Angelman Syndrome by MS-MLPA

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe developmental delay, intellectual disability, speech impairment, seizures and characteristic behavior with an inappropriate happy demeanor with easily provoked laughter, short attention span, smiling and excitability. Individuals with AS plateau at a developmental level of 24 to 30 months, and cognitive performance usually shows severe functional impairment. Most children with AS have reduced need for sleep, and frequent awakening at night is common (Lossie et al. 2001). Fifty percent of the children develop microcephaly (Occipital Frontal Circumference < 2SD) by the age of 12 months. All children with AS have some component of hyperactivity with seemingly ceaseless activity. Language impairment is severe with most individuals lacking speech entirely, but a few develop single-word vocabularies. A large subset of children with AS qualify for a comorbid diagnosis of autism. A subset of AS patients also have hypopigmentation of the skin, hair and eyes, attributable to haploinsufficiency of OCA2 gene (due to maternal deletion) located close to UBE3A gene. The prevalence of AS is one in 12,000-20,000.

Differential diagnoses with several overlapping features with AS include Mowat-Wilson syndrome, X-linked Christianson syndrome, Rett syndrome, Pitt-Hopkins syndrome, 2q23.1 deletion syndrome, and Phelan-McDermid syndrome.

The AS and related Prader-Willi syndrome (PWS) region is localized to a 5-6 Mb genomic region on the proximal long arm of chromosome 15 (15q11.2-q13). This region contains several genes that are differentially expressed depending on whether the region is inherited from the father or the mother, i.e., some genes in this regions are expressed only from the paternal chromosome and some expressed only from the maternal chromosome. This differential gene expression is achieved by differential methylation (imprinting) patterns on the paternal and maternal chromosomes. The PWS paternally-only expressed region contains five protein coding genes (MKRN3, MAGEL2, NDN, NPAP1, and SNURF-SNRPN), one ORF (C15orf2), and a family of small nucleolar RNA (snoRNA) genes. The AS maternally-only expressed region contains one gene UBE3A. Deletion mapping in PWS/AS patients identified two small regions of deletion overlap (SRO) that define two critical imprinting centers (IC). PWS-SRO is a 4.3 kb region that lies at the 5’ end of the bicistronic SNURF-SNRPN, and has CpG islands encompassing the promoter, exon 1 and intron 1 of SNURF-SNRPN. AS-SRO is 880bp in size and maps 35 kb proximal to the SNURF-SNRPN exon 1.

AS is caused by absence of a functional UBE3A gene on the maternally inherited chromosome. Loss of maternally expressed genes at 15q11.2-13 can arise from several different genetic mechanisms. Approximately 65-75% of the AS patients have a recurrent deletion of the 15q11.2-13 on the maternally inherited chromosome, ~3-7% have a paternal uniparental disomy (UPD) of chromosome 15, 3% have imprinting defect (ID) i.e., mutations within the imprinting control region that establishes a paternal methylation pattern on the maternal chromosome (approximately 10-15% of the AS individuals with ID have a very small deletion in the AS IC), and 5-10% of AS patients have point mutations in the maternally inherited UBE3A gene (Williams et al. 2010; Dagli et al. 2012).

Around 10% of the patients with a clinical diagnosis of AS have no discernable abnormality at the 15q11.2-q13 region. Some of these patients include mosaic cases of imprinting defects, but a majority result from unidentified defects in UBE3A or mutations in other genes.

Alternatively, absence or loss of expression of paternally expressed genes at 15q11.2-13 lead to PWS.

Methylation analysis (Nygren et al. 2005) of the PWS/AS IC is by far the most efficient starting point for a genetic diagnosis of AS based on a clinical suspicion alone, as it can be used for all three classes of molecular defects (deletion, UPD and Imprinting defect). Additionally, methylation analysis differentiates between PWS and AS for patients with 15q11.2-q13 deletion without the need for the analysis of parental samples. Methylation analysis makes use of the differentially methylated maternal and paternal chromosome region at 15q11.2-q13 to identify maternal-only, paternal-only or bi-parental inheritance.

At PreventionGenetics we use commercially available methylation-specific Multiplex Ligation-dependent Probe Amplification (MS-MLPA) for the diagnosis of PWS and AS (Procter et al. 2006). This method combines both DNA methylation analysis and copy-number analysis across the entire PWS/AS region. MS-MLPA contains 32 dosage-sensitive probes (for copy number detection) specific for the 15q11.2 region, and five probes determine the DNA methylation status at differentially methylated sites in 15q11.2. Additionally, the dosage-sensitive probes cover SNORD116, one of the snoRNA gene clusters in the PWS/AS region (Ramsden et al. 2010).

The advantages of MS-MLPA method include;

1) Combined detection of both copy-number and methylation status.

2) Can differentiate between PWS caused by paternal deletion or UPD/Imprinting defect.

3) Detection of methylation status at five distinct differentially methylated regions (instead of one locus).

4) Detection of small deletions encompassing IC and SNORD116 gene cluster.

5) Detects more than 78% of the AS cases.

AS patients with normal methylation analysis of the 15q11.2-q13 region should reflex to UBE3A gene sequencing followed if necessary by UBE3A deletion/duplication analysis via aCGH.