JRD and test development

RENAL DYSPLASIA (RD) DNA TESTING
Discovery of the Mutation and development of a direct DNA test for RD.

What is RD?

Renal dysplasia (RD) is an important category of kidney diseases in canines. Dysplasia is defined as abnormal growth or development of cells or organs. In the case of RD the kidney fails to develop properly during embryogenesis in the womb. At birth immature structures consisting of undifferentiated fetal cells or tissue types are found in the kidney, and are persistent throughout the life of the animal.

RD can present itself with a wide range of symptoms and pathological findings. Definitive diagnosis of RD is done by a wedge biopsy which reveals dysplastic lesions, including abnormal ducts, and glomeruli. Individuals with an abnormal biopsy can be asymptomatic, showing no signs of the disease. On the other hand, they may present with classic signs of chronic end stage renal failure, or somewhere between these two extremes. Given this broad spectrum of symptoms affected individuals often go unnoticed, and remain in the breeding population. This is why development of a genetic test was necessary for the management and elimination of this disease.

RD a Closer Look

What you see is not always what you get. For those breeders that deny that RD is a problem in their kennel, you would see it if you had done biospies on you dogs.

Below is a summary of an article by Dr. Kenneth C. Bovee (click on link for actual reference)

In October of 2003, Dr. Kenneth C. Bovee from the University of Pennsylvania published his findings in the Shih tzu from a 10 year study involving 143 dogs and 52 matings. His findings clearly show that the majority of breeding stock had some level of fetal glomerul, and estimates from this study indicated that the prevalence of this defect (meaning biopsy positive fetal glomeruli) was probably about 85% in the breed, however the actual clinical cases that manifested severe renal dysfunction was low.

Other critical conclusion from this 10 year study was that animals with a low perentage of fetal glomeruli could produce those with renal disease and even the breeding of 0% fetal glomeruli (biopsy negative) adults resulted in offspring with 1-3% fetal glomeruli. The apparent low incidence of disease was a danger to the breeding population as seemingly normal adults could go undetected in the breeding population, and produce clinically affected offspring. Further, while using biopsy data to try and control this disease in the breeding population limited to some degree the production of severely affected progeny, this was not entirely successful in eliminating the transmission of biopsy positive offspring from the parents.

Further Dr. Bovee speculated, based on these findings that the mode of inheritance was not a simple recessive, and could follow a pattern of dominant with incomplete penetrance.

Thus the development of a genetic test was imperative to control this disease in this breed as well as others.

What Breeds Have RD?

Many breeds of dogs are afflicted with RD, and this has been documented in veterinary text books, as well as case reports and articles in the scientific literature. Of note is that RD in these breeds share a common phenotype, characterized by immature glomeruli, and/or tubules and persistent mesenchyme.

RD is largely thought of as a health problem in Lhasa apsos, and Shih Tzus. This is not true!

How is RD inherited?

The mode of inheritance of RD has been widely debated, as this disease can present itself with a wide range of symptoms and pathological findings. Definitive diagnosis of RD is done by a wedge biopsy which reveals dysplastic lesions, including abnormal ducts, and glomeruli. Individuals with an abnormal biopsy can be asymptomatic, showing no signs of the disease, On the other hand, they may present with classic signs of chronic end stage renal failure, or somewhere between these two extremes. Given this broad spectrum of symptoms affected individuals often go unnoticed, and remain in the breeding population. This is why development of a genetic test is critical to the management and elimination of this disease. Further a genetic test can be used to show the mode of inheritance .
Through pedigree studies, the mode of inheritance was finally revealed as Dominant with Incomplete Penetrance.

Development of a Genetic test at DOGenes .

During the development of the fetus, certain genes act in a pathway or cascade to direct the development of the various organs and structures that make up the body (think of it as dominos: if one domino is missing the other ones wont perform their task in the array). Since every cell contains two copies of every gene, one non-mutated copy may be sufficient to complete to cascade (recessive inheritance). The human genome contains about 20,000 to 25,000 genes, and the canine genome is likely to contain about the same number. Of these several hundred or More... may be dedicated to the maturation of specific organs. Not all of these tissue specificÓ genes are absolutely essential to the development of specific cell types, as has been shown in Òknockout miceÓ, where genes have been completely eliminated with no observable effect on the animal. In the case of renal dysplasia in model organisms such as the mouse, mutated transcription factors (genes that code for proteins that turn on other genes) or growth factors (genes that code for proteins that promote the growth of cells), have been implicated in causing the disease.

One approach to discover genes that are cause genetic diseases is to use candidate genes known to cause specific diseases in model organisms, like the mouse, rats, zebra fish or even the lowly fruit fly.

