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Ovarian cancer (OC) is the deadliest gynecological malignancy, having a high mortality rate due to its asymptomatic nature, chemoresistance, and recurrence. However, the proper mechanistic knowledge behind these phenomena is still inadequate. Cancer recurrence is commonly observed due to cancer stem cells which also show chemoresistance. We aimed to decipher the molecular mechanism behind chemoresistance and stemness in OC. Earlier studies suggested that PITX2, a homeobox transcription factor and, its different isoforms are associated with OC progression upon regulating different signaling pathways. Moreover, they regulate the expression of drug efflux transporters in kidney and colon cancer, rendering chemoresistance properties in the tumor cell. Considering these backgrounds, we decided to look for the role of PITX2 isoforms in promoting stemness and chemoresistance in OC cells. In this study, PITX2A/B has been shown to promote stemness and to enhance the transcription of ABCB1. PITX2 has been discovered to augment ABCB1 gene expression by directly binding to its promoter. To further investigate the regulatory mechanism of PITX2 gene expression, we found that TGFβ signaling could augment the PITX2A/B expression through both SMAD and non-SMAD signaling pathways. Collectively, we conclude that TGFβ1-activated PITX2A/B induces stem-like features and chemoresistance properties in the OC cells. Graphical illustration depicts that TGFβ1 enhances PITX2A/B expression through ERK and SMAD signaling pathway. Then, PITX2A/B bind ABCB1 promoter and therefore induce stemness and chemoresistance properties in OC cells. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Since the prognosis of patients with pancreatic cancer is very poor and there is a lack of treatment methods, this study is performed to investigate the function of PITX2 in pancreatic stellate cells (PSCs) in the progression of pancreatic cancer. Scientific hypotheses are proposed according to bioinformatics analysis and tissue microarray analysis. Stable knockdown of PITX2 in PSCs is achieved through lentiviral infection. The relative expressions of PITX2, α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are measured in wild-type PSCs and PITX2-knockdown PSCs. Proliferative capacity is measured by EdU assay. After coculture with PSCs, the proliferation, invasion and migration capacity of pancreatic cancer cells are tested. EMT and Wnt/β-catenin downstream genes of pancreatic cancer cells are investigated to reveal the potential mechanism. Bioinformatics analysis reveals that the PITX2 gene is highly expressed in stromal cells in pancreatic cancer and is correlated with squamous-type PDAC. Analysis of PDAC tissue microarray further demonstrates that high PITX2 level in stromal cells is correlated with poor prognosis in PDAC. After stable knockdown of PITX2 in PSCs, the relative protein levels of α-SMA, vimentin, CTNNB1, AXIN1 and LEF1 are decreased, and the proliferative capacity of PSCs is also decreased. After coculture with PSCs, in which PITX2 expression is downregulated, the proliferation, invasion and migration capacities of pancreatic cancer cells are inhibited. Thus, our results show that PITX2-silenced PSCs inhibit the growth, migration and invasion of pancreatic cancer cells via reduced EMT and Wnt/β-catenin signaling.

Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or p-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency. [ABSTRACT FROM AUTHOR]

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/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. [ABSTRACT FROM AUTHOR]

Background: As observed in recent genetic studies, PITX2 is one of the most popular genes with atrial fibrillation; single nucleotide polymorphism (rs2200733) at chromosome 4q25 (near PITX2) is found to be strongly associated with atrial fibrillation, but it has a difference among Chinese Han population. The basic aim of conducting this study is to find the correlation between PITX2 gene polymorphism and the risk of atrial fibrillation and to identify the possibility for early diagnosis of silent atrial fibrillation and high-risk atrial fibrillation. Methods: The study included 98 cases of atrial fibrillation patients and 88 non-atrial fibrillation patients in Affiliated Hospital of Yangzhou University were enrolled in a case-control study. The single nucleotide polymorphism of rs2200733 at 4q25 near PITX2 was genotyped by polymerase chain reaction-restriction fragment length polymorphism analysis. Results: A total of 98 patients with atrial fibrillation were genotyped, and the following frequencies were included in genotype percentages (44.9%, 50%, and 5.1%) while distribution of significant single nucleotide polymorphism rs2200733 consisted (29.55%, 53.41%, and 17.05%) which showed (χ2 = 9.159, P =.01). There was no significant difference in TC genotype frequency (P =.642), frequency of T allele (χ2 = 7.447, P =.006), and T allele was 1.806 times that of the control group (odds ratio = 1.806, 95% CI = 1.179-2.766, P =.006). According to logistic regression analysis, following results were concluded for TC genotype (odds ratio = 3.128, 95% CI = 1.053-9.287, P =.04), or TT genotype (odds ratio = 5.077, 95% CI = 1.653-15.595, P =.005) increased the risk of atrial fibrillation. Conclusions: The genotype and allele frequency distribution of rs2200733 (T/C) near PITX2 is different in the atrial fibrillation group and the control group. The T allele is a risk factor for atrial fibrillation. Compared with the CC genotype, the TT genotype increased the risk of atrial fibrillation.

Atrial fibrillation (AF) is the most common cardiac arrhythmia worldwide; however, the underlying causes of AF initiation are still poorly understood, particularly because currently available models do not allow in distinguishing the initial causes from maladaptive remodeling that induces and perpetuates AF. Lately, the genetic background has been proven to be important in the AF onset. iPSC-derived cardiomyocytes, being patient- and mutation-specific, may help solve this diatribe by showing the initial cell-autonomous changes underlying the development of the disease. Transcription factor paired-like homeodomain 2 (PITX2) has been identified as a key regulator of atrial development/differentiation, and the PITX2 genomic locus has the highest association with paroxysmal AF. PITX2 influences mitochondrial activity, and alterations in either its expression or function have been widely associated with AF. In this work, we investigate the activity of mitochondria in iPSC-derived atrial cardiomyocytes (aCMs) obtained from a young patient (24 years old) with paroxysmal AF, carrying a gain-of-function mutation in PITX2 (rs138163892) and from its isogenic control (CTRL) in which the heterozygous point mutation has been reverted to WT. PITX2 aCMs show a higher mitochondrial content, increased mitochondrial activity, and superoxide production under basal conditions when compared to CTRL aCMs. However, increasing mitochondrial workload by FCCP or β-adrenergic stimulation allows us to unmask mitochondrial defects in PITX2 aCMs, which are incapable of responding efficiently to the higher energy demand, determining ATP deficiency.

