QTLs Associated with Gonadal Fat Mass
Characteristics of the B6.ApoE−/− and C3H.ApoE−/− parents and the F2 BXH.ApoE−/− generation on a Western diet are summarized in Table 1. Gonadal fat mass differed significantly between the sexes in F2 (p < 10−4) and in the parental C3H.ApoE−/− (p < 0.05), but not in B6.ApoE−/− mice. Gonadal fat mass was the fat pad collection that represented the most animals and the most accurate collections and was thus chosen for further analysis. Broad sense heritability (h 2) calculated as (σ2 Total − σ2 Parental)/σ2 Total for the gonadal fat mass trait was 54% for females and 36% for males, which is in close agreement with previous reports [18,19] and demonstrates significant heritability of gonadal fat mass.
Table 1  Characteristics of the BXH.ApoE−/− Cross A total of 334 F2 mice were genotyped at an average 1.5 cM density using 1,032 single nucleotide polymorphisms (SNPs) spanning all 19 autosomes. QTL analysis for several clinical traits (clinical QTLs [cQTLs]), including the unadjusted raw values for gonadal fat mass, was performed using a single marker regression approach (justified by the high-density of markers, making interval mapping unnecessary). In order to test specifically for sex effects of linkage, we included additive, dominant, sex, sex-additive, and sex-dominant parameters in our calculations (see Materials and Methods). A stepwise regression procedure was used to determine whether the addition of the final two terms significantly improved the linear regression model, conditional on realizing a significant additive QTL effect. We performed permutation analyses over all gene expression traits, estimating false discovery rates (FDRs) at different logarithm of odds (LOD) score thresholds and assessing the overall rate of QTL detection. From these analyses we constructed receiver operating characteristic (ROC)-like curves to demonstrate that our straightforward method has significantly increased power to detect QTLs compared to QTL mapping methods that do not incorporate sex and genotype–sex interactions (Figure S1). It is clear from the ROC curves that the sex and sex-interaction terms add significantly to the detection of QTL for the gene expression traits. Using previously described conventions [20], QTL models without the final two interaction terms (sex*add and sex*dom) have a suggestive threshold of 3.0 (p < 1 × 10−3) and a significant threshold of 4.3 (p < 5 × 10−5, genome-wide p < 0.05). QTL models incorporating only the sex*add interaction term in addition to the additive terms have one extra degree of freedom that leads to a corresponding increase in the LOD score thresholds to 3.5 (suggestive) and 4.9 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively. QTL models incorporating both sex*add and sex*dom interaction terms possess two extra degrees of freedom, with a corresponding increase in LOD score thresholds to 4.0 (suggestive) and 5.4 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively.
One suggestive (Chromosome 1) and four significant (Chromosomes 3, 5, 11, and 19) cQTLs for the gonadal fat mass trait were identified (Figure 1A; Table 2). Four out of the five cQTLs showed statistically significant better fits with the full model incorporating the interaction terms sex*add and sex*dom, compared to the model including only the additive terms. Interestingly, the cQTL over Chromosome 11 did not improve, suggesting that the additional terms did not contribute to improved detection of this locus. Table 2 summarizes the position and LOD score of maximal linkage for each cQTL. While the focus of this study was the gonadal fat mass trait, it is noted that a genome scan for the adiposity trait resulted in cQTLs at the same locations, with very similar LOD scores and sex dependence (unpublished data).
Figure 1  Genome Scan for Gonadal Fat Mass
(A) Animals were genotyped at an average 1.5 cM density using 1,032 SNPs polymorphic between the parental strains. LOD scores computed using sex as an additive covariate (black) failed to detect significant linkage. A genome scan accounting for interactions between sex and QTL (red) showed evidence for suggestive linkage on Chromosome 1 and significant linkage on Chromosomes 3, 5, 11, and 19. Dashed and solid lines are thresholds for suggestive (p < 1 × 10−3) and significant linkage (p < 5 × 10−5), respectively.
(B) Genome scans for gonadal fat mass using different models over mouse Chromosome 5. Scans for fat mass using all animals with (black) and without (green) sex as an additive covariate failed to detect significant linkage. Females analyzed alone (magenta) showed evidence for suggestive linkage (p < 2 × 10−4). When both sexes were analyzed to account for sex effects (red), a significant QTL was realized (p < 10−6).
For clarity, only the model incorporating both the “sex*add” and “sex*dom” terms is shown in red, although additional models incorporating the terms separately were also computed.
Table 2  cQTLs for Gonadal Fat Mass Results from the various regression models used to determine linkage for the Chromosome 5 cQTL are depicted in Figure 1B. Analysis of all animals with and without sex as a covariate failed to demonstrate evidence of linkage on Chromosome 5. When females were analyzed alone, a suggestive LOD score of 3.7 was realized (p = 2 × 10−4); males analyzed alone did not demonstrate evidence for linkage. However, using all 334 animals and adding the interaction terms to the QTL model significantly improved sensitivity, and a cQTL with a maximum LOD of 7.56 (p = 1.7 × 10−6) was realized.
Given the improved detection of four of the five cQTLs when sex-additive and sex-dominant interaction terms were considered, we hypothesized that the main genotype effect of these cQTLs on the gonadal fat mass trait would differ between the sexes (Figure 2). Indeed, cQTLs located on Chromosomes 1, 3, and 5 showed opposing effects on fat mass, or sex antagonism. The effect of the cQTL on Chromosome 11 was in the same direction in both males and females, but was sex-biased toward a larger effect in females (R 2 = 0.091 in females versus R 2 = 0.046 in males), confirming the minimal sex specificity of this cQTL. The cQTL on Chromosome 19 showed a sex-specific effect in females, with no effect in males.
Figure 2  Effect of Genotype on Fat Mass
Homozygous B6 (BB), C3H (CC), or heterozygous (BC) genotype at all five QTL positions, separated by sex, are shown. The underlying genotypic effects of the QTLs on fat mass differ between the sexes. Coefficients of determination (R 2) are shown along with associated ANOVAs *p < 0.05, **p < 0.01, ***p < 0.001. Overall, all five cQTLs for gonadal fat mass were biased toward a larger effect in females. Assuming purely additive effects of each genotype, these cQTLs account for approximately 42% of the variation in female F2 mice and 13% in males, consistent with the narrow sense heritability estimates for this trait and again demonstrating significant differences in the regulation and heritability of the gonadal fat trait between the sexes.