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A Primer on the Genetics of Copper Toxicosis.
By
Shirley Davies.
Contributors Julie Cummings and Sheila Baldwin.

In this primer I have endeavoured to give the information necessary to understand the thinking on CT in its simplest form. The view of the genetics has evolved and changed over many years as more information has become available.
(To aid the reader the correct terminology is provided, together with a check copy minus the terminology, at the end).
The Basics of Genetics.
All the information needed to build a new puppy, how it should look when it is fully grown and how to make the puppy work as a living being is carried in the DNA, a term everyone these days has heard of.

The DNA is organised into structures called chromosomes which are found in the nucleus of each cell.

Diagram 1. A typical body cell.

A typical body cell has a nucleus, cytoplasm surrounding the nucleus with various bodies in it which do the day to day work of the cell and a cell membrane which holds the cell together and controls what enters and leaves the cell.

All chromosomes occur in pairs which look identical under the microscope, with the exception of the sex chromosome pair where the male “Y” chromosome is somewhat smaller than the female “X” chromosome.

Diagram 2. Body cells have 2 matching sets of chromosomes and sex cells – sperm and eggs- have 1 set. Human body cells contain 46 chromosomes,
22 pairs plus a sex chromosome pair as shown below.

In the dog there are 39 pairs of chromosomes making 78 chromosomes in total. We humans have 23 pairs making 46 chromosomes. The number of chromosomes must remain constant or there may be problems as a result, for example many of the symptoms of Down’s Syndrome are due to the presence of an extra chromosome.
This constant number becomes important when the sex cells [eggs and sperm] are made. If they contained all the chromosomes then each would have 78 chromosomes and when the egg was fertilised by the sperm there would then be 156 chromosomes in the cell produced, twice as many as needed!
So, when eggs and sperm are produced the cell divides in a special way which puts one of each chromosome pair into each sex cell, so eggs and sperm have only 39 chromosomes. Which one of each pair gets into a sex cell is completely random.

Diagram 3. When an egg and a sperm combine during the process of fertilization the first cell of a new individual from which
all other cells arise acquires its unique combination of genetic information.

(Gamete is the name given to sex cells and Zygote the name given to the first cell after fertilization.)

A sperm with 1 set of chromosomes + an egg with 1 set of chromosomes = the first cell with 2 sets of chromosomes, which will divide and divide to build up into all the cells of the puppy.
It is because of this process of fertilization that one set of chromosomes is received from each parent.
Each egg or sperm contains 1 form of a gene. ( the name given to alternative forms of a gene is alleles).
Every puppy thus has a half set of chromosomes from the dam and a half set of chromosomes from the sire but it is complete chance which chromosome of each pair gets into each sex cell. This allows a random mixture of the original sire and dams chromosomes in each sex cell.
When the egg and sperm join together the 39 pairs are restored, though these are now “shuffled”.

Chromosomes and genes
Chromosomes consist of very long strands of DNA. If you could imagine a gold chain with beads at intervals along it this would give a very basic model of how a chromosome is made up. The areas of chain represent between gene DNA the purpose of which is not fully understood. The beads would represent the areas of DNA which perform a function, these are the “genes”.

Each gene has a purpose. The easiest to understand are genes which control one characteristic such as eye colour. There is a form of the gene for eye colour which can produce brown eyes and a form which can produce blue eyes. We must remember here, though, that very few genes work completely in isolation and the eye colour gene is modified by others which allow, for example, hazel eyes to exist.
One example of a gene which has only two outcomes is that which controls the ability to roll your tongue…can you roll yours? Some people can, some cannot.. try it on some friends! Another is the ability to smell freesias, some people cannot smell freesias.

Many other characteristics, such as height, are controlled by many genes working together.

The way genes eventually show their effect in a dog or any organism is also influenced by the environment. If, for example, a dog with two of the defective forms of copper gene were given a diet almost totally lacking in copper, it could appear to have a normal liver when biopsied..

How single genes work.
Genes work in pairs, one from the dam and one from the sire.
Sometimes a gene gets damaged, what is called a “mutation” occurs. The kind of damage can include losing part of the gene, a DELETION mutation.
When a mutation happens this may have the effect of stopping the gene doing its job, a “bad” gene. We could call the working form of the gene the “good” gene.
Sometimes the “bad” mutated gene, which will produce an unwanted, even lethal affect, is what is called a Dominant gene…a bossy gene which always gets its own way about how the puppy will be. This kind of bad dominant gene, because its effect will always show up, usually quickly dies out.
More usually the changed gene is a “recessive” gene and if there is only one the other unchanged, dominant gene will show its effect because it is the bossy type! A dog with one of each type is a CARRIER.
Only when two of the recessives, the shy retiring types, are present dare they show their effect! An AFFECTED. Only Bedlington puppies with a recessive, damaged copper gene of some kind on both the chromosomes will develop copper toxicosis.
If the puppy has no damaged copper genes at all it will be a “CLEAR”.

