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 II la 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 restricti on fragment length polymorphisms indicated that the two probands carri ed Dce/dCe and Dce/DcE genotypes, respectively, Sequence analysis of R h cDNAs showed that their erythroid cells expressed both D and CE tran scripts, Nevertheless, the D transcripts were found to contain four nu cleotide changes scattered in three exons: nt455 A-to-C (exon 3), nt60 2 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 ch aracteristic of RhCE polypeptides: 152 Asn-to-Thr, 201 Thr-to-Arg, 218 IIe-to-Met, and 223 Phe-to-Val, The 152Thr and 223Val residues were p redicted to reside in proximity to the third and fourth extracellular loops, respectively, Together, these results establish a correlation o f the four amino acid changes in the RhD protein with the expression o f 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 t o have originated from CE by templated micro-conversion event(s). The identification of a specific nt736 C-to-G transversion in CE in the tw o probands suggests that 245Val may involve the expression of VS antig en. (C) 1997 Wiley-Liss, Inc.