic diagnostic group given the presence or Case Report: The 15 month old girl born from a consanguineous absence of clinical and/or Laboratory Findings. The emedicine/medscape marriage, presenting with macrocephaly consulted to our clinic with differential diagnosis table for inborn errors of metabolism was used. The an early diagnosis of canavan disease. MRI showed diffuse signal semi-quantitative data in the table was converted to quantitative weights. abnormality and swelling of the cerebral white matter, with evidence The resulted values and the frequency of each metabolic group in popula- of highly increased white matter water content, anterior temporal and tion were applied for calculating different probabilities. frontal cysts. The clinical features were characteristic of the disease, Results: The calculated probabilities for patients with typical presentations of consisting of an early-onset macrocephaly.We have analyzed the exons inherited metabolic disorders (Intoxication, Energy deficient or chronic pro- and flanking intronic regions of the gene MLC1 by sequence analysis gressive presentations) were according to our expectations; however, the at a genomic level in the patient. We have found that the patient is calculated probabilities for patients with chronic non-progressive global devel- homozygous for the following variant in the gene MLC1 (c.836 T>C, opmental delay without specific organ signs do not match the estimations. pLeu279Pro). Both parents carry the same c.836 T>C,pLeu279Pro Conclusions: It seems that this approach, potentially, is an effective meth- variant. Megalencephalic leucoencephalopathy with subcortical cysts odology to facilitate clinical decision making if patient selection is done in have been diagnosed based on clinical and radiological basis and an appropriate manner. confirmed by molecular genetic analysis. P-479 A CHILD WITH BRITTLE CORNEA SYNDROME PRESENTING WITH OSTEOPOROSIS, VERTEBRAL COMPRESSION FRACTURES AND A NEW HOMOZYGOUS MUTATION Öztürk Y1 , Guinta C2 , Spencer H3 , Erdur B1 , Çakmakçı H4 , Kavukçu S1 1Dokuz Eylul Univ, Dept Pediatr, İzmir, Turkey 2Children's Hospital, Univ Zurich, Zurich, Switzerland 3Univ of Manchester, Manchester, United Kingdom 4Dokuz Eylul Univ Dept Radiol, İzmir, Turkey

Brittle cornea syndrome (BCS) is characterized by blue sclerae, corneal fragility, keratoconus or keratoglobus, hyperelasticity of the skin and hypermobility of the joints. The BCS is sometimes confused with the kypho- scoliotictypeofEhlers–Danlos syndrome (EDS VI) because of the similarities such as rupture of the eye and the joint and skin manifestations, as well as the autosomal recessive mode of inheritance. In patients with BCS lysyl hydroxylase activity and urinary ratio of total pyridinolines are normal in contrast to Ehler-Danlos type VIA. Here we describe a 10 years- old boy with BCS admitted for vertebral compression fractures and a new homozygous mutation in the ZNF469 gene. Osteoporosis and fractures are not recognized as the cardinal features of BCS, however, in limited number of cases, osteoporotic bone thinning and fractures have been reported due to the decrease in the collagen content of the bones and reduced physical activity in EDS type VI. We propose that children with BCS need further examination for osteoporosis and fractures; andphysical and pharmacological strategies for reducing fractures should also be considered. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S169

