Paternal RHD zygosity determination in Tunisians: evaluation of three molecular tests

The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD.

RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction - sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced.

Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile).

RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD.