| The incidence of hepatocellular carcinoma(HCC) in the world in a variety of malignant tumors in the sixth place is the 12 th leading cause of death worldwide, with a total of 748 300 new cased reported annually; and HCC are the third-leading cause of death, with a total of 695 900 deaths every year. HCC patients in China is accounted for more than 50% of the world, with more than 50% of new cases of the world in China each year. With the rapid development of molecular biology, virology and genetics, many studies have shown that the occurrence and development of HCC are the result of multi-factor, multi-step and multi-gene interactive participation. The host genetic factors, such as single nucleotide polymorphisms(SNP) and gene methylation, also play a role in the occurrence and development of the HCC. It has great significance for early prevention and treatment of HCC to clear the risk factors and pathogenesis of HCC, as a major task faced by the public health.Chronic HBV infection is the crucial risk of HCC in China, and nearly 80%-90% of HCC patients with HBV index was positive. China has about 100 million chronic HBV infected people, which is about one-third of the world. And it is estimated that the number of HCC patients in China is more than half of the world, of which 30 million are active HBV patients. Researchers provided evidence that about 25% to 40% of HBV chronic infection will eventually progress for HCC. The HBV infection, there are different disease outcomes of the disease: asymptomatic HBs Ag carriers(ASCs) or occult hepatitis, acute and chronic hepatitis B(CHB), liver cirrhosis(LC) and HCC. These different liver diseases are mainly related to the virus, the environment and host genetic factors.More and more studies during recent years discovered that SNP is closely correlative with various genetic susceptibility and different outcomes of multiple diseases. The present results of research in the domestic and foreign focus on the relationships between the polymorphism of patatin-like phospholipase domain-containing 3(PNPLA3) and liver disease. For example, several studies have found that polymorphisms of PNPLA3 rs738409 have some effect in the process of a lot of liver disease, such as ALD, LC and so on, and play a role in promoting the development of hepatic steatosis and fibrosis for chronic hepatitis B(CHB) patients. Valenti L, et al. found PNPLA3 rs2294918 associated with nonalcoholic fatty liver disease(NAFLD) in 2013. The study of Pan Q, et al. found that PNPLA3 rs738408 influenced the occurrence of CHB. Recent studies found that rs738409 and rs2281135 of PNPLA3 were risk factors for LC. Then gene polymorphism of PNPLA3 may be an influence factor of genetic susceptibility to HBV infection and development of the diseases after HBV infection. So it have the important significance and value to explore the relation between PNPLA3 and predisposition to HCC.In this study, we investigated a large epidemiological study to explore distribution characteristics of PNPLA3 gene polymorphism(rs738408, rs738409, rs2294918, rs2294919 and rs2281135) in all group of Chinese population and the role of a potential functional SNPs of PNPLA3 and its interaction with HBV infection and HBV-related HCC. Part one: Genotype distribution of PNPLA3 gene polymorphismsObjective:To explore distribution characteristics of PNPLA3 gene polymorphism(rs738408, rs738409, rs2294918, rs2294919 and rs2281135) in all group of Chinese population.Methods:2410 eligible cases were divided into different groups, including 370 cases of healthy controls, 248 cases of natural clearance, 1371 cases of chronic HBV infection(691 cases of CHB and 680 cases of liver cirrhosis(LC)) and 421 cases of HBV-related HCC. General conditions of research object were acquired using questionnaires, and peripheral blood was collected to extract DNA for SNP classification by Sequenom. Student’s t-test or Wilcoxon test was used to compare between continuous data. Pearson chisquare test was used to evaluate the differences in categorical variables between groups. IBM SPSS 22.0 statistical software was used for data analysis.Results:1 Demographic and laboratory parameters of the subjectsThere were significant differences among studied groups regarding age, sex, smoke and drink(age: c2 <=001.0,803.485 P; sex: 001.0,446.532c<= P; smoke: 001.0,599.432c<= P; drink: 001.0,325.352c<= P). Moreover, in comparison among CHB, LC and HCC patients groups, there were significant differences as regards TBIL, DBIL, IBIL, TP, ALB, GLB, A/G, ALT, Albumin, ALT, AST and ALT/AST(all P<0.05).2 Subject characteristics stratified by genotypes of PNPLA3There is significant difference for PNPLA3 rs738408 in age concentrations(P=0.048), the age of TC genetype is higher than CC/TT genetype. But, we didn’t find any statistically significant differences in other