A unique TGFBI protein in granular corneal dystrophy types 1 and 2.
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ABSTRACT:
SUBMITTER: Han YP
PROVIDER: S-EPMC5552038 | biostudies-other | 2012 Nov
REPOSITORIES: biostudies-other
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Project description:PurposeSpecific components of transforming growth factor-beta-induced protein (TGFBIp) responsible for amyloid deposits in lattice corneal dystrophy (LCD) have not been delineated. LCD has been associated with various TGFBIp mutations such as R124C, L518P, and L527R. Using recombinant TGFBIp, this study was undertaken to identify TGFBIp components potentially contributing to the protein deposits in LCD.MethodsRecombinant wild-type (WT) TGFBIp and four mutants (R124C, R124H, L518P, and L527R) were generated in HEK293FT cells. WT and mutant TGFBIp were collected from crude cell lysates or purified from culture media. Immunoblot analyses were performed with four different anti-TGFBIp antibodies raised against various regions of TGFBIp.ResultsConsistent with the authors' previous findings, purified recombinant proteins are more prone to polymerize than crude cell lysates. As expected, all monomers and polymers of TGFBIp WT and mutants were detected by these antibodies. However, the authors noted WT and TGFBIp mutants showed differential reactivities with these antibodies. A 47-kDa band was detected in purified 2-tag proteins of L518P by all four antibodies. A unique 43-kDa band was detected in both 1-tag cell lysates and purified proteins of R124C by the authors' custom-made antibody (KE50) and a commercial anti-TGFBIp.ConclusionsBased on its universal reactivity with various antibodies, the authors surmise that the 47-kDa protein is a ubiquitous TGFBIp fragment derived from the N-terminus of the L518P mutant. The fact that the 43-kDa protein fragment was present primarily in R124C and R124H but not in WT implicates its potential role in the protein deposits of LCD.
| S-EPMC3253538 | biostudies-literature
Project description:Protein aggregation in the outermost layers of the cornea, which can lead to cloudy vision and in severe cases blindness, is linked to mutations in the extracellular matrix protein transforming growth factor-β-induced protein (TGFBIp). Among the most frequent pathogenic mutations are R124H and R555W, both associated with granular corneal dystrophy (GCD) characterized by the early-onset formation of amorphous aggregates. The molecular mechanisms of protein aggregation in GCD are largely unknown. In this study, we determined the crystal structures of R124H, R555W, and the lattice corneal dystrophy-associated A546T. Although there were no changes in the monomeric TGFBIp structure of any mutant that would explain their propensity to aggregate, R124H and R555W demonstrated a new dimer interface in the crystal packing, which is not present in wildtype TGFBIp or A546T. This interface, as seen in both the R124H and R555W structures, involves residue 124 of the first TGFBIp molecule and 555 in the second. The interface is not permitted by the Arg124 and Arg555 residues of wildtype TGFBIp and may play a central role in the aggregation exhibited by R124H and R555W in vivo. Using cross-linking mass spectrometry and in-line size exclusion chromatography-small-angle X-ray scattering, we characterized a dimer formed by wildtype and mutant TGFBIps in solution. Dimerization in solution also involves interactions between the N- and C-terminal domains of two TGFBIp molecules but was not identical to the crystal packing dimerization. TGFBIp-targeted interventions that disrupt the R124H/R555W crystal packing dimer interface might offer new therapeutic opportunities to treat patients with GCD.
