Objectives of the Presentation

 Review the known genetic tests in the domestic cat

 Review disease heterogeneity and its consequences on genetic tests

 Review nuances of testing in domestic cats breeds

 Review common testing methods and their accuracy

Overview of the Issue

 A variety of genetic tests has become available for the domestic cat in the past few years. Because many genetic tools have been developed, such as genetic markers and DNA sequence from the cat sequencing project, new discoveries will continue at a rapid pace. Finding the genes that cause interesting characteristics and sometimes undesired diseases is becoming a rapid and more efficient process. Once a mutation is identified for a gene that causes a particular coat color or disease, a service laboratory, either associated with the investigator who found the mutation, or an independent commercial laboratory, will establish a genetic test for that mutation to offer to the public. Nearly a dozen laboratories around the world now offer the genetic test for polycystic kidney disease in cats. All the laboratories may be technically very good and accurate, but, not all of them "know their cats". Hence, this presentation will address some of the "issues" with genetic tests, particularly in regards to cat breeds.

Additional Detail

The unique breed development for the cat suggests that the cat is not just a small dog. The domestic cat is one of 36 extant species of felids. Several small wildcat species, namely African, Asian and European wildcats, were available in all the early farming areas as the potential progenitors of the domestic cat. As humans transitioned from hunter-gatherers to farmers, villages produced refuse piles and grain stores, attracting mice and rats, a primary prey species for the small wildcat. Thus, cats actively participated in their owner domestication, both humans and felines developing a symbiotic, mutual tolerance. But, when does the cat begin to seek human affection and companionship and when does man develop the first controlled cat breeding programs still remains to be discovered? Regardless of where or when cat breeding developed, domestication of the cat is one of the most recent for our companion animals, distinctive from the much earlier domestication of the worlds other favorite companion animal, the domestic dog. Thus, the dynamics of genetic variation across cat breeds is likely significantly different from species that have more ancient domestication events and longer breed histories.

Random bred and feral cats represent the overwhelming majority of cats throughout the world, not fancy breed populations. Considering the worldwide distribution of cats, the USA has the highest proportion of purebred cats. The first documented cat show that judged cats on their aesthetic value occurred in London, England at the Crystal Palace in 1871. This first competition presented a handful of breeds, including the Persian, Abyssinian and Siamese. The first cat registry developed in the United States, The Cat Fanciers Association (CFA), by 1905, with the Maine Coon being an additional American breed. A majority of breeds have been developed in the past fifty years and many listed breeds have not been developed into viable populations. Persian cats and their related breeds, such as Exotics, a shorthaired Persian variety, are the most popular cat breeds worldwide, and represent an overwhelming majority of purebred cats. Although not all cats produced by breeders are registered, perhaps only 20 - 30%, the Cat Fanciers Association (CFA), one of the largest cat registries worldwide, generally registers approximately 40,000 total purebreds annually. Approximately 16,000 - 20,000 are Persians and approximately are 3,000 Exotics, thus, the Persian group of cats represents over 50% of the cat fancy population. Common breeds that generally have at least one thousand annual registrants are Abyssinians, Maine Coon cats and Siamese. Other popular breeds include Birman and Burmese. Most of these popular breeds also represent the oldest and most established cat breeds worldwide, thus genetic tools and SNP studies should primarily focus on domestic cats and a handful of fancy cat breeds.

