Genetic Testing: The Secret World Of Genes
Anyone who reads this magazine likely agrees that horses are amazing creatures. Valued for their speed, their beauty, and their grace, not to mention their generosity of spirit toward humans, horses are a continuing marvel even to those of us who work with them every day. And now, as researchers delve into the secrets of the DNA strands that make horses what they are, we're discovering anew just how miraculous they are--on a molecular level.
ANNE M. EBERHARDT
We now have at our disposal a great deal of information about the genetics of coat color.
Every species of living thing on the planet has a genetic code, which is a characteristic number and array of chromosomes, hidden in every cell, that supply the directions for the precise workings of the organism's metabolic function, development, and reproduction. On these chromosomes are genes, the term used to describe sections of the DNA spiral that are responsible for creating individual traits, some obvious to the eye, others invisible but no less crucial. Genes can vary in size from just a couple of molecules in the DNA strand, to large and complex sections with thousands upon thousands of subunits.
Genes come in pairs, one from each parent, and the various forms of each gene are called alleles. An organism might be homozygous for a certain trait (meaning both of the alleles are the same), or heterozygous (meaning the two alleles are different). A dominant gene is one that will create a certain physical characteristic whether it's present in single or double form; a recessive only expresses itself when it's present in homozygous form and not overruled by the presence of a dominant gene. While this is the genetic definition of dominant and recessive, unfortunately, in an animal as genetically complicated as the horse, it isn't as straightforward as it sounds.
Although observations of a horse's physical characteristics (his phenotype) have provided us with some information about his genetic make-up, and allowed us to make some predictions about the heritability of certain dominant and recessive traits, much of what we know about equine genetics has been guesswork. Even when we've studied breeding and registration records going back 20 generations, we've gotten an incomplete picture--because horses with genetic defects that affected their health usually weren't registered, because mares and stallions tend to be registered more often than geldings, because some individuals remained unregistered when they didn't meet the standards of the breed registry, or because their owners didn't consider them worth the economic outlay. But huge advances in research, delving down to the molecular level, have given us the hope of a far more complete picture.
The Equine Gene Mapping Project, an on-going international effort involving universities in more than a dozen countries, including Australia, England, France, and the United States, has made remarkable progress since it was begun in the early 1990s. Its goal: to identify at least 300 "markers" (like pushpins on a map) that will give researchers a reference plan for the location and character of the approximately 70,000 genes located on a horse's 32 paired chromosomes.
Once we can reliably locate the genes that create a horse's various traits, it's a straightforward step to being able to test for those traits, and calculate the likelihood of a horse passing certain characteristics on to his or her offspring. Heritability could be just a matter of interest, for instance, when a mare owner would like to know what the chances are of getting a palomino foal from the pairing of her mare and a certain stallion. It might be a matter of life and death, as when a breeder is trying to determine whether her Arabian stallion is a carrier of the gene for Combined Immunodeficiency Disease (CID), an invariably fatal condition. Once, it would take several matings, each one a roll of the dice, to determine an answer about a horse's genetic design. These days, we're able to go straight to the source, and decode the language of DNA itself.
Gene mapping for horses has had a slow start, relatively speaking. Geneticists already have established maps for several other species, including sheep, pigs, chickens, cattle, and mice. Human gene mapping also is nearing completion, with thousands of markers identified. Researchers currently are able to map more than 4,000 genetic abnormalities or diseases in humans, thanks to the multi-billion-dollar Human Genome Project, which in many ways is driving equine research.
One of the most interesting discoveries of recent years is that the DNA record doesn't vary that much from species to species, at least among vertebrates. There is some basic genetic material that is common to all species, and genes that "express" to produce similar traits tend to be in roughly the same location relative to other sets of genes when you compare one animal to another. For instance, the gene that creates a certain coat color in mice will often have a counterpart (called a homologue) that does the same thing in a horse's DNA. This lucky coincidence has provided an invaluable shortcut for researchers, who have been able to use species with more complete DNA records as templates for the Equine Gene Mapping Project. Mice, as it turns out, are an especially good model for equine coat color genes (of which at least 10 are now known), and many genetic diseases horses suffer, including CID, HYPP, and even the overo "lethal white" syndrome, all have parallels in humans or mice!
