SIMD Abstracts

SIMD Abstracts

Molecular Genetics and Metabolism 81 (2004) 153–186 SIMD Abstracts Table of Contents Awards . . . . . . . . . . . . . ...

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Molecular Genetics and Metabolism 81 (2004) 153–186

SIMD Abstracts

Table of Contents Awards . . . . . . . . . . . . . . . . . . . . . . . . . . . .


Invited Speakers . . . . . . . . . . . . . . . . . . . . . .


Oral Presentations . . . . . . . . . . . . . . . . . . . .


Poster Presentations . . . . . . . . . . . . . . . . . . .


Abstracts for publication only . . . . . . . . . . . . .


1096-7192/$ – see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.01.002


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

Awards Emmanuel Shapira Award Winners Judith Fleming Elena Tartaglini

Travel Award Winners Ljubica Caldovic Lina Correa-Cerro Regina E Ensenauer Marsha Fearing Ozlem Goker-Alpan Zongchao Han Renee Kinman John Koepke Vidya Krishnamurthy Hiroki Morizono Srinivas Narayan Joann Nguyen Dinesh Rakheja Christineh Sarkissian Andrea Schenone Dashuang Shi Wen-Hann Tan Elena Tartaglini Jamie Walker Chunli Yu

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186


Invited Speakers J. Muenzer C. Kalle N. Blau D. Matern J. van Hove K. Wisniewski A. Messing Y. Furukawa J. Garbern E. Sidransky M. Haffner D. Matern K. Strauss D. Koeberl A. Strauss P. Rinaldo C. Scriver G. Vladutiu P. Acosta D. Frazier R. Singh C. Trahms *Abstract provided.

Enzyme replacement therapy for lysosomal storage disorders Human gene therapy—risks and benefits BH4 therapy for phenylketonuria Acylcarnitine analysis in clinical practice New neurometabolic disorders including creatine disorders Neuronal ceroid lipofuscinosis Alexander disease* Dopa-responsive dystonia* Pelizaeus-Merzbacher and other PLP-related disorders Gaucher disease* Developing treatments for inborn errors: incentives available to the clinician Short chain acyl CoA dehydrogenase deficiency: what is it? how should we diagnose and treat it? Glutaric acidemia type I: treatment and outcome Diagnosis and management of new inborn errors of metabolism identified through tandem mass spectroscopy The changing spectrum of fatty acid oxidation disorders post-newborn screening* Error prevention in the prenatal diagnosis of disorders of organic acid and fatty acid metabolism* PKU is a problem in biology* The relationship between metabolic muscle disorders and cholesterol lowering drugs* Methods of evaluating protein status in patients with inborn errors of metabolism* MCAD deficiency: diagnosis and management. The North Carolina experience* D/G Galactosemia: to treat or nor to treat? Models of transition from pediatric to adult care*


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

Alexander disease. Albee Messing, University of Wisconsin at Madison. Alexander disease is a rare but fatal leukodystrophy that typically affects young children. The neuropathology is characterized by formation of protein aggregates, termed ‘‘Rosenthal fibers,’’ within astrocytes of the central nervous system. Patients with onsets after infancy but similar accumulation of Rosenthal fibers have previously been diagnosed as suffering from juvenile or adult variants of the same disease. Recent genetic studies demonstrate that nearly 90% of patients with infantile Alexander disease, and at least some proportion of juvenile and adult patients, carry heterozygous missense mutations in the coding region of the gene encoding glial fibrillary acidic protein (GFAP), the major intermediate filament protein of astrocytes. Most cases occur sporadically, with no apparent family history, and result from de novo mutations showing 100% penetrance. Familial forms of the disease do occur, particularly involving adults, where the possibilities of reduced penetrance and/or germline mosaicism cannot be excluded. Limited genotype–phenotype correlations exist, but only for the two most commonly affected amino acids. Dopa-responsive dystonia. Yoshiaki Furukawa. Movement Disorders Research Laboratory, Centre for Addiction and Mental Health-Clarke Division, Toronto, Ont., Canada. Dopa-responsive dystonia (DRD) is a clinical syndrome characterized by childhood-onset dystonia and a dramatic and sustained response to low doses of levodopa. There are at least three causative genes for DRD: (1) the GCH1 gene on chromosome 14q22.1–q22.2, which encodes GTP cyclohydrolase I (GTPCH), the first enzyme in the biosynthetic pathway for tetrahydrobiopterin (BH4; the cofactor for tyrosine hydroxylase [TH]), (2) the TH gene on 11p15.5, coding for the enzyme TH that catalyzes the rate-limiting step in the catecholamine biosynthesis, and (3) an as yet undefined gene on 14q13 (DYT14). Many patients with DRD, including apparently sporadic patients, have shown dominantly inherited GCH1 mutations. However, using conventional genomic DNA sequencing of GCH1, no mutations in either the coding region or the splice sites of this gene were identified in approximately 40% of DRD families. Only several families with autosomal recessive TH-deficient DRD and one pedigree with autosomal dominant DRD linked to the DYT14 locus have been reported. Thus, notwithstanding the discovery of the three causative loci for DRD, a therapeutic trial with low doses of levodopa is still the most practical approach to the diagnosis of this treatable disorder. Analyses of pterins and neurotransmitter metabolites in CSF appear to be useful for the diagnosis of GTPCH-deficient DRD and of TH-deficient DRD (the mild form of TH deficiency). Neuropathological findings (no Lewy bodies and a normal population of cells with reduced melanin in the substantia nigra) in DRD patients with GTPCH dysfunction were similar to those in a patient with DYT14 dystonia. There have been no reports of autopsied patients with THdeficient DRD. Neurochemical data suggest that striatal dopamine reduction in GTPCH-deficient DRD is caused not only by decreased TH activity resulting from a low cofactor (BH4) level but also by actual loss of TH protein without nerve terminal loss. This TH protein reduction in the striatum may be due to a diminished regulatory effect of BH4 on stability (rather than expression) of TH molecules or to a dysfunction of TH protein transport from the substantia nigra to the striatum. The extent of striatal TH protein loss could contribute to gender-related incomplete penetrance of GCH1 mutations in GTPCH-deficient DRD families. Findings of the precise mechanism of striatal TH protein loss in this major form of DRD, the actual status of dopaminergic systems in TH-deficient DRD, and the new causative gene on the DYT14 locus will better define the pathogenesis of DRD. Gaucher disease. Ellen Sidransky. NSB, NIMH and MGB, NHGRI, Bethesda, MD, USA. Gaucher disease is the recessively inherited deficiency of the enzyme glucocerebrosidase and the most common sphingolipidosis. There are

both non-neurologic and neuronopathic forms, with a continuum of diverse clinical manifestations ranging from death in utero to asymptomatic octogenarians. Type 1, or non-neuronopathic Gaucher disease, is by far the most common form, associated with hepatosplenomegaly, anemia, thrombocytopenia and skeletal involvement. Clinical, molecular and biochemical studies in humans and animals have been used to enhance our understanding of heterogeneity in Gaucher disease and to develop therapies. Genotype/phenotype studies reveal significant genotypic heterogeneity among clinically similar patients, and vastly different phenotypes among patients with the same mutations. The region surrounding glucocerebrosidase on 1q21 is particularly gene rich and there is a highly homologous pseudogene sequence 15 kb downstream. The role of recombination events within and around the glucocerebrosidase locus and the function and relationships between contiguous genes are being explored. Studies on subgroups of patients with Gaucher disease and atypical manifestations, including parkinsonism, myoclonic epilepsy, cardiac involvement, and collodion skin, seek to define other genetic or environmental factors contributing to the phenotypes. Gaucher disease is also an example where heterozygosity for a Mendelian disorder may be a risk factor for a complex disease, based on the association between Gaucher disease and parkinsonism, which became evident from the recognition of rare patients with early onset, treatment-refractory parkinsonism. In a series of seventeen such patients, there were many shared clinical features, but multiple different genotypes, including the common ‘‘non-neuronopathic’’ N370S mutation. Lewy bodies, a hallmark finding in Parkinson disease, were seen in brain regions specifically associated with Gaucher disease. Although parkinsonism is relatively common, deriving from multiple different causes, the shared clinical and neuropathologic findings in this subgroup suggest a related etiology. Family studies of probands with Gaucher disease suggested that the incidence of parkinsonism might be more frequent in obligate heterozygotes. This further prompted an examination of the glucocerebrosidase gene in probands with Parkinson disease. Complete sequencing of the glucocerebrosidase gene was performed in autopsy samples from individuals who died carrying the diagnosis of Parkinson disease and several had alterations in glucocerebrosidase. These studies provide evidence that altered glucocerebrosidase may contribute to a vulnerability to parkinsonism. Moreover, this research demonstrates that the study of rare, single gene disorders like Gaucher disease can provide a window into the etiology of more common, multifactorial genetic diseases. The changing spectrum of fatty acid oxidation disorders post-newborn screening. Arnold W. Strauss,a Ute Spiekerkoetter,a Li Ding,a Chonan Tokunaga,a Tom Zykovitz,b Deborah Marsden,c Piero Rinaldod, and Michael Bennette. aVanderbilt Children’s Hospital, bNew England Newborn Screening Laboratory, cBoston Children’s Hospital, dMayo Clinic, and eDallas Children’s Medical Center, USA. Tandem mass spectrometry (MS/MS) screening of newborn blood spots for numerous metabolic disorders, including those secondary to mutations in several enzymes of the fatty acid oxidation (FAO) pathway has been implemented in several American states and European countries for several years. The results of MS/MS screening for FAO, especially short-chain (SCAD), medium-chain (MCAD), and very-long-chain (VLCAD) acyl-CoA dehydrogenase deficiencies have substantially altered the care and prognosis of individuals with these disorders. Several conclusions are possible from these studies. First, MCAD deficiency occurs in about 1:15,000 live births in the US, and the common, A985G mutation in exon 11 accounts for fewer mutant alleles than originally believed. Many missense mutations in MCAD deficiency appear to be mild. Some apparently simple A985G heterozygotes have borderline positive newborn screens, suggesting that molecular confirmation is essential for treatment. Second, VLCAD deficiency is much more common than suspected, with about 1 case per 45,000 births; and molecular analysis suggests that the T848C mutation, altering valine-242 to alanine is quite common.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 Preliminary results suggest that MS/MS screening for VLCAD deficiency is sensitive, but that secondary samples may be interpreted as normal, even when mutations in the VLCAD gene are present. Third, trifunctional protein (TFP) or long-chain 3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency can be ascertained by MS/MS, but care in interpretation is necessary. Fourth, some FAO patients become critically ill before newborn screening is likely to be completed. We conclude that MS/MS newborn screening for FAO should become univeral, but that many issues related to sensitivity, specificity and prognosis remain to be studied. Error prevention in the prenatal diagnosis of disorders of organic acid and fatty acid metabolism. Piero Rinaldo, Si Houn Hahn, and Dietrich Matern. Biochemical Genetics Laboratory, Mayo Clinic College of Medicine, Rochester, MN, USA. Genetic counseling of a couple seeking a prenatal diagnosis of disorders of organic acid and fatty acid metabolism is essential to identify risk factors and provide a risk/benefit assessment. Tangible risks are the possibility of pregnancy loss as a consequence of the sampling procedure and particularly the potential fallacy of experimental procedures. An adequate understanding of the index case is critical: the performance of a prenatal diagnosis under different circumstances is prone to mistakes and is strongly discouraged. Other pre-requisites include evidence that the disorder is expressed in fetal tissue(s) and fluids, reliance on laboratories with sufficient experience in performing the analysis and interpreting the results, availability of a second and independent method, and approval of testing costs, which may be substantial. Methods used for prenatal diagnosis of IEM have different requirements in terms of timing, sample collection, and options for independent confirmation. Whenever applicable, molecular analysis should be the method of choice. Chorionic villus sampling (CVS) is performed earlier in the pregnancy and allows either molecular analysis or direct enzymatic analysis, which could be verified later in cultured cells. Amniocentesis is performed later in pregnancy (16–19 weeks), is a safer procedure and more importantly provides both amniocytes and cell free supernatant which can be used for direct metabolite analysis. The possibility of contamination with cells of maternal origin needs to be prevented and actively monitored. For these reasons, a variety of combinations of genotyping, enzyme assay, and metabolite analysis represent the correct approach to the prenatal diagnosis of IEM. When this is not technically feasible, at least two separate laboratories should perform the same test independently to minimize the risk of incorrect results. The report should include quantitative results matched against a statistically significant range of normal controls, evidence of quality control (results of duplicate analysis, simultaneous negative and positive controls), and a summary of the laboratory’s overall experience (number of cases tested, results obtained in affected and unaffected fetuses, respectively) with the prenatal diagnosis of that particular disorder. Finally, prenatal diagnoses of unaffected fetuses should be confirmed by biochemical testing of the newborn. PKU is a problem in biology. Charles R. Scriver. McGill University, Montreal, Canada. The histories of disease-causing PAH alleles are in the histories of human populations. Worldwide newborn screening for PKU has provided a platform for a study of human genetics—at the PAH locus. Such alleles are rare in African populations and do not seem to appear until the Out-of-Africa diaspora. (The latter is reflected in the distribution of polymorphic PAH haplotypes in modern human populations). Why is this so for the disease-causing alleles? Was there a founder effect or a selective advantage? The phenomena of demic expansion, radiation, and range expansion overseas, and of genetic drift and recurrent mutation are all reflected at the human PAH locus. The PKU/HPA phenotype reflects allelic heterogeneity. Most diseasecausing alleles are rare, and only a few are prevalent, a paradigm now


found at most human loci harboring pathogenic mutations. The majority (63%) of PAH alleles are missense. They tend to cause misfolding of the subunit with aggregation and removal in the proteasome. Might pharmacologic chaperones stabilize these mutant protein forms? Is 6R-BH4 acting accordingly in BH4-responsive PAHdeficient phenotypes? HPA also reflects locus heterogeneity. Mutations affecting enzymes in the biosynthesis and recycling of BH4 impair the catalytic role of cofactor in the phenylalanine hydroxylating reaction. Proteins involved in the total reaction (hydroxylation and recycling) appear to form an interactive network. Might there be oligogenic modulation of phenylalanine homeostasis in this circumstance? PKU/ HPA is a multifactorial phenotype. Mutations at the human PAH locus may affect integrity and function of the enzyme but exposure to dietary phenylalanine (an essential amino acid) is necessary to generate the HPA phenotype. The multifactorial nature of PKU permits therapy by restriction of dietary phenylalanine. It is a difficult treatment, particularly if continued into adult life. Alternative controls of the phenylalanine pool size (with phenylalanine ammonia lyase, for example) may become therapeutic options. Gene therapy is not yet an attractive option. Phenotype cannot be robustly predicted from PAH genotype as once anticipated—at least with regard to the degree of HPA, the excellence of cognitive outcome, etc. Modifiers of metabolic and cognitive phenotypes seem to be at work, some of which are beginning to reveal them. Accordingly 75 years after the discovery of the PKU phenotype, and 20 years after the discovery of HPA causing genotypes, it is clear that both a mutant PAH genotype and the genomic background need to be considered—an old theme in genetics and biology. What is the corresponding medical message? Know the patient and the phenotype not just the PAH genotype. The relationship between metabolic muscle disorders and cholesterollowering drugs. Georgirene D. Vladutiu and Paul J. Isackson. The State University of NY at Buffalo and The Women’s and Children’s Hospital of Buffalo, Buffalo, NY, USA. Over 5 million people in the US currently are treated with cholesterol-lowering drugs. The number of individuals taking these medications will increase to 36 million within 5 years. Side effects of HMG CoA reductase inhibitors include hepatotoxicity in 1% and severe myopathy with rhabdomyolysis in 0.1 to 0.2% of patients. The frequency of severe myopathic outcomes exceeds that found in the untreated general population by 10–20-fold. Up to 6% of treated patients experience either milder generalized myalgia unrelated to activity or exercise-induced muscle pain with elevated plasma CK. We hypothesize that the prevalence of combined or single inherited metabolic gene defects is higher than expected from the general population among patients who suffer from statin myopathies. Furthermore, since manifesting carriers for metabolic myopathies exist, we expect symptoms in carriers may also be triggered by statins. Muscle biopsies or whole blood were evaluated from more than 90 patients with statin myopathies for enzyme deficiencies or mutations, respectively, which cause common metabolic myopathies. Mutation analyses were performed for carnitine palmitoyltransferase (CPT) II deficiency, myoadenylate deaminase deficiency, and myophosphorylase deficiency. In support of our hypothesis, a significantly increased prevalence of selected metabolic myopathies was found among patients with statin myopathies. While the majority of patients with a 10-fold elevation of plasma CK had evidence for a defined underlying metabolic myopathy, affected individuals also were found among those with a wide range of CK levels including normal plasma CK. Significant secondary deficiencies also were found for certain enzymes, e.g., >50% of muscle biopsies evaluated for CPT II activity demonstrated significant reductions in activity. Correspondingly, lipid storage was present in 1/3 of these. Combined partial defects in enzymes of pertinent metabolic pathways may result in unique phenotypes or contribute to increased susceptibility to myotoxic agents such as statins. Association studies are underway using SNPs in genes either


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

known to have an impact on HMG CoA reductase inhibition by statins or that are found in metabolic pathways in which associated genetic defects have been identified from our studies. In conclusion, we have found that patients with statin-induced myopathies are at increased risk for having underlying metabolic muscle diseases and, in some cases, carrier status alone appears to contribute to the increased risk. Intrapathway genetic associations have also been found that may prove to compound the risk. Methods of evaluating protein status in patients with inborn errors of metabolism. Phyllis B. Acosta. Ross Products Division, Abbott Laboratories, Columbus, OH, USA. Objective: To describe some approaches to evaluating protein status of patients with an inborn error of metabolism using locally available physical, clinical, and laboratory data. Methods: Literature and clinical research forms review. Methods locally available for evaluating protein (and energy) status include physical growth, plasma concentrations of albumin, transthyretin (TT), and retinol binding protein (RBP) and dietary intakes of protein and energy. Results: Twenty-nine percent of patients with an organic acidemia (OA) and almost 41% of patients with a urea cycle enzyme defect (UCED) were found to be <5th centile in length at study initiation while 24 and 18% of patients with an OA or a UCED, respectively, were <5th centile in length at end of a 6-month study. Both patients with a UCED and patients with maple syrup urine disease (MSUD) showed physical symptoms of protein deficiency when only 1 amino acid was deficient in the diet. Forty-three percent of OA patients and 36% of UCED patients had both albumin and TT concentrations indicative of protein deficiency at baseline. At study end, 14% of OA patients and no UCED patients had both albumin and TT below reference values. During study, protein intakes were <100% of RDI in 31% of OA patients and 40% of UCED patients. Conclusions: Physical growth; other physical symptoms; laboratory measures of albumin, TT, and RBP; and nutrient intakes of protein and energy are all helpful local measurements useful in determining protein-energy malnutrition in patients with an inborn errors of metabolism. Applying these local measures to patients with an OA or a UCED has shown that a significant number are malnourished. Diagnosis and management of MCAD deficiency: the North Carolina experience. Dianne Frazier,a Shawn McCandless,a David Millington,b Shu Chaing,c Susan Weavil,c and Joseph Muenzera. aUniversity of North Carolina, Chapel Hill, NC, USA; bDuke University, Durham, NC, USA; cNorth Carolina Public Health Laboratory, Raleigh, NC, USA. In 77 months of tandem mass spectrometry newborn screening (MS/MS), the North Carolina Newborn Screening (NC NBS) Program identified 61 infants with medium chain acyl CoA dehydrogenase deficiency (MCADD) with an incidence of 1:12,354. All newborns whose initial C8 carnitine was P0.72 lM and C8:C10 ratio was P3.0 were referred for evaluation and confirmatory testing. The diagnostic testing included: a repeat newborn screen, plasma acyl carnitine profile, carnitine (total, free, and acyl) levels, urine organic acids, and MCAD DNA testing. Using these MS/MS cutoffs, there was only one false positive. This newborn had a normal acyl carnitine profile, urine organic acid analysis and was found to be a carrier for the common mutation. (985G > A). Several infants, who had samples taken after the immediate newborn period, and were on MCT-containing infant formulas, had abnormal levels of C8 and C8:C10, and MCADD was ruled out through confirmatory testing. Infants at risk for MCADD because of an older affected sibling had NBS samples taken from cord blood and at 12, 24, and 48 h. Affected infants had abnormal MS/MS profiles on all samples. Of the

infants for whom DNA testing was completed, 66% had 2 copies of the common mutation (985G > A), and 90% had at least one copy. At least 9 other mutations have been identified. Four infants had significant hypoglycemia in the newborn period. All these infants were homozygous for 985G > A, and were in a semi-fasted state awaiting adequate breast milk. The most important aspect of the treatment protocol for MCADD patients is to avoid fasting, especially during intercurrent illness. Caregivers were given instructions in glucose monitoring, a source of glucose gel, and an ‘‘emergency letter’’ to present at the ED, should inadequate intake become an issue. All infants were continued on their preparatory formula (except any containing MCT oil) or breast milk, with no reduction in fat content. All infants were given carnitine at a dosage of 50 mg/kg. Later counseling was given regarding a ‘‘heart-healthy’’ diet, with approximately 30% of kcal from fat after the first year, avoidance of excessive kcal, and the use of extra carbohydrates during extreme exercise. There were several documented hypoglycemic events among the MCADD infants and children. All responded to IV glucose and had no neurological sequelae. These and all the other MCADD patients had normal developmental evaluations at ages 2 and 5. Models of transition from pediatric to adult care. Cristine M. Trahms. University of Washington, Seattle, WA, USA. Problem: Young adults with inherited metabolic disorders are medically fragile thus the transition from pediatric to adult-based care is difficult. Methods: Identified transition models as: (1) a separate adult metabolic clinic, (2) transitioning care to primary care physicians in the community with consultation from the pediatric team, (3) bridging the pediatric and adult care clinics with staff overlap. Using these models required: (1) identifying barriers to care in adult based care systems; (2) developing strategies to empower young adults to have a significant role in their own health care; and (3) supporting primary care providers to understand and provide care for this group. Goals of transition: I. To identify Barriers to care. Barriers to transition are in many ways ‘fear of the unknown’ by families and care providers. Young people indicated they were ‘ready’ to transition but fearful about the process. Primary care providers indicated that without a plan for transition and support, they were reluctant to accept these adults with metabolic disorders into their practice. Building a network with a plan and a process to ensure communication between clients, families, primary care providers and the transition team, is essential. II. To prepare young adults to assume responsibility for the their management through self-care and self-advocacy. A transition protocol was developed to assess individual status. The protocol includes neuropsychological testing which supports families as they work with the transition team to establish timelines and refine expectations for independence. III. To prepare community-based health care providers to incorporate these young adults into their practices. The primary care providers were interested in specific information about disorders and treatment and requested a summary format for medical records. IV. To prepare parents to support and assist in the transition process. The Internet was used as a vehicle of communication between the transition team and families, primary care providers, and clients. This appears to be an effective way for individuals to share concerns and receive ready responses. Conclusions: Effective transition programs must be carefully organized and take advantage of the resources available. There is no one ideal transition model; all have advantages and disadvantages. For our center, a ‘centralized’ direct service model has been most effective for patients with metabolic disorders. Our emphasis is on encouraging young adults to assume responsibility for their own health care.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186


Travel Award Recipient Oral Presentations 1 2 3

L. Caldovic L. Correa-Cerro R. Ensenauer


M. Fearing


O. Goker-Alpan


C. Sarkissian


Z. Han


R. Kinman

9 10

H. Morizono S. Narayan

11 12

J. Nguyen J. Walker

Expression of wild and mutant mammalian N-acetylglutamate synthase. Development, characterization, and treatment of a hypomorphic SLOS mouse model. Novel phenotype of isovaleric acidemia associated with a common mutation identified in patients diagnosed by newborn screening. Maternal liver diseases (MLD) in the pregnancies of infants with the spectrum of fatty acid oxidation defects (FAOD) compared to matched population controls. Altered lysosomal targeting in fibroblasts from patients with acute neuronopathic Gaucher disease. Intravenous recombinant phenylalanine ammonia lyase treatment reduces brain phenylalanine in PKU mouse. Preliminary study of hammerhead ribozyme-mediated knockdown of rat PDC E1a mRNA in vitro. Potential of triheptanoate emulsion for the parenteral and enteral treatment of long-chain fatty acid oxidation disorders: metabolic studies in normal rats. Homology modeling of the human mitochondrial ornithine transporter. CLN3L, a novel alternatively spliced protein related to the batten disease protein is over expressed in CLN3L3)/) mice and in Batten disease. Glucocerebrosidase mutations in two cohorts with Parkinson disease. Glucocerebrosidase isoforms in brains of patients with Gaucher disease.


