Bibliographie

Os autores descrevem um caso típico de síndrome de Angelman. A paciente apresenta atraso de desenvolvimento neuropscomotor deficiência mental, macrostomia, dentes espaçados, convulsões, ausência de fala, andar com a base alargada e instável, crises de risos. Os estudos citogenéticos e moleculares revelaram deleção do segmento 15q11q13 de origem materna, confirmando o diagnóstico clínico de síndrome de Angelman.

The authors describe the case of a typical Angelman syndrome patient. The proband presents developmental delay, mental retardation, macrostomia, wide-spaced teeth, seizures, absent speech, jerky gait and paroxysms of laughter. The cytogenetic and molecular studies showed a maternal deletion of 15q11q13. These results are in agreement with the clinical diagnosis of Angelman syndrome.

A 10 1/2-month-old boy was found to have an unbalanced karyotype, 45,XY,der(8)t(8;15) (p23.3;q13). One of 83 analyzed cells also contained an unidentified small marker. Fluorescence in situ hybridization (FISH) using cosmid probes for SNRPN, D15S10, and GABRB3 for the Prader-Willi syndrome (PWS)/Angelman syndrome (AS) critical region were not present on the derived chromosome. The child had some physical findings compatible with monosomy 8p. The mother also was a balanced carrier for the translocation. She also had 2/80 cells with an additional small marker chromosome, similar in size to the extra chromosome in the one cell of the propositus. FISH using an 8 paint did not show the reciprocal exchange on the der(15) but was demonstrated by using an 8p telomeric probe. At 18 months of age the child has some manifestations of AS. Earlier diagnosis may have been masked by the 8p- phenotype, or related to difficulty in diagnosing AS in infants.

Genomic imprinting is an epigenetic mechanism resulting in the preferential expression of the maternal or paternal alleles of a specific subset of genes in the mammalian genome. A key but relatively unexplored question is how imprints are established in the germline. New observations on two classical imprinting disorders, the Prader-Willi (PWS) and Angelman (AS) syndromes, offer the first genetic insight into this process. Molecular analysis of imprinting mutations that interfere with the appropriate establishment of the maternal and paternal epigenotypes has led to the identification of imprinted transcripts that could be involved in switching imprints in the germlines.

Angelman syndrome (AS) is a complex neurological disorder with different genetic aetiologies. It is not known whether the clinical features vary depending on the genetic mechanism. We report four patients with AS owing to uniparental disomy (UPD). There were two males and two females, with a mean age of 8 years (range 7 to 11 years). All patients had a happy disposition, hyperactive behaviour, and the characteristic facial phenotype of AS, but in three there was a normal head circumference, two had epilepsy, ataxic movements were mild in three, the mean age of onset of walking was 2.4 years, and there was some sign language in all four patients. Our cases add further weight to the previously reported impressions of a milder phenotype in cases of AS resulting from UPD than in deleted AS patients. Patients suspected of having AS, but who are considered atypical, warrant DNA testing.

The E6-AP gene (UBE3A) encodes an E3 ubiquitin-protein ligase that binds the human papillomavirus E6 oncoprotein and catalyzes the ubiquitination of p53. Recent studies have also established that mutations in E6-AP are the genetic basis of the Angelman syndrome in humans. In this study we present the genomic structure of the coding region of E6-AP and an analysis of a set of five E6-AP mRNAs with the potential to encode three protein isoforms of the E6-AP protein (isoforms I, II, and III) that differ at their extreme amino-termini. These transcripts were expressed in a variety of different cell lines examined.

