Causes of and diagnostic approach to methylmalonic acidurias.

Several mutant genetic classes that cause isolated methylmalonic acidurias (MMAuria) are known based on biochemical, enzymatic and genetic complementation analysis. The mut(0) and mut(-) defects result from deficiency of MMCoA mutase apoenzyme which requires adenosyl-cobalamin (Ado-Cbl) as coenzyme. The cblA, cblB and the variant 2 form of cblD complementation groups are linked to processes unique to Ado-Cbl synthesis. The cblC, cblD and cblF complementation groups are associated with defective methyl-cobalamin synthesis as well. Mutations in the genes associated with most of these defects have been described. Recently a few patients have been described with mild MMAuria associated with mutations of the MMCoA epimerase gene or with neurological symptoms due to SUCL mutations. A comprehensive diagnostic approach involves investigations at the level of metabolites, genetic complementation analysis and enzymatic studies, and finally mutation analysis. MMA levels in urine range from 10-20 mmol/mol creatinine in mild disturbances of MMA metabolism to over 20000 mmol/mol creatinine in severe MMCoA mutase deficiency, but show considerable overlap and are of limited value for differential diagnosis. The underlying defect in isolated MMAuria can be characterized in cultured skin fibroblasts using several assays, e.g. conversion of propionate to succinate, specific activity of MMCoA, cobalamin adenosyltransferase assay, cellular uptake of CN-[(57)Co] cobalamin and its conversion to cobalamin coenzymes and complementation analysis. The reliable characterization of patients with isolated MMAuria pinpoints the correct gene for mutation analysis. Reliable classification of these patients is essential for ongoing and future prospective studies on treatment and outcome.

Familial giant cell hepatitis associated with synthesis of 3 beta, 7 alpha-dihydroxy-and 3 beta,7 alpha, 12 alpha-trihydroxy-5-cholenoic acids.

Urinary bile acids from a 3-mo-old boy with cholestatic jaundice were analyzed by ion exchange chromatography and gas chromatography-mass spectrometry (GC-MS). This suggested the presence of labile sulfated cholenoic acids with an allylic hydroxyl group, a conclusion supported by analysis using fast atom bombardment mass spectrometry (FAB-MS). The compounds detected by FAB-MS were separated by thin layer chromatography and high performance liquid chromatography. The sulfated bile acids could be solvolyzed in acidified tetrahydrofuran, and glycine conjugates were partially hydrolyzed by cholylglycine hydrolase. Following solvolysis, deconjugation, and methylation with diazomethane, the bile acids were identified by GC-MS of trimethylsilyl derivatives. The major bile acids in the urine were 3 beta,7 alpha-dihydroxy-5-cholenoic acid 3-sulfate, 3 beta,7 alpha,12 alpha-trihydroxy-5-cholenoic acid monosulfate, and their glycine conjugates. Chenodeoxycholic acid and cholic acid were undetectable in urine and plasma. The family pedigree suggested that abnormal bile acid synthesis was an autosomal recessive condition leading to cirrhosis in early childhood.

Mutations in exon 3 of the glycogen debranching enzyme gene are associated with glycogen storage disease type III that is differentially expressed in liver and muscle.

Glycogen storage disease type HI (GSD-III), an autosomal recessive disease, is caused by deficient glycogen debranching enzyme (GDE) activity. Most GSD-III patients are GDE deficient in both liver and muscle (type IIIa), and some GSD-III patients have GDE absent in liver but retained in muscle (type IIIb). The molecular basis for this enzymatic variability is largely unknown. In the present study, the analysis of the GDE gene in three GSD-IIIb patients by single-strand conformation polymorphism (SSCP), DNA sequencing, restriction analysis, and family studies, revealed each of them as being a compound heterozygote for two different mutations. The first mutant alleles in all three patients involved mutations in exon 3 at amino acid codon 6 of the GDE protein. Two had an AG deletion at nucleotides 17 and 18 of the GDE cDNA (17delAG) which resulted in change of subsequent amino acid sequence and a truncated protein (25X); the other had a C to T transition at nucleotide 16 of the cDNA which changed a Glutamine codon to a stop codon (Q6X). The 17delAG mutation was also found in 8 of the 10 additional GSD-IIIb patients. The Q6X mutation was found in one of the remaining two GSD-IIIb patients. These two mutations were not found in any of the 31 GSD-IIIa patients, 2 GSD-IIId patients, nor 28 unrelated normal controls. The second mutant alleles in each of the three GSD-IIIb patients were R864X, R1228X, and W68OX. The R864X and R1228X were not unique for GSD-IIIb as they were also found in GSD-IIIa patients (frequency of 10.3% and 5.2% in Caucasian patients, respectively). Our data demonstrated that both IIIa and IIIb had mutations in the same GDE gene and established for the first time the molecular basis of GSD-III that differentially expressed in liver and muscle. The striking and specific association of exon 3 mutations with GSD-IIIb may provide insight into mechanisms controlling tissue-specific expression of the GDE gene. The identification of exon 3 mutations has clinical significance as well because it distinguished GSD-IIIb from IIIa hence permitting diagnosis from a blood sample rather than a more invasive muscle biopsy.

