Complex diseases; can we really “find the genes”?

Many breeds have been pinning their hopes on finding the genetic mutations responsible for diseases and health issues with the expectation that breeders will be able to test their way out of problems.

In some breeds, we have been “fortunate” to be able to identify so-called simple mutations from which DNA tests have been developed. In theory, these enable breeders to make informed decisions before breeding from a dog and bitch so that no “affected” puppies are born. It is, of course, important that we know how these single genetic mutatioons directly correlate with the clinical manifestation of the disease. There is also the potential unintended consequence of a reduction in overall genetic diversity in these breeds which may result from removing Affected (and sometimes, Carriers) from the breeding population.

I’ve written before about a couple of examples where “simple” recessive mutations may, in fact, subsequently turn out not to be so simple. One example is Cord1 PRA in Miniature Dachshunds where we now know there is a second mutation (MAP9) which influences the age of onset of blindness. This second mutation helps explain why some Cord1 Affected dogs don’t suffer retinal degeneration until old age (if at all). The other example is the POMC mutation which was associated with obesity in Labradors. The mutation is also found in Flatcoated Retrievers, but this breed is not noted for having an issue with obesity.

In the case of so-called complex diseases (e.g. Hip Dysplasia, Epilepsy, BOAS) there has been an assumption that multiple genes are involved in these conditions as well as environmental factors.

The search for simple and complex genetic explanations for canine diseases has been accelerated by the development of Genome Wide Association Studies (GWAS). These are large-scale investigations of genetic disease that aim to identify genetic variants scattered throughout the whole canine genome. The canine genome is a sequence of 2.4 billion letters of DNA (G, A, C and T), so the scale of these studies is truly enormous and requires massive computing power. In human genetic research, the number and scale of GWAS have been growing year by year. In 2016, of more than 400 published studies, around 50 involved studying the genomes of samples of more than 100,000 people. A similar situation has occurred in dogs. Last year, a team from Cornell University published a canine GWAS paper based on a sample of more than 4200 dogs from 150 breeds as well as mixed breeds. They tracked down two loci linked to Elbow Dysplasia and one for Hip Dysplasia. They also identified loci associated with epilepsy and lymphoma.

There has, however, been some debate about the extent to which GWAS in humans has actually led to useful clinical applications. For example, they may not fully explain the genetic familial risk of common diseases and there is a small size effect for many of the identified associations. They have also proved to be of limited value in predicting disease risk. All these shortcomings, of course, would mitigate against GWAS being of much practical use to dog breeders.

A new omnigenic model

A paper published in the journal Cell in June this year adds further challenge to the idea that there are relatively simple, causal, links between genetic variation and disease. In their paper, geneticists Boyle, Li and Pritchard from Stanford University suggest that many genetic variants identified by GWAS have no specific biological relevance to diseases. Their view is that common illnesses could, in fact, be linked to hundreds of thousands of DNA variants. Their conclusion is that, for complex traits, association signals from a GWAS tend to be spread across the whole genome, including near many genes without any obvious connection to the disease. They also state that most heritability can be explained by effects on genes outside core pathways. They called this an “omnigenic model” whereby most genes matter for most things!

“There’s been this notion that for every gene that’s involved in a trait, there’d be a story connecting that gene to the trait,” says Pritchard. But he thinks that’s only partly true because genes don’t work in isolation. They influence each other in networks so, if there is a variant in one gene, it could well change a whole gene network. All this suggests that the search for simple genetic causes of complex diseases will continue to be challenging and breeders are unlikely to have new DNA tests for these conditions anytime soon. Of course, GWAS may well continue to help identify simple recessive mutations and it is important to remember that the paper is critical of the value of GWAS in human studies where the population structures are likely to be rather different to pedigree dogs with their closed gene pools and high levels of inbreeding.

I recently saw a comment by Carol Beuchat (Institute of Canine Biology) that 70% of genetic disorders in dogs are caused by recessive mutations. We also need to know the extent to which these have an impact on canine welfare as many of them could be relatively trivial. Developing yet more DNA tests for some of these would actually make life more difficult for breeders. Given that many of the high welfare-impact diseases are in the remaining 30% of complex conditions, it’s going to be virtually impossible to “breed away” from the “bad genes”.

