TGFBI Research Articles

Repeat Femtosecond-Assisted Anterior Lamellar Keratoplasty for Recurrence of TGF B I Dystrophy in Eyes After Previous FALK.

To report the management of recurrent TGF BI dystrophy after prior femtosecond-assisted anterior lamellar keratoplasty (FALK) with repeat FALK. Clinical and histopathological study of 2 eyes of 2 patients with a recurrence of TGFBI dystrophy. Patient 1 had Reis-Buckler corneal dystrophy, and patient 2 had granular corneal dystrophy GCD type 1. Patient 1 had FALK 8 years ago, when she was 23 years old. Patient 2 had FALK 7 years ago at the age of 24 years. Slit-lamp examination showed recurrence in the subepithelial layer of the anterior lamellar graft as confluent chalky white granular deposits. Anterior segment optical coherence tomography highlighted the deposits in the subepithelial region of the anterior lamellar graft. The anterior lamellar graft with deposits was removed and replaced with another graft created using femtolaser dissection of a healthy donor. The parameters for femtosecond laser-assisted donor dissection was similar to the size and depth as the previously used donor. The best-corrected visual acuity was restored to 20/30 in patient 1 and 20/25 in patient 2. The histology of the anterior lamellar graft showed eosinophilic deposits between the epithelium and the Bowman layer in both samples. In addition, the corneal sample from patient 2 revealed Bowman layer breach at some places and few deposits at 1 edge of the lamellar graft. Repeat FALK with a healthy donor is effective in the management of recurrence of deposits. The histology of the recurrence in the anterior lamellar graft revealed eosinophilic deposits predominantly between the epithelium and Bowman layer.

Mimicking TGFBI Hot-Spot Mutation Did Not Result in Any Deposit Formation in the Zebrafish Cornea

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.

MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL

You have accessJournal of UrologyCME1 Apr 2023MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL Jasper C. Bash, Lyndsey E. Shorey-Kendrick, Carol B. Hannah, Charles A. Easley, Eliot Spindel, Jamie O. Lo, and Jason C. Hedges Jasper C. BashJasper C. Bash More articles by this author , Lyndsey E. Shorey-KendrickLyndsey E. Shorey-Kendrick More articles by this author , Carol B. HannahCarol B. Hannah More articles by this author , Charles A. EasleyCharles A. Easley More articles by this author , Eliot SpindelEliot Spindel More articles by this author , Jamie O. LoJamie O. Lo More articles by this author , and Jason C. HedgesJason C. Hedges More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cannabis use has been associated with male infertility, but the etiology is not well understood. Seminal fluid is comprised of multiple factors (e.g. proteins), that influence fertility. Our objective was to determine the impact of chronic delta-9-tetrahydrocannabinol (THC) use on the seminal fluid proteome in a rhesus macaque (RM) model. METHODS: 6 adult RMs were given a daily THC edible over 280 days (∼4 spermatogenesis cycles) with serial non-sedated semen collection. Differential expression of proteins in the seminal fluid at baseline (pre-THC), high-THC dose (2.5 mg/7 kg/day), and post-THC discontinuation for 140 days (∼2 spermatogenesis cycles) was evaluated. Proteomic analysis was performed by liquid chromatography tandem mass spectrometry followed by statistical analysis to identify differentially expressed proteins. RESULTS: Proteomic analysis resulted in 1,395 quantifiable proteins (Figure 1A). The average percent identity was 88.6% (SD=14.9%). All RM proteins were matched to human proteins with 99.4% of the RM proteins (1,386) exceeding an identity cutoff of 44.3% (average minus 3 SDs). 7 proteins reached FDR significance in any contrast, thus we focused downstream analyses on nominally significant candidate proteins. Ingenuity Pathway Analysis (Figure 1B) revealed that high-THC exposure was associated with dysregulation of pathways related to “Remodeling of Epithelial Adherens Junctions”, “Axonal Guidance Signaling”, and “Glucocorticoid Receptor Signaling”. THC discontinuation for 140 days was associated with decreased “LXR/RXR Activation”, “Acute Phase Response Signaling”, and “Production of Nitric Oxide and ROS in Macrophages”. 5 proteins dysregulated with THC and significantly restored to baseline included products of CHIT1, FGG, MMP9, TGFBI, and CFB genes (Figure 1D). CHIT1 has been associated with oligozoospermia and MMP9 protein with sperm motility. CONCLUSIONS: This study demonstrated the translational strength of the RM model to study male reproductive health; there was significant seminal fluid proteome homology between humans and RMs. THC-exposed RMs are likely to exhibit proteins in their seminal ejaculate that are involved in regulatory processes that could potentially contribute to male infertility. Source of Funding: OHSU Faculty SEED Award and ASRM Pilot and Exploratory Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasper C. Bash More articles by this author Lyndsey E. Shorey-Kendrick More articles by this author Carol B. Hannah More articles by this author Charles A. Easley More articles by this author Eliot Spindel More articles by this author Jamie O. Lo More articles by this author Jason C. Hedges More articles by this author Expand All Advertisement PDF downloadLoading ...

