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PubMed1989

Metabolic myopathies: evaluation by graded exercise testing.

Exertional muscle pain and fatigue are common complaints; some patients with these symptoms have a metabolic myopathy. We have performed graded exercise testing with analysis of expired ventilation on 13 individuals with various kinds of metabolic myopathies. Their results differed from normal and reflected the underlying biochemical abnormality. Patients with disorders of the mitochondrial electron transport chain demonstrated marked limitations in aerobic metabolism and a greatly reduced maximum oxygen consumption. During intense exertion, normal individuals increase carbon dioxide generation due to buffering of lactic acid. This did not occur in patients with McArdle disease, in whom the respiratory exchange ratio (carbon dioxide production/oxygen consumption) did not rise above 1.0 at maximum exercise. These results indicated a deficit in anaerobic metabolism. Pyruvate dehydrogenase complex allows pyruvate produced from carbohydrate metabolism to enter the citric acid cycle. Patients with this enzyme deficiency showed an initially normal pattern followed by an abrupt cessation in carbohydrate dependent aerobic metabolism at higher work loads. During high-intensity exercise, progressive anaerobic metabolism was not accompanied by additional oxygen consumption. Finally, results from a patient with carnitine palmitoyl transferase deficiency revealed an early dependence on carbohydrate metabolism. The ventilatory threshold occurred at a low percentage of maximal oxygen consumption, reflecting the limited availability of lipid substrates for aerobic metabolism. Detection of some muscle metabolic abnormalities can be made on small biopsy specimens. However, definitive diagnosis of the defect nearly always requires studies on fresh or frozen muscle tissue obtained by an open biopsy. The decision on how the tissue should be processed and which metabolic studies should be performed frequently needs to be made before the biopsy is obtained. Thus, a noninvasive method to initially characterize patients with potential metabolic disorders is useful. Exercise testing with expired gas analysis can indicate the presence of a metabolic myopathy and results can then be used to direct the appropriate biochemical evaluations.

Adolescent↗
PubMed1996

Autoantibodies to M2 mitochondrial autoantigens in normal human sera by immunofluorescence and novel assays.

Primary biliary cirrhosis (PBC) is characterized by the presence of antimitochondrial antibodies (anti-M2), directed against the E2 subunits of the 2-oxo-acid dehydrogenase complexes (2-OADC), chiefly pyruvate dehydrogenase complex (PDC-E2). We present here a detailed study, based on a large panel of normal sera, of the specificity of tests for anti-M2 by immunofluorescence and for anti-PDC by other assays for the diagnosis of PBC. The assays for anti-PDC included immunoblotting with bovine heart mitochondria, ELISA using recombinant PDC-E2 and an enzyme inhibition assay using purified porcine PDC. The positivity rates for normal sera were 0 (0/170), 2 (4/201), 1.5 (3/198) and 0% (0/186) for immunofluorescence, immunoblotting, ELISA and the enzyme inhibition assay, respectively. The seven positive reactions detected either by immunoblotting (n = 4) or ELISA (n = 3) were negative by the other three assays and in no instance did biochemical indices give any indication of chronic liver disease. Thus, as judged by reactivity with normal sera, the specificity of a positive test for the antibody to the major M2 autoantigen (PDC-E2) is 100% for immunofluorescence and the enzyme inhibition assay, 98% for immunoblotting and 98.5% for ELISA.

Autoantibodies↗
PubMed1994

Prenatal diagnosis of pyruvate dehydrogenase E1 alpha subunit deficiency.

Pyruvate dehydrogenase (PDH) E1 alpha subunit deficiency is an X-linked inborn error of metabolism affecting males and females with equal frequency. The diagnosis is usually based on determination of enzyme activity, although this may present difficulties in some females because of X-inactivation patterns favouring expression of the normal X chromosome. This is a particular problem for prenatal diagnosis using chorionic villus cells where normal enzyme assay results do not necessarily exclude the diagnosis and confirmatory X-inactivation analysis may be complicated by variable methylation of active and inactive X chromosomes. We describe prenatal diagnosis in two pregnancies in a family following diagnosis of a PDH E1 alpha deficient male. The first prenatal diagnosis was performed by enzyme assay, but by the time of the subsequent pregnancy, the underlying mutation in the affected male had been identified and direct gene analysis was possible. This study highlights the limitations of diagnosis of PDH E1 alpha deficiency based on measurement of the gene product and illustrates the need for mutation analysis in affected individuals.

