Interleukin 10 rs 1800872 T > G Polymorphism was Associated with an Increased Risk of Esophageal Cancer in a Chinese Population

Esophageal cancer is the eighth most common cancer and sixth leading cause of cancer associated death worldwide (Shinomiya et al., 1999; Parkin et al., 2005; Jemal et al., 2008). The 5 years survival rate for esophageal cancer patients is very poor and accounts only 12.3% (Berrino et al., 2007). The most frequent subtype of esophageal cancer, esophageal squamous cell carcinoma (ESCC), accounts for >90% of cases (Macfarlane et al., 1994). ESCC has multi-factorial etiology, besides environmental risk factors, accumulated evidence has shown genetic factors might play an important role in the ESCC carcinogenesis, including single nucleotide polymorphisms (SNPs) (Wu et al., 2011). Chronic inflammation in esophageal tissues may play a role in ESCC development and multiple genes that play critical roles in inflammatory pathways may be associated with ESCC risk. Interleukin 9 (IL9) was originally described as a growth


Introduction
Esophageal cancer is the eighth most common cancer and sixth leading cause of cancer associated death worldwide (Shinomiya et al., 1999;Parkin et al., 2005;Jemal et al., 2008).The 5 years survival rate for esophageal cancer patients is very poor and accounts only 12.3% (Berrino et al., 2007).The most frequent subtype of esophageal cancer, esophageal squamous cell carcinoma (ESCC), accounts for >90% of cases (Macfarlane et al., 1994).ESCC has multi-factorial etiology, besides environmental risk factors, accumulated evidence has shown genetic factors might play an important role in the ESCC carcinogenesis, including single nucleotide polymorphisms (SNPs) (Wu et al., 2011).
Chronic inflammation in esophageal tissues may play a role in ESCC development and multiple genes that play critical roles in inflammatory pathways may be associated with ESCC risk.
Interleukin 9 (IL9) was originally described as a growth

RESEARCH ARTICLE
Interleukin 10 rs1800872 T>G Polymorphism was Associated with an Increased Risk of Esophageal Cancer in a Chinese Population Jia-Ming Sun 1& , Qiong Li 1& , Hai-Yong Gu 2 , Yi-Jang Chen 3 , Ji-Shu Wei 4 , Quan Zhu 3 *, Liang Chen 3 * factor for a subset of murine T cell clones (Uyttenhove et al., 1988).IL9 is a pleiotropic cytokine in the T helper cell type 1 (Th1):Th2 pathway.Now, it was well known that a specialized subset termed Th9 cells selectively produce IL9 (Goswami et al., 2011).CDKN2A is a major susceptibility gene for cutaneous malignant melanoma (CMM).IL9 single nucleotide polymorphisms (SNPs) might have stronger risks of CMM in CDKN2A-positive families (Yang et al., 2009).IL10 has anti-inflammatory and immunosuppressive effects by decreasing the production of pro-inflammatory mediators, IL10 has also been shown to regulate the differentiation and proliferation of several immune cells (Couper et al., 2008).The IL10 gene is located on chromosome 1q31-32.Studies have shown that IL10 may be involved in the pathogenesis of many types of cancers (Howell et al., 2007).The maturation of Th1 cells from the naive CD4+ T cell pool is regulated by IL12 (Trinchieri et al., 2003), IL12 exhibits an immunoregulatory impact on T and NK cells by inducing IFN-γ biosynthesis from both cell types, augmenting their proliferation and cytotoxicity (Trinchieri et al., 2003).IL12 displays anti-angiogenic activity, which may antagonize the pro-angiogenic signals whose presence has been demonstrated during the progression of malignancies (Imagawa et al., 2004).IL13 play a central role in allergy by stimulating IgE synthesis (Akdis et al., 2006), show strong antitumor activity in mice, and inhibit proliferation of astrocytoma and low-grade glioma in human cell lines (Liu et al., 2000).
On the basis of the biological and pathologic significance of IL9, IL10, IL12A, IL12B and IL13, it is possible that functional genetic variations in these genes may contribute to the development of ESCC.The objective of this investigation was to evaluate the association between IL9 rs31563 C>T, IL9 rs31564 G>T, IL10 rs1800872 T>G, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T genotypes and ESCC susceptibility in a hospital-based case-control study.We performed genotyping analyses for the six SNPs with 380 ESCC cases and 380 controls in a Chinese population.

Ethical approval of the study protocol
This hospital-based case-control study was approved by the Review Board of Jiangsu University (Zhenjiang, China).All subjects provided written informed consent to be included in the study.

Study subjects
Three-hundred and eighty subjects with esophageal cancer were consecutively recruited from the Affiliated People's Hospital of Jiangsu University and Affiliated Hospital of Jiangsu University (Zhenjiang, China) between October 2008 and November 2009.All cases of esophageal cancer were diagnosed as ESCC by pathologic means.The exclusion criteria were patients who previously had: cancer; any metastasized cancer; radiotherapy or chemotherapy.The controls were patients without cancer frequency-matched to the cases with regard to age (±5 years) and sex recruited from the two hospitals mentioned above during the same time period.Most of the controls were admitted to the hospitals for the treatment of trauma.
Each subject was personally questioned by trained interviewers using a pre-tested questionnaire to obtain information on demographic data (e.g., age, sex) and related risk factors (including tobacco smoking and alcohol consumption).After the interview, 2-mL samples of venous blood were collected from each subject.Individuals who smoked one cigarette per day for >1 year were defined as "smokers".Subjects who consumed ≥3 alcoholic drinks a week for >6 months were considered to be "alcohol drinkers".

