Evaluation of Mitochondrial Function on Pyruvate Dehydrogenase Complex Deficient Patient-derived Cell Lines.

INTRODUCTION: Pyruvate Dehydrogenase Complex (PDC) is a pivotal gatekeeper between cytosolic glycolysis and mitochondrial oxidative phosphorylation, playing important role in aerobic energy metabolism. Most PDC deficiency, cases being caused by mutations in PDHA1 encoding the α subunit of the rate-limiting E1 enzyme, which is characterized by abnormal phenotypes caused by energy deprivation at peripheral/central nervous systems and muscular tissues. This study aims to evaluate the potential therapeutic effect of arginine and thiamine in ameliorating mitochondrial function in patient-derived cultured cells. MATERIALS AND METHODS: PDC-deficient cell lines, carrying three different PDHA1 variants, were cultured in the absence and presence of arginine and/or thiamine at therapeutical levels, 4 mM and 100 µM, respectively. Mitochondrial bioenergetics profile was evaluated using the Seahorse extracellular flux analyzer. RESULTS: In physiological conditions, control cells presented standard values for all parameters evaluating the mitochondrial function, no differences being observed after supplementation of culture medium with therapeutic levels of arginine and/or thiamine. However, PDC-PDHA1 deficient cell lines consumed less oxygen than the control cells, but arginine and thiamine supplementation increased the basal respiration for values similar or higher than the control cell line. Moreover, arginine and thiamine treatment highlighted an inefficient oxidative phosphorylation carried out by PDC-deficient cell lines. Finally, this treatment showed an increased oxygen consumption by enzymes other than those in the respiratory chain, thus proving the dependence of these mutant cell lines on cytosolic sources for ATP production, namely glycolysis. CONCLUSIONS: This study showed that arginine and thiamine, at therapeutical levels, increase the basal oxygen consumption rate of PDC-deficient cell lines, as well as their ATP-linked respiration. This parameter measures the capacity of the cell to meet its energetic demands and, therefore, its increase reveals a higher electron flow through the respiratory chain, which is coupled to elevated oxidative phosphorylation, thus indicating an overall increased robustness in mitochondrial- related bioenergetics.

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.

Pyruvate dehydrogenase complex deficiency: updating the clinical, metabolic and mutational landscapes in a cohort of Portuguese patients.

BACKGROUND: The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible decarboxylation of pyruvate into acetyl-CoA. PDC deficiency can be caused by alterations in any of the genes encoding its several subunits. The resulting phenotype, though very heterogeneous, mainly affects the central nervous system. The aim of this study is to describe and discuss the clinical, biochemical and genotypic information from thirteen PDC deficient patients, thus seeking to establish possible genotype-phenotype correlations. RESULTS: The mutational spectrum showed that seven patients carry mutations in the PDHA1 gene encoding the E1α subunit, five patients carry mutations in the PDHX gene encoding the E3 binding protein, and the remaining patient carries mutations in the DLD gene encoding the E3 subunit. These data corroborate earlier reports describing PDHA1 mutations as the predominant cause of PDC deficiency but also reveal a notable prevalence of PDHX mutations among Portuguese patients, most of them carrying what seems to be a private mutation (p.R284X). The biochemical analyses revealed high lactate and pyruvate plasma levels whereas the lactate/pyruvate ratio was below 16; enzymatic activities, when compared to control values, indicated to be independent from the genotype and ranged from 8.5% to 30%, the latter being considered a cut-off value for primary PDC deficiency. Concerning the clinical features, all patients displayed psychomotor retardation/developmental delay, the severity of which seems to correlate with the type and localization of the mutation carried by the patient. The therapeutic options essentially include the administration of a ketogenic diet and supplementation with thiamine, although arginine aspartate intake revealed to be beneficial in some patients. Moreover, in silico analysis of the missense mutations present in this PDC deficient population allowed to envisage the molecular mechanism underlying these pathogenic variants. CONCLUSION: The identification of the disease-causing mutations, together with the functional and structural characterization of the mutant protein variants, allow to obtain an insight on the severity of the clinical phenotype and the selection of the most appropriate therapy.

Molecular basis and clinical presentation of classic galactosemia in a Croatian population.

BACKGROUND: Classic galactosemia is an autosomal recessive disorder of galactose metabolism caused by severely decreased activity of galactose-1-phosphate uridylyltransferase (GALT) due to pathogenic mutations in the GALT gene. To date more than 330 mutations have been described, with p.Q188R and p.K285N being the most common in Caucasian populations. Although acute manifestations can be fully avoided by a galactose-restricted diet, chronic complications, such as neurological ones, cannot be prevented in a significant number of patients despite compliance with the dietary treatment. METHODS: A cohort of 16 galactosemic Croatian patients, including one pair of siblings, was studied. Molecular characterization was performed by direct sequence analysis of the GALT gene. RESULTS: Sixteen patients were analyzed and only four different mutations were detected. As expected, p.Q188R and p.K285N were common, accounting for 40% and 37% of unrelated alleles, respectively. The third mutation accounting for 20% of mutant alleles was p.R123X causing a premature stop codon, is thus considered to be severe, which is in accordance with the phenotype presented by the homozygous patient described here. The fourth mutation p.E271D was found in a single allele. More than half of our patients manifested some chronic neurological complications. CONCLUSIONS: This is the first report on mutational and phenotypic spectra of classic galactosemia in Croatia that expands the knowledge on the mutational map of the GALT gene across Europe and reveals the genetic homogeneity of the Croatian population.

Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: Null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells.

Human pyruvate dehydrogenase complex (PDC) catalyzes a key step in the generation of cellular energy and is composed by three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. We report on a young adult female patient who has PDC deficiency associated with a compound heterozygosity in PDHX encoding the E3-binding protein. Additionally, in the patient and in all members of her immediate family, a full-length testis-specific PDHA2 mRNA and a 5'UTR-truncated PDHA1 mRNA were detected in circulating lymphocytes and cultured fibroblasts, being both mRNAs translated into full-length PDHA2 and PDHA1 proteins, resulting in the co-existence of both PDHA isoforms in somatic cells. Moreover, we observed that DNA hypomethylation of a CpG island in the coding region of PDHA2 gene is associated with the somatic activation of this gene transcription in these individuals. This study represents the first natural model of the de-repression of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to the somatic activation of this gene as a potential therapeutic approach for most forms of PDC deficiency.

Data supporting the co-expression of PDHA1 gene and of its paralogue PDHA2 in somatic cells of a family.

This article presents a dataset proving the simultaneous presence of a 5'UTR-truncated PDHA1 mRNA and a full-length PDHA2 mRNA in the somatic cells of a PDC-deficient female patient and all members of her immediate family (parents and brother). We have designed a large set of primer pairs in order to perform detailed RT-PCR assays allowing the clear identification of both PDHA1 and PDHA2 mRNA species in somatic cells. In addition, two different experimental approaches were used to elucidate the copy number of PDHA1 gene in the patient and her mother. The interpretation and discussion of these data, along with further extensive experiments concerning the origin of this altered gene expression and its potential therapeutic consequences, can be found in "Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells" (A. Pinheiro, M.J. Silva, C. Florindo, et al., 2016) [1].