Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase.

The pyruvate dehydrogenase multienzyme complex (PDHc) connects glycolysis to the tricarboxylic acid cycle by producing acetyl-CoA via the decarboxylation of pyruvate. Because of its pivotal role in glucose metabolism, this complex is closely regulated in mammals by reversible phosphorylation, the modulation of which is of interest in treating cancer, diabetes, and obesity. Mutations such as that leading to the αV138M variant in pyruvate dehydrogenase, the pyruvate-decarboxylating PDHc E1 component, can result in PDHc deficiency, an inborn error of metabolism that results in an array of symptoms such as lactic acidosis, progressive cognitive and neuromuscular deficits, and even death in infancy or childhood. Here we present an analysis of two X-ray crystal structures at 2.7-Å resolution, the first of the disease-associated human αV138M E1 variant and the second of human wildtype (WT) E1 with a bound adduct of its coenzyme thiamin diphosphate and the substrate analogue acetylphosphinate. The structures provide support for the role of regulatory loop disorder in E1 inactivation, and the αV138M variant structure also reveals that altered coenzyme binding can result in such disorder even in the absence of phosphorylation. Specifically, both E1 phosphorylation at αSer-264 and the αV138M substitution result in disordered loops that are not optimally oriented or available to efficiently bind the lipoyl domain of PDHc E2. Combined with an analysis of αV138M activity, these results underscore the general connection between regulatory loop disorder and loss of E1 catalytic efficiency.

Pyruvate dehydrogenase deficiency. / Pyruvatdehydrogenase-mangel.

Pyruvate dehydrogenase deficiency is a rare mitochondrial disease leading to energy deficiency in the cells. This particularly affects the central nervous system, resulting in a broad range of neurological symptoms.

The spectrum of pyruvate dehydrogenase complex deficiency: clinical, biochemical and genetic features in 371 patients.

CONTEXT: Pyruvate dehydrogenase complex (PDC) deficiency is a genetic mitochondrial disorder commonly associated with lactic acidosis, progressive neurological and neuromuscular degeneration and, usually, death during childhood. There has been no recent comprehensive analysis of the natural history and clinical course of this disease. OBJECTIVE: We reviewed 371 cases of PDC deficiency, published between 1970 and 2010, that involved defects in subunits E1α and E1ß and components E1, E2, E3 and the E3 binding protein of the complex. DATA SOURCES AND EXTRACTION: English language peer-reviewed publications were identified, primarily by using PubMed and Google Scholar search engines. RESULTS: Neurodevelopmental delay and hypotonia were the commonest clinical signs of PDC deficiency. Structural brain abnormalities frequently included ventriculomegaly, dysgenesis of the corpus callosum and neuroimaging findings typical of Leigh syndrome. Neither gender nor any clinical or neuroimaging feature differentiated the various biochemical etiologies of the disease. Patients who died were younger, presented clinically earlier and had higher blood lactate levels and lower residual enzyme activities than subjects who were still alive at the time of reporting. Survival bore no relationship to the underlying biochemical or genetic abnormality or to gender. CONCLUSIONS: Although the clinical spectrum of PDC deficiency is broad, the dominant clinical phenotype includes presentation during the first year of life; neurological and neuromuscular degeneration; structural lesions revealed by neuroimaging; lactic acidosis and a blood lactate:pyruvate ratio ≤20.

Reprogramming of aerobic glycolysis in non-transformed mouse liver with pyruvate dehydrogenase complex deficiency.

