PHOX2B Research Articles

Common PHOX2B poly-alanine contractions impair RET gene transcription, predisposing to Hirschsprung disease

HSCR is a congenital disorder of the enteric nervous system, characterized by the absence of neurons along a variable length of the gut resulting from loss-of-function RET mutations. Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy characterized by impaired response to hypercapnia and hypoxemia caused by heterozygous mutations of the PHOX2B gene, mostly polyalanine (polyA) expansions but also missense, nonsense, and frameshift mutations, while polyA contractions are common in the population and believed neutral. HSCR associated CCHS can present in patients carrying PHOX2B mutations. Indeed, RET expression is orchestrated by different transcriptional factors among which PHOX2B, thus suggesting its possible role in HSCR pathogenesis. Following the observation of HSCR patients carrying in frame trinucleotide deletions within the polyalanine stretch in exon 3 (polyA contractions), we have verified the hypothesis that these PHOX2B variants do reduce its transcriptional activity, likely resulting in a down-regulation of RET expression and, consequently, favouring the development of the HSCR phenotype. Using proper reporter constructs, we show here that the in vitro transactivation of the RET promoter by different HSCR-associated PHOX2B polyA variants has resulted significantly lower compared to the effect of PHOX2B wild type protein. In particular, polyA contractions do induce a reduced transactivation of the RET promoter, milder compared to the severe polyA expansions associated with CCHS+HSCR, and correlated with the length of the deleted trait, with a more pronounced effect when contractions are larger.

Neuroanatomical Analysis of a Conditional Knockin Mutant PHOX2B Mouse Model

BackgroundCentral Congenital Hypoventilation Syndrome (CCHS) is a rare disorder characterized by autonomic dysfunction, including abnormal respiration. Symptoms of CCHS include the inability to establish a regular respiratory rhythm, inadequate responses to hypoxemia and hypercapnia, heart rate abnormalities, and impaired bowel function. Mutations in paired‐like homeobox 2b (PHOX2B) are known to be causative for CCHS. We previously demonstrated that mice harboring an eight‐nucleotide deletion in PHOX2B (d8‐PHOX2B) displayed diffuse hindbrain pathology and perinatal lethality due to lack of spontaneous respiration. In this study, we are further exploring which brainstem circuits are required at birth for proper respiratory function and whether specific circuits are lost or insufficiently matured in mice expressing d8‐PHOX2B.MethodsWe are crossing several Cre driver mice with d8‐PHOX2B mice to selectively express d8‐PHOX2B in hindbrain neural nuclei known to be affected by mutant PHOX2B expression. Histological examination of brainstems from these mice along with unbiased quantification will allow us to determine how the morphology and number of cells within these neural populations is altered.Preliminary resultsAtoh1‐cre; d8‐PHOX2B mice were generated and assessed for perinatal viability, as Atoh1 is a transcription factor known to be involved in the development of medullary respiratory centers. At birth Atoh1‐cre; d8‐PHOX2B pups appeared healthy and displayed spontaneous respiration. Histological examination of hindbrains from these pups revealed no neuropathology, with evident intact locus coeruleus, facial nucleus, and retrotrapezoid nucleus. These preliminary results suggest that the expression of Phox2b in the Atoh1 lineage is not required for normal hindbrain development and respiration at birth.Support or Funding Information1R01HL132355‐01A1

Normal sleep on mechanical ventilation in adult patients with congenital central alveolar hypoventilation (Ondine\u2019s curse syndrome)

