Influence of Interleukin-18 and Interleukin-17 Receptor A gene polymorphisms on the risk of thyroid cancer among Chinese Han population

Background Thyroid cancer (TC) is a common endocrine pathology with an increasing incidence worldwide. It has been reported that high genetic impact is involved in the pathogenesis of TC, as well as cytokines, especially the interleukins play a crucial role in it. This study was designed to detect the association of IL-18 and IL-17RA polymorphisms with TC risk. Methods This case-control study conducted 365 TC patients and 503 healthy controls from Chinese Han population. Three selected SNPs (IL-18 rs360718, IL-18 rs1946519, and IL-17RA rs4819554) were genotyped to investigate the possible association of the polymorphisms with the risk of TC. Multifactor dimensionality reduction (MDR) was used to analyze the interactions of SNP-SNP. Results IL-17RA rs4819554 was associated with the decreased risk of TC under dominant model (OR = 0.76, 95% CI = 0.56-0.99, p = 0.04). IL-18 rs360718 significantly decreased the risk at age ≤ 44 years (C vs. A, OR = 0.63, 95% CI = 0.41-00.97, p = 0.033; CC vs. AA, OR = 0.12, 95% CI = 0.01-0.91, p = 0.040). On the contrast, among people older than 44 years, IL-18 rs1946519 (C vs. A, OR = 1.77, 95% CI = 1.03-3.06, p = 0.040) shown an increased risk with TC. MDR analysis revealed a positive interaction between the SNPs. Conclusion The present study firstly demonstrated that IL-18 rs360718, rs1946519 and IL-17RA rs4819554 polymorphisms might be related to thyroid cancer. The results were significantly worthy of further validation by larger studies. This case-control study aimed to investigated the potential effects of IL-18 and IL-17RA gene polymorphisms on TC in Chinese Han population. Our findings suggested that IL-17RA rs4819554 was associated with the decreased risk of TC. Especially in patients who were older than 44 years old, a modestly statistical relevance between IL-17RA rs4819554 and decreasing risk effect of TC deserved further research. Additionally , IL-18 rs360718 was detected to shown a significantly decreased risk with TC in patients under 44 years of age, whereas rs1946519 had an effect of increased risk with TC in patients 44 years and older. To our knowledge, this is the first study to report the effect of these SNPs in TC individuals.


Introduction
Thyroid cancer (TC), a common endocrine malignancy, accounting for more than 90% of endocrine cancers, had constantly increased worldwide recent years [1]. TC led the fifth most common cancer in women in the United States [2], while the estimates of cancer incident cases and deaths were 143.9 thousand and 6500 respectively in China [3]. Although the exact pathophysiologic mechanisms of TC remained elusive, accumulating evidence indicated that the inter-individual genetic factors, especially the single nucleotide polymorphisms (SNPs) in tumor-associated genes took essential part in the genetic susceptibility to TC [4][5][6][7].
Cytokines were molecules that contribute to regulate activation, growth, and differentiation of some target cells [8]. Importantly, they were pro-and anti-inflammatory mediators that played a crucial role in the induction and effector phases of the inflammatory and immune responses, and functioned as a regulator in development and growth of both normal and neoplastic thyroid cells [9]. Interleukin-18 (IL-18), a pleiotropic pro-inflammatory cytokine that induced interferon-gamma (IFN-γ) production and was involved in T helper type 1  The current case-control study was obtained the permission by the Review Board of the Hainan General Hospital. All subjects provided written inform consent to be included in the study.

Study participants
A total of 365 TC patients (mean age: 43.98 ± 15.17) and 503 healthy control subjects (mean age: 44.16 ± 12.37) were enrolled in this case-control study. All patients were Chinese Han adults and recruited from the Hainan General Hospital. The inclusion criteria for the patients were: patients who were recently diagnosed and identified as TC, according to diagnostic imaging and histopathological examination. The healthy controls without medical history of any type of cancer were randomly recruited from the health checkup at the same time.

SNPs selection and genotyping
We identified two single nucleotide polymorphisms (SNPs) in IL-18 and one in IL-17RA with a minor allele frequency (MAF) > 0.05 in Chinese Han population from the NCBI dbSNP database (http://www.ncbi.nlm.nih.gov/projects/SNP) and the 1000 Genomes Projects (http://www.internationalgenome.org/). The 5mL blood samples from all participants were collected in vacutainers which contained Ethylenediaminetetraacetic acid (EDTA). To extract gDNA, the GoldMag whole blood genomic DNA purification kit (GoldMag Co. Ltd., Xi'an, China) was used and then, the Importantly, we randomly selected about 10% of the samples to repeat genotyping for the quality control, the reproducibility was > 99%.

