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A natural mutator allele shapes mutation spectrum variation in mice

Abstract

Although germline mutation rates and spectra can vary within and between species, common genetic modifiers of the mutation rate have not been identified in nature1. Here we searched for loci that influence germline mutagenesis using a uniquely powerful resource: a panel of recombinant inbred mouse lines known as the BXD, descended from the laboratory strains C57BL/6J (B haplotype) and DBA/2J (D haplotype). Each BXD lineage has been maintained by brother–sister mating in the near absence of natural selection, accumulating de novo mutations for up to 50 years on a known genetic background that is a unique linear mosaic of B and D haplotypes2. We show that mice inheriting D haplotypes at a quantitative trait locus on chromosome 4 accumulate C>A germline mutations at a 50% higher rate than those inheriting B haplotypes, primarily owing to the activity of a C>A-dominated mutational signature known as SBS18. The B and D quantitative trait locus haplotypes encode different alleles of Mutyh, a DNA repair gene that underlies the heritable cancer predisposition syndrome that causes colorectal tumors with a high SBS18 mutation load3,4. Both B and D Mutyh alleles are present in wild populations of Mus musculus domesticus, providing evidence that common genetic variation modulates germline mutagenesis in a model mammalian species.

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Fig. 1: Accumulation of homozygous singletons over many generations of laboratory inbreeding.
Fig. 2: A QTL on chromosome 4 for the germline C>A mutation rate.
Fig. 3: Context-dependent C>A mutation enrichment in lines with the D haplotype at the C>A QTL.
Fig. 4: Nonsynonymous differences between B and D Mutyh alleles segregate in both wild and inbred mouse strains and appear to be ancestral in DBA/2J.

Data availability

BXD mutations and other data files necessary to reproduce the manuscript are available at https://github.com/tomsasani/bxd_mutator_manuscript (archived at Zenodo (https://doi.org/10.5281/zenodo.5941048)). A VCF file containing variant calls from the sequenced BXDs is available in the European Nucleotide Archive with project accession PRJEB45429. The germline mutation calls from TOY-KO triple knockout mice24 are available as supplementary data file 1 from https://doi.org/10.1038/srep04689. The SBS18 COSMIC mutation signature data are available at the COSMIC web page: https://cancer.sanger.ac.uk/cosmic/signatures/SBS/SBS18.tt. The strain-private mutation data from Dumont17 are available as supplementary data from the following: https://doi.org/10.1093/molbev/msz026. The wild mouse data from Harr et al.35 are available at https://wwwuser.gwdg.de/~evolbio/evolgen/wildmouse/, as described in the manuscript at https://doi.org/10.1038/sdata.2016.75. The mm10/GRCm38 reference genome used for these analyses is version GCA_000001635.2, and can be obtained at https://hgdownload.soe.ucsc.edu/goldenPath/mm10/bigZips/mm10.fa.gz.

Code Availability

All code used for data analysis and figure generation is deposited at https://github.com/tomsasani/bxd_mutator_manuscript (archived at Zenodo (https://doi.org/10.5281/zenodo.5941048)).

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Acknowledgements

We thank B. Dumont, M. Gymrek, M. Mortazavi, A. Clark, D. Rothschild, U. Arora, M. Maksimov, H. Chen and J. Sebat for contributing helpful feedback as part of the BXD Genome Sequencing Consortium; M. Przeworski for providing comments on a manuscript draft; Y.-Y. Ren for assistance with preliminary genotype calling; members of the Harris and Pritchard laboratories for additional helpful discussions; the staff of HudsonAlpha—S. Levy and team—for DNA library preparation and sequencing, and for providing us with great support on data transfer; staff at the UT ISAAC facility for storage and processing of all sequence-associated data files; A. Centeno for assisting with the upload of phenotype data to GeneNetwork; C. Lutz and A. Valenzuela and J. Ingels at UTHSC for DNA sample acquisition, handling and assistance. We acknowledge support from NIH T32 Postdoctoral Training Grant 5T32HG000035-25 (to T.A.S.), a University of Tennessee Center for Integrative and Translational Genomics grant (to R.W.W., D.G.A. and L.L.), NIH Biological Mechanisms of Healthy Aging Training Grant T32AG066574 (to A.C.B.), NIH NIDA grant P50DA037844 (to A.A.P.), NIH NIDA grant U01DA051234 (to A.A.P.), NIH NIGMS grant 1R01GM123489 (to R.W.W.), NIH NIDA grant P30 DA044223 (to R.W.W.), NIH R01 HG008140 (to J.K.P.), NIH NIGMS grant 1R35GM133428-01 (to K.H.), a Searle Scholarship (to K.H.), a Sloan Research Fellowship (to K.H.), a Burroughs Wellcome Fund Career Award at the Scientific Interface (to K.H.) and a Pew Biomedical Scholarship (to K.H.).

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Contributions

T.A.S., D.G.A., A.A.P., R.W.W., J.K.P. and K.H. contributed to study conceptualization and design. D.G.A., L.L., J.K.P., A.A.P. and R.W.W. contributed to data curation, including maintenance of the BXD family, DNA sequencing and data warehousing. T.A.S., D.G.A., K.H. and A.C.B. contributed to formal data analysis. T.A.S. and K.H. wrote the original draft of the manuscript. T.A.S., D.G.A., A.C.B., A.A.P., R.W.W., J.K.P. and K.H. contributed to review and editing of the manuscript.

