UBE3A Mutation Research Articles

Genotype-phenotype correlation over time in Angelman syndrome: Researching 134 patients

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function of maternal UBE3A. The major cause of AS is a maternal deletion in 15q11.2-q13 and the minor causes are a UBE3A mutation, uniparental disomy (UPD) and imprinting defect (ID). Previous reports suggest that all patients with AS exhibit developmental delay, movement or balance disorders, behavioral characteristics and speech impairment. In contrast, a substantial number of AS patients with a UBE3A mutation, UPD or ID were reported not to show these consistent features and to show age dependent change of their features. In this study, we investigated 134 patients with AS, including 57 patients with a UBE3A mutation and 48 patients with UPD or ID. Although developmental delay was present in all patients, 20% of patients with AS caused by UPD or ID did not exhibit movement or balance disorders. Differences were also seen in hypopigmentation and seizures, depending on the causes. Moreover, patients with a UBE3A mutation, UPD or ID tended to show fewer of the specific phenotypes depending on their age. Especially, in patients with UPD or ID, easily-provoked laughter and hyperactivity tended to become more pronounced as they aged. Therefore, the clinical features of AS based on cause and age should be understood and genetic testing should not be limited to patients with the typical clinical features of AS.

A case of an Angelman-syndrome caused by an intragenic duplication of UBE3A uncovered by adaptive nanopore sequencing

Adaptive nanopore sequencing as a diagnostic method for imprinting disorders and episignature analysis revealed an intragenic duplication of Exon 6 and 7 in UBE3A (NM_000462.5) in a patient with relatively mild Angelman-like syndrome. In an all-in-one nanopore sequencing analysis DNA hypomethylation of the SNURF:TSS-DMR, known contributing deletions on the maternal allele and point mutations in UBE3A could be ruled out as disease drivers. In contrast, breakpoints and orientation of the tandem duplication could clearly be defined. Segregation analysis in the family showed that the duplication derived de novo in the maternal grandfather. Our study shows the benefits of an all-in-one nanopore sequencing approach for the diagnostics of Angelman syndrome and other imprinting disorders.

Evaluation of an In-House Genetic Testing Method for Confirming Prader-Willi and Angelman Syndromes in Sri Lanka.

Prader-Willi syndrome (PWS, MIM 176,270) and Angelman syndrome (AS, MIM 105,830) are caused by imprinting defects of chromosome 15q11-13, with loss of maternal gene expression causing AS and paternal gene expression causing PWS. The diagnosis, once established in most cases by using a methylation-specific PCR test, enables appropriate therapeutic interventions and avoids the need for further investigations. Genetic testing for PWS/AS is limited in Sri Lanka (and in other low- and middle-income countries), mainly because parents are unable to pay for testing as these are not funded by the health service. Ninety cases (46 female) with clinical features suggesting PWS (n = 37) and AS (n = 53), referred by a pediatric endocrinologist and a pediatric neurologist, were recruited. Clinical information and blood samples were obtained following informed consent. DNA was extracted and methylation-specific PCR (MS-PCR) was performed following bisulfite modification of DNA by using an in-house method and a kit. Results were validated using known positive controls. Parent-child trio DNA samples were used in cases with confirmed PWS and AS to determine if the disease was due to a deletion or uniparental disomy. The cost of the MS-PCR testing of the two modification methods and the microsatellite analysis was determined. Among the suspected PWS cases, 19/37 were positive, while 5/53 of the suspected AS cases were positive. The lower identification rate of AS is probably related to the overlap of clinical features of this condition with other disorders. The kit-based modification method was more reliable, less time-consuming, and cost-effective in our laboratory. The kit-based modification followed by MS-PCR described in this study enables more affordable genetic testing of suspected PWS/AS cases, and this is likely to improve patient care by targeting appropriate therapy for the affected cases. Parental genetic counselling is made possible regarding the low recurrence risk, especially where a deletion or uniparental disomy is confirmed. In MS-PCR, negative cases with a strong clinical suspicion of AS, UBE3A mutation testing is required. In addition, imprinting center mutation/deletion testing may also be needed in strongly clinically suspected, MS-PCR negative PWS and AS cases.

