IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and risk of gastric cardiac adenocarcinoma
- Authors:
- Published online on: October 23, 2017 https://doi.org/10.3892/mco.2017.1469
- Pages: 1101-1106
Abstract
Introduction
Gastric cardiac adenocarcinoma (GCA) is one of the most common malignant tumors and among the leading causes of cancer-related mortality worldwide. Genetic as well as environmental factors may be included in the etiology of GCA (1). Single-nucleotide polymorphisms (SNPs), which account for >90% of genetic variations, may affect gene function (2). Individual differences in disease susceptibility have been suggested to be affected by SNPs (3).
Interleukin 18 (IL18), initially described as an interferon (IFN)-γ-inducing factor, may upregulate several cytokines, including IFN-γ, tumor necrosis factor-α and IL1β (4). IL18 participates in innate as well as acquired immunity and is largely produced by activated macrophages, Kupffer cells and dendritic cells (5,6). IL18 also promotes T helper cell type 1 (Th1) differentiation. The major functions of IL18 include production of IFN-γ by activated T lymphocytes and natural killer (NK) cells (5), and enhancement of T- and NK-cell maturation (7). IL18 may also induce proinflammatory cytokines and chemokines (8,9) and augment Fas ligand-mediated and perforin-dependent cytotoxicity of T and NK cells (10–12). Furthermore, IL18-deficient mice exhibited a reduction in IFN-γ production, NK-cell activity impairement and defective Th1 response (13).
As one of the main products of inflammasomes, IL18 exerts profound effects on carcinogenesis and tumor progression (14). The IL18 receptor comprises the IL18 receptor accessory protein (IL18RAP) and IL18 receptor 1 (IL18R1) protein (15,16).
Upon binding to its receptor IL18R1 protein, IL18 triggers the recruitment of IL18RAP and initiates signalling. As regards preventing colon adenocarcinoma development, IL18 plays a role in MyD88-mediated signalling (17). IL18RAP has been shown to be crucial for IL18 signalling, forms the signalling chain of this receptor complex and results in the production of IFN-γ (18). The two subunits of the IL18 receptor are mainly expressed on Th1 cells in response to interferon-α and/or IL12 (19). IL18RAP was also recently found to be associated with inflammatory bowel disease (20). The IL18R1 and IL18RAP genes have been found to be associated with atherosclerosis and its cardiovascular complications (21).
IL18 rs360719 polymorphism leads to loss of the octamer (OCT)-1 transcription factor binding site. OCT-1 is known to be a ubiquitously expressed factor and is involved in the regulation of several genes; it may also repress the expression of certain cytokines (22). Allele A of rs917997, a SNP 1.5 kb downstream of IL18RAP, was found to be strongly associated with coeliac disease susceptibility (23). This allele is also correlated with lower mRNA levels in whole blood. Furthermore, the IL18RAP GA haplotype of rs13015714 and rs917997 exhibited the strongest association with coeliac disease (23). Recently, genome-wide association studies have identified IL18RAP SNP rs917997, located on chromosome 2, as protective against type 1 diabetes (T1D) (24). The SNP, a C-to-T substitution in the 3′-untranslated region of IL18RAP, has been associated with decreased gene expression in the peripheral blood (25).
Interleukin 28B (IL28B) rs8099917 T>G polymorphism has been found to be associated with the response to IFN-γ-based antiviral therapy during the natural course of hepatitis C virus infection and following liver transplantation (26,27).
The IL18 receptor comprises IL18RAP and IL18R1. IL18RAP has been shown to be crucial for IL18 signalling, resulting in the production of IFN-γ. Allele A of IL18RAP rs917997 was found to be strongly associated with coeliac disease susceptibility (23). This allele also exerted a significant allele dosage effect on IL18RAP mRNA expression in whole blood. The IL18RAP rs917997 C allele is strongly associated with a protective effect in Barrett's esophagus and esophageal adenocarcinoma, and the CC genotype at IL18RAP locus rs917997 was associated with a protective effect against esophageal disease, which is in accordance with our results in stratification analyses (28).
In this study, we hypothesized that these SNPs may alter the individual risk of GCA. Thus, genotyping analyses for the four SNPs were performed in a Chinese Han population of 243 GCA cases and 476 control subjects.
Patients and methods
Ethics approval of the study protocol
This study was approved by the Institutional Review Board of Jiangsu University (Zhenjiang, China). All the subjects included in the study provided written informed consent and the study protocol complied with the World Medical Association Declaration of Helsinki regarding ethical conduct of research involving human subjects and/or animals.
