PHOX2B Mutations Research Articles

METTL3-induced lncARSR aggravates neuroblastoma tumorigenic properties through stabilizing PHOX2B

Neuroblastoma (NB), the most common extracranial solid tumor in pediatric patients, manifests with considerable variability across multiple primary sites. Despite this, the extent of genetic heterogeneity within these tumor foci and the identification of consistent oncogenic drivers remains largely unexplored. Of particular interest, genetic mutations in PHOX2B have been linked to familial cases of NB, yet the underlying molecular mechanisms are not fully delineated. In our research, we focus on unraveling the role of a novel functional long non-coding RNA (lncRNA) associated with PHOX2B in the context of NB. Using NB cell models with overexpressed PHOX2B, combined with lncRNA microarray analysis, we discovered that lncARSR is significantly upregulated in response to PHOX2B overexpression. Subsequent biological assays demonstrated that lncARSR promotes both the proliferation and metastasis of NB cells. Further molecular investigations revealed that lncARSR plays a crucial role in stabilizing PHOX2B expression within NB cells. Moreover, we identified that the expression of lncARSR is regulated by methylation through methyltransferase-like 3 (METTL3), which itself is positively correlated with PHOX2B expression. Rescue experiments underscored the functional importance of METTL3, lncARSR, and PHOX2B in NB cells. In summary, our findings provide new insights into the molecular functions of PHOX2B in the progression of neuroblastoma and propose a novel therapeutic target for this aggressive malignancy.

Identification of a histone deacetylase inhibitor as a therapeutic candidate for congenital central hypoventilation syndrome

Congenital central hypoventilation syndrome (CCHS), a rare genetic disease caused by heterozygous PHOX2B mutations, ischaracterized by life-threatening breathing deficiencies. PHOX2B is a transcription factor required for the specification of the autonomic nervous system, which contains, in particular, brainstem respiratory centers. In CCHS, PHOX2B mutations lead to cytoplasmic PHOX2B protein aggregations, thus compromising its transcriptional capability. Currently, the only available treatment for CCHS is assisted mechanical ventilation. Therefore, identifying molecules with alleviating effects on CCHS-related breathing impairments is of primary importance. A transcriptomic analysis of cells transfected with different PHOX2B constructs was used to identify compounds of interest with the CMap tool. Using fluorescence microscopy and luciferase assay, the selected molecules were further tested invitro for their ability to restore the nuclear location and function of PHOX2B. Finally, an electrophysiological approach was used to investigate exvivo the effects ofthe most promising molecule on respiratory activities of PHOX2B-mutant mouse isolated brainstem. The histone deacetylase inhibitor SAHA was found to have low toxicity invitro, to restore the proper location and function of PHOX2B protein, and to improve respiratory rhythm-relatedparameters exvivo. Thus, our results identify SAHA as a promising agent to treat CCHS-associated breathing deficiencies.

Congenital central hypoventilation syndrome in Chinese population: Analysis of three new cases and review of the literature.

Congenital central hypoventilation syndrome (CCHS) is a rare autosomal dominant disease that is mainly caused by PHOX2B mutations. The purpose of this study is to analyze and summarize the clinical and genetic characteristics of CCHS patients in the Chinese population from our study and previous literature. The potential pathogenic gene mutations of CCHS were identified and verified by next generation sequencing combined with Sanger sequencing, fluorescent probe PCR and capillary electrophoresis. The clinical characteristics and gene mutations of CCHS cases in Chinese population were summarized from our study and previous literature to explore the genotype-phenotype correlations. We identified 48 CCHS cases including three new cases from our report in China. Overall, 77.1% of the patients had PHOX2B polyalanine repeat expansion mutations (PARMs), and the remaining 22.9% had 10 distinct PHOX2B non-polyalanine repeat expansion mutations (NPARMs). Compared to those with PARMs, patients with NPARMs were more likely to have premature birth (54.5% vs. 2.8%, p < 0.001) and lower birth weight (33.3% vs. 3.2%, p = 0.030), with statistical significance. The patients with PARMs were more likely to have cardiovascular defects (64.9% vs. 27.3%, p = 0.063), cerebral hemorrhage (29.7% vs. 9.1%, p = 0.322) and seizures (37.8% vs. 9.1%, p = 0.151) than those with NPARMs, with no statistical significance. CCHS patients with PHOX2B NPARMs were more likely to have premature birth and low birth weight, while PHOX2B PARMs tended to be positively associated with the risk of cardiovascular defects, cerebral hemorrhage and seizures in Chinese population.