BackgroundThe purpose of this study was to describe the sleep structure (especially slow wave sleep) in adults with congenital central hypoventilation syndrome (CCHS), a rare genetic disease due to mutations in the PHOX2B gene. Fourteen patients aged 23 (19.0; 24.8) years old (median [1rst-3rd quartiles]) with CCHS underwent a sleep interview and night-time attended polysomnography with their ventilatory support. Their sleep variables were compared to those collected in 15 healthy control subjects matched for age, sex and body mass index.ResultsThe latency to N3 sleep was shorter in patients (26.3 min [24.0; 30.1]) than in controls (49.5 min [34.3; 66.9]; P = 0.005), and sleep onset latency tended to be shorter in patients (14.0 min [7.0; 20.5]) than in controls (33.0 min [18.0; 49.0]; P = 0.052). Total sleep time, sleep stage percentages, sleep fragmentation as well as respiratory and movement index were within normal ranges and not different between groups.ConclusionsNormal sleep in adult patients with CCHS and adequate ventilator support indicates that the PHOX2 gene mutations do not affect brain sleep networks. Consequently, any complaint of disrupted sleep should prompt clinicians to look for the usual causes of sleep disorders, primarily inadequate mechanical ventilation. Shorter N3 latency may indicate a higher need for slow wave sleep, to compensate for the abnormal respiratory-related cortical activity during awake quiet breathing observed in patients with CCH.

Anesthetic Considerations for Patients With Congenital Central Hypoventilation Syndrome: A Systematic Review of the Literature.

Congenital central hypoventilation syndrome (CCHS) is a form of sleep-disordered breathing characterized by a diminished drive to breathe during sleep, despite progressive hypercapnia and hypoxia. The condition results from mutations in the paired-like homeobox 2B (PHOX2B) gene. The aim of this review was to conduct a systematic search of the current data on CCHS as it relates to perioperative considerations and to discuss the classification, prevalence, pathophysiology, presentation, genetics, and management of the condition. A systematic search of Medline, EMBASE, Cochrane Database of Systematic Reviews, and the Cochrane Central Register of Controlled Trials was done up to October 2015. The results were limited to human studies published in the English language. Study titles and abstracts were screened to identify studies relating to CCHS relevant to anesthetic care. All study designs including randomized controlled trials, observational studies, case reports, or case series were included. The searches yielded 165 articles, of which 45 were relevant to perioperative considerations. There were 15 relevant case reports categorized as pertaining to the following: (1) novel presentations of the condition after sedation/anesthesia; (2) anesthetic techniques used in patients with established CCHS; and (3) patients with CCHS who experienced anesthetic complications. Review of the case reports showed that patients ranged from neonates up to 59 years of age. Novel presentations of the disease after sedation or anesthesia for minor procedures often led to diagnosis. The sequelae of undiagnosed CCHS led to complications, such as hypoxia, desaturations, apneas, seizures, unplanned intensive care admissions, prolonged hospital stays, and long-term tracheostomies. There appeared to be few postoperative complications in patients with known CCHS. Anesthesiologists need to be aware of undiagnosed late-onset CCHS and include this condition in the differential diagnosis of patients with unexplained postoperative respiratory depression. Anesthetic techniques should minimize the use of agents that further depress respiration postprocedure and ensure adequate monitoring to detect postoperative apneas.

Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system.

The enteric nervous system (ENS) of the gastrointestinal tract controls many diverse functions, including motility and epithelial permeability. Perturbations in ENS development or function are common, yet there is no human model for studying ENS-intestinal biology and disease. We used a tissue-engineering approach with embryonic and induced pluripotent stem cells (PSCs) to generate human intestinal tissue containing a functional ENS. We recapitulated normal intestinal ENS development by combining human-PSC-derived neural crest cells (NCCs) and developing human intestinal organoids (HIOs). NCCs recombined with HIOs in vitro migrated into the mesenchyme, differentiated into neurons and glial cells and showed neuronal activity, as measured by rhythmic waves of calcium transients. ENS-containing HIOs grown in vivo formed neuroglial structures similar to a myenteric and submucosal plexus, had functional interstitial cells of Cajal and had an electromechanical coupling that regulated waves of propagating contraction. Finally, we used this system to investigate the cellular and molecular basis for Hirschsprung's disease caused by a mutation in the gene PHOX2B. This is, to the best of our knowledge, the first demonstration of human-PSC-derived intestinal tissue with a functional ENS and how this system can be used to study motility disorders of the human gastrointestinal tract.

