A novel mutation in the promotor region in a family with a mild form of retinoblastoma indicates the location of a new regulatory domain for the RB1 gene

We describe a family segregating the retinoblastoma phenotype where the affected individuals have only tumours and where linkage analysis has identified unaffected mutant gene carriers. DNA from members of this 'low penetrance' pedigree was subjected to an exon-by-exon SSCP analysis of the RB1 gene. No mutations were found in the 27 exons of the coding region but an SSCP band shift was seen for PCR products covering the RB1 promotor region. Sequencing identified a G → C change within a GGGCGG motif which is the core of the recognition sequence of the SP1 transcription factor. Electromobility shift assays demonstrated that SP1 does not bind to oligomers from this region of the RB1 promotor but bandshifts were seen for an, as yet, unidentified protein(s) which was not seen using an oligomer containing the G → C mutation. Thus, identification of a naturally occurring mutation in a family with only 'mild' phenotypes has identified another regulatory sequence in the RB1 promotor which binds an endogenous cellular protein(s). Identification of this protein should allow a better understanding of the control of expression of the RB1 gene.