General information
The SALSA MLPA Probemix P047 RB1 is a research use only (RUO) assay for the detection of deletions or duplications in the RB1 gene and of methylation status of the RB1 gene promoter and imprinted locus in a DNA sample.

Retinoblastoma (RB; OMIM180200) is an embryonic neoplasm of retinal origin developing in early childhood and often bilaterally. The incidence of RB is estimated between 1:15000 and 1:20000 live births (Moll et al. 1997, Seregard et al. 2004). Retinoblastomas occur in two forms: hereditary and non-hereditary (sporadic), representing about 40 and 60% of all RB cases, respectively. In hereditary RB, germline mutations causing RB1 gene loss or inactivation are inherited in autosomal dominant pattern and predispose to RB development with high penetrance. Over 80% of hereditary RB is caused by de novo RB1 mutations arising during embryonic development. Bilateral and unilateral hereditary RB represent about 25-30% and 10-15% of all RB cases, respectively. None of the sporadic RBs is bilateral. Bilateral RB patients also have a predisposition for secondary cancers with the highest risk for osteosarcomas. In about 1.5% of unilateral RB cases somatic MYCN amplification (but no RB1 pathogenic variants) was detected (Rushlow et al. 2013).

The RB1 gene (27 exons) located on 13q14.2 spans about 180 kb of genomic DNA and is a well characterized tumour suppressor gene encoding a ubiquitously expressed nuclear protein involved in cell cycle regulation, cellular differentiation and survival. Point mutations, small and large deletions, as well as promoter methylation in the RB1 gene affect the function of retinoblastoma-associated protein (pRB). Over 500 pathogenic germline variants were identified in the RB1 gene (https://databases.lovd.nl/shared/variants/RB1/). Moreover, additional clinical features were described for deletions at 13q14 encompassing the RB1 gene (Mitter et al. 2011), such as RB1 gene deletions spanning to the PCDH8 gene have been shown to play an important role in psychomotor delay in RB patients (Castera et al. 2013, Mitter et al. 2011). Contiguous loss of MED4, which is located centromeric to RB1 is thought to contribute to synthetic lethality in cells with RB1 homozygous loss (Dehainault et al. 2014).

pRB loss, predominantly via heterozygous deletion of RB1 gene, is also a common abnormality for various cancer types, including breast cancer, lung cancer, prostate cancer and osteosarcoma, and is often associated with poor survival (reviewed in Mandigo et al. 2021).

RB1 inactivation by methylation of promoter region (CpG106) has been shown in 8-15% RB (Dommering et al. 2014, Greger et al. 1989, Price et al. 2014) and in other cancers (Sahi et al. 2014, Simpson et al. 2000). Moreover, an imprinted locus in intron 2 of the RB1 gene (CpG85) - methylated at the maternal allele, has been identified (Kanber et al. 2009), and recently the importance of the methylation of this imprinted locus in hepatocellular carcinoma has been shown (Anwar et al. 2014).

More information is available at https://www.ncbi.nlm.nih.gov/books/NBK1452/.

This SALSA 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 MLPA Probemix P047-E2 RB1 contains 57 (MS-)MLPA probes with amplification products between 129 and 500 nucleotides (nt). This includes 35 probes for RB1 covering all exons, except exon 15 which is located at a close distance to the adjacent exons, of which five MS-MLPA probes target the RB1 promoter region (CpG106) and three MS-MLPA probes target the imprinted CpG island in intron 2 (CpG85). These MS-MLPA probes contain an HhaI recognition site and provide information on the methylation status of selected GCGC sites in CpG106 and CpG85.

In addition, 13 reference probes are included that are not affected by HhaI digestion and target relatively copy number stable regions in various cancer types. 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 are available online (https://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 (see table below). More information on how to interpret observations on these control fragments can be found in the (MS-)MLPA General Protocol and online at https://www.mrcholland.com.