Highly sensitive assessment of neuroblastoma minimal residual disease in ovarian tissue using RT-qPCR-A strategy for improving the safety of fertility restoration.

Ovarian tissue cryopreservation (OTC) is the only option available to preserve fertility in prepubertal females with neuroblastoma (NB), a childhood solid tumor that can spread to the ovaries, with a risk of reintroducing malignant cells after an ovarian graft. We set out to determine whether the analysis of TH (tyrosine hydroxylase), PHOX2B (paired-like homeobox 2b), and DCX (doublecortin) transcripts using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) could be used to detect NB contamination in ovarian tissue. Analyses were performed on benign ovarian tissue from 20 healthy women between November 2014 and September 2015 at the University Hospital of Clermont-Ferrand. Pericystic benign ovarian tissues were collected and contaminated with increasing numbers of human NB cells (cell lines IMR-32 and SK-N-SH) before detection using RT-qPCR. TH and DCX transcripts were detected in uncontaminated ovarian tissue from all the donors, hampering the detection of small numbers of tumor cells. By contrast, PHOX2B was not detected in any uncontaminated ovarian fragment. PHOX2B levels were significantly increased from 10 NB cells. Our study is the first to evaluate minimal residual disease detection using NB mRNAs in human ovarian tissue. Only PHOX2B was a reliable marker of NB cells contaminating ovarian tissue. These results are encouraging and offer hope in the near future for grafting ovarian tissue in women who survive cancer, whose fertility has been jeopardized by treatment, and who could benefit from OTC without oncological risk.

Congenital central hypoventilation syndrome: a bedside-to-bench success story for advancing early diagnosis and treatment and improved survival and quality of life.

The "bedside-to-bench" Congenital Central Hypoventilation Syndrome (CCHS) research journey has led to increased phenotypic-genotypic knowledge regarding autonomic nervous system (ANS) regulation, and improved clinical outcomes. CCHS is a neurocristopathy characterized by hypoventilation and ANS dysregulation. Initially described in 1970, timely diagnosis and treatment remained problematic until the first large cohort report (1992), delineating clinical presentation and treatment options. A central role of ANS dysregulation (2001) emerged, precipitating evaluation of genes critical to ANS development, and subsequent 2003 identification of Paired-Like Homeobox 2B (PHOX2B) as the disease-defining gene for CCHS. This breakthrough engendered clinical genetic testing, making diagnosis exact and early tracheostomy/artificial ventilation feasible. PHOX2B genotype-CCHS phenotype relationships were elucidated, informing early recognition and timely treatment for phenotypic manifestations including Hirschsprung disease, prolonged sinus pauses, and neural crest tumors. Simultaneously, cellular models of CCHS-causing PHOX2B mutations were developed to delineate molecular mechanisms. In addition to new insights regarding genetics and neurobiology of autonomic control overall, new knowledge gained has enabled physicians to anticipate and delineate the full clinical CCHS phenotype and initiate timely effective management. In summary, from an initial guarantee of early mortality or severe neurologic morbidity in survivors, CCHS children can now be diagnosed early and managed effectively, achieving dramatically improved quality of life as adults.

Congenital central hypoventilation syndrome: diagnostic and management challenges.

Congenital central hypoventilation syndrome (CCHS) is a rare genetic disorder with failure of central control of breathing and of the autonomic nervous system function due to a mutation in the paired-like homeobox 2B (PHOX2B) gene. Affected patients have absent or negligible ventilatory sensitivity to hypercapnia and hypoxemia, and they do not exhibit signs of respiratory distress when challenged with hypercarbia or hypoxia. The diagnosis of CCHS must be confirmed with PHOX2B gene mutation. Generally, the PHOX2B mutation genotype can aid in anticipating the severity of the phenotype. They require ventilatory support for life. Home assisted ventilation options include positive pressure ventilation via tracheostomy, noninvasive positive pressure ventilation, and diaphragm pacing via phrenic nerve stimulation, but each strategy has its associated limitations and challenges. Since all the clinical manifestations of CCHS may not manifest at birth, periodic monitoring and early intervention are necessary to prevent complications and improve outcome. Life-threatening arrhythmias can manifest at different ages and a normal cardiac monitoring study does not exclude future occurrences leading to the dilemma of timing and frequency of cardiac rhythm monitoring and treatment. Given the rare incidence of CCHS, most health care professionals are not experienced with managing CCHS patients, particularly those with diaphragm pacers. With early diagnosis and advances in home mechanical ventilation and monitoring strategies, many CCHS children are surviving into adulthood presenting new challenges in their care.

Alanine Expansions Associated with Congenital Central Hypoventilation Syndrome Impair PHOX2B Homeodomain-mediated Dimerization and Nuclear Import

Heterozygous mutations of the human PHOX2B gene, a key regulator of autonomic nervous system development, lead to congenital central hypoventilation syndrome (CCHS), a neurodevelopmental disorder characterized by a failure in the autonomic control of breathing. Polyalanine expansions in the 20-residues region of the C terminus of PHOX2B are the major mutations responsible for CCHS. Elongation of the alanine stretch in PHOX2B leads to a protein with altered DNA binding, transcriptional activity, and nuclear localization and the possible formation of cytoplasmic aggregates; furthermore, the findings of various studies support the idea that CCHS is not due to a pure loss of function mechanism but also involves a dominant negative effect and/or toxic gain of function for PHOX2B mutations. Because PHOX2B forms homodimers and heterodimers with its paralogue PHOX2A in vitro, we tested the hypothesis that the dominant negative effects of the mutated proteins are due to non-functional interactions with the wild-type protein or PHOX2A using a co-immunoprecipitation assay and the mammalian two-hybrid system. Our findings show that PHOX2B forms homodimers and heterodimerizes weakly with mutated proteins, exclude the direct involvement of the polyalanine tract in dimer formation, and indicate that mutated proteins retain partial ability to form heterodimers with PHOX2A. Moreover, in this study, we investigated the effects of the longest polyalanine expansions on the homeodomain-mediated nuclear import, and our data clearly show that the expanded C terminus interferes with this process. These results provide novel insights into the effects of the alanine tract expansion on PHOX2B folding and activity.

Retracted: PHOX2B Is Associated with Neuroblastoma Cell Differentiation

Neuroblastoma is a common pediatric malignancy that accounts for ∼15% of tumor-related deaths in children. The tumor is generally believed to originate from neural crest cells during early sympathetic neurogenesis. As the degree of neuroblastoma differentiation has been correlated with clinical outcome, clarifying the molecular mechanisms that drive neuroblastoma progression and differentiation is important for increasing the survival of these patients. In a previous study, the authors identified paired-like homeobox 2b (PHOX2B) as a key mediator of neuroblastoma pathogenesis in a TH-MYCN mouse model. In the present study, they aimed to define whether PHOX2B is also associated with proliferation and differentiation of human neuroblastoma cells. PHOX2B expression in neuroblastoma cells was evaluated by immunoblot analyses, and the effects of PHOX2B on the proliferation of neuroblastoma cells in vitro were determined using clonogenic and sphere formation assays. Xenograft experiments in NOD/SCID mice were used to examine the in vivo response to PHOX2B knockdown. Their data demonstrated that PHOX2B acts as a prognostic marker in neuroblastoma and that retinoic acid-induced neuronal differentiation downregulates PHOX2B expression, thereby suppressing the self-renewal capacity of neuroblastoma cells and inhibiting tumorigenicity. These findings confirmed that PHOX2B is a key regulator of neuroblastoma differentiation and stemness maintenance and indicated that PHOX2B might serve as a potential therapeutic target in neuroblastoma patients.

