IL1 Receptor Antagonist Gene IL1-RN Variable Number of Tandem Repeats Polymorphism and Cancer Risk: A Literature Review and Meta-Analysis

IL1 receptor antagonist (IL1RA) and IL1beta (IL1β), members of the pro-inflammatory cytokine interleukin-1 (IL1) family, play a potential role against infection and in the pathogenesis of cancers. The variable number of tandem repeats (VNTR) polymorphism in the second intron of the IL1 receptor antagonist gene (IL1-RN) and a polymorphism in exon 5 of IL1B (IL1B+3954C>T, rs1143634) have been suggested in predisposition to cancer risk. However, studies have shown inconsistent results. To validate any association, a meta-analysis was performed with 14,854 cases and 19,337 controls from 71 published case–control studies for IL1-RN VNTR and 33 eligible studies contained 7,847 cases and 8917 controls for IL1B +3954. Odds ratios (ORs) with 95% confidence intervals (CIs) were calculated from comparisons to assess the strength of the association. There was significant association between the IL1-RN VNTR polymorphism and the risk of cancer for any overall comparison. Furthermore, cancer type stratification analysis revealed that there were significantly increased risks of gastric cancer, bladder cancer and other cancer groups. Infection status analysis indicated that the H. pylori or HBV/HCV infection and IL1-RN VNTR genotypes were independent factors for developing gastric or hepatocellular cancers. In addition, a borderline significant association was observed between IL1B+3954 polymorphism and the increased cancer risk. Although some modest bias could not be eliminated, this meta-analysis suggested that the IL1-RN VNTR polymorphisms may contribute to genetic susceptibility to gastric cancer. More studies are needed to further evaluate the role of the IL1B+3954 polymorphism in the etiology of cancer.


Introduction
Interleukins (ILs) are pro-inflammatory cytokines produced by monocytes, macrophages and epithelial cells. The interleukin-1 (IL1) family consists of the cytokines IL1alpha (IL1a), IL1beta (IL1b) and a specific receptor antagonist (IL1RA) [1]. The genes encoding this family are mapped on chromosome 2q14 [2] and include three related genes, IL1A, IL1B and IL1-RN, which encode IL1a, IL1b and IL1RA respectively. IL1B may play an essential role in the carcinogenic process. Several IL1B gene single nucleotide polymorphisms have been described to be associated with cancers to date [3]. Previously, we have reported the polymorphism in the promoter of IL1B(IL-1B -31T.C, rs1143627) was a low-penetrance protective factor for the development of cancer [4]. In addition, a polymorphism in exon 5 of IL1B(IL1B +3954C.T, rs1143634) has been found to affect IL1B gene expression [5] and to be associated with occurrence of cancers [6,7]. IL1RA competitively binds to IL1 receptors with IL1a and IL1b. Naturally occurring anti-inflammatory IL1RA is a 16 to 18 kD protein that competes with the binding of IL1 to its receptor. In the second intron of the IL1-RN gene, there exists a variable number of tandem repeats (VNTR) with an 86 base pair nucleotide sequence as its repeating element. This region contains three potential protein binding sites: an interferon a silencer A, an interferon b silencer B and an acute phase response element, and thus may be of functional relevance [8]. It has been reported that these three binding sites affect the control of cell proliferation activity, resulting in potential regulation of IL1RA production [5,8]. El-Omar et al. [9] first reported positive association between gastric cancer risk and these VNTR polymorphisms in IL1-RN. Another study revealed that IL1-RN VNTR genotypes were highly correlated with cellular function. The IL-1RN alleles were coded as previously described: allele 1, four repeats; allele 2, two repeats; allele 3, five repeats; allele 4, three repeats and allele 5, six repeats. The IL-1RN alleles were further divided into two categories: long genotype (L: including alleles 1, 3, 4, and 5) and short genotype (2: allele 2 only). The genotypes were classified as LL, 2L, and 22 [10].
A large number of genetic susceptibility studies have been carried out to investigate the associations between the IL1B+3954 and IL1-RN polymorphism and cancers of different tissue origins, but inconsistent results have been obtained. These inconsistent findings might be caused by a range of factors such as heterogeneity by cancer subtype, limited sample size, and geneenvironment interactions. To further assess and validate the association of the IL1B +3954 and IL1-RN VNTR polymorphism with cancer risk, a comprehensive review and analysis of published data from different study groups is urgently needed. In this study, we have extensively reviewed literature and performed a metaanalysis based on all eligible case-control published data to evaluate the association between the IL1B+3954 and IL1-RN VNTR polymorphism and cancer susceptibility.

