PDHA1 Research Articles

A putative exonic splicing enhancer in exon 7 of thePDHA1 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.

Pyruvate Dehydrogenase Complex Deficiency Caused by Ubiquitination and Proteasome-mediated Degradation of the E1β Subunit

Congenital deficiencies of the human pyruvate dehydrogenase (PDH) complex are considered to be due to loss of function mutations in one of the component enzymes. Here we describe a case of PDH deficiency associated with the PDH E1beta subunit (PDHB) gene. The clinical phenotype of the patient was consistent with reported cases of PDH deficiency. Cultured skin fibroblasts demonstrated a 55% reduction in PDH activity and markedly decreased immunoreactivity for PDHB protein, compared with healthy controls. Surprisingly, nucleotide sequence analyses of cDNAs corresponding to the patient PDH E1alpha (PDHA1) and PDHB genes revealed no pathological mutations. Moreover, the relative expression level of PDHB mRNA and the rates of transcription and translation of the PDHB gene were normal. However, PDC activity could be restored in cells from this patient following treatment with MG132, a specific proteasome inhibitor, and normal levels of E1beta could be detected in MG132-treated cells. Similar results were obtained following treatment with Tyr-phostin 23 (Tyr23), a specific inhibitor of epidermal growth factor receptor-protein-tyrosine kinase (EGFR-PTK), which also restored E1beta protein levels to those in cells from healthy subjects or from patients with PDHA1 deficiency. The index patient's cells contained a high basal level of EGFR-PTK activity that correlated with the high level of ubiquitination of cellular proteins, although the total EGFR protein levels were similar to those in cells from Elalpha-deficient subjects and healthy subjects. These data indicate that PDH deficiency in our patient involves a post-translational modification in which EGFR-PTK-mediated tyrosine phosphorylation of the E1beta protein leads to enhanced ubiquitination followed by proteasome-mediated degradation. They also provide a novel mechanism accounting for congenital deficiency of the PDH complex and perhaps other inborn errors of metabolism.

Two silent substitutions in the PDHA1 gene cause exon 5 skipping by disruption of a putative exonic splicing enhancer

Background Synonymous mutations within exons may cause aberrant splicing by disrupting exonic splicing enhancer (ESE) motifs in the vicinity of non consensus splice sites. Mutational analysis of PDHA1 revealed only one silent single nucleotide substitution in exon 5 in two unrelated boys and a girl (c.483C>T and c.498C>T variants, respectively). For both patients, pyruvate dehydrogenase complex activity was low and the immunoreactive E1alpha protein was defective in cultured fibroblasts. Methods and results One of the boys was a somatic mosaic for the c.483C>T variant, as shown by the variable ratio of mutant to normal alleles in fibroblast, lymphocyte and single hair root DNA. Transcript analysis in fibroblasts from the three patients revealed the presence of both normal and truncated cDNAs, with the splicing out of exon 5 predicted to result in a frame shift and premature termination (p.Arg141AlafsX11). The treatment of fibroblasts with emetine before harvesting to prevent nonsense mRNA-mediated decay increased the amount of mutant mRNA. In silico analysis revealed that each variant disrupted a putative SRp55 binding site and that the intron 5 donor splice site (5′ss) contained a weak splicing signal. Transient transfection of COS-7 or Hela cells with hybrid minigene constructs containing wild-type or mutant PDHA1 exon 5, followed by RT-PCR demonstrated that each variant resulted in the incomplete inclusion of PDHA1 exon 5, and that this defect was corrected following the restoration of a perfect consensus sequence for the 5′ splice site by site-directed mutagenesis. Conclusion These two synonymous mutations expand the spectrum of rare PDHA1 splicing mutations, all of which are located in non canonical splice sites.

Down-regulation of expression of rat pyruvate dehydrogenase E1α gene by self-complementary adeno-associated virus-mediated small interfering RNA delivery

Mutations in the E1α subunit gene (PDHA1) of the pyruvate dehydrogenase complex (PDC) are common causes of congenital lactic acidosis. An animal model of E1α deficiency could provide insight into the pathological consequences of mutations and serve to test potential therapies. Small interfering RNAs (siRNAs) were designed to cleave the messenger RNA (mRNA) of the E1α subunit and were tested in vitro to assess the feasibility of producing a gene knockdown in rats. HEK 293 cells were co-transfected with a rat PDHA1 expression vector and eight naked siRNAs that specifically targeted rat E1α mRNA. Quantitative PCR (qPCR) analyses showed that four siRNAs reduced rat PDHA1 RNA levels up to 85% by 24 h and up to 65% by 56 h, compared to negative and positive controls. Since oligonucleotide-mediated siRNA delivery provided only transient suppression, we next selected two siRNA candidates and generated self-complementary, double-stranded adeno-associated virus (scAAV) vectors (serotypes 2 and 5) expressing a rat short hairpin siRNA expression cassette (scAAVsi-PDHA1). Rat lung fibroblast (RLF) cultures were infected with scAAVsi-PDHA1 vectors. The RLF PDHA1 mRNA level was reduced 53–80% 72 h after infection and 54–70% 10 days after infection in RLF cultures. The expression of E1α and the specific activity of pyruvate dehydrogenase were also decreased at 10 days after infection in RLF cultures. Thus, scAAV siRNA-mediated knockdown of PDHA1 gene expression provides a strategy that may be applied to create a useful animal model of PDC deficiency.