Study subjects
The 243 GCA patients included in the case group were consecutively recruited at the Affiliated People's Hospital of Jiangsu University and the Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October, 2008 and July, 2010. In all the cases, a definite diagnosis was based on pathological examination. Tumor-node-metastasis (TNM) stage was defined according to the 7th edition of the Union for International Cancer Control TNM staging guidelines (29). The exclusion criteria included patients who previously had any primary or metastatic cancer and received radiotherapy or chemotherapy. In this study, 476 cancer-free controls were also included during the same time period, of whom 380 controls were recruited from the two aforementioned hospitals and were matched to the cases for age (±5 years) and sex; another 96 controls were recruited from hospitals in the city of Changzhou (which is adjacent to Zhenjiang) (30). The majority of the controls were admitted to the hospitals to receive treatment for trauma.
Each patient was personally questioned by a trained investigator to obtain information on demographic data (e.g., age and sex) and related environmental risk factors (including tobacco use and alcohol consumption), using a pre-tested questionnaire. A 2-ml venous blood sample was collected from each subject after the investigation. Individuals who smoked one cigarette per day for >1 year were classed as smokers, whereas subjects who consumed ≥3 alcoholic drinks per week for >6 months were defined as alcohol drinkers.
DNA extraction and genotyping analysis
Blood samples were collected from the subjects using vacutainers and drawn into tubes lined with EDTA. The QIAamp DNA Blood Mini kit (Qiagen, Berlin, Germany) was used to extract genomic DNA from peripheral blood (31). Sample DNA (10 ng) was amplified by polymerase chain reaction (PCR) according to the manufacturer's recommendations. As previously described, a custom-by-design 48-Plex SNPscan™ kit (Genesky Biotechnologies, Inc., Shanghai, China) was used to determine the genotypes of the four SNPs (32–35). This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies, which was mainly based on double ligation and multiplex fluorescence PCR. To guarantee genotyping quality, repeated analyses were accomplished by randomly selecting 4% of samples with high DNA quality.
Statistical analyses
Differences in the distribution of demographic characteristics, selected variables and genotypes of the IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G variants between the cases and controls were estimated using the χ2 test. The associations between the four SNPs and susceptibility to GCA were evaluated by calculating the odds ratios (ORs) and their 95% confidence intervals (CIs) using logistic regression analyses for crude and adjusted ORs after adjusting for age, sex, smoking and drinking status. The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit χ2 test to compare the observed genotype frequencies to those expected among the control group. All the statistical analyses were performed with SAS 9.1.3 software (SAS Institute, Inc., Cary, NC, USA).
Results
Characteristics of the study population
The characteristics of the cases and controls included in this study are summarized in Table I. The cases and controls appeared to be adequately matched for age and sex, as suggested by the χ2 tests (P=0.923 and P=0.197, respectively). As shown in Table I, no significant difference in drinking status was observed between the cases and the controls (P=0.217), but the smoking rate was higher in GCA patients compared with that in control subjects (P=0.004). Lymph node metastasis information was available in 220 (90.5%) of the 243 GCA patients and regional lymph node metastasis was present in 122 (55.5%) cases. TNM stage data were available in 196 (80.7%) of the 243 patients (stage I, 24; stage II, 34; stage III, 101; and stage IV, 37 patients) (Table I). The primary information for the four genotyped SNPs is presented in Table II. For the four SNPs, genotyping was successful, ranging from 96.94 to 97.50% in the 719 samples. The concordance rates of repeated analyses were 100%. Minor allele frequency (MAF) in our controls was similar to the MAF for Chinese subjects in the database for all four SNPs (Table II). The observed genotype frequencies for these four polymorphisms in the controls were all in HWE (Table II).
