Daniel F. Freitag

Interleukin-6 receptor pathways in coronary heart disease: a collaborative meta-analysis of 82 studies.

Summary 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. Funding British Heart Foundation; UK Medical Research Council; UK National Institute of Health Research, Cambridge Biomedical Research Centre; BUPA Foundation.

Inflammatory cytokines and risk of coronary heart disease: new prospective study and updated meta-analysis

AIMS Because low-grade inflammation may play a role in the pathogenesis of coronary heart disease (CHD), and pro-inflammatory cytokines govern inflammatory cascades, this study aimed to assess the associations of several pro-inflammatory cytokines and CHD risk in a new prospective study, including meta-analysis of prospective studies. METHODS AND RESULTS Interleukin-6 (IL-6), IL-18, matrix metalloproteinase-9 (MMP-9), soluble CD40 ligand (sCD40L), and tumour necrosis factor-α (TNF-α) were measured at baseline in a case-cohort study of 1514 participants and 833 incident CHD events within population-based prospective cohorts at the Danish Research Centre for Prevention and Health. Age- and sex-adjusted hazard ratios (HRs) for CHD per 1-SD higher log-transformed baseline levels were: 1.37 (95% CI: 1.21-1.54) for IL-6, 1.26 (1.11-1.44) for IL-18, 1.30 (1.16-1.46) for MMP-9, 1.01 (0.89-1.15) for sCD40L, and 1.13 (1.01-1.27) for TNF-α. Multivariable adjustment for conventional vascular risk factors attenuated the HRs to: 1.26 (1.08-1.46) for IL-6, 1.12 (0.95-1.31) for IL-18, 1.21 (1.05-1.39) for MMP-9, 0.93 (0.78-1.11) for sCD40L, and 1.14 (1.00-1.31) for TNF-α. In meta-analysis of up to 29 population-based prospective studies, adjusted relative risks for non-fatal MI or CHD death per 1-SD higher levels were: 1.25 (1.19-1.32) for IL-6; 1.13 (1.05-1.20) for IL-18; 1.07 (0.97-1.19) for MMP-9; 1.07 (0.95-1.21) for sCD40L; and 1.17 (1.09-1.25) for TNF-α. CONCLUSIONS Several different pro-inflammatory cytokines are each associated with CHD risk independent of conventional risk factors and in an approximately log-linear manner. The findings lend support to the inflammation hypothesis in vascular disease, but further studies are needed to assess causality.

Association of Cardiometabolic Multimorbidity With Mortality.

IMPORTANCE The prevalence of cardiometabolic multimorbidity is increasing. OBJECTIVE To estimate reductions in life expectancy associated with cardiometabolic multimorbidity. DESIGN, SETTING, AND PARTICIPANTS Age- and sex-adjusted mortality rates and hazard ratios (HRs) were calculated using individual participant data from the Emerging Risk Factors Collaboration (689,300 participants; 91 cohorts; years of baseline surveys: 1960-2007; latest mortality follow-up: April 2013; 128,843 deaths). The HRs from the Emerging Risk Factors Collaboration were compared with those from the UK Biobank (499,808 participants; years of baseline surveys: 2006-2010; latest mortality follow-up: November 2013; 7995 deaths). Cumulative survival was estimated by applying calculated age-specific HRs for mortality to contemporary US age-specific death rates. EXPOSURES A history of 2 or more of the following: diabetes mellitus, stroke, myocardial infarction (MI). MAIN OUTCOMES AND MEASURES All-cause mortality and estimated reductions in life expectancy. RESULTS In participants in the Emerging Risk Factors Collaboration without a history of diabetes, stroke, or MI at baseline (reference group), the all-cause mortality rate adjusted to the age of 60 years was 6.8 per 1000 person-years. Mortality rates per 1000 person-years were 15.6 in participants with a history of diabetes, 16.1 in those with stroke, 16.8 in those with MI, 32.0 in those with both diabetes and MI, 32.5 in those with both diabetes and stroke, 32.8 in those with both stroke and MI, and 59.5 in those with diabetes, stroke, and MI. Compared with the reference group, the HRs for all-cause mortality were 1.9 (95% CI, 1.8-2.0) in participants with a history of diabetes, 2.1 (95% CI, 2.0-2.2) in those with stroke, 2.0 (95% CI, 1.9-2.2) in those with MI, 3.7 (95% CI, 3.3-4.1) in those with both diabetes and MI, 3.8 (95% CI, 3.5-4.2) in those with both diabetes and stroke, 3.5 (95% CI, 3.1-4.0) in those with both stroke and MI, and 6.9 (95% CI, 5.7-8.3) in those with diabetes, stroke, and MI. The HRs from the Emerging Risk Factors Collaboration were similar to those from the more recently recruited UK Biobank. The HRs were little changed after further adjustment for markers of established intermediate pathways (eg, levels of lipids and blood pressure) and lifestyle factors (eg, smoking, diet). At the age of 60 years, a history of any 2 of these conditions was associated with 12 years of reduced life expectancy and a history of all 3 of these conditions was associated with 15 years of reduced life expectancy. CONCLUSIONS AND RELEVANCE Mortality associated with a history of diabetes, stroke, or MI was similar for each condition. Because any combination of these conditions was associated with multiplicative mortality risk, life expectancy was substantially lower in people with multimorbidity.

