DIRECT SEQUENCE ANALYSIS OF THE RETINOBLASTOMA GENE

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This proposal describes the use of enzymatic amplification and direct DNA sequencing techniques to identify mutations in the human retinoblastoma gene. These methods will be used to find point mutations or other genetic changes that are too small to be detected by conventional methods such as Southern blotting. Approximately 80% of the disease-causing mutations that occur in the retinoblastoma gene fall into this category and very little is known about these mutations. A primary goal of the project is to define the spectrum of oncogenic point mutations that occur in the retinoblastoma gene. This will be accomplished by analyzing DNA from various sporadic or hereditary tumors, and from the blood of individuals predisposed to retinoblastoma or other cancers. Knowledge of the spectrum of mutations that occur in this gene will help define critical functional domains of the retinoblastoma protein and will identify possible hotspots for mutation. In addition, this mutational spectrum will include both germinal and somatic point mutations in the same gene as they have occurred in vivo. Hence the proposed studies represent a unique opportunity for an unbiased comparison of germinal versus somatic point mutations at a single human locus. An important direct benefit of the proposed studies, and a specific aim of the proposal, will be application of these techniques for DNA-based diagnosis and genetic counseling of both hereditary and nonhereditary forms of retinoblastoma. Because this gene is characterized by a high new mutation rate, "linked marker" or RFLP-based diagnosis are often ineffective because there is no previous family history of the disease. We believe that establishment of protocols for the routine identification of diagnostic point mutations in this complex gene represents an important step forward in DNA-based diagnosis of retinoblastoma, and may serve as a model for diagnosis of other genetic disorders with high new mutation rates. Finally, it is proposed to extend these studies to examine the role of point mutations of the retinoblastoma gene in several families with atypical, low-penetrance hereditary retinoblastoma and in families at high risk for breast cancer. It is argued that these are excellent candidate syndromes for unusual point mutations of the retinoblastoma gene and that identification of predisposing mutations in either group would be of great interest. We believe that the proposed studies will have not only specific direct benefits related to the diagnosis of retinoblastoma but are broadly relevant to questions elated to carcinogenesis and to the origins of human genetic disease.