PHOX2B is a suppressor of neuroblastoma metastasis.

Paired like homeobox 2B (PHOX2B) is a minimal residual disease (MRD) marker of neuroblastoma. The presence of MRD, also referred to as micro-metastases, is a powerful marker of poor prognosis in neuroblastoma.Lung metastasis is considered a terminal event in neuroblastoma. Lung micro-metastatic neuroblastoma (MicroNB) cells show high expression levels of PHOX2B and possess a less malignant and metastatic phenotype than lung macro metastatic neuroblastoma (MacroNB) cells, which hardly express PHOX2B.In vitro assays showed that PHOX2B knockdown in MicroNB cells did not affect cell viability; however it decreased the migratory capacity of the MicroNB-shPHOX2B cells. An orthotopic inoculation of MicroNB-shPHOX2B cells into the adrenal gland of nude mice resulted in significantly larger primary tumors and a heavier micro-metastatic load in the lungs and bone-marrow, than when control cells were inoculated.PHOX2B expression was found to be regulated by methylation. The PHOX2B promoter in MacroNB cells is significantly more methylated than in MicroNB cells. Demethylation assays using 5-azacytidine demonstrated that methylation can indeed inhibit PHOX2B transcription in MacroNB cells.These pre-clinical data strongly suggest that PHOX2B functions as a suppressor of neuroblastoma progression.

Maternal Congenital Central Hypoventilation Syndrome in Pregnancy: A Case Report

Background: Congenital Central Hypoventilation Syndrome is an exceedingly rare condition which may predispose to compromised placental function and intrauterine growth restriction. Labor is best managed in an intensive care unit. Congenital Central Hypoventilation Syndrome (CCHS) is an exceedingly rare autosomal dominant condition with about 160-180 cases diagnosed worldwide. It is characterized by adequate ventilation while awake and alveolar hypoventilation during sleep and if untreated the affected individual may survive only one to two months of age. Given the extreme rarity of the condition ideal antepartum and intrapartum management is largely undefined. Case: A 28 year-old primigravida with CCHS was followed from 16 weeks gestational age through delivery and post partum course. Amniocentesis confirmed that the fetus was not affected. During the antepartum course there was a progressive lag in fetal growth and placental weight was found to be much less than the third percentile. At night mechanical ventilation was utilized and a normal spontaneous vaginal delivery was achieved in the intensive care unit. Conclusion: CCHS is an exceedingly rare diagnosis. During pregnancy multispecialty interaction is critical for both maternal and fetal wellbeing. Fetal and placental growth is likely impacted by a hypoxic environment even with nightly mechanical ventilation. Amniocentesis and PHOX2B gene testing should be highly considered even if termination is not an option as it allows critical preparation for the newborn caregiver and other hospital personnel.

Impaired neural structure and function contributing to autonomic symptoms in congenital central hypoventilation syndrome.

Congenital central hypoventilation syndrome (CCHS) patients show major autonomic alterations in addition to their better-known breathing deficiencies. The processes underlying CCHS, mutations in the PHOX2B gene, target autonomic neuronal development, with frame shift extent contributing to symptom severity. Many autonomic characteristics, such as impaired pupillary constriction and poor temperature regulation, reflect parasympathetic alterations, and can include disturbed alimentary processes, with malabsorption and intestinal motility dyscontrol. The sympathetic nervous system changes can exert life-threatening outcomes, with dysregulation of sympathetic outflow leading to high blood pressure, time-altered and dampened heart rate and breathing responses to challenges, cardiac arrhythmia, profuse sweating, and poor fluid regulation. The central mechanisms contributing to failed autonomic processes are readily apparent from structural and functional magnetic resonance imaging studies, which reveal substantial cortical thinning, tissue injury, and disrupted functional responses in hypothalamic, hippocampal, posterior thalamic, and basal ganglia sites and their descending projections, as well as insular, cingulate, and medial frontal cortices, which influence subcortical autonomic structures. Midbrain structures are also compromised, including the raphe system and its projections to cerebellar and medullary sites, the locus coeruleus, and medullary reflex integrating sites, including the dorsal and ventrolateral medullary nuclei. The damage to rostral autonomic sites overlaps metabolic, affective and cognitive regulatory regions, leading to hormonal disruption, anxiety, depression, behavioral control, and sudden death concerns. The injuries suggest that interventions for mitigating hypoxic exposure and nutrient loss may provide cellular protection, in the same fashion as interventions in other conditions with similar malabsorption, fluid turnover, or hypoxic exposure.

