Blood cells RNA biomarkers as a first long‐term detection strategy for EPO abuse in horseracing

Abstract

Recombinant human erythropoietins (rHuEPOs) are glycoproteins drugs, produced by the pharmaceutical industry to restore production of red blood cells by stimulating human bone marrow for which this pathology has been diagnosed. It is suspected that these molecules are diverted as doping agents in horseracing to enhance oxygen transport and aerobic power in racehorses. Although indirect double-blotting or direct liquid chromatography-mass spectrometry (LC-MS) methods have been developed to confirm the presence of rHuEPO in a sample, the short detection time (48 h) is still a problem for doping control. In this context, gene profiling investigation through Serial Analysis of Gene Expression (SAGE) has been conducted on seven thoroughbreds treated with Eprex. This functional genomic method has been performed from total blood cells collected from each animal to assess the mRNA expression consecutive to rHuEPO injections. Sample pooling was chosen as a powerful, cost-effective, and rapid means of identifying the most common and specific changes in terms of gene expression profile and to eliminate individual variation. Consequently, three SAGE libraries were constructed, before, during, and after Eprex treatment. More than 71 440 mRNA signatures were observed and subjected to statistical analysis; 49 differentially expressed genes were identified and analyzed by qPCR. From the selected gene list, were defined as potential biomarkers in terms of their low inter-individual variation and capacity as strong markers of rHuEPO administration up to 60 days after the beginning of the doping period. In this paper, a new strategy is proposed to the horseracing industry to prevent rHuEPO abuse.

UPLC-MS/MS Method for the Identification of Recombinant Human Erythropoietin Analogues in Horse Plasma and Urine

“…Furthermore, the use of an internal standard with rhEPO LC-MS/MS assays has not been reported. In addition to IEF and LC-MS/MS methods of detection, a novel assay based on blood cell RNA biomarkers has recently been published, which provides an interesting possibility for the future detection of rhEPO use once further validation experiments using larger populations of animals have been conducted [30].…”

Optimization of Extraction of Circulating RNAs from Plasma – Enabling Small RNA Sequencing

“…Besides profiling of clinical samples for disease-specific biomarkers, the screening for distinct and convincing RNA signatures is an applied method in the surveillance of food safety and anti-doping control systems in food-delivering livestock as well as in human or equine competitive sports [5], [14], [15]. Including small RNAs in the screening for differentially expressed genes on the transcriptional level could lead to multiple RNA biomarkers that are more robust, reliable and failure-safe.…”

Intelligence‐based anti‐doping from an equine biological passport

“…The search for suitable EBP biomarkers will undoubtedly further increase the multidisciplinary scope of equine anti‐doping science – from small‐molecule chemistry to molecular biology. An example of this has been reported by Bailly‐Choruiberry et al with changes in the expression of eight target genes following administration of recombinant human erythropoietin to thoroughbred horses. More generally, Reichel presented OMICS‐strategies and methods in the fight against doping , highlighting the relationship between genomics, transcriptomics, proteomics and metabolomics for identifying novel biomarkers and the applicability of each of these for longitudinal ‘passport’ monitoring.…”