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Am J Hum Genet. Jun 2005; 76(6): 1008–1022.
Published online Apr 21, 2005. doi:  10.1086/430721
PMCID: PMC1196439

Heterozygous Mutations of OTX2 Cause Severe Ocular Malformations


Major malformations of the human eye, including microphthalmia and anophthalmia, are examples of phenotypes that recur in families yet often show no clear Mendelian inheritance pattern. Defining loci by mapping is therefore rarely feasible. Using a candidate-gene approach, we have identified heterozygous coding-region changes in the homeobox gene OTX2 in eight families with ocular malformations. The expression pattern of OTX2 in human embryos is consistent with the eye phenotypes observed in the patients, which range from bilateral anophthalmia to retinal defects resembling Leber congenital amaurosis and pigmentary retinopathy. Magnetic resonance imaging scans revealed defects of the optic nerve, optic chiasm, and, in some cases, brain. In two families, the mutations appear to have occurred de novo in severely affected offspring, and, in two other families, the mutations have been inherited from a gonosomal mosaic parent. Data from these four families support a simple model in which OTX2 heterozygous loss-of-function mutations cause ocular malformations. Four additional families display complex inheritance patterns, suggesting that OTX2 mutations alone may not lead to consistent phenotypes. The high incidence of mosaicism and the reduced penetrance have implications for genetic counseling.


Abnormalities in the ocular development program can lead to a variety of major structural defects of the eye that present at birth (Graw 2003). At the severe end of the spectrum are anophthalmia (absence of the eye [MIM 206900]) and extreme microphthalmia (small eye [MIM 309700]), which are likely to result from a variety of developmental pathologies (Morrison et al. 2002). Other common malformations include sclerocornea (MIM 181700); anterior chamber malformations, including aniridia (MIM 106210), colobomata (MIM 120200), and congenital cataracts (MIM 116700); and disorders of early retinal differentiation. Diagnostically useful subclasses within these groups include optic-fissure closure defects (colobomata affecting iris, optic nerve, retina, and choroid) (Morrison et al. 2002), disorders of early retinal differentiation (Leber congenital amaurosis [LCA {MIM 204000}] and Walker-Warburg syndrome [MIM 236670]) (Dobyns et al. 1989), and disorders of lens induction or differentiation (congenital cataract/aphakia [MIM 177075]) (Jamieson et al. 2002). Each of these can be isolated or associated with microphthalmia and/or anophthalmia.

Ocular malformations display a broad range of inheritance patterns. Classic aniridia is almost always caused by PAX6 (MIM 607108) haploinsufficiency and is transmitted with high penetrance and expressivity (Hanson 2003). In contrast, anophthalmia and severe microphthalmia show some evidence of familial recurrence but usually no clear Mendelian transmission pattern (Morrison et al. 2002). This is likely to be a reflection of several potentially interactive factors: oligogenic causation, gene-environment interactions, and stochastic variation in development. There is increasing recognition that, although non-Mendelian inheritance patterns make gene identification more difficult, they are ultimately very revealing about the complex molecular networks that contribute to normal development (Ming and Muenke 2002; van Heyningen and Yeyati 2004).

Several genes for anophthalmia, microphthalmia, and coloboma (RAX [MIM 601881], CHX10 [MIM 142993], PAX6, MAF [MIM 177075], and SOX2 [MIM 184429]) have been identified by different methods, ranging from the candidate-gene approach to positional identification, often using informative chromosomal rearrangements (Azuma et al. 1999; Ferda-Percin et al. 2000; Jamieson et al. 2002; Fantes et al. 2003; Graw 2003; Voronina et al. 2004). Apart from SOX2, which appears to account for ~10% of severe anophthalmias (Fantes et al. 2003; Ragge et al. 2005), none of these can be considered a major causative gene, because each examined cohort revealed mutations in a very small proportion of affected individuals. We decided to search for additional causative mutations, using a candidate-gene approach.

OTX2 (MIM 600037), a bicoid-type homeodomain gene, is a vertebrate ortholog of the Drosophila gene orthodenticle (Otd), which is required for anterior brain, eye, and antenna formation (Finkelstein and Boncinelli 1994) and for regulating the development of photoreceptors and the expression of rhodopsin (Tahayato et al. 2003). Mammals have three Otx genes: Otx1, Otx2, and the more diverged Crx, which is known as “Otx5” in lower vertebrates (Germot et al. 2001). All three genes are expressed in the developing eye—Crx almost exclusively so. Human CRX (MIM 602225) mutations have been reported in retinal degenerations, including cone-rod dystrophy (MIM 120970), late-onset pigmentary retinopathy, and LCA (Freund et al. 1997; Sohocki et al. 1998; Rivolta et al. 2001). Mouse Otx1 and Otx2 are expressed in developing neural and sensory structures, including the brain, ear, nose, and eye (Simeone et al. 1993; Martinez-Morales et al. 2001). The spatiotemporally complex expression pattern is reflected in the large flanking region that is occupied by enhancers required for the regulation of Otx2 expression (Kurokawa et al. 2004a, 2004b). Ectopic expression studies in Xenopus suggest that Otx2 operates early, interacting with a network of eye-field transcription factors, including Rx1, Pax6, and Six3 (Zuber et al. 2003). During eye morphogenesis, initial expression in the entire optic vesicle becomes restricted to the presumptive retinal pigment epithelium (RPE) (Simeone et al. 1993; Bovolenta et al. 1997; Martinez-Morales et al. 2001), where OTX2 protein interacts with the transcription factor MITF (MIM 156845), leading to the activation of target genes, including tyrosinase (Martinez-Morales et al. 2003). Later, OTX2 is increasingly expressed in neural retinal cells, including postmitotic neuroblasts (Bovolenta et al. 1997; Nishida et al. 2003). Conditional ablation of Otx2 in neural retina leads to loss of Crx expression and failure of photoreceptor development (Nishida et al. 2003).

Homozygous Otx2-knockout embryos have gastrulation defects and die at midgestation with severe brain anomalies (Acampora et al. 1995; Ang et al. 1996). Heterozygotes show highly variable phenotypes—ranging from acephaly; through micrognathia, anophthalmia, and microphthalmia; to normal—depending on genetic background (Acampora et al. 1995; Matsuo et al. 1995; Ang et al. 1996). Recurrent interstitial deletions of human chromosome 14q22-q23 have been consistently associated with anophthalmia (Brewer et al. 1998). No definitive analysis of OTX2 has been reported in these cases, but the gene maps to the 14q22 genomic region and is a strong candidate for mutation studies in anophthalmia and related eye anomalies.

We therefore undertook mutation analysis of the OTX2 gene. The common 289-aa isoform is transcribed from a 5-kb genomic region encompassing three exons. The protein has a homeodomain and a 192-aa proline-, serine-, and threonine-rich C-terminal region, which contains a highly conserved SIWSPA peptide sequence and a tandemly duplicated OTX tail, all motifs shared with OTX1 (MIM 600036) and CRX (Germot et al. 2001). Unrelated patients (n=333) with a spectrum of ocular malformations were assessed by direct sequencing. Brain magnetic resonance imaging (MRI) data were obtained for two affected individuals and for an unaffected mutation carrier, and older diagnostic images from other patients were reassessed. The OTX2 expression pattern was studied in early human embryos for comparison with the known mouse pattern.

Material and Methods

Patients and Controls

Patients with ocular malformation spectrum defects (n=333) were recruited, with informed consent and with appropriate ethics review committee approvals. Detailed case histories of individuals with OTX2 coding-region sequence changes are available in appendix A (online only). Control DNAs (n=164) from apparently healthy adults were anonymized; they matched the patients in ethnicity (apart from patient 7, who was of African origin [see the “Results” section]). We sequenced 94 DNAs in full, and a further 70 partially, over the region of the proline missense mutations.

DNA Preparation

Genomic DNA was prepared from peripheral blood by use of a Nucleon DNA extraction kit (Tepnel Life Sciences). Buccal cell lysates were prepared from mouthwash samples by resuspending the cell pellet in 100 μl of 0.05 M NaOH, heating in a boiling water bath for 20 min, and then neutralizing with 12.5 μl of 1 M Tris (pH 7.6).

