One in al million - An incredible genetic circumstance creates a unique DNA puzzle to solve
"Excluded". For the second time, Denise Charpilloz had sent in hair from her 2004 foal out of her mare Sharp One for DNA testing and parentage verification. And for the second time, the Veterinary Fenetics Laboratory at the University of California-Davis excluded not only Dunbars Gold as the foal's sire, but also Sharp One as the foal's dam. Every now and then, people make mistakes when they submit mane or tail hair samples for DNA testing on al foal. It usually happens when people accidentally mix hair if they're collecting samples from more than one horse. Although very rare, sometimes mares can actually switch foals of the wrong stallion's se´men is unintenionally shipped and used to breed a mare. All those scenarios result in a DNA test that "excludes" a mare or stallion as a foal's parent. Usually, it doesn't take long to figure out what went wrong. But Denise, who lives in Vancouver, Washington, knew there were no mistakes here. "I saw it being born!" she say. "I didn't mix it up wiht any other foals; there were no other foals!" And Dunbars Gold's owner, Carole Dunbar, had only one stallion from whom to ship semen. AQHA's registration department turned back to the lab's geneticists and asked if they could give the case another look.
Puzzling Pattern
The case landed on the desk of Cecilia Penado, the lab's assiciate director of service and genomic research and development. Cecilia immediately noticed that Dunbars Gold and Sharp One were brindles. It's a very rare coat pattern in horses", she says, though it is common in dogs and cattle. People have not been very successful reproducing this pattern through breeding, and we've never really had much information on the genetics of it because it is so rare." In fact, of the more than 4.7 million American Quarter Horses that have been registered with AQHA (excluding appendix horses), the registration department has a list of only 15 horses that have exhibited some form of the brindle pattern. Because "brindle" is not an official AQHA color, Dunbars Gold and Sharp One are registered as red duns, which is their base color. Her interest sparked, Cecilia began reviewing the lab's testing on the horses. "Wen I looked up the stallion, I found that we had tested him three times before we established his (DNA) type," she says. The first two tests the lab ran were on hair samples. "At that time, there was something odd about his type," she says. "It looked like the hair samples came from two different animals, like the hair had been mixed together. We requested a second sample and had the same problem." Convinced the hair samples were getting contaminated, the lab requested a blood sample from the horse for the third test. "When the blood sample came in, we got a perfectly good type that would be consistent with one animal," she says. The blood test results also had some things in common with the hair test results. The lab used the DNA type obtained from blood for the parentage verification on Sharp One's 2004 foal (the first of the stallion's foals to be tested for parentage verification). but in her review, something else caught Cecilia's attention. "The oddest thing about the stallion's blood sample results was that the DNA type for sex-linked markers were typical of a female and not a male," she says. "There was no evidence of a Y choromosome." Cecilia decided to retest Dunbars Gold. "We went back to the original hair samples and used a single hair for the DNA test," she says. "And we performed several of these single-hair tests. Some of the tests yielded a perfectly good type for a male individual and some a good type for a female individual." Some of the restults also showed two DNA types within a single hair root. "At that point I thought, this horse is chimeric," Cecilia says.
Chimerism
A chimera is an idividual formed from two different cell lines. Scientists believe it happens when two non-identical twin embryos (fertilized eggs) fuse into one embryo very early in their development. The embryo develops into a normal, complete individual that has two different DNA types. He or she might have kidneys that developed from one DNA type and a heart of skin cells from the other type. Extremely rare, chimerism has been documented in other species, including cats and humans. "Dunbars Bold has one cell line that is female and one cell line that is male," Cecilia explains. "The cell lines have slighty different DNA types, but both qualify to his sire, so there's not a parentage issue involved. It's almost clear that there were two embryos produced and they fused." Although chimeric in his skin and hair, Dunbars Gold's reproductive organs were apparently formed by his male DNA type. "It's an interesting biological developmental event. He went on to develop as a completely normal male," Cecilia says. The lab has now tested several of his foals, and they were all produced bayhis male cell line. When Cecilia turned her attention to Sharp One, she again made note of the rare brindle coat pattern the mare had in common with Dunbars Gold. If Dunbars Gold was chimeric in his skin and hair, could there be a link between the equine brindle coat pattern and chimerism? What if the problem the lab was having qualifying Sharp One as the foal's dam was also due to chimerism? Like Dunbars Gold, Sharo One's DNA type had also been initially established from hair samples. However, Cecilia says there was no evidence of chimerism in those hair samples. "I wondered if she could be the opposite of Dunbars Gold; if she might show chimerism in her blood, but not in her hair," Cecilia says. When the lab tested Sharp One's blood, that's exactly what it found: two different cell lines (and DNA types) in her blood. However, both of her cell lines were female. The lab then tested Sharp One's body hair taken from the darker and lighter areas of her brindle pattern. "We were able to identify the two cell lines in the different (body hair) patches," Cecilia says. When the lab compered the 2004 foal's DNA type to Sharp One's 2003 foal, they found that each came from her two different female lines. "Unlike Dunbars Gold, she is chimeric in her (reproductive organs)," Cecilia says. "She is producing two different types of eggs, the can be from one or the other cell line, which is a very interesting situation. Once wer put the puzzle together, we were able to qualify the foal without any problem."
The Pieces Fit
"We now have evidence of chimerism from two different brindle horses," Cecilia says. "We think that the brindle pattern in some horses could be explained by this very rare event, where two embryos fuse early in their development and go on to make one individual with different cell lines." Cecilia is quick to point out that there is eveidence of a type of brindling pattern in horses that appears to be inherited, linked to a coat pattern gene, as it is in dogs and cattle. "We can't say that it is always embryo fusion that leads to the brindling pattern in horses," she says. "But for the very classic, clear brindling pattern like we see in Dunbars Gold and Sharp One, I wouldn'd be surprised if that was caused by chimerism. If it results from embryo development, then there is no genetic control, and you can't really breed for it. Unless there is a gene controlling something that makes it more likely for the fusion to occur, such as something that increases the likelihood of a twin pregency." Cecilia thinks you would only see outward evidence of chimerism in the coat pattern if the two embryos that fused had genes coding for contrasting coat colors. "For example, if two bay embryos fused, or two chestnuts, you wouldn't see anything," she explains. "I think you'd see the brindle pattern if you had a combination of a base color with one dilution gene. In this case it was the dun gene. Both (Dunbars Gold and Sharp One) have dun in their background." "It has been a great learing experience," Cecilia ads. "It is shedding a little bit of light on the brindling pattern."
Warboel
