Imprinting Defects Research Articles

Flat and highly flexible composite stamps for nanoimprint, their preparation and their limits

To reduce the overall costs, replica stamps have become more and more relevant in the past years. These replicas often are composite stamps with at least two layers, a structured top layer and a backplane. In most cases, the composite stamps are rigid, which limits their use for rolling processes or low-pressure imprints. Flexible composite stamps avoid these limitations. However, due to the flexibility, preparation-induced bending may occur. A concept to avoid such a bending will be introduced and verified by the preparation of a flat and flexible composite stamp consisting of a structured OrmoStamp top layer and a polydimethylsiloxan (PDMS) backplane. To increase the adhesion between both layers, two measures will be adopted: one relies on an increase in the surface energy of the PDMS and the other one on an increase in the interface area. Finally, these flat and highly flexible composite stamps are tested in a critical imprint situation, where particles are dusted to the substrate before imprint. Imprint defects and stamp damage due to these particles will be examined.

Prader-Willi syndrome and atypical submicroscopic 15q11-q13 deletions with or without imprinting defects

We report a 20 year follow up on a Caucasian female, now 26 years of age, with Prader-Willi syndrome (PWS) harboring an atypical 15q11-q13 submicroscopic deletion of 100–200 kb in size first detected in 1996 involving the imprinting center, SNRPN gene and surrounding region. PWS is a rare complex disorder caused by the loss of paternally expressed genes in the 15q11-q13 region. With high resolution chromosomal microarray and methylation – specific MLPA analysis, we updated the genetic findings on our patient and found a 209,819bp deletion including the SNURF-SNRPN gene complex which includes the imprinting center and the SNORD116 region. We compared with four other similarly reported individuals in the literature with atypical submicroscopic deletions within this region but without imprinting center involvement to better characterize the specific genetic lesions causing PWS clinical findings. Clinically, our patient met the diagnostic criteria of PWS including infantile hypotonia, a poor suck with feeding difficulties, global developmental delays and later food foraging, childhood obesity, small hands and skin picking. Small atypical deletions of comparable sizes were seen in the 15q11-q13 region in all five cases and similar behavioral/physical characteristics were found despite an imprinting defect in our patient. These results further support an overlapping critical deletion region involving the non-coding snoRNA SNORD116 in common in the five individuals playing a key role in contributing to the PWS phenotype.

Angelman syndrome - insights into a rare neurogenetic disorder.

Angelman syndrome is a rare neurogenetic disorder that is characterized by microcephaly, severe intellectual deficit, speech impairment, epilepsy, EEG abnormalities, ataxic movements, tongue protrusion, paroxysms of laughter, abnormal sleep patterns, and hyperactivity. Angelman syndrome results from loss of function of the imprinted UBE3A (ubiquitin-protein ligase E3A) gene on chromosome 15q11.2-q13. This loss of function can be caused by a mutation on the maternal allele, a 5-7 Mb deletion of the maternally inherited chromosomal region, paternal uniparental disomy of chromosome 15, or an imprinting defect. The chromosomal deletion tends to cause the most severe symptoms, possibly owing to co-deletion of GABA receptor genes. UBE3A mutations and imprinting defects can be associated with a high risk of recurrence within families. Disruption of UBE3A function in neurons seems to inhibit synapse formation and experience-dependent synapse remodelling. Clinical diagnosis of Angelman syndrome in infants and young children is sometimes difficult, but can be verified by genetic tests. At present, treatment of symptoms such as seizures is the only medical strategy, but genetic therapies aimed at activating the silent copy of UBE3A on the paternal allele are conceivable.

Compromised oocyte quality and assisted reproduction contribute to sex-specific effects on offspring outcomes and epigenetic patterning.