In the case of CT there is a “good”/ dominant, and a “bad”/recessive form of the gene involved.

It is possible to predict what genes a puppy will get from its parents.
If we look at a single gene inheritance pattern we can predict the various possible combinations of dominant and recessive genes

Dominant forms of genes are usually represented by CAPITAL letters and recessive forms of genes by small letters.

Letters are used to represent the form of the genes.

For Copper Toxicosis we might therefore have ‘C’ representing the Dominant form of the gene, which is in this case the working, normal, form of the gene.
and ‘c’ representing the recessive form of the gene, the abnormal form of the gene which does not work properly, if at all.

Three combinations of the forms of the gene are possible. These combinations are called the “genotypes”.
CC is used to represent a dog with two dominant forms of the gene.
Such a dog is called by breeders a “clear”. (The correct term is “homozygous normal”).
Cc is used to represent a dog with one Dominant and one recessive form of the gene, it is called a carrier since it carries the abnormal form of the gene hidden by the Dominant one. (The correct name is “heterozygous normal”).
cc is used to represent an “affected”. (The correct term is “homozygous recessive”.)

If an individual has the genotype CC then all the eggs or sperm made by it will contain the C form of the gene.
If an individual is Cc then half the eggs or sperm will be C and the other half c.

The possible outcomes in the puppies from a cross of two dogs where only one gene is being looked at can be worked out using a Punnet square to calculate all possible combinations of the eggs with sperm with C representing the dominant characteristic and c the recessive:
Where parents which have two of the same form of the gene are crossed a Punnet square is not needed.
Eg.Parents: Sire CC x cc Dam

All sperm from this sire will be ‘C’ and all eggs from the dam will be ‘c’
All puppies in the first generation (F1) will be Cc, they will all be “carriers”

Cross between two carriers: Sire Cc x Cc Dam

Half the sperm will have a ‘C’ and half a ‘c’.
Half the eggs will have a ‘C’ and half a ‘c’.

Punnet Square.

Sperm

Eggs

The Punnet square shows three types of offspring are possible.
CC two functioning dominant forms of the gene
Cc one functioning dominant form and one recessive non-functioning form
cc two non-functioning forms of the gene.
These are called the possible GENOTYPES.

These three genotypes will produce two different outcomes in the puppies.
‘CC’ puppies will have completely normal livers and ‘Cc’ puppies(carriers) will also have normal livers as the Dominant, functioning, form of the gene is present.
The unlucky ones are the ‘cc’ genotypes as these puppies have two non-functioning forms of the gene (affecteds). Their livers will not be able to do something the liver cells need to do to get rid of any excess copper taken into the body.
These translations of the GENOTYPE into how the liver cells behave produce the PHENOTYPE. In Copper Toxicosis there is a normal phenotype where the liver cells work to send excess copper out of the body, and an abnormal phenotype where the liver cells cannot somehow do some part of the processing of excess copper leading to it remaining in the liver.
You will see that there are three “normal” phenotypes to one abnormal phenotype. Phenotypic ratio: 3:1

Single gene crosses, like the one shown above where the parents differ from one another in one characteristic will produce close to a 3:1 phenotypic ratio if sufficient offspring are looked at.
In 200 offspring the results might be:
147 normal phenotypes: 53 abnormal phenotypes
142 normal phenotypes : 58 abnormal phenotypes.
The ratio is approximately 3:1.
However, in a litter of four puppies this is not likely to show up, there could be 4 normal phenotypes or four abnormal phenotypes, 2 of each, or, 3 normal and 1 abnormal. At this small number of puppies the laws of chance come into play, so the more puppies that are looked at in any test mating of an individual dog or bitch the better.

If an individual posses the dominant phenotype they have two possible genotypes, CC or Cc. Test mating using what is called a back cross can predict whether the Genotype is CC “clear”
or Cc a “carrier”. There are many different forms of test mating available to use, but none gives 100% certainty that the dog or bitch being test mated is “clear” because of the laws
of chance or “Probability”.

Diagram 4. Dominant CC, or as shown above ‘BB’ individuals will always produce offspring with the same kind of phenotype.

However, genes are not inherited in isolation as multiple genes are carried on a single chromosome. Let us consider a cross where the parents differ from one another by possessing different forms of the gene for 2 different genes, R and Y.

Parents: Sire RRYY x rryy Dam
Sperm RY ry Eggs
First generation all RrYy, all carriers

A cross of two such carriers: RrYy x RrYy
Would produce possible sperm RY, Ry, rY and ry
And possible eggs RY, Ry, rY and ry

Sperm Eggs	RY	Ry	rY	ry
RY	RRYY	RRYy	RrYY	RrYy
Ry	RRYy	RRyy	RrYy	Rryy
rY	RrYY	RrYy	rrYY	rrYy
ry	RrYy	Rryy	rrYy	rryy

There will be a variety of phentoypes produced, some resembling neither parents phenotypes.
There are 16 possible combinations of the eggs and sperm involved and when you consider that we are only looking at 2 genes here and in reality hundreds of genes are transmitted together you can see how complex the process of inheritance actually is!

