Baby food and bedtime: Evidence for opposite phenotypes from different genetic and epigenetic alterations in Prader-Willi and Angelman syndromes.

American College of Medical Genetics Statement on Diagnostic Testing for Uniparental Disomy

Epilepsy in Angelman syndrome

The Sins of the Fathers and Mothers: Genomic Imprinting in Mammalian Development

The molecular basis of Angelman syndrome and Prader-Willi syndrome is well established, and genetic testing for these disorders is clinically available. Imprinting abnormalities account for up to 4% of patients with Angelman and Prader-Willi syndromes. Deletions of the imprinting center region are the molecular abnormality observed in a subset of Angelman and Prader-Willi syndrome cases with imprinting defects. Genetic testing of imprinting center deletions in patients with Angelman and Prader-Willi syndrome is not readily available. Such testing is important for the diagnostics of Angelman and Prader-Willi syndrome because it allows for more accurate diagnosis and recurrence risk prediction in families. Here we describe the development, validation, and implementation of a real time quantitative polymerase chain reaction (PCR)-based assay for imprinting center deletion detection in patients with Angelman and Prader-Willi syndrome, which we have incorporated into our genetic testing strategy for these disorders. To date we have tested, on a clinical basis, five patients with either Angelman or Prader-Willi syndrome in whom an imprinting center defect was implicated and found a deletion in one patient that was determined to be familial.

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

Back to table of contents Previous article Next article Images in NeuroscienceFull AccessThe Human Genome: Detecting Chromosomal Deletions: Angelman and Prader-Willi SyndromesDeborah J. Morris-Rosendahl, PH.D., and Eike Back, M.D., Deborah J. Morris-RosendahlSearch for more papers by this author, PH.D., and Eike BackSearch for more papers by this author, M.D., Frieburg, GermanyPublished Online:1 Mar 2002https://doi.org/10.1176/appi.ajp.159.3.372AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InEmail Angelman syndrome and Prader-Willi syndrome are two related but clinically and genetically distinct neurogenetic syndromes, characteristically caused by deletion of the human chromosomal region 15q11-q13. Clinical features of Angelman syndrome include severe mental retardation with absence of speech, epileptic seizures, ataxia, inappropriate bursts of laughter, unusually happy disposition, hyperactivity, and micro- and brachycephaly. Patients with Prader-Willi syndrome show infantile hypotonia, mild to moderate mental retardation, hyperphagia with subsequent obesity, hypogonadism, short stature, mild facial dysmorphism, and characteristic behavior. In Angelman syndrome the chromosomal deletions are exclusively of the maternal chromosome, whereas in Prader-Willi syndrome the deletions are of paternal origin, i.e., the absence of any paternal contribution to the 15q11-q13 region. Both syndromes can result either from deletions or from uniparental disomy, in which two chromosomes 15 are inherited from a single parent, instead of one chromosome from each parent.The detection of chromosomal deletions has become routine in both prenatal and postnatal diagnosis with the use of fluorescence in situ hybridization, a process that vividly paints chromosomes or portions of chromosomes with fluorescent molecules. In situ hybridization is a powerful and versatile tool for the detection and localization of nucleic acid sequences (the constituents of genes) in cell preparations. The technique is based on the hybridization (attraction and complexing) of a labeled and complementary DNA or RNA probe to immobilized chromosomal preparations. Although radioactively labeled DNA probes were formerly used for this purpose, commercially available fluorescent probes are now available for diagnosis. Fluorescence in situ hybridization is routinely used to detect chromosomal rearrangements and deletions, including those associated with chromosomal microdeletion syndromes, such as Angelman syndrome or Prader-Willi syndrome.Address reprint requests to Dr. Tamminga, Maryland Psychiatric Research Center, University of Maryland, P.O. Box 21247, Baltimore, MD 21228; [email protected] (e-mail). Image courtesy of the authors. FigureFluorescence in situ hybridization image showing the deletion of chromosomal region 15q11-q13 that causes Angelman syndrome. Two control probes—CEP 15 and LSI PML (Vysis, Downers Grove, Ill.)—are included in the probe mixture to highlight the short arms around the centromeric region (CEP 15 on 15p11.2, blue-green signals) and long arms (LSI PML on 15q22, orange-pink signals) of chromosome 15 and to detect possible chromosomal translocations. The absence of one of the orange-pink signals on one chromosome 15 (q11-q13, white arrow) indicates the deletion of the small nuclear ribonucleoprotein-associated polypeptide N locus in this 3-year-old male Angelman syndrome patient. FiguresReferencesCited byDetailsCited byNone Volume 159Issue 3 March 2002Pages 372-372 Metrics PDF download History Published online 1 March 2002 Published in print 1 March 2002

