启东肝癌高发区乙肝病毒流行株全基因分析

Full-genome sequence analysis of hepatitis B virus isolated from hepatocellular carcinoma in high incidence area Qidong

目的:通过对江苏启东地区乙型肝炎病毒(HBV)全基因序列的分析,探讨该地区肝癌高发的分子病毒学病因。方法:以蛋白酶K消化后,酚/氯仿抽提7例肝炎和7例肝癌患者血清中DNA。应用聚合酶链反应(PCR),扩增血清中HBV基因全长,克隆至T载体后行全自动测序。以PHYLIP软件构建的系统进化树判断HBV的基因型;以Clastal W软件对序列进行突变分析。结果:14例标本中有12例为C基因型,2例为B基因型。肝癌和肝炎组中HBV基因型类别分布无差异。有5例HBV发生了PreS2的缺失突变,其中肝癌标本4例(57.1%),肝炎标本仅1例(14.3%)。HBV基因组中常见的点突变为PreS1区的nt.3116及核心启动子区的nt.1762/1764,发生率高达78.6%(11/14)。与肝炎组相比,肝癌中点突变发生率显著增高的位点为前C区nt.1899 G→A的突变(P=0.01)及核心启动子区nt.1653 C→T的突变(P=0.05)。结论:启东地区HBV以C基因型为主;启东HBV基因组中可能存在着与肝癌相关的点突变和缺失突变。

Objective ：To investigate the genotype of hepatitis B virus （HBV） in Qidong area and further characterize the sequence properties of HBV isolated from hepatocellular carcinoma （HCC） and chronic hepatitis （CH） patients. Methods：DNA was extracted from serum samples of 7 HCC and 7 CH patients by conventional proteinase K digestion/phenol extraction method. The full-genome of HBV was amplified by polymerase chain reaction （PCR） using serum DNA as the template. PCR product was cloned into Tvector and DNA sequence was obtained by ABI auto-sequencing. The genotypes of HBV were determined by the Phylogenetic tree using PHYLIP software based on the entire HBV sequences and mutation analysis was carried out by the Clastal W software. Results：Among the total 14 HBV strains, 12 belonged to genotype C and 2 belonged to genotype B. No significant difference of genotype distribution existed between HBV and HCC groups. Deletion in PreS2 region was observed in 4 （57.1%） HCC patients and 1 （ 14.3% ） CH patient. The mutational hotspots, which incidence was as high as 78.6% （ 11/14）, were located at nt. 3116 （ PreS1 region） and nt. 1762/1764 （ core promoter/X region）. Compared with HBV isolated from of CH patients, two point mutations, i.e. nt. 1899A in PreC region （ P = 0.01 ） and nt. 1653T in core promoter/X region （ P = 0.05 ）, occurred more frequently in HCC patients. Conclusion ： The prevalent HBV genotype in Qidong area is genotype C. HCC-related point mutation and deletion of HBV may exist in Qidong genomes.