| S-EPMC8220419 | biostudies-literature
Project description:To describe 2 unrelated families with multiple members demonstrating a less commonly recognized vortex pattern of corneal deposits confirmed to be granular corneal dystrophy type 1 (GCD1) after identification of the p.(Arg555Trp) mutation in the transforming growth factor ?-induced gene (TGFBI).A slit-lamp examination was performed on individuals from 2 families, one of Mexican descent and a second of Italian descent. After DNA extraction from affected individuals and their unaffected relatives, TGFBI screening was performed.Eight of 20 individuals in the Mexican family and 20 of 55 in the Italian family demonstrated corneal stromal opacities. Seven of the 8 affected individuals in the Mexican family and 4 of the 20 affected individuals in the Italian family demonstrated a phenotype characterized by a "sea fan" or vortex pattern of superficial stromal corneal deposits originating from the inferior aspect of the cornea. Screening of TGFBI in both families revealed a heterozygous missense mutation [p.(Arg555Trp)] in exon 12, confirming the diagnosis of GCD1.Our findings demonstrate that GCD1 may present with a vortex pattern of anterior stromal deposits. Although this pattern of dystrophic deposits is not recognized by clinicians as a typical phenotype of GCD1, it is consistent with the production of the majority of the TGFBI protein by the corneal epithelium.
| S-EPMC5298206 | biostudies-literature
Project description:The progressive degeneration of granular corneal dystrophy type 2 (GCD2) corneal fibroblasts is associated with altered mitochondrial function, but the underlying mechanisms are incompletely understood. We investigated whether an imbalance of mitochondrial dynamics contributes to mitochondrial dysfunction of GCD2 corneal fibroblasts. Transmission electron microscopy revealed several small, structurally abnormal mitochondria with altered cristae morphology in GCD2 corneal fibroblasts. Confocal microscopy showed enhanced mitochondrial fission and fragmented mitochondrial tubular networks. Western blotting revealed higher levels of MFN1, MFN2, and pDRP1 and decreased levels of OPA1 and FIS1 in GCD2. OPA1 reduction by short hairpin RNA (shRNA) resulted in fragmented mitochondrial tubular networks and increased susceptibility to mitochondrial stress-induced apoptosis. A decrease in the mitochondrial biogenesis-related transcription factors NRF1 and PGC1α was observed, while there was an increase in the mitochondrial membrane proteins TOM20 and TIM23. Additionally, reduced levels of mitochondrial DNA (mtDNA) were exhibited in GCD2 corneal fibroblasts. These observations suggest that altered mitochondrial fission/fusion and biogenesis are the critical molecular mechanisms that cause mitochondrial dysfunction contributing to the degeneration of GCD2 corneal fibroblasts.
| S-EPMC10048436 | biostudies-literature
Project description:Both granular and lattice deposits are present in Avellino corneal dystrophy (ACD), primarily associated with the R124H mutation of transforming growth factor-?-induced (TGFBIp). We investigated the presence of these deposits in other TGFBI mutations and the use of Thioflavin-T (ThT), a fluorescent amyloid stain for characterizing corneal amyloid deposits.Surgical corneal specimens of 3 unrelated patients clinically diagnosed with ACD were studied. Corneal sections from normal individuals and patients with prior lattice corneal dystrophy (LCD) were used as controls. Histochemical studies were performed with Congo red and Masson trichrome stains, and fluorescent imaging with scanning laser confocal microscopy was performed for ThT and anti-TGFBIp antibody staining.Clinical and histopathological findings supported the diagnoses of ACD in these 3 cases in whom granular deposits stained with Masson trichrome and lattice deposits stained with ThT and Congo red showed birefringence and dichroism as expected. However, genotyping revealed a heterozygous R124C mutation in each case. In addition to classical stromal deposits, unique subepithelial TGFBIp aggregates, which stain with neither ThT nor trichrome, were observed. In control LCD sections, stromal deposits were stained with ThT but not with trichrome, confirming lack of granular deposits.Our results demonstrate that both granular and lattice corneal deposits can be associated with R124C mutation in addition to the more common R124H mutation. An additional feature of nonhyaline, nonamyloid, TGFBIp subepithelial deposits might substantiate the categorization of such cases as a variant form of ACD. This study further validates ThT staining for detection of amyloid TGFBIp deposits.
