UNASSIGNED: To describe the clinical and genetic findings of an Axenfeld-Rieger syndrome (ARS) family with a new PITX2 splicing mutation.
UNASSIGNED: A Chinese ARS family with five affected individuals was recruited. Exome sequencing was performed on the proband and the variant (C.253-9C > A) in PITX2 gene was detected as a pathogenic mutation. Sanger sequencing was performed for verification and cosegregation analysis. Real-time polymerase chain reaction (RT- PCR) and Western blotting were performed to verify the expression of the pathogenic gene.
UNASSIGNED: All the patients showed abnormalities in the anterior segment of both eyes including posterior embryotoxon, corectopia, iris dysplasia, and iridocorneal tissue adhesions. In addition, they all presented systemic features, including maxillary hypoplasia, underbite, hypodontia, conical teeth. Only III-7 showed obvious umbilical skin. In the PITX2 family, we identified a novel heterozygous splicing mutation (C.253-9C > A) which was confirmed by Sanger sequencing to be completely cosegregated with the ARS phenotype. Real-time quantitative PCR and Western results showed that PITX2 mRNA and protein expression were significantly lower in patients compared with unrelated normal controls.
UNASSIGNED: In the ARS pedigree, we summarized the variable phenotype, described a novel PITX2 splicing mutation which expand the genetic spectrum of ARS. We further confirmed the possibility of development of ARS induced by this PITX2 gene deficiency.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is a rare kind of anterior segment dysgenesis (ASD). The most common ocular features of ARS are posterior embryotoxon and iris hypoplasia, while some patients may manifest as corneal opacity and edema. However, the current understanding of how ARS affects the cornea is still incomplete. This study reports a novel histopathological finding of ARS, complicating corneal abnormalities, including congenital corneal opacity and irreversible endothelial decompensation.
METHODS: This retrospective study included 6 eyes of 3 ARS patients, 5 of which underwent keratoplasty for irreversible endothelial decompensation from May 2016 to January 2019. No eye had a history of surgery. We reviewed the data of epidemiology, clinical manifestations and histopathologic examinations.
RESULTS: Five eyes developed irreversible endothelial decompensation, among which 4 were born with corneal opacity. One eye exhibited transparent cornea but showed a continuous loss of endothelial cells in the absence of surgery and elevated intraocular pressure thereafter. Anterior segment optical coherence tomography photographs showed that anterior synechia existed in the area with corneal opacities, where we found the interlayer splitting of the Descemet membrane inserted by hypoplastic iris and a basement membrane-like structure under a light microscope.
CONCLUSIONS: Anterior synechia might be associated with corneal abnormalities in ARS patients. The novel histopathologic finding revealed the internal relation between anterior segment dysgenesis and would help explore the inner mechanism of corneal abnormalities in ARS.

PITX2 and FOXC1 are the most common pathogenic genes associated with Axenfeld-Rieger syndrome (ARS). In this study, we aimed to explore the variation spectrum of PITX2 and FOXC1 and their associated phenotype based on data from our study and previously reported literatures. Whole exome sequencing was performed on eight probands in our study. Multistep bioinformatic and co-segregation analyses were performed to detect pathogenic variants. Genotype-phenotype correlations of PITX2 and FOXC1 and the differences between them were determined. We detected three variants of FOXC1 and two variants of PITX2 in five unrelated families with ARS. Macular retinoschisis had been observed in AR1 with variant in PITX2 and it is not reported before. Additionally, a review of published literature and our study led to the identification of 593 families with variants of PITX2 or FOXC1, including 316 families with heterozygous variants in FOXC1, 251 families with heterozygous variants in PITX2, 13 families with variants in double genes, seven families with homozygous or compound heterozygous variants in FOXC1, and six families with variants in ADAMTS17, PRDM5, COL4A1 or CYP1B1. Significant differences were observed between the prevalence of missense and in-frame, truncation, and large deletion variants in PITX2 (32.00%, 42.67%, and 25.33%, respectively) and FOXC1 (34.49%, 35.13%, 30.38%, respectively) (p = 1.16E-43). Enrichment and frequency analyses revealed that missense variants were concentrated in the forkhead domain of FOXC1 (76.14%) and homeodomain of PITX2 (87.50%). The percentage of Caucasians with variants in FOXC1 was significantly higher than that of PITX2 (p = 2.00E-2). Significant differences between PITX2 and FOXC1 were observed in glaucoma (p = 3.00E-2), corectopia (p = 3.050E-6), and polycoria (p = 5.21E-08). Additionally, we observed a significant difference in best-corrected visual acuity (BCVA) between FOXC1 and PITX2 (p = 3.80E-2). Among all the family members with PITX2 or FOXC1 variants, the prevalence of systemic abnormalities was significantly higher in PITX2 than in FOXC1 (89.16% vs. 58.77%, p = 5.44E-17). In conclusion, macular retinoschisis as a novel phenotype had been observed in patient with variant in PITX2. Significant differences were detected in phenotypes and genotypes between PITX2 and FOXC1.

