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Keywords:

  • Axenfeld-Rieger;
  • PITX2;
  • FOXC1;
  • anterior segment dysgenesis;
  • digenic inheritance

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Disease-causing mutations affecting either one of the transcription factor genes, PITX2 or FOXC1, have been previously identified in patients with Axenfeld-Rieger syndrome (AR). We identified a family who segregate novel mutations in both PITX2 (p.Ser233Leu) and FOXC1 (c.609delC). The most severely affected individual, who presented with an atypical phenotype of corneal opacification, lens extrusion, persistent hyperplastic primary vitreous (PHPV), and subsequent bilateral retinal detachment, inherited mutations in both genes, whereas the single heterozygous mutations caused mild AR phenotypes. This is the first report of such digenic inheritance. By analyzing cognate targets of each gene, we showed that FOXC1 and PITX2 can independently regulate their own and each other's target gene promoters and do not show synergistic action in vitro. Mutation in either gene caused reduced transcriptional activation to different extents on the FOXO1 and PLOD1 promoters, whereas both mutations in combination showed the lowest level of activation. These data show how the compensatory activity of one factor, when the other is impaired, may lessen the phenotypic impact of developmental anomalies, yet reduced activity of both transcription factors increased disease severity. This suggests an under-reported mechanism for phenotypic variability whereby single mutations cause mild AR phenotypes, whereas digenic inheritance increases phenotypic severity. Hum Mutat 32:1144–1152, 2011. ©2011 Wiley-Liss, Inc.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Axenfeld-Rieger syndrome (AR) is a group of conditions characterized by specific abnormalities of the anterior segment of the eye (anterior segment dysgenesis). The condition exhibits considerable clinical and genetic heterogeneity, which often complicates diagnostic classification. Affected individuals exhibit a spectrum of anterior segment phenotypes that occur in isolation or combination with variable clinical severity. These are iris hypoplasia, corectopia (displacement of the pupil), polycoria (hole formation in the iris appearing as multiple pupils), posterior embryotoxon, and abnormal iris strands connecting the iridocorneal angle to the trabecular meshwork. Patients may also have systemic extraocular features including facial dysmorphism (maxillary hypoplasia, hypertelorism, telecanthus, prominent forehead), dental anomalies (hypodontia, microdontia), and/or redundant periumbilical skin. The condition is associated with raised intraocular pressure and an increased incidence of glaucoma affecting approximately 50% of patients has been reported [Idrees et al., 2006b; Sowden, 2007; Tumer and Bach-Holm, 2009].

AR segregates in an autosomal dominant manner. Heterozygous mutations and copy number changes of the developmental transcription factor genes PITX2 (MIM# 601542) and FOXC1 (MIM# 601090) have been identified in AR patients [Mears et al., 1998; Semina et al., 1996; Tumer and Bach-Holm, 2009]. Phenotypic overlap is associated with mutations in these two genes, which account for approximately 40% of cases [D'haene et al., 2010]. Two other loci on chromosomes 13q14 and 16q24 have been suggested by linkage analysis and chromosomal abnormalities but the underlying genes have not yet been identified [Phillips et al., 1996; Stathacopoulos et al., 1987; Werner et al., 1997]. Mutations in PITX2 or FOXC1 have also been rarely reported in association with primary congenital glaucoma and Peters' anomaly (congenital corneal opacity and iris-corneal adhesions, with or without lens adhesion to the cornea; MIM# 604229) [Honkanen et al., 2003; Nishimura et al., 1998; Perveen et al., 2000]. The incidence of glaucoma in AR patients with identified PITX2 or FOXC1 mutations has been reported to be 75%, rising to 100% in patients with duplications of FOXC1. These patients also respond poorly to medical or surgical pressure lowering interventions [Strungaru et al., 2007]. It is not uncommon for a single identified genetic mutation segregating within a family with AR to show variable phenotypic expression between different individuals [Honkanen et al., 2003; Idrees et al., 2006a; Komatireddy et al., 2003; Perveen et al., 2000]. For example, we have previously reported a single family with three affected individuals carrying an identical PITX2 mutation, exhibiting a range of phenotypes from iris hypoplasia and normal intraocular pressure to polycoria, corneal opacity, and glaucoma [Idrees et al., 2006a].

PITX2 is a Bicoid-related homeodomain transcription factor involved in regulating the development of different tissues of the anterior segment, as well as several nonocular tissues including the branchial arches, heart, and pituitary [Gage et al., 1999; Kitamura et al., 1999]. The PITX2 gene produces three major protein-coding isoforms (PITX2A, PITX2B, PITX2C), each containing an identical homeodomain and C-terminal domain, differing only at the N-terminal region. The common C-terminal domain contains a highly conserved 14 amino acid OAR (otp, aristaless, and rax) domain (residues 233–246 in PITX2A; NP_700476.1) shown to be involved in mediating PITX2 protein interactions [Amendt et al., 1999; Berry et al., 2006; Semina et al., 1996]. A fourth isoform, PITX2D, has also been reported to suppress the activity of the other PITX2 isoforms [Cox et al., 2002]. FOXC1 is a member of the Forkhead (FOX) transcription factor family, characterized by a conserved 110 amino acid DNA-binding domain, and involved in the regulation of a diverse range of developmental processes [Lehmann et al., 2003]. Functional analysis of disease-causing mutations [Footz et al., 2009; Kozlowski and Walter, 2000; Saleem et al., 2001; Saleem et al., 2003], combined with reports of deletions, duplications, and chromosomal abnormalities affecting these genes in patients with overlapping clinical phenotypes has shown that correct gene dosage of both PITX2 and FOXC1 is essential for normal development [Engenheiro et al., 2007; Lehmann et al., 2000; Lehmann et al., 2002; Nishimura et al., 2001].

