Risk free simultaneous prenatal identification of fetal Rhesus D status and sex by multiplex real-time PCR using cell free fetal DNA in maternal plasma

Questions under study: Pregnancies with a Rhesus constellation still presen t a considerable obstetric problem. Tn addition, pregnancies with male Rhes us D fetuses are more severely affected by haemolytic disease of the newbor n, requiring more transfusions in utero and having a three fold higher mort ality than female Rhesus D fetuses. Furthermore, almost 150 X-linked geneti c deficiencies have now been characterised, increasing the need for prenata l sex determination in pregnancies at risk for such a disorder. In order ex amine these two important fetal loci in a risk free manner, we have develop ed a novel multiplex real-time PCR assay for the analysis of extracellular fetal DNA in maternal plasma. Methods: Cell free DNA was isolated from 34 maternal plasma samples and exa mined by a multiplex real-time PCR assay for the Rhesus D gene and the SRY locus on the Y chromosome. Results: Our study showed that we were able to genotype 12/13 Rhesus D male s correctly. All 5 Rhesus d males were correctly identified. In addition a 100% concordance was found in the 16 samples obtained from pregnancies with female Rhesus D or Rhesus d fetuses. Conclusions: By developing a novel multiplex real-time PCR assay we present the first report describing the determination of multiple fetal loci from cell free DNA in maternal plasma by these means. As this assay is suitable for automation, our data, therefore, suggest that such assays provide a goo d basis for the clinical examination of multiple fetal loci, in particular Rhesus D status or fetal sex, anti can be performed effectively using real- time multiplex PCR assays.