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Morteza Seifi, Tim Footz, Michael A Walter; Characterization of FOXC1 mutations in patients with Axenfeld-Rieger syndrome. Invest. Ophthalmol. Vis. Sci. 2016;57(12):798. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations of the forkhead transcription factor gene FOXC1 underlie Axenfeld-Rieger syndrome (ARS), a disorder characterized by a wide range of the anterior chamber anomalies and increased prevalence of glaucoma. In this study, three FOXC1 missense mutations (T368N, H128R and C135Y) in patients with ARS were characterized.
Site-directed mutagenesis was performed to introduce the mutations into the FOXC1 complementary DNA. The effects of mutations on structure and function of the FOXC1 protein were determined by investigating the subcellular localization and expression levels of mutant proteins through immunofluorescence and immunoblotting, respectively. Electrophoretic mobility shift assay (EMSA) and luciferase reporter assay were employed to identify DNA binding and transactivational abilities of the FOXC1 mutant proteins.
Molecular analyses of the mutations indicated that, although T368N had the similar capability as wild-type to localize to the nucleus, H128R and C135Y showed a significant decrease (nearly 60% reduction). In addition, the results showed that whereas T368N and H128R had normal expression and protein half-life, C135Y exhibited low levels of expression and shortened protein half-life. The DNA binding affinity and the transactivation properties of T368N were similar to those of wild-type. Thus, interestingly, T368N mutation had no structural nor functional effect on the FOXC1 protein.
This study demonstrates that the missense mutations in FOXC1 (H128R and C135Y) cause ARS via different mechanisms including disruption of nuclear localization, gene expression and altered protein half-life. The T368N mutation, indistinguishable from wild type FOXC1 in our analyses, is likely a rare non-disease causing variant. Our study clearly demonstrates the necessity of functional testing to determine the pathogenicity of missense variants of FOXC1.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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