Molecular basis of the D variant phenotypes DNU and D II allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

The discovery of Rh partial D variant red cells by discrepant reactions with different monoclonal anti‐D has demonstrated the range of Rh D epitopes that have arisen due to alterations in Rh D protein structure. There are two current classification systems, one which uses a nine epitope model (epD1–epD9) whereas a more recent model proposes 30 different epitopes. We describe here the molecular basis of two D variants which lack epD4 and epD9 namely the DNU and D II phenotypes. These would have both been originally classified as D II phenotype individuals, but we have revealed subtle differences in the serological profile of these erythrocytes. Such a differential reactivity and determination of the molecular bases of these phenotypes allows us to predict critical amino acids for epD3, epD4 and epD9 expression. The DNU phenotype arises from a single point mutation in the RHD gene resulting in a single amino acid change (Gly353Arg). Sequence analysis of exon 7 of the RHD gene derived from the D II propositus indicates that there is a single point mutation in this exon resulting in a single amino acid change (Ala354Asp). It is likely that this point mutation gives rise to the D II phenotype. Both mutations result in the change to Rh D‐specific residues. Our results indicate that the following amino acids are crucial for epD3a (Asp 350 ), epD3b (Asp 350 +Gly 353 ), epD4a (Gly 353  + Ala 354 ), epD4b (Ala 354 ), epD9a (Asp 350  + Gly 353  + Ala 354 ) and epD9b (Asp 350  + Ala 354 ) expression. All of these amino acids reside on the predicted sixth external domain of the Rh D protein, so it is possible that epD3, 4 and 9 are continuous epitopes.