Genetic associations with expression for genes implicated in GWAS studies for atherosclerotic cardiovascular disease and blood phenotypes

Abstract

Genome-wide association studies (GWAS) have uncovered many genetic associations for cardiovascular disease (CVD). However, data are limited regarding causal genetic variants within implicated loci. We sought to identify regulatory variants (cis- and trans-eQTLs) affecting expression levels of 93 genes selected by their proximity to SNPs with significant associations in prior GWAS for CVD traits. Expression levels were measured by qRT-PCR in leukocytes from 1846 Framingham Heart Study participants. An additive genetic model was applied to 2.5 million imputed SNPs for each gene. Approximately 45% of genes (N = 38) harbored at least one cis-eSNP after a regional multiple-test adjustment. Applying a more rigorous significance threshold (P < 5 × 10(-8)), we found the expression level of 10 genes was significantly associated with more than one cis-eSNP. The top cis-eSNPs for 7 of these 10 genes exhibited moderate-to-strong association with ≥ 1 CVD clinical phenotypes. Several eSNPs or proxy SNPs (r(2) = 1) were replicated by other eQTL studies. After adjusting for the lead GWAS SNPs for the 10 genes, expression variances explained by top cis-eSNPs were attenuated markedly for LPL, FADS2 and C6orf184, suggesting a shared genetic basis for the GWAS and expression trait. A significant association between cis-eSNPs, gene expression and lipid levels was discovered for LPL and C6orf184. In conclusion, strong cis-acting variants are localized within nearly half of the GWAS loci studied, with particularly strong evidence for a regulatory role of the top GWAS SNP for expression of LPL, FADS2 and C6orf184.

Figure 1.

Regional plots and box plots for gene LPL and FADS2 top cis-SNPs whose signals were attenuated markedly after adjusting for the lead GWAS SNPs. Top panel: observed –log₁₀(P) and recombination rates by chromosomal position for all associated SNPs near rs6993414 near LPL on chromosome 8 (A) and rs968567 near FADS2 on chromosome 11 (B). Association plots were conducted with the use of R. Top SNP of interest and P-values in each region are indicated (red diamonds). Color coding indicates the strength of LD of each SNP with the top SNP in each region (measured by r²). The light blue line represents the estimated recombination rates. Gene annotations are shown as dark green lines. Middle panel: linear regression analysis of normalized log transformed ΔC_T expression values (ΔC_T =CT_gene − mean CT_{3 housekeeping genes}) for gene LPL with genotypes of SNP rs6993414, giving a P-value of 1.21 × 10⁻⁵² and beta-coefficient of −1.83 (C), and for gene FADS2 with genotypes of SNP rs968567, giving a P-value of 5.72 × 10⁻⁷³ and β-coefficient of −1.53 (D). Bottom panel: regional plots for eQTL associations (red triangles) and GWAS clinical traits associations (blue circles). Colors indicate LD with the peak eSNPs (red diamonds). LPL cis-associations versus. GWAS HDL results (E), and FADS2 cis-associations versus. GWAS HDL results (F).

Figure 2.

Regional plots and box plots for gene ABO top cis-SNPs whose signal was not attenuated after adjusting for the lead GWAS SNPs. (A) Observed −log₁₀(P) and recombination rates by chromosomal position for all associated SNPs near rs8176731 near ABO on chromosome 9. Top SNP of interest and P-values in each region are indicated (red diamonds). Color coding indicates the strength of LD of each SNP with the top SNP in each region (measured by r²). The light blue line represents the estimated recombination rates. Gene annotations are shown as dark green lines. (B) Linear regression analysis of normalized log transformed ΔC_T expression values (ΔC_T = CT_gene − mean CT_{3 housekeeping genes}) for gene ABO with genotypes of SNP rs8176731, giving a P-value of 1.12 × 10⁻⁹¹ and β-coefficient of −1.43. (C) ABO cis-associations versus. GWAS vWF results. Regional plots for eQTL associations (red triangles) and GWAS clinical traits associations (blue circles). Colors indicate LD with the peak eSNPs (red diamonds).