by Margret Casal, med. vet, PhD Section of Medical Genetics Veterinary Hospital of the University of Pennsylvania  There are two ways to go about finding the gene that causes epilepsy in the Irish Wolfhound. The first method (A) is simpler, but as will become evident, cannot be used for this breed at this time. The second method (B) requires more time, extensive pedigrees, blood samples from many affected and normal dogs, and finally quite a bit of mathematical know-how. The type of epilepsy found in Irish Wolfhounds must be described in great detail, which includes clinical findings, MRI results, necropsy results, and, if possible, the knowledge of biochemical processes in the brain leading to epilepsy. If all of this has been investigated, the disease can be compared to the different forms of epilepsy that exist in humans. Plan A comes into play, if a type of epilepsy in humans matches that found in the Irish Wolfhound, and the gene responsible for the disease in humans is known (=has been sequenced and mutations in that gene have been shown to cause epilepsy). Let's say that the affected dogs have the same chemical imbalances in the brain as do some human epileptic patients and that the gene that is mutated to cause this disease is known to be the stretch of glass beads (the gene) between Normal, IL and Urbana, IL. In this case, we can "just" go and look at the same stretch in dogs, which may be slightly different, but very similar. Now imagine that you are in an airplane high enough that you can see both Normal and Urbana, you would never be able to see the glass beads and analyze them. This is where the PCR reaction comes in: we get the inhabitants of Normal to run towards Urbana, while placing the exact same glass bead next to every glass bead they pass. At the same time, we let the people from Urbana run towards Normal also making copies of every glass bead they come across (Figure 3). If 30 inhabitants from each city do this, we would have a million copies in no time. This we can see from the airplane and we would be able to see the differences in the dog gene versus the human gene. Finally, if we do this for the normal dog gene, we could write down the exact sequence of the glass beads and then compare it to that sequence obtained from an affected dog. Thus, we would have found the "mutation" and all dogs could be tested and bred or neutered accordingly to eliminate the disease from the Irish Wolfhound population. But - alas - we do not know the gene in the humans, so we don't know where to look in the dog (i.e. we have to search the whole stretch of glass beads between NYC and San Francisco). Plan B requires blood samples from many affected and normal Irish Wolfhounds and their pedigrees, and in fact that is what we have already received from you. Total DNA is extracted from each blood sample and we "run" a large panel of markers on each sample. Then we look for the presence of markers that are common in all of the normal dogs, and variations of these markers that are only present in the affected dogs (see Part 2). When we find the same marker that is always present in a normal dog, we know it has to be close to the actual normal gene, because the marker generally gets passed on with the gene during conception because of its close proximity. While this may sound simpler than plan A, it actually requires a tremendous amount of very repetitive work. Imagine the DNA as a chain of glass beads again and imagine that the gene we are looking for lies between Normal and Urbana. BUT we don't actually know that. Now imagine that we have 50 DNA samples from normal dogs, 50 samples from affected dogs, and 200 markers (That's 20,000 PCR reactions to perform and analyze!). These markers and their variations are defined, short stretches of DNA (glass beads) that bind to their counterparts on the total DNA (the whole chain from NYC to SF). Once we make their binding to the DNA visible, we look to see which markers are always present in the normal or affected dogs. These markers are then said to be "segregating" with those diseases, i.e. inherited or passed on with the diseases. After we have bound the markers to the DNA samples that you have sent us and compared the results to the pedigrees and the clinical findings, we calculate which markers always show up in non-epileptic dogs but never in affected dogs. Once we have found a marker that segregates with the gene of interest, we begin sequencing the DNA around this marker, until we find the gene. Then we look for mutations within this gene to develop a test that can be used in the future to determine normal, carrier, and affected dogs. Ultimately, the production of epileptic Irish Wolfhounds will be prevented if the parents are tested before they are used for breeding. Figure 3: The polymerase chain reaction (PCR) is one of the most commonly used tools in molecular biology. With every cycle of copying, the number of genes already copied is doubled; i.e. four copies are made from two, eight copies from four, 16 from 8, and so forth. By the time one reaches 2
For inquiries regarding submission of blood samples, contact: Anne Janis PO Box 928, Fayetteville, GA 30214  |