Foxc1 and Pitx2 have been shown to be coexpressed within the same tissues during embryonic eye development in the mouse, although differences in spatial and temporal expression patterns also occur. At embryonic day (E) 11.5, the two transcription factors are coexpressed within the periocular mesenchyme and in the presumptive cornea of the developing anterior segment. By E16.5, coexpression is localized to cells within specific ocular structures, including the corneal endothelium, presumptive iridocorneal angle, and choroid [Berry et al., 2006]. There are also specific regions where both genes are not coexpressed. For example, at E11.5–E12.5 cells surrounding the optic stalk only express FOXC1 whereas later in development cells of the corneal stroma express only PITX2 [Berry et al., 2006]. Within cells expressing both PITX2 and FOXC1, the proteins have been shown to colocalize in restricted nuclear domains suggesting they are capable of regulating common genetic pathways [Berry et al., 2006]. These data support the idea that PITX2 and FOXC1 work in tandem to regulate anterior segment development.

We now report a family with several affected members presenting with variable ocular phenotypes. Screening of PITX2 and FOXC1 shows that mutations in both genes are cosegregating with disease in the family. The most severely affected individual, who showed an atypical disease presentation, is the only member of the family to inherit mutations in both FOXC1 and PITX2. This is the first report of digenic inheritance of mutations in both these genes in a single individual. To substantiate the proposal that the severe phenotype is due to the presence of both mutations, we compared transcription factor activities singly and in combination to mimic the heterozygous and the digenic scenarios in vitro. We show for the first time that FOXC1 and PITX2 are both capable of independently regulating transcription from the same promoters of target genes that are expressed during anterior segment development. We show that the lowest transactivation activity is observed when both mutations are present together. These data support a mechanism by which mutation of either gene results in similar ocular phenotypes as they regulate common target genes, whereas digenic inheritance of mutations dramatically influences phenotypic severity as the compensatory mechanisms are impaired. This finding is particularly important as mutation screening is typically halted after the identification of a single pathogenic mutation within a family and supports the need for sequencing both genes in AR families and other anterior segment developmental anomalies.

Patients and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Patient Details

The proband was referred to the ophthalmic service at age 10 days with bilateral anterior segment developmental anomalies. Examination revealed the right lens extruding through the cornea with peripheral corneal vascularization (Fig. 1A and B) and a thinned cornea centrally with peripheral corneal vascularization on the left. Both eyes exhibited raised intraocular pressure, 26 mmHg in the left eye and 28 mmHg in the right (right eye pressure measured at the limbus), measured using a Perkins tonometer (Keeler,Windsor, UK). Tectonic penetrating keratoplasty (PKP) was performed within the next few days, antiglaucoma medication of betaxolol 0.25% and dorzolamide 2% twice daily having been started in both eyes. At right PKP, the lens was also removed as it extruded at the time of trephination, though a thin tissue still covered the lens (thought to be Descemet's membrane) and she was left aphakic in the right eye. Corneal transplantation was performed as per previously published details [Nischal, 2003]. The left eye underwent PKP 2 weeks later and the left lens was spared as it was not extruding. Histologically, there was insufficient tissue on the right side to make a comprehensive description. The left cornea showed focal thickening of the corneal epithelium with basal epithelial vacuolation and Bowman's layer missing in most places. The corneal stroma showed separation of the collagen fibers associated with accumulation of mucopolysaccharide-like ground substance (Alcian blue-positive). A central pseudocystic space associated with plump reactive cells but no true lining was present, also observed by high-frequency ultrasound (Fig. 1C and D). The surrounding stroma was markedly fibrotic and showed vascularization. Posteriorly, Descemet's membrane and the endothelium were absent.

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Figure 1. Clinical pictures of family showing range of ocular phenotypes. A and B: Right eye of proband showing the lens extruding through the cornea with peripheral corneal vascularization. C: Ultrasound biomicroscopy of the left eye of the proband showing abnormal thickened cornea with a central corneal cyst (asterisk). D: Histological section of the anterior left host cornea from the proband showing a central cystic space within the stroma (asterisk). Focally thickened corneal epithelium indicated by arrowheads. E and F: Right and left eye, respectively, of the father of the proband showing iris hypoplasia and posterior embryotoxon (arrowheads). G and H: Right and left eye, respectively, of the mother of the proband who exhibits iris heterochromia and bilateral posterior embryotoxon.

