Genetic analysis of leukocyte type-I interferon production and risk of coronary artery disease

Objective: Patients with systemic lupus erythematosus (SLE) are genetically predisposed to enhanced production of the type-I interferon IFN-α, and are also at elevated risk of developing atherosclerosis compared to healthy subjects. We aimed to test whether or not genetic predisposition to increased type-I interferon production affects risk of coronary artery disease (CAD). Approach and results: Using a list of 11 SNPs from the results of genome-wide association studies for SLE, which we hypothesised would be enriched in variants that regulate type-I IFN production, we identified a genetic risk score (GRS) based on 3 SNPs (rs10516487, rs3131379 and rs7574865) which correlated significantly with production of IFN-α by human peripheral leukocytes stimulated with CpG-oligonucleotide (n=60, P=1.50x10-5 ). These SNPs explained 27.8% of variation in the CpG-oligonucleotide induced IFN-α response and were also associated with Toll-like receptor (TLR)7/8- and TLR9-dependent IFN-α and IFN-β responses, but were not associated with inflammatory cytokine production in response to TLR4 stimulation, or risk of CAD in 22,233 cases and 64,762 controls (OR 1.00, 95%CI 0.98-1.02) using Mendelian randomization-based analyses. CAD risk was also not associated with the full panel of 11 SLE SNPs, or loci responsible for the monogenic type-I interferonopathies Aicardi-Goutières syndrome and Spondyloenchondrodysplasia with immune dysregulation. Conclusion: The results argue against the potential utility of drugs targeting type-I interferon production for CAD. The use of genetic variants that modify leukocyte signalling pathways, rather than circulating biomarkers, as instruments in Mendelian randomization analyses may be useful for studies investigating causality of other candidate pathways of atherogenesis

Genomic prediction of coronary heart disease.

AIMS: Genetics plays an important role in coronary heart disease (CHD) but the clinical utility of genomic risk scores (GRSs) relative to clinical risk scores, such as the Framingham Risk Score (FRS), is unclear. Our aim was to construct and externally validate a CHD GRS, in terms of lifetime CHD risk and relative to traditional clinical risk scores. METHODS AND RESULTS: We generated a GRS of 49 310 SNPs based on a CARDIoGRAMplusC4D Consortium meta-analysis of CHD, then independently tested it using five prospective population cohorts (three FINRISK cohorts, combined n = 12 676, 757 incident CHD events; two Framingham Heart Study cohorts (FHS), combined n = 3406, 587 incident CHD events). The GRS was associated with incident CHD (FINRISK HR = 1.74, 95% confidence interval (CI) 1.61-1.86 per S.D. of GRS; Framingham HR = 1.28, 95% CI 1.18-1.38), and was largely unchanged by adjustment for known risk factors, including family history. Integration of the GRS with the FRS or ACC/AHA13 scores improved the 10 years risk prediction (meta-analysis C-index: +1.5-1.6%, P < 0.001), particularly for individuals ≥60 years old (meta-analysis C-index: +4.6-5.1%, P < 0.001). Importantly, the GRS captured substantially different trajectories of absolute risk, with men in the top 20% of attaining 10% cumulative CHD risk 12-18 y earlier than those in the bottom 20%. High genomic risk was partially compensated for by low systolic blood pressure, low cholesterol level, and non-smoking. CONCLUSIONS: A GRS based on a large number of SNPs improves CHD risk prediction and encodes different trajectories of lifetime risk not captured by traditional clinical risk scores

Genome-wide haplotype analysis of cis expression quantitative trait loci in monocytes.

