摘要：
目的 探讨乙型肝炎病毒(hepatitis B virus, HBV)感染者持续低水平表达乙型肝炎表面抗原(hepatitis B surface antigen, HBsAg)的S基因序列特征.方法 收集2016年2月至2017年12月解放军第九〇三医院和杭州市江干区人民医院的非活动性HBsAg 携带者1 308 例的血清样本,根据HBsAg血清表达水平分为高水平组(≥10 IU／mL)和低水平组(＜10 IU／mL).对低水平组患者进行HBV S基因测序,另外在低水平组年龄和血清学模式( HBeAg阴性)相匹配的基础上,在高水平组患者中随机选择(分层抽样)100例进行HBV S基因测序,并对低水平HBsAg组患者的HBV S基因序列和高水平HBsAg组患者的HBV参考S基因序列进行比较分析.数据为正态分布的结果以均数±标准差表示,采用t检验;非正态分布的结果以M(QR)表示,采用Mann-Whitney U检验;卡方检验或Fisher精确检验比较两组间的连续变量和分类变量.结果 低水平组血清标本276份;高水平组血清标本1 032份,其中HBsAg、HBeAg、抗-HBc阳性257份,HBsAg、抗-HBe和抗-HBc阳性753份,HBsAg和抗-HBc阳性22份.对276例低水平组中126例感染者进行了成功的HBV S基因测序,根据低水平组年龄情况,选取高水平组内HBeAg阴性的随机标本100例,对其中94例感染者进行了基因分型和血清分型.结果显示,高水平组(94例)和低水平组(126例)在HBV血清学标志物、HBV DNA水平和HBV基因型分布差异均有统计学意义(均P＜0.05).建立的非活动性HBeAg携带者中B型和C型的参考序列与上海市、成都市、武汉市、云南省、北京市报道的B和C基因型序列具有很高的同源性(99.6%～100.0%),与日本、韩国的NCBI基因型B和C参考序列具有很高的同源性(98.2%～99.6%),但与一些远离中国地区患者具有较低的同源性(加拿大和印度尼西亚分别为98.2%和98.7%).低水平组B基因型小蛋白(即主要蛋白,SHB)的氨基酸突变数为71,热点突变数为19,均高于高水平组的39和8,比较差异均有统计学意义(χ2 值分别为12.303 和4.766,均P＜0.05).低水平组的氨基酸突变位点分布在主要亲水区(major hydrophilic region , MHR)的两侧(氨基酸残基40-49和198-220). C基因型两组间氨基酸突变数和热点突变数差异均无统计学意义(χ2 值分别为0.383和0.409,均P＞0.05).在B基因型中,低水平组有12个单位点突变,4个双位点共突变,其中1个单位点突变(S210R)和3个双位点共突变(G44E／V+T45P／I、G44E／V+L49P／R、N40S+I208T)不是热点突变;高水平组有2个单位点突变和2个双位点共突变,两组突变频率差异有统计学意义(χ2 ＝7.533,P＝0.006).在C基因型中,低水平组有5个单位点突变(T5A、A45T、T47A／K、Q101R、I126S／T),高水平组有1个单位点突变(N3S),两组突变频率差异有统计学意义(χ2 ＝47.914,P＝0.000).结论 MHR两侧多个区域和多个位点的显著突变(包括共突变)可能是导致非活动性HBsAg携带者HBsAg表达水平低的原因之一.%Objective To reveal the characteristics of S gene sequence of hepatitis B surface antigen (HBsAg) in hepatitis B virus (HBV)-infected patients with low HBsAg level.Methods From February 2016 to December 2017, 1 308 serum samples of inactive HBsAg carriers were collected from the 903rd Hospital of PLA and Hangzhou Jianggan District People′s Hospital.The cases were divided into high-level group and low-level group according to the level of serum HBsAg (10 IU／mL) expression.The HBV S gene was sequenced in patients with low-level HBsAg expression.In addition, in patients with high-level HBsAg, 100 patients were randomly selected (stratified sampling) for HBV S gene sequencing based on the matching of age and serological pattern (hepatitis B e antigen [HBeAg] negative) of low-level HBsAg group.A comparative analysis was conducted between HBV S gene sequences from inactive HBsAg carrier in low HBsAg expression group and the HBV reference S gene sequences from inactive HBsAg carrier in high HBsAg expression group .The results of normal distribution data were expressed as Mean ±SD, and analyzed using t-test.The results of non-normal distribution data were expressed by M(QR), and analyzed using Mann-Whitney U test.Chi-square test or Fisher exact test was used to compare continuous variables and classification variables between the two groups .Results There were 276 serum samples from the low level group and 1 032 serum samples from the high level group , including 257 HBsAg／HBeAg／anti-HBc-positive cases, 753 HBsAg／anti-HBe／anti-HBc-positive cases, and 22 HBsAg／anti-HBc-positive cases.Successful HBV S gene sequencing was performed on 126 out of 276 patients in the low-level HBsAg group.According to the age inthe low-level HBsAg group, 100 samples with negative HBeAg in the high-level HBsAg group were randomly selected , among which 94 patients were genotyped and hemotyped.The results showed that there were statistically significant differences in HBV serological markers , HBV DNA level and HBV genotype distribution between the high level group (94 cases) and the low level group (126 cases) (all P＜0.05).The ASC-R-B and ASC-R-C genotypes reported in this study had high homology (99.6%-100.0%) with those reported in Shanghai , Chengdu, Wuhan, Yunnan and Beijing of China , and high homology (98.2%-99.6%) with those reported in Japan and Korea of NCBI genotype B and C reference sequences, but had low homology with patients far away from China (98.2% in Canada and 98.7% in Indonesia).In genotype B of the low level group , the amino acid mutation number of SHB protein was 71, and the hot spot mutation number was 19, both higher than those in the high level group (39 and 8, respectively). The difference was statistically significant (χ2 ＝12.303 and 4.766, respectively, both P＜0.05).Amino acid mutation sites in the low HBsAg group were mainly distributed on both sides of the major hydrophilic region (MHR) (amino acid residues 40 -49 and 198 -220).There were no significant differences in amino acid mutation number and hot spot mutation number between the two groups of C genotype (χ2 ＝0.383 and 0.409, respectively, both P＞0.05).For genotype B, 12 single point mutations and 4 dual co-mutations were found in low level group.Among them, one single point mutation (S210R) and 3 dual co-mutations (G44E／V+T45P／I, G44E／V+L49P／R and N40S+I208T) were not hot spot mutations , while 2 dual co-mutations and 2 single point mutations were found in high level group.The difference between two groups was statistical significant (χ2 ＝7.533,P ＝0.006).For genotype C, 5 single point mutations ( T5A, A45T, T47A／K, Q101R and I126S／T) were found in low level group and 1 single point mutation (N3S) in high level group.The difference in mutation frequency between two groups were statistical significant (χ2 ＝47.914,P＝0.000).Conclusions Significant mutations in multiple regions and at multiple sites ( including co-mutations) on both sides of the MHR may be one of the causes of low HBsAg expression level in this population .