Congenital central hypoventilation syndrome: analysis of PHOX2B gene mutation in a case

OBJECTIVE To report on the phenotype of an infant with central hypoventilation syndrome (CCHS) and result of PHOX2B gene mutation analysis for the purpose of genetic counseling and prenatal diagnosis. METHODS Clinical data of an infant with CCHS was collected and analyzed. Potential mutation of PHOX2B gene was analyzed by amplified fragment length polymorphism (amp-FLP) and DNA sequencing. RESULTS The patient had typical clinical features of CCHS including frequent hypoventilation during sleeping, hypoxemia and hypercapnia which could be corrected by continuous ventilatory support. She also had repeated bruising and was difficult-to-wean, but without any cardiac, pulmonary, neuromuscular or brainstem lesions. DNA sequencing and amp-FLP of the PHOX2B gene showed that the patient has carried a polyalanine expansion repeat mutation (PARM) in exon 3. A 27 bp duplication was confirmed in the repeat sequence of 20 alanines by cloned and sequenced. This has led to an expansion of the repeat tract to 29 alanines. The genotype was therefore 20/29. CONCLUSION A patient with CCHS has been described. Mutation screening of PHOX2B gene can be used as an important support for diagnosis and genetic counseling for such patients.

Shox2 is required for the proper development of the facial motor nucleus and the establishment of the facial nerves.

BackgroundAxons from the visceral motor neurons (vMNs) project from nuclei in the hindbrain to innervate autonomic ganglia and branchial arch-derived muscles. Although much is known about the events that govern specification of somatic motor neurons, the genetic pathways responsible for the development of vMNs are less well characterized. We know that vMNs, like all motor neurons, depend on sonic hedgehog signaling for their generation. Similarly, the paired-like homeobox 2b (Phox2b) gene, which is expressed in both proliferating progenitors and post-mitotic motor neurons, is essential for the development of vMNs. Given that our previous study identified a novel role for the short stature homeobox 2 (Shox2) gene in the hindbrain, and since SHOX2 has been shown to regulate transcription of islet 1 (Isl1), an important regulator of vMN development, we sought to determine whether Shox2 is required for the proper development of the facial motor nucleus.ResultsUsing a Nestin-Cre driver, we show that elimination of Shox2 throughout the brain results in elevated cell death in the facial motor nucleus at embryonic day 12.5 (E12.5) and E14.5, which correlates with impaired axonal projection properties of vMNs. We also observed changes in the spatial expression of the vMN cell fate factors Isl1 and Phox2b, and concomitant defects in Shh and Ptch1 expression in Shox2 mutants. Furthermore, we demonstrate that elimination of Shox2 results in the loss of dorsomedial and ventromedial subnuclei by postnatal day 0 (P0), which may explain the changes in physical activity and impaired feeding/nursing behavior in Shox2 mutants.ConclusionsCombined, our data show that Shox2 is required for development of the facial motor nucleus and its associated facial (VII) nerves, and serves as a new molecular tool to probe the genetic programs of this understudied hindbrain region.Electronic supplementary materialThe online version of this article (doi:10.1186/s12868-015-0176-0) contains supplementary material, which is available to authorized users.

