Characterisation of hepatitis B virus X protein mutants in tumour and non-tumour liver cells using laser capture microdissection

Massimo Iavarone, Jean Baptiste Trabut, Oona Delpuech, Françoise Carnot, Massimo Colombo, Dina Kremsdorf, Christian Bréchot, Valérie Thiers

Research output: Contribution to journalArticle

Abstract

Background/Aims: 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.

Original languageEnglish
Pages (from-to)253-261
Number of pages9
JournalJournal of Hepatology
Volume39
Issue number2
DOIs
Publication statusPublished - Aug 1 2003

Keywords

  • Hepatitis B
  • Hepatocellular carcinoma
  • Laser capture microdissection
  • X protein

ASJC Scopus subject areas

  • Gastroenterology

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