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Research Notes: PWS Abstracts - 2005[ 2007 | 2006 | 2005 | 2004 | 2003 | 2002 | 2001 | 2000 | 1999 | 1998 | 1997 | 1996 | 1995 | 1994 | 1993 | 1992 | 1991 | 1990 | 1980-1989 | 1979 and prior ] J Pediatr. 2005 Dec. Objectives: To determine the prevalence and type of sleep-disordered breathing among patients with Prader-Willi syndrome (PWS) and its relationship to such neurobehavioral abnormalities as mental retardation, obsessive-compulsive behavior, and conduct disorders. Study design: Polysomnography (PSG) studies were conducted in 13 unselected subjects with PWS (age 1.5 to 28 years). PSG results were compared with tests of behavior and cognition (Development Behavior Checklist [DBC], Auditory Continuous Performance Test [ACPT], and Wechsler Intelligence Scale appropriate for age). Results: Nine of 13 (69%) subjects had > 10 apneas and hypopneas per hour of sleep. Apart from a 2-year-old subject with normal body weight who demonstrated severe central hypopnea in rapid eye movement sleep, the sleep-breathing disturbance was due to upper airway obstruction. Age-adjusted body mass index was associated with more severe hypoxemia during sleep (min SaO2, r = -.87, P < .005) and more sleep disruption (arousals/hour of sleep, r = .62, P < .05; sleep efficiency, r = -.66, P < .05). Increasing severity of obstructive sleep apnea (OSA) or sleep disturbance was associated with daytime inactivity/sleepiness and autistic-relating behavior (DBC) and with impulsiveness (ACPT). Unexpectedly, sleep hypoxemia appeared to be predictive of increased performance IQ. Conclusions: OSA is prevalent among subjects with PWS and is associated with increased body mass, daytime inactivity/ sleepiness, and some behavioral disturbances. Psychiatr Genet. 2005 Dec. Autism spectrum disorders (ASDs) have been linked with maternally derived duplications/triplications of chromosome 15q11-13 and therefore might occur more frequently in people with Prader-Willi syndrome (PWS) when due to uniparental disomy (UPD), than in other forms of chromosomal abnormality involving this region [i.e. deletion (DEL) forms of PWS and DEL+UPD forms of Angelman's syndrome -(AS)]. Twelve studies regarding ASD in PWS and AS were reviewed. It was noteworthy that among the genetically confirmed UPD and DEL cases of PWS and AS, the rate of ASD was 25.3% (38/150; range 0-36.5%) in PWS and 1.9% in AS (2/104; range 0-100%) (Fisher's exact P<0.0001). Among the subset of cases with confirmed UPD or DEL, the rate of ASD in the UPD cases of PWS was significantly higher (20/53) than in the remaining combined samples (i.e. DEL PWS+UPD AS+DEL AS cases; 20/201) (Fisher's exact P<0.0001). ASD in UPD PWS cases (20/53) compared with DEL PWS cases (18/97) was also statistically significant (Fisher's exact P=0.0176). Thus, the limited available evidence supported the prediction that overexpression of maternally imprinted genes in 15q11-13 confers a risk for ASD. Further research will be required to confirm these findings. Growth Horm IGF Res. 2005 Dec. Prader-Willi syndrome (PWS) is a complex genetic disease, clinically characterised by short stature, abnormal body composition, with more body fat than lean body mass, hyperphagia and obesity. Partial growth hormone (GH) deficiency is common, and GH treatment to PWS children and adults has shown beneficial effects on body composition. In this study, we have evaluated indices of GH's lipolytic effect in 6 PWS adults analysing glycerol, lactate and glucose in dialysate from microdialysis in subcutaneous abdominal adipose tissue. The patients were four men and two women, 19-37 years old; all hypogonadal. BMI was 24.2-49.1, mean 35.9 kg/m(2). All had normal serum insulin levels. They received GH therapy (Genotropin Pfizer) during 12 months and doses were individually titrated to normal serum IGF-I for age. Immediately before treatment start and at 12 months, 30-36 h after the last GH injection, sampling of dialysate was carried out at night (11 p.m. to 7 a.m.), as well as after intravenously injection of a standardised GH dose (0.8 mg). At baseline individual mean night time glycerol and lactate were similar to levels in adults without PWS (160.7-278.1 micromol/L and 0.80-3.99 mmol/L, respectively), and did not change with 12 months GH treatment. Glucose levels were normal, except in a patient with diabetes, and did not change during the study. Compared to baseline the immediate effect of GH injection resulted in a significant increase in glycerol levels after 12 months. In conclusion, night time lipolytic response in this small group of PWS adults seemed normal and did not change after 12 months GH treatment. On the other hand short-term GH induced lipolysis increased, indicating normal lipolytic response in PWS.
Endocrinology. 2005 Oct. Prader-Willi syndrome (PWS) has a biphasic clinical phenotype with failure to thrive in the neonatal period followed by hyperphagia and severe obesity commencing in childhood among other endocrinological and neurobehavioral abnormalities. The syndrome results from loss of function of several clustered, paternally expressed genes in chromosome 15q11-q13. PWS is assumed to result from a hypothalamic defect, but the pathophysiological basis of the disorder is unknown. We hypothesize that a fetal developmental abnormality in PWS leads to the neonatal phenotype, whereas the adult phenotype results from a failure in compensatory mechanisms. To address this hypothesis and better characterize the neonatal failure to thrive phenotype during postnatal life, we studied a transgenic deletion PWS (TgPWS) mouse model that shares similarities with the first stage of the human syndrome. TgPWS mice have fetal and neonatal growth retardation associated with profoundly reduced insulin and glucagon levels. Consistent with growth retardation, TgPWS mice have deregulated liver expression of IGF system components, as revealed by quantitative gene expression studies. Lethality in TgPWS mice appears to result from severe hypoglycemia after postnatal d 2 after depletion of liver glycogen stores. Consistent with hypoglycemia, TgPWS mice appear to have increased fat oxidation. Ghrelin levels increase in TgPWS reciprocally with the falling glucose levels, suggesting that the rise in ghrelin reported in PWS patients may be secondary to a perceived energy deficiency. Together, the data reveal defects in endocrine pancreatic function as well as glucose and hepatic energy metabolism that may underlie the neonatal phenotype of PWS. Excerpts from the full text article: Introduction Prader-Willi Syndrome (PWS) is characterized by a distinct biphasic clinical phenotype. Infants show failure to thrive, including severe muscle hypotonia, respiratory and feeding difficulties, and hypogonadotropic hypogonadism (1), whereas children and adults display short stature, mild to moderate mental retardation with behavioral abnormalities, hyperphagia, and severe obesity (2, 3, 4). Indeed, much of the pathology that results in morbidity and mortality in PWS, including type 2 diabetes and cardiovascular disease, is secondary to obesity (3). A number of endocrine abnormalities occur in children and adults with PWS, including GH (3, 5, 6, 7, 8, 9), IGF-I, and IGF binding protein (IGFBP)-3 deficiencies (3, 8), and insulin levels are relatively decreased compared with individuals with common obesity (10, 11). In contrast, fasting levels of circulating ghrelin, a gut hormone believed to act on hypothalamic arcuate nucleus neurons to modulate feeding (12), are grossly elevated in children and adults with PWS, which may play a major role in the hyperphagia and obesity (13, 14, 15). Although the hormonal and clinical abnormalities in PWS have led to the idea of a primary hypothalamic origin for the disorder (2, 9, 16), hormonal and metabolic studies have not been reported in newborns with PWS and the precise pathophysiology for the disorder remains unknown. PWS is caused by loss of function of a unique set of 10 known imprinted, paternally expressed genes from human chromosome 15q11-q13 (4). Seventy-five percent of cases are associated with a 4- to 5-Mb deletion of this region that encompasses the imprinted genes and a set of five or nine nonimprinted loci (4, 17). The other 25% of PWS cases typically have maternal uniparental disomy or, rarely, an imprinting defect (4). The PWS imprinted candidate genes include three intronless genes (NDN, MAGEL2, and MKRN3), a complex polycistronic locus (SNURF-SNRPN), and five subfamilies of box C/D snoRNAs (4). SNRPN encodes a core spliceosomal protein in postnatal neurons (18). Although some biochemical properties for the other PWS-region gene products are known or have been proposed based on sequence homologies, the precise functions of these genes and the links with clinical aspects of the syndrome are not known. The PWS chromosome region is conserved in mouse chromosome 7C, and three PWS mouse models have been generated with maternal uniparental disomy, an imprinting defect (ID), or a paternally derived chromosome deletion. These models share a very similar phenotype that models the first stage of the human syndrome and includes hypotonia, respiratory difficulties, failure to thrive, and early postnatal lethality (19, 20, 21). Because there is reduced fetal movement and a severe clinical course in the newborn with PWS, we hypothesize a fetal developmental abnormality in PWS leading to the neonatal failure to thrive with the adult metabolic phenotype resulting from a failure in compensatory mechanisms. To begin to test this hypothesis, in the present study, we use a transgenic PWS (TgPWS) deletion mouse model to evaluate endocrinological and metabolic abnormalities in late fetal life and during the failure-to-thrive period. TgPWS mice display fetal and neonatal growth retardation associated with insulin/IGF axis abnormalities. Because the fetal changes predate the postnatal failure to thrive, this may identify a primary mechanism. Subsequently endocrine and metabolic deficiencies peak at postnatal day (P)2, leading to dramatic hypoglycemia and severe failure to thrive in TgPWS neonates, mimicking the clinical course in untreated infants with PWS. [...] Results Metabolic and endocrine evaluations Growth. TgPWS mice have a transgene insertion/deletion transmitted through CD-1 TgAS (transgenic Angelman syndrome mouse model) males, with half of the offspring being