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The forkhead transcription factor FOXE3 is critical for vertebrate eye development. Recessive and dominant variants cause human ocular disease but the full range of phenotypes and mechanisms of action for the two classes of variants are unknown. We identified FOXE3 variants in individuals with congenital eye malformations and carried out in vitro functional analysis on selected alleles. Sixteen new recessive and dominant families, including six novel variants, were identified. Analysis of new and previously reported genetic and clinical data demonstrated a broad phenotypic range with an overlap between recessive and dominant disease. Most families with recessive alleles, composed of truncating and forkhead-domain missense variants, had severe corneal opacity (90%; sclerocornea in 47%), aphakia (83%) and microphthalmia (80%), but some had milder features including isolated cataract. The phenotype was most variable for recessive missense variants, suggesting that the functional consequences may be highly dependent on the type of amino acid substitution and its position. When assessed, aniridia or iris hypoplasia were noted in 89% and optic nerve anomalies in 60% of recessive cases, indicating that these defects are also common and may be underrecognized. In dominant pedigrees, caused by extension variants, normal eye size (96%), cataracts (99%) and variable anterior segment anomalies were seen in most, but some individuals had microphthalmia, aphakia or sclerocornea, more typical of recessive disease. Functional studies identified variable effects on the protein stability, DNA binding, nuclear localization and transcriptional activity for recessive FOXE3 variants, whereas dominant alleles showed severe impairment in all areas and dominant-negative characteristics.

Microphthalmia, coloboma, and aniridia are congenital ocular phenotypes with a strong genetic component but often unknown cause. We present a likely causative novel variant in MAB21L1, c.152G>T p.(Arg51Leu), in two family members with microphthalmia and aniridia, as well as novel or rare compound heterozygous variants of uncertain significance, c.184C>T p.(Arg62Cys)/c.‐68T>C, and c.658G>C p.(Gly220Arg)/c.*529A>G, in two additional probands with microphthalmia, coloboma and/or cataracts. All variants were predicted as damaging by in silico programs. In vitro studies of coding variants revealed normal subcellular localization but variable stability for the corresponding mutant proteins. In vivo complementation assays using the zebrafish mab21l2 Q48Sfs*5 loss‐of‐function line demonstrated that though overexpression of wild‐type MAB21L1 messenger RNA (mRNA) compensated for the loss of mab21l2, none of the coding variant mRNAs produced a statistically significant rescue, with p.(Arg51Leu) showing the highest degree of functional deficiency. Dominant variants in a close homolog of MAB21L1, MAB21L2, have been associated with microphthalmia and/or coloboma and repeatedly involved the same Arg51 residue, further supporting its pathogenicity. The possible role of p.(Arg62Cys) and p.(Gly220Arg) in microphthalmia is similarly supported by the observed functional defects, with or without an additional impact from noncoding MAB21L1 variants identified in each patient. This study suggests a broader spectrum of MAB21L1‐associated disease., MAB21L1 alleles in microphthalmia and aniridia.

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a−/− single homozygous and foxc1−/− double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/−;foxc1b−/−) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

Background The male-abnormal 21 like (MAB21L) genes are important in human ocular development. Homozygous loss of MAB21L1 leads to corneal dystrophy in all affected individuals along with cataracts and buphthalmos in some. The molecular function and downstream pathways of MAB21L factors are largely undefined. Results We generated the first mab21l1 zebrafish mutant carrying a putative loss-of-function allele, c.107delA p.(Lys36Argfs*7). At the final stages of embryonic development, homozygous mab21l1c.107delA fish displayed enlarged anterior chambers and corneal thinning which progressed with age. Additional studies revealed increased cell death in the mutant corneas, transformation of the cornea into a skin-like epithelium, and progressive lens degeneration with development of fibrous masses in the anterior chamber. RNA-seq of wild-type and mutant ocular transcriptomes revealed significant changes in expression of several genes, including irf1a and b, stat1, elf3, krt17, tlr9 and loxa associated with immunity and/or corneal function. Abnormal expression of lysyl oxidases have been previously linked with corneal thinning, fibrosis, and lens defects in mammals, suggesting a role for loxa misexpression in the progressive mab21l1c.107delA eye phenotype. Conclusions Zebrafish mab21l1 is essential for normal corneal development, similar to human MAB21L1. The identified molecular changes in mab21l1c.107delA mutants provide the first clues about possible affected pathways. This article is protected by copyright. All rights reserved.

Complex microphthalmia is characterized by small eyes with additional abnormalities that may include anterior segment dysgenesis. While many genes are known, a genetic cause is identified in only 4–30% of microphthalmia, with the lowest rate in unilateral cases. We identified four novel pathogenic loss-of-function alleles in PRR12 in families affected by complex microphthalmia and/or Peters anomaly, including two de novo, the first dominantly transmitted allele, as well as the first splicing variant. The ocular phenotypes were isolated with no additional systemic features observed in two unrelated families. Remarkably, ocular phenotypes were asymmetric in all individuals and unilateral (with structurally normal contralateral eye) in three. There are only three previously reported PRR12 variants identified in probands with intellectual disability, neuropsychiatric disorders, and iris anomalies. While some overlap with previously reported cases is seen, non-syndromic developmental ocular anomalies are a novel phenotype for this gene. Additional phenotypic expansions included short stature and normal development/cognition, each noted in two individuals in this cohort, as well as absence of neuropsychiatric disorders in all. This study identifies new associations for PRR12 disruption in humans and presents a genetic diagnosis resulting in unilateral ocular phenotypes in a significant proportion of cases.

PURPOSE: Ocular coloboma is caused by failure of optic fissure closure during development and recognized as part of the microphthalmia, anophthalmia, and coloboma (MAC) spectrum. While many genes are known to cause colobomatous microphthalmia, relatively few have been reported in coloboma with normal eye size. METHODS: Genetic analysis including trio exome sequencing and Sanger sequencing was undertaken in a family with two siblings affected with bilateral coloboma of the iris, retina, and choroid. RESULTS: Pathogenic variants in MAC genes were excluded. Trio analysis identified compound heterozygous donor splice site variants in CDON, a cell-surface receptor known to function in the Sonic Hedgehog pathway, c.928+1G>A and c.2650+1G>T, in both affected individuals. Heterozygous missense and truncating CDON variants are associated with dominant holoprosencephaly (HPE) with incomplete penetrance and Cdon−/− mice display variable HPE and coloboma. A homozygous nonsense allele of uncertain significance was recently identified in a consanguineous patient with coloboma and a second molecular diagnosis. CONCLUSIONS: We report the first compound heterozygous variants in CDON as a cause of isolated coloboma. CDON is the first HPE gene identified to cause recessive coloboma. Given the phenotypic overlap, further examination of HPE genes in coloboma is indicated.

