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

BackgroundPathogenic mitochondrial (mt)DNA variants cause neuromuscular disease with highly variable severity and phenotypic presentation, the reason for which is poorly understood. Cells are thought to tolerate the presence of pathogenic mtDNA variants up to a threshold proportion with little or no functional consequence, developing significant respiratory complex defects above this threshold. We developed a robust method to identify deficient muscle fibres, applied it to biopsies from 17 patients carrying the common m.3243A>G variant and examined the relationship between respiratory deficiency and m.3243A>G level in hundreds of single skeletal muscle fibres. We hypothesised that single-cell between-patient differences may explain the vast clinical heterogeneity of mtDNA disease. ResultsImmunohistochemical measurements of respiratory complexes I and IV and unsupervised machine learning identified muscle fibres with respiratory deficiency; the pattern of deficiency and proportion of deficient fibres (range 0-64%) varies between patients. 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. Estimated thresholds do not differ between patients, but sections with narrow m.3243A>G distributions have a lower proportion of deficient fibres. ConclusionsInter-individual differences in respiratory complex deficiency in muscle fibres from patients with m.3243A>G are more complex than previously thought and may be driven by differential segregation and expansion of mtDNA molecules. Our quantitative observations could constrain the range of feasible mechanisms responsible for phenotypic diversity in mitochondrial disease.