Eighty seven referrals with Prader-Willi syndrome and 49 with Angelman's syndrome were studied. High resolution cytogenetics was performed on all probands. Molecular studies, performed on the proband and both parents in each case, utilised multiple probes from within and distal to the 15(q11-13) region in order to establish the presence of DNA deletion or uniparental disomy. In addition, FISH, with probes at D15S11 and GABR beta 3 from the Prader-Willi syndrome/Angelman's syndrome region, was performed on a subset of 25 of these patients. In the referral group with Prader-Willi syndrome, 62 patients had a normal karyotype and 25 were deleted on high resolution cytogenetics. Twenty nine were found to be deleted with DNA techniques. In the Angelman's syndrome group, 37 had a normal karyotype and 12 were deleted on high resolution cytogenetics while 26 were deleted on molecular studies. The diagnosis was reassessed in 35 referrals with Prader-Willi syndrome and 11 with Angelman's syndrome following a non-deleted, non-disomic result. Of individuals who were neither deleted nor disomic on DNA studies, a false positive rate of 11.4% (4/35) for Prader-Willi syndrome and 16.7% (2/12) for Angelman's syndrome was found for a cytogenetically detected deletion. The false negative rate for deletion detected on high resolution cytogenetics was 19.5% (12/62) for Prader-Willi syndrome and 35% (13/37) for Angelman's syndrome. Thus high resolution cytogenetics was shown to be unreliable for deletion detection and should not be used alone to diagnose either syndrome. There were no discrepancies with FISH in 25 cases when FISH was compared with the DNA results, indicating that FISH can be used reliably for deletion detection in both syndromes.

Newborn screening for Angelman syndrome (AS), Prader-Willi syndrome (PWS), and chromosome 15 duplication syndrome (Dup15q) may lead to benefit from early diagnosis and treatment. To examine the feasibility of newborn screening for these chromosome 15 imprinting disorders at population scale. In this diagnostic study, the validation data set for the first-tier SNRPN test, called methylation-specific quantitative melt analysis (MS-QMA), included 109 PWS, 48 AS, 9 Dup15q, and 1190 population control newborn blood spots (NBS) and peripheral tissue samples from participants recruited from January 2000 to December 2016. The test data set included NBS samples from 16 579 infants born in 2011. Infants with an NBS identified as positive for PWS, AS, or Dup15q by the first-tier test were referred for droplet digital polymerase chain reaction, real-time polymerase chain reaction, and low-coverage whole-genome sequencing for confirmatory testing. Data analyses were conducted between February 12, 2015, and August 15, 2020. In the validation data set, the median age for the 77 patients with PWS was 3.00 years (IQR, 0.01-44.50 years); for the 46 patients with AS, 2.76 years (IQR, 0.028 to 49.00 years); and for the 9 patients with Dup15q, 4.00 years (IQR, 1.00 to 28.00 years). Thirty-eight patients (51.4%) in the PWS group, 20 patients (45.5%) in the AS group, and 6 patients (66.7%) in the Dup15q group who had sex reported were male. The validation data set showed MS-QMA sensitivity of 99.0% for PWS, 93.8% for AS, and 77.8% for Dup15q; specificity of 100% for PWS, AS, and Dup15q; positive predictive and negative predictive values of 100% for PWS and AS; and a positive predictive value of 87.5% and negative predictive value of 100% for Dup15q. In the test data set of NBS samples from 16 579 infants, 92 had a positive test result using a methylation ratio cut-off of 3 standard deviations from the mean. Of these patients, 2 were confirmed to have PWS; 2, AS; and 1, maternal Dup15q. With the use of more conservative PWS- and AS-specific thresholds for positive calls from the validation data set, 9 positive NBS results were identified by MS-QMA in this cohort. The 2 PWS and 2 AS calls were confirmed by second-tier testing, but the 1 Dup15q case was not confirmed. Together, these results provided prevalence estimates of 1 in 8290 for both AS and PWS and 1 in 16 579 for maternal Dup15q, with positive predictive values for first-tier testing at 67.0% for AS, 33.0% for PWS, and 44.0% for combined detection of chromosome 15 imprinting disorders for the validation data set. The findings of this diagnostic study suggest that it is feasible to screen for all chromosome 15 imprinting disorders using SNRPN methylation analysis, with 5 individuals identified with these disorders out of 16 579 infants screened.

