[show abstract][hide abstract] ABSTRACT: BACKGROUND: Persistent inflammation has been proposed to contribute to various stages in the pathogenesis of cardiovascular disease. Interleukin-6 receptor (IL6R) signalling propagates downstream inflammation cascades. To assess whether this pathway is causally relevant to coronary heart disease, we studied a functional genetic variant known to affect IL6R signalling.
METHODS: In a collaborative meta-analysis, we studied Asp358Ala (rs2228145) in IL6R in relation to a panel of conventional risk factors and inflammation biomarkers in 125,222 participants. We also compared the frequency of Asp358Ala in 51,441 patients with coronary heart disease and in 136,226 controls. To gain insight into possible mechanisms, we assessed Asp358Ala in relation to localised gene expression and to postlipopolysaccharide stimulation of interleukin 6.
FINDINGS: The minor allele frequency of Asp358Ala was 39%. Asp358Ala was not associated with lipid concentrations, blood pressure, adiposity, dysglycaemia, or smoking (p value for association per minor allele ≥0·04 for each). By contrast, for every copy of 358Ala inherited, mean concentration of IL6R increased by 34·3% (95% CI 30·4-38·2) and of interleukin 6 by 14·6% (10·7-18·4), and mean concentration of C-reactive protein was reduced by 7·5% (5·9-9·1) and of fibrinogen by 1·0% (0·7-1·3). For every copy of 358Ala inherited, risk of coronary heart disease was reduced by 3·4% (1·8-5·0). Asp358Ala was not related to IL6R mRNA levels or interleukin-6 production in monocytes.
INTERPRETATION: Large-scale human genetic and biomarker data are consistent with a causal association between IL6R-related pathways and coronary heart disease.
[show abstract][hide abstract] ABSTRACT: To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom ∼50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with ∼2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 × 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 × 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 × 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 × 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 × 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.
The American Journal of Human Genetics 01/2012; · 11.20 Impact Factor
[show abstract][hide abstract] ABSTRACT: To identify genetic factors contributing to type 2 diabetes (T2D), we performed large-scale meta-analyses by using a custom approximately 50,000 SNP genotyping array (the ITMAT-Broad-CARe array) with approximately 2000 candidate genes in 39 multiethnic population-based studies, case-control studies, and clinical trials totaling 17,418 cases and 70,298 controls. First, meta-analysis of 25 studies comprising 14,073 cases and 57,489 controls of European descent confirmed eight established T2D loci at genome-wide significance. In silico follow-up analysis of putative association signals found in independent genome-wide association studies (including 8,130 cases and 38,987 controls) performed by the DIAGRAM consortium identified a T2D locus at genome-wide significance (GATAD2A/CILP2/PBX4; p = 5.7 x 10(-9)) and two loci exceeding study-wide significance (SREBF1, and TH/INS; p < 2.4 x 10(-6)). Second, meta-analyses of 1,986 cases and 7,695 controls from eight African-American studies identified study-wide-significant (p = 2.4 x 10(-7)) variants in HMGA2 and replicated variants in TCF7L2 (p = 5.1 x 10(-15)). Third, conditional analysis revealed multiple known and novel independent signals within five T2D-associated genes in samples of European ancestry and within HMGA2 in African-American samples. Fourth, a multiethnic meta-analysis of all 39 studies identified T2D-associated variants in BCL2 (p = 2.1 x 10(-8)). Finally, a composite genetic score of SNPs from new and established T2D signals was significantly associated with increased risk of diabetes in African-American, Hispanic, and Asian populations. In summary, large-scale meta-analysis involving a dense gene-centric approach has uncovered additional loci and variants that contribute to T2D risk and suggests substantial overlap of T2D association signals across multiple ethnic groups.
