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Lancet. Mar 31, 2012; 379(9822): 1214–1224.
PMCID: PMC3316968
The interleukin-6 receptor as a target for prevention of coronary heart disease: a mendelian randomisation analysis
The Interleukin-6 Receptor Mendelian Randomisation Analysis (IL6R MR) Consortium
Members listed at end of paper
This document was posted here by permission of the publisher. At the time of the deposit, it included all changes made during peer review, copy editing, and publishing. The U. S. National Library of Medicine is responsible for all links within the document and for incorporating any publisher-supplied amendments or retractions issued subsequently. The published journal article, guaranteed to be such by Elsevier, is available for free, on ScienceDirect, at: http://dx.crossref.org/10.1016/S0140-6736(12)60110-X
Background
A high circulating concentration of interleukin 6 is associated with increased risk of coronary heart disease. Blockade of the interleukin-6 receptor (IL6R) with a monoclonal antibody (tocilizumab) licensed for treatment of rheumatoid arthritis reduces systemic and articular inflammation. However, whether IL6R blockade also reduces risk of coronary heart disease is unknown.
Methods
Applying the mendelian randomisation principle, we used single nucleotide polymorphisms (SNPs) in the gene IL6R to evaluate the likely efficacy and safety of IL6R inhibition for primary prevention of coronary heart disease. We compared genetic findings with the effects of tocilizumab reported in randomised trials in patients with rheumatoid arthritis.
Findings
In 40 studies including up to 133 449 individuals, an IL6R SNP (rs7529229) marking a non-synonymous IL6R variant (rs8192284; p.Asp358Ala) was associated with increased circulating log interleukin-6 concentration (increase per allele 9·45%, 95% CI 8·34–10·57) as well as reduced C-reactive protein (decrease per allele 8·35%, 95% CI 7·31–9·38) and fibrinogen concentrations (decrease per allele 0·85%, 95% CI 0·60–1·10). This pattern of effects was consistent with IL6R blockade from infusions of tocilizumab (4–8 mg/kg every 4 weeks) in patients with rheumatoid arthritis studied in randomised trials. In 25 458 coronary heart disease cases and 100 740 controls, the IL6R rs7529229 SNP was associated with a decreased odds of coronary heart disease events (per allele odds ratio 0·95, 95% CI 0·93–0·97, p=1·53×10−5).
Interpretation
On the basis of genetic evidence in human beings, IL6R signalling seems to have a causal role in development of coronary heart disease. IL6R blockade could provide a novel therapeutic approach to prevention of coronary heart disease that warrants testing in suitably powered randomised trials. Genetic studies in populations could be used more widely to help to validate and prioritise novel drug targets or to repurpose existing agents and targets for new therapeutic uses.
Funding
UK Medical Research Council; British Heart Foundation; Rosetrees Trust; US National Heart, Lung, and Blood Institute; Du Pont Pharma; Chest, Heart and Stroke Scotland; Wellcome Trust; Coronary Thrombosis Trust; Northwick Park Institute for Medical Research; UCLH/UCL Comprehensive Medical Research Centre; US National Institute on Aging; Academy of Finland; Netherlands Organisation for Health Research and Development; SANCO; Dutch Ministry of Public Health, Welfare and Sports; World Cancer Research Fund; Agentschap NL; European Commission; Swedish Heart-Lung Foundation; Swedish Research Council; Strategic Cardiovascular Programme of the Karolinska Institutet; Stockholm County Council; US National Institute of Neurological Disorders and Stroke; MedStar Health Research Institute; GlaxoSmithKline; Dutch Kidney Foundation; US National Institutes of Health; Netherlands Interuniversity Cardiology Institute of the Netherlands; Diabetes UK; European Union Seventh Framework Programme; National Institute for Healthy Ageing; Cancer Research UK; MacArthur Foundation.
Inflammation is implicated in atherogenesis,1 but a causal association with a specific inflammatory mediator has not been established. Interleukin 6, an inflammatory cytokine produced mainly by T cells, macrophages, and adipocytes, promotes inflammatory responses via the membrane-bound or circulating soluble interleukin-6 receptor (IL6R) on monocytes, hepatocytes, and endothelial cells2 (appendix p 26). Similarly to C-reactive protein and fibrinogen, whose synthesis is stimulated by IL6R signalling, high circulating concentrations of interleukin 6 were associated with increased risk of coronary heart disease events in prospective observational studies.3–5 Despite exclusion of C-reactive protein and fibrinogen as causal mediators, on the basis of mendelian randomisation studies6,7 IL6R signalling could be an important therapeutic target for prevention of coronary heart disease.
Tocilizumab, a monoclonal antibody that blocks both membrane-bound and circulating IL6R, has anti-inflammatory actions that extend beyond reductions in C-reactive protein and fibrinogen concentrations.8,9 Tocilizumab is licensed for treatment of rheumatoid arthritis10–12 and has been shown to reduce articular inflammation and promote disease remission.13,14 However, adequately powered, long-term trials of tocilizumab on risk of cardiovascular disease have not yet been undertaken.
Randomised trials in patients with rheumatoid arthritis revealed that tocilizumab increases total, HDL, and LDL cholesterol and triglycerides,15,16 yet whether these lipid changes are on-target or off-target effects of tocilizumab, or whether they reflect a non-specific alleviation of suppressed inflammation as reported with other anti-inflammatory rheumatoid arthritis treatments, is uncertain.17 Whether the potentially proatherogenic increases in LDL cholesterol are offset by potentially antiatherogenic effects of reduced inflammation, or by the increase in HDL cholesterol, is also uncertain. Recognising that patients with rheumatoid arthritis are at increased risk of cardiovascular disease by virtue of their autoimmune disease and related vascular pathological changes18 and that tocilizumab is intended as a long-term treatment, the US Food and Drug Administration has mandated randomised controlled trials examining the cardiovascular effects of tocilizumab in patients with rheumatoid arthritis12 (NCT01331837 and NCT00535782). However, these ongoing trials will not answer the question of whether IL6R blockade will modify risk of coronary heart disease in the general population.
