Hypocretin/Orexin: A Molecular Link Between Sleep, Energy Regulation, and Pleasure

Four patients with clinically and genetically confirmed Prader-Willi syndrome (PWS) underwent nocturnal polysomnograpy (PSG), multiple sleep latency test (MSLT), human leukocyte antigens (HLA) typing and estimation of cerebrospinal fluid (CSF) hypocretin-1 (Hcrt-1) level to investigate if a role of hypothalamic dysfunction and sleep disturbance might be functionally connected through the hypocretin (orexin) system. In all four patients physical examination confirmed extreme obesity (increasing with age) with dysmorphogenetic features. Excessive daytime sleepiness (EDS) was manifested in only two subjects without any imperative feature. None of the patients under study suffered from cataplexy. Nocturnal PSG revealed fragmented sleep with low efficiency, the hypopnea and apnea indexes increasing from borderline up to very high values in direct proportion to the patients' age. MSLT latency was shortened in two patients with clinically expressed EDS, only one sleep onset rapid eye movements (REM) period (SOREM) was found. HLA typing showed DQB1*0602 positivity in two patients; the further two were negative. Mean value of CSF Hcrt-1 in the patients group was down to 164 +/- 46.8 pg/ml (in comparison with 265.8 +/- 48.8 pg/ml in 10 young healthy subjects, P=0.02). The deficiency of CSF Hcrt-1 level correlated in PWS patients with their EDS severity.

An Obese Female with Prader-Willi Syndrome and Daytime Sleepiness

Context Insulin sensitivity is higher in patients with Prader-Willi syndrome (PWS) than in body mass index-matched obese controls (OCs). Factors contributing to the heightened insulin sensitivity of PWS remain obscure. We compared the fasting levels of various hormones, cytokines, lipids, and liver function tests in 14 PWS patients and 14 OCs with those in 14 age- and gender-matched lean children (LC). We hypothesized that metabolic profiles of children with PWS are comparable with those of LC but different from those of OCs. Results Leptin levels were comparable in PWS patients and OCs, suggesting comparable degrees of adiposity. Glucose levels were comparable among groups. However, fasting insulin concentrations and homeostasis model assessment insulin resistance index were lower in PWS patients than in OCs (P < 0.05) and similar to LC. Moreover, high-density lipoprotein levels were lower and triglycerides higher in OCs (P < 0.05) but not PWS patients. Total adiponectin, high-molecular-weight (HMW) adiponectin and the HMW to total adiponectin ratio were higher in PWS patients (P < 0.05) than in OCs and similar to LC. High-sensitivity C-reactive protein and IL-6 levels were higher in OCs than in PWS patients or LC (P < 0.05). Nevertheless, PAI-1 levels were elevated in both OC and PWS patients. There were no group differences in glucagon-like peptide-1-(7-36)-amide, macrophage chemoattractant protein-1, TNFα, IL-2, IL-8, IL-10, IL-12p40, IL-18, resistin, total or low-density lipoprotein cholesterol, aspartate aminotransferase, or aminotransferase. Conclusions The heightened insulin sensitivity of PWS patients relative to OCs is associated with higher levels of adiponectin and lower levels of high-sensitivity C-reactive protein and IL-6. Future studies will determine whether PWS children are protected from obesity comorbidities such as type 2 diabetes, hyperlipidemia, and nonalcoholic fatty liver disease.

Prader-Willi syndrome (PWS) is associated with hyperphagia and obesity, without effective pharmacological treatment. Exenatide, recently developed for treatment of type 2 diabetes, induces appetite suppression and weight loss with common side effects. The objective of the study was to investigate the initial safety and effectiveness of exenatide in adult PWS subjects compared with obese controls (OBESE). Eight PWS and 11 OBESE patients underwent standardized meal studies after a single sc injection of 10 μg exenatide or placebo in a single-blinded, crossover design. Glucose, insulin, C-peptide, glucagon, peptide YY (PYY; total)/PYY (3-36), glucagon-like peptide-1, and ghrelin (total) were measured fasting and postprandially. Appetite and satiety were assessed by visual analog scales. Energy expenditure (EE) was measured by indirect calorimetry. Side effects were screened during and for 24 h after the meal. PWS and OBESE patients were matched for gender, age, body mass index, and central/total body fat. In both groups, exenatide increased satiety and lowered glucose and insulin levels but increased insulin secretion rate. Side effects were absent in PWS but common in OBESE patients. During the meal, PYY (total) and ghrelin were elevated in PWS patients. Exenatide decreased PYY (total) and glucagon-like peptide-1, whereas ghrelin remained unchanged. Energy expenditure was unchanged by exenatide. Our pilot study demonstrates that exenatide is well tolerated in PWS patients. It increases satiety independently of measured appetite hormones, exerting glucose lowering, and insulinotropic effects similarly in PWS and OBESE patients. Larger prospective studies should investigate whether chronic exenatide administration will reduce hyperphagia and overweight in PWS patients without side effects.

