Human D^VI Category Erythrocytes: Correlation of the Phenotype With a Novel Hybrid RhD-CE-D Gene But Not an Internally Deleted RhD Gene

To the Editor:

The Rh blood group system is highly complex due to the occurrence of a large number of antigenic polymorphisms. Of these, the D category VI (D^VI) is a well-recognized partial D antigen and is divided into two subtypes according to its association with C/c and E/e antigens.1 Type I refers to the D^VIccEe association and type II the D^VICCee association. It was suggested that the former is correlated with a deletion of exons 4 through 6 in the RhD gene and the latter with the formation of a RhD-CE-D hybrid gene retaining exons 4 through 6 of the RhCE gene.2 Here evidence is presented that D^VI type I is defined by a novel RhD-CE-D hybrid gene but not an internally deleted RhD gene and that its CE portion contains exons 4 and 5 originating from a RhE allele.

D^VIccEe blood was obtained from a mother (T.S.) and her daughter (V.S.). Analysis of Rh gene structure and expression was performed as described.3 Haplotyping by Sph I RFLPs4 indicated a D^VIcE/dce genotype for both subjects. Although no gross change was found in CE genes, the absence of two Sph I bands in the D gene was associated with the appearence of a 10.8-kb new fragment (Fig 1A). Next, exons retained in the new fragment were mapped and their identity was determined by polymerase chain reaction (PCR) coupled with diagnostic restriction digestion and DNA sequencing.3 The results showed that in both T.S. and V.S., exons 4 and 5 are CE-specific (Fig 1A, Bcl I and Taq I panels), whereas other exons including exon 6 (Fig 1A, Rsa I panel) are from both D and CE genes. Thus, the D^VI type I variant is associated with the occurrence of a D-CE-D hybrid gene, but not a simple deletion of exons 4 to 6 in the RhD gene.2 This hybrid gene most likely originated from a genetic recombination event by which exons 4 and 5 of CE were transferred to replace the D counterpart (Fig 1B).

To further characterize this hybrid gene and its expression, Rh cDNAs were obtained using a novel approach that exploits the sequence difference between D and CE transcripts to avoid their coamplification.3 Surprisingly, rather than detecting a single truncated cDNA species,2 the D and CE cDNAs from T.S. and V.S. were found to be comparable to that of controls in size and amount (gels not shown). To establish the transcript structure, the nucleotide sequences of D and CE cDNAs from T.S. and V.S. were determined. In agreement with the results of genotyping and phenotyping, the sequence analysis showed the expression of three full-length transcripts. The CE cDNAs included both Rhce and RhcE forms as indicated by the coexistence of allelic differences.5 However, normal RhD transcript was not found in the erythroid cells of both D^VI individuals. Instead, a hybrid transcript was identified which has the same arrangement of the proposed D-CE-D gene (Fig 1B).

The D^VI type I transcript maintains a normal open reading frame and thus should encode a protein with 417 amino acid residues (Fig 2). Immunoblotting with a D-specific monoclonal antibody confirmed the expression of this Rh protein in the erythrocyte membranes (data not shown). The deduced sequence differs from the normal D sequence by 14 amino acids from positions 169 through 267 (Fig 2). Sequence comparison also shows that the CE portion of the D^VI type I protein must be from the RhE allele because a proline (Pro) occurs at position 226. Assessment of this protein by hydropathy plot6 suggests a model for its membrane disposition where the RhE portion would contribute to the formation of three membrane spans (C6-C8) plus the entire fourth extracellular loop (Fig 2). Whether the placement of a RhE portion in the context of D might elicit a new antigenicity is not known at present. Nevertheless, it is of interest to note that this partial D category exhibits a very weak D expression, yet there was no apparent reduction of its transcript or protein in the erythrocytes from both individuals. These observations, together with the definition of a new hybrid arrangement, suggest strongly that the amino acid sequence and its variations encoded by exons 4 and 5 of the RhD gene represent a minimal requirement for the exhibition of D antigen.