Analysis Of Factors And Mechanisms Involved In The Failure Of Mother-to-child Transmission Of Hepatitis B Virus

Objective:Hepatitis B virus(HBV)infection can cause chronic hepatitis B,which may progress to hepatic fibrosis and Hepatocellular carcinoma,posing a serious threat to human health and becoming a global public health problem.The main mode of transmission of hepatitis B is vertical transmission from mother to child.Although newborns are born with a combination of hepatitis B vaccine and hepatitis B immunoglobulin for blockade,some infants still experience blockade failure resulting in HBV infection.The causes and mechanisms of failure of mother-to-child transmission of HBV are not yet fully understood,but mainly focus on maternal characteristics(maternal HBV DNA,maternal HBeAg status and mode of delivery,etc.)and genetic predisposition.This study was conducted to investigate the causes and mechanisms influencing mother-to-child transmission of HBV.In this study,we investigated the relationship between PARP15 polymorphism and mother-to-child transmission of hepatitis B,for exploring the correlation and molecular mechanism between PARP 15 and HBV infection,in order to provide a scientific basis for avoiding mother-to-child HBV infection.Methods:Part Ⅰ:Failure factors of mother-to-child blockade of HBV1.The subjects were HBsAg positive mothers and their children in the Affiliated Hospital of Guizhou Medical University from December 2019 to December 2022.According to whether the children were HBsAg positive or not,they were divided into blocking failure group and success group.2.Blood samples were collected from HBsAg-positive mothers to complete hepatitis B virus genotype testing.3.A case-control study was conducted to retrospectively collect the age,ethnicity,number of births,serum ALT,serum AST,serum HBeAg status and serum HBVDNA quantification of HBsAg-positive mothers as well as the use of antiviral drugs during pregnancy.Information was collected for both groups of children,including age,sex,mode of delivery,feeding method,first dose of hepatitis B vaccination within 12 hours after birth,hepatitis B immunoglobulin vaccination within 12 hours after birth,hepatitis B vaccination at 1 month of age and 6 months of age.Logistic regression was used for data analysis.Part Ⅱ:Association of candidate gene polymorphic loci such as PARP15 with mother-to-child transmission of HBV1.The children who failed to block hepatitis B from mother to child were taken as the case group,and the children who succeeded in blocking were taken as the control group.The case-control study was used to collect the general information of the two groups and the blood samples were collected of the two groups.2.Thirteen SNPs on seven genes associated with HBV infection through literature review were selected as candidate SNPs including PARP9 gene(rs 1560352,rs1107377),PARP15 gene(rs6796228),TMEM191 gene(rs583208,rs4822376,rs34592890),BAK1 gene(rs72879617,rs9272770,rs35597441,rs9348920),UBE2L3 gene(rs4821116),HLA-DOB gene(rs9394104),C4A gene(rs9272714).SNaPshot sequencing was used to genotype the candidate SNPs.The association between the above genetic polymorphism loci and the risk of mother-to-child transmission of hepatitis B was analysed by chi-square test and multi-factor logistic regression models.Part Ⅲ:Functional validation of the PARP15 gene promoter region rs6796228T/C and its mechanism affecting HBV infectionThe second part of the study identifies locus T/C of the PARP15 gene rs6796228 as a risk factor for mother-to-child transimission of hepatitis B.We will investgate the relationship between PARP15 polymorphism and mother-to-child transmission of hepatitis B,for exploring the correlation and molecular mechanism between PARP15 and HBV infection1.The study population was the same as in Part Ⅱ.The general condition and levels of ALT,AST,HBeAg status and HBV DNA of the case group were collected.2.Relationship between TT risk gene and PARP15 expression at rs6796228 locus:mRNA expression of PARP15 in PBMC of control group with CC and TT genotype was detected by RT-qPCR;PARP15 protein expression in peripheral serum of control group with CC and TT genotype was detected by Elisa.3.Validation of T/C function