Molecular heterogeneity for bovine maple syrup urine disease

In Poll Herefords, it is known that maple syrup urine disease results from a nonsense mutation in codon -6 of the gene for the Elα subunit of branched-chain α-keto acid dehydrogenase. The disease also occurs in Poll Shorthorns, but its molecular basis in this breed has not yet been determined. Allele-specific hybridization and allele-specific amplification, both based on the Poll Hereford mutation, failed to detect the mutant allele in Poll Shorthorn heterozygotes, and detected the normal allele in affected Poll Hereford-cross-Poll Shorthorn calves. These results demonstrate between breed molecular heterogeneity for bovine maple syrup urine disease.     

Molecular characterization of maple syrup urine disease patients from Tunisia

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.     

Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding

The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease.     

Evaluation of a heterozygote test for maple syrup urine disease in leucocytes and cultured fibroblasts

Summary Methods are given in detail to assay branched chain keto acid oxidases in native leucocytes and fibroblasts. In peripheral blood these enzymes are located preferentially in lymphocytes.The intraindividual variation of enzyme activities in leucocytes is reduced by correcting for the number of lymphocytes. In contrast, interindividual variation remains unchanged. Consequently, an overlap between enzyme activities of control persons and heterozygotes for classic maple syrup urine disease still exists. For explanation multiple alleles and influence of genetic background on enzyme activities are invoked.Arguments are given for the simultaneous defect of the three branched chain keto acid oxidases in classic maple syrup urine disease.Furthermore some new observations on the intermittent type of maple syrup urine disease are given.Tests for heterozygosity in fibroblasts are complicated because of environmental influences in cultures which are not fully understood at present. However, the enzymatic defect is clearly demonstrated in fibroblasts of patients with the classic type and the intermittent type of maple syrup urine disease.

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Zusammenfassung Die Methoden zur Testung der Oxidasen für die verzweigtkettigen -Ketosäuren in Leukocyten und Fibroblasten werden beschrieben. Im peripheren Blut sind diese Enzyme bevorzugt in den Lymphocyten lokalisiert.In den Leukocyten wird die intraindividuelle Variation der Enzymaktivitäten durch Berücksichtigung des Differentialblutbildes verringert. Die interindividuelle Variation bleibt dagegen unverändert. — Für die Enzymaktivitäten von Normalpersonen und Eltern von Patienten mit klassischer Ahornsirupkrankheit bleibt damit ein Überlappungsbereich bestehen. Als mögliche Erklärung werden multiple Allelie und multifaktorielle Determinierung von Enzymaktivitäten diskutiert.Bisher gewonnene Ergebnisse lassen vermuten, daß bei der klassischen Form der Ahornsirupkrankheit alle drei Oxidasen für die verzweigtkettigen -Ketosäuren defekt sind. Neuere Untersuchungen über die intermittierende Form der Ahornsirupkrankheit werden mitgeteilt.Die Erkennung von Heterozygoten in Testen mit Fibroblasten ist erschwert, da die Abhängigkeit der Aktivität der -Ketosäure-Oxidasen von den Kulturbedingungen noch nicht genügend geklärt ist. Es ist dagegen möglich, Patienten mit der klassischen und der intermittierenden Form der Ahornsirupkrankheit durch enzymatische Teste an Fibroblasten zu erkennen.

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Dedicated to Prof. Dr. K. H. Schäfer at the occasion of his 60th birthday.

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This work was supported in part by Deutsche Forschungsgemeinschaft and Stiftung Volkswagenwerk.

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U. L. is recipient of a training grant from Stiftung Volkswagenwerk.     