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

1. Expression of wild type and mutant mammalian N-acetylglutamate synthase. Ljubica Caldovic, Hiroki Morizono, Maria Panglao, Xiaolin Yu, Dashuang Shi, and Mendel Tuchman. Children’s Research Institute, Children’s National Medical Center, The George Washington University, Washington DC, USA. N-acetylglutamate synthase (NAGS, EC catalyzes the formation of N-acetylglutamate (NAG) which is an obligatory allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme of the urea cycle. This enzyme is localized in the mitochondrial matrix of the liver and intestinal cells and has a potential regulatory role in ureagenesis by supplying NAG for modulating CPSI activity and thus ureagenesis flux. The complete coding sequence of the human NAGS gene was recently identified in our laboratory. The human NAGS protein is 534 amino acids long and is 85% identical to the mouse NAGS. The N-terminus of the mature mouse NAGS protein was determined by expression in insect cells of the preprotein in insect cells followed by purification and protein sequencing. Two processing sites for the mitochondrial processing peptidase were identified. One processing site is after amino acid 50, yielding mature NAGS protein and the second site is after amino acid 85, yielding the conserved domain NAGS. Coding sequences for the mature human NAGS and conserved domain NAGS were cloned by PCR amplification from the liver cDNA library. Both purified proteins had arginine-responsive NAGS catalytic activity. Patients who had undiagnosed hyperammonemia were chosen for initial screening based on the results of their clinical and biochemical tests. Exons and intron/ exon boundaries of the NAGS gene were sequenced from genomic DNA. Deleterious mutations (W324Ter and 1025delG), resulting in the absence of functional NAGS was found in two patients. In the third patient, sequencing of the NAGS gene revealed two different mutant alleles. One mutation is in the acceptor splice site of intron 4 (AG to AC) and the other changes an amino acid (R509Q) that is part of a conserved motif close to the C-terminus of the NAGS protein. The recombinant R509Q mutant NAGS has approximately a twofold lower specific activity than the wild-type protein. The fourth patient was a compound heterozygote for two amino acid substitutions: V173E and T431I. Both recombinant mutant proteins have a hundredfold lower specific activity than the wild-type NAGS. Biochemical characterization of the wild type and R509Q mutant proteins is in progress. 2. Development, characterization, and treatment of a hypomorphic SLOS mouse model. Lina S. Correa-Cerro,a C.A. Wassif,a L. Kratz,b R.I. Kelly,b and F.D. Portera. aNICHD, National Institutes of Health, Bethesda, MD, USA; bThe Kennedy Krieger Institute, Baltimore, MD, USA. Introduction: The Smith–Lemli–Opitz syndrome (SLOS) is an autosomal recessive, multiple malformation syndrome due to mutation of the 7-dehydrocholesterol reductase gene (DHCR7). DHCR7 reduces 7-dehydrocholesterol (7-DHC) to cholesterol. SLOS patients typically have decreased cholesterol and increased 7-DHC levels. Clinical manifestations are facial abnormalities, mental retardation, and limb defects including 2–3 toe syndactyly. The most common missense mutation in DHCR7 is 278C > T (T93M). Dietary cholesterol supplementation has been used to treat SLOS patients and small trials of simvastatin therapy to decrease 7-DHC levels have been reported. Objective: To investigate therapeutic interventions for SLOS. Methods: We generated a hypomorphic SLOS mouse model by ‘‘knocking-in’’ a T93M mutation using targeted homologous recombination in embryonic stem cells. Results: T93M homozygous and T93M/null are viable, fertile, and appear to have normal growth. Phenotypically T93M/T93M and T93M/null mice have mild dilatation of the third and lateral ventricles, and T93M/null mice have 2–3 toe syndactyly. Two to three toe syndactyly is the most common physical finding in SLOS patients. Sterol profiles analyzed by Gas Chromatography/Mass Spectrometry of brain, liver, and kidney in both one-

day- and six-week-old mice showed elevated 7-DHC. As expected, 7DHC levels were higher in T93M/null compared to T93M/T93M mice. To experimentally evaluate dietary cholesterol supplementation we compared T93M/null and wild-type control mice at 8 weeks of age on a regular versus cholesterol supplemented diet. After 5 months, no survival or pathological differences were found. Sterol analysis of tissues showed biochemical improvement in some peripheral tissues; however, brain sterol levels were not changed. Neuromuscular testing (vertical pole and hanging wire) indicated that cholesterol supplementation might improve neuromuscular status. Treatment of T93M/null mice with simvastatin at doses of 10, 20 or 30 mg/kg/day by subcutaneous injection at 2–5 months of age for 3 weeks significantly decreased 7-DHC levels in serum, and in some peripheral tissues. Notably, simvastatin therapy also decreased dehydrocholesterol levels in the brain. Conclusion: We are describing the development of a viable SLOS mouse model, and have demonstrated a beneficial effect of both cholesterol and simvastatin therapy in this mouse model. 3. Novel phenotype of isovaleric acidemia associated with a common mutation identified in patients diagnosed by newborn screening. Regina E Ensenauer,a,b J. Vockley,a S. Gru¨nert,c B.K. Burton,d J.M. Willard,a J.O. Sass,c P. Rinaldo,a,b and D. Materna,b. a Departments of Medical Genetics and bLaboratory Medicine and Pathology, Mayo Clinic College of Medicine, Rochester, MN, USA; cUniversity Children’s Hospital Freiburg, Germany; dDepartment of Genetics, Children’s Memorial Hospital, Chicago, IL, USA. Objective: Isovaleric acidemia (IVA) manifests as either acute neonatal encephalopathy or recurrent episodes of vomiting and lethargy, and developmental delays. Highly heterogeneous mutations in the IVD gene have been characterized. We report the occurrence of a common mutation among patients diagnosed by newborn screening (NBS) that is associated with a previously unrecognized mild phenotype of IVA. Methods: Sixteen newborns were identified with C5 acylcarnitine (C5AC) elevations by NBS using MS/MS. Follow up testing included plasma acylcarnitine, urine organic acid/acylglycine, and urine acylcarnitine analyses. Isovalerylcarnitine concentrations of culture medium of fibroblasts or lymphoblasts were determined following incubation with palmitate, carnitine and isotopically labeled valine, and isoleucine. Sequencing of PCR products of all 12 exons of the IVD gene was performed. The presence of the 932C > T mutation was determined by PCR and restriction enzyme analysis. Results: Sixteen IVD gene mutations were identified; 12 of them were new. Notably, the 932C > T (A282V) mutation was found in 14 of 32 (44%) of mutant alleles, including 4 homozygous cases. All were healthy at the time of diagnosis. This allele was not present in 100 randomly selected subjects with normal NBS results. In families harboring the 932C > T mutation, screening of older and asymptomatic siblings led to the identification of 3 additional cases. However, 2 other patients who also are compound heterozygous for the 932C > T mutation and a second mutant allele had mild developmental delay when diagnosed with IVA at age 4 years. In patients harboring the 932C > T mutation, urine IVG (median: 80 mmol/mol creat., range: 6–281; controls: <10) and C5AC (median: 25 mmol/mol creat., range: 4–605; controls: <7.5) concentrations were lower than in 18 symptomatic patients (IVG median: 1222 mmol/mol creat., range: 685–4541; C5AC median: 1495 mmol/mol creat., range: 11–3323). In vitro acylcarnitine profiling clearly differentiated patients from normal controls. Cell cultures of the 4 newborns who are homozygous for the 932C > T mutation produced isovalerylcarnitine concentrations from 0.2 to 0.4 lmol/g protein (controls: median 0.04, range 0.03–0.05), whereas 94% (15/16) of the symptomatic IVA patients showed levels >0.4 lmol/g protein (median: 0.7; range: 0.3–1.3). Conclusions: Patients with IVA diagnosed by NBS and carrying a 932C > T mutant allele can exhibit a milder, potentially asymptomatic phenotype. This is a major departure from our understanding of the natural history of the disorder, with implications for management and

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 genetic counseling. Follow up urine IVG analysis and in vitro acylcarnitine profiling appear to be of prognostic value in IVA. 4. Maternal liver diseases in the pregnancies of infants with the spectrum of fatty acid oxidation defects compared to matched population controls. Marsha K. Fearing,a,b,c Harvey L. Levy,a,c Louise E. Wilkins-Haug,a,d Cecilia Larson,e and Vivian E. Shiha,b. aHarvard Medical School, bMassachusetts General Hospital, cChildren’s Hospital Boston, dBrigham and Women’s Hospital, and eNew England Newborn Screening Program, Boston, MA, USA. Objectives: Infant fatty acid oxidation defects (FAOD) are rare conditions, occurring in 1:12,000 births. Increasingly, fetal long chain FAODs are associated with rare maternal pregnancy complications, including acute fatty liver of pregnancy (AFLP) and hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome. Maternal liver disease (MLD) in the general population has a low prevalence rate of only 0.1–1.5% for AFLP and 0.6–1.0% for HELLP syndrome. Given the paucity of these conditions, elucidating the true epidemiological relationship is difficult. The lack of literature comparing the entire spectrum of FAOD and pregnancy outcomes compared to the population led us to perform the following study. Methods: Fifty case infants with fatty acid oxidation defects (FAOD) were identified in the New England region, either clinically or by expanded panel MS/MS newborn screening. For each affected infant, 25 controls were selected for each case, matched by date of birth and hospital setting, generating a total of 1300 infant–mother pairs. The pairs were phenotyped for antenatal, intrapartum and neonatal characteristics. The pairs were analyzed using a conditional logistic regression model. Results: Case and control infants analyzed were similar with respect to mean gestational age = 38.2 (SD ± 2.1) weeks, and 37.8 (SD ± 3.6) weeks, mean birth weight = 3264 (SD ± 577) g and 3308 (SD ± 446) g; and maternal age 30.2 (SD ± 5) years and 28.4 (SD ± 6) years for the FAOD and control infants, respectively. In the antenatal period, MLD was noted in 16% of all FAOD pregnancies (equally represented in long versus short-medium chain defects) compared to 0.88% in the general population (OR = 20.4; 95% CI = 7.8–53.2). Isolated preeclampsia without hepatic involvement was not significantly different between the case (6%) and control pregnancies (6.1%). Post-natal results included elevated rates of neonatal jaundice [FAOD 36%, control 8% (OR 6.25; CI = 3.42–11.4)]. Subgroup analysis of all FAOD infants revealed 32% had long chain defects and 68% had a medium or short chain defects, without a significant difference in demographic characteristics. Conclusions: MLD is significantly higher across the entire spectrum of FAOD demonstrating an 18.1-fold increase in the pregnancies of FAOD neonates compared to our control population. Notably, the prevalence is equally high in the pregnancies of infants with short and medium chain defects and not isolated to those infants with long chain FAOD, implicating the entire spectrum of the acylcarnitine intermediates. 5. Altered lysosomal targeting in fibroblasts from patients with acute neuronopathic Gaucher disease. Ozlem Goker-Alpan, Eduard Orvisky, Barbara Stubblefield, and Ellen Sidransky. Section on Molecular Neurogenetics, NIMH, and Medical Genetics Branch, NHGRi, NIH, Bethesda, MD, USA. Objective: To determine whether aberrant glucocerebrosidase alters proper targeting to the lysosomes in fibroblasts from patients with Gaucher disease and if so, whether any correlation exists between abnormal protein trafficking and patient phenotypes. Background: Gaucher disease (GD), the inherited deficiency of glucocerebrosidase (GC) results in deposition of glucocerebroside in various organs, including the nervous system. The disease has been classified into three major groups depending upon the presence and progression of nervous system involvement and the age of onset: type 1, nonneuronopathic; type 2, acute neuronopathic; and type 3, subcaute neuronopathic GD. The gene for human GC is located on chromosome 1q21 and most of


the disease-causing alleles are missense mutations leading to the synthesis of GC with decreased catalytic function and/or stability. GC is a peripheral membrane glycoprotein, and proper targeting to the lysosomes is crucial for the normal catalytic activity. Unlike other lysosomal hydrolases, GC is targeted to the lysosomes via mannose-6phosphate receptor-independent pathway. The mechanisms of folding, processing, and targeting of GC to the lysosomes still remain unknown and how the aberrant GC is targeted might impact upon disease severity and progression. Methods: Colocalization experiments were performed in fibroblast cell lines from five patients with type 2 and one with type 1 GD and compared to three normal fibroblast cell lines at a similar passage number. Double immunofluorescence staining was carried out using different antibodies against the different cellular components in the protein trafficking pathway, together with anti-GC antibodies. The slides were viewed with a Zeiss Axiovert 100 scanning confocal microscope. Genotyping and enzymatic essays were performed as previously described. Results: The genotypes encountered in the type 2 GD cases varied, but mostly included missense/missense or missense/null alleles. The genotype for the type 1 GD case was N370S/ N370S. The enzymatic activity ranged from 1 to 7% in type 2, and was 14% of the control in type1 fibroblasts. In all the cell lines from patients with type 2 GD, GC colocolized with the early endosomal marker but not with any of the lysosomal markers. Conclusions: GC is not properly targeted to the lysosomes in cell lines from patients with acute neuronopathic GD. This defect may play a role in the poor correlation between enzymatic activity and phenotype in these patients. Exploration of the cellular processes involving the mutant GC will provide insights into normal cellular trafficking machinery. 6. Intravenous recombinant phenylalanine ammonia lyase treatment reduces brain phenylalanine in PKU mouse. Christineh N. Sarkissian,a Me´lanie Hurtubise,a Charles A. O’Neill,b Steven Striepeke,b Dan Oppenheimer,b Paul Fitzpatrick,b and Charles R. Scrivera. a Department of Biology and Human Genetics, McGill University, Montre´al, Que., Canada; bBioMarin Pharmaceutical Inc., Novato, California, USA. PKU can be effectively managed with strict application of a low phenylalanine (phe) diet. However, compliance is difficult; and restriction for life, as currently recommended, is probably unrealistic. A new potential treatment using recombinant phenylalanine ammonia lyase (rPAL) is under development. rPAL eliminates phe by converting phe into trans-cinnamic acid and ammonia. Here we report that intravenous administration of rPAL reduces both plasma and brain phe levels in the PKU mouse model. The effect of varying the dose and the time course response was analyzed following soluble rPAL administration via the intravenous route. A dose response effect, 24 h post-treatment, on plasma phenylalanine was apparent (control = 2049.33 ± 75.5 lM; 0.75 IU rPAL = 706.8 ± 57.8 lM; 3.75 IU rPAL = 55.9 ± 6.3 lM; p < 0.012). This effect was also evident in the brain phenylalanine counterparts (control = 745.1 ± 24.05 lM; 0.75 IU rPAL = 559.18 ± 51.07 lM; 3.75 IU rPAL = 161.7 ± 31.37 lM; p < 0.015). Diminished phenylalanine levels remained evident up to 48 h post-treatment at the higher dose of rPAL; plasma (control = 2171.7 ± 149.53 lM; 3.75 IU rPAL = 767.0 ± 192.46 lM; p < 0.0013) and brain (control = 688.0 ± 17.95 lM; 3.75 IU rPAL = 459.84 ± 57.49 lM; p < 0.009). This reduction in whole brain phe is not accounted for by the change in intra-vascular phe content. Our findings further support the rationale for rPAL treatment via intravenous injection, which favorably alters the flux and equilibrium of phe between the brain and other body compartments. 7. Preliminary study of hammerhead ribozyme-mediated knockdown of rat PDC E1a mRNA in vitro. Zongchao Han,a Joshua C. Harrison,a J.R. James Thomas,b Alfred S. Lewin,b and Peter W. Stacpoolea,c,d. a Department of Medicine (Division of Endocrinology and Metabolism), bMolecular Genetics and Microbiology, cBiochemistry and


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

Molecular Biology, and dGeneral Clinical Research Center, University of Florida, College of Medicine, Gainesville, FL 32610, USA. Objective: Pyruvate dehydrogenase complex (PDC) deficiency is usually caused by mutations in the E1a subunit of the PDC that give rise to congenital forms of lactic acidosis. Insight into the biochemical and clinical phenotype of this disease has been hampered by lack of a viable animal model. Induction of complete deficiency of PDC E1a by gene knockout techniques was lethal to mouse embryos, indicating the critical importance of PDC in early growth and development. We employed an in vitro RNA interference strategy to initiate our attempt to create an animal model of E1a deficiency. Methods: Two hammerhead ribozymes, Rz422 (50 -AUC AAC UGA UGA GCC GUU CGC GGC GAA ACC CGC-30 ) and Rz1431 (50 -GCA UAC UGA UGA GCC GUU CGC GGC GAA AGC CUC-30 ), which cleave the rat PDHA1 mRNA at position 422 and 1431, were designed using the Mfold program. Ribozymes considered to cleave the mRNA of the E1a subunit were test in vitro and further cloned into an AAV based vector, p21NewHp, to assess the validity of producing a gene knockdown in rats. Using pUF5-GFP to obtain optimal transfection efficiency (same promoter as p21NewHp) produced prior transfections in rat lung fibroblasts (RLFs). Cultured RLFs were transfected with p21NewHp-Rz422 or p21NewHp-Rz1431 containing DNA that coded for one of the two ribozymes. PDC E1a mRNA quantitation was performed by RT-PCR. PDC enzyme activity was measured by the rate decarboxylation of 1-14C-pyruvate. Results: Using RT-PCR on the RNA extracts, we found the ribozymes decreased the E1a mRNA 16–24%, compared to control. Transfection efficiency, measured by GFP expression, was approximately 20%, which suggests that the ribozymes were 60–80% effective in cleaving the target mRNA. PDC specific activity was 50–60% lower in ribozyme-treated, compared to control, cultures. Conclusion: An in vitro ribozyme-mediated gene knockdown of E1a was achieved and was associated with a decrease in PDC activity. A similar strategy maybe practical for creating organspecific PDC deficiency in vivo. 8. Potential of triheptanoate emulsion for the parenteral and enteral treatment of long-chain fatty acid oxidation disorders: metabolic studies in normal rats. Renee P. Kinman,a,b Kathryn A. Jobbins,a Katherine Thomas,a Takhar Kasumov,a Jillian Adams,a Lisa N. Brunengraber,a and Henri Brunengrabera. aDepartments of Nutrition and bPediatrics, Case Western Reserve University, Cleveland, OH, USA. Current treatment of long-chain fatty acid oxidation disorders is often ineffective, with patients developing cardiac, muscular, neurologic, and hepatic dysfunction. It is hypothesized that these complications could result in part from increased cellular leakage of citric acid cycle intermediates, leading to defects in energy production. A novel approach to circumvent this loss was recently introduced which utilizes triheptanoin, a medium chain, odd-carbon triglyceride not normally present in the diet (Roe et al, JCI 110:259–269, 2002). Triheptanoin is hydrolyzed to heptanoate, which is partially oxidized in the liver to C5 ketone bodies. These are then exported, along with part of the heptanoate, to peripheral tissues where they are converted to acetylCoA and propionyl-CoA. C5 ketone bodies can be used by brain, heart, and skeletal muscle. Propionyl-CoA is a very effective anaplerotic substrate, and can compensate for any increased cataplerosis. In this study, replacement of dietary trioctanoin by triheptanoin resulted in a rapid, sustained clinical improvement in all three patients tested. Based upon these initial results, we performed in vivo studies to further characterize triheptanoin’s metabolic effects. Rats, anesthetized with sodium pentobarbital, had catheters placed into the jugular vein and ventral tail artery. Triheptanoin was infused into the jugular vein for 90 min at 0, 10, 20, 30, and 40% of the total daily caloric requirement, and arterial samples obtained at 0, 60, 70, 80, and 90 min. In one group of rats, triheptanoin was infused intraduodenally, rather than

parenterally, at the 40% dose. Steady state heptanoate and C5 ketone body concentrations increased in a dose-response manner. In the 0% control group, steady state C4 ketone body concentrations also increased. Surprisingly, this was prevented by the 10 or 20% infusion, while higher infusion rates led to decreased C4 ketone body concentrations, presumably due to inhibition of adipose tissue lipolysis. Intraduodenal infusion resulted in complete hepatic clearance of heptanoate and the highest C5 ketone body concentrations. Thus, parenteral infusion of triheptanoin produced primarily heptanoate, whereas enteral infusion produced mainly C5 ketone bodies. Interestingly, intraduodenal infusion resulted in no significant decrease in C4 ketone bodies from basal, suggesting parenteral triheptanoin, rather than enteral, might be the best therapy in an acute lipolytic crisis. 9. Homology modeling of the human mitochondrial ornithine transporter. Hiroki Morizono,a Joelle Woolston,b Marco Columbini,b Dashuang Shi,a and Mendel Tuchmana. aChildren’s Research Institute, Children’s National Medical Center, 111 Michigan Ave NW, Washington DC 20010, USA; bDepartment of Biology, University of Maryland, College Park, MD 20742, USA. Defects in the human mitochondrial ornithine transporter (ORNT) cause hyperornithemia, hyperammonemia, and homocitrullinuria (HHH) syndrome. This disorder was first described in 1969 by Shih and colleagues, since then, approximately 50 cases have been reported. The range of severity is variable, ranging from normal development to severe mental retardation. Several mutations have been identified in patients, (G27E, P126R, E180K, delF188) and how these changes affect the biochemical properties of the protein has only just begun to be studied in detail. Bioinformatic analysis shows that ORNT is a member of the mitochondrial carrier family based on conserved regions of its primary structure. The three-dimensional structure of a related member of this family, the bovine mitochondrial ADP/ATP carrier was recently solved. The ornithine transporter and the ADP/ ATP carrier share 33.3% similarity and 22.7% identity. The coordinates of this structure were used as a basis for homology modeling of the ornithine transporter. The two primary sequences were aligned using the Needleman Wunsch algorithm, and a homology model constructed based on the satisfaction of spatial restraints. Ten gaps and insertions were required based on the alignment, however, these primarily occur in loop regions and at the ends of helices. Loop optimization was performed using conjugate gradients and molecular dynamics with simulated annealing. Refinement of the homology model is in progress. The structure shows a pore made from six transmembrane helices, and the side located in the mitochondrial matrix is predicted to contain an additional three smaller helices. The ˚ ; the same size outer diameter of the transporter is approximately 40 A as the concave active site face of ornithine transcarbamylase. Support for the hypothesis that ornithine transport is directly coupled to citrulline formation through protein:protein interactions between the transporter and transcarbamylase is seen in this docking model. It is clear that the spatial segregation between the cytosol and mitochondria requires the participation of transmembrane transporters of the metabolites for proper function. Therefore, it becomes important to consider the urea cycle to consist of more 5 enzymes, and analysis of the properties of the ornithine transporter may prove it to be an important regulatory or rate limiting step in ammonia detoxification. 10. CLN3L, a novel alternatively spliced protein related to the Batten disease protein is over expressed in CIN3)/) mice and in Batten disease. Srinivas B. Narayan,a Johanne V. Pastor,a and Michael J. Bennettb,*. aDepartment of Pathology and Children’s Medical Center of Dallas and bDepartment of Pathology and Pediatrics, Children’s Medical Center of Dallas, University of Texas Southwestern Medical Center at Dallas, TX 75390, USA. *This abstract is also available to view as poster 72.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 Background: Batten disease is a severe autosomal recessive neurodegenerative disease, which results from mutations in the Cln3 gene. The clinical course is progressive and characterized by loss of vision, seizures, loss of motor function, and behavioral alterations leading to a vegetative state and early death. A 1.02 kb deletion comprising of exons 7 and 8, resulting in the removal of nucleotides 461–677 accounts for the majority of patients with Batten disease. Knowledge of the tissue distribution of CLN3P is essential in understanding the underlying pathology in Batten disease. Methods: CLN3L mRNA was cloned and expressed in Escherichia coli expression system containing arabinose promoter sequence (pBAD/TOPO expression kit, Invitrogen Life Technologies). Protein expression was induced using various concentrations of arabinose ranging from 0.00002 to 20%. Northern analysis for the CLN3 and CLN3L mRNA was performed using Neverfail human northern blots from RNWAY (Seoul, Korea). Hybridization was done using DIG-labeled cDNA probe generated from the plamid containing full length CLN3L gene. RT-PCR analysis was done using purified poly(A) mRNA from brain samples using poly(dT) oligomer for first strand synthesis and subsequently using specific primers. Immnunohistochemistry and western immunoblot analysis was performed using the specific CLN3P and CLN3LP antibodies. Discussion: We have demonstrated the presence of a novel 33 kDa protein (CLN3LP) in normal human, bovine, and wild type mouse brain. The 33 kDa protein, which is overexpressed in Batten disease and Cln3)/) mouse brain, binds to the antibody raised against peptide sequence of CLN3P and results in aberrant CLN3P localization studies. We have been able to show that our novel protein is identical to that recognized previously in Batten disease and Cln3)/) brain. Presence of mRNA for 33 kDa protein was confirmed using reverse transcription PCR in both wild type and knock mouse models. Northern blot analysis results showed an approximately 3.0 kb transcript corresponding to CLN3L from various human tissues. CLN3 mRNA (1.6 kb transcript) was also seen with similar distribution in all the lanes. Immunohistochemical analysis showed the presence of CLN3P and CLN3LP in both human and mouse brains. Western immunoblot analysis using specific antibodies against the CLN3P and CLN3LP further confirmed the presence of these proteins in normal human, bovine, and mouse brain tissues. Conclusion: These studies strongly suggest the presence of alternate spliced products in Batten disease. We submit that the previous reports of CLN3P tissue distribution and intracellular localization should now be reanalyzed in order to determine the true tissue expression of CLN3P. 11. Glucocerebrosidase mutations in two cohorts with Parkinson disease. Joann M. Nguyen,a Jamie M. Walker,a Alicia Lwin,a Ozlem Goker-Alpan,a Eduard Orvisky,a John Hardy,b Andrew Singleton,b and Ellen Sidranskya. aSection on Molecular Neurogenetics, NIMH, and Medical Genetics Branch, NHGRI, NIH, Bethesda, MD, USA; bLaboratory of Neurogenetics, NIA, NIH, USA. Objective: Parkinsonian manifestations have been noted in a rare subset of patients with Gaucher disease, the inherited deficiency of glucocerebrosidase. Brains from patients with Gaucher disease and early onset, treatment-refractory parkinsonism show brainstem-type Lewy bodies specifically associated with brain regions also affected by Gaucher disease, including the CA4–CA2 hippocampal layers. Family studies have revealed an increased incidence of parkinsonism in obligate Gaucher carriers. Such data suggests that alterations in glucocerebrosidase may contribute to a vulnerability to parkinsonism. To explore this association, we examined the glucocerebrosidase gene in subjects with Parkinson disease. Methods: The glucocerebrosidase gene was se-


quenced in 57 brain samples obtained from five different tissue banks from subjects with a primary diagnosis of Parkinson disease. A second series of lymphocyte DNA samples obtained by the National Institute of Aging from 80 patients diagnosed with early onset were also examined. Results: In the first series of 57 brain samples, alterations in the glucocerebrosidase gene were identified in 12 (21%), including eight with mutations (N370S, L444P, K198T, and R329C), of which two were N370S homozygotes, and four with polymorphisms (T369M and E326K). Among the 28 younger Parkinson probands who died before age 75, 27% were Gaucher carriers or homozygotes. Among the second series of 80 patients diagnosed with early onset parkinsonism, there were seven (9%) that had glucocerebrosidase alterations, including four with mutations (L444P, N370S, and two recombinant alleles) and three with polymorphisms (T369M and E326K). Interestingly, all four samples with glucocerebrosidase mutations are from a subset of 33 samples obtained from Venezuela. Conclusion: These results suggest that mutations in glucocerebrosidase may be a risk factor for the development of parkinsonism. Thus, the study and treatment of Gaucher disease could have implications directly relevant to patients with parkinsonism, a more common disorder. 12. Glucocerebrosidase isoforms in brains of patients with Gaucher disese. Jamie M. Walker,a E. Orvisky,a B.M. Martin,b and E. Sidranskya. a Section on Molecular Neurogenetics, NIMH, NIH, USA and Medical Genetics Branch, NHGRI, NIH, USA; bLaboratory of Neurotoxicology, NIMH, NIH, Bethesda, MD, USA. Objective: Isoforms of glucocerebrosidase from different tissues of patients with Gaucher disease and controls were studied to determine whether neuronopathic patients have a distinct form of glucocerebrosidase in the brain. Previous studies have demonstrated that glucocerebrosidase deficiency in both patients and mice with Gaucher disease leads to the accumulation of both glucocerebroside and glucosylsphingosine in all visceral organs. However, only neuronopathic patients appear to store glucosylsphingosine in the brain, regardless of genotype. This suggested that the mutant glucocerebrosidase present in the brains of neuronopathic patients might have unique properties. Methods: Protein extracts from multiple tissues from both controls and patients with differing Gaucher phenotypes were examined by the standard 4-methylumbelliferyl glucocerebrosidase assay for enzyme activity. Western blots using a polyclonal anti-glucocerebrosidase antibody were prepared from brain, liver, and spleen samples from controls and patients to compare isoforms of glucocerebrosidase. Results: Western blots performed on protein extracts from normal brains consistently showed two glucocerebrosidase isoforms, one at 56 kDa and one at 59 kDa, as compared to liver and spleen, which had the 56 kDa form and various higher molecular weight forms. After Nglycosidase F digestion, all samples demonstrated only the lowest 56 kDa form of glucocerebrosidase, regardless of the tissue source, suggestive that the 56 kDa form is unglycosylated. Brain samples from patients with all three types of Gaucher disease predominantly had the 56 kDa form, and all were deficient in the functional 59 kDa form. Residual enzymatic activity was compared in different tissues from the same patient. Generally the activity was depressed consistently in various tissues, with the highest activity found in the liver. Conclusions: While the Western patterns differed between patients and controls, we were unable to distinguish the neuronopathic forms from the nonneuronopthic forms of Gaucher disease based on the glucocerebrosidase isoforms. It is possible that the mutant protein is misfolded and incorrectly glycosylated. This leads to improper intracellular interactions with other proteins and cell components of neurons and eventual cell death.


