Investigation of pseudogenes RHDΨ and RHD -CE-D hybrid gene in D-negative blood donors by the real time PCR method

IntroductionThe Rh system is the most polymorphic and immunogenic of all systems of blood groups. Currently more than 49 antigens were identified with five major antigens D, C, c, E, e. Knowledge of the molecular basis of the Rh system permitted the understanding of both the mechanism of Rh phenotype on the antigen variants of RHD and RHCE In Caucasians the primary mechanism of D-negative phenotype is the complete deletion of RHD gene, while the black Africans is the presence of pseudogene and gene hybrid RHD-CE (3–7)-D.ObjectiveTo determine the prevalence gene pseudogene and hybrid gene and standardization of molecular techniques in method of Taqman on real-time PCR for RHD genotyping.Patients and methods203 samples of D-negative donor were used to establish and validate the effectiveness of RHD genotyping in real-time PCR using Taqman technology. The extraction was performed using a commercial kit QIAmp DNA mini kit. Samples exon 10 and 7 positive were submitted to amplification of exon 5, confirming the pseudogene RHDΨ, whereas exon 10 + exon 7 – for the hybrid gene (C) cdes and mutation C733G (Leu245Val) of the RHCE gene.ResultsTwenty-five (12.3%) samples were positive, 14 amplified for both exons 10 and 7 while in 11 only for the exon 10. When extended the screening using exon 10, 7 and 5, only 06 amplified. The pseudogene was present in 07 samples (3.5%) and the hybrid RHD-CE (3–7) in 04 (1.97%), while in 177 (87.2%) of Rh negative donors were RHD gene deletion. In 07 samples not amplified for exon 3 had mutated and the mutation C733G antigen.ConclusionThe prevalence of pseudogene was 3.5% and the gene hybrid RHD-CE of 1.9%. This approach for real-time PCR as a complementary tool is technically feasible and the results of this study helped develop a new strategy for RHD genotyping.