FPWR 2020 Virtual Research Symposium Poster Session

Prader-Willi Syndrome (PWS) is a genetic neurodevelopmental disease with a complex pathological phenotype including respiratory alterations, present from birth to adulthood. NECDIN is one of the genes invalidated in PWS, and Necdin-KO mice is the only mouse model reproducing the respiratory alterations observed in patients. They include apneas and a blunted respiratory response to hypoxia and hypercapnia. We showed previously that from embryonic day (E) 10, Necdin-KO mice exhibit anatomical and functional alteration of serotoninergic (5-HT) neurons, involved in the genesis of respiratory alterations. We hypothesized that restoring the expression of Necdin in 5-HT neurons, and/or in all neurons, would prevent the onset of respiratory alterations. We performed in utero intracerebroventricular injection in Necdin-KO embryos (Ndn tm1.1Mus) either at E12.5 or at E14.5 with viruses inducing the expression of Necdin, either only in 5-HT neurons (AAV2-TPH2-Necdin) or pan-neuronally (AAV9-CBA-Necdin). We then assessed the expression of Necdin at the post-natal day P7 by immunohistochemistry. The AAV9-CBA-Necdin induced brain-wide Necdin expression, showing the efficacy of the combination of this virus serotype, the promoter used, and the injection age and procedure. Unfortunately, in utero injection of AAV2-TPH2-Necdin under the same conditions did not induce Necdin expression. We aim next at determining whether pan-neuronal Necdin expression in utero prevents the development of 5-HT and respiratory alterations in Necdin-KO mice. This preliminary work will provide new mechanistic insights on the respiratory phenotype in PWS. Also, it is the first step to develop a proof-of-concept gene therapy approach that could be extended to all genes involved in PWS.

Julie Buron <julie.buron@inserm.fr>

Institut de Neurobiologie de la Méditerranée (INMED), INSERM, Aix-Marseille University, Marseille, France

CSTI-500 is a novel triple monoamine reuptake inhibitor, similar in its mechanism of action to tesofensine, but with a different pharmacological profile across the three transporters. CSTI-500 exhibits comparable binding affinity to dopamine (DAT) and norepinephrine (NET) transporters but with greater affinity for the serotonin transporter (SERT) at the therapeutic level of known SSRIs; tesofensine appears to have lower affinity for SERT and higher affinity for DAT and NET. CSTI-500’s SSRI-level activity is expected to help address the compulsive eating behavior and other neuropsychiatric symptoms in Prader-Willi syndrome (PWS), and this is in addition to the potential therapeutic benefit of CSTI-500 on hyperphagia. A double blind, placebo-controlled, Phase 1 (Single Ascending Dose + Multiple Ascending Dose [MAD] studies) clinical trial in healthy volunteers has been conducted to assess the safety, tolerability, pharmacokinetics and pharmacodynamics (PET imaging) of CSTI-500. The drug was well tolerated up to 100 mg single dose and 60 mg multiple dose in healthy males. Overall, CSTI-500 is generally well tolerated, and the adverse events profile was consistent with this class of drugs. There was moderate pharmacokinetic variability and the observed increases in key variables were dose-proportional, with a Tmax of ~2 hours and a half-life of ~50 hours. The PET imaging study confirmed the target engagement of CSTI-500 in human brain. An average of ~1.2% body weight reduction per week was observed in the MAD study. Overall, this data provides a solid basis for further investigation of CSTI-500 in indications such as PWS and hypothalamic injury-induced obesity.

Roman Dvorak <roman@consynance.com>

1ConSynance Therapeutics, Inc (USA), 2Bristol-Myers Squib (USA), 3Quintiles AB (Sweden), 4Uppsala University Hospital (Sweden) (Affiliations 2, 3 and 4 are at the time the studies were conducted)

The efficacy of Acceptance and Commitment Training (ACT), delivered on-line to a group of fathers who have adolescents with PWS, is assessed. ACT teaches coping strategies to decrease perception of stress and increase cognitive flexibility, resulting in an increase in valued action and allocation of resources to improve family function. Fathers were recruited via social media and met criteria for participation. Baseline questionnaires assessed parental demographics, family function, level of stress and burden in both parents. Also, the degree of behavioral dysfunction attributed to the PWS phenotype (PWS Behavior Questionnaire) in the adolescent was assessed by both parents. Parental stress and demographics were measured using the Zarit Burden Interview. A parental stress questionnaire (Cohen PSS-10), a 10-item, self-report questionnaire that uses a 7-point Likert scale for each item, was obtained at baseline, after sessions 2 and 4, at the conclusion of treatment, and 6 weeks post treatment. Psychological flexibility and parenting satisfaction were measured using the Parental Acceptance and Action Questionnaire (6-PAQ) ), a 15-item, self-report questionnaire using a 7-point Likert scale for each item. The 6-PAQ was administered at baseline, at the conclusion of treatment and 6 weeks post treatment. The Moos Family Environment Scales, a measure of family functioning, were administered at baseline and 6 weeks post-treatment. This preliminary report describes the level of stress, family function, degree of acceptance, and phenotypic characteristics in 4 families who have an adolescent with PWS. Parents with adolescent children with PWS are stressed, family function is distressed, and fathers display difficulties being mindfully present with the adolescent, emotionally reactive to the adolescent, and accepting of their adolescent’s degree of dysfunction. These parenting characteristics may be amenable to change through ACT. It was difficult to recruit subjects for participation in this pilot study. This may reflect the underlying difficulty engaging fathers in the family process of parenting an adolescent with PWS.

Janice Forster <janiceforstermd@aol.com>

PLEA, FPWR

PATH for PWS (Paving the way for Advances in Treatments and Health) is an on-going 4-year, non-interventional, natural history study of participants with PWS in the United States, Canada, Australia, and New Zealand. PATH is being conducted through the Global PWS Registry (www.pwsregistry.org). PATH is aimed at: developing a better understanding of the serious medical events individuals with PWS experience; identifying what factors may increase or decrease risk for serious medical events; and tracking the changes of PWS over time. Patient/caregiver reported data is collected at 6-month intervals. PATH questionnaires include capturing both Thrombotic and Non-Thrombotic Serious Medical Events, PWS-Profile, Food Safety Zone, and the Hyperphagia Questionnaire for Clinical Trials (HQ-CT).

HQ-CT was developed specifically for assessing behaviors associated with hyperphagia in the PWS population, and has been widely used as a primary endpoint in Phase 2 and 3 clinical trials. Here, will we report on initial baseline HQ-CT data for more than 600 PATH for PWS study participants. Ages of the participants range from 5-61 years old, with 60% under the age of 18, and 40% 18 years and older. Gender demographics are 52% female, 48% male. Geographically, the participants predominantly reside in the United states (88.72%), followed by Canada (7.56%), Australia (2.78%) and New Zealand (<1%). Baseline analysis of HQ-CT score will be provided by age of participant, PWS genetic subtype, and BMI. This data will provide a foundation for understanding hyperphagic behaviors and variability in HQ-CT scores over time in a broad PWS population.

Jessica Bohonowych <jessica.bohonowych@fpwr.org>

1Foundation for Prader-Willi Research, Walnut, California; 2University of Florida, Gainesville, Florida; 3University of Colorado, Denver, Colorado

Background Emotional outbursts (also known as temper outbursts) are prevalent and have a significant negative impact for people with Prader-Willi syndrome, and for people with other neurodevelopmental conditions. Emotional outbursts may manifest via different pathways that are related to specific setting events and triggers. This study aimed to identify these contextual pathways transdiagnostically through a newly designed caregiver-report questionnaire.

Methods 268 caregivers of young people (6-25 years) with neurodevelopmental conditions – including PWS – completed the Emotional Outburst Questionnaire online. Potential pathways were identified by examining the patterns of setting events and triggers related to emotional outbursts through factor and cluster analyses.

Results Six contextual factors were derived from the Emotional Outburst Questionnaire. Based on these factors, the responses were classified into three clusters, which may represent potential pathways of emotional outbursts. The three clusters were characterised by the increased likelihood of outbursts: 1) across all setting events and triggers; 2) under safe setting events; 3) across setting events. Whilst there were some associations between diagnoses and cluster membership, the heterogeneity of diagnoses within each cluster supported the transdiagnostic approach.

Conclusion The mechanisms of these potential pathways might be related to: 1) sensory processing difficulties; 2) masking of emotions in unsafe environments; 3) differences in threat perception. Establishing the potential pathways of emotional outbursts may facilitate the identification of the context-dependent cognitive and emotional differences that distinguish these pathways. These cognitive and emotional differences may consequently inform the development of pathway-specific intervention strategies. In future work we hope to examine how such transdiagnostic pathways may intersect with PWS phenotypic features, which may support further personalisation of intervention strategies.

Acknowledgements/Funding: Thank you to all parents and caregivers who took part in the research, as well as the organisations, including FPWR, PWSA-UK, and PWS Australia, who have helped promote the study.

Justin Chung <justincychung@googlemail.com>

Centre for Applied Psychology, University of Birmingham

Background. Prader-Willi Syndrome (PWS) is a genetic disorder that results from lack of paternally derived imprinted material on chromosome 15q11-q13. This region is also implicated in the etiology of Autism Spectrum Disorder (ASD), and not surprisingly, ASD is frequently diagnosed in association with PWS. The factor structure of the Aberrant Behavior Checklist (ABC), a commonly used rating scale that assesses maladaptive behaviors, has been validated in children with ASD (it was originally developed for children with developmental disabilities).The factor structure of the ABC in children with PWS has not yet been examined. A factor analysis may determine whether the ABC-ASD subscales are valid in PWS, or if different PWS-specific subscales emerge. The factor structure of the ABC may help to better define the PWS phenotype and also provide a more precise PWS-specific outcome measure for use in clinical research studies.

Objectives. To conduct a Principal Component analysis (PCA) to examine the factor structure of the ABC in children with PWS.

Methods. Primary caregivers of 23 children with PWS completed the ABC. The ABC contains 58-items that load onto five separate factors; 1. Irritability; 2. Social Withdrawal; 3. Stereotypic Behavior; 4. Hyperactivity/Noncompliance; and 5. Inappropriate Speech. In this study, a PCA on the ABC was performed using an ordinary least squares estimation with oblique Promax rotation.

Results. PCA supported the retention of 6 factors determined on the basis of the eigenvalue >1 rule, examination of the scree plot and clinical meaningfulness. Compared to the factor structure of the ABC in ASD, different factors occurred in the PWS sample and are as follows: Factor 1. Hyperactivity- the noncompliant items from the original ASD Hyperactivity/Noncompliant subscale were removed; Factor 2. Irritability- Similar to the ASD Irritability factor except the three self-injurious behaviors were removed; Factor 3. Noncompliant/Nonresponsive- this is a new factor that emerged and consists of the noncompliant items from the original Hyperactivity/Noncompliant factor and items from the ASD Lethargy /Social Withdrawal factor; Factor 4. Repetitive Motor, Speech, and Self-Injurious Behaviors- this is another new factor that materialized and consists of items from the original Stereotypic Behavior and Inappropriate Speech factors, and the self-injurious behavior items from the ASD Irritability factor, Factor 5. Lethargy- consists of items from the ASD Lethargy /Social Withdrawal and the Stereotypic Behavior factors, and Factor 6. Social Withdrawal- consists of items from the Lethargy /Social Withdrawal factor.