P-480 P-482 INBORN ERRORS OF METABOLISM (IEM) AND ASSOCIATED STEREOTACTIC RADIO FREQUENCY ABLATION: THERAPEUTIC METABOLIC DISTURBANCES DETECTED IN A COHORT OF OPTION FOR LIVER TUMORS IN INHERITED METABOLIC GREEK PATIENTS WITH AUTISM SPECTRUM DISORDER (ASD) DISORDERS Spilioti MG1 , Trama D2 , Michailidi E3 ,SpanouS2 , Frysira H4 , Karall D1 , Bale R2 , Albrecht U1 , Niedermayr K1 , Widmann G2 , Scholl- Haidopoulou A2 , Metaxas S5 , Asprangathou D2 , Bondi E6 , Tsalkidis Bürgi S1 AJ7 , Kardaras P3 , Wevers R8 , Jakobs C9 , Evangeliou AE2 , Gibson KM10 1Pediatrics I, Medical University, Innsbruck, Austria 1AHEPA hospital, First Dept of Neurolog, Thessaloniki, Greece 2Dept of Radiology, Medical University, Innsbruck, Austria 2Papageorgiou Hospital, dept of Pediatr, Thessaloniki, Greece 3Hippokration Hospital, 3d Dept of Pediat, Thessaloniki, Greece Introduction: Glycogen storage disease type I (GSD Ia) and tyrosinemia 4Agia Sophia Children's Hospital, Athens, Greece type I (TYR I) are recessively inherited disorders, caused by deficiency of 5Papageorgiou Hospital, HNO Dept, Thessaloniki, Greece glucose-6-phosphatase and of fumarylacetoacetase. GSD Ia can be compli- 6Papageorgiou Hospital, Dept of Psychiatr, Thessaloniki, Greece cated by formation of liver adenomas (with risk of malignancy), TYR I by 7University Hospital of Alexandroupolis,, Alexandroupolis, Greece development of hepatocellular carcinoma. Standard procedure is observa- 8University Hospital Nijmegen,, Nijmegen, Netherlands tion of adenomas and eventually operative removal. Stereotactic radiofre- 9Akademical University Hospital Amsterdam, Amsterdam, Netherlands quency ablation (SRFA) is a local curative ablation method, which uses the 10Clin Pharmacol, Wash State Univ, Spokane, WA, United States application of high frequency alternating current between multiple probe positions in tissue and skin electrodes, causing targeted heat of >60 °C and Background: Numerous pathologies (Fragile X, Rett syndromes, congen- controlled tumor destruction (1). Biopsies for histology can precede the ital infection, perinatal damage, etc) have been discussed as etiologies procedure (2). associated with autism, and an expanding literature has demonstrated fre- Patients: SRFA was applied for removal of a single large liver adenoma in a quent associations between inborn errors of metabolism and autism. 22 year-old patient with GSD Ia and a suspicious lesion in a 16 year-old Methods: We screened for IEM in 187 autistic children (105 males; vs 82 patient with TYR I and imminent AFP elevation with good results. Large females, ages 4 -14 years old). scars were avoided, in the TYR I patient elevated AFP promptly returned to Results: Five diagnoses included:Lesch Nyhan syndrome (2), succinic semi- normal values. aldehyde dehydrogenase deficiency (2) and phenylketonuria (1) . Additionally, Conclusion: The use of SRFA technique could have immediate and strong we observed metabolic disturbances in many that suggested an IEM, including consequences in the treatment guidelines for patients with inherited meta- elevated urinary 3-hydroxyisovaleric acid (12 patients, two of whom also bolic disorders with liver involvement. manifested elevated methylcitrate and lactate in sera) and abnormal glucose- References: loading tests (30 patients). In the latter , 16/30 patients manifested increased sera (1) Widmann G. Cardiovasc Intervent Radiol, 2011 Jun 14 beta hydroxybutyrate production, 4/30 had a paradoxical increase of sera lactate, (2) Schullian P. Comput Aided Surg, 2011;16:181-7 14/30 manifested increased serum lactate, and 5/30 had decreased serum ketones. Twelve of 187 patients demonstrated nonspecific MRI pathology, while 25/187 had abnormal EEG findings. Finally, family history was positive for 22/ 187 patients and consanguinity was documented in 12/187 families. Conclusions: Our study lends further support to systematic evaluation of IEM in patients with ASD, as recently suggested for inherited disorders of carnitine biosynthesis in autism (Celestino-Soper et al (2012) Molec Genet Metab 105: 308).