The picture is further complicated by some characteristics being controlled by multiple genes, for example height, and some where the Dominant gene is not totally dominant so that an intermediate offspring is produced, for example some flowers are red and white and produce mixtures of red and white in future generations, but sometimes red and white flowers may produce pink flowers in the next generation, an intermediate of both parents’ phenotypes. Variation also arises from chromosomes lining up in a random order when eggs and sperm are being formed and from genes crossing from one chromosome to another during this process. There are also many characteristics where the environment has a profound effect on the eventual way they appear in the phenotype, e.g. in the case of CT affected dogs could appear unaffected when biopsied because their diet has been very low in copper,
or they have had de-coppering medication, but the dog is never-the-less genetically an affected.

In order to use genetics to identify and test for inherited characteristics we must be able to identify specific genes as being responsible for certain characteristics. This is a huge task when you consider the number of individual genes that an organism possesses. It is essential to compare chromosomes from ‘normal’ organisms and those which have two of the ‘defective form of the gene’ to find the exact location of the gene responsible for a specific characteristic.

How does all this apply to what has been learned about CT over the last twenty years?
Way back in the 1970s experiments were carried out which suggested that CT was caused by a single recessive gene.
Since affecteds can be identified by a liver biopsy even if they are not “sick” it was possible for breeders to remove affecteds as quickly as possible from their bloodlines. This action alone will reduce the number of recessive, non-functioning CT genes in the gene pool markedly and fairly quickly.

Affecteds were assumed to be ‘cc’.
The biopsied dogs which were unaffected were, however, either carriers (Cc) or “clears” (CC). As we have seen it is possible to do special crosses which allow a degree of certainty that the animal “test mated” is a “clear”, i.e. ‘CC’.
However, no-one can ever be 100% certain that the tested dog/bitch is “clear” there is always the possibility of being wrong in that assumption no matter how many puppies are produced that are unaffected. The more unaffected puppies that are produced, the more certain the breeder can be that the animal being “test mated” is probably a “clear”. A number of dogs and bitches were identified as “clears” in this way. The favoured test cross was between a known affected ‘cc’ and a biopsied unaffected, either ‘CC’ or ‘Cc’.
Other breeders chose different methods of trying to rid their lines of CT genes, or, sadly did nothing at all.
Some simply opted for mating their bitches, biopsied or unbiopsied, to “clear” dogs. In some instances known carrier bitches were mated to dogs claimed to be ‘clear’.

Because there is always a slight uncertainty associated with test mating to affecteds (or to known carriers) and some not even biopsied, everyone wanted a completely sure method of identifying carriers and clears. Some would not biopsy their dogs on the grounds that, for example, it was “cruel” and those who do biopsy would always have also much preferred a non-invasive method of identifying affecteds and unaffecteds, i.e. a method which does not involve liver biopsies.
Mike Herrtage and his team, way back in the late 1980s, proved conclusively that liver biopsy was the only certain method of distinguishing between affecteds and unaffecteds. Blood test, urine tests etc were all dismissed as not accurate in dogs which were not sick, but never-the-less were “copper toxicosis cases” as proven by their copper values and the changes which were observed in affected livers at the cellular level. Most of the dogs examined then were not sick, as this does not usually appear until the dog is at least 2years old. Biopsy results have been shown not to be accurate until the dog is at least 6 months old. Breeders wanted a method of diagnosing CT at a much earlier age, and most importantly something to identify carriers and clears.
The hunt was therefore on, for a DNA test.

In the mid 1990s Vetgen in the USA produced a Linked Marker (C04107) Test.
A DNA marker is some piece of DNA which varies in some way in different individuals. A linked marker is assumed to be close to the gene but not within it. In the case of the C04107 linked marker discovered by Vetgen, it differed in having two different lengths of DNA. It was believed to be close to, but not within, a gene causing CT.
The marker, to be useful, must be found to have one form which tracks through pedigrees with a strong degree of “linkage” to the defective form of the gene thought to be causing the problem and another form which tracks through pedigrees closely associated with the normal, dominant form of the gene. The only way to achieve this is by using the biopsy results to conclusively show which dogs in the pedigrees are affected and unaffected. In other words, most of the time where a CT biopsied ‘affected’ appears in a pedigree, since we know that animal must have two defective forms of the gene, i.e. be ‘cc’, then one of the two alternate lengths of DNA must more often be found “linked” to these than not. For C04107 the length most often linked to the defective form of the gene was called “2”. The 2 marker is most commonly passed on together with a defective ‘c’ form of the gene. But not always!
Complicated mathematics together with pedigree analysis was used. Wherever a biopsied affected dog appears in a pedigree, and given that there is only one gene operating to cause CT, then that animal must have the genotype ‘cc’.
In the majority of cases it was found that ‘cc’ dogs/bitches had two ‘2’ linked markers, i.e. were 2:2 and ‘cc’ but NOT ALWAYS.
From the beginning in the mid 1990s Vetgen cautioned that there were some affecteds which were not 2:2 and cc.
This was thought to be a consequence of the marker being “linked” to the gene as opposed to within it.
The other marker with the different length of DNA was called “1”.
Some of the known affected dogs were 1:1 and ‘cc’ or 1:2 and ‘cc’.

