AHT Spinone Day, Italian Spinone Club Of Great Britain

Last Updated on November 17, 2014 : :

If you wish to share any of the information in this article, please link directly to the page. Written permission is required to copy or reproduce the content in part or in full by any other method.

On Friday 7th November, the Animal Health Trust hosted a Spinone Open Day at their premises near Newmarket, Suffolk. Our Speakers for the day were Dr Luisa De Risio, Head of Neurology, Dr Cathryn Mellersh, Head of Canine Genetics and Dr Davide Berlato, Head of Oncology.

Cathryn outlined the plan for the day and talked about the 4 ingredients that are needed for successful research, Clinicians to identify robust cases and analyse the enormous amount of data collected, the Genetics Team to undertake the search for those elusive genes, Money to fund the process and of course DNA samples from affected and unaffected dogs. The Spinone community were complimented for their support in this research project.

Luisa then took us through what had taken place to date. Some of the results were published at an international conference in September but today we would be shown some new findings that were soon to be published. She explained that all cases used in the study were kept anonymous and only identified by number.

Epilepsy has a strong genetic component but there are other reasons for seizures and it is important to understand this. That is why so much work has been necessary to identify which cases are genetic and which are due to other factors. If DNA was included for dogs that are seizing for other reasons, it would muddy the water and make it harder to investigate the gene(s) responsible.

Idiopathic Epilepsy (IE) is described as recurrent seizures with no underlying cause other than a strongly suspected or confirmed genetic or familial basis.

The goals of the study were to:

* Investigate the prevalence in the UK

* Investigate the clinical characteristics and predictors of treatment response and survival. Ours is the first study in the UK to investigate this aspect.

Data Collection

All owners of UK KC registered dogs born between 2000 and 2011 were invited to complete a Phase 1 questionnaire asking for basic information. Because of Data Protection, the Kennel Club had to issue the invitations and about a quarter of these couldn’t be sent out because owners had ticked the box requesting no further mailings from the KC.

From those that were sent out, about a third responded. This phase was left open for several months whilst we tried to obtain maximum response. The Breed Clubs and Spinone community in general worked hard to encourage participation and whilst we had hoped for an ambitious 50% response, a third was still excellent.

The Clinical Team then went through each of the Phase 1 questionnaires and the medical records of Spinoni that experienced one or more seizures to identify those Spinoni with IE according to rigorous definitions. Where owners had given consent for further contact, a letter was sent out to owners of Spinoni with IE inviting them to complete a more detailed 110 question questionnaire which provided valuable information.

When the questionnaires were returned, the team then undertook a statistical analysis to identify any predictors which might help evaluate survival and seizure remission.

The main things to report are:

IE is more prevalent in the Spinone than in general dog population in UK and therefore a genetic component of IE in the Spinone breed is very likely.

The average age at 1st seizure is slightly over 3 years. For breeders this has a massive implication as dogs may be bred from before developing the condition. So predicting the likelihood of a dog developing IE would be invaluable.

Males were overrepresented.

The Spinone seems to have a severe type of IE. If treatment is started after a 2nd seizure, this increases their and long term prognosis and chance of survival. Dogs that experience cluster seizures are less likely to survive so it is important to try and prevent cluster seizures.

Treatment by a neurology specialist (as opposed to a GP vet) was also recommended, an MRI scan is not always necessary, sometimes a detailed consultation and blood tests are all that is needed.

Medication is more effective if it is tailored to the individual dog as not all dogs’ metabolism is the same. Luisa has first hand experience where doing this has reduced the frequency of seizures and significantly improved dog and dog’s owner quality of life. Only a third of affected Spinoni had been seen by a specialist.

Luisa thanked the Breed Clubs and Anita, Julia and Richard from her team.

Pexion Study

The AHT are undertaking a collaborative study with the RVC to investigate the impact of Pexion on an epileptic dog’s behaviour and welfare.

If you have an IE dog that is being treated with Pexion and would like to take part in the study, you can contact Dr Rowena Packer via email at rpacker@rvc.ac.uk or go online to complete the survey at https://www.surveymonkey.com/s/rvcepilepsypexionstudy.

Questions asked

Q) Why do you think the severity is greater than other breeds?

A) The mutation(s) underlying epilepsy in different breeds are probably different, and the protein(s) that are ‘broken’ in the Spinoni unfortunately lead to a severe form of disease

Q) Is it possible that CA could be having an impact.

A) The 2 diseases are very different neurologically. There is no evidence that the CA mutation predisposes dogs to epilepsy, but can test once the epilepsy mutation(s) is found.

The genetics of treatment response is another potential area of work.

Cathryn then took us through what is happening next.

As a result of Luisa’s research, we have confirmed that IE is more prevalent in the Spinone than in general dog population. It is associated with a higher mortality rate and has a severe expression in the breed. Because of this, a means of prevention would be highly beneficial.

If inherited traits are more common in one breed, compared to another, they are predicted to have a genetic component. IE is therefore likely to be caused by mutations in one or more genes in the Spinone. If the AHT can find the most important mutations, they can hopefully design a DNA test that breeders can use to reduce the prevalence if IE in the Spinone.

The genetics of canine IE are poorly understood. Only 2 mutations have been identifed to date that are associated with types of canine epilepsy, both of which are clinically different to epilepsy in the Spinoni. Disease mutations are usually breed specific so it is not the same mutation in different breeds. Therefore when looking for the mutations we have to start from scratch.

A quick lesson in DNA basics showed us the enormity of the task ahead, to find a tiny needle(s) in a giant haystack(s).