The Pyruvate Dehydrogenase Complex (PDC), a key enzyme in glucose metabolism, catalyzes an irreversible oxidative decarboxylation reaction of pyruvate to acetyl-CoA, linking the cytosolic glycolytic pathway to mitochondrial tricarboxylic acid cycle and oxidative phosphorylation. Earlier we reported a down-regulation of several key hepatic lipogenic enzymes and their upstream regulators in liver-specific PDC-deficient mouse (L-PDCKO model by deleting the Pdha1 gene). In this study we investigated gene expression profiles of key glycolytic enzymes and other proteins that respond to various metabolic stresses in liver from L-PDCKO mice. Transcripts of several, such as hexokinase 2, phosphoglycerate kinase 1, pyruvate kinase muscle-type 2, and lactate dehydrogenase B as well as those for the nonglycolysis-related proteins, CD-36, C/EBP homologous protein, and peroxisome proliferator-activated receptor γ, were up-regulated in L-PDCKO liver whereas hypoxia-induced factor-1α, pyruvate dehydrogenase kinase 1 and Sirtuin 1 transcripts were down-regulated. The protein levels of pyruvate kinase muscle-type 2 and lactate dehydrogenase B were increased whereas that of lactate dehydrogenase A was decreased in PDC-deficient mouse liver. Analysis of endoplasmic reticulum and oxidative stress indicators suggests that the L-PDCKO liver showed evidence of the former but not the latter. These findings indicate that (i) liver-specific PDC deficiency is sufficient to induce "aerobic glycolysis characteristic" in mouse liver, and (ii) the mechanism(s) responsible for these changes appears distinct from that which induces the Warburg effect in some cancer cells.

Structural and functional impact of clinically relevant E1α variants causing pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

Somatic mosaicism for PDHA1 mutation in a male with pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex deficiency is a clinically heterogeneous disorder. Most cases are due to mutations in an X-linked PDHA1 gene encoding the E1alpha subunit of the multienzyme complex. Females with mutations in the PDHA1 gene may be asymptomatic or have a milder phenotype as a result of skewed X-inactivation, while males are typically more severely affected. We report a case of PDHA1 mosaicism in a male patient who had a milder phenotype.

Mutational study in the PDHA1 gene of 40 patients suspected of pyruvate dehydrogenase complex deficiency.

We screened for PDHA1 mutations in 40 patients with biochemically demonstrated PDHc deficiency or strong clinical suspicion, and found changes with probable pathological significance in 20. Five patients presented new mutations: p.A169V, c.932_938del, c.1143_1144 ins24, c.1146_1159dup and c.510-30G> A, this latter is a new undescribed cause of exon 6 skipping. Another four mutations have been found, and previously reported, in our patients: p.H113D, p.P172L, p.Y243del and p.Y369Q. Eleven patients presented seven known mutations: p.R127Q, p.I166I, p.A198T, p.R263G, p.R302C, p.R378C and c.1142_1145dup. The latter three were found in more than one unrelated patient: p.R302C was detected in a heterozygous girl and a mosaic male, p.R378C in two males and finally, c.1142_1145dup in three females; only one in 20 mothers was found to be a carrier (p.R263G). Apart from those 20 patients, the only alteration detected in one girl with clear PDHc and PDH-E1 deficiency was the silent change c.396A> C (p.R132R), and other eight PDHc deficient patients carry combinations of known infrequent polymorphisms that are overrepresented among our 20 unsolved patients. The importance of these changes on PDH activity is unclear. Investigations in the other PDHc genes are in course in order to elucidate the genetic defect in the unresolved patients.

An overview of a cohort of South African patients with mitochondrial disorders.

Mitochondrial disorders are frequently encountered inherited diseases characterized by unexplained multisystem involvement with a chronic, intermittent, or progressive nature. The objective of this paper is to describe the profile of patients with mitochondrial disorders in South Africa. Patients with possible mitochondrial disorders were accessed over 10 years. Analyses for respiratory chain and pyruvate dehydrogenase complex enzymes were performed on muscle. A diagnosis of a mitochondrial disorder was accepted only if an enzyme activity was deficient. Sixty-three patients were diagnosed with a mitochondrial disorder, including 40 African, 20 Caucasian, one mixed ancestry, and two Indian patients. The most important findings were the difference between African patients and other ethnicities: respiratory chain enzyme complexes CI+III or CII+III deficiencies were found in 52.5% of African patients, being of statistical significance (p value = 0.0061). They also presented predominantly with myopathy (p value = 0.0018); the male:female ratio was 1:1.2. Twenty-five (62.5%) African patients presented with varying degrees of a myopathy accompanied by a myopathic face, high palate, and scoliosis. Fourteen of these 25 also had ptosis and/or progressive external ophthalmoplegia. No patients of other ethnicities presented with this specific myopathic phenotype. Caucasian patients (16/20) presented predominantly with central nervous system involvement. Of the South African pediatric neurology patients, Africans are more likely to present with myopathy and CII+III deficiency, and Caucasian patients are more likely to present with encephalopathy or encephalomyopathy.

