Development of a Transgenic Mouse with R124H Human TGFBI Mutation Associated with Granular Corneal Dystrophy Type 2

Yamazoe, Katsuya; Yoshida, Shigeo; Yasuda, Motoaki; Hatou, Shin; Inagaki, Emi; Ogawa, Yasushi; Tsubota, Kazuo; Shimmura, Shigeto

doi:10.1371/journal.pone.0133397

Cited by 19 publications

(26 citation statements)

References 21 publications

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“…In Tgfbi R124C/R124C mice, more than 70% of eyes developed corneal opacity by 40 weeks of age ( Table 1). The frequency of corneal opacity in our mutant mice was nearly four times greater than that in previously established mouse models 8 . On the other hand, no corneal opacity was observed in Tgfbi R124C/WT mice up to 20 weeks of age, and the frequency of corneal opacity by 40 weeks was lower than that in Tgfbi R124C/R124C mice.…”

Section: Discussioncontrasting

confidence: 56%

“…Establishing clinically relevant mouse models of TGFBI corneal dystrophy would be very helpful to understand the mechanisms or pathophysiology underlying corneal opacity. Two animal models of TGFBI corneal dystrophy have been established via introduction of human mutant TGFBI genes into mice 6,8 . However, these models were generated by artificial induction of human genetic mutations, and the frequency of corneal opacity observed was insufficient to explain the patient phenotype.…”

Section: Discussionmentioning

confidence: 99%

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Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Kitamoto

Taketani

Fujii

et al. 2020

Sci Rep

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Mutations in transforming growth factor-beta-induced (TGFBI) gene cause clinically distinct types of corneal dystrophies. To delineate the mechanisms driving these dystrophies, we focused on the R124C mutation in TGFBI that causes lattice corneal dystrophy type1 (LCD1) and generated novel transgenic mice harbouring a single amino acid substitution of arginine 124 with cysteine in TGFBI via ssODNmediated base-pair substitution using CRISPR/Cas9 technology. Eighty percent of homozygous and 9.1% of heterozygous TGFBI-R124C mice developed a corneal opacity at 40 weeks of age. Hematoxylin and eosin and Masson trichrome staining showed eosinophilic deposits in subepithelial corneal stroma that stained negative for Congo-red. Although amyloid deposition was not observed in TGFBI-R124C mice, irregular amorphous deposits were clearly observed via transmission electron microscopy near the basement membrane. Interestingly, we found that the corneal deposition of TGFBI protein (TGFBIp) was significantly increased in homozygous TGFBI-R124C mice, suggesting a pathogenic role for the mutant protein accumulation. Furthermore, as observed in the LCD1 patients, corneal epithelial wound healing was significantly delayed in TGFBI-R124C mice. In conclusion, our novel mouse model of TGFBI-R124C corneal dystrophy reproduces features of the human disease. This mouse model will help delineate the pathogenic mechanisms of human corneal dystrophy. Corneal dystrophy is a hereditary disease that causes corneal opacity; 22 types of corneal dystrophy are currently classified according to the International Committee for Classification of Corneal Dystrophies (IC3D)0 1. Among these, transforming growth factor-beta-induced (TGFBI) corneal dystrophies occur most frequently in East Asian populations 2. TGFBI corneal dystrophies are caused by a point mutation, or insertion or deletion of some frames, in TGFBI, which is located on chromosome 5q31 1,2. Various mutations in TGFBI cause corneal opacities with different phenotypes, such as granular corneal dystrophy (GCD), lattice corneal dystrophy (LCD), Reis-Bücklers corneal dystrophy (RBCD), and Thiel-Behnke corneal dystrophy (TBCD) 3. There are genotype-phenotype correlations in TGFBI corneal dystrophy; for example, the R124H mutation causes GCD type2 (GCD2), and the R124C mutation causes LCD type1 (LCD1) 2. Even though we know that the corneal opacities are caused by accumulated TGFBI protein (TGFBIp) 4 , the mechanism or pathophysiology is not yet clearly understood 5. Animal models help elucidate the pathophysiology of corneal dystrophies. Previously, Bustamante et al. used a lentiviral vector to generate knock-in mice overexpressing the human R555W mutation, which causes GCD type1 (GCD1) 6. However, R555W mutant mice did not show any corneal phenotype according to this report 6. Tgfbi-knockout mice also failed to show corneal abnormalities even after systemic depletion of Tgfbi expression 7. Recently, Yamazoe et al. established an R124H mutant transgenic mouse model showing corneal opacities 8. Ho...

