拉米夫定耐药的HBV反转录酶区基因突变模式分析

摘要

目的：分析慢性乙型肝炎（CHB）患者在拉米夫定（LAM ）治疗过程中出现耐药的 HBV 反转录酶区基因的突变模式及临床特征，指导临床用药。方法采用巢式聚合酶链反应（PCR）方法对2009年1月至2011年12月广州市第八人民医院肝病门诊或住院治疗的 LAM 初治发生耐药的105例 CHB 患者血清 HBV 聚合酶基因反转录酶区进行扩增，对 PCR 产物进行测序，分析 LAM 耐药时 HBV 聚合酶基因的不同突变模式及患者临床特征。结果105例患者诊断为 LAM 耐药，98例患者检测到 LAM 相关的 HBV 聚合酶基因突变，总体突变模式共8种，其中95例患者存在 YMDD 基因突变，占96．9％。 rtM204I 单点突变患者42例，占43．9％；rtM204V 单点突变患者3例，占3．1％，二者比较差异有统计学意义（χ2＝21．899，P ＜0．05），提示 rtM204位点（YMDD ）点突变模式以rtM204I 点突变为主。 rtM204V 以联合 rtL180M 组合突变的模式为主。 LAM 耐药时，3种主要突变模式 rtM204I 、rtL180M ＋ rtM204I 、rtL180M ＋ rtM204V 患者的年龄、性别比例、血清 HBV DNA 载量、ALT 水平、肝硬化发生率及HBeAg 阳性率比较，差异均无统计学意义（P＞0．05）。 HBV 聚合酶区基因突变患者与伴生化学突破患者的 HBV DNA 载量明显高于 HBV-P-RT 区基因未突变和未发生生化学突破的患者，差异均有统计学意义（P＜0．05）。结论 YMDD 基因突变是 LAM 耐药后 HBV 聚合酶基因突变的主要模式，且突变患者的 HBV DNA 载量明显增高，检查耐药患者基因序列，可指导临床合理用药。%Objective To analyse the mutation patterns of HBV reverse transcriptase region gene and the clin-ical features in chronic hepatitis B (CHB) patients with lamivudine(LAM ) resistance for guiding clinical medication . Methods 105 cases of CHB with LAM resistance were chosen among the outpatients and inpatients in our hospital from January 2009 to December 2011 .The serum HBV polymerase gene reverse transcriptase region was amplified by nest PCR ,and the PCR products were directly sequenced .The different mutation patterns of the HBV polymerase gene and the clinical features after the emergence of LAM-resistance were retrospectively analyzed .Results 105 pa-tients were diagnosed as LAM resistance ,98 patients were found to have LAM-resistant-associated mutations in the polymerase gene and the overall mutation modes had 8 kinds .Among them ,95 cases had the YMDD motif mutation , accounting for 96 .9% .RtM204I point mutation was in 42 cases ,accounting for 43 .9% ;rtM204V point mutation was in 3 cases ,accounting for 3 .1% ,both comparison ,χ2 = 21 .899 ,P< 0 .05 ,the difference was statistically significant , suggesting rtM204 loci(YMDD) point mutation mode was given priority to rtM 204I point mutation .RtM204V was given priority to joint rtL180M combination mutation model .In LAM resistance ,the comparison of three main muta-tion patterns rtM204I ,rtL180M + rtM204I ,rtL180M + rtM204V ,proportion of patient′s age and gender ,serum HBV DNA load ,ALT level and incidence of cirrhosis of the liver and HBeAg positive rate had no statistical significance (P> 0 .05) .HBV DNA load in the patients with HBV polymerase gene mutations and the patients with associated chemical breakthrough was obviously higher than that in the patients without HBV-P-RT gene mutation and bio-chemical breakthrough ,the difference had statistical significance (P < 0 .05) .Conclusion YMDD motif mutation is the main mode of HBV polymerase gene mutation in CHB patients with LAM resistance .HBV DNA load in the mu-tation patients is significantly increased .Testing the gene sequences in the patients with LAM resistance can guide clinical rational drug use .