Fine mapping of a linkage peak with integration of lipid traits identifies novel coronary artery disease genes on chromosome 5

Abstract

Background: Coronary artery disease (CAD), and one of its intermediate risk factors, dyslipidemia, possess a demonstrable genetic component, although the genetic architecture is incompletely defined. We previously reported a linkage peak on chromosome 5q31-33 for early-onset CAD where the strength of evidence for linkage was increased in families with higher mean low density lipoprotein-cholesterol (LDL-C). Therefore, we sought to fine-map the peak using association mapping of LDL-C as an intermediate disease-related trait to further define the etiology of this linkage peak. The study populations consisted of 1908 individuals from the CATHGEN biorepository of patients undergoing cardiac catheterization; 254 families (N = 827 individuals) from the GENECARD familial study of early-onset CAD; and 162 aorta samples harvested from deceased donors. Linkage disequilibrium-tagged SNPs were selected with an average of one SNP per 20 kb for 126.6-160.2 MB (region of highest linkage) and less dense spacing (one SNP per 50 kb) for the flanking regions (117.7-126.6 and 160.2-167.5 MB) and genotyped on all samples using a custom Illumina array. Association analysis of each SNP with LDL-C was performed using multivariable linear regression (CATHGEN) and the quantitative trait transmission disequilibrium test (QTDT; GENECARD). SNPs associated with the intermediate quantitative trait, LDL-C, were then assessed for association with CAD (i.e., a qualitative phenotype) using linkage and association in the presence of linkage (APL; GENECARD) and logistic regression (CATHGEN and aortas).

Results: We identified four genes with SNPs that showed the strongest and most consistent associations with LDL-C and CAD: EBF1, PPP2R2B, SPOCK1, and PRELID2. The most significant results for association of SNPs with LDL-C were: EBF1, rs6865969, p = 0.01; PPP2R2B, rs2125443, p = 0.005; SPOCK1, rs17600115, p = 0.003; and PRELID2, rs10074645, p = 0.0002). The most significant results for CAD were EBF1, rs6865969, p = 0.007; PPP2R2B, rs7736604, p = 0.0003; SPOCK1, rs17170899, p = 0.004; and PRELID2, rs7713855, p = 0.003.

Conclusion: Using an intermediate disease-related quantitative trait of LDL-C we have identified four novel CAD genes, EBF1, PRELID2, SPOCK1, and PPP2R2B. These four genes should be further examined in future functional studies as candidate susceptibility loci for cardiovascular disease mediated through LDL-cholesterol pathways.

Figure 1

Quantitative Associations by Base Pair Position. This figure displays the results of association of SNPs with LDL cholesterol traits in the GENECARD, CATHGEN and aorta samples, with -log10 of the p-value (Y-axis) versus the base pair position of the SNP (X-axis). The five candidate genes are labeled with their approximate positions indicated by a horizontal bar.

Figure 2

Qualitative Associations by Base Pair Position. This figure displays the results of association of SNPs with CAD/atherosclerosis in the GENECARD, CATHGEN and aorta samples, with -log10 of the p-value (Y-axis) versus the base pair position of the SNP (X-axis). The five candidate genes are labeled with their approximate positions indicated by a horizontal bar; other select genes with significant results are labeled and have no horizontal bar.

Figure 3

Analytical Strategy for 'Parallel Analysis' of Chromosome 5q31 Region. This figure details the study design, cardiovascular cohorts and analytic techniques used, and the number of unique genes containing SNPs with significant associations (indicated underneath the method used). The union of quantitative results (α) indicates the subset of genes shared by the two methods. The qualitative trait (CVD) was analyzed in GENECARD, CATHGEN, and aortas and the total number of unique genes containing a significant SNP in any of those analyses is indicated. The commonality of the genes between the quantitative and qualitative analyses (N = 9, PRELID2, SPOCK1, EBF1, PPP2R2B, DMXL1, DTWD2, GABRG2, GLRA1, and RP11-166A12.1) is indicated by ω.