Association between the IL1B, IL1RN polymorphisms and COPD risk: A meta-analysis

The interleukin-1 (IL-1) gene polymorphisms have been implicated in chronic obstructive pulmonary disease (COPD) risk, but results are controversial. We aimed to conduct a meta-analysis to address this issue. Odds ratio (OR) and 95% confidence interval (CI) were used to investigate the strength of the association. The meta-analysis revealed no association between the IL1B (−511), (−31), (+3954) polymorphisms and COPD risk. However, stratification by ethnicity indicated that the T allele carriers of the IL1B (−511) polymorphism and the C allele carriers of the IL1B (−31) variant were associated with an increased risk for developing COPD in East Asians (OR = 1.61, 95% CI: 1.13–2.31, Pz = 0.009 and OR = 1.55, 95% CI: 1.14–2.11, Pz = 0.006, respectively). The meta-analysis revealed a significant association between the IL1RN (VNTR) polymorphism and COPD risk in all study subjects and East Asians under homozygote model (22 vs. LL: OR = 3.16, 95% CI: 1.23–8.13, Pz = 0.017 and OR = 3.20, 95% CI: 1.13–9.12, Pz = 0.029, respectively). Our meta-analysis suggests that the IL1B (−511), (−31) and IL1RN (VNTR) polymorphisms are associated with COPD risk in East Asians. There is no association between the IL1B (+3954) polymorphism and COPD risk. Further studies should be performed in other ethnic groups besides East Asians.

C hronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease characterized by the gradual progression of irreversible airflow obstruction and increased inflammation in the airways and lung parenchyma 1 . It is a leading cause of chronic morbidity and mortality worldwide. Although cigarette smoking has been showed to be a major environmental risk factor for COPD, only 10-20% of smokers develop COPD, implying that apart from environmental features, additional risk factors such as genetic variation contributes to COPD susceptibility 2 . Genetic linkage studies, candidate gene association studies and genome-wide association studies (GWAS) have identified genes that may have roles in the pathogenesis of COPD, including microsomal epoxide hydrolase (EPHX1), glutathione S-transferase (GST), interleukin-6 (IL-6), iron-responsive element binding protein 2 (IREB2), matrix metallopeptidase 9 (MMP9) and transforming growth factor-b (TGFb) 3,4 . A better understanding of the genetic architecture of the disease will be important in helping to unravel the pathogenetic mechanism and develop novel therapeutic strategies for COPD.
IL-1 is a pro-inflammatory cytokine and key contributor to immune responses. IL-1 occurs in two forms, IL-1A and IL-1B, both of which bind to the IL-1 receptor 5 . The IL-1 receptor antagonist (IL-1RA) does not transmit any signal and functions as a cell bound inhibitor to both IL-1A and IL-1B 5 . The loci for human IL1A, IL1B, and IL1RN gene are found as a cluster on chromosome 2q12 to 2q14 6 . The genes are arranged with IL1A situated 5-prime, and then IL1B and finally IL1RN 3-prime. Several common polymorphisms within the IL-1 gene complex have been described, including single nucleotide polymorphisms (SNPs) in IL1B at position 2511 (rs16944) and 231 (rs1143627) in the promoter region and at position 13954 (rs1143634) in exon 5, and a penta-allelic polymorphism representing a variable number of an 86-bp tandem repeat in intron 2 (rs2234663) of the IL1RN gene 7,8 . Many genetic association studies have been conducted to investigate the relationship of these variants with COPD risk; however, small sample size and varying population characteristics led to conflicting results. To validate the potential association between the IL1B, IL1RN polymorphisms and COPD risk, we performed a meta-analysis of data reported in 12 case-control studies from 11 publications.