Vetgen therefore cautioned that breeders should continue to BIOPSY to uncover the affecteds which had ‘1’ markers to allow these to be removed from the gene pool.

For the linkage to be good enough to provide a test which will help identify affecteds, carriers and clears in a pedigree the linkage must be shown to be between one type of marker and the defective form of the gene in the vast majority of instances, in this case between the “2” and “c”.

At first this seemed to hold good. Most affected dogs were “2:2”, but not all, some 2:2s were biopsied normal and therefore could not be affected and pedigree/family tree analysis proved some 2:2s were in fact free of CT causing genes.

Most 1:1s were thought to be “clears”, i.e. CC, but increasing numbers of biopsied affected 1:1s have come to light. A biopsied affected 1:1 must be ‘cc’.

Most carriers were thought to be 1:2 Cc, where the 2 and the ‘c’ were linked and the 1 and the C were linked. Again, more and more 1:2 but biopsied affecteds have surfaced over the last
ten or so years.
A 1:2 biopsied affected must be cc, i.e. have two defective copies of a CT producing gene.

A 1:1 biopsied affected must be cc, i.e. have two defective forms of a CT producing gene.

Using the Laws of Genetics, it is categorically correct to say that ALL PARENTS AND ALL OFFSPRING of biopsied affecteds are themselves at least carriers of the defective form of the gene represented by “c”.
ALL have either passed on or received at least one recessive “c” form of a CT causing gene.

Originally the existence of 2 markers linked to “C”s and 1 markers linked to “c”s was explained by pieces of the chromosomes being swapped over while eggs and or sperm were being produced in some ancestors of the dogs concerned. (Swapping pieces of chromosomes is called “recombination”).

Diagram 5 The diagram shows how a white and a black piece of chromosome have swapped over to the opposite chromosomes, a “recombination” event.

In this way it was thought that the linkage between the marker and the gene had changed so that you could have 1 and ‘c’ or 2 and ‘C’.
However, various family trees cast doubt on such swapping (recombination) of genes and markers being the explanation for 1:1 and 1:2 biopsied affecteds.

The discovery of COMMD1.
Some years later saw academic papers out of the University of Utrecht and the University of Alberta announcing the discovery of a gene originally called MURR1, then later re-named COMMD1.

It was further discovered that the linked marker C04107 was inside this gene.

Bart van de Sluis from the University of Utrecht et al published details of a deletion mutation found in the defective form of the gene COMMD1 and stated that this was the gene involved in CT.
AHT developed a method of demonstrating the presence or absence of the deletion and this was applied as a test for CT caused by a defective COMMD1.

Prof. Diane Cox’s paper (University of Alberta in Canada) dismissed COMMD1 as the gene causing CT but little notice was taken of her work.

Breeders now believed dogs with no deletions were “clear” of CT genes, i.e. CC
those with one deletion were carriers of the CT gene, i.e. Cc
and those with 2 deletions were affecteds and had two copies of the CT causing form of the gene, i.e. cc.

The breakages in chromosomes which allow recombination to occur usually occur in the “between genes” regions of chromosomes, it is virtually unknown to have recombination within genes. This therefore ruled out recombination as an explanation for the existence of 1:1 and 1:2 biopsied affecteds.

Where the existing “numbers” were already known ALL 1:1s have had no deletions. All subsequently tested 1:1s have had no deletions.
1:1s do not have deletions!

Not all 1:2s had one deletion, some had no deletions.

Not all 2:2s had two deletions, some had one and some had none.
It was therefore thought that the deletion test identified:
“clear” 2:2 plus C:C
carrier 2:2s, 2 plus C and 2 plus c
and “clear” 1:2s which must have been 2 plus “C” as they did not have any deletions.
The deletion test showed up “2”s which did not have damaged COMMD1s.
Some “2s”are not associated with deletions, they are within fully working COMMD1 genes without deletions!

In conclusion, COMMD1 is not the main CT causing gene.
By applying the facts stated here, try to explain a dog with no deletions which is never-the-less unequivocally biopsied affected if COMMD1 is the CT causing gene. (This dogs’ COMMD1s would have to be normal since they are intact, working forms of the gene, they are undamaged …..do not have the deletion!)

It can’t be done!

