Wolf-Hirschhorn syndrome candidate 1 (Whsc1) methyltransferase signals via a Pitx2-miR-23/24 axis to effect tooth development.

Wolf-Hirschhorn syndrome (WHS) is a developmental disorder attributed to a partial deletion on the short arm of chromosome 4. WHS patients suffer from oral manifestations including cleft lip and palate, hypodontia, and taurodontism. WHS candidate 1 (WHSC1) gene is a H3K36-specific methyltransferase that is deleted in every reported case of WHS. Mutation in this gene also results in tooth anomalies in patients. However, the correlation between genetic abnormalities and the tooth anomalies has remained controversial. In our study, we aimed to clarify the role of WHSC1 in tooth development. We profiled the Whsc1 expression pattern during mouse incisor and molar development by immunofluorescence staining and found Whsc1 expression is reduced as tooth development proceeds. Using real-time quantitative reverse transcription PCR, Western blot, chromatin immunoprecipitation, and luciferase assays, we determined that Whsc1 and Pitx2, the initial transcription factor involved in tooth development, positively and reciprocally regulate each other through their gene promoters. miRNAs are known to regulate gene expression posttranscriptionally during development. We previously reported miR-23a/b and miR-24-1/2 were highly expressed in the mature tooth germ. Interestingly, we demonstrate here that these two miRs directly target Whsc1 and repress its expression. Additionally, this miR cluster is also negatively regulated by Pitx2. We show the expression of these two miRs and Whsc1 are inversely correlated during mouse mandibular development. Taken together, our results provide new insights into the potential role of Whsc1 in regulating tooth development and a possible molecular mechanism underlying the dental defects in WHS.

Complex balanced intrachromosomal rearrangement involving PITX2 identified as a cause of Axenfeld-Rieger Syndrome.

Axenfeld-Rieger Syndrome (ARS) type 1 is a rare autosomal dominant condition characterized by anterior chamber anomalies, umbilical defects, dental hypoplasia, and craniofacial anomalies, with Meckel's diverticulum in some individuals. Here, we describe a clinically ascertained female of childbearing age with ARS for whom clinical targeted sequencing and deletion/duplication analysis followed by clinical exome and genome sequencing resulted in no pathogenic variants or variants of unknown significance in PITX2 or FOXC1. Advanced bioinformatic analysis of the genome data identified a complex, balanced rearrangement disrupting PITX2. This case is the first reported intrachromosomal rearrangement leading to ARS, illustrating that for patients with compelling clinical phenotypes but negative genomic testing, additional bioinformatic analysis are essential to identify subtle genomic abnormalities in target genes.

HMGN2 represses gene transcription via interaction with transcription factors Lef-1 and Pitx2 during amelogenesis.

The chromatin-associated high mobility group protein N2 (HMGN2) cofactor regulates transcription factor activity through both chromatin and protein interactions. Hmgn2 expression is known to be developmentally regulated, but the post-transcriptional mechanisms that regulate Hmgn2 expression and its precise roles in tooth development remain unclear. Here, we demonstrate that HMGN2 inhibits the activity of multiple transcription factors as a general mechanism to regulate early development. Bimolecular fluorescence complementation, pull-down, and coimmunoprecipitation assays show that HMGN2 interacts with the transcription factor Lef-1 through its HMG-box domain as well as with other early development transcription factors, Dlx2, FoxJ1, and Pitx2. Furthermore, EMSAs demonstrate that HMGN2 binding to Lef-1 inhibits its DNA-binding activity. We found that Pitx2 and Hmgn2 associate with H4K5ac and H3K4me2 chromatin marks in the proximal Dlx2 promoter, demonstrating Hmgn2 association with open chromatin. In addition, we demonstrate that microRNAs (miRs) mir-23a and miR-23b directly target Hmgn2, promoting transcriptional activation at several gene promoters, including the amelogenin promoter. In vivo, we found that decreased Hmgn2 expression correlates with increased miR-23 expression in craniofacial tissues as the murine embryo develops. Finally, we show that ablation of Hmgn2 in mice results in increased amelogenin expression because of increased Pitx2, Dlx2, Lef-1, and FoxJ1 transcriptional activity. Taken together, our results demonstrate both post-transcriptional regulation of Hmgn2 by miR-23a/b and post-translational regulation of gene expression by Hmgn2-transcription factor interactions. We conclude that HMGN2 regulates tooth development through its interaction with multiple transcription factors.

Pitx2-Sox2-Lef1 interactions specify progenitor oral/dental epithelial cell signaling centers.

