肝癌患者体内乙型肝炎病毒基因组BCP/Pre C区基因突变多样性分析

Analyses of the Genetic Diversities and Mutations of the Hepatitis B Virus Genome BCP/Pre C Region

为研究肝癌患者组织样本中HBV DNA核心启动子区(BCP区)和前C区(Pre C区)的基因突变多样性及其突变规律。收集第四军医大学附属西京医院2015年收治的192例HBV阳性的肝癌患者组织样本,PCR扩增HBV BCP/Pre C区DNA片段并进行测序分析,从测序失败的样本中随机抽取21例,采用构建单克隆文库后测序的方法进行分析。结果显示37.89%(72/190)的HBV阳性肝癌患者体内HBV病毒因呈现多种突变株混合感染的特点而导致PCR产物直接测序失败,经单克隆测序揭示,每一例失败样本的HBV DNA准种池中至少有2~11种突变株共同存在;突变株中缺失突变和插入突变的发生率高达80.95%;其它突变形式按照频率从高到低分别为A1762T/G1764A双突变90.48%,G1756C/T1803A/Δ(1 757~1 765)/Δ(1 824~1 832)四联突变80.95%,T1753C/A1762T/G1764A三联突变57.14%,A1762T/G1764A/G1896A三联突变42.86%,G1756C/Δ(1 757~1 765)双突变28.57%,T1753C/A1762T/G1764A/G1896A四联突变23.81%。由此可见,肝癌患者体内HBV病毒具有BCP/Pre C区DNA突变的多样性,这些缺失与插入突变是导致序列移码与PCR产物测序失败的直接原因。研究结果为HBV持续感染及基因突变检测、相关机制研究和个体化防治奠定了基础。

We wished to study the genetic diversities and mutations of the basic core promoter （BCP） and pre-C region of the hepatitis B virus （HBV） in liver-cancer tissues. One hundred and ninety-two tissue samples were collected from patients suffering from hepatocellular carcinoma （HCC） and HBV infection in 2015 in Xijing Hospital. Twenty-one cases were selected, of which direct sequencing of the polymerase chain reaction （PCR）-amplified products of BCP/pre-C region was unsuccessful. Cloning and sequencing allowed the DNA sequences of the BCP/pre-C region to be analyzed. Sequencing showed infection with mixed mutants of HBV in 37.89% of HBV-positive HCC patients, and that HBV DNA in each sample contained 2- 11 types of mutations. The mutation rate of deletion and insertion was 80. 95%. Other mutations in descending order by mutation rate were a： A1762T/G1764A combined mutation （90.48%） ; G1756C/T1803A/△（1757 - 1765）/△ （1824 - 1832） combined mutation （80. 95%） ; T1753C/A1762T/ G1764A combined mutation （57. 14%） ; A1762T/G1764A/G1896A combined mutation （42. 86%） ; G1756C/A（1757~1765） combined mutation （28. 57%）; T1753C/A1762T/G1764A/G1896A combined mutation （23.81%）. The sequencing failure of PCR products may have been correlated directly with the deletion and insertion mutations of HBV DNA. These findings lay the foundation for further studies on HBV mutations, persistent infection, and the mechanism of personalized medicine.