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Genes Associated With Fracture Risk in Thoroughbred Horses Have Novel Roles in Osteogenesis.
Abstract: Bone fractures in Thoroughbred racehorses are a major welfare problem. Genetic factors contribute to fracture risk. Cell models have previously identified 112 differentially expressed genes in bone-forming osteoblasts derived from horses at high and low genetic risk of fracture. However, 42 of these genes have no published role in bone. In this study, we identified novel roles for a subset of these genes in bone formation. Twenty-six of the 42 genes were expressed in Saos2 cells during basal culture and/or after 21 days of osteogenic culture. Five of these genes (ADSSL1, CABP1, ENO2, SPARCL1 and UCP2) were then stably overexpressed and knocked down, and their effect on osteogenesis was measured. Gene overexpression resulted in significant decreases in Saos2 cell viability and decreased expression of osteogenic genes under basal cell culture, but after 21 days of osteogenic culture there were few significant changes in osteogenic gene expression, collagen deposition or matrix mineralisation. Knockdown of SPARCL1 resulted in total cell death, whereas knockdown of ADSSL1, CABP1, ENO2 and UCP2 resulted in decreased cell viability but limited significant changes in osteogenic gene expression under basal cell culture. However, following osteogenic culture, gene knockdown induced widespread changes in osteogenic gene expression, decreased collagen deposition and increased matrix mineralisation. ADSSL1, CABP1, ENO2 and UCP2 were all expressed at significantly lower levels in osteoblasts from genetically high-risk horses. Taken together, this work demonstrates novel roles for fracture-associated genes in bone formation and matrix mineralisation suggesting these processes may be altered in genetically susceptible horses.
© 2026 The Author(s). Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.
Publication Date: 2026-07-08 PubMed ID: 42418226PubMed Central: PMC13344464DOI: 10.1002/age.70166Google Scholar: Lookup The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
Summary
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Genes linked to fracture risk in Thoroughbred horses have been found to influence bone formation and mineralisation, suggesting these biological processes may be disrupted in horses genetically predisposed to fractures.
Background and Rationale
- Thoroughbred racehorses commonly suffer bone fractures, which pose significant welfare concerns.
- Genetic factors have been identified as contributors to fracture susceptibility.
- Previous studies using cell models revealed 112 genes differentially expressed between horses at high and low genetic risk of fracture.
- Of these 112 genes, 42 had no known roles in bone biology.
- This study focused on exploring whether these 42 genes have novel functions related to bone formation (osteogenesis) and mineralisation.
Gene Expression Analysis
- Researchers tested 26 of the 42 genes for expression in Saos2 cells, a human osteosarcoma cell line used as a model for bone-forming osteoblasts.
- These genes were analyzed under two conditions:
- Basal cell culture (normal growth conditions).
- After 21 days in osteogenic culture to induce differentiation and bone formation characteristics.
Functional Testing by Gene Manipulation
- Five genes of interest (ADSSL1, CABP1, ENO2, SPARCL1, and UCP2) were selected for detailed functional studies.
- For each gene, researchers performed:
- Stable overexpression (increasing gene activity).
- Knockdown (reducing gene activity) experiments.
- Effects on osteogenesis were measured by assessing:
- Cell viability (survival and proliferation of Saos2 cells).
- Expression of osteogenic marker genes.
- Collagen deposition (important for bone matrix formation).
- Matrix mineralisation (calcium deposition indicative of bone strength).
Results: Overexpression Effects
- Overexpressing the five genes mostly decreased cell viability during basal culture conditions.
- Osteogenic gene expression levels were generally reduced with overexpression under basal conditions.
- However, after 21 days of osteogenic induction, overexpression caused few significant changes in:
- Osteogenic gene expression.
- Collagen deposition.
- Matrix mineralisation.
Results: Knockdown Effects
- Knocking down SPARCL1 led to total cell death, highlighting its essential role in cell survival.
- Knocking down ADSSL1, CABP1, ENO2, and UCP2 reduced cell viability during basal culture but did not significantly alter osteogenic gene expression at this stage.
