Quantification and sequencing of somatic deleted mtDNA in single cells: evidence for partially duplicated mtDNA in aged human tissues

Single-cell PCR of the whole mitochondrial genome provides detailed informa tion about intracellular clonal expansions of deleted mitochondrial DNA (De lta mtDNA), which contribute to aging of the muscle and possibly other tiss ues. Analysis of similar to 1400 cells from heart, diaphragm and skeletal m uscle from 20 individuals without mitochondrial disease revealed that up to 25% of cells in a tissue sample may bear clonally expanded Delta mtDNA. Se quence analysis of >50 clonal Delta mtDNA reveals that about half of them l ack the light strand origin of replication. This observation is puzzling si nce these molecules must have retained the ability to replicate in order to be able to undergo clonal expansion. We present evidence that such Delta m tDNA molecules may in fact exist in the cell as partially duplicated mtDNA (pdmtDNA) previously described in certain mtDNA disorders. In contrast to t he 'originless' Delta mtDNA, the corresponding pdmtDNA do possess a light s trand origin required for their propagation. Most pdmtDNA also possess an e xtra heavy strand origin, which may result in higher replication rate and t hus provide a mechanism for expansion. Importantly, pdmtDNA are indistingui shable from Delta mtDNA in PCR assays routinely used to detect somatic mtDN A deletions in tissues of normally aged individuals. These results indicate that a substantial proportion of age-related mtDNA deletions reported in t he literature may exist as or be derived from pdmtDNA.