Foreward

This study reports on a Californian project conducted with some practical help from me upon the brindle gene. In the main it has involved UK Boxers, but some American and Continental dogs are included. The original objective was to find the gene responsible for brindle coat colour/pattern, but as the study developed it has also begun to take on the question of how the brindle striping pattern itself is created, how the gene is regulated to give its observed effects. The article has been quite difficult to write for two reasons. First the material it presents has been reported earlier in a series of updates - in the British dog press. Therefore, slotting the various elements of the progress together in this single article has been awkward; it was not quite the way results came through, and the study is not even yet complete. Second, the report covers some complex elements of genetics from the meiotic process and chromosomal recombination to deep molecular biology and its technology. Clearly there was no way I could include all this genetics background in this article so I have attempted to skim over the detail, hoping that I can highlight the interesting points, give a ‘flavour’ of the excitement the results generated, and illustrate the significance of some of the surprise revelations. I should also point to the importance of a brindle-to-fawn mutation that should be of uniquely valuable for identifying how and where within the gene the brindle and fawn forms differ. More such mutations could be very useful and so, for this purpose, can I stress that further breeder help, notably from American breeders, would be greatly appreciated.

I had one further reason for writing this article for a Boxer magazine. As everyone knows, Boxers are having a bad time with certain health issues, notably with heart problems and with cardiomyopathy in particular. For this reason there is often talk on internet groups on the possibilities of finding the cardiomyopathy gene. The study reported here describes the equivalent molecular search for the brindle gene and this can therefore be thought as a rough model for the procedures involved for any gene search. You will read of the time scale involved and difficulties encountered with this simple well-known gene. Its inheritance is clear-cut, its effects can easily be distinguished from the alternative fawn, these effects can be distinguished at any age, an environmental influence does not exist, and there is only the one genetic cause for brindle. By comparison, the inheritance of cardiomyopathy is not totally clear, affected animals are recognised by the their diagnosis but one cannot reliably distinguish those dogs that do not carry the gene, the affects may not be expressed until late in life, environmental causes of cardiomyopathy are known and, from human studies, there is the expectation of more than one gene independently being responsible for this complex disease. Different forms of cardiomyopathy are certainly recognised in different breeds of dogs, and there is even some evidence that two genetic forms of the disease exist in UK Boxers. I do not wish to dampen enthusiasm for cardiomyopathy research but it will be a huge and difficult project to find the gene/s involved.

 

Background to this study on brindle

An American colleague of mine, Greg Barsh, who also is a mouse geneticist, has in recent years turned his attention to dog coat colour genetics (see his website: http://barshlab.stanford.edu/DogCoatColorIndex.htm). We have been in correspondence arguing about some of the problems with these genes as presented in C.C. Little’s classic book on dog coat colour genetics (Little was also a mouse geneticist). And there was also the seemingly incomprehensible finding from my early youth when I had, by ‘mistake’, a mixed (two sire) litter of fawn Boxers and fawn/red Boxer x Irish Setter crossbreds. (According to Little the crossbreds should have been black but I won’t explain here). However in April last year Greg wrote to me asking if I would like to collaborate in a genetic study to find the gene for brindle. It was an academic project of no commercial significance, the object of which was to resolve a problem in Little’s original work that has remained unresolved for all the years. The result would be of no major practical application for dog breeding - although identification of the gene would provide a test that would distinguish brindles that are true-breeding (‘dominant’ brindles) from those that carry fawn. I thought it would be nice to be involved and maybe find out something new pertaining to Boxers, and some other breeds of course.

