Registration

Wednesday, September 6: 6:00-8:00 pm

Cocktail Reception: Ariel’s Grotto

Wednesday, September 6: 8:00 pm...

Social Outing: Movie and Fireworks on the beach

Breakfast on site: Your choice

9:00-9:45 am

Thomas Keens, M.D., Children's Hospital, Los Angeles, recently retired after 40 years as a Pediatric Pulmonologist at Children’s Hospital Los Angeles (CHLA), is among a handful of experts in the world experienced in treating children with CCHS.  Dr. Keens, together with an interdisciplinary team of specialists, has been at the forefront of inovation in diagnosing and treating CCHS for more than four decades. The life saving advances developed at CHLA have allowed CCHS patients to lead long, fulfilling lives.

From Discovery to Advancements: Tracing the History of Congenital Central Hypoventilation Syndrome
Dr. Keens will trace the evolution of Congenital Central Hypoventilation Syndrome (CCHS) from its earliest observations to current molecular advancements. His presentation aims to deepen understanding, celebrate scientific achievements, and inspire further progress. He examines the initial perplexity and challenges faced by medical professionals, highlights key breakthroughs, and recognizes the contributions of clinicians and researchers. Personal stories of resilience from individuals with CCHS underscore the importance of ongoing research, education, and support networks. Together, we honor the past, embrace the present, and strive for a brighter future for those affected by CCHS.

9:45- 10:00 am

Break

10:00-10:40 am

Patrice Guyenet, Ph.D., University of Virginia, is Professor of Pharmacology, emeritus, with a career spanning 42 years on the faculty of the University of Virginia School of Medicine. Born in France, he obtained his basic sciences degree at Ecole Normale Supérieure, Paris (1967-1971). He earned a Ph.D. in neuropharmacology at INSERM, College de France, Paris, (1972-1976, mentor J Glowinski), followed by postdoctoral studies in neurophysiology at Yale University (1976-1978; mentor GK Aghajanian). Dr. Guyenet’s research focuses primarily on the autonomic nervous system and the neurol control of breathing in mammals with a special emphasis on central respiratory chemoreceptors.

The retrotrapezoid nucleus, lynchpin of CO2 homeostasis
Defining reticular formation nuclei and their physiological role has always been a challenge, particularly within the network that regulates breathing. The RTN is a rare example of a cell group with a well-defined developmental lineage and a specialized and clearly defined function. The final fate of the RTN (~700 neurons in mice) seems determined by E12.5 by the cumulative effect of four lineage-specific genes (egr-2, Phox2b, Lbx1, atoh-1). Prenatally RTN (dubbed e-Pf) possesses H+-dependent group pacemaker properties that wane around birth in rodents (the pfRG) and disappear in adulthood. The adult RTN produces an excitatory drive to the respiratory pattern generator (RPG) that is metered to achieve CO2 homeostasis by adjusting alveolar ventilation. This property is well illustrated during moderate hypoxia when the activity of RTN is down regulated by the exact amount needed to offset the increased respiratory drive from the carotid bodies. Like CO2, RTN regulates breathing frequency only when the RPG is autorhythmic (i.e. slow wave sleep, quiet resting and anesthesia but not during REM sleep). RTN drives breathing frequency, inspiration and active expiration via projections to the entire RPG. RTN operates via glutamatergic/peptidergic transmission. Its activity is pH regulated via neuronal pH sensors (Task-2; GPR4) and paracrine mechanisms. Importantly, RTN is also regulated by countless synaptic inputs. Complex, probably inhibitory inputs from phase-spanning RPG neurons, could conceivably underlie RTN’s participation to various respiratory oscillators. Strong inhibitory inputs from lung afferents may help prevent lung overinflation. Excitatory inputs from the hypothalamus (orexin), noradrenergic nuclei may upregulate RTN activity during arousal and stress. A partially pH-regulated serotonergic input may upregulate RTN during hyperthermia. Finally, RTN are excited during exercise, perhaps via input from the spinal locomotor network. In short, RTN is a pH-regulated hub whose output to the RPG appears essential to match alveolar ventilation with the metabolic production of CO2. During this presentation, gray or even controversial areas requiring considerable further research will also be pointed out.