PCR and Sequencing

The human OTX2 cDNA (GenBank accession number NM_172337) is encoded by three exons that are located in BAC AL161757 (GenBank) at positions 59950–60217 (exon 1), 61214–6138 (exon 2), and 6322–64868 (exon 3). The cDNA numbering is derived from the human OTX2 cDNA (GenBank accession number NM_172337). We designed four genomic amplicons to cover the OTX2 coding region and some flanking intronic sequences in each case: amplicon 1 contained exon 1, amplicon 2 contained exon 2, and overlapping amplicons 3a and 3b covered the coding part of exon 3. PCR primers are shown in table 1. PCRs were performed in 96-well plates. For amplicons 1 and 2, 50 ng genomic DNA was amplified in a volume of 50 μl containing 1× Qiagen HotStarTaq PCR buffer, 1.5 mM MgSO4, 5 μl PCR enhancer buffer (Invitrogen), 0.2 mM dNTPs, 250 nM forward primer, 250 nM reverse primer, and 2 U of HotStarTaq polymerase. For amplicons 3a and 3b, the PCR enhancer buffer was omitted and was replaced with 10 μl of Q solution (Qiagen). Thermal cycling was performed using an MJ PTC225 machine. PCR conditions were 1 cycle of 95°C for 15 min; 35 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 1 cycle of 72°C for 5 min.

Table 1
PCR Primers for Amplification of the OTX2 Coding Region

PCR products were run on a 2% agarose gel to ensure adequate yield and to check for the absence of nonspecific products. Unincorporated primers and dNTPs were removed by incubating 5 μl of PCR product with 1 μl of ExoSapIT (exonuclease I and shrimp alkaline phosphatase enzyme mix [USB]) for 45 min at 37°C, followed by 20 min at 80°C to inactivate the enzyme.

Direct Sequencing

Sequencing of PCR products was performed in a 96-well plate format, as described elsewhere (Aijaz et al. 2004), with the use of forward and reverse primers as shown in table 1. Individual sequence traces were inspected using Chromas software (Technelysium). Multiple sequence traces were aligned and compared using the phredPhrap program from the Consed package (see the Laboratory of Phil Green Web site). All changes were verified by performing a second PCR and sequencing reaction on the patient’s DNA. Family members were analyzed if samples were available.

RNA In Situ Hybridization

The expression pattern of OTX2 was investigated by nonradioactive RNA in situ hybridization in human embryos from Carnegie stages (CS) 14–22, which correspond approximately to mouse Theiler stages 18–23 (E11.25–E15.25). Human embryos were obtained from the Medical Research Council/Wellcome Trust Human Developmental Biology Resource, with full ethical approval. Preparation of embryo sections and nonradioactive RNA in situ hybridization was performed as described elsewhere (Lai et al. 2003).

The template for the OTX2 probe was a 1.1-kb fragment that was PCR amplified from exon 3 with the use of primers hOTX2A (5′-GCG GAT CCA GTG CTC CTG TGT CTA TCT GGA G-3′) and hOTX2D (5′-GCG AAT TCC AGA GGT GGA GTT CAA GGT TGC-3′) and that was cloned into pGEM-3Z. The construct was linearized with BamHI and was transcribed with T7 RNA polymerase to synthesize the digoxygenin-labeled antisense riboprobe.

MRI Analysis

MRI scanning was approved by the local institutional ethics committees. All patients scanned for the present study provided written informed consent. Patient 5 and his unaffected mother underwent MRI scanning. MRI acquisition parameters were TE = 4.2, TI = 450, TR = 15, NEX = 1, flip angle = 20°, acquisition matrix 256 × 128, and field of view 24 cm; 124 contiguous slices were produced, with voxel dimension 0.9375 mm × 0.9375 mm × 1.5 mm. An equivalent sequence was used for patient 4B. These data were reformatted in multiple planes to allow careful examination of regions of interest. Additionally, signal changes were inspected on T2 and FastFLAIR sequences (T2 and PD sequence: TE1 = 30, TE2 = 120, TR = 2000, NEX = 1, acquisition matrix 256 × 128, field of view 24 cm × 18 cm, and slice thickness 5 mm contiguous; FastFLAIR sequence: TE1 = 152, TE2 = 2200, TR = 10002, NEX = 1, acquisition matrix 256 × 128, field of view 24 cm, and slice thickness 5 mm contiguous). Patient 4B underwent similar high-resolution T1-weighted MRI scanning. For patients 1, 2, and 6, existing MRI data were reviewed. For patient 3A, CT scan data were reviewed.

Psychometric Testing

Patient 5 and his mother underwent standard neuropsychometric testing, omitting any tests requiring visual function. This component of the study was also approved by the local institutional ethics committee and was undertaken after written informed consent was obtained.


OTX2 Mutation Analysis

Of the 333 patients analyzed, 8 showed sequence variation in the OTX2 coding region. The changes are distributed throughout the ORF (fig. 1). Additional family members were analyzed, if samples were available, for a total of 11 affected individuals in eight families (fig. 1). Detailed case histories of affected individuals are presented in appendix A (online only), and the phenotypes are summarized in table 2. In the course of the study, we uncovered evidence of mosaicism; therefore, when possible, we also analyzed oral epithelial DNA (obtained by mouthwash or buccal scrape).

Figure  1
OTX2 mutation analysis in eight families with congenital eye malformations. Top, Schematic diagram of the human OTX2 cDNA, showing the homeodomain (“HD”) (red rectangle); SIWSPA motif (dark blue bar); SGQFTP motif (orange bar); and tandemly ...
Table 2
Summary of Clinical Features[Note]

Patient 1, with bilateral anophthalmia, has a de novo frameshifting insertion (c.635insGC) at codon 155 in exon 3, the A in the SIWSPA motif (figs. (figs.11 and and22 and table 2). Subsequent out-of-frame translation of 22 amino acids is predicted to terminate at a TGA codon, beginning at c.701. Neither the parents nor the unaffected brother carry the mutation (fig. 1). Identical results were obtained with buccal cell DNA from all four individuals.

Figure  2
Facial views of eight patients with OTX2 coding-region sequence changes: patient 1 (A), patient 3A (B), patient 4A (C), patient 4B (D), patient 5 (E), patient 6 (F), patient 7 (G), and patient 8 (H). Patients 5 and 7 are wearing prostheses in both eyes. ...

Patient 2, with bilateral optic-nerve aplasia (MIM 165550) and microphthalmia (fig. 3 and table 2), has a de novo transversion, c.436C→G, in exon 3 (fig. 1). This is predicted to produce an arginine-to-glycine (R89G) change at position 52 of the homeodomain. Neither parent carries the change in lymphocyte DNA (fig. 1).

Figure  3
Ocular findings for patient 2 at age 6 wk. The right eye (A) and left eye (B) are shown, both with persistent pupillary membrane and tunica vasculosa lentis and a clear lens. Ultrasound scans of the right (C) and left (D) orbits show small eyes. In panel ...

Patient 3A, with severe bilateral microphthalmia, has an N-terminal domain frameshifting mutation, c.252delC, in codon 27 of exon 1 (figs. (figs.11 and and22 and table 2). Out-of-frame translation is predicted until the first base of a termination codon is encountered at c.320 in exon 2. A fetus (patient 3B [fig. 1]), terminated after ultrasound diagnosis of affected status, also carried the same mutation. The phenotypically normal mother of patients 3A and 3B was found to have the mutation at low levels in blood and buccal DNA before the existence of the affected fetus was revealed. The unaffected father and maternal grandparents do not have the mutation in blood DNA (fig. 1). These data indicate that the mother is a gonosomal (gonadal and somatic) mosaic carrier of the mutation (Edwards 1989).

Siblings 4A and 4B—diagnosed with microphthalmia and severe developmental delay and with LCA, respectively (figs. (figs.22 and and44 and table 2)—carry a transversion, c.708T→A, in exon 3 (fig. 1). The tyrosine-to-termination (TAT→TAA) change at codon 179 is in the C-terminal domain. Their mother (patient 4C) is a gonosomal mosaic for the Y179X mutation (fig. 1). She has a later-onset phenotype resembling pigmentary retinopathy (fig. 4).