TgPWS and half being WT. Despite their relatively normal appearance at birth, TgPWS mice were significantly growth retarded at E18 (P = 0.0007), and after birth they did not grow at the same rate as their WT littermates [F(3, 1) = 444, P < 0.0001]. Consequently, at P5 TgPWS pups reached only half the weight of their WT littermates (Fig. 2A). At E18, placental weights did not differ between genotypes (TgPWS, 0.11 ± 0.007 g, n = 5; WT, 0.13 ± 0.005 g, n = 9; P = 0.14). Similar postnatal growth patterns for a small number of PWS ID mice have been reported (20), except that the majority of mutants in that study died within 48 h (20). In contrast, in our study the majority (80%) of TgPWS mice survived beyond P2 but then usually died between P5 and P7... The difference is likely due to the use of inbred (20) vs. outbred (our study) strains of mice, with the earlier lethality potentially associated with Ndn deficiency and a respiratory deficit that is pronounced in some inbred strains of mice (27). Body composition. From P1 to P4, TgPWS contained a greater proportion of body water than the WT mice [F(3, 1) = 10.53, P = 0.001] (Fig. 2B). At P4, percent water content was the highest in TgPWS, compared with WT littermates (P = 0.04). Body protein percentage was unchanged from P1 to P3, but by P4 protein content was higher in TgPWS vs. WT (P = 0.01) (Fig. 2C). Triglycerides, comprising the majority of total body fat content, did not differ at birth (P1). However, overall body triglyceride percentage was decreased in TgPWS [F(3, 1) = 40.87, P = 0.0001] and was most pronounced at P2 (P = 0.0003) (Fig. 2D). Liver glycogen. Liver glycogen content was similar at E18 and P1 (Fig. 3A) between TgPWS and WT pups. Both groups of mice had a significant reduction in hepatic glycogen content between P1 and P2 (–76 and –91% for WT and TgPWS, respectively; P < 0.0003). Overall, postnatal WT pups had more glycogen in liver than did TgPWS pups [F(3, 1) = 13.6, P = 0.004]. This difference was most pronounced at P2 and P3 (P < 0.01). Plasma glucose. There was no difference in plasma glucose levels between TgPWS and WT pups at E18 or P1 (Fig. 3B). However, at P2 glucose concentration dropped in TgPWS pups and thereafter was significantly lower, compared with WT mice [F(3, 1) = 100.7, P = 0.0001] (Fig. 3B). Plasma insulin. Plasma insulin levels were significantly lower in TgPWS vs. WT mice in fetal life at E18 (P = 0.04) and from P1 to P4 [F(3, 1) = 44.9, P = 0.0001] (Fig. 3C). Insulin levels were extremely low (<0.15 ng/ml, the detection limit of the assay) in 75 and 100% of TgPWS pups at P2 and P3, respectively. Plasma glucagon. The concentration of plasma glucagon was also lower in TgPWS, compared with WT mice [F(3, 1) = 7.4, P = 0.008] (Fig. 3D). Similar to insulin levels, glucagon was also very low (<20 pg/mg, the detection limit of the assay) in TgPWS plasma from P2 to P4. Corticosterone. Corticosterone was significantly higher in TgPWS vs. WT mice at all postnatal days [F(3, 1) = 9.31, P = 0.003] but not at E18 (Fig. 3E). The normal fall in corticosterone levels that occurs after birth was blunted in TgPWS mice (P < 0.0001). Ghrelin. There was no difference in plasma ghrelin levels between TgPWS and WT mice at P1 and P2, but for P3, P4 and P5, ghrelin concentration was significantly higher in TgPWS pups (P = 0.0003) (Fig. 3F). Triglycerides. Between P1 and P4, plasma triglyceride concentrations in TgPWS pups were 35–59% of those in WT pups [F(3, 1) = 91.2, P = 0.0001] (Fig. 4A). Between P1 and P2, there were substantial increases in both WT (+182%; P = 0.0001) and TgPWS (+387%; P = 0.0001) pups. Overall plasma free fatty acids were also significantly reduced in TgPWS mice [F(3, 1) = 19.8, P = 0.0001] and were most profoundly reduced on P1 and P4 (P < 0.05) (Fig. 4B). Concentrations of free fatty acids increased substantially between P1 and P2 in both WT (+95%; P = 0.0001) and TgPWS (+159%; P = 0.0001) pups. In contrast, plasma levels of ketones were higher in TgPWS than in WT pups from P1 to P4 [F(3, 1) = 6.9, P = 0.01] (Fig. 4C). Liver lipids. Paralleling the increase in plasma triglyceride and free fatty acid levels, liver lipid content also increased from P1 to P2 in both WT and TgPWS pups (Fig. 4D). However, this increase was markedly blunted in TgPWS mice (97%, P = 0.0001, and 47%, P < 0.0001, respectively). Overall, between P1 and P4, liver lipid contents were higher in WT pups than TgPWS pups [F(3, 1) = 10.5, P < 0.002]. Liver ATP. Liver ATP content of WT pups did not change between P1 and P4 (Fig. 4E). There was also no difference between WT and TgPWS pups on P1. In contrast, in TgPWS pups, liver ATP content significantly increased from the first to the second day of life in TgPWS mice (P < 0.02) but then fell back to P1 levels by P3. Overall, liver ATP contents were higher in TgPWS pups than WT pups [F(3, 1) = 7.3, P < 0.01]. Between P1 and P4, liver phosphorylation potential decreased gradually in WT pups (P = 0.0001) (Fig. 4F), most likely due to a gradual increase in liver inorganic phosphate content (P = 0.0001) (data not shown). Overall, TgPWS pups had higher liver phosphorylation potential than did WT pups [F(3, 1) = 6.0, P < 0.02], which was highest at P2 and P3 (P < 0.01). Hepatic IGF system gene expression We examined hepatic expression of the genes encoding IGF-I, IGFBP-3, and IGFBP-1 because plasma levels are often reported abnormal in humans with PWS (3, 8, 28, 29). QRT-PCR analysis showed a 2.6-fold decreased level of Igf1 mRNA at P1 (P = 0.002) and a 2.2-fold decreased level at P5 (P = 0.006) but no difference at E18 in TgPWS vs. WT liver (Fig. 5A). Hepatic mRNA levels of Igfbp3, which encodes the protein that binds nearly 75% of circulating IGF-I (30), did not differ between TgPWS and WT mice at all three time points (Fig. 5B). In contrast, Igfbp1 mRNA levels were consistently up-regulated in postnatal TgPWS mice, by 2.3-fold, compared with WT at P1 (P = 0.02), and 5.3-fold at P5 (P = 0.002). However, there was no difference between TgPWS and WT at E18 (P = 0.8) (Fig. 5C). Discussion We measured metabolic and hormonal parameters in a TgPWS deletion mouse model in fetal life and early postnatal life to assess the abnormalities that underlie the neonatal phenotype and premature lethality in these mice. We found a constellation of abnormalities in TgPWS mice that are consistent with the clinical presentation in newborns or children with PWS, including growth retardation, insulin/IGF-I deficiencies, and hyperghrelinemia. Additional findings of abnormal glucose and energy homeostasis and pancreatic insufficiency, beginning in fetal life, provide new insights into the pathophysiological basis of PWS. The most surprising finding of our study was the deficit in insulin and glucagon observed in fetal and postnatal TgPWS mice, suggesting that this model of PWS in mice induces a primary pancreatic defect. To determine whether TgPWS mice have defects in pancreatic development and function, we are currently examining islet architecture and ß- and -cell mass and performing analyses of global gene expression in the pancreas, compared with WT mice. Our findings have important implications for the pathophysiology of type 2 diabetes that is so often observed in patients with PWS. It has generally been hypothesized that diabetes in this population is secondary to profound obesity; however, a number of studies have demonstrated that insulin levels are actually much lower than expected for the degree of the increase in body mass index (3, 10, 11). Glucose levels were normal in fetal TgPWS mice, suggesting that placental function is normal in these mice. The fetal requirement for glucose is met almost, if not entirely, by transplacental transport from the mother to the fetus (31, 32). At birth, there is an abrupt loss of the maternal supply of substrates and nutrients and the newborn has to mobilize glucose and other substrates to meet its energy needs. This is achieved primarily through breakdown of glycogen. A number of factors initiate liver glycogenolysis at birth; however, the precise mechanisms have not been completely elucidated. Whereas insulin and glucagon play a role in the breakdown of glycogen, increased plasma catecholamine levels have been suggested to be the primary mediators of the sudden increase in hepatic glucose output (33). This hypothesis is consistent with our findings of low levels of insulin and glucagon at birth, but a normal decrease in hepatic glycogen content in the first day of life in TgPWS mice. Because of the rapid depletion of glycogen, liver glycogenolysis can support glucose homeostasis for only a short period of time, and maintenance of the newborn's blood glucose levels requires active liver gluconeogenesis. TgPWS mice develop severe hypoglycemia by d 2 of life that is likely due to impaired gluconeogenesis. Phosphoenolpyruvate carboxykinase, the rate-limiting enzyme of gluconeogenesis, is not turned on until several hours after birth and is activated by a decrease in the insulin to glucagon ratio (fall in insulin and rise in glucagon) and an increase in glucocorticoid levels. In response to hypoglycemia, corticosterone levels were appropriately higher in TgPWS mice, but the mutant mice were unable to increase glucose production. The low levels of glucagon and limited substrate supply are likely to be among the primary causes of the inability of the TgPWS mouse to mount an adequate gluconeogenic response. It will be important in future studies to examine the expression levels of genes encoding critical gluconeogenic enzymes or the levels and/or activity of phosphoenolpyruvate carboxykinase or pyruvate carboxylase, for example, that could be altered in the TgPWS mouse. The low levels of glucagon may also explain the relatively low levels of triglycerides and free fatty acids observed in P1 TgPWS pups. The surge of glucagon that normally occurs after birth induces mobilization of lipids from peripheral tissues. After birth, milk provides a relatively high-fat diet, and lipids are the main energy source of the newborn. However, due to severe failure to thrive, TgPWS mice are not able to suckle normally and lipid levels continue to be low. Because ketone concentrations are actually higher in TgPWS mice, it is unlikely that these mice have fatty acid oxidation defects. Fetal TgPWS mice are significantly growth retarded, and growth rates remain abnormally low after birth. PWS patients