A machine-learning method for biobank-scale genetic prediction of blood group antigens.

A key element for successful blood transfusion is compatibility of the patient and donor red blood cell (RBC) antigens. Precise antigen matching reduces the risk for immunization and other adverse transfusion outcomes. RBC antigens are encoded by specific genes, which allows developing computational methods for determining antigens from genomic data. We describe here a classification method for determining RBC antigens from genotyping array data. Random forest models for 39 RBC antigens in 14 blood group systems and for human platelet antigen (HPA)-1 were trained and tested using genotype and RBC antigen and HPA-1 typing data available for 1,192 blood donors in the Finnish Blood Service Biobank. The algorithm and models were further evaluated using a validation cohort of 111,667 Danish blood donors. In the Finnish test data set, the median (interquartile range [IQR]) balanced accuracy for 39 models was 99.9 (98.9-100)%. We were able to replicate 34 out of 39 Finnish models in the Danish cohort and the median (IQR) balanced accuracy for classifications was 97.1 (90.1-99.4)%. When applying models trained with the Danish cohort, the median (IQR) balanced accuracy for the 40 Danish models in the Danish test data set was 99.3 (95.1-99.8)%. The RBC antigen and HPA-1 prediction models demonstrated high overall accuracies suitable for probabilistic determination of blood groups and HPA-1 at biobank-scale. Furthermore, population-specific training cohort increased the accuracies of the models. This stand-alone and freely available method is applicable for research and screening for antigen-negative blood donors.

Epigenetic dissection of human blood group genes reveals regulatory elements and detailed characteristics of KEL and four other loci.

BACKGROUND: Blood typing is essential for safe transfusions and is performed serologically or genetically. Genotyping predominantly focuses on coding regions, but non-coding variants may affect gene regulation, as demonstrated in the ABO, FY and XG systems. To uncover regulatory loci, we expanded a recently developed bioinformatics pipeline for discovery of non-coding variants by including additional epigenetic datasets. METHODS: Multiple datasets including ChIP-seq with erythroid transcription factors (TFs), histone modifications (H3K27ac, H3K4me1), and chromatin accessibility (ATAC-seq) were analyzed. Candidate regulatory regions were investigated for activity (luciferase assays) and TF binding (electrophoretic mobility shift assay, EMSA, and mass spectrometry, MS). RESULTS: In total, 814 potential regulatory sites in 47 blood-group-related genes were identified where one or more erythroid TFs bound. Enhancer candidates in CR1, EMP3, ABCB6, and ABCC4 indicated by ATAC-seq, histone markers, and co-occupancy of 4 TFs (GATA1/KLF1/RUNX1/NFE2) were investigated but only CR1 and ABCC4 showed increased transcription. Co-occupancy of GATA1 and KLF1 was observed in the KEL promoter, previously reported to contain GATA1 and Sp1 sites. TF binding energy scores decreased when three naturally occurring variants were introduced into GATA1 and KLF1 motifs. Two of three GATA1 sites and the KLF1 site were confirmed functionally. EMSA and MS demonstrated increased GATA1 and KLF1 binding to the wild-type compared to variant motifs. DISCUSSION: This combined bioinformatics and experimental approach revealed multiple candidate regulatory regions and predicted TF co-occupancy sites. The KEL promoter was characterized in detail, indicating that two adjacent GATA1 and KLF1 motifs are most crucial for transcription.

A deep learning approach to prediction of blood group antigens from genomic data.

BACKGROUND: Deep learning methods are revolutionizing natural science. In this study, we aim to apply such techniques to develop blood type prediction models based on cheap to analyze and easily scalable screening array genotyping platforms. METHODS: Combining existing blood types from blood banks and imputed screening array genotypes for ~111,000 Danish and 1168 Finnish blood donors, we used deep learning techniques to train and validate blood type prediction models for 36 antigens in 15 blood group systems. To account for missing genotypes a denoising autoencoder initial step was utilized, followed by a convolutional neural network blood type classifier. RESULTS: Two thirds of the trained blood type prediction models demonstrated an F1-accuracy above 99%. Models for antigens with low or high frequencies like, for example, Cw, low training cohorts like, for example, Cob, or very complicated genetic underpinning like, for example, RhD, proved to be more challenging for high accuracy (>99%) DL modeling. However, in the Danish cohort only 4 out of 36 models (Cob, Cw, D-weak, Kpa) failed to achieve a prediction F1-accuracy above 97%. This high predictive performance was replicated in the Finnish cohort. DISCUSSION: High accuracy in a variety of blood groups proves viability of deep learning-based blood type prediction using array chip genotypes, even in blood groups with nontrivial genetic underpinnings. These techniques are suitable for aiding in identifying blood donors with rare blood types by greatly narrowing down the potential pool of candidate donors before clinical grade confirmation.