Dogs with one or no deletions should have normal livers because when there is no deletion the gene COMMD1 will be working normally.
This 1:1 dog does not have a normal liver.

It has been shown to be affected by liver biopsy.
But its COMMD1 genes are working properly.

This combination is impossible IF COMMD1 is the CT causing gene

The simple and only possible answer is the gene causing CT is not COMMD1 in these dogs.

The actual gene still has to be found, meanwhile the only certain method of knowing a dog has or has not got CT is to biopsy it before breeding from it.

There may be more than one gene involved, the evidence of the huge variation in the different ways the dogs present as phenotypes…(dogs who live with huge amounts of copper with no apparent affect on them, dogs who die young with relatively small amounts of copper, some who may be a bit “sickly/poor doers”), suggests that more than one gene may be involved. As we saw in the case of only two genes this immediately makes things much more difficult to follow through pedigrees in the quest for eradication of CT!!!

The role of COMMD1 is not yet clear.
Prof. Diane Cox believes it may have some modifying effect on the main, as yet undiscovered, CT causing gene or genes.
We will need to be patient a little longer before the full picture emerges and finally achieve a completely foolproof test for any and all CT causing genes!

The “Proper” terminology.
The different forms of a gene are called alleles.
The genes a dog has are called its genotype
The way a dog is observed to be, either by looking or by a form of testing, is called its phenotype.
A carrier is called a heterozygote.
A carrier where there is a dominant gene has the dominant phenotype and is said to be heterozygous normal.
A “clear” has two functioning dominant genes, correctly termed homozygous normal.
The biopsied affected dogs have two recessive, defective forms of the gene (alleles) and are said to be homozygous recessive.
During cell division chromosomes sometimes break and attach to opposite chromosomes. This is called recombination. It occurs in the “between gene DNA”,
the inter-genic DNA , and virtually never within a gene.
A mutation is the name given to changes in DNA or chromosomes, a deletion mutation occurs when a large piece of the DNA disappears somehow, the gene can therefore not carry out its job as it is incomplete, defective, damaged..

A Primer on the Genetics of Copper Toxicosis.

In this primer I have endeavoured only to give the information necessary to understand the thinking on CT and how the view of the genetics has evolved and changed over many years as more information has become available.
(The correct terminology is given once in this format, a further copy onto which you can write the correct terminology appears at the end).

The Basics of Genetics.
All the information needed to build a new puppy, how it should look when it is fully grown and how to make the puppy work as a living being is carried in the DNA, a term everyone these days has heard of.

The DNA is organised into structures called chromosomes which are found in the nucleus of each cell.

Diagram 1. A typical body cell.

Diagram 2. Body cells have 2 matching sets of chromosomes and ______________ – sperm and eggs- have 1 set.
Human body cells contain 46 chromosomes, 22 pairs plus a sex chromosome pair as shown below.

In the dog there are 39 pairs of chromosomes making 78 chromosomes in total. We humans have 23 pairs making 46 chromosomes. The number of chromosomes must remain constant or there may be problems as a result, for example many of the symptoms of Down’s Syndrome are due to the presence of an extra chromosome.
The constant number becomes important when the ______________, eggs and sperm, are made. If they contained all the chromosomes then each would have 78 chromosomes and when the egg was fertilised by the sperm there would then be 156 chromosomes in the cell produced, twice as many as needed!
So, when eggs and sperm are produced the cell divides in a special way which puts one of each chromosome pair into each sex cell, so eggs and sperm have only 39 chromosomes. Which one of each pair gets into a sex cell is completely random.

Diagram 3. It is when an egg and a sperm combine during the process of fertilization that the first cell of a
new individual from which all other cells arise acquires its unique combination of genetic information.

(Gamete is the name given to ______________ and Zygote the name given to the _________ after fertilization.)

Each gene has a job to do. The easiest to understand are genes which control one characteristic such as eye colour. There is a gene for eye colour with a form which can produce brown eyes and a form which can produce blue eyes. We must remember here, though, that very few genes work completely in isolation and the eye colour gene is modified by others which allow, for example, hazel eyes to exist.
One example of a gene which has only two outcomes is that which controls the ability to roll your tongue…can you roll yours? Some people can, some cannot.. try it on some friends! Another is the ability to smell freesias, some people cannot smell freesias.

Many other characteristics, such as height, are controlled by many genes working together.

How single genes work.
Genes work in pairs, one from the dam and one from the sire.
Sometimes a gene gets damaged, what is called a “mutation” occurs. The kind of damage can include losing part of the gene, a ____________mutation.
When a mutation happens this may have the effect of stopping the gene doing its job, a “bad” gene. We could call the working form of the gene the “good” gene.
Sometimes the “bad” mutated gene, which will produce an unwanted, even lethal affect, is what is called a Dominant gene…a bossy gene which always gets its own way about how the puppy will be. This kind of bad dominant gene, because its effect will always show up, usually quickly dies out.
More usually the changed gene is a “recessive” gene and if there is only one the other unchanged, dominant gene will show its effect because it is the bossy type! A dog with one of each type is a _________________.
Only when two of the recessives, the shy retiring types, are present dare they show their effect! An _________________. Only Bedlington puppies with a recessive, damaged copper gene of some kind on both the chromosomes will develop copper toxicosis.
If the puppy has no damaged copper genes at all it will be a “CLEAR”.