Females with PDHA1 gene mutations: A diagnostic challenge

Biochemical analysis was performed in muscle tissue and in fibroblasts of four unrelated females consecutively diagnosed with a ‘de novo’ point mutation in the PDHA1 gene. Pyruvate dehydrogenase E1 subunit deficiency was confirmed in the muscle sample of all patients, however, in three out of four cases the activity of the pyruvate dehydrogenase complex in fibroblasts showed a normal activity. A skewed inactivation was confirmed of the maternal X chromosome in fibroblasts in all children. Due to the possibility of a skewed X inactivation pattern enzyme measurements in fibroblasts are not always reliable for the diagnosis of a PDHc defect in females. Based on the overlapping features of PDHc deficiency with those of the disorders of the oxidative phosphorylation we suggest performing a fresh muscle biopsy for detailed biochemical analysis in females with a suspected pyruvate dehydrogenase deficiency, followed by molecular genetic analysis of the PDHA1 gene.

A Korean Female Patient with Thiamine-responsive Pyruvate Dehydrogenase Complex Deficiency Due to a Novel Point Mutation (Y161C)in the PDHA1 Gene

Pyruvate dehydrogenase complex (PDHC) deficiency is mostly due to mutations in the X-linked E1α subunit gene (PDHA1). Some of the patients with PDHC deficiency showed clinical improvements with thiamine treatment. We report the results of biochemical and molecular analysis in a female patient with lactic acidemia. The PDHC activity was assayed at different concentrations of thiamine pyrophosphate (TPP). The PDHC activity showed null activity at low TPP concentration (1×10-3 mM), but significantly increased at a high TPP concentration (1 mM). Sequencing analysis of PDHA1 gene of the patient revealed a substitution of cysteine for tyrosine at position 161 (Y161C). Thiamine treatment resulted in reduction of the patient's serum lactate concentration and dramatic clinical improvement. Biochemical, molecular, and clinical data suggest that this patient has a thiamine-responsive PDHC deficiency due to a novel mutation, Y161C. Therefore, to detect the thiamine responsiveness it is necessary to measure activities of PDHC not only at high but also at low concentration of TPP.

First characterization of a large deletion of the PDHA1 gene

Pyruvate dehydrogenase complex (PDC) deficiency is one of the major recognized causes of congenital lactic acidosis. The most common form is due to PDHA1 gene (Xp22.12) defects. Here, we report the case of a Polynesian girl presenting with delayed neurological development, cortical atrophy, and posterior corpus callosum agenesis. Elevated lactate and pyruvate levels in blood and cerebrospinal fluid suggested PDC deficiency. However, PDC activity was within the normal range in lymphocytes and the direct sequencing of the 11 exons and intron–exon junctions of the PDHA1 gene did not show any changes. Long-range PCR amplification of the whole gene (16 kb) from blood DNA revealed a heterozygous deletion of ≈4.2 kb. Fine mapping of the deletion breakpoint was achieved using purified long-range PCR products for restriction enzyme analysis and direct sequencing. The deletion removed a 4227 bp region covering part of intron 5 to part of intron 9 [g.10145_14371del4227]. The deletion breakpoint contained a short direct repeat (GTAG), which may be derived either from the upstream or the downstream homologous sequence. The presence of a GAG triplet and inverted repeats in the vicinity of the deletion suggest replication slippage at a polymerase α arrest site. This is the first time that a large intragenic deletion of the PDHA1 gene has been characterized.

Pyruvate dehydrogenase deficiency presenting as dystonia in childhood

Two individuals with pyruvate dehydrogenase (PDH) deficiency due to missense mutations in the gene for the E1α subunit (PDHA1) presented during childhood with dystonia. The first patient, a male, presented at age 4 years with dystonia affecting the lower limbs, which responded to treatment with combined carbidopa and levodopa. The second patient, a female, was first investigated at age 6 years because of a dystonic gait disorder. In both patients, the main clue to the biochemical diagnosis was a raised concentration of lactate in the cerebrospinal fluid. PDH activity was significantly reduced in cultured fibroblasts in both cases. Dystonia is a previously unrecognized major manifestation of PDH deficiency and is of particular interest as the mutations in the PDHA1 gene in these patients have both been identified previously in individuals with typical presentations of the condition.

