Association Of VEGFA Gene Polymorphism With Susceptibility To HBV-related Chronichepatitis Cirrhosis And Hepatocellular Carcinoma

Objective:To detect the genetic polymorphisms of rs10434,rs3025040 and rs833061 of the Vascular endothelial growth factor A(Vascular endothelial growth factor A)gene to explore its relationship with Hepatitis B virus(HBV)-related hepatitis B(CHB),liver cirrhosis(Liversoncirrhosis,LC)and hepatocellular carcinoma(HCC)susceptibility,provides relevant theoretical basis for disease prevention and diagnosis.Methods:The study subjects were divided into control group,HBV-related chronic hepatitis group,HBV-related cirrhosis group and HBV-related liver cancer group,and the inclusion and exclusion criteria were formulated.Collect the relevant clinical data and EDTA-2K anticoagulated whole blood of each group of research subjects.After DNA extraction and purification of the anticoagulated whole blood,Using SNa Pshot technology to test the genotypes of the DNA fragments at the three locus of VEGFA gene rs10434,rs3025040 and rs833061,at the same time,Sanger sequencing for sampling verification,Hardy-Weinberg equilibrium(HWE)is used to assess whether each group of research subjects comes from a genetically balanced population,and infer whether there is a population representative.Calculate the distribution frequency of the genotype and alleles at each site based on the sequencing results.After adjust for age and/or gender,use binary logistic regression analysis to calculate the odds ratio(OR)and 95%confidence interval(Confiduntial interval,CI),and then to explore the relationship between single nucleotide polymorphism(SNP)at the three locus of VEGFA gene and the genetic susceptibility of HBV-related chronic hepatitis,liver cirrhosis and liver cancer.The haplotypes of VEGFA gene rs10434,rs3025040,and rs83361 were constructed by SHEsis online software,and the relationship between the haplotypes and the genetic susceptibility of HBV-related chronic hepatitis,liver cirrhosis and liver cancer was analyzed.Results:1.A total of 659 subjects were included in the study,including 159 cases in the control group,162 cases in the HBV-related chronic hepatitis group,165cases in the HBV-related liver cirrhosis group,and 173 cases in the HBV-related liver cancer group(hereinafter referred to as hepatitis group,liver cirrhosis group and liver cancer group).The age of the four groups is skewed distribution data analyzed by normality test,so the non-parametric Kruskal-Wallis H test was used to compare the ages in each groups.The result was H=35.034,P<0.05,and the difference was statistically significant.Further multiple comparisons found that the age difference between the hepatitis group and the control group,liver cancer group,and liver cirrhosis group was statistically significant(P<0.05).The R*C contingency tableX~2 test was used for the gender comparison of the four groups of study subjects,the result wasX~2=3.422,P=0.331,the difference was not statistically significant.2.Multiplex SNa Pshot show that VEGFA gene rs10434 locus has three genotypes GG,AG and AA,rs3025040 locus has three genotypes CC,CT and TT,and rs833061 locus has three genotypes of TT,CT and CC.The results of sanger sequencing are consistent with multiplex Snapshot.3.The genotype distribution frequencies of the three locus of VEGFA gene rs10434,rs3025040 and rs833061 in the control group and each case group all conformed to the Hardy-Weinberg equilibrium(P>0.05).The genotypes and allele distribution frequencies of the three loci were not statistically different between the control group and each case group(P>0.05).4.For each comparison group(control group vs.hepatitis group,control group vs.liver cirrhosis group,control group vs.liver cancer group),after set a reference and adjust for gender and age factors,binary Logstic regression analysis found that,under the genotype model and the allele model,the genotype or allele carrying frequency of the three locus rs10434,rs3025040 and rs833061 were not statistically different in each comparison group(P>0.05).5.Grouped by gender,used binary Logstic regression analysis for each comparison group,set a reference,and adjusted for age factors,found that in the female group,the AA genotype and A allele which at the rs10434 locus of the VEGFA gene are statistically different between the control group and the hepatitis group(AA genotype:P=0.030,OR=11.853,95%CI:1.266-110.960;A allele:P=0.009,OR=2.805,95%CI:1.297-6.066),AG genotype and A alleles are statistically different between the control group and the cirrhosis group(AG genotype:P=0.019,OR=3.423,95%CI:1.227-9.552;A allele:P=0.005,OR=3.009,95%CI:1.385-6.538).In the male group,there was no statistically significant difference in the genotype or allele carrying frequency of this locus under the two models in each comparison group(P>0.05).6.Grouped by age,used binary Logstic regression analysis for each comparison group,set a reference,and adjusted for gender and age factors,found that in the group of≥50 years old,AG genotype at rs10434 of the VEGFA gene may statistically significant difference between the control group and the liver group(P=0.046,OR=1.854,95%CI:1.011-3.401),and in the<50 years group,under the genotype model and the allele model,there is no statistically significant difference in the genotype or allele carrying frequency of this locus in each comparison group(P>0.05).Regardless of whether it is a group of<50years old or a group of≥50 years old,and under the genotype model and the allele model,there is no statistical difference(P>0.05)in the genotype or allele carrying frequency of the two locus of VEGFA gene rs3025040 and rs833061.7.In each comparison group,the SHEsis online software was used to construct haplotypes at the three locus of VEGFA gene rs10434,rs3025040 and rs833061 respectively,and a total of six haplotypes(CCA,CCG,CTG,TCA,TCG,TTG)were constructed.,statistical analysis found that while CCG,CTG and TCG,the difference between the control group and the cirrhosis group was statistically significant(CCG:P=0.037,OR=1.525,95%CI:1.024-2.272;CTG:P=0.047,OR=0.457,95%CI:0.207-1.006;TCG:P=0.023,OR=0.695,95%CI:0.507-0.952),while the other haplotypes showed no significant difference between the control group and the case group(P>0.05).Conclusion:1.The AA genotype and A allele at the rs10434 locus of the VEGFA gene may increase the risk of HBV-related chronic hepatitis in women,and the AG genotype and A allele may increase the risk of HBV-related cirrhosis in women,AG genotype may increase the risk of HBV-related liver cancer in people≥50years of age.2.The genetic polymorphisms at rs3025040 and rs833061 of the VEGFA gene may have no relationship with the risk of HBV-related chronic hepatitis,liver cirrhosis and liver cancer.3.The haplotype CCG constructed at the three locus of VEGFA gene rs10434,rs3025040 and rs833061 may increase the risk of HBV-related cirrhosis;CTG and TCG two haplotypes may reduce the risk of HBV-related cirrhosis.