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(Investigative Ophthalmology and Visual Science. 2006;47:3846-3852.)
© 2006 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0343
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Novel Mutations of FOXC1 and PITX2 in Patients with Axenfeld-Rieger Malformations

Nicole Weisschuh,1 Paul Dressler,2 Frank Schuettauf,3 Christiane Wolf,1 Bernd Wissinger,1 and Eugen Gramer4

1From the Molecular Genetics Laboratory and 3Department II, University Eye Hospital, Tübingen, Germany; the 2Department of Prosthodontics, School of Dental Medicine, Julius-Maximilians University, Würzburg, Germany; and the 4University Eye Hospital, Würzburg, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. To determine the prevalence of FOXC1 and PITX2 mutations and to assess clinical phenotypes in a cohort of German patients with Axenfeld-Rieger malformations.

METHODS. All coding exons of the FOXC1 and PITX2 genes were amplified by PCR from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by restriction fragment length polymorphism (RFLP) analysis.

RESULTS. Sequence variants were identified by DNA sequencing in 15 of 19 cases. Mutation screening identified four potentially pathogenic FOXC1 mutations causing amino acid substitutions (P79R, Y115S, G149D, and M161V) that were not present in 100 control subjects. In addition, two different 1-bp deletions causing a frameshift and subsequent premature stop codon were identified in two subjects. One patient harbored a FOXC1 nonsense mutation (S48X). Mutation screening also identified two potentially pathogenic PITX2 mutations (P64L and P64R) in two index patients that were excluded in 100 healthy control subjects.

CONCLUSIONS. The findings in the present study clearly demonstrate that FOXC1 and PITX2 mutations are responsible for a significant proportion of Axenfeld-Rieger malformations in Germany.

Axenfeld-Rieger (AR) malformations comprise a series of clinically and genetically heterogeneous conditions. Affected individuals display a spectrum of classic ocular anomalies such as iris hypoplasia; a prominent Schwalbe line; adhesion of iris and cornea, microcornea, and corneal opacity, and increased intraocular pressure (IOP). In addition to the ocular phenotype, systemic features may also be associated with the disorder, including maxillary hypoplasia, hypodontia, microdontia, umbilical abnormalities, hearing defects, and congenital cardiac or kidney abnormalities.1 These syndromic features are seen with incomplete penetrance and variable expressivity. Because of the severe changes in eye morphology, glaucoma develops in roughly half of all patients. The mode of inheritance is autosomal dominant and the incidence of the disease is estimated to be approximately 1:200,000.2 Until now, four genetic loci have been associated with AR, including the genes FOXC1 and PITX2 located on 6p25 and 4q25, respectively.3 4 A third locus was mapped to 13q14, but the gene has not yet been identified.5 In addition, an isolated case of Rieger syndrome has been reported to harbor a deletion in the PAX6 gene.6

FOXC1 belongs to the forkhead family of transcription factors which comprises at least 43 members7 that act as critical regulators of embryogenesis, cell migration, and cell differentiation.8 9 Mutations of the FOXC1 gene have been identified as the underlying cause in a variety of anterior segment disorders.3 10 11 12 13 14 15 16 17 18 19 20 The mutation spectrum comprises frameshift and nonsense as well as missense mutations in the forkhead domain (for an overview, see Table 1 ). The observation of interstitial duplications and deletions of the FOXC1 gene in patients with anterior segment dysgenesis indicates the importance of a stringent control of FOXC1 expression and function.13 21 22


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  		TABLE 1. Summary of FOXC1 Mutations Reported to Date

 
PITX2 encodes a bicoid-like homeodomain transcription factor and is expressed very early during tooth development.23 From experimental data it seems likely that the molecular basis of tooth anomalies in AR is the inability of mutant PITX2 to activate genes involved in tooth morphogenesis,24 and it has been shown that expression of PITX2 in the neural crest is also necessary for optic stalk and anterior segment development.25 To date, 30 mutations of the PITX2 gene have been associated with AR4 26 27 28 29 30 31 32 33 and other cases of anterior segment malformations, such as iridogoniodysgenesis,34 iris hypoplasia,35 and Peters’ anomaly36 (Table 2) . Very recently, FOXC1 and PITX2 were shown to interact physically, and this interaction may be an explanation for the similar phenotypes caused by mutations of the two genes.37