Evidence that CD36 is expressed on red blood cells and constitutes a novel blood group system of clinical importance.

BACKGROUND AND OBJECTIVES: Polymorphic molecules expressed on the surface of certain blood cells are traditionally categorized as blood groups and human platelet or neutrophil antigens. CD36 is widely considered a platelet antigen (Naka) and anti-CD36 can cause foetal/neonatal alloimmune thrombocytopenia (FNAIT) in CD36-negative pregnant women. CD36 is used as a marker of differentiation in early erythroid culture. During the experimental culture of CD34+ cells from random blood donors, we observed that one individual lacked CD36. We sought to investigate this observation further and determine if CD36 fulfils the International Society of Blood Transfusion criteria for becoming a blood group. MATERIALS AND METHODS: Surface markers were monitored by flow cytometry on developing cells during the erythroid culture of CD34+ cells. Genetic and flow cytometric analyses on peripheral blood cells were performed. Proteomic datasets were analysed, and clinical case reports involving anti-CD36 and foetal anaemia were scrutinized. RESULTS: Sequencing of CD36-cDNA identified homozygosity for c.1133G>T/p.Gly378Val in the CD36-negative donor. The minor allele frequency of rs146027667:T is 0.1% globally and results in abolished CD36 expression. CD36 has been considered absent from mature red blood cells (RBCs); however, we detected CD36 expression on RBCs and reticulocytes from 20 blood donors. By mining reticulocyte and RBC datasets, we found evidence for CD36-derived peptides enriched in the membrane fractions. Finally, our literature review revealed severe cases of foetal anaemia attributed to anti-CD36. CONCLUSIONS: Based on these findings, we conclude that CD36 fulfils the criteria for becoming a new blood group system and that anti-CD36 is implicated not only in FNAIT but also foetal anaemia.

A Bioinformatically Initiated Approach to Evaluate GATA1 Regulatory Regions in Samples with Weak D, Del, or D- Phenotypes Despite Normal RHD Exons.

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.

ABO Genotyping finds more A2 to B kidney transplant opportunities than lectin-based subtyping.

ABO compatibility is important for kidney transplantation, with longer waitlist times for blood group B kidney transplant candidates. However, kidneys from non-A1 (eg, A2) subtype donors, which express less A antigen, can be safely transplanted into group B recipients. ABO subtyping is routinely performed using anti-A1 lectin, but DNA-based genotyping is also possible. Here, we compare lectin and genotyping testing. Lectin and genotype subtyping was performed on 554 group A deceased donor samples at 2 transplant laboratories. The findings were supported by 2 additional data sets of 210 group A living kidney donors and 124 samples with unclear lectin testing sent to a reference laboratory. In deceased donors, genotyping found 65% more A2 donors than lectin testing, most with weak lectin reactivity, a finding supported in living donors and samples sent for reference testing. DNA sequencing and flow cytometry showed that the discordances were because of several factors, including transfusion, small variability in A antigen levels, and rare ABO∗A2.06 and ABO∗A2.16 sequences. Although lectin testing is the current standard for transplantation subtyping, genotyping is accurate and could increase A2 kidney transplant opportunities for group B candidates, a difference that should reduce group B wait times and improve transplant equity.

Truncated glycosyltransferase coding regions in novel ABO alleles give rise to weak A or B blood group expression and discrepant typing results.

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.

A large cohort study of the effects of Lewis, ABO, 13 other blood groups, and secretor status on COVID-19 susceptibility, severity, and long COVID-19.