Gene mapping can provide answers for a multitude of questions about heritability. For instance, we might be able to identify a gene, or genes, that would be a sure indicator of superior speed in a Standardbred, or "gaitedness" in a Tennessee Walker or Paso Fino. We might be able to institute a high degree of predictability of conformation in the offspring of a certain bloodline. Color breeders would be able to choose breeding stock with the best possible chance of producing a tobiano, a buckskin, or a blanketed Appaloosa. And it's possible we might be able to identify a gene that predisposes foals to Developmental Orthopedic Disease (DOD), or older horses to heaves or navicular disease, or, at least, determine whether genetics plays a role in those (or other) conditions. On top of all this, gene mapping also can provide us with insights into evolution on a chromosomal level, as we begin to understand how some changes in DNA sequence (mutations) make a breed or a species more, or less, successful than it was before.
On a more prosaic level, gene mapping will allow us to make a far more accurate determination of the parentage of an individual horse, an increasingly important requirement of many breed registries. Blood typing, the current method used by most registries, is reliable, but DNA testing can provide a far more definitive, and informative, result, potentially telling us more than just the likelihood that a foal is sired by a particular stallion. The possibilities are, to say the least, tantalizing.
Gene Mapping Project
Gus Cothran, PhD, director of the Equine Blood Typing Research Laboratory, Veterinary Sciences Department, University of Kentucky, says that the Equine Gene Mapping Project now has identified some 160 genetic markers. While the project is by no means complete, he says, two papers that provide the first fairly comprehensive maps of the equine genome have been accepted by academic journals and are awaiting publication. "Before the year is out, or very early in 1999, we should have the first good maps of the (DNA of the) horse in the literature.
"The map is progressing rather well," he adds. "We achieved our goal of 150 (markers) pretty close to when we thought we would, and 300 looks well within reach. Three hundred markers should give us adequate coverage for almost anything we want to look for."
Genetic markers for the map are called "microsatellites"--which Cothran describes as subunits that are repeated a number of times. "Microsatellites are not necessarily genes, although they may be incorporated in a gene," he explains. "They're just repeating sequences which are easy to spot and can be used as signposts to help us find genes we're interested in. If you have a trait you'd like to locate a gene for (in horses), you would find the coding genes close to a marker, and compare that to the human gene map, which is close to complete. From the comparison, you can pick out a candidate gene for the trait."
The International Equine Gene Mapping Project is involved in linkage mapping, which is designed to provide researchers with a ready comparison of the correlating sections of DNA between different species.
Meanwhile, at both the University of California/Davis and the University of Kentucky, another kind of mapping is simultaneously underway. Ann Bowling, PhD, Executive Associate Director of the Veterinary Genetics Laboratory at UC Davis, explains that her lab is working on synteny mapping of the equine genome, a method that helps researchers identify which genes are located together on a single chromosome. Through an elaborate system which uses a panel of cells created by fusing horse cells with those from mice (a somatic hybrid), synteny mapping is making it easier to locate specific genes on specific chromosomes.
Cothran mentions that there is a third level of activity in gene mapping going on in an independent project at the Animal Health Trust in Newmarket, Great Britain. "It's fascinating work," he says. A group of research mares there are being bred by artificial insemination, with the embryos harvested soon after for their DNA material. In this way, a "family" of genetic results of specific matings is being built up very quickly, allowing researchers to examine the results without the time and expense of raising the foals. All the information is contained within the DNA of even the tiniest embryo.
Bowling notes that although the human genome likely will be completely sequenced within the next five years (with the help of several billion dollars in research funding), the Equine Gene Mapping Project will probably never be absolutely complete. Research on horses, she points out, tends to be under-funded, thanks both to the cost of maintaining research animals and the perception that the horse industry in North America has a relatively minor economic impact when compared to livestock like cattle or swine. Still, on small research budgets, equine geneticists already have uncovered some fascinating information about the molecular makeup of horses. We'll highlight just a few of them.
A Coat Of Many Colors
It's often been said that "a good horse is never a bad color," but for many breeders, some colors are better than others. Certain coat colors or patterns have been prized and selected for over many centuries. Those who breed paints and pintos would like a guarantee of spotted foals every spring; Appaloosa breeders, too, are looking for "good color," with no hint of pinto coloration or graying to dilute the characteristic coat patterns. Those who breed palominos are hoping for that 100% probability of a golden colt or filly. And sometimes, the "wrong" color can be an indication of an outcross somewhere in a horse's parentage, since many breed registries forbid pinto coloration, for example, or limit the extent of a horse's white markings. It should be kept in mind that the molecular description of the genetics for coat colors for the most part is in its infancy and much research will come from Bowling's initial work in this field.