A recently developed extension to the mendelian randomisation paradigm19,20—mendelian randomisation for drug target validation—uses variants in a gene encoding a drug target to profile the mechanism-based effects of pharmacological modification of that target and to distinguish on-target from off-target actions.21 By providing randomised evidence for the likely effectiveness of a new treatment in human beings without the potential risks of exposure to a novel drug or the cost of a randomised trial, this approach could aid prioritisation of targets for drug development. We applied mendelian randomisation to examine whether IL6R modulation is likely to reduce risk of coronary heart disease in the general population. We first evaluated the legitimacy of single nucleotide polymorphisms (SNPs) in the IL6R gene (Ch1q21.3) as indicators of the mechanism-based effect of pharmacological interference in IL6R signalling (appendix p 26). We then undertook a large-scale collaborative genetic association analysis of IL6R variants with coronary heart disease events and stroke and examined safety endpoints, including infections and common cancers.
Treatment trials and other studies of tocilizumab
Following PRISMA guidelines22 (appendix p 28), we searched Medline using PubMed for randomised trials, cohorts, or meta-analyses comparing tocilizumab (4 or 8 mg/kg) with placebo in human beings (appendix pp 1, 7). Details of extracted data and methods used to synthesise and combine estimates of trial results are reported in the appendix.
Genetic association studies
We included individual participant data for up to 133 449 participants of European ancestry from 40 studies (appendix pp 1–7, 11–12). Data from this de-novo analysis were pooled with previously published information about the association of IL6R variants with clinical events. We gathered phenotypic data across several studies (appendix pp 13–14) for analysis of associations between IL6R genotype and interleukin 6, C-reactive protein, fibrinogen, and major blood lipid fractions.
The primary event endpoint for the genetic analysis was all fatal and non-fatal coronary heart disease events (consisting of myocardial infarction and coronary revascularisation; appendix pp 15–16). Secondary efficacy endpoints were all-cause stroke, and all fatal and non-fatal cardiovascular disease (consisting of myocardial infarction, coronary revascularisation, and stroke). These disease outcomes are analogous to the efficacy outcomes in an orthodox randomised trial.
On the basis of published associations of interleukin-6 concentrations with disease outcomes other than coronary heart disease, safety endpoints reported in tocilizumab trials, and standard safety endpoints for cardiovascular intervention trials, we investigated the association of IL6R variants with all-site cancer, major cancer subtypes (breast and colorectal), respiratory infection, and liver enzyme concentrations. We obtained safety data from de-novo investigations of IL6R variants, from estimates of the association of any safety outcome with IL6R variants reported in the National Human Genome Research Institute genome-wide association study catalogue,23 and from other reported associations of published genome-wide association studies (appendix p 17). We also estimated the association between IL6R variants and other established risk factors for cardiovascular disease including blood pressure and type 2 diabetes.
SNP selection, genotyping, and quality control
Using the HumanCVD BeadChip,24 we genotyped 4489 individuals of European ancestry in the Whitehall II study. From the 42 SNPs located within 55 kb of IL6R present on the array, we selected a subset of SNPs for further analysis in other datasets on the basis of four factors: (1) the statistical strength of association with interleukin-6 concentration; (2) linkage disequilibrium (LD) between SNPs in populations of European ancestry using Human HapMap Phase 3 Build 36 data, to reduce redundancy; (3) previous disease and biomarker associations of SNPs in this region (appendix p 27); and (4) a minor allele frequency (MAF) threshold of greater than 0·3. Data were excluded if the allele call rate was less than 90% or the Hardy-Weinberg equilibrium (HWE) χ2 p value was less than 0·001 in any study.
Statistical analysis
Genotypes were coded as 0, 1, and 2, indicating the number of variant allele copies. The analysis was done with an additive model suggested by IL6R associations with circulating interleukin-6 concentrations in the index Whitehall II Study. Owing to skewed distributions, values of interleukin 6, C-reactive protein, fibrinogen, and triglycerides were analysed on the natural logarithmic scale.
Using individual participant-level data, we estimated the mean difference in interleukin 6, C-reactive protein, and fibrinogen between genotype groups for each SNP. Additionally, for these inflammatory markers, the major lipid fractions, and other biomarkers, we fitted univariate linear regression models within each study to investigate evidence of a linear association between the biomarker and possession of each additional copy of the minor allele. All analyses were done within each study according to a common analysis plan implemented with a standardised Stata (version 11.1) program, adapted for SPSS and PLINK in some studies. Where possible, we repeated analyses in prespecified subgroups (appendix pp 5–6).
To assess association of SNPs with disease endpoints, we estimated unadjusted odds ratios (OR) per minor allele within each study using logistic regression models. In studies for which relevant data were available (27 studies, 97 300 participants), we estimated the per-allele OR for coronary heart disease events, stratified where appropriate by prespecified characteristics (appendix pp 5–6) within each study, and by study design. Within-study estimates were combined with inverse-variance weighted fixed-effects meta-analysis. We used I2 to quantify between-study heterogeneity.25 In subgroup analyses, we tested for heterogeneity between strata using the meta-analysis χ2 test for heterogeneity.
Role of the funding source
The funding sources had no role in study design, in the collection, analysis, and interpretation of data, in the writing of the report, or in the decision to submit for publication. The corresponding author (DIS) and co-senior authors (ADH and JPC) had full access to all data in the study and had final responsibility for the decision to submit for publication.