Functional IL6R 358Ala Allele Impairs Classical IL-6 Receptor Signaling and Influences Risk of Diverse Inflammatory Diseases

Inflammation, which is directly regulated by interleukin-6 (IL-6) signaling, is implicated in the etiology of several chronic diseases. Although a common, non-synonymous variant in the IL-6 receptor gene (IL6R Asp358Ala; rs2228145 A>C) is associated with the risk of several common diseases, with the 358Ala allele conferring protection from coronary heart disease (CHD), rheumatoid arthritis (RA), atrial fibrillation (AF), abdominal aortic aneurysm (AAA), and increased susceptibility to asthma, the variants effect on IL-6 signaling is not known. Here we provide evidence for the association of this non-synonymous variant with the risk of type 1 diabetes (T1D) in two independent populations and confirm that rs2228145 is the major determinant of the concentration of circulating soluble IL-6R (sIL-6R) levels (34.6% increase in sIL-6R per copy of the minor allele 358Ala; rs2228145 [C]). To further investigate the molecular mechanism of this variant, we analyzed expression of IL-6R in peripheral blood mononuclear cells (PBMCs) in 128 volunteers from the Cambridge BioResource. We demonstrate that, although 358Ala increases transcription of the soluble IL6R isoform (P = 8.3×10−22) and not the membrane-bound isoform, 358Ala reduces surface expression of IL-6R on CD4+ T cells and monocytes (up to 28% reduction per allele; P≤5.6×10−22). Importantly, reduced expression of membrane-bound IL-6R resulted in impaired IL-6 responsiveness, as measured by decreased phosphorylation of the transcription factors STAT3 and STAT1 following stimulation with IL-6 (P≤5.2×10−7). Our findings elucidate the regulation of IL-6 signaling by IL-6R, which is causally relevant to several complex diseases, identify mechanisms for new approaches to target the IL-6/IL-6R axis, and anticipate differences in treatment response to IL-6 therapies based on this common IL6R variant.

PhenoScanner: a database of human genotype–phenotype associations

Abstract Summary: PhenoScanner is a curated database of publicly available results from large-scale genetic association studies. This tool aims to facilitate ‘phenome scans’, the cross-referencing of genetic variants with many phenotypes, to help aid understanding of disease pathways and biology. The database currently contains over 350 million association results and over 10 million unique genetic variants, mostly single nucleotide polymorphisms. It is accompanied by a web-based tool that queries the database for associations with user-specified variants, providing results according to the same effect and non-effect alleles for each input variant. The tool provides the option of searching for trait associations with proxies of the input variants, calculated using the European samples from 1000 Genomes and Hapmap. Availability and Implementation: PhenoScanner is available at www.phenoscanner.medschl.cam.ac.uk. Contact: jrs95@medschl.cam.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Fifteen new risk loci for coronary artery disease highlight arterial-wall-specific mechanisms

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Although 58 genomic regions have been associated with CAD thus far, most of the heritability is unexplained, indicating that additional susceptibility loci await identification. An efficient discovery strategy may be larger-scale evaluation of promising associations suggested by genome-wide association studies (GWAS). Hence, we genotyped 56,309 participants using a targeted gene array derived from earlier GWAS results and performed meta-analysis of results with 194,427 participants previously genotyped, totaling 88,192 CAD cases and 162,544 controls. We identified 25 new SNP–CAD associations (P < 5 × 10−8, in fixed-effects meta-analysis) from 15 genomic regions, including SNPs in or near genes involved in cellular adhesion, leukocyte migration and atherosclerosis (PECAM1, rs1867624), coagulation and inflammation (PROCR, rs867186 (p.Ser219Gly)) and vascular smooth muscle cell differentiation (LMOD1, rs2820315). Correlation of these regions with cell-type-specific gene expression and plasma protein levels sheds light on potential disease mechanisms.