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Section: Discussioncontrasting

confidence: 56%

Section: Discussionmentioning

confidence: 99%

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Kitamoto

Taketani

Fujii

et al. 2020

Sci Rep

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“…Yamazoe et al () reported a transgenic mouse model of GCD2 caused by the p.Arg124His variant, showing granular with or without lattice deposits in the center of the cornea. Histology was similar to that of human‐affected corneas.…”

Section: Tgfbi Expression Role and Pathology In The Eye Of Humans Amentioning

confidence: 99%

“…Histology was similar to that of human‐affected corneas. As not all transgenic animals had corneal opacities, the authors concluded that epigenetic and/or environmental factors could influence the phenotype in mice and humans (Yamazoe et al, ). As mentioned above, our p.Arg124Leu knock‐in animal failed to show any corneal deposits, either at the heterozygous or homozygous state (data not shown).…”

Section: Tgfbi Expression Role and Pathology In The Eye Of Humans Amentioning

confidence: 99%

Mutation update:TGFBIpathogenic and likely pathogenic variants in corneal dystrophies

et al. 2019

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Human transforming growth factor β‐induced (TGFBI), is a gene responsible for various corneal dystrophies. TGFBI produces a protein called TGFBI, which is involved in cell adhesion and serves as a recognition sequence for integrins. An alteration in cell surface interactions could be the underlying cause for the progressive accumulation of extracellular deposits in different layers of the cornea with the resulting changes of refractive index and transparency. To this date, 69 different pathogenic or likely pathogenic variants in TGFBI have been identified in a heterozygous or homozygous state in various corneal dystrophies, including a novel variant reported here. All disease‐associated variants were inherited as autosomal‐dominant traits but one; this latter was inherited as an autosomal recessive trait. Most corneal dystrophy‐associated variants are located at amino acids Arg124 and Arg555. To keep the list of corneal dystrophy‐associated variant current, we generated a locus‐specific database for TGFBI (http://databases.lovd.nl/shared/variants/TGFBI) containing all pathogenic and likely pathogenic variants reported so far. Non‐disease‐associated variants are described in specific databases, like gnomAD and ExAC but are not listed here. This article presents the most recent up‐to‐date list of disease‐associated variants.

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“…There have been several previous attempts to generate a suitable transgenic animal model, either to knock-in or knock-out TGFBI gene, and to evaluate the pathologic role of the mutant protein [54][55][56] . All the generated animal models were not very successful to express the disease phenotype or in the survival of the animals.…”

Section: Discussionmentioning

confidence: 99%

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp

Venkatraman

Murugan

Lin

et al. 2020

Sci Rep

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Protein aggregation has been one of the leading triggers of various disease conditions, such asAlzheimer's, Parkinson's and other amyloidosis. TGFBI-associated corneal dystrophies are protein aggregation disorders in which the mutant TGFBIp aggregates and accumulates in the cornea, leading to a reduction in visual acuity and blindness in severe cases. Currently, the only therapy available is invasive and there is a known recurrence after surgery. In this study, we tested the inhibitory and amyloid dissociation properties of four osmolytes in an in-vitro TGFBI peptide aggregation model. The 23-amino acid long peptide (TGFBIp 611-633 with the mutation c.623 G>R) from the 4th FAS-1 domain of TGFBIp that rapidly forms amyloid fibrils was used in the study. Several biophysical methods like Thioflavin T (ThT) fluorescence, Circular Dichroism (CD), fluorescence microscopy and Transmission electron microscopy (TEM) were used to study the inhibitory and amyloid disaggregation properties of the four osmolytes (Betaine, Raffinose, Sarcosine, and Taurine). The osmolytes were effective in both inhibiting and disaggregating the amyloid fibrils derived from TGFBIp 611-633 c.623 G>R peptide. The osmolytes did not have an adverse toxic effect on cultured human corneal fibroblast cells and could potentially be a useful therapeutic strategy for patients with TGFBIp corneal dystrophies. Cytotoxicity of osmolytes on cultured human corneal fibroblast (HCF). Real-time live-cell imagingby IncuCyte. For real-time observation of the effect of the osmolytes on cultured human corneal fibroblast, 3000 cells/well were seeded in 96-well plates and placed in an incubator at 37 °C for 24 h to proliferate. Cells were then incubated with varying concentrations of the osmolytes (0.1 mM, 1 mM, 10 mM, 100 mM and 1000 mM) in triplicates and images were captured with IncuCyte ZOOM System (Essen BioScience Inc., Research Instruments, Singapore). Frames were then captured at 4-h intervals from 4 separate regions/well using a 10× objective for 20 h to observe cell toxicity. Scientific RepoRtS |(2020) 10:4011 | https://doi.

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Development of a Transgenic Mouse with R124H Human TGFBI Mutation Associated with Granular Corneal Dystrophy Type 2

Cited by 19 publications

References 21 publications

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Generation of mouse model of TGFBI-R124C corneal dystrophy using CRISPR/Cas9-mediated homology-directed repair

Mutation update:TGFBIpathogenic and likely pathogenic variants in corneal dystrophies

Effect of osmolytes on in-vitro aggregation properties of peptides derived from TGFBIp

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