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Fundoscopy of both eyes showed bilateral posterior persistent fetal vasculature or persistent hyperplastic primary vitreous (PHPV). The corneal transplants remained clear and the child developed the ability to fix and follow small toys and navigate unfamiliar surroundings. Her best corrected visual acuity reached 1.0 LogMAR equivalent using the Cardiff acuity test. At the age of 2 years and 2 months, the posterior PHPV progressed to bilateral total tractional retinal detachments. Despite attempted vitreoretinal surgery, her eyesight and globes could not be saved and both eyes became phthisical. Her corneal grafts remained clear until most recent follow-up, age 4 years. She had no evidence of any other syndromic features.

The father of the proband had iris hypoplasia, prominent anteriorly displaced Schwalbe's line (posterior embryotoxon), and peripheral iridocorneal adhesions (Fig. 1E and F), all consistent with a diagnosis of AR with no posterior abnormalities. He also presented with hypertelorism, downslanting palpebral fissures, malar hypoplasia, mild retrognathia, and unusual dentition. His umbilicus was unremarkable and there were no other systemic abnormalities. During the course of these investigations, the parents had a second child who was examined at age 2 months. He also had signs of AR similar to his father: posterior embryotoxon with adherent iris strands peripherally and marked iris hypoplasia without corneal opacity and with normal intraocular pressures. The mother, who was previously not known to be affected, was examined at first presentation and found to have isolated bilateral posterior embryotoxon and iris heterochromia. All else was unremarkable. Ethical committee approval was obtained from the Institute of Child Health/Great Ormond Street Hospital for Children Joint Research Ethics Committee. Informed written consent was obtained from the parents prior to DNA analysis.

Mutation Analysis of PITX2 and FOXC1

PCR primer pairs were designed to amplify the coding regions of the PITX2 and FOXC1 genes. Primer sequences were as detailed in Supp. Table S1. PCR products treated with Shrimp Alkaline Phosphatase (GE Healthcare, Chalfont St. Giles, UK) and Exonuclease I (New England Biolabs, Hitchin, UK) were directly sequenced using BigDye Terminator v1.1 sequencing chemistry (Applied Biosystems, Life Technologies, Paisley, UK) using a 3730XL DNA Analysis System (Applied Biosystems). Sequences were analyzed using Sequencher v4.8 software (Gene Codes Corp., Ann Arbor, MI) and compared to the reference sequence for PITX2A (NM_153427.2) or FOXC1 (NM_001453.2), respectively. Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence as indicated in the text, according to journal guidelines (www.hgvs.org/mutnomen). The initiation codon is codon 1.

Plasmid Constructs

Full-length PITX2A coding sequence (IMAGE Consortium Clone ID 3914727) [Lennon et al., 1996] was obtained from Geneservice Ltd. (Cambridge, UK) and subcloned into pcDNA3.1(+) vector (Invitrogen, Life Technologies, Paisley, UK). The c.698C>T (p.Ser233Leu) mutation was introduced using the QuikChange Site-Directed Mutagenesis Kit (Stratagene; Agilent Technologies UK Ltd, Stockport, UK) according to the manufacturer's instructions. PCR products comprising the entire FOXC1 coding sequence were generated from the proband using the following primers: F 5′-GGCATGCAGGCGCGCTACTCCGTG-3′, R 5′-GAGGGTGTGT-CAAAACTTGC-3′. Purified PCR products were cloned into pCDNA3.1(+) and sequenced to distinguish wild-type (WT) and c.609delC clones. PCR primers were designed to amplify an 845-bp fragment from the proximal promoter of the FOXO1 gene from c.-130 to c.-923 relative to the A of the ATG translation initiation codon as +1 in the reference sequence (NM_002015.3), and a 1,697-bp fragment of the PLOD1 promoter (c.-139 to c.-1,735; NM_000302.3) were generated from human genomic DNA using the following primers: FOXO1F 5′-CGATTCCCCACGTCGTTC-3′, FOXO1R 5′-ACGGAAACTGGGAGGAAG-3′, PLOD1F 5′-CACTCCTCTG-GAGCCTAATC-3′, PLOD1R 5′-CTCGCAGGGCTGGAAACT-3′. Purified PCR products of promoter sequences were cloned directly upstream of the luciferase gene in pGL41.0 (Promega, Southampton, UK).

Electrophoretic Mobility Shift Assay

FOXC1 and PITX2A WT and mutant proteins were synthesized using the TnT T7 Coupled Reticulocyte Lysate System (Promega). EMSAs were performed using the following radiolabeled DNA probes, double stranded by annealing to their complementary oligos: Bicoid 5′-GATCCAAATAATCCCAACAGA-3′; FOX BS 5′-GATCCAAAGTAAATAAACAACAGA-3′; FOXO1 5′-GATCCA-AAGTAGTAAACAAAGTACAGA-3′; PLOD1H 5′-GATCCTCACA-CCTGTAATCCCAGC-3′; PLOD1J 5′-GATCCACATGCCTGTAA-TCCCAGC-3′. Ten microliters of in vitro translated protein was added to 50 pmol of probe in a total 40-μl binding reaction containing 7.5 mM Tris, 37.5 mM KCl, 3.25% glycerol, 30 μg bovine serum albumin, 75 μM dithiothreitol, 750 μM ethylenediaminetetra-acetic acid (EDTA). Binding reactions were incubated at room temperature for 20 min prior to electrophoresis through an 8% nondenaturing polyacrylamide gel containing 0.5× tris-borate-EDTA (TBE) and 1% glycerol in 0.5× TBE, 0.5% glycerol at 18 V/cm.

