Objective

The etiologic basis of transient left ventricular apical ballooning, a novel cardiac syndrome, is not clear. Among the proposed pathomechanisms is coronary vasospasm. Long-term ST segment analysis may detect vasospastic episodes but has not been reported.

Methods

30 consecutive patients with transient left ventricular apical ballooning, left ventricular dysfunction and normal or near-normal coronary arteries were investigated. A 24-hour Holter ECG was obtained after emergency admission. ST segment analysis was performed automatically in 2 leads and confirmed by visual inspection. Criteria for an ischemic event were: 1. ST elevation or 2. horizontal or down-sloping ST segments ≥1 min duration and ≥100 µV J+80 point deviation corrected for baseline ST-deviation.

Results

Patients presented with ST segment elevation (n = 19) and/or T wave inversion (n = 20) on admission ECG. Ejection fraction was 50±12%. No transient ST elevations were observed during Holter ECG analysis. In 3 patients, 8 transient episodes of ST depression were recorded. Durations of episodes varied between 75s and 790s (mean 229s). Maximal ST deviation averaged −191±71 µV. Ischemic burden was −1 to −22 mVs (mean −8 mVs). 27 patients showed no ischemic events.

Conclusions

ST segment analysis of 24 h Holter recordings revealed minor ischemic events in only 10% of patients with transient left ventricular apical ballooning. Overall, ST segment changes were not indicative of recurrent coronary spasm playing a major role in the genesis of transient left ventricular apical ballooning.

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 ∼2,1×109 haplotypic combinations. 2,650 probes demonstrated haplotypic p-values >104-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. 1000G 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 1000G imputed cis eQTL analysis before commencing functional studies at disease-associated loci.

Author Summary

In order to assess whether gene expression variability could be influenced by the presence of more than one cis-acting SNP, we have conducted a systematic genome-wide search for haplotypic cis eQTL effects in a sample of 758 individuals and replicated the findings in an independent sample of 1,374 subjects. In both studies, genome-wide monocytes expression and genotype data were available. We identified 105 genes whose monocyte expression was under the influence of multiple cis-acting SNPs. About 75% of the detected genetic effects were related to independent additive SNP effects and the last quarter due to more complex haplotype effects. Of note, 24 of the genes identified to be affected by multiple cis eSNPs have been previously reported to reside at disease-associated loci. This could suggest that such multiple locus-specific genetic effects could contribute to the susceptibility to human diseases.

Dihydropyridines (DHP), which nifedipine is a member of, preferentially block Ca2+ channels of different cell types. Moreover, influx of Ca2+ through L-type Ca2+ channels (LTCCs) activates Ca2+ signaling pathways, which in turn contribute to numerous cellular processes. Although LTCCs are expressed in undifferentiated cells, very little is known about its contributions to the transcriptional regulation of mesodermal and cardiac genes. This study aimed to examine the contribution of LTCCs and the effect of nifedipine on the commitment of pluripotent stem cells toward the cardiac lineage in vitro. The murine embryonic stem (ES, cell line D3) and induced pluripotent stem (iPS, cell clone 09) cells were differentiated into enhanced green fluorescence protein (EGFP) expressing spontaneously beating cardiomyocytes (CMs). Early treatment of differentiating cells with 10 µM nifedipine led to a significant inhibition of the cardiac mesoderm formation and cardiac lineage commitment as revealed by gene regulation analysis. This was accompanied by the inhibition of spontaneously occurring Ca2+ transient and reduction of LTCCs current density (ICaL) of differentiated CMs. In addition, nifedipine treatment instigated a pronounced delay of the spontaneous beating embryoid body (EB) and led to a poor surface localization of L-type Ca2+ channel α1C (CaV1.2) subunits. Contrary late incubation of pluripotent stem cells with nifedipine was without any impact on the differentiation process and did not affect the derived CMs function. Our data indicate that nifedipine blocks the determined path of pluripotent stem cells to cardiomyogenesis by inhibition of mesodermal commitment at early stages of differentiation, thus the proper upkeep Ca2+ concentration and pathways are essentially required for cardiac gene expression, differentiation and function.

Aims

Lipoprotein-associated phospholipase A2 (Lp-PLA2) generates proinflammatory and proatherogenic compounds in the arterial vascular wall and is a potential therapeutic target in coronary heart disease (CHD). We searched for genetic loci related to Lp-PLA2 mass or activity by a genome-wide association study as part of the Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium.

