Sequence Analysis And Correlation Research Of Pre-S/S Gene In Asymptomatic Carriers With Low Level HBsAg Expression

Objective: To investigate the characteristics of HBV Pre-S/S gene sequence and amino acid variation in asymptomatic hepatitis B carriers(ASCs)with low level HBsAg,further accumulate and improve the molecular epidemiological data of ASCs with low level HBsAg,and provide theoretical basis for the prevention of HBV transmission and the effective clearance of low level HBsAg.Methods: 1.Using chemiluminescence immunoassay(CMIA)screening of three hospitals over 18 adult health examination in 23130 cases(male 10543,female 12587)of HBsAg positive ASCs cases,retrospectively collected medical records,HBV serum markers(HBsAg,anti-HBs,HBeAg,anti-HBe,anti-HBc)and biochemical indexes were measured in patients who met the inclusion criteria,HBV DNA level was measured by real-time fluorescent quantitative PCR.High and low HBsAg levels were grouped by HBsAg 10IU/mL to analyze the molecular epidemiological characteristics of the low level HBsAg group.2.276 patients in the low level HBsAg group and 100 patients in the high level HBsAg group(randomly selected according to the principle of age matching)were sequenced by nested PCR with segmented amplification of HBV Pre-S/S genes and Sanger sequencing.After sequencing,Seqman program in DNAstar software was used for sequence splicing,and blast was used for sequence alignment.3.MEAG6.0 was used to construct phylogenetic tree and HBV genotyping of the two groups of samples,and the correlation among serology,virology and HBV genotype characteristics of the two groups samples was analyzed.4.The reference sequence of ASCs Pre-S/S gene was constructed based on the obtained HBV Pre-S/S gene sequence of high level HBsAg group,and homology analysis and verification were conducted with the reference sequence of NCBI recommended genotypes.Finally,MEAG6.0 was used for sequence comparison to analyze the expression of amino acids in ASCs Pre-S/S region.Results: 1.The serological results of the low level HBsAg group were as follows: Age(55.09±16.45)years old,HBV DNA concentration(1.32±1.60)log10IU/mL,HBV DNA positive rate of 45.65%(126/276),B genotype(104 cases,82.54%),adw serotype(106 cases,84.13%),M2(HBsAg/anti-HBe/anti-HBc,97.1%)serological model.The mean age was higher than that of the high level HBsAg group(P<0.05),the HBV DNA level and the positive rate of HBV DNA were lower than that of the high level HBsAg group(P<0.05),there was no statistical difference in the sex composition ratio and ALT between the two groups(P>0.05),and there were statistical differences in age,HBV serological marker composition ratio,HBV genotype composition ratio and HBV serotype composition ratio between the two groups(P<0.05).2.126 patients in the low level HBsAg group were successfully sequenced in the Pre-S/S region of HBV,and 94 patients in the randomly selected 100 patients in the high level HBsAg group were sequenced.The Pre-S/S reference sequence of ASCs genotypes B and C in eastern China was constructed on the basis of the sequencing results of high level groups and had a high homology with the Pre-S/S genotypes B and C of ASCs reported in most regions of China.3.Unreported mutation sites in the Pre-S gene region of the low level HBsAg group included: 30 unit-point mutations,16 double-site combined mutations,5 three-site combined mutations,and 5 deletion mutations in Pre-S2 gene.As well as 7 meaningful unit point mutations and 17 hot spot mutations,the number of amino acid mutation sites in Pre-S(Pre-S1,Pre-S2)protein was higher than that in the high level HBsAg group(P<0.05),and the percentage of characteristic amino acid mutations(unreported mutations and meaningful mutations)in Pre-S1 gene(B:55.77%,C:72.72%)was higher than that in Pre-S2 gene(B:38.46%,C:45.45%)(P<0.05).In addition,some hot spots in the Pre-S region mutated T76 A,P15L,Y21 T and V32 A,Lack of mutations in the Pre-S2?2-5,Pre-S2?12-14,Pre-S2?5-22,Pre-S2?9-22,Pre-S2 ? 12-22 in structural loci associated with viral replication and T,B cell identification table.4.Five unit-point mutations,40 double-site associated mutations,13 three-site associated mutations and 1 four-site associated mutations were found in the S region of the low level HBsAg group.There were 19 meaningful unit point mutations,7 meaningful double-site associated mutations(P<0.05)and 25 hot spot mutations.These characteristic amino acid unit point and multi-site associated mutations accounted for a high proportion in the low level HBsAg group(B genotype: 82.7%,86/104,C genotype: 63.6%,14/22),and the mutation hotspots were mainly distributed on both sides of MHR,which were amino acid sites 40-49 and 198-220.Conclusions: The ASCs HBV Pre-S/S gene with low HBsAg infection has multiple unreported mutations(including joint mutations),meaningful mutations,and deletion mutations with high mutation frequency,Pre-S/S gene mutations may be associated with HBV replication defects,which may be the cause of the observed low expression levels of HBsAg.