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. [ABSTRACT FROM AUTHOR]

Total bilateral Limbal Stem Cell Deficiency is a pathologic condition of the ocular surface due to the loss of corneal stem cells. Cultivated oral mucosa epithelial transplantation (COMET) is the only autologous successful treatment for this pathology in clinical application, although abnormal peripheric corneal vascularization often occurs. Properly characterizing the regenerated ocular surface is needed for a reliable follow-up. So far, the univocal identification of transplanted oral mucosa has been challenging. Previously proposed markers were shown to be co-expressed by different ocular surface epithelia in a homeostatic or perturbated environment. In this study, we compared the transcriptome profile of human oral mucosa, limbal and conjunctival cultured holoclones, identifying Paired Like Homeodomain 2 (PITX2) as a new marker that univocally distinguishes the transplanted oral tissue from the other epithelia. We validated PITX2 at RNA and protein levels to investigate 10-year follow-up corneal samples derived from a COMET-treated aniridic patient. Moreover, we found novel angiogenesis-related factors that were differentially expressed in the three epithelia and instrumental in explaining the neovascularization in COMET-treated patients. These results will support the follow-up analysis of patients transplanted with oral mucosa and provide new tools to understand the regeneration mechanism of transplanted corneas. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and a major cause of stroke and morbidity. The strongest genetic risk factors for AF in humans are variants on chromosome 4q25, near the paired-like homeobox transcription factor 2 gene PITX2. Although mice deficient in Pitx2 (Pitx2+/−) have increased AF susceptibility, the mechanism remains controversial. Recent evidence has implicated hyperactivation of the cardiac ryanodine receptor (RyR2) in Pitx2 deficiency, which may be associated with AF susceptibility. We investigated pacing-induced AF susceptibility and spontaneous Ca2+ release events in Pitx2 haploinsufficient (+/−) mice and isolated atrial myocytes to test the hypothesis that hyperactivity of RyR2 increases susceptibility to AF, which can be prevented by a potent and selective RyR2 channel inhibitor, ent -verticilide. Compared with littermate wild-type Pitx2+/+, the frequency of Ca2+ sparks and spontaneous Ca2+ release events increased in permeabilized and intact atrial myocytes from Pitx2+/− mice. Atrial burst pacing consistently increased the incidence and duration of AF in Pitx2+/− mice. The RyR2 inhibitor ent -verticilide significantly reduced the frequency of spontaneous Ca2+ release in intact atrial myocytes and attenuated AF susceptibility with reduced AF incidence and duration. Our data demonstrate that RyR2 hyperactivity enhances SR Ca2+ leak and AF inducibility in Pitx2+/− mice via abnormal Ca2+ handling. Therapeutic targeting of hyperactive RyR2 in AF using ent -verticilide may be a viable mechanism-based approach to treat atrial arrhythmias caused by Pitx2 deficiency. [Display omitted] • Pitx2 deficiency (+/-) causes RyR2 hyperactivity, Ca2+ spark leak and AF susceptible. • Increased spontaneous Ca2+ release in Pitx2+/- atrial cardiomyocytes was prevented by RyR2 inhibition with ent -verticilide. • Adrenergic stimulation aggravated AF susceptibility in Pitx2+/- mice. • RyR2 inhibition with ent -verticilide effectively prevented pacing-induced AF. [ABSTRACT FROM AUTHOR]

The bicoid‐related transcription factor 2 (Pitx2) plays a crucial role in the development of many organs and tissues by affecting the mitotic cell cycle. Postnatal testis development is related to mitosis and meiosis in multiple cell types, but the role of Pitx2 gene in seasonal inhibition of testicular development remains unknown in rodents. We analyzed PITX2 protein and Pitx2 mRNA expression features using both laboratory and wild male Rattus norvegicus caraco. In postnatal testicle of laboratory colony, we found that PITX2 was expressed in Leydig cells, pachytene spermatocytes, round spermatids, and elongating spermatids rather than spermatogonia and leptotene/zygotene spermatocytes. Pitx2b expression significantly increased along with the occurrence of pachytene spermatocytes and round spermatids, while decreased along with the processes of elongated spermatids. In wild male rats with similar testes weight, a significantly suppressed Pitx2b expression occurred with an active meiotic stage in the inhibited testes in autumn and winter, compared with the normally developing testes in spring and summer. These results indicate that Pitx2b expression suppression plays a crucial role in the seasonal inhibition of testis development. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

BackgroundAtrial fibrillation (AF) is a major cause of morbidity with a high prevalence among the elderly and has an established genetic disposition. Surgery is a well-known risk factor for AF; however, it is currently not recognized how much common genetic variants influence the postoperative risk. The purpose of this study was to identify Single Nucleotide Polymorphisms associated with postoperative AF.MethodsThe UK Biobank was utilized to conduct a Genome-Wide Association Study (GWAS) to identify variants associated with AF after surgery. An initial discovery GWAS was performed in patients that had undergone surgery with subsequent replication in a unique non-surgical cohort. In the surgical cohort, cases were defined as newly diagnosed AF within 30 days after surgery. The threshold for significance was set at 5 × 10−8.ResultsAfter quality control, 144,196 surgical patients with 254,068 SNPs were left for analysis. Two variants (rs17042171 (p = 4.86 × 10−15) and rs17042081 (p = 7.12 × 10−15)) near the PITX2-gene reached statistical significance. These variants were replicated in the non-surgical cohort (1.39 × 10−101 and 1.27 × 10−93, respectively). Several other loci were significantly associated with AF in the non-surgical cohort.ConclusionIn this GWAS-analysis of a large national biobank, we identified 2 variants that were significantly associated with postoperative AF. These variants were subsequently replicated in a unique non-surgical cohort. These findings bring new insight in the genetics of postoperative AF and may help identify at-risk patients and guide management.

