Mechanisms of imprinting of the Prader-Willi/Angelman region.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two distinct neurodevelopmental disorders, each caused by several genetic and epigenetic mechanisms involving the proximal long arm of chromosome 15. Lack of a functional paternal copy of 15q11-q13 causes PWS; lack of a functional maternal copy of UBE3A, a gene within 15q11-q13, causes AS. This region of chromosome 15 contains a number of imprinted genes that are coordinately regulated by an imprinting center (PWS/AS-IC) that contains two functional elements, the PWS-SRO and the AS-SRO. A chromosome lacking the PWS-SRO has the maternal state of gene activity and epigenetic modification after either maternal or paternal transmission; a chromosome lacking the AS-SRO but containing the PWS-SRO has the paternal state of gene activity and epigenetic modification after either maternal or paternal transmission. The maternal state of chromosome 15q11-q13 is associated with methylation of the PWS-SRO, while the paternal state is associated with lack of methylation of the PWS-SRO. Although most models of PWS/AS region imprinting assume that the PWS-SRO is methylated during oogenesis and that this methylation of the maternal PWS-SRO is maintained after fertilization, several lines of evidence suggest that the maternal PWS-SRO is in fact not methylated until after fertilization. Imprinting defects affecting the PWS/AS region can arise from failure to demethylate the PWS-SRO in the male germ line, from failure to methylate the maternal PWS-SRO, or from failure to maintain PWS-SRO methylation after fertilization.

Maternal disruption of Ube3a leads to increased expression of Ube3a-ATS in trans.

Angelman syndrome (AS) is a neurogenetic disorder characterized by severe mental retardation, 'puppet-like' ataxic gait with jerky arm movements, seizures, EEG abnormalities, hyperactivity and bouts of inappropriate laughter. Individuals with AS fail to inherit a normal active maternal copy of the gene encoding ubiquitin protein ligase E3A (UBE3A). UBE3A is transcribed predominantly from the maternal allele in brain, but is expressed from both alleles in most other tissues. It has been proposed that brain-specific silencing of the paternal UBE3A allele is mediated by a large (>500 kb) paternal non-coding antisense transcript (UBE3A-ATS). There are several other examples of imprinting regulation involving antisense transcripts that share two main properties: (i) the sense transcript is repressed by antisense and (ii) the interaction between sense and antisense occurs in cis. We show here that, in a mouse model of AS, maternal transmission of Ube3a mutation leads to increased expression of the paternal Ube3a-ATS, suggesting that the antisense is modulated by sense rather than the reciprocal mode of regulation. Our observation that Ube3a regulates expression of Ube3a-ATS in trans is in contrast to the other cases of sense-antisense epigenetic cis-interactions and argues against a major role for Ube3a-ATS in the imprinting of Ube3a.

From electrophysiology to chromatin: a bottom-up approach to Angelman syndrome.

Angelman syndrome is one of the most studied human diseases related to a gene that is expressed on the maternal chromosome only in at least some brain cells. It is caused by inactivation of the UBE3A gene in the brain due to various abnormalities of the 15q11-q13 chromosome inherited from the mother. It is characterized by severe developmental delay, seizures, virtual absence of speech, motor impairment, and a particular behavioral phenotype. Studies of cortical, electromyographic and cerebellar electrophysiology in patients with Angelman syndrome and a mouse model revealed unique rhythmic neurophysiological activities in the cerebral cortex, cerebellar cortex, and muscles. The oscillatory patterns may be linked to molecular pathophysiology of the syndrome involving dysregulation of synaptic neurotransmission through UBE3A-related modulation of functional GABAA receptor complexes.

Angelman syndrome 2005: updated consensus for diagnostic criteria.

In 1995, a consensus statement was published for the purpose of summarizing the salient clinical features of Angelman syndrome (AS) to assist the clinician in making a timely and accurate diagnosis. Considering the scientific advances made in the last 10 years, it is necessary now to review the validity of the original consensus criteria. As in the original consensus project, the methodology used for this review was to convene a group of scientists and clinicians, with experience in AS, to develop a concise consensus statement, supported by scientific publications where appropriate. It is hoped that this revised consensus document will facilitate further clinical study of individuals with proven AS, and assist in the evaluation of those who appear to have clinical features of AS but have normal laboratory diagnostic testing.

Clinical outcome of infants with confined placental mosaicism and intrauterine growth restriction of unknown cause.