10:45-11:25 am

Douglas Bayliss, Ph.D., University of Virginia, received B.Sc./M.Sc. degrees in Human Kinetics/Human Biology from the University of Guelph in Ontario, Canada, and a Ph.D. in Physiology from the University of North Carolina, Chapel Hill. After pursuing postdoctoral studies in Physiology and Biophysics at the University of Washington in Seattle, he accepted a faculty position in the Department of Pharmacology in the School of Medicine at the University of Virginia in Charlottesville. Dr. Bayliss is now the Joseph and Frances Larner Professor and Chair of Pharmacology at the University of Virginia. Dr. Bayliss has a longstanding interest in understanding how brain cell activity is regulated by neurotransmitters and their receptors, signaling pathways and ion channel effectors. A current research focus is on a select group of brainstem respiratory neurons that sense CO2 to regulate breathing, and the cellular and molecular basis for their intrinsic activity and chemosensitivity. These neurons are implicated in some of the cardinal clinical signs of CCHS, and work from the Bayliss laboratory has identified critical molecular mediators for CO2 sensing and cellular excitability in those neurons.

Molecular and cellular mechanisms for tonic activity, CO2 sensitivity and neuromodulation in Phox2b-expressing neurons of the retrotrapezoid nucleus
Congenital central hypoventilation syndrome (CCHS) is a neurodevelopmental disorder that results from mutations in the transcription factor, PHOX2B. The syndrome is characterized primarily by alveolar hypoventilation, as the name implies, with reduced respiratory drive and blunted reflex regulation of breathing and arousal by elevated CO2 or reduced O2. The mechanisms that account for these clinical signs have not been determined. In rodent models, substantial experimental evidence based on numerous complementary approaches has implicated a select group of Phox2b-expressing brainstem respiratory neurons located in the retrotrapezoid nucleus (RTN) for the reduced respiratory drive, and especially the blunted ventilatory and arousal effects of CO2. These RTN neurons are directly sensitive to CO2 (via H+, as proxy) and their ongoing activity reflects a host of feedforward and feedback inputs that are integrated to modulate overall respiratory drive. In this presentation, we will describe our work that has characterized the molecular phenotype of RTN neurons in mice, identifying unique markers for these cells and determining different cellular and ionic mechanisms that account for their baseline activity, intrinsic CO2/H+ sensitivity, neuromodulation and peptidergic excitatory transmission to respiratory centers. We will also discuss ongoing studies that seek to define the output pathways of RTN neurons in wild type and CCHS mouse models, particularly the molecular characteristics of specific target neuron groups associated with breathing and autonomic function. This work will highlight how these important Phox2b-expressing RTN neurons act as a chemosensitive and integrative respiratory control center regulating breathing and arousal functions, the disruption of which likely account for the cardinal pathophysiological features of CCHS.

11:30 am-12:10 pm

Yingtang Shi, M.D., University of Virginia, received her B.Sc./M.Sc. degrees in Biology/Physiology from the East China Normal University in Shanghai, and a M.D. degree in Human Anatomy and Histoembryology from Shanghai JiaoTong University School of Medicine. Dr. Shi undertook postdoctoral training in the Department of Neuroscience at the University of Virginia before moving to the Department of Pharmacology where she is an Assistant Professor of Research. Dr. Shi has a long-term interest in molecular and developmental neurobiology; she is currently investigating brainstem neurons that contribute to central respiratory control during development, physiological challenge, and disease conditions, with a specific focus on the role of the Phox2b transcription factor in mouse respiratory chemoreceptor neurons.

Molecular physiology of Phox2b-expressing brainstem neurons
Congenital central hypoventilation syndrome (CCHS) arises from mutations in the transcription factor, PHOX2B, which is necessary for the development and function of key brainstem nuclei involved in autonomic and respiratory control systems. Accordingly, alveolar hypoventilation, reduced respiratory and arousal reflexes, and dysautonomia are key clinical signs associated with CCHS. The mechanisms that account for the range of respiratory and autonomic signs associated with CCHS have not been determined, and the gene regulatory function of Phox2b in relevant brainstem nuclei has not been explored. We developed an adeno associated virus (AAV)-mediated approach to deplete Phox2b expression in specific brainstem neurons based both on cell selective expression of Cre recombinase and precise neuroanatomical targeting of virus injection. Specifically, we targeted retrotrapezoid nucleus (RTN) respiratory chemoreceptor neurons using Nmb-Cre mice, adrenergic C1 neurons and noradrenergic locus coeruleus neurons using Dbh-Cre mice, and nucleus tractus solitarius neurons using Phox2b mice. In this presentation, we will describe our ongoing studies to examine effects of Phox2b depletion in these different cells groups on: a) gene expression, as assessed by single cell molecular approaches; b) target neuron connections and function; and c) regulation of breathing and arousal by elevated CO2 or reduced O2. Our preliminary observations suggest that Phox2b is required for expression in the RTN of key genes involved in CO2-stimulated breathing, specifically GPR4 and TASK-2. In addition, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase, was downregulated after Phox2b depletion in C1 and LC neurons. We plan to present an unbiased expression analysis from single cell transcriptomic experiments that will identify genes and cell signaling pathways differentially regulated by Phox2b in these CCHS-relevant neurons, and the consequences of Phox2b depletion on breathing and arousal reflexes. We hope this work will yield new insights into the gene regulatory functions of Phox2b in specific brainstem cell groups relevant to CCHS and uncover novel cellular and/or molecular targets to examine for potential therapeutic intervention.

