The association between interleukin-6 gene -174G/C single nucleotide polymorphism and sepsis: an updated meta-analysis with trial sequential analysis

Background This article intends to explore the association between interleukin-6 gene (IL-6) -174 G/C single nucleotide polymorphism (SNP) and the risk and mortality of sepsis by conducting this updated meta-analysis with trial sequential analysis. Methods References were made to PubMed, Web of Science, China National Knowledge Infrastructure for studies available by September 2018. Each publication was screened for its eligibility and data accessible. Statistical analysis was conducted on Stata 14.1 and TSA software 0.9.5.10 Beta Results Twenty studies (including 3282 cases and 4926 controls) and eight studies (including 610 cases and 1856 controls) were respectively enrolled in the analysis on the association between IL-6-174 G/C polymorphism and the risk and mortality of sepsis. The results did not present any association between IL-6-174 G/C polymorphism and the risk and mortality of sepsis. An exception was that IL-6-174 G/C polymorphism was correlated with worse outcome in non-adults in recessive model, co-dominant model (CC vs. GG) and allelic model, while trial sequential analysis revealed it could be a false positive result nevertheless. Conclusions IL-6-174 G/C polymorphism is not associated with the risk and mortality of sepsis. Trial sequential analysis showed that a large sample size was needed to get a more reliable result of the association between IL-6-174 G/C polymorphism and sepsis in non-adults. Electronic supplementary material The online version of this article (10.1186/s12881-019-0766-2) contains supplementary material, which is available to authorized users.


Background
Sepsis is a systemic multiorgan dysfunction secondary to the dysregulated host response to infection as suggested in the latest definition for sepsis (Sepsis-3.0) and has a strong correlation with intensive care unit (ICU) admission and in-hospital mortality [1,2]. The recognition and management of sepsis has become a major issue in critical care medicine.
The alteration of immune function has been considered a key factor in the pathogenesis of sepsis [3]. The host immune system generates a series of substances such as cytokines in response to an infection or injury. IL-6 is a pro-inflammatory cytokine which is involved in the inflammatory reaction at the early stage of sepsis. It serves as a biomarker of the sepsis, and elevated serum IL-6 level indicates the deterioration of the disease and higher tendency to death [4,5]. IL-6 blockade therapy is beneficial to the prognosis of sepsis by blocking systemic inflammatory response [6].
IL-6 gene, located at 7p15.3 is responsible for the regulation of the transcriptional activity during inflammation reaction. The possible association between its − 174 G/C polymorphism (rs1800795) at promoter region and the risk and mortality of sepsis has been widely studied. However, the findings are varied among different studies. IL-6-174 C allele is thought to block the norepinephrine-induced transcription factor binding to IL-6 gene promotor and thus to lower the gene expression, which is favorable for inflammation-related diseases [7]. A meta-analysis by Chauhan M et al. in 2008 did not support the association between − 174 G/C polymorphism and the risk of sepsis in very low birth weight (VLBW) infants [8]. After that, a meta-analysis in 2013 demonstrated that IL-6-174 G/C polymorphism did not have a link with the risk and mortality of sepsis at any age and ethnicity groups [9]. In recent years, more studies on this topic have been published. A study found that IL-6-174 G allele was associated with early-onset sepsis in Saudi infants [10]. However, Mao Z et al. and Feng B et al. thought it was IL-6-174 C allele rather than G allele that contributed to the risk of sepsis induced by pneumonia [11,12]. When it came to the mortality of septic patients, Lorente L et al. discovered better survival of septic patients with CC genotype [13]. Jimenez-Sousa MA et al. found the possible association between IL-6-174 CC genotype and a higher septic shock-related mortality in patients who underwent major surgery [14]. These studies have controversial findings, which made us curious how they would influence the results if they were included in a new meta-analysis. Thus, an updated meta-analysis was made, which was intended to add valuable information to the future studies.

Searching strategy
We searched PubMed, Web of Science and China National Knowledge Infrastructure (CNKI) for available studies published before September 2018. The keywords for searching were a combination of "IL-6, interleukin-6, rs1800795, -174G/C, polymorphism, sepsis, septicemia, septic shock". Meanwhile, references of the relevant literature reviews were screened to identify potentially relevant publications.

