Augustine Blood Group System and Equilibrative Nucleoside Transporter 1.

Augustine (AUG) is a blood group system comprising four antigens: AUG1, AUG2 (Ata), and AUG4 are of very high frequency; AUG3 is of very low frequency. These antigens are located on ENT1, an equilibrative nucleoside transporter encoded by SLC19A1. AUG antibodies are of clinical relevance in blood transfusion and pregnancy: anti-AUG2 have caused haemolytic transfusion reactions; the only anti-AUG3 was associated with severe haemolytic disease of the fetus and newborn. ENT1 is present in almost all human tissues. It facilitates the transfer of purine and pyrimidine nucleosides and is responsible for the majority of adenosine transport across plasma membranes. Adenosine transport appears to be an important factor in the regulation of bone metabolism. The AUGnull phenotype (AUG:-1,-2,-3,-4) has been found in three siblings, who are homozygous for an inactivating splice-site mutation in SLC29A1. Although ENT1 is very likely to be absent from all cells in these three individuals, they were apparently healthy with normal lifestyles. However, they suffered frequent attacks of pseudogout, a form of arthritis, in various joints with multiple calcifications around their hand joints. Ectopic calcification in the hips, pubic symphysis, and lumbar discs was present in the propositus. The three AUGnull individuals had misshapen red cells with deregulated protein phosphorylation, but no anaemia or shortening of red cell lifespan. Defective in vitro erythropoiesis in the absence of ENT1 was confirmed by shRNA-mediated knockdown of ENT1 during in vitro erythropoiesis of CD34+ progenitor cells from individuals with normal ENT1. Nucleoside transporters, such as ENT1, are vital in the uptake of synthetic nucleoside analogue drugs, used in cancer and viral chemotherapy. It is feasible that the efficacy of these drugs would be compromised in patients with the extremely rare AUGnull phenotype.

International Society of Blood Transfusion Working Party on Red Cell Immunogenetics and Blood Group Terminology: Report of the Dubai, Copenhagen and Toronto meetings.

BACKGROUND AND OBJECTIVES: The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session. METHODS: As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented. RESULTS AND CONCLUSIONS: Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies.

Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type and ectopic mineralization.

The Augustine-negative alias At(a-) blood type, which seems to be restricted to people of African ancestry, was identified half a century ago but remains one of the last blood types with no known genetic basis. Here we report that a nonsynonymous single nucleotide polymorphism in SLC29A1 (rs45458701) is responsible for the At(a-) blood type. The resulting p.Glu391Lys variation in the last extracellular loop of the equilibrative nucleoside transporter 1 (ENT1; also called SLC29a1) is known not to alter its ability to transport nucleosides and nucleoside analog drugs. Furthermore, we identified 3 individuals of European ancestry who are homozygous for a null mutation in SLC29A1 (c.589+1G>C) and thus have the Augustine-null blood type. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis as recently suggested by the skeletal phenotype of aging Slc29a1(-/-) mice. Our results establish Augustine as a new blood group system and place SLC29A1 as a new candidate gene for idiopathic disorders characterized with ectopic calcification/mineralization.

Variants of RhD--current testing and clinical consequences.

Anti-D (-RH1) of the Rh blood group system is clinically important as it causes haemolytic transfusion reactions and haemolytic disease of the fetus and newborn. Although most people are either D+ or D-, there is a plethora of D variants, often categorized as either weak D or partial D. These two types are inadequately defined and the dichotomy is potentially misleading. DVI is the D variant most commonly associated with anti-D production and UK guidelines recommend that patients are tested with anti-D reagents that do not react with DVI. Weak D types 1, 2, and 3 are seldom, if ever, associated with alloanti-D production, so a policy recommendation would be to treat patients with those D variants as D+, to preserve D- stocks, whereas patients with all other D variants would be treated as D-. All donors with D variant red cells, including DVI, should be treated as D+.

Critical band 3 multiprotein complex interactions establish early during human erythropoiesis.