Figure  4
Ocular findings in family 4. Patient 4B at age 28 years: right fundus (A) and left fundus (B), both with pale optic disks, atrophic maculae, and thin retinal vessels (arrowheads); right iris (C) and left iris (D). The left iris has peripheral anterior ...

Patient 5, with bilateral anophthalmia, has a frameshifting deletion, c.288delCC, in codons 39–40 in exon 2, at the start of the homeodomain (figs. (figs.11 and and22 and table 2). This mutation is predicted to generate a 46-aa nonsense peptide before reaching a TAA termination codon, beginning at c.429 in exon 3. The phenotypically normal mother of this patient has full representation of the same mutation in both lymphocyte and buccal DNA, indicating that she is probably a nonmosaic mutation carrier. The unaffected father, brother, and sister do not carry the mutation (fig. 1).

Patient 6, with bilateral anophthalmia, has the transition c.466C→T in exon 3 (figs. (figs.11 and and22 and table 2). Glutamine (CAA) is substituted by a termination codon (TAA) at the second amino acid of the C-terminal domain (Q99X). The phenotypically normal father shows full expression of the same mutation in his blood DNA, suggesting that he is a constitutive mutation carrier. The unaffected mother does not have the mutation (fig. 1).

Patient 7, with bilateral microphthalmia and left sclerocornea, has the transversion c.568C→A in exon 3 (figs. (figs.11 and and22 and table 2). This results in replacement of proline (CCC) with threonine (ACC) at position 133 (P133T). The patient’s phenotypically normal mother and brother have the same change, but the patient’s unaffected father and sister do not (fig. 1). Identical results were obtained with DNA from blood and oral epithelium from all five family members. P133 occurs at the end of a highly conserved hexapeptide motif, SGQFTP, in the C-terminal domain of vertebrate Otx2, Otx1, and Crx proteins (Germot et al. 2001).

Patient 8, with unilateral anophthalmia, has the transversion c.571C→G in exon 3 (figs. (figs.11 and and22 and table 2). This replaces proline (CCC) with alanine (GCC) at amino acid position 134 (P134A). The patient’s similarly affected mother does not have this variant in blood or oral epithelium. The father was not available for analysis. The patient’s unaffected maternal grandmother and maternal great-grandmother do not have the change, nor does a maternal half brother (fig. 1).

Ninety-four control individuals (188 chromosomes) were examined by direct sequencing for the presence of all the coding-region variants. An additional 70 individuals were sequenced for the region containing prolines 133 and 134. None of the changes was observed in the controls. Ethnic matching was not feasible for family 7, who are of African origin, and therefore no conclusion can be drawn about the frequency of this change in the population from which patient 7 comes.

We detected three SNPs during the course of the present study. All were located in noncoding regions: two newly reported sites (submitted to NCBI dbSNP) in the first intron, 268+12 C/T (dbSNP accession number ss35522250) and 269–70 C/A (dbSNP accession number ss35522251), and one already-known variant near the start of the 3′ UTR (c.1050 G/A) (dbSNP accession number rs171978).

MRI Data and Neuropsychometric Testing of OTX2 Mutation Carriers

Three of the patients with OTX2 mutations have severe learning disabilities; these disabilities are associated, in two patients, with frequent seizures (table 2). These neurological phenotypes, together with the extensive expression of OTX2 in the brain, indicated that MRI scans and neuropsychometric testing might be informative, as shown for PAX6 (Sisodiya et al. 2001).

Patient 4B, patient 5, and the unaffected mother of patient 5 had MRI scans. Patient 5 and his mother also underwent neuropsychometric testing. Original diagnostic MRI data were reviewed for patients 1, 2, and 6, and CT data were reviewed for patient 3A. For patients 2 and 3A, only ocular data were available.

In four of the six patients for whom data are available, the optic nerves and optic chiasm are abnormal (fig. 3 and table 2). The nerves and chiasm are absent in patients 2 and 6 and are hypoplastic in patient 5. In patient 1, the optic nerve is absent on the anophthalmic side and is hypoplastic on the microphthalmic side; the chiasm is thin.

Patients 3A and 5 both had slightly enlarged lateral ventricles. A striking cerebral anomaly is evident in patient 6, who has bilateral abnormal hippocampal morphology (fig. 5). A similar hippocampal malformation was seen, on review, in patient 1.

Figure  5
Coronal MRIs from patient 5 (T1-weighted image [A]), a normal control (T1-weighted image [B]), and patient 6 (T2-weighted image [C]). The left side of each image is the right side of the patient. In panels A and C, the hippocampi are circled. Panel B ...

Patient 5 functions intellectually just within the average ability range (Wechsler Adult Intelligence Scale–Revised [WAIS-R]: verbal IQ [VIQ] 91). His verbal memory was good and was in keeping with his intellectual level (verbal learning and recall 25th–50th percentile). His performance on tests of executive skills was average. His 55-year-old mutation-carrying mother has no eye phenotype and functions intellectually at a level comparable to that found for her son (VIQ 96). Her memory was not as good as that of her son: after correcting for age, her scores were in the 10th–25th percentile. Executive skills were average.

Patient 2 was described as having cognitive and language skills at least age-appropriate for a blind child when a neurodisability assessment was performed at age 4.5 years.

RNA In Situ Hybridization

The developing human eye at CS16–CS19, like that of the mouse at a corresponding age, shows strong OTX2 expression in the RPE layer and weaker OTX2 expression in the neural retina (fig. (fig.6F,6F, ,6G,6G, and and6H6H and data not shown). Mice have low-level expression in the E11 neural retina, which becomes restricted to the neuroblastic layer by E17.5 and which remains present in the inner nuclear layer into the postnatal period (Martinez-Morales et al. 2001; Nishida et al. 2003).

Figure  6
Expression pattern of the human OTX2 gene in the developing head (A–E) and eye (F–H). Panels show representative results of nonradioactive in situ hybridization with a human OTX2 probe. All panels (apart from panel C, which shows a sense ...

OTX2 is widely expressed throughout the telencephalon and mesencephalon at CS14, with a sharp cutoff at the midbrain-hindbrain boundary (fig. 6A). OTX2 expression was observed in the lamina terminalis and the floor of the telencephalon at CS16 (fig. (fig.6B6B and and6C),6C), sites that have also been reported in mouse (Simeone et al. 1993; Mallamaci et al. 1996; Simeone 2000). At CS22, OTX2 was expressed in the choroid plexus, the dorsal thalamus, and the roof of the mesencephalon (fig. 6E). In the developing nasal structures, OTX2 is seen in the olfactory epithelium of the nasal pits at CS18 (fig. 6D), again mirroring the mouse patterns.


Using a candidate-gene approach, we found a wide spectrum of human phenotypes to be associated with OTX2 coding-region sequence changes. The phenotypes were, however, significantly different from those in the mouse knockouts (Acampora et al. 1995; Matsuo et al. 1995; Ang et al. 1996). The inheritance patterns in the families are diverse.

Spectrum of OTX2 Mutations and Genetic Mechanisms

We found eight different heterozygous OTX2 exonic changes in the patient cohort. Five of the eight changes are predicted to cause premature protein truncation either through direct creation of a stop codon or through a frameshift (fig. 1 and table 2). The only mutation predicted to lead to haploinsufficiency through nonsense-mediated decay (reviewed by Holbrook et al. [2004]) is in family 3, in which a stop codon is predicted in exon 2. In the other four families (1, 4, 5, and 6), termination is predicted in the last exon—making it likely that the truncated protein is expressed and that it may function in a dominant negative manner (Nussbaum et al. 2001).

The R89G de novo missense change found in patient 2 causes substitution of an invariant homeodomain arginine that is normally in contact with DNA phosphate residues. Missense mutations of the homologous residue have been identified in seven different homeodomain proteins, three of them (PITX2 [MIM 601542], SIX3 [MIM 603714], and CRX) associated with eye disease (Swaroop et al. 1999; D’Elia et al. 2001; Laflamme et al. 2004).