Clinical studies have revealed an increased incidence of growth and genomic imprinting disorders in children conceived using assisted reproductive technologies (ARTs), and aberrant DNA methylation has been implicated. We propose that compromised oocyte quality associated with female infertility may make embryos more susceptible to the induction of epigenetic defects by ART. DNA methylation patterns in the preimplantation embryo are dependent on the oocyte-specific DNA methyltransferase 1o (DNMT1o), levels of which are decreased in mature oocytes of aging females. Here, we assessed the effects of maternal deficiency in DNMT1o (Dnmt1Δ1o/+) in combination with superovulation and embryo transfer on offspring DNA methylation and development. We demonstrated a significant increase in the rates of morphological abnormalities in offspring collected from Dnmt1Δ1o/+ females only when combined with ART. Together, maternal oocyte DNMT1o deficiency and ART resulted in an accentuation of placental imprinting defects and the induction of genome-wide DNA methylation alterations, which were exacerbated in the placenta compared to the embryo. Significant sex-specific trends were also apparent, with a preponderance of DNA hypomethylation in females. Among genic regions affected, a significant enrichment for neurodevelopmental pathways was observed. Taken together, our results demonstrate that oocyte DNMT1o-deficiency exacerbates genome-wide DNA methylation abnormalities induced by ART in a sex-specific manner and plays a role in mediating poor embryonic outcome.

Defect tracking for nanoimprint lithography using optical surface scanner and scanning electron microscope

Fast optical surface scanners are used in combination with high-resolution scanning electron microscopes to facilitate the identification and tracking of nanoimprint defects. We have confirmed that hard particles cause permanent template damages during imprint, resulting in repeating imprint defects. Since contaminants encountered during imprint are dominated by hard metal oxide particles capable of causing such damage, stringent pre-imprint substrate screening is a critical requirement in a manufacturing environment.

Imprinting Defects in Osteosarcoma: DNA- and Chromatin-Modifying Drugs Hold Promise for Osteosarcoma Therapy

Imprinting Defects in Osteosarcoma: DNA- and Chromatin-Modifying Drugs Hold Promise for Osteosarcoma Therapy

A twin sibling with Prader-Willi syndrome caused by type 2 microdeletion following assisted reproductive technology: A case report.

Prader-Willi syndrome (PWS) is a neurobehavioral imprinting disorder, which arises due to an absence of paternally expressed genes within the 15q11.2-q13 region. This occurs via one of the three main genetic mechanisms, as follows: Deletion of the paternally inherited 15q11.2-q13 region, maternal uniparental disomy and imprinting defect. Recent studies have reported an association between imprinting disorders and assisted reproductive technologies (ART). The current study presents a 6-year-old female patient who is a dizygotic twin, in which one was born with de novo microdeletion at 15q11.2-q13.1 following in vitro fertilization. The patient had characteristic facial features including narrow bifrontal diameter, strabismus, downturned mouth, feeding problems and generalized hypotonia during infancy, developmental delay, mental retardation and rapid weight gain. Based upon phenotypic resemblance and the medical records, methylation-specific multiplex ligation-dependent probe amplification and array-based comparative genome hybridization analyses demonstrate type 2 microdeletion between breaking point 2 (BP2) and BP3, which occur from MKRN3 through HERC2 at 15q11.2-q13.1. To the best of our knowledge, the present study is the first to report a PWS case born following ART reported in South Korea. In addition to previous studies, the present study contributes to the consensus regarding genotype-phenotype comparisons in this respect.

Prader-Willi Syndrome: A Case Report

Prader–Willi Syndrome (PWS) is a rare multi-systemic genetic disorder, in which 7 or some subset of genes on chromosome 15 are unexpressed or deleted on the paternal chromosome, resulting from failed expression of paternally inherited genes on chromosome 15q11–13. The majority of individuals with PWS (70%) have a paternally derived deletion of 15q11–13, Twenty-five percent have maternal disomy of chromosome 15 (an absence of the normally active paternally inherited genes on the long arm of chromosome 15) and 2-5% have imprinting defects (certain genes or groups of genes are expressed differently depending on the gender of the parent from which they were inherited). The main objective of this report is to further characterize the dental problems along with some of the general features of this syndrome and to emphasize the need of early involvement of dental practitioners for the overall management of these patients.