Many breeds are derived from an older breed, forming breed families. Approximately fifteen breeds can be considered "foundation" or "natural" breeds, implying that many other breeds have been derived from these foundation cats. Derived breeds are often single gene variants, such as longhaired and shorthaired varieties, or even a no haired variety, as found in the Devon Rex and Sphynx grouping. Color variants also tend to demarcate breeds, such as the "pointed" variety of the Persian, known as the Himalayan by many cat enthusiasts and as a separate breed by some associations. Many cat breeds originated from single gene traits, such as folded ears of the Scottish Fold and dorsally curled pinnae of the American Curl, and then later developed into a more conformationally unique breed. The newly identified spontaneous mutations are recognized often in random bred cat populations, followed by morphological molding with various desired breed combinations. Thus, many new and some established breeds have allowable outcrosses to influence the "type" and to support genetic diversity in the breed foundation. Persians have a highly desired brachycephalic head type, thus they tend to influence many breeds. Breeds desiring the dolichocephalic type often outcross with the Siamese family of cats. The outcrosses that are valid for any breed can vary between cat registries and the same breed may have a different name depending on the country. For example, the Burmese registered by the Governing Council of the Cat Fancy (GCCF) in the United Kingdom and the Feline International Federations (FIFe) in Europe are known as the Foreign Burmese breed in the USA and these cat "breeds" have significantly different craniofacial type between the countries. The Havana Brown has developed into a distinctive breed in the USA, also with a significantly different craniofacial structure that its foundation breeds, the Siamese and Oriental Shorthairs. However, in Europe, the chestnut color variety of the Oriental Shorthair is similar to the Havana Brown. But, some breeds, such as Korats and Turkish Vans, have very similar standards across most all countries and registries. Oddly, some cat breeds are actually hybrids between clearly different species of cats and the domestic cat. Asian leopard cats are part of the foundation of the Bengal breed, which is a highly popular breed worldwide, but not registered by the CFA. Serval hybrids, known as Savannahs, and Jungle cat hybrids, known as Chaussies, are also growing in popularity. Hence, genomic tools should give some attention to these three cat species to support disease studies within these hybrid cat breeds.

Thirty-four mutations involving 24 genes have been identified in the cats that confer non-wildtype conditions or phenotypes. Ten of these mutations affect coat color, fur length or blood type and each of the resulting phenotypes segregate in random bred cat populations. In Table 1 are all the known genetic mutations in the cat that have been published or are in peer-review. In the case of diseases, usually they present in a specific breed and the disease is generally only associated with that breed. But you have to know your cats! Some breeds are allowed to outcross with others and some are legal or illegally used to help refine the "look" of another breed. Siamese and Persians both have a host of other cat breeds that they have influenced. Hence, any mutation found in one breed can be found in others if cross breeding has occurred. And, cats are all over the world and the rules between registries and associations are not always the same. Hence, an outcross that may be legal for TICA may be illegal for the CFA or perhaps the GCCF. Thus, testing laboratories need to understand some of these dynamics so that they know a test is valid for a given breed in any part of the world.

Why does one care if a test is valid? The concern is an issue called "disease heterogeneity". As owners, breeders AND veterinarians, we see a clinical presentation that is abnormal in our cats. However, any of us can quickly jump to conclusions. Cats have a lot of renal failure, it is NOT all caused by polycystic kidney disease (PKD). Cats have lots of cardiac diseases; it is NOT all hypertrophic cardiomyopathy (HCM). Even when there is diagnoses of HCM, we now know that it is not all caused by the same mutation. Herein lines the concern! An unknowing veterinarian, owner or breeder may want a cat to have a genetic test for HCM or PKD because the cat has clinical signs. The test comes back negative. This does NOT mean the cat does not have HCM or PKD if the test has not been proven in that selected breed. The PKD test works for Persians and related cat breeds. NOT for all breeds! A laboratory may very well run the test for you, but then you are on your own to understand the meaning of a negative test. This is why a test is generally listed for a specific breed. Until enough cats from a particular breed come forward with clinical data, such as ultrasound diagnoses and genetic test results, a test cannot be valid for all breeds. I get occasional reports of Maine Coon cats in Europe suspected of having PKD. Did someone breed them with Persians in Europe? Our lab will help valid this issue by doing free testing IF the clinical diagnosis can be provided.

Besides knowing your cats, one has to know their genetics also. The protein sequence for the "points' mutation in cats is the following:

The letters on the top line are the single letter codes for amino acids: A is alanine, R is arginine, G is glycine, and so on. There are twenty amino acids. The top line is the sequence for a non-pointed cat. The second line is humans, the third is the Siamese cat, which is pointed, and the last line is the dog. You can see that some places have different amino acids between the species, more between cats and humans than to dog. These are normal differences between humans, dogs and cats. The change of a glycine to an arginine in the cat is what makes a cat have "points".

But, in the DNA sequence, three nucleotides join together to code for one amino acid, kind of like an area code of a phone number. Hence, the DNA sequence is three times longer than the protein sequence. There are four nucleotides (A is adenine, G is guanine, C is cytosine and T is thymidine) that make up DNA. But, the trick is that the coding of amino acids is redundant! The amino acid glycine can be coded by the DNA sequence GGG, GGC, GGA, or GGT. So the DNA sequence in the area around the point's mutations reads like this:

CCTGGGAAT for the non-pointed cat

CCTAGGAAT for the pointed cat

The single nucleotide change of the guanine (G) to the adenine (A) changes the amino acid from glycine to arginine and this makes the cats have points when both copies of their DNA has the same change.