[show abstract][hide abstract] ABSTRACT: Coronary artery disease (CAD) has a significant genetic contribution that is incompletely characterized. To complement genome-wide association (GWA) studies, we conducted a large and systematic candidate gene study of CAD susceptibility, including analysis of many uncommon and functional variants. We examined 49,094 genetic variants in ∼2,100 genes of cardiovascular relevance, using a customised gene array in 15,596 CAD cases and 34,992 controls (11,202 cases and 30,733 controls of European descent; 4,394 cases and 4,259 controls of South Asian origin). We attempted to replicate putative novel associations in an additional 17,121 CAD cases and 40,473 controls. Potential mechanisms through which the novel variants could affect CAD risk were explored through association tests with vascular risk factors and gene expression. We confirmed associations of several previously known CAD susceptibility loci (eg, 9p21.3:p<10(-33); LPA:p<10(-19); 1p13.3:p<10(-17)) as well as three recently discovered loci (COL4A1/COL4A2, ZC3HC1, CYP17A1:p<5×10(-7)). However, we found essentially null results for most previously suggested CAD candidate genes. In our replication study of 24 promising common variants, we identified novel associations of variants in or near LIPA, IL5, TRIB1, and ABCG5/ABCG8, with per-allele odds ratios for CAD risk with each of the novel variants ranging from 1.06-1.09. Associations with variants at LIPA, TRIB1, and ABCG5/ABCG8 were supported by gene expression data or effects on lipid levels. Apart from the previously reported variants in LPA, none of the other ∼4,500 low frequency and functional variants showed a strong effect. Associations in South Asians did not differ appreciably from those in Europeans, except for 9p21.3 (per-allele odds ratio: 1.14 versus 1.27 respectively; P for heterogeneity = 0.003). This large-scale gene-centric analysis has identified several novel genes for CAD that relate to diverse biochemical and cellular functions and clarified the literature with regard to many previously suggested genes.
[show abstract][hide abstract] ABSTRACT: Raised blood pressure (BP) is a major risk factor for cardiovascular disease. Previous studies have identified 47 distinct genetic variants robustly associated with BP, but collectively these explain only a few percent of the heritability for BP phenotypes. To find additional BP loci, we used a bespoke gene-centric array to genotype an independent discovery sample of 25,118 individuals that combined hypertensive case-control and general population samples. We followed up four SNPs associated with BP at our p < 8.56 x 10(-7) study-specific significance threshold and six suggestively associated SNPs in a further 59,349 individuals. We identified and replicated a SNP at LSP1/TNNT3, a SNP at MTHFR-NPPB independent (r(2) = 0.33) of previous reports, and replicated SNPs at AGT and ATP2B1 reported previously. An analysis of combined discovery, and follow-up data identified SNPs significantly associated with BP at p < 8.56 x 10(-7) at four further loci (NPR3, FIFE, NOS3, and SOX6). The high number of discoveries made with modest genotyping effort can be attributed to using a large-scale yet targeted genotyping array and to the development of a weighting scheme that maximized power when meta-analyzing results from samples ascertained with extreme phenotypes, in combination with results from nonascertained or population samples. Chromatin immunoprecipitation and transcript expression data highlight potential gene regulatory mechanisms at the MTHFR and NOS3 loci. These results provide candidates for further study to help dissect mechanisms affecting BP and highlight the utility of studying SNPs and samples that are independent of those studied previously even when the sample size is smaller than that in previous studies.
American Journal of Human Genetics. 01/2011; 89(6):688-700.
[show abstract][hide abstract] ABSTRACT: Genetic markers can be used as instrumental variables, in an analogous way to randomization in a clinical trial, to estimate the causal relationship between a phenotype and an outcome variable. Our purpose is to extend the existing methods for such Mendelian randomization studies to the context of multiple genetic markers measured in multiple studies, based on the analysis of individual participant data. First, for a single genetic marker in one study, we show that the usual ratio of coefficients approach can be reformulated as a regression with heterogeneous error in the explanatory variable. This can be implemented using a Bayesian approach, which is next extended to include multiple genetic markers. We then propose a hierarchical model for undertaking a meta-analysis of multiple studies, in which it is not necessary that the same genetic markers are measured in each study. This provides an overall estimate of the causal relationship between the phenotype and the outcome, and an assessment of its heterogeneity across studies. As an example, we estimate the causal relationship of blood concentrations of C-reactive protein on fibrinogen levels using data from 11 studies. These methods provide a flexible framework for efficient estimation of causal relationships derived from multiple studies. Issues discussed include weak instrument bias, analysis of binary outcome data such as disease risk, missing genetic data, and the use of haplotypes.
