prader - willi syndrome ( pws ) is a congenital disease caused by the loss of rna expression from an imprinted region on chromosome 15 . patients with pws typically undergo several stages beginning as infants when they are characterized by severe infantile hypotonia with poor suck and failure to thrive . this stage is followed by early childhood onset obesity and hyperphagia . the patients exhibit developmental delay and/or mild intellectual disability . other features include short stature , hypogonadism , thick saliva , obsessive - compulsive behavior , and hormonal imbalances.1,2 recently , several patients with prader - willi - like phenotypes have been described ( fig . these patients had microdeletions affecting two clusters of small nucleolar rnas ( snornas ) , snord115 and snord116 . this indicates that the loss of snornas plays a central role in the etiology of the disease ( fig . 1a).35 the molecular mechanisms that link the loss of two clusters of snornas to the disease are currently under investigation . loss of snord115 could explain hyperphagia observed in pws patients , as snord115 has been shown to regulate alternative splicing of the serotonin receptor 2c . snord115 helps to generate the most active form of the receptor.68 no molecular mechanisms have been established thus far demonstrating the role of snord116 in the generation of pws . it is known , however , that snord116 influences the expression of micrornas hosted in the second exon of the serotonin receptor 2c.9 snord115 and snord116 do not express canonical c / d box snornas , but are likely processed into shorter rnas , termed psnornas,10,11 that do not associate with the proteins typically found in c / d box snornas.10,12 canonical c / d box snornas function in 2-o - methylation of non - coding rnas and form a defined ribonuclear complex with the proteins fibrillarin , nop56/58 and 15.5 kd ( nhp2l1 ) . currently , there are no mouse models available that faithfully replicate the human phenotype13 and molecular investigations rely on human material . since snord115 is exclusively , and snord116 mainly , expressed in the brain , tissue deposited in brain banks is crucial for molecular analysis aimed to find the cause of the disease . the patient s cause of death was pulmonary embolism at the age of 56 years ( year of birth 1950 ) . she was described as morbidly obese with a weight of 140 kg ( 308 lbs ) and height of 157.5 cm ( 5 ft , 2 in ) with a body mass index ( bmi ) of 56.3 . she had a life long history of learning difficulties , carrying a diagnosis of mental retardation . it was necessary to lock food sources and as such , the refrigerator had a chain and lock on it . medical records are sparse , but she was reported to suffer from anxiety and have frequent falls . the frequent falls may have been due to low muscle tone and decreased muscle mass that is described in pws . additionally , the anxiety may have contributed to her behavior problems and are usually described in pws patients . this patient also had obstructive sleep apnea ( osa ) with chronic co2 retention , also described in pws patients . morbid obesity is also associated with osa , however , pws patients have a low muscle tone in addition to obesity that increases odds for osa . this diagnosis was based on clinical criteria since , at the time , routine diagnostic tests were not available . the brain was removed after a post mortem interval of 41 hrs and showed no gross abnormalities . microscopic examination showed gliosis and deposition of dark granular material in the cerebral cortical molecular layer and in the midbrain . the dentate gyrus showed focal thinning and dispersion of neurons , some of which showed degenerative features . the patient s medical history included depression , mental retardation , morbid obesity , hypothyroidism , diabetes mellitus , history of falls , chronic co2 retention secondary to obesity , hypercholesterolemia , recent urinary tract infections , and meningioma . the donor had two sisters with the same phenotype , one of whom is deceased . the brain of patient 4876 was donated to the nichd brain and tissue bank ( baltimore , md ) . as a probe , we used a uniformly radiolabeled riboprobe against chr15 : 25 , 318 , 253 - 25 , 318 , 349 for snord115 and chr15 : 25 , 494 , 345 - 25 , 494 , 426 for snord116 in hg19 for snord115 and chr15 : 25 , 494 , 345 - 25 , 494 , 426 for snord116 . the rnase protections were performed as previously described.14 rna was isolated from brain using rneasy lipid tissue kit ( qiagen , hilden ) according to manufactory protocol . rna expressing constructs to make rnase protection probes are available through addgene ( cambridge ma ) . as controls , we used rna obtained from non prader - willi subject tissues from the nichd brain bank ( table 1 ) . as shown in figure 1b , patient 4876 expressed snord115 and snord116 to the same degree as the controls . performing an rnase protection assay with next , the expression of small nuclear ribonucleoprotein polypeptide n ( snrpn ) that hosts snord115 and snord116 in their 3 utr was analyzed ( fig . ( to exon # 4 ) snrpn - r tggctttaaccacctcttgg ( to exon # 5 ) . again , there was expression of snrpn in patient 4876 , but not in pws controls . quantitative ( q ) rt - pcr was conducted for each of the brain tissue samples using 400 ng of total rna , with a net final rna concentration per reaction of 20 ng/l . then , 2.5 l of the cdna product ( approximately 50 ng of cdna ) was used for quantitative