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 information about intracellular clonal expansions of deleted mitochondrial DNA (DeltamtDNA), which contribute to aging of the muscle and possibly other tissues. Analysis of approximately 1400 cells from heart, diaphragm and skeletal muscle from 20 individuals without mitochondrial disease revealed that up to 25% of cells in a tissue sample may bear clonally expanded DeltamtDNA. Sequence analysis of >50 clonal DeltamtDNA reveals that about half of them lack the light strand origin of replication. This observation is puzzling since these molecules must have retained the ability to replicate in order to be able to undergo clonal expansion. We present evidence that such DeltamtDNA molecules may in fact exist in the cell as partially duplicated mtDNA (pdmtDNA) previously described in certain mtDNA disorders. In contrast to the 'originless' DeltamtDNA, the corresponding pdmtDNA do possess a light strand origin required for their propagation. Most pdmtDNA also possess an extra heavy strand origin, which may result in higher replication rate and thus provide a mechanism for expansion. Importantly, pdmtDNA are indistinguishable from DeltamtDNA in PCR assays routinely used to detect somatic mtDNA deletions in tissues of normally aged individuals. These results indicate that a substantial proportion of age-related mtDNA deletions reported in the literature may exist as or be derived from pdmtDNA.