Hallo, mal wieder ich.
Ich hab Frau Whiteley angeschrieben und die Erlaubnis, ihre Antworten hier einzustellen. Leider momentan nur in Englisch, da ich noch keine Zeit zum Übersetzen gefunden habe.
Dear Mrs. Mary Whiteley,
I am Benita Brühl-Hanke from Germany, Airedale-Terrier-breeder kennel “vom Goetschetal”.
I hope I do not disturb and nerve you but I`d like to get some more special information about the test. If you allow I`d like to put your answers online in our forum/blog.
First the problem:
A lot of people, among them Prof. Dr. Tosso Leeb from Switzerland, are sure, that the test developed by DOGenes is not valid. To his/their opinion the making-of of that test is not according to scientific standards for developing genetic tests. Therefore one needs 150 sick/ill animals in addition their families and one healthy control group of animals. Because of that there is strong suspicion that this test is not valid/ sensitive for Airedale Terriers and suspicion becomes stronger because at German Boxer breed there were (?) for some test results false results(positive and negative) , too.
That is argumentation of those people, who think about the money-making of test-labs.
You wrote to Kathrin, that there are up to 16th of July 27 Airedales tested from Europe, among them are 3 carriers, all others are homozygous mutant.
I know that from Germany up to now you tested about 30 dogs but all are homozygous.
Now my questions:
Could you give a statement to allegation, that the test you offer shouldn`t be valid? Could you tell us how did you create this test? Why are you sure that it works?
If we have no clears in Germany up to now and the dogs are all from different bloodlines there should be a much higher degree of ill puppies and dogs according to statement that cases of illness should be 5% on JRD. How can this be? That is reason for hesitation, too.
Why test is called RD-test and not JRD-test?
Can you tell us where do the carriers come from?
Sorry, but your answers are very important for discussion and therefore to get and give sureness we do the right.
I`m looking forward to hearing from you, soon.
Best regards
Yours, sincerely
Benita Brühl-Hanke
Dear Benita,
I am still working on the information request, but I will answer one question right away.
Mary Whiteley
Why test is called RD-test and not JRD-test?
The disease should be referred to as renal dysplasia only. This is because MOST of the clinical cases are 5 years and older, so the 5% that you refer to are in adults. The majority of animals with one or two copies of a mutant allele are sub-clinical and will live a normal life with respect the kidney function.
The cases in puppies are rare.
Dear Benita,
Here is my response to your email.
From your email.
“A lot of people, among them Prof. Dr. Tosso Leeb from Switzerland, are sure, that the test developed by DOGenes is not valid. To his/their opinion the making-of of that test is not according to scientific standards for developing genetic tests. Therefore one needs 150 sick/ill animals in addition their families and one healthy control group of animals.”
As to this point from Dr. Leeb and others they seem to be suggesting that the only acceptable experimentation would be the technology known as GWAS (Genome Whole Association Studies). While I have the impression that the canine research community appears to have embraced this technology as the choice for discovery of disease causing mutation, in fact the scientific community as a whole is less optimistic.
Note: I do agree with this technology in general but not in this case study for RD. I believe that it has tremendous potential to open up new hope to discovering disease causing mutations, however the biology of the disease needs to be explained along with the results of a GWAS.
GWAS has not evolved to the point where mapping of genetic traits even with that are complex or inherited with low penetrance are not considered routine and successful. This notion is supported in many scientific articles (examples given below) that state that while associations are being reported all the time, the issue remains to determine how many of these are synthetic, and not causal.
Wray NR, Purcell SM, Visscher PM (2011). Synthetic associations created by rare variants do not explain most GWAS results. PLoS Biol.;9(1):e100057.)
Hirschhorn et al. (Genetics in Medicine Vol4 No2 (45-61) reports that most associations are not robust. In this study of 166 associations studies repeated more than 3 times, only 6 showed a reproducible result. Hirschhorn suggests that irreproducibility of these associations may be due to linkage disequilibrium, gene-gene environmental interactions and weak genetic effects and lack of power to resolve these factors that may lead to irreproducibility.
The number of subjects suggested by Dr. Leeb and others (150 sick/ill animals in addition their families and one healthy control group of animals) are based on requirements for GWA studies, however in the case of renal dysplasia in breeds with a high frequency of mutant alleles and sub-clinical animal subjects this technology does not make sense. Therefore, I respectfully disagree with the opinion of Dr. Leeb and others that GWAS is appropriate for breeds with a high incidence of disease and therefore a high frequency of disease causing mutations and a mode of inheritance that has variable penetrance.
