Analyses of a Novel L130F Missense Mutation in FOXC1

LABORATORY SCIENCES Analyses of a Novel L130F Missense Mutation in FOXC1

Yoko A. Ito, BSc; Tim K. Footz, MSc; Tara C. Murphy, MSc; Winnie Courtens, MD; Michael A. Walter, PhD

Objective: To understand how the novel L130F muta- duced molecular weight compared with the wild-type pro- tion, found in 2 patients with Axenfeld-Rieger syn- tein, suggesting that the mutant and wild-type proteins drome, disrupts function of the forkhead box C1 pro- may be differentially phosphorylated. The L130F protein (FOXC1). tein also had a significantly impaired capacity to localize to the nucleus, bind DNA, and transactivate reporter Methods: Sequencing DNA from patients with Axenfeld- genes. Rieger syndrome identified a novel missense mutation that results in an L130F substitution in the FOXC1 gene. Conclusions: The disease-causing L130F mutation fur- Site-directed mutagenesis was used to introduce the L130F ther demonstrates that helix 3 of the forkhead domain mutation into the FOXC1 complementary DNA. The level is important for the FOXC1 protein to properly localize of L130F protein expression was determined by means to the nucleus, bind DNA, and activate gene expression. of immunoblotting. We determined the mutant protein’s ability to localize to the nucleus, bind DNA, and Clinical Relevance: The inability of FOXC1 to func- transactivate a reporter construct. tion owing to the L130F mutation provides further insight into how disruptions in the FOXC1 gene lead to hu- Results: The FOXC1 L130F mutant protein is ex- man Axenfeld-Rieger syndrome. pressed at levels similar to those of wild-type FOXC1. The L130F protein, however, migrated at an apparent re- Arch Ophthalmol. 2007;125:128-135

HE FORKHEAD BOX (FOX) lar defects, patients with AR can have sys- family of transcription fac- temic defects, including maxillary tors, including FOXC1, hypoplasia, hypodontia, and a protrud- share an evolutionarily con- ing umbilicus.10-12 served 110–amino acid se- Correct FOXC1 expression is crucial for quence known as the forkhead domain embyrogenesis and, in particular, for the T1 (FHD). This DNA-binding motif is com- normal development of the skeletal, car- posed of 1 minor and 3 major ␣-helixes and diovascular, urogenital, and ocular tis- 2 ␤-sheets.1 The 2 ␤-sheets form 2 loops that sues.13-17 Furthermore, FOXC1 contin- wrap around the DNA, giving the FHD its ues to be expressed in several adult tissues, characteristic winglike structure.2 including the eyes, brain, heart, and kid- Patients with FOXC1 mutations map- neys.16 Because the spatial and temporal ping to chromosome 6p25 are affected with patterns for FOXC1 expression have been human Axenfeld-Rieger (AR) syn- observed to coincide with the differentia- drome.3,4 This genetic disease is transmit- tion of specific tissues,16 mutations in the ted in an autosomal dominant manner and FOXC1 gene usually result in gross mor- is highly penetrant. The FOXC1 protein phological defects. is expressed in many of the developing For normal development, not only does ocular tissues in the anterior chamber of FOXC1 need to be expressed in the ap- Author Affiliations: the mouse eye.5 Mutations in FOXC1 re- propriate spatial and temporal patterns, but Departments of Ophthalmology sult in malformations in the anterior cham- the level of FOXC1 expression must be and Medical Genetics, ber of the human eye that include iridogo- strictly regulated.18 Thus, AR syndrome can University of Alberta, niodysgenesis, iris hypoplasia, corectopia, result from either a loss-of-function mu- Edmonton, Alberta (Mss Ito polycoria, a prominent Schwalbe line, and tation, in which FOXC1 expression is less and Murphy, Mr Footz, and 6-8 Dr Walter); and Department of iridocorneal tissue adhesions. The most than the critical lower threshold of 80% Medical Genetics, University serious consequence of AR syndrome is of wild-type activity levels, or a mutation Hospital Antwerp, Antwerp, that approximately 50% of patients de- causing FOXC1 expression to exceed the Belgium (Dr Courtens). velop glaucoma.9 In addition to the ocu- upper threshold of 150%.18 No strong

