studies

1028A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Disclosures:
Kazuaki Chayama - Consulting: AbbVie; Grant/Research Support: Ajinomoto,
Astellas, Torii, Tsumura, Aska, Bayer, Zeria, Daiichi Sankyo, Dainippon Sumitomo,
Eisai, Eli Lily, Janssen, Kowa, Mitsubishi Tanabe, MSD, Nippon Kayaku,
Nippon Shinyaku, Otsuka, Roche, Takeda, Toray; Speaking and Teaching:
Ajinomoto, AbbVie, Abott, Astellas, AstraZeneca, Aska, Bayer, BMS, Chugai,
Daiichi Sankyo, Dainippon Sumitomo, Eisai, J & J, Jimro, Miyarisan, MSD, Nihon
Kayaku, Olympus
The following authors have nothing to disclose: Yuji Ishida, Chihiro Yamasaki,
Ami Yanagi, Yasumi Yoshizane, Kazuyuki Fujikawa, Koichi Watashi, Hiromi
Abe, Takaji Wakita, Nelson Hayes, Chise Tateno
1680
Modeling Hepatits B Virus infection in stem cell derived
hepatocytes reveals role of perturbations in NTCP genotype
and expression in HBV permissiveness
Angela Frankel, Robert E. Schwartz; Weill Cornell Medical College,
New York, NY
Worldwide, hepatitis B virus (HBV) infection is the most common
viral hepatitis having infected over two billion people and
chronically infecting more than 400 million, putting them at
increased risk to develop cirrhosis and hepatocellular carcinoma.
HBV research has been hampered by the virus’s narrow
host range and cellular tropism for hepatocytes, which has led
to a paucity of robust and reliable infectious systems for HBV
study. Current model systems to study HBV include hepatoma
cell lines which do not faithfully recapitulate adult hepatocyte
phenotype or function as they have undergone a variety of
genetic and metabolic changes. As a consequence, major components
of the viral entry process and viral life cycle - including
the establishment and persistence of a nuclear cccDNA pool
- and many aspects of virus-host interactions have been poorly
understood. We have recently shown that micropatterned
co-cultures of primary human hepatocytes with stromal cells
and inducible pluripotent stem cells differentiated into hepatocyte-like
cells (iHeps) are permissive to HBV infection. iHeps
become permissive only during late stages of differentiation,
following the activation of the HBV transcription machinery,
and the appearance of the recently discovered HBV receptor
sodium-taurocholate co-transporter polypeptide (NTCP). Mutations
of NTCP have been shown to severely impair bile acid
uptake but the impact that particular variants play in HBV infection
is largely unknown. In particular the NTCP Ser267Phe variant
has recently been shown to be associated with resistance
to the development of chronic HBV in a recently completed
genome-wide association study. We hypothesized that mutations
in NTCP may be directly responsible for this association.
A gene editing approach using CrispR (and guide RNA’s targeting
the NTCP locus) along with homology directed repair
enabled the production of induced pluripotent stem cells (iPS)
with variant mutations in NTCP in the genomic NTCP locus.
Wild-type and variant NTCP iPS were differentiated into iHeps
and then inoculated with HBV containing serum. NTCP variants
had variable permissiveness for HBV infection. In particular the
Ser267Phe mutation impacts NTCP expression and HBV permissiveness
to HBV infection. These results exemplify the utility
of a physiologically relevant infectious system for studying HBV
interactions with relevant host cell genetics and physiology.
Combining these systems with gene editing technologies will
enable the dissection of clinically relevant mutations in physiologically
relevant human infectious systems.
Disclosures:
The following authors have nothing to disclose: Angela Frankel, Robert E.
Schwartz
1681
Genetic association study failed to confirm an association
between the NTCP S267F mutation and persistence
of hepatitis B virus or development of hepatocellular
carcinoma in the Japanese population
Daiki Miki 1,2 , Hidenori Ochi 1,2 , C. Nelson Hayes 1,2 , Hiromi Abe 1,2 ,
Sakura Akamatsu 1,2 , Atsushi Ono 1,2 , Takashi Nakahara 1,2 , Yizhou
Zhang 1,2 , Keiichi Masaki 1,2 , Hatsue Fujino 1,2 , Eisuke Miyaki 1,2 ,
Hiromi Kan 1,2 , Takuro Uchida 1,2 , Nobuhiko Hiraga 1,2 , Masataka
Tsuge 1,2 , Tomokazu Kawaoka 1,2 , Michio Imamura 1,2 , Yoshiiku
Kawakami 1,2 , Hiroshi Aikata 1,2 , Kazuaki Chayama 1,2 ; 1 Laboratory
for Digestive Diseases, RIKEN Center for Integrative Medical
Sciences, Hiroshima, Japan; 2 Department of Gastroenterology and
Metabolism, Hiroshima University Hospital, Hiroshima, Japan
Background & Aims: Sodium taurocholate cotransporting
polypeptide (NTCP) has long been known as a liver bile acid
transporter but was also recently identified as a hepatocyte
receptor for hepatitis B virus (HBV). The NTCP S267F mutation
has been reported to reduce taurocholate transporting activity
and result in defective HBV receptor function. This amino acid
substitution corresponds to a single nucleotide polymorphism
(SNP), rs2296651 is found in the Asian population but not in
European, African and American populations, according to the
1000 Genomes database. Moreover, this SNP frequency varies
widely even among Asian populations: 12% (Chinese Dai),
11% (Kinh (Vietnam)), 8% (Southern Han Chinese), 3% (Han
Chinese), and 2% (Japanese). In this study, we investigated
the importance of NTCP in HBV persistence and the development
of HBV-induced hepatocellular carcinoma (HCC) by
focusing on the S267F mutation using a large scale case-control
association analysis. Methods: We genotyped SNPs in
2,687 Japanese patients with chronic HBV infection (631 with
HCC and 2,056 without HCC) and 5,489 control individuals
without liver disease or cancer. Results: We found that minor
allele (T) frequencies of rs2296651 were 0.013 and 0.010 in
controls and chronic HBV patients, respectively. We observed
3 minor allele homozygotes (TT genotype) in controls. In contrast,
we did not observe any HBV patients with TT genotype.
However, we found no significant differences in rs2296651
frequencies between chronic HBV patients and controls in any
genetic models. We also compared chronic HBV patients with
and without HCC and found that their T allele frequencies were
0.007 and 0.011, respectively. Finally, we found no significant
association between the SNP genotype and development
of HCC with or without adjustment for age and gender. These
results might be partially affected by a low allele frequency
of rs2296651. Therefore, we examined another NTCP SNP,
rs4646287, which was reported as an intronic SNP associated
with HBV-induced HCC susceptibility in the Han Chinese
population and is more frequent in Japanese population than
rs2296651. Previously reported risk allele (A) frequencies of
rs4646287 were 0.144 and 0.142 in Japanese HBV patients
with and without HCC, respectively, and we again found no
significant association between SNP genotype and development
of HCC. Conclusions: We found no genetic association
between the S267F mutation of NTCP and HBV persistence or
the development of HBV-induced HCC in the Japanese population.
Further genetic analysis in populations with a higher
frequency of rs2296651 would improve understanding about
the role of NTCP in chronic HBV infection.