Hope for the future

The AHT’s “Give a dog a Genome” project is a current example of Whole Genome Sequencing (WGS) which has the potential to avoid some of the shortcomings of non-sequencing GWAS. Here, by sequencing the genomes of different breeds, the AHT hopes to identify the variations that exist within the canine genome. Having built a database of “neutral variants” from healthy dogs, the genomes of dogs affected by particular diseases can be compared. The different variants between healthy and unhealthy dogs potentially lead to the identification of the associated disease mutation.

The AHT can already claim some success for their WGS work; on their website, they showcase the development of the DNA tests for cerebellar ataxia in Hungarian Vizslas and primary open angle glaucoma in the PBGV. The Vizsla genome sequence can now be used as a control sequence in future studies of inherited diseases in other breeds. With more than 70 breeds participating in the Give a dog a Genome project, the AHT expects to see many more useful and practical developments like those in the Vizsla and PBGV.

I’m sure all the breeds who are participating in the GDG project will recognise the scale and complexity of this project. I hope they see it as a longer-term opportunity to address health issues. In the meantime, they need to look for and implement other strategies that address the root causes of disease in pedigree dogs; closed stud books and high levels of inbreeding.

 

[There’s a presentation – pdf- by Boyle, Li and Pritchard here]

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The sins of the father are laid upon his children

An article that has generated a lot of discussion is Dr. Carol Beuchat’s “Three key strategies to reduce genetic disorders in dogs” published on her Institute of Canine Biology blog. Carol starts by saying “In many breeds, dodging genetic disorders is becoming a significant problem because troublesome recessive mutations can be widespread in the population.”. She goes on to discuss something I have pointed out before in my articles; that is the futility of many breeders’ determination to adopt a “search and destroy” strategy to eliminate genetic mutations by finding ever more DNA tests. There are many more genetic mutations than there are DNA tests and even if we had tests for all of them, it would be impossible for breeders to make breeding decisions to prevent every risk.

Her stark conclusion is that “We can spend millions on research and testing to battle genetic diseases in dogs, but we cannot win this fight unless we change the breeding strategies that produce the problems in the first place.”

The three strategies in Carol’s article are:

• Increase the number of breeding animals
• Eliminate Popular Sires
• Use strategic outcrossing to reduce inbreeding

I’ve written about the first two of these in the past. If a wider range of stud dogs is used, or more puppies are used in breeding programmes, then this will have a positive impact on breed populations (I talked about encouraging owners of “pet” puppies to breed with them). We would then have more individual dogs and bitches producing the next generation. The second point is actually a specific dimension of the first one. Popular Sires disproportionately contribute their genes to the next generation, with all the consequent risks of doubling-up on deleterious recessive mutations in later generations. Every breeder who jumps on the bandwagon by using a Popular Sire can be held responsible for genetic problems that pop up as a “surprise” subsequently. In reality, these problems should not be a surprise because that’s exactly what you’d expect when recessive mutations combine down the line.

“The sins of the father are to be laid upon the children” is a quote from Shakespeare’s Merchant of Venice. The dog breeder’s equivalent should be “The sins of the Popular Sire are laid upon his puppies and his puppies’ puppies”.

The FCI’s International Breeding Strategies paper recommends “As a general  recommendation, no dog should have more offspring than equivalent to 5% of the number of puppies registered in the breed population during a five-year period. The size of the breed population should be looked upon not only on national but also on international level, especially in breeds with few individuals.”

In an earlier article where I discussed the Kennel Club’s 2015 paper on genetic diversity, Carol’s third point was also raised. Her suggestion about “strategic outcrossing” could be as simple as planning matings with dogs from distinct sub-populations such as imports (but beware the Popular Import Sire), different coat/variety, or different disciplines (e.g. working/show).

I’d perhaps add a fourth suggestion to Carol’s three and it’s a very simple one that every breeder could adopt immediately: Use the KC’s MateSelect to choose breeding combinations that result in litters with a COI below the breed’s current median COI. This will (slowly) help to reduce a breed’s overall level of inbreeding. There is, of course, my previously noted caveat that the COI data on MateSelect may only be for 3 generations for imported dogs which may result in an under-estimate of the COI of any planned matings.

