Molecular mechanisms of Axenfeld-Rieger syndrome

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DESCRIPTION (provided by applicant): This application represents a continuation of our studies of a role and function of a to/co/cf-related transcription factor PITX2 during human development, particularly in respect to the formation of ocular structures. Mutations in the PITX2 gene cause Axenfeld-Rieger syndrome (ARS) associated with glaucoma in humans and arrest in eye development in Pitx2-/- knockout mice. In our previous studies we characterized Pitx2 transcripts and genomic sequences in several species. Human PITX2 sequences were screened for mutations in patients with Axenfeld-Rieger syndrome and other similar conditions and multiple mutations have been identified. We also initiated generation of a knock-in mouse carrying dominant-negative mutation in Pitx2 gene identical to the mutation found in a patient with a severe form of ARS. These studies form basis for our further investigation of PITX2 role(s) in development that are specifically aimed at: 1) To elucidate molecular mechanisms underlying Pitx2 regulation during ocular development. Under this aim, regions involved in regulation of Pitx2 and factors interacting with these sequences will be identified; 2) To explore pathological effects of PITX2 inactivation by characterization of mutant mice and human cell lines carrying K88E dominant-negative mutation in the PITX2 gene. Creation of ARS animal model associated with Pitx2 mutations will facilitate studies of mechanisms of Pitx2's action in development, identification of modifiers and evaluation of therapeutic agents. Because of the fact that Pitx2 knockout mice generally do not demonstrate Axenfeld-Rieger features while homozygous -/- animals die before birth due to multiple defects, comprehensive studies of Axenfeld-Rieger anomalies and glaucoma are not possible in these animals. We anticipate that Pitx2(K88E) mice carrying dominant-negative mutation in Pitx2 gene will result in a better model to study A-R syndrome. Identification of downstream targets of PITX2 will be performed by analysis of the differential gene expression in cells/ tissues expressing wild type and mutant forms of PITX2 and microarrays. Mutation analysis in human patients will be performed to identify factors playing the most critical role in the human eye. We contend that these studies will advance our knowledge of the mechanisms of normal eye development and understanding of glaucoma at the molecular level.