Lack of an Association Between Interleukin-6 −174G/C Polymorphism and Circulating Interleukin-6 Levels in Normal Population: A Meta-Analysis

Abstract

Interleukin-6 (IL-6) signaling may play a causal role in the development of coronary heart disease. However, the relationship between IL-6 genotypes and plasma levels of IL-6 appears to be complex. To help clarify the inconsistent findings, we conducted a meta-analysis of the published genetic association studies of the −174 G/C polymorphisms in the IL-6 gene and the circulating IL-6 levels in a normal population. In this meta-analysis, no significant association of IL-6 −174G/C polymorphism and circulating IL-6 levels in a normal population was observed. However, when compared among GG, GC, and CC genotypes, heterogeneity existed among the studies. Sensitivity analysis revealed that, the independent study by Shen et al. influenced the heterogeneity in the homozygous and heterozygous comparison. Although Shen et al.'s study was excluded, no significant association was observed between IL-6 −174G/C polymorphism and circulating IL-6 levels in a normal population [homozygous comparison (GG vs. CC): the pooled standard mean difference (SMD) was −0.01, 95% confidence interval (CI): −0.1–0.08; heterozygous comparison (GC vs. GG or CC): the pooled SMD (GG vs. GC) was −0.05, 95%CI: −0.11–0.01, and the pooled SMD (CC vs. GC) was 0.03, 95%CI: −0.03–0.1]. Under the dominant model, the pooled SMD was −0.05, 95%CI: −0.11–0.01). The meta-analysis provides evidence that the −174G/C polymorphism in the IL-6 gene is not significantly associated with circulating IL-6 levels in a normal population.

Introduction

I nflammation is a key component of atherosclerotic disease, and genes coding for inflammatory cytokines are therefore candidates for predisposition to risk of coronary heart disease. Interleukin-6 (IL-6) is a multifunctional cytokine produced by many different cell types, including immune cells, endothelial cells, fibroblasts, myocytes, and adipose tissue, mediating inflammatory as well as stress-induced responses. Similarly to C-reactive protein and fibrinogen, whose synthesis is stimulated by IL-6R signaling, high circulating concentrations of IL-6 were associated with increased risk of coronary heart disease events in prospective observational studies. (Ridker et al., 2000; Danesh et al., 2008) Recently, using Mendelian randomization analysis, Hingorani and Casas (2012) proved that IL-6R signaling has a causal role in the development of coronary heart disease.

The circulating concentrations of IL-6 are likely to be influenced by several environmental and genetic factors, including a polymorphic site (−174G/C) in the IL-6 gene promoter (Fishman et al., 1998). Although the presented evidence favors the proinflammatory character of the −174GG genotype, some other data may raise doubts about the universality of this phenomenon. For example, in the studies of Humphries et al. (2001), it was the −174C allele carriers that had the increased risk of higher systolic blood pressure and coronary heart disease. In addition, it was the −174C allele carriers that produced in vivo the highest amount of IL-6 and had less favorable outcomes of coronary revascularization surgery than the GG homozygotes (Bittar et al., 2005).

We hypothesized that variation in the promoter region of the IL-6 gene, if of functional consequence, would influence IL-6 gene transcription and thus affect plasma or tissue levels of IL-6, which would in turn influence plasma levels of key coronary heart disease risk factors and thus coronary heart disease risk itself.

However, the relationship between IL-6 genotypes and plasma levels of IL-6 appears to be complex. Several studies have suggested that a common variant (−174G/C) located in the promoter of the IL-6 gene could play a significant role in regulating the level of circulating and locally produced IL-6 (Liu et al., 2006). In the first report of 102 healthy subjects, the −174CC group had the lowest levels (Fishman et al., 1998); the Fishman's study showed that the −174 G/C polymorphisms in the IL-6 gene could increase the plasma concentrations of IL-6, but in subsequent large studies, the −174C allele has been associated with higher levels of IL-6 (Brull et al., 2001; Jones et al., 2001). Several large studies (Bennet et al., 2003; Qi et al., 2006; van Oijen et al., 2006; Danesh et al., 2008), with the largest (Herbert et al., 2006), including 1,526 Caucasians, did not find evidence for association. However, other studies with the largest Cardiovascular Health Study (CHS) with 4,714 elderly Caucasians (72 years median age) and a high prevalence of T2DM (28%) showed borderline significantly higher IL-6 levels for CC-genotype subjects compared to GG (p=0.04) (Walston et al., 2007).

