Disorders of mitochondrial DNA (mtDNA) maintenance have emerged as an important cause of human genetic disease, but demonstrating the functional consequences of de novo mutations remains a major challenge. We studied the rate of depletion and repopulation of mtDNA in human fibroblasts exposed to ethidium bromide in patients with heterozygous POLG mutations, POLG2 and TK2 mutations. Ethidium bromide induced mtDNA depletion occurred at the same rate in human fibroblasts from patients and healthy controls. By contrast, the restoration of mtDNA levels was markedly delayed in fibroblasts from patients with compound heterozygous POLG mutations. Specific POLG2 and TK2 mutations did not delay mtDNA repopulation rates. These observations are consistent with the hypothesis that mutations in POLG impair mtDNA repopulation within intact cells, and provide a potential method of demonstrating the functional consequences of putative pathogenic alleles causing a defect of mtDNA synthesis.
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