The analysis of approximately 135,000 individuals more than doubled the number of loci with CAD association, yielding 13 previously unidentified loci and confirming at least 10 previously reported loci. CARDIoGRAM also found that very few of the established and new loci appear to act through traditional risk factors. In fact, the majority of these loci reside in gene regions not previously thought to be involved in the pathogenesis of CAD. Finally, a substantial proportion of the CAD risk variants were also strongly associated with various other human disease traits in GWAS demonstrating a potential pleotropic effect of these gene regions.
Ten of the 12 loci (PCSK9 and SHB3 did not achieve genome-wide significance on initial meta-analysis) previously associated with CAD at a genome-wide significance level surpassed the same threshold of significance in CARDIoGRAM in their initial meta-analysis (SORT1, MIA3, WDR12, MRAS, PHACTR1, LPA, 9p21, CXCL12, LDLR, MRPS6). Of note, at least four of these loci are related to low density lipoprotein (LDL) cholesterol. Next, following meta-analysis, they selected 23 new loci with a significance level of P < 5 × 10−6 for follow up genotyping. Of these 23 loci, 13 replicated using the a priori definition of a validated locus (showing independent replication after Bonferroni correction and also achieving P < 5 × 10−8 in the combined discovery and replication data). These 13 loci (PPAP2B, ANKS1A, TCF21, ZC3HC1, ABO, CYP17A1/CNNM2/ NT5C2, ZNF259/APOA5-A4-C3-A1,COL4A1/COL4A2, HHIPL1, ADAMTS7, SMG6/SRR, RASD1/SMCR3/PEMT, UBE2Z/GIP/ATP5G1/SNF8) had had risk allele frequencies ranging from 0.13 to 0.91 and moderate effect sizes with odds ratios ranging from 1.06–1.17 demonstrating the power of their study. Interestingly, given that the consortium had access to angiography, they performed various subgroup analyses starting with age of onset of CAD, as well as ascertainment of CAD which yielded the following findings: 1) 20 out of 22 loci with P < 5 × 10−8 (known and new loci combined; for one locus, age subgroups were not available) had higher odds ratios for early onset than for late onset CAD (P = 1.2 × 10−4 for observed versus expected); 2) the odds ratios for most individual SNPs tended to be slightly greater for cases with angiographically proven CAD than for cases with unknown angiographic status (P = 0.019 for observed versus expected); 3) analyses in males and females revealed no sex-specific effects for any risk alleles (P = 0.4 for observed versus expected).
Next, to better understand the biology of these novel loci, the authors examined whether novel risk alleles were associated with traditional CAD risk factors, were coding variants, had functional significance or associated with other human disease traits. They found that 3 of the risk allele on chromosome 11q23.3 (rs964184, ZNF259, APOA5-APOA4-APOC3-APOA1 gene region) was associated with increased low-density lipoprotein (LDL) cholesterol and decreased high-density lipoprotein (HDL) cholesterol, that the risk allele on chromosome 9q34.2 (rs579459, ABO) was associated with increased LDL and total cholesterol, and that variant rs12413409 on chromosome 10q24.32 representing the CYP17A1-CNNM2-NT5C2 gene region was associated with hypertension.
Furthermore, the lead SNP at 4 of the 13 new loci were either non-synonymous coding variants or were in high linkage disequilibrium (LD) with such SNPs. Variants at 7q32.2 (rs11556924) and 15q25.1 (rs3825807) encoded changes in ZC3HC1 (p.Arg363His) and ADAMTS7 (p.Ser214Pro), respectively, whereas the lead SNP at 14q32.2 (rs2895811) was in strong LD (r2 = 0.82) with the p.Val691Ala variant in HHIPL1. Lastly, the lead SNP at 17q21.32 (rs46522) is in strong LD (r2 = 0.94) with two potential functional variants in GIP: p.Ser103Gly (rs2291725) and a variant influencing the splice site of intron 3 (rs2291726) leading to a truncated transcript.
In terms of expression, 3 of the new CAD risk variants showed convincing association with regional gene expression (cis effect) by either representing the most significant expressed SNP in the region or by being in high LD (r2 ≥ 0.85) with the strongest expressed SNP in the region: rs12190287 at 6q23.2 (TCF21), rs12936587 at 17p11.2 (RASD1, SMCR3 and PEMT) and rs46522 at 17q21.32 (UBE2Z). They also interrogated new loci in a genome-wide map of allelic expression imbalance which provided further support for the expression quantitative trait locus findings at the 17q21.32 locus which yielded strong evidence for cis effects for the 17p13.3 locus lead SNP (rs216172) on the expression of SMG6.
Finally, the authors identified 5 new loci (9q34 (ABO), 10q24 (CYP17A1), 11q23 (ZNF259/APO A5-A4-C3-A1), 15q25 (ADAMTS7) and 17p13(SMG6, SRR)) where the CAD risk variant was fully or strongly correlated (r2 > 0.8) with variants that have previously been associated with many other traits or diseases ranging from expected traits such as LDL cholesterol, HDL cholesterol, triglycerides to aneurysms, soluble levels of adhesion molecules, coagulation factor VIII and von Willebrand factor (at P < 5 × 10−8) demonstrating that a subset of the new CAD risk loci appear to have pleiotropic effects.
The authors state that their study focused on common risk variants, and by assuming a heritability of 40% for CAD, the lead SNPs of previously established loci combined with the loci discovered in their study explain approximately 10% of the additive genetic variance of CAD, suggesting that many other common susceptibility variants of similar or lower effects and/or rare variants contribute to risk of CAD.