Statistics in Medicine 03/2010; 29(12):1298-311. · 2.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Flash-frozen myocardium samples provide a valuable means of correlating clinical cardiomyopathies with abnormalities in sarcomeric contractile and biochemical parameters. We examined flash-frozen left-ventricle human cardiomyocyte bundles from healthy donors to determine control parameters for isometric tension (P(o)) development and Ca(2+) sensitivity, while simultaneously measuring actomyosin ATPase activity in situ by a fluorimetric technique. P(o) was 17 kN m(-2) and pCa(50%) was 5.99 (28 degrees C, I = 130 mM). ATPase activity increased linearly with tension to 132 muM s(-1). To determine the influence of flash-freezing, we compared the same parameters in both glycerinated and flash-frozen porcine left-ventricle trabeculae. P(o) in glycerinated porcine myocardium was 25 kN m(-2), and maximum ATPase activity was 183 microM s(-1). In flash-frozen porcine myocardium, P(o) was 16 kN m(-2) and maximum ATPase activity was 207 microM s(-1). pCa(50%) was 5.77 in the glycerinated and 5.83 in the flash-frozen sample. Both passive and active stiffness of flash-frozen porcine myocardium were lower than for glycerinated tissue and similar to the human samples. Although lower stiffness and isometric tension development may indicate flash-freezing impairment of axial force transmission, we cannot exclude variability between samples as the cause. ATPase activity and pCa(50%) were unaffected by flash-freezing. The lower ATPase activity measured in human tissue suggests a slower actomyosin turnover by the contractile proteins.
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Estrogen receptor alpha (ERalpha) mediates beneficial actions in the cardiovascular system, including on cholesterol metabolism and endothelial function. Genetic variations in ERalpha may therefore influence these phenotypes. METHODS AND RESULTS: We identified a single nucleotide polymorphism ERNE T>C, in a negative transcriptional element upstream of ERalpha, that occurred with a minor allele frequency of 3.1%. When cloned upstream of a basic promoter and luciferase reporter gene, and transfected in a hepatic cell line, ERNE (T) was associated with a significant reduction in promoter activity compared with control (44+/-16%; p<0.05; n=3). The negative regulation by the ERNE wild-type sequence was abolished by inclusion of the variant C allele (103+/-17%). CONCLUSION: The disruption of the ERalpha negative transcriptional regulation element by this newly identified polymorphism may influence estrogen-sensitive phenotypes in vivo.
International journal of cardiology 01/2009; 144(1):86-8. · 7.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Estrogen receptor alpha (ERalpha) mediates beneficial actions on endothelial nitric oxide synthase (eNOS) and cholesterol metabolism. Genetic variations in the promoter of the ERalpha may therefore influence vascular function. We have identified a single nucleotide polymorphism (T>C) in the transcriptional element "ERNE" upstream of ERalpha which abolished the negative effect of this element in luciferase reporter assays and was associated with reduction in LDL cholesterol in a small association study. We have now examined for the association of this putative functional polymorphism with endothelial function. Endothelial-dependent relaxation (EDR) was measured in organ bath preparations of human saphenous vein obtained from 101 individuals (81 males and 20 females) undergoing coronary artery bypass surgery. The presence of the variant C allele was associated with enhanced EDR independently of hypercholesterolaemia, smoking and diabetes, as well as sex (ANOVA for ACh induced relaxation: p=0.033). In males, the presence of the C allele was associated with a 225% augmentation of endothelial-dependent relaxation compared to wild-type (55.5+/-10%; n=3 vs. 24.7+/-1%; n=78; p<0.001). In summary, a polymorphism in the ERalpha negative transcriptional element which results in increased transcription in vitro is associated with substantial augmentation of endothelial-dependent vasorelaxation.