Dihydrolipoamide dehydrogenase (DLD) deficiency in a Spanish patient with myopathic presentation due to a new mutation in the interface domain.

We present a 32-year-old patient who, from age 7 months, developed photophobia, left-eye ptosis and progressive muscular weakness. At age 7 years, she showed normal psychomotor development, bilateral ptosis and exercise-induced weakness with severe acidosis. Basal blood and urine lactate were normal, increasing dramatically after effort. PDHc deficiency was demonstrated in muscle and fibroblasts without detectable PDHA1 mutations. Ketogenic diet was ineffective, however thiamine gave good response although bilateral ptosis and weakness with acidosis on exercise persisted. Recently, DLD gene analysis revealed a homozygous missense mutation, c.1440 A>G (p.I480M), in the interface domain. Both parents are heterozygous and DLD activity in the patient's fibroblasts is undetectable. The five patients that have been reported with DLD-interface mutations suffered fatal deteriorations. Our patient's disease is milder, only myopathic, more similar to that due to mutation p.G229C in the NAD(+)-binding domain. Two of the five patients presented mutations (p.D479V and p.R482G) very close to the present case (p.I480M). Despite differing degrees of clinical severity, all three had minimal clues to DLD deficiency, with occasional minor increases in α-ketoglutarate and branched-chain amino acids. In the two other patients, hypertrophic cardiomyopathy was a significant feature that has been attributed to moonlighting proteolytic activity of monomeric DLD, which can degrade other mitochondrial proteins, such as frataxin. Our patient does not have cardiomyopathy, suggesting that p.I480M may not affect the DLD ability to dimerize to the same extent as p.D479V and p.R482G. Our patient, with a novel mutation in the DLD interface and mild clinical symptoms, further broadens the spectrum of this enzyme defect.

A cognitively normal PDH-deficient 18-year-old man carrying the R263G mutation in the PDHA1 gene.

Pyruvate dehydrogenase (PDH) is a crucial multienzyme system linking glycolysis to the tricarboxylic acid cycle by catalysing the decarboxylation of pyruvate to acetyl-CoA. Deficiency in pyruvate dehydrogenase is most commonly secondary to mutations in the X-linked PDHA1 gene encoding the E1 alpha subunit. There is a wide range of clinical presentations from severe neonatal lactic acidosis to chronic encephalopathy (Leigh syndrome). In recent years, a small subset of patients was recognized with less severe involvement, presenting initially only with intermittent symptoms, mainly of ataxia. Most of these patients remain stable for a number of years before developing progressive neurological deterioration around puberty at the latest. There does not appear to be a reliable correlation between genotype, phenotype, or enzyme activity. This makes counselling in a clinical setting challenging. We report a case with a previously known common mutation in PDHA1 (R263G) with an excellent outcome at 18 years of age. Previous patients with this mutation have presented with mental retardation and/or Leigh syndrome, while our patient's clinical outcome is exceptional. He is cognitively normal and has normal brain MRI. His management includes a stringent carbohydrate-free diet, as well as supplementation with thiamine, carnitine and vitamin E. This case further broadens the clinical spectrum, including now an example of a cognitively normal adult with PDH deficiency.

Four novel PDHA1 mutations in pyruvate dehydrogenase deficiency.

