Analyses of a novel L130F missense mutation in FOXC1.

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.

The wing 2 region of the FOXC1 forkhead domain is necessary for normal DNA-binding and transactivation functions.

PURPOSE: To determine the biochemical defects that underlie Axenfeld-Rieger malformations, to determine a functional role for wing 2 in FOXC1, and to understand how mutations in this region disrupt FOXC1 function. METHODS: Sequencing DNA from patients with Axenfeld-Rieger malformation resulted in the identification of two novel missense mutations (G165R and R169P) in wing 2 of FOXC1. Site-directed mutagenesis was used to introduce these mutations, as well as previously reported mutation (M161K), into the FOXC1 cDNA. These FOXC1 mutants were evaluated to determine their ability to localize to the nucleus, bind DNA and activate gene expression. RESULTS: Two novel missense mutations were identified in unrelated patients, in wing 2 of the FOXC1 forkhead domain. Because there had been no previous biochemical analysis, the mutation M161K was also investigated. All three mutant proteins localized correctly to the nucleus. The G165R mutation maintained wild-type levels of DNA binding; however, both the M161K and R169P mutations displayed reduced DNA binding ability. Biochemical analysis showed that all three mutations disrupt FOXC1's transactivation ability. CONCLUSIONS: Biochemical analysis of mutations G165R and R169P and of a previously reported mutation, M161K, demonstrate the functional significance of wing 2. M161K and R169P disrupt DNA binding of FOXC1, consistent with the hypothesis that wing 2 is necessary for DNA binding. The results also suggest that wing 2 plays a role in gene activation. These results provide the first insights into how mutations in wing 2 disrupt FOXC1 function.

Identification and analysis of a novel mutation in the FOXC1 forkhead domain.

PURPOSE: To determine the genetic and biochemical defects that underlie Axenfeld-Rieger malformations, identify the pathogenic mutation causing these malformations, and understand how these mutations alter protein function. METHODS: FOXC1 was amplified from a proband with Axenfeld-Rieger malformations and the proband's mother. PCR products were sequenced to identify the pathogenic mutation. Site-directed mutagenesis was used to introduce this mutation into the FOXC1 cDNA. A synthetic mutation at the same position was also introduced, and both natural and synthetic proteins were tested for their ability to localize to the nucleus, bind DNA, and transactivate gene expression. RESULTS: A novel missense mutation (L86F) was identified in FOXC1 in this family. The mutation is located in alpha-helix 1 of the forkhead domain. Biochemical assays showed that the L86F mutation does not affect nuclear localization of FOXC1, but reduces DNA binding and significantly reduces transactivation. The severity of the disruption to FOXC1 protein activity does not appear to correspond well with the severity of the phenotype in the patient. Analogous studies using a L86P, a known alpha-helix breaker, severely disrupts FOXC1 function, revealing the importance of helix 1 in FOXC1 structure and function. CONCLUSIONS: A novel mutation in helix 1 of the FOXC1 forkhead domain has been identified and the importance of position 86 in FOXC1 activity demonstrated. These studies also identified the role of helix 1 in FOXC1 function and provide further evidence for the lack of strong genotype-phenotype correlation in FOXC1 pathogenesis. Normal development appears to be dependent on tight upper and lower thresholds of FOXC1 activity.