Horse Genetics

Racing Australia monitors developments in Thoroughbred breeding trends to ensure that breeders are kept informed of important findings.

This paper describes the discovery of two very rare, but fatal, genetic disorders in Thoroughbred horses in other parts of the world.

This report was compiled by the Australian Stud Book and Equine Genetics Research Centre and supported by the Racing Australia Research & Development Fund.

You can also find a downloadable Fact Sheet and a link to a short presentation below.

Genetic disorders in horses

Recently, two fatal recessive genetic disorders have been identified in Thoroughbred horses in other parts of the world. In this document, we provide the background information and tools you might need to ensure you do not encounter an affected foal. To understand the impact of these disorders, you do not need to be an expert in genetics.

What is DNA & genes?

DNA is a molecule found in most cells of living things. Genes are sections of DNA that tell the body how to make proteins. Genes occur in matching pairs, with one of each pair inherited from each parent. Consequently, most cells carry two copies of most genes.

What is DNA variation?

Whilst most DNA is identical across horses, it is normal that there is also some variation in its sequence. A lot of this variation has no observable effect. Other variants contribute to differences between individuals that we can see, like coat colour, size and speed.

However, some DNA variation can be harmful and cause illness. Genetic disorders can occur when something goes wrong with the DNA sequence in a gene.

What are ‘recessive’ genetic disorders?

Some variants only cause illness if both copies of the gene are affected. In these cases, there is no noticeable effect if a horse has only one copy of the harmful variant. There is only an impact if the foal inherits the harmful variant from both its parents, meaning it has two copies. Because the illness appears only sporadically when two carriers of the harmful variant are mated, these disorders are considered hidden or ‘recessive’ genetic disorders.

By the time we become aware of these variants, they are often already carried by a significant number of horses. Once they are established in the population, these variants are often called mutations.

The next section describes the two recessive genetic disorders that have been documented in Thoroughbreds in the US and UK.

Fragile Foal Syndrome 1 (FFS1)

FFS1 prevents the normal formation of collagen. Affected foals have hyperextensible (lax and stretchy) leg joints and very fragile skin which tears easily. The condition is obvious at birth and newborn foals are immediately euthanised as it is untreatable. FFS1 is also suspected to cause early embryonic loss so it may manifest as poor fertility. More research needs to be done to confirm this.

The DNA mutation causing FFS1 was originally identified in 2013. Foals need to inherit a copy of the mutation from both of their parents to be affected by the disorder. Horses with only one copy of the mutation are not affected.

This disorder was originally called “Warmblood Fragile Foal Syndrome” because it occurred most frequently in Warmbloods. Between 11 and 18 percent of Warmbloods are carriers. However, it is now known that many breeds carry the mutation (Reiter et al. 2021). The latest studies have shown that the FFS1 mutation is found at lower frequencies (17/716; 2.4% and 25/1789; 1.8%) in American Thoroughbreds (Bellone et al. 2019; Elcombe et al 2022).

The EGRC tested anonymous samples from 550 Thoroughbreds for the FFS1 mutation, finding 7 carriers. The Australian carrier frequency of 1.3% is considerably lower than that observed in US Thoroughbreds.

A case of an affected Thoroughbred foal born in the UK has recently been documented (Grillos et al. 2021) so it has since been proposed the disorder should be re-named Fragile Foal Syndrome Type 1.

Equine Familial Isolated Hypoparathyroidism (EFIH)

EFIH prevents the foal from using calcium properly. This causes low calcium in the blood, resulting in painful muscle contractions, seizures, and eventually death. Foals with this disorder are born without parathyroid glands, which is why their bodies can’t process calcium.

The disorder has been fatal in all documented cases so far (n = 9). The symptoms of EFIH are unspecific, so it is possible that foals have been born with EFIH in Australia, but the cause was not recognised.

The mutation causing EFIH was identified in 2020 and affected foals must inherit a copy of the mutated gene from each parent (Rivas et al. 2020).

Unlike FFS1, EFIH has only been identified in Thoroughbreds. The mutation was first published in 2020, where it was found in 3/82 randomly selected Thoroughbreds (3.7% carrier frequency). A larger study found that 28/1789 (1.6%) Thoroughbreds were carriers (Elcombe et al 2022).

Testing of 880 anonymous Thoroughbreds by the EGRC found that 12 carried the mutation (1.4% carrier frequency).

Conclusions

The FFS1 and EFIH DNA mutations are present in the Australian Thoroughbred population. However, both mutations are found at very low frequencies.

