The Coton de Tulear is still remarkably free of genetic disease.  There are perhaps 45-55 known genetic diseases in Cotons (compared to hundreds in other more common breeds).  The known incidence of any particular genetic problem is low, in the 1-5% range.  Often in other breeds, the incidence of one or more diseases can be greater than 50% in the population and presents a real problem to long term breed vitality.  The known incidence of von Willebrands disease in Cotons is so low that most people have never heard of it.  It is good to be aware of possible health problems but these health articles are not meant to convey that there is a particular health problem in the breed.  ACC breeders maintain a lifelong educational commitment to learning as much about the breed as possible.  It's always a good idea to ask the breeders you approach for puppies if they health test their dogs and what they can tell you about any potential problems in their lines.

Von Willebrands Disease in Cotons

Von Willebrands Disease:  Part I       Diane Callison as told to Ron Hiskes

This article appeared in the Spring, 2006 issue of American Coton Quarterly

Diane Callison is a Coton breeder in Northern California.  Diane is a founding member and a Board Director of the American Coton Club.

Von Willebrand’s disease (VWD) is an inherited bleeding disorder common in many breeds of dogs - German Shepherds, Doberman Pinschers, Shetland Sheepdogs, Chesapeake Bay Retrievers, German Shorthaired Pointers, Golden Retrievers, Standard Poodles, and Scottish Terriers.  Blood clotting is a complex phenomenon and VWD is caused by a deficiency in one of the clotting factors called the VWDf or von Willebrand’s Disease Factor.  Severe spontaneous bleeding is rare, but hemorrhage may follow any type of surgery or even cutting into the quick of a toenail.  Acute symptoms include nosebleeds, bruising or passed blood in feces and urine.  Von Willebrands also predisposed dogs for dying under anaesthesia. 

Inheritance in most breeds is thought to be autosomal dominance with incomplete penetrance, meaning it can be passed directly from one parent dog to the next generation in varying degrees.  It is not sex linked.  In some breeds it is known to be caused by a recessive gene and both parents must be carriers for there to be affected puppies. There are three distinct types of von Willebrands disorder (Type I being the most common and Type II and III being less common and more severe) and severity of the disease can vary within each type.  The good news is that carriers and affected dogs can be determined from a simple blood test, the VWD Factor Antigen test which measures the percentage of clotting factor in a dog’s blood.  Clotting factors of  70% or above indicate a normal dog, 50-70% is borderline and indicative of a probable carrier, and clotting factors less than 50% indicate the dog is either a carrier or affected.   Dogs with VWDf less than 15% are definitely affected, although they may not exhibit symptoms.

What does this have to do with Cotons?  Unfortunately there have been four dogs identified as affected, and six, possibly seven, as carriers out of a total of 18 dogs tested in the last few months.  We don’t yet have all the information to make conclusions about the mode of inheritance since some key dogs in the puzzle have not yet been tested, but we felt it was important to present the information we know so far about this disorder and the dogs involved.

It started late last year when a Coton puppy named Daisy, bred by Diane Callison in California, was being prepped for a spay surgery and her vet noticed excessive bleeding from the IV site.  The surgery was cancelled until tests could be run.  A blood test revealed a VWDf of 33% on the first test and 25% on the second.  Diane notified the owners of the other five puppies in the litter and requested they be tested.  Of those puppies, two more were found to be affected, two are carriers and one is normal/clear.  In addition, the mother dog, Olivia of Purple Sage Cotons, was tested and came in at 35%, positive as a carrier.  Olivia is three years old, this was a first litter, and she had never exhibited any unusual symptoms.  The sire of the puppy, Vitalize of Woodland Cottage, was tested.  He is 8 years old and has sired numerous puppies without any indication of von Willebrands disorder in those pups.  His first test came back in the indeterminate range—63%.  A retest resulted in a value of 44%, leading us to believe he is a carrier.  It must be stated that the clotting factor depends on a number of variables, and can vary based on things such as whether the dog is calm or stressed just before the blood draw, whether a female is in a heat cycle, even how much to eat the dog has had in the hours prior to the test, but if the test is administered properly, the results should be accurate.  The test, called the ELISHA test, was developed by Dr. Jean Dodds, a leading veterinarian and expert in this and other canine blood disorders.

In the quest to further understand the origin of this disorder, Olivia’s mother, Ueleme of Woodland Cottage, was tested.  Ueleme came in at 44%—also a carrier.  At this writing we have not been able to get any information about Olivia's father, Shining Star.  Olivia’s sister, Ani, also owned by Diane Callison and a full sister to Olivia, was tested and had a VWD of 44%.  Ani’s litter of three puppies from 2004, were all tested, and two are clear but one of those puppies was found to be affected (15%) although he has shown no symptoms.   The sire of this litter is currently unavailable to be tested but his dam has been and is clear, and his sire is in the process of being tested.

Two very popular Coton sires are prominent on both sides of the pedigrees of the carrier dogs.  Ithalique appears on both sides of the pedigree but we were fortunate enough to get him tested thanks to his very cooperative current owner, Noreen Fitzgerald.  Ithalique is tested as normal and so we can rule him out as contributor to this disorder.

Qalamity of Woodland Cottage is also prominent in the pedigree.  He is the the grandsire of the three affected puppies in the original litter through Vitalize, and is also the great-grandsire on the dam’s side, through Shining Star, which makes him related to the fourth affected puppy as well.  Unfortunately Qalamity is deceased and we can’t get his test results. At this writing we are attempting to have Vitalize’s dam tested.  Ruling her out as a carrier would mean Qualmity is most likely a carrier and that the mode of inheritance of von Willebrands in Cotons is autosomal dominant.

