The identification and characterization of genes in a candidate region for the Huntington’s disease gene

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease affecting approximately 1/10,000 individuals. Progressive psychiatric disturbances, involuntary movement disorders, termed chorea, and dementia are clinical hallmarks of HD while, neuropathologically, a progressive loss of neurons throughout the central nervous system and in particular within the caudate nucleus of the basal ganglia is observed in HD. The gene responsible for HD was localized to chromosome band 4p16.3 in 1983, by genetic linkage and in situ hybridization studies. This thesis describes the delineation of a 50 kb HD gene candidate region, within chromosome 4p16.3, and the identification and characterization of two novel human genes mapping within that 50 kb interval. The chromosomes of a patient reported to carry a new mutation at the HD locus and those from 11 of her siblings were haplotyped revealing a distal 4pl6.3 recombination event in the affected individual. Because both new mutations at the HD locus, and distal 4p16.3 recombination events are rare, a hypothesis was formulated relating the recombination event to the occurrence of HD in the patient. In a test of this hypothesis a chromosome walk was performed to identify the site of postulated unequal recombination. In the course of the chromosome walk two restriction fragment length polymorphisms, informative in the sibship, were identified enabling a 500 kb HD candidate region, approximately 700 kb from the 4p telomere, to be narrowed to 80 kb. The molecular characterization of the 80 kb region ultimately reduced the interval to 50 kb and, moreover, revealed three putative CpG islands. Zoo blot analysis identified phylogenetically conserved DNA associated with two of these CpG islands suggesting the presence of encoded genes. The subsequent probing of cDNA libraries and Northern blots led to the identification and characterization of two genes encoding for the n-subunit of rod photoreceptor cGMP phosphodiesterase (PDEB) and a novel regulatory myosin light chain (MYL5). No evidence for an unequal recombination, within the 50 kb interval, was revealed by Southern blot analysis. Similarly, single strand conformational polymorphism (SSCP) analysis of PDEB and MYL5 genes failed to reveal evidence for the postulated mutation. These findings strongly suggest that the 4p16.3 recombination event is unrelated to the occurrence of HD in this sporadic case.Medicine, Faculty ofMedical Genetics, Department ofGraduat