Epithelial signaling centers control epithelial invagination and organ development, but how these centers are specified remains unclear. We report that Pitx2 (the first transcriptional marker for tooth development) controls the embryonic formation and patterning of epithelial signaling centers during incisor development. We demonstrate using Krt14Cre /Pitx2flox/flox (Pitx2cKO ) and Rosa26CreERT/Pitx2flox/flox mice that loss of Pitx2 delays epithelial invagination, and decreases progenitor cell proliferation and dental epithelium cell differentiation. Developmentally, Pitx2 regulates formation of the Sox2+ labial cervical loop (LaCL) stem cell niche in concert with two signaling centers: the initiation knot and enamel knot. The loss of Pitx2 disrupted the patterning of these two signaling centers, resulting in tooth arrest at E14.5. Mechanistically, Pitx2 transcriptional activity and DNA binding is inhibited by Sox2, and this interaction controls gene expression in specific Sox2 and Pitx2 co-expression progenitor cell domains. We demonstrate new transcriptional mechanisms regulating signaling centers by Pitx2, Sox2, Lef1 and Irx1.

Enamel defects of Axenfeld-Rieger syndrome and the role of PITX2 in its pathogenesis.

OBJECTIVES: 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. SUBJECTS AND 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/ß-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/ß-catenin signaling pathway.

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.

Are birds pseudoteeth and denticulations related to touch papilla? An investigation in parrot, goose, and chicken.

We first studied the morphology and the development of goose denticulations, which develop mainly by a ripple process, and the touch papillae of the bill tip organ, which appears through an evagination process at the end of the beak. During their development, we observed the specific expression of PAX9, PITX2, and BMP4, while SHH was expressed mainly in the basal layer of the epithelium in a non-specific manner. Adult goose denticulations are associated with numerous columns. The goose denticulations and columns were filled with numerous Herbst and Grandry corpuscles, as well the touch papillae of the bill tip organ. Histological analysis of adult parrot pseudoteeth revealed that the osseous pseudoteeth were extended by similar columns filled with Herbst and Grandry corpuscles. We also examined adult and embryonic chicken beaks. During ontogeny, we observed a process of rostral evagination with folding associated with discrete ripples in the anterior part of the beak rudiment, in which PAX9, PITX2, and BMP4 are expressed. In the corresponding adult areas, there were numerous sensory corpuscles with rostral columns, which were similar to the features observed in goose. These observations support the hypothesis that pseudoteeth and denticulations constitute sensory organs, and that the touch papillae exhibit some similarities with pseudoteeth.

Mechanistic Insights into Axenfeld-Rieger Syndrome from Zebrafish foxc1 and pitx2 Mutants.

Axenfeld-Rieger syndrome (ARS) encompasses a group of developmental disorders that affect the anterior segment of the eye, as well as systemic developmental defects in some patients. Malformation of the ocular anterior segment often leads to secondary glaucoma, while some patients also present with cardiovascular malformations, craniofacial and dental abnormalities and additional periumbilical skin. Genes that encode two transcription factors, FOXC1 and PITX2, account for almost half of known cases, while the genetic lesions in the remaining cases remain unresolved. Given the genetic similarity between zebrafish and humans, as well as robust antisense inhibition and gene editing technologies available for use in these animals, loss of function zebrafish models for ARS have been created and shed light on the mechanism(s) whereby mutations in these two transcription factors cause such a wide array of developmental phenotypes. This review summarizes the published phenotypes in zebrafish foxc1 and pitx2 loss of function models and discusses possible mechanisms that may be used to target pharmaceutical development and therapeutic interventions.

Genetic linkage between altered tooth and eye development in lens-ablated Astyanax mexicanus.

The phenotype of lens-ablated Mexican tetra (Astyanax mexicanus) compared to wild-type surface fish has been described and includes, among other effects, eye degeneration, changes in tooth number and cranial bone changes. Here, we investigate the spatiotemporal expression patterns of several key genes involved in the development of these structures. Specifically, we show that the expression of pitx2, bmp4 and shh is altered in the eye, oral jaw, nasal pit and forebrain in these lens-ablated fish. Furthermore, for the first time, we show altered pitx2 expression in the cavefish, which also has altered eye and tooth phenotypes. We thus provide evidence for a genetic linkage between the eye and tooth modules in this fish species. Furthermore, the altered pitx2 expression pattern, together with the described morphological features of the lens-ablated fish suggests that Astyanax mexicanus could be considered as an alternative teleost model organism in which to study Axenfeld-Rieger syndrome (ARS), a rare autosomal dominant developmental disorder that is associated with PITX2 and which has both ocular and non-ocular abnormalities.

Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development.

