Molecular and structural characterization of a novel high‐prevalence antigen of the Augustine blood group system

Background An antibody directed against a high‐prevalence red blood cell (RBC) antigen was detected in a 67‐year‐old female patient of North African ancestry with a history of a single pregnancy and blood transfusion. So far, the specificity of the proband's alloantibody remained unknown in our immunohematology reference laboratory. Study Design and Methods Whole‐exome sequencing (WES) was performed on the proband's DNA. The reactivity to the SLC29A1‐encoded ENT1 adenosine transporter was investigated by flow cytometry analyses of ENT1‐expressing HEK293 cells, and RBCs from Augustine‐typed individuals. Erythrocyte protein expression level, nucleoside‐binding capacity, and molecular structure of the proband's ENT1 variant were further explored by western blot, flow cytometry, and molecular dynamics calculations, respectively. Results A missense variant was identified in the SLC29A1 gene, which encodes the Augustine blood group system. It arises from homozygosity for a rare c.242A > G missense mutation that results in a nonsynonymous p.Asn81Ser substitution within the large extracellular loop of ENT1. Flow cytometry analyses demonstrated that the proband's antibody was reactive against HEK‐293 cells transfected with control but not proband's SLC29A1 cDNA. Consistent with this finding, proband's antibody was found to be reactive with At(a‐) (AUG:–2), but not AUG:–1 (null phenotype) RBCs. Data from structural analysis further supported that the proband's p.Asn81Ser variation does not alter ENT1 binding of its specific inhibitor NBMPR. Conclusion Our study provides evidence for a novel high‐prevalence antigen, AUG4 (also called ATAM after the proband's name) in the Augustine blood group system, encoded by the rare SLC29A1 variant allele AUG*04 (c.242A > G, p.Asn81Ser).