Abstract
BACKGROUND
Mutations in the PITX2 homeobox gene are known to contribute to Axenfeld‐Rieger syndrome (ARS), an autosomal‐dominant developmental disorder. Although most mutations are in the homeodomain and result in a loss of function, there is a growing subset in the C‐terminal domain that has not yet been characterized. These mutations are of particular interest because the C‐terminus has both inhibitory and stimulatory activities.
METHODS
In this study we used a combination of in vitro DNA binding and transfection reporter assays to investigate the fundamental issue of whether C‐terminal mutations result in gain or loss of function at a cellular level.
RESULTS
We report a new frameshift mutation in the PITX2 allele that predicts a truncated protein lacking most of the C‐terminal domain (D122FS). This newly reported mutant and another ARS C‐terminal mutant (W133Stop) both have greater binding than wild‐type to the bicoid element. Of interest, the mutants yielded ∼5‐fold greater activation of the prolactin promoter in CHO cells, even though the truncated proteins were expressed at lower levels than the wild‐type protein. The truncated proteins also had greater than wild‐type activity in 2 other cell lines, including the LS8 oral epithelial line that expresses the endogenous Pitx2 gene.
CONCLUSIONS
The results indicate that the PITX2 C‐terminal domain has inhibitory activity and support the notion that ARS may also be caused by gain‐of‐function mutations. Birth Defects Research (Part A), 2006. © 2006 Wiley‐Liss, Inc.