The Csa blood group antigen was identified more than 50 years ago but its genetic basis has yet to be elucidated. All our recent genomic investigation has failed to resolve the genetic basis of this enigmatic antigen. By investigating the association of the HNA-3a/b polymorphism (rs2288904-G/A) in SLC44A2 with clinical features of Sickle Cell Disease (SCD), we incidentally discovered that rare subjects with the homozygous HNA-3b/b genotype also carry the uncommon Cs(a-) phenotype. We genotyped this SNP in a cohort of 25 Cs(a-) subjects and found that all of them showed a HNA-3b/b genotype. This result suggests that the high-prevalence allele with rs2288904 (HNA-3a
455G) encoding Arg152 encodes the high-prevalence Csa. Accordingly, anti-Csa does not react with SLC44A2null RBCs, SLC44A2 knockout K562 cells, and K562 cells expressing HNA-3b, confirming that the Csa and Csb antigens are carried on this protein. Furthermore, mass spectrometry analysis of SLC44A2 from neutrophils and RBCs, along with serological investigation, showed that, despite HNA-3a and Csa having the same genetic basis, anti-HNA-3a and anti-Csa recognize different epitopes on the SLC44A2 protein. Overall, our data resolve the genetic bases of the Cs(a-) and Cs(b-) blood phenotypes, with new insights on the anti-HNA-3a specificity.