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

Poster List 13 14 15

SIMD SIMD J. Abdenur

16 17

J. Abdenur G. Arnold

18 19

B. Barshop I. Bernardini

20 21 22 23 24

N. Blau N. Blau S. Bruni P. Chakraborty A. Chakrapani


L. Clarke


D. Corzo


D. Corzo


D. Corzo


G. Cox

30 31

R. DeBerardinis L. Fiori


D. Grange

33 34 35

A. Griffin C. Harding P. Harmatz


A. Helip-Wooley


K. Huntington


W. Introne


J. Isaacs

40 41 42

K. Ker R. Kinman S. Kleppe

43 44

R. Kleta J. Koepke

SIMD BUSINESS SIMD BUSINESS Combined enzyme replacement treatment (ERT) and bone marrow transplant (BMT) for MPS-I. Complex carbohydrates in the treatment of LCHAD deficiency. Outcome of confirmatory studies in infants referred for abnormal newborn Screening (NBS) for MCAD in New York State. Analysis of coenzyme Q10 by tandem mass spectrometry. Clinical, biochemical, and molecular findings in a free sialic acid storage disease patient of moderate severity. Cerebral folate deficiency: an underestimated neurometabolic condition. Importance of CSF in the diagnosis of pediatric neurotransmitter diseases. Disease progression in Scheie syndrome: case report. An atypical case of leukoencephalopathy with vanishing white matter. Hurler syndrome (MPS1H): enzyme replacement therapy pre-bone marrow transplantation. Aldurazyme (laronidase) enzyme replacement therapy for MPS I: 72-week extension data. Enzyme replacement therapy (ERT) for infantile onset pompe disease: long term follow-up results. Mental development in patients with infantile onset pompe disease (IOPD) treated with enzyme replacement therapy (ERT). Natural history of infantile onset pompe disease (IOPD): results from a retrospective chart review study. Aldurazyme (laronidase) enzyme replacement therapy in MPS I: preliminary data in children less than 5 years of age. Safety and efficacy of penicillamine therapy in pediatric cystinuria. Incidence of BH4-responsiveness in a population of PAH-deficient hyperphenylalaninemic infants. Sialidosis presenting with severe fetal hydrops and associated with two novel mutations in lysosomal a-neuraminidase. Hemizygosity at 10q23 in a family with Hermansky–Pudlak syndrome type 1. Low therapeutic threshold for hepatocyte transplantation in murine phenylketonuria. Pharmacokinetic profile of recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) enzyme replacement therapy in patients with mucopolysaccharidosis VI (Maroteaux–Lamy syndrome). Abnormal vesicle formation and trafficking in Hermansky–Pudlak syndrome (HPS) cells. A case management database profiling patient utilization of medical food for the treatment of phenylketonuria (PKU): a model for review of patient adherence to therapy recommendations, nutritional adequacy, indices of control and treatment costs. Expanded newborn screening for medium chain acyl-CoA dehydrogenase (MCAD) deficiency in western New York. Maternal homocystinuria: impact of gestation, birth, breast feeding on total homocysteine using dietary management and low dose heparin. Urine sulfite assay: how important is the ‘‘freshness’’ of urine samples? Sodium phenylbutyrate toxicity: case report and potential mechanism. Atypical clinical and biochemical presentations in a patient with molybdenum cofactor deficiency. FISH analysis of the common 57-kb deletion in cystinosis. Hepatocellular carcinoma (HCC) as a complication of glycogen storage disease type III.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

45 46 47

V. Krishnamurthy U. Lichter-Konecki N. Longo

48 49

M. Merideth E. Naylor


K. Okajima

51 52

S. Palmer M. Park

53 54 55 56

V. Proud D. Rakheja D. Rakheja C. Rangel


A. Schenone


J. Sharer


D. Shi


S. Sparks


S. Sparks

62 63

C. Stuart M-S. Sun

64 65

P. Suwannarat W-H. Tan


C. Tifft


C. Tifft


M. Torres


S. Young


C. Yu


Hepatocellular carcinoma in glycogen storage disease type Ia: a case series. A new prospective multi center study of treatment and outcome in urea cycle disorders. Tyrosine residues affecting sodium stimulation of carnitine transport in the OCTN2 carnitine/organic cation transporter. Clinical and molecular findings in a discordant twin with Bartter syndrome type II. Newborn and high risk screening for glutaric aciduria-type 1: results of primary tandem mass spectrometry and 2nd-tier molecular testing on over 1.8 million specimens. Somatic mosaicism in a male with an exon skipping mutation in PDHA1 of the pyruvate dehydrogenase complex results in a mild phenotype. Barth syndrome with presumed secondary carnitine palmitoyltransferase II deficiency. The cystinotic phenotype may be caused by cystine-induced apoptosis due to alterations of cytochrome C and/or PKC-d. Clinical significance of DG galactosemia: literature review and meta-analysis. CLN3P, the Batten disease protein, localizes to membrane lipid raft domains. Glutaric acidemia type I can be missed by plasma acylcarnitine analysis. 3-Methylchrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry in a Mexican neonate. Detection of inherited metabolic disorders (IMD) in cord blood samples by tandem mass spectrometry (TMS). Homocitrullinuria revisited: evaluating the significance of homocitrulline excretion in disease and normal metabolism. Fusing N-acetylglutamate synthase to green fluorescent protein and ornithine transcarbamylase to increase solubility and prevent aggregation. Hereditary inclusion body myopathy due to mutations in GNE: epimerase activity and treatment strategies. Transient sulfite oxidase deficiency and S-sulfocysteinuria due to cholestatic liver disease. Renal glucosuria due to SGLT2 mutations. generation of a conditional knock-in mouse model with deficiency of UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase (GNE) to mimic hereditary inclusion body myopathy. Therapeutic use of nitisinone in alkaptonuria. Isolated sulfite oxidase deficiency—report of a case in a newborn and review of the literature. Elevated chitotriosidase activity in CSF in patients with GM1 and GM2 gangliosidosis: a surrogate marker of disease progression? Elevated chitotriosidase activity in CSF in patients with Tay-Sachs disease: a surrogate marker of disease progression? Inborn errors of metabolism, newborn screening by tandem mass spectrometry in a Mexican population. A comparison of mass spectrometry methods for the measurement of creatine and guanidinoacetate in plasma. 10 years’ diagnostic experience in galactosemia caused by galactose-1-phosphate uridyltransferase (GALT) deficiency using combined biochemical and molecular approaches.


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13. SIMD BUSINESS. 14. SIMD BUSINESS. 15. Combined enzyme replacement treatment and bone marrow transplant for MPS-I. Jose E. Abdenur,a G. Young,b T. Zadeh,c K. Jones,a and D. Nugentb. aDivisions of Metabolic Disorders, b Hematology, and cChildren’s Hospital of Orange County and the Genetics Center, Orange CA, USA. MPS-I, a-iduronidase (IDU) deficiency has a wide spectrum of clinical presentations. The most severe cases appear in infancy with inguinal hernias, coarse facies, upper respiratory infections, progressive joint, and visceral involvement and developmental delay. Two treatments are possible: enzyme replacement treatment (ERT) and bone marrow transplant (BMT). ERT has few side effects, rapidly decreases the excretion of urinary glycosaminoglycans (GAG’s) and improves the visceral abnormalities, but does not prevent neurological deterioration. BMT has high morbidity and mortality, but it is considered the treatment of choice for patients under 2 year of age, with no or little CNS involvement. BMT may take several weeks to begin (search for donors and patient preparation) and several more weeks are needed until engraftment is achieved. During that period ERT could be administered to improve patient’s clinical condition. We report successful combined treatment of a patient with MPS-I by ERT and BMT. Patient: Medical history was significant for repaired inguinal hernias and frequent upper respiratory infections. At 7 months he was referred to Genetics due to coarse facies and motor delay. He was also found to have hepatomegaly, kyphosis, and obstructive apneas. IDU activity was in the affected range. Methods: ERT was given weekly 12· prior to BMT, and continued 4· thereafter at 0.58 mg/k/dose, via central catheter. (Aldurazyme Genzyme). BMT with unrelated adult bone marrow was performed using a standard protocol with busulfan, cytoxan, and anti-thymocyte globulin (ATG) as preparative regimen followed by cyclosporine and methotrexate to prevent GVHD. During ERT, enzyme levels were measured in leukocytes at the trough, (D.Wenger, Jefferson Univ, PA). GAG’s were measured at Mayo Clinic in first morning urine. Results: There was a clear improvement (decreased hepatomegaly and no respiratory infections) prior to BMT No adverse events were noted with ERT prior or after BMT. Engraftment was documented within 14 days after BMT. Complications included rapid engraftment pulmonary syndrome, which required treatment with albumin, diuretics, and ventilatory support for 7 days. ERT was not provided during that week. The patient fully recovered, was discharged home on day 45 post-BMT is now transfusion independent, and demonstrates trilineage engraftment ten weeks after transplant. Enzyme levels were 0.17 nmol/h/mg protein at diagnosis, 5.3–7.2 during ERT (10–20% of normal) and rose to 49.4–59.8 (normal) during ERT + BMT. Urinary GAG’s were 73.8 mg/mmol Creat. at diagnosis (normal <17.5), decreased to 22–26 during ERT and remained at similar levels during ERT + BMT. Conclusions: Combined ERT and BMT is a new alternative for the treatment of MPS-I. This approach might improve the patient’s tolerance to the side effects of the BMT. Further studies with large number or patients are needed. 16. Complex carbohydrates in the treatment of LCHAD deficiency. Jose E. Abdenur,a S. Lavorgna,b A.B. Schenone,b A. Guinle,b L. Jorge,b B. Andresen,c N. Gregersen,c and N. Chamolesb. aDivision of Metabolic Disorders, Children’s Hospital of Orange County, CA, USA; bFESEN, Buenos Aires, Argentina; cAarhus University, Denmark. LCHAD deficiency is a fatty acid oxidation defect associated with hypotonia, cardiomyopathy, abnormal LFTs and hypoketotic hypoglycemia. Rational of dietary treatment is to decrease accumulated substrates by restricting long chain fats and preventing lypolysis and to provide enough energy. These goals can be achieved with a low fat, high CHO diet, supplemented with MCT. Additionally frequent

feedings and/or the use of complex carbohydrates (CC) are indicated. There is little information regarding the length of fasting periods that can be considered to be safe and the response to different diets. Goal: To evaluate the biochemical response to the dietary treatment with CC in a patient with LCHAD deficiency. Patient and methods: A 6-monthold infant presented with marked hypotonia, hepatomegaly, cardiomyopathy, hypoglycemia, and abnormal LFTs. Initial results revealed markedly decreased free carnitine levels and a marginal increment of C16-OH-acylcarnitine (C16-OH-AC). Mutation analysis confirmed the diagnosis of LCHAD. Treatment with carnitine (50 mg/kg/day) and a high carbohydrate, low fat diet supplemented with MCT was able to revert the clinical abnormalities. The metabolic response to short fasting periods (4–10 h) was assessed at different ages, with diets of equal caloric intake and distribution but different carbohydrate composition: glucose polymers (GP) only, vs. GP + corn starch (CS) at 1, 1.5, and 2 g/kg/dose. Levels of blood glucose, lactate, CK, and AC were obtained every 2 h during the tests. Results: During the GP test there was a marked elevation of C16-OH-AC at 8 h (0.93 lMol, nl < 0.08). These levels decreased to 0.44 with CS at 1 g/kg. Further decrease was achieved with higher doses, reaching 0.24 and 0.31 lMol at 8 and 10 h, respectively when 2.0 g/kg was used. CK was elevated only when GP alone were used. Patient is currently 5.5 years old, has normal growth, development, and cardiovascular exam. Conclusions: (1) AC is a very sensitive and useful marker to monitor dietary management of LCHAD deficiency. (2) The use of CS provides source energy as long acting CHO, preventing the lypolysis associated to short fasting periods in LCHAD deficiency. 17. Outcome of confirmatory studies in infants referred for abnormal newborn screening for MCAD in New York State. Georgianne L. Arnold, Jane DeLuca, Beth Urbonas, and Wendy Introne. University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. Objective: To describe the outcome of confirmatory studies on infants referred for abnormal newborn screens for MCAD in the first year of testing. Methods: New York State added MCAD to its newborn screening (NBS) panel on October 1, 2001. Infants with C8 levels of >0.29 undergo DNA analysis for the common MCAD mutation, and those with C8 levels >0.39 or with C8 levels >0.29 with one or two mutations are reported as presumptive positive. All screen positive infants in our region are seen in the IMD clinic for follow-up studies (except low birth weight (LBW) infants in regional NICUs). Confirmatory testing is sent including plasma acylcarnitine and carnitine, and urine acylglycine and organic acids. Results: Fortythree infants were referred for abnormal NBS for MCAD. Eleven had low birth weight (LBW) less than 1500 gm. Nine of the infants were older than 3 weeks, and all nine were LBW infants who had a normal initial screen for C8 and who were receiving intravenous intralipid at the time of the second screen. Four infants were diagnosed with MCAD. All had initial C8 levels >3.6 on the state screen (range 3.6– 37.4), and one was homozygous for the common mutation. Two had normal urine organic acid (OA) analyses and two had OA analyses suggestive of MCAD. Plasma acylcarnitine and urine acylglycine analyses were diagnostic on all 4 infants, plasma carnitine levels were not helpful. Ten of the unaffected infants had some abnormalities identified on follow-up testing. Three unaffected LBW infants (one carrier) had increased dicarboxylic acids on OA and another had elevated 3OH propionate but normal acylcarnitine and acylglycine. A normal BW noncarrier infant had multiple abnormalities on OA with elevated glutaric acid on acylcarnitine and normal acylglycine (followup studies not suggestive of glutaric acidemia). Another noncarrier infant had mild elevation of C8 and C10 with normal ratio and normal acylglycine, fibroblast analysis found mild impairment of long chain fatty acid oxidation of unknown significance. Lastly a carrier infant had mild persistent elevation of C8 on acylcarnitine analysis with normal acylglycine and is undergoing clarification of genotype studies

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 and enzyme activity. Conclusions: Mild persistent abnormalities in urine organic acids or plasma acylcarnitine analyses are relatively frequent among infants with abnormal newborn screens for MCAD. LBW infants on intravenous intralipid commonly have elevated dicarboxylic acids. Some normal BW carrier and non-carrier infants have mild elevations of C8 of uncertain significance. 18. Analysis of coenzyme Q10 by tandem mass spectrometry. Jon A. Gangoiti and Bruce A. Barshop. Division of Biochemical Genetics, Department of Pediatrics, UCSD School of Medicine, La Jolla, CA, USA. Background: Coenzyme Q10 (ubiquinone; CoQ10) depletion has been described in some inborn errors of metabolism presenting with mitochondrial encephalomyopathy and ataxia. CoQ10 deficiency may be also present in metabolic disease, neurodegenerative disease, mitochondrial cardiomyopathy, and possibly other conditions associated with increased oxidative stress. Objective: To develop a method of analysis of CoQ10 in plasma, tissue homogenates, fibroblasts, and subcellular fractions by tandem mass spectrometry exploring the different ionization modes, electrospray (ESI) and atmospheric pressure chemical ionization (APCI). Methods: For total plasma CoQ10, oxidation with p-benzoquinone was carried out, followed by a single-step extraction in 1-propanol. For the other specimen types, quantitative hexane extraction from a sodium dodecylsulfate-treated aqueous homogenate after protein precipitation was used. Coenzyme Q9 (CoQ9) was used as internal standard. The quantitative determination involves reversed phase-HPLC and multiple reaction monitoring tandem mass spectrometry from a common product ion fragment, the benzylium ion at m/z 197, measured at the third quadrupole. The ammonium adducts of the molecular ions (m/z 884.1 and m/z 812.9, for CoQ10 and CoQ9) were monitored in the first quadrupole when ESI was used. The protonated pseudo-molecular ions (m/z 863.5 and m/z 796) were monitored using APCI. Results: After optimisation of the source and lens parameters, the method can assay total human plasma CoQ10 over an analytical range of 0.01–11 lg/mL. Intra-assay repeatability showed a CV of 1.6–5.5% across the range. Intermediate (interassay) precision was 3.9–8%. Analytical recoveries were 99 ± 4%. The limit of quantitation using a criterion of 15% precision was found to be 0.01 lg/mL. The method allows ubiquinol detection using a transition m/z 865.3 to m/z 197. Reduced (ubiquinol) and oxidized (ubiquinone) forms could be resolved chromatographically, but our routine practice was to measure total CoQ10 as ubiquinone after oxidation. The use of appropriate scanning functions to detect synthetic intermediates, provided they are present at detectable concentrations, is under active investigation. Conclusions: Tandem mass spectrometry is suitable for monitoring CoQ10 concentrations in clinical samples. The method proved to be specific, sensitive, precise, accurate, robust and fast enough to allow for high throughput sample analysis. 19. Clinical, biochemical, and molecular findings in a free sialic acid storage disease patient of moderate severity. Isa Bernardini,a Robert Kleta,a,b Richard P. Morse,c Eduard Orvisky,d Donna Krasnewich,a Joseph Alroy,e Angelo A. Ucci,f David A. Wenger,g and William A. Gahla,b. aSection on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; b Office of Rare Diseases, Intramural Program, Office of the Director, National Institutes of Health, Bethesda, MD, USA; c Department of Pediatrics, Dartmouth-Hitchcock Medical Center, Lebanon, NH, USA; dNSB, National Institute of Mental Health, National Institutes of Health, Bethesda, MD, USA; eDepartments of Pathology Tufts University Schools of Medicine and Veterinary Medicine, and Tufts-New England Medical Center, Boston, MA, USA; fDepartment of Pathology Tufts University School of Medicine, and Tufts-New England Medical Center, Boston, MA, USA; gDepartment of Neurology, Jefferson Medical College, Philadelphia, PA, USA.


The allelic autosomal recessive lysosomal storage disorders Salla disease and infantile free sialic acid storage disease (ISSD) result from mutations in SLC17A5. This gene codes for sialin, a lysosomal membrane protein that transports the charged sugar, N-acetylneuraminic acid (sialic acid), out of lysosomes. ISSD has a severe phenotype with infantile onset, while the Finnish variant, Salla disease, has a milder phenotype with later onset. Both disorders cause developmental delay, and ISSD is generally fatal in early childhood. We describe a 30-month old non-Finnish, Caucasian child with global developmental delay of postnatal onset, language and motor skills stagnant at a 3–4 month level, hypotonia, and mild but progressive coarsening of facial features. Urinary excretion of free sialic acid was elevated 4.5 times above control. EM of a skin biopsy revealed enlarged secondary lysosomes consistent with oligosaccharide storage. Free sialic acid in fibroblasts was 3.8 ± 0.9 nmol/mg protein (concurrent normal controls, 0.5 ± 0.1); differential centrifugation indicated a lysosomal location. Genomic analysis revealed compound heterozygosity for two new SLC17A5 mutations. In exon 2, a heterozygous c291 G > A mutation leads to skipping of exon 2; cDNA analysis showed a 197-bp deletion. The exon 2 skipping causes termination after 32 amino acids instead of 495. A second mutation in exon 9, c1226 G > A, G409E, changes a conserved glycine to glutamate within a transmembrane region. Each mutation was present in one parent. This child’s clinical manifestations of a lysosomal free sialic acid storage disease are consistent with her sialin mutations and biochemical findings. The differential diagnosis of postnatal developmental delay should include free sialic acid storage disorders such as ISSD and Salla disease. 20. Cerebral folate deficiency: an underestimated neurometabolic condition. Nenad Blau,a Th Opladen,a and VTh Ramaekersb. aDivision of Clinical Chemistry and Biochemistry, University Children’s Hospital, Zu¨rich, Switzerland; bDivision of Pediatric Neurology, University Hospital Aachen, Aachen, Germany. Objective: For normal functioning, the central nervous system requires folate stores which depend on folate homeostasis and intact transport mechanisms across the choroid plexus. Cerebral folate deficiency can result from disturbed folate transport or from increased utilization of folates. Methods: Recently, we identified a number of children with suspected cerebral folate deficiency by simultaneous measurement of CSF monoamine metabolites, pterins, and 5-methyltetrahydrofolate (5MTHF) using HPLC. Results: Cerebral folate deficiency (low 5MTHF in CSF) was found in 31 patients with a recently described neurometabolic syndrome presenting with irritability, psychomotor retardation, cerebellar ataxia, spastic paraplegia, dyskinesias, and occasional seizures (Neuropediatrics 2002;33:301– 308), in 8 girls with Rett syndrome (Neurology 2003;61:506–514), and in 3 children with Aicardi–Goutie`res syndrome variant (Neurology 2003;61:642–648). Treatment with folinic acid (0.5–2.0 mg/kg/d) proved beneficial in most patients. Conclusions: Although the exact causes for these conditions still need further clarification, additional CSF analysis in the context of salient clinical features seems important to diagnose cerebral folate deficiency. Cerebral folate depletion should be corrected as early as possible by oral administration of folinic acid (Leucovorine). 21. Importance of CSF in the diagnosis of pediatric neurotransmitter diseases. Nenad Blau. Division of Clinical Chemistry and Biochemistry, University Children’s Hospital, Zu¨rich, Switzerland. Objective: Although many neurometabolic disorders can be diagnosed through serum, urine, and/or CT/MRI investigations, CSF is the only informative sample for the differentiation of pediatric neurotransmitter diseases. Methods: We investigated CSF from patients with suspected impaired biogenic amine neurotransmission by HPLC of amino acids, neurotransmitter metabolites, pterins, and folates. Results: Preanalytical factors such as time of puncture, fraction, blood


