BACKGROUND AND OBJECTIVES:Noninvasive fetal antigen genotyping can assist the management of immunized pregnant women, and fetal RHD genotyping can be used as a screening assay to guide restricted use of Rh prophylaxis. Based on a survey, we present an overview of the implementation of noninvasive fetal antigen genotyping into clinical practice. MATERIALS AND METHODS:A survey was developed and sent out to the members of the International Society of Blood Transfusion (ISBT) working party on Red Cell Immunogenetics and Blood Group Terminology and to participants of the international noninvasive fetal RHD genotyping programme from the Danish Institute for External Quality Assurance for Laboratories in the health sector (DEKS). The survey contained four sections: fetal RHD screening, fetal RHD genotyping for RhD immunized women, fetal genotyping of other antigen targets than RhD and a section for collecting topics for future collaboration. The survey data were evaluated by the core author group. RESULTS:Fifty-two survey responders were from 22 countries. Fetal RHD screening was implemented by 73%, foremost using real-time PCR with in-house assays, primarily, or commercially available kits. Most laboratories use the same assay for RhD immunized women. Thirty-eight percent of the responders test for other antigen targets than RhD, using either real-time PCR, droplet digital PCR (ddPCR) or DNA sequencing. There was an interest in collaborations on topics across methodology, technology, strategy and health care regulations. CONCLUSION:In general, we found that noninvasive fetal blood group antigen genotyping is well implemented. However, our results are biased towards high-income countries, Europe and laboratories already running noninvasive fetal antigen genotyping.

Background and Objectives The International Society of Blood Transfusion (ISBT) Working Party (WP) on Red Cell Immunogenetics and Blood Group Terminology (RCI&BGT) held six business meetings between December 2021 and June 2024. This report describes the new blood group systems and antigens ratified during these meetings.Materials and Methods Candidate systems and antigens were reviewed according to serological, genetic and biological evidence. This evidence was matched against defined criteria, and the acknowledged systems/antigens were assigned a unique identifier.Results Four new systems, ER (ISBT 044), CD36 (ISBT 045), ATP11C (ISBT 046) and MAL (ISBT 047), were ratified. CD36 and ATP11C were de novo entries, while ER and MAL systems resolved the genetic basis for the Er and AnWj antigens. Thirteen antigens were added to existing systems: one each to LU (005), YT (011), SC (013), LW (016), KN (022), GLOB (028); an antithetical pair to KEL (006); two antigens to RHAG (030); and three to CTL2 (039). Two CTL2 antigens defined the hitherto unresolved antithetical antigens, Csa/Csb, whose genetic basis coincides with those of variants responsible for the HNA-3a/3b neutrophil antigens, respectively.Conclusion As of June 2024, the ISBT has acknowledged 47 blood group systems, comprising 366 antigens. The WP continues to ratify new systems and antigens, which are available on the ISBT website (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Background - The Rh blood group system is highly complex, polymorphic, and immunogenic. The presence of RHD gene variants in RhD negative pregnant women is a challenge in fetal RHD genotyping as it may influence the antenatal management of anti-D prophylaxis. The aim of this study was to determine the efficiency of a non-invasive single-exon approach in the obstetric population of Western Sweden in a 31-month follow up. The frequency and type of maternal RHD variants were explored and the relation to the ethnicity was elucidated. Discrepant results between fetal RHD genotyping and serological blood group typing of newborns were investigated and clarified. Materials and methods - RHD exon 4 was analysed with quantitative real-time PCR technique in a total of 6,948 blood samples from RhD negative women in early pregnancy. All cases with suspected maternal RHD gene and discrepant results observed in newborn samples, were further investigated using both serological and molecular technologies. Results - A total of 43 samples (0.6%) had inconclusive fetal genotyping result due the presence of a maternal RHD gene. These findings were in most cases (>66%) observed in pregnant women of non-European ancestry. Additionally, two novel RHD alleles were found. Seven discrepant results between fetal RHDgenotype and serological RhD type of the newborns, were shown to be related to D antigen variants in newborns. Assay sensitivity was 99.95%, specificity 100%, and accuracy 99.97%. Discussion - The single-exon approach for fetal RHD screening early in pregnancy is an appropriate choice in the population of Western Sweden, with a very low frequency of inconclusive results caused by the presence of maternal RHD gene variants. Due to the high sensitivity, specificity, and accuracy of the test, serological typing of neonates born to RhD negative women has no longer been performed at our laboratory since June 2023.

