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Comparison of Bioinformatics Prediction, Molecular Modeling, and Functional Analyses of FOXC1 Mutations in Patients
Morteza Seifi1, Tim Footz1, Sherry A M Taylor1
1Department of Medical Genetics, Faculty of Medicine & Dentistry, University of Alberta, Edmonton, Alberta, Canada.
Abstract:
Mutations in the forkhead box C1 gene (FOXC1) cause Axenfeld-Rieger syndrome (ARS). Here, we investigated the effect of four ARS missense variants on FOXC1 structure and function, and examined the predictive value of four in silico programs for all 31 FOXC1 missense variants identified to date. Molecular modeling of the FOXC1 forkhead domain predicts that c.402G> A (p.C135Y) alters FOXC1's structure. In contrast, c.378A> G (p.H128R) and c.481A> G (p.M161V) are not predicted to change FOXC1's structure. Functional analysis indicates that p.H128R reduced DNA binding, transactivation, nuclear localization, and has a longer protein half-life than normal. p.C135Y significantly disrupts FOXC1's DNA binding, transactivation, and nuclear localization. p.M161V reduces transactivation capacity without affecting other FOXC1 functions. C.1103C> A (p.T368N) is indistinguishable from wild-type FOXC1 in all tests, consistent with being a rare benign variant. Comparison of these four variants, plus 18 previously characterized FOXC1 missense variants, with predictions from four commonly used in silico bioinformatics programs indicated that sorting intolerant from tolerant (SIFT), polymorphism phenotyping (PolyPhen-2), and MutPred can sensitively identify as pathogenic only FOXC1 mutations with significant functional defects. This information was used to predict, as disease-causing, nine additional FOXC1 missense variations. Importantly, our results indicate SIFT, PolyPhen-2, and MutPred can reliably be used to predict missense variant pathogenicity for forkhead transcription factors.
Insights
Mutations in the forkhead box C1 (FOXC1) gene cause Axenfeld-Rieger syndrome (ARS). In silico tools like SIFT and PolyPhen-2 can reliably predict the pathogenicity of FOXC1 missense variants, aiding in diagnosing rare genetic disorders.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- Mutations in the forkhead box C1 (FOXC1) gene are the primary cause of Axenfeld-Rieger syndrome (ARS), a developmental disorder.
- Understanding the functional impact of FOXC1 missense variants is crucial for diagnosing ARS and related conditions.
Purpose of the Study:
- To investigate the structural and functional effects of four ARS-associated FOXC1 missense variants.
- To evaluate the predictive accuracy of in silico programs for classifying FOXC1 missense variants.
Main Methods:
- Molecular modeling was used to predict structural changes in the FOXC1 forkhead domain.
- Functional assays assessed DNA binding, transactivation, nuclear localization, and protein half-life.
- In silico programs (SIFT, PolyPhen-2, MutPred) were used to predict variant pathogenicity and compared with experimental data.
Main Results:
- The p.C135Y variant significantly disrupted FOXC1 structure, DNA binding, transactivation, and nuclear localization.
- The p.H128R variant impaired DNA binding, transactivation, and nuclear localization, but increased protein half-life.
- The p.M161V variant reduced transactivation, while p.T368N showed no significant functional impact.
- SIFT, PolyPhen-2, and MutPred accurately identified pathogenic variants with significant functional defects and predicted nine additional disease-causing variations.
Conclusions:
- FOXC1 missense variants have diverse impacts on protein structure and function, ranging from severe disruption to benign effects.
- In silico tools, particularly SIFT, PolyPhen-2, and MutPred, are reliable for predicting the pathogenicity of FOXC1 missense variants.
- These predictive tools can aid in the clinical assessment of patients with suspected ARS and other FOXC1-related disorders.