12:20-1:20 pm

Lunch: sponsored by Avery BioMedical @Yachtsman Steakhouse, Beach Club Resort

1:30-2:10 pm

Dan Mulkey, Ph.D., University of Connecticut, is Associate Head of Physiology & Neurobiology, University of Connecticut. Dr. Mulkey’s research centers on understanding the cellular and molecular basis of respiratory control.  His work includes identifying chemosensitive neurons in the retrotrapezoid nucleus (RTN), characterizing mechanisms controlling basal activity and transmitter modulation of RTN neurons, and discovering the role of RTN astrocytes in breathing control. Specifically, Dr, Mulkey’s lab uses a combination of electrophysiology, fluorescent imaging and genetic approaches to identify ion channels that regulate activity and neurotransmitter modulation of neurons that control respiratory function.  They use similar approaches to understand how disruption of these mechanisms contributes to life threatening breathing problems in disease states including Rett syndrome, Pitt Hopkins syndrome and epilepsy.  His findings have advanced our understanding of respiratory regulation and hold promise for improving respiratory disorder management.

Expression of an encephalopathy-associated Kcnq2 gain-of-function variant in Phox2b neurons suppresses respiratory activity
The recurrent KCNQ2 gain of function mutation R201C has been identified in patients with neonatal epileptic encephalopathy, a primary feature of this condition is severe hypoventilation reminiscent of congenital central hypoventilation syndrome. Despite this, little is known about roles of KCNQ2 in control of breathing. The retrotrapezoid nucleus (RTN) is an important site of respiratory chemoreception - mechanism by which the brain regulates breathing in response to changes in tissue CO2/pH- and previous work showed that Kcnq channels regulate output of the RTN. Therefore, we consider the RTN a potential substrate responsible for Kcnq2 related breathing abnormalities. To test this, we used the cre-loxP system to conditionally express Kcnq2R201C/+ in Phox2b-exprsssing neurons (Phox2bcre/+::Kcnq2R201C/+; Kcnq2 GOF). We find that Kcnq2 GOF mice exhibit a respiratory phenotype similar to humans with that same mutation including hypoventilation in room air and a blunted ventilatory response to CO2/H+. At the cellular level, expression of Kcnq2R201C/+ in chemosensitive RTN neurons resulted in cell autonomous hyperpolarization and diminished excitability. Interestingly, we also found that Kcnq2 function in RTN neurons is regulated by a H+-myo-inositol cotransporter (HMIT) that imports myo-inositol to support production of phosphatidylinositol 4,5-bisphosphate, a cofactor required for Kcnq2 channel activity. For example, HMIT is preferentially expressed by RTN neurons and at the cellular level exposure to myo-inositol strongly inhibits activity of RTN chemoreceptors by a Kcnq-dependent mechanism. Moreover, at the whole animal level, conditional deletion or knockdown of Slc2a13 (the gene encoding HMIT) from Phox2b+ neurons increased the ventilatory response to CO2/H+, as expected for loss of Kcnq2 channel activity. Together, these results identify Kcnq2 channels as important regulators of RTN chemoreceptors, and suggest HMIT is an important component of this mechanism.

2:15-2:55 pm

Silvia Pagliardini, Ph.D. University of Alberta, is an Associate Professor in the Department of Physiology, an Associate Adjunct Professor in the Department of Anesthesiology & Pain Medicine and the Neuroscience Honors undergraduate program coordinator at the University of Alberta. Her research lies at the intersection between respiratory physiology and neuroscience, focusing on the neuronal mechanisms responsible for respiratory rhythmogenesis and the effects of sleep on respiratory control. More recently her lab started investigating the effect of sex hormones on respiratory control in health and rodent models of central hypoventilation. Dr. Pagliardini is also the Team Lead on a CIHR funded Team grant project aiming to investigate the effect of cannabinoids on sleep and breathing. Her research program is funded by CIHR, NSERC and the Canadian Lung Association.

Etonogestrel promotes respiratory recovery in a model of chemoreflex impairment
The powerful effect of hypercapnia to stimulate breathing has long been recognized and the impairment in the response is one of the main symptoms in congenital central hypoventilation syndrome (CCHS). The Phox2b expressing neurons in the retrotrapezoid nucleus (RTN) are critical to this response, since their ablation greatly diminishes respiratory sensitivity to hypercapnia. It has long been postulated that female sex hormones have stimulatory effects on the respiratory neural networks. Yet, these results are not universal and hormonal treatment in various respiratory disorders of central origin is controversial. Here, we demonstrate that chronic administration of a potent progestin drug (Etonogestrel) restored CO2-chemoreflexes in adult female rats with medium, but not large, RTN lesions, which may shed light on ambiguity in previous studies and supports a role of progestins in CO2-chemoreflex potentiation.

