An integrative genetics approach to identify candidate genes regulating BMD: combining linkage, gene expression, and association

Abstract

Numerous quantitative trait loci (QTLs) affecting bone traits have been identified in the mouse; however, few of the underlying genes have been discovered. To improve the process of transitioning from QTL to gene, we describe an integrative genetics approach, which combines linkage analysis, expression QTL (eQTL) mapping, causality modeling, and genetic association in outbred mice. In C57BL/6J x C3H/HeJ (BXH) F(2) mice, nine QTLs regulating femoral BMD were identified. To select candidate genes from within each QTL region, microarray gene expression profiles from individual F(2) mice were used to identify 148 genes whose expression was correlated with BMD and regulated by local eQTLs. Many of the genes that were the most highly correlated with BMD have been previously shown to modulate bone mass or skeletal development. Candidates were further prioritized by determining whether their expression was predicted to underlie variation in BMD. Using network edge orienting (NEO), a causality modeling algorithm, 18 of the 148 candidates were predicted to be causally related to differences in BMD. To fine-map QTLs, markers in outbred MF1 mice were tested for association with BMD. Three chromosome 11 SNPs were identified that were associated with BMD within the Bmd11 QTL. Finally, our approach provides strong support for Wnt9a, Rasd1, or both underlying Bmd11. Integration of multiple genetic and genomic data sets can substantially improve the efficiency of QTL fine-mapping and candidate gene identification.

FIG. 1

Genome scan results for BMD in BXH F₂ mice. (Top) LOD score plots for BMD using an additive model adjusting for sex, cross direction, and weight. The dashed horizontal lines represent the permutationally derived significant (p < 0.05) and suggestive (p < 0.63) LOD thresholds. (Bottom) LOD score plots for BMD using a sex interaction model adjusting for sex, cross direction, weight, and sex × QTL. The dashed horizontal lines represent the permutationally derived significant (p < 0.05) and suggestive (p < 0.63) LOD thresholds.

FIG. 2

Sex-specific BMD QTL analysis in BXH F₂ mice. LOD score plots for BMD using an additive model (adjusting for cross direction and weight) in males (solid line) and females (dashed line) separately. The dashed horizontal lines represent the permutationally derived significant (p < 0.05) and suggestive (p < 0.63) LOD thresholds for males (male and female thresholds were only slightly different).

FIG. 3

Effect plots for BMD QTLs in BXH F₂ mice. Genotypic contrasts are plotted for each of the nine BMD QTLs identified in Fig. 1. The mean (± SE) BMD residuals (mg/cm²) (after adjusting for body weight and cross direction) are plotted for B6/B6 homozygotes (black line), B6/C3H heterozygotes (wide dashed line), and C3H/C3H homozygotes (narrow dashed line) for each sex and QTL.

FIG. 4

Chromosome 11 (Bmd11) SNPs are associated with whole body BMD in MF1 mice. Each panel contains the association results plotted as the negative log₁₀ of the ANOVA p value (−logP) for MF1 markers overlapping each of the nine BXH BMD QTL regions. Individual SNPs are represented by vertical bars. The solid horizontal lines represent the nominal p < 0.001 level of significance. The empiric p value for the most significant association on Chr 11 (determined by permutation) was p = 0.06. The 95% bootstrap CI for the Bmd11 association (2.8 Mbp) is highlighted by the dark horizontal line at the top of the Bmd11 (Chr 11) panel.

FIG. 5

An integrative genetics approach identified Wnt9a and Rasd1 as candidate QTGs for Bmd11. (Top) LOD score profile for Bmd11 in BXH F₂ mice. The 1.5 LOD-drop SI extended from 25.7 to 64.1 Mbp (defined by vertical lines). (Middle) Frequency of SNPs polymorphic between C57BL/6J and C3H/HeJ per 25 kbp bin across Bmd11. A total of 15 genes with prioritized local eQTL peaks were located within the Bmd11 SI interval (●) (Supplemental Table 1). (Bottom) Negative log₁₀ of the ANOVA p value (−logP) for each Bmd11 SNP in MF1 mice. The three most associated SNPs had empiric p values of p = 0.06. The 95% bootstrap CI for the Bmd11 association (2.8 Mbp) is highlighted by the dark horizontal line at the top of the Bmd11 (Chr 11) panel. Six of the genes with eQTL were predicted to be causal using NEO (●). Wnt9a and Rasd1 were the only genes located in the region, regulated by local eQTLs, and predicted to be causal. The x-axis for all panels is in base pairs.