also have low birth weight and reduced weight gain in infancy (34, 35, 36). Placental insufficiency cannot be implicated because placental weights are normal for TgPWS animals. The growth restriction in TgPWS mice starts in fetal life and is not due to GH deficiency that occurs postnatally in PWS patients (3, 8, 9) because GH does not regulate fetal growth (37, 38). Additionally, levels of hepatic Igf1 and Igfbp1 mRNA were normal in TgPWS at E18, so these are unlikely to play a role in growth retardation of the TgPWS fetus. However, we cannot completely rule out a causative role for the IGF axis because we did not measure Igf1 receptor levels. In contrast, because insulin is significantly reduced in the TgPWS fetus, decreased activity of insulin signaling pathways (39) may be responsible for the growth restriction observed in fetal and newborn TgPWS mice. Nevertheless, because PWS patients have obesity-independent GH and IGF-I deficiencies (3, 4, 5, 6, 7, 8, 9, 10, 28), a pituitary defect could contribute to or underlie postnatal growth retardation of PWS infants and children. Analysis of hepatic gene expression revealed significantly deregulated Igf1 and Igfbp1 expression but normal Igfbp3 expression in TgPWS newborn mice. These changes correlate with known hormonal regulation of these genes. For example, whereas Igfbp3 production is primarily regulated by GH (29), insulin and/or corticosterone are implicated in the hepatic transcriptional regulation of Igfbp1 and Igf1 (40, 41, 42, 43), and these genes are therefore deregulated in the expected manner in hypoinsulinemic TgPWS mice at P1 and P5. Indeed, by P5 there is a dramatic increase in Igfbp1 mRNA in TgPWS on progression of failure to thrive. Because hepatic overexpression of Igfbp1 in two mouse models have been correlated with postnatal growth retardation (44, 45), this may contribute to failure to thrive in the TgPWS mouse. Nevertheless, despite the known alteration in insulin, IGF-I and IGFBP-1 and -3 in individuals with PWS (3, 8, 28, 29), the hormonal and metabolic status of newborns with PWS is unknown, and thus, it is not clear what role the insulin-IGF axis might play in the failure-to-thrive component of PWS in the human, but it is expected that this will be revealed from retrospective or prospective patient studies. Body composition measurements revealed that postnatal TgPWS mice maintain the same proportion of whole-body water and protein content as do their WT littermates, but whole-body fat (triglyceride) levels decrease after P2, a pattern similar to that seen in animals under conditions of energy restriction (46). This is not unexpected for TgPWS pups, given their hypotonia and failure to thrive. The absence of dehydration in TgPWS mice is consistent with the presence of milk in their stomachs at each age (21), although the amount is usually visibly less than for WT littermates. Nevertheless, intake and nutrient uptake have as yet not been examined in TgPWS mice. Although the milk intake is sufficient to prevent dehydration, a reduced intake may explain the decrease in liver lipids, plasma triglycerides, and plasma free fatty acids as well as whole body fat. Another model generated by gene targeting of the imprinting center (Fig. 1) is PWS ID mice, which disrupts a genetic element controlling imprinting and active expression of all the paternally expressed genes in the PWS domain (20). Recently it was found that these PWS ID mice generally survive on an FVB strain but not other genetic backgrounds, with surviving mice being small and not displaying hyperphagia or obesity (47). However, if the PWS hyperphagia and obesity phase results from a compensatory attempt to overcome a metabolic basis for neonatal failure to thrive, as we propose, the lack of the latter phenotype in the PWS ID mice on the FVB strain (47) leads to the prediction that these mice would not develop hyperphagia and obesity. In contrast, TgPWS deletion mice did not survive on the FVB background, even with removal of most WT littermates (Ohta, T., and R. D. Nicholls, unpublished data). An increase in plasma ghrelin levels occurs in postnatal TgPWS mice and appears to begin at the onset of severe hypoglycemia but is not directly coincident with hypoinsulinemia. These findings are consistent with known regulators of ghrelin expression and secretion because both glucose and insulin have been shown to suppress ghrelin levels (48). By P5, ghrelin levels in TgPWS mice are approximately 3-fold higher than in WT littermates, suggesting that high ghrelin levels in TgPWS might be a physiological adaptive mechanism in an attempt to increase feeding via its actions on the arcuate nucleus (49) to ameliorate the rapidly worsening failure to thrive. However, either this signal is unrecognized due to an unknown mechanism, or it may be too late to elicit a physiological response. The finding of high ghrelin levels in TgPWS mice echoes observations in PWS children and adults, in whom plasma ghrelin levels are 2.5- to 4.5-fold higher than those in normal lean and obese controls (13, 14, 15). Although these studies suggested that this orexigenic hormone could be a mediator for the hyperphagia observed in PWS, our mouse data are consistent with an alternative hypothesis that PWS patients metabolically do not sense the degree of adipose tissue, and hence their lean body mass is in a starvation state that induces ghrelin production (50, 51). In accordance with severe hypoglycemia and hypoinsulinemia, postnatal TgPWS pups show evidence of increased fat oxidation, compared with their WT littermates, as revealed by lower plasma levels of triglycerides and free fatty acids as well as increased ketogenesis. Consistent with this, hepatic energy status (ATP content and phosphorylation potential) was higher in TgPWS vs. WT at P2 and P3, possibly as a result of compensatory beta-oxidation due to impaired glucose homeostasis. In adults, it has been proposed that low liver ATP levels stimulates food intake through signals to the central nervous system via vagal sensory neurons (52, 53). Because a decrease in liver ATP in rats is associated with increased eating (24, 54), it is possible that a negative feedback signal of elevated hepatic ATP in TgPWS pups may contribute to a decrease in feeding behavior as part of the failure to thrive process. At least in the newborn rat, it appears that the vagal afferent connections are intact and sufficiently mature (55, 56). In contrast, leptin neural pathways are not mature until closer to the time of weaning (57, 58), suggesting that leptin signaling abnormalities do not underlie the TgPWS phenotype. In conclusion, we characterized the endocrine and metabolic profile of a TgPWS deletion mouse model with fetal growth retardation and neonatal failure to thrive. Most surprising among the deficiencies found in TgPWS mice were fetal insulin and glucagon insufficiency, suggestive of a primary pancreatic defect. It will be important to further examine the mechanisms that might contribute to the phenotype in TgPWS mice and PWS newborns and children. Furthermore, many of the findings in TgPWS mice, such as hypoinsulinemia, low glucose and IGF-I, and high IGFBP-1 and ketones, are also consistently seen in small-for-gestational-age infants in the human (59). The TgPWS mouse therefore serves as a useful PWS and small-for-gestational-age animal model for further investigation, and additional studies on animal models and similar studies on PWS newborns will shed new understanding on the underlying pathophysiological basis for this syndrome. Pediatr Int. 2005 Oct. Background: Thermoregulation problems, resulting in hypo- or hyperthermia, have been infrequently reported in children with Prader Willi syndrome (PWS), yet their clinical details remained unknown. Methods: The clinical characteristics of three infants with PWS are reported. Results: Etiologies of high fever could not be identified in three children with PWS. One of these children was also admitted to the intensive care unit with extremely high body temperature in a life-threatening condition, similar to septic shock, without a plausible explanation. Conclusion: Hyperthermia may be a part of the clinical spectrum in young infants with PWS and should be carefully monitored, since it may cause life-threatening complications. J Clin Endocrinol Metab. 2005 Oct. CONTEXT: Adult patients with Prader-Willi syndrome (PWS) are prone to develop obesity, GH deficiency (GHD), and their related complications, with cardiopulmonary failure explaining more than half of PWS fatalities. OBJECTIVE AND STUDY PARTICIPANTS: This study was undertaken to examine the effect of GHD and sleep breathing disorders on cardiovascular risk factors and heart features of 13 PWS (age 26.9 +/- 1.2 yr) and 13 age-, gender-, and body mass index-matched obese individuals (age 26.2 +/- 0.8 yr). RESULTS: Compared with controls, PWS patients had lower GH response to arginine+GHRH, IGF-I levels, triglycerides, total and LDL-cholesterol, insulin, and insulin resistance measured by a homeostatic model approach. Dual-energy x-ray absorptiometry, abdominal computed tomography scans, and polysomnography revealed a greater fat mass, similar abdominal fat, but greater sleep breathing disorders in PWS than obese subjects. Echocardiography showed no systolic or diastolic alteration, although PWS had lower left ventricle (LV) mass (135.7 +/- 7.7 vs. 163.5 +/- 8.4 g, P < 0.05) and near significantly lower values of LV end-diastole diameter (P = 0.08), compared with obese controls. Baseline radionuclide angiography documented comparable values of systolic and diastolic values between groups. However, adrenergic stimulation with dobutamine caused a lower increase of LV ejection fraction (71.9 +/- 1.9 vs. 76.3 +/- 1.2%, P < 0.05) and heart rate (103 +/- 6.9 vs. 128 +/- 2.8 beats/min, P < 0.05) in PWS than obese individuals. By multivariate analysis, nocturnal oxygen desaturation and IGF-I levels were main significant predictors of LV mass and heart rate in PWS patients. CONCLUSIONS: PWS differs from simple obesity by a healthier metabolic profile, impaired nocturnal breathing, decreased heart geometry, and systolic and chronotropic performance. GHD and the predictive role of IGF-I on structural and functional heart parameters suggest a GH/IGF-I-mediated control of cardiac risk in PWS. J Child Psychol Psychiatry. 