Asymmetric Dimethylarginine and Cardiovascular Risk: Systematic Review and Meta-Analysis of 22 Prospective Studies

Background Asymmetric dimethylarginine (ADMA) inhibits the production of nitric oxide, a key regulator of the vascular tone, and may be important in the development of cardiovascular disease (CVD). Our aim was to reliably quantify the association of ADMA and its isomer symmetric dimethylarginine (SDMA) with the risk of CVD outcomes in long-term cohort studies. Methods and Results Data were collated from 22 prospective studies involving a total of 19 842 participants, which have recorded 2339 CVD, 997 coronary heart disease, and 467 stroke outcomes during a mean follow-up of 7.1 years. In a comparison of individuals in the top with those in the bottom third of baseline ADMA values, the combined risk ratios were 1.42 (95% confidence interval: 1.29 to 1.56) for CVD, 1.39 for coronary heart disease (1.19 to 1.62), and 1.60 for stroke (1.33 to 1.91). Broadly similar results were observed according to participants’ baseline disease status (risk ratios for CVD: 1.35 [1.18 to 1.54] in general populations; 1.47 [1.16 to 1.87] in individuals with pre-existing CVD; and 1.52 [1.26 to 1.84] in individuals with pre-existing kidney disease) and by different study characteristics, including geographical location, sample type, assay method, number of incident outcomes, and level of statistical adjustment (all P values>0.05). In contrast, in 8 prospective studies involving 9070 participants and 848 outcomes, the corresponding estimate for SDMA concentration was 1.32 (0.92 to 1.90) for CVD. Conclusions Available prospective studies suggest associations between circulating ADMA concentration and CVD outcomes under a broad range of circumstances. Further research is needed to better clarify these associations, particularly in large general population studies.

Using multivariable Mendelian randomization to disentangle the causal effects of lipid fractions.

Background Previous Mendelian randomization studies have suggested that, while low-density lipoprotein cholesterol (LDL-c) and triglycerides are causally implicated in coronary artery disease (CAD) risk, high-density lipoprotein cholesterol (HDL-c) may not be, with causal effect estimates compatible with the null. Principal Findings The causal effects of these three lipid fractions can be better identified using the extended methods of ‘multivariable Mendelian randomization’. We employ this approach using published data on 185 lipid-related genetic variants and their associations with lipid fractions in 188,578 participants, and with CAD risk in 22,233 cases and 64,762 controls. Our results suggest that HDL-c may be causally protective of CAD risk, independently of the effects of LDL-c and triglycerides. Estimated causal odds ratios per standard deviation increase, based on 162 variants not having pleiotropic associations with either blood pressure or body mass index, are 1.57 (95% credible interval 1.45 to 1.70) for LDL-c, 0.91 (0.83 to 0.99, p-value  = 0.028) for HDL-c, and 1.29 (1.16 to 1.43) for triglycerides. Significance Some interventions on HDL-c concentrations may influence risk of CAD, but to a lesser extent than interventions on LDL-c. A causal interpretation of these estimates relies on the assumption that the genetic variants do not have pleiotropic associations with risk factors on other pathways to CAD. If they do, a weaker conclusion is that genetic predictors of LDL-c, HDL-c and triglycerides each have independent associations with CAD risk.

Natriuretic peptides and integrated risk assessment for cardiovascular disease: an individual-participant-data meta-analysis

