University of Kentucky study shows association between mRNA biomarkers and catastrophic injuries in Thoroughbred racehorses, a positive step forward in the development of a pre-race screening tool.

First appeared in Trainer Magazine: https://issuu.com/anderson-co/docs/nat-60_issuu_v1

Catastrophic injuries in Thoroughbred racehorses is a top-of-mind concern for the global racing industry and its fans. That sentiment is shared by researchers at the University of Kentucky and their collaborators, who are working to learn more about changes happening at a cellular level that might indicate an injury is lurking before it becomes career or life ending.

Could it be possible to identify an early marker or signal in horses at risk of catastrophic injury, allowing for intervention before those injuries happen? And, if yes, might this type of detection system be one that could be implemented cost effectively on a large scale?

According to Allen Page, DVM, PhD, staff scientist and veterinarian at UK’s Gluck Equine Research Center, the short answer to both questions is that it looks promising.

To date, attempts to identify useful biomarkers for early injury detection have been largely unsuccessful. However, the use of a different biomarker technology, which quantifies messenger RNA (mRNA), was able to identify 76% of those horses at risk for a catastrophic injury. 

An abstract of this research was recently presented at the American Association of Equine Practitioners’ annual meeting in December 2020 and the full study published Jan. 12 in the Equine Veterinary Journal (https://beva.onlinelibrary.wiley.com/journal/20423306 ). In this initial research, which looked at 21 different mRNA markers selected for their roles in encoding proteins associated with inflammation, bone repair and remodeling, tissue repair and general response to injury, three markers showed a large difference in mRNA levels between injured and non-injured horses.

For almost four years, Page and his University of Kentucky colleagues have been analyzing blood samples from almost 700 Thoroughbred racehorses. The samples, collected by participating racing jurisdictions from across the United States, have come from both catastrophically injured and non-injured horses in a quest to better understand changes that might be happening at the mRNA level and if there are any red flags which consistently differentiate horses that suffer a catastrophic injury.

According to Page, the ultimate hope is to develop a screening tool that can be used pre-race to identify horses at increased risk for injury. The results of this study, which was entirely funded by the Kentucky Horse Racing Commission’s Equine Drug Research Council, suggest that analysis of messenger RNA expression could be an economical, effective and non-invasive way to identify individual racehorses at risk for catastrophic injury.

Joining Page in the research from UK’s Gluck Center are Emma Adam, BVetMed, PhD, DACVIM, DACVS, assistant professor, research and industry liaison, and David Horohov, PhD, chair of the Department of Veterinary Science, director of the Gluck Center and Jes E. and Clementine M. Schlaikjer Endowed Chair.

Previous research has shown that many catastrophic injuries occur in limbs with underlying and pre-existing damage, leading to the theory that these injuries occur when damage accumulation exceeds the healing capacity of the affected bones over time. Since many of these injuries have underlying damage, it is likely that there are molecular markers of this that can be detected prior to an injury.

The identification of protein biomarkers for these types of injuries has been explored in previous research, albeit with limited success. The focus of this project, measuring messenger RNA, had not yet been explored, however. The overall objective was to determine if horses that had experienced a catastrophic injury during racing would show increased inflammatory mRNA expression at the time of their injury when compared to similar horses who were not injured.

The genetic acronyms: A primer on DNA, RNA, mRNA and PCR

This research leverages advances made in genetics during the last several decades, both in a greater understanding of the field as well as in applying that knowledge to specific issues facing the equine industry, including catastrophic breakdown in racehorses.

The genetic code of life is made up of genes and regulatory elements encoded by DNA, or deoxyribonucleic acid, which is found in the nucleus of cells in all living organisms. It is arranged in a double helix structure, similar to a twisted ladder. The rungs of that ladder are nucleotide base pairs and the ordering of those base pairs results in the specific genetic code called a gene. The genetic code in the genes and the DNA tell the body how to make proteins.

RNA, ribonucleic acid, is created by RNA polymerases, which read a section of DNA and converts it into a single strand of RNA in a process called transcription. While all types of RNA are involved in building proteins, mRNA is the one that actually acts as the messenger because it is the one with the instructions for the protein, which is created via a process called translation. In translation, mRNA bonds with a ribosome, which will read the mRNA’s sequence. The ribosome then uses the mRNA sequence as a blueprint in determining which amino acids are needed and in what order. Amino acids function as the building blocks of protein (initially referred to as a polypeptide). Messenger RNA sequences are read as a triplet code where three nucleotides dictate a specific amino acid.  After the entire polypeptide chain has been created and released by the ribosome, it will undergo folding based on interactions between the amino acids and become a fully functioning protein.

While work looking at inflammation often involves measuring proteins, Page and his collaborators opted to focus on mRNA due to the limited availability of reagents available to measure horse proteins and concerns about how limited the scope of that research focus would be. Focusing on mRNA expression, however, is not without issues.

According to Page, mRNA can be extremely difficult to work with. “A normal blood sample from a horse requires a collection tube that every veterinarian has with them. Unfortunately, we cannot use those tubes because mRNA is rapidly broken down once cells in tubes begin to die. Luckily, there are commercially-available blood tubes that are designed solely for the collection of mRNA,” he said.