Methods and results

In meta-analyses of findings from five population-based studies, comprising 13 664 subjects, variants at two loci (PLA2G7, CETP) were associated with Lp-PLA2 mass. The strongest signal was at rs1805017 in PLA2G7 [P = 2.4 × 10−23, log Lp-PLA2 difference per allele (beta): 0.043]. Variants at six loci were associated with Lp-PLA2 activity (PLA2G7, APOC1, CELSR2, LDL, ZNF259, SCARB1), among which the strongest signals were at rs4420638, near the APOE–APOC1–APOC4–APOC2 cluster [P = 4.9 × 10−30; log Lp-PLA2 difference per allele (beta): −0.054]. There were no significant gene–environment interactions between these eight polymorphisms associated with Lp-PLA2 mass or activity and age, sex, body mass index, or smoking status. Four of the polymorphisms (in APOC1, CELSR2, SCARB1, ZNF259), but not PLA2G7, were significantly associated with CHD in a second study.

Conclusion

Levels of Lp-PLA2 mass and activity were associated with PLA2G7, the gene coding for this protein. Lipoprotein-associated phospholipase A2 activity was also strongly associated with genetic variants related to low-density lipoprotein cholesterol levels.

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.

We aimed to assess whether pri-miRNA SNPs (miSNPs) could influence monocyte gene expression, either through marginal association or by interacting with polymorphisms located in 3'UTR regions (3utrSNPs). We then conducted a genome-wide search for marginal miSNPs effects and pairwise miSNPs × 3utrSNPs interactions in a sample of 1,467 individuals for which genome-wide monocyte expression and genotype data were available. Statistical associations that survived multiple testing correction were tested for replication in an independent sample of 758 individuals with both monocyte gene expression and genotype data. In both studies, the hsa-mir-1279 rs1463335 was found to modulate in cis the expression of LYZ and in trans the expression of CNTN6, CTRC, COPZ2, KRT9, LRRFIP1, NOD1, PCDHA6, ST5 and TRAF3IP2 genes, supporting the role of hsa-mir-1279 as a regulator of several genes in monocytes. In addition, we identified two robust miSNPs × 3utrSNPs interactions, one involving HLA-DPB1 rs1042448 and hsa-mir-219-1 rs107822, the second the H1F0 rs1894644 and hsa-mir-659 rs5750504, modulating the expression of the associated genes.

As some of the aforementioned genes have previously been reported to reside at disease-associated loci, our findings provide novel arguments supporting the hypothesis that the genetic variability of miRNAs could also contribute to the susceptibility to human diseases.

To date, few mutations are described to underlie highly-elevated HDLc levels in families. Here we sequenced the coding regions and adjacent sequence of the LIPG, CETP, and GALNT2 genes in 171 unrelated Dutch Caucasian probands with HDLc≥90th percentile and analyzed segregation of mutations with lipid phenotypes in family members. In these probands, mutations were most frequent in LIPG (12.9%) followed by GALNT2 (2.3%) and CETP (0.6%). A total of 6 of 10 mutations in these three genes were novel (60.0%), and mutations segregated with elevated HDLc in families. Interestingly, the LIPG mutations N396S and R476W, which usually result in elevated HDLc, were unexpectedly found in 6 probands with low HDLc (i.e., ≤10th percentile). However, 5 of these probands also carried mutations in ABCA1, LCAT, or LPL. Finally, no CETP and GALNT2 mutations were found in 136 unrelated probands with low HDLc. Taken together, we show that rare coding and splicing mutations in LIPG, CETP, and GALNT2 are enriched in persons with hyperalphalipoproteinemia and segregate with elevated HDLc in families. Moreover, LIPG mutations do not overcome low HDLc in individuals with ABCA1 and possibly LCAT and LPL mutations, indicating that LIPG affects HDLc levels downstream of these proteins.

Summary

Background

High plasma HDL cholesterol is associated with reduced risk of myocardial infarction, but whether this association is causal is unclear. Exploiting the fact that genotypes are randomly assigned at meiosis, are independent of non-genetic confounding, and are unmodified by disease processes, mendelian randomisation can be used to test the hypothesis that the association of a plasma biomarker with disease is causal.