P-481 P-483 GLUT1 DEFICIENCY: A MILD PHENOTYPE IN A 4 YEARS OLD INBORN ERRORS OF METABOLISM IN ASIA FEMALE WITH EARLY ONSET ABSENCES AND ATAXIA Wasant P1 Mastrangelo Mario1 , Celato Andrea1 , Galosi Serena1 , Leuzzi Vincenzo1 1Fac Med Siriraj Hosp, Mahidol Univ, Bangkok, Thailand 1Div Child Neurol Sapienza Univ Rome, Rome, Italy The study of inherited metabolic disorders (IMD) in Asia first started in Japan in Introduction: Glucose (GLUT1) transporter 1 deficiency (OMIM 606777) is 1960's. The Japanese Society for Inborn Errors of Metabolism was established in a polymorphic syndrome including an epileptic encephalopathy, early onset 1965, currently has 700 members. Research and treatment of IMD e.g. enzyme absences, complex movement disorders and developmental delay. replacement therapy for lysosomal storage disorders (LSD) was started in 1996, Case report: We present the case of a 4 year old female with episodes of liver transplantation in 1990's. Development of IMD in Japan is considered the recurrent staring since the age of 3 months and fluctuating ataxic gait when most advanced in Asia. As for other developed countries in Asia e.g. Korea and awakening since the age of 2 years and 6 months. Symptoms were exacerbated Taiwan - the facilities for biochemical and molecular diagnosis have been at awakening and with fasting. Psychomotor development was only mildly available in Korea since early 1990's; treatment of IMD included special formula impaired. Electroencephalogram evidenced diffuse epileptiform discharges with for diet therapy, organ transplantation and enzyme replacement therapy for LSD the prominent involvement of the anterior regions. Seizures have been well also are available. Genetic Health Act in 1985 and Prevention and treatment of controlled with valproic acid. Brain MRI was negative. Lumbar puncture rare disease Act in 2010 play important role in improving quality of life of IEM showed a CSF/blood glucose ratio of 0,28. Molecular investigation on SLC2A1 patients in Taiwan. As for developing countries - Malaysia, Philippines and gene revelaed a heterozygosis for the mutation c.274 C>T (p.Arg92Trp). Thailand where small number of clinical geneticists and metabolic specialists are Discussion: GLUT1 deficiency have an expanding phenotype. Our experi- available; however, numerous cases of IMD have been identified. Diagnostic ence evidences that the pattern of GLUT 1 deficiency includes different laboratory facilities are also being developed at the tertiary care centers. Collab- ranges of clinical severity. This is probably due to differences in the func- orations with more advanced countries in Asia (Japan, Korea, Taiwan) and tional impairment of the transporter. United States, Europe and Australia have been very active. S170 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

P-484 A-043 MCRN INHERITED METABOLIC DISORDERS CLINICAL STUDIES DYSMORPHIC FEATURES - AS A DIAGNOSTIC SIGN OF GROUP INBORN ERRORS OF METABOLISM Hendriksz C1 Plaiasu V.1 , Ochiana D.1 , Avram P.1 , Stambouli D.2 1University of Manchester, Manchester, United Kingdom 1IOMC Prof.dr.Alfred Rusescu, Bucharest, Romania 2Cytogenomic Medical Laboratory, Bucharest, Romania The National Institute for Health Research (NIHR) Medicines for Children Research Network (MCRN) is part of the NIHR Clinical Research Network Early diagnosis of inborn errors of metabolism (IEMs) can prevent severe (NIHR CRN), which supports research to make patients, and the NHS, disease progression. The clinical features accompanying metabolic diseases better. seldom permit a specific diagnosis. A number of metabolic disorders are The Inherited Metabolic Disorders Clinical Studies Group (CSG) is one of associated with dysmorphic manifestations. They may be apparent at birth or 15 CSGs which have been established by the MCRN. We are supported by develop progressive with age. The mechanism of the dysmorphism occurring as the UK Lysosomal Storage Disorders Patient Collaborative Group and are a result of IEM varies from one disorder to another. There are types of inherited responsible for developing/overseeing a portfolio of clinical trials and metabolic diseases in which dysmorphism might be expected to be prominent. related studies covering the spectrum of major disease areas in paediatrics We present few cases of our work with features suggestive of a metabolic and adults affected by ultra orphan inherited metabolic disorders. disorder: Membership of our group includes active researchers (clinical and other a) Amino Acids disorders (homocystinuria, oculocutaneous albinism) professions), parents/carers, nurses, charity representatives and formulation b) Peroxisomal disorders (Zellweger syndrome, rhizomelic chondrodyspla- experts. We provide expert advice to help NHS/academic researchers and sia punctata) companies develop high quality research proposals ready for grant sub- c) Disorders of sterol synthesis (Smith-Lemli-Opitz syndrome) mission. Our support is provided free of charge. d) Lysosomal storage disorders: mucopolysaccharidoses and mucolipidoses Seeking an opinion from the CSG is a helpful step to take at an early stage, e) Congenital hypothyroidism although you may contact us at any stage as you develop your research Inborn errors of metabolism may be associated with consistent pattern of proposal. birth defects, suggesting that metabolic derangement in utero disrupt the Our study portfolio includes 24 studies, predominantly sponsored by indus- normal process of fetal development. Clinical examination is the first step in try, with UK units supporting the successful recruitment to these studies. a practical approach to raise IEM suspicion and to facilitate a proper To submit a proposal (at any stage), please email [email protected]. metabolic evaluation. It is important to detect IEM for some reasons: accurate counseling, avoiding metabolic decompensation and improving follow-up of patients, existing of particular treatments.

