September 2006
Volume 47, Issue 9
Free
Glaucoma  |   September 2006
Novel Mutations of FOXC1 and PITX2 in Patients with Axenfeld-Rieger Malformations
Author Affiliations
  • Nicole Weisschuh
    From the Molecular Genetics Laboratory and
  • Paul Dressler
    Department of Prosthodontics, School of Dental Medicine, Julius-Maximilians University, Würzburg, Germany; and the
  • Frank Schuettauf
    Department II, University Eye Hospital, Tübingen, Germany; the
  • Christiane Wolf
    From the Molecular Genetics Laboratory and
  • Bernd Wissinger
    From the Molecular Genetics Laboratory and
  • Eugen Gramer
    University Eye Hospital, Würzburg, Germany.
Investigative Ophthalmology & Visual Science September 2006, Vol.47, 3846-3852. doi:10.1167/iovs.06-0343
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Nicole Weisschuh, Paul Dressler, Frank Schuettauf, Christiane Wolf, Bernd Wissinger, Eugen Gramer; Novel Mutations of FOXC1 and PITX2 in Patients with Axenfeld-Rieger Malformations. Invest. Ophthalmol. Vis. Sci. 2006;47(9):3846-3852. doi: 10.1167/iovs.06-0343.

      Download citation file:


      © 2015 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements

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 members 7 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  
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 AR 4 26 27 28 29 30 31 32 33 and other cases of anterior segment malformations, such as iridogoniodysgenesis, 34 iris hypoplasia, 35 and Peters’ anomaly 36 (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  
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
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
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 4and examples of the anterior segment phenotypes are shown in Figure 1
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 ). 
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. 
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 before 39 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. 
Discussion
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 family 16 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 study 13 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 lateralization 47 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. 
Table 1.
 
Summary of FOXC1 Mutations Reported to Date
Table 1.
 
Summary of FOXC1 Mutations Reported to Date
References Nucleotide Change Protein Change
Nishimura et al.3 c.153-163del Nonsense
c.335T→C Missense (F112S)
c.378C→G Missense (I126M)
c.392C→T Missense (S131L)
Mears et al.10 c.93-102del Nonsense
c.245G→C Missense (S82T)
c.261C→G Missense (I87M)
Swiderski et al.11 c.210delG Nonsense
Mirzayans et al.12 c.67C→T Nonsense (Q23X)
Nishimura et al.13 c.99-108del Nonsense
c.116-123del Nonsense
c.1512del Nonsense
c.265insC Nonsense
c.26-47ins Nonsense
c.236C→T Missense (P79L)
Kawase et al.14 c.286insG Nonsense
c.272T→G Missense (I91S)
c.380G→A Missense (R127H)
Suzuki et al.15 c.235C→A Missense (P79T)
Panicker et al.16 c.482T→A Missense (M161K)
Saleem et al.17 c.255GC→TT Missense (L86F)
Komatireddy et al.18 c.4C→T Nonsense (Q2X)
c.367C→T Nonsense (Q123X)
Mortemousque et al.19 c.272T→C Missense (I91T)
Murphy et al.20 c.494G→C Missense (G165R)
c.506G→C Missense (R169P)
Table 2.
 
Summary of PITX2 Mutations Reported to Date
Table 2.
 
Summary of PITX2 Mutations Reported to Date
References Nucleotide Change Protein Change
Semina et al.4 c.744T→A Missense (L54Q)
c.785A→C Missense (T68P)
c.855G→C Missense (R91P)
c.981G→A Nonsense (W133X)
IVS3(−11)A→G Splice site
IVS3(+5)G→C Splice site
Kulak et al.34 c.789G→A Missense (R69H)
Alward et al.35 c.833C→T Missense (R84W)
Doward et al.36 IVS3(−2)A→T Splice site
Perveen et al.26 c.845A→G Missense (K88E)
c.851C→T Missense (R90C)
IVS2(−1)G→C Splice site
c.1083insC Nonsense
c.868-869delAA Nonsense
c.939delA Nonsense
c.1235-1236TA→AAG Nonsense
Priston et al.27 c.830G→C Missense (V83L)
c.713-733dupl Nonsense
Borges et al.28 c.1272delG Nonsense
Philips39 c.774C→T Missense (P64L)
c.852G→C Missense (R90P)
c.896C→G Missense (L105V)
c.906A→C Missense (N108T)
Wang et al.29 c.717-720delACTT Nonsense
Brooks et al.30 c.1261delT Nonsense
Lines et al.31 c.697delG Nonsense
IVS3(−1)G→T Nonsense
c.998delC Nonsense
Saadi et al.32 c.959delC Nonsense
Idrees et al.33 c.710C→T Missense (R43W)
Table 3.
 