OBJECTIVE: To investigate the detailed ultrastructural patterns of dental abnormalities affected by Axenfeld-Rieger syndrome (ARS) with a heterozygous microdeletion involving paired-like homeodomain 2 (PITX2) and explored the underlying molecular mechanisms driving enamel defects.
METHODS: Sanger sequencing, genomic quantitative PCR analysis, and chromosomal microarray analysis (CMA) were used to screen the disease-causing mutation in one ARS proband. An exfoliated tooth from an ARS patient was analyzed with scanning electron microscopy and micro-computerized tomography. A stable Pitx2 knockdown cell line was generated to simulate PITX2 haploinsufficiency. Cell proliferation and ameloblast differentiation were analyzed, and the role of the Wnt/β-catenin pathway in proliferation of ameloblast precursor cells was investigated.
RESULTS: An approximately 0.216 Mb novel deletion encompassing PITX2 was identified. The affected tooth displayed a thinner and broken layer of enamel and abnormal enamel biomineralization. PITX2 downregulation inhibited the proliferation and differentiation of inner enamel epithelial cells, and LiCl stifmulation partially reversed the proliferation ability after Pitx2 knockdown.
CONCLUSIONS: Enamel formation is disturbed in some patients with ARS. Pitx2 knockdown can influence the proliferation and ameloblast differentiation of inner enamel epithelial cells, and PITX2 may regulate cell proliferation via Wnt/β-catenin signaling pathway.

Mutations in the Forkhead Box C1 (FOXC1) are known to cause autosomal dominant hereditary Axenfeld-Rieger syndrome, which is a genetic disorder characterized by ocular and systemic features including glaucoma, variable dental defects, craniofacial dysmorphism and hearing loss. Due to late-onset of ocular disorders and lack of typical presentation, clinical diagnosis presents a huge challenge. In this study, we described a pathogenic in-frame variant in FOXC1 in one 5-year-old boy who is presented with hypertelorism, pupil deformation in both eyes, conductive hearing loss, and dental defects. By whole exome sequencing, we identified a 3 bp deletion in FOXC1, c.516_518delGCG (p.Arg173del) as the disease-causing variant, which was de novo and not detected in the parents, and could be classified as a \"pathogenic variant\" according to the American College of Medical Genetics and Genomics guidelines. After confirmation of this FOXC1 variant, clinical data on Axenfeld-Rieger syndrome-associated clinical features were collected and analyzed. Furthermore, Although the affected individual present hearing loss, however, the hearing loss is conductive and is reversible during the follow-up, which might not linke to the FOXC1 variant and is coincidental. Routine examination of FOXC1 is necessary for the genetic diagnosis of hypertelorism-associated syndrome. These findings may assist clinicians in reaching correct clinical and molecular diagnoses, and providing appropriate genetic counseling.