While researching von Willebrands disorder we were able to track down another litter in 2002 born in Oregon which had von Willebrands-affected puppies – two of whom died shortly after being neutered and a third who had a bleeding incident and nearly died after surgery.   The dam of this litter was tested and is a carrier but is now deceased.  The sire was never tested and we are unable to locate him.  So far, no commonalities have been found between the pedigrees of Diane’s and this earlier litter.  But this may meant the disorder is far more widespread than anyone realizes.

It is Diane’s recommendation that breeders consider having a Willebrands Factor Antigen test done on all their breedable dogs.  She believes we can stop this disorder before it becomes a widespread problem in Cotons, and by breeding only dogs testing as normal, we can eliminate it entirely.  We will keep you informed of any more developments regarding the mode of inheritance.  Diane has been in contact with VetGen laboratory in Michigan which has developed a DNA test for the disorder in several breeds of dog.  VetGen has DNA samples from two of her affected dogs and they are looking for a match with a known marker in other breeds, hopefully expediting a DNA test for the disorder in the future. 

Diane Callison wrote to Dr. Jean Dodds, who developed the ELISHA test for von Willebrand’s disease at Cornell University.

Dr. Dodds,
My veterinarian suggested I write you, and I was also encouraged by the vets at UC Davis after an appointment with them earlier this week to write.  I have been a breeder of Coton de Tulear dogs for over 10 years.  Recently I had a litter with von Willebrands affected puppies. 

It was the first time I had heard of it in our breed.  Since that time I discovered a litter from 2002 which also apparently had vWD puppies.  We haven't found anything in common in the pedigrees of the two litters going back four generations but we will continue to look.  None of my puppies have died, and apparently only one has been recognized as a bleeder.  Two puppies in the earlier litter died and one had an episode of severe bleeding.

In my litter there were six puppies - one has not been tested.  The ELISHA test results were 117, 62, 65, 25, and 7%  The 7% puppy had been neutered before we knew there might be a problem with apparently no ill effects.  His test will be repeated in case it was not accurate.  The 25% dog was recognized before she was spayed as having excessive bleeding and testing was done twice.  She's since been spayed with plasma transfusions before and after.

The dam of this litter tested at 35%.  Three full sisters have also been tested - 115, 83, and 44%  The sire of the litter tested at 63% and his test has just been repeated at UC Davis because we were again uncertain if the first test was done accurately.  If he would test in the normal range, we could see this as an autosomal dominant mode of inheritance which would make it more clearcut.

The dam's dam has been tested - also 44%.  We have not been able to test the dam's sire yet.  The owner is not cooperative.   The dam's maternal grandmother was placed as a pet and has disappeared somewhere in the UK.  We're still searching.  The maternal grandsire has been tested and is clear at 101%.  This is a very good thing since he is a very popular sire in our limited gene pool.  The sire's dam is in Norway will be tested.  The sire's sire is deceased.

I hope that makes the genetic pedigree as clear as we have information for so far.

So my questions are - and I appreciate your reading this far...
1.  Is there any possibility of the disorder resurfacing in later generations if we breed the normal offspring to other normal offspring?

2.  Do you suppose that my litter is primarily Type I  and the earlier litter was Type II  since those puppies obviously had more bleeding problems?  How important is it to make the distinction, and can the two types appear in the same litter, or are they distinct disorders?

3.  How significant are the scores of the dogs who test in the mid-60's?  Should we attempt to regulate breeding strictly to dogs who test above 70%?

4.  Do you have any specific recommendations that I can pass on to other Coton breeders?

Several of us who are involved with this litter are attempting to get all the information we can before presenting it to the larger Coton breeder community on our club web site www.cotondogs.com and as an article in our The American Coton Quarterly.  Any advice, suggestions, comments you can make would be greatly appreciated.      Diane

Dear Diane:    Sorry that you've had this unfortunate situation, but I applaud your diligence and perseverance ! It's hard to be sure whether this is an incomplete dominant expression of vWD [the most common form -- dominant with variable penetrance of the trist amongst offspring of carrier heterozygotes], but it appears to be so.  In a rare breed like the Coton, it would be highly unlikely that two subtypes of vWD co-exist -- even though one litter had more bleeding than the other.  Breeding is best done only with dogs testing truly normal, so in your situation, would avoid breeding any dog in the 50-70% range.  As the situation unravels, you may be able to go back and pick up very desirable borderline dogs and breed them only to mates with high levels [preferably over 90%].  The defect should not surface again later on IF only truly normal to normal offspring are bred , although there can be some overlap between the normal and carrier population. Years ago, we identified by tracking his offspring, a Scottie carrying the type 3 recessive form of vWD [produced true zero affected offspring] with a vWF:Ag level as high as 77% and never testing below 50%. So, misclassification can occur.  One just has to be persistent in breeding away from the trait.   Best wishes. 

Jean

Hi Dr Dodds,
Thank you very much for your prompt reply.  Now I have another question.... To recap:  I had a litter of Coton de Tulear puppies with vWD.  All their test results are now in - 117%, 62%, 65%, 25%, 8% and 7%.  The mother tested at 35%.  The sire's original test was 63% but because we felt that result was ambiguous and we were't totally confident that the blood draw was done properly, we had him re-tested, this time at UC Davis where we thought we could get the most accurate results.  To our surprise this most recent test showed a 45%.  This is really confusing.  I was under the assumption that incorrect blood draws and bad handling would result in a lower percentage score - a false positive -  rather than one that was more towards normal.  Maybe that's not a correct assumption?  We do know that when the sire was tested the second time he was in some degree of pain due to a back injury.  Could this have caused such a dramatic drop in his result?  Would you recommend a third test?

