Expression of the GBGT1 Gene and the Forssman Antigen in Red Blood Cells in a Palestinian Population.

BACKGROUND: The Forssman antigen (FORS1 Ag) is expressed on human red blood cells (RBCs). We investigated its presence on RBCs from Palestinian subjects and Swedish subjects by serological testing and by sequencing part of exon 7 of the GBGT1 gene, which encodes Forssman synthase. MATERIALS AND METHODS: Blood samples from Palestinian subjects (n = 211 adults and n = 73 newborns) and from Swedish subjects (n = 47 adults) were analyzed in the study. RBCs from the Palestinian samples were typed for the FORS1 Ag using a monoclonal anti-Forssman antibody. The GBGT1 gene was genotyped by DNA sequencing (all adult samples) or by using amplification refractory mutation system PCR (newborn samples). RESULTS: All of the studied samples were negative for the FORS1 Ag by serologic typing. DNA sequencing of the 3' end of exon 7 of the GBGT1 gene, which includes Arg296, showed that all samples had the wild-type Arg296 sequence, which is associated with an inactive form of Forssman synthase. We detected four single nucleotide polymorphisms in the adult samples; two were silent (p.Tyr232=, p. Gly290=), and two were missense (p. Arg243Cys, p. Arg243His). The allele frequencies ranged from 0.2 to 3.6%. The p. Arg243Cys SNP was a novel SNP that was detected in one Palestinian sample. CONCLUSION: Our results confirmed the allelic diversity of GBGT1 and identified a novel nucleotide polymorphism in this gene, p. Arg243Cys. Our results also confirmed that the FORS blood group system is a low-frequency system.

Prevalence of antibodies to a new histo-blood system: the FORS system.

BACKGROUND: In 1987, three unrelated English families were reported with a putative blood subgroup called Apae. Swedish researchers later found evidence leading to abolishment of the Apae subgroup and establishment instead of the FORS blood group system (System 31 - ISBT, 2012). It is important to know the prevalence of antibodies in order to make the best decisions in transfusion medicine. Cells expressing the Forssman saccharide, such as sheep erythrocytes, are needed to detect the anti-Forssman antibody. The aim of this study was to define the prevalence of human anti-Forssman antibody. MATERIALS AND METHODS: Plasma samples from 800 individuals were studied. Sheep erythrocytes or Forssman "kodecytes" were mixed with the plasma samples using the tube technique. Plasma from an Apae individual was used as a negative control and monoclonal anti-Forssman antibody (M1/22.25.8HL cell line supernatant) was used as the positive control. RESULTS: Of the 800 individuals tested, one was negative for the presence of anti-Forssman antibody. We compared the anti-Forssman antibody reaction pattern between genders and found that males have weaker reactions than females, both at room temperature (p=0.026) and at 37 °C (p=0.043). We also investigated the reaction pattern of anti-Forssman antibody in relation to ABO and Rh blood group types without finding any significant differences. DISCUSSION: Sheep erythrocytes are suitable for searching for human anti-Forssman antibody. The quantity of anti-Forssman antibodies in plasma is higher in females than in males. In the population (n=800) studied here, we found one individual lacking the anti-Forssman antibody. These results contribute to the data already published, confirming that FORS is a rare blood group.

Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system.

In analogy with histo-blood group A antigen, Forssman (Fs) antigen terminates with α3-N-acetylgalactosamine and can be used by pathogens as a host receptor in many mammals. However, primates including humans lack Fs synthase activity and have naturally occurring Fs antibodies in plasma. We investigated individuals with the enigmatic ABO subgroup A(pae) and found them to be homozygous for common O alleles. Their erythrocytes had no A antigens but instead expressed Fs glycolipids. The unexpected Fs antigen was confirmed in structural, serologic, and flow-cytometric studies. The Fs synthase gene, GBGT1, in A(pae) individuals encoded an arginine to glutamine change at residue 296. Gln296 is present in lower mammals, whereas Arg296 was found in 6 other primates, > 250 blood donors and A(pae) family relatives without the A(pae) phenotype. Transfection experiments and molecular modeling showed that Agr296Gln reactivates the human Fs synthase. Uropathogenic E coli containing prsG-adhesin-encoding plasmids agglutinated A(pae) but not group O cells, suggesting biologic implications. Predictive tests for intravascular hemolysis with crossmatch-incompatible sera indicated complement-mediated destruction of Fs-positive erythrocytes. Taken together, we provide the first conclusive description of Fs expression in normal human hematopoietic tissue and the basis of a new histo-blood group system in man, FORS.

