title: Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection.
abstract: OBJECTIVE
  The analysis of hepatitis B virus (HBV) X protein genetic variability and is correlation with liver disease severity have only been addressed, so far, on whole liver extracts. We have studied, therefore, the HBV X protein (HBx) gene sequence in morphologically well-characterised tumour and non-tumour liver cells from patients with HBV-related hepatocellular carcinoma.
  
  
  METHODS
  Using laser capture microdissection (LCM), we picked up six to eight groups of tumour and non-tumour hepatocytes in serial frozen sections from six patients. After global DNA preamplification followed by HBx-specific polymerase chain reaction, the HBx gene was sequenced in each group of microdissected cells. We also validated the quantification of HBV-DNA in microdissected hepatocytes using HBV Amplicor.
  
  
  RESULTS
  Heterogeneous mutations in HBx gene were found in distinct cirrhotic nodules and tumour areas from the same patient. Mutations at aa 127, 130 and 131 were frequently detected but there was no distinct point mutation profile between tumour and non-tumour samples. In contrast, deletions in HBx gene, which were found in five/six patients, were more frequent in tumour-derived sequences (6/18) than in non-tumour-derived sequences (1/20).
  
  
  CONCLUSIONS
  We have shown that LCM provides a direct insight of intrahepatic HBV infection. Using this technique, we demonstrated the persistence of distinct HBx encoding sequences in clonally expanding cells, thus supporting the hypothesis that HBx deletions may be implicated in liver carcinogenesis.
authors.affiliation: Centre National de Référence des Hépatites Virales, Institut Pasteur/CHU Necker, 156 rue de Vaugirard, 75730, Paris Cedex 15, France.
authors.forename: Massimo
authors.initials: M
authors.lastname: Iavarone
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Trabut
authors.forename: Oona
authors.initials: O
authors.lastname: Delpuech
authors.forename: Françoise
authors.initials: F
authors.lastname: Carnot
authors.forename: Massimo
authors.initials: M
authors.lastname: Colombo
authors.forename: Dina
authors.initials: D
authors.lastname: Kremsdorf
authors.forename: Christian
authors.initials: C
authors.lastname: Bréchot
authors.forename: Valérie
authors.initials: V
authors.lastname: Thiers
mesh_terms: D000328:Adult; D000368:Aged; D000595:Amino Acid Sequence; D006528:Carcinoma, Hepatocellular / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000032:analysis; D017353:Gene Deletion; D006509:Hepatitis B / Q000473:pathology / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007834:Lasers; D008113:Liver Neoplasms / Q000473:pathology / Q000821:virology*; D008297:Male; D042282:Microdissection; D008875:Middle Aged; D008969:Molecular Sequence Data; D017354:Point Mutation; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D004279:DNA, Viral; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein