Mammalian mitochondrial DNA accumulates insertions and deletions with age in energetically demanding tissues.

De novo mtDNA indels accumulate with age in somatic tissues with high energetic demand or high proliferation across three mammalian species.

• Three mammalian species studied: mouse, macaque, and human.
• Tissues examined included brain, skeletal muscle (high energetic demand), and liver (high proliferation).
• Highly accurate duplex sequencing was used to detect de novo mutations at low frequency against the high mtDNA copy number background.
• The accumulation pattern is similar to that previously described for de novo nucleotide substitutions in mtDNA.

In oocytes, mtDNA indels accumulate slower with age than nucleotide substitutions, or do not accumulate at all.

• This contrasts with somatic tissues where indels accumulate rapidly with age.
• The germline (oocyte) appears to have a protective mechanism or lower indel mutation rate compared to somatic tissues.
• The slower or absent accumulation in oocytes has implications for understanding maternal inheritance of mtDNA mutations.
• This finding illuminates why age-related mtDNA indel accumulation may affect reproduction later in life.

The age-related increase in mtDNA indel frequency is driven mostly by deletions rather than insertions.

• Deletions were identified as the primary contributor to the overall increase in indel frequency with age.
• This pattern was observed across species and tissues studied.
• The predominance of deletions is consistent with known mechanisms of mtDNA instability during aging.

Short tandem repeats (STRs) are highly enriched for mtDNA indels, implicating DNA replication slippage as a major driver of indel formation.

• STRs showed strong enrichment for indel mutations in mtDNA.
• DNA replication slippage at repetitive sequences is implicated as the major mechanistic driver.
• This finding applies across the mammalian species and tissues examined.
• The association with STRs provides a mechanistic explanation for the genomic distribution of de novo mtDNA indels.

For some species and tissues, indels are depleted at protein-coding sequences, but indels that are multiples of 3 bp are not overrepresented.

• Depletion of indels at protein-coding regions suggests purifying selection against frameshift mutations in mtDNA.
• However, the absence of overrepresentation of indels that are multiples of 3 bp (which would preserve the reading frame) contrasts with what might be expected if in-frame indels were selectively tolerated.
• The pattern was not consistent across all species and tissues examined.

This study represents the most detailed characterization of de novo small indels in mtDNA to date, enabled by duplex sequencing technology.

• Conventional next-generation sequencing technologies have low accuracy for detecting de novo mutations against the high mtDNA copy number background.
• Duplex sequencing provides highly accurate detection of rare de novo mutations.
• The study spans multiple age groups, three mammalian species, and both somatic and germline tissues.
• Results provide parameters for models of mtDNA evolution and inform molecular mechanisms for human genetic diseases.