Congenital aniridia is a severe autosomal dominant congenital panocular disorder, mainly associated with pathogenic variants in the PAX6 gene. The objective of the study was to investigate the mutational and clinical spectra of congenital aniridia in a cohort of 117 patients from Russia. Each patient underwent detailed ophthalmological examination. From 91 unrelated families, 110 patients were diagnosed with congenital aniridia and 7 with WAGR syndrome (Wilms tumor, Aniridia, Genitourinary anomalies, and mental Retardation syndrome). The clinical presentation in aniridia patients varied from the complete bilateral absence of the iris (75.5%) to partial aniridia or iris hypoplasia (24.5%). Additional ocular abnormalities were consistent with previous reports. In our cohort, we saw a previously not described high percentage of patients (45%) who showed non-ocular phenotypes. Prevalence of deletions coherent with WAGR syndrome appeared to be 19.4% out of sporadic patients. Among the other aniridia cases, PAX6 deletions were identified in 18 probands, and small intragenic changes were detected in 58 probands with 27 of these mutations being novel and 21 previously reported. In 3 families mosaic mutation was transmitted from a subtly affected parent. Therefore, PAX6 mutations explained 96.7% of aniridia phenotypes in this study with only 3 of 91 probands lacking pathogenic variants in the gene.

The genetic basis of congenital glaucoma with systemic anomalies is largely unknown. Whole exome sequencing (WES) in 10 probands with congenital glaucoma and variable systemic anomalies identified pathogenic or likely pathogenic variants in three probands; in two of these, a combination of two Mendelian disorders was found to completely explain the patients' features whereas in the third case only the ocular findings could be explained by the genetic diagnosis. The molecular diagnosis for glaucoma included two cases with compound heterozygous or homozygous pathogenic alleles in CYP1B1 and one family with a dominant pathogenic variant in FOXC1; the second genetic diagnosis for the additional systemic features included compound heterozygous mutations in NPHS1 in one family and a heterozygous 18q23 deletion in another pedigree. These findings show the power of WES in the analysis of complex conditions and emphasize the importance of CYP1B1 screening in patients with congenital glaucoma regardless of the presence/absence of other systemic anomalies.

Pediatric cataract represents an important cause of pediatric visual impairment. While both genetic and environmental causes for pediatric cataract are known, a large proportion remains idiopathic. The purpose of this review is to discuss genes involved in isolated pediatric cataract, with a focus on variable inheritance patterns within genes. Mutations in over 52 genes are known to cause isolated pediatric cataract, with a major contribution from genes encoding for crystallins, transcription factors, membrane proteins, and cytoskeletal proteins. Interestingly, both dominant and recessive inheritance patterns have been reported for mutations in 13 different cataract genes. For some genes, dominant and recessive alleles represent distinct types of mutations, but for many, especially missense variants, there are no clear patterns to distinguish between dominant and recessive alleles. Further research into the functional effects of these mutations, as well as additional data on the frequency of the identified variants, is needed to clarify variant pathogenicity. Whole exome sequencing continues to be successful in identifying novel genes associated with congenital cataract but is hindered by the extreme genetic heterogeneity of this condition. The large number of idiopathic cases suggests that more genes and potentially novel mechanisms of gene disruption remain to be identified.

The PITX2 (paired-like homeodomain 2) gene encodes a bicoid-like homeodomain transcription factor linked with several human disorders. The main associated congenital phenotype is Axenfeld-Rieger syndrome, type 1, an autosomal dominant condition characterized by variable defects in the anterior segment of the eye, an increased risk of glaucoma, craniofacial dysmorphism and dental and umbilical anomalies; in addition to this, one report implicated PITX2 in ring dermoid of the cornea and a few others described cardiac phenotypes. We report three novel PITX2 mutations—c.271C > T, p.(Arg91Trp); c.259T > C, p.(Phe87Leu); and c.356delA, p.(Gln119Argfs*36)—identified in independent families with typical Axenfeld-Rieger syndrome characteristics and some unusual features such as corneal guttata, Wolf-Parkinson-White syndrome, and hyperextensibility. To gain further insight into the diverse roles of PITX2/pitx2 in vertebrate development, we generated various genetic lesions in the pitx2 gene via TALEN-mediated genome editing. Affected homozygous zebrafish demonstrated congenital defects consistent with the range of PITX2-associated human phenotypes: abnormal development of the cornea, iris and iridocorneal angle; corneal dermoids; and craniofacial dysmorphism. In addition, via comparison of pitx2M64* and wild-type embryonic ocular transcriptomes we defined molecular changes associated with pitx2 deficiency, thereby implicating processes potentially underlying disease pathology. This analysis identified numerous affected factors including several members of the Wnt pathway and collagen types I and V gene families. These data further support the link between PITX2 and the WNT pathway and suggest a new role in regulation of collagen gene expression during development.

Anophthalmia and microphthalmia (A/M) are developmental ocular malformations defined as the complete absence or reduction in size of the eye. A/M is a highly heterogeneous disorder with SOX2 and FOXE3 playing major roles in dominant and recessive pedigrees, respectively; however, the majority of cases lack a genetic etiology. We analyzed 28 probands affected with A/M spectrum (without mutations in SOX2/FOXE3) by whole-exome sequencing. Analysis of 83 known A/M factors identified pathogenic/likely pathogenic variants in PAX6, OTX2 and NDP in three patients. A novel heterozygous likely pathogenic variant in PAX6, c.767T>C, p.(Val256Ala), was identified in two brothers with bilateral microphthalmia, coloboma, primary aphakia, iris hypoplasia, sclerocornea and congenital glaucoma; the unaffected mother appears to be a mosaic carrier. While A/M has been reported as a rare feature, this is the first report of congenital primary aphakia in association with PAX6 and the identified allele represents the first variant in the PAX6 homeodomain to be associated with A/M. A novel pathogenic variant in OTX2, c.651delC, p.(Thr218Hisfs*76), in a patient with syndromic bilateral anophthalmia and a hemizygous pathogenic variant in NDP, c.293 C>T, p.(Pro98Leu), in two brothers with isolated bilateral microphthalmia and sclerocornea were also identified. Pathogenic/likely pathogenic variants were not discovered in the 25 remaining A/M cases. This study underscores the utility of whole-exome sequencing for identification of causative mutations in highly variable ocular phenotypes as well as the extreme genetic heterogeneity of A/M conditions.