27 - Rare Focal Deletions Involving SNRPN and UBE3A Genes

Prader-Willi (PWS) and Angelman (AS) syndromes are genetic disorders with multiple well-known mechanisms. Approximately 70% of individuals with PWS or AS have a ~4 Mb deletion of 15q11-q13, while the remainder have UPD15, an imprinting defect or, in the case of UBE3A, a sequence variant. Rarely have focal deletions been reported. We report here on three such unusual cases from our clinical microarray experience. Case 1 was a 2-1/2 year old boy with global developmental delay, short stature, microcephaly, failure to thrive and possible hearing loss. This boy, his healthy mother and maternal grandfather had a 36 kb deletion involving only noncoding exons 1 and 2 of UBE3A (NM_130839). Although previously unreported, this deletion is considered likely pathogenic based upon the AS phenotype in the proband and the inheritance pattern in this family. Case 2 was a 2-1/2 year old girl with epilepsy referred for testing on our childhood epilepsy panel which includes sequencing and copy number analysis of 58 relevant genes. Exon-level array showed an intragenic deletion of exons 2–4 of UBE3A. Methylation -specific MLPA (MS-MLPA) showed normal imprinting. Parental testing may be informative in assisting diagnosis since only maternal inheritance would be consistent with AS. Case 3 was the phenotypically normal father of a boy with PWS and a known SNRPN deletion. Array showed that the father had a 109 kb deletion of exons 4–12 of SNRPN (NM_022808) as well as the upstream reading frame (SNURF). MS-MLPA showed that this deletion was on the maternally derived chromosome. Although rare, intragenic or gene deletion of either UBE3A or SNRPN rather than the common 4 Mb deletion can be observed with PWS and AS. An assay which provides exon level coverage of these genes is important when interrogating this region.

Visualizing allele-specific expression in single cells reveals epigenetic mosaicism in an H19 loss-of-imprinting mutant.

Imprinting is a classic mammalian epigenetic phenomenon that results in expression from a single parental allele. Imprinting defects can lead to inappropriate expression from the normally silenced allele, but it remains unclear whether every cell in a mutant organism follows the population average, which would have profound implications for human imprinting disorders. Here, we apply a new fluorescence in situ hybridization method that measures allele-specific expression in single cells to address this question in mutants exhibiting aberrant H19/Igf2 (insulin-like growth factor 2) imprinting. We show that mutant primary embryonic mouse fibroblasts are comprised of two subpopulations: one expressing both H19 alleles and another expressing only the maternal copy. Only in the latter cell population is Igf2 expression detected. Furthermore, the two subpopulations are stable in that cells do not interconvert between the two expression patterns. Combined small input methylation analysis and transcriptional imaging revealed that these two mutant subpopulations exhibit distinct methylation patterns at their imprinting control regions. Consistently, pharmacological inhibition of DNA methylation reduced the proportion of monoallelic cells. Importantly, we observed that the same two subpopulations are also present in vivo within murine cardiac tissue. Our results establish that imprinting disorders can display striking single-cell heterogeneity in their molecular phenotypes and suggest that such heterogeneity may underlie epigenetic mosaicism in human imprinting disorders.

Deletion of the Snord116/SNORD116 Alters Sleep in Mice and Patients with Prader-Willi Syndrome.

Sleep-wake disturbances are often reported in Prader-Willi syndrome (PWS), a rare neurodevelopmental syndrome that is associated with paternally-expressed genomic imprinting defects within the human chromosome region 15q11-13. One of the candidate genes, prevalently expressed in the brain, is the small nucleolar ribonucleic acid-116 (SNORD116). Here we conducted a translational study into the sleep abnormalities of PWS, testing the hypothesis that SNORD116 is responsible for sleep defects that characterize the syndrome. We studied sleep in mutant mice that carry a deletion of Snord116 at the orthologous locus (mouse chromosome 7) of the human PWS critical region (PWScr). In particular, we assessed EEG and temperature profiles, across 24-h, in PWScr (m+/p-) heterozygous mutants compared to wild-type littermates. High-resolution magnetic resonance imaging (MRI) was performed to explore morphoanatomical differences according to the genotype. Moreover, we complemented the mouse work by presenting two patients with a diagnosis of PWS and characterized by atypical small deletions of SNORD116. We compared the individual EEG parameters of patients with healthy subjects and with a cohort of obese subjects. By studying the mouse mutant line PWScr(m+/p-), we observed specific rapid eye movement (REM) sleep alterations including abnormal electroencephalograph (EEG) theta waves. Remarkably, we observed identical sleep/EEG defects in the two PWS cases. We report brain morphological abnormalities that are associated with the EEG alterations. In particular, mouse mutants have a bilateral reduction of the gray matter volume in the ventral hippocampus and in the septum areas, which are pivotal structures for maintaining theta rhythms throughout the brain. In PWScr(m+/p-) mice we also observed increased body temperature that is coherent with REM sleep alterations in mice and human patients. Our study indicates that paternally expressed Snord116 is involved in the 24-h regulation of sleep physiological measures, suggesting that it is a candidate gene for the sleep disturbances that most individuals with PWS experience.