Pitx2 is a transcription factor responsible for establishment of the right–left axis and development of the gut and pituitary. In mouse embryos, Pitx2 is expressed in the greater curvature of the stomach and midgut. Previously, Pitx2 was studied in pituitary neuroendocrine tumours but not in other NETs. Pitx2 expression was immunohistochemically assessed in whole sections and tissue microarrays in a cohort of 224 neuroendocrine neoplasms, and was analysed in 29 cases. The cohort included 18 cauda equina NETs, 38 paragangliomas, 98 cases of primary visceral NETs from different organs, 23 metastases of visceral NETs and 47 neuroendocrine carcinomas (NECs). Pitx2 expression was observed in 29.5% (29 of 98) NETs and 14.9% (7 of 47) NECs, but was not observed in any paraganglioma or cauda equina NET. Pitx2 was observed only in tumours of midgut‐derived organs, including the small intestine (100%, 20 of 20), appendix (88.9%, eight of nine) and large intestine (9.1%, one of 11 – only caecal NET). The NETs of remaining locations were negative. Pitx2 was 96.7% sensitive and 100% specific for NETs of midgut origin. In NECs, Pitx2 positivity was observed in goblet cell adenocarcinoma (75%, three of four), medullary thyroid carcinoma (42.9, three of seven) and one Merkel cell carcinoma (25%, one of four). In metastatic NETs, Pitx2 was observed in all the tumours originating in the small intestine (n = 17) or caecum (n = 1). No positivity was observed in tumours from other locations (four pancreas, one lung). We observed no correlation between immunoreactivity and mRNA expression. Thus, Pitx2 immunohistochemistry can be helpful in assessing the midgut origin of NETs. [ABSTRACT FROM AUTHOR]

Paired-like homeodomain 2 (PITX2) functions as a transcription factor to participate in vertebrate embryogenesis, and dysregulated PITX2 expression was associated with the progression of various cancers. The functional role of PITX2 in tumorigenesis of liver cancer remains unknown. Western blot analysis showed that expression levels of PITX2 were enhanced in the liver cancer tissues and cells. siRNAs targeting PITX2 induced downregulation of PITX2 in liver cancer cells. siRNA-induced knockdown of PITX2 decreased liver cancer cell viability and proliferation, while promoting cell apoptosis by increasing cleaved-PARP, cleaved caspase 3, and cleaved caspase 9. The knockdown of PITX2 repressed liver cancer cell migration and invasion. In conclusion, elevated PITX2 expression was associated with liver cancer progression through repression of cell apoptosis and promoting cell proliferation and metastasis, and silencing of PITX2 might serve as a potential therapeutic strategy for the treatment of liver cancer.

Abstract Background Breast cancer chemoresistance is attributed to a wide variety of mechanisms, including autophagy. Transcription factor EB (TFEB) has been recently identified and characterized as one major regulator of autophagy and lysosomal genesis. Objective This study aims to evaluate the prognostic impact of TFEB and its pathway in breast cancer chemoresistance. Methods This retrospective study analyzes the expression of TFEB, CARM1, SIRT1, and Beclin-1 and the methylation of PITX2 in breast carcinoma. A group of breast cancer patients treated with chemotherapy, who relapsed within 12 months from treatment initiation, were compared to a sub-cohort of chemo-treated patients who did not recur within 12 months of follow-up. The expression of TFEB, CARM1, SIRT1, and Belcin-1 was analyzed using immunohistochemistry or RT-PCR on formalin-fixed paraffin-embedded samples. PITX2 methylation was tested with the diagnostic CE-marked kit Therascreen PITX2 RGQ PCR. In the final model, 136 cases of chemo-treated breast cancer were included. Results A higher TFEB and Beclin-1 expression correlate with shorter survival in patients with chemo-treated invasive breast cancer (respectively HR 3.46, CI.95 1.27–9.47, p

The hypothalamus is a key vertebrate brain region involved in survival and physiological functions. Understanding hypothalamic organization and evolution is important to deciphering many aspects of vertebrate biology. Recent comparative studies based on gene expression patterns have proposed the existence of hypothalamic histogenetic domains (paraventricular, TPa/PPa; subparaventricular, TSPa/PSPa; tuberal, Tu/RTu; perimamillary, PM/PRM; and mamillary, MM/RM), revealing conserved evolutionary trends. To shed light on the functional relevance of these histogenetic domains, this work aims to interpret the location of developed cell groups according to the prosomeric model in the hypothalamus of the catshark Scyliorhinus canicula, a representative of Chondrichthyans (the sister group of Osteichthyes, at the base of the gnathostome lineage). To this end, we review in detail the expression patterns of ScOtp, ScDlx2, and ScPitx2, as well as Pax6-immunoreactivity in embryos at stage 32, when the morphology of the adult catshark hypothalamus is already organized. We also propose homologies with mammals when possible. This study provides a comprehensive tool to better understand previous and novel data on hypothalamic development and evolution. [ABSTRACT FROM AUTHOR]

IntroductionAtrial fibrillation (AF) is a heritable disease, and the paired-like homeodomain transcription factor 2 (PITX2) gene is highly associated with AF. We explored the differences in the circumferential pulmonary vein isolation (CPVI), which is the cornerstone procedure for AF catheter ablation, additional high dominant frequency (DF) site ablation, and antiarrhythmic drug (AAD) effects according to the patient genotype (wild-type and PITX2+/− deficient) using computational modeling.MethodsWe included 25 patients with AF (68% men, 59.8 ± 9.8 years of age, 32% paroxysmal AF) who underwent AF catheter ablation to develop a realistic computational AF model. The ion currents for baseline AF and the amiodarone, dronedarone, and flecainide AADs according to the patient genotype (wild type and PITX2+/− deficient) were defined by relevant publications. We tested the virtual CPVI (V-CPVI) with and without DF ablation (±DFA) and three virtual AADs (V-AADs, amiodarone, dronedarone, and flecainide) and evaluated the AF defragmentation rates (AF termination or changes to regular atrial tachycardia (AT), DF, and maximal slope of the action potential duration restitution curves (Smax), which indicates the vulnerability of wave-breaks.ResultsAt the baseline AF, mean DF (p = 0.003), and Smax (p < 0.001) were significantly lower in PITX2+/− deficient patients than wild-type patients. In the overall AF episodes, V-CPVI (±DFA) resulted in a higher AF defragmentation relative to V-AADs (65 vs. 42%, p < 0.001) without changing the DF or Smax. Although a PITX2+/− deficiency did not affect the AF defragmentation rate after the V-CPVI (±DFA), V-AADs had a higher AF defragmentation rate (p = 0.014), lower DF (p < 0.001), and lower Smax (p = 0.001) in PITX2+/− deficient AF than in wild-type patients. In the clinical setting, the PITX2+/− genetic risk score did not affect the AF ablation rhythm outcome (Log-rank p = 0.273).ConclusionConsistent with previous clinical studies, the V-CPVI had effective anti-AF effects regardless of the PITX2 genotype, whereas V-AADs exhibited more significant defragmentation or wave-dynamic change in the PITX2+/− deficient patients.

