Variant ABO Research Articles

Regulation of ABO blood group antigen expression by miR-331-3p and miR-1908-5p during hematopoietic stem cell differentiation.

The ABO blood group system is the most important factor in clinical transfusion medicine and is implicated in a number of human diseases. ABO antigens are not confined to red blood cells (RBCs) and are widely expressed in a variety of human cells and tissues. To date, many alleles with variant ABO expression have been identified and in many cases traced to one of the >250 reported genetic variations in the respective glycosyltransferase. The role of microRNAs (miRNAs) in the regulation of blood group antigens during erythropoiesis has not been addressed, however. Here, we show that miR-331-3p and miR-1908-5p directly target the mRNA of glycosyltransferases A and B. Expression levels of miR-331-3p and miR-1908-5p inversely correlated with levels of blood group A antigen. In addition, we found that overexpression of these miRNAs in hematopoietic stem cells led to a significantly reduced number of blood group A antigens per RBC. Simultaneous targeting of the transcription factor (TF) SP1 by miR-331-3p further enhanced these effects. The targeting rendered SP1 incapable of binding to the ABO gene promoter, causing further downregulation of blood group A antigen expression by up to 70%. Taken together, expression changes in these miRNAs may account for rare cases of weak A/B phenotypes that genetic variations in the glycosyltransferase coding region cannot explain. These results also suggest an explanation for the disappearance of ABH antigens during carcinogenesis and point to new therapeutic targets in ABO mismatched organ transplantation.

Comparative analysis of asexual and sexual stage Plasmodium falciparum development in different red blood cell types

BackgroundRed blood cell (RBC) polymorphisms are suggested to influence the course of Plasmodium falciparum malaria. Whereas some variants have been found to be protective, others have been found to enhance parasite development. This study evaluated the effect of variant haemoglobin (Hb) and ABO blood groups on P. falciparum merozoite invasion, multiplication rates as well as gametocyte development.MethodsApproximately 2.5 mL of venous blood was collected from each participant. Flow cytometry was used to determine the in vitro merozoite invasion rates of NF54 parasites into the blood of 66 non-parasitaemic individuals with variant Hb genotypes (HbSS, HbSC) and blood groups (A, B, O), which were then compared with invasion into HbAA blood. The ex vivo asexual parasite multiplication and gametocyte production rates of parasites from 79 uncomplicated malaria patients with varying Hb genotypes (HbAS, HbAC and HbAA) were also estimated using microscopy.ResultsMerozoite invasion rates were significantly reduced by about 50% in RBCs containing HbSS and HbSC relative to HbAA cells. The presence of blood group O and B reduced the invasion rates of HbSS by about 50% and 60%, respectively, relative to HbSC but the presence of blood group A removed the inhibitory effect of HbSS. The initial parasite densities in uncomplicated malaria patients with Hb genotypes HbAS and HbAC cells were similar but significantly lower than those with genotype HbAA. The ex vivo parasite multiplication rate, gametocytaemia and gametocyte conversion rates followed a similar trend but did not reach statistical significance (p > 0.05).ConclusionsParasite invasion rate into erythrocytes is dependent on both erythrocyte blood group antigen and haemoglobin genotype as blood group O and B provided protection via reduced merozoite invasion in RBCs containing HbSS relative to HbSC. Regardless of haemoglobin type, greater than 70% malaria patients had circulating ring stage parasites that differentiated into stage II gametocytes in 4 days.

The potential role of bacteria in pancreatic cancer: a systematic review.

Pancreatic cancer is a lethal and devastating disease in the worldwide. Recognized risk factors for pancreatic cancer include cigarette smoking, obesity, type II diabetes and chronic pancreatitis. Other factors such as variant ABO blood type and Helicobacter pylori may also play an important role in pancreatic carcinogenesis. Recently, growing evidence suggests that the association between bacteria and pancreatic cancer is positive and related immune/inflammation activation and increased nitrosamine exposure may be its potential mechanism. Interestingly, it is debatable whether the relationship of bacteria and pancreatic cancer is causative, reactive or parallel and future studies are in progress. Here we review recent progress in pancreatic cancer and its related bacteria.