concentrations(age, sex, smok, drink, TBIL, DBIL, IBIL, TP, ALB, GLB, A/G, ALT, Albumin, ALT, AST and ALT/AST)(all P>0.05).3 Genotype distribution of five SNP alleles of PNPLA3 in different populationThe genotype distributions of PNPLA3 rs 738408 and rs2294918 were significant different among studied groups of genotype in the total and female population(all P<0.05). The genotype distributions of rs2294918 were significant different among studied groups of genotype in the male population(P=0.026).Conclusions:1 The success rates of genotyping detection were more than 97%, indicating that the entire experimental process had quality control operations properly and reasonable steps.2 The results of the demographic and laboratory parameters of the patients groups and control subjects shown that age, sex, smoke and drink may affect the HBV infection and occurance of HBV-related HCC. Therefore, future research will consider these factors as control factors to analyze PNPLA3 with HBV infection susceptibility and relevance of HBV-related HCC.3 The results of the genotype distributions of PNPLA3 rs 738408 and rs2294918 showed that there were significant different among studied groups of genotype in the total and female population. The genotype distributions of rs2294918 were significant different among studied groups of genotype in the male population. IBM SPSS 22.0 statistical software was used for data analysis. Part two: Association between PNPLA3 gene polymorphisms and risk of hepatitis B virus infectionObjective:To explore the role of potential functional SNPs of PNPLA3(rs738408, rs738409, rs2294918, rs2294919 and rs2281135) and its interaction with HBV infection.Methods:2410 eligible cases were divided into case group(without HBV infection) and control group(with HBV infection), case group including 370 cases of healthy controls and 248 cases of natural clearance, control group including 691 cases of CHB, 680 cases of liver cirrhosis(LC) and 421 cases of HBVrelated HCC. General conditions of research object were acquired using questionnaires, and peripheral blood was collected to extract DNA for SNP classification by Sequenom. Pearson chi-square test was used to evaluate the differences in categorical variables between groups. Unconditional logistic regression was conducted to calculate odds ratio(OR) and 95% confidence intervals(95% CI), adjusting sex, age, alcohol drinking and smoking. LD and haplotype block analysis was used to investigate the LD of PNPLA3 SNPs. IBM SPSS 22.0 statistical software and Haploview 4.2 software were used for data analysis.Results:1 Genotype distribution of five SNP alleles of PNPLA3The percentage of PNPLA3 rs738408 TC, TT genotype and T allele were significantly lower in the subjects without HBV infection than in those with HBV infection(TC vs. CC: OR=0.558, 95% CI=0.405-0.770; TT vs. CC: OR=0.659, 95% CI=0.472-0.919; T vs. C: OR=0.638, 95% CI=0.502-0.811). The PNPLA3 rs2294918 AG genotype were more prevalent in the subjects with HBV infection(OR=1.363, 95% CI=1.092-1.702), but AG genotype were less prevalent in the subjects with HBV infection(OR=0.511, 95% CI=0.354-0.736).2 Multivariate analysis of predicting factors for HBV infectionThe subjects those with the PNPLA3 rs738408 TC/TT genotype would have lower odds of infection of HBV(OR=0.059, 95% CI=0.028-0.125, P<0.001 for TC; OR=0.020, CI=0.007-0.053, P<0.001 for TT); the subjects with rs738409 GG genotype would have a 27.550(95% CI: 10.024-75.718, P<0.001) times higher odds of with HBV infection; rs2294918 AA genotype reduced the risk of HBV infection(OR=0.354, 95% CI: 0.210-0.599, P<0.001); rs2294919 TC genotype would have a 0.598(95% CI: 0.429, 0.833; P=0.002) times lower odds of having HBV infection, whereas the values of sex, age, smoke and drink covariates were fixed.3 Gene-environment interactionGene-environment interaction of PNPLA3 SNPs and smoke/drink was not discovered, considering sex, age, drinking and smoking.4 LD and haplotype block analysisThe haplotype AC, GT and GC of the haplotype block of rs2281135 and rs2294919 had no significant difference between subjects with HBV infection and without HBV infection.Conclusions:1 PNPLA3 rs738408 TC or TT genotype and T allele, and rs2294918 AA genotype significantly reduced the risk of HBV infection. In contrast, the PNPLA3 rs2294918 AG genotype increased the risk of HBV infection.2 PNPLA3 rs738408 TC or TT, rs738409 GG, rs2294918 AA and rs2294919 TC had relationship with HBV infection, considering age, sex, smoke and drink at the same time.3 Gene-environment interaction of PNPLA3 SNPs and smoke/drink was not discovered for the occurance of HBV infection.4 The haplotype AC, GT and GC of the haplotype block of rs2281135 and rs2294919 had no significant difference between subjects with HBV infection and without HBV infection. Part three: Association between PNPLA3 gene polymorphisms and risk of hepatitis B