The knowledge of the molecular mechanisms that regulate embryonic myogenesis from early myogenic progenitors to myoblasts, as well as the emergence of adult satellite stem cells (SCs) during development, are key concepts to understanding the genesis and regenerative abilities of the skeletal muscle. Several previous pieces of evidence have revealed that the transcription factor Pitx2 might be a player within the molecular pathways controlling somite-derived muscle progenitors’ fate and SC behavior. However, the role exerted by Pitx2 in the progression from myogenic progenitors to myoblasts including SC precursors remains unsolved. Here, we show that Pitx2 inactivation in uncommitted early myogenic precursors diminished cell proliferation and migration leading to muscle hypotrophy and a low number of SCs with decreased myogenic differentiation potential. However, the loss of Pitx2 in committed myogenic precursors gave rise to normal muscles with standard amounts of SCs exhibiting high levels of Pax7 expression. This SC population includes few MYF5+ SC-primed but increased amount of less proliferative miR-106b+cells, and display myogenic differentiation defects failing to undergo proper muscle regeneration. Overall our results demonstrate that Pitx2 is required in uncommitted myogenic progenitors but it is dispensable in committed precursors for proper myogenesis and reveal a role for this transcription factor in the generation of diverse SC subpopulations.

Ferroptosis inhibits tumor progression in pancreatic cancer cells, while PITX2 is known to function as a pro-oncogenic factor in various tumor types, protecting them from ferroptosis and thereby promoting tumor progression. In this study, we sought to investigate the regulatory role of PITX2 in tumor cell ferroptosis within the context of pancreatic cancer. We conducted PITX2 knockdown experiments using lentiviral infection in two pancreatic cancer cell lines, namely PANC-1 and BxPC-3. We assessed protein expression through immunoblotting and mRNA expression through RT-PCR. To confirm PITX2 as a transcription factor for GPX4, we employed Chromatin Immunoprecipitation (ChIP) and Dual-luciferase assays. Furthermore, we used flow cytometry to measure reactive oxygen species (ROS), lipid peroxidation, and apoptosis and employed confocal microscopy to assess mitochondrial membrane potential. Additionally, electron microscopy was used to observe mitochondrial structural changes and evaluate PITX2's regulation of ferroptosis in pancreatic cancer cells. Our findings demonstrated that PITX2, functioning as a transcription factor for GPX4, promoted GPX4 expression, thereby exerting an inhibitory effect on ferroptosis in pancreatic cancer cells and consequently promoting tumor progression. Moreover, PITX2 enhanced the invasive and migratory capabilities of pancreatic cancer cells by activating the WNT signaling pathway. Knockdown of PITX2 increased ferroptosis and inhibited the proliferation of PANC-1 and BxPC-3 cells. Notably, the inhibitory effect on ferroptosis resulting from PITX2 overexpression in these cells could be countered using RSL3, an inhibitor of GPX4. Overall, our study established PITX2 as a transcriptional regulator of GPX4 that could promote tumor progression in pancreatic cancer by reducing ferroptosis. These findings suggest that PITX2 may serve as a potential therapeutic target for combating ferroptosis in pancreatic cancer. [ABSTRACT FROM AUTHOR]

Facial asymmetry is a feature that occurs to a greater or lesser extent in the general population. As its severity is usually slight, facial asymmetry may not be noticeable to the patient. However, there are cases when severe facial asymmetry not only affects the facial aesthetics by distorting facial proportions, but also contributes to problems related to the function of the stomatognathic system. The nodal signalling pathway appears to be of particular importance in the process of mandibular asymmetry, as it affects not only structures formed from the first pharyngeal arch, but also other organs, such as the heart and lungs. Following the evaluation of the available literature, the inheritance of mandibular asymmetry is a very complex and multifactorial process, and the genes whose altered expression appears to be a more important potential aetiological factor for asymmetry include PITX2, ACTN3, ENPP1 and ESR1. This systematic review attempts to systematise the available literature concerning the impact of signalling pathway disruption, including the disruption of the nodal signalling pathway, on the development of mandibular asymmetry. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aim: Reduced left atrial PITX2 is associated with atrial cardiomyopathy and atrial fibrillation. PITX2 is restricted to left atrial cardiomyocytes in the adult heart. The links between PITX2 deficiency, atrial cardiomyopathy and atrial fibrillation are not fully understood., Methods and Results: To identify mechanisms linking PITX2 deficiency to atrial fibrillation, we generated and characterized PITX2-deficient human atrial cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) and their controls. PITX2-deficient hiPSC-derived atrial cardiomyocytes showed shorter and disorganised sarcomeres and increased mononucleation. Electron microscopy found an increased number of smaller mitochondria compared to the control. Mitochondrial protein expression was altered in PITX2-deficient hiPSC-derived atrial cardiomyocytes. Single-nuclear RNA-sequencing found differences in cellular respiration pathways and differentially expressed mitochondrial and ion channel genes in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2 repression in hiPSC-derived atrial cardiomyocytes replicated dysregulation of cellular respiration. Mitochondrial respiration was shifted to increased glycolysis in PITX2-deficient hiPSC-derived atrial cardiomyocytes. PITX2-deficient human hiPSC-derived atrial cardiomyocytes showed higher spontaneous beating rates. Action potential duration was more variable with an overall prolongation of early repolarization, consistent with metabolic defects. Gene expression analyses confirmed changes in mitochondrial genes in left atria from 42 patients with atrial fibrillation compared to 43 patients in sinus rhythm. Dysregulation of left atrial mitochondrial (COX7C) and metabolic (FOXO1) genes was associated with PITX2 expression in human left atria., Conclusions: In summary, PITX2 deficiency causes mitochondrial dysfunction and a metabolic shift to glycolysis in human atrial cardiomyocytes. PITX2-dependent metabolic changes can contribute to the structural and functional defects found in PITX2-deficient atria., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Background Androgenetic alopecia, also known as male pattern baldness, is a common form of hair loss influenced by environmental, hormonal, and genetic factors. According to recent research, the PITX2 gene may play a key role in the pathophysiology of androgenetic alopecia (AGA). Objective This study examines the association between genetic variants of the PITX2 gene and AGA risk. Methods The genomic DNA was extracted from peripheral blood samples collected from 70 male AGA patients and 60 non-androgenetic alopecia controls. The isolated DNA was quantified and the genotype for three PITX2 polymorphisms (rs2200733, rs10033464, and rs13143308) was identified using TaqMan assays. The statistical analysis was done to determine the allele frequency of genetic variants between AGA and non-AGA groups. Result The demographic profile of the study population showed that the AGA and non-AGA groups differed in age. The AGA group had higher blood pressure, a higher prevalence of smoking, alcohol consumption, metabolic syndrome, insulin resistance, and a higher incidence of family history. Through genetic analysis, significant correlations were found between AGA risk and specific PITX2 polymorphisms, significantly with the rs2200733 allele (OR = 6.08, p < 0.001*), the rs1003464 G allele (OR = 2.02, p < 0.019*) and the rs13143308 showed GT genotype (OR = 4.26, p < 0.001*). Conclusion Based on our findings, the PITX2 polymorphisms may play a vital role in the development of AGA. This study also found the interactions between genetic and environmental factors in AGA pathogenesis.

