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L. F. Porter, B. Gibson, C. J. MacEwen, N. D. Ebenezer, W. H. I. McLean; The Highly Disruptive Mutation L132P in the Keratin 12 Gene is Associated With an Unusually Severe Presentation of Meesmann Epithelial Corneal Dystrophy. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1003.
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Meesmann epithelial corneal dystrophy (MECD; OMIM #122100) is caused by dominant-negative mutations in keratins K3 or K12. These intermediate filament proteins form the main stress bearing cytoskeleton of anterior corneal epithelial keratinocytes, disruption of which leads to corneal epithelial microcystic fragility. Here we aim to establish whether molecular genetic and biochemical analysis may explain the consistent and unusually severe presentation of MECD in three unrelated kindreds.
We performed direct mutation analysis of K3 and K12 genes and generated protein expression constructs, by cloning full-length RT-PCR products from human corneal keratinocyte cDNA into expression vector pCR3.1. Mutation L132P, identified in our families, and the most common European MECD mutation, R135T, which produces a subtle phenotype, were introduced into wild-type K12 clones by site-directed mutagenesis. Co-transfection studies of both wild-type K3 and K12 constructs, and wild-type K3 and both mutant versions of K12 were performed to study intermediate filament network formation. By measuring percentage of transfected cells showing filaments versus aggregates at 24 and 48 hours post-transfection, we obtained a measure of pathogenicity of particular mutations on keratin assembly.
The genetic defect in all three families was found to be a novel missense mutation L132P in the helix 1A domain of the K12 polypeptide. Protein expression studies confirmed that L132P is more disruptive to keratin assembly than mutation R135T. Six replicate experiments demonstrated that expression of either K12 mutants resulted in a much lower percentage of filamentous keratin than wild-type K12 (p<0.001). Interestingly, L312P produced a consistently lower percentage of filamentous keratin than R135T (p<0.05), suggesting that the L132P is a more strongly dominant-negative mutation than R135T.
We describe a novel missense mutation L132P in the helix 1A domain of the K12 polypeptide responsible for cases of MECD with phenotypes easily confused with more severe corneal dystrophies. We developed an assay system for assessing pathogenicity of K3 or K12 mutations. We identified a possible molecular basis for the more severe clinical presentation, with implications for the counselling and management of such individuals in the future.
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