Research On The Mutations Of The PreS/S Gene In CHB Patients HAD Impact On The Amounts Of Circulating HBsAg After NAs Treatment

Research background:HBV cccDNA is the original template of HBsAg, while the available antiviral drugs can not eradicate intrahepatic HBV cccDNA. The method of quantitative checking HBV cccDNA is liver biopsy which is guided by ultrasound, but there are risks and contraindictions of liver biopsy. So HBsAg which is closely related to HBV cccDNA was proposed as a surrogate marker for HBV cccDNA and used as criteria of end point treatment. Researches in our team show that 14% of CHB patients with NAs treatment over 10 years got HBsAg seroconversion. An international research found that HBV cccDNA is slightly related to serum HBsAg titers. Therefore, are there any factors affecting HBsAg expression? Whether PreS/S gene mutations alter the structure and function of HBsAg? Will HBsAg mutants escape detection by common screening tests? Recently, the related reports just explore it at transverse section and these results are not concordant.Objective:We aimed to evaluate whether mutations of the preS/S gene in CHB patients had any impact on the amounts of circulating HBsAg after NAs treatment.Method:Study patients:70 cases which were selected from our antiviral treatment cohort were included in this study according to the following criteria: (1)All subjects are CHB patients and their diagnosis meet the criteria from The guideline of prevention and treatment of CHB in 2010.(2)Patient got virological response(VR) within 6 months after NAs antiviral treatment and maintain VR over than 3 years.(3)All patients have done liver biopsy at baseline and their liver samples have been stored in refrigerator. (4)During the following up, there is no virological breakthrough.(5)No evidence of HBV carriers, LC or HCC based on clinical criteria and ultrasound examination at baseline.(6)Patients with immunodepressant and interferon treatment were removed. Laboratory method: we collected serum samples and liver tissue prior NAs treatment and serum samples at the second year and the fourth year during NAs treatment. Specific primer of HBV cccDNA and rolling circle amplification (RCA) method were used to amplify intrahepatic HBV cccDNA. Real-time PCR method was applied in amplifing PreS/S gene from HBV cccDNA, and the PCR products were sent to biocompany for gene senqucing. The sequenced results were matched to the standard sequence which was found from the website NCBI for finding the mutations in PreS/S. Serum rcDNA were extracted by water boiling, then specific primers and real-time PCR method has been used to check HBV DNA genotype. Quantitative detect serum HBsAg by Roche reagent. All study patients were divided into two groups-those patients’serum HBsAg level decreaseing extent less than 2LogIU/mL belong to group A from the second year to the fourth year after treatment, and the rest of them belong to group B. Statistic method:All statistic tests were two-sided, and performed by using the Statistical Program for Social Sciences(SPSS 13.0 for Windows, SPSS, Chicago, IL, USA). Student’s t test and Fisher’s exact test were used to analyse the factors that may affect HBsAg levels after NAs treatment. Apply Chi-square test to analyse whether PreS/S gene mutation has influence on HBsAg levels after NAs treatment and the factors that may affect PreS/S gene mutation. A P value of less than 0.05 was considered as statistically significant.Results:11 cases (15.71%) had PreS deleted mutation,5 happened in PreS1 region,6 happened in PreS2 region. The rate of deletion in the PreS region among C genotype patients is higher than that in B genotype patients (25%VS5.8%, P=0.028). Common point mutations’ratio in PreS1 gene among A group is significantly lower than that in B group (52.63% VS 90.19%, P<0.05). The rate of general point mutation in the region of PreS2 among A group is higher than that among B group (52.63% VS 84.31%, P=0.006).The rate of sI126 amino acid mutation among A group is lower than B group (5.26% VS 33.33%, P<0.05)Colclusion:The deletions in the PreS region are more often found in patients with HBV genotype C than in those with genotype B. C2988 and A96 point mutations in HBV cccDNA PreS region may have influence on the expression of HBsAg. sI126 amino acid mutation may affect the screening test of HBsAg and lead to the results lower than the true value, which suggest that not only the decreasing of HBsAg level influenced by the amount of HBV cccDNA and its transcription activity, but also by HBV cccDNA preS/S gene mutation.