Episode 208: The Power of Genotype & Phenotype in Dog Breeding

Discover the limits and benefits of a dog s genetic evaluation versus its phenotypic diagnostics. Both can be used effectively to have a powerful impact on your bloodline.

Watch the video version of this presentation here.

Transcript

Nicole Engelman  00:04

Welcome to the Good Dog Pod. Join us every other Wednesday when we discuss all things dogs, from health and veterinary care to training and behavior science, as well as the ins and outs of Good Dog and how our platform can help you successfully run your breeding program. Follow us and join Good Dog's mission to build a better world for our dogs and the people who love them. 

Thank you all so much for joining us for another webinar. Today we have our guest Dr. Victor J. Stora, who's going to be presenting on the power of genotype and phenotype in dog breeding. Dr. Stora joins us to share how we can discover the limits and benefits of a dog's genetic evaluation versus its phenotypic diagnostics and how both can be used effectively to have a powerful impact on your bloodlines. So we're really excited to have him joining us again today. We're also excited to partner with Purina to bring you this webinar and to bring it to life. As I mention every time we host one of these webinars, we have already hosted a ton this year, and I just want to remind everyone again that you can always find all recordings in our Good Breeders Center, in case this is your first time joining us, and this webinar is also being recorded, and it will be added there, and the recording will also be in our newsletter. So don't worry about missing anything. Or if you want to rewatch this, the recording will make its way to you. And if you are joining us for the first time, we just wanted to share a little bit about who we are at Good Dog. Good Dog is on a mission to build a better world for our dogs and the people who love them, by advocating for dog breeders like yourselves, educating the public and promoting canine health and responsible dog ownership. We're a secure online community that's created for responsible breeders like yourselves to connect with quality, Good Dog applicants from across the country and find homes for your puppies. We help breeders run all aspects of their breeding programs, everything from getting your new litter of puppies listed, connecting with high quality buyers, getting payments for those puppies, sending them off to their new homes, and all of those smaller details that happen in between a litter of puppies being born and going off to their forever homes. We're really here to support breeders throughout every step of that. We also have a number of free educational resources, events just like this, health related discounts to help your programs thrive in other ways too. So if you're not yet part of our community, we would love to have you join and learn more about our mission, and you can do that at GoodDog.com/join. That's a little bit about us. And then I just wanted to wrap things up by sharing a little bit more about Dr. Stora and his background in canine health before we pass things over to him. 

Dr. Stora graduated from LSU School of Veterinary Medicine and then completed an internship in small animal medicine at Virginia–Maryland College of Veterinary Medicine. He went on to a residency in reproduction, medical genetics, and pediatrics at Ryan Veterinary Hospital at the University of Pennsylvania that was sponsored by the American Kennel Club, AKC Canine Health Foundation and the Theriogenology Foundation. He specializes in canine genetic counseling and andrology, and has special interest in canine and feline internal medicine, critical care, dermatology, and behavior. He currently works at GoodVets Downtown Brooklyn. Dr. Stora is a diplomat of the American College of Theriogenology and a member of the American Veterinary Medical Association, Society for Theriogenology, and American Society for Andrology. He's a longtime breeder of Shetland Sheepdogs under the House Astoria kennel name, which is a very cool fact about him. So with that, Dr. Stora, I will pass things over to you for our presentation.

Dr. Victor Stora DVM, DACT  03:49

Hello, I'm Dr. Stora, like we just announced. I want to talk about basic sciences. So there'll be a basic portion of it, just to kind of refresh stuff about genetics, what terminology I use and what that would be in the entire presentation. And I do have a special interest in genetic oncology and tumor genetics. So I did a previous presentation for Good Dog that was in oncology, and you could see that. So okay, this is about me again. I was a fellow at UPenn in their genetics lab at the end of my residency. I am at GoodVets Brooklyn Heights now, and I have my own consulting service that we're starting. It's under Astoria Veterinary Oncology. What I want to go through first is how dogs were domesticated, and then just talk about how modern science came about, comparing the two, because you can then see where there may be discrepancies, or why, where there's a lag period behind what we know, and you know, when we started breeding dogs. I want to go through the basics of genetics, how you can identify a genetic disease in your kennel, why purebred dogs seem to be plagued by genetic diseases, and why we test. Also understanding health testing as a tool, not just to eliminate dogs from breeding, and understanding the bias risk association and publications for your use in your program, and how you can look at those and determine if they may help you in your breeding program because they're verified scientific resources.

