PITX2 Mutations Research Articles

In Silico Characterization of Pathogenic Homeodomain Missense Mutations in the PITX2 Gene.

Paired homologous domain transcription factor 2 (PITX2) is critically involved in ocular and cardiac development. Mutations in PITX2 are consistently reported in association with Axenfeld-Rieger syndrome, an autosomal dominant genetic disorder and atrial fibrillation, a common cardiac arrhythmia. In this study, we have mined missense mutations in PITX2 gene from NCBI-dbSNP and Ensembl databases, evaluated the pathogenicity of the missense variants in the homeodomain and C-terminal region using five in silico prediction tools SIFT, PolyPhen2, GERP, Mutation Assessor and CADD. Fifteen homeodomain mutations G42V, G42R, R45W, S49Y, R53W, E53D, E55V, R62H, P65S, R69H, G75R, R84G, R86K, R87W, R91P were found to be highly pathogenic by both SIFT, PolyPhen2 were further functionally characterized using I-Mutant 2.0, Consurf, MutPred and Project Hope. The findings of the study can be used for prioritizing mutations in the context of genetic studies.

Whole genome sequencing in families with oligodontia.

Tooth agenesis (TA) is among the most common malformations in humans. Although several causative mutations have been described, the genetic cause often remains elusive. Here, we test whether whole genome sequencing (WGS) could bridge this diagnostic gap. In four families with TA, we assessed the dental phenotype using the Tooth Agenesis Code after intraoral examination and radiographic and photographic documentation. We performed WGS of index patients and subsequent segregation analysis. We identified two variants of uncertain significance (a potential splice variant in PTH1R, and a 2.1 kb deletion abrogating a non-coding element in FGF7) and three pathogenic variants: a novel frameshift in the final exon of PITX2, a novel deletion in PAX9, and a known nonsense variant in WNT10A. Notably, the FGF7 variant was found in the patient, also featuring the WNT10A variant. While mutations in PITX2 are known to cause Axenfeld-Rieger syndrome 1 (ARS1) predominantly featuring ocular findings, accompanied by dental malformations, we found the PITX2 frameshift in a family with predominantly dental and varying ocular findings. Severe TA predicts a genetic cause identifiable by WGS. Final exon PITX2 frameshifts can cause a predominantly dental form of ARS1.

Ophthalmological Manifestations of Axenfeld-Rieger Syndrome: Current Perspectives.

Axenfeld-Rieger syndrome (ARS) is a rare congenital disease that is primarily characterized by ocular anterior segment anomalies but is also associated with craniofacial, dental, cardiac, and neurologic abnormalities. Over half of cases are linked with autosomal dominant mutations in either FOXC1 or PITX2, which reflects the molecular role of these genes in regulating neural crest cell contributions to the eye, face, and heart. Within the eye, ARS is classically defined as the combination of posterior embryotoxon with iris bridging strands (Axenfeld anomaly) and iris hypoplasia causing corectopia and pseudopolycoria (Rieger anomaly). Glaucoma due to iridogoniodysgenesis is the main source of morbidity and is typically diagnosed during infancy or childhood in over half of affected individuals. Angle bypass surgery, such as glaucoma drainage devices and trabeculectomies, is often needed to obtain intraocular pressure control. A multi-disciplinary approach including glaucoma specialists and pediatric ophthalmologists produces optimal outcomes as vision is dependent on many factors including glaucoma, refractive error, amblyopia and strabismus. Further, since ophthalmologists often make the diagnosis, it is important to refer patients with ARS to other specialists including dentistry, cardiology, and neurology.

Effects of hydroxychloroquine on atrial electrophysiology in in silico wild-type and PITX2+/- atrial cardiomyocytes.

