Bozdayi A M, Uzunalimoğlu O, Türkyilmaz A R, Aslan N, Sezgin O, Sahin T, Bozdayi G, Cinar K, Pai S B, Pai R, Bozkaya H, Karayalçin S, Yurdaydin C, Schinazi R F
Institute of Hepatology, Ankara University, Ankara, Turkey.
J Viral Hepat. 2003 Jul;10(4):256-65. doi: 10.1046/j.1365-2893.2003.00435.x.
The emergence of drug-resistant virus in hepatitis B virus (HBV) patients treated with lamivudine is well documented. In this study, we determined the mutations occurring in the tyrosine-methionine-aspartate-aspartate (YMDD) amino acid motif of the HBV DNA polymerase gene, as well as upstream and downstream of this region, in patients with breakthrough virus during lamivudine therapy. Thirty-one Turkish patients (20 patients HBeAg positive, 11 patients HBeAg negative and anti-HBe positive) with chronic HBV infection who completed at least 104 weeks of lamivudine treatment were investigated. All patients received lamivudine, (150 mg/day), for 104 weeks, with or without 4 months of interferon (IFN) combination. HBV-specific sequences were amplified by polymerase chain reaction (PCR) from sera of patients with breakthrough virus, and the PCR products were directly analysed by sequencing. Breakthrough virus was detected in seven of the 31 patients (22.6%) between 9 and 18 months of therapy. Of the seven patients, six were HBeAg positive at baseline, and four had a double mutation consisting of rtM204V and rtL180M, while two had an rtM204I change. In one patient, two base substitutions at rt204 (ATG --> AGT; T to G and G to T) lead to a methionine to serine change (YMDD --> YSDD). This novel DNA pol mutation was detected at month 18 of lamivudine treatment. In addition, this new variant had the rtL180M mutation and a 12 base pair deletion in the pre-S1 region between nucleotides 43-54. The YSDD mutation was still present 6 months after lamivudine discontinuation. In vitro transfection studies also confirmed that the YSDD strain is resistant to lamivudine. In conclusion, the results indicate that, in addition to a Met --> Val and Met --> Ile change in YMDD, a Met --> Ser change at rt204 (YMDD --> YSDD) associated with the rtL180M change can also emerge during lamivudine treatment, which confers lamivudine resistance in vivo and in vitro, leading to virological breakthrough and ALT increases.
拉米夫定治疗的乙型肝炎病毒（HBV）患者中出现耐药病毒已有充分记录。在本研究中，我们确定了拉米夫定治疗期间出现病毒突破的患者中，HBV DNA聚合酶基因酪氨酸-甲硫氨酸-天冬氨酸-天冬氨酸（YMDD）氨基酸基序及其上下游区域发生的突变。对31例完成至少104周拉米夫定治疗的慢性HBV感染的土耳其患者（20例HBeAg阳性，11例HBeAg阴性且抗-HBe阳性）进行了调查。所有患者接受拉米夫定（150mg/天）治疗104周，联合或不联合4个月的干扰素（IFN）。通过聚合酶链反应（PCR）从病毒突破患者的血清中扩增HBV特异性序列，并对PCR产物进行直接测序分析。在治疗9至18个月期间，31例患者中有7例（22.6%）检测到病毒突破。在这7例患者中，6例基线时HBeAg阳性，4例有rtM204V和rtL180M的双重突变，2例有rtM204I改变。1例患者rt204处两个碱基替换（ATG→AGT；T到G和G到T）导致甲硫氨酸变为丝氨酸（YMDD→YSDD）。这种新的DNA聚合酶突变在拉米夫定治疗第18个月时被检测到。此外，这个新变体有rtL180M突变，且在43-54位核苷酸之间的前S1区域有12个碱基对缺失。停用拉米夫定6个月后，YSDD突变仍然存在。体外转染研究也证实YSDD毒株对拉米夫定耐药。总之，结果表明，除了YMDD中甲硫氨酸变为缬氨酸和甲硫氨酸变为异亮氨酸的改变外，rtM204处甲硫氨酸变为丝氨酸（YMDD→YSDD）并伴有rtL180M改变，在拉米夫定治疗期间也可能出现，这在体内和体外均赋予拉米夫定耐药性，导致病毒学突破和ALT升高。