The pyruvate dehydrogenase (PDH) complex is a mitochondrial multienzyme that catalyses the irreversible oxidative decarboxylation of pyruvate to acetyl-CoA. We report four novel PDHA1 mutations in patients with pyruvate dehydrogenase deficiency. Analysis of PDH activity showed decreased activity in fibroblasts from all four patients, around 16-52% of mean control, similar to what has been found in previous studies. Two of the mutations were missense mutations: c.616G>A (p.Glu206Lys) and c.457A>G (p.Met153Val), one was a 3 bp in-frame deletion: c.429_431delAGG (p.Gly143del), and one was a 65 bp duplication: c.900-6_958dup65. cDNA analysis of the 65 bp duplication showed a small amount of normal transcript in addition to the transcript corresponding to the duplication. The small amount of normal transcript likely explains the survival of the patient, who was a boy. The duplication and one of the missense mutations were associated with decreased amounts of E(1)α And E(1)ß protein on western blot analysis, whereas the other two mutations were associated with normal amounts. This study adds four novel mutations to the around 90 reported mutations in PDHA1 (HGMD PDHA1 mutation database). The phenotypes of patients with PDH deficiency have been divided into three groups: a neonatal form with severe lactic acidosis, a form observed only in males and characterized by episodes of ataxia with relapses associated with hyperlactataemia, and an infantile form with hypotonia, lethargy, onset of seizures or dystonia, psychomotor retardation, in some cases Leigh-like lesions and mild to moderate hyperlactataemia. The four patients reported here all belong to the latter group, which is the largest.

A putative exonic splicing enhancer in exon 7 of the PDHA1 gene affects splicing of adjacent exons.

A nonsense mutation (c.729C>A, Y243X) in exon 7 of the PDHA1 gene in a patient with pyruvate dehydrogenase deficiency results in aberrant splicing of the primary transcript with production of stable mRNAs which lack either both exons 6 and 7 or exon 7 alone. Transfection and expression of genomic constructs covering exons 5 to 8 of the mutant PDHA1 gene reproduced this aberrant splicing in vitro. The same pattern of abnormal splicing was found when a silent mutation was introduced at the same position. Both the nonsense and silent mutations alter a strong consensus site for the binding of SRp40, suggesting that they may interfere with an exonic splicing enhancer in exon 7 of the gene. However, this appears to affect splicing of not only exon 7, but also the adjacent upstream exon. The splice acceptor site of intron 5 has weak homology to the consensus sequence and this may contribute to the combined splicing defect.

Characterization of a missense mutation at histidine-44 in a pyruvate dehydrogenase-deficient patient.

Genetic defects in pyruvate dehydrogenase complex (PDC) cause lactic acidosis, neurological deficits, and often early death. Most mutations of PDC are localized in the alpha subunit of the pyruvate dehydrogenase (E1) component. We have kinetically characterized a patient's missense mutation alphaH44R in E1alpha by creating and purifying three recombinant human E1s (alphaH44R, alphaH44Q, and alphaH44A). Substitutions at histidine-15 resulted in decreased V(max) values (6% alphaH44R; 30% alphaH44Q; 90% alphaH44A) while increasing K(m) values for thiamine pyrophosphate (TPP) compared to wild-type (alphaH44R, 3-fold; alphaH44Q, 7-fold; alphaH44A, 10-fold). This suggests that the volume of the residue at site 15 is important for TPP binding and substitution by a residue with a longer side chain disrupts the active site more than the TPP binding site. The rates of phosphorylation and dephosphorylation of alphaH44R E1 by E1-kinase and phospho-E1 phosphatase, respectively, were similar to that of the wild-type E1 protein. These results provide a biochemical basis for altered E1 function in the alphaH44R E1 patient.

Thiamine-responsive pyruvate dehydrogenase deficiency in two patients caused by a point mutation (F205L and L216F) within the thiamine pyrophosphate binding region.

The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit. We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.

Deficiency of pyruvate dehydrogenase activity in pancreatic islets of diabetic GK rats.

