By Dr Jeff Sampson, KC Genetics Co-ordinator
Genetics of epilepsy in the Tervueren
Idiopathic, or primary, epilepsy is the form of epilepsy that is believed to be inherited in over 30 different breeds of dog. Although inheritance is suspected in all of these breeds, definitive data showing a precise mode of inheritance is only available for a small number of these breeds. In the Keeshond, data suggest a very simple single autosomal recessive gene disorder. In most other breeds where genetic studies have been undertaken, including the Tervueren, the picture looks more complicated than that seen in the Keeshond.
As many of you will know, epilepsy in the Belgian Tervueren has been the focus of research work at Davis, University of California for some time now. Both Tom Famula and Anita Oberbauer have been supported by the Canine Health Foundation (an American Trust Fund closely associated with the AKC) to study epilepsy in the Tervueren. Their initial work on analysing pedigrees in which one or more of the offspring had been diagnosed with idiopathic epilepsy indicated that although this is a heritable disorder governed by a number of different genes, there exists a single gene which has a very large effect on the incidence of epilepsy in the Tervueren. The epileptic condition influenced by this particular gene is inherited as an autosomal recessive.
So, what does ‘autosomal recessive’ mean? The adjective ‘autosomal’ means that the gene is present on one of the chromosomes known as an autosome. The dog has 39 pairs of chromosomes, 38 different autosomes and a pair of sex chromosomes (the X and Y chromosomes that determine the sex of a dog). The fact that it is recessive means that a dog has to have two copies of the recessive mutation before it is clinically affected. A dog that has one recessive mutant version of this gene and one normal version of the gene (remember dogs have two copies of each and every gene) will not be clinically affected with epilepsy, but will, genetically, be a carrier. Both parents of an affected dog (affected because it has two mutant versions of this major gene) will be carriers of the major gene involved.
Let’s look at this in a little bit more detail. Present estimates suggest that around 30,000 to 40,000 different genes are required to specify the dog. Each of these different genes will be distributed along one of the 39 chromosomes that make up the dog genome. Each autosome contains anywhere between 1000 and 2000 different genes arranged side by side along the length of the chromosome. Running along the length of each chromosome is a single molecule of DNA , the chemical that genes are made from. Chromosomes are often likened to beads on a string. It is not the best analogy, but it will do. The string running along the length of a necklace represents the DNA molecule, each bead represents a stretch of DNA that comprises a given gene. There are 38 pairs of autosomes and two sex chromosomes that contain each one of the 30,000 or 40,000 different genes. Each gene is present in two copies, one on each of the chromosome pairs.
Genes are simply genetic plans, the information being stored in the chemical structure of the DNA that makes up the gene. The plan embedded in each gene is used to make a protein and it is the activity of proteins, either working individually or in groups, that determines the characteristics of dogs. So there are in excess of 40,000 different proteins required for a perfectly functioning dog. (Some genes actually contain a plan for more than one protein). Occasionally, the chemical structure in the DNA making up a gene is changed, a process known as mutation. Generally speaking, the majority of mutations in DNA are neutral and have little, if any, consequence. Some mutations are actually beneficial giving rise to an improved genetic plan. The whole of evolutionary theory is based on the generation of new mutations that offer a new competitive edge to individuals that carry such mutations. Other mutations are deleterious because they impair the genetic plan so much that the protein produced doesn’t function correctly. If the protein that is mutated plays a crucial role in a biological process, then an inherited disease might ensue.
That is clearly what is happening with epilepsy in the Tervueren. This major gene that has been discovered clearly contains a plan for a protein that plays a crucial role in the brain, probably involved in regulating the electrical activity that balances excitatory and inhibitory responses. Some time in the dim and distant past this particular gene has undergone a mutation that has either inactivated the protein or severely altered its activity so that it can no longer balance electrical activity under certain circumstances, causing affected individuals to fit. The original dog in which the mutation occurred will have passed on the mutant gene to half of its offspring who, in turn will have passed on the mutation to approximately half of their offspring, and so on. Because the original mutation was a recessive mutation, the early recipients of the mutant gene would have been carriers and therefore clinically unaffected. So, unbeknown to Tervueren breeders, the frequency of the mutant gene would slowly build up in the population, as will have the number of unidentified carriers. Eventually, the carrier frequencies will have got so high that there is a reasonable chance that two carriers will, through bad luck, be mated and then there will be a chance of producing one or more epileptic puppies.
