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Invest Ophthalmol Vis Sci 2004;45: E-Abstract 694.
© 2004 ARVO


Analysis of PITX2 mutations reveals thresholds of PITX2 activity associated with anterior segment dysgenesis

F. Idrees1,2, K. Kozlowski1, S. Fraser3, P. Khaw4, J.C. Sowden2 and M.A. Walter1

1 Department of Ophthalmology and Medical Genetics, University of Alberta, Edmonton, AB, Canada
2 Developmental Biology Unit, Institute of Child Health, London, United Kingdom
3 Glaucoma Unit, Sunderland Eye Infirmary, Sunderland, United Kingdom
4 Glaucoma and Wound Healing Unit, Institute of Ophthalmology, London, United Kingdom

Commercial Relationships: F. Idrees, None; K. Kozlowski, None; S. Fraser, None; P. Khaw, None; J.C. Sowden, None; M.A. Walter, None.

Grant Identification: Support: RCOphth , Royal Society of Medicine, British Council, UCL, CIHR


Purpose: Investigate four mutant PITX2 proteins and determine their ability to bind DNA and activate gene expression. Refine the threshold of activity necessary for PITX2 to function normally.

Methods: Four PITX2 missense mutations underlying Axenfeld–Rieger malformations, two within and two outside the homeodomain, were studied. Underlined residues indicate positions from the ATG start codon in exon 1. The first mutant protein R5W (R43W) was found in a family with three generations affected by severe Axenfeld–Rieger Syndrome (Idrees et al. Invest Ophthalmol Vis Sci 2002;43: E–Abstract 3402). The other 3 mutations studied have been reported in the literature and their positions are R90C (R52C in the homeodomain) L105V and N108T. Site–directed mutagenesis was used to introduce these changes to the PITX2 cDNA. Epitope–tagged PITX2 mutant constructs were expressed in COS–7 cells, yielding the appropriate 35kDa product by Western analysis. The effect of each missense mutation on PITX2–DNA binding was tested by electrophoretic mobility shift assays (EMSAs) and the transactivational ability of the mutant protein was tested using a luciferase reporter assay. Immunofluorescence was used to investigate subcellular localization of recombinant Xpress–tagged PITX2. Computer models were employed to study the possible interaction of the mutant proteins with DNA.

Results: R5W has consistently failed to produce a stable protein. None of the 4 mutants inhibit the ability of PITX2 to translocate to the nucleus. L105V transactivates slightly higher than wild–type and binds to DNA. R52C and N108T transactivate at near wild type PITX2 levels and bind to DNA.

Conclusions: This is the first time an unstable PITX2 mutant protein has been reported. In addition this is the first study investigating the function of missense PITX2 mutations outside the homeobox. The experiments show that missense mutations in PITX2 are able to disrupt the interaction of PITX2 with DNA and the transactivation potential of PITX2. This study provides the second report of a disease causing allele due to a hyperactive PITX2 mutant. The in vivo control of PITX2 activity may be very tightly regulated with both an under or hyperactivity of a mutant protein resulting in disease.

Keywords: transcription factors • mutations • protein structure/function

 © 2004, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}