We investigated the role of islet pyruvate dehydrogenase (PDH) enzyme activity and fatty acid oxidation in the impaired insulin secretion in spontaneously diabetic GK rats. Blood glucose levels were elevated in 2- to 3-month-old GK rats (8.7 +/- 0.5 vs. 6.5 +/- 0.3 mM in control Wistar rats; P < 0.01), whereas serum insulin levels were comparable to those in control rats. Insulin and DNA contents were similar in freshly isolated islets from GK and control rats, whereas insulin responses to 27 mM glucose from GK islets were reduced by 52%. The effect of acetate or pyruvate on insulin responses evoked by succinate monomethylester (SAM) were compared to indirectly assess deficient generation of acetyl-coenzyme A from pyruvate. Acetate potentiated SAM-induced insulin secretion similarly in GK and control islets, whereas 10 mM pyruvate (which supplies acetyl-coenzyme A through PDH enzyme activity) failed to normally potentiate insulin secretion in GK islets (92% of SAM-induced response in GK vs. 154% in control islets). The PDH activity (active form) was decreased in GK islets by 35% (P < 0.001). The proportion of active form PDH to total PDH activity was reduced in GK islets (56% vs. 71% in control islets; P < 0.01). The activity of PDH kinase (which inactivates PDH by phosphorylation) was increased in GK islets, the rate of ATP-dependent inactivation of PDH was -0.29 +/- 0.02 vs. -0.19 +/- 0.02/min in control islets (P < 0.05). Culturing GK islets for 48 h at 5.5 mM glucose failed to correct the impaired insulin response to glucose and the decreased PDH activity. Serum FFA levels and islet triglyceride contents did not differ between GK and control rats. Etomoxir (1.0 and 10 microM), a carnitine palmitoyl transferase I inhibitor, failed to enhance glucose-induced insulin release in GK islets. The following conclusions were reached: 1) a kinase-mediated decrease in PDH activity in islets of GK rats may in part account for the decreased ratio of oxidized to utilized glucose and impaired insulin release in these islets; and 2) impaired insulin release in the GK rats is not linked to an inhibitory influence of islet fatty acid oxidation.

Dichloroacetate stabilizes the mutant E1alpha subunit in pyruvate dehydrogenase deficiency.

OBJECTIVE: To determine whether dichloroacetate (DCA) treatment can increase pyruvate dehydrogenase (PDH) activity in PDH-deficient cell lines harboring pathogenic mutations in the PDH E1alpha gene. BACKGROUND: PDH deficiency is a nuclear-encoded mitochondrial disorder and a major recognized cause of neonatal encephalomyopathies associated with primary lactic acidosis. Over the last decade, DCA has been used therapeutically, but it has not been clear which patients might benefit. Recent studies suggest that chronic DCA treatment may act by increasing the stability of mutant E1alpha polypeptide. The relative effects of DCA treatment on PDH-deficient cell lines with E1alpha mutations primarily affecting polypeptide stability or catalytic activity were determined and the mechanism of enhancement of residual PDH activity explored. METHODS: The effect of chronic 5-day DCA treatment on PDH activity was assessed in PDH-deficient cell lines containing the R378H, R141Q, K387(FS), and R302C E1alpha mutations. PDH subunit turnover and steady-state E1alpha levels before and after DCA treatment were measured in the R378H mutant line. RESULTS: Chronic DCA treatment resulted in 25% (p = 0.0434), 31% (p = 0.0014) increases in PDH activity in the K387(FS) and R378H cell lines, both of which are associated with decreased mutant polypeptide stability. In the R378H mutant cell line, chronic DCA treatment increased steady-state E1alpha levels and slowed the rate of E1alpha turnover twofold. In contrast, PDH activity did not change in the chronically DCA-treated R302C mutant line, in which the mutant polypeptide has normal stability and reduced catalytic activity. CONCLUSIONS: Chronic DCA treatment can increase PDH activity in PDH-deficient cell lines harboring mutations that affect E1alpha stability, suggesting a biochemical criterion by which DCA-responsive patients might be selected.

