PITX2, located on chromosome 4 at q25, was cloned in 1996.
1 It contains a homeobox, a 60-amino-acid domain originally described in fruit fly homeotic or embryonic patterning genes, that is responsible for binding to DNA, localizing PITX2 to the cell nucleus, and for protein-protein interactions. PITX2 is expressed in the developing eye, jaw, and umbilicus, as well as the heart and limb buds.
PITX2 mutations have been found in patients with a range of anterior segment dysgenesis phenotypes, including IH, IGD, or AR malformations.
1 2 3 4 5 Members of my laboratory have assayed the affect on PITX2 function of different
PITX2 mutations found in patients.
5 6 We found that
PITX2 mutations can alter PITX2 nuclear localization, DNA binding, and transactivation and that the differences in amounts of functional PITX2 protein may be the basis of the spectrum of IH, IGD, and AR malformation phenotypes. DNA-binding ability, reporter gene transactivation, and nuclear localization of recombinant PITX2 proteins containing missense mutations of the PITX2 homeodomain identified in patients with IH, IGD, and AR malformations were assayed.
5 6 Missense mutations of the PITX2 homeodomain identified in patients with the IH (R46W), IGD (R31H), or AR malformation (L16G; T30P; R53P) were introduced into recombinant
PITX2 cDNA by site-directed mutagenesis. PITX2 mutant proteins expressed in COS-7 cells were determined to be stable and to localize to the nucleus; however, the R53P AR mutant also displayed cytoplasmic staining. Analysis of the five mutant PITX2 proteins by electrophoretic mobility shift and transactivation assays demonstrated reduced activity of the IH and IGDS mutant PITX2 proteins, with the IH mutant retaining the most activity in both studies, although the mutant PITX2 proteins from patients with AR malformations were shown to have no DNA-binding or transactivation abilities. Thus, the wide range of phenotypic consequences due to mutations of
PITX2 appears to correlate with the amount of residual PITX2 activity (
Fig. 1 , top).