Advanced age increases frequencies of de novo mitochondrial mutations in macaque oocytes and somatic tissues

Mutations in mitochondrial DNA (mtDNA) contribute to multiple diseases. However, how new mtDNA mutations arise and accumulate with age remains understudied because of the high error rates of current sequencing technologies. Duplex sequencing reduces error rates by several orders of magnitude via independently tagging and analyzing each of the two template DNA strands. Here, using duplex sequencing, we obtained high-quality mtDNA sequences for somatic tissues (liver and skeletal muscle) and single oocytes of 30 unrelated rhesus macaques, from 1 to 23 y of age. Sequencing single oocytes minimized effects of natural selection on germline mutations. In total, we identified 17,637 tissue-specific de novo mutations. Their frequency increased ∼3.5-fold in liver and ∼2.8-fold in muscle over the ∼20 y assessed. Mutation frequency in oocytes increased ∼2.5-fold until the age of 9 y, but did not increase after that, suggesting that oocytes of older animals maintain the quality of their mtDNA. We found the light-strand origin of replication (OriL) to be a hotspot for mutation accumulation with aging in liver. Indeed, the 33-nucleotide-long OriL harbored 12 variant hotspots, 10 of which likely disrupt its hairpin structure and affect replication efficiency. Moreover, in somatic tissues, protein-coding variants were subject to positive selection (potentially mitigating toxic effects of mitochondrial activity), the strength of which increased with the number of macaques harboring variants. Our work illuminates the origins and accumulation of somatic and germline mtDNA mutations with aging in primates and has implications for delayed reproduction in modern human societies.

Mitochondria produce energy and are involved in myriad other cellular functions (reviewed in ref. 1). The mammalian mitochondrial DNA (mtDNA) is a small (∼16.6 kb in humans), circular, maternally transmitted molecule, which harbors 37 genes encoding 13 proteins (which form oxidative phosphorylation subunits), 22 transfer RNAs (tRNAs), and 2 ribosomal RNAs (rRNAs; reviewed in ref. 2). mtDNA is present in hundreds to thousands of copies per somatic cell and in >100,000 copies in an oocyte (3).The germline nucleotide substitution rate of mtDNA is an order of magnitude higher than that of nuclear DNA (4, 5). Germline mutations increase in frequency with paternal and maternal age in nuclear DNA of humans (6) and macaques (7); however, whether they accumulate with maternal age in mtDNA of primates has been understudied. Such age-related accumulation was suggested based on the analysis of human pedigrees (4, 8) without the direct examination of germline cells and, thus, might have been influenced by selection. An investigation of mutation accumulation in the oocytes of females of different ages is needed to settle this question unequivocally.The direct examination of mtDNA mutations in oocytes has been challenging due to methodological limitations. Most studies either focused on a limited number of mtDNA sites (e.g., refs. 9, 10) or used sequencing methods with high error rates (e.g., refs. 11, 12). Recently, an age-related increase of mtDNA mutations in mouse oocytes was demonstrated with duplex sequencing (13). However, we still do not know definitively whether the frequency of mtDNA mutations increases with age in primate oocytes. Answering this question is critical due to the association of mtDNA mutations with human genetic diseases (reviewed in ref. 14) and because of frequently delayed reproduction in modern human societies. Examining mutations in human oocytes presents multiple logistical and ethical challenges, requiring one to turn to a primate model.The rhesus macaque is an excellent model organism to study mtDNA mutations in relation to aging due to 1) the high similarity between macaque and human mtDNA, innate defenses against oxidative damage (15), and age-related decline in metabolic rate (16); and 2) the possibility of collecting oocytes from macaques starting at a young age. For humans, oocyte collection is mainly restricted to the reproductive lifespan, when in vitro fertilization procedures are performed.Here, we analyzed mutations in single oocytes and somatic tissues of rhesus macaques over an age span of >20 y, including samples from animals who have not reached sexual maturity (occurring at ∼3 y; ref. 17), as well as from animals up to the age of 23 y, covering the whole reproductive lifespan (macaques reach menopause at the age of ∼25 y; ref. 18). To measure de novo mutations, we used highly accurate duplex sequencing (19), allowing one to distinguish bona fide DNA variants from artifacts (sequencing and PCR errors, or DNA lesions) by barcoding double-stranded sequencing templates and achieving error rates <10⁻⁷. With this method, first, single-strand consensus sequences (SSCSs) are formed for reads originating from each of the two template strands separately. Next, a duplex consensus sequence (DCS) is formed from the two SSCSs. True DNA variants are expected to be present in both SSCSs and, thus, in the DCS. Using this method, we directly measured the frequency of de novo germline and somatic mutations across the whole mtDNA in macaques, demonstrating their accumulation with age. We identified variant hotspots, analyzed the effect of selection, and examined the dependence of allele frequencies of inheritable mtDNA heteroplasmies on age.