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contamination, storage conditions, and medications may influence the concentration of metabolites. We investigated almost 4000 CSF samples and diagnosed 147 patients with PTPS deficiency, 75 with DHPR deficiency, 8 with GTPCH deficiency, 5 with SR deficiency, 19 with DRD (Segawa), 8 with TH deficiency, and 5 with AADC deficiency. Conclusions: Many clinical signs and symptoms are not specific for a single neurometabolic disorder and extensive biochemical investigations are essential for the early diagnosis and appropriate treatment. CSF investigations should be considered in patients presenting with hypokinesia, distal chorea, myoclonic epilepsy, dystonia, oculogyric crises, hypersalivation, temperature instability, aggressive behavior, or mental retardation. 22. Disease progression in Scheie syndrome: case report. Stefano Bruni,a Patrizia Salvatori,a Marco Radice,b and Giovanni V. Coppaa. aIstituto di Clinica Pediatrica, Universita` Politecnica delle Marche, Italy; bGenzyme s.r.l., Italy. MPS I is a genetic disorder resulting from a deficiency of the lysosomal enzyme, a-L -iduronidase, with subsequent accumulation of glycosaminoglycans (GAGs) throughout the body. The disease encompasses a broad clinical spectrum, ranging from the most severe form, Hurler syndrome, to the attenuated forms, Hurler-Scheie (intermediate) and Scheie (less severe) syndromes. Scheie syndrome is not well-described in the literature and is often incorrectly referred to as being ‘‘mild.’’ Due to the progressive course of MPS I, Scheie patients often experience significant disease-related complications that worsen over time and result in permanent disability. The patient described in this case report is a 53-year-old female whose first symptoms began at the age of 3 with joint involvement. Juvenile rheumatoid arthritis was suspected, and she was treated with steroids. At 25, orthopaedic manifestations were identified consisting of dysplasia of the femoral head and the acetabulum. Treatment with gold salts and NSAIDs was ineffective. At 35, the patient’s lack of stamina led to the suspicion of spinal cord compression. She was also diagnosed with valvular heart disease and 7 years later underwent major valve surgery (aortic and mitral replacement and tricuspid plasty). In her early 40s, loss of vision from corneal clouding and retinal degeneration became apparent. She soon became non-ambulatory and since 42, has been wheelchair-bound. Over the years, the patient has been continuously examined by numerous physicians and treated ineffectively with aggressive rheumatologic drug therapies. Finally, at 53 she was diagnosed with Scheie MPS I, on the basis of the electrophoretic characterization of urinary mucopolysaccharides, the dosage of total urinary GAGs through colorimetric method (DMB method) and the dosage of enzymatic activity of a-L -iduronidase with fluorometric substrate. In July 2003 she started enzyme replacement therapy (ERT) with Aldurazyme. At the first check-up three months later a reduction of urinary mucopolysaccharides was unmistakable. The patient reports a reduction of joint pains and also the woman’s mood seems slightly better. Classification of patients with the ‘‘mild form of MPS I’’ or Scheie syndrome, is misleading as patients can develop severe disease manifestations that lead to significant disabilities. Prompt recognition of MPS I-related signs and symptoms and diagnosis of patients with attenuated forms of MPS I is needed in order to evaluate the effects of early intervention with ERT for preventing and/or slowing disease progression. 23. An atypical case of Leukoencephalopathy with vanishing white matter. Pranesh Chakraborty,a Jean Michaud,a Raffy Gorospe,b and Michael T. Geraghtya. aChildren’s Hospital of Eastern Ontario, Ottawa, Ont., Canada; bChildren’s National Medical Center, Washington, DC, USA. Objective: To describe a young boy with ‘‘Leukoencephalopathy with vanishing white matter’’ (VWM) manifesting atypical features of this disease. Methods: Clinical, laboratory, neuro-imaging, and preand post-mortem pathology findings are described. Results: This

previously healthy and developmentally appropriate 10-month-old boy developed ataxia with a rapidly progressive impairment of his level of consciousness following a viral illness. He died two months after the onset of symptoms. MRI of the head demonstrated a severe leukoencephalopathy with subcortical cysts in the anterior temporal, frontal, and parietal lobes as seen in patients with ‘‘Megalencephalic Leukoencephalopathy with Subcortical Cysts’’ (MLC). His head circumference was normal. There initially was relative sparing of the internal capsule, brainstem, and corpus callosum. There was no biochemical evidence of a lysosomal storage, peroxisome biogenesis, respiratory chain, or fatty acid oxidation disease. Urine organic acids, plasma acylcarnitine profile, and plasma amino acids were normal. CSF protein was elevated (>1.1 g/L), as was the CSF glycine (41 lM). Plasma glycine concentration, and liver glycine cleavage enzyme activity was normal. Liver and muscle biopsies pre-mortem revealed marked lipid accumulation. Autopsy findings included lipid accumulation in the central nervous system, as well as systemically in skeletal muscle, liver, kidney, and autonomic nervous system. There was moderate to severe neuronal loss and gliosis throughout the central nervous system with apoptotic features in the cerebral cortex. There was a severe diffuse leukodystrophy with numerous cystic foci most prominent in the anterior frontal lobes, anterior temporal lobes, and parieto-occipital areas. There was a mild peripheral neuropathy involving mostly the small myelinated fibres and dominated by myelin degeneration. The diagnosis of VWM was confirmed by the detection of mutations in the EIF2B4 gene (P242L and an exon 5 2 bp deletion), and his mother was found to be a carrier of only the exon 5 mutation. Analysis of the MLC1 gene was normal. Conclusions: This case broadens the described phenotype of VWM, illustrates a overlap between neuro-imaging findings in VWM and MLC, suggests that VWM is not an isolated central nervous system disease, and provides further evidence that elevated CSF glycine in VWM is not due to a deficiency of the glycine cleavage enzyme. 24. Hurler syndrome (MPS1H): enzyme replacement therapy pre-bone marrow transplantation. Joy Wright,a Jayashree Motwani,a George Gray,a Alan Cooper,b Ed Wraith,b Sarah Lawson,a Phil Darbyshire,a and Anupam Chakrapania. aBirmingham Children’s Hospital, Birmingham, UK; bWillink Biochemical Genetics Unit, Manchester, UK. Hematopoietic stem cell transplantation (HSCT) is used to treat patients with Hurler syndrome (MPS1H), the severe form of aiduronidase deficiency. The procedure is associated with high mobidity and mortality due to systemic complications of MPS1H. We report the first patient with MPS1H to have undergone recombinant enzyme replacement therapy (ERT) prior to HSCT. Case Report: The male infant presented with facial dysmorphism and developmental delay aged 15 months. MPS1H was suspected from the abnormal pattern of urine GAGs; the diagnosis was confirmed by enzyme analysis and mutation analysis. Hepatosplenomegaly, cardiac valvular abnormalities, and cardiomyopathy were present at the time of diagnosis but there was no evidence of sleep apnea. ERT with recombinant aiduronidase 100 IU/kg as weekly infusions was commenced at 18 months of age whilst awaiting HSCT. A total of 14 infusions were given pre-HCST and a further 4 infusions after HCST. There were no adverse reactions. The urine GAGs (quantitated by dimethylbenzidine) were nearly normal after 4 weeks of ERT and continue to remain normal 3 months after HCST. At the time of the transplant his cardiomyopathy and hepatosplenomegaly had completely resolved. He received a successful matched unrelated donor transplant 14 weeks after commencing ERT following conditioning with busulphan, cyclophosphamide and fludarabine. The post transplant course was uncomplicated. He was fully donor chimeraic 2 weeks after transplantation and the leucocyte a-iduronidase levels have been at the lower end of normal since. Conclusion: Based on these very promising results, we recommend pre-transplant ERT as it significantly reduces clinical

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 symptoms. A predicted benefit should be a reduction in transplantrelated morbidity and mortality. 25. Aldurazyme (laronidase) enzyme replacement therapy for MPS I: 72-week extension data. Lorne A. Clarke,a James E. Wraith,b Michael Beck,c Edwin H. Kolodny,d Gregory M. Pastores,d and Joseph Muenzere. aUniversity of British Columbia, Vancouver, Canada; bUniversity of Manchester, Manchester, UK; cUniversity of Mainz, Mainz, Germany; dNew York University, New York, NY, USA; eUniversity of North Carolina, Chapel Hill, NC, USA. Objective: To evaluate the long-term efficacy and safety of Aldurazyme (recombinant human a-L -iduronidase, laronidase, rhIDU) for the treatment of MPS I. Methods: All 45 patients from a 26-week Phase 3 randomized, double-blind, placebo-controlled study were enrolled into a Phase 3 open-label extension study. Patients receiving rhIDU throughout both portions of the study (N = 22; total rhIDU exposure = 98 weeks) were designated rhIDU/rhIDU, while those crossing over from placebo to rhIDU (N = 23; total rhIDU exposure = 72 weeks) were designated Placebo/rhIDU. Changes in forced vital capacity (FVC), the 6-min walk test (6MWT), liver volume, and urinary glycosaminoglycan (GAG) level were evaluated from baseline (start of double-blind phase) or entry (start of open-label phase) based on initial treatment group. Safety data are summarized descriptively. Results: Both groups showed relative improvements in mean percent of predicted FVC calculated using baseline height measurements (rhIDU/ rhIDU, 10.2 percentage points, p = 0.001; Placebo/rhIDU, 4.9 percentage points, p = 0.019), which corresponded to small changes in the mean percent of predicted FVC calculated using current height measurements ()0.2 and )0.7 percentage points, respectively). These data suggest stabilization of the rate of lung volume growth with treatment, as opposed to the expected decline over time in percent of predicted FVC for untreated patients. In the 6MWT, the rhIDU/ rhIDU and Placebo/rhIDU groups showed mean increases of 43.0 m (p = 0.015) and 27.6 m (p = 0.190). Shoulder and knee range of motion improved in both groups by approximately 5–10. Overall, mean liver volume decreased by approximately 20% with normalization in 75% of patients, and mean urinary GAG levels decreased by approximately 70% with normalization in 38% of patients. All patients experienced at least one adverse event, most of which were related to underlying disease. Approximately 50% of patients experienced mild to moderate infusion-related reactions, most commonly rash and flushing (5 patients each), that were managed by adjustment of the infusion rate and/or medication. A single patient experienced two laronidase-related serious adverse events consisting of dyspnea and anaphylaxis. At week 72, 87% of patients were seropositive for antibodies to laronidase. Conclusions: Long-term enzyme replacement therapy of MPS I patients with laronidase is associated with a favorable risk-benefit profile. Patients demonstrated sustained improvements in clinically relevant endpoints that are related to the efficient clearance of GAG substrate. (Sponsored by BioMarin/Genzyme LLC) 26. Enzyme replacement therapy for infantile onset Pompe disease: long term follow-up results. P. Kishnani,a M. Nicolino,b T. Voit,c C.H. Tsai,d G. Herman,e J. Waterson,f R.C. Rogers,g J. Levine,h A. Amalfitano,a J. Charrow,i G. Tiller,j B. Schaefer,k E. Kolodny,l D. Corzo,m and Y.T. Chena. aDuke Univ. Med. Cr, Durham, NC, USA; bHopital de Brousse, Lyon, France; cUniv Hos, Essen, Germany; dChildren’s Hosp., Denver, CO, USA; eChildren’s Hosp., Columbus, OH, USA; fChildren’s Hosp., Oakland, CA, USA; fGreenwood Genet. Cr., Greenville, SC, USA; gChildren’s Hosp., Boston, MA, USA; hChildren’s Mem. Hosp., Chicago, IL, USA; iVanderbilt Univ. School of Med., Nashville, TN, USA; j Univ. Nebraska Med. Cr., Omaha, NE, USA; kNew York Univ. School of Med., New York, NY, USA; lGenzyme Corp., Cambridge, MA, USA. Background: Pompe disease is an autosomal recessive disorder caused by a deficiency of acid a-glucosidase (GAA). The infantile form


presents in the first few months of life with rapidly progressive hypotonia, generalized muscle weakness, and hypertrophic cardiomyopathy. Death usually occurs by one year of age. Methods: Two clinical trials explored the safety and efficacy of ERT with CHO-cell derived rhGAA in patients with infantile onset Pompe disease. Three patients in the first trial initially received rhGAA at 5 mg/kg IV twice weekly. Eight patients in the second trial received rhGAA at 10 mg/kg IV weekly. Currently, patients receive 10 mg/kg IV weekly or 20 mg/kg IV every two weeks. Results: Four patients are alive (mean age 37.5 months; range 30–52 months); mean duration of ERT is 34.3 months (range 27–50 months). All four surviving patients have demonstrated marked decrease in LVMI, do not require the use of cardiac medications, remain ventilator-free, are ambulatory, and fed by mouth. In the four surviving patients hearing has remained normal. Seven patients are deceased. Deaths were attributed to disease progression and unrelated to rhGAA. Mean age of death was 30 months (range 18–50 months); mean duration of ERT was 23.5 months (range 3.8–46 months). This subgroup of patients had also shown decrease in LVMI, although skeletal muscle response was variable. All patients treated with rhGAA developed anti-rhGAA antibodies. All patients experienced infusion-associated reactions. The majority of the infusion-associated reactions were mild and managed symptomatically. Conclusions: ERT in infantile onset Pompe disease appears to be safe and well tolerated. ERT-treated patients have demonstrated prolonged survival and marked improvement in cardiomyopathy, although skeletal muscle response has been variable. ERT has thus changed the natural history of infantile onset Pompe disease. Long-term follow-up of patients is required to further evaluate the safety and efficacy of rhGAA. Additional studies are needed to identify predictors of skeletal muscle response to treatment. 27. Mental development in patients with infantile onset Pompe disease treated with enzyme replacement therapy. Alison Skrinar,a Deya Corzo,a and Priya Kishnanib. aGenzyme Corporation, Cambridge, MA, USA; bDuke University Medical Center, Durham, NC, USA. Background: Pompe disease is an autosomal recessive disorder caused by a deficiency of the lysosomal enzyme acid a-glucosidase (GAA). Assessment of mental development is an important component of evaluating the response to ERT in IOPD. Methods: An openlabel study of eight patients with IOPD is ongoing. A summary of clinical efficacy results is presented. Preliminary results of mental development evaluations conducted using the Bayley Scale of Infant Development, 2nd edition (BSID-II) are discussed. Results: Mean duration of treatment for all patients is 20.4 months (range 3.8–30.5 months). Three patients are currently alive (at 35.1, 31.8, and 30.3 months of age, respectively). The three surviving patients have demonstrated marked improvement in cardiomyopathy, require no ventilatory assistance, are currently ambulatory, and fed by mouth. Five patients are deceased. Deaths were attributed to disease progression and were unrelated to rhGAA. Mean age at death was 23 months (range 14.7–33.8 months). At baseline, mean chronological age for all patients was 6.0 months (range 2.5–14.6 months) with a mean BSID-II mental performance age equivalent of 4.5 months (range <1–14 months). After a mean duration of treatment of 15.2 months (range 3.2–25.2 months), the mean chronological age was 20.1 months (range 8.2–29.5 months) with a mean BSID-II mental performance age equivalent of 15.6 months (range 7–23 months). Improvement in raw scores from baseline was observed in all patients indicating the continued acquisition of cognitive, language, and personal/social development skills. The changes BSID-II raw scores corresponded to increases in mental performance age equivalents. In spite of these gains, normalized standard scores of mental performance (BSID-II mental development index, MDI) indicate that IOPD patients were not functioning at the same level of age-matched normally developing peers. Conclusions: In patients with IOPD treated with rhGAA, an increase in BSID-II raw scores corresponded to gains in mental performance age equivalents. This finding indicates a continuous


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acquisition of new skills, although the improvement does not place these patients at the same functional level of age-matched normally developing children. The increasing complexity of items involving integrated oral-motor and fine-motor skills required for the administration of BSID-II to older children may contribute to the lag in MDI scores between normally developing children and patients with IOPD treated with ERT over time. There is a limited value in comparing IOPD patients and normally developing children with MDI scores alone. However, progress in the acquisition of new skills can be effectively monitored by evaluating changes in raw scores and mental performance age equivalents. 28. Natural history of infantile onset Pompe disease: results from a retrospective chart review study. Deya Corzo,a W.L. Hwu,b Hannah Mandel,c Marc Nicolino,d Florence Yong,a and Priya Kishnanie. aGenzyme Corporation, Cambridge, MA, USA; bNational Taiwan University Hospital, Taipei, Taiwan, ROC; cRambam Medical Center, Haifa, Israel dHopital de Brousse, Lyon, France; eDuke University Medical Center, Durham, NC, USA. Background: Pompe disease is an autosomal recessive disorder caused by a deficiency of acid a-glucosidase (GAA). Reports on the natural history of IOPD are based on data from studies conducted in a relatively small number of patients. To fully characterize the disease, a retrospective chart review was conducted. Methods: This is a multinational, multicenter, historical cohort study. Inclusion required of onset of symptoms by 12 months of age and documentation of GAA deficieny or GAA mutation(s). Data collected from medical charts included demographics, family history, progression of signs, and symptoms of the disease, diagnostic and other ancillary evaluations, and treatment modalities. Results: 300 cases were screened; 168 cases from 33 study sites in 9 countries met all eligibility criteria. 93 cases (55%) were born after 1995 and 75 cases (45%) were born before 1995. Median age ± SD (months) at presentation of first symptoms was 2.0 ± 2.5 (n = 166); at diagnosis, 4.7 ± 8.8 (n = 165); at first ventilator use, 5.9 ± 6.3 (n = 165), and at death, 8.7 ± 1.1 (n = 163). By 12 months of age mortality was 78%. By 18 months of age mortality reached 88%. The most common sign/symptoms of the disease were cardiomegaly (92%, n = 154), hypotonia (88%, n = 148), cardiomyopathy (88%, n = 147), respiratory distress (78%, n = 131), failure to thrive (53%, n = 89), congestive heart failure (50%, n = 84), and pneumonia (45%, n = 76). At least 20% of patients (n = 49%) were ventilated. Ninety percentage (n = 151) were treated with one or more medications, 77% (n = 130) with nutritional support, and 93 (55%) with other supportive therapies. Conclusions: This is the largest retrospective case review study performed to date in IOPD. Results from this study support earlier literature reports with regard to the fatal course of the disease and the rapidity of the disease progression. In spite of widespread use of different therapeutic modalities mortality has changed little across the decades. Although a small group of patients survived past 12 months of age, IOPD is still a rapidly progressive and fatal disease, with the majority of patients dying before 12 months of age. 29. Aldurazyme (laronidase) enzyme replacement therapy in MPS I: preliminary data in children less than 5 years of age. Gerald F. Cox,a N. Guffon,b J.E. Wraith,c and K. Walton-Bowend. aClinical Research and dBiostatistics, Genzyme Corporation, Cambridge, MA, USA; bDepartment of Pediatrics, Debrousse Hospital, Lyon, France; cWillink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, UK. Objective: To evaluate the safety, efficacy, and pharmacokinetics of Aldurazyme in young children with severe MPS I (Hurler syndrome). Methods: Approximately 20 MPS I patients less than 5 years old are currently being enrolled in a Phase 2 open-label study of Aldurazyme administered at 0.58 mg/kg (100 U/kg) once weekly for 12 months. Most patients are expected to have the severe form of the disorder. Patients must have declined or not undergone hematopoietic stem cell transplantation to be eligible. Efficacy is being evaluated by changes in

urinary glycosaminoglycan (GAG) excretion, liver volume, upper respiratory care requirements, sleep apnea, hearing, vision, growth velocity and cardiac status. Pharmacokinetics is being assessed at Baseline and at Weeks 26 and 52 of treatment. Safety monitoring includes physical examination, vital signs, ECG, clinical laboratory testing, immunogenicity testing, and adverse event (AE) reporting. Serious adverse events (SAEs) and infusion-associated reactions (IARs) are reported by investigators to Genzyme Pharmacovigilance. Results: An interim analysis of urinary GAG levels and AEs was performed after the first 12 patients received 26 weeks of treatment. All patients underwent Port-a-Cath placement to facilitate infusions. Large reductions in urinary GAG excretion (>40%) occurred in 7/10 patients by Week 13 and 9/9 patients by Week 26. The mean decrease in urinary GAG level (± standard deviation) from Baseline to Week 26 was 57.05% (±13.71). Mean urinary GAG levels (± standard deviation) at Baseline and at Week 26 (543.35 ± 170.66, 235.7 ± 117.29) were much higher than the levels of Aldurazyme-treated patients in the Phase 3 study who were older and had attenuated disease (188.9 ± 60.93, 81.3 ± 26.36). From study entry to the point of this analysis, patients had been treated for a median of 34 weeks (range 14– 43). In general, therapy was well tolerated. Only 3 patients out of 12 experienced in total 5 IARs over a period of 3–11 months reporting commonly encountered symptoms such as chills (5), fever (4), tachycardia (2), hypertension (2), vomiting (1), and paleness (1). All IARs were managed by temporary interruption or slowing of the infusion and administration of antihistamines and/or antipyretics. Conclusions: The initial safety profile and pharmacodynamic response of Aldurazyme in severe patients less than 5 years old is similar to that of older patients with attenuated disease. The impact of higher substrate burden and the potential need for adjusting the dose in some young severe patients is under investigation. 30. Safety and efficacy of penicillamine therapy in pediatric cystinuria. Ralph DeBerardinis, Sayaka Yamazaki, and Paige Kaplan. Section of Biochemical Genetics and Metabolic Disease, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Cystinuria is an autosomal recessive defect in transport of the amino acids cystine, ornithine, arginine, and lysine. Its major morbidity, urolithiasis, results from decreased cystine resorption in the proximal renal tubule, causing urinary cystine concentration to exceed its solubility threshold. In addition to renal colic, patients are at longterm risk for renal failure. Attempts to lower stone formation include increasing fluid intake and urinary alkalinization. However, the most effective treatment appears to be penicillamine, which forms disulfides with cystine and decreases crystallization. Penicillamine is routinely used in adults, but concern for toxicities (skin eruptions, fevers, proteinuria, arthralgias, marrow suppression, etc.) has limited its use in children, although the incidence of toxicity has not been studied in an American pediatric population. We present our experience in treating cystinuric children with penicillamine. We used a strategy involving gradual initiation of the dose, starting with 5 mg/kg/day and increasing to 20 mg/kg/day over 4–6 weeks. Baseline studies included blood urea nitrogen (BUN), creatinine, liver function tests, and a complete blood count (CBC). While initiating treatment, patients checked urine for protein daily, and had a CBC every two weeks. Using this protocol, we initiated penicillamine therapy in 11 patients (5 male, 6 female; mean age at stone presentation 5.6 years; mean age at penicillamine initiation 7.4 years). No patients experienced a significant penicillamine-associated toxicity during the initiation phase, and all had reduction in urinary cystine concentration after initiation (mean reduction 54%; range 5–85%). Ten of the eleven patients were compliant with subsequent treatment. We followed them for a total of 746 months (range 3-189) on penicillamine, with periodic assessments of urinary cystine concentration, CBC, BUN, creatinine, and liver function. During this time, only two patients had significant side effects; a 22year-old woman who developed elastosis perforans serpiginosa after 15

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 years; and a 6-year-old boy with generalized amino aciduria after three months. The woman had normal serum copper and bone density. Of note, only 1 of the 10 patients suffered a stone event while on penicillamine therapy; however, this patient was intermittently noncompliant. Our data suggest that penicillamine, if initiated gradually, is a safe and effective treatment for children with cystinuria. 31. Incidence of BH4-responsiveness in a population of PAH-deficient hyperphenylalaninemic infants. L. Fiori, B. Fiege, M. Beaga, E. Riva, and M. Giovannini. Department of Paediatrics, San Paolo Hospital, University of Milan, Italy. Introduction: Hyperphenylalaninemia (HPA) is a common inborn error of metabolism due to deficiency of phenylalanine-hydroxylase (PAH) or one of the enzymes involved in the metabolism of its cofactor, tetrahydrobiopterin (BH4). Recently forms of PAH-deficiency HPA responsive to oral administration of BH4 by lowering plasma phenylalanine levels have been described. BH4 might represent a valid alternative to dietary treatment in BH4-responsive patients. Aim of the study: Determination of the incidence of BH4 responsiveness among all patients born from January 2000 until September 2003 in Lumbardy (Italy) detected through neonatal screening for HPA. Identification of PAH-deficient HPA patients who might benefit from pharmacological treatment with BH4 as alternative to diet therapy. Evaluation of genotype-phenotype correlation in BH4-responsiveness. Patients and methods: All patients born in Lumbardy between January 2000 and September 2003 and affected by HPA (79 subjects) were investigated for BH4 responsiveness. BH4 deficiency was excluded at diagnosis by BH4 loading test, urinary pterins analysis and determination of DHPR activity on blood spot. The BH4 loading test was performed according to a standardized protocol; 6R-tetrahydrobiopterin (20 mg/kg), was administered to patients and plasma samples were collected at time 0, 4, 8, and 24 h. In patients with basal plasma phe levels lower than 360 lmol/L, a combined phe and BH4 loading test was performed: 100 mg phe/kg body weight were administered orally 3 h before the BH4 load. Results: The incidence of BH4responsiveness among our PAH-deficient patients is 77.2%. The majority of responsive patients are affected by mild hyperphenylalaninemia, a smaller percentage of patients is affected by mild phenylketonuria (PKU) and moderate PKU. No responsiveness was found among patients affected by classical PKU. Conclusions: BH4-responsiveness seems to be an important feature, not only in MHP but even in some patients affected by mild and moderate PKU, who undergo a dietary treatment. The mechanisms underlying BH4 responsiveness have to be further elucidated. Pharmacological security and high costs of BH4 therapy are limiting the introduction of pharmacological therapy in PAH deficient hyperphenylalaninemia. 32. Sialidosis presenting with severe fetal hydrops and associated with two novel mutations in lysosomal a-neuraminidase. Dorothy K. Grange,a Y. Campos,c Allesandra D’Azzo,c Enid Gilbert-Barness,d Aaron Hamvas,a David J. Loren,a Frances V. White,b and Lance Wyblee. a Department of Pediatrics and bDepartment of Pathology, Washington University School of Medicine, St. Louis, MO, USA; cDepartment of Genetics, St. Jude Children’s Research Hospital, Memphis, TN, USA; dDepartments of Pathology and e Department of Pediatrics, University of South Florida, Tampa, FL, USA. Sialidosis is a lysosomal storage disease caused by mutations in the lysosomal a-neuraminidase gene (NEU1) and is characterized by accumulation of sialylated oligosaccharides in tissues and body fluids. There is wide variability in age of onset and severity of symptoms. We report a case of congenital sialidosis with severe non-immune hydrops fetalis. The patient was the 2.7 kg product of a 34 week gestation. Fetal edema, pleural effusions and ascites were detected by ultrasound at 18 weeks, and peritoneal-amniotic and pleural-amniotic shunts were placed at 24 and 30 weeks gestation, respectively. At birth, he was


diffusely edematous with massive hydroceles. Marked hepatosplenomegaly was present. Despite maximal support, the patient had persistent respiratory failure and expired at 10 weeks of age. There was twofold elevation of bound sialic acid in the plasma. Urine oligosaccharide analysis revealed massive excretion of sialic acid. aNeuraminidase activity in skin fibroblasts was <1% of the control value. Histologic examination of the placenta showed foamy material within trophoblasts, and peripheral blood monocytes contained cytoplasmic vacuoles. A lung biopsy showed alveolar septal widening, prominent pneumocyte hyperplasia and muscular thickening of arterial walls. There was vacuolated cytoplasm of the alveolar macrophages, histocytic-like cells in alveolar septae, and endothelial cells. Electron microscopy of lung revealed numerous cytoplasmic single membrane bound vacuoles consistent with secondary lysosomes, present in macrophages, mesenchymal cells, and endothelial cells. Analysis of the NEU1 gene revealed compound heterozygosity for two novel mutations. There was a G to A substitution at nucleotide 45 in exon 1, which results in a premature stop codon (Trp15stop), and a G to A substitution at nucleotide 1022 in the last codon of exon 5 which interferes with splicing of intron 5 and is predicted to result in the insertion of 32 amino acids into the protein. Although these mutations have not previously been described, both would be predicted to cause a severe phenotype; one is a null allele, and the other would likely result in an abnormally folded protein that may remain localized to the endoplasmic reticulum or Golgi, or may be prematurely degraded before it reaches the lysosome. Histologic examination of lung, peripheral blood monocytes and placental tissue provided clues to the diagnosis of a storage disorder in this case. Placenta and peripheral blood leukocytes are readily accessible tissues that may be useful in the rapid investigation for lysosomal storage disorders in non-immune fetal hydrops. 33. Hemizygosity at 10q23 in a family with Hermansky–Pudlak syndrome type 1. Ashley Griffin, Paul Anderson, David Claassen, William A. Gahl, and Marjan Huizing. Section on Human Biochemical Genetics, MGB, NHGRI, NIH, Bethesda, MD, USA. Hermansky–Pudlak syndrome (HPS) is a rare autosomal recessive disorder characterized by oculocutaneous albinism, a platelet storage pool deficiency and, in some patients, lysosomal accumulation of ceroid lipofuscin. HPS is associated with defects in the biosynthesis and/or processing of melanosomes, platelet dense bodies, and lysosomes. We studied the molecular and clinical aspects of a patient with the HPS-1 subtype, caused by mutations in the HPS1 gene, localized on chromosome 10q23.1–23.3. This patient appeared to be homozygous for the S369delC in exon 13, resulting in no RNA on Northern blot. PCR amplification and direct sequencing of exon 13 from gDNA showed that her mother’s gDNA was heterozygous for this mutation, while her father’s gDNA did not show this mutation. Her brother has neither HPS nor the S396delC mutation, but has cardiomyopathy (Wolf–Parkinson–White-like syndrome). These findings made us investigate hemizygosity in this family. Since Southern blotting was not informative, we performed real-time PCR (quantitative PCR) to study the area of interest. Real-time PCR demonstrated that only one allele of the HPS1 gene is present in the patient, her father, and her brother. We will continue to use real-time PCR to determine the breakpoint in the 10q23 area. Additionally, we will determine if the brother’s syndrome is caused by another gene in the deletion area. 34. Low therapeutic threshold for hepatocyte transplantation in murine phenylketonuria. Cary O. Harding,a Kelly Hamman,a Heather Clark,a Eugenio Montini,a Muhsen Al-Dhalimy,a Markus Grompe,a and Milton Finegoldb. aOregon Health and Science University, Portland, OR, USA; bTexas Children’s Hospital, Houston, TX, USA. Objective: Determine the minimal number of phenylalanine hydroxylase (PAH) positive hepatocytes and minimal level of PAH activity