Clinical Data and Phenotypes of Subjects/Pedigrees with AR
Table 3.
 
Clinical Data and Phenotypes of Subjects/Pedigrees with AR
Patient Family History IOP Max [mm Hg] (OD;OS) Excavation of Optic Disk BCVA (OD;OS) Visual Field Defect* (OD;OS) Corneal Changes Ocular Findings Dental Anomalies Other Findings Mutation
1 Glaucoma 23;23 −OU 20/20;20/20 0;0 PE OU, iridocorneal adhesions OU Hypertelorismus, umbilicus FOXC1:L246fsx68
2 Glaucoma, AR 45;52 +OU 20/25;HM 3;4 Corneal opacity OS IH OU FOXC1:N503fsx15
3 50;44 +OD 20/60;20/20 3;0 Microcornea OD PE OU, iridocorneal adhesions OU, corectopia OU FOXC1:S48X
4 Glaucoma 40;55 +OU 20/80;20/200 4;2 Iridocorneal adhesions OU, IH OU Micrognathia FOXC1:P79R
5.1 Glaucoma, AR 50;60 +OU PL;20/25 3;4 PE OU, IH OU, iridocorneal adhesions OU Middle-ear deafness FOXC1:Y115S
5.2 Glaucoma, AR 30;41 +OU 20/16;20/25 1;2 Megalocornea OU PE OU, IH OU, iridocorneal adhesions OU FOXC1:Y115S
6 AR 33;28 −OU 20/40;20/30 NA PE OU, iridocorneal adhesions OU, corectopia OU Heart defect, hypospadia FOXC1:G149D
7.1 Glaucoma, AR 16;16 NA NA 0;0 PE OU, IH OU, iridocorneal adhesions OU, corectopia OU Umbilicus, middle-ear deafness FOXC1:M161V
7.2 Glaucoma, AR 17;18 NA 20/20;20/20 0;0 Megalocornea OU PE OU, IH OU, iridocorneal adhesions OU FOXC1:M161V
8.1 Glaucoma, AR 36;20 −OU 20/20;20/30 2;0 PE OU, IH OU, iridocorneal adhesions OU Hypodontia, microdontia Micrognathia, umbilicus PITX2:P64L
8.2 Glaucoma, AR 30;22 +OS 20/20;20/20 NA PE OU, IH OU, iridocorneal adhesions OU Microdontia Umbilicus PITX2:P64L
8.3 Glaucoma, AR 35;35 −OU NA 0;0 PE OU, IH OU, iridocorneal adhesions OU Hypodontia, microdontia Micrognathia, umbilicus PITX2:P64L
8.4 Glaucoma, AR 15;16 −OU 20/20;20/20 0;0 PE OU, IH OU, iridocorneal adhesions OU Microdontia, taurodontism Micrognathia, umbilicus PITX2:P64L
8.5 Glaucoma, AR 16;16 −OU 20/20;20/20 0;0 PE OU, IH OU, iridocorneal adhesions OU, corectopia OU Hypodontia, microdontia Umbilicus PITX2:P64L
9 33;20 +OU HM;20/40 NA;0 IH OU Hypodontia, microdontia Micrognathia, umbilicus PITX2:P64R
10 30;32 +OU PL;20/50 NA PE OU, iridocorneal adhesions OU, corectopia OU Hypertelorismus, heart defect, middle-ear deafness None
11 AR 21;21 −OU 20/40;20/50 NA Corneal opacity OU PE OU, iridocorneal adhesions OU None
12 Glaucoma 28;28 +OU 20/20;20/30 0;0 PE OU, IH OU Hypodontia None
13 Glaucoma NA +OU 20/16;20/16 0;2 Iridocorneal adhesions OU, IH OU Hypodontia None
Table 4.
 