Sometimes an interest in color heritability is more than academic or economic. In the case of overo-patterned paints, there is a condition called aganglionosis, more commonly called "lethal white," which sometimes crops up when breeders match two overo horses. A foal with a double dose of the overo gene is born white and appears normal, but it is unable to pass food through its digestive tract due to a lack of nerve cells in its intestine and soon dies. A similar condition in humans is called Hirschsprung's disease and is associated with a white "forelock." There are also at least three white spotting genes in mice that can be lethal when they're homozygous. Because of the possibility of lethal white, there has been a pressing need to understand the genetics of the overo coat pattern, and to be able to test for the gene for lethal white.
Fortunately, we now have at our disposal a great deal of information about the genetics of equine coat colors, much of it uncovered by Bowling and her team at UC Davis. It turns out that all of the various colors horses can be are created by the presence, or absence, of two pigments--eumelanin (black/brown) and phaeomelanin (red/yellow). These two pigments can be influenced by a number of genes that can modify the basic coat color, diluting it or producing patterns. There also is an allele for the absence of color.
Most color genes are dominant, but one of the most common, the gene that controls red (chestnut) coloration, is recessive. When a horse is homozygous for the red gene (designated as ee), he will possess no black hairs on his body or "points" (the mane, tail, ear tips, muzzle, and lower legs). Depending on the modifier genes present, a horse could be chestnut, sorrel, palomino, red dun, gray, cremello, or even white, with the added possibility of roan, Appaloosa, or paint patterning if those genes are present.
On the other hand, if a horse's genetic code contains the "black factor," E, he'll have black hairs in his points and possibly his body color as well. He could be black, bay, or brown, or if there are other modifiers present he could be buckskin, dun, grulla, perlino, gray, or white (or any of the spotted-coat variations). A horse which is homozygous EE doesn't have the red factor in his genetic makeup and thus cannot produce red foals. This can be an important detail in breeds where the color black is prized (as it is in the Arabian). Because the "red factor" is a simple recessive gene, it has been fairly straightforward to develop a test for it. This test recently has become available, and with this test, a breeder can discover whether or not a horse with black points is homozygous (EE) or heterozygous (Ee). The best chance of producing black horses will come when two EE horses are mated. The red factor test also can be a tip-off as to parentage--for example, two chestnut parents cannot produce a horse with black points, such as a bay.
It's less simple to try and detect whether black will breed true. Researchers originally compared the black coat color in horses to a gene called "agouti" in mice, but it's now known that black is controlled by more than one gene. Being homozygous EE isn't enough to ensure that a horse is black; he must also be free of body-color modifiers that might make him a buckskin or grulla instead.
Another gene, designated as A (for agouti), appears to control the distribution of black hair. In combination with the presence of the E gene, it will confine the black hair to the points, to produce a bay horse. If the alternative gene, a, is present instead, it and the E gene will produce a horse with a uniform black color. In most breeds, the a gene is quite unusual, so true black horses are a rare occurrence (except in breeds such as the Percheron, Friesian, and Canadien, which select for it). As of yet, there is no reliable test available to discover the "black factor."
Another interesting color gene is G, which causes graying. Breeders have known for centuries that a gray can only be produced in a mating with at least one gray parent, making G a simple dominant. Horses with the G allele, whether they are homo- or heterozygous (GG or Gg), are born dark and gradually fade to gray, then to white, as they age. Because these horses get lighter as they get older, they can "lose" patterned markings such as tobiano or overo spotting, or Appaloosa markings (which is the reason the Appaloosa registry forbids gray horses).
Tobiano coloration also is controlled by a simple dominant gene, designated TO or P (for "piebald," the European term for a tobiano whose dark spots are black). Tobiano spotting features large patches of color on a basically white background. Horses marked this way usually have white legs and dark faces (except for markings such as a blaze). In order to achieve tobiano coloring, at least one parent must be tobiano, and the chances are better if both parents have the coat pattern. Fortunately, there seems to be no risk of lethal white occurring as it does with overo spotting. Any lab performing blood-typing can perform blood testing for the tobiano gene, although Cothran notes that the procedure looks for two linked genes associated with tobiano coloration, rather than the tobiano gene itself, and the test is not 100% reliable. Despite this, the lab receives two or three requests a week for the test (which costs about $31 and takes a couple of weeks to yield results).