Methods

We performed two mendelian randomisation analyses. First, we used as an instrument a single nucleotide polymorphism (SNP) in the endothelial lipase gene (LIPG Asn396Ser) and tested this SNP in 20 studies (20 913 myocardial infarction cases, 95 407 controls). Second, we used as an instrument a genetic score consisting of 14 common SNPs that exclusively associate with HDL cholesterol and tested this score in up to 12 482 cases of myocardial infarction and 41 331 controls. As a positive control, we also tested a genetic score of 13 common SNPs exclusively associated with LDL cholesterol.

Findings

Carriers of the LIPG 396Ser allele (2·6% frequency) had higher HDL cholesterol (0·14 mmol/L higher, p=8×10−13) but similar levels of other lipid and non-lipid risk factors for myocardial infarction compared with non-carriers. This difference in HDL cholesterol is expected to decrease risk of myocardial infarction by 13% (odds ratio [OR] 0·87, 95% CI 0·84–0·91). However, we noted that the 396Ser allele was not associated with risk of myocardial infarction (OR 0·99, 95% CI 0·88–1·11, p=0·85). From observational epidemiology, an increase of 1 SD in HDL cholesterol was associated with reduced risk of myocardial infarction (OR 0·62, 95% CI 0·58–0·66). However, a 1 SD increase in HDL cholesterol due to genetic score was not associated with risk of myocardial infarction (OR 0·93, 95% CI 0·68–1·26, p=0·63). For LDL cholesterol, the estimate from observational epidemiology (a 1 SD increase in LDL cholesterol associated with OR 1·54, 95% CI 1·45–1·63) was concordant with that from genetic score (OR 2·13, 95% CI 1·69–2·69, p=2×10−10).

Interpretation

Some genetic mechanisms that raise plasma HDL cholesterol do not seem to lower risk of myocardial infarction. These data challenge the concept that raising of plasma HDL cholesterol will uniformly translate into reductions in risk of myocardial infarction.

Funding

US National Institutes of Health, The Wellcome Trust, European Union, British Heart Foundation, and the German Federal Ministry of Education and Research.

Background

More accurate coronary heart disease (CHD) prediction, specifically in middle-aged men, is needed to reduce the burden of disease more effectively. We hypothesised that a multilocus genetic risk score could refine CHD prediction beyond classic risk scores and obtain more precise risk estimates using a prospective cohort design.

Methods

Using data from nine prospective European cohorts, including 26,221 men, we selected in a case-cohort setting 4,818 healthy men at baseline, and used Cox proportional hazards models to examine associations between CHD and risk scores based on genetic variants representing 13 genomic regions. Over follow-up (range: 5–18 years), 1,736 incident CHD events occurred. Genetic risk scores were validated in men with at least 10 years of follow-up (632 cases, 1361 non-cases). Genetic risk score 1 (GRS1) combined 11 SNPs and two haplotypes, with effect estimates from previous genome-wide association studies. GRS2 combined 11 SNPs plus 4 SNPs from the haplotypes with coefficients estimated from these prospective cohorts using 10-fold cross-validation. Scores were added to a model adjusted for classic risk factors comprising the Framingham risk score and 10-year risks were derived.

Results

Both scores improved net reclassification (NRI) over the Framingham score (7.5%, p = 0.017 for GRS1, 6.5%, p = 0.044 for GRS2) but GRS2 also improved discrimination (c-index improvement 1.11%, p = 0.048). Subgroup analysis on men aged 50–59 (436 cases, 603 non-cases) improved net reclassification for GRS1 (13.8%) and GRS2 (12.5%). Net reclassification improvement remained significant for both scores when family history of CHD was added to the baseline model for this male subgroup improving prediction of early onset CHD events.

Conclusions

Genetic risk scores add precision to risk estimates for CHD and improve prediction beyond classic risk factors, particularly for middle aged men.

