Analysis of clinical phenotype and genetic variants in a Chinese pedigree affected with Angelman syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the genetic etiology for a Chinese pedigree affected with Angelman syndrome (AS).@*METHODS@#The proband with phenotypes suggestive of AS was subjected to copy number variation sequencing (CNV-seq), methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) and high-throughput next generation sequencing (NGS). Variant of the UBE3A gene was verified among family members by Sanger sequencing and bioinformatic analysis.@*RESULTS@#NGS revealed that the proband has carried a heterozygous variant of the UBE3A gene, namely c.1517G>A (p.R506H). The variant has co-segregated with the disease in the pedigree. Multiple amino acid sequence alignment showed that the site of mutant residue is conserved among nine homologous species. The variant was predicted to be deleterious by bioinformatic analysis.@*CONCLUSION@#A novel variant of the UBE3A gene has been identified in a Chinese pedigree affected with AS. Above finding has further expanded the spectrum of UBE3A gene variants and phenotypes of AS, which also facilitated molecular diagnosis and genetic counseling for the family.

Análisis de variación del número de copias y de patrones de metilación en la región 15q11-q13 / Variation analysis of the number of copies and methylene patterns in region 15q11-q13

La región q11-q13 del cromosoma 15 humano es proclive a sufrir alteraciones genéticas. Algunos genes de la región presentan expresión parental diferencial monoalélica, regulada por imprinting (EI). Errores en la regulación del EI, disomías uniparentales (DSU), así como también el cambio en el número de copias genómicas (CNV) producidos por sitios susceptibles de quiebre cromosómico (BP), producen alteraciones en esta región. Las enfermedades más frecuentes asociadas son el síndrome de Prader-Willi, el síndrome de Angelman y el síndrome de microduplicación 15q11-q13. En el presente trabajo analizamos la región 15q11-q13 por Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA) en 181 muestras de ADN derivadas a nuestro servicio de análisis genético molecular. En este trabajo mostramos que, de las 181 muestras, 39 presentaron alteraciones detectables por MS-MLPA. El 61.5% (24/39) de esas alteraciones detectadas fueron deleciones, el 5.1% (2/39) duplicaciones y el 33.3%(13/39) DSU/EI. Los CNV fueron 4 veces más frecuentes que las DSU/EI (OR = 4; IC 95%: 1.56-10.25) consistente con la literatura. Entre los CNV, dos casos atípicos permiten postular posibles sitios BP que no han sido informados en la literatura previamente.

Human chromosome 15q11-q13 region is prone to suffer genetic alterations. Some genes of this region have a differential monoallelic imprinting-regulated expression pattern. Defects in imprinting regulation (IE), uniparental disomy (UPD) or copy number variation (CNV) due to chromosomal breakpoints (BP) in 15q11-q13 region, are associated with several diseases. The most frequent are Prader-Willi syndrome, Angelman syndrome and 15q11-q13 microduplication syndrome. In this work, we analyzed DNA samples from 181 patients with phenotypes which were compatible with the above-mentioned diseases, using Methyl specific-multiplex ligation-dependent probe amplification (MS-MLPA). We show that, of the 181 samples, 39 presented alterations detectable by MS-MLPA. Of those alterations, 61.5% (24/39) were deletions, 5.1% (2/39) duplications and 33.3% (13/39) UPD/IE. The CNV cases were 4 times more frequent than UPD/IE (OR= 4; IC 95%: 1.56-10.25), consistent with the literature. Among the CNVs, two atypical cases allow to postulate new possible BP sites that have not been reported previously in the literature.

Del fenotipo neuropsiquiátrico al cuidado clínico del síndrome de Angelman: descripción de siete casos / Neuropsychiatrie phenotype of Angelman syndrome and clinical care: report of seven cases

El síndrome de Angelman es un trastorno neurogenético debido a la falta o reducción en la expresión del gen UBE3A en el cromosoma 15, el cual codifica la proteína ubiquitina ligasa E3A, que tiene un papel integral en el desarrollo sinóptico y la plasticidad neuronal. Se manifiesta por retraso en el neurodesarrollo o discapacidad intelectual, comportamiento característico y epilepsia. Se describen las características clínicas de siete pacientes con deleción del cromosoma 15q11-13 y su manejo integral. Por la expectativa de vida, es importante conocer y manejar las comorbilidades de forma interdisciplinaria para lograr mejorar la calidad de vida de los afectados. Se realiza una revisión de la literatura sobre la aproximación integral al diagnóstico y cuidado clínico a largo plazo de los pacientes con síndrome de Angelman.

