20
In the Lab
Throughout this book we have referred to the fascinating genetic research carried out on dogs and wolves. Much of it has been done by scientists eager to use dogs as a proxy for humans in medical research. That was the primary drive behind the Dog Genome project. Other scientists like Robert Wayne and Elaine Ostrander have had a long-standing interest in dogs and their evolution from wolves. They have both contributed to the genome project, but as the supergroups, like that from the Broad Institute, have turned their attention to the next project that deserves the application of the dazzling array of technological weaponry at the Institute’s disposal, the dog enthusiasts among them have been busy applying the genome results to projects that benefit dogs rather than humans. One such research group, headed by Dr Cathryn Mellersh, is based at the Animal Health Trust (AHT) Laboratories near Newmarket in Cambridgeshire, and she kindly agreed to my visit. Dr Mellersh had completed her PhD at Leicester University. She then spent a few years in Seattle with Dr Ostrander before coming back to the UK to work at the Trust.
The Suffolk town of Newmarket is the epicentre of British flat racing and home to the National Stud, the headquarters of the UK thoroughbred breeding industry. The Stud was built up by William Paul Walker, the son of a wealthy brewer and horse breeder who was concerned at the UK’s shortage of thoroughbred stallions to re-supply the cavalry regiments. Beginning with Walker’s bloodstock in 1915, the National Stud has grown to become the centre for supplying a comprehensive range of services to the thoroughbred horse-breeding industry. The Stud has been owned since 2008 by the Jockey Club, the equine equivalent of the Kennel Club. It was the natural home for the Animal Health Trust, a veterinary charity founded in 1942 and devoted to cats, dogs and, of course, horses.
The Trust is located in the spacious grounds of a leafy estate on the outskirts of Newmarket. On my way to the laboratories I passed horses both in spacious paddocks and being led around the grounds. There were dogs too, playing on the greensward under the watchful eye of their handlers. The whole place exuded a feeling of prosperous calm.
It was a pleasure for me to be talking with Dr Cathryn Mellersh, head of the AHT laboratory, about the nitty-gritty of laboratory genetics, which I had always loved. Lazing on the floor of her office were her two pet dogs Libby and Tess who, she told me, she had found in a rescue centre. She wasn’t sure but she thinks they might have belonged to some travellers and used for coursing until they became too old. It’s not uncommon for dogs that are no longer needed to be tied to a fence or a lamp-post and abandoned. It was immediately clear to me that I was in the presence not only of a scientist but also a dedicated ‘dog person’. Cathryn had a deep desire to help dogs, not just to use them as a tool for medical research.
The primary focus of research at the AHT is to tackle the health issues caused by inbreeding, the perennial problem of pedigree dogs. As we’ve covered in earlier chapters, closed breeds are vulnerable to recessively inherited diseases because whereas carriers are usually entirely free of symptoms, the homozygous animals are affected.
In 2008, an investigative documentary called Pedigree Dogs Exposed was shown on BBC1, the main terrestrial channel in the UK. This documentary claimed that the inbreeding inherent in maintaining breed standards of pedigree dogs, especially in the show ring, had adversely affected the welfare of several breeds. Screening of the film caused a public outcry and precipitated a crisis for the Kennel Club amid a deluge of negative publicity. Commercial sponsors withdrew their support and the BBC seriously considered terminating coverage of the annual Crufts dog show, the jewel in the crown of the Kennel Club. It was essential for the Kennel Club to show the world that they were well aware of the problems of inbreeding and that they were doing something about it. Soon after this embarrassing exposé, Kennel Club support for genetics at AHC increased substantially, allowing Dr Mellersh and her team to expand their research programme into inherited canine diseases. I do not want to dwell on Pedigree Dogs Exposed here. Enough has been said and written elsewhere.
The ultimate ambition of Cathryn’s research is to develop diagnostic DNA tests for recessive disorders and use these to eliminate the mutant genes from pedigree breeds.1 However, as she explained, it isn’t all that simple. Even getting a DNA sample is not straightforward. For example, vets in the UK are not allowed to take blood samples just for research, even if the owner consents. Also these pets belong to people and families and the right approach is needed to secure their agreement. All this takes time, and if a blood sample is needed as a source of DNA it cannot be taken until the dog is having other blood tests, at which time a few extra drops can be saved for research use. This is a difficulty I hadn’t appreciated at all before I visited Dr Mellersh. As I can now see, it poses a formidable obstacle.
As we touched on in earlier chapters, there are different ways of finding mutations. These days the standard route is through saturation mapping with a panel of SNPs to find which of them segregates with the disease. This requires a group of at least six unrelated dogs who suffer from the disorder under investigation. For some rare diseases in rare breeds, it is difficult to find enough owners who are prepared to allow blood to be taken from their dogs. To sidestep this issue, Dr Mellersh now sequences the entire genome of individual affected dogs using DNA collected with a mouth swab, though this approach too has brought its own problems.