Anorectal malformation (ARM) is the most common congenital digestive tract anomaly in newborns, and children with ARM often have varying degrees of underdevelopment of the pelvic floor muscles (PFMs). To explore the effects of RARα and Pitx2 on the development of rat PFMs, we constructed a rat ARM animal model using all-trans retinoic acid (ATRA), and verified the expression of RARα and Pitx2 in the PFMs of fetal rats. Additionally, we used rat myoblasts (L6 cells) to investigate the regulatory roles of RARα and Pitx2 in skeletal muscle myoblast differentiation and their interactions. The results indicated a significant decrease in the expression of RARα and Pitx2 in the PFMs of fetal rats with ARM. ATRA can also decrease the expression of RARα and Pitx2 in the L6 cells, while affecting the differentiation and fusion of L6 cells. Knocking down RARα in L6 cells reduced the expression of Pitx2, MYOD1, MYMK, and decreased myogenic activity in L6 cells. When RARα is activated, the decreased expression of Pitx2, MYOD1, and MYMK and myogenic differentiation can be restored to different extents. At the same time, increasing or inhibiting the expression of Pitx2 can counteract the effects of knocking down RARα and activating RARα respectively. These results indicate that Pitx2 may be downstream of the transcription factor RARα, mediating the effects of ATRA on the development of fetal rat PFMs., Competing Interests: Declaration of Competing Interest We declare that there are no conflicts of interests., (Copyright © 2024. Published by Elsevier Inc.)

Background and Objectives: Recurrence of atrial fibrillation (AF) within six months after sinus rhythm restoration with direct current cardioversion (DCC) is a significant treatment challenge. Currently, the factors influencing outcome are mostly unknown. Studies have found a link between genetics and the risk of AF and efficacy of rhythm control. The aim of this study was to examine the association between eight single-nucleotide variants (SNVs) and the risk of AF development and recurrence after DCC. Materials and Methods: Regarding the occurrence of AF, 259 AF cases and 108 controls were studied. Genotypes for the eight SNVs located in the genes CAV1, MYH7, SOX5, KCNN3, ZFHX3, KCNJ5 and PITX2 were determined using high-resolution melting analysis and confirmed with Sanger sequencing. Six months after DCC, a telephone interview was conducted to determine whether AF had recurred. A polygenic risk score (PRS) was calculated as the unweighted sum of risk alleles. Multivariate regression analyses were performed to assess SNV and PRS association with AF occurrence and recurrence after DCC. Results: The risk allele of rs2200733 (PITX2) was significantly associated with the development of AF (p = 0.012, OR = 2.31, 95% CI = 1.206–4.423). AF recurred in 60% of patients and the allele generally associated with a decreased risk of AF of rs11047543 (SOX5) was associated with a greater risk of AF recurrence (p = 0.014, OR = 0.223, 95% CI = 0.067–0.738). A PRS of greater than 7 was significantly associated (p = 0.008) with a higher likelihood of developing AF after DCC (OR = 4.174, 95% CI = 1.454–11.980). Conclusions: A higher PRS is associated with increased odds of AF recurrence after treatment with DCC. PITX2 (rs2200733) is significantly associated with an increased risk of AF. The protective allele of rs11047543 (SOX5) is associated with a greater risk of AF recurrence. Further studies are needed to predict the success of rhythm control and guide patient selection towards the most efficacious treatment. [ABSTRACT FROM AUTHOR]

Background: Breast cancer chemoresistance is attributed to a wide variety of mechanisms, including autophagy. Transcription factor EB (TFEB) has been recently identified and characterized as one major regulator of autophagy and lysosomal genesis.Objective: This study aims to evaluate the prognostic impact of TFEB and its pathway in breast cancer chemoresistance.Methods: This retrospective study analyzes the expression of TFEB, CARM1, SIRT1, and Beclin-1 and the methylation of PITX2 in breast carcinoma. A group of breast cancer patients treated with chemotherapy, who relapsed within 12 months from treatment initiation, were compared to a sub-cohort of chemo-treated patients who did not recur within 12 months of follow-up. The expression of TFEB, CARM1, SIRT1, and Belcin-1 was analyzed using immunohistochemistry or RT-PCR on formalin-fixed paraffin-embedded samples. PITX2 methylation was tested with the diagnostic CE-marked kit Therascreen PITX2 RGQ PCR. In the final model, 136 cases of chemo-treated breast cancer were included.Results: A higher TFEB and Beclin-1 expression correlate with shorter survival in patients with chemo-treated invasive breast cancer (respectively HR 3.46, CI.95 1.27-9.47, p < 0.05 and 7.11, CI.95 2.54-19.9). TFEB, CARM1, and SIRT1 are positively correlated with Beclin-1. The protein expression of SIRT1 is significantly associated with TFEB and CARM1 so that a very low SIRT1 expression (lower than the first quartile of the H-score distribution) correlates with a low expression of TFEB and CARM1 and with longer survival. SIRT1 seems to have a lower H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. Beclin-1 and TFEB seem to have a higher H-score in the basal-like and HER2-enriched tumors than the luminal subtypes. PITX2 methylation analysis was feasible only in 65% of the selected samples, but no significant differences between cases and controls were found, and there was also no correlation with the expression of the TFEB pathway.Conclusions: TFEB, SIRT1, and Beclin-1 seem to have a potential prognostic significance in patients with chemo-treated breast cancer, likely because of their role in the regulation of autophagy. In addition, no correlation between TFEB and PITX2 methylation was found, likely because they perform two different roles within the autophagy process. [ABSTRACT FROM AUTHOR]

