Identification of four new PITX2 gene mutations in patients withAxenfeld-Rieger syndrome

摘要

Purpose: Axenfeld Rieger syndrome (ARS) is an autosomal dominantinherited disorder affecting development of the ocular anterior chamber,abdomen, teeth and facial structures. The PITX2 gene is a major geneencoding a major transcription factor associated with ARS.Methods: ARS patients were collected from six unrelated families.Patients and their families were ophthalmologically phenotyped and theirblood was collected for DNA extraction. We screened the coding region ofhuman PITX2 gene by direct sequencing. The consequences of themutations described were investigated by generating crystallographicrepresentations of the amino acid changes. In order to better understandthe occurrence of glaucoma in ARS patients, we studied the PITX2gene expression in human embryonic and fetal ocular tissue sections.Results: We identified four novel PITX2 genetic alterations infour unrelated families with ARS. These mutations included two nonsensemutations (E55X and Y121X), an eight nucleotides insertion (1251 insCGACTCCT) and a substitution (F58L), in familial and sporadic cases ofARS. We also showed for the first time that PITX2 is expressed atearly stages of the human embryonic and fetal periocular mesenchyme, aswell as at later stages of human development in the fetal ciliary body,ciliary processes, irido corneal angle and corneal endothelium. Thehuman fetal eye PITX2 gene expression pattern reported here for thefirst time provides a strong basis for explaining the frequentoccurrence of glaucoma in patients affected by PITX2 gene mutations.Conclusions: Two mutations identified affect the homeodomain (E55Xand F58L). The E55X nonsense mutation is likely to alter dramaticallythe DNA-binding capabilities of the PITX2 homeodomain. Furthermore,there is a complete loss of the carboxy-terminal part of the PITX2protein beyond the site of the mutation. The phenylalanine F58 is knownto contribute to the hydrophobic network of the homeodomain. Thecrystallographic representations of the mutation F58L show that thismutation may change the conformation of the helical core. The F58Lmutation is very likely to modify the homeodomain conformation andprobably alters the DNA binding properties of PITX2. The other mutations(Y121X and the eight-nucleotide insertion (1251 ins CGA CTC CT) CGA CTCCT, at position 224 in PITX2A) result in partial loss of the C-terminaldomain of PITX2. Pitx2 synergistically transactivates the prolactinpromoter in the presence of the POU homeodomain protein Pit-1. Pitx2activity is regulated by its own C-terminal tail. This region contains ahighly conserved 14-amino-acid element involved in protein-proteininteractions. The C-terminal 39-amino-acid tail represses DNA bindingactivity and is required for Pitx2 interactions with other transcriptionfactors, for Pitx2-Pit-1 interaction and Pit-1synergism. Pit-1interaction with the Pitx2 C terminus masks the inhibitory effect andpromotes increased DNA binding activity. Thus, the partial or completeloss of the C terminus tail can lead to decreased or absent DNA bindingactivity and trigger severe ARS phenotypes. Our in situ hybridizationresults obtained on human embryonic and fetal ocular tissue sectionsconstitute the first molecular histological data providing anexplanation for the occurrence of precocious glaucoma in human patientsaffected by ARS caused by PITX2 mutations. Further structural andbiochemical studies are needed for understanding the wide spectrum ofclinical phenotypes caused by the increasing number of new PITX2mutations found in ARS affected patients.