3:00-3:40pm

Javier Oroz Garde, Ph.D., Institute for Physical Chemistry Spanish Research Council, is a “Ramón y Cajal” scientist from the Blas Cabrera Institute for Physical Chemistry (CSIC) in Madrid, Spain. His group is focused on the structural biology of prion-like polymorphic proteins which undergo structural transitions that may induce cell degeneration and are responsible for human diseases. In particular, Dr. Oroz is interested in understanding the structural impact of aberrant polyAlanine expansions in PHOX2B to uncover CCHS molecular pathophysiology. Providing the template structures which prime PHOX2B loss of function in CCHS is instrumental for the search of compounds that could restore protein functionality.

The structure of expanded PHOX2B reveals novel pathways for its malignancy in CCHS
Abnormal trinucleotide repeat expansions alter protein conformation causing malfunction and contribute to a significant number of incurable human diseases. Due to the repetitive, polymorphic and dynamic nature of expanded homorepeats, their structural study is highly challenging. Indeed, only sparse structural insights are available on homorepeat-expanded proteins related with disease, which hinders the design of effective therapeutics. For PHOX2B, polyAlanine repeat expansions in a 20-alanine tract are associated with CCHS. PHOX2B aggregation triggered by long polyalanine expansions is one of the fundamental mechanisms proposed to explain PHOX2B dysfunction in CCHS. However, the structural basis of the aggregation-prone PHOX2B mutants is still unknown. To help understand the pathogenic nature of polyalanine expansions in PHOX2B, we have determined the structure and dynamic properties of PHOX2B C-terminal fragment by Nuclear Magnetic Resonance spectroscopy. Interestingly, while polyalanine expansions do not affect PHOX2B main structural conformations, they promote nascent conformations that prompt a length-dependent liquid to solid transition within biomolecular condensates that capture wild-type PHOX2B. The direct observation of the nascent polymorphs in expanded PHOX2B leads us to propose unbalanced phase separation as a novel pathophysiological mechanism in homorepeat expansion diseases, paving the way towards the search of therapeutics modulating biomolecular condensates in CCHS.

3:40- 3:55 pm

Break

3:55-4:35pm

Avraham Ashkenazi, Ph.D.,Tel Aviv University, is a senior lecturer/assistant professor position at Tel Aviv University. Dr. Ashkenazi’s long-term scientific goal is to identify mechanisms that contribute to neuron survival and exploit them to fight neurological disorders. Dr. Ashkenazi's laboratory combines stem cell technology, animal models, and biochemical and cellular approaches to reveal protein degradation mechanisms in tri-nucleotide repeat disorders and in Parkinson’s disease.

Regulation of Proteostasis by PA Expansion
Dr. Ashkenazi will discuss polyalanine expansions and the ubiquitin cellular system. Trinucleotide repeats present within coding regions of the genome encode single amino acid repeats. Expansion mutations of trinucleotide repeats within translated sequences of either glutamine or alanine amino acids have now been linked to a growing number of neurological disorders. The vast majority of cases are sporadic and de novo in-frame duplications have been reported. Disease-causing expansions vary in length, depending upon the gene in question, with the severity of the associated clinical phenotype generally increasing with the length of the expanded tract. The exact functional or structural role of alanine repeats (polyalanine) within wild-type mammalian proteins is unclear. In the native state, we showed that a polyalanine motif in the ubiquitin-conjugating E2 enzyme UBE2Z/USE1 contributed to USE1 ubiquitin loading by the E1 ubiquitin-activating enzyme, UBA6. In addition, our results identify a domain in UBA6 that recognizes polyalanine-containing proteins. Under disease conditions, we showed that UBA6 preferentially interacts with different polyalanine expanded disease-causing proteins including mutant PHOX2B, thereby competing with USE1 binding. These pathological interactions disrupted downstream ubiquitin signaling to target proteins including the E3-ubiquitin ligase, E6AP that is involved in neurodevelopment. Since similar effects could be seen in autonomic CCHS patient-derived neurons, we suggest that this represents a previously undescribed vulnerability caused by polyalanine expansion mutations.

