Molecular-Genetic Diagnostics of Angelman Syndrome – The Bulgarian Experience

AbstractObjective: The aim of the study was to determine the molecular mechanisms of mutagenesis in Bulgarian patients with Angelman syndrome (AS). AS is a severe neurodevelopmental disorder caused by loss of expression in brain of the maternally inherited UBE3A gene as a result of various 15q11.2-q13 alterations.Material and Methods: In total 24 patients (11 boys, 13 girls) from 22 unrelated families with suspected clinical diagnosis AS were analysed. We used methylation specific PCR, multiplex ligation-dependent probe amplification, methylation sensitive MLPA, and direct sequencing of the UBE3A gene.Results: In 9 families (41%) pathogenic mutations were detected, which confirmed the clinical diagnosis on а molecular-genetic level. In 4 (44%) of these families we found 15q11-q13 region deletion with breakpoints BP1-BP3 or BP2-BP3. In 1 (11%) of the families we found imprinting defect: deletion of the AS-SRO regulatory region (part of the PWS-AS imprinting center). In 1 (11%) of the families we detected a rare finding – paternal uniparental disomy of chromosome 15. In 3 (33%) of the families diff erent point mutations in the UBE3A gene were detected: two novel missence mutations c.488T > C; p.Leu163Ser and c.1832A > T; p.Gln611Leu, and one known frameshift mutation c.2576_2579delAAGA; p.Lys859Argfs*4.Conclusion: The obtained results helped us to develop a systematic diagnostic algorithm in order to provide proper diagnosis for the patients with AS. Combining excellent knowledge of the molecular mechanisms of mutagenesis and proper molecular-genetic testing approaches is a cornerstone in the management of AS patients, ensuring AS families would receive both adequate genetic counseling and prophylaxis of the disease in the future.

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Loss of nuclear UBE3A activity is the predominant cause of Angelman syndrome in individuals carrying UBE3A missense mutations

Human Molecular Genetics ◽

10.1093/hmg/ddab050 ◽

2021 ◽

Author(s):

Stijn N V Bossuyt ◽

A Mattijs Punt ◽

Ilona J de Graaf ◽

Janny van den Burg ◽

Mark G Williams ◽

...

Keyword(s):

Catalytic Activity ◽

Angelman Syndrome ◽

Neurodevelopmental Disorder ◽

Critical Role ◽

Rapid Assessment ◽

Missense Mutations ◽

Catalytic Potential ◽

Ubiquitin Ligase Activity ◽

Ube3a Gene

Abstract Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion (~75%) or mutation (~10%) of the UBE3A gene, which encodes a HECT type E3 ubiquitin protein ligase. Although the critical substrates of UBE3A are unknown, previous studies have suggested a critical role of nuclear UBE3A in AS pathophysiology. Here we investigated to what extent UBE3A missense mutations disrupt UBE3A subcellular localization as well as catalytic activity, stability and protein folding. Our functional screen of 31 UBE3A missense mutants revealed that UBE3A mislocalization is the predominant cause of UBE3A dysfunction, accounting for 55% of the UBE3A mutations tested. The second major cause (29%) is a loss of E3-ubiquitin ligase activity, as assessed in an E. coli in vivo ubiquitination assay. Mutations affecting catalytic activity are found not only in the catalytic HECT domain, but also in the N-terminal half of UBE3A, suggesting an important contribution of this N-terminal region to its catalytic potential. Together, our results show that loss of nuclear UBE3A E3 ligase activity is the predominant cause of UBE3A-linked Angelman syndrome. Moreover, our functional analysis screen allows rapid assessment of the pathogenicity of novel UBE3A missense variants which will be of particular importance when treatments for AS become available.

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Angelman Syndrome due to a Maternally Inherited Intragenic Deletion Encompassing Exons 7 and 8 of the UBE3A Gene

Cytogenetic and Genome Research ◽

10.1159/000480030 ◽

2017 ◽

Vol 152 (3) ◽

pp. 132-136

Author(s):

Athina Ververi ◽

Lily Islam ◽

Beverley Bewes ◽

Louise Busby ◽

Caroline Sullivan ◽

...