The morphogenesis of left-right (LR) asymmetry is a crucial phase of organogenesis. In the digestive tract, the development of anatomical asymmetry is first evident in the leftward curvature of the stomach. To elucidate the molecular events that shape this archetypal laterality, we performed transcriptome analyses of the left versus right sides of the developing stomach in frog embryos. Besides the known LR gene pitx2, the only gene found to be expressed asymmetrically throughout all stages of curvature was single-minded 2 (sim2), a Down Syndrome-related transcription factor and homolog of a Drosophila gene (sim) required for LR asymmetric looping of the fly gut.We demonstrate that sim2 functions downstream of LR patterning cues to regulate key cellular properties and behaviors in the left stomach epithelium that drive asymmetric curvature. Our results reveal unexpected convergent cooption of single-minded genes during the evolution of LR asymmetric morphogenesis, and have implications for dose-dependent roles of laterality factors in nonlaterality- related birth defects. [ABSTRACT FROM AUTHOR]

The successful implantation of embryos is crucial for pregnancy in mammals. This complex process is inevitably dependent on the development of the endometrium. The paired‐like homeodomain transcription factor 2 (PITX2) is involved in a variety of biological processes, but whether it is involved in embryo implantation has not been reported. In this study, we aimed to investigate uterine expression and regulation of PITX2 during implantation. We found that PITX2 was elevated in the human endometrium in the secretory phase. The results of the pregnant mouse models showed that PITX2 expression was spatiotemporal in mouse endometrial tissue throughout peri‐implantation period, and it was significantly upregulated at the time of implantation. Interestingly, PITX2 was mainly localized to the glandular epithelium cells on D2.5‐3.5 of pregnancy, while D5.5‐6.5 was largely expressed in stromal cells. In vitro, PITX2 regulated endometrial cells proliferation, migration, invasion, and other functions through the Wnt/β‐catenin signaling pathway. In addition, a significant decrease in the rate of embryo implantation was observed after injecting PITX2 small interfering RNA into the uterine horn. These results demonstrate the effects of PITX2 on the physiological function of endometrial cells and embryo implantation, suggesting a role in the endometrial regulatory mechanism during implantation. [ABSTRACT FROM AUTHOR]

Background: PITX2 DNA methylation has been shown to predict outcomes in high-risk breast cancer patients after anthracycline-based chemotherapy. To determine its prognostic versus predictive value, the impact of PITX2 DNA methylation on outcomes was studied in an untreated cohort vs. an anthracycline-treated triple-negative breast cancer (TNBC) cohort. Material and Methods: The percent DNA methylation ratio (PMR) of paired-like homeodomain transcription factor 2 (PITX2) was determined by a validated methylation-specific real-time PCR test. Patient samples of routinely collected archived formalin-fixed paraffin-embedded (FFPE) tissue and clinical data from 144 TNBC patients of 2 independent cohorts (i.e., 66 untreated patients and 78 patients treated with anthracycline-based chemotherapy) were analyzed. Results: The risk of 5- and 10-year overall survival (OS) increased continuously with rising PITX2 DNA methylation in the anthracycline-treated population, but it increased only slightly during 10-year follow-up time in the untreated patient population. PITX2 DNA methylation with a PMR cutoff of 2 did not show significance for poor vs. good outcomes (OS) in the untreated patient cohort (HR = 1.55; p = 0.259). In contrast, the PITX2 PMR cutoff of 2 identified patients with poor (PMR >2) vs. good (PMR ≤2) outcomes (OS) with statistical significance in the anthracycline-treated cohort (HR = 3.96; p = 0.011). The results in the subgroup of patients who did receive anthracyclines only (no taxanes) confirmed this finding (HR = 5.71; p = 0.014). Conclusion: In this hypothesis-generating study PITX2 DNA methylation demonstrated predominantly predictive value in anthracycline treatment in TNBC patients. The risk of poor outcome (OS) correlates with increasing PITX2 DNA methylation. [ABSTRACT FROM AUTHOR]

Sensorimotor transformation, a process that converts sensory stimuli into motor actions, is critical for the brain to initiate behaviors. Although the circuitry involved in sensorimotor transformation has been well delineated, the molecular logic behind this process remains poorly understood. Here, we performed high-throughput and circuit-specific single-cell transcriptomic analyses of neurons in the superior colliculus (SC), a midbrain structure implicated in early sensorimotor transformation. We found that SC neurons in distinct laminae expressed discrete marker genes. Of particular interest, Cbln2 and Pitx2 were key markers that define glutamatergic projection neurons in the optic nerve (Op) and intermediate gray (InG) layers, respectively. The Cbln2+ neurons responded to visual stimuli mimicking cruising predators, while the Pitx2+ neurons encoded prey-derived vibrissal tactile cues. By forming distinct input and output connections with other brain areas, these neuronal subtypes independently mediated behaviors of predator avoidance and prey capture. Our results reveal that, in the midbrain, sensorimotor transformation for different behaviors may be performed by separate circuit modules that are molecularly defined by distinct transcriptomic codes.