Clinical Application of an Innovative Multiplex-Fluorescent-Labeled STRs Assay for Prader-Willi Syndrome and Angelman Syndrome.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurodevelopmental disorders caused by absence of paternally or maternally expressed imprinted genes on chr15q11.2-q13.3. Three mechanisms are known to be involved in the pathogenesis: microdeletions, uniparental disomy (UPD) and imprinting defects. Both disorders are difficult to be definitely diagnosed at early age if no available molecular cytogenetic tests. In this study, we identified 5 AS patients with the maternal deletion and 26 PWS patients with paternal deletion on chr15q11-q13 by using an innovative multiplex-fluorescent-labeled short tandem repeats (STRs) assay based on linkage analysis, and validated by the methylation-specific PCR and array comparative genomic hybridization techniques. More interesting, one of these PWS patients was confirmed as maternal uniparental isodisomy by the STR linkage analysis. The phenotypic and genotypic characteristics of these individuals were also presented. Our results indicate that the new linkage analysis is much faster and easier for large-scale screening deletion and uniparental disomy, thus providing a valuable method for early diagnosis of PWS/AS patients, which is critical for genetic diagnosis, management and improvement of prognosis.

Genome-wide DNA methylation analysis of pseudohypoparathyroidism patients with GNAS imprinting defects

BackgroundPseudohypoparathyroidism (PHP) is caused by (epi)genetic defects in the imprinted GNAS cluster. Current classification of PHP patients is hampered by clinical and molecular diagnostic overlaps. The European Consortium for the study of PHP designed a genome-wide methylation study to improve molecular diagnosis.MethodsThe HumanMethylation 450K BeadChip was used to analyze genome-wide methylation in 24 PHP patients with parathyroid hormone resistance and 20 age- and gender-matched controls. Patients were previously diagnosed with GNAS-specific differentially methylated regions (DMRs) and include 6 patients with known STX16 deletion (PHPΔstx16) and 18 without deletion (PHPneg).ResultsThe array demonstrated that PHP patients do not show DNA methylation differences at the whole-genome level. Unsupervised clustering of GNAS-specific DMRs divides PHPΔstx16 versus PHPneg patients. Interestingly, in contrast to the notion that all PHP patients share methylation defects in the A/B DMR while only PHPΔstx16 patients have normal NESP, GNAS-AS1 and XL methylation, we found a novel DMR (named GNAS-AS2) in the GNAS-AS1 region that is significantly different in both PHPΔstx16 and PHPneg, as validated by Sequenom EpiTYPER in a larger PHP cohort. The analysis of 58 DMRs revealed that 8/18 PHPneg and 1/6 PHPΔstx16 patients have multi-locus methylation defects. Validation was performed for FANCC and SVOPL DMRs.ConclusionsThis is the first genome-wide methylation study for PHP patients that confirmed that GNAS is the most significant DMR, and the presence of STX16 deletion divides PHP patients in two groups. Moreover, a novel GNAS-AS2 DMR affects all PHP patients, and PHP patients seem sensitive to multi-locus methylation defects.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0175-8) contains supplementary material, which is available to authorized users.

Imprinting defects at human 14q32 locus alters gene expression and is associated with the pathobiology of osteosarcoma.

Osteosarcoma is the most common primary bone malignancy affecting children and adolescents. Although several genetic predisposing conditions have been associated with osteosarcoma, our understanding of its pathobiology is rather limited. Here we show that, first, an imprinting defect at human 14q32-locus is highly prevalent (87%) and specifically associated with osteosarcoma patients < 30 years of age. Second, the average demethylation at differentially methylated regions (DMRs) in the 14q32-locus varied significantly compared to genome-wide demethylation. Third, the 14q32-locus was enriched in both H3K4-me3 and H3K27-me3 histone modifications that affected expression of all imprinted genes and miRNAs in this region. Fourth, imprinting defects at 14q32 - DMRs are present in triad DNA samples from affected children and their biological parents. Finally, imprinting defects at 14q32-DMRs were also observed at higher frequencies in an Rb1/Trp53 mutation-induced osteosarcoma mouse model. Further analysis of normal and tumor tissues from a Sleeping Beauty mouse model of spontaneous osteosarcoma supported the notion that these imprinting defects may be a key factor in osteosarcoma pathobiology. In conclusion, we demonstrate that imprinting defects at the 14q32 locus significantly alter gene expression, may contribute to the pathogenesis of osteosarcoma, and could be predictive of survival outcomes.