International journal of cardiology 01/2009; 143(2):207-8. · 7.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: It is uncertain whether the novel single nucleotide polymorphisms (SNPs) that have recently been associated with coronary artery disease (CAD) in genome-wide studies also influence carotid atheroma and stroke risk. The mechanisms of their association with CAD are unknown; relationships to other cardiovascular phenotypes may give mechanistic clues. Carotid artery intima-media thickness (CIMT) is a subclinical marker of atherosclerosis associated with stroke. We investigated association of reported CAD risk variants with CIMT, and with other intermediate phenotypes that may implicate causative pathways.
We studied 1425 members of 248 British Caucasian families ascertained through a hypertensive proband. We genotyped CAD risk SNPs on chromosomes 9 (rs1333049, rs7044859, rs496892, rs7865618), 6 (rs6922269) and 2 (rs2943634) using TaqMan. Merlin software was used for family-based association testing.
No significant association was found between genotype at any SNP and CIMT in 846 individuals with acceptable measurements. Nor were SNPs significantly associated with blood pressure, obesity, cholesterol, CRP, interleukin-6, TNF-alpha, or leptin.
These novel CAD variants are not associated with CIMT and do not appear to mediate the risk of atherothrombosis through known risk factors.
[show abstract][hide abstract] ABSTRACT: Myosin-binding protein C (MyBPC) is proposed to take on a trimeric collar arrangement around the thick filament backbone in cardiac muscle, based on interactions between cardiac MyBPC domains C5 and C8. We have now determined, using yeast two-hybrid and in vitro binding assays, that the C5:C8 interaction is not dependent on the 28-residue cardiac-specific insert in C5. Furthermore, an interaction of similar affinity occurs between domains C5 and C8 of fast skeletal muscle MyBPC, but not between these domains of the slow skeletal muscle protein. These data have implications for the role and quaternary structure of MyBPC in skeletal muscle.
[show abstract][hide abstract] ABSTRACT: E40K and E54K mutations in alpha-tropomyosin cause inherited dilated cardiomyopathy. Previously we showed, using Ala-Ser alpha-tropomyosin (AS-alpha-Tm) expressed in Escherichia coli, that both mutations decrease Ca(2+) sensitivity. E40K also reduces V(max) of actin-Tm-activated S-1 ATPase by 18%. We investigated cooperative allosteric regulation by native Tm, AS-alpha-Tm, and the two dilated cardiomyopathy-causing mutants. AS-alpha-Tm has a lower cooperative unit size (6.5) than native alpha-tropomyosin (10.0). The E40K mutation reduced the size of the cooperative unit to 3.7, whereas E54K increased it to 8.0. For the equilibrium between On and Off states, the K(T) value was the same for all actin-Tm species; however, the K(T) value of actin-Tm-troponin at pCa 5 was 50% less for AS-alpha-Tm E40K than for AS-alpha-Tm and AS-alpha-Tm E54K. K(b), the "closed" to "blocked" equilibrium constant, was the same for all tropomyosin species. The E40K mutation reduced the affinity of tropomyosin for actin by 1.74-fold, but only when in the On state (in the presence of S-1). In contrast the E54K mutation reduced affinity by 3.5-fold only in the Off state. Differential scanning calorimetry measurements of AS-alpha-Tm showed that domain 3, assigned to the N terminus of tropomyosin, was strongly destabilized by both mutations. Additionally with AS-alpha-Tm E54K, we observed a unique new domain at 55 degrees C accounting for 25% of enthalpy indicating stabilization of part of the tropomyosin. The disease-causing mechanism of the E40K mutation may be accounted for by destabilization of the On state of the thin filaments; however, the E54K mutation has a more complex effect on tropomyosin structure and function.
Journal of Biological Chemistry 06/2007; · 4.65 Impact Factor