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required to correct hyperphenylalaninemia in Pahenu2 mice, a model of human phenylketonuria (PKU). Hypothesis: Phenylalanine (PHE) clearance is primarily determined by the total amount of PAH activity present in liver and is not limited by the absolute number of PAHexpressing hepatocytes. Method: Hyperphenylalaninemic Pahenu2 mice were injected intrasplenically with 105 primary hepatocytes, either wild type hepatocytes expressing normal PAH activity or Pahenu2/+ cells with partial PAH deficiency, to develop a series of animals with varying numbers of PAH positive hepatocytes and amounts of liver PAH activity. To allow a selective growth advantage for the donor hepatocytes, recipient mice also lacked fumarylacetoacetate hydrolase (FAH) activity due to a targeted deletion of the FAH gene(FahDexon5)s. Following liver repopulation, serum PHE was measured and compared to the % liver repopulation measured by quantitative PCR and to total liver PAH activity. Results: Serum PHE levels decreased from 1650 ± 350 to 279 ± 138 lM (range 84–569 lM; normal = 114 ± 12 lM) 3–5 weeks after hepatocyte transplantation in fourteen treated mice. Just 2% repopulation was associated with a decrease in serum PHE to 569 lM, while serum PHE decreased to 84 lM in an animal with 21% PAH positive hepatocytes. Complete correction of serum PHE (<200 lM) required at least 10% repopulation with PAH positive hepatocytes. Above 10% repopulation, there was a strong negative correlation between PAH activity and the final serum PHE level. At repopulation levels <10%, this correlation is weak, suggesting that factors other than PAH activity alone play a role in phenylalanine clearance when repopulation frequency is low. Conclusion: Hepatocyte transplantation under selective growth conditions corrects hyperphenylalaninemia in murine PKU and is a useful model for the investigation of physiologic factors in liver that affect PHE clearance. The threshold for a therapeutic effect in murine PKU from hepatocyte transplantation is low; hyperphenylalaninemia significantly decreases with as few as 2% PAH positive hepatocytes. 35. Pharmacokinetic profile of recombinant human N-acetylgalactosamine 4-sulfatase (rhASB) enzyme replacement therapy in patients with mucopolysaccharidosis VI (Maroteaux–Lamy syndrome). Paul Harmatz,a Chester Whitley,b Robert Steiner,c Barabara Plecko,d Paige Kaplan,e John Waterson,a David Ketteridge,f Roberto Giugliani,g Ida Schwartz,g Nathalie Guffon,h Clara Sa Miranda,i Laura Keppen,j E.Leao Teles,k William G. Kramer,l and John J. Hopwoodf. aChildren’s Hospital and Research Center at Oakland, CA, USA; bUniversity of Minnesota Medical School, USA; c Oregon Health and Science University, USA; dUniversity Kinderklinik Graz, Austria; eChildren’s Hospital Philadelphia, USA; f Women’s and Children’s Hospital, Adelaide, Australia; gHospl de Clinicas de Porto Alegre, Brazil; hHospital Edouard Herriot, France; iMedica Jacinto de Magalhaes, Portugal; jUniversity of South Dakota, SD, USA; kHosp de Sao Joao, Porto, Portugal; l Kramer Consulting LLC, N. Potomac, MD, USA. Introduction: rhASB has been studied in: a randomized, doubleblind, two-dose Phase I/II study (n = 7; age 7–16 years), and a Phase II, open-label, single dose study (n = 10; age 6–22 years). Objective: To evaluate the pharmacokinetics profile of rhASB and the impact of antibody development. Methods: PK analysis was performed for Phase I/II at Weeks 1, 2, 12, 24, 83, 84, and 96 and Phase II at Weeks 1, 2, 12, and 24. Infusions were administered over 4 h using a ramp-up protocol. ASB was measured at 0–6.5 h. PK parameters were calculated using non-compartmental methods. Antibody to rhASB was determined by ELISA from baseline to Week 24 for Phase II and to Week 96 for Phase I/II. Results: For the Phase II study, the mean values for AUC0)t and AUC¥ increased from Week 1 to Week 2, but remained unchanged at Weeks 12 and 24. Although not significantly different, mean values for plasma clearance (CL) and volume of distribution (Vz) decreased at 24 weeks, consistent with the trend toward a higher AUC¥. Mean t1/2 was stable ranging from 15 to 19 min. Eight of 10 patients in Phase II developed antibody to rhASB.

By Week 24, Ab levels stabilized or decreased in 6 of 8 patients, but continued to increase in 2 patients. The patient with the highest antibody titer at Week 24 also had low ASB concentrations at Week 24. The Phase II PK results are consistent with the Phase I/II patients receiving 1 mg/kg. Phase I/II PK results at Weeks 83, 84, and 96 are similar to Week 24. Six patients in the Phase I/II study developed antibody to rhASB, 4 of 6 remained low, 2 of 6 increased to higher levels. Patient (#43) who developed high antibody titers had high ASB concentration. By Week 72, antibody had decreased in all patients and #43 had plasma ASB values consistent with other patients. Antibody levels did not appear to influence urine glycosaminoglycan (GAG) concentrations and were not associated with any infusion-related reactions. Conclusions: PK parameters for rhASB remain stable over time after several months of treatment. Antibody to rhASB develops in most patients, but decreases over time. Antibody formation may influence PK parameters during the early phases of treatment, but does not appear to significantly impact biochemical efficacy. (Sponsored by BioMarin Pharmaceutical Inc., Novato, CA). 36. Abnormal vesicle formation and trafficking in Hermansky–Pudlak syndrome cells. Amanda Helip-Wooley, M. Huizing, H. Dorward, D. Claassen, R. Hess, and W.A. Gahl. Medical Genetics Branch, NIH, Bethesda, USA. HPS is a disorder of abnormal biogenesis of lysosomes and related organelles. Seven genes are now identified causing HPS in humans (HPS-1 to -7). Some HPS-gene products are part of distinct protein complexes, including adaptor complex-3 (AP-3) and five different BLOCs (Biogenesis of Lysosome-related Organelles Complexes). We examined 129 patients with HPS to extensively characterize each subtype. All patients have oculocutaneous albinism and a platelet storage pool deficiency, i.e., absent dense bodies. HPS-1 and HPS-4 patients occasionally exhibit granulomatous colitis and fatal pulmonary fibrosis, but no pulmonary disease has been observed in the other subtypes. HPS-2 patients show persistent neutropenia and childhood infections. HPS-3 patients, and perhaps HPS-5 and HPS-6 patients, have milder disease, with occasional granulomatous colitis. HPS-2 encodes the b 3A subunit of AP-3, involved in trans-Golgi protein sorting and export of transmembrane cargo proteins to lysosomes. HPS7, dysbindin, is a ubiquitously expressed protein that binds to a- and b-dystrobrevins, components of the dystrophinassociated protein complex in muscle and nonmuscle cells. HPS7 is also a component of BLOC-1. The HPS1, 3, 4, 5, and 6 proteins all have unknown function. HPS1 and HPS4 interact as part of BLOC3, -4, and -5. HPS5 and HPS6 also interact and form BLOC-2. No HPS3 interactions have been shown. We characterized the distribution of integral lysosomal membrane proteins LAMP1, LAMP2, and CD63 in HPS-1 through HPS-6 fibroblasts. In HPS-1 and HPS-4 fibroblasts, a dense aggregation of LAMP staining occurs in the perinuclear region with little or no staining in the cell periphery. HPS-3, -5, and -6 display a phenotype similar to HPS-1 and HPS-4, but milder, with LAMP staining not as tightly restricted to the perinuclear region. In HPS-2 fibroblasts, lack of functional AP-3 results in mis-sorting of lysosomal membrane proteins and increased LAMP staining on the cell membrane. In HPS-2 melanocytes, tyrosinase remains in the peri-nuclear region while tyrosinase-related protein (TRP1) migrates with melanosomes to the dendrites. These cellular phenotypes can provide insight into the basic defect in each HPS subtype, and allow for in vitro investigations into corrective therapies. In addition, the use of LAMP immunofluorescence could be a useful diagnostic tool in ascertaining HPS subtypes. 37. A case management database profiling patient utilization of medical food for the treatment of phenylketonuria: a model for review of patient adherence to therapy recommendations, nutritional adequacy, indices of control and treatment costs. Kathleen Huntington, Cary Harding, Brian Hansen, and Sam Sanchez. Child Development

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 and Rehabilitation Center, Doernbecher Children’s Hospital, Oregon Health and Science University, Portland, OR, USA. Objective: To determine through the use of a case management database whether patient usage of medical food parallels therapy recommendations for maintenance of the plasma phenylalanine (PHE) level within treatment range for different age group specifications. A secondary objective was to develop a method for evaluating nutritional adequacy without relying on self-reporting through the mechanism of food record review and to develop a more comprehensive appreciation for minimum treatment costs to support appropriate therapy intervention. Methods: Since 1994 individual patient orders for all medical foods including both medical protein and low protein substitute products indicated for the treatment of inborn errors of metabolism have been entered into a case management database that was conceptually developed and implemented by Information Services at CDRC and programmed in C Language. Indices of biochemical control including those determined by inhibition bacterial assay, quantitative amino acid analysis and tandem mass spectroscopy have been entered on all patients monitored for blood PHE levels. Biochemistries including complete blood count, ferritin, essential fatty acids, and urine methylmalonic acid levels for evaluation of vitamin B12 status have also been entered. These entries profile patients in all age groups throughout the life span. Entered inventory data includes cost of unit, size of unit, number of units per case, protein and calorie content per gram of product. Results: Audits of individual patient orders provide feedback on patient adherence in terms of appropriate use of medical protein options and the suggested energy intake from all medical foods which are compared against the frequency of testing and average PHE results, nutritional biochemical review and growth assessment. These audits can be segmented into specific time periods as well as the entire ordering history of any or all patients included in the database. Profiles on treatment costs can be established since the database program also maintains inventory records on vendor orders, stock on hand and units shipped. Conclusion: A case management database can be an effective clinical supervision tool. By providing a comprehensive review of a patient’s ordering history, phenylalanine control and a suggested profile of medical protein and energy intake, one can determine a more realistic characterization of therapy intervention as well as general treatment costs for patients across the lifespan. 38. Expanded newborn screening for medium chain acyl-CoA dehydrogenase deficiency in western New York. Wendy J. Introne, Jane M. DeLuca, Beth Urbonas, Erin Lagoe, Eileen Blakely, and Georgianne L. Arnold. University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. In October, 2002, New York State implemented screening for medium chain acyl-CoA dehydrogenase (MCAD) by tandem mass spectrometry (TMS). Per state protocol, infants with C8 levels >0.29 are genotyped, and those with one or more mutations or with C8 levels >0.39 regardless of mutation status are reported to the PMD and nearest Inherited Metabolic Disorders clinic as presumptive positive. Prior to the initiation of the state screen our clinic followed 5 MCAD patients, one of whom was neurologically devastated and two of whom were his asymptomatic siblings. In our first year, our metabolic center received 43 referrals for abnormal newborn screens for MCAD. With rare exceptions we were unable to arrange to have confirmatory studies sent through the PMD, thus all infants not in the NICU were seen in our IMD clinic and families traveled an average of 67 miles for evaluation. Of the 43 referrals, 4 individuals were identified as affected and 21 as carriers due to the presence of the K304E mutation. One infant was found to be homozygous for K304E mutation, the other three are compound heterozygotes with one of the three carrying two novel mutations. Interestingly, one unusually brittle patient clinically is a compound heterozygote for K304E with one mutation still undetected. The average initial C8 level for affected individuals was


15.25 lmol/l (3.67–37.37 lmol/l) while the average initial C8 level for all unaffected individuals was 0.43 lmol/L (0.28–1.0 lmol/L), and when infants with birth weight less than 1500 g were excluded the mean C8 was 0.39 lmol/L (0.29–0.49 lmol/L). On confirmatory testing, C8 levels decreased in all four affected patients (mean 4.45 lmol/L, range 1.98–6.3 lmol/L), but none of the levels fell to the unaffected range. Conclusions: Currently, the positive predictive value of the NY state screen is 10%. The burden on families and regional metabolic centers is significant. However one infant was recognized just in time to save her from early decompensation, and in only one year our affected population has nearly doubled. Thus this disorder has been under-diagnosed despite our efforts to educate local physicians, and this screen is clearly lifesaving. 39. Maternal homocystinuria: impact of gestation, birth, breast feeding on total homocysteine using dietary management and low dose heparin. Janet S. Isaacs, Susan E. Sparks, Pim Suwannarat, Uta Lichter-Konecki, Cynthia J. Tifft, and Gerard T. Berry. Department of Pediatrics, The George Washington University School of Medicine and Children’s National Medical Center, Washington, DC, USA. Objective: To maintain total homocysteine at less than 50 lmol/L in a patient with treated B6-responsive homocystinuria during pregnancy, delivery, the post-partum period, and breast-feeding. Methods: Total homocysteine, plasma amino acids, dietary intake of natural and methionine-deficient protein and kilocalories, and weight were monitored during three pregnancies. Results: Pre-pregnancy total homocysteine was well controlled at about 30 mmol/L with a low protein diet (45 g/day or 0.8 g/kg) supplemental B6, folic acid and calcium, but without a methionine-deficient formula. All pre-pregnancy vitamin and mineral supplements were continued during each pregnancy and the natural protein tolerance was increased to 55 g/day. Methioninedeficient formulas and supplemental cystine (400 mg qd) were initiated based on rising total homocysteine and low cystine levels during the second trimester of each pregnancy. Cystadane (3 g bid) was added in the third trimester for the first and second pregnancies. Total homocysteine tripled within 24 h after delivery despite continuation of the low protein diet. The peak total homocysteine elevation was at post-partum day 4 and 6 in pregnancies two and three, and later, near post-partum day 35 in the first pregnancy. In the third post-partum period, total homocysteine declined from 94 to 57 lmol/L within 48 h when adequate caloric intake was documented. In the three pregnancies, total homocysteine returned to pre-pregnancy levels in 4–6 weeks after delivery, regardless of breast-feeding status. For each delivery the patient was supported with adequate fluids (1.5 times maintenance) that continued into the post-partum period. Lovenox was prescribed shortly after delivery to minimize the risk of thromboembolism and continued until total homocysteine was under 50 lmol/L. All three infants were born at term without congenital anomalies, however, the second infant developed viral endocarditis and died at day 6 of life. No connection to homocystinuria has been identified. Conclusions: The catabolism of delivery resulted in high total homocysteine levels, but the rate of decline was faster when adequate caloric intake was stressed. Our experience suggests that vitamin and cystine supplementation, dietary adjustments and close monitoring throughout pregnancy, delivery, and the post-partum period are of value in maternal homocystinuria. 40. Urine sulfite assay: how important is the ‘‘freshness’’ of urine samples? Katie Ker and Murray Potter. McMaster University, Hamilton, Ont., Canada. Objective: Urine sulfite dipstick tests used in diagnosing sulfite oxidase deficiency and molybdenum cofactor deficiency must be performed on ‘‘fresh’’ urine due to the rapid oxidation of sulfite to sulfate. The objective of this study was to determine the length of time urine might be stored before producing false-negative results


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considering the detection limit of two sulfite tests, the initial urine sulfite concentration, and the rate of oxidation at various temperatures. Methods: Normal urine was spiked with sulfite to produce initial concentrations of 10, 25, and 250 mg/L. Aliquots of each solution were tested at baseline and stored at room temperature, refrigerated, or frozen. Two methods for testing sulfite levels were used: Sulfite Test Paper and Quantofix sulfite test sticks, both from Macherey–Nagel and distributed by Aldert Chemicals. Aliquots of each concentration and temperature were tested using both methods at 8 h, 1, 2, 3, and 4 days, and the remaining frozen aliquots were tested at 1, 2, and 3 weeks. Results: Sulfite Test Paper results for the 10 mg/L solution were negative for samples at each temperature for all times tested, even at baseline. For the 25 mg/L solution, room temperature samples were positive for up to 2 days, refrigerated samples were positive for up to 4 days, and frozen samples were positive for up to 1 week. For the 250 mg/L solution, results were positive for samples at each temperature for all times tested. The Quantofix test sticks were unable to detect sulfite in the 10 mg/L solution. For the 25 mg/L solution, sulfite levels greater than 10 mg/L were detected for up to 8 h for room temperature samples, 3 days for refrigerated samples, and 4 days for frozen samples. For the 250 mg/L solution, no decrease in sulfite concentration was detected for samples at each temperature for all times tested. Conclusion: The two tests have comparable detection limits of 10 mg/L. Samples with initial sulfite concentrations of 25 mg/ L should be tested before 8 h if stored at room temperature, 3 days if refrigerated, and 4 days if frozen. For samples with higher initial concentrations, the freshness of the sample is less critical. There is little in the literature about actual urine sulfite levels in affected patients, however, clinicians could use this information when considering the validity of a negative result. 41. Sodium phenylbutyrate toxicity: case report and potential mechanism. Renee P. Kinman,a,b Takhar Kasumov,a Charles L. Hoppel,c,d Henri Brunengraber,a and Douglas S. Kerrb. aDepartments of Nutrition, bPediatrics, cPharmacology, and dMedicine, Case Western Reserve and University, Cleveland, OH, USA. Sodium phenylbutyrate (PB) is a highly effective drug used in the treatment of patients with hyperammonemia resulting from inborn errors of urea synthesis. However, its clinical usefulness is limited by occasional episodes of toxicity at high doses, including neurologic dysfunction and electrolyte abnormalities, and pancreatitis. We report here the case of an 18-year-old female who experienced what we believe to be a life-threatening toxic reaction to PB that resolved following a marked decrease in her PB dose from 500 to 125 mg kg)1 day)1. Complications included a metabolic encephalopathy, pancreatitis, lactic acidosis, pancytopenia, persistent tachycardia, multiple electrolyte abnormalities (including severe hypokalemia), anorexia, dysarthria, and exacerbation of peripheral neuropathy, and resulted in a prolonged recovery time (>2 months). Because of recurrent attacks of pancreatitis, PB was ultimately discontinued several months later and the patient placed on sodium benzoate. Plasma acylcarnitine profiles obtained when she was on high dose PB showed normal carnitine, lownormal free carnitine, high-normal total acylcarnitine:free acylcarnitine ratio (including high levels of C12–C18 acylcarnitines), and prominent phenylacetylcarnitine. Urine organic acid analysis revealed high concentrations of lactic, pyruvic, phenylacetic, and phenylbutyric acids. These were greatly diminished following the 4-fold decrease in PB dosing, and completely resolved following replacement of PB with sodium benzoate. Urinary acylcarnitine profiles at this time revealed that benzoylcarnitine accounted for 50% of the excreted acylcarnitines. The high levels of long-chain acylcarnitines, combined with the increased lactic and pyruvic acids, suggest that PB metabolism may interfere with oxidative metabolism. It remains unclear at this time whether (1) the observed carnitine insufficiency is secondary to production of an excess of acylcarnitines (e.g., phenylacetylcarnitine and phenylbenzoylcarnitine), (2) PB toxicity is associated with

interference with oxidative metabolism, and (3) PB toxicity may result from accumulation of certain intermediary or end metabolites of PB, or some other cause. 42. Atypical clinical and biochemical presentation in a patient with molybdenum cofactor deficiency. Soledad Kleppe, M. Shinawi, J. Hunter, and F. Scaglia. Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA. Molybdenum cofactor deficiency (MCD) (MIM#252150) is a rare progressive neurodegenerative disorder that usually is considered in the evaluation of patients with intractable neonatal seizures. The observed progressive demyelinating encephalopathy is most probably due to the accumulation of toxic sulphite levels, however the underlying pathomechanism is still unknown. We present a 9-monthold Mexican-American infant with MCD who exhibited an unusual phenotype with a static encephalopathy, dysmorphic features, and no seizure disorder. The proband was born to non-consanguineous parents after an uneventful pregnancy. At the age of 2 weeks, he presented with an abnormal neurological exam consistent with spastic quadriparesis, opisthotonos, nystagmus, and irritability. The patient was initially referred for evaluation of a possible respiratory chain (RC) defect due to persistent lactic acidosis. Brain MRI revealed cerebral atrophy, gliotic white matter, and thinned corpus callosum. A significant lactate peak was visualized on MR spectroscopy. The patient was found to have a significantly decreased total plasma homocysteine, and low serum and urine uric acid that prompted the work-up for MCD. The diagnosis was confirmed with an abnormal urine purine profile and elevated sulfocysteine in urine. The finding of low total plasma homocysteine in this patient suggests, in conjunction with previous reports, that hypohomocysteinemia may be a general feature of MCD although the possible role in the pathogenesis is unknown. Peripheral lactic acid elevation and increased lactate peak of brain MRS have been observed in two reported cases of MCD. Sulfite oxidase interacts with the mitochondrial RC since it catalyzes the oxidation of cytochrome c by sulfite. Hence, sulfite oxidase deficiency could secondarily affect the mitochondrial RC. The atypical clinical presentation of this infant points towards a considerable phenotypic variability in MCD, expanding the clinical spectrum of this condition. 43. FISH analysis of the common 57-kb deletion in cystinosis. Claude Bendavid,a Robert Kleta,a Robert Long,a Maia Ouspenskaia,a Bassem R. Haddad,b Maximilian Muenke,a and William A. Gahla. a Medical Genetics Branch, NHGRI, NIH, Bethesda, MD, USA; b Georgetown University Medical Center, Washington DC, USA. Cystinosis is an autosomal recessive lysosomal storage disorder caused by mutations in CTNS. Most mutations in this lysosomal transporter protein lead to a renal tubular Fanconi syndrome in infancy, to renal glomerular failure at age 10, and to other complications in early adulthood. CTNS resides on chromosome 17p13, spans 25 kb of genomic DNA, and has 12 exons, the last 10 of which code for the 367 amino acid protein cystinosin. The most prevalent CTNS mutation, present in 50% of patients of Northern European extraction, is a 57,257-bp deletion removing exons 1–10, the promoter region and an adjacent gene of unknown function called CARKL. This founder mutation is thought to have arisen in Germany 500 AD. This study describes the generation of a FISH probe for this deletion. Using the sequenced BAC RP11-235E17 containing CTNS and CARKL, we designed three 10-kb PCR amplicons resulting in a 30kb contig inside the 57-kb deleted region. Those 3 PCR products were cloned in TOPO XL plasmid and were labeled with Cy3 and FITC. They were tested separately and combined on normal chromosome spreads with a Cy5 labeled control probe hybridizing in 17q23. In a blinded study using multiplex PCR to test for the presence and absence of the 57-kb deletion, the probe’s contigs successfully identified 12 lymphoblastoid cell lines as either singly or doubly deleted, or