Conclusions. Six modified ABC factors were derived from a PCA in a sample of children with PWS: 1. Hyperactivity; 2. Irritability; 3. Noncompliant/Nonresponsive; 4. Repetitive Motor, Speech, and Self-Injurious Behaviors; 5. Lethargy; and 6. Social Withdrawal. The pattern of factor modifications between the PWS and original ASD sample is consistent with clinical observation and our understanding of the neurobiology of children with PWS. Firstly, an independent Lethargy subscale in the PWS sample is not surprising given that individuals with PWS have low muscle tone and often present with sleep apnea. Secondly, the Noncompliant/Nonresponsive factor may be demarcating behaviors that are associated with food seeking, which is the hallmark behavior in PWS. Lastly, a new PWS-specific Repetitive Motor, Speech, and SIB factor may represent behaviors that transpire when rigid behaviors, which are highly prevalent in PWS, are interfered with and/or when the child experiences a deviation from expectations. Future studies should utilize larger samples to assess the reliability and validity of these PWS-specific ABC factors.

Bonnie Taylor, PhD <botaylor@montefiore.org>

Montefiore Medical Center-Albert Einstein College of Medicine. Department of Psychiatry and Behavioral Sciences

The current COVID-19 pandemic and restrictions has had broad impacts on daily living and access to mental health services for everyone including people with Prader-Willi Syndrome. Very little is known about the risk and protective factors for mental health during and after prolonged threats, like the pandemic, which continues to unfold. CRISIS is an online survey, developed to rapidly evaluate the impact of the pandemic and restrictions on the mental health, behaviour and well-being of typically developing youth. An adaption, CRISIS-AFAR was developed for use with youth with neurodevelopmental disabilities. Our research group further adapted the survey, CRISIS-AFAR-PWS for people with PWS. The CRISIS survey assess the following domains: (1) background and demographics; (2) physical and mental health three months prior to the pandemic; (3) COVID-19 exposure and infection status; (4) life changes due to the pandemic; (5) concerns and worries associated with COVID-19 and (7) behaviour factors. The PWS adaption included medical and behaviour questions specific to PWS. Data collection for this study is currently ongoing. Preliminary data from the survey that is specific to PWS is presented.

Louise Gallagher <LGALLAGH@tcd.ie>

Department of Psychiatry, Trinity College Dublin

Background: Hyperphagia leading to severe obesity with increased morbidity and mortality is the major manifestation of Prader-Willi syndrome. Caring for these individuals in a home environment is challenging and stressful for caregivers and families. Residential hostels specifically for PWS adults offer programs of diet, exercise, and vocational opportunities, but long-term effects of PWS hostel living on have not been reported. Objectives: Study changes in body mass index (BMI) for PWS adults living in residential hostels and compare BMI and mortality with age-matched controls living with their families at home. Patients and methods: The study included all 34 individuals (18 men) ages >17 years with genetically confirmed PWS living in residential hostels. BMI was recorded at the time of yearly clinic visits and compared to 23 PWS adults (10 men) living at home. Results: BMI on entering the hostel was 36.3±11.0 kg/m2, decreased to 27.0±5.6 kg/m2 (p<0.001) after 6.9±3.9 years, and rose slightly to 28.8±5.1 kg/m2 BMI of 23 PWS adults living at home was 36.8±12.7 kg/m2 while for hostel residents in the same age range BMI was 27.9±7.1 kg/m2 (p=0.008). From 2008 to 2019, there were five deaths, mainly due to complications of obesity, among PWS individuals ages 18-40 years living at home, compared to one death (a 43year-old man) among hostel residents.) Conclusions: Adults with PWS living in hostels lose weight, maintain BMI values in a normal to mildly overweight range, and have lower mortality in contrast to individuals in a family home environment.

Harry Hirsch <hirschmd@gmail.com>

(1) Multidisciplinary Prader-Willi syndrome clinic, Neuropediatric Unit, Department of Pediatrics, Shaare Zedek Medical Center, Jerusalem, Israel; (2)Herman Dana Division of Child and Adolescent Psychiatry, Hadassah-Hebrew University Medical Center, Jerusalem, Israel; (3) Seymour Fox School of Education, The Hebrew University, Jerusalem, Israel; (4) Hebrew University School of Medicine, Jerusalem, Israel

The objective of this study was to investigate lobule-specific cerebellar structural alterations relevant to clinical behavioral characteristics of Prader-Willi syndrome (PWS). We performed a case-control study of 21 Japanese individuals with PWS (age; median 21.0, range 13–50 years, 14 males, 7 females) and 40 age- and sex-matched healthy controls with typical development. Participants underwent 3-Tesla magnetic resonance imaging. Three-dimensional T1-weighted images were assessed for cerebellar lobular volume and adjusted for total intracerebellar volume (TIV) using a spatially unbiased atlas template to give a relative volume ratio. A region of interest analysis included the deep cerebellar nuclei. A correlation analysis was performed between the volumetric data and the clinical behavioral scores derived from the standard questionnaires for global intelligence assessment in paired subgroups. In individuals with PWS, TIV was significantly reduced compared with that of controls. Decreased relative lobular volume ratios were observed in posterior inferior lobules with age, sex, and TIV as covariates (Crus I, Crus II, lobules VIIb, VIIIa, VIIIb, and IX). However, increased ratios were found in the dentate nuclei in individuals with PWS. The altered lobular volume ratios showed negative correlations with hyperphagic and autistic characteristics and positive correlations with obsessive and intellectual characteristics. This study provides the first objective evidence of topographic patterns of volume differences in cerebellar structures consistent with clinical behavioral characteristics in individuals with PWS and strongly suggests a cerebellar contribution to altered functional brain connectivity in PWS.