 | Table II.Primary information for IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms. |
Associations between IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and risk of GCA
The genotype distributions of IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G in the cases and the controls are presented in Table III. In the single locus analyses, the genotype frequencies of IL18 rs360719 A>G were 74.47% (AA), 24.26% (AG) and 1.28% (GG) in the cases and 76.24% (AA), 22.46% (AG) and 1.30% (GG) in the control subjects; the differences were not statistically significant (P=0.868). When the IL18 rs360719 AA homozygote genotype was used as the reference group, the AG genotype was not associated with the risk for GCA (AG vs. AA: adjusted OR=1.08, 95% CI: 0.75–1.57, P=0.679); the GG genotype was not associated with the risk for GCA (GG vs. AA: adjusted OR=1.04, 95% CI: 0.25–2.29, P=0.956). In the dominant model, the IL18 rs360719 AG/GG variants were not associated with the risk for GCA, compared with the IL18 rs360719 AA genotype (adjusted OR=1.08, 95% CI: 0.75–1.56, P=0.680). In the recessive model, when the IL18R1 rs13015714 AA/AG genotypes were used as the reference group, the GG homozygote genotype was not associated with risk for GCA (adjusted OR=1.02, 95% CI: 0.25–4.20, P=0.976) (Table III). No association was observed between the IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and the risk for GCA (Table III).
| Table III.Logistic regression analyses of associations between IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms and risk of GCA. |
Discussion
This study was conducted to demonstrate the associations of IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G with the susceptibility to GCA in a high-risk Chinese population. Our findings revealed that the variants IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G were not associated with the risk for GCA.
IL18 promotes cell death and tumor progression by activation of immune response and NK cells and may be used in anticancer gene therapy. IL18 rs360719 polymorphism leads to loss of the OCT-1 transcription factor binding site. IL18RAP rs917997 C>T was found to be strongly associated with susceptibility to coeliac disease (23). IL18RAP rs917997 C>T was a protective factor in T1D (24). IL18RAP rs917997 C>T in the 3′-untranslated region of IL18RAP has been associated with decreased gene expression in the peripheral blood (25). IL28B rs8099917 T>G polymorphism has been associated with the response to IFN-γ-based antiviral therapy during the natural course of hepatitis C virus infection and after liver transplantation (26,27). In the present study, no significant associations with GCA were observed.
The frequencies of genetic polymorphisms often vary between ethnic groups (36). In the present study on Chinese subjects, the allele frequency of IL18 rs360719 G was 0.125 among the 476 control subjects, which is consistent with that of the Chinese Han (0.142) and the African American populations (0.152), but significantly lower compared with that of the European population (0.292) in the SNP database. The allele frequency of IL18R1 rs13015714 T was 0.512 among the 476 control subjects, which is consistent with that of the Chinese Han population (0.547), but significantly lower compared with that of the European (0.796) and Sub-Saharan African populations (0.894) in the SNP database. Similarly, the allele frequency of IL18RAP rs917997 T was 0.495 among the 476 control subjects, which is consistent with that of the Chinese Han population (0.488), but significantly higher comapred with that of the European (0.208) and Sub-Saharan African populations (0.049). Finally, the allele frequency of IL28B rs8099917 G was 0.048 among the 476 control subjects, which is consistent with that of the Chinese Han population (0.035), but significantly lower compared with that of the European (0.167) and Mexican populations (0.310) in the SNP database (http://www.ncbi.nlm.nih.gov/SNP).
Several limitations must be addressed in this case-control study. First, the patients and controls were enrolled from hospitals and may not represent the general population. Second, the moderate sample size limited the statistical power of our study, particularly in stratification analyses, and further well-designed two-stage fine-mapping studies are required to confirm our findings. Third, detailed information on cancer metastasis and survival were not available, which restricted further analyses on the role of IL18R1 rs13015714 G>T and IL18RAP rs917997 C>T polymorphisms in GCA progression and prognosis.
In conclusion, our study provides evidence that IL18 rs360719 A>G, IL18R1 rs13015714 G>T, IL18RAP rs917997 C>T and IL28B rs8099917 T>G polymorphisms do not contribute to the risk of GCA. However, the power of our analysis was low, with a limited sample size, therefore allowing us to draw only preliminary conclusions. Future larger studies including other ethnic populations are required to confirm our findings.
Acknowledgements
The authors would like to thank Dr Da Ding and Dr Yan Liu (Genesky Biotechnologies, Inc., Shanghai, China) for the technical support. This study was supported in part by the National Natural Science Foundation of China (grant nos. 81370001, 81371927, 81101889 and 81000028), the Jiangsu Province Natural Science Foundation (grant nos. BK2010333 and BK2011481), the Social Development Foundation of Zhenjiang (grant no. SH2010017), the Changzhou Young Talents and Science-Technology Foundation of Health Bureau (grant no. QN201102), the Affiliated People's Hospital of Jiangsu University Fund (grant no. Y200913) and the Jiangsu University Clinical Medicine Science and Technology Development Fund (grant no. JLY20120004).