Identification of Eligible Studies
We searched the PubMed and Embase databases for all relevant reports (updated to May 23, 2012) by using the key words 'interleukin-1 receptor antagonist', 'IL1', 'polymorphism', or 'cancer'. The search was limited to English language papers and human subject studies. In addition, studies were identified by a manual search of the reference lists of reviews and retrieved studies. When more than one of the same or overlapping populations by different investigators or overlapping data by the same authors were found, only the most recent or complete study was used for this meta-analysis. Studies, regardless of sample size, were included if they met the following criteria: (i) the study was of the IL1B+3954 and IL1-RN VNTR polymorphism and cancer risk, (ii) it was a case-control designed study and (iii) it contained available genotype frequencies ( Figure S1).

Data Extraction
Two individuals (Ying Zhang and Changming Liu) extracted all data independently complying with the selection criteria. For each study, the following characteristics were extracted: the first author's last name, year of publication, country of origin, ethnicity, matching numbers of genotyped cases and controls, the source of control groups (population-or hospital-based controls), genotyping methods and cancer type. Different ethnic descents were categorized as Caucasian, Asian or mixed, which included more than one ethnic descent. One study included the information for genotypes of LL/2L and 22, without data for LL and 2L genotypes, so we were only able to calculate the OR for the comparison between alleles 2 and L [11]. In addition, the infection status of cases and controls in the enrolled studies were also extracted for the IL1-RN VNTR subgroup analysis.

Statistical Analysis
The IL1B+3954 and IL1-RN VNTR polymorphisms were tested for associations with cancer susceptibility based on different genetic models. The meta-analysis examined the overall association of the IL1B+3954 and IL1-RN VNTR polymorphism with the risk of cancer measured by odds ratios (ORs) with 95% confidence intervals (CIs). To contrast the wild-type homozygote (IL1B+3954-CC or IL1-RN-L/L), we first estimated the risks of the IL1B+3954 -CT, -TT and IL1-RN -2/L, -2/2 genotypes on cancers, then evaluated the risk of cancer under a dominant model(IL1B+3954:T/T+C/T versus CC; IL1-RN:2/2+2/L versus L/ L). In addition, recessive model associations were also estimated (IL1B+3954: T/T versus C/C+C/T; IL1-RN: 2/2 versus L/L +2/ L). Stratified analyses were also performed by ethnicity (Asian, Caucasian and Mix), cancer types (if only one cancer type contained fewer than three individual studies it was combined into the 'Other Cancers' group), moreover, subgroup analysis was carried by infection status for IL1-RN VNTR polymorphism.
The statistical significance of the pooled OR was determined with the Z test, a P value of,0.05 was considered significant. The heterogeneity between studies was evaluated by the chi-square based Q statistical test [12], heterogeneity was considered significant for P,0.05. A fixed-effect model using the Mantel-Haenszel method and a random-effects model using the DerSimonian and Laird method were used to pool the results [13]. The random-effects model was used when there was heterogeneity in the results of the studies; otherwise, the fixedeffect model was used. Sensitivity analyses were performed to assess the stability of the results, namely, a single study in the metaanalysis was deleted each time to reflect the influence of the individual data set to the pooled OR. To test for publication bias, Funnel plots and Egger's linear regression test were applied [14].
All statistical tests for this meta-analysis were performed with STATA version 10.0 (Stata Corporation College Station, TX), and SAS (version 9.1; SAS Institute, Cary, NC).

