Non-invasive fetal RHD exon 7 and exon 10 genotyping using real-time PCR testing of fetal DNA in maternal plasma

Objective: In this prospective study, we assessed the feasibility of foetal RHD genotyping by analysis of DNA extracted from plasma samples of Rhesus (Rh) D-negative pregnant women using real-time PCR and primers and probes targeted toward exon 7 and 10 of RHD gene. Methods: We analysed 24 RhD-negative pregnant woman and 4 patients with weak D phenotypes at a gestational age ranging from 11th to 38th week of gestation and correlated the results with serological analysis of cord blood after the delivery. Results: Non-invasive prenatal foetal RHD exon 7 genotyping analyses of maternal plasma samples was in complete concordance with the serological analysis of cord blood in all 24 RhD-negative pregnant women delivering 12 RhD-positive and 12 RhDnegative newborns. RHD exon-10-specific PCR amplicons were not detected in 2 out of 12 studied plasma samples from women bearing RhD-positive foetus, despite the positive amplification in RHD exon 7 region observed in all cases. In 1 case red cell serology of cord blood revealed that the mother had D-C-E-c+e+ C-w- and the infant D+C-E-c+e+ C-w+ phenotypes. RhD exon 10 real-time PCR analysis of cord blood was also negative. These findings may reflect that DCw- paternally inherited haplotype probably possesses no RHD exon 10. In another case no cord blood sample has been available for additional studies. The specificity of both RHD exon 7 and 10 systems approached 100% since no RhD-positive signals were detected in women currently pregnant with RhD-negative foetus (n = 8). Using real-time PCR and DNA isolated from maternal plasma, we easily differentiated pregnant woman whose RBCs had a weak D phenotype (n = 4) from truly RhD-negative patients since the threshold cycle (CT) for RHD exon 10 or 7 amplicons reached nearly the same value like CT for control P-globin gene amplicons detecting the total DNA present in maternal plasma. However in these cases foetal RhD status cannot be determined. Conclusion: Prediction of foetal RhD status from maternal plasma is highly accurate and enables implementation into clinical routine. We suggest that safe non-invasive prenatal foetal RHD genotyping using maternal plasma should involve the amplification of at least two RHD-specific products. Copyright (c) 2005 S. Karger AG, Basel.