Genetic investigation of the ubiquitin-protein ligase E3A gene as putative target in Angelman syndrome.

Angelman syndrome (AS) is caused by maternal chromosomal deletions, imprinting defects, paternal uniparental disomy involving chromosome 15 and the ubiquitin-protein ligase UBE3A gene mutations. However the genetic basis remains unclear for several patients. To investigate the involvement of UBE3A gene in AS and identifying new potential genes using exome sequencing. We established a cohort study in 50 patients referred to Farhat Hached University Hospital between 2006 and 2021, with a strong suspicion of AS and absence of chromosomal aberrations. The UBE3A gene was screened for mutation detection. Two unrelated patients issued from consanguineous families were subjected to exome analysis. We describe seven UBE3A variants among them 3 none previously described including intronic variants c.2220+14T>C (intron14), c.2507+43T>A (Exon15) and insertion in Exon7: c.30-47_30-46. The exome sequencing revealed 22 potential genes that could be involved in AS-like syndromes that should be investigated further. Screening for UBE3A mutations in AS patients has been proven to be useful to confirm the diagnosis. Our exome findings could rise to new potential alternative target genes for genetic counseling.

Clinical Exome Sequencing in Patients with Undifferentiated General Developmental Delay and Intellectual Disabilities

Introduction. Each fifth neurodevelopmental disorder is diagnosed in massively parallel sequencing only.
 Objective: to present the experience of exome sequencing in children with undifferentiated general developmental delay and intellectual disabilities.
 Materials and methods. We assessed 33 patients (19 males and 14 females) at the age of 4.5 2.4 years with general developmental delay and intellectual disabilities. We studied patients' medical and family histories and their neurological statuses as well as the findings of neuropsychological testing and clinical exome sequencing.
 Results.The effectiveness of clinical exome sequencing in the sample was 39.4% (MECP2, WDR45, SYNJ1, ADAR, PMM2, SHANK3, KMT5B, UBE3A, PTPN11, CTNNB1, and MTOR mutations). The pathogenic variants were significantly more prevalent in the patients with motor development delay, 53.8% of patients (P .05). Most incident conditions included insomnias (46.2%), autism spectrum disorders (38.5%), developmental regression (38.5%), and epilepsy (38.5%). Genetic disorders were more common in the female patients .
 Conclusion. With the study results, we can suppose that ca. 40% of patients with undifferentiated general developmental delay and intellectual disabilities had genetic disorders and, therefore, needed further evaluation with molecular genetic testing.

A review on Angelman syndrome

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Targeting immune cell types of tumor microenvironment to overcome resistance to PD-1/PD-L1 blockade in lung cancer.

Lung cancer is the common malignant tumor with the highest mortality rate. Lung cancer patients have achieved benefits from immunotherapy, including immune checkpoint inhibitors (ICIs) therapy. Unfortunately, cancer patients acquire adaptive immune resistance, leading to poor prognosis. Tumor microenvironment (TME) has been demonstrated to play a critical role in participating in acquired adaptive immune resistance. TME is associated with molecular heterogeneity of immunotherapy efficacy in lung cancer. In this article, we discuss how immune cell types of TME are correlated with immunotherapy in lung cancer. Moreover, we describe the efficacy of immunotherapy in driven gene mutations in lung cancer, including KRAS, TP53, EGFR, ALK, ROS1, KEAP1, ZFHX3, PTCH1, PAK7, UBE3A, TNF-α, NOTCH, LRP1B, FBXW7, and STK11. We also emphasize that modulation of immune cell types of TME could be a promising strategy for improving adaptive immune resistance in lung cancer.

An Analysis of Phenotype and Genotype in a Large Cohort of Chinese Children with Angelman Syndrome.

