EVALUATION FOR THE CANINE DILATED CARDIOMYOPATHY MUTATION

Dilated Cardiomyopathy Mutation (DCM) is a form of heart disease in the Doberman Pinscher dog. It is inherited and our laboratory has identified a mutation responsible for the gene in some Doberman Pinscher. However, it should be noted that in human beings with the same disease, there are many different genetic mutations which can cause this disease. We do not yet know if this is the only mutation in the Doberman Pinscher or if there will be many different mutations. Please keep in mind that we are continually learning about this disease and recommendations will be altered as we obtain more information.

Currently our interpretation of the test is:

Negative results:

The absence of the mutation in this dog, DOES NOT mean that it will never develop the disease. It means that it does not have the only known mutation that can cause the disease in the dog at this time.

Positive Results:

Dogs that are positive for the test will not necessarily develop significant heart disease and die from the disease. Some dogs will develop a very mild form of the disease and will live quite comfortably, some may need treatment.

Importantly, breeding decisions should be made carefully. At this time we have do not yet know what percentage of Doberman Pinscher will be positive for the mutation. However, removal of a significant number of dogs from the breeding population could be very bad for the Doberman Pinscher breed. Remember that dogs that carry this mutation also carry other important good genes that we do not want to lose from the breed.

Positive Heterozygous (1 copy of the mutated gene and 1 copy of a normal gene) Dogs that are positive heterozygous should be carefully evaluated for signs of disease (Holter monitor and an echocardiogram). If abnormalities are detected, possible treatment options should be discussed with your veterinarian. Adult dogs that do not show signs of disease and that have other positive attributes could be bred to mutation negative dogs. Puppies may be screened for the mutation and over a few generations, mutation negative puppies may be selected to replace the mutation positive parent and gradually decrease the number of mutation positive dogs in the population.

Positive Homozygous (2 copies of the mutated gene). We recommend not breeding the homozygous dogs. Dogs that are homozygous for the mutation appear to have more significant disease and will certainly pass on the mutation.

Veterinary Cardiac Genetics Laboratory PO Box 2310, Pullman, WA 99165-2310 509-335-6038 b" FAX: 509-335-6038 b" vcgl@vetmed.wsu.edu _________________________________________________________

To Test or Not to Test

I have read with long expected dismay some of the recent posts *against* employment/application of the test for the recently discovered genetic mutation responsible for causing cardiomyopathy in the Doberman breed. Why would we advocate against investigating every even potentially promising avenue with which to help our dogs? Hmmm.....Here are some thoughts for what they are worth.

First off, Human history 101: Org had to discover fire, then Org's descendents had to discover the wheel, the internal combustion engine and a veritable Boatload of other things over the eons before those descendents found themselves sitting on their pretty little cans amidst creature comforts perusing the internet. A succinct summary of eons worth of *bigger picture* history.

We find ourselves today *discovering fire* for our dogs. For those who believe this genetic discovery is our very first fire- our first beginning tool with which to ultimately eradicate the greatest health scourge our breed has suffered since its creation- they should test. The information we will get from results of thousands of tested dogs and their progressive cardiac health will be simply invaluable to help answer many important questions. Is/are there another gene(s) that we need to search for? What are the modifying factors- are they other genes? Are they environmental influences? Both?

Those who don't believe this is *our fire*, should simply not test. For pet owners in this category, not a tremendous biggie. For breeders in this category, as someone else said, *time will cull*- with each passing year the market for puppies from untested parents will increasingly approach microscopic in magnitude. So have *not* at it. Actually, those who don't *believe in it* would be smart and savvy to test as well, because there is no other way to *disprove* it than to identify incongruities between test results and physical symptoms/lack thereof. But that is of course a personal choice.

But we should be extremely wary of any facades presented against testing based ultimately on deep rooted psychological desires *not to know*. It IS OK for owners of non-reproducing dogs to not want to know and follow that path, it is simply though sadly a loss of useful information for breed health (and, with at very best only a 50% chance your dog will not develop cardiomyopathy, are those odds really worth the loss of useful information for the breed?). It is disserving of the breed for those reproducing it to not test, regardless of what breeding decisions they make.