We identified six short-term randomised trials (12–52 week duration) evaluating 4 mg/kg or 8 mg/kg tocilizumab in 2891 patients with rheumatoid arthritis (weighted mean age 52·3 years; 19% male; appendix p 28).8,9,26–29 C-reactive protein was the most widely reported inflammation marker and its weighted mean concentration at baseline was 28·2 (SD 1·9) mg/L. Tocilizumab treatment (4, 8, and 16 mg/kg in randomised or observational studies every 4 weeks) was associated with a dose-dependent reduction in C-reactive protein concentration (appendix p 29). The 8 kg/mg dose reduced fibrinogen, increased interleukin 6 and soluble IL6R, and increased LDL and HDL cholesterol (table).
Table
Table
Summary effects of tocilizumab (8 mg/kg) and the IL6R rs7529229 variant on inflammatory, lipid, hepatic, and haematological biomarkers
40 studies contributed genotype and phenotype data for the de-novo genetic analysis of IL6R SNPs from a total of 133 449 individuals with mean age at recruitment of 59 (range 26–75) years, of whom 49% were women. Additional characteristics of study participants are described in appendix pp 11–16, 30–33. 12 of the 42 SNPs in the region of the IL6R locus on the HumanCVD BeadChip met chip-wide significance (p<1×10−5) for their association with circulating interleukin 6 in the Whitehall II study (appendix pp 20–21). We selected three SNPs (rs7529229, rs4845371, rs12740969) based on MAF greater than 0·3, β coefficient greater than 0·9 log interleukin-6 concentration per allele, previously reported associations, and low-redundancy LD structure (appendix p 27). The rs7529229 variant was in strong LD (r2=0·92 in the Whitehall II study) with a non-synonymous variant (rs8192284, also annotated as rs2228145, which did not meet our initial selection criteria) previously reported to be associated with increased proteolytic cleavage of the soluble IL6R from its membrane-bound form30,31 (see appendix p 26 for mechanistic details) and became our lead SNP for the analysis. Where rs7529229 was not genotyped, a proxy SNP was used (defined on the basis of r2≥0·90 with rs7529229 in individuals of European ancestry; appendix pp 18–19). Information about rs4845371 and rs12740969 is reported in subsidiary analyses. 40 studies (133 449 participants) provided data for rs7529229, 18 studies (52 475 participants) for rs4845371, and 19 studies (59 126 participants) for rs12740969. All studies met the prespecified quality control threshold criteria for call rate, HWE, and MAF (appendix pp 18–19).
The IL6R rs7529229 SNP displayed additive associations with circulating concentrations of interleukin 6, C-reactive protein, and fibrinogen (figure 1, table). Circulating interleukin-6 concentration increased with each additional copy of the minor allele at rs7529229 (relative increase in geometric mean log interleukin-6 concentration per allele 9·45%, 95% CI 8·34–10·57; p=8·41×10−68), whereas the concentrations of C-reactive protein and fibrinogen decreased per minor allele (relative decrease in geometric mean log C-reactive protein 8·35%, 95% CI 7·31–9·38, and fibrinogen 0·85%, 0·60–1·10, per minor allele). The associations with interleukin 6 and C-reactive protein were consistent across study-specific subgroups (appendix pp 34–37) with no evidence of genotype-by-subgroup interaction (p>0·05 for all analyses). Concentration of soluble IL6R increased per minor allele (table). The functional rs8192284 variant showed associations with interleukin 6, C-reactive protein, and fibrinogen that were directionally concordant with those of rs7529229 in the Whitehall II study (appendix p 24). No significant association was noted between the rs7529229 SNP and concentration of total, LDL, and HDL cholesterol or triglycerides in analyses including up to 114 615 individuals (table).
Figure 1
Figure 1
Association of the IL6R rs7529229 variant with (A) interleukin 6, (B) C-reactive protein, and (C) fibrinogen concentration
The blood markers interleukin 6, soluble IL6R, C-reactive protein, fibrinogen, and total, LDL, and HDL cholesterol were available in both genetic studies and tocilizumab treatment trials allowing a direct comparison of IL6R genotype and IL6R blockade (table). The minor allele of rs7529229 and treatment with tocilizumab showed directionally concordant effects; each was associated with reduced C-reactive protein and fibrinogen and increased interleukin 6 and soluble IL6R (table, figure 2). Tocilizumab treatment increased circulating total, HDL, and LDL cholesterol, and triglycerides, but the IL6R rs7529229 SNP, by contrast, showed no significant association with any of these lipid fractions (table, figure 2). In randomised trials, tocilizumab increased concentrations of albumin and haemoglobin and decreased erythrocyte sedimentation rate (ESR), platelet count, and serum amyloid A (table). The effect of rs7529229 was directionally concordant with that of tocilizumab on albumin, haemoglobin, and platelet count (table, figure 2). Data for ESR were unavailable in the genetic studies, but plasma viscosity (reflected by ESR) was lower in carriers of the rs7529229 minor allele (mean difference per allele −2·16×10−3 mPa.s, 95% CI −3·86×10−4 to −3·94×10−3, p=0·02; five studies, 15 589 individuals). Absence of data for serum amyloid A in the genetic analysis precluded comparison with tocilizumab treatment. In comparison of tocilizumab treatment with the rs7529229 variant, the direction of effect was concordant for nine of the ten biomarkers (table, figure 2), and greater than expected under the null hypothesis of no concordance (binomial test, p=0·01).
