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Y. Ito, F. Berry, T. Footz, T. Murphy, M. A. Walter; Molecular Characterization of a Novel FOXC1 Mutation Indicates That Altered FOXC1 Phosphorylation May Be Associated With Axenfeld-Rieger Malformations. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5597.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the Forkhead Box transcription factor gene, FOXC1, cause human Axenfeld-Rieger (AR), which is characterized by malformations to the anterior segment of the eye and an elevated risk to develop glaucoma. A novel L130F missense mutation was identified in two related individuals with AR syndrome. In this study a molecular analysis was carried out to determine the functional consequences of the FOXC1 L130F mutation.
The L130F mutation was introduced into the FOXC1 cDNA by site-directed mutagenesis. The levels of L130F protein expression and the mutant protein’s ability to localize to the nucleus, bind DNA, and transactivate a reporter gene were determined.
Immunoblotting indicated that the L130F mutant FOXC1 protein was expressed at levels similar to the wild type protein. FOXC1 is known to be a phosphorylated protein. Interestingly, altered migration of FOXC1 L130F immunoreactive bands as compared to the wild type FOXC1 revealed that the L130F protein is differently phosphorylated than the wild type protein. Further analyses revealed that the L130F FOXC1 mutant protein also had a reduced ability to localize to the nucleus, bind DNA, and transactivate a luciferase reporter gene.
Functional analyses of the L130F mutant FOXC1 protein reinforce the importance of helix 3 for normal FOXC1 activity and indicate that L130F is one of the most disruptive FOXC1 mutations studied to date. These analyses also suggest that altered phosphorylation of FOXC1 is a disease-causing mechanism underlying AR. Despite the severe disruptions to FOXC1 function, the two related individuals in whom the L130F mutation was identified had variable phenotypic consequences. Thus, stochastic events during development that may result in variable expression of downstream target genes of FOXC1, as well as environmental factors appear to contribute to the phenotypic variability in AR malformation.
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