Partial s antigen expression in GP(B-A-B) hybrid glycophorins and the corresponding in vitro expression study

Abstract

Background - The s antigen expression is mainly determined by a single nucleotide polymorphism at c.143C (p.Thr48) on the exon 4 of GYPB gene. Several mutations on the GYPB gene have been reported to cause aberrant s antigen expression. GP.Mur has an extra 31-amino acid insertion encoded by the active compound GYP(B-A) exon 3, which closely locates at the upstream of p.Thr48. It has been reported to cause altered s antigen expression.

Materials and methods - Serologic testing and flow cytometry analysis were performed to detect s antigen expression on RBCs of GP(B-A-B) hybrid glycophorins, including GP.Mur, GP.Bun and GP.HF. Several mutant plasmids based on the different sites between GYPB and GYP(B-A-B) alleles were constructed and transfected into HEK293T cells for in vitro expression, to reveal the key amino acids for the aberrant s antigen expression.

Results - Serologic testing and flow cytometry assay showed the RBCs of GP.Mur homozygotes reacted positively with IgG anti-s (P3YAN3) but negatively with IgM anti-s (P3BER). Flow cytometry analysis also revealed half level of s antigen expressed on the RBCs of GP.Mur, GP.Bun and GP.HF heterozygotes with ss genotype compared to S-s+ controls when detected by IgM anti-s (P3BER). Furthermore, in vitro expression study showed that p.Asn45 is critical for the epitope expression of s antigen detected by IgM anti-s (P3BER).

Discussion - The results demonstrated partial s antigen expression on GP(B-A-B) RBCs. In addition to p.Thr48, p.Asn45 is also important for the epitope expression of s antigen detected by IgM anti-s (P3BER). To avoid false negative serologic typing, it is recommended to use several different clones of monoclonal anti-s for the correct s typing, especially in the regions with high frequency distribution of GP(B-A-B) hybrid glycophorins.