Band 3, the major anion transport protein of human erythrocytes, forms the core of a multiprotein complex in the erythrocyte membrane. Here we studied the spatiotemporal mechanisms of band 3 multiprotein complex assembly during erythropoiesis. Significant pools of intracellular band 3 and Rh-associated glycoprotein (RhAG) were found in the basophilic erythroblast. These intracellular pools decreased in the polychromatic erythroblast, whereas surface expression increased and were lowest in the orthochromatic erythroblast and reticulocytes. Protease treatment of intact cells to remove extracellular epitopes recognized by antibodies to band 3 and RhAG was used to study surface delivery kinetics and intracellular complex composition from the proerythroblast stage to the enucleated reticulocyte. Newly synthesized band 3 and protein 4.2 interact initially in the early stages of the secretory pathway and are found associated at the plasma membrane from the basophilic stage of erythropoiesis. Although we could successfully coimmunoprecipitate Rh with RhAG from plasma membrane pools at a similar stage, no intracellular interaction between these proteins was detectable. Knockdown of RhAG during early erythropoiesis was accompanied by a concomitant drop in membrane expression of Rh polypeptides. These data are consistent with assembly of major components of the band 3 macrocomplex at an early stage during erythropoiesis.

Fetal RHD genotyping in maternal plasma at 11-13 weeks of gestation.

OBJECTIVE: To examine the feasibility of fetal RHD genotyping at 11-13 weeks' gestation from analysis of circulating cell-free fetal DNA (ccffDNA) in the plasma of RhD negative pregnant women using a high-throughput robotic technique. METHODS: Stored plasma (0.5 ml) from 591 RhD negative women was used for extraction of ccffDNA by a robotic technique. Real-time quantitative polymerase chain reaction (PCR) with probes for exons 5 and 7 of the RHD gene was then used to determine the fetal RHD genotype, which was compared to the neonatal RhD phenotype. RESULTS: In total there were 502 (85.7%) cases with a conclusive result and 84 (14.3%) with an inconclusive result. The prenatal test predicted that the fetus was RhD positive in 332 cases and in all of these the prediction was correct, giving a positive predictive value of 100% (95% CI 96.8-100). The test predicted that the fetus was RhD negative in 170 cases and in 164 of these the prediction was correct, giving a negative predictive value for RhD positive fetuses of 96.5% (95% CI 93.7-99.2). CONCLUSION: The findings demonstrate the feasibility and accuracy of non-invasive fetal RHD genotyping at 11-13 weeks with a high-throughput technique.

The majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing the contribution of CD34(+) cells and significantly increasing the erythroblast yield from peripheral blood samples.

The study of human erythropoiesis in health and disease requires a robust culture system that consistently and reliably generates large numbers of immature erythroblasts that can be induced to differentiate synchronously. We describe a culture method modified from Leberbauer et al. (2005) and obtain a homogenous population of erythroblasts from peripheral blood mononuclear cells (PBMC) without prior purification of CD34(+) cells. This pure population of immature erythroblasts can be expanded to obtain 4x10(8) erythroblasts from 1x10(8) PBMC after 13-14 days in culture. Upon synchronized differentiation, high levels of enucleation (80-90%) and low levels of cell death (<10%) are achieved. We compared the yield of erythroblasts obtained from PBMC, CD34(+) cells or PBMC depleted of CD34(+) cells and show that CD34(-) cells represent the most significant early erythroid progenitor population. This culture system may be particularly useful for investigating the pathophysiology of anemic patients where only small blood volumes are available.

Investigating the key membrane protein changes during in vitro erythropoiesis of protein 4.2 (-) cells (mutations Chartres 1 and 2).

