(P-IG-39) RHD*DEL44 [RHD-CE(4-9)-D] is associated with lack of RhD antigen and expression of altered C antigen

Background/Case Studies: Two blood donors (D1 and D2) were submitted for molecular testing to obtain an extended phenotype. Donor D1 typed D- on several donations. The HemoID DQS panel (Agena Bioscience) predicted both donors to type C-. RBCs from both donors typed C+ using serological methods with two sources of anti-C.

Study

Design/Methods: Genomic DNA was isolated from peripheral blood using standard methods. RH genotyping was performed using RHCE and RHD BeadChip (Immucor). RNA was extracted from red blood cells. cDNA was prepared by standard methods. Amplicons from RHD and RHCE transcripts were generated and cloned into plasmids. Sanger sequencing of plasmid DNA was performed, and results were aligned to the reference sequences using Sequencher software (GeneCodes).

Results/Findings: RHCE BeadChip predicted both donors to type C- c+, concordant with HemoID DQS. D1 was predicted to carry RHCE*ce and RHCE*ce733G alleles while D2 was predicted to be homozygous for RHCE*ce. RHCE cDNA analysis confirmed these alleles with no additional genetic variation present. RHD BeadChip testing of both donors yielded low signal for exon 4-9 analytes. RHD cDNA analysis detected an RHD transcript in both donors with coding sequences in exons 4-9 replaced with RHCE sequences. This allele is listed in the ISBT RHD allele table as RHD*DEL44 with alias of RHD-CE(4-9)-D.

Conclusions: High-resolution cDNA sequencing detected the presence of an RHD-CE(4-9)-D hybrid allele in two blood donors. The reported phenotype of this hybrid allele is equivocal – one study used medium-resolution testing of a sample with a Del phenotype and assigned this allele (Li et al. Vox Sang 2009;97:139-146). Another study predicted the phenotype of this allele to be D- (Gassner et al. Transfusion 2005;45:527-538). The RHD*03N.01 allele that is common in individuals of African descent is a hybrid allele in which exons 4-7 of RHD are replaced with exons 4-7 of RHCE. That allele is associated with a phenotype of D- and C antigen expression. Given the phenotype of that allele, it would be expected that this allele carrying RHCE exons 4-9 would not express the D antigen and would express an altered C antigen. The RHD-CE(4-9)-D allele is reported to be associated with Del phenotype which is surprising since the RHD-CE(4-7)-D allele is associated with D- phenotype. The RHD-CE(4-9)-D allele has no C phenotype reported however RHD-CE(4-7)-D allele is associated with expression of C antigen. The fact that both of these donors types D- and C+ supports that the RHD-CE(4-9)-D allele phenotype should be revised to D- and C+.

Importance of research: Currently, the RHD*DEL44 allele is assigned a Del phenotype. However, this allele encodes a D- and C+ phenotype. It is critical that alleles are catalogued with correct phenotype information and that if prior information is found to be false, that the data be presented such that the information can be corrected.