B LOOD G ROUP R EVIEW May the FORS be with you: a system sequel A.K. Hult and M.L. Olsson This article is an update of the review of the FORS system 100 years after Professor Forssman (Fig. 1) of Lund, Sweden, published in Immunohematology in 2017 (Hult AK, Olsson ML. discovered it in animals by immunizing rabbits with extracts The FORS awakens: review of a blood group system reborn. of tissue from guinea pigs or horses to obtain the so-called Immunohematology 2017;33:64–72). This update incorporates the most recently presented knowledge on this still enigmatic Forssman antiserum that, when given to sheep, would lyze system and its genetic, enzymatic, and immunological aspects. their RBCs.5 Detailed studies were published by two groups4,6 Further insight into the genetic variation and allele frequencies explaining why rare humans and certain animal species of the GBGT1 locus has been reported, and screening studies regarding the prevalence of naturally occurring anti-FORS1 express this antigen while most humans and some animal in human plasma have been performed and presented. More species lack it, and these discoveries constitute the foundation basic knowledge on the specificity of the gene product, the of the continued studies summarized here. Forssman synthase, has been obtained in several detailed studies, and its relation to the homologous ABO gene has been investigated. Taken together, we summarize recently added The GBGT1 Gene and Its Alleles information about the carbohydrate-based FORS blood group system (International Society of Blood Transfusion number 031). The knowledge about different alleles in the FORS blood Immunohematology 2020;36:14–18. group system was extended by extraction of blood group gene data from the whole genome sequences accessible through Key Words: FORS, Forssman, GBGT1 Erythrogene,7 which is based on data from the third and final phase of the 1000 Genomes Project.8 These data were The FORS blood group system was reviewed in this used, and genetic variation at the GBGT1 locus was studied journal1 and elsewhere2 and was acknowledged by the by Hult et al.9 The two most common alleles (GBGT1*01N.01, International Society of Blood Transfusion in 2012.3 The GBGT1*01N.02) constitute 89 percent of all alleles and, of discovery that human red blood cells (RBCs) from rare the 66 remaining alleles reported in Erythrogene, there were families could express the Forssman glycolipid4 was made six different alleles containing c.363C>A (rs35898523), of Fig. 1 Left: Professor John Forssman (1901–1933), professor of pathology and chief physician at the Lasarrette in Lund, Sweden, together with Arvid Lindau, his later successor. Right: Professor John Forssman in action at the health center in Lund, often called “the seaman’s home in Lund” thanks to his clientele, who often came from Skåne's port cities. Source: The South Swedish Society for the History of Medicine. 14 IMMUNOHEMATOLOGY, Volume 36, Number 1, 2020 FORS system update which GBGT1*02N (allele frequency 3.6%) was the most of the coding region were sequenced, it is not known whether common. This single nucleotide polymorphism (SNP) gives this GBGT1 allele has any additional mutations or what the rise to a severely truncated protein predicted to lack the effects of p.Arg243Cys are. whole enzymatically active domain. Four healthy individuals homozygous for this nonsense mutation were identified, and Antibodies it was shown that, although the gene product is predicted to be severely truncated, transcripts were detected, and the Additional examination of the FORS blood group system expressed protein can be detected intracellularly in hema- has mainly focused on the presence and level of the naturally topoietic cells. It has been hypothesized that the Forssman occurring antibodies seen in the majority of all humans. The synthase has acquired an alternative function10 because it question is what the clinical relevance of these antibodies may appears to have degenerated more slowly than confirmed be. Because most blood centers do not have access to RBCs pseudogenes in the glycosyltransferase 6 (GT6) enzyme with the very rare FORS1+ phenotype, different approaches family. Although this is slightly speculative and not yet have been used to screen for anti-FORS1 and to estimate its proven, the presence of “true null” individuals showed for the prevalence: first time that the GBGT1-encoded function, whatever it is, is 1. Screening with FORS1+ RBCs (only in one study).9 dispensable in humans. 2. Kodecytes as described by Frame et al.12 have been used in two studies in which Function-Spacer-Lipid (FSL) FORS1 Antigen Expression constructs expressing the Forssman pentasaccharide (FSL-FS) were uploaded to RBCs then used for screening.13 The reactivation of the Forssman synthase (globoside 3. Ovine RBCs are known to express the Forssman antigen 3-α-N-acetylgalactosaminyl transferase, EC no. 2.4.1.88) is at very high levels (also shown by Svensson et al.4 in the due to a missense mutation, c.887G>A (p.Arg296Gln), in exon first publication regarding this blood group system, in 7 of the GBGT1 gene.4 After searching databases, another which we used sheep RBCs as a positive control for testing missense mutation, c.886C>T, affecting the same codon and with monoclonal anti-Forssman); if used for screening also giving rise to an amino acid change (p.Arg296Trp), was of human plasma, however, the possible presence of reported. This mutation is also rare and mainly (approximately xenoantibodies has to be taken into account. 