Genetics and Expression of Glycosphingolipids in the P1PK and GLOB Histo-Blood Group Systems

Abstract: The glycosphingolipid (GSL) antigens in the clinically important P1PK and GLOB blood group systems are present on erythrocytes as well as other tissues and are so-called histo-blood group antigens. Blood group antigens of carbohydrate nature are products of enzymes, so-called glycosyltransferases, adding sugars in a step-wise manner to an acceptor substrate. Presence or absence of the P1PK and GLOB antigens gives rise to the common P1 and P2 (P1−) phenotypes as well as the rare but clinically important p, P1k and P2k phenotypes. The aim of this thesis was to explore the molecular genetics underlying the expression of the antigens in the P1PK and GLOB histo-blood group systems by analyzing the genes responsible for their expression, A4GALT and B3GALNT1, respectively. Investigation of potential regulatory regions of the A4GALT identified a SNP in a novel exon, denoted exon 2a, which correlates with the P1/P2 phenotypes. A P1/P2-discriminating SNP enabled genotypic prediction of P1 status in >200 samples. Presence of one or two P1 allele(s) resulted in increased A4GALT transcript levels and more Pk and P1 antigens on erythrocytes, compared to homozygosity for a P2 allele. The A4GALT-encoded enzyme α1,4Gal-T was thereby linked to synthesis of the two α1,4Gal-terminating antigens, Pk and P1, and consequently the former P blood group system was renamed to P1PK based on these findings. Critical mutations in the A4GALT open reading frame (ORF), give rise to the PP1Pk-negative p phenotype, whilst B3GALNT1-null alleles result in the P-deficient P1k/P2k phenotypes. P1/P2 genotyping revealed that the p phenotype can arise on both P1 and P2 alleles, whilst the presence or absence of P1 antigen was predicted in 92% of the P1k/P2k samples. In addition, a novel genetic explanation underlying the p phenotype was identified, where three different alleles were shown to have an intact A4GALT ORF but instead had large deletions comprising the promoter and non-coding exons 1 and 2a. The number of p- and P1k/P2k-inducing mutations was increased by these studies as new alterations were identified in A4GALT and B3GALNT1, respectively. Interestingly, p phenotype cells express a GSL named x2 that was shown here to be absent on erythrocytes of the P1k/P2k phenotypes. This structure was recently acknowledged as a blood group antigen under the name PX2, since anti-PX2 is present in P1k/P2k plasma. PX2 is a β1,3GalNAc-elongation of paragloboside, a neolacto series GSL. Overexpression and knock-down of B3GALNT1 in a cellular system revealed that β1,3GalNAc-T1 encoded by this gene is responsible for both P and PX2 antigen expression. As a consequence of these findings, PX2 was removed from the GLOB blood group collection to join P as the second antigen in the GLOB blood group system. This thesis work further emphasizes the molecular and genetic diversity of the P1PK and GLOB histo-blood group systems. It also highlights the fact that these glycosyltransferases are more versatile than previously thought. For instance, both α1,4Gal-T (Pk and P1) and β1,3GalNAc-T1 (P and PX2) are able to synthesize more than one GSL antigen by utilizing more than one acceptor. Future work will hopefully gain further knowledge of these GSL antigens and their extensions, as well as their function in health and disease.