Disentangling Erythropoietin-specific Gene Expression From Confounding Factors: A Transcriptomic Approach For Anti-doping

PURPOSE: Recombinant human erythropoietin (rHuEPO) is a banned substance used to enhance physical performance by stimulating red blood cell production. Detecting its misuse remains challenging as its effects can mimic those of exercise, altitude training, and illness. This study aims to identify gene expression changes specific to rHuEPO, helping to distinguish it from other confounders. METHODS: Gene expression was analysed in endurance trained individuals who received rHuEPO. RNA sequencing (MGI DNBSEQ, Illumina NextSeq 500) and microarray technology were used to identify differentially expressed genes (DEGs). Functional analyses were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. These findings were compared with previous rHuEPO studies and datasets from altitude exposure studies. Identified DEGs were also cross-referenced with our previous established datasets GEPREP and previous studies to determine whether they were uniquely associated with rHuEPO or shared with adaptations to exercise, hypoxia, inflammation or other. RESULTS: 16,738 genes (MGI RNA-seq) and 16,581 genes (Illumina RNA-seq) were identified in differential gene expression analyses. RNA sequencing demonstrated greater sensitivity than microarrays. TMOD1, XK, RBM38, SNCA, SELENBP1, BCL2L1, BPGM, FECH, FAM46C, MARCH8, SIAH2, OSBP2, and E2F2 were consistently found in rHuEPO studies but not in altitude exposure studies, suggesting their potential as specific biomarkers for detecting rHuEPO use.VEGFA, HIF1A, and IL6 exhibited overlapping expression between EPO, exercise, and hypoxia, underscoring the challenge of distinguishing doping from natural adaptations. CONCLUSION: Our findings enhance the ability to differentiate rHuEPO doping from natural physiological responses by identifying unique gene expression signatures. The consistency of these gene markers across multiple EPO studies strengthens their potential as anti-doping biomarkers. While transcriptomic analysis provides strong evidence for EPO specific gene expression, definitive proof requires further validation through laboratory-based functional assays. This research contributes to more reliable anti-doping strategies, improves our understanding of gene expression responses to doping and environmental stimuli.