I suggest the tenets of our approach to using the new DNA test for Doberman cardiomyopathy should include the following.

  1. Every dog tested provides IMPORTANT INFORMATION for the future well being of the breed.

  2. There is NO GUILT in a positive result. We inherited the situation with Doberman cardiomyopathy; all we can do now is work to resolve it/eradicate it from the breed.

  3. There is NO GUILT in breeding a dog that tests positive at this time. We COULD NOT HAVE ASKED FOR a more reasonable, reasoned approach than that advocated by Dr. Meurs (and others) than that we NOT lose countless great things about our dogs!!! The importance of NOT losing the spectacular top show dog conformations, the awesome working aptitude of great dogs, the splendid breed temperament of many dogs, etc. is recognized to be foremost as we carefully begin to work away from the devastation of cardiomyopathy. Testing and receiving a positive result does NOT mean you can not or should not breed that dog. It means you have *a goal to work toward with future generations*, so that our dogs do not continue to suffer and die from this horrible affliction endlessly into the future.

    The public should be educated that the Doberman community responsible for maintaining the grandness of this magnificent breed understands that the principles of our breeding program henceforth must be a cautious, studied, careful move away from cardiomyopathy positive dogs lest the breed suffer even worse devastation via sudden, severe *genetic bottlenecking*. For this reason, the availability of *cardio clear* puppies will continue to be limited for a number of years; additionally, because we do not still have all the answers re: genetic and/or environmental modifiers of this variable penetrance genetic flaw, positive testing puppies may not develop the disease and negative testing puppies may do so- UNTIL WE KNOW MORE VIA ONGOING TESTING/EVALUATION.

This is a first tool. It represents a beginning approach. One person likened it to how one goes about putting together a jigsaw puzzle- first you work on putting together the border, then on all the other pieces of, for example, the yellow flowers or the clouds in the blue sky, etc. Completion of the puzzle will take longer and be more difficult if one just randomly takes unrelated pieces and tries to shove them together. It would be unlikely and nearly magical if a wild, random approach resulted in quick completion of the puzzle.

There is no *magical* resolve to be had for the situation we face with cardiomyopathy. How nice it would be to have all the answers, every single detail of every single part of the puzzle of Doberman cardiomyopathy, all dropped neatly in our laps gift wrapped and all, but its not going to happen. It's frustrating to have to contend with something analagous to a jigsaw puzzle, where we must work still to put all the pieces together even after being presented with them. But that's often the reality of scientific discovery, especially with complex situations, and that is the situation here.

We must start somewhere, and starting with a *first tool*- with part of the border of the jigsaw puzzle given us- is most logical. If one or more of the pieces of that border were put together incorrectly, it's our job to find that. If we don't work at the puzzle, we can never do that. If those first few pieces were put together correctly, then it is our job to put together the rest of the puzzle- WITH JOINT DETERMINATION.

Just because something is complicated, just because something is not One Hundred Percent black and white from the start, just because something does not serve up every single last answer on a silver platter, does NOT mean it is not real, does NOT mean it is not in fact an important part of a larger, factual, accurate picture. Anyone preferring to discount this finding/test because it does not Immediately provide us All the answers for 100% certain is certainly welcome to do so. Those who wish to be an active part of the ultimate solution for our beloved breed, will step in to do so.

Fear is an inappropriate reaction here, however well concealed. Viewing things in a *vacuum* is likewise an inappropriate reaction. Employing this new tool with careful, dedicated, open minded, joint resolve is the only appropriate approach.

We begin here. For our Dobes.

Kathy Davieds DVM

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Negative - no copy of the identified gene for DCM. It might still possible to get DCM, IF there is another, as yet unidentified, cause

Positive Heterozygous -One copy of the gene- more likely to get DCM than negative, but less the positive homo

Positive Homozygous - Both copies, Most likely to develop DCM BUT, due to incomplete penetrance ( a dominant gene that, despite being dominant, doesn't, manifest itself ) both the the Positive Het and Positve Hom might still NOT get DCM.