“One of the early concerns people had about this project when we talked with them was whether we were going to try to link catastrophic injuries to the presence or absence of certain genes and familial lines. Not only was that not a goal of the study, the samples we obtain make that impossible,” Page said. “Likewise for testing study samples for performance enhancing drugs. The tubes do an excellent job of stabilizing mRNA at the expense of everything else in the blood sample.”

In order to examine mRNA levels, the project relied heavily on the ability to amplify protein-encoding genes using a technique called the Polymerase Chain Reaction (PCR). By using a variety of techniques, samples from the project were first converted back to DNA, which is significantly more stable than mRNA, and then quantified using a specialized machine that is able to determine the relative amount of mRNA that was initially present in the individual samples. While it is easy to take for granted the abilities of PCR, this Nobel Prize winning discovery has forever changed the face of science and has enabled countless advances in diagnostic testing, including those used in this study.

The research into mRNA biomarkers

Catastrophic racing and training injuries have long been a target for researchers due to the high societal and welfare impacts on the racing industry. With the nearly universal requirement for necropsies on horses that succumb to these injuries, work by researchers has demonstrated that most horses with catastrophic injuries have pre-existing damage in their legs. This pre-existing damage presents an opportunity to detect injuries before they occur, whether that be with advanced imaging or less invasive techniques, such as screening of blood for injury biomarkers. 

Horses eligible for inclusion in the study were Thoroughbreds entered into any race in one of five participating jurisdictions from September 2017 to June 2020. To look at the mRNA, these jurisdictions collected specific blood samples either pre-race or post-race from a selection of non-injured horses or immediately from a horse after a catastrophic injury. Once collected, samples were sent to the Gluck Center where they were analyzed using PCR. The names of horses and sample types (injured, pre-race or post-race) were kept from the researchers until the samples had been fully analyzed.

Once the names and dates of samples were revealed, public records were then used to learn more about each horse. Information examined included the horse’s sex, age, race type and whether non-injured horses raced again within three months of the sampled race. For horses who had been catastrophically injured, necropsy results were used to categorize the type of musculoskeletal injury that occurred.

“Out of the 21 markers (genes) that were measured, three of them immediately stood out as being able to predict injury. The three individual markers of interest were Insulin-like Growth Factor 1 (IGF-1), Matrix Metalloproteinase-2 (MMP2) and IL-1 Receptor Antagonist (IL1RN). Taken together, the changes seen in all three of these markers suggest that there is increased inflammation in the injured horses and that the inflammation arises from bone, just as was suspected,” Page said.

“Based only on these three markers, we were able to correctly identify horses at risk for injury 76% of the time and exclude horses for being at risk 88% of the time,” Page said. “Obviously, we want to maximize those numbers as much as possible, so while there’s room for improvement, this is significantly better than any other option currently available.”

One of the limitations of the study was that horses were only sampled once, so there was no ability to identify changes in individual horses over a period of time. Once horses start being sampled repeatedly on a regular basis with this testing, Page said he believes the ability to identify at-risk horses will improve dramatically.

What does the future hold?

“Since the ultimate hope is to develop a commercially-viable screening tool that can be used pre-race to identify horses at increased risk for injury, we anticipate adding multiple other markers with a new study that is just getting started,” Page said.

As part of the new study, also funded by the Kentucky Horse Racing Commission, Page and two Gluck Center colleagues, James MacLeod, VMD, PhD, John S. and Elizabeth A. Knight chair and director of UK Ag Equine Programs, and Ted Kalbfleisch, PhD, associate professor, plan to utilize RNA-sequencing, a relatively new technology, to expand their search to all of the approximately 22,000 protein-coding genes horses have. This will dramatically increase the likelihood that they will be able to identify additional markers for horses at risk of injury. They plan to do this by using the large number of samples that have already been collected, further leveraging their initial research and decreasing the amount of time it will take to complete their new study.

“We are really excited about this new project and the promise that it holds,” Page said. “In our first study, we drove the data because we had to select which mRNA markers we wanted to examine. In our new study, the RNA-sequencing data is really what will be driving us.”

While that project is ongoing, Page and his colleagues continue to refine and improve upon the various laboratory steps required to isolate and analyze mRNA. Guided by the hope of providing the racing industry with a high-throughput screening tool, the group has employed multiple robotic platforms that can already handle 100 samples per day and be easily scaled up to handle more.

“As a researcher, I see it as being my job to provide practical and reliable solutions to the horse racing industry,” Page said. “I know that change can be scary, but we can all agree that something needs to change to help better protect racehorses and the jockeys who ride them. Ultimately, the racing industry will decide when it wants to give this screening tool a chance. I’m confident that, when the industry is ready, we will be too.”

The full study published in the Equine Veterinary Journal can be found here: https://doi.org/10.1111/evj.13423

Holly Wiemers, MA, APR, is the communications and managing director for UK Ag Equine Programs. Additional information provided by Allen Page, DVM, PhD, staff scientist and veterinarian at UK’s Gluck Equine Research Center and Emma Adam, DVM, PhD, DACVIM, DACVS, who is based at the University of Kentucky Gluck Equine Research Center and Veterinary Diagnostic Lab and is responsible for research and serves as a veterinary industry liaison.