2005 Oct. BACKGROUND: Studies of chromosome 15 abnormality have implicated over-expression of paternally imprinted genes in the 15q11-13 region in the aetiology of autism. To test this hypothesis we compared individuals with Prader-Willi syndrome (PWS) due to uniparental disomy (UPD--where paternally imprinted genes are over-expressed) to individuals with the 15q11-13 deletion form of the syndrome (where paternally imprinted genes are not over-expressed). We also tested reports that PWS cases due to the larger type I (TI) form of deletion show differences to cases with the smaller type II (TII) deletion. METHOD: Ninety-six individuals with PWS were recruited from genetic centres and the PWS association. Forty-nine individuals were confirmed as having maternal UPD of chromosome 15 and were age and sex matched to 47 individuals with a deletion involving 15q11-13 (32 had the shorter (T II) deletion, and 14 had the longer (TI) deletion). Behavioural assessments were carried out blind to genetic status, using the Autism Diagnostic Observation Schedule (ADOS), the Autism Diagnostic Interview (ADI), the Autism Screening Questionnaire (ASQ), the Children's Yale-Brown Obsessive-Compulsive Scale (CY-BOCS), the Vineland Adaptive Behaviour Scales (VABS), and measurements of intellectual ability, including the Wechsler and Mullen Scales and Raven's Matrices. RESULTS: UPD cases exhibited significantly more autistic-like impairments in reciprocal social interaction on questionnaire, interview and standardised observational measures. Comparison of TI and TII deletion cases revealed few differences, but ability levels tended to be lower in the TI deletion cases. CONCLUSIONS: Findings from a large study comparing deletion and UPD forms of Prader-Willi syndrome were consistent with other evidence in indicating that paternally imprinted genes in the 15q11-13 region constitute a genetic risk factor for aspects of autistic symptomatology. These genes may therefore play a role in the aetiology of autism. By contrast with another report, there was no clear-cut relationship between the size of the deletion and the form of cognitive and behavioural phenotype. J Clin Endocrinol Metab. 2005 Sep. Context: Narcoleptic patients with cataplexy have a general loss of hypocretin (orexin) in the lateral hypothalamus, possibly due to an autoimmune-mediated degeneration of the hypocretin neurons. In addition to excessive daytime sleepiness, Prader-Willi syndrome (PWS) patients may show narcolepsy-like symptoms, such as sleep-onset rapid eye movement sleep and cataplexy, independent of obesity-related sleep disturbances, which suggests a disorder of the hypocretin neurons. Objective: We hypothesized that the narcolepsy-like symptoms in PWS are caused by a decline in the number of hypocretin neurons. Design: We estimated the number of hypocretin neurons in postmortem hypothalami using immunocytochemistry and an image analysis system. Setting: This study was conducted at the Netherlands Institute for Brain Research. Patients: Eight PWS adults, three PWS infants, and 11 controls were studied. Main outcome measure: The total number of hypocretin neurons in the lateral hypothalamus was measured. Results: There was no significant difference in the total number of hypocretin-containing neurons among the seven PWS patients (in whom sufficient hypothalamic material was available to quantify total cell number) and seven age-matched controls, either in adults or in infants. A significant decline with age was found in adult PWS patients (r = -0.9; P = 0.037). Conclusions: We conclude that a decrease in the number of hypocretin neurons does not play a major role in the occurrence of narcolepsy-like symptoms in PWS. Nucleic Acids Res. 2005 Aug 22. The imprinted SNRPN locus is a complex transcriptional unit that encodes the SNURF and SmN polypeptides as well as multiple non-coding RNAs. SNRPN is located within the Prader-Willi and Angelman syndrome (PWS/AS) region that contains multiple imprinted genes, which are coordinately regulated by a bipartite imprinting center (IC). The SNRPN 5' region co-localizes with the PWS-IC and contains two DNase I hypersensitive sites, DHS1 at the SNRPN promoter, and DHS2 within intron 1, exclusively on the paternally inherited chromosome. We have examined DHS1 and DHS2 to identify cis- and trans-acting regulatory elements within the endogenous SNRPN 5' region. Analysis of DHS1 by in vivo footprinting and chromatin immunoprecipitation identified allele-specific interaction with multiple regulatory proteins, including NRF-1, which regulates genes involved in mitochondrial and metabolic functions. DHS2 acted as an enhancer of the SNRPN promoter and contained a highly conserved region that showed allele-specific interaction with unphosphorylated RNA polymerase II, YY1, Sp1 and NRF-1, further suggesting a key role for NRF-1 in regulation of the SNRPN locus. We propose that one or more of the regulatory elements identified in this study may also contribute to PWS-IC function. Excerpt from the full text article: In addition, we have identified by sequence analysis a conserved potential NRF-1 binding site in the NDN promoter region, which coincides with a sequence that is in vivo footprinted on the paternal NDN allele only. The fact that NRF-1 may be regulating at least some of the genes in the PWS/AS region is interesting because of the involvement of NRF-1 in the regulation of genes related to mitochondrial biogenesis and function, metabolism (including growth factor receptors and factors involved in glucose homeostasis), DNA replication and transcriptional regulation. This suggests that genes in the AS/PWS region and genes that function in metabolism and in cellular energetics may be co-regulated through the common transcriptional regulator NRF-1. This would further suggest a potential link between energy metabolism and aspects of the PWS phenotype (e.g. obesity and growth factor deficiency). However, the resting metabolic rate of PWS patients does not seem to differ from that of normal obese individuals. Am J Med Genet A. 2005 Jul 1. Patients with Prader-Willi syndrome (PWS) are recognized to have a tendency of sudden, unexpected death (SED), but its exact cause is unknown because of paucity of such case reports. Since growth hormone (GH) treatment was applied to PWS patients worldwide, several cases of death have been reported. However, whether the therapy is directly related to their SED remains unknown, too. We collected 13 deceased PWS patients (Group A, aged 9 months to 34 years) who had never received GH therapy, and seven deceased patients (Group B, all boys aged 0.7-15 years) having received the therapy from the registration in PWS-patient-support associations and from the literature, respectively. We then compared the cause of SED between the two groups. Irrespective of GH therapy, SED of infants under age 1 year was associated with milk aspiration or hypothalamic dysregulation of respiration, while SED of patients in early childhood or adolescence occurred at sleeping in association with preceding viral infections. In contrast, SED of four adult (>20 years of age) patients who never received GH therapy was associated with complications, such as leg cellulites and pulmonary embolism, secondary to massive obesity and diabetes mellitus (DM). Two Group-B patients (aged 14 and 20 years) without any obesity-related or diabetes-related complications died of drowning in a bath tub, and their drowning death could be related to poor respiratory control. These findings indicated that the cause of SED is not essentially different between PWS patients with and without GH treatment. Deceased PWS patients may have had underlying respiratory dysregulation and hypothalamic dysfunction, and GH therapy might have led to certain obstructive respiratory disturbances that exacerbated the respiratory conditions. This will call clinicians' attention when using GH in PWS patients, for example, careful determination of the dose of GH and careful monitoring of patient's respiratory conditions, especially in male obese patients with respiratory problems. Acta Paediatr. 2005 Jul. Reports on sudden death in Prader-Willi syndrome (PWS) patients after the start of growth hormone (GH) treatment have been published recently. We observed a 4.7-y-old girl who showed a continuous increase in pulmonary artery pressure and died of cardiorespiratory failure 7 wk after GH therapy had been initiated, and a 9.3-y-old girl with additional trisomy 21 who died during a minor respiratory infection 6 mo after GH had been started. Both patients were overweight (weight for height 127% and 224%, respectively). GH-induced fluid retention may have occurred in the younger girl. In contrast to the reported cases, our PWS patients were female. CONCLUSION: Our cases illustrate the difficulty of differentiation between possible GH side effects and the natural course of disease, in particular with respect to obesity-related comorbidity and mortality. J Clin Endocrinol Metab. 2005 Jul. CONTEXT: Determinants of insulin resistance in Prader-Willi syndrome (PWS) are not completely understood. The discovery of several adipokines with relevant effects on insulin resistance and cardiovascular complications of metabolic syndrome offered new tools of investigation of insulin resistance in PWS. OBJECTIVE: The purpose of this study was to measure serum resistin and mRNA in adipose tissue of patients with PWS, those with simple obesity, and healthy controls and correlate resistin levels with anthropometric and biochemical features. DESIGN: Twenty-eight adult PWS patients, 29 obese patients, and 25 healthy controls were studied. Anthropometric variables were measured and fasting serum and plasma were collected for measurement of resistin, adiponectin, leptin, lipid profile, glucose, and insulin. RESULTS: Serum resistin and resistin mRNA expression in adipose tissue was significantly higher in PWS patients, compared with both healthy lean controls and obese patients. Moreover, on regression analysis resistin was significantly correlated with body mass index, whereas no significant association was found between resistin and homeostasis model assessment index. A weak association between resistin and adiponectin was found in the PWS group only. However, on multivariate analysis only the correlation between resistin and body mass index remained significant. CONCLUSIONS: These results support a link between circulating resistin and obesity in humans but do not support a role for resistin in human insulin resistance. Pediatr Int. 2005 Jun. No abstract available. J Endocrinol Invest. 