Serotonin and the ventilatory effects of etonogestrel, a gonane progestin, in a murine model of congenital central hypoventilation syndrome.

Congenital Central Hypoventilation Syndrome, a rare disease caused by PHOX2B mutation, is associated with absent or blunted CO2/H+ chemosensitivity due to the dysfunction of PHOX2B neurons of the retrotrapezoid nucleus. No pharmacological treatment is available. Clinical observations have reported non-systematic CO2/H+ chemosensitivity recovery under desogestrel. Here, we used a preclinical model of Congenital Central Hypoventilation Syndrome, the retrotrapezoid nucleus conditional Phox2b mutant mouse, to investigate whether etonogestrel, the active metabolite of desogestrel, led to a restoration of chemosensitivity by acting on serotonin neurons known to be sensitive to etonogestrel, or retrotrapezoid nucleus PHOX2B residual cells that persist despite the mutation. The influence of etonogestrel on respiratory variables under hypercapnia was investigated using whole-body plethysmographic recording. The effect of etonogestrel, alone or combined with serotonin drugs, on the respiratory rhythm of medullary-spinal cord preparations from Phox2b mutants and wildtype mice was analyzed under metabolic acidosis. c-FOS, serotonin and PHOX2B were immunodetected. Serotonin metabolic pathways were characterized in the medulla oblongata by ultra-high-performance liquid chromatography. We observed etonogestrel restored chemosensitivity in Phox2b mutants in a non-systematic way. Histological differences between Phox2b mutants with restored chemosensitivity and Phox2b mutant without restored chemosensitivity indicated greater activation of serotonin neurons of the raphe obscurus nucleus but no effect on retrotrapezoid nucleus PHOX2B residual cells. Finally, the increase in serotonergic signaling by the fluoxetine application modulated the respiratory effect of etonogestrel differently between Phox2b mutant mice and their WT littermates or WT OF1 mice, a result which parallels with differences in the functional state of serotonergic metabolic pathways between these different mice. Our work thus highlights that serotonin systems were critically important for the occurrence of an etonogestrel-restoration, an element to consider in potential therapeutic intervention in Congenital Central Hypoventilation Syndrome patients.

Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia.

Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O2 and CO2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2bΔ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2bΔ8, Atoh1cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b+/TH--expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2bΔ8 mutation in Atoh1-expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.

Chapter 2 - Central respiratory chemoreception

Brain PCO2 is sensed primarily via changes in [H+]. Small pH changes are detected in the medulla oblongata and trigger breathing adjustments that help maintain arterial PCO2 constant. Larger perturbations of brain CO2/H+, possibly also sensed elsewhere in the CNS, elicit arousal, dyspnea, and stress, and cause additional breathing modifications. The retrotrapezoid nucleus (RTN), a rostral medullary cluster of glutamatergic neurons identified by coexpression of Phoxb and Nmb transcripts, is the lynchpin of the central respiratory chemoreflex. RTN regulates breathing frequency, inspiratory amplitude, and active expiration. It is exquisitely responsive to acidosis in vivo and maintains breathing autorhythmicity during quiet waking, slow-wave sleep, and anesthesia. The RTN response to [H+] is partly an intrinsic neuronal property mediated by proton sensors TASK-2 and GPR4 and partly a paracrine effect mediated by astrocytes and the vasculature. The RTN also receives myriad excitatory or inhibitory synaptic inputs including from [H+]-responsive neurons (e.g., serotonergic). RTN is silenced by moderate hypoxia. RTN inactivity (periodic or sustained) contributes to periodic breathing and, likely, to central sleep apnea. RTN development relies on transcription factors Egr2, Phox2b, Lbx1, and Atoh1. PHOX2B mutations cause congenital central hypoventilation syndrome; they impair RTN development and consequently the central respiratory chemoreflex.

