Germline mutation rates reflect the complex interplay between DNA proofreading and repair pathways, exogenous sources of DNA damage, and life-history traits. For example, parental age is an important determinant of mutation rate variability; in many mammalian species, the number of germline de novo mutations observed in offspring increases as a function of paternal and maternal age [@PMID:28959963;@PMID:31549960;@PMID:35771663;@PMID:32804933;@PMID:31492841]. Rates of germline mutation accumulation are also variable across human families [@PMID:26656846;@PMID:31549960], likely due to either genetic variation or differences in environmental exposures. Although numerous protein-coding genes contribute to the maintenance of genome integrity, genetic variants that increase germline mutation rates, known as mutator alleles, have proven difficult to discover in mammals.
The dearth of observed germline mutators in mammalian genomes is not necessarily surprising, since alleles that lead to elevated germline mutation rates would likely have deleterious consequences and be purged by negative selection if their effect sizes are large [@PMID:27739533]. Moreover, germline mutation rates are relatively low, and direct mutation rate measurements require whole-genome sequencing data from both parents and their offspring. As a result, large-scale association studies — which have been used to map the contributions of common genetic variants to many complex traits — are not currently well-powered to investigate the polygenic architecture of germline mutation rates [@PMID:31964835].
Despite these challenges, less traditional strategies have been used to identify a small number of mutator alleles in humans, macaques [@doi:10.1101/2023.03.27.534460], and mice. By focusing on families with rare genetic diseases, a recent study discovered two mutator alleles that led to significantly elevated rates of de novo germline mutation in human genomes [@PMID:35545669]. Other groups have observed mutator phenotypes in the germlines and somatic tissues of adults who carry cancer-predisposing inherited mutations in the POLE/POLD1 exonucleases [@PMID:34594041;@PMID:37336879]. Candidate mutator loci were also found by identifying human haplotypes from the Thousand Genomes Project with excess counts of derived alleles in genomic windows [@PMID:28095480].
In mice, a germline mutator allele was recently discovered by sequencing a large family of inbred mice [@PMID:35545679]. Commonly known as the BXDs, these recombinant inbred lines (RILs) were derived from either F2 or advanced intercrosses of C57BL/6J and DBA/2J, two laboratory strains that exhibit significant differences in their germline mutation spectra [@PMID:33472028;@PMID:30753674]. The BXDs were maintained via brother-sister mating for up to 180 generations, and each BXD therefore accumulated hundreds or thousands of germline mutations on a nearly-homozygous linear mosaic of parental B and D haplotypes. Due to their husbandry in a controlled laboratory setting, the BXDs were largely free from confounding by environmental heterogeneity, and the effects of selection on de novo mutations were attenuated by strict inbreeding [@doi:10.1146/annurev.ecolsys.39.110707.173437].
In this previous study, whole-genome sequencing data from the BXD family were used to map a quantitative trait locus (QTL) for the C>A mutation rate [@PMID:35545679].
Germline C>A mutation rates were nearly 50% higher in mice with D haplotypes at the QTL, likely due to genetic variation in the DNA glycosylase Mutyh that reduced the efficacy of oxidative DNA damage repair.
Pathogenic variants of Mutyh also appear to act as mutators in normal human germline and somatic tissues [@PMID:35803914;@PMID:30753674].
Importantly, the QTL did not reach genome-wide significance in a scan for variation in overall germline mutation rates, which were only modestly higher in BXDs with D alleles, demonstrating the utility of mutation spectrum analysis for mutator allele discovery.
Close examination of the mutation spectrum is likely to be broadly useful for detecting mutator alleles, as genes involved in DNA proofreading and repair often recognize particular sequence motifs or excise specific types of DNA lesions [@PMID:32619789].
Mutation spectra are usually defined in terms of
Although mutation spectrum analysis can enable the discovery of mutator alleles that affect the rates of specific mutation types, early implementations of this strategy have suffered from a few drawbacks.
For example, performing association tests on the rates or fractions of every
Here, we present a new mutation spectrum association test, called "aggregate mutation spectrum distance," that minimizes multiple testing burdens and mitigates the challenges of sparsity in de novo mutation datasets. We leverage this method to re-analyze germline mutation data from the BXD family and find compelling evidence for a second mutator allele that was not detected using previous approaches. The new allele appears to interact epistatically with the mutator that was previously discovered in the BXDs, further augmenting the C>A germline mutation rate in a subset of inbred mice. Our observation of epistasis suggests that mild DNA repair deficiencies can compound one another, as mutator alleles chip away at the redundant systems that collectively maintain germline integrity.