The D gene is a dilution factor or modifier, which can have an influence on the body color of both red (ee) and black (Ee or EE) horses. When D is present, a horse coded for chestnut body color becomes a red or a claybank dun, while black coloration becomes grulla or mouse dun. Horses with the D gene also have dark points, dorsal stripes, and sometimes shoulder striping and zebra stripes on the legs. If a horse is coded dd, however, his coat color will be undiluted.
Likewise, the C gene has an allele called Ccr which can dilute red pigment to yellow on both the body and the points. When Ccr is present, horses coded for chestnut (ee) become palomino, with the mane and tail turning flaxen or white. A bay would become a buckskin, the black color of his points unaffected. Ccr does not affect genetically black horses when it's present in heterozygous form. In homozygous form, however (CcrCcr), just about any coat color becomes diluted to a very pale cream with pink skin and blue eyes, usually called cremello or perlino. It usually takes a mating between two "dilute" horses, such as palominos or buckskins, to produce a homozygous cremello. The alternative, CC (with no cr) is a fully pigmented horse.
Researchers also have identified a gene that codes for roaning, logically designated R, and a Z gene, which codes for the rare color called "silver dapple," found only in a few breeds (Shetlands, miniatures, Rocky Mountain horses, Icelandics, and Dutch warmbloods, among them). The Z gene is dominant, but only expresses itself when E is present (in other words, the horse is not coded to be a homozygous chestnut). Variations include a silver dapple black, silver dapple bay, and a silver dapple buckskin, which is often mistaken for a dappled palomino.
One of the more complicated colors, from a genetic point of view, is the overo paint pattern so popular among breeders of registered Paint horses. While breeders have long known that when you cross two overos, you generally have a 50% chance of overo-spotted offspring (along with a 25% chance of a solid-colored foal, and a 25% chance of homozygous lethal white), there have always been, in the Quarter Horse breed, horses called crop-outs--those which arrive with overo spotting despite no history of the pattern in their parents or their ancestors. Furthermore, studbook investigations have shown that some stallions, when bred to solid-colored mares, buck the statistics by producing substantially more overos than solids (although so far, no overo stallion has been shown to sire 100% color).
Early on in her studies of Paint coloration, Bowling speculated that overo spotting might be controlled by several genes. Further investigation has revealed that there might indeed be more than one kind of overo and that blending of these ovaro patterns is common. The type most often associated with the incidence of lethal white is what breeders call a "frame overo," in which the white markings usually are confined to the head and the sides of the body, "framed" by color. A pure "frame" overo rarely has white on its legs, ventral abdomen, or dorsal line. The type of overo sometimes described as "sabino" (typified by the snowflake-edged splotches of white, and significant roaning, often seen on Clydesdales and Shires) now appears to be a separate type of spotting with its own genetic coding. Unlike the frame overo gene, sabino is fully dominant, so at least one parent must be sabino in order to achieve a sabino foal.
Another spotting pattern lumped by the registries under overo is a pattern called "splash white," which features a continuous pattern of white on all four legs, chest, and completely over the head. There also is a relatively rare spotting pattern designated F, which produces a "white head splash" pattern (occasionally accompanied by neck and belly white as well). It's seen only in certain northern European breeds (the F stands for Finland, where it was first recognized), and it is a homozygous recessive.
Some spotted horses might in fact possess several spotting genes all working together. Such horses are called compound heterozygotes (also known as Paint blends, as discussed above) by geneticists, but breeders might describe them with names like tovero (a combination of overo and tobiano characteristics). It's suspected that compound heterozygous stallions are in fact the most successful producers of color. They can sire a high percentage of spotted foals on both spotted and solid-colored mares.
Fortunately for breeders, who might find it difficult to define their horse's genetic makeup by observation, there now is a test for the overo gene that is known to produce lethal white. The test is useful to breeders not only to help them avoid producing lethal white foals, but also to help them in identify potential pedigree sources of the overo pattern that could be useful in their breeding programs. If you do have a frame overo, according to the testing, it's safest to breed that horse to solid-colored counterparts. However, in order to avoid a lethal white entirely you should test both parents.
