D variants at the RhD vestibule in the weak D type 4 and Eurasian D clusters

One branch of the RHD phylogenetic tree is represented by the weak D type 4 cluster of alleles with F223V as the primordial amino acid substitution. F223V as well as a large number of further substitutions causing D variants are located at the extracellular RhD protein vestibule, which represents the entrance to the transmembraneous channel of the RhD protein. RHD and RHCE nucleotide sequences were determined from genomic DNA and cDNA. D epitope patterns were established with commercial monoclonal anti-D panels. The RHD alleles DOL-1 and DOL-2 had the two amino acid substitutions M170T (509T > C) and F223V (667T > G) in common. DOL-2 harbored the additional substitution L378V (1132C > G). Both alleles were observed in Africans and are probably evolutionary related. DMI carried M170I (510G > A), which differed from the DOL-typical substitution. DFW and DFL harbored the substitutions H166P (497A > C) and Y165C (494A > G). The antigen densities of DOL-1, DFL, and DFW were only moderately reduced. DOL-1 and DOL-2 belong to the weak D type 4 cluster of RHD alleles. Together with DMI, DFL, and DFW they represent D variants with amino acid substitutions located at extracellular loops 3 or 4 lining the RhD protein vestibule. These substitutions were of minor influence on antigen density while adjacent substitutions in the transmembraneous section caused weak D antigen expression. All these D variants were partial D and alloanti-D immunizations have been observed in DOL-1, DMI, and DFL carriers. The substitution at position 170 causes partial D although located deep in the vestibule.