Platform Presentations: Abstracts 101–153

Molecular Genetics and Metabolism 98 (2009) 1–16

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Platform Presentation 1: Lysosomal Storage Disorders Sunday, 30 August 2009 16:00-17:30 101 THE FIRST REPORT OF THE MPS VI CLINICAL SURVEILLANCE PROGRAM (CSP) C J Hendriksz1, V Valayannopoulos2, E L Teles3, E Miebach4, P Harmatz5, G M Pastores6, R Steiner7 1Birmingham Children's Hospital, Birmingham, United Kingdom, 2Centre de Référence des Maladies Métaboliques, Hôpital NeckerEnfants Malades, Paris, France, 3Hospital Sao Joao Dept Pediatria Alameda, Porto, Portugal, 4Universitäts-Kinderklinik, Mainz, Mainz, Germany, 5Children's Hospital Oakland, Oakland, CA, United States, 6New York University School of Medicine, New York, New York, United States, 7Oregon Health Science University, Portland, Oregon, United States Objectives: To evaluate clinical outcomes in the MPS VI CSP. Methods: The MPS VI (Mucopolysaccharidosis VI, Maroteaux-Lamy syndrome) CSP is a voluntary, multicenter, multinational, long-term observational program which tracks the specific clinical outcomes of patients with MPS VI. Cumulative clinical data collected from September 12, 2005 through to March 20, 2009 on 101 patients were analysed. Results: 101 patients (40 US and 61 EU) were enrolled in the MPS VI CSP, including 95 patients on enzyme replacement therapy (ERT) with galsulfase (Naglazyme®). One patient had a successful hematopoietic stem cell transplant (HSCT); one patient had non-grafting HSCT and is now receiving Naglazyme. The median age of the enrolled subjects is 14 years (range 0 - 60). Sixteen patients are under the age of 5 and 34 are aged 18 or older. Of the 95 patients who received ERT, 86 received the recommended Naglazyme dose of 1 mg/kg administered once a week, 5 patients received doses other than the recommended dose, and for the remaining 4 patients no dosing information was available. In 75 patients, enrollment into CSP occurred after ERT was started. Of the enrolled patients, 24 had received infusions while participating in a previous clinical study. Since the start of the program there have been 6 discontinuations from the CSP, 4 deaths, and 2 lost to follow up. A decrease in urinary glycosaminoglycans (uGAG) was seen after Naglazyme treatment in 47 evaluable patients, with uGAG results at baseline and at 1 or more follow–up visits. An increase in both height and weight, together with improved endurance as measured by 12 minute walk test and 3 minute stair climb test was observed. No changes, indicative of improvement or deterioration, were noted on examination of the cardiac, ophthalmological and audiology data. Antibodies were detected in 34/37 evaluable patients, with pre- and post- treatment serum antibody results. The mean time to sero-conversion was 5.6 months. No correlation between antibody level and changes in uGAG levels was observed. Based on medical history and MRI exams, 47 patients had cervical spinal stenosis or symptoms of spinal cord compression. A total of 44 serious adverse events were reported for 30 patients. Eleven patients experienced one or more Naglazyme infusion related reactions. Conclusions: This is the first report of the MPS VI CSP, which indicates that treatment with Naglazyme appears to provide clinical benefit and is well-tolerated. Cervical spinal stenosis appears to be a frequently observed pathology in MPS VI patients. Further reports on untreated patients, those given ERT and those with HSCT will provide information of the natural history of MPS VI and the impact of therapy, including ERT and/or HSCT, on disease course. 102 IMPACT OF ANTIBODY FORMATION FOR ENZYME REPLACEMENT THERAPY FOR LYSOSOMAL STORAGE DISEASES AND IMMUNE TOLERANCE INDUCTION FOR INFUSED ENZYME T Ohashi1,2, S Iizuka1, Y Eto3, H Ida1,2 1Dep. of Gene Therapy, Insititute of DNA Medicine, The Jikei University School of Medicine, Tokyo, Japan, 2Dep. of Pediatrics, The Jikei University School of Medicine, Tokyo, Japan, 3Dep. of Genetic Disease, The Jikei University School of Medicine, Tokyo, Japan Enzyme replacement therapies (ERT) for 6 lysosomal storage diseases including Gaucher disease, Fabry disease, Pompe disease, mucoplysaccharidosis type I, II and VI, are approved in developed countries. Although their efficacy was proven in many clinical trials, a couple of limitations were turned out. Among them, negative impact of neutralizing antibody to enzyme was one of major limitation in ERT. First, we compared the reduction of urinary globotriaosylceramide from human Fabry patients between seronegative and seropositive patients. Normalization of urinary globotriaosylceramide level in seropositive patients was significantly delayed or not achieved by ERT. The antibody which was developed during Fabry ERT has neutralizing activity toward purified enzyme in various degrees. Once enzyme was reacted with such antibodies, the cellular up take of enzyme was significantly reduced in Fabry fibroblasts and various organs in Fabry model mice. From these observations, we concluded that development of antibody during Fabry ERT had negative impact upon biochemical improvement of Fabry ERT. Moreover, the negative impact of antibody formation was also reported in ERT for mucopolysaccharidosis type I, VI and Pompe disease. Among then, antibody formation clearly affect the clinical outcome in infantile onset Pompe disease. To overcome these obstacles, we tried to induce immune tolerance to infused enzyme. Because of reason stated above, we choose alpha-Glucosidase (GAA) which is used for Pompe ERT. In this study, we tried two methods to induce immune tolerance. First, we administered anti-CD3 antibody. Anti-CD 3 antibody was reported to induce immune tolerance to infused coagulation factor VIII in hemophilia and approved for rejection of renal transplantation in human. We intravenously injected low and high dose of anti-CD3 antibody to Balb/c mice

followed by GAA immunization. Low dose anti-CD 3 successfully induced immune tolerance to enzyme but high dose of anti-CD 3 antibody did not. Although anti-CD 3 antibody induced immune tolerance, there are some side effect such as fever and chilling. Ideally, tolerance induction in ERT for lysosomal diseases should be long lasting and antigen specific. So, we tried oral tolerance induction, which might be safe and antigen specific. We orally administrated low and high dose of GAA to Balb/c mice and C57BL/6 mice, followed by GAA immunization. Both orally administered high dose and low dose of GAA successfully induced immune tolerance in both mouse strains. But the stronger immune tolerance was achieved in C57BL/6 mice by high dose oral GAA administration. We currently tested how long immune tolerance persists. In conclusion, parenteral administration of anti-CD 3 antibody and oral administration of GAA might be translated to clinics to induce immune tolerance ERT for lysosomal storage diseases. 103 THE FABRY OUTCOME SURVEY (FOS): 2-YEAR DATA SHOWING EFFECTIVENESS OF ENZYME REPLACEMENT THERAPY WITH AGALSIDASE ALFA IN CHILDREN U Ramaswami1, R Parini2, G Pintos-Morell3, G Kalkum4, M Beck4 1Addenbrooke's University Teaching Hospital, Cambridge, United Kingdom, 2San Gerardo Hospital, Monza, Italy, 3University Hospital Germans Trias i Pujol, Badalona, Spain, 4University of Mainz, Mainz, Germany Background: Long-term enzyme replacement therapy (ERT) in Fabry disease, a progressive X-linked lysosomal storage disorder, may potentially reduce the risk of premature death due to the cardiac and renal manifestations of the disease. Early signs of organ damage, such as increased left ventricular mass (LVM), and impaired renal function, may be present in children. However, limited data exist on the long-term effectiveness of ERT in patients <18 years of age. Methods: As of March 2009, the Fabry Outcome Survey (FOS), which provides long-term data on the natural history of Fabry disease and the effects of ERT with agalsidase alfa (Replagal®; Shire HGT), had enrolled 130 males and 145 females aged <18y, of whom 64 boys (median age at start of ERT, 12.6y; 10th–90th percentile, 6.6–17.1y) and 34 girls (median age at start of ERT, 15.2y; 10th–90th percentile, 9.7–17.1y) have been treated for at least 6m. Data were analysed at baseline and after 6, 12 and 24m of ERT. Results: ERT was well tolerated, and no IgE antibodies were detected. Of the 98 patients treated for at least 6m, data on the effectiveness of agalsidase alfa were available for 37 boys and 32 girls at 24m. Of these, 22 boys and 17 girls had data at baseline and 24m. Many of the children with signs and symptoms at baseline were symptom free at 24m. In children who were symptomatic at baseline, there were significant decreases in the prevalence of heat and cold intolerance, hearing abnormalities, gastrointestinal pain and disturbance, chronic pain and pain attacks at 12 and 24m of ERT (p< 0.01). Similarly, in both boys and girls with signs of disease at baseline (neurological, hearing-related and cardiac), the prevalence of signs was significantly reduced at 12 and 24m (p< 0.01). Baseline and 24m follow-up cardiac data (n=17) showed mean decreases in cardiac dimensions, both overall and in children stratified by sex and age at baseline (0–4, 5–9, 10–14, 15–17y) (p=NS). Early and sustained reductions in cardiac dimensions were demonstrated in 9 children with data at 6, 12 and 24m (p=NS). Baseline and 24m follow-up renal data showed renal function stabilization across all age groups. Conclusions: These data demonstrate the short- and medium-term efficacy of ERT with agalsidase alfa in children. Longer term follow-up is required to show whether the benefit of early treatment is maintained into adulthood. 104 BELGIAN FABRY STROKE STUDY (BEFAS): A NATIONAL, MULTICENTER, PROSPECTIVE STUDY ON THE PREVALENCE OF FABRY DISEASE IN YOUNG PATIENTS WITH STROKE F J Eyskens1, P P De Deyn2 1Metabolics and Endocrinology, CEMA, Antwerp, Belgium, 2Neurology, ZNA-Middelheim, Antwerp, Belgium Ischemic and hemorrhagic CVA occurs in Fabry disease, mainly due to small vessels disease and dolichoectasia of posterior circulation. The frequency of cerebrovascular events (stroke and transient ischemic attack or TIA) was reported higher in woman (27%) than in man (12%). Although the mean age at onset of cerebral vascular events was 28.8 years in males and 43.4 years in females, a TIA was reported in a boy aged 12 years and a woman of 25 years. Stroke recurrence is higher than in the general population and has a poor prognosis in general. Rationale of searching for Fabry disease (FD) in stroke patients: Stroke is the third potentially life-threatening complication of FD and has been described in 13 to 25% of Fabry patients. Recently, Rolfs et al. diagnosed FD in 4% of young patients with cryptogenic stroke. The incidence of stroke in Belgium is 1 in 18,975 inhabitants per year, of which 4% occurs before the age of 50 years and 10% between 50 and 60 years. If 4% of young stroke patients suffers from FD, as suggested by Rolfs’ study, this would imply that about 50 strokes per year in Belgium could be attributable to FD. This is in strong contrast to the current prevalence of FD in Belgium where only 55 patients are known to have this disease (1 in 180,000). Moreover, in the Middelheim Fabry Study (MiFaS) we were unable to identify any patient with FD in a population of 103 patients aged 60 or less with cryptogenic stroke. Therefore, the objective of a national, multi-center study was to prospectively assess the prevalence of FD in patients aged 16 to 60 years with TIA or stroke. Methods: Diagnosis of Fabry disease in patients with stroke: Hemizygous males: measurement of alpha-galactosidase A levels in blood spots, followed by enzymatic analysis in leucocytes. Genetic confirmation by genetic sequencing in male patients with deficient enzyme activitity. Heterozygous females: given the

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possibility of false normal enzymatic activity in females, DNA sequencing of the α-GAL gene was preferred. Inclusion criteria: First or recurrent stroke (ischemic or hemorrhagic) or transient ischemic attack < 60 years; blood sampling within 90 days after last event. Exclusion criteria: Known diagnosis of Fabry disease; unable to provide informed consent. Results: Over a period of 2 years, 1,000 patients with cryptogenic stroke were enrolled in the study. Four female patients, heterozygous for known mutations in the α-galactosidase gene were diagnosed. All patients had no signs of organ involvement besides stroke, TIA and/or headaches. Conclusion: The presented study shows a high prevalence of Fabry disease in women presenting with stroke at an early age. Selective screening for Fabry disease in this population at risk is indicated. 105 THE CANADIAN FABRY DISEASE INITIATIVE: A RANDOMIZED CONTROLLED TRIAL OF AGALSIDASE THERAPY IN FABRY DISEASE S M Sirrs1, M L West2, G Flowerdew2, K Lemoine2, D Bichet3, R Casey4, J Clarke5,6, C Auray-Blais6 1Adult Metabolic Diseases Clinic, Vancouver General Hospital, Vancouver, BC, Canada, 2University of Dalhousie, Halifax, Nova Scotia, Canada, 3University of Montreal, Montreal, Quebec, Canada, 4University of Calgary, Calgary, Alberta, Canada, 5Sick Kids Hospital, Toronto, Ontario, Canada, 6 University of Sherbrooke, Sherbrooke, Quebec, Canada Background: The Canadian Fabry Disease Initiative (CFDI) is a multicentre study which aims to study all patients in Canada with Fabry disease (FD) between ages 5 and 85 (Clinical Trial Registration protocol # NCT 00455104). We report clinical features of CFDI participants at 18-24 months from enrollment and information on mutation analysis from the CFDI. Methods: All Canadian patients with FD are eligible for inclusion. There are 3 patient cohorts: cohort 1a: those previously treated with enzyme replacement therapy (ERT) to continue ERT; cohort 1b: ERT naïve patients randomized 1:1 to agalsidase-beta 1.0 mg/kg q2wks or agalsidase-alfa 0.2 mg/kg q2wks; and cohort 1c: ERT naïve patients who do not meet the Canadian criteria for ERT or who refuse ERT. Variables studied include quality of life, pain, Mainz severity score index, eGFR, proteinuria, cardiac events, heart function, LVMI, incidence of stroke/TIAs, hospitalization and death to determine the natural history, and clinical outcomes with ERT. Sub-studies examining urine globotriaosylceramide (GB3) and antibody to agalsidase are included. Results: Over 2 years, 230 patients (91 males, 139 females, age 41.2±14.7 yrs) have been enrolled: 81 (35.2%) in cohort 1a, 36 (15.7%) in cohort 1b and 113 (49.1%) in cohort 1c. As expected males had more severe disease than females with a higher MSSI (30.4 vs. 17.7 p<0.0001), earlier age at diagnosis 25.5 vs. 34.4 yrs, p=0.0021), lower age (39.0 vs. 43.8 yrs p=0.05), lower eGFR (82.3 vs. 90.5 p=0.0146) and greater urine protein/creatinine (0.71 vs. 0.35 mg/mmol p=0.0294). In cohort 1b, females predominated (63.9%) over males (36.1%) but were equally distributed between the agalsidase treatment groups. Baseline

clinical features in cohort 1b did not differ in the two arms except for older age at diagnosis (42.9 vs. 21.9 p=0.0038) and age at baseline (52.0 vs. 39.4 p=0.0029) in the agalsidase-beta group compared with the agalsidase-alfa group. Mutation analysis (n=178) shows 31 different missense, 7 stop, 1 deletion and 6 unclassifiable mutations. Four of the mutations were novel. The A143P c.427G>C mutation of the large Nova Scotia kindred is most prevalent (40%). Urine GB3 levels were lower with ERT (1561.7 vs. 125.1) and with female gender. Outcomes data in cohort 1b show no differences at 18 months between patients on agalsidasealfa and agalsidase-beta. Further data collected over the next 5 years will inform governments as to the effectiveness of ERT in FD and form the basis for a decision about future ERT funding in Canada. Conclusion: In patients followed for 18 months, no significant differences in clinical outcomes can be determined between the two forms of agalsidase. 106 IMPLICATIONS OF INDUCED PLURIPOTENT STEM (IPS) CELLS ON MECHANISTIC STUDY FOR LYSOSOMAL STORAGE DISEASES XL Meng1,2, JS Shen1,2, S Kawagoe2, T Ohashi1,2, Y Eto2 1Department of Gene Therapy, The Jikei University School of Medicine, Tokyo, Japan, 2Research Center for Genetic Diseases, The Jikei University School of Medicine, Tokyo, Japan Mucopolysaccharidosis VII (MPS VII) is a lysosomal storage disorder caused by a deficiency in the enzyme activity of beta-glucuronidase. The mechanisms of the cellular and organ dysfunction relating to glycosaminoglycans accumulation are largely unknown. Induced pluripotent stem (iPS) cells are disease-specific pluripotent cells could be generated in vitro and differentiated to a variety of cell types for mechanistic studies. Since iPS cells have the pluripotency similar to embryonic stem (ES) cells, they are valuable tools for the study of early embryogenesis when disease-specific ES cells are not available or favored. The most severe type of MPS VII presents as hydrops fetalis indicating an embryonic developmental failure in utero. The murine model of MPS VII, gus mps/gus mps, also showed partial embryonic lethality possibly occurs before embryonic day 9.5. In order to study the role of beta-glucuronidase deficiency in early embryogenesis we generated iPS cells from MPS VII mouse (MPS-miPS) by retroviral transfection of Oct3/4, Sox2 and Klf4 to tail-tip fibroblasts. The MPS-miPS cells expressed ES cell markers and differentiated into all three germ layers. When subject to differentiation MPS-miPS cells showed significantly decreased efficiency of embryoid body (EB) formation. In MPS VII EBs, two-fold hyaluronan content was detected. The expression of the receptor for hyaluronan, CD44, was increased and a decrement in E-Cadherin expression was observed. When cultured with the conditioned media containing human beta-glucuronidase, efficiency of EB formation was improved. Results indicate that abnormal accumulation of hyaluronan play an important role in early embryogenesis possibly through regulating adhesion molecules.

Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Platform Presentation 2: Expanded Newborn Screening Sunday, 30 August 2009 16:00-17:30 107 LONG-TERM OUTCOME IN CHILDREN WITH INBORN ERRORS OF METABOLISM DETECTED BY EXPANDED NEWBORN SCREENING IN SOUTH-WEST GERMANY G Gramer1, P Burgard1, G Haege1, J Fang-Hoffmann1, F Trefz2, K O Schwab3, E Mengel4, U Wendel5, M Leichsenring6, G F Hoffmann1, M Lindner1 1University Hospital for Paediatric and Adolescent Medicine, Heidelberg, Germany, 2Children’s Hospital, Reutlingen, Germany, 3University Children’s Hospital, Freiburg, Germany, 4 University Children’s Hospital, Mainz, Germany, 5University Children’s Hospital, Düsseldorf, Germany, 6University Children’s Hospital, Ulm, Germany Newborn screening (NBS) using tandem mass spectrometry (MSMS) was started in Germany in 1998 and established nationwide in 2004. To date published data on the quality of screening processes and long-term outcome of patients detected are scarce. Process parameters until confirmation of diagnoses and start of treatment were assessed for the Heidelberg Newborn Screening Centre. Long-term outcome was evaluated by laboratory, clinical and psychological examinations. The study was approved by the Institutional Review Board of the medical faculty of the University of Heidelberg. 1,034,593 neonates were screened between 1998 and 2009. 407 neonates were referred for confirmatory analysis. The most common diagnoses were Phenylketonuira /mild Hyperphenylalaninemia (PKU/MHP) (191, 46.9%), MediumChain-Acyl-CoA-Dehydrogenase (MCAD) deficiency (85, 20.9%) and other Fatty acid oxidation disorders (FAOD) (31, 7.6%). The other disorders accounted for less than 5% of referrals individually. 142 patients could not be included in the study on long-term outcome (consent rejected 14, MHP 95, lost to follow-up 12, deceased 5, included in other studies 6, false positive 4, maternal disease 6). The deceased patients suffered from LongChain-Acyl-CoA-Dehydrogenase deficiency (2 patients), Multiple Acyl-CoA Dehydrogenase Deficiency (MAD, 1 patient), Tyrosinemia I (1 patient) and Nonketotic Hyperglycinemia (1 patient). Mean age at first blood sampling was 3.2 days (SD 3.2), at positive report from the screening lab 7 days (SD 5.4). Treatment was started at 16 days (SD 27), definite confirmation of diagnoses was obtained at a mean of 23 days (SD 34). Outcome data for patients suffering from PKU and fatty acid oxidation disorders are excellent so far. Patients with organic acidurias, MAD, maple syrup urine disease (MSUD) and galactosemia may show early symptoms and to a significant proportion various neurological or mental deficits. Variants/non-diseases are detected. Follow-up studies of patients detected in NBS are essential to prove the beneficial effect of newborn screening programs using MSMS. Only national or even international collaboration will provide sufficient patient numbers for rarer disorders. 108 NEWBORN SCREENING BY TANDEM MASS SPECTROMETRY: A COHORT STUDY COMPARING OUTCOME IN SCREENED AND CLINICALLY DIAGNOSED PATIENTS AT SIX YEARS OF AGE B Wilcken1,2, M Haas3, P Joy1, V C Wiley1, F Bowling4, K H Carpenter1, J Christodoulou1, D Cowley4, C Ellaway1, J M Fletcher5, E P Kirk6, B Lewis7, J McGill8, H Peters9, J Pitt9, E Ranieri5, J Yaplito-Lee9, A Boneh9 1The Children's Hospital at Westmead, Westmead (Sydney), New South Wales, Australia, 2 Paediatrics, University of Sydney, Sydney, NSW, Australia, 3University of Technology Sydney, Sydney, NSW, Australia, 4Mater Hospital, Brisbane, Qld, Australia, 5Adelaide Women's & Children's Hospital, Adelaide, SA, Australia, 6 Sydney Children's Hospital, Randwick, NSW, Australia, 7Princess Margaret Hospital, Perth, Western Australia, Australia, 8Brisbane Children's Hospital, Brisbane, Queensland, Australia, 9Murdoch Children's Research Institute, Melbourne, Victoria, Australia Background: Tandem mass spectrometry newborn screening is now widespread but there are no reports of the long-term overall clinical outcome. We assessed the clinical outcome at 6 years of age in an Australia-wide study. Methods: We included all babies with inborn errors other than phenylketonuria detected clinically or by newborn screening born from 1994 to 1998 (1,017,800, all unscreened) and 1998-2002 (461,500 screened, 533,400 unscreened) and recorded intellectual and physical abilities, school placement, other medical problems, growth, treatment, diet and hospital admissions. We analysed three patient groups: those presenting clinically or dying in the first 5 days of life, patients presenting later or diagnosed by screening, and those with disorders now thought substantially benign. Additionally we analysed separately medium-chain acyl-CoA dehydrogenase deficiency and all other disorders. Findings: Inborn errors, excluding phenylketonuria, were diagnosed in 116 of 1,551,200 unscreened babies (7.5/100,000 births) and 70 of 461,500 screened babies (15.2/100,000 births). Diagnostic rate was almost double in the screened cohort. Excluding medium-chain acyl-CoA dehydrogenase deficiency, 21 of 50 (42%) unscreened patients with metabolic disorders diagnosed after 5 days of life died or had a significant intellectual or physical handicap (1.35/100,000 population) compared with 2 of 28 (7%) of the screened cohort (0.43/100,000; odds ratio 3.1; 95% CI 0.73 – 13.32). On a population basis an extra 44 patients would be expected in the unscreened cohort. Considering the likely morbidity or mortality among the expected number of never-diagnosed unscreened patients there would be a significant difference. School placement was more favourable for screened patients. Growth distribution was normal in all cohorts, and other medical problems, diet, medications and other treatment were not different. Interpretation: Screening by tandem mass spectrometry provides a better outcome for

patients at 6 years, with fewer deaths and fewer clinically significant disabilities. 109 EXPERIENCE OF 13 YEARS USING TANDEM MASS SPECTROMETRY IN CHILE V Cornejo1, E Raimann1, JF Cabello1, A Valiente1 1INTA, University of Chile, Santiago, RM, Chile Since 1996 the Metabolic Diseases Laboratory (LEM) of INTA, University of Chile, has used Tandem Mass Spectrometry (MS/MS), as screening method for Inborn Errors of Metabolism. 19,753 blood samples on filter paper were analyzed, of these 6,885 (34.9%) correspond to samples from newborns and 12,868 (65.1%) to older patients with clinical symptoms. Of the total neonatal samples 2.7% were abnormal, whereas in older individuals 5.1% presented some alteration in the results. In 17.2% of the altered samples, a diagnosis was confirmed: 11 with Propionic Acidemia, 7 Methylmalonic Acidemia, 5 Isovaleric Acidemia, 11 Maple Syrup Urine Disease, 4 Citrullinaemia, 1 Ornithine-transcarbamylase Deficiency, 1 Argininosuccinic Aciduria, 3 Medium-chain AcylCoA dehydrogenase Deficiency (MCAD), 1 Long-chain AcylCoA dehydrogenase deficiency (LCAD), 1 Carnitine Palmitoyl Transferase Deficiency, 56 Phenylketonuria and 80 Hyperphenylalaninaemias, 6 Glutaric Aciduria Type 1, 3 Short-chain AcylCoA dehydrogenase deficiency (SCAD), 2 Urea Cycle Disorders, 1 Tyrosinaemia, 1 Ketotiolase Deficiency, 1 Very long-chain AcylCoA dehydrogenase deficiency (VLCAD) and 1 Homocystinuria. The diagnosis was confirmed with alternate biochemical or molecular methods. All the diagnosed patients were admitted to the Follow Up Program for Inborn Errors of Metabolism receiving the treatment according to their pathology. The MS/MS allows for the diagnosis of Aminoacidopathies, Organic Acidurias and defects of Fatty Acid Oxidation. 110 EXPANSION OF NEWBORN SCREENING FOR METABOLIC DISORDERS IN THE NETHERLANDS: RESULTS OF THE FIRST 2 YEARS G Visser1, M G M de Sain2, H J Blom3, A M Bosch4, C C A Boelen5, M F Mulder6, M E Rubio-Gozalbo7, M Williams8, M M C de Vries9, F J van Spronsen10 1Metabolic diseases, Wilhelmina Children's Hospital, Utrecht, Utr, Netherlands, 2Laboratory of Metabolic Diseases, Wilhelmina Children's Hospital, Utrecht, Utr, Netherlands, 3 Clinical Chemistry Metabolic Unit, VU Medical Center, Amsterdam, NH, Netherlands, 4Pediatrics, AMC, Amsterdam, NH, Netherlands, 5Pediatrics, LUMC, Leiden, ZH, Netherlands, 6Pediatrics, VU Medical Center, Amsterdam, NH, Netherlands, 7Pediatrics, UMCM, Maastricht, Limburg, Netherlands, 8Neonatology and Metabolic Disease, Erasmus MC, Rotterdam, ZH, Netherlands, 9Dept. of Metabolic and Endocrine Disorders, UMCN, Nijmegen, Gld, Netherlands, 10 Pediatrics, UMCG, Groningen, Gr, Netherlands Introduction: As of 1 January 2007 the Dutch newborn screening program for metabolic disorders in the Netherlands expanded from 1 (phenylketonuria (PKU)) to 14 disorders (biotinidase deficiency, galactosemia, glutaric aciduria type 1 (GA1), HMG-CoA-lyase deficiency, homocystinuria, holocarboxylase synthase deficiency, isovaleric acidemia, long-chain hydroxyacyl CoA dehydrogenase (LCHAD) deficiency (MTP), maple syrup urine disease (MSUD), medium chain acyl CoA dehdrogenase (MCAD) deficiency, 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiency, tyrosinemia type I, very-long-chain acyl CoA dehydrogenase (VLCAD) deficiency). Objective: To describe and to evaluate the results of the expansion of the Dutch newborn screening programme for metabolic disorders. Methods: Data were collected from all newborns with an abnormal metabolic screening result indicative for a metabolic disorder during the period 1 January 2007 to 31 December 2008. The prevalence of diagnosed metabolic diseases was compared with retrospective data from the Dutch Diagnosis Registry for Metabolic Diseases (DDRMD). Results: In 2007 and 2008 abnormal results were detected in 469 and 259 newborns respectively (0.26% and 0.14% of all newborns). The first 3 screening months resulted in a very high number of false positive results for detection of galactosemia, due to the choice of screening solely on galactose. Thereafter, this screening method was adapted by adding also screening on GALT. In addition, screening for tyrosinemia type 1 had to be stopped temporarily due to a high risk of false negative results by solely screening on tyrosine. In 75 and 102 (0.04% and 0.05% of all newborns) the diagnosis was a true positive (ratio of true to false positives 1:6.4 and 1:2.6 in 2007 and 2008 respectively). MCAD deficiency was most often found, with an incidence of 1:7500. Homocystinuria and holocarboxylase synthase deficiency are thus far not found. Conclusion: The number of patients diagnosed in both 2007 and 2008 was higher than predicted on the basis of retrospective data. This was largely due to detection of milder forms of enzyme deficiencies, which are not yet known to lead to clinical symptoms. Finally, the implementation plan was not properly developed resulting in the absence of structural financial backing for registration and long-term follow-up. Nonetheless, it is obvious that the expansion of the screening programme has lead to considerable health benefits as a result of early detection. 111 SPECTRUM OF MEDIUM CHAIN ACYL COA DEHYDROGENASE (MCAD) MUTATIONS IDENTIFIED FROM NEWBORN SCREENING OF 1.56 MILLION INFANTS FROM THE UK B S Andresen1,2, J Orton3, J Leonard3, C Dezateux3 1Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 2Research Unit for Molecular Medicine, Aarhus University, Aarhus, Denmark, 3Institute of Child Health, University College London, London, United Kingdom On behalf of the UK Collaborative Study of Newborn Screening (UKCSNS) for MCADD.

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Background: Newborn screening for medium-chain acyl-CoA dehydrogenase deficiency (MCADD) - the most common defect of fatty acid oxidation - has been implemented in a large number of countries. Homozygosity for the prevalent mutation c.985A>G is found in approx. 55% of those identified through newborn screening compared with 80% of those presenting clinically. Few studies report other genotypes encountered following newborn screening in ethnically diverse populations. We report the spectrum of MCAD mutations identified within a large scale prospective study carried out in the UK. Methods: Over a 4 year period (1.3.2004 – 28.2.2008), 1.56 million babies (approx. 80% White, 10% Asian [Indian subcontinent] and 5% Black) were screened at age 510 days using electrospray tandem mass spectrometry analysis of underivatised blood spot samples to quantitate octanoylcarnitine (C8). Presumptive positive infants (avg. C8 ≥0.5µmol/L) were investigated with repeat C8, urinary organic acids and tested for c.985A>G, with mutation screening of the entire MCAD gene for those not homozygous (HMZ) for c.985A>G. An independent expert panel assigned those with persistent biochemical abnormalities who were HMZ or compound heterozygous (HTZ) for known disease-causing genotypes such as c.985A>G or c.799G>A as having MCADD of ‘definite’ phenotype. Those homo- or hetero-allellic for genotypes not observed in clinically presenting patients were assigned as MCADD of 112 OUTCOME OF 3-MCC DEFICIENCY DIAGNOSED BY NEWBORN SCREENING G L Arnold1, P Suwannarat2, B Graham3, U Lichter-Konecki4, A M Bosch5, K Cusmano-Ozog6, G Enns6, E Wright7, J Thomas7, B C Lanpher8, N Owen8, M H Lipson9, R Cerone10, P Levy11, S Poetzsch12, L J Wong3, J A Neidich13, D Z Salazar13, A Dezsofi14 1Pediatrics, University of Rochester SOMD, Rochester, NY, United States, 2Children's Hospital of PIttsburgh, Pittsburgh, PA, United States, 3Baylor College of Medicine, Houston, TX, United States, 4Children's National Medical Center, Washington, DC, United States, 5University Hospital of Amsterdam, Amsterdam, Netherlands, 6Stanford SOM, Palo Alto, CA, United States, 7University of Colorado SOM, Denver, CO, United States, 8Vanderbilt SOM, Nashville, TN, United States, 9Kaiser Permanente, Sacramento, CA, United States, 10Istituto G. Gaslini, Genova, Italy, 11Albert Einstein COM, Bronx, NY, United States, 12Ottovon-Guericke University, Magdeburg, Germany, 13Quest Diagnostics, San Juan Capistrano, CA, United States, 14Semmelweis University, Budapest, Hungary Purpose: 3-MCC deficiency is detectable on expanded newborn screening (NBS), but there is significant uncertainty regarding the clinical significance of this disorder. We reviewed 110 cases of 3-MCC deficiency diagnosed after positive NBS in order to better understand the outcome of infants diagnosed with this disorder by NBS. Method: Coauthors were solicited on metab-l and submitted 110 cases. The majority of cases did not undergo either enzyme assay or molecular testing for confirmation of the diagnosis, but the 37 confirmed cases were examined in more detail. Results: Of confirmed cases, the mean BW was 3244gm, mean C5OH on NBS was 5.32 μM, and mean C5OH on follow-up was 3.50. Organic acid (OA) analysis noted elevated 3OH-isovaleric acid (3-HIVA) in 88%, and 3-methycrotonylglycine (3MCG) in 80%; detection of both metabolites was associated with higher C5OH levels and lower residual enzyme activity (p<0.05). Mean enzyme activity in blood or fibroblasts was 21.4% of normal. The NBS C5OH level was negatively correlated to residual enzyme activity (r=-0.58, p<0.002). Reported neonatal symptoms included tachypnea (1), ketosis (1), lethargy improved by protein restricted diet (1), and fussiness (1). In later infancy and early childhood, symptoms that were seen with illness or fasting included: hypoglycemia (1), lethargy and seizure with fever (1), altered mental status (1), elevated lactate (1), and behavior changes and tantrums with fasting (1). Poor feeding was reported in 6 and FTT in 1. Low free carnitine developed in 55%. Neurodevelopmental findings included Down syndrome (1), schizencephaly (1), microcephaly/seizures/MR (1), Asperger syndrome (1), autism (plus multiple VSD, 1), and gifted (1). Discussion: Although the majority of infants diagnosed with 3-MCC deficiency by NBS appear asymptomatic, these finding demonstrate a wide clinical variability. Long-term prospective follow-up is essential to better define any potential metabolic and neurological phenotype of this disorder, and to determine the extent to which newborn screening may alter outcome.

‘uncertain’ phenotype. Results: 152 children were confirmed as MCADD (birth prevalence: 1 per 10,000) of whom 82 (54%) were c.985A>G HMZ. 107 children were assigned as MCADD of ‘definite’ phenotype and 45 as MCADD of ‘uncertain’ phenotype. Seven newborns of Asian ethnicity were HMZ for a new c.946-6T>G mutation, not previously reported in patients with clinical illness, although studies of minigenes and patient cells showed a severe mRNA splicing defect. Conclusions: Newborn screening identifies children with persistent biochemical abnormalities and a diverse spectrum of mutations which differs between ethnic groups. Evaluation of persistent biochemical abnormalities in newborns from minority ethnic groups with few reported clinical cases may be difficult, especially where new genotypes or those not previously associated with clinical illness are identified. Initial screening with a panel of ‘frequent’ mutations is unlikely to be useful in multiethnic populations such as those encountered in the UK. Acknowledgement: Funded by the UK Department of Health and the National Screening Committee.

Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Platform Presentation 3: Mitochondrial Disorders Sunday, 30 August 2009 16:00-17:30 113 A NEW GENETIC CAUSE OF PRIMARY COENZYME Q10 DEFICIENCY S Rahman1,2, A J Duncan1, M Bitner-Glindzicz1, B Meunier3, H Costello1, I P Hargreaves4, L C López5, M Hirano5, C M Quinzii5, M I Sadowski6, A Singleton7, P T Clayton1 1Clinical and Molecular Genetics Unit, UCL Institute of Child Health, London, United Kingdom, 2MRC Centre for Neuromuscular Diseases, National Hospital for Neurology, London, United Kingdom, 3Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France, 4Neurometabolic Unit, National Hospital for Neurology, London, United Kingdom, 5Columbia University Medical Center, New York, NY, United States, 6Division of Mathematical Biology, National Institute for Medical Research, London, United Kingdom, 7Laboratory of Neurogenetics, National Institute of Aging, National Institutes of Health, Bethesda, MD, United States Background: Coenzyme Q10 is a mobile lipophilic electron carrier located in the inner mitochondrial membrane and shuttles electrons from complexes I and II to complex III in the respiratory chain. Defects of coenzyme Q10 biosynthesis represent one of the few treatable mitochondrial diseases. These disorders are clinically and genetically heterogeneous, and the genetic basis has been identified in only a minority of patients. Methods: We aimed to identify the genetic basis of primary coenzyme Q10 deficiency in a patient who presented with neonatal lactic acidosis and later developed multisystem disease including intractable seizures, global developmental delay, hypertrophic cardiomyopathy and renal tubular dysfunction. In view of the rarity of coenzyme Q10 deficiency, we hypothesised that the disease-causing gene might lie in a region of ancestral homozygosity by descent. We searched for regions of homozygosity by performing high density genome-wide SNP genotyping of genomic DNA using the Illumina HumanHap550 array. Results: Sixteen regions of homozygosity >1.5Mb were identified in the affected infant. Two of these regions included the loci of two of 16 candidate genes implicated in human coenzyme Q10 biosynthesis (COQ9 and CABC1). Sequence analysis demonstrated a homozygous stop mutation affecting a highly conserved residue of COQ9, and the transcript was subject to nonsense mediated decay. CABC1 sequence was wild type. Cultured skin fibroblasts from the patient had a coenzyme Q10 biosynthetic rate of 11% of normal controls, and accumulated an abnormal metabolite that we believe to be a biosynthetic intermediate. Site-directed mutagenesis targeting the equivalent residue in the yeast Saccharomyces cerevisiae abolished respiratory growth. Discussion: We report a new genetic cause of primary coenzyme Q10 deficiency and confirm that COQ9 is involved in the biosynthesis of coenzyme Q10 in humans. 114 HUMAN TESTIS-SPECIFIC PDHA2 GENE: DEMETHYLATION OF A CpG ISLAND IN THE OPEN READING FRAME CORRELATES WITH TRANSCRIPTIONAL ACTIVITY I Rivera1, A Pinheiro2, I Faustino2, M J Silva2, R Sá3, M Sousa3,4, A Barros4,5, I Tavares de Almeida2 1iMed, Faculty of Pharmacy University of Lisbon, Lisbon, Portugal, 2iMed, Faculty Pharmacy University Lisbon, Lisbon, Portugal, 3Laboratory Cell Biology, Institute Biomedical Sciences Abel Salazar, Porto, Portugal, 4Center Reproductive Genetics Alberto Barros, Porto, Portugal, 5Department of Medical Genetics, Faculty of Medicine, University of Porto, Porto, Portugal Background: DNA methylation is an important epigenetic modification that has profound roles in gene expression and, in particular, is thought to be crucial for regulation of tissue-specific genes in animal cells. The pivotal E1α subunit of human Pyruvate Dehydrogenase Complex, an essential and rate-limiting enzyme system in energy metabolism, is encoded by two distinct genes: PDHA1 gene, located on chromosome X is expressed in somatic tissues, whereas PDHA2 gene, located on chromosome 4, is exclusively expressed in postmeiotic spermatogenic cells. Several authors presented a strong correlation between the methylation status and transcriptional activity of several other testis-specific genes. Accordingly, the present study was designed to analyse the methylation pattern of PDHA2 gene in order to gain insight into the regulatory mechanisms governing its expression. Objective: Elucidate the role of DNA methylation as an epigenetic mechanism controlling the regulation of PDHA2 gene expression in human tissues, namely its repression in somatic tissues and its activation in testicular germ cells. Methods: Genomic DNA was isolated from human somatic tissues (circulating lymphocytes and gastric cells) and from testis, including isolated fractions haploid and diploid germ cells. After primer design with appropriate software, it was performed the sodium bisulfite PCR sequencing of the PDHA2 promoter and coding regions. Total RNA of the same tissues was isolated, reverse transcribed and PDHA2 transcript was amplified with specific primers and analysed by agarose gel electrophoresis. Results and Conclusion: The analysis of the genomic sequence of the PDHA2 gene revealed the presence of 61 CpG sites whose distribution matches the criteria for the presence of two CpG islands. Sequence analysis of both CpG islands upon bisulfite treatment displayed several differences, either between islands or among tissues. In particular, the methylation pattern one of the CpG islands revealed a perfect correlation with transcriptional activity of the PDHA2 gene either in testis or in somatic tissues. Surprisingly, it is the full demethylation of the CpG island located in the coding region that seems to play a crucial role upon PDHA2 gene transcription in testis. Work supported by FCT (SFRH/BD/31264/2006; SFRH/BD/23616/2005;

POCI/SAU-MMO/57052/2004; POCI/SAU-MMO/60709/60555/59997/2004; UMIB) 115 A NON-ENZYMATIC FUNCTION OF 17-BETA-HYDROXYSTEROID DEHYDROGENASE TYPE 10 IS REQUIRED FOR MITOCHONDRIAL INTEGRITY AND CELL SURVIVAL J Zschocke1,2, K Rauschenberger1, K Schöler1, J O Sass3, S Sauer4, Z Djuric5, C Rumig6, N I Wolf4,7, J Okun4, S Kölker4, H Schwarz8, H Runz1, A Nümann1, N Shafqat9, K L Kavanagh9, G Hämmerling6, R J A Wanders10, J H P Shield11, U Wendel12, P Nawroth5, G F Hoffmann4, C R Bartram1, B Arnold6, A Bierhaus5, U Oppermann9, H Steinbeisser1 1Inst Human Genetics, Heidelberg Univ, Germany, 2 Human Genetics, Medical Univ Innsbruck, Austria, 3Univ Children's Hospital, Freiburg, Germany, 4Dept. Pediatrics, Heidelberg Univ, Germany, 5Dept. Medicine I, Heidelberg Univ, Germany, 6German Cancer Res Centre, Heidelberg, Germany, 7VU Univ Med Centre, Amsterdam, Netherlands, 8Max Planck Inst Dev Biol, Tübingen, Germany, 9Structural Genomics Unit, Oxford Univ, United Kingdom, 10Acad Med Center, Amsterdam Univ, Netherlands, 11Royal Hosp Sick Children, Bristol, United Kingdom, 12Univ Children's Hospital, Düsseldorf, Germany 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MHBDD) is generally thought to be primarily a disorder of isoleucine metabolism. However, the clinical picture of progressive neurodegeneration usually without metabolic crises is markedly different from other classical organic acidurias. The disease-causing gene is HSD17B10 and encodes 17-beta-hydroxysteroid dehydrogenase type 10 (HSD10), a protein also implicated in the pathogenesis of Alzheimer disease. Here we report that clinical symptoms in patients with MHBDD are not correlated with residual enzymatic activity in vivo, confirmed through extensive in vitro analyses of wild type and mutated HSD10. Loss-of-function and rescue experiments in Xenopus embryos and cells derived from conditional Hsd17b10-/- mice demonstrate that a property of HSD10 independent of its enzymatic activity is essential for structural and functional integrity of mitochondria. Impairment of this function in neural cells causes apoptotic cell death whilst the enzymatic activity of HSD10 is not required for cell survival. Our findings indicate that the symptoms in patients with mutations in the HSD17B10 gene are unrelated to accumulation of toxic metabolites in the isoleucine pathway and, rather, related to the disturbance of a non-enzymatic mitochondrial function. MHBD (HSD10) deficiency is more appropriately regarded as a primary mitochondrial disorder, and alternative therapeutic approaches to an isoleucinerestricted diet are required. Secondary HSD10 dysfunction e.g. due to its interaction with amyloid beta may also play an important role in other neurodegenerative disorders such as Alzheimer disease. 116 METABOLOMIC ANALYSIS OF DILATED CARDIOMYPOATHY AND ATAXIA (DCMA) SYNDROME REVEALS ALTERATIONS IN OXIDATIVE, ENERGY-PRODUCING PATHWAYS F F Snyder1,2, D S Sinasac2, E Fung2, S D Hodges2, K B Mantik2, A Parenta2, D M Scheuchner2, R E Casey1, A Khan1, F P Bernier1 1Medical Genetics, University of Calgary, Calgary, Alberta, Canada, 2Biochemical Genetics Lab, Alberta Children's Hospital, Calgary, Alberta, Canada Metabolomics aims to utilize undirected, global metabolite profiling in physiological fluids to identify characteristic patterns that both differentiate, and inform about, various biological states. Here we apply a metabolomic survey to DCMA, a recently described autosomal recessive syndrome in the Canadian Dariusleut Hutterites, characterized by dilated cardiomyopathy and cerebellar ataxia and increased plasma and urine 3-methylglutaconic and 3-methylglutaric acids. Additional variable features of DCMA include growth failure, mild developmental delay and male genital anomalies. We previously mapped and identified a common splice-site mutation within the DNAJC19 gene in all identified Hutterite DCMA patients (Davey et al. J. Med. Genet. 2006, 43; 285). The DNAJC19 protein is an ortholog of yeast Tim14 and therefore also likely functions as a member of the protein import translocase of the inner mitochondrial membrane. Although we would predict that mutations altering the function of DNAJC19 would affect mitochondrial protein import, and hence general mitochondrial function, direct evidence for mitochondrial deficits in DCMA has proven elusive. Cellular ATP pools and adenylate energy-charge ratios in fibroblasts from DCMA patients are unchanged from controls. Similarly, a mitochondrial function-dependent, fibroblast proliferation assay also showed no differences between DCMA and controls. Using a principal component analysis of metabolite profiles that included 35 plasma acylcarnitines and 110 urine organic acids, however, revealed nearly complete class segregation between DCMA patients and controls. Loading plots identified minor but statistically significant increases in C12, C14, and C16 long chain acylcarnitine species in DCMA patients (p=<0.01). In addition, significant increases in tricarboxyllic-acid-cycle intermediates, principally malic, fumaric and succinic acids, also contributed to the separations between DCMA and controls (p=<0.001). Overall, the greatest metabolite changes identified in DCMA patients were found in those TCA cycle intermediates that are substrates for enzymatic steps that are either energetically neutral (i.e., reversible) or, as in the case of malate dehydrogenase, that are energetically unfavorable, with a positive free energy. These observations constitute the first evidence of impairments in oxidative, energy-producing pathways in patients with DCMA. 117 HEALTHY BABY GIRL BORN FOLLOWING PRE-IMPLANTATION GENETIC DIAGNOSIS FOR MITOCHONDRIAL DNA M.8993T>G MUTATION D R Thorburn1,2, L Wilton3, S Stock-Myer4 1Mitochondrial & Metabolic Research, Murdoch Childrens Research Institute, Melbourne, VIC, Australia, 2Department of Paediatrics, University of Melbourne, Melbourne, VIC, Australia, 3Genetic &

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Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Molecular Research Laboratory, Melbourne IVF, Melbourne, VIC, Australia, 4 Melbourne IVF, Melbourne, VIC, Australia We describe the use of Pre-implantation Genetic Diagnosis (PGD) for prevention of mitochondrial disease caused by the m.8993T>G mutation. This maternally inherited mutation results in Leigh Disease when the mutant DNA is in high proportion in the cell (high mutant load). The proband died at 2 years of age with a m.8993T>G mutant load of >95% in various tissues. The healthy mother had a blood mutant load of 35% and had prenatal diagnosis in two subsequent pregnancies. On both occasions CVS mutant load was >95% and the pregnancies were terminated. In order to diagnose the embryos, a quantitative fluorescent PCR-RFLP test was used to determine the mutant load from a number of different cell types. This involved the simultaneous amplification of mitochondrial and nuclear DNA from a single cell. The mitochondrial DNA was amplified using primers surrounding the mutation followed by restriction enzyme digestion of the fluorescently labeled products. Nuclear DNA was co-amplified using primers to determine gender of the embryos, and to confirm the absence of any maternal (or other) contamination. Once this test was established, it was used over two cycles of IVF and PGD to diagnose ten embryos. Of these ten embryos, six had very high mutant loads and it was predicted that they would result in affected children if implanted. Three embryos had a moderate to low mutant load (30-40%) and were predicted to have a low risk of disease. The final embryo had an extremely low mutant load of 2.5% and this embryo was transferred resulting in a pregnancy and a healthy baby girl with a mutant load of approximately 4% in cord blood. In addition to diagnosing the embryos, we were able to determine the mutant load in an additional 7 oocytes, as well as examine a number of individual cells from the affected embryos to determine if the mutant load was consistent between all cells (blastomeres) arising from the same embryo. A total of 54 cells could be tested from the 10 embryos and the mutant load was consistent between all sister blastomeres, strongly supporting the concept that the mutant load measurement from a single blastomere is representative of the entire embryo. From all the oocytes and embryos tested, 12/17 (70%) had a very high mutant load, showing that PGD gave this couple a much greater chance of having a healthy child than they would have had otherwise. This is only the second reported live birth following PGD for a mitochondrial DNA mutation, and the only reported study looking at the distribution of mutant load of m.8993T>G between sister blastomeres. We conclude that PGD can be an extremely useful reproductive option for carriers of mitochondrial DNA mutations. 118 TARGETED GENE ANALYSIS OF MITOCHONDRIAL DISORDERS BY NEXT GENERATION SEQUENCING V Vasta1, S B Ng2, E H Turner2, J Shendure2, SH Hahn1,3 1Seattle Children's Research Institute, Seattle, WA, United States, 2Genome Science, University of

Washington, Seattle, WA, United States, 3Pediatrics, University of Washington, Seattle, WA, United States Background: Mitochondrial disorders are the most common group of metabolic disorders with an estimated minimum prevalence of 1 in 5,000. The variability in clinical presentation and underlying causative mutations, either in nuclear or mitochondrial genes, make the diagnosis very challenging. There is no reliable diagnostic screening or diagnostic biomarker available that is both sensitive and specific in all cases of mitochondrial diseases. Mutations have been found in approximately 150 nuclear genes in patients with mitochondrial disorders; however, a large number of nuclear genes causing diseases are still unknown. Here, we developed a novel targeted genome re-sequencing approach to develop a comprehensive clinical diagnostic tool for mitochondrial disorders. Methods: Simultaneous sequencing of the entire mtDNA genome and the exons of 364 nuclear genes, which include the genes with mutations previously identified in patients and candidate genes possibly related to mitochondrial disorders, was performed using sequence capture technology coupled to next-generation sequencing by Illumina Genome Analyzer. The total target size is 0.6 Mb, for over 3000 exons from 364 genes and the entire mtDNA genome (16.6Kb). A custom array was designed containing 244,000 oligos. The targeted sequences were tiled at a very high density (i.e. 40 probes per 100 bp intervals). One HapMap sample and two positive control samples were analyzed. Read-lengths of up to 36 bp were obtained with perbase accuracies on the order of 99%. The sequence reads were aligned to the human reference genome using Mapping Quality scores (Maq) software. Results: The majority of the targets were covered by sequencing reads. A coverage of ≥8X with quality score ≥30 was observed for 92% of the targets. A variable range of coverage was achieved across the targeted areas and a small portion of targets (5%) was not covered by sequence reads. Known polymorphisms in HapMap samples were correctly identified. All pathogenic mutations blindly tested in positive samples were 100% concordant. Of the variants identified, 95% were present in dbSNP while 5% represented new variations. The non-synonymous new variants were all in heterozygote status and mostly predicted to be benign except a few genes. The depth of sequencing coverage of mtDNA was extremely high allowing the detection of low levels of polymorphisms, indicating that it may be feasible to detect pathogenic mtDNA mutations in the presence of low level heteroplasmy. Conclusion: The use of next generation sequencing technology holds a great promise as a tool for screening mitochondrial disorders. Interpreting the unknown alterations within the candidate genomes may be challenging. Technical issues with uniformity and coverage of targeted regions require further improvement. Given the current limit of diagnostic testing, as the number of genes potentially responsible for mitochondrial disorders is vast, this new diagnostic approach will allow the identification of new culprit genes in patients, thus expanding and re-defining the spectrum of mitochondrial disorders.

Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Platform Presentation 4: Various Inborn Errors of Metabolism Tuesday, 1 September 2009 13:30-15:30 119 IMPACT OF INBORN ERRORS OF METABOLISM ON PAEDIATRIC MORTALITY: A POPULATION BASED STUDY H Goel1, A Lusher2, A. Boneh1 1Victorian Clinical Genetic Services, Murdoch Children Research Institute, Royal Children's Hospital, Melbourne, Australia, 2Health Information Services, Royal Children’s Hospital, Melbourne, Australia Background: Inborn errors of Metabolism (IEM) are an important group of paediatric disorders, causing significant morbidity and mortality. Despite the recognition of the impact of specific disorders on mortality (e.g. urea cycle disorders), there is little population based information about their impact on paediatric mortality. We present data on paediatric mortality due to IEM, over 10 years, in Victoria, Australia. Methods: Patients with IEM are managed by a centralised service at the Victorian Clinical Genetics Services and the Royal Children’s Hospital. Information about these patients was retrieved from databases of these organisations, using the OMIM and the ICD-9-CM and ICD-10-AM coding systems, respectively. Paediatric mortality data (between 1997 to 2006) were retrieved from the Victorian Perinatal Data Collection Unit (http://health.vic.gov.au/perinatal). Information was cross validated between the databases. Only patients who were residents of Victoria were included. Patients were stratified according to their age at death: neonatal (birth weight >1000gm [1997-2000] or gestation >32 weeks [2001-2006]), 29-364 days, 14, 5-9 and 10-14 years of life. Results: Between 1997-2006, 100 children died of an IEM, including 24 (24%) neonates. Post neonatal child mortality in Victoria (excluding accidental, non accidental and acquired diseases,) averaged at 61/year. In average, 7.6 post-neonatal deaths/year were due to IEM (12.4 %). Neonatal mortality, excluding obstetric or infectious causes, averaged at 43/year. In average, 2.4 neonatal deaths/year were due to IEM (5.5% of the total/year). Over all, the most common mortality causing IEM were the mitochondrial respiratory chain disorders (MRCD), causing 36/100 deaths (36%). The most common age-related mortality causing IEM were: MRCD in the neonatal period; lysosomal storage disorders (LSD) at 29-364 days; MRCD at 1-4yrs, LSD at 5-9yrs, and MRCD at 10-14yrs. Discussion: This is first population based study assessing the impact of IEM on Paediatric mortality. Despite their rarity, IEM are a major cause of Paediatric mortality, ranging, depending on age, between 5.5-12.4% of non-environmental causes. Our results suggest that MRCD have been underestimated as a major cause of child mortality (or possibly under-diagnosed), surpassing urea cycle disorders and organic acidaemias. These results represent the minimum impact of IEM on paediatric mortality as it is most likely that some patients counted on the overall Victorian mortality database, have not been appropriately diagnosed with IEM. 120 LENTIVIRUS MEDIATED NEONATAL GENE THERAPY FOR SEVERE INFANTILE HYPOPHOSPHATASIA S Yamamoto1,2, H Orimo1, T Matsumoto1, S Narisawa3, J Millan3, T Shimada1 1 Biochemistry and Molecular Biology, Nippon Medical School, Bunkyouku, Tokyo, Japan, 2Pediatric Dentistry, Nihon Univ. Graduate School Search of Dentistry at Matsudo, Matsudo, Chiba, Japan, 3Burnham Institute for Medical Research, La Jolla, CA, United States Hypophosphatasia (HPP) is an inherited skeletal disease caused by mutations of the gene encoding tissue-nonspecific isozyme of alkaline phosphatase (TNALP) and features rickets or osteomalacia. The clinical severity of HPP is varies widely; the most severe to the mildest forms are prenatal, infantile, childhood, adult, and odontohypophosphatasia. TNALP is attached to the outside plasma membrane via a GPI anchor and expressed in mainly liver, bone and kidney. Absence of TNALP activity results in extracellular accumulation of natural substrates such as inorganic pyrophosphate (PPi), pyridoxal 5′phosphate (PLP), and phosphoethanolamine (PEA). Since PPi at high concentrations is a strong inhibitor of hydroxylapatite crystal growth, mineralization of systemic bones and teeth are inhibited in HPP patients. Pyridoxine-responsive seizures are observed in some severe cases. There is no established medical treatment. TNALP knock-out mice phenotypically mimic to infantile HPP. They are born with a normally mineralized skeleton but develop apparent richets and die between day 12 and 16, suffering severe skeletal hypomineralization and epileptic seizures. Recently, Millan et. al., demonstrated that TNALP knockout mice could be treated by daily subcutaneous injection of a bonetargeted form of TNALP in which a deca-aspartate sequence is linked to the C terminal end of soluble TNALP. Based on this preclinical data, clinical trials of enzyme replacement therapy for patients with adult and infantile HPP initiated. Here, we examined a feasibility of gene therapy to treat HPP model mice using lentiviral vector expressing a bone-targeted TNALP. The vector (5.0x107 TU in 100 µl) was injected into the jugular vein of newborn HPP mice at 0 and 3 days after birth. The ALP activity in plasma of treated animals increased to extremely high levels (33.0 U/ml vs normal control 0.14 U/ml at 30 days old) and persisted for at least 6 months. Lentivrus vector preferentially transduced the liver, heart, and lung. No seizure was observed in treated mice. Localization of ALP activity in bone tissues was demonstrated by histochemical staining. X-ray evaluation and histological examination showed that mineralization in bones is significantly improved by gene therapy. Most strikingly, treated HPP mice lived for more than 8 months with normal physical activity and healthy appearance, while non-treated mice died by 20 days. These results clearly showed, for the first time, that severe infantile HPP in TNALP knockout mice can be treated by a single injection of lentiviral vector during the

neonatal period. We suggest that lentiviral mediated neonatal gene therapy is an important option to treat hypophosphatasia. 121 INTERIM RESULTS OF ADULTS AND INFANTS WITH HYPOPHOSPHATASIA TREATED WITH BONE -TARGETTED HUMAN RECOMBINANT ALKALINE PHOSPHATASE ENB-0040 C R Greenberg1, A Mhanni1, D Catte2, P Kishnani3, T Weber3, M McGinn4, S Craig5, N Salman6, M Hamdan7, W McAlistair8, H Landy9, M P Whyte10 1University of Manitoba, Winnipeg, Manitoba, Canada, 2Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada, 3Duke University, Durham, North Carolina, United States, 4Royal Belfast Hospital, Belfast, United Kingdom, 5Royal Maternity Hospital, Belfast, United Kingdom, 6Tawan- Johns Hopkins Hospital, Dubai, United Arab Emirates, 7Tawan-Johns Hopkins Hospital, Dubai, United Arab Emirates, 8Washinton University, St. Louis, Missouri, United States, 9Enobia Pharma, Cambridge, MA, United States, 10Shriners Hospital for Children, St Louis, Missouri, United States Hypophosphatasia (HPP) is a heritable form of rickets or osteomalacia caused by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP). Severity reflects age at presentation and ranges from stillbirth with profound rickets to osteomalacia with pathological fractures in adults. There is no established medical treatment. ENB-0040 is a bone-targeted, human recombinant TNSALP fusion protein that preserved skeletal mineralization and survival in a mouse model of lethal HPP1. Clinical trials began in Winnipeg in the summer of 2008. Study 1 was a 1 month, open-label protocol and tested repeat doses. Adult patients received an IV ENB-0040 infusion of 3 mg/kg followed by weekly SC injections of 1 mg/kg (n=3 Winnipeg patients) or 2 mg/kg (n=3 U.S. patients). Study 2 is a 6-month, open-label protocol and involves 5 patients with potentially lethal HPP (ages 6-36 mo). Herein, we report the results of 12 weeks of treatment with ENB-0040 in the first 5 affected infants with HPP to be enrolled and treated (Study 2). Three infants with profound rickets have shown marked radiographic, respiratory and functional improvement, 1 has shown respiratory and functional improvement and 1 has shown functional gains. There have been no drug-related serious adverse events (SAEs) and no patient has developed anti-ENB-0040 antibodies. SC injections have been well tolerated and appear to provide a viable mode of administering enzyme replacement therapy in patients with HPP. This study is ongoing and longer term treatment studies are about to begin in children and adults to assess the effect of EB-0040 on bone mineralization in these populations. 1 Millan JL et al., J Bone Mineral Res 2008; 23: 777 Sources of Support: Enobia Pharma, Montreal Canada 122 HYPERLIPIDEMIA AFFECTS THE EFFICACY OF THE ACE-INHIBITORS ON MICROALBUMINURIA: AN ITALIAN MULTICENTER STUDY ON GLYCOGEN STORAGE DISEASE TYPE 1 D Melis1, M Cozzolino1, V Gaudieri1, F. Balivo1, R Parini2, M Rigoldi2, M. Giovannini3, S Paci3, C. Dionisi Vici4, F Deodato4, C Castana5, M Caserta5, G Cappuccio1, A.B. Burlina6, L Giordano6, R Della Casa1, G Andria1, G Parenti1,7 1 Department of Pediatrics, Federico II University, Naples, Italy, 2Ospedale S. Gerardo, Monza, MI, Italy, 3Ospedale S. Paolo, Milano, Italy, 4Ospedale Pediatrico Bambino Gesù, Roma, Italy, 5Ospedale Civico e Benfratelli G. Di Cristina e M. Ascoli, Palermo, Italy, 6Università di Padova, Padova, Italy, 7Telethon Institute of Genetics and Medicine (TIGEM), Napoli, Italy Glycogen storage disease type 1 (GSD1) is caused by the deficiency of either glucose-6-phosphatase (type Ia) or glucose-6-phosphate translocase (type 1b). Renal damage is one of the most serious and frequent complications of GSD1. Although the pathophysiology of renal disease in GSD1 is still partially understood, its natural history is well known. Like in diabetic nephropathy, a period of “silent” glomerular hyperfiltration evolves, through a phase of microalbuminuria, to proteinuria and ultimately to chronic renal failure. Aim of the study: to investigate the correlations between metabolic control, progression of renal disease and efficacy of ACEinhibitors. Patients and Methods: Sixty-one patients (41 GSD1a, 20 GSD1b) were enrolled from six Italian referral centers for metabolic disease. A retrospectiveprospective observational, multicentre study was performed over a period corresponding to the past 15 years of follow-up. The following parameters were analyzed: glomerular filtration rate (GFR), microalbuminuria and proteinuria, glucose, cholesterol, triglycerides, bicarbonate, lactic acid and uric acid serum levels. The MannWhitney test was used to compare the evolution of renal damage in patients who started and who did not use ACE-inhibitors; the Wilcoxon rank and the chisquare tests were used to analyze the evolution of each parameter of renal damage and its relation to metabolic control, respectively. Results: A significant improvement of GFR was observed only during the first year of ACE-inhibitors treatment (p=0.04). No significant effect was observed on both microalbuminuria and proteinuria. The lack of an improvement of microalbuminuria during ACE-inhibitors treatment was associated to the presence of severe hyperlipidemia (chi2 6.6, p=0.01). Moreover, among the parameters of metabolic control, serum glucose levels inversely correlated with GFR (p=0.01); triglyceride and cholesterol serum levels correlated with the rate of progression of microalbuminuria (p=0.0001; p=0.02, respectively) and proteinuria (p=0.001). Conclusions: The results of the present study suggest the key role of metabolic control on the progression of renal damage in GSD1. According to our results, the presence of dyslipidemia seems to affect the efficacy of ACE-inhibitors in GSD1 patients. Acknowledgment: The study was supported in part by the Italian Agency of Drug (AIFA).

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123 GALACTOSAEMIA, A SINGLE GENE DISORDER WITH EPIGENETIC CONSEQUENCES D Coman1, D Murray2, J Byrne3, P Bagaglia2, P Doran2, E P Treacy1 1National Centre for Inherited Metabolic Disorders, Childrens University Hospital, Dublin, Ireland, 2 UCD CRC, Mater Misericordiae Hospital, Dublin, Ireland, 3Dublin-Oxford Glycobiology Lab, Conway Institute, UCD, Dublin, Ireland Long term outcomes of Galactosaemia (Gal) remain disappointing. It is unclear if the complications result mainly from prenatal-neonatal toxicity or if there is continuous neurodegeneration related to persistent glycoprotein and glycolipid synthesis abnormalities. In a 25 year outcome analysis of sib pairs diagnosed and treated at our centre following newborn screening, we have noted a very high complication rate with the suggestion of progressive neurodegeneration in a number of highly compliant patients (J Hughes et al, 2009). Using gene Affymetrix U133A plus 2.0 expression arrays we identified the gene expression profile of T lymphocytes isolated from 3 adult Irish Gal patients (Q188R/Q188R) and one paediatric African patient (S135L/S135L), maintained on a galactose restricted diet ( intake < 30 mg/day). Compared with controls, analysis showed significant dysregulation of multiple cell signalling pathways including MAPK signalling, signalling regulating the actin cytoskeleton, focal adhesion and ubiquitin mediated proteolysis. A number of significantly altered genes (p < 0.05) were shortlisted for further investigation. These included among others SPARC (osteonectin), a protein important to bone calcification and S100A8 (S100 calciumbinding protein), a major calcium and zinc binding protein both highly upregulated in T lymphocytes of the Gal cohort. Quantitative real time PCR independently confirmed upregulation of these genes. Comparing patients with severe vs. mild CNS outcomes expression array analysis also displayed aberrations in several pathways including MAPK signalling, regulation of actin cytoskeleton, calcium signalling and cell adhesion. The N-glycan serum and IgG glycosylation status of 9 treated Gal patients was compared with healthy control serum and IgG using a newly developed quantitative high-throughput analytical HPLC platform. Increased levels of agalactosylated and monogalactosylated structures and decreases in digalactosylated structures in patients were identified and quantified. Tri- and tetra-antennary glycans present in the healthy serum glycome were undetectable in many of the patients. We propose that the persistent abnormal glycosylation of serum glycoproteins seen in treated patients with the concurrent microarray data indicates persisting metabolic dyshomeostasis and gene dysregulation in ‘treated’ Galactosaemia. Strict restriction of dietary galactose is clearly life saving in the neonatal period; long term severe galactose restriction may contribute to the ongoing systemic abnormalities observed. Glycan profiling may prove useful in patient monitoring for dietary changes. 124 LIVER NETWORK PATHWAY DIFFERENCES AT GESTATION DAY 19 IN A MOUSE MODEL OF AN INBORN ERROR OF METABOLISM, GKD, SUGGESTS A MOONLIGHTING FUNCTION FOR THE GK PROTEIN N Maclennan1, A Presson1, S Horvath1, E RB McCabe1 1UCLA, Los Angeles, CA, United States Glycerol kinase deficiency (GKD) results in an increase in glycerol. The dogma is that maternal enzyme should protect the fetus in utero by removing glycerol. Work from us and others suggest the GK protein has moonlighting functions beyond its enzyme activity. We proposed to identify liver gene expression and networks disrupted by Gyk KO prior to birth to determine if network alterations are seen prenatally, which would suggest moonlighting functions beyond metabolizing the small molecule, glycerol. We performed microarray expression analysis on embryonic day (e) 19 KO mouse pup livers and compared gene expression levels to those of dol3 livers. Gene filtering (dChip) identified 650 differentially expressed, mapped genes (1.2 fold change) at e19. Ingenuity Pathways Analysis (IPA) revealed differential gene expression in novel network pathways at e19. The top enrichment category for e19 liver expression was apoptosis (p=2.8E-5). Pathways significantly altered at e19 between WT and KO were metabolic disease, apoptosis and cell cycle, and cell to cell signaling and interaction (p=3.4E-14). Gyk was in the metabolic disease and cellular development pathway (p=1.9E-12) at e19. Glucocorticoid receptor signaling and protein ubiquitination were significant canonical pathways at e19 (p=3.8E-2). IPA confirmed prenatal alterations of several known postnatal network pathways. Intersection of e19 and dol3 KO networks revealed similarities and differences in hepatic gene expression in an apoptosis canonical pathway. Plasma glycerol levels were significantly different between KO and WT at both e19 and dol3, but e19 KO glycerol did not exceed dol3 WT levels. In conclusion, we show enrichment of apoptosis genes and glucocorticoid receptor signaling, proposed moonlighting functions of GK in prenatal life in Gyk KO mice. Plasma glycerol levels at e19 and dol3 suggest maternal enzyme is protecting the fetus by removing glycerol, but not protecting the fetus from loss of GK’s moonlighting functions. These alterations may be responsible for onset of GKD pathogenesis by disruption of these moonlighting functions. 125 MOLECULAR ANALYSIS OF 54 PATIENTS WITH PYRIDOXINE DEPENDENT EPILEPSY DUE TO ANTIQUITIN DEFICIENCY B R Plecko1, K Paul1, E Struys2, E Paschke1, S G Stöckler-Ipsiroglu3, W Erwa4, C Jakobs2 1Pediatrics, Medical University Graz, Graz, Styria, Austria, 2Clinical Chemistry, Metabolic Unit, Vrije Universiteit Amsterdam, Amsterdam, Netherlands, Netherlands, 3Division of Biochemical Diseases, Children's and Womens Health Center, Vancouver, British Columbia, Canada, 4Laboratory for Clinical Chemistry, Medical University Graz, Graz, Styria, Austria Introduction: Most cases of pyridoxine dependent epilepsy (MIM#266100) are