Angelman syndrome (AS) is a neurodevelopmental genetic disorder, but there has been limited analysis of a large cohort of Chinese children with Angelman syndrome. This study aims to assess the phenotype and genotype of Chinese children with Angelman syndrome. We retrospectively analyzed data through a detailed online survey combined with an on-site study. Furthermore, phenotype analysis stratified by deletion and non-deletion groups was carried out. The responses of family members of 695 individuals with AS revealed that 577 patients (83.02%) had maternal deletions, 65 patients (9.35%) carried UBE3A mutations, 31 (4.46%) patients had UPD15pat (one patient with UPD15pat constituted by a mosaic), 10 patients (1.44%) had imprinting defects and 12 (1.58%) patients only showed abnormal methylation without further detection. We identified 50 different pathogenic variants in this cohort, although 18 of these variants were unreported. Recurrent variant c.2507_2510del (p.K836Rfs*4) was found in 7 patients. In the deletion group, patients were diagnosed at an earlier age, had a more severe clinical phenotype, a higher rate of epilepsy with more multiple seizure types, and more frequently combined medication. Strabismus and sleep disturbances were both common in deletion and non-deletion groups. The top three resources invested in caring for AS children are: daily involvement in patient care, rehabilitation cost, and anti-epileptic treatment. Our study showed the genetic composition of Chinese children with 83.02% of maternal deletions, and the mutation spectrum for UBE3A variants was expanded. Developmental outcomes are associated with genotype, and this was confirmed by deletion patients having a worse clinical phenotype and complex epilepsy.

Generation of an induced pluripotent stem cell line from a patient with Angelman syndrome carrying UBE3A mutation

Angelman syndrome (AS) is a neurodevelopmental disorder caused by abnormal expression or function defects of the UBE3A gene in the maternal chromosome region 15q11-13. In order to study the pathogenesis of Angelman syndrome and further search for its effective treatment, we established a human induced pluripotent stem cells (iPSCs) from an AS patient carrying the mutation p.Asp563Gly of UBE3A gene at maternal 15q11.2-q13. The established patient-derived iPSC showed normal karyotype, expressed pluripotency markers, and had the capacity to differentiate into three germ layers.

Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

The mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

Investigation of age-related facial variation among Angelman syndrome patients

Angelman syndrome (AS) is one of the common genetic disorders that could emerge either from a 15q11–q13 deletion or paternal uniparental disomy (UPD) or imprinting or UBE3A mutations. AS comes with various behavioral and phenotypic variability, but the acquisition of subjects for experiment and automating the landmarking process to characterize facial morphology for Angelman syndrome variation investigation are common challenges. By automatically detecting and annotating subject faces, we collected 83 landmarks and 10 anthropometric linear distances were measured from 17 selected anatomical landmarks to account for shape variability. Statistical analyses were performed on the extracted data to investigate facial variation in each age group. There is a correspondence in the results achieved by relative warp (RW) of the principal component (PC) and the thin-plate spline (TPS) interpolation. The group is highly discriminated and the pattern of shape variability is higher in children than other groups when judged by the anthropometric measurement and principal component.

Identification of Novel UBE3A Mutation Causing Angelman Syndrome

The Angelman Syndrome (AS) is neurodevelopmental disease associated with maternal disruption of the UBE3A gene and is mainly characterized by global developmental delay, sever mental retardation with absence of speech, seizures, dysmorphic facial features, and distinct behavioral profile. In this study a pedigree with one affected member with neurodevelopmental disease who was a result of an unconsanguineous marriage were investigated by Whole Exome Sequencing (WES). DNA was extracted from whole blood and library was prepared using Agilent V6 capturing system. WES was performed on Illumina HiSeq 4000 platform. Genome Analysis Toolkit (GATK) was used for variant calling. Classification of selected variants was done based on ACMG guideline for variant interpretation 2015. WES revealed that the proband has previously unreported nonsense variant (c.2459T>G) in UBE3A gene that causes the substitution of Leu (TTA) with stop codon (TGA), confirming the diagnosis of Angelman syndrome. The patient had delayed motor development, speech impairment, an attention deficit, and an abnormal electroencephalogram (EEG), but no seizures by the age of 2 years. This study emphasis the role of WES in the early diagnosis and better management for AS patient.

