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Jeanfrancois Brunet - One of the best experts on this subject based on the ideXlab platform.
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A PHOX2B::FLPo transgenic mouse line suitable for intersectional genetics.
Genesis, 2013Co-Authors: Marierose Hirsch, Jeanfrancois Brunet, Fabien D'autréaux, Susan M Dymecki, Christo GoridisAbstract:PHOX2B is a transcription factor expressed in the central and peripheral neurons that control cardiovascular, respiratory, and digestive functions and essential for their development. Several populations known or suspected to regulate visceral functions express PHOX2B in the developing hindbrain. Extensive cell migration and lack of suitable markers have greatly hampered studying their development. Reasoning that intersectional fate mapping may help to overcome these impediments, we have generated a BAC transgenic mouse line, P2b::FLPo, which expresses codon-optimized FLP recombinase in PHOX2B expressing cells. By partnering the P2b::FLPo with the FLP-responsive RC::Fela allele, we show that FLP recombination switches on lineage tracers in the cells that express or have expressed PHOX2B, permanently marking them for study across development. Taking advantage of the dual-recombinase feature of RC::Fela, we further show that the P2b::FLPo transgene can be partnered with Lbx1(Cre) as Cre driver to generate triple transgenics in which neurons having a history of both PHOX2B and Lbx1 expression are specifically labeled. Hence, the P2b::FLPo line when partnered with a suitable Cre driver provides a tool for tracking and accessing genetically subsets of PHOX2B-expressing neuronal populations, which has not been possible by Cre-mediated recombination alone.
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PHOX2B expression in the taste centers of fish
The Journal of Comparative Neurology, 2012Co-Authors: Eva Coppola, Fabien Dautreaux, Marc Nomaksteinsky, Jeanfrancois BrunetAbstract:The homeodomain transcription factor PHOX2B controls the formation of the sensory-motor reflex circuits of the viscera in vertebrates. Among PHOX2B-dependent structures characterized in rodents is the nucleus of the solitary tract, the first relay for visceral sensory input, including taste. Here we show that PHOX2B is expressed throughout the primary taste centers of two cyprinid fish, Danio rerio and Carassius auratus, i.e., in their vagal, glossopharyngeal, and facial lobes, providing the first molecular evidence for their homology with the nucleus of the solitary tract of mammals and suggesting that a single ancestral PHOX2B-positive neuronal type evolved to give rise to both fish and mammalian structures. In zebrafish larvae, the distribution of PHOX2B²⁺ neurons, combined with the expression pattern of Olig4 (a homologue of Olig3, determinant of the nucleus of the solitary tract in mice), reveals that the superficial position and sheet-like architecture of the viscerosensory column in cyprinid fish, ideally suited for the somatotopic representation of oropharyngeal and bodily surfaces, arise by radial migration from a dorsal progenitor domain, in contrast to the tangential migration observed in amniotes.
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autonomic neurocristopathy associated mutations in PHOX2B dysregulate sox10 expression
Journal of Clinical Investigation, 2012Co-Authors: Mayumi Nagashimada, Jeanne Amiel, Jeanfrancois Brunet, Hiroshi Ohta, Kazuki Nakao, Toshihiro Uesaka, Delphine Trochet, Teruhiko Wakayama, Hideki EnomotoAbstract:The most common forms of neurocristopathy in the autonomic nervous system are Hirschsprung disease (HSCR), resulting in congenital loss of enteric ganglia, and neuroblastoma (NB), childhood tumors originating from the sympathetic ganglia and adrenal medulla. The risk for these diseases dramatically increases in patients with congenital central hypoventilation syndrome (CCHS) harboring a nonpolyalanine repeat expansion mutation of the Paired-like homeobox 2b (PHOX2B) gene, but the molecular mechanism of pathogenesis remains unknown. We found that introducing nonpolyalanine repeat expansion mutation of the PHOX2B into the mouse PHOX2B locus recapitulates the clinical features of the CCHS associated with HSCR and NB. In mutant embryos, enteric and sympathetic ganglion progenitors showed sustained sex-determining region Y (SRY) box10 (Sox10) expression, with impaired proliferation and biased differentiation toward the glial lineage. Nonpolyalanine repeat expansion mutation of PHOX2B reduced transactivation of wild-type PHOX2B on its known target, dopamine β-hydroxylase (DBH), in a dominant-negative fashion. Moreover, the introduced mutation converted the transcriptional effect of PHOX2B on a Sox10 enhancer from repression to transactivation. Collectively, these data reveal that nonpolyalanine repeat expansion mutation of PHOX2B is both a dominant-negative and gain-of-function mutation. Our results also demonstrate that Sox10 regulation by PHOX2B is pivotal for the development and pathogenesis of the autonomic ganglia.