The mutations found in patient 1 (635insGC) and patient 2 (R89G) arose de novo. In siblings 3A and 3B (252delC) and siblings 4A and 4B (Y179X), the sequence changes were inherited from mothers who were found to carry, at a low level in blood DNA, the same mutation as their offspring. In both cases, the mother transmitted the mutation to two affected children, providing strong evidence that these mothers are gonosomal mosaics with a high frequency of mutant cells in their germ lines (Edwards 1989). The parents of the mosaic individual should not have the mutation, as observed in family 3 (fig. 1). Gonosomal mosaic individuals may or may not express clinical features themselves, depending on the proportion and distribution of mutant cells in their body. The mosaic mother in family 3 has undergone a full eye examination, including high-resolution multifocal electroretinogram (ERG), and is ophthalmologically normal. The mosaic mother in family 4 (patient 4C) has progressive retinal dystrophy (fig. 4E and 4F), with a self-reported history of undefined visual problems from an early age. Typically, an affected mosaic individual has a milder phenotype than his or her constitutive offspring (Zlotogora 1998). This seems to be true for patient 4C, since she has retinal pathology without the structural eye defects observed in her children.

Families 1–4 fit a model in which OTX2 haploinsufficiency (loss of function in one allele) alone causes structural eye malformations. However, families 5 and 6 provide evidence of incomplete penetrance, since predicted protein-truncating mutations identified in bilateral anophthalmic children are also found in an unaffected mother and father, respectively (fig. 1 and table 2).

Results from families 7 and 8 are difficult to interpret. Both have missense substitutions at adjacent highly conserved proline residues, but the functional consequences of these sequence changes are unknown. Both families show discordance of phenotype and genotype. For family 7, no ethnically matched controls were available, so the frequency of the P133T change in the population of origin of family 7 is unknown. In family 8, the affected son has the P134A change, but his very similarly affected mother does not, making it highly unlikely that this sequence variant is a major contributor to the phenotype. We shall now focus on the loss-of-function mutations.

Two different models, based on recent insights from other human genetic diseases, may explain incomplete penetrance. First, if OTX2 is the only locus involved, the level of gene expression may be affected by stochastic and/or genetic factors. This model assumes that an abnormal phenotype will be manifest if OTX2 expression falls below a certain critical threshold level and if loss of function of one copy reduces gene expression levels close to this threshold. Stochastic fluctuations in transcription levels, possibly influenced by environmental factors but independent of genetic background, may reduce expression levels below the threshold. Evidence of this comes from the observation that genetically identical Otx2-knockout mice have highly variable phenotypes (Matsuo et al. 1995). Genetic factors at the mutant locus—specifically, regulatory polymorphisms of the normal allele—can also determine penetrance by controlling transcription levels (Gouya et al. 2002; Vithana et al. 2003). If the mutant allele is in trans to a strongly expressing normal allele, transcription is maintained above the threshold, which explains the clinically unaffected status of constitutive mutation carriers, but a weakly expressing normal allele leads to expression below the threshold required for normal development, and the abnormal phenotype is revealed (Gouya et al. 2002). The human OTX2 locus spans >500 kb and has numerous distant enhancers that could be targets for regulatory polymorphisms (Kurokawa et al. 2004b).

Second, digenic or oligogenic inheritance may manifest as incomplete penetrance (Ming and Muenke 2002; van Heyningen and Yeyati 2004). An example of digenic inheritance is provided by a form of retinitis pigmentosa in which affected individuals have heterozygous mutations in both the ROM1 (MIM 180721) and RDS (MIM 179605) genes, which encode related components of an oligomeric photoreceptor protein. Mutations at only one locus do not lead to an abnormal phenotype; penetrance is dependent on cosegregation of both mutations (Kajiwara et al. 1994). Perhaps more relevant to the developmental complexity of the eye and brain is holoprosencephaly (HPE [MIM 142945 and MIM 157170]), a disease that frequently shows non-Mendelian segregation patterns (Ming and Muenke 2002). The complex inheritance patterns and range of phenotypes we observe for OTX2 are similar to those reported for SHH (MIM 600725) and SIX3, the major contributors toward HPE types 3 and 2, respectively (Nanni et al. 1999; Wallis et al. 1999). Whereas some HPE-associated SHH and SIX3 mutations have arisen de novo, others have been inherited from completely unaffected carrier parents (Nanni et al. 1999; Wallis et al. 1999). The possibility that interaction with modifier gene variants at other loci may be required for full penetrance is supported by the observation that, in three reported cases, affected individuals carry mutations in two HPE genes (SHH with TGIF [MIM 602630] or ZIC2 [MIM 603073]) (Nanni et al. 1999; Ming and Muenke 2002). Most HPE genes are involved in developmental signaling, and it is possible that reduction in the dosage of one protein can be buffered but two cannot, so that expression of an abnormal phenotype may depend on mutations in more than one gene (Ming and Muenke 2002).

Of six families with loss-of-function mutations, four have mutation-carrier parents (families 3, 4, 5, and 6 [fig. 1]), three of whom are unaffected. Two parents are probably constitutive mutation carriers, and two are gonosomal mosaics. This presents challenges for genetic counseling of families with OTX2 mutations. Estimating the risk of having an affected child is extremely difficult. The relationship between levels of somatic and germline mosaicism is unpredictable. Little is known about either the precise stage at which the germ line is determined in humans or the number of precursor cells recruited. However, the number of cells ancestral to the germ line in mammals is thought to be small, so an individual who is gonosomal mosaic is likely to have a significant proportion of germ cells carrying the mutation (Zlotogora 1998; Leuer et al. 2001). Consequently, a relatively high recurrence risk is predicted for the offspring of gonosomal mosaic individuals, even if the proportion of mutant alleles in blood DNA is low. This has been observed in practice by others (Zlotogora 1998; Leuer et al. 2001) and in the present study, with both gonosomal mosaic mothers passing the mutation to two affected offspring. Constitutive-mutation-carrier parents are expected to be at 50% risk of having an affected child; however, recurrence was observed only in families with a mosaic parent. The lack of penetrance of OTX2 mutations must also be taken into account; the recurrence risk will presumably be lower if, as we suggest, other factors are required for manifestation of an abnormal phenotype. The relevant factors are, however, already present in families with one or more affected offspring.

Unaffected parents may be constitutive or mosaic mutation carriers; therefore, it is essential to test them for any OTX2 mutation identified in affected probands. However, accurate estimates of the recurrence risk await a molecular definition of the factors that determine penetrance of these mutations.

Phenotypic Variability

OTX2 loss-of-function mutations are associated with a broad spectrum of ocular phenotypes, ranging from bilateral anophthalmia to mild microphthalmia with retinal abnormalities. Asymmetry between the two sides, also noted in other genetic ocular malformations (Fantes et al. 2003; Ragge et al. 2005), is frequently seen. The spectrum of neurological phenotypes is broad, ranging from severe developmental delay to normal cognitive development. Variable expressivity is illustrated by the different phenotypes of siblings 4A and 4B.

The OTX2 transcription pattern in human embryos is very similar to that described for mouse at the stages examined. This is not always the case (Fougerousse et al. 2000). The highly conserved expression pattern indicates conserved transcriptional control mechanisms. Analysis of the Otx2 loci in human, mouse, Xenopus, zebrafish, and Fugu demonstrated that many of the distant enhancers that control Otx2 expression are functionally conserved (Kurokawa et al. 2004a, 2004b). These elements, spread over hundreds of kilobases of genomic DNA, are possible targets for mutations/variations that might influence phenotypic severity and penetrance.

The ocular expression pattern of OTX2 correlates well with the range of eye phenotypes observed in patients with loss-of-function mutations. Otx2 interacts with Mitf to specify the RPE, which is essential for normal eye development (Martinez-Morales et al. 2003). Early RPE defects cause gross structural abnormalities of the eye (Raymond and Jackson 1995; Graw 2003). The anophthalmia and severe microphthalmia phenotypes in our cohort may result directly from loss of OTX2 function in the developing RPE.