A recent discussion about Carol’s article among KC Breed Health Coordinators largely centred on the uphill struggle they have to persuade their breed communities to read any of these articles, let alone begin to change their behaviour when breeding. One BHC said she was exhausted trying to explain and “apparently, you have to have been breeding for the usual 40 years to have any idea”. Another said “I sense that many breeders don’t believe what they do read or are being told by scientists/geneticists and if they do, they find it difficult to apply the information to what they are doing. Sometimes this might be because there is still a deep-seated feeling of mistrust – especially when it comes to anything sent out from the KC – or because they just don’t understand it.”

I’ve argued before that breed health improvement is a change management issue, not a scientific one. It requires individual breeders and buyers to change their behaviour. We have to keep nudging people in the right direction by sharing data, evidence and practical examples of what actions can be taken. Remember, “without data, you’re just another person with an opinion” [Dr. W Edwards Deming – statistician].

The gene genie is truly out of the bottle

Recently, I’ve had some fascinating conversations with canine geneticists about emerging technologies and upcoming research projects.

It seems that it is now possible to identify new, simple recessive mutations from the DNA of a single affected dog. Not long ago, laboratories like Cathryn Mellersh’s at the AHT would typically be asking Breed Clubs to come up with cheek swab samples from 50 affected and 50 unaffected animals. For many breeds, that was probably quite a challenge and virtually impossible in the numerically small breeds. Back in the days when the AHT was looking for the mutation that caused PRA in Miniature Longhaired Dachshunds, they even had to have their own research colony of dogs. By the way, “back in the days” is just over 10 years ago.

I was told of an example where a new genetic mutation that causes a painful eye condition has been identified in a particular breed and a new DNA test can be offered to breeders. There have only ever been a couple of clinical cases reported. That may be because it is incredibly rare in the breed population, or perhaps it simply goes undiagnosed or unrecognised during BVA/KC/ISDS eye examinations. This is a breed where several other DNA tests are already available for eye conditions and breeders are expected to make use of other available screening programmes for hips and elbows.

Should the breeders be encouraged to use the new test? Should the Breed Clubs carry out a research screening exercise on a suitably random, but statistically significant, sample number of dogs? Should DNA samples that have been submitted for the other eye conditions be re-screened for the new mutation?

One danger is that breeders will find themselves faced with yet more expense; the good breeders will want to do the right thing and the bad breeders will carry on regardless (and make a bigger “profit” on the sale of their puppies).

The risk of not screening a suitable sample of the breed is that there is no way of knowing how high the mutation frequency is in the population. Although there may be currently very few known Affected dogs, if it turns out that there are a large number of Carriers in the population, then there is certainly a risk of more clinically affected dogs appearing in the future. If a Popular Sire is a Carrier, then there’s an even greater risk to the breed.

The other complication for many breeds comes when there are multiple tests available and any given pair of dogs in a planned mating could be Clear, Carrier or Affected for different DNA tests, plus have a range of different screening statuses for their clinical tests. Just how do you decide if it is “safe” to mate two animals together? I’m sure a number of breeds are  already finding themselves in this situation. Presumably, the use of Estimated Breeding Values and Genetic Breeding Values holds out some hope for them, but these are very dependent on having enough data available from people participating in the the various screening programmes.

Give a dog a genome

Perhaps the most exciting news in January was the AHT’s announcement that they plan to sequence the genome of 50 different breeds. They have £50,000 of funding from the KC Charitable Trust and want Breed Clubs to match this. I’m aware there has already been a flurry of activity on various breed Facebook pages to set up fundraising activities.

We already know the potential value of this type of project because a Cornell University team has performed a large, across-breed genome-wide association study (GWAS) in dogs, uncovering variants associated with everything from body size and fur traits to dog diseases such as epilepsy, cancer, and dysplasia. They have pre-empted the AHT’s project and published their results in January.

The Cornell researchers genotyped more than 4,200 dogs for the GWAS, focusing on a dozen common dog traits and diseases. The analyses included dogs from 150 breeds and 170 mixed breeds, as well as 350 village dogs from 32 countries. This is clearly on a much larger scale than the AHT plans, but their results certainly add weight to the case for the AHT looking at breeds in the UK.

For example, the researchers used data for dogs from 82 breeds — 113 cases and 633 controls — to track down two loci linked to elbow dysplasia. One locus had stronger effects on elbow dysplasia risk in Golden Retrievers and English Setters, the other showed closer ties to the trait in Labrador Retrievers and German Shepherds. Along with the across-breed analyses, the team did several within-breed association studies, including a lymphoma analysis in Golden Retrievers, a search for idiopathic epilepsy in Irish Wolfhounds, and a Boxer-centered analysis of granulomatous colitis.

