Detection of exon 5 c.577del variant of human erythropoietin gene in whole blood, dried blood spots and urine samples for doping control

Abstract

Background: In doping control, the presence of the exon5 c.577del variant in the human erythropoietin gene may be a confounding factor in the interpretation of the results from the analytical method currently in force for the detection of human recombinant erythropoietin, based on immunoelectrophoresis on SDS-PAGE and/or SAR-PAGE. This variant, determining the transcription of a higher molecular weight protein, can erroneously suggest the presence of recombinant erythropoietin in a biological sample, causing the possibility of a false positive result. Although the variant was now identified only in East Asian populations and with a very low frequency, it can threaten the reliability of current anti-doping tests.Methods: We have implemented a genetic test to identify the presence of this variant in the biological samples that are presently collected for anti-doping analysis (whole blood, urine, and dried blood spots). The test is based on the Sanger sequencing of the human erythropoietin gene exon 5, where the c.577del variant falls.Results and Discussion: The method has a specificity of 100% and allows identification of the possible presence of the variant starting from 100 pg of genomic DNA extracted from each biological sample. The efficacy of the test has been confirmed by the analysis of real samples from subjects showing and not showing the exon c.577del variant.

Dried blood spots for erythropoietin analysis: Detection of micro‐doses, EPO c.577del variant and comparison with in‐competition matching urine samples

“…Their EPO profiles unambiguously showed a double‐band, confirming that 2 spots of 20‐μL Whatman DMPK‐C DBS could be used as an alternative to serum or plasma to identify or exclude the expression of this EPO gene variant by an athlete. As DBS has made its proof for genetic analyses, one DBS could alternatively or additionally be used for genotyping the EPO c.577del gene fragment 10,14 …”

“…In case of a heterozygous expression of the variant, two EPO bands are present 9 . If sensitivity is sufficient, DBS could also be appropriate for this additional analysis alternatively to a serum/plasma sample or used to perform the sequencing of the EPO gene 10 …”

Detection of Gene Doping Using Dried Blood Spots from a Mouse Model with rAAV9 Vector-Mediated Human Erythropoietin Expression as a Pilot Study

“…Therefore, automating most processes in doping testing is desirable as well. There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18].…”

“…Moreover, this approach not only promises considerable cost reductions but also the possibility for long-term sample preservation. As an example, experimental results have been reported for detecting steroids or genetic polymorphism using DBSs, and it has been shown that DBSs look promising for the future [8,9].…”