| Objective: Through sequence alignment analysis of viral S gene in HBV-related HCC,we can understand the role of viral S gene mutation in HBV-related HCC in the process of HBV-induced HCC,and provide theoretical basis for revealing the pathogenesis of HBV-related HCC.Methods: A total of 204 samples were collected and divided into high level(n=60,HBs Ag level>10 IU/ml)and low level(n=144,HBs Ab level ≤ 10 IU/ml)HBs Ag groups.General laboratory tests,detection of the HBs Ag immune complex(HBs Ag-CIC),and sequencing of samples between the two groups were conducted for sequence comparison,analysis of mutations related to the main hydrophilic region(MHR)of the HBV S gene,and prediction of changes in the protein properties and functions of HBs Ag caused by the MHR hot spot mutation of the S gene.The viral S gene sequence in HBV related HCCs was downloaded through Gen Bank,and a total of219 HBV related HCC samples and 22 reference sequences containing HBV A-H genotypes were included.Bioedit software was used to manually proofread and sequence splice the sample sequences included in the study,Cluster X multiple sequence alignment software was used to perform multiple sequence alignment,and phylogenetic evolution trees were constructed to genotype 219 samples.The S gene nucleotide sequence alignment database was constructed using B genotype samples from 41 HBV related HCCs obtained by genotyping and 52 asymptomatic HBV infected individuals with low level HBs Ag obtained by sequencing.Amino acid comparison analysis was conducted on the self built database through MEGA X software,and the amino acid mutation sites and frequencies of the S gene between the two groups were statistically analyzed.Results: Compared with the high level HBs Ag group,the low level HBs Ag group had epidemiological characteristics such as older age,more B genotypes,serological patterns dominated by HBs Ag/anti HBe/anti HBC positive,and low HBV DNA replication(P<0.05).Comparing the positive rates of HBs Ag-CIC under different serological patterns and genotypes between the two groups,it was found that the low level HBs Ag group had a higher positive rate of HBs Ag-CIC(P<0.05);There was a weak correlation between HBs Ag-CIC and HBs Ag or HBV DNA between the two groups(r=0.32,0.27,0.41,0.48;P<0.05).The results of HBV S gene sequencing between the two groups showed that the MHR hot spot mutations of S gene of genotype B were T126 A,M133L/T/S and F134L/T/I;The MHR hot spot mutations of S gene of genotype C were Q101 R and I126S/T.The mutation rate of MHR mutation of S gene in HBs Ag-CIC positive patients was higher in low-level HBs Ag group(P<0.05).Through gene sequence analysis of 41 HBV-related HCC samples and 52 asymptomatic HBV samples of low level HBs Ag,a total of 18 amino acid hot spots were found,including 9significant amino acid mutations between the two groups.In the asymptomatic HBV group,the mutation frequency of G10K/R,N40 S,V47E/G,M133S/T/L,M198 I,S204N/R,I208 T was higher than that of the HBV-related HCC group,and the difference was statistically significant(P<0.05).In the HBV-related HCC group,the L21 S mutation frequency of the hot spot mutation was higher than that of the asymptomatic HBV group,and the difference was statistically significant.Conclusion: Multiple characteristic mutations in the S gene occur in asymptomatic HBV infection with low levels of HBs Ag.Mutations in T126 A,M133L/T/S,and F134L/T/I may be one of the causes of low expression and asymptomatic HBs Ag in HBV patients.L21 S is the hot spot mutation site of virus S gene in HBV-related HCC,and L21 S may be a high risk factor for HCC in HBV infected persons. |
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