BACKGROUND: Previous studies have reported Blood type O to confer a lower risk of SARS-CoV-2 infection, while secretor status and other blood groups have been suspected to have a similar effect as well. STUDY DESIGN AND METHODS: To determine whether any other blood groups influence testing positive for SARS-CoV-2, COVID-19 severity, or prolonged COVID-19, we used a large cohort of 650,156 Danish blood donors with varying available data for secretor status and blood groups ABO, Rh, Colton, Duffy, Diego, Dombrock, Kell, Kidd, Knops, Lewis, Lutheran, MNS, P1PK, Vel, and Yt. Of these, 36,068 tested positive for SARS-CoV-2 whereas 614,088 tested negative between 2020-02-17 and 2021-08-04. Associations between infection and blood groups were assessed using logistic regression models with sex and age as covariates. RESULTS: The Lewis blood group antigen Lea displayed strongly reduced SARS-CoV-2 susceptibility OR 0.85 CI[0.79-0.93] p < .001. Compared to blood type O, the blood types B, A, and AB were found more susceptible toward infection with ORs 1.1 CI[1.06-1.14] p < .001, 1.17 CI[1.14-1.2] p < .001, and 1.2 CI[1.14-1.26] p < .001, respectively. No susceptibility associations were found for the other 13 blood groups investigated. There was no association between any blood groups and COVID-19 hospitalization or long COVID-19. No secretor status associations were found. DISCUSSION: This study uncovers a new association to reduced SARS-CoV-2 susceptibility for Lewis type Lea and confirms the previous link to blood group O. The new association to Lea could be explained by a link between mucosal microbiome and SARS-CoV-2.

Genetic prediction of 33 blood group phenotypes using an existing genotype dataset.

BACKGROUND: Accurate blood type data are essential for blood bank management, but due to costs, few of 43 blood group systems are routinely determined in Danish blood banks. However, a more comprehensive dataset of blood types is useful in scenarios such as rare blood type allocation. We aimed to investigate the viability and accuracy of predicting blood types by leveraging an existing dataset of imputed genotypes for two cohorts of approximately 90,000 each (Danish Blood Donor Study and Copenhagen Biobank) and present a more comprehensive overview of blood types for our Danish donor cohort. STUDY DESIGN AND METHODS: Blood types were predicted from genome array data using known variant determinants. Prediction accuracy was confirmed by comparing with preexisting serological blood types. The Vel blood group was used to test the viability of using genetic prediction to narrow down the list of candidate donors with rare blood types. RESULTS: Predicted phenotypes showed a high balanced accuracy >99.5% in most cases: A, B, C/c, Coa /Cob , Doa /Dob , E/e, Jka /Jkb , Kna /Knb , Kpa /Kpb , M/N, S/s, Sda , Se, and Yta /Ytb , while some performed slightly worse: Fya /Fyb , K/k, Lua /Lub , and Vel ~99%-98% and CW and P1 ~96%. Genetic prediction identified 70 potential Vel negatives in our cohort, 64 of whom were confirmed correct using polymerase chain reaction (negative predictive value: 91.5%). DISCUSSION: High genetic prediction accuracy in most blood groups demonstrated the viability of generating blood types using preexisting genotype data at no cost and successfully narrowed the pool of potential individuals with the rare Vel-negative phenotype from 180,000 to 70.

A novel nonsense variant in RHAG underlies a Nordic Rhnull phenotype.

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.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology Report of Basel and three virtual business meetings: Update on blood group systems.

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 (http://www.isbtweb.org/working-parties/red-cell-immunogenetics-and-blood-group-terminology/).

Recommendation for validation and quality assurance of non-invasive prenatal testing for foetal blood groups and implications for IVD risk classification according to EU regulations.

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.

Platelets inhibit erythrocyte invasion by Plasmodium falciparum at physiological platelet:erythrocyte ratios.