This study aimed to identify the disease-causing gene of three Chinese families with glaucoma. Whole exome sequencing was performed on the probands and detected three different variants (c.405C>A (p.Cys135Ter), c.851G>T (p.Ser284Ile), and c.392C>T (p.Ser131Leu)) in FOXC1 as a causative gene of glaucoma, and Sanger sequencing was performed for verification and cosegregation analysis. Three in silico tools all predicted these two missense variants to be probably disease-causing. Western blot analysis, immunofluorescence, and dual-luciferase assay were further used to evaluate the effect of FOXC1 missense variants, and demonstrated that the two variants resulted in decreased transactivation activity of FOXC1 although the variants had no effect on the protein amount and the nucleus subcellar localization of FOXC1 compared with the wild type, which implies that both of two variants may be probably pathogenic. In this study, we reported two novel FOXC1 variants as well as a reported variant and the phenotypes associated to these variants, which expands the spectrum and relevant phenotypes of FOXC1 variants. Additionally, the functional analysis of FOXC1 variants provides further insight into the possible pathogenesis of anterior segment anomaly related to FOXC1.

OBJECTIVE: Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder characterized by ocular anterior segment abnormalities. In the current study, we describe clinical and genetic findings in a Chinese ARS pedigree.
METHODS: An ARS pedigree was recruited and patients were given comprehensive ophthalmic examinations and general physical examinations. DNA from the proband II:2 was used for exome sequencing. Sanger sequencing was utilized to identify and validate PITX2 variations. qPCR and western blotting were performed to detect PITX2 expression in immortalized peripheral blood lymphocytes.
RESULTS: All affected family members showed typical ocular abnormalities, including iris atrophy, corectopia, shallow anterior chamber, complete or partial angle closure, and advanced glaucoma. They also exhibited systemic anomalies, such as microdontia, hypodontia, and redundant periumbilical skin. A heterozygous splice-site variation c.390 + 1G > A in PITX2, which might lead to a truncated PITX2 protein (p.Val131IlefsX127), was found in the proband. Sanger sequencing validated that the variation completely co-segregated with the ARS phenotype within this family and was absent in 100 unrelated controls. Western blotting revealed that the nuclear PITX2 protein was significantly decreased in patients compared with controls. Nonetheless, there was no significant difference in the total PITX2 protein level, consistent with qPCR results showing no alteration in PITX2 mRNA levels in the patient group.
CONCLUSIONS: PITX2 c.390 + 1G > A (p.Val131IlefsX127) was a novel genetic etiology of the ARS pedigree. The mutation leads to decreased nuclear PITX2, indicating lower transcriptional activity.

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

Axenfeld-Rieger syndrome is characterized by a spectrum of anterior segment dysgenesis involving neural-crest-derived tissues, most commonly secondary to mutations in the transcription factor genes PITX2 and FOXC1.
Single retrospective case report.
A full-term infant presented at 5 weeks of age with bilateral Peters anomaly and Axenfeld-Rieger syndrome, with development of atypical features of progressive corneal neovascularization and proliferative vitreoretinopathy. Despite surgical interventions, the patient progressed to bilateral phthisis bulbi by 22 months of age. Genetic testing revealed a novel de novo p.Leu212Valfs*39 mutation in PITX2, leading to loss of a C-terminal OAR domain that functions in transcriptional regulation.
It is important to consider mutations in PITX2 in atypical cases of anterior segment dysgenesis that also present with abnormalities in the angiogenesis of the anterior and posterior segments.

Axenfeld-Rieger syndrome (ARS) is a disorder affecting the anterior segment of the eye and causing systemic malformations, and follows an autosomal-dominant inheritance pattern. The aim of the present study was to identify the underlying cause of ARS in a Chinese family. Genomic DNA was extracted from the peripheral blood of the subjects from a family with ARS. The pathogenic variant was identified by targeted next-generation sequencing and confirmed by Sanger sequencing. A novel heterozygous mutation of the forkhead box (FOX)C1 gene (c.1494delG, p.G499Afs*20) was detected in all affected members of the family, while no mutation was identified in the unaffected members or in the 150 normal controls. The affected members exhibited typical ocular and craniofacial anomalies. The results of the present study demonstrated that a novel deletion in exon 1 of the FOXC1 gene caused ARS in this Chinese family.