Non-invasive fetalRHD genotyping on DNA isolated from maternal plasmain RhD negative pregnancies

Overview

In this prospective study, we assessed the feasibility of fetal RHD genotyping by analysis of DNAextracted from maternal plasma samples collected from RhD negative pregnant women using RQ-PCRand primers and probes targeted toward exon 7 and exon 10 of RHD gene.We analysed 46 samples from39 RhD negative pregnant women including 5 sensitised (1x anti-D, 2x anti-D and anti-C, 2x non-specificantibodies in enzymatic assay) and correlated the results with serological analysis of cord blood afterthe delivery. Non-invasive prenatal fetal RHD exon 7 genotyping analysis of maternal plasma sampleswas in complete concordance with the analysis of cord blood in all 20 RhD negative pregnant womendelivering 12RhDpositive and8RhDnegativenewbornsuntilnow.RHDexon 10 specificPCRampliconswere not detected in 2 out of 12 studied plasma samples from women bearing RhDpositive fetus, despitethe positive amplification in RHD exon 7 region observed in all cases. We supposed that false negativeresults might be caused by a partly deletion or aberrant structure of the 3´ untranslated exon 10 regionof RHD gene where specific primers and probe should anneal. The specificity of both RHD exon 7 andexon 10 systems approached 100% since no RHD positive signals were detected in women currentlypregnant with RhD negative fetus (n = 8). Prediction of fetal Rhesus D status from maternal plasma ishighly accurate and enables implementation into clinical routine. We suggest that safe non-invasiveprenatal fetal RHD genotyping using maternal plasma should involve the amplification of at least twoRHD specific products. Fetal RHD genotyping may allow the identification of fetuses at risk ofhaemolytic disease of newborn. However,we suggest to apply a non-invasive prenatal RHD genotyping assay in the routine testing of allRhDnegativewomen involving both sensitised as well as nonsensitizedpregnancies, which is the majority.

Key words:
fetal DNA, haemolytic disease of newborn, real-time PCR, maternal plasma, RHD gene