Molecular determination of RHD zygosity

Abstract

Determining the RhD zygosity of a partner is valuable for the management of hemolytic disease of the newborn (HDN) related to anti-D. Zygosity is typically predicted using Rh phenotype. However, this may not be satisfactory in all ethnic groups. Our goal was to develop a robust clinical assay for RhD zygosity using molecular techniques that is suitable for use in both Caucasian and African-American populations. We used quantitative fluorescent PCR to detect RhD exons 5 and 7 with an internal control, exon 7 from the RhCE gene. PCR products were quantified by an automated sequencer. The ratio of exon 5 or 7 amplicon to the internal control determined RhD copy number. Accuracy was assessed using 17 Caucasian and 11 African-American samples whose zygosity was predicted from an Rh phenotype and 11 HDN paternal samples predicted heterozygous after the fetus tested RhD-negative. The assay accurately distinguished the 2 genotypes. Ratios for the DD and Dd genotypes for exon 5 and exon 7 were distinct and non-overlapping (Table). Common variant alleles such as DVI and the RhD pseudogene are recognized when the RhD copy number of exon 5 and exon 7 are discordant. All 17 Caucasian samples were concordant with serology. We found 2 discrepancies out of 11 African-American samples; however, the serology prediction for both cases was only 50-58%. All results were concordant for the 11 HDN paternal heterozygous samples. This molecular assay accurately assesses RhD zygosity and helps predict the risk that a fetus will inherit RhD.Tabled 1Exon Ratios Determine RhD Copy NumberExon 5Exon 5Exon 5Exon 7Exon 7Exon 7Copy#MeanRange3SDMeanRange3SDDD1.051.00-1.120.091.271.07-1.430.30Dd0.530.45-0.570.090.680.55-0.770.18 Open table in a new tab