IL-18 gene polymorphisms were associated with risk of chronic obstructive pulmonary disease in a Chinese Han population

Chronic obstructive pulmonary disease (COPD) is a kind of lung disease with high morbidity and mortality. Genetic polymorphisms of IL18 have been associated with respiratory system disease such as asthma, pulmonary tuberculosis, and lung cancer; however, little information is found about the association between IL18 polymorphisms and risk of COPD. We investigated the association between single nucleotide polymorphisms (SNPs) in IL18 and COPD risk in a case–control study that included 300 COPD cases and 300 healthy controls. Five SNPs were selected and genotyped using the Sequenom MassARRAY platform. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using unconditional logistic regression after adjusting for gender and age. In the genotype model analysis, we determined that rs2043055 polymorphism had an increased effect on the risk of COPD (GG versus AA: OR = 5.29; 95% CI = 1.15–24.35; p = 0.006). In the genetic model analysis, we identified four SNPs associated with COPD risk under recessive model. The “GG” genotype of rs2043055 and rs187238 were associated with increased risk of COPD (rs2043055: OR = 5.13, 95% CI = 1.12-23.49, p = 0.021; rs187238: OR = 4.99, 95% CI = 1.08–23.06, p = 0.025). Additionally, the “CC” genotype of rs1946519 was associated with increased risk of COPD (OR = 2.31; 95% CI = 1.03–5.19; p = 0.038). By contrast, the “TT” genotype of rs1946518 was associated with decreased risk of COPD (OR = 0.58; 95% CI = 0.35–0.98; p = 0.039). Our data shed new light on the association between IL18 polymorphisms and risk of COPD in a Chinese Han population. www.impactjournals.com/oncotarget/ Vol.9, (No.1), Supplement 1, pp: s109-s116


INTRODUCTION
In recent years, with the developing of industry and worsening of air condition, more and more people suffer from chronic respiratory disease.Chronic obstructive pulmonary disease (COPD) is a type of chronic respiratory disease with high morbidity and mortality.In clinical medicine, COPD is distinguished by chronic airflow limitation, which mainly caused by local inflammation of the respiratory system and systemic inflammatory [1].Epidemiologic studies pointed out that smoking is an important environmental risk factor for COPD, whereas only a fraction of smokers eventually developed to COPD [2].COPD is a complicated disease which influenced by multiple genes and interaction with environmental factors [3].Previous literatures have identified several susceptibility genes contribute to the development of COPD, including SCGB1A1, CHRNA5, VEGF-A, FAM13A, SETD7 and so on [4][5][6].However, this is not enough to explain the hereditary susceptibility of COPD.Researchers still concentrate on work on identifying more susceptibility genes for this disease.

Research Paper
Interleukin-18 , also known as interferon (IFN)-γ inducing factor, belongs to the IL-1 cytokine superfamily [7].IL-18 is a pro-inflammatory cytokine which could modulates the Th1/Th2 response together with other factors [8], and further activates innate immunity and inflammatory response in human body [9].Previous studies have identified a lot of single nucleotide polymorphism (SNPs) in IL18 gene associated with several disease.For example, rs187238 and rs1946518, in the promoter of the IL18, have close correlation with a wide range of disease in different populations, including multiple sclerosis, Crohn's disease, pulmonary tuberculosis, asthma, hepatitis B virus-related cirrhosis, breast, lung and liver cancer [10][11][12][13][14][15][16].To date, genetic polymorphisms of IL18 have been associated with respiratory system disease such as asthma, pulmonary tuberculosis, and lung cancer; however, the correlation between IL18 polymorphisms and risk of COPD is still unclear.
In this study, we selected five SNPs in IL18: rs2043055, rs187238, rs1946518, rs1946519 and rs5744224, and investigated their association with COPD risk in a Chinese Han population.

RESULTS
A total of 300 COPD patients and 300 healthy controls were recruited in the study.The distribution of gender, age and smoking status of the patient and control groups are described in Table 1 The basic characteristics of patient and control groups are well matched (p > 0.05).
All SNP call rates exceeded 98.0%, which was considered high enough to perform association analyses.The basic information of the IL18 polymorphisms (rs2043055, rs187238, rs1946518, rs1946519 and rs5744224) are listed in Table 2, including gene, band, position, alleles and minor allele frequency (MAF).All SNPs accord with Hardy-Weinberg equilibrium (HWE) in the controls (p > 0.05).No significant differences were observed in the MAFs of SNPs between COPD patients and healthy controls.
The genotypes frequencies of the IL18SNPs and their associations with risk of COPD are shown in Table 3. Notably, for rs2043055, compared with the AA genotype, the frequency of GG genotype was significantly different between cases and controls (GG versus AA: OR = 5.29; 95% CI = 1.15-24.35;p = 0.006), which suggested that the GG genotype of rs2043055 may be a risk genotype for development of COPD.
Finally, the relationship of IL18 haplotypes with the risk of developing COPD was also evaluated.Figure 1 showed the linkage disequilibrium (LD) block in IL18 constructed by rs2043055, rs187238, rs1946518, rs1946519 and rs5744224 in chromosome 11.The association analysis results between different haplotypes and COPD risk was shown in Table 5.However, no haplotype was observed significantly associated with COPD risk after the adjustment.