P-485 A-044 HOW USEFUL ARE CSF INVESTIGATIONS FOR MAKING A L-CARNITINE DEFICIENCY IN BETA-THALASSEMIA MAJOR DIAGNOSIS IN CHILDREN WITH NEUROLOGIC SYMPTOMS? Kalkan Ucar S1 ,Yılmaz Karapınar D2 , Balkan C2 , Kavaklı K2 , Bozdemir Scholl-Bürgi S1 , Lagler FB2 , Druck C1 , Haberlandt E1 , Baumann M1 , E3 , Parıldar Z3 , Habif S3 , BayindırO3 , Coker M1 Rostásy K1 , Deisenhammer F3 , Loacker L4 , Albrecht U1 , Karall D1 1Div Metab Dis, Ege Univ Child Hosp, Izmir, Turkey 1Pediatrics I, Medical University, Innsbruck, Austria 2Div Ped Hem, Ege Univ Child Hosp, Izmir, Turkey 2Institute IEM, Paracelsus Med Univ, Salzburg, Austria 3Div Med Bioch, Ege Univ Child Hosp, Izmir, Turkey 3Dept Neurology, Medical University, Innsbruck, Austria 4ZIMCL, Medical University, Innsbruck, Austria Background: The role of carnitine is to facilitate the transport of long- chain fatty acids esters through the mitochondrial membranes. The intra- Aim: This retrospective study aimed to evaluate the diagnostic value of a mitochondrial pool of carnitine enhances the oxidation of short-chain fatty structured lumbar puncture procedure in children with neurologic symptoms acids and possibly of branched keto-acids. The aim of this study was to (grouped as e.g. epilepsy, mental retardation, dystonia and neurodegeneration). measure the serum carnitine levels with homozygous beta-thalassemia Methods: The lumbar puncture protocol included routine CSF parameters patients and clarify the reasons of deterioration at carnitin levels using acyl (protein, albumin, immune globulins, glucose, lactate) and metabolic inves- carnitine, aminoasids, organic asids and oxidative parameters. tigations (amino acids, neurotransmitters). Simultaneously, a venous puncture Methods: In the present study, serum carnitine levels were measured in 48 was performed (1, 2). Additionally, sparse CSF was frozen for further inves- children with homozygous beta thalassemia and 11 healthy subjects. A tigations (e.g. TAU protein, oligoclonal bands). The procedure was considered decrease in serum free carnitine levels (17.08±7.22 mmol/L) was found. diagnostic, when it helped to establish a (previously not considered) diagnosis Results: Isoleucine, Phenilalanine, Tyrosine, Aspartate, Glutamate, Glycin, and helpful, when it helped to exclude differential diagnosis. Alanine, Serine, Argnine and Ornitine levels in patients with homozygous Results: 207 of 271 investigations performed 2003 to 2011 fulfilled inclu- beta thalassemia were lower (p<0.05) and Taurine, Glutamine, Treonine, sion criteria: patient age<18 years; no follow up puncture, adequate pre- Citrulline, Valine, Leucine, Hystidine, Tryptophan, OH-Prolin, Proline ve analytic handling. In 26 patients (13 %) the procedure was diagnostic, a Lysine levels were higher compare