Reverse Transcription PCR

RNA was extracted from microdissected human fetal cornea and whole eye using Trizol reagent (Invitrogen) following the manufacturers' protocol. First strand cDNA synthesis was performed with 50-ng total RNA using M-MLV Reverse Transcriptase (Promega) and random hexamer oligonucleotide primers. Negative control reactions were also performed in the absence of M-MLV reverse transcriptase enzyme. Second strand synthesis was performed using gene-specific primers. For all genes investigated, exonic primers were designed to flank an exon–intron boundary with the exception of the single-exon gene FOXC1. Primer sequences and expected product sizes from cDNA and genomic DNA are listed in Supp. Table S2.

Cell Culture and Transient Transfection

Human embryonic kidney 293 cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum at 37°C in humidified air containing 5% CO2. Transient transfection assays were performed using Lipofectamine 2000 reagent (Invitrogen) following the manufacturer's instructions. Briefly, 1.5 × 10 cells/well were seeded into a 96-well tissue culture plate. Cells were transfected with 20 ng of relevant luciferase reporter and 15 ng of each expression construct either individually or in combination as indicated in Figure 4. pRLSV40 (Promega) was cotransfected in all experiments to control for transfection efficiency, and the total amount of transfected DNA was normalized to 120 ng/well by the addition of empty expression vector. Cells were harvested 24 h following transfection and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega), using a BMG Fluostar Optima microplate reader. Luciferase activity for each expression vector was determined by normalization to the level of baseline luciferase activity detected after cotransfection of empty pcDNA3.1 expression vector and the relevant reporter construct. Data are presented as mean ± SD from three independent experiments each performed in triplicate. Two-way Student's t-test was used to determine significant difference between means.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Molecular Genetic Analysis of PITX2 and FOXC1

The proband presented with complex bilateral anterior segment developmental anomalies including the presence of a corneal cyst (see “Patient Details” above for clinical history; Fig. 1). The father of the proband showed a typical AR phenotype and her mother showed isolated posterior embryotoxon and irides heterochromia. A second affected child displayed a similar AR phenotype to his father. We screened the four available members of the family for coding mutations in both the FOXC1 and PITX2 genes. The proband was found to be heterozygous for a deletion of a single cytosine in FOXC1 (c.609delC), predicted to result in a frameshift and subsequent disruption of the FOXC1 coding sequence (p.Ala204ArgfsX111). Analysis of the parents revealed that this mutation was inherited from her heterozygous father as her mother was found to have a normal FOXC1 coding sequence. Screening of PITX2 showed that the proband and her mother were heterozygous for a cytosine to thymine transition at position c.698 with respect to the PITX2A transcript (NM_153427.2, c.698C>T). This affects a highly conserved serine residue within the OAR domain converting it to leucine in all three known protein-coding isoforms (p.Ser233Leu, PITX2A; p.Ser279Leu, PITX2B; p.Ser286Leu, PITX2C). The second affected child was found to have the same genotype as his father, and has inherited the paternally derived FOXC1 c.609delC mutation but not the PITX2 variant from his mother (see Supp. Fig. S1).

Functional Analysis of FOXC1 and PITX2 Mutations Using DNA-Binding Assays

The FOXC1 c.609delC mutation is predicted to prematurely truncate the FOXC1 protein downstream of the DNA-binding forkhead domain. Since FOXC1 is a single-exon gene, it is likely that the mutant transcript would escape nonsense-mediated decay mechanisms [Maquat, 2004, 2005]. In order to assess the ability of the mutant FOXC1 protein to bind to DNA, we utilized DNA probes comprising sequences that FOXC1 has previously been shown to bind: a consensus FOX protein family DNA-binding sequence (FOX BS) [Saleem et al., 2004] and a known FOXC1 binding site from the proximal promoter of the FOXO1 gene, previously identified as a putative target of FOXC1 regulation [Berry et al., 2008]. Electrophoretic mobility shift assays (EMSA) showed that WT FOXC1 was capable of binding to both probes, whereas the mutant protein could not (see Fig. 2).