Hydroxychloroquine (HCQ) is commonly used in the treatment of autoimmune diseases and increases the risk of QT interval prolongation. However, it is unclear how HCQ affects atrial electrophysiology and the risk of atrial fibrillation (AF). We quantitatively examined the potential atrial arrhythmogenic effects of HCQ on AF using acomputational model of human atrial cardiomyocytes. We measured atrial electrophysiological markers after systematically varying HCQ concentrations. The HCQ concentrations were positively correlated with the action potential duration (APD), resting membrane potential, refractory period, APD alternans threshold, and calcium transient alternans threshold (p < 0.05). By contrast, HCQ concentrations were inversely correlated with the maximum upstroke velocity and calcium transient amplitude (p < 0.05). When the therapeutic concentration (Cmax) of HCQ was applied, HCQ increased APD90 by 1.4% in normal sinus rhythm, 1.8% in wild-type AF, and 2.6% in paired-like homeodomain transcription factor2 (PITX2)+/- AF, but did not affect the alternans thresholds. The overall in silico results suggest no significant atrial arrhythmogenic effects of HCQ at Cmax, instead implying apotential antiarrhythmic role of low-dose HCQ in AF. However, at an HCQ concentration of fourfold Cmax, arapid pacing rate of 4 Hz induced prominent APD alternans, particularly in the PITX2+/- AF model. Our in silico analysis suggests a potential antiarrhythmic role of low-dose HCQ in AF. Concomitant PITX2 mutations and high-dose HCQ treatments may increase the risk of AF, and this potential genotype/dose-dependent arrhythmogenic effect of HCQ should be investigated further.

Craniofacial and dental features of Axenfeld-Rieger syndrome patients with PITX2 mutations.

We aimed to characterize the genetic basis and craniofacial and dental features of Finnish patients with Axenfeld-Rieger syndrome (ARS). Mutational analyses of seven patients in five families were performed by sequencing or comparative genomic hybridization. Phenotypic analysis was based on both clinical and radiographic examinations, as well as on medical data. Lateral cephalometric radiographs of five patients were analysed using Viewbox 3.1-Cephalometric Software. The cephalometric values were compared to Finnish population-standard values of the same age and gender. Two frameshift mutations and three whole gene deletions were detected in five families. Class III skeletal relationship with retrognathic maxilla and mildly retrognathic mandible were detected in all five patients studied. Significant differences compared with the control values were in SNA (P=.0014), ANB (P=.0043) and SNB angles (P=.013). Five patients had anterior crossbite. Six patients showed tooth agenesis. The average number of missing teeth (third molars excluded) was 9 (range 0-15). The tooth agenesis rate was 52% in maxilla and 26% in mandible. Maxillary central and lateral permanent incisors were most often missing (rate 71% equally) while no one lacked canines or first molars in mandible. Two patients had a supernumerary mandibular permanent incisor. Six patients had either taurodontic and/or single-rooted molars. Our results suggest that class III skeletal relationship with maxillary and mandibular retrognathism, anterior crossbite, maxillary incisor agenesis and taurodontic, even pyramidal, roots are common determinants of ARS caused by PITX2 mutations.

Generation of two PITX2 knock-out human induced pluripotent stem cell lines using CRISPR/Cas9 system

PITX2 is a homeobox gene located in the human 4q25 locus and is commonly associated with atrial fibrillation (AF). Here, we generated two PITX2 knock-out human induced pluripotent stem cell (iPSC) lines using CRISPR/Cas9 genome editing. The edited iPSCs maintained fullpluripotency, normal karyotype and spontaneousdifferentiation capability. This cell line provides a suitable model for investigating the physiopathologyof PITX2 mutation in atrial fibrillation.

Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation.

Mutations in PITX2 cause Axenfeld–Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.

Genetics and epidemiology of aniridia: Updated guidelines for genetic study.

Aniridia is a panocular disease characterized by iris hypoplasia, accompanied by other ocular manifestations, with a high clinical variability and overlapping with different abnormalities of the anterior and posterior segment. This review focuses on the genetic features of this autosomal dominant pathology, which is caused by the haploinsufficiency of the PAX6 gene. Mutations causing premature stop codons are the most frequent among the wider mutational spectrum of PAX6, with more than 600 different mutations identified so far. Recent advances in next-generation sequencing (NGS) have increased the diagnostic yield in aniridia and contributed to elucidate new etiopathogenic mechanisms leading to PAX6 haploinsufficiency. Here, we also update good practices and recommendations to improve genetic testing and clinical management of aniridia using more cost-effective NGS analysis. Those new approaches also allow studying simultaneously both structural variants and point-mutations in PAX6 as well as other genes for differential diagnosis, simultaneously. Some patients with atypical phenotypes might present mutations in FOXC1 and PITX2, both genes causing a wide spectrum of anterior segment dysgenesis, or in ITPR1, which is responsible for a distinctive form of circumpupillary iris aplasia present in Gillespie syndrome, or other mutations in minor genes. Since aniridia can also associate extraocular anomalies, as it occurs in carriers of PAX6 and WT1 microdeletions leading to WAGR syndrome, genetic studies are crucial to assure a correct diagnosis and clinical management, besides allowing prenatal and preimplantational genetic testing in families.