Next Generation Sequencing-Based Fetal ABO Blood Group Prediction by Analysis of Cell-Free DNA from Maternal Plasma

Introduction: ABO blood group incompatibility between a pregnant woman and her fetus as a cause of morbidity or mortality of the fetus or newborn remains an important, albeit rare, risk. When a pregnant woman has a high level of anti-A or anti-B IgG antibodies, the child may be at risk for hemolytic disease of the fetus and newborn (HDFN). Performing a direct prenatal determination of the fetal ABO blood group can provide valuable clinical information. Objective: Here, we report a next generation sequencing (NGS)-based assay for predicting the prenatal ABO blood group. Materials and Methods: A total of 26 plasma samples from 26 pregnant women were tested from gestational weeks 12 to 35. Of these samples, 20 were clinical samples and 6 were test samples. Extracted cell-free DNA was PCR-amplified using 2 primer sets followed by NGS. NGS data were analyzed by 2 different methods, FASTQ analysis and a grep search, to ensure robust results. The fetal ABO prediction was compared with the known serological infant ABO type, which was available for 19 samples. Results: There was concordance for 19 of 19 predictable samples where the phenotype information was available and when the analysis was done by the 2 methods. For immunized pregnant women (n = 20), the risk of HDFN was predicted for 12 fetuses, and no risk was predicted for 7 fetuses; one result of the clinical samples was indeterminable. Cloning and sequencing revealed a novel variant harboring the same single nucleotide variations as ABO*O.01.24 with an additional c.220C>T substitution. An additional indeterminable result was found among the 6 test samples and was caused by maternal heterozygosity. The 2 indeterminable samples demonstrated limitations to the assay due to hybrid ABO genes or maternal heterozygosity. Conclusions: We pioneered an NGS-based fetal ABO prediction assay based on a cell-free DNA analysis from maternal plasma and demonstrated its application in a small number of samples. Based on the calculations of variant frequencies and ABO*O.01/ABO*O.02 heterozygote frequency, we estimate that we can assign a reliable fetal ABO type in approximately 95% of the forthcoming clinical samples of type O pregnant women. Despite the vast genetic variations underlying the ABO blood groups, many variants are rare, and prenatal ABO prediction is possible and adds valuable early information for the prevention of ABO HDFN.

ABO antigen levels on platelets of normal and variant ABO blood group individuals

Although differential expression of ABO antigens on platelets is related to recipients’ platelet recovery and neonatal alloimmune thrombocytopenia in ABO-incompatible transfusion and pregnancy, the expression of ABO antigens on platelets in normal and variant ABO blood group individuals is rarely reported. Here, we analyzed the expression of ABO antigens on platelets in a cohort of 515 individuals with normal and variant ABO. The variants were also genotyped for ABO and/or FUT1 loci to distinguish inherited or acquired ABO variants. The results showed that ABO antigens expression on individual platelets was relatively stable during the storage period in vitro. The frequency of low-, moderate-, and high-expression phenotype in normal individual was 59.6%, 35.5%, and 4.9%, respectively. The platelets from para-Bombay and most inherited ABO subgroups express very low levels of ABO antigens, but the acquired ABO variants and the B3 subgroup express normal or abnormally high levels of ABO antigens on platelets. The data extend the information of antigens expression on platelets, which may help to improve the strategy of platelets transfusion.