Kenichi Yamada <yamadak@bri.niigata-u.ac.jp>

University of Niigata, Brain Research Institute

BACKGROUND: Prader-Willi Syndrome (PWS) is a neurogenetic syndrome characterised by the onset of hyperphagia in childhood leading to morbid obesity. On-going clinical trials investigating the potential of medications to treat hyperphagia rely on caregiver-report questionnaires and BMI as outcome measures. Objective and accurate measures of hyperphagia are urgently needed for evaluating treatment effects. Eye-tracking provide an opportunity to use a non-invasive and developmentally appropriate method of investigating attentional biases to food that may have potential as a biomarker for hyperphagia. The aims of the present study were (1) to develop a preferential looking paradigm that would measure changes in attentional bias for food pre and post (lunchtime) meal consumption, and (2) to validate it in a healthy population.

METHODS: Participants (N=34) were shown 10 visual displays pre-lunch (hunger condition) and post-lunch (satiety condition) that comprised of a target food stimulus among 4 heterogeneous objects as distractors. Attentional bias for food was investigated across the two conditions by comparing the total duration of fixations, number of fixations and time to first fixation when participants were hungry versus satiated. RESULTS: A two-way repeated measures ANOVA showed statistically significant differences in the total duration of time spent looking at food compared to distractor categories in both the hungry and satiated conditions. Participants also looked at food stimuli significantly longer when hungry compared to when satiated. (F(1,33) =14.26, p < .01).

CONCLUSION: This study provides preliminary evidence for the use of eye-tracking as a promising objective marker of hyperphagia. The next steps in this research will involve validating the paradigm in children and individuals with PWS.

Sarah-Marie Feighan <feighans@tcd.ie>

Department of Psychiatry

The smallest genomic region causing Prader-Willi Syndrome (PWS) deletes the non-coding RNA SNORD116 cluster; however, the function of SNORD116 remains a mystery. Previous work by Burnett and colleagues revealed the tantalizing possibility that NHLH2 expression was downregulated in PWS patients and stem cells. As Nhlh2 knockout mice share many of the same phenotypes with PWS patients, this possibility suggested that NHLH2 could be a gene regulatory target of SNORD116. In silico RNA:RNA modeling identified several potential interaction domains between SNORD116 and NHLH2 mRNA. One of these interaction domains was highly conserved in most vertebrate NHLH2 mRNAs examined. A construct containing the Nhlh2 mRNA, including its 3’-UTR, linked to a c-myc tag was transfected into the hypothalamic cell line N29/2 in the presence and absence of exogenously-expression Snord116. Nhlh2 mRNA expression was upregulated only in the presence of Snord116. Furthermore, use of actinomycin D to stop new transcription in N29/2 cells demonstrated that the upregulation occurred through increased stability of the Nhlh2 mRNA. In silico modeling also revealed that a single nucleotide variant (SNV) in the NHLH2 mRNA could destroy the NHLH2:SNORD116 interaction. Indeed, use of an Nhlh2 mRNA construct containing this SNV reduces the ability of Snord116 to increase Nhlh2 mRNA levels. For the first time, these data identify a motif and mechanism for SNORD116-mediated regulation of NHLH2, clarifying the mechanism by which deletion of the SNORD116 snoRNAs locus leads to PWS phenotypes. We now can extend these findings by identifying other proteins or factors involved in SNORD116-mediated posttranscriptional regulation, identifying additional target mRNAs, and characterizing SNVs in NHLH2 and other target mRNAs can lead to PWS-like phenotypes, including human obesity, and infertility

Deborah Good <goodd@vt.edu>

Translational Biology, Medicine and Health Graduate Program, Virginia Tech; Biocomplexity Institute & Initiative, University of Virginia; Department of Human Nutrition, Foods, and Exercise, Virginia Tech

INTRODUCTION/BACKGROUND Evidence suggests that individuals with PWS are at higher risk of experiencing thrombotic events compared to the general population. Currently, there is no laboratory test to screen for risk of thrombosis in the PWS population. The goals of this study were to determine if asymptomatic individuals with PWS have elevated levels of D-dimer, a fibrin degradation product, compared to the normal population, and to evaluate whether D-dimer testing can serve as a sensitive and specific tool to identify those at risk for blood clots.

METHODS Participants in the PATH for PWS (Paving the way for Advances in Treatments and Health) natural history study who reside in the United States were given the option to consent to participate in the D-dimer sub-study. Participants were enrolled remotely and completed testing at a local LabCorp location close to their residence. After testing was completed, the findings were made available through PWN Health for inclusion in the analysis. All Participants received a copy of the findings and a $100 Amazon eGift card for participation in the sub-study. Those with abnormal test results were contacted by PWN Health to review risk factors for blood clots and referred for additional evaluation and testing as needed. All results were uploaded Global PWS Registry account by the Study Coordinator.

RESULTS/DISCUSSION The remote approach to laboratory testing allowed rapid enrollment and completion of the target number of 200 tests. The D-dimer sub study enrolled 310 Participants, and of these, 215 Participants provided a blood sample to measure D-dimer levels. One sample was compromised and excluded. In total, 14.95% of the Participants had results above the normal limits (n=32) and 3.7% (n=8) had findings more than 2x the normal limit. One Participant with an elevated D-dimer level was found to have a blood clot. Overall, the specificity of the D-dimer as a screen for blood clots in PWS is low.