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Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in PHOX2B Mutants Targeting Retrotrapezoid Nucleus Neurons
The Journal of Neuroscience, 2009Co-Authors: Veronique Dubreuil, Muriel Thoby-brisson, Gilles Fortin, Alexandre Pattyn, Murielle Rallu, Karin Persson, Jeanfrancois Brunet, Carmen Birchmeier, Christo GoridisAbstract:The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as PHOX2B-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional PHOX2B mutations, which target different subsets of PHOX2B-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described PHOX2B(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the PHOX2B-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.
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PHOX2B in respiratory control: lessons from congenital central hypoventilation syndrome and its mouse models.
Respiratory physiology & neurobiology, 2009Co-Authors: Jeanne Amiel, Nelina Ramanantsoa, Gilles Fortin, Veronique Dubreuil, Jorge Gallego, Jeanfrancois Brunet, Christo GoridisAbstract:PHOX2B is a master regulator of visceral reflex circuits. Its role in the control of respiration has been highlighted by the identification of heterozygous PHOX2B mutations as the cause of Central Congenital Hypoventilation Syndrome (CCHS), a rare disease defined by the lack of CO(2) responsiveness and of breathing automaticity in sleep. PHOX2B(27Ala/+) mice that bear a frequent CCHS-causing mutation do not respond to hypercapnia and die in the first hour after birth from central apnoea. They are therefore a reliable animal model for CCHS. Neurons of the retrotrapezoïd nucleus/parafacial respiratory group (RTN/pFRG) were found severely depleted in these mice and no other neuronal loss could be identified. Physiological experiments show that RTN/pFRG neurons are crucial to driving proper breathing at birth and are necessary for central chemoreception and the generation of a normal respiratory rhythm. To date, the reason for the selective vulnerability of RTN/pFRG neurons to PHOX2B protein dysfunction remains unexplained.
Hermann Rohrer - One of the best experts on this subject based on the ideXlab platform.
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neuroblastoma PHOX2B variants stimulate proliferation and dedifferentiation of immature sympathetic neurons
The Journal of Neuroscience, 2010Co-Authors: Tobias Reiff, Konstantina Tsarovina, Afsaneh Majdazari, Mirko Schmidt, Isabel Del Pino, Hermann RohrerAbstract:Neuroblastoma is a pediatric tumor that is thought to arise from autonomic precursors in the neural crest. Mutations in the PHOX2B gene have been observed in familial and sporadic forms of neuroblastoma and represent the first defined genetic predisposition for neuroblastoma. Here, we address the mechanisms that may underlie this predisposition, comparing the function of wild-type and mutant PHOX2B proteins ectopically expressed in proliferating, embryonic sympathetic neurons. PHOX2B displays a strong antiproliferative effect, which is lost in all PHOX2B neuroblastoma variants analyzed. In contrast, an increase in sympathetic neuron proliferation is elicited by PHOX2B variants with mutations in the homeodomain when endogenous PHOX2B levels are lowered by siRNA-mediated knockdown to mimic the situation of heterozygous PHOX2B mutations in neuroblastoma. The increased proliferation is blocked by Hand2 knockdown and the antiproliferative PHOX2B effects are rescued by Hand2 overexpression, implying Hand2 in PHOX2B-mediated proliferation control. A PHOX2B variant with a nonsense mutation in the homeodomain elicits, in addition, a decreased expression of characteristic marker genes. Together, these results suggest that PHOX2B mutations predispose to neuroblastoma by increasing proliferation and promoting dedifferentiation of cells in the sympathoadrenergic lineage.
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the bhlh transcription factor hand2 is essential for noradrenergic differentiation of sympathetic neurons
Development, 2006Co-Authors: Marsha E Lucas, F Muller, Roland Rudiger, Paul D Henion, Hermann RohrerAbstract:The basic helix-loop-helix transcription factor Hand2 , together with Ascl1, Phox2a, PHOX2B and Gata2/Gata3 , is induced by bone morphogenetic proteins in neural crest-derived precursor cells during sympathetic neuron generation. Hand2 overexpression experiments and the analysis of its function at the Dbh promotor implicated Hand2 in the control of noradrenergic gene expression. Using the zebrafish hand2 deletion mutant hands off , we have now investigated the physiological role of hand2 in the development of sympathetic ganglia. In hands off mutant embryos, sympathetic precursor cells aggregate to form normal sympathetic ganglion primordia characterized by the expression of PHOX2B, phox2a and the achaete-scute family member zash1a/ascl1 . The expression of the noradrenergic marker genes th and dbh is strongly reduced, as well as the transcription factors gata2 and tfap2a ( Ap-2 α). By contrast, generic neuronal differentiation seems to be unaffected, as the expression of elavl3 (HuC) is not reduced in hands off sympathetic ganglia. These results demonstrate in vivo an essential and selective function of hand2 for the noradrenergic differentiation of sympathetic neurons, and implicates tfap2a and gata2 as downstream effectors.