Introduction: With over 360 blood group antigens in systems recognized, there are antigens, such as RhD, which demonstrate a quantitative reduction in antigen expression due to nucleotide variants in the non-coding region of the gene that result in aberrant splicing or a regulatory mechanism. This study aimed to evaluate bioinformatically predicted GATA1-binding regulatory motifs in the RHD gene for samples presenting with weak or apparently negative RhD antigen expression but showing normal RHD exons. Methods: Publicly available open chromatin region data were overlayed with GATA1 motif candidates in RHD. Genomic DNA from weak D, Del or D- samples with normal RHD exons (n = 13) was used to confirm RHD zygosity by quantitative PCR. Then, RHD promoter, intron 1, and intron 2 regions were amplified for Sanger sequencing to detect potential disruptions in the GATA1 motif candidates. Electrophoretic mobility shift assay (EMSA) was performed to assess GATA1-binding. Luciferase assays were used to assess transcriptional activity. Results: Bioinformatic analysis identified five of six GATA1 motif candidates in the promoter, intron 1 and intron 2 for investigation in the samples. Luciferase assays showed an enhancement in transcription for GATA1 motifs in intron 1 and for intron 2 only when the R-2 haplotype variant (rs675072G>A) was present. GATA1 motifs were intact in 12 of 13 samples. For one sample with a Del phenotype, a novel RHD c.1-110A>C variant disrupted the GATA1 motif in the promoter which was supported by a lack of a GATA1 supershift in the EMSA and 73% transcriptional activity in the luciferase assay. Two samples were D+/D- chimeras. Conclusion: The bioinformatic predictions enabled the identification of a novel DEL allele, RHD c.1-110A>C, which disrupted the GATA1 motif in the proximal promoter. Although the majority of the samples investigated here remain unexplained, we provide GATA1 targets which may benefit future RHD regulatory investigations.

BACKGROUND:Correct ABO blood-group matching between donor and patient is crucial for safe transfusions. We investigated the underlying reason causing inconclusive ABO serology in samples referred to our laboratory.STUDY DESIGN AND METHODS:Flow cytometric analysis, ABO genotyping, and sequencing were used to characterize ABO-discrepant blood samples (n = 13). ABO gene variants were inserted in a GFP-containing bicistronic vector to assess A/B expression following overexpression in HeLa cells.RESULTS:Seven novel alleles with nonsense mutations predicted to truncate the encoded ABO glycosyltransferases were identified. While these variants could represent O alleles, serology showed signs of ABO glycosyltransferase activity. ABO*A1.01-related alleles displayed remarkably characteristic percentages of A-positive cells for samples with the same variant: c.42C>A (p.Cys14*; 10%), c.102C>A (p.Tyr34*; 31%-32%, n = 2), c.106dup (p.Val36Glyfs*21; 16%-17%, n = 3) or c.181_182ins (p.Leu61Argfs*21; 12%-13%, n = 2). Transfection studies confirmed significantly decreased A expression compared to wild type. The remaining variants were found on ABO*B.01 background: c.1_5dup (pGly3Trpfs*20), c.15dup (p.Arg6Alafs*51) or c.496del (p.Thr166Profs*26). Although the absence of plasma anti-B was noted overall, B antigen expression was barely detected on erythrocytes. Overexpression confirmed decreased B in two variants compared to wildtype while c.1_5dup only showed a non-significant downward trend.CONCLUSION:Samples displaying aberrant ABO serology revealed seven principally interesting alleles. Despite the presence of truncating mutations, normally resulting in null alleles, low levels of ABO antigens were detectable where alterations affected ABO exons 1-4 but not exon 7. This is compatible with the previously proposed concept that alternative start codons in early exons can be used to initiate the translation of functional ABO glycosyltransferase.