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 genotype-phenotype correlation has been established, reverse, 5Ј-gca ctc gtt gag cga gaa gtt gtg gcg gat gct-3Ј. Poten- which suggests that the process for developing a particu- tial mutant constructs were sequenced with the 3130ϫl ge- lar phenotype in a patient with a mutation of the FOXC1 netic analyzer. Confirmed mutants were subcloned into the gene is a complex one.19 FOXC1 pcDNA4 His/Max vector and resequenced. We have identified a novel FOXC1 missense mutation, L130F, in 2 related individuals with AR syndrome. CELL CULTURE The hydrophobic L130 residue is located in helix 3 of We cultured COS-7 cells and HeLa cells in Dulbecco modified the FHD. Helix 3 is referred to as the recognition helix Eagle medium and 10% fetal bovine serum at 37°C. because it interacts with the major groove of DNA.20 Pre- vious studies have found that missense mutations that IMMUNOBLOT ANALYSIS AND occur within helix 3 disrupt DNA binding and subse- 21 CALF INTESTINAL ALKALINE quent transcriptional activation. Thus, we performed PHOSPHATASE TREATMENT a molecular analysis to examine how the disease- causing L130F mutation disrupts FOXC1 function. Mo- The COS-7 cells (106 cells per 100-mm plate) were transfected lecular analyses of FOXC1 missense mutations such as with4µgofFOXC1 tagged with Xpress epitope (Invitrogen) L130F will provide further insight into how disruptions using commercially available reagent (Fugene 6; Roche, India- in FOXC1 lead to human AR syndrome and will give new napolis, Ind). Forty-eight hours after transfection, the pro- possibilities for the development of treatments for this teins were extracted and resolved on a 10% sodium dodecyl disease. sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins were detected by immunoblotting using an anti- Xpress antibody (1:10 000 dilution; Invitrogen) against the METHODS pcDNA4 His/Max vector–encoded N-terminal Xpress tag and visualized with chemiluminescent substrate (Supersignal West REPORT OF PATIENTS Pico; Pierce Biotechnology, Rockford, Ill). Protein extracts were incubated with 20 U of calf intestinal alkaline phosphatase (In- This research adhered to the tenets of the Declaration of vitrogen) with or without 11µM sodium vanadate for 1 hour Helsinki. Patient samples and information were collected with in a 37°C water bath. An equal amount of 2ϫSDS-PAGE load- informed consent as specified by the University of Alberta Eth- ing buffer was added, and the proteins were then resolved on ics Board. The L130F mutation was identified in a white woman a 10% SDS-PAGE gel and visualized by immunoblotting as de- (patient 1) and her son (patient 2). Patient 1 was diagnosed as scribed. having AR syndrome at 27 years of age, after her son was diagnosed as having AR syndrome and glaucoma at 2 months of IMMUNOFLUORESCENCE age. Patient 1 had no dental or facial abnormalities. An ophthalmological examination revealed that patient 1 had iris hy- The COS-7 cells (2ϫ105 cells per 35-mm well) were grown and poplasia, a prominent Schwalbe line, and peripheral anterior transfected directly on coverslips with 1 µg of Xpress-epitope– synechiae, but no