A Systematic Study On HBV-ACLF Associated Genetic Factors And Its Functional Pathway Interaction Network

Caused by acute exacerbation of chronic hepatitis B（CHB）,HBV related acute-on chronic liver failure（HBV-ACLF）is a severe life-threatening syndrome in China and Southeast Asia.The disease exhibits high mortality and a lack of effective treatment.Studies have proved that the genetic diversity of host and pathogen may be highly related to the pathogenic process of HBV-ACLF.Potential association sites can be found in gene promoters,lnc RNAs,micro RNAs,and protein-coding genes in the human genome.The variation of surface antigen-related pre-S/S gene and C promoter/pre-C gene is associated with fulminant hepatitis,acute exacerbation,and chronic liver disease.Comprehensive analysis of genetic factors and their functions in HBV-ACLF will deepen the understanding of the pathogenic mechanism and intervention targets.A genome-wide association study（GWAS）based on a multicenter,large sample,and high marker density is one of the effective strategies to identify relevant genetic loci.Microarray typing technology mainly includes two types of markers:single nucleotide polymorphism（SNP）and genomic copy number variation（CNV）.In the previous study,our group conducted SNP-based GWAS on 780 HBV-ACLF related cohorts and successfully identified a major associated SNP（rs3129859）on chromosome 6.HBV-ACLF is a complex disease.A single genetic susceptibility locus cannot comprehensively explain the genetic mechanism of the disease.More major or minor effective loci need to be identified.However,only a few SNP associates were reported.As the main kind of genetic variation,CNV also shows high diversity in the human population,but direct evidence of the association between host CNVs and HBV-ACLF remains unknown.In the pre-experiment,we found that the endogenous superantigen reaction mediated by a human endogenous retrovirus（HERV）may be involved in HBV-ACLF.HBV-ACLF patients reveal a high number of up-regulated HERV elements.Functional analysis showed that its transcription and regulation mechanism was similar to that of EBV activating human endogenous superantigen HERV K18.Interestingly,this phenomenon also appears in severe patients with COVID-19,which indicates that the two severe diseases may be similar in the mechanism of activating superantigen.Endogenous superantigen can cause inflammatory storms by activating a huge number of T cells.We observed the possibility of HBV activating host HERV to produce superantigen reaction in the pathology study of HBV-ACLF,which needs further study and verification.Therefore,to widely predict HBV-ACLF related genetic factors,this study will analyze the effects of SNV,CNV,HERV activation on HBV-ACLF.Meanwhile,HBV-ACLF specific expression genes and the related key regulatory pathways were found by transcriptome analysis.Finally,all genetic factors and regulatory pathways were clustered to construct the HBV-ACLF pathology-related regulatory network.The main analysis contents are as follows:（1）Collecting HBV-ACLF and ASC samples,and genotyping the samples with SNP and CNV;（2）GWAS studies were carried out on SNP and CNV respectively to obtain potential HBV-ACLF associated genetic regions;（3）Prediction of HBV-ACLF differentially expressed genes and verification of HERV superantigen activation process.The transcriptome data of two severe patients（HBV-ACLF and COVID-19）were analyzed.Study of the expression pattern of HERV in severe and mild patients;Predict the specific expression genes and their functional pathways of severe patients,and analyze whether these pathways are involved in superantigen regulation;（4）Analyze the quasispecies characteristics of pathogens and predict the potential superantigen activation mutation sites.HBV-ACLF and COVID-19 have similarities in activating superantigen.（5）Construction of interaction network between HBV-ACLF regulatory pathway and associated genetic factors.All related genetic factors in this study were summarized,and the regulatory network was constructed through protein-protein interaction analysis.The main results are listed as follows:1.SNP-based GWAS results of HBV-ACLF.The study cohort consisted of 693HBV-ACLF patients and 672 asymptomatic HBV carriers（As C）,As C.A total of 15associates were identified.The associated region includes three protein-coding genes（HLA-DRB1,CCDC88B,and ZNF507）and four lnc RNA genes（NLGN1-AS1,KB-1568E2.1,NAMA,and RP11-168o22.1）.2.CNV-based GWAS results of HBV-ACLF.The study cohort consisted of 389HBV-ACLF patients and 391 As C controls.HBV-ACLF patients are prone to 100-200kb fragment deletion,and related genes are enriched in the leukocyte transendothelial migration pathway(p-value=4.68×10^-3),which contains five cell adhesion