BACKGROUND: Protein 4.2 deficiency caused by mutations in the EPB42 gene results in hereditary spherocytosis with characteristic alterations of CD47, CD44 and RhAG. We decided to investigate at which stage of erythropoiesis these hallmarks of protein 4.2 deficiency arise in a novel protein 4.2 patient and whether they cause disruption to the band 3 macrocomplex. DESIGN AND METHODS: We used immunoprecipitations and detergent extractability to assess the strength of protein associations within the band 3 macrocomplex and with the cytoskeleton in erythrocytes. Patient erythroblasts were cultured from peripheral blood mononuclear cells to study the effects of protein 4.2 deficiency during erythropoiesis. RESULTS: We report a patient with two novel mutations in EPB42 resulting in complete protein 4.2 deficiency. Immunoprecipitations revealed a weakened ankyrin-1-band 3 interaction in erythrocytes resulting in increased band 3 detergent extractability. CD44 abundance and its association with the cytoskeleton were increased. Erythroblast differentiation revealed that protein 4.2 and band 3 appear simultaneously and associate early in differentiation. Protein 4.2 deficiency results in lower CD47, higher CD44 expression and increased RhAG glycosylation starting from the basophilic stage. The normal downregulation of CD44 expression was not seen during protein 4.2(-) erythroblast differentiation. Knockdown of CD47 did not increase CD44 expression, arguing against a direct reciprocal relationship. CONCLUSIONS: We have established that the characteristic changes caused by protein 4.2 deficiency occur early during erythropoiesis. We postulate that weakening of the ankyrin-1-band 3 association during protein 4.2 deficiency is compensated, in part, by increased CD44-cytoskeleton binding.

Blood groups: the past 50 years.

Since the first issue of TRANSFUSION in 1961, there has been a tremendous expansion in not only the number of blood group antigens identified but also in our knowledge of their biochemical basis, function, and more recently, associated DNA changes. As certain techniques became available, our ability to discover and elucidate blood group antigens and appreciate their contribution to biology became possible. In particular, Western blotting, monoclonal antibodies, cloning, and polymerase chain reaction-based assays have led to an explosion of our knowledge base. The study of blood groups has had a significant effect on human genetics where they serve as useful markers in genetic linkage analyses. Indeed blood groups have provided several "firsts" in certain aspects of genetics. Blood group-null phenotypes, as natural human knockouts, have provided valuable insights into the importance of red blood cell membrane components. This review summarizes key aspects of the discovery of blood groups; the inconsistent terminology that has arisen; and the contribution of blood groups to genetics, safe transfusion, transplantation, evolution, and biology.

The molecular genetics of blood group polymorphism.

Over 300 blood group specificities on red cells have been identified, many of which are polymorphic. The molecular mechanisms responsible for these polymorphisms are diverse, though many simply represent single nucleotide polymorphisms (SNPs). Other mechanisms include the following: gene deletion; single nucleotide deletion and sequence duplication, which introduce reading-frame shifts; nonsense mutation; intergenic recombination between closely linked genes, giving rise to hybrid genes and hybrid proteins; and a SNP in the promoter region of a blood group gene. Examples of these various genetic mechanisms are taken from the ABO, Rh, Kell, and Duffy blood group systems. Null phenotypes, in which no antigens of a blood group system are expressed, are not generally polymorphic, but provide good examples of the effect of inactivating mutations on blood group expression. As natural human 'knock-outs', null phenotypes provide useful clues to the functions of blood group antigens. Knowledge of the molecular backgrounds of blood group polymorphisms provides a means to predict blood group phenotypes from genomic DNA. This has two main applications in transfusion medicine: determination of foetal blood groups to assess whether the foetus is at risk from haemolytic disease and ascertainment of blood group phenotypes in multiply transfused, transfusion-dependent patients, where serological tests are precluded by the presence of donor red cells. Other applications are being developed for the future.

The use of maternal plasma for prenatal RhD blood group genotyping.

Alloimmunization to the blood group antibody anti-RhD (anti-D) is the most common cause of hemolytic disease of the fetus and newborn. Knowledge of fetal D type in women with anti-D makes management of the pregnancy much easier and avoids unnecessary procedures in those women with a D-negative fetus. Fetal D typing can be performed by detection of an RHD gene in cell-free DNA in the plasma of D-negative pregnant women. The technology involves real-time quantitative polymerase chain reactions targeting exons 4, 5, and 10 of RHD, with the exons 4 and 10 tests performed as a multiplex. Testing for SRY in multiplex with the RHD exon 5 test provides an internal control for the presence of fetal DNA when the fetus is male. Fetal D typing has become the standard of care in England in pregnant women with a significant level of anti-D.