Testing the evolutionary conservation of vocal motoneurons in vertebrates

Medullary motoneurons drive vocalization in many vertebrate lineages including fish, amphibians, birds, and mammals. The developmental history of vocal motoneuron populations in each of these lineages remains largely unknown. The highly conserved transcription factor Paired-like Homeobox 2b (Phox2b) is presumed to be expressed in all vertebrate hindbrain branchial motoneurons, including laryngeal motoneurons essential for vocalization in humans. We used immunohistochemistry and in situ hybridization to examine Phox2b protein and mRNA expression in caudal hindbrain and rostral spinal cord motoneuron populations in seven species across five chordate classes. Phox2b was present in motoneurons dedicated to sound production in mice and frogs (bullfrog, African clawed frog), but not those in bird (zebra finch) or bony fish (midshipman, channel catfish). Overall, the pattern of caudal medullary motoneuron Phox2b expression was conserved across vertebrates and similar to expression in sea lamprey. These observations suggest that motoneurons dedicated to sound production in vertebrates are not derived from a single developmentally or evolutionarily conserved progenitor pool.

A Phox2b BAC Transgenic Rat Line Useful for Understanding Respiratory Rhythm Generator Neural Circuitry.

The key role of the respiratory neural center is respiratory rhythm generation to maintain homeostasis through the control of arterial blood pCO2/pH and pO2 levels. The neuronal network responsible for respiratory rhythm generation in neonatal rat resides in the ventral side of the medulla and is composed of two groups; the parafacial respiratory group (pFRG) and the pre-Bötzinger complex group (preBötC). The pFRG partially overlaps in the retrotrapezoid nucleus (RTN), which was originally identified in adult cats and rats. Part of the pre-inspiratory (Pre-I) neurons in the RTN/pFRG serves as central chemoreceptor neurons and the CO2 sensitive Pre-I neurons express homeobox gene Phox2b. Phox2b encodes a transcription factor and is essential for the development of the sensory-motor visceral circuits. Mutations in human PHOX2B cause congenital hypoventilation syndrome, which is characterized by blunted ventilatory response to hypercapnia. Here we describe the generation of a novel transgenic (Tg) rat harboring fluorescently labeled Pre-I neurons in the RTN/pFRG. In addition, the Tg rat showed fluorescent signals in autonomic enteric neurons and carotid bodies. Because the Tg rat expresses inducible Cre recombinase in PHOX2B-positive cells during development, it is a potentially powerful tool for dissecting the entire picture of the respiratory neural network during development and for identifying the CO2/O2 sensor molecules in the adult central and peripheral nervous systems.

MiR-204 mediates post-transcriptional down-regulation of PHOX2B gene expression in neuroblastoma cells

Neuroblastoma (NB) is a rare childhood cancer of the peripheral sympathetic nervous system and accounts for approximately 10% of all pediatric tumors. Heterozygous PHOX2B mutations have been found in association with NB development in familial, sporadic and syndromic cases. In addition, the PHOX2B gene is widely over-expressed both in tumor samples and NB cell lines. Post-transcriptional gene regulation is known to be involved in mRNA stability and, in NB, microRNAs (miRNAs) seem to be responsible for altered expression of genes driving differentiation, apoptosis, and migration. To assess the possible impact of post-transcriptional regulation in NB cell lines, we have focused on the PHOX2B mRNA stability by both in silico analysis and functional studies on its 3′untranslated region (3′UTR). PHOX2B gene expression has resulted under post-transcriptional control, as suggested by: i) instability of PHOX2B mRNA, demonstrated by short mRNA half-life levels in both IMR32 and LAN-1 cell lines, ii) role of the PHOX2B-3′UTR, confirmed by the activity of proper reporter constructs, and iii) miRNA-204, shown to enhance the PHOX2B 3′UTR mediated down-regulation of the reporter construct activity. Finally, miRNA-204 has resulted to decrease the stability of the PHOX2B mRNA at different extents in the presence of different SNP rs1063611 alleles. Therefore, post-transcriptional down-regulation of the PHOX2B gene takes place in NB cell lines and miRNA-204 participates in such a 3′UTR mediated control.

