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

Abstract

The D antigen is a mosaic comprising at least 30 epitopes. Partial Rh D phenotypes occur when there is absence of one or more of these epitopes, with the remainder expressed. The D(VI) phenotype is the most common 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 transfusion with D-positive blood (if typed as D-positive using polyclonal typing reagents) or by fetuses which have all of the D antigen. This situation can give rise to severe hemolytic disease of the newborn (HDN). The molecular basis of the D(VI) phenotype has previously been proposed to occur by two different genetic mechanisms, one (in individuals of D(VI)Ccee phenotype) where a gene conversion event generates a hybrid RHD-RHCE-RHD gene; the second (in individuals of D(VI)ccEe phenotype) was proposed to be caused by a partial RHD gene deletion. We present evidence that in four D(VI)Ccee phenotypes studied, this phenotype is not generated by a partial RHD gene deletion, but occurs by a similar mechanism to the D(VI)Ccee phenotypes. In two individuals we have found hybrid RHD-RHCE-RHD transcripts in both D(VI)Ce and D(VI)cE haplotypes. These differ in that the D(VI)Ce transcripts are derived from an RHD gene where exons 4-6 have been replaced with RHCE equivalents (encoding Ala226); the D(VI)cE transcripts are derived from an RHD gene where exons 4 and 5 are replaced by RHCE equivalents (encoding Pro226). We provide direct evidence that Rh D(VI) polypeptides are expressed 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 domains of all Rh polypeptides. Our results illustrate that these domains are present on all Rh D(VI) proteins studied, and suggest that Rh D(VI) polypeptide species studied here exist as full-length Rh proteins.