Genome-Wide Association Study Identifies TLL1 Variant Associated With Development of Hepatocellular Carcinoma After Eradication of Hepatitis C Virus Infection.
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.



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).



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.



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.
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.
Kentaro
K
Matsuura
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Hiromi
H
Sawai
Center for Information Biology, National Institute of Genetics, Mishima, Japan.
Kazuho
K
Ikeo
Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Shintaro
S
Ogawa
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.
Etsuko
E
Iio
Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan.
Masanori
M
Isogawa
Division of Gastroenterology and Hepatology, Otakanomori Hospital, Kashiwa, Japan.
Noritomo
N
Shimada
Clinical Research Center, National Nagasaki Medical Center, Omura, Japan.
Atsumasa
A
Komori
Department of Gastroenterology and Hepatology, Ogaki Municipal Hospital, Ogaki, Japan.
Hidenori
H
Toyoda
Department of Gastroenterology and Hepatology, Ogaki Municipal Hospital, Ogaki, Japan.
Takashi
T
Kumada
Third Department of Internal Medicine, Nara Medical University, Kashihara, Japan.
Tadashi
T
Namisaki
Third Department of Internal Medicine, Nara Medical University, Kashihara, Japan.
Hitoshi
H
Yoshiji
Department of Internal Medicine, Hokkaido University Graduate School of Medicine, Sapporo, Japan; Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan.
Naoya
N
Sakamoto
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan; Center for Interprofessional Education, Tokyo Medical and Dental University, Tokyo, Japan.
Mina
M
Nakagawa
Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan; Department of Liver Disease Control, Tokyo Medical and Dental University, Tokyo, Japan.
Yasuhiro
Y
Asahina
Division of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Musashino, Japan.
Masayuki
M
Kurosaki
Division of Gastroenterology and Hepatology, Musashino Red Cross Hospital, Musashino, Japan.
Namiki
N
Izumi
First Department of Internal Medicine, University of Yamanashi, Chuo, Japan.
Nobuyuki
N
Enomoto
Division of Gastroenterology, Japanese Red Cross Nagoya Daini Hospital, Nagoya, Japan.
Atsunori
A
Kusakabe
Department of Hepatology, Steel Memorial Yahata Hospital, Kitakyushu, Japan.
Eiji
E
Kajiwara
Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Yoshito
Y
Itoh
Division of Gastroenterology, Department of Medicine, Kurume University, Kurume, Japan.
Tatsuya
T
Ide
Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
Akihiro
A
Tamori
Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
Misako
M
Matsubara
Department of Hepatology, Graduate School of Medicine, Osaka City University, Osaka, Japan.
Norifumi
N
Kawada
Department of Surgery and Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Ken
K
Shirabe
Department of Gastroenterology, Gifu Municipal Hospital, Gifu, Japan.
Eiichi
E
Tomita
Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
Masao
M
Honda
Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa, Japan.
Shuichi
S
Kaneko
Department of Hepatology and Pancreatology, Kawasaki Medical School, Kurashiki, Japan.
Sohji
S
Nishina
Department of Gastroenterology, Gifu University Graduate School of Medicine, Gifu, Japan.
Atsushi
A
Suetsugu
Department of Gastroenterology and Metabology, Ehime University Graduate School of Medicine, Toon, Japan.
Yoichi
Y
Hiasa
Department of Gastroenterology, Yamagata University Faculty of Medicine, Yamagata, Japan.
Hisayoshi
H
Watanabe
Department of Gastroenterology and Hepatology, Juntendo University Shizuoka Hospital, Shizuoka, Japan.
Takuya
T
Genda
Department of Gastroenterology and Hepatology, Yamaguchi University Graduate School of Medicine, Ube, Japan.
Isao
I
Sakaida
Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan.
Shuhei
S
Nishiguchi
Department of Hepatology, Kagawa Prefectural Central Hospital, Takamatsu, Japan.
Koichi
K
Takaguchi
Department of Medicine, Shinshu University School of Medicine, Matsumoto, Japan.
Eiji
E
Tanaka
Department of Gastroenterology, Gifu Prefectural General Medical Center, Gifu, Japan.
Junichi
J
Sugihara
Department of Surgery, The University of Tokushima, Tokushima, Japan.
Mitsuo
M
Shimada
Division of Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Yasuteru
Y
Kondo
Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
Yosuke
Y
Kawai
Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
Kaname
K
Kojima
Department of Integrative Genomics, Tohoku Medical Megabank Organization, Tohoku University, Sendai, Japan.
Masao
M
Nagasaki
Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Katsushi
K
Tokunaga
Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan. Electronic address: ytanaka@med.nagoya-cu.ac.jp.
Yasuhito
Y
Tanaka
D000367:Age Factors; D000368:Aged; D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D002251:Carbon Tetrachloride; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D016022:Case-Control Studies; D002794:Choline / Q000008:administration & dosage; D048909:Diabetes Complications / Q000150:complications; D005234:Fatty Liver / Q000209:etiology / Q000235:genetics*; D005260:Female; D055106:Genome-Wide Association Study; D055166:Hepatic Stellate Cells / Q000378:metabolism; D019698:Hepatitis C, Chronic / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007438:Introns; D008103:Liver Cirrhosis / Q000235:genetics / Q000378:metabolism; D008113:Liver Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000821:virology; D008297:Male; D051379:Mice; D008875:Middle Aged; D020641:Polymorphism, Single Nucleotide; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D012307:Risk Factors; D012709:Serum Albumin / Q000378:metabolism; D012737:Sex Factors; D000072230:Sustained Virologic Response; D055394:Tolloid-Like Metalloproteinases / Q000235:genetics*; D000509:alpha-Fetoproteins / Q000378:metabolism
D000998:Antiviral Agents; D012333:RNA, Messenger; D012709:Serum Albumin; D000509:alpha-Fetoproteins; D002251:Carbon Tetrachloride; D055394:Tolloid-Like Metalloproteinases; C484478:TLL1 protein, human; D002794:Choline
Genetics; Liver Cancer; Metalloprotease; Mutation