Abstract 3030: Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences

Abstract Background: Neuroblastoma is a childhood cancer that arises from the sympathetic nervous system. Approximately 1% of patients have a family history of disease with causal germline mutations in neuroblastoma predisposition genes, ALK or PHOX2B. Recent genome-wide association studies (GWAS) of sporadic cases have identified over two dozen susceptibility loci, and next-generation sequencing studies estimate that 8-10% of children with cancer harbor a rare pathogenic (P) or likely pathogenic (LP) germline variant in a cancer predisposition gene (CPG). To date, the heritability and clinical significance of germline P/LP variants remains unknown due to a lack of parental germline DNA. Methods: Through the Gabriella Miller Kids First program, we performed whole genome sequencing (WGS) at 30x depth on patient-parent triads (n=457) and dyads (n=99), along with WGS and exome sequencing on matched tumor DNA (n=336) and RNA-sequencing on matched tumor RNA (n=207). Rare variants (allele frequency &amp;lt;0.1% in ExAC, 1000 Genomes, and gnomAD) in a predefined set of CPGs (n=197) were annotated, and pathogenicity was assessed using ClinVar, InterVar, and manual review. Gene-based enrichment was performed by comparison to gnomAD WGS data (n=143,068), excluding TCGA samples. Heritability was evaluated using family-based variant calling. Tumor DNA sequencing was analyzed for second hits or somatic enrichment of the germline P/LP variant. Tumor RNA sequencing was interrogated to identify expressed P/LP variants and preferential allelic expression. Ongoing analyses are utilizing polygenic risk scores from our GWAS study to inform tumor penetrance. Results: We observed 78 P/LP germline variants in CPGs in 75 probands (13.5%). Of the 72 probands with trio data, 95% of the P/LP variants were inherited (54% maternal, 46% paternal). Several CPGs showed enrichment of P/LP variants, including BARD1 (p=0.001; OR 16.16; 95% CI 3.21-50.45). We observed one canonical ALK mutation (R1275Q), but no PHOX2B mutations. For patients with corresponding tumor samples, all germline P/LP variants were observed in the tumor DNA. Germline P/LP variants were associated with high-risk disease as defined by risk group (high vs. low/intermediate, p=0.005), INRG stage (stage M vs stage L1/L2/MS, p=0.024), and MYCN amplification status (amplified vs not amplified, p=0.016). Conclusions: Here we show that approximately 13.5% of patients with neuroblastoma harbor a rare P/LP germline variant in a CPG, and the vast majority of these are inherited. Neuroblastoma patients with P/LP germline variants are more likely to have high-risk disease and several variants suggest potential therapeutic opportunities. Work is ongoing to understand the genetic factors that explain why parents harboring the same P/LP variant did not develop neuroblastoma, and to determine the genetic counseling implications of these data. Citation Format: Emily Blauel, Zalman Vaksman, Alex Lee, Rebecca Kaufman, Laura Egolf, Andrew Olshan, John Maris, Diskin Sharon. Heritability of cancer predisposition gene mutations in 556 neuroblastoma patients with paired parental DNA whole genome sequences [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3030.