There is no observable effect of these variants on horses that carry one copy and there is nothing wrong with horses that carry these DNA mutations.

The EGRC DOES NOT SUPPORT the prohibition of carriers from breeding. These horses only have a problem with one copy of one gene. If they have made it to the breeding population, they clearly have more than 20,000 other perfectly good genes so they should not be prevented from mating because of these DNA variants with hidden effects.

The only danger is if two carriers are mated, there is a one in four chance of the resultant foal being affected by the disorder. Based on the published frequencies, we would expect to see one foal affected by either FFS1 or EFIH born every two years. Therefore, these disorders are very rare.

What to do if you are concerned your horse is a carrier

Contact the EGRC (geneticslab@racingaustralia.horse) if you suspect you have an affected foal. The EGRC will provide free DNA testing for the foal and any available parents in these cases. All enquiries and DNA test results will be kept confidential. You do not need to provide the names of the parents of the foal.

A foal with suspected FFS1 will likely require immediate euthanasia. The EGRC can arrange with the attending veterinarian what sample would be the most suitable to take from the foal to confirm diagnosis. Blood is the best sample (ideally in an EDTA (purple) vacutainer; although LithHep (green) also works), but we can work with what you are able to obtain at the time.

DNA testing can also confirm a definitive diagnosis of EFIH, allowing for informed decision making.

The EGRC offers routine DNA testing for these disorders. DNA testing costs $48 (incl GST) for one disorder and $65 (incl GST) for both. Tests for Thoroughbreds can be ordered by emailing the EGRC (geneticslab@racingaustralia.horse).

For the 2023 breeding season, the EGRC is offering free testing of mares that have proven difficult to get in foal, lost foals for an undiagnosed reason, or have had foals that may have been affected by either FFS1 or EFIH. Contact the EGRC if you have a mare that fits any of these criteria and you would like to have them tested. This testing is sponsored by the Racing Australia Research and Development Fund.

Please contact the EGRC if you have any other concerns about these disorders.

References

Reiter et al. (2021). Distribution of WFFS1 mutation (PLOD) in different breeds from Europe and US. Genes 11:1518 doi.org/10.3390/genes11121518.

https://www.mdpi.com/2073-4425/11/12/1518

Bellone et al. (2019). Warmblood fragile foal syndrome type 1 mutation (PLOD1 .2032G>A) is not associated with catastrophic breakdown and has a low allele frequency in the Thoroughbred breed. Equine Veterinary Journal 52:411-414. DOI: 10.1111/evj.13182

https://beva-onlinelibrary-wiley-com./doi/pdf/10.1111/evj.13182

Elcombe et al. (2023). Prevalence of the RAPGEF5 c.2624C>A and PLOD1 c.2032G>A variants associated with equine familial isolated hypoparathyroidism and fragile foal syndrome in the US Thoroughbred population (1988–2019). Equine Veterinary Journal 55:666–671. DOI: 10.1111/evj.13883. Not available without subscription.

Grillos et al. (2021). First reported case of fragile foal syndrome type 1 in the Thoroughbred caused by PLOD1c2032GA. Equine Veterinary Journal 54:1086-1093. DOI: 10.1111/evj.13547. Not available without subscription.

Rivas et al. (2020). A nonsense variant in Rap Guanine nucleotide Exchange Factor 5 (RAPGEF5) is associated with equine familial isolated hypoparathyroidism in Thoroughbred foals. PLoS Genetics. 16:e1009028. doi.org/10.1371/journal.pgen.1009028

https://journals.plos.org/plosgenetics/article/file?id=10.1371/journal.pgen.1009028&type=printable

The base coat colours of the horse are chestnut, bay and black. All other colours and patterns are created by genes that modify or add some form of white pattern to the base colours.

Coat colour is determined by the relative amounts of the pigment’s eumelanin and pheomelanin, which are both types of melanin. Eumelanin is a dark pigment responsible for the black coat colour and brown based hues, while pheomelanin is red and in horses causes the chestnut coat colour.

The amount and distribution of these two pigments on the coat is determined by two genes: ASIP and MC1R.

Firstly, we will consider chestnut coat colour, which is determined by the gene called MC1R, of the melanocortin 1 receptor. The symbol for the alleles of this gene are ‘E’ and ‘e’. E stands for extension, one of the alternate names for this genetic test, which is also sometimes called red factor or red/black.

Chestnut horses have a single base change in both copies of their MC1R gene that triggers the pigment cells to only produce phaeomelanin. Horses carrying two copies of this recessive ‘e’ variant will only have red pigment and will be chestnut. This action is independent of the ASIP gene. This horse would have the genotype ‘ee’ at this locus.

