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目的调查慢性乙型肝炎患者血清HBV前C/基本核心启动子(BCP)区的突变情况,分析各种突变对HBeAg表达的影响。方法抽提患者血清DNA,采用改进的巢式PCR技术,扩增HBV前C/BCP区基因,对PCR产物进行DNA测序。用NTI软件比对结果,根据文献报道,选取1753、1762、1764、1862、1896和1899共6个位点进行突变分析,并重点分析不同突变对患者血清HBeAg阳性率和病情的影响。结果在234例中6个位点均无突变者为74例(31.6%),其血清HBeAg阳性率为74.3%(55/74)。在234例中6个位点检出至少1种突变的病例为160例(68.4%),突变形式包括4种单一位点突变和21种组合形式的突变;检出G1896A突变73例(31.2%),其中36例检出有共存未突变序列,37例仅检出突变序列,后者血清HBeAg的阳性率为18.9%(7/37)。检出G1896A以外其他形式突变的有87例,HBeAg阳性率为63.2%(55/87);其中以A1762T+G1764A最为常见,HBeAg阳性率为69.4%(34/49)。在1753、1862位点上检出4种特殊碱基突变,前C区有基因片段插入或缺失的有2例。结论多数慢性乙型肝炎患者在HBV前C/BCP区可检出突变,突变形式多样,其中G1896A突变样本血清HBeAg阳性率显著下降,而其他突变对其影响较小。应用DNA序列测定法分析HBV前C/BCP区基因突变,获得的信息全面,对临床评估病情进展和实施抗病毒治疗有参考价值。
Objective To investigate the mutations of pre-HBV precore / basic core promoter (BCP) in patients with chronic hepatitis B and analyze the influence of various mutations on HBeAg expression. Methods Serum DNA was extracted from the patients. The improved nested PCR technique was used to amplify the HBV pre-C / BCP region and the DNA was sequenced. NTI software was used to compare the results. According to the literature, six mutations of 1753, 1762, 1764, 1862, 1896 and 1899 were selected for mutation analysis. The impact of different mutations on the serum HBeAg positive rate and severity of the disease were analyzed. Results There were 74 cases (31.6%) with no mutation in 6 of 234 cases. The positive rate of HBeAg in serum was 74.3% (55/74). Among the 234 cases, 160 cases (68.4%) were detected in at least one of the six loci, including four single locus mutations and 21 combinations of mutations. G1896A mutation was detected in 73 cases (31.2% ), Of which 36 cases detected coexistence of unmutated sequences, 37 cases detected only mutation sequence, the latter positive rate of serum HBeAg was 18.9% (7/37). There were 87 cases of other mutations except G1896A, the positive rate of HBeAg was 63.2% (55/87). Among them, A1762T + G1764A was the most common, and the positive rate of HBeAg was 69.4% (34/49). In the 1753,1862 sites detected four kinds of special base mutation, pre-C region gene fragment insertion or deletion in 2 cases. Conclusions Most patients with chronic hepatitis B can detect mutations in the pre-HBV C / BCP region. The mutation types are diverse. The positive rate of HBeAg in serum of G1896A mutant patients is significantly decreased, while the other mutations have little effect on it. DNA sequence analysis of pre-HBV C / BCP gene mutations, the information obtained comprehensive, clinical evaluation of disease progression and the implementation of antiviral therapy with reference value.