05:26

So first, when wolves became dogs, dogs have the levels of morphologic and behavioral diversities of any species. They have lots of repeats in their genome that allow for selection bias, or phenotypic selection—what you do when you breed, you choose based on looks, also by their abilities. But what you're choosing is these large repeat sections in DNA and then kind of sculpting them to what you want. They were the first domesticated species, and they have the largest phenotypic or outwardly appearance of any of the domestic species. You know, when you look at cattle, pretty much everything is a cow or cattle. And if you look at dogs, if you have a Great Dane and a Chihuahua, and you showed that to somebody who's never seen anything like that before, they would think that they're two different species because they're so different in almost all of their traits. When dogs were domesticated, they noted that probably this happened in Southeast Asia early on with rice cultivation, and they now are the first domesticated species, and they accompany us on every continent on Earth. We know that probably the latest dog fossils that were found near human settlements was about 15,000 to 20,000 years before now. There were more distinct changes in the wolf. What they look at are head distinctions. So dogs that had distinct head transformations from the wolf: they have widened snouts and decreased to sizes compared to that. It's hard to see all the other things, like their body color and stuff like that from preserved remains. Initially, dogs were used for cooperative hunting techniques, then became more versatile based on their use for living near humans and cultivating our culture, basically. So when we started breeding dogs, it was about the early 19th century, in the Victorian era, when there was a lot of money and affluence during these times where we see the first confirmation in working dog events popping up, and that's because they had the ability to do these, you know, to actually spend time working on their lines, or working on kennels and changing dogs’ appearances to what we start to see today. What happened on the other side when we look at sciences and molecular biology, we all know, Gregor Mendel was the first one who started to look at heredity and the ability or change of organisms, and their link to probably some sort of code (because he didn't know at the time it was DNA). So that was not published until the early 20th century, which is many, many years after dog breeding had started, or domestication had started. The early 20th century is when they see the chromosomal theory of inheritance, which is just saying that there are physical pieces inside organisms that are chromosomes. But they didn't know that at the time, but they said that there are physical pieces that hold DNA, and they're the basis of what makes us, us. And so the molecular basis and understanding of genetics and hereditary distinction didn't happen until the mid 20th century. Later on, when we get into heavier science, DNA wasn't identified until 1953. And then technologies to look at DNA and to screen for disease. The technology is called recombinant DNA. Technology is looking at the ability to manipulate DNA, or RNA, or any of the like, to actually identify pieces, you know, or decipher the code—that wasn't there until 1970, so health testing couldn't happen until much later than you would probably think. And most of that health testing was very crude and had started from that small point. So we started selecting dogs, or started to take dogs that we thought were helpful and would benefit us all the way back to 11,000 years/11,500 years, and then, from now, from today, more modern dog breeding began about 180 years ago. So there's a large gap between when all of these, you know, more modern based sciences have the ability to help us understand what's going on when we breed dogs. Genes are the basic physical and functional unit. They're a code of four letters that DNA encompasses. And when we talk about that, that's a unit. The unit may have something called alleles, which means the variation of them; there can be different forms of a gene. And then chromosomes are DNA that's been tightly coiled, and they're around these proteins called histones. They support the structure. When we talk about the loci, that’s the area on a chromosome that a gene may lie.