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essential role of gata transcription factors in sympathetic neuron development
Development, 2004Co-Authors: Konstantina Tsarovina, Alexandre Pattyn, Jeanfrancois Brunet, Jutta Stubbusch, F Muller, Jacqueline Van Der Wees, Christoph Schneider, Hermann RohrerAbstract:Sympathetic neurons are specified during their development from neural crest precursors by a network of crossregulatory transcription factors, which includes Mash1, PHOX2B, Hand2 and Phox2a. Here, we have studied the function of Gata2 and Gata3 zinc-finger transcription factors in autonomic neuron development. In the chick, Gata2 but not Gata3 is expressed in developing sympathetic precursor cells. Gata2 expression starts after Mash1, PHOX2B, Hand2 and Phox2a expression, but before the onset of the noradrenergic marker genes Th and Dbh, and is maintained throughout development. Gata2 expression is affected in the chick embryo by Bmp gain- and loss-of-function experiments, and by overexpression of PHOX2B, Phox2a, Hand2 and Mash1. Together with the lack of Gata2/3 expression in PHOX2B knockout mice, these results characterize Gata2 as member of the Bmp-induced cluster of transcription factors. Loss-of-function experiments resulted in a strong reduction in the size of the sympathetic chain and in decreased Th expression. Ectopic expression of Gata2 in chick neural crest precursors elicited the generation of neurons with a non-autonomic, Th-negative phenotype. This implies a function for Gata factors in autonomic neuron differentiation, which, however, depends on co-regulators present in the sympathetic lineage. The present data establish Gata2 and Gata3 in the chick and mouse, respectively, as essential members of the transcription factor network controlling sympathetic neuron development.
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Interaction of Mash1 and PHOX2B in sympathetic neuron development.
Molecular and cellular neurosciences, 2004Co-Authors: Matthias Stanke, Jutta Stubbusch, Hermann RohrerAbstract:The transcription factors Mash1 and PHOX2B are both essential for sympathetic neuron development. To understand in more detail their function and interaction, PHOX2B and Mash1 were ectopically expressed in vivo, in peripheral nerve precursors. Here, we demonstrate that the PHOX2B-induced generation of ectopic noradrenergic neurons in chick peripheral nerve involves the induction of Cash1, the chick homolog of Mash1. All Phox2-induced neurons coexpress the noradrenergic marker genes TH and DBH. Conversely, Mash1 induces neuronal differentiation characterized by the expression of generic neuronal genes SCG10, Hu and NF160; however, only a subpopulation of these neurons also displays an autonomic, noradrenergic phenotype. This context-dependent action of Mash1 implicates autonomic codeterminants, required for noradrenergic differentiation in response to Mash1. In contrast, PHOX2B coordinates generic and noradrenergic gene expression, recruiting Mash1/Cash1, which may have a major function in the control of pan-neuronal gene expression during noradrenergic neuron development.
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bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons
Neuron, 1999Co-Authors: Carolin Schneider, Helmut Wicht, Jana Enderich, Michael Wegner, Hermann RohrerAbstract:Abstract Bone morphogenetic proteins (BMPs) induce autonomic neurogenesis in neural crest cultures and stimulate sympathetic neuron development when overexpressed in vivo. We demonstrate that inhibition of BMPs in the chick embryo by the BMP antagonist Noggin prevents sympathetic neuron generation. In Noggin-treated embryos, the noradrenergic marker genes tyrosine hydroxylase ( TH ) and dopamine-β-hydroxylase ( DBH ), panneuronal neurofilament 160 ( NF160 ) and SCG10 genes, and the transcriptional regulators PHOX2B and Phox2a are not expressed in sympathetic ganglia. Whereas initial ganglion development is not affected, the expression of the basic helix–loop–helix transcription factor Cash-1 is strongly reduced. These results demonstrate that BMPs are essential for sympathetic neuron development and establish Cash-1 and Phox2 genes as downstream effectors of BMPs in this lineage.
Debra E Weesemayer - One of the best experts on this subject based on the ideXlab platform.
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congenital central hypoventilation syndrome severe disease caused by co occurrence of two PHOX2B variants inherited separately from asymptomatic family members
American Journal of Medical Genetics Part A, 2019Co-Authors: Yakov Sivan, Casey M Rand, Lawrence J Jennings, Lili Zhou, Elizabeth Berrykravis, Debra E Weesemayer, Amy ZhouAbstract:Congenital Central Hypoventilation Syndrome (CCHS) is a rare disease characterized by autonomic nervous system dysregulation. Central hypoventilation is the most prominent and clinically important presentation. CCHS is caused by mutations in paired-like homeobox 2b (PHOX2B) and is inherited in an autosomal dominant pattern. A co-occurrence of two asymptomatic PHOX2B variants with a classical CCHS presentation highlights the importance of clinical PHOX2B testing in parents and family members of all CCHS probands. Despite being an autosomal dominant disease, once a polyalanine repeat expansion mutation has been identified, sequencing of the other allele should also be considered.