I am kind of at a standstill about whether or not to recommend testing to people who may or may not have carrier dogs if the results can be so widely divergent.  I have been in contact with the VetGen lab in Michigan who have offered to take DNA from my two lowest scoring puppies and try to find a match with markers for other breeds.  This is a long-shot, but we may get lucky.  Interestingly, and you probably know this as well, of the breeds for whom there are known DNA markers, all are apparently - according to VetGen - caused by a recessive gene.  Now that our sire has tested in the carrier range, I'm leaning in that direction for Cotons.

In the meantime, though, I was planning to try to put something coherent together to present to other breeders, but I'm just not sure what to recommend.  One of my fellow breeders suggested I ask if you would be interested in writing something for our breed magazine.  I'm sure you're a very busy individual and I would not want to impose, but we would be extremely grateful if you could make some recommendations about where to start with testing and how to proceed with breeding.

Thanks in advance,   Diane

Dear Diane: von Willebrand Factor is synthesized exclusively by endothelil cells that line blood vessels throughout the body. It is therefore an "acute phase " reacting protein that is increased by any stress events the body is responding to.  Thus, the difference between 63% (equivocal) and 45% (carrier) is not significantly different, and in your case it makes sense because you would tend to get affected bleeders pups with two carrier offspring. Bad processing of samples will tend to lower results but I cannot comment on the way these two samples were processed, because I don't know the specifics.  All labs that do this testing in the world use the test we developed and patented in Albany, NY. Regardless, proper processing of samples is necessary -- and we and my colleagues at Cornell have literature about how samples should be prepared.  We don't know whether any of the DNA tested breed markers will work in the CT -- as only the Scottish Terrier vWD genetic marker has been validated in the refereed scientific literature. None of the other breeds for which screening is advertised have been validated in the refereed scientific literature -- so they may or may not identify the genes or just reflected a linkage marker. Perhaps we should chat on the phone about all of this.  Jean

Meanwhile, Diane contacted Dr. Duffendack of VetGen Laboratory in Ann Arbor, Michigan.  VetGen has done DNA research of VWD in other breeds. 

Diane reports:

John Duffendack of VetGen asked me to call him so I just spent about half an hour talking with him.  Very interesting.  He believes Dr Dodds is incorrect about the gene being autosomal dominant with incomplete penetrance because in the 10 breeds VetGen maintains they have been able to find the marker for, all are recessive. (Of course there are 50 or so breeds with vWD!)   VetGen is sending me two DNA kits.  I will take samples from the two lowest scoring pups on the Cornell test and send them into VetGen.In about a month they will have run all the tests they can and maybe we'll get lucky. He did say not to get hopes up because they fail to find a match far more often than they find one.

This is all the factual information available at present.  VWD may be prevalent in the breed or may not be.  Few if any pet Cotons in Europe are spayed or neutered.  If they never have any surgery, it is difficult to know how prevalent this disease is in Europe. 

In any case, this is one of the few or only diseases in which there is a number found in a simple blood test costing just over $100, which can determine if a Coton is clear, a carrier or affected.  Information about a DNA test involving a simple mucosal cheek swab for Coton de Tulear, will be published as soon as more information becomes available.

Coton breeders and dog breeders in general are notoriously defensive about hereditary diseases and in particular, hereditary disease which has a possibility of being in their dogs or their lines.  The paradigm has been that secrecy is the best policy except for sharing among a few close friends to make sure YOUR dogs are free from

disease.

Babies have to stumble and fall before they walk.  When they realize the floor way down there is not that scary they make great strides.  Similarly, it’s hard for Coton breeders to openly share health information.  Once we realize that sharing information about hereditary genes in our lines makes us heroes in the eyes of our peers instead of goats, we will make great strides.

Once we get past being defensive about our dogs and our lines and our reputations, the Coton Community working as a whole can solve the problems facing the breed.  Will you do your part?      —editor

One Genetic Pedigree Line for von Willebrands Disease --squares are males, circles are females.

this article appeared in the Summer 2006 issue of American Coton Quarterly

Diane Callison is a Coton breeder in Northern California.  Diane is a founding member and a Board Director of the American Coton Club.

Note:  The following series of articles were written by VetGen personnel to report the results of their research on Doberman Pinschers.  Although the dogs referred to in the articles are Dobes, the results and conclusions are equally applicable to Coton de Tulear based on the DNA research done by VetGen in 2006 on Cotons.

In the Spring, 2006 issue of ACQ we identified the first known Cotons with Von Willebrand’s disease (vWD), an inherited bleeding disorder common in many other breeds of dogs.  VWD is caused by a deficiency in one of the clotting factors in blood called the vWf or von Willebrand’s

Disease factor.  Hemorrhage may follow any type of surgery or even cutting into the quick of a toenail of a Coton with a vWf deficiency.

The Cotons were identified with a special blood test analyzed at Cornell University which determines the amount of clotting factor in the blood.  This test for vWD is unique in that a blood test can be used to determined if a dog is clear, a carrier or affected for this inherited disease  It is useful but imprecise and is sometimes said to be quite inaccurate. 

Although a blood test is helpful, it would be wonderful to have DNA test to determine unequivocally whether a particular Coton is a carrier of vWD.  A researcher at VetGen Laboratories in Ann Arbor, Michigan, agreed in March of 2006 to work with us to determine a DNA marker for the disease.  Dr. Duffendack unexpectedly passed away last month, but the research was completed at the lab using cheek swabs supplied by volunteer Coton owners.  