The structural basis of blood group A-related glycolipids in an A3 red cell phenotype and a potential explanation to a serological phenomenon.

Glycolipids from the red cells of a rare blood group A subgroup individual, expressing the blood group A(3) phenotype with the classical mixed-field agglutination phenomenon, A(2(539G>A))/O(1) genotype, and an unusual blood group A glycolipid profile, were submitted to a comprehensive biochemical and structural analysis. To determine the nature of blood group A glycolipids in this A(3) phenotype, structural determination was carried out with complementary techniques including proton nuclear magnetic resonance (1D and 2D), mass spectrometry (MS) (nano-electrospray ionization/quadrupole time-of-flight and tandem mass spectrometry) and thin layer chromatography with immunostaining detection. As expected, total blood group A structures were of low abundance, but contrary to expectations extended-A type 2 and A type 3 glycolipids were more dominant than A hexaglycosylceramides based on type 2 chain (A-6-2 glycolipids), which normally is the major A glycolipid. Several para-Forssman (GalNAcß3GbO(4)) structures, including extended forms, were identified but surmised not to contribute to the classic mixed-field agglutination of the A(3) phenotype. It is proposed that the low level of A antigen combined with an absence of extended branched glycolipids may be the factor determining the mixed-field agglutination phenomenon in this individual.

Expression of carbohydrate xenoantigens on porcine peripheral nerve.

BACKGROUND: The use of thin easily revascularized cutaneous nerve autografts, which has been the gold standard, or the alternative use of nerve allografts or artificial grafts for nerve reconstructing have all their pros and cons. Nerve xenotransplantation may offer a potential alternative. In a potential pig to human nerve xenograft transplantation set-up several porcine antigen barriers have to be considered such as carbohydrate antigens system like the blood group A/O, the Galalpha1-3Gal (alphaGal) and the Hanganutziu-Deicher (HD) antigens. The swine leukocyte protein antigens system may also have to bee considered. The knowledge of the antigen expression on pig peripheral nerves is today limited. The present study describes the distribution of glycolipid based carbohydrate xenoantigens in ischiadicus nerve from blood group A and O pigs. METHODS: Glycolipid fractions were separated on thin layer chromatography plates and immunostained with human AB sera, biotinylated Griffonia simplicifolia isolectin B4, monoclonal antibodies reacting with the HD antigen and with blood group A antigens based on different core saccharide structures. In addition, the subcellular distribution of alphaGal and HD antigens were studied by light- and electron-microscopical immunohistochemistry. The total amount of neutral glycolipids was 15 mg/g tissue for both blood group A and O nerves with mono-glycosylceramides as the dominating component. RESULTS AND CONCLUSIONS: The total amount of acidic glycolipids (gangliosides and sulpholipids) was 9 mg/g tissue for both the blood group O and A nerves with sulphatides as the dominating components. Analyses of the glycolipid fractions showed strong expression of both the alphaGal and the HD antigens in nerves from both blood group A and O pigs. In addition, small amounts of blood group A antigens were expressed in nerves from blood group A pigs. Staining of neutral glycolipids from blood group A pigs using monoclonal antibodies reacting with A antigen having different core structures suggested that the A epitope expressed on pig ischiadicus nerves is based on the type 1 core chain structure. Light and electron microscopical studies on the alphaGal and HD-antigen distribution revealed that the neural cells were alphaGal antigen negative. Endothelial cells of blood vessels, and lymphatic and perineural cells expressed alphaGal antigen. Both endothelial cells and myelinized axons revealed positively labelled for the HD antigen.