| Summary: | The dominantly inherited cancer syndrome multiple endocrineneoplasia type 2A (MEN 2A) is characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and parathyroid hyperplasia. The related syndrome MEN 2B comprises MTC, pheochromocytoma and mucosalneuromas. Genes responsible for these cancers map to the pericentromericregion of chromosome 10, as do loci responsible for dominantly inherited MTC without additional clinical features, and MEN 2A associated with askin disorder, cutaneous lichen amyloidosis. The relationship between the genes responsible for these diseases is not known. Nothing is known of the biochemical basis for MEN 2; a positional cloning strategy to identify MEN2 gene(s) is therefore warranted. This thesis makes contributions to several steps of a strategy to identify MEN2. The first objective was to clone DNA markers more closely linked toMEN2A than the known flanking markers, FNRB and RBP3. Repeat element-mediated polymerase chain reaction (REM-PCR), a method of cloning human DNA fragments from hybrid DNA sources, was developed for this purpose. Primers directed to the 3' ends of human Alu and Ll elements provided the basis for amplification of human-specific DNA fragments; DNA from a somatic cell hybrid highly enriched for the MEN2Aregion was used as template. This technique yielded thirty-three REM-PCR clones, two of which mapped to intervals near MEN2A. One of these, pC11/A1S-6-c23, defines a polymorphic locus, D10S94.
Somatic cell hybrid mapping of pC11/A1S-6-c23, combined with the results of linkage studies conducted by Drs. Nancy Simpson and Paul Goodfellow, served to localize D10S94 to 10811.2 between the centromere and RBP3. D10S94 is very tightly linked to MEN2A and was not idemonstrated to recombine with the disease locus in these studies. It was used as a starting point for large scale cloning and mapping efforts directed to the identification of genes. Long range restriction mapping by pulsed field gel electrophoresis revealed a dense cluster of CpG islands at D10S94. Genes potentially associated with these CpG islands represent candidate genes for MEN2. To determine whether relatively large sequence alterations, which could serve to direct gene cloning efforts to a small region, were associated with D10S94, a survey of the 180-kb CpG island-rich region at this locus was undertaken in the genomes of MEN 2A and MEN 2B patients. Long range restriction mapping by field inversion gel electrophoresis demonstrated that no alterations of 3.3 kb or more were present at DlOS94 in these patients. Yeast artificial chromosomes (YACs) identified in collaboration with Drs. Ken Kidd and Jay Lichter, expanded DlOS94 and, with genomic long range restriction mapping, established physical linkage to six other DNA markers near MEN2. Recent published reports identify very closely linked flanking markers and refine the minimal region to which MEN2A is localized. The genomic long range restriction map and YAC contig span the entire refined MEN2A candidate region. This map and contig can now serve as a guide and a cloning source, respectively, for experiments designed to identify genes within this region, and an intensive search for potentially disease-causing mutations to identify genes important in the etiology of MEN 2.
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