ABO splice-site variants encoding weak A and B phenotypes complicate pre-transfusion ABO blood group diagnostics

Background

Genetic variations in the ABO gene can cause discrepancies in routine blood group typing such as weak ABO antigen expression that need further investigations. This study aimed to characterize two novel mutations located at exon-intron junctions of the ABO gene resulting in weak A and Bel phenotypes in patients.

Methods

For serologic antigen typing and reverse grouping standard gel matrix techniques (Bio-Rad) were used and adsorption-elution studies of the patients' red blood cells were done using monoclonal and human antisera. Genomic DNAs were isolated by a column-based method on an automated DNA isolation system (Qiagen). The seven exons of ABO and its' regulatory regions, as well as FUT1 and FUT2 genes were amplified and sequenced by Sanger sequencing (AB3500). Total RNA was isolated (Qiagen) and cDNA synthesis was performed using Superscript III First-Strand Synthesis System and oligo-(dT) primers (Invitrogen). The transcripts detected were further investigated by allele-specific sequencing and translated by the in-silico translation tool ExPASy.

Results

Serologically sample 1 showed a weak A antigen expression with two distinct cell populations. Allele-specific sequencing identified a missense variant at the junction of intron 1 and exon 2 (c.28+12978T>C) compared to the ABO*A1.01 allele. Sample 2 was negative for A and B antigens indicating an O phenotype. Reverse grouping resulted in positive agglutination reactions with A1 and A2 test erythrocytes and a lack of anti-B. An insertion of an additional T nucleotide at the 5´-end of intron 1 (c.28+3-4insT) resulting in an ABO*B1-like allele was found. Both alleles were inherited with ABO*O.01.01 alleles in trans. Aberrant ABO transcript variants were detected and changes in the translated transferases were predicted.

Conclusion

The two novel mutations located at the splice-sites of ABO disrupt RNA splicing and result in the synthesis of aberrant ABO gene transcripts. Altered glycosyltransferase activity may be responsible for weak phenotypes as observed. Analysis of ABO mRNA can help to explain serological ABO blood grouping discrepancies found in routine testing. The gene variants characterized in this study add to the growing blood group gene databases and may help to improve pre-transfusion blood group diagnostics.

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