title: PCR restriction fragment length polymorphism in detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine.
abstract: OBJECTIVE
  To analyse the emergence of YMDD motif (tyrosine-methionine-aspartate-aspartate) variants in patients with hepatitis B treated with lamivudine.
  
  
  METHODS
  The amino acid substitution from methionine or isoleucine at the YMDD motif at the HBV polymerase gene is a main mutation resistant to lamivudine treatment. Generated from a fragment of domain C of the polymerase gene, patients' HBV DNA, which had been positive previously became positive again ever since it had been negative during lamivudine therapy. Variants were detected by cleavage of the products of the three PCRs with following enzymes: FokI, SspI, Alw441. The results of PCR-RELP were analysed by 8.4% polypropylene acidemide gel electrophoresis. PCR-RFLP assay was compared to direct sequencing.
  
  
  RESULTS
  HBV DNA was positive again in 33 patients and positive for one year in 2 patients. YMDD variants were detected in serum 14 of 35 patients, YIDD variants in 4, YVDD variants in 6, and YI/MDD variants in 1; all were in concordance with the results of direct sequencing. The samples of other 3 patients showed YI/VDD mutations, as shown by direct sequencing. The results of PCR-RFLP assay of the mixed sera of YIDD and YVDD variants were similar to those sera of YI/VDD variants.
  
  
  CONCLUSIONS
  PCR-RFLP is suitable for rapid detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine.
authors: Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. xieyuanjun@mail.hz.zj.cn
authors: Dan-Hong
authors: DH
authors: Yang
authors: Wei-Feng
authors: WF
authors: Liang
authors: Yuan-Jun
authors: YJ
authors: Xie
authors: Nian-Feng
authors: NF
authors: Zhao
authors: Jun
authors: J
authors: Fan
mesh_terms: D000293:Adolescent; D000328:Adult; D020816:Amino Acid Motifs; D001224:Aspartic Acid / Q000235:genetics; D001483:Base Sequence; D002648:Child; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D004351:Drug Resistance / Q000235:genetics; D014644:Genetic Variation; D006509:Hepatitis B / Q000188:drug therapy* / Q000821:virology; D006515:Hepatitis B virus / Q000378:metabolism; D006801:Humans; D019259:Lamivudine / Q000627:therapeutic use*; D008715:Methionine / Q000235:genetics; D008875:Middle Aged; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D018894:Reverse Transcriptase Inhibitors / Q000627:therapeutic use*; D014443:Tyrosine / Q000235:genetics
chemical_list: D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; D001224:Aspartic Acid; D014443:Tyrosine; D008715:Methionine; D004259:DNA-Directed DNA Polymerase