Identicication Of Chronic Hepatitis B Virus Susceptible Gene By Transcriptome Wide Association Study

Background: HBV infection is one of the major infectious diseases affecting global public health.At least 2 billion people worldwide have been infected with HBV,and 350-400 million people are chronic HBV infection.These patients mainly distributed in the Asia-Pacific region.According to the latest 2017 Global Burden of Disease(2017 GBD)report,the number of deaths caused by HBV infection is about 799,000 per year,ranking among the top five deaths caused by infectious diseases.In China,about 300,000 people die of cirrhosis and hepatocellular carcinoma(HCC)due to HBV infection each year.Therefore,chronic HBV infection is still one of the major public health problems in China for a long time.Persistent HBV infection is a multifactorial disease that affected by viruses,environmental and genetic factors.Chronic HBV infection rate is different from ethnics,In Asia,there is a higher prevalence than other regions.Even after Aians move to regions with lower prevalence of chronic HBV infection,the prevalence of chronic HBV infection among them was still higher than indigenous inhabitant,which indicates that genetic factors may play a role in the susceptibility of chronic HBV infection.Single nucleotide polymorphism(SNP)is one of the most common accesses to study germline variants.Genome-wide association study(GWAS)has become one of the most efficient research tool in molecular epidemiology,and with the rapid deveploment of genomic technologies,GWAS has been widely used and scuuess in exploring genetic risk loci for complex diseases.Recent years,genome-wide association studies(GWAS)have provided evidence that genetic variants could contribute to persistent HBV infection,four loci identified by GWAS highlighting the essential role of the immune response to HBV.Expression quantitative trait loci(e QTLs),genetic variants identified by GWASs are more likely to be associated with the expression(m RNA)levels of one or more genes.Chronic HBV infection is a risk factor for HCC.Therefore,it is important to investigate the association between susceptible genetic variants of persistent HBV infection and HBV-related HCC risk.However,the majority of the GWAS-identified variants were located in the noncoding regions,and the causal genes were difficult to identify.In addition,the stringent criteria and single variant test greatly limited the power of GWAS.The Genotype-Tissue Expression(GTEx)database provides RNA-seq data from more than 50 human tissues,which provides a feasibility for the association study of the whole transcriptome level of specific tissues.A transcriptome-wide association study(TWAS)was a powerful study designed to pinpoint susceptibility genes by integrating the effects of multiple single nucleotide polymorphism(SNPs)and transcriptome information.For example,Recent studies identified causal genes and related SNPs by conducting TWAS on cancer,schizophrenia,hypertension,attention deficit hyperactivity disorder and so on.TWAS could identify new susceptible regions and potential target genes and also provide a basis for the identification of susceptible genes in GWAS reporting regions.Aim: We aimed to identify the potential causal genes by integrating genotypes data and transcriptome data from GTEx database and GWAS imputationed data by our previous study.Coexpression-based gene set enrichment analysis(GSEA)was applied to infer the function of the identified genes.To identify the causal single nucleotide polymorphisms(SNPs)of HBV infection risk,we conducted the expression quantitative trait loci(e QTL)-based conditional analysis in the regions around 1 Mb of these genes.To assess the association of novel SNPs in HBV-HCC.Subjects and methods: Here,we conducted the first TWAS of persistent HBV infection by integrating GWAS data from 951 HBV carriers and 937 HBV cleared controls and expression data from 97 liver tissues.We performed a two-stage study,in the first stage,we integrated RNA sequencing data of liver tissues and high-density genotyping data from the Genotype-Tissue Expression(GTEx)project with our previous GWAS data to conduct a transcriptome-wide association study(TWAS)on HBV infection.Firstly,the screening of cis-heritable genes by a genetic relatedness matrix of genome-wide complex trait analysis(GCTA)from GTEx data.Then,the genetic expression of 2587 cis-heritable genes were predicted by restricted maximum likelihood(REML)of genome-wide efficient mixed-model association(GEMMA)in our GWAS data with 951 HBV carrier cases and 937 HBV cleared controls.Next,we investigated the associations between predictive expression levels and persistent HBV infection risk.Coexpression-based gene set enrichment analysis(GSEA)was applied to infer the function of the identified genes.To identify the causal single nucleotide polymorphisms(SNPs)of HBV infection risk,we conducted the expression quantitative trait loci(e QTL)-based stepwise logistic regression in the regions around 1 Mb of these genes.The second stage,1538 HBV-related hepatocellular carcinoma(HCC)cases and 1465 persistent HBV infection controls were collected to determine whether the effect of these variants on HBV-related HCC as well,which were examined by the additive model in logistic regression analysis.Results: We identified eight genes associated with HBV infection.In the classic human leukocyte antigen(HLA)region,three novel genes HLA-DOB(Z =-3.72,P = 2.00 × 10-7),BAK1(Z =-3.95,P = 7.84 × 10-5)and C4A(Z =-3.64,P = 2.70×10-4);as well as two genes(HLA-DPA1 and HLA-DPB1),as reported by previous GWAS.In the non-HLA region,including two immune-related genes at newly identified loci,PARP9(Z = 3.69,P = 2.20 × 10-4)at 3q21.1 and a pseudogene AC138783.12(Z = 3.57,P = 3.60 × 10-4)at 7q11.23.At 22q11.21,we identified TMEM191A(Z = 3.55,P = 3.80 × 10-4).After further conditional analysis in those regions,we identified 14 variants independently associated with persistent HBV infection.Among those variants,the additive model showed that three SNPs associated with HBV-related HCC risk(rs9394104,OR = 1.23,95%CI: 1.10-1.37,P = 3.97 ′ 10-4;rs9272714,OR = 1.29,95%CI: 1.12-1.42,P = 1.19 ′ 10-4;rs9272770: OR = 1.23,95%CI: 1.07-1.42,P = 4.75 ′ 10-4).Conclusion: We identified six new genes(PARP9,AC138783.12,HLA-DOB,C4 A,BAK1 and TMEM191A)associated with persistent HBV infection,besides the known HLA-DPA1 and HLA-DPB1.Additionally,we determined 14 variants independent of previously reported SNPs,and three of them were associated with HBV-related HCC.By integrating transcriptome data,our study not only identified new susceptibility loci of persistent HBV infection but also determined the potential target genes at reported loci,which provided insight into the genetic etiology of persistent HBV infection and related HCC.