NOW THE FACTS
Dr. Bovee from the University of Pennsylvania laid the groundwork for understanding renal dysplasia in a comprehensive study in the Shih tzu breed.
Wedge renal biopsy data was collected from 52 matings and 143 dogs. Results of this study showed that the severity of the disease was highly variable amongst individuals. The severity of the disease was reflected in the percentage of fetal glomeruli found in the renal wedge biopsy. The mode of inheritance did not follow a simple Mendelian pattern. The proposed mode in inheritance by Bovee was autosomal dominant with incomplete penetrance.
He further predicted that the prevalence of this defect is approximately 85% in this breed in North America indicating a high frequency of mutant alleles in this population. By defect Bovee is not referring to dogs with chronic renal failure but an abnormal wedge biopsy with fetal glomeruli present in adults.
Here is how the numbers were broken down by Bovee:
“The prevalence of renal dysplasia in this breed is very high in North America. In a study of 74 random dogs evaluated with wedge renal biopsy, only 16% were free of any histological evidence of the disease. Among the remaining, about 52% had 1-5% fetal glomeruli, while 20% were moderately affected with 6-15% fetal glomeruli. The remaining 12% had more than 15% fetal glomeruli. These findings suggest that the genetic character of the disease is very high and variable in this breed. Because many dogs are mildly affected and escape detection in the absence of renal biopsy, the question arises as to the genetic transmission of the defect in normal appearing dogs.”
The total from this study of 74 dogs with an abnormal renal wedge biopsy is:
Abnormal biopsy: 84%
Normal biopsy: 14%
Here is what we know from our research from a pedigree of 52 Lhasa apsos. Renal wedge biopsies performed on 35 of these.
There were 35 Lhasa apsos in this family with both genotypic and biopsy data. Of these 34 carried at least one of the mutant alleles, 25 had biopsy findings consistent with renal dysplasia, and 9 had normal biopsy results. The other individual was homozygous for wild type alleles and had a normal renal wedge biopsy. Mating of two of the normal biopsy Lhasa apsos in this study resulted in a puppy with an abnormal biopsy. It is important to note a one of these Lhasa apsos in the study had a wedge biopsy with 40% fetal glomeruli as a puppy, and this dog lived to be 8 years old.
The take home message from this research is that this disease is not a problem limited to puppies. The majority of animals that die from renal dysplasia are adults between 5 and 10 years of age.
Why these unusual cases in puppies occurs in not yet understood. The mutant alleles for RD may be susceptible to influences of other genes or environmental factors that play an unknown but important role in the development of RD in dogs.
Bovee also pointed out that mating Biopsy normal parents resulted in an abnormal renal wedge biopsy in the offspring.
Our results:
Abnormal biopsy: 74%
Normal Biopsy: 26%
Therefore this data and along with that of Bovee suggests a genetic bottleneck in both the Lhasa apso and Shih tzu breeds.
So the question remains. In populations with a high frequency of mutant alleles corresponding to a high frequency of affected animals (ie: with an abnormal renal wedge biopsy but not necessarily showing clinical signs of renal failure), how do you choose 150 sick/ill dogs for your study, and who are the controls within that population????? Are you looking for animals with chronic renal failure, and are you willing to do renal wedge biopsies on these 150 dogs and their relatives. How do you find a control “line” that is free of the mutation in a purebred population with a disease showing a genetic bottleneck? Unlike human populations there are no suitable controls.
As a control group in our study we used the German shepherd dog breed that is not known to have a high incidence of RD.
Here are the results of this control.
German shepherd dog analysis
Further, a few cases were recently identified in a population of German shepherd dogs in Great Britain. The pathology reports were obtained for two littermates that were under 6 months old. The diagnosis was Juvenile nephropathy, a term which is equivalent to renal dysplasia. Of twenty random dogs tested for the mutation, one carrier of a mutant allele was identified. The sire of this dog was one of the animals that had produced RD affected puppies, and was inferred as having at least one copy of a mutant allele based on examination of the remainder of the littermates of the carrier dog that was identified. The dam also had to be at least a carrier as some of the puppies were homozygous for mutant alleles. The rest of the German Shepherds in this initial survey tested as wt/wt.
So how does this relate to Airedale terriers?
According to the Kennel Club health surveys the third leading cause of death in adult Lhasa apsos (adults) is chronic renal failure (accounting for 7 % of deaths). The third leading cause of death in Airedale terriers (adults) is bladder rupture and chronic kidney failure accounting for 8.3% of deaths.
