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, EPD4 AND EPD9 TO THE 6TH EXTERNAL DOMAIN OF THE RH D PROTEIN

The discovery of Rh partial D variant red cells by discrepant reaction s 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 diff erent epitopes. We describe here the molecular basis of two D variants which lack epD4 and epD9 namely the DNU and D-II phenotypes. These wo uld have both been originally classified as D-II phenotype individuals , but we have revealed subtle differences in the serological profile o f these erythrocytes. Such a differential reactivity and determination of the molecular bases of these phenotypes allows us to predict criti cal amino acids for epD3, epD4 and epD9 expression. The DNU phenotype arises from a single point mutation in the RHD gene resulting in a sin gle amino acid change (Gly353Arg). Sequence analysis of exon 7 of the RHD gene derived from the D-II propositus indicates that there is a si ngle point mutation in this exon resulting in a single amino acid chan ge (Ala354Asp). It is likely that this point mutation gives rise to th e D-II phenotype. Both mutations result in the change to Rh D-specific residues. Our results indicate that the following amino acids are cru cial 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 p ossible that epD3, 4 and 9 are continuous epitopes.