In the case of CT there is a “good”/ dominant, and a “bad”/recessive ______________ involved.

Dominant forms of genes are usually represented by CAPITAL letters and recessive forms of genes by small letters.

Letters are used to represent the ________s.

For Copper Toxicosis we might therefore have ‘C’ representing the Dominant ____________, which is in this case the working, normal, _________________.
and ‘c’ representing the recessive _________, the abnormal _________________ which does not work properly, if at all.

Three combinations of the forms of the gene are possible. These combinations are called the “___________”.
CC is used to represent a dog with two dominant forms of the gene.
Such a dog is called by breeders a “clear”. (The correct term is “homozygous normal”).
Cc is used to represent a dog with one Dominant and one recessive _________, it is called a _____________ since it carries the abnormal _____________ hidden by the Dominant one. (The correct name is “heterozygous normal”).
cc is used to represent an “_________________”. (The correct term is “homozygous recessive”.)

If an individual has the genotype CC then all the eggs or sperm made by it will contain the C ____________.
If an individual is Cc then half the eggs or sperm will be C and the other half c.

The possible outcomes in the puppies from a cross of two dogs where only one gene is being looked at can be worked out using a Punnet square to calculate all possible combinations of the eggs with sperm with C representing the dominant characteristic and c the recessive:
Where parents which have two of the same _______________ are crossed a Punnet square is not needed.
Eg.Parents: Sire CC x cc Dam

All sperm from this sire will be ‘C’ and all eggs from the dam will be ‘c’
All puppies in the first generation (F1) will be Cc, they will all be “_________________s”

Cross between two __________s: Sire Cc x Cc Dam

Half the sperm will have a ‘C’ and half a ‘c’.
Half the eggs will have a ‘C’ and half a ‘c’.

Punnet Square.

Sperm

Eggs

These three genotypes will produce two different outcomes in the puppies.
‘CC’ puppies will have completely normal livers and ‘Cc’ puppies(____________s) will also have normal livers as the Dominant, functioning, ___________ is present.
The unlucky ones are the ‘cc’ genotypes as these puppies have two non-functioning forms of the gene (_________________s). Their livers will not be able to do something the liver cells need to do to get rid of any excess copper taken into the body.
These translations of the GENOTYPE into how the liver cells behave produce the P___________. In Copper Toxicosis there is a normal phenotype where the liver cells work to send excess copper out of the body, and an abnormal p__________ where the liver cells cannot somehow do some part of the processing of excess copper leading to it remaining in the liver.
You will see that there are three “normal” phenotypes to one abnormal phenotype. Phenotypic ratio: 3:1

If an individual posses the dominant phenotype they have two possible genotypes, CC or Cc. Test mating using what is called a back cross can predict whether the Genotype is CC “clear” or Cc a “_________________”. There are many different forms of test mating available to use, but none gives 100% certainty that the dog or bitch being test mated is “clear” because of the laws of chance or “Probability”.

Diagram 4. Dominant CC, or as shown above ‘BB’ individuals will always produce offspring with the same kind of phenotype.

Parents: Sire RRYY x rryy Dam
Sperm RY ry Eggs
First generation all RrYy, all _________________s

A cross of two such _________________s: RrYy x RrYy
Would produce possible sperm RY, Ry, rY and ry
And possible eggs RY, Ry, rY and ry

Sperm Eggs	RY	Ry	rY	ry
RY	RRYY	RRYy	RrYY	RrYy
Ry	RRYy	RRyy	RrYy	Rryy
rY	RrYY	RrYy	rrYY	rrYy
ry	RrYy	Rryy	rrYy	rryy

There will be a variety of ___________ produced, some resembling neither parents phenotype.
There are 16 possible combinations of the eggs and sperm involved and when you consider that we are only looking at 2 genes here and in reality hundreds of genes are transmitted together you can see how complex the process of inheritance actually is!

The picture is further complicated by some characteristics being controlled by multiple genes, for example height, and some where the Dominant gene is not totally dominant so that an intermediate offspring is produced, for example some flowers are red and white and produce mixtures of red and white in future generations, but sometimes red and white flowers may produce pink flowers in the next generation, an intermediate of both parents’ phenotypes. Variation also arises from chromosomes lining up in a random order when eggs and sperm are being formed and from genes crossing from one chromosome to another during this process. There are also many characteristics where the environment has a profound effect on the eventual way they appear in the phenotype, e.g. in the case of CT _______________ dogs could appear _________________ when biopsied because their diet has been very low in copper, or they have had de-coppering medication, but the dog is never-the-less genetically an _________________.

