The Study On The Relationship Between HBV X Gene Promoter Methylation In Liver Tissues And Clinical In Chronic Hepatitis B Patients

ObjectiveTo detect HBV X gene promoter methylation status in liver tissues of patients with chronic hepatitis B, and to explore the relationship between HBV X gene promoter methylation in Liver tissues and clinical.MethodsWe collected liver tissue and serum samples from 45 patients with chronic hepatitis B in Tianjin infectious diseases hospital in July 2009-March 2010, including HBeAg-pos 30 cases, HBeAg-neg 15 cases, aged 16-65 years old. All cases are in line the diagnostic criteria for chronic hepatitis B of "chronic hepatitis B prevention and treatment guidelines," in 2005. Serum ALT, AST, TBiL were detected for all patients. Quantification of the HBsAg, HBeAg in sera was quantified using electrochemical Chemiluminescence immunoassay. The expression of HBsAg, HBcAg in liver was using immunohistochemistry. Liver tissue inflammatory activity grading (G) and degree of fibrosis staging (S) were evaluated. Quantitative PCR were used to examine HBV DNA in Sera. HBV DNA CpG Island was analyzed by MethPrimer Software, while using High Resolution Melting (HRM) to exam the methylation status of HBV X gene promoter.Results1. The result of liver tissue specimens immunohistochemistry in 45 chronic hepatitis B patients:HBsAg-pos:35 cases, HBsAg-neg:10 cases; HBcAg-pos: 23 cases, HBcAg-neg:22 cases.2.21 cases were detected methylation in 45 cases of liver tissues. (21/45,46.7%), Including HBeAg-pos 10 cases (10/21,47.6%), HBsAg-pos 11 cases (11/21, 52.4%), HBcAg-pos 7 cases (7/21,33.3%); 24 cases were not detected methylation, Including HBeAg-pos 20 cases (20/24,83.3%), HBsAg-pos 24 cases (24/24,100%), HBcAg-pos 16 cases (16/24,66.7%). Compared with unmethylated group, the positive rate of HBeAg, HBsAg, HBcAg in methylated group was lower, there was statistically significance, respectively. (P<0.05).3. There was no statistically significance on sex, age, levels of serum ALT, AST, TBiL, HBV DNA, HBsAg, HBeAg between methylated and unmethylated group, respectively (P>0.05).4. All of 21 methylated cases, Compared the methylation levels of HBeAg-neg group (61.45±18.27) with that of HBeAg-pos group (34.10±16.03), there was statistically significance (t=-3.631, P<0.01); the HBV DNA level of HBeAg-neg group (4.32±1.57), the HBV DNA level of HBeAg-pos group (7.03±1.57), there was statistically significance (t'=5.100, P<0.01).5. All of 21 methylated cases, there was negative correlation between serum HBV DNA level and methylation density of HBV DNA (r=-0.832, P<0.01). There was negative correlation between serum HBeAg level and methylation density of HBV DNA (rs=-0.657,P<0.05). And there was negative correlation between methylation density of HBV DNA and age (rs=-0.706, P<0.05). There was no correlation between methylation density of HBV DNA, HBsAg quantification and ALT, AST, TBiL (rs=0.165,0.432,0.24,0.291,P>0.05).Conclusion1. The CpG island 2 of HBV DNA is situated at 1228-1665nt of HBV genotype C(AB540585) sequences and situated at 1246-1672nt of HBV genotype B(AB540582) sequences. Island 2 overlaps with the HBV enhancerⅠandⅩgene promoter, and is adjacent to the enhancerⅡ/core gene promoter region.2. Compared with unmethylated group, the positive rate of HBeAg in serum, HBsAg, HBcAg in liver of methylated group was lower. All of 21 methylated cases, the methylation level of HBeAg-neg group was higher than HBeAg-pos group.and the HBV DNA level of HBeAg-neg group was lower than HBeAg-pos group. There was negative correlation between serum HBeAg, HBV DNA level and methylation density. Our data show that HBVⅩgene promoter CpG island methylation may inhibit the expression of HBeAg in serum, the expression of HBsAg, HBcAg in liver tissue and the HBV DNA replication.3. There was no statistically significance on sex, age, levels of serum ALT, AST, TBiL, HBV DNA, HBsAg, HBeAg between methylated and unmethylated group, respectively. However, There was negative correlation between methylation density of HBV DNA and age in methylated group. As the growth of the age, methylation levels decreased. There was no correlation between methylation density and HBsAg, ALT, AST, TBiL level.