Investigating negative selection of pathogenic mtDNA variants in the blood

Abstract

The most common cause of adult mitochondrial disease is the pathogenic mitochondrial DNA (mtDNA) variant m.3243A>G. This variant is heteroplasmic, meaning that it does not affect all mtDNA molecules. The level of m.3243A>G in post-mitotic tissues remains stable throughout life. In mitotic tissues such as the blood, the level of pathogenic mtDNA is consistently decreased in comparison to post-mitotic levels and further declines with age. This work aims to characterise this decline in different cell types and understand the mechanism underlying this process. By investigating m.3243A>G level within 15 cell populations from 26 individuals, I observed enhanced mutation loss in six cell subtypes; this included all cells investigated within the T-cell compartment. Single cell analysis in six individuals supported these findings; all T-cell subsets exhibited an increased proportion of cells with low mutation levels compared to progenitor, myeloid and B-cell groups. This shift was more marked in memory compared to naïve T-cells and followed consistent trends between patients throughout maturation. Notably, this pattern was also observed in samples carrying the m.8344A>G variant, which remains stable in whole blood with age, but not for any investigated protein-coding variants. Flow cytometry data revealed a lower proportion of T-cells in m.3243A>G patients compared to controls and higher T-cell expression of exhaustion marker PD-1, suggesting that m.3243A>G impacts blood homeostasis. Parallel sequencing of mtDNA and the transcriptome revealed high levels of cellular stress and abnormal interleukin signalling in T-cells with high m.3243A>G heteroplasmy, indicating decreased fitness of these cells. This was supported by in vitro studies which demonstrated increased cell death in patient T-cells following stimulation. Understanding selection can give an invaluable insight into disease prognosis and help to identify therapeutic targets. These findings lay the foundations for further exploration into cellular fitness as a driver of negative selection in the blood.