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Background and Objectives: The chimaerism phenomenon constitutes a significant mechanism underlying ABO phenotype discrepancies; however, its detection has technical challenges. In the current study, we explored different techniques to establish the chimaeric status of ABO blood types., Materials and Methods: Fifteen individuals with possible chimaeric ABO blood type, as suggested by standard tube or column agglutination method and RBC adsorption-elution test, were enrolled in the study. The red blood cells from 11 investigated subjects showed mix-field agglutination with anti-A or anti-B in blood typing; weak A or B antigens on the other four individuals' RBCs were detected by adsorption-elution tests. The genetic study was conducted with PCR-SSP genotype, DNA sequencing of the ABO gene, STR analysis and ddPCR., Results: The genetic chimaeric status was confirmed in four (27%) individuals by SSP test alone. The ABO gene sequencing identified an additional ABO allele and enabled chimaerism detection in 10 (67%) subjects. The STR analyses established the chimaerism status in 13 (87%) individuals. In the two cases where neither of the tests mentioned above had positive findings, the ddPCR was adopted, and microchimaerism, with an extremely low degree of chimaerism (0.77% and 0.12%), was revealed. The ddPCR revealed the unequal haplotypes (29.5% B vs. 70.5% O) in one subject and distinguished this B/O-O/O chimaera from certain B subgroups (B/O genotype without any mutation) like B 3 ., Conclusion: The ABO blood type chimaerism can be genetically established by comprehensive molecular methods, including PCR-SSP/DNA sequencing, STR and ddPCR, which is particularly sensitive for the detection of microchimaerism., (© 2022 International Society of Blood Transfusion.)

Background: 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 A el and B el phenotypes., Materials and Methods: 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., Results: We identified a mutation c. 955C>G (p. L319V) of A allele in an A el subject and a mutation c. 272T>C (p. L91P) of B allele in a B el 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., Conclusions: 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 A el phenotype.

Objective: To explore the molecular basis for an A subtype of the ABO blood group., Methods: The forward and reverse typing of the ABO blood group were identified by gel card and test tube methods. The ABO gene of the patient was detected by PCR-sequence specific primer (PCR-SSP). Exons 1 to 7 of the ABO gene was amplified by PCR and sequenced. The ABO gene was also subjected to subclone sequencing for haplotype analysis., Results: The patient's red cells showed weak agglutination with anti-A but non-agglutination with anti-B. The patient's serum showed 1+ agglutination with A cells and 4+ agglutination with B cells. Based on above serological characteristics, the patient was defined as Aw subtype of the ABO blood group. Sequencing analysis showed that the patient was heterozygous for c.106G>T, c.188G>A, c.189C>T, c.220C>T, c.297A>G, c.467C>T, c.543G>C, c.646T>A, c.681G>A, c.771C>T, c.829G>A, in addition with a c.261G deletion. Combined with the result of subclone sequencing, the ABO genotype of the patient was determined as ABO*AW.33. new/O.01.02, which harbored c.467C>T and c.543G>C variants compared with ABO*A1.01 and c.543G>C variant compared with ABO*A1.02. The novel allele has been submitted to GenBank with an accession number of MK302122., Conclusion: A novel allele of Aw33 subtype has been identified with its GTA transferase gene harboring c.467C>T and c.543G>C variants compared with A1.01.

Background: 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., Material and Methods: 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., Results: 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., Conclusion: 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.

Background: The amino acid substitutions caused by ABO gene mutations are usually predicted to impact glycosyltransferase's function or its biosynthesis. Here we report an ABO exonic missense mutation that affects B-antigen expression by decreasing the mRNA level of the ABO gene rather than the amino acid change., Study Design and Methods: Serologic studies including plasma total GTB transfer capacity were performed. The exon sequences of the ABO gene were analyzed by Sanger sequencing. B 310 cDNA with c.28G>A (p.G10R) mutation was expressed in HeLa cells and total GTB transfer capacity in cell supernatant was measured. Flow cytometry was performed on these HeLa cells after transfection, and agglutination of Hela-B weak cells was also examined. The mRNA of the ABO gene was analyzed by direct sequencing and real-time reverse transcriptase-polymerase chain reaction. A minigene construct was prepared to evaluate the potential of splicing., Results: While plasma total GTB transfer capacity was undetectable in this B 3 -like individual, the relative percentage of antigen-expressing cells and mean fluorescence index of the B weak red blood cells (RBCs) were 19 and 14% of normal B RBCs, respectively. There was no significant difference of total GTB transfer capacity in cell supernatant and B-antigen expression on cell surfaces between HeLa cells transfected with B 310 cDNA and B cDNA. The mRNA expression level of B 310 in peripheral whole blood was significantly reduced. The amount of splicing is significantly lower in c.28G>A construct compared to that in wild-type construct after transfection in K562 cells., Conclusion: ABO c.28G>A mutation may cause B 3 -like subgroup by affecting RNA splicing of the ABO gene., (© 2017 AABB.)

Background: Identifying genetic variants of the ABO gene may reveal new biologic mechanisms underlying variant phenotypes of the ABO blood group. We report the molecular genetic analysis of 322 apparently unrelated ABO subgroup individuals in an estimated 2.1 million donors., Study Design and Methods: We performed phenotype investigations by serology studies, analyzed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated promoter activity by reporter assays., Results: In 62 rare ABO alleles, we identified 29 novel ABO subgroup alleles in 43 apparently unrelated subgroup individuals and their four available pedigrees. Of these alleles, one was a deletion-mutation allele, four were hybrid alleles, and 24 were point-mutation alleles. Most of the point mutations were detected in Exons 6 to 7, while several others were also detected in Exons 1 to 5 or splicing regions. One ABO promoter mutation, -35 to -18 del, was found and verified to reduce promoter activity, as determined by dual luciferase assays. Two mutations, 7G>T and 52C>T, carrying the premature terminal codons E3X and R18X in the 5'-region, were found to be associated with the very weak ABO subgroups "Ael" and "Bel.", Conclusion: Twenty-nine ABO subgroup alleles were newly linked to different kinds of ABO variations. We provide the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes. We also described the first naturally occurring ABO alleles with premature terminal codons in the 5'-region that led to Ael and Bel phenotypes., (© 2013 American Association of Blood Banks.)