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. We have completed the third genetic linkage map of the domestic cat, which was generated in a large multi-generational domestic cat pedigree maintained by the Nestl -Purina PetFood Company (n=483 informative meioses). Previous first and second generation maps of the cat were generated in an interspecies pedigree between the domestic cat and the Asian leopard cat. Whereas, this gave us the opportunity to map and integrate Type I (coding loci) and Type II (polymorphic microsatellites) loci, the current map allows us to address whether the hybrid nature of the previous pedigree had an impact on recombination values. The third generation map is composed entirely of microsatellite loci. Approximately 625 loci are ordered in the current map. Linkage groups have been assembled and ordering of loci within the linkage groups is complete. The current map demonstrates considerable improvement over previous maps. All of the cat's 18 autosomes are now spanned by single linkage groups; marker density is more than doubled, providing a 5 cM resolution. There is also greatly expanded cover of the X chromosome, with some 75 microsatellite loci. The pseudoautosomal (PAR) and nonPAR regions of the X-chromosome are spanned by separate linkage groups. Approximately 85% of the markers contained in the genetic linkage map are also mapped in the most current radiation hybrid (RH) map of the cat. This provides critical reference and integration with coding (Type I) loci. Whereas the third generation map is composed entirely of microsatellite loci, the access to extended genomic regions flanking the repeat motif, enabled by the recent cat 2X whole genome sequence,, and the whole genome sequence (WGS) of the dog have enabled us to obtain identifiable orthologues in the canine and human genome sequences for over 95% of the microsatellites. Essentially, practically every microsatellite acts as a virtual Type 1 locus. Combined with the cat RH map, these genomic tools provide us with the comparative reference to other mammalian genomes which is critical for linkage and association mapping. Mapping and Characterization of Genes Associated with Inherited Disease Pathology in Cat Pedigrees with Homology to Human Hereditary Disease: 1. Autosomal recessive retinitis pigmentosa (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 Narfstrm 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. Linkage mapping and sequence analysis of retinally expressed genes identified a splice junction mutation which introduces a premature stop codon in CEP290, a gene recently implicated in retinal degeneration in human Joubert syndrome and Lebers congenital ameurosis (LCA), a frequent cause of human retinal atrophy. The cat model provides an important model of mutation in a primary ciliary protein gene, which have gained attention recently due to their implication in human hereditary disease (polycystic kidney disease, retinitis pigmentosa and recently kidney cancer). The cat model will be important in both functional studies and as a gene therapy model. 2. Four Independent Mutations in the Feline Fibroblast Growth Factor 5 Gene Determine the Long-Haired Phenotype in Domestic Cats. The hair follicle provides a uniquely mammalian model in which to study the complex genetic regulation between stem and stromal cells during self-renewal and terminal differentiation of a tissue. Genetic modulation of the hair follicle cycle can affect hair length, providing a rapid means for significant phenotypic change under either artificial or natural selection. A linkage scan in the Nestl-Purina pedigree, which is segregating for the long hair phenotype, demonstrated significant linkage to a genomic region flanking a strong candidate gene for the long hair phenotype, fibroblast growth factor 5 (FGF5). Sequence analyses of FGF5 in a survey of unrelated cats from 14 short and 12 long-haired breeds revealed 4 separate mutations predicted to disrupt the biological activity of the FGF5 protein. Pedigree analyses of 2 colonies of non-breed cats containing both short and long-haired individuals demonstrated that different combinations of paired mutant FGF5 alleles segregated with the long-haired phenotype in an autosomal recessive manner. Association analyses of over 350 genotyped breed and non-breed cats were consistent with mutations in the FGF5 gene causing the long-haired phenotype in an autosomal recessive manner. 3. Completion of a Population Genetic Database of Domestic Cat Breeds to be Utilized in a Domestic Cat Short Tandem Repeat (STR) Forensic Typing System and validation of the STR typing system. Under support from the National Institutes of Justice, we have completed the development of a microsatellite forensic typing system and genetic database of cat breeds for genetic individualization of cat specimens. A robust multiplex amplification protocol has been validated and genotyped in a panel of 1040 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. We have determined that the high incidence of null alleles which we observed for one of the loci in cat breeds of Southeast Asian origin is due to a deletion in a primer site. 4. Assessment of Patterns of Molecular Genetic Variation Among Cat Breeds. Genetic variation in cat breeds was assessed utilizing a panel of STR loci genotyped in 38 cat breeds, and 284 single nucleotide polymorphisms (SNPs) genotyped in 24 breeds. Population structure in cat breeds generally reflects their recent ancestry and absence of strong breed barriers between some breeds. There is a wide range in the robustness of population definition. Most of the variation in cat breeds was observed within a breed population (83.7%), versus 16.3% of the variation observed between populations. An understanding of the relationship of cat breeds is important in future studies of hereditary disease to determine the likelihood that the causative mechanisms of similar pathologies may be common by descent in related breeds