Statin therapy reduces the risk of coronary heart disease (CHD), however, the person-to-person variability in response to statin therapy is not well understood. We have investigated the effect of genetic variation on the reduction of CHD events by pravastatin. First, we conducted a genome-wide association study of 682 CHD cases from the Cholesterol and Recurrent Events (CARE) trial and 383 CHD cases from the West of Scotland Coronary Prevention Study (WOSCOPS), two randomized, placebo-controlled studies of pravastatin. In a combined case-only analysis, 79 single nucleotide polymorphisms (SNPs) were associated with differential CHD event reduction by pravastatin according to genotype (P<0.0001), and these SNPs were analyzed in a second stage that included cases as well as non-cases from CARE and WOSCOPS and patients from the PROspective Study of Pravastatin in the Elderly at Risk/PHArmacogenomic study of Statins in the Elderly at risk for cardiovascular disease (PROSPER/PHASE), a randomized placebo controlled study of pravastatin in the elderly. We found that one of these SNPs (rs13279522) was associated with differential CHD event reduction by pravastatin therapy in all 3 studies: P = 0.002 in CARE, P = 0.01 in WOSCOPS, P = 0.002 in PROSPER/PHASE. In a combined analysis of CARE, WOSCOPS, and PROSPER/PHASE, the hazard ratio for CHD when comparing pravastatin with placebo decreased by a factor of 0.63 (95% CI: 0.52 to 0.75) for each extra copy of the minor allele (P = 4.8×10−7). This SNP is located in DnaJ homolog subfamily C member 5B (DNAJC5B) and merits investigation in additional randomized studies of pravastatin and other statins.

Background

Calcium (Ca2+) handling proteins are known to play a pivotal role in the pathophysiology of cardiomyopathy. However little is known about early changes in the diabetic heart and the impact of insulin treatment (Ins).

Methods

Zucker Diabetic Fatty rats treated with or without insulin (ZDF ± Ins, n = 13) and lean littermates (controls, n = 7) were sacrificed at the age of 19 weeks. ZDF + Ins (n = 6) were treated with insulin for the last 6 weeks of life. Gene expression of Ca2+ ATPase in the cardiac sarcoplasmatic reticulum (SERCA2a, further abbreviated as SERCA) and phospholamban (PLB) were determined by northern blotting. Ca2+ transport of the sarcoplasmatic reticulum (SR) was assessed by oxalate-facilitated 45Ca-uptake in left ventricular homogenates. In addition, isolated neonatal cardiomyocytes were stimulated in cell culture with insulin, glucose or triiodthyronine (T3, positive control). mRNA expression of SERCA and PLB were measured by Taqman PCR. Furthermore, effects of insulin treatment on force of contraction and relaxation were evaluated by cardiomyocytes grown in a three-dimensional collagen matrix (engineered heart tissue, EHT) stimulated for 5 days by insulin. By western blot phosphorylations status of Akt was determed and the influence of wortmannin.

Results

SERCA levels increased in both ZDF and ZDF + Ins compared to control (control 100 ± 6.2 vs. ZDF 152 ± 26.6* vs. ZDF + Ins 212 ± 18.5*# % of control, *p < 0.05 vs. control, #p < 0.05 vs. ZDF) whereas PLB was significantly decreased in ZDF and ZDF + Ins (control 100 ± 2.8 vs. ZDF 76.3 ± 13.5* vs. ZDF + Ins 79.4 ± 12.9* % of control, *p < 0.05 vs control). The increase in the SERCA/PLB ratio in ZDF and ZDF ± Ins was accompanied by enhanced Ca2+ uptake to the SR (control 1.58 ± 0.1 vs. ZDF 1.85 ± 0.06* vs. ZDF + Ins 2.03 ± 0.1* μg/mg/min, *p < 0.05 vs. control). Interestingly, there was a significant correlation between Ca2+ uptake and SERCA2a expression. As shown by in-vitro experiments, the effect of insulin on SERCA2a mRNA expression seemed to have a direct effect on cardiomyocytes. Furthermore, long-term treatment of engineered heart tissue with insulin increased the SERCA/PLB ratio and accelerated relaxation time. Akt was significantly phosphorylated by insulin. This effect could be abolished by wortmannin.

Conclusion

The current data demonstrate that early type 2 diabetes is associated with an increase in the SERCA/PLB ratio and that insulin directly stimulates SERCA expression and relaxation velocity. These results underline the important role of insulin and calcium handling proteins in the cardiac adaptation process of type 2 diabetes mellitus contributing to cardiac remodeling and show the important role of PI3-kinase-Akt-SERCA2a signaling cascade.

Background

Recent genome-wide association studies (GWAS) have identified several novel loci that reproducibly associate with CAD and/or MI risk. However, known common CAD risk variants explain only 10% of the predicted genetic heritability of the disease, suggesting that important genetic signals remain to be discovered.

