CT Seminar Report


Notes of a Seminar held on 2nd November 2008


A few CD copies of the proceedings of the seminar are still available.  Copies can be obtained from David Taylor who can be contacted by telephone at 01909 515815.



Preliminary Comments.


These notes are just a summary of the presentations given at the seminar and are not a transcript.  They need to be read in conjunction with the articles on CT that appear on the Bedlington Terrier Health Group web site.  


At the moment the only DNA test available for detecting whether a dog is unaffected, affected or a carrier is the COMMD1 test available in the UK from the Animal Health Trust or from VetGen in the USA.  Because a number of anomalies have been identified this test cannot be regarded as 100% accurate.  Current research suggests a strong probability that there is one or more other bad genes that cause or contribute to CT.  At this time the only way of identifying whether a dog is affected or unaffected by CT is by means of a liver biopsy.  However, a biopsy will not tell you whether a dog is a carrier.  The seminar explored the history, pathology and genetics of CT in the Bedlington Terrier.




David Cavill.


The Seminar was introduced and chaired by David Cavill, a well-known international judge, who has written a number of dog oriented books and currently publishes Our Dogs newspaper.  David is also Chairman of Southern Counties Canine Association and Show Manager for their annual championship show.




Professor Mike Herrtage – The history of CT from the vet’s perspective.


Professor Herrtage is a highly respected and experienced vet and academic.  He is professor of small animal medicine in the University of Cambridge and Dean of the Cambridge Veterinary School.  He spoke about the history of CT in the Bedlington Terrier from when it was first identified in 1975.


The first reported case of CT was in the USA in 1975 followed by reports from Finland (1983), Australia (1983) and the UK by Dr Susan Haywood in 1984.


What is known about CT is that it is an inherited condition and an autosomal recessive disease (that is it is caused by a dog inheriting the same defective gene from each parent – a dog with only one defective gene will be a carrier) manifested by an accumulation of copper in the liver with an inability to excrete excess copper in the bile. The condition is not present at birth, but results from a gradual build up of copper.  Dogs are not therefore normally biopsied until they are at least 12 months old.


In human beings there is a similar autosomal recessive disease called Wilson’s Disease which can manifest itself from 3 to 50 years of age.  This disease also involves an accumulation of copper in the liver with an inability to excrete the excess in bile.  There are therefore strong similarities with CT in the Bedlington, but there are also differences.  In human beings Wilson’s Disease can be manifested by accumulation of copper that can affect the kidneys, the brain, the eyes and other tissues.  The defective gene causing Wilson’s Disease was identified in 1993.


Copper accumulation causes chronic hepatitis.  The sudden release of copper from damaged liver cells can result in a breakdown of red blood cells, but the liver can suffer up to 70% damage before the disease manifests itself through symptoms.


Professor Herrtage described three categories of CT.


1. Acute CT


This usually occurs to either sex when the dog is between 2 and 6 years old. It is often precipitated by stress and manifests itself by the sudden onset of depression, lethargy and anorexia.  Despite intensive therapy the dog usually dies after 1 to 2 days.


2. Chronic CT


This usually occurs in middle aged to old dogs. It is slowly progressive.  The clinical signs are similar to those of acute CT but less severe.  There will be chronic weight loss and a build up of fluid in the abdomen.


3. Clinically asymptomatic CT


This is probably the largest group of CT affected dogs and it is important that they are identified if they are to be used for breeding.  The dog may have raised liver enzymes concentrations, but these are not specific and can be raised in other conditions where there are no clinical signs of liver disease.


The only way to identify whether such a dog is affected by CT is by means of a liver biopsy.  Without a biopsy there is always a risk of breeding from an affected animal.  Even with a biopsy there remains a risk until complete genetic screening is available, as a biopsy will not show whether an animal is a carrier.


Professor Herrtage first described the various ways in which a biopsy can be performed.  He then went on to describe an earlier study of 62 clinically asymptomatic dogs, 21 of which were found to be affected.  The evidence was that copper concentration tended to increase with age usually peaking at about 6 years of age.


Treatment can help CT affected dogs and Professor Herrtage referred to the drugs that have been used with considerable success citing the case of 2 dogs where treatment resulted in a dramatic reduction in the copper concentration in the liver.  He also referred to the use of zinc sulphate that can inhibit the absorption of copper.


Professor Herrtage concluded his presentation with the development of a DNA test.  This was first developed in the USA when the old micro-satellite marker test was introduced in 1997.  This was widely welcomed as it identified unaffected, affected and carriers, removing the risks associated with the system of biopsies.  However some discrepancies were noted between micro-satellite marker test results and liver biopsy results in the UK as early as 1998. In 2002 as a result of research at the University of Utrecht the defective gene that was thought to be the primary cause of CT was identified, and a DNA test was soon developed.  This eventually was launched by the Animal Health Trust as the COMMD1 test.  The good news was that the micro-satellite marker was within the COMMD1 gene.


