Identification of Disease Genes such as the cause of Trapped Neutrophil Syndrome in Border Collies.

(original contribution by Alan Wilton, PhD, genetics researcher at University of New South Wales)

Genetic diseases are a problem in most domestic animals including all pedigree dogs.  The reason is due to the extensive use of champion animals, which spreads any hidden genetic defect carried by that animal through the population making it common.  We all have a few defective genes but since we have two copies and one is still functioning, it does not cause a problem.  However, a common defect combined with mating of related individuals results in the genetic defect showing up in the offspring.  Affected animals will have the champion as a common ancestor on both sides and inherit the same gene defect from both the maternal and paternal lines.  They have inherited two identical copies of the gene, both coming from the champion.  This can be good to fix some desirable characters in a line but is a disaster if it happens for genes that are defective and cause disease.

In Border Collies there are three common diseases that have arisen through this process.  They are collie eye anomaly, CEA, which is a defect in the development of the eye, neuronal ceroid lipofuscinosis (NCL), which is a nerve degenerative disease with onset of symptoms about 16 months, and trapped neutrophil syndrome (TNS), which is a defect in the immune system.  Other diseases with a presumed genetic cause are known but generally rare.  For example, cerebellar abiotrophy, which is a nerve degenerative disease that shows as ataxia (difficulty with coordination and movement), narrow angle glaucoma, which is a blockage of the ducts in the eye causing pressure in the eye and blindness, epilepsy, which is another nerve disorder resulting in seizures (fits),  osteochondritis dissecans (OCD) which results in lifting of cartilage from bone at joints, and several others.

CEA is very common in most collie breeds.  The affect is rarely severe but can result in detached retinas and blindness.  It results from a deletion of a large chunk of DNA near the sonic hedgehog gene.  This missing piece of DNA results in a disruption to normal development.  The mutation was discovered by researchers at Cornell and a DNA test is available through Optigen in US.

NCL is a particularly devastating disease as it is a slow, sure wasting disease that strikes after 15 months and the owners and breeders can only watch the dog lose his mind and control of his body.  The mutation is a single change at one of the thousands of bases that make up the CLN5 gene  [1].  Mutations in CLN5 cause Batten disease in humans. NCL was a common problem in Australian and New Zealand lines.  It presumably arose due to a mutation in a dog in NZ and became common because of the popular sire effect i.e. was carried by a champion dog.  The mutation was discovered after 10 years of research at UNSW.  DNA testing is now provided by UNSW and the test is licensed to other labs.  The test can detect carrier status of NCL, i.e. dogs that carry one copy of the defect and will not show any signs of the disease.  This means breeders can test their dogs prior to mating to prevent production of affected pups.  Carriers can still be used if mated to CL clear dogs and the progeny are tested to identify which are clear and to be used in future breeding programs.  DNA testing has made it possible for breeders to drastically reduce the incidence of CL carriers in the population.  NCL cases have also been reported in Japan, US and Europe in Border collies.

TNS was first described in Border collies in New Zealand by Frazer Allen and Boyd Jones [2].  At least 12 affected litters had been reported and it was suspected that a large number have gone unreported.  The inheritance of the disease is as a recessive disorder i.e. similar to inheritance of NCL with asymptomatic carriers producing litters with a few affected pups.  The Australian/NZ TNS pedigrees all have the same common ancestor on both sides of each pedigree, which is consistent with this animal being a carrier for the defect and as a popular stud dog made it common in the population.  This is also consistent with the recessive inheritance of the disease. 

The physiological defect in TNS is failure of the neutrophils to migrate out of the bone marrow, where they form and mature, and into the blood stream where they can protect against infection.  The consequence of low neutrophil count is high susceptibility to infections.  Gut infections are common in some TNS pups resulting in lack of nutrients and poor growth.  Some animals show a poor response to vaccination being unable to deal with the introduced microbes.

The DNA test for TNS was developed at UNSW in 2007.  There is a very small piece of DNA missing from a very large gene .  Little is known about the function of the gene but the results of mutations are obvious in the dog and in humans.  DNA testing has shown that the mutation is found at high frequencies (>5%) in all Border collie populations around the world, including show dogs and working dogs in UK and Australia.  This indicates the mutation has been present since the beginning of the Border collie breed and may even be found in other collie breeds.  DNA testing has shown that a major cause of loss of pups close to birth is this genetic defect, fading/failing puppy syndrome.  Antibiotics and steroids can be used to help alleviate symptoms and allow some dogs to live long lives.  The disease symptoms vary with the type of infection contracted so it was difficult to recognise that the rare problems with raising puppies was genetically related.  The DNA test for TNS is offered exclusively by UNSW and funds raised from testing is used for research into other dog diseases.