These studies showed conflicting results regarding the effect of −174G/C IL-6 polymorphism on plasma IL-6 levels and circulating IL-6 levels. To help clarify the inconsistent findings, we conducted a meta-analysis of the published genetic association studies of the −174 G/C polymorphisms in the IL-6 gene and the circulating IL-6 levels in the normal population.

Method

Publication search

This review was performed according to the Quality of Reporting of Meta-Analysis (QUORUM) guidelines (Moher et al., 1999). Three online electronic databases (PubMed, Embase, Web of Science) were searched using the terms: “IL-6” or “IL 6” or “IL6” or “interleukin6” or “interleukin 6” or “interleukin-6” paired with “174C,” “174G,” “rs1800795,” respectively (last search was updated on 30 April, 2013). In addition, the references of the candidate articles were examined in an effort to identify eligible studies that may not have been identified by the initial search. Abstracts or unpublished reports were not included and only the studies published in the English language were considered in the present meta-analysis.

Inclusion and exclusion criteria

For inclusion in the present meta-analysis, the identified articles had to provided all the following criteria: (1) studies on the IL-6 −174G/C polymorphism of a normal population (people without disease, especially disease related to inflammation and obviously influenced the IL-6 levels, such as coronary heart disease, diabetes mellitus, hypertension, nephropathy, obesity, metabolic syndrome, cancer, pregnancy, Alzheimer's disease, and stroke); (2) published case–control studies; (3) cohort studies; (4) studies published as full-length articles in English; (5) studies with data for −174 genotype in the IL-6 gene promoter; and (6) studies with sufficient data for estimating the mean differences of circulating IL-6 or with 95% confidence interval (CI). The exclusion criteria included (1) no control population; (2) no usable data reported; and (3) duplicates.

Data extraction

Two investigators independently extracted data (Huang and Wang) and discrepancies were resolved by group discussion. For each study, the following information was extracted: name of the first author, year of publication, ethnic origin of the studied population, number in −174 genotype groups, male percentage, mean ages in −174 genotype groups, genotyping method, and the IL-6 assay method. The studies' baseline and exclusion criteria were inspected to be sure they were adapted to our definition of a normal population. If multiple published reports from the same study population were available, we included only the one with the largest sample size and complete data.

Statistical analysis

The mean differences were pooled through a random effects model, using the inverse variance approach when heterogeneity existed among these studies. Otherwise, a fixed effects model was adopted. Heterogeneity between studies was assessed by the Q-test and I ² statistic, where p<0.10 and I ²>30% indicated evidence of heterogeneity. For −174G/C polymorphism, the association between the allele and the circulating IL-6 levels, as well as homozygous comparison (GG vs. CC), heterozygous comparison (GC vs. GG or CC), dominant genetic model (CC+GC vs. GG), and recessive genetic model (CC vs. GG+GC) were explored. Forest plots were used to demonstrate the differences between studies and to provide an estimate of the overall result. Sensitivity analysis (one-study removed test) was performed to assess the impact of each study on the combined effect of the present meta-analysis. The single given study is excluded, when it is omitted in the sensitivity analysis and the estimate value is outside the pooled 95% CI. The Egger's and Begg's graphical methods were used to provide the diagnosis of the potential publication bias (Egger et al., 1997). All statistical analyses were performed using Stata software (version 12.0; Stata Corporation, College Station, TX) and RevMan software (version 5.2; Cochrane Collaboration). All p values were for a two-sided analysis and values of p<0. 05 were considered statistically significant.

Result

Characteristics of studies

The study selection process is detailed in Figure 1. Based on our preliminary search criteria, a total of 19 publications were eligible. Among these articles, thirteen publications were homozygous (CC vs. GG) or heterozygous (GC vs. GG or CC) comparisons, five publications were dominant genetic model (CC+GC vs. GG), and only one publication was recessive genetic model (CC vs. GG+GC). However, only 18 studies were included as the recessive genetic model study (only one publication was excluded in the final meta-analysis), comprising in total of 9432 homozygous subjects, 8670 heterozygous subjects, and 6528 subjects with dominant genetic model. All studies were case control in design. Table 1 shows the main characteristics of these studies. For homozygous comparison, 13 studies were available (all Caucasian), including 5971 subjects with GG genotype and 3461subjects with CC genotype. Three studies considered that the −174CC group had the lowest IL-6 levels, but nine studies suggested there was no association between the IL-6−174G/C polymorphism and IL-6 levels. Furthermore, one study found an association of the IL-6 −174G/C polymorphism and an increased level of IL-6. For the dominant genetic model (CC+GC vs. GG), five studies were available (four Caucasian, one North Indian), including 6528 subjects with CC/GC genotype and 3719 subjects with GG genotype.