Gamma-Aminobutyric acid type A receptors (GABA(A)-Rs) mediate the bulk of rapid inhibitory synaptic transmission in the central nervous system. The beta3 subunit is an essential component of the GABA(A)-R in many brain regions, especially during development, and is implicated in several pathophysiologic processes. We examined mice harboring a beta3 gene inactivated by gene targeting. GABA(A)-R density is approximately halved in brain of beta3-deficient mice, and GABA(A)-R function is severely impaired. Most beta3-deficient mice die as neonates; some neonatal mortality, but not all, is accompanied by cleft palate. beta3-deficient mice that survive are runted until weaning but achieve normal body size by adulthood, although with reduced life span. These mice are fertile but mothers fail to nurture offspring. Brain morphology is grossly normal, but a number of behaviors are abnormal, consistent with the widespread location of the beta3 subunit. The mice are very hyperactive and hyperresponsive to human contact and other sensory stimuli, and often run continuously in tight circles. When held by the tail, they hold all paws in like a ball, which is frequently a sign of neurological impairment. They have difficulty swimming, walking on grids, and fall off platforms and rotarods, although they do not have a jerky gait. beta3-deficient mice display frequent myoclonus and occasional epileptic seizures, documented by electroencephalographic recording. Hyperactivity, lack of coordination, and seizures are consistent with reduced presynaptic inhibition in spinal cord and impaired inhibition in higher cortical centers and/or pleiotropic developmental defects.

The Prader-Willi (PWS) and Angelman (AS) syndromes are two clinically distinct syndromes which result from lack of expression of imprinted genes within chromosome 15q11-q13. These two syndromes result from 15q11-q13 deletions, chromosome 15 uniparental disomy (UPD), imprinting centre mutations and, for AS, probable mutations in a single gene. The differential phenotype results from a paternal genetic deficiency in PWS patients and a maternal genetic deficiency in AS patients. Within 15q11-q13, four genes (SNRPN, IPW, ZNF127, FNZ127) and two expressed sequence tags (PAR1 and PAR5) have been found to be expressed only from the paternally inherited chromosome, and therefore all must be considered candidate genes involved in the pathogenesis of PWS. A candidate AS gene (UBE3A) has very recently been identified. The mechanisms of imprinted gene expression are not yet understood, but it is clear that DNA methylation is involved in both somatic cell expression and inheritance of the imprint. The presence of DNA methylation imprints that distinguish the paternally and maternally inherited alleles is a common characteristic of all known imprinted genes which have been studied extensively, including SNRPN and ZNF127. Recently, several PWS and AS patients have been found that have microdeletions in a region upstream of the SNRPN gene referred to as the imprinting centre, or IC. Paternal IC deletions in PWS patients and maternal IC deletions in AS patients result in uniparental DNA methylation and uniparental gene expression at biparentally inherited loci. The IC is a novel genetic element which controls initial resetting of the parental imprint in the germline for all imprinted gene expression over a 1.5-2.5 Mb region within chromosome 15q11-q13.

Angelman's syndrome is a rare genetic disorder characterized by developmental delay, craniofacial abnormalities, ataxia, paroxysmal laughter, and seizures. The diagnosis is suspected in infants who have the characteristic clinical features and electroencephalographic (EEG) abnormalities and is confirmed by the genetic identification of a maternally derived 15q11-13 deletion. We report on 3 patients with genetically confirmed Angelman's syndrome who had the characteristic clinical and EEG features. The EEGs demonstrated high-amplitude 2- to 3-Hz delta activity, with intermittent spike-and-slow-wave discharges maximal in the occipital region in 2 patients and generalized sharp-and-slow-wave discharges, occipital spikes, and electrographic status epilepticus during slow-wave sleep in the other patient. The findings of generalized high-amplitude delta slowing and occipital spike-and-wave discharges, facilitated by eye closure, in children with developmental delay and seizures suggest the diagnosis of Angelman's syndrome and should lead to genetic testing.