THIS IS WHY WE TEST_ if we get negative dogs that still get DCM - then we will have proof that there is another cause...... and need more research to identify any and all the others that still might be out there killing our dogs. Some day we will be working on what causes the incomplete penetrance and if there anything that we can do to influence it.

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Some Insights into the DCM Mutation--From your local biochemist
There have been quite a few questions about the nature of the mutation Dr. Meurs has found ---the designation that is proper to use, and what might be the actual heart metabolism consequences of the mutation, what might possibly be done to help dogs that have it. I know there are quite a few medical and other professionals that would appreciate some more detail so I have tried to provide some kind of explanation. However, keep in mind that translating a lot of genetic and biochemical nomenclature into clearer language without making a mistake is not so easy. I have contacted Dr. Meurs for the accuracy of the following and she has enthusiastically endorsed it.

Some questions are what do you mean by the various kinds of positives. The enzyme that is deficient is pyruvate dehydrogenase kinase 4 (the 4 is the type that is found in cardiac muscle which is why Dr. Meurs knew right away that it had something to do with Doberman hearts).

Positive heterozygous is one mutant and one normal copy. Positive homozygous is two mutant copies. Given that this is a gene with incomplete penetrance we cannot know exactly the consequences of this. As a biochemist I have been researching the underlying defect and its consequences and I can tell you that it is not simple and it probably accounts for why the mutation is not lethal from birth. If the enzyme mutated were absent entirely from birth then dogs would not make it past infancy in most case of homozygous. This was shown in mice knock outs (genetically manipulated mice where the gene for the enzyme is entirely gone). This is a splice mutation like the vWD (although in this case the heritance does not seem to be the same) but we can probably conclude that a LITTLE of the enzyme is made but not enough and this was shown by Dr. Meurs electrophoresis experiments where the enzyme was detected in heart tissue extracts. Just like "AFFECTED" vWD dogs, a splice mutation is likely to leak a little and some of the right protein may be made even in homozygotes (two bad copies) and more but NOT normal levels in heterozygotes (one bad copy and one normal copy). Read about the vWD mutation as there are SOME *but not all* similarities.

This opens the possibility that some kind of external factors (diet, supplements, etc.) may give us a handle to help dogs. Just to give you an idea of the complications involved is that this enzyme INHIBITS the use of glucose interconversion to acetate by another enzyme (pyruvate dehydrogenase) --the products of this pathway end up as acetyl-CoA that is used by the mitochondria for energy in the heart muscle. Ordinarily this pathway produces only about 10% of the energy needed by the heart (in humans, dogs could be different) with most of the energy coming from fatty acids. Now with not enough of this regulatory enzyme this pathway could go run away. So far, I have not been able to find papers where the consequence of a runaway pathway are clear as this situation is unusual. But we do know that in diabetes where there is too much glucose getting in to the heart muscle that would get into this pathway that the body produces MORE of this regulatory enzyme to dampen down this pathway so too much of this pathway is not a good thing.

I will keep researching the consequences of this mutation and so will the veterinary cardiologists but there is some reason to hope that we might be able to help dogs or at least understand better what is happening in their hearts.

Linda C. Kurz, Ph.D.
Washington University School of Medicine
Department of Biochemistry and Molecular Biophysics

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The PDK4 Mutation and DCM

The PDK4 mutation pyruvate deydrogenase kinase 4:
The enzyme missing in positive homozygous Dobermans and present in lowered quantities in positive heterozygous Dobermans is not just a simple protein and has nothing to do with a nutritional protein deficiency as might be concluded from your article. Pdk4 is one of several enzymes that REGULATE energy in tissues. In heart, most of the energy comes from acetyl-CoA transported into the mitochondria from the cytoplasm. Acetyl-CoA is an energy molecule that eventually is converted to the energy molecule ATP you find out about in high school biology. The enzyme providing the acetyl CoA that is transported into the heart's energy factories (mitochondria) is pyruvate dehydrogenase. This is the PD part of the name of the mutation. The end product of GLUCOSE metabolism is acetyl CoA but the adult heart usually mostly uses fatty acids for energy that are more directly converted to acetyl-CoA. So the heart cell gets glucose and fatty acids from the bloodstream and converts both to acetyl CoA that are transported into the mitochondria energy factories. Only in the neonatal heart is glucose the major source of energy of the heart. It is perfectly correct to view the mitochondria as little energy factories. Pyruvate dehydrogenase activity is REGULATED by two other enzymes that put a phosphate group on the protein or remove it. This enzyme is the kinase part of the name -that means it puts a phosphate on. Putting a phosphate group on, inhibits the action of pyruvate dehydrogenase conversion of glucose to acetyl-CoA. and that is pdk4 that is the enzyme affected by the mutation. There is another enzyme that removes the phosphate to allow pyruvate dehydrogenase to become active again. So in the absence of pdk4, we have a potential runaway pathway.