FIG. 1.

Flow diagram of the article selection process for Interleukin-6 (IL-6) −174G/C polymorphism and circulating IL-6 levels.

Table 1.

Main Characteristics of the Studies Included in the Meta-Analysis

Reference	Year	Ethnicity	Subjects, n GG/GC/CC	Gender component in GG/GC/CC (%male)	Mean age, y GG/GC/CC	Genotyping method	Plasma IL-6 assay method
Studies compared GG,GC,CC
Fishman et al., 1998	1998	Caucasian	35/45/22	NA	NA	RFLP	ELISA
Basso et al., 2002	2002	Caucasian	98/97/86	NA	NA	SSCP	ELISA
Kubaszek et al., 2003	2003	Caucasian	32/55/37	65.6/69/62.1	52/52/51	SSCP	ELISA
Bennet et al., 2003	2003	Caucasian	278/500/235	Male	NA	DASH	ELISA
			120/254/113	Female
Illig et al., 2004	2004	Caucasian	205/326/112	57.9/55.8/57.5	65.5/65.2/64.1	PCR	ELISA
Mohlig et al., 2004	2004	Caucasian	150/311/103	61.3/58.5/56.3	55.2/56.2/54.4	PCR	ELISA
Cardellini et al., 2005	2005	Caucasian	142/112/21	29.6/32.1/33.3	39/37/39	RFLP	ELISA
Shen et al., 2008	2008	Caucasian	128/358/230	NA	NA	TaqMan	ELISA
Sie et al., 2008	2008	Caucasian	1390/1830/629	42/43/41	72/72/73	TaqMan	ELISA
Corella et al., 2009	2009	Caucasian	303/324/94	NA	68.9/69.1/68.5	TaqMan	ELISA
Mysliwska et al., 2009	2009	Caucasian	51/68/51	NA	NA	RFLP	ELISA
Sanderson et al., 2009	2009	Caucasian	1429/1988/748	NA	NA	TaqMan	ELISA
Studies compared GG+GC and CC
			(GG+GC)/CC	(GG+GC)/CC	(GG+GC)/CC
Fernandez-Real et al., 2000	2000	Caucasian	21/11	Male: 10/5	37.5/36.8	RFLP	ELISA
Studies compared CC+GC and GG
			(CC+GC)/GG	(CC+GC)/GG	(CC+GC)/GG
Kelberman et al., 2004	2004	North Europe	156/62	NA	NA	PCR	ELISA
		South Europe	95/103
Moleres et al., 2009	2009	Caucasian	293/211	NA	14.6/14.5	TaqMan	Flow cytometry
Sanderson et al., 2009	2009	Caucasian	2736/1429	NA	NA	TaqMan	ELISA
Kanoni et al., 2010	2010	Caucasian	414/405	43/46.3	72.6/73.6	RFLP	Sandwich immunoassay
Gupta et al., 2011	2011	North Indian	98/80	All female	28.6/27.49	RFLP	ELISA

NA, not available; RFLP, restriction fragment length polymorphism; PCR, polymerase chain reaction; DASH, dynamic allele-specific hybridization; SSCP, single-strand conformation polymorphism; ELISA, enzyme-linked immunosorbent assay.

Meta-analysis

No significant association of IL-6 −174G/C polymorphism and circulating IL-6 levels in a normal population was observed (Figs. 2 –4 and Table 2). However, heterogeneity was observed among the studies when compared among the GG, GC, and CC genotypes (Table 2). Sensitivity analysis revealed that, for IL-6 −174 G/C polymorphism, the independent study by Shen et al. influenced the heterogeneity in the homozygous and heterozygous comparison (Figs. 5 –7). After Shen et al.'s study was excluded, the remaining studies' heterogeneity became acceptable (Table 2). When all 12 studies were pooled into the meta-analysis, there was no evidence of an association between IL-6 −174G/C polymorphism and circulating IL-6 levels; for homozygous comparison (GG vs. CC): the pooled standard mean difference (SMD) was −0.01, 95%CI: −0.1–0.08, I ²=63.6%, p=0.001(Fig. 8); for heterozygous comparison (GC vs. GG or CC), the pooled SMD (GG vs. GC) was −0.05, 95%CI: −0.11–0.01, I²=47.6%, p=0.024 (Fig. 9) and the pooled SMD (CC vs. GC) was 0.03, 95%CI: −0.03–0.1, I ²=43.6%, p=0.041 (Fig. 10). Under dominant model, five eligible studies had been identified: the pooled SMD was −0.05, 95%CI: −0.11–0.01, I ²=33.2%, p=0.175 (Fig. 11), there was no association between IL-6 −174G/C polymorphism and circulating IL-6 levels. Under recessive genetic model, only one eligible study had been identified, and therefore it was excluded from the analysis.

