Weak ABO Research Articles

Serological and molecular characterization of novel ABO variants including an interesting B(A) subgroup.

ABO grouping is the most important pretransfusion testing that is directly related to the safety of blood transfusion. A weak ABO subgroup is one of the important causes of an ABO grouping discrepancy. Here, we investigated the characterization of four novel ABO variants including a novel B(A) subgroup. RBCs were phenotyped by standard serology methods. The full coding regions of the ABO gene and the erythroid cell-specific regulatory elements in intron one were sequenced. The effect of the possible splice site variant was predicted by Alamut software. The 3D structural modeling of three relative B(A) enzymes (p.Met214Thr, p.Met214Val, and p.Met214Leu) were performed by PyMOL software. Four novel ABO alleles were identified with weak ABO expression in this study, in which two would lead to premature terminations, and two resulted in amino acid changes. In silico analysis revealed that the splice site variant c.155G>T had the potential to alter splice transcripts. 3D structural view shown that the variant amino acid position 214 was spatially adjacent to the donor recognition pocket residues (266Met and 268Ala) and just next to the 211DVD213 motif. The size of the side chain of Thr and Val is the smallest, Leu is medium, and Met is the largest, and the size changes in the critical position 214 may affect the donor recognition pocket. Four ABO subgroup alleles were newly linked to different kinds of ABO variants and the possible mechanism through which they produce weak ABO subgroups was analyzed in silico.

Molecular genetic analysis of two novel a allele to cause Ax phenotype in Chinese

BackgroundMutations of ABO gene may cause the dysfunction of ABO glycosyltransferase (GT) that can result in weak ABO phenotypes. Here, we identified two novel weak ABO subgroup alleles and explored the mechanism that caused Ax phenotype. Materials and methodsThe ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the mutations was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. ResultsThe results of serological showed the subject RJ23 and RJ52 both were Ax phenotypes. The novel A alleles, Avar-1 and Avar-2 were identified according to the gene analysis. Both Avar-1 and Avar-2 harbored recombinant heterozygous alleles, specifically A2.05 and O.01.02. These alleles showcased substitutions at positions c.106G > T, c.189C > T, c.220C > T, and c.1009A > G in their respective exons. It is worth noting that the crossing-over regions of these two alleles differed from each other. In vitro expression study showed that GTA mutant impaired H to A antigen conversion, and the mutant did not affect the production of GTA though the Western bolt. In silico analysis showed that GTA mutant may change the local conformation and the stability of GT. ConclusionsThe Avar-1 and Avar-2 alleles were identified, which could cause the Ax phenotype through changing the local conformation and reducing stability of the GTA.

Prevalence and distribution of serological characteristics of weak ABO subgroups in the Chinese population

ObjectivesThis study aimed to determine the true frequency of weak ABO subgroups and to investigate the serological characteristics of various subgroup alleles in the Chinese population. MethodsA total of 2,945,643 blood samples were collected from January 2009 to December 2017. After initial screening and re-examination using automatic blood group analyzers, all ABO-discrepant samples were confirmed by standard serological analysis and molecular detection by DNA sequencing. The true frequency of weak ABO subgroups was determined by the number of ABO subgroup donors and the missed detection rate. The ABO antigen expressions corresponding to subgroup alleles were analyzed by the agglutination intensity of red blood cells. ResultsThe detection rate of ABO subgroups was 0.031 % (927/2,945,643). Considering the missed detection rate (27.81 %), the true frequency of ABO subgroups in the Chinese population was 0.044 %. The three most common genetic variations among blood donors in Shanghai were BA.04, BW.12 and BA.02. BW.03 showed the weakest B antigen expression (6.00 ± 1.97) and Bvar-1 the strongest (9.20 ± 1.10). ConclusionMany ABO subgroups were missed. BA.04 was found to be the most common subgroup allele in the Chinese population. Different ABO subgroup alleles exhibit different ABO antigen expression patterns.