Recent studies have identified a new class of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) patients who have biparental inheritance, but neither the typical deletion nor uniparental disomy (UPD) or translocation. However, these patients have uniparental DNA methylation throughout 15q11-q13, and thus appear to have a mutation in the imprinting process for this region. Here we describe detailed clinical findings of five AS imprinting mutation patients (three families) and two PWS imprinting mutation patients (one new family). All these patients have essentially the classical clinical phenotype for the respective syndrome, except that the incidence of microcephaly is lower in imprinting mutation AS patients than in deletion AS patients. Furthermore, imprinting mutation AS and PWS patients do not typically have hypopigmentation, which is commonly found in patients with the usual large deletion. Molecular diagnosis of these cases is initially achieved by DNA methylation analyses of the DN34/ZNF127, PW71 (D15S63), and SNRPN loci. The latter two probes have clear advantages in the simple molecular diagnostic analysis of PWS and AS patients with an imprinting mutation, as has been found for typical deletion or UPD PWS and AS cases. With the recent finding of inherited microdeletions in PWS and AS imprinting mutation families, our studies define a new class of these two syndromes. The clinical and molecular identification of these PWS and AS patients has important genetic counseling consequences.

The suitability of new dynamic system analysis was investigated to compare postural control in Prader-Willi syndrome (PWS) and Down syndrome (DS) patients. Time-domain, frequency-domain parameters and fractal dimension (FD) of centre of pressure (CoP) were computed in maintaining normal standing on a force platform in 20 DS and 13 PWS patients, compared to 26 obese (obese control group, OCG) and 20 healthy individuals (healthy control group, HCG). DS and PWS showed greater displacements along both directions and longer sway path (SP) parameter than HCG and OCG, with statistical differences between PWS and DS for anteroposterior displacement and SP. DS used higher frequency strategy when compared to PWS, OCG and HCG. Both DS and PWS were characterised by greater values of FD than OCG and HCG, with higher values in DS. The analyses in frequency domain and of the dynamic nature of CoP suggest that DS patients are characterised by a more complex and irregular signal than PWS patients.

Abnormalities of ventilatory control may play a significant role in the pathophysiology of sleep-disordered breathing in patients with the Prader-Willi syndrome (PWS). We measured rebreathing hypercapnic and hypoxic ventilatory responses (HCVR and HPVR, respectively) during wakefulness in 8 nonobese PWS (NOB-PWS) and 9 obese PWS (OB-PWS) patients and compared their results with those from 24 healthy nonobese control (NOB-CON) and 10 obese control (OB-CON) subjects. The slope of HCVR was similar in NOB-PWS patients and NOB-CON subjects (NS). However, HCVR was significantly lower in OB-PWS patients than in OB-CON subjects (P < 0.02). In PWS patients, the mean point of origin of the positive slope of HCVR occurred at a significantly higher end-tidal PCO2 than in either control group. During isocapnic hypoxic challenges, six PWS patients had no significant HPVR. In the remainder, mean slopes of HPVR were -0.80 +/- 0.06 l.min-1.%arterial O2 saturation-1 in five NOB-PWS patients and -0.68 +/- 0.15 l.min-1.%arterial O2 saturation-1 in six OB-PWS patients. These responses were significantly decreased compared with those in the control groups (P < 0.006). We conclude that NOB-PWS patients have normal HCVR, which is blunted in OB-PWS patients. Furthermore, isocapnic HPVR is either absent or markedly reduced in PWS patients. The severity of abnormality of the HPVR is independent of the degree of obesity. We postulate that the primary abnormality of ventilatory control in PWS affects peripheral chemoreceptor pathways.

