Thiamine pyrophosphokinase deficiency in encephalopathic children with defects in the pyruvate oxidation pathway

Abstract

Thiamine pyrophosphate (TPP) is an essential cofactor of the cytosolic transketolase and of three mitochondrial enzymes involved in the oxidative decarboxylation of either pyruvate, α-ketoglutarate or branched chain amino acids. Thiamine is taken up by specific transporters into the cell and converted to the active TPP by thiamine pyrophosphokinase (TPK) in the cytosol from where it can be transported into mitochondria. Here, we report five individuals from three families presenting with variable degrees of ataxia, psychomotor retardation, progressive dystonia, and lactic acidosis. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate but normal pyruvate dehydrogenase complex activity in the presence of excess TPP. A reduced concentration of TPP was found in the muscle and blood. Mutation analysis of TPK1 uncovered three missense, one splice-site, and one frameshift mutation resulting in decreased TPK protein levels.

Figure 1

Thiamine Metabolism in Mammalian Cells The following abbreviations are used: TPP, thiamine pyrophosphate; PDHC, pyruvate dehydrogenase complex; α-KGDH, α-ketoglutarate dehydrogenase; BCKDH, branched chain α-keto acid dehydrogenase.

Figure 2

Pedigrees of the Five Affected Individuals from Three Families

Figure 3

Investigations of Cofactor Dependency of Pyruvate Dehydrogenase Complex Activity Pyruvate dehydrogenase complex was measured in the in the absence of thiamine pyrophosphate and showed a decrease in the affected individuals compared to controls (A). An even more pronounced decrease was found in the ratio of PDHC activities under TPP-unsupplemented versus TPP-supplemented (0.8 mmol/l) assay conditions (B).

Figure 4

Sequence Analysis of TPK1 Sequence analysis revealed compound heterozygous mutations c.[148A>C]+[501+4A>T] p.[Asn50His]+[Val119_Pro167del] in the individuals P1 and P2 (Figure 2) in TPK1 (RefSeq NM_022445.3). In the individuals P3 and P4 a homozygous mutation c.119T>C p.Leu40Pro was found. In the individual P5 compound heterozygous mutations c.[179_182delGAGA]+[656A>G] p.[Arg60LysfsX52]+[Asn219Ser] were found. The expression of the missense mutations is shown in the cDNA of the affected individuals.

Figure 5

Phylogenetic Conservation of the Identified Thiamine Phyrophosphokinase Mutations Alignment by ClustalW shows phylogenetic conservation of missense mutations found in the thiamine pyrophosphokinase in the affected individuals (P1–P5, cf. Figure 2).

Figure 6

Immunoblot Analysis of Muscle Extracts A decrease in the amount of the active isoform a (27.3 kDa) was found in all affected individuals with a TPK antibody (A). An antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control for cytosolic protein (B).