The long-term objectives of the proposed research are to obtain polymorphic DNA probes near to and flanking the Huntington Disease (HD) locus on the short arm of human chromosome 4, and to use these markers to isolate candidate genes coding for HD. Flanking markers will not only be required for developing strategies for isolating the gene, but will also be immediately useful as a tool for improving the accuracy of presymtomatic diagnosis of the disease. Isolation of candidate genes provide a direct means of identifying the primary defect that results in the disease, and may provide insights into the development of an effective treatment. A physical map of the 10,000 kb surrounding the HD locus will be constructed by first transferring that region of chromosome 4 into a hamster cell by somatic cell genetics methods, followed by cloning of the human DNA from the hybrid cell into a series of overlapping yeast artificial chromosomes. The order of the overlapping fragments will be determined by physical methods, generating a physical map o the region at 200-500 kb resolution. This map will allow us to identify a small number of probes, approximately 10-15, spaced 500-1,000 kb apart throughout the HD region. These probes will then be tested for recombination with HD by using genomic DNA from individuals with the disease. This procedure will establish a physical map of the region and identify those few DNA probes that are closest to and flanking the HD gene, as defined by recombination. As opposed to a random screening of probes, this more directed approach minimizes the number of probes that need to be studied. Four separate but complementary approaches will be used to flanking probes. These are: 1) isolation of exon sequences conserved between mammalian species; 2) isolation of GC-rich HTF islands; 3) isolation of probes homologous to mRNA made in whole brain and caudate nucleus; and 4) development of an animal model of HD.
