慢性乙型肝炎、乙型肝炎肝硬化及原发性肝癌患者HBV-X基因突变分析

Study of HBV- X gene mutation among patients with HBV- related chronic hepatitis,liver cirrhosis,and primary liver cancer

目的研究慢性HBV感染者,如慢性乙型肝炎(CHB)、乙型肝炎肝硬化(LC)、原发性肝癌(PLC)患者血清中HBV-X基因序列的突变与肝癌发生的关系。方法收集2011-2013年间于重庆医科大学附属第二医院就诊的慢性HBV感染者血清共89例,从血清中提取HBV DNA,扩增全长HBV-X基因序列,经测序后与已知HBV-X基因相应序列比较该患者体内HBV-X基因变异位点以及变异形式,并用卡方检验、单因素方差分析处理数据,NCBI的genotype工具测定基因型。结果所有患者均属于B/C基因型,HBeAg阳性患者中B基因型占46.2%,C基因型占53.8%;HBeAg阴性患性中B基因型占81.2%,C基因型占18.8%(P=0.001)。在PLC组中,启动子(BCP)区的突变显著高于CHB、LC(69.2%vs34.4%和61.3%,P<0.05),且nt1821位点存在明显的T碱基的缺失(88.5%vs 53.1%和71%,P=0.014)。在CHB、LC中,C基因型BCP的双突变率显著高于B基因型(61.5%vs 15.8%,P=0.007;83.3%vs 47.4%,P=0.045),HBV DNA低病毒载量(≤106拷贝/ml)中BCP的突变率较高病毒载量(>106拷贝/ml)更显著(81.3%vs 47.9%,P=0.015)。结论 BCP区的双突变及nt1821位点的缺失可能与PLC的发生密切相关。

Objective To study the relationship between hepatocarcinogenesis and the mutation in X gene among patients with chronic hepa-titis B virus （HBV）infection,such as chronic hepatitis B （CHB）,liver cirrhosis （LC）and primary liver cancer （PLC）.Methods The serum samples from 89 patients with chronic HBV infection who visited the Second Affiliated Hospital of Chongqing Medical University from 201 1 to 2013 were collected.PCR was used to amplify the X gene of HBV DNA extracted from the serum samples.After sequencing,the HBV-X genome was compared with those reported in GenBank to find the variable sites and variant forms.Chi -square and one -way ANOVA were used for the statistical analysis afterwards,whereas genotypes were determined by the genotyping tool of the National Center for Biotechnology Information.Results All patients were genotype B or C.Among HBeAg-positive patients,46.2% were genotype B,and 53.8% were genotype C;among HBeAg-negative patients,81.2%were genotype B,and 18.8%were genotype C （P=0.001）.PLC pa-tients had a significantly higher risk of mutation in the basic core promoter （BCP）region than the CHB and LC groups （69.2%vs 34.4%and 61.3%,P〈0.05）;in addition,an evident T-base deficiency was observed at nt1821 site （88.5% vs 53.1% and 71%,P=0.014）.Among CHB and LC patients,those with genotype C had a significantly higher risk of BCP double mutation than those with geno-type B （61.5%vs 15.8%,P=0.007;83.3%vs 47.4%,P=0.045）.The incidence of BCP double mutation was significantly higher in the low-viral load group （≤10^6 copies/ml）than in the high-viral load group （〉106 copies/ml）（81.3% vs 47.9%,P=0.015）. Conclusion The BCP double mutation and T-base deficiency at nt1821 site may play important roles in the development of PLC.