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germline mosaicism of PHOX2B mutation accounts for familial recurrence of congenital central hypoventilation syndrome cchs
American Journal of Medical Genetics Part A, 2012Co-Authors: Casey M Rand, Lawrence J Jennings, Kelvin Panesar, Lili Zhou, Elizabeth Berrykravis, Debra E WeesemayerAbstract:Congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by alveolar hypoventilation and autonomic dysregulation, is caused by mutations in the PHOX2B gene. Most mutations occur de novo, but recent evidence suggests that up to 25% are inherited from asymptomatic parents with somatic mosaicism for these mutations. However, to date, germline mosaicism has not been reported. This report describes a family with recurrence of PHOX2B mutation-confirmed CCHS due to germline mosaicism. The first occurrence was a baby girl, noted on day 2 of life to have multiple episodes of apnea, bradycardia, and cyanosis while breathing room air. PHOX2B gene testing confirmed the diagnosis of CCHS with a heterozygous polyalanine repeat expansion mutation (PARM); genotype 20/27 (normal 20/20). Both parents tested negative for this mutation using fragment analysis (limit of detection <1%). Upon subsequent pregnancy [paternity confirmed using short tandem repeat (STR) analysis], amniocentesis testing identified the PHOX2B 20/27 genotype, confirmed with repeat testing. Elective abortion was performed at 21.5 weeks gestation. Testing of abortus tissue confirmed amniocentesis testing. The PHOX2B 20/27 expansion was not observed in a paternal sperm sample. This case represents the first reported family with recurrence of PHOX2B mutation-confirmed CCHS without detection of a parental carrier state or mosaicism, confirming the previously hypothesized possibility of germline mosaicism for PHOX2B mutations. This is an important finding for genetic counseling of CCHS families, suggesting that even if somatic mosaicism is not detected in parental samples, there is still reason for careful genetic counseling and consideration of prenatal testing during subsequent pregnancies. © 2012 Wiley Periodicals, Inc.
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variable human phenotype associated with novel deletions of the PHOX2B gene
Pediatric Pulmonology, 2012Co-Authors: Casey M Rand, Lawrence J Jennings, Elizabeth Berrykravis, Debra E Weesemayer, Nicole S Kravis, Pallavi P PatwariAbstract:Background Clinical testing for PHOX2B mutations is widely used for patients with any symptoms suggestive of hypoventilation (with/without anatomic/physiologic autonomic dysregulation), though not necessarily with the congenital central hypoventilation syndrome (CCHS) phenotype. Consequently, a multitude of referrals for clinical PHOX2B testing (fragment analysis of the 20 polyalanine repeat region and/or sequencing of entire coding region) have no identifiable mutation. Whole gene deletions/duplications have recently been identified as a common disease-causing mechanism, but have not been reported in a clinical population referred for PHOX2B testing. The objective of this study was to determine if PHOX2B exon or whole gene deletion/duplication would be identified in a subset of patients referred for PHOX2B testing. The objective of this study was to determine if PHOX2B exon or whole gene deletion/duplication would be identified in a subset of patients referred for PHOX2B testing. Hypothesis: We hypothesized that PHOX2B exon or whole gene deletion or duplication would be identified in a subset of cases who were referred for genetic testing but not found to have a PHOX2B mutation with currently available clinical PHOX2B testing. Methods: Genomic DNA samples from patients that tested negative for PHOX2B mutations using fragment analysis and/or sequencing, and control samples, were screened for PHOX2B exon deletions/duplications by multiplex ligation-dependent probe amplification with confirmation by array comparative genomic hybridization. Results: Deletions of/in PHOX2B were identified in 4/250 patients and 0/261 controls. The deletions ranged from 6,216 base pairs (involving only PHOX2B exon 3) to 2.6 megabases (involving all of PHOX2B and 12 other genes). The case with PHOX2B partial exon 3 deletion had a CCHS-compatible phenotype (hypoventilation, Hirschsprung disease). Phenotypes for the other three cases, all PHOX2B whole-gene deletions, were varied including: (1) apparent life threatening event, (2) full CCHS necessitating artificial ventilation with ganglioneuroblastoma, and (3) hypoventilation during sleep. Family studies of two of the four probands showed these deletions to be maternally inherited; the mothers also had phenotypic findings of autonomic dysfunction. Conclusions: PHOX2B exon or whole gene deletion should be considered as another mechanism of disease which may include CCHS, Hirschsprung disease, and/or tumors of neural crest origin, although the genotype-phenotype relationship requires further clarification. Pediatr Pulmonol. 2012; 47: 153-161. (C) 2011 Wiley Periodicals, Inc.
