Abstract
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 ssODN-mediated 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.
Highlights
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)[01]
Generation of mouse model of transforming growth factor-beta-induced (TGFBI)-R124C corneal dystrophy using single-stranded donor oligodeoxynucleotide (ssODN)-mediated basepair substitution introduced via CRISPR/Cas[9]
The mechanisms driving the formation of corneal opacity in TGFBI corneal dystrophy are largely unknown[5]
Summary
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)[01]. Yamazoe et al established an R124H mutant transgenic mouse model showing corneal opacities[8]. These mice harbour an artificial human TGFBI mutant gene cassette and do not reflect the complexity www.nature.com/scientificreports of the human disease. It is imperative to develop physiologically relevant animal models to decipher the mechanisms driving TGFBI corneal dystrophy. Single-nucleotide polymorphism (SNP)-based animal models of human disease are a useful and physiologically relevant approach that reflects point mutations or substitutions in the human genome. To understand the effect of TGFBI mutation in corneal dystrophy, we generated mutant mice with the Tgfbi R124C mutation, which is the representative mutation causing LCD1, using the CRISPR/Cas[9] approach. We examined how the expression of this phenotype affected corneal integrity and wound healing in this mouse model
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