Methods
Search strategy and study identification. We conducted electronic searches, not limited to the English language, of PubMed, Embase, Scopus, Cochrane database, China National Knowledge Infrastructure (CNKI) and Wanfang databases to identify relevant studies reporting on the association between the IL1B, IL1RN polymorphisms and COPD risk in the medical literature from January 1990 up to the end of June 2014. Search terms included ''chronic obstructive pulmonary disease'', ''COPD'', ''interleukin-1'', ''IL-1'', ''polymorphism'', ''genetics'', and ''association''. References from retrieved papers were also considered. Association studies were included in this meta-analysis if they met the following criteria: 1) studies on human subjects; 2) studies written in English or Chinese; 3) case-control design; 4) sufficient data for examining an odds ratio (OR) with 95% confidence interval (CI). The major exclusion criteria were as follows: 1) studies on animal populations; 2) no usable data reported; 3) duplicate data; 4) studies presenting deviation from Hardy-Weinberg equilibrium (HWE) in the control group. All relevant publications identified through the search were scanned on the basis of title, keywords and abstract, and were rejected in the initial screening if the paper clearly did not meet the inclusion criteria. Where a title/abstract could not be rejected with certainty, the full text of the publications was obtained for evaluation.
Data extraction. Two independent investigators extracted data from the published studies and entered them in a customized database. Disagreement was resolved by the evaluation of a third reviewer and discussion until a consensus was reached. For each study, the following data were collected: first author's name, country, year of publication, ethnicity, total number of cases and controls, characteristics of the control group, and genotypic frequencies of the IL-1 polymorphisms.
Quality score assessment. The quality of selected studies was evaluated by scoring according to Newcastle Ottawa Scale (NOS) (www.ohri. ca/programs/ clinical_epidemiology/oxford.asp). This scale consists of three parts relating to selection, comparability and ascertainment of exposure. Quality scores ranged from 0 to 9 and studies were regarded as ''high quality'' if the score was $ 5. Otherwise, studies was regarded as ''low quality''.
Data analysis. Data were entered and analyzed with the Stata version 11.0. We used raw data of genotypic distribution, without adjustment for calculation of the studyspecific estimates of OR and 95% CI. Dominant, recessive and homozygote models were evaluated. Z-test was used for assessing the significance of the pooled OR, with P,0.05 considered statistically significant. Between-study heterogeneity was evaluated with Cochran's Q-test statistics among the studies, with significance level set at 0.10. The DerSimonian Laird random-effects model rather than the Mantel-Haenszel fixed-effect model was employed to calculate the pooled OR and 95% CI 9,10 . For the IL1RN (VNTR) polymorphism, L signifies any long allele embracing allele 1, 3, 4, or 5. We created a funnel plot to assess potential publication bias by plotting natural logarithm of individual study effect size against the standard error of the natural logarithm of individual study effect size. We also assessed publication bias using the Begg's test and Egger's test. The HWE law states that if two alleles, T and C, with frequencies p and q51-p, are in equilibrium in a population, then the proportion of people with genotypes TT, TC and CC will be p 2 , 2pq and q 2 . HWE in the control group for each study was tested by using a web-based programme (http://www.oege. org/software/hwe-mr-calc.html). Since we only utilized previously published data, we did not obtain approval of an ethics committee or written informed consent.

Results
Included studies. Figure 1 showed the process of identifying eligible studies. 487 publications were identified from the initial keywords search. After review, 474 were excluded. Overall, 12 studies from 11 publications met our inclusion criteria with a total of 1530 COPD patients and 1524 controls [11][12][13][14][15][16][17][18][19][20][21] . Table 1 summarized the characteristics of the studies included in the meta-analysis. In terms of ethnicity, eight studies including 1055 cases and 1199 controls were performed in East Asians 11,12,[14][15][16][17]19,20 , two studies containing 169 cases and 97 controls were undertaken in Arabians 12,21 , one study with 102 cases and 20 controls was conducted in Europeans 13 , and one study including 204 cases and 208 controls was performed in South Asians 18 . The publication of Hegab et al. contained an Egyptian study and a Japanese study, respectively 12 . It was noteworthy that we did not extract data for the IL1B (2511), (231) polymorphisms from the Liu et al. study 17 , since they further evaluated these two polymorphisms using larger sample numbers in another study 19 . In addition, the data for the IL1B (231) SNP from the Liu et al. study 19 and the data for the IL1RN (VNTR) polymorphism from the Hegab et al. study 12 , the Lee et al. study 16 , and the Shukla et al. study 18 presented deviations from HWE in controls, respectively, so we did not include them in the final analysis.
Individual polymorphism meta-analysis. Table 2 showed genotypic distribution of the four polymorphisms. The genotype-wise meta-analytic results for each of the four polymorphisms were showed in Table 3.
For the IL1B (13954) polymorphism, four studies from three publications with 406 cases and 359 controls were included in the meta-analysis 11,12,17 . Available data did not suggest an association between this polymorphism and COPD risk in all study subjects in dominant model (OR51. 16 (Table 3). In subgroup analysis according to ethnicity, we also did not find an association between this SNP and COPD risk in East Asians ( Table 3). Because of limited availability of published results, we were unable to perform subgroup analysis in Arabians, Europeans and South Asians, respectively. Between-study heterogeneity for the genotype-wise ORs was not found (Table 3).
Publication bias. Since publication bias was hard to detect when the number of studies were small, we selected the IL1B (2511) polymorphism to assess publication bias (nine studies included). The shape of the funnel plot seemed symmetrical (Fig. 5). Both Begg's test and Egger's test suggested no evidence of publication bias (P51.000 and P50.865, respectively).