Summary Background Guidelines for primary prevention of cardiovascular diseases focus on prediction of coronary heart disease and stroke. We assessed whether or not measurement of N-terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration could enable a more integrated approach than at present by predicting heart failure and enhancing coronary heart disease and stroke risk assessment. Methods In this individual-participant-data meta-analysis, we generated and harmonised individual-participant data from relevant prospective studies via both de-novo NT-proBNP concentration measurement of stored samples and collection of data from studies identified through a systematic search of the literature (PubMed, Scientific Citation Index Expanded, and Embase) for articles published up to Sept 4, 2014, using search terms related to natriuretic peptide family members and the primary outcomes, with no language restrictions. We calculated risk ratios and measures of risk discrimination and reclassification across predicted 10 year risk categories (ie, <5%, 5% to <7·5%, and ≥7·5%), adding assessment of NT-proBNP concentration to that of conventional risk factors (ie, age, sex, smoking status, systolic blood pressure, history of diabetes, and total and HDL cholesterol concentrations). Primary outcomes were the combination of coronary heart disease and stroke, and the combination of coronary heart disease, stroke, and heart failure. Findings We recorded 5500 coronary heart disease, 4002 stroke, and 2212 heart failure outcomes among 95 617 participants without a history of cardiovascular disease in 40 prospective studies. Risk ratios (for a comparison of the top third vs bottom third of NT-proBNP concentrations, adjusted for conventional risk factors) were 1·76 (95% CI 1·56–1·98) for the combination of coronary heart disease and stroke and 2·00 (1·77–2·26) for the combination of coronary heart disease, stroke, and heart failure. Addition of information about NT-proBNP concentration to a model containing conventional risk factors was associated with a C-index increase of 0·012 (0·010–0·014) and a net reclassification improvement of 0·027 (0·019–0·036) for the combination of coronary heart disease and stroke and a C-index increase of 0·019 (0·016–0·022) and a net reclassification improvement of 0·028 (0·019–0·038) for the combination of coronary heart disease, stroke, and heart failure. Interpretation In people without baseline cardiovascular disease, NT-proBNP concentration assessment strongly predicted first-onset heart failure and augmented coronary heart disease and stroke prediction, suggesting that NT-proBNP concentration assessment could be used to integrate heart failure into cardiovascular disease primary prevention. Funding British Heart Foundation, Austrian Science Fund, UK Medical Research Council, National Institute for Health Research, European Research Council, and European Commission Framework Programme 7.Summary Background Guidelines for primary prevention of cardiovascular diseases focus on prediction of coronary heart disease and stroke. We assessed whether or not measurement of N-terminal-pro-B-type natriuretic peptide (NT-proBNP) concentration could enable a more integrated approach than at present by predicting heart failure and enhancing coronary heart disease and stroke risk assessment. Methods In this individual-participant-data meta-analysis, we generated and harmonised individual-participant data from relevant prospective studies via both de-novo NT-proBNP concentration measurement of stored samples and collection of data from studies identified through a systematic search of the literature (PubMed, Scientific Citation Index Expanded, and Embase) for articles published up to Sept 4, 2014, using search terms related to natriuretic peptide family members and the primary outcomes, with no language restrictions. We calculated risk ratios and measures of risk discrimination and reclassification across predicted 10 year risk categories (ie, Findings We recorded 5500 coronary heart disease, 4002 stroke, and 2212 heart failure outcomes among 95 617 participants without a history of cardiovascular disease in 40 prospective studies. Risk ratios (for a comparison of the top third vs bottom third of NT-proBNP concentrations, adjusted for conventional risk factors) were 1·76 (95% CI 1·56–1·98) for the combination of coronary heart disease and stroke and 2·00 (1·77–2·26) for the combination of coronary heart disease, stroke, and heart failure. Addition of information about NT-proBNP concentration to a model containing conventional risk factors was associated with a C-index increase of 0·012 (0·010–0·014) and a net reclassification improvement of 0·027 (0·019–0·036) for the combination of coronary heart disease and stroke and a C-index increase of 0·019 (0·016–0·022) and a net reclassification improvement of 0·028 (0·019–0·038) for the combination of coronary heart disease, stroke, and heart failure. Interpretation In people without baseline cardiovascular disease, NT-proBNP concentration assessment strongly predicted first-onset heart failure and augmented coronary heart disease and stroke prediction, suggesting that NT-proBNP concentration assessment could be used to integrate heart failure into cardiovascular disease primary prevention. Funding British Heart Foundation, Austrian Science Fund, UK Medical Research Council, National Institute for Health Research, European Research Council, and European Commission Framework Programme 7.

Genetic invalidation of Lp-PLA2 as a therapeutic target: large-scale study of five functional Lp-PLA2-lowering alleles

Aims Darapladib, a potent inhibitor of lipoprotein-associated phospholipase A2 (Lp-PLA2), has not reduced risk of cardiovascular disease outcomes in recent randomized trials. We aimed to test whether Lp-PLA2 enzyme activity is causally relevant to coronary heart disease. Methods In 72,657 patients with coronary heart disease and 110,218 controls in 23 epidemiological studies, we genotyped five functional variants: four rare loss-of-function mutations (c.109+2T > C (rs142974898), Arg82His (rs144983904), Val279Phe (rs76863441), Gln287Ter (rs140020965)) and one common modest-impact variant (Val379Ala (rs1051931)) in PLA2G7, the gene encoding Lp-PLA2. We supplemented de-novo genotyping with information on a further 45,823 coronary heart disease patients and 88,680 controls in publicly available databases and other previous studies. We conducted a systematic review of randomized trials to compare effects of darapladib treatment on soluble Lp-PLA2 activity, conventional cardiovascular risk factors, and coronary heart disease risk with corresponding effects of Lp-PLA2-lowering alleles. Results Lp-PLA2 activity was decreased by 64% (p = 2.4 × 10–25) with carriage of any of the four loss-of-function variants, by 45% (p < 10–300) for every allele inherited at Val279Phe, and by 2.7% (p = 1.9 × 10–12) for every allele inherited at Val379Ala. Darapladib 160 mg once-daily reduced Lp-PLA2 activity by 65% (p < 10–300). Causal risk ratios for coronary heart disease per 65% lower Lp-PLA2 activity were: 0.95 (0.88–1.03) with Val279Phe; 0.92 (0.74–1.16) with carriage of any loss-of-function variant; 1.01 (0.68–1.51) with Val379Ala; and 0.95 (0.89–1.02) with darapladib treatment. Conclusions In a large-scale human genetic study, none of a series of Lp-PLA2-lowering alleles was related to coronary heart disease risk, suggesting that Lp-PLA2 is unlikely to be a causal risk factor.