Methods and Results

We performed a discovery meta-analysis of 5 GWASs involving 13,949 subjects (7123 cases, 6826 controls) imputed at approximately 5 million SNPs using pilot 1000 Genomes based haplotypes. Promising loci were followed up in an additional 5 studies with 11,032 subjects (5211 cases, 5821 controls). A novel CAD locus on chromosome 6p21.3 in the major histocompatibility complex (MHC) between HCG27 and HLA-C was identified and achieved genome wide significance in the combined analysis (rs3869109; pdiscovery=3.3×10−7, preplication=5.3×10−4 pcombined=1.12×10−9). A sub-analysis combining discovery GWASs showed an attenuation of significance when stringent corrections for European population structure were employed (p=4.1×10-10 versus 3.2×10-7) suggesting the observed signal is partly confounded due to population stratification. This gene dense region plays an important role in inflammation, immunity and self cell recognition. To determine whether the underlying association was driven by MHC class I alleles, we statistically imputed common HLA alleles into the discovery subjects; however, no single common HLA type contributed significantly or fully explained the observed association.

Conclusions

We have identified a novel locus in the MHC associated with CAD. MHC genes regulate inflammation and T cell responses that contribute importantly to the initiation and propagation of atherosclerosis. Further laboratory studies will be required to understand the biological basis of this association and identify the causative allele(s).

Background

Recent genome-wide association studies (GWAS) have identified several novel loci that reproducibly associate with CAD and/or MI risk. However, known common CAD risk variants explain only 10% of the predicted genetic heritability of the disease, suggesting that important genetic signals remain to be discovered.

Methods and Results

We performed a discovery meta-analysis of 5 GWASs involving 13,949 subjects (7123 cases, 6826 controls) imputed at approximately 5 million SNPs using pilot 1000 Genomes based haplotypes. Promising loci were followed up in an additional 5 studies with 11,032 subjects (5211 cases, 5821 controls). A novel CAD locus on chromosome 6p21.3 in the major histocompatibility complex (MHC) between HCG27 and HLA-C was identified and achieved genome wide significance in the combined analysis (rs3869109; pdiscovery=3.3×10−7, preplication=5.3×10−4 pcombined=1.12×10−9). A sub-analysis combining discovery GWASs showed an attenuation of significance when stringent corrections for European population structure were employed (p=4.1×10−10 versus 3.2×10−7) suggesting the observed signal is partly confounded due to population stratification. This gene dense region plays an important role in inflammation, immunity and self cell recognition. To determine whether the underlying association was driven by MHC class I alleles, we statistically imputed common HLA alleles into the discovery subjects; however, no single common HLA type contributed significantly or fully explained the observed association.

Conclusion

We have identified a novel locus in the MHC associated with CAD. MHC genes regulate inflammation and T cell responses that contribute importantly to the initiation and propagation of atherosclerosis. Further laboratory studies will be required to understand the biological basis of this association and identify the causative allele(s).

Summary

Background

A sexual dimorphism exists in the incidence and prevalence of coronary artery disease—men are more commonly affected than are age-matched women. We explored the role of the Y chromosome in coronary artery disease in the context of this sexual inequity.

Methods

We genotyped 11 markers of the male-specific region of the Y chromosome in 3233 biologically unrelated British men from three cohorts: the British Heart Foundation Family Heart Study (BHF-FHS), West of Scotland Coronary Prevention Study (WOSCOPS), and Cardiogenics Study. On the basis of this information, each Y chromosome was tracked back into one of 13 ancient lineages defined as haplogroups. We then examined associations between common Y chromosome haplogroups and the risk of coronary artery disease in cross-sectional BHF-FHS and prospective WOSCOPS. Finally, we undertook functional analysis of Y chromosome effects on monocyte and macrophage transcriptome in British men from the Cardiogenics Study.

Findings

Of nine haplogroups identified, two (R1b1b2 and I) accounted for roughly 90% of the Y chromosome variants among British men. Carriers of haplogroup I had about a 50% higher age-adjusted risk of coronary artery disease than did men with other Y chromosome lineages in BHF-FHS (odds ratio 1·75, 95% CI 1·20–2·54, p=0·004), WOSCOPS (1·45, 1·08–1·95, p=0·012), and joint analysis of both populations (1·56, 1·24–1·97, p=0·0002). The association between haplogroup I and increased risk of coronary artery disease was independent of traditional cardiovascular and socioeconomic risk factors. Analysis of macrophage transcriptome in the Cardiogenics Study revealed that 19 molecular pathways showing strong differential expression between men with haplogroup I and other lineages of the Y chromosome were interconnected by common genes related to inflammation and immunity, and that some of them have a strong relevance to atherosclerosis.