BACKGROUND AND OBJECTIVES:The extremely rare Rhnull phenotype is characterized by the absence of all Rh antigens on erythrocytes. It is divided into the regulator and amorph types based on the underlying genetic background. The more common regulator type depends on critical variants silencing RHAG, which encodes RhAG glycoprotein, necessary for RhD/RhCE expression. Rhnull cells have altered expression of glycophorin B and LW glycoprotein.MATERIALS AND METHODS:Four unrelated Rhnull individuals were investigated. Serological testing was performed according to standard blood bank practice. RHD/RHCE and S/s allele-specific Polymerase chain reaction (PCR) genotyping was done on genomic DNA using in-house PCR assays. RHAG, and in some cases also RHD/RHCE, were sequenced. Initial s phenotyping results triggered additional serological investigation.RESULTS:Anti-Rh29 was identified in all four individuals. Extended typing with anti-S and anti-s showed that the three samples predicted to type as s+ failed to react with 2 of 5 anti-s. Sequence analysis of all 10 RHAG exons and the immediate intron/exon boundaries revealed a single nucleotide variant in the 3'-end of intron 6, c.946 -2a>g in all samples. RHD/RHCE showed no alterations.CONCLUSION:A novel Nordic Rhnull allele was identified. In addition, it was shown that s+ Rhnull red blood cells are not only U- but also have qualitative changes in their s antigen expression.

The high-prevalence blood group antigen, Sda, had been puzzling blood bankers and transfusionists for at least a decade when it was reported in 1967. The characteristic mix of agglutinates and free red blood cells (RBCs), caused by anti-Sda, is seen with the RBCs from 90 percent of individuals of European descent. However, only 2-4 percent of individuals are truly Sd(a-) and may produce anti-Sda. The antibodies, generally considered insignificant, may cause hemolytic transfusion reactions with high-expressing Sd(a+) RBCs (e.g., the unusual Cad phenotype, which can also be polyagglutinable). The Sda glycan, GalNAcβ1-4(NeuAcα2-3)Gal-R, is produced in the gastrointestinal and urinary systems, while its origin on RBCs is more controversial. According to current theory, Sda is likely to be passively adsorbed in low amounts, except in Cad individuals, where it has been found on erythroid proteins and at higher levels. The long-standing hypothesis that B4GALNT2 encodes the Sda synthase was confirmed in 2019, since homozygosity for a variant allele with rs7224888:C produces a non-functional enzyme associated with most cases of the Sd(a-) phenotype. Thereby, the SID blood group system was acknowledged as number 038 by the International Society of Blood Transfusion. Although the genetic background of Sd(a-) was settled, questions remain. The genetic background of the Cad phenotype has not yet been determined, and the source of the RBC-carried Sda is unknown. Furthermore, the interest of Sda stretches beyond transfusion medicine. Some tantalizing examples are lowered antigen levels in malignant tissue compared with normal tissue and interference with infectious agents like Escherichia coli, influenza virus, and malaria parasites.