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congenital central hypoventilation syndrome from past to future model for translational and transitional autonomic medicine
Pediatric Pulmonology, 2009Co-Authors: Debra E Weesemayer, Casey M Rand, Lawrence J Jennings, Elizabeth Berrykravis, Pallavi P Patwari, Darius A Loghmanee, Isabella CeccheriniAbstract:The modern story of CCHS began in 1970 with the first description by Mellins et al., came most visibly to the public eye with the ATS Statement in 1999, and continues with increasingly fast paced advances in genetics. Affected individuals have diffuse autonomic nervous system dysregulation (ANSD). The paired-like homeobox gene PHOX2B is the disease-defining gene for CCHS; a mutation in the PHOX2B gene is requisite to the diagnosis of CCHS. Approximately 90% of individuals with the CCHS phenotype will be heterozygous for a polyalanine repeat expansion mutation (PARM); the normal allele will have 20 alanines and the affected allele will have 24-33 alanines (genotypes 20/24-20/33). The remaining approximately 10% of individuals with CCHS will have a non-PARM (NPARM), in the PHOX2B gene; these will be missense, nonsense, or frameshift. CCHS and PHOX2B are inherited in an autosomal dominant manner with a stable mutation. Approximately 8% of parents of a CCHS proband will be mosaic for the PHOX2B mutation. A growing number of cases of CCHS are identified after the newborn period, with presentation from infancy into adulthood. An improved understanding of the molecular basis of the PHOX2B mutations and of the PHOX2B genotype/CCHS phenotype relationship will allow physicians to anticipate the clinical phenotype for each affected individual. To best convey the remarkable history of CCHS, and to describe the value of recognizing CCHS as a model for translational and transitional autonomic medicine, we present this review article in the format of a chronological story, from 1970 to the present day.
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characterization of dermatoglyphics in PHOX2B confirmed congenital central hypoventilation syndrome
Pediatrics, 2006Co-Authors: Emily S Todd, Elizabeth Berrykravis, Debra E Weesemayer, Nicole M Scott, Seth M Weinberg, Jean M Silvestri, Anna S Kenny, Susan A Hauptman, Lili ZhouAbstract:OBJECTIVE. Individuals with congenital central hypoventilation syndrome have characteristic variants in the PHOX2B gene (primarily polyalanine expansion mutations). The PHOX2B gene acts as a transcriptional activator in the promotion of pan-neuronal differentiation in the autonomic nervous system during early embryologic development, with a primary role in the sympathetic noradrenergic phenotype in vertebrates. Because sympathetic innervation has been hypothesized to affect the development of dermatoglyphic pattern types, we hypothesized that individuals with PHOX2B-confirmed congenital central hypoventilation syndrome would have characteristic dermatoglyphic patterning and that the dermatoglyphic phenotype would be related to the disease-defining PHOX2B genotype. METHODS. Dermatoglyphic pattern type frequency, left/right symmetry, and genotype/phenotype correlation were assessed for 33 individuals with PHOX2B-confirmed congenital central hypoventilation syndrome and compared with published control data. RESULTS. Dermatoglyphic pattern type frequencies were altered in congenital central hypoventilation syndrome cases versus controls. In particular, there was an increase of arches in females and ulnar loops in males, with the largest differences for the left hand and for individuals with both congenital central hypoventilation syndrome and Hirschsprung disease. Dissimilarity scores between the congenital central hypoventilation syndrome and congenital central hypoventilation syndrome + Hirschsprung disease cases were not significantly different, nor were dissimilarity scores between all of the female and all of the male cases. No significant association was found between the number of polyalanine repeats in the PHOX2B genotypic category and dermatoglyphic pattern frequencies in the congenital central hypoventilation syndrome study groups. CONCLUSIONS. These results represent the first report describing specific dermatoglyphic patterning in congenital central hypoventilation syndrome and suggest a relationship between PHOX2B and the expression of dermatoglyphic pattern types. An expanded congenital central hypoventilation syndrome data set to include the full spectrum of PHOX2B mutations is necessary to further delineate the role of PHOX2B in dermatoglyphic patterning.
Isabella Ceccherini - One of the best experts on this subject based on the ideXlab platform.
