Cat Genetics 101 – Part 2
Feline Genetic Testing
By Lucy Drury, MT(ASCP)SC, and Roger Brown, DVM
In our last installment, we covered some of the basic terminology and concepts in that mysterious world of genetics. Next, we will cover some of the basics of feline genetic testing: what is currently available, how the tests are performed, some dos and don’ts, and what to do with the information once you have it. If you haven’t read the prior article and genetics is a new and/or confusing topic to you, you may want to review Part I, as that information will be helpful in understanding Part II.
Genetic Testing: What is it?
Genetic testing, fundamentally, is an attempt to determine an organism’s genotype, particularly when observing that the phenotype (how the organism appears) does not reveal all that one needs or wants to know about the genotype. The ability to test for a specific genetic trait, though, has only been available to the general public for less than twenty years, and is the product of the investment – over the last 70+ years – of billions of dollars and countless hours by researchers focused on deciphering the genetic code.
Prior to the emergence of gene sequencing technologies, one could only rely on recording the phenotypes of the parents and offspring and then infer the genotype from controlled reproduction through multiple generations. However, the last half of the 20th century saw explosive advances in the field of genetics, starting with the discovery in the 1940s that genetic material is made of deoxyribonucleic acid (DNA) and that DNA is composed of four nucleotide bases, adenosine (A), thymine (T), cytosine (C), and guanine (G). In the late 1940s and 1950s, thanks to the work of Pauling, Franklin, Watson, Crick, and many other molecular biologists, the molecular structure of DNA was determined along with the relationship of the nucleotides (base pairs A-T and C-G). This paved the way for the development of technology that enabled the sequencing of the base pairs in the DNA strand (DNA sequencing) starting in the 1970s. This in turn allowed the matching of a specific sequence of base pairs on a specific position on a chromosome, which then enabled the identification of genes and single-nucleotide polymorphisms (sNPs), which are variances in a single nucleotide at a specific position. 2,9
An understanding of how these genes function in the synthesis of specific proteins in the organism, combined with observation of expressed traits and diseases, allows researchers to map specific genes and sNPs to the observed traits and diseases. While initial testing methods were labor-intensive and only found in research laboratories, recent advances in technology have exponentially reduced the cost and testing time and made it economically feasible for commercial laboratories to offer the testing. While there are currently many methods of genetic testing used today, with the “gold-standard” being direct DNA sequencing, it is still labor-intensive and expensive. As mentioned in Part I, the emergence of Polymer Chain Reaction (PCR) technology in 1983 revolutionized genetic research and eventually made determining specific genetic traits and mutations relatively easy and cheap. About a decade ago, sNP array testing methodologies were developed. At a high level, this methodology was created by combining DNA array testing (a technology used to detect multiple DNA sequences of interest simultaneously) and PCR technology (which amplifies the sNPs of interest). Array chips could then be developed that tested for all the sNPs of interest to a specific population, such as the array developed by Neogen’s GeneSeek laboratory for CFA. sNP array testing further reduced the complexity and cost of genetic testing.1,3,4
How Does It Work?
While a detailed description of genetic testing methodologies is well beyond the scope of this article, there are a few bits of information useful to the cat fancier and breeder. The core technology of most commercially available genetic testing is Polymerase Chain Reaction (PCR) . PCR testing, at a high level, takes a single or a few copies of a specific DNA sequence and amplifies it so it may be detected. However, it is important to understand that PCR is an underlying technology made up of many specific testing procedures, which vary greatly in the types of results they produce, their sensitivity, and their accuracy. For example, there are end-point and real-time PCR tests. An end-point test will only tell you IF a particular sequence is present; a real-time test will tell you how much of that sequence is present. How much is present is generally not as important in genetic testing as it is when PCR is used to diagnose what microorganisms may be plaguing your cat (a topic for another day), but the sensitivity and accuracy are extremely important.7
What is within the scope of this article is how to obtain the best possible test result. While there are many factors that can impact the overall quality, most of them are not in the cat fancier’s control. There are three factors, however, that can be controlled and which are vitally important.
