Detection Method Of HBV-DNA Integration Sites And Frequencies In Liver Tissues Of Patients With Chronic Hepatitis B At End-Stage Liver Disease And The Association With Clinical Characteristics

Background&Aims: Pregenomic RNA(pgRNA) is the viral intermediate of Hepatitis B Virus(HBV),which can form covalently closed circular DNA(ccc DNA)during reverse transcription.Meanwhile,it can also reverse transcript into doublestranded linear DNA(dsl DNA).A fraction of dsl DNA can go into the cell nucleus and integrate into the human genome.HBV-DNA integration not only affects the clinical cure of chronic hepatitis B(CHB),but also is closely related to the occurrence and development of chronic hepatitis B related end-stage liver diseases.There have been several methods to detect the HBV-DNA integration in human genome,but they have their inherent advantages and disadvantages in sensitivity,specificity,cost and so on.Taking the above aspects into consideration,Inverse Nested PCR(Inv PCR)detection method and mathematical and statistical analysis method were used to obtain HBV-DNA integration sites and integration frequencies in liver tissues,so as to investigate the clinical features of HBV-DNA integration in liver tissues of patients with chronic hepatitis B at end-stage liver diseases.Methods: Firstly,DNA in liver tissues of different patients with chronic hepatitis B at end-stage liver diseases was extracted,and the non-conserved region of S gene was amplified by the designed primers,and the genotypes of HBV infected by different patients were determined by BLAST comparison.Different restriction enzymes(RE)and primers were selected according to different HBV genotypes.Inverted DNA fragments were obtained through repeated enzyme digestion and ligation of extracted liver tissue DNA.Both ends of the inverted DNA fragments are segments of HBV,and in the middle of the inverted DNA fragments are sequences of the human genome.After repeated digestion and ligation,the reverse nested DNA fragment was purified and condensed.Virus cell DNA junctions can be proved to exist using agarose gel electrophoresis,then cut the VCJs glue,recover and sequence them,and we can get the integration sites of HBV-DNA.Different integration fragments corresponded to different integration sites.Count the number of each integration site at different dilution degrees,and 7 values can be obtained for each integration site.Combined with the amount of DNA loss,data simulation can be conducted.The range of integration frequency of each integration site can be obtained by taking 95%confidence interval,and the maximum integration frequency of each integration site can be obtained.The method was validated in Hep G2.2.15 cells and Hep G2 cells.The integration sites and frequencies of HBV-DNA in the liver tissues of patients with chronic hepatitis B at End-Stage Liver Disease were detected,so as to analyze the clinical characteristics of the integration sites and frequencies of these patients.Results:1.Integration site of Hep G2.2.15 cells HBV-DNA integration site in Hep G2.2.15 cells was successfully detected.HBV-DNA is integrated in SHANK2,which is located in chromosome11.This gene encodes a protein that is a member of the Shank family of synaptic proteins.2.HBV-DNA Integration Sites and Frequencies in Liver Tissues of Patients with Chronic Hepatitis B at End-Stage Liver Disease and the Association with Clinical Characteristics.This study examined the integration of HBV-DNA in the liver tissues of 8patients with chronic hepatitis B at end-stage liver disease.7 patients had TBXAS1(thromboxane A synthase 1)integration,6 patients had JAG2(jagged canonical Notch ligand 2)integration,4 patients had MVK(mevalonate kinase)integration,and 3patients had TERT(telomerase reverse transcriptase)integration in liver tissues.HBV-DNA was integrated in ADCY5(adenylate cyclase 5)and RP11-594G13 on chromosome 3,TERT and CTD-2345P7 on chromosome 5,MVK and HNRNPABP1(heterogeneous nuclear ribonucleoprotein A/B pseudogene 1)on chromosome 12,SYN2(synapsin II)and SC22 CB on chromosome 22,TBXAS1 on chromosome 7,HPSE2(heparanase 2)on chromosome 10,SHANK2 on chromosome 11,RP11-594G13 on chromosome 13,JAG2 on chromosome 14,and D-loop of mitochondrial DNA,respectively.Patients aged less than 50 had a tendency to have more integration sites than patients aged more than 50(5.500±1.658 VS 2.000±0.0,p=0.079).Patients with decompensated cirrhosis tended to have more integration sites than those with compensated cirrhosis(2.000±0.0 VS 4.800±1.463,p=0.20).The mean maximum integration frequency of patients with age more than 50 tended to be higher than that of patients with age less than 50(1021±582.7 VS 5658±3274,p=0.21).The mean maximum integration frequency of patients with differentiated HCC tended to be higher than that of patients with poorly differentiated HCC(10165±4757 VS 392.0±269.5,p=0.071).The differences were not statistically significant due to the limited sample size,so further sample size expansion was needed for further research.Conclusions: According to the method provided in the literature,The method of detecting HBV-DNA integration sites and calculating the integration frequency in liver tissue of patients with chronic hepatitis B at end-stage liver diseases was successfully established and stabilized in our laboratory.And the method was preliminarily applied to the study of HBV-DNA gene integration in patients with chronic hepatitis B at end-stage liver disease.We used Inv PCR to dectet that the HBV-DNA integration site on the nuclear genome was SHANK2 in Hep G2.2.15 cells.This study detected previously reported gene integration site,such as TERT,and found new integration sites,such as MVK,TBXAS1,JAG2,etc.TBXAS1 was a host gene locus that was easily integrated by HBV DNA.The frequency of HBV-DNA integration in host genes may be related to the age of >50 years old,the decompensation stage of cirrhosis,and the pathological classification of liver cancer.It is still necessary to further expand the sample size for further study.