Background: Although catheter ablation is an effective therapy for atrial fibrillation (AF), the most common cardiac arrhythmia encountered in clinical practice, AF ablation generates inflammation and oxidative stress in the early postoperative period predisposing to recurrence of AF. Isolevuglandins (IsoLGs) are reactive lipid mediators of oxidative stress injury that rapidly react with endogenous biomolecules to compromise their function. 2-Hydroxybenzylamine (2-HOBA), a potent small molecule scavenger of IsoLGs, sequesters the reactive species as inert adducts. This mechanism, coupled with reported safety in humans, supports the investigation of 2-HOBA as a novel therapeutic to reduce AF caused by oxidative stress, such as that which occurs after catheter ablation. Accordingly, we seek to test the hypothesis that treatment with 2-HOBA will decrease early recurrence of AF and other atrial arrhythmias following AF ablation by decreasing IsoLG adducts with native biomolecules.Methods: The proposed trial will randomly assign 162 participants undergoing cryo- or radiofrequency catheter ablation for AF to 2-HOBA (N = 81) or placebo (N = 81). Individuals will begin the study drug 3 days prior to ablation and continue for 28 days. Participants will be given a wearable smartwatch capable of detecting and recording atrial arrhythmias. They will be instructed to record ECGs daily with additional ECGs if they experience symptoms of AF or when alerted by the smartwatch AF detection alarm. The primary clinical endpoint will be an episode of AF, atrial tachycardia, or atrial flutter lasting 30 s or more within 28 days post-AF ablation. Secondary measures will be the change in IsoLG adduct levels from blood samples collected immediately pre-ablation and post-ablation and reduction in AF burden as calculated from the smartwatch.Discussion: The proposed trial will test the hypothesis that 2-HOBA reduces post-ablation atrial arrhythmias through sequestration of reactive IsoLG species. The results of this study may improve the understanding of the role of IsoLGs and oxidative stress in AF pathogenesis and provide evidence to advance 2-HOBA and related compounds as a new therapeutic strategy to treat AF.Trial Registration: ClinicalTrials.gov NCT04433091 . Registered on June 3, 2020. [ABSTRACT FROM AUTHOR]

Background: Although atrial fibrillation ablation is increasingly used for rhythm control therapy, antiarrhythmic drugs (AADs) are commonly used, either alone or in combination with ablation. The effectiveness of AADs is highly variable. Previous work from our group suggests that alterations in atrial resting membrane potential (RMP) induced by low Pitx2 expression could explain the variable effect of flecainide.Objective: The purpose of this study was to assess whether alterations in atrial/cardiac RMP modify the effectiveness of multiple clinically used AADs.Methods: The sodium channel blocking effects of propafenone (300 nM, 1 μM), flecainide (1 μM), and dronedarone (5 μM, 10 μM) were measured in human stem cell-derived cardiac myocytes, HEK293 expressing human NaV1.5, primary murine atrial cardiac myocytes, and murine hearts with reduced Pitx2c.Results: A more positive atrial RMP delayed INa recovery, slowed channel inactivation, and decreased peak action potential (AP) upstroke velocity. All 3 AADs displayed enhanced sodium channel block at more positive atrial RMPs. Dronedarone was the most sensitive to changes in atrial RMP. Dronedarone caused greater reductions in AP amplitude and peak AP upstroke velocity at more positive RMPs. Dronedarone evoked greater prolongation of the atrial effective refractory period and postrepolarization refractoriness in murine Langendorff-perfused Pitx2c+/- hearts, which have a more positive RMP compared to wild type.Conclusion: Atrial RMP modifies the effectiveness of several clinically used AADs. Dronedarone is more sensitive to changes in atrial RMP than flecainide or propafenone. Identifying and modifying atrial RMP may offer a novel approach to enhancing the effectiveness of AADs or personalizing AAD selection. [ABSTRACT FROM AUTHOR]

Purpose: To determine the common gene mutation in patients with primary congenital glaucoma (PCG) in the Southeast region of Turkey via genetic analysis and to evaluate whether there were other gene mutations in these patients. Methods: A total of 25 patients with PCG were included in this study. We performed sequence analysis including all exons of cytochrome p450 1B1 (CYP1B1), myocilin (MYOC), forkhead box C1 (FOXC1), and paired-like homeodomain 2 (PITX2) genes of the obtained samples. Further, we analyzed the results using the Nextgen analysis program. Results: The CYP1B1 gene mutation was detected in 20 (80%) of 25 patients, and FOXC1 gene mutation was detected in one (4%) patient. The mutation site of nine (45%) of the 20 CYP1B1 genes was found in the second exon. The pathogenic variant (p.Gly61Glu) was observed in 12 (60%) patients (in the first and second exons); the mutation type of six (50%) of these patients was homozygous. The mutation site of one patient with FOXC1 gene mutation was found to be in the first exon; its pathogenic variant was p.Met400lle. The mutation type in this gene was observed to be heterozygous. Lastly, there were no mutations in the MYOC, FOXC1, and PITX2 genes in combination with the CYP1B1 gene mutation. Conclusion: The most common cause of PCG in our region is the CYP1B1 gene mutation, and the most frequent pathogenic variant is c.182G > A (p.Gly61Glu). We also determined that the CYP1B1 gene mutation was alone and did not occur with other gene mutations (MYOC, FOXC1, and PITX2). [ABSTRACT FROM AUTHOR]

Metastasis is a major obstacle to better prognosis in patients with hepatocellular carcinoma (HCC). Mesenchymal‐epithelial transition (MET) is the driving force for metastatic colonization in which E‐cadherin re‐expression is a critical procedure. It has been reported that the loss of paired‐related homeobox transcription factor 1 (PRRX1) is required for cancer cell metastasis. However, the role of PRRX1 in MET and how its downregulation triggers E‐cadherin re‐expression are unknown. In this study, we performed a systematic, mechanistic study regarding the role of PRRX1 in MET of HCC. We observed PRRX1 downregulation in HCC tissues, which correlated with early metastasis and short overall survival. Overexpression of PRRX1 induced epithelial‐mesenchymal transition (EMT), but did not promote metastasis formation, while knockdown of PRRX1 promoted metastasis and colonization of circulating HCC cells as shown in animal model. PRRX1 protein levels reversely correlated with E‐cadherin levels in HCC cell lines. PRRX1 knockdown promoted E‐cadherin re‐expression and cell proliferation and inhibited cell invasion and migration. The microarray results showed that PRRX1 deficiency regulated extracellular matrix (ECM) interaction, focal adhesion, TGF‐β signaling and cancer pathways. PRRX1 knockdown upregulated paired‐like homeodomain 2 (PITX2) and inhibited catenin beta 1 (CTNNB1) and SNAIL family zinc finger 2 (SLUG). Silencing of PITX2 reversed CTNNB1 and SLUG inhibition and E‐cadherin re‐expression. PITX2 upregulation increased miR‐200a and miR‐200b/429, which further inhibited the transcription of CTNNB1 and SLUG, respectively, thus abrogating the inhibitory effect on E‐cadherin. In conclusion, our data showed that the downregulation of PRRX1 induced E‐cadherin re‐expression through PITX2/miR‐200a/CTNNB1 and PITX2/miR‐200b/429/SLUG pathway. [ABSTRACT FROM AUTHOR]

Abstract 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.

