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.
OBJECTIVE
Pregnancies with a Rhesus constellation still present a considerable obstetric problem. In addition, pregnancies with male Rhesus D fetuses are more severely affected by haemolytic disease of the newborn, requiring more transfusions in utero and having a three fold higher mortality than female Rhesus D fetuses. Furthermore, almost 150 X-linked genetic deficiencies have now been characterised, increasing the need for prenatal sex determination in pregnancies at risk for such a disorder. In order examine these two important fetal loci in a risk free manner, we have developed 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 examined 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 males 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 good basis for the clinical examination of multiple fetal loci, in particular Rhesus D status or fetal sex, and can be performed effectively using real-time multiplex PCR assays.
Zhong
X Y
XY
Laboratory for Prenatal Medicine, Department of Obstetrics and Gynaecology, University of Basel, Switzerland.
Holzgreve
W
W
Hahn
S
S
D004247:DNA / Q000097:blood* / Q000235:genetics; D004899:Erythroblastosis, Fetal / Q000175:diagnosis* / Q000235:genetics; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D008297:Male; D008431:Maternal-Fetal Exchange / Q000502:physiology*; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011262:Pregnancy Trimester, Second; D011296:Prenatal Diagnosis; D011993:Recombinant Fusion Proteins; D012203:Rh Isoimmunization / Q000175:diagnosis* / Q000235:genetics; D012204:Rh-Hr Blood-Group System; D018570:Risk Assessment; D019849:Sex Determination Processes
D015514:Oncogene Proteins, Fusion; D011993:Recombinant Fusion Proteins; D012204:Rh-Hr Blood-Group System; C117760:RhD fusion protein, human; C026410:Rho(D) antigen; D004247:DNA