We are pleased to report that in a remarkably short time, VetGen has found a marker and has a DNA test available for the Coton de Tulear breed.  This marks the first time a DNA test for any disease has been developed for Cotons.  We hope it will be the first of many.

The reason for the unprecedented  success of this effort is that VetGen has done research already on a number of other breeds and has found similarities.  vWD in Cotons has been found to be identical to the disease found in Doberman Pinschers, for which the research has already been done.

Dr. Duffendack had planned to write an article for this issue of ACQ presenting his research results, but instead we present information by VetGen researchers on the Doberman Pinscher.  The results of the studies apply equally to Coton de Tulear since the same marker has been identified for both breeds.

The Mutation Discovered and a DNA Test Developed

by George J. Brewer, Professor, Department of Human Genetics and Internal Medicine, University of Michigan Medical School, Co-Founder of VetGen LLC

Our research team is very excited about our discovery of the mutation that causes von Willebrand's disease (vWD) in the Doberman Pinscher. Credit for the discovery must include my colleagues, Dr.'s Patrick Venta, Vilma Yuzbasiyan-Gurkan, and William Schall, of the College of Veterinary Medicine at

Michigan State University, and to Dr. Jianping Li, who works in my laboratory at the University of Michigan as well as at VetGen LLC, and who did all the DNA sequencing. This discovery is a nice example of the productive cooperation between the two universities and the company mentioned, as well as four funding organizations that provided support, The Doberman Pinscher Foundation of America, Inc., The Orthopedic Foundation for Animals, the Morris Animal Foundation, and the American Kennel Club.

The mutation itself has some interesting aspects. For one thing, precisely the same mutation has occurred in some human patients with vWD. It is a little unusual to see mutations be identical across species. This shows how closely we are related to our canine brethren! Second, the mutation is of a type such that completely normal von Willebrand's factor (vWf) is made about 5-10% of the time. Technically, the mutation is called a splice site mutation, with alternative splicing occurring about 90-95% of the time. That jargon won't mean much to the average Doberman breeder or owner, but let me explain what is happening in layperson language. It may be useful for the Doberman fancy to understand the mutation to a certain extent, because its nature explains why it was so confusing to understand for a long time, and it also explains why affected Dobermans have a milder disease than, say, affected Scotties.

To try to understand the effects of this mutation, let's use an analogy common to general experience. Imagine that a freight train is supposed to go from point A to point B following a railroad track. There is a point where a sidetrack goes to point C. However, normally the train never goes to point C, because the switch to point C, connecting the track up to the main track, is never thrown. Then the switch breaks (this is the mutation) such that the lock holding the switch from connecting the track to point C is no longer effective. The switch can now jiggle back and forth, sending some trains to point B, and others to point C. As freight trains rumble towards the switch, 95% of the time it jiggles over and causes the train to end up at point C. This is useless because point C ends at a cliff. The trains rumble over the cliff and are never heard from again. A minority of the time, maybe about 5%, the switch jiggles the other way and the trains end up at their normal destination. So, only 5% of the freight is delivered.

This is exactly what happens in the Doberman affected animal. These animals have two doses (two trains in the above example) of the mutated gene. Each gene is capable of making 5-10% of normal vWf (that is, going down the main track to point B), because the normal splice site is used a little. The 90-95% of the time the mutated splice site is used (going down the side track to point C), no useful vWf is produced. Since each of the two mutated genes is producing 5-10% of normal vWf the affected Doberman ends up with twice that, or 10-20% of normal vWf in their blood.

So, one of the mysteries of Doberman vWD that has puzzled scientists for years, how affected dogs can end up with a small amount of completely normal vWf, is cleared up by understanding this type of mutation. A second mystery is also cleared up. Doberman owners and breeders have had their dogs tested for vWf for years using the protein assay of vWf, and have often discovered low values in dogs without a bleeding history, even at surgery. The reason is, such dogs have 10-20% of normal vWf. If the bleeding stress isn't too great, the 10-20% of normal vWf that is present can prevent undue bleeding. Part of the time uneventful surgery fits that criterion, and unusual bleeding does not occur.

I hasten to add that this should not be taken to mean that vWD in the Doberman is clinically harmless. The literature is full of reports of Doberman's bleeding and dying from vWD. There are a number of factors, known and unknown, which will affect the clinical outcome in a given case. First coagulation factors, such as vWf, are consumed during blood clotting. The more the bleeding, from injury or surgery, the more the consumption, and the more likely the limited supply of vWf in an affected Doberman will be used up, leading to renewed bleeding, now from vWf deficiency. Second there is also variation in the amount of vWf in affected Dobermans. A dog with a 5% value is at greater risk than one with 15%. Of course, other factors, such as other coagulation and tissue factors that we aren't measuring, will certainly vary from one affected dog to another, and change the risk of bleeding up or down in a given situation.

The Doberman breeder and owner should view vWD as a significant health risk, and a fault, and strive to get rid of the mutated gene. The discovery of the mutation, and the recent development of a DNA test, now provides just that opportunity.

Another mystery about Doberman vWD that we now understand better is the actual frequency of vWD in Dobermans. Dobermans have been said to have a 70% plus frequency of this disease, but that is not correct. It's more on the order of 35% affected, with an additional large group being carriers, but free of any bleeding risk. The disease is an "autosomal recessive", which means that affected animals have two doses of the mutated gene, and a mild to moderate risk of bleeding, for reasons explained earlier. Based on very preliminary data, we believe the mutant gene has a frequency of about 0.6 (60% of the genes are mutant) which translates into about 36% of all Dobermans being homozygous affected (two doses of the abnormal gene and at risk for bleeding), 48% being carriers (one abnormal and one normal gene, no risk of bleeding), and 16% being

homozygous clear (two doses of the normal gene). If the gene frequency turns out to be closer to 0.5, the frequencies for

affected will be 25%, carriers 50%, and clear 25%. Of course, our small sample comes from a limited region of the country. The gene frequencies may vary some in different parts of the country,   but the bottom line will remain the same. This is a very common disease and a very common mutant gene.