In the developing neural retina, OTX2 regulates CRX, and CRX mutations cause LCA and retinal degeneration (Sohocki et al. 1998; Rivolta et al. 2001; Nishida et al. 2003). The retinal phenotypes in patients 4B (with an original diagnosis of LCA) and 4C (resembling pigmentary retinopathy) may be due to disturbed OTX2 regulation of CRX function.

The optic nerve and chiasm were reduced or absent in four of the six patients for whom MRI, CT, or ultrasound data were available (patients 1, 2, 5 and 6 [fig. 3 and table 2]). Optic-nerve aplasia, which is very rare, was observed bilaterally in patients 2 and 6, with absence of the chiasm, and unilaterally on the anophthalmic side in patient 1, who also shows a thin chiasm. Hypoplasia of optic nerves and chiasm was observed in patient 5. Although OTX2/Otx2 is expressed in the optic stalk and subsequently in the optic-nerve sheath, it is not expressed in the optic nerve itself (fig. 6) (Simeone et al. 1993; Bovolenta et al. 1997; Martinez-Morales et al. 2001). Hypoplasia of the optic nerve and chiasm may therefore be a secondary effect of abnormal retinal development. The optic nerve is formed when retinal ganglion-cell axons exit the globe. The majority of axons, encased in the optic sheath, project to the contralateral side of the brain, forming the optic chiasm in the ventral diencephalon (Rasband et al. 2003). Otx2 is expressed in retinal ganglion cells (Bovolenta et al. 1997), so OTX2 mutations may compromise the development of the optic nerve and the formation of the chiasm.

Pronounced hippocampal malformation was observed (fig. 5C) in patient 6, with bilateral anophthalmia, severe developmental delay, and seizures beginning in infancy. The hippocampus is derived from the alar plate, which develops from an OTX2-expressing domain of the neural plate (Fernandez-Garre et al. 2002). Patient 4A was the other individual in the cohort with severe developmental delay and seizures with teenage onset (table 2). Unfortunately, no MRI information is available for this patient. However, a hippocampal abnormality similar to that in patient 6 was observed in patient 1, a young child with severe developmental and speech delay, like patient 4B, but currently without seizures. The association of hippocampal malformation with seizures remains to be confirmed as additional patients are identified.

It is interesting to note that the ocular defects reported in heterozygous Otx2-knockout mice were usually accompanied by severe craniofacial malformations (Matsuo et al. 1995). Our data show that heterozygous OTX2 mutations in humans are associated with structural eye malformations in the absence of gross craniofacial abnormalities. However, the phenotypic composition of our study cohort leads to biased ascertainment of OTX2 mutations in ocular malformation cases. A broader human mutation study might reveal OTX2 mutations associated with craniofacial malformation syndromes or seizure-associated brain defects in the absence of eye anomalies.

We have presented evidence that heterozygous loss-of-function mutations in the OTX2 gene make a major contribution to the genetic basis of anophthalmia and microphthalmia in a small but significant proportion of patients. Two notable findings of our study are the lack of penetrance of OTX2 mutations and the high incidence of gonosomal mosaicism. The factors that influence de novo mutagenesis in the early embryo are unknown; it is unclear at present whether some genes or genomic regions are more susceptible than others following fertilization or whether gonosomal mosaicism is more common than previously recognized and has simply been underascertained. Of five nucleotide substitutions in our cohort, four are transversions and one is a transition: this unusual bias (Nachman and Crowell 2000) may hint at novel mutational mechanisms. Elucidation of the factors that influence OTX2 penetrance is a high priority and will assist with genetic counseling; in addition, further studies will help to unravel the complex genetic interactions that are responsible for the development of the human eye.


We gratefully acknowledge the cooperation of the patients and their families. We thank Marie Restori of Moorfields Eye Hospital, for performing ocular ultrasound; Alison Condie of the Wellcome Trust Clinical Research Facility, Edinburgh, for technical assistance; John Stevens, for expert neuroradiology; and the Medical Research Council/Wellcome Trust–funded Human Developmental Biology Resource. N.K.R. is a Senior Surgical Scientist and is supported by the Academy of Medical Sciences/The Health Foundation and the Medical Research Fund, Oxford. I.M.H. is supported by a Career Development Award from the U.K. Medical Research Council.

Appendix A: Patient Histories

Patient 1 (see figs. figs.11 and and22 and table 2), a male child, was born with right anophthalmia and left microphthalmia at 38 wk of gestation with a birth weight of 2.9 kg, after a normal pregnancy. Parental ages at the patient's birth were 21 years (mother) and 22 years (father). After confirmation of an absent right globe on ultrasound, he was fitted with a right conformer at age 2 mo. He had some vision present in the left eye and was also noted to have a persistent pupillary membrane, iris coloboma, high hypermetropia (+7.50D), a large chorioretinal coloboma, and an axial length of 12.3 mm. Horizontal palpebral apertures were 12 mm (right) and 16 mm (left). He was fitted with a conformer R socket at age 18 mo. He received a right dermis fat graft with mucous membrane graft at age 2 years. He then developed a total retinal detachment on the left that was inoperable. He had no family history of eye or systemic anomalies. However, his mother had a hypopigmented area around the macula on the right and a hypopigmented fundus or RPE disturbance in the inferonasal fundus. She had no refractive error and no functional deficit.

MRI scan at age 1 mo showed partial agenesis of the corpus callosum, with a normal-sized pituitary gland. Recent review of this original scan suggested that the right optic nerve was absent and the left optic nerve was small. The chiasm was thin. The hippocampi appeared to be malformed bilaterally (similar to patient 6 [fig. 5]). The patient's pituitary function was within normal limits. Even though his growth hormone levels were borderline normal, his growth was poor. By age 2.5 years, he had fallen from the 25th–50th percentile for height and weight to under the 3rd percentile. By age 4 years, he had marked developmental delay, with speech and language delay (10 words), which he lost and later regained. General examination showed generalized hypotonia; he was able to walk very short distances independently, with a wide gait. He had increased joint laxity and a small head circumference of 48 cm (<0.4th percentile) but no other abnormal neurological signs. Assessment by an educational psychologist showed development at the 12-mo level at age 4 years.

Patient 2 (see figs. figs.11 and and33 and table 2), a male child with bilateral microphthalmia, was born to unrelated parents with normal eyes. MRI at age 3 wk showed bilateral optic-nerve aplasia and asymmetrically microphthalmic globes. The chiasm was not visualized, with mild thinning of the corpus callosum apparent anteriorly. There was no neuroradiological evidence of a neuronal migrational defect. Ocular examination revealed that horizontal corneal diameters were 6.5 mm (right) and 8.0 mm (left). Both eyes had persistent pupillary remnants with tunica vasculosa lentis and clear lenses. The right fundus was a uniform orange, with a paler area near the center. No retinal vessels were identified in either eye. The left fundus was hypopigmented, with a granular appearance and a few choroidal vessels. ERG responses were very low, and visual evoked potential responses were absent (fig. 3). The only systemic abnormalities seen at birth were midline dermoid cysts on the forehead and nasal bridge.

MRI at age 2 years indicated that both globes were asymmetrical and abnormal; a prominent pituitary stalk was seen but no optic chiasm. Structures that could be thin optic nerves or their sheaths were seen in the orbits. The corpus callosum appeared normal at the time of the examination.

Developmental assessment at age 4.5 years showed a Wechsler Preschool and Primary Scale of Intelligence–Revised score of 7, which would be low normal intelligence if the patient were sighted; however, verbal comprehension, vocalization, and expressive language scores were high, equivalent to a 4-year-old sighted child or a 5-year-old blind child. Cognitive and language skills were judged to be at least age-appropriate for a blind child of this age.

This patient has an unaffected older sister, and his mother had a stillborn infant at 38 wk of gestation, a year before patient 2 was born. No postmortem examination information is available about the stillborn infant.

Patient 3A (see figs. figs.11 and and22 and table 2), a female child, was born at term to parents who are second cousins. Birth weight was 2.7 kg. A CT scan shortly after birth showed bilateral rudimentary globes with muscle attachments and a structure, probably the hypoplastic optic nerve, exiting each eye centrally. A thickened septum pellucidum and slightly enlarged lateral ventricles were observed. The horizontal palpebral apertures were 7 mm (right) and 9 mm (left). A maternally transmitted polymorphic chromosome variant inv(2)(p11.2;q13)mat was found upon karyotype analysis but was considered to be of no clinical significance.

