Research On The Pathogenicity And Pathogenesis Of Novel Variants Related To Systemic Autoinflammatory Diseases

Objectives:Haploinsufficiency of A20(HA20)is a newly described rare monogenic systemic autoinflammatory disease(SAID)caused by high-penetrance heterozygous variants in TNFAIP3.HA20 patients manifest as recurrent oral and genital ulcers,fevers,gastrointestinal lesions,arthritis,and skin lesions since adolescence.With overlapping phenotypes and a lack of reliable diagnostic criteria,most HA20 patients were initially diagnosed with autoimmune diseases and autoinflammatory diseases other than HA20.Therefore,an accurate genetic diagnosis of TNFAIP3 is recommended when the clinical phenotype is highly suggestive of HA20.However,due to the low incidence and short history of research on HA20,the spectrum of pathogenic variants in HA20 is very limited,and most of them are frameshift mutations and nonsense mutations that cause truncation effects.Hence,the reference for HA20 genetic diagnosis in clinical practice is far from enough especially when missense mutations and intronic mutations are detected.Here we aimed to explore the pathogenic significance of novel TNFAIP3 variants found in potential HA20 pedigrees,providing genetic evidence for clinical diagnosis and prenatal diagnosis.Methods:A prospective study of pedigrees that were highly suspected of HA20 according to clinical phenotypes was conducted at the adult SAID center,Department of Rheumatology and Clinical Immunology,Peking Union Medical College Hospital.Based on pedigree analysis,whole-exome sequencing(WES)was performed to screen potential variants,whose pathogenic significance and possible pathogenic mode were predicted by software.Then we designed and performed in vitro functional experiments to detect the expression of A20 and the level of key signaling pathways,nuclear factor-kappa B(NFkB)and nucleotide-binding oligomerization domain-like receptor family,pyrin domain containing 3(NLRP3)inflammasome.Results:Between 2018 and 2021,three pedigrees highly suggestive of HA20 were included in our study and three TNFAIP3 variants,A547T,c.1906+2T>G,and R271*were identified.Among them,R271*had been confirmed to be pathogenic by Zhou et al and the pedigree with R271*variant was diagnosed with HA20.The pathogenicity of the other two novel variants remained to be verified.Affected by the COVID-19 pandemic,we failed to obtain biological samples from the pedigree with the A547T variant to evaluate its contribution to HA20.Since c.1906+2T>G was predicted by Mutation Taster as a possible intronic mutation by affecting the splice site,we used BDGP and NetGene2 to search for other candidate splicing sites in the intron 7 where the mutation was located.Reverse transcription-polymerase chain reaction(RT-PCR)and agarose gel electrophoresis confirmed the presence of an aberrant spliceosome with complete retention of intron 7,with greater concentrations than the wild-type spliceosome.Western blot(WB)and enzyme-linked immunosorbent assay(ELISA)were followed to verify the insufficient expression of A20 protein and exaggerated NF-kB and NLRP3 inflammasome signaling pathways.Thus,a novel TNFAIP3 variant,c.1906+2T>G,was validated to be pathogenic and served as evidence for the clinical diagnosis of HA20.Conclusion:A novel heterozygous TNFAIP3 intronic variant,c.1906+2T>G,resulted in insufficient expression of A20 protein by aberrant splicing,leading to hyperactive NF-kB and NLRP3 inflammasome signaling pathways.Our study initially validated the pathogenicity of TNFAIP3 c.1906+2T>G and confirmed its clinical diagnostic value in HA20.Objectives:NOD2 variants present a clear pathogenic significance in several kinds of autoinflammatory diseases,including Blau syndrome(BS)and Crohn’s disease(CD),and show certain regularity.The NOD2 variants located in the LRRs domain are thought to be susceptible to CD and belong to loss-of-function(LOF)mutations.And the pathogenic NOD2 variants related to BS are located in the NACHT domain,which is regarded as gain-of-function(GOF)mutations.However,more and more genetic analysis,in vitro functional experiments,and disease animal models have put forward different opinions on the location and pathogenesis of variants associated with disease phenotype.The purpose of this study is to explore the pathogenicity and pathogenesis of novel NOD2 variants related to NOD2-associated autoinflammatory diseases,to enrich the genetic evidence of NOD2-associated autoinflammatory diseases,and provide more experimental evidence for the controversial and unclear pathogenesis of NOD2-associated autoinflammatory diseases.Methods:A prospective study to include highly suspected NOD2-associated autoinflammatory diseases according to clinical phenotypes was conducted at the adult SAID center,Department of Rheumatology and Clinical Immunology,Peking Union Medical College Hospital.Based on pedigree analysis,whole-exome sequencing(WES)was performed to search potential variants.The variants were screened by searching Chinese and foreign literature,hereditary disease databases,variation databases,and"population" scale sequencing databases.Finally,we designed and performed in vitro functional experiments to explore the pathogenicity and pathogenesis of novel NOD2 variants related to NOD2-associated autoinflammatory diseases.Results:G319E,a novel NOD2 variant located in the NACHT domain,was carried by a patient with Crohn’s disease and his symptom-free mother.Based on the dose effect of pathogenic genes common in Crohn’s disease,we assumed that the proband’s mother had fewer changes in the pathogenic pathway although she did not manifest as Crohn’s disease.KEGG enrichment analysis showed that the peripheral blood mononuclear cells(PBMCs)derived from the group of G319E variant were significantly enriched in the inflammatory bowel disease(IBD)pathway under the stimulation of PGN or LPS,which provided preliminary support for our hypothesis and grouping.The differentially expressed genes(DEGs)in the IBD pathway were mapped to other significantly enriched signaling pathways,and the Th17 cell differentiation and JAK-STAT signaling pathway caught our attention.Based on the gene dose effect hypothesis,IL-10,IL-23A and IL-17A were screened as the key DEGs.The stimulation and culture of PBMCs and monocyte-derived dendritic cells(MoDCs)in vitro confirmed the disability of IL10 production in the G319E variant group.Immunohistochemical(IHC)analysis of the involved intestinal tissue showed a high level of IL-17A expression.Results of in vitro functional experiments and the effective therapeutic response of the probands to IL12/23 p40 monoclonal antibody both confirmed that the impairment of IL-10/IL-17A balance in the G319E variant group is the key pathogenic pathway.Using the luciferase report detection system,the inhibitory effect of the G319E variant on the IL-10 promoter was demonstrated again and showed a dose-dependent effect.Protein structure prediction and co-immunoprecipitation(Co-IP)analysis showed that the interaction between the protein encoded by the G319E variant and Atg16L1 was significantly stronger than that between WT and Atg 16L1.By co-transfecting Atg 16L1 and NOD2 at different ratios,we found that Atg16L1 affected the regulation of IL-10 promoter by NOD2.When Atg16L1 was co-expressed with NOD2 at a 1:10 ratio,the activity of the IL-10 promoter was the lowest when WT and G319E co-expressed at a 1:1 ratio.However,the enhanced interaction between the G319E variant and Atg16L1 did not affect the formation of autophagosomes and the antigen presentation ability of MoDCs.Conclusion:The NOD2 G319E variant related to Crohn’s disease might play a pathogenic role by inhibiting the transcriptional activity of IL-10,resulting in the imbalance of IL-10/IL-17A.The enhanced interaction between the protein encoded by the G319E variant and Atg16L1 might be involved in the pathogenesis of Crohn’s disease but did not affect autophagy related to NOD2.