Characteristics of the Studies
For IL1-RN VNTR polymorphism, a total of 71 eligible studies met the preset inclusion criteria, in which 14,854 cases and 19,337 controls were included for the pooled analysis (Table S1). All studies were case-control studies, including 37 studies on gastric cancer, 6 on hepatocellular cancer, 4 on cervical cancer, 4 on breast cancer, 4 on lung cancer, and 16 on those categorized into other cancers. There were 40 studies of Asian descendents, 29 of Caucasian descendents and two with mixed ethnicity [15,16]. Cancers were diagnosed histologically or pathologically in most studies. To analyze the VNTR in the second intron of IL1-RN, polymerase chain reaction (PCR) was performed in most studies, direct sequencing was used by two studies [17,18], and the denaturing high performance liquid chromatography (DHPLC) was applied in one study [19]. In addition, most of the controls were sex-and age-matched for the case groups, of which 49 were population based and 22 were hospital based. In addition, 20 studies investigated the interactions between the polymorphism and helicobacter pylori (H. pylori), hepatitis B virus (HBV) or hepatitis C virus (HCV) infection status.
For IL1B+3954 polymorphism, 33 eligible studies contained 7,847 cases and 8,917 controls were enrolled for the pooled risk analysis (Table S2). Among the 33 studies, 14 studies discussed the risk of gastric cancer, 3 studies discussed the risk of oral cancer, 3 studies focused the risk of lung cancer, and 13 studies categorized in to other cancer. There were 14 studies of Asian descendents, 18 studies of Caucasian descendents, and one study with mixed ethnicity.

IL1-RN VNTR Polymorphism and H.pylori, HBV/HCV Infection Status
The genotype distribution of the IL1-RN VNTR genotype among cases and infection-matched controls was available in twenty studies that investigated gastric cancer infected by H. pylori and hepatocellular cancer infected by the hepatitis B or hepatitis C virus. Carriers of the 2 allele had higher cancer risk than those with the IL1-RN-L allele among gastric cancer studies, and the subgroup analysis results revealed that the L allele is a risk of gastric cancer independent of H.pylori infection. However, there was no association between the IL1-RN VNTR polymorphism and cancer risk in hepatocellular cancer studies as illustrated in Table 3.

Publication Bias
Begg's funnel plot and Egger's test were performed to assess the publication bias of the currently available literature. The shape of the funnel plots did not reveal any evidence for obvious asymmetry in all comparison models. Then, the Egger's test was used to provide statistical evidence for funnel plot symmetry. The results still did not show any evidence of publication bias (IL1-RN VNTR: t = 1.84, P = 0.071 for 2L versus LL, Figure 2; IL1B+3954: t = 0.82, P = 0.420 for CT versus CC, Figure 3).