There are now available extensive genetic tools for identifying genetic defects in the dog.  [3].  The sequencing of the dog genome means that the code for every gene is readily available.  Many genetic markers are available to help locate genetic diseases as they are passed from one generation to the next [4, 5, 6].  What took 10 years to do for CL can now be done much more efficiently.  The causes of many genetic traits are quickly being identified in dogs.  Tests are being made available for everything from coat colour to genetic diseases.  In the long term test may even be available for behavioural traits.

The main resource required for any research is the affected subjects.  In the past large families with the disease were needed but now new testing methods can identify disease gene locations with 10 to 20 affected dogs and a similar number of unaffected for comparison.  No progress can be made without DNA extracted from affecteds for the disease of interest.  DNA banking, which is storage of DNA/blood/tissue for use in future research, is a good idea for any disease that is likely to become a problem.  It gives researchers the opportunity to jump straight in when their attention is drawn to the problem instead of having o wait until enough cases come along to start work.

There are two main approaches to identifying disease genes.  One is linkage analysis where the inheritance of the genetic defect and genetic markers are traced through pedigrees and used to identify the location of the disease-causing gene on the chromosomes.  The other is to look for regions of homozygosity in affected animals.  That is, regions that have inherited the same copy of a gene from the common ancestor from both mother and father, and both copies are identical and carry the genetic defect.  Dogs have 39 pairs of chromosomes made up about 2,300,000,000 DNA bases.  An average gene uses 5,000 bases from a region about 30,000 bases long and the gene defect is likely a change in a single one of those bases.  So, it is a little like looking for a needle in a haystack even when you know where to look.

The more samples that are available from affecteds and their relatives, the easier the search for the gene will be.  Pathology on affected animals to confirm the exact nature of the diseases is important so all animals in the study have the same genetic disease.  Once the resource has been built up, SNP chips, which will examine 50,000 or more variable sites on the genome, can be used to locate the disease gene.  This whole genome analysis has been successfully used to find disease genes in humans in studies funded by the Wellcome Trust and others..  While this type of research is not cheap and requires substantial research funds, it is very effective.  When combined with new high throughput sequencing technologies that can sequence an entire genome of an animal like a dog in a few weeks it is very powerful.   It will undoubtedly lead to the discovery of the causes of more genetic diseases and more test DNA tests becoming available for every breed of dog.  Responsible breeders will check their breeding stock to ensure they do not produce animals suffering from these avoidable diseases.

1.    Melville SA, Wilson CL, Chiang CS, Studdert VP, Lingaas F, Wilton AN. (2005). A mutation in canine CLN5 causes neuronal ceroid lipofuscinosis in Border collie dogs. Genomics. 86: 287-294

2.    Allan FJ, Thompson KG , Jones BR , Burbidge HM , McKinley RL. (1996). Neutropenia with a probable hereditary basis in Border collies. New Zealand Veterinary Journal. 44: 67-72

3.    http://www.ncbi.nlm.nih.gov/genome/guide/dog/index.html

4.    Brasch GS. How the dog got its spots  (2007) Nature Genetics 39:1304-1306

5.    Karlsson, E.K. et al. (2007) Nature Genetics 39, 1321–1328

6.    Ostrander EA, Krugylak L. (2000). Unleashing the canine genome. Genome Reasearch. 10: 1271-1274

Alan Wilton

School of Biotechnology and Biomolecular Sciences

University of New South Wales

Sydney  NSW  2052

http://www.babs.unsw.edu.au/directory.php?personnelID=14

Canine DNA Testing

http://www.babs.unsw.edu.au/canine_genetics_facility.php

TNS information for Border Collie breeders.

TNS stands for Trapped Neutrophil Syndrome.  It is an immune deficiency in Border collies.  It is an inherited disorder that is very common in all populations of Border Collies with more than 10% of both working and show dogs carrying the defective gene and capable of having affected puppies.

*TNS is a condition where the bone marrow produces neutrophils but they are not released into the bloodstream. This results in an impaired immune system that cannot fight infections.

*Symptoms are variable, many of the reported TNS puppies have been born looking normal but others have been born small.

*Some puppies with TNS have been small and fine boned with narrow heads at some point but this may not be evident until approx 16 weeks.

*A common first sign is a bad reaction to vaccinations with signs of fever.

*Blood tests may show an abnormally low segmented neutrophil level but TNS can only be definitely diagnosed by bone marrow biopsy or DNA test.

*Any puppy that shows any signs of infection or failure to thrive is a possible case of TNS.

