Mitochondria are unique organelles, containing their own, maternally inherited genome. Human mitochondrial DNA (mtDNA) is small, circular and intronless, encoding 37 genes, precisely 13 proteins of the respiratory chain, 22 transfer RNAs (tRNAs) and 2 ribosomal RNAs. Each mitochondrion contains several mtDNA copies. Mitochondrial DNA deletions are pathogenic mutations that remove various portions of mtDNA genome, leading to shorter mtDNA molecules. In patients, there are two classes of mtDNA deletions: single, large-scale deletions that are present from the birth, whereas multiple deletions accumulate with age secondarily to mutations in nuclear genes involved in mtDNA maintenance. Every deleted mtDNA, coexists with other wild type mtDNAs, in an intracellular pool of mitochondrial genomes, determining the condition known as heteroplasmy. Pathogenic mtDNA mutations may become prevalent in certain cells; this process is defined as intracellular clonal expansion.
This study investigates clonal expansion patterns of mtDNA deleted genomes, applying for the first time the droplet-digital polymerase chain reaction (ddPCR) approach on single muscle cells collected by laser-capture microdissection from muscle biopsies of patients with different paradigms of mitochondrial disease with single and multiple mtDNA deletions accumulation. The results of this study indicate different patterns of accumulation of clonally expanded mtDNA deletions in patients with single and multiple deletions. It was found that single deletions patients have clonally expanded deletion in all single muscle cell populations, suggesting that the original deletion event occurred at early stage of embryonic development or even along the maternal germline transmission. Importantly, we distinguish localized clonal expansion of mtDNA deletions in patients with mutations in the OPA1 fusion gene. In conclusion, the ddPCR is a promising new technique for the investigation of clonal expansion by accurate of mtDNA deletions by accurate quantifying the mtDNA heteroplasmy levels