Carriers of the mutant vWD gene are at no risk of bleeding from vWD, but of course, will transmit the mutant gene to their offspring 50% of the time. Roughly, the ranges of vWf factor levels are 5 to 20% for affected, 30-100% for carriers, and 50-130% for homozygous normal. Note the major overlap between carriers and normals for vWf levels. This overlap accounts for the extreme unreliability of the vWf assay in trying to identify Doberman carriers of vWD.

The new DNA test for Doberman vWD is offered by VetGen LLC (3728 Plaza Drive, Suite 1, Ann Arbor, Michigan 48108; (734) 669-8440, (800) 4-VETGEN; Fax (734) 669-8441). It is very easy to do the test. You can order the test kit from VetGen by phone or letter. Each test kit contains three soft brushes including collection instructions. Following the instructions, the dog owner brushes the inside of the dog's mouth. Some of the cells lining the inside of the mouth stick to the brush, and provide the DNA for the test. No blood is required. The brushes are replaced in their envelope and mailed back to VetGen. Each vWD DNA test costs $140. VetGen will supply test results within two weeks of receiving the DNA.

Test results will come back as "clear," "carrier," or "affected." As stated earlier, clear means both vWf genes are normal,

carrier means one is normal and one is defective, and affected means both genes are defective. It is important to realize that this DNA test is very different from the old protein-based factor assay. The DNA test is definitive and final, a lifelong, permanent determination of the vWD status of each dog tested as

contrasted to the factor assay, in which the levels could change drastically over time. We can now say in hindsight that the old test probably correctly identified some affected Dobermans (values under 20), but it is completely unreliable for carrier

detection.

What should a breeder do with the test results, once they are obtained, in terms of breeding decisions? The problem facing the Doberman breeder is that it appears that only 15 to 20% of Dobermans are clear of the vWD gene. If one breeds mostly clear to clear, it narrows the breeding pool so much that there is risk of losing some of the Doberman's genetic heritage, i.e., some of the genes determining valuable positive characteristics of the Doberman might be lost, or highly diluted. Therefore, as a first priority, we advise breeding clear to clear and clear to carrier, at least for the next two or three generations. Over time, as the frequency of clear dogs increases, it should be possible to breed mostly clear to clear, and to eventually eliminate the mutant vWD gene.

As a second priority, we suggest that it is reasonable to breed carrier to carrier, if an acceptable clear dog is not available for breeding. This type of mating will produce 25% clear, 50% carrier, and 25% affected, on average. The puppies should be tested and the affected puppies not used for breeding.

Breeding carrier to affected and affected to affected should be avoided if at all possible. The first breeding produces 50% affected on average, and the second produces 100% affected animals. In my opinion, there should be two initial objectives of the Doberman vWD breeding program. One objective should be to produce as few affected animals as possible, because each is a health risk. That doesn't mean we believe affected Doberman puppies should be put down. Most of them can live normal lives. If possible, we believe it would be a good idea to neuter affected animals. The second objective of the breeding program should be to gradually reduce the gene and disease frequency. The kinds of breedings involving the mating of an affected, as listed at the first of this paragraph, tend to increase the disease gene frequency, whereas clear to clear and clear to carrier breedings tend to decrease frequency.

To further raise the awareness and standards of Doberman breeders, VetGen is helping the Orthopedic Foundation for Animals (OFA) establish a vWD registry for Dobermans. By registering the results of the vWD DNA test on their dogs, breeders stand to benefit at the point of sale when selling either carrier or clear puppies as established by the vWD DNA test.

In summary, ... breeders are now in the advantageous position of being able to begin eliminating one of the significant diseases in their breed, because of the discovery of the mutation producing vWD in this breed, and the development of a vWD DNA test by VetGen. The test is remarkably easy to get done, and is reasonably priced, considering that it is a definitive lifetime determiner of the vWD genetic type of the dog tested. We urge... breeders to get their breeding stock tested, so that we can get on with eliminating this disease.

Autosomal Recessive Diseases …

There are three possible test results: Clear, Carrier, and Affected. Below is a description of what each result means to you as a breeder.

CLEAR   This finding indicates that the gene is not present in your dog. Therefore, when used for breeding, a Clear dog will not pass on the disease gene.

CARRIER   This finding indicates that one copy of the disease gene is present in your dog, but that it will not exhibit disease symptoms. Carriers will not have medical problems as a result. Dogs with Carrier status can be enjoyed without the fear of developing medical problems but will pass on the disease gene 50% of the time.

 AFFECTED   This finding indicates that two copies of the disease gene are present in the dog. These dogs always have a potential to bleed given the right circumstance and will always pass on the disease gene (mutation) to their progeny. Always inform your veterinarian and consult with him/her regarding this test result.

Breeding Strategies...

VetGen's DNA test findings can be extremely valuable when developing and implementing your breeding plans. The chart provided below outlines the implications of various breeding pair combinations. Remember, it is always best to breed "Clear to Clear". If followed by all breeders, these strategies will ensure a significant reduction in the frequency of the targeted disease gene in future generations of dogs. However, to maintain a large enough pool of good breeding stock, it may be necessary for some breeders to breed "Clear" to "Carriers" (see below).

