Linkage analysis at the retinoblastoma locus (RB1) is essential for identifying individuals at risk and to offer adequate genetic counseling in familial retinoblastoma. It can also be used to detect large deletions involving RB1, which accounts for 15% of the genetic alterations in hereditary retinoblastoma. These studies are usually carried out with lengthy Southern blot analyses of relatively uninformative restriction fragment length polymorphisms. The authors report an alternative, reliable protocol for genotyping the RB1 locus using two pairs of highly informative intragenic and flanking microsatellites linked closely to the RB1 gene, and analysis of the fluorescent-labeled polymerase chain reaction products with automatic sizing technology. This methodology has successfully identified high risk carriers in five of the five pedigrees of familial retinoblastoma studied. In addition, gross deletions affecting the RB1 gene were identified in two of 12 sporadic bilateral retinoblastomas, and loss of heterozygosity at the RB1 locus has been detected in one of three osteosarcomas using the same experimental protocol. The described protocol is simpler and faster than conventional Southern blot methodologies and can identify a larger number of informative cases.

Supported by grants from Fondo de Investigación Sanitaria (FIS 98/1349) and the Comunidad de Madrid (08.1/0034.1/98), Madrid, Spain.

Peer Reviewed