Associations of IL-4, IL-4R, and IL-13 Gene Polymorphisms in Coal Workers' Pneumoconiosis in China: A Case-Control Study

Background The IL-4, IL-4 receptor (IL4R), and IL-13 genes are crucial immune factors and may influence the course of various diseases. In the present study, we investigated the association between the potential functional polymorphisms in IL-4, IL-4R, and IL-13 and coal workers' pneumoconiosis (CWP) risk in a Chinese population. Methods Six polymorphisms (C-590T in IL-4, Ile50Val, Ser478Pro, and Gln551Arg in IL-4R, C-1055T and Arg130Gln in IL-13) were genotyped and analyzed in a case-control study of 556 CWP and 541 control subjects. Results Our results revealed that the IL-4 CT/CC genotypes were associated with a significantly decreased risk of CWP (odds ratio (OR) = 0.74, 95% confidence interval (CI) = 0.58–0.95), compared with the TT genotype, particularly among subgroups of age <65 years (OR = 0.68, 95%CI = 0.46–0.99) and dust exposure years ≥26 years (OR = 0.69, 95%CI = 0.50–0.94). Moreover, the polymorphism was significantly associated with risk of CWP patients with stage I. In addition, a combined effect was observed in a dose-dependent manner with increasing numbers of risk variant alleles (P trend = 0.023), and individuals with 11–12 risk alleles had a 47% higher risk of CWP than those with 0–8 risk alleles (OR = 1.47, 95% CI = 1.05–2.05). Conclusions Our results suggest that the IL-4 C-590T polymorphism is involved in the etiology of CWP and susceptibility to this disease. Larger studies are warranted to validate our findings.


Introduction
Coal workers' pneumoconiosis (CWP) is a lung disease caused by the inhalation and deposition of occupational coal mine dust in the lungs. The incidence and rate of CWP progression is related to the amount of respirable coal dust or silica to which coal miners were exposed during their work [1]. In China, workers exposed to coal dust or silica have increasing morbidity and mortality annually [2]. Although the exact mechanisms leading to CWP are yet to be elucidated, current evidence suggests that CWP is characterized by chronic pulmonary inflammation and fibrotic nodular lesions that usually lead to progressive fibrosis [3]. CWP can be diagnosed among workers who have worked in poor workplace environments two to three decades ago even though their work environments have been improved [4]. Therefore, identification of new genetic factors for CWP would improve diagnosis of patients at risk and help determine effective prophylactic intervention.
The IL-4 and IL-13 cytokines share many structural and functional similarities, as well as a common receptor component, IL-4 receptor (IL4R), located on chromosome 16p11. IL-4 plays important roles in the differentiation of T cells, eosinophilic inflammation, and isotype switching in B cells from IgM to IgE [5]. It reduces the production of proinflammatory cytokines and destructive enzymes by monocytes [6]. IL-4 is also a key factor in the polarization of T helper cells toward Th2 differentiation [7]. IL-13 is a central immune regulator of many allergic characteristics, including IgE synthesis, mucus hypersecretion, airway hyperreactivity, and fibrosis [8], which is found to be overexpressed in the lungs of patients and in murine models [9]. IL-13 shares overlapping biological functions with IL-4, and mediates its effect through the IL-4R [10]. Therefore, polymorphisms in IL-4, IL-4R, and IL-13 genes may constitute a common etiologic pathway in CWP patients. To determine whether polymorphisms in IL-4, IL-4R, and IL-13 genes contribute to the development of CWP, we genotyped the IL-4 C-590T (rs2243250 in the IL-4 promoter region), IL-4R Ile50Val (rs1805010 in the IL-4R exon 5), IL-4R Ser478Pro (rs1805015 in the IL-4R exon 12), IL-4R Gln551Arg (rs1801275 in the IL-4R exon 12), IL-13 C-1055T (rs1800925 in the IL-13 promoter region) and IL-13 Arg130Gln (rs20541 in the IL-13 exon 4) polymorphisms and investigated the association between these genetic variants and CWP risk in a Chinese population.

Study subjects
The detailed methods of recruiting study subjects for this study have been described previously [11]. Briefly, 556 CWP patients and 541 controls were recruited in an ongoing study from five coal mines of Xuzhou Mining Business Group Co. Ltd., China, starting in January 2006. The high kilovolt chest X-ray and physical examinations were performed for reconfirming the diagnoses based on the China National Diagnostic Criteria for Pneumoconiosis (GBZ 70-2002), which is the same as the 1980 International Labour Organization (ILO) Classification of Pneumoconiosis in the judgment of opacity profusion. The pneumoconiosis patients were classified into stage I, stage II and stage III according to the size, profusion, and distribution range of opacities on chest X-ray. The chest X-rays were assessed by two independent physicians (Z Song and X Jia). The controls were selected from the same coal mines, who were matched for age (within 5 years), dust exposure period, and job type. Subjects were excluded if they had clinical evidence of autoimmunity diseases; had immunosuppressive or immunostimulatory therapy, or were subjected to radiotherapy. The epidemiological survey was done by face-to-face interviewers in order to obtain correct information including age, dust exposure period, job types, smoking status and others. The questionnaire was blinded regarding the case or control status of participants. After the interview, a 5 mL venous blood sample was collected from each subject. Informed consent was obtained from all subjects and the authorization was given by the Institutional Review Board of Nanjing Medical University.