Background and Objectives Non-invasive assays for predicting foetal blood group status in pregnancy serve as valuable clinical tools in the management of pregnancies at risk of detrimental consequences due to blood group antigen incompatibility. To secure clinical applicability, assays for non-invasive prenatal testing of foetal blood groups need to follow strict rules for validation and quality assurance. Here, we present a multi-national position paper with specific recommendations for validation and quality assurance for such assays and discuss their risk classification according to EU regulations. Materials and Methods We reviewed the literature covering validation for in-vitro diagnostic (IVD) assays in general and for non-invasive foetal RHD genotyping in particular. Recommendations were based on the result of discussions between co-authors. Results In relation to Annex VIII of the In-Vitro-Diagnostic Medical Device Regulation 2017/746 of the European Parliament and the Council, assays for non-invasive prenatal testing of foetal blood groups are risk class D devices. In our opinion, screening for targeted anti-D prophylaxis for non-immunized RhD negative women should be placed under risk class C. To ensure high quality of non-invasive foetal blood group assays within and beyond the European Union, we present specific recommendations for validation and quality assurance in terms of analytical detection limit, range and linearity, precision, robustness, pre-analytics and use of controls in routine testing. With respect to immunized women, different requirements for validation and IVD risk classification are discussed. Conclusion These recommendations should be followed to ensure appropriate assay performance and applicability for clinical use of both commercial and in-house assays.

The authors declare that there is no conflict of interest.

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.

Weak expression of ABO antigens is encountered in the clinical laboratory occasionally, and subgroups of A are more commonly observed in Europeans than subgroups of B. To date, weakly expressing B variant phenotypes have been associated with 38 different alleles according to ISBT (https://www.isbtweb.org/resource/001aboalleles.html). This number is an underrepresentation since there have been several reports of aberrant B expression due to variant alleles since the last update of the ISBT allele table. The current study was initiated by an unusual blood group typing result in a 55-year-old male patient of Czech origin and previously reported as an abstract.1 Blood grouping was performed according to standard blood banking practice, initially using an automatic analyzer (Galileo, Immucor) followed by confirmation with manual gel (BioRad; DG-Gel) and tube agglutination techniques. Initial genotyping analysis was done using a PCR-SSP kit (Innotrain), microarray (BloodChip Reference, Progenika) and subsequently verified by expanded PCR-ASP and PCR-RFLP as described previously.2, 3 ABO exons 1–7 and splice sites were amplified and analyzed, together with the product(s) of PCR-ASP for exons 6–7, by Sanger sequencing.4 A single nucleotide variation (SNV) was detected, and the localization of the affected amino acid is visualized in a 3D-model of ABO glycosyltransferase by Cn3D (v.4.3.1, www.ncbi.nih.gov) and a detailed view obtained by AlphaFold.5, 6 Flow cytometry testing with monoclonal ABO reagents was performed as described previously.7 The proband's red blood cells (RBCs) initially typed as group O but the plasma typing gave negative or weak reactions with test RBCs of group B, depending on the method used, Table 1. An ABO*B.01/O.01.01 genotype was revealed, normally consistent with group B. Screening for selected A and B subgroup allele markers was negative.2 After informed consent, samples from family members were drawn and further investigation was performed. In samples from the proband, his sister and niece, sequence analysis revealed heterozygosity for a SNV in ABO exon 7, c.662G>A (no rs number available) in an otherwise normal ABO*B.01 allele. Significantly weakened B antigen expression was observed in all three individuals. An overview of serological testing and genetic results is shown in Table 1. SNV c.662G>A encodes an amino acid change, p.Gly221Asp. The glycine residue is completely evolutionarily conserved among the members of the GT6 family of glycosyltransferases8 and centrally located in the enzyme, seven amino acids away from the DVD motif (pp. 211–213) that coordinates the Mn2+ ion and the UDP part of the UDP-galactose donor substrate (Figure 1A). However, it is not directly interfering with the catalytic site. Instead, the change of the small neutral glycine to the bulkier and charged aspartic acid is predicted to abolish selected hydrogen bonds and is therefore hypothesized to destabilize the protein conformation (Figure 1B).5, 6 Other family members were also tested but did not display the phenotype nor the genotype (Figure 2A). Flow cytometric analysis showed very weak B antigen expression on RBCs from the proband and his sister and notably stronger expression on RBCs from the niece (Figure 2B). While the same mutation was present in all three individuals, the niece had also inherited a normal ABO*A1.01 allele in trans. The increased B antigen expression in this individual is consistent with the phenomenon of allelic enhancement.7 Subsequent to the family study, an unrelated person, also of Czech origin, with the same SNV (genotype ABO*B.01/O.01.01) and identical phenotype to the proband, was identified. This study revealed an inherited ABO variant affecting an invariant residue in the catalytic domain and results in weakened B expression. The activity of the altered B glycosyltransferase clearly demonstrates a paradoxical dependence on the allele in trans, that is, allelic enhancement when a full-length, competitive glycosyltransferase is present. In addition to our findings, this SNV has been abstract published9 and shown to give rise to a similar phenotype in a Caucasian blood donor. A GenBank record (KU206323.1) reports the same SNV but without any information regarding the phenotype and ethnic background. In clinical routine practice, this SNV gives rise to a phenotype that causes a discrepant typing result or the very weak B antigen expression may even go undetected by routine serological methods and cause erroneous ABO determination. This work has been supported by the ALF program from the Swedish government and regional county councils (grant no. ALFSKANE-446521 to M.L.O.) to university healthcare in Region Skåne and by the Ministry of Health, Czech Republic - conceptual development of research organization Institute of Haematology and Blood Transfusion IHBT CZ00023736. The authors have no conflicts of interest.