Chloride-dependent inhibition of vesicular glutamate uptake by alpha-keto acids accumulated in maple syrup urine disease

Maple syrup urine disease is a metabolic disorder caused by mutations of the branched chain keto acid dehydrogenase complex, leading to accumulation of alpha-keto acids and their amino acid precursors in the brain. We now report that alpha-ketoisovaleric, alpha-keto-beta-methyl-n-valeric and alpha-ketoisocaproic acids accumulated in the disease inhibit glutamate uptake into rat brain synaptic vesicles. The alpha-keto acids did not affect the electrochemical proton gradient across the membrane, suggesting that they interact directly with the vesicular glutamate carrier. Chloride anions have a biphasic effect on glutamate uptake. Low concentrations activate the uptake (0.2 to 8 mM), while higher concentrations are inhibitory. The alpha-keto acids inhibited glutamate uptake by a new mechanism, involving a change in the chloride dependence for the activation of glutamate uptake. The activation of glutamate uptake by low chloride concentrations was shifted toward higher concentrations of the anion in the presence of alpha-keto acids. Inhibition by alpha-keto acids was abolished at high chloride concentrations (20 to 80 mM), indicating that alpha-keto acids specifically change the stimulatory effect of low chloride concentrations. High glutamate concentrations also reduced the inhibition by alpha-keto acids, an effect observed both in the absence and in the presence of low chloride concentrations. The results suggest that in addition to their possible pathophysiological role in maple syrup urine disease, alpha-keto acids are valuable tools to study the mechanism of vesicular transport of glutamate.     

Study on established lymphoid cells in maple syrup urine disease. Correlation with clinical heterogeneity

Summary Branched-chain keto acid dehydrogenase complex (BCKAD) was measured in lymphoid cells established from five patients with maple syrup urine disease (MSUD) and six control subjects. Two other MSUD lymphoid cell lines obtained from The Human Genetic Mutant Cell Repository were used as references. One day after subculture, the cells grew logarithmically up to 4–5 days. With this cell growth, BCKAD activity increased greatly in controls, but not in MSUD cells. The maximum BCKAD activity of MSUD cells was less than 7% and 13%–16% of the control in classic and variatant types, respectively. Leucine added to culture medium at the concentration of 10–20 mM significantly inhibited cell growth in MSUD cells alone, and with increasing concentration and impaired enzyme activity in a cell line, the effect became more prominent. The effects of isoleucine and valine were mild and did not differ between control and MSUD cells.This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (56480191; Matsuda)     

Inhibition of brain energy metabolism by the alpha-keto acids accumulating in maple syrup urine disease

Neurological dysfunction is a common finding in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of brain damage in this disorder are poorly known. In the present study, we investigated the effect of the in vitro effect of the branched chain alpha-keto acids (BCKA) accumulating in MSUD on some parameters of energy metabolism in cerebral cortex of rats. [14CO(2)] production from [14C] acetate, glucose uptake and lactate release from glucose were evaluated by incubating cortical prisms from 30-day-old rats in Krebs-Ringer bicarbonate buffer, pH 7.4, in the absence (controls) or presence of 1-5 mM of alpha-ketoisocaproic acid (KIC), alpha-keto-beta-methylvaleric acid (KMV) or alpha-ketoisovaleric acid (KIV). All keto acids significantly reduced 14CO(2) production by around 40%, in contrast to lactate release and glucose utilization, which were significantly increased by the metabolites by around 42% in cortical prisms. Furthermore, the activity of the respiratory chain complex I-III was significantly inhibited by 60%, whereas the other activities of the electron transport chain, namely complexes II, II-III, III and IV, as well as succinate dehydrogenase were not affected by the keto acids. The results indicate that the major metabolites accumulating in MSUD compromise brain energy metabolism by blocking the respiratory chain. We presume that these findings may be of relevance to the understanding of the pathophysiology of the neurological dysfunction of MSUD patients.     

Intracellular levels and metabolism of leucine and alpha-ketoisocaproate in normal and maple syrup urine disease fibroblasts

The interdependence of intra- and extracellular leucine and KICA concentration was studied in cultured MSUD and control fibroblasts. Intracellular KICA levels were measurable only after leucine load and were 1/20th to 1/40th that of leucine. In cells exposed to high KICA concentrations (2 mmole/liter) KICA accumulation followed linear kinetics, finally overwhelming the transamination capacity. Transamination of KICA to leucine became saturated at 2-6 mmole/liter of extracellular KICA. MSUD and control fibroblasts differed slightly in intracellular KICA concentrations, and only at high concentrations of leucine in medium. Release of KICA into the medium after leucine loading was found to be slightly increased in MSUD fibroblasts.     

