Abstract
Abstract: :
Purpose: To determine the range of mutations in the RB1 gene in patients with retinoblastoma. Methods: Exons and flanking regions of the RB1 gene were amplified by PCR using genomic DNA isolated from peripheral blood leukocytes and tumors. A total of 37 patients were studied, tumors were obtained from 23 patients while blood samples were obtained from all patients. PCR products were screened for mutations either by direct sequencing (21 patients) or by single-strand conformation polymorphism (SSCP) followed by sequencing (16 patients). SSCP was carried out using denatured PCR products, which were electrophoresed on polyacrylamide gels containing 5% glycerol at 4C and at room temperature in the absence of glycerol. Sequencing was done on an automated sequencer. Results: Out of 37 patients, 20 had bilateral and 17 had unilateral retinoblastoma. 3 patients with bilateral disease had affected siblings, while all others had sporadic disease. Mutations were detected in 13/37 patients, 8 with bilateral and 5 with unilateral disease. Screening of all 27 exons by direct sequencing revealed mutations in 7/21 patients. In an analysis of 16 patients by SSCP of exons 10-24, mutations were found in 5 /16 patients. One patient was found to have a deletion between exons 12 and 24. Out of 13 patients, 12 had mutations that result in premature termination, while one had a missense mutation. Conclusion: Using a combination of approaches that are aimed at screening exons, mutations could be found in about one-third (13/37) of patients. The data suggest a) that a higher proportion of mutations may reside in exons 10-24 of the RB1 gene with some mutations being recurrent b) methods that are designed to detect other types of alterations are needed to identify the full range of mutations in these patients.
Keywords: 569 retinoblastoma • 480 mutations • 420 genetics