Abstract
Variants of the enzyme transketolase which possess reduced affinity for its cofactor thiamine pyrophosphate (high apparent Km) have been described in chronic alcoholic patients with Wernicke-Korsakoff syndrome. Since the syndrome has been shown to be directly related to thiamine deficiency, it has been hypothesized that such transketolase variants may represent a genetic predisposition to the development of this syndrome. To test this hypothesis, human transketolase cDNA clones were isolated, and their nucleotide and predicted amino acid sequence were determined. Transketolase was found to be a single copy gene which produces a single mRNA of approximately 2100 nucleotides. Additionally, the nucleotide sequence of the transketolase coding region in fibroblasts derived from two Wernicke-Korsakoff (WK) patients was compared to that of two nonalcoholic controls. Although nucleotide and predicted amino acid differences were detected between fibroblast cultures and the original cDNAs and among the cultures themselves, no specific nucleotide variations, which would encode a variant amino acid sequence, were associated exclusively with the coding region from WK patients. Thus, allelic variants of the transketolase gene cannot account for the biochemically distinct forms of the enzyme found in these patients nor be considered as a mechanism for genetic predisposition to the development of Wernicke-Korsakoff syndrome. Instead, the underlying mechanism must be extragenic and may be a result of differences in post-translational processing/modification of the transketolase polypeptide.
Highlights
None differences between the sequences preseinnt the cDNA clones and those present in WK (A and H) and non-WK(E and F) fibroblast cultures, aswell as those differences found between these two "classes" of fibroblast cultures,were identified position158, T --* A in cultures A, H, E, and F, resulting in the aminoacid change Ser-30+
Culture, no amino acid change; position 1346, G + C in each residue is deleted from their published nucleotide sequence resulting in a frameshift and
The relative locations of the above nucleotide and apparent aminoacid alterations are indicated in Fig. 4, A and B, by underlined residues and thusaberrant amino acid sequence. While these regions occur at positions where we found compression of the bands in the sequencing gel, the presence of these G residues has been confirmed by several independent experiments in our laboratory
Summary
The following nucleotide variations were observed during the sequence comparisons of the various TK cDNAs with TK coding regions in fibroblasts derived from Wernicke-Korsakoff (cultures A and H) Rnd non-Wernicke-Korsakoff (cultures E andF) individuals. (humanliverandfrontalcortex)andfromHelacells was virtuallyidentical.,thetransketolasevariants(chromatographic or biochemical) which have been implicatedas a predisposing factor for the developmeonf tWernicke-Korsakoff syndrome[7,8,9,56]shouldbeviewedeither as allelic variations of a single gene or as arising from different posttranslational modifications. Imental details are provided under “Experimental Procedures” and To address the possibility that allelic variations are responin the text.
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