Interpretation

The human Y chromosome is associated with risk of coronary artery disease in men of European ancestry, possibly through interactions of immunity and inflammation.

Funding

British Heart Foundation; UK National Institute for Health Research; LEW Carty Charitable Fund; National Health and Medical Research Council of Australia; European Union 6th Framework Programme; Wellcome Trust.

Summary

Background

We tested whether genetic factors distinctly contribute to either development of coronary atherosclerosis or, specifically, to myocardial infarction in existing coronary atherosclerosis.

Methods

We did two genome-wide association studies (GWAS) with coronary angiographic phenotyping in participants of European ancestry. To identify loci that predispose to angiographic coronary artery disease (CAD), we compared individuals who had this disorder (n=12 393) with those who did not (controls, n=7383). To identify loci that predispose to myocardial infarction, we compared patients who had angiographic CAD and myocardial infarction (n=5783) with those who had angiographic CAD but no myocardial infarction (n=3644).

Findings

In the comparison of patients with angiographic CAD versus controls, we identified a novel locus, ADAMTS7 (p=4·98×10−13). In the comparison of patients with angiographic CAD who had myocardial infarction versus those with angiographic CAD but no myocardial infarction, we identified a novel association at the ABO locus (p=7·62×10−9). The ABO association was attributable to the glycotransferase-deficient enzyme that encodes the ABO blood group O phenotype previously proposed to protect against myocardial infarction.

Interpretation

Our findings indicate that specific genetic predispositions promote the development of coronary atherosclerosis whereas others lead to myocardial infarction in the presence of coronary atherosclerosis. The relation to specific CAD phenotypes might modify how novel loci are applied in personalised risk assessment and used in the development of novel therapies for CAD.

Funding

The PennCath and MedStar studies were supported by the Cardiovascular Institute of the University of Pennsylvania, by the MedStar Health Research Institute at Washington Hospital Center and by a research grant from GlaxoSmithKline. The funding and support for the other cohorts contributing to the paper are described in the webappendix.

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns—independent component analysis—to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

Author Summary

One major expectation from the transcriptome in humans is to help characterize the biological basis of associations identified by genome-wide association studies. Here, we take advantage of recent technical and methodological advances to examine the influence of natural genetic variability on >12,000 genes expressed in the monocyte, a blood cell playing a key role in immunity-related disorders and atherosclerosis. By examining 1,490 European population-based subjects, we identify three regions of the genome reproducibly associated with specific patterns of gene expression. Two of these regions overlap genetic variants previously known to be involved in the susceptibility to type 1 diabetes, celiac disease, and hypertension. Genes whose expression is modulated by these genetic variants may act as mediators in the causal relationship linking the variability of the genome to complex disease. These findings illustrate how integration of genetic and transcriptomic data at an epidemiological scale can help decipher the genetic basis of complex diseases.

We previously identified two closely linked quantitative trait loci (QTL) on distal chromosome 1 contributing to major variations in plasma cholesterol and triglyceride levels in an intercross derived from C57BL/6 (B6) and C3H/HeJ (C3H) apolipoprotein E-deficient (apoE−/−) mice. Soat1, encoding sterol o-acyltransferase 1, is a functional candidate gene located underneath the proximal linkage peak. We sequenced the coding region of Soat1 and identified four single nucleotide polymorphisms (SNPs) between B6 and C3H mice. Two of the SNPs resulted in amino-acid substitutions (Ile147Val and His205Tyr). Functional assay revealed an increased enzyme activity of Soat1 in peritoneal macrophages of C3H mice relative to those of B6 mice despite comparable protein expression levels. Allelic variants of Soat1 were associated with variations in plasma cholesterol and triglyceride levels in an intercross between B6.apoE−/− and C3H.apoE−/− mice. Inheritance of the C3H allele resulted in significantly higher plasma lipid levels than inheritance of the B6 allele. Soat1 variants were also significantly linked to major variations in plasma esterified cholesterol levels but not with free cholesterol levels. Trangenic expression of C3H Soat1 in B6.apoE−/− mice resulted in elevations of plasma cholesterol and triglyceride levels. These results indicate that Soat1 is a QTL gene contributing to hyperlipidemia.

