Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xg^a expression and resolves the Xg blood group system

The Xg^a blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xg^a expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xg^a allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P 5 2.0 3 102²²). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a2) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xg^a genotyping possible.