Summary Paragraph Somatic variation contributes to biological heterogeneity by modulating cellular proclivity to differentiate, expand, adapt, or die. While large-scale sequencing efforts have revealed the foundational role of somatic variants to drive human tumor evolution, our understanding of the contribution of mutations to modulate cellular fitness in non-malignant contexts remains understudied. Here, we identify a mosaic synonymous variant (m.7076A&gt;G) in the mitochondrial DNA (mtDNA) encoded cytochrome c-oxidase subunit 1 gene ( MT-CO1 , p.Gly391=), which was present at homoplasmy in 47% of immune cells from a healthy donor. Using single-cell multi-omics, we discover highly specific selection against the m.7076G mutant allele in the CD8 + effector memory T cell compartment in vivo , reminiscent of selection observed for pathogenic mtDNA alleles 1, 2 and indicative of lineage-specific metabolic requirements. While the wildtype m.7076A allele is translated via Watson-Crick-Franklin base-pairing, the anticodon diversity of the mitochondrial transfer RNA pool is limited, requiring wobble-dependent translation of the m.7076G mutant allele. Notably, mitochondrial ribosome profiling revealed altered codon-anticodon affinity at the wobble position as evidenced by stalled translation of the synonymous m.7076G mutant allele encoding for glycine. Generalizing this observation, we provide a new ontogeny of the 8,482 synonymous variants in the human mitochondrial genome that enables interpretation of functional mtDNA variation. Specifically, via inter- and intra-species evolutionary analyses, population-level complex trait associations, and the occurrence of germline and somatic mtDNA mutations from large-scale sequencing studies, we demonstrate that synonymous variation impacting codon:anticodon affinity is actively evolving across the entire mitochondrial genome and has broad functional and phenotypic effects. In summary, our results introduce a new ontogeny for mitochondrial genetic variation and support a model where organismal principles can be discerned from somatic evolution via single-cell genomics.

Codon affinity in mitochondrial DNA shapes evolutionary and somatic fitness