Introduction

This is a brief summary of where I have got to in my attempt to find markers or even a causative gene linked to JKD. The study was started by extensive arm-twisting of me by Bruce Cattanach, who slowly eroded my resistance to help him over a period of about 6 months. It was with the deepest sadness that I learned of his death, earlier this year. Nonetheless, as a result of his urging and energy, we began collecting samples from boxers that were variously unaffected, affected or related to affected dogs and I now have over 1200 samples. The following is a summary of where the study is at and, as far as possible, what it has shown.

Phase 1: ‘microsatellites’

Microsatellites are not, as one student mistakenly thought, small devices that can be attached to animals to see where they go! No, they are genetic ‘markers’. A marker is any genetic trait that can be traced through the generations. Some markers have visible consequences. For example, a gene that affects eye colour has several alternative forms (= alleles) and can make people with blue, brown or green eyes. However, most markers used by geneticists are ‘silent’ in the sense that you cannot tell what alleles a person or dog carries just by looking at them. To find out the alleles a dog carries you need to read their genetic material, their DNA. Microsatellites are short bits of DNA that are both very variable (it is as if any one can have the equivalent of 5, 10 or even 20 different eye colours), and they occur scattered frequently throughout our genetic material.

Imagine a microsatellite with 10 different alleles that sits on dog chromosome 1. We know where each microsatellite sits because the entire genome, the book of life, for dogs has been read and published. Now imagine a stud dog who carries alleles 3 and 7. If he has 20 offspring and 5 have JKD, and all the puppies with JKD have inherited allele 3, this is unexpected just by chance. It is like tossing a coin 5 times and getting 5 ‘heads’. Consequently, this observation provides some evidence that this particular microsatellite sits somewhere near a gene influencing the disease. It is not proof, far from it, but it is an indication. If we then find that a related family also has puppies with JKD and these also carry allele 3, the evidence strengthens. Across the whole sample set we may end up with enough evidence to be confident that having JKD and inheriting a particular bit of chromosome 1 happen much more often that they should be chance. The most likely reason is that a gene that makes JKD more likely has a faulty copy that is ‘marked’ by allele 3, it likely lies on the same bit of chromosome 1.

In my initial study I analysed all the dog samples for 64 microsatellites, chosen to be evenly spaced across the book of life. Each one was tested to see whether it carried an allele or alleles that were found unusually often in dogs with JKD. One microsatellite stood out, where dogs with the disease were three or four times more likely to carry particular ‘risk’ alleles. The pattern was not perfect, but it was strong enough to be reasonably confident that an influential gene lay nearby. Even more so, Bruce told me of one important stud that he was sure was homozygous (both its copies of the gene have the same risk allele). I tested this prediction and he was right! This dog had 13 offspring for which I had samples and all 13 carried the risk allele. This alone is enough evidence to be more or less sure that the stud was homozygous, but I then found an archive sample of the stud himself and I was able to show directly that he was indeed homozygous.

In conclusion, this was the part of the study I initially agreed to do for Bruce and from it I was able to conclude that there was probably an influential gene somewhere ‘close’ to this microsatellite on chromosome 1. This is good, in that dogs have almost 40 chromosomes so the target region has been narrowed, but less good in that chromosome 1 is the biggest chromosome, and likely carries more than 1,000 genes! There is still a lot of searching to do.

Phase 2: the pedigrees

As part of my dog database I was keen to make full use of the excellent pedigree information that is available. One of the big problems with pedigree dogs is that a cluster of cases reported by a breeder could have many different causes: is it that the breeder is unusually keen and efficient to publicise cases? Is it a genetic effect inherited from their prize stud? Is it an environmental effect linked to how the dogs are reared? Is it a combination of genetic and environmental effects? There are many possible explanations.

Having spent untold hours tracking down parents and parents of parents, I managed to establish my own database with at least 5 generations all the JKD affected dogs in Bruce’s database as well as those form which I have been given samples. This database is useful for conducting quite sophisticated analyses and one of these is to test for an effect of inbreeding. Inbreeding depression is the adverse effect that can occur when relatives mate, and occurs in all species. However, pedigree dogs are problematic because they are almost all inbred to some extent. Moreover, inbreeding depression is not easy to predict. If two pairs of siblings have litters, one litter may be more or less fine and the other very sickly, it depends on what harmful genes lie hidden in the parents.

To test for an effect of inbreeding in a fair way, we need to find some way of controlling for the fact that each family is different. I chose to do this by comparing JKD puppies with their parents. Specifically, I used the deep pedigrees to calculate 4 generation inbreeding coefficients for each affected puppy and each of its two parents. I then asked whether the puppy is more or less inbred than the average of its parents. This controls as well as we can for the fact that breeders differ in the extent to which they avoid inbreeding. If inbreeding does impact JKD then we would expect, on average, JKD pups to be slightly more inbred than their parents. This is exactly what I find. The effect is small but statistically significant and it adds weight to the data from Phase 1 that indicates at least some genetic effect. Also, I believe (though have not yet tested) that breeders are increasingly trying to avoid inbreeding. If so, pups should on average be less inbred than their parents and the effect I found then actively bucks the trend making more significant.

Phase 3: searching for the gene

I never intended to go beyond the microsatellite analysis but got carried along on a combination of Bruce’s persuasive pestering and the enthusiasm of all the breeders Bruce put me in contact with. I therefore felt the need to try to find the / a gene itself. This is not trivial! From Phase 1 I was reasonably confident that at least one informative gene lies towards one end of chromosome 1. The problem is that I have no way of knowing whether my marker lies a long way from a gene with a very strong impact on JKD or lies close to a gene which only influences whether a dog develops the disease slightly. The two options, along with anything in between, would give similar patterns in the data.

In theory, if I can assay a lot more genetic markers from the same general region of chromosome 1, I should be able to home in on the gene. The closer a marker is to the gene, the stronger should be its association with JKD. Imagine chromosome 1 is a book. Using the published version of the book, I chose to read a random sentence from each chapter. Unfortunately, the reading (= sequencing = reading the DNA sequence) is very pricey. As a result, I developed a cunning but highly complicated way to get as much reading done for each pound spent. The good news is that the scheme worked and I have managed to read some 70% of dogs. The bad news is twofold: (1) too many dogs failed to be read and (b) at some point I made a mistake such that part of the data are scrambled. To resolve this, I need to do a partial repeat so that I can discover where the mistake lies. Sorry, but mistakes happen. I hope to do this repeat when lockdown has eased a lot more.

Summary

I have reasonably strong evidence that there is at least some genetic component to JKD. This is seen both in the weak linkage to a marker on chromosome 1 and the weak impact of inbreeding. Unfortunately, my analysis so far does not tie the problem down much further. My best guess is that JKD lies somewhere between 80% genetic / 20% environmental and 20% genetic / 80% environmental. I think it highly unlikely that there is one single dominant gene for several reasons: (a) the weak linkage I found; (b) the fact that there is a strong sex bias in affected dogs; (c) the existence of unaffected dogs that carry two risk alleles. The whole problem is made more complicated by the fact that diagnosing the disease is not easy. It is entirely possible that there is one form of the disease that has a strong genetic basis and another that is mainly environmental.