Glossary
Abbreviations
Abbreviations:
|
CI |
confidence interval |
|
GCA |
gastric cardiac adenocarcinoma |
|
IL18 |
interleukin 18 |
|
OR |
odds ratio |
|
SNP |
single-nucleotide polymorphism |
References
|
Zhang L, Du C, Guo X, et al: Interleukin-8-251A/T polymorphism and Helicobacter pylori infection influence risk for the development of gastric cardiac adenocarcinoma in a high-incidence area of China. Mol Biol Rep. 37:3983–3989. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Hu S, Song QB, Yao PF, et al: No relationship between IL-1B gene polymorphism and gastric acid secretion in younger healthy volunteers. World J Gastroenterol. 11:6549–6553. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Zhang W, Li C, Wang J and He C: Functional polymorphisms in FAS/FASL system contribute to the risk of occurrence but not progression of gastric cardiac adenocarcinoma. Hepatogastroenterology. 59:141–146. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Yoshimoto T, Tsutsui H, Tominaga K, et al: IL-18, although antiallergic when administered with IL-12, stimulates IL-4 and histamine release by basophils. Proc Natl Acad Sci USA. 96:pp. 13962–13966. 1999, View Article : Google Scholar : PubMed/NCBI | |
|
Okamura H, Tsutsi H, Komatsu T, et al: Cloning of a new cytokine that induces IFN-gamma production by T cells. Nature. 378:88–91. 1995. View Article : Google Scholar : PubMed/NCBI | |
|
Stoll S, Jonuleit H, Schmitt E, et al: Production of functional IL-18 by different subtypes of murine and human dendritic cells (DC): DC-derived IL-18 enhances IL-12-dependent Th1 development. Eur J Immunol. 28:3231–3239. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Nakanishi K, Yoshimoto T, Tsutsui H and Okamura H: Interleukin-18 regulates both Th1 and Th2 responses. Annu Rev Immunol. 19:423–474. 2001. View Article : Google Scholar : PubMed/NCBI | |
|
Micallef MJ, Ohtsuki T, Kohno K, et al: Interferon-gamma-inducing factor enhances T helper 1 cytokine production by stimulated human T cells: synergism with interleukin-12 for interferon-gamma production. Eur J Immunol. 26:1647–1651. 1996. View Article : Google Scholar : PubMed/NCBI | |
|
Puren AJ, Fantuzzi G, Gu Y, Su MS and Dinarello CA: Interleukin-18 (IFNgamma-inducing factor) induces IL-8 and IL-1beta via TNFalpha production from non-CD14+ human blood mononuclear cells. J Clin Invest. 101:711–721. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Tsutsui H, Nakanishi K, Matsui K, et al: IFN-gamma-inducing factor up-regulates Fas ligand-mediated cytotoxic activity of murine natural killer cell clones. J Immunol. 157:3967–3973. 1996.PubMed/NCBI | |
|
Dao T, Ohashi K, Kayano T, Kurimoto M and Okamura H: Interferon-gamma-inducing factor, a novel cytokine, enhances Fas ligand-mediated cytotoxicity of murine T helper 1 cells. Cell Immunol. 173:230–235. 1996. View Article : Google Scholar : PubMed/NCBI | |
|
Dao T, Mehal WZ and Crispe IN: IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells. J Immunol. 161:2217–2222. 1998.PubMed/NCBI | |
|
Takeda K, Tsutsui H, Yoshimoto T, et al: Defective NK cell activity and Th1 response in IL-18-deficient mice. Immunity. 8:383–390. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Zitvogel L, Kepp O, Galluzzi L and Kroemer G: Inflammasomes in carcinogenesis and anticancer immune responses. Nat Immunol. 13:343–351. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Born TL, Thomassen E, Bird TA and Sims JE: Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signalling. J Biol Chem. 273:29445–29450. 1998. View Article : Google Scholar : PubMed/NCBI | |
|
Torigoe K, Ushio S, Okura T, et al: Purification and characterization of the human interleukin-18 receptor. J Biol Chem. 272:25737–25742. 1997. View Article : Google Scholar : PubMed/NCBI | |
|
Salcedo R, Worschech A, Cardone M, et al: MyD88-mediated signalling prevents development of adenocarcinomas of the colon: role of interleukin 18. J Exp Med. 207:1625–1636. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Cheung H, Chen NJ, Cao Z, Ono N, Ohashi PS and Yeh WC: Accessory protein-like is essential for IL-18-mediated signalling. J Immunol. 174:5351–5357. 2005. View Article : Google Scholar : PubMed/NCBI | |
