Thousands of Genes Show a Sex-Specific Correlation with Gonadal Fat Mass
For each of the 23,574 oligonucleotides represented on the array, we computed a linear regression analysis to test for association between the trait “gonadal fat mass” and each transcript abundance measure, incorporating the terms “gene,” “sex,” and “gene-by-sex,” where the “gene-by-sex” parameter tests for sex-specific correlation between a gene and the trait. As before, a stepwise regression procedure was used to determine if the addition of the interaction term significantly improved the model fit (see Materials and Methods). Multiple testing was addressed by controlling for the FDR. Distribution of the p-values obtained from these 23,574 correlations is shown in Figure 5A. At FDR = 0.01, 4,613 genes were significantly correlated with gonadal fat mass. Of these genes, 4,524 (98%) showed significant “gene-by-sex” effects, supporting the high degree of sex specificity in the genetic regulation of this trait. A complete list of all genes correlated with gonadal fat mass is provided in Table S2.
Figure 5  Properties of Transcripts Significantly Correlated with Gonadal Fat Mass
(A) Distribution of p-values for trait–gene correlations between transcripts and gonadal fat mass. At FDR = 0.01, 4,613 transcripts are significantly correlated with the trait.
(B) Number of eQTLs generated by the 4,613 genes significantly correlated with gonadal fat mass. Of these, 1,130 genes possessed at least one significant eQTL.
(C) Distribution of 1,478 eQTLs significantly correlated with gonadal fat mass across the genome in 2-cM bins.
(D) Identification of genomic regions enriched for eQTLs correlated with gonadal fat mass. The x-axis represents genome position in 2-cM bins, and the y-axis represents the −log10 Fisher exact test p-value for enrichment of eQTLs in overlapping 6-cM bins. The dashed line corresponds to p = 0.05 after correction for multiple comparisons. One significantly enriched region on Chromosome 19 is shown. The Chromosome 19 (40-cM) hotspot is coincident with a cQTL for gonadal fat mass and is highlighted in red. Of the 4,613 genes correlated with gonadal fat mass, 1,130 generate 1,478 significant eQTLs (Figure 5B). The colocalization of eQTLs for these correlated genes with the cQTL for the fat mass trait provides useful implications for the possible role of these genes. Whether the eQTLs are cis or trans determines what that role may be. Genes that show significant correlation with the gonadal fat mass trait and that have cis-eQTLs coincident with the fat mass cQTLs are potential candidate genes for the trait (i.e., they may contain a genetic variation in that gene that is the cause of the trait cQTL). Table 5 summarizes the genes that possess these properties for each cQTL, increasing evidence for these genes as potential candidates. As addressed below, given the complex multiorgan regulation of adipose tissue mass, it is unlikely that the genetic regulation of all five loci resides in the liver. However, some may involve the liver, and even for those that do not, the liver transcriptional variation may reflect that of the relevant tissue.
Genes that show significant correlation with gonadal fat mass and have trans-eQTLs coincident with the fat mass cQTL cannot be candidates directly responsible for the trait, as they are physically located elsewhere in the genome. However, they are potentially involved in the pathway(s) leading from the causative gene to the expression of the fat mass trait (i.e., their transcription is closely regulated by the causative gene at the locus). All of the five fat mass cQTLs have colocalizing trans-eQTLs for correlated genes. However, for a trait such as fat mass that is regulated by multiple tissues and organs, it is unlikely that all five fat mass cQTLs are primarily driven by liver gene expression. As an approach to this problem, we hypothesized that those cQTLs that are most closely associated with liver gene expression would show an overrepresentation of colocalized eQTL for correlated genes, while those loci primarily controlled by other tissues would not have shown this pattern.
To assess this, we first determined the distribution of these 1,478 eQTLs across the genome in 2-cM bins as shown in Figure 5C. In order to see if there exist any hotspots for these eQTLs, we tested eQTLs with p < 0.001 for enrichment along the genome in overlapping 2-cM bins against the distribution of all liver eQTLs (Figure 4A) using a Fisher exact test. Figure 5D shows the significance of enrichment reported as −log10 of the enrichment p-value across the genome. One locus on Chromosome 19 was significantly enriched for eQTLs of transcripts correlated with the gonadal fat mass trait. As anticipated, there was an overlap of a correlated eQTL hotspot and a fat mass cQTL, specifically Chromosome 19 at 40 cM. This suggests that the genetic regulation of fat mass for the Chromosome 19 locus is more closely tied to liver gene expression than are the other four fat mass cQTLs.
The effect of the trans-eQTLs at the Chromosome 19 locus on gene expression is summarized in Figure 6. Twenty-nine trans-eQTLs colocalize to Chromosome 9 at 40 cM, suggesting that 29 genes correlated with gonadal fat mass are regulated in trans by a polymorphism at this position. The proportion of gene expression levels controlled by this locus (approximated as the coefficients of determination R 2) differs between males and females for the majority of the transcripts (as in Figure 6A), and for Chromosome 19 (Figure 6B), females demonstrated greater genetic regulation of gene expression than males. This substantial female bias is significantly higher than would be expected to arise by chance for the Chromosome 19 locus (p < 0.001 by χ2). This locus corresponds to one of the four sex-biased cQTLs for gonadal fat mass reported in this study, and the significant sex specificity of both the cis and trans genetic regulation of liver genes correlated with fat mass supports the functional significance of this locus in this organ.
Figure 6  The Effects of Sex on Trans-eQTL Correlated with Gonadal Fat Mass
(A) Example of the effect of genotype at a trans locus on gene expression. Presence of homozygous B6 (BB), C3H (CC), or heterozygous (BC) genotype at a trans locus affects transcript MMT00016118 levels (reported as mlratio) in a sex-specific manner, with effects detectable only in females. Coefficients of determination (R 2, or proportion variance explained) are shown along with associated ANOVA p-values. Several trans-eQTLs correlated with gonadal fat mass localize to regions overlapping with cQTLs for this trait, specifically, to Chromosome 19, 40 cM.
(B) For Chromosome 19, the vast majority of these correlated trans-eQTLs are biased toward larger effects on gene expression in females (red lines). The effect of any given trans-eQTL is approximated as R 2 determined in a manner similar to that depicted in (A).