Meng Zhu1, Caiwang Yan2, Chuanli Ren3, Xiaodan Huang4, Xun Zhu2, Haiyong Gu5, Meilin Wang6, Shouyu Wang7, Yong Gao8, Yong Ji9, Xiaoping Miao10, Ming Yang11, Jinfei Chen12, Jiangbo Du2, Tongtong Huang2, Yue Jiang2, Juncheng Dai2, Hongxia Ma2, Jianwei Zhou7, Zhaoming Wang2, Zhibin Hu2, Guozhong Ji4, Zhengdong Zhang6, Hongbing Shen13, Yongyong Shi14, Guangfu Jin15. 1. Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China; State Key Laboratory of Reproductive Medicine, International Joint Research Center for Environment and Human Health, Nanjing Medical University, Nanjing, China. 2. Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China. 3. Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China; Clinical Medical Testing Laboratory, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, Yangzhou, China. 4. Institute of Digestive Endoscopy and Medical Center for Digestive Diseases, Second Affiliated Hospital of Nanjing Medical University, Nanjing, China. 5. Department of Cardiothoracic Surgery, Affiliated People's Hospital of Jiangsu University, Zhenjiang, China. 6. Department of Environmental Genomics, Jiangsu Key Laboratory of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, Nanjing Medical University, Nanjing, China. 7. Department of Molecular Cell Biology and Toxicology, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China. 8. Department of Medical Oncology, The Affiliated Huai'an First People's Hospital of Nanjing Medical University, Huai'an, China. 9. Department of Cardiothoracic Surgery, The Affiliated Wuxi People's Hospital of Nanjing Medical University, Wuxi, China. 10. MOE Key Lab of Environment and Health, School of Public Health, Tongji Medical College, Huazhong University of Science & Technology, Wuhan, China. 11. Shandong Provincial Key Laboratory of Radiation Oncology, Cancer Research Center, Shandong Cancer Hospital Affiliated to Shandong University, Shandong Academy of Medical Sciences, Jinan, China. 12. Department of Oncology, Nanjing First Hospital, Nanjing Medical University, Nanjing, China. 13. Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China. Electronic address: hbshen@njmu.edu.cn. 14. Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Bio-X Institutes, Shanghai Jiao Tong University, Shanghai, China. Electronic address: shiyongyong@gmail.com. 15. Department of Epidemiology and Biostatistics, Jiangsu Key Lab of Cancer Biomarkers, Prevention and Treatment, Collaborative Innovation Center for Cancer Medicine, School of Public Health, Nanjing Medical University, Nanjing, China; State Key Laboratory of Reproductive Medicine, International Joint Research Center for Environment and Human Health, Nanjing Medical University, Nanjing, China. Electronic address: guangfujin@njmu.edu.cn.
Abstract
BACKGROUND & AIMS: Several genetic variants have been associated with gastric cancer risk, although these account for only a fraction of cases of gastric cancer. We aimed to identify low-frequency and other genetic variants that determine gastric cancer susceptibility. METHODS: We performed exome array analysis of DNA in blood samples from 1113 patients with gastric cancer, collected at hospitals from 2006 to 2010 in China, and 1848 individuals without cancer (controls) undergoing physical examinations. Among 71,290 variants analyzed (including 25,784 common variants), 24 variants were selected and replicated in an analysis of DNA in blood samples from 4687 additional cases of gastric cancer and 5780 controls. We compared expression of candidate genes in tumor vs normal gastric tissues using data from TCGA and performed functional annotation analyses. An immortalized human gastric epithelial cell line (GES1) and 7 human gastric cancer lines were used to express transgenes, knock down gene expression (with small interfering RNAs), disrupt genes (using the CRISPR/Cas9 system), or assess expression of reporter constructs. We measured cell proliferation, colony formation, invasion, and migration, and assessed growth of xenograft tumors in nude mice. RESULTS: A low-frequency missense variant rs112754928 in the SPOC domain containing 1 gene (SPOCD1; encoding p.Arg71Trp), at 1p35.2, was reproducibly associated with reduced risk of gastric cancer (odds ratio, 0.56; P = 3.48 × 10-8). SPOCD1 was overexpressed in gastric tumors, and knockout of SPOCD1 reduced gastric cancer cell proliferation, invasive activity, and migration, as well as growth of xenograft tumors in nude mice. We also associated the variant rs1679709 at 6p22.1 with reduced risk for gastric cancer (odds ratio, 0.80; P = 1.17 × 10-13). The protective allele rs1679709-A correlated with the surrounding haplotype rs2799077-T-rs2799079-C, which reduced the enhancer activity of this site to decrease expression of the butyrophilin subfamily 3 member A2 gene (BTN3A2). BTN3A2 is overexpressed in gastric tumors, and deletion of BTN3A2 inhibited proliferation, migration, and invasion of gastric cancer cells. CONCLUSIONS: We have associated variants at 1p35.2 and 6p22.1 with gastric cancer risk, indicating a role for SPOCD1 and BTN3A2 in gastric carcinogenesis.
BACKGROUND & AIMS: Several genetic variants have been associated with gastric cancer risk, although these account for only a fraction of cases of gastric cancer. We aimed to identify low-frequency and other genetic variants that determine gastric cancer susceptibility. METHODS: We performed exome array analysis of DNA in blood samples from 1113 patients with gastric cancer, collected at hospitals from 2006 to 2010 in China, and 1848 individuals without cancer (controls) undergoing physical examinations. Among 71,290 variants analyzed (including 25,784 common variants), 24 variants were selected and replicated in an analysis of DNA in blood samples from 4687 additional cases of gastric cancer and 5780 controls. We compared expression of candidate genes in tumor vs normal gastric tissues using data from TCGA and performed functional annotation analyses. An immortalized human gastric epithelial cell line (GES1) and 7 humangastric cancer lines were used to express transgenes, knock down gene expression (with small interfering RNAs), disrupt genes (using the CRISPR/Cas9 system), or assess expression of reporter constructs. We measured cell proliferation, colony formation, invasion, and migration, and assessed growth of xenograft tumors in nude mice. RESULTS: A low-frequency missense variant rs112754928 in the SPOC domain containing 1 gene (SPOCD1; encoding p.Arg71Trp), at 1p35.2, was reproducibly associated with reduced risk of gastric cancer (odds ratio, 0.56; P = 3.48 × 10-8). SPOCD1 was overexpressed in gastric tumors, and knockout of SPOCD1 reduced gastric cancer cell proliferation, invasive activity, and migration, as well as growth of xenograft tumors in nude mice. We also associated the variant rs1679709 at 6p22.1 with reduced risk for gastric cancer (odds ratio, 0.80; P = 1.17 × 10-13). The protective allele rs1679709-A correlated with the surrounding haplotype rs2799077-T-rs2799079-C, which reduced the enhancer activity of this site to decrease expression of the butyrophilin subfamily 3 member A2 gene (BTN3A2). BTN3A2 is overexpressed in gastric tumors, and deletion of BTN3A2 inhibited proliferation, migration, and invasion of gastric cancer cells. CONCLUSIONS: We have associated variants at 1p35.2 and 6p22.1 with gastric cancer risk, indicating a role for SPOCD1 and BTN3A2 in gastric carcinogenesis.