Clearly, dogs that contain two normal, unmutated copies of this gene will be able to make active protein and will therefore be clinically unaffected. A carrier will have one normal copy of the gene and one mutated copy of the gene. The normal copy of the gene in the carrier will allow it to make sufficient protein, hence carriers will be clinically normal. Only the dog that receives two copies of the mutant gene will be affected because both of its plans are mutated so it has no chance of making normal protein.
Let’s now take a look at how this mutation is inherited. The diagram below represents a mating between two carriers of this major gene involved in epilepsy. In the diagram the normal gene is represented as a white circle and the recessive mutant version as a black circle. So, both parents have one normal gene and one mutant gene; they are carriers but they are clinically normal. From such a mating we would expect to get 25% normal offspring (two white circles), 50% carrier offspring (one white and one black circle) and 25% clinically affected offspring (two black circles). Where do these estimates come from?
When the bitch produces eggs, each egg receives just one copy of each and every maternal gene. This is achieved by putting one complete set of maternal chromosomes into each egg. This means that the carrier bitch produces two types of egg with respect to this major gene involved in epilepsy: one egg carrying the normal version of the gene (white circle) and one carrying the mutant version (black circle). Similarly, each sperm produced in the male has a complete set of paternal genes, achieved by placing a complete set of paternal chromosomes into the sperm head. So, again, there will be two types of sperm
produced by the carrier dog: one type carrying the normal gene version and the other carrying the mutant gene version.
Fertilisation of eggs by sperm is a purely random process. So, consider egg A in the diagram below, it could be fertilised either by a sperm carrying a normal gene or a sperm carrying the recessive mutant gene. If egg A is fertilised by a carrying a normal gene, the fertilised egg will have two normal copies of the gene. This single fertilised cell is the source of all of the cells in the puppy that develops and the genes in this puppy are identical to those present in the fertilised egg. Therefore the puppy that develops from this fertilisation will be clinically and genetically clear of epilepsy because it has two normal versions of the gene in question. If egg A was fertilised by a sperm carrying the mutant gene, the puppy that develops from this will be a carrier. Now look at egg B, which carries a mutant version of the gene. If egg B is fertilised by a sperm carrying a normal version of the gene, the ensuing puppy will be a carrier. The problem arises when egg B is fertilised by a sperm carrying a mutant version of the gene. The subsequent fertilised egg will now have two copies of the mutant gene and the puppy that develops from this fertilised egg will be clinically affected because it has two mutant genes.
Thus in a carrier mated to a carrier you would expect one clear to two carriers to one affected, and this is where the 25%:50%:25% comes from. It is worth pointing out that these are what you would expect to happen. Reality can often be quite different. Because of the total randomness of the process of fertilisation in any individual litter you might experience significant deviation from these expected ratios.
Progress from now on should be quite rapid. Anita Oberbauer at Davis, University of California has now been awarded a new grant from the Canine Health Foundation to develop genetic markers for idiopathic epilepsy in the Tervueren. What does this actually mean? Although we know this major gene now exists, we don’t know what it is, neither do we know where it is on one of those 38 different autosomes. Using the newly developed genetic map of the dog, Anita Oberbauer hopes to be able to localise the gene to one small region of one of those chromosomes. The genetic map of the dog is made up of a series of special DNA markers, each marker uniquely identifying just one region of one of the chromosomes. The map contains 100s of different markers ensuring that each chromosome is evenly decorated with DNA markers. What Oberbauer is trying to find out is which of these markers is always co-inherited with the mutant version of the gene. DNA regions that are always co-inherited are physically close to one another on a chromosome. Markers that are co-inherited with the mutant version of the gene are said to be linked to the gene.