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  		TABLE 2. Summary of PITX2 Mutations Reported to Date

 
The purpose of this study was to determine the prevalence of FOXC1 and PITX2 mutations in a cohort of German patients with AR malformations.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Ascertainment of Patients and Clinical Evaluation
Written informed consent was obtained from all subjects, and the study was approved by the ethics committees of the University Hospital Tübingen and the University Hospital Würzburg and conducted in accordance with the Declaration of Helsinki.

Ophthalmic examinations included slit lamp biomicroscopy, gonioscopy, and measurement of intraocular pressure (IOP), visual acuity, and visual fields. Diagnosis of hypodontia was based on panoramic radiographs. Other diagnoses were obtained from the patients’ attending specialists.

Mutation Detection by Direct Sequencing
Patient DNA was extracted from peripheral blood lymphocytes using a standard salting-out procedure. Individual exons of the PITX2 gene were amplified by polymerase chain reaction (PCR) using appropriate amplification protocols. Amplification of the single FOXC1 exon was performed with a set of four overlapping primers. Primer pairs for amplification and sequencing are available on request. PCR fragments were purified (ExoSAP-IT enzyme cleanup; USB, Cleveland, OH) and sequenced with dye-termination chemistry (Big Dye Termination chemistry; Applied Biosystems [ABI], Weiterstadt, Germany), and the products were separated on a DNA capillary sequencer (3100 Genetic Analyzer; ABI).

Cloning of the FOXC1 Gene
In two patients, we observed a heterozygous 1-bp deletion of the FOXC1 gene. The corresponding PCR fragments of the FOXC1 gene were cloned in a cloning vector (TA; Invitrogen, Carlsbad, CA) to confirm the nature of the mutation. Ligation and cloning were performed in accordance with the manufacturer’s protocol.

Detection of Nucleotide Variants by PCR/RFLP
Missense mutations detected in this study were assessed by analysis of 100 normal control subjects (200 chromosomes) applying PCR/restriction fragment length polymorphism (RFLP) assays. The respective fragments harboring the missense mutations were amplified from the affected patients and from control subjects. An aliquot of each amplicon was digested with the appropriate restriction enzyme (New England Biotechnology, Beverly, MA). All restriction digests were analyzed on a 4% agarose gel.

The C->T transition at codon 64 (P64L) of PITX2 results in the loss of a NciI restriction site. Screening for the P64R sequence variant in PITX2 was performed by amplification of a PCR fragment using mismatch primers that introduce an EcoO109I restriction site in the mutant allele. The T->C transition at codon 115 (Y115S) of FOXC1 results in the gain of an SmaI restriction site, whereas the G->C transversion at codon 149 (G149D) leads to the loss of a TseI site. The A->G transversion at codon 161 (M161V) results in the loss of an NlaIII recognition site. Screening for the P79R missense change in FOXC1 was performed by amplification of a PCR fragment using mismatch primers that introduce an NlaIV restriction site in the mutant allele. Detailed PCR and RFLP protocols are available on request.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
A total of 19 patients within 13 families with anterior segment dysgenesis were screened for FOXC1 and PITX2 mutations. All patients exhibited abnormal ocular findings demonstrating phenotypes including diagnoses of Rieger and Axenfeld anomalies. In several of the patients a wide variety of nonocular manifestations were noted, including dental and cardiac defects. The clinical features and mutations are summarized in Tables 3 and 4 and examples of the anterior segment phenotypes are shown in Figure 1 .