*There is no cure for TNS and it appears to always be fatal eventually.  Antibiotic and steroid treatment can help affected dogs live a relatively active life.

*TNS is an autosomal recessive condition.

*The symptoms are extremely variable and will depend on the bacteria that the pup encounters. There may also be other genes that effect the disease expression.

*Some dogs do not show symptoms until later in life.  Older puppies & young adult dogs diagnosed with immune system problems may have TNS so they should also be tested with the DNA test

*There is no evidence that carriers of the TNS defect have increased immune problems.

DNA testing

In early 2007, Jeremy Shearman in Dr Wilton’s lab, identified the genetic defect that causes TNS and developed a simple DNA test to identify carriers.  The test amplifies DNA extracted from blood using the Polymerase Chain Reaction.  The CL test in Border collies developed in Dr Wilton’s lab and CEA test from Optigen work in a similar way.  Application of the TNS test has shown that TNS is widespread through the Border Collie breed (Table 1).  Proportions of TNS carriers from testing are an overestimate of the population frequency because dogs related to carriers are preferentially tested.  It is estimated that 10% to 15% of Border collies worldwide are TNS carriers.  In a randomly selected sample of Borders tested from Norway 14% carried the TNS mutation.  Testing has confirmed the TNS mutation in UK, US, Japan, Europe and Scandinavia as well as Australia and New Zealand (Table 1).

Breeders can now test for TNS before breeding to avoid mating two carriers and risk producing affected puppies.  Puppies from matings between a carrier and a TNS clear will produce (on average) half carrier pups and half clear.  Puppies can be tested at a few weeks of age from a blood spot on an FTA card (provided by Dr Wilton, Table 2).

 Dr Wilton’s lab, where the research was done, is the only lab that can do TNS testing currently.  Some other labs will collect samples and send them to UNSW for testing.  Blood samples on FTA cards are the preferred sample for testing.  FTA cards can be easily sent with request forms via airmail.  Collection kits for sampling can be obtained by providing your postal address.  A kit for pricking the pups skin to get a drop of blood is the preferred method of puppy testing.  Mouth swabs are another way to sample pups but they are not as reliable for testing. The sample brush (swab) must pick up enough cells from the lining of the cheek to work and there are often problems.  Blood on FTA cards can also have problems if the card is flooded with blood and overloaded.  Blood should be applied sparingly to FTA cards so that the salts on the card can preserve the DNA.

Request forms for TNS testing (available from Border Collie Health and other websites) ask for supporting evidence to confirm that the sample supplied is from the dog listed on the form.  This is usually done by a vet reading a microchip or a tattoo.  Testing can be done without this (for example for puppies) but the results may not be accepted by official organisations.

            About 5000 Border Collies have been tested so far.  The high incidence of carriers (>10%) in all lines suggests that the causative mutation goes right back to the beginning of the breed and may even be found in other collie breeds, like CEA.  For this reason, it is recommended that all lineages be tested.  Once the parents have been tested clear for TNS it is not necessary to test the pups, they can only inherit the genetic defect from a carrier parent.  Both parents of any affected dog must be carriers.  Many lines that have shown no sign of the disease may still have carriers, and this includes ISDS lines and Australian working dog lines. 

            UNSW cannot give Clear by Parentage Certificates for DNA tests because we are not the holders of the registered parentage information.  In many countries, the breed clubs that register the dogs maintain a database of test results include the information on registration certificates and provide Clear by Parentage certificates for puppies of registered litters where the parents have been tested.  We could prove paternity with DNA testing using the ISAG Paternity Testing markers but this would cost as much as testing the pups directly for disease.

            Our lab also developed the CL test and offers DNA testing for it.  This test has been successfully applied to reduce the incidence of CL carriers in the population.  Most of the carriers identified in our research on samples submitted for TNS testing were known as CL carriers but occasional CL cases are still appearing in Australia from backyard breeders (Table 2).  It is hoped that DNA testing will also enable breeders to reduce carrier rate for TNS.  However numbers of samples being tested are dropping off with 1930 tested in 2007 but only 1150 in 2008.

            Current research in the lab is focussing on Cerebellar Abiotrophy, which causes ataxia, and glaucoma, which have been a rare problem with several recent cases in Borders collies.  The CA is likely to be due to a different gene mutation than the one we are working on in kelpies where it is a common problem.  DNA testing for inherited diseases in dogs is an effective way to improve the breed by managing the breeding population and slowly removing the carriers for inherited diseases without reducing the gene pool or destroying lines that carry desired traits.

Alan Wilton

School of Biotechnology, University of NSW 2052

Email:  a.wilton@unsw.edu.au