PCR restriction fragment length polymorphism in detection of YMDD variants of viral polymerase in hepatitis B virus patients treated with lamivudine.
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.
Yang
Dan-Hong
DH
Department of Infectious Diseases, First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China. xieyuanjun@mail.hz.zj.cn
Liang
Wei-Feng
WF
Xie
Yuan-Jun
YJ
Zhao
Nian-Feng
NF
Fan
Jun
J
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
D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; D001224:Aspartic Acid; D014443:Tyrosine; D008715:Methionine; D004259:DNA-Directed DNA Polymerase