title: Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV.
abstract: OBJECTIVE
  HBV has two forms of genomic DNA, relaxed-circular DNA (rcDNA) and duplex-linear DNA (dlDNA). Compared to rcDNA, dlDNA has been demonstrated to integrate more frequently into host cellular chromosomes, which may have oncogenic consequences. However, the dlDNA proportion relative to total HBV DNA and its clinical significance in patients remain to be investigated.
  
  
  METHODS
  Based on the structural difference between rcDNA and dlDNA, we developed a peptide nucleic acid (PNA)-mediated quantitative real-time PCR (qPCR) clamping assay to measure the proportions of dlDNA in total HBV DNA in sera obtained from patients with chronic hepatitis B (CHB), liver cirrhosis (LC) or LC-developed hepatocellular carcinoma (HCC). The factors that influence the proportion of dlDNA were also investigated.
  
  
  RESULTS
  The average dlDNA proportion was approximately 7% in the sera of chronic HBV-infected patients and was elevated in CHB patients with abnormal levels of alanine aminotransferase. The sera dlDNA proportions increased to approximately 14% and 20% in the patients with LC and HCC, respectively. Interferon-α treatment slightly increased the dlDNA proportion in the responders; and nucleotide analogue therapy spuriously elevated the proportion. Moreover, treatment of human hepatoma cells supporting HBV replication with inflammatory cytokines significantly altered the dlDNA proportion in vitro.
  
  
  CONCLUSIONS
  Using a novel PNA-mediated qPCR clamping assay, we first showed that serum dlDNA proportions progressively increased during the development of HBV-related liver diseases. The dlDNA proportion can be regulated by inflammatory cytokines, suggesting an association among inflammation, increased production of HBV dlDNA and development of HCC.
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Xing-Liang
authors.initials: XL
authors.lastname: Zhao
authors.affiliation: Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, P.R. China.
authors.forename: Jian-Rong
authors.initials: JR
authors.lastname: Yang
authors.affiliation: Department of Hepato-Biliary-Pancreatic Surgery, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, Zhejiang, P.R. China.
authors.forename: Sheng-Zhang
authors.initials: SZ
authors.lastname: Lin
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Hui
authors.initials: H
authors.lastname: Ma
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China Department of Microbiology and Immunology, Drexel Institute for Biotechnology and Virology Research, Drexel University College of Medicine, Doylestown, Pennsylvania, USA.
authors.forename: Fang
authors.initials: F
authors.lastname: Guo
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Rui-Feng
authors.initials: RF
authors.lastname: Yang
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Heng-Hui
authors.initials: HH
authors.lastname: Zhang
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Jin-Chao
authors.initials: JC
authors.lastname: Han
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Lai
authors.initials: L
authors.lastname: Wei
authors.affiliation: Peking University People's Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, Beijing, P.R. China.
authors.forename: Xiao-Ben
authors.initials: XB
authors.lastname: Pan
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D006528:Carcinoma, Hepatocellular / Q000097:blood / Q000473:pathology / Q000821:virology*; D004279:DNA, Viral / Q000097:blood*; D018450:Disease Progression; D005260:Female; D006515:Hepatitis B virus / Q000235:genetics*; D019694:Hepatitis B, Chronic / Q000097:blood / Q000473:pathology / Q000821:virology*; D006801:Humans; D008103:Liver Cirrhosis / Q000097:blood / Q000473:pathology / Q000821:virology*; D008113:Liver Neoplasms / Q000097:blood / Q000473:pathology / Q000821:virology*; D008297:Male; D008875:Middle Aged; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D004279:DNA, Viral
keywords: CYTOKINES; HEPATITIS B; HEPATOCELLULAR CARCINOMA; INFLAMMATION; POLYMERASE CHAIN REACTION