Abstract Background The homeodomain transcription factor, PITX2 is associated with tumorigenesis of multiple cancers. In this research, we aimed to study the expression, function and mechanism of PITX2 in lung adenocarcinoma (LUAD). Methods The TCGA dataset was used to analyze the expression and clinical significance of PITX2 in LUAD. The expression of PITX2 in tumor samples and LUAD cell lines was examined by quantitative real-time PCR (qRT-PCR) and western blotting. Small interfering RNAs (siRNAs) were constructed to knockdown PITX2 and to determine the physiological function of PITX2 in vitro. Xenograft model was used to confirm the role of PITX2 in vivo. Results PITX2 was overexpressed in LUAD and patients with high level of PITX2 had a worse overall survival and an advanced clinical stage. Knockdown of PITX2 inhibited cell proliferation, migration and invasion of LUAD cells. Further study revealed that the oncogenic role of PITX2 was dependent on activating Wnt/β-catenin signaling pathway, especially by transcriptionally regulating the Wnt gene family member, WNT3A. Lastly, we identified miR-140-5p as a negative mediator of PITX2 by binding its 3′UTR and ectopic expression of miR-140-5p inhibited progression of LUAD cells via suppressing the expression of PITX2. Conclusions Up-regulation of PITX2 acts as an oncogene in LUAD by activating Wnt/β-catenin signaling pathway, suggesting that PITX2 may serve as a novel diagnostic and prognostic biomarker in LUAD.

Background: The efficacy of antiarrhythmic drugs (AAD) can vary in patients with atrial fibrillation (AF), and the PITX2 gene affects the responsiveness of AADs. We explored the virtual AAD (V-AAD) responses between wild-type and PITX2+/−-deficient AF conditions by realistic in silico AF modeling.Methods: We tested the V-AADs in AF modeling integrated with patients' 3D-computed tomography and 3D-electroanatomical mapping, acquired in 25 patients (68% male, 59.8 ± 9.8 years old, 32.0% paroxysmal type). The ion currents for the PITX2+/− deficiency and each AAD (amiodarone, sotalol, dronedarone, flecainide, and propafenone) were defined based on previous publications.Results: We compared the wild-type and PITX2+/− deficiency in terms of the action potential duration (APD90), conduction velocity (CV), maximal slope of restitution (Smax), and wave-dynamic parameters, such as the dominant frequency (DF), phase singularities (PS), and AF termination rates according to the V-AADs. The PITX2+/−-deficient model exhibited a shorter APD90 (p < 0.001), a lower Smax (p < 0.001), mean DF (p = 0.012), PS number (p < 0.001), and a longer AF cycle length (AFCL, p = 0.011). Five V-AADs changed the electrophysiology in a dose-dependent manner. AAD-induced AFCL lengthening (p < 0.001) and reductions in the CV (p = 0.033), peak DF (p < 0.001), and PS number (p < 0.001) were more significant in PITX2+/−-deficient than wild-type AF. PITX2+/−-deficient AF was easier to terminate with class IC AADs than the wild-type AF (p = 0.018).Conclusions: The computational modeling-guided AAD test was feasible for evaluating the efficacy of multiple AADs in patients with AF. AF wave-dynamic and electrophysiological characteristics are different among the PITX2-deficient and the wild-type genotype models.

Background: The efficacy of antiarrhythmic drugs (AAD) can vary in patients with atrial fibrillation (AF), and the PITX2 gene affects the responsiveness of AADs. We explored the virtual AAD (V-AAD) responses between wild-type and PITX2 +/−-deficient AF conditions by realistic in silico AF modeling. Methods: We tested the V-AADs in AF modeling integrated with patients' 3D-computed tomography and 3D-electroanatomical mapping, acquired in 25 patients (68% male, 59.8 ± 9.8 years old, 32.0% paroxysmal type). The ion currents for the PITX2 +/− deficiency and each AAD (amiodarone, sotalol, dronedarone, flecainide, and propafenone) were defined based on previous publications. Results: We compared the wild-type and PITX2 +/− deficiency in terms of the action potential duration (APD90), conduction velocity (CV), maximal slope of restitution (Smax), and wave-dynamic parameters, such as the dominant frequency (DF), phase singularities (PS), and AF termination rates according to the V-AADs. The PITX2 +/−-deficient model exhibited a shorter APD90 (p < 0.001), a lower Smax (p < 0.001), mean DF (p = 0.012), PS number (p < 0.001), and a longer AF cycle length (AFCL, p = 0.011). Five V-AADs changed the electrophysiology in a dose-dependent manner. AAD-induced AFCL lengthening (p < 0.001) and reductions in the CV (p = 0.033), peak DF (p < 0.001), and PS number (p < 0.001) were more significant in PITX2 +/−-deficient than wild-type AF. PITX2 +/−-deficient AF was easier to terminate with class IC AADs than the wild-type AF (p = 0.018). Conclusions: The computational modeling-guided AAD test was feasible for evaluating the efficacy of multiple AADs in patients with AF. AF wave-dynamic and electrophysiological characteristics are different among the PITX2 -deficient and the wild-type genotype models. [ABSTRACT FROM AUTHOR]

Purpose: 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. [ABSTRACT FROM AUTHOR]

Axenfeld‐Rieger syndrome is a genetic condition characterized by ocular and systemic features and is most commonly caused by variants in the FOXC1 or PITX2 genes. Facial dysmorphism is part of the syndrome but the differences between both genes have never been systematically assessed. Here, 11 facial traits commonly reported in Axenfeld‐Rieger syndrome were assessed by five clinical geneticists blinded to the molecular diagnosis. Individuals were drawn from the Australian and New Zealand Registry of Advanced Glaucoma in Australia or recruited through the Genetic and Ophthalmology Unit of l'Azienda Socio‐Sanitaria Territoriale Grande Ospedale Metropolitano Niguarda in Italy. Thirty‐four individuals from 18 families were included. FOXC1 variants were present in 64.7% of individuals and PITX2 variants in 35.3% of individuals. A thin upper lip (55.9%) and a prominent forehead (41.2%) were common facial features shared between both genes. Hypertelorism/telecanthus (81.8% vs 25.0%, p = 0.002) and low‐set ears (31.8% vs 0.0%, p = 0.036) were significantly more prevalent in individuals with FOXC1 variants compared with PITX2 variants. These findings may assist clinicians in reaching correct clinical and molecular diagnoses, and providing appropriate genetic counseling. [ABSTRACT FROM AUTHOR]