caused by alterations in the (ALDH7A1) antiquitin gene. α-aminoadipic semialdehyde (AASA) in urine serves as a specific diagnostic marker and remains elevated while on pyridoxine treatment. Pipecolic acid (PA) in plasma, a second biomarker, is less specific as it is also increased in peroxisomal disorders. We analysed the genotype and biochemical findings in a cohort of 54 patients with proven antiquitin deficiency. Methods: Samples of 54 patients, including 7 pairs of affected siblings, were sent from 35 different European and 1 Canadian center. AASA and PA were analysed as described (1, 2). Molecular analysis was performed by sequencing of all 18 exons and flanking introns of the antiquitin gene as described (3). Results: AASA in urine was elevated in 28/28 and ranged from 90 mmol/mol creatinine pre-treatment to 2,4 while on pyridoxine; normal <1.0. AASA in plasma was elevated in 32/35 cases ranging from 14 to 0.22 µmol/l; normal <0.2. AASA in plasma was undetectable in 2 patients and within normal range (0.15 µmol/l) in 1 patient with two proven mutations of the antiquitin gene. PA in urine was markedly elevated in 6/6 pre-treatment samples but normalized during infancy when on pyridoxine. PA in plasma was elevated in 37/39 cases ranging from 71 to 17.6 µmol/l pre-treatment and from 32 to 2.8 µmol/l while on pyrdoxine; normal values are age dependent (4); over 5y normal <2.6). 2 patients with 2 proven antiquitin mutations had normal PA in plasma while on pyridoxine. By molecular sequencing of the antiquitin gene 104 of 108 mutant alleles were identified, including 13 novel mutations (8 missense, 3 deletions, 1 splice site, and 1 intronic). Pathogenicity was suggested by localization in conserved domains, cosegregation within the family and absence in >100 control alleles. 27 patients, including 4 pairs of siblings, were homozygous with a total of 9 different mutations in 23 families. Conclusion: AASA in urine serves as the most reliable biomarker of antiquitin deficiency, while AASA and PA in plasma may fail to detect single patients when on pyridoxine. As shown by repeated measurement in 10 patients, biomarkers decline over the first 2-3 years of life despite a constant dose of pyridoxine. We identified 13 novel mutations within the ALDH7A1 gene, but could not establish a clear correlation of genotype and biochemical findings. Mills et al. Nat Med (2006) 12:307-309, Kok et al. Clin Chim Acta (1987) 168:143152, Plecko et al. Human Mutation (2007) 28:19-26, Kelley et al. In Hommes FA, ed. Techniques in diagnostic human biochemical genetics. Wiley Liss (1991) 205-218 126 ERYTHROPOIETIC PROTOPORPHYRIA: MOLECULAR ANALYSIS REVEALS PATIENTS WITH X-LINKED CAUSATIVE MUTATIONS IN THE ERYTHROID-SPECIFIC 5’-AMINOLEVULINATE SYNTHASE 2 GENE D O Doheny1, I Nazarenko1, M Balwani1, L Liu2, R J Desnick1 1Genetics & Genomic Sciences, Mount Sinai School of Medicine, New York, NY, United States, 2Medicine, Liver Diseases, Mount Sinai School of Medicine, New York, NY, United States Erythropoietic Protoporphyria (EPP), the most common erythropoietic porphyria, is an inherited disorder characterized by acute photosensitivity and painful cutaneous lesions. Autosomal recessive EPP is biochemically diagnosed by elevated levels of ertythrocyte protoporphyrins (PROTO) with free-PROTO greater than zinc-PROTO. Most cases result from mutations in the ferrochelatase (FECH) gene that causes the markedly deficient activity of ferrochelatase, the last enzyme in the heme biosynthetic pathway. To date, 119 FECH mutations have been described (Human Gene Mutation Database, 2009), as well as the common hypomorphic IVS3-48T>C allele. Recently, an X-linked variant of EPP was described with similar symptoms, elevated erythrocyte PROTO with equal proportions of free-PROTO and Zn-PROTO, and no FECH mutations; however, causative mutations were identified in the Cterminus of the erythroid-specific 5’-aminolevulinate synthase 2 (ALAS2) gene at Xp11.21 (Whatley et al, 2008). We performed FECH mutation analysis on 22 unrelated patients with biochemical and/or clinical evidence suggestive of EPP, identifying FECH mutations in 15 patients. Among these, six had novel mutations, including one deletion (c.1052delA), one insertion (c.215-216insT), two splice-site (c.1077-1G>T, c.314+1T>G), two nonsense (c.832C>T (p.Q278X), c.181C>T (p.Q61X)), of the seven cases with no detectable FECH mutation, we identified three patients with ALAS2 lesions. Of these, a 19 year old male had photosensitivity since childhood, elevated PROTO with equal proportions of free-PROTO and Zn-PROTO, and a fluorescence peak at 633nm consistent with EPP. The patient’s mother reported mild photosensitivity. Molecular studies revealed a novel ALAS2 frameshift mutation, c.1734delG, in exon 11. The two other patients, a 7 year old male and 3 month old female, had the previously reported ALAS2 c.1706-1709delAGTG mutation. These findings indicate that X-linked EPP is not rare among EPP patients (14% of index cases; 43% of FECH-negative cases) and that ALAS2 mutation analysis should be undertaken in patients with EPP symptoms of acute photosensitivity who have elevated erythrocyte PROTO, high Zn-PROTO, and no detectable FECH lesion.

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Platform Presentation 5: Amino Acid Disorders Tuesday, 1 September 2009 13:30-15:30 127 THE EFFECT OF LOW-MOLECULAR-WEIGHT COMPOUNDS ON THE IN VITRO POLYMERIZATION/FIBRIL FORMATION PROCESS OF MUTANT G46S-HPAH J Leandro1,2, N Simonsen2, I Tavares de Almeida1, P Leandro1, T Flatmark2 1 Metabolism and Genetics Group, Research Institute for Medicines and Pharmaceutical Sciences (iMed.UL), Faculdade de Farmácia UL, Lisboa, 1649-003, Portugal, 2Department of Biomedicine, University of Bergen, Bergen, Norway Human phenylalanine hydroxylase (hPAH; E.C.1.14.16.1) catalyzes the conversion of L-phenylalanine (L-Phe) to L-tyrosine (L-Tyr) in the presence of the natural cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and dioxygen. A deficient activity of this homotetrameric non-heme iron dependent enzyme leads to the autossomal recessive disorder known as phenylketonuria (PKU; OMIM 261600) and related forms of hyperphenylalaninemia (HPA), which present an average incidence in Caucasians of 1:10000. To date, more than 500 different mutations have been identified in the human pah gene, with missense mutations accounting for ~60% of the observed nucleotide changes. Characterization of several mutant hPAH forms revealed that the majority of the studied proteins result in enzyme forms with reduced stability in vitro and in vivo which will be degraded more rapidly. Therefore, PKU is now being classified as a protein misfolding disease. Among the identified hPAH mutant proteins the G46S form, localized in the regulatory domain of hPAH and associated with a severe PKU phenotype, when expressed in E. coli with MBP as a fusion partner (MBP-pep(Xa)-G46S-hPAH) is recovered as a soluble tetrameric/dimeric enzyme (in addition to soluble aggregates) presenting a normal catalytic efficiency (tetrameric form). Interestingly, when MBP was cleaved off (by factor Xa) the enzyme (G46S-hPAH) showed abnormal physico-chemical properties with a strong propensity to form catalytically inactive higher-order oligomers and polymers. In the present work we have studied the effect of low-molecular weight compounds on the in vitro aggregation/polymerization process of G46S hPAH. Our results show that the G46S-hPAH self-association is a multiphasic process (lag phase, growth phase, deposition phase) and results in the formation of amyloid-like fibrils. Phosphorylation of Ser16 has a protective effect on the self-association (extended lag phase and lower rate of polymerization) whereas L-Phe increases the rate of selfassociation and BH4 has no effect on the process. The action of low-molecular-weight compounds (chemical chaperones) on aggregation has a mix effect on the selfassociation: glycerol inhibits this process whereas trimethylamine N-oxide (TMAO) and epigallocatechin-3-gallate (EGCG) increase the rate of polymerization. Elucidation of the mechanism of G46S-hPAH polymerization is expected to have an impact on our understanding of PKU as a protein misfolding disorder. (J Leandro was supported by FCT, grant SFRH/BD/19024/2004) 128 GENOTYPES AND BH4-RESPONSIVENESS IN 600 TURKISH PKU PATIENTS C Heintz1, S Dobrowolski2, M Demirkol3, T Miller4, T Baykal3, G Gokçay3, B Thony1, N Blau1 1Children's Hospital Zurich, Zurich, Switzerland, 2Department of Pathology, University of Utah, Salt Lake City, Utah, United States, 3Nutrition and Metabolism, Istanbul Faculty of Medicine, Istanbul, Turkey, 4Idaho Technology, Salt Lake City, Utah, United States Background: The prevalence of phenylketonuria (PKU) varies by country and ethnic group. In Turkey there is a prevalence of 1 in 6’500 births, but the information about the genotype distribution and BH4-responsiveness within different PKU variants is limited. Methods: We genotyped 600 Turkish PKU patients, 166 with MHPA, 120 with mild PKU, and 265 with classic PKU, and in a total of 466 patients a tetrahydrobiopterin (BH4) loading test was performed (20 mg/kg b.w.). In all patients BH4 deficiency was also investigated by urinary pterins and dihydropteridine reductase activity measurement. Details on DNA mutation and loading test data will be tabulated in the BIOPKU database of BH4-responsive genotypes (www.biopku.org). Results: A total of 88 different mutations were found; the most common missense mutations being c.1066-11G>A (21.8%), p.P281L (9.5%), p.R261Q (9.5%), p.L48S (7.6%), p.A300S (6.6%), and p.R408W (6.4%) and the most common deletions being c.1089del6 (1.6%), c.590-611del22 (1.6%), and c.164delT (1.2%). Twenty novel mutations were detected (10 missense, 5 splice site, and 5 deletion/insertions) and in 9 patients atypical genotypes with three mutations was found (6 with p.R155H in cis). The most common genotypes were c.1066-11G>A/c.1066-11G>A (14%), p.R261Q/p.R261Q (4.7%), and p.P281L/p.P281L (4.5%). About 22% of the tested patients were BH4-responsive (62% MHPA, 21% mPKU, 17% cPKU). In 14 patients mutations were found in BH4-metabolizing genes (8 DHPR, 5 PTPS, and 1 PCD). Conclusions: This is the first report of a large-scale genotype assessment in a population of ethnically Turkish PKU patients, with a high prevalence of the severe classic phenotype (48%). These data will serve, in conjunction with the BIOPKU database, as a basis for development of algorithms for the genotype-based prediction of BH4-responsiveness. 129 LONGITUDINAL FOLLOW-UP OF TETRAHYDROBIOPTERIN (BH4) THERAPY IN PATIENTS WITH BH4 DEFICIENCY IN JAPAN H Shintaku1, M Ohwada2, T Kitagawa3 1Pediatrics, Osaka City University Graduate School of Medicine, Osaka, Japan, 2Pediatric Nutrition, Kagawa Nutrition University, Sakado, Saitama, Japan, 3Tokyo Health Service Association, Sinjuku-ku, Tokyo,

Japan Since tetrahydrobiopterin (BH4) therapy was begun for BH4 deficiency in 1981 in Japan, 29 patients with BH4 deficiency have received BH4. In Japan, Sapropterin dihydrochloride (Biopten: a synthetic formulation of the active 6R-isomer of BH4) was approved for the treatment of hyperphenylalaninemia based on BH4 deficiency in 1992. Afterwards, all of BH4 deficiency came to be treated with Biopten. We studied all patients with BH4 deficiency for 28 years and evaluated the efficacy and safety of BH4 therapy from the newborn to the adulthood. Patients: In all of 29 patients with BH4 deficiency, 26 were 6-pyruvoyl tetrahydropterin synthase (PTPS) deficiency and 3 were dihydropteridine reductase (DHPR) deficiency. Male were 15 and female were14. As for the age category, under 3 years old were one, and between 3 and 15 years old were 7, and between 16 and 29 years old were 9, and more than 30 years old were 12. As for Biopten start age, under 1 year old were 14, and between 1 and 6 years old were 5, and between 7 and 15 years old were 2, and more than 16 years old were 8. As for the dosage for one day, less than 5mg/kg were 17, and more than 5mg/kg were 12. As for the combined medicine, levodopa was 26 patients, and 5-HTP was 13 patients. It was one that used together the phenylalanine restricted diet. Therapeutic efficacy: In all of 29 patients judgement of family doctor was "availability" with the efficacy, and most cases maintained a normal range in serum Phe value. In PTPS deficiency serum phenylalanine levels were kept within control range (0.7-3.0 mg/dl) with the doses (1.5-13 mg/kg) of BH4 without diet therapy. In DHPR deficiency 2 patients showed serum phe levels of 2.4 and 4.6 mg/dl with the doses of BH4 of 3.8 and 2.2 mg/kg respectively, and one had slightly high serum phenylalanine levels (9.9-15.5 mg/dl) with the dose (16.9 mg/kg) of BH4, which was still remain control range. Safety: There was only one event of adverse effect in one patient and the incident was 3.8%. It was a side effect of "the unfamiliarity" without mention in precautions for use in one case of slight "both leg edemas". However this patient (57 years old / a woman) had hypothyroidism and her leg edema was improved after supplementation of thyroid hormone. Therefore it was thought that safety did not have the new problem. One patient treated with a long-term continuous administration of Biopten carried out the childbirth, and both the mother and child was in good health, and it was confirmed about the birth child to be healthy. Conclusion: BH4 therapy in BH4 deficiency was very effective to maintain serum phenylalanine levels within control range and very safe from a baby to an adult for a long term. There were no serious adverse events that were thought to be related to Biopten treatment at least for 17 years. 130 LARGE NEUTRAL AMINO ACIDS AND LATE DIAGNOSED PHENYLKETONURIA: THE CALIFORNIA LATE TREATMENT PROJECT K D Moseley1, S Yano2, C Azen3, R G Boles4, R Koch5 1Pediatrics, University of Southern California/USC Keck School of Medicine, Los Angeles, CA, United States, 2 Pediatrics, University of Southrn California, USC/Keck School of Medicine, Los Angeles, CA, United States, 3Medicine, University of Southern California/USC Keck School of Medicine, Los Angeles, CA, United States, 4Medical Genetics, Childrens Hospital Los Angeles, USC/Keck School of Medicine, Los Angeles, CA, United States, 5Pediatrics, University of Southern California, USC/Keck School of Medicine, Los Angeles, CA, United States Introduction: Many individuals with Phenylketonuria (PKU) who were born before newborn screening are severely affected and remain untreated since it was surmised that the damage was already done and it was too late to treat. However, recent experience has suggested that proper treatment can reduce negative behaviors, improve overall health and reduce costs of care. Methods: The aim of this one year prospective observational study was to assess the impact of treatment with the large neutral amino acid therapy (LNAA) for one year on health outcomes, behaviors and cost of care in late-or never-treated PKU adults. Ten late diagnosed, severely affected individuals with PKU were evaluated. Baseline and end of study testing included laboratory tests and psychological evaluations. Blood phenylalanine (phe) and tyrosine (tyr) levels and behavior were evaluated monthly. Cost benefit analysis compared the cost of care in state institutions versus community placement, as well as any decrease in medications. Results: While the blood phe remained relatively stable there were significant increases in tyr and declines in phe/tyr ratios. Increased blood tyr levels were significantly associated with less aggression towards others and improvement on the Vineland Daily Living Scale. Cost comparisons of psychotropic medications were also obtained before and after the study revealed a cost savings of 50%. Annual cost for an individual living in a California state institution versus living in a community setting decreased approximately 52% as four individuals were moved from the institution to community care. Summary: Using LNAA to treat individuals with PKU who were previously untreated resulted in an improvement in behavior as well as cost savings. 131 N-CARBAMYLGLUTAMATE ACCELERATES UREAGENESIS IN PATIENTS WITH HYPERAMMONEMIA 1 2 2 2 2 1 1 N Ah Mew , E Daikhin , I Payan , I Nissim , M Yudkoff , M Tuchman Children's Research Institute, Children's National Medical Center, Washington, DC, United States, 2Children's Hospital of Philadelphia, Philadelphia, PA, United States The urea cycle is responsible for nitrogen regulation and balance in humans. Disruption of normal urea cycle function leads to hyperammonemia, neurological sequelae and can result in death. Current treatment for hyperammonemia is based on dietary protein restriction and ammonia-scavengers, but these interventions commonly fail to avert brain damage. In this study, we investigate the use of NCarbamylglutamate (NCG), a new therapeutic agent for a subset of hyperammonemic conditions which are caused by a deficiency of N-acetylglutamate (NAG). Nacetylglutamate (NAG) is the allosteric activator for the carbamyl phosphate synthase

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I (CPSI) reaction. CPSI is the first and rate-limiting enzyme of the urea cycle. A shortage of NAG causes decreased flux through CPSI and is responsible for hyperammonemia in many conditions, including NAG synthase deficiency, propionic acidemia (PA) and methylmalonic acidemia (MMA). Though orally administered NAG is hydrolyzed in the gut, N-carbamylglutamate (NCG) is a stable analog of NAG and activates CPSI in vitro. We demonstrate through stable isotope studies that a 3-day trial of oral NCG improves urea cycle function and decreases plasma ammonia levels in patients with PA or MMA. Thus, NCG is a useful adjunct in the treatment of acute hyperammonemia of various metabolic etiologies that are due to a lack of NAG. We also demonstrate that in two subjects with NAG synthase deficiency, NCG can restore ureagenesis and is therefore curative for patients with this condition. 132 HYPERCITRULLINEMIA, HYPERCHOLESTEROLEMIA AND AUGMENTED OXIDATIVE STRESS IN THE PATIENTS WITH CITRIN DEFICIENCY DURING THE SILENT PERIOD Y Okano1, H Nagasaka2, Y Shigematsu3, H Tsukahara3, T Momoi4, T Yorifuji5, T Ohura6, T Miida7, K Hirano8, K Kobayashi9, T Saheki10, M Takayanagi2 1Pediatrics, Osaka Citu University Graduate School of Medicine, Osaka, Japan, 2Division of Metabolism, Chiba Children's Hospital, Chiba, Japan, 3Pediatrics, Fukui University School of Medicine, Eiheiji-cho, Fukui, Japan, 4Pediatrics, Japanese Red Cross Society, Wakayama Medical Center, Wakayama, Japan, 5Pediatrics, Kyoto University Graduate School of Medicine, Kyoto, Japan, 6Pediatrics, Sendai City Hospital, Sendai, Japan, 7Clinical and Laboratory Medicine, Juntendo University, Tokyo, Japan, 8Cardiovascular Medicine, Osaka University Graduate, Suita, Japan, 9 Molecular Metabolism and Biochemical Genetics, Kagoshima University Graduate School of Medicine and Dental Science, Kagoshima, Japan, 10Heath Science, Tokushima Bunri University, Tokushima, Japan Citrin is an aspartate glutamate carrier located on the inner membrane of mitochondria, and plays a role in the malate-aspartate NADH shuttle and urea synthesis. Citrin deficiency due to SLC25A13 mutation leads to an increased NADH/NAD+ ratio in the cytosol, and the failure of aspartate supply from the mitochondria to the cytoplasm. Citrin deficiency is characterized a dynamic change of clinical symptoms by age. In adult, adult-onset type II citrullinemia shows frequent attacks of hyperammonemia, mental derangement, and sudden attacks of unconsciousness. In neonatal and infantile periods, neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD) shows cholestatic jaundice, hypercitrullinemia, suppressed gluconeogenesis, and is usually resolved by the age of 1 year. The patients in the silent period between NICCD and CTLN2 have a peculiar fondness for protein- and fat-rich foods. However, metabolic profiles in the silent period remain unknown. We examined oxidative stress markers and profiles of amino acids, carbohydrates, and lipids in 20 asymptomatic children with citrin deficiency aged 1– 10 years. The affected children showed high blood levels of ornithine (p<0.001) and citrulline (p<0.01) despite normal plasma ammonia levels, compared to healthy children. Blood levels of nitrite/nitrate, metabolites of nitric oxide (NO), and asymmetric dimethylarginine inhibiting NO production from arginine were not different between these two groups. Blood glucose, galactose, pyruvate, and lactate levels after 4–5 h fasting were not different between these groups, but the affected group showed a significantly higher lactate-to-pyruvate ratio. Low-density and high-density lipoprotein cholesterol levels in the affected group were 1.5 times higher than those in the controls. Plasma oxidized low-density lipoprotein apparently increased in the affected children; their levels of urinary oxidative stress markers such as 8-hydroxy2’-deoxyguanosine and acrolein-lysine were significantly higher than those in the controls. The present study showed that children with citrin deficiency at the silent stage had urea cycle dysfunction and hypercholesterolemia, and raised a possibility that they were persistently in oxidative stress. Further study is required to know whether such sustaining metabolic abnormalities might render the development of CTLN2. 133 QUANTIFICATION OF THERAPEUTIC SUCCESS AFTER LIVER CELL TRANSPLANTATION (LCT) FOR NEONATAL UREA CYCLE DISORDERS (UCD) J Meyburg1, A Burlina2, J M Nuoffer3, M Lindner4, H Kriegbaum5, J Schmidt6, M Ott7, J Poeschl8, G F Hoffmann1 1General Pediatrics, University Children's Hospital, Heidelberg, Germany, 2University Children's Hospital, Padova, Italy, 3University Children's Hospital (Inselspital), Berne, Switzerland, 4General Pediatrics, University Children's Hospital, Heidelberg, Baden-Wuerttemberg, Germany, 5Research & Development, Cytonet GmbH, Weinheim, Germany, 6Department of Visceral and Transplantation Surgery, Heidelberg, Germany, 7Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany, 8Neonatology, University Children's Hospital, Heidelberg, Germany Background: LCT may be a promising alternative to whole organ transplantation for severe hepatic-based inborn errors of metabolism. Several children have been treated with LCT since its first use in an OTC deficient boy in 1997. However, quantification of therapeutic success has been an unsolved problem so far. Methods: Two boys with neonatal UCD (patient 1: CPS1 deficiency, patient 2: OTC deficiency) underwent orthotopic liver transplantation 15 and 10 months resp. after allogeneic LCT with cryopreserved liver cells. Immediately after explantation of the original liver, 40 resp 80 tissue samples were taken from patient 1 resp. 2), according to topographic protocols and immediately frozen in liquid nitrogen. Enzyme activities were measured after thawing and homogenisation of the samples. Results: In patient 1, a liver biopsy prior to LCT showed 0% CPS1 activity. In the explanted liver, an overall enzyme activity of 4.5 ± 2.7% was found. The distribution