We performed a meta-analysis of 14 genome-wide association studies of coronary artery disease (CAD) comprising 22,233 cases and 64,762 controls of European descent, followed by genotyping of top association signals in 60,738 additional individuals. This genomic analysis identified 13 novel loci harboring one or more SNPs that were associated with CAD at P<5×10−8 and confirmed the association of 10 of 12 previously reported CAD loci. The 13 novel loci displayed risk allele frequencies ranging from 0.13 to 0.91 and were associated with a 6 to 17 percent increase in the risk of CAD per allele. Notably, only three of the novel loci displayed significant association with traditional CAD risk factors, while the majority lie in gene regions not previously implicated in the pathogenesis of CAD. Finally, five of the novel CAD risk loci appear to have pleiotropic effects, showing strong association with various other human diseases or traits.

Background

Venous Thrombosis (VT) is a common multifactorial disease associated with a major public health burden. Genetics factors are known to contribute to the susceptibility of the disease but how many genes are involved and their contribution to VT risk still remain obscure. We aimed to identify genetic variants associated with VT risk.

Methodology/Principal Findings

We conducted a genome-wide association study (GWAS) based on 551,141 SNPs genotyped in 1,542 cases and 1,110 controls. Twelve SNPs reached the genome-wide significance level of 2.0×10−8 and encompassed four known VT-associated loci, ABO, F5, F11 and FGG. By means of haplotype analyses, we also provided novel arguments in favor of a role of HIVEP1, PROCR and STAB2, three loci recently hypothesized to participate in the susceptibility to VT. However, no novel VT-associated loci came out of our GWAS. Using a recently proposed statistical methodology, we also showed that common variants could explain about 35% of the genetic variance underlying VT susceptibility among which 3% could be attributable to the main identified VT loci. This analysis additionally suggested that the common variants left to be identified are not uniformly distributed across the genome and that chromosome 20, itself, could contribute to ∼7% of the total genetic variance.

Conclusions/Significance

This study might also provide a valuable source of information to expand our understanding of biological mechanisms regulating quantitative biomarkers for VT.

Background

The biological functions of epicardial adipose tissue (EAT) remain largely unknown. However, the proximity of EAT to the coronary arteries suggests a role in the pathogenesis of coronary artery disease (CAD). The objectives of this study were to identify genes differentially regulated among three adipose tissues, namely EAT, mediastinal (MAT) and subcutaneous (SAT) and to study their possible relationships with the development of cardiovascular diseases.

Methods and Results

Samples were collected from subjects undergoing coronary artery bypass grafting surgeries. Gene expression was evaluated in the three adipose depots of six men using the Illumina® HumanWG-6 v3.0 expression BeadChips. Twenty-three and 73 genes were differentially up-regulated in EAT compared to MAT and SAT, respectively. Ninety-four genes were down-regulated in EAT compared to SAT. However, none were significantly down-regulated in EAT compared to MAT. More specifically, the expression of the adenosine A1 receptor (ADORA1), involved in myocardial ischemia, was significantly up-regulated in EAT. Levels of the prostaglandin D2 synthase (PTGDS) gene, recently associated with the progression of atherosclerosis, were significantly different in the three pairwise comparisons (EAT>MAT>SAT). The results of ADORA1 and PTGDS were confirmed by quantitative real-time PCR in 25 independent subjects.

Conclusions

Overall, the transcriptional profiles of EAT and MAT were similar compared to the SAT. Despite this similarity, two genes involved in cardiovascular diseases, ADORA1 and PTGDS, were differentially up-regulated in EAT. These results provide insights about the biology of EAT and its potential implication in CAD.

Background

The stability of atherosclerotic plaques determines the risk for rupture, which may lead to thrombus formation and potentially severe clinical complications such as myocardial infarction and stroke. Although the rate of plaque formation may be important for plaque stability, this process is not well understood. We took advantage of the atmospheric 14C-declination curve (a result of the atomic bomb tests in the 1950s and 1960s) to determine the average biological age of carotid plaques.

