B l o o d G r o u p R e v i e w 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 frequencies explaining why rare humans and certain animal species of the GBGT1 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 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 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.

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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 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 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 6 (GT6) 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 theGBGT1- 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 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 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 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. The authors The effect of different substitutions and/or deletions in showed, among other findings, that equine pericardia display the ABO and GBGT1 genes derived from both humans and

Fig. 2 Schematic depiction of the biosynthetic pathway of the FORS1 antigen in comparison to the A blood group. The glycosyltransferase model shown is in both cases based on the ABO sequence, which is homologous to the GBGT1 gene; that is, it represents in this case both the Forssman synthase (Fs) and the blood group A transferase. More details about the similarities and differences between the ABO- and GBGT1-derived can be found in the article by Svensson et al. and on the corresponding cover of Blood. (Svensson L, Hult AK, Stamps R, et al. Forssman expression on human erythrocytes: biochemical and genetic evidence of a new histo-blood group system. Blood 2013;121:1459–68.) RBCs = red blood cells.

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mice has been tested and evaluated in a series of articles by GBGT1 mutations have been described in databases of cancer- Yamamoto and colleagues.18–21 The aim has been to provide associated alterations, and it has not been formally excluded more insight into the specificity of glycosyltransferases that such changes may contribute to acquired synthesis of included in the GT6 family and how the specificity of one can Forssman glycolipid. be changed to the other’s in an experimental setting. Taken together, there have been interesting additions The positions in the ABO glycosyltransferase mainly recently to the knowledge about this enigmatic blood group responsible for donor specificity (i.e., those that differentiate system. At the same time, more general lessons are being directly between donor substrates UDP-GalNAc or UDP-Gal learned, for example, extending our understanding about the to give rise to blood group A or B, respectively) are codons 266 specificity of glycosyltransferases and genetic diversification. and 268 (Leu266Gly267Gly268 in A and Met266Gly267Ala268 in B, respectively). The corresponding sequence in the GBGT1 gene Disease Associations is GlyGlyAla, and this motif is well conserved throughout evolution and was hypothesized to be important for Forssman The connection between Forssman and disease has expression.22 In many of the experiments performed by mainly focused on the presence of the Fs antigen in different Yamamoto’s laboratory, variants of this tripeptide of amino cancers, and also, in analogy with many other carbohydrate acids have been tested in different settings, sometimes with structures, it can be used as a receptor by various pathogens. additional deletions and substitutions.15,19,22 The effect of these In recent years, it has become increasingly more common changes regarding donor sugar and acceptor specificity was to do exome sequencing—to use different chip assays and tested and evaluated. In their setup, the system was pushed microarray detection where a multitude of different genes to investigate whether it would be possible to encode “dual” and their expression can be examined. In that context, the specificities—that is, can different, syntheticABO constructs GBGT1 gene has been highlighted in a few publications even (not yet found in humans) give rise to FORS1 expression in though the correlation to the diseases remains to be verified. addition to either A or B in transfected cell lines, where such Villegas-Ruiz and Juarez-Mendez25 examined recombinant enzyme sequences are overexpressed. In this in cell lines and malignant ovarian tumors with the aim to way, it was shown that the specificity of the GT can be changed find molecular targets as markers in ovarian cancer. Different but, when forced to synthesize another structure in this in pathways were examined, and the gene expression of GBGT1 vitro setting, the level of antigen expression is generally low,18 was (among many other genes) correlated between malignant although exceptions may exist. ovarian tumors and ovarian cell lines. GBGT1 was shown to The Forssman antigen was shown in several studies to be downregulated in the most significant signaling pathway be present in cancerous tissue, as discussed in the previous examined. review in this journal.1 To date, it is still unclear how this is In a family study focusing on celiac disease, the achieved, or, more relevant (and interesting), whether this c.363C>A SNP (rs35898523) was present in a majority of antigen is synthesized by the Forssman synthase (since the the participants,26 but the significance and meaning of this is GBGT1 gene was deemed a pseudogene, and the product unclear. For instance, it is possible that linkage disequilibrium in the vast majority of humans is inactive when it comes to with nearby genetic markers may explain this and similar making FORS1 on RBCs). The background of the elusive, so- findings. Finally, it was recently reported that an upregulation called incompatible A present in some forms of cancer has of GBGT1 was noted in samples from patients with myocardial been debated, but no answer has yet been found. This A-like infarction, and the authors suggested this to be one of six activity was shown to occur in individuals devoid of blood markers used as a diagnostics tool.27 group A alleles,23 but it has also been suggested that this A-like antigen may in fact be the Forssman antigen.24 It was Summary suggested that differential splicing and/or substitutions in the ABO gene may cause the ABO glycosyltransferase to make the In conclusion, more basic knowledge on the FORS blood Forssman structure. Hypothetically, this is very interesting, group system and its relation to ABO has been obtained, but the splicing variants tested in these studies have not been and practical issues on how to screen for anti-Fs have been detected in nature nor have these variants been identified investigated. In addition, the GBGT1 gene is also implicated in various databases.18,19 On the other hand, 89 different in correlation to different types of disease, although causality