The first is proper sample collection. Typically, buccal cells from the inside of the cat’s cheek are collected on a cotton swab, folded up in clean paper, and sent off to the lab. It is essential to be sure they are properly labeled and do not come into contact with samples from other cats. Many labs also accept blood collected by your vet. Make sure you follow exactly each laboratory’s instructions for collecting the sample. Don’t assume that the process is the same for every laboratory. For example, some labs want you to use a specific brand of cotton swab; others don’t care what you use. While it may seem to you that the lab demanding the particular type of swab is being overly particular, they may be using a different process, and their process is only reliable and tested with a particular kind of swab. Don’t take short cuts – that’s your hard-earned money being spent on the test! If you do what they tell you and the result comes out wrong, they can’t blame you. If you don’t do what they tell you, it’s your fault by default. One other tip to help ensure the accuracy of your test: Wear a new, unused pair of disposable gloves when you collect your sample and handle your testing materials, and change them if you are testing multiple cats. You are measuring substances that are present in extremely small quantities, and you can easy contaminate your sample if you are in a multi-cat household. Try to think about all the things that could produce cross-contamination, such as shared food bowls, grooming each other, your handling them, floating hairs in the air … and try to eliminate them as much as possible at least 30 minutes or more before sample collection.
The second factor is choosing your laboratory. They are not all created equal. What testing procedure are they using? How long have they been in business? What are the qualifications of their staff? What resources are available if you have questions? Do they have a veterinarian on staff, and does that veterinarian have experience with genetics and laboratory testing? Don’t be afraid to ask questions. Even in the hands of experienced professionals, genetic testing is not foolproof and you should understand what those limitations are. A sign of a good laboratory is one that knows and will tell you the limitations of their testing methods, particularly the percentage of false positives and negatives. This is critical to know when interpreting results, particularly when the results don’t make sense.
Which leads us to our third factor: Question and potenially repeat all tests producing questionable results, no matter how reputable your lab is. If necessary, send them to a different laboratory for confirmation. If you take away just one point from this article, it should be this: No single lab test is 100% accurate. It might be 99.9999 percent accurate, but there are many potential sources of error before, during, and after testing. We briefly discussed pre-test contamination; other potential sources of error are a technician or the equipment having a bad day while testing, or results being mixed up and matched to the wrong cat after testing. A good lab will be willing to repeat or confirm the test results at no charge if the results are questionable.
Who and Why?
So, who should do genetic testing on their cats and why should they do it? There is no easy answer to this question, particularly for health markers. If you ask breeders for their opinion, you will likely get answers ranging from “Don’t waste your money testing, just buy clean lines,” to “Test everyone for everything.” The answer also depends on what your plans are for a particular cat and where it came from. If you are an exhibitor who is buying a cat just to show or buying a pet (each with a well-documented pedigree free of any genetic issues) from a quality, reputable breeder, then you probably are wasting your money testing the cat. However, if you are a serious breeder planning to buy a new foundation cat, or if you have specific traits you want to breed for, or if you can’t bear the thought of selling someone a kitty only to have it die before it’s a year old from a genetic abnormality, you would definitely want to invest in genetic screening before you purchase the new kitty. What tests you choose will depend on the breed. For example, inherited polycystic kidney disease (PKD) is prevalent in Persians and breeds out-crossed with Persians, but rare in others, so Persian breeders would definitely want to test for PKD.7 Knowing the blood type to avoid breeding incompatibilities is more important in some breeds than in others. If the cats in your breed all have the same blood type, then it would probably be a waste to test for the blood type, unless you have some questions about the pedigree.4
One might question the need to test a breeding cat if the pedigree is well-documented and free of genetic problems. In interviewing a number of breeders, there were two general schools of thought. Let’s use PKD in Persians and their kin as an example. The PKD1 gene mutation that is associated with the disease is dominant and has high penetrance, meaning it only takes one copy to produce disease and a cat with the gene almost always develops the disease. There are some cases of healthy cats with the mutation, but they are very rare. Two copies of the gene are worse than one, and a fetus that is homozygous for the mutation almost never goes full-term.4 So wouldn’t it be safe to assume the lines are clean if some of the parents have been tested and no cases have manifested themselves for multiple generations?