Identification of the Potential Molecular Mechanism of TGFBI Gene in Persistent Atrial Fibrillation.

Transforming growth factor beta-induced protein (TGFBI, encoded by TGFBI gene), is an extracellular matrix protein, widely expressed in variety of tissues. It binds to collagens type I, II, and IV and plays important roles in the interactions of cell with cell, collagen, and matrix. It has been reported to be associated with myocardial fibrosis, and the latter is an important pathophysiologyical basis of atrial fibrillation (AF). However, the mechanism of TGFBI in AF remains unclear. We aimed to detect the potential mechanism of TGFBI in AF via bioinformatics analysis. The microarray dataset of GSE115574 was examined to detect the genes coexpressed with TGFBI from 14 left atrial tissue samples of AF patients. TGFBI coexpression genes were then screened using the R package. Using online analytical tools, we determined the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Ontology (GO) annotation, and protein-protein interaction (PPI) network of TGFBI and its coexpression genes. The modules and hub genes of the PPI-network were then identified. Another dataset, GSE79768 was examined to verify the hub genes. DrugBank was used to detect the potential target drugs. In GSE115574 dataset, a total of 1818 coexpression genes (769 positive and 1049 negative) were identified, enriched in 120 biological processes (BP), 38 cellular components (CC), 36 molecular functions (MF), and 39 KEGG pathways. A PPI-network with average 12.2-degree nodes was constructed. The genes clustered in the top module constructed from this network mainly play a role in PI3K-Akt signaling pathway, viral myocarditis, inflammatory bowel disease, and platelet activation. CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 were identified and finally verified as the hub genes, mainly enriched in pathways like leukocyte transendothelial migration, PI3K-Akt signaling pathway, viral myocarditis, rheumatoid arthritis, and platelet activation. Pegcetacoplan, ocriplasmin, and carvedilol were the potential target drugs. We used microdataset to identify the potential functions and mechanisms of the TGFBI and its coexpression genes in AF patients. Our findings suggest that CXCL12, C3, FN1, COL1A2, ACTB, VCAM1, and MMP2 may be the hub genes.

An Arg124Cys mutation in transforming growth factor β-induced gene associated with lattice corneal dystrophy type I in a Chinese pedigree.

Purpose:To identify a clinical and genetic form of a large Chinese family with an autosomal-dominant lattice corneal dystrophy type I (LCD I).Methods:The patients’ eyes were examined on the basis of slit-lamp microscopy, and other clinical records were also collected. Genomic DNA was extracted from peripheral leukocytes of the affected patients and their unaffected family members. Each previous reported mutation of the transforming growth factor β-induced gene (TGFBI) gene was amplified by touch-down polymerase chain reaction and directly sequenced to verify the disease-causing mutation.Results:Typical clinical features of LCD I were found by slit-lamp photography in these affected Chinese pedigrees. A heterozygous single base-pair transition from C to T (c.418 C > T), leading to amino acid substitution Arg124Cys (R124C) in the encoded TGFBI protein, was detected in all of the eighteen affected patients. The same mutation was not found in unaffected family members.Conclusion:The R124C mutation hot spot, which was relatively rare in China, was responsible for LCD I in the large family. Molecular genetic analysis of TGFBI gene can offer an accurate diagnosis of patients with lattice corneal dystrophies in the clinical treatment.

