Genomics is a field of genetics focusing on the overall organization of genes, including their order, distance, and structure, on the chromosomes of a given species. Several early prominent geneticists recognized that many phenotypic traits influence the appearance of an individual. Coat colors and fur types, including those of the cat, had simple modes of inheritance and followed the same inheritance rules noted by Gregor Mendel in his studies with pea plants. One of the first traits ever mapped to a specific chromosome in any species—the first genomics—was the Orange coat color of cats, which was recognized to be sex-linked and therefore on the X chromosome. Since that time, the inheritance patterns of many phenotypic traits in the cat have been defined, the loci localized (mapped) to chromosomes, and now the causative genes and mutations are rapidly being identified (see Chapter 52: Genetics of Feline Diseases and Traits). Early gene mapping studies indicated the cat has a genome organization more similar to that of humans than to the mouse or the domestic dog.¹ Since the limitations of mouse models in human studies have been realized and the cost of genomic and genetic resource development is within a feasible range, genomic studies in the cat have significantly advanced. Advances in the genetic tools and resources support the investigation of feline health problems, directly improving the health of the cat and facilitating the use of the cat as a biomedical model for human disease. This chapter presents an overview of the evolution of genetic tools for the domestic cat and highlights their use and value for improving feline health. A glossary of terms is found in Box 51.1.

Early studies of chromosomes revealed the domestic cat has 18 autosomal chromosomes and the XY sex chromosome pair, implying the cat genome has 38 overall chromosomes (Fig. 51.1). Cat chromosomes are clearly defined by size, centromeric position, distinctive banding patterns with Giemsa staining, the size of the short (p) and long (q) arms of each chromosome, and the presence of only a few small acrocentric chromosomes, which have no p arms and are traditionally hard to distinguish from one another. Various cytogenetic techniques, such as R-, RBG-banding, and fragile site studies have also helped distinguish and characterize the cat chromosomes. For example, cats do not have a significant fragile X site on the X chromosome as is found in humans and is associated with intellectual disabilities. Although a sequential numbering of the chromosomes has been proposed,² the historic classification of chromosomes into morphologic groups has been retained in the cat. Hence, cats have three large metacentric chromosomes (A1 to A3), four large subtelomeric chromosomes (B1 to B4), two medium-size metacentrics (C1 and C2), four small subtelomerics (D1 to D4), three small metacentrics (E1 to E3), and two small acrocentrics (F1 and F2). The X chromosome is midsize and subtelomeric, similar to chromosome B4 (Fig. 51.1).

Domestic cats have a chromosomal architecture highly representative of all felids and ancestral for most carnivores and are a good representative for the Carnivora order.3 Early chromosome staining recognized some major alterations in the felid genome, particularly the Robertsonian translocation of F1 and F2 to form chromosome C3 in the ocelot lineage of cats from South America (2 N = 36). Minor pericentric inversions, additions, or deletions of the small chromosomes cause variation in the felid karyotype. However, new data from the assembly of the entire genome of a growing number of cat species has revealed that some suspected pericentric inversions are actually expansions of repeated sequences of DNA.⁴

Chromosomal differences are present in some cats with reduced fertility and intersex cats, especially via the loss of one of the sex chromosomes. Karyotypic and now gene-based assays are common methods to determine if a cat with ambiguous genitalia or a poor reproductive history has a chromosomal abnormality. Karyotypic studies of male tortoiseshell cats have shown they are often mosaics or chimeras, being XX/XY in all or some tissues. Other significant chromosomal abnormalities causing common syndromes are not well documented in the cat.

The variation of cat chromosomal sizes is sufficient to allow easy sorting of chromosomes using a technique called flow sorting. The DNA in the flow-sorted pools of each chromosome can be individually dye labeled. The dye-labeled DNA from each chromosome can then be hybridized to chromosomes of another species, such as humans, to provide a view of which chromosomes in the two species have the same DNA (Fig. 51.2).⁵ For example, the p arm of human chromosome 1 (1p) is largely composed of the same genes that are on the cat chromosome C1, whereas human chromosome 1q is composed of genes that are found on cat chromosome F1. The chromosome painting technique can also be performed reciprocally where cat chromosomes are painted onto human mitotic chromosome spreads and human chromosomes onto cat mitotic chromosome spreads, revealing the high conservation of chromosomal arrangement of cat to humans, especially compared to mice. Thus, chromosome painting gives an excellent overview of cat genome organization, which greatly facilitates candidate gene approaches because the location of specific genes can be anticipated in cats from comparison with the genetic map of humans. This additional confirmation of conservation to human genome organization further supported development of genetic resources for the cat as a valuable animal model for human disease.