Discussion
In this meta-analysis, the VNTR polymorphism in intron 2 of IL1-RN was associated with increased risk for developing cancers of interest. Results from stratified analyses by cancer type indicated that the 2L genotype was associated with the increased risk of gastric cancer, and with increased cancer in Asian populations. Infection status analysis indicated that the 2 allele was a risk allele for gastric cancer independent of H. pylori status, but not hepatocellular cancer with HBV or HCV infection. For IL1B+3954 polymorphism borderline significant risk association was observed for cancer risk.
In the present study, this meta-analysis indicated a significant association of the IL1-RN VNTR polymorphism with gastric cancer, but not with breast cancer, hepatocellular cancers, cervical cancer or lung cancer. Our results suggested that the IL1-RN VNTR polymorphism may play different roles in the pathogenesis of different cancer types. Our findings are supported by evidence that the immune response is essential for gastric cancer development. It has been shown the 86 bp VNTR polymorphism of the IL1-RN has potential roles in regulating the immune response. The IL1-RN-2 allele had a high circulating IL1RA level and an even more elevated IL1b level [34]. Binding of IL1RA to the IL1 receptor inhibited IL1mediated signaling, resulting in a strengthened and prolonged inflammatory response [35]. Moreover, El-Omar et al. found that the gastric cancer risk genotypes IL1-RN-2/2 were associated with decreased acid-secretion capacity [5]. Xue et al. reported IL1-RN -2 allele was associated with an increased risk of developing gastric carcinoma and even more significantly with noncardia gastric carcinoma or with intestinal-type gastric carcinoma [36], indicated IL1-RN VNTR polymorphism associated with different cancer risk according to the subtypes of gastric cancer and histological types. Furthermore, Wang et al. [37] has also revealed that IL1-RN -2 allele was associated with an increased risk of gastric cancer among Caucasians. Interestingly, in the cancer subtype analysis, we observed that the carriage of the 2 allele was found to act as a protective factor for breast cancer risk (Table 1). Our findings were not in Table 2. Stratification analyses of genetic susceptibility of IL1B+3954 polymorphism to cancer risk.  accordance with some previous reports, which indicated inammatory response might not be the major factor for the pathology of breast cancer [38,39,40]. However, other studies have suggested inflammatory response was closely related to breast cancer. It has been shown that IL1b may combine with the estrogen receptor-alpha, resulting in the enhancement of transcriptional activation in breast cancer cells [41]. Furthermore, it has been regarded that high levels of IL1b in peripheral blood is known to increase the level of leptin [42], which correlates with total body fat and stimulates the growth of mammary epithelium [43,44,45]. It is commonly known that H. pylori plays important roles in the pathology of gastric cancer [46]. Similarly, HCV and HBV are involved in the pathology of hepatocellular cancer [47]. It is important to perform the GxE analysis to evaluate the IL1-RN VNTR polymorphism effects for gastric cancer and hepatocellular cancer. Our results revealed that there was no interactions between the IL1-RN VNTR polymorphism and H.pylori infection in gastric cancer, suggesting that the risks for developing gastric carcinoma conferred by the H.pylori and IL1-RN VNTR genotypes may be independent. This may be due to different pathogenesis mechanisms underlying H.pylori-negative gastric cancer in contrast to H.pylori-positive gastric cancer [48]. The similar results were also reported by Wang et al. [37].
IL1b is well known to be a pro-inammatory factor and mediate several immune responses in HCV infection [49]. Several independent studies provided evidence of associations between chronic hepatocellular cancer and chronic HCV or HBV infection [50]. However, our results from HBV/HCV infection matched studies that have shown negative interaction between the IL1-RN VNTR polymorphism and HBV/HCV infection in predisposition to hepatocellular cancers. More studies are needed to further evaluate the role of this polymorphism in virus-infected hepatocellular cancers.
IL1B+3954 polymorphism is a coding synonymous variant located in exon 5 of IL1B. The transition from cytosine to thymine does not change amino acid coding but may lead to an inactivation of the original splicing donor site. The alternative splicing results in a premature stop codon or exon skipping and produces a truncated protein that is likely to be rapidly degraded or functionally inactive [51]. IL1B+3954 polymorphism has been reported to influence the production of IL-1b protein [5,51].
Meta-analysis results revealed IL1B+3954 had a borderline significant association with increased cancer risk. The pooled results were affected by individual studies, therefore, more studies should be cumulated to evaluate the role of the IL1B+3954 polymorphism in the etiology of cancer.
Although meta-analyses are robust, our study still has some limitations. First, most of the selected studies focused on gastric cancer. The relatively small sample size of studies in other stratified groups may lead to reduced statistical power. Second, although all eligible studies were summarized, the total sample size might have not been enough to make a convincing conclusion. When stratified analysis of tumor type, ethnicity or infection status was performed, the number of each subgroup was smaller. Third, our meta-analysis included no evaluation of potential gene-gene interactions and limited gene-environment interactions due to the lack of relevant published data. Because cancer is a complex disease, genetic factors may play a limited impact on the pathogenesis of cancers. In the results of our present metaanalysis, the IL1-RN VNTR had only moderate genetic susceptibility effects (ORs less than 2). More studies on large-scale genegene and gene-environment interactions are needed to further evaluate and elucidate the human genetic predisposition to various cancers.
In conclusion, this meta-analysis suggests that the IL1-RN VNTR polymorphism may contribute to genetic susceptibility to gastric cancer, which is closely related to the immunological response. IL1B+3954 has a borderline significant association with increased cancer risk. More studies are needed to further evaluate the role of the IL1B+3954 polymorphism in the etiology of cancer.