April 2009
Volume 50, Issue 13
ARVO Annual Meeting Abstract  |   April 2009
Novel Mutations of the Transcription Factor FOXL2 and Functional Effects in BPES Patients
Author Affiliations & Notes
  • P. Sundaresan
    Genetics, Aravind Med Res Foundation, Madurai, India
  • J. Nallathambi
    Genetics, Aravind Med Res Foundation, Madurai, India
  • K. Usha
    Orbit Clinic, Aravind Eye Hospital, Madurai, India
  • S. Krishnaswamy
    Centre of Excellence in Bioinformatics, School of Biotechnology, Madurai Kamaraj University, Madurai, India
  • V. A. Reiner
    Genetics and Development, INSERM, Institut Cochin, 24 rue du Faubourg St-Jacques, Paris, France
  • Footnotes
    Commercial Relationships  P. Sundaresan, None; J. Nallathambi, None; K. Usha, None; S. Krishnaswamy, None; V.A. Reiner, None.
  • Footnotes
    Support  ICMR, New Delhi, India
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 2315. doi:
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      P. Sundaresan, J. Nallathambi, K. Usha, S. Krishnaswamy, V. A. Reiner; Novel Mutations of the Transcription Factor FOXL2 and Functional Effects in BPES Patients. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2315.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Mutational screening and functional analysis of the transcription factor FOXL2, involved in cranio-facial and ovarian development lead to the Blepharophimosis-Ptosis-Epicanthus Inversus Syndrome (BPES) in human.

Methods: : We analyzed nine patients affected with BPES, aged from 1 to 9 years old from unrelated families and diagnosed at the Aravind Eye Hospital, Madurai, India. After genomic DNA extraction from whole blood samples FOXL2 was PCR-amplified and directly sequenced the entire ORF using an ABI 3100 sequencer. The FOXL2 mutations encoding the p.Leu108Pro, p.Ser217Cys and p.Ala253fs mutant proteins were cloned into thepcDNA3.1/CT-GFP topoTA cloning vector to produce recombinant fusionproteins in frame with the Green Fluorescent Protein (GFP). To assess subcellular localization/aggregation, cells were transfected in three independent experiments, using the calcium phosphate method . The protein subcellular localization and aggregation were observed and scored using standard and confocal fluorescence microscopy. The transcriptional activity of three FOXL2 mutants (p.Leu108Pro, p.Ser217Cys and p.Ala253fs) was assessed using the Dual-Luciferase Reporter Assay System. In silico 3D modeling of FOXL2 FKH mutations were made.

Results: : We describe nine mutations in the open reading frame of FOXL2. Six of them are novel: c.292T>A (p.Trp98Arg), c.323T>C (p.Leu108Pro), c.650C>G (p.Ser217Cys) and three frameshifts). We have performed localization and functional studies for three of them. We have observed a strong cytoplasmic mislocalization induced by the missense mutation p.Leu108Pro located in the forkhead (FKH) domain of FOXL2. Interestingly, the novel mutation p.Ser217Cys, mapping between the FKH and the polyalanine domain of FOXL2 and producing a mild eyelid phenotype, led to normal localization and transactivation. We have also modeled the structure of the FKH domain to explore the potential structural impact of the mutations reported here and other previously reported ones. This analysis shows that mutants can be sorted into two classes: those that potentially alter protein-protein interactions and those that might disrupt the interactions with DNA.

Conclusions: : Our findings reveal new insights into the molecular effects of FOXL2 mutations, especially those affecting the FKH binding domain.

Keywords: genetics • gene/expression • mutations 

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