Dogs have a lot of bacteria in their mouths, bacteria with their own DNA. If there’s too much of this contamination, it can interfere with the selectivity of the sequencing reactions and you end up with DNA sequences most of which come from the bacteria and not the dog. To combat this difficulty Cathryn’s lab does a preliminary sequence on the swab DNA and rejects any samples that are less than 90 per cent dog. The bacterial sequences still get read but can be weeded out during the subsequent computational manoeuvres.
As you can see, in laboratory science, solving practical issues such as these can make the difference between success and failure. The students and researchers I valued most highly in my Oxford lab were not necessarily the very brightest but the ones who could get experiments to work.
Cathryn then took me through some of her recent successes. Many of these have yet to be published and it would be wrong of me to disclose them here. However, I am able to mention some examples that have appeared in print. In 2014 one of Cathryn’s team was contacted by a neurologist who had just seen a Hungarian Vizsla, a medium-sized hunting dog, in his clinic, with an unusual ataxia, the general name for a disorder affecting movement. The Vizsla is a rare breed in Britain so there was little chance of rounding up enough of them for a gene association study. Cathryn decided instead to go for a complete genome sequence of the one dog. She reckoned that the affected dog would be homozygous for the mutant gene and that this gene would not be found in any other breed. The trouble was that there could be very many other variants in the Vizsla apart from the mutant gene, and so it transpired. The DNA sequence picked out over three hundred variants, any one of which could be the culprit. Cathryn and her team patiently went through all three hundred variants until they found a gene that, from the deduced amino-acid sequence of the protein it encoded, fitted the bill for involvement in ataxia. Further experiments confirmed this.
The early success with the Vizsla persuaded the lab that whole genome sequencing was the way to go, and the AHT launched their ‘Give a Dog a Genome’ initiative. Breeders and enthusiasts from around the country were asked to share the cost of a genome sequence for a dog in their favourite breed. The initiative was very popular, and continues to be a great success, steadily increasing the number of gene sequences available for comparison in any of the lab’s genome projects.
The AHT team next found the mutation for an eye disorder in a Giant Schnauzer. They followed the same strategy as for the Vizsla but with the added advantage of being able to sequence both carrier parents. This narrowed the search to variants that were homozygous in the affected dog and heterozygous in both parents. This filtered out many irrelevant sequences and made the final search considerably easier.
In another example of the success of the genome sequencing process, the AHT team found a cysteine–tyrosine substitution mutation in a form of recessive spinocerebellar ataxia in the Parson Russell Terrier.2 This was the very same gene that was reported in four cases of cerebellar ataxia in humans. In this instance the researchers were guided to this gene by Cathryn’s dog work, a rare example of success in line with the aspirations of the Dog Genome project. The mutation in humans is not the same as in dogs, which is no surprise, and will have arisen completely independently.
There is, however, one case where the mutation in dogs and humans is not only within the same gene but is the very same mutation. There is a common form of a recessively inherited eye disease called progressive rod-cone retinal degeneration (PRCD) in several dog breeds including Labrador Retriever with what I hope by now is a familiar inheritance pattern. The mutation was eventually identified, after a long hunt, in a newly discovered gene of unknown function which was named PRCD after the disease it caused. Just like the Parson Russell mutation, it is a single base change that substitutes a tyrosine for a cysteine in the protein product. As we found out in the last chapter, replacing a cysteine can interfere with the three-dimensional structure of the encoded protein and eliminates its function.
The human mutation was initially discovered in a Bangladeshi woman and has since been found in many other people. In dogs exactly the same mutation is found in all of the following breeds: Australian Cattle and Stumpy-Tailed Cattle Dog, American and English Cocker Spaniels, American Eskimo, Chesapeake Bay Retriever, Chinese Crested, Entebucher Mountain Dog, Finnish and Swedish Lapphund, Hungarian Kuvasz, Lapponian Herder, Labrador Retriever, Miniature Poodle, Nova Scotia Duck Tolling Retriever, Portuguese Water Dog, Silky Terrier and Toy Poodle.
I’ve given you the list in full to illustrate how many breeds with apparently little connection to each other in function or appearance must nonetheless be related back to a single dog through an impossibly complicated network of ancestors.