A novel frameshift mutation in the PITX2 gene in a family with Axenfeld-Rieger syndrome using targeted exome sequencing.

BACKGROUND: Axenfeld-Rieger syndrome (ARS) is an autosomal dominant genetic disorder that is characterized by specific abnormalities of the anterior segment of the eye. Heterozygous mutations in two developmental transcription factor genes PITX2 and FOXC1 have been identified within ARS patients, accounting for 40 to 70% of cases. Our purpose is to describe clinical and genetic findings in a Chinese family with ARS. METHODS: An ARS family with three affected members was recruited. The patients underwent a series of complete ophthalmologic examinations, general physical examination and dental radiography. DNA samples of proband II-1 were used for targeted exome sequencing of the FOXC1 and PITX2 genes. Sanger sequencing was used to validate the variation in PITX2. Quantitative real-time PCR was carried out to detect the expression of PITX2 in patients and normal controls. RESULTS: All affected members showed iris atrophy, corectopia, shallow anterior chamber, complete or partial angle closure, and advanced glaucoma. In addition, they revealed systemic anomalies, including microdontia, hypodontia, and redundant periumbilical skin. A novel heterozygous frameshift variation, c.515delA, in PITX2 was found in the proband, which might lead to a truncated PITX2 protein (p.Gln172ArgfsX36). Sanger sequencing validated that the variation completely cosegregated with the ARS phenotype among this family, but was absent in 100 unrelated controls. Quantitative real-time PCR analysis revealed that the mRNA expression of PITX2 was significantly decreased in patients compared with that in unrelated normal controls. CONCLUSIONS: PITX2 c.515delA (p.Gln172ArgfsX36) was the genetic etiology of our pedigree. The mutation led to decreased PITX2 gene expression and a truncated mRNA transcript.

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

OBJECTIVES: To investigate the mutations in patients with Axenfeld-Rieger syndrome (ARS) and the pattern of PITX2-related tooth agenesis. METHODS: 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. RESULTS: 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%). CONCLUSIONS: We identified a novel 4q25 microdeletion including PITX2 and three novel PITX2 mutations, and statistically analyzed the PITX2-related tooth agenesis pattern.

Mutations in zebrafish pitx2 model congenital malformations in Axenfeld-Rieger syndrome but do not disrupt left-right placement of visceral organs.

Pitx2 is a conserved homeodomain transcription factor that has multiple functions during embryonic development. Mutations in human PITX2 cause autosomal dominant Axenfeld-Rieger syndrome (ARS), characterized by congenital eye and tooth malformations. Pitx2(-/-) knockout mouse models recapitulate aspects of ARS, but are embryonic lethal. To date, ARS treatments remain limited to managing individual symptoms due to an incomplete understanding of PITX2 function. In addition to regulating eye and tooth development, Pitx2 is a target of a conserved Nodal (TGFß) signaling pathway that mediates left-right (LR) asymmetry of visceral organs. Based on its highly conserved asymmetric expression domain, the Nodal-Pitx2 axis has long been considered a common denominator of LR development in vertebrate embryos. However, functions of Pitx2 during asymmetric organ morphogenesis are not well understood. To gain new insight into Pitx2 function we used genome editing to create mutations in the zebrafish pitx2 gene. Mutations in the pitx2 homeodomain caused phenotypes reminiscent of ARS, including aberrant development of the cornea and anterior chamber of the eye and reduced or absent teeth. Intriguingly, LR asymmetric looping of the heart and gut was normal in pitx2 mutants. These results suggest conserved roles for Pitx2 in eye and tooth development and indicate Pitx2 is not required for asymmetric looping of zebrafish visceral organs. This work establishes zebrafish pitx2 mutants as a new animal model for investigating mechanisms underlying congenital malformations in ARS and high-throughput drug screening for ARS therapeutics. Additionally, pitx2 mutants present a unique opportunity to identify new genes involved in vertebrate LR patterning. We show Nodal signaling-independent of Pitx2-controls asymmetric expression of the fatty acid elongase elovl6 in zebrafish, pointing to a potential novel pathway during LR organogenesis.

The Pitx2:miR-200c/141:noggin pathway regulates Bmp signaling and ameloblast differentiation.