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  		TABLE 3. Clinical Data and Phenotypes of Subjects/Pedigrees with AR

 

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  		TABLE 4. Summary of FOXC1 and PITX2 Mutations

 

Figure 1
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  		FIGURE 1. Typical ocular phenotypes of patients described in the study. (A) Gonioscopic appearance of iridocorneal adhesions extending from a prominent Schwalbe’s line to the peripheral iris in patient 3. (B) Patient 8.5 showed a hypoplastic iris revealing the underlying pupillary sphincter muscle. (C) External view of mild corectopia and hypoplasia of the iridic stroma in patient 3.

 
Complete sequencing of the coding exons revealed seven sequence alterations in the single FOXC1 exon as well as two nucleotide changes in exon 4 of PITX2. All the changes were present in the heterozygous state. Eight of the nine mutations we found are described for the first time (Table 4 , Fig. 2 ).


Figure 2
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  		FIGURE 2. Protein sequence alignments of FOXC1 and PITX2 transcription factor family members and location of missense mutations identified in the DNA-binding domains. (A) Forkhead domains of human FOXC1 and related human FOX protein sequences. (B) Homeodomain alignment of human PITX2 and related human homeodomain-containing proteins. Shaded boxes: conserved residues among protein family members. Amino acid substitutions identified in this study are shown in bold. Protein secondary structures were predicted by Swiss PdbViewer (http://www.expasy.org/ provided in the public domain by the Swiss Institute of Bioinformatics, Geneva, Switzerland).

 
Two different 1-bp deletions were detected in FOXC1: one at nucleotide position 738 (patient 1) and the other at position 1511 (patient 2). Both deletions represent frameshift mutations that are predicted to result in premature translation termination after another 68 and 15 amino acids, respectively. In both cases, the corresponding PCR fragment of FOXC1 was cloned to obtain appropriate sequencing results of wild-type and mutant alleles (Fig. 3) . One patient (patient 3) was found to harbor a C->A transversion at nucleotide position 143. This nucleotide change causes a nonsense mutation and thus a truncated protein (S48X) lacking the forkhead DNA-binding domain.


Figure 3
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  		FIGURE 3. Sequence analysis of patients with FOXC1 deletions (top row: mutant allele, bottom row: wild-type allele). (A) c.738delG (B) c.1511delT.

 
In addition, four patients were found to harbor mutations in the FOXC1 gene leading to amino acid substitutions: a C->G transversion at nucleotide position 236, leading to a substitution of proline with arginine (P79R, patient 4); a T->C transition at nucleotide position 339, leading to a substitution of tyrosine with serine (Y115S, patients 5.1 and 5.2); a G->A transition at nucleotide position 446 that replaces glycine with aspartate (G149D, patient 6); and a G->A transition at nucleotide position 481 that replaces methionine with valine (M161V, patients 7.1 and 7.2). None of these four missense mutations was observed in 100 ethnically matched control subjects (n = 200 chromosomes).

Mutation screening also revealed two sequence variants in the PITX2 gene. Five members of a family with AR (patients 8.1 to 8.5) were found to harbor a C->T transition at nucleotide position 774. This missense mutation P64L in the homeodomain of PITX2 has already been described before39 and was found to segregate in the family (Fig. 4) . Of note, we also identified another missense mutation at the same nucleotide position, but in this case (patient 9) we observed a C->G substitution that causes a proline-to-arginine transversion (P64R). Both mutations were excluded in 100 ethnically matched control subjects. In patients 10 to 13 sequencing revealed no changes either in the FOXC1 or in the PITX2 gene.


Figure 4
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  		FIGURE 4. Phenotypic variability in a family with the PITX2 missense change P64L (patients 8.1 to 8.5). All family members exhibited posterior embryotoxon, iris hypoplasia, and iridocorneal adhesions.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Mutations in dominantly inherited disorders may be pathogenic either due to haploinsufficiency or because of dominant-negative effects. Nonsense or frameshift mutations result in truncated and therefore nonfunctional proteins. The disease-causing effects of such mutations are readily understandable. The effect of missense mutations, in contrast, depends on their location and the functional effect of the exchange. That may range from subtle effects to functionally null mutations, but may be even more deleterious than nonsense or frameshift mutations by provoking a dominant-negative effect in case the affected protein does oligomerize or interact with DNA or other proteins. In our screening of patients with AR we were able to identify nonsense and frameshift as well as missense mutations. Altogether, we found nine different mutations in FOXC1 and PITX2, eight of which were novel and are described for the first time.