FIG. 2.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for homozygous comparison (GG vs. CC).

FIG. 3.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for heterozygous comparison (GG vs. GC).

FIG. 4.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for heterozygous comparison (GC vs. CC).

FIG. 5.

Sensitivity analysis of studies of the IL-6 −174G/C polymorphism for homozygous comparison (GG vs. CC).

FIG. 6.

Sensitivity analysis of studies of IL-6 −174G/C polymorphism for heterozygous comparison (GC vs. CC).

FIG. 7.

Sensitivity analysis of studies of IL-6 −174G/C polymorphism for heterozygous comparison (GG vs. GC).

FIG. 8.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for homozygous comparison (GG vs. CC) after Shen et al.'s study was excluded.

FIG. 9.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for heterozygous comparison (GG vs. GC) after Shen et al.'s study was excluded.

FIG. 10.

Meta-analysis of studies of the IL-6 −174G/C polymorphism for heterozygous comparison (GC vs. CC) after Shen et al.'s study was excluded.

FIG. 11.

Meta-analysis of studies of the IL-6 −174G/C polymorphism in the dominant genetic model (GC+CC vs. GG).

Table 2.

The Effect of the IL-6-174 G/C Polymorphism and Circulating Interleukin-6 Levels

Genetic model (number of studies)	Main effects of IL-6-174 G/C polymorphism and circulating interleukin-6 levels
	Analysis model	Std. mean difference (95% CI)	p-Value	I²	Egger's test
GG vs. CC (13)	Random	0.23 (0 to +0.45)	0.000	95.7%	0.116
GG vs. GC (13)	Random	0.08 (−0.05 to +0.22)	0.000	92.2%	0.111
GC vs. CC (13)	Random	0.20 (−0.01 to 0.41)	0.000	95.7%	0.374
GG vs. GC+CC (5)	Random	−0.06 (−0.1 to −0.02)	0.0003	33.2%	0.48

	Main effects of IL-6-174 G/C polymorphism and circulating interleukin-6 levels after removed the study influenced the heterogeneity
GG vs. CC (12)	Random	−0.01 (−0.1 to 0.08)	0.001	63.6%	0.062
GG vs. GC (12)	Random	−0.07 (−0.11 to 0.01)	0.024	47.6%	0.135
GC vs. CC (12)	Random	0.03 (−0.03 to 0.1)	0.041	43.6%	0.282

For the studied IL-6 promoter polymorphisms, the visual examinations of the shape of funnel plots did not reveal any evidence of obvious asymmetry, suggesting the absence of publication bias. Subsequently, the Egger's test was used to provide statistical evidence of funnel plot symmetry and, as expected, the results showed no evidence of publication bias (Table 2).

Discussion

In this meta-analysis of a large normal population, we have demonstrated that the presence of the minor G allele at the IL-6 −174G/C single-nucleotide polymorphism (SNP), reportedly responsible for higher IL-6 expression, was not significantly associated with circulating IL-6 levels. However, because of the high heterogeneity, we were not able to determine a definitive conclusion. Therefore, we conducted a sensitivity analysis to identify the source of the heterogeneity, and the independent study by Shen et al. influenced the heterogeneity in the homozygous and heterozygous comparison. We found their participants were all with higher fasting plasma total TG, VLDL, and large VLDL. It has been suggested that increased adiposity may lead to higher IL-6 levels and worse insulin resistance (Fernandez-Real et al., 2000). It is likely that the known relationship between inflammation, obesity, and insulin resistance in humans is involved (Donath and Shoelson, 2011). Obesity is associated with a proinflammatory state (Madec et al., 2010), and maybe such a proinflammatory state influenced the heterogeneity in our meta-analysis.

Many studies have demonstrated associations of IL-6 −174G/C polymorphism with obesity, insulin resistance, dyslipidemia, and cardiovascular disease (CVD) (Fernandez-Real et al., 2000; Humphries et al., 2001; Stephens et al., 2004), and both in vitro and in vivo studies have indicated that the −174G/C polymorphism alters the IL-6 gene transcription rate and protein expression (Terry et al., 2000).