Given this information, it is not surprising that the frequency of the mutant alleles is high in Airedale terriers, since the majority of animals with one or two copies of mutant alleles are sub-clinical.
HOW WAS THIS TEST CREATED?
Another approach to discover disease causing mutations is candidate gene selection based on other the disease in other model organisms. In the case of RD a candidate gene was chosen from the mouse model of RD that most closely resembled RD in dogs. This was the Cox-2 (Cyclooxgenase-2) gene. This was a logical choice as a gene to study since Cox-2 deficient mice have renal abnormalities and a pathology that has striking similarities to RD in dogs.
A cluster of allelic variants was located in the Promoter region of the canine Cox-2 gene. These are the DNA sequences that drive the production of the protein product. The chemical composition of these alleles is consistent with altering gene expression, and there are examples in the scientific literature that implicates DNA with this type of chemical structure to alter gene expression.
Further, the mode of inheritance is typical for allelic variants in the promoter region, and therefore supports the finding in this study.
In addition to the dogs in the Lhasa apso breed an additional 22 clinical cases from various breeds were examined. Regardless of the breed or severity of disease, all of these had one or two copies of the Cox-2 allelic variants. This included breeds with a relatively low frequency of the disease.
The focus of mutation research has recently shifted to looking for disease causing mutations in the non- coding regions that regulate gene expression such as the ones for RD. Scientists have increasingly been looking for mutations in promoter and regulatory region of genes identified by linkage or GWAS in cases where the biology of suitable candidate genes relates to the phenotype but there are no disease causing mutations in the coding regions of the gene. The RD research represents an example of this emerging science.
My area of expertise is gene expression during embryogenesis, and that is why I understand these findings, and stand behind the results and the test.
FINAL REMARKS
RD is inherited as "dominant with incomplete penetrance. This means that animals with one or two copies of the mutation are at risk for developing the disease and/or passing this on to their progeny. The risk in the case of RD is low meaning that most carriers or homozygotes for the mutation are sub-clinical.
What does the genetic test tell you?
The DNA test results are reported as follows:
a) Carrier - (one copy of the RD mutation)
b) Homozygous mutant allele = Homozygote (two copies of the RD mutation)
c)
c) Clear - No copies of the RD mutation are present.
With a) or b) results the animal is potentially affected by RD or may be clinically normal. However, clinically normal animals can pass the disease on to their offspring.
For breeds with a high frequency of mutant alleles it is extremely important that the breeding decisions are not solely based on an RD result. The overall traits of both parents must be considered. The goal is to breed these mutations out of the population without destroying the gene pool.
For example:
1. My dog is a clear. Examine this dogs good and bad traits. Can he/she be bred to a carrier in your kennel that can complement their traits? Yes. At this time, many breeds with RD have a very high frequency of the mutation, and in order to protect the gene pool, this type of breeding is necessary.
You keep the clear puppy from this cross that has the traits from both parents that you were hoping to get.
2. My dog is a carrier. Ideally, this animal should be bred to a clear with traits that would complement this animal. - Clear progeny from this cross can be kept for future breeding. There is a 50% chance in this case of producing a clear in the first generation. If no other options exist, this animal can be bred to another carrier. In this case, your chances of producing a clear for your next generation are 25%. There is a 25% chance that an animal that is homozygous for the mutant allele will be produced from this breeding.
3. My dog is homozygous for the mutant allele, but otherwise is sound in body and temperament, and brings positive trait to the breed. This is a two-step breeding to get a clear. This dog should be bred to a clear, if possible or a carrier if necessary. A carrier puppy from the first generation of breeding can now be used in the second generation to produce clears as in example 2.
DOWN THE ROAD, your ultimate goal is to breed clear to clear so that you have eliminated RD from your kennel without having compromised the gene pool.
Can you tell us where do the carriers come from?
DOGenes does not give out this kind of information on the test results, however breeders are welcome to put their results on our genetic testing database at:
http://www.dogenes.com/testingdatabasemembers.html
Membership to this database is free.
I hope that this message will help the breeders better understand this disease.
Sincerely,
Mary Whiteley PhD,
DOGenes Inc.
Reference:
Whiteley MH, Bell JS, Rothman DA. Novel allelic variants in the canine cyclooxgenase-2 (cox-2) promoter are associated with renal dysplasia in dogs. PLoS One. 2011 Feb 8;6(2)
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bingo!
