234例慢性乙型肝炎患者HBV前C/BCP区突变分析

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。

Mutation of hepatitis B virus precore/basal core promoter in 234 patients with chronic hepatitis

Objective To investigate the occurrence of hepatitis B virus （HBV）precore/basal core promoter（BCP） mutations and its correlation on serum HBeAg expression in patients with chronic hepatitis B（CHB）. Methods Sera were collected for extraction of HBV DNA. The precore/BCP gene fragment was amplified by an in-house nested PCR assay and analyzed by direct PCR-produet sequencing. Based on reference literatures,6 mutation sites which possibly influencing HBeAg expression,secretion,or HBV replication were selected as follows：1753,1762,1764,1862,1896 and 1899. The data were processed by NTI software. Serum HBeAg was detected by enzyme-linked immunosorbent assay. Results Seventyfour （31.6%） of 234 patients enrolled in the study were mutation-negative at all 6 sites. Serum HBeAg positive rate of these cases was 74.3%. One hundred and sixty （68.4%） of the 234 patients were detected with various mutations at the sites,including 4 single-site and twenty-one multiple-site mutations. Among the total,73（31.2%） cases contained G1896A with or without coexistence of wild-type sequence at the sites,of whom 37 cases without coexistent wild-type sequence at the site had 18.9% HBeAg positive rate. Preeore/BCP mutations other than G1896A were detected in 87 patients whose HBeAg positive rate was 63.2%（55/87）. A1762T＋G1764A was the most frequent mutation in patients positive with other than G1896A mutations,and the positive HBeAg was documented in 69.4%（34/49） of these patients. Four unusual substitutions were detected at site 1753 and 1862. Two cases were detected with insertion or deletion in precore/BCP region. Conclusion HBV precore/BCP mutations exist in most CHB patients in diverse forms. The patients with G1896A mutation had a significantly lower positive rate of serum HBeAg,while other mutations detected had little influence on HBeAg secretion. Screening HBV precore/BCP mutations by DNA sequencing could obtain rich information and thus is valuable for evaluating disease progression and administrating anti-viral therapy in clinical practice.