Congenital central hypoventilation syndrome with hyperinsulinemia in an infant

Congenital central hypoventilation syndrome (CCHS) is a rare disorder that typically presents in the newborn period and is characterized by alveolar hypoventilation and symptoms of autonomic nervous system dysregulation. We describe an infant with CCHS who developed hyperinsulinism, which is an uncommon association. She was born by semi-elective Caesarean section at 37 weeks of gestation after a pregnancy complicated by polyhydramnios. Apgar scores were 7and 9 (at 1 and 5 min) and birth weight was 3550 g. She was intubated and ventilated within 24 hours for frequent apnoea’s and desaturations. Multiple extubation attempts on the following days failed because of progressive respiratory acidosis. She underwent tracheostomy at 2 months of age. CCHS was diagnosed in the first month of life, after a polyalanine repeat expansion of the PHOX2B gene (20/26genotype) was detected. At 7 months of age, she was admitted to hospital with seizures associated with hypoglycaemia. A critical sample (glucose 2.5mmol/L) revealed an inappropriately raised plasma insulin of 3.0 mU/L, growth hormone 7mu/L, cortisol 470 mmol/L, and serum ketones <0.1g/L. Urine and serum metabolic screen was normal, as was an EEG. Diazoxide was commenced at 5 mg/kg/day along with hydrochlorothiazide at 1mg/kg/d. These relatively low doses were used in view of parental concerns of potential drug side effect of hirsutism. The dose had to be increased to 7.5mg/kg/day as hypoglycaemic episodes persisted. Capillary blood glucose testing and continuous glucose monitoring showed no further hypoglycaemia at this dose. Sequence analysis of the KCNJ11 and ABCC8 genes implicated in congenital hyperinsulinism was undertaken and no pathogenic mutation was found. The mechanisms underlying abnormal glycaemia in CCHS are not fully understood. Multiple hypotheses have been postulated including the co-expression of the dopamine beta hydroxylase gene mutation with PHOX 2B mutation. Further, disordered autonomic homeostasis as seen in PHOX2B mutations is thought to impact the regulation of insulin and glucagon secretion. Our case highlights the importance of considering hyperinsulinism in the differential diagnosis of hypoglycaemia in infants with CCHS. Written informed consent was obtained from the patient's parent or guardian for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

Identification of novel pathways and molecules able to down-regulate PHOX2B gene expression by in vitro drug screening approaches in neuroblastoma cells

PHOX2B is a transcription factor involved in the regulation of neurogenesis and in the correct differentiation of the autonomic nervous system. The pathogenetic role of PHOX2B in neuroblastoma (NB) is supported by mutations in familial, sporadic and syndromic cases of NB and overexpression of PHOX2B and its target ALK in tumor samples and NB cell lines.Starting from these observations, we have performed in vitro drug screening approaches targeting PHOX2B overexpression as a potential pharmacological means in NB.In particular, in order to identify molecules able to decrease PHOX2B expression, we have evaluated the effects of 70 compounds in IMR-32 cell line stably expressing the luciferase gene under the control of the PHOX2B promoter. Curcumin, SAHA and trichostatin A showed to down-regulate the PHOX2B promoter activity which resulted in a decrease of both protein and mRNA expressions. In addition, we have observed that curcumin acts by interfering with PBX-1/MEIS-1, NF-κB and AP-1 complexes, in this work demonstrated for the first time to regulate the transcription of the PHOX2B gene.Finally, combined drug treatments showed successful effects in down-regulating the expression of both PHOX2B and its target ALK genes, thus supporting the notion of the effectiveness of molecule combination in tumor therapy.