Targeted Expression of TGFBIp Peptides in Mouse and Human Tissue by MALDI-Mass Spectrometry Imaging

Stromal corneal dystrophies are a group of hereditary disorders caused by mutations in the TGFBI gene. The mutant TGFBIp is prone to protein aggregation and the mutant protein gets deposited in the cornea, leading to severe visual impairment. The mutations lead to a corneal specific protein aggregation suggesting the involvement of tissue-specific factors. The exact molecular mechanism of the process of tissue-specific protein aggregation remains to be elucidated. Differential proteolysis of mutant TGFBIp is a critical component of the disease pathology. The differential proteolysis gives rise to shorter peptides that are highly aggregation-prone and initiate the aggregation cascade. Analyzing the proteolytic processing of the different TGFBIp mutant may provide insight to aid in understanding the amyloid aggregation mechanism. We developed a MALDI-MSI methodology to identify expression and spatial localization of TGFBIp peptides in the cornea. Corneal tissue samples were collected from both control and dystrophic patients (with 2 different mutations), embedded in OCT and sectioned. The sections were trypsin digested and subjected to mass spectrometry imaging using a targeted approach to detect TGFBIp. MALDI-MSI identified peptides from TGFBIp that co-localized with the amyloid corneal deposits. In addition to the relative abundance data, the specific location of the peptides across the corneal sections as molecular signatures was also identified. Spatial distribution and intensity of the TGFBIp peptides showed differences between diseased and control models but also between the two LCD phenotypes. The TGFBIp peptide with m/z of 787.474 and m/z of 1179.579 showed increased expression in both LCD mutants compared to the controls. The peptide with m/z of 929.5 showed increased expression in the LCD phenotype with H626R mutation while the peptide with m/z of 1315.802 was abundant in the sample with R124C mutation. This initial report of 2D spatial protein signature and localization of TGFBIp may be expanded to other mutations to understand the proteolytic patterns of TGFBIp in different mutations.

Deterioration of Avellino corneal dystrophy in a Chinese family after LASIK.

To reveal the importance of TGFBI gene screening for candidates with a family history of corneal disease or granular opacities in corneal stroma before refractive surgery. A 37-year-old male (proband) underwent bilateral laser-assisted in situ keratomileusis (LASIK) in 2002, with right vision decreased significantly in 2006. The proband and other 32 members of the family underwent a detailed ophthalmic examination, including vision acuity, intraocular pressure, slit-lamp photograph, fundus examination, optical coherence tomography (OCT) of cornea, and in vivo confocal microscope (IVCM) and peripheral blood was used for genomic DNA extraction. Seventeen TGFBI gene exons were analyzed via polymerase chain reaction amplification and direct sequencing. Slit-lamp, IVCM, and OCT images showed that a large amount of dense and confluent granular opaque were seen at the interfaces of the flap and remnant stromal bed in right and light degree in left eye. Sanger sequencing showed that there was a 371G>A mutation (CGC>CAC) in exon 4, which indicated that he harbored a heterozygote R124H mutation, identifying the diagnosis of Avellino corneal dystrophy (ACD). Among the other 32 family members, 6 of them harbored the identical mutation to that in the proband. ACD will worsen and recur after LASIK. Preoperative gene-screening for TGFBI mutations is important in diagnosing ACD.

Genetic Screening Revealed Latent Keratoconus in Asymptomatic Individuals.

PurposeTo adopt molecular screening in asymptomatic individuals at high risk of developing keratoconus as a combinative approach to prevent subclinical patients from post-refractive surgery progressive corneal ectasia.MethodsIn this study, 79 Chinese and nine Greek families with keratoconus were recruited, including 91 patients with clinically diagnosed keratoconus as well as their asymptomatic but assumptive high-risk first-degree relatives based on underlying genetic factor. Mutational screening of VSX1, TGFBI, and ZEB1 genes and full clinical assessment including Pentacam Scheimpflug tomography were carried out in these individuals.ResultsFive variants in VSX1 and TGFBI genes were identified in three Chinese families and one Greek family, and four of them were novel ones. Surprisingly, ultra-early corneal changes in Belin/Ambrosio Enhanced Ectasia Display of Pentacam corneal topography together with co-segregated variants were revealed in the relatives who had no self-reported symptoms.ConclusionsVariants of VSX1 and TGFBI genes identified in both the clinically diagnosed and subclinical patients may cause the keratoconus through an autosomal dominant inheritance pattern, with different variable expressivity. Combining genetic with Belin/AmbrosioEnhanced Ectasia Display can be used to identify patients with latent keratoconus. This study indicates that genetic testing may play an important supplementary role in re-classifying the disease manifestation and evaluating the preoperative examination of refractive surgery.

