Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment
Detection of YMDD mutant using a novel sensitive method in chronic liver disease type B patients before and during lamivudine treatment
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DOI：
10.1016/s0168-8278(02)00145-9
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发表时间：
2002-08-01
影响因子：
25.7
通讯作者：
Minami, M
中科院分区：
文献类型：
--
作者：
Kirishima, T;Okanoue, T;Minami, M
背景/目的：据报道，拉米夫定耐药的B型肝炎病毒（HBV）出现在长期服用拉米夫定的患者中。在非拉米夫定治疗的慢性B型肝炎患者中，没有报道存在酪氨酸-甲硫氨酸-天冬氨酸-天冬氨酸（YMDD）突变。在本研究中，我们开发了一种敏感的检测方法，并将其应用于检测YMDD突变体。方法：我们开发了肽核酸（PNA）介导的聚合酶链反应钳检测B型肝炎病毒DNA聚合酶基因的YMDD基序的突变。结果：用该方法可检测出0.01-0.001%的变异病毒与10（5）-10（9）个拷贝的野生型病毒共存。在临床突破前7个月检测到YMDD突变，比常规限制性片段长度多态性方法提前6个月。比色法在18例抗-HBe抗体阳性的慢性B型肝炎患者中，有4例检测到YMDD突变：YMDD +酪氨酸-缬氨酸-天冬氨酸（YVDD）2例，YMDD +酪氨酸-异亮氨酸-天冬氨酸（YIDD）2例，而在HBe抗原阳性的患者中未检测到YMDD突变。这是一个有效的监测过程中或拉米夫定治疗前的患者，可以提供更多的洞察慢性肝炎B患者的治疗策略。(C)2002年欧洲肝脏研究协会。由Elsevier Science B. V.出版，版权所有。
Background/Aims: The emergence of lamivudine-resistant hepatitis B virus (HBV) was reported in patients with prolonged lamivudine administration. There was no report of the existence of tyrosine-methionine-aspartate-aspartate (YMDD) mutant in non-lamivudine treated chronic hepatitis B patients. In the present study, we developed a sensitive assay and applied it to the detection of YMDD mutant.Methods: We developed peptide nucleic acid (PNA) mediated polymerase chain reaction clamping for detecting mutations in a YMDD motif of the hepatitis B virus DNA polymerase gene. We studied YMDD mutants in a patient with HBV DNA breakthrough longitudinally and in non-lamivudine treated patients (36 patients).Results: We could detect as little as 0.01-0.001% of mutant viruses coexisting in 10(5)-10(9) copies of wild-type viruses using this assay. YMDD mutant was detected 7 months before clinical breakthrough, which was 6 months earlier than using the conventional restriction fragment length polymorphism. assay. YMDD mutants were also,detected in four of 18 anti-HBe antibody positive untreated chronic hepatitis type B: YMDD + tyrosine-valine-aspartate-aspartate (YVDD) in two patients and YMDD + tyrosine-isoleucine-aspartate-aspartate (YIDD) in two patients, however, none in HBe antigen positive patients.Conclusions: We developed a highly sensitive assay for detecting YMDD mutants. This is an effective procedure for monitoring patients during or before lamivudine treatment and can provide more insight into the therapeutic strategies for chronic hepatitis B patients. (C) 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.