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The PITX2 transcription factor was identified as a causative gene for Axenfeld-Rieger syndrome (ARS; Mendelian Inheritance in Man no. 180500) by Semina et al.1 ARS is a rare autosomal dominant disorder that affects anterior eye development2 and can occur with or without systemic findings. The ocular findings include structural anomalies of the anterior chamber angle and aqueous drainage structures. Nonocular features typically include facial, dental, and umbilical defects. Our recent study3 showed that ARS is associated with an approximately 75% risk for glaucoma in patients with FOXC1 or PITX2 defects. Glaucoma, the second leading cause of blindness throughout the world,4–6 is the most important clinical consequence of ARS.7,8 Glaucoma is often associated with elevated intraocular pressure (IOP), as regulated by the ciliary body (the site of aqueous humor production) and the limbal region (which includes the trabecular meshwork), the principal site of aqueous humor outflow.4,5 The disease can continue to progress in patients even when IOP is significantly reduced. Research conducted in the past two decades has revealed that oxidative stress is involved in glaucoma pathogenesis.9–16 Identifying genes involved in adult anterior segment function or responses to oxidative stress will greatly improve our understanding of glaucoma pathology and will result in finding better treatment options. The PITX2 protein contains a conserved 60 amino acid homeodomain of the paired-bicoid class that is responsible for DNA binding, localization to the nucleus, and protein-protein interaction.17 Point mutations in PITX2 have been demonstrated to be loss-of-function mutations affecting DNA binding and transcriptional transactivation.1,18–27 Tight control of PITX2 expression is required for normal development of the eye because too much or too little activity of this transcription factor results in anterior segment defects and glaucoma.24,28 We hypothesize that altered regulation and expression of PITX2 target genes in adult tissues may have a significant impact on the development and progression of ARS-associated glaucoma and may reveal the cellular pathways affected in primary glaucoma phenotypes. We previously designed a hormone-inducible transcription factor expression system to discover target genes for FOXC129 and have now adapted this system to identify PITX2 target genes in ocular cells. We report here the identification and characterization of the Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) as a gene directly regulated by PITX2. Its protein product, NaDC3, is implicated as an important transporter of neuronal metabolites30 and may regulate the uptake of glutathione (GSH) to protect against ocular oxidative stress.31 We demonstrate for the first time that PITX2 and SLC13A3 are necessary for cellular responses to oxidative insult from hydrogen peroxide.

