The ABO gene locus is highly polymorphic and the main cause for ABO subtype alleles is one or several single nucleotide substitutions in exons 6 or 7, which encode for the catalytic domain of the A and B glycosyltransferases. Identification of the underlying genetic background reveals the mechanisms of altered phenotypes. Therefore, two patient samples were characterized molecularly due to discrepant serological results in the ABO blood group system

ABO phenotyping was performed by standard gel column agglutination testing (Grifols; BioRad) and tube technique. Reverse typing was done using commercial A, B, and O test cells (Grifols, BioRad). Genomic DNA was extracted from peripheral EDTA blood samples using a commercial DNA isolation kit (EZ1 DSP DNA Blood Kit, Qiagen AG). Samples were further characterized by using a sequence specific primer (SSP)-PCR kit detecting common ABO variants (ABO-Type variant, BAG Diagnostics). ABO exons including adjacent flanking intronic regions were amplified and sequenced using published and in-house primers. Allele specific sequencing was performed to identify the haplotype of the ABO alleles. The effect of the amino acid substitutions on the glycosyltransferases A and B was predicted using PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/).

The erythrocytes of the first proband showed a weak resp. 1+ agglutination with monoclonal anti-A and anti-AB, and no agglutination with anti-B. The erythrocytes of the second proband showed a 1+ resp. no agglutination with monoclonal anti-B and anti-AB, a 1+ resp.3+ agglutination with polyclonal anti-B and anti-AB and no agglutination with anti-A. By SSP-PCR the single nucleotide variations known for alleles ABO*A2.01 and ABO*O.01.02 (proband 1) resp. ABO*B.01 and ABO*O.01.02 (proband 2) could be detected. Sequencing of ABO revealed heterozygous substitutions c.407C>A (p.Thr136Lys, proband 1) and c.860C>T (p.Ala287Val, proband 2) in exon 7. Allele-specific sequencing confirmed that the substitutions were located on the ABO*A2.01 or ABO*B.01 allele respectively.

Here we report two novel ABO variant alleles, ABO*A2.01.407A and ABO*B1.860T. Both substitutions have not been reported on the underlying allelic backgrounds to the best of our knowledge. According to PolyPhen-2 the substitutions are predicted to be probably damaging (PolyPhen-2 score 0.999) (allele ABO*A2.01.407A) or possibly damaging (PolyPhen-2 score 0.923) (allele ABO*B1.860T).

I have no conflict of interest to disclose.