Genetic analysis of a mouse cross implicates an anti-inflammatory gene in control of atherosclerosis susceptibility

Abstract

Nearly all genetic crosses generated from Apoe^-/- or Lldlr^-/- mice for genetic analysis of atherosclerosis have used C57BL/6 J (B6) mice as one parental strain, thus limiting their mapping power and coverage of allelic diversity. SM/J-Apoe ^-/- and BALB/cJ-Apoe ^-/- mice differ significantly in atherosclerosis susceptibility. 224 male F₂ mice were generated from the two Apoe ^-/- strains to perform quantitative trait locus (QTL) analysis of atherosclerosis. F₂ mice were fed 5 weeks of Western diet and analyzed for atherosclerotic lesions in the aortic root. Genome-wide scans with 144 informative SNP markers identified a significant locus near 20.2 Mb on chromosome 10 (LOD score: 6.03), named Ath48, and a suggestive locus near 49.5 Mb on chromosome 9 (LOD: 2.29; Ath29) affecting atherosclerotic lesion sizes. Using bioinformatics tools, we prioritized 12 candidate genes for Ath48. Of them, Tnfaip3, an anti-inflammatory gene, is located precisely underneath the linkage peak and contains two non-synonymous SNPs leading to conservative amino acid substitutions. Thus, this study demonstrates the power of forward genetics involving the use of a different susceptible strain and bioinformatics tools in finding atherosclerosis susceptibility genes.

Figure 1

Frequency distributions of atherosclerotic lesion areas in male F₂ mice derived from BALB-Apoe^−/− and SM-Apoe^−/− mice. Left panel: the distribution of untransformed atherosclerotic lesion areas; right panel: the distribution of square-root transformed atherosclerotic lesion areas. Graphs were created using a plotting function of J/qtl software.

Figure 1

Frequency distributions of atherosclerotic lesion areas in male F₂ mice derived from BALB-Apoe^−/− and SM-Apoe^−/− mice. Left panel: the distribution of untransformed atherosclerotic lesion areas; right panel: the distribution of square-root transformed atherosclerotic lesion areas. Graphs were created using a plotting function of J/qtl software.

Figure 2

Genome-wide scans to search for main effect QTLs influencing atherosclerotic lesion areas in male F₂ mice. Chromosomes 1 through 20 are represented numerically on the X-axis. Each minor tick on the X axis represents one informative SNP. The Y-axis represents the LOD score. Two horizontal dashed lines denote genome-wide thresholds for suggestive (P=0.63) and significant (P=0.05) linkage. The upper panel (A) shows a genome-wide scan for atherosclerotic lesion areas using the non-parametric mode, and the lower panel (B) shows a genome-wide scan for atherosclerotic lesion areas using the parametric mode. Graphs were created by J/qtl

Figure 2

Genome-wide scans to search for main effect QTLs influencing atherosclerotic lesion areas in male F₂ mice. Chromosomes 1 through 20 are represented numerically on the X-axis. Each minor tick on the X axis represents one informative SNP. The Y-axis represents the LOD score. Two horizontal dashed lines denote genome-wide thresholds for suggestive (P=0.63) and significant (P=0.05) linkage. The upper panel (A) shows a genome-wide scan for atherosclerotic lesion areas using the non-parametric mode, and the lower panel (B) shows a genome-wide scan for atherosclerotic lesion areas using the parametric mode. Graphs were created by J/qtl

Figure 3

Interval mapping graphs of chromosomes 10 (left panel) and 9 (right panel) for atherosclerotic lesion areas created by MapManager QTX. The black plot represents LOD scores calculated at 1-cM intervals, the red plot denotes the effect of BALB alleles, and the blue plot represents the effect of SM alleles. The histogram in the plot estimates the confidence interval for a QTL. Two green vertical lines denote genome-wide significance thresholds for suggestive or significant linkage (P=0.63 and P=0.05, respectively).

Figure 3

Interval mapping graphs of chromosomes 10 (left panel) and 9 (right panel) for atherosclerotic lesion areas created by MapManager QTX. The black plot represents LOD scores calculated at 1-cM intervals, the red plot denotes the effect of BALB alleles, and the blue plot represents the effect of SM alleles. The histogram in the plot estimates the confidence interval for a QTL. Two green vertical lines denote genome-wide significance thresholds for suggestive or significant linkage (P=0.63 and P=0.05, respectively).

Figure 4

Comparison of selected Tnfaip3 protein sequences in three mammal species, including rat, human, B6 and BALB mice. Amino acid residues that are different among the species are highlighted.

Figure 5

3D structure of Tnfaip3 protein for B6 (left panel) and BALB (right panel) mice predicted by RaptorX software. The predicted 3D structure is noticeably different between the two mouse strains.

Figure 5

3D structure of Tnfaip3 protein for B6 (left panel) and BALB (right panel) mice predicted by RaptorX software. The predicted 3D structure is noticeably different between the two mouse strains.

Figure 6

Real-time analyses of Tnfaip3 and Gapdh mRNA expression in the liver of B6 and BALB Apoe^−/− mice fed a Western diet. Results were expressed as a ratio of Tnfaip3 to Gapdh in real-time PCR cycle threshold (Ct) values of 4 individual mice per strain. * P < 0.05.

Figure 7

Correlations of atherosclerotic lesion sizes with plasma levels of HDL, non-HDL cholesterol, triglyceride, and glucose in the F₂ mice. Each point represents values of an individual F₂ mouse. The correlation coefficient (R) and significance (P) are shown.