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1031-1034delTAAC (Leu125Stop): a novel familial UBE3A mutation causing Angelman syndrome in two siblings showing distinct phenotypes
© De Molfetta et al.; licensee BioMed Central Ltd. 2012
Received: 19 December 2011
Accepted: 13 December 2012
Published: 20 December 2012
More than 50 mutations in the UBE3A gene (E6-AP ubiquitin protein ligase gene) have been found in Angelman syndrome patients with no deletion, no uniparental disomy, and no imprinting defect.
We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows a milder phenotype presenting the typical AS features.
We hypothesized that the mutation Leu125Stop causes both severe and milder phenotypes. Potential mechanisms include: i) maybe the proband has an additional problem (genetic or environmental) besides the UBE3A mutation; ii) since the two siblings have different fathers, the UBE3A mutation is interacting with a different genetic variant in the proband that, by itself, does not cause problems but in combination with the UBE3A mutation causes the severe phenotype; iii) this UBE3A mutation alone can cause either typical AS or the severe clinical picture seen in the proband.
Angelman syndrome (AS) is a neuro-genetic disorder characterized by intellectual and developmental delay, sleep disturbance, seizures, jerky movements, frequent laughter or smiling and a happy disposition . The incidence of AS is estimated to be between 1/10,000 and 1/20,000  and is inherited in an autosomic dominant trait, modified by imprinting, or inherited by imprinting . Analysis of parent-specific DNA methylation pattern in the 15q11-13 chromosome region detects approximately 77% of individuals with AS, including those cases with a deletion (approximately 70%), uniparental disomy (1-2%), or an imprinting defect (3-5%); fewer than 1% of individuals have a cytogenetically chromosome rearrangement and UBE3A sequencing detects mutations in approximately 5-10% of the patients . In 10-15% of the cases the molecular exam is normal with no deletions, uniparental disomy, imprinting defects or UBE3A mutations . Recently, it was demonstrated that the Angelman syndrome protein Ube3A is a neuronal activity-regulated protein that controls synaptic function by ubiquitinating and degrading the synaptic protein Arc. In the absence of Ube3A, elevated levels of Arc accumulate in neurons resulting in the excessive internalization of AMPA receptors at synapses and impaired synaptic function . We report a brother and sister who was referred to our laboratory in order to investigate a clinical suspicion of AS. As the analysis of the differential parental specific DNA methylation within the 15q11-13 region was normal, we investigated the UBE3A gene in order to screen for mutations causing AS. We here describe a novel UBE3A frameshift mutation in two siblings who have inherited it from their asymptomatic mother. Despite carrying the same UBE3A mutation, the proband shows a more severe phenotype whereas his sister shows the typical AS features associated to a milder phenotype.
The genomic DNA was extracted from leukocytes from peripheral blood samples, using Super Quick-gene-rapid DNA isolation (Promega), following the manufacturer’s instructions.
Methylation analysis using Methylation Sensitive High Resolution Melting (MS-HRM) assay
For methylation analysis of the SNRPN gene, DNA conversion using bisulfite and cleanup of 2 μg of genomic DNA was carried out using EpiTect Bisulfite Kit (Qiagen), following the manufacturer’s instructions. Afterwards the DNA conversion, each sample was run in duplicate by the MS-HRM assay to determine the methylation pattern within the 15q11-13 region using primers and protocol described by White et al. (2007) . The MS-HRM analysis was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) using MeltDoctor HRM Master Mix (Applied Biosystems) according manufacturer’s instructions. The PCR reaction conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 58°C for 1 minute and 95°C for 1 minute, followed by the standard melting curve.
PCR and mutation screening using High Resolution Melting (HRM) assay
In order to screen for UBE3A mutations, we used the primers described by Malzac et al. (1998)  and the HRM technique. The HRM analysis was performed in a 7500 Fast Real-Time PCR System (Applied Biosystems) using MeltDoctor HRM Master Mix (Applied Biosystems) according manufacturer’s instructions. Each sample was run in duplicate and approximately 20 ng of genomic DNA was amplified in a total volume of 20 μl containing 5 μM of each primer, deionizated water, and 10μl of MeltDoctor. master mix (Applied Biosystems). The PCR reaction conditions were 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds, 55°C for 1 minute and 95°C for 1 minute, followed by the standard melting curve.
Cloning and DNA sequencing
DNA fragments amplified by PCR were subjected to direct sequencing in an automatic capillary sequencing system ABI 3130 Genetic Analyser using BigDyeR Terminator v3.1 Cycle Sequencing kit, following the manufacturer’s instructions. The sequencing results were analyzed through FinchTV version 1.4.0 (Geospiza Inc. 2004–2006); the sequences obtained were compared with reference from the GenBank (NM_130.839.1). In order to proper characterize the mutation, the same DNA fragments amplified by PCR were cloned into TOPOR vector using TOPO TA cloning (Invitrogen) according to manufacturer’s instructions.
In addition, as shown in Figure 3A, we observed three differential methylation patterns. This result may be associated to the phenotypic variability observed between patients, due to the differential methylation pattern along the SNRPN analyzed region containing 21 CpG dinucleotides. To test this hypothesis we performed the bisulfite sequencing analysis of the patients, their mother and a normal control (Additional file 1). All the samples showed the same heterozygous CpG methylation pattern as expected in cases of AS patients with biparental inheritance (Additional file 1). We also observed that all cytosine in CpG dinucleotides were heterozygous (T/C) with a small T peak height compared to the C peak height. The bisulfite conversion was efficient since all the isolated cytosine was fully converted into thymine. No mutation was observed in the same region. The imbalance observed between the methylated allele (observed as the C peak in the chromatogram) and the non-methylated allele (observed as the T peak in the chromatogram) is due to the peripheral blood cell heterogeneity where CpG methylation status is differentially maintained in the PWS/AS region [18, 19].
It is worthy to discuss that this is the first paper using HRM to search for mutations within the UBE3A gene. HRM is a relatively new technique, which is being widely used as a method for screening for mutations . Therefore, it provides very simple solutions for genotyping as this technique combines high sensitivity and specificity making it useful for personalized DNA diagnostics.
Written informed consent was obtained from the patient’s mother for publication of this Case Report and any accompanying images. A copy of the written consent is available for review by the Series Editor of this journal.
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