Establishing Blood Group Genotyping to Resolve ABO Discrepancies in Iran

ABO discrepancies are recognized when the reactions obtained in the forward type do not "match" the reactions obtained in the reverse type. Discrepant results are often caused by a variant ABO gene. Molecular analysis is required to confirm the type of subgroups and discrepancy. In this study ABO genotyping was performed on a series of blood donors and patients to determine their definite blood groups. We examined 100 samples with ABO discrepancies from blood donors and patients referred to Tehran Blood Transfusion Center between October 2015 and August 2016. ABO genotyping was performed on all samples with allele specific PCR for differentiation of A, B and O alleles. Exon 6 and 7 of ABO gene were sequenced to confirm the results. The genotyping of donor/patients samples with discrepant results of ABO blood typing consisted of 61 cases of A2 and A2B, 3 cases of B 302 and 4 cases of Aw06. Genotyping of 6 samples that had extra antibody in their serum (AB blood group) confirmed the cell type reaction results. 6 samples that had shown a very weak reaction with anti-AB (similar to O blood group) and had no anti-A in their serum were genotyped as O 1 O 2. Blood group genotyping laboratory provides an efficient service for evaluation of ABO discrepancies and resolve the problems encountered in serology reactions.

Molecular genetic analysis and structure model of a rare B(A)02 subgroup of the ABO blood group system

BackgroundSerological analysis of ABO blood group has been widely applied in transfusion medicine. However, ABO subgroups with different expression of blood group antigens sometimes cannot be determined by serological methods. Therefore, genotyping is useful to understand the variant ABO phenotypes. Material and MethodsExon 6 to exon 7 and adjacent introns of the ABO gene from a donor with ABO typing discrepancy were amplified and sequenced. Cloning sequencing was also performed to identify the allele. To explore the effect of mutation, three dimensional model of mutant p.Pro234Ala was built and optimized. ResultsThe variant B (c. 700C > G) allele expressed an AweakB phenotype with anti-A in his serum with a ABO*B(A)02/O02 heterozygote genotype. Cloning sequencing confirmed that the c.700C > G single nucleotide polymorphism was associated with a B101 allele. Three dimensional molecular modeling suggested that p.Pro234Ala might affect the conformation of His233, Met266 and Ala268, which were known as critical residues for donor recognition. ConclusionABO genotyping is needed for correct identification subgroups to improve accuracy evaluation of blood typing and increase the safety of blood transfusion. Alteration of DNA sequence in the ABO gene resulted in amino acid substitutions and led to a weak or missing expression of ABO antigens.

A Novel ABO Gene Variant Leads to Discrepant Results in Forward/Reverse and Molecular Blood Grouping

SummaryBackground: Discrepant results in antigen and reverse ABO blood typing are often caused by a variant ABO gene. Molecular analysis can help to characterize such variants. Here, we describe the identification of a novel ABO gene variant in a patient with aberrant ABO phenotype and discrepant genotyping results. Methods: A patient with discrepant results in automated forward and reverse ABO phenotyping was further investigated by serological (gel and tube technique) and molecular (commercial and inhouse PCR-SSP, DNA sequencing) methods. A PCR-SSP system was established to screen the novel mutation in 1,820 blood donors. Results: Standard serological tests confirmed blood group O, however, only anti-B isoagglutinins were present. A monoclonal anti-AB antibody detected very weak agglutination in gel technique. Standard ABO genotyping using PCR-SSP led to discrepant results (O¹/O¹ or O¹/A) depending on the test system used. ABO exon re-sequencing identified a novel missense mutation in exon 6 at position 248A>G (Asp83Gly) in the binding region of PCR-SSP primers for the detection of 261G alleles. Blood donors with regular ABO blood groups were all negative for the 248G allele designated Aw34. Conclusion: The novel ABO gene variant Aw34 is associated with very weak A antigen expression and absent anti-A isoagglutinins. The mutation is located in exon 6 close to the O¹-specific 261G deletion in the binding region of PCR-SSP primers. Presumably, depending on the primer concentration used in commercial ABO genotyping kits, the mutation could lead to a false-negative reaction.

Variant ABO blood group alleles, secretor status, and risk of pancreatic cancer: results from the pancreatic cancer cohort consortium.

