Human RhD^el Is Caused by a Deletion of 1,013 bp Between Introns 8 and 9 Including Exon 9 of RHD Gene

To the Editor:

The Rh system is genetically controlled by two different but highly homologous genes on chromosome 1p34-36. The RHCE gene encodes a different RhCcEe polypeptide and the RHD gene encodes the D polypeptide, and there are a large number of antigenic polymorphisms between these two peptides. Of these, the RhD^elis characterized as RhD⁻ by using a conventional serological test, but it does show absorption and elution of anti-D.1 The molecular basis of RhD^el is not known.

The blood of 21 D^el (21.6%) of 102 serological RHD⁻ patients was obtained after an absorption and elution test. A modified polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the polymorphisms between RHCE and RHD genes was used to analyze the D^el gene structure,2 and the results showed that there was no difference between the RhD and RhD^el gene except that at the BspHI site of exon 9, the D^el gene lacked theBspHI site that was similar with the RHCE gene. Haplotyping by Sph I bands showed no gross difference between RHD and RHD^el genes3 (data not shown).

To further characterize the D^el gene and its expression, a nested reverse tranascriptase-polymerase chain reaction (RT-PCR) method was used to amplify the different region between RHD and D^el genes.4 The RNA was extracted from red blood cells and white blood cells, and the RT-PCR was performed as described.5 The first PCR amplified the RHD or D^el gene from the exon 7 to exon 10 region (using upstream primer P₀: 5′-TCCCCACAGCTCCATCATGGG-3′ and downstream primer P₄: 5′-GTATTCTACAGTGCATAATAAATGGTG-3′, both are RHD gene-specific primers). The PCR products were subjected to nested-PCR: using an RHD specific upstream primer P₁: 5′-CATTGTGCTGCTGGTGCTTG-3′, and downstream primer P₂: 5′-CTGTCAGGAGACCAGACGTG-3′ to amplify part of exon 7 and exon 8; using P₁ and downstream primer P₃: 5′-CTTCCAGAAAACTTGGTCATC-3′ to amplify from exon 7 to exon 9; using primer P₁ and P₄ to amplify from exon 7 to exon 10. The results showed that the D and D^el genes were similar at exon 7 and 8, but there was no nested RT-PCR product for D^el gene for the primers P₁ and P₃, and the nested PT-PCR product of D^el gene for the primers P₁ and P₄ was shorter than the product of normal D gene. Direct sequencing of the nested RT-PCR products of D and D^el genes showed that there was an exon 9 deletion of D^el gene (Fig1A).

To characterize the breakpoint of D^el gene, a nested PCR method was used to amplify the breakpoint region. For the first PCR, an RHD gene-specific downstream primer (primer P₄) and a nonspecific upstream primer (5′-GATTGGCTTCCAGGTCCTCC-3′) were used to amplify part of the RhD or RhD^el gene from exon 8 to 3′ noncoding region using genomic DNA. For the second PCR, two nonspecific RH gene primers were used (upstream primer 5′-TCAGCATTGGGGAACTCAGC-3′, and downstream primer: 5′-GCCTTGTTTTTCTTGGATG-3′) to amplify from part of exon 8 to exon 10. The PCR products were subjected to direct sequencing or subcloning sequencing analysis. The results showed that the D^el had a 1,013-bp deletion between introns 8 and 9, including whole exon 9 (Fig 1B).

The D^el gene transcript maintains a normal open reading frame and thus should encode a protein with 463 amino acid residues with a new C-terminal extension from codon 384 as compared with the normal D protein of 417 amino acid residues (Fig2). Although the D^el show some D activity after an absorption and elution test, a case of Rh⁻ with D^el activity patient was transfused with RHD⁺ blood did develop anti-D antibody by a traditional serological test several weeks after transfusion. From this point of view, RhD^el should be recognized as a type of RhD⁻, and whether the D^el blood transfused to RhD⁺ or other types of RhD⁻cases will develop anti-D^el antibody needs further study.