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causative and common PHOX2B variants define a broad phenotypic spectrum
Clinical Genetics, 2020Co-Authors: T. Bachetti, Isabella CeccheriniAbstract:Paired Like homeobox 2B (PHOX2B) is a gene crucial for the differentiation of the neural lineages of the autonomic nervous system (ANS), whose coding mutations cause congenital central hypoventilation syndrome (CCHS). The vast majority of PHOX2B mutations in CCHS is represented by expansions of a polyalanine region in exon 3, collectively defined PARMs (PolyAlanine Repeat Mutations), the minority being frameshift, missense and nonsense mutations, defined as NPARMs (Non-PARMs). While PARMs are nearly exclusively associated with isolated CCHS, most of NPARMs is detected in syndromic CCHS, presenting with neuroblastoma and/or Hirschsprung disease. More recently, evidence of a complex role of PHOX2B in the pathogenesis of a wider spectrum of ANS disorders has emerged. Indeed, common and hypomorphic PHOX2B variants, including synonymous, polyalanine-contractions, gene deletions may influence the occurrence of either apparent life-threatening event (ALTE), Sudden Infant Death Syndrome (SIDS), neuroblastoma, or isolated HSCR, likely through small effects on PHOX2B expression levels. After an introduction to the role of PHOX2B in the ANS development, causative mutations, common variants, and gene expression deregulation of the PHOX2B gene are discussed, though the involvement of synonymous variants and contractions requires further confirmations with respect to ANS disorders and molecular mechanisms underlying the PHOX2B phenotypic heterogeneity.
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desogestrel down regulates PHOX2B and its target genes in progesterone responsive neuroblastoma cells
Experimental Cell Research, 2018Co-Authors: Silvia Cardani, Simona Di Lascio, Debora Belperio, Roberta Benfante, Isabella Ceccherini, Erika Di Biase, Diego FornasariAbstract:The paired-like homeobox 2B gene (PHOX2B) encodes a key transcription factor that plays a role in the development of the autonomic nervous system and the neural structures involved in controlling breathing. In humans, PHOX2B over-expression plays a role in the pathogenesis of tumours arising from the sympathetic nervous system such as neuroblastomas, and heterozygous PHOX2B mutations cause Congenital Central Hypoventilation Syndrome (CCHS), a life-threatening neurocristopathy characterised by the defective autonomic control of breathing and involving altered CO2/H+ chemosensitivity. The recovery of CO2/H+ chemosensitivity and increased ventilation have been observed in two CCHS patients using the potent contraceptive progestin desogestrel. Given the central role of PHOX2B in the pathogenesis of CCHS, and the progesterone-mediated effects observed in the disease, we generated progesterone-responsive neuroblastoma cells, and evaluated the effects of 3-Ketodesogestrel (3-KDG), the biologically active metabolite of desogestrel, on the expression of PHOX2B and its target genes. Our findings demonstrate that, through progesterone nuclear receptor PR-B, 3-KDG down-regulates PHOX2B gene expression, by a post-transcriptional mechanism, and its target genes and open up the possibility that this mechanism may contribute to the positive effects observed in some CCHS patients.
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transcriptional dysregulation and impairment of PHOX2B auto regulatory mechanism induced by polyalanine expansion mutations associated with congenital central hypoventilation syndrome
Neurobiology of Disease, 2013Co-Authors: Simona Di Lascio, T. Bachetti, Elena Saba, Roberta Benfante, Isabella Ceccherini, Diego FornasariAbstract:The PHOX2B transcription factor plays a crucial role in autonomic nervous system development. In humans, heterozygous mutations of the PHOX2B gene lead to congenital central hypoventilation syndrome (CCHS), a rare disorder characterized by a broad variety of symptoms of autonomic nervous system dysfunction including inadequate control of breathing. The vast majority of patients with CCHS are heterozygous for a polyalanine repeat expansion mutation involving a polyalanine tract of twenty residues in the C-terminus of PHOX2B. Although several lines of evidence support a dominant-negative mechanism for PHOX2B mutations in CCHS, the molecular effects of PHOX2B mutant proteins on the transcriptional activity of the wild-type protein have not yet been elucidated. As one of the targets of PHOX2B is the PHOX2B gene itself, we tested the transcriptional activity of wild-type and mutant proteins on the PHOX2B gene promoter, and found that the transactivation ability of proteins with polyalanine expansions decreased as a function of the length of the expansion, whereas DNA binding was severely affected only in the case of the mutant with the longest polyalanine tract (+13 alanine). Co-transfection experiments using equimolar amounts of PHOX2B wild-type and mutant proteins in order to simulate a heterozygous state in vitro and four different PHOX2B target gene regulatory regions (PHOX2B, PHOX2A, DBH, TLX2) clearly showed that the polyalanine expanded proteins alter the transcriptional activity of wild-type protein in a promoter-specific manner, without any clear correlation with the length of the expansion. Moreover, although reduced transactivation may be caused by retention of the wild-type protein in the cytoplasm or in nuclear aggregates, this mechanism can only be partially responsible for the pathogenesis of CCHS because of the reduction in cytoplasmic and nuclear accumulation when the +13 alanine mutant is co-expressed with wild-type protein, and the fact that the shortest polyalanine expansions do not form visible cytoplasmic aggregates. Deletion of the C-terminal of PHOX2B leads to a protein that correctly localizes in the nucleus but impairs PHOX2B wild-type transcriptional activity, thus suggesting that protein mislocalization is not the only mechanism leading to CCHS. The results of this study provide novel in vitro experimental evidence of a transcriptional dominant-negative effect of PHOX2B polyalanine mutant proteins on wild-type protein on two different PHOX2B target genes.