Criteria for inclusion and exclusion
The publications fulfilling the following criteria were included: an original study evaluating the association between IL-6-174 G/C polymorphism and the risk and/or mortality of sepsis; objects in each study were from same epoch; including a case group of sepsis; including a control group; including precise sample size of IL-6-174 G/C polymorphism of case and control groups which could be directly extracted or calculated according to the information available. Excluded were: repetition of the published studies; a meta-analysis or a review; study with insufficient or incorrect data; study with only G or C allele carriers. The screening process is illustrated in a flow chart (Fig. 1). All publications were identified by Fig. 1 Flow diagram of the study selection process two reviewers independently. If two reviewers had opposite views, a third reviewer would be consulted for a decision.

Quality assessment and data extraction
The quality of included studies was evaluated according to Newcastle-Ottawa Scale (NOS) by two researchers [15]. Available data were extracted by two authors independently. Disagreements were dealt with by a third reviewer. The following information was extracted from studies: first author, year of publication, country of the study and features of case and control groups such as ethnicity, age group, type of case and controls. Some studies discussed both the risk and mortality of sepsis. In this case, the information was collected respectively.

Statistical analysis
The role of minor allele C in the risk and mortality of sepsis was targeted. Thus an analysis was made using dominant model (GC + CC versus GG), recessive model (CC versus GC + GG), co-dominant model (GC versus GG and CC versus GG) and allelic model (C versus G). While some studies only yielded a sum of number of genotypes, they were enrolled in analyses just under certain genetic models [16][17][18][19]. Statistical analysis was conducted on Stata 14.1. Each control group underwent goodness-of-fit χ 2 -test for evaluating Hardy-Weinberg equilibrium (HWE). When P > 0.05, the objects were under Hardy-Weinberg equilibrium and within a same Mendelian population. Homogeneity of the studies was tested. When P < 0.1 or I 2 > 50%, a high level of heterogeneity between studies was envisaged and random-effect model was adopted; when P > 0.1 and I 2 < 50%, there was no significant heterogeneity between studies, and fixed-effect model was used. The effect of IL-6-174 G/C on the risk or mortality of sepsis was measured by P value, odd ratio (OR) and 95% confidence interval (CI). P < 0.05 was reviewed as statistical significance. In the analysis on sepsis risk, subgroup analyses based on age group, ethnicity, restricted healthy controls were conducted. Subgroup analysis on age and ethnicity in the mortality analysis was also made. If no information of age group was given in a study, we considered it would be as a study on adult. The majority of the nation of study was taken as the ethnicity of the study population in case that it was not specifically mentioned. The objects in three studies were mainly Caucasians, so they were included in the subgroup analysis of the Caucasians as the number of non-Caucasians were very small [16,20,21]. Meanwhile, sensitivity analysis was conducted by repeating analysis after omitting one study each time to estimate the effect of quality of studies on the final result. Publication bias was evaluated by Egger's test. Once P > 0.05 in regression test, no obvious publication bias would exist.

Trial sequential analysis
Meta-analysis might be affected by type I error due to the increased risk of random error and repeated significance testing. Trial sequential analysis (TSA) was a useful tool to verify the reliability of the results from meta-analysis by estimating the required information size (RIS) (sample size of included studies) and calculating the threshold for statistical significance [22]. A type I error of 5%, power of 80%, relative risk reduction of 20% were defined and control event proportion was an average of each included study. If the Z-curve crossed RIS line, the result of meta-analysis would be conclusive. If the Z-curve crossed the O'Brien-Fleming boundary or futility boundary, the conclusions could be made even before it crossed the RIS line that IL-6-174G/C polymorphism have or did not have a correlation with sepsis, respectively. TSA was conducted in the genetic model with the most included studies. If each genetic model had the same number of included studies, TSA would be conducted in allelic model. Meta-analysis which presented a significant result in the pool analysis was also tested under TSA. TSA was conducted on TSA software 0.9.5.10 Beta.

