Exome sequencing as a tool to discover genes for heart attack risk and blood lipids

Myocardial infarction (MI), the leading cause of death in the United States, is a heritable phenotype, and the role of inheritance is greatest when MI occurs early in life. Genome-wide association studies (GWAS) have identified at least 30 common variants associated with MI, but the modest proportion of overall heritability that these account for suggests that variants that are low in frequency (0.5 to 5% frequency) or rare (<0.5% frequency) may contribute to the risk of early onset MI (EOMI). To test the hypothesis that rare coding mutations contribute to EOMI risk, we are sequencing the protein-coding region - the exome - of about 1,100 cases with EOMI (MI in men ≤50 and women ≤60) and about 1,100 controls free of MI. Using next-generation sequencing, we have targeted 32.7 Mb at 188,260 exons from 18,560 genes. In the first 970 exomes sequenced, we have generated about 6 billion bases of sequence per individual. Each targeted base was read, on average, 146 times; for each individual, approximately 87% of all bases were covered with at least 20× depth. We performed burden-of-rare-variant tests, carried out single SNP (single nucleotide polymorphism) association tests and imputed exomic variants into completed MI GWAS datasets. In the burden-of-variant tests, we found an excess of rare mutations (all nonsynonymous with a minor allele frequency <1% (T1) or 5% (T5)) in several genes, including CHRM5 (P = 0.0001 for T1), DKK2 (P = 0.0003 for T5) and LRIG2 (P = 0.0002 for T5). In single SNP association tests, we rediscovered a known nonsense mutation in PCSK9 that confers protection against MI (0 in cases and 6 in controls, in 466 cases and 504 controls). In imputation using EOMI exomes as the reference panel, we rediscovered the association of a known low-frequency missense SNP in LPA (I4399M, 2% allele frequency, P < 5 × 10^–8). We are replicating findings from the discovery study by using three approaches: Sanger sequencing in independent samples (500 cases and 500 controls) of specif c genes with the signal based on a burden of rare mutations; genotyping of 212 low-frequency SNPs in >10,000 independent MI cases and controls; and imputation of exomic variants into >35,000 MI cases and controls with GWAS data. These replication results should provide insight into the role of rare variants in conferring MI risk and the role of exome sequencing in understanding the inherited basis of complex traits.