Sd a is a high-frequency carbohydrate histo-blood group antigen, GalNAcβ1-4(NeuAcα2-3)Galβ, implicated in pathogen invasion, cancer, xenotransplantation and transfusion medicine. Complete lack of this glycan epitope results in the Sd(a−) phenotype observed in 4% of individuals who may produce anti-Sd a . A candidate gene ( B4GALNT2 ), encoding a Sd a -synthesizing β-1,4- N -acetylgalactosaminyltransferase (β4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sd a deficiency was never elucidated. Experimental and bioinformatic approaches were used to identify and characterize B4GALNT2 variants in nine Sd(a−) individuals. Homozygosity for rs7224888:T > C dominated the cohort (n = 6) and causes p.Cys466Arg, which targets a highly conserved residue located in the enzymatically active domain and is judged deleterious to β4GalNAc-T2. Its allele frequency was 0.10–0.12 in different cohorts. A Sd(a−) compound heterozygote combined rs7224888:T > C with a splice-site mutation, rs72835417:G > A, predicted to alter splicing and occurred at a frequency of 0.11–0.12. Another compound heterozygote had two rare nonsynonymous variants, rs148441237:A > G (p.Gln436Arg) and rs61743617:C > T (p.Arg523Trp), in trans . One sample displayed no differences compared to Sd(a+). When investigating linkage disequilibrium between B4GALNT2 variants, we noted a 32-kb block spanning intron 9 to the intergenic region downstream of B4GALNT2 . This block includes RP11-708H21.4 , a long non-coding RNA recently reported to promote tumorigenesis and poor prognosis in colon cancer. The expression patterns of B4GALNT2 and RP11-708H21.4 correlated extremely well in >1000 cancer cell lines. In summary, we identified a connection between variants of the cancer-associated B4GALNT2 gene and Sd a , thereby establishing a new blood group system and opening up for the possibility to predict Sd(a+) and Sd(a‒) phenotypes by genotyping.

P1 and P are glycosphingolipid antigens synthesized by the -encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P (P1P) and P (P1P) phenotypes. transcript levels are higher in P individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of , particularly rs5751348, correlate with P/P status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 () haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates expression. Intriguingly, -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of did not affect transcript levels. Instead, protein pull-down experiments using but not oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds alleles selectively, and knockdown of significantly decreased transcription. These data indicate that RUNX1 regulates and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

This update on the P1PK blood group system (Hellberg Å, Westman JS, Thuresson B, Olsson ML. P1PK: the blood group system that changed its name and expanded. Immunohematology 2013;29:25–33) provides recent findings concerning the P1PK blood group system that have both challenged and confirmed old theories. The glycosphingolipids can no longer be considered the sole carriers of the antigens in this system because the P1 antigen has been detected on human red blood cell glycoproteins. New indications suggest that P1Pk synthase activity truly depends on the DXD motif, and the genetic background and molecular mechanism behind the common P1 and P2 phenotypes were found to depend on transcriptional regulation. Transcription factors bind the P1 allele selectively to a motif around rs5751348 in a regulatory region of A4GALT, which enhances transcription of the gene. Nonetheless, unexplained differences in antigen expression between individuals remain.

BACKGROUND The P1 antigen was first described in 1927 and belongs to the P1PK histo‐blood group system, together with Pk and NOR. The A4GALT‐encoded 4‐α‐galactosyltransferase synthesizes these antigens and has been considered to extend glycolipids exclusively. However, contradicting studies have been published regarding the presence of P1 on human glycoproteins. In other species, P1 occurs on glycoproteins. Furthermore, human ABH antigens occur on both glycolipids and glycoproteins and are biochemically related to P1. Thus, we hypothesized that P1 is present on RBC glycoproteins in humans. STUDY DESIGN AND METHODS RBCs of known P1/P2 status (phenotype and rs8138197 genotype) were used. The RBC surface glycans were modified with α‐galactosidases, papain, and/or peptide‐N‐glycosidase F. RBC membrane proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis/immunoblot. A new P 1/P 2‐allelic discrimination assay based on rs5751348 was validated. RESULTS P1 occurs on various glycoproteins, seen as smearlike patterns in anti‐P1‐stained immunoblots with RBC membranes of P1 but not P2 or p phenotype. There was a significant difference between the staining of P 1‐homozygous and P 1‐heterozygous RBCs (P 1P 1 > P 1P 2), as well as intragenotypic variation. Immunoblotting banding patterns show major carriers at approximately 50 and 100 kDa. P1 staining was lost after treatment of RBCs with α‐galactosidase of broad Galα‐1,3/4/6‐specificity. Peptide‐N‐glycosidase F treatment reduced the P1 signal, while papain or α‐1,3‐specific galactosidase did not. P 1/P 2 status was confirmed by a new rs5751348 assay. CONCLUSION Our data indicate that the P1 antigen can reside on human RBC glycoproteins. Glycosidase studies suggest that at least part of the epitopes occur on N‐glycans.

The Sda histo-blood group antigen (GalNAcβ1-4(NeuAcα2-3)Galβ-R) is implicated in various infections and constitutes a potential biomarker for colon cancer. Sd(a−) individuals (2–4% of Europeans) may produce anti-Sda, which can lead to incompatible blood transfusions, especially if donors with the high-expressing Sd(a++)/Cad phenotype are involved. We previously reported the association of B4GALNT2 mutations with Sd(a−), which established the SID blood-group system. The present study provides causal proof underpinning this correlation. Sd(a−) HEK293 cells were transfected with different B4GALNT2 constructs and evaluated by immunostaining and glycoproteomics. The predominant SIDnull candidate allele with rs7224888:T>C (p.Cys406Arg) abolished Sda synthesis, while this antigen was detectable as N- or O-glycans on glycoproteins following transfection of wildtype B4GALNT2. Surprisingly, two rare missense variants, rs148441237:A>G and rs61743617:C>T, found in a Sd(a−) compound heterozygote, gave results similar to wildtype. To elucidate on whether Sd(a++)/Cad also depends on B4GALNT2 alterations, this gene was sequenced in five individuals. No Cad-specific changes were identified, but a detailed erythroid Cad glycoprotein profile was obtained, especially for glycophorin-A (GLPA) O-glycosylation, equilibrative nucleoside transporter 1 (S29A1) O-glycosylation, and band 3 anion transport protein (B3AT) N-glycosylation. In conclusion, the p.Cys406Arg β4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.