Novel mutation in the TGFBI gene in a Moroccan family with atypical corneal dystrophy: a case report

BackgroundCorneal dystrophies (CDs) are a heterogeneous group of bilateral, genetically determined, noninflammatory bilateral corneal diseases that are usually limited to the cornea. CD is characterized by a large variability in the age of onset, evolution and visual impact and the accumulation of insoluble deposits at different depths in the cornea. Clinical symptoms revealed bilateral multiple superficial, epithelial, and stromal anterior granular opacities in different stages of severity among three patients of this family. A total of 99 genes are involved in CDs. The aim of this study was to identify pathogenic variants causing atypical corneal dystrophy in a large Moroccan family and to describe the clinical phenotype with severely different stages of evolution.Case presentationIn this study, we report a large Moroccan family with CD. Whole-exome sequencing (WES) was performed in the three affected members who shared a phenotype of corneal dystrophy in different stages of severity. Variant validation and familial segregation were performed by Sanger sequencing in affected sisters and mothers and in two unaffected brothers. Whole-exome sequencing showed a novel heterozygous mutation (c.1772C > A; p.Ser591Tyr) in the TGFBI gene. Clinical examinations demonstrated bilaterally multiple superficial, epithelial and stromal anterior granular opacities in different stages of severity among three patients in this family.ConclusionsThis report describes a novel mutation in the TGFBI gene found in three family members affected by different phenotypic aspects. This mutation is associated with Thiel-Behnke corneal dystrophy; therefore, it could be considered a novel phenotype genotype correlation, which will help in genetic counselling for this family.

Hsa_circRNA6448-14 promotes carcinogenesis in esophageal squamous cell carcinoma.

Circular RNAs (circRNAs) play important roles in cancer progression. hsa_circRNA6448-14 originates from exon 5 to exon 11 of the TGFBI gene. We investigated the roles of hsa_circRNA6448-14 in esophageal squamous cell carcinoma (ESCC) with microarrays and quantitative real-time polymerase chain reaction (qRT-PCR), Kaplan-Meier analysis, loss-of-function and gain-of-function assays, and pull-down assays for miRNA binding. The hsa_circRNA6448-14-miRNA-mRNA network was drawn using Circos. hsa_circRNA6448-14 was significantly upregulated in ESCC tissues and cell lines. As a diagnostic biomarker, hsa_circRNA6448-14 had an area under the curve (AUC), sensitivity, and specificity of 0.906, 82.9%, and 85.5%, respectively. hsa_circRNA6448-14 upregulation was correlated with poor differentiation, advanced pTNM stage, poor disease-free survival (DFS), and poor overall survival (OS). Elevated hsa_circRNA6448-14 promoted cell proliferation, migration, invasion, and inhibited apoptosis in vitro. hsa_circRNA6448-14 functioned as a miRNA sponge to competitively bind miR-455-3p, and hsa_circRNA6448-14 expression negatively correlated with that of miR-455-3p. hsa_circRNA6448-14 promoted carcinogenesis in ESCC, suggesting that hsa_circRNA6448-14 could serve as a diagnostic and prognostic biomarker for ESCC.

Pharmaceutical modulation of the proteolytic profile of Transforming Growth Factor Beta induced protein (TGFBIp) offers a new avenue for treatment of TGFBI-corneal dystrophy