Searching results
354 studies were initially acquired from databases and 4 studies were from references of publications. After exclusion of irrelevant studies, review and meta-analysis as well as repetitions, 29 studies were identified for full-text review. 5 studies were further excluded after full-text review because of inadequate data for analysis [23][24][25][26][27]. Eventually, 24 studies fulfilled all criteria for inclusion, including 16 studies on the risk of sepsis, 4 studies on the mortality and 4 studies on both topics [10-14, 16-21, 28-40]. All included studies had a NOS score ≥ 6 (see Additional file 1: Table S1). Totally, there were 3282 cases and 4926 controls for the analysis on sepsis risk together with 610 cases and 1856 controls for the analysis on mortality. Main characteristics of all studies included as well as the genotype distributions of IL-6-174 G/C polymorphism were shown in Table 1.
The association between IL-6-174 G/C polymorphism and the risk of sepsis The results were shown in Table 2 (Fig. 2). When limiting control group to healthy ones or Mendelian population, the results remained unchanged. In the subgroup analysis on non-adults, adults and Caucasians, no association between IL-6-174 G/C polymorphism and the risk of sepsis was found, neither.
The association between IL-6-174 G/C polymorphism and the mortality of sepsis The results were shown in Table 2

Publication bias and sensitivity analysis
No obvious publication bias was found in every section of this meta-analysis in Egger's test. The results and graphs of Egger's tests were shown in Table 2 and supplementary figures (see Additional file 2). The results of sensitivity analysis were shown in Table S2 (see Additional file 1). The results of meta-analysis on the association between IL-6-174 G/C polymorphism and the mortality of non-adult (CC vs. GG and allelic model) were changed after omitting certain studies. Additionally, some relevant studies might influence the results of study were also tested in our analyses. An analysis after omitting two studies including elder children from non-adults group was also made, the results being unchanged [32,33] (see Additional file 1: Table S3). Moreover, an study was included in the meta-analysis by Gao et al. but excluded in our study [26]. Thus an analysis was carried out and proved that the exclusion of this study did not influence the results of our meta-analysis (see Additional file 1: Table S4).

Trial sequential analysis
In the meta-analysis on the risk of sepsis, TSA was conducted on analysis of overall population (dominant and analysis involving healthy controls (allelic model) and Mendelian population (allelic model). Z-curves crossed the futility boundary and reached the required sample size in those analysis. The exception was the analysis on non-adult group, where Z-curve crossed neither conventional test boundary nor the futility boundary (Fig. 3). In the meta-analysis on the mortality of sepsis, TSA was conducted on analysis of overall population (allelic model), non-adults (recessive model, codominant model: CC vs. GG and allelic model), adults (allelic model) and Caucasians (allelic model). Although Z-curves crossed the conventional test boundary in the analyses on non-adults, they did neither cross O'Brien--Fleming boundary nor cross the RIS boundary (see Additional file 2). Z-curves in other TSAs crossed futility boundary and RIS boundary.

