Portal de Periódicos da CAPES

Homozygous Nonsense Mutation in the FOXE3 Gene as a Cause of Congenital Primary Aphakia in Humans

2006; Elsevier BV; Volume: 79; Issue: 2 Linguagem: Inglês

10.1086/505654

1537-6605

Sophie Valleix, Florence Niel, Brigitte Nédelec, Marie‐Paule Algros, C. Schwartz, B. Delbosc, Marc Delpech, B Kantelip,

Intraocular Surgery and Lenses

Congenital primary aphakia (CPA) is a rare developmental disorder characterized by the absence of lens, the development of which is normally induced during the 4th–5th wk of human embryogenesis. This original failure leads, in turn, to complete aplasia of the anterior segment of the eye, which is the diagnostic histological criterion for CPA. So far, the genetic basis for this human condition has remained unclear. Here, we present the analysis of a consanguineous family with three siblings who had bilateral aphakia, microphthalmia, and complete agenesis of the ocular anterior segment. We show that a null mutation in the FOXE3 gene segregates and, in the homozygous state, produces the mutant phenotype in this family. Therefore, this study identifies—to our knowledge, for the first time—a causative gene for CPA in humans. Furthermore, it indicates a possible critical role for FOXE3 very early in the lens developmental program, perhaps earlier than any role recognized elsewhere for this gene. Congenital primary aphakia (CPA) is a rare developmental disorder characterized by the absence of lens, the development of which is normally induced during the 4th–5th wk of human embryogenesis. This original failure leads, in turn, to complete aplasia of the anterior segment of the eye, which is the diagnostic histological criterion for CPA. So far, the genetic basis for this human condition has remained unclear. Here, we present the analysis of a consanguineous family with three siblings who had bilateral aphakia, microphthalmia, and complete agenesis of the ocular anterior segment. We show that a null mutation in the FOXE3 gene segregates and, in the homozygous state, produces the mutant phenotype in this family. Therefore, this study identifies—to our knowledge, for the first time—a causative gene for CPA in humans. Furthermore, it indicates a possible critical role for FOXE3 very early in the lens developmental program, perhaps earlier than any role recognized elsewhere for this gene. Human aphakia is a rare congenital eye disorder in which the lens is missing. It has been histologically subdivided into primary and secondary forms, in accordance with the severity of defects of the ocular tissues, whose development requires the initial presence of a lens.1Beebe DC Coats JM The lens organizes the anterior segment: specification of neural crest cell differentiation in the avian eye.Dev Biol. 2000; 220: 424-431Crossref PubMed Scopus (161) Google Scholar, 2Gould DB Smith RS John SW Anterior segment development relevant to glaucoma.Int J Dev Biol. 2004; 48: 1015-1029Crossref PubMed Scopus (130) Google Scholar, 3Graw J Cataract mutations and lens development.Prog Retin Eye Res. 1999; 18: 235-267Crossref PubMed Scopus (80) Google Scholar, 4Manschot W Primary congenital aphakia.Arch Ophthalmol. 1963; 69: 551-577Crossref PubMed Scopus (20) Google Scholar Congenital primary aphakia (CPA) results from an early developmental arrest, around the 4th–5th wk of gestation in humans, that prevents the formation of any lens structure and leads to severe secondary ocular defects, including a complete aplasia of the anterior segment of the eye.4Manschot W Primary congenital aphakia.Arch Ophthalmol. 1963; 69: 551-577Crossref PubMed Scopus (20) Google Scholar, 5Barishak YR Embryology of the eye and its adnexae.Dev Ophthalmol. 1992; 24: 1-142Crossref PubMed Google Scholar In contrast, in secondary aphakic eyes, lens induction has occurred, and the lens vesicle has developed to some degree but finally has progressively resorbed perinatally, leading, therefore, to less-severe ocular defects. Few cases fulfill the diagnostic criteria for CPA, and the most famous cases histologically documented are those described by Manschot in 1963.4Manschot W Primary congenital aphakia.Arch Ophthalmol. 1963; 69: 551-577Crossref PubMed Scopus (20) Google Scholar So far, the underlying genetic causes of CPA have remained obscure, but they undoubtedly are diverse, since lens formation is dependent upon complex interactions of numerous tissues deriving from surface ectoderm, neural ectoderm, and neural crest mesenchyme.6Graw J The genetic and molecular basis of congenital eye defects.Nat Rev Genet. 2003; 4: 876-888Crossref PubMed Scopus (168) Google Scholar However, a major critical step in lens development is lens induction, a multistep process that leads to the formation of the lens placode.7Lang RA Pathways regulating lens induction in the mouse.Int J Dev Biol. 2004; 48: 783-791Crossref PubMed Scopus (140) Google Scholar Many genes, including transcription factors, homeobox genes, and signaling molecules, have been shown to be involved in the control of each stage of lens development. Although many of these components have been well defined and assembled in a provisional genetic pathway, numerous questions remain concerning the temporal and spatial coordination of their expression.7Lang RA Pathways regulating lens induction in the mouse.Int J Dev Biol. 2004; 48: 783-791Crossref PubMed Scopus (140) Google Scholar, 8Ashery-Padan R Gruss P Pax6 lights-up the way for eye development.Curr Opin Cell Biol. 2001; 13: 706-714Crossref PubMed Scopus (183) Google Scholar, 9Dimanlig PV Faber SC Auerbach W Makarenkova HP Lang RA The upstream ectoderm enhancer in Pax6 has an important role in lens induction.Development. 2001; 128: 4415-4424PubMed Google Scholar, 10Fitzpatrick DR van Heyningen V Developmental eye disorders.Curr Opin Genet Dev. 2005; 15: 348-353Crossref PubMed Scopus (74) Google Scholar, 11Goudreau G Petrou P Reneker LW Graw J Loster J Gruss P Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.Proc Natl Acad Sci USA. 2002; 99: 8719-8724Crossref PubMed Scopus (74) Google Scholar, 12Kamachi Y Uchikawa M Tanouchi A Sekido R Kondoh H Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.Genes Dev. 2001; 15: 1272-1286Crossref PubMed Scopus (289) Google Scholar Here, we have investigated a consanguineous family with three children (IV.1, IV.3, and IV.4) who have bilateral aphakia in association with microphthalmia, complete aplasia of the anterior segment of the eye, absence of iris, and retinal dysplasia (figs. 1A, 2B, and 3A). All the family members underwent a complete ophthalmologic examination that did not reveal ocular abnormalities in either parent. In addition, all three affected children were referred to a pediatrician for an examination of associated systemic anomalies, but none were detected, in particular cerebral malformations or dysmorphic features (figs. 2C, 2D, 3C, and 3D). In this pedigree, there was no relevant family history of ocular phenotype, and, since the parents were consanguineous, this aphakia was most probably inherited in a recessive manner. The first proband (IV.1) of this kindred was born with bilateral aphakia and microphthalmia and exhibited sclerocornea (data not shown). This child died of infant sudden death syndrome at age 3 mo, for unknown reasons. Infectious and toxic causes were ruled out, and chromosomal analysis showed a normal karyotype. An autopsy was performed and did not reveal systemic malformation or neurological distortion, and both eyes were removed for histological examination. Histological examination of all sections of both eyes from proband IV.1 indicated absence of lens or any residual lens structures associated with a complete aplasia of the anterior segment, absence of iris, no ciliary body, no trabecular meshwork or Schlemm's canal, and dysplasia of the retina (fig. 4A–4C). All these severe anterior ocular defects fulfill the diagnostic criteria of CPA, rather than that of secondary congenital aphakia.4Manschot W Primary congenital aphakia.Arch Ophthalmol. 1963; 69: 551-577Crossref PubMed Scopus (20) Google Scholar The corneal epithelium appeared thinner and consisted of three cell layers, rather than four or five. The corneal stroma contained cells with fibroblastic configuration and was abnormally vascularized (fig. 5A). The corneal endothelium was not observed in either eye, and, after periodic acid schiff staining, neither Bowman's layer nor Descemet's membranes were detected (fig. 5A). Instead of a corneal endothelium at the posterior surface of the corneal stroma, there was a mass of a highly vascularized fibromuscular tissue, which was covered by a pigmented epithelium that occasionally formed some rudimentary pseudofolds, evoking the aspect of ciliary processes (fig. 5A and 5B). By immunochemistry, this pigmented epithelium stained positively for vimentin antibody, but it was unstained with desmin, α-smooth muscle actin, and CK7/CK20 antibodies, for a cytoskeletal profile suggestive of a ciliary epithelium (fig. 5C and 5D). The layer of smooth muscular cells at the posterior face of the corneal stroma was of variable thickness, and these cells were found to be positive for anti–α-smooth muscle actin and antidesmin antibodies and negative for antivimentin antibody (figs. 4B and 5D). Immunohistochemical analysis with HMB45 antibody, which marks melanocyte cells, was performed, and the choroid and a small number of cells within the vascular fibromuscular tissue adherent to the posterior face of the corneal stroma stained positive (fig. 5E and 5F). In comparison with human fetal eyes at 7, 13.5, and 17.5 wk of development, we found that ciliary muscle and choroid were the only ocular structures positive for HMB45 antibody (data not shown). The retina appeared dysplastic, with rosette-like structures suggesting that the absence of lens most probably disturbed but did not prevent the invagination of the optic vesicule (fig. 4A and 4C).13Ashery-Padan R Marquardt T Zhou X Gruss P Pax6 activity in the lens primordium is required for lens formation and for correct placement of a single retina in the eye.Genes Dev. 2000; 14: 2701-2711Crossref PubMed Scopus (438) Google Scholar However, outside the rosettes, the retina showed an apparently normal architecture, in which all neuronal nuclear layers were recognizable, reinforcing the idea that retinogenesis is relatively normal in the absence of lens.14Hyer J Kuhlman J Afif E Mikawa T Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation.Dev Biol. 2003; 259: 351-363Crossref PubMed Scopus (88) Google Scholar This observation also indicates that the lens is required for proper optic cup morphogenesis, therefore supporting the view that contact between the optic vesicle and the prelens ectoderm, but not the lens per se, triggers the optic vesicle to form a cup.14Hyer J Kuhlman J Afif E Mikawa T Optic cup morphogenesis requires pre-lens ectoderm but not lens differentiation.Dev Biol. 2003; 259: 351-363Crossref PubMed Scopus (88) Google ScholarFigure 2Clinical phenotypes of patient IV.3. A, MRI (T2-weighted image) sagittal section at 28.5 wk of gestation. The fetal eyeball appears as a white sphere (arrow), and the lens, normally visible inside as a black and well-circumscribed sphere, is absent. B, Aspect of eyes of proband IV.3 at the age of 1 mo, revealing bilateral microphthalmia with aphakia in the right eye and extreme microphthalmia of the left eye, preventing correct examination. C and D, Axial and sagittal sections of cerebral MRI (T1-weighted images) at the age of 4 years, showing absence of lens, asymmetric eyeballs, and normal appearance of brain structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3Clinical phenotypes of patient IV.4, the affected daughter. A, Ocular appearance showing bilateral and symmetric microphthalmia. B, Higher magnification of her left orbit, illustrating the sclerocornea. C and D, Axial and sagittal sections of cerebral MRI (T1-weighted images) at the age of 3 years, showing absence of lens, symmetric eyeballs, and normal appearance of brain structures.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Histologic aspect of eyes from proband IV.1. A, Photograph representing section through the entire eye (hematoxylin-eosin staining [HES]). Note the absence of lens, with a empty cavity most probably corresponding to the vitreous (*), the aplasia of the anterior chamber (arrow), the dysplastic retina, and the presence of the optic nerve (magnification 4×). c = Cornea; on = optic nerve; r = retina. B, Histological photograph of the anterior segment of the eye, stained with anti–α-smooth muscular actin antibody. The anterior chamber space, including iris and ciliary body, is not formed, and the posterior face of the corneal stroma is lined by a layer of muscular smooth cells, a fibroconnective tissue, and a continuous layer of pigmented cells (*). pe = Pigmented epithelium. C, Abnormal folds of the retina with rosette-like structures in which the retinal lamination is disturbed (HES; magnification 20×).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Histologic and immunohistochemical analyses of eyes from proband IV.1. A, HES (left panel) and immunohistochemical staining with anti–α-smooth muscular actin (right panel) of the posterior corneal stroma, showing absence of corneal endothelium and Descemet's membrane, vascularized corneal stroma (arrowhead), and the different abnormal tissues that adhered to the posterior face of the cornea (magnification 10× and 20×, respectively). The arrow points to the site where corneal endothelium and Descemet's membrane normally take place in the cornea. cs = Corneal stroma; fvt = fibrovascular tissue; pe = pigmented epithelium; smc = smooth muscular cells. B, HES showing the pigmented epithelium that occasionally forms some folds resembling ciliary processes, with higher magnification (20×) shown in the box. C, HES (panel a) and immunohistochemical staining with antivimentin (panel b), anti–α-smooth actin (panel c), and anti-CK7 (panel d) antibodies of the pigmented ciliary epithelium. Only vimentin immunohistochemical staining is positive (magnification 40×). D, Immunohistochemical staining with antidesmin antibody (magnification 40×). Only smooth muscular cells lining the posterior face of the corneal stroma are positive. E and F, Immunohistochemical positive staining with anti-HMB45 antibody in the choroid and in some cells (arrow) within the ectopic fibromuscular tissue adherent to the cornea.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In the same family, two other children were born with bilateral aphakia, which was confirmed by magnetic resonance imaging (MRI) (figs. 2C, 2D, 3C, and 3D). Ophthalmologic examination of proband IV.3 revealed asymmetric microphthalmia, a right megalocornea, and normal intraocular pressure (IOP) (fig. 2B). However, at the age of 3 years, IOP progressed in the patient's right eye, which led to severe buphthalmos requiring trabeculectomy. In contrast to her brother, proband IV.4 presented at birth with symmetric microphthalmia with aphakia and sclerocornea, with an IOP of 10 mm Hg in both eyes (fig. 3A and 3B). At the present time, these children, aged 10 and 9 years, are in good health and have no intellectual or neurologic impairment. During the third and the fourth pregnancies, prenatal ultrasonography and MRIs successively performed at 16, 19, and 28.5 wk of gestation revealed absence of lens or capsular remnants in both eyes for probands IV.3 and IV.4, indicating that either the lens had never been formed or was resorbed very early during embryogenesis (fig. 2A). The probability that both lenses had formed and were bilaterally resorbed completely and symmetrically in both affected children is highly unlikely. Furthermore, the complete aplasia of the anterior segment of the eye, documented histologically in both aphakic eyes of proband IV.1, argues in favor of an absence of lens formation during embryogenesis. Since the karyotypes of the three infants were normal and the size of the family did not allow linkage analysis, we attempted the strategy of the candidate gene to identify the genetic cause of this congenital malformation. Therefore, on the basis of either animal models or spatial and temporal pattern of expression, we studied several genes, including PAX6,15Hanson IM Fletcher JM Jordan T Brown A Taylor D Adams RJ Punnett HH Van Heyningen V Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.Nat Genet. 1994; 6: 168-173Crossref PubMed Scopus (423) Google ScholarPITX3 and its promoter,16Semina EV Ferrell RE Mintz-Hittner HA Bitoun P Alward WL Reiter RS Funkhauser C Daack-Hirsch S Murray JC A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD.Nat Genet. 1998; 19: 167-170Crossref PubMed Scopus (317) Google Scholar, 17Semina EV Murray JC Reiter R Hrstka RF Graw J Deletion in the promoter region and altered expression of Pitx3 homeobox gene in aphakia mice.Hum Mol Genet. 2000; 9: 1575-1585Crossref PubMed Scopus (155) Google ScholarSIX3,18Hsieh YW Zhang XM Lin E Oliver G Yang XJ The homeobox gene Six3 is a potential regulator of anterior segment formation in the chick eye.Dev Biol. 2002; 248: 265-280Crossref PubMed Scopus (22) Google ScholarBMP7,19Wawersik S Purcell P Rauchman M Dudley AT Robertson EJ Maas R BMP7 acts in murine lens placode development.Dev Biol. 1999; 207: 176-188Crossref PubMed Scopus (218) Google ScholarSOX2,12Kamachi Y Uchikawa M Tanouchi A Sekido R Kondoh H Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.Genes Dev. 2001; 15: 1272-1286Crossref PubMed Scopus (289) Google ScholarFOXC1,20Honkanen RA Nishimura DY Swiderski RE Bennett SR Hong S Kwon YH Stone EM Sheffield VC Alward WL A family with Axenfeld-Rieger syndrome and Peters anomaly caused by a point mutation (Phe112Ser) in the FOXC1 gene.Am J Ophthalmol. 2003; 135: 368-375Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar and FOXE321Blixt A Mahlapuu M Aitola M Pelto-Huikko M Enerback S Carlsson P A forkhead gene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle.Genes Dev. 2000; 14: 245-254PubMed Google Scholar, 22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar, 23Ormestad M Blixt A Churchill A Martinsson T Enerback S Carlsson P Foxe3 haploinsufficiency in mice: a model for Peters' anomaly.Invest Ophthalmol Vis Sci. 2002; 43: 1350-1357PubMed Google Scholar, 24Semina EV Brownell I Mintz-Hittner HA Murray JC Jamrich M Mutations in the human forkhead transcription factor FOXE3 associated with anterior segment ocular dysgenesis and cataracts.Hum Mol Genet. 2001; 10: 231-236Crossref PubMed Scopus (153) Google Scholar (MIM 601094). After receiving informed consent from the family, we analyzed the entire coding region and intron-exon boundaries of these genes by bidirectional direct sequencing, and all these genes were excluded as being causative, except for FOXE3. In both living affected children (IV.3 and IV.4), we identified a homozygous nucleotide transversion, C→A, at nucleotide position 720, according to the sequence published by Semina et al.24Semina EV Brownell I Mintz-Hittner HA Murray JC Jamrich M Mutations in the human forkhead transcription factor FOXE3 associated with anterior segment ocular dysgenesis and cataracts.Hum Mol Genet. 2001; 10: 231-236Crossref PubMed Scopus (153) Google Scholar (GenBank accession number AF275722). This nucleotide variation predicts the substitution of the cytosine (TGC) at position 240 in the resulting amino acid sequence by a nonsense codon (TGA; C240X) (fig. 1B). This nucleotide variation was not found in 100 chromosomes from control individuals, which suggests that it is not a common SNP. Sequence analysis confirmed that both parents carried this mutation in the heterozygous state, which supports the hypothesis that the transmission of the phenotype is compatible with autosomal recessive inheritance (fig. 1A and 1B). This mutation results in the truncation of the 80 C-terminal aa that represent more than one-quarter of the total protein length of 319 aa. The functional consequences of premature termination codon (PTC) mutations are commonly expected to be null alleles, because these mutant mRNA are likely to be detected by RNA surveillance and to be degraded by nonsense-mediated decay (NMD).25Holbrook JA Neu-Yilik G Hentze MW Kulozik AE Nonsense-mediated decay approaches the clinic.Nat Genet. 2004; 36: 801-808Crossref PubMed Scopus (470) Google Scholar This mechanism prevents the accumulation of truncated proteins that could interfere with the function of the normal protein or that could have toxic effects in the cell, thereby protecting many heterozygous carriers of genes with PTC mutations from manifesting dominantly inherited disease phenotypes. Here, both carrier parents had normal ocular examination, which supports the hypotheses that mRNA containing C240X has most probably been subjected to NMD and that the congenital aphakia in the children results from FOXE3 haploinsufficiency. Elsewhere, two human FOXE3 missense mutations were identified in the heterozygous state in two families independently presenting anterior segment dysgenesis (ASD) with cataract or Peters' anomaly.23Ormestad M Blixt A Churchill A Martinsson T Enerback S Carlsson P Foxe3 haploinsufficiency in mice: a model for Peters' anomaly.Invest Ophthalmol Vis Sci. 2002; 43: 1350-1357PubMed Google Scholar, 24Semina EV Brownell I Mintz-Hittner HA Murray JC Jamrich M Mutations in the human forkhead transcription factor FOXE3 associated with anterior segment ocular dysgenesis and cataracts.Hum Mol Genet. 2001; 10: 231-236Crossref PubMed Scopus (153) Google Scholar Of these, one was a single-nucleotide frameshift insertion, 15 bp upstream of the stop codon in FOXE3, that replaced the last 5 aa of the predicted protein with 116 novel residues.24Semina EV Brownell I Mintz-Hittner HA Murray JC Jamrich M Mutations in the human forkhead transcription factor FOXE3 associated with anterior segment ocular dysgenesis and cataracts.Hum Mol Genet. 2001; 10: 231-236Crossref PubMed Scopus (153) Google Scholar The second missense mutation reported in FOXE3 gave rise to a nonconservative amino acid substitution (Arg90Leu) located in the DNA-binding domain, without impairing the fixation of FOXE3.23Ormestad M Blixt A Churchill A Martinsson T Enerback S Carlsson P Foxe3 haploinsufficiency in mice: a model for Peters' anomaly.Invest Ophthalmol Vis Sci. 2002; 43: 1350-1357PubMed Google Scholar These two FOXE3 missense mutations were found in the heterozygous state and caused a dominantly inherited lens phenotype milder than that observed in our family, which had a recessively inherited CPA. The fact that currently reported FOXE3 mutations result in variable ocular anterior segment phenotypes and distinct inheritance patterns highlights the complexity of this disease condition. Ocular ASD is a phenotypic and genetically heterogeneous condition that has also been associated with mutations in PAX6, FOXC1, PITX3, or PITX2.15Hanson IM Fletcher JM Jordan T Brown A Taylor D Adams RJ Punnett HH Van Heyningen V Mutations at the PAX6 locus are found in heterogeneous anterior segment malformations including Peters' anomaly.Nat Genet. 1994; 6: 168-173Crossref PubMed Scopus (423) Google Scholar, 16Semina EV Ferrell RE Mintz-Hittner HA Bitoun P Alward WL Reiter RS Funkhauser C Daack-Hirsch S Murray JC A novel homeobox gene PITX3 is mutated in families with autosomal-dominant cataracts and ASMD.Nat Genet. 1998; 19: 167-170Crossref PubMed Scopus (317) Google Scholar, 20Honkanen RA Nishimura DY Swiderski RE Bennett SR Hong S Kwon YH Stone EM Sheffield VC Alward WL A family with Axenfeld-Rieger syndrome and Peters anomaly caused by a point mutation (Phe112Ser) in the FOXC1 gene.Am J Ophthalmol. 2003; 135: 368-375Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar All these genes have not been exhaustively screened in the patients with FOXE3 mutations reported by Semina et al.22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar or Ormestad et al.23Ormestad M Blixt A Churchill A Martinsson T Enerback S Carlsson P Foxe3 haploinsufficiency in mice: a model for Peters' anomaly.Invest Ophthalmol Vis Sci. 2002; 43: 1350-1357PubMed Google Scholar It is, therefore, not excluded that the presence of additional variants in genes having related functions in the development of the anterior segment of the eye could likely influence the penetrance and expressivity of ocular phenotypes observed in patients with FOXE3. At the present time, the small number of patients reported with FOXE3 mutations limits an accurate genotype-phenotype correlation, which will await the complete understanding of the pathogenic mechanism of FOXE3 mutations. The involvement of FOXE3 in this family is interesting, in different regards. First, it is a gene whose expression is restricted to prelens and lenticular tissues, which provides the opportunity to better define the direct role of the lens on the formation of the diverse structures of the eye.21Blixt A Mahlapuu M Aitola M Pelto-Huikko M Enerback S Carlsson P A forkhead gene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle.Genes Dev. 2000; 14: 245-254PubMed Google Scholar, 22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar In this family, the disease phenotype is perfectly restricted to ocular tissues, as is expected for this gene. Similarly to Foxe3−/− mice, the brain structures of our patients have not shown any malformations, although Foxe3 shows transient expression in discrete regions of the developing midbrain between E8.5 and E12.0 of mouse embryogenesis.21Blixt A Mahlapuu M Aitola M Pelto-Huikko M Enerback S Carlsson P A forkhead gene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle.Genes Dev. 2000; 14: 245-254PubMed Google Scholar, 22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar Secondly, our data emphasize the role of FOXE3 as essential for early developmental stages of the lens—an unexpected finding—while the molecular cascades that result in the formation of a lens are being elucidated.6Graw J The genetic and molecular basis of congenital eye defects.Nat Rev Genet. 2003; 4: 876-888Crossref PubMed Scopus (168) Google Scholar, 7Lang RA Pathways regulating lens induction in the mouse.Int J Dev Biol. 2004; 48: 783-791Crossref PubMed Scopus (140) Google Scholar, 26Grainger RM Embryonic lens induction: shedding light on vertebrate tissue determination.Trends Genet. 1992; 8: 349-355Abstract Full Text PDF PubMed Scopus (183) Google Scholar In the mouse, two spontaneous Foxe3 missense mutations predicted to be null alleles cause the recessive dysgenetic lens (dyl) mouse phenotype.21Blixt A Mahlapuu M Aitola M Pelto-Huikko M Enerback S Carlsson P A forkhead gene, FoxE3, is essential for lens epithelial proliferation and closure of the lens vesicle.Genes Dev. 2000; 14: 245-254PubMed Google Scholar, 22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar The dyl mice, which are viable and appear normal, have defects in lens vesicle closure and separation, which implies that Foxe3 is an essential regulator of this event. Finally, mice deficient in Foxe3 through targeted gene disruption show lens abnormalities similar to those described for the dyl mice.27Medina-Martinez O Brownell I Amaya-Manzanares F Hu Q Behringer RR Jamrich M Severe defects in proliferation and differentiation of lens cells in Foxe3 null mice.Mol Cell Biol. 2005; 25: 8854-8863Crossref PubMed Scopus (68) Google Scholar Despite these two mouse Foxe3 models, the murine lens phenotype remains milder than that observed in our aphakic children carrying the C240X nonsense mutation in the human gene counterpart. Usually, haploinsufficiency mutations in murine and in human orthologous genes result in similar phenotypes, and a large number of animal models have contributed to the understanding of human diseases. However, interspecies differences exist, and it is accepted that mice are often more tolerant of haploinsufficiency for developmental genes than are humans.10Fitzpatrick DR van Heyningen V Developmental eye disorders.Curr Opin Genet Dev. 2005; 15: 348-353Crossref PubMed Scopus (74) Google Scholar One possibility to explain these phenotypic differences may be the existence in the mouse of an additional Foxe gene family member, which may be functioning partly redundantly to Foxe3. Indeed, appreciable differences have been noted elsewhere between ocular phenotypes of certain human and murine forkhead orthologues, whose mechanisms have not been clarified.28Lehmann OJ Tuft S Brice G Smith R Blixt A Bell R Johansson B Jordan T Hitchings RA Khaw PT John SW Carlsson P Bhattacharya SS Novel anterior segment phenotypes resulting from forkhead gene alterations: evidence for cross-species conservation of function.Invest Ophthalmol Vis Sci. 2003; 44: 2627-2633Crossref PubMed Scopus (44) Google Scholar Alternatively, the possibility remains that our FOXE3 nonsense mutation does not result in total loss of function, and we cannot formally exclude the possibility that this mutation could result, in fact, in partial loss of function. The human FOXE3 gene contains a single coding-exon genomic structure, and some intronless genes have been reported to be insensitive to NMD.25Holbrook JA Neu-Yilik G Hentze MW Kulozik AE Nonsense-mediated decay approaches the clinic.Nat Genet. 2004; 36: 801-808Crossref PubMed Scopus (470) Google Scholar Consequently, it remains possible that PTC mutations in FOXE3 could escape RNA surveillance and could produce truncated mutant proteins. In this scenario, absence of lens in our patients could result in the production of a truncated protein that is still capable of binding DNA but is devoid of the 80 C-terminal aa. FOXE3 is a transcription factor that acts by switching on and off expression of other genes and that must be correctly activated or repressed at the appropriate time during lens development. However, the targets of FOXE3 are not known, and it is not known whether FOXE3 could act synergistically with other partner(s), as Pax6 and Sox2 do, both of which form a co–DNA-binding complex that regulates initiation of lens development.12Kamachi Y Uchikawa M Tanouchi A Sekido R Kondoh H Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.Genes Dev. 2001; 15: 1272-1286Crossref PubMed Scopus (289) Google Scholar The C240X mutation lies outside the forkhead domain of FOXE3, but it is included in a 26-aa motif (PEPPCCAAPDAAAAAFPPCAAAASPPLYS) that is highly conserved between mouse and human, which suggests that this motif is, most probably, important for the normal function of FOXE3 (fig. 1C). We could conceive that these residues might be essential, for example, to exert the transactivation effects of FOXE3 in its interactions with some unidentified partners, even though DNA binding still occurred. Thus, a full understanding of the potential role of FOXE3 in early stages of lens development would require the identification of genes that may be directly controlled by FOXE3 at this specific developmental stage and the elucidation of the mechanisms by which FOXE3 exerts its actions. However, in the developing eye of the mouse, the onset of Foxe3 expression begins very early in the presumptive lens ectoderm, and its expression is first detected as early as E.8.75, a period when the Pax6 ectoderm enhancer becomes active and directs Pax6 expression in the surface ectoderm (SE).7Lang RA Pathways regulating lens induction in the mouse.Int J Dev Biol. 2004; 48: 783-791Crossref PubMed Scopus (140) Google Scholar, 22Brownell I Dirksen M Jamrich M Forkhead Foxe3 maps to the dysgenetic lens locus and is critical in lens development and differentiation.Genesis. 2000; 27: 81-93Crossref PubMed Scopus (100) Google Scholar, 29Williams SC Altmann CR Chow RL Hemmati-Brivanlou A Lang RA A highly conserved lens transcriptional control element from the Pax-6 gene.Mech Dev. 1998; 73: 225-229Crossref PubMed Scopus (112) Google Scholar During lens placode formation at E.9.5, Foxe3 expression is upregulated in the SE, in conjunction with transcription factors Six3, Sox2/Sox3, and Prox1.8Ashery-Padan R Gruss P Pax6 lights-up the way for eye development.Curr Opin Cell Biol. 2001; 13: 706-714Crossref PubMed Scopus (183) Google Scholar, 11Goudreau G Petrou P Reneker LW Graw J Loster J Gruss P Mutually regulated expression of Pax6 and Six3 and its implications for the Pax6 haploinsufficient lens phenotype.Proc Natl Acad Sci USA. 2002; 99: 8719-8724Crossref PubMed Scopus (74) Google Scholar, 12Kamachi Y Uchikawa M Tanouchi A Sekido R Kondoh H Pax6 and SOX2 form a co-DNA-binding partner complex that regulates initiation of lens development.Genes Dev. 2001; 15: 1272-1286Crossref PubMed Scopus (289) Google Scholar Then, Foxe3 expression becomes confined to the lens vesicle as this structure detaches from the SE (E10.5), a process that fails in both dyl and Foxe3−/− mice. Therefore, the temporal expression pattern of Foxe3 highly supports a possible role for Foxe3 in earlier stages of lens development upstream of events such as lens vesicle separation or lens lineage proliferation. A unique demonstration in support of this hypothesis is the identification here—to our knowledge, for the first time—of a nonsense mutation in FOXE3 that cosegregates with familial CPA. Although identifications of human disease-causing genes have been often guided by study of their murine orthologues, the family in this article illustrates that the converse approach might also provide a means of identifying a novel phenotype that has not yet been revealed in mice. We are indebted to the children and their parents for participating in this study. We thank C. Fromentin, P. Roth, and C. Moret, for their help with clinical and radiological investigations, and Susan Saint-Just, for her helpful assistance with English.