Experimental evidence for human mitochondrial DNA (mtDNA) recombination was recently obtained in an individual with paternal inheritance of mtDNA and in an in vitro cell culture system. Whether mtDNA recombination is a common event in humans remained to be determined. To detect mtDNA recombination in human skeletal muscle, we analyzed the distribution of alleles in individuals with multiple mtDNA heteroplasmy using single-cell PCR and allele-specific PCR. In all ten individuals who carried a heteroplasmic D-loop mutation and a distantly located tRNA point mutation or a large deletion, we observed a mixture of four allelic combinations (tetraplasmy), a hallmark of recombination. Twelve of 14 individuals with closely located heteroplasmic D-loop mutation pairs contained a mixture of only three types of mitochondrial genomes (triplasmy), consistent with the absence of recombination between adjacent markers. These findings indicate that mtDNA recombination is common in human skeletal muscle.
Recently, somatic recombination of human mitochondrial DNA (mtDNA) was discovered in skeletal muscle. To determine whether recombinant mtDNA molecules can be transmitted through the germ line, we investigated two families, each harboring two inherited heteroplasmic mtDNA mutations. Using allele-specific polymerase chain reaction and single-cell and single-molecule mutational analyses, we discovered, in both families, all four possible allelic combinations of the two heteroplasmic mutations (tetraplasmy), the hallmark of mtDNA recombination. We strongly suggest that these recombinant mtDNA molecules were inherited rather than de novo generated somatically, because they (1) are highly abundant and (2) are present in different tissues of maternally related family members, including young individuals. Moreover, the comparison of the complete mtDNA sequence of one of the families with database sequences revealed an irregular, nontreelike pattern of mutations, reminiscent of a reticulation. We therefore propose that certain reticulations of the human mtDNA phylogenetic tree might be explained by recombination of coexisting mtDNA molecules harboring multiple mutations.
We explored whether muscle disuse (i.e. bed rest) contributes to mitochondrial (mt)DNA deletions in skeletal muscle. mtDNA deletions have been associated with various degenerative processes (e.g. sarcopenia) and sedentary behavior. Human muscle tissue from a small sample (n=3) taken before and after a 28‐day bed rest were subjected to mutational analysis using, a restriction digestion / long range digital PCR approach. Total DNA was digested with SnaBI, an enzyme with a single restriction site located in an area commonly removed in deleted mtDNA molecules. DNA was exposed to long‐range PCR capable of amplifying most of the mitochondrial genome. Restriction digestion rendered mtDNA un‐amplifiable, except for molecules with deletions. The actual number of molecules with deletions and types thereof were determined using highly diluted DNA samples, reaching single molecule per reaction, in multiple independent PCR reactions, so that molecules could be directly counted (“digital PCR”). We found an increase in the fraction of molecules with mtDNA deletions after bedrest, relative to intact wild type molecules. This finding suggests that disuse results in skeletal muscle mtDNA deletions. Further studies are needed to determine the association of these deletions with physiological and metabolic changes seen with disuse or sedentary behavior. Grant Funding Source: Supported by Ellison Medical Foundation, NSBRI‐NPFR00301, GCRC M01 RR00
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