Appaloosa coat patterns are an area not yet fully explored from a genetic point of view, says Cothran, although an investigation by University of Kentucky graduate student Rebecca Terry currently is underway. Early indications are that all of the various Appaloosa patterns, from blanket to leopard to varnish roan, are controlled by a single dominant gene. Cothran elaborates, "We have a strong suspicion as to the location of the Appaloosa gene, as well...it's probably in the same area as the tobiano and roan genes."
The silver dapple gene also is being explored by undergraduate student Shawn Phillips, who has an interest in Rocky Mountain horses (a breed in which the color is prevalent).
Testing For Genetic Disease
One of the most important applications of genetic testing is its use in detecting and diagnosing genetically linked diseases and abnormalities. Usually the result of a "typographical error" when DNA is reproduced--an addition (the most common type), substitution, deletion, or scrambling of the order of the individual codons--most abnormalities, or mutations, are never seen because they create an animal which isn't viable and is resorbed or perishes long before birth.
Occasionally, though, a mutation occurs that allows life to develop to adulthood. If the animal possessing that mutation is used for breeding, there's the chance that it could pass on its defect--or improvement--to the population. New mutations are cropping up all the time; researchers postulate that the process explains much about how species change and adapt to their environment over the millennia.
Bowling points out, "It's not realistic to talk about 'wiping out' genetic disease in horses, because mutations are always happening. No species of animal is ever completely free of defects, and horsepeople need to start thinking in those terms. We can't get rid of everything. The trick is that you don't want to match up a sire and dam who have the same deleterious genes."
Hyperkalemic periodic paralysis, or HYPP, is the mutation that opened up a world of possibilities in terms of the detection and diagnosis of genetically linked diseases. HYPP is a disease that can be traced back to a single Quarter Horse sire, Impressive. Due to a "typo" the horse suffered at conception (although he never demonstrated any disease symptoms himself), some of Impressive's descendants have inherited a genetic defect that disrupts the way sodium channels (tiny gateways in the membranes of muscle cells) open and close. Because the gateways tend to get stuck on "open," they can allow an uncontrolled flow of sodium ions into the cells. This changes the voltage current of the cells, causing weakness and/or twitching. High levels of potassium also tend to build up in the blood when these disruptions occur.
Cases of HYPP can be mild or severe, depending on how many sodium channels are affected. At its worst, the condition can be fatal if respiratory failure or cardiac arrest occurs. More often, an HYPP horse suffers attacks of weakness and staggering, which can be exacerbated by stress. He often is exercise-intolerant as well. The severity of the disease is influenced by whether a horse has inherited a single or double dose of the defective gene, called H. Homozygous horses, as a rule, are far more severely affected than heterozygous ones.
On a molecular level, the HYPP mutation is the result of a single nucleotide error in the DNA strand. Within the gene that controls sodium channels in muscle cells, the amino acid leucine is substituted for the correct one, phenylalanine. In other words, one single misplaced base pair in the DNA sequence causes "all hell to break loose."
Because of Impressive's popularity as a stud, HYPP now is widespread among registered Quarter Horses and in other breeds that accept Quarter Horse lineage (such as the Paint and Appaloosa). Fortunately, once the initial shock of the disease's discovery wore off, the American Quarter Horse Association became pro-active, vowing to wipe the mutation from the breed's makeup by a program of vigorous testing for any Impressive-descended horse. HYPP proved to be the first equine disease traceable to a single bloodline by genetic analysis, thanks to Eric Hoffman, PhD, a human geneticist who had already worked on the human version of the disease. He identified the HYPP gene in 1992 at the University of Pittsburgh. It was also one of the first diseases for which a reliable test was developed.
In the HYPP test, as in most DNA tests, genetic material is extracted from a blood, hair, or tissue sample. The gene coding for the muscle cell's sodium ion channels is amplified (copied using PCR, polymerese chain reaction), cut out using enzymes that cut specific DNA sequences, separated by electrophoresis, then stained and examined. The HYPP test is virtually 100% accurate. A horse which has tested negative for the gene bears the notation HYPP N/N on his registration papers; one which tests positive is N/H or H/H and should not be used for breeding purposes.
Although HYPP was the first equine disease to be identified, beyond the shadow of a doubt, as genetic, researchers expect it will not be the last. Close on the heels of the development of the HYPP test in the early 1990s was a test for combined immunodeficiency, a devastating condition in Arabians that breeders long had suspected of being heritable. In CID, foals are born without a functioning immune system. They appear normal at first, but once their colostral immunity wears off, they succumb to the first bacterial or viral infection to invade their systems. For decades, the only way to identify a carrier of the CID gene was for the horse to produce a CID foal.