10:37

So when we look at genes and DNA and heredity, we talk about homozygosity a lot. Homozygosity means that there are identical genes in the same loci. These two genes are exactly the same. And so if we looked at it like there are types of ice cream, you get one from the maternal side, one from the paternal side. And homozygosity means they're the same flavor on both sides, so there's no change. That means that there's no backup copy, should something go wrong, because they're the same. When we breed dogs, it's called artificial selection. So what we're doing is we're taking what we like based on what we see. There's no way I can tell you that there's no risk that something will happen or something maybe new will pop up. The only way to get variants of a gene or alleles is through mutations. Mutations are the only way to change a gene. So mutations may not always be something called deleterious or damaging to the organism, but they may not do anything, or they may actually change what something looks like. So you always have to weigh the risks and potentials, and the risks should never outweigh the benefits. When dog breeding, we look a lot about lineage, which is helpful, because there are strict breeding records that are maintained by different kennel clubs. In America, we have the American Kennel Club. There's the United Kennel Club. There are kennel clubs in Europe. These organizations maintain the records for breeding. They are what allow us to look many generations back, which is actually easier in dogs than in humans, because I know a lot of people may use ancestry, but it's not always easy to find all your ancestors, because there's so much global movement of humans and there's global movement of dogs, but we generally kept records of who is bred to who and what they produced and what is continuing from their lines. When we breed dogs, we use line breeding, which is technically inbreeding. It's a form of it. We use that because there's a predictability associated with it. Inbreeding is using two related individuals. Outcrossing would mean they have no ancestral relation whatsoever. Line breeding, we call line breeding, because generally that's considered a softer form of inbreeding, whereas incest is strictly to more immediate familial relations, like brother/sister, father/daughter, something along those lines, that's considered incestuous. Probably want to stay away from that. That's a very easy way to generally collect genes that are exactly the same. Everyone probably knows what this looks like, but this is the pedigree and how we all get this certificate. This is very difficult for me to read. It's not easy for me to understand where certain dogs may be used again and again. When we look at pedigrees, there's a determination we can do, which is how inbred a dog is by looking at the coefficient of inbreeding, which is the nodes—this is a large formula on the bottom—but you're just looking for basically nodes or loops, where you can get the same dog back to each other again; the more loops, the more inbred, then the higher the number. That's basically what that means. But then there's the whole formula on the bottom of what we actually look at. It just means the sum of all of the nodes is equal to how inbred an individual is.

13:57

It's easier to do that from a transformation of the pedigree into more of a medical pedigree, where circles are females and squares are males. Then you can start to link them together. Each square and each circle is a discrete or its own individual. It's not a repetitive individual. It's only that one animal. So again, every circle or every square is a different individual. When you look at this, or expand the pedigree to back into generations, you can start to identify nodes. So nodes are what I said, like there's a loop. So that square, that top square, which is a male, we can link the female on the bottom, that's her grandfather, but is also on her other side, her great grandfather. So there is an inbreeding coefficient there, because there's a loop that we can see, and there are multiple nodes. And then this is just a tool. It's not an exact science. It's not an exact number. It's just a predictability number. It's not something that is saying that you have to be at this number exactly, or everything will go wrong. All genetics is, is probability. There's very little hard, fast rules in genetics, and it's very difficult for me to say you can't keep a certain dog based on most of its genes, because we generally want to keep most of the individual's ability to breed, because that means that we're keeping a larger population of alleles or variants of genes. So in scientific liturgy, you want your inbreeding coefficient to be below 12% on an eighth generation pedigree, but that's just a number. That's not something that you have to like strive for, and we have better ways of looking at it now, because this is an estimation, whereas we have abilities to form (and I'll talk about that later) gene arrays, where you can actually see the inbreeding coefficient, because they look at multiple different areas in the genome and they say, “Well, these are all the same. So then this individual is this percent inbred based on actual looking at the DNA.” The problem that we get with homozygosity, or the more you inbreed, is it's a vortex. So if you line breed very hard, or you use lots of incestuous inbreeding, the increase in homozygosity will eventually lower your ability to have puppies, because their reproduction starts to go. The ability for them to cycle, or for sperm quality starts to dramatically plummet. This has been linked to the loss of variability. The problem with decreasing homozygosity is then you start to lose what you're aiming for, what you're looking for. There's more variation. They're not as uniform as you would want them to be. But again, like the more you use inbreeding or line breeding, the heavier you go, the more likely you are to kind of fall into difficulty with reproduction because of this loss of this variation. They're linked again. And this can be shown in clonal populations, because in mice, when you do research, you want them to have a specific disease. The old way was to take a mouse with this specific disease and breed it to its family members, until only mice were normal, but they had that one disease variation, or that one variation that they were predictably getting and they could then do certain diagnostics or tests or experiments with. This only happened one in 1000 times to make this specific mouse, because ultimately they would get to a reproductive blockade where they would not be able to reproduce anymore because they were breeding incestuously so hard to get that mouse that's only that one variation, that one change, that they would become infertile at some point. 

