The cause of slow greying

It has been thirteen years since I made a series of posts about a group of late-greying horses. At the time, an unusual PRE stallion, Comico IV, was generating a lot of interest. He was unusual in that, unlike most older greys, he had not yet turned entirely white. The pattern of progression of his greying was also distinctive. 

A good friend reached out to say that late greying and failure to turn completely white are known to occur in Connemaras. She shared pictures she took of her mare’s grandsire, Canal Laurinston. I have run that archived post and one about a Mangalarga with a greying pattern like Comico. I am returning to them because recent studies have discovered why horses like these are different. 

Speed of greying

One reason grey persisted in domestic horses is that people found white horses attractive. In breeds like the Boulonnais and the Lipizzaner, the goal is often a color like the one at the top of this post. One way to achieve this is to breed grey to grey consistently; homozygous greys turned white more rapidly and completely than heterozygous greys.

Other breeders are interested in a slower change. Instead of white, the desired phenotype is often the attractive dappling pattern seen on this Eriskay Pony. Unlike white-grey, getting greys that stabilize at this point is impossible. However, horses that take longer to turn white spend more time in the dappled phase. Here, the opposite is true. Crossing to non-greys produces horses that take longer to turn white. 

This means that grey has a dosage effect: two copies have a stronger effect than one.

But what about horses like the Connemaras? Earlier studies had shown that the slow greys and all other greys had the same mutation. In fact, because the gene where the mutation was found, STX17, had not previously been implicated in changes in pigment, transgenic zebrafish were created to test the idea. Since there seemed little doubt scientists had the correct mutation for grey, and the slow greys tested as having that mutation, the assumption was that some other change in their DNA was modifying the grey.

Advances in technology

The slow greys did have a somewhat different form of STX17. To explain what is different, and why it wasn’t apparent at first, it helps to know a little about the physical nature of the mutation for grey. There are many ways to change – to mutate – DNA. If you think of DNA using the familiar analogy of computer coding, then the possibilities for change are similar to how you might change text. You could swap out a letter, delete portions, or insert a carriage return that truncated a sentence. You could also duplicate portions of the text. That’s the type of change that makes a horse grey.

Grey horses have a portion of their STX17 gene duplicated. That portion is repeated twice, for a total of three copies. To help you visualize this, here are the three different options for traditional greying in horses: non-grey, heterozygous grey, and homozygous grey.

STX17 with the "fast grey" mutation

Researchers have known that grey was caused by repetition in STX17, but the fact that it was a tripling of that segment is a recent discovery. That’s because the technology for finding repeating portions of the DNA is relatively new. Getting a precise count of copies is newer still. Using that technology, researchers found that the standard form of grey gives a horse a total of four copies (heterozygous) or six copies (homozygous) of the relevant segment. The more copies the horse has, the more rapidly (and completely) it turns white – and the greater the horse’s risk for developing melanomas. That’s why homozygous greys are more likely to develop cancer than heterozygous greys.

What makes the Connemaras and other late greying horses different is that they have two repetitions rather than three. A horse heterozygous for this alternate form of grey has three copies, while a homozygous one has four. Like heterozygous horses with traditional grey (Gg), they have fewer copies of the segment and, therefore, turn white more slowly and have a reduced risk for melanoma. 

Three variants at STX17

This discovery means there are three different options for grey in horses: non-grey (g1), slow grey (G2), and fast grey (G3). Non-grey is the wild, unmutated version.

Fast grey (G3) was the original mutation, and slow grey (G2) was a later change to it. It is thought that slow grey arose when one of the duplications was lost, and this occurred relatively recently. (In contrast, G3 is known to be an ancient mutation.) The slow grey variant (G2) was found in a family of Connemaras descending from Hagen’s D’Arcy. He carried one of each variant (G2/G3) for a total of five copies of the segment. With that many copies, he greyed out in the traditional fashion, which is apparent in his video below.

However, a portion of his offspring were slow greying. It seems likely this came from his sire, Hagens O’Chief. In the link, you can see how dark he was nine years old. His photos as an aged stallion have that same unusual not-quite-white color that Canal Laurinston had. O’Chief’s dam Ashfield Fancy is listed as bay, but this photo shows she was likely another late grey. There are more photos of members from this family in the extended data from one of the papers published on the discovery. A Japanese Thoroughbred family was also included in the study but was not identified by name. 

 Testing for copy numbers determined that slow greying was also present in at least seven other breeds. These included the Andalusian, Miniature Horse, Marchador, Mustang, Quarter Horse, Walking Horse, and Welsh Pony. It is particularly interesting that the Andalusian and the Marchador were among the breeds where the unusual slow greys were first identified. Although the identities are not given in the supplemental information, it does seem likely that the Comico (PRE) and Unico (Marchador) lines might be the source of those samples.

What this says about variation in greys

The idea that differing copy numbers change whether the hair turns white offers a possible explanation for some variations among grey horses. One researcher suggested that fleabites could come from a somatic loss of one of the copies. (Somatic changes occur in an animal’s body cells rather than its reproductive cells.) That would explain why heterozygous greys are far more likely to be fleabitten than homozygous greys. The cells of a heterozygous grey have fewer overall copies, so losing one is more likely to change the outcome.

It is also true that mutations involving tandem repeats like this are unstable. If you look at the three different variants, the repeating section changes the length of the STX17 gene. That means that when the cells replicate, the two genes aren’t always going to be perfectly aligned. This can lead to mistakes where an extra duplication is lost or gained. That instability and the fact that copy numbers change the rate of change - and the endpoint - might explain timing variations like bloody shoulders and chubari spots.

Further reading

If you would like to read more about these discoveries, here are links to the studies.

White horses – non-coding sequences drive premature hair greying and predisposition to melanoma

Of the articles discussing slow-greying, this is the one I would recommend for a good overview. The language is pretty straight-forward for a scientific journal.

An intronic copy number variation in Syntaxin 17 determines speed of greying and melanoma incidence in Grey horses This pre-press article has images of the Connemaras and Thoroughbreds in the supporting documents. It is has more detail and is more technical than the previous article.