| [Objectives] To explore the risk factor and protect factor of hepatocellular carcinoma in Xiamen and to investigate the association between genotype and haplotype of MBL2 polymorphisms and the HCC risk. Biological assay was performed to determine the functional of MBL2 haplotypes, in addition, the relationship between MBL2 haplotypes and hepatitis B virus mutation was also studied.[Methods] 1. A 1:1 matched case-control study was conducted, in total, 315 HCC cases and identical number of healthy control were enrolled and investigated by using questionnaire. ELISA was performed to determine the HBV infection status. Univariate and multivariate logistic analysis were performed to calculate the ORs and 95%CI.Random forest was employed to identify the main effect factor of HCC risk among Xiamen population. Stratification analysis by HBV infection was performed to estimate the impact of HCC risk of all factors.2. High resolution melting method was used to MBL genotyping among HCC cases and healthy controls. The ORs and 95%CI of MBL2 genotypes and alleles were estimated by using conditional univariate regression. The frequency of MBL2 haplotypes were calculated by using Haplo.stats, and generalized linear model was applied to calculate the HCC risk of MBL2 haplotypes.3. Sequences contain MBL2 haplotypes were acquired by PCR and cloned to p GL3-Basic vector, and then transinfected to 293 T cell. The luciferase activity was determined to estimate the function of MBL2 haplotypes.4. The MBL2 genotyping and haplotype construction were performed among 50 treatment naive patients with chronic HBV infection, and deep sequencing was conducted in HBV isolates acquire from subjects. The mutation numbers, mutation frequency and quasispecies complexity estimated by Shannon's entropy were compared based on the MBL2 haplotypes.[Results] The conditional univariate regression suggested that HBs Ag positive, indoor decoration within 10 years, salty food intake, smoked food intake, mouldy food, ETS at home and at work, alcohol consumption and liver disease history were positively associated with HCC risk; Use of refrigerator, fruit, tea and coffee consumption were identified as the protective factors of HCC. The random forest suggested that the MDG of liver disease history, tea, alcohol, and fruit consumption were 42.31, 28.63, 26.99 and21.43, respectively.2. After the adjustment of education, HL genotype of-550H/L polymorphism was not significantly associated with HCC risk, however, LL genotype was significantly associated with HCC risk(AOR=1.61, 95%CI:1.00-2.57); The HCC risk of YX and XX genotype of-221 Y/X polymorphism were 1.51(95%CI:1.03-2.21) and 5.67(95%CI:1.82-17.67) when comparing with YY genotype. No significance was found in the +4 P/Q genotype and HCC risk. The AORs for Non B/B genotype and BB genotype of codon 54 polymorphism were 1.48(95%CI:1.01-2.17) and 3.99(95%CI:1.55-10.30),respectively. No mutation in codon 52 D and codon 57 were observed in our subjects which is consistent with the genetic characteristic of Asian population. The haplotype analysis indicated that the top 3 haplotypes with high frequency were LYB, HYB and HXB in order, and the frequency were 39.3%, 32.1%, and 12.5%, respectively.Generalized linear model was applied to estimate the HCC risk of haplotypes, and the OR for HYB and LXB were 0.64(95%CI:0.49-0.85) and 2.75(95%CI:1.13-6.64),respectively.3. Four types of MBL2 promoter were constructed by synthesis, and have been transfected into 293 T cells to observe the effect of luciferase transcription via p GL3-Basic vector. The analysis of variance suggested that the luciferase activity mean of promoter HX was 5509.00 which is lower than promoter HY and LY, however, it was higher than promoter LX, and significance was detected.4. The PHASE was use to construct MBL2 haplotypes among 50 treatment naive patients with chronic HBV infection, and comparison on HBV deep sequencing data was performed between MBL2 haplotype groups. T test demonstrated that the difference on the mutation number on core, X, HBs Ag, Long S and Middle S region were significant. Mann-Whitney U test indicated that the frequency of A1499 G was significantly higher in High MBL2 group, while T1653, T1674,T1753, A1726 T and G1764 A have higher frequency in medium/low MBL2 group. Shannon entropy was calculated to estimate the quasispecies complexity of regions in HBV genome, the comparison found that the complexity in C and X region were significantly higher in medium/low MBL2 group when comparing with high MBL2 group.[Conclusions] 1. HBV infection, ETS at work, alcohol consumption and liver disease history were identified as the risk factor of HCC, while fruit and tea consumption were protective. The random forest suggested that HBV infection is the factor with main effect in developing HCC. During the investigation, we found that unhealthy lifestyle was prevalent in some degree, therefore, health education must be conducted.2. The-550 H/L,-221 Y/X and codon 54 polymorphism were associated with HCC risk,and the haplotype analysis showed that HYB haplotype was protective while LXB haplotype was identified as HCC risk factors. Moreover, the frequency of HXB haplotype was 12.5% which was different with corresponding figure of foreign populations, it could be a genetic risk factor contributed to the high prevalence of HCC among Xiamen population.3. The dual luciferase reporter gene system indicated that the promoter HY has the highest luciferase activity, while LY was identified as the second, the activity of promoter HX was significantly lower than that of above-mentioned promoters, however,it was higher than promoter LX with significance.4. MBL2 haplotypes were capable of affecting the mutation number of specific regions,the frequency of single nucleotide mutation and the quasispecies complexity of C and X region, and consequently, lead to the progression of hepatitis B and alteration of HCC risk. |
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