Cells, Cultured↗
PubMed1993

Neuropathological findings of a patient with pyruvate dehydrogenase E1 alpha deficiency presenting as a cerebral lactic acidosis.

Neuropathological findings are reported of a 6-month-old female child with a "cerebral" lactic acidosis. A mutation in the pyruvate dehydrogenase (PDH) E1 alpha gene was found. Gross examination of the brain revealed a severe thinning of the cerebral parenchym, a marked hydrocephalus sparing the aqueduct and fourth ventricle, agenesis of the corpus callosum and heterotopic noduli of gray matter in subependymal regions. Microscopical examination showed heterotopic inferior olives, absent pyramids and focal neuroglial overgrowth into meninges. In addition some heterotopia of Purkinje cells and dysplasia of the dentate nuclei were observed. There was a marked vascular proliferation with many thin-walled, congestive vessels in the cerebral and cerebellar white matter, and to a lesser extent in the striatum. To our knowledge these cerebellar and vascular abnormalities have not been reported before in patients with "cerebral" lactic acidosis. The combination of these neuropathological findings might be characteristic for PDH deficiency and more specifically for its E1 alpha subtype. Neuropathological examination could lead to the retrospective diagnosis of PDH E1 alpha deficiency in those cases where biochemical investigations were not or incompletely performed. This may have potential implications for genetic counseling.

Acidosis, Lactic↗
PubMed1991

Polymorphisms in the human X-linked pyruvate dehydrogenase E1 alpha gene.

Pyruvate dehydrogenase E1 alpha deficiency is an X-chromosome-linked disorder, often with fatal consequences. We have searched for genetically useful polymorphisms in or near this gene. No restriction fragment length polymorphisms were detected using a battery of 36 different restriction enzymes and probing with a full-length cDNA fragment, or two single-copy genomic fragments located within intron 8, and 15 kb 3' of the coding region, respectively. The chemical cleavage method was then applied to the detection of base changes in or near the gene. One polymorphism was found in exon 8 of the coding region. However, no base changes were detected in intron 3 or in the part of intron 8 covered by fragment gB2. Three blocks of microsatellite DNA containing variable numbers of CA-repeats were isolated from the 5' end of the gene and characterized. Length polymorphisms in these microsatellite DNAs were analysed using the polymerase chain reaction. Although the three loci are tightly linked, the polymorphisms appear not to be in disequilibrium, making them useful markers in linkage studies of the pyruvate dehydrogenase E1 alpha gene. Of 31 females analysed 12(39%) were heterozygous for at least one length polymorphism of the three (CA)n alleles.

Autoradiography↗
PubMed1992

Mutation of E1 alpha gene in a female patient with pyruvate dehydrogenase deficiency due to rapid degradation of E1 protein.

A mutation of an insertion of 4 bp in the gene for the alpha subunit of pyruvate dehydrogenase (E1 alpha) was found in a female with pyruvate dehydrogenase deficiency due to the rapid degradation of alpha and beta subunit proteins of pyruvate dehydrogenase. This mutation caused a frameshift that altered the amino acid sequence and created a premature stop codon. This 4-bp insertion has been found in an unrelated female patient with E1 alpha deficiency. It is rare that the same mutation is found in unrelated patients with this rare inborn error of metabolism. Furthermore, short deletions or duplications in the E1 alpha gene of patients with E1 alpha deficiency have been found only in exons 10 and 11. These exons may be hot spots for the mutations by the recombinational processes. This patient was heterozygous for the normal and a mutant allele. However, in most of the cultured skin fibroblasts from this patient, the mutant allele was expressed. These observations suggest that the X chromosome containing the normal allele was predominantly inactivated so that she developed lactic acidaemia and neurological abnormalities despite being heterozygous. The mutant alpha subunit protein failed to form a stable structure of pyruvate dehydrogenase, so that both alpha and beta subunit proteins were degraded rapidly.

Alleles↗
PubMed1983

Revised assays for the investigation of congenital lactic acidosis using 14C keto acids, eliminating problems associated with spontaneous decarboxylation.