Isolation of DNA and genotyping by a custom-by-design 48-Plex SNPscan TM Kit
Blood samples were collected from patients using Vacutainers and transferred to tubes lined with ethylenediamine tetra-acetic acid (EDTA).Genomic DNA was isolated from whole blood with the QIAamp DNA Blood Mini Kit (Qiagen, Berlin, Germany) (Gu et al., 2012).Sample DNA (10 ng) were amplified by PCR according to the manufacturer's recommendations.The SNP genotyping work was performed using a custom-bydesign 48-Plex SNPscan TM Kit (Genesky Biotechnologies Inc., Shanghai, China) as previously described (Chen et al., 2012).This kit was developed according to patented SNP genotyping technology by Genesky Biotechnologies Inc., which was based on double ligation and multiplex fluorescence PCR.For quality control, repeated analyses were done for 4% of randomly selected samples with high DNA quality.

Statistical Analyses
Differences in the distributions of demographic characteristics, selected variables, and genotypes of the IL9 rs31563 C>T, IL9 rs31564 G>T, IL10 rs1800872 T>G, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T variants between the cases and controls were evaluated using the χ 2 test.The associations between IL9 rs31563 C>T, IL9 rs31564 G>T, IL10 rs1800872 T>G, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T genotypes and risk of ESCC were estimated by computing the ORs and their 95% CIs using logistic regression analyses for crude ORs and adjusted ORs when adjusting for age, sex, smoking and drinking status.The Hardy-Weinberg equilibrium (HWE) was tested by a goodness-of-fit χ 2 test to compare the observed genotype frequencies to the expected ones among the control subjects.All statistical analyses were performed with SAS 9.1.3(SAS Institute, Cary, NC, USA).

Characteristics of the study population
Characteristics of cases and controls included in the study are summarized in Table 1.The cases and controls appeared to be adequately matched on age and sex as suggested by the χ 2 tests (P = 0.056 and P = 0.346, respectively).As shown in Table 1, no significant difference was detected on drinking status between the cases and the controls (P = 0.183), but smoking rate was higher in ESCC patients than in control subjects (P = 0.014).The primary information for six genotyped SNPs was in Table 2.For the six SNPs, the genotyping was successful ranging from 94.87% to 97.11% in all 760 samples.The concordance rates of repeated analyses were 100%.Minor allele frequency (MAF) in our controls was similar to MAF for Chinese in database for all six SNPs (Table 2).The observed genotype frequencies for these six polymorphisms in the controls were all in HWE (Table 2).

Associations between six polymorphisms and risk of ESCC
The genotype distributions of IL9 rs31563 C>T, IL9 rs31564 G>T, IL10 rs1800872 T>G, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T in the cases and the controls are shown in Table 3.In the single locus analyses, the genotype frequencies of IL10 rs1800872 T>G were 45.5% (TT), 45.8% (TG), and 8.7% (GG) in the case patients and 52.3% (TT), 38.6% (TG), and 9.0% (GG) in the control subjects, and the difference was not statistically significant (P = 0.140).When the IL10 rs1800872 TT homozygote genotype was used as the reference group, the TG genotype was associated with a significantly increased risk for ESCC (TG vs. TT: OR 1.36, 95% CI 1.00-1.85,P = 0.049).When the IL10 rs1800872 TT homozygote genotype was used as the reference group, the GG genotype not associated with the risk for ESCC (GG vs. TT: OR 1.11, 95% CI 0.65-1.89,P = 0.707).In the recessive model, when the IL10 rs1800872 TT/TG genotypes were used as the reference group, the GG homozygote genotype was not associated with the risk for ESCC (OR 0.96, 95% CI 0.57-1.60,P = 0.875).In the dominant model, the IL10 rs1800872 TG/GG variants were associated with a borderline statistically increased risk of ESCC, compared with the IL10 rs1800872 TT genotype (TG/GG vs. TT: OR 1.32, 95% CI 0.98-1.76,P = 0.067) (Table 3).After adjusting for age, sex, smoking and drinking, a borderline statistically increased risk of ESCC was observed both in the heterozygote comparing model (TG vs. TT: adjusted OR 1.36, 95% CI 1.00-1.85,P = 0.053) and the dominant model (TG/GG vs. TT: adjusted OR 1.31, 95% CI 0.97-1.75,P = 0.078) (Table 3).
None of the IL9 rs31563 C>T, IL9 rs31564 G>T, IL12A rs2243115 T>G, IL12B rs3212227 T>G and IL13 rs1800925 C>T polymorphisms achieved a significant difference in the genotype distributions between cases and controls (Table 3).Logistic regression analyses revealed that the five polymorphisms were not associated with the risk of ESCC (Table 3).

Stratification analyses of IL10 rs1800872 T>G polymorphisms and risk of ESCC
To evaluate the effects of IL10 rs1800872 T>G genotypes on ESCC risk according to different age, sex, smoking and alcohol drinking status; we performed the stratification analyses.No significantly risk of ESCC associated with the IL10 rs1800872 T>G polymorphism was evident among any subgroups (Table 4).