Twenty years of painstaking research, and a lot of blind alleys and dead ends, finally yielded the exact location of the CID mutation in 1996, when a team led by Dr. Katheryn Meek, at the University of Texas Southwestern Medical Center, found a five base-pair deletion in the DNA of CID-affected horses and known CID carriers. It soon was confirmed that CID was an autosomal (not sex-linked) recessive defect. From there, it was a simple process to develop a CID test for breeders, a test that when launched in the summer of 1997, was expected to revolutionize the Arabian industry, or destroy it, depending on who you talked to.
John Duffendack, president and CEO of VetGen, the Michigan-based company that made available the commercial CID test, notes that a year later, "the CID test is doing quite well. It's becoming more accepted now that the initial scare is over, and we've tested several thousand Arabians in the past 12 months. The carrier rate is working out to be about 16-18%. Testing is still voluntary--it's not legislated by the breed registry--but I think most conscientious breeders want to know, and they want to eliminate the defect from the breed."
The CID test is particularly simple for owners or veterinarians to access; the VetGen labs can analyze either leukocytes, isolated from a blood sample, or a swab taken from the inside of a horse's cheek, by the same basic techniques described earlier. Generally speaking, tissue samples are better for DNA analysis than blood samples. Blood must be carefully handled and analyzed quickly, but tissue samples, properly harvested and stored (under refrigeration), are more durable. Once extracted, a DNA sample will "keep virtually forever," says Cochran.
Bowling notes that the lab at UC Davis recently developed a method of extracting DNA from the bulb at the root of a strand of horse hair, "because we recognized the problem of shipping blood or tissue long distances in the United States. The distances became a major concern when you're dealing with a sample which must be kept under refrigeration. Hair is clean and neat and doesn't degrade, and it's easy for the owner to obtain without having to call a veterinarian out. Furthermore, it can be easily stored indefinitely. The HYPP test is now being performed using hair, and the CID test would work this way as well."
Other Genetic Defects
Several other genetically linked conditions are currently the focus of research to identify their locations on the DNA strand, and to develop a reliable testing procedure. For example, the University of Kentucky genetics lab has been collecting tissue samples from Miniature horses exhibiting signs of dwarfism "for a couple of years now," says Cochran.
"There are lots of types of dwarfism in humans, so we need to narrow down the candidates that would be most likely to parallel what goes on in horses. So far, our investigation has shown that the (gene) which causes about 80% of dwarfism in humans is not the one which causes the same condition in horses or cattle. We have to keep looking. But we do know that in horses, dwarfism seems to be a recessive trait."
Genetic research also might yield some answers for an alarming condition called epitheliogenesis imperfecta, or EI, which occurs in Belgians, Quarter Horses, and Saddlebreds (some researchers suspect that up to 80% of Belgians carry the gene). EI foals are born with large areas of their skin, oral membranes, and sometimes their hooves, missing. They usually die within hours or days, starving because their delicate mouth parts hurt too much to allow nursing, or suffering massive infections as a result of the large swathes of unprotected tissue. A similar condition in humans is called epidermolysis bullosa, but Cochran says that initial studies have indicated that EI probably isn't controlled by the single gene originally suspected. That's a mixed blessing, he says, "because it was a large and complex gene, and would have been hard to work with." The studies are continuing.
Yet another genetically linked condition for which there likely will be a reliable test for soon is anterior segment dysgenesis, or ASD, which has a high frequency among Rocky Mountain Horses. Horses which are heterozygous for the ASD defect tend to develop cysts in their eyes, while homozygotes can range anywhere from almost unaffected, to having no eyes at all (this, fortunately, is rare). In any case, the horse's vision is definitely compromised. A candidate gene has been identified at Michigan State University, and it's expected that we might soon know a lot more about this unusual condition.