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congenital central hypoventilation syndrome genotype phenotype correlation in parents of affected children carrying a PHOX2B expansion mutation
Clinical Genetics, 2010Co-Authors: Stefano Parodi, Isabella Ceccherini, C Vollono, Maria Pia Baglietto, Martina Balestri, M Di Duca, P A Landri, Giancarlo Ottonello, Maria Roberta CilioAbstract:Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder. Although most CCHS associated PHOX2B mutations occur de novo, about 10% of the cases are inherited from apparently asymptomatic parents, thus confirming variable expressivity and incomplete penetrance of PHOX2B mutations. Three asymptomatic parents of children affected with CCHS, and found to carry the same PHOX2B expansion mutations as their siblings, were studied by overnight polysomnography and somatic mosaicism analysis. In one case, significant sleep breathing control anomalies were detected, while the other two resulted in normal. In tissue-specific allele studies, mosaicism with a comparatively low mutant allele proportion was showed in the two unaffected adult carriers. Accurate polysomnography and assessment of the degree of somatic mosaicism should be conducted in asymptomatic carriers of PHOX2B mutations, as they may unmask subclinical but significant anomalies.
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congenital central hypoventilation syndrome from past to future model for translational and transitional autonomic medicine
Pediatric Pulmonology, 2009Co-Authors: Debra E Weesemayer, Casey M Rand, Lawrence J Jennings, Elizabeth Berrykravis, Pallavi P Patwari, Darius A Loghmanee, Isabella CeccheriniAbstract:The modern story of CCHS began in 1970 with the first description by Mellins et al., came most visibly to the public eye with the ATS Statement in 1999, and continues with increasingly fast paced advances in genetics. Affected individuals have diffuse autonomic nervous system dysregulation (ANSD). The paired-like homeobox gene PHOX2B is the disease-defining gene for CCHS; a mutation in the PHOX2B gene is requisite to the diagnosis of CCHS. Approximately 90% of individuals with the CCHS phenotype will be heterozygous for a polyalanine repeat expansion mutation (PARM); the normal allele will have 20 alanines and the affected allele will have 24-33 alanines (genotypes 20/24-20/33). The remaining approximately 10% of individuals with CCHS will have a non-PARM (NPARM), in the PHOX2B gene; these will be missense, nonsense, or frameshift. CCHS and PHOX2B are inherited in an autosomal dominant manner with a stable mutation. Approximately 8% of parents of a CCHS proband will be mosaic for the PHOX2B mutation. A growing number of cases of CCHS are identified after the newborn period, with presentation from infancy into adulthood. An improved understanding of the molecular basis of the PHOX2B mutations and of the PHOX2B genotype/CCHS phenotype relationship will allow physicians to anticipate the clinical phenotype for each affected individual. To best convey the remarkable history of CCHS, and to describe the value of recognizing CCHS as a model for translational and transitional autonomic medicine, we present this review article in the format of a chronological story, from 1970 to the present day.
Christo Goridis - One of the best experts on this subject based on the ideXlab platform.
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A PHOX2B::FLPo transgenic mouse line suitable for intersectional genetics.
Genesis, 2013Co-Authors: Marierose Hirsch, Jeanfrancois Brunet, Fabien D'autréaux, Susan M Dymecki, Christo GoridisAbstract:PHOX2B is a transcription factor expressed in the central and peripheral neurons that control cardiovascular, respiratory, and digestive functions and essential for their development. Several populations known or suspected to regulate visceral functions express PHOX2B in the developing hindbrain. Extensive cell migration and lack of suitable markers have greatly hampered studying their development. Reasoning that intersectional fate mapping may help to overcome these impediments, we have generated a BAC transgenic mouse line, P2b::FLPo, which expresses codon-optimized FLP recombinase in PHOX2B expressing cells. By partnering the P2b::FLPo with the FLP-responsive RC::Fela allele, we show that FLP recombination switches on lineage tracers in the cells that express or have expressed PHOX2B, permanently marking them for study across development. Taking advantage of the dual-recombinase feature of RC::Fela, we further show that the P2b::FLPo transgene can be partnered with Lbx1(Cre) as Cre driver to generate triple transgenics in which neurons having a history of both PHOX2B and Lbx1 expression are specifically labeled. Hence, the P2b::FLPo line when partnered with a suitable Cre driver provides a tool for tracking and accessing genetically subsets of PHOX2B-expressing neuronal populations, which has not been possible by Cre-mediated recombination alone.