In order to use genetics to identify and test for inherited characteristics we must be able to identify specific genes as being responsible for certain characteristics. This is a huge task when you consider the number of individual genes that an organism possesses. It is essential to compare chromosomes from ‘normal’ organisms and those which have two of the ‘defective _____________’ to find the exact location of the gene responsible for a specific characteristic.

How does all this apply to what has been learned about CT over the last twenty years?
Way back in the 1970s experiments were carried out which suggested that CT was caused by a single recessive gene.
Since ______________s can be identified by a liver biopsy even if they are not “sick” it was possible for breeders to remove ______________s as quickly as possible from their bloodlines. This action alone will reduce the number of recessive, non-functioning CT genes in the gene pool markedly and fairly quickly.

_________________s were assumed to be ‘cc’.
The biopsied dogs which were _________________ were, however, either ________________ (Cc) or “clears” (CC). As we have seen it is possible to do special crosses which allow a degree of certainty that the animal “test mated” is a “clear”, i.e. ‘CC’.
However, no-one can ever be 100% certain that the tested dog/bitch is “clear” there is always the possibility of being wrong in that assumption no matter how many puppies are produced that are un_____________. The more un________________ puppies that are produced, the more certain the breeder can be that the animal being “test mated” is probably a “clear”. A number of dogs and bitches were identified as “clears” in this way. The favoured test cross was between a known _______________ ‘cc’ and a biopsied _________________, either ‘CC’ or ‘Cc’.
Other breeders chose different methods of trying to rid their lines of CT genes. Some, sadly, did nothing at all.
Some simply opted for mating their bitches, biopsied or unbiopsied, to “clear” dogs. In some instances known ______________ bitches were mated to dogs claimed to be ‘clear’.

Because there is always a slight uncertainty associated with test mating to ______________s (or to known _______________s) and some were not even biopsied, everyone wanted a completely sure method of identifying __________s and clears. Some would not biopsy their dogs on the grounds that, for example, it was “cruel” and those who do biopsy would always have much preferred a non-invasive method of identifying ______________s and un_________________s, i.e. a method which does not involve liver biopsies.
Mike Herrtage and his team, way back in the late 1980s, proved conclusively that liver biopsy was the only certain method of distinguishing between__________s and un____________s. . Blood test, urine tests etc were all dismissed as not accurate in dogs which were not sick, but never-the-less were “copper toxicosis cases” as proven by their copper values and the changes which were observed in affected livers at the cellular level. Most of the dogs examined then were not sick, as this does not usually appear until the dog is at least 2years old. Biopsy results have been shown not to be accurate until the dog is at least 6 months old. Breeders wanted a method of diagnosing CT at a much earlier age, and most importantly something to identify _________________s and clears.
The hunt was therefore on, for a DNA test.

In the mid 1990s Vetgen in the USA produced a Linked Marker (C04107) Test.
A DNA marker is some piece of DNA which varies in some way in different individuals. A linked marker is assumed to be close to the gene but not within it. In the case of the C04107 linked marker discovered by Vetgen, it differed in having two different lengths of DNA. It was believed to be close to, but not within, a gene causing CT.
The marker, to be useful, must be found to have one form which tracks through pedigrees with a strong degree of “linkage” to the defective _____________ thought to be causing the problem and another form which tracks through pedigrees closely associated with the normal, dominant _____________. The only way to achieve this is by using the biopsy results to conclusively show which dogs in the pedigrees are ________________ and un_________________. In other words, most of the time where a CT biopsied ‘________________’ appears in a pedigree, since we know that animal must have two defective forms of the gene, i.e. be ‘cc’, then one of the two alternate lengths of DNA must more often be found “linked” to these than not. For C04107 the length most often linked to the defective _________ was called “2”. The 2 marker is most commonly passed on together with a defective ‘c’ __________. But not always!
Complicated mathematics together with pedigree analysis was used. Wherever a biopsied _______________ dog appears in a pedigree, and given that there is only one gene operating to cause CT, then that animal must have the genotype ‘cc’.
In the majority of cases it was found that ‘cc’ dogs/bitches had two ‘2’ linked markers, i.e. were 2:2 and ‘cc’ but NOT ALWAYS.
From the beginning in the mid 1990s Vetgen cautioned that there were some _________________s which were not 2:2 and cc.
This was thought to be a consequence of the marker being “linked” to the gene as opposed to within it.
The other marker with the different length of DNA was called “1”.
Some of the known _______________ dogs were 1:1 and ‘cc’ or 1:2 and ‘cc’.

Vetgen therefore cautioned that breeders should continue to BIOPSY to uncover the _________________s which had ‘1’ markers to allow these to be removed from the gene pool.

