Impact of IL-4 polymorphisms on head and neck cancer susceptibility in the Chinese Han population

Background: Head and neck cancer (HNC) is the sixth most frequent malignancy in the world. Interleukin 4 (IL-4) has been proved to be closely related to the occurrence and development of cancer. The objective of this research was to assess the association of IL-4 genetic polymorphisms with HNC risk in Chinese Han population. Methods: Six single nucleotide polymorphisms (SNPs) of IL-4 gene were successfully genotyped using Agena MassARRY in 535 HNC patients and 538 healthy controls. Under multiple genetic models, stratification analysis and haplotype analysis, odds ratio (OR) and 95% confidence interval (CI) were calculated to assess the association between SNPs and HNC risk. Results: The polymorphism, rs2243250, in the IL-4 gene was significantly associated with an increased HNC risk in the allele model (P < 0.05). In the genetic model, the polymorphisms rs2243250, rs2243267 and rs2243270 in IL-4 gene conferred an increased risk of HNC (P < 0.05). Gender stratification analysis showed that in males, rs2243250, rs2243267, rs2243270 and rs2243289 were associated with an increased risk of HNC (P < 0.05), while rs2243283 was associated with a decreased risk of HNC in males (P < 0.05). Besides, we have not found any association between six SNPs in IL-4 gene and HNC risk in females. Further haplotype analysis revealed that the haplotype CTGAC was significantly associated with a decreased risk of HNC. Conclusion: This study provides an evidence for polymorphism of IL-4 gene associated with the development of HNC in the Chinese Han population. with an increased risk of HNC, while rs2243283 was associated with a decreased risk of HNC in males. Haplotype analysis revealed that the haplotype CTGAC was significantly associated with a decreased risk of HNC. These results suggest that polymorphisms of IL-4 gene may play an important role in the risk of HNC in the Chinese Han population.


Introduction
Head and neck cancer (HNC) is found in the oral cavity, pharynx, nasal cavity, paranasal sinuses, larynx, and salivary glands1, ranking the sixth most frequent malignancy worldwide [1]. Although HNC is treated with a comprehensive treatment involving radiation, chemotherapy, and surgical treatment modalities, its mobility remains high.
HNC has a mortality rate of 40 -50% and is detected in 600,000 cases annually worldwide, accounting for ~3.8% of global cancer cases and ~3.6% of all cancer-associated mortality [2,3]. The etiology of HNC is multifactorial, and epidemiological evidence suggests that traditional risk factors such as tobacco, alcohol use, occupational exposure and use of external substances are well-known causal factors for HNC development [4]. However, more evidences demonstrated that genetic factors also play more important roles in the pathogenesis of HNC, and further studies have confirmed that the genetic susceptibility of HNC was closely related to the mutations of several genes [5].
Inflammation is a complex response at the cellular and tissue levels that can promotes tumorigenesis by stimulating angiogenesis [6], damaging DNA [7] and stimulating cell proliferation [8]. Interleukin 4 (IL-4) is an anti-inflammatory immunosuppressive cytokin, and is produced by activated CD4+ T cells, mast cells, and basophils [9]. IL-4 is responsible for inducing the production of IgE by B cells, and it can antagonize the function of IFN-γ and inhibit the activation of macrophages [10]. There is some evidence that IL-4 signaling promotes malignancy through its immunosuppressive effect on T cells, increased proliferation of cancer cells, and resistance to apoptosis [11,12]. Vasilena et al.
reported that IL-4 can promote cancer growth and invasion by inducing the cathepsin activity in tumor-associated macrophages (TAM) both in vivo and in vitro [13]. It also has been reported that IL-4 was related to humoral immune response, and may regulate the growth of head and neck (SCCHN) cell lines by a paracrine mechanism [14]. The serum samples of patients with HNSCC were detected to contain abnormal levels of inflammatory cytokines, such as lower IFN-γ and IL-13 levels, compared with the control mean level [15]. The expression of IL-4 was up regulated in several human malignancies including breast, colon, pancreatic, ovarian, and lung cancer [16,17]. Besides, there is evidence receptors for interleukin 4 (IL-4R) are preferentially expressed on the surface of a variety of solid tumors including SCCHN [18,19]. IL-4R-directed cytotoxin has pronounced antitumor activity against SCCHN in animal models of human disease [20]. All the above studies indicated that IL-4 gene is closely related to the susceptibility of HNC.
Furthermore, the polymorphism of IL-4 is associated with genetic susceptibility to a variety of diseases. However, few studies have examined the association of IL-4 polymorphisms with the risk of HNC. Therefore, we conducted a case-control study to investigate the relationship between IL-4 polymorphisms (rs2243250, rs2227284, rs2243267, rs2243270, rs2243283 and rs2243289) and the susceptibility to HNC in the Chinese Han population. Our results may provide new insights into understanding the mechanisms of HNC and contribute to the early prevention and targeted treatment of HNC.

Study subjects
Using a case control design, 267 patients with newly diagnosed HNC and 302 healthy controls were recruited. All the subjects were recruited from the First Affiliated Hospital of Xi'an Jiaotong University. All participants were genetically unrelated Chinese Han population. None of the patients had received radiotherapy, chemotherapy, or endocrine therapy before blood collection. Patients with HNC were selected from the Department of Oncology. The HNC diagnoses of all patients were confirmed by histopathological analysis.
Patients with a history of other types of cancers or who underwent radiotherapy or chemotherapy were excluded were excluded in this study. The control group was healthy individuals, and had no history of tumor, no genetic family history of tumors, and no tumor was found during the physical exam. Participants were selected without restrictions on age, gender, or disease stage.

