Objective: To understand how the novel L130F mutation, found in 2 patients with Axenfeld-Rieger syndrome, disrupts function of the forkhead box C1 protein (FOXC1).
Methods: Sequencing DNA from patients with Axenfeld-Rieger syndrome identified a novel missense mutation that results in an L130F substitution in the FOXC1 gene. Site-directed mutagenesis was used to introduce the L130F mutation into the FOXC1 complementary DNA. The level of L130F protein expression was determined by means of immunoblotting. We determined the mutant protein's ability to localize to the nucleus, bind DNA, and transactivate a reporter construct.
Results: The FOXC1 L130F mutant protein is expressed at levels similar to those of wild-type FOXC1. The L130F protein, however, migrated at an apparent reduced molecular weight compared with the wild-type protein, suggesting that the mutant and wild-type proteins may be differentially phosphorylated. The L130F protein also had a significantly impaired capacity to localize to the nucleus, bind DNA, and transactivate reporter genes.
Conclusions: The disease-causing L130F mutation further demonstrates that helix 3 of the forkhead domain is important for the FOXC1 protein to properly localize to the nucleus, bind DNA, and activate gene expression.
Clinical relevance: The inability of FOXC1 to function owing to the L130F mutation provides further insight into how disruptions in the FOXC1 gene lead to human Axenfeld-Rieger syndrome.