Introduction: Prader Willi syndrome is a genetic disorder with multisystem involvement. Bariatric laparoscopic sleeve gastrectomy surgery (LSG) has been proposed as a mean of weight control in Prader Willi Syndrome (PWS) patients. In this case-series, we are questioning the effectiveness of bariatric gastric sleeve surgery as a long-term solution for weight control. Aims and Objectives: There is some controversy around sleeve gastrectomy as a management strategy in PWS to control OSA. We aimed at analysing long term usefulness of LSG in successfully alleviating obesity related OSA. Therefore, we followed up four patients in our centre with PWS who had LSG and evaluated their growth parameters along with symptoms of sleep disordered breathing. Results: Conclusions: We believe that LSG has a limited and transient success for obesity management in PWS patients, especially in the absence of a multidisciplinary team which can provide behavioural therapy, nutritional plan, and psychosocial support. Further studies are needed to compare the long term outcome of nutritional counselling alongside gastric sleeve surgery.

We have evaluated serum leptin concentrations in two forms of genetic obesity. The subjects examined were eight women with Down syndrome and eight women with Prader-Willi syndrome. All patients were in the reproductive age range and were obese (body mass index ≥ 21 kg/m2). Plasma leptin values ,analyzed as a function of body mass index showed a statistically significant correlation in both Prader-Willi (r = 0.985; p < 0.001) and Down syndrome patients (r = 0.943; p < 0.001). Obese Down syndrome women exhibited significantly lower leptin values (10.8 ± 1.1) as compared to patients with Prader-Willi syndrome (31 ± 2.6; p < 0.01). The linear correlation between leptin and insulin in the two groups of patients was not statistically significant. The data suggested that obesity in Prader-Willi subjects could be caused by failure of leptin to reach its target in the brain ,as a consequence of defects in the receptor or in postreceptor processing ,whereas data on obese patients with Down syndrome could be due to a different pathogenetic origin.

Polisomnographic phenotype is linked with genetic findings in Prader–Willi syndrome

Prader-Willi syndrome (PWS) is a genetic disorder associated with baseline respiratory impairment caused by multiple contributing etiologies. While this may be expected to increase the risk of severe COVID-19 infections in PWS patients, survey studies have suggested paradoxically low disease severity. To better characterize the course of COVID-19 infection in patients with PWS, this study analyses the outcomes of hospitalizations for COVID-19 among patients with and without PWS. The National Inpatient Sample, an all-payors administrative claims database of hospitalizations in the United States, was queried for patients with a coded diagnosis COVID-19 in 2020 and 2021. Hospitalizations for patients with PWS compared to those for patients without PWS using Augmented Inverse Propensity Weighting (AIPW). There were 295 (95% CI: 228-362) COVID-19 hospitalizations for individuals with PWS and 4,112,400 (95% CI: 4,051,497-4,173,303) for individuals without PWS. PWS patients had a median age of 33 years compared to 63 for those without PWS. Individuals with PWS had higher baseline rates of obesity (47.5% vs. 28.4%). AIPW models show that PWS diagnosis is associated with increased hospital length of stay by 7.43 days, hospital charges by $80,126, and the odds of mechanical ventilation and in-hospital death (odds ratios of 1.79 and 1.67, respectively). PWS patients hospitalized with COVID-19 experienced longer hospital stays, higher charges, and increased risk of mechanical ventilation and death. These results suggest that PWS should be considered a risk factor for severe COVID-19, warranting continued protective measures and vaccination efforts. Further research is needed to validate coding for PWS and assess the impact of evolving COVID-19 variants and population immunity on this vulnerable population.