DNA Extraction
The peripheral blood samples from HNC patients and controls were collected into vacutainers containing ethylene diaminetetraacetic acid (EDTA) and stored at -80C until further analysis. GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi'an City, China) was used to extract genomic DNA from peripheral blood leukocytes in whole blood samples according to the manufacturer's agreement, and spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, USA) was used to determine the purity and concentration of the extracted DNA.

Statistical Analysis
Differences in the characteristics of the case and control study populations were analyzed using χ 2 tests for categorical variables and Welch's t-tests for continuous variables. The lower frequency alleles were considered to be the risk allele in all analyses. Departure from Hardy Weinberg equilibrium (HWE) of genotypic distribution of each SNP in controls was analyzed using Fisher s exact test. Pearson's χ2 test was applied to compare the allele and genotype frequencies between the HNC patients and healthy subjects. Odds ratios (ORs) and 95% confidence interval (95% CI) were calculated using unconditional logistic regression analysis after adjusting for age and sex. Genotypic model analysis

Demographic and clinical characteristics
A case control study containing 535 HNC patients with a mean age of 46.80 ± 15.08 and 538 healthy controls with a mean age of 46.94 ± 13.79 was performed. Table 2 listed the demographics and clinical information of the cases and controls enrolled in the present study. Gender, age, nasopharyngeal cancer and thyroid cancer were not statistically significant in the cases and controls (P > 0.05), while lymph node metastasis and staging were significantly different between the cases and controls (P < 0.05). Table 3 summarizes the basic characteristics of the SNPs in the study population, and shows that all the six SNPs from controls were in accordance with HWE (P > 0.05). The average SNPs call rate was more than 95% in cases and controls. We compared the differences in frequency distributions of alleles between cases and controls compared by Pearson's χ 2 test and found only the minor allele "C" of rs2243250 from IL4 was significantly associated with an increased risk of HNC in the study population (P = 0.044, OR = 1.23, 95% CI = 1.01 -1.50).
The three other SNPs (rs2227284, rs2243283 and rs2243289) were not found to be associated with HNC risk under the four genetic models (P > 0.05).

Discussion
Genetic studies have provided insight into numerous diseases, including HNC. In the present study, we investigated the correlations between six SNPs in IL-4 gene and the risk of HNC. Our results indicate that the polymorphisms rs2243250, rs2243267, and rs2243270 in IL-4 gene were associated with an increased risk of HNC. Gender stratification analysis showed that in males, rs2243250, rs2243267, rs2243270 and rs2243289 were associated with an increased risk of HNC, while rs2243283 was associated with a decreased risk of HNC in males. Haplotype analysis revealed that the haplotype CTGAC was significantly associated with a decreased risk of HNC. These results suggest that polymorphisms of IL-4 gene may play an important role in the risk of HNC in the Chinese Han population.
The IL-4 gene has been mapped ton the long arm of chromosome 5 within a cluster of other cytokine genes in human beings. Meanwhile, IL-4 gene variants may affect the occurrence of cancer by change promoter activity of IL-4 gene and conformation of IL-4R information is available about the polymorphic sites in IL-4 and HNC susceptibility. In our study, we found that rs2243250, rs2243267 and rs2243267 were significantly associated with an increased risk of HNC. Rosenwasser et al. [41] report that T allele of IL-4 rs2243250 (C/T) polymorphism can increase gene transcription of IL-4 and thereby increases IL-4 gene expression. One may speculate that this mechanism may be related to our results, suggesting that carriers of the T allele were associated with an increased risk of HNC. However, it has also been reported that IL-4-590 C>T (rs2243250) polymorphisms did not influence the development of HNC, which was contrary to our findings [42]. Therefore, the correlation between IL-4-590 C>T (rs2243250) polymorphisms and HNC risk needs to be further verified in a larger number of samples.
However, there were still some deficiencies in this study. Firstly, some bias in population selection should not be ignored. Firstly, all participants were recruited from the same hospital, so inherent selection and information bias was the unavoidable problems.
Secondly, the number of cases in our study was not large enough to rule out false

Ethics approval and consent to participate
The study was conducted in accordance with the Declaration of Helsinki and was approved by the ethical committee of the Hospital of the First Affiliated Hospital of Xi'an Jiaotong University, China. Written informed consent was obtained from all individual participants included in the study.

Consent for publication
Written informed consent was obtained from the patient for publication of this report.

Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.     P a -value calculated by logistic regression analysis;

Tables
P b -value calculated by logistic regression analysis with adjustments for gender and age; * P-value < 0.05 indicates statistical significance.   P b -value calculated by Wald test and adjusted by gender and age; * P-value < 0.05 indicates statistical significance. Figure 1 The haplotype block map constructed by candidate SNPs in IL-4. Block 1 includes rs2243250, rs2227284, rs2243267, rs2243270 and rs2243283; the linkage disequilibrium between two SNPs is indicated by standardized D' (red boxes).