Corneal dystrophies are a group of genetically inherited disorders with mutations in the TGFBI gene affecting the Bowman’s membrane and the corneal stroma. The mutant TGFBIp is highly aggregation-prone and is deposited in the cornea. Depending on the type of mutation the protein deposits may vary (amyloid, amorphous powdery aggregate or a mixed form of both), making the cornea opaque and thereby decreases visual acuity. The aggregation of the mutant protein is found to be specific with a unique aggregation mechanism distinct to the cornea. The proteolytic processing of the mutant protein is reported to be different compared to the WT protein. The proteolytic processing of mutant protein gives rise to highly amyloidogenic peptide fragments. The current treatment option, available for patients, is tissue replacement surgery that is associated with high recurrence rates. The clinical need for a simple treatment option for corneal dystrophy patients has become highly essential either to prevent the protein aggregation or to dissolve the preformed aggregates. Here, we report the screening of 2500 compounds from the Maybridge RO3 fragment library using weak affinity chromatography (WAC). The primary hits from WAC were validated by 15N-HSQC NMR assays and specific regions of binding were identified. The recombinant mutant proteins (4th FAS-1 domain of R555W and H572R) were subjected to limited proteolysis by trypsin together with the lead compounds identified by NMR assays. The lead compounds (MO07617, RJF00203 and, BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the R555W mutant and compounds (RJF00203 and BTB05094) were effective to delay/prevent the generation of amyloidogenic peptides in the H572R mutant. Thus the lead compounds reported here upon further validation and/or modification might be proposed as a potential treatment option to prevent/delay aggregation by inhibiting the formation of amyloidogenic peptides in TGFBI-corneal dystrophy.

Point mutation in the TGFBI gene: surface-enhanced infrared absorption spectroscopy (SEIRAS) as an analytical method

A novel method to detect DNA mutations based on gold nanoparticles is described. This bioconjugate was prepared by conjugation of the TGFBI gene on the surface of gold nanoparticles. The surface plasmon resonance band observed in the ultraviolet–visible spectrum of the gold nanoparticles showed a shift to a lower energy after the surface was covered with the TGFBI gene. Surface-enhanced infrared spectroscopy (SEIRAS) showed the absorption bands of the bioconjugate due to their proximity to the nanoparticles. The SEIRAS effect was able to detect small and specific changes in the sequence of the TGFBI gene using interactions of the bioconjugates. The study was performed on patients clinically diagnosed with lattice corneal dystrophy (LCD). To our knowledge, this is the first report of FTIR spectroscopy employed to analyse this gene. We used genotyping results previously obtained by next-generation DNA sequencing technology to ensure that the analysed samples had the mutation. Our results show highly reproducible signals, which could be used for analytical studies of mutations in the genome.

Epigenetic silencing of TGFBI confers resistance to trastuzumab in human breast cancer

BackgroundAcquired resistance to trastuzumab is a major clinical problem in the treatment of HER2-positive (HER2+) breast cancer patients. The selection of trastuzumab-resistant patients is a great challenge of precision oncology. The aim of this study was to identify novel epigenetic biomarkers associated to trastuzumab resistance in HER2+ BC patients.MethodsWe performed a genome-wide DNA methylation (450K array) and a transcriptomic analysis (RNA-Seq) comparing trastuzumab-sensitive (SK) and trastuzumab-resistant (SKTR) HER2+ human breast cancer cell models. The methylation and expression levels of candidate genes were validated by bisulfite pyrosequencing and qRT-PCR, respectively. Functional assays were conducted in the SK and SKTR models by gene silencing and overexpression. Methylation analysis in 24 HER2+ human BC samples with complete response or non-response to trastuzumab-based treatment was conducted by bisulfite pyrosequencing.ResultsEpigenomic and transcriptomic analysis revealed the consistent hypermethylation and downregulation of TGFBI, CXCL2, and SLC38A1 genes in association with trastuzumab resistance. The DNA methylation and expression levels of these genes were validated in both sensitive and resistant models analyzed. Of the genes, TGFBI presented the highest hypermethylation-associated silencing both at the transcriptional and protein level. Ectopic expression of TGFBI in the SKTR model suggest an increased sensitivity to trastuzumab treatment. In primary tumors, TGFBI hypermethylation was significantly associated with trastuzumab resistance in HER2+ breast cancer patients.ConclusionsOur results suggest for the first time an association between the epigenetic silencing of TGFBI by DNA methylation and trastuzumab resistance in HER2+ cell models. These results provide the basis for further clinical studies to validate the hypermethylation of TGFBI promoter as a biomarker of trastuzumab resistance in HER2+ breast cancer patients.

Paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS): clinical findings in twelve patients including recurrence after keratoplasty.