The purpose of this study was to know a role of confined placental mosaicism (CPM) in perinatal outcome and postnatal growth and development of infants with intrauterine growth restriction (IUGR). We selected 50 infants with IUGR (<-2.0 SD) from 3,257 deliveries in a regional medical center during the past 10-year period, and carried out cytogenetic and molecular analyses in their placenta and cord blood. Of the 50 infants, 8 had CPM (CPM group) and were composed of five single (CPM2, 7, 13, 22, and 22), one double (CPM7/13), and one quadruple trisomy (CPM2/7/15/20), and one partial monosomy [del(2)(p16)]. The origin of an extra chromosome of trisomy was maternal in six cases of CPM, paternal in one, and undetermined in one. Uniparental disomy in disomic cell lines was ruled out in all these mosaics. We also compared clinical parameters for perinatal outcome between CPM group and infants without evidence of CPM (non-CPM group), such as maternal and gestational age, birth weight, Apgar score, cord blood pH, gender, and uterine artery patterns by Doppler ultrasonography, as well as weight, height, and developmental quotient (DQ) by Denver Developmental Screening Test at age 12 months. Phenotypic abnormalities were noted in two infants with CPM and three infants of non-CPM group: One with CPM22 had ASD and hypospadias, one with CPM7/13 had Russell-Silver syndrome (RSS), and one without CPM had polydactyly, and two without CPM had RSS. All but one infant with CPM are alive at age 12 months. Among the clinical parameters, the detection rate of a notch waveform pattern of the uterine artery was significantly higher in the CPM group (P < 0.05). However, no significant difference was noted in perinatal outcome of pregnancy and in DQ at age 12 months between the two groups. Interestingly, short stature (<-2 SD) at age 12 months was more frequently seen in CPM group (7/8 infants with CPM vs. 8/15 infants without CPM), although no statistically significant difference was obtained. The information obtained will be useful for perinatal care and genetic counseling for infants with IUGR and CPM.

Imprinting centers, chromatin structure, and disease.

Two regions that best exemplify the role of genetic imprinting in human disease are the Prader-Willi syndrome/Angelman syndrome (PWS/AS) region in 15q11-q13 and the Beckwith-Wiedemann syndrome (BWS) region in 11p15.5. In both regions, cis-acting sequences known as imprinting centers (ICs) regulate parent-specific gene expression bidirectionally over long distances. ICs for both regions are subject to parent-specific epigenetic marking by covalent modification of DNA and histones. In this review, we summarize our current understanding of IC function and IC modification in these two regions.

Sleep disturbances in Ube3a maternal-deficient mice modeling Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a severe neurodevelopmental disorder with electroencephalographic (EEG) abnormalities and sleep disturbances. It results from lack of the functional maternal allele of UBE3A, which encodes a ubiquitin-protein ligase. Different mechanisms of UBE3A inactivation correlate with clinical phenotypes of varying severity; the majority of cases of AS are due to a de novo maternal deletion of the 15q11-q13 region. METHODS: Ube3a maternal-deficient mice (Ube3a m-/p+) were generated in a C57Bl/6J background. This study compares cortical EEG and architecture of the sleep-waking cycle in adult Ube3a m-/p+ mice compared with those of age-matched WT (m+/p+) mice, under baseline conditions or after 4-h sleep deprivation (SD). RESULTS: Ube3a m-/p+ mice exhibited: reduced slow-wave sleep (SWS) amount with increase waking (W) at the dark/light transitions; increased SWS and W episode numbers; and deterioration of paradoxical sleep (PS) over 24 h [amount: -44%; episode duration: -46%; episode number: -40%; theta peak frequency (TPF) acceleration: 7.6 Hz vs. 7.0 Hz in WT mice]. Characteristic paroxysmal EEG discharges are observed during W and SWS associated with synchronous muscle bursting activity during hypoactive W. During the recovery period following SD, Ube3a m-/p+ mice exhibited no rebound either in slow-wave activity (+89% in WT) or in delta-power spectra but a slight rebound in PS amount (+20%). CONCLUSIONS: These data validate the mouse model produced by null mutation of the maternal Ube3a gene and provide useful results to investigate and better understand the molecular basis of sleep disturbances in AS patients.

Role of histone methyltransferase G9a in CpG methylation of the Prader-Willi syndrome imprinting center.

Imprinted genes in mammals are often located in clusters whose imprinting is subject to long range regulation by cis-acting sequences known as imprinting centers (ICs). The mechanisms by which these ICs exert their effects is unknown. The Prader-Willi syndrome IC (PWS-IC) on human chromosome 15 and mouse chromosome 7 regulates imprinted gene expression bidirectionally within an approximately 2-megabase region and shows CpG methylation and histone H3 Lys-9 methylation in somatic cells specific for the maternal chromosome. Here we show that histone H3 Lys-9 methylation of the PWS-IC is reduced in mouse embryonic stem (ES) cells lacking the G9a histone H3 Lys-9/Lys-27 methyltransferase and that maintenance of CpG methylation of the PWS-IC in mouse ES cells requires the function of G9a. We show by RNA fluorescence in situ hybridization (FISH) that expression of Snrpn, an imprinted gene regulated by the PWS-IC, is biallelic in G9a -/- ES cells, indicating loss of imprinting. By contrast, Dnmt1 -/- ES cells lack CpG methylation of the PWS-IC but have normal levels of H3 Lys-9 methylation of the PWS-IC and show normal monoallelic Snrpn expression. Our results demonstrate a role for histone methylation in the maintenance of parent-specific CpG methylation of imprinting regulatory regions and suggest a possible role of histone methylation in establishment of these CpG methylation patterns.

Biochemical analysis of Angelman syndrome-associated mutations in the E3 ubiquitin ligase E6-associated protein.

Angelman syndrome is a severe neurological disorder characterized by mental retardation, absent speech, ataxia, seizures, and hyperactivity. The gene affected in this disorder is UBE3A, the gene encoding the E6-associated protein (E6AP) ubiquitin-protein ligase. Most patients have chromosomal deletions that remove the entire maternal allele of UBE3A. However, a small subset of patients have E6AP point mutations that result in single amino acid changes or short in-frame deletions that still allow translation of a full-length protein. By studying these point mutations in E6AP, we found a strong correlation between Angelman-associated mutations and a loss of E3 ubiquitin ligase activity. Interestingly the point mutations affect E6AP activity in different ways. Some mutant proteins cannot form thiol ester intermediates with ubiquitin, others retain the thiol ester formation activity but cannot efficiently transfer ubiquitin to a substrate, and still others are unstable in cells. Our results suggest that the loss of E6AP catalytic activity and likely the improper regulation of E6AP substrate(s) are important in the development of Angelman syndrome.

Neurobehavioral and electroencephalographic abnormalities in Ube3a maternal-deficient mice.

Angelman syndrome (AS), characterized by motor dysfunction, mental retardation, and seizures, is caused by several genetic etiologies involving chromosome 15q11-q13, including mutations of the UBE3A gene. UBE3A encodes UBE3A/E6-AP, a ubiquitin-protein ligase, and shows brain-specific imprinting, with brain expression predominantly from the maternal allele. Lack of a functional maternal allele of UBE3A causes AS. In order to understand the causal relationship between maternal UBE3A mutations and AS, we have constructed a mouse model with targeted inactivation of Ube3a. The inactive allele contains a lacZ reporter gene for analysis of brain-specific imprinting. Maternal, but not paternal, transmission of the targeted allele leads to beta-galactosidase activity in hippocampal and cerebellar neurons. Maternal inheritance of the Ube3a mutant allele also causes impaired performance in tests of motor function and spatial learning, as well as abnormal hippocampal EEG recordings. As predicted from the dependence of UBE3A-mediated ubiquitination of p53 on HPV E6 protein, our maternal-deficient mice show normal brain p53 levels.

SNURF-SNRPN and UBE3A transcript levels in patients with Angelman syndrome.

The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the control of an imprinting center (IC). The paternally expressed SNURF-SNRPN gene hosts several snoRNA genes and overlaps the UBE3A gene, which is encoded on the opposite strand, expressed - at least in brain cells - from the maternal chromosome only, and affected in patients with Angelman syndrome (AS). In contrast to SNURF-SNRPN, imprinted expression of UBE3A is not regulated by a 5' differentially methylated region. Here we report that splice forms of the SNURF-SNRPN transcript overlapping UBE3A in an antisense orientation are present in brain but barely detectable in blood. In contrast, splice forms that do not overlap with UBE3A are of similar abundance in brain and blood. The tissue distribution of the splice forms parallels that of the snoRNAs encoded in the respective parts of the SNURF-SNRPN transcript. Using a quantitative PCR assay, we have found that the ratio of SNURF-SNRPN/UBE3A transcript levels is increased in blood cells of AS patients with an imprinting defect, but not in AS patients with a UBE3A mutation or an unknown defect. Our findings are compatible with the assumption that imprinted UBE3A expression is regulated through the SNURF-SNRPN sense- UBE3A antisense transcript.