Genotyping
Genomic DNA was isolated from peripheral blood lymphocytes using the conventional phenol-chloroform method. Genotyping was performed using the TaqMan method with ABI 7900HT Real Time PCR system according to the manufacturer's instructions (Applied Biosystem, Foster city, CA). The sequences of primer and probe for each SNP are available on request. A total of 10 negative controls and 8 duplicates were included for each SNP as a quality control measure. About 10% of the samples were randomly selected for repeated genotyping for confirmation and the results were 100% concordant. Genotyping was performed by two persons independently in a blinded fashion.

Statistical analyses
Differences in the distributions of demographic characteristics, selected variables, and frequencies of genotypes of IL-4, IL-4R, and IL-13 polymorphisms between the CWP cases and controls were evaluated by using the Student's t-test (for continuous variables) or x 2test (for categorical variables). Hardy-Weinberg equilibrium (HWE) was tested using a goodness-of-fit x 2 -test. The associations between genotypes and CWP were estimated by computing odds ratios (ORs) and their 95% confidence intervals (CIs) from unconditional logistic regression analysis with the adjustment for possible confounders. For the stratified analysis, the age and dust-exposure cutoff used in this study were according to the median of age and dust-exposure year of the recruited patients and controls. The statistical power was calculated by using the PS software (http://biostat.mc.vanderbilt. edu/twiki/bin/view/Main/PowerSampleSize). EM algorithm in SAS 9.1 PROC HAPLOTYPE was used to infer haplotype frequencies based on observed genotypes. All tests were two-sided by using the SAS software (version 9.1; SAS Institute, Inc., Cary, NC), unless indicated otherwise.

Results
The distributions of selected characteristics between CWP patients and control subjects are shown in Table 1. Briefly, there was no significant difference in the distribution of age (P = 0.304),  exposure years (P = 0.452), and job types (P = 0.106), between the cases and controls. However, there were more ever smokers (50.9%) among the cases than among the controls (41.4%) (P = 0.002). Specially, light smokers (#20 pack-years) had a 2.62fold (95% CI = 1.93-3.57) increased risk. Furthermore, the stages from I to III of the cases were 56.7%, 34.2%, and 9.1%, respectively. The primary information and allele frequencies observed are listed in Table 2. All genotyped distributions of control subjects were consistent with those expected from the Hardy-Weinberg equilibrium except for Ser478Pro polymorphism in the IL-4R exon 12 (P = 0.029). The minor allele frequency (MAF) of all the six polymorphisms was consistent with that reported in the HapMap database. As shown in Table 3, only the genotype frequencies of C-590T polymorphism in the IL-4 promoter region were significantly different between the cases and controls (P = 0.049 and 0.034 for genotype and allele, respectively). But this significance disappeared after the Bonferroni correction. Logistic regression analysis revealed that the IL-4 C-590T CT genotype, but not the CC genotype, In the stratification analyses, we found that individuals with the IL-4 CT/CC genotypes had a significant decreased risk of CWP than those with the TT genotype, and this decreased risk was more evident among subgroups of age ,65 years (OR = 0.68, 95%CI = 0.46-0.99) and dust exposure years $26 years (OR = 0.69, 95%CI = 0.50-0.94) ( Table 4). In addition, significant associations were observed between the genotypes and patients with stage I (OR = 0.70, 95%CI = 0.52-0.95). However, no statistical evidence was found for the gene-environment interaction (data not shown).
Considering potential interactions of these cytokine gene polymorphisms on risk CWP, we combined these six polymorphisms based on the numbers of variant (risk) alleles (i.e., IL-4 rs2243250 T, IL-4R rs1805010 C, IL-4R rs1805015 T, IL-4R rs1801275 A, IL-13 rs1800925 C, and IL-13 rs20541 G alleles). As shown in Table 5, individuals with multiple risk alleles had a higher risk of CWP, compared with those with 0-8 risk alleles, with a dose-dependent manner with increasing numbers of risk variant alleles conferring increasing risk (P trend = 0.023). Specifically, individuals carrying 11-12 risk alleles had a significantly higher risk of CWP than those with 0-8 risk alleles (OR = 1.47, 95% CI = 1.05-2.05).
Haplotypes analysis of IL-4R and IL-13 polymorphisms was performed. However, the distributions of the haplotypes of IL-4R and IL-13 between the CWP cases and controls were not significantly different, and the haplotypes had no apparent relationship with risk of CWP (data not shown).