To describe the ocular findings of 12 subjects with paraproteinemic keratopathy associated with monoclonal gammopathy of undetermined significance (MGUS). Ocular examination included corneal spectral domain optical coherence tomography. In three individuals with an initial diagnosis of a lattice or Thiel-Behnke corneal dystrophy, the TGFBI gene was screened by conventional Sanger sequencing. We confirmed a diagnosis of MGUS by systemic examination and serum protein electrophoresis in 12 individuals (9 males and 3 females), with a mean age at presentation of 52.2years (range 24-63years) and mean follow-up 6.4years (range 0-17years). The best-corrected visual acuity (BCVA) at presentation ranged from 1.25 to 0.32. In all individuals, the corneal opacities were bilateral. The appearances were diverse and included superficial reticular opacities and nummular lesions, diffuse posterior stromal opacity, stromal lattice lines, superficial and stromal crystalline deposits, superficial haze and a superficial ring of hypertrophic tissue. In one individual, with opacities first recorded at 24years of age, we documented the progression of corneal disease over the subsequent 17years. In another individual, despite systemic treatment for MGUS, recurrence of deposits was noted following bilateral penetrating keratoplasties. The three individuals initially diagnosed with inherited corneal dystrophy were negative for TGFBI mutations by direct sequencing. A diagnosis of MGUS should be considered in patients with bilateral corneal opacities. The appearance can mimic corneal dystrophies or cystinosis. In our experience, systemic treatment of MGUS did not prevent recurrence of paraproteinemic keratopathy following keratoplasty.

Generation of TGFBI knockout ABCG2+/ABCB5+ double-positive limbal epithelial stem cells by CRISPR/Cas9-mediated genome editing.

Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced (TGFBI) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI-targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.

Phenotype‐genotype correlation of p.R124S mutation in granular type 1 corneal dystrophy of Tunisian origin

Purpose: We report the clinical features and phenotype‐genotype correlation in a large Tunisian family with granular corneal dystrophy type I. Methods: Twenty‐seven members of the Tunisian family underwent a complete ophthalmologic examination. A histopathological examination was performed on corneal specimens of four patients with advanced stage of corneal dystrophy. DNA extraction and direct Sanger sequencing of the exons 4 and 12 of TGFBI gene was performed for forty‐two members. Results: The diagnosis of granular corneal dystrophy type I was clinically and genetically confirmed. Sequencing of exon 4 of TGFBI gene revealed the p.Arg124Ser mutation in heterozygous and in homozygous status in patients with different clinical severity. Visual acuity was severely affected in the homozygous patients leading to a first penetrating keratoplasty at 20 years of age in 3 cases, and at 8 years in one case. Recurrence occurred rapidly, began in the seat the corneal stitches and remained superficial up to 40 years after the graft. In heterozygous cases, visual acuity ranged from 6/10 to 10/10. Corneal opacities were deeper and predominating in the stromal center. Conclusions: Our study describes for the first time phenotype‐genotype correlation in a large Tunisian family with granular corneal dystrophy type I and illustrates clinical presentation of homozygous p.Arg124Ser mutation. These results help to understand pathophysiology of the disease and profile of relapse after keratoplasty.

Analysis of TGFBI gene mutation in a pedigree affected with corneal dystrophy

To detect potential mutation in a large Chinese pedigree affected with congenital corneal dystrophy. Two patients from the pedigree were subjected to whole exome sequencing to determine the candidate gene. Suspected mutation was verified in 13 additional members by directional Sanger sequencing. Ccorrelation between genotype and phenotype was explored. A missense mutation, c.1877A>C (p.His626Pro), was detected in exon 14 of the TGFBI gene in 8 patients from the pedigree, but not in five unaffected members and 100 unrelated healthy controls. Respectively, the mutation was predicted as "affecting protein function", "probably damaging" and "disease causing" by SIFT, PolyPhen-2 and MutationTaster. The c.1877A>C mutation of the TGFBI gene probably underlies the disease in this pedigree.