OBJECTIVE: To evaluate the effect of platelet:erythrocyte (P:E) ratios on Plasmodium falciparum erythrocyte invasion. BACKGROUND: Recent reports have shown that platelets are directly involved in the immune response towards P. falciparum during erythrocyte invasion. However, the literature both supports and conflicts with a role for platelets in limiting invasion. Also, the effect of platelet numbers on invasion (parasitemia) has not been thoroughly investigated. METHODS/MATERIALS: The P. falciparum strains FCR3S1.2 and W2mef were cultured with group O erythrocytes. The cultures were synchronised and supplemented with pooled platelets at P:E ratios ranging from 1:100 to 1:2. Parasitemia was measured at 40 h by flow cytometry and by microscopy of blood smears. RESULTS: A linear relationship was observed between reduced invasion and increased platelet numbers at P:E ratios ranging from 1:100 to 1:20. However, this effect was reversed at lower ratios (1:10-1:2). Microscopic evaluation revealed aggregation and attachment of platelets to erythrocytes, but not specifically to parasitised erythrocytes. CONCLUSION: We have shown that under physiological P:E ratios (approx. 1:10-1:40), platelets inhibited P. falciparum invasion in a dose-dependent manner. At ratios of 1:10 and below, platelets did not further increase the inhibitory effect and, although the trend was reversed, inhibition was still maintained.

Glycoproteomic and Phenotypic Elucidation of B4GALNT2 Expression Variants in the SID Histo-Blood Group System.

The Sda histo-blood group antigen (GalNAcß1-4(NeuAcα2-3)Galß-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 ß4GalNAc-T2 variant causes Sda-deficiency in humans, while the enigmatic Cad phenotype remains unresolved, albeit further characterized.

Network pharmacology of triptolide in cancer cells: implications for transcription factor binding.

Background Triptolide is an active natural product, which inhibits cell proliferation, induces cell apoptosis, suppresses tumor metastasis and improves the effect of other therapeutic treatments in several cancer cell lines by affecting multiple molecules and signaling pathways, such as caspases, heat-shock proteins, DNA damage and NF-ĸB. Purpose We investigated the effect of triptolide towards NF-ĸB and GATA1. Methods We used cell viability assay, compare and cluster analyses of microarray-based mRNA transcriptome-wide expression data, gene promoter binding motif analysis, molecular docking, Ingenuity pathway analysis, NF-ĸB reporter cell assay, and electrophoretic mobility shift assay (EMSA) of GATA1. Results Triptolide inhibited the growth of drug-sensitive (CCRF-CEM, U87.MG) and drug-resistant cell lines (CEM/ADR5000, U87.MGΔEGFR). Hierarchical cluster analysis showed six major clusters in dendrogram. The sensitive and resistant cell lines were statistically significant (p = 0.65 × 10-2) distributed. The binding motifs of NF-κB (Rel) and of GATA1 proteins were significantly enriched in regions of 25 kb upstream promoter of all genes. IPA showed the networks, biological functions, and canonical pathways influencing the activity of triptolide towards tumor cells. Interestingly, upstream analysis for the 40 genes identified by compare analysis revealed ZFPM1 (friend of GATA protein 1) as top transcription regulator. However, we did not observe any effect of triptolide to the binding of GATA1 in vitro. We confirmed that triptolide inhibited NF-κB activity, and it strongly bound to the pharmacophores of IκB kinase ß and NF-κB in silico. Conclusion Triptolide showed promising inhibitory effect toward NF-κB, making it a potential candidate for targeting NF-κB.

Allele-selective RUNX1 binding regulates P1 blood group status by transcriptional control of A4GALT.

