Signatures of replication timing, recombination, and sex in the spectrum of rare variants on the human X chromosome and autosomes

Abstract

The sources of human germline mutations are poorly understood. Part of the difficulty is that mutations occur very rarely, and so direct pedigree-based approaches remain limited in the numbers that they can examine. To address this problem, we consider the spectrum of low-frequency variants in a dataset (Genome Aggregation Database, gnomAD) of 13,860 human X chromosomes and autosomes. X-autosome differences are reflective of germline sex differences and have been used extensively to learn about male versus female mutational processes; what is less appreciated is that they also reflect chromosome-level biochemical features that differ between the X and autosomes. We tease these components apart by comparing the mutation spectrum in multiple genomic compartments on the autosomes and between the X and autosomes. In so doing, we are able to ascribe specific mutation patterns to replication timing and recombination and to identify differences in the types of mutations that accrue in males and females. In particular, we identify C > G as a mutagenic signature of male meiotic double-strand breaks on the X, which may result from late repair. Our results show how biochemical processes of damage and repair in the germline interact with sex-specific life history traits to shape mutation patterns on both the X chromosome and autosomes.

Keywords: DNA damage and repair; X chromosome vs. autosome; germline mutation spectrum; sex differences in mutation; signatures of replication and recombination.

Fig. 1.

(A) A schematic of genomic compartments on the X chromosome and autosomes. Three compartments on the X chromosome are depicted: the PAR, regions of the X that escape inactivation (E), and regions of the X that undergo inactivation (I). Also depicted are 2 hypothetical autosomal compartments with distinct biochemical properties (c1 and c2). Analyses include pairwise comparisons of mutational patterns between autosomal compartments and between the X chromosome and autosomes. (B) The frequency spectrum of variants (n = 120,521,915) in the 13,860 chromosomes analyzed. Over two-thirds of mutations are present at 3 copies or less in the sample.

Fig. 2.

The effect of replication timing on the mutation spectrum at different scales, using replication timing scores from the LCL cell line. (A) Comparison of the spectrum of 96 mutation types in late replicating (score ≤ −0.5) autosomal regions relative to early replicating (score ≥ 0.5) autosomal regions. Positive and negative effects have been separately ordered by effect size from left to right; only the top 50 significant positive and negative effects are shown for legibility. The size of the circle reflects the number of mutations of that type. (B) For each of 6 mutational classes, the enrichment in 1-Mb windows relative to all other autosomal windows combined, ordered by the mean replication timing. Positive replication timing scores indicate earlier than average replication. Autosomal windows are shown in solid light gray circles; windows on the X chromosome have been overlaid in black hollow circles. (C) For each of 6 mutational classes, enrichment on individual autosomes and X relative to all other autosomes combined, ordered by the mean replication timing. Positive replication timing scores indicate earlier than average replication.

Fig. 3.

Comparison of the mutation spectrum on the X chromosome and autosomes. The PAR and CpG sites are excluded from this analysis. Only significant differences are shown. Positive and negative effects have been separately ordered by effect size from left to right. The size of the circle reflects the number of mutations of that type. (A) Enrichment of mutation types on the X chromosome relative to autosomes. (B) Enrichment of mutation types in the genic compartment of the X chromosome that escapes inactivation, relative to genic regions on autosomes. Hollow circles represent mutation types enriched (or depleted) in both the escaped (i.e., active) and inactive compartments of the X relative to autosomes. (C) Enrichment of mutation types in the genic X chromosome compartment that undergoes X inactivation, relative to genic regions on autosomes. Hollow circles represent mutation types enriched (or depleted) in both the escaped (i.e., active) and inactive compartments of the X relative to autosomes. The larger number of significant differences in C compared to B likely reflects at least in part the approximately 5-fold greater amount of data in the inactive versus the active genic regions of the X chromosome.

Fig. 4.

The mutation spectrum on the X and autosomes matched for average replication timing. The PAR and CpG sites are excluded from this analysis. (A) Comparison of the mutation spectrum on the X chromosome and autosomes matched for average replication timing. Only significant differences are shown. Positive and negative effects have been separately ordered by effect size from left to right. Hollow circles represent mutation types also enriched (or depleted) in both the active and inactive compartments of the X relative to autosomes (Fig. 3 B and C), and in both early and late replicating regions of the X relative to autosomes (SI Appendix, Fig. S14). Crosses denote mutation types reported as significant sex differences by ref. . (B) The X-autosome spectrum for 6 mutation classes, controlling for mean replication timing (in red), compared to known male-female differences from ref. (in black). Solid points are statistically significant differences at the 5% level, accounting for multiple tests.

Fig. 5.

Distribution of C > G mutations in genomic compartments relative to autosomes. CpG sites are excluded from these analyses. (A) The mutation spectrum on PAR1 relative to autosomes. Only significant differences are shown. Positive and negative effects have been separately ordered by effect size from left to right. (B) Enrichment of C > G mutations in DMC1 hotspots of varying intensity on autosomes. For each estimate, the 95% confidence interval from a binomial test is represented by the vertical bars (and is sometimes too small to be apparent). The horizontal black bar shows the reference, namely autosomes outside DMC1 hotspots and excluding regions rich in clustered de novo mutations (identified by ref. 29). (C) Enrichment of C > G mutations in DMC1 hotspots of varying intensity on the X chromosome, relative to autosomes outside DMC1 hotspots and excluding autosomal regions rich in clustered de novo mutations. For each estimate, the 95% confidence interval from a binomial test is represented by the vertical bars. The reference, same as for B, is denoted by a horizontal black bar.