Refining the Behavioral Phenotype of Angelman Syndrome: Examining Differences in Motivation for Social Contact Between Genetic Subgroups.

Angelman syndrome (AS) is caused by loss of information from the 15q11.2-13 region on the maternal chromosome with striking phenotypic difference from Prader–Willi syndrome in which information is lost from the same region on the paternal chromosome. Motivation for social contact and sensory seeking behaviors are often noted as characteristics of the phenotype of AS and it has been argued that the strong drive for social contact supports a kinship theory interpretation of genomic imprinting. In this study we developed an experimental paradigm for quantifying the motivation for social contact in AS and examined differences across the genetic subtypes that cause AS [deletion, imprinting centre defect (ICD), uniparental disomy and UBE3A mutation]. Using single case experimental designs we examined the rate of acquisition of behavioral responses using operant learning paradigms for 21 children with AS whilst systematically varying the nature of social and sensory reinforcement. Variability in rates of acquisition was influenced by the nature of rewarding stimuli. Across the total sample both sensory stimuli and social contact could increase the rate of rewarded behavior with difference between children in the most effective reward. A striking difference in the rewarding properties of social contact across genetic subtypes was evidenced by non-deletion genetic causes of AS showing significantly higher rates of responding than the deletion cause in the social reinforcement paradigm. The results indicate that reinforcer assessment can beneficially inform behavioral interventions and that within syndrome variability in the behavioral phenotype of AS is likely driven by genetic difference. The non-deletion cause of AS, and particularly the ICD group, may be the optimal group for further study of genomic imprinting.

An Angelman syndrome substitution in the HECT E3 ubiquitin ligase C-terminal Lobe of E6AP affects protein stability and activity.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.

Genetic interaction screen for severe neurodevelopmental disorders reveals a functional link between Ube3a and Mef2 in Drosophila melanogaster

Neurodevelopmental disorders (NDDs) are clinically and genetically extremely heterogeneous with shared phenotypes often associated with genes from the same networks. Mutations in TCF4, MEF2C, UBE3A, ZEB2 or ATRX cause phenotypically overlapping, syndromic forms of NDDs with severe intellectual disability, epilepsy and microcephaly. To characterize potential functional links between these genes/proteins, we screened for genetic interactions in Drosophila melanogaster. We induced ubiquitous or tissue specific knockdown or overexpression of each single orthologous gene (Da, Mef2, Ube3a, Zfh1, XNP) and in pairwise combinations. Subsequently, we assessed parameters such as lethality, wing and eye morphology, neuromuscular junction morphology, bang sensitivity and climbing behaviour in comparison between single and pairwise dosage manipulations. We found most stringent evidence for genetic interaction between Ube3a and Mef2 as simultaneous dosage manipulation in different tissues including glia, wing and eye resulted in multiple phenotype modifications. We subsequently found evidence for physical interaction between UBE3A and MEF2C also in human cells. Systematic pairwise assessment of the Drosophila orthologues of five genes implicated in clinically overlapping, severe NDDs and subsequent confirmation in a human cell line revealed interactions between UBE3A/Ube3a and MEF2C/Mef2, thus contributing to the characterization of the underlying molecular commonalities.

Neurodevelopmental profile of siblings with Angelman syndrome due to pathogenic UBE3A variants.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by a lack of expression of the maternally inherited UBE3A gene on chromosome 15. Individuals with AS due to a UBE3A mutation are more likely to have siblings who also have AS compared with those with AS due to other cytogenetic/molecular mechanisms, but it is unknown whether the developmental outcome of siblings who have AS is similar. Through an ongoing AS Natural History Study, we identified seven pairs of siblings with AS due to a UBE3A mutation. We compared the neurodevelopment of the first-born and second-born siblings with AS participants who have a UBE3A mutation and have either typically developing siblings or no siblings. Second-born AS participants due to a UBE3A mutation were more likely to be diagnosed at an earlier age. With the exception of higher expressive language scores among the second-born participants, no other differences were observed in the developmental and adaptive functioning skills across the different groups. The presence of an older sibling with the same neurodevelopmental disorder is associated with an earlier age of diagnosis and may be associated with an improvement in expressive language skills; the developmental outcome of siblings with AS due to a UBE3A mutation is otherwise comparable.

