Kentaro Matsuura1, Hiromi Sawai2, Kazuho Ikeo3, Shintaro Ogawa4, Etsuko Iio1, Masanori Isogawa4, Noritomo Shimada5, Atsumasa Komori6, Hidenori Toyoda7, Takashi Kumada7, Tadashi Namisaki8, Hitoshi Yoshiji8, Naoya Sakamoto9, Mina Nakagawa10, Yasuhiro Asahina11, Masayuki Kurosaki12, Namiki Izumi12, Nobuyuki Enomoto13, Atsunori Kusakabe14, Eiji Kajiwara15, Yoshito Itoh16, Tatsuya Ide17, Akihiro Tamori18, Misako Matsubara18, Norifumi Kawada18, Ken Shirabe19, Eiichi Tomita20, Masao Honda21, Shuichi Kaneko21, Sohji Nishina22, Atsushi Suetsugu23, Yoichi Hiasa24, Hisayoshi Watanabe25, Takuya Genda26, Isao Sakaida27, Shuhei Nishiguchi28, Koichi Takaguchi29, Eiji Tanaka30, Junichi Sugihara31, Mitsuo Shimada32, Yasuteru Kondo33, Yosuke Kawai34, Kaname Kojima34, Masao Nagasaki34, Katsushi Tokunaga2, Yasuhito Tanaka35. 1. Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; Department of Gastroenterology and Metabolism, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. 2. Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 3. Center for Information Biology, National Institute of Genetics, Mishima, Japan. 4. Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. 5. Division of Gastroenterology and Hepatology, Otakanomori Hospital, Kashiwa, Japan. 6. Clinical Research Center, National Nagasaki Medical Center, Omura, Japan. 7. Department of Gastroenterology and Hepatology, Ogaki Municipal Hospital, Ogaki, Japan. 8. Third Department of Internal Medicine, Nara Medical University, Kashihara, Japan. 9. Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan; Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan. 10. Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan; Center for Interprofessional Education, Tokyo Medical and Dental University, Tokyo, Japan. 11. Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan; Department of Liver Disease Control, Tokyo Medical and Dental University, Tokyo, Japan. 12. Division of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Musashino, Japan. 13. First Department of Internal Medicine, University of Yamanashi, Chuo, Japan. 14. Division of Gastroenterology, Japanese Red Cross Nagoya Daini Hospital, Nagoya, Japan. 15. Department of Hepatology, Steel Memorial Yahata Hospital, Kitakyushu, Japan. 16. Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan. 17. Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Japan. 18. Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan. 19. Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan. 20. Department of Gastroenterology, Gifu Municipal Hospital, Gifu, Japan. 21. Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan. 22. Department of Hepatology and Pancreatology, Kawasaki Medical School, Kurashiki, Japan. 23. Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu, Japan. 24. Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Japan. 25. Department of Gastroenterology, Yamagata University Faculty of Medicine, Yamagata, Japan. 26. Department of Gastroenterology and Hepatology, Juntendo University Shizuoka Hospital, Shizuoka, Japan. 27. Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan. 28. Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan. 29. Department of Hepatology, Kagawa Prefectural Central Hospital, Takamatsu, Japan. 30. Department of Medicine, Shinshu University School of Medicine, Matsumoto, Japan. 31. Department of Gastroenterology, Gifu Prefectural General Medical Center, Gifu, Japan. 32. Department of Surgery, The University of Tokushima, Tokushima, Japan. 33. Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan. 34. Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan. 35. Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. Electronic address: ytanaka@med.nagoya-cu.ac.jp.