In order to assess whether gene expression variability could be influenced by several SNPs acting in cis, either through additive or more complex haplotype effects, a systematic genome-wide search for cis haplotype expression quantitative trait loci (eQTL) was conducted in a sample of 758 individuals, part of the Cardiogenics Transcriptomic Study, for which genome-wide monocyte expression and GWAS data were available. 19,805 RNA probes were assessed for cis haplotypic regulation through investigation of ~2,1 × 10(9) haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >10(4)-fold smaller than the best single SNP p-value. Replication of significant haplotype effects were tested for 412 probes for which SNPs (or proxies) that defined the detected haplotypes were available in the Gutenberg Health Study composed of 1,374 individuals. At the Bonferroni correction level of 1.2 × 10(-4) (~0.05/412), 193 haplotypic signals replicated. 1000 G imputation was then conducted, and 105 haplotypic signals still remained more informative than imputed SNPs. In-depth analysis of these 105 cis eQTL revealed that at 76 loci genetic associations were compatible with additive effects of several SNPs, while for the 29 remaining regions data could be compatible with a more complex haplotypic pattern. As 24 of the 105 cis eQTL have previously been reported to be disease-associated loci, this work highlights the need for conducting haplotype-based and 1000 G imputed cis eQTL analysis before commencing functional studies at disease-associated loci

Powerful Identification of Cis-regulatory SNPs in Human Primary Monocytes Using Allele-Specific Gene Expression

A large number of genome-wide association studies have been performed during the past five years to identify associations between SNPs and human complex diseases and traits. The assignment of a functional role for the identified disease-associated SNP is not straight-forward. Genome-wide expression quantitative trait locus (eQTL) analysis is frequently used as the initial step to define a function while allele-specific gene expression (ASE) analysis has not yet gained a wide-spread use in disease mapping studies. We compared the power to identify cis-acting regulatory SNPs (cis-rSNPs) by genome-wide allele-specific gene expression (ASE) analysis with that of traditional expression quantitative trait locus (eQTL) mapping. Our study included 395 healthy blood donors for whom global gene expression profiles in circulating monocytes were determined by Illumina BeadArrays. ASE was assessed in a subset of these monocytes from 188 donors by quantitative genotyping of mRNA using a genome-wide panel of SNP markers. The performance of the two methods for detecting cis-rSNPs was evaluated by comparing associations between SNP genotypes and gene expression levels in sample sets of varying size. We found that up to 8-fold more samples are required for eQTL mapping to reach the same statistical power as that obtained by ASE analysis for the same rSNPs. The performance of ASE is insensitive to SNPs with low minor allele frequencies and detects a larger number of significantly associated rSNPs using the same sample size as eQTL mapping. An unequivocal conclusion from our comparison is that ASE analysis is more sensitive for detecting cis-rSNPs than standard eQTL mapping. Our study shows the potential of ASE mapping in tissue samples and primary cells which are difficult to obtain in large numbers

Lifelong reduction of LDL-cholesterol related to a common variant in the LDL-receptor gene decreases the risk of coronary artery disease--a Mendelian Randomisation study.

Background Rare mutations of the low-density lipoprotein receptor gene (LDLR) cause familial hypercholesterolemia, which increases the risk for coronary artery disease (CAD). Less is known about the implications of common genetic variation in the LDLR gene regarding the variability of cholesterol levels and risk of CAD. Methods Imputed genotype data at the LDLR locus on 1 644 individuals of a population-based sample were explored for association with LDL-C level. Replication of association with LDL-C level was sought for the most significant single nucleotide polymorphism (SNP) within the LDLR gene in three European samples comprising 6 642 adults and 533 children. Association of this SNP with CAD was examined in six case-control studies involving more than 15 000 individuals. Findings Each copy of the minor T allele of SNP rs2228671 within LDLR (frequency 11%) was related to a decrease of LDL-C levels by 0.19 mmol/L (95% confidence interval (CI) [0.13–0.24] mmol/L, p = 1.5×10−10). This association with LDL-C was uniformly found in children, men, and women of all samples studied. In parallel, the T allele of rs2228671 was associated with a significantly lower risk of CAD (Odds Ratio per copy of the T allele: 0.82, 95% CI [0.76–0.89], p = 2.1×10−7). Adjustment for LDL-C levels by logistic regression or Mendelian Randomisation models abolished the significant association between rs2228671 with CAD completely, indicating a functional link between the genetic variant at the LDLR gene locus, change in LDL-C and risk of CAD. Conclusion A common variant at the LDLR gene locus affects LDL-C levels and, thereby, the risk for CAD

Association of Rare and Common Variation in the Lipoprotein Lipase Gene With Coronary Artery Disease.

