[Diagnosis of a predisposition of retinoblastoma at the DNA level]

Retinoblastoma is a malignant tumor that arises in the eyes of children at a frequency of about 1 in 15,000 live births in Japan. Thirty to 40 percent of the patients with retino-blastoma have a heritable predisposition to this tumor. This predisposition is determined by a germinal mutation in the RB gene, which was located at q14 region of chromosome 13 and isolated in 1986. Retinoblastoma will develop in about 90 percent of the persons who carry a mutated RB allele. Thus, it is necessary to develop diagnostic tests that can identify persons who have inherited the mutation. To this end, RBcDNA clones or several polymorphic markers which link to the RB gene have been identified and used for the diagnosis of retinoblastoma predisposition and five RFLP markers within the RB gene have been shown to be useful (Wiggs et al. 1988). Recently we have developed a simple, rapid method for detecting aberrations of RBmRNA including single nucleotide substitutions directly, using polymerase chain reaction-single strand conformation polymorphism analysis (PCR-SSCP) in combination of reverse transcriptase (RT) reaction (RT-PCR-SSCP). For this analysis, we obtain mRNA from 10 ml of peripheral blood and entire procedure including time for isolating mRNA can be completed within 48 hours. By this way we could detect a point mutation of RB gene transcript in peripheral blood cells from two of eight hereditary retinoblastoma patients. We conclude that it is practicable and clinically useful to use this RT-PCR-SSCP analysis of RB gene transcript for direct determination of the risk of retinoblastoma.