Mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) in patients with a pyruvate dehydrogenase complex deficiency

Defects in the pyruvate dehydrogenase (PDH) complex are an important cause of primary lactic acidosis, a frequent manifestation of metabolic disease in children. Clinical symptoms can vary considerably in patients with PDH complex deficiencies, and almost equal numbers of affected males and females have been identified, suggesting an autosomal recessive mode of inheritance of the disease. However, the great majority of PDH complex deficiencies result from mutations in the X-linked pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1). The major factors that contribute to the clinical variation in E1alpha deficiency and its resemblance to a recessive disease are developmental lethality in some males with severe mutations and the pattern of X-inactivation in females. To date, 37 different missense/nonsense and 39 different insertion/deletion mutations have been identified in the E1alpha subunit gene of 130 patients (61 females and 69 males) from 123 unrelated families. Insertion/deletion mutations occur preferentially in exons 10 and 11, while missense/nonsense mutations are found in all exons. In males, the majority of missense/nonsense mutations are found in exons 3, 7, 8 and 11, and three recurrent mutations at codons R72, R263 and R378 account for half of these patients with missense/nonsense mutations (25 of 50). A significantly lower number of females is found with missense/nonsense mutations (25). However, 36 females out of 55 affected patients have insertion/deletion mutations. The total number of female and male patients is thus almost the same, although a difference in the distribution of the type of mutations is evident between both sexes. In many families, the parents of the affected patients were studied for the presence of the PDHA1 mutation. The mutation was never present in the somatic cells of the father; in 63 mothers studied, 16 were carriers (25%). In four families, the origin of the new mutation was determined to be twice paternal and twice maternal.     

Mutations and polymorphisms in the pyruvate dehydrogenase E1 alpha gene.

We present an update on mutations and polymorphisms in the human X chromosome located pyruvate dehydrogenase E1 alpha gene. A total of 20 different mutations are tabulated. The mutations include deletions, insertions, and point mutations. Certain sequences seem particularly prone to mutation. Most of the mutations are found in exons 10 and 11. Furthermore, four of the mutations are seen in unrelated patients. Little is known about how the mutations affect the structure or function of the pyruvate dehydrogenase complex.     

Mutations in the X-linked E1{alpha} subunit of pyruvate dehydrogenase leading to deficiency of the pyruvate dehydrogenase complex

Human PDH complex deficiency is an extremely heterogeneous diseasein its presentation and clinical course. In an investigationat the level of the gene into ten cases of PDH complex (E₁)deficiency, we found that all had mutations in the coding sequenceof the X-linked E₁     

Deficiency of pyruvate dehydrogenase caused by novel and known mutations in the E1alpha subunit

Pyruvate dehydrogenase (PDH)-complex deficiency (OMIM 312170) is a clinically heterogeneous disorder, with phenotypes ranging from fatal lactic acidosis (LA) in the newborn to chronic neurological dysfunction. To date, over 80 different mutations have been identified in the PDHA1 gene in patients with PDH complex deficiency, which are thus thought to contribute to the PDH deficient phenotype. We have identified 14 additional patients with total PDH complex deficiency, all of whom were found to contain mutations within the PDHA1 gene (E(1)alpha subunit). The mutations include both missense mutations and duplications. Eight of these patients had novel mutations, and the remaining had mutations that have been identified previously in PDH complex deficient patients, with residual fibroblast activity ranging from 2.4 to 69% of control values. The nature of these mutations illustrates the variability in phenotype for a given gene defect, with intermittent ataxia being the mildest presentation, Leigh syndrome being the most common and severe neonatal LA the most severe.     

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.     

