| Objective to investigate the association between HBV preS mutation and theprogression of clinical infections (liver fibrosis and liver cirrhosis), identifyingsignificant mutation for further functional analysis.Methods (1) Cross-sectional comparison: Total of469patients with chronicHBV infection were enrolled, including352patients with chronic hepatitis B (CHB),and117patients with decompensated liver cirrhosis (dLC). All CHB patientsunderwent liver biopsy, among whom119patients were (F)0–1and233patientswere F2–4. Serum HBV DNA were extracted for Pre-S gene amplification andsubjected to sequencing to identify significant individual mutation.(2) To Construct vector containing preS deletion viral genome and counterpartwild-type (WT) genome. Viral gemome with frequently-detected preS deletion(nt16–54) was taken as template to construct pTriEx-preS2-deleted and WT plasmids.The engineering constructs were respectively transfected HepG2cells. Cells wereharvested72hours after transfection. Intracellular replicative intermediate andpgRNA levels were assessed for determing and replication capacity and viraltransicriptional level. Supernatant HBsAg, PreS2, PreS1, and HBeAg levels wereassesed to evaluate viral protein expression level..(3) To compare preS mutation between serum HBV DNA and intrahepaticHBV cccDNA in six patients..Results (1) Cross-sectional analysis showed (i) Occrrrence of total preS deletionmutations: among the469patients,59patients (12.6%) were detected with preSdeletion, including34patients (9.7%) in CHB group and25patients (21.4%) in dLCgroup, with statistical significant difference between groups (P=0.002). For theCHB patients that with preS deletion,5patients (4.2%) were F0–1and29patients (12.4%) were F2–4(P=0.013).(ii) Occruuence of preS start code mutation:55patients (11.7%) were detected with start code mutation, including30(8.5%) in CHBgroup and25(21.4%) in dLC group (P <0.001). For the CHB patients that with startcode mutation,5patients (4.2%) were F0-1and25patients (10.7%) were F2–4(P=0.043).(iii) Characteristics of preS individual mutations: preS mutations weredetected on31sites of preS gene. The detection rates of mutations like T2931etc.were significantly higher in dLC patients than in CHB patients, in genotype Bpatients than in genotype C patients.(iv) Multivarate analysis: age, genotype, preSdeletion, preS2start code mutation and T2857, C2931, G2950, C3116,A3120, andA109mutations were independent risk factors for dLC; genotype was an independentrisk factor for fibrosis progression (F2–4) in CHB patietns.(2) Functional analysis, showed that no difference was found in supernatantHBsAg, HBeAg and preS protein levels of HepG2cells transfected withpTriEx-preS2-deleted or pTriEx-preS2-wildtype replicon. The HBV DNA level insupernatant and intracellular replicative intermediate level were slightly higher, butnot statistically different in pTriEx-preS2-deleted replicon transfected HepG2cells,compared with pTriEx-preS2-wildtype replicon transfected HepG2cells. However,secreted preS2protein in supernatant was significantly lower inpTriEx-preS2-deleted replicon transfected HepG2cells. Intracellular HBV RNAanalysis showed that no difference in3.5kb pgRNA,2.4kb mRNA, and2.1kb mRNAbetween the HepG2cells that transfected with preS-deleted replicon or wildtypereplicon.(3) Comparison of preS sequences from serum HBV DNA and intrahepaticHBVcccDNA of the six patient showed that preS deletion could be detected in both serumand liver tissue sample.Conclusion: along with the disease progression, the detection rate of preSdeletion significantly increased. PreS deletion was closely associated with clinicaloutcome of HBV infection (namely, liver fibrosis). Supernatant preS2protein levelsignificantly reduced in HepG2cells transfected with preS2-deleted replicon compared with the wildtype replicon. HBV preS deletion could be recoreded inintrahepactic HBV cccDNA pool. |