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 undeleted. For laboratories with FISH capabilities but no molecular diagnostics, this technique can accurately diagnose the 57-kb deletion in CTNS. In addition, an accurate prenatal diagnosis is possible even in the event of a heterozygous maternal contamination. 44. Hepatocellular carcinoma as a complication of glycogen storage disease type III. John Koepke,a Donald Frush,b Anne Boney,a Deeksha Bali,a YT Chen,a and Priya Kishnania. aDepartment of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, NC, USA; bDepartment of Radiology, Duke University Medical Center, Durham, NC, USA. Glycogen storage disease type III (GSD III) is caused by deficiency of glycogen debranching enzyme, resulting in accumulation of abnormal glycogen in various organs. Hepatomegaly, hypoglycemia, short stature, variable myopathy, and cardiomyopathy characterize GSD III. Liver cirrhosis, liver failure, and hepatocellular carcinoma (HCC) has been reported, but are not recognized long-term complications of GSD III. In our series of 29 GSD III patients, age range (2–68, mean age 16.9) HCC was diagnosed in the 2 oldest at the ages of 54 and 67. Case reports: We report 2 Caucasian male patients with GSD IIIa followed at our center for 19 and 32 years, respectively,. Both demonstrated progressive myopathy and were wheelchair bound by ages 43 and 52, respectively. Serial liver functions, including a fetoprotein (AFP) were normal, and hepatitis B and C screening performed in one of the patients was also normal. Serial imaging exams of the liver showed stable volume and the development of cirrhosis in both patients, one patient developed a 2 cm adenoma at the age of 53 and an additional 9 mm adenoma at the age of 56, these lesions remained stable over the next 7 years. Both patients were lost to follow-up for 1 and 5 years, respectively. At the time of their return they were found to have metastatic HCC, while still maintaining normal liver functions including synthetic functions. Discussion: As life expectancy in GSD III increases, the natural history of the disease is unfolding. Historically, progressive myopathy was considered the major cause of morbidity. There have been a few reports of liver cirrhosis and HCC. These two patients developed severe myopathy, and had stable liver cirrhosis at the time of HCC development. The pathophysiology of the progression to HCC in GSD III is unclear. Possible factors could be abnormal glycogen structure leading to fibrosis, cirrhosis, and end stage liver disease. Currently there are no guidelines for monitoring liver disease in GSD III patients. Our cases demonstrate that current monitoring practices are not sufficient for proper health surveillance. We believe more stringent liver surveillance is needed including more frequent use of imaging techniques that allow accurate assessment of liver volume, architecture, and signs of cirrhosis. Furthermore, identification of risk factors, such as exposure to hepatitis B or C, amongst others should be included and orthotopic liver transplant should be considered as a treatment option in the care of individuals with GSD III. 45. Hepatocellular carcinoma in glycogen storage disease type Ia: a case series. Vidya Krishnamurthy,a Luis Franco,a Deeksha Bali,a David Weinstein,b Pamela Arn,c Bryan Clary,d Y.T. Chen,a and Priya Kishnania. aDepartment of Pediatrics, Division of Medical Genetics, Duke University Medical Center, Durham, USA; b Division of Endocrinology, Children’s Hospital, Boston, USA; c Nemours Children’s Clinic, Jacksonville, USA; dDepartment of Surgery, Duke University Medical Center, Durham, USA. We present eight patients (6 males, 2 females) with hepatocellular carcinoma (HCC) as a long term complication of Glycogen Storage Disease Type I (GSDI). Age of GSDI diagnosis ranged from birth to 7 years (mean age 7 months). Age when cornstarch/frequent feeds was initiated ranged from birth to 39 years. Five patients started treatment by age 1, 2 by age 7, and 1 by age 15. Seven patients were followed by a metabolic physician; one was lost to follow up until age 33. All patients were non compliant and had irregular follow up. GSD Ia mutations


detected in these patients were Homozygous Q347X (2) ; Compound heterozygote Q347X/35X (1); Homozygous R83C (1); Compound heterozygous R83C/unknown (1); homozygous for G727T (1) and 2 patients had no mutational analysis done. Hepatic adenomas of varying sizes were diagnosed at age ranges 13–45 years (mean age 19) in 6 patients. In three patients adenoma status prior to HCC diagnosis was unknown (one hepatitis B positive with liver cirrhosis, one with adenomas at HCC diagnosis and in one prior adenoma status unclear). Age at HCC diagnosis ranged from 19 to 50 years (mean age 41). Two patients had positive hepatitis serologies (1 HepB, 1 Hep C). Follow up a-fetoprotein (AFP) in 6 patients and Carcinoembryonic Antigen (CEA) in 7 patients remained normal even after HCC diagnosis. Delayed initiation of currently accepted therapy and poor compliance.was common to all patients. 6 patients had pre-existent HCA’s, prior to or at time of HCC development, whereas in 2 patients HCA status was unknown. Progression to HCC is likely to be a part of an adenoma–carcinoma sequence rather than a de novo carcinogenesis. Mean duration between onset of adenomas and HCC development for 5 patients in whom data was available was 13 years (range 3–32). This series confirms HCC as a long term complication in patients with GSD Ia and hepatic adenomas. Current guidelines recommend 3 monthly abdominal ultrasounds with AFP and CEA levels once patients develop adenomas. Abdominal CT/MRI is advised when adenomas are poorly defined, of large size or have increasing growth. Risk factors such as hepatitis B/C should be included in the evaluation of GSD I patients. We question the reliability of AFP and CEA as a marker for HCC development. HCA management should be aggressive and include Percutaneous ethanol injection, radio frequency ablation, transcatheter arterial embolization, and liver transplantation to prevent HCC. 46. A new prospective multi center study of treatment and outcome in urea cycle disorders. Uta Lichter-Konecki, Mark L. Batshaw, Stephen Cederbaum, George Diaz, Brendan Lee, Greta Seashore, Marshall Summar, Marc Yudkoff, and Mendel Tuchman. Children’s National Medical Center (CNMC), Washington, DC, USA. The objective of the study is to determine the natural history, efficacy of the current state of the art treatment, and neuropsychological outcome of patients with urea cycle disorders (UCDs). The clinical goal of the study is to determine the optimal therapy for patients with UCDs. Its research goal is to contribute to the understanding of the underlying pathophysiology of UCDs. This study is funded by a NIH Rare Disease Clinical Research Center grant given to CNMC and 6 collaborating institutions (Baylor College of Medicine, UCLA, Children’s Hospital of Philadelphia, Vanderbilt University, Yale University, and Mt. Sinai School of Medicine). The primary goals of the RDCRC are to: (1) Establish a registry and nation-wide network of regional centers for the diagnosis, treatment and clinical research in UCD; (2) Conduct a longitudinal study to determine the natural history of UCD; (3) Conduct a clinical trial of an investigational new drug, N-carbamyl-L -glutamate, for treatment of these disorders; (4) Conduct a demonstration/pilot project to develop a novel method for measuring in vivo ureagenesis in UCD using 13C acetate that will be important for diagnosis and classification of patients and for evaluation of treatment efficacy; (5) Train graduate students, pediatric residents, clinical fellows and junior faculty members in the field of inborn errors of metabolism; and (6) Develop and maintain UCD website content that will: (a) include guidelines for health care providers regarding diagnosis and treatment; (b) provide information to the lay public regarding consultation and treatment at major centers; and (c) provide links to recent scientific literature for interested investigators. This initiative is being undertaken in close collaboration with the National Urea Cycle Disease Foundation (NUCDF), the leading public advocacy organization for this group of diseases.


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47. Tyrosine residues affecting sodium stimulation of carnitine transport in the OCTN2 carnitine/organic cation transporter. Nicola Longo, and Amat di San Filippo C. Division of Medical Genetics, Department of Pediatric, University of Utah, Salt Lake City, UT, USA. Primary carnitine deficiency is disorder of fatty acid oxidation caused by mutations in the sodium-dependent carnitine/organic cation transporter OCTN2. Studies with tyrosyl-group modifying reagents support the involvement of tyrosine residues in sodium binding by sodiumcoupled transporters. Here we report two new patients with carnitine deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (E452) previously shown to affect sodium stimulation of carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in CHO cells, increased the concentration of sodium required to half-maximally stimulate carnitine transport from 14.8 ± 1.8 mM to 34.9 ± 5.8 mM (p < 0.05), while Y447C completely abolished carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal carnitine transport in Y449F, but resulted in markedly impaired carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate carnitine transport to 57.8 ± 7.4 mM (p < 0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium, indicating that their effect on carnitine transport was specific and likely associated with the impaired sodium stimulation of carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to carnitine. Confocal microscopy of OCTN2 transporters tagged with the green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, while Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2. 48. Clinical and molecular findings in a discordant twin with Bartter syndrome type II. Melissa Merideth,a,b Michael Freundlich,c Caroline Stuart,a William A. Gahl,a,b and Robert Kletaa,b. aSection on Human Biochemical Genetics, MGB, NHGRI, NIH (DHHS), Bethesda, MD, USA; bOffice of Rare Diseases, Intramural Program, Office of the Director, NIH (DHHS), Bethesda, MD, USA; cDepartment of Pediatrics, University of Miami, Miami and Joe DiMaggio Childrens Hospital, Hollywood, FL, USA. Bartter syndrome type II is an autosomal recessive renal salt losing nephropathy caused by mutations in KCNJ1. It has significant morbidity if not recognized and treated. KCNJ1 codes for an integral membrane potassium channel protein in the thick ascending limb (TAL) in the loop of Henle. Mutations in SLC12A1 (Na2ClK cotransporter), CLCNKB (chloride channel), and BSND (chloride channel subunit) lead to Bartter syndromes type I, III, and IV, respectively. KCNJ1 resides on chromosome 11q24, spans 29 kb of genomic DNA, and has 5 exons. Five alternatively spliced variants code for 3 different isoforms. Transcript variant 2 (also named ROMK2) is expressed in the kidney and codes for a 372 amino acid protein. Mutations in this splice variant, derived from exons 1 and 5 of KCNJ1, give rise to Bartter syndrome type II. Here we report the clinical and molecular findings in a boy with polyhydramnios who was born significantly smaller than his same sex twin born without polyhydramnios. Based on the prenatal findings, a twin–twin transfusion syndrome was suspected, but eventually excluded by the finding of a dichorionic, diamniotic, non-fused twin placenta. Shortly after birth the affected twin displayed marked hyperkalemia (6–7 mmol/L), hyponatremia (129 mmol/L), azotemia (BUN 44 mg/dl), natriuresis (sodium 118 mmol/L), and polyuric renal failure (creatinine 2.0 mg/ dl), which gradually changed to hypokalemia (3 mmol/L) following sodium chloride supplementation. Due to the defective potassium

channel, potassium cannot be secreted into the lumen of the TAL resulting in hyperkalemia. Since about 20% of NaCl reabsorption in this nephron segment is handled by the concerted action of SLC12A1, CLCNKB, BSND, and ROM-K2, a furosemide-like diuresis takes place, leading eventually to compensatory hyperaldosteronism. The latter causes increased sodium (and water) reabsorption more distally in exchange for potassium secretion. Since this compensatory mechanism is absent before birth, affected neonates present initially with hyperkalemia, followed by hypokalemia shortly thereafter. This finding is typical of neonates affected by Bartter syndrome type II. The child was compound heterozygous for two missense mutations in ROM-K2, a known R311W and a novel L359R mutation. During infancy, the patient developed polyuria, growth retardation (weight and height < 3rd percentiles), hypoosmolar urine, hypercalciuria, nephrocalcinosis, metabolic alkalosis, and elevated prostaglandins. Treatment with a COX-2 inhibitor, rofecoxib, improved all laboratory abnormalities and anthropometric delay. In summary, we present a rare but important cause of discordant growth in twins, early neonatal non-oliguric hyperkalemia and hypokalemia in infancy. 49. Newborn and high risk screening for glutaric aciduria-type 1: results of primary tandem mass spectrometry and 2nd-tier molecular testing on over 1.8 million specimens. Edwin W. Naylor, D.H. Chace, J.C. DiPerna, T.A. Kalas, R.A. Banas, Z. Lin, and P. Murphy. Pediatrix Screening, Inc., 90 Emerson Lane, Bridgeville, PA, USA. Glutaric aciduria-type 1 (GA-1) is a disorder in organic acid metabolism due to a deficiency in glutaryl-CoA dehydrogenase. Newborn are clinically normal but develop an acute neurological crisis generally precipitated by an infectious illness. Early pre-symptomatic treatment can prevent the onset of serious clinical symptoms in over 75% of patients. We introduced the use of tandem mass spectrometry (MS/MS) for routine newborn screening for inborn errors of amino, organic, and fatty acid disorders in late 1992 and picked up our first cases of GA-1 in 1993. Through December 31, 2003 we had screened over 1.8 million newborns and high risk patients using MS/MS. We have detected 26 newborns through prospective screening and also detected or confirmed 40 cases sent to us for high risk screening. The glutarylcarnitine means and ranges for both newborns < 4days of age and older cases 5 days to 44 years of age were calculated as were the means and ranges for the C5/C16 ratios for newborns and older cases. We routinely test for the presence of the common Amish mutation (A421V) as well as the common Latin American mutation (R402W) in the original filter paper blood specimen as part of our 2ndtier testing program using real time PCR and FRET analysis with the Roche LightCycler. Several of the unknown mutations have been sequenced. 19 of our 26 newborns were homozygous for the A421V mutation; 2 were A421V compound heterozygotes; and the mutation was not identified in 4 cases. Among the 40 older high risk patients 10 were A421V homozygotes; 8 were A421V compound heterozygotes; 1 was an R402W homozygote; 2 were R402W compound heterozygotes; 4 were compound heterozygotes with two other known mutations; and in 16 the mutation was not identified. 50. Somatic mosaicism in a male with an exon skipping mutation in PDHA1 of the pyruvate dehydrogenase complex results in a mild phenotype. K. Okajima, E. Sieverding, M.L. Warman, and D.S. Kerr. Center for Inherited Disorders of Energy Metabolism, Rainbow Babies and Children’s Hospital and Department of Genetics, Case Western Reserve University, Cleveland Ohio, USA. Patient: This 5-year-old male has hypotonia, moderate developmental delay and seizures. His growth and brain MRI are normal. Lactate is normal to mildly elevated in blood (1.5–2.9 mM) and CSF (3.5–4.4 mM) with a normal lactate/pyruvate ratio. Pyruvate dehydrogenase complex (PDC) activity is low in skin fibroblasts and in skeletal muscle (27 and 37% of control, respectively) and within the

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 normal range in lymphocytes (60% of control). PDHA1 (E1) activity is low in skin fibroblasts and in skeletal muscle. DLAT (E2) activity and DLD (E3) activity are normal. Standard karyotype and FISH revealed a normal male (46, XY). Methods: Genomic DNA from skin fibroblasts, lymphocytes, buccal swab, and hair roots was amplified by PCR followed by direct sequencing. Entire cDNA from skin fibroblasts was sequenced after RT-PCR. Western blot and immunohistochemistry were done with skin fibroblasts using a commercially available anti-PDHA1 monoclonal antibody. All procedures were done in duplicate. Results: PDHA1 cDNA analysis from skin fibroblasts revealed two populations of mRNA; one species was wild-type, the other lacked exon 6 (c.511-603 del). Genomic DNA from skin fibroblasts contained nearly equal amounts of wild-type sequence and sequence harboring g.592G > A in exon 6. Flanking introns had wild-type sequence. The mosaic ratio varied among tissues analyzed. Mutant:normal ratio in skin fibroblasts and buccal swab was nearly 1:1, while lymphocytes and hair roots showed much lower ratios. Western blotting detected normal molecular weight PDHA1 and no smaller bands. Immunohistochemistry suggested a mosaic pattern for PDHA1 expression levels. Discussion: PDHA1 is an Xlinked gene; thus, only single sequence should be present in males. Our data indicate this patient is a somatic mosaic for a leaky exon 6 skipping mutation. Mosaicism due to aneuploidy, chimerism, or trisomic rescue were excluded by karyotype and polymorphic marker analysis. The 592G residue is conserved among all eukaryotes including yeast. 592G > A may lead to exon skipping by creating a novel SRp40 exonic splice enhancer site. This is the second report of a PDHA1 exon 6 skipping mutation in males, the first being in two male cousins with Leigh syndrome and a hemizygous silent mutation (g.555A > G; G185G) which caused leaky exon 6 skipping. We hypothesize that a reduced amount of normal PDHA1 in a subset of cells which contain the g.592G > A mutation accounts for the biochemical and clinical phenotype, in our patient. 51. Barth syndrome with presumed secondary carnitine palmitoyltransferase II deficiency. Susan E. Palmer, Richard L. Boriack, and Michael J. Bennett. University of Oklahoma Health Science Center, Oklahoma City, OK, USA; Children’s Medical Center, University of Texas Southwestern, Dallas, TX, USA. Barth Syndrome is an X-linked disorder characterized by dilated cardiomyopathy, skeletal myopathy, growth, and developmental problems, neutropenia, and 3-methylglutaconic aciduria. We present a case whose initial presentation suggested a diagnosis of carnitine palmitoyltransferase II (CPT2) deficiency. Case Report: The medical history of this almost 4-year-old male includes cardiogenic shock at 6 weeks of age. He was found to have a dilated hypertrophic cardiomyopathy with pericardial effusion. The family history was unremarkable. Testing of amino acids, organic acids, carnitines, and acylcarnitines was non-diagnostic; CPK levels are unknown. Fatty acid oxidation studies from fibroblasts revealed a modest reduction of palmitate oxidation with normal myristate oxidation. CPT2 activity was reduced to approximately half of controls. At age 2, repeat organic acid analysis revealed excessive excretion of 3-methylglutaconic acid. The cardiomyopathy showed some improvement and stabilized, but failure to thrive, mild developmental delay, and mild muscular weakness with hypotonia persisted. At age 3, he developed recurrent fevers and intermittent neutropenia was found which later became somewhat chronic. Analysis of the TAZ1 gene revealed a previously reported G fi A mutation at base 934 of mRNA (gly 216 arg). CPK and carnitine levels were normal at that time. Discussion: Barth Syndrome and CPT2 deficiency are both diseases of the inner mitochondrial membrane, which are considered to be genetically distinct. The acyltransferase encoded by CPT2 facilitates the transport of long-chain fatty acids across the mitochondrial membranes into the matrix. The causative gene for Barth syndrome, G4.5 or TAZ1, encodes a family of tafazzin proteins, which play a role in the


deficiency of cardiolipin observed in these patients. Cardiolipin, as the major glycerophospholipid of the inner mitochondrial membrane, is believed to be essential to the stability of the membrane’s structure and function. Various respiratory-chain abnormalities in fibroblasts from other individuals with Barth syndrome have been reported in the literature. A less-specific indicator of inner membrane and oxidation phosphorylation dysfunction is the 3-methylglutaconic aciduria. CPT2 deficiency in this proband is suggestive of a yet another secondary functional defect of an inner mitochondrial membrane enzyme complex associated with the cardiolipin deficiency of Barth Syndrome. 52. The Cystinotic phenotype may be caused by cystine-induced apoptosis due to alterations of cytochrome C and/or PKCd. Margaret A. Park and Jess G. Thoene. Hayward Genetics Center, Tulane University, New Orleans, LA, USA. Cystinosis is an autosomal recessive disorder caused by a defect in the lysosomal cystine transporter. The defect is characterized by cystine accumulation, short stature, corneal crystals, retinopathy, renal Fanconi syndrome, the swan neck deformity and end stage real failure by age ten if untreated. We have recently found that cystinotic fibroblasts undergo apoptosis at a greater rate than normal fibroblasts in response to three different apoptotic stimuli, and that this is due to the lysosomal cystine storage. Lysosomal membrane permeabilization is an early apoptotic event, occurring upstream of mitochondrial involvement. Lysosomal cystine enhanced apoptosis (LCEA) is dependent on p53, as HeLa S3 cells (which do not express p53), do not show increased apoptosis when loaded with cystine dimethylester (CDME), a compound which causes lysosomes to accumulate cystine. We hypothesize that lysosomal cystine released during apoptosis, enhances the activity of a protein involved in the apoptotic cascade. We have thus far studied cytochrome C (CyC) and PKCd, which fit these criteria. Cytochrome C, a heme-containing mitochondrial protein, is released to form the apoptosome during the mitochondrial membrane permeability pore formation phase of apoptosis. It is most active in apoptosome formation in its ferric form. We find that cystine, in a 1:2 molar ratio oxidizes CyC as determined by scanning spectrophotometry. This could increase apoptosome formation and hence the rate of apoptosis. When microinjected into renal proximal tubule epithelial cells, oxidized CyC induces apoptosis at a greater rate than in its reduced form. PKCd is a protein kinase C isoform. It is proapoptotic when activated by caspase-3. We show that 35S-cystine forms a disulfide bond with PKCd, and at 1.0, 2.0, and 4.0 mM, cystine enhances activity of PKCd, 3-fold. Microinjection of PKCd, that was pre-incubated with 4.0 mM cystine induces 47.7% apoptosis in renal proximal tubule epithelial cells, compared to a rate of 18.9% for cells microinjected with native PKCd. LCEA and hence the cystinotic phenotype, may be caused by oxidation of CyC and/or cysteinylation of PKCd by lysosomally released cystine. 53. Clinical significance of DG galactosemia: literature review and meta-analysis. V.K. Proud, L. Lawson, M.A. Barnes, M.E. Kent, and H.A. Creswick. Division Medical Genetics and Center for Pediatrics Research, Department of Pediatrics, Children’s Hospital of The King’s Daughters, Eastern Virginia Medical School, Norfolk, VA 23507-1921, USA. Duarte/galactosemia (D/G) heterozygotes make up the majority of abnormal newborn screening results for galactosemia. With approximately 25% galactose-1-phosphate uridyltransferase (GALT) activity, one could predict that heterozygotes should be at minimal risk for medical problems. However, many heterozygotes are treated for the first year or two and concerns have been raised about long-term problems such as premature ovarian failure or cancer in females. Methods: Comprehensive literature review and meta-analysis were performed to determine whether DG heterozygotes in the literature have medical complications. 701 abstracts were reviewed to identify


