HETEROGENEITY OF BLOOD-GROUP RHE VARIANTS REVEALED BY SEROLOGICAL ANALYSIS AND MOLECULAR ALTERATION OF THE RHCE GENE AND TRANSCRIPT

After testing red cells from 12 RhE variants with a panel of anti-E mo noclonal antibodies (MoAbs), four patterns of reactivity were detected indicating that the MoAbs may recognize four distinct E epitopes desi gnated epE1, epE2, epE3 and epE4. The variants were classified into fo ur categories (cat EI to EIV) which carried epE1 and epE2, epE1 and ep E4, epE1, epE3 and epE4, and all four epitopes, respectively Molecular analysis of the transcripts and genomic DNA of the variants from cat EI, EII and Em displayed three distinct genetic alterations, Cat EI va riants exhibited a point mutation (T500A) in exon 4 of the RHCE gene t hat resulted in a Met167Lys substitution in the third extracellular lo op of the RhcE protein. Cat EII variant carried a hybrid gene structur e characterized by replacement of exons 1-3 (or 2-3) of the RHCE gene by their specific counterparts in the RHD gene. This latter variant wa s also associated with a weak expression of the RhC antigen. In cat EI II variants there was a partial DNA exchange of exon 5 sequences (nt 6 97 and 712) between the RHCE and the RHD genes, generating a hybrid Rh cE-D-cE protein carrying the Glu233 and Val238 substitutions. The ser ological and molecular studies of the RhE variants indicated that: (i) the RhE antigen is a mosaic composed of at least four epitopes and pr oline at position 226 is necessary but not sufficient for the full exp ression of the E antigen, (ii) the lack of RhE epitope(s) is associate d with heterogenous molecular alterations of the RHCE gene. and (iii) aminoacids located on the third and fourth extracellular loops of the RhCE polypeptide are critical for some RhE epitopes expression.