Figure 2
Figure 2
Associations of the minor allele of the IL6R SNP rs7529229 and tocilizumab (8 mg/kg) versus placebo with commonly reported biomarkers
We also examined the association of IL6R variants with coronary heart disease. In a meta-analysis of 34 studies (25 458 coronary heart disease cases, 100 740 controls) the OR for the primary outcome (all fatal and non-fatal coronary heart disease events; appendix pp 15–16) per minor allele at rs7529229 was 0·95 (95% CI 0·93–0·97, p=1·53×10−5). There was low heterogeneity between studies (I2=10%, 95% CI 0–41) and the effect estimates were consistent in prospective and case-control studies, including previously published data32 (figure 3). In a subset of 97 300 individuals (27 studies) for whom relevant data were available, the association of rs7529229 with the primary outcome (14 360 cases and 82 940 controls) was consistent in stratified analyses (appendix pp 1–7) with no evidence for effect modification by any of these subgroups (appendix pp 40–41).
Figure 3
Figure 3
Association of IL6R rs7529229 with risk of fatal and non-fatal coronary heart disease
Associations of rs7529229 with risk of fatal or non-fatal stroke (OR 0·98, 95% CI 0·95–1·02, p=0·30) in 6904 cases and 90 512 controls (27 studies) and with fatal or non-fatal cardiovascular disease events combined (OR 0·98, 95% CI 0·95–1·00, p=0·05) in 17 595 cases and 76 321 controls (26 studies) were suggestive but not compelling (figure 4). Up to three of six randomised trials of tocilizumab reported the incidence of cardiac or vascular events, or both, with median follow-up of 24 weeks (appendix pp 22–23). However, imprecise endpoint definition and the small number of events prevented comparison with genetic studies.
Figure 4
Figure 4
Association of IL6R rs7529229 with secondary and safety endpoints
In an analysis of safety endpoints in tocilizumab trials, data suggested an increased risk of infection (OR 1·30, 95% CI 1·07–1·58) and increased concentrations of hepatic enzymes alanine transaminase and aspartate transaminase (table) with tocilizumab treatment compared with placebo (appendix pp 22–23). By contrast with evidence for tocilizumab, genetic analyses (although in a relatively small subset) did not reveal any association with concentrations of aspartate transaminase (table) or in log γ-glutamyl transferase (relative difference in geometric mean per allele −0·64%, 95% CI −1·95 to 0·69, p=0·34; seven studies, 15 641 individuals). Our genetic experiment did not include infection as an outcome and published evidence for the IL6R rs7529229 variant was scarce.33 Genome-wide association studies of tuberculosis34 and meningococcal disease35 have not reported associations of variants in IL6R with risk of those outcomes.
Neither the evidence from tocilizumab trials nor the genetic studies to date have suggested an association of IL6R blockade with increased risk of cancer. The pooled OR for development of any cancer was 0·42 (95% CI 0·06–2·88; four cases and 1196 controls) for tocilizumab treatment in randomised trials, and was 0·98 (95% CI 0·93–1·03; 5376 cases and 57 123 controls) for the IL6R rs7529229 variant. In published genome-wide association studies and new look-ups, the IL6R rs7529229 variant showed no association with breast cancer (OR 1·01, 95% CI 0·94–1·10) or colorectal cancer (OR 1·03, 95% CI 0·96–1·12; figure 4; appendix p 17).
The IL6R rs7529229 variant was associated with lowered systolic blood pressure (per-allele β coefficient −0·21 mm Hg, 95% CI −0·37 to −0·05, p=0·01) and diastolic blood pressure (per-allele β coefficient −0·11 mm Hg, 95% CI −0·20 to −0·02, p=0·02) in 33 studies (112 979 individuals). There was suggestive evidence that the rs7529229 variant was associated with reduced risk of type 2 diabetes (OR 0·97, 95% CI 0·94–1·00, p=0·06) in 12 859 cases and 86 807 controls (figure 4), although this exploratory finding needs further investigation.
Our study provides strong evidence in human beings for a causal role of a specific inflammatory mechanism (ie, IL6R signalling) in coronary heart disease (panel). A common polymorphism in IL6R marking a non-synonymous variant (p.Asp358Ala) with known functional consequences30,36 was associated with differences in circulating concentrations of soluble IL6R, interleukin 6, C-reactive protein, and fibrinogen that were directionally concordant with those reported in trials of IL6R blockade with tocilizumab. Meta-analysis of 34 studies including 25 458 coronary heart disease cases and 100 740 controls suggested the same IL6R rs7529229 variant was associated with reduced odds of coronary heart disease events. This finding suggests that targeting of IL6R could provide a novel therapeutic approach to prevention of coronary heart disease.
Panel. Research in context
Systematic review
We searched Medline via PubMed for “tocilizumab AND (“coronary heart disease” OR “myocardial infarction”), for “interleukin-6 receptor AND (“coronary heart disease” OR “myocardial infarction”), and for “IL6R” AND (“coronary heart disease” OR “myocardial infarction”) up to Jan 8, 2012. Local and systemic inflammation is implicated in atherosclerosis but, as yet, there are no licensed approaches for prevention of cardiovascular disease that target inflammatory mechanisms. Mendelian randomisation studies suggest that two intensively studied biomarkers of inflammation associated with cardiovascular disease, C-reactive protein and fibrinogen, are unlikely to be causally related to atherosclerosis. High circulating interleukin-6 concentration is associated with increased risk of coronary heart disease in observational studies and preliminary evidence suggests a variant in the gene for its receptor (IL6R) might be associated with reduced risk of coronary heart disease.31 Tocilizumab, a monoclonal antibody targeting the interleukin-6 receptor (IL6R), is licensed for treatment of rheumatoid arthritis, but whether IL6R blockade reduces risk of coronary heart disease is unknown.