The human eye is a complex organ whose development requires extraordinary coordination of developmental processes. The conservation of ocular developmental steps in vertebrates suggests possible common genetic mechanisms. Genetic diseases involving the eye represent a leading cause of blindness in children and adults. During the last decades, there has been an exponential increase in genetic studies of ocular disorders. In this review, we summarize current success in identification of genes responsible for microphthalmia, anophthalmia, and coloboma (MAC) phenotypes, which are associated with early defects in embryonic eye development. Studies in animal models for the orthologous genes identified overlapping phenotypes for most factors, confirming the conservation of their function in vertebrate development. These animal models allow for further investigation of the mechanisms of MAC, integration of various identified genes into common developmental pathways and finally, provide an avenue for the development and testing of therapeutic interventions.

Anophthalmia/microphthalmia (A/M) is a developmental ocular malformation defined as complete absence or reduction in size of the eye. A/M is a heterogenous disorder with numerous causative genes identified; however, about half the cases lack a molecular diagnosis. We undertook whole exome sequencing in an A/M family with two affected siblings, two unaffected siblings, and unaffected parents; the ocular phenotype was isolated with only mild developmental delay/learning difficulties reported and a normal brain magnetic resonance imaging (MRI) in the proband at 16 months. No pathogenic mutations were identified in 71 known A/M genes. Further analysis identified a shared heterozygous mutation in COL4A1, c.2317G>A, p.(Gly773Arg) that was not seen in the unaffected parents and siblings. Analysis of 24 unrelated A/M exomes identified a novel c.2122G>A, p.(Gly708Arg) mutation in an additional patient with unilateral microphthalmia, bilateral microcornea and Peters anomaly; the mutation was absent in the unaffected mother and the unaffected father was not available. Mutations in COL4A1 have been linked to a spectrum of human disorders; the most consistent feature is cerebrovascular disease with variable ocular anomalies, kidney and muscle defects. This study expands the spectrum of COL4A1 phenotypes and indicates screening in patients with A/M regardless of MRI findings or presumed inheritance pattern.

Anophthalmia/microphthalmia (A/M) represent severe developmental ocular malformations. Currently, mutations in known genes explain less than 40% of A/M cases. We performed whole-genome copy number variation analysis in 60 patients affected with isolated or syndromic A/M. Pathogenic deletions of 3q26 (SOX2) were identified in four independent patients with syndromic microphthalmia. Other variants of interest included regions with a known role in human disease (likely pathogenic) as well as novel rearrangements (uncertain significance). A 2.2-Mb duplication of 3q29 in a patient with non-syndromic anophthalmia and an 877-kb duplication of 11p13 (PAX6) and a 1.4-Mb deletion of 17q11.2 (NF1) in two independent probands with syndromic microphthalmia and other ocular defects were identified; while ocular anomalies have been previously associated with 3q29 duplications, PAX6 duplications, and NF1 mutations in some cases, the ocular phenotypes observed here are more severe than previously reported. Three novel regions of possible interest included a 2q14.2 duplication which cosegregated with microphthalmia/microcornea and congenital cataracts in one family, and 2q21 and 15q26 duplications in two additional cases; each of these regions contains genes that are active during vertebrate ocular development. Overall, this study identified causative copy number mutations and regions with a possible role in ocular disease in 17% of A/M cases.

Anterior segment dysgenesis (ASD) disorders encompass a spectrum of developmental conditions affecting the cornea, iris, and lens and are generally associated with an approximate 50% risk for glaucoma. These conditions are characterized by both autosomal dominant and recessive patterns of inheritance often with incomplete penetrance/variable expressivity. This article summarizes what is known about the genetics of ASD disorders and reviews recent developments.Mutations in Collagen type IV alpha-1 (COL4A1) and Beta-1,3-galactosyltransferase-like (B3GALTL) have been reported in ASD patients. Novel findings in other well known ocular genes are also presented, among which regulatory region deletions in PAX6 and PITX2 are most notable.Although a number of genetic causes have been identified, many ASD conditions are still awaiting genetic elucidation. The majority of characterized ASD genes encode transcription factors; several other genes represent extracellular matrix-related proteins. All of the involved genes play active roles in ocular development and demonstrate conserved functions across species. The use of novel technologies, such as whole genome sequencing/comparative genomic hybridization, is likely to broaden the mutation spectrums in known genes and assist in the identification of novel causative genes as well as modifiers explaining the phenotypic variability of ASD conditions.

FOXE3 forkhead transcription factor is essential to lens development in vertebrates. The eyes of Foxe3/foxe3-deficient mice and zebrafish fail to develop normally. In humans, autosomal dominant and recessive mutations in FOXE3 have been associated with variable phenotypes including anterior segment anomalies, cataract and microphthalmia. We undertook sequencing of FOXE3 in 116 probands with a spectrum of ocular defects ranging from anterior segment dysgenesis and cataract to anophthalmia/microphthalmia. Recessive mutations in FOXE3 were found in four of 26 probands affected with bilateral microphthalmia (15% of all bilateral microphthalmia and 100% of consanguineous families with this phenotype). FOXE3-positive microphthalmia was accompanied by aphakia and/or corneal defects; no other associated systemic anomalies were observed in FOXE3-positive families. The previously reported c.720C>A (p.C240X) nonsense mutation was identified in two additional families in our sample and therefore appears to be recurrent, now reported in three independent microphthalmia families of varied ethnic backgrounds. Several missense variants were identified at varying frequencies in patient and control groups with some apparently being race-specific, which underscores the importance of utilizing race/ethnicity-matched control populations in evaluating the relevance of genetic screening results. In conclusion, FOXE3 mutations represent an important cause of nonsyndromic autosomal recessive bilateral microphthalmia.

The human eye is a complex organ whose development requires extraordinary coordination of developmental processes. The conservation of ocular developmental steps in vertebrates suggests possible common genetic mechanisms. Genetic diseases involving the eye represent a leading cause of blindness in children and adults. During the last decades, there has been an exponential increase in genetic studies of ocular disorders. In this review, we summarize current success in identification of genes responsible for microphthalmia, anophthalmia, and coloboma (MAC) phenotypes, which are associated with early defects in embryonic eye development. Studies in animal models for the orthologous genes identified overlapping phenotypes for most factors, confirming the conservation of their function in vertebrate development. These animal models allow for further investigation of the mechanisms of MAC, integration of various identified genes into common developmental pathways and finally, provide an avenue for the development and testing of therapeutic interventions.