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and mechanisms remain to be established to appreciate the 17. Eirin-Lopez JM, Rebordinos L, Rooney AP, Rozas J. The birth- relevance of FORS in disease. and-death evolution of multigene families revisited. Genome Dyn 2012;7:170–96. 18. Cid E, Yamamoto M, Yamamoto F. Blood group ABO gene- References encoded A transferase catalyzes the biosynthesis of FORS1 antigen of FORS system upon Met69Thr/Ser substitution. 1. Hult AK, Olsson ML. The FORS awakens: review of a blood Blood Adv 2018;2:1371–81. group system reborn. Immunohematology 2017;33:64–72. 19. Yamamoto M, Cid E, Yamamoto F. ABO blood group A 2. Brito S, Hesse C, Mendes F. FORS, a new histo-blood group: a transferases catalyze the biosynthesis of FORS blood group current review. Med Res Innov 2017;1:1–5. FORS1 antigen upon deletion of exon 3 or 4. Blood Adv 3. Storry JR, Castilho L, Daniels G, et al. International Society of 2017;1:2756–66. Blood Transfusion Working Party on red cell immunogenetics 20. Cid E, Yamamoto M, Yamamoto F. Amino acid substitutions and blood group terminology: Cancun report (2012). Vox Sang at sugar-recognizing codons confer ABO blood group system- 2014;107:90–6. related alpha1,3 Gal(NAc) transferases with differential 4. Svensson L, Hult AK, Stamps R, et al. Forssman expression enzymatic activity. Sci Rep 2019;9:846. on human erythrocytes: biochemical and genetic evidence of a 21. Yamamoto M, Tarasco MC, Cid E, Kobayashi H, Yamamoto F. new histo-blood group system. Blood 2013;121:1459–68. ABO blood group A transferase and its codon 69 substitution 5. Forssman J. Die Herstellung hochwertiger spezifisher synthesize FORS1 antigen of FORS blood group Schafhämolysin ohne Verwendung von Schaftblut: Ein Beitrag system. Sci Rep 2019;9:9717. Zur Lehre von heterolofer Antikörperbildung. Biochem Zeit 22. Yamamoto M, Cid E, Yamamoto F. Crosstalk between ABO 1911;37:78–115. and Forssman (FORS) blood group systems: FORS1 antigen 6. Yamamoto M, Cid E, Yamamoto F. Molecular genetic basis of synthesis by ABO gene-encoded glycosyltransferases. Sci Rep the human Forssman glycolipid antigen negativity. Sci Rep 2017;7:41632. 2012;2:975. 23. Grunnet N, Steffensen R, Bennett EP, Clausen H. Evaluation 7. Möller M, Jöud M, Storry JR, Olsson ML. Erythrogene: a of histo-blood group ABO genotyping in a Danish population: database for in-depth analysis of the extensive variation in 36 frequency of a novel O allele defined as O2. Vox Sang blood group systems in the 1000 Genomes Project. Blood Adv 1994;67:210–5. 2016;1:240–9. 24. Hakomori S, Wang SM, Young WW Jr. Isoantigenic expression 8. Genomes Project Consortium, Auton A, Brooks LD, Durbin of Forssman glycolipid in human gastric and colonic mucosa: RM, et al. A global reference for human genetic variation. its possible identity with "A-like antigen" in human cancer. Nature 2015;526:68–74. Proc Natl Acad Sci U S A 1977;74:3023–7. 