低水平HBsAg表达的慢性无症状HBV携带者Pre-S基因序列分析

Sequence analysis of Pre-S gene in asymptomatic chronic HBV carriers with low-level HBsAg

目的探讨低水平HBsAg表达的慢性无症状HBV携带者(Asymptomatic chronic HBV carriers,ASCs)Pre-S基因序列特征及持续表达的分子机制。方法收集2015年2月至2016年12月浙江大学医学院附属第一医院、杭州市第六人民医院、解放军第九〇三医院的654份ASCs血清标本和临床资料,根据HBsAg水平分为低水平HBsAg组(≤10 IU/mL)和高水平HBsAg组(>10 IU/mL),并且在与低水平HBsAg组年龄匹配的基础上,从高水平组血清标本中随机抽样100份,对138例低水平HBsAg患者和100例高水平HBsAg患者进行Pre-S/S基因组扩增与测序,构建系统进化树确定基因亚型,以高水平HBsAg组的HBV Pre-S基因成功测序结果为基础建立中国东部地区ASCs主要基因型的Pre-S基因参考序列,并对低水平HBsAg组的ASCs Pre-S基因序列进行对比分析。采用SPSS 12.01统计软件对数据进行分析。结果138例低水平HBsAg组Pre-S/S成功测序63例,其中B基因型52例,C基因型11例;100例高水平HBsAg组标本中Pre-S/S成功测序94例,其中B基因型48例,C基因型46例。通过序列比较分析发现,B基因型中,低水平HBsAg组Pre-S蛋白共发现81个氨基酸突变位点,其中有意义突变4个,分别为Pre-S1区F56I/V、T76A/N/P,Pre-S2区P15L/S/T、Y21T/F/H/N;高水平HBsAg组Pre-S蛋白发现47个氨基酸突变位点,其中有意义突变3个,为Pre-S1区L34F、V49A和P59S/L;B基因型中低水平HBsAg组的Pre-S蛋白氨基酸突变位点总数高于高水平HBsAg组,差异具有统计学意义(χ2=14.008,P<0.05)。C基因型中,低水平HBsAg组Pre-S蛋白发现19个氨基酸突变位点,其中有意义突变3个,分别为Pre-S1区W66V/G和A79V,Pre-S2区V32A;高水平HBsAg组Pre-S蛋白发现39个氨基酸突变位点,其中有意义突变2个,为Pre-S1区A79V和Pre-S2区T49I;C基因型中Pre-S蛋白氨基酸突变位点总数在两组间差异有统计学意义(χ2=7.571,P<0.05)。结论ASCs Pre-S基因存在有意义的突变,这些突变可能与低水平HBsAg持续表达相关。

Objective To analyze the sequence of Pre-S gene in asymptomatic chronic HBV carriers (ASCs) with low-level HBsAg. Methods The serum samples were collected from 654 ASCs in the First Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou Sixth People’s Hospital and the 903th Hospital of PLA. According to the level of HBsAg, ASCs were divided into low-level HBsAg group (≤10 IU/mL) and high-level HBsAg group (>10 IU/mL). The pre-S/S gene amplification and sequencing were performed in 138 ASCs with low-level HBsAg and 100 age-matched ASCs with high-level HBsAg. A phylogenetic tree was constructed to determine the genotype, based on the successful sequencing results of Pre-S gene in the high level HBsAg group, the Pre-S gene reference sequences of the main ASCs genotypes in Eastern China were established. The sequence of Pre-S gene in low-level HBsAg group was analyzed and compared with the reference sequences. SPSS 12.01 statistical software was used to analyze the data. Results Sixty-three cases of Pre-S/S were successfully sequenced in 138 ASCs of low-level HBsAg group, including 52 cases of B genotype and 11 cases of C genotype. Among the 100 cases of high-level HBsAg group, 94 cases of Pre-S/S were successfully sequenced, including 48 cases of B genotype and 46 cases of C genotype. The sequence analysis indicated that in the B genotype, 81 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 4 significant mutations: F56I/V, T76A/N/P in the Pre-S1 region, P15L/S/T and Y21T/F/H/N in the Pre-S2 region;while 47 amino acid mutation sites were found in Pre-S protein of high-level HBsAg group, including 3 significant mutations: L34F, V49A and P59S/L in Pre-S1 region. The total number of amino acid mutation sites in the low-level HBsAg group of B genotype was higher than that of the high-level HBsAg group (χ2=14.008, P<0.05). In the C genotype, 19 amino acid mutation sites were found in the Pre-S protein of the low-level HBsAg group, including 3 significant mutations: W66V/G and A79V in the Pre-S1 region, V32A in the Pre-S2 region;while 39 amino acid mutation sites were found in Pre-S protein of the high-level HBsAg group, including 2 significant mutations: A79V in Pre-S1 region and T49I in Pre-S2 region. The total number of amino acid mutation sites of Pre-S protein in the C genotype was significantly different between the two groups (χ2=7.571, P<0.05). Conclusion Significant mutations in Pre-S gene may be associated with the persistent expression of low-level HBsAg in ASCs.