Prader-Willi syndrome (PWS) is widely recognised as the most common cause of syndro-mal obesity in children. The syndrome is also characterised by a range of neurological andendocrine anomalies, which occur at varying degrees of severity in each patient leading toconsiderable phenotype variability. The genetic basis for PWS has been well dened andinvolves the loss of expression of paternally expressed genes within chromosome 15q11-q13. Genetic heterogeneity occurs with some patients harbouring interstitial deletions,others presenting with uniparental disomy and a small contingency harbouring imprintingdefects or chromosomal translocations. Limited knowledge exists regarding the biochem-ical and metabolic mechanisms occurring in this syndrome. This thesis aims to improveon this limited knowledge by evaluating protein glycosylation and protein ubiquitinationin a cohort of PWS children.The protein glycosylation pathway was targeted based on a previous (unpublished)nding of abnormal transferrin isoelectric focusing (IEF) proles in PWS and also on theunderstanding that a number of glycosylated proteins are integral components of PWSpathophysiology. Seven out of 25 PWS patients were reported to have the presence oflow-intensity protein bands migrating with monosialo- and/or asialo- transferrin. Thispattern represents a carbohydrate-decient transferrin pattern and had not previouslybeen reported in PWS patients. The chromosome 15q11-q13 loci, responsible for PWS,was examined for candidate genes to explain the transferrin IEF anomaly. This led to theidentication of two genes with putative roles as E3 ubiquitin ligase enzymes and hence,the studies on protein ubiquitination in PWS. Genotyping experiments were performedto determine the deletion sizes in patients harbouring deletions as they are known to vary.These experiments were to provide an improved platform for genotype-phenotype studies.Tryptic digests of anity puried transferrin were analysed by LC-ESI MS/MS forN-glycosylation studies in PWS. O-glycosylation was studied through an IEF assay ofserum apolipoprotein C-III, which harbours a single core 1 mucin type O-glycan. Serumubiquitination was assessed through the development of an ELISA to detect protein-ubiquitin conjugates and also through SDS-PAGE analysis of anity puried protein-ubiquitin conjugates. Chromosomal deletion sizes were dened by the development of aSYBR Green I based real-time PCR assay utilising genomic DNA samples.PWS patients harbouring deletions were divided into two groups based on the real-time PCR results. The deletion sizes diered by approximately 1 Mb due to dierentproximal deletion breakpoints. All deletions encompassed the cluster of imprinted genesresponsible for the PWS phenotype. The transferrin MS/MS analysis suggested that PWSpatients did not harbour detectable abnormalities in transferrin glycosylation. Results ofthe apoC-III IEF experiments indicated that 40 % of PWS patients had higher thannormal levels of the hyposialylated glycoform. These ndings did not correlate with thegenetic mechanism, or a range of clinical features of the disorder. The ubiquitin studiesdid not show any dierences in protein-ubiquitin conjugates between the PWS group orthe control cohort.A proteomic evaluation of the transferrin LC-ESI MS/MS data was performed basedon an unexpected nding during these experiments. One of the glycopeptides was foundto cleave at an internal histidine residue and this was observed in both PWS patientsand the control samples. To determine whether additional peptides within transferrinharboured unusual tryptic cleavage sites, the LC-ESI MS/MS datasets were searchedagainst sequence databases for peptide identication. These studies identied severalother peptides with non lysine or arginine residues unexplainable through alternativedigest agents.A proposed mechanism to relate the results of the investigations performed duringthis thesis to the genetic and biochemical aspects of PWS involves an error in the normalprotein quality control pathway. Loss of function of the putative E3 ligase enzymessituated within the PWS loci could result in an increased pool of abnormally folded orincompletely modied proteins, as was observed in this study.

Hypocretins (Hcrts) are recently discovered peptides linked to the human sleep disorder narcolepsy. Humans with narcolepsy have decreased numbers of Hcrt neurons and Hcrt-null mice also have narcoleptic symptoms. Hcrt neurons are located only in the lateral hypothalamus (LH) but neither electrolytic nor pharmacological lesions of this or any other brain region have produced narcoleptic-like sleep, suggesting that specific neurons need to be destroyed. Hcrt neurons express the Hcrt receptor, and to facilitate lesioning these neurons, the endogenous ligand hypocretin-2/orexin B (Hcrt2) was conjugated to the ribosome-inactivating protein saporin (SAP). In vitro binding studies indicated specificity of the Hcrt2-SAP because it preferentially bound to Chinese hamster ovary cells containing the Hcrt/orexin receptor 2 (HcrtR2/OX(2)R) or the Hcrt/orexin receptor 1 (HcrtR1/OX(1)R) but not to Kirsten murine sarcoma virus transformed rat kidney epithelial (KNRK) cells stably transfected with the substance P (neurokinin-1) receptor. Administration of the toxin to the LH, in which the receptor is known to be present, eliminated some neurons (Hcrt, melanin-concentrating hormone, and adenosine deaminase-containing neurons) but not others (a-melanocyte-stimulating hormone), indicating specificity of the toxin in vivo. When the toxin was administered to the LH, rats had increased slow-wave sleep, rapid-eye movement (REM) sleep, and sleep-onset REM sleep periods. These behavioral changes were negatively correlated with the loss of Hcrt-containing neurons but not with the loss of adenosine deaminase-immunoreactive neurons. These findings indicate that damage to the LH that also causes a substantial loss of Hcrt neurons is likely to produce the multiple sleep disturbances that occur in narcolepsy.