Some genetic defects are sex-linked--that is, they are associated with the X chromosome. Two examples of these occasionally crop up as an explanation for infertility in broodmares--XO Gonadal Dysgenesis, in which a mare is missing one X chromosome (in other words, she has a total of 63 chromosomes, not 64), and XY Sex Reversal, sometimes called testicular feminization, in which a horse has the outward appearance of a mare, but is genetically male. Both have parallels in humans. The XO defect is known as Turner's Syndrome in women. It's also possible for a horse to have extra chromosomes (65 instead of 64); this has been demonstrated in a few cases of small, unthrifty foals and, like Downs syndrome in people, is associated with foals born to older mares. The first extra-chromosome foal to be identified with certainty happened, by sheer coincidence, to be born to a 24-year-old mare belonging to Bowling. That filly, now entering middle age, remains small and suffers from a number of angular limb deformities and stiff gaits. Since then, a couple of other foals with an extra chromosome have been identified.
Question Of Genetics
Unfortunately, it's not always clear whether a disease or defect has a genetic basis. Witness a condition called degenerative suspensory ligament desmitis, or DSLD, in which a healing error forces the body to repair torn or strained suspensory ligament tissue with non-stretchable cartilage. A horse with DSLD ends up with ankles that sink toward the ground. This horse often tries to dig holes in the ground to stand with his toes down to relieve his discomfort. Although the condition has been found in many breeds, it appears to be particularly common in Peruvian Pasos, and many researchers strongly suspect the existence of a DSLD gene (especially since there is a candidate gene in the human code). But not all breeders admit there is an increased incidence, and some actively resist the idea of further exploration, perhaps for fear the results will devalue their breeding stock.
Conditions that are suspected to have a genetic link, and probably will be testable in the future, include developmental orthopedic disease (DOD), heaves, cerebellar hypoplasia, cryptorchidism (undescended testicles), parrot mouth, and club foot. Even sweet itch, a hypersensitivity to a type of gnat, and cataracts, might end up having a genetic element.
Testing In The Future
Like computer technology, which seems to change so fast that your current home system is always a little out of date, advances in genetic testing are proceeding faster than they can be implemented on a practical level. At the moment, says Cochran, the University of Kentucky labs are involved in parentage testing for approximately 30 different breed registries in North, Central, and South America, and the labs at UC/Davis "are doing at least that many"...not to mention many other university-based and privately operated laboratory companies. That parentage testing is based largely on blood-typing, a technology that pre-dates true DNA testing and that categorizes factors on the red blood cell surfaces, much as blood typing in humans designates us as type O, A, B, or AB, and as positive or negative.
Blood-typing is perfectly adequate for some purposes; not only can it provide a fairly reliable determination of parentage (based on the idea that a foal's blood type must be inherited from the sire and dam), but it's also useful for detecting problems like neonatal isoerytholysis (NI, also called hemolytic disease or foal jaundice). NI is similar to Rh incompatibility in expectant mothers and babies--if a foal's blood group is incompatible with the mare's, he will become weak, anemic, and jaundiced within six hours to five days after drinking his first colostrum,and might require blood transfusions of thoroughly washed red blood cells from his dam in order to survive. A mare which has had one NI foal is likely to produce another, so most veterinarians recommend testing her blood three weeks prior to foaling, with a blood grouping test that can indicate whether she is carrying an incompatible foal. (Disaster can be quickly averted if the answer is yes, by having an alternate colostrum source on hand to feed the newborn.)
Blood-typing also can provide an indirect indicator of a color gene. The tobiano spotting pattern is strongly associated with the presence of certain protein variants in the blood--so analyzing the blood for those factors can help you determine whether your tobiano horse is homozygous and likely to pass on his coloration. The current tobiano test is based on an analysis of these two linked genes.
However, blood typing doesn't provide nearly as much information, or do it as accurately, as DNA testing. The procedure, which uses a process called allele-specific polymerase chain reaction, or ASPCR, can be performed on any source of nuclear DNA extracted from a cellular source. A few registries already have made the switch from blood to DNA testing, most notably the AQHA. It moved from blood typing to DNA testing for all registered Quarter Horses two years ago. Other registries are considering the move, but just as you might hesitate to buy a new piece of electronics equipment for fear something better is just on the horizon, some are holding back, waiting for new developments that might make DNA testing simpler and easier.
"The question," says Cochran, "is whether the DNA testing technology we have now is the best we can do, and I suspect not. And if we switch (a breed registry) to another technology, we have to re-type all the parents from scratch, because different markers are used. It's debatable whether that's worth doing.
"The upcoming technology is going to be better within five years, no question. And it has the potential to be quite inexpensive," he adds.
