| Gastric cancer is one of the most common malignant tumors. The age-standardized rates of incidence and mortality of gastric cancer in China occupy the second and third position in all malignant tumors, resulting in a heavy burden to people’s health. Gastric cancer is a complex, multistep and multifactorial process. It’s the result from joint action of the external environment factors and inherent genetic factors. Despite more than 50% of people in the world infected with H. pylori, only less than 3% of infected individuals suffering from gastric cancer. This suggests that the susceptibility of individuals responding to environmental exposure is different. Now it is generally believed that this susceptibility is mainly influenced by individual genetic factors. Therefore, screening high-risk populations with genetic susceptibility to gastric cancer is one of the effective strategies to control the high incidence of gastric cancer.With the advent and improvement of the high throughput technology, genomewide association study(GWAS) has become a powerful tool to study the genetic susceptibility of many complex diseases such as cancer. In recent years, there have been several gastric cancer GWASs in China, Japan and South Korea population finding that 1q22, 6p21.1, 7p15.3, 8q24.3, 10q23 and others associated with susceptibility to gastric cancer. We preliminary conducted a three stages GWAS of non-cardia gastric cancer, finding that 3q13.31(rs9841504) and 5p13.1(rs13361707) are the Chinese susceptibility regions of non-cardia gastric cancer. These new gastric cancer susceptibility loci will provide biomarkers for high-risk population screening in the near future.Functional study found that rs4072037, located in 2nd exon of MUC1(1q22), can influence the expression and physiological function of MUC1 in the gastric epithelial cells by controlling the alternative splicing at the 5’ end of this exon. The rs2294008 T>C, located in 1st exon of PSCA(8q24.3) can reduce PSCA expression of gastric cells by weakening the transcription activity of PSCA. The gastric cancer susceptibility loci found by GWAS will provide new clues to clarify mechanism of gastric cancer, and lay a foundation for screening out gastric cancer potential therapeutic targets.Though some new gastric cancer susceptibility loci GWAS found, they can explain only a small fraction of the gastric cancer susceptibility. With the high statistical efficiency, GWAS reduces the false positive results effectively, but may cover a large number of susceptible regions at the same time. Therefore, deep analysis based on the existed GWAS data is expected to further clarify the gastric cancer susceptibility. Meanwhile, there’s nearly none functional experiment on the gastric cancer susceptibility regions except 1q22 and 8q24.3. The biology mechanisms of other gastric cancer susceptibility regions haven’t yet been elucidated. Further functional studies are needed.In our previous GWAS of non-cardia gastric cancer, we genotyped 1033 noncardia gastric cancer cases and 2285 healthy controls(565 cases and 1162 controls from Nanjing, Jiangsu; 468 cases and 1123 controls from Beijing) with Affymetrix 6.0 Chips. In this study, we selected single nucleotide polymorphisms(SNPs) met the following criteria, which were not concerned in the previous GWAS: 1) having the same direction risks in the Nanjing and Beijing samples; 2) not the previous GWAS reported and verified loci and their high LD(r2>0.3) loci; 3)(1.0×10-5<P≤1.0×10-3 for all samples and P≤1.0×10-2 for both Nanjing and Beijing samples) or(P≤1.0×10-5 for all samples, P≤0.01 for Nanjing/Beijing samples and 0.01<P≤0.05 for Beijing/Nanjing samples); 4) having clear genotyping clusters; 5) the SNP with the lowest P value when multiple SNPs were observed in strong LD(r2>0.8). Finally, 11 SNPs were survived from the criteria and validated in two independent populations. The first-stage validation was performed in 1154 non-cardia gastric cancer cases and 1600 healthy controls from Jiangsu with Sequenom Mass ARRAY system. For the second-stage validation, we verified SNPs, significant in the first-stage validation, in 1687 non-cardia gastric cancer cases and 904 healthy controls from Wuhan and Beijing with the Taq Man platform.We conducted a system function prediction and an experimental verification on gastric cancer susceptibility loci that we found: 1) score these SNPs and their high LD(r2≥0.5) SNPs in Regulome DB 1.1 database, select SNPs with 1-3 points; 2) predict the effect of transcription factor binding ability when the base of SNP changes in TFsearch 1.3, Alibaba 2.1 and JASPAR 5.0_ALPHA databases, select the SNPs with consistent results as potential functional SNPs; 3) construct recombinant plasmid and conduct dual luciferase reporter gene experiment in Hela, BGC-823 and HGC-27 cell lines to verify the bioinformatics prediction results.We found that rs6882769 A>G at 5p13.1 can significantly reduce the non-cardia gastric cancer risk at GWAS scan stage [OR(95%CI): 0.72(0.62-0.83)] and two validations [for the first-stage validation, OR(95%CI): 0.79(0.70-0.90); for the second-stage validation, OR(95%CI): 0.81(0.71-0.93)]. After merging the data of three stages, rs6882769 G allele can reduce 20% risk of non-cardia gastric cancer [OR(95%CI): 0.78(0.72-0.84), P=2.15×10-10]. There’s no significant heterogeneity in populations with different age, gender, smoking and drinking status within stratified analysis. The SNP rs6882769 and rs13361707, which is 144 kb far apart and reported previously, are both at 5p13.1. The two loci have a certain LD correlation(r2=0.249). After conditional logistic analysis in the GWAS scan phase, we found they were independent susceptibility loci of non-cardia gastric cancer [rs6882769: OR(95%CI): 0.76(0.65-0.89), P=2.92×10-4; rs13361707: OR(95%CI): 1.31(1.15-1.50), P=3.12×10-5]. Our study verified 5p13.1 is the Chinese people non-cardia gastric cancer susceptibility region.To reveal the biological functions of the non-cardia gastric cancer susceptibility loci at 5p13.1, we conducted a system function prediction on rs6882769, rs13361707 and their high LD(r2>0.5) SNPs. The results showed rs59133000, which was complete linkage disequilibrium(r2=1.00) with rs13361707, was located in the binding region of transcription factor NF-κB(p65). The SNP rs59133000 was located in the PRKAA1 upstream region with the promoter transcription activity. We constructed report gene plasmids containing PRKAA1 upstream sequence with different rs59133000 alleles. After dual luciferase report gene experiment in Hela, BGC-823 and HGC-27 cell lines, we found the fluorescein expression level of the plasmid with rs59133000 T was significantly lower than the one with rs59133000 C(P<0.001). The rs59133000 T>C can significantly enhance PRKAA1 transcription.Based on our previous GWAS of non-cardia gastric cancer and current independent validation, we found rs6882769 was a new independent susceptibility locus of non-cardia gastric cancer in Chinese, and validated 5p13.1 was a susceptibility region of non-cardia gastric cancer in Chinese. Meanwhile, we also found rs59133000(5p13.1) T>C can increase the transcription of PRKAA1 significantly, which may affect the formation of non-cardia gastric cancer. This study further revealed the genetic susceptibility of gastric cancer in Chinese, provided clues to clarify the gastric cancer pathogenic mechanisms of the functional loci at 5p13.1, and provided important bases for genetic variations at 5p13.1 as biomarks to screen high-risk population, diagnose early and treat individualized. |
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