title: Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases.
abstract: OBJECTIVE
  PreS2-defective hepatitis B virus (HBV) variants may emerge during chronic HBV infection. These variants carry mutation(s) at the ATG-start-codon and/or in-frame deletion into the preS2 genomic region and are commonly detected by sequencing analyses. We evaluated the prevalence of these variants in a large series of chronic HBV infected patients through non-sequencing molecular approaches.
  
  
  METHODS
  We examined HBV isolates from 110 HBV carriers: 15 were inactive carriers (IC); 50 had chronic hepatitis (CH); 25 were cirrhotics; 19 had hepatocellular carcinoma (HCC). The entire preS2 genomic region was amplified by PCR technique. The amplicons were processed: (A) through electrophoresis on acrylamide gel to reveal deleted genomes; (B) through electrophoresis on agarose gel after digestion by NlaIII enzyme that cuts the wild ATG-start-codon but not the mutated one.
  
  
  RESULTS
  We detected preS2 variants in 56/110 cases (51%). In particular, we found preS2-defective mutants in 2/15 IC, 25/50 CH, 13/26 cirrhotics, and 16/19 HCC. The presence of these variants was thus significantly associated with active infection and liver disease (P<0.002). Moreover, among cases with liver disease preS2-mutants were more prevalent in HCC patients (P<0.02).
  
  
  CONCLUSIONS
  Our non-sequencing molecular methods are sensitive and specific, and simplify the identification of all preS2 HBV variant forms. Infection by these variants is significantly associated with active infection and HCC.
authors.affiliation: Unità di Epatologia Clinica e Biomolecolare, Dipartimento di Medicina Interna, Policlinico Universitario di Messina, 98124 Messina, Italy. raimondo@unime.it
authors.forename: Giovanni
authors.initials: G
authors.lastname: Raimondo
authors.forename: Lucy
authors.initials: L
authors.lastname: Costantino
authors.forename: Gaia
authors.initials: G
authors.lastname: Caccamo
authors.forename: Teresa
authors.initials: T
authors.lastname: Pollicino
authors.forename: Giovanni
authors.initials: G
authors.lastname: Squadrito
authors.forename: Irene
authors.initials: I
authors.lastname: Cacciola
authors.forename: Santa
authors.initials: S
authors.lastname: Brancatelli
mesh_terms: D006528:Carcinoma, Hepatocellular / Q000235:genetics; D002353:Carrier State / Q000503:physiopathology; D018387:Codon, Initiator / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005787:Gene Frequency; D005821:Genetic Techniques; D014644:Genetic Variation; D016679:Genome, Viral; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000235:genetics; D006801:Humans; D008103:Liver Cirrhosis / Q000235:genetics; D008107:Liver Diseases / Q000453:epidemiology / Q000235:genetics / Q000503:physiopathology* / Q000821:virology*; D008113:Liver Neoplasms / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D015995:Prevalence; D011498:Protein Precursors / Q000235:genetics*; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index
chemical_list: D018387:Codon, Initiator; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen