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Canine Leucocyte Adhesion Deficiency
testing in the Irish Setter
by Jeff Sampson, the Kennel Club Genetics Co-ordinator

The KC/Animal Health Trust (AHT) DNA Screening Programme for CLAD in the Irish Setter began in 1999 with the identification of the gene mutation responsible for CLAD by research workers at the University of Uppsala in Sweden. This same group subsequently developed a DNA test specific for this mutation that became available at the AHT to Irish Setter breeders in the United Kingdom.

Owners wishing to have their dogs tested submit blood samples directly to the AHT where the DNA test is carried out. The result of the test is then returned to the owner in the form of a certificate, a copy of which, after a one month holding period, is sent to the Kennel Club where the dog’s DNA result is added to its registration details on the Registration Database, published in the Breed Records Supplement (BRS) and also added to the tables of DNA results that are present on the Health Pages of the KC website. There are three possible outcomes to this test; a dog can be clear, a carrier or affected . Fortunately, the programme has not revealed a single genetically ‘affected’ dog, but approximately 18% of the tested dogs have been shown to be carriers, many of which have now been removed from the breeding population.

Recent re-examination of samples submitted from dogs early in this scheme (those submitted up to early 2000) has revealed two dogs that were originally diagnosed as clear that unfortunately turned out to be carriers on re-testing. The owners of these two dogs have been notified and the KC and the AHT are working closely with them to address the problems that these re-diagnoses will cause. It has to be stressed that no blame can be attached to these two owners who submitted their dogs for testing and acted in good faith using the original result, which was clear in both cases.

Below is a series of questions/ answers that is designed to answer breeders’ questions that may arise as a result of these discoveries.

Why were the samples re-tested in the first place?

The samples submitted to the scheme in 1999/ early2000, 520 in total (figure supplied by the AHT), were tested using the DNA test developed at Uppsala University. During this first year of the scheme scientists at the AHT discovered that this test was not sufficiently robust and could give incorrect diagnoses, particularly when the DNA prepared from an individual dog was of less than ideal quality for the test. As a result, a small number of these early dogs’ samples, originally diagnosed as carriers, were reclassified as clear on re-testing.

Those involved with this early phase of testing will recall these dogs and those involved were advised at the time and the results of the re-tests were openly published. This anomaly certainly could not have been predicted at the start of testing and only became apparent when significant samples had been tested on a routine basis.

Following this discovery, the scientific staff at the AHT developed a far more robust DNA testing procedure based on DNA sequencing. Soon after, the University of Uppsala also stopped using their initial test and adopted a DNA sequencing test as a replacement. The revised sequence-based DNA test has been used by the AHT for all subsequent CLAD tests in the Irish Setter and Irish Red & White Setter.

As a result of this, the AHT resolved, on their own initiative and at their own cost, to repeat the first 520 tests with the more robust DNA sequencing test. It is a result of this re-testing programme that the two dogs originally diagnosed as clear have now been shown to be carriers.

Why has it taken so long to complete this re-testing?

The re-testing, which had to be completed alongside other commitments, involved applying the new testing method to stored DNA samples. The DNA sequencing test requires DNA of a higher-quality than the original testing method, and some additional work to achieve optimisation of the procedures was necessary to achieve satisfactory quality for these archived DNAs.

Have all 520 samples now been retested?

All of the 520 samples have now been retested but there are 25 of these retests that have been unsuccessful, despite several attempts to sequence the DNA samples. Their original CLAD test results therefore still need to be confirmed in these 25 dogs. The AHT will continue to attempt to sequence their existing DNA samples from these dogs; in some cases we will be able to use the DNA test results of their progeny, if sufficient exist, to confirm their original test results. In some cases, it may be necessary to ask owners to resubmit fresh samples that the AHT will test free of charge. If necessary, owners will be contacted, hopefully within the next two weeks, with a request for a fresh sample and an explanation of why it has been necessary to make the request.

Can we still have confidence in the DNA test for CLAD?

Everyone involved in the present testing programme would say the answer to this question is a very definite ‘YES’, and I wholeheartedly agree with this. The DNA sequencing test for CLAD developed at the AHT is far more robust and reliable than the original test and gives a very definitive answer because it directly reads the DNA sequence in the two gene copies that a dog possesses and indicates whether these sequences are normal or mutant. It is not susceptible to the problems of less than ideal quality of DNA that affected the original test in the early days because, if the DNA is sub-optimal, then there is no DNA sequence that can be read. What gives greatest confidence is that around one thousand dogs have now been tested using the new sequencing test; in many cases the progeny of tested parents have been tested and not one anomaly has been identified.

How will the identification of the problem with these two dogs affect the on-going control scheme for CLAD in the Irish Setter?

The two dogs in question have in fact been used for breeding and produced litters and there are now second-generation progeny that have been registered. The AHT will contact owners of all first and second generation progeny and offer free DNA testing for these dogs.

Fortunately, the dogs have been mated to either DNA tested clear dogs or hereditarily clear dogs in producing first and second generation progeny. However, some of these progeny may well now be carriers, rather than the expected ‘hereditarily clear’ status. The free testing offered by the AHT to these progeny will identify any carriers in this group.

Carriers will not become clinically affected but their status is important in the on-going breed control scheme. Carriers can be mated, provided they are mated to either a DNA tested dog or an hereditarily clear dog, and their progeny are DNA tested. Presently there is a deadline of July 2005 beyond which the Kennel Club will not register an Irish Setter unless it is either DNA tested clear or hereditarily clear of CLAD. Any new carriers that are now identified as a result of these re-diagnoses will of course now be much closer to this 2005 deadline and the Kennel Club will have to re-evaluate the CLAD situation as it relates to registration of dogs affected by this ‘mis-diagnosis’. The Kennel Club will discuss the impact of these changed diagnoses on the present control scheme in collaboration with the breed clubs and decide on the best way forward to continue the control scheme without prejudicing the future breeding potential of dogs caught by this particular problem.

To summarise

It is extremely unfortunate that these two early errors have occurred, but it is better that they have come to light, rather than remaining undetected, because we can now identify and test the dogs concerned and their progeny. The Kennel Club and the AHT will offer whatever help they can to the owners of these two dogs and the new owners of any progeny that have been produced that are identified as carriers.

Unfortunate as they undoubtedly are, these two results should not dent breeders’ confidence in the DNA testing programme; the mis-diagnoses resulted from an early test that, in hind sight, had unforeseen technical problems. As stated above, there is every reason to have confidence in the DNA sequencing test that has been used to test the majority of Irish Setters that have gone through the scheme.

This DNA testing scheme has identified many carriers within the breeding population and the control scheme that was introduced has already reduced the carrier frequency. The testing programme and control scheme is working and offers the only realistic chance to reduce the frequency of the CLAD mutation in the Irish Setter breed, to the point of irrelevance.