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A PCR test for the detection of hypermethylated alleles at the retinoblastoma locus
  1. Institut für Humangenetik, Hufelandstrasse 55, D-45122 Essen, Germany

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Editor—The development of retinoblastoma, a childhood malignancy of the eye, is initiated by mutations in both alleles of the retinoblastoma gene (RB1). Mutations changing the nucleotide sequence at this locus and chromosomal mechanisms resulting in loss of heterozygosity (LOH) are the most common events responsible for gene inactivation.1However, in the RB1 gene, as well as in other tumour suppressor genes includingBRCA1, p15, andp16, hypermethylation of 5′ regulatory regions may also cause gene inactivation and, consequently, tumour development.2-6 Methylation of the CpG rich island at the 5′ end of the RB1 gene is observed in some 10% of unilateral sporadic retinoblastomas.3 ,4 ,7Recently, the methylation pattern of tumours with hypermethylatedRB1 alleles was analysed in detail by the bisulphite genomic sequencing technique.8 In most of these tumours, most CpG sites were methylated (75-100%). In one of seven tumours only, the density of methylated CpG sites was more variable, ranging from 25% to 62.5%.

Mutation analysis in tumours from patients with sporadic unilateral retinoblastoma is required for accurate risk prediction in relatives.1 Consequently, we have devised a methylation specific PCR assay (MSP)5 ,9 for rapid and reliable detection of methylation at the RB1promoter. Bisulphite treatment of denatured DNA converts all unmethylated cytosines to uracil leaving methylated cytosines in CpG dinucleotides unaltered. After …

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