Glucose is going into the heart and is converted to acetyl-CoA in a potentially runaway manner. We know that this is bad for a couple of reasons. First of all, mice with a knock-out mutation (cannot make any PDK4 at all) die shortly after birth. Secondly, in a diabetic where there is too much glucose getting to the heart, the body cranks up the amount of pdk4 to keep pyruvate dehydrogenase from cranking out the mitochondrial energy molecule acetyl CoA. Exactly what happens inside the mitochondria when there is too much fuel so to speak is not known. Maybe it gets broken down because it can't be used in productive ways and generates toxins that end up destroying the mitochondria. Dr. Meurs reports destruction of mitochondria in DCM hearts. I am sure that the exact details of how this runaway pathway leads to mitochondrial disruption is under active investigation by Dr. Meurs (confirmed by her). There is another complication. The reason that there is a 4 on the name of the enzyme is that there are 3 other forms of the enzyme --these are called isozymes. In heart, the main one present is isozyme 4 (PDK4) but there are small amounts of the others. In skeletal muscle tissue, pdk4 is also the major enzyme regulating this pathway and I had asked Dr. Meurs in the Webinar (and in later short conversations) exactly what this might mean. She noted that many CHF patients had wasted skeletal muscle and she was actively investigating whether a pdk4 deficiency might have a more general role in this rather than it just being a function of the poor heart function of these dogs. It is certainly true that there is a protein deficiency, but it is really a particular enzyme that is involved that is only a small amount of the total protein present. So talking about a general protein deficiency is an oversimplification.

Another part of the story is the nature of the mutation that LEADS to deficiency in the regulatory enzyme PDK4. It is a splice mutation somewhat similar to that causing VWD. Splice mutations are leaky and a very small amount of PDK4 is probably made even in homozygous positive animals. A small amount of VW factor is made in homozygous affected vW dogs. Is a small amount of PDK4 that leaks enough? It seems that it is for some part of a dog's life or like the knock out mice, they would die shortly after birth.

Dominant mutation with incomplete penetrance: There is a real life example in the so-called Dutch eye disease PHPV/PHTVL. In this disease there is variable penetrance of a dominant mutation and this has been proven by breeding studies. A dog with only one tiny spot can produce blind puppies and there might be blind puppies in one litter and none in the other with a different mate. Clearly, unknown factors affect the amount of penetrance --how much the bad gene affects the dog is penetrance. In some European countries where there is a lot of this mutation, breeders have taken to breeding animals with low grades of penetrance. In the case of DCM, a breeder could argue that his heterozygous dog has a pedigree filled with long-lived dogs and that therefore he could breed this dog with the risk that enough modifier genes are present that his get will not develop the disease till very old age or not at all. Of course, I probably would not buy such a puppy but I can see that this argument has some validity.

I believe we have to use every tool at our disposal to eliminate the terrible disease and to be absolutely transparent in our dealings with puppy buyers. Those who do not test or test and do not tell are not acting in the best interests of our breed, I probably would not purchase a puppy that is homozygous or heterozygous positive unless the pedigree was filled with long-lived dogs. Afterall, longevity is the bottom line.

Dr. Meurs is working on finding wtether there is another common mutation and is collecting samples from negative dogs with a diagnosis of DCM.

Linda Kurz, Ph.D.
UDC Administrator of Records
UDC Health Committee, Co-Chair
UDC President