thymidine-5--triphosphate and Mitochondrial-Encephalomyopathies

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is a severe human disease caused by mutations in TYMP, the gene encoding thymidine phosphorylase (TP). It belongs to a broader group of disorders characterized by a pronounced reduction in mitochondrial DNA (mtDNA) copy number in one or more tissues. In most cases, these disorders are caused by mutations in genes involved in deoxyribonucleoside triphosphate (dNTP) metabolism. It is generally accepted that imbalances in mitochondrial dNTP pools resulting from these mutations interfere with mtDNA replication. Nonetheless, the precise mechanistic details of this effect, in particular, how an excess of a given dNTP (e.g., imbalanced dTTP excess observed in TP deficiency) might lead to mtDNA depletion, remain largely unclear. Using an in organello replication experimental model with isolated murine liver mitochondria, we observed that overloads of dATP, dGTP, or dCTP did not reduce the mtDNA replication rate. In contrast, an excess of dTTP decreased mtDNA synthesis, but this effect was due to secondary dCTP depletion rather than to the dTTP excess in itself. This was confirmed in human cultured cells, demonstrating that our conclusions do not depend on the experimental model. Our results demonstrate that the mtDNA replication rate is unaffected by an excess of any of the 4 separate dNTPs and is limited by the availability of the dNTP present at the lowest concentration. Therefore, the availability of dNTP is the key factor that leads to mtDNA depletion rather than dNTP imbalances. These results provide the first test of the mechanism that accounts for mtDNA depletion in MNGIE and provide evidence that limited dNTP availability is the common cause of mtDNA depletion due to impaired anabolic or catabolic dNTP pathways. Thus, therapy approaches focusing on restoring the deficient substrates should be explored.

Topics: Animals; Cell Culture Techniques; Deoxycytosine Nucleotides; DNA Replication; DNA, Mitochondrial; Fibroblasts; Humans; Mice; Mitochondria, Liver; Mitochondrial Encephalomyopathies; Thymine Nucleotides

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder associated with multiple mutations in mitochondrial DNA, both deletions and point mutations, and mutations in the nuclear gene for thymidine phosphorylase. Spinazzola et al. (Spinazzola, A., Marti, R., Nishino, I., Andreu, A., Naini, A., Tadesse, S., Pela, I., Zammarchi, E., Donati, M., Oliver, J., and Hirano, M. (2001) J. Biol. Chem. 277, 4128-4133) showed that MNGIE patients have elevated circulating thymidine levels and they hypothesized that this generates imbalanced mitochondrial deoxyribonucleoside triphosphate (dNTP) pools, which in turn are responsible for mitochondrial (mt) DNA mutagenesis. We tested this hypothesis by culturing HeLa cells in medium supplemented with 50 microM thymidine. After 8-month growth, mtDNA in the thymidine-treated culture, but not the control, showed multiple deletions, as detected both by Southern blotting and by long extension polymerase chain reaction. After 4-h growth in thymidine-supplemented medium, we found the mitochondrial dTTP and dGTP pools to expand significantly, the dCTP pool to drop significantly, and the dATP pool to drop slightly. In whole-cell extracts, dTTP and dGTP pools also expanded, but somewhat less than in mitochondria. The dCTP pool shrank by about 50%, and the dATP pool was essentially unchanged. These results are discussed in terms of the recent report by Nishigaki et al. (Nishigaki, Y., Marti, R., Copeland, W. C., and Hirano, M. (2003) J. Clin. Invest. 111, 1913-1921) that most mitochondrial point mutations in MNGIE patients involve T --> C transitions in sequences containing two As to the 5' side of a T residue. Our finding of dTTP and dGTP elevations and dATP depletion in mitochondrial dNTP pools are consistent with a mutagenic mechanism involving T-G mispairing followed by a next-nucleotide effect involving T insertion opposite A.

Topics: Base Pairing; Blotting, Southern; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyguanine Nucleotides; Deoxyribonucleotides; DNA, Mitochondrial; Gastrointestinal Diseases; Gene Deletion; HeLa Cells; Humans; Mitochondria; Mitochondrial Encephalomyopathies; Point Mutation; Polymerase Chain Reaction; Thymidine; Thymine Nucleotides