Interpretation
Applying the mendelian randomisation principle, we found that a variant in the IL6R gene had effects on biomarkers of inflammation and related pathways (including interleukin 6, C-reactive protein, fibrinogen, haemoglobin, albumin, and others) that are directionally concordant with those of tocilizumab treatment reported by randomised trials. In keeping with that expected for common alleles, the size of the genetic effect was small compared with that of the drug. Nevertheless, the same genetic variant was associated with a lowered risk of coronary heart disease in a sample of 25 458 coronary heart disease cases and 100 740 controls (odds ratio per minor allele 0·95, 95% CI 0·93–0·97, p=1·53×10−5). This mendelian randomisation analysis suggests that IL6R signalling is involved in coronary heart disease and that the IL6R could be a valuable target for the prevention or treatment of coronary heart disease.
Although the IL6R rs7529229 variant was associated with reduced circulating C-reactive protein and fibrinogen concentrations, this study should not be interpreted as a mendelian randomisation analysis investigating causality of C-reactive protein or fibrinogen in coronary heart disease. Previous large mendelian randomisation studies using SNPs in the genes encoding C-reactive protein and fibrinogen6,7,32,37–39 suggested that neither is a causal mediator of coronary heart disease. Therefore, other consequences of reduced interleukin-6 signalling could be responsible for the association with decreased risk of coronary heart disease that we identified. Specific mendelian randomisation analyses for interleukin 6 have not yet been done, largely because SNPs in the gene encoding interleukin 6 (IL6, Ch7p15.3) that reliably associate with circulating interleukin-6 concentration have not been identified.40,41 By contrast, the present study provides an example of a different type of mendelian randomisation analysis: one used to validate a drug target.
Although the association of the IL6R variant with raised concentrations of interleukin 6 and reduced coronary risk noted in this study might seem paradoxical, the pattern is consistent with pharmacological blockade of IL6R with tocilizumab. The finding can be explained by reduced IL6R signalling in carriers of the variant allele, which leads to attenuation of downstream consequences of interleukin 6 (of which a reduction in C-reactive protein and fibrinogen concentrations are but two), and an accumulation or release of feedback inhibition of the upstream ligand (interleukin 6) and its soluble receptor.42
Randomised trials of tocilizumab in patients with rheumatoid arthritis reported increases in blood lipid fractions.15,16 By contrast, carriage of the IL6R rs7529229 minor allele was not associated with changes in any major blood lipid fraction. Evidence suggesting individuals carrying the rs7529229 variant were more likely to use lipid-lowering drugs than were non-carriers was weak (OR per minor allele 1·02, 95% CI 0·99–1·06, p=0·24); the absence of association with blood lipids was consistent between users and non-users of these drugs (heterogeneity χ2 p=0·15).
There are several potential explanations for the discordance in effects on blood lipids between tocilizumab treatment and IL6R genotype. First, randomised trials of tocilizumab were done in patients who had higher levels of background inflammation (baseline mean C-reactive protein 28·4 mg/L) than did participants in the genetic studies sampled from general populations (geometric mean C-reactive protein 1·8 mg/L). The effects of tocilizumab on lipids might be mechanism-based but only manifest on a background of substantial systemic inflammation (which, in many conditions, is associated with reduced circulating lipid concentration43) and therefore not detectable at low levels of inflammation seen in healthy individuals. Second, there might be differences between the lifelong effect of genetic variants in IL6R and the short-term, later-life exposure to tocilizumab. Third, the effect of pharmacological blockade and genetic variation on classic signalling through the membrane-bound IL6R versus trans-signalling via the soluble receptor might also differ. Tocilizumab binds both the soluble and membrane-bound receptors inhibiting classical and trans pathways,44,45 but the functional polymorphism tagged by rs7529229 (rs8192284) results in increased soluble IL6R concentration through increased proteolytic cleavage of the membrane-bound receptor46–48 and possibly a reduction in the number of functioning membrane-bound receptors.30,36 Membrane-bound IL6R mediates interleukin-6 signalling in hepatocytes and some leucocyte populations, whereas the soluble receptor acts on a diverse range of cell types including megakaryocytes and endothelial cells;2,48 both mechanisms rely on the ubiquitously expressed signal transducer, gp130. IL6R-mediated effects on blood lipids might also need a suprathreshold change in IL6R signalling, which might be achieved by pharmacological inhibition but not by natural genetically-mediated changes in the concentration or function of the IL6R. Finally, the possibility remains that the lipid-related effects of tocilizumab are an off-target action.
The association of the IL6R rs7529229 variant with lowered risk of coronary heart disease provides robust evidence of a role for inflammation in pathogenesis of coronary heart disease that is consistent with previously reported findings based on the IL6R rs4537545 SNP (in LD with rs7529229, r2=1·00),32 although our analysis included more than twice the number of cases. The effect estimates obtained from our de-novo analysis of largely prospective studies and the previous analysis based mainly on case-control studies were highly consistent with no evidence of heterogeneity in the effect estimates obtained from prospective, case-control, or cross-sectional studies. Furthermore, we did not identify heterogeneity in the genetic effects in individuals stratified by prevailing concentrations of non-HDL cholesterol or by lipid-lowering drug use, generating the hypothesis that the effects of IL6R blockade could be additive to those of established lipid-based interventions.
The randomised trials of tocilizumab designed to examine drug efficacy in rheumatoid arthritis were fairly small and of short duration. Cardiac and vascular safety endpoints were reported as part of the safety assessment, but only one trial28 reported myocardial infarctions. Absence of detail on the definitions of safety outcomes in the remaining randomised trials made assessment of the effect of tocilizumab on risk of coronary events too imprecise to be valuable. Infections were the most commonly reported adverse events in tocilizumab trials.15 We lacked data for infectious events in the genetic studies; however, published evidence from candidate gene studies have not suggested an IL6R association with risk of respiratory infection.33 Risk of incident infection would be an important safety consideration in any trial of IL6R inhibition for prevention of coronary heart disease.