Subjects with non-O ABO blood group alleles have increased risk of pancreatic cancer. Glycosyltransferase activity is greater for the A(1) versus A(2) variant, whereas O01 and O02 variants are nonfunctioning. We hypothesized: 1) A(1) allele would confer greater risk than A(2) allele, 2) protective effect of the O allele would be equivalent for O01 and O02 variants, 3) secretor phenotype would modify the association with risk. We determined ABO variants and secretor phenotype from single nucleotide polymorphisms in ABO and FUT2 genes in 1,533 cases and 1,582 controls from 12 prospective cohort studies. Adjusted odds ratios (OR) for pancreatic cancer were calculated using logistic regression. An increased risk was observed in participants with A(1) but not A(2) alleles. Compared with subjects with genotype O/O, genotypes A(2)/O, A(2)/A(1), A(1)/O, and A(1)/A(1) had ORs of 0.96 (95% CI, 0.72-1.26), 1.46 (95% CI, 0.98-2.17), 1.48 (95% CI, 1.23-1.78), and 1.71 (95% CI, 1.18-2.47). Risk was similar for O01 and O02 variant O alleles. Compared with O01/O01, the ORs for each additional allele of O02, A(1), and A(2) were 1.00 (95% CI, 0.87-1.14), 1.38 (95% CI, 1.20-1.58), and 0.96 (95% CI, 0.77-1.20); P, O01 versus O02 = 0.94, A(1) versus A(2) = 0.004. Secretor phenotype was not an effect modifier (P-interaction = 0.63). Among participants in a large prospective cohort consortium, ABO allele subtypes corresponding to increased glycosyltransferase activity were associated with increased pancreatic cancer risk. These data support the hypothesis that ABO glycosyltransferase activity influences pancreatic cancer risk rather than actions of other nearby genes on chromosome 9q34.

Aberrant intracellular trafficking of a variant B glycosyltransferase

Little is known about the mechanism by which amino acid polymorphisms outside the catalytically active cleft of ABO glycosyltransferases cause weak ABO phenotypes. Extensive ABO phenotyping and genotyping were performed to classify the blood of a healthy blood group O donor with weak isoagglutinins. ABO antigen and glycosyltransferase expression profiles were then studied in eukaryotic transfection experiments, and the topology of ABO glycosyltransferase was analyzed. The donor's red blood cells were retyped as A(weak), and his serum contained weakly reactive anti-A and anti-B. Sequence analysis revealed two novel ABO alleles. A donor splice-site mutation detected at the exon 6/intron 6 junction of an ABO*A101 allele was predicted to result in skipping exon 6 in the mRNA. The other haplotype displayed a single 688G>C substitution predicting a Gly230Arg exchange in the catalytic domain in an otherwise normal ABO*B101 allele. The transfection studies revealed very weak expression of B antigen by the novel ABO*B allele. According to the topologic analysis, steric hindrance due to the Gly230Arg exchange may cause conformational changes in the variant B transferase. Compared to the wild-type B transferase, the transfected cells exhibited lower-level protein expression and intracellular dislocation. This study provides first evidence that aberrant trafficking of variant ABO transferases may be involved in the formation of weak ABO phenotypes.

A weak blood group A phenotype caused by a translation‐initiator mutation in the ABO gene

Weak blood group A and B phenotypes are correlated with ABO glycosyltransferases exhibiting single-amino-acid changes and/or C-terminal modifications. A healthy donor diagnosed as having weak A antigen expression and his two children were subjected to extensive ABO typing. HeLa cells were used to transfect ABO expression plasmids. The donor's red blood cells were type A(weak)B and his serum sample contained weakly reactive anti-A(1) antibodies. A single T>C transition identified at the +2 position of the start codon of an ABO*A101 allele predicted the disruption of this methionine codon. In the transfection studies, a significant reduction of A activity was observed on HeLa cells transfected with a plasmid containing the variant ABO*A allele. Coexpression of the respective antithetical ABO*B101 wild-type construct further reduced cell surface A antigen expression. Similar expression results were obtained with ABO constructs in which the Met(1) start codon and five alternative start sites at codons 20, 26, 43, 53, and 69 had successively been interrupted. The donor's weak blood group A phenotype most likely resulted from expression of an N-truncated A transferase triggered by alternative translation start sites in the transmembrane domain or stem region.