Abstract
BACKGROUND & AIMS: There is still a risk for hepatocellular carcinoma (HCC) development after eradication of hepatitis C virus (HCV) infection with antiviral agents. We investigated genetic factors associated with the development of HCC in patients with a sustained virologic response (SVR) to treatment for chronic HCV infection. METHODS: We obtained genomic DNA from 457 patients in Japan with a SVR to interferon-based treatment for chronic HCV infection from 2007 through 2015. We conducted a genome-wide association study (GWAS), followed by a replication analysis of 79 candidate single nucleotide polymorphisms (SNPs) in an independent set of 486 patients in Japan. The study end point was HCC diagnosis or confirmation of lack of HCC (at follow-up examinations until December 2014 in the GWAS cohort, and until January 2016 in the replication cohort). We collected clinical and laboratory data from all patients. We analyzed expression levels of candidate gene variants in human hepatic stellate cells, rats with steatohepatitis caused by a choline-deficient L-amino acid-defined diet, and a mouse model of liver injury caused by administration of carbon tetrachloride. We also analyzed expression levels in liver tissues of patients with chronic HCV infection with different stages of fibrosis or tumors vs patients without HCV infection (controls). RESULTS: We found a strong association between the SNP rs17047200, located within the intron of the tolloid like 1 gene (TLL1) on chromosome 4, and development of HCC; there was a genome-wide level of significance when the results of the GWAS and replication study were combined (odds ratio, 2.37; P = 2.66 × 10-8). Multivariate analysis showed rs17047200 AT/TT to be an independent risk factor for HCC (hazard ratio, 1.78; P = .008), along with male sex, older age, lower level of albumin, advanced stage of hepatic fibrosis, presence of diabetes, and higher post-treatment level of α-fetoprotein. Combining the rs17047200 genotype with other factors, we developed prediction models for HCC development in patients with mild or advanced hepatic fibrosis. Levels of TLL1 messenger RNA (mRNA) in human hepatic stellate cells increased with activation. Levels of Tll1 mRNA increased in liver tissues of rodents with hepatic fibrogenesis compared with controls. Levels of TLL1 mRNA increased in liver tissues of patients with progression of fibrosis. Gene expression levels of TLL1 short variants, including isoform 2, were higher in patients with rs17047200 AT/TT. CONCLUSIONS: In a GWAS, we identified the association between the SNP rs17047200, within the intron of TLL1, and development of HCC in patients who achieved an SVR to treatment for chronic HCV infection. We found levels of Tll1/TLL1 mRNA to be increased in rodent models of liver injury and liver tissues of patients with fibrosis, compared with controls. We propose that this SNP might affect splicing of TLL1 mRNA, yielding short variants with high catalytic activity that accelerates hepatic fibrogenesis and carcinogenesis. Further studies are needed to determine how rs17047200 affects TLL1 mRNA levels, splicing, and translation, as well as the prevalence of this variant among other patients with HCC. Tests for the TLL1 SNP might be used to identify patients at risk for HCC after an SVR to treatment of HCV infection.
BACKGROUND & AIMS: There is still a risk for hepatocellular carcinoma (HCC) development after eradication of hepatitis C virus (HCV) infection with antiviral agents. We investigated genetic factors associated with the development of HCC in patients with a sustained virologic response (SVR) to treatment for chronic HCV infection. METHODS: We obtained genomic DNA from 457 patients in Japan with a SVR to interferon-based treatment for chronic HCV infection from 2007 through 2015. We conducted a genome-wide association study (GWAS), followed by a replication analysis of 79 candidate single nucleotide polymorphisms (SNPs) in an independent set of 486 patients in Japan. The study end point was HCC diagnosis or confirmation of lack of HCC (at follow-up examinations until December 2014 in the GWAS cohort, and until January 2016 in the replication cohort). We collected clinical and laboratory data from all patients. We analyzed expression levels of candidate gene variants in human hepatic stellate cells, rats with steatohepatitis caused by a choline-deficient L-amino acid-defined diet, and a mouse model of liver injury caused by administration of carbon tetrachloride. We also analyzed expression levels in liver tissues of patients with chronic HCV infection with different stages of fibrosis or tumors vs patients without HCV infection (controls). RESULTS: We found a strong association between the SNP rs17047200, located within the intron of the tolloid like 1 gene (TLL1) on chromosome 4, and development of HCC; there was a genome-wide level of significance when the results of the GWAS and replication study were combined (odds ratio, 2.37; P = 2.66 × 10-8). Multivariate analysis showed rs17047200 AT/TT to be an independent risk factor for HCC (hazard ratio, 1.78; P = .008), along with male sex, older age, lower level of albumin, advanced stage of hepatic fibrosis, presence of diabetes, and higher post-treatment level of α-fetoprotein. Combining the rs17047200 genotype with other factors, we developed prediction models for HCC development in patients with mild or advanced hepatic fibrosis. Levels of TLL1 messenger RNA (mRNA) in human hepatic stellate cells increased with activation. Levels of Tll1 mRNA increased in liver tissues of rodents with hepatic fibrogenesis compared with controls. Levels of TLL1 mRNA increased in liver tissues of patients with progression of fibrosis. Gene expression levels of TLL1 short variants, including isoform 2, were higher in patients with rs17047200 AT/TT. CONCLUSIONS: In a GWAS, we identified the association between the SNP rs17047200, within the intron of TLL1, and development of HCC in patients who achieved an SVR to treatment for chronic HCV infection. We found levels of Tll1/TLL1 mRNA to be increased in rodent models of liver injury and liver tissues of patients with fibrosis, compared with controls. We propose that this SNP might affect splicing of TLL1 mRNA, yielding short variants with high catalytic activity that accelerates hepatic fibrogenesis and carcinogenesis. Further studies are needed to determine how rs17047200 affects TLL1 mRNA levels, splicing, and translation, as well as the prevalence of this variant among other patients with HCC. Tests for the TLL1 SNP might be used to identify patients at risk for HCC after an SVR to treatment of HCV infection.
Authors: Wen-Yue Liu; Mohammed Eslam; Kenneth I Zheng; Hong-Lei Ma; Rafael S Rios; Min-Zhi Lv; Gang Li; Liang-Jie Tang; Pei-Wu Zhu; Xiao-Dong Wang; Christopher D Byrne; Giovanni Targher; Jacob George; Ming-Hua Zheng Journal: J Clin Transl Hepatol Date: 2021-02-22