We report phenotypic and genetic analyses of a recessive, larval lethal zebrafish mutant, bal(a69), characterized by severe eye defects and shortened body axis. The bal(a69) mutation was mapped to chromosome 24 near the laminin alpha 1 (lama1) gene. We analyzed the lama1 gene sequence within bal(a69) embryos and two allelic mutants, bal(arl) and bal(uw1). Missense (bal(a69)), nonsense (bal(arl)), and frameshift (bal(uw1)) alterations in lama1 were found to underlie the phenotypes. Extended analysis of bal(a69) ocular features revealed disrupted lens development with subsequent lens degeneration, focal cornea dysplasia, and hyaloid vasculature defects. Within the neural retina, the ganglion cells showed axonal projection defects and ectopic photoreceptor cells were noted at inner retinal locations. To address whether ocular anomalies were secondary to defects in lens differentiation, bal(a69) mutants were compared to embryos in which the lens vesicle was surgically removed. Our analysis suggests that many of the anterior and posterior ocular defects in bal(a69) are independent of the lens degeneration. Analysis of components of focal adhesion signaling complexes suggests that reduced focal adhesion kinase activation underlies the anterior segment dysgenesis in lama1 mutants. To assess adult ocular phenotypes associated with lama1 mutations, genetic mosaics were generated by transplanting labeled bal cells into ocular-fated regions of wild-type blastulas. Adult chimeric eyes displayed a range of defects including anterior segment dysgenesis and cataracts. Our analysis provides mechanistic insights into the developmental defects and ocular pathogenesis caused by mutations in laminin subunits.

The human eye is a complex organ whose development requires extraordinary coordination of developmental processes. Multiple genes responsible for the proper development and maintenance of the vertebrate eye have been identified and shown to be involved in a variety of debilitating ocular conditions. Genetic diseases involving the eye represent a leading cause of blindness in children and adults. This article summarizes current genetic approaches and their application to studies of ocular disease.

Axenfeld–Rieger syndrome (ARS) is a rare autosomal dominant inherited disorder affecting the development of the eyes, teeth and abdomen. The syndrome is characterised by complete penetrance but variable expressivity. The ocular component of the ARS phenotype has acquired most clinical attention and has been dissected into a spectrum of developmental eye disorders, of which open-angle glaucoma represents the main challenge in terms of treatment. Mutations in several chromosomal loci have been implicated in ARS, including PITX2, FOXC1 and PAX6. Full-spectrum ARS is caused primarily by mutations in the PITX2 gene. The homeobox transcription factor PITX2 is produced as at least four different transcriptional and splicing isoforms, with different biological properties. Intriguingly, PITX2 is also involved in left–right polarity determination, although asymmetry defects are not a feature of ARS. In experimental animal models and in cell culture experiments using PITX2, abundant evidence indicates that a narrow window of expression level of this gene is vital for its correct function.

The human PITX3 gene encodes a bicoid-like homeodomain transcription factor associated with a variety of congenital ocular conditions, including anterior segment dysgenesis, Peter's anomaly, and cataracts. We identified a zebrafish pitx3 gene encoding a protein (Pitx3) that possesses 63% amino acid identity with human PITX3. The zebrafish pitx3 gene encompasses approximately 16.5 kb on chromosome 13 and consists of four exons, which is similar to the genomic organization of other pitx genes. Expression of the zebrafish pitx3 gene was studied by in situ mRNA hybridization and RT-PCR. The pitx3 transcripts were detected throughout development with the greatest level of expression occurring in the developing lens and brain at 24 hpf. In adults, the highest expression was detected in the eye. Morpholinos were used to knockdown expression of the Pitx3 protein and a control morpholino that contains five mismatched bases was used to confirm the specificity of the phenotypes. The morphants had small eyes, misshapen heads and reduced jaws and fins relative to controls. The morphants exhibited abnormalities in lens development and their retinas contained pyknotic nuclei accompanied by a reduction in the number of cells in different neuronal classes. This suggests the lens is required for retinal development or Pitx3 has an unexpected role in retinal cell differentiation or survival. These results demonstrate zebrafish pitx3 represents a true ortholog of the human PITX3 gene and the general function of the Pitx3 protein in lens development is conserved between mammals and the teleost fish.

We have generated embryonic stem (ES) cells and transgenic mice with green fluorescent protein (GFP) inserted into the Pitx3 locus via homologous recombination. In the central nervous system, Pitx3-directed GFP was visualized in dopaminergic (DA) neurons in the substantia nigra and ventral tegmental area. Live primary DA neurons can be isolated by fluorescence-activated cell sorting from these transgenic mouse embryos. In culture, Pitx3–GFP is coexpressed in a proportion of ES-derived DA neurons. Furthermore, ES cell-derived Pitx3–GFP expressing DA neurons responded to neurotrophic factors and were sensitive to DA-specific neurotoxin N-4-methyl-1, 2, 3, 6-tetrahydropyridine. We anticipate that the Pitx3–GFP ES cells could be used as a powerful model system for functional identification of molecules governing mDA neuron differentiation and for preclinical research including pharmaceutical drug screening and transplantation. The Pitx3 knock-in mice, on the other hand, could be used for purifying primary neurons for molecular studies associated with the midbrain-specific DA phenotype at a level not previously feasible. These mice would also provide a useful tool to study DA fate determination from embryo- or adult-derived neural stem cells., This work is supported by grants from the UK Medical Research Council (MRC) and an MRC–Stem Cell Science UK Ltd Collaborative PhD studentship (S.M.), and a CONCYTEA Mexico Studentship (A.J.-B.).

Specific DNA-binding transcription factors (TFs) are critical components of gene regulatory networks (GRNs) that govern lens growth and differentiation. Lens placode formation is controlled by Pax6, Six3, and Sox2. Lens vesicle formation and its separation from the surface ectoderm are regulated by AP-2α, Foxe3, and retinoic acid/retinoid-activated nuclear receptors. The lens precursor cells exit cell cycle via a coordinated action of Gata3, Pitx3, and Prox1. Lens fiber cell differentiation is controlled by ATF4/CREB2, c-Maf, Gata3, Hsf4, Pax6, Pitx3, Prox1, and Sox1. Lens morphogenesis requires intricate temporal and spatial control of gene expression that is executed through multiple links between TFs and extracellular signaling, including BMP, FGF, Notch, and Wnt pathways. Disrupted function of lens-preferred TFs results in multiple lens developmental abnormalities that range from the lack of lens formation, lost of primordial lens (aphakia), incomplete separation of the lens vesicle from the surface ectoderm (corneal-lenticular stalk), disrupted lens fiber cell differentiation, and retention of subcellular organelles including the nuclei. These developmental abnormalities are not compatible with lens transparency and result in congenital cataracts. These cataracts affect 1:2,500 individuals and are difficult to manage due to the age of the child and eye growth considerations, possible associated microcornea or microphthalmia, and risks of glaucoma and amblyopia. For example, up to ~40 % of children with bilateral cataracts develop glaucoma following surgery, particularly when cataracts are removed early in life. This review describes normal and abnormal lens formation through the perspective of TFs preferentially expressed in the lens, their contributions to the human pediatric cataracts, and current strategies in human genetic and clinical studies to identify novel cataract-associated genes to better understand molecular basis of these defects and its treatments.