Lisa Matesevac <lisa.matesevac@fpwr.org>

Foundation for Prader-Willi Research

Lack of SNORD116 (HBII-85) gene cluster expression underlies the Prader-Willi syndrome (PWS) phenotypic features. SNORD116 family consists of 29 homologous small nucleolar RNAs (snoRNAs), which display no apparent sequence complementary to canonical RNA targets and are thus considered orphan snoRNAs with yet unknown function. Ectopic overexpression of SNORD116 in HEK293T, a cell line in which this gene is not endogenously expressed, resulted in perturbation of more than 200 protein coding genes, implying a role for SNORD116 in regulating mRNA stability. Whether the SNORD116 RNAs target mRNAs directly or indirectly (i.e., by regulating trans‐acting factors) remains to be resolved. Identifying SNORD116 RNA interactome is thus a crucial step in understanding this snoRNA’s biological function. We use psoralen cross-linking to capture transient RNA-RNA interactions in live cells endogenously or ectopically expressing SNORD116. Upon enrichment of extracted and partially degraded cross‐linked RNAs, the interacting RNA arms are proximity-ligated, cross-links are reversed and the RNA hybrids are subjected to massive parallel sequencing, thereby allowing the detection of interacting events. To increase the number of rare SNORD116-containing reads, we PCR-amplified such hybrids from the initial library prior to sequencing. Using this approach, we identified 1694 potential interacting partners of SNORD116 in the NTERA‐2 cell line, some of which were previously found to be differentially expressed in PWS hypothalami as compared to control samples.

Tomaž Bratkovič <tomaz.bratkovic@ffa.uni-lj.si>

1 - University of Ljubljana, Faculty of Pharmacy, Ljubljana, Slovenia; 2 - Jožef Stefan Institute, Department of Biotechnology, Ljubljana, Slovenia; 3 - The Francis Crick Institute, Bioinformatics and Computational Biology Laboratory, London, UK; 4 - The Francis Crick Institute, RNA Regulatory Networks Laboratory, London, UK

Prader-Willi syndrome (PWS) is an imprinting disorder that is caused by the absence of paternal-only expressed genes in the Prader-Willi Critical Region (PWCR). To date, three molecular mechanisms have been described as the cause of PWS, including deletion in the PWCR, uniparental disomy 15, and imprinting defect. Here we report the first case of PWS associated with a de-novo point mutation of the SNRPN gene. The 10-year-old girl presented with clinical features that were consistent with PWS, including infantile hypotonia and feeding difficulty, developmental delay, cognitive impairment, excessive eating, central obesity, skin picking, behavior issues, and sleep abnormality. Dysmorphic features included mild ptosis, downslanting palpebral fissures, prominent incisors and widely-spaced teeth. Karyotype and Fragile X DNA testing were negative. Whole exome sequencing revealed a de novo mosaic nonsense mutation of the SNRPN gene (c.73C>T, p.R25X) in 10.38% of buccal cells. Parental testing was normal. Methylation study did not detect abnormal methylation pattern in the SNRPN locus. Although SNRPN has long been speculated as the PWS ‘gene’, prior studies have only identified rare patients with deletions of the imprinting center including the SNRPN locus. This case provides conclusive evidence for a new disease-causing mechanism for PWS caused by a point mutation in the SNRPN gene. Exome or genomic sequencing is recommended for all patients with a suggestive phenotype who test negative with methylation testing for PWS.

Virginia Kimonis <vkimonis@hs.uci.edu>

1Department of Pediatrics, University of California, Irvine, CA, Children’s Hospital of Orange County, Orange, CA.,2 Dept. of Pediatrics, Cedars Sinai Medical Center & David Geffen School of Medicine at UCLA, 3Pediatric Endocrinology, Cedars Sinai Medical Center, Los Angeles, CA, 4 Department of Psychiatry & Behavioral Sciences and Pediatrics, University of Kansas, Med Ctr, KS

Introduction: Negative emotional and behavioural responses to altered routines or expectations – resistance to change - is common in individuals with PWS. It is a major trigger of behaviour problems which may be disabling in their impact on the individual and their family. Growing evidence suggests that flexibility early-in-life may reduce later resistance by enhancing the development of task-switching, a cognitive process that appears to contribute to the effective management of change. This study aims to develop an intervention, which systematically increases variability in children’s environments in a way that provides necessary structure for managing current behaviour challenges, alongside necessary flexibility for appropriate cognitive training.

Methods: An iterative collaborative design process was conducted with professionals (n=12) and families of children between 5-12 years with a diagnosis of PWS (n=15) or another neurodevelopmental disorder linked to resistance to change (n=21). Design specifications that would allow the intervention to meet families’ needs were identified via individual interviews with caregivers (n=36). These criteria were used to create a paper prototype of the intervention, which was refined via focus groups with caregivers (n=13). A functional online prototype was created and tested at home by families in three stages (1-2 weeks) with iterative improvements being integrated throughout (n=12). Semi-structured individual interviews and questionnaires assessed acceptability and feasibility for all caregivers involved in testing. Focus groups with parents and children from three families further informed on these issues.

Results: All participants rated ‘agreement’ or ‘strong agreement’ on the potential benefits for their child, that it was an acceptable way to manage difficulties with flexibility, and it would likely improve behaviours around change. Participants reported that the ‘game-like’ experience was exciting, it helped children manage changes they historically difficult changes, and that motivation was maintained.

Conclusions: Participants had an overall positive reaction to the intervention with reports that the integrated strategies supported parental confidence and management of challenges. However, to increase long-term engagement children should have the capacity to personalize graphics, and attainment of short-term goals should be noticeable for parents. With refinements, the intervention has the capacity to provide families with remote-access to evidence-based behaviour principles, which can have an immediate positive impact on the experience of children and their families.