molecules（CAM）genes.GWAS predicted 17 HBV-ACLF related CNVs.The peak associate is at 1p36.13(P-value=1.99×10^-4),the fragment size is～38 kb,and contains the protein-coding gene MST1L.The second highest signal is at 6p22.1(3.45×10^-4),with a length of～33 kb and covering a long noncoding RNA gene HCG4B.3.Prediction of HBV-ACLF differentially expressed genes and verification of HERV superantigen activation process.HBV-ACLF specific up-regulated genes are enriched in 24KEGG signal pathways（including 96 specific high expressed genes）.HERV K elements in severe patients with HBV-ACLF and COVID-19 are highly active（K5 and k59 subclasses are active in two kinds of severe patients）,which may be related to the activation of endogenous superantigens by pathogens.The functions of specifically up-regulated genes in two severe diseases contain superantigen-related regulatory pathways,such as PI3K-Akt signaling pathway,MAPK signaling pathway,Ras signaling pathway,and so on.4.Quasispecies analysis and prediction of potential activation sites of HERV.The average quasispecies entropy（SN）of severe patients with HBV-ACLF and COVID-19 was significantly lower than that of the control（t-test p-value=0.0021,and 0.068respectively）.The abundance distribution of HBV quasispecies showed a gradient change,while the composition of SARS-Cov-2 quasispecies showed a trend of"master and dusty"(the average abundance of the master mutant was 0.70,and the abundance of other mutants was no more than 2.55×10^-3).The quasispecies pre-S/S protein of HBV-ACLF patients showed 22 specific amino acid variations,of which W233U only appeared in HBV-ACLF.Accordingly,the spike protein of quasispecies in severe COVID-19 patients contains 16amino acid variants.5.Construction of interaction network for HBV-ACLF associated genetic factors.A total of 119 HBV-ACLF associated genes were obtained,including 96 specifically up-regulated genes and 23 GWAS-based genes.Through the construction of a protein-protein interaction network,10 key regulatory genes were found:KRAS,GAPDH,LYN,NFKBIA,Akt3,Jak1,TNF,and CHUK.The results of regulatory pathway interaction show that TNF signaling pathway and chemokine signaling pathway contain the most number of edges（6）,followed by Ras（5）and PI3K Akt（3）signaling pathways.HBV-ACLF has the most up-regulated genes in the hematopoietic cell-related pathway（24）,followed by the PI3K-Akt signaling pathway（17）.Four of the seven pathways shared by the two severe diseases are clustered together,including PI3K-Akt,TNF,Rap1,and osteoclast function-related pathways.GWAS associated genes MST1L and DEFB can participate in the regulation of TNF and chemokine pathways.Conclusions:The current study systematically analyzed genetic impacts（SNV,CNV,and HERV）on HBV-ACLF.The range of HBV-ACLF related genetic factors was expanded.Associated genetic factors or genes participate in the pathogenic process mainly through three aspects:response of immune cells（HLA-DRB1,CCDC88B）,proinflammatory response（MST1L,DEFB,HCG4B）,and specific activation of HERV.The gene CCDC88B containing associatied SNP may participate in the pathological process of HBV-ACLF by affecting T cell immune response and pro-inflammatory effect.More copies of MST1L,DEFB,and HCG4B genes caused by an associated CNV may enhance the inflammatory response during HBV infection.HERV K elements are highly active in patients with HBV-ACLF and a similar phenomenon also occurs in severe patients with COVID-19.The functional pathway of up-regulated genes in severe patients（HBV-ACLF and COVID-19）includes the superantigen mediated regulation pathway.Through superantigen reaction,the HERV K elements are likely to participate in the development of HBV-ACLF and severe COVID-19 diseases.The seven common pathways of up-regulated expression of the two severe diseases represent the common pathological process,which may be important to the regulation of superantigen（PI3K-Akt,TNF,Rap1,and osteoclast function-related pathways etc.）.Specific mutations that occurred in the quasispecies surface antigen protein provide a reference for the prediction of the core sequence of activation sites.Finally,HBV-ACLF associated genetic factors can interact with 24 key regulatory pathways and together form a HBV-ACLF related genetic linkage map.TNF and chemokine signaling pathways may play a key role in the functional network and can interact with associated genes of MST1L and DEFB.In summary,the current study widely predicted the genetic association factors of HBV-ACLF,which improves the understanding of the genetic mechanism of HBV-ACLF.In addition,our study novelly found that activating endogenous superantigen may be one of the genetic factors of HBV-ACLF,which may provide a new field for the study of HBV-ACLF or severe infectious diseases.