Molecular determination of RHD zygosity: predicting risk of hemolytic disease of the fetus and newborn related to anti-D.

Objective: Development of an accurate molecular method for paternal RHD zygosity to predict risk to a fetus for hemolytic disease of the fetus and newborn (HDFN) related to anti-D.

Methods: Quantitative fluorescence polymerase chain reaction (QF-PCR) was used to detect RHD exons 5 and 7, using RHCE exon 7 as an internal control. The genotype and zygosity were determined from the peak area ratios of RHD exon 5 or 7 to RHCE exon 7. We tested 25 Caucasian and 25 African American (AA) samples whose zygosity was predicted from the Rh phenotype and an alternate molecular method. In addition, we tested 71 paternal samples from prenatal cases where fetal testing was performed.

Results: RHD/RHCE ratios clearly distinguished the RHD/D and RHD/d genotypes. RHD variants were recognized when RHD exon 5 copy number was discordant with exon 7. The molecular assay identified eight cases where the phenotype incorrectly assigned zygosity and we observed three false-negatives in the hybrid Rhesus box assay. The prenatal results were consistent with the zygosity determined for the paternal samples in our study.

Conclusions: This QF-PCR method accurately determines RHD zygosity in Caucasians and AAs and will help predict the risk that a fetus will inherit RHD.