glaucoma. Patient 2 was diagnosed as hav- tagged FOXC1. The COS-7 cells were also transfected with the ing AR syndrome because he had corectopia and hypertelor- pcDNA4 His/Max vector as a control experiment. Twenty- ism. He had a slight excess of skin at the umbilical region. Re- four hours after transfection, the localization of the FOXC1 pro- sults of the ophthalmological examination disclosed a posterior tein was visualized by incubating the coverslips with anti- embryotoxon. Patient 2 also had an intraocular pressure of 14 Xpress antibody and antimouse Cy3–conjugated secondary mm Hg, but abnormally high cup-disc ratios of 0.85 and 0.80 antibody. The position of the nucleus was visualized by stain- for the left and right eyes, respectively. Both patients were bi- ing with 4Ј,6-diamidine-2-phenylindole. First, a total of 480 laterally affected. The maternal grandparents did not have the cells transfected with wild-type FOXC1 were scored for nuclear L130F mutation, indicating a de novo mutation in patient 1. or cytoplasmic staining or both. Then, a total of 623 cells transfected with L130F were scored. MUTATION DETECTION ELECTROPHORETIC MOBILITY SHIFT ASSAY The FOXC1 gene was amplified as previously described.2 Poly- merase chain reaction products were gel purified, extracted on The amount of L130F protein in the COS-7 cell extracts was separation columns (Qiagen, Valencia, Calif), and sequenced equalized to wild-type FOXC1 levels by inspection of the pro- directly by using a phosphorus 33–labeled terminator cycle se- teins detected by immunoblotting. The protein extracts were quencing kit (Amersham Biosciences, Baie d’Urfe, Quebec). In incubated for 30 minutes at room temperature with 1.25mM addition, the polymerase chain reaction products were se- dithiothreitol, 0.3 µg of sheared salmon sperm DNA, 0.125 µg ϫ quenced using a 3130 l genetic analyzer (Applied Biosys- of poly dI/dC (Sigma-Aldrich Corp, St Louis, Mo), and 80 000 tems Inc, Foster City, Calif) to generate chromatograms and counts per minute of phosphorus 32 (32P)–deoxycytidine tri- to confirm the observations from the manual sequencing ex- phosphate–labeled double-stranded DNA containing the fol- periments. lowing FOXC1 binding site (shown underlined): forward, 5Ј- gatccaaagtaaataaacaacaga-3Ј; reverse, 5Ј-gatctctgttgtttatttactttg- PLASMID 3Ј.18 After prerunning the 6% polyacrylamide gel containing Tris-glycine-EDTA buffer for 15 minutes, the electrophoretic mo- The FOXC1 pcDNA4 His/Max B (Invitrogen, Carlsbad, Calif) bility shift assay (EMSA) reaction products were subjected to elec- has been described previously.18 Site-directed mutagenesis was trophoresis. As a control, the COS-7 cells were transfected with performed using a mutagenesis kit (QuickChange; Strate- the pcDNA4 His/Max vector. Also, a mock EMSA reaction was gene, La Jolla, Calif) with the addition of 10% dimethylsulfox- carried out with just the 32P-deoxycytidine triphosphate–labeled ide. The mutagenic primer sequences for L130F were as fol- double-stranded DNA to ensure that the EMSA was specific for lows: forward, 5Ј-agc atc cgc cac aac ttc tcg ctc aac gag tgc-3Ј; the ability of the FOXC1 protein to bind to DNA.