DISCUSSION
Previous studies have identified several SNPs associated with COPD; however , the results is still not enough to explain the heredity of COPD.In the present study, we found that the "GG" genotypes of rs2043055 and rs187238, and the "CC" genotype of rs1946519 are significantly associated with increased risk of COPD.Additionally, the "TT" genotype of rs1946518 was associated with decreased risk of COPD.These results shed new light on the genetic predisposition for COPD.
IL-18, a member of the pro-inflammatory cytokine superfamily, is a crucial mediator in immunoreaction in human body.Several SNPs in the promoter region of IL18 have been identified associated with the expression of IL-18.Single nucleotide changes will cause the change of transcription factor binding site [17].For example, rs187238 (-137C>G) and rs1946518 (-607C>A) disrupt the H4TF-1 nuclear factor and cAMP-responsive element binding protein binding sites, respectively [18].The breakdown of transcription factor binding sites will further cause the abnormal immune status in human body.We demonstrated that genetic polymorphisms of IL18 were associated with risk of COPD, which may also due to the abnormal immune status in COPD patients.Based on the above explanation, we speculated that the SNPs in IL18 may cause the abnormal immune status, and further related to the underlying pathogenesis of COPD.
A total of five SNPs were investigated in this study, including rs2043055, rs187238, rs1946518, rs1946519 and rs5744224.Among these SNPs, rs2043055 was found to be associated with insulin resistance [19], and risk of chronic chagas disease [20], ischemic stroke [21] and tuberculosis [22].We for the first time reported that rs2043055 was associated with COPD risk, which need to be confirmed in further study with a larger sample size.Rs187238 and rs1946518 were extensively studied, and identified to have association with several type of disease in different populations, including multiple sclerosis [10], alopecia areata [23], hepatocellular carcinoma [24], chronic hepatitis and cirrhosis [25], recurrent miscarriage [26], coronary artery disease [27], type I diabetes [28], tuberculosis [11] and asthma [13].It is noteworthy that the C allele of rs1946518 has been found associated with 1.48-fold increased risk of COPD in male smokers [29].We found the "CC" genotype of rs1946519 was associated with 2.31-fold increased risk of COPD, which is consistent with previous results.Little information is found about rs1946519 and rs5744224, these two SNPs were only found to be associated with risk of cervical cancer in Chinese literature.In our study, we found the "TT" genotype of rs1946518 was associated with decreased risk of COPD, which suggested this SNP is an important susceptibility locus for disease and need to be confirmed in further studies.
Some limitations should to be considered in our study.First, all the samples were recruited from a same hospital, which may not represent the common population.Second, COPD is a complex genetic disease and influenced by a range of genes.In addition to the five SNPs we investigated, other SNPs may also influence the development of COPD.Therefore, the results identified here need to be further confirmed in a large sample size and different populations.
In sum, the current data showed that IL18 polymorphisms have close correlation with COPD risk in a Chinese Han population.Further studies will focus  on the verification of the association in other populations, and the functional role of these SNPs in the development of COPD.

Study participants
For the current analysis, we established a casecontrol study of 300 COPD patients and 300 healthy controls.The diagnosis of COPD was confirmed according to the criteria established by the National Heart, Lung and Blood Institute/World Health Organization Global Initiative for Chronic Obstructive Lung Disease (GOLD) [1].The entry criteria for COPD cases were post-bronchodilator forced expiratory volume in 1 second (FEV1) less than 80% predicted and FEV1/forced vital capacity less than 70%.The control group was randomly selected healthy individuals, which included current or ex-smoker with no known disease, no history of any lung disease, and no airflow limitation.All participants in our study were recruited between September 2013 and September 2016 at Xi'an Hospital of Traditional Chinese Medicine, People's Republic of China.
All of the participants provided written informed consent.The Human Research Committee for Approval of Research Involving Human Subjects, Xi'an Hospital of Traditional Chinese Medicine, approved the use of human blood samples in this study.

SNP selection and genotyping
In this study, five SNPs in IL18 were selected from previous study for analysis [18,19,30].The lower frequency alleles were coded as the minor allele.All of the SNPs had minor allele frequencies (MAFs) >5% in the HapMap Chinese Han Beijing population.Genomic DNA was isolated from whole blood samples using the GoldMag-Mini Purification Kit (GoldMagCo.Ltd.Xi'an, China), and DNA concentrations were measured using the NanoDrop2000 (Thermo Scientific, Waltham, Massachusetts, USA).Sequenom Massarray Assay Design 3.0 softwarewas used to design a multiplexed SNP Mass EXTENDED assay [31][32][33].Genotyping was performed

Table 1 : Characteristics of cases and controls in this study
a p value was calculated from Pearson's chi-square tests.b p value was calculated by Welch's t-tests.

Table 2 : Allele frequencies in cases and controls and odds ratio estimates for COPD
SNP: single nucleotide polymorphism, Alleles A/B: Minor/major alleles; MAF, minor allele frequency; OR: odds ratio, CI: confidence interval, HWE: Hardy-Weinberg equilibrium P values were calculated using two-sided Chi-squared test and adjusted by gender, age and smoking status * p ≤ 0.05 indicates statistical significance

Table 3 : Genotypes frequencies of the SNPs and their associations with risk of COPD
SNP: Single nucleotide polymorphism; OR: odds ratio; 95%CI: 95% confidence interval.P values were calculated by unconditional logistic regression analysis with adjustments for age and gender.* p ≤ 0.05 indicates statistical significance.

Table 4 : Association between SNPs and risk of COPD in multiple inheritance models (adjusted by gender, age and smoking status)
ORs, odds ratios; CI: confidence interval; AIC: Akaike's Information criterion; BIC: Bayesian Information criterion.* p value ≤ 0.05 indicates statistical significance.

Table 5 : Haplotype frequencies of SNPs in the IL18 gene and the association with COPD risk in case and control subjects
values were calculated from two-sided Chi-squared test/Fisher's exact test, and adjusted by gender, age and smoking status; * p ≤ 0.05 indicates statistical significance.