Positional cloning strategies and the Huntington's disease gene.

Abstract

Positional cloning is an approach that allows the molecular isolation of the genes involved in human inherited diseases, in the absence of functional information about the gene product. Briefly, the approach involves (1) finding a genetic marker whose alleles are coinherited with disease symptoms in affected families (termed a "linked" marker), (2) generating more markers in the area to refine the genetic candidate region through the identification of chromosomal recombinations, (3) cloning the genomic DNA within the candidate region, (4) isolating genes from the genomic clones, and (5) determining which gene harbors mutations that cause the disease. This work contributed to the successful identification of the gene involved in Huntington's disease (HD), an autosomal dominant, neurodegenerative disorder. As a result of this work, an overlapping set of yeast artificial chromosomes (YACs) was isolated, each clone containing genomic DNA from the HD candidate region. The insert size and approximate physical location of each YAC was determined. Over 50 ends of critical YACs were rescued and mapped against the entire YAC set. Overlap relationships proved this set of YACS to form a contiguous group which spans the entire HD candidate region. A novel technique (called "island rescue PCR") was developed which allowed the rapid isolation of genes directly from these YACs. Four genes were isolated from the HD region and characterized. One of these genes was human $\alpha$-adducin, which was previously mapped to the HD region. Island rescue PCR was also applied to the BRCA1 candidate region (which contains a gene involved in early onset familial breast and ovarian cancer). Five novel genes were isolated from the BRCA1 region and characterized. During the interval, the HD gene was identified. The predicted sequence gave little functional insight into disease mechanism. Ongoing efforts to characterize the HD gene are described, including: (1) in situ hybridization to determine gene expression patterns in both human and rodent systems, (2) identifying alternatively spliced isoforms by PCR with 32 overlapping primer pairs on reverse transcribed mRNA from various tissues including brain subregions, and (3) antibody generation against fusion proteins to determine localization of the HD gene product.