Discussion
The IL1B and IL1RN polymorphisms have been implicated in the pathogenesis of COPD and have been investigated in numerous casecontrol association studies. However, the results are controversial, possibly because single studies may have been underpowered. Given the amount of accumulated data and the still equivocal role of the IL-1 gene polymorphisms in the etiology of COPD, we performed the present meta-analysis of 12 case-control studies for a total of 1530 COPD patients and 1524 controls to investigate the association between the four most studied IL1B and IL1RN polymorphisms and COPD. The main findings of the present meta-analysis are: (1) the T allele carriers of the IL1B (2511) polymorphism are associated with an increased risk for developing COPD in East Asians; (2) the C allele carriers of the IL1B (231) polymorphism are associated with an increased risk for COPD in East Asians; (3) compared with the LL homozygotes, the 22 homozygotes of the IL1RN (VNTR) polymorphism are associated with an increased risk for developing COPD in East Asians; and (4) there is no significant association between the IL1B (13954) polymorphism and COPD risk. IL-1B is a pro-inflammatory cytokine which is synthesized by a variety of cell types, including blood monocytes and tissue macrophages. IL-1B is thought to be an important mediator in cigarette smoke-induced inflammation and COPD. In BALB/c mice, IL-1B production was significantly up-regulated in lung homogenates after smoke exposure 22 . In human subjects, elevated IL-1B levels were observed among smokers 23 and in primary explant cultures of bronchial epithelial cells derived from COPD patients 24 . IL-1B expression in COPD neutrophils was correlated with disease severity as measured by forced expiratory volume in 1 second (FEV1) 25 . In addition, sputum IL-1B was showed to be a potential biomarker for bacteriaassociated exacerbations of COPD 26 . IL-1B plays a central role in the regulation of immune responses and inflammatory processes, including promotion of the movement of inflammatory cells from the blood to inflamed tissues, regulation of the extracellular matrix, induction of the expression of a variety of inflammatory mediators such as IL-6, IL-8 and tumor necrosis factor-a(TNF-a), and promotion of the differentiation of inflammatory cells 5 . Since IL-1B regulates a variety of inflammatory processes, any alteration in its blood or tissue concentration may significantly affect these processes. The IL1B (2511) T and (231) C alleles are associated with higher      IL-RA binds to the IL-1 receptor and acts as a competitive inhibitor of IL-1B. Allele 2 (two repeats) of the IL1RN (VNTR) polymorphism is associated with increased IL-1ra levels 29 . In addition, allele 2 strongly enhances in vitro production of IL-1B and plays a pivotal role in determining the balance between secreted IL-1B and IL-1RA 30 . The IL1-RA/IL-1B ratio is critical in determining the severity of inflammatory responses. The IL1RN allele 2 has been showed to be associated with a low IL1-RA/IL-1B ratio, thereby leading to a more prolonged and more severe pro-inflammatory immune response 31 .The frequency of this allele was found to be increased in diseases of autoimmune or inflammatory nature, including ankylosing spondylitis 32 , diabetic nephropathy 33 , and ulcerative colitis 34 . Our meta-analysis showed that the IL1RN allele 2 homozygotes had an increased risk for COPD, suggesting a significant pathogenetic effect of the IL1RN allele 2 in COPD.
There are several limitations to this meta-analysis. First, betweenstudy heterogeneity was found in some pooled analyses. We were unable to further identify the exact sources of heterogeneity by metaregression analysis in that there were limited relevant data provided by the included studies. Some potentially relevant factors, such as ethnicity, genotyping method, gender, phenotype of the disease may account for heterogeneity. We further performed subgroup analysis according to ethnicity, which greatly reduced heterogeneity. Second, we only included publications written in English or Chinese in the present meta-analysis, which may have missed potentially eligible studies published in other languages. Third, few included studies shown by the point estimate of the odds ratio, and a horizontal line denotes the 95% confidence interval. The pooled odds ratio is represented by a diamond. The area of the grey squares reflects the weight of the study in the meta-analysis.  reported the percentage of smokers in COPD patients and controls. Because of limited availability of published data, we were unable to evaluate the effect of smoking on the association between the IL1 gene polymorphisms and COPD. Fourth, there has been considerable strength of linkage disequilibrium (LD) in the IL-1 gene cluster. Feakes et al. found by family studies significant LD between the IL1B (2511) and IL1RN (VNTR) polymorphisms 35 . Joos et al. found that the IL1B (2511) T allele in combination of the IL1RN allele 2 was protective against a rapid decline in lung function in smokers 36 . These results suggest that haplotype analysis, as well as genotype analysis, may be necessary in evaluating genetic effects of the IL-1 gene cluster on COPD. We did not address this issue in our metaanalysis since few included studies performed haplotype analysis and there were discrepancies in constructing haplotypes among them 12,16,18 . Association studies may provide more insights for the role of the IL-1 gene in COPD if researchers can investigate haplotype association.
In summary, our meta-analysis showed that the IL1B (2511), (231) and IL1RN (VNTR) polymorphisms are associated with COPD risk in East Asians. There is no significant association between the IL1B (13954) polymorphism and COPD risk. Further studies with large sample size are needed to establish a more definitive conclusion. In addition, more studies should be performed in other ethnic groups besides East Asians.