Keyword(s):

Developmental Delay ◽

Angelman Syndrome ◽

Array Cgh ◽

De Novo ◽

Uniparental Disomy ◽

Genetic Mechanism ◽

Paternal Allele ◽

Speech Impairment ◽

Intragenic Deletion ◽

Ube3a Gene

Angelman syndrome (AS) is characterised by developmental delay, lack of speech, seizures, a characteristic behavioural profile with a happy demeanour, microcephaly, and ataxia. More than two-thirds of cases are due to an approximately 5-Mb interstitial deletion of the imprinted region 15q11.2q13, which is usually de novo. The rest are associated with point mutations in the UBE3A gene, imprinting defects, and paternal uniparental disomy. Small intragenic UBE3A deletions have rarely been described. They are usually maternally inherited, increasing the recurrence risk to 50%, and may be missed by conventional testing (methylation studies and UBE3A gene sequencing). We describe a boy with AS due to an 11.7-kb intragenic deletion. The deletion was identified by array-CGH and was subsequently detected in his affected first cousin and unaffected maternal grandfather, mother, and aunt, confirming the silencing of the paternal allele. The patient had developmental delay, speech impairment, a happy demeanour, microcephaly, and an abnormal EEG, but no seizures by the age of 4 years. Delineation of the underlying genetic mechanism is of utmost importance for reasons of genetic counselling, as well as appropriate management and prognosis. Alternative techniques, such as array-CGH and MLPA, are necessary when conventional testing for AS has failed to identify the underlying genetic mechanism.

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Angelman Syndrome-Affected Individual with a Numerically Normal Karyotype and Isodisomic Paternal Uniparental Disomy of Chromosome 15 due to Maternal Robertsonian Translocation (14;15) by Monosomy Rescue

Cytogenetic and Genome Research ◽

10.1159/000490838 ◽

2018 ◽

Vol 156 (1) ◽

pp. 9-13 ◽

Cited By ~ 1

Author(s):

Nuria C. Bramswig ◽

Karin Buiting ◽

Natalie Bechtel ◽

Bernhard Horsthemke ◽

Kevin Rostasy ◽

...

Keyword(s):

Angelman Syndrome ◽

Robertsonian Translocation ◽

Chromosome Region ◽

Neurodevelopmental Disorder ◽

Uniparental Disomy ◽

Normal Karyotype ◽

Early Embryo ◽

Affected Individual ◽

Chromosome 15 ◽

Paternal Uniparental Disomy

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deletion of the maternally inherited 15q11q13 region, paternal uniparental disomy 15 [upd(15)pat], an imprinting defect of the maternal chromosome region 15q11q13, or a pathogenic mutation of the maternal UBE3A allele. Predisposing factors for upd(15)pat, such as nonhomologous robertsonian translocation involving chromosome 15, have been discussed, but no evidence for this predisposition has been published. In the present study, chromosomal analysis was performed in a child with AS, both parents, and the maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was employed on DNA of the index individual, and microsatellite analysis was carried out on DNA of the index individual and his parents. The cytogenetic analysis showed that the mother and maternal grandfather are carriers of a rob(14;15). The index individual has a numerically normal karyotype, but MS-MLPA and microsatellite analyses confirmed the clinical diagnosis of AS and revealed a pattern highly suggestive of isodisomic upd(15)pat. This is the first report of an AS-affected individual with isodisomic upd(15)pat and a numerically normal karyotype that most likely results from a rob(14;15)-associated meiotic error in the maternal germline followed by monosomy 15 rescue in the early embryo.

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Genotype–Phenotype Correlations in Angelman Syndrome

Genes ◽

10.3390/genes12070987 ◽

2021 ◽

Vol 12 (7) ◽

pp. 987

Author(s):

Lili Yang ◽

Xiaoli Shu ◽

Shujiong Mao ◽

Yi Wang ◽

Xiaonan Du ◽

...

