Impact of the X Chromosome and sex on regulatory variation

Abstract

The X Chromosome, with its unique mode of inheritance, contributes to differences between the sexes at a molecular level, including sex-specific gene expression and sex-specific impact of genetic variation. Improving our understanding of these differences offers to elucidate the molecular mechanisms underlying sex-specific traits and diseases. However, to date, most studies have either ignored the X Chromosome or had insufficient power to test for the sex-specific impact of genetic variation. By analyzing whole blood transcriptomes of 922 individuals, we have conducted the first large-scale, genome-wide analysis of the impact of both sex and genetic variation on patterns of gene expression, including comparison between the X Chromosome and autosomes. We identified a depletion of expression quantitative trait loci (eQTL) on the X Chromosome, especially among genes under high selective constraint. In contrast, we discovered an enrichment of sex-specific regulatory variants on the X Chromosome. To resolve the molecular mechanisms underlying such effects, we generated chromatin accessibility data through ATAC-sequencing to connect sex-specific chromatin accessibility to sex-specific patterns of expression and regulatory variation. As sex-specific regulatory variants discovered in our study can inform sex differences in heritable disease prevalence, we integrated our data with genome-wide association study data for multiple immune traits identifying several traits with significant sex biases in genetic susceptibilities. Together, our study provides genome-wide insight into how genetic variation, the X Chromosome, and sex shape human gene regulation and disease.

Figure 1.

Differential expression variance within the sexes. (A) Comparison of genes with significant sex-specific expression variance (FDR 5%) and sex-specific expression (FDR 5%) on the autosomes and the X Chromosome in the DGN. To test for differences in mean expression and variance, the number of males and females were matched (n = 274). One-sided Fisher's exact test P-values above bars indicate significance of higher expression or variance on the X Chromosome relative to autosomes. (B) Proportion of variance explained (PVE) by genotype on the X Chromosome in males and females. To test for the PVE, the number of males and females were matched (n = 274). We tested all genes on the X Chromosome and genes with a cis-eQTL (Bonferroni adjusted P-value < 0.05).

Figure 2.

Characterization of eQTLs on the X Chromosome. (A) Proportion of genes with an eQTL at different FDR thresholds discovered in the joint (n = 922), (B) female (n = 648), and (C) male (n = 274) populations. (**) P-value < 1 × 10⁻¹⁵, (*) P-value < 0.05, Bonferroni adjusted χ² test. (D) Comparison of eQTL effect size between autosomes and the X Chromosome for common variants (MAF ≥ 0.05) and low-frequency variants (0.01 < MAF < 0.05). The difference in effect size is statistically significant for both common ([*] P-value = 0.0382, two-sided Wilcoxon rank-sum test) and low-frequency variants ([**] P-value = 1.21 × 10⁻⁷).

Figure 3.

Discovery of sex-interacting eQTLs. (A) Quantile-quantile (QQ) plot describing the sex-interacting eQTL association P-values for SNPs tested within 1 Mb of genes on the X Chromosome (orange) and the autosomes (green) with 95% confidence interval (gray). (B) Sex-interacting eQTL (q-value = 0.0198) for DNAH1 (dynein, axonemal, heavy chain 1), a protein-coding gene involved in microtubule motor activity, ATPase activity, and sperm motility.

Figure 4.

Discovery of sex-specific chromatin accessibility regions. (A) QQ plot for tests of differential chromatin accessibility between the sexes. 95% genome-wide confidence interval in gray. (B) Enrichment of genes with differential expression between the sexes (FDR 5%) with differential chromatin accessibility (varying thresholds) 40 kb upstream. (**) P-value < 10⁻⁵, (*) P-value < 10⁻³, Fisher's exact test. (C) Proportion of sex-interacting eQTL genes (P-value < 0.05) in differential chromatin accessibility regions (varying thresholds). (*) P-value < 5.0 × 10⁻², Fisher's exact test. (D) Chromatin accessibility peak located at Chr 7: 95,063,722–95,064,222 and 5000 bp upstream and downstream. This region has differential chromatin accessibility between males and females (nominal P-value = 4.1 × 10⁻⁴, Q-value = 7.5 × 10⁻²). (E) Sex-interacting eQTL for PON2 and rs35903871 located at Chr 7: 95,063,972 (nominal P-value = 8.0 × 10⁻³).

Figure 5.

Disease associated variants with sex-biased eQTLs. Proportion of independent (LD-pruned) variants with higher eQTL effect sizes in females for GWAS variants of traits in ImmunoBase. Each trait variant tested had to be a significant eQTL variant (nominal P-value < 0.001). Red data points indicate traits with eQTL effect sizes that are significantly different between sexes (Bonferroni adjusted P-value < 0.05). (ATD) Autoimmune thyroid disease, (CEL) celiac disease, (JIA) juvenile idiopathic arthritis, (MS) multiple sclerosis, (PBC) primary biliary cirrhosis, (PSO) psoriasis, (RA) rheumatoid arthritis, (RND) random eQTL variants, (T1D) type 1 diabetes.