Demyelination of the brain is associated with methionine adenosyltransferase I/III deficiency.

Individuals deficient in hepatic methionine adenosyltransferase (MAT) activity (MAT I/III deficiency) have been demonstrated to contain mutations in the gene (MATA1) that encodes the major hepatic forms, MAT I and III. MAT I/III deficiency is characterized by isolated persistent hypermethioninemia and, in some cases, unusual breath odor. Most individuals with isolated hypermethioninemia have been free of major clinical difficulties. Therefore a definitive diagnosis of MAT I/III deficiency, which requires hepatic biopsy, is not routinely made. However, two individuals with isolated hypermethioninemia have developed abnormal neurological problems, including brain demyelination, suggesting that MAT I/III deficiency can be deleterious. In the present study we have examined the MATA1 gene of eight hypermethioninemic individuals, including the two with demyelination of the brain. Mutations that abolish or reduce the MAT activity were detected in the MATA1 gene of all eight individuals. Both patients with demyelination are homozygous for mutations that alter the reading frame of the encoded protein such that the predicted MATalpha1 subunits are truncated and enzymatically inactive. The product of MAT, S-adenosylmethionine (AdoMet), is the major methyl donor for a large number of biologically important compounds including the two major myelin phospholipids, phosphatidylcholine and sphingomyelin. Both are synthesized primarily in the liver. Our findings demonstrate that isolated persistent hypermethioninemia is a marker of MAT I/III deficiency, and that complete lack of MAT I/III activity can lead to neurological abnormalities. Therefore, a DNA-based diagnosis should be performed for individuals with isolated hypermethioninemia to assess if therapy aimed at the prevention of neurological manifestations is warranted.

Recent advances in amino acid and organic acid metabolism.

This paper focuses on the three areas in this field in which there have been advances in amino acid and organic acid metabolism. These are the description of glutamine synthetase deficiency, the elucidation of the mechanism of pyridoxine-dependent convulsions, and a hypothesis to explain the neurological complications of some organic acidaemias.

Neurological implications of urea cycle disorders.

The urea cycle disorders constitute a group of rare congenital disorders caused by a deficiency of the enzymes or transport proteins required to remove ammonia from the body. Via a series of biochemical steps, nitrogen, the waste product of protein metabolism, is removed from the blood and converted into urea. A consequence of these disorders is hyperammonaemia, resulting in central nervous system dysfunction with mental status changes, brain oedema, seizures, coma, and potentially death. Both acute and chronic hyperammonaemia result in alterations of neurotransmitter systems. In acute hyperammonaemia, activation of the NMDA receptor leads to excitotoxic cell death, changes in energy metabolism and alterations in protein expression of the astrocyte that affect volume regulation and contribute to oedema. Neuropathological evaluation demonstrates alterations in the astrocyte morphology. Imaging studies, in particular (1)H MRS, can reveal markers of impaired metabolism such as elevations of glutamine and reduction of myoinositol. In contrast, chronic hyperammonaemia leads to adaptive responses in the NMDA receptor and impairments in the glutamate-nitric oxide-cGMP pathway, leading to alterations in cognition and learning. Therapy of acute hyperammonaemia has relied on ammonia-lowering agents but in recent years there has been considerable interest in neuroprotective strategies. Recent studies have suggested restoration of learning abilities by pharmacological manipulation of brain cGMP with phosphodiesterase inhibitors. Thus, both strategies are intriguing areas for potential investigation in human urea cycle disorders.

Guideline for the diagnosis and management of glutaryl-CoA dehydrogenase deficiency (glutaric aciduria type I).

