Investigating the mechanism of clonal expansion of deleted mtDNA species

Abstract

Mitochondrial DNA deletions are a primary cause of inherited and sporadic mitochondrial disease, whilst somatic mtDNA deletions contribute to the focal respiratory chain deficiency observed in post-mitotic cells associated with ageing and neurodegenerative disorders. As mtDNA deletions only cause cellular pathology at high levels of heteroplasmy, an mtDNA deletion formed within a cell must accumulate by a process known as clonal expansion to levels which result in biochemical dysfunction. The mechanism driving clonal expansion remains uncertain; this research aimed to investigate clonally expanded mtDNA deletions in sporadic and inherited mitochondrial myopathies in order to elucidate this mechanism. A number of different approaches were taken to assess the mechanism driving accumulation of mtDNA deletions. The effect of the mtDNA deletion size on clonal expansion was first investigated by assessing the longitudinal spread of mtDNA deletions in single muscle fibres isolated from patients presenting with mtDNA maintenance disorders; no relationship was found to exist between mtDNA deletion size and the area over which the mutation has accumulated. A longitudinal study was carried out using tissue acquired over a 13-year period from a single patient with a sporadic mtDNA deletion, to identify whether the mtDNA deletion heteroplasmy level continued to increase over time, as would be expected if the mutation displayed a selective advantage over wildtype mtDNA – however, both the genetic and biochemical defect were found to be stable over time in this patient. A subsequent study aimed to identify a correlation between mtDNA deletion size and heteroplasmy levels at the whole tissue level in a cohort of patients with sporadic single mtDNA deletions, but no evidence was found to suggest that larger mtDNA deletions accumulate to higher levels of heteroplasmy. Finally, single cytochrome c oxidase- deficient muscle fibres were investigated using single-molecule PCR to assess whether clonal expansion of multiple mtDNA deletions could be observed in single cells. Evidence of multiple clonally expanded mtDNA species was found in approximately 40% of all examined fibres, with no correlation between mtDNA deletion size and level of accumulation. Each of these four studies has highlighted accumulation by random genetic drift to be the most likely mechanism for clonal expansion of mtDNA deletions in human muscle; no evidence has been found to support the presence of a selective advantage for mtDNA deletion species over wildtype mtDNA.