Greg’s earlier work had shown that brindle was one of three forms or alleles of a single gene. The bottom recessive is the colour we know in Boxers as fawn or red, but in some other breeds is called sable. As all Boxer people know brindle is dominant to fawn, so that brindles can carry fawn but not the reverse. However there is also the third form of the gene, black, that is no longer found in Boxers but exists in other breeds such as Great Danes. Greg has provisionally assigned the symbol K to this gene. K stands for kuro, the Japanese word for black. I can’t imagine why he went Japanese on this but one reason was that the symbol B was already taken up by the brown locus. But this is just a little historical aside. The main point is that the three alleles at this newly named K locus are: black, or kuro (K), brindle (k>br), and fawn (k) and the order of dominance is K, which is dominant over k>br, which is dominant over k. Black dogs can therefore be genetically K/K (true breeding black), or K/k>br (black carrying brindle), or K/k (black carrying fawn). Brindles can be k>br/k>br (true breeding brindles) or k>br/k (brindles carrying fawn). Fawns can only be k/k and do not carry brindle or black.

 

The start of the study

At this point (May 2004) Greg handed over to his associate, Sophie Candille who has been on the email with me several times a week ever since. Greg’s earlier work had identified which chromosome was involved and the approximate position on this chromosome. This had been achieved using DNA (microsatellite) markers, whose positions in the chromosome were established, and looking for co-inheritances of different forms of the markers with brindle and with fawn. Genes/markers that lie close together tend to be co-inherited together. For those that are more widely separated, there can be an exchange between chromosomes, which causes the genes to be shuffled up in the process of distribution to the sperm and eggs.

For the initial study, the idea was to screen brindle and fawn progeny derived from brindle x fawn matings and check for the frequency of exchange between the K locus and those of the markers. To do this, Sophie sent me ‘brushes’ to obtain cheek swabs for her DNA preparation and analysis and I started the marathon collection of samples from around the summer shows. After obtaining material from 90 dogs, as permitted by tolerant exhibitors, these were mailed to Sophie in California. By now it was July 2004.

 

First results

The first results, using 7 different markers, came back within a few weeks. Of the 90 samples/dogs checked, one form of one particular marker, which we could call ‘X’, was almost always found with brindle and another form, which we could call ‘Y’, was almost always found with fawn and the same was seen in a smaller sample of American Boxers. The other markers did not show such a tight co-inheritance with our gene of interest (K); they must lie further away. In total, only three exceptions were found with the key marker, this giving a first estimate of how close this marker is to brindle; and closeness identifies the gene’s location on the chromosome. It was close enough that with reasonable confidence we could tell which of our brindle dogs carried fawn and which did not. This was of immediate interest for quite a number of breeders. But there was still the ‘error rate’ of 3 exceptions in 90 (3.3%).

 

The exceptions

The three ‘exceptions’ include two brindles that had a fawn parent and therefore must carry fawn. But, on the basis of the key marker they should not have been brindle at all. They carried marker Y and should have been fawn, but genetically they were now k>br - Y./k –Y. The third exception was a fawn that typed as a brindle; she carried marker X and genetically was now expected to be k – X/k - Y. There appeared to have been an exchange of markers. But was this the correct interpretation? And if so, when did these marker - gene exchanges occur? In a parent, or grandparent, or a yet earlier generation? And in the case of the two brindles, did they derive from two independent events, or from a single event in some common ancestor? The two brindles were related, so the latter was a possibility, but one of them had a French sire and this raised further possibilities.

 

Information on the origins of the breed

There was another outcome from the study at this early stage. The findings told us something about the very origins of the Boxer breed. It seems that the seven DNA markers in our fawns can be grouped in several distinct ways to suggest that several different fawn-bearing chromosomes deriving from different ancestral dog groups contributed to the modern Boxer. By contrast, it seemed that there may have been only one brindle chromosome source – or only one has survived.

 

A new approach

This latter aspect of the study prompted an alternative method of finding how close the DNA markers were to brindle. It involves similar studies in other breeds as well as in unrelated groups of Boxers from different countries. Thus closely related breeds/families may have a similar grouping of the markers while more distantly related breeds/families may differ significantly. For this reason, this first shipment of swabs to America included some from Mastiffs and Bullmastiffs, courtesy of some interested and enthusiastic exhibitors at a major all-breed sho, and some other breeds are also being looked at.

