Isolation and Chromosomal Localization of a Cornea-Specific Human Keratin 12 Gene and Detection of Four Mutations in Meesmann Corneal Epithelial Dystrophy

Abstract

Keratin 12 (K12) is an intermediate-filament protein expressed specifically in corneal epithelium. Recently, we isolated K12 cDNA from a human corneal epithelial cDNA library and determined its full sequence. Herein, we present the exon-intron boundary structure and chromosomal localization of human K12. In addition, we report four K12 mutations in Meesmann corneal epithelial dystrophy (MCD), an autosomal dominant disorder characterized by intraepithelial microcysts and corneal epithelial fragility in which mutations in keratin 3 (K3) and K12 have recently been implicated. In the human K12 gene, we identified seven introns, defining eight individual exons that cover the coding sequence. Together the exons and introns span approximately 6 kb of genomic DNA. Using FISH, we found that the K12 gene mapped to 17q12, where a type I keratin cluster exists. In this study, four new K12 mutations (Arg135Gly, Arg135Ile, Tyr429Asp, and Leu140Arg) were identified in three unrelated MCD pedigrees and in one individual with MCD. All mutations were either in the highly conserved alpha-helix-initiation motif of rod domain 1A or in the alpha-helix-termination motif of rod domain 2B. These sites are essential for keratin filament assembly, suggesting that the mutations described above may be causative for MCD. Of particular interest, one of these mutations (Tyr429Asp), detected in both affected individuals in one of our pedigrees, is the first mutation to be identified within the alpha-helix-termination motif in type I keratin.

Conditional Deletion of the Mouse Klf4 Gene Results in Corneal Epithelial Fragility, Stromal Edema, and Loss of Conjunctival Goblet Cells

“…Mutations in cornea-specific keratin-3 or keratin-12 gene are associated with fragility of the corneal epithelium, a feature of Meesmann's corneal dystrophy observed in the Klf4CN mice ( Fig. 2 and 3) (33,35,49). We have shown that keratin-12 gene expression is downregulated in the Klf4CN corneas and that KLF4 binds and activates the keratin-12 gene promoter (Fig.…”

Characterization of Structural Changes in Vimentin Bearing an Epidermolysis Bullosa Simplex-like Mutation Using Site-directed Spin Labeling and Electron Paramagnetic Resonance

“…Mutation of this site has proven causative for human diseases in the Type I cytokeratins (e.g. skin blistering and corneal dystrophies) but also in the Type III IF protein GFAP as well (1,3,5,14,16,19,21,22,25,33). Thus this specific site appears to be critical to normal assembly in multiple IF proteins from more than one class, suggesting that the data derived from study of this mutation may be broadly 191 dimer forms, while the 348:348 dimer does not, as assessed by EPR using these residues as reporters for activity at these sites.…”

“…Commonly, a point mutation in the IF gene led to an Arg 3 His (12) or Arg 3 Cys mutation in this motif (12,21). Subsequently, mutations in the cornea-specific keratins K3 and K12 at the same LNDR sequence were shown to segregate with a corneal dystrophy phenotype (22)(23)(24). Most recently, the same region in GFAP has been shown to be the site of mutations leading to the neurodegenerative Alexander disease (25).…”

Severe Meesmann’s epithelial corneal dystrophy phenotype due to a missense mutation in the helix-initiation motif of keratin 12

“…16,25 K3 and K12 are important components of the intermediate filament cytoskeleton of the corneal epithelium, 21,22,26 and mutations within either the KRT12 or KRT3 genes cause MECD. 7,27 The KRT12 mutations that cause MECD are found in both the helix-initiation and helix-termination motif, while mutations of KRT3 are currently only described in the helix-termination motif (Figure 4). The most common mutation associated with MECD in the European population is a founder effect change in K12, Arg135Thr, that results in relatively mild disease, 28 but a more severe phenotype has been associated with mutations within both the helixinitiation 16,28,29 and helix-termination motif.…”