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Figure 2. DNA-binding analysis of wild-type and mutant FOXC1 and PITX2A proteins. A: Electrophoretic mobility shift assay (EMSA) showing that wild-type FOXC1 is capable of binding to DNA probes comprising a forkhead domain consensus binding sequence (FOX BS; left) and a site within the proximal promoter of the FOXO1 gene (right; arrow). A nonspecific complex is observed in all lanes including in vitro transcribed empty expression vector (empty). The protein product of the FOXC1 c.609delC mutation has lost the ability to bind DNA. Neither the wild-type or p.Ser233Leu PITX2A proteins are able to bind to either FOXC1 binding site. B: EMSA showing that both wild-type and mutant PITX2A are capable of binding to probes comprising a consensus Bicoid homeodomain binding site, as well as two elements within the proximal promoter of the PLOD1 gene (labeled H and J from reference [Hjalt et al., 2001] (arrow)). FOXC1 is unable to bind to any of these three sites. (C) In vitro translation of FOXC1 and PITX2 proteins incorporating [35S]-L-methionine showing equivalent amounts of protein synthesized in each reaction. Bands of the expected size of 35 kDa are observed for both PITX2A proteins. Wild-type FOXC1 shows a band at the expected size of 57 kDa, the smaller band from the product of the FOXC1 c.609delC mutation is as expected due to premature truncation of the mutant protein.

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Similar EMSA experiments using WT and p.Ser233Leu PITX2A proteins were performed using several probes containing targets for PITX2: a consensus Bicoid binding site, in addition to two probes comprising known PITX2 binding elements identified from the promoter of the PLOD1 gene (elements H and J identified by [Hjalt et al., 2001]), previously identified as a putative target of PITX2 regulation. As shown in Figure 2, both WT and p.Ser233Leu PITX2A proteins were capable of binding to all three of these probes. Production of equivalent amounts of in vitro translated protein was confirmed by performing parallel reactions incorporating [35S] L-methionine followed by electrophoresis on a denaturing 10% SDS polyacrylamide gel (see Fig. 2C).

Transcriptional Activation by Wild-Type and Mutant PITX2 and FOXC1

We next tested the ability of the mutant PITX2 and FOXC1 proteins to independently activate transcription as compared to their respective WT proteins in luciferase reporter assays. Rather than artificial consensus binding sequences, the proximal promoters of two target genes, previously reported to be regulated by PITX2 and FOXC1, respectively, were selected for analysis in order to increase the biological relevance of the in vitro study. These were an 845-bp fragment of the proximal promoter of FOXO1, including the FOXC1 binding site tested by EMSA, and 1,697 bp of the proximal promoter of PLOD1 including both H and J PITX2 binding sites [Hjalt et al., 2001]. To confirm that these genes were relevant for understanding the phenotypic presentation in the family under investigation, we examined their expression in the developing human eye. We detected FOXO1 and PLOD1 transcripts, together with PITX2 and FOXC1, by RT-PCR in RNA extracted from microdissected developing human cornea between 8 and 11 weeks of development as well as whole eye at 18 weeks (stages at which tissue was available; Fig. 3). This is in line with previous reports of Foxc1 and Pitx2 expression during murine anterior segment development [Berry et al., 2006]. To investigate synergistic or compensatory relationships between the two transcription factors, their ability to transactivate both target promoters was tested.

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Figure 3. Expression of PLOD1 and FOXO1 in the developing human cornea. RT-PCR analysis of microdissected human embryonic cornea at 8, 9, and 11 weeks of development in addition to 18-week whole eye. Parallel reactions performed in the absence (−) of reverse transcriptase are shown as a negative control. Transcripts of both PLOD1 and FOXO1 as well as PITX2 and FOXC1 were detected at all stages investigated. PAX6 is included as a positive control for all cDNA samples.

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Cotransfection of a reporter construct containing the FOXO1 promoter together with an expression construct containing WT FOXC1 showed an approximate 4-fold (3.8 ± 0.6) increase in reporter gene activation compared to control experiments using empty expression vector. The mutant c.609delC FOXC1 construct failed to show any appreciable activation of reporter gene expression compared to WT FOXC1 (P < 0.0001; see Fig. 4A). Cotransfection of a WT PITX2A expression construct showed that PITX2A could also independently activate transcription of the FOXO1 promoter, to a significantly greater extent than WT FOXC1 (5.2 ± 0.8 vs. 3.8 ± 0.6 fold; P = 0.0007). Cotransfection of the p.Ser233Leu PITX2A construct with the FOXO1 reporter showed a significantly reduced level of reporter gene expression compared to WT PITX2A (3.3 ± 0.6 fold; P < 0.0001; Fig. 4A), implying that the c.698C>T mutation represents a hypomorphic PITX2 allele. Neither WT PITX2 nor FOXC1 were able to activate transcription from the promoter-less luciferase reporter construct (data not shown). As EMSA using both WT and p.Ser233Leu PITX2A proteins showed that neither is capable of binding to the same 20-bp element in the FOXO1 promoter as WT FOXC1 (or to the consensus FOX BS sequence, Fig. 2), these data indicate that PITX2 action on the FOXO1 promoter is independent of this motif.