Axenfeld\u2010Rieger syndrome combined with a foveal anomaly in a three\u2010generation family: a case report

BackgroundAxenfeld-Rieger syndrome (ARS) is a rare autosomal dominant eye disorder that can also affect other organs of the human body. The condition is primarily characterized by the anterior segmental abnormalities of the eye. Here, we present an observational case series of a three-generation family with ARS and unexpected foveal anomaly.Case presentationA 33-year-old woman was admitted to an Ophthalmology Clinic in Bialystok for left eye congenital cataract surgery. The patient (proband) was diagnosed with visual deterioration, multiple defects of iris, corectopia, displacement of the Schwalbe’s line, and phenotypic characteristics of ARS. A perimetric examination indicated peripheral visual field loss and signs typical for glaucoma. Based on the phenotypic symptoms and genetic test, the patient was diagnosed with Axenfeld Rieger Syndrome. However, the optical coherence tomography of the macula showed foveal anomaly (absence of the physiological pit), which is not typically associated with this genetic disorder. The patient’s family history revealed that her two daughters were undergoing treatment for congenital glaucoma, and one of the daughters also had foveal anomaly the same as her mother. Interestingly, an examination of the patient’s mother showed typical phenotypic features of ARS such as a defect of the iris, posterior embryotoxon, and coloboma, as well as foveal anomaly. A genetic test confirmed PITX2 mutation in both, proband’s two daughters and mother.ConclusionsThis study highlights the occurrence of ARS with unusual ophthalmic features such as foveal anomaly (absence of the physiological pit) in a three-generation family. Although ARS is known to represent the developmental defects of the anterior segment of the eye, it is very important to perform fundus evaluation to identify associated posterior segment anomalies that may affect visual acuity. The presence of ocular defects not typically associated with ARS suggests a wide spectrum of mutations within PITX2 gene which are required to identify in order to determine genotype- phenotype correlation in ARS affected individuals.

MON-257 Axenfeld Rieger Syndrome: An Uncommon Cause of Growth Hormone Deficiency

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder presenting with abnormal eye development, which leads to glaucoma related blindness in 50% of individuals. Associated mutations affect the transcription factors pituitary homeobox 2 gene (PITX2) and forkhead box C1 gene (FOXC1). Three types of ARS have been described. PITX2 mutation causes ARS type I, which is associated to systemic malformations, including dental hypoplasia, redundant periumbilical skin, and growth hormone deficiency (GHD).This is the case of a 28-year-old male diagnosed with GHD during childhood. He was referred to a pediatric-endocrinologist at age 10 due to short stature. Evaluation showed a height-for-age curve below the 10th percentile. Physical examination with prominent forehead, decrease visual acuity, maxillary hypodontia, umbilical hernia, and delayed sexual maturity. Testing with reduced IGF-1 and delayed bone age. Clonidine GH stimulation confirmed the diagnosis of GDH. He was treated with somatropin, until linear growth decreased to ½ inch per year at age 16. GHD etiology was never established. At age 26, he developed progressive decrease in visual acuity. Ophthalmology evaluation disclosed polycoria, megalocornea and increase intraocular pressure, suggestive of ARS. Patient was referred to our endocrinology clinics for follow up of previous diagnosis of GHD. Based on clinical findings and history, sequence analysis and deletion/duplication testing of FOXC1, PAX6 and PITx2 were performed, with results positive for pathogenic variant PITX2, Exon 5, c.363_364delinsAA. Assessment of pituitary hormone axis was normal, and no persistent GHD found. No family members exhibited clinical signs of ARS.Axenfeld-Rieger syndrome is a rare genetic disease. PITX homeodomain transcription factors are critical for the development of the anterior segment of the eye and pituitary. Most mutations in PITX2 affect DNA binding and transactivation that leads to defects in cell proliferation and differentiation of the Rathke’s pouch. As a result, GHD may ensue. ARS patients are usually diagnosed during childhood after the development of vision abnormalities. The diagnosis remains primarily clinical upon identification of ocular abnormalities in the iris and cornea, and increased intraocular pressure. Systemic changes are rare findings in ARS, but may include face and tooth abnormalities and isolated growth hormone deficiency. Genetic diagnosis is based on identification of mutations. An adequate management of ARS requires a multidisciplinary approach. Although ophthalmologists usually diagnose this condition, some patients initially present with isolated growth impairment. This may lead to a delay in ophthalmologic evaluation and management. Thus, in patients with GHD of unknown etiology, it is important to have a high index of suspicion of ARS in order to decrease morbidity from vision loss.