Improved methods using 14C keto acids for the investigation of patients with congenital lactic acidoses are described. The addition of rat serum to assay media reduces the spontaneous decarboxylation of [1-14C] and [2-14C] pyruvate and alpha-[1-14C]ketoglutarate to low levels. A study of the stability of pyruvate dehydrogenase in fibroblasts has shown that the activity is rapidly lost when cell membranes are broken unless homogenisation is done gently at -15 degrees C. Under these conditions broken cell preparations may be stored for up to 3 hours without loss of activity. Freezing and thawing results in unpredictable changes in pyruvate dehydrogenase activity. A quality control solution containing pyruvate dehydrogenase activity has been prepared which is stable for at least 6 months (coefficient of variation = 7.7%). Normal values for pyruvate dehydrogenase in fibroblasts range from 0.59 to 1.26 nmol . min-1 . mg-1 protein (mean = 0.98, n = 8) and pyruvate dehydrogenase deficient fibroblasts can be detected with confidence.

Acidosis↗
PubMed1994

Clinical diversity of pyruvate dehydrogenase deficiency.

Clinical features, magnetic resonance, and biochemical studies are reported in 7 children with pyruvate dehydrogenase (PDH) deficiency. These findings confirm the diverse clinical presentation of this condition, although neurological abnormalities are consistent features. Imaging results are also varied. Six of the children were investigated with proton magnetic resonance spectroscopy and lactate was demonstrated in brain in all patients. Regional variation in the lactate signal was observed in those patients in whom 2 regions were examined. Advances in molecular genetics have provided some explanations for the clinical variation in pyruvate dehydrogenase deficiency.

Brain↗
PubMed2000

Recombinant adeno-associated virus vector-based gene transfer for defects in oxidative metabolism.

Defects in oxidative metabolism may be caused by mutations either in nuclear genes or in mitochondrial DNA (mtDNA). We tested the hypothesis that recombinant adeno-associated virus (rAAV) could be used to complement mtDNA mutations. AAV vector constructs were designed to express the reporter gene encoding green fluorescent protein (GFP), fused to a targeting presequence that directed GFP to be translocated into mitochondria. These vectors mediated expression of mitochondrial-localized GFP, as indicated by fluorescence microscopy and electron microscopy, in respiring human embryonic kidney 293 cells and nonrespiring mtDNA-deficient (rho 0) cells. However, when sequences encoding hydrophobic segments of proteins normally encoded by mtDNA were inserted between the presequence and GFP, mitochondrial import failed to occur. In similar experiments, a fusion was created between pyruvate dehydrogenase (PDH) E1 alpha subunit, a nuclear-encoded mitochondrial gene with its own targeting presequence, and GFP. With this construct, expression of GFP was observed in mitochondria in vitro and in vivo. We conclude that the hydrophobicity of mtDNA-encoded proteins limits their ability to be transported from the cytoplasm. However, rAAV-based gene therapy may hold promise for gene therapy of PDH deficiency, the most common biochemically proven cause of congenital lactic acidosis.

Acidosis, Lactic↗
PubMed1994

Pyruvate dehydrogenase deficiency. Clinical presentation and molecular genetic characterization of five new patients.

Fibroblast cultures from five patients with early onset severe encephalopathy and lactic acidosis were studied for evidence of pyruvate dehydrogenase (PDH) deficiency. Three males had significantly reduced activity (0.29-0.45 nmol/mg protein/min versus normal controls 0.7-1.1 nmol/mg protein/min); two females had PDH activity within the normal range. However, as the majority of cases of PDH deficiency result from defects in the X-linked E1 alpha subunit and both females had biased patterns of X-inactivation (making it impossible to rule out the possibility that they were heterozygous for an E1 alpha gene defect) molecular genetic studies were performed. cDNA from the male patients was sequenced and mis-sense mutations found: Y243N (T-->A) in exon 7, D315A (G-->A) in exon 10 and R378H (G-->A) in exon 11. Single-strand conformation polymorphism analysis of amplified genomic DNA fragments and sequencing revealed a mis-sense mutation M282L (A-->C) in one female and a frameshift mutation caused by insertion of T (R288ins) in the other. Adding to recent descriptions of new mutations, this report emphasizes the allelic heterogeneity of the condition. The identification of mutations in females with a suggestive clinical phenotype, even when peripheral fibroblasts do not show deficient PDH activity, illustrates the importance of molecular analysis of this disease.

Base Sequence↗