862 Volume-Assured Pressure Support improves outcomes in a patient with Congenital Central Hypoventilation Syndrome

IntroductionCongenital Central Hypoventilation Syndrome (CCHS) is a condition caused by a mutation of the PHOX2B gene and an incidence of 1 in 50,000 live births. Clinically the condition is characterized by autonomic nervous system dysfunction, the most prominent feature of which is the failure of respiratory homeostasis during sleep. In patients severely affected, life-long ventilatory support is required. This might start as early as the newborn period. Subsequent adjustments are required due to their growth and development. The role of Volume-Assured Pressure Support (VAPS) ventilation in treatment of CCHS was only described in a couple reports before.Report of case(s)A 17-year-old female patient born at term and diagnosed with CCHS at birth at our center with a PHOX2B mutation confirmed. Her daytime ventilatory support was weaned at age 18 months and the tracheostomy was removed at age 10 years old. She relocated to another state, was lost to follow-up, and returned this year for adjustments of her ventilator whose settings had not been adjusted for several years. She was on a Trilogy 100 ventilator, in pressure-controlled mode with settings of EPAP 5, IPAP 20, and a rate of 22/min without supplemental oxygen. Her measured weight is 69kg, body mass index (BMI) 27. The patient complained of difficulty breathing while on those settings and reported decreased desire to use the machine. The patient was empirically switched to VAPS due to titration availability limitations during the COVID-19 pandemic. Initial AVAPS settings were: EPAP 4–7, PS 3–12, breath rate 16/min, TV 350mL. Upon implementing the changes, the patient reported improved comfort and increased usage. Average minute ventilation decreased from 10.5 to 5.8 L/min, patient triggered breaths increased from 1.1% to 12.3%, average breaths per minute decreased from 22.0/min to 16.2/min, the average peak flow 31.8 to 26.2L/min, tidal volume decreased from 463 to 355mL.ConclusionAVAPS ventilation can be successfully used in managing patients with CCHS, and it might be superior to pressure-controlled mode in certain cases, improving patient comfort and compliance.Support (if any)Department of Internal Medicine, University of New Mexico School of Medicine, Albuquerque, NM.

563 Effect of PHOX2B Genotype and Age on Heart Rate Variability in Pediatric Patients with Congenital Central Hypoventilation Syndrome

Abstract Introduction Congenital Central Hypoventilation Syndrome (CCHS) is a rare neurocristopathy causing a wide array of cardiac autonomic nervous system (ANS) abnormalities (abnormal heart rate (HR) regulation, decreased HR variability (HRV), prolonged sinus pauses). CCHS is caused by mutations throughout the PHOX2B gene. Different types and locations of PHOX2B mutations have been associated with different levels of CCHS cardiac phenotype severity. We hypothesized that HRV in CCHS will be influenced by age and PHOX2B genotype. Methods PHOX2B mutation-confirmed CCHS patients in our cohort followed longitudinally with serial Holter monitoring were included. HRV measures were extracted from Holter monitoring reports and analyzed using linear mixed-effect models. Additional analyses explored which variables predicted sinus pauses and cardiac pacemaker implantation using linear and logistic regression. Only data preceding implantation of a cardiac pacemaker were included in analysis. Results 97 patients (0–18 years old) were enrolled in the study: 21 with the 20/25 genotype, 21 with the 20/26 genotype, 26 with the 20/27 genotype, 7 with genotypes ranging from 20/28-20/33, and 22 with non-polyalanine repeat expansion mutations (NPARMs). There were 965 total observations (Holters). The following HRV metrics were analyzed: min- and max-HR, longest-RR, SDNN, ASDNN5, SDANN5, and RMSDD. Statistically significant differences were observed between PHOX2B genotypes for min-HR and longest-RR (ps&amp;lt;0.05). Age by genotype interactions emerged such that certain metrics in HRV had different developmental trajectories by genotype (ps&amp;lt;0.05). Min-HR and longest-RR predicted eventual cardiac pacemaker implantation (AUCs&amp;gt;0.78). As for predictors of sinus pauses, different HRV metrics emerged as predictors across PHOX2B genotypes suggesting their utility in predicting cardiac pacemaker implantation dependent on genotype. Conclusion HRV in patients with CCHS is dependent on age and PHOX2B genotype, with significant differences in HRV metrics between patients with PHOX2B genotypes 20/25, 20/26, and 20/27 and the patients with 20/28-20/33 genotypes as well as those with NPARMs. Consistent with treatment guidelines, min-HR and longest-RR were predictive of eventual cardiac pacemaker implantation. Differences in the HRV metrics which predict sinus pauses as well as the overall group differences observed and trajectory-based differences may improve anticipatory management by earlier risk identification for prolonged sinus pauses in CCHS. Support (if any) None

