Study On Host Genetic Factors In Fulminant Hepatitis B And Diagnosis Of Inherited Liver Diseases

Objectives:Fulminant hepatitis B is an extremely rare but highly fatal outcome of acute hepatitis B virus(HBV)infection.The first part of study was conducted to investigate possible host genetic factors associated with pathogenesis of fulminant hepatitis B,which have not been studied before.Mice experiments were performed to verify roles of selected candidate gene in fulminant hepatitis B.DNA sequencing and mutation analysis are important method for diagnosis of Mendelian inherited liver diseases.The second part of study was taken to analyze corresponding gene mutations in type I glycogen storage disease and Wilson’s disease to confirm the diagnosis and find out novel mutations.Methods:In the first part of study,genomic DNA samples from 10 unrelated fulminant hepatitis B patients were collected to perform massively parallel sequencing of the whole exomes.Mutations identified were filtered through a bioinformatics analysis pipeline designed to select for uncommon variations most likely to affect protein functions.The candidate fulminant hepatitis B-associated mutations were confirmed using Sanger DNA sequencing and checked for presence in 282 HBV-infected patients of non-fulminant hepatitis B outcomes,including asymptomatic self-limited infection,acute hepatitis B and chronic hepatitis B.Functional characterization of TLR2 mutation was studied using HEK293 cell transfected with plasmids expressing wild-type and mutant TLR2.A HBV-transfected mouse model was established by hydrodynamic injection of replication-competent HBV plasmids into the tail veins of Tlr2-/-mice and C57BL/6 mice.The serum specimens were collected for assays of hepatitis B surface antigen,hepatitis B e antigen,HBV DNA and serum ALT at indicated time points after injections.In the second part of study,direct DNA sequencing of the coding region and splicing-sites in the G6PC gene for type I glycogen storage disease and the ATP7B gene for Wilson’s disease.Novel mutations were directly sequenced in 200 chromosomes from 100 unrelated healthy control subjects,and the novel mutation identified in patient with type I glycogen storage disease was further confirmed by digestion with Rsa I restriction endonuclease.Results:We identified twelve novel protein-altering variations in the first part of study that were found exclusively in fulminant hepatitis B patients and targeted eleven genes:AOAH,APPL1,C5,CYLD,IL1RL1,ITSN2,NPC1,TLR2,TNFRSF17,UNG and XRCC5.All twelve variations were heterozygous,including one possible compound heterozygosis.At least six genes of these affected genes are known or suspected to be involved in NF-κB signaling pathways.An identical TLR2 single nucleotide variation causing F679I mutation in the Toll/Interleukin-1 receptor(TIR)domain of TLR2 protein was found in two fulminant hepatitis B patients and demonstrated to be a loss-of-function mutation in in vitro analysis.Moreover,Tlr2 deficient mice had obvious higher levels of serum ALT compared to C57BL/6 mice after injections of HBV plasmids.In the second part of study,we revealed a novel no-stop mutation,p.*358Yext*43 in the G6PC gene and four novel mutations(I116T,A982T,G1030D,W1353*)in the ATP7B gene.The diagnosis of eleven patients with Wilson’s disease was confirmed with mutation analysis.Conclusions:For the first part of study,novel host genetic variations were identified for the first time in fulminant hepatitis B patients using whole-exome sequencing.More than half of the affected genes are involved in NF-κB signaling,suggesting the importance of this pathway in the pathogenesis of fulminant hepatitis B.The occurrence of an identical loss-of-function TLR2 SNV in two fulminant hepatitis B patients strongly indicates TLR2’s involvement in host defense for anti-HBV.The suggestion was further verified in mice experiments.For the second part of study,the definitive diagnoses of type I glycogen storage disease and Wilson’s disease for the patients were confirmed by clinical manifestations,biochemical examinations and/or histological analysis of liver biopsy sample combined with mutation analyses of the G6PC gene and the ATP7B gene respectively.The identified novel mutations in this study expand the spectrum of mutations in these genes.