Summary of FOXC1 and PITX2 Mutations
Table 4.
 
Summary of FOXC1 and PITX2 Mutations
Patient Nucleotide Change Predicted Amino Acid Alteration Present in 100 Controls Reported Before
FOXC1
 1 c.738delG L246fsx68 NA No
 2 c.1511delT N503fsx15 NA No
 3 c.143C→A S48X NA No
 4 c.236C→G P79R No No
 5 c.339T→C Y115S No No
 6 c.446G→A G149D No No
 7 c.481A→G M161V No No
PITX2
 8 c.774C→T P64L No Philips39
 9 c.774C→G P64R No No
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.
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.
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).
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).
Figure 3.
 
Sequence analysis of patients with FOXC1 deletions (top row: mutant allele, bottom row: wild-type allele). (A) c.738delG (B) c.1511delT.
Figure 3.
 
Sequence analysis of patients with FOXC1 deletions (top row: mutant allele, bottom row: wild-type allele). (A) c.738delG (B) c.1511delT.
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.
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.
 
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. 
ShieldsMB, BuckleyE, KlintworthGK, ThresherR. Axenfeld-Rieger Syndrome: a spectrum of developmental disorders. Surv Ophthalmol. 1985;29:387–409. [CrossRef] [PubMed]
GorlinRJ, PindborgJ, CohenMM. Syndromes of the head and neck. Syndromes with Unusual Dental Findings. 1976;649–651.McGraw-Hill New York.
NishimuraDY, SwiderskiRE, AlwardWL, et al. The forkhead transcription factor gene FKHL7 is responsible for glaucoma phenotypes that map to 6p25. Nat Genet. 1998;19:140–147. [CrossRef] [PubMed]
SeminaEV, ReiterR, LeysensNJ, et al. Cloning and characterization of a novel bicoid-related homeobox transcription factor gene, RIEG, involved in Rieger syndrome. Nat Genet. 1996;14:392–399. [CrossRef] [PubMed]
PhillipsJC, del BonoEA, HainesJL, et al. A second locus for Rieger syndrome maps to chromosome 13q14. Am J Hum Genet. 1996;59:613–619. [PubMed]
RiiseR, StorhaugK, Brondum-NielsenK. Rieger syndrome is associated with PAX6 deletion. Acta Ophthalmol Scand. 2001;79:201–203. [CrossRef] [PubMed]
KatohM, KatohM. Human FOX gene family. Int J Oncol. 2004;25:1495–1500. [PubMed]
SasaiN, MizusekiK, SasaiY. Requirement of FoxD3-class signaling for neural crest determination in Xenopus. Development. 2001;128:2525–2536. [PubMed]
KaufmannE, KnochelW. Five years on the wings of fork head. Mech Dev. 1996;57:3–20. [CrossRef] [PubMed]
MearsAJ, JordanT, MirzayansF, et al. Mutations of the forkhead/winged-helix gene, FKHL7, in patients with Axenfeld-Rieger anomaly. Am J Hum Genet. 1998;63:1316–1328. [CrossRef] [PubMed]
SwiderskiRE, ReiterRS, NishimuraDY, et al. Expression of the Mf1 gene in developing mouse hearts: implication in the development of human congenital heart defects. Dev Dyn. 1999;216:16–27. [CrossRef] [PubMed]
MirzayansF, GouldDB, HeonE, et al. Axenfeld-Rieger syndrome resulting from mutation of the FKHL7 gene on chromosome 6p25. Eur J Hum Genet. 2000;8:71–74. [CrossRef] [PubMed]
NishimuraDY, SearbyCC, AlwardWL, et al. A spectrum of FOXC1 mutations suggests gene dosage as a mechanism for developmental defects of the anterior chamber of the eye. Am J Hum Genet. 2001;68:364–372. [CrossRef] [PubMed]
KawaseC, KawaseK, TaniguchiT, et al. Screening for mutations of Axenfeld-Rieger syndrome caused by FOXC1 gene in Japanese patients. J Glaucoma. 2001;10:477–482. [CrossRef] [PubMed]