The parents subsequently had a healthy daughter. A third pregnancy was terminated because of fetal abnormality (patient 3B). Transabdominal ultrasound at 21 wk of gestation revealed abnormal brain anatomy, with increased distance between the anterior horns of the lateral ventricles and dilation of the posterior horns, indicative of agenesis of the corpus callosum. Cerebellar hemispheres were separated by a nonreflective space. Face, nose, palate, and lips were unremarkable. The eyes were abnormal. After termination 5 d later, histological examination of the ~21-wk-old fetus showed bilateral microphthalmia, with axial lengths of 9.5 mm and 5.8 mm (normal axial length for gestational age is around 10 mm [Fledelius and Christensen 1996]). The rectus muscle and parts of the lacrimal gland were present. Both globes contained lenses. The cornea showed nonkeratinized squamous epithelium of reduced thickness. Bowman’s layer was absent, with subepithelially vascularized connective tissue over a layered stroma of reduced thickness. Descemet’s membrane, which usually develops in week 8, was absent. The hypoplastic chamber angle lacked the trabecular network, which was probably also abnormal. Adjacent to the cornea was the lens with tunica vasculosa lentis, which starts to regress at ~22 wk, and an intact capsule with regular anterior lens epithelium. Some peripheral lens fibers were swollen. The retina was not vascularized, and it extended to the pars plicata with zonular fibers. Posteriorly, the retina showed many tubular rosette formations. The structure of the vitreous body was described as normal. The optic nerve was present with a central retinal artery. In the anterior part of the optic fascicle, there was a colobomatous pit with adjacent fat tissue. The other, larger globe had a colobomatous pit in which the RPE was missing. DNA isolated from this fetus was found to carry the same mutation as the affected child, confirming that the mother must be a gonosomal mosaic.

The mosaic mother was examined carefully for eye anomalies. Her eye structure and vision were found to be entirely normal. A high-resolution (363 hexagons) multifocal ERG revealed no functional deficits. The mother believed that she had been exposed to agricultural chemicals in the rural area where she lived during her pregnancy with patient 3 and the terminated fetus.

Patient 4A (see figs. figs.11 and and22 and table 2), a female, was born at 40 wk of gestation to unrelated apparently healthy parents. She was diagnosed as blind at age 6 mo, after her mother voiced concerns about her nonresponsiveness at age 3 mo. At age 19 mo, she was reported to have bilateral colobomata with microphthalmia. Her lenses were normal. On assessment at age 7 years, she was noted to have severe learning difficulties. She had absent speech and had lost the vocabulary of four or five words she developed as a younger child. Apart from microphthalmia and bilateral fifth finger clinodactyly, she has no specific malformations; her physical health is good. However, since age 14 years, she has suffered from regular tonic clonic seizures which are partly controlled with sodium valproate; seizure frequency varies from several per day to a couple per month. She suffers some valproate side effects. At a recent reassessment, she was noted to have bilateral enophthalmos. Both eyes are microphthalmic. The corneas measure ~4 mm in each eye. The irises are present. She has small, eccentric, downwardly displaced, slitlike pupils. The left eye has a dense cataract. She does not have sclerocornea. Further examination was not possible.

Patient 4B (figs. (figs.1,1, ,2,2, and and44 and table 2), a male (full brother of patient 4A), was born at 39 wk of gestation. He has no significant dysmorphic features. He was, however, visually impaired from early childhood and was given a diagnosis of LCA. Consistent with this diagnosis, pale optic disks, thin retinal vessels, atrophic maculae, and large clumps of pigment in the midperiphery were observed on examination at age 26 years. ERG is absent, and he has nystagmus. His eyes are borderline microphthalmic (corneal diameters 11 mm, axial lengths 20 mm). He has bilateral peripheral anterior synechiae (adhesions between the iris and the cornea). His visual acuity at age 22 years was 6/60 in each eye. An MRI scan performed at age 25 years revealed no notable abnormalities in brain structure. The axial length measured from the scan again showed minimal microphthalmia. In addition to visual problems, he has unilateral right-sided hearing loss. He also had a midline lumbar epidermoid tumor removed at age 8 years.

Patient 4C (figs. (figs.11 and and44 and table 2), the mother of patients 4A and 4B, was thought to have some mild, acquired visual problems, but she was found to be a mosaic carrier of the same OTX2 mutation that was found in her two affected children. A more detailed eye examination revealed retinal dystrophy comparable to that found in her son. It is difficult to determine whether the retina is segmentally affected, but there are strongly pigmented areas and adjacent unpigmented ones (fig. 4). She has night blindness. Visual field testing showed very extensive peripheral-field loss bilaterally. No ERG data are available. Visual acuities are R 6/60, L 6/12. She has a right divergent squint. The anterior segment is unaffected. Her current phenotype would be most readily classified as pigmentary retinopathy. The optic disks are pale, and the vessels are of reasonably normal caliber, with widespread atrophy of the RPE layer and choroid and with ghost vessels visible in the choroid. There are patches of pigment clumping around the disk and the macula. There are no other physical or cognitive problems. She managed adequately in mainstream school and, subsequently, in the general social environment.

Patient 5 (see figs. figs.1,1, ,2,2, and and55 and table 2), a male, was born at 42 wk of gestation with a birth weight of 4.3 kg, with bilateral anophthalmia. The pregnancy was normal, apart of mild hypertension and proteinuria. The mother took Debendox in 2nd and 3rd mo of gestation. A maternal great-grandfather was reported to have had hemophilia and “small eyes” but had no visual problems.

The patient commenced socket expansion at age 1 year but was unable to keep the socket expanders in at age 2 years. He had bilateral mucous membrane grafts to the sockets at age 5 years and age 10 years. His general development was delayed: he talked at age 3 years and walked at age 4 years. He has lax joints, right-leg hypotonia, and a palatal tooth. At age 10 years, his height was in the 3rd percentile, and his head circumference was between the 3rd and 10th percentiles.

On examination at age 24 years, he had bilateral anophthalmia with no visible ocular remnants. He had a left upper-lid entropion and has scars from previous medial canthal z-plasties. He has hypotelorism, with an intercanthal distance of 28 mm. Horizontal palpebral apertures are 18 mm. Ultrasound revealed buried ocular remnants of 4.4 mm axial length on the right and 4.9 mm on the left. General physical examination revealed a moderately high arched palate; reduced muscle tone generally and more marked reduction in the lower limbs, with normal coordination, increased reflexes, and clonus of lower limbs, with the right limb more severely affected than the left; no heart murmurs; testes reportedly normal; normal puberty; and slightly square ears. His hearing is normal. A recent MRI scan revealed anophthalmia; the optic nerves are present but small. The orbits are small. The chiasm is present but extremely thin. The chiasmatic recess of the third ventricle is absent. The anterior commissure is thin. The lateral ventricles are slightly enlarged.

The mother of patient 5 is healthy, with normal eye-examination results. A recent MRI was unremarkable, with normal optic nerves and chiasm. She carries the same OTX2 mutation as her affected son (fig. 1).

Patient 6 (see figs. figs.1,1, ,2,2, and and55 and table 2), a male child, was born at 38 wk gestation, with a weight of 2.7 kg, to healthy unrelated parents. He had an intracerebral bleed in the newborn period, with subsequent periventricular leucomalacia and hydrocephalus (not requiring surgical intervention). Microphthalmia was noted neonatally, and prostheses were fitted shortly after birth and were worn until age 5 years. A seizure disorder was present from birth. A cranial MRI at age 10.9 years revealed asymmetry of the lateral ventricles. The optic nerve was bilaterally absent, and no chiasm was seen. All eye muscles were present, with the rectus muscle shifted medially. Bilateral orbital cysts were found during examination under anesthesia.

The father, who was found to be an unaffected carrier of the son’s protein truncation mutation (fig. 1), was found to have normal eye structures and vision. The mother reported being exposed to wood varnish and solvents during pregnancy.