For the linkage to be good enough to provide a test which will help identify _________________s, _________________s and clears in a pedigree the linkage must be shown to be between one type of marker and the defective ____________ in the vast majority of instances, in this case between the “2” and “c”.

At first this seemed to hold good. Most ______________ dogs were “2:2”, but not all, some 2:2s were biopsied normal and therefore could not be ____________ and pedigree/family tree analysis proved some 2:2s were in fact free of CT causing genes.

Most 1:1s were thought to be “clears”, i.e. CC, but increasing numbers of biopsied _____________ 1:1s have come to light.
A biopsied ______________ 1:1 must be ‘cc’.

Most _____________s were thought to be 1:2 Cc, where the 2 and the ‘c’ were linked and the 1 and the C were linked. Again, more and more 1:2 but biopsied _______________s have surfaced over the last ten or so years.
A 1:2 biopsied ______________ must be ‘cc’, i.e. have two defective copies of a CT producing gene.

A 1:1 biopsied _______________ must be ‘cc’, i.e. have two defective forms of a CT producing gene.

Using the Laws of Genetics, it is categorically correct to say that ALL PARENTS AND ALL OFFSPRING of biopsied _______________s are themselves at least _______________s of the defective _____________ represented by “c”.
ALL have either passed on or received at least one recessive “c” form of a CT causing gene.

Diagram 5 The diagram shows how a white and a black piece of chromosome have swapped over to the opposite chromosomes, a “recombination” event.

It was further discovered that the linked marker __________ was inside this gene.

Bart van de Sluis from the University of Utrecht et al published details of a deletion mutation found in the defective ________________ COMMD1 and stated that this was the gene involved in CT.
AHT developed a method of demonstrating the presence or absence of the deletion and this was applied as a test for CT caused by a defective COMMD1.

Prof. Diane Cox’s paper (University of Alberta in Canada) dismissed COMMD1 as the gene causing CT but little notice was taken of her work.

Breeders now believed dogs with no deletions were “clear” of CT genes, i.e. CC
those with one deletion were _________________s of the CT gene, i.e. Cc
and those with 2 deletions were ________________s and had two copies of the CT causing _________, i.e. cc.

Where the existing “numbers” were already known ALL 1:1s have had no deletions. All subsequently tested 1:1s have had no deletions.
1:1s do not have deletions!

Not all 1:2s had one deletion, some had no deletions.

Not all 2:2s had two deletions, some had one and some had none.
It was therefore thought that the deletion test identified:
“clear” 2:2 plus C:C
_________________ 2:2s, 2 plus C and 2 plus c
and “clear” 1:2s which must have been 2 plus “C” as they did not have any deletions.
The deletion test showed up “2”s which did not have damaged COMMD1s.
Some “2s”are not associated with deletions, they are within fully working COMMD1 genes without deletions!

In conclusion, COMMD1 is not the main CT causing gene.
By applying the facts stated here, try to explain a dog with no deletions which is never-the-less unequivocally biopsied ______________ if COMMD1 is the CT causing gene. (This dogs COMMD1s would have to be normal since they are intact, working forms of the gene, they are undamaged …..do not have the deletion!)

It can’t be done!.

Dogs with one or no deletions should have normal livers because when there is no deletion the gene COMMD1 will be working normally.
This 1:1 dog does not have a normal liver.

It has been shown to be _________________ by liver biopsy.
But its COMMD1 genes are working properly.

This combination is impossible IF COMMD1 is the CT causing gene

The simple and only possible answer is the gene causing CT is not COMMD1 in these dogs.

The actual gene still has to be found, meanwhile the only certain method of knowing a dog has or has not got CT is to biopsy it before breeding from it.

There may be more than one gene involved, the evidence of the huge variation in the different ways the dogs present as phenotypes…(dogs who live with huge amounts of copper with no apparent affect on them, dogs who die young with relatively small amounts of copper, some who may be a bit “sickly/poor doers”), suggests that more than one gene may be involved. As we saw in the case of only two genes this immediately makes things much more difficult to follow through pedigrees in the quest for eradication of CT!!!

The “Proper” terminology.
The different forms of a gene are called alleles.
The genes a dog has are called its genotype
The way a dog is observed to be, either by looking or by a form of testing, is called its phenotype.
A _________________ is called a heterozygote.
A _________________ where there is a dominant gene has the dominant phenotype and is said to be heterozygous normal.
A “clear” has two functioning dominant genes, correctly termed homozygous normal.
The biopsied _________________ dogs have two recessive, defective forms of the gene (alleles) and are said to be homozygous recessive.
During cell division chromosomes sometimes break and attach to opposite chromosomes. This is called recombination. It occurs in the “between gene DNA”, the inter-genic DNA , and virtually never within a gene.
A mutation is the name given to changes in DNA or chromosomes, a deletion mutation occurs when a large piece of the DNA disappears somehow, the gene can therefore not carry out its job as it is incomplete, defective, damaged..