T cell differentiation drives the negative selection of pathogenic mtDNA variants

Pathogenic mitochondrial (mt)DNA single nucleotide variants are the most common cause of adult mitochondrial disease. Whilst levels of the most common heteroplasmic variant (m.3243A>G) remain stable in post-mitotic tissues, levels in mitotic tissues, such as blood, decrease with age. Given differing division rates, longevity and energetic requirements within haematopoietic lineages, we hypothesised that variant level decline is driven by cell-type specific mitochondrial metabolic requirements. To address this, we coupled cell sorting with mtDNA sequencing to investigate mtDNA variant levels within progenitor, myeloid and lymphoid lineages from 26 individuals harbouring pathogenic mtDNA variants. We report that whilst the level of m.3243A>G declines with age in all analysed cell types, the T-cell lineage shows a significantly greater decline. This was confirmed for a second pathogenic tRNA variant; m.8344A>G, indicating that this phenomenon is not limited to m.3243A>G. High-throughput single cell analysis revealed that decline is driven by increasing proportions of cells that have cleared the variant genome, following a hierarchy that follows the current orthodoxy of T-cell differentiation and maturation. This work identifies the unique ability of T-cell subtypes to selectively purify their mitochondrial genomes, and identifies pathogenic mtDNA variants as a new means to track blood cell differentiation status. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by a Wellcome Career Re-entry Fellowship to SJP (204709/Z/16/Z); a MRC DiMeN DTP studentship to IGF, the Wellcome Centre for Mitochondrial Research (203105/Z/16/Z) and the UK NHS England Specialist Commissioners which funds the Highly Specialised Service for "Rare Mitochondrial Disorders of Adults and Children" in Newcastle upon Tyne. MC and PM are supported by Wellcome Trust Investigator Award 219562Z/19Z. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethical approval was granted by the Newcastle and North Tyneside Research Ethics Committee (REC:19/LO/0117; Understanding the decline in levels of the mitochondrial DNA mutation m.3243A>G within blood cell subtypes). Additional control samples were obtained from the Dendritic Cell Homeostasis in Health and Disease study (REC:08/H0906/72) and the MAGMA study (REC:17/NE/0015). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Underlying data and supporting analytic code for the manuscript can be accessed via