Congenital central hypoventilation syndrome and ventilatory responses during cardiopulmonary exercise testing.

Previous studies have shown evidence of hypoxemia and hypercapnia during cardiopulmonary exercise test (CPET) evaluation in children with congenital central hypoventilation syndrome (CCHS). However, there are no longitudinal studies which compared CPET findings to polysomnogram (PSG) or PHOX2B mutation, to date. To describe the longitudinal CPET findings in a cohort of children with CCHS and correlate the findings to the PSG results. This retrospective study was conducted in children with CCHS followed in the Long-term Ventilation Program at SickKids, Toronto, Canada between September, 2013 and January, 2020. CCHS genetic mutation, age of diagnosis, ventilatory support, family history, disease associations of CCHS, CPETs, and PSG parameters were recorded and analyzed. A total of nine patients with CCHS (46 CPETs and 46 PSGs) were enrolled. Four (44.4%) children had polyalanine repeat mutations. The mean (SD) age at the time of diagnosis and duration of ventilatory usage were 3.2 ± 3.4 years and 11.5 ± 2.8 years, respectively. All abnormal CPETs had hypercapnia in at least 1 phase of the exercise test. Hypercapnia (12/46; 26.1%) at peak of exercise was the most common abnormality. None of the children experienced an oxygen desaturation below 90%. End-tidal CO2 (PetCO2 ) at rest and at peak exercise in the CPETs were significantly correlated with PSG TcCO2 while PetCO2 at anaerobic threshold was correlated with CO2 in pre-PSG capillary blood gas. Nocturnal hypoventilation may impact the CPET results in CCHS children. Serial CPETs should be considered standard clinical care for all CCHS children.

Neonatal apneic phenotype in a murine congenital central hypoventilation syndrome model is induced through non-cell autonomous developmental mechanisms.

Congenital central hypoventilation syndrome (CCHS) represents a rare genetic disorder usually caused by mutations in the homeodomain transcription factor PHOX2B. Some CCHS patients suffer mainly from deficiencies in CO2 and/or O2 respiratory chemoreflex, whereas other patients present with full apnea shortly after birth. Our goal was to identify the neuropathological mechanisms of apneic presentations in CCHS. In the developing murine neuroepithelium, Phox2b is expressed in three discrete progenitor domains across the dorsal‐ventral axis, with different domains responsible for producing unique autonomic or visceral motor neurons. Restricting the expression of mutant Phox2b to the ventral visceral motor neuron domain induces marked newborn apnea together with a significant loss of visceral motor neurons, RTN ablation, and preBötzinger complex dysfunction. This finding suggests that the observed apnea develops through non‐cell autonomous developmental mechanisms. Mutant Phox2b expression in dorsal rhombencephalic neurons did not generate significant respiratory dysfunction, but did result in subtle metabolic thermoregulatory deficiencies. We confirm the expression of a novel murine Phox2b splice variant which shares exons 1 and 2 with the more widely studied Phox2b splice variant, but which differs in exon 3 where most CCHS mutations occur. We also show that mutant Phox2b expression in the visceral motor neuron progenitor domain increases cell proliferation at the expense of visceral motor neuron development. We propose that visceral motor neurons may function as organizers of brainstem respiratory neuron development, and that disruptions in their development result in secondary/non‐cell autonomous maldevelopment of key brainstem respiratory neurons.