Prader-Willi syndrome (PWS) is a complex developmental genetic disorder associated with hypotonia, poor feeding in neonates, onset of hyperphagia in early childhood, and shorter overall life expectancy. Prior epidemiology studies of PWS have examined smaller populations, with limited research in a US population. The aim of this study was to provide a contemporary estimate of PWS prevalence and annual all-cause mortality in the US using a large administrative medical claims dataset.Methods: PWS patients were identified between 2012-2014 via the presence of ≥2 claims with a diagnosis code for PWS on medical claims provided by IQVIA™ Health Plan Claims Data and CMS Medicare fee-for-service claims. Patients were grouped into age bands including: 0-2, 3-8, 9-17, 18-26, 27-49, and ≥50. PWS prevalence and mortality rates were calculated for 2014, then 2018 US census data was used to project rates for the total US population. The presence of select diagnoses and procedures suggestive of a life-threatening event (e.g., mechanical ventilation) with a patient’s prompt disenrollment defined as death in the IQVIA data; vital status is indicated in Medicare data.Results: Overall US diagnosed PWS prevalence was 2.7 per 100k persons (or 1 per 37,037), a prevalence of 8,870 patients in the US in 2018. Diagnosed PWS prevalence 3.9, 5.2, 4.5, 4.2, 2.5, and 1.1 per 100k persons respectively for age bands 0-2, 3-8, 9-17, 18-26, 27-49, and ≥50. The median age of PWS patients was 21 years. The mortality rate was highest among diagnosed PWS patients aged 0-2 years and lowest among those aged 9-17 years and the overall mortality rate was 2.7%. For all respective age bands 0-2, 3-8, 9-17, 18-26, 27-49, and ≥50, the all-cause mortality rate was 5.4%, 3.0%, 1.4%, 2.1%, 2.4%, and 4.5%. The observed median age of death was 23 years (IQR 6-36)Conclusions: The diagnosed PWS prevalence of 1 per 37,037 persons estimated for the 2018 US population is comparable to the other reported US prevalence estimate. As the current study describes diagnosed patients, it likely represents a lower bound of true PWS prevalence. Annual PWS mortality is ≥3 times higher than the overall US population (2.7% vs 0.8%). This rate appears unchanged from mortality estimates reported for PWS populations in the last several decades despite significant advances in genetic testing and the availability of growth hormone therapies in the US. Aggressive management of serious comorbid conditions, especially in younger PWS patients, should be a clinical priority.

Background:Prader-Willi syndrome (PWS) is a genetic disorder associated with baseline respiratory impairment caused by multiple contributing etiologies. While this may be expected to increase the risk of severe COVID-19 infections in PWS patients, survey studies have suggested paradoxically low disease severity. To better characterize the course of COVID-19 infection in patients with PWS, this study analyzes the outcomes of hospitalizations for COVID-19 among patients with and without PWS.Methods:The National Inpatient Sample, an all-payors administrative claims database of hospitalizations in the United States, was queried for patients with a coded diagnosis COVID-19 in 2020 and 2021. Hospitalizations for patients with PWS compared to those for patients without PWS using Augmented Inverse Propensity Weighting (AIPW).Results:There were 295 (95% CI: 228 to 362) COVID-19 hospitalizations for individuals with PWS and 4,112,400 (95% CI: 4,051,497 to 4,173,303) for individuals without PWS. PWS patients had a median age of 33 years compared to 63 for those without PWS. Individuals with PWS had higher baseline rates of obesity (47.5% vs. 28.4%). AIPW models show that PWS diagnosis is associated with increased hospital length of stay by 7.43 days, hospital charges by $80,126, and the odds of mechanical ventilation and in-hospital death (odds ratios of 1.79 and 1.67, respectively).Conclusions:PWS patients hospitalized with COVID-19 experienced longer hospital stays, higher charges, and increased risk of mechanical ventilation and death. PWS should be considered a risk factor for severe COVID-19, warranting continued protective measures and vaccination efforts. Further research is needed to validate coding for PWS and assess the impact of evolving COVID-19 variants and population immunity on this vulnerable population.