Prenatal prediction of fetal Rh C, c and E status by amplification of maternal cfDNA and deep sequencing

Background The Rh blood group system has considerable clinical importance. The C, c, and E antigens are targets of alloantibodies. Anti‐C, anti‐c or anti‐E alloreactive antibodies produced in pregnant women can cause anemia of a fetus carrying the corresponding antigens. Aims Based on NGS technology, we have developed a noninvasive diagnostic assay to predict the fetal blood group of C, c or E antigens by sequencing cell‐free DNA (cfDNA) during pregnancy. Materials and methods The SNVs underlying either the C, c or E antigens were PCR amplified and sequenced using NGS on a MiSeq instrument. The DNA sequences encoding the C, c or E antigen were counted, as were the number of total sequences. Based on the percentage of fetally derived target SNVs inherited from the father, the fetal blood group could be predicted. Results The results of 55 consecutive RHCE prenatal analyses with postnatal serological blood group determination of 30 newborns showed no discordant results. A threshold discerning positive from negative samples was set at 0.05% specific reads. Discussion Noninvasive, prenatal prediction of fetal blood groups by sequencing cfDNA for the detection of low‐level RHCE*C, RHCE*c and RHCE*E sequences was established as an accurate and robust assay applicable for use in clinical settings. Key Points What's already known about this topic? Other methods as described in the manuscript have been used for prenatal prediction of blood group antigens, but NGS‐based methods have been used only in a few instances. NGS has several advantages: an enormous depth of analysis and obtaining actual DNA sequence data as opposed to a signal, for example, a real‐time PCR method What does this study add? This study verifies that our NGS‐based method is applicable in prenatal predictions and constitutes an important diagnostic tool for obstetricians handling complicated cases of maternal blood group immunization