Now we can use CRISPR-Cas9, which are gene editing techniques that are very, very targeted, that they can change the genetics of an individual as an embryo—well at the one cell stage. So we have different effects that reduce our genetic diversity, especially in breeding dogs. Probably the one that's the most notable is popular sire effect. Then we have population bottlenecks, founder effects and selection pressures. Whereas a bottleneck is kind of like a vast reduction in dogs like parvo virus that killed approximately half of the dog population when it became a virus. So that would be a bottleneck event, and hopefully they recover from that. But again, you're losing everything that went with those dogs, all of that genetic variation that went with them or died with them, basically. So popular sire effect is the overuse of a particular sire in a breeding program, which happens often, because we have a sire that wins many awards or many dog shows, and thus then increases the uniformity in a given population, because if he's used so much in everybody's kennel or everybody's breeding program, across the board, or across all of these different channels, then it's hard to find individuals that are unrelated, should disease or should issues come from breeding to that male, it kind of infects everything, almost like a disease, in a way, because then you can't kind of get away from it, or it's harder to get away from it. When you start to breed with that male, there's going to be unmasked recessive diseases that they carry. It's just what are they and how hard did an entire population or an entire group breed to that one male? And it's very easy, because we can ship semen across the country now. You can ship chilled semen from Europe, from some countries. So it's not that there's a geographical blockade anymore. It has this ability to go or do whatever you need to. This is again, a man-made phenomenon. It doesn't tarnish the dog's reputation. So we're not saying that the dog is bad or the dog is wrong. It’s something that we select for. So in 1915 this was actually looked at as something that was good. But again, in 1915 there were geographical blockades to the ability of males to mate. So if a sire was very good, then you would have areas of a breed where they were able to breed to this male, and they could then show this male's like, why we would use them, you know, what his virtues are. So this would ultimately create pockets of little areas where that male would be more used, and then you could see his virtues. And then eventually that could peter out into the population. But again, it was a slower effect, not very fast. Whereas, if someone wins a national now, they can then be ship chilled to the entire country in the next coming days.

Dr. Victor Stora DVM, DACT  20:51

They look at Fox Terriers. I was in Scottish Terriers when they were looking at them initially, and then these were only producing maybe three to five litters in each channel near them annually. They weren't producing as many as we can have now, which can be the production of three to five litters per month, or even more, for popular sires, because they can, again, be shipped everywhere. They can be used as much as you need. So a story that really shows what can happen is when we look at quarter horses. In the 1600s, these horses were bred to run a quarter of a mile, they have very, very pronounced muscularity, and that's a hallmark of the breed. When one particular sire exemplified these traits, they didn't know that they were breeding for a specific disease because it wasn't really showing. The overuse of this sire showed a significant increase in something called hyperkalemic periodic paralysis, whereas you need potassium for your muscle fibers to move or nerves to conduct, and so if the potassium is too high in the blood for too long, it can cause these twitching or fasciculations or tremors in muscles and ultimately collapse or paralysis. And this would happen in horses because their muscles were larger, but they didn't realize at the time that they were breeding into their population this disorder. This is actually a disorder that's not a homozygous disorder. It is actually the dominant disorder. So you just need one copy of this to actually have the problem. In 1984/1986 he was looked at as actually being the sire of 10% of the entire registered quarter horse breed. So that means that 90% were unrelated to him, but 10% were related to him. And then from 6,000 blood samples, when they looked at that at Davis in 1989, 22 were thoroughbreds; 978 quarter horses were identified; 43 quarter horses were all positive homozygotes for this, and all of them were quarter horses. And then they realized that all of these quarter horses that were positive were descendants of Impressive. So this is another medical pedigree. The top one is Impressive. If the shape is colored in, that individual has the disease. So you can see that generation one was mated to multiple different females, and then generation two came out, where there was some identification, or some showing that there's disease there. And then as we go down and down and into the generations, the number of individuals with disease just dramatically increases. It's because in the beginning it's kind of silent. It's not as easy to observe or identify because it's kind of gray. You know, you see this problem, but you don't link it to actual genetics, or link it to an individual. You just identify that there's something going on that's wrong. And it's not until the latest generation, or the one on the bottom where you're looking at it and you're looking back, and everyone's related to this one individual, and you kind of have not as easy of a way out. It's not that you can just breed based on virtues and looks. You have to actually look for their genetics in the background, because something is going on, and then you need to be vigilant in identifying that so that you can use it as a tool. Because it’s not necessarily that they can't breed. In this case, yes, they can't really breed because it's dominant. Unless the population was somewhat endangered or restricted to that level, then I say that that's okay, but because it's dominant, you're going to have a larger portion with that is disease than if it was a recessive disease, like we all think of. Again, like I talked about, the first generation from apostasis is masked. You really can't see it as much. And then in generation two, there are hints that something might be going on. Generation three as well, you may see something. And then generation four or beyond, there's something wrong, and everyone's trying to figure out what's wrong and why, and you're trying to kind of get out of this hole, because something is not right, and it's hindering your ability to breed dogs and your ability to produce what you need.

