Elucidating Genetic and Biochemical Aspects of the P1 and Sda Carbohydrate Histo-Blood Group Antigens

Sammanfattning: Human histo-blood groups are inherited polymorphic variants that occur in the molecular structures on the humanred blood cell (RBC) surface. Introducing foreign RBCs into a recipient lacking an antigen may activate the humoraldefence leading to a hemolytic transfusion reaction. Antigenic differences can also cause hemolytic disease of thefetus and newborn (HDFN). Blood group antigens are implicated as receptors in pathogen invasion and theirexpression are often altered in cancerous tissues. Blood group antigens are carried by protein or carbohydratestructures. Carbohydrate antigens are synthesized stepwise by glycosyltransferases and are carried onglycosphingolipids or glycoproteins anchored into the RBC membrane. The aim of this work was to elucidate themolecular genetic mechanisms behind the P1 and Sda antigens, as well as to study their glycan structures. The P1antigen belongs to the P1PK blood group system. Silencing of A4GALT causes the null phenotype (Pk–, P1–) of thissystem. However, the consequence of the genetic differences between the P1 (Pk+, P1+) and P2 (Pk+, P1–)phenotypes, i.e. the molecular mechanism underlying how P1 antigen is expressed, has remained unknown.Additionally, there have been divided views regarding the molecular carriers of the P1 antigen, Galα1-4Galβ1-4GlcNAc-R. The Sda antigen GalNAcβ1-4(NeuAcα2-3)Gal-R was associated with the B4GALNT2 gene already in2003. However, the genetic basis of the Sd(a–) phenotype was never revealed.Through EMSA experiments the Runt-related transcription factor 1 (RUNX1) was identified to bind P1 allelesspecifically, dependent on rs5751348 in A4GALT. Knock-down of RUNX1 decreased the A4GALT mRNA levels,establishing its effect as a P1/P2-discriminating factor. Based on these findings a genotyping assay was implementedat the Nordic Reference Laboratory for Genomic Blood Group Typing in Lund, Sweden. P1 was also established tobe carried on glycoproteins in N-glycan conjugates, in addition to glycosphingolipids.Sequencing of B4GALNT2 in nine Sd(a–) individuals identified the missense mutation rs7224888 as highlyassociated with the phenotype. Additionally, the splice-site polymorphism rs72835417, and the rare missensevariants rs148441237 and rs61743617 were encountered in the Sd(a–) cohort. In silico studies identified a closecorrelation between expression of B4GALNT2 and the cancer-associated lncRNA RP11-708H21.4 locus, locateddirectly downstream of the gene. Finally, the Sd(a–) associated SNP rs7224888 was shown to abolish Sda synthaseactivity in over-expression experiments. The epitope was evaluated with DBA lectin binding, fluorescencemicroscopy, enzyme immunoblots and mass spectrometry. The latter confirmed that the glycotransferase utilizessubstrates on both on N- and O-glycan elongation.Understanding the molecular mechanism underlying the P1 antigen as well as defining the genetic background ofthe Sd(a–) phenotype has enabled genotyping approaches for clinical practice. Additionally, the confirmation ofB4GALNT2 expressing the Sda synthase, has allowed the International Society of Blood Transfusion (ISBT) to movethe Sda antigen from the series of high-frequency antigens to its own, new blood group system designated SID, no.038.