One novel single nucleotide polymorphism c.424A>G on A1.02 allele in ABO glycosyltransferases leads to Aweak phenotype

BackgroundThe dysfunction of the ABO glycosyltransferase (GT) enzyme, which is caused by mutations in the ABO gene, can lead to weak ABO phenotypes. In this study, we have discovered a novel weak ABO subgroup allele and investigated the underlying mechanism to causing its Aweak phenotype. Materials and methodsThe ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of ABO gene. The role of the novel single nucleotide polymorphism (SNP) was evaluated by 3D model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. ResultsThe results of serological showed the subject was Aweak phenotype. A novel SNP c.424A > G (p. M142V) based on ABO∗A1.02 was identified, and the genotype of the subject was AW-var/O.01 according to the gene analysis. In silico analysis showed that the SNP c.424A > G on the A allele may change the local conformation by damaging the hydrogen bonds and reduce the stability of GT. In vitro expression study showed that SNP p.M142V impaired H to A antigen conversion, although it did not affect the generation of A glycosyltransferase (GTA). ConclusionOne novel AW allele was identified and the SNP c.424A > G (p.M142V) can cause the Aweak phenotype through damaging the hydrogen bonds and reducing stability of the GTA.

Incidence of acute haemolytic transfusion reaction among ABO-incompatible recipients transfused with A3 blood: A case series.

ABO antigens are among the most immunogenic, but the haemolytic risks of ABO incompatibilities involving a donor with a weak ABO phenotype are little documented. This retrospective case series assessed the incidence of acute haemolytic transfusion reaction (AHTR) among ABO-incompatible recipients of A3 blood in Québec (Canada). Transfusion safety officers reported laboratory AHTR indicators measured ≤24 h pre- and post-transfusion. Because the AHTR case definition of Québec's Hemovigilance System (QHS) leaves significant room for clinical judgement, a two-step approach was used to assess potential cases: Step 1 consisted in a highly sensitive-but unspecific-initial screen that identified all candidate cases per QHS case definition, and Step 2 consisted in a detailed review of candidate cases by two haematologists. Nine donors initially typed as Group B (N = 1) or O (N = 8) were subsequently found to display an A3 B or A3 O phenotype. Eighty-one recipients received ABO-incompatible blood, including 53 (65.4%) with interpretable data. Of these, 29 (54.7%) were classified as candidate cases after Step 1. Following Step 2, no conclusive evidence of AHTR was found: Abnormal pre- versus post-transfusion changes appeared modest, within normal range, insufficient to ascertain AHTR, or were consistent with a pre-existing condition unrelated to AHTR. Two candidate cases had a QHS-reported transfusion reaction; both were unrelated to AHTR. In this case series, no conclusive evidence of serious AHTR was found among ABO-incompatible recipients who were inadvertently transfused with A3 blood.

Identification of a novel variant c.761C>T on ABO*B.01 gene in ABO glycosyltransferases associated with Bweak phenotype.

ABO antigens are produced from H antigen by the activity of glycosyltransferase enzyme encoded by the ABO gene. Variants in the ABO gene can produce a weak ABO phenotype. In this study, we identify a novel ABO*BW allele and investigate the underlying mechanism leading to the Bweak phenotype. The ABO phenotype and genotype of the sample were determined using serological and direct DNA sequencing methods. We assessed the impact of the novel variant by three-dimensional modelling to predict protein stability changes (ΔΔG), and carried out an in vitro expression assay. The total glycosyltransferase transfer capacity in the supernatant of transfected cells was also examined. Serological analysis confirmed the Bweak phenotype in the subject, and gene sequencing identified a novel variant c.761C>T (p.A254V) on the ABO*B.01 allele, resulting in a BW-var/O.01.02 genotype. In silico analysis suggested that the p.A254V variant on the B allele may reduce the stability of glycosyltransferase B (GTB), as indicated by the ΔΔG values. In vitro expression studies showed that the variant p.A254V impaired H to B antigen conversion, although it did not affect the expression of GTB. We identified a novel BW allele and demonstrated that the variant c.761C>T (p.A254V) can cause the Bweak phenotype by reducing the stability of GTB.