25:18

So this is a small population of a recessive disorder. I made the shapes where they have one good gene and one bad gene. So like, that's why they're half-half. So the white is good, the black is bad, or the black is disease related, not bad. So we'll mate this male to four females who don't have a similar allele, and then generation one comes out, and this is a recessive disorder, so they have to have two copies, so they have to be a fully colored-in shape for them to have or show disease. So none of these are showing disease at this time because none are colored in. And then we'll mate these to other individuals that are not related to our founder, and again, you'll not see anything. So generation two, you see nothing, because it's a recessive disorder. You need two disease related alleles to show and so none of these individuals in F1 or F2 are showing any sort of signs of issues. Then if we line breed, or we go back and we breed, the pairing in the middle, where there is an inbreeding coefficient, there's a loop there, then there's a female that is a heterozygote that's bred to a male that's a heterozygote. And so then you start to see a female that has a disease or problem, and then on the other side, you see a male that has a disease with a problem, and also his litter mates do carry, or some of them carry a copy of this disease gene. So for two generations, you didn't see anything, or no one saw anything. And it wasn't until step three where something happened, but likely it's not identified that this may be genetic at this time, because only two dogs in six have a problem, and it's maybe just a one-off thing, or just be written off as something that is just wrong with them, and not with an entire population or a breed. And so I talked about this a lot. So the problem with popular sire effect is that males can be used unrestricted to geographical locations. There's no limit. There's no limit of restrictions. Some other countries do recommend or try to police in a way that there's no more than 5% of the entire population of a breed that comes out that's related to each other. So that it dampens the ability of popular sire effect to some degree, but it's hard to police that, and it's possibly unethical to make that a mandate. So when I talk about genetics, I talk about genetic hygiene, which is using genetics in your kennel to help you and not hinder you. You can manage this, because in your lines, you don't have to have each line exactly related to each other. Yes, they should look like your dogs or your breed, but they don't have to be identical. So if you have a variation in lines in your kennel, you can avoid this. So if you have the ability, you want to have somewhat distinct lineages, maybe at least three, I say, is probably beneficial. Maybe two if you're in a smaller kennel, so that you limit the ability, or limit one line to having being bred to this or the winner of the national or popular sire, versus having your entire kennel related, because then you will fall into a pit hole, and should something go wrong, you really can't get out of that, because if everyone's related, and then everyone you want to breed to is related, or it's hard to see in the breed club, unrelated individuals, then you're kind of stuck, and it's going to be very difficult. When we look at founder effect—popular sire effect is a derivation of founder effect—but this can happen in the beginning of a dog breed, because you take a subset of the population. So we have the circle with all different colors of alleles and all different variations that you can possibly have of different traits. And essentially all dogs will exhibit this phenomenon, because you need to have a certain founding number to create a breed. But that founding number is usually small because those are the only individuals that have the traits that are so like that they can be called a breed. So we don't want to use the blue ones. We don't want to use the gray ones. We just want the light pink one. So all of the variation that we possibly had went away, but these are the only individuals that actually look like each other or possess the traits that I want, and so my founding population is only five individuals.

29:59

So should you have a problem, like I said, you want to be able to outcross within your breed, which is not really an outcross, but it is the only way you can do something is to breed to another lineage, basically, of distinct lines. So that's called hybrid vigor. If you have two inbred lines, and you immediately outcross, you really remove this ability, or remove your problem fairly quickly, because if the male on the left has a very uniform genome, he has two chromosomes that are identical on either side, and the female on the right has some variation, but otherwise has chromosomal relations. When you breed those two together, since they're distinct lines, you kind of completely reversed your inbreeding, because you're going to get so much different variation from breeding those two individuals together; there's no crossover events of them. 