Patient 7 (see figs. figs.11 and and22 and table 2), a female child, was born at 42 wk of gestation after an uneventful pregnancy by emergency cesarean section because of failure to progress and previous cesarean section. Birth weight was 5 kg. She had a right cataract and microphthalmia, with a microcornea (left sclerocornea, ~12–14 mm axial length). She had right cataract surgery at age 4 mo, after which the right eye became phthisical. She has no perception of light. She has no family history of eye or systemic anomalies. She has no other medical problems and is within the average range developmentally, compared with fully sighted peers.

Examination results of her mother, father, and two siblings were entirely normal. The carrier mother’s vision was 6/6 in both eyes, with normal anterior segments, intraocular pressures, and fundi. The carrier brother’s ocular examination results were entirely normal.

Patient 8 (see figs. figs.11 and and22 and table 2), a male child, was born with left anophthalmia, to a mother who also had left anophthalmia. He was born after a normal pregnancy at 42 wk of gestation by ventouse extraction, with a birth weight of 3.4 kg and a head circumference of 39 cm (10th percentile). At age 1 year, his head circumference was 44.6 cm (<3rd percentile). There were no neonatal concerns and no other illnesses. His early development was slightly delayed, with mild speech delay. He had a balloon expander inserted at age 2 years, a left mucous-membrane graft at age 4 years, and a lateral canthal sling at age 5 years. At age 8 years, concerns were expressed about attention-deficit/hyperactivity disorder. He attends a normal school but has a special-needs teacher for 15 h a week. His right visual acuity is 6/7.5. He has a posterior subcapsular cataract, with a normal fundus appearance. He has a left anophthalmic socket, with no visible remnant, and evidence of previous mucous membrane grafts. He is mildly brachycephalic and has square-topped simple ears with crumpled helices. Chromosomal analyses of this boy and his mother were normal. His half brother (same mother) (fig. 1) has learning difficulties.

Electronic-Database Information

Accession numbers and URLs for data presented herein are as follows:

dbSNP, http://www.ncbi.nlm.nih.gov/SNP/ (for 268+12 C/T [accession number ss35522250], 269–70 C/A [accession number ss35522251], and c.1050 G/A [accession number rs171978])
GenBank, http://www.ncbi.nlm.nih.gov/Genbank/ (for human OTX2 cDNA [accession number NM_172337] and BAC AL161757)
Laboratory of Phil Green, http://www.phrap.org/index.html
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for anophthalmia, extreme microphthalmia, sclerocornea, aniridia, colobomata, congenital cataracts, LCA, Walker-Warburg syndrome, congenital cataract/aphakia, PAX6, RAX, CHX10, MAF, SOX2, OTX2, CRX, cone-rod dystrophy, MITF, OTX1, optic-nerve aplasia, PITX2, SIX3, ROM1, RDS, HPE, SHH, TGIF, and ZIC2)


