HBsAg无症状携带者乙型肝炎病毒核心基因启动子突变的研究

目的了解HBsAg无症状携带者乙型肝炎病毒(HBV)核心基因启动子双突变(nt1762 A→T,nt1764 G→A)发生情况及其与基因型、病毒量和HBeAg的关系。方法 HBsAg无症状携带者从隆安研究队列中选择,用套式聚合酶链反应(nested PCR)对研究对象血清HBV核心基因启动子、S基因扩增、序列分析及基因分型,用HBV引物和双标记的TaqMan探针扩增和定量病毒DNA。结果观察开始时核心基因启动子为野毒株的109例观察对象,3年后双突变平均年发生率为2.8%;观察开始时已发生nt1762或nt1764点突变的59例对象,3年后另一个位点也发生突变的平均年发生率为6.8%,两率差异有统计学意义(χ2=5.109 0,P<0.05)。nt1762 A→T突变组HBeAg阳性率(45.5%,10/22)与nt1764 G→A突变组HBeAg阳性率(10.8%,4/37)差异有统计学意义(χ2=9.149,P<0.05)。20例基因型B未发生双突变,重组基因型年双突变率为4.8%(2/14),基因型C年突变率为3.1%(7/75),各基因型间突变率差异无统计学意义(P>0.05)。5例双突变发生后HBeAg仍然阳性者病毒量有下降趋势,而另5例突变发生后HBeAg阴性者其病毒量有升有降。结论 HBV核心基因启动子双突变年发生率较高,这两个位点中的任何一个发生突变是双突变的过渡形式,双突变与基因型无关,nt1764 G→A突变与HBeAg阴性有关。

Evolution of core promoter gene mutations in asymptomatic HBV carriers

Objective To understand the evolution of HBV core promoter double-nucleotide mutations（nt1762 A→T,nt1764 G→A） and their association with genotypes,viral load and HBeAg detection.Methods The participants were selected from the HBV asymptomatic carriers of Longan cohort.The core promoter and S gene of HBV were amplified from serum samples by nested PCR and genotyped by DNA sequencing.The HBV viral load was measured by a dual-labeled TaqMan probe assay.Results The annual double-nucleotide mutation rate of core promoter was 2.8%（9/109） for the wild type of HBV strains during a three-year follow-up.The annual single nucleotide mutation rate of core promoter gene nt1762 or nt1764 would be 6.8%（12/59） if the HBV strains began with the single mutation of core promoter gene nt1764 or nt1762.The annual double-nucleotide mutation rate of HBV core promoter was significantly higher in the strains with the single mutation than in wild strains（χ2=5.109 0,P0.05）.The positive rate of HBeAg was significantly higher in patients with HBV nt1762T site mutation than in patients with HBV nt1764A mutation（χ2=9.149,P0.05）.No double nucleotide mutations were found in subjects with HBV genotype B.The prevalence rates of double-nucleotide mutations were 3.1%（7/75） and 4.8%（2/14）,respectively for HBV genotype C and recombinant genotypes.The decreasing trend of viral load was observed in 5 subjects with serum HBeAg negative conversion after the double-site mutations.Conclusion The annual incidence rate of HBV core promoter double-nucleotide mutations is high.The single nucleotide mutation is the transitional form of the double-nucleotide mutations.The G→A mutation of HBV core promoter nt1764 is associated with serum HBeAg negative conversion.