Age-related accumulation of de novo mitochondrial mutations in mammalian oocytes and somatic tissues

Abstract

Mutations create genetic variation for other evolutionary forces to operate on and cause numerous genetic diseases. Nevertheless, how de novo mutations arise remains poorly understood. Progress in the area is hindered by the fact that error rates of conventional sequencing technologies (1 in 100 or 1,000 base pairs) are several orders of magnitude higher than de novo mutation rates (1 in 10,000,000 or 100,000,000 base pairs per generation). Moreover, previous analyses of germline de novo mutations examined pedigrees (and not germ cells) and thus were likely affected by selection. Here, we applied highly accurate duplex sequencing to detect low-frequency, de novo mutations in mitochondrial DNA (mtDNA) directly from oocytes and from somatic tissues (brain and muscle) of 36 mice from two independent pedigrees. We found mtDNA mutation frequencies 2- to 3-fold higher in 10-month-old than in 1-month-old mice, demonstrating mutation accumulation during the period of only 9 mo. Mutation frequencies and patterns differed between germline and somatic tissues and among mtDNA regions, suggestive of distinct mutagenesis mechanisms. Additionally, we discovered a more pronounced genetic drift of mitochondrial genetic variants in the germline of older versus younger mice, arguing for mtDNA turnover during oocyte meiotic arrest. Our study deciphered for the first time the intricacies of germline de novo mutagenesis using duplex sequencing directly in oocytes, which provided unprecedented resolution and minimized selection effects present in pedigree studies. Moreover, our work provides important information about the origins and accumulation of mutations with aging/maturation and has implications for delayed reproduction in modern human societies. Furthermore, the duplex sequencing method we optimized for single cells opens avenues for investigating low-frequency mutations in other studies.

Fig 1. Two mouse pedigrees included in the study.

At the time of sample collection, the age of the mothers was approximately 10 mo, whereas the age of the pups was approximately 20 d. Red numbers are individual IDs. Black numbers in bold indicate the number of single oocytes sequenced per individual, and gray numbers in parentheses indicate the number of oocytes included in a pool per individual (only one oocyte pool was included per individual). (A) Pedigree with four mothers (sisters: G136, G137, G139, and G140) and 11 pups. This pedigree is further referred to as G126, based on the ID of the grandmother (who was not included in the study). For simplicity, mouse G136 is also referred to as a mother despite her not having pups. Pups of G137 were born in two litters (S1 Table), separated by a large gap on the figure. (B) Pedigree with four mothers (sisters: G131–G134) and 17 pups. This pedigree is further referred to as G129. Pups of G132 were born in two litters (S1 Table). ID, identifier; un, unknown number of oocytes included in a pool.

Fig 2. Mutations along the mtDNA molecule in pups and mothers.

Distributions of (A) somatic and (B) germline mutations along the mitochondrial genome. Dot size represents the number of molecules (i.e., the number of DCSs) in which a mutation was observed. The majority of mutations were found only in a single molecule; therefore, the shown MAF might be inaccurate in some samples. Among the mutations observed in at least two DCSs (for which MAF can be estimated more accurately), only 11 had MAF >1%—they all occurred in the germline (two mutations in single oocytes of pups, eight mutations in single oocytes of mothers, and one mutation in oocyte pool of a mother; S3 Table). The dashed blue line marks the beginning of the D-loop region. The raw data for the information depicted in this figure are available at https://github.com/makovalab-psu/mouse-duplexSeq. DCS, duplex consensus sequence; MAF, minor allele frequency; mtDNA, mitochondrial DNA.

Fig 3. Nucleotide substitutions accumulate with age.

Higher median mutation frequencies were observed in brain, muscle, single oocytes, and oocyte pools of mothers compared with those of pups. Permutation test p-values (one-sided test based on medians, corrected for multiple testing) are shown with the respective sample sizes (n). In the calculation of mutation frequencies for single oocytes, the numbers of mutations and sequenced nucleotides were combined across all oocytes analyzed for a mouse. Considering only samples sequenced at a DCS depth ≥100× does not change the results qualitatively (S5B Fig). The raw data for the information depicted in this figure are available at https://github.com/makovalab-psu/mouse-duplexSeq. DCS, duplex consensus sequence.

Fig 4. Mutation patterns in pups and mothers.

(A) Tissue-specific mutation frequencies (computed as total number of tissue-specific mutations divided by the product of mtDNA region length and mean DCS depth) for pups and mothers in different mtDNA regions: D-loop (877 bp), protein-coding (11,331 bp), tRNA (1,499 bp), and rRNA (2,536 bp) sequences. Mutation frequencies for noncoding sequences outside of the D-loop (57 bp) are shown in S6 Table. (B) Tissue-specific frequencies of different mutation types for pups and mothers. C>T/G>A mutations separated by CpG and non-CpG sites are shown in S8 Fig. Mutation frequency bars are shown with 95% Poisson confidence intervals. Differences between mothers and pups in each category were tested using the Fisher’s exact test; stars indicate p-values (*p < 0.05, **p < 0.01, ***p < 0.001). “Oocytes” comprise single oocytes and oocyte pools in both (A) and (B). The raw data for the information depicted in this figure are available at https://github.com/makovalab-psu/mouse-duplexSeq. CpG, 5′-cytosine-phosphate-guanine-3′; DCS, duplex consensus sequence; mtDNA, mitochondrial DNA.

Fig 5. Correlations of allele frequencies for inherited heteroplasmies in somatic tissues and oocytes.

(A) MAF of heteroplasmies in brain versus muscle for pups. (B) MAF of heteroplasmies in brain versus muscle for mothers. (C) Mean MAF of heteroplasmies in single oocytes versus somatic tissues (averaged between brain and muscle) for pups. (D) Mean MAF of heteroplasmies in single oocytes versus somatic tissues (averaged between brain and muscle) for mothers. In (C-D), single oocytes (not oocyte pools) were used to obtain approximately equal numbers of oocytes for individual mothers and pups. Dot size indicates the number of sampled oocytes for each pup or for each mother. The gray bands on all plots represent confidence intervals around the regression line, and the dashed lines are the 1:1 relationship. The raw data for the information depicted in this figure are available at https://github.com/makovalab-psu/mouse-duplexSeq. MAF, minor allele frequency.