Data analysis
SPSS 20.0 (SPSS Inc., IBM, USA) statistical software and PLINK software were used for this study. The categorical variables were evaluated using Pearson's.χ 2 test, and student's t-test was performed to analyze the differences in the age distribution between the cases and controls. Hardy-Weinberg equilibrium (HWE) was tested by χ 2 test for each SNP which was selected in this study. χ 2 test or Fisher's exact test were used to compare the genotype and allele frequencies between cases and controls. For respective genotype, we used logistic regression analysis to assess the relevance of selected SNPs with TC risk by odds ratios (ORs) and 95% confidence intervals (CIs) based on previous methods [15]. Haploview software (version 4.2) and PLINK software were used for analyzing the Linkage disequilibrium (LD) and haplotype [16]. Multifactor dimensionality reduction (MDR) (version 3.0.2) was performed to evaluate the interactions between SNP and SNP in the TC risk [17].A twotailed p-value <0.05 was statistically significant for all the analyses.

Characteristic of the study participants
IL-17RA and IL-18 polymorphisms were analyzed in 365 patients (97 men and 268 women) and 503 unrelated disease controls (137 men and 366 women). The characteristic of participants included in this study were listed in Table 1. The cases and controls appeared to be adequately matched on age and sex as suggested by the student-t and χ 2 tests respectively (p = 0.857, p = 0.877).

Genotyping of the SNPs in IL-18 and IL-17RA
SNPs in IL-18 ( rs360718 and rs1946519) and IL-17RA (rs4819554) were successfully genotyped. The details of these SNPs and potential function predicted by HaploReg database about them were presented in Table 2. The Hardy-Weinberg equilibrium (HWE) tests for the IL-18 and IL-17RA polymorphisms were shown in Table 2 To evaluate the associations between IL-18, IL-17RA variants and the TC risk, five inheritance models (Allele, Genotype, Dominant, Recessive and Log-additive) adjusted for potential confounding variables (age, and gender) were applied. The results shown in Table3 indicated that IL-17RA rs4819554 polymorphism was associated with the decreased risk of thyroid cancer under dominant model (GG-GA vs. AA, OR = 0.76, 95% CI = 0.56-0.99, p = 0.040). However, IL-18 rs360718 and rs1946519 polymorphisms presented no statistically difference in genotype frequencies between the TC patients and the healthy controls (p ≥ 0.05). Logistic regression analyses indicated that none of these two SNPs in IL-18 were associated with any change of susceptibility to TC ( Table 3).
On the contrast, among people older than 44 years, IL-17RA rs4819554 (G vs A, OR = 0.76, 95%CI = 0.58-1.00, p = 0.047) was associated with the modestly decreased risk of TC which need to be further investigated (Table 4). IL-18 rs1946519 (C vs. A, OR = 1.77, 95% CI = 1.03-3.06, p = 0.040) shown an increased risk. Also, the results of stratification analysis by gender were listed in Table 4. There was no statistically significant difference between man and woman.
Furthermore, we researched the linkage disequilibrium (LD) and haplotype analyses of the SNPs in IL-18. The reconstructed LD plot was presented in Figure 2, and the LD block was comprised of two SNPs including IL-18 rs360718 and IL-18 rs1946519. The frequencies distribution of haplotypes in the cases and controls were showed in Table 5. The results indicated an association of CA haplotype with a modestly increased risk of TC which needed to be further detected in the future (OR = 1.34,95% CI = 1.00-1.78, p = 0.047).

Discussion
This case-control study aimed to investigated the potential effects of IL-18 and IL-17RA gene polymorphisms on TC in Chinese Han population. Our findings suggested that IL-17RA rs4819554 was associated with the decreased risk of TC. Especially in patients who were older than 44 years old, a modestly statistical relevance between IL-17RA rs4819554 and decreasing risk effect of TC deserved further research. Additionally, IL-18 rs360718 was detected to shown a significantly decreased risk with TC in patients under 44 years of age, whereas rs1946519 had an effect of increased risk with TC in patients 44 years and older. To our knowledge, this is the first study to report the effect of these SNPs in TC individuals.    p values were calculated by logistic regression analysis with adjustments for age and gender.
p < 0.05 means the data is statistically significant.  p values were calculated using Pearson's χ 2 tests with and without adjustment by gender and age.
p < 0.05 indicates statistical significance.   Haplotype block map for two SNPs in IL-18 gene.