Leslie A. Lyons, PhD
Department of Population Health & Reproduction, School of Veterinary Medicine, University of California-Davis, Davis, CA, USA
Objectives of the Presentation
Genetic considerations of FIP pathogenesis
Genetic considerations of the host genetics
Genetic resources-SNP arrays for the cat
FIP Case-Control Design
Overview of the Issue
Feline infectious peritonitis (FIP) is a complex disease of cats that has both environmental and genetic components controlling its pathogenesis. Both random breed and purebred cats get the disease, both the young and the old. The environmental components of the disease include viral exposure, stress and maternal immunity. The cat's genetic constitution controls its immune system and its overall susceptibility or resistance to disease. FIP has a complex pathogenesis and different genes and mutations may influence each step of the process. Therefore, FIP studies need to focus on identifying a homogeneous group of cases and controls for investigation. The positive selection of genes in cat breeds may simplify the genetic constitution of a cat, lowering the number of cases and controls need in a genome-wide association study for FIP. As with pathogenesis, different genes and mutations may influence FIP susceptibility / resistance within different breeds or populations. The developing SNP-based DNA array for the domestic cat will strongly support case-control genome-wide association studies in the cat, however, sufficient numbers of cases and controls will need to be ascertained. FIP studies will require concerted and consistent interactions with investigators, veterinarians, and breeders. Worldwide and coordinated sample collection schemes and diagnostic criteria need to be developed and promoted.
Feline enteric coronavirus (FECV) infectious precedes the development of FIP, however, FECV is necessary but not sufficient to cause FIP in all infected cats.1 Environmental components such as stress interact with the cats immune system, making the cat more susceptibly to FIP disease progression. Cats are far more susceptible to stress than they let on, changing the housing situation, overcrowding, the smell of dirty litter boxes or scent marking toms, all have been shown to elicit a stress response in cats.2 Overcrowding not only leads to stress, but may also increase viral loads in the immediate environment due to poor husbandry standards when having an overabundance of cats. Exchanges of cats can bring different and novel viral strains into a cattery or shelter, causing new challenges to an immune system of the cat. Thus, chronic re-infection with the same or different strains of the virus can stress the immune system, further exasperating the environmental stresses to the hosts defense mechanisms.
The cat's immune defense mechanisms also include its genetic constitution. Studies in humans made prominent headlines when genes affecting HIV infection were discovered that clearly elucidated genes which are shown to control viral susceptibility / resistance and disease progression.3,4 At each step during FECV infection and FIP pathogenesis, a gene could be affecting the success or failure or the defenses to ward off the invading virus. Cats could have genes that influence initial susceptibility to all FECV or perhaps a specific strain of initial virus infection. A different gene could influence viral replication and viral load in the gut, slowing viral mutations and the evolution of FECV to FIP causing strains. Entry of a FIP-causing strain into the macrophage could be a separate genetic component, as well as the strains future replication and the subsequent damage to the immune system. Hence, every event that is a step in the pathogenesis of FIP could have a genetic influence that is also impacted by the strain(s) of the virus invading the host.
Another consideration as to the genetics of the host is the age of the cat at the time of infection. The age of the cat has already been shown to influence the pathogenesis of FIP since more young cats develop "wet" FIP and older cats tend to develop "dry" FIP. Maternal immunity helps to protect young kittens, thus, susceptibility / resistance may also be influenced by a combination of the maternal genetic constitution as well as the kitten's individual genes. As the kitten matures, so does its immune system, somewhat changing throughout a lifetime, thus, age of onset of disease and the kitten's rearing environment may need to be considered, including cross-fostering to other queens. The pathogenesis to these two different FIP disease states warrants treatment as different forms of disease, thus, independent genetic studies.