at rs6796228 locus:PROMO HOME PAGE website predicts transcription factors bound to the promoter region of the PARP15 gene;Dual luciferase reporter gene assay was to verify the effect of T/C transition on PARP15 gene promoter activity;SNaPshot sequencing was to identify cells carrying rs6796228TT genotype and cells carrying rs6796228CC genotype;Chromatin immunoprecipitation was used to determine the effect of T/C transformation on transcription factor binding capacity.4.Correlation of PARP15 with HBV infection:PARP15 mRNA expression in PBMC of children in case and control groups was detected by RT-qPCR;PARP15 protein expression in peripheral serum of children in case and control groups was detected by Elisa;The correlation between PARP15 protein and HBVDNA,ALT and AST levels in peripheral serum of children in the case group was analyzed.5.PARP15 gene promotes HBV replication and transcription:HEPG2.2.15 cell line with HBV replication function and HuH7 cell line constructed by transfection with HBV plasmid were selected.(1)Transfection of siPARP15 or siRNA into into HEPG2.2.15 cell lines and HuH7 cell lines with HBV replication function.PARP15siRNA transient transfection efficiency in HEPG2.2.15 and HuH7 cell lines was detected by Western Blot.HBVDNA and HBVRNA expression in HEPG2.2.15 and HuH7 cells after PARP15 silencing were detected by RT-qPCR;HBsAg levels in the supernatant of HEPG2.2.15 and HuH7 cells after PARP15 silencing were measured by Elisa.(2)PARP15 plasmid or vector was transfected into HEPG2.2.15 cell line and HuH7 cell line with HBV replication function.Transient transfection efficiency of PARP15 expression plasmids in HEPG2.2.15 and HuH7 cells was detected by Western Blot.HBVDNA and HBVRNA levels in HEPG2.2.15 and HuH7 cells after PARP15 overexpression were detected by RT-qPCR;HBsAg levels in the supernatant of HEPG2.2.15 and HuH7 cells after PARP15 overexpression were measured by Elisa.Results:Part Ⅰ:Failure factors of mother-to-child blockade of HBV1.A total of 229 HBsAg-positive mothers and their 229 children were included in the study.124 children(case group)failed to block hepatitis B and 105 children(control group)successfully blocked hepatitis B.2.HBV genes detected in HBsAg-positive mothers were type B,C,D and untyped,with type B and C predominating.HBV genotype of mothers in case group was predominantly type C,while the control group was predominantly type B,P<0.05.The percentage of mothers with serum HBeAg positive in the case group was higher than in the control group(P<0.001).The serum HBVDNA levels of mothers in the case group were higher than in the control group(P<0.001).3.There was no statistical difference between the case group and control group in age,sex,production mode,feeding mode,hepatitis B vaccine and hepatitis B immunoglobulin vaccination.4.Multifactorial regression analysis showed that maternal HBeAg positivity during pregnancy(OR=7.670,95%CI:2.193-26.820,P<0.001)and HBVDNA level during pregnancy(OR=1.514,95%CI:1.291-1.774,P<0.0001)were independent risk factors for failure of hepatitis B mother-to-child transmission.Part Ⅱ:Association of candidate gene polymorphic loci such as PARP15 with mother-to-child transmission of HBV1.A total of 229 study subjects were included in this study,124 children in the case group(HBsAg positive)and 105 children in the control group(HBsAg negative).There was no difference in age and gender between the two groups.2.BAK1(rs9272770,rs35597441)was genotyped at frequencies below 95%,C4A(rs9272714)and HLA-DOB(rs9394104)did not meet the Hardy-Weinberg equilibrium test.The final candidate genes and SNPs included in the analysis were:PARP15 gene(rs6796228),PARP9 gene(rs1560352,rs1107377),TMEM191 gene(rs583208,rs4822376,rs34592890),BAK1 gene(rs72879617,rs9348920)and UBE2L3 gene(rs4821116).3.The distribution of TT and CC genotypes at rs6796228 of PARP15 gene differed between the case and control groups,P<0.05.After logistic regression analysis correcting for child sex,age,maternal HBVDNA level and HBeAg status,the risk of mother-to-child transmission of hepatitis B infection was higher in children carrying the TT genotype than with the CC genotype by 106%(OR 2.06,95%CI:1.013-3.206,P=0.042).In the dominant model,children