4:40-5:20pm

Diego Fornasari, M.D., Ph.D., University of Milan, is an Associate Professor of Pharmacology in the Department of Medical, Biotechnology, and Translational Medicine, University of Milan. He is the Director of SBBL, the virtual Biomedical Library of Regione Lombardia, as well as Director of the Postgraduate School in Clinical Pharmacology and Toxicology, University of Milan. Dr. Fornasari has extensive experience in gene expression regulation in neuronal as well as non-neuronal cell models, with particular emphasis on transcription regulation of autonomic nervous system development and function. His scientific interests have been concentrated on PHOX2A and PHOX2B, two master genes that play crucial roles in regulating not only the development but also the functions of the Autonomic Nervous System (ANS). Under Dr, Fornasari’s  supervision, his laboratory has implemented new technological approaches that have allowed critical questions about the biology of PHOX2B and its role in Congenital Central Hypoventilation Syndrome (CCHS) to be addressed. Recently, they have successfully generated induced pluripotent stem cell (iPSC) lines from two CCHS patients, both carrying +5 Ala mutations but showing different clinical onsets (early or late). Through independent work and collaborations, they have published several significant papers that have challenged the prevailing notion of CCHS as an incurable developmental disorder. Instead, Dr. Fornasari has been advocating the potential for pharmacological intervention to alleviate symptoms and reduce the risk of death during sleep. Due to his extensive studies on CCHS, Dr. Fornassari has been appointed a member of the scientific committee of the Italian Association of CCHS families.

New research strategies and novel potential therapeutic targets in Congenital Central Hypoventilation Syndrome
Mutations in the PHOX2B gene cause Congenital Central Hypoventilation Syndrome (CCHS), impacting the Autonomic Nervous System (ANS) development. In vivo and in vitro studies indicate that the disease results from a combination of loss of function, dominant-negative effect, and/or toxic gain of function of the mutated proteins. Presently, there are no pharmacological treatments available for CCHS or its symptoms. However, a fortuitous clinical observation revealed that the progestin desogestrel might improve respiratory functions and reduce risks during sleep for CCHS patients. Our in vitro and in vivo research demonstrated that desogestrel downregulates the expression of both wild-type and mutant PHOX2B proteins, along with some of their target genes. This suggests that the clinical effect may be attributed to limiting the toxic impact of the mutant protein.
Human-induced pluripotent stem cell (hiPSC) technology is essential to overcome the limitations of current in vivo and in vitro models for Congenital Central Hypoventilation Syndrome (CCHS) and to create patient-specific cell models. This personalized disease-in-a-dish approach allows researchers to explore molecular and cellular defects induced by CCHS-causing mutations and provides a platform for drug discovery and screening for potential therapies. Recent research has revealed a natural antisense long non-coding RNA, PHOX2B-AS1, in the PHOX2B gene locus, which influences the production of PHOX2B protein. The discovery of PHOX2B-AS1 offers the prospect of a novel therapeutic strategy targeting its expression to mitigate the toxic effects of mutant proteins during the differentiation of autonomic neurons derived from CCHS patients. The presentation will review recent advances in therapeutic approaches to CCHS and explore the potential of PHOX2B-AS1 as a therapeutic target.

6:30 PM

Dinner on site: Your choice

8:00 PM...

Social Outing: Movie and Fireworks on the beach; evening treat provided

Breakfast on site: your choice

9:00-10:00 am

Poster Session:Cape Cod Lobby
(Set up 8:00-9:00)

10:15-10:55 am

Gordon Mitchell, Ph.D., University of Florida, received his PhD in Developmental and Cell Biology from the University of California at Irvine in 1978, followed by postdoctoral work at the Max Planck Institute for Experimental Medicine in Göttingen, Germany, where he worked with Dr. Peter Scheid. He then moved to the University of Wisconsin-Madison where he became a faculty member in 1981. After progressing to full professor in 1992; he served as department Chair for 17 years before leaving to join the University of Florida as a Preeminence Professor of Neuroscience in the Department of Physical Therapy and the McKnight Brain Institute. There he founded the Breathing Research and Therapeutics Center and the Training Program of the same name. Dr. Mitchell has received numerous research awards, including a MERIT Award from the National Institutes of Health, is a Fellow of the American Physiological Society, and was the UW Steenbock Professor in Behavioral and Neural Science. He has delivered multiple award lectures, including the Society for Neuroscience Special Lecturer (2008), Julius H. Comroe Distinguished Lecturer (APS, 2014), and Guyton Distinguished Lectureship (2014). His research concerns (cellular and network) mechanisms of respiratory and non-respiratory motor plasticity, particularly spinal plasticity induced by intermittent hypoxia, and translation of that work to develop novel therapies for humans living with chronic spinal cord injury, ALS and other clinical disorders that compromise movement.