We need to be clear though; these are not “DNA tests” for these particular conditions, but they are an important finding not only for canine health, but also for helping to understand similar complex diseases in people. This ties closely to the increasingly common messages about “one medicine” that many people will have heard Professor Noel Fitzpatrick speak about on his SuperVet TV programmes. Noel’s work on treating canine patients is attracting attention among human surgeons and there are, no doubt, many more potential examples where veterinary and human medicine and surgery can learn from each other.

One medicine

One of the earliest examples of that cooperation and learning was the search for the Lafora gene in Miniature Wirehaired Dachshunds. Nearly 15 years ago Dr. Berge Minassian from the SickKids Hospital in Toronto visited owners of Mini Wires in the UK and began a collaboration that led to the discovery of the gene and mutation which causes Lafora.

Lafora Disease is a late-onset myoclonic form of epilepsy that affects humans and dogs. In people, the disease, which is progressive, usually results in death in the teenage years. In Miniature Wirehaired Dachshunds, just under 10% of UK dogs have been identified as Affected by the DNA test that Dr. Minassian’s team developed.

In January, he returned to the UK to seek their support in the development of a therapy which has been shown to be effective in mice and which, it is hoped, will benefit humans and dogs. The Wirehaired Dachshund Club’s Lafora Team will be working with Berge and Dr. Clare Rusbridge to recruit a group of clinically affected Mini Wires, plus a control group of dogs, to run the trial with the new therapy over a 2 year period once funding and ethical approvals have been confirmed.

This could be a landmark moment in what is a unique collaboration between breeders, veterinary medicine and human medicine. The potential to develop a viable therapy for Lafora Disease would be life-changing, both for the people affected by the disease and for affected dogs.

The gene genie is truly out of the bottle and it opens up opportunities to benefit people and dogs that could barely have been imagined just a few years ago.

Time for individual Breed Improvement Strategies?

Quite a lot has already been written about the KC’s Genetics and Diversity reports with a range of comments from “good news” to “it’s the end of the line for some pedigree dogs”. No prizes for guessing which commentators were at opposite ends of that particular spectrum!

From my perspective, the availability of more data is always good news. There are, however, challenges. Firstly, what we have is a report (or a series of reports at breed level) and it will only have value if somebody can make use of it. It needs to be read, understood and acted upon by the people within individual breeds.

There is a clear role here for Breed Health Coordinators and their associated Health Committees. They need to take the report for their breed and distil it into some key messages using language that will be accessible to breeders and owners.

Each report includes data on 25 years of registrations, trends in Coefficients of Inbreeding, Effective Population Size and the use of Popular Sires. Taken in the round, rather than cherry-picking individual elements of the data, provides a unique insight into the current situation faced by each breed. A breed with growing registrations, but declining EPS and increasing COI will need a different response to a numerically small breed with stable registrations and an already high average COI, but with a variety of recent imports.

What is the picture for your breed?

A potentially useful technique from the world of Systems Thinking is General Morphological Analysis (GMA). This is a method for structuring and analysing complex problems and can be used for developing scenarios, for example when considering options for improvement. It’s also helpful when looking at the relationship between ends (e.g. COI, EPS) and means (breeding strategies).

Taking the data from the KC reports and developing a GMA matrix could result in something like this for “ends”. Each column is for a set of data in the diversity reports and each row describes a range of results that might be found for a breed (e.g. colouring the text to show current status for an individual breed):

Registration Trend	COI (Current Mean)	COI Trend	EPS	Popular Sire Use
Declining (>25 p.a.)	>25%	Increasing	0-25	Extensive; increasing
Declining (5-24 p.a.)	12-24%	Static	26-50	Extensive; static
Static	6-11%	Decreasing	51-75	Extensive; decreasing
Increasing (5-24 p.a.)	2-5%		76-100	Moderate; increasing
Increasing (>25 p.a.)	0-1%		>100	Moderate; static
				Moderate; decreasing
				Negligible; increasing
				Negligible; static
				Negligible; decreasing

In practice, this needs to be developed collaboratively, with involvement of the interested parties (genetics experts and breeders) to agree the criteria and “levels” that describe the current situation for any breed.

What actions are needed in your breed?