Background: Congenital cataracts are clinically and genetically heterogeneous with more than 45 known loci and 38 identified genes. They can occur as isolated defects or in association with anterior segment developmental anomalies. One of the disease genes for congenital cataract with or without anterior segment dysgenesis (ASD) is PITX3, encoding a transcription factor with a crucial role in lens and anterior segment development. Only five unique PITX3 mutations have been described, of which the 17-bp duplication c.640-656dup, p.(Gly220Profs*95), is the most common one and the only one known to cause cataract with ASD. The aim of this study was to perform a genetic study of the PITX3 gene in five probands with autosomal dominant congenital cataract (ADCC) and ASD, to compare their clinical presentations to previously reported PITX3-associated phenotypes and to functionally evaluate the PITX3 mutations found. Methods. Sanger sequencing of the coding region and targeted exons of PITX3 was performed in probands and family members respectively. Transactivation, DNA-binding and subcellular localization assays were performed for the PITX3 mutations found. Ophthalmological examinations included visual acuity measurement, slit-lamp biomicroscopy, tonometry and fundoscopy. Results: In four Belgian families with ADCC and ASD the recurrent 17-bp duplication c.640-656dup, p.(Gly220Profs*95), was found in a heterozygous state. A novel PITX3 mutation c.573del, p.(Ser192Alafs*117), was identified in heterozygous state in a Belgo-Romanian family with a similar phenotype. Functional assays showed that this novel mutation retains its nuclear localization but results in decreased DNA-binding and transactivation activity, similar to the recurrent duplication. Conclusions: Our study identified a second PITX3 mutation leading to congenital cataract with ASD. The similarity in phenotypic expression was substantiated by our in vitro functional studies which demonstrated comparable molecular consequences for the novel p.(Ser192Alafs*117) and the recurrent p.(Gly220Profs*95) mutations. 2014 Verdin et al. licensee BioMed Central Ltd., SCOPUS: ar.j, info:eu-repo/semantics/published

We report here the identification of a new human homeobox gene, PITX3, and its involvement in anterior segment mesenchymal dysgenesis (ASMD) and congenital cataracts in humans. The PITX3 gene is the human homologue of the mouse Pitx3 gene and is a member of the RIEG/PITX homeobox gene family. The protein encoded by PITX3 shows 99% amino-acid identity to the mouse protein, with 100% identity in the homeodomain and approximately 70% overall identity to other members of this family. We mapped the human PITX3 gene to 10q25 using a radiation-hybrid panel. A collection of 80 DNA samples from individuals with various eye anomalies was screened for mutations in the PITX3 gene. We identified two mutations in independent patients. A 17-bp insertion in the 3'-end of the coding sequence, resulting in a frame shift, occurred in a patient with ASMD and cataracts, and a G--A substitution, changing a codon for serine into a codon for asparagine, in the 5'-end of the gene occurred in a patient with congenital cataracts. Both mutations cosegregate with the disease phenotype in families, and neither were found in up to 300 control individuals studied. Further expression analysis of Pitx3 in the mouse supports a unique role in early ocular development, with later expression extending to the midbrain, tongue, incisors, sternum, vertebrae and limbs. These data strongly suggest a role for PITX3 in ASMD and cataracts and provide new evidence of the contribution of the RIEG/PITX gene family to the developmental program underpinning normal eye formation.

We recently reported the positional cloning of a homeobox gene involved in the pathogenesis of Rieger syndrome, RIEG1 , and its mouse homolog, Rieg1 . Rieg1 (also independently described as Pitx2) is highly homologous to the Ptx1/Potx gene product, suggesting that there may be additional members of this novel Pitx family. The Pitx genes play an important role in eye, tooth, pituitary and umbilical region development as evidenced by Rieger syndrome and iris hypoplasia phenotypes, resulting from mutations in the RIEG1 gene and by expression studies. In order to characterize further the Pitx gene family we searched mouse cDNA libraries to identify additional members. A new gene was isolated which encodes a homeoprotein with strong homology to the other Pitx proteins and 97-100% identity in the homeodomain itself, suggesting that this is a third member of the family, Pitx3 . In whole mount in situ hybridization on mouse embryos ranging from 8.5 to 11.5 days post-coitum (d.p.c.), Pitx3 mRNA was seen only in the developing lens starting at day 11. Hybridization on cross-sections revealed strong signals in the lens vesicle in 11 d.p.c. embryos and throughout the lens, particularly in the anterior epithelium and equator region in 15 d.p.c. embryos. Pitx3 was mapped close to aphakia on mouse chromosome 19. The aphakia homozygous mouse is characterized by small eyes lacking a lens, which fail to develop beyond 11 d.p.c. These data make Pitx3 a strong candidate gene for the aphakia phenotype in the mouse and suggest a role for the human homolog in congenital lens malformations.

We report analysis of the ocular lens phenotype of the recessive, larval lethal zebrafish mutant,lama1a69/a69. Previous work revealed that this mutant has a shortened body axis and eye defects including a defective hyaloid vasculature, focal corneal dysplasia, and loss of the crystalline lens. While these studies highlight the importance of lamininα1 in lens development, a detailed analysis of the lens defects seen in these mutants was not reported. In the present study, we analyze the lenticular anomalies seen in thelama1a69/a69mutants and show that the lens defects result from the anterior extrusion of lens material from the eye secondary to structural defects in the lens capsule and developing corneal epithelium associated with basement membrane loss. Our analysis provides further insights into the role of the lens capsule and corneal basement membrane in the structural integrity of the developing eye.

Pediatric cataracts are observed in 1–15 per 10,000 births with 10–25 % of cases attributed to genetic causes; autosomal dominant inheritance is the most commonly observed pattern. Since the specific cataract phenotype is not sufficient to predict which gene is mutated, whole exome sequencing (WES) was utilized to concurrently screen all known cataract genes and to examine novel candidate factors for a disease-causing mutation in probands from 23 pedigrees affected with familial dominant cataract. Review of WES data for 36 known cataract genes identified causative mutations in nine pedigrees (39 %) in CRYAA, CRYBB1, CRYBB3, CRYGC (2), CRYGD, GJA8 (2), and MIP and an additional likely causative mutation in EYA1; the CRYBB3 mutation represents the first dominant allele in this gene and demonstrates incomplete penetrance. Examination of crystallin genes not yet linked to human disease identified a novel cataract gene, CRYBA2, a member of the βγ-crystallin superfamily. The p.(Val50Met) mutation in CRYBA2 cosegregated with disease phenotype in a four-generation pedigree with autosomal dominant congenital cataracts with incomplete penetrance. Expression studies detected cryba2 transcripts during early lens development in zebrafish, supporting its role in congenital disease. Our data highlight the extreme genetic heterogeneity of dominant cataract as the eleven causative/likely causative mutations affected nine different genes, and the majority of mutant alleles were novel. Furthermore, these data suggest that less than half of dominant cataract can be explained by mutations in currently known genes.