Importance: The activity of lipoprotein lipase (LPL) is the rate-determining step in clearing triglyceride-rich lipoproteins from the circulation. Mutations that damage the LPL gene (LPL) lead to lifelong deficiency in enzymatic activity and can provide insight into the relationship of LPL to human disease. Objective: To determine whether rare and/or common variants in LPL are associated with early-onset coronary artery disease (CAD). Design, Setting, and Participants: In a cross-sectional study, LPL was sequenced in 10 CAD case-control cohorts of the multinational Myocardial Infarction Genetics Consortium and a nested CAD case-control cohort of the Geisinger Health System DiscovEHR cohort between 2010 and 2015. Common variants were genotyped in up to 305 699 individuals of the Global Lipids Genetics Consortium and up to 120 600 individuals of the CARDIoGRAM Exome Consortium between 2012 and 2014. Study-specific estimates were pooled via meta-analysis. Exposures: Rare damaging mutations in LPL included loss-of-function variants and missense variants annotated as pathogenic in a human genetics database or predicted to be damaging by computer prediction algorithms trained to identify mutations that impair protein function. Common variants in the LPL gene region included those independently associated with circulating triglyceride levels. Main Outcomes and Measures: Circulating lipid levels and CAD. Results: Among 46 891 individuals with LPL gene sequencing data available, the mean (SD) age was 50 (12.6) years and 51% were female. A total of 188 participants (0.40%; 95% CI, 0.35%-0.46%) carried a damaging mutation in LPL, including 105 of 32 646 control participants (0.32%) and 83 of 14 245 participants with early-onset CAD (0.58%). Compared with 46 703 noncarriers, the 188 heterozygous carriers of an LPL damaging mutation displayed higher plasma triglyceride levels (19.6 mg/dL; 95% CI, 4.6-34.6 mg/dL) and higher odds of CAD (odds ratio = 1.84; 95% CI, 1.35-2.51; P < .001). An analysis of 6 common LPL variants resulted in an odds ratio for CAD of 1.51 (95% CI, 1.39-1.64; P = 1.1 × 10-22) per 1-SD increase in triglycerides. Conclusions and Relevance: The presence of rare damaging mutations in LPL was significantly associated with higher triglyceride levels and presence of coronary artery disease. However, further research is needed to assess whether there are causal mechanisms by which heterozygous lipoprotein lipase deficiency could lead to coronary artery disease

The Metabochip, a Custom Genotyping Array for Genetic Studies of Metabolic, Cardiovascular, and Anthropometric Traits

Genome-wide association studies have identified hundreds of loci for type 2 diabetes, coronary artery disease and myocardial infarction, as well as for related traits such as body mass index, glucose and insulin levels, lipid levels, and blood pressure. These studies also have pointed to thousands of loci with promising but not yet compelling association evidence. To establish association at additional loci and to characterize the genome-wide significant loci by fine-mapping, we designed the "Metabochip," a custom genotyping array that assays nearly 200,000 SNP markers. Here, we describe the Metabochip and its component SNP sets, evaluate its performance in capturing variation across the allele-frequency spectrum, describe solutions to methodological challenges commonly encountered in its analysis, and evaluate its performance as a platform for genotype imputation. The metabochip achieves dramatic cost efficiencies compared to designing single-trait follow-up reagents, and provides the opportunity to compare results across a range of related traits. The metabochip and similar custom genotyping arrays offer a powerful and cost-effective approach to follow-up large-scale genotyping and sequencing studies and advance our understanding of the genetic basis of complex human diseases and traits

A genome-wide association study reveals variants in ARL15 that influence adiponectin levels.

