A p.Leu319Pro missense mutation in ABO likely results in a non-deletional O allele

Background

ABO is the most important blood group system in transfusion medicine. A glycosyltransferase attaching N-acetyl-galactosamine or galactose to specific sugar chains of glycoproteins and glycolipids is responsible for the expression of antigen A or B, respectively. Typical for the null phenotype is a single base deletion in the corresponding ABO gene (c.261delG). In addition, several missense mutations are known that also lead to an O phenotype; often referred to as non-deletional O alleles.

Methods

Here, we present a case of a male patient with a discrepancy between forward and reverse typing of ABO. Antigen determination was performed by hemagglutination with both, a column technique (Ortho Biovue) and a gel card technique (Bio-Rad ID-System). Commercially available red blood cells were used for detection of isoagglutinins (i.e. anti-A and anti-B). Initial genotyping was performed by commercial SSP-PCR (ABO subtype, innotrain). For sequencing of ABO a direct taq-cycle method was chosen. To detect weakly expressed antigens, a combined adsorption-elution test was performed: adsorption with a human anti-A at 4 °C, followed by heat elution. Additionally, antigen A expression on red blood cells was measured by flow cytometry.

Results

Forward typing of the patients own red blood cells (RBC) revealed no expressed A or B antigen, respectively. The serum of the patient contained a strongly reacting anti-B and a weak anti-A1. Reverse typing with A2 RBC was negative. Genotyping by SSP-PCR suggested an A1 phenotype of the patient"s RBC. However, haplotype specific sequencing of ABO revealed a deletional O allele (ABO*O.01.02) and an A1-Allel with an additional nucleotide exchange in exon 7 (c.956C) leading to an amino acid substitution (p.Leu319Pro). An AEL phenotype was excluded by a negative result in a combined adsorption-elution test. Flow cytometry confirmed these findings: anti-A binding was comparable with well known RBC of the O2 phenotype. Figure 1 shows a pedigree.

Conclusion

Typically, RBC of individuals with an O phenotype do not express any A antigen. However, there is evidence that non-deletional O alleles have a residual activity of glycosyltransferase A. In our case we could not elute any potentially adsorbed anti-A. But, anti-A1 and anti-A in the serum were strongly diminished; a typical result for non-deletional O alleles. Interestingly, the predicted aminoacid exchange p.L319P is not in close proximity to direct enzyme activity or substrate binding sites.

Offenlegung Interessenkonflikt:

All authors declare no conflicts of interest.