MOLECULAR ANALYSIS OF RH TRANSCRIPTS AND POLYPEPTIDES FROM INDIVIDUALS EXPRESSING THE D-VI VARIANT PHENOTYPE - AN RHD GENE DELETION EVENT DOES NOT GENERATE ALL D(VI)CCEE PHENOTYPES

The D antigen is a mosaic comprising at least 30 epitopes. Partial Ph D phenotypes occur when there is absence of one or more of these epito pes, with the remainder expressed. The D-VI phenotype is the most comm on of the partial D phenotypes, lacking most D antigen epitopes (ep D) (epD1, 2, 5-8 using the 9-epitope model or epD 1-4,7-22, 26-29 using the 30-epitope model). D-VI mothers may become immunized by transfusio n with D-positive blood (if typed as D-positive using polyclonal typin g reagents) or by fetuses which have all of the D antigen. This situat ion can give rise to severe hemolytic disease of the newborn (HDN). Th e molecular basis of the D-VI phenotype has previously been proposed t o occur by two different genetic mechanisms, one (in individuals of D( VI)Ccee phenotype) where a gene conversion event generates a hybrid RH D-RHCE-RHD gene; the second (in individuals of D(VI)ccEe phenotype) wa s proposed to be caused by a partial RHD gene deletion. We present evi dence that in four D(VI)Ccee phenotypes studied, this phenotype is not generated by a partial RHD gene deletion, but occurs by a similar mec hanism to the D(VI)Ccee phenotypes. In two individuals we have found h ybrid RHD-RHCE-RHD transcripts in both (DCe)-Ce-VI and D(VI)cE haploty pes. These differ in that the (DCe)-Ce-VI transcripts are derived from an RHD gene where exons 4-6 have been replaced with RHCE equivalents (encoding Ala(226)); the D(VI)cE transcripts are derived from an RHD g ene where exons 4 and 5 are replaced by RHCE equivalents (encoding Pro (226)) We provide direct evidence that ph D-VI polypeptides are expres sed at the erythrocyte surface as full-length polypeptide products. We have used immunoprecipitation experiments using anti-D reactive with D-VI erythrocytes followed by immunoblotting the immune complexes with rabbit sera immunoreactive to the fourth external and C-terminal doma ins of all Ph polypeptides. Our results illustrate that these domains are present on all Ph D-VI proteins studied, and suggest that Ph D-VI polypeptide species studied here exist as full-length Ph proteins. (C) 1997 by The American Society of Hematology.