A goal of our research is to characterize genetic organization in the domestic cat and to develop genomic resources facilitating and establishing Felis catus as a useful animal model contributing to our understanding of human hereditary disease analogues, neoplasia, genetic factors associated with host response to infectious disease and mammalian genome evolution. In order to map and characterize genes associated with inherited pathologies in the domestic cat which mirror inherited human conditions we have focused on increasing microsatellite density in the genetic map of the cat to increase resolution for mapping of genes associated with inherited and infectious disease. The following projects are directed toward these goals: 1. Generation of a Second Generation Integrated Type I and Type II Genetic Linkage Map of the Cat. A second generation genetic linkage map of the cat has been generated which is a first integration of coding and polymorphic loci from the previous feline linkage map. The integrated map consists of 328 markers including 81 Type I loci and 247 Type II loci (240 autosomal and 7 X-linked microsatellites). Forty-seven linkage groups were identified, all of which have been mapped to a cat chromosome. The average intragroup inter-marker distance equals 8 centimorgans (cM) and we estimate the genetic length of the sex-averaged map at 3300 cM. The map confirms a remarkable conservation of genome conservation with human previously observed in physical and radiation-hybrid maps of the cat as well as reciprocal chromosome paints of human and cat. Additionally, the map provides a valuable resource for mapping phenotypic variation in the species and relating it to gene maps of other mammals, including human. 2. Isolation, Characterization of Additional Polymorphic Microsatellite Loci from Genomic DNA of the Domestic Cat, which have been Incorporated into the Radiation Hybrid (RH) Map of the Cat, (a Third Generation Integrated Type I and Type II Genetic RH Map of the Cat). A 1169 locus third generation domestic cat RH map has been generated from the incorporation of 335 unique Type II microsatellite loci isolated and characterized from the domestic cat genome to the 834 locus 2nd generation map. With a total number of 614 short tandem repeats (STRs) mapped in the feline genome, microsatellite coverage now spans every autosome with an average spacing of one STR every 4 cM. These loci now provide a useful tool for undertaking full-genome scans to identify genes associate with phenotypes of interest, such as those relating to hereditary disease, coat color, patterning and morphology. 3. Construction of a Third Generation Genetic Linkage Map of the Cat. We are currently focused on completion of a third generation full genome genetic linkage map of microsatellites in the cat, with marker resolution of approximately 3 cM. This map will provide us with a sufficient resolution to perform linkage-mapping exercises in several cat pedigrees of interest segregating for disease phenotype and phenotypes of other biological interest. Approximately 700 microsatellites, described above, are being genotyped in a large multi-generation domestic cat pedigree maintained by the Nestle-Purina Pet Care Company (n=483 informative meioses). Based on previous mapping of a majority of these loci in the cat radiation-hybrid panel, we know that the loci are well distributed across the cat genome. 4. Mapping and Characterization of Genes Associated with Inherited Disease Pathology in Cat Pedigrees with Homology to Human Hereditary Disease. Autosomal recessive retinitis pigmentosa (arRP) arRP is a genetically and clinically heterogeneous and progressive degenerative disorder of the retina, leading usually to severe visual handicap in adulthood. Our collaborator, Dr. Kristina Narfstrom at the Missouri College of Veterinary Medicine, maintains a colony of Abyssinian cats with progressive retinal atrophy (rdAc), a slowly progressive degeneration process of the rod and cone systems with similarities to classical human RP. The Abyssinian cat has the potential of becoming a new and important animal model in the study of hereditary visual cell disease processes. In order to identify and characterize the gene, which gives rise to feline rdAc, our approach is to map the gene using classical linkage mapping with a full genome scan of Dr. Narfstroms's pedigree. As initial screening of the pedigree with 254 microsatellite loci exhibited that the colony was highly inbred, Dr. Narfstrom out bred members of the pedigree with controlled matings to increase the average marker heterozygosity. To date, a total of 36 backcross individuals have been generated from 18 out bred F1, and more individuals are being generated. Simulation results (SIMLINK) indicate that the likelihood of detecting linkage to the disease locus in a pedigree structure/size similar to Dr. Narfstrom's with 45 or 60 back-crossed individuals is 0.88 and 0.91, respectively, at q = 0.1 (lod score = 5.5, 7.1, respectively) (average heterozygosity of microsatellites = 0.75). Spinal Muscular Atrophy Degeneration of lower motor neurons in the spinal cord that causes neurogenic muscle atrophy, the so-called spinal muscular atrophies (SMAs), are a large category of inherited disorders, but the molecular basis or linkage of a genetic locus has been established in only a few. The SMAs are categorized generally by pattern of inheritance and by distribution of muscle atrophy. In collaboration with Dr. John Fyfe (Michigan State), who maintains a pedigree of Maine Coon cats segregating for SMA Type III, we are investigating the molecular basis of this new and unique model of inherited motor neuron degeneration in cats. Our prior linkage analysis has indicated that disease phenotype is unlinked to the feline SMN locus. A full genome scan of microsatellites is currently being performed in his affected pedigree. Once linkage is established to a cat chromosome, comparative gene mapping will be utilized to identify candidate genes for the pathology. 5. Completion of an STR Forensic Typing System for Genetic Individualization of Domestic Cat Specimens. Under support from the National Institutes of Justice, we are completing the development of a microsatellite forensic typing system and genetic database of cat breeds for genetic individualization of cat specimens. A panel of 11 tetra-nucleotide repeat microsatellite loci has been isolated, mapped in the cat RH map and examined for Mendelian inheritance and mutation rate. A robust multiplex amplification protocol has been developed, validated and genotyped in a panel of approximately 1200 cats representing 38 recognized breeds in the United States. The multiplex demonstrates high discriminating power in cat breeds, exhibiting average locus heterozygosity across the 38 breeds of 0.71. A real-time polymerase chain reaction (PCR) based method has been developed for quantifying domestic cat genomic DNA, which is sensitive to 10 femtograms.