HAP1 is an in vivo UBE3A target that augments autophagy in a mouse model of Angelman syndrome

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal mutation and paternal imprinting of the gene encoding UBE3A, an E3 ubiquitin ligase. Although several potential target proteins of UBE3A have been reported, how these proteins regulate neuronal development remains unclear. We performed a large-scale quantitative proteomic analysis using stable-isotope labeling of amino acids in mammals (SILAM) in mice with maternal Ube3a mutation. We identified huntingtin (Htt)-associated protein (HAP1), a protein that is involved in Huntington's disease (HD), as a new target of UBE3A. We demonstrate that HAP1 regulates autophagy at the initiation stage by promoting PtdIns3K complex formation and enhancing its activity. HAP1 also co-localized with MAP1LC3 (LC3) and other proteins involved in autophagosome expansion. As a result, HAP1 increased autophagy flux. Strikingly, knocking down of HAP1 alleviated aberrant autophagy in primary neurons from AS mice. Concordantly, treatment of AS neurons with an autophagy inhibitor alleviated the reduction in density of dendritic spines. Furthermore, autophagy inhibition in AS mice partially alleviated a social interaction deficit as shown in open field test. Thus, our results identify HAP1 as an in vivo UBE3A target that contributes to deregulated autophagy and synaptic dysfunction in the central nervous system of AS mouse.

A behavioral test battery for mouse models of Angelman syndrome: a powerful tool for testing drugs and novel Ube3a mutants

BackgroundAngelman syndrome (AS) is a neurodevelopmental disorder caused by mutations affecting UBE3A function. AS is characterized by intellectual disability, impaired motor coordination, epilepsy, and behavioral abnormalities including autism spectrum disorder features. The development of treatments for AS heavily relies on the ability to test the efficacy of drugs in mouse models that show reliable, and preferably clinically relevant, phenotypes. We previously described a number of behavioral paradigms that assess phenotypes in the domains of motor performance, repetitive behavior, anxiety, and seizure susceptibility. Here, we set out to evaluate the robustness of these phenotypes when tested in a standardized test battery. We then used this behavioral test battery to assess the efficacy of minocycline and levodopa, which were recently tested in clinical trials of AS.MethodsWe combined data of eight independent experiments involving 111 Ube3a mice and 120 wild-type littermate control mice. Using a meta-analysis, we determined the statistical power of the subtests and the effect of putative confounding factors, such as the effect of sex and of animal weight on rotarod performance. We further assessed the robustness of these phenotypes by comparing Ube3a mutants in different genetic backgrounds and by comparing the behavioral phenotypes of independently derived Ube3a-mutant lines. In addition, we investigated if the test battery allowed re-testing the same animals, which would allow a within-subject testing design.ResultsWe find that the test battery is robust across different Ube3a-mutant lines, but confirm and extend earlier studies that several phenotypes are very sensitive to genetic background. We further found that the audiogenic seizure susceptibility phenotype is fully reversible upon pharmacological treatment and highly suitable for dose-finding studies. In agreement with the clinical trial results, we found that minocycline and levodopa treatment of Ube3a mice did not show any sign of improved performance in our test battery.ConclusionsOur study provides a useful tool for preclinical drug testing to identify treatments for Angelman syndrome. Since the phenotypes are observed in several independently derived Ube3a lines, the test battery can also be employed to investigate the effect of specific Ube3a mutations on these phenotypes.