A report on a modified protocol for flow cytometry-based assessment of blood group erythrocyte antigens potentially suitable for analysis of weak ABO subgroups.

Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non-negligible levels. We sought to establish an FC method that could prevent antibody-induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC-FC analysis of AB expression. We optimized fixatives, multicolor-staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, Bm and Ael , were analyzed with the established protocol. The optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co-staining of leukocyte- and erythrocyte-markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the Bm sample a small RBC population exhibiting weak B antigen expression. The assay also proved it feasible to identify a small population (0.04%) of RBCs weakly expressing the A antigen in the Ael sample confirmed as harboring a rare c.816dupG ABO variant allele. The RBC-FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.

Analysis of ABO subgroups which result in ABO discrepancies in Iranian blood donors

The ABO blood group system plays a vital role in the blood transfusion and transplantation medicine [ [1] Hosseini‐Maaf B. Hellberg Å. Chester M.A. Olsson M.L. An extensive polymerase chain reaction–allele‐specific polymorphism strategy for clinical ABO blood group genotyping that avoids potential errors caused by null, subgroup, and hybrid alleles. Transfusion. 2007; 47: 2110-2125 Crossref PubMed Scopus (43) Google Scholar ]. It consists of the major A, B, O alleles and many of subtypes with weak expression of A or B antigens on red blood cells. The results of both forward and reverse ABO typing are necessary for ABO blood group typing, as they are both necessary for interpreting the results [ [2] Arumugam P. Hamsavardhini S. Ravishankar J. Bharath R. Resolving ABO discrepancies by serological workup‐an analysis of few cases. Int J Res Med Sci. 2017; 5: 893-900 Crossref Google Scholar ]. ABO discrepancies may be due to technical or human errors, weak subgroups, the transient expression of antigens in some disease like carcinomas and mixed-filled agglutination after massive or chronic transfusion [ 3 Kaur G. Kaur P. Basu S. Kaur R. Blood group discrepancies at a tertiary care centre–analysis and resolution. Int J Lab Hematol. 2014; 36: 481-487 Crossref PubMed Scopus (18) Google Scholar , 4 Meny G. Recognizing and resolving ABO discrepancies. Immunohematology. 2017; 33: 76-81 Crossref PubMed Google Scholar , 5 Storry J.R. Condon J. Hult A.K. Harrison A. Jørgensen R. Olsson M.L. An age‐dependent ABO discrepancy between mother and baby reveals a novel A weak allele. Transfusion. 2015; 55: 422-426 Crossref PubMed Scopus (7) Google Scholar ]. The incidence of ABO discrepancies among blood donors was reported from 0.02 % to 0.06 %, Also the ABO subgroups are considered the most common cause of ABO discrepancies [ 3 Kaur G. Kaur P. Basu S. Kaur R. Blood group discrepancies at a tertiary care centre–analysis and resolution. Int J Lab Hematol. 2014; 36: 481-487 Crossref PubMed Scopus (18) Google Scholar , 6 Sharma T. Garg N. Singh B. ABO blood group discrepancies among blood donors in Regional Blood Transfusion Centre GTB Hospital, Delhi, India. Transfus Apher Sci. 2014; 50: 75-80 Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar , 7 Makroo R.N. Kakkar B. Agrawal S. Chowdhry M. Prakash B. Karna P. Retrospective analysis of forward and reverse ABO typing discrepancies among patients and blood donors in a tertiary care hospital. Transfus Med. 2019; 29: 103-109 Crossref PubMed Scopus (10) Google Scholar ]. Weak ABO subgroups are categorized as type II (Weak/missing antigen) or type IV (due to presence of Anti–A1) ABO discrepancy [ [8] Jain A. Gupta A. Malhotra S. Marwaha N. Sharma R.R. An ABO blood grouping discrepancy: Probable B (A) phenotype. Transfus Apher Sci. 2017; 56: 459-460 Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar ]. Mistyped A and B weak blood group as O and B(A) as cisAB and AxB during routine serologic tests can have serious consequences, such as hemolytic adverse transfusion reactions [ [9] Jiao L.X. Zhang J.Y. Chen L. Yu X.L. Han Y. Yang F. et al. Gene identification of rare B (A) blood group. Transfus Apher Sci. 2017; 56: 855-857 Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar ].

