Discovery of c.577del in EPO: Investigations into endogenous EPO double‐band detected in blood with SAR‐PAGE

Abstract

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.

Detection of de‐N‐glycosylated EPO with SDS‐PAGE: A complementary confirmation procedure for recombinant EPO in blood samples

“…De‐N‐glycosylation was conducted according to a previously described method 8,12 . Same as the immunopurification protocol for intact EPO, diluted sample from 500 μL of serum/plasma was passed through a MAIIA column first.…”

“…De-N-glycosylation was conducted according to a previously described method. 8,12 Same as the immunopurification protocol for intact EPO, diluted sample from 500 μL of serum/plasma was passed through a MAIIA column first. After washing, the column was eluted with 40 μL of denaturing buffer (50 mM ammonium bicarbonate, 1% SDS, and 1% β-mercaptoethanol).…”

“…The MW of its encoded protein EPO_p.Arg193AspfsTer28 (VAR‐EPO) would increase by 3.3 kDa relative to WT‐EPO as a result of the 27 AA‐length extension, resulting in an MW similar to that of rEPO. Both WT‐EPO and VAR‐EPO will be naturally present in urine and blood samples from heterozygous variant carriers, giving rise to an EPO pattern (double‐band in blood and a “mixed” WT‐EPO and VAR‐EPO band in urine) that cannot be robustly distinguished from the EPO pattern seen in samples from individuals who indeed use rEPO by the current SAR‐PAGE or SDS‐PAGE method 8 …”

“…Both WT-EPO and VAR-EPO will be naturally present in urine and blood samples from heterozygous variant carriers, giving rise to an EPO pattern (double-band in blood and a "mixed" WT-EPO and VAR-EPO band in urine) that cannot be robustly distinguished from the EPO pattern seen in samples from individuals who indeed use rEPO by the current SAR-PAGE or SDS-PAGE method. 8 WADA has published an annex to the TD2022EPO (Annex B) to guide how to deal with samples with rEPO positive findings after confirmation. 9 Currently, we can determine whether the relevant athlete is a c.577del carrier by additional DNA sequencing after a series of investigations and further analysis.…”

“…Our previous research has proved the deduction that after de-N-glycosylation, the MW of WT-EPO and rEPO is the same, which is nearly 5 kDa lower than that of the VAR-EPO when tested by SDS-PAGE. 8 Based on this, our goal was to test the detection of de-N-glycosylated EPO as a complementary method of confirmation for rEPO, which can distinguish the rEPO and VAR-EPO directly.…”