Inherited diseases in dogs

n this article I want to explore the genetic basis of disease in dogs and explain how recent advances have set the scene for a whole new set of tools which will enable breeders to breed genetically healthier animals. There are a number of ways to approach discussing genetics, but the way that I like to start is by discussing the basis of the characteristics displayed by all living things. Let's use the dog as our example. Everything about your dog: the way it looks and the way it behaves, for example, are the result of a complex group of molecules in its body called proteins. It is the activity of these proteins, either working individually or in groups, that determines every characteristic of the dog. What about an example to show you how proteins determine characteristics? There are many potential examples, but let us consider a rather complex, but crucial, characteristic - the ability to see.

It turns out that dogs see in very much the same way that we see. In fact, at many levels, our dogs are very similar to us in the kinds of characteristics they display; their similarities to their owners far and away outnumber their differences. The ability to see resides in the cells that make up the retina. Light enters the eye through the lens and is focused onto the retina at the back of the eye. The retinal cells perceive that they have received a light stimulus and produce a nerve impulse, which goes down the optic nerve to the brain where it is decoded and turned into a picture. Just how do the retinal cells convert light into what is in fact an electrical impulse, which passes down the optic nerve? Well they are able to do this because they contain a specialized group of proteins whose job it is to convert light impulses into nerve impulses. The light impulse activates a protein in the retinal cell called rhodopsin. Rhodopsin is normally unable to perform its function unless it is activated by the energy of the light. You could imagine that rhodopsin is switched on by the light. Once activated, rhodopsin then activates another protein which, in turn, activates a third protein and so on until the final protein activated in the pathway, a protein called cyclic GMP phosphodiesterase, generates a nerve impulse.

In the example above I have just used two proteins which switch between being active and inactive, in reality there are many more proteins in the pathway leading from light perception to nerve impulse generation inthe eye.

Clearly dogs are highly complex in the vast array of characteristics they display and this must be reflected in the number and complexity of the different proteins present in the cells of the dog's body. No one really knows precisely how many different proteins are present in the cells of the dog, but a conservative estimate would be around 100,000. Where do all these different proteins come from? Well, like us,

Dogs start their life as a single fertilized egg, called a zygote, which is the result of a fusion between an egg from the bitch and a sperm from the dog. This zygote contains few, if any, of those 100,000 proteins which are present in the adult dog. As the single cell grows and divides, first to produce the embryo and then, after birth and further growth, the adult, the new cells of the body begin to make the proteins as and when they are needed. However, the cells require plans in order to assemble this vast array of proteins at the right time and in the correct part of the body. The fertilized egg doesn't have any of the actual proteins, but it does have a complete set of plans that will allow the developing animal to manufacture the proteins. These plans are called genes and there is a gene for every different protein, hence there must be around I00,000 different genes.

Genes are made up of a chemical, which I am sure you have all heard of Deoxyribosenucleicacid, or DNA for short. A gene comprises a stretch of DNA that holds the plan for the synthesis of a unique protein. The plan is stored in its chemical structure. Each cell of the dog's body contains a complete set of plans for all of the proteins that are required. In fact it is more complex than that, because, perhaps for safety reasons, each cell contains two copies of each plan. This DNA is stored in special structures called chromosomes and a complete set of plans in the dog is represented by 38 different chromosomes; two sets would be 76 chromosomes and that is the number of chromosomes in each canine cell. In addition, each cell has two very special chromosomes that determine the sex of the dog. Every cell in the bitch has two identical X chromosomes, every cell in the dog has one X chromosome and one Y chromosome. So every cell in the dog's body contains 2 sets of 38 chromosomes plus two sex chromosomes, making 78 in total. The only exceptions are red blood cells that have lost all of the chromosomes during their maturation and the reproductive cells, eggs and sperm, which just contain one set of each chromosome, as we will see later.

So, when you look at your Rhodesian Ridgebacks, what you see is the result of a complex interaction between the genes present on these chromosome sets. Obviously, every Rhodesian Ridgeback will have an essentially common set of chromosomes because at one level they all look alike. However, there must be very subtle differences in the combinations to make one animal have slightly more desirable Ridgeback qualities than another. When you select a stud dog for your prized bitch, you are looking for a particular set of qualities/characteristics which you would like to introduce into your own line. What you are actually selecting when you choose your stud dog is a combination of genes that, hopefully, will give you those extra, desired qualities in your puppies. Perhaps now will be a good time to look at how these genetic plans are inherited.

In terms of inheritance, we only have to look at two cell types: those that give rise to the eggs in the bitch and those that give rise to the sperm. Both cell types mature from specialized precursor cells in either the bitch or the dog. In many ways these precursor cells are similar to all other cells in the body, certainly with respect to the numbers of chromosomes. Each contains two copies of all the chromosomes, that is 78 chromosomes. Such cells are said to be diploid and contain 2n chromosomes. Now, clearly something very special must happen to this chromosome number as the sex cells mature, otherwise, if a mature egg and a mature sperm both had 2n chromosomes, then after fusion the resultant fertilized egg would have 4n.

Since every cell develops from this single fertilized egg, every new cell in the developing animal would have 4n chromosomes. When such animals reach reproductive age they would produce 4n eggs and 4n sperm which, after fusion would give rise to 8n cells. On this scenario, at every new generation the chromosome number would double, which is clearly not what happens. The reason that it doesn't happen is that as the egg and sperm precursor cells mature they undergo a very special type of cell division that results in the reduction of the chromosome complement from two sets to one. Eggs and sperm are what we call haploid and have n chromosomes. Following fertilization, the normal diploid, 2n, state is re-established.

OK, let's get back to the main theme of this paper, which is probing the genetic basis of disease. The problems arise when one or more of the genetic plans are altered such that the protein that is made is different. These alterations, called MUTATIONS, alter the chemical structure of the DNA in a particular gene thereby changing the plan. The consequences are that the new plan is altered so drastically that it cannot make the desired protein. Consider a mutation in the gene for Rhodopsin that resulted in Rhodopsin that could no longer be activated by light. Obviously, the result of this mutation would be blindness because the light would not initiate the cascade that leads to nerve impulse generation. Not all changes in the genes are necessarily detrimental. As I have said, although all Ridgebacks will have essentially the same genetic complement, some will have more highly regarded, and therefore more sought after, characteristics and so the genes that lie behind these characteristics must be slightly different in that subset of animals that display the desired qualities. However, what I want to deal with are those mutations that are deleterious and result in disease states.

How mutations arise in the first place is still something of a mystery. They certainly seem to occur at random and are very much out of our control. We know of certain external factors that contribute to the mutation rate. Radiation is one example of an agent that clearly induces mutations. X-rays, UV and the radiation that is released during radioactive decay, all can be shown experimentally to induce mutations in DNA, i.e. alter the structure in a particular region of DNA thereby altering the plan in the gene represented by that stretch of DNA. Presently, there is an extremely interesting study being performed at Leicester University by Russian scientists working in collaboration with Professor Alec Jeffreys. You will all remember the nuclear accident that occurred at Chernobyl in Russia some years ago. Many Russians are alive who were exposed to excessively large doses of radiation because of their proximity to the explosion. The experiments involve analysing DNA from these individuals and measuring their mutation rates, compared to a matched set of Russians who were not exposed. The findings are startling. Those exposed can be shown to have hugely increased mutation rates over their normal controls. So, mutations can clearly be caused by environmental stimuli. However, they may also be a natural consequence of life! Every time a cell divides, and there is a phenomenal amount of cell division in our body, all of the DNA in the cell has to be replicated so that each daughter cell receives 2n copies of the chromosomes. This really does require immense fidelity to accurately replicate the entire DNA and we have special proof-reading systems to ensure that the process is as error free as possible, but inevitably mistakes are made and mutations can ensue.

Once an error has been made, either as a result of some environmental insult, or simply an error in replication, it is fixed forever in the DNA. If the mutation occurs in any of the vast majority of the cells in the body, it could well have serious consequences for that individual. For example, it is now generally accepted that in the progression from normal cell to cancer cell, the cell that changes has to acquire a number of mutations in its DNA which result in pushing the affected cell into uncontrolled growth mode. Clearly catastrophic for the individual in which this has occurred, but when that particular individual dies, the mutations that caused the cancer die with it. The more severe problem for the population is when the mutation occurs in the cells that form the precursors of the eggs and the sperm, because once fixed there the mutation can be passed onto offspring via eggs and sperm, thus causing an inherited disease.