Ideal Breeding Pair. Puppies will not have the disease gene (neither as Carrier nor as Affected).

Breeding Is Safe. No Affected puppies will be produced. However, some or all puppies will be Carriers. Accordingly, it is recommended that Carrier dogs which are desirable for breeding be bred with Clear dogs in the future, which will produce 50% carrier and 50% clear animals, to further reduce the disease gene frequency. These offspring should be tested by VetGen's test for this defective gene, and if possible, only the clear animals in this generation should be used.

High Risk Breeding. Some puppies are likely to be Carriers and some puppies are likely to be Affected. Even though it is possible that there will be some clear puppies when breeding "Carrier to Carrier", in general, neither this type of breeding pair nor "Carrier to Affected" are recommended for breeding.

Breeding Not Recommended.  -  All puppies will be genetically and medically affected

 

posted on [DOBERWORLD-L] listserv Date: Tue, 19 Nov 1996 12:17:14 -0800  From: "James W. Anable, Jr."

(Editors Note:  this information should also apply to Cotons according to the research performed March-June 2006)

Subject: READ THIS--Dr. Venta's response to vWD questions List members,

Hi Jim,

My colleagues finally had a chance to comment and make changes to this letter to you, and so it is now a joint effort. So here it is.

As you know, VetGen is now offering DNA tests for von Willebrand's disease (vWD) in Scotties, Shelties, and Dobes, and Kristi, of VetGen, is beginning to post information about these tests. The responses indicate some confusion, particularly about the Doberman. You have asked me, as one of the investigators who discovered the mutation, to clarify the genetics and usefulness of the new DNA tests. This is a fairly long letter, and I will apologize for its length now, but I felt that much of what is presented is information that you and other interested parties will want. I have not followed the posts for the lists, so I hope that you will forgive me if I might repeat something that has already been said a million times.

A key part of this message that you must understand is that we now know the precise DNA mutations and why and how they cause vWD in these three breeds. So all past hypotheses and speculations in the Merck Vet Manual and elsewhere, which were based upon the old protein-based factor assay, are out the window. Ignore them--they are past history. Now that we have the mutations in Scotties, Shelties, and Dobes, we can speak from fact not speculation regarding these three breeds. We are working on other breeds as well, but we cannot promise the date at which we will find any of the other mutations (although, of course, we hope it will be sooner rather than later).

The bottom line of what is given below is as follows: (1) vWD in Doberman pinschers is a true clinical disease in which affected animals are predisposed to have abnormally (and sometimes fatally) prolonged bleeding times. (2) The Dobe disease is recessively inherited, contrary to what some previous research had suggested in the past. (3) Carriers are unlikely to have bleeding problems but affected (that is, homozygous mutant) animals are at a significant risk of serious bleeding problems, if they undergo surgery or sustain moderate trauma. Penetrance is *far* less important than was inferred from the dominant, incompletely penetrant model. (4) If this one mutant gene was eliminated from the breed, vWD would become a very rare disease, indeed, in Dobes.

I will begin by describing the disease in two other breeds, because I believe that this will lead into the Dobe situation very well. Both the Scottie and Sheltie have the severe Type 3 form of the disease. The Sheltie may be a rare exception to the rule, that better than 99% of any simply inherited disease in a breed is caused by one mutation. In other words, while the major and most severe form of vWD in Shelties is Type 3, there is a possibility that a minor portion of the vWD problem is due to Type 1 vWD. This is according to data developed by Dr. Jean Dodds and her colleagues (Brooks et al., 1992--see below for complete references). We are working to see if this is true (it seems likely). Type 2 vWD, by the way, has only been seen in only two breeds of dogs, German shorthaired pointers and German wirehair pointers, so we will ignore it, in this letter. Both the Scottie and Sheltie Type 3 vWDs are caused by mutations that prevent *any* von Willebrand factor (vWF) from being produced. The technical term for these mutations are "single base deletions." These diseases are recessive, so that both copies of the gene that a dog possesses must be mutant before the animal has a bleeding problem. Carriers almost never have bleeding problems (Johnson et al., 1988).

The Doberman pinscher mutation, on the other hand, is Type 1 but it is *recessively* inherited! Most human Type 1 vWD is inherited in a dominant, incompletely penetrant mode. There are two things that made the Dobe vWD appear to be inherited in a fashion like the human disease. (1) The Dobe mutation is what geneticists refer to as "leaky." That is, the mutant gene makes a small amount of normal vWF protein. The amount made by each mutant gene is about 5% of the total normal amount. A normal gene would make 50% (so that two genes produce 100%). (2) The frequency of the mutant gene in the Doberman pinscher breed is greater than 60%! The parent of an affected animal can be also be affected, due to the high gene frequency (thus, the apparent dominant inheritance), but this is not always the case (thus, the apparent incomplete penetrance). This result was easily (and understandably) misinterpreted as the dominant, incompletely penetrant mode of inheritance as seen in humans. In human genetics, it is assumed that each genetic disease is rare, and one would not expect the parents of "affecteds" to also be affected if the disease was recessive. With animals, rarity of a disease gene cannot always be assumed, as illustrated by Dobe vWD. Other researchers have also presented data and arguments that Dobe vWD is actually a recessive disease (Moser et al., 1996; Johnson et al., 1988). The identification of the mutation fully explains it. Homozygotes for the disease in Dobermans do *not* die in utero. The mode of inheritance with other breeds, such as the German shepherd dog, could still be dominant, incompletely penetrant. We simply are not certain of the inheritiance pattern for other breeds at this time.