The Bloodgen Project of the European Union, 2003-2009.

The Bloodgen project was funded by the European Commission between 2003 and 2006, and involved academic blood centres, universities, and Progenika Biopharma S.A., a commercial supplier of genotyping platforms that incorporate glass arrays. The project has led to the development of a commercially available product, BLOODchip, that can be used to comprehensively genotype an individual for all clinically significant blood groups. The intention of making this system available is that blood services and perhaps even hospital blood banks would be able to obtain extended information concerning the blood group of routine blood donors and vulnerable patient groups. This may be of significant use in the current management of multi-transfused patients who become alloimmunised due to incomplete matching of blood groups. In the future it can be envisaged that better matching of donor-patient blood could be achieved by comprehensive genotyping of every blood donor, especially regular ones. This situation could even be extended to genotyping every individual at birth, which may prove to have significant long-term health economic benefits as it may be coupled with detection of inborn errors of metabolism.

New mutations in C1GALT1C1 in individuals with Tn positive phenotype.

Tn polyagglutination results from inactivating mutations in C1GALT1C1, an X-borne gene encoding a core 1 beta3-galactosyltransferase-specific molecular chaperone (cosmc) required for the functioning of T-synthase (beta 1,3-galactosyltransferase), a glycosyltransferase essential for the correct biosynthesis of O-glycans. This study found novel inactivating mutations (Glu152Lys, Ser193Pro and Met1Ile) in the coding sequence of C1GALT1C1 in three Tn positive individuals and a complete lack of C1GALT1C1 cDNA expression was observed in an additional Tn positive individual. In addition, expression of ST6GALNAC1, which encodes (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1, 3)-N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 and gives rise to sialyl-Tn antigen, was present at comparable levels in normal and Tn-positive human erythroblasts. Expression studies of wild-type and Tn positive C1GALT1C1 cDNA in the Jurkat cell line confirmed that the amino acid substitutions observed in Tn are inactivating. Analysis of the transcriptome of cultured normal and Tn positive erythroblasts revealed numerous differences in gene expression. Reduced transcript levels for fatty acid binding protein 5 (FABP5) and plexin D1 (PLXND1), and increased levels for aquaporin 3 (AQP3) were confirmed by quantitative real-time polymerase chain reaction. These data show that alteration of O-glycan structures resulting from T-synthase deficiency is accompanied by altered expression of a wide variety of genes in erythroid cells.

Two MER2-negative individuals with the same novel CD151 mutation and evidence for clinical significance of anti-MER2.

BACKGROUND: MER2 (RAPH1), the only antigen of the RAPH blood group system, is located on the tetraspanin CD151. Only four examples of alloanti-MER2 are known. We report here two new examples of alloanti-MER2, in women of Pakistani and Turkish origin, one of whom showed signs of a hemolytic transfusion reaction (HTR) after transfusion of 3 units of red cells (RBCs). STUDY DESIGN AND METHODS: Standard serologic methods were used. A monocyte monolayer assay (MMA) was used to assess the potential clinical significance of one of the antibodies. All exons and flanking intronic sequences of CD151 were amplified and sequenced. A homology model for CD151 second extracellular loop (EC2) was constructed based on the crystal structure of CD81. RESULTS: RBCs of both patients did not react with alloanti-MER2, and neither of their antibodies reacted with MER2-negative RBCs. The MMA results suggested that the antibody that appeared to have caused an HTR had the potential to be clinically significant. Both patients were homozygous for a 511C>T mutation in CD151 encoding an Arg171Cys change. This change did not result in any significant structural rearrangement in the protein model. CONCLUSIONS: Two MER2-negative patients with anti-MER2 are homozygous for the same novel mutation encoding an amino acid substitution in the EC2 of CD151. One of the antibodies may have been responsible for an HTR, and crossmatch-compatible RBCs should be recommended for transfusion to patients with anti-MER2.

Blood group antibodies and their significance in transfusion medicine.