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379 399 CCCCGGAAA CCCTTC CC T AA C

Patient 2 L130F

379 399 CCCCGGAAA CTT CC CC T AA C C/T ∗

Figure 1. Identification of the L130F mutation in the FOXC1 (forkhead box C1) gene. The chromatogram shows the genomic DNA sequence of an unaffected individual and patient 2. Patient 2 has a heterozygous C-to-T transition that results in a leucine-to-phenylalanine change at codon position 130.

WT FOXCI L130F RESULTS

83 kDa The FOXC1 gene was screened for mutations by direct sequence analysis of polymerase chain reaction products of patient DNA. A heterozygous C-to-T transition at codon position 130 (388CϾT; L130F) that results in a leucine-to-phenylalanine change was detected in patients 1 and 2 (Figure 1). Sequencing of the patients’ 62 kDa DNA and 100 healthy control chromosomes confirmed that the L130F mutation is not present in the healthy population. Figure 2. The L130F mutation in the FOXC1 (forkhead box C1) gene does not Immunoblotting indicated that a plasmid containing affect protein stability. The Xpress (Invitrogen, Carlsbad, Calif ) a complementary DNA encoding FOXC1 with the L130F epitope–tagged wild-type (WT) FOXC1 and L130F, transfected into COS-7 cells, were detected by immunoblotting. The L130F protein is expressed at mutation and transfected into COS-7 cells was capable levels similar to those of WT FOXC1 protein. Both occurred as a doublet at of expressing the L130F protein, which was approxi- approximately 65 kDa. The protein size marker is indicated to the left. mately the same size as the wild-type protein (approximately 65 kDa) (Figure 2). Thus, the relative stability DUAL LUCIFERASE ASSAY of L130F protein expression was similar to that of the wild-type protein. However, the L130F construct repeat- The HeLa cells (4ϫ104 cells per 15-mm well) were cotrans- edly produced a pattern of immunoreactive degradation fected with 100 ng of the FOXC1 pcDNA4 His/Max construct, products that differed from the wild-type construct pat- 20 ng of the pGL3-TK construct with 6ϫFOXC1 binding sites,18 tern (data not shown). Similar to the wild-type sample, and 1 ng of the pRL-TK control plasmid. Forty-eight hours af- the L130F protein bands occurred as a doublet (Figure 2). ter transfection, the luciferase assays were carried out using the However, 1 band was slightly shifted so that it had a lower dual luciferase assay kit (Promega Corp, Madison, Wis). Each molecular weight than either of the wild-type bands, sug- experiment was done in triplicate and was performed 3 times. As a control, the HeLa cells were transfected with the pcDNA4 gesting that the L130F protein was modified differently His/Max empty vector instead of the FOXC1 pcDNA4 His/ than the wild-type protein. Because FOXC1 is known to 23 Max construct. be phosphorylated, we examined whether the L130F mutation affected protein phosphorylation. When the L130F protein was incubated with calf intestinal alka- MODELING line phosphatase, the higher-molecular-weight band was In silico mutagenesis of the FOXC2 FHD model was performed eliminated (Figure 3). When the L130F protein was in- using the Swiss-PdbViewer (http://ca.expasy.org/spdbv/), and the cubated with the phosphatase inhibitor sodium vana- models were evaluated with the atomic nonlocal environment as- date, the doublet was restored. This finding indicated that sessment (ANOLEA) Swiss model server (http://swissmodel phosphorylation of the L130F protein was responsible .expasy.org/anolea/), as described previously.22 for the occurrence of the immunoreactive doublet bands.

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NaVO3 – – + + – – + + CIP – + – + – + – +

Figure 3. The L130F mutation in the FOXC1 (forkhead box C1) gene alters phosphorylation of wild-type (WT) FOXC1 protein. In this immunoblot, the disappearance of the higher-molecular-weight bands on incubation with calf intestinal alkaline phosphatase (CIP) and their appearance with the inhibition of CIP

by sodium vanadate (NaVO3) indicated that the WT FOXC1 and L130F proteins are both phosphorylated.

Nucleus Nucleus and Cytoplasm Cytoplasm

WT FOXCI 92.5% 7.1% 0.4%

L130F 33.7% 44.3% 22.0%

Figure 4. The L130F mutation in the FOXC1 (forkhead box C1) gene disrupts efficient nuclear localization of the FOXC1 protein. The L130F proteins, visualized by Cy3 fluorescence (red) during microscopy, showed reduced localization to the nucleus, visualized by 4Ј,6-diamidine-2-phenylindole staining (blue), compared with wild-type (WT) FOXC1. A total of 480 cells and 623 cells were counted for WT FOXC1 and L130F, respectively.