Hemolytic disease of the fetus and newborn (HDFN), as well as fetal and neonatal alloimmune thrombocytopenia (FNAIT), represent two important disease entities that are caused by maternal IgG antibodies directed against nonmaternally inherited antigens on the fetal blood cells. These antibodies are most frequently directed against the RhD antigen on red blood cells (RBCs) or the human platelet antigen 1a (HPA-1a) on platelets. For optimal management of pregnancies where HDFN or FNAIT is suspected, it is essential to determine the RhD or the HPA-1a type of the fetus. Noninvasive fetal RhD typing is also relevant for identifying which RhD-negative pregnant women should receive antenatal RhD prophylaxis. In this review, we will give an overview of the clinical indications and technical challenges related to the noninvasive analysis of fetal RBCs or platelet types. In addition, we will discuss the ethical implications associated with the routine administration of antenatal RhD to all pregnant RhD-negative women and likewise the ethical challenges related to making clinical decisions concerning the mother that have been based on samples collected from the (presumptive) father, which is a common practice when determining the risk of FNAIT.

Background and Objectives Under the ISBT, the Working Party (WP) for Red Cell Immunogenetics and Blood Group Terminology is charged with ratifying blood group systems, antigens and alleles. This report presents the outcomes from four WP business meetings, one located in Basel in 2019 and three held as virtual meetings during the COVID-19 pandemic in 2020 and 2021. Materials and Methods As in previous meetings, matters pertaining to blood group antigen nomenclature were discussed. New blood group systems and antigens were approved and named according to the serologic, genetic, biochemical and cell biological evidence presented. Results Seven new blood group systems, KANNO (defined numerically as ISBT 037), SID (038), CTL2 (039), PEL (040), MAM (041), EMM (042) and ABCC1 (043) were ratified. Two (039 and 043) were de novo discoveries, and the remainder comprised reported antigens where the causal genes were previously unknown. A further 15 blood group antigens were added to the existing blood group systems: MNS (002), RH (004), LU (005), DI (010), SC (013), GE (020), KN (022), JMH (026) and RHAG (030). Conclusion The ISBT now recognizes 378 antigens, of which 345 are clustered within 43 blood group systems while 33 still have an unknown genetic basis. The ongoing discovery of new blood group systems and antigens underscores the diverse and complex biology of the red cell membrane. The WP continues to update the blood group antigen tables and the allele nomenclature tables. These can be found on the ISBT website ().