So let's look at DNA testing. The homozygosity is a prediction of inbreeding, and we're looking at the risk of that. So we're going to expand our understanding, or our looking at homozygosity to using actual tests and DNA abilities to identify problems. When we have a population of disease incidence is high, we have to test because that overrules the use of the inbreeding coefficient, because the test is going to be the more determining factor of what is going on versus a predictable number. So everyone has their breed in their breed club, and their breed club should have, at some level, the ability to look at their overall health in that breed and identify what diseases or what traits, or what things you want to look at when you're continuing to breed, so that you can breed with awareness. There's also on the OFA, there's CHICC data, there's AKC identification. This is ultimately a branch of that. And then you can kind of go there and see what you maybe should be testing for, or what is recommended. You get a list, and then they'll basically show you what is highly recommended, what is maybe conditionally recommended, and what is like completely optional. So we have like two right now that I know of. There's the optimal selection panel for Mars and the Embark panel. These test for heterozygosity. So these are almost like chips that have different genes on them, and then your dog's genes, or DNA, is plated over this array of different genes. And when your dog has the same gene that's on the chip, it's going to bind so it binds to that area. So then the computer will analyze the chip and identify where the DNA has bound, and it'll look and say that, you know, there's two different variations of this gene, the DNA only bound to one side, so that dog only has one variation. The power of it only comes from how many people or how many have used that specific DNA test, because the data has to be available across the board for increasing the power of something. 

So this is one of my dogs, and this is an inbreeding coefficient, or identification of inbreeding based on the actual DNA test itself. So they give you the inbreeding coefficient, which is 44%, then it looks at these two different classes of white blood cells, basically. And it tries to say, if there's diversity, most breeds are locked in their diversity of white blood cells. There may be some linkage of autoimmune diseases to that, but it's hard to get diversity there. You can. There are some breeds you can, but it's not as easy as everyone would like. So basically, this was what the chip would tell you. They would look at the areas of a chromosome or DNA, and it would say the areas that are inbred. And so when you look at them like it's easy, when it's a picture, it's easy to see. You can see the chromosomes. They're in color, and the outcross areas are gray or there's variation, and the yellows are where there's no variation in the genes. So basically, it's homozygous at those sites. There's no variation there. This is probably pretty common for a purebred dog, this type of array of the chromosomes.

34:31

So health testing standards may give you a false sense of security. There's nothing wrong. But if they come up negative for all the diseases that you can test for, doesn't mean they should be bred as much as possible, because everyone walks around with DNA based markers in their genome that ultimately they're not good, so they can hinder your ability to live. But most animals, the individuals with that, will have a secondary copy that is pumping out the protein that works well, and so you don't really see that, so there's no problem. But if you unmask this problem by using sire effect or founder effect, then you'll see disease more. Because you can't test for everything, there's always going to be some risk. Okay, so what I talk to people about when I look at their counseling is you have to give up the notion that there's no risk alleles, because there are, and you'll find them, but we can deal with them. And then there are ways that we go about that, but you're going to find something, and there's always going to be things that pop up. So as long as everyone remains vigilant, and everyone kind of provides each other with more openness and open data, then you'll be able to get around these issues. When we look at phenotypic diagnostics, there's no known marker. So this is not a test where we're testing DNA. We're testing what we can see this is hip dysplasia, and that's a hip dysplastic dog on the right. And then also, you know, these more complex diseases, like dry eye, which is KCS, or keratoconjunctivitis sicca, those are difficult to identify because there's multiple genes likely that are playing a role in this, as well as the environment. We didn't really talk about that, but the environment plays a large impact on usually, if diseases will come out, or if genes will show themselves, because there are other layers of how genetics work that we want to really get into. But the environment can play a role in locking or unlocking certain genes. When we use phenotypic diagnostics, there's a normalization of animals participating. So there's a bell curve, meaning that some have a problem and some are better than others. And when we look at them, animals that are average to above average, you know, are chosen, and that shifts the curve more towards less severe disease, or limiting disease, because when we look at issues or traits, there can be a severity to them. They don't always black and white. They can be like hip dysplasia has an array of issues. It's not just very cut and dry. They can have dysplastic hips and be mild versus moderate versus severe. So the phenotypic diagnostics can shift how severe something is, as well as if you'll see issues. When we look at molecular diagnostics, these are tests where we can actually use the DNA, like I said before, and we identify physically if there's a problem, without a doubt. We're not looking at a picture of X-rays. We're looking at the actual gene itself. There are two types. There are ones where we can identify the exact gene itself and the exact mutation that's wrong. And then there are others that we use areas of the DNA that are linked to the disease, or the gene that we don't want, or that gene. They're in physically close proximity, so when we test for it, often, if we find that, it comes up positive for that marker, likely the disease gene is next to it or near it, and so it has likely been bred into the next individual. The wild type is the non disease and then this is the one where we have the disease gene. And so when we look for the disease gene, we're looking for the ATATAT tag that's in front of it, because we don't know exactly the sequence after. We just know that there's a disease gene and that the disease gene’s there and it exists, but ultimately we can't see that part. So we're looking for that, that marker, so that we can find the disease gene. It’s a suspicion of disease with these. It's not definitive, because you can't find the mutations. You just breed with awareness, and you have to report test faults to laboratories, because when DNA for reproduction reassorts itself, that physical marker might actually be cut and move away from your disease gene, so it might move away from it where you're not actually going to see it with that disease. So if you pick up that marker, it might not have the disease, and the disease gene may be lost to it.