Rare Blood Groups in ABO, Rh, Kell Systems - Biological and Clinical Significance.

Background: The frequency of ABO, Rh and Kell blood group antigens differs among populations of different ethnic ancestry. There are low-frequency antigens (<1%) and high-frequency antigens (>90%). A rare blood group is defined as the absence of a high-frequency antigen in the general population, as well as absence of multiple frequent antigens within a single or multiple blood group systems. Aim: To perform red blood cell typing and to calculate the antigen and phenotype frequencies, in order to identify rare blood group donors within the clinically most important АВО, Rh and Kell systems. Material and Methods: АВО, Rh (D, C, E, c, e) and Kell (K) antigen typing was performed using specific monoclonal sera and microplate technique, while Cellano (k) typing was performed with a monoclonal anti-k, antihuman globulin and column agglutination technique. Weak ABO subgroups were determined using the absorption elution method or molecular genotyping (PCR-SSP). Results: ABO antigen frequency is: A (40.89%), O (34.22%), B (16.97%), AB (7.92%) and weak ABO subgroups (0, 009 %). The established genotypes were AxO1 (0, 0026%) and AxB (0, 001%). Rh antigen frequency is: D (85.79%), C (71.7%), c (76.0%), E (26.0%) and е (97.95%). The most common Rh pheno-type is the DCcee (32.7%) while the rarest phenotype is the DCCEE phenotype (0. 003%). The prevalence of K and k antigen is 7.5% and 99.94%, respectively. The frequency of the rare phenotype K+k- is 0.06%. Conclusion: Large scale phenotyping of blood group antigens enables the identification of blood donors with rare blood groups for patients with rare phenotypes or with antibodies to high-frequency antigens and to frequent antigens within one or more blood group systems.

Association between diminished miRNA expression and the disease status of AML patients: Comparing to healthy control

BackgroundChanges in ABO blood type caused by a gradual decrease in antigen expression have been found in patients with acute myeloid leukemia (AML). Studies have indicated that alteration of ABO gene methylation accounts for 50% of acquired weak ABO antigen expression in patients with leukemia. However, the molecular mechanisms contributing to the remaining 50% of cases are unknown. We hypothesize that deregulation of miRNA is correlated with weak ABO antigen expression in patients with AML. MethodsBlood samples of 19 patients with AML and 12 healthy controls were collected, in which the blood type was not changed in these AML patients. Flow cytometric analysis was applied to measure the ABO antigen expression titer among AML patients and controls. A total of 18 leukemia-related miRNAs were analyzed via quantitative real-time polymerase chain reactions. ResultsWe found that miRNA profiles were correlated with the AML patients, especially in those who had constant or weakened ABO antigen expressions. Compared with healthy controls, the miR-16 and miR-451 expression were significantly lower in either AML cases with weak ABO antigen expressions (p = 0.003, p = 0.028, respectively) or AML cases with constant ABO antigen expressions (p = 0.043, p = 0.040, respectively). Although not statistically significant, decreasing trends in the miR-451 and miR-16 expressions in the AML patients with weakened ABO were observed compared to those with constant ABO antigens. The weak ABO antigen expression might correlate with miRNAs, especially miR-16 and miR-451. ConclusionThis study indicated that decreasing in miR-16 and miR-451 was associated with AML and AML with weakened ABO expression. In the future, we will continue to include more cases and exclude the others factor influencing ABO antigen expression, promoter methylation and oxidative stress, to replicate the results of this study and investigate the underlying mechanism of decreasing miR-16 and miR-451 in AML patients with varied ABO antigen expression levels.

Analysis of Blood Group Discrepancy in Healthy Blood Donors at a Tertiary Care Referral Hospital from Eastern India: A Retrospective Study.