The cat’s importance in human health, comparative genomics, and evolutionary studies supported the decision of the National Institutes of Health—National Human Genomics Research Institute to produce a low coverage (2×) sequence of the cat genome.1^a Genome coverage describes how many times the same DNA sites are sequenced. As the same sites are sequenced more times, the coverage becomes deeper, and the determination of the sequence is more accurate. Led by the Broad Institute and AgenCourt, approximately 327,037 DNA variants termed single nucleotide polymorphisms (SNPs) were identified in the sequence from one highly inbred Abyssinian cat named Cinnamon.²⁰ Because this was a small-scale sequencing effort, only approximately 65% of the euchromatin (gene-coding) sequence was identified. The sequence assembly suggested the identification of 20,285 feline genes that have counterparts (orthologs) in the human genome. This sequencing effort reiterated the conservation between human and cat chromosomal organization by identifying 133,499 regions of conservation and by identifying additional introgression sites of endogenous retroviruses, such as FeLV.^21,22

Since then, improvements in the cat genome assembly have greatly enhanced the accuracy and utility of the resource. More in-depth sequencing provides a more accurate picture of the relationships of genes on a chromosome. The euchromatin coverage has improved to approximately 90% to 95%. The better coverage implies that a more cat-specific genetic sequence will be known for any gene of interest. Mutation screening methods will be more efficient, leading to the identification of causative mutations more rapidly. In addition to more genetic variation being identified in the Abyssinian cat used for the feline genome project, additional cats were partially sequenced as well. Four representatives from six breeds (Birman, Maine Coon, Norwegian Forest Cat, Egyptian Mau, Japanese Bobtail, Turkish Van) were sequenced. A pool of wild cats was also included, as well as four random-bred cats from Southeast Asia. In addition, Hill’s Pet Nutrition supported a private sequencing effort which included sequencing of single cats from five different breeds (Persian, Siamese, Ragdoll, Cornish Rex, Burmese), a western random-bred cat, and an African wild cat.²³ These combined sequencing efforts helped identify the normal genetic variation that is found across cat breeds and populations, especially the SNPs.

As techniques for genome sequencing have improved, the cat genome assembly has also continually improved, producing versions 8.0 and 9.0.^24,25 In addition, the use of hybrid animals, such as the F1 Bengals from a domestic and leopard cat cross, have greatly improved the efforts to piece together the cat genome.⁴ A new reference assembly, termed Fca126, is now available, based on the maternal parental random bred cat of a F1 Safari cat, which is a domestic cat bred to a Geoffroy’s cat (Leopardus geoffroyi).^b This newest genome assembly of the cat is on par with the quality of the human genome, and has many complete chromosomes, reading the exact DNA sequence from telomere to telomere.

An important by-product of the DNA sequencing effort is the identification of normal genetic variation in the cat genome SNPs (i.e., single nucleotide variants). The SNPs can be demonstrated to be specific to one breed or common across many breeds and populations. The genome assembly allows the determination of the positioning of the SNPs across the genome. A resource called a DNA array or DNA chip can then be produced that contains assays for the highly polymorphic and evenly spaced SNPs; thus, these arrays can assess the entire genome of the cat in one experiment. Hill’s Pet Nutrition provided funding to develop a cat DNA array with approximately 63,000 SNPs.26 Each DNA chip, which is about the size of a microscope slide, has 12 regions and each region is used to test one cat. Each region has the assays for approximately 63,000 SNPs. The major benefit of the arrays is that they allow assessment of the entire genome; this is known as a genome-wide association study (GWAS). Because the SNPs are at such a high density, the cats used for a GWAS can be from a population and not direct relatives. Thus, individual cases of diseases or traits can be examined from a population or across breeds and populations; cases (cats with the trait) and controls (cats without the trait) are required. In addition, because there is less concern for the mode of inheritance of the trait, a GWAS can be performed even with traits that may have complex inheritance but with a high heritability or high relative risk in a population. Fewer cases are required to investigate a recessive trait, more for a dominant trait, and even more for complex traits that cause an increased relative risk—the lower the relative risk, the more cases required.

A second factor, linkage disequilibrium (LD), is considered when determining the number of cases and controls required for a GWAS. Linkage disequilibrium is often different among breeds, as seen in dogs and horses, and generally nearly absent in large random-bred populations, such as humans and random-bred cats. The lower the LD, the lower the power of the SNPs to identify an association with a trait of interest. Burmese and Birman cats have extended LD whereas Persians have shortened LD, implying more Persian cats would be needed for the same study than Burmese cats.²⁷ Thus, if LD is low, either more SNPs are needed or more cases and controls are required to have effective association studies using LD. Because the chip will have a defined set of SNPs, the LD estimates will predict the number of cases and controls for a study. If the population has lower LD, more samples will be required.

Cat array studies have already proven successful. Using less than 20 cases and controls, a heritable craniofacial defect²⁸ and hypokalemia have been identified in Burmese cats.²⁹ The mutation in Scottish Folds that causes the folded ears and is associated with osteochondrodysplasia³⁰ as well as the rare AB blood type common in Ragdolls have been identified based on analyses of data from the 63 K array.³¹ The arrays are a strong resource because DNA from buccal swabs is sufficient for analyses and only cases and related controls are required, not every member of an affected pedigree. Thus, this low-density DNA array for the cat has proven extremely powerful for diseases with Mendelian inheritance.

