Study of PITX3 mutations: spectrum, frequency and mechanisms.

Abstract

Abstract: : Purpose: PITX3 encodes homeodomain protein that plays important role in eye development. Mutations in PITX3 result in ocular phenotypes in humans and mouse. In order to better characterize role of PITX3 in human disorders, we screened patients with various ocular anomalies for mutations in PITX3. Molecular defects of identified mutations were analyzed by biochemical techniques to reveal mechanisms of action. Methods: DNA samples were screeened by direct nucleotide sequencing of PITX3–PCR fragments. For protein studies, wt and mutant PITX3 cDNAs were cloned into pcDNA vector to express protein with a C–terminus c–Myc tag. The TK–Bic–Luciferase reporter containing thymidine kinase minimal promoter with four bicoid elements was used to test for PITX3 transcriptional activity in human lens epithelial and corneal endothelial cells. Results: A total of 160 DNA samples were screened including anterior segment dysgenesis/cataract (48), Axenfeld–Rieger anomaly (60), and various ocular anomalies associated with other systemic defects (52). No new mutations in PITX3 have been identified. Therefore PITX3 mutations in our sample are represented by the previously reported C–terminal 17–bp insertion identified in three families with anterior segment dysgenesis/cataract (ASMD) and S14N missense mutation in N–terminal region found in a single family with cataract and glaucoma (ADCC). Biochemical studies demonstrated that PITX3 mutations impaired transcriptional activity. As compared to wild–type PITX3, transcriptional activities of ASMD and ADCC mutants were decreased to 71% and 80%, respectively. The artificial PITX3–K111E protein, in which the lysine at position 50 of the homeodomain was mutated, suppressed transcriptional activity of wtPITX3 in a dose–dependent manner. Studies of protein localization and binding activities are underway. Conclusions:PITX3 mutations were found in ∼8% of patients with isolated anterior segment/cataract anomaly, no mutations were found in other ocular phenotypes. There is a paucity of PITX3 mutations with no mutations found in the homeodomain region. At the same time, preliminary analysis of known PITX3 mutations demonstrated that mutant proteins suffered relatively mild alterations of their function. Therefore mutations in homeodomain are likely to result in more severe phenotypes that are yet to be discovered. The artificial K111E mutant demonstrated dominant negative effect and therefore can be used to abolish normal function of PITX3 in vitro.

Keywords: genetics • cataract • anterior segment