Acknowledgements/Funding: Project funded by the Medical Research Council, United Kingdom and the FPWR.

Siobhan Blackwell <siobhan.blackwell@nhs.net>

University of Birmingham, School of psychology

Atypical responses to sensory stimuli are considered as a core aspect and early life marker of neurodevelopmental disorders.We hypothesized that animal models of Schaaf-Yang and Prader willi syndromes might present sensory alteration during neonatal period. During the first week of life, sensory integrity is instrumental and among the various stimuli arising from the external world, sensing any reduction of the ambient temperature is particularly relevant for neonatal pups. Indeed, unlike their homeothermic adult counterparts, neonates are poikilothermic and should be keep in close contact with the mother in order to keep their body temperature. In the absence of their warmth-giving mother and being exposed to cool temperatures, neonates generate ultrasonic vocalizations (USV). We thus explored thermosensory-evoked call reactivities to coolness environment (15°C) in mice knock-out for Magel2. We found that coolness exposure decreases the latency to emit the first calls (sensory reactivity) in WT, while this latency is clearly elevated in Magel2 KO neonates. This lack of reactivity is temperature-dependent since WT and mutant neonates behave similarly when isolated at room temperature. The responsive rate to coolness (i.e. the proportion of pups responsive to cooling) is markedly lower in Magel2 KO than WT neonates and gets worse from P1 to P6. Investigations of body surface temperature and brown adipose tissue reveal normal nonshivering thermogenesis in mutant pups. In addition, thermosensory neurons of the Grueneberg ganglion (a main thermosensor region located at the tip of the nose of neonate) are normally active when exposed to cool stimuli. However, biochemical and electrophysiological analyses demonstrate abnormal activities in the preoptic area; a main central region that integrates peripheral thermal information. Interestingly, intranasal injection of oxytocin can rescue this sensory deficit. Moreover, inactivation in WT neonates of hypothalamic oxytocin neurons through DREAAD technique reproduces the coolness response failure observed in mutants. All together these results highlight impairments of thermal reactivity in a mouse model of Schaaf-Yang and Prader willi syndromes during early life period and reveal that the oxytocinergic system regulates this innate sensory-motor behavior.

Valery Matarazzo <valery.matarazzo@inserm.fr>

INMED (INSERM / Aix-Marseille University)

Prader–Willi syndrome (PWS) has been described as “a genetic obesity secondary to an impaired satiety response”. This satiety deficit prolongs eating, and after a meal hunger returns sooner compared to controls, leading to consumption of very large meals, overeating and severe obesity – the major cause of PWS morbidity and mortality. Thus, developing an effective treatment that prevents the dramatic overeating of PWS is essential. Satiety signals are activated by accumulation of food in the upper gut and excite neurons in the caudal solitary tract nucleus (cNTS). These neurons form the main hub for distributing satiety information throughout the brain. The overlap in brain areas between the widely distributed satiety circuit and expression of genes associated with hyperphagia and obesity in PWS raised the possibility that any of these brain areas could be affected by PWS mutations, including the cNTS. Therefore, we hypothesized PWS mutations compromise satiety signaling by the cNTS to contribute to increased meal size and hyperphagia. To examine this, Snord116 wild-type (wt) and null (paternal allele knocked out, maternal silenced) mice were each divided into 2 gps matched on food intake (FI) and body weight (BW) and trained to eat a large meal to excite cNTS neurons: For 5 consecutive days mice were food-deprived overnight and fed a palatable diet for 1 hr each subsequent morning (Ensure Vanilla, 1.48 Kcal/ml), and then fed their maintenance chow pellets for 3 hr each afternoon. Ensure consumption and BW were measured each day. On day 5, one gp from each genotype was given Ensure for 1 hr as on previous mornings (FED-null n=9, FED-wt n=7), and the other gp was not given Ensure at this time (NONFED-null n=7, NONFED-wt n=8). 30 min after Ensure removal mice were perfused with fixative, the medulla frozen, cross-sectioned at 30m, and immunostained for c-Fos. cNTS cells that exhibited nucleolar c-Fos staining were counted in all viable sections containing the area postrema (AP). BW (null 22.1 + 0.8 g, wt 24.5 + 1.27 g) and the amount of the large meal consumed (null 3.6 + 0.7 g, wt 3.5 + 0.8 g) were similar in null and wt mice. There were increases in the number of cNTS, AP and dorsal vagal motor nucleus (DMV) neurons excited in FED vs NONFED mice after a meal as assessed by c-Fos expression, but these numbers were similar in Snord116 null and wt mice (Figure; mean # c-Fos cells/section + SEM is plotted). This suggests excitation of cNTS neurons during a meal is normal in snord116 null mice. Thus, the deficit in satiety signaling may be downstream of the cNTS. These findings do not support the hypothesis that satiation signaling in the cNTS is reduced in Snord116 null- as compared with wt mice and thus such a decrease in satiation signaling does not appear to explain the increased meal size of Snord116 null mice.