In addition to increases in blood lipids, there were infrequent reports of raised hepatic enzymes in trials of tocilizumab, although the magnitude of these changes did not increase with prolonged exposure.15 We noted no association of the lead IL6R SNP with aspartate transaminase. Analysis of data from genetic studies in this collaboration and those reported in the literature (including genome-wide association studies and other large-scale studies) did not reveal increased risk of common cancers. Our safety profiling of IL6R blockade in the genetic experiment included the effect of IL6R variants on established cardiovascular risk factors such as type 2 diabetes and blood pressure. The findings were suggestive of associations of the rs7529229 SNP with reduced risk of type 2 diabetes and lowered systolic and diastolic pressures, although these need further investigation.
This large-scale analysis provides reliable genetic evidence for the role of a specific inflammatory pathway in the development of coronary heart disease in humans. Comparison of the genetic findings with data from randomised trials of tocilizumab supports further evaluation of IL6R inhibition as a therapeutic strategy for prevention of coronary heart disease. Other monoclonal antibodies against IL6R are now in advanced development and small molecules with activity at IL6R have also been reported.49 An ongoing trial of the anti-interleukin-1β monoclonal antibody canakinumab for reduction of coronary heart disease risk (NCT01327846) underlines the potential of inflammatory pathways as targets for cardiovascular prevention and supports a need for a trial of IL6R inhibition for prevention of coronary heart disease events.
Correspondence to: Dr Daniel I Swerdlow, Genetic Epidemiology Group, Research Department of Epidemiology and Public Health, UCL Institute of Epidemiology and Health Care, University College London WC1E 6BT, UK
Acknowledgments
DIS is supported by a UK Medical Research Council (MRC) doctoral training award, and acknowledges the support of the UCL MBPhD programme. MVH is supported by a MRC population health scientist fellowship (G0802432). JE is supported by the National Institutes of Health (NIH), the MRC, and the British Heart Foundation (BHF; grant number RG/10/12/28456). R Sofat is supported by a BHF (Schillingford) clinical training fellowship (FS/07/011). CC and APeasey are supported by the Wellcome Trust and the US National Institute on Aging. HAI is supported by the Coronary Thrombosis Trust, Northwick Park Institute for Medical Research, and the UCLH/UCL Comprehensive Biomedical Research Centre. SJB is supported by the US National Heart, Lung, and Blood Institute (NHLBI; HHSN268200900009C) and National Human Genome Research Institute (U01HG005152). R Houlston is supported by Cancer Research UK (C1298/A8362, Bobby Moore Fund for Cancer Research UK). AW, DK, and R Hardy are supported by the MRC. JWS is supported by a Diabetes UK clinical training fellowship and was supported at the time of creation of UDACS by Diabetes UK. J A Hubacek is supported by IKEM (Czech Republic) project number 00023001. MGM is supported by the MRC. RW is supported by the National Institute for Healthy Ageing (grant 05060810). J Price is supported by the BHF, Chest, Heart and Stroke Scotland, the Wellcome Trust, and the MRC. J Peto is supported by Cancer Research UK (C150/A5660). CLW is supported by NIH/NHLBI (subcontract no. 5215810-55000000041). SEH and PJT are supported by the BHF (RG 08/008 and PG/07/133/24260). M Kivimaki is supported by the National Institute on Aging, MRC, BHF, NHLBI, and the Academy of Finland. FWA is supported by a clinical fellowship from the Netherlands Organisation for Health Research and Development (ZonMw grant 90700342). ADH is supported by a BHF senior fellowship (FS 05/125). This work is supported by the British Heart Foundation (grant number RG/10/12/28456). CARe is supported by contract no. HHSN268200625226C from the NIH/NHLBI, and subcontract no. 5215810-55000000041 to CLW. A full listing of the grants and contracts that have supported CARe is provided at http://public.nhlbi.nih.gov/GeneticsGenomics/home/care.aspx. MESA and the MESA SHARe project are conducted and supported by the NHLBI in collaboration with MESA investigators. Support is provided by grants and contracts N01 HC-95159, N01-HC-95160, N01-HC-95161, N01-HC-95162, N01-HC-95163, N01-HC-95164, N01-HC-95165, N01-HC-95166, N01-HC-95167, N01-HC-95168, N01-HC-95169, and RR-024156. The HAPIEE studies are supported by the Wellcome Trust (064947/Z/01/Z and 081081/Z/06/Z), the National Institute on Aging (1R01 AG23522-01), and the MacArthur Foundation. The ISGS-SWISS studies were supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Department of Health and Human Services; project number Z01 AG000954-06; ISGS (R01 NS42733) and SWISS (R01 NS39987) had been funded by the US National Institute of Neurological Disorders and Stroke. The inclusion of Baltimore Longitudinal Study of Aging samples as controls was supported by the Intramural Research Program of the National Institute on Aging, NIH, Department of Health and Human Services; project number Z01 AG000015-50. The EPIC-Netherlands study was funded by the “Europe against Cancer” Programme of the European Commission (SANCO); the Dutch Ministry of Public Health, Welfare and Sports (formerly Ministry of Welfare, Public Health and Culture); the Dutch Cancer Society; ZonMW the Netherlands Organisation for Health Research and Development; and World Cancer Research Fund. The IMPROVE study was supported by the European Commission (QLG1-CT-2002-00896), the Knut and Alice Wallenberg Foundation, the Swedish Heart-Lung Foundation, the Swedish Research Council (projects 8691 and 0593), the Torsten and Ragnar Söderberg Foundation, the Strategic Cardiovascular Programme of the Karolinska Institutet and the Stockholm County Council and the Stockholm County Council (project 562183). Genetics research in the PREVEND study