Background and objectives Fetal RHD genotyping of cell-free maternal plasma DNA from RhD negative pregnant women can be used to guide targeted antenatal and postnatal anti-D prophylaxis for the prevention of RhD immunization. To assure the quality of clinical testing, we conducted an external quality assessment workshop with the participation of 31 laboratories. Materials and methods Aliquots of pooled maternal plasma from gestational week 25 were sent to each laboratory. One sample was fetal RHD positive, and a second sample was fetal RHD negative. A reporting scheme was supplied for data collection, including questions regarding the methodological setup, results and clinical recommendations. The samples were tested blindly. Results Different methodological approaches were used; 29 laboratories used qPCR and two laboratories used ddPCR, employing a total of eight different combinations of RHD exon targets. Fetal RHD genotyping was performed with no false-negative and no false-positive results. One inconclusive result was reported for the RHD positive sample. All clinical conclusions were satisfactory. Conclusion This external quality assessment workshop demonstrates that despite the different approaches taken to perform the clinical assays, fetal RHD genotyping is a reliable laboratory assay to guide targeted use of Rh prophylaxis in a clinical setting.

CONCLUSIONS: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.

Objectives To carry out genetic and serological analyses of a Swiss blood donor and a Danish patient carrying an aberrant ABO phenotype with weak A expression. Background ABO is the most clinically important blood group system but also one of the most complex. The system antigens are determined by carbohydrate structures generated by A and B glycosyltransferases encoded by the ABO gene. Genetic variants of ABO may encode a glycosyltransferase with reduced activity, leading to weak expression of A antigen. Methods Samples from two individuals were examined using genetic testing and extended immunohaematological evaluation, including standard serological methods, flow cytometry and analysis of plasma glycosyltransferase activity. Results Both individuals were serologically determined to be A(weak)B. Genetic testing revealed that both were heterozygous for a novel ABO*A1.01-like allele with an in-frame duplication of 21 nucleotides in exon 7 (c.543_563dup), leading to the insertion of seven amino acids (QDVSMRR). Flow cytometric testing of native red blood cells (RBCs) showed very weak A antigen expression. This was in accordance with the enzyme activity test. Conclusion In summary, we describe a novel A allele with a duplication of 21 nucleotides in exon 7 that significantly decreases the enzyme activity and leads to very weak expression of A antigen. (200 words)

Background and objectives There is concern regarding the lack of prevention of unnecessary transfusion of RhD negative red cells and unnecessary administration of Rh immunoglobulin (RhIG) to pregnant women. In this study, performance of ID RHD XT, a genotyping assay for identification of six RHD allelic variants and human platelet antigens HPA-1a/1b was assessed. Materials and methods Whole blood samples presenting weak, discrepant or inconclusive D phenotype results were genotyped with ID RHD XT and compared to reference molecular tests. Candidacy for RhIG prophylaxis was determined by analysing samples from pregnant women. Hands-on time to complete the procedures was measured. Results Overall, 167 samples were tested (55 donors, 56 patients, 52 pregnant women and four newborns). Agreement between ID RHD XT and the reference method was 100% (51% weak D type 1, 2 or 3; 35 center dot 5% weak D Types 1, 2 or 3 not detected; 4% RHD deletion; 1% RHD*Pseudogene; 1% RHD*DIIIa-CE(3-7)-D; and 4% no amplification variant detected for RHD genotype; and 64% HPA-1a/a; 30% HPA-1a/b; and 3% HPA-1b/b for HPA-1 genotype). Call rate was 98 center dot 2%. ID RHD XT identified 40% of the pregnant women that would not have required RhIG prophylaxis. Overall hands-on time was 25-45 min to process a batch of 24 samples, and four hours for total assay time. Conclusion ID RHD XT yielded reproducible results for RHD typing in serologically weak D phenotype individuals. ID RHD XT was proven useful for the correct management of patients with RhD serological discrepancies and the rational use of RhIG in pregnancy.

Sd(a) is a high-frequency carbohydrate histo-blood group antigen, GalNAc beta 1-4(NeuAc alpha 2-3)Gal beta, 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 beta-1,4-N-acetylgalactosaminyltransferase (beta 4GalNAc-T2), was cloned in 2003 but the genetic basis of human Sda 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 beta 4GalNAc-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.