testing for equine genetic disorders. what, why & how?

Testingfor

Equine Genetic Disorders

What, Why and How?©2016 Arreton Arabians

©2016 Arreton Arabians

Genetic Disorders• are the result of inheritable abnormalities (mutations) in a

horse’s genes

• we will be discussing disorders that result from the mutation of single genes (defined sections of chromosomes)

Chromosomes occur in pairs (except in sperm and ova), and are strings of genes (thus, genes occur in pairs too).

Genes are made up of DNA

At conception, the embryo receives one half of each pair of chromosomes from the sire and the other half of the pair from the dam


Sperm + egg = zygote embryo

Homo means ‘same’ Hetero means ‘different’

Homozygous means that the zygote received two identical copies of a gene (one from the sperm and the other from the egg). They may be two mutant copies, or two normal copies of the gene.

Heterozygous means that the zygote received one normal copy of the gene from one parent and one mutant copy from the other parent.

Some definitions


Genetic Disorders can be autosomal and recessive (autosomal means that the

gene responsible is present on any chromosome other than the X/Y chromosomes)

autosomal and dominant

sex-linked (the gene is located on the X/Y chromosomes)

I don’t know of any sex-linked disorders that have been identified in the equine, but a human example is Haemophilia; which results in failure of the blood to clot.


Autosomal Dominant Disorders

• A dominant mutation does precisely what we should expect: it dominates

• Even if a horse inherits one normal copy of the gene, and only ONE mutant copy, the

mutation will dominate and the horse will be affected by the condition.

• Some conditions are termed ‘incomplete dominant’. In these cases, a homozygous (having two mutant copies of the gene) horse will be more seriously affected by the condition than a heterozygous horse (who has one mutant copy and one normal copy of the gene).

• Examples of dominant genetic conditions of horses are MH (Malignant Hyperthermia) HYPP (Hyperkalemic Periodic Paralysis)


Autosomal Recessive Disorders

• For a horse to be affected by an autosomal recessive genetic disorder it has to inherit

TWO mutant copies of the gene responsible (one copy from each parent).

• Heterozygous horses (who have one normal copy and one mutant copy) are unaffected by the condition because any effect of the mutation is suppressed by the normal copy of the gene. These horses are also called ‘Carriers’ – they carry the mutation, but it is not expressed.

• Carrier horses are physiologically normal and exhibit no symptoms of the disorder.

• Examples of recessive autosomal conditions are Lavender Foal Syndrome (LFS) Hereditary Equine Regional Dermal Asthenia (HERDA)


Inheritance of Autosomal Recessive Conditions (1)

• Is independent of the gender of the progeny.

• An affected parent (carrying two mutant copies of the gene) will always pass on the mutation to every one of its progeny.

• Every time a carrier (one normal copy of the gene and one mutant copy) is mated there is a 50% probability that the mutation will be inherited by the resultant foal.


Inheritance of Autosomal Recessive Conditions (2)• If a carrier is put to a mate that is clear (has two normal copies of the gene), the

progeny will always be either clear or carriers; never affected. No problem.

• If a carrier is bred to a carrier there is a 25% chance that the foal will be affected by the disorder (inherit two mutant

copies of the gene). BIG problem. a 50% chance that the foal will be a carrier (inherit a mutant copy of the gene from

one parent, and a normal copy from the other). a 25% chance that the foal will be clear of the disorder (inherit a normal copy of

the gene from each parent).

• It is important to understand that every mating of carrier to carrier has a 25% chance of resulting in an affected foal. If one affected foal is bred, it does not mean that the next three foals from the same mating will be unaffected or carriers.


Inheritance of Autosomal Recessive Conditions (3)

N = normal copy of the gene n = mutated copy of the gene

NN = unaffected horse Nn/nN = carrier nn = affected horse

Carrier Sire Carrier DamNn Nn

Possible progeny NN Nn nN nn

Potential progeny from each mating:1 in 4 (25%) chance of a homozygous normal foal2 in 4 (50%) chance of a carrier1 in 4 (25%) chance of an affected foal


Genetic conditions that can be tested for include:Condition Abbreviation Dominant/Recessive Breed first identified in

Malignant Hyperthermia MH Autosomal Dominant Quarter Horse

Polysaccharide Storage Myopathy Type 1 PSSM1 Autosomal Dominant Several

Foal Immunodeficiency Syndrome FIS Autosomal Recessive Fell Pony

Overo Lethal White Syndrome OLWS Autosomal Recessive Several

Cerebellar Abiotrophy CA Autosomal Recessive Arabian

Severe Combined Immune Deficiency SCID Autosomal Recessive Arabian

Lavender Foal Syndrome LFS Autosomal Recessive Arabian

Hereditary Equine Regional Dermal Asthenia HERDA Autosomal Recessive Quarter Horse

Hyperkalemic Periodic Paralysis HYPP Autosomal Dominant Quarter Horse

Glycogen Branching Enzyme Deficiency GBED Autosomal Recessive Several

Hoof Wall Separation Disease HWSD Autosomal Recessive Connemara

Junctional Epidermolysis Bullosa JEB1/JEB2 Autosomal Recessive Draught Horses/Saddlebreds

Friesian Dwarfism Autosomal Recessive Friesian

Friesian Hydrocephalus Autosomal Recessive Friesian

Warmblood Fragile Foal Syndrome WFFS Autosomal Recessive Warmblood


Why test for recessive conditions?