Immunofluorescent microscopy was performed to de- DNA complexes that formed increased as the amount of termine whether the L130F protein was able to localize protein was increased (Figure 5). In contrast, the L130F to the nucleus. Only 33.7% of the L130F proteins local- protein showed a greatly reduced capacity to bind DNA, ized exclusively to the nucleus, compared with 92.5% for even when the amount of protein added to the EMSA re- the wild-type proteins (Figure 4). The COS-7 cells trans- action was increased (Figure 5), indicating that this mu- fected with the pcDNA4 His/Max vector showed no stain- tation significantly disrupts the normal DNA-binding ca- ing with Cy3 (data not shown), indicating that the im- pacity of the FOXC1 protein. munofluorescence observed was specific for the FOXC1 The ability of the L130F protein, expressed in the HeLa protein. These data indicate that the L130F mutation cells, to activate expression of a luciferase reporter con- severely disrupts the ability of the FOXC1 protein to taining 6 consensus FOXC1 binding sites was deter- localize to the nucleus. mined. Wild-type FOXC1 was able to activate expres- The DNA-binding ability of the L130F protein, ex- sion of the luciferase reporter (Figure 6). The pressed in the COS-7 cells, was determined by EMSA re- transactivation potential of the mutant protein was results. For the wild-type protein, the amount of protein- duced 3-fold compared with wild-type FOXC1 (Figure 6).

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 over the FHDs; the only differences are that FOXC1 con- WT FOXCI L130F tains aspartate residues at positions 96 and 117, whereas Probe pcDNA4 1 × 2 × 4 × 1 × 2 × 4 × FOXC2 has glutamate residues. In this homology model, L130 was mutated to phenylalanine in silico to predict structural defects in the L130F molecule via the ANOLEA mean force potential calculations. We compared the results with those for I87M (Figure 7) and indicate that, although the side chains of I87 and L130 are normally ∗ involved in the same hydrophobic cluster, mutations at these positions may produce different effects. When I87 was changed to a methionine residue, the ANOLEA scores for M87, I104, I126, L130, F136, and W152 were all affected, whereas the effect of L130F was limited to positions 87, 130, and 152. Although recombinant FOXC1 harboring a mutation at position I87 does not produce a stable protein,25 recombinant FOXC1 harboring an L130F mutation was recoverable in whole-cell extracts (Figure 2). Thus, the sum of the ANOLEA energy differences for any single mutation model does not necessarily predict the degree of stability of the expressed recombinant protein. Nevertheless, the model is able to predict that the Figure 5. The L130F mutation in the FOXC1 (forkhead box C1) gene impairs L130F mutation will result in severe disruptions to FOXC1 DNA binding. The wild-type (WT) FOXC1 and L130F proteins were incubated function. with phosphorus 32-deoxycytidine triphosphate–labeled double-stranded DNA containing FOXC1-binding sites. Unlike WT FOXC1, which formed protein-DNA complexes (*), the electrophoretic mobility shift assay showed, COMMENT with this autoradiogram, that the L130F protein was unable to bind to DNA even at high concentrations. The disruption of FOXC1 function by the novel disease- causing L130F mutation demonstrates the importance of helix 3 in FOXC1 function. Helix 3 of the FOXC1 protein interacts with the major groove of DNA and confers 6 × FOXCI BS TK Luciferase DNA-binding specificity. However, molecular modeling predicts that the L130 residue does not make direct contact with DNA (data not shown). Rather, the L130 Empty Vector residue is thought to be oriented toward the hydrophobic core and forms a pocket with other residues, including I87, I104, I126, F136, and W152.25 Missense muta- WT FOXCI tions that substitute a differently charged amino acid, such as R127H, appear to disrupt the electrostatic charge of the FHD and thus greatly reduce the affinity of the L130F mutant protein for DNA.19 Missense mutations such as I126E and I126K, which introduce a hydrophilic residue 0 20 40 60 80 100 120 into the hydrophobic core, also disrupt FOXC1-DNA Relative Luciferase Activity, % interactions.25 Although the interaction of the phenylalanine residue in place of the leucine residue at codon Figure 6. The L130F mutation in the FOXC1 (forkhead box C1) gene impairs position 130 preserves the neutrally charged and hydro- transcriptional activation. The L130F protein transactivated the luciferase phobic nature of this position, the EMSA results indicate reporter with 6ϫ FOXC1 binding sites (BS) (above the graph) at residual levels. The data show mean luciferase values, normalized to Renilla that the L130F mutation nevertheless reduces the ability luciferase, from a representative experiment carried out in triplicate. Error of the mutant protein to bind DNA (Figure 5). The phe- bars are the standard error of the mean. WT indicates wild type. nylalanine residue is bulkier and thus it is likely that the L130F mutation disrupts the helix 3 structure so that The transactivation potential of L130F is comparable to this helix can no longer fit into the major groove of the the transactivation potential of the empty expression vec- DNA. As a result, helix 3 of the FOXC1 protein may tor, indicating that the L130F mutation severely dis- no longer be able to interact with DNA. The L130F rupts the ability of the FOXC1 protein to activate a re- mutation also reduces the transactivation potential to porter gene. residual levels (Figure 5). This is consistent with the Finally, molecular modeling of the FOXC1 FHD was EMSA results that indicate that the L130F protein can- performed to predict which amino acid residue contacts not bind DNA because DNA binding is a prerequisite would be disrupted by the L130F mutation. The first for transactivation. model layer of the nuclear magnetic resonance–solved The FOXC1 protein is thought to be tightly regu- structure file of FOXC224 was used as a homology model lated by posttranslational modifications.26 One of the ways for FOXC1 because of its near-perfect sequence identity that the FOXC1 protein is regulated is by phosphoryla-