Novel PITX2 mutations identified in Axenfeld-Rieger syndrome and the pattern of PITX2-related tooth agenesis.

To investigate the mutations in patients with Axenfeld-Rieger syndrome (ARS) and the pattern of PITX2-related tooth agenesis. Whole-exome sequencing (WES) and copy number variation (CNV) array were used to screen the mutations in four ARS probands. After Sanger sequencing and quantitative polymerase chain reaction (qPCR) validation, secondary structure prediction and dual-luciferase assay were employed to investigate the functional impact. Eighteen PITX2-mutated patients with definite dental records were retrieved from our database and literatures, and the pattern of PITX2-related tooth agenesis was analyzed. A novel de novo segmental deletion of chromosome 4q25 (GRCh37/hg19 chr4:111, 320, 052-111, 754, 236) encompassing PITX2 and three novel PITX2 mutations c.148C>T, c.257G>A, and c.630insCG were identified. Preliminary functional studies indicated the transactivation capacity of mutant PITX2 on Distal-less homeobox 2 (DLX2) promoter was compromised. The maxillary teeth showed significantly higher rate of agenesis (57.94%) than the mandibular teeth (44.05%). The most often missing teeth were upper lateral incisors (83.33%) and upper second premolars (69.44%). Teeth with the least agenesis rate were the lower second molars (19.44%) and lower first molars (8.33%). We identified a novel 4q25 microdeletion including PITX2 and three novel PITX2 mutations, and statistically analyzed the PITX2-related tooth agenesis pattern.

Novel PITX2 Mutations including a Mutation Causing an Unusual Ophthalmic Phenotype of Axenfeld-Rieger Syndrome.

Purpose The aims of this study were to examine novel mutations in PITX2 and FOXC1 in Chinese patients with anterior segment dysgenesis (ASD) and to compare the clinical presentations of these mutations with previously reported associated phenotypes. Methods Twenty-six unrelated patients with different forms of ASD were enrolled from our paediatric and genetic eye clinic. The ocular manifestations of both eyes of each patient were recorded. Genomic DNA was prepared from venous leukocytes. All coding exons of PITX2 and FOXC1 were amplified by polymerase chain reaction (PCR) from genomic DNA and subjected to direct DNA sequencing. Analysis of mutations in control subjects was performed by heteroduplex single-strand conformation polymorphism (SSCP) analysis. Results Sequence analysis of the PITX2 gene revealed four mutations, including c.475_476delCT (P.L159VfsX39), c.64C > T (P.Q22X), c.296delG (P.R99PfsX56), and c.206G > A (P.R69H). The first three mutations were found to be novel. The c.475_476delCT (P.L159VfsX39) mutation, located at the 3′ end of the PITX2-coding region, was identified in a Chinese Axenfeld-Rieger syndrome (ARS) patient who presented with an unusual severe phenotype of bilateral aniridia. The clinical characteristics, including the severity and manifestations of the patient's phenotype, were compared with reported PITX2-associated aniridia phenotypes of ARS in the literature. Conclusions These results expand the mutation spectrum of the PITX2 gene in patients with ARS. The PITX2 gene may be responsible for a significant portion of ARS with additional systemic defects in the Chinese population. This is the first reported case of a mutation at the 3′ end of the PITX2-coding region extending the phenotypic consequences to bilateral aniridia. The traits of ARS could display tremendous variability in severity and manifestations due to the dominant-negative effect of PITX2. Our results further emphasize the importance of careful clinical and genetic analysis in determining mutation-disease associations and may lead to a better understanding of the role of PITX2 in ocular development.