Abstract A031: Androgen deprivation therapy-induced TGFBI signaling promotes EMT and bone metastasis of prostate cancer

Abstract Metastatic progression in patients with prostate cancer is common despite pharmacologic inhibition of androgen receptor signaling. This drug resistance is associated with increased signaling through the transforming growth factor-β (TGF-β) signaling pathway. We found the type I/II/IV collagen-binding protein transforming growth factor-β (TGF-β)-induced (TGFBI) is an important factor in the epithelial-to-mesenchymal transition (EMT) and malignant progression of prostate cancer. We identified that an androgen-responsive gene, SPDEF, navigates between AR and TGFβ signaling during prostate cancer progression to metastasis though a physical interaction with the promoter region of the TGFBI gene. We show that loss of AR-regulated SPDEF induces TGFBI expression in prostate cancer cells, leading to drastic and aggressive physiologic changes. Furthermore, we confirmed the negative correlation between SPDEF and TGFβ singling in prostate cancer patients and demonstrated that ADT can reduce SPDEF and increase TGFβ signaling. Our results support an inhibitory role of the AR on TGFβ signaling through SPDEF-mediated suppression of TGFBI, and that disruption of this pathway promotes metastatic progression in prostate cancer. Citation Format: Yen-Nien Liu, Wei-Yu Chen, Yuan-Chin Tsai, Hsiu-Lien Yeh, Florent Suau, Jiaoti Huang. Androgen deprivation therapy-induced TGFBI signaling promotes EMT and bone metastasis of prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A031.

Analysis of TGFBI gene mutation in a Chinese pedigree affected with lattice corneal dystrophy

To explore the clinical features and mutation of TGFBI gene in a Chinese pedigree affected with lattice corneal dystrophy (LCD). Genomic DNA was extracted from 35 members including 11 patients from the pedigree. The 17 exons and splicing region of introns of the TGFBI gene were amplified by PCR. The products were directly sequenced and compared with GenBank database to identify potential mutation. Bioinformatic analysis was carried out to predict the effect of mutation on proteins. A heterozygous mutation (p.R124C) was found in exon 4 of the TGFBI gene in all patients from the pedigree but not among unaffected members. The mode of inheritance of corneal dystrophy in this pedigree was identified as autosomal dominant. Bioinformatics analysis predicted that the p.R124C mutation may be functionally deleterious. The phenotype of corneal dystrophy in the pedigree was determined to be LCD I type. The p.R124C mutation of the TGFBI gene probably underlies the pathogenesis of LCD in this Chinese pedigree. Genetic testing can facilitate proper diagnosis of this type of corneal dystrophy.

Clinical observation and gene mutation detection in a pedigree of retinal lattice degeneration combined with granular corneal dystrophy

Objective To investigate the clinical manifestations and gene mutation of a pedigree with retinal lattice degeneration and granular corneal dystrophy (GCD) type 2. Methods Ten members in 3 generations of a pedigree with retinal lattice degeneration and GCD2 were included in the study, including 6 patients (3 males and 3 females) and 4 healthy family members. All members underwent visual acuity, slit lamp microscope, three-mirror lens, fundus color photography, optical coherence tomography, and corneal endothelial cells counting. Genomic DNA was extracted from peripheral venous blood (2 ml) from all the subjects and their spouses, who had no related inherited diseases. The next generation sequencing method was used to detect the mutation sites of transforming growth factor β (TGFBI), and all results underwent Sanger verification. Results Among the 12 eyes of 6 patients, the visual acuity was FC/20 cm-1.0. In the superficial central corneal stroma, snowflake-like deposits were observed in three cases (6 eyes), and a small amount of granular deposits were observed in three cases (6 eyes). Corneal endothelial cell counts were normal. Retinal lattice degeneration were observed in 3 cases, 6 eyes (including 3 cases of rhegmatogenous retinal detachment in 4 eyes); retinal thinning without obvious lattice degeneration in 4 eyes of 2 patients. Nystagmus in 1 patient and fundus examination showed no significant abnormalities. DNA sequencing results showed that the proband and 4 patients had missense mutation of TGFBI gene in exon 4 c.371G> A, the mutation site corresponding to the amino acid change encoded by TGFBI gene No. 124 Amino acids, from arginine to histidine (p.R124H). Patients with this mutation have varying degrees of clinical phenotype. Conclusions The mutation of c.701G> A (p.R124H) in TGFBI gene is the causative gene of GCD in this pedigree. The patients with this mutation have different clinical phenotypes. Key words: Corneal dystrophies, hereditary; Retinal degeneration; Mutation; Gene, TGFBI