The adipocyte-derived protein adiponectin is highly heritable and inversely associated with risk of type 2 diabetes mellitus (T2D) and coronary heart disease (CHD). We meta-analyzed 3 genome-wide association studies for circulating adiponectin levels (n = 8,531) and sought validation of the lead single nucleotide polymorphisms (SNPs) in 5 additional cohorts (n = 6,202). Five SNPs were genome-wide significant in their relationship with adiponectin (P< or =5x10(-8)). We then tested whether these 5 SNPs were associated with risk of T2D and CHD using a Bonferroni-corrected threshold of P< or =0.011 to declare statistical significance for these disease associations. SNPs at the adiponectin-encoding ADIPOQ locus demonstrated the strongest associations with adiponectin levels (P-combined = 9.2x10(-19) for lead SNP, rs266717, n = 14,733). A novel variant in the ARL15 (ADP-ribosylation factor-like 15) gene was associated with lower circulating levels of adiponectin (rs4311394-G, P-combined = 2.9x10(-8), n = 14,733). This same risk allele at ARL15 was also associated with a higher risk of CHD (odds ratio [OR] = 1.12, P = 8.5x10(-6), n = 22,421) more nominally, an increased risk of T2D (OR = 1.11, P = 3.2x10(-3), n = 10,128), and several metabolic traits. Expression studies in humans indicated that ARL15 is well-expressed in skeletal muscle. These findings identify a novel protein, ARL15, which influences circulating adiponectin levels and may impact upon CHD risk. The adipocyte-derived protein adiponectin is highly heritable and inversely associated with risk of type 2 diabetes mellitus (T2D) and coronary heart disease (CHD). We meta-analyzed 3 genome-wide association studies for circulating adiponectin levels (n = 8,531) and sought validation of the lead single nucleotide polymorphisms (SNPs) in 5 additional cohorts (n = 6,202). Five SNPs were genome-wide significant in their relationship with adiponectin (P≤5×10−8). We then tested whether these 5 SNPs were associated with risk of T2D and CHD using a Bonferroni-corrected threshold of P≤0.011 to declare statistical significance for these disease associations. SNPs at the adiponectin-encoding ADIPOQ locus demonstrated the strongest associations with adiponectin levels (P-combined = 9.2×10−19 for lead SNP, rs266717, n = 14,733). A novel variant in the ARL15 (ADP-ribosylation factor-like 15) gene was associated with lower circulating levels of adiponectin (rs4311394-G, P-combined = 2.9×10−8, n = 14,733). This same risk allele at ARL15 was also associated with a higher risk of CHD (odds ratio [OR] = 1.12, P = 8.5×10−6, n = 22,421) more nominally, an increased risk of T2D (OR = 1.11, P = 3.2×10−3, n = 10,128), and several metabolic traits. Expression studies in humans indicated that ARL15 is well-expressed in skeletal muscle. These findings identify a novel protein, ARL15, which influences circulating adiponectin levels and may impact upon CHD risk

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.)

Cystatin C and Cardiovascular Disease: A Mendelian Randomization Study.

BACKGROUND: Epidemiological studies show that high circulating cystatin C is associated with risk of cardiovascular disease (CVD), independent of creatinine-based renal function measurements. It is unclear whether this relationship is causal, arises from residual confounding, and/or is a consequence of reverse causation. OBJECTIVES: The aim of this study was to use Mendelian randomization to investigate whether cystatin C is causally related to CVD in the general population. METHODS: We incorporated participant data from 16 prospective cohorts (n = 76,481) with 37,126 measures of cystatin C and added genetic data from 43 studies (n = 252,216) with 63,292 CVD events. We used the common variant rs911119 in CST3 as an instrumental variable to investigate the causal role of cystatin C in CVD, including coronary heart disease, ischemic stroke, and heart failure. RESULTS: Cystatin C concentrations were associated with CVD risk after adjusting for age, sex, and traditional risk factors (relative risk: 1.82 per doubling of cystatin C; 95% confidence interval [CI]: 1.56 to 2.13; p = 2.12 × 10(-14)). The minor allele of rs911119 was associated with decreased serum cystatin C (6.13% per allele; 95% CI: 5.75 to 6.50; p = 5.95 × 10(-211)), explaining 2.8% of the observed variation in cystatin C. Mendelian randomization analysis did not provide evidence for a causal role of cystatin C, with a causal relative risk for CVD of 1.00 per doubling cystatin C (95% CI: 0.82 to 1.22; p = 0.994), which was statistically different from the observational estimate (p = 1.6 × 10(-5)). A causal effect of cystatin C was not detected for any individual component of CVD. CONCLUSIONS: Mendelian randomization analyses did not support a causal role of cystatin C in the etiology of CVD. As such, therapeutics targeted at lowering circulating cystatin C are unlikely to be effective in preventing CVD