Discussion
The association between IL-6-174 G/C polymorphism and the risk or mortality of sepsis has been widely studied, and previous meta-analyses have been conducted in 2008 and 2013. Five more studies have been published in recent years. Because of the controversial findings, an updated meta-analysis is necessary to bring some new insights into this topic. This study has the largest sample so far to address this issue. What's more, trial sequential analysis was carried out in order to quantify the statistical reliability of the results. No association between IL-6-174 G/C polymorphism and the risk or mortality of sepsis in the overall population was found. This is similar to the finding by Gao et al. [9]. Heterogeneity in each study might be possible, which could arise from age, ethnicity or type of controls. However, subgroup analyses based on those factors did not present any association, either. Trial sequential analysis revealed that the results Fig. 2 Forest plot of the association between IL-6-174 G/C polymorphism and the risk of sepsis under dominant model. The horizontal line represents the 95% confidence interval. And its length shows the range of the confidence interval and the size of the square in the middle shows the weight of the study. The diamond (and broken line) represents the overall summary estimate, with confidence interval given by its width. The unbroken vertical line is at the null value (OR = 1.0). CI, confidence interval were reliable, except for the cases concerning non-adults group. Located at promotor region of IL-6 gene, − 174 G/C was found to be associated with the lower serum IL-6 level and lower inflammatory response subsequently [12,27,41]. Some previous studies reported the association between IL-6-174G/C polymorphism and the risk and mortality of sepsis [10-12, 16, 29, 31, 32, 34, 39, 40]. However, our results again failed to present any association. The reason could be: Firstly, the incidence and development of sepsis was involved in many factors such as gender, pathogens, gene polymorphism, environment effect and medical procedure. IL-6-174 G/C polymorphism might influence the transcriptional activity and lower the inflammatory reaction, it did not get the point that it could influence the development of sepsis nevertheless. Secondly, IL-6-174 G/C polymorphism might have an impact on the certain groups of people and pathological states, which were not within the subgroups of this study. Thirdly, IL-6 had many promotor SNPs such as -597G/A, −572G/C, −373A n T n and -174G/C, these polymorphic sites did not regulate the gene function independently [42]. And the influence of linkage disequilibrium could not be underestimated.
It's noteworthy that C allele was associated with poor outcomes of sepsis in non-adults in recessive model, codominant model (CC vs. GG) and allelic model. However, TSA revealed that this result was not reliable as Z-curve crossed neither the conventional test boundary nor the futility boundary. Although many studies demonstrated C allele led to a lower IL-6 expression, a study on neonates showed serum IL-6 level was higher in C carriers [43]. It was also found that IL-6 has an age-associated increase during systemic inflammation so that young persons should have a comparatively low IL-6 level during sepsis [44]. We hypothesized that higher IL-6 level induced by C allele could be more sensitive in very young persons and lead to aggravation of sepsis. This suggested different pathogenesis between non-adults and adults. It's acknowledged that pediatric sepsis had different definitions, clinical presentations and management from adult sepsis. Maternal status also had an impact on newborns [45]. Thus the effect of IL-6-174 G/C polymorphism on the inflammatory Most included studies in our analysis were concerned with the Caucasians, rather than the Asians. Previous analysis also present that the frequency of IL-6 -174G/C polymorphism were rare in Asian population that resided in Korea, Japan and Shandong province and Guangxi province of China [23,24,46,47]. However, two studies included in our analysis reported possible association between IL-6 -174G/C polymorphism and the risk of sepsis in the Chinese Han population in Henan province [11,14]. This suggested that IL-6 -174G/C polymorphism was distributed in Asians that resided in certain areas and might have an influence on the pathogenesis of sepsis.
As the types of controls were varied, it was considered that the status of controls could influence the results. In some studies, the case group was not comprised of septic patients only. Being compared to healthy controls and to mendelian population, the association with the risk of sepsis was not found, nevertheless. The case group of one study included a part of patients diagnosed as systemic inflammatory response syndrome (SIRS) or multiple organ disorder syndrome (MODS) [32]. And control group of one study consisted of ICU patients with no or mild sepsis [17] Because no exact number of the patients with or without sepsis was available, total group was included in the analysis. However, the results remained unchanged after omitting these studies in sensitivity analysis. In subgroup analysis of non-adults on the risk of sepsis, most of studies discussed age groups defined as infant or neonate. Only two studies addressed children under the age of 19 [32,33]. After excluding these two studies including elder children, the results remained unchanged. In addition, one study that were included in the meta-analysis by Gao et al. was not included in ours, which was excluded due to limited information to obtain precise data and extreme small sample size of case group (4 patients with GG or GC genotype) [26]. And we have proven that the exclusion of this study did not affect the result of study.
Our study has two strengths. Firstly, to avoid our analysis to be biased from publication, database, inclusion criteria and language, strict criteria were laid out for enrolling studies and publications in the Chinese language were also searched. Publication bias was tested in Egger's tests. Secondly, TSA was adopted to test the reliability of results and provide the research field which required more studies.
There were several limitations in this study. First, as the definition for sepsis changed, it was unlikely that the enrolled studies would have same definition because our analysis included studies within a large time range. Second, the majority of the objects studied were Caucasians, more studies were needed to further the study on other ethnicity groups. Third, three studies were not under Hardy-Weinberg equilibrium, although the results were not changed after omitting those studies. In addition, we could not get some details of the objects such as treatment and lifestyle, which prevented us from seeking more factors influencing the results.

Conclusions
In conclusion, we did not find the evidence of the association between IL-6-174 G/C polymorphism and the risk or mortality of sepsis. Trial sequential analysis indicated that large sample size was needed to get a more reliable result for the association between IL-6-174 G/C polymorphism and the risk and mortality of sepsis in non-adults. More studies on the molecular mechanism and interactions between SNPs were needed.

Additional files
Additional file 1: Supplementary tables. Table S1. Results of quality assessment of included studies using Newcastle-Ottawa Scale. Table S2. Summary of the studies with changed results after sensitivity analysis. Table S3. Results of meta-analysis on non-adults after excluding two studies discussing elder children. Table S4. Results of meta-analysis after including the study by Reiman.