Anterior segment dysgenesis (ASD) encompasses a broad spectrum of developmental conditions affecting anterior ocular structures and associated with an increased risk for glaucoma. Various systemic anomalies are often observed in ASD conditions such as Axenfeld-Rieger syndrome (ARS) and De Hauwere syndrome. We report DNA sequencing and copy number analysis of PITX2 and FOXC1 in 76 patients with syndromic or isolated ASD and related conditions. PITX2 mutations and deletions were found in 24 patients with dental and/or umbilical anomalies seen in all. Seven PITX2-mutant alleles were novel including c.708_730del, the most C-terminal mutation reported to date. A second case of deletion of the distant upstream but not coding region of PITX2 was identified, highlighting the importance of this recently discovered mechanism for ARS. FOXC1 deletions were observed in four cases, three of which demonstrated hearing and/or heart defects, including a patient with De Hauwere syndrome; no nucleotide mutations in FOXC1 were identified. Review of the literature identified several other patients with 6p25 deletions and features of De Hauwere syndrome. The 1.3-Mb deletion of 6p25 presented here defines the critical region for this phenotype and includes the FOXC1, FOXF2, and FOXQ1 genes. In summary, PITX2 or FOXC1 disruptions explained 63% of ARS and 6% of other ASD in our cohort; all affected patients demonstrated additional systemic defects with PITX2 mutations showing a strong association with dental and/or umbilical anomalies and FOXC1 with heart and hearing defects. FOXC1 deletion was also found to be associated with De Hauwere syndrome.

The PITX3 bicoid-type homeodomain transcription factor plays an important role in lens development in vertebrates. PITX3 deficiency results in a spectrum of phenotypes from isolated cataracts to microphthalmia in humans, and lens degeneration in mice and zebrafish. While identification of downstream targets of PITX3 is vital for understanding the mechanisms of normal ocular development and human disease, these targets remain largely unknown. To isolate genes that are directly regulated by PITX3, we performed a search for genomic sequences that contain evolutionarily conserved bicoid/PITX3 binding sites and are located in the proximity of known genes. Two bicoid sites that are conserved from zebrafish to human were identified within the human promoter of the major intrinsic protein of lens fiber, MIP/AQP0. MIP/AQP0 deficiency was previously shown to be associated with lens defects in humans and mice. We demonstrate by both chromatin immunoprecipitation and electrophoretic mobility shift assay that PITX3 binds to MIP/AQP0 promoter region in vivo and is able to interact with both bicoid sites in vitro. In addition, we show that wild-type PITX3 is able to activate the MIP/AQP0 promoter via interaction with the proximal bicoid site in cotransfection experiments and that the introduction of mutations disrupting binding to this site abolishes this activation. Furthermore, mutant forms of PITX3 fail to produce the same levels of transactivation as wild-type when cotransfected with the MIP/AQP0 reporter. Finally, knockdown of pitx3 in zebrafish affects formation of a DNA-protein complex associated with mip1 promoter sequences; and examination of expression in pitx3 morphant and control zebrafish revealed a delay in and reduction of mip1 expression in pitx3-deficient embryos. Therefore, our data suggest that PITX3 is involved in direct regulation of MIP/AQP0 expression and that the alteration of MIP/AQP0 expression is likely to contribute to the lens phenotype in cataract patients with PITX3 mutations.

Purpose The role of SRY-Box 2 (SOX2) in anophthalmia/microphthalmia (A/M) is well known, with 10%–20% of A/M explained by mutations in SOX2. SOX2 plays roles in the development of both the posterior and anterior segment structures of the eye and relies on interactions with tissue-specific partner proteins to execute its function, raising the possibility that SOX2 mutations may result in varying ocular phenotypes. Recent data has identified a missense mutation in SOX2 in an extended pedigree with phenotypes as varied as A/M, isolated iris hypoplasia, iris and chorioretinal coloboma, pupil defects, and hypermetropia, suggesting a broader phenotypic spectrum associated with SOX2 mutations. Methods Screening of SOX2 was completed in 89 patients with a variety of ocular anomalies, including 28 with A/M and 61 with normal eye size and anterior segment dysgenesis (28), cataract (14), isolated coloboma (5), or other eye disorders (14). Results The recurrent de novo frameshift mutation c.70del20 was identified in one patient with microphthalmia and syndromic anomalies consistent with SOX2 anophthalmia syndrome; the mutation frequency in our A/M population (4%) was lower than previously reported; it is likely that extensive utilization of clinical SOX2 testing has led to a bias toward SOX2-negative A/M cases in our research cohort. No disease-causing mutations were identified in patients with non-microphthalmia phenotypes. Conclusions The recurrent c.70del20 mutation accounts for 21% of all independent SOX2 mutations reported to date. Due to the increased use of clinical SOX2 testing, the frequency of SOX2 mutations identified in research A/M populations will likely continue to decrease. Mutations in SOX2 do not appear to be a common cause of ocular defects other than anophthalmia/microphthalmia.

SOX2 represents a High Mobility Group domain containing transcription factor that is essential for normal development in vertebrates. Mutations in SOX2 are known to result in a spectrum of severe ocular phenotypes in humans, also typically associated with other systemic defects. Ocular phenotypes include anophthalmia/microphthalmia (A/M), optic nerve hypoplasia, ocular coloboma and other eye anomalies. We screened 51 unrelated individuals with A/M and indentified SOX2 mutations in the coding region of the gene in 10 individuals. Seven of the identified mutations are novel alterations, while the remaining three individuals carry the previously reported recurrent 20-nucleotide deletion in SOX2, c.70del20. Among the SOX2-positive cases, seven patients had bilateral A/M and mutations resulting in premature termination of the normal protein sequence (7/38; 18% of all bilateral cases), one patient had bilateral A/M associated with a single amino acid insertion (1/38; 3% of bilateral cases), and the final two patients demonstrated unilateral A/M associated with missense mutations (2/13; 15% of all unilateral cases). These findings and review of previously reported cases suggest a potential genotype/phenotype correlation for SOX2 mutations with missense changes generally leading to less severe ocular defects. In addition, we report a new familial case of affected siblings with maternal mosaicism for the identified SOX2 mutation, which further underscores the importance of parental testing to provide accurate genetic counseling to families.

