拉米夫定耐药患者乙型肝炎病毒多聚酶区基因突变的检测分析  

Analysis of mutations at hepatitis B virus P gene in lamivudine-refractory patients with chronic hepatitis B

作　　者：许彪[1,2] 成军[3] 徐东平[1] 李晓东[1] 毛远丽[4] 王海滨[4] 马洪滨[4] 胡瑾华[2] 王业东[5] 

机构地区：[1]解放军第三○二医院全军传染病研究所病毒性肝炎研究室,北京100039 [2]解放军第三○二医院全军传染病研究所肝衰竭治疗研究中心,北京100039 [3]北京地坛医院 [4]解放军第三○二医院全军传染病研究所临床检验中心,北京100039 [5]解放军第三○二医院院办,北京100039

出　　处：《肝脏》2008年第2期104-107,共4页Chinese Hepatology

基　　金：首都特色临床医学技术发展研究基金(Z07050700690702)

摘　　要：目的建立以基因测序检测乙型肝炎病毒(HBV)多聚酶区基因突变的方法,并分析拉米夫定耐药者HBV多聚酶区基因主要耐药突变的分布情况。方法采用巢式PCR方法对174例拉米夫定耐药患者血清HBV多聚酶区进行扩增,对PCR产物进行直接测序,将YMDD突变阳性与阴性的病例分别进行HBV耐药基因突变的分析和比较。结果本研究154例YMDD突变阳性的患者中有10种突变类型,其中以rt M204I突变最多见,为46例(29.9%),其次为rt L180M/M204V(26.6%)、rt L180M/M204I(17.5%)、rt V173/L180M/M204V(13.0%)、rt M204V(2.6%)、rt L180M/M204V/M204I(5.8%)、rt V173L/L180M/M204V/M204I(1.3%)、rt V173L/M204I rt M204I/M204V(1.3%)、rt M204I/M204V(1.3%)。另外,rt M204V联合rt L180M或rt V173L的突变率较rt M204I联合rt L180M或rt V173L显著增高。从rt L180M突变角度来看,rt L180M与rt M204V的联合突变率最高,其次为rt L180M联合rt M204I突变,rt L180M与rt L173M的联合突变率最低。从rt V173L突变角度来看,rt V173L联合rt M204V或rt L180M突变的发生率均高于rt V173L联合rt M204I的突变率,且rt V173L突变多联合rt M204V和rt L180M突变同时发生。结论拉米夫定耐药株的氨基酸突变复杂多样,需要对拉米夫定耐药多个相关位点进行检测。Objective To develop a method to detect mutation at hepatitis B virus （HBV） P gene RT region by DNA direct sequencing, and analyze distribution of major drug-resistant mutation at HBV P gene in patients resistant to lamivudine. Methods The HBV P gene RT region from 174 serum samples of hepatitis B patients resistant to lamivudine was amplified by nest PCR, and PCR products were directly sequenced. Then, the drug resistant mutation at HBV gene of cases with YMDD mutation and those without YMDD mutation were analyzed and compared respectively. Results There are 10 forms of resistant mutation in 154 cases harboring YMDD mutation in this study. They are rtM204 （29. 9%）, rtL180M/M204V （26. 6%）, rtL180M/M204I （ 17. 5%）, rtV173/L180M/M204V （ 13. 0%）, rtM204V （ 2.6% ）, rtL18OM/M204V/M204I （ 5. 8%）, rtV173L/L180M/M204V/M204I（1.3%）, rtV173L/M204IrtM204I/M204V（1.3%） and rtM204I/M204V（1.3%）. In addition, the mutation rate of rtM204V combined with rtL180M or rtV173L is conspicuously higher than the rate of rtM204I with rtL180M or rtV173L, respectively. From the view of rtL180M mutation, the mutation rate of rtL180M associated with rtM204V is the highest, followed by the next high mutation rate is rtL180M with rtM204I, and then rtL180M with rtL173M. From the view of rtV173L mutation, mutation rate of rtV173 combining with rtM204V or rtV173 combining with rtL180M is higher than that of rtV173 with rtM204I. Moreover, rtV173L mutation is most often associated with the rtM204V plus with rtL180M. Conclusion The mutation forms of lamivudine-resistant viral strains are complex. The fact requires us to assay the multiple sites at HBV P gene RT region which are resistant to lamivudine.

关 键 词：肝炎病毒 乙型 变异 拉米夫定 YMDD 

分 类 号：R512.62[医药卫生—内科学] R450[医药卫生—临床医学]