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Figure 4. In vitro analysis of PITX2 and FOXC1 mutations. A: Luciferase reporter assay showing that both wild-type PITX2A and FOXC1 are capable of independently activating transcription of a reporter construct containing an 845-bp fragment of the FOXO1 proximal promoter. Transfection of the FOXC1 c.609delC construct results in complete loss of reporter gene activation to background levels, whereas, the PITX2A c.698C>T (p.Ser233Leu) mutation results in significant reduction of reporter gene activation compared to wild-type PITX2A. B: Luciferase reporter assay cotransfecting the FOXO1 promoter reporter construct with wild-type and mutant PITX2 and FOXC1 constructs (15 ng each) together in combination. WT FOXC1 together with mutant PITX2A rescues the activation level of the mutant PITX2 alone to the level of both wild-type proteins (compare bars 5 and 6 with bar 2). Mutant FOXC1 together with wild-type PITX2 shows a lower level of activity compared to the two wild-type proteins (compare bars 7 and 5). Activity is reduced yet further with the two mutant proteins in combination on the FOXO1 reporter (bar 8) but is still higher than mutant FOXC1 alone (bar 4). C: Luciferase reporter assay using a 1,697-bp fragment of the PLOD1 promoter showing that both wild-type FOXC1 and PITX2A are capable of activating the reporter at different levels. Both the mutant constructs show significantly reduced activation compared to their respective wild-type constructs. D: Luciferase reporter assay cotransfecting the PLOD1 promoter reporter construct with wild-type and mutant PITX2 and FOXC1 constructs (15 ng each) together in combination. The combination of mutant PITX2 with wild-type FOXC1 shows equivalent levels of activation to that of cotransfection with both wild-type constructs (bars 5 and 6). The addition of mutant FOXC1 to wild-type PITX2 shows a decreased level of reporter gene activation (bar 7), which does not significantly decrease further with both mutant constructs in combination (bar 8). Both combinations show higher levels than mutant FOXC1 alone. Assays shown in (A) and (B) are normalized relative to the level of activity of empty expression vector cotransfected with the FOXO1 reporter. Assays shown in (C) and (D) are normalized relative to the level of activity of empty expression vector cotransfected with the PLOD1 reporter. “Reporter” corresponds to transfections of the relevant reporter construct only with no expression vector. “Vector” corresponds to cotransfection of the relevant reporter construct with empty pcDNA3.1 expression vector. Data are presented as mean ± SD of three repeat experiments each performed in triplicate.

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Cotransfection of a reporter construct containing 1,697 bp of the proximal promoter of PLOD1 together with the WT PITX2A expression construct showed 6.5-fold (±0.5) activation of the reporter compared to empty expression vector, whereas the mutant p.Ser233Leu PITX2A showed a significantly reduced level of activation compared to WT (4.6 ± 0.5; P < 0.0001; Fig. 4C). Similar experiments cotransfecting the PLOD1 reporter with WT FOXC1 construct showed that FOXC1 is also capable of activating PLOD1 expression in vitro, albeit at a much lower level than PITX2A (2.3 ± 0.4 fold; P < 0.0001). The mutant c.609delC FOXC1 construct showed no appreciable transcriptional activation of this reporter (see Fig. 4C). Similar to the effect we observed for the FOXO1 promoter, WT FOXC1 cannot bind to the same PITX2 recognition sites (Fig. 2), suggesting that FOXC1 is capable of independently activating the PLOD1 promoter in vitro. Equivalent expression levels of both proteins generated from WT and mutant expression construct were determined by Western blotting of whole cell lysates from transfected cells (data not shown).

Transcriptional Activation by Wild-Type and Mutant PITX2A and FOXC1 in Combination

Our observations that both PITX2 and FOXC1 can potentially activate transcription from both the FOXO1 and PLOD1 promoters in vitro, together with expression of both target genes and transcription factors in the developing cornea, suggests a mechanism by which both PITX2 and FOXC1 may be involved in coregulating expression of downstream genes in cells in which both proteins are expressed. In order to investigate whether the two proteins act independently or synergistically on these reporter constructs in vitro, we cotransfected equal amounts of WT or mutant FOXC1 and PITX2A constructs together to compare the activity of different combinations on each promoter. First, on the FOXO1 promoter, the combination of WT PITX2A and WT FOXC1 gave comparable levels of activation compared to an equivalent amount of WT PITX2A alone (5.5 ± 1.0 vs. 5.2 ± 0.8 fold) suggesting the activity of the two proteins was redundant. We next cotransfected WT FOXC1 with the hypomorphic p.Ser233Leu PITX2A, which restored the level of reporter gene activation to that of the two WT proteins (5.6 ± 0.8 vs. p.Ser233Leu PITX2A allele alone 3.3 ± 0.6, P < 0.0001; see Fig. 4B) indicating WT FOXC1 was able to compensate for reduced PITX2 activity. Conversely, the combination of WT PITX2A and mutant c.609delC FOXC1 produced a significant reduction in reporter gene expression compared to the two WT constructs (3.0 ± 0.5 vs. 5.5 ± 1.0 fold change; P < 0.0001), or to WT PITX2A alone (P < 0.0001). Cotransfection of both mutant constructs on the FOXO1 promoter reporter led to significantly lower levels of reporter gene activation than any other combination tested (2.24 ± 0.35; P = 0.002 vs. WT PITX2A and c.609delC FOXC1) suggesting the compensatory mechanism of one protein replacing lost activity of the other was impaired.

