Retinoblastoma (RB; MIM180200) is an embryonic neoplasm of retinal origin. It almost always develops in early childhood and is often bilateral. Retinoblastoma is associated with loss or inactivation of the RB1 gene. The RB1 gene is a negative regulator of cell cycle and proliferation. The incidence of RB is estimated between 1:15000 and 1:20000 live births (Moll AC et al., 1997. Br J Ophthalmol. 81:559-62; Seregard S et al., 2004. Ophthalmology. 111:1228-32). Bilateral and unilateral hereditary RB represent about 25-30% and 10-15% of all RB cases, respectively. None of the nonhereditary RBs (representing 55-65% of all RB cases) is bilateral. Bilateral RB patients also have a predisposition (230-500x increased risk) for osteogenic sarcoma.
Several heterozygous deletions in the RB1 gene have been characterised in germline (blood-derived) DNA. In tumours, homozygous deletions of the RB1 locus have been demonstrated in sporadic cases of leiomyosarcoma, malignant fibrous histiocytoma and undifferentiated sarcoma, in the absence of any history of retinoblastoma. Loss of heterozygosity of the RB1 locus is frequent in high grade astrocytomas but not in low grade gliomas. Deletion of exons 13-17 is frequently observed in various types of tumours, including retinoblastoma, breast cancer, and osteosarcoma, and the presence of a potential 'hotspot' for recombination in the region has been predicted. RB1 gene deletions spanning to the PCDH8 gene have been shown to play an important role in psychomotor delay in RB patients (Castera L et al., 2013. Eur J Hum Genet. 21:460-4). In recent years an alternative mechanism of RB1 inactivation by means of methylation of promoter region (CpG106) has been demonstrated in some cancers (Simpson DJ et al., 2000. Cancer Res. 60:1211-6; Sahi H et al., 2014. APMIS. 122:1157-66). Moreover, an imprinted region in intron 2 of the RB1 gene (CpG85) has been identified (Kanber D et al., 2009. PLoS Genet. 5(12):e1000790), and recently the importance of the methylation of this imprinted region in hepatocellular carcinoma has been shown (Anwar SL et al., 2014. J Pathol. 233:392-401).
The RB1 gene (27 exons) spans about 180 kb of genomic DNA and is located on 13q14.2. This P047-E1 RB1 probemix contains probes for 26 of the 27 RB1 exons. No probe is present for exon 15 which is located at a close distance to the adjacent exons. Five probes are present for exon 1, two for exon 12 and two for exon 27. The exon 1 probes target CpG106 and allow determination of the methylation status of the RB1 promoter region that is unmethylated in most blood-derived DNA samples. Upon HhaI digestion, the peak signal obtained in unmethylated samples will be very low or absent for these five probes. In contrast, these probes do generate a signal when tested on methylated human DNA in vitro. We have no data showing that methylation detected by a particular probe directly influences the corresponding mRNA levels. Furthermore, three MS-MLPA probes are present for the imprinted CpG island CpG85 in intron 2 and provide information about the methylation status of this region. As these three probes target an imprinted region, one allele is methylated and the other is unmethylated in healthy individuals. As compared to reference probes that do not contain a HhaI site, the signal of the MS-MLPA probes in the imprinted region is reduced by approximately 50% upon HhaI digestion in DNA samples from normal individuals.
Furthermore, this probemix contains flanking probes in the close proximity of RB1 (48 kb upstream; 35 kb downstream) as well as a probe for the DLEU1 gene and two probes for the PCDH8 gene, 1.6 Mb and 4.5 Mb downstream from RB1, respectively. In addition, 13 reference probes are included in this probemix, detecting several different autosomal chromosomal locations, which are relatively stable in human cancers and are not affected by HhaI digestion.
This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences of RB1 gene and to detect aberrant methylation of the RB1 gene promoter and imprinted region in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test. This probemix can be used to detect the methylation status of the promoter and imprinted region of RB1 gene. Methylation levels can be different for different tissues. If possible, use identically treated test and reference samples (same tissue type and extraction method).