Intended purpose
The SALSA MLPA Probemix ME028 Prader-Willi/Angelman is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of copy number variations and methylation status of the 15q11 region in genomic DNA isolated from human peripheral whole blood specimens, or in case of extracted DNA from prenatal samples, from (1) (un)cultured amniotic fluid obtained in week 16 of the pregnancy or later and free from blood contamination, (2) (un)cultured chorionic villi free from maternal contamination (copy number only), (3) fetal blood. ME028 Prader-Willi/Angelman is intended to confirm a potential cause for and clinical diagnosis of Prader-Willi syndrome (PWS), Angelman syndrome (AS), and 15q11 duplication syndrome. In rare cases, this product can also be used for carrier testing of at-risk family members.

Methylation changes and copy number variations (CNVs) found with multiple consecutive probes detected with ME028 Prader-Willi/Angelman should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. In the majority of patients, defects in the 15q11 region are copy number changes, but point mutations can occur which will not be detected by MLPA. For AS, it is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of the UBE3A gene.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, parental evaluation, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹ Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
² To be used in combination with a SALSA MLPA Reagent Kit, Coffalyser.Net analysis software and SALSA HhaI.

Clinical background
Genomic imprinting is the monoallelic expression of genes, dependent on the parental origin of the chromosome. It plays a role in growth and development. Differentially methylated regions (DMRs) act as imprinting control regions to regulate the expression of imprinted genes. Imprinting disorders like Prader-Willi syndrome (PWS) and Angelman syndrome (AS) originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al. 2013).

PWS and AS are distinct neurogenetic disorders, both usually caused by chromosomal deletions on chromosome 15q11 or by uniparental disomy (UPD). In UPD, both copies of a chromosome are inherited from a single parent. These 15q11 chromosomal alterations result in an aberrant expression profile of gene loci that are subject to imprinting. Absence of a paternal allele of chromosome 15q11, due to a chromosomal deletion of (part of) the paternal allele or the presence of two imprinted copies due to maternal UPD, results in PWS. The absence of the maternal copy of the same region or paternal UPD causes AS. Table 4 contains an overview of the expected copy number changes and methylation profiles in PWS/AS patients with deletions or aberrant methylation. Rare disorders with similar clinical features as PWS but different genetic etiology have been identified and are often referred to as PWS-like disorders. One of these PWS-like syndromes is caused by MAGEL2 truncating mutations and was renamed from PWS-like syndrome to Schaaf-Yang syndrome (OMIM #615547).

Paternally expressed genes in the 15q11 PWS/AS region are MKRN3, MAGEL2, NDN, SNRPN and the snoRNA cluster; UBE3A is maternally expressed. The PWS-AS Imprinting Centre (IC) (located upstream of the SNURF-SNRPN gene) contains both the PWS-SRO (smallest region of deletion overlap) and the AS-SRO. The AS-SRO is required for the PWS/AS region to have the maternal pattern of epigenetic modification and gene expression only if the chromosome has an intact PWS-SRO. The PWS-SRO is a 4.1 kb region that includes the SNRPN promoter. The PWS-SRO is unconditionally required for the PWS/AS region to have the paternal pattern of epigenetic modification and gene expression.

Finally, a rare cause of PWS is a small deletion within the SNORD116 cluster, downstream of SNRPN (Sahoo et al. 2008). However, this deletion will only be of interest when they are absent in parental samples. Additional probes for the 15q11 region are present in the SALSA MLPA Probemix P343 Autism-1 and the SALSA MLPA Probemix P336 UBE3A, please note that these are research use only (RUO) assays.

Additionally, maternal duplications of the PWS/AS critical region of the 15q11.2-q13.1 region cause the 15q11 duplication syndrome characterized by developmental delay, intellectual disability, hypotonia, and seizures. The extra copy is most commonly a maternal isodicentric 15q11.2-q13.1 supernumerary chromosome (80% of cases) or a maternal interstitial 15q11.2-q13.1 duplication (20% of cases).

The database of genomic variants mentions that copy number changes in the breakpoint region BP1-BP2 (NIPA1 and TUBGCP5) and u1B-u1B* (SNRPN exons 1 and 2) region have been found in healthy individuals (see http://dgv.tcag.ca/dgv/app/home). According to Stefansson et al. (2008), a deletion of the BP1-BP2 region is present in 0.19% of normal individuals and in 0.55% of schizophrenia patients. More probes for this BP1-BP2 region can be found in the SALSA MLPA Probemix P211 HSP region (RUO).

Update of the EMQN/ACGS best practice guidelines for molecular analysis of Prader-Willi and Angelman syndromes | European Journal of Human Genetics (nature.com)
https://www.ncbi.nlm.nih.gov/books/NBK1330/ (PWS), https://www.ncbi.nlm.nih.gov/books/NBK1144/ (AS), and https://www.ncbi.nlm.nih.gov/books/NBK367946/ (15q11 Duplication Syndrome).

Probemix content
The SALSA MLPA Probemix ME028-D1 Prader-Willi/Angelman contains 49 (MS-)MLPA probes with amplification products between 129 and 481 nucleotides (nt). 36 probes are target probes of which eight contain a HhaI recognition site and provide information on the methylation status of the 15q11 chromosomal region. All probes present will also give information on copy number changes in the analysed sample. In addition, 11 reference probes are included that are not affected by HhaI digestion and detect genes located outside the 15q11 region. Also, two digestion control probes are included in this probemix indicating whether or not restriction endonuclease digestion in the MS-MLPA reaction was complete. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MS-MLPA General Protocol and online at www.mrcholland.com.