to the controls (p<0.05). group mainly represented by inborn errors of metabolism (11/26) and Malondialdehyde, one of the most frequently used indicators of lipid inflammatory diseases (11/26). In 73 (35 %) patients it was helpful. peroxidation was increased 1,52±0,40 nmol/mL in thalasemic group com- Conclusion: Lumbar puncture was diagnostic in about 50 % of neuro- pare to the controls 1.32±0.22 nmo/mL (p<0.05). pediatric patients caused by metabolic (11/22) or inflammatory diseases Conclusions: In patients with homozygous beta-thalassemia, the (11/24). Moreover it was helpful in establishing or excluding diagnosis reduction of serum carnitine levels might secondary to reduced hepatic especially in epilepsy and developmental retardation of unknown etiology. carnitine biosynthesis, increased oxidative stress and subcellular iron (1) Scholl-Bürgi et al Pediatrics 2008, (2) Karall et al Neuropediatrics 2012 deposition. J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182 S171

Author Index

İncecik, F P-009 Almon-Brill, E P-262 Azar, NB P-276 Baumgartner, M P-023, P-087 Şener, E A-042 Alston, CL P-305 Azarsız, E A-037 Baumgartner, MR P-451, O-029 Żuber, Z P-399 ALTAY, I.Suheyla A-040 Azen, C O-003, P-036 Bavdekar, A P-270, P-407, P-410 A, Dursun P-382 Altay, S P-143, P-008 Bachański, M. P-340 Bax, BE P-411 A, Guzel P-382 Alvelos, M A-033 Baczyńska, A P-331 Bay, L. P-106 A, Tokatli P-382 Alvorcem, LM P-002 Badminton, MN P-124 Baydakova, GV P-166 Abbink, G E M P-477 Amaral, AU P-117, P-132, P-345 Badrinath, S O-061 Bayindır, O A-044 Abbot, V P-305 Amato, D O-032, P-262 Baggot, M P-445 Béard, E P-092 Abdullah, IS P-197 Ambreen, M O-015 Bahceci, S P-322 Beblo, S P-035, P-424 Abeling, N P-366 Amir, I P-017 Bahi-Buisson, N P-388, P-029 Beck, HC O-031 Abiose, A P-232 Anazi, S P-185 Bähr, L P-133 Beck, M P-210, P-240 Abu-Horwitz, A P-013 Andersen, H.S. O-070 Bain, MD P-411 Beco, A P-312 Abuhoul, L P-110 Andersen, J.R. P-052 Bainbridge, K P-445, P-446, P-246, Bedir, T P-371 Abulhoul, L O-016, P-138, P-167, Andrade, VM P-024 P-210 Beermann, F P-133, P-365 P-373 Andresen, B.S. O-070 Bakker, J P-032 Behulova, D P-070, A-027 Abulhoul, L. P-045, P-080 Andresen, BS O-043, P-158 Bakker, J.A. P-193 Behúlová, D P-353 Ackerley, C O-044 Andria, G P-047, P-168, P-173, Bakker, JA P-095 Belanger, A P-135 Adamowicz, M O-059 P-189 Bakouh, N P-094 Belanger-Quintana, A P-060, Adams, S O-010 Anikster, Y P-013, P-058, P-192 Bal, M P-179, P-105, P-047 P-064, P-065 Adelmann, K O-002 Anjema, K O-005, P-065 Bal, MO P-038, A-013, P-449 Bélanger-Quintana, A P-067, P-068 AERTS, H P-257 Aoki, K P-081, P-128, A-014, Balcı, M A-015 Beleza-Meireles, A P-316 Agalou, S P-305 P-442 Balcells, S P-284 Bell, L P-397 Agostini, C A-008, P-345 Aral, A P-352 Balci, MC P-289, P-343, P-419 Belmatoug, N