Well, maybe. It depends on how lucky you feel and how well documented those lines really are. Remember the earlier comments on laboratory errors. Can you ever really be sure those lines are clean? What if a test produced a false negative result for the mutation, and the breeder produced a litter before the disease manifested itself symptomatically, and then sold that kitten to you? If you can trace the pedigree six generations back and find no problems, yes, the odds are very, very low that the kitten has the mutation. A single screen used to cost several hundred dollars to perform, so it may have been worth it to study the pedigree instead. Currently, the cost is no more than a couple bags of litter, and the cost of being wrong could be very high in terms of vet bills, having a cat you can’t breed, or unhappy kitten buyers if you did breed it before you knew the cat had the mutation. Being on the safe side is a better option when mitigating the risk by using relatively inexpensive testing to allow you to move forward with a clear breeding program.
On the other hand, what about genetic testing when the penetrance is incomplete, meaning there are a significant number of cats that may carry a mutation, but never express it? An example of this is the gene identified in Maine Coon Cats as a marker for hypertrophic cardiomyopathy (HCM). Your cat may test positive for the HCM gene but never develop the disease. Conversely, your cat may develop the disease and not test positive for this particular mutation. Since echocardiograms can be performed on the cat to detect heart disease, should you even bother with genetic testing for HCM?6
Again, well … maybe. Echocardiograms can run upwards of $450 a scan, and HCM, whether it be from inherited factors or environmental factors (e.g., toxins or infectious agents), may not show up on the scan for a year, two years or even six years. That cat may have produced dozens of kittens and passed on the mutation to multiple generations by the time the disease manifests itself. So, again, if the cost of the test is relatively low, why not get it done? While it can’t guarantee your cat won’t develop HCM or other heart disease down the road, most experts would not recommend you breed a kitty that tests positive for any mutation associated with an undesirable trait or disease (even if the penetration is incomplete) … It is just not worth the risk. What you should do if the echocardiogram has abnormalities with or without evidence of disease is outside the scope of this discussion. The main conclusion to draw from this discussion is that there is very little downside to doing genetic testing, and much to be gained. The results may not be desirable and very disappointing, but it is better to know than NOT know in nearly every case.
When Should You Do It?
When should testing be performed? If you’ve decided to move forward with genetic testing, there is no time like the present, particularly if you are selecting your parents for that next litter of kittens. If you are buying the kitten or cat from another breeder, you should ask the breeder to have the testing done at a reputable lab prior to purchasing the kitten. If they won’t do it themselves, ask for a genetic guarantee. While a cat can be tested at any age when using a blood sample, if you are using a cheek swab, it may be better to wait until the kitten is weaned. With an older kitten, handling is generally easier and you can swab the cheek more effectively so that you can get enough cells (likely avoiding contamination from the mother’s DNA). Generally, each genetic marker will only need to be tested once in a lifetime, unless there is reason to believe the first test was performed incorrectly. As mentioned earlier, there is probably no need to genetically test a cat that you have no plans to breed as long as the cat is from a reputable breeder with healthy lines, particularly if the parents have been genetically tested. Testing does offer peace of mind before you buy the kitten, no matter your reason for choosing it.
In Our Next Installment….
We will discuss new genetic markers that will be coming to a laboratory near you soon!
References:
- Bumgarner, R. 2013. Overview of DNA Microarrays: Types, Applications, and Their Future. Current Protocols in Molecular Biology. 101:22.1:22.1.1–22.1.11
Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4011503/ - Wikipedia. (2017, Oct 16).
Retrieved from https://en.wikipedia.org/wiki/DNA. - DNA Sequencing. Wikipedia. (2017,Oct16).
Retrieved from https://en.wikipedia.org/wiki/DNA_sequencing. - DNA Tests Available. Neogen GeneSeek. (2017, Oct16).
Retrieved from http://www.catdnatest.org/index.shtml. - “Initial sequence and comparative analysis of the cat genome.” Genomic Research. 2007 Nov; 17(11): 1675–1689.Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2045150/.
- Lyons, L. “Feline Genetics: Clinical Applications and Genetic Testing.” Topics in Companion Animal Medicine. 2010 Nov; 25(4): 203–212.
Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3073564/. - Polymerase Chain Reaction. Wikipedia. (2017, Oct 16)
Retrieved from https://en.wikipedia.org/wiki/Polymerase_chain_reaction. - The Discovery of the Molecular Structure of DNA – The Double Helix: A Scientific Breakthrough. (2017, Oct 16).
Retrieved from https://www.nobelprize.org/educational/medicine/dna_double_helix/readmore.html
Originally published in Cat Talk Magazine