Background: Knowledge about paternal RHD hemi- or homozygosity is of clinical interest in alloimmunized pregnant women. D negativity in white persons is usually caused by deletion of the RHD gene. Recently, the physical structure of the RH locus and the mechanism causing the deletion of the RHD gene have been explored, enabling RHD zygosity determination in white persons by specific detection of a hybrid Rhesus box characteristic for the RHD- locus.
Study design and methods: RHD zygosity was determined in 402 samples from five different ethnic groups by polymerase chain reaction (PCR)-restriction fragment length polymorphism and by a newly developed real-time quantitative PCR. The Rhesus boxes of samples showing discrepancies between both tests were cycle sequenced.
Results: In nonwhite persons, several mutated Rhesus boxes exist that hamper zygosity determination by detection of the RHD- locus. Such mutated Rhesus boxes in D+RHD homozygous black persons have a frequency of 0.22. In white persons, no mutated Rhesus boxes were encountered so far.
Conclusions: Owing to the high frequency of the mutated Rhesus boxes, zygosity determination by detection of the RHD- locus is not feasible in nonwhite persons. The cosegregation of variant RHD genes (RHDpsi and (C)cdes) with specific mutated Rhesus boxes yields more insight into the evolutionary events concerning variant RHD genes and mutated Rhesus boxes.