Acampora D, Mazan S, Lallemand Y, Avantaggiato V, Maury M, Simeone A, Brulet P (1995) Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation. Development 121:3279–3290 [PubMed]
Aijaz S, Clark BJ, Williamson K, van Heyningen V, Morrison D, Fitzpatrick D, Collin R, Ragge N, Christoforou A, Brown A, Hanson I (2004) Absence of SIX6 mutations in microphthalmia, anophthalmia, and coloboma. Invest Ophthalmol Vis Sci 45:3871–3876 [PubMed]
Ang SL, Jin O, Rhinn M, Daigle N, Stevenson L, Rossant J (1996) A targeted mouse Otx2 mutation leads to severe defects in gastrulation and formation of axial mesoderm and to deletion of rostral brain. Development 122:243–252 [PubMed]
Azuma N, Yamaguchi Y, Handa H, Hayakawa M, Kanai A, Yamada M (1999) Missense mutation in the alternative splice region of the PAX6 gene in eye anomalies. Am J Hum Genet 65:656–663 [PMC free article] [PubMed]
Bovolenta P, Mallamaci A, Briata P, Corte G, Boncinelli E (1997) Implication of OTX2 in pigment epithelium determination and neural retina differentiation. J Neurosci 17:4243–4252 [PubMed]
Brewer C, Holloway S, Zawalnyski P, Schinzel A, FitzPatrick D (1998) A chromosomal deletion map of human malformations. Am J Hum Genet 63:1153–1159 [PMC free article] [PubMed]
D’Elia AV, Tell G, Paron I, Pellizzari L, Lonigro R, Damante G (2001) Missense mutations of human homeoboxes: a review. Hum Mutat 18:361–374 [PubMed]
Dobyns WB, Pagon RA, Armstrong D, Curry CJ, Greenberg F, Grix A, Holmes LB, Laxova R, Michels VV, Robinow M, Zimmerman RL (1989) Diagnostic criteria for Walker-Warburg syndrome. Am J Med Genet 32:195–210 [PubMed]
Edwards JH (1989) Familiarity, recessivity and germline mosaicism. Ann Hum Genet 53:33–47 [PubMed]
Fantes J, Ragge NK, Lynch SA, McGill NI, Collin JR, Howard-Peebles PN, Hayward C, Vivian AJ, Williamson K, van Heyningen V, FitzPatrick DR (2003) Mutations in SOX2 cause anophthalmia. Nat Genet 33:461–463 [PubMed]
Ferda-Percin E, Ploder LA, Yu JJ, Arici K, Horsford DJ, Rutherford A, Bapat B, Cox DW, Duncan AMV, Kalnins VI, Kocak-Altintas A, Sowden JC, Trabousli E, Sarfarazi M, McInnes RR (2000) Human microphthalmia associated with mutations in the retinal homeobox gene CHX10. Nat Genet 25:397–401 [PubMed]
Fernandez-Garre P, Rodriguez-Gallardo L, Gallego-Diaz V, Alvarez IS, Puelles L (2002) Fate map of the chicken neural plate at stage 4. Development 129:2807–2822 [PubMed]
Finkelstein R, Boncinelli E (1994) From fly head to mammalian forebrain: the story of otd and Otx. Trends Genet 10:310–315 [PubMed]
Fledelius HC, Christensen AC (1996) Reappraisal of the human ocular growth curve in fetal life, infancy, and early childhood. Br J Ophthalmol 80:918–921 [PMC free article] [PubMed]
Fougerousse F, Bullen P, Herasse M, Lindsay S, Richard I, Wilson D, Suel L, Durand M, Robson S, Abitbol M, Beckmann JS, Strachan T (2000) Human-mouse differences in the embryonic expression patterns of developmental control genes and disease genes. Hum Mol Genet 9:165–173 [PubMed]
Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A, Scherer SW, Tsui LC, Loutradis-Anagnostou A, Jacobson SG, Cepko CL, Bhattacharya SS, McInnes RR (1997) Cone-rod dystrophy due to mutations in a novel photoreceptor-specific homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell 91:543–553 [PubMed]
Germot A, Lecointre G, Plouhinec JL, Le Mentec C, Girardot F, Mazan S (2001) Structural evolution of Otx genes in craniates. Mol Biol Evol 18:1668–1678 [PubMed]
Gouya L, Puy H, Robreau AM, Bourgeois M, Lamoril J, Da Silva V, Grandchamp B, Deybach JC (2002) The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH. Nat Genet 30:27–28 [PubMed]
Graw J (2003) The genetic and molecular basis of congenital eye defects. Nat Rev Genet 4:876–888 [PubMed]
Hanson I (2003) PAX6 and congenital eye malformations. Pediatr Res 54:791–796 [PubMed]
Holbrook JA, Neu-Yilik G, Hentze MW, Kulozik AE (2004) Nonsense-mediated decay approaches the clinic. Nat Genet 36:801–808 [PubMed]
Jamieson RV, Perveen R, Kerr B, Carette M, Yardley J, Heon E, Wirth MG, van Heyningen V, Donnai D, Munier F, Black GC (2002) Domain disruption and mutation of the bZIP transcription factor, MAF, associated with cataract, ocular anterior segment dysgenesis and coloboma. Hum Mol Genet 11:33–42 [PubMed]
Kajiwara K, Berson EL, Dryja TP (1994) Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science 264:1604–1608 [PubMed]
Kurokawa D, Kiyonari H, Nakayama R, Kimura-Yoshida C, Matsuo I, Aizawa S (2004a) Regulation of Otx2 expression and its functions in mouse forebrain and midbrain. Development 131:3319–3331 [PubMed]
Kurokawa D, Takasaki N, Kiyonari H, Nakayama R, Kimura-Yoshida C, Matsuo I, Aizawa S (2004b) Regulation of Otx2 expression and its functions in mouse epiblast and anterior neuroectoderm. Development 131:3307–3317 [PubMed]
Laflamme C, Filion C, Labelle Y (2004) Functional characterization of SIX3 homeodomain mutations in holoprosencephaly: interaction with the nuclear receptor NR4A3/NOR1. Hum Mutat 24:502–508 [PubMed]
Lai CSL, Gerrelli D, Monaco AP, Fisher SE, Copp AJ (2003) FOXP2 expression during brain development coincides with adult sites of pathology in a severe speech and language disorder. Brain 126:2455–2462 [PubMed]
Leuer M, Oldenburg J, Lavergne JM, Ludwig M, Fregin A, Eigel A, Ljung R, Goodeve A, Peake I, Olek K (2001) Somatic mosaicism in hemophilia A: a fairly common event. Am J Hum Genet 69:75–87 [PMC free article] [PubMed]
Mallamaci A, Di Blas E, Briata P, Boncinelli E, Corte G (1996) OTX2 homeoprotein in the developing central nervous system and migratory cells of the olfactory area. Mech Dev 58:165–178 [PubMed]
Martinez-Morales JR, Dolez V, Rodrigo I, Zaccarini R, Leconte L, Bovolenta P, Saule S (2003) OTX2 activates the molecular network underlying retina pigment epithelium differentiation. J Biol Chem 278:21721–21731 [PubMed]
Martinez-Morales JR, Signore M, Acampora D, Simeone A, Bovolenta P (2001) Otx genes are required for tissue specification in the developing eye. Development 128:2019–2030 [PubMed]
Matsuo I, Kuratani S, Kimura C, Takeda N, Aizawa S (1995) Mouse Otx2 functions in the formation and patterning of the rostral head. Genes Dev 9:2646–2658 [PubMed]
Ming JE, Muenke M (2002) Multiple hits during early embryonic development: digenic diseases and holoprosencephaly. Am J Hum Genet 71:1017–1032 [PMC free article] [PubMed]
Morrison D, FitzPatrick D, Hanson I, Williamson K, van Heyningen V, Fleck B, Jones I, Chalmers J, Campbell H (2002) National study of microphthalmia, anophthalmia, and coloboma (MAC) in Scotland: investigation of genetic aetiology. J Med Genet 39:16–22 [PMC free article] [PubMed]
Nachman MW, Crowell SL (2000) Estimate of the mutation rate per nucleotide in humans. Genetics 156:297–304 [PMC free article] [PubMed]
Nanni L, Ming JE, Bocian M, Steinhaus K, Bianchi DW, Die-Smulders C, Giannotti A, Imaizumi K, Jones KL, Campo MD, Martin RA, Meinecke P, Pierpont ME, Robin NH, Young ID, Roessler E, Muenke M (1999) The mutational spectrum of the sonic hedgehog gene in holoprosencephaly: SHH mutations cause a significant proportion of autosomal dominant holoprosencephaly. Hum Mol Genet 8:2479–2488 [PubMed]
Nishida A, Furukawa A, Koike C, Tano Y, Aizawa S, Matsuo I, Furukawa T (2003) Otx2 homeobox gene controls retinal photoreceptor cell fate and pineal gland development. Nat Neurosci 6:1255–1263 [PubMed]
Nussbaum RL, McInnes RR, Willard HF (2001) Genetics in medicine, 6th ed. W.B. Saunders, Philadelphia, p 54
Ragge NK, Lorenz B, Schneider A, Bushby K, de Sanctis L, de Sanctis U, Salt A, Collin JRO, Vivian AJ, Free SL, Thompson P, Williamson K, Sisodiya SM, van Heyningen V, FitzPatrick DR (2005) SOX2 anophthalmia syndrome. Am J Med Genet. http://www3.interscience.wiley.com/cgi-bin/fulltext/110437025/HTMLSTART/ (electronically published April 5, 2005; accessed April 20, 2005) [PubMed]
Rasband K, Hardy M, Chien C-B (2003) Generating X: formation of the optic chiasm. Neuron 39:885–888 [PubMed]
Raymond SM, Jackson IJ (1995) The retinal pigmented epithelium is required for development and maintenance of the mouse neural retina. Curr Biol 5:1286–1295 [PubMed]
Rivolta C, Berson EL, Dryja TP (2001) Dominant Leber congenital amaurosis, cone-rod degeneration, and retinitis pigmentosa caused by mutant versions of the transcription factor CRX. Hum Mutat 18:488–498 [PubMed]
Simeone A (2000) Positioning the isthmic organizer where Otx2 and Gbx2 meet. Trends Genet 16:237–240 [PubMed]
Simeone A, Acampora D, Mallamaci A, Stornaiuolo A, D’Apice MR, Nigro V, Boncinelli E (1993) A vertebrate gene related to orthodenticle contains a homeodomain of the bicoid class and demarcates anterior neuroectoderm in the gastrulating mouse embryo. EMBO J 12:2735–2747 [PMC free article] [PubMed]
Sisodiya SM, Free SL, Williamson KA, Mitchell TN, Willis C, Stevens JM, Kendall BE, Shorvon SD, Hanson IM, Moore AT, van Heyningen V (2001) PAX6 haploinsufficiency causes cerebral malformation and olfactory dysfunction in humans. Nat Genet 28:214–216 [PubMed]
Sohocki MM, Sullivan LS, Mintz-Hittner HA, Birch D, Heckenlively JR, Freund CL, McInnes RR, Daiger SP (1998) A range of clinical phenotypes associated with mutations in CRX, a photoreceptor transcription-factor gene. Am J Hum Genet 63:1307–1315 [PMC free article] [PubMed]
Swaroop A, Wang QL, Wu W, Cook J, Coats C, Xu S, Chen S, Zack DJ, Sieving P (1999) Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function. Hum Mol Genet 8:299–305 [PubMed]
Tahayato A, Sonneville R, Pichaud F, Wernet MF, Papatsenko D, Beaufils P, Cook T, Desplan C (2003) Otd/Crx, a dual regulator for the specification of ommatidia subtypes in the Drosophila retina. Dev Cell 5:391–402 [PubMed]
van Heyningen V, Yeyati PL (2004) Mechanisms of non-Mendelian inheritance in genetic disease. Hum Mol Genet 13 Spec No 2:R225–R233 [PubMed]
Vithana EN, Abu-Safieh L, Pelosini L, Winchester E, Hornan D, Bird AC, Hunt DM, Bustin SA, Bhattacharya SS (2003) Expression of PRPF31 mRNA in patients with autosomal dominant retinitis pigmentosa: a molecular clue for incomplete penetrance? Invest Ophthalmol Vis Sci 44:4204–4209 [PubMed]
Voronina VA, Kozhemyakina EA, O’Kernick CM, Kahn ND, Wenger SL, Linberg JV, Schneider AS, Mathers PH (2004) Mutation in the human RAX homeobox gene in a patient with anophthalmia and sclerocornea. Hum Mol Genet 13:315–322 [PubMed]
Wallis DE, Roessler E, Hehr U, Nanni L, Wiltshire T, Richieri-Costa A, Gillessen-Kaesbach G, Zackai EH, Rommens J, Muenke M (1999) Mutations in the homeodomain of the human SIX3 gene cause holoprosencephaly. Nat Genet 22:196–198 [PubMed]
Zlotogora J (1998) Germ line mosaicism. Hum Genet 102:381–386 [PubMed]
Zuber ME, Gestri G, Viczian AS, Barsacchi G, Harris WA (2003) Specification of the vertebrate eye by a network of eye field transcription factors. Development 130:5155–5167 [PubMed]

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