Observational Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Dec 21, 2015; 21(47): 13360-13367
Published online Dec 21, 2015. doi: 10.3748/wjg.v21.i47.13360
Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation
Hong Kim, Seok-Hyun Hong, Seoung-Ae Lee, Jeong-Ryeol Gong, Bum-Joon Kim
Hong Kim, Seok-Hyun Hong, Seoung-Ae Lee, Jeong-Ryeol Gong, Bum-Joon Kim, Department of Biomedical Sciences, Microbiology and Immunology, and Liver Research Institute, Seoul National University College of Medicine, Seoul 110-799, South Korea
Author contributions: Kim H and Hong SH contributed equally to this work; Kim BJ was the guarantor and designed the study; Kim H and Hong SH participated in the acquisition, analysis, and interpretation of the data, and drafted the initial manuscript; Lee SA, Gong JR revised the article critically for important intellectual content.
Supported by a National Research Foundation (NRF) of Korea grant funded by the Korean government (Ministry of Education, Science, and Technology, MEST), Grant No. 2013-005810.
Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of Seoul National University Hospital (Seoul) (IRB No. 1404-070-572).
Informed consent statement: All study participants, or their legal guardian, provided informed written consent prior to study enrollment.
Conflict-of-interest statement: There are no conflicts of interest to report.
Data sharing statement: No additional data are available.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Bum-Joon Kim, Professor, Department of Biomedical Sciences, Microbiology and Immunology, and Liver Research Institute, Seoul National University College of Medicine, Seoul 110-799, South Korea. kbumjoon@snu.ac.kr
Telephone: +82-2-7408316 Fax: +82-2-7430881
Received: June 8, 2015
Peer-review started: June 11, 2015
First decision: July 10, 2015
Revised: July 17, 2015
Accepted: September 30, 2015
Article in press: September 30, 2015
Published online: December 21, 2015
Processing time: 190 Days and 0.5 Hours
Abstract

AIM: To develop a Fok-I nested polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) method for the detection of hepatitis B virus X region (HBx) V5M mutation.

METHODS: Nested PCR was applied into DNAs from 198 chronic patients at 2 different stages [121 patients with hepatocellular carcinoma (HCC) and 77 carrier patients]. To identify V5M mutants, digestion of nested PCR amplicons by the restriction enzyme Fok-I (GGA TGN9↓) was done. For size comparison, the enzyme-treated products were analyzed by electrophoresis on 2.5% agarose gels, stained with ethidium bromide, and visualized on a UV transilluminator.

RESULTS: The assay enabled the identification of 69 patients (sensitivity of 34.8%; 46 HCC patients and 23 carrier patients). Our data also showed that V5M prevalence in HCC patients was significantly higher than in carrier patients (47.8%, 22/46 patients vs 0%, 0/23 patients, P < 0.001), suggesting that HBxAg V5M mutation may play a pivotal role in HCC generation in chronic patients with genotype C infections.

CONCLUSION: The Fok-I nested PRA developed in this study is a reliable and cost-effective method to detect HBxAg V5M mutation in chronic patients with genotype C2 infection.

Keywords: Hepatitis B virus; X antigen; Polymerase chain reaction-restriction fragment length polymorphism analysis; V5M mutation; Hepatocellur carcinoma

Core tip: In the present study, we developed a reliable and cost-effective Fok-I nested polymerase chain reaction-restriction fragment length polymorphism analysis (PRA) method for the detection of V5M from chronic patients with genotype C2 infection. In addition, our epidemiological data based on the Fok-I nested PRA method strongly support the previous reports that V5M may play a very pivotal role in hepatocarcinogenesis, at least in chronic patients infected with genotype C2.