- After 21 days of osteogenic culture, knockdown of these genes induced:
- Extensive changes in osteogenic gene expression, indicating disrupted bone formation pathways.
- Reduced collagen deposition, impairing the bone matrix structure.
- Increased matrix mineralisation, suggesting altered regulation of mineral deposition.
Expression Levels in High-Risk Horses
- The four genes ADSSL1, CABP1, ENO2, and UCP2 were found to be expressed at significantly lower levels in osteoblasts derived from horses genetically predisposed to fractures.
- This correlation supports the idea that reduced expression of these genes may contribute to the compromised bone quality seen in high-risk animals.
Conclusions and Implications
- This study identifies novel roles for fracture-associated genes in regulating bone formation and matrix mineralisation processes.
- The altered gene expression and subsequent effects on osteoblast function and bone matrix in genetically high-risk horses could underlie their increased fracture susceptibility.
- Findings expand understanding of the genetic and cellular mechanisms influencing bone health in Thoroughbred horses.
- Potential future applications include genetic screening and targeted therapeutic strategies to improve bone strength and reduce fracture risk.
Cite This Article
APA
Ross AC, Lumsden ES, Flood C, Dudhia J, Psifidi A, Guest DJ.
(2026).
Genes Associated With Fracture Risk in Thoroughbred Horses Have Novel Roles in Osteogenesis.
Anim Genet, 57(4), e70166.
https://doi.org/10.1002/age.70166 Publication
Researcher Affiliations
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
- Department of Clinical Sciences and Services, Centre for Vaccinology and Regenerative Medicine, The Royal Veterinary College, Hatfield, Herts, UK.
MeSH Terms
- Animals
- Horses / genetics
- Osteogenesis / genetics
- Fractures, Bone / genetics
- Fractures, Bone / veterinary
- Osteoblasts / metabolism
- Horse Diseases / genetics
Grant Funding
- Alborada Trust
Conflict of Interest Statement
The authors are affiliated with The Royal Veterinary College, which holds patent WO 2015/019097 “Predictive Method for Bone Fracture Risk in Horses” in relation to this work. This patent claims a method of predicting fracture risk in horses using one or more genetic variations from within the associated region on ECA18.
References
This article includes 44 references
- Addison WN, Azari F, Sørensen ES, Kaartinen MT, McKee MD. Pyrophosphate Inhibits Mineralization of Osteoblast Cultures by Binding to Mineral, Up‐Regulating Osteopontin, and Inhibiting Alkaline Phosphatase Activity. Journal of Biological Chemistry 282: 15872–15883.
- Bailey S, Karsenty G, Gundberg C, Vashishth D. Osteocalcin and Osteopontin Influence Bone Morphology and Mechanical Properties. Annals of the New York Academy of Sciences 1409: 79–84.
- Berezovska O, Yildirim G, Budell WC. Osteocalcin Affects Bone Mineral and Mechanical Properties in Female Mice. Bone 128: 115031.
- Bertrand S, Fuentealba J, Aze A. A Dynamic History of Gene Duplications and Losses Characterizes the Evolution of the SPARC Family in Eumetazoans. Proceedings of the Royal Society B: Biological Sciences 280: 20122963.
- Blott SC, Swinburne JE, Sibbons C. A Genome‐Wide Association Study Demonstrates Significant Genetic Variation for Fracture Risk in Thoroughbred Racehorses. BMC Genomics 15: 147.
- Bogers SH, Rogers CW, Bolwell C, Roe W, Gee E, McIlwraith CW. Quantitative Comparison of Bone Mineral Density Characteristics of the Distal Epiphysis of Third Metacarpal Bones From Thoroughbred Racehorses With or Without Condylar Fracture. American Journal of Veterinary Research 77: 32–38.
- Chen L, Jacquet R, Lowder E, Landis WJ. Refinement of Collagen–Mineral Interaction: A Possible Role for Osteocalcin in Apatite Crystal Nucleation, Growth and Development. Bone 71: 7–16.