DNA Basics

DNA is a complicated molecule, think of it as beads or letters on a string (4 different ones). Every trait not determined by the environment comes from the DNA. Laid out, a dog’s DNA would stretch from Lands End to John O’Groats and back again.

A dog has about 30,000 genes, each of which codes for a protein. 97% of the DNA doesn’t directly code for a protein. We don’t know what it does but it has been maintained through evolution. The mutation for CA came from a non-coding section of DNA.

For the dog, there are 2.5 billion beads/letters in the DNA. A mutation is a bit like a spelling mistake – extra letters, missing letters, wrong letters. When we start looking we don’t know which type it will be or how many letters will be wrong. Lots of mutations are caused by just one letter being wrong. The CA mutation was a small piece of DNA that was repeated hundreds of times instead of 10 to 20 times.

The dog has 38 chromosomes, each cell has 2 copies of each chromosome, but each sperm and egg cell only have 1 copy of each chromosome. On fertilisation, the chromosome from the egg and the chromosome from the sperm end up in the cell so they are then a pair. Every time a cell divides, the DNA is copied.

A large amount of DNA is the same in all Spinoni. This is why we need the clinical expertise to make sure it is the same disease and the same breed to conclude it is caused by the same mutation(s). DNA is then taken from affected dogs and unaffected dogs. We then look for a piece of DNA that is similar in all the affected dogs and different in the unaffected dogs. That region is then looked at more closely.

The approach we are taking is to do a Genome Wide Association Study (GWAS) which looks at all the DNA. This looks at the markers that are distributed over the whole genome. Doing it this way we are not making a guess and can find the mutation(s) regardless of which gene it is in. One experiment will cover 172,000 markers. It is quite expensive at £130 per sample and there need to be enough affected cases and controls to show that any similarities are statistically significant and not just due to chance.

For a simple recessive mutation in 1 gene, 12 cases and 12 controls is enough. A simple dominant mutation would ideally need 50 cases plus 50 controls. A complex mutation across 2, 3 or 4 genes needs 100 cases plus 100 controls.

The results are plotted in a plot that looks like the Manhatten skyline – hence its name a Manhatten plot. Each dot represents 1 marker, colour coded by chromosome. The bigger the number the more certain this marker is associated with this disease.

Example Manhatten Plot (Human – 22 chromosomes. A dog has 38 chromosomes)

“Manhattan Plot” by M. Kamran Ikram et al – Ikram MK et al (2010) Four Novel Loci (19q13, 6q24, 12q24, and 5q14) Influence the Microcirculation In Vivo. PLoS Genet. 2010 Oct 28;6(10):e1001184. doi:10.1371/journal.pgen.1001184.g001. Licensed under CC BY 2.5 via Wikimedia Commons.

The Manhatten plot tells us which chromosome(s) to start looking in. The plot for the chromosome is then tipped on its side and we hope to see a peak which will give us an associated region (s). In the Lands End to John O’Groats analogy we have identified one or more pieces of road that may be 3 miles long where a mutation could be.

At some point it comes down to sequencing (or ‘reading’) 2-3 million letters DNA letters in a small number of affected and unaffected dogs, to find the one that is incorrect.

Preliminary analysis of the first GWAS data should be completed by the end of January. The best result we could have is a single significantly associated region. A nice single tall skyscraper on our Manhatten plot. The next best result would be more than one significantly associated regions and/or more than 1 suggestive region. With either of those results the regions will be followed up and the DNA sequenced.

A not so good result would be no significant or suggestive regions. If that happens we will need to obtain samples from more affected and unaffected dogs, then undertake a further GWAS which can be added to the first set of data and see if that changes the peaks either by pushing some up higher or removing others.

We need to keep collecting samples as the more samples we have the better, especially if a 2nd or 3rd GWAS is necessary. So if you know of anyone who has a UK Spinone with seizures, please ask them to get in touch with the AHT and provide a DNA swab plus medical history. These may be dogs that initially completed the first questionnaire and have since suffered a seizure or dogs that were too young to be included. Or they may not have known about the study.

We also need Controls. Dogs need to be over 7 and never had a seizure. If the AHT already hold your dog’s DNA please email Bryan.mclaughlin@aht.org.uk with confirmation that the dog has lived to over 7 and not ever had a seizure.

Once the mutation(s) are found, a DNA test is likely to take several years to achieve. It is more likely to be a risk score rather than a Clear / Carrier / Affected result as we had with CA. It will probably not be possible to give a definitive risk to any dog.

In the meantime the AHT have provided the following general breeding advice, although it is very difficult to provide robust advice for such a complex trait. Please bear in mind that following these guidelines is no guarantee of producing a litter that will be free of epilepsy.

Our gene pool is very small and we should not be excluding dogs or lines from being bred without good reason.

If a dog or bitch has produced a single confirmed epileptic , then the dog/bitch’s reproductive life doesn’t necessarily have to be terminated. But it is prudent

i) not to repeat the same mating that has produced an epileptic,

ii) not to breed the dog/bitch with a mate that has any common ancestors within at least 3 generations and

iii) if he/she produces an epileptic in a 2nd litter not to breed from him/her again.

For dogs/bitches that have not produced a confirmed epileptic themselves but are related to an epileptic within one or two generations then the same advice applies:

i) do not breed the dog/bitch with a mate that has any common ancestors within at least 3 generations and

ii) if he/she produces an epileptic in a 2nd litter not to breed from him/her again.

A dog or bitch that has suffered from seizures should not be used for breeding.

Please do not ask the AHT for breeding advice, for reasons of confidentiality they cannot give out this sort of advice.

When studying pedigrees, please look at affected and unaffected pedigrees. Don’t just focus on affected pedigrees.

The more we can share information openly, the better.

Cancer Research Update to follow.