In the translational flywheel: therapeutic acute intermittent hypoxia to improve breathing
Although the intermittent hypoxia experienced by people with obstructive sleep apnea is widely understood to elicit pathology, “low-dose” intermittent hypoxia has many beneficial effects (without detectable pathology) and is emerging as a promising therapeutic modality to restore breathing and non-respiratory motor function in people living with chronic spinal cord injury, ALS and other clinical disorders that compromise breathing and other movements. In this lecture, I will briefly review mechanisms whereby low-dose (therapeutic) acute intermittent hypoxia (tAIH) elicits spinal neuroplasticity in the phrenic motor system (phrenic long-term facilitation), and studies leading to the realization that tAIH elicits similar plasticity in non-respiratory motor systems. These foundational discoveries will be discussed in the context of ongoing efforts to harness tAIH to restore breathing and limb function in humans living with chronic spinal cord injury. Pilot clinical trials have informed the need for new additional experiments using animal models to develop greater understanding concerning: 1) optimization of tAIH protocols (depth & duration of hypoxia, interval duration, episodes/day & number/pattern of days); 2) biomarkers to identify individuals most/least likely to benefit from tAIH; and 3) combinatorial treatments to amplify/extend therapeutic benefits of tAIH. Recent discoveries in this “translational flywheel” will be featured to help guide the next round of clinical trials. Applications to other neuromuscular disorders (e.g. ALS, MS, TBI, stroke) will be considered.

11:00-11:40 am

Jan-Marino (Nino) Ramirez, Ph.D., Seattle Children's Research Institute. The Pathology of Dysautonomia: Lessons Learned from the Clinic and Basic Neuroscience (Remote)

11:45 AM-12:25 pm

James Oakley, Keep Me Breathing. Breathing Pacer Development

12:30-1:30 pm

Lunch on site: Your choice

1:30-2:10 pm

Nathan Beckouche, Ph.D., AtmosR, is a biologist who received his training at Pierre and Marie Curie University in Paris, where he completed his PhD. After a decade of academic research focused on cardiology and vascular biology, he transitioned to Alexion Pharmaceuticals, where he began working on rare diseases. During this time, he had the opportunity to collaborate with the French CCHS patient organization, serving on their scientific board. Dr. Beckouche's initial involvement with the CCHS community led to his pivotal role in the establishment of the biotech company AtmosR. This innovative organization is dedicated to the development of treatments for rare central nervous system (CNS) disorders, with a specific focus on CCHS. Through his work at AtmosR, Dr. Beckouche continues to contribute to the advancement of therapeutic options for individuals living with CCHS and other rare CNS disorders.

Drug screening discovery process for CCHS
Dr. Beckouche will discuss AtmosR’s innovative drug discovery process for CCHS. AtmosR employs an iterative process involving in vitro screening using FRAP technology to restore the molecular function of mutated PHOX2B. Promising compounds are evaluated in mouse models to assess their effectiveness in restoring the response to hypercapnia. Advanced proprietary molecules have been identified and are progressing through preclinical development. AtmosR also seeks collaborations to expand their pipeline with additional molecules. Their goal is to develop new therapies for CCHS and improve the lives of affected individuals.

2:15-2:55 pm

Casey Rand, MSDS, CCRP, Lurie Children's Hospital, is the Research Director at the Center for Autonomic Medicine in Pediatrics, Ann and Robert H. Lurie Children’s Hospital of Chicago and Stanley Manne Research Institute. Casey has 20 years of experience working in pediatric autonomic rare disease research.  His work on CCHS has ranged from laboratory investigations to understand the genetic underpinnings, to designing innovative clinical assessments of autonomic function to better understand patient stability, to technology development to improve patient monitoring and treatment.  Currently, Mr. Rand is helping lead an NIH-funded investigation of physiologic biomarkers of CCHS disease stability and progression, with a vision toward developing a platform for empowering future clinical trials.