0887 Diaphragm Pacer Malfunctions Requiring Surgical Repair in CCHS Patients

Abstract Introduction Congenital Central Hypoventilation Syndrome (CCHS) is a genetic disorder that results in the loss of autonomic ventilatory control, and patients require ventilatory support during sleep or both sleep and wakefulness. One method of ventilatory support is diaphragm pacing (DP), where electrodes surgically placed on the phrenic nerve are connected to subcutaneously implanted receivers that communicate with external antennas and transmitter. There are limited data on the frequency of DP malfunctions that require surgical revision. Methods We reviewed the records of 24 CCHS patients ventilated by DP followed at CHLA from 1990-2019. Records were examined for demographics, PHOX2B mutation, pacing duration/day, date and type of malfunctions, age and time since implantation at malfunction occurrence, and repair success rate. Results All 24 patients had thoracoscopic electrode placement. 17/24 (71%) of patients used DP while asleep; 3/24 (13%) during wakefulness only. 4/24 (17%) were not currently using their pacers. 10/24 (42%) patients required at least one surgical intervention (Age at implantation 9 ± 4.6 (SD) years; age at malfunction 12.5 ± 7.4 years). The average time from pacer implantation to malfunction was 3.8 ± 3.5 years. Malfunctions included defective receivers (6), insulation leaks (1), defective electrodes (4), and hardware infection (1). Of 12 unique component repairs, 6/12 (50%) involved changing receivers, 1/12 (8%) involved repairing an insulation leak, 4/12 (33%) involved replacing the electrodes and receivers, and 1/12 (8%) involved hardware extraction. Of the 12 malfunctions, 10 (83%) had successful surgical revision. 2/12 (17%) repairs were not attempted. While awaiting surgical revision, patients were successfully ventilated by unilateral DP. Conclusion Nearly half of CCHS patients on DP experienced malfunctions within 11 years of implantation. The most common DP repair was receiver replacement. Patients who are waiting for repair often successfully ventilate while pacing unilaterally. Support None

Dissecting the Contribution of Specific Hindbrain Cell Populations in the Phenotypic Characterization of a Mouse Model of Central Congenital Hypoventilation Syndrome

Hindbrain respiratory neuron networks undergo critical developmental maturation during the late embryonic, perinatal and post‐natal periods in mammals. Thus, abnormalities in neuron network development may contribute to autonomic respiratory dysfunction. Central Congenital Hypoventilation Syndrome (CCHS) is a rare genetic disorder characterized by an inability to sense and respond to altered levels of CO2 or O2 (Chemosensation). CCHS patients present with a spectrum of central respiratory control pathophysiologies, ranging from deficiency in chemosensation, to most severe manifestations of neonatal apnea that requires life‐sustaining mechanical ventilation. Heterozygous Mutations of the transcription factor PHOX2B, classified as either poly‐alanine repeat (PARM) and non‐poly‐alanine repeat (NPARM), have been linked to the development of CCHS. In order to explore CCHS pathophysiology, a novel transgenic mouse expressing an inducible NPARM Phox2b mutation (Phox2bΔ8) was developed and characterized by Nobuta and collaborators (Nabuta et al. 2015). Following this study and aiming to reveal the contribution of specific hindbrain populations to CCHS, we have generated two additional mouse models in which of Phox2bΔ8 expression is restricted to cell populations derived from either NKX2.2+ or OLIG3+ progenitors. Expression of Phox2bΔ8 on NKX2.2+ derived cells resulted in a strong apneic phenotype on newborn pups. Detail examination of this mouse model hindbrains showed deficiencies in a spectrum of rhombomeric nuclei not developmentally derived from NKX2.2+‐expressing progenitors, indicating that the developmental neuropathologies identified result through non‐cell‐autonomous mechanisms. Additionally, we find that these pathologies result from early rhombomeric patterning defects in embryonic ventral neural progenitor zones, which showed reduced endogenous PHOX2B expression. On the other hand, preliminary results indicate that expression of Phox2bΔ8 on OLIG3+ derived cells result in a subtle deficit in chemosensation, but we have been unable to find any neuropathological change in the hindbrain. Together, our results help to dissect the contributions of different cell populations in the hindbrain and to further clarify the different manifestations and severity of chemosensation and respiratory deficits seen in human CCHS patientsSupport or Funding InformationNIH R01HL132355This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adult With PHOX2B Mutation and Late-Onset Congenital Central Hypoventilation Syndrome.

