Study On The Correlation Between HBV Genotype And The Clinical Characteristics Of Hepatocellular Carcinoma

Background Hepatitis B virus (HBV) is a global problem of health, with an estimated 350 million chronic carriers around the world. Chronic infection of HBV is an important factor in the development of liver cirrhosis (LC) and hepatocellular carcinoma (HCC) in our country. HBV has circular and partially double-stranded DNA genome containing 4 overlapping open reading frames: C, S, P and X gene, translate nueclear shell protein, outer membrane protein, DNA polymerase, and transactivated X protein.Recently researches show that the incidence of the HBV mutation is 10 times than other DNA approximately, HBV gene mutation relate with the clinical prognosis of the disease, the virulence, replication capacity, change of epitope of the host immuno-response, and the drug-resistance of anti-viral drug of the mutant are more severe than before. Some sequences' mutation will interfere some other genes' express because of the overlapping of different open frames and the regular sequences within the reading frame, such as promoter, enhancer, etc. It is important that the research the mutation infection on biological function, rather than sub-genesegment research. The recently researches indicate that the damage of the liver cell is not induced by HBV directly, but caused by the immuno-response of the host aim at the antigen which expressed by the infected liver cell.According to the differences of HBV surface protein's epitope, HBV was divided into 9 subtypes in serology, but it is not sufficient to represent the heterogeneity of the viral gene. HBV is marked off into A-H, 8 genotypes based on the heterogeneity of the whole virus gene sequence no less than 8%. The genotypes have a characteristic geographic distribution, and have related with the progress and prognosis of the disease, but we have insufficient research between HBV genotype distribution and its clinical domestically, especially on the correlation of HBV genotype and HCC. It is important the establish a simple, rapid, and more feasible genotyping method, to commence a largescale epidemiology survey, then to research the correlation of HBV genotype and HCC. Objective To investigate the correlation of the mutation of HBV and its virulence, establish a sufficient amplification PCR and cloning the whole HBV gene via low titer serum samples; to establish a multi-pair type-specific primers method to genotyping the HBV; to research the HBV genotype distribution of HCC patients in HuBei province; and analysis the correlation of HBV genotypes and HCC clinical characteristics.Methods We designed the primers, differentiated the genotype using the type-specific primers method. Selection the sample of the genotype C, purification, enzymatic, filtration, and DNA polymerase. The obtained positive clone sequenced by CEQ2000XL automatic sequence. We analysis the homologies and restrictive endonuclease map of the HBV sequence which published in Genebank. We drawed the phylogenetic tree of HBV genotype B and C according to maximum likelihood method, and identified by the nearest neighbour method. A total of 297 HBV DNA-positive serum samples include 52 asymptomatic HBV carriers (ACS), 56 chronic hepatitis (CHB), 32 fulminant hepatic failure (FHF), 22 liver cirrhosis (LC), and 135 HCC patients. Use the type-specific primers genotyping the HBV based on PCR, compare the distribution of HCC and hepatitis, furthermore, analysis the genotype distribution of HBV DNA-positive in HCC patients, analysis the correlations between HBV genotypes and HBV DNA level, HBeAg positive rate, the levels of ALT, AST, T-BIL and AFP, pathology classification of HCC, and the survival rate, etc.Results The type-specific primers method is a simple and precise HBV genotyping method. In the 297 HBV DNA-positive patients, 218(73.4%) were genotype B, 65(21.9%) were genotype C, and 14(4.7%) were BC combination type, none was detected in A, D, E genotype. We compared the copy standard of CH and HCC patients, found that the HBV DNA leveland HBeAg positive rate of HCC patients are much lower than CHB. In the 135 patients of HCC, 102(75.6%) were genotype B, 26(19.2%) were genotype C, and 7(5.2%) were BC combination type. HBV genotypes have no correlation with age and genders, genotype C has a higher HBV DNA level and HBeAg positive rate than genotype B, ALT, AST, T-BIL and AFP levels have significant differences between genotype C and B, genotype C's are higher. The 1 year survival rate are 84.42%(B) and 69.57%(C), the tumor pathology classification have no significance between 3 group. Conclusion We confirmed that type-specific primers PCR genotyping HBV is a simple, rapid and precise method; the method can be used in epidemiology investigate; there are B, C and BC combination genotype in Hubei province, and genotype B is the major genotype in the province, genotype C has a higher HBV DNA level than B, and have a worse liver function damage, lower survival rate.The innovation of the thesis1. Confirmed that type-specific primers PCR genotyping HBV is a simple, rapid and precise method.2. Analysis the distribution of HBV genotype in Hubei, and found that genotype B is the major genotype in the province, and correlated with the activation of the disease.3. Researched the correlation of HBV genotypes and the clinical characteristics of HCC patient systematically in China, for the first time.The significance of the thesis1. Confirmed that type-specific primers PCR genotyping HBV is a simple, rapid and precise method. We can research the biological significance of HBV gene mutation on gene standard.2. Comprehended the HBV genotype distribution of HCC patients in Hubei, give a conference for the further research of HBV genotype in HCC patients in China.3. Give the HBV genotype and clinical characteristics of HCC patients investigate a pilot research, and the foundation of the biological characteristics and mechanism of HBV and HCC.