Objective ABO typing constitutes cell grouping and serum grouping. The discrepancy may arise in ABO typing due to a mismatch in cell grouping and serum grouping. It may be due to technical errors, missing or weak ABO antibodies (type I), weak ABO subgroups (type II), Rouleaux formation (type III), or other miscellaneous reasons (type IV). This study was carried out to determine the prevalence and cause of ABO blood group discrepancy in donor samples at our center. Methods A retrospective study of ABO blood group typing of blood donors was conducted at our center. The blood group typing was routinely performed using gel cards and a microcentrifuge system (Tulip Diagnostics(P) Ltd, Goa, India). If any discrepancy in ABO typing was noted, the test was repeated using the conventional tube technique. After sorting clerical/technical error, the causes of discrepancy were analyzed and resolved using anti-A 1 , anti-H, anti-AB, and other immunohematological tests like antibody screening and identification, saliva inhibition test, adsorption-elution studies. Results A total of 12,715 (98.6% males and 1.4% females) donor samples were tested. The number of ABO discrepancies detected were 15 (0.12%). The discrepancies were characterized as type I (6 cases; 40%), type II (1 case; 6.7%), type III (0 cases; 0%), and type IV (8 cases; 53.3%). Three cases, each of anti-M and anti-Le b , were detected in the study population. A single case of A 3 , a subgroup of A blood group, was found during the study. Conclusion The prevalence of ABO group discrepancy was 0.12% at our center. Discrepancy arising during ABO typing of blood donor must be resolved before reporting ABO blood group to minimize the recipient's chances of transfusion reaction. The serum grouping is equally crucial as cell grouping for reporting the ABO group of an individual.

A Novel Mutation Eliminates GATA-1 and RUNX1-Mediated Promoter Activity in Galactosyltransferase Gene

Introduction: Mutations in the promoter region and exons of ABO gene may cause changes in the expression of blood group antigens, often showing a weak ABO phenotype. Here, we identified a novel weak ABO subgroup allele that caused B_el phenotype and explored its mechanisms. Methods: The ABO phenotype of subjects (Chinese Han nationality) was classified by serological method. The plasma activity of erythrocyte glycosyltransferase was detected by the phosphate coupling method. ABO subtype genotyping was performed by PCR-SSP and exon sequencing. The activity of the promoter was evaluated by a dual-luciferase reporter assay. Results: We identified a mutation exon 1 c.15_16insTGTTG of the B allele in a B_el subject. Genealogical investigation showed that the mutation was inherited from her mother. The mutation was located in the promoter region of the ABO gene. The dual-luciferase reporter assay showed that the mutation inactivated GATA-1 and RUNX1-mediated activity of the ABO gene promoter, leading to a decrease in the expression and activity of B glycosyltransferase. Conclusion: A novel B_var ABO subgroup allele was identified. The novel mutation can reduce the promoter activity that activated by GATA-1 and RUNX1, subsequently causing the B_el phenotype.

Molecular basis of weak A subgroups in the Korean population: Identification of three novel subgroup-causing variants in the ABO regulatory regions.

Recent studies on Chinese and Japanese populations have shown that weak ABO subgroups could be caused by variants in the major regulatory regions of ABO, the proximal promoter, +5.8-kb site, and CCAAT-binding factor/NF-Y binding site. We investigated the molecular basis of weak A subgroups in the Korean population. This study included 11 samples suspected to have a weak A subgroup. These samples were subjected to sequencing analysis of ABO exons 6 and 7. If no subgroup-causing variants were detected in this region, exons 1-5 and three major regulatory regions were sequenced. Sequencing analysis of exons 6 and 7 detected two known subgroup alleles (ABO*AW.10, n=5; ABO*AEL.02, n=2). The remaining four samples contained a sequence variant in the proximal promoter (g.4944C>T, n=1; g.4954G>T, n=1) or +5.8-kb site (g.10843T>C, n=1; g.10935C>T, n=1). Notably, three of the four variants (g.4944C>T, g.4954G>T, and g.10843T>C) have not been reported previously in weak ABO subgroups. This study provides the first evidence that alterations in the proximal promoter and + 5.8-kb site could account for a substantial proportion of weak A subgroups in the Korean population.