| S-EPMC2965268 | biostudies-literature
Project description:AimTo evaluate the feasibility of promoting genetic detection for granular corneal dystrophy type 2 (GCD2) by a questionnaire conducted among citizens in five cities in China.MethodsThe data were collected by questionnaire, and analyzed by Chi-square test and one-tailed t test in IBM SPSS statistics.ResultsBased on the survey data on the awareness of GCD2 genetic detection in this study and the positive predictive analysis report of the citizens in five cities in China, the vast majority (84.2%) of respondents had never heard of it and did not know that GCD2 patients have been prohibited from performing excimer surgery that can deteriorate GCD2 patients' condition even leading to blindness. Though 3.4% of patients understood GCD2 very much, they have no idea that GCD2 could not be 100% accuracy diagnosed by the conventional inspection methods.ConclusionIt is feasible and necessary to use GCD2 genetic detection as an excimer preoperative examination project. In order to promote the development of detection project, a few improvements should be carried out in terms of the promoting efforts, costs, and research progress.
| S-EPMC7708363 | biostudies-literature
Project description:PurposeTo report the clinical and molecular features of a distinct form of transforming growth factor-β-induced (TGFBI) gene-linked corneal dystrophy exhibiting a new granular corneal dystrophy type I (CDGG1) phenotype.MethodsA complete ophthalmologic examination was performed in all individuals of a Chinese family in which autosomal dominant transmission of the disease had been observed. DNA was obtained from the peripheral blood leukocytes of each participating subject. Genetic analyses included keratin 3 (KRT3), keratin 12 (KRT12), and TGFBI polymerase chain reaction (PCR) amplification and automated nucleotide sequencing of exons from the genomic DNA.ResultsThe corneal phenotype in this pedigree was characterized by multiple bilateral dot-like, circular opacities at different corneal depths, with some of the affected individuals only having opacities in the epithelium, which is different from the typical CDGG1 phenotype. TGFBI analysis revealed a heterozygous point mutation at exon 12 (c.1663C>T) in all of the affected individuals, predicting a p.R555W missense mutation.ConclusionsThe phenotype which resulted from the TGFBI R555W mutation in this family is distinct from that observed in the typical case of CDGG1. We propose this disorder should be classified as a new phenotype of CDGG1, and this finding demonstrates the importance of gene diagnosis in the corneal dystrophies.
| S-EPMC3025098 | biostudies-literature
Project description:PURPOSE: With advances in phenotyping tools and availability of molecular characterization, an increasing number of phenotypically and genotypically diverse inherited corneal dystrophies are described. We aimed to determine the underlying causative genetic mechanism in a three-generation pedigree affected with a unique anterior membrane corneal dystrophy characterized by early onset recurrent corneal erosions, small discrete focal opacities at the level of Bowman layer and anterior stroma, anterior stromal flecks, and prominent corneal nerves. METHODS: Twenty affected and unaffected members of a three-generation family were examined and extensively clinically characterized including corneal topography and in vivo confocal microscopy, and biological specimens were collected for DNA extraction. Mutational analysis of two corneal genes (TGFBI [Transforming Growth factor-beta induced] and ZEB1 [zinc finger E box-binding homeobox 1]) was undertaken, in addition to testing with the Asper Corneal Dystrophy gene chip (Asper Ophthalmics, Tartu, Estonia). Subsequent Genotyping To 11 Known Corneal Gene Loci (COL8A2 [Collagen, Type VIII, Alpha-2], TACSTD2 [Tumor-Associated Calcium Signal Transducer 2], PIP5K3 [Phosphatidylinositol-3-Phosphate 5-Kinase, Type III], GSN [Gelsolin], KERA [Keratocan], VSX1 [Visual System Homeobox Gene 1], COL6A1 [Collagen, Type VI, Alpha-1], MMP9 [Matrix Metalloproteinase 9], KRT3 [Keratin 3]), and two putative loci, 3p14-q13 and 15q22.33-24) was undertaken using polymorphic markers, and haplotypes constructed. Multipoint linkage analysis was performed to generate LOD scores and produce LOD plots across the candidate intervals. RESULTS: No pathogenic sequence variations were detected in TGFBI or ZEB1 of the proband nor on the Asper Corneal Dystrophy gene chip (302 mutations in 12 genes). Multipoint linkage analysis of 11 known corneal genes and loci generated negative LOD plots and was able to exclude all genes tested including PIP5K3. CONCLUSIONS: Exclusion of linkage to candidate corneal loci combined with an absence of pathogenic mutations in known corneal genes in this pedigree suggest a different genetic causative mechanism in this dystrophy than the previously documented corneal genes. This unique phenotype of an anterior membrane dystrophy may therefore provide an opportunity to identify a new gene responsible for corneal disease.