throughout the liver was inhomogeneous with absent enzyme activity in some areas and clusters up to 30.8%. In patient 2, the mean OTC activity after LCT was 15.6 ± 6.9%. Similar to patient 1, distribution in the liver was irregular reaching peak values of 32.9%. Discussion: In both explanted livers, a distinct increase in enzyme activity could bedetected about one year after LCT. Based on dose calculations, the measured activities were surprisingly high. Therefore, it can be speculated that the donor cells not only engrafted permanently, but may have proliferated in the host liver and/or substantially upregulated their urea cycle function. Because the increase in enzyme activity was paralleled by clinical stabilisation in both children, these findings can be regarded as the first solid quantification of human LCT. Further studies are needed now to investigate the possible impact of LCT on treatment strategies for severe neonatal UCD. 134 COMBINATORIAL GENE THERAPY AND NO SUPPLEMENTATION FOR PHENOTYPIC CORRECTION OF ARGININOSUCCINIC ACIDURIA O A Shchelochkov1, A Erez1, N Brunetti-Pierri1, S C S Nagamani1, Y Chen1, D Palmer1, N S Bryan2, B Lee1,3 1Baylor College of Medicine, Houston, TX, United States, 2Brown Foundation Institute of Molecular Medicine, UT Health Science Center at Houston, Houston, TX, United States, 3Howard Hughes Medical Institute, Chevy Chase, MD, United States Argininosuccinic aciduria (ASA) is the second most common disorder of ureagenesis caused by deficiency of argininosuccinate lyase (ASL). Biochemically, ASA is characterized by an elevation of citrulline and argininosuccinic acid, as well as depletion of plasma arginine. Despite the arginine supplementation and control of hyperammonemia, patients frequently develop mental retardation and hepatic dysfunction. It has been suggested that intracellular depletion of arginine, the substrate for nitric oxide (NO) production, and subsequent NO dysregulation could be in part responsible for these findings. To address the question of NO contribution to ASA phenotype, we generated a novel hypomorphic ASA mouse, performed in vivo stable isotope studies in ASA patients, and conducted experiments on human Asl-/fibroblasts. In vitro studies with ASA patient’s fibroblasts and in vivo stable isotope infusion showed that despite the normal levels of arginine, ASA patients were unable to utilize arginine for NO production. In contrast, ASS-deficient patients were able to use arginine for NO generation emphasizing the importance of ASL for NO generation. The utility of restoring the NO biology was supported by improvement in survival and optimization of NO production in hypomorphic ASA mice treated with arginine (an essential amino acid in ASA) and sodium nitrite (to recapitulate NO biochemistry), which usually die around age 4 month when untreated. To assess the utility of liver transplantation in the treatment of ASA, we created a mouse model by performing a liver-targeted Asl gene transfer to ASA mice. We used a helper-dependent adenoviral vector carrying mouse Asl under control of a liverspecific promoter. Mice treated with the Asl vector carrying a liver-specific promoter showed dramatic improvement in their physical growth and statistically significant weight difference already 10 days after injection (p = 0.001) and 100% survival at age 158 days. In contrast, ASA mice, which received saline only and arginine/nitrite therapy in the first 5 weeks of life, expired by age 103 days. Plasma amino acids from ASA mice treated with Asl vector showed a normalization of ASA, citrulline, glutamine compared to the wild type. We report here that in ASA, NO dysregulation is involved in disease pathogenesis. Restoration of normal NO and ASA biology in ASL deficiency by combining NO donors and Asl gene transfer offers a new approach to the treatment of this disorder.

Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Platform Presentation 6: Fatty Acid Oxidation Disorders Tuesday, 1 September 2009 16:00-17:30 135 SEARCHING FOR FACTOR X IN SHORT-CHAIN ACYL-COA DEHYDROGENASE DEFICIENCY (SCADD) – A PROTEOMIC SCREENING OF FIBROBLASTS FROM C.625G>A HOMOZYGOUS PATIENTS C Bak Pedersen1, M Kjeldsen1, S Vang1, J Palmfeldt1, V Stenbroen1, N Gregersen1 1 Research Unit for Molecular Medicine, Aarhus University Hospital, Skejby, Aarhus N, Denmark Short-chain acyl-CoA dehydrogenase deficiency (SCADD) is an autosomal recessive inborn error of mitochondrial fatty acid oxidation. SCADD leads to the accumulation of butyrylcarnitine and ethylmalonic acid in blood and urine, respectively. A range of clinical symptoms has been recognized in SCADD patients, including failure to thrive, metabolic acidosis, hypoglycemia, developmental delay, seizures, and neuromuscular symptoms such as myopathy and hypotonia. Molecular analysis of the ACADS gene in affected individuals has identified a large number of inactivating mutations as well as two common susceptibility variations (c.511C>T and c.625G>A), which are also relatively frequent in the general population. Even though the susceptibility variations define the ACADS genotype in about 90% of SCADD patients, they cannot by themselves be sufficient for the development of clinical symptoms – additional genetic or environmental factors are required. Several attempts have been conducted but SCADD predisposing factors have not yet been identified. In the present study, we used a proteomic based approach to search for disease predisposing factors. Fibroblast mitochondria from four c.625G>A homozygous patients were subjected to global semi-quantitative protein screening using nano-LCMS/MS and compared to c.625G/G and c.625A/A control pools. We identified around 300 mitochondrial proteins in each sample of which 241 were represented in both patient and control samples. Among these ‘mitochondrial core proteins’, 88 could be assigned to well-known functional pathways, i.e. fatty acid oxidation, protein quality control, antioxidants, amino acid metabolism, citric acid cycle, respiratory chain, and apoptosis. Based on these pathways, protein profiles have been developed and potential changes in mitochondrial functionality was evaluated. One of the most interesting findings was a >2-fold reduced expression of the mitochondrial superoxide dismutase (SOD2 or MnSOD) in two of the patient samples. This has been confirmed by western blotting and lead to the suggestion that patients with SCADD may have a reduced mitochondrial antioxidant capacity thus being more susceptible to oxidative stress. 136 AFFINITY PROTEIN PULLDOWN OF SHORT-CHAIN HYDROXYACYLCOA DEHYDROGENASE REVEALS CONVERGENCE OF MULTIPLE MITOCHONDRIAL PATHWAYS S B Narayan1, L Tan1, C Bierl2, S R Master2, C A Stanley3, M J Bennett1 1Pathology and Labratory Medicine, The Childrens Hospital of Philadelphia, Philadelphia, PA, United States, 2Pathology and Labratory Medicine, University of Pennsylvania, Philadelphia, PA, United States, 3Department of Endocrinology, The Childrens Hospital of Philadelphia, Philadelphia, PA, United States Introduction: Mitochondrial short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD) encoded by the HADH gene catalyzes the third step in the β-oxidation cycle for medium and short chain L-3-hydroxyacyl-CoAs. Purified SCHAD has broad chain-length specificity and is highly active against C4-C8 hydroxyacyl-CoA’s. Deficiency of this enzyme has been described in a few patients with hypoglycemia associated with hyperinsulinism with accumulation of plasma 3-hydroxybutyrylcarnitine and urinary 3-hydroxyglutaric acid. The hypoglycemia in SCHAD can be controlled successfully by treatment with the KATP channel agonist, diazoxide. A number of patients have been described with enzymatic SCHAD deficiency but without deleterious mutations in HADH which coupled to the unusual observation of hyperinsulinism in SCHAD deficient patients and the HADH knock out mice suggested to us that intramitochondrial SCHAD activity is regulated by other proteins. Method: Protein pull down experiments were performed using, an Aminolink plus Immobilization Kit (Pierce, Rockford, IL, US) and recombinant SCHAD. Recombinant SCHAD was produced in our laboratory using E.Coli containing a hadh expression sequence in a pET 20 (b+) plasmid with a C-terminal His Tag (Novagen, USA). SCHAD was linked to the agarose resin which contains aldehyde functional groups that react with the primary amine groups on the protein. Liver homogenates from both wild type and SCHAD knock out mice were mixed with an equal volume of binding buffer and incubated with the SCHAD-linked amino link resin at 4oC overnight. The resin was spun down after incubation and washed with washing buffer and bound proteins were eluted with of eluting buffer. Proteomic analysis of pull down proteins: The protein extract was subjected to mass spectrometric analysis. The procedure involved digestion of the soluble protein using trypsin followed by tandem mass spectrometric analysis of the peptide sequences. Identified sequences were matched to the proteins using Scaffold protein identification software v 2.0. Results: Mass spectrometric analysis of the protein extract from the pull down assay using SCHAD protein as bait revealed the presence of two peptides from glutamate dehydrogenase, confirming a previously published study. In addition a number of positive peptide hits for the following mitochondrial proteins were made. Carbamoyl phosphate synthetase-1, mitochondrial trifunctional protein, beta subunit, aspartate amino transferase, 3-ketoacyl-CoA thiolase, 2, 4-dienoyl-CoA reductase, ATP

synthase, beta subunit, ATP synthase, alpha subunit. GDH- association was a confirmed by Western blot and confirmation of the additional proteins is under way. Conclusion: Our studies suggest that SCHAD binds specifically to several other proteins within the mitochondria including others involved with fatty acid oxidation. There is convergence of several metabolic pathways that are generally regarded as independent. The metabolic and physiological relevance of these protein associations requires further study in order to identify the mechanism by which this regulates metabolic functions within the cell. 137 GENOTYPE-METABOLITE CORRELATIONS AND OUTCOME IN MCAD DEFICIENCY DIAGNOSED BY NEWBORN SCREENING IN NEW YORK STATE G L Arnold1, P A Galvin-Parton2, R Erbe3, E DeVincentis4, D Kronn5, S Mofidi5, M Wasserstein6, J E Pellegrino7, P A Levy8, D J Adams9, C A Saavedra-Matiz10, T J Nichols10 1Pediatrics, University of Rochester SOMD, Rochester, NY, United States, 2 Pediatrics, State University of New York at Stony Brook, Stony Brook, NY, United States, 3Pediatrics and Medicine, State University of New York at Buffalo, Buffalo, NY, United States, 4Women and Children's Hospital of Buffalo, Buffalo, NY, United States, 5Pediatrics, New York Medical College, Valhalla, NY, United States, 6 Genetics and Genomic Sciences, Mount Sinai SOM, New York, NY, United States, 7 Pediatrics, State University of New York Uptate Medical University, Syracuse, NY, United States, 8Pediatrics, Albert Einstein College of Medicine, Bronx, NY, United States, 9Pediatrics, Albany Medical Center, Albany, NY, United States, 10Wadsworth Laboratory, New York State DOH, Albany, NY, United States Purpose: Medium chain acyl CoA carboxylase deficiency (MCADD) is disorder of fatty acid oxidation affecting about 1/15,000 infants. Outcome has been greatly improved by newborn screening (NBS), but rare deaths and uncertainty regarding optimal management persist. We reviewed the outcome of a cohort of infants with MCADD to identify markers associated with enhanced risk for poor outcome. Method: Chart review from all 8 New York State metabolic treatment centers of all infants with MCADD diagnosed by the New York State NBS program from Oct. 2002 to November 2008. Results: We identified 53 cases of MCADD. The mean C8 level on NBS was 10.7 µM and C8/C2 ratio was 0.38; on confirmatory diagnostic testing the mean C8 was 3.7 µM and hexanoylglycine was 20.6 µM. Organic acid analysis was diagnostic in only 53% of cases. Twenty infants had two copies of the common mutation c.985A>G, 19 had one, and 14 had no copies. Genotype did not predict the number of symptomatic episodes. ANOVA analysis between 2, 1 or 0 copies of the c.985A>G mutation found no difference in NBS C8 levels, but C8/C2 was 0.58, 0.37 and 0.09 respectively (p<0.016). Thirteen patients reported at least one symptomatic episode in later infancy or childhood (lethargy or hypoglycemia with intercurrent illness); these patients had higher C8 level on NBS (19.2 µM vs 7.3, p<0.0008) and higher NBS C8/C2 level (0.59 vs 0.26, p<0.003). The lowest NBS C8 level associated with a later onset symptomatic episode was 1.12 µM, but eleven of the thirteen with later infantile or childhood symptomatic episode had initial NBS C8 level >10 µM. Patients with NBS C8 levels of >20 µM had multiple symptomatic episodes (2), a single symptomatic episode (4), prior sibling death (1), or no symptomatic episodes (4). Discussion: No single biochemical or genotypic marker was protective from symptomatic episodes, thus fasting prevention is important for all affected patients. However these data suggest that at least some infants having a profound elevation of NBS C8 may be manifesting a more significant underlying intolerance for metabolic stress. Additional studies are needed to determine if these infants merit enhanced precaution beyond routine prevention of fasting (for example, night-time cornstarch). 138 DIAGNOSIS OF MATERNAL CARNITINE DEFICIENCY THROUGH NEWBORN SCREENING N-C Lee1, Y-H Chien1, N Leung-Sang Tang2, P-C Chiu3, A-C Huang4, W-L Hwu1 1 Pediatrics and Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan, 2Department of Chemical Pathology, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong, People's Republic of China, 3Department of Pediatrics, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, 4Medical Genetics, National Taiwan University Hospital, Taipei, Taiwan Background: Primary carnitine deficiency is an autosomal recessive fatty acid oxidation defect caused by mutations in the SLC22A5 gene, which encodes the highaffinity carnitine transporter OCTN2. Patients usually present with hypoketotic hypoglycemia, hepatic encephalopathy, hypotonia, dilated cardiomyopathy, and sudden death. Tandem mass spectrometry analysis of dry blood spot may lead to the detection of this disorder at newborn stage. Material and Methods: From Jan 2002 to Dec 2008, 549,596 newborns were screened by the National Taiwan University Hospital Newborn Screening Center. The cut off value for free carnitine (C0) was 3.5 standard deviations or 0.02th percentile. The confirmatory tests include also an acylcarnitine profile analysis for the mothers. Result: Seventy-two cases were asked for a second blood spot and 12 cases entered the confirmatory process. Four cases were found to have persistent low free carnitine levels. They were treated immediately by carnitine 100 mg/kg/day and there is no symptom in any of them at the time when we prepared the abstract. Four cases had a transient depression of free carnitine, while their mothers were found to have very low free carnitine and decreases in long-chain acylcarnitines. All 4 mothers were put on carnitine supplementation 50 mg/kg/day. One mother had dilated cardiomyopathy at the diagnosis of carnitine deficiency, and her cardiac function improved after treatment. Conclusion: Newborn screening allows the detection for both babies and mothers with severe carnitine deficiency. Early treatment may prevent them from disease attack.