• Are you willing to take the risk of breeding an affected foal?

• If you test your breeding stock and avoid mating carrier to carrier you will never run that risk.

• Don’t be complacent: the mutant genes are not restricted to the breeds in which they were first identified. CA was, for example, found in three breeds tested as part of a study: a Welsh Pony, two Trakheners, and 3 Bashkir Curlies

Why would you not test?????????


Who to test, and for what?• Genes do not discriminate. There is the same probability of a mutation being

inherited by the foal, no matter the breed of the other parent.

• The mutations responsible for LFS, CA and SCID were first identified in Arabians but are not restricted to that breed. Arabians have been used to develop and strengthen many breeds, all of which may carry the mutations.

• Test your own breeding stock, and when considering a potential mate for a carrier, check the extended pedigree and consider the possibility that they may be a carrier of the same condition. If the owner of the prospective mate refuses to disclose test results or to permit testing, you might consider looking elsewhere.

• Carrying a recessive disorder does not affect a horse’s conformation, temperament or performance, and should not be considered shameful, nor should the horse be considered unworthy, BUT they should be bred responsibly.


How to test• Contact an accredited testing laboratory that offers the tests you require.

• Usually they will require about thirty hairs pulled from the mane or tail, which must have the hair root attached. If you pull the hair straight out, the root will almost always come with it. Cut hair is of no use for testing.

Hair shaft (contains very little, if any, DNA)

Hair root (lots of cells containing DNA)


What happens to the hair when it gets to the testing lab? (1)

• DNA is extracted from the hair root, not from the hair itself, which is why it is important to ensure that you pull hairs cleanly and ensure that the roots are attached.

• The extracted DNA contains every gene that the horse from which the hair was pulled carries.

• The extracted DNA is mixed with a variety of chemicals, at least two of which are uniquely matched to the gene being investigated, and a dye that makes the gene fluoresce.


What happens to the hair when it gets to the testing lab? (2)

• During repeated cycles of heating and cooling, the gene of interest is copied multiple times if it is present in the sample, to generate enough material to be analysed.

A thermal cycler. The samples are in the tiny tubes

• Tests are designed so that both the normal and mutated gene of interest will be copied if they are present.

• Tests are also designed so that the size of the copies of the normal gene is different to the size of the copies of the mutant gene.


What happens to the hair when it gets to the testing lab? (3)• Next, the mixture, containing copies of the normal and defective genes (if present), is

applied to a thin slab of gel, and an electric current is applied

An electrophoresis tank. The samples are added to small wells at the top of the gel and an electric current applied.

• The copied genes migrate through the gel under the influence of the electric current, separating out according to size.

• The smaller the size of the copied gene, the faster and further it moves through the gel

• The DNA fluoresces and is visible under UV light


Interpreting Test Results

Stylised image of a DNA gel exposed to UV light

normal gene

Defective or mutant gene

Unaffected: Carrier: Affected:Normal gene only Normal and mutant gene Mutant gene only


Genetic disorders could be excluded from the gene pool if breeders

excluded carriers from their breeding programmes BUT• Carriers of autosomal recessive conditions are physiologically

normal and only identified as being ‘flawed’ if they are subjected to genetic testing (or breed affected progeny).

• Many fine examples of true to type and form mares and stallions would then be excluded from breeding programmes, and already limited gene pools would become even more so.

• As more and more genes are identified it is possible that we will eventually find a genetic ‘flaw’ in every horse tested.

Why Breed From Carriers of Recessive Disorders?

Waiting for the perfect horse?


Responsible Breeding of Carriers of Autosomal Recessive Conditions:

• Subject breeding stock to genetic testing for all relevant conditions.

• Do not ever breed a carrier to another carrier of the same genetic disorder.

• Do not assume that conditions such as SCID and LFS are restricted to Arabians. The original mutations occurred many generations ago and Arabians have been used to improve several breeds since then.

• Owners of carriers should ensure that any prospective breeding partners are tested and found clear, no matter what breed they may be.

testing for equine genetic disorders. what, why & how?

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