SuzukiT, TakahashiK, KuwaharaS, WadaY, AbeT, TamaiM. A novel (Pro79Thr) mutation in the FKHL7 gene in a Japanese family with Axenfeld-Rieger syndrome. Am J Ophthalmol. 2001;132:572–575. [CrossRef] [PubMed]
PanickerSG, SampathS, MandalAK, ReddyAB, AhmedN, HasnainSE. Novel mutation in FOXC1 wing region causing Axenfeld-Rieger anomaly. Invest Ophthalmol Vis Sci. 2002;43:3613–3616. [PubMed]
SaleemRA, MurphyTC, LiebmannJM, WalterMA. Identification and analysis of a novel mutation in the FOXC1 forkhead domain. Invest Ophthalmol Vis Sci. 2003;44:4608–4612. [CrossRef] [PubMed]
KomatireddyS, ChakrabartiS, MandalAK, et al. Mutation spectrum of FOXC1 and clinical genetic heterogeneity of Axenfeld-Rieger anomaly in India. Mol Vis. 2003;9:43–48. [PubMed]
MortemousqueB, Amati-BonneauP, CoutureF, et al. Axenfeld-Rieger anomaly: a novel mutation in the forkhead box C1 (FOXC1) gene in a 4-generation family. Arch Ophthalmol. 2004;122:1527–1533. [CrossRef] [PubMed]
MurphyTC, SaleemRA, FootzT, RitchR, McGillivrayB, WalterMA. The wing 2 region of the FOXC1 forkhead domain is necessary for normal DNA-binding and transactivation functions. Invest Ophthalmol Vis Sci. 2004;45:2531–2538. [CrossRef] [PubMed]
LehmannOJ, EbenezerND, JordanT, et al. Chromosomal duplication involving the forkhead transcription factor gene FOXC1 causes iris hypoplasia and glaucoma. Am J Hum Genet. 2000;67:1129–1135. [CrossRef] [PubMed]
LehmannOJ, EbenezerND, EkongR, et al. Ocular developmental abnormalities and glaucoma associated with interstitial 6p25 duplications and deletions. Invest Ophthalmol Vis Sci. 2002;43:1843–1849. [PubMed]
HjaltTA, SeminaEV, AmendtBA, MurrayJC. The Pitx2 protein in mouse development. Dev Dyn. 2000;218:195–200. [CrossRef] [PubMed]
EspinozaHM, CoxCJ, SeminaEV, AmendtBA. A molecular basis for differential developmental anomalies in Axenfeld-Rieger-Syndrome. Hum Mol Genet. 2002;11:743–753. [CrossRef] [PubMed]
EvansAL, GagePJ. Expression of the homeobox gene Pitx2 in neural crest is required for optic stalk and ocular anterior segment development. Hum Mol Genet. 2005;14:3347–3359. [CrossRef] [PubMed]
PerveenR, LloydIC, Clayton-SmithJ, et al. Phenotypic variability and asymmetry of Rieger syndrome associated with PITX2 mutations. Invest Ophthalmol Vis Sci. 2000;41:2456–2460. [PubMed]
PristonM, KozlowskiK, GillD, et al. Functional analyses of two newly identified PITX2 mutants reveal a novel molecular mechanism for Axenfeld-Rieger syndrome. Hum Mol Genet. 2001;10:1631–1638. [CrossRef] [PubMed]
BorgesAS, SusannaR, Jr, CaraniJC, et al. Genetic analysis of PITX2 and FOXC1 in Rieger Syndrome patients from Brazil. J Glaucoma. 2002;11:51–56. [CrossRef] [PubMed]
WangY, ZhaoH, ZhangX, FengH. Novel identification of a four-base-pair deletion mutation in PITX2 in a Rieger Syndrome family. J Dent Res. 2003;82:1008–1012. [CrossRef] [PubMed]
BrooksBP, MoroiSE, DownsCA, et al. A novel mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome. Ophthalmic Genet. 2004;25:57–62. [CrossRef] [PubMed]
LinesMA, KozlowskiK, KulakSC, et al. Characterization and prevalence of PITX2 microdeletions and mutations in Axenfeld-Rieger malformations. Invest Ophthalmol Vis Sci. 2004;45:828–833. [CrossRef] [PubMed]
SaadiI, ToroR, KuburasA, SeminaE, MurrayJC, RussoAF. An unusual class of PITX2 mutations in Axenfeld-Rieger syndrome. Birth Defects Res A Clin Mol Teratol. 2006;76:175–181. [CrossRef] [PubMed]