Dobe carriers should produce 55% of normal vWF, on average (5% from the mutant gene and 50% from the normal gene). However, other biological variables can affect the amount of factor found in the blood. These variables include thyroid hormone level, estrous, liver status (diseased or not), etc. Variation can also be produced by inappropriate handling of the blood sample or some variability in the protein-based tests themselves. These variations for concentration of the protein in the blood can make an animal appear to be a carrier on one day and homozygous normal (clear) on the next (which value does a breeder believe?). This is why the protein-based tests are not as

useful as they might otherwise be. The DNA-based tests are completely different, because they detect the genetic change at the gene level, which does not vary. There are only three possible results from the DNA-based test. An animal is either clear, a carrier, or affected. Re-testing is pointless, because the result will always be the same for a given animal. So one test is good for the life of the animal. Incidentally, we have also set up the test so that it is noninvasive (you swab the inside of the dog's mouth with a small, soft brush), convenient (you can send the brush by regular mail--no need to refrigerate), and you can test at any age, even young puppies.

Dobe *carriers* with abnormally long bleeding times are not common. Dodds, Johnson and Stokol et al. have all reported that animals do not usually bleed excessively when they have factor levels greater than 36% of normal (Dodds, 1982; Johnson et al., 1988; Stokol et al., 1995). Carriers will occasionally go below this level, but usually not by very much. Surveys of fairly large numbers of animals have been conducted, and the data appear to show the expected trimodel distributions for number of animals vs. factor concentrations (Dodds and Covey, 1981; Moser et al., 1996; Johnson et al., 1988; Stokol et al., 1995). By extrapolating the overlapping curves it can be seen that carriers do not dip into the danger range more than occasionally. Therefore, the fear that an animal who tests as a carrier might someday suddenly become a severe bleeder because of the dominant, incompletely penetrant scenario is completely negated. Occasional carriers might have bleeding times that are prolonged, but these are the exceptions. Clear animals will never bleed abnormally, due to hereditary vWD (the removal of the disease gene should be, after all, the eventual goal).

Fortunately, even affected Dobes usually do not bleed spontaneously (unlike the case for Scotties, and perhaps Shelties). If they did, there probably would have been a stronger natural selective pressure to remove the disease gene. However, with surgery or moderate trauma, these dogs are at risk for serious bleeding problems (there are numerous reports in the scientific literature addressing this fact, and I am sure that there have been numerous anecdotal reports in this forum as well). So the disease and its causative gene are something that breeders should most certainly want to remove from their breeding programs. This will have to be done with care, however, because we do not believe that it is in the best interest of the breed to limit the gene pool by breeding only clear to clear. By following the guidelines that Kristi at VetGen posted previously (also available at http://www.vetgen.com/), it should be possible to allow the desirable genes to separate from the disease gene over a few generations, while at the same time preventing the occurrence of affected animals.

The mutation that we have found accounts for essentially all of the vWD seen in Dobermans. It is always possible that a rare mutation in combination with the common mutation would cause a bleeder. However, this should be very rare, because the rate of occurrence of *new* mutations for most genes is between one in one hundred thousand to one in a million per generation (Crow, 1993 and references contained therein). If the mutation we have found is eliminated from the breed, von Willebrand's disease will also be eliminated from the breed (ignoring those one in a million new mutations that can never be prevented). The same is true for specific lines, as well. Breed out the disease gene (which can now be detected) and the disease will be gone from the line.

If some portion of this letter needs clarification, please let me know and I will do my best to do so. The other primary investigators for this research are Vilma Yuzbasiyan-Gurkan, Ph.D. and William Schall, DVM at Michigan State University, and George Brewer, MD and Jianping Li in the Department of Human Genetics at the University of Michigan. I hope that you find this letter useful and, once again, Jim, I apologize for its length.

Sincerely,

Patrick Venta

Department of Veterinary Medicine

Michigan State University

References:

Brooks, M., W.J. Dodds, and S.L. Raymond. Epidemiological features of von Willebrand's disease in Doberman pinschers, Scottish terriers, and Shetland sheepdogs. Journal of the American Veterinary Medical Association 200:1123-1127 (1992).

Crow, J.F. How much do we know about spontaneous human mutation rates? Evironmental and Molecular Mutagenesis 21:122-129 (1993).

Dodds, W.J. Detection of genetic defects by screening programs. AKC Gazette pp. 56-60 (June 1982).

Dodds, W.J. and J.S. Covey. Canine von Willebrand's disease. AKC Gazette pp 53-55 (April, 1981).

Johnson, G.S., M.A. Turrentine, and K.H. Kraus. Canine von Willebrand's disease. A heterogeneous group of bleeding disorders. Veterinary Clinics of North America: Small Animal Practice 18:195-229 (1988).

Moser, J., K.M. Meyers, and R.H. Russon. Inheritance of von Willebrand factor deficiency in Doberman pinschers. Journal of the American Veterinary Medical Association 209:1103-1106 (1996).

Stokol, T., B.W. Parry, and P.D. Mansell. von Willebrand's disease in Dobermann dogs in Australia. Australian Veterinary Journal 72:257-262 (1995).

