Towards Personalized Medicine Of Hepatitis B Virus Infectious Disease Based On Genetic Mutation Detection And Analysis

Infection with hepatitis B virus (HBV) is a serious health issue worldwide as well asthe most serious infectious disease in China. It has more than30years of Chinese medicalhistory. HBV genome is composed of double-stranded incomplete annular DNA,containing about3,200nucleotides. And can be mainly divided into P, C, X, S. HBVDNA has high level of mutability, and thus forms several genotypes of HBV DNA. Giventhat HBV infection frequently leads to the development of anti-viral resistance and/or theprogression to liver cirrhosis and hepatocellular carcinoma (LC/HCC), which in turn isclosely associated with HBV genotypes and genetic mutations, we profiled HBVgenotypes as well as genetic mutations in HBV patients from northwest China andanalyzed their associations with the development of drug resistance and LC/HCC. Ourultimate goal is to establish the individualized diagnosis platform for hepatitis Bvirus gene. It is found that the HBV genetic has new characteristics as well as biomarkersin disease progression. Experiment1: Bioformatics analyzing of hepatitis B virus and primers designUsing the bioinformatics analysis method, established the foundation for further genesequencing for the8genotypes of hepatitis B virus A~H. The way of genotyping,detection of resistance gene mutation and process characteristics mutation as well asclinical value estimate are based on amplifying S region, P region and BCP-PreC/C region.At the same time sample testing proved that P (S) region can be genotyped, while havingresistance detective feasibility and stability of highly conserved.Experiment2: Studying of correlation of HBV P region mutations causing drug-resistanceUsing bioinformatics analysis and Sanger sequencing method, the statistical analysisof HBV P region of HBV samples from northwest China in the groups of untreated na ve,nucleoside analogue (NA)-resistant, liver cirrhosis and hepatocellular carcinoma(LC/HCC), were analyzed by the individual gene diagnosis software HBV drug guide,genotyping software HBV liner, as well as NCBI genotyping tool. First we confirmed thatHBV liner can genotype hepatitis B virus; secondly we made sure HBV drug guide canexactly diagnose gene mutations. The statistical analysis showed that C genotype is themain genotype in Northwest China HBV patients (59.31%), the second is genotype B(38.35%), genotype D is less (2.34%), haven`t found genotype A, The drug resistancemutations in P region under the pressure of nucleoside analogue drug presented at theproportion of rt169(43.40%), rt180(47.17%), rt181(1.89%), rt202(5.67%), rt204(32.08%), rt236(3.77%), rt250(1.89%). These mutations have correlations with drugresistances, especially rt169is relative to ETV, rt180is to LVD and ETV as well LDT,rt204has direct effect to LVD and LDT. Natural mutations in P region were found at theaverage level of99%in B genotype among the3groups, rtI169and rtL180F are relativeto LVD resistance; both may relate to long-term evolution under the drug pressures andhave natural genetic variation, so as to potential genetic variability. Furthermore, thesephenomenon’s prompt that the hepatitis B virus in China may have the possibility ofgenetic drift. Experiment3: Studying of correlative mutations between HBV BCP-PreC/C regionmutations and clinical representationUsing bioinformatics analysis and Sanger sequencing method, the HBV BCP-PreC/Cregion of HBV samples from northwest China in the groups of untreated na ve, nucleosideanalogue (NA)-resistant, liver cirrhosis and hepatocellular carcinoma (LC/HCC), wereanalyzed by the individual gene diagnosis software HBV drug guide, and combined withclinical value for the statistics of gene and clinical presentations. It is confirmed that HBVdrug guide has great feasibility and advantage in analyzing BCP-PreC/C region. We foundthat1762/1764mutations have high level of mutations in the group of untreated na ve andLC/HCC. The average is99%. Statistical difference was found between mutant and wildas the condition of ALT and AST limits as40U/L by contrast with other sites, P<0.05.1762/1764mutant are positively correlated with liver injury;1896/1899mutationsassociate with HBeAg false negative, which was confirmed by statistical analysis in thegroup of untreated na ve and the clinical value limits1PEIU/ml, P<0.0001. G1799Cmutations was first found highly correlated with liver cirrhosis and hepatocellularcarcinoma (LC/HCC) in B genotype of Chinese hepatitis B virus. Statistical difference ispresented between mutant and wild1799, P<0.0001.G1799C is positively related tohepatocarcinogenesis genesis. Because of its correlation of X protein variation, G1799Cmay become the marked site for LC/HCC caused by HBV infection.