Methodology/Principal Finding

The cores of carotid plaques were dissected from 29 well-characterized, symptomatic patients with carotid stenosis and analyzed for 14C content by accelerator mass spectrometry. The average plaque age (i.e. formation time) was 9.6±3.3 years. All but two plaques had formed within 5–15 years before surgery. Plaque age was not associated with the chronological ages of the patients but was inversely related to plasma insulin levels (p = 0.0014). Most plaques were echo-lucent rather than echo-rich (2.24±0.97, range 1–5). However, plaques in the lowest tercile of plaque age (most recently formed) were characterized by further instability with a higher content of lipids and macrophages (67.8±12.4 vs. 50.4±6.2, p = 0.00005; 57.6±26.1 vs. 39.8±25.7, p<0.0005, respectively), less collagen (45.3±6.1 vs. 51.1±9.8, p<0.05), and fewer smooth muscle cells (130±31 vs. 141±21, p<0.05) than plaques in the highest tercile. Microarray analysis of plaques in the lowest tercile also showed increased activity of genes involved in immune responses and oxidative phosphorylation.

Conclusions/Significance

Our results show, for the first time, that plaque age, as judge by relative incorporation of 14C, can improve our understanding of carotid plaque stability and therefore risk for clinical complications. Our results also suggest that levels of plasma insulin might be involved in determining carotid plaque age.

Background

Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed.

Methods and Results

CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2×10−20).

Conclusion

CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI.

The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10−8 M (30 µg/l), 6 · 10−7 M (300 µg/l) or 2-chloro-N6-cyclopentyladenosine (CCPA) 10−6 M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/−87 pmol/g vs. 173+/−18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation–induced NE release in SHR (S2/S1 = 0.90±0.08 with capadenoson 6 · 10−8 M, 0.54±0.02 with 6 · 10−7 M), but not in Wistar hearts (S2/S1 = 1.05±0.12 with 6 · 10−8 M, 1.03±0.09 with 6 · 10−7 M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [35S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/−2% A1-receptor stimulation). These results suggest that partial adenosine A1-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release.

Objective

Turnover of the extracellular matrix in all solid organs is governed mainly by a balance between the degrading matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs). An altered extracellular matrix metabolism has been implicated in a variety of diseases. We investigated relations of serum levels of MMP-9 and TIMP-1 to mortality risk from an etiological perspective.

Design

The prospective Uppsala Longitudinal Study of Adult Men (ULSAM) cohort, followed from 1991–1995 for up to 18.1 years. A random population-based sample of 1,082 71-year-old men, no loss to follow-up. Endpoints were all-cause (n = 628), cardiovascular (n = 230), non-cardiovascular (n = 398) and cancer mortality (n = 178), and fatal or non-fatal myocardial infarction (n = 138) or stroke (n = 163).

Results

Serum MMP-9 and TIMP-1 levels were associated with risk of all-cause mortality (Cox proportional hazard ratio [HR] per standard deviation 1.10, 95% confidence interval [CI] 1.03–1.19; and 1.11, 1.02–1.20; respectively). TIMP-1 levels were mainly related to risks of cardiovascular mortality and stroke (HR per standard deviation 1.22, 95% CI 1.09–1.37; and 1.18, 1.04–1.35; respectively). All relations except those of TIMP-1 to stroke risk were attenuated by adjustment for cardiovascular disease risk factors. Relations in a subsample without cardiovascular disease or cancer were similar to those in the total sample.

Conclusion

In this community-based cohort of elderly men, serum MMP-9 and TIMP-1 levels were related to mortality risk. An altered extracellular matrix metabolism may be involved in several detrimental pathways, and circulating MMP-9 or TIMP-1 levels may be relevant markers thereof.

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10−6, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10−5. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10−3, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10−3, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10−4, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10−13, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.

This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.

Author Summary

Dilated cardiomyopathy is a severe disease of the heart muscle and often leads to chronic heart failure, eventually with the consequence of cardiac transplantation. Identification of genetic disease markers in at-risk persons could play an important role in preventive health care. Several mutations in familial forms of the disease are described. Here, we examine the role of common genetic variants on the sporadic form of dilated cardiomyopathy. By screening about 2,000 candidate genes previously related to cardiovascular disease in more than 1,900 cases and 3,600 controls, we show that a polymorphism in the HSPB7 gene (rs1739843) is strongly associated with susceptibility to dilated cardiomyopathy. We also show that the effect on disease risk is present in both German and French cohorts. Therefore, this study is an important step towards revealing insight in the genetic background of the sporadic form of dilated cardiomyopathy.