The discovery of almost universally present naturally occurring antibodies in blood plasma led to the discovery of the ABO blood group system which remains, more than 100 years later, the most important and clinically significant of all blood groups. Blood group antibodies play an important role in transfusion medicine, both in relation to the practice of blood transfusion and in pregnancy, but not all are clinically significant. Clinically significant antibodies are capable of causing adverse events following transfusion, ranging from mild to severe, and of causing hemolytic disease of the fetus and newborn following placental transfer from mother to fetus. Assessing the clinical significance of antibodies relies heavily on mode of reactivity and historical data relating to specificity; functional assays are sometimes employed. The principals of methodology for blood typing and antibody identification have changed little over the years, relying mainly on serological methods involving red cell agglutination. The recent advent of blood typing using DNA technology, although still in relative infancy, will surely eventually supersede serology. However, deciding on the clinical significance of an antibody when compatible blood is not immediately available is likely to remain as one of the most common dilemmas facing transfusion practitioners.

Fetal blood group genotyping: present and future.

Prediction of fetal blood group from DNA is usually performed when the mother has antibodies to RhD, to assess whether the fetus is at risk from hemolytic disease of the fetus and newborn (HDFN). Over the last five years RhD testing on fetal DNA in maternal plasma has been introduced. At the International Blood Group Reference Laboratory (IBGRL) we employ real-time quantitative polymerase chain reaction (RQ-PCR) to detect RHD exons 4, 5, and 10, which also reveals RHDpsi. SRY and, in RhD-negative (RhD-) females, eight biallelic polymorphisms are incorporated in an attempt to provide an internal positive control. Since 2000 we have tested 533 pregnancies for RhD. In 327 pregnancies where the RhD of the infant is known, we had one false-positive and one false-negative result. In 2004 we introduced fetal typing from DNA in maternal plasma for K, Rhc, and RhE, which represent single nucleotide polymorphisms (SNPs) on the KEL and RHCE genes. We have begun trials on an automated method for fetal RhD typing from DNA in maternal plasma. This is designed to test fetal RhD in all pregnant RhD- women, to identify the 40% with an RhD- fetus so that antenatal RhD immunoglobulin (Ig) prophylaxis can be avoided. Similar trials have already been reported by Sanquin Research Laboratories in Amsterdam.

McLeod syndrome: life-long neuropsychiatric disorder due to a novel mutation of the XK gene.

A 50-year-old man presented with worsening, virtually lifelong, chorea and progressive behavioural disturbance, involving disinhibition and hoarding, over 10 years. Clinical assessment revealed chorea, dysarthria, areflexia, an inappropriately jovial, impulsive manner and neuropsychological evidence of frontosubcortical dysfunction. Investigation results included an elevated creatine kinase, caudate atrophy and hypoperfusion, acanthocytes in the peripheral blood and the McLeod phenotype. DNA studies demonstrated a single-base deletion at position 172 in exon 1 of the XK gene, giving rise to a premature stop codon at position 129 in exon 2.

The molecular genetics of blood group polymorphism.

Nearly 300 blood group specificities on red cells are known, many of which are polymorphic. The molecular mechanisms responsible for these polymorphisms are diverse, though the majority represent single nucleotide polymorphisms (SNPs) encoding amino acid substitutions. Other mechanisms include the following: gene deletion; single nucleotide deletion and sequence duplication, which introduce reading-frame shifts; nonsense mutation; intergenic recombination between closely-linked genes, giving rise to hybrid genes and hybrid proteins; and a SNP in the promoter region of a blood group gene. Examples of these genetic mechanisms are taken from the ABO, Rh, Kell, and Duffy blood group systems. Null phenotypes, in which no antigens of a blood group system are expressed, are not generally polymorphic, but provide good examples of the effect of inactivating mutations on blood group expression. As natural human 'knock-outs' they provide useful clues to the functions of blood group antigens. Knowledge of the molecular bases to blood group polymorphisms provides a means to predict blood group phenotype from genomic DNA with a high degree of accuracy. This currently has two main applications in transfusion medicine: for determining fetal blood groups to assess whether the fetus is at risk from haemolytic disease; and to determine blood group phenotypes in multiply transfused, transfusion-dependent patients, where serological tests are precluded by the presence of donor red cells. Other applications are being developed for the future.