Estimating the allele frequency threshold of the pathogenic mtDNA variant m.3243A>G tolerated by human myofibres

Cells are thought to tolerate the presence of heteroplasmic pathogenic mitochondrial (mt)DNA variants, up to a threshold proportion with little or no functional consequence, only developing a detectable oxidative phosphorylation (OXPHOS) defect once this threshold is exceeded. We hypothesised that, given the highly variable phenotypic presentation and severity of disease caused by the most common heteroplasmic pathogenic mtDNA variant m.3243A>G, this threshold may vary between patients. To estimate this threshold we examined the relationship between m.3243A>G level and deficiency of respiratory complexes I and IV in hundreds of single skeletal muscle fibres captured by laser microdissection from 17 patients carrying the m.3243A>G variant. We confirmed that tissue homogenate m.3243A>G level is a poor surrogate for the broad and complex distributions of m.3243A>G level in single cells from individual patients. We used unsupervised machine learning to characterise the biochemical status of muscle fibres, based on immunocytochemical measurements and estimate that the m.3243A>G threshold proportion above which myofibres develop a respiratory complex defect is 82.8% (IQR:2.7%). We do not find any significant difference in threshold between individuals, however we demonstrate that the pattern of respiratory complex defect and the distribution of m.3243A>G varies considerably between patients. This observation suggests that the pathogenesis of m.3243A>G is more diverse than previously thought.