A System Biology Biomarkers Approach to the Differentiation of Recombinant Human Erythropoietin Doping from Confounding Factors
The main purpose of two research projects funded by the World Anti-Doping Agency in 2008 and 2012 entitled "A gene-microarray based approach to the detection of recombination human erythropoietin (rHuEpo) doping in endurance athletes" and "A systems biology biomarker based approach to the detection of microdose rHuEpo doping" was to develop new improved methods based on gene expression profiles. On the basis of the data generated to date, blood gene expression profiles were profoundly altered during rHuEpo and for at least 4 weeks after administration leading to a "molecular signature" of rHuEpo doping. These most promising data provide the strongest evidence to date that gene biomarkers will add a new dimension to the Athlete Biological Passport in terms of sensitivity and specificity.
Given the promising results, it is now of paramount importance and of great urgency to evaluate the effects of major confounding factors on this "molecular signature" of rHuEpo doping. These include: 1. The effects of altitude. Altitude training is used by athletes for performance enhancement and has the potential to influence indices of rHuEpo doping. There are concerns about the risk of false positive as well as the misuse of altitude to mask blood doping practices. 2. The effects of prior exercise. Samples are frequently collected at sporting or training venues after intense exercise which could potentially influence the "molecular signature" of rHuEpo.
In order to provide a set of robust candidate genes that can be used for the detection of rHuEpo doping and a basis for recommendations on the collection of samples, this project will: 1. Compare blood gene expression profiles altered by rHuEpo with altitude exposure and determine genes that can be used to differentiate rHuEpo from altitude training; and
2. Assess the effects of prior exercise on blood gene expression after microdose rHuEpo.
All necessary confounder studies were successfully performed (and biobanked for future use) to address the ambitious objectives of this research. Results confirm the enhanced sensitivity of the blood transcriptomic approach over standard haematological markers to detect changes in response to rHuEpo and potentially ABT. Importantly, the confounder analysis clearly demonstrates a large and unique transcriptomic response to rHuEpo involving thousands of genes compared to the major confounders that include altitude and strenuous exercise. On the basis of these results, it can be concluded with greater confidence that “omics” technologies will significantly strengthen current anti-doping strategies. In particular, the use of molecular biomarkers with an improved detection window and high sensitivity and specificity to develop the transcriptionally enhanced ABP model for detecting blood doping. Collectively, the findings of the present research, interpreted in the context of the latest “omics” research in biomedical sciences, are most encouraging and confirm claims that a systems biology approach combining various “omics” signatures from genomics, transcriptomics, proteomics and metabolomics will inevitably provide a deeper understanding of the effects of Epo stimulating agents on erythropoiesis with unparalleled potential to improve current drug detection strategies with particular reference to blood doping.