Meta‐analysis: Interleukin 6 gene ‐174G/C polymorphism associated with type 2 diabetes mellitus and interleukin 6 changes

Abstract The gene coding interleukin 6 (IL‐6) is a promising candidate in predisposition to type 2 diabetes mellitus (T2DM). This study aimed to meta‐analytically examine the association of IL‐6 gene −174G/C polymorphism with T2DM and circulating IL‐6 changes across −174G/C genotypes. Odds ratio (OR) and standard mean difference (SMD) with 95% confidence interval (CI) were calculated. Twenty‐five articles were meta‐analysed, with 20 articles for T2DM risk and 9 articles for circulating IL‐6 changes. Overall, there was no detectable significance for the association between −174G/C polymorphism and T2DM, and this association was relatively obvious under dominant model (OR: 0.82, 95% CI: 0.56‐1.21). Improved heterogeneity was seen in some subgroups, with statistical significance found in studies involving subjects of mixed races (OR: 0.63, 95% CI: 0.46‐0.86). Begg's and filled funnel plots, along with Egger's tests revealed week evidence of publication bias. In genotype‐phenotype analyses, carriers of −174CC and −174CG genotypes separately had 0.10 and 0.03 lower concentrations (pg/mL) of circulating IL‐6 than −174GG carriers. Albeit no detectable significance for the association of −174G/C with T2DM, our findings provided suggestive evidence on a dose‐dependent relation between −174G/C mutant alleles and circulating IL‐6 concentrations, indicating possible implication of this polymorphism in the pathogenesis of T2DM.


| INTRODUC TI ON
Diabetes is a chronic metabolic disorder, and globally an estimated 422 million persons are affected by diabetes, mainly in low-and middle-income countries. 1 The most common is type 2 diabetes mellitus (T2DM), which accounts for 90% to 95% of all diabetes. T2DM is a complex, multifactorial disease, attributing to the interaction between genetic defects and environmental factors. 2,3 As a risk factor of nearly all-cause mortality, T2DM can affect people across different life stages. 4 So, early identification of persons at a higher risk for T2DM is of great clinical and public health importance.
It is well known that T2DM is a polygenic disease. Extensive efforts have been made to decipher the genetic basis of T2DM, especially with the advent of genome-wide association studies (GWASs). [5][6][7] Although over a hundred genetic variants in predisposition to T2DM have been characterized, only a modest portion of T2DM heritability can be interpreted. 8,9 One of the major challenges facing global geneticists is the inconsistent replication of candidate genes with biological implications across different populations. 10,11 The gene coding interleukin 6 (IL-6) is one such gene.
To shed some light upon these reasons and yield more information for future investigations, we here prepared a systematic review of published studies to meta-analytically examine the association of IL-6 gene −174G/C polymorphism with T2DM, as well as the changes of circulating IL-6 concentrations across −174G/C genotypes.
Meanwhile, the possible sources for between-study heterogeneity attributed to inconsistent observations were also interrogated.

| ME THODS
This meta-analysis was proceeded in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Metaanalyses (PRISMA) statement. 25 The PRISMA checklist is presented in Table S1.

| Search strategy
Public databases including Medline/PubMed, EMBASE (Excerpta Medica database) and Web of Science were reviewed to seek potentially qualified articles published prior to 8 September 2020.
In addition, the reference lists of major articles or reviews were scanned for potential missing articles. Search process was independently completed by two of us (Hao Cheng and Wenbin Zhu), by using same key terms aforementioned, and any conflicts were adjudicated by a third author (Chunjing Zhang). The results were integrated, and duplicates were removed from the final reference set.

| Eligibility criteria
Eligible articles needed to meet the following three criteria: (i) available genotype or allele counts of IL-6 gene −174G/C polymorphism in both T2DM patients and controls or available circulating IL-6 concentrations across the genotypes of −174G/C polymorphism; (ii) clear definition of T2DM according to official guidelines; (iii) the adoption of validated assaying methods to determine three −174G/C genotypes.
If the retrieved publication was a narrative or quantitative review, was an animal study, focused on diabetic complications, did not have valid control groups, lacked necessary genotype information or was published in the languages other than the English, this publication was excluded from this meta-analysis.

| Data extraction
From each qualified article, extracted data included first author's name, year of publication, race or ethnicity, disease status, T2DM diagnosis, control source, study design, matched condition, age, gender and body mass index, as well as, if available, haemoglobin A1c (HbA1c), fasting plasma glucose (FPG), postprandial glucose (PPG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDLC) and low-density lipoprotein cholesterol (LDLC). Data extraction process was independently finished by two of us (Hao Cheng and Wenbin Zhu), and disagreement was solved by a third author (Chunjing Zhang).

