Analysis For Clinical Implications Of Hepatitis B Virus S-gene Additional N-glycosylation Mutation In Patients With Coexisting HBsAg And AntiHBs

Objective: To analyze additional N-glycosylation mutation in major hydrophilic region(MHR) of HBV S gene in patients with coexisting HBsAg+antiHBs for revealing its characteristics and clinical implications of coexisting HBsAg+antiHBs. Methods: HBV S gene from 284 patients with coexisting HBsAg+antiHBs and 314 patients with single HBsAg was amplified respectively for sequence analysis. A chronic hepatitis B patient with coexisting HBsAg+anti HBs was found to harbor a novel double additional N-glycosylation mutation in MHR and was selected for further study. pcDNA3.1(-)-myc-His A and pTriEx-mod-1.1 HBV recombinant vectors harboring the novel mutant or control PreS/S genes and wild type were constructed and transfected in HepG2 cells respectively for the analysis of supernatant HBV DNA production and HBV mutants’ replication competence by real-time PCR, at the same time supernatant HBsAg was absolutely quantitatived, Western blotting and immunofluores-cence were used for detecting HBsAg- His fusion protein to analyze the effect of mutation on the HBsAg antigenicity. Resμlts: The detection rate of MHR N-glycosylation mutation in conexisting HBsAg+antiHBs group was significantly higher than that in single HBsAg group(11.3% and 2.9% P<0.01, respectively). In coexisting HBsAg+antiHBs cohort, hepatocellular carcinoma(HCC) occupied 46.9%(15/32) in patients with N-glycosylation mutation at the time of testing; by contrast, HCC occupied 22.6%(57/252) in those without N-glycosylation mutation(P<0.01). N-glycosylation mutational pattern of the novel strain was s116-118TST→NST+s131-133TSM→NST concomitant with s P120deletion+G145D mutation. The novel mutant occupied 98.0%, 2.0%, and 2.5% of viral clones respectively in three sequential serum samples. Mutant with single N-glycosylation mutation s130-132GTS→NSS without sP120deletion+G145D was detected in sample 2, occupying 17.6% of viral clones. Compared to the wild-type, the novel mutant had 31% increase of replication capacity, but decrease of HBsAg level. Immunofluorescence showed that elimination of the two additional N-glycosylation mutations only partly restored HBsAg detection by antiHBs, suggesting that sP120deletion+G145D mutation also attenuated HBsAg antiginicity. Conclusions: Additional N-glycosylation mutation in MHR of HBV S gene is associated with coexisting HBsAg+antiHBs, and the two parameters together might be a better risk factor for HCC occurrence. Combination of two additional N-glycosylation mutation, sP120 deletion and sG145 D mutation may co-play a role in silence of HBsAg antigenicity.