PHOX2B Mutation-Confirmed Congenital Central Hypoventilation Syndrome (CCHS): Validating Wireless Capture of Ambulatory Biomarkers for Clinical Trial Readiness
Congenital central hypoventilation syndrome (CCHS), a rare and severe neurocristopathy caused by PHOX2B gene mutations, is characterized by profound hypoventilation and impaired automatic control of breathing. Patients suffer from a spectrum of severe symptoms compatible with autonomic dysregulation. Currently, no pharmacologic interventions have been demonstrated to decrease disease burden in CCHS, and the limited treatment options available are highly invasive, burdensome and offer only palliative support. Long considered a congenital disease with little hope for long-term improvement, mounting evidence suggests that many aspects of the CCHS phenotype, including respiratory control, cardiovascular, autonomic, and neurocognitive function, are part of ongoing disease processes that develop over time and are sensitive to intervention. Recent CCHS cases have been reported who appeared normal until a precipitating factor, such as severe respiratory infection or anesthetic exposure. These cases indicate that at least some CCHS patients maintain the physiologic potential to function without artificial ventilatory support and offer hope that some severe aspects of CCHS could be reversed. Potential for pharmaceutical and device intervention has been highlighted by reports of successful off-label drug use in CCHS case reports, cellular models indicating potential for reversing CCHS-related pathogenic processes, advances in gene editing, and development and approval of drugs and devices in diseases with shared phenotypic presentation and/or pathogenic underpinnings that have game-changing potential in CCHS. The imminent potential for candidate therapeutics underscores a critical need for clinical trial readiness. In the absence of identified objective biomarkers, clinical trials to assess the safety and efficacy of potentially life-changing therapeutics will be disrupted, diminishing their chances of approval and near-term impact. Utilizing a suite of clinical data captured over decades of caring for CCHS patients, we have identified several potential biomarkers for CCHS that could serve as sensitive markers of disease progression and response to intervention. However, approval of these biomarkers for clinical trial readiness will require an expanded cohort of CCHS cases with longitudinal data on biomarker stability and sensitivity. Recent advances in wireless wearable technology have opened the door to advanced monitoring of physiology during activities of daily living in the patient’s own home. Such technology has high potential to empower data capture critical to clinical trial readiness in expanded cohorts of CCHS patients. This presentation will share our results from such technologies that have the capability to capture specific data streams necessary for validating candidate biomarkers identified in our clinical data set. This is a critical step in establishing a foundation for accelerating clinical trial readiness in CCHS, advancing potential for impactful intervention in the near-term. These time sensitive data constitute a first step toward allowing analytics not previously possible in this disorder to validate individual and composite respiratory, cardiovascular and cerebrovascular biomarkers for stability and sensitivity to disease progression. The overall aim of this ongoing research is to empower design, conduct, and interpretation of rigorous clinical trials to assess candidate therapeutics and technology, increasing their likelihood of success and potential to reduce morbidity and mortality in CCHS patients.

2:55-3:10 pm

Break

3:10-3:50 pm

Ajay Kasi, M.D., Children’s Healthcare of Atlanta, is a pediatric pulmonologist and assistant professor of pediatrics at Emory University and Children's Healthcare of Atlanta. He completed pediatric residency in Nicklaus Children's Hospital, Miami, FL and pediatric pulmonology fellowship in Children's Hospital Los Angeles. Since starting as a pediatric pulmonologist in Atlanta, he has established a CCHS and diaphragm pacing program. He has published on clinical aspects of caring for children with CCHS and includes trainees in his research projects.

Atypical Presentations in NPARMS 
PHOX2B NPARMs are generally associated with severe phenotypes requiring full-time assisted ventilation, Hirschsprung's disease, and neural crest tumor risk. However, recent studies have reported relatively milder phenotypes and atypical presentations with PHOX2B NPARMs including lack of Hypoventilation. In this presentation, we will review the atypical presentations and management in NPARM patients.

3:55-4:35 pm

Yakov Sivan, M.D., Tel Aviv University, is a Pediatric Pulmonologist, Intensivist & Sleep Medicine expert @ Sheba Medical Center in Israel (trained in CHLA 1987-1990, and Stanford 2005). 6 years ago, Dr. Sivan has started the 1st CCHS Center in Israel that together with an interdisciplinary team of specialists has established standards and provides care, follow-up and support at home to all patients in Israel and beyond. Consultations and education is being provided to all relevant interdisciplinary care-givers in Israel.

Congenital Central Hypoventilation Syndrome in Israel - Novel Findings from a New National Center
Dr. Sivan will present the Israeli experience concentrating on unique new findings in CCHS epidemiology, phenotype and genotype and treatment options, and will share dilemmas and possible contributions to research.

5:30-...

Social Outing: Epcot Food and Wine Festival

Breakfast on site: Your choice

9:00-9:40 am

Gad Vatine, Ph.D., Ben-Gurion University of the Negev, is an Associate Professor, The Physiology and Cell Biology Department and the Regenerative Medicine and Stem Cell (RMSC) Research Center, Ben-Gurion University of the Negev. Dr. Vatine received his Ph.D. in 2011 from Tell Aviv University where, he studied the mechanisms underlying light-entrainment of the circadian clock using zebrafish as a model. He followed this with a short postdoc at Bar-Ilan University where he established a zebrafish model for the rare psychomotor disability disorder MCT8-deficiency. Next, he joined the laboratory of Dr. Clive Svendsen at the Regenerative Medicine Institute at Cedars-Sinai where he pioneered the concept of disease modeling at blood-brain barrier (BBB) using patient-specific induced pluripotent stem cells (iPSCs). In 2017, he became an assistant professor at The Physiology and Cell Biology Department and the Regenerative Medicine and Stem Cell Research Center at Ben-Gurion University of the Negev. The Vatine lab generates personalized iPSC-based models to study mechanisms underlying various rare neurological disorders, and to test potential treatments. He is also the director of the BGU-iPS-core facility.