P-251 Aguzzi, R P-254 Ardila, Y.A A-036 Baldazzi, L P-038 Belostotsky, R O-072 Ahring, K.K. P-052 Arelin, M P-424 Bale, R P-482 Bembi, B P-260, P-276 Ahting, U O-040, O-045, P-301 Arenas, M P-076, P-098 Balestrat, S P-104 Ben Dridi, M-F P-407, P-410 Aiach, K P-202 Arias, A P-327, P-332 Balivo, F P-168 Ben Turkia, H P-407, P-410 Aicher, J P-310 Arias, C P-046 Baljosevic, I A-023 Benarrosh, A P-013 Aitkenhead, H P-386 Armstrong, LL P-465 Balkan, C A-044 Benistan, K P-232 Ajima, Masami P-307 Arning, EA O-004 Ballhausen, D P-391, P-107, O-027 Benjamin, ER P-248, O-065, P-242 Akay, G P-252 Arnoux, JB P-474, O-011 Balmer, C P-100 Benoist, JF P-388, P-298, P-005, Aksoy, A P-265 Arranz, JA O-039 Balsamo, A P-449 P-088, P-089, P-016 Aktuğlu Zeybek, AÇ A-021, Arriola, G P-027 Balwani, M O-033 Ben-Omran, T O-021 A-035, A-038 Arroyo, I P-290 Bandeira, A P-156 Benouachane, T P-222 Aktuğlu Zeybek, AC P-008 Arslan, A P-201 Banderali, G P-059 Bergmann, J P-179, P-194 Aktuglu Zeybek, AC P-143 Arslan, Duran A-032 Banikazemi, M O-061 Berniah, a P-269 Aktuglu-Zeybek, A.C. A-040 Artigalás, OA P-277 Barba-Romero, MA P-229 Bernstein, L P-431 Aktuglu-Zeybek, C P-192 Artuch, R P-027, P-144, P-290, Barbeito, L P-034 Bertin, N P-260 Al Harbi, T P-185 P-332, P-390 Barbier, M O-067, P-342 Bertini, Enrico P-369 AL SALMAH, M P-185 Ashmore, C O-008, P-414, P-416, Barbier, V P-474 Bertino, Sara P-377 Alarcão, A P-470 P-417, P-418, P-425 Barbuti, D P-391 Bertuletti, B P-104 Albersen, M P-033 Ashworth, J P-112 Barends, ME O-013 Besse, A P-299 Albersen, M. O-053 Aslam, R P-205, P-206 Baric, I O-046 Besse, A. P-337 Albrecht, U P-482, P-485 Asmari, A P-439 Bar-Joseph, I P-013 Bessou, P A-034 Alcaide, P P-090 Asprangathou, D P-480 Barone, R P-285 Betta, K P-046 Aldamiz, L O-039 Assoun, M O-010 Baronio, F P-038, P-105, A-013, Bettocchi, I A-013, P-038, P-449, Al-Dehaimi, AW P-439 Asteggiano, CG P-284 P-179, P-449 P-105, P-179 Al-Dirbashi, OY P-436 Ather, S. O-041 Barouki, R P-463 Bevington, M P-432 Alegra, TA P-277 Aubourg, P O-067, P-342 Barrera, L.A. A-036 Bevington, S P-432 Alehan, D P-395 Auray-Blais, C P-013, P-218 Barroso, M O-020, P-075, P-085 Bezrodnaya, AI A-039 Alehan, F P-296 Austin, S P-188 Barschak, AG P-227 Bhardwaj, N P-311 Alfadhel, M P-185 Austin, SL P-190 Barshop, B.A. O-026 Bhatia, KP O-071 Alfaiate, C P-316 Avila Arreguin, E P-254 Barshop, BA P-405, P-406 Bhattacharya, K P-191, P-310 Al-Hertani, W P-466 Avram, P. A-043 Barth, M P-069 Bialer, M O-031 Al-Hertani, WAH P-467 Aydin, A A-021, A-035, A-038, Barto, R P-016, P-088, P-089 Biancini, GB P-149, P-227, P-084 Ali, b P-269 A-040 Baruteau, J O-010 Biberoglu, G P-171, P-195, P-252 Alkuraya, FS P-185 Aydin, A P-008, P-143 Bastaki, F P-269 Biberoglu, Gursel P-271, P-140 Allen, RH O-022 Aydın, K P-258, P-302, P-477 Bastin, J O-070 Bichet, DG P-230 Alm, J P-067, P-068 Aydin, HI A-018 Battelino, T P-040, P-041, P-048 Bideci, Aysun P-140 Almeida, L A-033 Aylett, S-B P-383, P-387 Baumann, M P-485 Bie, A P-155 S172 J Inherit Metab Dis (2012) 35 (Suppl 1):S1–S182

