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Dorota Muszynska, Judith Lechner, Durga P. Dash, Jonathan E. Moore, David A. Simpson, Colin E. Willoughby; Functional Analyses and Expression Profiling of Keratoconic Keratocytes Harbouring a Heterozygous Missense Mutation in the Zinc Finger E-box binding gene 1 (ZEB1). Invest. Ophthalmol. Vis. Sci. 2012;53(14):4220.
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ZEB transcription factors regulate epithelial-to-mesenchymal transition and other cellular functions (stemness, senescence, proliferation and apoptosis). Mutations in ZEB1 cause posterior polymorphous corneal dystrophy (PPCD3; MIM#609141), Fuch’s endothelial corneal dystrophy (FECD6; MIM#613270) and keratoconus. The purpose of this study was to investigate the functional consequences of a ZEB1 mutation (p.Gln640His) in cultured corneal keratocytes from a keratoconic patient.
A patient with a novel heterozygous missense mutation, c.1920G>T in ZEB1 resulting in a nonsynonymous substitution of a glycine with histidine (p.Gln640His), a highly conserved residue in the homeodomain of ZEB1, underwent deep anterior lamellar keratoplasty for clinically severe keratoconus. Human corneal keratocytes harbouring the ZEB1 mutation were cultured directly from corneal stromal tissue collected from the patient. The phenotype of the cells was assessed on the basis of morphology and positive vimentin and CD34 immunofluorescence staining. Cellular localisation and protein expression level of the mutant ZEB1 was determined by immunohistochemical and Western blot analyses respectively. Micro-array analysis (Illumina HumanHT-12) of RNA extracted from the patient’s cultured corneal keratocytes versus controls was employed to identify alterations in transcriptional regulation induced by the mutant ZEB1. Differentially expressed genes were validated using real time quantitative PCR (RT-qPCR).
Mutant ZEB1 (p.Gln640His) localised to the nucleus of cultured corneal keratocytes from the patient and controls. Western blot analysis revealed no difference in patient versus control ZEB1 protein expression level. Micro-array analysis with RT-qPCR confirmation identified a number of genes that were differentially expressed in cultured human keratocytes harbouring the ZEB1 mutation, including members of collagen IV group.
The functional consequences of the ZEB1 mutation (p.Gln640His) on potential ZEB1 transcriptional targets provides significant insight into the gene pathways involved in keratoconus pathogenesis.
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