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I87 M87

L130 F130

60 L130 I87M L130F I87 50

30 Wild Type)

–

20 (Mutant

ANOLEA Energy Difference 10 I126 F136 W152 I104 0

–10 70 80 90 100 110 120 130 140 150 160 170 180 FOXC1 Residue Position

Figure 7. Molecular models and scatterplot of in silico analysis of the L130F mutation in the FOXC1 (forkhead box C1) gene. The FOXC2-derived homology model of FOXC1 shows the protein backbone (ribbon), mutated residues (gray), and unmutated residues (white). The wild-type and mutant-equivalent models were submitted to an atomic nonlocal environment assessment (ANOLEA) Swiss model server. Energy differences are in E/kT units, where E represents energy; k, the Boltzmann constant; and T, absolute temperature.

tion.23 Previous research has determined that the phos- cause enough localized structural distortion to prevent phorylated residues of FOXC1 lie within the inhibitory the linear amino acid sequence from folding properly. domain, which is located within amino acid residues Thus, the altered topology of the L130F protein may 215 through 366.23,27 Recently, the ERK1/2 mitogen- hinder the normal recognition and regulation by protein activated protein kinase–dependent phosphorylation of kinases. FOXC1 at the S272 residue was determined to stabilize Mutations in FOXC2 cause hereditary lymphedema FOXC1 by preventing the recruitment of degradation with distichiasis.21 The FHD of FOXC1 and FOXC2 have factors or, conversely, by recruiting stabilization fac- 98% sequence homology.21 An R121H missense muta- tors.26 Because leucine and phenylalanine are amino tion in helix 3 of the FOXC2 FHD displayed a similar mi- acids that cannot become phosphorylated, the L130F gration pattern to that of the L130F mutation in FOXC1.21 missense mutation will not directly affect the phospho- The R121H and L130F proteins displayed a faster mi- rylation state of the residue at position 130. However, gration than did wild-type FOXC2 and wild-type FOXC1, the L130F protein was found to migrate at an apparently respectively.21 Also, when treated with calf intestinal al- reduced molecular weight compared with the wild-type kaline phosphatase, R121H and L130F displayed mo- protein (Figure 2), suggesting that the mutant and wild- bilities equal to those of wild-type FOXC2 and wild- type proteins are differentially phosphorylated. The type FOXC1, respectively.21 In both cases, the mutant bulkier nature of the phenylalanine residue appears to proteins are predicted to not be phosphorylated to the

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©2007 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 full extent of the wild-type proteins. This similarity dem- mutation is also likely to disrupt protein function onstrates how small changes in helix 3 of the FHD can severely. A mutation of this conserved leucine residue is result in great changes by altering the overall structure likely to lead to adverse functional consequences in all of the FOXC2 or FOXC1 protein. FOX proteins. The immunofluorescence results indicate that most In this study, we report the identification of a novel of the L130F proteins are unable to localize to the nucleus missense mutation in FOXC1, L130F. The severe dis- (Figure 4), which is surprising because L130F is not in ruption of FOXC1 function as a result of the disease- the regions of FOXC1 known to be directly involved in causing L130F mutation is consistent with previously nuclear localization.23 Also, previous experiments have studied missense mutations located within helix 3.21 shown that, in many cases, substitution of a differently The L130F FOXC1 mutation is one of the most disrup- charged amino acid at any position disrupts the ability tive FOXC1 mutations studied because this mutation of the protein to localize to the nucleus to a greater ex- disrupts nuclear localization and impairs DNA bind- tent than a substitution involving amino acids with the ing, which subsequently impedes transcriptional acti- same charge.22 For example, a FOXC1 missense muta- vation. Thus, the analysis of the L130F missense tion involving 2 neutrally charged amino acid residues, mutation provides further insight into how disrup- I126A, resulted in the localization of 77% of the I126A tions in the FOXC1 FHD lead to human AR syndrome. proteins to the nucleus.25 However, when the I126 residue was replaced with a negatively charged glutamic acid Submitted for Publication: June 30, 2006; final revi- residue or a positively charged lysine residue, none of the 25 sion received September 13, 2006; accepted September mutant proteins localized to the nucleus. This was not 20, 2006. the case with the L130F mutation. Although leucine and Correspondence: Michael A. Walter, PhD, Department phenylalanine are both neutrally charged, immunofluo- of Medical Genetics, 839 Medical Sciences Bldg, Univer- rescence showed that the L130F mutation severely dis- sity of Alberta, Edmonton, Alberta, Canada T6G 2H7 rupted the normal localization of the protein to the ([email protected]). nucleus. Only 33.7% of the L130F proteins are able to Financial Disclosure: None reported. localize exclusively to the nucleus. Because of the L130F Funding/Support: This study was supported by grant mutation, the overall topology of the L130F protein may G118160216 from the Canadian Institute for Health Re- be altered in a manner that prevents the nuclear local- search (Dr Walter). ization signal and nuclear localization accessory signal Acknowledgment: We thank May Yu for tissue culture from being properly detected. However, the alteration in expertise, Wim Wuyts, MD, for providing phenotypic in- the phosphorylation pattern in the L130F protein may formation about the patients, and Fred Berry, PhD, for also contribute to the reduction in the transport of the many enlightening discussions. mutant protein to the nucleus, because phosphorylation appears to regulate the nuclear transport of many transcription factors.28,29 REFERENCES Consistent with findings from previous studies,3,4 a single missense mutation in FOXC1 identified within a 1. Kaufmann E, Knochel W. Five years on the wings of fork head. Mech Dev. 1996; 57:3-20. single family had variable phenotypic consequences. In 2. Clark KL, Halay ED, Lai E, Burley SK. Co-crystal structure of the HNF-3/fork head the case of the 2 related individuals with the L130F mu- DNA-recognition motif resembles histone H5. Nature. 1993;364:412-420. tation in FOXC1, both individuals were diagnosed as hav- 3. Mears AJ, Jordan T, Mirzayans F, et al. Mutations of the forkhead/winged-helix ing AR syndrome. However, the mother had a mild form gene, FKHL7, in patients with Axenfeld-Rieger anomaly. Am J Hum Genet. 1998; 63:1316-1328. of the disease, whereas her son was severely affected and 4. Nishimura DY, Swiderski RE, Alward WL, et al. The forkhead transcription fac- was diagnosed as having glaucoma at just 2 months of tor gene FKHL7 is responsible for glaucoma phenotypes which map to 6p25. age. Stochastic events during development are likely to Nat Genet. 1998;19:140-147. result in variable expression of downstream targets of 5. Kidson SH, Kume T, Deng K, Winfrey V, Hogan BL. The forkhead/winged-helix FOXC1 in regard to the timing, location, and level of ex- gene, Mf1, is necessary for the normal development of the cornea and formation of the anterior chamber in the mouse eye. Dev Biol. 1999;211:306-322. pression. Thus, although tight regulation of FOXC1 is 6. Lines MA, Kozlowski K, Walter MA. Molecular genetics of Axenfeld-Rieger essential for proper development, environmental fac- malformations. Hum Mol Genet. 2002;11:1177-1184. tors and modifier genes may also contribute to pheno- 7. Reese AB, Ellsworth RM. The anterior chamber cleavage syndrome. Arch typic variability.18 Ophthalmol. 1966;75:307-318. 8. Weatherill JR, Hart CT. Familial hypoplasia of the iris stroma associated with Investigation of the L130F mutation also gives impor- glaucoma. Br J Ophthalmol. 1969;53:433-438. tant insight into the likely effects of mutations of other 9. Shields MB, Buckley E, Klintworth GK, Thresher R. Axenfeld-Rieger syndrome: FOX genes. A mutation equivalent to L130F has been a spectrum of developmental disorders. Surv Ophthalmol. 1985;29:387-409. found in the FOXL2 gene,30 where a C-to-T transition at 10. Feingold M, Shiere F, Fogels HR, Donaldson D. Rieger’s syndrome. Pediatrics. codon position 106 (553CϾT; L106F) was detected in a 1969;44:564-569. 11. Fitch N, Kaback M. The Axenfeld syndrome and the Rieger syndrome. J Med Genet. patient with blepharophimosis-ptosis-epicanthus inver- 1978;15:30-34. 30 sus syndrome. The leucine residue at position 130 in 12. Chisholm IA, Chudley AE. Autosomal dominant iridogoniodysgenesis with as- FOXC1, which is equivalent to that at position 106 in sociated somatic anomalies: four-generation family with Rieger’s syndrome. Br FOXL2, is highly conserved in other FOX genes. There J Ophthalmol. 1983;67:529-534. 13. Hiemisch H, Monaghan AP, Schutz G, Kaestner KH. Expression of the mouse are at least 43 members in the human FOX gene fam- Fkh1/Mf1 and Mfh1 genes in late gestation embryos is restricted to mesoderm 31 ily, and this L130 residue is found in 24 of those derivatives. Mech Dev. 1998;73:129-132. genes.30 As for L130F, we predict that the L106F FOXL2 14. Kume T, Deng KY, Winfrey V, Gould DB, Walter MA, Hogan BL. The forkhead/

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ARCHIVES Web Quiz Winner

Archives Web Quiz Winner

ongratulations to the winner of our August quiz, C Elvis Ojaimi, MBBS, MMed(Ophth), Sydney, Aus- tralia. The correct answer to our August challenge was Vogt-Koyanagi-Harada disease with subretinal fibrosis. For a complete discussion of this case, see the Photo Es- say section in the September ARCHIVES (Khairallah M, Rao NA, Yahia SB, Zaouali S, Attia S. Pseudotumoral reti- nal pigment epithelium proliferation in a patient with Vogt-Koyanagi-Harada disease. Arch Ophthalmol. 2006; 124:1366-1367.)

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Be sure to visit the Archives of Ophthalmology Web site (http://www.archophthalmol.com) and try your hand at our Clinical Challenge Interactive Quiz. We invite visi- tors to make a diagnosis based on selected information from a case report or other feature scheduled to be published in the following month’s print edition of the ARCHIVES. The first visitor to e-mail our Web editors with the correct answer will be recognized in the print jour- nal and on our Web site and will also be able to choose one of the following books published by AMA Press: Clini- cal Eye Atlas, Clinical Retina,orUsers’ Guides to the Medi- cal Literature.

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