However, if the gene mutation is fixed in the breed and all dogs are homozygous, as in the hyperuraemia mutation in Dalmatians, then the advantage of being able to study carriers is lost. This situation could well become an insurmountable problem in behavioural traits where selection may well have driven the gene responsible to fixation. A breeder in the USA came up with an alternative and rather old-fashioned way of ridding the Dalmation breed of disease altogether. He took a Dalmatian and bred it with a Pointer. The offspring were all hyperuraemia carriers, but by crossing them with their littermates over successive generations he was able to produce a Dalmatian that looked identical to the original but did not carry the hyperuraemia mutation. Even experts could not tell them apart. Unfortunately, though, the experiment encountered stiff resistance, not because the dogs did not look like Dalmatians, but because the community of breed enthusiasts rejected them as ‘impure’. Happily this resistance has diminished over the years and these dogs, free of the threat of painful bladder stones, can now be registered as Dalmatians.
Such resistance shows only too well the fickleness of some dog owners who would let dogs suffer rather than be pleased that they can own a Dalmatian with all the characteristics of the breed save one.
Although the AHT would in a perfect world like to see the elimination of all carriers from a breed, Cathryn concedes that it will be a slow process. Some breeds have very high carrier rates, for example the Shar Pei, a breed that was reduced to single figures by the end of the Second World War: 40 per cent of Shar Peis are carriers for a type of glaucoma, and even though the mutation is known, it would be a mistake not to breed from any of them. Doing so too quickly would also lose valuable genetic diversity from the breed and only encourage other recessive diseases to develop. Cathryn recommends breeding from carriers with clear dogs for a couple of generations, then gradually cutting down on carriers. With a bit of luck, half of the dogs in these litters will be clear and some at least will have all the desirable characteristics of the breed. The main thing to avoid is producing dogs with the painful glaucoma by mating two carriers together.
The wisdom of this softly, softly approach becomes clearer as tests become available for more and more genetic diseases. In some breeds there can be up to half a dozen serious genetic diseases in circulation, making it difficult to find a dog that is clear of all of them. The advice is to test all the dogs you want to breed from and avoid crossing two carriers.
Inbred populations of all animals and plants are vulnerable to effects on health. In the wild, populations of several well-known animals have at one time or another been reduced to only a few individuals. If and when the population expands from this low number the familiar problems that we have encountered with pedigree dogs begin to show. Even without specific recessive diseases showing themselves, there is a generalised phenomenon called inbreeding depression which impacts biological fitness by reducing fertility, resistance to infection and overall survival rates. For example, the cheetah population crashed about 1,000 years ago. Cheetahs, being descended from the few survivors, are all related. They suffer from high infant and juvenile mortality, low fertility and poor breeding success.
For so long as pedigree dogs are bred, the genetic issues raised by inbreeding will persist. They can be controlled but they can never be entirely eliminated. If the will is there, they can be managed. Over the last years, zoos have risen to the challenge posed by inbreeding and developed breeding schemes, including regular exchanges, to minimise the inherent risks. This becomes absolutely essential when trying to rescue species from the verge of extinction. Below a certain level of diversity, inbreeding depression makes survival almost impossible.
In the past, the criteria for selection were principally based on a dog’s performance, with appearance being only a secondary consideration. Over the past 150 years this has changed as dogs have been bred to be as close as possible to the defined ‘Breed Standard’ in which appearance is paramount. The rewards of winning an important contest are considerable for the owner, with the possibility of lucrative stud fees to look forward to. These potential gains introduce a conflict of interest for owners. If, for example, a dog with all the right qualities is shown by a DNA test to be a carrier for a serious genetic disorder, should it be withdrawn from the competition? Owners might then be naturally wary of having their dogs tested at all, just in case. If the winner of a contest is indeed a carrier, whether or not the owner knows it, and it goes on to be a popular stud dog, then there will be large numbers of offspring, at least half of which will themselves be carriers, or worse. This happened to an Irish Setter that recently won Crufts ‘Best in Show’ and went on to have at least 1,000 offspring. It turned out to be a carrier for an inherited eye disease. Homozygotes don’t begin to go blind until they are about ten years old, by which time owners, and vets, are inclined to put this down to old age. I was surprised to hear from Cathryn that dogs can tolerate blindness much better than humans. She once owned a blind Retriever who could still find and retrieve a thrown ball purely by sound and smell. Nonetheless the issue of the popular sire who carries a genetic disease is a serious one.
Ultimately the success or failure in improving the genetic health of dogs depends on the breeders. The exactly analogous human situation is the successful elimination of Tay-Sach’s disease in the Ashkenazi Jewish population, achieved by leadership and determination. The Kennel Club can lead by example but it has no statutory powers. Over the past decade it has risen to the challenge of tackling the issues raised by the BBC documentary by, among other things, funding the Animal Health Trust research effort. The Kennel Club has put its money where its mouth is. It has to work to improve the welfare of dogs by persuasion, with very limited recourse to legislation. I don’t envy them their task.