The genes encoding the gamma-aminobutyric acid (GABA) type-A receptor subunits beta 3 (GABRB3), alpha 5 (GABRA5), and gamma 3 (GABRG3) map to chromosome 15q11-q13. The three genes are contained within roughly 800 kb of the distal part of the imprinted Prader-Willi and Angelman syndrome region. A 570-kb contig encompassing GABRB3 and GABRA5 has been constructed in P1, lambda phage, and PAC clones. GABRB3 spans 250 kb of DNA and is organized into 9 exons that range from 68 to 504 bp, while GABRA5 is encoded by 11 exons (65 to 924 bp in length) within 86 kb. The exon/intron borders for both genes have been characterized and, primers have been designed to amplify each of the individual exons. Two reference STR markers have been positioned in the contig. The reference STR for GABRB3 is in fact located at least 60 kb beyond the 3' terminus of GABRB3, while D15S97 is contained within intron 4 of GABRB3. The detailed physical map of this GABAA receptor subunit gene cluster should not only be useful in genetic studies of the 15q11-q13 region, but will also be important for investigating the evolution and expression of the GABAA receptor gene superfamily.

As a reversible epigenetic modification which can affect gene expression, DNA methylation has been an attractive candidate for the biochemical mechanism of genomic imprinting. Many correlations in mice and humans link allele-specific DNA methylation to the allele-restricted RNA expression which is the hallmark of imprinted genes. Moreover, abnormal DNA methylation accompanies the pathological functional imprinting of certain human genes on chromosome 11p15.5 in Wilms' tumors and in the Beckwith-Weidemann syndrome and on chromosome 15q11-13 in the Prader-Willi and Angelman syndromes. A role for DNA methylation in maintaining the transcriptional silence of imprinted alleles at some loci has been supported by pharmacological manipulation with 5-aza-2'-deoxycytidine and by experiments with methyltransferase deletion mice. Gametic differences in DNA methylation could also account for the initiation of imprints, but this remains unproven. Comprehensive physical models for the role of DNA methylation in imprinting must account not only for local allele-restricted gene expression but also for the existence of large chromosomal domains containing multiple coordinately imprinted genes.

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct clinical phenotypes resulting from maternal and paternal deficiencies, respectively, in human chromosome 15qll-q13. Although several imprinted, paternally expressed transcripts have been identified within the PWS candidate region, no maternally expressed gene has yet been identified within the AS candidate region. We have developed an integrated physical map spanning the PWS and AS candidate regions and localized two breakpoints, including a cryptic t(14;15) translocation associated with AS and a non-AS 15q deletion, which substantially narrow the AS candidate region to approximately 250 kb. Mapping data indicate that the entire transcriptional unit of the E6-AP ubiquitin-protein ligase (UBE3A) gene lies within the AS region. The UBE3A locus expresses a transcript of approximately 5 kb at low to moderate levels in all tissues tested. The mouse homolog of UBE3A was cloned and sequenced revealing a high degree of conservation at nucleotide and protein levels. Northern and RT-PCR analysis of Ube3a expression in mouse tissues from animals with segmental, paternal uniparental disomy failed to detect substantially reduced or absent expression compared to control animals, failing to provide any evidence for maternal-specific expression from this locus. Recent identification of de novo truncating mutations in UBE3A taken with these observations indicates that mutations in UBE3A can lead to AS and suggests that this locus may encode both imprinted and biallelically expressed products.

The most common of the heterogeneous group of the extra structurally abnormal chromosomes (ESACs) is the inv dup(15), whose presence results in tetrasomy 15p and partial tetrasomy 15q. Inv dup(15), containing the Prader-Willi/Angelman syndrome (PWS/AS) region, are constantly associated with phenotypic abnormalities and mental retardation. We report on four additional patients with inv dup(15), whose behavioral pattern, and neurologic and physical findings further delineate the phenotype of this neurogenetic syndrome. We also provide FISH analyses on chromosomes of the observed ESACs and discuss the role of a number of genes located within the tetrasomic region.

The analysis of allelic methylation differences in 15q11-q13 has been established as a valid test for the Angelman and Prader-Willi syndromes. Current tests use methylation-sensitive restriction enzymes and Southern blot analysis. Here we describe a single-tube PCR test. It is based on sodium bisulfite treatment of DNA, which converts unmethylated, but not methylated cytosine residues to uracil, and PCR primers specific for the maternal and the paternal allele. The method was validated in a blinded retrospective study on 87 DNA samples from normal controls and patients. Prospective studies by independent laboratories will be needed before this assay can replace Southern blot analysis in routine diagnostic procedures.

