title: Development of Fok-I based nested polymerase chain reaction-restriction fragment length polymorphism analysis for detection of hepatitis B virus X region V5M mutation.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: 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.
authors.forename: Hong
authors.initials: H
authors.lastname: Kim
authors.affiliation: 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.
authors.forename: Seok-Hyun
authors.initials: SH
authors.lastname: Hong
authors.affiliation: 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.
authors.forename: Seoung-Ae
authors.initials: SA
authors.lastname: Lee
authors.affiliation: 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.
authors.forename: Jeong-Ryeol
authors.initials: JR
authors.lastname: Gong
authors.affiliation: 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.
authors.forename: Bum-Joon
authors.initials: BJ
authors.lastname: Kim
mesh_terms: D054458:Amplified Fragment Length Polymorphism Analysis / Q000379:methods*; D044466:Asian People; D006528:Carcinoma, Hepatocellular / Q000175:diagnosis / Q000208:ethnology / Q000821:virology; D004252:DNA Mutational Analysis / Q000379:methods*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D018450:Disease Progression; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000150:complications / Q000175:diagnosis / Q000208:ethnology / Q000821:virology*; D006801:Humans; D008113:Liver Neoplasms / Q000175:diagnosis / Q000208:ethnology / Q000821:virology; D009154:Mutation; D010641:Phenotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D056910:Republic of Korea / Q000453:epidemiology; D015534:Trans-Activators / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Hepatitis B virus; Hepatocellur carcinoma; Polymerase chain reaction-restriction fragment length polymorphism analysis; V5M mutation; X antigen