Angelman syndrome is a neurogenetic disorder caused by a lack or reduction of expression of UBE3A located within chromosome 15, which codes for ubiquitin protein ligase E3A, which has a key role in synaptic development and neural plasticity. Its main features are developmental delay/intellectual disability, lack of speech, a characteristic behavioural profile, and epilepsy. We describe clinical features and management of seven cases with 15q11-13 deletion. Due to their life expectancy, knowing and managing its comorbidities is crucial to improve their quality of life. We review the diagnosis and long-term clinical care of patients with Angelman syndrome.

Molecular cytogenetic evaluation of chromosomal microdeletions: the experience of a public hospital in Southern Brazil

Introduction: During the past few decades, the number of diseases identified to be caused by chromosomal microdeletions has increased quickly, bringing a new and crucial role for cytogenetics on the diagnosis of these conditions. The purpose of this study was to identify and characterize chromosomal microdeletions associated with malformation syndromes and intellectual disability. Methods: We retrospectively evaluated a consecutive series of samples from a cohort of 598 subjects with clinical symptoms of a microdeletion syndrome, including the deletion of chromosomes 4p16.3, 5p15.2, 5q35, 7q11.23, 8q24.12, 15q11.2, 16p13.3, 17p13.3, 17p11.2,2, and 22q11.2, as investigated by fluorescence in situ hybridization (FISH). Array-based comparative genomic hybridization (array-CGH) was performed on 25 samples with microdeletions. Results: A total of 598 samples were evaluated from patients whose clinical phenotypes were most indicative of 22q11.2 deletion syndrome (29.10%), Prader-Willi syndrome (23.41%), Angelman syndrome (16.89%), and Williams-Beuren syndrome (14.72%). In 142 of the samples (23.75%), a chromosomal imbalance associated with phenotypic abnormalities was found. The deletion of 7q11.23 was the most frequent (8.03%), followed by del22q11.2 (5.68%) and del15q11.2 (5%). Conclusion: Our study reinforces the idea that the effort to improve the capacity to perform molecular cytogenetic investigations associated with a qualified clinical evaluation is crucial for the detection and precise characterization of submicroscopic chromosome deletions, bringing benefits to patients, relatives, and genetic counselors. It also contributes to the continuing education of cytogeneticists and to the knowledge of chromosomal rearrangements associated with genomic disorders.

case report of a 2.5-year-old girl with angelman syndrome [AS]

Angelman Syndrome [AS] is a genetically determined syndrome that has a unique behavioral phenotype. This syndrome is described as jerky ataxia and an unusual happy facial expression with pathological laughter. Severe mental retardation is a unique feature of the syndrome, together with microbrachycephaly and abnormal electroencephalographic findings with or without clinical seizures. The patients cannot speak or at most, they have a vocabulary consisting only of a few words. The genetic abnormality of AS has been located on chromosome 15q11-q13. Patients with As mostly have deletions on the maternally derived allele [75-80%] while some of them show paternal uniparental disomy [approximetly sign 2%] or a rare imprinting mutation developmental disorder caused by deletion of the maternally-inherited chromosome 15q11-13. A 2.5-year-old girl is presented. Clinical suspicion of AS was raised at the age of 27 months when she presented with mental retardation and epilepsy, absence of speech, inability to gait and paroxysmal episodes of laughter. Moreover, she had facial dysmorphic features such as microbrachycephaly, mid-facial hypoplasia, macrostomia and a prominent mandible. Chromosomal analysis revealed 46 xx with the deletion of 15q chromosome [15q11q13-snrpn/ic] Our patient met the classical phenotype and genotype of AS

Novel deletion of the E3A ubiquitin protein ligase gene detected by multiplex ligation-dependent probe amplification in a patient with Angelman syndrome

Angelman syndrome (AS) is a severe neurobehavioural disorder caused by failure of expression of the maternal copy of the imprinted domain located on 15q11-q13. There are different mechanisms leading to AS: maternal microdeletion, uniparental disomy, defects in a putative imprinting centre, mutations of the E3 ubiquitin protein ligase (UBE3A) gene. However, some of suspected cases of AS are still scored negative to all the latter mutations. Recently, it has been shown that a proportion of negative cases bear large deletions overlapping one or more exons of the UBE3A gene. These deletions are difficult to detect by conventional gene-scanning methods due to the masking effect by the non-deleted allele. In this study, we have used for the first time multiplex ligation-dependent probe amplification (MLPA) and comparative multiplex dosage analysis (CMDA) to search for large deletions affecting the UBE3A gene. Using this approach, we identified a novel causative deletion involving exon 8 in an affected sibling. Based on our results, we propose the use of MLPA as a fast, accurate and inexpensive test to detect large deletions in the UBE3A gene in a small but significant percentage of AS patients.

