Association Study Of Genetic Mutations And Diversity Of Hepatitis B Virus And Pathogenesis

In our country,chronic HBV infection is an important cause of liver cirrhosis(LC)or hepatocellular carcinoma(HCC).According to sequence diversity of HBV genomic DNA,10 genotypes have been identifed,which were reported to be associated with clinical manifestations of diseases.Moreover,mutations of HBV are correlation to the treatment of hepatitis and hepatocarcinogenesis.Thus,to genotype HBV and to detect related gene mutations in clinical are access to early diagnosis and personalized therapy for HCC.In our previous study,we developed and were patented a ?Hepatitis B Testing Platform for Personalized Diagnosis? containing the specific HBV DNA amplification module,HBV genotyping software and HBV gene mutation analysis software,with which HBV DNA can be amplified and aligned so as to HBV genotyping and mutation analysis.In the case-control study,we found the mutations in rt169/rt180 loci of reverse transcriptase region(RT)and in 1799 locus of BCP region.However the problems of instability ofDNA amplification,reading sequences of some DNAs and software compatibility were encountered when utilizing the platform.In this study,we upgraded the platform according to the principles of bioinformatics,and explored the cause of sequencing failure.Further,we explained the mutations of rt169/rt180 and 1799 in hepatocarcinogenesis.Part 1: Upgrade of the “Hepatitis B Testing Platform for Personalized Diagnosis”Aim: Solving the problems of instability of DNA amplification and software compatibility were encountered when utilizing the platform.Method: Choosing the standard sequences which meet the characteristics of epidemic strains in China.By analyzing the sequences,we find the sequence can be used by HBV genotyping and genotypic resistance detection.Analyzing the sequences to find the specific SNPs and design a new scoring algorithm.Based on the original software,we merge these functions into the new software.Then genotyping the HBV DNA sequences from the crowd of Chinese in Gen Bank by the new software and Genotyping tool software,respectively.Result: By analyzing the sequences,we find the sequence can be used by HBV genotyping and genotypic resistance detection,and we design the primers.HBVAnalyzer software completely has the functions of genotyping and geno mutation analysis.The genotyping results of HBVAnalyzer and Genotyping tool completely consistent.Part 2: Analysis of mutations in HBV RT regionAim: To investigate the relationship of HBV genotypes and virus resistance and clinical manifestation,and to explore the relationship of rt169/rt180 mutations and liver cancer.Method: Platform and primers are used to amplification and analysis of HBV RT sequences from 191 tissue samples,and to analysis gene mutations and to genotyping.Result: 190 cases were successfully amplified.The result of genotyping showed that C genotype is the main genotype(71.01%),the second is genotype B(28.40%).In the previous study,we found rt169 and rt180 had extremely high proportion of natural synonymous mutations in patients who infected genotype B HBV.In HCC patient,we alsofind rt169 and rt180 have natural synonymous mutations,and the mutation rates are 95.83% and 100%.And the peak figure shows mainly for hybrid mutant.The ratio of hybrid mutant in HCCs is similar to that in LC/HCCs,and the ratio of hybrid mutant in HCCs is higher than that in CHBs.Further evidence that with disease progression,the proportion of hybrid mutation gradually increased,prompt that the natural synonymous mutations of rt169/rt180 are associated with disease progression.Part 3: Analyzing the genetic diversity and mutations of HBV BCP/Pre C area.Aim: Looking for the reason and solution of how to interpret the HBV DNA sequences which are difficult to interpret.We want to reveal the correlation between complex mutations and genomic diversity,and exploring the correlation between 1799 mutation and liver cancer.Method: By building DNA libraries and monoclonal sequencing,we sequencing HBV BCP/Pre C area sequence from 21 samples which were sequencing failure.Platform is used to analysis the HBV BCP/Pre C sequences from the tissue samples,and to analysis gene mutations.Result: By cloning-sequencing,we found that that there are 144 types of mutations in 342 monoclonal sequences,there are 20 kinds of deletion and 7 kinds of insertion.And find the ?(1757-1765)/?(1824-1832)combined mutation,which is closely related with liver cancer.Every sample has at least 6 kinds of mutation.Show that the HBV genome diversity is produced by complex mutation.G1799C mutation rate of B genotypes is 85.71%,and mutation rate of C genotype is 100%,accord with the results of pilot study.Our research found combined mutation of G1799 C and 1762/1764 /1896/1899.Combined with the early data in the 1799 mutation and HBe Ag pseudo overcast and liver damage,We speculate that the G1799 C mutations enhance the capacity of HBV replication,reduce HBe Ag expression level,and to heighten degree of liver damage.