However, some subsequent articles did not confirm the association with IL-6 levels in healthy subjects, but many did see this in patients undergoing an inflammatory stimulus such as after coronary artery bypass surgery (CABG) (Brull et al., 2001). Five to seven days after CABG, the mean IL-6 levels rose sixfold in the whole cohort (p<0.0001) compared with baseline; the magnitude of the rise was also IL-6 genotype dependent: patients carrying −174C allele had IL-6 levels significantly higher than those of −174GG genotype (Wypasek et al., 2010). Kuo et al. (2008) showed that in patients with stable coronary artery disease (CAD), IL-6 and CRP were independently related to the presence of complex coronary plaque; however, the IL-6 genotypes were not related to serum IL-6 levels. A higher frequency of −174G/C IL-6 polymorphism together with increased IL-6 levels was observed in patients with extensive CAD (Mysliwska et al., 2006). In the Wypasek et al. (2010) study, neither procedure-related data during CABG nor major adverse cardiovascular events during the perioperative period were associated with −174G/C IL-6 polymorphism, but the intubation time or duration of intensive care was positively correlated with the magnitude of a postoperative increase in IL-6 levels.

This could be explained by the fact that it is the inflammatory processes, which influenced circulating IL-6 levels; the effects of −174G/C IL-6 polymorphism were modified by inflammation, but were not significantly associated with normal populations without inflammation.

The key question is whether there is a genetic predisposition in the disease under investigation to produce higher IL-6 levels, leading to a chronic proinflammatory state. Because IL-6 is involved in a multitude of inflammatory processes, it is possible that this SNP is directly related to other genes and/or other inflammatory pathways. In many cases, the increase in IL-6 levels is believed to be secondary to other inflammatory mediators such as IL-1 and TNF (Woo and Humphries, 2013). Our meta-analysis gave us a clue that the influence of IL-6 −174G/C polymorphism to IL-6 levels is minimal or it is not the IL-6 −174G/C polymorphism that directly influenced IL-6 levels; otherwise, they can be detected by our study.

In addition, circulating IL-6 may be less important than local IL-6 expression as studies have shown that locally produced cytokines possess important auto-/paracrine properties that influence the diverse functions of other tissues (Fried et al., 1998).

In addition, a polymorphism in the promoter sequence of the IL-6 gene (rs1800795; IL6 −174G/C) has been found to modulate individual sensitivity to the effects of threatening or adverse environments in activating IL-6 transcription (Cole et al., 2010). In people bearing the ancestral G allele of the polymorphism, adversity-induced activation of the GATA1 transcription factor leads to increased IL-6 gene transcription, and those individuals show increased risk of inflammation-related disease and mortality when confronted by extended periods of significant life adversity (Cole et al., 2010). In contrast, individuals bearing the C allele of the polymorphism are protected from such effects, because the GATA1 transcription factor cannot efficiently bind to the IL-6–174 locus after SNS-induced activation (Cole et al., 2010).

Furthermore, a single point measurement of plasma IL-6 concentrations may not adequately reflect an individual's long-term exposure of chronic inflammation. It is unknown whether average, peak, or inflammatory levels of IL-6 are relating to −174G/C polymorphism; and fasting IL-6 measurements may not be appropriate for examining the identified genotype–phenotype relationship (Herbert et al., 2006). The accurate measurement of IL-6 in serum is complicated by the fact that IL-6 levels vary in a diurnal manner in the healthy individual, so the time of sampling would affect the final results (Woo and Humphries, 2013). Unfortunately, none of the articles describing serum IL-6 levels provide data on the time of collection or time to storage and length of storage.

Several potential limitations of this meta-analysis should be considered. First, this meta-analysis only focused on articles published in English. Our data were derived mostly from subjects of Caucasian descent; therefore, it may be that results do not apply to cases from other ethnicities. Second, not all the control subjects were age and sex matched to cases, which was likely one of the causes for heterogeneity. Third, because the included studies are observational and confounding, biases might have affected the pooled estimates. Furthermore, large and well-designed studies are necessary to determine the relationship of this genetic polymorphism with IL-6 expression in a normal population. However, this is the first meta-analysis, based on a normal population, about the relationship between IL-6 −174G/C polymorphism and circulating IL-6 levels. The potential sources of heterogeneity in the meta-analysis were assessed.

In conclusion, the meta-analysis provides evidence that the −174G/C polymorphism in the IL-6 gene is not significantly associated with circulating IL-6 levels in a normal population.