Using the PLOD1 promoter reporter construct we observed that WT FOXC1 was able to restore the reduction in expression of the hypomorphic p.Ser233Leu PITX2A allele to a similar level to that of both WT proteins (5.4 ± 0.5 vs. 5.6 ± 0.5 fold activation; P = 0.3; see Fig. 4D), comparable to the effect seen on the FOXO1 promoter. The combination of WT PITX2A with the mutant c.609delC FOXC1 constructs showed significantly reduced reporter gene activation compared to both WT (3.3 ± 0.2; P < 0.0001). This is similar to the effect on the FOXO1 promoter, indicating WT PITX2 cannot entirely compensate for the severe loss of function c.609delC FOXC1 allele. The combination of both the FOXC1 and PITX2A mutants on the PLOD1 promoter showed no further significant decrease in expression of the reporter beyond this level (3.0 ± 0.4 fold activation; see Fig. 4D).

Taken together these data indicate that both FOXC1 and PITX2A are capable of acting independently, with differential effects, on the same promoters and in some cases can compensate for loss of activity of the other; furthermore, that different combinations of transcription factor alleles show a range of transactivation levels. Finally, we demonstrated that the two alleles segregating in the family cause a lower activity in combination, than either do singly, therefore providing an explanation for the severe phenotype observed in the proband who has mutations in both genes.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

AR caused by mutation of either PITX2 or FOXC1 exhibits considerable phenotypic heterogeneity between affected individuals particularly with regard to the abnormalities of the anterior segment of the eye, with or without other syndromic features. Notably, this can occur between different affected members within a single pedigree all reported to carry the same mutation [Honkanen et al., 2003; Idrees et al., 2006a; Komatireddy et al., 2003; Perveen et al., 2000]. Phenotypic variability also occurs between families carrying different mutations in the same gene so that it is not possible to clinically distinguish PITX2 and FOXC1 mutations. We now report a single family with several affected members cosegregating mutations in both PITX2 and FOXC1 with highly variable anterior segment phenotypes. Moreover, there is a correlation within the family between FOXC1/PITX2 genotype and phenotypic severity. The father and brother of the proband exhibited similar mild AR phenotypes without posterior segment abnormalities. Both individuals harbor the c.609delC FOXC1 mutation, which has considerably reduced function in vitro. The mother of the proband has the mildest phenotypic manifestation in the family and is heterozygous for a hypomorphic PITX2A p.Ser233Leu allele, which retains some function but is significantly reduced compared to WT PITX2A in our in vitro assays. This observation is consistent with previous studies showing that mutant proteins retaining partial function result in mild AR phenotypes, suggesting a relationship between disease severity and residual PITX2 activity [Footz et al., 2009; Kozlowski and Walter, 2000]. The proband is the most severely affected individual showing an atypical phenotype that is distinct from the other affected members of the family. She has severe abnormalities of the whole globe, involving both anterior and posterior segments. The fact that she is the only family member to have inherited both mutations, combined with our in vitro data, would suggest that the presence of mutations in both FOXC1 and PITX2 are the cause of her phenotypic severity and atypical presentation. This is the first report of digenic inheritance of mutations in both these genes in a single individual, whereby the combination of the two genetic hits creates a severe disease phenotype that is not apparent in individuals carrying only a single mutation [Ming and Muenke, 2002]. These findings indicate that digenic inheritance is important for understanding the anterior segment dysgeneses as has previously been shown for complex whole globe abnormalities involving PAX6 and OTX2 mutations [Henderson et al., 2007]. It has previously been postulated that FOXC1 and PITX2 are likely to act in a common genetic pathway crucial for normal development of the anterior segment of the eye, providing an explanation why mutation in either gene can result in clinically overlapping phenotypes [Berry et al., 2006]. The correlation we observe between the severity of the phenotype between different affected members within the family and presence of the null FOXC1 mutation (father and brother), the hypomorphic PITX2 mutation (mother) or both (proband) provides in vivo support to this mechanism.

