The use of RNA‐based 5'‐aminolevulinate synthase 2 biomarkers in dried blood spots to detect recombinant human erythropoietin microdoses

Abstract

The hematological module of the Athlete Biological Passport (ABP) is used for indirect detection of blood manipulations; however, the use of this method to detect doping, such as with microdoses of recombinant human erythropoietin (rhEPO), is problematic. For this reason, the sensitivity of ABP must be enhanced by implementing novel biomarkers. Here, we show that 5'-aminolevulinate synthase 2 (ALAS2) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing. Moreover, the sensitivity was sufficient to distinguish rhEPO administration from exposure to hypoxic conditions. Levels of mRNAs encoding carbonate anhydrase 1 (CA1) and solute carrier family 4 member 1 (SLC4A1) RNA, as well as the linear (L) and linear + circular (LC) forms of ALAS2 mRNA, were monitored for 16 days after rhEPO microdosing and during exposure to hypoxic conditions. ALAS2 mRNAs increased by 300% compared with the baseline values after rhEPO microdosing. Moreover, ALAS2 mRNAs were not significantly increased under hypoxic conditions. By contrast, CA1 mRNA was increased after both rhEPO microdosing and hypoxia, whereas SLC4A1 mRNA did not significantly increase under either condition. Furthermore, the analyses described here were performed using dried blood spots (DBSs), which provide advantages in terms of the sample collection, transport, and storage logistics. This study demonstrates that ALAS2 mRNA levels are sensitive and specific transcriptomic biomarkers for the detection of rhEPO microdosing using the hematological module of the ABP, and this method is compatible with the use of DBSs for anti-doping analyses.

Proof of Gene Doping in a Mouse Model with a Human Erythropoietin Gene Transferred Using an Adenoviral Vector

“…In this study, since the amount of circulating EPO hormone increased in our mouse model, we assumed that the RNAs in whole-blood samples from the model were showing the same fluctuations as those reported in the studies described above. The total RNA-seq results were largely consistent with the expression patterns described in previous reports [57][58][59] (Supplementary Figure S6). In particular, five genes (Bcl2l1, Epb42, Slc4a1, Tmod1, and Trim58) that were analyzed as ABP candidate parameters in the previous study by Wang et al [58] showed a clear increase of about 2-4 times the average value, which was statistically significant (Supplementary Figure S6).…”

“…A previous study reported the findings obtained after administration of recombinant hEPO (rhEPO) to humans and transcriptome analysis using whole-blood RNA [57]. The RNAs of ABP parameter candidates found in that study [57] were also investigated in detail in other studies and could be recognized as robust biomarker RNA candidates for detecting rhEPO-administered doping [58,59]. In this study, since the amount of circulating EPO hormone increased in our mouse model, we assumed that the RNAs in whole-blood samples from the model were showing the same fluctuations as those reported in the studies described above.…”

Biomarker analysis

“…DBS is a complementary approach to traditional urine and venous blood matrices, which can facilitate simpler sample collection and increase the number of samples available for biomarker analysis due to reduced costs. Loria et al 7 The theme of differentiation is further explored by Ishii et al 13 for a biomarker approach to distinguish nicotine administration from tobacco exposure in horses. Both trans-3 0 -hydroxycotinine and anatabine could be used as potential biomarkers in equine urine and plasma to indicate recent exposure to tobacco products in horses.…”

“…The potential use of dried blood spots (DBS) for biomarker analysis is presented by Rocca et al 6 and Loria et al 7 for improved detection of recombinant erythropoietins. DBS is a complementary approach to traditional urine and venous blood matrices, which can facilitate simpler sample collection and increase the number of samples available for biomarker analysis due to reduced costs.…”

“…DBS is a complementary approach to traditional urine and venous blood matrices, which can facilitate simpler sample collection and increase the number of samples available for biomarker analysis due to reduced costs. Loria et al 7 demonstrated that 5′‐aminolevulinate synthase 2 ( ALAS2 ) mRNAs are useful transcriptomic biomarkers to improve the indirect detection of rhEPO microdosing, which could distinguish exposure to hypoxic conditions and improve the sensitivity of the ABP.…”