Novel HBsAg mutations correlate with hepatocellular carcinoma, hamper HBsAg secretion and promote cell proliferation in vitro.
BACKGROUND
An impaired HBsAg-secretion can increase HBV oncogenic-properties. Here, we investigate genetic-determinants in HBsAg correlated with HBV-induced hepatocellular carcinoma (HCC), and their impact on HBsAg-secretion and cell-proliferation.


METHODS
This study included 128 chronically HBV-infected patients: 23 with HCC (73.9% D; 26.1% A HBV-genotype), and 105 without cirrhosis/HCC (72.4% D, 27.6% A) as reference-group. The impact of mutations on HBsAg-secretion was assessed by measuring the ratio [secreted/intracellular HBsAg] until day 5 post-transfection. The impact of mutations on cell-cycle advancement was assessed by flow-cytometry.


RESULTS
Two HBsAg mutations significantly correlated with HCC: P203Q (17.4% [4/23] in HCC vs 1.0% [1/105] in non-HCC, P=0.004); S210R (34.8% [8/23] in HCC vs 3.8% [4/105] in non-HCC, P <0.001); P203Q+S210R (17.4% [4/23] in HCC vs 0% [0/110] in non-HCC, P=0.001). Both mutations reside in trans-membrane C-terminal domain critical for HBsAg-secretion. In in-vitro experiments, P203Q, S210R and P203Q+S210R significantly reduced the ratio [secreted/intracellular HBsAg] compared to wt at each time-point analysed (P <0.05), supporting an impaired HBsAg-secretion. Furthermore, P203Q and P203Q+S210R increased the percentage of cells in S-phase compared to wt, indicating cell-cycle progression (P203Q:26±13%; P203Q+S210R:29±14%; wt:18%±9, P <0.01. Additionally, S210R increased the percentage of cells in G2/M-phase (26±8% for wt versus 33±6% for S210R, P <0.001).


CONCLUSIONS
Specific mutations in HBsAg C-terminus significantly correlate with HBV-induced HCC. They hamper HBsAg-secretion and are associated with increased cellular proliferation, supporting their involvement in HCC-development. The identification of viral genetic markers associated with HCC is critical to identify patients at higher HCC-risk that may deserve intensive liver monitoring, and/or early anti-HBV therapy.
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Romina
R
Salpini
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Matteo
M
Surdo
Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia.
Nadia
N
Warner
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Maria Francesca
MF
Cortese
Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia.
Danny
D
Colledge
Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia.
Sally
S
Soppe
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Maria Concetta
MC
Bellocchi
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Daniele
D
Armenia
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Luca
L
Carioti
Laboratory of Monitoring Antiviral Drugs, National Institute for Infectious Diseases (INMI) "Lazzaro Spallanzani" Rome, Italy.
Fabio
F
Continenza
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Domenico
D
Di Carlo
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Patrizia
P
Saccomandi
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.|Institut Pasteur, Unité de Biologie des Virus Entériques, Paris, France.
Carmen
C
Mirabelli
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Michela
M
Pollicita
Unit of Microbiology, "S. Pertini Hospital", Rome, Italy.
Roberta
R
Longo
Unit of Microbiology, "S. Pertini Hospital", Rome, Italy.
Sara
S
Romano
Unit of Microbiology, "S. Pertini Hospital", Rome, Italy.
Giuseppina
G
Cappiello
Unit of Microbiology, "S. Pertini Hospital", Rome, Italy.
Alberto
A
Spanò
Laboratoire de Microbiologie Fondamentale et Pathogénicité, Hôpital Pellegrin Tripode, Bordeaux, France.
Pascale
P
Trimoulet
Laboratoire de Microbiologie Fondamentale et Pathogénicité, Hôpital Pellegrin Tripode, Bordeaux, France.
Herve
H
Fleury
Department of Medicine and Aging Sciences, "SS Annunziata" Hospital, Chieti, Italy.
Jacopo
J
Vecchiet
Infectious Diseases Unit, "S Salvatore" Hospital, L'Aquila, Italy.
Nerio
N
Iapadre
Hepatology Unit, "S Giacomo" Hospital, Rome, Italy.
Angelo
A
Barlattani
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Ada
A
Bertoli
Hepatology Unit, "Regina Margherita" Hospital, Rome, Italy.
Terenzio
T
Mari
Hepato-Infectivology Unit, "S Andrea" Hospital, Rome, Italy.
Caterina
C
Pasquazzi
Hospital of Parma, Parma, Italy.
Gabriele
G
Missale
Tor Vergata University Hospital, Infectious Diseases Unit, Rome, Italy.
Cesare
C
Sarrecchia
Department of Biomedicine and Prevention, University of Rome "Tor Vergata" Rome, Italy.
Elisa
E
Orecchini
Department of Biomedicine and Prevention, University of Rome "Tor Vergata" Rome, Italy.
Alessandro
A
Michienzi
Tor Vergata University Hospital, Infectious Diseases Unit, Rome, Italy.
Massimo
M
Andreoni
Tor Vergata University Hospital, Hepatology Unit, Rome, Italy.
Simona
S
Francioso
Tor Vergata University Hospital, Hepatology Unit, Rome, Italy.
Mario
M
Angelico
Tor Vergata University Hospital, Hepatology Unit, Rome, Italy.
Jens
J
Verheyen
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Francesca
F
Ceccherini-Silberstein
Research and Molecular Development, Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia.
Stephen
S
Locarnini
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Carlo Federico
CF
Perno
Department of Experimental Medicine and Surgery, University of Rome "Tor Vergata" Rome, Italy.
Valentina
V
Svicher
D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000473:pathology* / Q000821:virology; D002453:Cell Cycle; D049109:Cell Proliferation; D005260:Female; D005787:Gene Frequency; D005838:Genotype; D006514:Hepatitis B Surface Antigens / Q000235:genetics* / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D019694:Hepatitis B, Chronic / Q000473:pathology* / Q000821:virology; D054884:Host-Pathogen Interactions; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology* / Q000821:virology; D008297:Male; D008875:Middle Aged; D015999:Multivariate Analysis; D009154:Mutation; D012307:Risk Factors
D006514:Hepatitis B Surface Antigens
HBsAg mutations; cell proliferation; hepatitis B; hepatitis B surface antigen; hepatocellular carcinoma