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. In the US the frequency of RB is approximately 1 in 23,000 live births (Macklin MT, 1960. Am J Hum Genet. 12:1-43). Bilateral and unilateral hereditary RB represents, respectively, about 25-30% and 10-15% of all RB cases. 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 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 was predicted. RB1 gene deletions spanning to PCDH8 gene are 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. PMID:24735260). Moreover, an imprinted region in intron 2 of 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 (Hong FD et al, 1989, Proc Nat Acad Sci. 86:5502-6) and is located on 13q14.2. This P047-D1 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 are locating on 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 include for exon 1. In contrast, when tested on methylated human DNAin vitro, these probes do generate a signal. We have no data showing that methylation detected by a particular probe directly influences the corresponding mRNA levels. Furthermore, four MS-MLPA probes are present for the imprinted CpG island CpG85 locating in intron 2 and provide information about the methylation status of this region. As these four probes target imprinted region, one allele is methylated, the other is unmethylated in normal control samples. 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 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 P047-D1 RB1 probemix is primarily intended for non-tumour DNA analysis. Nevertheless, the new reference probes target relatively stable chromosomal regions in most cancer types and allow analysis of tumour DNA as well.