carrying CT/CC had a reduced risk of MTCT compared to children carrying the TT genotype(OR 0.27,95%CI:1.7446.132,P=0.041);the additive model showed that the T/C variant at rs6796228 was associated with the risk of MTCT of HBV(OR 0.656,95%CI:0.464-0.928,P=0.017).The PARP9 gene(rs1560352,rs1107377),TMEM191 gene(rs583208,rs4822376,rs34592890),BAK1 gene(rs72879617,rs9348920),UBE2L3 gene(rs4821116)SNPs did not differ in genotype distribution between case and control groups.The above genotype SNPs were not associated with risk of hepatitis B mother-to-child infection.Part Ⅲ:Functional validation of the PARP15 gene promoter region rs6796228T/C and its mechanism affecting HBV infection1.124 HBsAg positive children,HBeAg(+)72 and HBeAg(-)52,ALT mean value 22.50U/L,AST mean value 23.45U/L,HBV DNA mean value 4.97 log10IU/mL.2.Relationship between TT risk gene and PARP15 expression at rs6796228 locus:PARP15 mRNA expression was higher in peripheral blood mononuclear cells of TT genotype than CC genotype in 60 control children by RT-qPCR,P=0.008;PARP15 protein expression in peripheral serum was higher in TT genotype than CC genotype in 60 control children by Elisa,P=0.002.3.Validation of T/C function at rs6796228 locus:PROMO HOME PAGE website predicts that the transcription factor binding to the PARP15 gene promoter region is Yin Yang Protein 1(YY1);Dual luciferase reporter gene results showed that in both Hep3B2.1-7 and HuH-7 hepatoma cells,construct pGL3-(-1262T)showed higher luciferase activity compared to construct pGL3-(-1262C)(P<0.01);SNaPshot identified HuH-7 cells carrying rs6796228 TT genotype and Hep3B2.1-7 cells carrying rs6796228 CC genotype;Chromatin immunoprecipitation assay showed increased binding of YY1 to the PARP15 promoter in HuH-7 cell lines carrying TT genotype compared to Hep3B2.1-7 cell lines carrying CC genotype(P<0.001).4.Correlation of PARP15 with HBV infection:PARP15 mRNA expression in peripheral blood mononuclear cells was higher in case group(n=54)than in control group(n=44)detected by RT-qPCR,P=0.0016;PARP15 protein expression in peripheral serum was higher in case group(n=54)than in control group(n=44)measured by Elisa,P=0.0041;Serum PARP15 protein levels were positively correlated with HBVDNA levels in HBV-infected children(r=0.404,P=0.002);Serum PARP15 protein levels were positively correlated with ALT levels(r=0.432,P=0.001);Serum PARP15 protein levels were positively correlated with AST levels(r=0.413,P=0.002).5.PARP15 gene promotes HBV replication and transcription:RT-qPCR and Elisa results showed that HBV mRNA and HBsAg levels were significantly higher in the HBV plasmid-constructed HuH7 cells than in the negative control group.(1)Western Blot showed that in HEPG2.2.15 and HuH7 cell lines,PARP15 protein expression was reduced in the silencing group compared to vector group;RT-qPCR showed that the levels of HBVDNA and HBVRNA were reduced in HEPG2.2.15 and HuH7 cell lines after PARP15 silencing compared to the vector group;Elisa showed that HBsAg levels were reduced in HEPG2.2.15 and HuH7 cell lines after PARP15 silencing compared to vector group.(2)Western Blot showed that in HEPG2.2.15 and HuH7 cell lines,PARP15 protein expression was increased in the overexpression group compared to vector group;RTqPCR showed that the levels of HBVDNA and HBVRNA were higher in HEPG2.2.15 and HuH7 cell lines after PARP15 overexpression than vector group;Elisa showed that HBsAg levels were higher in HEPG2.2.15 and HuH7 cell lines after PARP15 overexpression than vector group.Conclusions:1.Maternal HBeAg positivity and high HBV DNA levels during pregnancy are independent risk factors for failure of mother-to-child blockade of hepatitis B,HBV genotype C may be a risk factor for failure of mother-to-child blockade of hepatitis B.2.The PARP15 polymorphic locus rs6796228 was associated with mother-to-child transmission of HBV,and the TT genotype of rs6796228 was a susceptible genotype for HBV mother-to-child transmission.3.In this study,we found for the first time that rs6796228 in the promoter region of the PARP 15 gene can affect the binding of transcription factor YY1,regulate PARP15 gene expression,promote HBV replication and transcription,and increase the risk of HBV infection.