Mutation Survey of Candidate Genes and Genotype–Phenotype Analysis in 20 Southeastern Chinese Patients with Axenfeld–Rieger Syndrome

ABSTRACTPurpose/aim of the study: To conduct a survey spectrum of the PITX2, FOXC1, and PRDM5 genes to reveal genotype–phenotype correlations in a cohort of Southeastern Chinese patients with Axenfeld–Rieger syndrome (ARS).Materials and methods: A total of 20 probands with ARS were recruited in Southeast China. All patients underwent full ocular and systemic examinations. Sanger sequencing was used to analyze all coding regions of and regions adjacent to PITX2, FOXC1, and PRDM5 and 13 upstream regulatory elements of PITX2. Multiplex ligation-dependent probe amplification was performed to detect gross insertions and deletions in PITX2 and FOXC1. Quantitative polymerase chain reaction was used to detect copy number variations in regulatory elements of PITX2. A bioinformatics analysis was conducted to evaluate the pathogenicity of variants.Results: Eleven mutations, including eight novel mutations, were identified in PITX2. Seven of the mutations were truncations. A genotype–phenotype correlation analysis showed that 81.8% (9/11) of patients with mutations in PITX2 developed glaucoma before reaching 10 years old. The proportion of patients without detected mutations was only 33.3% (3/9, P = 0.0399). In patient G1399, ultrasound biomicroscopy revealed that the left eye exhibited a phenotype similar to aniridia with complete angle closure and a remaining stub of iris tissue.Conclusion: This is the first genetic study of a cohort of Southeastern Chinese patients with ARS. Eight novel mutations were detected, expanding the mutation spectrum of PITX2. PITX2 may be a major candidate gene for ARS in Southeastern Chinese patients. Truncations may be the primary mutation type in PITX2. Glaucoma onset may be earlier in patients with mutations in PITX2 than in those without mutations in PITX2 and FOXC1. A block of the anterior chamber angle by the end of the iris might represent the main factor influencing the development of glaucoma in ARS patients with an asymmetric aniridia phenotype.

Accurate prediction of functional, structural, and stability changes in PITX2 mutations using in silico bioinformatics algorithms

Mutations in PITX2 have been implicated in several genetic disorders, particularly Axenfeld-Rieger syndrome. In order to determine the most reliable bioinformatics tools to assess the likely pathogenicity of PITX2 variants, the results of bioinformatics predictions were compared to the impact of variants on PITX2 structure and function. The MutPred, Provean, and PMUT bioinformatic tools were found to have the highest performance in predicting the pathogenicity effects of all 18 characterized missense variants in PITX2, all with sensitivity and specificity >93%. Applying these three programs to assess the likely pathogenicity of 13 previously uncharacterized PITX2 missense variants predicted 12/13 variants as deleterious, except A30V which was predicted as benign variant for all programs. Molecular modeling of the PITX2 homoedomain predicts that of the 31 known PITX2 variants, L54Q, F58L, V83F, V83L, W86C, W86S, and R91P alter PITX2’s structure. In contrast, the remaining 24 variants are not predicted to change PITX2’s structure. The results of molecular modeling, performed on all the PITX2 missense mutations located in the homeodomain, were compared with the findings of eight protein stability programs. CUPSAT was found to be the most reliable in predicting the effect of missense mutations on PITX2 stability. Our results showed that for PITX2, and likely other members of this homeodomain transcription factor family, MutPred, Provean, PMUT, molecular modeling, and CUPSAT can reliably be used to predict PITX2 missense variants pathogenicity.

A novel PITX2 mutation in non-syndromic orodental anomalies.

To identify orodental characteristics and genetic aetiology of a family affected with non-syndromic orodental anomalies. Physical and oral features were characterised. DNA was collected from an affected Thai family. Whole-exome sequencing was employed to identify the pathogenic variants associated with inherited orodental anomalies. The presence of the identified mutation was confirmed by Sanger sequencing. We observed unique orodental manifestations including oligodontia, retained primary teeth, taurodont molars, peg-shaped maxillary central incisors, high attached frenum with nodule and midline diastema in the proband and her mother. Mutation analyses revealed a novel heterozygous frameshift deletion, c.573_574delCA, p.L193QfsX5, in exon 5 of PITX2A in affected family members. The amino acid alterations, localised in the transcriptional activation domain 2 in the C-terminus of PITX2, were evolutionarily conserved. Mutations in PITX2 have been associated with autosomal-dominant Axenfeld-Rieger syndrome and non-syndromic eye abnormalities, but never been found to cause isolated oral anomalies. This study for the first time demonstrates that the PITX2 mutation could lead to non-syndromic orodental anomalies in humans. We propose that the specific location in the C-terminal domain of PITX2 is exclusively necessary for tooth development.