Missense mutations outside the catalytic domain of the ABO glycosyltransferase can cause weak blood group A and B phenotypes

Only little is known about the impact of amino acid substitutions outside an enzyme's active site on A and B transferase activity. A panel of blood group A- and B-specific plasmids containing the six known missense mutations of the coding sequence upstream of exon 6 of the ABO gene were constructed. HeLa cells were used to transfect ABO expression plasmids. Expression of ABO variants containing single or multiple missense mutations in HeLa cells resulted in a significant decrease in the percentage of antigen-expressing cells (up to 29%) and in mean fluorescence intensity (MFI; up to 50%) compared to transfection with ABO*A101 or ABO*B101. Coexpression of the respective antithetical wild-type construct (ABO*A101 and ABO*B101, respectively) further reduced cell surface expression of variant ABO constructs in regard to the percentage of expressing cells (up to 53% decrease) and MFI (up to 59% decrease). Weak A and B subgroups can arise from transferases with amino acid changes in the N-terminal domain, particularly in AB phenotypes, where normal A1 or B1 glycosyltransferases compete for the same substrates.

Nondeletional ABO*O alleles express weak blood group A phenotypes

Owing to a single-base deletion, the vast majority of ABO*O alleles encode for a truncated and catalytically inactive ABO glycosyltransferase, leading to the generation of a premature stop codon. Less frequent nondeletional ABO*O alleles such as ABO*O03, in contrast, have nonsynonymous mutations that may abolish the protein's enzyme activity by altering its sugar-binding site. Extensive ABO phenotyping and genotyping were performed in healthy blood group O donors with weak anti-A isoagglutinins and their relatives as well as in blood group O donors selected for the presence of ABO*O03. HeLa cells were used to transfect ABO expression plasmids. Donors or relatives carrying ABO*O03 and/or its rare variant ABO*Aw08 in homozygous (n = 2) or heterozygous (n = 14) form showed weak A antigen expression detectable only by adsorption-elution (n = 15) or by monoclonal anti-A typing (n = 1). The serum samples of most donors (n = 13) contained weak anti-A; in the remaining donors, anti-A isoagglutinin reactivity was in the normal range. In the transfection studies, weak A antigen expression on HeLa cells transfected with plasmids containing ABO*O03 or ABO*Aw08 expression constructs was detectable only by adsorption-elution. The data provide evidence that nondeletional ABO*O03-like alleles produce detectable amounts of A antigens.

The nature of diversity and diversification at the ABO locus

AbstractIn this study we analyzed the complete genomic sequences, except intron 1, and 2 regulatory regions of 6 common (ABO*A101, ABO*A201, ABO*B101, ABO*O01, ABO*O02, and ABO*O03) and 18 rare ABO alleles, 3 of which were new. This was done by phylogenetic analysis and correlating sequence data with the ABO phenotypes. The study revealed multiple polymorphisms in noncoding regions. The intron-based phylogenetic analysis revealed 5 main lineages: ABO*A, ABO*B, ABO*O01, ABO*O02, and ABO*O03. The genomic sequences of most rare ABO alleles differed slightly from those of the common alleles. Singular mutations or hybrid alleles were most common, but a few exhibited mosaic sequence pattern containing multiple exon and/or intron motifs from other ABO lineages. Thus, both an accumulation of mutations as well as an assortment of the mutations by recombination seems to be responsible for the ABO gene diversity. The prevalence of replacement mutations indicates positive selection for allelic diversity. Phenotype-genotype correlation showed that sequence variations within the complete coding sequence can affect A- and B-antigen expression. All variant ABO*A/B alleles and one new ABO*O03-like allele were associated with weak ABO phenotypes. These findings are suggestive of the requirement of a comprehensive coding sequence database for sequence-based phenotype prediction.