The success of the human genome project has led to technologies that can be used by any species for genetic studies, including the domestic cat. In the effort to drive the cost of human whole genome sequencing (WGS) to under $1000 USD per genome,32 so too have the costs dropped for WGS in cats. With approximately 2 mL of whole blood and for a cost of about $700 USD, a WGS can be produced for an individual cat. However, a key to generating WGS that is important to the research community is to produce the data using the same techniques and technologies and to combine the data into one dataset to allow researchers access to each cat that is sequenced. To facilitate this key aspect of WGS, the 99 Lives Cat Genome Sequencing Initiative / Consortium ( e-Box 51.1) was developed. Over 90% of the sequenced genomes are 30× coverage, with a minimum of 20× coverage, and produced using only 100–150 base pair paired-end reads from PCR (polymerase chain reaction)-free libraries using Illumina HiSeq sequencing technology. The genome dataset is comprised of 284 domestic cats, including at least 22 breeds, a representative from the ten racial populations of cats, four parent-offspring trios, and a few affected sibling pairs. Over 40 investigators have contributed sequences to the consortium as well as three industry partners. Funding has been provided by various contributors for each investigator, the EveryCat Health Foundation (formerly the Winn Feline Foundation), the National Geographic Foundation, and industry. Nearly 60% of wild felid species are represented within the dataset including cats from the domestic cat, leopard cat, puma, lynx, caracal, and Panthera lineages. The 99 Lives dataset has led to the discover of DNA variants likely causal for congenita myasthenic syndrome in Devon Rex and Sphynx cats,³³ progressive retinal atrophy in Persians,³⁴ bob tail trait in Japanese and Kurilian bobtails and some PixieBobs,³⁴ as well as several other traits. The 99 Lives dataset has allowed the identification of a vast number of variants in the cat genome that can support the development of a high-density DNA array.

A highlight of the 99 Lives project is the demonstration of “precision medicine” in cats. In precision medicine, an individual’s genetic profile is used to customize health care, including medical decisions, practices, and/or products that are tailored to the individual patient. Genetic testing has been a part of human health care in the United States since the 1960s with the national implementation of the DNA mutation test for phenylketonuria. Newborn screening programs are routine in the United States and other countries. Each state provides testing that is most appropriate for their ethnic population base. Most people do not realize that they were tested for a battery of genetic diseases from blood samples collected shortly after birth. Such testing is now commercially available to the public through several companies that specialize in the detection of single gene trait mutations as well as analysis of genetic ancestry, such as 23andMe, FamilyTreeDNA, and the Genographic Project ( e-Box 51.1). Similar offerings are available for domestic cats. Many laboratories, such as the Veterinary Genetics Laboratory at the University of California, Davis ( e-Box 51.1), offer most of the genetic tests important for cat breeds as well as a cat ancestry test that can determine a nonpedigreed cat’s race and potential breed (see Chapter 50: A Short Natural History of the Cat and Its Relationship with Humans). In humans and cats, the future of individual health care will rely on the individual’s personal genome.

Whole genome sequencing for individual patients is becoming routine in human medicine and is likely to become a part of routine health care management within our lifetimes. Hence, the novel mutations that cause children to have what appear to be sporadic and random health issues and dysmorphologies may be readily detected. Similarly, WGS of individual cats has been initiated, suggesting the possibility of routine use for feline health management in the future. With more than 100,000 individual human genomes sequenced, the database of normal and detrimental genetic mutations is well-defined.³⁵

As mentioned earlier, a key aspect to WGS is using the same technologies and combining the genome sequences into one dataset. This large dataset identified all the DNA variants in cats, most of which are neutral variants that do not cause disease. By comparing just a few cats with disease to this database, all the neutral or non-causal variants in the “case” genome can be eliminated. Already the 99Lives dataset has proven successful by identifying causal mutations in two sibling cases of British shorthairs with an autoimmune lymphoproliferative syndrome.³⁶ A hallmark success was the identification of an undiagnosed lysosomal disease in a feline patient at the University of Missouri Veterinary Health Center. The genome of this cat with what was considered a rare, heritable disease, was compared to the genomes of 83 normal cats and a novel form of Niemann-Pick Type C was identified.³⁷ The cat was still alive when WGS clarified the diagnosis although, unfortunately, no treatment is available for this disease.

Disease resistances and susceptibilities are also important to the future of feline genetics. Susceptibility to feline immunodeficiency virus (FIV) and particularly to disease caused by feline coronavirus are likely to be of particular interest (see Chapter 52: Genetics of Feline Diseases and Traits). Although FIV has low morbidity and mortality rates in the cat, the genes influencing the cat’s tolerance of FIV could shed light on interactions in humans and other species with similar immune-compromising pathogens. Feline enteric coronavirus is nearly ubiquitous in domestic cats. As an enteric pathogen, the virus may cause some malaise and diarrhea, but it is otherwise innocuous. However, mutated viral forms cause feline infectious peritonitis (FIP), which has a nearly 100% mortality rate in domestic cats, regardless of race, color, or breed. Deciphering the genes involved with infection and disease progression for FIP would be a major advance in feline health (see Chapter 52: Genetics of Feline Diseases and Traits). The cat genome sequence and the DNA arrays will greatly facilitate these studies.