Truncated glycosyltransferase coding regions in novel ABO alleles give rise to weak A or B blood group expression and discrepant typing results

Background
Correct ABO blood-group matching between donor and patient is crucial for safe transfusions. We investigated the underlying reason causing inconclusive ABO serology in samples referred to our laboratory.
Study Design and Methods
Flow cytometric analysis, ABO genotyping, and sequencing were used to characterize ABO-discrepant blood samples (n = 13). ABO gene variants were inserted in a GFP-containing bicistronic vector to assess A/B expression following overexpression in HeLa cells.
Results
Seven novel alleles with nonsense mutations predicted to truncate the encoded ABO glycosyltransferases were identified. While these variants could represent O alleles, serology showed signs of ABO glycosyltransferase activity. ABO*A1.01-related alleles displayed remarkably characteristic percentages of A-positive cells for samples with the same variant: c.42C>A (p.Cys14*; 10%), c.102C>A (p.Tyr34*; 31%–32%, n = 2), c.106dup (p.Val36Glyfs*21; 16%–17%, n = 3) or c.181_182ins (p.Leu61Argfs*21; 12%–13%, n = 2). Transfection studies confirmed significantly decreased A expression compared to wild type. The remaining variants were found on ABO*B.01 background: c.1_5dup (pGly3Trpfs*20), c.15dup (p.Arg6Alafs*51) or c.496del (p.Thr166Profs*26). Although the absence of plasma anti-B was noted overall, B antigen expression was barely detected on erythrocytes. Overexpression confirmed decreased B in two variants compared to wildtype while c.1_5dup only showed a non-significant downward trend.
Conclusion
Samples displaying aberrant ABO serology revealed seven principally interesting alleles. Despite the presence of truncating mutations, normally resulting in null alleles, low levels of ABO antigens were detectable where alterations affected ABO exons 1–4 but not exon 7. This is compatible with the previously proposed concept that alternative start codons in early exons can be used to initiate the translation of functional ABO glycosyltransferase.