Genomic imprinting defect in Zfp57 mutant iPS cell lines

ZFP57 maintains genomic imprinting in mouse embryos and ES cells. To test its roles during iPS reprogramming, we derived iPS clones by utilizing retroviral infection to express reprogramming factors in mouse MEF cells. After analyzing four imprinted regions, we found that parentally derived DNA methylation imprint was largely maintained in the iPS clones with Zfp57 but missing in those without maternal or zygotic Zfp57. Intriguingly, DNA methylation imprint was lost at the Peg1 and Peg3 but retained at the Snrpn and Dlk1-Dio3 imprinted regions in the iPS clones without zygotic Zfp57. This finding will be pursued in future studies.

Duplex Methylation-Specific Semi-Quantitative Real-Time PCR for Cost-Effective &amp; Time-Efficient Diagnostic Screening of Chromosome 15 and 14 Imprinted Regions

Abstract Purpose: Our goal was to develop two-tier strategy based on in house-designed methylation specific-duplex polymerase chain reactions (MS-PCRs) that could serve as a relatively simple, cost effective, time efficient approach for molecular screening of imprinted regions on chromosomes 15 and 14. Patients and methods: Patients were referred to examination during infancy due to hypotonia and motor development delay. Duplex MS-PCRs were performed that enabled detection of methylated/unmethylated DNA in NDN and MEG3 CpG islands via plurality of detection channels on PCR instrument Rotor Gene 6000. Results and discussion: Both, copy number variations as well as methylation changes, were revealed by our in house-designed methodology by focusing NDN gene. No imprinting aberrations were yet discovered in MEG3 gene. Clinical features of the patients were compared. In agree with literature no typical facial features were observed in PWS patient with imprinting defect and AS UPD patient seems to have a relatively better development and language ability in comparison to deletional form of the disease. Conclusion: In conclusion we were able to establish new, throughput and robust diagnostic approach to PWS/AS.

Absence of Maternal Methylation in Biparental Hydatidiform Moles from Women with NLRP7 Maternal-Effect Mutations Reveals Widespread Placenta-Specific Imprinting.

Familial recurrent hydatidiform mole (RHM) is a maternal-effect autosomal recessive disorder usually associated with mutations of the NLRP7 gene. It is characterized by HM with excessive trophoblastic proliferation, which mimics the appearance of androgenetic molar conceptuses despite their diploid biparental constitution. It has been proposed that the phenotypes of both types of mole are associated with aberrant genomic imprinting. However no systematic analyses for imprinting defects have been reported. Here, we present the genome-wide methylation profiles of both spontaneous androgenetic and biparental NLRP7 defective molar tissues. We observe total paternalization of all ubiquitous and placenta-specific differentially methylated regions (DMRs) in four androgenetic moles; namely gain of methylation at paternally methylated loci and absence of methylation at maternally methylated regions. The methylation defects observed in five RHM biopsies from NLRP7 defective patients are restricted to lack-of-methylation at maternal DMRs. Surprisingly RHMs from two sisters with the same missense mutations, as well as consecutive RHMs from one affected female show subtle allelic methylation differences, suggesting inter-RHM variation. These epigenotypes are consistent with NLRP7 being a maternal-effect gene and involved in imprint acquisition in the oocyte. In addition, bioinformatic screening of the resulting methylation datasets identified over sixty loci with methylation profiles consistent with imprinting in the placenta, of which we confirm 22 as novel maternally methylated loci. These observations strongly suggest that the molar phenotypes are due to defective placenta-specific imprinting and over-expression of paternally expressed transcripts, highlighting that maternal-effect mutations of NLRP7 are associated with the most severe form of multi-locus imprinting defects in humans.