80%) found in individuals of African descent, but nothing is The two studies that have performed screening studies known about the resulting RBC phenotype. presented different prevalences of anti-FORS1 in human The consequences of this SNP were further examined plasma. These screening studies differ in the amount of FSL- by Hult et al.,9 who showed in transfection studies that this FS uploaded onto the RBCs; Jesus et al.13 used a significantly substitution, in contrast to p.Arg296Gln, does not give rise to higher concentration than Hult et al.9 The latter study adapted any detectable FORS1 expression. To date, the only mutation the amount of FSL-FS to mimic the antigen strength detected in the GBGT1 gene reported to reactivate the Forssman on native FORS1+ RBCs. Jesus et al.13 reported a prevalence synthase in humans and give rise to antigen expression is still of anti-FORS1 in plasma of almost 100 percent, which was c.887G>A. Although screening studies for this mutation have corroborated by Hult et al. when using kodecytes with a only been performed in small cohorts, it is quite safe to say that similarly high concentration of Forssman pentasaccharide, this phenotype is very rare. This supposition is also supported but this group also showed that the negative control (plasma by data from the Genome Aggregation Database (gnomAD), from FORS1+ individuals, which should lack anti-FORS1) was in which 5 of 282,088 GBGT1 alleles carried c.887G>A positive with these FORS1+ kodecytes with extremely high (rs375748588) and 32 of 281,956 alleles were positive for antigen density. This reaction could possibly be due to cross- c.886C>T (rs14104392). reactivity of polyclonal anti-A or anti-A,B present in human In a study by Abusibaa et al.,11 the presence of the FORS1 blood group O plasma. antigen on RBCs and/or the presence of the activating When screening with either a more physiologically mutation, c.887G>A, in a Palestinian and Swedish sample relevant amount of FSL-FS on kodecytes or using FORS1+ cohort were investigated. No sample with the FORS1+ RBCs, the prevalence of anti-FORS1 was approximately 10 phenotype was encountered. One previously unreported SNP percent, and it was also shown that there seems to be an ABO was found: c.727C>T (p.Arg243Cys); but because only parts restriction. The vast majority of blood group A individuals (in IMMUNOHEMATOLOGY, Volume 36, Number 1, 2020 15 A.K. Hult and M.L. Olsson particular A1) do not make anti-FORS1, which was verified the Forssman antigen, although it is unclear whether the by negative screening results with both FSL-FS kodecytes antigens are still present in the commercial product. and FORS1+ RBCs. The clinical significance of this antibody is still unknown to a large extent but may (in analogy with FORS Versus ABO other antibody specificities against carbohydrate blood group antigens, like anti-A, -B, -Pk, or -P) cause intravascular The GBGT1 gene is a member of the GT6 family, as is the hemolysis if FORS1+ blood units are transfused. ABO gene responsible for the expression of A and B on RBCs On the other hand, antibodies against P1, which is the and other tissues in humans. These genes are derived from same length as FORS1 (i.e., a pentasaccharide), typically do the same ancestral gene and are therefore, not unexpectedly, not cause hemolysis even if rare cases occur. In vitro hemolysis homologous. Forssman synthase and glycosyltransferase A is mainly observed with papain-treated FORS1+ RBCs.4 Thus, both result in the addition of the same terminal sugar, α-3- additional studies are needed to answer the question whether N-acetyl-d-galactosamine, although using different acceptor anti-FORS1 is of clinical relevance in pretransfusion testing. substrates as precursors (P and H, respectively) (Fig. 2). The Another aspect of these naturally occurring antibodies ABO gene and the GBGT1 gene are located at the boundaries has been discussed in association with xenotransplantation. of chromosomal fragments, which seem to have been inverted The use of bioprosthetic heart valves in a clinical setting may and translocated during evolution, and this structure may have pose a problem, as suggested by Barone et al.14 These animal- provided an opportunity for further divergence.15 The genes derived heart valves may display an array of potentially are also present in other species, but the tissue expression and antigenic carbohydrate structures that can possibly cause an gene functionality is species dependent.16,17 immunological reaction to that specific tissue.