The role of blood cell dynamics and intracellular selection in the decline of the heteroplasmic mitochondrial DNA mutation m.3243A>G in blood

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Mitochondrial diseases are caused by mutations in nuclear and mitochondrial (mt) genomes. Individuals carrying the most prevalent heteroplasmic mtDNA mutation, m.3243A>G, display a wide range of phenotypes from clinically silent to severe neurodegeneration. Disease severity is partly explained by the proportion of mutated mtDNA molecules (heteroplasmy) but the relationship is complicated, making patient counselling challenging. In mitotic tissues (e.g. blood), heteroplasmy declines over time. Simulations suggest this is linked to the number of cell divisions. Intracellular negative selection may also contribute but the mechanism for this is unknown. There is no cure for mitochondrial disease; understanding this process could lead to the identification of new therapeutic targets. Objectives 1. Determine the extent to which variation in the leukocyte composition of peripheral blood is associated with heteroplasmy level and decline and whether heteroplasmy in a specific blood cell type is better correlated with disease burden than that of whole blood. 2. Determine whether the mitotic history of memory T cell clones associates with heteroplasmy. 3. Investigate the role of intracellular selection in declining heteroplasmy. Novelty There appears to be natural variation in heteroplasmy in different types of leukocytes. Population dynamics of individual leukocyte populations may illuminate the mechanisms of heteroplasmy decline over time, a poorly understood process. Access to a unique, deeply-phenotyped patient cohort will enable the production of a large quantity of high quality data within the time-frame of a PhD. Timeliness Ethical approval for recruiting 100 patients has been granted (REC:19/LO/0117). Blood samples have been collected for 25; the remaining 75 will be recruited by late 2020, allowing the student to begin experiments immediately. Experimental Approach The student will design a panel of antibodies for fluorescence activated cell sorting, separating blood into subpopulations representing the major leukocyte developmental stages. m.3243A>G heteroplasmy and mtDNA copy number will be measured by pyrosequencing and real-time PCR. Within T cells, which have the longest mitotic history and lowest heteroplasmy, there is likely to be heterogeneity among individual cells. Single T cells can be tracked and expanded into clones, allowing the student to correlate heteroplasmy with mitotic history and manipulate the number of cell divisions by stimulating proliferation in vitro. Using data from the MitoCohort, the student will model the relationship between cellular sub-population heteroplasmy and disease burden. To investigate the role of active intracellular selection in declining levels, cells will be cultured in conditions designed to manipulate reliance on oxidative phosphorylation.