Characterization of DNA concentration in urine and dried blood samples to detect the c.577 deletion within the EPO gene

Nicolas Leuenberger; Nicolas Jan; Tiia Kuuranne; Vincent Castella

doi:10.1002/dta.3647

Characterization of DNA concentration in urine and dried blood samples to detect the c.577 deletion within the EPO gene

Drug Test Anal. 2024 Jan 21. doi: 10.1002/dta.3647. Online ahead of print.

Authors

Nicolas Leuenberger¹, Nicolas Jan¹, Tiia Kuuranne¹, Vincent Castella²

Affiliations

¹ Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.
² Forensic Genetics Unit, University Center of Legal Medicine, Lausanne-Geneva, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland.

PMID: 38247130
DOI: 10.1002/dta.3647

Abstract

The EPO gene variant, c.577del (VAR-EPO), was discovered in the Chinese population in 2021. The mutated protein is naturally present in urine from individuals heterozygous for the variant. Electrophoresis methods currently applied in anti-doping laboratories produce a pattern in samples from individuals carrying VAR-EPO that cannot be unambiguously distinguished from individuals who received recombinant EPO doses. Consequently, the analysis of blood samples is obligatory to facilitate interpretation of suspicious findings from urine samples. However, this complicates the process and delays the reporting. Objective of this study was to develop EPO c.577del detection in urine and dried blood samples (DBS) in order to facilitate and accelerate EPO results management. Moreover, estimation of the success rate of sequencing regarding concentration of DNA in urine and DBS was evaluated. Conclusive results regarding Sanger sequencing were obtained for all samples with DNA concentrations above 0.024 ng/μL DNA in 80% of urines samples from volunteers. The potential success of DNA sequencing rate in athletes' urines was investigated. A total of 191 urine samples were considered. DNA concentration exceeding 0.024 ng/μL was detected in 85% of the samples. Interestingly, in-competition samples had a significantly higher DNA concentration than out-of-competition male urine samples (0.330 vs. 0.084 ng/μL). Moreover, conclusive EPO sequences were obtained for 100% of DBS (cellulose and polymer matrices). In conclusion, method for detection of EPO gene variant was developed in urine and DBS. Characterization of DNA concentration was performed in order to evaluate the probability of success of sequencing EPO gene in anti-doping field.

Keywords: blood doping; dried blood samples; genetic sequencing.