Here is the part where you have to know your genetics! A mutation can occur at any nucleotide site. What if the CCT became CCC, the thymidine (T) changes to a cytosine (C)? This change DOES NOT alter the amino acid, it still codes for proline. This is called a silent mutation, it does not affect the protein. When hunting for important mutations, the silent mutations we find we skip, this is normal genetic variation found between individuals or species and it does not change the protein, hence that cat will not have a change it color or its health. BUT, this change can mess up a genetic test! We had a Siberian with yellow eyes that was dominant white. The genetic test suggested the cat was homozygous for the pointed mutation. If so, the cat had to have blue eyes, regardless of being dominant white! What went wrong with the test? In this case, the cat did have the normal but silent variant in the sequence around the mutation for points. The cats sequence looked like this (in this case, presented are the two different alleles for the same cat):

CCCGGGAAT

CCTAGGAAT

Notice the cat has only one allele for points, the bottom line, but the top line has the silent mutation. Because the sequence was not the normal sequence before the important mutation site, the test failed for that top allele and the cat looked like it was homozygous for points! A good laboratory knows this can happen and has other ways to detect these "problems".

The normal level of variation between cats is expected, far less than 1% of a sequence that codes for a protein. Herein lies the problem for hybrid cats! The evolutionary time between cat species is millions of years, not hundreds to thousands between cat breeds and populations. An Asian leopard cat had a common ancestor with the domestic cat about 6 million years ago! The bobcat about 8 million years ago, the Serval about 9.5 million years ago and there is not strong estimate for the Jungle cat yet, but it is less than the leopard cat. Plus, for some of these wild cats, different subspecies have been incorporated into the breed! What about their genetic variation? This means that the DNA sequence between a domestic cat and one of these wild species will have lots of unexpected differences, maybe several percentage difference, less for the Jungle cat, more for Serval. The genetic differences are most likely still silent, they do not cause a disruption to the amino acid or the resulting protein, but, they wreak havoc with genetic tests! So, a testing lab has to know there cats to anticipate this problem.

Most labs recognize that disease mutations are specific to breeds, but, not the coat colors! The coat color mutations occurred during the early domestication of the cat before the breeds were developed, so, all breeds tend to have the same mutation. This is true for all the coat color tests so far. BUT, what about Bengals, Chaussies and Savannahs? The normal DNA sequence around each one the mutations for coat colors needs to be evaluated in many cats from each species in order to find the normal, silent mutations that occur between the wild cats and the domestic cats. At any given gene, in a Bengal, one never knows if you have one leopard cat sequence or two! Thus, we do not know how accurate coat color tests are for hybrid cat breeds! If the domestic cat alleles are present, the test is perfect, or at least you only have to worry about normal cat to cat variation. But, you never know when one allele or both is from the leopard cat. Which, most of the time you are trying to select for cats to have more leopard cat DNA, so, inherently the breed is selected for the DNA sequences that may cause the tests to fail!

What can be done? For each genetic test, the DNA sequence in the given region must be analyzed in many individuals of the wild cat species. We must analyze the pure wild cat or perhaps the pure F1 (wild cat to pure domestic) since in any other generations, we would not know if the sequences were wild cat or domestic. In the F1, we know we have to have one of each. This is why we have made recent requests for DNA samples from the wild cats. This request is open and anyone with a pure wildcat or F1 is encouraged to send in samples to our laboratory!

Table 1. Cat Traits and Diseases with Known Mutations

UP are mutations that are unpublished to date.

Summary

Genetic testing is becoming more commonplace for domestic cat breeds. However, the zeal of cat breeders and the misunderstanding of disease diagnosis can lead to misinformation and improper interpretations of genetic test results. Testing laboratories need to understand the relationships of cats and possible outcrossing practices for different organizations and countries. Scientific rigor must be employed to develop a test to ensure its accuracy in different populations and associated breeds. Both the veterinarian and the breeder need to recognize these nuances in cats to ensure proper use of the genetic assays.

References/Suggested Reading

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