Recently, some athletes were repetitively found to have rEPO positive results, including a characterized double-band pattern in blood samples, in routine doping analysis. In contrast to previous findings from excretion studies, this double-band pattern showed the same relative intensity even when the samples were collected weeks (/months) apart. We therefore suspected that these "positive" doping control samples were related with a novel pathway of endogenous EPO production. Thus, follow-up investigations were warranted to characterize the origin of such analytical test results and to avoid the issuing of adverse analytical findings in the absence of rEPO by identifying the root cause of these "constantly positives." In this study, we designed and conducted a series of causal studies, including population screening of EPO profiles, exploration of EPO de-N-glycosylation, single nucleotide polymorphism (SNP) browsing in EPO, sequencing of EPO exons, genealogical analysis of the c.577del EPO variant, and finally expression and investigation of mutant EPO. In summary, we found that these "constantly positives" were related to endogenous EPO production associated with the c.577del EPO variant. The frequency of this variant was 0.39% in our Chinese population pool. The mutant EPO encoded by this variant is 27 amino acids longer than the wild-type. The molecular weight of this mutant EPO is approximately the same as that of rEPO, exhibiting a similar electrophoretic behavior. To prevent charges against carriers of the c.577del variant, a revised rEPO testing strategy has been implemented in the new version of TD EPO.
Frameshift variant c.577del in the EPO gene can result in the extension of the amino acid sequence of EPO by invalidating the termination codon. As the molecular weight of its encoded protein EPO (VAR-EPO) is similar to that of rEPO, the World Anti-Doping Agency has published Annex B to the TD2022EPO in order to protect athletes with variant c.577del from the suspicion of rEPO administrations. However, it is still necessary to develop a confirmation method for rEPO that can discriminate rEPO from VAR-EPO.Based on the glycosylated characteristic of EPO, we selected the detection of de-Nglycosylated EPO as a complementary confirmation method for rEPO in blood samples. All samples were analyzed for both intact EPO and de-N-glycosylated EPO with SDS-PAGE, including rEPO spiked samples and blank samples.The results showed that, after de-N-glycosylation, a single-band was detected in samples collected from non-variant carriers, no matter whether the sample was spiked with rEPO. In samples collected from variant carriers, a double-band was detected. The ratio of lower band to upper band increased significantly corresponding to the concentration of rEPO. We calculated a series of cut-off values by normality distribution function to identify the presence of rEPO. Neither false positive results in blank samples nor false negative results in spiked samples at the applicable Minimum Required Performance Levels were found. This indicates that this method could be adopted as a complementary confirmation method for rEPO in blood samples. A revised testing strategy was also proposed, which would discriminate rEPO directly without further investigation.
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