Systematic cell-based phenotyping of missense alleles empowers rare variant association studies: a case for LDLR and myocardial infarction.

A fundamental challenge to contemporary genetics is to distinguish rare missense alleles that disrupt protein functions from the majority of alleles neutral on protein activities. High-throughput experimental tools to securely discriminate between disruptive and non-disruptive missense alleles are currently missing. Here we establish a scalable cell-based strategy to profile the biological effects and likely disease relevance of rare missense variants in vitro. We apply this strategy to systematically characterize missense alleles in the low-density lipoprotein receptor (LDLR) gene identified through exome sequencing of 3,235 individuals and exome-chip profiling of 39,186 individuals. Our strategy reliably identifies disruptive missense alleles, and disruptive-allele carriers have higher plasma LDL-cholesterol (LDL-C). Importantly, considering experimental data refined the risk of rare LDLR allele carriers from 4.5- to 25.3-fold for high LDL-C, and from 2.1- to 20-fold for early-onset myocardial infarction. Our study generates proof-of-concept that systematic functional variant profiling may empower rare variant-association studies by orders of magnitude.

Loss-of-function mutations in APOC3, triglycerides, and coronary disease.

BACKGROUND: Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. METHODS: We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. RESULTS: An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). CONCLUSIONS: Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.).

Common variants in the haemostatic gene pathway contribute to risk of early-onset myocardial infarction in the Italian population.

Occlusive coronary thrombus formation superimposed on an atherosclerotic plaque is the ultimate event leading to myocardial infarction (MI). Therefore, haemostatic proteins may represent important players in the pathogenesis of MI. It was the objective of this study to evaluate, in a comprehensive way, the role of haemostatic gene polymorphisms in predisposition to premature MI. A total of 810 single nucleotide polymorphisms (SNPs) in 37 genes were assessed for association with MI in a large cohort (1,670 males, 210 females) of Italian patients who suffered from an MI event before the age of 45, and an equal number of controls. Thirty-eight SNPs selected from the literature were genotyped using the SNPlex technology, whereas genotypes for the remaining 772 SNPs were extracted from a previous genome-wide association study. Genotypes were analysed by a standard case-control analysis corrected for classical cardiovascular risk factors, and by haplotype analysis. A weighted Genetic Risk Score (GRS) was calculated. Evidence for association with MI after covariate correction was found for 35 SNPs in 12 loci: F5, PROS1, F11, ITGA2, F12, F13A1, SERPINE1, PLAT, VWF, THBD, PROCR, and F9. The weighted GRS was constructed by including the top SNP for each of the 12 associated loci. The GRS distribution was significantly different between cases and controls, and subjects in the highest quintile had a 2.69-fold increased risk for MI compared with those in the lowest quintile. Our results suggest that a GRS, based on the combined effect of several risk alleles in different haemostatic genes, is associated with an increased risk of MI.

Effects of PCSK9 genetic variants on plasma LDL cholesterol levels and risk of premature myocardial infarction in the Italian population.

The R46L variant in the proprotein-convertase subtilisin-kexin type 9 (PCSK9) gene was associated with reduced levels of LDL and total cholesterol and with a lower risk of coronary artery disease. We investigated the association of R46L with myocardial infarction (MI) in 1,880 Italian patients with premature MI and 1,880 controls. A trend toward a protective effect of the L46 allele was observed [odds ratio (OR) = 0.75, 95% confidence interval (CI) = 0.49-1.13; P = 0.17], although the association with MI was not significant. This is probably due to the combined effect of the low frequency of R46L among Italians and of the young age of the analyzed cohort for whom the impact of coronary atherosclerosis is less important. This hypothesis was indirectly confirmed by the significant association found after including 1,056 additional older controls (OR = 0.67, 95% CI = 0.46-0.97; P = 0.036). LDL cholesterol was significantly lower in L46 carriers (116.2 ± 34.7 mg/dl) than in noncarriers (137.4 ± 47.3 mg/dl; P = 0.00022); a similar reduction was observed for total cholesterol (191.7 ± 37.7 vs. 211.7 ± 49 mg/dl; P = 0.00019). Analysis of 23 additional polymorphisms in the PCSK9 region identified another single nucleotide polymorphism (SNP) (rs11206510) associated with cholesterol levels. We confirmed that the L46 allele not only decreases LDL cholesterol but also protects against MI. Moreover, we replicated the association of total and LDL cholesterol with the SNP rs11206510.

Association and functional analyses of MEF2A as a susceptibility gene for premature myocardial infarction and coronary artery disease.

BACKGROUND: Mutations in the MEF2A gene, coding for a member of the myocyte enhancer factor 2 family of transcription factors, have been reported in patients with coronary artery disease and myocardial infarction (MI). In particular, a 21-bp deletion and 3 missense mutations were demonstrated either to reduce MEF2A transcriptional activity or to impair its nuclear translocation. However, the association of MEF2A with coronary artery disease/MI was not confirmed in other studies. We analyzed the role of MEF2A in the pathogenesis of MI in 2008 Italian patients with premature MI and in 2008 controls. METHODS AND RESULTS: Mutational screening of exon 8 (containing all so-far reported point mutations) disclosed 5 novel and 2 previously described missense mutations. Microsatellite genotyping and sequencing revealed the presence of the 21-bp deletion (located in exon 12) in 5 cases and in none of the controls. Functional studies on mutant proteins showed no alteration, neither in the transactivating properties (all mutants) nor in the nuclear localization (21-bp deletion). Furthermore, an association analysis performed using 3 microsatellites at the MEF2A locus showed no significant association with MI. These results were confirmed in a replication study performed on an independent Italian population with coronary artery disease. CONCLUSIONS: All together, our data do not support MEF2A as a susceptibility gene for coronary artery disease/MI in the Italian population.

Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants.

We conducted a genome-wide association study testing single nucleotide polymorphisms (SNPs) and copy number variants (CNVs) for association with early-onset myocardial infarction in 2,967 cases and 3,075 controls. We carried out replication in an independent sample with an effective sample size of up to 19,492. SNPs at nine loci reached genome-wide significance: three are newly identified (21q22 near MRPS6-SLC5A3-KCNE2, 6p24 in PHACTR1 and 2q33 in WDR12) and six replicated prior observations (9p21, 1p13 near CELSR2-PSRC1-SORT1, 10q11 near CXCL12, 1q41 in MIA3, 19p13 near LDLR and 1p32 near PCSK9). We tested 554 common copy number polymorphisms (>1% allele frequency) and none met the pre-specified threshold for replication (P < 10(-3)). We identified 8,065 rare CNVs but did not detect a greater CNV burden in cases compared to controls, in genes compared to the genome as a whole, or at any individual locus. SNPs at nine loci were reproducibly associated with myocardial infarction, but tests of common and rare CNVs failed to identify additional associations with myocardial infarction risk.