39:13

And when we have the ability to find the exact mutation, this is the best one and the easiest one. They're breed specific. So sometimes, if you're breached or an individual that you're testing, and you use a large panel, and it has come up positive for a disease unrelated in that population, it may be there, but it may just be a false positive and not actually something that you need to really look at fervently. Homozygous individuals we look at and we call them 1-1s. Heterozygous is 1-2s. And then affected dogs, we call 2-2s, and sometimes that's reported on your results. So managing this, carriers and affected individuals actually can breed if it's a recessive disease—should the affected individual not be infertile or have an issue with breeding, because if you breed the affected dog with a known normal dog that is not a heterozygote, then the progeny will all be heterozygote, so they'll all have a copy of the disease gene, but they won't actually express it. So you can use an affected dog, but you have to know going forward that all of the progeny will have that disease gene, and you just have to breed away from it. But it's a way that you're keeping variation, or you're keeping maybe that affected dog’s other good genes within a population. The problem is that what most people want tests for, we don't have tests for right now, like cancer or epilepsy, hip dysplasia—there's no exact mutation test that's going to tell you exactly that that's going to be there. There is only an association that there's probably something going on, because we usually use phenotypic diagnostics, and because these diseases are prevalent in certain breeds, we know they're probably genetic, but we don't know the exact mutation that we can use that test to identify exactly. So we need to do judicious health testing, because if you do everything, it becomes overwhelming, and you'll keep nothing again. The benefits of health testing is disease incidence is high enough to warrant testing, and then, does this disease cause significant issues or death? And then if you have this disease in your kennel, and is there an effective treatment? So you have to look at everything together when you're breeding, because you can't just say, “Oh, this gene is bad. This dog has a disease that doesn't cause issues.” But if you can see it, then you can use that individual and breed it to other, like I said, non affected dogs, and then you'll have that population be split between every dog has one bad copy and one good copy. But you can go forward with that. 

The difficulty with phenotypic diagnostics is they usually impose the harshest penalties that they remove them from breeding at all times, but these are an observation only, and certain mild cases may be beneficial to keep versus not keeping at all. And it's because there's no clear, defined testing tool for this. So you have to breed with knowledge. You can't throw everyone away. Removing an individual breeding is not simple. How I look at this individual, and need to remove it. Like I said: Does the disease state cause significant morbidity and mortality? So does it cause a significant disease? Is it severe? Is that animal distressed? Or is it something that is not distressing at all and is a cosmetic defect? Then it doesn't matter. Our heterozygote is debilitating. Because, like I said, if we breed an affected to non affected that's normal-normal, and it's not debilitating, that's fine. 

Are there enough individuals in a population that justify removing this one in question? Does this individual fall in relation to the breed standard? Well, and then does the individual have genetics that are uncommon, so that sometimes may trump whether or not my dog is the perfect vision of the breed, if they have genes that I want, because then an F2 I may breed and get some individuals that have the genes that I want, and then I may go back in the next generation and breed it, or line breed so that I get more of what I want. But at least I captured something that is maybe not easy, like the changes in white blood cells I talked about before. So again, we're trying to create the image of a standard. There are many goals in that at play. Health is a forefront, but you also need to not lose your sight of the kennel goals in lieu of health testing, because that's easy, just to say eliminate instead of, you know, using everything we know as a tool.