Keyword(s):

Angelman Syndrome ◽

Cognitive Skills ◽

Molecular Mechanisms ◽

Uniparental Disomy ◽

Treatment Modalities ◽

Global Developmental Delay ◽

Loss Of Function ◽

Neurodevelopmental Disease ◽

Severe Intellectual Disability ◽

New Treatment

Angelman syndrome (AS) is a rare neurodevelopmental disease that is caused by the loss of function of the maternal copy of ubiquitin–protein ligase E3A (UBE3A) on the chromosome 15q11–13 region. AS is characterized by global developmental delay, severe intellectual disability, lack of speech, happy disposition, ataxia, epilepsy, and distinct behavioral profile. There are four molecular mechanisms of etiology: maternal deletion of chromosome 15q11–q13, paternal uniparental disomy of chromosome 15q11–q13, imprinting defects, and maternally inherited UBE3A mutations. Different genetic types may show different phenotypes in performance, seizure, behavior, sleep, and other aspects. AS caused by maternal deletion of 15q11–13 appears to have worse development, cognitive skills, albinism, ataxia, and more autistic features than those of other genotypes. Children with a UBE3A mutation have less severe phenotypes and a nearly normal development quotient. In this review, we proposed to review genotype–phenotype correlations based on different genotypes. Understanding the pathophysiology of the different genotypes and the genotype–phenotype correlations will offer an opportunity for individualized treatment and genetic counseling. Genotype–phenotype correlations based on larger data should be carried out for identifying new treatment modalities.

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Anxiety in Angelman Syndrome

American Journal on Intellectual and Developmental Disabilities ◽

10.1352/1944-7558-127.1.1 ◽

2022 ◽

Vol 127 (1) ◽

pp. 1-10

Author(s):

Stacey C. Grebe ◽

Danica L. Limon ◽

Morgan M. McNeel ◽

Andrew Guzick ◽

Sarika U. Peters ◽

...

Keyword(s):

Angelman Syndrome ◽

Neurodevelopmental Disorders ◽

Neurodevelopmental Disorder ◽

Chromosome 15 ◽

Limited Information ◽

Measurement Instruments ◽

Natural History Study ◽

Developmental Behavior ◽

Ube3a Gene ◽

Specific Symptoms

Abstract Angelman Syndrome (AS) is a neurodevelopmental disorder most commonly caused by the impaired expression of the maternal UBE3A gene on chromosome 15. Though anxiety has been identified as a frequently present characteristic in AS, there are limited studies examining anxiety in this population. Studies of anxiety in other neurodevelopmental disorders have found disorder specific symptoms of anxiety and age specific displays of anxiety symptoms. However, there is a consistent challenge in identifying anxiety in people with neurodevelopmental disorders given the lack of measurement instruments specifically designed for this population. Given the limited information about AS and anxiety, the aims of the current project were to (a) examine symptoms of anxiety in children with AS and (b) determine the correlates of anxiety in children with AS. Participants included 42 adult caregivers of youth with AS in the AS Natural History study who completed the Developmental Behavior Checklist (DBC). The results found that 26% of the sample demonstrated elevated symptoms of anxiety and established a relationship between elevated anxiety in youth with AS and higher levels of irritability, hyperactivity, self-absorbed behaviors, and disruptive/antisocial behaviors. Findings from this research provide a foundation for tailoring evidence-based assessments and treatments for youth with AS and anxiety.

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A placebo-controlled trial of folic acid and betaine in identical twins with Angelman syndrome

Orphanet Journal of Rare Diseases ◽

10.1186/s13023-019-1216-0 ◽

2019 ◽

Vol 14 (1) ◽

Author(s):

Julia Han ◽

Terry Jo Bichell ◽

Stephanie Golden ◽

Irina Anselm ◽

Susan Waisbren ◽

...

Keyword(s):

Folic Acid ◽

Angelman Syndrome ◽

Antisense Transcript ◽

Controlled Trial ◽

Neurodevelopmental Disorder ◽

Uniparental Disomy ◽

Identical Twins ◽

Clinical Phenotypes ◽

Severe Intellectual Disability ◽

The Brain

Abstract Background Angelman syndrome (AS) is a neurodevelopmental disorder that is caused by maternal genetic deficiency of a gene that encodes E6-AP ubiquitin-protein ligase (gene symbol UBE3A) mapping to chromosome 15q11-q13. AS leads to stiff and jerky gait, excess laughter, seizures, and severe intellectual disability. In some parts of the brain, the paternally inherited UBE3A gene is subject to genomic imprinting by the action of the UBE3A-antisense transcript (UBE3A-ATS) on the paternally inherited allele. Consequently, only the maternally inherited UBE3A gene is expressed in mature neurons. AS occurs due to deletions of the maternal 15q11 − 13 region, paternal uniparental disomy (UPD), imprinting center defects, mutations in the maternal UBE3A gene, or other unknown genetic malfunctions that result in a silenced maternal UBE3A gene in the specific imprinted regions of the brain. Results A potential treatment strategy for AS is to increase methylation of UBE3A-ATS to promote expression of the paternal UBE3A gene and thus ameliorate the clinical phenotypes of AS. We treated two sets of male identical twins with class I deletions with a 1 year treatment trial of either betaine and folic acid versus placebo. We found no statistically significant changes in the clinical parameters tested at the end of the 1 year trial, nor did we find any significant adverse events. Conclusions This study tested the hypothesis that by increasing the methylation of the UBE3A-antisense transcript in Angelman syndrome to promote expression of the silenced paternal UBE3A gene we may ameliorate the clinical phenotypes of AS. We treated two sets of identical twins with placebo versus betaine and folic acid. Although this study represented a novel approach to treating Angelman syndrome, the differences in the developmental testing results was not significant. This paper also discusses the value of monozygotic twin studies in minimizing confounding variables and its utility in conducting small treatment studies. Trial registration NCT00348933. Registered 6 July 2006.

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Molecular Basis and Clinical Aspects of Hereditary Megakaryocyte and Platelet Membrane Glycoprotein Disorders

Hämostaseologie ◽

10.1055/s-0038-1656647 ◽

1996 ◽

Vol 16 (02) ◽

pp. 114-138 ◽

Cited By ~ 6

Author(s):

R. E. Scharf

Keyword(s):

Genetic Basis ◽

Molecular Mechanisms ◽

Molecular Genetic ◽

Platelet Membrane ◽

Clinical Aspects ◽

Membrane Glycoprotein ◽

Membrane Glycoproteins ◽

Membrane Expression ◽

Receptor Complexes ◽

Platelet Membrane Glycoprotein

SummarySpecific membrane glycoproteins (GP) expressed by the megakaryocyte-platelet system, including GPIa-lla, GPIb-V-IX, GPIIb-llla, and GPIV are involved in mediat-ing platelet adhesion to the subendothelial matrix. Among these glycoproteins, GPIIb-llla plays a pivotal role since platelet aggregation is exclusively mediated by this receptor and its interaction with soluble macromolecular proteins. Inherited defects of the GPIIb-llla or GPIb-V-IX receptor complexes are associated with bleeding disorders, known as Glanzmann's thrombasthenia, Bernard-Soulier syndrome, or platelet-type von Willebrand's disease, respectively. Using immuno-chemical and molecular biology techniques, rapid advances in our understanding of the molecular genetic basis of these disorders have been made during the last few years. Moreover, analyses of patients with congenital platelet membrane glycoprotein abnormalities have provided valuable insights into molecular mechanisms that are required for structural and functional integrity, normal biosynthesis of the glycoprotein complexes and coordinated membrane expression of their constituents. The present article reviews the current state of knowledge of the major membrane glycoproteins in health and disease. The spectrum of clinical bleeding manifestations and established diagnostic criteria for each of these dis-orders are summarized. In particular, the variety of molecular defects that have been identified so far and their genetic basis will be discussed.

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Aberrant aggressive behavior in a mouse model of Angelman syndrome

Scientific Reports ◽

10.1038/s41598-020-79984-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lilach Simchi ◽

Hanoch Kaphzan

Keyword(s):

Social Behavior ◽

Mouse Model ◽

Aggressive Behavior ◽

Social Interactions ◽

Angelman Syndrome ◽

Case Reports ◽

Neurodevelopmental Disorder ◽

Therapeutic Interventions ◽

Affiliative Behavior ◽

Severe Difficulty

AbstractAngelman syndrome (AS) is a genetic neurodevelopmental disorder due to the absence of the E3-ligase protein, UBE3A. Inappropriate social interactions, usually hyper-sociability, is a part of that syndrome. In addition, clinical surveys and case reports describe aggressive behavior in AS individuals as a severe difficulty for caretakers. A mouse model for AS recapitulates most of the human AS phenotypes. However, very few studies utilized this mouse model for investigating affiliative social behavior, and not even a single study examined aggressive behavior. Hence, the aim of the herein study was to examine affiliative and aggressive social behavior. For that, we utilized a battery of behavioral paradigms, and performed detailed analyses of these behaviors. AS mice exhibited a unique characteristic of reduced habituation towards a social stimulus in comparison to their wild-type (WT) littermates. However, overall there were no additional marked differences in affiliative social behavior. In contrast to the mild changes in affiliative behavior, there was a striking enhanced aggression in the AS mice compared to their WT littermates. The herein findings emphasize the use of AS mouse model in characterizing and measuring inappropriate aggressive behavior, and suggests these as tools for investigating therapeutic interventions aimed at attenuating aggressive behavior.

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A Concerted Action of UBA5 C-Terminal Unstructured Regions Is Important for Transfer of Activated UFM1 to UFC1

International Journal of Molecular Sciences ◽

10.3390/ijms22147390 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7390

Author(s):

Nicole Wesch ◽

Frank Löhr ◽

Natalia Rogova ◽

Volker Dötsch ◽

Vladimir V. Rogov

Keyword(s):

Cellular Localization ◽

Molecular Mechanisms ◽

Biochemical Characterization ◽

Effective Interaction ◽

Regulatory Region ◽

Titration Calorimetry ◽

Enzymatic Reactions ◽

Cellular Processes ◽

Mechanism Of Interaction

Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.

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Maternal Uniparental Disomy of Chromosome 20 (UPD(20)mat) as Differential Diagnosis of Silver Russell Syndrome: Identification of Three New Cases

Genes ◽

10.3390/genes12040588 ◽

2021 ◽

Vol 12 (4) ◽

pp. 588

Author(s):

Pierpaola Tannorella ◽

Daniele Minervino ◽

Sara Guzzetti ◽

Alessandro Vimercati ◽

Luciano Calzari ◽

...

Keyword(s):

Growth Retardation ◽

Molecular Mechanisms ◽

Uniparental Disomy ◽

Postnatal Growth ◽

Molecular Diagnostic ◽

Growth Delay ◽

Molecular Defect ◽

Maternal Uniparental Disomy ◽

Feeding Difficulties ◽

Silver Russell Syndrome

Silver Russell Syndrome (SRS, MIM #180860) is a rare growth retardation disorder in which clinical diagnosis is based on six features: pre- and postnatal growth failure, relative macrocephaly, prominent forehead, body asymmetry, and feeding difficulties (Netchine–Harbison clinical scoring system (NH-CSS)). The molecular mechanisms consist in (epi)genetic deregulations at multiple loci: the loss of methylation (LOM) at the paternal H19/IGF2:IG-DMR (chr11p15.5) (50%) and the maternal uniparental disomy of chromosome 7 (UPD(7)mat) (10%) are the most frequent causes. Thus far, about 40% of SRS remains undiagnosed, pointing to the need to define the rare mechanisms in such a consistent fraction of unsolved patients. Within a cohort of 176 SRS with an NH-CSS ≥ 3, a molecular diagnosis was disclosed in about 45%. Among the remaining patients, we identified in 3 probands (1.7%) with UPD(20)mat (Mulchandani–Bhoj–Conlin syndrome, OMIM #617352), a molecular mechanism deregulating the GNAS locus and described in 21 cases, characterized by severe feeding difficulties associated with failure to thrive, preterm birth, and intrauterine/postnatal growth retardation. Our patients share prominent forehead, feeding difficulties, postnatal growth delay, and advanced maternal age. Their clinical assessment and molecular diagnostic flowchart contribute to better define the characteristics of this rare imprinting disorder and to rank UPD(20)mat as the fourth most common pathogenic molecular defect causative of SRS.

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