Shortly after the turn of the century, we can expect to take a hair sample from a horse, extract DNA from the hair bulb, float it in a solution and flood a microchip with hundreds of tiny "wells" on it, Cochran explains. On each chip will be DNA that has been denatured to a single strand--so when it encounters a match from the hair sample, it will bind to it. Through a simple chemical reaction, a match in a single well will produce a visible color change, making the chip simple and economical to read.
The possibilities of this new system are virtually limitless, helping to identify hundreds of traits and/or abnormalities in a single test.
"The microchip we envision will hold all of the parentage information for a horse, plus diagnostic data, all from a single hair sample," says Cothran. The face of things to come.
DNA Testing Helps Resurrect the Quagga
Once upon a time, horses had a strange-looking relative on the south African veldt--a pony-sized animal with striping over part of his body, like a zebra, but with pale brown hindquarters designed to help him camouflage on the dusty plains of the Karoo (the southern part of the present Orange Free State of South Africa). European immigrants designated him Equus quagga, then merrily hunted him to extinction, as they did many other species in the 18th and 19th Centuries. They used his carcass for food and his hide for harness, ropes, and even grain sacks. On Aug. 12, 1883, the last quagga on earth, a lonely old mare, died at the Artis Magistra Zoo in Amsterdam. It would be several years before anyone even realized that she had been the final representative of her kind.
The quagga was assumed to be lost forever--until German taxidermist Reinhold Rau, working for the South Africa Museum in Cape Town, found his curiosity piqued in the late 1960s when he was charged with restuffing and restoring several preserved quagga skins (of the 23 left in the world). The more he examined the quagga's characteristics, the more he became convinced that the beast was not a separate species to the zebra, but a sub-species of the Burchell's, or plains zebra, which survives in southern Africa and shows a great deal of variation in its stripes from region to region. The Burchell's (unlike its cousin, the Grevy's zebra) often has brown "shadow" striping between the main black stripes on its coat, especially on the hindquarters. As you go south toward the southernmost tip of the African continent, its hindquarter striping becomes less distinct.
Only DNA would tell the tale for sure. Miraculously, Rau was able to extract some tiny bits of blood and tissue found clinging to one of the preserved quagga skins. He sent them to California's San Diego Zoo, where there is a depository of rare animal tissues, for analysis. Although it is almost impossible for genetic material from a sample more than 100 years old to survive, the tissue fragments did contain enough mitochondrial DNA to allow the San Diego geneticists to amplify it and compare it with that of the Burchell's zebra. To everyone's excitement, it was a match. The quagga had not been a separate species, after all--it had just been the southernmost sub-species of zebra, living in a different habitat, but genetically one and the same.
Suddenly there was the possibility of bringing the quagga back from extinction, by selective breeding of Burchell's zebras who demonstrated "quagga-like" characteristics. A team of researchers ranging from taxonomists and veterinarians to conservationists and geneticists founded the Quagga Project in 1987, capturing nine Burchell's zebras with brown shadow striping and transporting them from the Etosha Game Reserve to the Nature Conservation Station near Cape Town, where they were separated into breeding groups. In December 1988, the first generation foal of the founding herd was born. The team estimated that it would take at least two, and possibly three to four generations before they would see if it was possible to resurrect the quagga through selective breeding. There was much excitement when several years later, the first of the second-generation foals arrived and quagga characteristics were clearly emerging.
Today, there are 53 animals in the breeding program; some are grazing on the slopes of Cape Town's Table Mountain, and a herd of 11, with striping on their shoulders and light brown hindquarters, was released in the spring of 1998 into their ancestral territory in the Karoo National Park. Future generations are expected to exhibit, more and more, the patterns of the century-old skins of their lost brethren.
The saga of the quagga is by no means a cure-all for extinction, for few other species are likely to be resurrected in this manner. But it certainly is a heartening application of genetic research, and the righting of a huge wrong inflicted over a century ago on an odd-looking equine who used to roam the arid plains.
About the Author
Karen Briggs is the author of six books, including the recently updated Understanding Equine Nutrition as well as Understanding The Pony, both published by Eclipse Press. She's written a few thousand articles on subjects ranging from guttural pouch infections to how to compost your manure. She is also a Canadian certified riding coach, an equine nutritionist, and works in media relations for the harness racing industry. She lives with her band of off-the-track Thoroughbreds on a farm near Guelph, Ontario, and dabbles in eventing.
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