P1 and Pk are glycosphingolipid antigens synthesized by the A4GALT-encoded α1,4-galactosyltransferase, using paragloboside and lactosylceramide as acceptor substrates, respectively. In addition to the compatibility aspects of these histo-blood group molecules, both constitute receptors for multiple microbes and toxins. Presence or absence of P1 antigen on erythrocytes determines the common P1 (P1+Pk+) and P2 (P1-Pk+weak) phenotypes. A4GALT transcript levels are higher in P1 individuals and single-nucleotide polymorphisms (SNPs) in noncoding regions of A4GALT, particularly rs5751348, correlate with P1/P2 status. Despite these recent findings, the molecular mechanism underlying these phenotypes remains elusive. The In(Lu) phenotype is caused by Krüppel-like factor 1 (KLF1) haploinsufficiency and shows decreased P1 levels on erythrocytes. We therefore hypothesized KLF1 regulates A4GALT expression. Intriguingly, P1 -specific sequences including rs5751348 revealed potential binding sites for several hematopoietic transcription factors, including KLF1. However, KLF1 binding did not explain P1 -specific shifts in electrophoretic mobility-shift assays and small interfering RNA silencing of KLF1 did not affect A4GALT transcript levels. Instead, protein pull-down experiments using P1 but not P2 oligonucleotide probes identified runt-related transcription factor 1 (RUNX1) by mass spectrometry. Furthermore, RUNX1 binds P1 alleles selectively, and knockdown of RUNX1 significantly decreased A4GALT transcription. These data indicate that RUNX1 regulates A4GALT and thereby the expression of clinically important glycosphingolipids implicated in blood group incompatibility and host-pathogen interactions.

Disruption of a GATA1-binding motif upstream of XG/PBDX abolishes Xga expression and resolves the Xg blood group system.

The Xga blood group is differentially expressed on erythrocytes from men and women. The underlying gene, PBDX, was identified in 1994, but the molecular background for Xga expression remains undefined. This gene, now designated XG, partly resides in pseudoautosomal region 1 and encodes a protein of unknown function from the X chromosome. By comparing calculated Xga allele frequencies in different populations with 2612 genetic variants in the XG region, rs311103 showed the strongest correlation to the expected distribution. The same single-nucleotide polymorphism (SNP) had the most significant impact on XG transcript levels in whole blood (P = 2.0 × 10-22). The minor allele, rs311103C, disrupts a GATA-binding motif 3.7 kb upstream of the transcription start point. This silences erythroid XG messenger RNA expression and causes the Xg(a-) phenotype, a finding corroborated by SNP genotyping in 158 blood donors. Binding of GATA1 to biotinylated oligonucleotide probes with rs311103G but not rs311103C was observed by electrophoretic mobility shift assay and proven by mass spectrometry. Finally, a luciferase reporter assay indicated this GATA motif to be active for rs311103G but not rs311103C in HEL cells. By using an integrated bioinformatic and molecular biological approach, we elucidated the underlying genetic basis for the last unresolved blood group system and made Xga genotyping possible.

Characterization of GYP*Mur and novel GYP*Bun-like hybrids in Thai blood donors reveals a qualitatively altered s antigen.

BACKGROUND AND OBJECTIVES: The Mi(a+) GP(B-A-B) hybrid phenotypes occur with a prevalence of 2%-23% across Southeast Asia. While the s antigen is alleged to be altered, no evidence for specific variants is known. Screening using a monoclonal IgM anti-s mistyped six S-s+ RBC units as S-s-. Further, alloanti-s was identified in an S+s+ patient. Our objective was to investigate the s antigen further. MATERIALS AND METHODS: DNA from 63 Thai blood donor samples PCR-positive for a GYP(B-A-B) hybrid was sequenced with primers spanning GYPB exons 3-4. Flow cytometry was used for semiquantitative analysis of s expression and correlated with the glycophorin genotype. RESULTS: DNA sequencing showed that GYP*Mur was carried by 56/63 samples (88·9%) of which 5/56 lacked normal GYPB: three of these were GYP*Mur homozygotes, one was a compound heterozygote carrying GYP*Mur and a GYP*Bun-like allele (designated GYP*Thai), and the fifth sample carried GYP*Mur and another GYP*Bun-like allele. Seven samples (7/63) were GYP*Thai heterozygotes. IgM monoclonal anti-s (P3BER) did not react with the s antigen carried by GP.Mur or GP.Bun, whereas two IgG anti-s showed enhanced reactivity. CONCLUSIONS: We confirmed that GYP*Mur is the most frequent variant in Thai blood donors and also identified GYP*Thai with a frequency of 1·1%. We showed that s antigen on Mi(a+) GP(B-A-B) hybrids is qualitatively altered and should be considered when selecting reagents for phenotyping where such hybrids are prevalent, endemically and in blood centres worldwide.

A novel ABO allele with a 21-bp duplication identified in two unrelated European individuals with weak A expression.

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 Aweak 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).