| S-EPMC2731832 | biostudies-literature
Project description:We investigated the clinical and genetic features of patients with severe phenotype of granular corneal dystrophy type 2 (GCD2) associated with compound heterozygosity in the transforming growth factor-β-induced (TGFBI) gene. Patients with severe GCD2 underwent ophthalmic examination (best-corrected visual acuity test, intraocular pressure measurement, slit-lamp examination, and slit-lamp photograph analysis) and direct Sanger sequencing of whole-TGFBI. The patient's family was tested to determine the pedigrees. Five novel mutations (p.(His174Asp), p.(Ile247Asn), p.(Tyr88Cys), p.(Arg257Pro), and p.(Tyr468*)) and two known mutations (p.(Asn544Ser) and p.(Arg179*)) in TGFBI were identified, along with p.(Arg124His), in the patients. Trans-phase of TGFBI second mutations was confirmed by pedigree analysis. Multiple, extensive discoid granular, and increased linear deposits were observed in the probands carrying p.(Arg124His) and other nonsense mutations. Some patients who had undergone phototherapeutic keratectomy experienced rapid recurrence (p.(Ile247Asn) and p.(Asn544Ser)); however, the cornea was well-maintained in a patient who underwent deep anterior lamellar keratoplasty (p.(Ile247Asn)). Thus, compound heterozygosity of TGFBI is associated with the phenotypic variability of TGFBI corneal dystrophies, suggesting that identifying TGFBI second mutations may be vital in patients with extraordinarily severe phenotypes. Our findings indicate the necessity for a more precise observation of genotype-phenotype correlation and additional care when treating TGFBI corneal dystrophies.
| S-EPMC7997986 | biostudies-literature
Project description:Granular corneal dystrophy type 2 (GCD2) is the most common form of transforming growth factor β-induced (TGFBI) gene-linked corneal dystrophy and is pathologically characterized by the corneal deposition of mutant-TGFBIp. The defective autophagic degradation of pathogenic mutant-TGFBIp has been shown in GCD2; however, its exact mechanisms are unknown. To address this, we investigated lysosomal functions using corneal fibroblasts. Levels of cathepsins K and L (CTSK and CTSL) were significantly decreased in GCD2 cells, but of cathepsins B and D (CTSB and CTSD) did not change. The maturation of the pro-enzymes to their active forms (CTSB, CTSK and CTSL) was inhibited in GCD2 cells. CTSL enzymes directly degraded both LC3 (autophagosomes marker) and mutant-TGFBIp. Exogenous CTSL expression dramatically reduced mutant-TGFBIp in GCD2 cells, but not TGFBIp in WT cells. An increased lysosomal pH and clustered lysosomal perinuclear position were found in GCD2 cells. Transcription factor EB (TFEB) levels were significantly reduced in GCD2 cells, compared to WT. Notably, exogenous TFEB expression improved mutant-TGFBIp clearance and lysosomal abnormalities in GCD2 cells. Taken together, lysosomal dysfunction in the corneal fibroblasts underlies the pathogenesis of GCD2, and TFEB has a therapeutic potential in the treatment of GCD2.
| S-EPMC7521267 | biostudies-literature