Axenfeld-Rieger syndrome (ARS) is a relatively rare developmental disorder characterized by maxillary hypoplasia, anterior segment defects of the eye (with glaucoma in ∼50%), and dental and umbilical abnormalities. The PITX2 homeodomain-containing transcription factor gene plays a major role in this condition, explaining approximately 40% of classic ARS.1–5 In addition, PITX2 mutations were shown to cause isolated ocular conditions,6–8 to be associated with additional brain anomalies,9,10 and possibly to contribute to other phenotypes such as omphalocele and VATER-like association.11 The function of Pitx2 is conserved in vertebrates. Total or conditional knockouts of Pitx2 result in severe developmental phenotypes in mice that include craniofacial, ocular, dental, brain, heart, lung, and other systemic defects consistent with the expression sites of Pitx2. With respect to Axenfeld-Rieger syndrome, Pitx2-deficient mice exhibit arrest of eye and tooth organogenesis, defective body wall closure and brain abnormalities.12–17 The defects observed in mice are associated with a complete loss of Pitx2 activity in the corresponding structures and, therefore, are noticeably more severe that what is reported in humans with heterozygous PITX2 mutations, whereas heterozygous mice are generally described as normal. This suggests a variable requirement of PITX2/Pitx2 for normal human/mouse embryogenesis, with human development more sensitive to correct PITX2 dosage. Human PITX2 mutations mainly result in a complete or partial loss of function, with mutations retaining some wild-type activity producing milder phenotypes.18–20 Some mutations identified in patients with Axenfeld-Rieger syndrome displayed very minor defects in DNA-binding and transactivation activities, again suggesting that normal human development is highly susceptible to alterations of PITX2 function.5 In addition, several human mutations associated with gain-of-function have been reported,21 consistent with the dramatic ocular and limb phenotypes seen in mice with Pitx2 overexpression during eye and forelimb development.22,23 Therefore, correct dosage of PITX2 is critical for normal development, which further underscores the importance of precise transcriptional regulation of its expression. Regulation of Pitx2 expression has been studied by several groups, but only a few cis-regulatory elements have been identified and examined in animal models. In mouse, Pitx2 appears to be a part of the Wnt/β-catenin/TCF/LEF and cAMP-CREB signaling pathways.24,25 Transcriptional activation of Pitx2 during eye development has been shown to be dependent on retinoic acid signaling26–28; the nature of this interaction, direct or indirect, has not yet been established. A few studies reported in vivo identification of Pitx2 enhancer elements, which facilitated upstream factor analysis. Asymmetric Pitx2 expression in the developing visceral organs was shown to require an intronic enhancer located in the proximity of the last exon,29,30 whereas a 7-kb fragment located 4.2 kb downstream of the last exon of Pitx2 was found to direct reporter expression in the oral ectoderm and Rathke's pouch.31 Finally, several regulatory elements associated with pitx2 expression in the stomodeum territory have been isolated in ascidians; the cis-sequences were either intronic or located within 2.5 kb of the 5′ or 3′ end of the gene.32 PITX2 is flanked by glutamyl aminopeptidase (aminopeptidase A) (ENPEP) located approximately 54 kb downstream and chromosome 4 open reading frame 32 (C4orf32) expressed sequence positioned approximately 1.5 Mb upstream of PITX2; thus, the 1.5-Mb sequence upstream of the gene is considered a gene desert. Gene deserts often contain conserved sequences involved in transcriptional regulation of nearby genes.33 The presence of PITX2 regulatory elements in this region were consistent with the previous reports of patients with Axenfeld-Rieger syndrome with translocation breakpoints that occurred within the distant upstream region and did not disrupt the coding region of PITX2.1,2,34,35 We describe identification of several novel regulatory sequences in the PITX2/pitx2 region, including several distant elements located within the gene desert upstream of PITX2. The activities of these elements appear to be consistent with PITX2/pitx2 expression, suggesting that they play a role in this gene's function. In addition, we report the identification of a de novo deletion located 106 to 108 kb upstream of PITX2 in a patient with Axenfeld-Rieger syndrome, thus providing additional support for the essential role of the identified regulatory elements in PITX2 function.

Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

BackgroundAxenfeld-Rieger syndrome (ARS) is associated with mutations in thePITX2gene that encodes a homeobox transcription factor. Several intronicPITX2mutations have been reported in Axenfeld-Rieger patients but their effects on gene expression have not been tested.MethodsWe present two new families with recurrentPITX2intronic mutations and usePITX2cminigenes and transfected cells to address the hypothesis that intronic mutations effect RNA splicing. ThreePITX2mutations have been analyzed: a G>T mutation within the AG 3' splice site (ss) junction associated with exon 4 (IVS4-1G>T), a G>C mutation at position +5 of the 5' (ss) of exon 4 (IVS4+5G>C), and a previously reported A>G substitution at position -11 of 3'ss of exon 5 (IVS5-11A>G).ResultsMutation IVS4+5G>C showed 71% retention of the intron between exons 4 and 5, and poorly expressed protein. Wild-type protein levels were proportionally expressed from correctly spliced mRNA. The G>T mutation within the exon 4 AG 3'ss junction shifted splicing exclusively to a new AG and resulted in a severely truncated, poorly expressed protein. Finally, the A>G substitution at position -11 of the 3'ss of exon 5 shifted splicing exclusively to a newly created upstream AG and resulted in generation of a protein with a truncated homeodomain.ConclusionThis is the first direct evidence to support aberrant RNA splicing as the mechanism underlying the disorder in some patients and suggests that the magnitude of the splicing defect may contribute to the variability of ARS phenotypes, in support of a gene dosage model of Axenfeld-Rieger syndrome.