The second challenge is that there is no “one size fits all” response. Having looked at the data sets for each of the 6 varieties of Dachshund, there are definitely different strategies required. Wires have benefited from numerous imports and have a relatively high EPS, but the breed has a history of Popular Sires. Smooths and Longs have declining registrations and could benefit from imports to increase their gene pool. Mini Longs are declining in popularity, have an increasing level of inbreeding and are also adversely affected by Popular Sires and this appears to be a worrying combination of factors. Mini Smooths have exploded in popularity in the past few years (TV adverts seem to be a causal factor here), but also have an issue with Popular Sires which could create a problem in the future.

For the Dachshunds, a recurring theme is the use of Popular Sires and, I suspect, that will be a theme in many other breeds. While the FCI guidance on Breeding Strategies (*) provides suggestions on how many litters/puppies any individual sire should have, this sort of approach is typically not welcomed in the UK. It seems unlikely that this type of “regulation” would be acceptable to, or popular with, UK breeders in most breeds. Whether any degree of self-regulation is likely to happen, I doubt. I fear that the desire to use the latest, greatest, import or top-winning dog will outweigh any considerations for the future viability of most breeds.

The KC’s website has a page devoted to “managing inbreeding and genetic diversity”. In theory, this could be developed into a GMA matrix for the “means” to address the “ends”. Each column represents “levers that can be pulled” to influence genetic diversity, with rows showing some of the available options. For example, here are some of the options (which range from the “denial” options to the “nuclear” ones!):

Manage Popular Sires	Use COIs before Breeding	Use Health Tests	Use DNA Tests	Use Sub- populations	Use a different breed
Don’t restrict use	Don’t consider litter COI	Don’t carry out health tests	Don’t carry out DNA tests	Inbreed to a line/ family	Don’t outcross to another breed
Provide guidance only	Breed above COI average	Ignore health test results	Don’t breed from Affected dogs	Breed to other lines	Outcross to another variety of the same breed
Recommend limits for use	Breed below COI average	Take health tests results into consideration	Don’t breed from Carrier dogs	Breed to dogs from another discipline (e.g. working)	Outcross to a different breed
Set rules for use			Only breed from Clear dogs	Breed to an imported dog
			Only mate Affecteds/ Carriers to Clears

Some of these are options that can be influenced or regulated by the KC and Breed Clubs, while others are choices available to individual breeders.

If you wait for the perfect set of data, you’ll wait a very long time!

A final challenge associated with the KC’s Genetic Diversity reports is that some people will simply criticise the data and argue that the conclusions are based on dodgy data! We’ve had this criticism before; we know the KC’s COI calculations are based on available pedigree information and, in the case of imported dogs, that may be from as little as 3 generations.

Tom Lewis and Sarah Blott countered that criticism with a letter to the dog press in December 2013. They said “We know that truncating the pedigree when calculating COIs leads to an underestimate of the rate of inbreeding in a breed. We can then be deceived into thinking the breed has an acceptable rate of inbreeding when, in fact, it does not.”

Overall, that one factor probably means COI values quoted in the reports are underestimates for those breeds where there have been multiple imported dogs. All the more reason to acknowledge the lack of genetic diversity in many breeds and agree, at breed level, what actions are required.

Unless breeders wake up to the implications of the past 25 years’ breeding strategies as demonstrated by the KC’s reports, we will see the inevitable consequences of Darwinism in action. Some breeds are already defined as “vulnerable”; the KC reports highlight others that really ought to be implementing conservation programmes. If we were looking at Pandas, Rhinos or Tigers there would be worldwide conservation programmes in place and global cooperation. Breeds such as the Otterhounds have already recognised this risk and are trying to do something about it.

It’s not the KC’s responsibility to make change happen; they have provided the data and can influence the direction of change, but it’s down to breed club communities and individual breeders to act now for the benefit of their breed.

* FCI Breeding Strategies: “As a general recommendation no dog should have more offspring than equivalent to 5% of the number of puppies registered in the breed population during a five year period.”

“Dachshunds and Data” featured in IFORS News December 2014

We have previously shared the “Dachshunds and Data” presentation Sophie Carr and Ian Seath made at the 2014 OR Society Conference.  An article based on their presentation has been published in the December 2014 edition of the International Federation of Operational Research Societies (IFORS) Newsletter.

Read the article on pages 19 and 20 here (online interactive magazine).

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