Familial medullary thyroid carcinoma (MCT) and multiple endocrine neoplasia type 2A (MEN 2A) are autosomal dominant familial cancer syndromes characterized principally by the presence of multifocal MTC. These disorders have been mapped by genetic linkage analysis to the pericentric region of chromosome 10, indicating that they are caused by different mutations at a single locus, or by mutations in closely linked loci. Since these disorders are among the clearest examples of genetically determined cancer in man, delineation of the mechanism of action of the MTC/MEN 2A gene(s) may provide important insights into basic genetic mechanisms of carcinogenesis. The overall goals of this program are to identify, clone, sequence, and determine the function of the MTC/MEN 2A gene(s), and to develop a sensitive and specific presymptomatic genetic diagnostic test for these disorders. These goals will be approached first by isolating additional pericentric chromosome 10 probes, examining these clones for dinucleotide (CA)n repeat polymorphisms and RFLPs, and genetically mapping the polymorphic clones. Second, both polymorphic and non-polymorphic probes will be physically mapped by chromosome in situ hybridization on interphase nuclei. This technique will permit direct identification of clones which lie between the genetically mapped loci known to flank the disease locus, and will physically orient these clones relative to these loci and the centromere. It will also order some clones within linkage groups and provide an estimate of the physical distance between them. Third, these clones will be partially sequenced and converted to sequence tagged sites (STSs). Oligonucleotide primers which define the STSs will be used in a polymerase chain reaction (PCR) based strategy to screen yeast artificial chromosome (YAC) libraries for homologous clones. These YAC clones will, in turn, yield additional STSs at their termini which will be used to screen for overlapping YAC clones by PCR. YACs homologous to probes mapped genetically and by in situ hybridization will serve as anchors for the development of a series of physically oriented and genetically linked YAC contigs, and provide the foundation for the construction of an integrated physical and genetic map of the region encompassing the disease locus. Fourth, the location of the MTC/MEN2 locus will be further defined by identifying genetic crossovers in the study families with the set of highly informative probes which will have been developed in the course of assembling the YAC contigs. Fifth, these probes will also make possible the development and application of a highly informative and accurate genetic test for familial MTC and MEN 2A, which in turn will also serve as a gold standard for the evaluation of biochemical and histopathological criteria used for the diagnosis of C-cell hyperplasia, a pre-malignant expression of these genes in the thyroid gland. Finally, the integrated genetic and physical map around the MTC/MEN2A locus will guide the search for expressed genetic sequences. These sequences will be isolated and correlated with tissue-specific expression. Our ultimate goal is to identify and characterize the MTC/MEN 2A gene(s).