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.

Inactivation of the Murine Pyruvate Dehydrogenase (Pdha1) Gene and Its Effect on Early Embryonic Development

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the α subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1flox8 allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1flox8 females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1Δex8. The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1Δex8 allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1Δex8 allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.

Determination of X-Chromosome Inactivation Status Using X-Linked Expressed Polymorphisms Identified by Database Searching

The large number of redundant sequences available in nucleotide databases provides a resource for the identification of polymorphisms. Expressed polymorphisms in X-linked genes can be used to determine the inactivation status of the genes, and polymorphisms in genes that are subject to inactivation can then be used as tools to examine X-chromosome inactivation status in heterozygous females. In this study, we have identified six new X-linked single-nucleotide polymorphisms and determined the inactivation status of these genes by examination of expression patterns in female cells previously demonstrated to have skewed inactivation, as well as by analysis of somatic cell hybrids retaining the inactive human X chromosome. Expression was seen from both alleles in females heterozygous for the RPS4X gene, confirming the previously reported expression from the inactive X chromosome. Expression of only a single allele was seen in females heterozygous for polymorphisms in the BGN, TM4SF2, ATP6S1, VBP1, and PDHA1 genes, suggesting that these genes are subject to X-chromosome inactivation.

X chromosome evidence for ancient human histories.

Diverse African and non-African samples of the X-linked PDHA1 (pyruvate dehydrogenase E1 alpha subunit) locus revealed a fixed DNA sequence difference between the two sample groups. The age of onset of population subdivision appears to be about 200 thousand years ago. This predates the earliest modern human fossils, suggesting the transformation to modern humans occurred in a subdivided population. The base of the PDHA1 gene tree is relatively ancient, with an estimated age of 1.86 million years, a late Pliocene time associated with early species of Homo. PDHA1 revealed very low variation among non-Africans, but in other respects the data are consistent with reports from other X-linked and autosomal haplotype data sets. Like these other genes, but in conflict with microsatellite and mitochondrial data, PDHA1 does not show evidence of human population expansion.

Nance-Horan syndrome: linkage analysis in 4 families refines localization in Xp22.31-p22.13 region.

Nance-Horan syndrome (NHS) is an X-linked disease characterized by severe congenital cataract with microcornea, distinctive dental findings, evocative facial features and mental impairment in some cases. Previous linkage studies have placed the NHS gene in a large region from DXS143 (Xp22.31) to DXS451 (Xp22.13). To refine this localization further, we have performed linkage analysis in four families. As the maximum expected Lod score is reached in each family for several markers in the Xp22.31-p22.13 region and linkage to the rest of the X chromosome can be excluded, our study shows that NHS is a genetically homogeneous condition. An overall maximum two-point Lod score of 9.36 (theta = 0.00) is obtained with two closely linked markers taken together. DXS207 and DXS1053 in Xp22.2. Recombinant haplotypes indicate that the NHS gene lies between DXS85 and DXS1226. Multipoint analysis yield a maximum Lod score of 9.45 with the support interval spanning a 15-cM region that includes DXS16 and DXS1229/365. The deletion map of the Xp22.3-Xp21.3 region suggests that the phenotypic variability of NHS is not related to gross rearrangement of sequences of varying size but rather to allelic mutations in a single gene, presumably located proximal to DXS16 and distal to DXS1226. Comparison with the map position of the mouse Xcat mutation supports the location of the NHS gene between the GRPR and PDHA1 genes in Xp22.2.

Tissue-specific expression of the human pyruvate dehydrogenase α (Pdha-1) / chloramphenicol acetyltransferase fusion gene in transgenic mice

A chimeric gene (−763/+33Pdha-1/CAT) containing −763/+33 nucleotides of the human pyruvate dehydrogenase gene (Pdha-1) and the structural gene of chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. CAT activity was detected predominantly in the brain followed in decreasing order by adipose tissue, spleen, heart, kidney and liver. Developmental expression of CAT activity in the testes was similar to that of the endogenous Pdha-1 subunit expression in the testes. Dietary regulation of the transgene was comparable to the expression of endogenous pyruvate dehydrogenase complex in kidney and adipose tissue. Thus, the −763/+33 bp segment of the human Pdha-1 gene is transcriptionally active in vivo and can direct the expression of CAT in several tissues.