A deletion of 15q11-q13 and uniparental disomy 15 lead to Prader-Labhart-Willi syndrome (PWS) or Angelman syndrome (AS) because this region contains genes expressed exclusively from the paternal (PWS) or maternal (AS) chromosome, respectively. DNA methylation plays a role in the control of imprinted gene expression, but so far only a few 5'-CG-3' dinucleotides within the recognition sites of the methylation sensitive enzymes have been studied. As part of a study on DNA methylation patterns in the human genome, we have applied the bisulfite protocol of genomic sequencing to study all 5'-CG-3' dinucleotides around exon 1 of SNRPN and at the D15S63 locus, which contains a start site for alternative SNRPN transcripts possibly involved in imprint switching during gametogenesis. At least 17 PCR products derived from single chromosomes of normal individuals as well as PWS and AS patients have been sequenced. We have found that cytosine residues outside 5'-CG-3' dinucleotides are always unmethylated. However, > 96% of all of the 23 5'-CG-3' dinucleotides around SNRPN exon 1 are methylated on the maternal chromosome and completely devoid of methylation on the paternal chromosome. This finding is in contrast to the D15S63 locus, where only the two Cfol/Hhal sites are methylated on the maternal chromosome at the same frequency as seen for the SNRPN segment. At the other five 5'-CG-3' dinucleotides, differential methylation is less pronounced, i.e. 45-70% on the maternal chromosome and 5-14% on the paternal chromosome. The differences between SNRPN and D15S63 methylation may reflect different biological functions of the alternative SNRPN transcripts. The systematic investigation of 5'-CG-3' methylation patterns as reported here will provide the basis for a PCR-based methylation test to diagnose PWS and AS.

In a 15 year old girl, referred for growth retardation, conventional cytogenetic analysis detected an abnormal 15q+ chromosome with extra material in the proximal region, inherited from her father and grandfather. Using various molecular cytogenetic techniques, including comparative genomic hybridisation, we showed that the extra chromatin resulted from in situ amplification of DNA sequences located between the loci D15Z1 and D15S18. On the basis of the clinical features of our patient and the late replication of the large amplified region, we searched for functional modifications in the adjacent Prader-Willi syndrome region.

Angelman syndrome (AS) most frequently results from large (> or = 5 Mb) de novo deletions of chromosome 15q11-q13. The deletions are exclusively of maternal origin, and a few cases of paternal uniparental disomy of chromosome 15 have been reported. The latter finding indicates that AS is caused by the absence of a maternal contribution to the imprinted 15q11-q13 region. Failure to inherit a paternal 15q11-q13 contribution results in the clinically distinct disorder of Prader-Willi syndrome. Cases of AS resulting from translocations or pericentric inversions have been observed to be associated with deletions, and there have been no confirmed reports of balanced rearrangements in AS. We report the first such case involving a paracentric inversion with a breakpoint located approximately 25 kb proximal to the reference marker D15S10. This inversion has been inherited from a phenotypically normal mother. No deletion is evident by molecular analysis in this case, by use of cloned fragments mapped to within approximately 1 kb of the inversion breakpoint. Several hypotheses are discussed to explain the relationship between the inversion and the AS phenotype.

The diagnosis of Angelman syndrome has seldom been made in infancy because the typical craniofacial dysmorphism and the typical outbursts of unprovoked laughter are not fully developed before the second and third year of life. Other features such as mental retardation or absence of language, though invariably present, are less obvious in the first year of life. We describe three children in whom consecutive electroencephalographic (EEG) studies show very large amplitude slow activity at 2-3/s, often rhythmic, usually occurring in prolonged runs and often more prominent posteriorly, sometimes with spikes or sharp-wave activity, and invariably associated with a diffuse rhythmic activity at 4-6/ s of 200 microvolts. The changes were present as early as six months of life. They preceded development of seizure and occurred much earlier than the craniofacial dysmorphology. It is concluded that methodical use of EEG in the elucidation of children with developmental disorder and knowledge of the characteristic EEG picture may help to identify patients with Angelman syndrome at an early age and before the clinical features become obvious.