PITX2 deficiency and associated human disease: insights from the zebrafish model.

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.

Axenfeld-Rieger syndrome.

Axenfeld-Rieger syndrome (ARS) is a clinically and genetically heterogeneous group of developmental disorders affecting primarily the anterior segment of the eye, often leading to secondary glaucoma. Patients with ARS may also present with systemic changes, including dental defects, mild craniofacial dysmorphism, and umbilical anomalies. ARS is inherited in an autosomal-dominant fashion; the underlying defect in 40% of patients is mutations in PITX2 or FOXC1. Here, an overview of the clinical spectrum of ARS is provided. As well, the known underlying genetic defects, clinical diagnostic possibilities, genetic counseling and treatments of ARS are discussed in detail.

4q25 microdeletion encompassing PITX2: A patient presenting with tetralogy of Fallot and dental anomalies without ocular features

Axenfeld-Rieger syndrome (ARS) is a heterogeneous clinical entity transmitted in an autosomal dominant manner. The main feature, Axenfeld-Rieger Anomaly (ARA), is a malformation of the anterior segment of the eye that can lead to glaucoma and impair vision. Extra-ocular defects have also been reported. Point mutations of FOXC1 and PITX2 are responsible for about 40% of the ARS cases. We describe the phenotype of a patient carrying a deletion encompassing the 4q25 locus containing PITX2 gene. This child presented with a congenital heart defect (Tetralogy of Fallot, TOF) and no signs of ARA. He is the first patient described with TOF and a complete deletion of PITX2 (arr[GRCh37]4q25(110843057-112077858)x1, involving PITX2, EGF, ELOVL6 and ENPEP) inherited from his ARS affected mother. In addition, to our knowledge, he is the first patient reported with no ocular phenotype associated with haploinsufficiency of PITX2. We compare the phenotype and genotype of this patient to those of five other patients carrying 4q25 deletions. Two of these patients were enrolled in the university hospital in Toulouse, while the other three were already documented in DECIPHER. This comparative study suggests both an incomplete penetrance of the ocular malformation pattern in patients carrying PITX2 deletions and a putative association between TOF and PITX2 haploinsufficiency.

Mutations of conserved non-coding elements of PITX2 in patients with ocular dysgenesis and developmental glaucoma.

Mutations in FOXC1 and PITX2 constitute the most common causes of ocular anterior segment dysgenesis (ASD), and confer a high risk for secondary glaucoma. The genetic causes underlying ASD in approximately half of patients remain unknown, despite many of them being screened by whole exome sequencing. Here, we performed whole genome sequencing on DNA from two affected individuals from a family with dominantly inherited ASD and glaucoma to identify a 748-kb deletion in a gene desert that contains conserved putative PITX2 regulatory elements. We used CRISPR/Cas9 to delete the orthologous region in zebrafish in order to test the pathogenicity of this structural variant. Deletion in zebrafish reduced pitx2 expression during development and resulted in shallow anterior chambers. We screened additional patients for copy number variation of the putative regulatory elements and found an overlapping deletion in a second family and in a potentially-ancestrally-related index patient with ASD and glaucoma. These data suggest that mutations affecting conserved non-coding elements of PITX2 may constitute an important class of mutations in patients with ASD for whom the molecular cause of their disease have not yet been identified. Improved functional annotation of the human genome and transition to sequencing of patient genomes instead of exomes will be required before the magnitude of this class of mutations is fully understood.

Primary congenital and developmental glaucomas.

Glaucoma is the leading cause of irreversible blindness worldwide. Although most glaucoma patients are elderly, congenital glaucoma and glaucomas of childhood are also important causes of visual disability. Primary congenital glaucoma (PCG) is isolated, non-syndromic glaucoma that occurs in the first three years of life and is a major cause of childhood blindness. Other early-onset glaucomas may arise secondary to developmental abnormalities, such as glaucomas that occur with aniridia or as part of Axenfeld-Rieger syndrome. Congenital and childhood glaucomas have strong genetic bases and disease-causing mutations have been discovered in several genes. Mutations in three genes (CYP1B1, LTBP2, TEK) have been reported in PCG patients. Axenfeld-Rieger syndrome is caused by mutations in PITX2 or FOXC1 and aniridia is caused by PAX6 mutations. This review discusses the roles of these genes in primary congenital glaucoma and glaucomas of childhood.