Molecular Analysis of Rh Transcripts and Polypeptides From Individuals Expressing the DVI Variant Phenotype: An RHD Gene Deletion Event Does Not Generate All DVIccEe 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 DVI 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). DVI 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 DVI phenotype has previously been proposed to occur by two different genetic mechanisms, one (in individuals of DVICcee phenotype) where a gene conversion event generates a hybrid RHD-RHCE-RHD gene; the second (in individuals of DVIccEe phenotype) was proposed to be caused by a partial RHD gene deletion. We present evidence that in four DVICcee phenotypes studied, this phenotype is not generated by a partial RHD gene deletion, but occurs by a similar mechanism to the DVICcee phenotypes. In two individuals we have found hybrid RHD-RHCE-RHD transcripts in both DVICe and DVIcE haplotypes. These differ in that the DVICe transcripts are derived from an RHD gene where exons 4-6 have been replaced with RHCE equivalents (encoding Ala226 ); the DVIcE 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 DVI polypeptides are expressed at the erythrocyte surface as full-length polypeptide products. We have used immunoprecipitation experiments using anti-D reactive with DVI 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 DVI proteins studied, and suggest that Rh DVI polypeptide species studied here exist as full-length Rh proteins.

Molecular basis of the D variant phenotypes DNU and DII allows localization of critical amino acids required for expression of Rh D epitopes epD3, 4 and 9 to the sixth external domain of the Rh D protein

“…Previous reports describing the molecular basis of D variant phenotypes have revealed that replacement of RHD exons by RHCE equivalents (probably by gene conversion) appears to be the most common mechanism by which these phenotypes arise. Assessment of lost epitopes and comparison with predicted Rh D protein sequence (obtained by sequence analysis of Rh D transcripts) in these D variants is complicated because they all involve multiple alterations to the Rh D protein sequence at predicted extracellular, intracellular and intramembranous positions (Avent et al, 1996a(Avent et al, , 1997Mouro et al, 1994, Rouillac et al, 1995a. For this reason, it is difficult to predict the direct involvement of Rh D specific residues in expression of the D antigen.…”

“…D IVa and D IVb , Rouillac et al, 1995a; D II and DNU described here). EpD3, 4 and 9 are conserved on D VI variant erythrocytes, which have alterations to exons 4-6 (Mouro et al, 1994;Avent et al, 1997). It is possible that the epitopes are formed solely by the sixth loop structure, and are thus continuous (Fig 3).…”

“…Most D variants appear to arise by gene conversion events. For example, CcD VI ee phenotype individuals (lack epD1, 2, 5, 6, 7, 8) have rearranged RHD genes where exons 4-6 are replaced by RHCE equivalents (Mouro et al, 1994;Avent et al, 1997); in cD VI E haplotype individuals there is a rearranged RHD gene where exons 4 and 5 are replaced by RHCE equivalents (Avent et al, 1996a(Avent et al, , 1997; D IVa and D IVb individuals (which lack epD1, 2, 3, 9 and epD1, 2, 3, 4, 9 respectively) involve a rearranged exon 3 and part of exon 7 ( D IVa ), and exons 7-9 (D IVb ) of the RHD gene (Rouillac et al, 1995a). The D VII variant phenotype appears to be due to a Leu110Pro amino acid substitution on the Rh D protein, which is located in the second external domain of the protein, and indicates that partial D antigen expression can also be due to point mutations in the RHD gene (Rouillac et al, 1995b).…”

Variants of RhD – current testing and clinical consequences

“…All encode the same changes in the external loops of the RhD protein and all encode a D antigen with epitope characteristics typical of DVI. The encoded phenotypes are not identical; they differ in D antigen strength and expression of the DVI-associated antigen BARC (Mouro et al, 1994;Avent et al, 1997;Wagner et al, 1998;Esteban et al, 2006). Likewise, there are seven types of RHD*DV, all encoding Glu233Gln, but all except RHD*DV type 4 have additional mutations in exon 5 (Rouillac et al, 1995;Omi et al, 1999;Hyodo et al, 2000;M€ uller et al, 2001).…”

Prenatal genotyping of RHD and SRY using maternal blood

“…The high complexity of the Rh system is partly a result of the presence of two genes that are inherited together. The RHD and RHCE genes share 96·8% homology at the nucleotide level, and molecular elucidation of the Rh phenotypic variants indicates that a substantial proportion of the variants are caused by homologous recombination between the two genes [17–19]. Additional point mutations add to the complexity.…”