is supported by the Dutch Kidney Foundation (Grant E033), the European Union project grant GENECURE (FP-6 LSHM CT 2006 037697), the NIH (grant LM010098), The Netherlands Organisation for Health Research and Development (NWO VENI grant 916.761.70), and the Dutch Inter University Cardiology Institute Netherlands (ICIN). The MedStar study was supported by the MedStar Health Research Institute and GlaxoSmithKline. The PROSPER study was supported by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° HEALTH-F2-2009-223004 PHASE. Measurement of interleukin 6 in PROSPER was funded by Chest, Heart and Stroke Scotland. The Women's Health Initiative was supported by the NHLBI (RFP-NHLBI-WH-11-10 - Women's Health Initiative Extension 2010–2015), US Department of Health and Human Services, clinical trial registration NCT00000611. The UCP study was funded by Veni grant Organization for Scientific Research (NWO), Grant no. 2001.064 Netherlands Heart Foundation (NHS), and TI Pharma Grant T6-101 Mondriaan. The Edinburgh Artery Study and AAA Trial were supported by the BHF. Aspirin and placebo for the AAA trial were supplied by Bayler PLC, who were not involved in the running of the trial or the analysis and writing up of the data. The Fenland Study is funded by the Wellcome Trust and the MRC. The National Survey of Health and Development is funded by the MRC. The MRC NSHD team are very grateful to the members of this birth cohort for their continuing interest and participation in the study. UDACS was funded by Diabetes UK. DNA extraction in the Caerphilly Prospective Study was funded by the MRC. The Whitehall II study has been supported by grants from the MRC; Economic and Social Research Council; BHF; Health and Safety Executive; Department of Health; NHLBI (HL36310), NIH; National Institute on Aging (AG13196), NIH; Agency for Health Care Policy Research (HS06516); and the John D and Catherine T MacArthur Foundation Research Networks on Successful Midlife Development and Socio-economic Status and Health. The British Regional Heart Study is a BHF research group and is supported by the BHF (RG/04/003). The views expressed in this publication are those of the authors and not necessarily those of the funding bodies. Samples from the English Longitudinal Study of Ageing DNA Repository (EDNAR), received support under a grant (AG1764406S1) awarded by the National Institute on Aging. ELSA was developed by a team of researchers based at the National Centre for Social Research, University College London and the Institute of Fiscal Studies. The data were collected by the National Centre for Social Research. The developers and funders of ELSA and the archive do not bear any responsibility for the analyses or interpretations presented here. The Northwick Park Heart Study II was supported by the MRC, the NIH (grant NHLBI 33014), and Du Pont Pharma (Wilmington, USA). The British Women's Health and Heart Study is supported by the UK Department of Health Policy Research Programme and the BHF. The CARe Consortium wishes to acknowledge the support of the NHLBI and the contributions of the research institutions, study investigators, field staff, and study participants in creating this resource for biomedical research (NHLBI contract number HHSN268200960009C). The following nine parent studies have contributed parent study data, ancillary study data, and DNA samples through the Massachusetts Institute of Technology - Broad Institute (N01-HC-65226) to create this genotype/phenotype database for wide dissemination to the biomedical research community: the Atherosclerosis Risk in Communities (ARIC) study, the Cardiovascular Health Study (CHS), the Cleveland Family Study (CFS), the Cooperative Study of Sickle Cell Disease (CSSCD), the Coronary Artery Risk Development in Young Adults (CARDIA) study, the Framingham Heart Study (FHS), the Jackson Heart Study (JHS), the Multi-Ethnic Study of Atherosclerosis (MESA), and the Sleep Heart Health Study (SHHS). The MESA investigators thank the other investigators, the staff, and the participants of the MESA study for their valuable contributions. We thank the Fenland Study investigators, study coordination team, and the epidemiology field, data, and technical teams as well as the study participants and general practice staff for help with recruitment. Biochemical assays were performed by the National Institute for Health Research, Cambridge Biomedical Research Centre, Core Biochemistry Assay Laboratory, and the Cambridge University Hospitals NHS Foundation Trust, Department of Clinical Biochemistry. The Ely Study was funded by the MRC, Diabetes UK, and Eastern Region NHS R&D. We are grateful to all those who participated and to the staff of the St Mary's Street surgery, Ely, and all those who worked on the study. The EPIC Norfolk Study is supported by programme grants from the MRC and Cancer Research UK and with additional support from the European Union, Stroke Association, BHF, Research into Ageing, Department of Health, the Wellcome Trust, and the Food Standards Agency. We acknowledge the contribution of the staff and participants of the EPIC-Norfolk Study. The portion of the ISGS-SWISS studies reported here used the high-performance computational capabilities of the Biowulf Linux cluster at the NIH (Bethesda, MD; http://biowulf.nih.gov). The EPIC-Netherlands investigators thank GBA, Statistics Netherlands, Dutch Cancer Registry, and the institute PHARMO for providing data for vital status, cause of death, and occurrence of cancer and other chronic diseases. Finally, we thank Jan van der Laan, Anneke Blokstra, Robert Jan de Klein, Ido Toxopeus, and Bernard Slotboom for their contribution to setting up this cohort. The IMPROVE investigators express their deep and sincere appreciation to all members of the IMPROVE group for their time and their extraordinary commitment. The Caerphilly Prospective study was undertaken by the former MRC Epidemiology Unit (South Wales) and was funded by the UK MRC.