- Chen Y, Liu Y, Zhang Y, Yu J, Tang L. Galectin‐1 Deletion in Mice Causes Bone Loss via Impaired Osteogenic Differentiation Potential of BMSCs. FASEB Journal 36: e22516.
- Choi IA, Umemoto A, Mizuno M, Park‐Min KH. Bone Metabolism—An Underappreciated Player. NPJ Metabolic Health and Disease 2: 12.
- Diegel CR, Hann S, Ayturk UM. An Osteocalcin‐Deficient Mouse Strain Without Endocrine Abnormalities. PLoS Genetics 16: e1008361.
- Ducy P, Desbois C, Boyce B. Increased Bone Formation in Osteocalcin‐Deficient Mice. Nature 382: 448–452.
- Gagliardi F, Narayanan A, Mortini P. SPARCL1 a Novel Player in Cancer Biology. Critical Reviews in Oncology/Hematology 109: 63–68.
- Georgopoulos SP, Parkin TD. Risk Factors for Equine Fractures in Thoroughbred Flat Racing in North America. Preventive Veterinary Medicine 139: 99–104.
- Hauschka PV, Wians FH Jr. Osteocalcin‐Hydroxyapatite Interaction in the Extracellular Organic Matrix of Bone. Anatomical Record 224: 180–188.
- Kemper AM, Drnevich J, McCue ME, McCoy AM. Differential Gene Expression in Articular Cartilage and Subchondral Bone of Neonatal and Adult Horses. Genes 10: 745.
- Kuemmerle JM, Theiss F, Okoniewski MJ. Identification of Novel Equine Tendon Markers Using RNA Sequencing. Genes 7: 97.
- Lazic SE, Clarke‐Williams CJ, Munafò MR. What Exactly Is ‘N’ in Cell Culture and Animal Experiments?. PLoS Biology 16: e2005282.
- Lipps G, Krauss G. Adenylosuccinate Synthase From : Homologous Overexpression, Purification and Characterization of the Recombinant Protein. Biochemical Journal 341, no. pt. 3: 537–543.
- Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real‐Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25, no. 4: 402–408.
- Loughridge AB, Hess AM, Parkin TD, Kawcak CE. Qualitative Assessment of Bone Density at the Distal Articulating Surface of the Third Metacarpal in Thoroughbred Racehorses With and Without Condylar Fracture. Equine Veterinary Journal 49: 172–177.
- Moriishi T, Ozasa R, Ishimoto T. Osteocalcin Is Necessary for the Alignment of Apatite Crystallites, but Not Glucose Metabolism, Testosterone Synthesis, or Muscle Mass. PLoS Genetics 16: e1008586.
- Nakamura A, Dohi Y, Akahane M. Osteocalcin Secretion as an Early Marker of In Vitro Osteogenic Differentiation of Rat Mesenchymal Stem Cells. Tissue Engineering. Part C, Methods 15: 169–180.
- Noordwijk KJ, Chen L, Ruspi BD. Metacarpophalangeal Joint Pathology and Bone Mineral Density Increase With Exercise but Not With Incidence of Proximal Sesamoid Bone Fracture in Thoroughbred Racehorses. Animals 13: 827.
- Palomino Lago E, Baird A, Blott SC. A Functional Single‐Nucleotide Polymorphism Upstream of the Collagen Type III Gene Is Associated With Catastrophic Fracture Risk in Thoroughbred Horses. Animals 14: 116.
- Palomino Lago E, Roberts SJ, Orriss IR, Lumsden ES, Guest DJ. Generation and Characterization of an Immortalized Equine Osteoblast Cell Line. Veterinary Journal 316: 106573.
- Palomino Lago E, Ross AKC, McClellan A, Guest DJ. Identification of a Global Gene Expression Signature Associated With the Genetic Risk of Catastrophic Fracture in iPSC‐Derived Osteoblasts From Thoroughbred Horses. Animal Genetics 56: e13504.
- Paracha N, Mastrokostas P, Kello E. Osteocalcin Improves Glucose Tolerance, Insulin Sensitivity and Secretion in Older Male Mice. Bone 182: 117048.