Mutations of the E1beta subunit gene (PDHB) in four families with pyruvate dehydrogenase deficiency

Pyruvate dehydrogenase complex (PDC) deficiencies are a major cause of primary lactic acidosis. Most cases result from mutations of the gene for the pyruvate dehydrogenase E1α subunit (PDHA1), with fewer cases resulting from mutations in genes for E3, E3-binding protein, E2, and the E1β subunit (PDHB). We have found four cases of PDHB mutations among 83 analyzed cases of PDC deficiency. In this series, PDHB mutations were found to be about 10% as frequent as PDHA1 mutations. All cases were diagnosed by low PDC activity, with normal E2 and E3 activities. These included a 6.5-year-old male (consanguineous, homozygous R36C); a neonatal female who died soon after birth, (compound heterozygous C306R/D319V), a 26-year-old female (heterozygous I142M/W165S), and a 13 month old female (consanguineous, homozygous Y132C) who is a sibling of a previously published case. Their ethnic background is diverse (Caucasian, Arab, and African American descent). All cases had lactic acidosis and developmental delay. Three cases had agenesis of the corpus callosum, seizures, and hypotonia; one died within the first year of life. These clinical findings are similar to those of PDHA1 deficiency, except that ataxia was more frequent in PDHA1 cases and consanguinity was found only in PDHB families. PDC activity in lymphocytes from six parents is normal, who all are heterozygous carriers for the respective mutations. Immunoreactivity of E1β was markedly reduced in one case and showed a slightly larger form of E1β in one case. Computer analysis predicts that: R36C affects the interaction of several amino acids resulting in conformational change, C306R affects interaction of the two β subunits, D319 is in the interface of E1 and E2, I142M affects conformation around a K ion affecting stability of the β subunit, W165S affects hydrophobic interaction between the β subunits, and Y132C affects interaction between the β subunits. All of these residues are conserved in E1β across species, and Y132 is also conserved in other TPP-requiring enzymes. These observations support the conclusion that these are pathogenic mutations.     

AAV3-mediated transfer and expression of the pyruvate dehydrogenase E1 alpha subunit gene causes metabolic remodeling and apoptosis of human liver cancer cells

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as “aerobic glycolysis”, or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1–10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.     

Characterization of cDNA clones for the alpha subunit of pyruvate dehydrogenase from Ascaris suum.

The pyruvate dehydrogenase complex of the adult parasitic nematode Ascaris suum functions in the reducing environment present in their anaerobic mitochondria. These organelles use fumarate and enoyl CoAs as terminal electron acceptors instead of oxygen. A lambda gt11 cDNA library was constructed from RNA isolated from adult ascarid muscle. Partial clones for the pyruvate dehydrogenase alpha subunit were isolated by screening the lambda gt11 library with a specific antiserum. Full-length clones (type I) were identified in a cDNA library prepared from RNA isolated from early embryos. During the hybridization screening, a second type of cDNA clone (type II) was identified. The nucleotide sequences of both clones are presented. The predicted amino acid sequences of the mature proteins are 91% identical to one another and about 55% identical to the predicted sequences of the alpha subunit of human pyruvate dehydrogenase. Northern blots were used to examine the expression of both mRNAs in various larval stages and in tissues of the adult. Type I sequences are found mainly in adult muscle. Type II sequences are abundant in third-stage larvae as well as in adult muscle.     

Arginine 302 mutations in the pyruvate dehydrogenase E1α subunit gene: Identification of further patients and in vitro demonstration of pathogenicity

Three further patients with mutations in the codon for arginine 302 of the E1α subunit of the pyruvate dehydrogenase complex have been identified. Mutations in this codon have now been found in nine patients with pyruvate dehydrogenase deficiency in seven unrelated families, in sharp contrast to the great majority of other PDH E1α mutations which have been described in single individuals only. Because of the relatively high frequency of this mutation and because very few PDH E1α mutations have been demonstrated to be causative, we have established a system for analysing the consequences of defined mutations using transfection of normal and mutant PDH E1α cDNA into transformed human fibroblasts which have no endogenous E1α mRNA or protein. Using this test system, we have demonstrated that the R302C mutation results in the production of PDH E1α protein which is devoid of enzymic activity. Hum Mutat 12:114–121, 1998. © 1998 Wiley-Liss, Inc.     