Contributors
ADH, JPC, and DIS were responsible for the original study idea. FWA, CC, JAC, F Drenos, F Dudbridge, JE, TF, YG, MVH, KBK, ML, YRL, JL, SPM, MAN, RP, TS, DIS, and PvdH were responsible for data management and analysis. PH, KWL, J Palmen, TS, and DIS undertook additional genotyping. AdB, SB, GJdB, BAC, FWA, M Bacaviciene, SJLB, DB, EJ Benjamin, YBS, SJB, M Bobak, JMAB, EJ Brunner, MSB, JJC, PAD, GDS, UdF, JACD, JMD, MD, CBE, SEE, IF, NGF, FGRF, LF, TF, JG, YvdG, RTG, HH, AH, J Hardy, R Hardy, R Houlston, J A Hubacek, SEH, PdJ, JWJ, BJK, OK, M Kivimaki, RK, DK, M Kumari, LAL, DAL, GL, JL, SM, JEM, MGM, IML, JFM, RWM, AHMvdZ, GN, NCOM, AP, APeasey, J Peto, HP, J Price, SR, APR, JGR, NS, R Schnabel, BS, AS, JWS, MS, PJT, AT, NJT, I Tomlinson, TT, RTM, ET, I Tzoulaki, PvdH, YTvdS, WMM Verschuren, M Voevoda, NJW, CLW, RW, PHW, JCW, JGW, and AW provided tabular or individual participant data. FWA, EJ Benjamin, YBS, M Bobak, EJ Brunner, JPC, GDS, F Dudbridge, HH, AH, ADH, MVH, SEH, HAI, BJK, M Kivimaki, KBK, CL, DAL, SPM, RP, HP, J Price, SR, APR, NS, TS, R Sofat, DIS, PJT, NJT, WMM Verschuren, JCW, and JGW interpreted the findings and wrote the report.
The Interleukin-6 Receptor Mendelian Randomisation Analysis (IL6R MR) Consortium
Daniel I Swerdlow, Michael V Holmes, Karoline B Kuchenbaecker, Jorgen E L Engmann, Tina Shah, Reecha Sofat, Yiran Guo, Christina Chung, Anne Peasey, Roman Pfister, Simon P Mooijaart, Helen A Ireland, Maarten Leusink, Claudia Langenberg, KaWah Li, Jutta Palmen, Philip Howard, Jackie A Cooper, Fotios Drenos, John Hardy, Michael A Nalls, Yun Rose Li, Gordon Lowe, Marlene Stewart, Suzette J Bielinski, Julian Peto, Nicholas J Timpson, John Gallacher, Malcolm Dunlop, Richard Houlston, Ian Tomlinson, Ioanna Tzoulaki, Jian'an Luan, Jolanda M A Boer, Nita G Forouhi, N Charlotte Onland-Moret, Yvonne T van der Schouw, Renate B Schnabel, Jaroslav A Hubacek, Ruzena Kubinova, Migle Baceviciene, Abdonas Tamosiunas, Andrzej Pajak, Roman Topor-Madry, Sofia Malyutina, Damiano Baldassarre, Bengt Sennblad, Elena Tremoli, Ulf de Faire, Luigi Ferrucci, Stefania Bandenelli, Toshiko Tanaka, James F Meschia, Andrew Singleton, Gerjan Navis, Irene Mateo Leach, Stephan J L Bakker, Ron T Gansevoort, Ian Ford, Stephen E Epstein, Mary Susan Burnett, Joe M Devaney, J Wouter Jukema, Rudi G J Westendorp, Gert Jan de Borst, Yolanda van der Graaf, Pim A de Jong, Anke-Hilse Maitland-van der Zee, Olaf H Klungel, Anthonius de Boer, Pieter A Doevendans, Jeffrey W Stephens, Charles B Eaton, Jennifer G Robinson, JoAnn E Manson, F Gerry R Fowkes, Timothy M Frayling, Jackie F Price, Peter H Whincup, Richard W Morris, Debbie A Lawlor, George Davey Smith, Yoav Ben-Shlomo, Susan Redline, Leslie A Lange, Meena Kumari, Nick J Wareham, W M Monique Verschuren, Emelia J Benjamin, John C Whittaker, Anders Hamsten, Frank Dudbridge, J A Chris Delaney, Andrew Wong, Diana Kuh, Rebecca Hardy, Berta Almoguera Castillo, John J Connolly, Pim van der Harst, Eric J Brunner, Michael G Marmot, Christina L Wassel, Steve E Humphries, Philippa J Talmud, Mika Kivimaki, Folkert W Asselbergs, Mikhail Voevoda, Martin Bobak, Hynek Pikhart, James G Wilson, Hakon Hakonarson, Alex P Reiner, Brendan J Keating, Naveed Sattar, Aroon D Hingorani*, Juan Pablo Casas*. *Joint senior authors. See appendix pp 45–49 for affiliations.
Conflicts of interest
SEE is named as an inventor on a licensing agreement being processed for the following patent: method of using physiological markers to estimate cardiovascular risk (relates to using three biomarkers to predict cardiovascular risk). SEH has received travel/accommodation/meeting expenses from the European Atherosclerosis Society. JEM is listed as a coinventor on a patent held by Brigham and Women's Hospital that relates to inflammatory biomarkers in diabetes prediction. MAN is employed by the US National Institutes of Health. NS is on a cardiovascular disease endpoints steering committee for a study of tocilizumab versus etanercept; Glasgow University is reimbursed for his time expended in relation to this study. RW is director of the Leyden Academy on Vitality and Ageing, is a principal investigator of EU FP6-funded consortium LifeSpan, and is a member of the EU FP7 funded consortium MYOAGE, SWITCHBOX, identifying determinants of longevity. JCW has been employed (90%) by GlaxoSmithKline since 2009, owns stock in GlaxoSmithKline, and has received expenses for attendance as an invited speaker at various academic meetings from meeting organisers. All other authors declare that they have no conflicts of interest.
Supplementary Material
Supplementary appendix
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