Bierau, J P-193, P-095 Boyd, S P-314 Byrne, B P-244, P-245 Ceglrek, U P-424 Bigger, BW O-063, O-064, P-400 Boyunaga, O P-371 Bzduch, V A-027 Celato, Andrea P-359, P-369, Bigot, S O-010 Bozdemir, E A-044 Bzdúch, V P-353 P-377, P-481 Bijarnia, S A-029, P-109, P-028 Bradbury, M O-025 Cabello, JF P-046 Celik, G A-030 Bijarnia - Mahay, S P-119, P-187 Braga, LM P-117 Cabete, A P-470 Cerone, R P-047, P-065 Bisset, WM O-007 Braissant, O O-027, P-092, P-107 Cabrera-Luque, J P-134 Cerutti, A P-318 Bistué Millón, MB P-284 Brand, M P-251 Cadoudal, M P-388 Cerutti, M P-398 Bittar, C P-219 Brandão, O P-156 Caillaud, C P-222 Ceylaner, S P-143 Blank, RD P-044 Brands, MMMG P-221 Çakir, N P-419, P-343 Chabli, A P-094 Blasco, H P-396 Branson, H P-466 Çakmakçı, H P-479 Chabraoui, L P-010, P-222 Blasco, J P-450 Brasil, S P-127 Caldeira Araújo, H P-075 Chadefaux-Vekemans, B P-463 Blaser, S P-466, O-044 Brassier, A O-011, P-029, P-474 Calvin, J P-121 Chadha-Boreham, H P-281 Blau, N P-057, P-063, P-064, Braulke, T O-034, P-126 Calvinho, P P-470 Chahal, S P-414, P-418 P-065, O-005 Braulke, TB P-277 Cameron, BH P-408 Chaiwatanasirikul, K P-326 Blaud, N P-375 Breda Klobus, A P-186 Cameron, JM O-044 Chakraborty, P P-436 Bleakley, C P-397 Breen, C O-018, O-033, P-023, Campistol, J O-039 Chakrapani, A O-009, O-017, Bliksrud, YT P-473 P-078, P-209, A-019 Campos, FJ P-401 O-035, P-071, P-170, P-255, Blom, H O-021 Breitkreutz, J P-077 Campos-Xavier, AB P-391 A-020, P-300, P-417 Blom, HJ P-016, P-086, P-088, Brendel, C O-051 Canda, E P-207, P-267 Chakrapani, A B P-139 P-089 Brenner, O P-058 Candan, Ş A-015 Champigneulle, A P-068 Bobek-Billewicz, B P-317 Briand, G P-019 Canpolat, Mehmet P-103 Champion, H O-010 Boddaert, N O-009, P-029 Brignol, N O-065 Cansever, MŞ A-021, A-035, Champion, M O-012, P-074 Boelen, CCA O-005 Brill-Almon, E O-032 A-038 Champion, MP O-017, P-121, Boenzi, S O-030 Briones, P O-039, P-144, P-290, Cansever, MS P-008, P-079, P-143 P-300, P-305 Boenzi, SB P-006 P-327, P-332, Cansever, MS A-040 Chan, E P-209 Boespflug-Tanguy, O P-298 Brivet, M P-298 Cantarini, ME P-105 Chan, H P-422 Boey, CCM P-197 Broissand, C P-474 Carbone, F P-189 Chan, WH P-112 Bogo, MR P-161 Bromirski, M P-437 Cardoso, M T A-009, A-009 Chandler, RJ P-120 Bolgert, F P-459 Brookes, KE P-400 Carducci, C P-367, P-367, P-042, Chandoga, J A-027 bompaire, F P-458 Broomfield, A P-268, P-071 P-042 Chandrasehkar, HS O-071 Bompaire, F P-459 Broomfield, AA O-016, O-035, Carducci, Carla P-359, P-369 Chang, HH O-065 Bonafe, L. 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