Acknowledgements/Funding: Funded by Foundation for Prader-Willi Research

Edward Fox <foxe@purdue.edu>

Purdue University / Psychological Sciences

Prader Willi syndrome patients may go through up to five main nutritional phases (0, 1, 2, 3, and 4) with phases 1 and 2 further sub-divided into two sub types each, as described by Miller JL et al., 2011. Each phase is generally associated with an age range. The relationship between nutritional phase, gender, PWS sub types and age has not been characterized more recently. Methods: We evaluated the self-reported data from the Global PWS Registry entered through September 2019. We charted the development of various nutritional phases (1a, 1b, 2a, 2b, 3, and 4) and compared this against PWS sub types, patient, age, and gender. Results: A total of 610 participants were included. Demographics of this cohort are: 84.43% white, 7.70% multi-ethnic, 2.13% Asian, 1.13% Black or African American, 0.33% American Indian or Alaska Native, 0.33% Native Hawaiian or Other Pacific Islander, and 1.97% Prefer not to answer/no answer (N/A), 49.51% are female and 49.84% are male; 298 have the deletion sub type, 198 have UPD, 13 have ID, 5 have translocation, 7 have other sub types, and 89 did not know or did not answer. Based on self reports, the percentage of participants who were last reported to be in Phase 1a were: 6.23%, 1b: 16.89%, 2a: 11.48%, 2b: 15.90%, 3: 37.70%, 4: 10.16% and 1.64% N/A. Overall, 23.72% of participants reported that they were diagnosed by a physician with the following distribution across phases: 62.62%, 20.49%, 17.70%, 20.49%, 17.54%, and 3.44% for Phases 1a, 1b, 2a, 2b, 3, and 4 respectively. Genders were generally balanced across phases except for Phase 1b and Phase 3 which had slightly more males than females (53% vs 47%, and 54% vs 46% respectively). The mean ages when the participant entered each phase were- 1a: 30 months (±6.6), 1b: 33.2 months (±13.3), 2a: 67.4 months (±70.7), 2b: 79.9 months (±73.5), 3: 90.1 months (±71.9), 4: 138.9 months (±123.9). A vast majority of participants (87%) had not reached Phase 4. Of those that did have Phase 4, 95.56% of them had the deletion or UPD sub type. These data will be re-evaluated to exclude the outliers related to age at entry into a phase and will be summarized.

Conclusions: The age distributions for nutritional phases in this analysis is slightly different from that previously described in literature. One main contributor may be the limited number of physician diagnoses. These findings are important in understanding the epidemiology and planning treatment approaches for different nutritional phases of PWS.

Kaveri Bhargava <kaverib@bu.edu>

Soleno Therapeutics Inc.

Background: Excessive daytime sleepiness (EDS) is a common symptom in patients with Prader-Willi Syndrome (PWS), with prevalence ranging from 52% to 100% (Clarke et al. 1989). The goal of the present study was to establish the content validity of the Epworth Sleepiness Scale for Children and Adolescents (ESS-CHAD), a measure frequently used to evaluate daytime sleepiness in clinical trials, by determining the accuracy by which it captures the caregiver perspective of EDS in PWS. Methods: This cross-sectional, qualitative interview study was conducted in the United States from April to June 2020 with 18 caregiver/patient dyads recruited by the Foundation for Prader-Willi Research. Dyads participated in video interviews led by a trained interviewer using a semi-structured interview guide. Thematic analysis of the transcripts was conducted to examine patterns in the data. Results: All caregivers were mothers of individuals with PWS-related EDS (mean age: 49 (range: 30-61) and 14 (range: 6-36) years, respectively) and 100% reported that their children experienced moderately to extremely troublesome daytime sleepiness. Both caregivers and their children described their experience with EDS as a chronic (100% and 94%, respectively) and constant (67% and 67%, respectively) issue. Caregivers reported that daytime sleepiness resulted in their child’s inability to concentrate (94%), bouts of irritability (44%), and that it occurred regularly (i.e. at least 3-4 times per week) during the afternoon (78%); when at school, when doing school work, or during any other time that required mental concentration (67%); at times of emotional stress (67%); when riding in the car (67%); during quiet activities (56%); during periods of inactivity or boredom (56%); and when outside of their normal routine (50%). Activities in which daytime sleepiness typically occurred were reflective of the 8 items on the ESS-CHAD. These activities were: riding in the car (67%); inactive periods including sitting quietly by themselves after lunch (67%) and lying down to rest in the afternoon (11%); quiet activities including sitting and reading (33%), sitting and watching a screen (33%), and sitting and talking to someone (11%); during or after eating (28%); and activities necessitating mental concentration including sitting in a classroom in the morning (11%). When asked to complete and provide feedback on the ESS-CHAD, caregivers found it to be an accurate representation of their children’s experiences with daytime sleepiness; moreover, caregivers demonstrated comprehension of the instrument’s instructions, items, and response options. Conclusions: This research provides evidence supporting the content validity of the ESS-CHAD. It further validates EDS as a core symptom of PWS from the perspective of caregivers.

Erika Brockfeld McClure <Erika.McClure@covance.com>

Covance, Inc

Prader Willi Syndrome (PWS) is a congenital neurodevelopmental disorder characterized by initial failure to thrive, short stature, hypogonadism, hypotonia, hyperphagia, early onset childhood obesity, behavioral problems, Excessive Daytime Sleepiness (EDS), and cognitive dysfunction. Neuroanatomical abnormalities have been found in the hypothalamus in patients with PWS, and many of the clinical manifestations of PWS, such as growth hormone deficiency, hypogonadism, hyperphagia, and sleep abnormalities, are consequences of hypothalamic dysfunction. Notably, the hypothalamus is a crucial brain region for regulating sleep-wake timing and stability, as well as hunger/satiety. EDS is an often-underreported symptom by patients with PWS and their families, and there is an unmet medical need for effective treatments to address this symptom. EDS and poor nighttime sleep are prevalent in PWS and begin in early childhood. These sleep disturbances interfere with learning, and adversely affect functioning and performance, contributing to behavioral disorders. EDS appears to be continuously present across a patient’s lifespan. Symptoms of “sleepy/tired/fatigue” are amongst the most common and impactful symptoms which negatively affect the lives of people with this syndrome.