Somatic Mosaicism as Modulator of the Global and Intellectual Phenotype in Epimutated Angelman Syndrome Patients

Angelman Syndrome (AS) is due to the loss of function of the single UBE3A gene, mapping to chromosome 15q11-q13 and encoding the E6AP ubiquitin ligase. Expression of UBE3A is subject to genomic imprinting which is restricted to the brain, where only the maternal allele is transcribed. AS pathogenetic mechanisms include deletion of the maternal 15q11-13 chromosomal region, chromosome 15 paternal uniparental disomy (UPD), Imprinting Defects (ImpD) leading to silencing of the maternal allele and intragenic mutations of the maternal UBE3A allele. From our AS cohort we sorted out for detailed clinical-molecular characterization six mosaic cases, five with ImpD epimutations and one with patUPD15. This latter case referred for intellectual disability and fortuitously solved by SNP array, is, to our knowledge, the unique patient reported with mosaic patUPD of this imprinted region. Somatic epimutation mosaicism represents a challenge for both clinical and molecular diagnostics. The described patients, referred to our center either for uncertain AS or simply for intellectual disability, could be molecularly characterized by applying a multi-method approach including Methylation-Sensitive PCR and MS-MLPA without a strict cut off. The percentage of normal cells detected ranged up to 40%. We confirm the mild phenotype reported in mosaic AS ImpD and provide a detailed analysis of IQ. Mild mental retardation, with significant difficulties in language expression, but only mildly impaired performance skills, together with pathognomonic EEG, is a cue not to overlook in mosaic AS patients. Mosaic epimutations should be searched also in patients with minor AS features and presenting only with intellectual disability.

Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival.

BackgroundAberrant DNA methylation at imprinted loci is an important molecular mechanism contributing to several developmental and pathological disorders including cancer. However, knowledge about imprinting defects due to DNA methylation changes is relatively limited in hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide. Therefore, comprehensive quantitative DNA methylation analysis at imprinted loci showing ~50 % methylation in healthy liver tissues was performed in primary HCC specimens and the peritumoural liver tissues.ResultsWe found frequent and extensive DNA methylation aberrations at many imprinted loci in HCC. Unsupervised cluster analysis of DNA methylation patterns at imprinted loci revealed subgroups of HCCs with moderate and severe loss of methylation. Hypomethylation at imprinted loci correlated significantly with poor overall survival (log-rank test, p = 0.02). Demethylation at imprinted loci was accompanied by loss of methylation at LINE-1, a commonly used marker for global DNA methylation levels (p < 0.001). In addition, we found that loss of methylation at imprinted loci correlated with the presence of a CTNNB1 mutation (Fisher’s exact test p = 0.03). Re-analysis of publically available genome-wide methylation data sets confirmed our findings. The analysis of benign liver tumours (hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH)), the corresponding adjacent liver tissues, and healthy liver tissues showed that aberrant DNA methylation at imprinted loci is specific for HCC.ConclusionsOur analyses demonstrate frequent and widespread DNA methylation aberrations at imprinted loci in human HCC and identified a hypomethylated subgroup of patients with shorter overall survival.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-015-0145-6) contains supplementary material, which is available to authorized users.

Clinical, Molecular, and Neurophysiological Features in Angelman Syndrome

Angelman syndrome (AS) is a neurodevelopmental disorder associated with a unique type of epilepsy. AS is caused by loss of function of the ubiquitin protein ligase E3A (UBE3A) gene. Most cases of AS (70%) are caused by an approximate 5 Mb deletion in chromosome 15q11-q13 of the maternally derived allele; other causes include paternal uniparental disomy of chromosome 15 (5%), imprinting defects (5%), and UBE3A mutations (10%), with the genetic cause yet to be determined in the remaining 10% of cases. Many seizure types are present in AS, with myoclonic, absence, and tonic–clonic seizures being common. Characteristic findings of electroencephalography in AS include persistent rhythmic theta activity, anterior dominant rhythmic high-amplitude delta (2–3 Hz) activity or spikes and slow waves, and posterior dominant spikes and sharp waves mixed with 3 to 4 Hz high-amplitude slow waves. UBE3A has been shown to regulate both the glutamatergic and gamma-aminobutyric acidergic systems; further dissection of the molecular mechanisms of UBE3A action in mouse models and individuals with AS would facilitate a greater understanding of the pathophysiology of epilepsy.