At this point in the study it was clear that brindle and the one key marker were very close and it was even possible that not all the ‘exceptions’ were genetic exchanges of the kind sought. The linkage of the gene and marker could therefore be even tighter than indicated. This was great in the sense that it gave a first approximation of the location for the gene but it was now clear that to obtain a yet better positioning with the family screen approach huge numbers of dogs would be required.

A modified approach was needed. It was calculated that a more efficient means of assessing the tightness of the linkage to the marker, and therefore of identifying its position on the chromosome, would be through random screening across families, across different sections of the breed, and across different breeds. This would demonstrate how close the gene and marker were by their co-inheritance throughout the evolution of the breeds. But, the basis for the ‘exceptions’ also still needed to the resolved.

 

New results

The exceptional brindle with the French sire fitted in well with the across-the-breed survey. By chance I was going to the major Italian show near Florence that September 2004 and therefore took my swab sticks for the new screen. As a result not only was I able to get samples from the French sire on one of the brindle exceptions, and another son, but also from dogs coming from a range of European countries. A little later, UK breeders, Leslie Boyle and Brian Peters, also provided me with swabs from their Italian imports and some descendents. The findings were quite stunning. Like the one brindle exception with the French sire, many of the brindle Continental dogs had the form of the marker associated with fawn in British dogs, ie they were k>br – Y, not k<br – X.

 

Implications

The above finding has three implications:

1. Our key marker has no application for detecting which brindles carry fawn in Continental dogs.
2. In terms of the history of the Boxer, the British/American-Continental difference suggests that the post-war foundation stock of British/American lines derived either from a narrow base of Continental Boxers, or that the gene – marker exchange had occurred subsequently in some influential Continental line.
3. More importantly for the brindle study itself, the findings indicate that the marker is readily separable from the gene and therefore the chromosome region defined may still be too big for direct DNA screening for the brindle gene. Further evidence to indicate that the gene and marker were still quite distant from each other was confirmed in the surveys of other breeds (Danes, Mastiffs, Bullmastiffs, Akitas)

 

The fawn exception

The pedigree check on the exceptional fawn immediately suggested a completely different explanation for its marker typing. The animal traced back within two generations to a dog called Faerdorn Joker In The Pack. Those who had been in the breed long enough in the UK remembered this dog and the excitement he caused in his day. He was the only fawn sired by the well-established ‘dominant (homozygous) brindle’, Tyegarth Blue Kiwi. A DNA analysis done at the time on Joker and sibs, and also on Kiwi and some other dogs showed that everything was consistent with Kiwi being Joker’s sire; and Joker indeed bred like a fawn, never producing brindles to fawn bitches. The conclusion therefore was that he had directly inherited a new brindle-to-fawn mutation from his sire, Kiwi. Thus, in the current study, rather than representing an exchange between the DNA marker and brindle, the exceptional fawn may have inherited the brindle-to-red mutation from the great-grandfather. Evidence of this might be obtained by checking the marker type in the parents and earlier ancestors in line back to Joker.

Such a screen quickly showed that the mutation hypothesis was the most probable explanation. While the fawn sire of the exceptional fawn bitch typed as a normal fawn, k – Y, the fawn dam, as also another fawn daughter to a different sire, typed as brindle, k - X. And, while the dam’s sire and the key grand-sire, Joker, were no longer available, another Joker son was found to be k – X. In effect, the data on this family, obtained with the key marker suggested there had not been a brindle – marker exchange, rather the brindle gene itself had mutated. The k>br had become k while the key marker was still X. The brindle-to-fawn mutation hypothesis was strongly supported, but further evidence was needed.