Patients with PWS exhibit EDS, yet the sleep abnormalities in PWS are distinct from those of narcolepsy. People with PWS typically have a mean latency to fall asleep of only 7.2 minutes on the Mean Sleep Latency Test (MSLT), compared with over 15 minutes in healthy individuals, and 5 minutes in patients with narcolepsy. Sleep paralysis and hypnagogic/hypnopompic hallucinations are uncommon in PWS. The genetic markers associated with narcolepsy (e.g., variations in the human leukocyte antigen [HLA] complex) are not present in patients with PWS. In narcolepsy type 1, hypocretin levels are usually very low (≤110 pg/mL), while people with PWS have moderately low CSF hypocretin levels (192 pg/ml on average) compared to about 320 pg/ml in controls. Notably, in patients with PWS, higher scores on the Epworth Sleepiness Scale (ESS) have been correlated with lower CSF hypocretin levels, suggesting that decreased levels of hypocretin may contribute to EDS observed in patients with PWS. Preclinical and clinical data support a role for hypocretin in the pathophysiology of PWS. Both MAGEL2-null and SNORD116-null mouse models have disrupted hypocretin systems.

Given the role of the hypothalamus in PWS, the role of hypocretin in sleep-wake state stability, and the potential synergy between hypocretin and histamine signaling in the brain, there is a strong scientific rationale why pitolisant a histamine 3 receptor antagonist/inverse agonist, which enhances histamine release in the brain and has been shown to promote wakefulness in patients with narcolepsy, may improve EDS in patients with PWS. Pace (2018) investigated whether pitolisant, a histamine 3 receptor antagonist/inverse agonist, which enhances histamine release in the brain and has been shown to promote wakefulness in patients with narcolepsy, had an effect on REM sleep in SNORD116-null mice. Administration of pitolisant reduced REM sleep during the active phase in the SNORD116-null mice, but not in the control mice, suggesting the reduction in REM sleep may be from H3-receptor blockade and increased histamine levels in the brain, which in turn stimulate the release of norepinephrine and serotonin. Harmony Biosciences is conducting a randomized, double-blind, placebo-controlled phase 2 clinical trial to evaluate the safety, efficacy and appropriate dosing of pitolisant in the treatment of EDS in patients with PWS. The proposed Phase 2 trial will also explore the effect of pitolisant on other symptoms of PWS, including cognitive impairment and abnormal behavior.

Albena Patroneva <apatroneva@harmonybiosciences.com>

Harmony Biosciences, LLC, Plymouth Meeting, PA, USA

Smchd1 is an epigenetic repressor, targetable by small molecules and known to play a role in silencing PWS cluster genes on the maternal allele in mice. Removal of Smchd1 has been shown to result in activation of PWS genes at the proximal end of the cluster from the silent maternally inherited allele without affecting Ube3a at risk of causing Angelman Syndrome. Using a reporter mouse model for Magel2 expression we have shown that in line with previous data, postnatal deletion of Smchd1 can result in reactivation of maternally inherited PWS genes in vivo in the brain. This adds support to the concept of targeting SMCHD1 as a potential therapeutic approach for the treatment of PWS. Although much is known about Smchd1 function at the PWS cluster in mice, there is limited information about whether it plays the same role in humans. Using CRISPR-Cas9 technology we have created a system to knock out SMCHD1 in human cells. Through application of this system in patient-derived cell lines we have begun to investigate whether SMCHD1 also acts to silence maternally inherited PWS genes in humans. From this we aim to increase understanding of molecular mechanisms at work within the PWS cluster and to confirm viability of SMCHD1 as a target for epigenetic therapy of PWS.

Megan Iminitoff <iminitoff.m@wehi.edu.au>

The Walter and Eliza Hall Institute of Medical Research, Epigenetics and Development Division

Preliminary research has shown that children with PWS have impaired pretend play abilities and parent-child interactions, which may be markers of larger social cognitive deficits characteristic of the disorder. Targeting skill building through play and parent-child interactions in children with PWS may strengthen adaptive skills, socioemotional understanding, and the ability to engage in and appropriately interact in social situations while also decreasing rigid and repetitive behaviors. Telehealth methodology allows for the reduction of barriers such as distance and cost of in-person trials. Here we report efficacy findings from our ongoing examination of the Play-based Remote Enrichment To ENhance Development (PRETEND) parent training program for parents of preschoolers with PWS, which is an 8-week program delivered via videoconferencing that includes once weekly sessions, 30-45 minutes in length for parents of children aged 3-5 years. Twenty preschool-aged children and their parents participated in this study, and children were randomly assigned either to an intervention group or a waitlist control group. At baseline and outcome, measures were used to directly assess children’s pretend play skills and cognitive skills, as well as parent-child interactions in play. Parents also reported on their children’s social cognitive skills and measures of their own stress related to their children. The PRETEND-Preschool intervention program consists of 1) individual parent sessions to review play and related cognitive, emotional and behavioral skills, 2) coaching-play sessions in which the interventionist uses remote live-coaching with the parent playing with their child, and 3) play-based homework assignments for the parent and child. Results indicated that the intervention participants with UPD demonstrated meaningful improvement above and beyond that of the waitlist control participants on multiple play variables including increasing Imagination, Organization, Symbolic play, and Affect frequency, while the DEL subgroup demonstrated fewer improvements across play variables. Overall, a greater percentage of children in the UPD subgroup showed improvement across all variables of the APS-P. These results are the first to support preliminary efficacy of this play-based, remote intervention in preschool-aged children with PWS, and indicate important future directions for targeting differential areas of intervention for the two PWS subtypes.

Ellen A. Doernberg <exd185@case.edu>

Case Western Reserve University, Department of Psychological Sciences