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thirty-three (33) clinical articles. Data collected included ten biochemical and nine clinical parameters. Results: In 8 articles with 41 individuals only 18 patients were evaluated for clinical problems and in 9 articles with 305 patients with grouped data, no patients were reported to have medical complications. 16 articles contained only author opinions. In 19 articles focusing on DG heterozygotes, 7/19 had data that found no abnormal phenotype, another 7/19 found clinical abnormalities, and 5 articles neither supported nor denied medical complications. Among the 33 articles, four referred to case reports of cataracts, liver dysfunction, or decreased ovarian function, while one reported increased frequency of Duarte alleles in families with ovarian cancer. Biochemical studies predicted mixed results depending on parameters studied but the urinary galactitol levels and labeled galactose breath analysis suggested potentially significant abnormalities. Interestingly, an inverse relationship was found between age and enzyme activity, while complementing data demonstrated positive correlation between age and urinary galactitol. Altogether one-half of 102 patients from grouped data have biochemical abnormalities. Conclusions: There are a limited number of studies with little patientspecific data and only one study with long term follow up that did not show medical problems. All of the reports suggesting clinical complications are based on single case reports or anecdotes. Biochemical studies however, do suggest potential for medical problems. This analysis strongly supports the need to establish a registry of DG heterozygotes, to follow them longitudinally and to perform robust data analysis to determine if they are at risk, what enzyme levels or other biochemical parameters define that risk, and what dietary intervention, if any, is needed. 54. CLN3P, the Batten disease protein, localizes to membrane lipid raft domains. Dinesh Rakheja,a Johanne V. Pastor,a Srinivas B. Narayan,a and Michael J. Bennett,b. aDepartment of Pathology, b Department of Pathology and Pediatrics, Children’s Medical Center of Dallas and the University of Texas Southwestern Medical Center, Dallas, TX, USA. Background: Batten disease or juvenile neuronal ceroid lipofuscinosis (JNCL) is the most common inherited pediatric neurodegenerative disease. This autosomal recessive disease results from mutations in the CLN3 gene on chromosome 16p12.1. CLN3 encodes a 438 amino acid protein (CLN3P), predicted to have 5–7 transmembrane domains, the function of which is not known at present. During immunoprecipitation of CLN3P from brain tissue of wild-type mice, we discovered that the immunoprecipitate consisted of multiple proteins besides CLN3P. To explain this aberration, we hypothesized that CLN3P resides on membrane lipid raft domains. Materials and methods: We analyzed the immunoprecipitate for cholesterol and fatty acids using gas chromatography–mass spectrometry, and for flotillin-1 (a raft-associated protein) by Western blot. Lipid rafts were isolated from brain synaptosomes of wild-type mice using sucrose gradient centrifugation, sonication, and iodixanol gradient centrifugation; and from normal bovine brain tissues using Triton X-100 treatment followed by sucrose gradient centrifugation. Western blot was performed to look for the presence of CLN3P in the lipid raft and other fractions. Results: The CLN3P immunoprecipitate was found to contain cholesterol, fatty acids, and flotillin-1. Conventionally isolated lipid rafts from bovine brains and mouse brain synaptosomes were shown to contain CLN3P. Discussion: The recognition that CLN3P is located on lipid raft domains is a significant step towards understanding its function. 55. Glutaric acidemia type I can be missed by plasma acylcarnitine analysis. Dinesh Rakheja,a Vivian K. Jones,a and Michael J. Bennettb. aDepartment of Pathology and bPediatrics, Children’s Medical Center of Dallas and the University of Texas Southwestern Medical Center, Dallas, TX, USA. Glutaric acidemia type I (GA-I), an inborn error of metabolism, is characterized by the accumulation in a patient’s urine, of glutaric,

glutaconic, and 3-hydroxyglutaric acids. These diagnostic metabolites can be detected by gas chromatography–mass spectrometry. The basis for screening for asymptomatic GA-I is the detection of glutarylcarnitine in dried blood-spots using tandem mass spectrometry. We recently received samples from three patients with symptomatic GA-I, diagnosed by the characteristic urine organic acid profiles. The glutarylcarnitine levels in their plasma samples were either normal or only marginally elevated while the glutarylcarnitine levels in their urine samples were markedly elevated. Therefore, in the screening of asymptomatic individuals, the presence of normal levels of plasma glutarylcarnitine may be falsely reassuring. We suggest that urinary acylcarnitine levels be a part of the diagnostic protocol, and that laboratories providing newborn screening based upon blood spot acylcarnitine profiles provide information regarding the lack of sensitivity of the assay for GA-I. 56. 3-Methylchrotonyl-CoA carboxylase deficiency detected by tandem mass spectrometry in a Mexican neonate. E. Rangel Co´rdova,a L. Martı´nez de Villarreal,a R. Torres Sepu´lveda,a A. Sa´nchez Pen˜a,a I. Ibarra Cicero´n,b M. Vela Amieva,b and J. Villarreal Pe´rezc. a Departamento de Gene´tica Facultad de Medicina, U.A.N.L.; b Departamento de Gene´tica de la Nutricio´n, Instituto Nacional de Pediatrı´a; cServicio de Endocrinologı´a y Metabolismo, Hospital Universitario, U.A.N.L. Introduction: Isolated biotin-resistant 3-methylchrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism that seems to be the most frequent organic aciduria detected in tandem mass spectrometry-based neonatal screening programs. The phenotype is variable, ranging from neonatal onset with severe neurological involvement to asymptomatic adults. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing subunits and smaller b-subunits. Here, we report a clinical case detected after the addition of tandem mass spectrometry (MS/MS) to the Newborn Screening Program (NSP) in Nuevo Leo´n, a state located at Northeastern Mexico. Material and methods: In January 2002, Nuevo Leo´n started the first NSP in Mexico using the MS/MS. By August 2003 around 30,000 neonates born at public hospitals had been screened. We present the follow up of a female full term baby with MCC deficiency, detected during this period. The mother has the antecedent of two miscarriages. Birth weight of 3730 g. and height of 52 cm. Neonatal physical examination was normal and was discharged from the hospital at 24 h after the blood spot sample was taken. An elevation of C5OH was reported in the first as well as in the second sample taken at 3 weeks. The baby was referring to the Genetics’ department. The urine organic acids reported high levels of methylchrotonyl-glycine. The mother was negative. The treatment was immediately started, mainly of avoiding fasting, a diet rich in carbohydrates and was giving carnitine 100 mg/kg/day. The baby is growing well and asymptomatic. We need to test and confirm the enzyme deficiency by lymphoblast or fibroblast culture assay. Conclusions: The estimated incidence of 3-MCC deficiency in our population is 1 in 30,000. The use of MS/MS will detect additional inborn errors of metabolism not traditionally included in newborn screening. Long-term follow-up is needed to determine the outcome of presymptomatically diagnosed patients with 3-MCC deficiency. 57. Detection of inherited metabolic disorders in cord blood samples by tandem mass spectrometry. Andrea B. Schenone, Jose´ E. Abdenur, Adolfo Guinle, Andrea Fuertes, and Nestor A. Chamoles. Fundacio´n para el Estudio de las Enfermedades Neurometabo´licas (FESEN), Uriarte 2383, Buenos Aires (1425), Argentina. Objective: To evaluate the possibilities of detecting IMD’s by TMS in samples obtained prior to 48 h. Material and Methods: We studied 10 children with a positive family history of an IMD’s (PKU (1), MSUD (3), IVA (2), PPPA (2), GA1 (1), and CPTII (1)). Families of these patients did not request prenatal diagnosis.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 After inform concern, samples were obtained from cord blood, 12, 24, and 48 h in filter paper (S&S 903). All children were closely monitored in the neonatal intermediate care unit and were asymptomatic, on breast feedings at the time of sampling. For those patients with positive results, specific treatment for their disease was started as soon as the diagnosis was available (within 24 h). Additionally, 21 samples from cord blood of normal (not at risk) children were analyzed. Methods analysis was performed as described by Millington et al. using a Quattro II, triple quadrupole mass spectrometer (Micromass, UK), with electrospray injection and MassLynx software. Results: Abnormal results were obtained in 5 out of the 10 patients, within the first 24 h. In four of them the analytes were elevated in cord blood. Results were as follows (Normal values: maximum value n = 28). PKU: IVA: MCAD: CPT II:

Phe: IVC: C8: C16:

117.34 lM 10.56 lM 0.32 lM 20.02 lM

(NV < 95.88) (NV < 0.45) (NV < 0.14) (NV < 8.13)

Phe/Tyr: IVC/C2: C8/C10: C16/C2:

2.12 0.71 3.65 6.79

(NV < 1.90) (NV < 0.26) (NV < 1.53) (NV < 1.83).

The remainder patient, affected with MSUD, showed normal values in cord blood. Leu: 208.20 lM (VN < 271.40) and Leu: 3.82 (NV < 5.75). Levels were abnormal at 12 h Leu: 444.22 lM (NV < 288.54) Leu/Fen: 7.37 (NV < 4.8).All other patients had normal results throughout the study and were proved to be non-affected thereafter. Conclusions: Our results suggest that analysis of AC and AA by TMS might be useful to detect organic acidemias and fatty acid oxidation defects from cord blood. For the amino acid disorders, possibilities of detection during the first 24 h may still be difficult, depending on severity of the disease and protein intake. Further studies, with a larger number of patients are necessary to determine the best strategy of sampling in newborn screening programs with TMS. 58. Homocitrullinuria revisited: evaluating the significance of homocitrulline excretion in disease and normal metabolism. J. Daniel Sharer, Tisa H. Harper, Natalie Plotkina, Rani H. Singh, and Chunli Yu. Division of Medical Genetics, Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA. Introduction: Elevated excretion of homocitrulline (HC; e-aminocarbamoyl-lysine) is a novel feature of the hyperammonemia, hyperornithinemia, and hyperhomocitrullinuria (HHH) syndrome, a rare urea cycle disorder associated with deficient transport of ornithine across the inner mitochondrial membrane. However, the contribution of this metabolite with respect to HHH pathophysiology is unclear. HC associated with this disease appears to derive from alternative carbamylation of lysine (in the absence of ornithine), suggesting the possibility that more widespread, potentially harmful protein carbamylation occurs in the mitochondrial matrix under these conditions. Objective: To investigate the significance of elevated HC in HHH pathogenesis by (a) developing an effective method for measurement of urinary homocitrulline and (b) defining normal homocitrulline excretion for comparison with affected individuals in order to define pathologic levels. Methods: A modification of standard amino acid ion-exchange/ninhydrin chromatography was developed in order to better resolve urinary homocitrulline from methionine. Isocratic column development at a reduced temperature in the initial separation phase resulted in clean baseline resolution of the two peaks, allowing rapid quantitation of HC. This technique was used to examine HC excretion in urine samples from a broad control group (ranging from 2 months to 30 years of age) as well as a male patient diagnosed with HHH syndrome. Results: Low-level HC excretion was consistently detectable in the control samples (mean: 34 nmoles/mg creatinine; range: 0–117 nmoles/mg). The affected patient in this study presented at 4 years of age with mild cognitive impairment, mild peripheral neuropathy, and an aversion to protein-rich foods. The urinary [HC] at presentation was 259 nmoles/mg; [orotate] was 310 nmoles/mg and plasma [ornithine] was 512 uM. Following dietary modifications, the


patient’s HC levels dropped to 90 nmoles/mg (orotate = 4 nmoles/mg; ornithine = 387 uM). Conclusions: HC appears to be a normal constituent of human urine, and may be present in relatively large amounts without any obvious pathology, though control levels are generally significantly lower than in HHH syndrome. Contribution of dietary HC (particularly from milk products) is unknown, but may be significant. While an association between elevated HC excretion and HHH syndrome is evident, defining abnormal HC levels will necessitate additional evaluation of affected individuals. Continuing in vitro studies will seek to investigate the effects of chronic HC exposure and possible protein carbamylation on organelle/cell function. 59. Fusing N-acetylglutamate synthase to green fluorescent protein to increase solubility and prevent aggregation. Dashuang Shi,a Xiaolin Yu,a Ljubica Caldovic,a Hiroki Morizono,a Norma Allewell,b and Mendel Tuchmana. aChildren’s National Medical Center, Washington, DC, USA; bUniversity of Maryland, College Park, MD, USA. N-Acetylglutamate synthase (NAGS) catalyzes the formation of Nacetylglutamate (NAG) from acetyl-CoA and glutamate. Mammalian NAGS catalyzes the production of NAG, an essential allosteric activator of carbamoyl phosphate synthetase I CPSI), the first and rate limiting enzyme of the urea cycle. Bacterial NAGS provides the first intermediate for arginine biosynthesis. NAGS deficiency in human causes hyperammonemia due to the secondary deficiency of CPSI deprived of its cofactor NAG. There is no structural information on NAGS from any species, related to low solubility of this protein. We have purified Escherichia coli, human and mouse NAGS to homogeneity using Hitrap Chelating HP and Hiload Superdex 200 gel filtration columns. However, upon concentrating these proteins they aggregated preventing further crystallization effort. In order to overcome this problem, we fused the NAGS genes to the green fluorescent protein (GFP) gene. The fusion constructs were transformed into E. coli cells and were overexpressed. In order to facilitate the purification of NAGS–GFP fusion, poly-histidine tag was inserted within the linker between NAGS and GFP. The NAGS–His6– GFP fusion was purified to homogeneity using Cobalt-affinity and anion-exchange columns. The activity assays indicated that NAGS activity is not affected in NAGS–GFP fusion proteins and response to L arginine similar to NAGS alone. The solubility of the fusion proteins is significantly increased. The NAGS–GFP fusion proteins have been concentrated to approximately 10 mg/ml and are now suitable for crystallization, in contrast to that the concentration of soluble NAGS alone could not reach 1 mg/ml even after an extensive screen for a suitable buffer mixture. The fusion proteins are also more stable than NAGS alone since the decline in activity of NAGS upon storage was slower than that for NAGS alone. In conclusion, the solubility and aggregation problems of NAGS can be overcome by fusing NAGS to GFP. 60. Hereditary inclusion body myopathy due to mutations in GNE: epimerase activity and treatment strategies. Susan E. Sparks,a Molly Lalor,a Eduard Orvisky,b Marjan Huizing,a Donna Krasnewich,a M.-S. Sun,a Marinos Dalakas,c and William A. Gahla. a MGB/NHGRI, bNSB/NIMH, and cNINDS, NIH, DHHS, Bethesda, MD, USA. Hereditary inclusion body myopathy (HIBM) is an autosomal recessive disorder characterized by adult-onset, progressive distal, and proximal muscle weakness sparing the quadriceps muscles. It is caused by mutations in either or both the epimerase and kinase domains of GNE, which encodes uridine diphosphate-N-acetylglucosamine 2epimerase/N-acetylmannosamine kinase (UDP-GlcNAc 2-epimerase/ ManNAc kinase). This bifunctional and rate-limiting enzyme is involved in the biosynthesis of sialic acid (N-acetylneuraminic acid). The epimerase converts UDP-GlcNAc to N-acetylmannosamine (ManNAc) and the kinase converts ManNAc to ManNAc-6-phosphate. Sialic acid,


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the terminal sugar on cell surface oligosaccharides, is important in cell–cell interactions and signal transduction. We employed cultured fibroblasts as a model system to investigate epimerase activity in cells from three HIBM patients. Patient #1, with two epimerase domain mutations (G455T and C787T), had 38% of control epimerase activity as measured by conversion of [3H]UDP-GlcNAc to [3H]ManNAc. Patient #2, with one epimerase mutation (T698C) and one kinase mutation (C1943T), had epimerase activity 48% of control. Patient #3, with homozygous kinase mutations (T2186C), had epimerase activity 83% of control. This demonstrates decreased epimerase activity in patients who have mutations only in the kinase domain, not the epimerase domain. Abnormal glycosylation of a-dystroglycan, with resultant abnormal ligand interactions, causes several muscular dystrophies, including Fukuyama’s congenital muscular dystrophy, Muscle–Eye–Brain disease, and Walker–Warburg syndrome variant. This led us to investigate the a-dystroglycan glycosylation status in HIBM patients. Immunostaining of skeletal muscle tissue of three HIBM patients showed a dramatically decreased immunoreaction to both VIA4-1 and IIH6 a-dystroglycan antibodies, thought to detect the O-mannosylated glycoconjugates on a-dystroglycan. Antibodies to the core a-dystroglycan, to b-dystroglycan and to laminin a2 showed a normal pattern compared to control muscle. Western blot analysis performed on muscle tissue from the same patients showed decreased signals for VIA4-1 and IIH6 compared to control, while b-dystroglycan signals were normal, as expected. Defective glycoconjugate sialylation in a patient with HIBM was also demonstrated by using the lectin WGA-FITC, which recognizes sialic acid end-groups on glycoproteins. In normal fibroblasts, immunofluorescence showed a punctated pattern throughout the cells, but in HIBM fibroblasts, there was only residual staining around the Golgi. In patient cells grown in media containing free sialic acid or ManNAc were corrected to a normal pattern. Supplementation with ManNAc or free sialic acid may provide therapy for patients with HIBM. 61. Transient sulfite oxidase deficiency and S-sulfocysteinuria due to cholestatic liver disease. Susan E. Sparks, Brian P. Brooks, Janet S. Isaacs, Jaimie Higgs, Miriam D. Bloom, Kenneth N. Rosenbaum, and Gerard T. Berry. Department of Pediatrics, The George Washington University School of Medicine and Children’s National Medical Center, Washington, DC, USA. Objective: To present evidence for the first time that S-sulfocysteinuria secondary to acquired hepatic sulfite oxidase deficiency may occur in a patient with cholestatic liver disease. Methods: A male infant with truncal hypotonia and intermittent hypertonia of extremities, sparse hair, craniofacial dysmorphisms, hypertrophic cardiomyopathy, poor feeding, neonatal jaundice, and abnormal liver associated test results underwent evaluation for an underlying biochemical genetic disease. Eventually, it became apparent that a likely diagnosis was the cardiofacio-cutaneous syndrome. However, as part of the metabolic workup the urine S-sulfocysteine excretion was 750 lmol/g creatinine (normal <24; affected individuals with isolated sulfite oxidase deficiency or molybdenum cofactor deficiency is 200–2000) and plasma urate was 0.7 mg/dl (normal 1.8–5.1). The patient subsequently had a liver biopsy for analysis of sulfite oxidase enzyme activity, sequential analyses of urinary S-sulfocysteine, urate, hypoxanthine/xanthine, and plasma urate levels. Results: The bile stained hepatic tissue enzyme analyses revealed 25% residual sulfite oxidase activity, 48% xanthine oxidase activity, and 31% residual glucose-6-phosphatase activity (control enzyme). Upon placement on a low protein, methionine and cysteine restricted diet for one month, the S-sulfocysteine level decreased to 59 lmol/g creatinine. When placed on a normal diet for two months, the urine S-sulfocysteine levels were 32 lmol/g creatinine. The urine xanthine/hypoxanthine levels were always normal. The hypouricemia persisted and was associated with increased urinary urate excretion indicating that the hypouricemia was secondary to renal urate wastage. Conclusion: S-Sulfocysteinuria may occur in

patients with cholestatic liver disease because of a secondary deficiency in hepatic sulfite oxidase activity. Its relevance to pathophysiology remains to be determined. 62. Renal glucosuria due to SGLT2 mutations. Caroline Stuarta, Robert Kletaa,b, and William A. Gahla,b. aSection on Human Biochemical Genetics, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA; bOffice of Rare Diseases, Intramural Program, Office of the Director, National Institutes of Health, Bethesda, MD, USA. Renal glucose reabsorption is the conditio-sine-qua-non for the body’s energy homeostasis and one of the main principles in renal physiology. Glucose is filtered by the glomerulus and more than 99% reabsorbed within the proximal tubule through a sophisticated coupling of luminal secondary active glucose transporters, such as SGLT1 and SGLT2, with basolateral passive glucose transporters, such as GLUT2. SGLT1 and SGLT2 are thought to accomplish renal tubular glucose reabsorption in a sequential manner. In the early proximal tubule, the low affinity, high capacity transporter SGLT2, with a sodium to glucose coupling of 1:1, reabsorbs the bulk of the filtered glucose. In the late proximal tubule, the high affinity, low capacity transporter SGLT1, with a sodium to glucose coupling of 1:2, completes the reabsorption of glucose, removing virtually all of this sugar from the urine. Human ‘‘knock-outs’’ of the SGLT1, SGLT2, and GLUT2 genes are represented by the disorders glucose–galactose malabsorption, renal glucosuria, and Fanconi–Bickel syndrome, respectively. Currently, all known cases of isolated renal glucosuria are due to mutations in SGLT2. Mutations in GLUT1, which is expressed in the cells of the blood–brain barrier, give rise to the clinical picture of infantile seizures due to hypoglycorrhachia. This disorder is inherited in an autosomal dominant fashion, in contrast to the autosomal recessively inherited glucose–galactose malabsorption, renal glucosuria, and Fanconi–Bickel syndromes. Isolated renal glucosuria results from mutations in SGLT2, which codes for an active transporter specific for D -glucose and expressed in the luminal membrane of the renal proximal tubule. In affected individuals, glucosuria leads to pursuit of hyperglycemia to exclude defects in glucose metabolism, and to investigation of renal proximal tubular function to exclude renal Fanconi syndrome. We present clinical and molecular data regarding a 19-year-old woman with isolated glucosuria; daily urinary glucose wasting was 9.1 g, measured on a 24-h collection. She was compound heterozygous for two new SGLT2 mutations, one of which was previously considered a polymorphism. The consequences of isolated renal glucosuria due to SGLT2 mutations are not profound. Glucose wasting in our patient cost her only 36 kcal/day, and even severely affected patients lose only a few hundred kilocalories in wasted glucose. Rather, the danger lies in mistaking isolated renal glucosuria for a more serious condition, such as diabetes mellitus, and pursuing it with invasive interventions. The key to avoiding this complication is recognition of the disorder itself. 63. Generation of a conditional knock-in mouse model with deficiency of UDP-N-acetylglucosamine 2 epimerase/N-acetylmannosamine kinase to mimic hereditary inclusion body myopathy. Mao-Sen Sun,a Marjan Huizing,a Susan Sparks,a Donna Krasnewich,a Pamela L. Schwartzberg,b Chandrasekharappa Settara,c and William A. Gahla. aSection of Human Biochemical Genetics, Medical Genetics Branch, NHGRI, NIH, DHHS, Bethesda, MD, USA; bGenetic Disease Research Branch, NHGRI, NIH, DHHS, Bethesda, MD, USA; cGenome Technology Branch, NHGRI, NIH, DHHS, Bethesda, MD, USA. Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive, autosomal-recessive neuromuscular disorder. HIBM is caused by a deficiency in UDP-N-acetylglucosamine 2

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 epimerase/N-acetylmannosamine kinase (GNE), the key enzyme of sialic acid biosynthesis. Mutations in GNE gene have been found in patients with HIBM. We generated two GNE conditional knock-in mice to mimic human HIBM. Most HIBM patients carry GNE missense mutations in the kinase, the epimerase, or both domains. Additionally, a previous study showed that a GNE knock-out mouse is lethal. These findings let us to generate two mouse models. One mouse has a missense mutation in exon 3 (D176V) (epimerase domain) and another mouse with a missense mutation in exon 12 (M712T) (kinase domain). Crossbreeding of these mice will provide us with a compound heterozygous model. We first created a point mutant (D176V) in exon 3 of mouse GNE. The mouse genomic clone containing the 50 part of GNE was isolated from a BAC library (129S6/SvEvTac) by overlapping oligo hybridization. A 5-kb BamHI fragment containing exon 3 was used to create the missense mutation, and inserted into the pPNT/ DL target vector containing a Neor, HSV-tk, and PGK poly(A) additional site in the appropriate locations. Three Lox P sites were inserted upstream of exon 3 and just before and after the Neor gene. We are currently in the process of Southern blotting to screen 129S6/SvEvTac mouse ES cells. When generated, these models will help us investigate the pathogenesis of this disorder, the mechanisms of GNE function, glycoprotein metabolism, and future therapeutic approaches. 64. Therapeutic use of nitisinone in alkaptonuria. P. Suwannarat,a I. Bernardini,a E. Tsilou,b B.I. Rubin,b M.B. Perry,c L. Gerber,c K. O’Brien,a and W.A. Gahla. aSection on Human Biochemical Genetics, MGB, NHGRI, NIH, DHHS, Bethesda, MD, USA; b Ophthalmic Genetics and Visual Function Branch, NEI, NIH, DHHS, Bethesda, MD, USA; cRehabilitation Medicine Department, Warren G. Magnuson Clinical Center, NIH, DHHS, Bethesda, MD, USA. Alkaptonuria is an autosomal recessive disorder due to deficiency of the enzyme homogentisic acid dioxygenase in the tyrosine degradation pathway. It is characterized by daily excretion of gram quantities of urinary homogentisic acid (HGA), arthritis, and ochronosis. No effective therapy exists. However, 2-(2-nitro-4-trifluoromethylbenzoyl)1,3-cyclohexadine (nitisinone) inhibits 4-hydroxyphenylpyruvate dioxygenase, the enzyme that produces HGA. We have enrolled nine patients with alkaptonuria, five females and four males, for a 3–4 month nitisinone trial. We titrated the dose of nitisinone to lower HGA excretion to below 0.5 gm/day. The doses were 0.35 mg bid for 1 week and 1.05 mg bid for 3 months. The initial average HGA level, 4.2 gm/day fell to 80–300 mg/day. Plasma tyrosine concentrations, initially 60 lM, rose to 400–900 lM. Weekly eye examinations revealed no signs of corneal toxicity. After 5 weeks of treatment, patient #1 passed several kidney stones but remained on study. After 11 weeks, patient #3, who previously had asymptomatic aortic stenosis, developed symptoms of lightheadedness and dyspnea on exertion. She was removed from the study and received valve replacement. Patient #4 developed elevated ALT/AST levels on day 3 of treatment; nitisinone was discontinued on day 5 as required by protocol. Levels peaked on day 9, with ALT 117 U/L and AST 94 U/ L, and returned to normal 3 weeks after stopping nitisinone. Patient #7 also developed elevated ALT/AST levels, detected after 5 weeks of nitisinone, with peak ALT 144 U/L and AST 56 U/L, normalizing 10 days after stopping treatment. Both patients developing liver toxicity were males with prior histories of heavy alcohol intake, and were on additional medications including NSAIDs and narcotics for pain. Patient #4 was also receiving a statin. Currently, two patients have completed 12 and 13 weeks of nitisinone therapy, respectively. During the last week, each received only 40 g of protein per day, and plasma tyrosine decreased to 300 lM. The remaining patients will complete treatment in the near future providing additional data. A long-term clinical trial is planned to determine the benefit of nitisinone in preventing joint deterioration and providing pain relief.