The identification of markers linked to the mutant gene will immediately locate the mutant gene to one small region of one of those 38 chromosomes. The next stage of Oberbauer’s research will be to search this small region until she finds the major gene involved in epilepsy in the Tervueren. The ultimate goal of course is to find and identify the major gene responsible. However, the identification of DNA markers linked to the disease gene will be a major landmark for the Tervueren in respect of epilepsy. The identification of linked markers will permit the development of a simple DNA test that will be able to identify carriers. This will be what we call a ‘linked marker DNA test’. Once the gene is identified then scientists will be able to develop the ‘Rolls Royce’ of DNA tests, a test actually based on the mutation identified in the gene (a mutation based, or gene based, DNA test). Whereas linked marker tests have an inherent inaccuracy in them, usually less than 1 or 2%, gene based tests offer 100% accuracy. So, the immediate future will see the development of a linked marker test for epilepsy in the Tervueren. In the fullness of time this linked marker test will evolve into a gene based DNA test, once the gene in question has been identified.
There is now a real possibility that in the not too distant future Tervueren breeders will have a DNA test that will be able to identify carriers of this major gene involved in epilepsy. This, of course, will be a very significant step forward. Breeders will be able to use the test to pre-screen all potential breeding stock. If a carrier is identified in this process, the breeder will know it and will be able to choose a genetically compatible mate. The breeder will know that if a carrier is mated to a dog that has DNA tested clear, then about half of the offspring will be carriers and half will be clears. Furthermore, the existence of the DNA test means that all of the progeny can be DNA tested to identify those that are carriers and those that are clear. The availability of the DNA test means that breeders will be able to mate carriers with confidence and also identify genetically clear progeny from carriers that can form the basis of future breeding programmes. The availability of a DNA test ensures that owners can mate their carrier dog with confidence. They can choose a mate using traditional values of breed type and temperament, but then back up, or fine tune, their selection using DNA testing.
The arrival of a DNA test for epilepsy in the Tervueren will not be quite the ‘god send’ that other DNA tests have been in other breeds, for example the DNA test for CLAD in the Irish Setter, because, remember what you are testing for is a major gene involved in epilepsy, not the only gene. However, the arrival of a DNA test for this major gene will permit breeders to make very significant in-roads into controlling epilepsy through selective breeding. There may still be the odd surprise in litters, even when DNA testing has been undertaken. However, this should not dampen breeder enthusiasm, being able to identify carriers of this major genetic mutation, if not all of them, will ultimately lead to a very significant reduction in the frequency of this condition in the breed.
Is there anything that Tervueren breeders can be doing whilst waiting for the American research to come up with the ‘goods’? I think there is. The value of any DNA testing is to use it on the breeding stock, these, after all, are the source of future generations. It is their genetic status that needs to be known. For a long time now I have felt that breed clubs would benefit by generating blood banks of all of their breeding stock. These blood samples would represent DNA archives of those dogs that have contributed to the development of a breed. If the Tervueren had such a bank of DNAs, these would be the first to be tested with any new DNA test. Since these are the parents of future generations, knowing what each dog’s individual genetic status is will identify those offspring who need to be tested. For example, if a litter has been produced from two genetically clear parents, their progeny will not need to be tested because they will also be genetically clear. Generating such a bank of blood is not amazingly difficult. All you would need is approximately 5ml of blood collected into a special tube that contains chemicals that prevent blood clotting and then stored in a simple domestic freezer. Tervueren breeders could easily begin collecting and storing bloods from dogs that have produced litters. Obviously, the availability of these DNAs will be very helpful for the epilepsy problem when a DNA test does become available, but the store will represent a true DNA archive that will be invaluable for identifying genes that cause other inherited diseases in the Tervueren.