1. Primary Lens Luxation Cathryn Mellersh Animal Health Trust 22 nd November, 2009

2. Collaboration & Acknowledgements David Sargan (University of Cambridge) David Gould (Davies Veterinary Specialists) AHT Ophthalmologists & BVA Eye Panelists Owners and breeders who have donated DNA samples from their dogs Kennel Club Charitable Trust, American Kennel Club Canine Health Foudation, Breed Clubs & individuals.

3. Talk Layout Brief overview of Primary Lens Luxation. DNA – what it is, what it does, and how mutations in DNA cause inherited disease. How a mutation progresses through a pedigree. How a mutation is tracked down PLL mutation and DNA test –what the results mean –mutation frequency in the population –general advice Vitreal leakage

4. Primary Lens Luxation zonules In the normal eye the lens is held in place by numerous fibres, called zonules. Lens luxation occurs when all of these fibres are broken and the lens becomes loose within the eye, where it can move forwards or (less commonly) backwards in the eye. Zonules

5. PLL Anterior lens luxation Enucleated eye, due to anterior lens luxation and secondary glaucoma.

6. PLL Primary Lens Luxation is a well-recognised, painful & potentially blinding condition known to affect many terrier and terrier-type breeds. autosomal recessiveBelieved to be inherited as an autosomal recessive condition in all breeds studied. Difficult to eliminate from at risk breeds because: –Clinically unaffected carriers can pass mutation on to offspring –Condition is mid-late onset, so affected dogs often bred with prior to onset of clinical signs DNA test required

7. DNA is a very long molecule & is found in virtually every cell of the body. The canine genome consists of approximately 2,500,000,000 nucleotides of DNA If each nucleotide was 1mm long the canine genome would stretch from Land’s End to John O’Groats and back. ALL the DNA is copied every time a cell divides DNA is responsible for every aspect of you and your dog that is not controlled by the environment. DNA

8. Coding DNA AAUGGGGGAUUUCCC mRNA A T V W T Protein ACTGAAACCCCTTGG AATGGGGGATTTCCC DNA Transcription Translation

9. DNA DNA is a code/blueprint for every physical characteristic of a dog that is not determined by the environment. The code is determined by the order of nucleotides along the DNA. ALL 2,500,000,000 nucleotides are copied every time a cell divides. MUTATIONSMistakes that arise are called MUTATIONS. Most mutations that arise are repaired whereas a tiny minority persist. Most mutations have no effect, whereas some can be advantageous. Some mutations have a deleterious effect & cause inherited disease. *

10. Chromosomes Egg Sperm fertilization 38 pairs of autosomes & 1 pair sex chromosomes 38 autosomes & 1 X chromosome38 autosomes & 1 X or Y chromosome X Y

11. Spontaneous Mutation X

12. Recombination X

13. Propagation X

14. Inheritance of Mutation Down a Pedigree ParentsG-Parents G G-ParentsG G G-Parents

15. Shared Region of DNA Affected

16. Mutation Identification Affected Unaffected

17. PLL research Research to identify mutation responsible for PLL initiated ~ 10 years ago. DNA from affected & unaffected dogs was analysed to identify region of the genome that was homozygous in all affected dogs, and different in the unaffected dogs. –MBTS40 cases & 11 controls –LHs22 cases & 18 controls –X breeds5 cases …to cut a very long story short we identified the mutation responsible for PLL in Miniature Bull Terriers & Lancashire Heelers in summer 2009. It is a single nucleotide substitution. *

18. Miniature Bull Terrier Lancashire Heeler Jack Russell Terrier Parson Russell Terrier Patterdale Terrier Sealyham Terrier Chinese Crested Dog Tibetan Terrier Breeds carrying PLL mutation Jagd Terrier Volpino Italiano Australian Cattle Dog Yorkshire Terrier

19. Between Breeds

20. DNA Test Results DNA test developed and made available October 19th, 2009. Clear Carrier Genetically Affected

21. PLL DNA Test http://www.aht.org.uk/genetics_pll.html Kits for taking cheek swabs are available by phoning +44 (0)1638 555621 or via e-mail to swabrequest@aht.org.uk.swabrequest@aht.org.uk Further information dnatesting@aht.org.ukdnatesting@aht.org.uk

22. PLL DNA Test Results CLEAR: these dogs have two normal copies of DNA. Our research has demonstrated clear dogs will not develop PLL as a result of the mutation we are testing for, although we cannot exclude the possibility they might develop PLL due to other causes, such as trauma or the effects of other, unidentified mutations. GENETICALLY AFFECTED: these dogs have two copies of the mutation and will almost certainly develop PLL during their lifetime. We advise that all genetically affected dogs have their eyes examined by a veterinary ophthalmologist every 6 months, from the age of 18 months, so the clinical signs of PLL are detected as early as possible.

23. Carriers CARRIER: these dogs have one copy of the mutation and one normal copy of DNA. Our research has demonstrated that carriers have a very low risk of developing PLL. The majority of carriers do not develop PLL during their lives but a small percentage do. We currently estimate that between 2% – 20% of carriers will develop the condition, although we believe the true percentage is nearer to 2% than 20%. We do not currently know why some carriers develop the condition whereas the majority do not, and we advise that all carriers have their eyes examined by a veterinary ophthalmologist every 6- 12 months, from the age of 2, throughout their entire lives. Current best estimate = 2.5% *

24. Mutation Frequency We collected DNA from ‘random’ MBTs to estimate the mutation frequency 133Samples from 133 dogs collected We removed all full & half-siblings – left with 62 dogs that didn’t share a sire or a dam –Clear31 (50%) –Carrier29 (47%) –Affected2 (3%)

25. MBTs Tested Tests RequestedTests CompletedClear%Carrier%Affected% Australia 27200%2100%00% Czech Republic 33133%267%00% Eire 10 Estonia 1100%1100%00% Finland 53133%267% 0% France 42 0%2100% 0% Germany 57301137%1757%27% Hungary 95120%480% 0% Martinique 11 0%1100% 0% Spain 63 0%267%133% Sweden 20 UK 68432763%1535%12% Ukraine 30 187934144%4852%44%

26. Advice Normally for recessive conditions we advise breeders to mate their carriers to tested, clear dogs. Puppies don’t have to be DNA tested unless they will be bred with. NOT all the same advice for PLL. Owners DO need to breed with their carriers, because the mutation frequency is so high. But all puppies that are the offspring of at least one carrier parent should be DNA tested. Dogs carrying the mutation need to be clinically monitored throughout their lives. *

27. Free DNA Test Results participated in the researchAHT offers a free DNA test result to owners of all dogs that participated in the research. Dogs that were used were: – clinically affected dogs –unaffected dogs over the age of 5yo, with clear eye examination reports –Subset of random dogs 546 546 free DNA test results have been given out 197 197 dogs used in research 41 affected dogs 23 unaffected dogs >5yo 133 ‘random’ dogs 23 We have been unable to deliver a free DNA test result for 23 dogs *

28. Vitreal Leakage As zonules rupture vitreous can leak into the anterior chamber of the eye – vitreal leakage. In some countries evidence of vitreal leak age is sufficient for a dog to fail an eye examination and be classed as PLL affected We have examined samples from several dogs now, including a small number of MBTs, that have clinical evidence of vitreal leakage but that are clear of the PLL mutation. Vitreal leakage alone is not diagnostic of PLL, although many dogs with vitreal leakage will progress to develop LL.

29. Thank You… … to all the owners who have provided DNA from their dogs and supplied us with information. … to everybody who has made a financial donation to the research. We couldn’t have done it without you.