IdreesF, Bloch-ZupanA, FreeSL, et al. A novel homeobox mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome associated with brain, ocular, and dental phenotypes. Am J Med Genet B Neuropsychiatr Genet. 2006;141:184–191.
KulakSC, KozlowskiK, SeminaEV, PearceWG, WalterMA. Mutation in the RIEG1 gene in patients with iridogoniodysgenesis syndrome. Hum Mol Genet. 1998;7:1113–1117. [CrossRef] [PubMed]
AlwardWL, SeminaEV, KalenakJW, et al. Autosomal dominant iris hypoplasia is caused by a mutation in the Rieger syndrome (RIEG/PITX2) gene. Am J Ophthalmol. 1998;125:98–100. [CrossRef] [PubMed]
DowardW, PerveenR, LloydIC, RidgwayAE, WilsonL, BlankGC. A mutation in the RIEG1 gene associated with Peters’ anomaly. J Med Genet. 1999;36:152–155. [PubMed]
BerryFB, LinesMA, OasJM, FootzT, UnderhillDA, GagePJ, et al. Functional interactions between FOXC1 and PITX2 underlie the sensitivity to FOXC1 gene dose in Axenfeld-Rieger syndrome and anterior segment dysgenesis. Hum Mol Genet. 2006;15:905–919. [CrossRef] [PubMed]
AulhornE, KarmeyerH. Frequency distribution in early glaucomatous visual field defects. Doc Ophthalmol Proc Ser. 1977;14:75–83.
PhillipsJC. Four novel mutations in the PITX2 gene in patients with Axenfeld-Rieger syndrome. Ophthalmic Res. 2002;34:324–326. [CrossRef] [PubMed]
SaleemRA, Banerjee-BasuS, MurphyTC, BaxevanisA, WalterMA. Essential structural and functional determinants within the forkhead domain of FOXC1. Nucleic Acids Res. 2004;32:4182–4193. [CrossRef] [PubMed]
KozlowskiK, WalterMA. Variation in residual PITX2 activity underlies the phenotypic spectrum of anterior segment developmental disorders. Hum Mol Genet. 2000;9:2131–2139. [CrossRef] [PubMed]
ChiY-I. Homeodomain revisited: a lesson from disease-causing mutations. Hum Genet. 2005;116:433–444. [CrossRef] [PubMed]
StrommeP, SundetK, MorkC, CassimanJ-J, FrynsJ-P, ClaesS. X linked mental retardation and infantile spasms in a family: new clinical data and linkage to Xp11.4-Xp22.11. J Med Genet. 1999;36:374–378. [PubMed]
GrippKW, WottonD, EdwardsMC, et al. Mutations in TGIF cause holoprosencephaly and link NODAL signalling to human neural axis determination. Nat Genet. 2000;25:205–208. [CrossRef] [PubMed]
PernasettiF, MilnerRDG, Al AshwalAAZ, et al. Pro239ser: a novel recessive mutation of the Pit-1 gene in seven Middle Eastern children with growth hormone, prolactin, and thyrotropin deficiency. J Clin Endocr Metab. 1998;83:2079–2083. [PubMed]
ChaneyBA, Clark-BaldwinK, DaveV, MaJ, RanceM. Resolution structure of the K50 class homeodomain PITX2 bound to DNA and implications for mutations that cause Rieger syndrome. Biochemistry. 2005;44:7497–7511. [CrossRef] [PubMed]
YoshiokaH, MenoC, KoshibaK, et al. Pitx2, a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. Cell. 1998;94:299–305. [CrossRef] [PubMed]
LoganM, Pagan-WestphalSM, SmithDM, PaganessiL, TabinCJ. The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell. 1998;94:307–317. [CrossRef] [PubMed]
PiedraME, IcardoJM, AlbajarM, Rodriguez-ReyJC, RosMA. Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell. 1998;94:319–324. [CrossRef] [PubMed]
WalterMA. PITs and FOXes in ocular genetics: the Cogan lecture. Invest Ophthalmol Vis Sci. 2003;44:1402–1405. [CrossRef] [PubMed]
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×