2005 Jun. A few cases of death worldwide during GH treatment in pediatric patients with Prader-Willi syndrome (PWS) have been recently described. The evaluation of further cases is needed to better identify possible causal mechanism(s), as well as to suggest some additional guidelines for prevention. We report the death of 2 additional children with genetically confirmed PWS in the first months of GH therapy. Case 1: This 3.9-yr-old girl was born at 39 weeks gestation. Low GH response to two stimulation tests was observed. GH administration was started at the age of 3.5 yr (0.33 mg/kg per week), when the patient was at 130% of her ideal body weight (ibw). Hypertrophy of adenoids was previously demonstrated. Snoring and sleep apnea were present before GH treatment, and did not increase during therapy. Four months later she died at home suddenly in the morning. Case 2: This patient was a 6.3-yr-old boy. He was born at term after an uneventful pregnancy. At the age of 6 yr, his weight was at 144% of his ibw. He showed reduced GH secretion during provocation tests, and GH therapy was started (0.20 mg/kg per week). The previously reported nocturnal respiratory impairment had worsened after beginning GH administration. Tonsils and adenoids hypertrophy were noted. At the age of 6.3 yr he died at home in the morning following an acute crisis of apnea. These additional cases seem to confirm that some children with PWS may be at risk of sudden death at the beginning of GH therapy. J Intellect Disabil Res. 2005 Jun. BACKGROUND: Apart from a pervasive eating disorder, the Prader-Willi (PWS) syndrome is characterized by a distinct behavioural profile comprising maladaptive behaviours, obsessive-compulsive traits and skin picking, all included in the PWS behavioural phenotype. In this study, we present a further delineation of this characteristic behavioural profile by screening for indices of executive dysfunctions related to attention-deficit/hyperactivity disorder (ADHD), immature compulsive-like adherence to sameness and skin picking, and how these features aggregate into symptom constellations in children and adolescents with PWS. METHOD: Parents of 58 individuals with PWS (aged 5-18 years) participated by completing Childhood Routines Inventory (CRI) and Conners' Parent Rating Scale (CPRS-48). RESULTS: Results showed that indices of ADHD and excessive insistence on sameness were common, comorbid and of early onset. They were both associated with conduct problems. Skin picking, appearing as a single and comorbid symptom, was less associated with childlike compulsions and ADHD-related problems. CONCLUSIONS: Findings are discussed in terms of further research in executive dysfunctions in PWS. Int J Legal Med. 2005 May. A case of sudden cardiac death in a 3-year-old young male affected by Prader-Willi syndrome, clinically diagnosed and confirmed by means of DNA methylation, is presented. The infant suddenly collapsed at home and was taken apparently unconsciousness by his mother to the emergency clinic where he was pronounced dead. A complete postmortem examination was performed and the histological findings led to the definition of cardiac death with a typical picture of contraction band necrosis. Pulmonary hypoxic alterations are frequently reported as the primary cause of death in PWS cases. In this fatal case according to the macroscopic and microscopic findings, the cause of death was most likely cardiac and possibly related to contraction band necrosis linked with ventricular fibrillation and sudden death. J Clin Endocrinol Metab. 2005 May. The cause of the unique elevation in fasting plasma levels of the orexigenic gastric hormone ghrelin in many patients with Prader-Willi syndrome (PWS) is unclear. We measured fasting and postprandial plasma ghrelin in nonobese (n = 16 fasting and n = 8 postprandial) and obese non-PWS adults (n = 16 and 9), adults with genetically confirmed PWS (n = 26 and 10), and patients with hypothalamic obesity from craniopharyngioma tumors (n = 9 and 6). We show that 1) plasma ghrelin levels decline normally after food consumption in PWS, but there is still fasting and postprandial hyperghrelinemia relative to the patient's obesity (2.0-fold higher fasting ghrelin, 1.8-fold higher postprandial ghrelin, adjusting for percentage of body fat); 2) the fasting and postprandial hyperghrelinemia in PWS appears to be at least partially, but possibly not solely, explained by the concurrent relative hypoinsulinemia and preserved insulin sensitivity for the patient's obesity (residual 1.3- to 1.6-fold higher fasting ghrelin, 1.2- to 1.5-fold higher postprandial ghrelin in PWS, adjusting for insulin levels or homeostasis model assessment of insulin resistance); 3) hyperghrelinemia and hypoinsulinemia are not found in craniopharyngioma patients with hypothalamic obesity, and indeed, these patients have relative hyperinsulinemia for their obesity; and 4) there is no deficiency of the anorexigenic intestinal hormone peptide YY(3-36) in PWS contributing to their hyperghrelinemia. J Intellect Disabil Res. 2005 Apr. BACKGROUND: Prader-Willi syndrome (PWS) is a multisystem genetic disorder characterized by short stature, muscular hypotonia, hyperphagia, obesity, maladaptive behaviour, hypogonadism and partial growth hormone (GH) deficiency (GHD). Severe GHD of other aetiologies has been shown to affect mood and quality of life negatively, and there are reports of improvements with GH replacement. We have studied cognitive, emotional, physical and social parameters in PWS adults at baseline, during and after GH treatment. PATIENTS AND METHODS: Nineteen patients, 9 females and 10 males, median age 25 years, mean BMI 35 kg/m2 participated in this study. Approximately half of the group had GHD. All patients fulfilled the clinical criteria for PWS and 13 had a positive genotype. The patients were randomized to 6 months of treatment with either GH [1.6 IU/day (0.53 mg/day)] or placebo, followed by 12 months of active GH treatment. Treatment was then stopped, and the patients were followed for an additional period of 6 months. A test battery for general cognitive evaluation and a computer-based measurement of reaction time, motor speed and fluency were employed at baseline, after 6 months and at the end of GH treatment. At the same time intervals, a self-evaluation questionnaire was answered at the end of each test session. Other questionnaires reflecting the patients' cognitive, emotional, physical and social status were answered by relatives/caretakers at baseline and at 3 and 6 months following cessation of GH treatment. RESULTS: Baseline cognitive level was estimated to be moderately to mildly impaired; IQ range was 40-90. The results from some of the cognitive and the motor performance tests improved significantly after 6 and 18 months of GH treatment. According to the questionnaires, both the patients and the relatives/caretakers evaluated physical status rather negatively at baseline, but still, impairments in both physical and social status and overall functioning were observed when GH treatment was discontinued. The self-evaluation did not change in any aspect during GH treatment. CONCLUSIONS: In this pilot study of an adult PWS cohort, we were able to document beneficial effects in mental speed and flexibility and in motor performance during GH treatment. Impairment was seen in physical and social status as well as overall functioning, when GH treatment stopped. Studies of larger cohorts are needed to further elucidate the role of GH treatment in this group of patients. Int J Mol Med. 2005 Apr. Ghrelin and peptide YY (PYY) are peptides generally produced by the gastrointestinal organs which are involved in appetite regulation via highly specialized centers in the brain. Abnormal plasma ghrelin and PYY levels compared with controls have been reported for subjects with Prader-Willi syndrome (PWS) which is characterized by infantile hypotonia, poor suck reflex and failure to thrive followed by hyperphagia and marked obesity in early childhood. We studied gene expression of ghrelin, peptide YY, and their receptors (i.e., GHS-R1a, GHS-R1b, and NPY2R) in six different brain regions (frontal cortex, temporal cortex, visual cortex, pons, medulla, and hypothalamus) obtained from three subjects with PWS, two individuals with Angelman syndrome, and six controls to determine if expression of these genes is detectable in different regions of the brain in subjects with and without PWS. In general, expression of these genes using RT-PCR was detected in all subjects and no obvious differences were seen in their pattern of expression between subjects with or without PWS. Additional studies including quantitative gene expression measurements will be required to further evaluate the role of these genes in the eating disorder seen in PWS. Clin Genet. 2005 Mar. Prader-Willi syndrome (PWS), the most common genetic cause of marked obesity in humans, is usually due to a de novo paternally derived chromosome 15q11-q13 deletion or maternal disomy 15 [(uniparental disomy (UPD)]. Obesity is due to energy imbalance, but few studies have examined fat patterning and obesity-related factors in subjects with PWS (deletions and UPD) compared with subjects with simple obesity. We examined for differences in fatness patterning and lipid, leptin, and glucose and insulin levels in subjects with simple obesity and PWS and adjusted for gender, age, and body mass index (BMI). Fasting peripheral blood samples and cross-sectional magnetic resonance image scans at the level of the umbilicus were obtained in 55 subjects ranging in age from 10.4 to 49 years: 20 PWS deletion, 17 PWS UPD, and 18 obese controls. Subcutaneous fat area (SFA) and intra-abdominal visceral fat area (VFA) were calculated. No significant difference was seen between the PWS deletion subjects or PWS UPD subjects for fatness measurements or leptin levels. Twenty-three of 37 PWS subjects met the criteria for obesity (BMI > 95th percentile). No significant differences were observed for SFA and VFA between the PWS subjects judged to be obese and control subjects with simple obesity. There was an overall trend for decreased VFA in the PWS subjects but not significantly different. VFA was significantly positively correlated with both fasting insulin and total cholesterol in PWS deletion subjects but not in PWS UPD subjects or obese controls. Fasting insulin level was significantly lower in the obese PWS subjects compared with subjects with simple obesity, and insulin sensitivity (QUICKI) was significantly higher in PWS subjects with obesity. Homeostasis model assessment (HOMA) and QUICKI values were correlated and in opposite directions with triglycerides in the obese PWS subjects but not in the obese controls. Subjects in each group were stratified according to published criteria on the basis of their level of visceral fat (e.g. > or = 130 cm(2)) to assess the influence of VFA on metabolic abnormalities. In the obese PWS subjects, the fasting triglyceride, glucose, and insulin levels, and HOMA value were significantly elevated, while the QUICKI value was significantly lower in those with VFA > or = 130 cm(2). Such significant differences were not seen in the obese control group. Our results indicate that VFA may be regulated differently in PWS subjects compared to individuals with simple obesity. Insulin resistance is lower in PWS subjects and insulin sensitivity is higher compared with obese controls. PWS subjects with increased VFA may be at a higher risk of obesity-related complications compared to PWS subjects without increased VFA. Brain Dev. 2005 Mar. Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurobehavioral syndromes that are caused by deficiency of gene expression from paternally or maternally derived homologues on chromosome 15q11-q13, respectively. Clinical and genetic heterogeneities are common in both syndromes and they are now regarded as 'sister genetic imprinting syndromes'. This study aimed to describe and compare the electroclinical characteristics of seizures between PWS and AS, and to try to explore the possible mechanisms of epileptogenesis in these two syndromes. Fifty patients with genetically documented PWS and 18 patients with a putative diagnosis of AS were included in this study. These patients were diagnosed on the basis of characteristic physical findings and their neurobehavioral phenotype, as well as cytogenetic and molecular studies. Epileptic seizures were present in 16 of 18 patients with AS, but in only eight of 50 patients with PWS. Using electroencephalography (EEG), the most characteristic findings for AS were rhythmic 2-3 Hz delta waves of high-amplitude that were maximal over the frontal regions, and 3-4 Hz spikes and sharp wave runs posteriorly. These were never seen in PWS. Patients with AS had a much higher incidence of seizures with characteristic EEG findings, similar to those seen in mice that are deficient in a single gene (UBE3A) that displays regional brain-specific imprinting in humans and mice. In this series, cases with no detectable cytogenetic or molecular defect at the AS locus displayed similar AS phenotype, seizure severity and EEG abnormalities compared to those with such a defect. Thus, the UBE3A gene is presumed to be potentially involved in the epileptogenesis of AS. It is also possible that UBE3A and another gene located nearby, gamma-aminobutyric receptorbeta3 subunit, may interact in some way, and result in the severe epilepsy seen with AS. Some patients with PWS and AS share the common EEG features of persistent high-amplitude 4-6 Hz activity in recordings during sleep, and while awake. The significance of such EEG findings needs further experience to clarity. Hum Mol Genet. 2005 Mar 1. Necdin and Magel2 are related proteins inactivated in Prader-Willi syndrome (PWS), a sporadic chromosomal deletion disorder. We demonstrate that necdin and Magel2 bind to and prevent proteasomal degradation of Fez1, a fasciculation and elongation protein implicated in axonal outgrowth and kinesin-mediated transport, and also bind to the Bardet-Biedl syndrome (BBS) protein BBS4 in co-transfected cells. The interactions among necdin, Magel2, Fez1 and BBS4 occur at or near centrosomes. Centrosomal or pericentriolar dysfunction has previously been implicated in BBS and may also be important in the features of PWS that overlap with BBS, such as learning disabilities, hypogonadism and obesity. Morphological abnormalities in axonal outgrowth and fasciculation manifest in several regions of the nervous system in necdin null mouse embryos, including axons of sympathetic, retinal ganglion cell, serotonergic and catecholaminergic neurons. These data demonstrate that necdin mediates intracellular processes essential for neurite outgrowth and that loss of necdin impinges on axonal outgrowth. We further suggest that loss of necdin contributes to the neurological phenotype of PWS, and raise the possibility that co-deletion of necdin and the related protein Magel2 may explain the lack of single gene mutations in PWS. Excerpts from the full text article: Introduction NDN (encoding necdin) and MAGEL2 are two of four protein-coding genes inactivated in individuals with Prader–Willi syndrome (PWS), a sporadic chromosome deletion disorder marked by profound neonatal hypotonia, global developmental delay, hypoventilation, childhood onset hyperphagia, obesity and hypogonadism (1). Hypothalamic dysfunction underlies many aspects of this disorder and, at least one of the inactivated PWS genes, is predicted to be critical for the normal development and function of the hypothalamus (2). Decreased fetal movements, decreased myelination, hypoplasia of cortical commissures and enlarged lateral ventricles are found in fetuses and young children with PWS (3,4). The partial resolution of both neonatal hypotonia and failure to thrive suggested to us that a delay in the maturation of neuronal circuits might underlie the early course of PWS. In adults, additional abnormalities include chronically elevated levels of the orexigenic hormone ghrelin, elevated levels of serotonin metabolites and, to a lesser extent, dopamine metabolites in the cerebrospinal fluid (2,5) and peripheral insensitivity to pain. Defects in parasympathetic innervation of the gut and/or failure of serotonergic neuronal transmission could contribute to the voracious appetite of individuals with PWS. Simultaneous deletion of all PWS-equivalent genes in mice is usually lethal in the first postnatal week because of hypoventilation and failure to thrive (6,7). Necdin and MAGEL2 are part of a multiprotein family related by a MAGE homology domain. This family also includes MAGED1 (NRAGE), which interacts with the p75 neurotrophin receptor and facilitates nerve growth factor (NGF)-mediated apoptosis through a Jun kinase-dependent pathway (8,9). Expression of murine Magel2 is highest in neurons of the developing hypothalamus, particularly the suprachiasmatic nucleus and supraoptic tract. Magel2 null mice have not been described. Murine Ndn is expressed in many but not all postdifferentiation stage neurons (10,11), and also in developing muscle, skin and cartilage (11). A subset of necdin null mice exhibit a defect in respiratory rhythm generation in the medulla, causing hypoventilation with high neonatal mortality (12–15). A role for necdin in neuronal terminal differentiation is supported by experiments showing that necdin-transfected PC12 cells display increased differentiation and accelerated neurite extension in response to nerve growth factor (16), that ectopic necdin expression induces neurite outgrowth in neuroblastoma cells (17) and that repression of necdin in embryonic dorsal root ganglion cells suppresses their differentiation (18). We now present a novel role for necdin and Magel2 in neuronal function. We identified an interaction between both necdin and Magel2 and fasciculation and elongation (Fez) proteins implicated in centrosome-mediated cytoskeletal rearrangement after neuronal differentiation and in axonal outgrowth. We identified a second interaction of necdin and Magel2 with BBS4, another protein implicated in centrosome function. BBS4 is one of several genes mutated in Bardet–Biedl syndrome (BBS), a complex disorder in which affected individuals display learning disabilities, retinopathy and obesity, together with hypogonadism, cardiac, limb and kidney malformations (19). We show that necdin null embryos have defects in cortical commissural formation and axonal extension, bundling and pathfinding. These results suggest that necdin is required to facilitate the intracellular processes that underlie neurite and axonal outgrowth in embryonic neurons, leading us to propose that loss of necdin impairs these processes in necdin null mice and in PWS. Furthermore, if the functions of necdin and Magel2 are partially redundant in key neurons, their combined loss in PWS may abrogate this shared function. We postulate that PWS is one of an emerging class of neurodevelopmental disorders that includes BBS, schizophrenia and lissencephaly, which are in part caused by defects in centrosome function in cytoskeletal rearrangement during neurite extension. Results Necdin and Magel2 interact with fasciculation and elongation proteins Fez1 and Fez2 We performed a screen for cytoplasmic proteins interacting with necdin using the yeast two-hybrid Ras rescue system (16). We identified a necdin-interacting protein corresponding to amino acids 46–282 of the 353 amino acid Fez2 protein. Fez2 (UniGene Cluster Hs.258563), which is widely expressed, and Fez1 (UniGene Cluster Hs.79226), which is primarily expressed in the brain, are mammalian homologues of Caenorhabditis elegans and Drosophila melanogaster unc-76 (20–26). UNC-76 protein is found in the cell body and axonal cytoplasm of C. elegans neurons during development, and is essential for axon bundling (C. elegans) and transport mediated by the microtubule motor protein kinesin (D. melanogaster). The unc-76 mutation can be partially rescued by ectopic expression of human Fez1, suggesting retention of function between species (21). We confirmed the interaction of Fez1 and Fez2 (together called Fez1/2) with necdin by co-immunoprecipitation of transiently transfected epitope-tagged full-length proteins. [...] Necdin and Magel2 may increase Fez1 levels by increasing Fez1 transcription or stability. We first determined by RT–PCR that equivalent amounts of Fez1 RNA are present in cells transfected with pHAFez1 with or without pXNdn (data not shown). Co-transfected cells were then treated with each of three different proteasome inhibitors. In the presence of proteasome inhibitors, HAFez1 is 3-fold more abundant