Glutaryl-CoA dehydrogenase (GCDH) deficiency is an autosomal recessive disease with an estimated overall prevalence of 1 in 100 000 newborns. Biochemically, the disease is characterized by accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid, and glutarylcarnitine, which can be detected by gas chromatography-mass spectrometry of organic acids or tandem mass spectrometry of acylcarnitines. Clinically, the disease course is usually determined by acute encephalopathic crises precipitated by infectious diseases, immunizations, and surgery during infancy or childhood. The characteristic neurological sequel is acute striatal injury and, subsequently, dystonia. During the last three decades attempts have been made to establish and optimize therapy for GCDH deficiency. Maintenance treatment consisting of a diet combined with oral supplementation of L: -carnitine, and an intensified emergency treatment during acute episodes of intercurrent illness have been applied to the majority of patients. This treatment strategy has significantly reduced the frequency of acute encephalopathic crises in early-diagnosed patients. Therefore, GCDH deficiency is now considered to be a treatable condition. However, significant differences exist in the diagnostic procedure and management of affected patients so that there is a wide variation of the outcome, in particular of pre-symptomatically diagnosed patients. At this time of rapid expansion of neonatal screening for GCDH deficiency, the major aim of this guideline is to re-assess the common practice and to formulate recommendations for diagnosis and management of GCDH deficiency based on the best available evidence.

Komrower lecture: Treatment of inborn errors of metabolism: a review.

Although the treatment of a small number of inborn errors is very good, for the majority the outcome is less satisfactory. This review examines current treatment critically and suggests how the information on which decisions about the management of patients with inborn errors might be improved.

The aetiology of neurological complications of organic acidaemias--a role for the blood-brain barrier.

The blood-brain barrier (BBB) metabolically isolates the central nervous system (CNS) from the circulation and protects it against fluctuations of hydrophilic nutrients in plasma and from intoxication. Recent studies have shown that dicarboxylic acids (DCAs) are transported across the blood-brain barrier at very low rates. In organic acidaemias, neurological complications are common. We hypothesize that, as a result of the very limited efflux, in certain organic acidaemias there is pathological accumulation of DCAs (e.g. glutarate, 3-hydroxyglutarate, D-2- and L-2-hydroxyglutarate, methylmalonate) in the brain secondary to the metabolic block. At high concentrations some of these compounds may become neurotoxic. Treatment should be aimed at preventing the accumulation of these compounds using our understanding of the properties of the BBB.

A new species of wedgefish Rhynchobatus cooki (Rhinopristiformes, Rhinidae) from the Indo-West Pacific.

A new dwarf wedgefish, Rhynchobatus cooki sp. nov. is described from a single female from a Jakarta fish market (Indonesia) and 11 specimens collected at Jurong fish market (Singapore). First collected in 1934, the broader ichthyological community have been aware of this distinctive but little known ray since the late 1990's. Rhynchobatus cooki is the smallest of the wedgefishes (to 81 cm TL) and has the lowest vertebral count (fewer than 107 centra). It is also distinguishable from its congeners based on its long, hastate snout, very strongly undulate anterior pectoral-fin margin, coloration and aspects of its squamation. The dorsal coloration is mainly dark and distinctively marked with white blotches, spots and streaks, and has a dark cruciate marking on the interorbit and a prominent white border around the body margin. Unlike most other wedgefish species, the snout tip lacks dark blotches and there is no black pectoral-fin marking. It shares well-developed rostral spines with a much larger Atlantic species (Rhynchobatus luebberti), but these spines are confined to the snout tip (rather than being more numerous and extending in paired rows along the rostral ridges nearly to the eyes). No additional specimens have been observed since 1996, despite an increased recent effort to survey the chondrichthyan fauna of South-East Asia and collect biological data for species, raising concerns over its conservation status.

Exercise capacity and biochemical profile during exercise in patients with glycogen storage disease type I.

Glycogen storage disease type I (GSD-I) is an inherited disorder of carbohydrate metabolism. Hepatic glucose-6-phosphatase is deficient, leading to impaired gluconeogenesis and glycogenolysis. Patients prevent fasting hypoglycemia by frequent feeds of low glycemic index foods. Normal muscle does not contain glucose-6-phosphatase, and GSD-I is usually classified as a hepatic glycogenosis. However, clinical experience has suggested that patients have decreased cardiovascular fitness, but this had not been formally investigated. This paper reports the results of maximal treadmill cardiopulmonary exercise testing in adult patients with GSD-I. It documents a major reduction in exercise capacity in these patients and demonstrates biochemical aspects of exercise that are different from those of normal controls. All patients showed a reduction in exercise capacity, but there was a wide range of exercise tolerance. Additional work needs to address whether improved adherence to or intensification of therapy in adulthood will ameliorate exercise intolerance.