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Correspondence to Kelley Harris.

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Extended data figures and tables

Extended Data Fig. 1 Cross design for BXD RIL construction.

(a) BXDs derived from F2 crosses were subject to many generations of brother-sister mating in order to generate inbred RILs. The genomes of the parents of the BXD crosses (DBA/2J and C57BL/6J) are largely derived from Mus musculus domesticus. M. m. d. is Mus musculus domesticus, M. s. is Mus spretus, M. m. m. is Mus musculus musculus, and M. m. c. is Mus musculus castaneus. (b) To generate advanced intercross lines (AILs), pseudo-random pairs of F2 animals were crossed for N generations, and then subject to many generations of brother-sister mating to generate inbred RILs.

Extended Data Fig. 2 Generation times in the BXD lines.

a) The elapsed time since the founding of each BXD line (n = 130 biologically independent animals) was calculated by subtracting its initial breeding date from 2017. The elapsed number of years was then divided by the cumulative number of generations of inbreeding undergone by the line, to obtain an estimate of the line’s generation time in years. Boxplots are centered at the median of each distribution, with lower and upper hinges corresponding to the 25th to 75th percentiles (i.e., first and third quartiles), and whiskers extending to no further than 1.5 times the interquartile range from either hinge; data points outside of the range defined by the whiskers are displayed as individual points. b) A linear model predicting the C>A singleton fraction of each line as a function of both generation time and the line’s epoch of origin was trained using the BXD singleton mutations. C>A fraction is not significantly correlated with generation time (F = 0.055, DoF = 1, p = 0.815).

Extended Data Fig. 3 Singletons are enriched in highly conserved regions of the genome.

Cumulative distributions of phastCons conservation probabilities of either singletons (n = 47,659 mutations) or “fixed” variants (n = 81,186 mutations) that were randomly sampled from non-overlapping 50-kbp windows across the genome. The latter were present in a founder genome and inherited by all BXDs with the founder’s haplotype at that site. P-value of one-sided Kolmogorov-Smirnov test comparing distributions of phastCons scores is 3.8 x 10−53. Shaded area around each line indicates the bootstrap 95% confidence interval.

Extended Data Fig. 4 Results of QTL scans for other mutation rate phenotypes.

a) Using the same BXD lines and covariates as described in the Online Methods, a QTL scan was performed for the overall mutation rate of each line. The green dashed line indicates the genome-wide significance threshold using 1,000 permutations (Bonferroni-corrected alpha = 0.05/15). b)Using the same BXD lines and covariates as described in the Online Methods, QTL scans were performed for the rates and fractions of all mutation types other than C>A. Green and blue dashed lines indicate the genome-wide significance thresholds for the rate and fraction scans, respectively, using 1,000 permutations (Bonferroni-corrected alpha = 0.05/15).

Extended Data Fig. 5 3-mer mutation sequence contexts enriched in DBA/2J-like Mouse Genomes Project strains.

For each mutation type defined by its 3-mer sequence context, we can compute its Log-2 compositional enrichment in BXD strains with D vs. B haplotypes at the QTL on chromosome 4, as well as its log-2 compositional enrichment in Sanger Mouse Genomes Project strains that are D-like vs. B-like. These two odds ratios are correlated, indicating that the same mutational signature is enriched in the BXD D strains and the D-like Sanger MGP strains. Mutation types significantly enriched in BXDs with D haplotypes are colored red, outlined in black and labeled.

Extended Data Fig. 6 Site frequency spectra of C>A mutations in the four Mus species/subspecies on chromosome 4.

The site frequency spectra of M.m. domesticus, M.m. castaneus, M.m. musculus, and M. spretus were computed using a dataset of publicly available wild mouse genomes and a polarized version of the GRCm38/mm10 reference genome. Mmd is Mus musculus domesticus, Ms is Mus spretus, Mmm is Mus musculus musculus, and Mmc is Mus musculus castaneus.

Extended Data Fig. 7 Comparisons of singleton spectra between wild M.m.domesticus and other wild species.

Log-2 ratios of singleton fractions of each 3-mer mutation type in Mus musculus domesticus, compared to three other wild subspecies or species of Mus. Comparisons with Chi-square test of independence p-values < 0.05/96 are annotated with white circles.

Extended Data Fig. 8 Comparisons of singleton spectra between wild mouse populations in the genomic neighborhood of Mutyh.

Singleton fractions of each mutation type in each wild species or subspecies were computed in 50-kilobase pair windows in the QTL interval surrounding Mutyh (114.8 Mbp to 118.3 Mbp). The median absolute deviations of C>A fractions in the species or subspecies were: 0.00985 (Mmc), 0.0195 (Mmd), 0.0155 (Mmm), and 0.0214 (Ms).

Extended Data Table 1 Numbers and provenance of BXD lines analyzed in this manuscript
Extended Data Table 2 Mutyh missense mutations in the BXD family

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Sasani, T.A., Ashbrook, D.G., Beichman, A.C. et al. A natural mutator allele shapes mutation spectrum variation in mice. Nature 605, 497–502 (2022). https://doi.org/10.1038/s41586-022-04701-5

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