However subsequent research in Australia showed that the COMMD1 deletion was not the sole cause of CT.  For some time there had been speculation that CT is a polygenetic disease.  The identification of affected dogs which had tested 1.1 under the COMMD1 test supported this view.  The research continues.




Dr Susan Haywood – CT in the Bedlington Terrier – a pathological perspective.


Dr Haywood is Honorary Senior Fellow in the Faculty of Veterinary Science University of Liverpool specialising in pathology.  She has been involved with CT in Bedlingtons since 1983/4, and has studied CT in rats and sheep since the 1970s.  She divided her talk into three parts.


1. Understanding CT.



2. Diagnosis.


Dr Haywood uses 3 criteria for diagnosing whether a dog is affected by copper:



The first biopsy of a Bedlington conducted by Dr Haywood had a liver copper value of 10,728 μg/g and micronodular cirrhosis .  This was considered a typical or classical case of CT.  Dr Haywood then showed a series of slides of affected livers to illustrate the diagnostic criteria and disease progression from initial liver damage to cirrhosis.


3. Genetic causes of CT.


Dr Haywood is conducting research to try to determine whether there is a second gene that causes CT.  The outcome of her research is designed to enable the geneticists to undertake DNA sequencing, and she is working in close collaboration with Dr Cathryn Mellersh of the Animal Health Trust.


Dr Haywood recounted the history of the development of a DNA test from the microsatellite marker test to the launch of the COMMD1 test both of which categorise dogs as unaffected 1.1 [ no COMMD1 deletions], carriers 1.2 [1 COMMD1 deletion] and affected 2.2 [2 COMMD1 deletions].


How reliable is the test?  The microsatellite marker test was never regarded as totally reliable, but when it was discovered that the marker was within the COMMD1 gene the advice was that there was no need to retest 1.1 dogs as the result would be the same.  However subsequent events have cast even more doubt on the reliability of the test.  For some time there has been speculation that there is more than one gene causing or contributing to CT.  Dr Haywood and her colleagues in 2001 reported eight dogs in the UK with DNA markers 1-1 or 1-2 that were CT affected suggesting the possibility of a second gene or at least a second mutation on the COMMD1 gene.  This supposition was reinforced by reports from Professor Cox’s team in N. America (2003) and Hyun and co-workers in Australia (2004).


Dr Haywood then went on to examine whether there were clinico-pathological subtypes which reaffirmed the existence of more than a single gene responsible for CT.  Her autopsy studies support the existence of many dogs that are affected, but show no clinical signs of suffering from CT (Professor Herrtage’s asymptomatic dogs).  In contrast there are other cases of acute fatal disease in younger dogs often with quite moderate levels of liver copper.  Interestingly these latter cases consisted of dogs lacking the homozygous deletion [2-2] reinforcing the idea that genes other than COMMD1 must be involved.  The reason for these differences in disease expression is not known, but at present only a liver biopsy will disclose whether a dog is storing a level of copper that has the potential to seriously damage the liver. Copper values of 500 to 1000 μg/g have that potential.


As a result of the anomalies identified since the introduction of the COMMD1 test Dr Haywood set up a scheme, with financial support from the Kennel Club Charitable Trust and the generosity of Bedlington breeders, to try to identify more (1.1) or (1.2) CT affected dogs.  To date with the particular support of a few dedicated breeders she has identified 13 such dogs, some of which are closely related.  This is the minimum requirement for genotyping. However more unaffected (control) dogs are needed before her research can be handed to the geneticists to commence DNA sequencing.  An appeal was made for more help, details of which have been published in the canine press.


Dr Haywood concluded her talk on a controversial note.  She posed the question whether COMMD1 is a cause of CT or is perhaps no more than a marker.  She pointed out that there is no evidence for a direct role for COMMD1 in copper metabolism, a proposition that gains some support from current research reported at a recent copper conference attended by Dr Haywood.  Scientists there confirmed their attempt to create CT in a “knockout mouse” based on the COMMD1 gene but so far have not succeeded, posing the question whether COMMD1 is even a cause of CT at all.




Dr Cathryn Mellersh - Dog DNA and inherited disease.


Dr Mellersh is a geneticist leading the Canine Molecular Genetics Group at the Animal Health Trust.  For some time she has been leading research into the development of DNA tests for a range of inherited canine conditions in a number of different breeds.  She is working closely with Dr Susan Haywood.


Dr Mellersh divided her presentation into 3 headings



Mutations.


She started her presentation by giving a brief description of how genetics work.  The chromosome is the vehicle of DNA. A dog has 39 pairs including 1 pair of sex chromosomes.  The dog inherits one chromosome of each pair from each of its parents with the sex being determined by the sex chromosome that is inherited from the sire.


