EVIDENCE OF GENETIC DIVERSITY UNDERLYING RH-D-, WEAK-D (D-U), AND PARTIAL-D PHENOTYPES AS DETERMINED BY MULTIPLEX POLYMERASE CHAIN-REACTIONANALYSIS OF THE RHD GENE

The human blood group Ph antigens are expressed by proteins encoded by a pair of highly homologous genes located at chromosome 1p34-36. One of the genes (RHCE) encodes ph CcEe antigens, while the other (RHD) th e D antigen. Point mutations in the RHCE gene generate the C/c and E/e polymorphisms, while it has been shown that an RHD gene deletion can generate the D-negative phenotype, We have analyzed intron 4 of the RH CE and RHD genes and have defined the site of an RHD-specific deletion located in this intron. Using a multiplex RHD typing assay, which com bines a reverse polymerase chain reaction (PCR) primer, which straddle s this RHH-specific sequence, and a pair of primers located in exon 10 of the RHD gene, we have analyzed 357 different genomic DNA samples d erived from individuals expressing D+, D-, weak D, and partial D pheno types. Of these, we have noted a significant discordance with our mult iplex PCR assay in the D- phenotypes dCcee and dccEe (which have been previously described) and weak D phenotypes. Our results suggest that in five serologically D- individuals we have identified an apparently intact RHD gene. Sequence analysis of transcripts obtained from one of these individuals (of phenotype dCCee) illustrates the presence of fu ll-length RHD transcripts, which have a point mutation at nucleotide 1 21 (C --> T), which generates an in-frame stop codon (Gln41Stop). Thus , we describe a different molecular basis for generating the D- phenot ype to the complete RHD gene deletion described previously, We also sh ow that there are discordances with serotype and the multiplex assay i n weak D and partial D phenotypes, indicating that the underlying mole cular basis can be heterogeneous. Existing ph D PCR assays assume the complete absence of the RHD gene in D- phenotypes. We describe a diffe rent molecular basis for generating the D- phenotype to the complete R HD gene deletion described previously. (C) 1997 by The American Societ y of Hematology.