The consequences of a mutation vary considerably, as do the outcome of matings in which an inherited mutation is involved, depending on the type of mutation. We classify mutation into either DOMINANT or RECESSIVE. In order to understand these two different types of mutation I have to remind you that every cell is diploid which means that it has two copies of every chromosome and, consequently, two copies of every gene. A DOMINANT mutation is one in which the consequences of the mutation can be seen in cells even in the presence of a normal second copy of the affected gene. A RECESSIVE mutation is one whose effects are not seen in the presence of a normal second copy of the gene. Dominant mutations are readily spotted because each animal that has a dominant mutation will be affected with the disease that ensues. The ease of detection of animals that have dominant mutations means that the eradication of the disease gene through selective breeding programmes is relatively straightforward. Recessive mutations on the other hand pose an immense problem for breeders wishing to eliminate disease genes from particular lines and breeds. Unfortunately, of the 300 or so canine diseases that have either been shown, or are believed, to be inherited, 80% of them are the result of recessive mutations. The problem comes with the animal which has one copy of the recessive mutation and one normal copy. The animal is unaffected but has the ability to pass on the mutant gene onto its offspring. This animal is an unaffected carrier. Unfortunately, we do not know whether an individual is a carrier until it happens, by chance, to mate with another unsuspected carrier, then we will get affected pups in the litter. However, before carrier status comes to light, the animal can have been innocently used in mating programmes and passed on its recessive mutation to offspring, thereby increasing the frequency of the recessive mutation in the population. The problem can be particularly acute if it is a dog that is the carrier and that dog is a top winner. Because of the ability of a single dog to sire numerous pups, it could have passed on the deleterious mutation to large numbers of offspring before its carrier status was discovered. There is a phenomenon called 'The Popular Sire Effect' that describes this very problem.

As I have said above, carrier status only comes to light when two carriers are unknowingly mated. Statistically, the outcome of such a mating would be affected: carrier: totally normal in the ration 1: 2: 1. The important feature here is that ¼ of all offspring. Will be perfectly normal and carry two normal copies of the gene in question. Clearly using such animals in subsequent matings would free the line from the disease gene. I stress that these ratios are statistical and would only reach the theoretical values at high numbers of puppies. It is just the statistics, which says that if you flip a coin enough times, half the time it will come down heads and half the time it ill come down tails. That is not to say that on the way you will not get seven tails in a row. Carrier/carrier matings can still go undetected, particularly if the average litter size for the breed is small, because, just by chance the affected pool did not appear in the litter because of a statistical fluke.

Obviously, if we have ways to identify these recessively mutated genes, we will be able to identify carriers before they are used in breeding programmes and we will be able to screen litters from carrier/carrier crosses to identify the carrier offspring and, more importantly, the offspring that are perfectly normal with respect to the disease gene. It is for this reason that there is quite a deal of scientific activity directed at ways of identifying genes that cause inherited diseases in dogs. There are a number of experimental tricks that are available to allow us to identify disease causing genes. As canine geneticists, we are fortunate to be able to use the absolute wealth of information that human geneticists have been acquiring over the last decade, a period of unrivalled expansion in our understanding of the human gnome. One of the goals of human research is to identify every gene required to specify a human and to identify those genes which, when mutated, give rise to a genetic disease in man. As a result, we have managed to discover a great deal about a very large number of human genes and the diseases that are caused when they are mutated. This has a given rise to a general approach known as the CANDIDATE GENE APPROACH.