virus-related hepatocellular carcinomaObjective:To explore the role of potential functional SNPs of PNPLA3(rs738408, rs738409, rs2294918, rs2294919 and rs2281135) and its interaction with HBV HBV-related HCC.Methods:2410 eligible cases were divided into 5 groups, including 370 cases of healthy controls, 248 cases of natural clearance, 691 cases of CHB, 680 cases of liver cirrhosis(LC) and 421 cases of HBV-related HCC. General conditions of research object were acquired using questionnaires, and peripheral blood was collected to extract DNA for SNP classification by Sequenom. Pearson chi-square test was used to evaluate the differences in categorical variables between groups. Unconditional logistic regression was conducted to calculate odds ratio(OR) and 95% confidence intervals(95% CI), adjusting sex, age, alcohol drinking and smoking. LD and haplotype block analysis was used to investigate the LD of PNPLA3 SNPs. IBM SPSS 22.0 statistical software and Haploview 4.2 software were used for data analysis.Results:1 Genotype distribution of five SNP alleles of PNPLA3With the healthy population as control group, and HCC cases as case group, the percentage of PNPLA3 rs738408 TC genotype and T allele were significantly lower in the subjects with HCC than in those health control(OR=0.412, P <0.001 for TC; OR=0.538, P <0.001 for T). The PNPLA3 rs2294918 AG genotype were more prevalent in the subjects with HCC(OR=1.621, P =0.003). In contrast, the percentage of PNPLA3 rs2294919 TC genotype was significantly lower in the subjects with HCC than in those health control(OR=0.710, P =0.026).With the NC subjects as control group, and HCC cases as case group, the percentage of AG of rs2294918 at PNPLA3 was significantly higher in the subjects with HCC(OR=1.534, P =0.026), In contrast, the frequencies of AA was significantly lower than HCC subjects(OR=0.438, P =0.008).With the CHB or LC subjects as control group, and HCC cases as case group, we found no significant differences in genotypes of the five SNP alleles of PNPLA3.2 Multivariate analysis of predicting factors for HCCComparison between health control group and HCC, specifically, when compared with GG genotype, the subjects with the PNPLA3 rs2294918 AG genotype would have a 1.872(95% CI=1.256-2.792; P = 0.002) times higher odds of having HCC; but when compared with CC genotype, the subjects those with the PNPLA3 rs2294919 TC genotype would have a 0.605(95% CI=0.413-0.886; P=0.010) times lower odds of having HCC, whereas the values of the other covariates were fixed. No significant difference was found for the PNPLA3 rs2294918 and rs2294919 genotypes.Comparison between NC group and HCC, when compared with GG genotype, the subjects with the PNPLA3 rs2294918 AG genotype would have a 1.529(95% CI=1.004-2.329; P = 0.048) times higher odds of having HCC, whereas the values of the other covariates were fixed. No significant difference was found for the PNPLA3 other genotypes.At the same time, we found no significant differences of the PNPLA3 rs738408, rs738409, rs2294918, rs2294919 and rs2281135 genotypes in comparisons between CHB patients with HCC patients. Moreover, we didn’t find PNPLA3 rs738408, rs738409, rs2294918, rs2294919 and rs2281135 genotypes were influence factors of HCC in LC patients, considering sex, age, drinking and smoking.3 Gene-environment interactionThe gene-environment interaction of PNPLA3 rs738409 GC genotype and drinking in CHB had statistical significance(OR=0.556, 95% CI=0.309-0.998, P=0.049). Gene-environment interaction of PNPLA3 SNPs and smoke/drink was not discovered in other population.4 LD and haplotype block analysisSurprisingly, haplotype block LD mapping showed that SNPs of rs738409 and rs2281135 were in tight LD in a 7-kb sequence, and rs2294918 and rs2294919 were in tight LD in a 0-kb sequence. CA haplotype of the haplotype block of rs738409 and rs2281135 had significant difference between health control subjects and HCC patients(P=0.0193).Conclusions:1 PNPLA3 rs738408 TC genotype and T allele significantly reduced the risk of HBV-related HCC in the health subjects. Meanwhile, the PNPLA3 rs2294919 TC genotype protected health subjects from HBV-related HCC. The PNPLA3 rs2294918 AA genotype reduced the risk of HBV-related HCC in the NC subjects. In contrast, the PNPLA3 rs2294918 AG genotype increased the HBV-related HCC risk in healthy controls and NC subjects.2 PNPLA3 rs2294919 TC played a positive role and rs2294918 AG played a negative role for health subjects on suffering HBV-related HCC, considering age, sex, smoke and drink at the same time.3 Gene-environment interaction of PNPLA3 SNPs and smoke/drink was not discovered for the occurance of HBV-related HCC.4 CA haplotype of the haplotype block of rs738409 and rs2281135 had significant difference between health control subjects and HCC patients. |
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