- Price PA, Otsuka AA, Poser JW, Kristaponis J, Raman N. Characterization of a Gamma‐Carboxyglutamic Acid‐Containing Protein From Bone. Proceedings of the National Academy of Sciences of the United States of America 73: 1447–1451.
- Riggs CM. Fractures—A Preventable Hazard of Racing Thoroughbreds. Veterinary Journal 163: 19–29.
- Robling AG, Bonewald LF. The Osteocyte: New Insights. Annual Review of Physiology 82: 485–506.
- Rosanowski SM, Chang YM, Stirk AJ, Verheyen KLP. Risk Factors for Race‐Day Fatality in Flat Racing Thoroughbreds in Great Britain (2000 to 2013). PLoS One 13: e0194299.
- Ross AC, Feddersen L, Dudhia J, Psifidi A, Guest DJ. Modulating Galectin‐1 in Human Osteoblast‐Like Cells Alters Mineralisation and Influences the Expression of Genes Associated With Osteoblasts and Osteocytes. Bioscience Reports 46: BSR20253890.
- Rosset EM, Bradshaw AD. SPARC/Osteonectin in Mineralized Tissue. Matrix Biology 52–54: 78–87.
- Shaffer SK, To C, Garcia TC, Fyhrie DP, Uzal FA, Stover SM. Subchondral Focal Osteopenia Associated With Proximal Sesamoid Bone Fracture in Thoroughbred Racehorses. Equine Veterinary Journal 53: 294–305.
- Shi L, Wang D, Riggs CM, Qin L, Griffith JF. Statistical Analysis of Bone Mineral Density Using Voxel‐Based Morphometry—An Application on Proximal Sesamoid Bones in Racehorses. Journal of Orthopaedic Research 29: 1230–1236.
- Takeuchi T, Oyama M, Tamura M, Arata Y, Hatanaka T. Reduced Form of Galectin‐1 Suppresses Osteoclastic Differentiation of Human Peripheral Blood Mononuclear Cells and Murine RAW264 Cells In Vitro. Biomolecules 14: 121.
- Tian XY, Ma S, Tse G, Wong WT, Huang Y. Uncoupling Protein 2 in Cardiovascular Health and Disease. Frontiers in Physiology 9: 1060.
- Tozaki T, Kusano K, Ishikawa Y. A Candidate‐SNP Retrospective Cohort Study for Fracture Risk in Japanese Thoroughbred Racehorses. Animal Genetics 51: 43–50.
- Tozaki T, Miyake T, Kikuchi M. Heritability Estimates of Fractures in Japanese Thoroughbred Racehorses Using a Non‐Linear Model. Journal of Animal Breeding and Genetics 136: 199–204.
- Tsao YT, Huang YJ, Wu HH, Liu YA, Liu YS, Lee OK. Osteocalcin Mediates Biomineralization During Osteogenic Maturation in Human Mesenchymal Stromal Cells. International Journal of Molecular Sciences 18: 159.
- Welsh CE, Lewis TW, Blott SC, Mellor DJ, Stirk AJ, Parkin TD. Estimates of Genetic Parameters of Distal Limb Fracture and Superficial Digital Flexor Tendon Injury in UK Thoroughbred Racehorses. Veterinary Journal 200: 253–256.
- Wright I, Minshall G, Young N, Riggs C. Fractures in Thoroughbred Racing and the Potential for Pre‐Race Identification of Horses at Risk. Equine Veterinary Journal 56: 424–436.
- Xu W, Ni C, Wang Y, Zheng G, Zhang J, Xu Y. Age‐Related Trabecular Bone Loss Is Associated With a Decline in Serum Galectin‐1 Level. BMC Musculoskeletal Disorders 22: 394.
- Zhou Y, Zeng F, Richards GO, Wang N. ENO2, a Glycolytic Enzyme, Contributes to Prostate Cancer Metastasis: A Systematic Review of Literature. Cancers 16: 2503.
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