Human D IIIA Erythrocytes: RhD protein is associated with multiple dispersed amino acid variations

As a partial D antigen of the Rh blood group system, the D category IIIa phenotype occurs mainly in Blacks, but its molecular basis has not been defined. Here we describe studies of the D category D IIIa and VS+ red blood cells (RBC) from two unrelated probands by Southern blot, cDNA PCR, and nucleotide sequencing. Rh haplotyping by Sph I restriction fragment length polymorphisms indicated that the two probands carried Dce/dCe and Dce/DcE genotypes, respectively. Sequence analysis of Rh cDNAs showed that their erythroid cells expressed both D and CE transcripts. Nevertheless, the D transcripts were found to contain four nucleotide changes scattered in three exons: nt455 A‐to‐C (exon 3), nt602 C‐to‐G (exon 4), nt 654 C‐to‐G (exon 5), and nt667 T‐to‐G (exon 5). These variations resulted in the following amino acid substitutions characteristic of RhCE polypeptides: 152 Asn‐to‐Thr, 201 Thr‐to‐Arg, 218 Ile‐to‐Met, and 223 Phe‐to‐Val. The 152Thr and 223Val residues were predicted to reside in proximity to the third and fourth extracellular loops, respectively. Together, these results establish a correlation of the four amino acid changes in the RhD protein with the expression of D IIIa as a partial D antigen on the RBC membrane. Since the varied nucleotides identified in D IIIa all pre‐exist in CE , they are likely to have originated from CE by templated micro‐conversion event(s). The identification of a specific nt736 C‐to‐G transversion in CE in the two probands suggests that 245Val may involve the expression of VS antigen. Am. J. Hematol. 55:139–145, 1997. © 1997 Wiley‐Liss, Inc.