不同基因型慢性乙肝患者的耐药变异位点分析

Analysis of drug-resistant mutation sites in patients with different genotypes of chronic hepatitis B

目的通过研究江西省核苷(酸)类似物(NAs)下乙肝病毒逆转录区(RT区)的变异位点,分析乙肝病毒耐药原因。方法采集104例门诊接受NAs抗病毒治疗且疗程≥1年的患者血清,扩增乙型肝炎病毒(HBV) RT区可能耐药变异位点片段。收集出现应答不佳时和挽救治疗1月后血清HBV DNA、丙氨酸氨基转移酶(ALT)水平,分析挽救治疗的疗效。采用BioEdit软件包进行核酸与氨基酸序列的比对,分析患者RT区主要变异类型及在不同基因型患者中的分布。采用DataMonkey在线软件进行不同基因型的选择压力及正选择位点分析。结果挽救治疗后患者HBV DNA、ALT水平均下降,HBV DNA水平降低更加明显,但在不同基因型中无差异。104例患者中87例检测出与药物相关变异位点,其中14例(13.46%)出现多位点耐药变异(3个及以上位点);剩余17例中7例未检出耐药变异位点,10例为可能耐药变异位点。C基因型M204I+L180M位点变异率高于B基因型,差异有统计学意义。选择压力分析表明,两种基因型的选择压力无差别,并且都以中性选择为主,B基因型中rt207、rt229为正选择位点,C基因型中未发现。结论江西省慢性乙型病毒性肝炎患者B、C基因型最为常见,并且以M204I位点变异为主,在B基因型中检测出正选择位点,说明核苷(酸)类似物的广范使用,促使新的基因功能出现,也可能是出现多个新耐药位点的原因。

Objective To analyze the cause of drug resistance of hepatitis B virus by studying mutation sites the of HBV RT region under nucleoside(acid) analogues in Jiangxi Province. Methods Serum samples of 104 outpatients receiving NAs antiviral therapy for more than one year were collected, and fragments of possible drug-resistant mutation sites in HBV RT region were amplified. The levels of HBV DNA and ALT with poor response and salvage treatment for 1 month were collected to analyze the effect of salvage treatment. Nucleic acid and amino acid sequences were compared by BioEdit software package to analyze the main variant types in RT region of patients and their distribution with different genotypes. DataMonkey online software was used to analyze the selection pressure and positive selection sites of different genotypes. Results After salvage treatment, HBV DNA and ALT levels decreased, and HBV DNA levels decreased more significantly, but there was no difference among different genotypes. Of 104 patients, 87 were associated with drug resistancE-related mutations, 14(13.46%) were multi-site resistance mutations(3 or more sites);7 of the remaining 17 patients did not detect the drug resistance mutation,10 were possible drug resistance mutation sites. M204 I + L180 M site mutation rate in genotype C was higher than that of genotype B,and the difference was statistically significant. Analysis of selection pressure showed that there was no difference in selection pressure between the two genotypes,and neutral selection was the main choice. Rt207 and rt229 were positive selection sites in genotype B,but not in genotype C. Conclusion Genotypes B and C are most common types in chronic hepatitis B patients in Jiangxi Province,and the M204 I locus variation is dominant.Positive selection sites are detected in Genotype B,so the widespread use of nucleoside analogues promote the emergence of new gene functions,and may also be the cause of multiple new drug resistance sites.