pcr ( qpcr ) using taqman probes directed at the snord116 ( custom probe ) and snrpn ( applied biosystems ) target sites . a gapdh taqman probe ( applied biosystems ) as shown in figure 1d , patient 4876 had expression at snrpn that was more than any of the 3 control brain samples and a very robust expression at snord116 . in contrast , the two pws samples ( 4805 and 5324 ) had basically no expression at snrpn and snord116 . dna methylation analysis using methylation - specific multiplex ligation - dependent probe amplification ( ms - mlpa ) can be used to diagnose individuals with pws.1 we used 100 ng of dna for each of the brain tissue samples , according to the manufacturer s instructions ( mrc holland ; amsterdam , netherlands ) . our analysis showed that patient 4876 had approximately 57% dna methylation at mkrn3 , which would be typical for a normal control individual and was within the range of 3 control brain samples15 ( table 2 ) . dna methylation analysis at the snrpn locus could not be determined due to dna degradation from the aged brain sample . finally , we performed affymetrix exon junction array analysis using posterior hypothalamic rna . we compared genome wide gene expression of patient 4876 , with the two molecularly confirmed pws patients 5324 and 4805 , as well as the controls ( 5238 , 4782 , and 1539 ) ( table 1 ) . for the analysis , the mean values in the control and pws groups were compared with the values obtained for patient 4876 . the array analysis is shown in the supplementary files 1 and 2 as excel spreadsheets . the most distinct difference between 4876 and the two pws samples were rnas expressed from the pws critical region on chromosome 15 that showed more than a 20 fold change . this finding is supported by rnase protection of snord115 and snord116 , as well as the rt - pcr analysis of snrpn . the comparison between 4876 and control patients resulted in 18,050 changes with a significant change in expression . the high number of changes in both experiments likely indicates differences between individuals , rather than differences due to brain pathology . however , the rna from posterior hypothalamus of patient 4876 was more similar to pws subjects than to the control , as indicated by the lower numbers of fold changes . patient 4876 was diagnosed with pws because of typical clinical demonstrations such as hyperphagia , morbid obesity , short statue , low muscle tone , sleep apnea , and mental retardation . our genetic analysis is based on the copy number of genes in the genetic region causing the disease as well as expression of snord115 and snord116 snornas . due to the long post mortem interval of 41 hrs , the dna was damaged and did not yield complete dna methylation results for all genes of the pws critical region . similarly , the rna was degraded with an rna integrity number ( rin ) of 2.3 for patient 4876 . the low rin indicates a relatively high level of degradation of ribosomal rnas , as rin quantification relies on ratio of long ribosomal rna and short rnas . snornas appear to be fairly stable , as we could detect them using rnase protection analysis and qrt - pcr in rna with a high degree of degradation . in contrast to qpcr , rnase protection analysis can discriminate between related copies of snord115 and snord116 . in addition the technique allows absolute quantification of rna and can be performed using commercially available kits . thus using rnase protection and qrt - pcr , snord115 and snord116 a genome wide array analysis showed that in terms of rna expression , patient 4876 was more similar to pws subjects than the genetically normal controls . this similarity reflects the clinical phenotype where patient 4876 was clinically similar to pws resulting in misdiagnosis . due to the small sample number , which is an intrinsic problem of using post - mortem tissue of a rare disease , we can not make statistically supported analyses . however , the data suggest that some obesity syndromes have similarities with pws , although the basic molecular defect is clearly different . our findings point out that diagnosis of patients based on clinical phenotype may result in misdiagnosis . although clinical records are necessary and helpful , genetic analysis of tissues donated for research should be performed when feasible . supplementary file 1 . excel spread sheet showing the comparison between patient 4876 and two pws patients . distances heat map of gene expression of patient 4876 , pws patients , and normal subjects .
prader - willi syndrome ( pws ) is caused by the loss of rna expression from an imprinted region on chromosome 15 that includes snrpn , snord115 , and snord116 . 
 currently , there are no mouse models that faithfully reflect the human phenotype and investigations rely on human post - mortem material . during molecular characterization of tissue deposited in a public brain bank from a patient diagnosed with prader - willi syndrome , we found rna expression from snrpn , snord115 , and snord116 which does not support a genetic diagnosis of prader - willi syndrome . 
 the patient was a female , caucasian nursing home resident with history of morbid obesity ( bmi 56.3 ) and mental retardation . 
 she died at age of 56 from pulmonary embolism . snord115 and snord116 are unexpectedly stable in post mortem tissue and can be used for post - mortem diagnosis . 
 molecular characterization of pws tissue donors can confirm the diagnosis and identify those patients that have been misdiagnosed .