The Potential Significance of ABO Genotyping for Donor Selection in Kidney Transplantation

BackgroundThe ABO blood group system is clinically important in kidney transplantation, but ABO genotyping fails to attract sufficient attention in some countries and regions. We identified one case of early graft dysfunction due to an ABO genotype mismatch. Here, we performed ABO genotyping in blood samples, analyzed grouping discrepancies, and investigated the weak A subgroup frequency in kidney transplantation candidates.MethodsBlood samples from 302 uremic patients with grouping discrepancies and 356 uremic patients with type A blood were analyzed using standard serologic serotyping techniques. The ABO genotypes and alleles were analyzed by polymerase chain reaction sequence-specific primer (PCR-SSP) and sequence-based typing (PCR-SBT).ResultsAll 302 uremic patients with grouping discrepancies carried weak ABO subgroup alleles and 77.48% carried irregular ABO antibodies. The discrepancy rate between serotyping and genotyping was 42.38%, and the mismatching rate of donor selection according to serotype reached 88.74%. And 2.53% of 356 uremic patients with type A blood were determined to be in the weak A subgroup, which was a higher percentage than that observed in the healthy Chinese population (0.53%) by serological screening, but much lower than that observed in Caucasians (20%).ConclusionWe revealed the high risk of blood type misjudgment and genetically ABO-mismatched transplantation if serological test was performed only in blood-group typing. Improved precision of ABO genotyping is crucial for successful kidney transplantation and reasonable organ allocation.

Preparation of Glutaraldehyde-Treated Red Blood Cells Expressing Weak ABO and RhD Antigens for Use in Tube Methods

Preparation of Glutaraldehyde-Treated Red Blood Cells Expressing Weak ABO and RhD Antigens for Use in Tube Methods

Two novel mutations p. L319V and p. L91P in ABO glycosyltransferases lead to Ael and Bel phenotypes.

Mutations of the ABO gene may cause the dysfunction of ABO glycosyltransferase (GT) that can result in weak ABO phenotypes. Here, we identified two novel weak ABO subgroup alleles and explored their mechanisms that caused Ael and Bel phenotypes. The ABO phenotyping and genotyping were performed by serological studies and direct DNA sequencing of the ABO gene. The role of the novel mutations were evaluated by a three-dimensional model, predicting protein structure changes, and in vitro expression assay. The total glycosyltransferase transfer capacity in supernatant of transfected cells was examined. We identified a mutation c. 955C>G (p. L319V) of A allele in an Ael subject and a mutation c. 272T>C (p. L91P) of B allele in a Bel subject. In silico analysis showed that the mutation p. L319V of the A allele and p. L91P of the B allele may change the local conformation of GT and impair the catalysis of H to A or B antigen conversion. In vitro expression study showed that mutation p. L319V impaired H to A antigen conversion, although it did not affect the expression of glycosyltransferase A. Two novel "el"-type ABO subgroup alleles were identified. Both of the two novel mutations can change the local conformation of GTs and reduce protein stability. GTA mutation p. L319V can impair the conversion from H to A antigen and causes the Ael phenotype.

ABO blood grouping discrepancies in the donor population

Background and ObjectivesABO discrepancies occur when the reactions in forward grouping are not corroborative to those in the reverse grouping due to weak subgroups of A and/or B, missing or weak ABO antibodies or unexpected alloantibodies. We aimed to determine the prevalence of ABO discrepancies in our donor population and to know the causes by using a standard classification.Materials and MethodsIt was a retrospective study on donor samples. For all the discrepant samples, the ABO and RhD grouping was repeated using tube technique. Adsorption–elution testing was done for detecting weak subgroups of A and B. Antibody screen (3‐cell) and identification (11‐cell) were done by gel technique (Bio‐Rad, Switzerland).ResultsWe detected 93 (0·064%) ABO discrepancies out of the total 144279 donor samples tested. The ABO discrepancies were due to weak anti‐B antibody (33/93; 35·5%), weak anti‐A antibody and weak subgroups of A (24 each; 25·8% each) and weak subgroups of B (5/93; 5·4%). Agglutination with O cells was observed in 7 (7·5%) samples at room temperature testing. The alloantibodies identified in these samples were anti‐M (5) and anti‐Lea in (1), while the remaining 1 sample was of ‘Bombay’ (Oh) phenotype. The autologous control was negative for all the samples.ConclusionThe prevalence of ABO discrepancies in our donor population is 0·064%.