A research paper in 2000 reported on a second chestnut allele. It is called ‘e^a’ and acts in the same way as the normal e allele does. It is located only a couple of basepairs downstream of the e allele, so it is linked to it. It is also very rare, only being identified in a few breeds including German Black Forest horses. Because it is linked to the normal e allele, we don’t offer a test for this specific variant.

If the horse is not chestnut, it must have one or two ‘E’ alleles, allowing the cells to produce eumelanin. This permits the presence of black or dark pigment in its coat. The distribution of black is dependent on another gene called agouti-signaling protein (ASIP), which is often just called agouti. The two variants of agouti are ‘A’ and ‘a’.

If a horse has one or two ‘A’ alleles, the black colouring is restricted to the points of the horse, and it is bay. The genotype of a bay horse is E-/A-. The dashes indicate unknown alleles. If the horse is not chestnut, and only has the recessive ‘a’ ASIP allele, it will be black. Its genotype will be E-/aa.

While we have a good understanding of how these base colours are inherited, there is still a lot that is unknown. Genetically speaking, there are no brown horses, they are either dark bay or faded black. So why do some horses look brown? Recent research has indicated that there are other genes that can alter the base colours to create dark bay and liver chestnut. This will be discussed in another article.

Why would you order these tests?

You may not be able tell your horses base coat colour if your horse is grey, double dilute, or has a white spotting pattern covering much of its coat. In that case, you would order both tests. Alternately, you might be interested to know if your chestnut horse can have a black foal (with the agouti test), if your black horse can have a chestnut foal (with the red factor test), or if your bay horse can have non-bay coloured foals (using both tests).   

The base coat colours: A bay horse with a red coat and black legs, mane and tail (E-/A-) on the left; a chestnut horse with a completely red coat (ee/--) in the centre, and a black horse (E-/aa) on the right.

Here you will find definitions for many commonly used genetic terms

Allele

Changes in the DNA sequence cause a different version of a gene, called an allele. Alleles are also often called variants.

Amino Acid

Amino acids are organic compounds made of nitrogen, carbon, hydrogen and oxygen. They are the basic unit of proteins. The DNA sequence in a gene encodes a string of amino acids which will undergo modifications to become a protein.

Autosome

A chromosome that is not a sex chromosome. Each horse normally has 31 pairs of autosomes, with one of each pair inherited from each parent. When the term autosomal is used to describe the mode of action of an allele, it means the allele is not sex-linked.

bp (base pair)

DNA is made up of a sugar phosphate backbone (string) and four nucleotides (bases) called adenine (A), guanine (G), cytosine (C), and thymine (T). DNA is double stranded so these bases are counted in pairs.

Carrier

A carrier of a genetic disorder has one copy of a recessive allele. The allele is recessive, so the horse does not show any signs of the disorder, but ‘carries’ the allele and can pass it on to their offspring.

Chimera

When two fertilised eggs fuse to create a single animal, that animal will have one set of cells with one set of DNA mixed in with other cells that contain a different set of DNA. Chimerism is extremely rare.

Chromosome

DNA is arranged in long strings called chromosomes. Each horse normally has 31 pairs of autosomes and two sex chromosomes, either XX (in females) or XY (in males).

Co-dominance

Because most genes occur on autosomes, each horse has two copies (alleles) of most genes. If the different alleles of a gene are co-dominant, it means that both exert their full effect on the animal. An example of co-dominance is in people of the AB blood type. They have alleles for both the A and B blood type and both are expressed.

Complex trait

A trait that is influenced by both genetic and non-genetic (environmental) effects.

Compound heterozygote

A compound heterozygote has two different alleles on the same gene. An example would be a horse that is SW1/SW5. They carry one copy of SW1 (a 10 base pair insertion in the MITF gene promotor) and one copy of SW5 (a deletion of 63,000 bases from the MITF gene). They have no copies of the wild type gene.

DNA

Deoxyribonucleic acid (DNA) is a molecule that contains hereditary material that is passed down generations. DNA is made up of a sugar phosphate backbone (string) and four nucleotides (bases) called adenine (A), guanine (G), cytosine (C), and thymine (T). DNA is double stranded.

DNA sequence

The order of nucleotides on a DNA strand.

Dominance

Because most genes occur on autosomes or the X chromosome, each horse has two copies (alleles) of each gene. Dominance is when one allele masks the effect of the second.