This was the approach used in the quest to find the mutation for RD in dogs. After DNA sequencing six candidate genes, the causative mutation was finally uncovered in a gene in Lhasa apsos, and Shih Tzus. This mutation was then discovered in other breeds with RD, and a direct genetic test is now available for many breeds afflicted with this disorder.

Through pedigree studies, the mode of inheritance was finally revealed as Dominant with Incomplete Penetrance.

Pedigree examples

Below are examples from Soft coated wheaten terrier pedigrees that shows that this mutation is inherited as dominant with incomplete penetrance. These are used in this article as they best demonstrate the mode of inheritance.

In the first example, two SCWT's that were breed were homozygous for the mutant allele. Therefore all of the offspring would have two mutant alleles. In this breeding a RD puppy was diagnosed at 4 months. The diagnosis was by biopsy, blood work, ultrasound and ultimately autopsy. Thirty six offspring in total were produced by this male with seven different partners, thus demonstrating the apparent low level of penetrance.

In another case a SCWT female that was a homozygous for the mutant allele and bred only once produced a litter of seven with one RD clinical puppy. RD in this case was determined by autopsy. Irrespective of the genotype of the sire (whether he was a carrier or homozygous for the mutant), all of the puppies would have at least one copy of the mutant allele. One of the siblings to the RD affected puppy was tested as homozygous mutant allele. Therefore the sire of this was at the very least a carrier.

In the final example, a female that was homozygous for the mutant allele was bred with a sire that was clear (wild type). Therefore, all of the offspring would be carriers of the mutant allele. One of the puppies in this litter was diagnosed with RD by autopsy. The sire, who was clear had produced 11 litters, prior to this one with no RD puppies reported. This example shows that carriers can develop RD, while in this first example above some animals that were homozygous for the mutant allele developed RD. Therefore, the inheritance was dominant, and therefore can be transmitted from one parent alone.

Finally as demonstrated in the above examples, not all animals that have the mutation (one or two copies), develop this disease. Thus, the mode of inheritance is shown to be dominant with incomplete penetrance.

How can the new test be used to eliminate this disorder from a breed
without compromising the gene pool?

Genetic tests are designed to manage and eventually eliminate disorders without compromising the diversity in a gene pool. If you have just found out that your dog carries the mutation for renal dysplasia, do not panic. Now you have the opportunity to manage and eliminate this disease. Thefrequency of this mutation is extremely high in many breeds. This mutationhas been elusive and impossible to eliminate prior to the development of a genetic test, as the disease appears sporadically because it is inherited withincomplete penetrance, meaning that an animal that carries this mutation mayor may not show clinical signs of the disease, but can still pass it on to the next generation.

As in any breeding you must consider the positive and negative traits of each partner, and how the parents traits can best balance and compliment each other.

All dogs (and living organisms) are carriers of multiple mutations.
If a genetic disease is produced in an animal, it is not necessarily the result of poor breeding practices, but is the nature of inheritance as a random event. Although the exact mutation rate for canines is difficult to determine, by extrapolation from other species, there is a good chance that every individual produced has a new mutation in some gene. Therefore, with every generation of breeding, new mutations arise, but since they are present at a low frequency, they are generally lost in subsequent breeding. There is no such thing as a perfect animal!

Chromosomes exist in cells in pairs, one from the sire and one from the dam. Dogs have 39 sets of chromosomes. Each set or pair is composed of two chromosomes, one from the sire, and one from the dam. In the case of a simple recessive mutation, one of the chromosomes, either from the sire or the dam, makes enough protein from for the animal to survive. Therefore, the Òwild typeÓ chromosome of the pair provides enough protein (gene product) to compensate for the chromosome that carries a mutation. In the case of a dominant mutation, only one copy of the chromosome carrying the mutation is necessary to produce disease.

With the identification of one of the many mutations that your animal carries,
you can now proceed to at least eliminate this identified mutation, and not inadvertently select for another deleterious mutation that your animal carries.

Wholesale elimination of carriers is the worst decision that you can
make as this would deplete the gene pool.

As in any breeding you must consider the positive and negative traits of each partner, and how the parents traits can best balance and complement each other.

What information does this DNA test actually tell the breeder?

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) Clear - No copies o f the RD mutation are present.

With a & b results above - the animal is potentially affected by RD or
could pass it on to its progeny.

How do I submit my dogs DNA for testing?

DOGenes uses cheek swabs for DNA testing.

We ask for three samples per dog. When you order a test we send you the cheeks swabs.

You take the samples and return them to us for testing.

Breeding Decisions

For breeds with a high frequency of the RD mutation:

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 an 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 a 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. All of the puppies in the first generation will be carriers. No need to DNA test at this point. 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.

Use Your Knowledge Wisely
Protect Your Gene Pool and Preserve Genetic Diversity in your breed

Mary H. Whiteley, Ph.D.
DOGenes Inc.