Polymorphisms Of UNG2 And Hepatitis B Virus In The Development Of HBV-related Liver Disease

Background: there are about 30% people have been infected with the hepatitis B virus in the world's population,in this population,about 350 million people are carriers of hepatitis B virus,of which about 80% of the liver cancer is caused by HBV infection.Therefore,hepatitis B virus infection is one of the most important infectious diseases in the world.Although in most countries,due to the prevalence of hepatitis B vaccine,acute HBV infection rates are gradually reduced,but HBV chronic infection related cancer and mortality is rising year by year.The incidence and mortality of HCC in China become the top ten.The prevention and treatment of liver cancer has become a major public health problem that needs to be solved urgently in China.The major risk factors for HCC include genetic factors,HBV or HCV chronic infection,liver cirrhosis,exposure to aflatoxin,excessive drinking and diabetes,etc..Uracil-DNA-Glycosylase(UNG)is a very important human DNA excision repair enzymes and DNA base excision repair pathway key molecules,it can identify APOBECs induced C > U mutation,and removal of uracil formed abasic site(AP site),causing a chain hydrolysis of a nucleic acid in mismatch repair.UNG enzyme divide nucleus type(UNG 2)and mitochondrial(UNG1).The activity of UNG2 is higher,and the function of hepatitis B virus ss DNA is stronger.Therefore,it is important to explore the role of UNG2 promoter polymorphisms and hepatitis B virus variants in the occurrence and development of HBV related liver disease,and to explore the genetic susceptibility of HCC in Chinese population.Objective:To explore and analyze the associationsof polymorphism of UNG2 rs3890995 with the risk of HBV natural clearance,chronic infection caused by HBV,LC and HCC.In addition,the study also analyze the associations polymorphism of UNG2 rs3890995 with HBV mutations(genotype B and C)and their interactions in progress of the occurrence and development of LC and HCC,to explore the further associations between factors of hereditary of susceptible population and related HBV mutations.According to the results above,on the basis of single nucleotide polymorphism and interrelated HBV mutations,it can provide epidemiological clues of predicting the occurrence and development of HCC at a large-scale population level.Methods:The study include 1012 healthy controls,302 HBV natural clearance,316 asymptomatic HBs Ag carriers,886 chronic hepatitis B patients,482 LC patients and 1165 HCC patients.Using MGB-fluorescent probe real-time quantitative PCR to typing the genotype of SNP of DNA samples above.Multiplex PCR was used to amplify the HBV gene and typed HBV by sequence alignment.We test the Hardy-Weinberg equilibrium on the internet,the website is http://ihg.gsf.de/ihg/ snps.html.After sorting out and inputting the data,the further analysis was used by software SPSS 16.0,the statistical methods include student's t-test,variance analysis,Chi-square test and unconditional logistic regression analysis,aiming to explore the associations with single nucleotide polymorphism of UNG2 and each stage of hepatitis b,virus mutations and multiplicative interactions.Results:1?Associations between polymorphism of UNG2 rs3890995 and each stage of HBV-related liver diseaseThe healthy controls isused as controls,when compared with T/T genotype of rs3890995,T/C genotype and increased the risk of HCC(AOR=1.206,95%CI=1.000-1.454;)and dominant model(TC+CC)could also increase the risk of HCC(AOR=1.226,95%CI=1.027-1.464,P=0.024)?When the non-LC patients were used as controls,T/C genotype and dominant model(TC+CC)could increase the risk of LC significantly(AOR=1.566,95% CI=1.240-1.987,P<0.001;AOR=1.393,95% CI=1.110-1.747,P=0.004).When carried with T/C or C/C genotype,there is no statistical difference between HBV chronic infection group and HCC group.When compared with HBV natural clearances patients,which carried T/C genotype have lower risk of non-LC HBV chronic infection than T/T genotype(AOR=0.460,95%CI=0.297-0.712,P<0.001),and decrease the risk of HCC(AOR=0.706,95%CI=0.527-0.946,P=0.020).Compared with T/T genotype,dominant model(TC+CC)could cause HBV clearance easily(AOR=0.563,95%CI=0.375-0.847,P=0.006).When stratified by gender,we know that in female,compared with healthy controls,there is no association between genotypes of rs3890995 and HCC.But in male,compared with T/T genotype,C/C genotype and dominant model could increase HCC risk significantly(AOR=1.469,95%CI=1.074-2.009,P=0.016,AOR=1.244,95%CI=1.021-1.515,P=0.030).Using HBV natural clearance patients as controls,female that carried T/C genotype have low risk of early liver disease(AOR=0.548,95%CI=0.349-0.859,P=0.009),this phenomenon is the same as dominant model(TC+CC)(AOR=0.595,95%CI=0.389-0.911,P=0.017).Besidespolymorphism of rs3890995 could not influence the risk of early liver hepatitis B disease.Using non-LC HBV infection patients as controls,in the female,T/C genotype and dominant model(TC+CC)could increase risk of LC significantly(AOR=2.334,95%CI=1.494-3.646,P<0.001;AOR=1.900,95%CI=1.234-2.927,P=0.004).But in male,patients that carried T/C genotype have higher risk of LC(AOR=1.332,95%CI=1.003-1.770,P=0.048).When stratified by different HBV subtype,we can know that in the patients that infected by genotype B HBV,the polymorphism of rs3890995 have no associations with HCC risk.However,for the non-HCC patients infected by genotype C HBV,T/C genotype have significant risk of HCC(AOR=1.366,95%CI=1.068-1.748,P=0.013).Though the C/C genotype of rs3890995 have no difference between healthy controls group and HCC group,but dominant model(TC+CC)could increase the risk of HCC from chronic hepatitis B(AOR=1.340,95%CI=1.063-1.690,P=0.013).Compared with non-LC HBV infection group,among the people infected by genotype C HBV,TC genotype increase LC risk significantly(AOR=1.435,95%CI=1.002-2.005,P=0.049).C/C genotype and dominant model(TC+CC)could not increase or decrease the risk of LC.2?The association between polymorphism of UNG2 rs3890995 and HBV mutations.To the patients infected by genotype B HBV,T/C genotype could influence with frequency of HBV mutations significantly but have a little association with frequency of HBV mutations in genotype C HBV patients.On the other hand,C/C genotype of rs3890995 could influence the frequency of HBV mutations of C HBV infected subjects significantly.In the B genotype HBV infected patients,after adjusted by gender and age,T/C genotype of rs3890995 increased the frequency of HBV mutation include G1899 A,T2931C,T3026 C,T109A,G2962 A,T109A and G3063 C.C/C genotype could increase the frequency of A1846 T,and dominant model(TC+CC)could increase the frequency of mutation of G1899 A.In C genotype HBV infected subjects,T/C genotype could increase frequency of pre S deletion mutation,decrease T52 C.C/C genotype could increase the frequencies of mutation such as A1652 G,C1673T,A1727 T,C1730G and C1799 G,decrease G1899 A at the same time.Dominant model(TC+CC)decrease the frequencies of mutation G1899 A and T52 C.3?Interaction between polymorphism of rs3890995 and HBV mutation during end-stage hepatitis B liver diease.(1)Interaction between polymorphism of rs3890995 and A1652 G mutation on HCC risk.In C genotype HBV infected patients,after adjusted by gender and age,compared with HBV infected but non-HCC patients as controls,T/T genotype coexist with A1652 G mutation decrease HCC risk(AOR=0.211,95%CI=0.084-0.525,P=0.001),and C/C genotype coexist with A1652 G mutation could also decrease HCC risk(AOR=0.582,95%CI=0.344-0.987,P=0.044).Besides,there is significant interaction between TC genotype of rs3890995 and A1652G(AOR=3.677,95%CI=1.111-12.169,P=0.033).(2)Interaction between polymorphism of rs3890995 and HBV pre S deletionmutation on HCC risk.In C genotype HBV infected patients,after adjusted by gender and age,compared with HBV infected but non-HCC patients,T/C genotype coexist with A1652 G mutation increase HCC risk(AOR=1.721,95%CI=1.341-2.209,P<0.001),and C/C genotype coexist with A1652 G mutation could also increase HCC risk(AOR=1.895,95%CI=1.216-2.951,P=0.005).Besides,when dominant model(TC+CC)coexist with pre S deletion,HCC risk could be increased(AOR=1.758,95%CI=1..397-2.212,P<0.001),and there is significant interaction between dominant model of rs3890995 and pre S deletion(AOR=1.961,95%CI=1.025-3.751,P=0.042).(3)Interaction between polymorphism of rs3890995 and HBV T1674 G mutation on LC risk.In C genotype HBV infected patients,after adjusted by gender and age,compared with HBV infected but non-LC patients,independent effect of T/C genotype increase LC risk(AOR=2.018,95%CI=1.195-3.045,P=0.009),and there is significant interaction between T/C genotype and T1674G(AOR=0.326,95%CI=0.119-0.896,P=0.030).Independent effect of dominant model(TC+CC)increase l C risk(AOR=1.748,95%CI=1.078-2.835,P=0.023).There is no interaction between dominant model of rs3890995 and T1674 G.(4)Interaction between polymorphism of rs3890995 and HBV C1730 G mutation on LC risk.In C genotype HBV infected patients,after adjusted by gender and age,compared with HBV infected but non-LC patients,T/C genotype and dominant model(TC+CC)that coexist with C1730 G mutation increase LC risk(AOR=2.537,95%CI=1.604-4.013,P<0.001;AOR=1.781,95%CI=1.264-2.509,P=0.001),and there is significant synergistic between T/C genotype and C1730G(AOR=3.513,95%CI=1.076-11.467,P=0.037)Conclusion:1?T/C genotype and dominant model(TC+CC)of rs3890995 could increase risk of LC and HCC.2?After stratified by gender,we found that in the male,C/C genotype and dominant model(TC+CC)could increase HCC risk,but TC genotype and dominant model(TC+CC)could increase LC risk in the female.3?TC genotype of rs3890995 could increase LC and HCC risk of C genotype HBV infected subjects,dominant model(TC+CC)increase HCC risk significantly.4?In B genotype HBV infected patients,influence of T/C genotype to the frequencies of HBV mutations was greater than C genotype HBV infected patients,and C/C genotype have significant associations with HBV mutations in C genotype HBV infected subjects.5?To the patients that infected by C genotype HBV,there was a significant interaction on HCC risk between TC genotype of rs3890995 and A1652 G,dominant model(TC+CC)and pre S deletion;meanwhile,there are also interaction on LC risk between TC genotype of rs3890995 and T1674 G,C1730G.6?There was a interaction between APOBEC3 B rs2267401 GG genotype and UNG2 rs3890995 CCgenotype in HCC development.