Embryonic lethal

A genotype that stops the embryo from developing is called embryonic lethal.

Epigenetics

Heritable changes due to modification of gene expression, for example through the addition of chemical groups or changes in tertiary structure of DNA.

Epigenome

All the modifications to the DNA that regulates the activity (expression) of all genes.

Exon

A gene is a DNA sequence that contains the instructions to create a protein. Often the instructions are not continuous, so genes are made up of coding regions called exons interspersed with non-coding regions (introns).

Gene

A sequence of DNA that contains the instructions to create a protein.

Gene expression

When a gene is turned on to make a protein. First the gene sequence is transcribed into RNA, then translated into a string of amino acids which are modified to create the protein.

Genetics

Genetics is the study of heredity, the process by which parents pass information onto their offspring. Genetics is also the study of the characteristics of particular genes.

Genome

All of the genetic material in an organism.

Genomics

The study of all of the genetic material in an organism, often in more than one individual.

Genotype

A description of the specific gene variants carried by in an animal.

GWA

Genome wide association analysis. A scan of the whole genome to identify regions associated to a particular trait.

Haplotype

A combination of alleles that are inherited together, usually because they sit close to each other on a chromosome.

Heredity

The reason for the similarity between parents and their offspring. The transmission of characteristics down generations.

Heterozygous

Having two different alleles at a particular locus or place on the genome.

Homozygous

Having the same alleles at a particular locus or place on the genome.

Incomplete dominance

Incomplete dominance is when an allele has a different effect when there are different numbers of the allele. It only has a partial effect if there is one copy compared to two. Cream dilution is an example of incomplete dominance.

Incomplete penetrance

If an allele has incomplete penetrance, not every animal with that allele will express its full effects. Some animals with the allele may never show signs of that trait. An example is the disorder MYHM.

Intron

A gene is a DNA sequence that contains the instructions to create a protein. Often the instructions are not continuous, so genes are made up of coding regions (exons) interspersed with regions called introns that do not code for protein.

Locus

The location of a particular marker or gene on a chromosome.

Marker

A DNA sequence (like a gene) or variant which has a known location.

Microsatellite

Microsatellites are areas of very short (1, 2, 3 or 4 bases) repeat sequences, e.g. CACACACACA. The numbers of repeats are highly variable and can be traced from parents to offspring. Microsatellite markers (also called short tandem repeats) are the markers analysed when creating a DNA profile or in parentage verification.

Mode of Inheritance

The way a trait is inherited and its action; eg autosomal recessive.

Modifier

A gene that modifies the action or product of another gene.

Mutation

A variation in the nucleotide sequence of DNA compared to the wild type (reference) sequence. The term mutation is often associated with a disease variant.

Non-coding DNA

Intergenic (between genes) and intronic (within genes) DNA that is not translated into a protein. It is assumed to have other functions to regulate gene expression.

Nucleotide

There are four types of nucleotides (bases) called adenine (A), guanine (G), cytosine (C), and thymine (T) that make up part of the DNA strand. The arrangement of these nucleotides in order is called the DNA sequence.

Phenotype

The appearance of the animal, what is observed. This includes behaviour and physiology as well as physical appearance.

Pleiotropy

When a single gene affects multiple phenotypic characteristics. An example is some Splashed White mutations which can affect both coat colour and hearing.

Promotor

Sequence at the beginning of a gene that contains motifs to which proteins bind to and turn on translation of the gene into RNA.

Polymorphism

A variation in the nucleotide sequence of DNA compared to the wild type (reference) sequence. The term polymorphism is often associated with a benign variant that doesn’t cause disease.

Recessive

One of the modes of action of an allele. A recessive trait needs two copies of the recessive allele to produce (express) the phenotype. For example, the chestnut coat colour is recessive to bay.

RNA

Ribonucleic acid (RNA) is similar to DNA but is a single stranded molecule. RNA contains uracil (U) instead of thymine (T). When a gene is switched on, the DNA in the gene is transcribed into a string of RNA, then RNA is translated into a string of amino acids before being processed into a completed protein.

SNP

Single nucleotide polymorphism. A type of mutation consisting of a single letter change in DNA sequence.

Transcription

The creation of RNA from DNA.

Translation

The creation of a protein from an RNA strand. First the RNA is translated into a string of amino acids, then this is processed and packaged into a mature protein.

WGS

Whole genome sequence. The whole genome sequence of a horse is approximately 2.7 billion base pairs long.

Wild-type

The wild type version of a gene is the original version that existed before a mutation occurred. It is the ancestral version.