| Statistical analyses
All statistical analyses were performed with the use of STATA software Release 14.1 (StataCorp,College Station,TX,USA).
Weighted odds ratio (OR) and its 95% confidence interval (95% CI) were calculated to assess the association between IL-6 gene −174G/C polymorphism and T2DM. In addition, changes in circulating IL-6 and the other laboratory biomarkers across −174G/C genotypes were expressed as standard mean difference (SMD) and 95% CI. Pooled OR and SMD were derived under the randomeffects model. The inconsistency index (I 2 ) was adopted to appraise between-study heterogeneity, which meant that the percentage of observed variability between studies that was due to heterogeneity instead of a chance finding. If the I 2 is over 50.0%, statistically significant heterogeneity is recorded. Subsidiary analyses were done to interrogate underlying sources for between-study heterogeneity.
Cumulative analyses and sensitivity analyses were carried out to appraise the risk of bias. The former measured the impact of the first publication on subsequent publications and the evolution of cumulative estimates over time. The latter removed one publication at a time to appraise the influence of a single publication on pooled estimates.

| Overall analyses: −174G/C polymorphism and T2DM
The overall association of IL-6 gene −174G/C polymorphism with T2DM was assessed under three different genetic models, as illustrated in Figure 2. The mutation of this polymorphism was related to a reduced risk of T2DM, albeit no detectable statistical significance.
There was statistically significant between-study heterogeneity across three genetic models, with the I 2 around 80% (P <.001).

| Subsidiary analyses: −174G/C polymorphism and T2DM
Due to significant heterogeneity in overall analyses, a wide panel of subsidiary analyses was carried out separately according to sample sizes, races, countries, diagnostic criteria of T2DM, disease status of patients with T2DM, matched statue and study designs (

| Cumulative and influential analyses: −174G/C polymorphism and T2DM
In cumulative analyses, there was no suggestion of significant influence from the first publication on subsequent publications for IL-6 gene −174G/C polymorphism associated with T2DM under three genetic models ( Figure S1). The influential analyses indicated no significant influence of any one studies on overall estimates under three genetic models ( Figure S2).

| Publication bias: −174G/C polymorphism and T2DM
Begg's plots and filled funnel plots are presented in Figure 3 for the association between IL-6 gene −174G/C polymorphism and T2DM under three genetic models. The Begg's funnel plots seemed symmetrical, and there was no statistical evidence of publication bias. In addition, there were no theoretically missing studies in filled funnel plots.

| Other circulating biomarkers across −174G/ C genotypes
The changes in other circulating biomarkers, including LDL, HDL, TC, TG, HbA1c, and FPG, across the genotypes of IL-6 gene −174G/C polymorphism are separately summarized in Figure S3.

| D ISCUSS I ON
This study was designed to meta-analytically examine the as-     53 In addition, IL-6 is considered to be involved in the development of inflammation, insulin resistance, as well as β-cell dysfunction. 23 The interaction between IL-6 and TNFα can exacerbate oxidative stress and reduce phosphorylation of endothelial nitric oxide synthase (eNOS), which may cause various complications. 54 On the basis of above evidence, it is reasonable to speculate that IL-6 gene is a possible candidate in susceptibility to the development of diabetes.
Several limitations should be acknowledged for the current meta-analysis. The first limitation lied in the analysis of only one polymorphism in IL-6 gene. The second limitation was that only retrieved articles in English were analysed in this study, and the 'grey' literature was not included. The exclusion of 'grey' literature from meta-analysis may result in an overestimate of an association impact by an average of 12%. 55 The third limitation was about publication bias. Although there was a low probability, the possibility of missing small or negative studies that had not yet been published was still existed. The fourth limitation was about heterogeneity. Although a set of auxiliary analyses had been conducted, the heterogeneity was still significant in some subgroups, which limited the interpretation of combined risk estimates.
Taken together, albeit no detectable significance between IL-6 gene −174G/C polymorphism and T2DM, our genotype-phenotype analyses provided suggestive evidence on a dose-dependent relation between the number of −174G/C mutant alleles and circulating IL-6 concentrations, indicating possible implication of IL-6 gene in the pathogenesis of T2DM. For practical reasons, our hope is that this meta-analysis will not represent just another endpoint of investigations, instead of a start to clarify the association of other genetic defects in IL-6 gene with the risk for T2DM, as well as to elucidate the underlying molecular mechanisms of circulating IL-6 concentrations in the onset and progression of T2DM.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data involved in this study are available upon reasonable request.