Evaluating the involvement of mtDNA variants in patients diagnosed with myalgic encephalomyelitis

Abstract

In mitochondrial research, many investigators have examined the association between mitochondrial DNA (mtDNA) variants in rare as well as common complex diseases. Previous studies at the CHM (NWU) detected three known pathogenic mtDNA variants (m.7497G>A, m.9185T>C and m.10197G>A) at low allele frequencies in a number of patients diagnosed with myalgic encephalomyelitis (ME). Since no diagnostic examinations or conclusive treatments currently exist for ME, an association between ME and known pathogenic variants, or a cumulative effect of rare non-synonymous variants (pathogenicity score) on ME, could provide valuable insights into understanding the causes of ME. Literature shows contradicting data regarding the role of mitochondrial dysfunction in ME, and while uncommon mtDNA deletions have been reported, the three known pathogenic mtDNA variants introduced here have not previously been observed in ME patients (but were later identified as sequencing artefacts in the duration of this study), nor has the combined effect of numerous rare non-synonymous variants on the mitochondrial bioenergetics of ME patients been assessed. To do this, cytoplasmic hybrid (cybrid) cells were developed by fusing ρ0 (mtDNA-depleted) cells with healthy control and ME patient‟s blood platelets (containing solely mtDNA). These cybrid cells were used for mitochondrial bioenergetic analyses, using a Seahorse XFe96 analyser, and for determination of the relative mtDNA copy number (RMCN), using real-time PCR. In addition, conditions for analysing selected cell lines (including the cybrids) using the Seahorse XFe96 analyser were optimized. While no apparent bioenergetic irregularities were observed in ME patient cybrids compared to healthy controls, an increased pathogenicity score appeared to be associated with a decrease in ATP production and a decreased electron transport system (ETS) capacity in ME patients. This new approach for investigating mtDNA variants and a common complex disease may provide new insights into the diagnostic and causative factors of ME.