Impaired endothelial function occurs in the systemic arteries of children with homozygous homocystinuria but not in their heterozygous parents.

OBJECTIVES: Because endothelial dysfunction is an early event in atherogenesis, we aimed to determine whether endothelial function is normal or impaired in the systemic arteries of children with homozygous homocystinuria or in those of heterozygous adults, or both. BACKGROUND: Homocystinuria is strongly associated with premature vascular disease in homozygotes, and even heterozygotes have been shown to be at increased risk from early atherosclerosis associated with hyperhomocystinemia. METHODS: We conducted noninvasive studies on the superficial femoral or brachial arteries of 9 children aged 4 to 17 years (mean 11) with homozygous homocystinuria and on the brachial arteries of 14 obligate heterozygous parents age 33 to 49 years (mean 41). Each subject was matched with two control subjects. Using high resolution ultrasound, we measured vessel diameter at rest, during reactive hyperemia (with flow increase causing endothelium-dependent dilation) and after sublingual administration of nitroglycerin (an endothelium-independent vasodilator). RESULTS: Flow-mediated dilation was observed in the control children (9 +/- 0.6%, range 6% to 14%) but was impaired in the children with homocystinuria (2.8 +/- 0.7%, range 0% to 7%, p < 0.0001). In contrast, nitroglycerin-mediated dilation was similar in both groups (15.7 +/- 1.6% vs. 13.1 +/- 1.2%, p = 0.27), indicating that the impaired flow-mediated dilation is secondary to endothelial dysfunction. In the heterozygous parents, both flow-mediated dilation and nitroglycerin responses (6.3 +/- 0.9%, 17 +/- 1.4%, respectively) were similar to control values (6.8 +/- 0.7%, 20.7 +/- 1.7%, p > 0.10). CONCLUSIONS: Children with homozygous homocystinuria had impaired endothelial function in the systemic arteries as early as 4 years of age, representing an early event in their premature vascular disease. However, endothelial function was preserved in the heterozygous adults.

A pathogenic glutamate-to-aspartate substitution (D296E) in the pyruvate dehydrogenase E1 subunit gene PDHA1.

In a patient with fatal neonatal lactic acidosis due to pyruvate dehydrogenase deficiency, the only potential mutation detected was c.888C>G in PDHA1, the gene for the E1alpha subunit of the complex. This would result in a substitution of glutamate for aspartate (D296E). Pathogenicity of this minor alteration in amino acid sequence was demonstrated by expression studies. By comparing the mutant sequence with the known structures of the E1 components of pyruvate dehydrogenase and the closely related branched chain alpha-ketoacid dehydrogenase, an explanation for the profound consequences of the mutation can be proposed.

The putative glucose 6-phosphate translocase gene is mutated in essentially all cases of glycogen storage disease type I non-a.

The purpose of this work was to test the hypothesis that mutations in the putative glucose 6-phosphate translocase gene would account for most of the cases of GSD I that are not explained by mutations in the phosphohydrolase gene, ie that are not type Ia. Twenty-three additional families diagnosed as having GSD I non-a (GSDIb, Ic or Id) have now been analysed. The 9exons of the gene were amplified by PCR and mutations searched both by SSCP and heteroduplex analysis. Except for one family in which only one mutation was found, all patients had two allelic mutations in the gene encoding the putative glucose 6-phosphate translocase. Sixteen of the mutations are new and they are all predicted to lead to non-functional proteins. All investigated patients had some degree of neutropenia or neutrophil dysfunction and the clinical phenotype of the four new patients who had been diagnosed as GSD Ic and the one diagnosed as GSD Id was no different from the GSD Ib patients. Since these patients, and the four type Ic patients from two families previously studied, shared several mutations with GSD Ib patients, we conclude that their basic defect is in the putative glucose 6-phosphate translocase and that they should be reclassified as GSD Ib. Isolated defects in microsomal Pi transporter or in microsomal glucose transporter must be very rare or have phenotypes that are not recognised as GSD I, so that in practice there are only two subtypes of GSD I (GSD Ia and GSD Ib).

