Identification of a Novel B(972T) Allele in a Blood Donor Associated with B(A) Phenotype

The ABO blood group system is one of the most important in the field of transfusion medicine. When a forward and reverse group are inconsistent, the ABO discrepancy should be investigated and resolved. These discrepancies can be due to ABO subgroups that can pose a risk to compatibility between a donor's red blood cells and a patient's plasma which could lead to a transfusion reaction. We investigated a volunteer blood donor who had previously been typed as Group B on multiple donations but had ABO grouping discrepancies on three separate donations. The most recent discrepancy was discovered by the hospital during ABO recheck of the unit, with the unit ultimately discarded. Using traditional tube testing, the donor's RBCs were tested with two sources of ABO grouping reagents (Seraclone [Bio-Rad] and Gamma-clone [Immucor]) and their plasma was tested against in-house prepared reagent RBCs. Genomic DNA was isolated from white blood cells (Qiagen). Sanger sequencing of ABO exons 6 and 7 and flanking intron regions and allele-specific sequencing to determine phasing of variants in exon 7 was performed. Serological testing of the donor's RBCs showed 1–2+ mixed field agglutination with Anti-A and strong 4+ agglutination with Anti-B and Anti-A,B from both sources. No mixed field was seen with Anti-B and Anti-A,B. The donor's plasma reacted strongly positive 4+ with A1 RBCs and non-reactive with A2 and B RBCs at immediate spin; however, the donor's plasma became strongly positive 3+ with the A2 RBCs after a room temperature incubation. Sanger sequencing results were consistent with ABO*O.01.01 (c.261delG) and ABO*B (c.297G, c.526G, c.657T, c.703A, c.796A, c.803C, c.930A) but also identified a novel heterozygous variant, c.972G >T (p.Leu324Phe) in exon 7. The variant was placed on B allele by allele-specific sequencing. We identified novel ABO*B allele with c.972G >T (p.Leu324Phe) variant associated with weak A and strong B expression and a probable B(A) phenotype in trans to ABO*O. Interestingly, B(A) transferases reported to date have variations at either p.214-235 or at key transferase positions p.266-268. Although p.324Phe is distant from those positions in the polypeptide, it is possible that protein folding brings it into close proximity. Furthermore, while this change has not been reported previously, a nearby variant, c.976G>T (p.Asp326Tyr), is associated with weak B phenotype (BW.30).