Myelin proteins: degradation in rat brain initiated by metabolites causative of maple syrup urine disease

Maple syrup urine disease (MSUD), an inborn error of metabolism in humans, is expressed as an inability to oxidatively decarboxylate the branched-chain alpha-keto acids derived from leucine, isoleucine and valine. Rats 14 days old were injected intracranially with a solution containing leucine, alpha-ketoisocaproate, and tracer amounts of 3H-lysine. Myelin isolated from these rat brains at 28 days of age had a washed dry weight 85 per cent of controls. The protein content of the myelin prepared from treated and control rats was identical, as were the specific activities of the individual proteins separated by polyacrylamide gel electrophoresis. Myelin protein from treated rats was deficient in myelin high molecular weight proteins including glycoproteins, and degradation products of these proteins were observed in myelin of treated rats. MSUD associated metabolites in man may initiate a process leading to the proteolytic degradation of myelin proteins, thereby producing abnormal myelin sheaths.     

Complementation analysis in lymphoid cells from five patients with different forms of maple syrup urine disease

Summary The possibility of genetic heterogeneity in maple syrup urine discase was investigated by measuring branchedchain ketoacid dehydrogenase in polyethylene glycol-induced heterokaryons of lymphoid cells. The lymphoid cell lines from five patients with varying forms of the disease were establisjed after incubation with Epstein-Barr virus. The results suggested that there are at least two genetic complementation groups in the disease.This study was supported by a scientific research grant from the Ministry of Education, Science, and Culture of Japan (56480191; Matsuda) and a research grant from the Ministry of Health and Welfare of Japan     

Defective decarboxylase in branched chain ketoacid oxidase multienzyme complex in classic type of maple syrup urine disease

Summary Enzyme kinetic studies on tissue extract from a newborn child who had died from the classic type of maple syrup urine disease (MSUD) revealed an altered decarboxylase moiety of the branched chain ketoacid oxidase multienzyme complex. Of two kinetically different decarboxylase components present in normal human controls the one with higher affinity for the substrate -ketoisovalerate was absent in our patient.

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Zusammenfassung Es wurden enzymkinetische Untersuchungen des verzweigtkettigen -Ketosäureoxidase-Komplexes in angereicherten Gewebeextrakten eines neugeborenen Kindes beschrieben, das an sogenannter klassischer Ahornsirupkrankheit gestorben ist. Während in normalen Kontrollen zwei kinetisch unterscheidbare Komponenten mit unterschiedlicher Substrataffinität nachweisbar sind, wurde im Gewebe des Patienten nur die Komponente mit geringerer Affinität zur -Ketoisovaleriansäure gefunden. Dieses Ergebnis deutet darauf hin, daß bei der klassischen Ahornsirupkrankheit das Teilenzym Decarboxylase im Multienzymkomplex der verzweigtkettigen Ketosäureoxidase defekt ist.

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This work was supported in part by grants of the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain alpha-ketoacid decarboxylase in maple syrup urine disease

Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-alpha, Y368C-alpha, and F364C-alpha, which occur in the E1alpha subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an alphabeta heterodimeric intermediate to a native alpha(2)beta(2) heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 m concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations.     

Management of maple syrup urine disease in Canada. Committee for improvement of Hereditary Disease Management.

Nine patients with classic maple syrup urine disease (MSUD) and four with variant forms are under care at five treatment centres in the network affiliated with the National Food Distribution Centre for the Treatment of Hereditary Metabolic Diseases (the "Food Bank"). Diagnosis was made by clinicians and not from mass screening programs. MSUD requires complex emergency treatment to prevent severe neurologic damage, but effective management is compatible with normal growth and development. Long-term treatment requires continuous monitoring of the response to diets restricted in branched-chain amino acids; semisynthetic diet products free of branched-chain amino acids, provided by the Food Bank, are essential. Centralized treatment programs reduce the cost of treatment and maximize the potential benefits. The leucine requirement for adequate somatic growth during infancy in MSUD was found to be 200 to 600 mg/d; this range is lower than that estimated for infants with an intact leucine catabolic outflow pathway. The requirements for isoleucine and valine in infancy were also found to be lower than published values for normal infants.