The Harry M. Zweig Memorial Fund for Equine Research

Generation of a Molecular Resource to Identify Gene Mutations Causing Inherited Equine Sterility and Infertility

- Dr. Vicki Meyers-Wallen

Meyers-WallenDr. Vicki Meyers-Wallen

In mammals, sexual development in the developing embryo is sequentially controlled at three levels: the sex chromosomes, the internal sex organs (gonads) and the external genitalia (phenotype). The sex chromosomes (XX or XY) indirectly control which type of gonad develops, such that XY embryos develop the male gonad (testes) and the XX embryos develop the female gonad (ovaries). However, in the early stages of development, the gonads are similar in male and female embryos and they require genetic signals to develop further. The molecular signal to develop testes is the gene SRY (sex determining region Y), which is located on the Y chromosome. The testes in turn secrete hormones, which induce development of male genitalia. Absence of SRY in XX (female) embryos allows the development of ovaries. A number of genes have now been identified that play a role in normal sexual development in humans and other mammals. Mutations in these genes cause inherited abnormal sexual development and sterility or infertility. Such disorders include cryptorchidism, testicular feminization, XY sex reversal, XX sex reversal and uterus masculinus, for which causative mutations affecting 15 genes have been identified (Table 1). These disorders have been reported in Thoroughbreds, American Standardbred, Quarter horses, French trotter and several pony breeds. Together, these disorders constitute the major cause of inherited infertility or sterility reported in horses. Sterility resulting from these disorders causes economic losses to the racing industry and owners of affected horses. Any extraordinary performance traits that sterile horses possess are lost to the gene pool and to the industry. Affected horses often need surgery to correct undesirable physical or behavioral characteristics, incurring further expenses for owners.

Whereas there is a national system to objectively record the incidence of inherited disorders in human beings, there is no such system for horses. The veterinary literature remains an unreliable gauge of how common these may be in horses of any breed. An ongoing problem in studying inherited disorders is that owners and veterinarians rarely follow up on tentative diagnoses or report inherited disorders in the veterinary literature. Rather than seeking a definitive diagnosis, owners cut financial losses by selling the horse, often without informing the breeder, who may not even be known to them. While this may be a suitable short term solution for an owner, it is not a good long term solution for the breed. At best, it allows these gene mutations to remain in the breeding population, and at worst, it can lead to an increase in mutation frequency in the breed when the mutation is carried by a popular sire or is frequent in a popular line. Some of these inherited disorders, like cryptorchidism, are sufficiently frequent and well known that case reports on this condition are no longer considered newsworthy by veterinary publications. Nevertheless, pedigree studies have been rare and the genetic mechanism for equine cryptorchidism remains unknown. Other disorders of abnormal sexual development, such as SRY-negative XX sex reversal, are anecdotally considered to be rare, yet this disorder has been reported in several breeds. This suggests that frequency gauged from literature reports represents the “tip of the iceberg.” This disorder may not be rare at all. An ancient mutation that arose before the development of horse breeds could be responsible for this condition in many breeds. Identification of the causative mutation for such disorders provides a definitive way to identify carrier and affected horses and establish how common these mutations are within breeds.

Presently, ultrasound and physical examination are used to tentatively diagnose these disorders in adult horses that are already being used in performance and breeding. In contrast, DNA based diagnostic tests can accurately diagnose carrier and affected horses before investments in training or breeding are made. Furthermore, they provide the means to eliminate these disorders from horse populations. An effective, long term strategy to reduce the frequency of these mutations is to identify carriers and avoid breeding them together. As long as carriers are bred only to tested noncarriers, desirable performance traits in carriers can be passed to the next generation without producing affected horses. However, to ultimately eliminate the mutation from a breed, those carriers without desirable performance traits must be removed from the breeding pool at each generation. We propose to develop a practical molecular diagnostic panel that can be used for this purpose, simply by the owner or veterinarian submitting a blood or hair sample for testing. This resource will be developed from the Horse Genome database website, where we will identify equine counterparts of genes known to cause abnormal sexual development in humans and other mammals. After designing primers that are small stretches of DNA unique to these genes, we will use the primers in polymerase chain reaction (PCR) to generate small DNA pieces of each equine gene (products). We will sequence the products to identify each base pair in their DNA sequence. We will validate the panel by testing a normal horse and confirming that their gene sequence is the same as in the Horse Genome database. Testing an affected horse’s DNA in the same manner can identify sequence differences (mutations) between the affected horse, the Horse Genome, and normal horses. To demonstrate this principle, we will test six horses affected with SRY-negative XX sex reversal, testing our hypothesis and illustrating the utility of this molecular diagnostic panel in identifying mutations.