1740例慢性乙型肝炎患者替诺福韦酯耐药可能相关突变分析

Mutations possibly associated with tenofovir disoproxil fumarate resistance in 1740 patients with chronic hepatitis B

目的通过大样本测序分析慢性乙型肝炎(CHB)患者HBV反转录酶(reverse transcriptase,RT)序列,明确与替诺福韦酯(TDF)耐药可能相关的突变在我国患者未使用该药前的检出状况,预测TDF在我国临床的应用前景。方法收集1740例CHB患者血清,提取血清中HBVDNA,采用巢式PCR方法扩增HBVRT基因,对PCR产物进行DNA测序,NTI软件比对结果。检测与核苷(酸)类似物耐药相关的RT区12个位点序列,重点分析了与TDF临床耐药可能相关的A194T突变以及体外证实可降低TDF活性的V214A、Q215S和A181V+N236T突变。结果540例(31%)患者分别检出了与拉米夫定(LAM)、阿德福韦(ADV)、恩替卡韦(ETV)和替比夫定(LdT)耐药相关突变;未检出A194T突变;34例(2.0%)患者检出V214A突变;3例(0.2%)检出Q215S突变;15例(0.9%)检出A181V+N236T(其中3例为A181V/T+N236T)突变。结论未检出与TDF临床耐药可能相关的A194T突变,提示目前我国CHB患者自然发生或应用核苷(酸)类似物后引起的TDF原发耐药的可能性很小,预测该药在我国治疗对目前四种抗HBV核苷(酸)类似物耐药的患者应具有较好疗效,但V214A和A181V+N236T突变是否影响TDF临床疗效仍值得关注。

Objective To analyze HBV reverse transcriptase （RT） sequences in patients with chronic hepatitis B （CHB） by large sample sequencing, and investigate prevalence of HBV mutations possibly associated with tenofovir disoproxil fumarate （TDF） resistance in CHB patients who are naive to TDF treatment, so as to predict the therapeutic efficacy of TDF in the clinical practice in China. Methods Serum samples from 1 740 CHB patients were collected, from which HBV DNA was extracted. HBV RT gene was amplifed by nested PCR. Direct DNA sequencing of PCR product was performed followed by computational comparison of the sequencing data using Vector NTI software. Mutations at 12 sites of RT gene associated with nucleos（t）ide analogue resistance were analyzed, with the emphasis on mutation A194T which was possibly associated with TDF resistance, as well as mutations V214A, Q215S and A181V＋N236T which were confirmed to reduce TDF susceptibility （/〉 10-fold ） in vitro. Results Five hundreds and forty patients （31.0%） were positive for mutations associated with lamivudine, adefovir, entecavir, and telbivudine resistance, while no single patient was positive for mutation A194T, 34 patients （2.0%） were positive for mutation V214A, 3（0.2%） for mutation Q215S, and 15（0.9%） for combined mutations of A181V and N236T, with 3 for combined mutations of A181V/T and N236T. Conclusions Mutation A194T possibly associated with TDF resistance is not found in all enrolled patients, suggesting the little chance of TDF primary resistance due to either natural occurrence or the induction of nucleos（t）ide analogues in CHB patients in China. Therefore, it is suggested that TDF has good efficacy on Chinese patients resistant to the present 4 anti-HBV nucleos（t）ide analogues, but the influence of mutations V214A and A181V＋N236T on the therapeutic efficacy of TDF needs to be evaluated in the future clinical management.