The mouse incisor is a remarkable tooth that grows throughout the animal's lifetime. This continuous renewal is fueled by adult epithelial stem cells that give rise to ameloblasts, which generate enamel, and little is known about the function of microRNAs in this process. Here, we describe the role of a novel Pitx2:miR-200c/141:noggin regulatory pathway in dental epithelial cell differentiation. miR-200c repressed noggin, an antagonist of Bmp signaling. Pitx2 expression caused an upregulation of miR-200c and chromatin immunoprecipitation assays revealed endogenous Pitx2 binding to the miR-200c/141 promoter. A positive-feedback loop was discovered between miR-200c and Bmp signaling. miR-200c/141 induced expression of E-cadherin and the dental epithelial cell differentiation marker amelogenin. In addition, miR-203 expression was activated by endogenous Pitx2 and targeted the Bmp antagonist Bmper to further regulate Bmp signaling. miR-200c/141 knockout mice showed defects in enamel formation, with decreased E-cadherin and amelogenin expression and increased noggin expression. Our in vivo and in vitro studies reveal a multistep transcriptional program involving the Pitx2:miR-200c/141:noggin regulatory pathway that is important in epithelial cell differentiation and tooth development.

A potential molecular pathogenesis of cardiac/laterality defects in Oculo-Facio-Cardio-Dental syndrome.

Pitx2 is the last effector of the left-right (LR) cascade known to date and plays a crucial role in the patterning of LR asymmetry. In Xenopus embryos, the expression of Pitx2 gene in the left lateral plate mesoderm (LPM) is directly regulated by Xnr1 signaling, which is mediated by Smads and FoxH1. Previous studies suggest that the suppression of Pitx2 gene in the left LPM is a potential cause of cardiac/laterality defects in Oculo-Facio-Cardio-Dental (OFCD) syndrome, which is known to be caused by mutations in BCL6 co-repressor (BCOR) gene. Recently, our work has revealed that the BCL6/BCOR complex blocks Notch-dependent transcriptional activity to protect the expression of Pitx2 in the left LPM from the inhibitory activity of Notch signaling. These studies indicated that uncontrolled Notch activity in the left LPM caused by dysfunction of BCOR may result in cardiac/laterality defects of OFCD syndrome. However, this Notch-dependent inhibitory mechanism of Pitx2 gene transcription still remains unknown. Here we report that transcriptional repressor ESR1, which acts downstream of Notch signaling, inhibits the expression of Pitx2 gene by binding to a left side-specific enhancer (ASE) region in Pitx2 gene and recruiting histone deacetylase 1 (HDAC1) to this region. Once HDAC1 is tethered, histone acetyltransferase p300 is no longer recruited to the Xnr1-dependent transcriptional complex on the ASE region, leading to the suppression of Pitx2 gene in the left LPM. The study presented here uncovers the regulatory mechanism of Pitx2 gene transcription which may contribute to an understanding of pathogenesis of OFCD syndrome.

A pituitary homeobox 2 (Pitx2):microRNA-200a-3p:ß-catenin pathway converts mesenchymal cells to amelogenin-expressing dental epithelial cells.

Pitx2, Wnt/ß-catenin signaling, and microRNAs (miRs) play a critical role in the regulation of dental stem cells during embryonic development. In this report, we have identified a Pitx2:ß-catenin regulatory pathway involved in epithelial cell differentiation and conversion of mesenchymal cells to amelogenin expressing epithelial cells via miR-200a. Pitx2 and ß-catenin are expressed in the labial incisor cervical loop or epithelial stem cell niche, with decreased expression in the differentiating ameloblast cells of the mouse lower incisor. Bioinformatics analyses reveal that miR-200a-3p expression is activated in the pre-ameloblast cells to enhance epithelial cell differentiation. We demonstrate that Pitx2 activates miR-200a-3p expression and miR-200a-3p reciprocally represses Pitx2 and ß-catenin expression. Pitx2 and ß-catenin interact to synergistically activate gene expression during odontogenesis and miR-200a-3p attenuates their expression and directs differentiation. To understand how this mechanism controls cell differentiation and cell fate, oral epithelial and odontoblast mesenchymal cells were reprogrammed by a two-step induction method using Pitx2 and miR-200a-3p. Conversion to amelogenin expressing dental epithelial cells involved an up-regulation of the stem cell marker Sox2 and proliferation genes and decreased expression of mesenchymal markers. E-cadherin expression was increased as well as ameloblast specific factors. The combination of Pitx2, a regulator of dental stem cells and miR-200a converts mesenchymal cells to a fully differentiated dental epithelial cell type. This pathway and reprogramming can be used to reprogram mesenchymal or oral epithelial cells to dental epithelial (ameloblast) cells, which can be used in tissue repair and regeneration studies.

Protein inhibitors of activated STAT (Pias1 and Piasy) differentially regulate pituitary homeobox 2 (PITX2) transcriptional activity.