Environmental influences, viral biotypes, maternal immunity, host genetics and immunity and disease pathogenesis all contribute to FIP being a complex disease trait in cats. Overcrowded shelters of random bred cats and overcrowded catteries of pure bred cats tend to have the higher incidences of FIP, suggesting the environmental components are significant influences in FIP pathogenesis. But the higher incidence in specific breeds may simplify the tangled web of disease development. By definition, cat breeds must share a sufficient genetic background that allows the production of individuals with specifically desired traits. These traits include coat colors, hair length and texture, body type and behavior. Because breeds generally work within a limited gene pool, accidentally, some undesired traits can become more prevalent within a breed, including a susceptibility or resistance to FIP. And since cats within a breed will have a more homogeneous genetic constitution than a bunch of random bred cats, identifying genes that cause susceptibility / resistance to FIP may take fewer individuals for a case-control based study. Some variation in breed susceptibility / resistance to FIP has already been noted by investigators.5,6
Positive selection for breed specific traits and linkage disequilibrium are the population dynamics that support the use of breeds in complex disease studies.7 When a specific trait is under positive selection, the mutation(s) that cause that trait generally become fixed within a breed, such as points in a Siamese or Birman.8 However, every gene on a chromosome does not "independently assort" during each generation, large blocks of genes are inherited as a unit from one generation to another. Thus, the genes and mutations residing in juxtaposition to the gene for the points coloration in Siamese and Birmans are also under position selection, hitch-hiking along with the truly desired traits, a selective sweep. This phenomenon is termed "linkage disequilibrium (LD)". Thus, if a gene that influences FIP pathogenesis is nearby, these mutations also accidently become fixed or more prevalent in a breed population and are in LD with a trait of interest. Over time, recombination during gametogenesis whittles away these inherited blocks to be smaller and smaller units. Overall, the more generations within a breed, the lower the LD. Generations can be influenced by time, older breeds having more generations, but also by population size and outcrossing, larger populations and more outcrossing leading to lower LD. The strength of the positive selection is also important and affects LD. If a breed must have a trait, selection is strong, if a trait is option, such as a color variant, the selection is weaker. Strong selection implies strong LD, longer blocks of DNA being inherited as a unit.
All things considered, a genetic study of FIP will likely entail several genetic studies of FIP. Different genes with influence different aspects of the disease pathogenesis, progression and status. The genetics of the virus, host and potentially the maternal immunity will need to be considered. The genes having the most major effects in one breed may be different than another breed and the given mutations may result in higher susceptibility or higher resistance. Working within a breed may simplify both the genetic and environmental components as breeders for a certain breed may have more common husbandry practices. The breed LD is under evaluation and should influence the design of the DNA array that will be developed for the domestic cat.
Genome-wide association studies (GWAS) combine the use of thousands of DNA markers with a case-control sampling process.9,10 The DNA array for the domestic cat will likely consist of ~100,000 (100k) single nucleotide polymorphisms (SNPs) that have an even distribution across the cat genome and sufficient polymorphism in a majority of breeds. A 100k SNP chip should be one of the best developed for companion animals, the dog and horse are already coveting or developing arrays of this higher density.
The LUPA project in Europe (http://www.eurolupa.org/) is a large international collaboration focusing on the investigation of complex diseases in the dog. Based on dog LD and SNP array size, the LUPA project suggests 20 cases and 20 controls for recessive (AR) trait studies, 50 cases and 50 controls for dominant (AD) traits, 100 cases and 100 controls for traits with a 5X increased relative risk in the population. Horse studies to date have been successful using slightly more individuals than the dog studies.11 Cat LD estimates are now being studies, but, knowing breed history and the selection processes in cats, cat LD can be anticipated to be less than for the dog, perhaps more similar to horses, thus, a higher density SNP array is required for the domestic cat.
Based on current knowledge for GWAS and cat LD, an FIP study should focus on a disease within specific breeds and will require over 100 cases and controls. As mentioned earlier, the cases need to be well defined as to their age, environment, disease type and as much of the viral genetic and environment as possible. The goal is to ascertain the most homogenous group of FIP cases that can be obtained. Likewise, controls need special attention as well. The individuals selected as controls should match the cases in as many aspects as possible, including potential exposure to the virus. The most consistent environments for cases and controls would be young kittens prior to departure from their rearing conditions. Thus, a case is a kitten that develops FIP, a control would be a sibling. If retrospective cases can be ascertained, the controls may now be in different households, and of course, older, but, they would still be considered a sufficient control. Half-siblings from the same cattery situation could also be considered as controls, as well as other older cats, particularly parents, in the household. If these older cats later succumb to FIP, disease would likely be due to different viral and genetic influences.
The ascertainment of FIP cases has begun but the enthusiasm needs to be re-enforced and spread to the worldwide cat community. No FIP case should be turned aside as who knows which breed will have an "outbreak" of FIP and which breeders become the most productively involved. FIP studies will require close and consistent interactions between the investigators, veterinarians, pathologists and the breeders to develop a homogeneous and sufficient group of cases and controls for a GWAS study. FIP genetics is complex, no one mutation or gene will influence all aspects of disease pathogenesis, but some genetic components will likely be deciphered in the coming years.
A genetic component could affect each step in FIP pathogenesis
Both host and virus genetics need to be considered independently and together
Case-control association studies are feasible but need to be focused on one aspect of pathogenesis
FIP studies will require concerted and consistent interactions with investigators, veterinarians, and breeders.
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