The kinship theory of genomic imprinting predicts that imprinted genes affect mother-child and child-child interactions. According to this theory paternally expressed genes will promote behaviours that increase costs of maternal investments and enable children to compete with siblings. Maternally expressed genes will promote behaviours that reduce the mother’s costs of child-rearing and enable children to engage in collaborative actions. Prader-Willi syndrome and Angelman syndrome are caused by the absence of expression of imprinted genes in 15q11-q13. Children with Prader-Willi syndrome lack the expression of paternally expressed genes; children with Angelman syndrome lack maternally expressed genes. The current paper discusses the role of imprinted genes in the development of communicative behaviours during the transition from breastfeeding to (consuming) solid food. Its focus is the possible role of imprinted genes in the development of empathy out of (reactive) crying, and in the development of behaviours necessary for joint action. Observed behavioural differences between children with Angelman and Prader-Willi syndrome, and data from mouse models on the effects of imprinted genes on brain development, are used to explore possible effects of imprinted genes. (Netherlands Journal of Psychology, 65, 78-88).

Electroclinical characteristics of seizures—comparing Prader-Willi syndrome with Angelman syndrome

Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are considered sister imprinting disorders. Although both AS and PWS congenital neurodevelopmental disorders have chromosome 15q11.3-q13 dysfunction, their molecular mechanisms differ owing to genomic imprinting, which results in different parent-of-the-origin gene expressions. Recently, several randomized controlled trials have been proceeded to treat specific symptoms of AS and PWS. Due to the advance of clinical management, early diagnosis for patients with AS and PWS is important. PWS is induced by multiple paternal gene dysfunctions, including those in MKRN3, MAGEL2, NDN, SNURF-SNPRPN, NPAP1, and a cluster of small nucleolar RNA genes. PWS patients exhibit characteristic facial features, endocrinological, and behavioral phenotypes, including short and obese figures, hyperphagia, growth hormone deficiency, hypogonadism, autism, or obsessive– compulsive-like behaviors. In addition, hypotonia, poor feeding, failure to thrive, and typical facial features are major factors for early diagnosis of PWS. For PWS patients, epilepsy is not common and easy to treat. Conversely, AS is a single-gene disorder induced by ubiquitin-protein ligase E3A dysfunction, which only expresses from a maternal allele. AS patients develop epilepsy in their early lives and their seizures are difficult to control. The distinctive gait pattern, excessive laughter, and characteristic electroencephalography features, which contain anterior-dominated, high-voltage triphasic delta waves intermixed with epileptic spikes, result in early suspicion of AS. Often, polytherapy, including the combination of valproate, levetiracetam, lamotrigine, and benzodiazepines, is required for controlling seizures of AS patients. Notably, carbamazepine, oxcarbazepine, and vigabatrin should be avoided, since these may induce nonconvulsive status epilepticus. AS and PWS presented with distinct clinical manifestations according to specific molecular defects due to genomic imprinting. Early diagnosis and teamwork intervention, including geneticists, neurologists, rehabilitation physicians, and pulmonologists, are important. Epilepsy is common in patients with AS, and after proper treatment, seizures could be effectively controlled in late childhood or early adulthood for both AS and PWS patients.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe mental retardation, motor disturbances, seizures, hyperactivity, attention deficits and an absence of speech. AS is caused by loss of functional ubiquitin protein ligase E3A (UBE3A) in neurons of the central nervous system. As an E3 ligase, UBE3A participates in the ubiquitin proteasome pathway by binding specific target proteins, which are finally marked by ubiquitination for degradation by the proteasome. Loss of UBE3A activity is thought to result in dysregulation of target proteins, which further interfere with neuron development and function. The UBE3A gene is located in the Prader-Willi syndrome / Angelman syndrome imprinted domain on chromosome 15q11.2-q13. Here, paternal UBE3A expression is silenced in neurons due to expression of the long-non-coding RNA SNHG14, which overlaps the UBE3A gene in antisense. Genetic or epigenetic alterations affecting the maternal UBE3A gene or its expression are sufficient to result in loss of functional UBE3A protein. To date, neither the pathomechanism explaining how missing UBE3A protein leads to symptoms, nor the overall reason for silencing of paternal UBE3A by SNHG14 expression is known. Research on AS is hampered by missing relevant functional human brain tissue. To overcome this limitation and gain insights into the cellular and molecular pathomechanism, we created an in vitro AS model based on neuronal differentiation of patient specific induced pluripotent stem cells (iPSCs). Initially, only one AS-iPSC line, carrying a large chromosomal deletion, was available. Therefore, we reprogrammed dermal fibroblasts from an AS patient carrying a three base pair deletion in the maternal UBE3A gene. Resulting iPSCs were extensively characterized and identified as being pluripotent. A second AS-iPSC line from a patient with an imprinting defect was also generated and tested for genetic and epigenetic integrity and pluripotency. Since paternal UBE3A silencing occurs late during neuron development, an accelerated neuronal differentiation protocol was applied. Neurons expressing TBR1, CUX2 and GAD67 were generated after 50 days of differentiation. During differentiation, silencing of paternal UBE3A was detected. Since UBE3A protein is reported to localize not only in the cytoplasm but also to the nucleus and some target proteins are known to function in regulation of gene expression, gene expression analysis by RNAseq of 50 days old neurons was performed. However, only minor differences between AS- and Ctrl-iPSC-derived neurons were detected, pointing to loss of ligase function as critical parameter for AS. In the future, our AS model will provide the perfect basis to answer open questions contributing to a better understanding of the AS pathomechanism.

