title: Novel approach to identifying the hepatitis B virus pre-S deletions associated with hepatocellular carcinoma.
abstract: OBJECTIVE
  To develop a novel non-sequencing method for the detection of hepatitis B virus (HBV) pre-S deletion mutants in HBV carriers.
  
  
  METHODS
  The entire region of HBV pre-S1 and pre-S2 was amplified by polymerase chain reaction (PCR). The size of PCR products was subsequently determined by capillary gel electrophoresis (CGE). CGE were carried out in a PACE-MDQ instrument equipped with a UV detector set at 254 nm. The samples were separated in 50 μm ID eCAP Neutral Coated Capillaries using a voltage of 6 kV for 30 min. Data acquisition and analysis were performed using the 32 Karat Software. A total of 114 DNA clones containing different sizes of the HBV pre-S gene were used to determine the accuracy of the CGE method. One hundred and fifty seven hepatocellular carcinoma (HCC) and 160 non-HCC patients were recruited into the study to assess the association between HBV pre-S deletion and HCC by using the newly-established CGE method. Nine HCC cases with HBV pre-S deletion at the diagnosis year were selected to conduct a longitudinal observation using serial serum samples collected 2-9 years prior to HCC diagnosis.
  
  
  RESULTS
  CGE allowed the separation of PCR products differing in size > 3 bp and was able to identify 10% of the deleted DNA in a background of wild-type DNA. The accuracy rate of CGE-based analysis was 99.1% compared with the clone sequencing results. Using this assay, pre-S deletion was more frequently found in HCC patients than in non-HCC controls (47.1% vs 28.1%, P < 0.001). Interestingly, the increased risk of HCC was mainly contributed by the short deletion of pre-S. While the deletion ≤ 99 bp was associated with a 2.971-fold increased risk of HCC (95%CI: 1.723-5.122, P < 0.001), large deletion (> 99 bp) did not show any association with HCC (P = 0.918, OR = 0.966, 95%CI: 0.501-1.863). Of the 9 patients who carried pre-S deletions at the stage of HCC, 88.9% (8/9) had deletions 2-5 years prior to HCC, while only 44.4%4 (4/9) contained such deletions 6-9 years prior to HCC.
  
  
  CONCLUSIONS
  CGE is a sensitive approach for HBV pre-S deletion analysis. Pre-S deletion, especially for short DNA fragment deletion, is a useful predictive marker for HCC.
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Zhi-Mei
authors.initials: ZM
authors.lastname: Zhao
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Gan
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Zhu
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Tao-Yang
authors.initials: TY
authors.lastname: Chen
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Jin-Bing
authors.initials: JB
authors.lastname: Wang
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Zhi-Gang
authors.initials: ZG
authors.lastname: Cao
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Geng-Sun
authors.initials: GS
authors.lastname: Qian
authors.affiliation: Zhi-Mei Zhao, Yan Jin, Yu Gan, Yu Zhu, Zhi-Gang Cao, Geng-Sun Qian, Hong Tu, State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200032, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Tu
mesh_terms: D000328:Adult; D000368:Aged; D006528:Carcinoma, Hepatocellular / Q000821:virology*; D016022:Case-Control Studies; D019075:Electrophoresis, Capillary; D005260:Female; D017353:Gene Deletion; D005838:Genotype; D006509:Hepatitis B / Q000150:complications / Q000175:diagnosis / Q000821:virology*; D006514:Hepatitis B Surface Antigens / Q000235:genetics*; D006801:Humans; D008113:Liver Neoplasms / Q000821:virology*; D008137:Longitudinal Studies; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011498:Protein Precursors / Q000235:genetics*; D018570:Risk Assessment; D012307:Risk Factors; D013056:Spectrophotometry, Ultraviolet; D013997:Time Factors
chemical_list: D006514:Hepatitis B Surface Antigens; D011498:Protein Precursors
keywords: Capillary gel electrophoresis; Hepatitis B virus; Hepatocellular carcinoma; Pre-S deletion; Sequence analysis