Footnotes

Acknowledgments

This study was supported by grants from the National Natural Science Foundation (No.81270127), China.

Disclosure Statement

No competing financial interests exist.

References

Basso

, Lowe

G.D.

, Rumley

, McMahon

A.D.

, Humphries

S.E.

2002. Interleukin-6-174G>C polymorphism and risk of coronary heart disease in West of Scotland coronary prevention study (WOSCOPS) Arterioscler Thromb Vasc Biol, 22:599–604.

Bennet

A.M.

, Prince

J.A.

, Fei

G.Z.

, Lyrenas

, Huang

, Wiman

, Frostegard

, Ud

2003. Interleukin-6 serum levels and genotypes influence the risk for myocardial infarction. Atherosclerosis, 171:359–367.

Bittar

M.N.

, Carey

J.A.

, Barnard

, Fildes

J.E.

, Pravica

, Yonan

, Hutchinson

I.V.

2005. Interleukin 6 G-174C polymorphism influences outcome following coronary revascularization surgery. Heart Surg Forum, 8:E140–145; discussion E145.

Brull

D.J.

, Montgomery

H.E.

, Sanders

, Dhamrait

, Luong

, Rumley

, Lowe

G.D.

, Humphries

S.E.

2001. Interleukin-6 gene -174g>c and -572g>c promoter polymorphisms are strong predictors of plasma interleukin-6 levels after coronary artery bypass surgery. Arterioscler Thromb Vasc Biol, 21:1458–1463.

Cardellini

, Perego

, D'Adamo

, Marini

M.A.

, Procopio

, Hribal

M.L.

, Andreozzi

, Frontoni

, Giacomelli

, Paganelli

et al. 2005. C-174G polymorphism in the promoter of the interleukin-6 gene is associated with insulin resistance. Diabetes Care, 28:2007–2012.

Cole

S.W.

, Arevalo

J.M.

, Takahashi

, Sloan

E.K.

, Lutgendorf

S.K.

, Sood

A.K.

, Sheridan

J.F.

, Seeman

T.E.

2010. Computational identification of gene-social environment interaction at the human IL6 locus. Proc Natl Acad Sci USA, 107:5681–5686.

Corella

, Gonzalez

J.I.

, Bullo

, Carrasco

, Portoles

, Diez-Espino

, Covas

M.I.

, Ruiz-Gutierrez

, Gomez-Gracia

, Aros

et al. 2009. Polymorphisms cyclooxygenase-2-765G>C and interleukin-6-174G>C are associated with serum inflammation markers in a high cardiovascular risk population and do not modify the response to a Mediterranean diet supplemented with virgin olive oil or nuts. J Nutr, 139:128–134.

Danesh

, Kaptoge

, Mann

A.G.

, Sarwar

, Wood

, Angleman

S.B.

, Wensley

, Higgins

J.P.

, Lennon

, Eiriksdottir

et al. 2008. Long-term interleukin-6 levels and subsequent risk of coronary heart disease: two new prospective studies and a systematic review. PLoS Med, 5:e78.

Donath

M.Y.

, Shoelson

S.E.

2011. Type 2 diabetes as an inflammatory disease. Nat Rev Immunol, 11:98–107.

10.

Egger

, Davey

S.G.

, Schneider

, Minder

1997. Bias in meta-analysis detected by a simple, graphical test. BMJ, 315:629–634.

11.

Fernandez-Real

J.M.

, Broch

, Vendrell

, Gutierrez

, Casamitjana

, Pugeat

, Richart

, Ricart

2000. Interleukin-6 gene polymorphism and insulin sensitivity. Diabetes, 49:517–520.

12.

Fernandez-Real

J.M.

, Broch

, Vendrell

, Richart

, Ricart

2000. Interleukin-6 gene polymorphism and lipid abnormalities in healthy subjects. J Clin Endocrinol Metab, 85:1334–1339.

13.

Fishman

, Faulds

, Jeffery

, Mohamed-Ali

, Yudkin

J.S.

, Humphries

, Woo

1998. The effect of novel polymorphisms in the interleukin-6 (IL-6) gene on IL-6 transcription and plasma IL-6 levels, and an association with systemic-onset juvenile chronic arthritis. J Clin Invest, 102:1369–1376.

14.

Fried

S.K.

, Bunkin

D.A.

, Greenberg

A.S.

1998. Omental and subcutaneous adipose tissues of obese subjects release interleukin-6: depot difference and regulation by glucocorticoid. J Clin Endocrinol Metab, 83:847–850.