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Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

139 QUANTITATION OF TISSUE ACYL-COAS IN BETA-OXIDATION DEFECTS USING TANDEM MASS SPECTROMETRY: A NOVEL METHOD A A Palladino1, J Chen2, S B Narayan2, A W Strauss3, P A Wood4, M J Bennett5, C A Stanley6 1Division of Endocrinology, The Children's Hospital of Philadelphia, Philadelphia, PA, United States, 2Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Philadelphia, PA, United States, 3Department of Pediatrics, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States, 4 Burnham Institute for Medical Research, Orlando, FL, United States, 5Pathology and Laboratory Medicine, University of Pennsylvania, The Children's Hospital of Philadelphia, Philadelphia, PA, United States, 6Division of Endocrinology, University of Pennsylvania, The Children's Hospital of Philadelphia, Philadelphia, PA, United States Objective: The primary accumulating metabolites in fatty acid oxidation defects are intramitochondrial acyl-CoAs. Typically, secondary metabolites such as acylcarnitines, acylglycines and dicarboxylic acids are measured to study these defects. Methods have not been available for tissue acyl-CoA measurement. Our objective was to develop a method to measure fatty acyl-CoA species that are present in various tissues of mice with fatty acid oxidation defects using tandem mass spectrometry. Methods: We have developed a method for the isolation and measurement of multiple chain-length acyl-CoA species in mouse tissues using direct injectionelectrospray ionization tandem mass spectrometry. Following the addition of internal standards of [C13C2] acetyl-CoA, [C13C8] octanoyl-CoA, and [C17] heptadecanoic CoA, tissue samples are twice homogenized in methanol: chloroform (2:1), and then undergo phase separation with equal volumes of chloroform and ammonium formate. The upper aqueous layer is saved and washed with an additional volume of chloroform. Methanol is removed by evaporation under a steady stream of nitrogen at room temperature. Solid phase extraction is then performed using a phenomenex Strata-X weak anion column. The column is conditioned with methanol then water. Samples are loaded followed by a 2% formic acid wash, then a methanol wash. Acyl-CoAs are eluted with 2% ammonium hydroxide then 5% ammonium hydroxide. The pooled eluates are dried down under a steady stream of nitrogen and reconstituted in 100μL 5% methanol and loaded onto the HPLC injector. No column is used in the HPLC system, but a C18 guard column is used to prevent backpressure. Data is acquired using the 506.9 neutral loss scan using the multiple reactionmonitoring (MRM) mode using a Waters Quattro Ultima mass spectrometer. Results: This method can identify all long- medium and short-chain acyl-CoA species in wild type mouse liver and muscle including 3-hydroxy and 3-keto species. We have also confirmed this method in liver of short-chain-acyl-CoA dehydrogenase (SCAD) knock-out mice. As expected, there is a significant increase in [C4] butyrylCoA species in the SCAD -/- mouse liver compared to wild type. Conclusions: This method will allow us to detect the buildup of fatty acyl-CoA metabolites in the various tissues of mice with other defects of fatty acid oxidation including LCHAD, TFP, VLCAD, LCAD, MCAD, CPT-I, and SCHAD deficiencies.

140 RESVERATROL AND SIRT1 ACTIVATORS, A NEW CLASS OF COMPOUNDS FOR PHARMACOLOGICAL RESCUE OF MITOCHONDRIAL ß-OXIDATION DEFECTS. 1 1 1 1 J Bastin , F Aubey , F Djouadi CNRS FRE 3210, Faculté Necker-Enfants Malades, Paris, France Resveratrol (RSV) is a natural polyphenol compound found in red grape and other plants, which is extensively studied for its potential anti-cancer and cardioprotective properties. Recent data suggest that RSV might impact mitochondrial energy metabolism in mice, and we therefore tested RSV effects in fibroblasts from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency. Addition of RSV in the culture medium stimulated residual Fatty Acid Oxidation (FAO) capacities (3H-palmitate oxidation) in a dose- (20µM to 80µM) and time-dependant manner in fibroblasts from patients with the mild form of CPT2 or VLCAD deficiency. Furthermore, complete correction of FAO was achieved in patient fibroblasts with different CPT2-deficient or VLCADdeficient genotypes, after treatment by RSV at 80µM for 48h. Quantitative PCR revealed a +30 to +60% CPT2 and VLCAD mRNA increase in response to RSV, while western-blot indicated a marked RSV-induced up-regulation of CPT2 or VLCAD protein in the treated patient cells. We investigated in parallel the level of oxidative stress using the DCFH-DA fluorescent probe, and found that several VLCAD-deficient patient cells exhibited abnormally high ROS production levels that returned to normal after exposure to RSV (80µM, 48h). Altogether, these preliminary data indicate that RSV can both correct FAO deficiency, and reduce oxidative stress, in ß-oxidation-deficient patient cells. The mechanisms of action of RSV on mitochondrial metabolism are not yet delineated but could be mediated by a cascade involving SIRT 1, a member of the NAD-dependant histone deacetylase family, and the alpha isoform of the PPAR Gamma Co-activator 1 (PGC-1α), a key player in mitochondrial energy metabolism regulation. FAO studies revealed that exposure of patient fibroblasts to a SIRT1 activator (CAY10591, Cayman chemicals) could mimic the stimulatory effects of RSV in FAO-deficient fibroblasts, whereas addition of SIRT1 inhibitor Sirtinol prevented the RSV-induced increase in FAO. In parallel, patient cells treated with siRNA directed against PGC-1α exhibited a markedly blunted response to RSV in FAO studies. These initial results suggest that the SIRT1/PGC-1 α signaling pathway represents a highly relevant therapeutic target for inborn ß-oxidation disorders, and identify natural or synthetic SIRT1 activators capable to rescue ß-oxidation defects in patient cells. This might open new avenues for pharmacological treatment of these disorders, and possibly of other mitochondrial enzyme deficiencies, since a large number of genes involved in mitochondrial oxidative metabolism could be transcriptionally controlled by PGC-1 α.

Abstracts / Molecular Genetics and Metabolism 98 (2009) 1–16

Platform Presentation 7: Organic Acid Disorders Tuesday, 1 September 2009 16:00-17:30 141 A ZEBRAFISH MODEL OF COBALAMIN C DEFICIENCY DISPLAYS GROWTH RETARDATION, DEVELOPMENT DEFECTS OF THE CENTRAL NERVOUS SYSTEM AND METABOLIC ABNORMALITIES J L Sloan1, T L Blake2, R J Chandler1, M S Tsai1, B P Brooks3, R B Sood2, C P Venditti1 1Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, United States, 2Zebrafish Core Facility, Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, United States, 3Ophthalmic Genetics and Visual Function Branch, National Eye Institute, Bethesda, MD, United States Cobalamin C deficiency (cblC) features a combined impairment of the cobalamin dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. It is caused by mutations in the MMACHC gene, which encodes an enzyme that is suspected to participate in cobalamin decyanation and intracellular trafficking. The clinical spectrum of cblC is wide and can feature prenatal manifestations, such as congenital microcephaly and intrauterine growth retardation (IUGR). To examine the phenotype caused by loss of function of this gene, we have created and studied a zebrafish model of cobalamin C deficiency. The zebrafish MMACHC gene was identified using informatics and cDNA cloning. FITC-tagged morpholinos targeting the cognate ATG and an exonic junction were designed and used to knock-down the zebrafish ortholog. Morphants began to display defects at 24 hours, including brain necrosis and diminished movement when compared to injected controls. The morphants had delayed hatching and were significantly smaller in size, had less blood, smaller heads and eyes, and were neurologically impaired compared to controls. By 96 hours, injected fish displayed pericardial edema. Both morpholinos produced a similar phenotype, which was dose-dependent. Additionally, histologic examination revealed a fatty liver. Further investigation using fish expressing GFP in the CNS revealed underdeveloped brains and absent Rohon-Beard neurons. Metabolic analysis of extracts derived from the morphants showed increased methylmalonic acid (MMA) and cystathionine, and MMA was increased in fish incubated with the precursor, propionic acid (PA). Furthermore, the mutant fish were sensitive to PA, such that they died earlier than the mutant fish not incubated with PA. Future studies will be conducted in fish lacking the p53 gene to confirm that the phenotype is not due to off-target effects. The model presented here is the first animal model of cblC and faithfully replicates some of the more severe findings observed in humans. Furthermore, it demonstrates the utility of zebrafish to easily examine aspects of metabolic diseases that will be difficult to study in other organisms, such as embryonic manifestations, and should facilitate the exploration of the underlying pathophysiological mechanisms and testing of new therapies for cblC. 142 IDENTIFICATION OF A PUTATIVE LYSOSOMAL COBALAMIN EXPORTER MUTATED IN THE CBLF DEFECT OF VITAMIN B12 METABOLISM F Rutsch1, S Gailus1, I R Miousse2, T Suormala3, C Sagné4, M R Toliat5, G Nürnberg5, T Wittkampf1, I Buers6, A Sharifi4, M Stucki7, C Becker5, M Baumgartner7, H Robenek6, T Marquardt1, W Höhne8, B Gasnier4, D Rosenblatt2, B Fowler3, P Nürnberg5,9 1General Pediatrics, Muenster University Children's Hospital, Muenster, Germany, 2MUHC McGill University, Montreal General Hospital, Montreal, QB, Canada, 3University Children's Hospital, Basel, Switzerland, 4Centre National de la Recherche Scientifique - UPR 1929, Paris, France, 5Cologne Center for Genomics and Institute for Genetics, Cologne, Germany, 6Leibniz Institute for Arteriosclerosis Research, Muenster, Germany, 7University Children's Hospital, Zurich, Switzerland, 8Charité University Hospital, Berlin, Germany, 9Cologne Excellence Cluster on Cellular Stress Responses and Aging-associated Diseases (CECAD), Cologne, Germany Vitamin B12 (cobalamin) is essential for normal human development and survival. Before serving as a substrate for the coenzymes 5’adenosylcobalamin and methylcobalamin in the mitochondria and cytoplasm, cobalamin must pass through lysosomes. In the cblF defect of vitamin B12 metabolism, transcobalamin-bound cobalamin is endocytosed into cells, but free cobalamin accumulates in lysosomes presumably because of defective lysosomal export. Accordingly, synthesis of the two cobalamin coenzymes is deficient. Microcell-mediated transfer of wild-type human chromosomes into immortalized fibroblasts from a cblF patient increased the incorporation of label from [14C]propionate towards normal values in clones isolated after the transfer of chromosome 6. Using high-density SNP arrays, we performed homozygosity mapping in 12 unrelated patients with cblF disease from different outbred populations and identified LMBRD1 as a positional candidate gene on chromosome 6q13. LMBRD1 encodes LMBD1, a 61.4 kDa protein with 9 putative transmembrane helices, which shows significant homology to the lipocalin membrane receptor LIMR. Four different frameshift mutations leading to premature termination codons and down-regulation of the transcript resulted in a loss of function of both LMBRD1 alleles in the patients. 18 of the 24 disease chromosomes analysed carried the same mutation embedded into a common haplotype of 1.34 Mb. Transfection of cblF patient fibroblasts with the LMBRD1 wild-type construct rescued cobalamin coenzyme synthesis and function. EGFP-tagged LMBD1 colocalized with the lysosomal membrane marker LAMP1. This identifies the gene for cblF LMBRD1 and suggests that LMBD1 is a lysosomal membrane transporter for cobalamin. 143 NEURAL DAMAGE IN PROPIONIC ACIDAEMIA: A MAGNETIC RESONANCE SPECTROSCOPY WINDOW TO DISEASE PATHOGENESISJ

E Davison1,2, L MacPherson1, N Davies1,2, A Chakrapani1, Y Sun2, C Hendriksz1, P McKiernan1, A C Peet1,2, P Gissen1,2 1Birmingham Children's Hospital NHS Foundation Trust, Birmingham, West Midlands, United Kingdom, 2College of Medical & Dental Sciences, University of Birmingham, Birmingham, West Midlands, United Kingdom Introduction: Propionic acidemia (PA) is characterised by acute metabolic decompensations, commonly followed by residual neurological damage to basal ganglia, resulting in progressive neurological impairment. The pathogenesis of this damage is not fully understood but ammonia toxicity resulting in astrocyte glutamine accumulation and secondary energy metabolism impairment are the proposed causes. We employed magnetic resonance spectroscopy (MRS) to investigate metabolic changes in well and acutely sick children with PA. Patients and Methods: 8 children (2 females, mean age 5.7y) with PA had 11 MRS scans. 2 of the patients had received orthotopic liver transplant (OLT). Scans were obtained in 3 children during acute encephalopathic episodes. MRS was performed when MRI scan was clinically indicated on GE or Siemens 1.5T scanner. Point resolved spectroscopy technique was used with 2cm sided voxel in basal ganglia (BG) and parieto-occipital white matter (WM). Quantitation of metabolites was achieved by LCModel software. MRS results were compared by independent-sample t-test with a large cohort of children (n=149 BG, n=114 WM) with various metabolic or suspected neurodegenerative pathologies. Results: There was no significant difference in mean NAA, Choline, Creatine or inositol between the PA cohort and comparator group. Lactate was detected reliably in 6 out of 8 including 1 post-OLT. In one acutely encephalopathic child lactate was not detected but the 1.3ppm area was dominated by other large signal peaks. Glutamate (Glu), glutamine(Gln) and glutamate+glutamine (Glx) were lower in the PA cohort in BG and WM, reaching statistical significance in BG for Gln and Glx. Glu, Gln and Glx were lower in scans obtained during acute encephalopathy (trend to significance). In 3 children with scans during acute encephalopathy, a complex prominent signal was returned in range 0.8-1.4ppm, with peaks at 0.9, 1.3 and 1.4ppm. Peaks were larger in BG than WM. Summary and Conclusions: In agreement with previous studies, lactate was detected in PA patients even during metabolic stability, however in contrast to previous findings we demonstrated reduced Glu and Gln particularly during acute encephalopathy. Peaks detected at 0.8-1.3ppm during encephalopathy may represent mobile lipids liberated by acute cell damage/necrosis or accumulation of other cerebrotoxic metabolites including branch chain amino acids. These data demonstrate that MRS is able to offer insight in to real-time in vivo metabolic derangements, and is feasible to be employed in children with acute encephalopathy. 144 MATERNAL RIBOFLAVIN DEFICIENCY, MIMICKING NEONATALONSET GLUTARIC ACIDURIA TYPE 2, IS CAUSED BY MUTATION OF THE RIBOFLAVIN TRANSPORTER, GPR172B J Christodoulou1, G Ho1, A Yonezawa2, S Masuda2, K-I Inui2, K G Sim1, K Carpenter1, J J Mitchell3, W J Rhead4, R Olsen5, G Peters1 1Western Sydney Genetics Program, Children's Hospital at Westmead, Westmead, NSW, Australia, 2Kyoto University Hospital, Kyoto, Japan, 3Montreal Children’s Hospital, Montreal, Quebec, Canada, 4Children’s Hospital of Wisconsin, Milwaukee, Wisconsin, United States, 5 Aarhus University Hospital, Skejby, Denmark Riboflavin, or vitamin B2, is a precursor to flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), both used in biological oxidation-reduction systems. We have previously reported a case of a newborn female who had clinical and biochemical features of multiple acyl-CoA dehydrogenation deficiency (MADD; glutaric aciduria type 2), which was rapidly corrected by riboflavin supplementation. Further investigations revealed that the mother was persistently riboflavin deficient, suggesting that a possible genetic defect in riboflavin transport in the mother was the cause of the transient MADD seen in the infant. The G protein-coupled receptor 172B (GPR172B) has recently been shown to be a riboflavin transporter RFT1 in humans and rats. This transporter is highly expressed in the small intestine, and even more so in the placenta, and is therefore a likely candidate gene for the riboflavin deficiency in the mother. The GPR172B gene was analysed in the mother. Using quantitative real-time PCR of genomic DNA, a deletion in GPR172B spanning exons 2 and 3 was identified in one allele from the mother, and two missense sequence variations, c.209A>G [p.Q70R] and c.886G>A [p.V296M] were identified in the other allele. The former variation has an allele frequency of 0.973 in the HapMapCEU (European) dataset, whilst the latter has an allele frequency of 0.136. In vitro functional studies of the two missense variations using a HEK 293-based assay showed that riboflavin transport is not affected by these variations, either individually or together. This is the first instance of a genetic defect in the GPR172B gene potentially causing riboflavin deficiency. We postulate that haplo-insufficiency of this riboflavin transporter causes mild riboflavin deficiency, and when coupled with nutritional riboflavin deficiency in pregnancy, resulted in the transient but exquisitely riboflavinresponsive clinically significant disease seen in her newborn infant. 145 GENE EXPRESSION IN LYMPHOBLASTOID CELL LINES FROM PATIENTS WITH PROPIONIC ACIDEMIA K A Chapman1, R S Spielman2 1Genetics, The Children's Hospital of Philadelphia, Philadelphia, PA, United States, 2Genetics, University of Pennsylvania, Philadelphia, PA, United States Propionic acidemia (PA, OMIM #606054) is an inborn error of metabolism that is the consequence of the inability of propionyl CoA carboxylase to convert propionyl CoA to methylmalonyl CoA and leads to ketoacidosis, metabolic encephalopathy, movement disorders and strokes in patients. Lymphoblastoid cell lines (LCLs)

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derived from patients with PA and their carrier parents (Coriell Institute for Medical Research, Camden, NJ) were assayed for gene expression using the Affymetrix Human Gene 1.0 ST microarray (Santa Clara, CA). The change in gene expression due to this inborn alteration in the cellular biochemical milieu was then compared to expression found in LCLs from apparently healthy sex-matched control (CEPH) individuals. In LCLs from PA patients, changes in gene expression were noted in transcripts from genes which have Gene Ontology (GO) biological process terms (http://www.geneontology.org/) consistent with transcription, ion transport, signal transduction, and immune response. Obligate carrier (heterozygous) parents also had differential expression in several of these same transcripts compared to healthy controls. This finding implies that carrying a mutation in one allele of the genes for the enzyme influences gene expression levels. 146 CHARACTERIZATION OF THE MITOCHONDRIAL PROTEOME IN CULTIVATED CELLS FROM PATIENTS SUFFERING FROM ETHYLMALONIC ENCEPHALOPATHY 1 2 2 2 1 J Palmfeldt , V Stenbroen , S Vang , N Gregersen Research Unit for Molecular Medicine, Clinical Medicine, Aarhus, Denmark, 2Clinical Medicine, Aarhus, Denmark Ethylmalonic encephalopathy (EE) is a serious autosomal recessive disease with

multiple symptoms. Clinically the disease exhibits a span of characteristics (neurodevelopmental delay and regression, petechiae, orthostatic acrocyanosis and chronic diarrhea) resulting in death in the first decade of life. The biochemical findings of EE are also complex: hyperlactic acidaemia, elevated concentrations of C4 and C5 plasma acylcarnitine, and urinary excretion of ethylmalonic acid (EMA) and C4–6 acylglycines. Furthermore, the specific activity of cytochrome c oxidase has been found to be reduced in skeletal muscle. First recently results at molecular level have been presented, so that the biochemical function of ETHE1-protein now better is understood. However, although the ETHE1 protein and its involvement in sulfur metabolism, together with the symptoms, have been described we are far from being able to explain the complex etiology of EE. Since EE have multiple effects we have chosen a large scale technique –MS based proteomics -to map the consequences of EE at the protein level. The aim was to find molecular clues to the effects of mutation in ETHE1. The strategy was to enrich mitochondria from cultivated skin fibroblasts and perform mass spectrometry based quantitative proteomics to compare protein levels in homozygote patient with those of the patient’s heterozygote parents. We performed quantitative proteomics and quantified almost two hundred mitochondrial proteins and mapped the proteins into functional categories. Preliminary results indicate perturbations of the protein levels of oxidative stress protection and components of respiratory complex IV.