|
Sareneva T, Julkunen I and Matikainen S: IFN-alpha and IL-12 induce IL-18 receptor gene expression in human NK and T cells. J Immunol. 165:1933–1938. 2000. View Article : Google Scholar : PubMed/NCBI | |
|
Zhernakova A, Festen EM, Franke L, et al: Genetic analysis of innate immunity in Crohn's disease and ulcerative colitis identifies two susceptibility loci harboring CARD9 and IL18RAP. Am J Hum Genet. 82:1202–1210. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Thompson SR and Humphries SE: Interleukin-18 genetics and inflammatory disease susceptibility. Genes Immun. 8:91–99. 2007. View Article : Google Scholar : PubMed/NCBI | |
|
Sanchez E, Palomino-Morales RJ, Ortego-Centeno N, et al: Identification of a new putative functional IL18 gene variant through an association study in systemic lupus erythematosus. Hum Mol Genet. 18:3739–3748. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Koskinen LL, Einarsdottir E, Dukes E, et al: Association study of the IL18RAP locus in three European populations with coeliac disease. Hum Mol Genet. 18:1148–1155. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Smyth DJ, Plagnol V, Walker NM, et al: Shared and distinct genetic variants in type 1 diabetes and celiac disease. N Engl J Med. 359:2767–2777. 2008. View Article : Google Scholar : PubMed/NCBI | |
|
Zhernakova A, Elbers CC, Ferwerda B, et al: Evolutionary and functional analysis of celiac risk loci reveals SH2B3 as a protective factor against bacterial infection. Am J Hum Genet. 86:970–977. 2010. View Article : Google Scholar : PubMed/NCBI | |
|
Suppiah V, Moldovan M, Ahlenstiel G, et al: IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet. 41:1100–1104. 2009. View Article : Google Scholar : PubMed/NCBI | |
|
Eurich D, Boas-Knoop S, Ruehl M, et al: Relationship between the interleukin-28b gene polymorphism and the histological severity of hepatitis C virus-induced graft inflammation and the response to antiviral therapy after liver transplantation. Liver Transpl. 17:289–298. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Babar M, Ryan AW, Anderson LA, et al: Genes of the interleukin-18 pathway are associated with susceptibility to Barrett's esophagus and esophageal adenocarcinoma. Am J Gastroenterol. 107:1331–1341. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Kwon SJ: Evaluation of the 7th UICC TNM Staging System of gastric cancer. J Gastric Cancer. 11:78–85. 2011. View Article : Google Scholar : PubMed/NCBI | |
|
Cheng J, Zhang H, Zhuang C, et al: Peptidylarginine deiminase type 4 and methyl-CpG binding domain 4 polymorphisms in Chinese patients with rheumatoid arthritis. J Rheumatol. 39:1159–1165. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Gu H, Ding G, Zhang W, et al: Replication study of PLCE1 and C20orf54 polymorphism and risk of esophageal cancer in a Chinese population. Mol Biol Rep. 39:9105–9111. 2012. View Article : Google Scholar : PubMed/NCBI | |
|
Wei J, Zheng L, Liu S, et al: MiR-196a2 rs11614913 T>C polymorphism and risk of esophageal cancer in a Chinese population. Hum Immunol. 74:1199–1205. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Zheng L, Yin J, Wang L, et al: Interleukin 1B rs16944 G>A polymorphism was associated with a decreased risk of esophageal cancer in a Chinese population. Clin Biochem. 46:1469–1473. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Li Q, Yin J, Wang X, et al: B-cell Lymphoma 2 rs17757541 C>G polymorphism was associated with an increased risk of gastric cardiac adenocarcinoma in a Chinese population. Asian Pac J Cancer Prev. 14:4301–4306. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Sun JM, Li Q, Gu HY, et al: Interleukin 10 rs1800872 T>G polymorphism was associated with an increased risk of esophageal cancer in a Chinese population. Asian Pac J Cancer Prev. 14:3443–3447. 2013. View Article : Google Scholar : PubMed/NCBI | |
|
Spielman RS, Bastone LA, Burdick JT, Morley M, Ewens WJ and Cheung VG: Common genetic variants account for differences in gene expression among ethnic groups. Nat Genet. 39:226–231. 2007. View Article : Google Scholar : PubMed/NCBI |