F179. Notched delta, epilepsy and Angelman’s syndrome genetics

Introduction Angelman syndrome (AS) is a neurogenetic disorder resulting from a deletion of the maternal 15q11.2-13.1 region, mutation of maternal UBE3A gene, paternal uniparental disomy of chromosome 15, or methylation imprinting defect. Epilepsy affects the majority of the patients. Notched delta (ND) is characterized by ill-defined mixed spikes among a diffuse delta slowing, giving a “notched” appearance seen in AS. The aim of this study was to evaluate the prevalence of ND and epilepsy among the known genetic mechanisms. Methods Patients with AS seen at the Mayo Clinic Medical Genetics Clinics between September 2006 and September 2017 were identified. Medical records were reviewed for epilepsy history and EEG findings. The first 4 available EEG reports were reviewed for presence of ND. Patients were classified into 4 genetic groups: 1. microdeletion, 2.uniparental disomy (UPD), 3.UBE3A mutation, or 4.methylation defect. Patients without research consent (n = 1), inadequate epilepsy or EEG records (n = 3) were excluded. The age at seizure onset and the prevalence of ND was calculated in each genetic group. Fisher exact tests and nonparametric Wilcoxon rank sum test were used. Results Twenty-four patients were included (male 42%, median age 14.1 years, IQR 8.4–25.8). Genetic etiology was microdeletion 13, UPD 1, UBE3A mutation 4, and methylation defect 6. Epilepsy was present in 21/24 (88%) patients. There was no difference in the prevalence of epilepsy among the different genetic groups (microdeletion 12/13, UPD 1/1, UBE3A mutation 3/4, methylation defect 5/6, p > 0.05). Patients without seizure were younger than those with epilepsy (No seizure median age 7.4 years, IQR 2.6–8.4 vs Epilepsy 17.3 years, IQR 9.6–27.5, p = 0.03). Among epilepsy patients, the median age of seizure onset was 2 years, IQR 0.5–3.7. Patients with microdeletion have earlier age of seizure onset compared to the other groups: microdeletion, n = 12, median age of seizure onset 1.3 years, IQR 0.5–2.0; UPD, n = 1, age of seizure onset 3.0 years; UBE3A mutation, n = 3, median age of seizure onset 4.3 years, IQR 2.7–5.2; methylation defect, n = 5, median age of seizure onset, 3.8 years IQR 1.7–5.1; p = 0.03. ND was recognized in 15/24 (62%) patients at any time, including 8/13 chromosomal microdeletion, 1/1 UPD, 2/4 UBE3A mutation, 4/6 methylation defect. The median age at which ND was first recognized was 9.2 years, IQR 2.83–18.2. Prevalence of epilepsy was no different between patients with or without ND (13/15 patient ND and 8/9 patients without ND, p > 0.05). Conclusion Epilepsy was invariably present in the majority of the different genetic groups of AS patients, with earlier age of onset in the chromosome 15 microdeletion group. Patients with seizures were older than those without which may suggest an age related increased risk for developing epilepsy. ND was recognized in nearly two-thirds of all patients, with no specific distribution among the different genetic etiologies and did not influence the prevalence of epilepsy.

Clinical and genetic analysis of two unrelated patients with Angelman syndrome and novel UBE3A mutations

To explore the genetic cause for two familial Angelman syndrome cases and correlation between the clinical phenotypes and their genetic basis. Karyotyping analysis and microarray assay were carried out to exclude chromosome anomalies and uniparental disomy. The UBE3A gene was analyzed for potential point mutations, deletions, insertions and splice site mutations. Reverse transcription PCR was used to evaluate splicing mutation of the RNA transcripts. DNA sequencing showed the proband of family 1 has carried a novel maternal UBE3A splice acceptor site mutation, resulting in a guanine-to-cytosine transversion (IVS15-1G>C). Reverse transcription PCR revealed the proband and his mother both carried heterozygous mutant transcripts with loss of 54 and 59 nucleotides in exon 16, respectively. The proband displayed severe mental retardation, ataxia, seizures and inappropriate laughter. The siblings of family 2 has carried a novel maternal missense mutation in exon 16 of the UBE3A gene (c.2540C>T). She also presented with mental retardation, absent speech, mild ataxia and inappropriate laughter. The novel IVS15-1G>C and c.2540 C>T mutations of the UBE3A gene probably underlie the AS in the two families. Compared with small-scale mutations, larger fragments mutations can produce more severe phenotypes.