Síndrome de Angelman: diagnóstico precoz / Angelman syndrome: early diagnosis

Se describen dos niños con Síndrome de Angelman, diagnosticados antes de los dos años de edad. La sospecha clínica basada en la presencia de retraso global del desarrollo, ausencia de lenguaje, temblores y/o movimientos anormales y risa fácil, permitió plantear el diagnóstico y solicitar el estudio genético confirmatorio. El diagnóstico precoz es importante ya que evita exámenes innecesarios, permite informar a los padres de su evolución a futuro, dar asesoría genética y un tratamiento apropiado al paciente.

A further case of a Prader-Willi syndrome phenotype in a patient with Angelman syndrome molecular defect

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are distinct human neurogenetic disorders; however, a clinical overlap between AS and PWS has been identified. We report on a further case of a patient showing the PWS phenotype with the AS molecular defect. Despite the PWS phenotype, the DNA methylation analysis of SNRPN revealed an AS pattern. Cytogenetic and FISH analysis showed normal chromosomes 15 and microsatellite analysis showed heterozygous loci inside and outside the 15q11-13 region. The presence of these atypical cases could be more frequent than previously expected and we reinforce that the DNA methylation analysis is important for the correct diagnosis of severe mental deficiency, congenital hypotonia and obesity

Diagnosis of patients with Prader-Willi and Angelman Syndromes: the importance of an overall investigation

Seventy-two patients with clinical diagnoses of Prader-Willi (PWS; n = 28 patients) or Angelman syndromes (AS; n = 44 patients) were submitted to chromosome analysis, SNRPN-SNURF exon 1 methylation assay, and microsatellite genotyping. Analysis of the methylation pattern confirmed the PWS diagnosis in 18 out of 28 patients and the AS diagnosis in 20 out of 44 patients. FISH and microsatellite analysis detected a deletion in 30 patients (14 PWS and 16 AS). Eight patients had normal FISH results (4 PWS and 4 AS); microsatellite markers showed that these patients had a uniparental disomy (UPD). Based on this study, we propose a strategy for the routine diagnosis of these syndromes that consists of the following steps: 1) methylation analysis, which does not require parental samples; 2) microsatellite genotyping of patient and parents to differentiate deletions, UPD and imprinting mutations; and 3) FISH for otherwise uninformative cases, and whenever parental samples are not available. Of the 34 patients whose PWS or AS diagnoses were not confirmed by laboratory tests, five presented a small extra marker chromosome, identified in three of them as an inv dup(15). One AS patient carried a balanced t(15;15) translocation associated with paternal UPD. Therefore G-banded chromosome analysis should be performed on all such patients, to detect possible structural rearrangements

Genomic imprinting and human chromosome 15

Genomic imprinting is a reversible phenomenon that affects the expression of genes depending on their parental origin. The best characterized human disorders resulting from an alteration of the imprinting process are Angelman and Prader-Willi syndromes. They are due to the lack of active maternal or paternal genes, respectively, from chromosome region 15q11q13. Most cases arise via interstitial deletions. We review evidence that other common cytogenetic alterations of this region, interstitial and supernumerary duplications, could be the reciprocal products of the deletions and are also affected by the imprinting phenomenon, given the predominance of maternally-derived duplications in patients ascertained due to developmental delays or autistic features.

Genomic imprinting: genetic mechanisms and phenotypic consequences in Prader-Willi and Angelman syndromes

O segmento cromossômico 15q11-q13 é de grande interesse em Genética Humana uma vez que diversos rearranjos estruturais têm sido descritos nessa regiäo (deleçöes, duplicaçöes e translocaçöes) resultando em fenótipos diferentes como os das síndromes de Prader-Willi (PWS) e Angelman (AS), que foram as primeiras doenças humanas a serem relacionadas com a expressäo diferencial de alelos parentais (imprinting genômico). Contrário às leis de Mendel onde a herança parental da informaçäo genética näo influencia a expressäo gênica, o imprinting genômico é caracterizado por modificaçöes no DNA que produzem diferentes fenótipos dependendo da origem parental da mutaçäo. A manifestaçäo clínica da PWS aparece quando ocorre a perda de genes com expressäo exclusivamente paterna, e a AS resulta da perda do gene com expressäo exclusivamente materna. Diferentes mecanismos genéticos podem resultar em PWS ou AS como deleçöes, dissomia uniparental ou mutaçöes no processo de imprinting. Em pacientes com AS existe uma classe adicional de pacientes com mutaçäo no gene UBE3A. Estudos de pacientes com PWS e AS podem ajudar no entendimento do processo de imprinting e, assim, outras regiöes do genoma com características similares podem ser localizadas, e diferentes síndromes podem ter seus mecanismos genéticos elucidados.

Síndrome de Angelman. Presentación de un caso clínico / Angelman syndrome. A clinical case

El síndrome de Angelman (SA) consiste en un conjunto de alteraciones neurológicas que incluyen trastornos en el aprendizaje y el lenguaje, presentando hiperactividad y retraso mental. El comportamiento de los pacientes afectados es característico, ríen con facilidad y en ocasiones sin sentido, ya que su risa no se asocia con episodios de alergría. Son sociables y habitualmente parecen contentos. Su etiología es una de las formas no clásicas de herancia, conocida como disomía uniparenteral; a pesar de ser relativamente frecuente, pocas veces se diagnostica. La descripción clínica de esta enfermedad podrá servir para identificar a un mayor número de afectados, presentamos el caso clínico de un paciente con síndrome de Angelman y recomendamos a un grupo de contacto que existe en nuestro país

Clinical-neurologic, cytogenetic and molecular aspects of the Prader-Willi and Angelman syndromes

The Prader-Willi syndrome (PWS) and the Angelman syndrome (AS) are human neurogenetic disorders involving the imprinting mechanism, at the 15q11-13 chromosome region. The predominant genetic defects in PW are 15q11-13 deletions of paternal origin and maternal chromosome 15 uniparental disomy. In contrast, maternal deletions and paternal chromosome 15 uniparental disomy are associated with a different neurogenetic disorder, the AS. In both disorders, these mutations are associated with parent-of-origin specific methylation at several 15q11-13 loci. We studied 5 patients suspect of PWS and 4 patients suspect of AS who were referred to the Medical Genetics Unit at the Universsity Hospital of Medical School from Ribeirao Preto. Our objective was to establish the correct clinical and etiological diagnosis in these cases. We used conventional cytogenetics, methylation analysis with the probe KB17 (CpG island of the SNRPN gene) by Southern blotting after digestion with the Xba I and Not I restriction enzymes. We studied in patients and their parents the segregation of the (CA)(n) repeats polymorphisms by PCR, using the primers 196 and IR4-3R. All the patients had normal conventional cytogenetical analysis. We confirmed 3 cases of PWS: one by de novo deletion, one by maternal chromosome 15 uniparental disomy and one case with no defined cause determined by the used primers. We confirmed 2 cases of AS, caused by de novo deletion at the 15q11-13 region, and one case with normal molecular analysis but with strong clinical characteristics.

Síndrome de Angelman: causa frequentemente näo reconhecida de deficiência mental e epilepsia: relato de caso / Angelman syndrome: a frequently undiagnosed cause of mental retardation and epilepsy. Case report

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

Genomic imprinting relevant to genetic diseases.

Genomic imprinting is a new concept proposed to explain unusual observations in early mammalian development, the occurrence of certain genetic diseases, genetic anticipation or incomplete penetrance, and tumorigenesis. The basic mechanism of the imprinting has remained obscure, although DNA-methylation, chromatin structure, and/or DNA replication may have a role. Genomic imprinting is a biological phenomenon determined by an evolutionally acquired, underlying system that may control harmonious development and growth in mammals. It is also relevant to the occurrence of some genetic disorders in man.

Estudo clínico, citogenético e molecular nas síndromes de Prader-Willi e Angelman / Clinical, cytogenetical and molecular studies in Prader-Willi and Angelman syndromes

A Síndrome de Prader-Willi (SPW) e a Síndrome de Angelman (SA) säo doenças neurogenéticas consideradas como exemplos do fenômeno de impressäo genômica, em seres humano, estando relacionadas com alteraçöes envolvendo a regiäo cromossômica 15q11-13. As alteraçöes genéticas predominantes, na SPW, säo deleçöes na regiäo 15q11-13, de origem paterna e dissomia uniparental materna e, na SA, encontram-se deleçöes na regiäo 15q11-13 materna e dissomia uniparental paterna. Estudamos 5 pacientes com suspeita clínica de SPW e 4 pacientes com suspeita clínica de SA, atendidos no Setor de Genética Médica do Hospital das Clínicas da FMRP-USP, com o objetivo de estabelecer o diagnóstico clínico e etiológico nessa amostra. Para isso, utilizamos citogenética convencional, estudo de metilaçäo por Southern blotting com a sonda KB17 (ilha CpG do gene SNRPN), após digestäo com as enzimas de restriçäo Xba I e Not I, e análise de polimorfismos de repetiçäo (CA)n por PCR, usando os primers 196 e IR4-3R. Dos 9 pacientes avaliados, todos tiveram avaliaçäo citogenética convencional normal. Foram confirmados molecularmente 1 caso de SPW por deleçäo nova, 1 caso de SPW por dissomia uniparenteal materna e 1 caso de SPW em que a causa genética näo pode ser esclarecida pela análise de polimorfismo com os primers usados. Foram confirmados, molecularmente, 2 casos de SA, ambos por deleçäo nova na regiäo 15q11-13 e, 1 caso de SA, cuja clínica é extremamente sugestiva, teve resultado molecular normal, podendo-se sugerir uma mutaçäo de ponto no gene responsável pela SA.