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Akita Friends
VKH Forum - Detecting Genes
by Jamie Haight, Bea Pitts, Bev Lewis, Dr. Sophia Kaluzniacki, Les Ray
 
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May I take the opportunity to intervene here, with an article which has been written in very recent weeks by the KC Canine Genetics Co-ordinator with the Animal Health Trust here in England. The article was originally written specifically for my own breed club's newsletter, but Dr Sampson has very kindly given me permission to reproduce it in the hope of better understanding how the genes that cause these inherited diseases are detected.

- Bea Pitts


How Do We Detect Genes That Cause Inherited Disease In Dogs?

By Dr Jeff Sampson 
- KC Canine Genetics Co-ordinator Animal Health Trust,
Newmarket, England

The dog's DNA molecule contains between 50,000 and 100,000 genes, no one knows the precise number. As you know, these genes are the genetic plans that control every characteristic displayed by the dog, its appearance, its behavior and so on. Unfortunately, the plans embedded in genes can become altered by a process called mutation. If this happens, and the gene controls a crucial function, then the result could well be a disease. Since the mutation makes an irreversible change in the gene, if the mutated gene is passed into either an egg or sperm, then we have an inherited disease because offspring will inherit the potential for the disease as part of either the maternal DNA or the paternal DNA that is present in the fertilized egg.

The majority of canine inherited diseases are caused by a recessive mutation. A puppy needs to inherit two copies of a recessive mutation, one from its mother and one from its father, before it is affected by the disease. Animals that have just one copy of a recessive mutation are not affected by the disease, but they are carriers who can pass on the mutation to their offspring. When two carriers are mated, on average, 25% of the puppies will be affected, 50% will be carriers and the final 25% will be clear. It stands to reason, therefore, if we can find a way of identifying carriers this will allow breeders to make more informed choices as to which two animals they mate, avoiding, wherever possible, carrier/carrier matings. Carriers could be identified if only we have a way of identifying the mutant genes that cause inherited disease. A carrier will be the apparently normal individual who does not have the disease, but has one copy of the mutant gene and one copy of the normal gene. (Animals affected with the disease will have two copies of the mutant gene and clear animals will have two copies of the normal gene.)

So, how can we identify mutant genes that cause inherited disease? There are a number of ways available to scientists. The first is a method that we call the Candidate Gene Approach. This method relies on being able to identify diseases in man and mouse that are identical to the canine disease in question. We know very much more about the genes in man and mouse, and it is very likely that the mutations that cause similar diseases in these two species will be the genes involved in the canine disease. Let's look at a specific example, Progressive Retinal Atrophy (PRA) in the Irish Setter. We have known for some time that this particular disease is very similar to a disease in man known as Retinitis Pigmentosa and a disease in mouse caused by the rde mutant gene. When the same gene was shown to be involved in the disease in man and mouse, it was an obvious step to ask whether this same gene was responsible for PRA in the Setter. Satisfyingly, the same gene was also responsible for Setter PRA, so it had been identified. A similar approach has recently successfully identified another PRA mutation, this time in the Cardigan Welsh Corgi.

The candidate gene approach is a wonderful short cut to the identification of gene mutations involved in canine disease. Unfortunately, it is not always possible to identify candidate genes. We therefore need another approach to identifying mutant genes that cause disease. Over the last ten years, scientists world-wide have been collaborating on something called the Canine Genome Project with the aim of providing more efficient means of identifying individual genes. You can imagine that the canine DNA molecule is a very long necklace with the beads representing the individual genes; so you have a necklace with approximately 100,000 beads side by side. The mutant gene being sought would be just one of these beads, but we have no idea which one. The canine genome project set out to begin to describe this necklace in some detail. Specifically, it set out to identify particular beads along the length of the necklace so that we could tell, by looking at these beads, precisely where we are at any point along the necklace. The beads that have been identified as part of the genome project are called markers.

How does this help in identifying mutant genes? Well, for each mutation we know it will be one of the beads on the necklace, but we will not know which one and where it is on the necklace. The markers that have been developed by the genome project are placed at evenly spaced intervals along the length of the necklace, and each marker identifies a unique position. So, although we don't know from the genome project where they are on the necklace, we can immediately go to the region of the necklace that contains the mutant gene.

Being able to identify the markers either side of the mutant gene is the key to being able to identify the mutant gene itself. During reproduction the fertilized egg contains a complete necklace, but sections of it have come from the mother and sections from the father. The point is that beads that are close together will be on the same section of the necklace and will always be co-inherited. This is how we identify markers that are either side of the mutant gene; we look in the DNA of those animals known to have inherited the mutant gene and ask which of the available markers are always present when the mutant gene is present. By doing this carefully we will identify those markers that are either side of the mutant gene.

In practice, we need an extended family where we know that the disease gene is present. For a disease known to be caused by a single recessive gene, we need approximately fifty dogs (grandparents, parents and offspring, both clinically affected and clinically clear) where somewhere in the region of 10-12 dogs are clinically affected. We need to be able to take DNA from each of the dogs, most easily obtained from a small blood sample, and then use the DNA for analysis. What we then attempt to do is identify the markers that are always present when the mutant gene is present. There are approximately 300 markers that are spaced along the dog's necklace and we systematically work through these 300 markers. Eventually we will identify markers that are always present in DNA samples with the mutant gene. This means that they are physically close to each other in the canine genetic necklace.

Once identified, these markers, now known as linked markers, will form the basis of a reliable test for the disease gene. Not a 100% reliable test, but one which will detect the disease gene in 98 out of 100 cases. For most instances this is sufficient to be used to begin to reduce the frequency of the disease gene by selective mating; it will in effect detect 98 out of 100 carriers. More importantly, the linked markers will provide an experimental short cut to the identification of the mutant gene itself. Once this has been identified then a test with 100% reliability can be devised. It is hard to put precise timings and costs on this kind of research. However, most people feel that it will take no more than two years, and probably less, to identify markers linked to disease genes, at a cost of about 30,000 -  40,000. With a bit of luck and a following wind, it will probably be as long again before the disease gene itself is identified. "Guesstimates" of the time required  to go from linked markers to gene identification are very rough at the moment and could well be a bit optimistic. The point is, however, that identification of flanked markers will in the vast majority of cases provide a test that will allow significant in roads to be made toward reducing the frequency of disease genes in particular breeds.

 
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