Non-sequencing molecular approaches to identify preS2-defective hepatitis B virus variants proved to be associated with severe liver diseases.
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.
Raimondo
Giovanni
G
Unità di Epatologia Clinica e Biomolecolare, Dipartimento di Medicina Interna, Policlinico Universitario di Messina, 98124 Messina, Italy. raimondo@unime.it
Costantino
Lucy
L
Caccamo
Gaia
G
Pollicino
Teresa
T
Squadrito
Giovanni
G
Cacciola
Irene
I
Brancatelli
Santa
S
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
D018387:Codon, Initiator; D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors; C050807:presurface protein 2, hepatitis B surface antigen