Sequence Analysis Of HBV In Primary Hepatomas Patients Infected With HBV

Objectives To study the relationship between the development of hepatocellular carcinoma(HCC)and HBV gene characteristics among the HCC patients with hepatitis B virus(HBV)infection.Methods During the year of 2012-2015,we collecting HBV related HCC patients as HCC group from the HCC monitoring network in Hainan Tibetan Autonomous Prefecture,Qinghai province,Longan County,Guangxi pro vince,Zhengding County,Hebei province,Huangpu District,Shanghai city,Kaifeng City,Henan province and Xiangtan,city,Hunan province in China,and chronic hepatitis B(CHB)patients without HCC and liver cirrhosis(LC)as the control group from national hepatitis B monitoring room in the same area.Serological HBV markers of Serum samples were detected by Enzyme-linked immunosorbent assay(Elisa).HBV Deoxyribonucleic acid(DNA)of Serum samples were extracted by QI Aamp DNA Blood Mini Kit(QIAGEN company)and HBV gene were amplified by nested polymerase chain reaction(PCR).DNAstar(SeqMan)software was used for sequences assembly and then blast with 49 standard reference strains obtained from GeneBank to made phylogenetic trees for determining genotype by MEGA6 software.C1653T,T1753V,A1762T/G1764A,G1896A and PreS deletion mutation and amino acids sequence of PreS region translated by the standard codon were analyzed by Bioedit software.Data were analyzed using the Statistical Program for Social Science,version 16.0 for Windows(SPSS).Results In the HCC group,546 serum samples were collected,of which 61.2%(334/546)cases were positive for HBsAg,.In the control group,25 cases were collected in each area,150 cases in total,and All were positive for HBsAg.86 samples in HCC group and 39 samples in control group were amplified the sequence of S region and BCP/Precore region of HBV.and a significantly higher proportion of over 50 years patients was detected in HCC group than control group(80.23%[69/86]vs 33.34%[13/39];?2=26.153,P=0.000,OR=8.118;95%CI=3.465-19.019).as well as GenetypeC(81.40%[70/86]vs58.97%[23/39];?2=9.961,P=0.002,OR=4.261,95%CI=1.667-10.889).No significant difference were detected of the sex and HBeAg status in HCC group than control group.A significantly higher prevalence of HBV pre-S deletions was detected in HCC group than control group(34/86[39.53%]vs8/39[20.51%];x2=4.350,P=0.037,OR=2.534;95%CI=1.041-6.166).furthermore,mutations with PreS2 deletions only were found to be statistically associated with the development of HCC?(?2=4.747,P=0.029,OR=3.506,95%CI=1.084-11.340).Loss of PreS1 and PreS2 start codons were detected in HBV isolates from both HCC group and control group.Our study also found that PreS deletions occurred commonly in the human serum albumin receptor region(88.10%[37/42])and the S promoter region(38.10%[16/42]),but few in the hepatocyte binding site(11.90%[5/42])and the CCAAT box(9.52%[4/42]).The prevalence of A1762T and A1762T/G1764A in HCC group were 74.42%and 72.09%,respectively,and in control group were 53.85%and 53.85%respectively.The statistical differences of them were significant.No significant difference was found in G1764A,G1896A,T1753V mutation between the two groups.The prevalence of A1762T and A1762T/G1764A in>50 years group were higher than that of<50 years group.The prevalence of A1762T?G1764A and A1762T/G1764A of subjects who infected genotype C were higher than those infected genotype B.On the contrary,the prevalence of G1896A of subjects who infected genotype C were lower than that of genotype B.It was found that ? 50years[Adjusted odds ratio(AOR)=9.349,95%CI=3.329-26.257,P=0.000],genotype C(AOR=28.875,95%CI=4.897-170.258,P=0.000)and G1896A mutation(AOR=7.648,95%CI=1.564-37.4,P=0.012)were independently associated with HCC.The risk for suffer from HCC of>50 years group,genotype C group and G1896A group were 9.349,28.875 and 7.648 times compared with<50 years group,genotype B group and without G1896A mutation group,respectively.However,sex,HBeAg,PreS deletion,A1762T,G1764A,T1753V,and A1762T/G1764A can not be considered as independently associated with HCC,as compared with the subjects of control group.Conclusions The proportion of over 50 years old and genotype C were significantly higher in HCC group than control group;A significantly higher prevalence of HBV PreS deletion,A1762T and A1762T/G1764A were detected in HCC group than control group.The population of ?50 years or genotype C had a higher prevalence of A1762T,A1762T/G1764A,? 50years,genotype C,G1896A were independently associated with HCC and have a higher risk with development of HCC,as compared with the subjects of control group.