The SALSA MS-MLPA
Probemix ME028 Prader-Willi/Angelman is a research use only (RUO)
assay for the detection of aberrant methylation of one or more sequences of the 15q11 chromosomal region. This probemix can also be used to detect deletions/duplications in the aforementioned chromosomal region.
Genomic imprinting is the monoallelic expression of genes, dependent on the parental origin of the chromosome. It plays a role in growth and development. 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. The 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.
Paternally expressed genes in the 15q11 PWS/AS region are: MKRN3
and the snoRNA cluster; UBE3A
is only 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, these deletions will only be of interest when they are absent in parental samples. Additional probes for the 15q11 region are present in the P343 Autism-1 and the P336 UBE3A probemixes.
Additionally, maternal duplications of the Prader-Willi/Angelman 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 BP1-BP2 (NIPA1
) and u1B-u1B* (SNRPN
exons 1 and 2) regions 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 P211 HSP region probemix.
More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1330/
(AS), and https://www.ncbi.nlm.nih.gov/books/NBK367946/
(15q11 Duplication Syndrome).
The SALSA MS-MLPA Probemix ME028-C1 Prader-Willi/Angelman contains 47 (MS−)MLPA probes with amplification products between 129 and 481 nt. Six MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of the 15q11 chromosomal region. All probes will also give information on copy number changes in the analysed sample. In addition, 11 reference probes are included which are not affected by HhaI digestion and detect genes located outside the 15q11 region. Also, two digestion control probes are included in this probemix that indicate 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 is available online (www.mlpa.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.mlpa.com