Protein inhibitors of activated STAT (Pias) proteins can act independent of sumoylation to modulate the activity of transcription factors and Pias proteins interacting with transcription factors can either activate or repress their activity. Pias proteins are expressed in many tissues and cells during development and we asked if Pias proteins regulated the pituitary homeobox 2 (PITX2) homeodomain protein, which modulates developmental gene expression. Piasy and Pias1 proteins are expressed during craniofacial/tooth development and directly interact and differentially regulate PITX2 transcriptional activity. Piasy and Pias1 are co-expressed in craniofacial tissues with PITX2. Yeast two-hybrid, co-immunoprecipitation and pulldown experiments demonstrate Piasy and Pias1 interactions with the PITX2 protein. Piasy interacts with the PITX2 C-terminal tail to attenuate its transcriptional activity. In contrast, Pias1 interacts with the PITX2 C-terminal tail to increase PITX2 transcriptional activity. The E3 ligase activity associated with the RING domain in Piasy is not required for the attenuation of PITX2 activity, however, the RING domain of Pias1 is required for enhanced PITX2 transcriptional activity. Bimolecular fluorescence complementation assays reveal PITX2 interactions with Piasy and Pias1 in the nucleus. Piasy represses the synergistic activation of PITX2 with interacting co-factors and Piasy represses Pias1 activation of PITX2 transcriptional activity. In contrast, Pias1 did not affect the synergistic interaction of PITX2 with transcriptional co-factors. Last, we demonstrate that Pias proteins form a complex with PITX2 and Lef-1, and PITX2 and ß-catenin. Lef-1, ß-catenin, and Pias interactions with PITX2 provide new molecular mechanisms for the regulation of PITX2 transcriptional activity and the activity of Pias proteins.

Axenfeld-Rieger syndrome: further clinical and array delineation of four unrelated patients with a 4q25 microdeletion.

Axenfeld-Rieger syndrome (ARS) is an autosomal dominant disorder with variable expressivity. It is characterized by dysgenesis of the anterior segment of the eye together with dental, cardiac, and umbilical anomalies. There is a high incidence of secondary high tension glaucoma. It is a genetically heterogeneous condition due to deletion or mutations of FOXC1 (6p25) or PITX2 (4q25). We report on four unrelated patients with overlapping microdeletions encompassing PITX2 at 4q25. We compare the genotypes and phenotypes of these newly described ARS patients and discuss the involvement of contiguous genes. Patients 1, 2, and 3 had mild learning difficulties, not typically seen in patients with ARS. We implicate the adjacent neuronally expressed genes; NEUROG2, UGT8, NDST3, and PRSS12 as potentially causal. Our findings support the use of microarray analysis in ARS patients for full prognostic information in infants presenting with ARS-like phenotypes.

Homeobox Genes in Odontogenic Lesions: A Scoping Review.

BACKGROUND: Homeobox genes play crucial roles in tooth morphogenesis and development and thus mutations in homeobox genes cause developmental disorders such as odontogenic lesions. The aim of this scoping review is to identify and compile available data from the literatures on the topic of homeobox gene expression in odontogenic lesions. METHOD: An electronic search to collate all the information on studies on homeobox gene expression in odontogenic lesions was carried out in four databases (PubMed, EBSCO host, Web of Science and Cochrane Library) with selected keywords. All papers which reported expression of homeobox genes in odontogenic lesions were considered. RESULTS: A total of eleven (11) papers describing expression of homeobox genes in odontogenic lesions were identified. Methods of studies included next generation sequencing, microarray analysis, RT-PCR, Western blotting, in situ hybridization, and immunohistochemistry. The homeobox reported in odontogenic lesions includes LHX8 and DLX3 in odontoma; PITX2, MSX1, MSX2, DLX, DLX2, DLX3, DLX4, DLX5, DLX6, ISL1, OCT4 and HOX C in ameloblastoma; OCT4 in adenomatoid odontogenic tumour; PITX2 and MSX2 in primordial odontogenic tumour; PAX9 and BARX1 in odontogenic keratocyst; PITX2, ZEB1 and MEIS2 in ameloblastic carcinoma while there is absence of DLX2, DLX3 and MSX2 in clear cell odontogenic carcinoma. CONCLUSIONS: This paper summarized and reviews the possible link between homeobox gene expression in odontogenic lesions. Based on the current available data, there are insufficient evidence to support any definite role of homeobox gene in odontogenic lesions.

The clinical and genetic findings in a Chinese family with Axenfeld-Rieger syndrome.

Objecive: To describe the clinical and genetic findings of an Axenfeld-Rieger syndrome (ARS) family with a new PITX2 splicing mutation. Methods: 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. Results: 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. Conclusion: 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.