E-mail response by VETGEN to Questions raised on various mailing lists, regarding von Willebrand's Disease (vWD Type I)

Comparison of the Elisa Assay test and DNA testing for vWD Type I

Doberman breeders have used the Elisa test for a number of years, only to find that it was inadequate to say the least, and found it useless for making breeding decisions.  The Elisa test is not capable of determining the genetic status of Clear (does not carry the mutant gene), can not pass on the disease to it's progeny, Carrier (carries one copy of the mutant gene), is not likely to bleed from cause of vWD, but is capable of passing on the mutation to its progeny, no matter what the vWf % level is at any given time, or, Affected (carries 2 copies of the mutant gene) does always have a potential to bleed given the right circumstance, and will always pass on the disease (mutation) to it's progeny.  The Elisa test capability is to determine the vWf (von Willebrand's factor) on the day of testing and is variable to a large degree, yielding mostly different results on the same dog subject to conditions on the day.    Therefore the Elisa test has no value in determining if a dog is an affected, carrier or clear.

The only test that can provide this information definitively is the genetic DNA test, (a once a lifetime test).  There are no variables, no grades and no percentages.  A carrier for instance is simply a carrier and not less or more a carrier (no matter what the vWf is at any given time), a clear is simply genetically clear and not more or less clear (simply does not have the mutations).  The Elisa test on the other hand is so variable that even in the higher percentages a clear definitive status cannot be determined. I would like to point to a Correlation Graph between Elisa and DNA tested dogs.  Correlation Graph

The information for this graph was voluntarily provided to VetGen by Doberman owners, from various Doberman

e-mail lists, who had tested their dogs with the Elisa test and then as the DNA test became available tested their dogs with the DNA test.  It is easy to see the great overlap between the Elisa and DNA test, particularly in the carrier region.

In the event of a pending surgery for a vWD DNA tested genetically affected dog, the test of value is a bleeding time test immediately prior to the surgery, thus enabling the owner and veterinarian to be prepared and make arrangements to combat any possible bleeding episode.

A DNA tested Carrier or Clear dog does not require this precaution regarding concerns of bleeding from vWD.

Blood Clotting & vWD

Type I vWD is a splice site mutation, a characteristic of a mutation at a splice site, which in this case is such that the protein vWf (von Willebrand's factor) is produced some of the time, but not all of the time. vWD affected dogs have two copies of the mutated gene. Each one of these genes is capable of producing 5-10% of normal vWf, because the mutated splice site works correctly just some of the time. For 90-95% of the time when the mutated splice site is used, no useful vWf is produced. Since each of the two mutated genes is producing 5-10% of normal vWf the affected Doberman ends up with twice that amount, or 10-20% of normal vWf in their blood. This means that these dogs are able to produce enough protein (clotting factor) to get by, even through surgery or whelping until such time that the small amount of vWf is used up through stress or trauma, may this be surgical or injury, at which time a dog can becomes a bleeder. For further information please read Dr. G. Brewer's article at:  Dr. G. Brewer vWD Report  

Also a very explanatory article is the 
Temporal Variation and Factors affecting measurement of Canine von Willebrand Factor

Here is another very interesting link. See:  Concept of DNA Sequence The picture on the web page says it all... The loops are the introns which are left out of the coding regions (in green) when RNA is produced (in red) which goes on to produce the protein of interest, relating to vWD, in this case vWf. The places where the gene is reconnected, leaving out the loops, is called the splice site. When it is mutated and doesn't work, it leaves one of the loops in the coding portion and a meaningless protein is produced.

Further, the question of clotting time and bleeding time tests was raised and I might add comments regarding this subject here as well.  There are 2 specific tests used by veterinarians to evaluate bleeding time and clotting time.

1. BMBT (Buccal Mucosa Bleeding Time).

This test is performed under anesthesia where a section of the lip is restricted and is a calibrated (done to exact specifications) test and the bleeding time is measured. For reference see: PubMed Abstract
Buccal mucosa bleeding times of healthy dogs and of dogs in various pathologic states, including thrombocytopenia, uremia, and von Willebrand's

Disease.  Jergens AE, Turrentine MA, Kraus KH, Johnson GS. Comparative Hemostasis Laboratory, College of Veterinary Medicine, University of

Missouri, Columbia 65211.

2. Clotting time test.

A sample of blood is collected and placed in a tube with a certain chemical and the time the blood takes to clot is measured. In cases where this method is used they are not testing for vWD or Hemophilia. Rather they are looking for things like rat poison etc.

Inge Craik, 
GenTest
Australian Representative for VetGen
Saturday, April, 6, 2002

The first two Cotons have had their DNA tested for von Willebrand’s disease as part of the research program. Both - Jespersen's Bani Thani and Idris Mongo Khan of North Pole Cotons - were thought to be free of the disease, but Idris Mongo Khan was determined to be a carrier. This was a surprise. Subsequently, Mongo's daughter, Kiki, was determined to be a carrier.  There is a common ancestor for Mongo and for a litter from an Oregon breeder, two of whose puppies died of massive hemorrhage during neutering. Ghost du Royaume de la Mesange is sire of Mongo's sire Jordi du Mas of Sunnyside and of Isaty's du Mas of Sunnyside who is the granddam of the carrier sire of the other affected litter. Having an open health registry with links to the European clubs which also publish their health information allows rapid dissemination of vital data for everyone to use to the breed’s advantage. Let’s all pledge to cooperate in this effort.

Since the research has been published, many Coton breeders and owners have volunteered to have their Cotons tested as part of an ongoing program to determine how widespread this disease is in Cotons.  Two of the Cotons in the diagram above have been determined to be carriers,  Beany Baby and Gucci.  This explains the carriers and affected in their lines.  Tidelidee has been tested and found to be clear.  We will publish a list of Cotons that have been certified to be clear in the near future.

We thank all those who instantly and graciously volunteered to have their Cotons tested, initially with the blood test and later with the definitive DNA test.  Without your full selfless cooperation and support this work could not have been brought so quickly to a successful conclusion.