慢性HBV感染患者bcp/pc区点突变模式、pres区缺失及其临床意义

目的阐明HBV(hepatitis B virus,HBV)感染患者不同临床阶段bcp/pc(basic core promoter,BCP/precore,PC)点突变模式及pres区缺失突变规律,并探讨其临床意义。方法慢性HBV感染患者180例,其中,无症状HBV携带者13例,慢性乙型肝炎者75例,HBV相关肝硬化及肝癌分别为62、30例。Qiagen法提取血清HBV-DNA,常规PCR扩增目的基因,纯化PCR产物ABI377DNA自动测序仪直接双向测序。直接测序失败者,回收目的 DNA与PMD-18T载体连接,克隆质粒双向测序。DNAStar软件包的SeqMan软件进行生物信息分析。结果 Bcp/pc区点突变包括nt1753、nt1762、nt1764、nt1776、nt1803、nt1846、nt1896。HBV携带者、慢性乙型肝炎、HBV相关肝硬化及肝癌患者nt 1762(nt 1764)点突变率分别为7.7%、68.0%、72.7%(68.0%)及90.9%(81.8%),肝癌患者G1896A突变频率占54.6%。A1762T+G1764A联合突变占36%;A1762T、G1764A、G1896A联合突变占11%;T1753A/C、A1762T、G1764A、G1896A发生率为7%。克隆测序显示,肝硬化及肝癌患者bcp区起始点A1727G点突变率分别为72%、63%。HBeAg阴性患者存在更多的基因变异(P=0.022),G1776A和G1896A突变是HBeAg阴性的独立预测因素(P<0.05)。Bcp区点突变与HBeAg阴性无明显关系。肝硬化和肝癌患者pres基因缺失突变频率最高,肝癌及肝硬化患者pres1、pres2及pres1+s2缺失频率分别为7.1%、71.4%、7.1%及41.2%、58.8%、29.4%(P<0.05)。结论 A1727G、A1762T、G1764A及A1762T/G1764A联合突变、pres缺失在HBV相关肝硬化及肝癌患者多见,可能是肝脏疾病进展的危险因素,G1776A和G1896A突变是HBeAg阴性的独立预测因素。Bcp/pc点突变及pres区缺失可能为肝癌发生的早期预测因素,值得进一步研究。

Mutation profiles of basic core promoter and precore and pres in patients with chronic hepatitis B virus infection and their clinical implications

ObjectiveTo analyze the mutation profiles and their implications of basic core promoter(BCP), precore(PC) and pres in patients with chronic hepatitis B virus(HBV) infection. MethodsTotally 180 patients with chronic HBV infection were included. Of them, 13 were asyptomatic hepatitis B virus carriers(CC), 75 were chronic hepatitis(CH) patients, 62 and 30 were cases with HBV related liver cirrhosis(LC) or carcinoma(HCC). Serum HBV DNA was extracted using QIAamp DNA MiniKit and the BCP/PC and PreS genes were amplified with routine PCR. The gene sequence was directly measured using ABI377 DNA sequencing machine. If sequencing failed, cloning products were also sequenced. The bioinformatics were analyzed with SeqMan software. ResultsMutation profiles of bcp/pc included nt 1753, nt 1762, nt 1764, nt 1776, nt 1803, nt1846 and nt 1896. The mutation frequency of nt 1762（nt 1764）in patients with CC, CH, LC and HCC were 7.7%, 68.0%, 72.7%（68.0%） and 90.9%（81.8%）, respectively. The frequency of G1896A mutation in HCC patients was 54.6%. A1762T+G1764A, A1762T+G1764A+G1896A or T1753A/C+A1762T+G1764A+G1896A was 36%,11%,7%,respectively. The mutation frequency of A1727G in LC and HCC patients were 72% and 63%. There were more mutations in HBeAg negative patients（P=0.022）. G1776A and G1896A mutation were independently predicted HBeAg loss(P＜0.05). The deletion frequency of PreS1, PreS2 and PreS1+S2 were 41.2%, 58.8%, 29.4% in LC patients and 7.1%, 71.4%, 7.1% in HCC patients(P＜0.05). Conclusion Mutations of A1727G, A1762T, G1764A and A1762T+G1764A and PreS deletion were more common in HBV related LC and HCC patients. G1776A and G1896A mutations were independently related to HBeAg loss. A1762T and G1764A mutations and PreS deletions may early predict HCC development, although this warrants further studies.