Discussion
In this case-control study, six functional polymorphisms in the IL-4, IL-4R, and IL-13 genes were investigated with respect to an association with risk of CWP in a Chinese population. We found that the IL-4 C-590T polymorphism in the promoter region was significantly associated with CWP, and the association was more evident in younger workers with a long exposure history.
CWP is a chronic inflammatory lung disease where various environmental and genetic factors can influence its phenotype. Genetic factors such as polymorphisms can contribute to the extent or severity of CWP. Many genetic studies in CWP patients have involved genes encoding for cytokines and their receptors [12,13,14]. Cytokines play a crucial role in the widely used immunological model that explain the increasing prevalence of Table 4. Stratification analyses between the genotypes of IL-4 C-590T polymorphism and CWP risk.  inflammatory diseases by an altered balance between Th1 and Th2 immune response [15]. IL-4 is a typical Th2 cytokine of decisive significance in regulating Th1/Th2 cytokine balance [16]. It plays an important role through the IL-4R [17]. IL-4R binds not only IL-4, but also IL-13. IL-4 is dominant mediator of Th2 cell differentiation, proliferation, and activity, whereas IL-13 has minimal effects on T cell function [18]. A recent study reported that a promoter polymorphism C-590T of IL-4 was associated with an increased gene expression with T allele [19]. The -590T promoter sequence showed greater binding to nuclear transcription factors from allergen stimulated Jurkat human T cells than that of -590C sequence, and alteration in electrophoretic mobility shift assay was observed [20,21]. Several association studies have demonstrated that the T allele was correlated to inflammatory disease, such as asthma [22,23]. In the present study, carriers of the TT genotype had an increased risk of CWP compared with the CT/CC genotypes. One possible explanation is that the T allele may result in increased anti-inflammatory cytokine production or as a pro-fibrogenic factor favoring the fibrotic nodular lesions of lung.
Polymorphisms leading to amino acid changes have been described for mouse and human IL-4R [24]. Three functional polymorphisms Ile50Val, Ser478Pro, and Gln551Arg in the exons of IL-4R are frequently linked, which were associated with low total IgE concentrations, and an increase in the phosphorylation of insulin receptor substrate molecules [25]. However, the effects of these polymorphisms on receptor signaling have been contradictory [26,27,28]. IL-13 binds with high affinity to IL-13 receptor a-1, which induces heterodimerization with IL-4R to form a complex identical to the type II receptor [29]. Functional characterization of IL-13 C-1055T polymorphism showed that T allele had opposite transcriptional effects, paralleled by distinct patterns of DNA-protein interactions at the IL-13 promoter [30], which has been shown to be associated with allergic asthma and abnormal IL-13 production [31]. Another IL-13 Arg130Gln polymorphism in exon 4 has been shown to be associated with high total serum IgE level [32]. However, our study found that individuals with IL-4R and IL-13 polymorphisms did not have an increased risk of developing CWP. The mechanism of inconsistent results is still unknown, which needs to be validated by other studies with different ethnic populations.
Interestingly, we found that the protective effects of IL-4 variant genotype were evident in younger workers with long exposure history. Although the exact molecular mechanisms underlying are unknown, it is possible that individuals in those subgroups more likely were less exposed to some risk factors involved in the etiology of CWP risk [33]. Hessel et al. performed a meta-analysis and showed that smoking was significantly associated with silicosis [34]. Animal studies also revealed that smoking could induce lung fibrosis [35]. In the present study, we also found a significant difference between smokers and non-smokers related to CWP risk (P = 0.002), but there was no difference between the smoking status and CWP patients with different IL-4 C-590T genotypes. This negative result in our study could be due to insufficient sample size. Interestingly, we found that the IL-4 polymorphism carriers had a significantly decreased risk of developing stage I CWP. These findings suggested that there might be different mechanisms underlying the early development of CWP and the subsequent progression of CWP [36], and the IL-4 polymorphism might affect these two mechanisms differently.
Several limitations of this study should be addressed. First, our study was hospital-base study design, we could not rule the possible of selection bias of subjects that may have been associated with a particular genotype. Second, our sample size is moderate, and the statistical power of the study is limited, especially for subgroup and interaction analyses. However, we have 80% power at 0.05 significance level to detect an OR of 1.50 or higher and 0.63 or lower with an exposure frequency of 20% under the current sample size Third, because our results were based on the statistical significance and the ORs were very weak, the significant association between the IL-4 C-590T polymorphism and CWP risk should be interpreted with caution. Further functional study is needed to validate our findings.
In conclusion, our present study indicated that the functional IL4 C-590 T polymorphism is associated with decreased risk of CWP in a Chinese population. Further validation studies with diverse populations are warranted to confirm our findings.