Specific cell populations within the developing eye have previously been shown to coexpress both FOXC1 and PITX2, whereas some cell types express only one or other but not both genes. This suggests that in some cells, regulation of target gene expression relies independently on either PITX2 or FOXC1 function and other cells may require both for normal target gene regulation. Previous in vitro studies, using artificial short consensus binding sequences (FOX BS and Bicoid), have suggested that PITX2 cannot transactivate FOXC1-target sequences directly, but can cooperatively regulate FOXC1-target genes and vice versa via direct protein–protein interactions [Berry et al., 2006]. In our investigations, rather than using tandemly repeated short consensus binding sequences linked to minimal viral promoters, we have instead tested cis-acting proximal promoter fragments (>0.8 and >1.6 kb) of recently identified target genes to explore the hypothesis that PITX2 and FOXC1 can compensate for each other's activity. Our in vitro data show for the first time that both factors have the potential to independently regulate the promoters of the same target genes and demonstrate different levels of activation depending on the particular target gene and the combination of PITX2 and/or FOXC1 alleles. Although we have focused our investigations on the PITX2A isoform, the affected amino acid occurs in a highly conserved region of a known functional domain [Amendt et al., 1999; Berry et al., 2006; Semina et al., 1996] and PolyPhen2 predictions [Adzhubei et al., 2010] suggest the identified mutation is “probably damaging” to the function of all known PITX2 isoforms. Both PITX2A and PITX2B have been reported to be specifically expressed within the developing eye during human embryonic development [Markintantova et al., 2008]. It has previously been demonstrated that different PITX2 isoforms have differential effects on transcriptional activation on different promoters, including the promoter of PLOD1, and that different PITX2 isoforms can also dimerize and function synergistically to regulate gene expression in a cell-specific manner [Cox et al., 2002]. Here we observed that the PITX2A isoform showed different levels of activation on different promoters, and the reduced function of the hypomorphic p.Ser233Leu allele could be compensated for by the presence of WT FOXC1 in vitro. This suggests further levels of complexity in the regulation of target genes in certain cell types depending on the presence or absence of FOXC1, or other as yet unknown factors, as well as on the isoform(s) of PITX2 present.

We used regions from the promoters of PLOD1 and FOXO1 to test the function of the mutations we identified, as there is evidence to suggest that these genes may be natural targets of PITX2 and FOXC1 regulation in the developing eye, which may be important for AR pathology. In silico analysis of the PLOD1 promoter fragment using MatInspector software (www.genomatix.de) also identifies a consensus Forkhead domain core-binding site (CAAAC) located 489-bp upstream of the ATG start codon. Similar analysis for the FOXO1 promoter fragment reveals two sites to which PITX2 could potentially bind between positions c.-381 to c.-402. We have shown that all four genes are expressed during a range of developmental stages in microdissected human fetal cornea. FOXC1 has previously been shown to bind to sites within the FOXO1 promoter by chromatin immunoprecipitation (ChIP) in human nonpigmented epithelial cells in vivo. Moreover, FOXO1 expression is reduced following downregulation of FOXC1 in cultured human trabecular meshwork cells and by using foxc1 antisense morpholinos in zebrafish, where the foxo1a homolog is also coexpressed in the developing anterior segment [Berry et al., 2008]. A recent genome-wide association study found single nucleotide polymorphisms at the FOXO1 locus to be associated with reduced central corneal thickness [Lu et al., 2010], an important factor for accurate intra-ocular pressure measurement. Furthermore, mutations in PITX2 have also been associated with significantly reduced central corneal thickness in humans and mice [Asai-Coakwell et al., 2006]. Mutations in PLOD1 cause Ehlers-Danlos syndrome type VI (MIM# 130050), a connective tissue disorder characterized by joint hypermobility, skin fragility, and hypotonia with ocular manifestations including microcornea, retinal detachment, and fragility of the globe [Hyland et al., 1992; Yeowell and Walker, 2000]. We have shown that PITX2A and FOXC1 are each capable of independently activating transcription from the PLOD1 and FOXO1 promoters in vitro. This is the first report demonstrating that regulation by both FOXC1 and PITX2 on naturally occurring promoter regions provides a redundant mode of regulation of their reciprocal putative target genes. Considered together these data support the idea that these compensatory genetic interactions are critical for normal human eye development.

In summary, we report a family segregating mutations in both FOXC1 and PITX2 with highly variable ocular phenotypes between different affected members. We observe a correlation between FOXC1/PITX2 mutant genotypes and mild to moderate to severe phenotypic severity. The digenic patient showed a severe atypical phenotype in line with the lowest activity of the combination of mutant proteins in vitro. These data indicate how hypomorphic mutations in either gene could potentially have a range of subtle effects affecting the dysregulation of target genes, which may produce very mild or subclinical phenotypes that could go undetected. For example, the mother showed only isolated posterior embryotoxon, which is described as occurring in the general population with a frequency of 8–15% [Waring et al., 1975]. Such alleles could dramatically influence phenotypic severity on the background of a second mutation. Mutations in PITX2 or FOXC1 are only reported to account for ∼40% of AR cases [D'haene et al., 2010], suggesting that modifying mutations in other as yet unidentified factors acting in the same pathway may also influence the extreme phenotypic heterogeneity observed between individuals with anterior segment developmental anomalies. This case also demonstrates the importance of detailed mutation analysis in patients and their families with complex and variable phenotypes, even after a single mutation is identified, for comprehensive understanding of the molecular etiology of the condition and accurate genetic counseling.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

We would like to thank the family for participating in this study. We would also like to thank the members of the Ophthalmology and Clinical Genetics Departments, Great Ormond Street Hospital for Children NHS Trust and Michael Walter, University of Alberta, Edmonton, Canada for helpful discussions. The human embryonic and fetal material was provided by the Joint MRC (grant# G0700089)/Wellcome Trust (grant# GR082557) Human Developmental Biology Resource (http://hdbr.org).

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  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Patients and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

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