Scientists first discovered the mutation that makes horses turn gray 13 years ago on a color-processing gene called syntaxin 17 (STX17) on Chromosome 25. Initial analyses suggested the gene mutated by “reprinting,” so to speak, an entire section of STX17. Using more modern technology, however, Polish researchers have determined this particular section doesn’t just get reprinted once, but twice—giving horses three copies of that genetic material on the same gene instead of one.

“This means that the tested mutation is a triplication rather than duplication, as was suggested before,” said Jakub Cieślak, PhD, of the Poznan University of Life Sciences, in Poland.

Previous study results have shown the gene—which manages a horse’s pigmentation–doesn’t function as it normally would when it has repeated sections.

How Genes—Including STX17—Mutate

Genes can mutate in different ways—sometimes by switching out certain lines of genetic material (base pairs), sometimes by copying a section of base pairs and tacking them onto the gene’s lineup of base pairs, and sometimes by deleting an entire section of base pairs.

In gray horses, the mutation is the kind where genetic material gets copied and tacked onto the long line of base pairs that make up the gene. Specifically, a very large section—a fragment measuring 4,600 base pairs long—of STX17 repeats, making the gene that much longer. Repeating a section of a gene can confuse it, in a sense, and alter normal gene expression. In this case that relates to coding for coat colors. Horses without the mutation develop gray hairs when they start getting old—like most humans. But horses with the mutation lose their color prematurely (even as young as 2 or 3 years old), and they gradually turn grayer as they age. By age 15, many of these horses—especially those that inherit the mutation from both parents—are entirely white, and most have also developed melanoma skin tumors, a likely consequence of their lack of pigmentation.

Using Modern Technology To Find STX17 Mutations

Unfortunately, testing for STX17 mutations isn’t easy, which can be frustrating for scientists and breeders alike. Although laboratories regularly test for many genetically inherited conditions in horses, the graying-with-age mutation has been challenging to analyze using standard techniques, mainly because the repeat section is so long and molecularly complex, Cieślak said.

Eager to find a better way, Cieślak and his fellow researchers tried a newer, digital technological process called droplet digital polymerase chain reaction (ddPCR). They used the technique to examine the STX17 mutation in 75 horses—including grays and nongrays—representing 12 breeds.

DNA analyses from the horses’ hair or blood using ddPCR easily picked up genotypes in both homozygous (mutation inherited from both parents) and heterozygous (mutation inherited from only one parent) gray horses, Cieślak said. The ddPCR technique also made it much easier to distinguish homozygous horses from heterozygous ones, compared to conventional polymerase chain reaction techniques.

Surprise Finding: STX17’s “Gray” Fragment Repeats Not Once, But Twice

During their study, the researchers stumbled across a “surprising” discovery, said Cieślak. They found that homozygous horses had six STX17 fragments that get repeated in the mutation—three for each of the horse’s two Chromosomes 25. (Chromosomes come in pairs.) Heterozygous horses had the fragment four times, and nongray horses only had their original two (one on each Chromosome 25)—meaning it hadn’t been repeated.

Surprised by their findings, the team ran additional and more complex tests, all of which confirmed their discovery, he said.

The results could lead not only to better identification of horses likely to gray early or pass on gray-with-age genes but also to a better understanding of how the mutation affects horses’ health.

“Since the tested variant is located within the regulatory region affecting gene expression and is associated with predisposition to melanoma tumors development, our results may be a good introduction for further functional studies on this topic,” Cieślak said.