Reply to Camprubí et al

It has recently been shown that apparently similar deletions of chromosome 15q occur commonly in the Prader-Willi and Angelman syndromes. The distinctness of the syndromes suggests that the deletions are not identical. To address this possibility, the specific bands involved and the sizes of the deletions were compared in seven patients with Prader-Willi syndrome and 10 patients with Angelman syndrome using high-resolution G-, Q-, and fluorescent R-banding techniques. The parental origin of the nine cases of Angelman syndrome for which parents were available for study was determined. The same proximal band was deleted (q11.2) in both syndromes. In general, the deletion in patients with Angelman syndrome was larger, though variable, and included bands q12 and part of q13. All of the studied deletions in patients with Angelman syndrome were of maternal origin. This contrasts with the predominant paternal origin of the deletion in patients with Prader-Willi syndrome. Two possible reasons for these observations are postulated: 1) the deleted regions are different at the cytologic and/or molecular level because of different exchange points in meiosis in males and females or to different mechanisms of breakage in males and females, resulting in differing breakpoints; 2) the deleted regions are essentially the same, but differential expression of the genes in the homologous chromosome 15 has occurred (imprinting).

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are distinct neurodevelopmental disorders with interrelated genetic mechanisms because genomic imprinting within the chromosome 15q11-13 region affects both the PWS and the AS locus. Methylation analysis is one method of distinguishing between the maternally and paternally inherited chromosome 15. Here we present clinical and molecular data on a large series of 258 referred patients, evaluated with methylation analysis: 115 with suspected PWS and 143 with suspected AS. In these patients, the clinical phenotype was graded into three groups: classical (group 1); not classical but possible (group 2); not classical and unlikely (group 3). For PWS, a fourth group consisted of hypotonic babies. DNA methylation analysis confirmed the diagnosis of PWS in 30 patients (26%) and AS in 28 patients (20%). For 21 PWS patients the mechanism was established: 15 had deletions, 4 had uniparental disomy (UPD) and 2 a presumed imprinting defect. Clinically all those with an abnormal methylation pattern had the classical phenotype and none of those with a normal methylation pattern had classical PWS. For 23 AS patients in whom a mechanism was established, 17 had a deletion, 3 had UPD and 3 had a presumed imprinting defect. There was greater clinical overlap in AS, with 26 classical AS patients having a normal methylation pattern while an abnormal methylation pattern was seen in one patient from group 2. In addition, there were a further 40 patients with a normal methylation pattern in whom AS was still a possible diagnosis. Our conclusion is that methylation analysis provides an excellent screening test for both syndromes, providing approximately 99% diagnosis for PWS and for AS, a 75% diagnostic rate, supplemented for the remaining 25% with an essential basic starting point to further investigations.