Background The homeodomain-containing transcription factor PITX3 was shown to be essential for normal eye development in vertebrates. Human patients with point mutations in PITX3 demonstrate congenital cataracts along with anterior segment defects in some cases when one allele is affected and microphthalmia with brain malformations when both copies are mutated. The functional consequences of these human mutations remain unknown. Results We studied the PITX3 mutant proteins S13N and G219fs to determine the type and severity of functional defects. Our results demonstrate alterations in DNA-binding profiles and/or transactivation activities and suggest a partial loss-of-function in both mutants with the G219fs form being more severely affected. No anomalies in cellular distribution and no dominant-negative effects were discovered for these mutants. Interestingly, the impairment of the G219fs activity varied between different ocular cell lines. Conclusion The G219fs mutation was found in multiple families affected with congenital cataracts along with anterior segment malformations in many members. Our data suggest that the presence/severity of anterior segment defects in families affected with G219fs may be determined by secondary factors that are expressed in the developing anterior segment structures and may modify the effect(s) of this mutation. The S13N mutant showed only minor alteration of transactivation ability and DNA binding pattern and may represent a rare polymorphism in the PITX3 gene. A possible contribution of this mutation to human disease needs to be further investigated.

The anterior segment of the vertebrate eye is a complex arrangement of interdependent tissues of different embryonic origins. Despite its critical role in normal vision, relatively little is currently known about the development of the anterior segment and its molecular determinants. All three members of the PITX homeobox-containing transcription factor gene family were found to be expressed in the anterior segment structures during embryonic development. Two of these genes, PITX2 and PITX3, were shown to be responsible for a spectrum of developmental anterior segment phenotypes associated with glaucoma, corneal opacities and cataracts in humans and mice. These findings demonstrate a requirement of PITX genes for normal development of the anterior segment of the eye and provide tools to study the molecular control of development of these structures.

The overall goal of this review is to highlight the power of zebrafish as a model system for studying complex diseases which involve multiple genetic loci. We are interested in identifying and characterizing genes implicated in the blinding condition of glaucoma. Glaucoma is a complex disease that often involves multiple genetic loci. Most disease causing and modifying genes for glaucoma remain unidentified. However, several genes that regulate various aspects of ocular development have been shown to associate with glaucoma. With zebrafish, forward and reverse genetic approaches can be combined in order to identify critical genetic interactions required for normal and pathological events in the development and maintenance of the eye.

The purpose of this study was to evaluate chromosomal regions previously linked to pathological myopia for linkage to juvenile myopia in a sample of myopic children and their families. Of 125 families with a myopic child participating in the Orinda longitudinal study of myopia, 53 submitted 221 buccal swab samples for genetic analysis. Myopia in proband children was defined as -0.75 D or more myopia in both meridians on cycloplegic autorefraction (1% tropicamide). Affected status in parents and siblings was obtained by survey. DNA was extracted from buccal mucosal cells, amplified by polymerase chain reaction (PCR), and then analyzed with seven markers for chromosome 12 and five markers for chromosome 18 in the regions previously associated with pathological myopia. LOD scores were not significant for any marker tested. The largest positive LOD score was 0.15 for GATA30F04. Model-free methods using a SimIBD approach suggested a possible linkage at one marker, GATA6H09 (P = 0.003), but these results were not supported by transmission disequilibrium test (TDT) analysis. The statistical power to detect LOD scores of > or =1.0, assuming homogeneity, was estimated at 93.2%. We found no confirmatory evidence of linkage between juvenile myopia and regions of chromosomes 12 and 18 previously associated with pathological myopia.

Mouse aphakia (ak) is a recessive phenotype that spontaneously occurs in the 129/Sv-SlJ strain and is characterized by small eyes that lack a lens. We have recently identified a homeobox-containing gene, Pitx3, and have shown that it is expressed in the developing lens and maps to chromosome 19 close to ak in mouse. Human PITX3 gene was found to underlie anterior segment dysgenesis and cataracts. We have now obtained the entire sequence of the mouse Pitx3 gene including 10 kb of the 5' region and 5 kb of the 3' region. Of several microsatellite repeat regions identified within the Pitx3 sequence, one was informative for linkage analysis. No recombination was observed between ak and the Pitx3 marker, indicating that these two loci are closely linked (0.2 +/- 0.2 cM). Additionally, Pitx3 transcripts were not detected in the ak/ak mice either in the lens placode or at later developmental stages of the lens by in situ hybridization. Since no differences were previously found between ak/ak and wild-type sequences in the Pitx3 coding region, we hypothesized that an etiologic mutation is located in the promoter or other regulatory regions. To test this hypothesis we studied the 5' flanking region of the Pitx3 gene. This analysis revealed a deletion of 652 bp located 2.5 kb upstream from the start point of the Pitx3 5' UTR sequence in ak/ak mice. The deletion co-segregated with the ak mutation and was not detected in 16 samples from 10 different mouse strains including the founder strains. Analysis of the 652 bp region identified sequences similar to consensus binding sites for transcription factors AP-2 and Maf that were shown to play a critical role in lens determination. These lines of evidence suggest that the abnormal ocular development in the aphakia mouse is due to the deletion upstream of the Pitx3 gene.

Axenfeld-Rieger syndrome (ARS) and iridogoniodysgenesis syndrome (IGDS) are clinically related autosomal dominant disorders which affect the anterior segment of the eye as well as non-ocular structures. ARS patients present with iris hypoplasia, a prominent Schwalbe line, adhesions between the iris stroma and the iridocorneal angle and increased intraocular pressure. IGDS is characterized by iris hypoplasia, goniodysgenesis and increased intraocular pressure. Each syndrome also presents with non-ocular features including maxillary hypoplasia, micro and anodontia, redundant periumbilical skin, hypospadius (in males), and each has been genetically linked to chromosome 4q25. RIEG1 , the gene responsible for the 4q25 ARS phenotype, recently has been cloned. RIEG1 encodes a novel member of the bicoid class of homeobox proteins known to be active as transcription factors. Mutational analysis has previously detected several mutations in this gene in ARS individuals. We have now detected a mutation in RIEG1 which segregates with the disease phenotype in a family with IGDS. This mutation is a G-->A transition altering an arginine residue to a histidine in a highly conserved location in the second helix of the homeobox of RIEG1. This mutation indicates that IGDS and ARS are allelic variants of the same disorder. This wide variability in clinical consequences of mutations at the RIEG1 4q25 locus implicates the RIEG gene broadly in ocular and craniofacial disorders.