Systematic analysis of the ABO gene diversity within exons 6 and 7 by PCR screening reveals new ABO alleles.

Mutations critical for ABO blood group phenotypes have predominantly been found in exons 6 and 7 of the ABO gene, both of which encode the catalytic domain of ABO glycosyltransferase. To design rapid and reliable ABO genotyping assays, a profound knowledge of the prevalent alleles is required and a reliable sequence database needs to be established. A PCR screening system was established consisting of 102 different PCRs, each specific for a single nucleotide (nt) variation. The primer mixes were developed to walk from the 5' to the 3' end of exons 6 and 7 of the ABO gene to screen for nt mutations at 50 known polymorphic sites. A total of 109 unrelated individuals with common and rare ABO characteristics were screened. All blood samples in which the PCR results were inconclusive or inconsistent with the ABO phenotypes were subjected to sequence analysis of exons 6 and 7. The results of PCR screening were conclusive and consistent with the ABO phenotypes in 90 cases. In the remaining 19 cases, PCR screening revealed unusual allele combinations or amplification results that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis. In these 19 cases, sequencing revealed new ABO alleles (one ABO*Ael allele, one ABO*B(A) allele and two ABO*O alleles) in two individuals with common and seven individuals with variant ABO phenotypes. This PCR screening strategy is an effective tool for obtaining deeper insight into the ABO gene diversity and diversification and may be useful to increase the quality of the ABO sequence database.

ABO and Rh(D) blood typing on the PK 7200 with ready-to-use kits

Abstract The performance of ready-to-use kits was evaluated on the PK 7200 blood grouping system. The Olymp Group (kit 1) and Olymp Group II (kit 2) containing anti-A, -B, -AB, and -D reagents were tested for first and second determinations of A, B, and D antigens. More than 500 RBC samples, including several variant ABO and D phenotypes, were evaluated for specificity, repeatability, reproducibility, and sensitivity. Specificity was tested with well-characterized reagent RBCs. Repeatability was established by at least 12 assays per run with three reagent RBCs, and reproducibility was established on one run per day for 5 days. No discrepancy was observed in ABO and D determinations with either kit. In repeatability, three discrepancies were found with group A and B RBCs with kit 1. In reproducibility, no discrepancies were observed. The kit 1 anti-A reagent detected A3 but not Ax RBCs and anti-AB detected both. A B3 RBC was detected by both kits. Among eight weak D phenotypes, six were positive with kit 1. With kit 2, only one of five weak D phenotypes was detected. Immunohematology 2003;19:54–56.

Molecular Genetic Analysis of Variant Phenotypes of the ABO Blood Group System

ABO is clinically the most important blood group system in transfusion medicine and includes many variant phenotypes. To understand the molecular genetic basis of this polymorphic system, we have analyzed genomic DNAs obtained from Japanese individuals possessing variant ABO phenotypes including A2, Ax, Ael, cis-AB, Bx, and Bel. By polymerase chain reaction-single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, we identified 11 different alleles. These alleles had nucleotide sequences different from those of the previously described 13 different alleles responsible for the common ABO phenotypes. Analysis of the nucleotide sequences of the alleles responsible for those variant phenotypes showed that the amino acid residues at position 266 and 268 may be crucial for transferase specificity, whereas those at positions 214, 216, 223, 291, and 352 may be critical for the activity level. Nine of the 11 alleles, responsible for the A2, Ax, Ael, cis-AB, Bx, and Bel phenotypes, were presumed to be generated from common ABO alleles by single nucleotide mutations such as nonsynonymous substitution, deletion, or insertion. Two other alleles, responsible for the A2 and Ael phenotypes, may have originated by recombination, gene conversionlike events or accumulation of nucleotide substitutions. Our data indicate that different alleles could cause the same ABO variant phenotypes, and that these alleles do not necessarily belong to a single evolutionary lineage.