The thinking behind this approach goes as follows. For a particular canine disease, attempt to find either a human or murine disease which looks clinically identical to the canine disorder and where we actually know the genetic lesion. This was the approach that was so successful in identifying the gene that cause Progressive Retinal Atrophy (PRA) in the Irish Setter. It had been known for some time that the pathology of PRA and the way the disease progressed in the Setter was similar, if not identical, to a Retinitis Pigmentosa disease in man and a disease caused by the rde mutation in mouse. The diseases looked identical. When the gene involved in both the Retinitis Pigmentosa in man and the rde mutation in mouse were shown to be the result of the same mutation in the same gene, namely the gene which contained the plan for the synthesis of the protein involved in the very last step of light transduction, the cyclic GNM phosphodiesterase gene. It became obvious that the same gene should be checked in the Setter. When the gene was studied in normal Setters, no mutation was seen, but in all of the affected Setters, the gene had undergone the same mutation seen in both man and mouse. Science was therefore fortunate in being able to use information provided by the human and murine geneticists to go directly for the gene that was the problem in the Irish Setter. Once the Setter mutation had been characterized, it was a relatively simple task to devise a test that would check the DNA of any Setter to see if it carried the normal gene or the mutated gene.

It is amazingly spectacular when the candidate gene approach works because it can short-circuit years of research and go directly to the cause- Unfortunately, candidate genes for other diseases in the dog are not easy to find. . Fortunately, the actual pathology of the disease may also give strong clues as to the affected gene. This was the case for a disease known as a-fucosidosis in Springer Spaniels. The disease is the result of a failure to metabolise certain sugars in the diet. Because of the pattern of the lesion, and what was known about similar diseases in man, again it was possible to pin point the likely candidate gene and verify that mutation were present in affected Spaniels and only affected Spaniels. So again, a DNA-based test could be devised to check for the presence of the defective gene in Springer Spaniels whose disease status was not known.

A final example of this kind of candidate gene approach comes from hemophilia in the Rotweiler. This disease is typified by the absence of a particular clotting factor in the plasma called von Willebrands protein. The gene for human von Willebrand protein has been known for some years and scientists in America were able to use information from the human gene to isolate the canine gene for von Willebrand protein. It was then a simple task to compare this gene in normal dogs and haemophilic dogs to demonstrate that the von Willebrand protein gene was mutated in haemophilic Rotweilers. A test for the mutant von Willebrand protein gene quickly followed.

All of these discoveries and the subsequent DNA-based tests were made in the dog because we had equivalent human disorders and genes that we could use for comparison. Sadly, that is not true for the vast majority of inherited disease in the dog. We can show that a mutation is at the base of the disease and we know that the gene involved will be somewhere in the affected dog's DNA, but locating it and identifying it is a real genetic needle in a haystack problem. Each cell of the dog has an immense amount of DNA. To put it into perspective, if we represented the DNA in a gene as a 10cm long stretch, then the entire DNA molecule inside the cell would be 150 kilometres long. The problem is that a mutant gene that causes an inherited disease could be a 10cm-long stretch at any position along the 150 kilometres! Let's look at it in a slightly different way. If we represent the mutant gene as a 10cm ruler, then it will be located somewhere between Leicester Station and St.Pancreas Station. What we face as geneticists is to find that ruler! Without help it is a seemingly impossible task.

Help is at hand, however. What if we were able to lay marker posts say every ½ kilometre and were then able to ascertain between which two marker posts the ruler could be found? With this information we would be able to go straight to a 500 metre stretch to start looking for the ruler, a much simplified task. Scientists have spent the last five years trying to lay down marker posts along the DNA molecule in the dog. We have yet to put a post every ½ kilometre, but we probably have one every 2-5 kilometres. How do we actually identify between which two marker posts the disease gene is located? We do this by following the inheritance of the disease gene in pedigrees and also the inheritance of the marker posts. A marker that is physically close to the disease gene on the DNA will always be co-inherited with the disease gene. In other words, whenever the marker is inherited so will be the disease gene. The presence of the marker is diagnostic of the presence of the disease gene. So when we have identified a close marker we can test if the marker is present in a DNA sample; if it is then the disease gene will also be there. This technique is known as 'genetic linkage analysis' and it is exactly this technology which has provided a test for the presence of the gene which causes copper toxicosis in the Bedlington Terrier.

I hope these words, perhaps a bit too long?, will shed some light into how we as scientists hope to contribute to the better breeding of pedigree dogs designed to identify, and hopefully eliminate, the genes that cause inherited disease in dogs.