New Transcriptomic Biomarkers for Detection of the Recombinant Human Erythropoietin (rHuEPO) MirCERA in Horses

Detection and monitoring of biomarkers related to doping is particularly suitable for the development of analytical strategies dedicated to indirect detection of banned substances. Previous studies in horses have already allowed the investigation of transcriptomic biomarkers in equine blood associated with reGH and rHuEPO administrations. Our most recent developments continue to focus on the discovery and monitoring of transcriptomic biomarkers for the control of ESAs, and a collaborative study with WADA-accredited doping control laboratories has recently been initiated to conduct a pilot study. In humans, three mRNAs (ALAS2, CA1, and SLC4A1) were previously observed to be differentially expressed after blood doping and were associated with immature red blood cells, the so-called circulating reticulocytes. In horses, circulating reticulocytes are rarely observed even after rHuEPO administration. With the improved primers that detect the equine orthologues of the human mRNAs from the ALAS2, CA1, and SLC4A1 genes, we can now report the first evidence of the detection of the three biomarkers in equine blood. In addition, an upregulation of the mRNA levels of the three genes was observed after analysis of blood samples collected from MirCERA-treated animals, with kinetics similar to those previously documented in humans. Our data suggest that ALAS2 and CA1 are promising indirect biomarkers for the detection of recombinant EPO abuse in horses, as observed in humans. ALAS2, CA1, and SLC4A1 mRNAs have previously been described as blood doping biomarkers in humans. A qPCR method was developed to detect the equine orthologues in equine whole blood, and their upregulation was observed after administration of a single dose of MirCERA. This work provides proof of concept for the use of ALAS2, CA1, and SLC4A1 in horses for doping control as an indirect method of detecting rHuEPO MirCERA abuse by monitoring transcriptomic biomarkers.image