than with DMSO alone (Fig. 1G), and is slightly more abundant than when co-expressed with XNdn (Fig. 1D). Similarly, co-transfection of pXNdn with pHAFez2 increases the amount of HAFez2, and proteasome inhibitors stabilize HAFez2 in the absence of necdin co-transfection (Fig. 1G). This suggests that necdin stabilizes Fez1/2 by preventing their degradation by the proteasomal pathway. Co-localization of necdin and Fez1 near centrosomes Endogenous Fez1 is present in SK-N-SH neuroblastoma cells in a punctate fashion in the cytosol, along organized filamentous structures (23). In cultured rat hippocampal neurons, Fez1 also colocalizes with F-actin in growth cones (23). Endogenous necdin is highly concentrated in the cytoplasm of differentiated neurons, and moves to the nucleus under specific conditions (27). [...] Interaction of necdin with BBS4 We next investigated whether necdin or Magel2 interact with other proteins located in centrosomes. One of the proteins implicated in the genetic disorder BBS, BBS4, localizes to basal bodies of ciliated cells and to centrosomes. BBS4 is proposed to act in intracellular microtubule-associated transport but not in the formation of the cilia themselves (19,28). Given the overlapping phenotypes of PWS and BBS and the common centrosomal localization of BBS4 and the necdin/Magel2-interacting protein Fez1, we explored the interactions among necdin, Magel2, Fez1 and BBS4. We co-transfected HEK293 cells with pMycBBS4 and/or pHAFez1. As expected, staining for both epitopes revealed an overlap in centrosomes, with HAFez1 also present in the cytoplasm (Fig. 2C). On co-transfection of pHABBS4 and pXpressNdn, both proteins were present in centrosomes, although transfected necdin is also widely distributed in the cell (Fig. 2D). Transfected Magel2 is present in a punctate pattern in the cytoplasm, and is also detected in an overlapping juxtanuclear location with transfected HABBS4 (Fig. 2E). [...] Axonal extension and bundling of primary sympathetic neurons are impaired in cell culture Because Fez-related proteins are essential for axonal elongation in C. elegans (21) and kinesin-dependent axonal transport in D. melanogaster (25) and because of the role of the centrosome in organizing microtubules during axonal outgrowth in postmitotic neurons (29), we investigated whether the absence of necdin caused a defect in neuronal fasciculation and elongation in the developing murine embryonic nervous system. We assayed superior cervical ganglion (SCG) neurons, which extend axons in a reproducible manner when plated in compartmented chambers (30); these neurons normally express necdin (11). In this assay, dissociated neurons are plated in the central compartment of a tissue culture dish, and their axons extend along collagen tracks and cross under grease barriers into side compartments that contain different culture medium. To observe differences in neuronal bundling, we isolated SCGs from control or necdin null embryonic day 17.5 (E17.5) mouse embryos, then dissociated and plated the neurons in compartmented culture dishes supplemented with rat serum in the center compartment and NGF in the side compartments. The necdin null ganglia were typically more difficult to dissect, and the neurons grew poorly in culture, with thinner, less bundled and more branched axons in surviving neurons (Fig. 3A). At higher magnification, we observed necdin null axons with varicosities and atypically localized thickenings, changes in direction without branching and backtracking towards the barrier. These anomalies are rarely seen in control axons (Fig. 3B). The necdin null growth cones are swollen rather than flattened, with ruffles as is typical of control growth cones (Fig. 3C). Overall, the outgrowth, bundling and morphology of axons are significantly compromised in necdin null SCG neurons. Axon projections and tracts are reduced or misrouted in necdin null mice The compartmented culture data established the importance of necdin for axon outgrowth in vitro. We then compared the histology of axon tracts in necdin null embryonic brain sections with those of control littermates. Thionin labels neuronal Nissl substance and cell nuclei, and is excluded from axonal tracts. On sagittal sections at E18.5, the axonal bundle leading into anterior commissure is typically reduced or missing in the necdin null embryos (Fig. 4A'). In contrast, the corpus callosum axonal bundle and the hippocampal commissure axonal bundle are variably affected in the mutant embryos. The lateral and fourth ventricles were consistently moderately enlarged in necdin null embryos, from E13.5 to birth. Abnormalities in the corpus callosum and fornix were also apparent by anti-neurofilament immunohistochemistry of transverse brain sections at E18.5 (Fig. 4B). To visualize the optic chiasm, we traced the trajectory of retinal ganglion cell (RGC) axons in E16.5 mice by placing DiA powder in the optic cup of the right eye, then 10 days later examining coronal sections through the telencephalon. The optic chiasm is larger and less compact as visualized by thionin staining in the necdin null embryos (Figs 4C and 5E), and the DiA labeling shows that most of the axons reach the contralateral side, as in the control (Fig. 4D). We then examined the fasciculus retroflexus (Fr), a long fasciculated tract that carries axons within its core from the medial habenula to the interpeduncular nucleus, and carries axons within its sheath from the lateral habenula to specific midbrain targets. In thionin-stained sagittal (Fig. 4E) and coronal (Fig. 4F) E18.5 control sections, the Fr is visualized as a single, non-staining bundle of axons. In contrast, the Fr is less tightly bundled in the necdin null (Fig. 4E') and appears as a cluster of non-staining ectopic circular axonal bundles (arrowheads in Fig. 4F') that surround what is normally a tight axonal bundle. This confirmed a fasciculation defect in both cultured and in vivo neurons, and led us to perform a more detailed immunohistological investigation of the necdin null brain. On thionin-stained sections of necdin null embryos from E14.5 onwards, an ectopic axonal bundle was detected in the anterior hypothalamic region dorsolateral to the optic chiasm and rostral to the zona incerta and lateral hypothalamus (Lh) (Fig. 5A' and B'). At E16.5, this bundle has not increased in its extent, the internal capsule is poorly defined and ectopic axonal whorls become evident in the striatum of the necdin null embryos (Fig. 5C'). We supposed that the ectopic bundle could be misrouted thalamocortical axons (Tca), which normally course ventrally from the thalamus, then rostrally through the internal capsule before extending dorsolaterally into the cortex (31), or alternatively could be misrouted RGC axons (32). At E16.5, the necdin null thalamic axons labeled with the L1 cell adhesion molecule do not form an internal capsule, and a reduced number of projections extend to the intermediate zone of the cortex (Fig. 5D', arrow). Both the ectopic bundle in the rostral lateral hypothalamus and the smaller ectopic whorls in the thalamus stain strongly with L1, which labels both thalamic efferent projections and RGC axons. RGC axons are normally fasciculated within the optic nerve, course through the optic chiasm at the base of the ventral hypothalamus and partially defasciculate as they grow over the diencephalon to reach the superior colliculus (32) (Fig. 5E). In the necdin null E16.5 embryos, DiA-labeled RGC axons reach the optic chiasm and extend partway along the lateral border of the thalamus, but we failed to detect any axonal labeling, with DiA extending into the superior colliculus on coronal sections (Fig. 5E') or on transverse sections. Some of the RGC axons deviate dorsomedially into the dorsal aspect of the ectopic patch (Fig. 5E'). In both the control and the necdin null brain, a minority of RGC axons does not cross at the optic chiasm but instead course along the ipsilateral aspect of the hypothalamus. In summary, the defects in the thalamus and hypothalamus of necdin null embryos include failures in both extension and routing of thalamocortical and retinal ganglion cell axons towards their respective targets. Developmental timing of the axonal defect We analyzed mid-gestation embryos to define the timing of the axonal defects, particularly the ectopic bundle in the developing hypothalamus. The hypothalamus is of interest because of its major role in endocrine function and appetite regulation, which are profoundly affected in PWS. Necdin and Fez1 are co-expressed in the developing hypothalamus by E10.5 (Fig. 6A–D). We then investigated whether the expression of early hypothalamic markers was perturbed in the necdin null mice. The obesity-related transcription factor Sim1 and Magel2 are expressed in regions overlapping with Ndn expression in the early hypothalamic neuroepithelium (11,33), and Sim1 is also expressed in the zona limitans. The expression of Sim1 and Magel2 in the necdin null embryo is comparable to control in serial sections through the hypothalamus at E12.5 (Fig. 6E–G). At E13.5, a misrouting of thalamocortical L1-positive axons is evident in the ventral part of the dorsal thalamus (Fig. 6H'), the region later to become part of the ectopic bundle shown in Figure 5C. Thus no evidence for defects in hypothalamic neuroepithelial differentiation was found at E12.5, but misrouting of hypothalamic axons was seen in necdin null embryos by E13.5. Serotonergic and catecholaminergic projections are reduced in the necdin null embryo The rostral and caudal projections of the serotonergic raphe nuclei participate in many functional systems, and are implicated in behavioral disorders including PWS (34). At E12.5, we detected a comparable number and placement of rostral serotonergic cells using RNA in situ hybridization with a probe for the serotonin transporter (SERT) in control and necdin null embryos (Fig. 7A). Immunohistochemistry with an anti-serotonin antibody reveals that while these cell bodies were indeed producing serotonin, the ascending fibers containing serotonin are not detectable in the mesencephalon of the necdin null embryo (Fig. 7B'). Furthermore, serotonin immunohistochemistry on sagittal sections at E15.5 revealed a continuing paucity of ascending serotonergic-positive fibers (Fig. 7C'). Immunohistochemistry with anti-tyrosine hydroxylase (TH) antibody at E15.5 normally detects the noradrenergic