65. Isolated sulfite oxidase deficiency—report of a case in a newborn and review of the literature. Wen-Hann Tan,a Florian S. Eichler,b Sadaf Hoda,c Melissa S. Lee,d P. Ellen Grant,e Kalpathy S. Krishnamoorthy,b and Vivian E. Shihc. aHarvard Medical School Genetics Training Program, Boston, MA, USA; bPediatric Neurology Unit, Massachusetts General Hospital, Boston, MA, USA; cDepartment of Neurology, Massachusetts General Hospital, Boston, MA, USA; dHarvard Combined Medicine/Pediatrics Program, Harvard Medical School, Boston, MA, USA; eDivision of Neuroradiology, Massachusetts General Hospital, Boston, MA, USA. Sulfite oxidase is an enzyme that catalyzes the oxidation of toxic sulfites to non-toxic sulfates in the final step of sulfur amino acids catabolism. We report a full-term newborn baby boy who presented with respiratory distress and poor feeding soon after birth. A highpitched cry and episodes of opisthotonic posturing were noted. On his third day of life, he developed generalized tonic–lonic seizures that evolved into refractory sub-clinical seizures after treatment with phenobarbital and fosphenytoin. Laboratory investigations were significant for the presence of urinary sulfites, elevation of urinary thiosulfate and sulfocysteine, low plasma homocysteine levels, absence of plasma cystine and normal serum uric acid, all of which were consistent with isolated sulfite oxidase deficiency. Of note, this is the only disorder in which plasma homocysteine levels are depressed. An EEG showed diffuse bilateral epileptiform discharges on a background of burst suppression pattern. Subsequent magnetic resonance imaging of his brain with spectroscopy (MRI/S) showed changes that resembled those seen in perinatal hypoxic–ischemic encephalopathy. He was started on a low methionine and low cysteine diet using a mixture of methionine and cysteine-free formula with regular infant formula. As sulfites have previously been shown to destroy thiamine in animal studies, he is receiving thiamine supplementation. In addition, he is receiving dextromethorphan to help antagonize sulfocysteine toxicity at the NMDA receptor. At four months of age, he is hypertonic with opisthotonus, and has severe developmental delay. However, he is still able to feed orally. He is maintaining his linear growth, but his head circumference is falling off the growth chart. He has not had any convulsions since two weeks of life. We reviewed the clinical and biochemical features in all 19 cases of isolated sulfite oxidase deficiency published in English. Among the six cases in which mutations in the SUOX gene were found, mutations leading to a prematurely truncated protein appeared to result in more severe phenotypes with early deaths than those mutations resulting in amino acid substitutions. 66. Elevated chitotriosidase activity in CSF in patients with GM1 and GM2 gangliosidosis: a surrogate marker of disease progression? Cynthia J. Tifft,a,b Joanne Kurtzberg,c and Richard L. Proiab. a Division of Genetics and Metabolism, Children’s National Medical Center, Washington, DC, USA; bGenetics of Development and Diseases Branch, NIDDK, NIH, Bethesda, MD, USA; c Department of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA. Background and objectives: Chitotriosidase, a human chitinase produced by activated macrophages, is markedly elevated in the plasma of patients with type 1 Gaucher disease, moderately elevated in some other lysosomal storage disorders, but not elevated in GM2 gangliosidosis. Activation of macrophage-derived microglial cells in the central nervous system (CNS) has been demonstrated in mouse models of GM2 and GM1 gangliosidosis and verified on autopsy samples of a human GM2 patient. The chitotriosidase activity was 200-fold elevated in the thalamic nuclei and brainstem of this patient. To determine whether chitotriosidase might be a useful marker of disease progression in GM1 and GM2 patients we analyzed enzyme activity in cerebrospinal fluid (CSF) from three patients with GM2 and one patient with GM1 gangliosidosis and compared them with CSF from 23 control subjects. Methods: Chitotriosidase activity in CSF samples was measured using 0.02 mM of 4methylumbelliferyl-chitotrioside as the substrate in citrate buffer, pH


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5.2. The results were expressed as nanomolar 4MU cleaved per milligram protein per minute. Results: In two symptomatic patients with GM2 gangliosidosis, ages 15 and 18 months, the chitotriosidase activity was elevated 48- and 38-fold greater than control subjects, respectively. In a third symptomatic patient with GM1 gangliosidosis the chitotriosidase level was 55-fold greater than controls. In contrast, the chitotriosidase level in a presymptomatic patient with GM2 gangliosidosis at 2 weeks of age was less than 2-fold elevated over control subjects. Conclusions: Substantial elevations in chitotriosidase activity were seen in the CSF of three symptomatic patients with GM1 or GM2 gangliosidosis as compared with controls, whereas the presymptomatic GM2 patient did not show an elevation. In the GM2 mouse model microglial activation has been shown to precede the onset of neurodegeneration. Elevations in chitotriosidase in the CSF of gangliosidosis patients may be an indication of microglial activation and a useful marker of disease progression. 67. Elevated chitotriosidase activity in CSF in patients with tay–Sachs disease: a surrogate marker of disease progression? Cynthia J. Tifft,a,b Joanne Kurtzberg,c and Richard L. Proiab. aDivision of Genetics and Metabolism, Children’s National Medical Center, Washington, DC, USA; bGenetics of Development and Diseases Branch, NIDDK, NIH, Bethesda, MD, USA; cDepartment of Pediatrics, Duke University Medical Center, Durham, NC 27710, USA. Background and objectives: Chitotriosidase, a human chitinase produced by activated macrophages, is markedly elevated in the plasma of patients with type 1 Gaucher disease, moderately elevated in some other lysosomal storage disorders, but not elevated in GM2 gangliosidosis. Activation of macrophage-derived microglial cells in the central nervous system (CNS) has been shown in mouse models of GM2 gangliosidosis and verified on autopsy samples of a human GM2 patient. The chitotriosidase activity was 200-fold elevated in the thalamic nuclei and brainstem of this patient. To determine whether chitotriosidase might be a useful marker of disease progression in GM2 patients we analyzed enzyme activity in cerebrospinal fluid (CSF) from three patients with Tay–Sachs disease and compared them with CSF from 23 control subjects. Methods: Chitotriosidase activity in CSF samples was measured using 0.02 mM of 4-methylumbelliferylchitotrioside as the substrate in citrate buffer, pH 5.2. The results were expressed as nanomolar 4MU cleaved per milligram protein per minute. Results: In two patients diagnosed symptomatically at 15 and 18 months the chitotriosidase activity was elevated 48- and 38-fold greater than control subjects, respectively. The third patient was diagnosed with Tay–Sachs disease prenatally and was asymptomatic at 2 weeks of age when the chitotriosidase activity was less than 2-fold elevated over control subjects. Substantial elevations in chitotriosidase activity were seen in the CSF of two symptomatic patients with Tay– Sachs disease as compared with controls, whereas the presymptomatic patient did not show an elevation. In the GM2 mouse model microglial activation has been shown to precede the onset of neurodegeneration. Elevations in chitotriosidase activity in the CSF of Tay–Sachs disease patients may be an indication of microglial activation and a useful marker of disease progression. 68. Inborn errors of metabolism, newborn screening by tandem mass spectrometry in a mexican population. M.R. Torres,a L.E. Villarreal,a C. Ruiz,a and P.J.Z. Villarrealb. aDepto de Gene´tica, fac. de Medicina and bServicios de Salud de Nuevo Leo´n, Monterrey Nuevo Leo´n, Mexico. Introduction: In March 2002 an expanded newborn screening (NS) program using MS/MS for the early detection of IEM was started in Nuevo Leo´n, Me´xico, by the Secretary of Health, to prevent infant mortality and disability. We report our results of the first 20 months of operation, detailing our experience, outcome data, and the problem faced. Methods: Dried blood spot specimens (DBS) have been collected

on S&S filter paper from newborns at 24 h of age, for analysis on Wallac MS2 triple quadrupole mass spectrometer (Perkin–Elmer). The acylcarnitines (AC) and aa (AA) were determinated in whole blood (lmol/L) against a mixture containing 12 deuterated stable isotopes for AC and AA, respectively (Neogram Kit). Results: We have screened 39,843 samples in a lapse of 20 months. Cut-off values were obtain from 10,000 samples. One thousand forty-five (2.6%) were positives and a second sample requested. Only 82.9% were resampled and six cases were positives. AA abnormal values accounted for more of the positives results in the first sample (tyrosine, aspartic acid, and citrulline). Free carnitine and 3-OH isovalerylcarnitine were the most common abnormal values. Newborns were clinically evaluated by a Pediatrician and confirmatory tests were performed. Confirmed cases were 1 hyperphenylalaninaemia, 1 citrullinemia, 1 homocystinuria, 1 tyrosinemia, and 1,3-methylcotonylCoA carboxilase deficiency and one case with high 3-OH isovalerylcarnitine levels. Conclusion: Our incidence is 1 in 5000. Implementation of MS/MS in screening programs allows to avoid early deaths, life threatening events, and permanent neurological disorders in infants with IEM. This is the first program in Me´xico and it is free for the low income population, which is unique in Latin Ame´rica. 69. A comparison of mass spectrometry methods for the measurement of creatine and guanidinoacetate in plasma. Sarah P. Young,a T.C. Wood,b L.S. Almeida,c G.S. Salomons,c C. Jakobs,c R.D. Stevens,a D.D. Koeberl,a and D.S. Millingtona. aDivision of Medical Genetics, Department of Pediatrics, Duke University Medical Center, Durham, NC, USA; bGreenwood Genetics Center, Greenwood, SC, USA; cVU University Medical Center, Amsterdam, Netherlands. Arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) deficiencies and creatine transporter defects result in syndromes characterized by cerebral creatine deficiency, mental retardation, and seizures. Diagnosis may be achieved by the demonstration of creatine deficiency in the brain using proton magnetic resonance spectroscopy, determination of guanidinoacetate, creatine and creatinine in urine and plasma, and by mutation analysis. We have developed and validated a stable isotope dilution LC-MS/MS method for the simultaneous measurement of underivatized creatine (CR), guanidinoacetate (GA), and creatinine in urine and plasma. Urine is directly analyzed, whereas plasma is deproteinized prior to analysis. Interfering compounds are separated from the analytes of interest using a TSK-Gel amide 80 column and the total cycle time is 6 min. This method was compared with previously published methods performed at the Greenwood Genetics Center, SC and VU University Medical Center, Amsterdam including an LC-MS/MS method that analyzes butylated derivatives of creatine and guanidinoacetate and a GC-MS method. Twenty deidentified plasma samples were analyzed using the three methods and results compared. Good agreement was observed between the underivatized method and both the butylated and GC-MS methods. Comparison of the underivatized and butylated methods: y = 0.79x + 0.35, r2 = 0.90, and Sy.x = 0.18 for GA and y = 0.9x + 3.5, r2 = 0.93, and Sy.x = 6.3 for CR. Comparison of underivatized and GC-MS methods: y = 1.1x + 0.03, r2 = 0.97, and Sy.x = 0.15 for GA and y = 0.86x + 1.64, r2 = 0.97, and Sy.x = 3.6 for CR. 70. 10 years’ diagnostic experience in galactosemia caused by galactose1-phosphate uridyltransferase deficiency using combined biochemical and molecular approaches. Chunli Yu,a Philip P. Dembure,a Kasinathan Muralidharan,a Genet Kiros,a Nick L. Hjelm,a Sharon Langley,a Rani H. Singh,a and Louis J. Elsasb. aDivision of Medical Genetics, Department of Human Genetics, Emory University School of Medicine, Atlanta, GA, USA; bThe Dr. John T. MacDonald Foundation Center for Medical Genetics, University of Miami, Miami, FL, USA.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 Introduction: Galactose-1-phosphate uridyltransferase (GALT) deficiency results in classical or variant forms of galactosemia. We propose that combining GALT biochemical phenotype along with GALT genotype can result in improved diagnosis and provide complete information to ensure high quality of care to the patients with galactosemia. Our GALT diagnostic panel includes complete biochemical phenotype (GALT activity, GALT isozyme, and red cell galactose-1-phosphate) as well as GALT molecular genotype. In this study, data were retrospectively collected for the past 10 years to determine whether this combined approaches improved the diagnosis of galactosemia. Methods: Red cell GALT activity was measured by conversion of [14C]galactose-1-phosphate and UDP-glucose to glucose-1-phosphate and [14C]UDP-galactose at 37 C and then analyzed by ion exchange paper chromatography. GALT isozymes were performed on isoelectric focusing gels to detect Duarte galactosemia (D/G) or homozygote Duarte (D/D). Red cell Gal-1-P is determined spectrophotometrically as galactose after hydrolysis by alkaline phosphatase. DNA analysis: multiplex PCR and Elisa analysis were used for codon changes for Q188R, N314D, S135L, K285N, L195L, and Y209C. Uncommon mutations were confirmed by sequencing of GALT gene coding regions. Results: From January 1994 to October 2003, 3278 samples were screened for galactosemia. We identified 356


(11%) G/G galactosemia, 514 (16%) D/G galactosemia, 84 (3%) Duarte homozygote (D/D), 835 (25%) galactosemia carriers (G/N), and 380 (12%) Duarte carriers (D/N). A total of 1413 G alleles were detected by genotyping for the common mutations (Q188R, S135L, K285N, L195P, and Y209C). These accounted for 68.5% of all G alleles. Simultaneous abnormal biochemical parameters (GALT activity and isozyme and red cell Gal-1-P) indicated the presence of G alleles. These G alleles were confirmed by direct GALT sequencing. Additionally, 113 less common mutations were detected. Among these less common mutations, two unique genotypes included: (1) the less common G alleles in cis with N314D created specific problems because they produced conflicts between the biochemical phenotype and GALT genotype. Eighteen of such double mutants in cis were: N314D ins T@ bp 41, N314D-A320T, N314D-H114L, N314D-P325L, N314DR204X, N314D-P185L, N314D-R148Q, N314D-E203K, and N314D-H315H. (2) Similarly the single base pair deletions as well as large deletions in GALT genes could also be miss-leading without biochemical phenotype. We identified a total of 28 G alleles due to deletions of the GALT gene. Conclusion: Combined biochemical and molecular approaches increase the detection of G alleles in patients with galactosemia and helps differentiate between polymorphisms or disease causing mutations.


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

Abstracts for Publication only 71 72 73 74 75

M. Cabrera-Salazar M. Dasouki M. Dasouki C. Ficicioglu M. McGovern

76 77

C. Prasad WJ. Rhead

Microvascular disease in young children with Fabry Disease. Carnitine therapy in patients with recurrent vomiting and non-diabetic ketoacidosis. Hepatocelluar carcinoma in an adolescent with Niemann–Pick C disease. Bilateral cataracts in a 38-year-old woman with D/G galactosemia: a case report. The natural history of type B Niemann–Pick disease: results from a 10-year longitudinal study. Non-immune hydrops: a marker for inborn errors of metabolism? Very-long-chain acyl-CoA dehydrogenase (VLCAD), 2-methylbutyryl-AD (2-MBAD), short chain-AD (SCAD), and 3-methylcrotonyl-CoA carboxylase (3-MCC) deficiencies: newborn screening detects clinically benign or very mild cases.

SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186 71. Microvascular disease in young children with Fabry Disease. Mario A. Cabrera-Salazar,a Erin O’Rourke,a Gustavo Charria,b and John A. Barrangera. aDepartment of Human Genetics, University of Pittsburgh, USA; bDepartment of neurology, University of Miami, USA. Fabry disease is a lysosomal storage disorder caused by deficiency of the lysosomal enzyme alpha galactosidase A (GLA). Deficient or absent levels of this enzyme produces accumulation of globotriaosylceramide (GL3) in the lysosomes of a variety of cells, causing the symptoms of Fabry disease. Such accumulation results in damage to kidney, myocardium and nervous system. Besides autonomic nervous system involvement, adult patients with Fabry disease present neuroradiologic findings that may be important such as increased signal intensity in the pulvinar on Tl weighted images, which is a common and sensitive finding in adults with Fabry disease. We report 2 children who have no clinical signs of central nervous system involvement despite MRI evidence of leukodystrophy. Case 1: An 11 year old Caucasian male with suggestive symptoms of Fabry disease was tested for GLA activity following a diagnosis of Fabry disease in his maternal grandfather. Decreased enzymatic activity confirmed the suspicion of Fabry disease and sequence analysis revealed the presence of an R227Q mutation in the GLA gene. Baseline audiologic exam, ECG, and echocardiogram were unremarkable. The ophthalmologic exam showed whorled corneal opacities in both eyes. An MRI of the brain showed several lesions characterized by increased signal (T2) in both basal ganglia and the subcortical white matter of both hemispheres, similar to those seen in microvascular diseases in adults. Subtle deep white matter hyperintensities adjacent to the atria and posterior bodies of the lateral ventricle were also observed. Case 2: An 8 year old Caucasian male was tested for Fabry disease following the diagnosis of Fabry disease in his maternal grandfather. Decreased activity of GLA confirmed the suspicion of Fabry disease and sequence analysis revealed the presence of an I96 mutation in the GLA gene. An MRI of the brain showed minimally increased signal in multiple areas in the subcortical white matter of both hemispheres, similar to those seen in microvascular disease in adults. Significance: Enzyme replacement therapy (ERT) has shown promising effectiveness in adults with Fabry disease who have neurovascular and cardiac disease. It is important for pediatric patients to be identified and to have access to management with ERT prior to the onset of irreversible complications. These case reports suggest that brain MRI in children with Fabry disease is useful in uncovering pathology that was not expected. These observations support vigorous evaluation of pediatric cases of Fabry disease and would favor early intervention with ERT. 72. Carnitine therapy in patients with recurrent vomiting and non-diabetic ketoacidosis. Majed J. Dasouki, Susan E. Hunter, Shannon M. McGuire, and Uttam Garg. Section of Medical Genetics and Molecular Medicine and Department of Pathology, The Children’s Mercy Hospitals and Clinics and the University of Missouri, School of Medicine, Kansas City, MO, USA. Objective: To study the effect of carnitine supplementation in four patients with recurrent cyclic vomiting and recurrent keto-acidosis. Methods: Two phenotypically normal children and two (7-year-old boy and 5-year-old girl) children with amyoplasia congenita had cyclic vomiting and recurrent keto-acidosis. Routine chemistry studies, plasma amino acids, urine organic acids, and plasma acylcarnitine profiles were done. Skeletal muscle histology, histochemistry, electron transport chain studies, as well as skin fibroblast b-ketothiolase and succinyl CoA:3 ketoacid transferase activities were examined. Oral carnitine supplementation at 50 mg/kg/day was given to all four patients. Results: Ketosis and metabolic acidosis was detected in all four patients during acute presentation. All metabolic studies performed failed to identify a specific metabolic defect in any of the four patients included in this study. The frequency of recurrent episodes of keto-acidosis decreased following the administration of carnitine.


Conclusion: Carnitine may be useful in preventing recurrent metabolic keto-acidosis in some patients with cyclic vomiting. 73. Hepatocelluar carcinoma in an adolescent with Niemann–Pick C disease. Majed J. Dasouki, William San Pablo, James Daniel, and Eugenio Taboada. Section of Medical Genetics and Molecular Medicine and Section of Gastroenterology and Department of Pathology, The Children’s Mercy Hospitals and Clinics and the University of Missouri, School of Medicine, Kansas City, MO, USA. Objective: to report the detection of hepatocelluar carcinoma in a 15year old African-American boy with Niemann–Pick type C disease. Method: Skin fibroblast cholesterol staining (with filipin) and cholesterol esterification studies were done at 7 years of age. At 15 years of age, routine chemistry, abdominal and chest imaging studies, and liver biopsy were performed for evaluation for weight loss, fever, and anemia. Results: Abnormal filipin staining and cholesterol esterification studies confirmed the diagnosis of NPC in this patient. He presented with Escherchia coli bacteremia and a liver mass. Moderately differentiated multi-focal hepatocelluar carcinoma was found on liver biopsy. Imaging studies showed pulmonary metastases and involvement of the inferior vena cava. Conclusion: This is the third case report of NPC with hepatocelluar carcinoma. Such reports suggest a possible carcinogenic role for the abnormal storage of cholesterol in patients with NPC. 74. Bilateral cataracts in a 38-year-old woman with D/G galactosemia: a case report Can Ficicioglu, Paige Kaplan, Stanton Segal, and Gerard T. Berry. University of Pennsylvania School of Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. Objective: To present evidence that cataracts may occur in an adult with D/G galactosemia, a common variant form, that is associated with elevated red blood cell galactose-1-phosphate, galactitol, galactonate, and increased urinary excretion of galactitol in early infancy during lactose ingestion. There is ongoing debate about the value of patients with this biochemical variant state having a lactose-restricted diet. Patient: A 38-year-old female with D/G galactosemia, who ingested cow’s milk daily and ice cream frequently, developed bilateral cataracts over a 1-year period of time at 38 years of age. The zonular cataracts necessitated bilateral lens surgery and removal as they were associated with poor vision and had matured rapidly. Results: The erythrocyte galactose-1-phosphate uridyltransferase activity was 5.5 lmol/h/g hemoglobin (26% of normal activity–normal range; 15.9–26.4). Isoelectric focusing pattern was compatible with the patient being a duarte/galactosemia compound heterozygote. The molecular analysis showed N314D/Q188R genotype. The concentration of urine galactitol was 13 mmol/mol creatinine (normal range; 3.0-1 8 for an adult on an unrestricted diet). Conclusion: Early development of cataracts in an adult can be due to D/G galactosemia. As cataracts have not been widely reported in association with the D/G condition, we speculate that an epigenetic effect(s)/modifier gene(s) is playing an important role in the pathogenesis of cataracts in this rare patient. Whether certain patients with D/G galactosemia should be on a lactose restricted diet will require further investigation. 75. The natural history of type B Niemann–Pick disease: results from a 10-year longitudinal study. Melissa P. Wasserstein, Robert J. Desnick, Edward H. Schuchman, Sabera Hossain, Sylvan Wallenstein, Carin Lamm, and Margaret M. McGovern. Mount Sinai School of Medicine, New York, NY, USA. Objective: Type B Niemann–Pick disease (NPD-B) due to acid sphingomyelinase (ASM) deficiency is an autosomal recessive lysosomal storage disorder. Because of the rarity of the disorder and the broad range of disease severity, little is known about the natural history of the disease, and how genotype influences phenotype. In this


SIMD Abstracts / Molecular Genetics and Metabolism 81 (2004) 153–186

study 29 patients with NPD-B had serial evaluations at least one year apart over a four to ten year period to determine disease progression and variability. Methods: Organ volumes, hematological indices, lipid concentrations, pulmonary function, and hepatic activity were studied. Genotype was determined by direct sequencing and compared to phenotypic severity for each patient. Results: All patients had splenomegaly (mean value: 12.7 multiples of normal (MN); range: 4.5–27.3 MN), and all but one had hepatomegaly (mean volume: 1.91 MN; range: 0.93–3.21 MN). At the initial visit, 39% had thrombocytopenia and 3% had leukopenia. Mean annual decreases in platelet count and leukocyte count were 7 · 103/mm3 and 100 · 103/mm3, respectively . The typical atherogenic lipid profile, which is characterized by low HDL-cholesterol and hypertriglyceridemia, was worse in older patients. Sixty-nine percent of patients had low DLCO, and more than a third had low FEV1, FVC, and FEV1/FVC at initial visit. All measurements of pulmonary function showed a gradual deterioration over time. Liver dysfunction was characterized by stable elevation of hepatic transaminases and bilirubin. Homozygotes for deltaR608, P323A, P330R, and G245S had milder disease than patients with all other genotypes. Conclusion: The natural history of NPD-B is characterized by hepatosplenomegaly with progressive hypersplenism, worsening atherogenic lipid profile, gradual deterioration in pulmonary function, and stable liver dysfunction. 76. Non-immune hydrops: a marker for inborn errors of metabolism? Chitra Prasad,a Victoria Siu,a,c Kathy Corley,a and Tony Rupara,b,c. aDepartment of Pediatrics and Medical Genetics, University of Western Ontario, London, Canada; bDepartments of Biochemistry and Pathology, University of Western Ontario, London, Canada; cChild and Parent Resource Institute, London Canada. With the advent of ultrasonography, it has been possible to detect hydrops fetalis antenatally. In recent years there have been an increasing number of case reports of metabolic disorders, particularly lysosomal storage disorders, presenting as hydrops fetalis such as mucopolysaccharidoses I, IVA, VII (1, 1, and 10 case reports each), type 2 Gaucher disease (11), sialidosis (10), GM1 gangliosidosis (4), galactosialidosis (10), Niemann–Pick disease type C (7), Farber disease (1), infantile free sialic acid storage disease (1), mucolipidosis II (I-cell disease) (1), and GSD IV (1). Other disorders that have been reported to cause hydrops are congenital disorders of glycosylation and carnitine deficiency. In the past 5 years at the Children’s Hospital of Western Ontario in London we have identified five patients with the following metabolic disorders presenting as hydrops: glycogen storage disease type IV, mevalonic acid kinase deficiency, type 2 Gaucher disease, and sialidosis (2). The diagnoses were confirmed enzymatically and mutations were characterized. The pathophysiology of hydrops in this group of disorders is not well defined. Possible mechanisms include the presence of cardiomegaly and possibly cardiomyopathy with congestive heart failure, profound anemia as a result of hypersplenism due to hepatosplenomegaly and bone marrow involvement, hypoproteinemia, and mechanical factors including an enlarged liver

compressing the inferior vena cava and causing ascites. Clinical assessment in cases of hydrops should include presence or absence of facial dysmorphism, hepatosplenomegaly, and skeletal involvement. Investigations for such disorders include clinical photographs, skeletal survey, echocardiogram, tissue sampling for histology and snap-frozen tissues for enzyme studies, skin biopsies for fibroblast assays and molecular studies, serum for transferrin isoelectric focusing, and urine for mucopolysaccharides, oligosaccharides, organic acid analysis, and sialic acid. Babies with lysosomal storage disorders that present with hydrops constitute the severe end of the disease spectrum with poor survival and prognosis. The correct diagnosis is essential to allow for appropriate management and assessment regarding long-term prognosis and accurate genetic counseling. In most situations there is a defined recurrence risk of ¼ based on autosomal recessive inheritance. Prenatal diagnosis is almost always possible when a precise diagnosis is made. In the future there may be specific therapies. Awareness of inborn errors of metabolism as a cause of hydrops on the part of obstetricians, neonatologists, and perinatologists is critical to ensure these disorders are not missed. Formal metabolic consultation is recommended to ensure and facilitate a complete work up by obtaining all the appropriate diagnostic tests. 77. Very-long-chain acyl-CoA dehydrogenase (VLCAD), 2-methylbutyryl-AD (2-MBAD), short chain-AD (SCAD), and 3-methylcrotonylCoA carboxylase (3-MCC) deficiencies: newborn screening detects clinically benign or very mild cases. W.J. Rhead,a A. White,a D. Allain,a H. Lindh,b K. Hanson,b S. van Calcar,b and J. Wolff b. a Medical College of Wisconsin, Milwaukee, WI, USA; bUniversity of Wisconsin, Madison, WI, USA. Expanded newborn screening with tandem mass spectrometry (MS/MS) detects serious organic acidemias and fatty acid oxidation disorders, such as propionic and methylmalonic acidemias and MCAD deficiency, and improves their clinical outcome. NBS programs also inform us of the natural course of less severe forms and variants of VLCAD, 2-MBAD, SCAD, and 3-MCC. In the initial 4.5 years of the WI NBS, we have detected one VLCAD, one undiagnosed long chain fatty acid defect (?LCHAD/MTP), 15 2MBAD, 11 SCAD, and 7 3-MCC infants. The LCHAD/MTP infant died suddenly of hypoglycemia before follow up, while the healthy VLCAD patient is homozygous for a mild mutation found to date only in infants detected by NBS. One 2-MBAD infant has had several admissions to the hospital for central apnea, not obviously related to this defect, while all the SCAD and 3-MCC infants remain normal developmentally and clinically. Long chain fatty acid disorders aside, these outcomes, coupled with the detection of asymptomatic older affected siblings in 2-MBAD and 3-MCC families, indicate the relatively innocuous nature of several of these conditions. However, during extreme metabolic decompensation or tissue catabolism, some disorders may still produce ketoacidosis, hypoglycemia and/or primary or secondary CNS damage. Following these infants and children over time will inform us of their natural histories and phenotypic variations.