Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing stud...Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.展开更多
OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a rec...OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a recently discovered novel OCT4 spliced variant,which has been considered as a putative marker of stemness.Compared with the OCT4B mRNA,OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it.As a result,the protein product of OCT4B1 mRNA could be truncated.Interestingly,we present here that OCT4B1 can indirectly produce the same protein products as OCT4B.We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA,and encode three protein isoforms.The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site.Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.展开更多
Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral bl...Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral blood mononuclear cells(PBMC)in VKH remains largely unknown.In this study,mRNA sequencing was conducted in PBMC from VKH patients with active uveitis before treatment(n=7),the same patients after prednisone combined with cyclosporine treatment(n=7)and healthy control subjects strictly matched with gender and age(n=7).We found 118 differentially expressed genes(DEGs)between VKH patients and healthy control subjects,and 21 DEGs between VKH patients before and after treatment.TRIB1 was selected as a potential biomarker to monitor the development of VKH according to the mRNA sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to predict the possible biological functions and signaling pathways of DEGs.Neutrophil degranulation,peptidase regulator activity,secretory granule membrane,cellular response to peptide,growth factor binding and cell projection membrane were enriched as GO annotations of DEGs.Arachidonic acid metabolism and mitogen-activated protein kinase(MAPK)signaling pathway were potential signaling pathways involved in pathogenesis and drug response of VKH.A protein–protein interaction(PPI)network was constructed by STRING,and colony stimulating factor 1 receptor(CSF1R)was identified as the hubgene of all DEGs by Cytoscape.The cell type presumed to contribute to the aberrant expression of DEGs was analyzed with the use of publicly available single-cell sequencing data of PBMC from a healthy donor and single-cell sequencing dataset of monocytes from VKH patients.Our findings may help to decipher the underlying cellular and molecular pathogenesis of VKH and may lead novel therapeutic applications.展开更多
基金We thank the families for participation in this study,and we thank Novogene Technology Co.,Ltd.,for the WES sequencing and analysis.This work was supported by the National Natural Science Foundation Project of China(82070951,82271078)the National Natural Science Foundation Key Program(81930023)+3 种基金The Innovative Research Group Project of Chongqing Education Commission(CXQT19015)the Innovation Supporting Plan of Overseas Study of Chongqing(cx2018010)the National Key Clinical Specialties Construction Program of China,the Chongqing Branch of the National Clinical Research Center for Ocular Diseases,the Chongqing Key Laboratory of Ophthalmology(CSTC,2008CA5003)the Program for Youth Innovation in Future Medicine,Chongqing Medical University(w0047).
文摘Vogt–Koyanagi–Harada(VKH)disease is a leading cause of blindness in young and middle-aged people.However,the etiology of VKH disease remains unclear.Here,we performed the first trio-based whole-exome sequencing study,which enrolled 25 VKH patients and 50 controls,followed by a study of 2081 VKH patients from a Han Chinese population to uncover detrimental mutations.A total of 15 de novo mutations in VKH patients were identified,with one of the most important being the membrane palmitoylated protein 2(MPP2)p.K315N(MPP2-N315)mutation.The MPP2-N315 mutation was highly deleterious according to bioinformatic predictions.Additionally,this mutation appears rare,being absent from the 1000 Genome Project and Genome Aggregation Database,and it is highly conserved in 10 species,including humans and mice.Subsequent studies showed that pathological phenotypes and retinal vascular leakage were aggravated in MPP2-N315 mutation knock-in or MPP2-N315 adeno-associated virus-treated mice with experimental autoimmune uveitis(EAU).In vitro,we used clustered regularly interspaced short palindromic repeats(CRISPR‒Cas9)gene editing technology to delete intrinsic MPP2 before overexpressing wild-type MPP2 or MPP2-N315.Levels of cytokines,such as IL-1β,IL-17E,and vascular endothelial growth factor A,were increased,and barrier function was destroyed in the MPP2-N315 mutant ARPE19 cells.Mechanistically,the MPP2-N315 mutation had a stronger ability to directly bind to ANXA2 than MPP2-K315,as shown by LC‒MS/MS and Co-IP,and resulted in activation of the ERK3/IL-17E pathway.Overall,our results demonstrated that the MPP2-K315N mutation may increase susceptibility to VKH disease.
基金supported by the National Basic Research Program of China (973 Program) (No 2006CB943601)the National Natural Science Foundation of China (NSFC) (No 90919042)
文摘OCT4 is one of the key transcription factors in maintaining the pluripotency and self-renewal of embryonic stem （ES） cells.Human OCT4 can generate two isoforms OCT4A and OCT4B by alternative splicing.OCT4B1 is a recently discovered novel OCT4 spliced variant,which has been considered as a putative marker of stemness.Compared with the OCT4B mRNA,OCT4B1 mRNA is generated by retaining intron 2 as a cryptic exon which contains a TGA stop codon in it.As a result,the protein product of OCT4B1 mRNA could be truncated.Interestingly,we present here that OCT4B1 can indirectly produce the same protein products as OCT4B.We have demonstrated that OCT4B1 mRNA can be spliced into OCT4B mRNA,and encode three protein isoforms.The splicing of OCT4B1 mRNA into OCT4B mRNA can be remarkably inhibited by the mutation of the classical splicing site.Our result suggests that OCT4B mRNA may originate from OCT4B1 mRNA by alternative splicing.
基金supported by National Natural Science Foundation Key Program,China(No.81930023)Natural Science Foundation Major International(Regional)Joint Research Project,China(No.81720108009)+2 种基金Chongqing Outstanding Scientists Project(2019),Chongqing Key Laboratory of Ophthalmology,China(No.CSTC,2008CA5003)Chongqing Science&Technology Platform and Base Construction Program,China(No.cstc2014ptsy10002)the Chongqing Chief Medical Scientist Project,China(2018).
文摘Vogt-Koyanagi-Harada disease(VKH)is a rare autoimmune disease characterized by diffuse and bilateral uveitis,alopecia,tinnitus,hearing loss,vitiligo and headache.The transcriptional expression pattern of peripheral blood mononuclear cells(PBMC)in VKH remains largely unknown.In this study,mRNA sequencing was conducted in PBMC from VKH patients with active uveitis before treatment(n=7),the same patients after prednisone combined with cyclosporine treatment(n=7)and healthy control subjects strictly matched with gender and age(n=7).We found 118 differentially expressed genes(DEGs)between VKH patients and healthy control subjects,and 21 DEGs between VKH patients before and after treatment.TRIB1 was selected as a potential biomarker to monitor the development of VKH according to the mRNA sequencing.Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis were performed to predict the possible biological functions and signaling pathways of DEGs.Neutrophil degranulation,peptidase regulator activity,secretory granule membrane,cellular response to peptide,growth factor binding and cell projection membrane were enriched as GO annotations of DEGs.Arachidonic acid metabolism and mitogen-activated protein kinase(MAPK)signaling pathway were potential signaling pathways involved in pathogenesis and drug response of VKH.A protein–protein interaction(PPI)network was constructed by STRING,and colony stimulating factor 1 receptor(CSF1R)was identified as the hubgene of all DEGs by Cytoscape.The cell type presumed to contribute to the aberrant expression of DEGs was analyzed with the use of publicly available single-cell sequencing data of PBMC from a healthy donor and single-cell sequencing dataset of monocytes from VKH patients.Our findings may help to decipher the underlying cellular and molecular pathogenesis of VKH and may lead novel therapeutic applications.