15.

Gupta

, Gupta

, Singh

A.K.

, Tiwari

, Agrawal

, Natu

S.M.

, Agrawal

C.G.

, Negi

M.P.

, Pant

A.B.

2011. Interleukin-6 G-174C gene polymorphism and serum resistin levels in North Indian women: potential risk of metabolic syndrome. Hum Exp Toxicol, 30:1445–1453.

16.

Herbert

, Liu

, Karamohamed

, Liu

, Manning

, Fox

C.S.

, Meigs

J.B.

, Cupples

L.A.

2006. BMI modifies associations of IL-6 genotypes with insulin resistance: the Framingham Study. Obesity (Silver Spring), 14:1454–1461.

17.

Hingorani

A.D.

, Casas

J.P.

2012. The interleukin-6 receptor as a target for prevention of coronary heart disease: a mendelian randomisation analysis. Lancet, 379:1214–1224.

18.

Humphries

S.E.

, Luong

L.A.

, Ogg

M.S.

, Hawe

, Miller

G.J.

2001. The interleukin-6-174 G/C promoter polymorphism is associated with risk of coronary heart disease and systolic blood pressure in healthy men. Eur Heart J, 22:2243–2252.

19.

Illig

, Bongardt

, Schopfer

, Muller-Scholze

, Rathmann

, Koenig

, Thorand

, Vollmert

, Holle

, Kolb

et al. 2004. Significant association of the interleukin-6 gene polymorphisms C-174G and A-598G with type 2 diabetes. J Clin Endocrinol Metab, 89:5053–5058.

20.

Jones

K.G.

, Brull

D.J.

, Brown

L.C.

, Sian

, Greenhalgh

R.M.

, Humphries

S.E.

, Powell

J.T.

2001. Interleukin-6 (IL-6) and the prognosis of abdominal aortic aneurysms. Circulation, 103:2260–2265.

21.

Kanoni

, Dedoussis

G.V.

, Herbein

, Fulop

, Varin

, Jajte

, Rink

, Monti

, Mariani

, Malavolta

et al. 2010. Assessment of gene-nutrient interactions on inflammatory status of the elderly with the use of a zinc diet score—ZINCAGE study. J Nutr Biochem, 21:526–531.

22.

Kelberman

, Hawe

, Luong

L.A.

, Mohamed-Ali

, Lundman

, Tornvall

, Aillaud

M.F.

, Juhan-Vague

, Yudkin

J.S.

, Margaglione

et al. 2004. Effect of Interleukin-6 promoter polymorphisms in survivors of myocardial infarction and matched controls in the North and South of Europe. The HIFMECH Study. Thromb Haemost, 92:1122–1128.

23.

Kubaszek

, Pihlajamaki

, Punnonen

, Karhapaa

, Vauhkonen

, Laakso

2003. The C-174G promoter polymorphism of the IL-6 gene affects energy expenditure and insulin sensitivity. Diabetes, 52:558–561.

24.

Kuo

L.T.

, Yang

N.I.

, Cherng

W.J.

, Verma

, Hung

M.J.

, Wang

S.Y.

, Liu

M.H.

, Chen

S.Y.

, Wang

C.H.

2008. Serum interleukin-6 levels, not genotype, correlate with coronary plaque complexity. Int Heart J, 49:391–402.

25.

Liu

, Berthier-Schaad

, Fallin

M.D.

, Fink

N.E.

, Tracy

R.P.

, Klag

M.J.

, Smith

M.W.

, Coresh

2006. IL-6 haplotypes, inflammation, and risk for cardiovascular disease in a multiethnic dialysis cohort. J Am Soc Nephrol, 17:863–870.

26.

Madec

, Chiarugi

, Santini

, Rossi

, Miccoli

, Ferrannini

, Solini

2010. Pattern of expression of inflammatory markers in adipose tissue of untreated hypertensive patients. J Hypertens, 28:1459–1465.

27.

Moher

, Cook

D.J.

, Eastwood

, Olkin

, Rennie

, Stroup

D.F.

1999. Improving the quality of reports of meta-analyses of randomised controlled trials: the QUOROM statement. Quality of Reporting of Meta-analyses. Lancet, 354:1896–1900.

28.

Mohlig

, Spranger

, Osterhoff

, Ristow

, Pfeiffer

A.F.

, Schill

, Schlosser

H.W.

, Brabant

, Schofl

2004. The polycystic ovary syndrome per se is not associated with increased chronic inflammation. Eur J Endocrinol, 150:525–532.