Mechanisms of expression of pyruvate dehydrogenase deficiency caused by an E1alpha subunit mutation.

OBJECTIVE: To characterize the biochemical mechanisms of expression of the pyruvate dehydrogenase (PDH) E1alpha subunit exon 10 R302C missense mutation. BACKGROUND: Mutations in the X-linked E1alpha subunit gene are responsible for most cases of PDH deficiency, an important cause of neurodevelopmental defects and neurodegeneration with primary lactic acidemia. Although the disease shows extreme allelic heterogeneity, the R302C mutation has been defined in several unrelated cases. METHODS: Cell lines expressing selectively either the mutant or wild-type E1alpha alleles against identical genetic backgrounds were generated from the fibroblasts of a female heterozygous for the R302C mutation. Enzyme activity, mRNA, polypeptide expression, and turnover were studied in each. RESULTS: The residual PDH activity was below measurable levels in the cell line (B5) expressing only the mutant allele and normal in the wild-type polypeptide expressing (A10) cell line, confirming that the R302C mutation alone is sufficient to cause a severe PDH deficiency. The mutant polypeptide was less stable than the wild-type polypeptide, but the steady-state level of the mutant E1alpha protein was reduced only two- to threefold. CONCLUSIONS: The primary mechanism of expression of the R302C mutation must be limitation of catalytic efficiency. We speculate that catalysis may be inhibited in the mutant polypeptide because conformational changes are induced near serine 300, a residue that is particularly important as a regulatory phosphorylation site in the wild-type polypeptide.

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.

Detection of pyruvate dehydrogenase E1 alpha-subunit deficiencies in females by immunohistochemical demonstration of mosaicism in cultured fibroblasts.

Deficiency of the E1 alpha-subunit of the pyruvate dehydrogenase (PDH) complex is an X-linked inborn error of metabolism and one of the major causes of lactic acidosis in children. Although most heterozygous females manifest symptoms of the disease, it is often difficult to establish the diagnosis as results based on measurement of total PDH activity, and E1 alpha-immunoreactive protein in patient fibroblasts may be ambiguous because of the variability in the pattern of X chromosome inactivation. We report the development of a set of monoclonal antibodies (MAbs) specific to four subunits of the PDH complex that can be used for detection of PDH E1 alpha deficiency. We also show that anti-E1 alpha and anti-E2 MAbs, when used in immunocytochemical analysis, can detect mosaicism in cell cultures from female patients in which as few as 2-5% of cells express the deficiency. This immunocytochemical approach, which is fast, reliable, and quantitative, will be particularly useful in identifying females with PDH E1 alpha-subunit deficiency as a precursor to mutation analysis.

Leigh syndrome: pyruvate dehydrogenase defect. A case with peripheral neuropathy.

Pyruvate dehydrogenase deficiency is one of the most common causes of encephalopathy associated with lactic acidosis and is known to account for congenital lactic acidosis, recurrent ataxia, and infantile Leigh syndrome. Hitherto, however, peripheral neuropathy has not been regarded as a presenting symptom of pyruvate dehydrogenase deficiency. Here, we report on a boy who presented peripheral neuropathy with severe limb hypotonia, absent deep-tendon reflexes, and reduced motor nerve conduction velocities at 8 months of age. Persistent hyperpyruvicemia with normal lactate/pyruvate molar ratios in plasma were highly suggestive of a pyruvate dehydrogenase deficiency, and the determination of pyruvate dehydrogenase activity in circulating lymphocytes led to the diagnosis of pyruvate decarboxylase (PDH-E1) deficiency in the proband. Based on this observation, we suggest that pyruvate dehydrogenase deficiency should be considered in the diagnosis of peripheral neuropathy in infancy, especially when associated with persistent hyperpyruvicemia, normal lactate/pyruvate molar ratios in plasma, and recurrent episodes of drowsiness and hypotonia of unknown origin.