Angelman/Prader Willi Syndrome by MS-MLPA

Angelman syndrome (AS) is a neurodevelopmental disorder characterized by severe developmental delay, intellectual disability, speech impairment, seizures, and characteristic behavior including 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 a reduced need for sleep, and frequent awakening at night is common (Lossie et al. 2001. PubMed ID: 11748306). Fifty percent of 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 has hypopigmentation of the skin, hair, and eyes, attributable to haploinsufficiency of OCA2 gene (due to maternal deletion) located close to the UBE3A gene. The prevalence of AS is one in 12,000-20,000.

Prader-Willi syndrome (PWS) is a multisystem disorder characterized by severe hypotonia, global developmental delay, feeding difficulties, and failure to thrive in the newborn period, followed by hyperphagia (excessive eating) and development of morbid obesity in late infancy and early childhood. In addition, delay in the development of motor milestones and language occurs, with all individuals showing some degree of cognitive impairment. Behavioral problems such as temper tantrums, stubbornness, manipulative behavior, and obsessive-compulsive features are common in early childhood, and psychosis is common in adolescents and adults (Gunay-Aygun et al. 2001. PubMed ID: 11694676). Older male and female patients manifest hypogonadism (genital hypoplasia, incomplete pubertal development, and infertility). Characteristic physical features include short stature, strabismus, almond-shaped palpebral fissures, narrow nasal bridge, thin upper lip with down-turned mouth, and scoliosis. Hypopigmentation of hair, eyes, and skin are seen in a third of the affected individuals. PWS has an estimated incidence of 1 in 15,000-25,000 live births.

The AS and related 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 region 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 SNURF-SNRPN exon 1. The CpG islands are unmethylated in the expressed chromosomes and methylated in the unexpressed chromosomes (Cassidy and Miller. 2012. PubMed ID: 22237428).

AS is caused by absence of a functional UBE3A gene on the maternally-inherited chromosome. Loss of maternally expressed genes at 15q11.2-q13 can arise from several different genetic mechanisms. Approximately 65-75% of the AS patients have a recurrent deletion of the 15q11.2-q13 on the maternally inherited chromosome, while ~3-7% have a paternal uniparental disomy (UPD) of chromosome 15. Three percent have an imprinting defect (ID) i.e., pathogenic variants 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). Five to ten percent of AS patients have point pathogenic variants in the maternally-inherited UBE3A gene (Williams et al. 2010. PubMed ID: 20445456; Dagli et al. 2012. PubMed ID: 22670133). Around 10% of the patients with a clinical diagnosis of AS have no discernable abnormality in 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 pathogenic variants in other genes.

PWS is caused by loss of function of five coding genes on the paternally inherited chromosome 15q11.2-q13. Loss of paternally-expressed genes at 15q11.2-q13 can arise from several different genetic mechanisms. Approximately 75-80% of the PWS patients have a recurrent deletion of the 15q11.2-q13 on the paternally-inherited chromosome, and ~20-25% have a maternal uniparental disomy (UPD) of chromosome 15. Less than 5% have an imprinting defect (ID) i.e., pathogenic variants within the imprinting control region that establishes a maternal methylation pattern on the paternal chromosome (approximately 10-15% of the PWS individuals with an ID have a very small deletion in the PWS IC), and < 1% have a structural chromosome abnormality (e.g., translocation) involving 15q11.2-q13.

Approximately 78% of AS cases and 99% of PWS cases will be detected by this assay. Because of simultaneous detection of both copy number and methylation status, MS-MLPA is able to differentiate between AS/PWS caused by maternal/paternal deletion and those caused by paternal/maternal UPD or ID. If no deletion is present, DNA polymorphism analysis will be necessary to differentiate between maternal/paternal UPD and ID. This method cannot detect AS caused by other molecular mechanisms, including UBE3A point pathogenic variants. MS-MLPA will pick up some IC deletions and deletions encompassing the SNORD116 gene cluster.

Methylation analysis (Nygren et al. 2005. PubMed ID: 16106041) 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 regions 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 AS and PWS (Procter et al. 2006. PubMed ID: 16690734). This method combines both DNA methylation analysis and copy-number analysis across the entire AS/PWS 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 AS/PWS region (Ramsden et al. 2010. PubMed ID: 20459762).The advantages of MS-MLPA method include:

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

2) Differentiation between AS/PWS caused by maternal/paternal deletion or UPD or 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) Detection of more than 78% of the AS cases and 99% of PWS 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.