RESUMEN
PURPOSE: Mutations in transforming growth factor beta-induced (TGFBI) protein are associated with a group of corneal dystrophies (CDs), classified as TGFBI-associated CDs, characterized by deposits in the cornea. Mouse models were not proper in several aspects for modelling human disease. The goal of this study was to generate zebrafish mutants to investigate the corneal phenotype and to decide whether zebrafish could be a potential model for TGFBI-associated CDs. METHODS: The conserved arginine residue, codon 117, in zebrafish tgfbi gene was targeted with Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 method. Cas9 VQR variant was used with two target-specific sgRNAs to generate mutations. The presence of mutations was evaluated by T7 Endonuclease Enzyme (T7EI) assay and the type of the mutations were evaluated by Sanger sequencing. The mutant zebrafish at 3 months and 1 year of age were investigated under the microscope for corneal opacity and eye sections were evaluated histopathologically with hematoxylin-eosin, masson-trichrome and congo red stains for corneal deposits. RESULTS: We achieved indel variation at the target sequence that resulted in p.Ser115_Arg117delinsLeu (c. 347_353delinsT) by nonhomology mediated repair in F1. This zebrafish mutation had the potential to mimic two disease-causing mutations reported in human cases previously: R124L and R124L + del125-126. Mutant zebrafish did not show any corneal opacity or corneal deposits at 3 months and 1 year of age. CONCLUSION: This study generated the first zebrafish model mimicking the R124 hot spot mutation in TGFBI-associated CDs. However, evaluations even at 1 year of age did not reveal any deposits in the cornea histopathologically. This study increased the cautions for modelling TGFBI-associated CDs in zebrafish in addition to differences in the corneal structure between zebrafish and humans.
Asunto(s)
Distrofias Hereditarias de la Córnea , Opacidad de la Córnea , Animales , Humanos , Ratones , Córnea/metabolismo , Distrofias Hereditarias de la Córnea/genética , Opacidad de la Córnea/metabolismo , Análisis Mutacional de ADN , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Mutación , Linaje , ARN Guía de Sistemas CRISPR-Cas , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismo , Pez Cebra/genéticaRESUMEN
Desmin is a muscle specific intermediate filament protein located in cytoplasm. Lamin B, on the other hand, is a nuclear intermediate filament protein. There are studies suggesting a possible interaction between desmin and lamin B yet there is no physical evidence. In the present study, we have shown for the first time a physical interaction between desmin and lamin B via reciprocal co-immunoprecipitation from muscle tissue of wild type AB zebrafish (Danio rerio, Hamilton). The interaction between desmin and lamin B might be a lead on a novel nucleocytoplasmic communication network.
Asunto(s)
Lamina Tipo B/genética , Animales , Núcleo Celular , Desmina , Proteínas de Filamentos Intermediarios , Pez CebraRESUMEN
PURPOSE: To identify pathogenic variations in carbohydrate sulfotransferase 6 (CHST6) and transforming growth factor, beta-induced (TGFBI) genes in Turkish patients with corneal dystrophy (CD). METHODS: In this study, patients with macular corneal dystrophy (MCD; n = 18), granular corneal dystrophy type 1 (GCD1; n = 12), and lattice corneal dystrophy type 1 (LCD1; n = 4), as well as 50 healthy controls, were subjected to clinical and genetic examinations. The level of antigenic keratan sulfate (AgKS) in the serum samples of patients with MCD was determined with enzyme-linked immunosorbent assay (ELISA) to immunophenotypically subtype the patients as MCD type I and MCD type II. DNA was isolated from venous blood samples from the patients and controls. Variations were analyzed with DNA sequencing in the coding region of CHST6 in patients with MCD and exons 4 and 12 in TGFBI in patients with LCD1 and GCD1. Clinical characteristics and the detected variations were evaluated to determine any existing genotype-phenotype correlations. RESULTS: The previously reported R555W mutation in TGFBI was detected in 12 patients with GCD1, and the R124C mutation in TGFBI was detected in four patients with LCD1. Serum AgKS levels indicated that 12 patients with MCD were in subgroup I, and five patients with MCD were in subgroup II. No genetic variation was detected in the coding region of CHST6 for three patients with MCD type II. In other patients with MCD, three previously reported missense variations (c. 1A>T, c.738C>G, and c.631 C>T), three novel missense variations (c.164 T>C, c.526 G>A, c. 610 C>T), and two novel frameshift variations (c.894_895 insG and c. 462_463 delGC) were detected. These variations did not exist in the control chromosomes, 1000 Genomes, and dbSNP. CONCLUSIONS: This is the first molecular analysis of TGFBI and CHST6 in Turkish patients with different types of CD. We detected previously reported, well-known hot spot mutations in TGFBI in the patients with GCD1 and LCD1. Eight likely pathogenic variations in CHST6, five of them novel, were reported in patients with MCD, which enlarges the mutational spectrum of MCD.