Whole Exon Sequencing Analysis In Patients With Chronic Active EBV Infection And Expression Of Caspase-8 In Liver Tissue

Part one: Analysis of clinicopathological features of chronic active EB virus infection in adults Objective: To Summarize the clinicopathological characteristics of adult patients with chronic active Epstein-Barr virus infection(CAEBV)in order to improve the understanding of the disease.Methods: A retrospective analysis was conducted 19 adult patients with CAEBV from January 2010 to December 2019.The data collected included basic information,clinical symptoms,laboratory examinations,histopathological results,therapeutic methods and follow-up data.Results: Of the 19 patients,there included 14 males and 5 females.The median age was 33 years old,ranging from 14 to 83 years old.The median time from symptom onset to first diagnosis was 8(3,28)months.The clinical manifestations were different,including recurrent fever(84.2%,16/19),liver dysfunction(78.9%,15/19),anemia(84.2%,16/19),thrombocytopenia(84.2%,16/19),hepatosplenomegaly(94.7%,18/19),lymphadenopathy(42.1%,8/19)and hemophagocytic lymphohistiocytosis(HLH)(52.6%,10/19).A total of 22 histopathological specimens were obtained from 19 patients.Most of the tissue biopsy specimens,including bone marrow(8),liver(6),spleen(1)and lymph node(1),showed the presence of lymphocyte infiltration of varying sizes,inconspicuous atypia,and varying numbers in the diseased tissue(68.2%,15/22).A few tissue biopsy specimens showed normal morphology(31.8%,7/22).Of the 22 tissue specimens,14 specimens were immunohistochemically stained.All specimens tested for CD3 and CD20 immunohistochemical staining were positive,and the number of CD3 positive cells was more than that of CD20 positive cells(100%,14/14).Most EBVencode small RNA(EBER)in situ hybridization tests were positive(85.7%,12/14),and more than half of the T cell receptor(TCR)gene rearrangement tests were monoclonal rearrangements(66.6%,,4/6).The mortality rate was 63%(12/19),and the median survival time was 20.75(8.25,36.5)months.The causes of death included multiple organ failure(50%,6/12),gastrointestinal bleeding(25%,3/12),sepsis(16.7%,2/12)and disseminated intravascular coagulation(8.3%,1/12).There was no significant correlation between the prognosis of the patients and whether they were treated with antivirus,immunosuppressive therapy,CAEBV diagnosis time and EBV DNA load(P<0.05).The occurrence of HLH has nothing to do with the time of CAEBV diagnosis and EBV DNA load(P<0.05).Conclusion:(1)CAEBV in adults was prevalent in young men.The clinical features were repeated fever,cytopenia,abnormal liver function,hepatosplenomegaly and HLH.The prognosis is poor.(2)Pathological characteristics showed that the lymphocytes infiltrated in the diseased tissue,and the positive rate of EBER and TCR gene monoclonal rearrangement were high.Part two: Expression of caspase-8 in liver tissue of patients with CAEBV Objective: To investigate the expression of caspase-8 in the liver tissue of patients with CAEBV.Methods: The liver tissue of CAEBV patients was assigned to the experimental group,and the normal liver tissue to the control group.The apoptosis index(AI)were detected by TUNEL and the expression level of caspase-8 were by immunohistochemical staining.Results: There were 4 cases either in the experimental group or in the control group.The AI of the liver tissue was 91.10% in the experimental group and 1.00% in the control group,with statistical significance(Z =-2.323,P <0.001).The positive score of liver tissue caspase-8 was 8.00 in the experimental group and 1.50 in the control group,with statistical significance(Z =-2.381,P <0.001).Conclusion: Caspase-8 was highly expressed in liver tissues of CAEBV patients,suggesting that apoptosis may be one of the important pathogenesis of CAEBV.Part three: Whole exon sequencing analysis in patients with CAEBV Objective: To sequence and analyze the gene mutations in adults with CAEBV in an attempt to explore the possible pathogenic genes.Methods: Taking adult patients with CAEBV as the research object,using all-exon sequencing technology and bioinformatics analysis to screen immune-related mutant genes.The gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis were performed according to the function of the mutant gene.The common genes enriched in the most significant pathways were selected for pathogenicity analysis to determine the most likely pathogenic genes.Results: 10 specimens were received.After preliminary screening,410 immune genes were obtained.GO enrichment analysis showed that immune genes were mainly expressed as receptor complexes on cell components(CC),molecular functions(MF)were mainly reflected in peptide binding,and biological functions(BP)were mainly reflected in lymphocyte activation.But the most significant enrichment was lymphocyte activation.KEGG pathway analysis showed that the most significant enrichment was the hematopoietic cell lineage.The lymphocyte activation pathway and hematopoietic lineage pathway contained 43 mutant genes.Pathogenicity analysis showed that ATM(c.8246A>T,p.K2749I)and HLADRB1(c.373C>T,p.Q125X)may be the pathogenic genes.Conclusion: Enrichment analysis showed that abnormal lymphocyte activation and proliferation may be one of the pathogenesis of CAEBV.ATM(c.8246A>T,p.K2749I)and HLA-DRB1(c.373C>T,p.Q125X)may be the pathogenic genes of CAEBV patients.