9. Hult AK, McSherry E, Moller M, Olsson ML. GBGT1 is 25. Villegas-Ruiz V, Juarez-Mendez S. Data mining for allelically diverse but dispensable in humans and naturally identification of molecular targets in ovarian cancer. Asian Pac occurring anti-FORS1 shows an ABO-restricted pattern. J Cancer Prev 2016;17:1691–9. Transfusion 2018;58:2036–45. 26. Szperl AM, Ricano-Ponce I, Li JK, et al. Exome sequencing in a 10. Xu H, Storch T, Yu M, Elliott SP, Haslam DB. Characterization family segregating for celiac disease. Clin Genet 2011;80:138– of the human Forssman synthetase gene: an evolving 47. association between glycolipid synthesis and host-microbial 27. Fan L, Meng H, Guo X, Li X, Meng F. Differential gene interactions. J Biol Chem 1999;274:29390–8. expression profiles in peripheral blood in Northeast Chinese 11. Abusibaa WA, Srour MA, Moslemi AR, et al. Expression of Han people with acute myocardial infarction. Genet Mol Biol the GBGT1 gene and the Forssman antigen in red blood cells 2018;41:59–66. in a Palestinian population. Transfus Med Hemother 2019 2019;46:450–4. Annika K. Hult, PhD (corresponding author), Postdoctoral 12. Frame T, Carroll T, Korchagina E, Bovin N, Henry S. Synthetic glycolipid modification of red blood cell membranes. Researcher, Department of Clinical Immunology and Transfusion Transfusion 2007;47:876–82. Medicine, Office of Medical Services, Region Skåne, and Division of 13. Jesus C, Hesse C, Rocha C, et al. Prevalence of antibodies to Hematology and Transfusion Medicine, Department of Laboratory a new histo-blood system: the FORS system. Blood Transfus Medicine, Lund University, BMC C14, SE-22184 Lund, Sweden, 2018;16:178–83. [email protected]; and Martin L. Olsson, MD, PhD, Medical 14. Barone A, Benktander J, Whiddon C, et al. Glycosphingolipids Director of the Nordic Reference Laboratory for Genomic Blood of porcine, bovine, and equine pericardia as potential immune Group Typing and Senior Consultant at the Department of Clinical targets in bioprosthetic heart valve grafts. Xenotransplantation Immunology and Transfusion Medicine, Office of Medical Services, 2018;25:e12406. Region Skåne, and Vice Dean of the Faculty of Medicine, Professor 15. Yamamoto F. Evolutionary divergence of the ABO and GBGT1 of Transfusion Medicine at the Department of Laboratory Medicine, genes specifying the ABO and FORS blood group systems Division of Hematology and Transfusion Medicine, Lund University, through chromosomal rearrangements. Sci Rep 2017;7:9375. Lund, Sweden. 16. Turcot-Dubois AL, Le Moullac-Vaidye B, Despiau S, et al. Long- term evolution of the CAZY glycosyltransferase 6 (ABO) gene family from fishes to mammals: a birth-and-death evolution model. Glycobiology 2007;17:516–28.

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