Molecular genetic analysis of weak ABO subgroups in the Chinese population reveals ten novel ABO subgroup alleles.

A weak ABO subgroup is one of the most important causes of an ABO blood grouping discrepancy. Here, we investigated the distribution of weak ABO subgroups in the Chinese population and identified ten novel weak ABO subgroup alleles. We performed phenotype investigations by serological studies, analysed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated the role of glycosyltransferase mutations by in silico analysis and in vitro expression assay. Three hundred and fifty-one individuals with a weak ABO subgroup were detected among 1.45 million blood-typed subjects. Ten novel weak ABO subgroup alleles were identified. Molecular modelling and analysis of GTA mutation p.L339P suggested that the mutation may change the local conformation of GTA and reduce its stability. The in vitro expression assay showed that A antigen expression and agglutination of HeLa cells transfected with GTA mutant p.L339P decreased significantly compared to those of cells transfected with wild-type GTA. Ten novel weak ABO subgroup alleles were identified in the Chinese population. GTA mutant p.L339P may lead to a weak A phenotype by changing the local conformation of GTA and reducing its stability.

Comparison of Weak ABO Antigen and Normal ABO Antigen in Patients with Acute Leukemia

To compare the differences between weak ABO antigen patients and normal ABO antigen patients with acute leukemia, and to explore the clinical significance of weak ABO antigen in acute leukemia. The ABO blood group was detected in 110 newly diagnosed acute leukemia patients(including 68 cases of AML and 42 cases of ALL) and 68 normal controls. Then the leukemia subtype, age, sex, laboratory test, risk status of leukemia patients, and DNA methylation of ABO promoter were compared between patients with weak and normal ABO antigen. The weak ABO antigen was found in patients with newly diagnosed acute leukemia, and was not found in ALL patients or normal group. No statistical differences were found in the distribution of ABO blood group, age, hepatosplenomegaly, lymphadenovarix, plt, precursor cell clusters derived from bone marrow, immunopheno-typing, LDH level, and risk status between AL patients of weak and normal ABO antigen groups (P>0.05). Compared with patients in normal ABO antigen group, the pateins in weak ABO antigen group had higher percentage of male(77.8% vs 30%), lower WBC(32.26×109/L vs 82.69×109/L) and Hb level(64.00 g/L vs 85.94 g/L) and higher DNA methylation level (18.91% vs 10.76%) (P<0.05). The cases of weak ABO antigen frequently appear in the male AML patients, the DNA methylation level of ABO gene promoter in patients with weak ABO antigen is significantly higher than that in patients with normal ABO antigen.

The p.R168Q mutation is associated with the Bw phenotype and a predicted decrease in the stability of the resulting ABO glycosyltransferase.

Mutation of ABO glycosyltransferase (GT) can cause protein stability changes that can result in a weak ABO phenotype. To explain the Bw phenotype of a novel ABO*Bw allele, a protein stability of the mutant GT, which enhances the information of the three-dimensional (3D) structural analysis, was calculated. ABO serology and genotyping were performed on a neonate and her five family members. A 3D structural analysis of the wild-type GTB and enzymes with a variety of mutations at Residue 168, along with predicted protein stability changes (ΔΔG) and flow cytometric analysis of ABO antigen expression on HeLa cells transfected with plasmids containing R168Q, R168L, and R168P mutants was also performed. A novel ABO*Bw allele (c.503G>A, p.R168Q) was discovered. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTB, and the ΔΔG values, which inversely correlated with the mean relative fluorescence intensity of ABO antigen expression, quantitatively explained the reduced ABO antigen expression. The predicted protein stability change of a mutant GT enzyme might be a useful and convenient approach to objectively and quantitatively explain the reduced ABO antigen expression.