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)), ep(D3b) (Asp(350)+Gly(353)), epD4a (Gly(353)+Ala(354)), epD4b (Ala(354)), epD9a (Asp(350)+Gly(350)+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.