隐匿性乙型肝炎病毒感染者病毒基因跨膜结构变异性分析

Analysis of transmembrane structural variability of viral genes in cryptic hepatitis B virus infection

目的分析隐匿性乙型肝炎病毒(HBV)感染(OBI)者血清样本中HBV S区基因跨膜结构(TM)突变导致的氨基酸替换现象,探讨造成HBV表面抗原(HBsAg)漏检的原因和临床意义。方法选取上海中医药大学附属第七人民医院检验科采集的28例OBI患者血清样本(OBI组)和10例慢性HBV感染(CHB)患者血清样本(CHB组)。采用聚合酶链反应(PCR)技术扩增OBI组和CHB组HBV S区基因,扩增产物分别进行克隆测序和直接测序,进而进行核苷酸序列比对分析。结果OBI组TM1~TM4共26个位点发生185次突变,突变率为3.1%(185/6052),TM1、TM2、TM3、TM4突变率分别为1.6%(16/1020)、2.2%(28/1292)、4.2%(97/2312)、3.1%(44/1428)。CHB组TM发生4次突变,突变率为0.4%(4/890),TM1、TM2、TM3均未发生突变,TM4突变率为1.9%(4/210)。OBI组TM突变率、TM4突变率均高于CHB组,差异均有统计学意义(χ^(2)=19.92,P<0.01;χ^(2)=0.89,P=0.03)。OBI组TM1、TM2、TM3、TM4亲水性突变率分别为62.5%(10/16)、71.4%(20/28)、38.1%(37/97)、20.5%(9/44),4者比较差异有统计学意义(P<0.01)。OBI组共发现7种精氨酸替换突变,分别为C85R、L162R、W163R、W172R、L186R、W191R、C211R。结论HBV S区基因TM突变导致疏水性氨基酸替换为亲水性氨基酸,会导致临床上HBsAg的漏检,产生OBI现象。

Objective To analyze the amino acid substitution phenomenon caused by mutation of transmembrane domain(TM)in S region of hepatitis B virus(HBV)in serum samples from occult HBV infection(OBI)patients,and to explore the reasons and clinical significance of the missed detection of HBV surface antigen(HBsAg).Methods The serum samples of 28 patients with OBI(OBI group)and the serum samples of 10 patients with chronic hepatitis B(CHB)infection(CHB group)were collected in Department of Medical Laboratory,Seventh People′s Hospital Affiliated to Shanghai University of Traditional Chinese Medicine were selected.Polymerase chain reaction(PCR)technology was used to amplify the HBV S region gene in OBI group and CHB group.The amplified products were subjected to both cloning sequencing and direct sequencing,respectively,then were subjected to nucleotide sequence alignment analysis.Results There were 185 mutations in 26 sites of TM1-TM4 in the OBI group,with a mutation rate of 3.1%(185/6052).The mutation rates of TM1,TM2,TM3 and TM4 were 1.6%(16/1020),2.2%(28/1292),4.2%(97/2312)and 3.1%(44/1428)respectively.There were 4 mutations in the CHB group,with a mutation rate of 0.4%(4/890).No mutation occurred in TM1,TM2 and TM3,and the mutation rate of TM4 was 1.9%(4/210).The mutation rate of TM and the mutation rate of TM4 in the OBI group were higher than those in the CHB group(χ^(2)=19.92,P<0.01;χ^(2)=0.89,P=0.03).The hydrophilic mutation rates of TM1,TM2,TM3 and TM4 in the OBI group were 62.5%(10/16),71.4%(20/28),38.1%(37/97)and 20.5%(9/44)respectively(P<0.01).Seven arginine substitution mutations were found in the OBI group,and they were C85R,L162R,W163R,W172R,L186R,W191R,and C211R respectively.Conclusion The mutation of TM in S region of HBV gene leads to the replacement of hydrophobic amino acids with hydrophilic amino acids,which can result in false-negative HBsAg clinical detection and the occurrence of OBI.