| [Background]Granular corneal dystrophy type 1(GCD1)is an autosomal dominant disorder characterized by bilateral,symmetrical,discrete,grayish-white "breadcrumb" granular deposits in the corneal stroma.The cornea between the particles and the periphery of the cornea is transparent.At present,corneal transplantation is still the main method for the treatment of this disease.However,there is a serious shortage of corneal donors worldwide,and some patients will still experience recurrence and deterioration after transplantation.It is urgent to explore new pathogenic mechanisms and intervention strategies.Previous research on the pathogenesis of GCD1 has mainly focused on the TGFBI gene and the related pathways affected by it.In the early stage of our group,a new mutation associated with the disease,namely,BMP3(c.A710G,p.E237G),was discovered by whole genome sequencing of an autosomal dominant family.[Objectives]1.To study the relationship between BMP3(c.A710G,p.E237G)mutation and type I granular corneal dystrophy.2.To reveal the role of BMP3(c.A710G,p.E237G)mutation in the pathogenesis of type I granular corneal dystrophy.3.To explore the mechanisms of BMP3(c.A710G,p.E237G)mutation in the pathogenesis of type I granular corneal dystrophy.[Methods]Based on the previously discovered novel pathogenic gene mutation associated with GCD1,namely,BMP3(c.A710G,p.E237G)mutation,this study first analyzed the pathogenicity of the mutation through bioinformatics structure and function prediction.BMP3 wild-type and BMP3(c.A710G,p.E237G)mutant plasmids were cloned and constructed,and wild-type and mutant BMP3 were expressed in corneal stromal keratocytes by lipofection and lentiviral vector.Then,stable transfected corneal stromal keratocytes overexpressing wild-type and mutant BMP3 were obtained by puromycin screening.The stable cell line was then used to study the role and mechanism of this mutation in GCD1 by Western blot,cell immunofluorescence,protein coimmunoprecipitation,mass spectrometry,transmission electron microscopy and RNA-seq.Peripheral blood mononuclear cells from mutation patients were obtained by a nonintegrating pluripotency transcription factor plasmid-induced method to establish patient-specific iPSCs and further induced to differentiate into corneal stromal keratocytes.Cell immunofluorescence and RNA-seq were used to study the effect of BMP3 mutation on the effect of iPSCs on the differentiation process of corneal stromal keratocytes and the phenotype of the differentiated corneal stromal keratocytes.[Results]1.Glutamate at position 237 of the BMP3 protein is highly conserved among species,and BMP3(c.A710G)mutation mutates glutamate at this position to glycine;PolyPhen-2 predicts that this mutation is likely to damage protein structure and function,trRosetta protein Structural prediction showed that mutant BMP3 had a different three-dimensional structure compared to wild-type.2.In corneal stromal keratocytes stably overexpressing BMP3-WT and BMP3-MUT,it was found that compared with the BMP-WT group,the BMP3-MUT group had higher BMP3 preprotein production,but there was no significant change in the amount of extracellular secretion.BMP3 and the endoplasmic reticulum marker Calnexin were colocalized,and BMP3(c.A710G,p.E237G)mutation did not affect its endoplasmic reticulum localization;BMP3-MUT did not affect the activation of Smad2/3 compared with the BMP3-WT group;BMP3-MUT and BMP3-WT have different protein binding profiles,including changes in protein folding-related protein profiles;Co-IP experiments showed that the ability of mutant BMP3 to bind to Calnexin and Calreticulin was lower than that of the wild-type control group;Western blot results showed that the endoplasmic reticulum stress,unfolded protein response-related markers PERK,PDI,CHOP and Erol-Lα in the BMP3-MUT group were significantly upregulated compared with the BMP3-WT group;electron microscopy showed that the endoplasmic reticulum in the BMP3-MUT group was significantly higher than that in the BMP3-WT group.At the fold change>2/1 or<1/2,a significant total of 1580 RNAs were identified,of which the increased Of the 490 genes,1,090 were decreased.Gene Ontology(GO)functional annotation and enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)pathway analysis were performed on the significantly differentially expressed genes,and the analysis found that BMP3 mutation had a significant impact on apoptosis,necrosis,lysosome and calcium signaling pathways.3.GCD1-specific iPSCs(G1-iPSCs)were successfully established using the peripheral blood of patients with BMP3(c.A710G,p.E237G)mutations,and their pluripotent stem cell characteristics and patient-derived identity were verified.Normal iPSCs(N1-iPSCs)and G1-iPSCs were successfully differentiated into corneal stromal keratocytes,and the G1 group had a weaker migration ability than the N1 group at the early stage of the differentiation process.RNA-seq analysis was performed on the corneal stromal keratocytes derived from the differentiation of the G1 group and its control N1 group.At the fold change>2/1 or<1/2,a significant total of 1387 genes were identified,of which 676 genes were increased and 711 genes were decreased.GO functional annotation and KEGG pathway analysis were performed on the significantly differentially expressed genes,and it was found that BMP3 mutation significantly affects the calcium ion signaling pathway.The regulation of the Hippo,PI3K-Akt,and HIF-1 signaling pathways is closely related to apoptosis,which is similar to the RNA-seq results in the stable corneal stromal keratocytes lines overexpressing BMP3-WT or BMP3-MUT.[Conclusions]BMP3(c.A710G,p.E237G)mutation is closely associated with the onset of type 1 granular corneal dystrophy;BMP3(c.A710G,p.E237G)mutation leads to folding and conformational abnormalities of BMP3 precursor protein,causing endoplasmic reticulum stress and an unfolded protein response,which may be involved in the development of granular corneal dystrophy;BMP3(c.A710G,p.E237G)mutation does not affect the final differentiation of iPSCs into corneal stromal keratocytes. |
