THE CYTOGENETICS LABORATORY

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Basic studies continue to concentrate primarily on Giemsa banded karyotyping of animals with sexual anomalies as well as syndromic disorders that suggest the presence of a chromosomal anomaly. Since the last progress report, a total of 19 cytogentic studies were performed on canine and feline cases referred to the genetics clinic or following consultations. Canine investigations included DiGeorge Syndrome, os clitoris, azoospermia, ichthyosis, a PCH-1 heterozygote, intersex, hypospadias extreme, and ataxia. A feline case of intersex was also examined. Novel disease presentations included canine soft tissue swelling, ichthyosis, ataxia, extreme joint laxity, perodontal disease, DiGeorge syndrome, cystrophic mineralization in limbs, HD and ED. Feline cases of spinal muscular atrophy, ostogenesis imperfecta, multiple fractures, cutaneous asthemia, and other connective tissue disorders were also examined. Studies include Western blotting using antibodies specific for different collagens and pulse chase assays for collagen type I function and assembly. The Western blots have been challenging as many of the antibodies are specific to species other than the dog and cat. The findings in animals with skin fragility are being summarized as part of a dermatology residency project and will be submitted for publication. Another primary role of the cytogenetic laboratory is tissue culture of models currently under investigation in the Referral Center. Affected cell lines have been added for canine MPS IIIB, MPS VI, PFK, X-linked anhydrotic ectodemal dysplaisa, epidermolysis bullosa, and lethal acrodermatitis in an effort to define cellular pathology, provide RNA and DNA for mutation analysis, and for use in future cross-correction studies. Fibroblast cultures were established from dogs with hypophosphatemia, Labrador myopathy, renal failure, possible MPS III, and pathologic fractures. Fibroblast cultures from cats were established for MPS VI, an undiagnosed lysosomal storage disease, pyruvate kinase deficiency, I-cell disease, portocaval shunt, GM2 gangliosidosis, neuromuscular disease, MPS VII, MPS I, and GM1 gangliosidosis. Methods have been developed for the use of fluorescence in situ hybridization (FISH) to define the physical location of genes on canine chromosomes. This approach uses gene probes labeled with fluorescent dyes. The labeled probes are hybridized to metaphase chromosomes and detected by fluorescence microscopy, allowing cloned genes and other markers to be mapped to specific chromosome locations. The FISH method provides a means of mapping the mutant genes involved in canine genetic diseases to their chromosomal locations, further increasing knowledge of the comparative medical genetics of these important models and enhancing their use as animal models of human genetic disease. In addition, the FISH studies add to the development of the canine genome map, increasing its value as a resource for finding new canine homologs of human genetic diseases. We have collaborated in a worldwide initiative to map the dog genome. As a part of this work, we have FISH-mapped a series of markers that serve as anchors for linkage groups on canine chromosomes. These linkage groups contain 341 mapped markers distributed over 37 canine autosomes and the X Chromosome. These gene markers simultaneously serve as anchor loci for microsatellite markers in the canine linkage map and establish the chromosomal locations of the linkage groups, allowing the canine gene regions to be lined up with the homologous regions of human and mouse chromosomes. This provides access to the rich source of information on evolutionarily conserved arrangements of genes on chromosomes in these other species, greatly facilitating attempts to locate and isolate canine genes by positional cloning methods.