The two 1-bp deletions and the stop codon mutation found in the FOXC1 gene are predicted to result in truncated proteins and are consistent with haploinsufficiency as the underlying cause of AR, whereas the four missense mutations of highly conserved amino acids could impair DNA binding and, possibly, nuclear localization of the FOXC1 protein. Two of the four missense mutations—Y115S and M161V—were shown to segregate in the corresponding families: Patient 5.1 has two daughters, one of them affected (patient 5.2). The latter also carries the Y115S mutation, whereas the unaffected daughter is homozygous for the wild-type allele (data not shown). Patient 7.1 has an affected daughter (patient 7.2) harboring the same mutation (M161V). The possible pathogenicity of this mutation is supported by the former report of an Indian family16 showing a T->A transversion at nucleotide position 482 with subsequent replacement of methionine with lysine at position 161.

For the P79R and G149D missense changes identified in our study, a family history was reported, but other family members were not available for clinical or genetic analysis. Of note, a former study13 reported a patient with Rieger syndrome harboring a C->T transition at nucleotide position 236, resulting in an substitution of lysine for proline at codon 79. Another patient in an independent report was found to demonstrate a C->A transversion at nucleotide position 235, subsequently replacing the proline residue with threonine.15 This is, to the best of our knowledge, the first reported instance of a FOXC1 mutation being found with three different mutation events at the same codon position.

Missense mutations that affect Y115 and G149 in the forkhead domain of FOXC1 have not been reported before. However, a multiple sequence alignment of different FOX family members shows considerable conservation of these residues, indicating functional importance (Fig. 2A) . For two of the missense mutations we identified—P79R and M161V—functional data of amino acid exchanges are available, although not for the same substitutions we observed. The biochemical analysis of a mutant FOXC1 protein harboring a replacement of methionine with lysine at amino acid position 161 has revealed a reduction in the ability of DNA binding, suggesting an essential role of the methionine residue for normal DNA binding.20 The functional consequences of substitutions of the proline residue at amino acid position 79 have been extensively studied. It has been shown that P79L and P79T, both having been observed in patients with AR,13 15 impair protein localization and transactivation capability of the protein whereas a P79K substitution had a less severe effect in vitro, reaching two thirds of the wild-type level in a transactivation assay.40 However, as this mutation has not been reported in patients with Axenfeld-Rieger malformation, no clinical data are available to judge this particular substitution. The proline-to-arginine substitution found in patient 4 in our study also introduces a positively charged amino acid residue at this site, implying a similar effect. Yet, because this patient shows a phenotype rather typical for Axenfeld-Rieger malformation it seems that this proline-to-arginine substitution has a more deleterious effect than was implied in the in vitro assay with the similar proline-to-lysine substitution.

Taken together, we have several facts that indicate the pathogenicity of the four missense mutations we found in the FOXC1 gene: Two mutations (Y115S and M161V) were shown to segregate in the corresponding families; two altered amino acid positions (79 and 161) had been reported to be essential for proper protein activity; each of the four is located in the DNA binding domain and affects conserved residues and furthermore was excluded in 100 ethnically matched control subjects.

