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.