The PITX2 transcription factor was identified as a causative gene for Axenfeld-Rieger syndrome (ARS; Mendelian Inheritance in Man no. 180500) by Semina et al.1 ARS is a rare autosomal dominant disorder that affects anterior eye development2 and can occur with or without systemic findings. The ocular findings include structural anomalies of the anterior chamber angle and aqueous drainage structures. Nonocular features typically include facial, dental, and umbilical defects. Our recent study3 showed that ARS is associated with an approximately 75% risk for glaucoma in patients with FOXC1 or PITX2 defects. Glaucoma, the second leading cause of blindness throughout the world,4–6 is the most important clinical consequence of ARS.7,8 Glaucoma is often associated with elevated intraocular pressure (IOP), as regulated by the ciliary body (the site of aqueous humor production) and the limbal region (which includes the trabecular meshwork), the principal site of aqueous humor outflow.4,5 The disease can continue to progress in patients even when IOP is significantly reduced. Research conducted in the past two decades has revealed that oxidative stress is involved in glaucoma pathogenesis.9–16 Identifying genes involved in adult anterior segment function or responses to oxidative stress will greatly improve our understanding of glaucoma pathology and will result in finding better treatment options. The PITX2 protein contains a conserved 60 amino acid homeodomain of the paired-bicoid class that is responsible for DNA binding, localization to the nucleus, and protein-protein interaction.17 Point mutations in PITX2 have been demonstrated to be loss-of-function mutations affecting DNA binding and transcriptional transactivation.1,18–27 Tight control of PITX2 expression is required for normal development of the eye because too much or too little activity of this transcription factor results in anterior segment defects and glaucoma.24,28 We hypothesize that altered regulation and expression of PITX2 target genes in adult tissues may have a significant impact on the development and progression of ARS-associated glaucoma and may reveal the cellular pathways affected in primary glaucoma phenotypes. We previously designed a hormone-inducible transcription factor expression system to discover target genes for FOXC129 and have now adapted this system to identify PITX2 target genes in ocular cells. We report here the identification and characterization of the Solute carrier family 13 sodium-dependent dicarboxylate transporter member 3 (SLC13A3) as a gene directly regulated by PITX2. Its protein product, NaDC3, is implicated as an important transporter of neuronal metabolites30 and may regulate the uptake of glutathione (GSH) to protect against ocular oxidative stress.31 We demonstrate for the first time that PITX2 and SLC13A3 are necessary for cellular responses to oxidative insult from hydrogen peroxide.

Axenfeld-Rieger syndrome (ARS) is a relatively rare developmental disorder characterized by maxillary hypoplasia, anterior segment defects of the eye (with glaucoma in ∼50%), and dental and umbilical abnormalities. The PITX2 homeodomain-containing transcription factor gene plays a major role in this condition, explaining approximately 40% of classic ARS.1–5 In addition, PITX2 mutations were shown to cause isolated ocular conditions,6–8 to be associated with additional brain anomalies,9,10 and possibly to contribute to other phenotypes such as omphalocele and VATER-like association.11 The function of Pitx2 is conserved in vertebrates. Total or conditional knockouts of Pitx2 result in severe developmental phenotypes in mice that include craniofacial, ocular, dental, brain, heart, lung, and other systemic defects consistent with the expression sites of Pitx2. With respect to Axenfeld-Rieger syndrome, Pitx2-deficient mice exhibit arrest of eye and tooth organogenesis, defective body wall closure and brain abnormalities.12–17 The defects observed in mice are associated with a complete loss of Pitx2 activity in the corresponding structures and, therefore, are noticeably more severe that what is reported in humans with heterozygous PITX2 mutations, whereas heterozygous mice are generally described as normal. This suggests a variable requirement of PITX2/Pitx2 for normal human/mouse embryogenesis, with human development more sensitive to correct PITX2 dosage. Human PITX2 mutations mainly result in a complete or partial loss of function, with mutations retaining some wild-type activity producing milder phenotypes.18–20 Some mutations identified in patients with Axenfeld-Rieger syndrome displayed very minor defects in DNA-binding and transactivation activities, again suggesting that normal human development is highly susceptible to alterations of PITX2 function.5 In addition, several human mutations associated with gain-of-function have been reported,21 consistent with the dramatic ocular and limb phenotypes seen in mice with Pitx2 overexpression during eye and forelimb development.22,23 Therefore, correct dosage of PITX2 is critical for normal development, which further underscores the importance of precise transcriptional regulation of its expression. Regulation of Pitx2 expression has been studied by several groups, but only a few cis-regulatory elements have been identified and examined in animal models. In mouse, Pitx2 appears to be a part of the Wnt/β-catenin/TCF/LEF and cAMP-CREB signaling pathways.24,25 Transcriptional activation of Pitx2 during eye development has been shown to be dependent on retinoic acid signaling26–28; the nature of this interaction, direct or indirect, has not yet been established. A few studies reported in vivo identification of Pitx2 enhancer elements, which facilitated upstream factor analysis. Asymmetric Pitx2 expression in the developing visceral organs was shown to require an intronic enhancer located in the proximity of the last exon,29,30 whereas a 7-kb fragment located 4.2 kb downstream of the last exon of Pitx2 was found to direct reporter expression in the oral ectoderm and Rathke's pouch.31 Finally, several regulatory elements associated with pitx2 expression in the stomodeum territory have been isolated in ascidians; the cis-sequences were either intronic or located within 2.5 kb of the 5′ or 3′ end of the gene.32 PITX2 is flanked by glutamyl aminopeptidase (aminopeptidase A) (ENPEP) located approximately 54 kb downstream and chromosome 4 open reading frame 32 (C4orf32) expressed sequence positioned approximately 1.5 Mb upstream of PITX2; thus, the 1.5-Mb sequence upstream of the gene is considered a gene desert. Gene deserts often contain conserved sequences involved in transcriptional regulation of nearby genes.33 The presence of PITX2 regulatory elements in this region were consistent with the previous reports of patients with Axenfeld-Rieger syndrome with translocation breakpoints that occurred within the distant upstream region and did not disrupt the coding region of PITX2.1,2,34,35 We describe identification of several novel regulatory sequences in the PITX2/pitx2 region, including several distant elements located within the gene desert upstream of PITX2. The activities of these elements appear to be consistent with PITX2/pitx2 expression, suggesting that they play a role in this gene's function. In addition, we report the identification of a de novo deletion located 106 to 108 kb upstream of PITX2 in a patient with Axenfeld-Rieger syndrome, thus providing additional support for the essential role of the identified regulatory elements in PITX2 function.