PURPOSE: To evaluate the evolution of epileptic seizures and EEG features in a large group of patients with Angelman syndrome (AS).
METHODS: Thirty-six patients with AS with a proven chromosome 15q11-13 deletion were retrospectively analyzed with regard to their epilepsy and EEG findings by examination of patient files and EEGs. AIJ EEGs were reviewed by one of the authors. A logistic regression model, with a follow-up from 1 to 39 years (mean, 15 years), was used for statistical analysis.
RESULTS: Epileptic seizures had occurred in 30 (83%) patients. In 43% of them, the initial symptoms of epilepsy were febrile convulsions in infancy. In childhood, epilepsy could start with almost any type of seizure. Atypical absences and myoclonic seizures prevailed in adulthood. Epileptic seizures were present in 92% of the adult patients. The most typical EEG findings were rhythmic triphasic delta waves of high amplitude with a maximum over the frontal regions, identified in 99 (66%) of 150 EEGs, and continuously or intermittently, in 30 (83%) of 36 patients with AS. In 47% it was present even before a clinical diagnosis of AS was considered. High-amplitude rhythmic 4-6/s slow activity, seen in 44 (29%) of 150 EEGs, was not present after the age of 12 years.
CONCLUSIONS: In contrast to previous reports suggesting a decreasing frequency of epileptic seizures with age, we found that 92% of the adult patients with AS continued to have epileptic seizures. The most typical EEG finding in AS, in both children and adults, was the presence of frontal triphasic delta waves. In mentally retarded patients, this EEG pattern should point the physician in the direction of AS.

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.

Based on cytogenetic observations, several syndromes have been previously identified as microdeletion-based disorders. In this review, recent progress is presented regarding whether one or multiple genes can be implicated in the pathogenesis of these segmentally aneusomic syndromes. The syndromes discussed include Angelman, Alagille, Williams, Langer-Giedeon, Prader-Willi, Smith-Magenis, Miller-Dieker, and DiGeorge/velocardiofacial or the 22q11 deletion syndromes. For Angelman and Alagille syndromes, single genes have been identified, whereas for Williams and Langer-Giedion syndromes, more than one gene can be implicated. Although there has been significant progress in dissecting the molecular basis for the other disorders, the ultimate answer regarding one versus several genes remains to be determined.

Uniparental disomy (UPD) is often the result of an aneuploid event masquerading under the features of diploidy. As such, it may never be recognized, being at 2 opposite phenotypic poles, harmless to the bearer, or, if harmful, eventually responsible for uncharacteristic although perhaps serious conditions. UPD can also be associated with problems such as recessiveness or mosaicism. This article considers the chances of unmasking UPD, in the course of CVS or AC prenatal diagnosis, by reviewing the main cytogenetic signals and major familial or personal antecedents raising its suspicion. Once suspected, the lead toward UPD may or may not be followed through appropriate molecular studies. UPD for either maternal or paternal chromosomes 13, 21 and 22 may not have consistent, common deleterious effects, while other identified UPD's are too rare to call. Unconditionally, main, consistent or near consistent damages to the phenotype have been traced to specific chromosome pairs such as 15 mat (Prader-Willi syndrome), 15 pat (Angelman syndrome), 11 pat (Wiedemann-Beck with syndrome), 14 mat and pat (multiple cogenital and developmental anomalies [MCDA]-several rather constant) and 7 mat (Russel-Silver [RS] and Growth-failure [GF]). The above problems all stem from an alteration of the normal, developmentally important genomic imprinting processes and most of them may recognize several etiopathogenic paths, other than UPD, none of which abides by straight Mendelian rules. In this very area, therefore, a new, non-traditional type of inheritance is confronting genetic counselling. In this paper, for want of appropriate semantic language, the neologism "likon" (or "laikon") is coined to make reference to the hemizygously expressed sequences of the genomic parts imprinted in the somatic tissues. Broadening the definition, the word is then applied to the 4 possible epigenotypes of imprinted domains, which depend on the parental sex-of-origin: germinally "resting" (R), or "acting" (A), to be made somatically silent, that is to say "unexpressed" (U), or transcribed and "expressed" (E), thus abbreviated as EA, ER, UA and UR. Entire pedigrees may then be analyzed accordingly in health and in disease. Examples are presented herewith.

Angelman syndrome (AS) is characterized by severe psychomotor retardation, speech impairment, happy disposition with bursts of laughter, ataxia, convulsions, and some distinct physical anomalies. Correct diagnosis of AS is important because of its clinical implications, and once the disease is confirmed, familial genetic counseling becomes crucial. We evaluated 22 patients with a putative diagnosis of AS by both clinical and molecular cytogenetic analysis. A deletion of the region 15q11-13 could be identified cytogenetically in 11 cases by high-resolution technique (group I). Four additional cases were confirmed by fluorescence in situ hybridization (FISH) study with D15S11, SNRPN, D15S10, and GABRB 3 [Prader-Willi syndrome (PWS)/AS region probes] (group II). The common deletion of GABRB 3 was documented in those AS cases (n = 15) by FISH. The other 7 cases exhibited no deletion over 15q11-13 at either the cytogenetic or molecular level (group III). We compared the following associated neurological disorders: convulsions and abnormal EEG, microcephaly, sleep and behavior problems, brain anomalies proved by image studies, sexual precocity with pineal tumor among the three groups, as well as other clinical conditions including congenital heart disease, obesity, scoliosis, and hypopigmentation. In the present study, the differences in neurological and facial characteristics were not distinct among these groups. However, the associated conditions were more frequently observed in the patients with deletion than in those without deletion. The EEG features of AS appear to be less sufficient in helping identify patients at an early age before the clinical features become obvious. Therefore, a region involved in the major As phenotypes may contain only one or more tightly contiguous genes around the GABRB 3 locus, which may explain the clinical heterogeneity in AS.

The tools of molecular biology will bring the field of human genetics into a new era by permitting the analysis of the genetic contribution to disease. Most single gene disorders, inherited in a Mendelian fashion, will be molecularly diagnosed. In addition, the genetic susceptibility of common, complex diseases such a schizophrenia can be clarified, even though the conditions are not inherited as Mendelian characteristics. The mapping of the human genome will increase the rate at which new disease genes are identified and isolated. Finally, the development of genetically engineered animal models will help to dissect the steps involved in physiological and pathophysiological processes and thereby enhance our understanding of complex biological systems.

Plasma gamma-aminobutyric acid (GABA) levels were measured in 14 subjects with Prader-Willi syndrome, 9 subjects with Angelman syndrome, and matched control subjects. Mean levels in both patient groups were 2 to 3 times higher than in nonretarded moderately obese or retarded nonobese control subjects. Levels in each patient group differed significantly from both control groups. Neither the two patient groups nor the two control groups differed. GABA levels seemed unrelated to genetic status (chromosome 15 deletion or disomy). These preliminary findings of elevated plasma GABA levels possibly represent a compensatory increase in presynaptic GABA release in response to hyposensitivity of a subset of GABA receptors and could produce increased postsynaptic activation of other normal GABA receptor subtypes, resulting in complex alterations of GABAergic function throughout the brain.

Inv dup(15) is the most common supernumerary marker chromosome in humans. To investigate the mechanism responsible for this frequent chromosome rearrangement, we characterized the breakpoints in 18 individuals with small inv dup(15) chromosomes [i.e., negative for the Prader-Willi (PWS)/Angelman syndrome (AS) critical region]. Since two proximal breakpoint regions ("hotspots") for PWS/AS deletions have been previously identified with the most proximal 15q markers D15S541/S542 and S543, we hypothesized that formation of the small inv dup(15) chromosomes may involve one or both of these breakpoint hotspots. By analysis with S542, both breakpoint regions were found to be involved in approximately equal frequencies. In ten cases, the inv dup(15) was negative for S542 (Class I), indicating the breakpoint is between the centromere and the most proximal marker on chromosome 15. For the other eight cases, S542 was positive by fluorescence in situ hybridization (5/5) and/or microsatellite analysis (7/7), but S543 was negative (Class II). These two breakpoint regions appear to be the same as the two proximal breakpoints reported in the common PWS/AS deletions. To initiate cloning and sequencing of the Class II breakpoint, the gap in the yeast artificial chromosome (YAC) contig between S541/S542 and S543 was filled by screening the CEPH YAC and mega-YAC libraries. YACs 705C2 and 368H3 were found to bridge this gap, and therefore contain the more distal breakpoint region. The finding of consistent breakpoints in small inv dup(15), like that found in PWS/AS deletions, provides strong evidence for hotspots for chromosome breakage in this region. In addition, our results show that two extra copies (tetrasomy) of the region from 15cen to the euchromatic region containing S542 are present in individuals with Class II breakpoints. Since most individuals carrying a small inv dup(15) are phenotypically normal, the euchromatin region included in the small inv dup(15) chromosomes does not appear to contain genes with clinically significant dosage effects.

Angelman syndrome (AS) is associated with maternal deletions of human chromosome 15q11-q13 and with paternal uniparental disomy for this region indicating that deficiency of an imprinted, maternally expressed gene within the critical interval is the likely cause of the syndrome. Although the gene for E6-AP ubiquitin-protein ligase (UBE3A) was mapped to the critical region for AS, evidence of expression from both parental alleles initially suggested that it was an unlikely candidate gene for this disorder. Because attempts to identify any novel maternally expressed transcripts were unsuccessful and because the UBE3A gene remained within a narrowed AS critical region, we searched for mutations in UBE3A in 11 AS patients without known molecular defects (large deletion, uniparental disomy, or imprinting mutation). This analysis tested the possibility that deficiency of an undefined, maternally expressed transcript or isoform of the UBE3A gene could cause AS. Four mutations were identified including a de novo frameshift mutation and a de novo nonsense mutation in exon 3 and two missense mutations of less certain significance. The de novo truncating mutations indicate that UBE3A is the AS gene and suggest the possibility of a maternally expressed gene product in addition to the biallelically expressed transcript. Intragenic mutation of UBE3A in AS is the first example of a genetic disorder of the ubiquitin-dependent proteolytic pathway in mammals. It may represent an example of a human genetic disorder associated with a locus producing functionally distinct imprinted and biallelically expressed gene products.

Angelman syndrome (AS), characterized by mental retardation, seizures, frequent smiling and laughter, and abnormal gait, is one of the best examples of human disease in which genetic imprinting plays a role. In about 70% of cases, AS is caused by de novo maternal deletions at 15q11-q13 (ref. 2). Approximately 2% of AS cases are caused by paternal uniparental disomy (UPD) of chromosome 15 (ref. 3) and 2-3% are caused by "imprinting mutations'. In the remaining 25% of AS cases, no deletion, uniparental disomy (UPD), or methylation abnormality is detectable, and these cases, unlike deletions or UPD, can be familial. These cases are likely to result from mutations in a gene that is expressed either exclusively or preferentially from the maternal chromosome 15. We have found that a 15q inversion inherited by an AS child from her normal mother disrupts the 5' end of the UBE3A (E6-AP) gene, the product of which functions in protein ubiquitination. We have looked for novel UBE3A mutations in nondeletion/non-UPD/non-imprinting mutation (NDUI) AS patients and have found one patient who is heterozygous for a 5-bp de novo tandem duplication. We have also found in two brothers a heterozygous mutation, an A to G transition that creates a new 3' splice junction 7 bp upstream from the normal splice junction. Both mutations are predicted to cause a frameshift and premature termination of translation. Our results demonstrate that UBE3A mutations are one cause of AS and indicate a possible abnormality in ubiquitin-mediated protein degradation during brain development in this disease.

Par en haut !
This way up !
Retour à la page d'accueil...
Back to Home Page...