The genetic code is the set of rules by which information encoded in genetic material (DNA or RNA sequences) is translated into proteins (amino acid sequences) by living cells.  DNA is in nearly every cell in the body and governs everything that is not controlled by the environment.

The genome is the term given to an organism’s complete set of genetic material.  However, the genome includes both the genes (coding) and the non-coding sequences of the DNA.  The vast majority of DNA is non-coding – only around 5% is coding.  DNA can be thought of as an enormously long string of nucleotides, or ‘letters’, with the entire canine genome consisting of approximately 2,500,000,000 letters.  This amount of ‘text’ is equivalent to around 2000 inch thick textbooks and is present in every cell of the body that contains DNA. Each time a cell divides all this genetic information is copied.


The proteins coded for by DNA have particular jobs and are interactive.  An inherited condition is caused by a mutation in at least one gene.  A mutation is the equivalent of a typing error in the copying of the gene which means it will no longer be the right shape and unable to interact in the correct way.  This may cause disease and lead to an inherited condition. Searching for a mutation that is responsible for an inherited disease is a mammoth task, because a mutation can be as small as a single incorrect letter of DNA. Inherited diseases can be caused by a single mutation in a single gene or by mutations in more than one gene.  When searching for a mutation (insertion or deletion) the geneticist does not know what he is looking for, and the search can take a long time before a defective gene is identified.


Genetic Markers.


Most DNA is outside the coding genes, and variations can occur in non-coding DNA without causing inherited disease.  Genetic markers are short regions of variable DNA; the two major types of genetic markers are micro-satellites and single nucleotide polymorphisms (SNPs)

Every individual has 2 complete copies of each micro-satellite (alleles) that can be different lengths.  If different they are referred to as heterozygous and if the same homozygous. Variable DNA allows the geneticist to tell chromosomes apart and which come from the mother and which from the father.


Identifying Mutations.


All mutations arise in a single founding dog, and over generations the defective gene will be copied many times and passed from one dog to another along with the genetic markers located on the DNA flanking the mutation.


The geneticist relies on the vet to identify affected dogs so that the geneticist can then search for DNA common to those dogs.  The suspicion is that there may be another gene other than COMMD1 that causes or contributes to CT.


In 2004 the genome sequencing of animals was completed and published.  Scientists now know a lot more about animal DNA, and this should facilitate a speedier identification of defective genes.


When designing a study the criteria are:



Dr Mellersh outlined various approaches to identifying the offending gene.


The candidate gene approach is to focus on genes known to cause similar diseases in other species such as that responsible for Wilson’s disease in humans.  The advantage of this approach is that it is possible to look at quite a modest number of genes quickly and cheaply.  The disadvantage is that if the wrong candidate gene is chosen this approach will not be successful.


Another approach is a whole genome scan.  You don’t need to know about the gene, but it is a very time consuming and expensive process.


The next question is which samples are available.  A pedigree based linkage analysis requires samples from affected dogs and large numbers of close relatives.


A case control study which uses affected and unrelated unaffected dogs is an alternative and useful approach, because it does not require the unaffected dogs to be closely related to the affected ones.  If it transpires a condition is the result of a single recessive gene then DNA samples from 12 affected dogs and 12 control dogs should be sufficient to locate the mutation.  If the offending gene turns out to be dominant a much larger sample will be required.  At some point it is necessary to start sequencing the DNA letter by letter, after which it should be possible to develop a test.


Given the prospect of another defective gene Dr Mellersh favours a process of starting from scratch.  First it will be necessary to formally exclude the COMMD1 gene by sequencing all of COMMD1 in a small number of affected dogs that are known to not carry the COMMD1 deletion to ensure there is not a second mutation in the same gene. Then it makes sense to exclude other known copper-disease genes and finally undertake a full genome scan with the 13 deletion free dogs that have been found to be affected and 13 control dogs.  Dr Mellersh would like to stress that 13 is the minimum number of samples required and the project would definitely benefit from more samples should they become available.




Questions.


A useful question and answer session was held at the conclusion of the seminar. These notes outline only 2 questions of particular importance to breeders.


It was suggested that a programme of biopsy followed by test mating should ensure safe breeding. Whilst this may be the case if there is only one defective gene, if the cause of CT proves to be polygenetic such an approach offers no guarantees, as a dog may inherit a different mutation from each parent, and breeders may unwittingly be breeding with a carrier.


The Panel was asked what advice should be given to people buying a puppy.  It was pointed out that the new Kennel Club Code of Ethics places great emphasis on breeders being entirely open with purchasers, and should not do or say anything that could be misleading especially to the uninformed.  The view of the Panel was that purchasers must be told that the COMMD1 test is not wholly reliable and relates only to one gene.  As there is a possibility of one or more other bad genes the only way at this time to tell whether a dog is affected by CT is by examination of a liver tissue sample obtained via a liver biopsy






Copper Toxicosis

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