Three Molecular Structures Cause Rhesus D Category VI Phenotypes With Distinct Immunohematologic Features

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Abstract
Rhesus D category VI (DVI) is the clinically most important partial D. DVI red blood cells were assumed to possess very low RhD antigen density and to be caused by twoRHD-CE-D hybrid alleles. Because there was no population-based work-up, we screened three populations in central Europe for DVI. Twenty-six DVI samples were detected and examined by exon-specific RHD polymerase chain reaction with sequence-specific primers (PCR-SSP). A new genotype, hereby designated D category VI type III, was characterized as a RHD-Ce(3-6)-D hybrid allele by sequencing of the cDNA, parts of intron 1, and by PCR-restriction fragment length polymorphism (PCR-RFLP) of intron 2. Rhesus introns 5 and 6 were sequenced and the 3′ breakpoints of all knownDVItypes shown to be distinct. We differentiated the 5′ breakpoints of DVItypeI andDVItype II by a newly devised RHD-PCR. Thus, the DVI phenotype originated in at least three independent molecular events. Each DVI type showed distinct immunohematologic features in flow cytometry. The number of RhD proteins accessible on the red blood cells' surface ofDVItype III was normal (about 12,000 antigens/cell; DVItypeI, 500;DVItype II, 2,400) based on the determination of an RhD epitope density profile. DVItype II and DVItype III occurred as CDe haplotypes, and DVItype I as a cDE haplotype.The distribution of the DVItypes varied significantly in three German-speaking populations. Genotyping strategies should take account of allelic variations in partial RhD. The reconsideration of previous serologic and clinical data for partial D in view of the underlying molecular structures may be worthwhile.