Transfection screening for defects in the PCCA and PCCB genes encoding propionyl-CoA carboxylase subunits

Propionic acidemia can result from mutations in the PCCA or PCCB genes encoding the alpha and beta subunits, respectively, of propionyl-CoA carboxylase. We have developed a method based on complementation of the enzyme defect using a lipid-mediated transient transfection of the normal human PCCA or PCCB cDNA into primary fibroblasts. We demonstrate the reliability of this method for identification of the defective PCC gene in order to unequivocally approach the mutational analysis in the corresponding PCCA and PCCB genes.     

The pyruvate dehydrogenase complex as a target for gene therapy

Here we review the rationale for considering the pyruvate dehydrogenase multienzyme complex (PDC) as a target for gene therapy for defects in mitochondrial energetics. PDC is entirely nuclear encoded and is situated in the mitochondrial inner membrane. The complex catalyzes the rate-determining step in aerobic carbohydrate metabolism and plays a critical role in the efficient conversion of substrate fuel into energy by cells. PDC activity is regulated in large part by reversible phosphorylation (inactivation) of its E1alpha subunit. Congenital defects in PDC are usually due to mutations in E1alpha and are typified by lactic acidosis, neurodegeneration and early death. Acquired deficiency in PDC has been implicated in the etiopathology of several other metabolic or neurodegenerative disorders. Recently, a vector using recombinant adeno-associated virus (rAAV) that contained a fusion protein of full-length E1alpha and the reporter gene green fluorescent protein was used to deliver wild type E1alpha into mitochondria after injection of the construct in vivo into the central nervous system of rats and in vitro into human cells. Transduction of cultured fibroblasts from a male patient with E1alpha deficiency led to partial restoration of PDC activity, as determined by decarboxylation of 14C-pyruvate. These data indicate that at least partial correction of PDC defects may be feasible by gene transfer. Furthermore, the combination of AAV-mediated delivery of E1alpha with pharmacologic activation (dephosphorylation) of the wild type enzyme subunit may provide an optimal therapeutic strategy for patients with acquired or congenital deficiencies in mitochondrial energy metabolism.     

Mutations in mitochondrial NADH dehydrogenase subunit 1 (mtND1) gene in colorectal carcinoma

Mitochondrial Subunit ND1 (mtND1) gene is involved in the first step of the electron transport chain of oxidative phosphorylation (OXPHOS). Alteration of the electron transport components by mutations in mtDNA may compromise the normal electron flow. This could lead to an increase of bifurcation and generation of superoxidase radicals and increase oxidative stress in various types of cancer cells. Genomic DNA was extracted from thirty matched primary colorectal tumour tissues and matching non-tumour tissues. Blood samples were obtained from twenty-five normal people. The mtNDI coding region was amplified by step-down PCR. The purified products were then subjected to direct sequencing and subsequently, the DNA sequences obtained were compared with the revised Cambridge Reference Sequence (rCRS) and MITOMAP. From the analysis, the mtND1 gene showed 11 (45.8%) different mutations and also 13 (54.2%) polymorphisms. The heteroplasmic mutation A4123A/G (I273I/V) might have a pathogenic significance as it fulfills various pathogenic criteria. Three mutations, T3394C (Y30H), A3434G (Y43C) and C3497T (A64V) which occur in a highly conserved region were likely to alter the structure and function of the ND1 protein. We suggest that these mutations, and in combination with the polymorphic variance in mtDNA, may cause slight changes that generate subtly higher levels of toxic reactive oxygen species (ROS).     

筛选介导化学合成siRNA转染原代肝癌细胞的最佳试剂

背景：理想的细胞转染试剂应具有高效安全的特点。 目的：筛选能够高效介导化学合成siRNA转染原代肝癌细胞的最佳转染试剂。 方法：应用Lipofectamine RNAiMAX,Lipofectamine 2000和DharmaFECT1转染试剂介导FAM-siRNA和多药耐药基因MDR1siRNA转染原代肝癌细胞,分别于转染6和48 h后应用流式细胞仪和实时荧光定量PCR检测转染效率,然后用MTT法检测3种转染试剂处理原代肝癌细胞24 h后的细胞毒性。 结果与结论：对于Lipofectamine RNAiMAX,Lipofectamine 2000和DharmaFECT1转染试剂介导的FAM-siRNA和MDR1 siRNA转染,流式细胞仪和实时荧光定量PCR仪检测出RNAiMAX转染效率最高(P < 0.05),分别为70.3%和71.5%。MTT法检测结果表明RNAiMAX对原代肝癌细胞没有表现出细胞毒性。结果提示,由于RNAiMAX介导的FAM-siRNA和MDR1 siRNA转染的效率最高,并且对细胞的毒性最小,所以RNAiMAX是最适合介导化学合成siRNA转染原代肝癌细胞的转染试剂。     

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

Quintana E, Gort L, Busquets C, Navarro‐Sastre A, Lissens W, Moliner S, Lluch M, Vilaseca MA, De Meirleir L, Ribes A, Briones P, PDH Working Group. 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.     

Oral tolerance and pyruvate dehydrogenase in patients with primary biliary cirrhosis

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the immunological destruction of intralobular bile ducts and serum anti-mitochondrial antibodies (AMA). Based upon previous work of oral tolerance and autoimmunity, we hypothesized that feeding the mitochondrial autoantigens of PBC would alter the clinical course and the level of antimitochondrial antibodies. The bovine pyruvate dehydrogenase complex (PDC) was purified and 5 mg fed in gelatin capsules to 6 patients with early stage PBC for 6 months. Antimitochondrial antibodies and liver biochemistries were measured at every 3 months for 12 months. The clinical trial was completed for all patients except for 1 who showed deterioration of pre-existing skin rash during treatment, which disappeared within 2 weeks after treatment was discontinued. However, after 1 year, neither the titers of AMAs nor liver biochemistries were significantly changed by this treatment. This is the first trial to test the efficacy of oral tolerance induction in PBC. However, the data, which limited in scope, did not demonstrate efficacy and further highlights the difficulties in showing continuing evidence of tolerance induction in autoimmunity.     

A novel mutation in the dihydrolipoamide dehydrogenase E3 subunit gene (DLD) resulting in an atypical form of alpha-ketoglutarate dehydrogenase deficiency

The alpha-ketoglutarate dehydrogenase complex (KGDC) catalyses the decarboxylation of alpha-ketoglutarate into succinyl-coenzyme A in the Krebs cycle. This enzymatic complex is made up of three subunits (E1, encoded by PDHA1; E2, encoded by DLST; and E3, encoded by DLD). The E3 subunit is common to two other enzymatic complexes, namely pyruvate dehydrogenase complex (PDC) and branched-chain ketoacid dehydrogenase complex (BCKDC). KGDC deficiency is a rare autosomal recessive disorder, most often presenting with severe encephalopathy and hyperlactatemia with neonatal onset. We found a KGDC deficiency in cultured skin fibroblasts from three siblings born to consanguinous parents. E3 subunit activity was shown to be deficient (20% of control values), despite the absence of usual clinical clues to E3 deficiency, i.e. accumulation of pyruvate and branched-chain amino acids in plasma and branched-chain alpha-ketoacids in urine. RT-PCR of E3 mRNA from the three patients, followed by sequencing, revealed an homozygous c.1444A>G substitution located in E3 exon 13, predictive of a p.R482G (or R447G in the processed gene product) substitution in a highly conserved domain of the protein. Only eleven E3 mutations have been reported so far. The only other case of E3 deficiency without clinical or biochemical evidences of PDC and BCKDC deficiencies has been ascribed to a c.1436A>T (p.D479V; or D444V in the processed gene product) mutation, very close to the mutation reported herein. Since c.1444A>G (p.R482G; or R447G in the processed gene product) and c.1436A>T (p.D479V; or D444V in the processed gene product) lie within the interface domain of E3 with E2 (KGDC and BCKDC) or the E3-binding protein (PDC), our data suggest that interaction of E3 with these other subunits differs in some extent among KGDC, PDC, and BCKDC.