General information
The SALSA MS-MLPA Probemix ME030 BWS/RSS is a research use only (RUO) assay for the detection of aberrant methylation of one or more sequences of the IC2 (KvDMR) and IC1 (H19DMR) domains in the 11p15 chromosomal region associated with Beckwith-Wiedemann syndrome (BWS) and Russell-Silver syndrome (RSS). This probemix can also be used to detect deletions/duplications in the aforementioned chromosomal regions. Additionally, two probes are included for the NSD1 gene which is associated with Sotos syndrome, a disease that has a similar phenotype. 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 originate from a disturbance in this monoallelic expression by disruption or epimutation of imprinted genes (Ishida et al. 2013).

BWS is a clinically heterogeneous overgrowth syndrome associated with an increased risk for embryonal tumour development. RSS is a genetically heterogeneous disorder involving both intrauterine and postnatal growth retardation. The incidence of both BWS and RSS is estimated to be approximately 1 in 10,000-15,000 newborns and around 85% of the cases are sporadic (Õunap 2016). These conditions are both caused by a genetic or epigenetic alteration within two domains of imprinted growth regulatory genes on chromosome 11p15, leading to deregulated expression of the imprinted genes within this region. Approximately 60-70% of the patients have imprinting abnormalities at one of two imprinted domains IC1 or IC2, and these changes are frequently mosaic (see Figure 1 in the Product Description for a scheme of the imprinted gene cluster). Other known causes of BWS and RSS are uniparental disomy (UPD), trisomy 11p15, mutations in the CDKN1C gene, as well as small deletions and translocations. About 10% of RSS cases are caused by maternal UPD for chromosome 7 (Õunap 2016).

This SALSA MS-MLPA Probemix ME030 BWS/RSS is capable of rapidly detecting most causes of BWS and RSS, as both copy numbers and methylation status of the 11p15 region can be determined. This MS-MLPA assay for BWS/RSS can also be useful for screening of childhood cancers, in particular Wilms’ tumour. A strong linkage between hypermethylation of the IC1 locus, but not IC2, has been described in these patients resulting in biallelic expression of the IGF2 gene (Maas et al., 2016). Because of similarities between BWS and Sotos syndrome, two probes for NSD1 are included.

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1394/ (BWS) and https://www.ncbi.nlm.nih.gov/books/NBK1324/ (RSS).

This SALSA MS-MLPA probemix is not CE/FDA registered for use in diagnostic procedures. Purchase of this product includes a limited license for research purposes.

Probemix content
The SALSA MS-MLPA Probemix ME030-C3 BWS/RSS contains 42 (MS-)MLPA probes with amplification products between 129 and 463 nucleotides (nt). 26 probes are specific for the BWS/RSS 11p15 region. Ten MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of the BWS/RSS 11p15 region. Two probes are specific for the NSD1 gene. All probes present will also give information on copy number changes in the analysed sample. In addition, 13 reference probes are included that are not affected by HhaI digestion and detect genes located outside the BWS/RSS 11p15 region. Also, one digestion control probe is 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.