Sequence analysis of the PITX2 gene demonstrated two sequence changes within the homeodomain. Patients 8.1 to 8.5 from one pedigree exhibited a C->T transition at nucleotide position 774, changing proline to leucine in codon 64 of the protein. This sequence change has been reported before in a pedigree with Axenfeld-Rieger.39 In our pedigree, the five affected mutation carriers show a certain degree of phenotypic variability (Fig. 4) , only the Axenfeld phenotype and the umbilical abnormalities seem to be highly penetrant. On the contrary, the dental phenotype varies among the family, with only some members showing hypodontia or micrognathia, although all members exhibit microdontia. It is also noteworthy that two of the younger family members show intraocular pressures that are within the lower range of the spectrum. The reason for this intrafamilial variation is unclear, but phenotypic variability has been described for PITX2 mutations.4 26 34 35 One might speculate that the phenotypic outcome in patients with Axenfeld-Rieger is also dependent on additional genetic and environmental influences.

Of note, we found another individual (patient 9) to harbor a nucleotide transversion at nucleotide position 774, but in this case a C->G transversion, causing the predicted missense change P64R. This particular change has not been reported yet. Experimental data show that mutant PITX2 proteins that harbor missense mutations in their homeodomain demonstrated reduced or even abolished DNA binding.41 Amino acid position 64 is located in the loop between helices 1 and 2 of the homeodomain (Fig. 2B) and is conserved considerably between homeodomain-containing proteins.42 Amino acid changes at this particular residue have been shown to have a disease-causing effect in the case of the homeodomain transcription factors ARX (x-linked myoclonic epilepsy),43 TGIF (holoprosencephaly),44 and PIT1 (pituitary hormone deficiency).45 Because of their rigid structure, proline residues provoke the orientation of helices and are therefore often found in loops. It may be that a replacement of this proline residue with leucine or arginine changes the orientation of the first and third helices and subsequently alters the stability of the homeodomain.46 Because the P64L missense mutation has been reported before in a pedigree with Axenfeld-Rieger and was not present in 100 ethnically matched control subjects, we consider it to be pathogenic, as well as the P64R mutation. It was striking that all our patients harboring PITX2 mutations exhibited a dental phenotype, but none of the patients who carried a FOXC1 mutation had dental anomalies. This result is consistent with those of previous studies showing that mutations in the PITX2 gene appear to be more strongly associated with dental findings than mutations in the FOXC1 gene.3 4 Earlier studies have shown that PITX2 activates the DLX2 gene which is required for tooth and craniofacial development.24

In several cases (e.g., patients 2 and 5.1), significant differences were noted in visual acuity between the two eyes due to unilateral glaucoma. This asymmetry may reflect the fact that PITX2 has been shown to be associated with genes involved in lateralization47 48 49 and in turn apparently interacts with FOXC1.37 It remains to be established whether this left-right difference is related to the functional importance of PITX2 in lateralization processes during development. Mutations in either of both genes may be responsible for the differences between the two sides in ocular development; however, a functional connection between the two gene products and laterality in the eye has not been reported.

In our cohort of patients with AR malformations, we were able to identify the potential disease-causing mutation in 70% of the index cases. This result is above the common calculation that mutations in FOXC1 and PITX2 are responsible for approximately 35% of cases.50 Taking into account that our screening protocol only applied sequencing, which does not reveal gross insertions or duplications, the prevalence of mutations in both genes may be even higher in our patient cohort. Nevertheless, the high prevalence of mutations in both genes may be accidental due to the relatively small number of patients; therefore, it seems likely that additional genes with yet unknown function in anterior segment development contribute to the spectrum of AR malformations.


   Acknowledgements
 
The authors thank Ulrich Schiefer and all participating assistant doctors of the glaucoma special ambulance unit of the University Eye Hospital Tübingen for substantial help with sample collection.


   Footnotes
 
Submitted for publication March 29, 2006; revised May 3, 2006; accepted June 26, 2006.

Disclosure: N. Weisschuh, None; P. Dressler, None; F. Schuettauf, None; C. Wolf, None; B. Wissinger, None; E. Gramer, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Nicole Weisschuh, University Eye Hospital, Molecular Genetics Laboratory, Roentgenweg 11, D-72076 Tübingen, Germany; nicole.weisschuh{at}uni-tuebingen.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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