Transcriptome analysis of patient-specific iPSC-based autonomic neurons
Mouse models with knocked-out or overexpressed Phox2b are helpful in understanding CCHS. However, differences between species highlight the need for human-relevant models. We created and fully characterized induced pluripotent stem cell (iPSC) lines from three CCHS patients with 20/25 or 20/27 polyalanine expansion mutations (PARMs) and two healthy control relatives (CTR). These iPSCs were differentiated into PHOX2B-positive autonomic neurons (iANs) expressing relevant cell markers and exhibiting spontaneous electrophysiological activity. Our findings revealed that cells derived from CCHS patients displayed a fraction of PHOX2B that did not translocate to the nucleus and instead mislocalized to the cytoplasm, indicating potential disease-relevant traits. To study PHOX2B-regulated molecular pathways, we used sNuc-seq analysis on a CTR line, identifying specific populations with PHOX2B expression. By comparing PHOX2B positive and negative cells, we discovered a list of differentially expressed genes (DEGs). To investigate the impact of PARMs on molecular pathways, we performed sNuc-seq analysis on two CCHS lines and two CTR lines. Preliminary data analysis indicates that each cell line is distributed differentially across clusters, suggesting that PARMs may influence the differentiation process. In conclusion, our patient-specific platform demonstrates the ability to unveil biochemical and molecular distinctions between CCHS and CTR cells, making it a valuable tool for CCHS research.

9:45-10:25 am

Isabella Ceccherini, Ph.D., IRCCS Giannina Gaslini Institute, started to work in 1988 in the Medical Genetics Unit (Gaslini Institute). Since then, she has acquired a broad scientific background, specific trainings and expertise in human molecular genetics, particularly neural crest development and its congenital defects, autoinflammatory disorders, genotype- phenotype correlation, complex inheritance, molecular pathogenesis. She has been using the Next Generation Sequencing (NGS) technology for the last 10 years to diagnose and gene search in rare diseases. She has been involved in the study of Congenital Central Hypoventilation Syndrome (CCHS) since 2002, assessing i) >100 PHOX2B mutations in patients, ii) relation between length of the PHOX2B polyAla tract with severity of the respiratory phenotype, age at onset, trans−activating activity, percentage of cells with PHOX2B cytoplasmic localization and aggregates. Somatic mosaicism was observed among asymptomatic parents of CCHS probands leading to a total of 25% of families with mutation inheritance versus 75% of de novo events. Finally, the drug geldanamycin (GA) was shown to effectively induce clearance of PHOX2B polyAla aggregates and rescue the PHOX2B ability to transactivate its target promoters. has been working on rare genetic diseases since 1988 at Istituto Giannina Gaslini, a pediatric hospital in Genova (Italy). Together with her lab group, she has set up molecular diagnostic protocols, performed >300 genetic tests for CCHS, detected somatic mosaicism in asymptomatic parents, investigated the pathogenic mechanisms of CCHS associated PHOX2B mutations, tested several candidate drugs for beneficial cellular effects. 

Multiomics approach to identify possible PHOX2B genetic modifiers responsible for phenotypic variability in CCHS
Congenital Central Hypoventilation Syndrome (CCHS) is a rare genetic disorder affecting breathing regulation, chronically managed by ventilator supports, tracheostomy or diaphragmatic pacers. It is caused by mutations in the PHOX2B gene. Mutations in PHOX2B result in abnormal protein function and impaired gene activation. Researchers have observed potential benefits in vitro of using Hsp90 inhibitors to modulate pathogenic cellular response. The severity of CCHS can vary, indicating the influence of other genetic factors. Further investigation is needed to identify and understand these modifiers and develop targeted therapeutic approaches of them. To this end, we investigated the phenotypic variability observed in CCHS patients in association with same causative PHOX2B mutations, applying transcriptomics to cells expressing the +13Ala mutant allele with or without treatments, thus allowing us to identify the pathways most involved in the molecular pathogenesis of PHOX2B polyalanine expansion mutations in CCHS. Whole exome sequencing was then conducted in affected child-parent pairs and late-onset patients with a 20/25 genotype, with the purpose to identify genetic variations in genes involved in protein homeostasis, which ensures the proper degradation of misfolded proteins and their aggregates through protein quality control mechanisms. These variations may potentially influence the severity of CCHS.

10:30 am - 12:00 pm

Workshops:
Research Group A: Cellular Mechanisms
Research Group B: Treatment Development/Future Technologies
Clinical Group: Patient Care

12:00-12:30 pm

Next Steps/Closing Remarks

Poster Session: Cape Cod Lobby