PHOX2B 20/27 polyalanine repeat mutation (PARM) in patients with congenital central hypoventilation syndrome (CCHS) is generally associated with full-time ventilator dependence, Hirschsprung disease, and increased risk for cardiac asystole. We follow a 14-year-old boy with CCHS PHOX2B 20/27 PARM who is full-time ventilator dependent via tracheostomy and has Hirschsprung disease. His mother, age 52 years, has a history of prolonged recovery from anesthesia and an elevated serum bicarbonate level of 45 mEq/L discovered on routine blood chemistry. PHOX2B gene mutation analysis was performed and showed an identical 20/27 PARM, diagnostic of CCHS. Late-onset CCHS has been reported in those with 20/24, 20/25 PHOX2B PARM, and in nonpolyalanine repeat mutations. This is the first report of a patient with PHOX2B 20/27 PARM with a mild phenotype diagnosed during adulthood. This unusual presentation supports the screening for PHOX2B mutations in parents of children with CCHS.

RAS-MAPK Pathway-Driven Tumor Progression Is Associated with Loss of CIC and Other Genomic Aberrations in Neuroblastoma.

Mutations affecting the RAS-MAPK pathway frequently occur in relapsed neuroblastoma tumors, which suggests that activation of this pathway is associated with a more aggressive phenotype. To explore this hypothesis, we generated several model systems to define a neuroblastoma RAS-MAPK pathway signature. Activation of this pathway in primary tumors indeed correlated with poor survival and was associated with known activating mutations in ALK and other RAS-MAPK pathway genes. Integrative analysis showed that mutations in PHOX2B, CIC, and DMD were also associated with an activated RAS-MAPK pathway. Mutation of PHOX2B and deletion of CIC in neuroblastoma cell lines induced activation of the RAS-MAPK pathway. This activation was independent of phosphorylated ERK in CIC knockout systems. Furthermore, deletion of CIC caused a significant increase in tumor growth in vivo These results show that the RAS-MAPK pathway is involved in tumor progression and establish CIC as a powerful tumor suppressor that functions downstream of this pathway in neuroblastoma.Significance: This work identifies CIC as a powerful tumor suppressor affecting the RAS-MAPK pathway in neuroblastoma and reinforces the importance of mutation-driven activation of this pathway in cancer. Cancer Res; 78(21); 6297-307. ©2018 AACR.

Desogestrel down-regulates PHOX2B and its target genes in progesterone responsive neuroblastoma cells

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.

Analysis of sporadic neuroblastic tumors reveals a novel PHOX2B mutation in neuroblastoma

Germline mutations of PHOX2B have been associated with neurocristopathies predisposing to neuroblastoma (NB). Genetic studies on NB have also shown that PHOX2B is rarely mutated in both sporadic and hereditary NB cases. In this study, we aimed to further evaluate the mutational status of PHOX2B in 120 additional neuroblastic tumors. Genomic DNA samples were isolated from tumor tissues of 104 neuroblastoma, 9 ganglioneuroblastoma, and 7 ganglioneuroma cases. Mutation analysis by direct sequencing of all PHOX2B exons revealed a novel missense mutation of c.94G > A (D32N) in exon 1 in an NB case. In addition, indel variations of c.1101_1118het-del18 and c.1098_1136het-del39 in exon 3 that encodes the second polyalanine tract were detected in an NB and a ganglioneuroma, respectively. Two different single nucleotide polymorphisms, c.552C > T and c.762A > C were found in five NB samples. Polyphen analysis revealed that the amino acid substitution due to the c.94G > A novel missense mutation is in the highly conserved residue among phylogenetically distant species and it has potentially a disease-causing effect.

Genetic susceptibility to neuroblastoma: current knowledge and future directions.

Neuroblastoma, a malignancy of the developing peripheral nervous system that affects infants and young children, is a complex genetic disease. Over the past two decades, significant progress has been made toward understanding the genetic determinants that predispose to this often lethal childhood cancer. Approximately 1-2% of neuroblastomas are inherited in an autosomal dominant fashion and a combination of co-morbidity and linkage studies has led to the identification of germline mutations in PHOX2B and ALK as the major genetic contributors to this familial neuroblastoma subset. The genetic basis of "sporadic" neuroblastoma is being studied through a large genome-wide association study (GWAS). These efforts have led to the discovery of many common susceptibility alleles, each with modest effect size, associated with the development and progression of sporadic neuroblastoma. More recently, next-generation sequencing efforts have expanded the list of potential neuroblastoma-predisposing mutations to include rare germline variants with a predicted larger effect size. The evolving characterization of neuroblastoma's genetic basis has led to a deeper understanding of the molecular events driving tumorigenesis, more precise risk stratification and prognostics and novel therapeutic strategies. This review details the contemporary understanding of neuroblastoma's genetic predisposition, including recent advances and discusses ongoing efforts to address gaps in our knowledge regarding this malignancy's complex genetic underpinnings.

Congenital heart disease and aortic arch variants associated with mutation in PHOX2B.

Congenital central hypoventilation syndrome (CCHS, OMIM 209880) is a rare autosomal dominant disorder caused by mutation in PHOX2B that manifests as a consequence of abnormal neural crest cell migration during embryogenesis. Unlike other neurocristopathies, however, its impact on the cardiovascular system has not been previously assessed. This study was an effort to characterize the association between congenital heart disease (CHD) and mutations in PHOX2B in patients with CCHS. A retrospective review of patients with CCHS in conjunction with functional analysis of PHOX2B mutations associated with CHD was performed. To substantiate functional implications of identified variants, we conducted protein structure analyses and in silico mutagenesis were conducted. The prevalence of CHD among patients with CCHS was significantly greater (30%; p < 0.001) than that of the current estimated prevalence of CHD. The majority of patients had anomalies involving the proximal aortic arch and/or proximal coronary arteries. Variants associated with CHD in this cohort appear to disrupt DNA binding of PHOX2B via alteration of its homeobox domain. This is the first report of an association between CHD and mutation in PHOX2B. Results are highly suggestive that alteration or elimination of the homeobox domain conveys significant risk for associated CHD or aortic arch variation.

Structural and functional differences in PHOX2B frameshift mutations underlie isolated or syndromic congenital central hypoventilation syndrome.

Heterozygous mutations in the PHOX2B gene are causative of congenital central hypoventilation syndrome (CCHS), a neurocristopathy characterized by defective autonomic control of breathing due to the impaired differentiation of neural crest cells. Among PHOX2B mutations, polyalanine (polyAla) expansions are almost exclusively associated with isolated CCHS, whereas frameshift variants, although less frequent, are often more severe than polyAla expansions and identified in syndromic CCHS. This article provides a complete review of all the frameshift mutations identified in cases of isolated and syndromic CCHS reported in the literature as well as those identified by us and not yet published. These were considered in terms of both their structure, whether the underlying indels induced frameshifts of either 1 or 2 steps (“frame 2” and “frame 3” mutations respectively), and clinical associations. Furthermore, we evaluated the structural and functional effects of one “frame 3” mutation identified in a patient with isolated CCHS, and one “frame 2” mutation identified in a patient with syndromic CCHS, also affected with Hirschsprung's disease and neuroblastoma. The data thus obtained confirm that the type of translational frame affects the severity of the transcriptional dysfunction and the predisposition to isolated or syndromic CCHS.