Age-Related Nuclear Mutation Profiling Across Somatic and Germline Tissues in MutaMouse Males

Abstract

Aging is associated with progressive declines in tissue function and increased susceptibility to chronic disease, but the molecular changes that accumulate across tissues during aging remain incompletely understood. Nuclear mutations are permanent genomic changes that can provide a cumulative record of DNA damage, repair, replication errors, and cellular population dynamics experienced by tissues over time. However, age-related nuclear mutagenesis remains difficult to characterize because spontaneous mutations are rare and existing evidence is fragmented across methods, species, tissues, and genomic compartments. Duplex Sequencing (DS) enables high-accuracy detection of rare mutations in endogenous DNA, but accurate mutation detection alone is not sufficient for biological interpretation. The overarching objective of this thesis was therefore to determine whether nuclear mutation frequency (MF) and mutation spectra change with age across MutaMouse male tissues in a tissue-dependent manner.

First, a simulation-based framework was developed to evaluate how DS study-design parameters influence the ability to detect differences in MF. Using empirical liver and bone marrow data from adult MutaMouse males as reference conditions, the analyses examined the effects of baseline MF, inter-animal variability, expected effect size, sample size, and informative duplex sequencing depth. As expected, power increased with larger sample sizes, greater sequencing depth, and larger expected effect sizes, but decreased as inter-animal variability increased. Increasing sequencing depth improved power only up to a point, after which additional informative duplex bases provided limited benefit relative to increasing the number of animals per group. Under the modeled conditions, sample sizes of 4-5 animals per group were sufficient to detect at least a 1.5-fold change in MF with feasible sequencing depth, whereas routine detection of smaller changes remained challenging.

Second, DS was used to characterize endogenous nuclear mutagenesis across liver, lung, bone marrow, and male germ cells from MutaMouse males spanning early to late life. These tissues were selected to represent distinct biological contexts, including differences in proliferative activity, metabolic demand, environmental exposure, and somatic versus germline identity. Early in life, MF was broadly similar across tissues. Across the full age range, however, tissue-specific trajectories emerged. MF increased with age in all three somatic tissues, with the unique-mutation estimate, MFMin, rising approximately 1.5-2.0-fold between 3 weeks and ≥78 weeks. Liver and lung showed gradual age-related accumulation, whereas bone marrow showed a delayed increase that became most apparent in late life. In contrast, male germ cells did not show a significant age-related increase in MF over the ages examined. Mutation spectra and genomic-context analyses further showed that tissue identity shaped broader mutational patterns, although higher-resolution signature-level analyses were limited by sparse mutation counts after stratification.

Overall, this thesis shows that age-related nuclear mutation accumulation in MutaMouse males is tissue-dependent rather than uniform across the organism. Somatic tissues showed detectable age-related increases in MF, whereas male germ cells did not show a significant increase over the ages examined. The simulation-based framework developed here further shows that high-accuracy mutation detection is most informative when paired with sufficient statistical power, especially when expected effects are modest or rare mutations are subdivided for higher-resolution analyses. These findings show that age-related nuclear mutagenesis depends on the tissue being studied and provide practical guidance for designing DS-based studies that can distinguish biological patterns from limitations of study design.