29.

Moleres

, Rendo-Urteaga

, Azcona

, Martinez

J.A.

, Gomez-Martinez

, Ruiz

J.R.

, Moreno

L.A.

, Marcos

, Marti

2009. Il6 gene promoter polymorphism (-174G/C) influences the association between fat mass and cardiovascular risk factors. J Physiol Biochem, 65:405–413.

30.

Mysliwska

, Wieckiewicz

, Hak

, Siebert

, Rogowski

, Szyndler

, Mysliwski

2006. Interleukin 6 polymorphism corresponds to the number of severely stenosed coronary arteries. Eur Cytokine Netw, 17:181–188.

31.

Mysliwska

, Zorena

, Mysliwiec

, Malinowska

, Raczynska

, Balcerska

2009. The -174GG interleukin-6 genotype is protective from retinopathy and nephropathy in juvenile onset type 1 diabetes mellitus. Pediatr Res, 66:341–345.

32.

, van Dam

, Meigs

J.B.

, Manson

J.E.

, Hunter

, Hu

F.B.

2006. Genetic variation in IL6 gene and type 2 diabetes: tagging-SNP haplotype analysis in large-scale case-control study and meta-analysis. Hum Mol Genet, 15:1914–1920.

33.

Ridker

P.M.

, Rifai

, Stampfer

M.J.

, Hennekens

C.H.

2000. Plasma concentration of interleukin-6 and the risk of future myocardial infarction among apparently healthy men. Circulation, 101:1767–1772.

34.

Sanderson

S.C.

, Kumari

, Brunner

E.J.

, Miller

M.A.

, Rumley

, Lowe

G.D.

, Marmot

M.G.

, Humphries

S.E.

2009. Association between IL6 gene variants -174G>C and -572G>C and serum IL-6 levels: interactions with social position in the Whitehall II cohort. Atherosclerosis, 204:459–464.

35.

Shen

, Arnett

D.K.

, Perez-Martinez

, Parnell

L.D.

, Lai

C.Q.

, Peacock

J.M.

, Hixson

J.E.

, Tsai

M.Y.

, Straka

R.J.

, Hopkins

P.N.

et al. 2008. The effect of IL6-174C/G polymorphism on postprandial triglyceride metabolism in the GOLDN studyboxs. J Lipid Res, 49:1839–1845.

36.

Sie

M.P.

, Mattace-Raso

F.U.

, Uitterlinden

A.G.

, Arp

P.P.

, Hofman

, Pols

H.A.

, Hoeks

A.P.

, Reneman

R.S.

, Asmar

, van Duijn

et al. 2008. The interleukin-6–174 G/C promoter polymorphism and arterial stiffness; the Rotterdam Study. Vasc Health Risk Manag, 4:863–869.

37.

Stephens

J.W.

, Hurel

S.J.

, Cooper

J.A.

, Acharya

, Miller

G.J.

, Humphries

S.E.

2004. A common functional variant in the interleukin-6 gene is associated with increased body mass index in subjects with type 2 diabetes mellitus. Mol Genet Metab, 82:180–186.

38.

Terry

C.F.

, Loukaci

, Green

F.R.

2000. Cooperative influence of genetic polymorphisms on interleukin 6 transcriptional regulation. J Biol Chem, 275:18138–18144.

39.

van

Oijen M

, Arp

P.P.

, de Jong

, Hofman

, Koudstaal

P.J.

, Uitterlinden

A.G.

, Breteler

M.M.

2006. Polymorphisms in the interleukin 6 and transforming growth factor beta1 gene and risk of dementia. The Rotterdam Study. Neurosci Lett, 402:113–117.

40.

Walston

J.D.

, Fallin

M.D.

, Cushman

, Lange

, Psaty

, Jenny

, Browner

, Tracy

, Durda

, Reiner

2007. IL-6 gene variation is associated with IL-6 and C-reactive protein levels but not cardiovascular outcomes in the Cardiovascular Health Study. Hum Genet, 122:485–494.

41.

Woo

, Humphries

S.E.

2013. IL-6 polymorphisms: a useful genetic tool for inflammation research. J Clin Invest, 123:1413–1414.

42.

Wypasek

, Undas

, Sniezek-Maciejewska

, Kapelak

, Plicner

, Stepien

, Sadowski

2010. The increased plasma C-reactive protein and interleukin-6 levels in patients undergoing coronary artery bypass grafting surgery are associated with the interleukin-6-174G>C gene polymorphism. Ann Clin Biochem, 47:343–349.