The effects of l-alanine supplementation in late-onset glycogen storage disease type II.

Myopathy in glycogen storage disease type II (GSD-II) is slowly progressive. Five subjects with the late-onset form of GSD-II (age range, 15 to 47 years) and seven healthy control subjects (age range, 28 to 55 years) were studied. Following alanine supplementation, resting energy expenditure decreased in patients with GSD-II (p < 0.05) compared to values seen in control subjects. Leucine flux decreased (p < 0.004), as did leucine oxidation, to levels lower than those observed in control subjects (p < 0.001). l-Alanine reduces protein turnover and catabolism in GSD-II.

Demyelination and decreased S-adenosylmethionine in 5,10-methylenetetrahydrofolate reductase deficiency.

We previously described demyelination in the brain and subacute combined degeneration of the spinal cord in a patient with 5,10-methylenetetrahydrofolate reductase deficiency. To assess the role of methionine, S-adenosylmethionine, folate, and neurotransmitter amine metabolism in the demyelination process, we measured these metabolites in CSF from this patient; the findings are compared with those obtained from three patients in whom neurologic deterioration had been halted by the administration of betaine. Folate concentrations were low, and amine and biopterin metabolism were abnormal in all patients. Methionine and S-adenosylmethionine concentrations were undetectable in the first patient. In those receiving betaine, methionine concentrations were proportional to the dose administered and S-adenosylmethionine concentrations were near normal. The results provide the first evidence for an association between defective S-adenosylmethionine metabolism and demyelination in humans.

Neurodevelopmental abnormalities and lactic acidosis in a girl with a 20-bp deletion in the X-linked pyruvate dehydrogenase E1 alpha subunit gene.

We describe a girl with developmental abnormalities of the CNS and a lactic acidosis whose cultured fibroblasts showed a profound deficiency of pyruvate dehydrogenase complex (PDHC) activity (patient = 0.14 nmol/mg protein per minute, controls = 0.7 to 1.1 nmol/mg protein per minute). Immunocytochemistry demonstrated the fibroblast culture to be mosaic, with 14% of cells expressing the PDHC E1 alpha subunit protein in normal amounts and the remaining 86% having no detectable immunoreactive activity. Direct sequencing of cDNA for the X-linked PDHC E1 alpha subunit established that the patient was heterozygous for a 20-bp deletion beginning in the codon for Ser300 of the derived amino acid sequence. The pattern of methylation at the DXS255 locus suggested predominant expression of the X chromosome carrying the mutant allele in the fibroblast culture. There was a good correlation between the residual PDHC activity, the proportion of cells with immunoreactive E1 alpha protein, and the X chromosome inactivation ratio, demonstrating the importance of X-inactivation for expression of this X-linked neurometabolic disease in females.

Hyperlipidaemia does not impair vascular endothelial function in glycogen storage disease type 1a.

Patients with glycogen storage disease type 1 (GSD-1) often have marked hyperlipidaemia with abnormal lipoprotein profiles. This metabolic abnormality improves, but is not fully corrected, with dietary therapy and therefore these patients may be at high risk for the development of atherosclerosis. Endothelial dysfunction is an early event in atherogenesis and can be detected in children and young adults at high risk. We studied endothelial function, using a non-invasive ultrasonographic method, in the brachial arteries of 6 adult GSD-1a patients (aged 23-33 years) with mean cholesterol of 7.9 mmol/l (range 4.7 to 14.6) and mean triglycerides of 9.1 mmol/l (range 4.1 to 21.3), and 12 age- and sex-matched normolipidaemic controls. Flow-mediated (endothelium-dependent) dilation was similar in patients and controls (8.2% vs. 10.5%; P = 0.20). Although the patient numbers are small, these results are consistent with the surprising lack of clinically evident atherosclerosis in GSD-1. The reasons these patients appear less susceptible to the damaging arterial effects of hyperlipidaemia are unknown. These results may have implications for others with secondary hyperlipidaemias.

Comparison of the functional significance of left ventricular hypertrophy in hypertrophic cardiomyopathy and glycogenosis type III.

A comparison of blood pressure response with exercise stress, thallium scintigraphy, and 24-hour electrocardiographic monitoring between 5 patients with left ventricular hypertrophy associated with glycogen storage disease type III and 10 matched patients with hypertrophic cardiomyopathy revealed normal results in the former group. These data highlight the importance of the etiology of left ventricular hypertrophy before the application of risk stratification.