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Defective Respiratory Rhythmogenesis and Loss of Central Chemosensitivity in PHOX2B Mutants Targeting Retrotrapezoid Nucleus Neurons
The Journal of Neuroscience, 2009Co-Authors: Veronique Dubreuil, Muriel Thoby-brisson, Gilles Fortin, Alexandre Pattyn, Murielle Rallu, Karin Persson, Jeanfrancois Brunet, Carmen Birchmeier, Christo GoridisAbstract:The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as PHOX2B-, Vglut2-, neurokinin1 receptor-, and Atoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional PHOX2B mutations, which target different subsets of PHOX2B-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously described PHOX2B(27Ala) mutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the PHOX2B-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.
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PHOX2B in respiratory control: lessons from congenital central hypoventilation syndrome and its mouse models.
Respiratory physiology & neurobiology, 2009Co-Authors: Jeanne Amiel, Nelina Ramanantsoa, Gilles Fortin, Veronique Dubreuil, Jorge Gallego, Jeanfrancois Brunet, Christo GoridisAbstract:PHOX2B is a master regulator of visceral reflex circuits. Its role in the control of respiration has been highlighted by the identification of heterozygous PHOX2B mutations as the cause of Central Congenital Hypoventilation Syndrome (CCHS), a rare disease defined by the lack of CO(2) responsiveness and of breathing automaticity in sleep. PHOX2B(27Ala/+) mice that bear a frequent CCHS-causing mutation do not respond to hypercapnia and die in the first hour after birth from central apnoea. They are therefore a reliable animal model for CCHS. Neurons of the retrotrapezoïd nucleus/parafacial respiratory group (RTN/pFRG) were found severely depleted in these mice and no other neuronal loss could be identified. Physiological experiments show that RTN/pFRG neurons are crucial to driving proper breathing at birth and are necessary for central chemoreception and the generation of a normal respiratory rhythm. To date, the reason for the selective vulnerability of RTN/pFRG neurons to PHOX2B protein dysfunction remains unexplained.
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forced expression of phox2 homeodomain transcription factors induces a branchio visceromotor axonal phenotype
Developmental Biology, 2007Co-Authors: Marierose Hirsch, Jeanfrancois Brunet, Joel C Glover, Heloise D Dufour, Christo GoridisAbstract:What causes motor neurons to project into the periphery is not well understood. We here show that forced expression of the homeodomain protein PHOX2B, shown previously to be necessary and sufficient for branchio-visceromotor neuron development, and of its paralogue Phox2a imposes a branchiomotor-like axonal phenotype in the spinal cord. Many Phox2-transfected neurons, whose axons would normally stay within the confines of the neural tube, now project into the periphery. Once outside the neural tube, a fraction of the ectopic axons join the spinal accessory nerve, a branchiomotor nerve which, as shown here, does not develop in the absence of PHOX2B. Explant studies show that the axons of Phox2-transfected neurons need attractive cues to leave the neural tube and that their outgrowth is promoted by tissues, to which branchio-visceromotor fibers normally grow. Hence, Phox2 expression is a key step in determining the peripheral axonal phenotype and thus the decision to stay within the neural tube or to project out of it.
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the role of PHOX2B in chromaffin cell development
Developmental Biology, 2005Co-Authors: Katrin Huber, Uwe Ernsberger, Nicole Karch, Christo Goridis, Klaus UnsickerAbstract:Abstract PHOX2B, a homeodomain transcription factor closely related to Phox2A, is expressed in peripheral and central noradrenergic neurons. In neural crest (NC) derivatives PHOX2B is restricted to sympathetic and parasympathetic ganglia, enteric neurons, and adrenal and extraadrenal chromaffin cells. Similar to MASH-1, PHOX2B has been implicated in synchronizing pan-neuronal and catecholaminergic phenotype-specific aspects of neurogenesis. The role of PHOX2B for the differentiation of the neuroendocrine NC derivatives, the adrenal medullary chromaffin cells, has not been explored. We have previously reported that in MASH-1-deficient mice most chromaffin cells are arrested at the early neuroblast stage and lack catecholaminergic differentiation. We show now that in PHOX2B knockout/lacZ knockin mice the maturation of presumptive chromaffin cells is arrested at an even earlier stage of development. The cells lack the catecholaminergic marker enzyme TH and fail to form a centrally located medulla. In contrast to MASH-1 (−/−) mice they do not express dHand, Phox2A, c-ret, neurofilament, neuron-specific tubulin, and NCAM and appear ultrastructurally more immature. Many of these cells die by apoptosis. Despite the complete lack of differentiation, few lacZ-positive adrenal cells can still be found at E16.5. We conclude that PHOX2B regulates very early events in the differentiation of adrenal chromaffin cells distinct to steps, which essentially require MASH-1.