43:43

We try now to get our veterinary students and everybody to work together more about using these tests and looking at these individuals and helping to counsel breeders and owners on this because this is really close to rehabilitating an endangered species, and it's not very well known, the genetics or genetic testing in dogs, because it's not something we're necessarily taught in veterinary school at that level. I'll just run through this quickly, because this is not about genetics, but this just shows how you can find data and how you can find things that may help you in your breeding program, that are true and not just something in like a magazine that may be someone wrote or someone who's not as knowledgeable as I would like, or anyone would like, and it's not significant data. Significance means there's a difference in the data and that it's actually beyond a certain point that we can say that this is definitely something that we need to be looking at. This is just a tree of what is the highest level to the lowest level. So anecdotal evidence is the lowest, a meta analysis is the highest. That looks at many, many studies actually, instead of just one study, and it starts to compile the data from multiple different studies. So you can get this information from your library, from Google Scholar, and then if you really want your veterinarian probably can get it for you, should you find an article that you need to read to help you in your breeding program. I like to work with breeders, and I try to foster that in my veterinary students and those that are under me, that come to work with me, and in general, it's more receptive than not. Thank you for your time, and we'll take some questions now.

Nicole Engelman  45:24

Awesome. Thank you so much. Dr. Stora, this was great. We do have just a few questions. I want to be sensitive of your time, because I know we're almost at the full hour. This is a bit of a lengthy question. Someone asked when a serious health condition appears in a litter, particularly one that isn't detectable through standard genetic panel testing, how should a breeder evaluate the risks of litter mates they've retained for their program? What factors should guide that decision when the mode of inheritance is unknown and it's unclear which parent may have contributed and what's the most responsible path forward, both for the breeding program and contributing to broader breed health data?

Dr. Victor Stora DVM, DACT  46:03

So in that case, I would look at your lines, and I would see if there's lots of relation between the two dogs that you had bred to. If there's a lot of relation, I would try to go to a kennel or something that's different from what you're used to, but at least it's different enough to where you can get some of that confidence, that you're more confident that you outcrossed a little bit. And so these should be varied, so you're looking at your new litters as like, okay, they may have something with them, but I outcrossed them. So all of these puppies may have that same problem that's not masked, but I'm going to not breed them, not heavily line breed them. I'm going to keep one, and I'm going to outcross it again. So you're going to kind of tease out that ability for a disease to infiltrate because you're not line breeding as hard as you would probably like, but you're moving away from it.

Nicole Engelman  46:58

Awesome. Thank you for answering that one. Now we have one specifically about merle genes causing blindness. Can Merle cause blindness if there is only one copy, and do the nature of the color of eyes 9so very light piercing eyes) indicate future blindness? Or is that not enough to tell? 

Dr. Victor Stora DVM, DACT  47:18

That’s not enough to tell. The merle gene, as a single gene DOESN'T cause blindness. But the problem is that you need pigment, or, like, blackness. You need that color. What the merle gene does is it doesn't allow pigment to move across the body like it should. So my dog was a merle. And so because it doesn't allow pigment movement appropriately, you get this splotchiness, but you need, like 50% which is what it's doing in the retina. And so if you don't have 50% then you have blindness or issues with that. But if you have other types of genes that hinder pigment movement, yes, then the merle gene, plus that, can cause blindness.

Nicole Engelman  47:55

Awesome. We do have one last question. This person is wondering or assuming that most dogs' genetics must be the same with only a small percentage differentiating individuals and breeds. What percentage of the genome is variable?

Dr. Victor Stora DVM, DACT  48:12

So the whole genome is variable, but how much is actually expressed is a very little bit. There are lots of areas in the genome that are just like gibberish for what it is. It doesn't code for anything. So that's kind of fail safe, because if there's mutations or there's damage in those areas, then nothing happens. So the coding regions can vary upon individual, upon, you know, breed or like species. So it's hard to exactly say.

Nicole Engelman  48:40

Awesome. Thank you for answering that. I think that was all of the questions we got, but this was such a great presentation. Thank you again for doing this for us, Dr. Stora. It was so great to have you back. Thank you so much to everyone who joined us as well. Thank you all so much for being here. Thank you again, Dr. Stora, and we'll see everyone at our next webinar. Bye, everyone.