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Platform Presentation: Nurses Research and Clinical Project Symposium Monday, 31 August 2009 15:30-17:30 147 METABOLIC NURSE CONSULTANT/GENETIC COUNSELLOR - A MODEL OF CARE FOR METABOLIC PATIENTS AND FAMILIES H J Upton1 1Metabolic Service, Murdoch Children's Research Institute, Parkville, Victoria, Australia Children with metabolic conditions and their families have diverse and complex needs requiring a multi-disciplinary approach to their care. The Metabolic Nurse/Genetic Counsellor (MN/GC) has a multifaceted role within the Metabolic Service. The MN/GC makes first contact with a family gathering relevant information and obtaining the family pedigree. Following participation with the physician in the consultation setting the MN/GC is responsible for organizing and undertaking investigative procedures, including loading tests, daily metabolic profiles and co-ordinating biopsy procedures in liaison with laboratories. The MN/GC participates with the physician in discussing the diagnosis, treatment and follow-up with the family. Telephone triage by the MN/GC for unwell patients enables early assessment and appropriate intervention. The MN/GC also functions as a case manager when hospitalization is necessary. Ongoing education of individuals/families and allied health professionals both in the hospital and community setting and assisting in research activities is also part of the MN/GC role. The establishment of the MN/GC role has provided an innovative and invaluable resource to children and families ensuring continuity of care and improved accessibility to health services. The trust and rapport developed through the nursing role also assists in the genetic counseling setting. 148 EXPLORING PARENTS’ IDEAS FOR IMPROVING EXPANDED NEWBORN SCREENING PRACTICES J M DeLuca1, M Kearney1 1School of Nursing, University of Rocheter, Rochester, NY, United States Purpose: Expanded newborn screening has brought many additional families into referral centers for diagnostic testing. However, little is known about families’ experiences of evaluation, especially during the testing process for conditions with uncertain prognosis or treatment. The goal of this qualitative study is to explore parents’ experiences of the newborn screening evaluation process. In this component of the study parents were asked to identify barriers to care and to describe what they thought might improve the screening process for families referred for abnormal screening results. Methods: Parents without prior newborn screening experiences whose full term healthy infants were referred to four metabolic treatment centers in New York State were invited to participate in the study. In-person and telephone interviews for parents were conducted during and after the newborn screening evaluation. The interviews were transcribed, coded, and analyzed using content analysis. Interviews from 11 families of a projected total of 30 were analyzed. Results: Parents offered a wide range of opinions and comments on how to improve the newborn screening process. In the initial conversation about the abnormal screening results offered by the primary care provider or metabolic treatment center, parents appreciated a clear, straightforward explanation. Parents preferred that this discussion be delivered by a known, trusted health care provider. Contradictory or outdated information from providers perplexed parents, “he was giving us information from what I understand was actually inaccurate.” If the providers’ knowledge about newborn screening was lacking, parents preferred that providers “just say they didn’t know.” One parent cited the need for ongoing contact with the

treatment center while waiting for confirmatory results, and recommended that genetics professionals contact families at least weekly until the results were back. One couple described their debate with their poorly informed lactation consultant who advised them not to offer a bottle to their sluggish infant who was later diagnosed with MCADD, “so if I didn’t give her a bottle on Sunday, I mean, things could have been very different for us.” This couple pressed for a swifter turnaround for screening results and improved communication between providers. A universal screening panel across all states was cited as important to most families so that no infant with a metabolic disorder would be missed, “you would hate to think just because I live in a particular state my son could have seizures and not know the cause of it.” Discussion: Parents who are closely involved in newborn screening are in a unique position to offer their ideas about what works and does not work in the newborn screening process. Parent can inform and help providers envision improvements in the clinical care of expanded newborn screening families. 149 IMPACT OF A HOME CARE WORKER IN PKU AND OTHER INHERITED METABOLIC DISORDERS A MacDonald1, H Gokmen-Ozel1, H Wooldridge1, S Chahal1, A Daly1, C Neville1 1 The Children's Hospital, Birmingham, United Kingdom In patients requiring special diets with Inherited Metabolic Disorders (IMD), the practical application of diet is the cornerstone to ensuring the provision of varied and palatable meals, thereby leading to good compliance and optimal treatment outcome. This may be difficult for some carers because many special diets require cooking skills, organizational ability, literacy skills, competent parenting skills and the ability to work creatively with food to make it appealing, appetizing and to replicate ‘normal’ meals. Previous cooking failures and apprehension lead to limited food dishes being prepared. This results in monotonous food choices being offered to children who then view their diet negatively; impacting on their compliance and metabolic control. Health professionals, particularly dietitians will try and offer support. However, they are commonly hospital based and may have inadequate time to help some families with all the necessary day to day practical skills they need in order to provide appropriate care. Therefore, a new role of a ‘home support worker,’ with direct experience of IMD child care, has been created to work with families in their own homes. Aim: to evaluate the role of the ‘home support worker’ to ensure it is supportive, effective, and ultimately improves life quality of patients. Methods: the part-time home care worker had a case load of 46 patients median age 4.7y (range 3 months-54y); and all were on special diets. Records were kept of all her activities and findings. In addition, a sub-section of 21 patients/carers (n=21) gave feedback about any changes they had made since receiving this additional help and also highlighted any aspects of the home support role they found valuable. Results: All the home visits involved special diet cooking (100%), and some had help with food shopping (13%), and parenting skills (38%). In the kitchen, it was common for families to lack cooking equipment, have non-functioning equipment or be unable to use it (38%). Some carers and patients were unaware of some suitable ingredients, particularly unusual vegetables. Carers/patients were shown how to prepare a median of 9 dishes or items. In the carer/patient feedback, a median of 4 (2-11) of these dishes were being prepared longer term. Nineteen (90%) carers/patients used a median of 3 (1 to 4) new cooking ingredients. Benefits identified by carers/patients were learning new cooking techniques (67%); ideas for meals and snacks (100%); use of unusual ingredients (86%); help with child feeding difficulties (38%); shown how to use kitchen equipment (33%) and advice on kitchen equipment to buy (38%). Many commented on how home cooking visits improved their confidence and how valuable practical demonstration was in their home environment. Conclusions: By working collaboratively with families, the home support worker helped carers/patients build confidence, developed special diet cooking ability, improved parenting skills and enhanced diet quality for patients.

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Platform Presentation: Dietitians Workshop Monday, 31 August 2009 11:00-12:30 150 POST GRADUATE TRAINING IN INHERITED METABOLIC DISORDERS (IMD) FOR DIETITIANS IN EUROPE A Macdonald1, K Ahring1, A Andresen1, M Boström1, A Clark1, M Dixon1, K Dokoupil1, K Eftring1, A Faria1, A Fekete1, M Ferreira de Almeida1, C Gibson1, H Gokmen-Ozel1, C Heidenborg1, P Jacobs1, R Lilje1, R Link1, K Motzfeldt1, M Robert1, J C Rocha1, E Sjöqvist1, K Van de Kerckhove1, M van Rijn1, H Vestergaard1, D Wurzenrainer1, H Zweers1 1European Metabolic Dietetic Group (EMDG), Birmingham, United Kingdom An agreed standard of post-graduate training should be an essential requirement for clinical dietitians and nutritionists working in Inherited Metabolic Disorders (IMD). For IMD dietitians within Europe, there is no consensus about their required knowledge, skills and competencies to ensure they are safe and effective practitioners. There is also no documentation about the training available for IMD dietitians in individual countries. Aim: To identify the provision of IMD dietetic training across Europe. Methods: Representatives from 12 European countries (Austria, Belgium, Denmark, Germany, Ireland, Netherlands, Norway, Portugal, Switzerland, Sweden, Turkey, UK) (all members of EMDG) all supplied written information outlining their IMD dietetic training. Results: The type of postgraduate training, and training validation was highly variable. Only two countries ran formal postgraduate courses (Germany and UK). The German course provides 70 hours of training over 10 days in 4 individual training blocks. Participants gain a postgraduate certificate after being tested and producing a thesis. The UK run an annual, IMD dietetic course at Masters level. This involves pre-course work, a 4 day taught component and post course work. Participants can choose to study for 20 Masters credits or for a postgraduate certificate only. Some European countries have <10 IMD dietitians so national, formal IMD dietetic courses are not justified. Instead, IMD dietitians in Sweden, Norway, Denmark, Austria, Switzerland, Turkey and the Netherlands obtain their training by in-house training, national and International meetings, shared meetings between dietitians in neighbouring countries, and visiting other IMD centres. They also may attend the formal training courses ran in Germany, UK or USA. In Belgium post graduate training differs according to area. In the French speaking community, dietitians receive a one year post specialisation course which includes 30 hours on IMD training; the Dutch speaking community receives no additional training. Southern Ireland, only have one IMD unit, with 9 IMD dietitians. Therefore, they run their own extensive in house training. Portugal offers IMD dietetic training at some of the IMD specialist centres. Conclusions: There are wide differences in post graduate IMD dietetic training opportunities. European countries should aim for core training standards and validation for the training of IMD dietitians. 151 MCT OIL BASED DIET REVERSES HYPERTROPHIC CARDIOMYOPATHY IN A PATIENT WITH VLCADD 1 2 1 1 Muhammad A Pervaiz , Fran Kendall , Rani H Singh Department of Human Genetics, Emory University School of Medicine, Decatur, GA, United States, 2Virtual Medical Practice, LLC, Atlanta, GA, United States Fatty acids are oxidized as a major source of fuel by some organs of the body especially during fasting, sustained aerobic exercise and stress. Very long-chain acyl-CoA dehydrogenase deficiency (VLCADD) is one of the genetic defects of mitochondrial fatty acid beta-oxidation presenting in early infancy or childhood manifesting with episodes of hypoketotic hypoglycemia and cardiomyopathy and may also be associated with hepatomegaly, hyperammonemia and high CPK because of rhabdomyolysis. If undiagnosed and untreated, VLCADD may be fatal secondary to the cardiac involvement. Treatment of patients with VLCADD is based on nutrition therapy and involves avoiding fasting, frequent feeding with a diet rich in carbohydrate and MCT and treating episodes of hypoglycemia with IV glucose and possibly L-carnitine. MCT does not require VLCAD for metabolism and has a primary therapeutic effect as a source of energy in patients with VLCADD. Some data suggest that MCT may help prevent hypoglycemia. We assessed the effect of replacing part of the fat in the diet of a 2.5 month old male who presented with vomiting and dehydration with MCT and essential fats. The patient had been found to have persistent elevation of liver function tests, hepatomegaly and pleural effusion. A chest x-ray revealed cardiomegaly and an EKG noted sinus rhythm with a right bundle branch block. Biventricular hypertrophy alongwith pericardial effusion was confirmed on an echocardiogram. Because of the cardiomyopathy, hepatomegaly and abnormal acylcarnitine profile and urine organic acids, he was diagnosed as having VLCADD.This was confirmaed via both enzymatic and molecular studies. He was begun on an MCT based formula with added essential fatty acids, uncooked cornstarch (around 1 year of age) and frequent feeds. Due to the aversion to the taste of the formula, G-tube was placed for improved compliance. By 7 months of age, the cardiomyopathy had reversed and by 14 months of age all cardiac medications were discontinued and hypotonia had improved such that physical therapy was no longer required. At 5 years of age he is at the 50th centile for height and weight and has normal development. Further clinical studies with a larger sample size are needed to determine optimal nutrition care for patients with these genetic metabolic disorders. 152 AN OUTDOOR ACTIVITY WEEKEND FOR PATIENTS WITH A METABOLIC DISORDER - A NOVEL APPROACH TO DIETETIC MANAGEMENT OF ADULT PATIENTS S A Adam1, J Wildgoose2, C Ferguson3 1Dietetics & Nutrition, Royal Hospital for

Sick Children, Glasgow, Lanarkshire, United Kingdom, 2Dietetics & Nutrition, St Lukes Hospital, Bradford, West Yorkshire, United Kingdom, 3Dietetics & Nutrition, Newcastle General Hospital, Newcastle, Newcastle Upon Tyne, United Kingdom The outdoor activity weekend is a popular method for supporting children with Phenylketonuria (PKU) but equivalent opportunities for adults do not exist in the United Kingdom. Given its success with children, we felt a similar activity for adults would prove valuable in their dietetic management. The weekend away was planned as an exciting opportunity to promote and support the continuation of the diet and was designed to give opportunities for patients to meet other people with the same condition, talk about their experiences in managing their diet, and to share ideas and support. The activity weekend was held over 3 days at an outdoor centre in the Lake District, England. Eleven adults with PKU and one adult with Homocystinuria from Glasgow, Newcastle, and West Yorkshire participated. They were aged between 17 and 37 years with an even mix of males and females. The weekend was split into discussion groups, cookery and leisure activities. For the discussion groups, we facilitated sharing of ideas and experiences on protein substitutes, their importance and the varieties available. They were informed of new low protein foods and how they could be incorporated into their diets. For the cooking activity, the patients were divided into teams for a low-protein cookery competition (there was a wide range of cooking skills in each team, some people’s skills were very limited but all could obtain advice from ourselves and the chefs from the centre). The leisure activities such as the high ropes challenges and gorge sliding were designed to build teamwork, self-esteem, and confidence. At the end of the weekend the patients were asked to complete evaluation forms. The results of the evaluation and our own experiences during the weekend demonstrated that it was a success. The patients reported learning the importance of continuing on their diet. Many had the opportunity to try new foods, learn new cookery skills, and exchange practical tips and ideas about their diets with their peers. The cookery teams worked well together and produced some delicious food which inspired confidence amongst the patients. The discussion groups provided an ideal forum for participants to share ideas and discuss the practical aspects of dietary treatment. The leisure activities promoted friendship and sharing across the group. We intend to follow-up the patients in due course to see if they have made any changes in their diet. If the weekend did indeed lead to long-term changes in the patients’ dietary choices then it demonstrates that such an event could prove to be a valuable treatment method for metabolic patients and for educating, engaging, and maintaining links with them. We thank the staff of the Derwent Hill Outdoor Education and Training Centre for their support through the weekend. Financial support was provided by the patients, Yorkhill Metabolic Fund, Vitaflo, and SHS International. 153 22 YEARS OF BREASTFEEDING IN PKU IN SOUTH AUSTRALIA A L Sweeney1, M J Netting2, D B Ketteridge3, J M Fletcher3,4 1Nutrition, Children's, Youth and Women's Health Service, Adelaide, South Australia, Australia, 2 Children's, Youth and Women's Health Service, Adelaide, South Australia, Australia, 3Metabolic Clinic, Children's, Youth and Women's Health Service, Adelaide, South Australia, Australia, 4Biochemical and Community Genetics, SA Pathology, Adelaide, South Australia, Australia Aim: To describe the feeding regime of infants diagnosed with phenylketonuria (PKU) by newborn screening in South Australia between 1987 and 2008 and to describe a case where a twin with PKU and her non-PKU sib were both successfully breast fed. Methods: Data were obtained from clinical records. Phenylalanine-free formula was fed first, followed by breastfeeding until satiety. The volume of phenylalanine-free formula was altered according to the infant’s blood-spot phenylalanine level, monitored twice weekly until age 3 months of age then weekly. Blood spot phenylalanine was measured by standard newborn screening techniques using Bacterial Inhibition assay between 1987 and 1999, and by tandem mass spectrometry from 1999. Case report: Both twins had been exclusively breastfed prior to diagnosis. The mother was keen to maintain breastfeeding. The nutritional challenge was to maintain adequate phenylalanine control and growth in the PKU infant, and adequate breast milk supply for the non-PKU twin. Breastfeeding was exclusive in the non-PKU infant until 4 months of age and continued in the PKU twin with supplemental phenylalanine free formula for 37 weeks. Results: 27 infants were diagnosed with PKU in this period, 13 between 1987 and 1998 and 14 between 1999 and 2008. 5 infants were on formula at diagnosis (4 19871998). 22 were being breastfed or receiving expressed breast milk and all 22 infants recommenced breastfeeding once phenylalanine levels were below 600umol/l. The overall median mean duration of breastfeeding supplemented with phenylalanine free formula was 27.4 weeks (range 0.7-65.7 weeks). Median duration of breast feeding was longer in the more recent cohort (32 weeks, compared to 19 weeks in the earlier cohort). All infants had acceptable metabolic control (aim < 350 µmol/L whole blood) and growth. Conclusion: breastfeeding in addition to a phenylalanine free formula provides safe and acceptable management for infants newly diagnosed with PKU.