 

New markers and discovery of the gene

To resolve the problem posed by the size of the gene-to-marker distance Sophie Candille introduced a new screen utilising a different type of DNA marker, the SNP (which stands for single nucleotide repeat). SNPs are common throughout the whole genome and their positions are known. And they are highly variable such that associations between the dense array of different SNP forms and brindle could be checked to allow the gene to be positioned more precisely. This new approach has been highly successful in so far as:

1. the region in which brindle lies has narrowed down substantially,
2. a screen in this defined region has actually identified a candidate gene,
3. one variation in this gene that distinguishes brindles from fawns has been recognised. In effect, I think one can now just about say that the brindle gene has been found, and
4. analysis of the exceptional fawn family indicated that the whole chromosome region typed with all markers as brindle. The brindle mutation hypothesis was now confirmed.

 

Mutations as tools for elucidation of the differences in the gene

Importantly the brindle-to-fawn mutation becomes an important tool for establishing precisely what change at the DNA level is responsible for the brindle – fawn gene difference. More such brindle-to-fawn mutations could be of the greatest help is defining exactly the nature of the genetic change. I have learned of a further case in Australia and efforts are currently been made to collect cheek swabs for analysis of this probable second mutation. Yet another has been reported in Italy, so the chase is on there too. Clearly brindle-to-fawn mutations are not nearly as rare as one might expect. This in itself tells us something about the gene. More examples are needed.

 

Mechanisms to create the brindle coat pattern

But, potentially, there is yet more information to come from this study. It may be remembered that brindle is only one of three forms of the gene. The top dominant is black (K) (long extinct in Boxers), the bottom recessive is fawn (k), and brindle (k>br) is the intermediate form that is recessive to black, and dominant to fawn. Now, is it not interesting that the intermediate brindle form is expressed as ‘black’ stripes on a ‘fawn’ background?

This could suggest that three mechanisms are involved in deriving the brindle phenotype.

1. The gene variably produces black or fawn areas in the coat.
2. The black and fawn areas are not irregular patches; there is some preset, if variable, pattern of striping that extends diagonally down the sides of the body and limbs. This pattern closely resembles one seen in mice with a hair follicle mosaicism (two different cell populations). In brindle dogs we indeed seem to have two populations of cells, those that lead to the formation of black hair and those that lead to the formation of fawn hair.
3. Third, there would seem to be a clonal element. By this I mean that black cells produce populations

of black cells only, and fawn cells produce populations of fawn cells only. Were this not the case one would not get the black striping on a fawn background, but just an overall ‘salt and pepper’ mix. Cell movement of necessity obscures the pattern in some parts of the body, and genetic background effects will swing the proportions of black and fawn from faint blacks stripes of the ‘feather’ brindle to the near whole black of the ‘black’ brindle. For me, such possibilities comprise the most interesting aspect of the whole study because not only will we have found the brindle gene but to misquote Rudyard Kipling, we should soon know "how brindles get their stripes".

 

Final analysis

It is now May 2005. A year’s effort has been put into the study but the original objective has been essentially achieved. The gene for brindle has been identified. What remains to be done to complete this element of the project is to ascertain the genetic basis of the difference between brindle and fawn, and indeed black. For the former, the key tool will be the brindle-to-fawn mutation as this will specify the region of the gene that makes the difference, brindle versus fawn.

Further mutations would help considerably with this part of the study and therefore we now put out a call for further possible cases. The search would be best confined to progeny of brindle males that have large numbers of progeny almost all of which are brindle. It is the exceptional singleton fawn progeny that are of interest and these will be very rare. A cheek swab from them, the parents and some sibs would be all that would be required. If anyone knows of such cases please let me know. The final report on this part of the study will be published formally in a specialist science journal after which I should be free to present actual details and final results to the dog press.

There is however also the question of how the brindle gene works and how the stripes are formed. This takes us into a different genetics speciality that goes under the title of epigenetics. How are genes switched on and off in different tissue and in different cell populations? I’ll bet this element of the study, if resolved, will "hit the headlines" in the world of genetics well beyond that involving dogs.