neurons of the brainstem and their rostral and caudal projections, and the mesencephalic dopaminergic neurons and their striatal, cortical and limbic projections. In a section adjacent to a thionin-stained section showing the ectopic thalamocortical bundle (Fig. 8A), we noted a severe disruption of the TH-positive projections in the nigrostriatal pathway and caudoputamen (Nsp) (Fig. 8B). In a section 600 µm rostral to that in Figure 8B, few TH-positive axons were detected in the necdin null section (Fig. 8C), but a section 600 µm caudal to the ectopic bundle had fairly normal TH staining of the cell bodies in the dorsal medial hypothalamus (Dmh) and the Nsp (Fig. 8D). Thus both the serotonergic and catecholaminergic projection systems are disrupted in the necdin null embryo. Discussion We propose that deficiency of necdin causes a delay or dysfunction in axonal extension, which accounts for the consistent finding of profound hypotonia, reduced myelination, enlarged ventricles and commissural defects described in fetuses and children with PWS. A defect in parasympathetic innervation of the stomach has been suggested in PWS (2), and parallels with the axonal defects we see in necdin null sympathetic neurons. Defects in serotonin pathways and altered responses to psychoactive drugs targeting dopaminergic pathways are consistently seen in PWS, mirroring the dysfunction in the late embryonic necdin null embryo. In this model, hypoventilation and central sleep apneas prevalent in individuals with PWS are also ascribed to necdin deficiency (15,35). We propose that necdin and the related, co-deleted protein Magel2 act in Fez and BBS4 centrosome-related activities that lead to cytoskeletal rearrangements during neurite outgrowth. Centrosomes are critically dependent on the function of BBS proteins (including BBS4) (19), providing a tantalizing mechanistic link between these two rare disorders that each result in impaired mental development and obesity (19). BBS4 is thought to transport the scaffold protein PCM1 to centrosomal satellites through interactions with the dynein microtubule-based molecular motor, allowing for formation of the centrosomal microtubule organizing center (19). In D. melanogaster, the interaction of the Fez orthologue UNC-76 with the molecular motor kinesin is essential for axonal transport (25), and loss of UNC-76 causes disruption of fast axonal transport and 'axon clogs' similar in appearance to the varicosities we describe in necdin null cultured sympathetic neurons. Necdin enhances neurite outgrowth in NGF-stimulated PC12 cells, whereas Fez1, when phosphorylated by PKC zeta, causes neurite outgrowth in PC12 cells (22). We identified a necdin- and Magel2-mediated protection of Fez proteins from proteasomal degradation, and co-localization of necdin and Fez to a juxtanuclear compartment overlapping centrosomes. Moreover, Ndn, Magel2 and Fez1 mRNAs are co-expressed in the embryonic ventral and caudal hypothalamus. Unfortunately, Magel2 null mice are not available to test whether specific hypothalamic neurons are dependent on Magel2 function for appropriate axonal extension. Together, our data support a model whereby up-regulation of necdin/Magel2 in postmitotic neurons stabilizes Fez proteins to facilitate centrosome-mediated cytoskeletal rearrangements required for axonal outgrowth and kinesin-mediated transport. The necdin null pathology also emulates a subset of neuronal patterning and specification mutant phenotypes that cause ectopic whorls and bundles (32,36). Although we have clearly shown an axonal extension defect in necdin null mice, it remains possible that some of the axonal misrouting and whorling defects are related to dysfunctional specification or patterning of the ventral telencephalon. However, the up-regulation of necdin in most postmitotic, differentiated neurons, the localization in proximal axons and cell bodies but not in distal axons in SCG cultures and the role of necdin in cytoskeletal rearrangements and the dysfunction of necdin null neurons in culture are more strongly suggestive of an intrinsic growth defect in cytoskeletal dynamics than of a direct role in patterning and/or specification of subsets of neurons during development. Necdin and Fez1 interact in a juxta–centrosomal compartment also containing the schizophrenia candidate protein DISC1 in a complex with the cytoplasmic dynein-regulating proteins LIS1 and NUDEL (37,38). Although we have not fully characterized the domains of Fez1/2 interacting with necdin, in the initial yeast two-hybrid screen necdin interacted with amino acids 46–282 of the 353 amino acid Fez2 protein, while the more C-terminal domain of this protein family is implicated in binding to the KHC tail of kinesin (25) and to DISC1 (23). LIS1 mutations cause neuronal migration defects and lissencephaly in humans and mice, through a disruption of dynein and microtubular dynamics mediated in part through centrosomes (37). This complex set of interactions points to overlapping dysfunctions in centrosome–microtubule dynamics in PWS, BBS, lissencephaly and possibly other psychiatric disorders. The possibility that concurrent deletion of necdin and Magel2 in PWS may abrogate the functional redundancy of these two related proteins in specific neurons in the hypothalamus provides a plausible explanation for the lack of single gene mutations in PWS. The neurons involved in the hypothalamic circuits implicated in PWS may indeed have delayed or insufficient axonal connections. Therefore, strategies applied early in postnatal life that functionally restore some of these connections could provide a novel line of therapy for infants diagnosed with PWS. [...] J Child Neurol. 2005 Feb. The purpose of this study was to examine the psychiatric characteristics of children with Prader-Willi syndrome in Korea, focusing particularly on their behavioral problems and obsessive-compulsive spectrum symptoms. Fourteen patients with Prader-Willi syndrome, together with their parents, underwent a psychiatric interview and parent questionnaire consisting of a Child Behavior Checklist. Twenty-four patients with mental retardation and 45 normal students were selected as control groups. Compared with the normal control group, the rates of inclusion in the clinical range and the mean scores with regard to social problems, thought problems, attention problems, delinquent behavior, aggressive behavior, externalizing problems, and total problems profiles were significantly higher (P < .01) in the Prader-Willi syndrome group according to the results of the Child Behavior Checklist. Compared with the mental retardation group, there was a statistically significant (P < .05) difference in the delinquent behavior profile. Comparing selected Child Behavior Checklist items related to obsessive-compulsive spectrum symptoms, the Prader-Willi syndrome group showed significantly more (P < .05) compulsion, skin picking, and stealing than the mental retardation or normal control groups. These findings suggest that children with Prader-Willi syndrome in Korea have many behavioral problems, including obsessive-compulsive spectrum symptoms, needing proper psychiatric attention and treatment. This is the first study in Korea to evaluate the psychiatric and behavioral characteristics of children with Prader-Willi syndrome. Am J Med Genet A. 2005 Jan 15. No abstract available. Eur J Neurol. 2005 Jan. Abstract: 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-narcolepsy. 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. Clin Genet. 2005 Jan. Prader-Willi syndrome (PWS) can result from a 15q11-q13 paternal deletion, maternal uniparental disomy (UPD), or imprinting mutations. We describe here the phenotypic variability detected in 51 patients with different types of deletions and 24 patients with UPD. Although no statistically significant differences could be demonstrated between the two main types of PWS deletion patients, it was observed that type I (BP1-BP3) patients acquired speech later than type II (BP2-BP3) patients. Comparing the clinical pictures of our patients with UPD with those with deletions, we found that UPD children presented with lower birth length and started walking earlier and deletion patients presented with a much higher incidence of seizures than UPD patients. In addition, the mean maternal age in the UPD group was higher than in the deletion group. No statistically significant differences could be demonstrated between the deletion and the UPD group with respect to any of the major features of PWS. In conclusion, our study did not detect significant phenotypic differences among type I and type II PWS deletion patients, but it did demonstrate that seizures were six times more common in patients with a deletion than in those with UPD. Horm Res. 2005. We describe a child with Prader-Willi syndrome (PWS) aged 3 years and 11 months who suddenly died 7 months after the initiation of GH therapy. The child never showed respiratory problems, but suffered from severe obesity. This case raises the question about the association between sudden death in children with PWS (with or without respiratory problems) and GH therapy, as already suspected in the recent past. We suggest that further epidemiological studies are required in order to determine more accurately the frequency of this causal connection and better understand its pathogenesis. Horm Res. 2005. Irrespective of GH treatment, children with Prader-Willi syndrome (PWS) suffer more frequently and more seriously from respiratory problems than healthy children. The pathogenesis of such respiratory problems in PWS seems to be multifactorial in origin, but mainly related to insufficiency of respiratory muscles and pharyngeal narrowness. Deaths of children with PWS are reported among GH treated as well as untreated children. Our data show that also disturbed body composition plays an important role in fatal outcomes, possibly enhancing the ventilation disorder. For several years, in our recommendations we have pointed out the secondary risks of increasing obesity. In addition, it is recommended for all children with PWS, in particular before institution of GH therapy, to have polysomnography and an otorhinolaryngologic examination performed, and tonsillectomy in the case of enlarged tonsils. Furthermore, upper airway infections should be treated aggressively. 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