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Identification and analysis of a novel mutation in the FOXC1 forkhead domain
Ramsey A Saleem1, Tara C Murphy, Jeffery M Liebmann
1Department of Medical Genetics, University of Alberta, Edmonton, Alberta, Canada.
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.
Insights
A novel mutation in the FOXC1 gene (L86F) causes Axenfeld-Rieger malformations by impairing DNA binding and gene activation. This study highlights the critical role of helix 1 in FOXC1 function and suggests a weak genotype-phenotype correlation.
Area of Science:
- Genetics
- Molecular Biology
- Developmental Biology
Background:
- Axenfeld-Rieger malformations are congenital developmental disorders.
- These malformations are associated with mutations in the FOXC1 gene, a key regulator of ocular development.
Purpose of the Study:
- To identify the genetic and biochemical basis of Axenfeld-Rieger malformations.
- To pinpoint the specific mutation in FOXC1 and elucidate its impact on protein function.
Main Methods:
- Sequencing of the FOXC1 gene in affected individuals and family members.
- Site-directed mutagenesis to create and study the identified FOXC1 mutation.
- Biochemical assays to assess nuclear localization, DNA binding, and transactivation activity of the mutant FOXC1 protein.
Main Results:
- A novel missense mutation, L86F, was identified in the FOXC1 gene of a patient with Axenfeld-Rieger malformations.
- The L86F mutation impairs FOXC1's DNA binding and transactivation capabilities while maintaining nuclear localization.
- The severity of the observed phenotype did not directly correlate with the degree of functional impairment caused by the mutation.
Conclusions:
- A new mutation in the FOXC1 forkhead domain (L86F) is linked to Axenfeld-Rieger malformations.
- The study underscores the importance of helix 1 within the FOXC1 protein for its function.
- Evidence suggests a limited correlation between genotype and phenotype severity in FOXC1-related disorders, with normal development requiring precise FOXC1 activity levels.
