ABO exon and intron analysis in individuals with the A(weak)B phenotype reveals a novel O(1v)-A(2 )hybrid allele that causes four missense mutations in the A transferase

BACKGROUND: Since the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood-group ABO locus, polymorphisms due to ethnic and/or phenotypic variations have been reported. Some subgroups have been explained at the molecular level, but unresolved samples are frequently encountered in the reference laboratory. RESULTS: ABO blood grouping discrepancies were investigated serologically and by ABO genotyping [duplex polymerase-chain-reaction (PCR) – restriction-fragment-length-polymorphism (RFLP) and PCR – allele-specific-primer (ASP) across intron 6] and DNA sequencing of the ABO gene and its proposed regulatory elements. Blood samples from five individuals living in Portugal, Switzerland, Sweden and the USA were analysed. These individuals were confirmed to be of Black ethnic origin and had the unusual A(weak)B phenotype but appeared to have the A(2)B genotype without previously reported mutations associated with weak A or B expression. Sequencing of this A allele (having 467C>T and 1061delC associated with the common A(2 )[A201] allele) revealed three mutations regularly encountered in the O(1v )[O02] allele: 106C>T (Val36Phe), 188G>A (Arg63His), 220C>T (Pro74Ser) in exons 3, 4 and 5, respectively. The additional presence of 46G>A (Ala16Thr) was noted, whilst 189C>T that normally accompanies 188G>A in O(1v )was missing, as were all O(1v)-related mutations in exons 6 and 7 (261delG, 297A>G, 646T>A, 681G>A, 771C>T and 829G>A). On screening other samples, 46G>A was absent, but two new O alleles were found, a Jordanian O(1 )and an African O(1v )allele having 188G>A but lacking 189C>T. Sequencing of introns 2, 3, 4 and 5 in common alleles (A(1 )[A101], A(2), B [B101], O(1), O(1v)and O(2 )[O03]) revealed 7, 12, 17 and 8 polymorphic positions, respectively, suggesting that alleles could be defined by intronic sequences. These polymorphic sites allowed definition of a breakpoint in intron 5 where the O(1v)-related sequence was fused with A(2 )to form the new hybrid. Intron 6 has previously been sequenced. Four new mutations were detected in the hybrid allele and these were subsequently also found in intron 6 of A(2 )alleles in other Black African samples. CONCLUSIONS: A novel O(1v)-A(2 )hybrid was defined by ABO exon/intron analysis in five unrelated individuals of African descent with the A(weak)B blood group phenotype

Genetic Characterisation of Human ABO Blood Group Variants with a Focus on Subgroups and Hybrid Alleles

ABO is the most important blood group system in transfusion medicine and transplantation immunology. The ABO blood groups differ by the presence or absence of antigens on RBCs and antibodies in plasma. Accurate determination of ABO status is critical. Genomic typing can increase the precision of blood group determination in complicated cases, e.g. when variant expression of A or B antigen is encountered. The overall aim of this study was to compare the molecular diversity of ABO alleles with various phenotypes, and to contribute to our knowledge of the ABO gene and encoded glycosyltransferases. Novel alleles (six Aweak, eleven Bweak, seven O) were identified containing single-point mutations. Structure/function studies explained the weakening of some B subgroup glycosyltransferases. Two new hybrid Ax alleles were characterised. Analysis of introns 2-5 revealed 44 previously unknown, allele-related polymorphisms that proved valuable allelic markers. These findings enabled localisation of cross-over regions in two other new hybrids: 1) an O1v allele fused with an A2 allele, 2) the novel O1bantu-A2 combination that explained the Abantu phenotype. Phylogenetic and population analyses indicated that O1bantu is a unique and distinct evolutionary lineage so far only found among individuals of African descent. Of clinical importance, a new approach to ABO genotyping was developed that identifies all common alleles, most null and weak A/B subgroups as well as hybrid alleles resulting from recombinational crossing-over events. In summary, 30 novel alleles were identified and characterized, representing 30% of all alleles reported since the start of this study in 2001

Blood grouping discrepancies between ABO genotype and phenotype caused by O alleles

Purpose of review In the modern transfusion service, analysis of the ABO allele underlying a donor or recipient's A or B subtype phenotype is becoming a mainstream adjunct to the serological investigation. Although an analysis of the ABO gene can be helpful in establishing the nature of the subtype phenotype, numerous confounding factors exist that can lead to a discrepancy between the genotype and the observed phenotype. Recent findings Although the most common group O alleles share a common crippling polymorphism, a growing number of alleles feature other polymorphisms that render their protein nonfunctional yet are similar enough to the consensus A allele that an errant phenotype would be predicted from the genotype, if the genotyping method was not specifically designed for their detection. Some of these O alleles might actually encode a protein with weak and variable A antigen synthetic ability. Summary ABO genotyping can be a powerful asset in the transfusion service, but a thorough knowledge of the confounding factors that can lead to genotype/phenotype discrepancies is required

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

Background: ABO genotyping is complicated by the remarkable diversity at the ABO locus. Recombination or gene conversion between common alleles may lead to hybrids resulting in unexpected ABO phenotypes. Furthermore, numerous mutations associated with weak subgroups and nondeletional null alleles should be considered. All known ABO genotyping methods, however, risk incorrect phenotype predictions if any such alleles are present. Study Design and Methods: An extensive set of allele-specific primers was designed to accomplish hybrid-proof multiplex polymerase chain reaction (PCR) amplification of DNA fragments for detection of ABO alleles. Results were compared with serologic findings and ABO genotypes defined by previously published PCR-restriction fragment length polymorphism/PCR-allele-specific polymorphism (ASP) methods or DNA sequencing. Results: Phenotypically well-characterized samples from blood donors with common blood groups and rare-subgroup families were analyzed. In addition to the commonly encountered alleles (A(1), A(1(467C > T)), A(2), B, O-1, O-1v, and O-2), the new method can detect hybrid alleles thanks to long-range amplification across intron 6. Four of 12 PCR-ASP procedures are used to screen for multiple infrequent subgroup and null alleles. This concept allows for a low-resolution typing format in which the presence of, for example, a weak subgroup or cis-AB/B(A) is indicated but not further defined. In an optional high-resolution step, more detailed genotype information is obtained. Conclusion: A new genotyping approach has been developed and evaluated that can correctly identify ABO alleles including nondeletional null alleles, subgroups, and hybrids resulting from recombinational crossing-over events between exons 6 and 7. This approach is clinically applicable and decreases the risk for erroneous ABO phenotype prediction compared to previously published methods

<it>ABO </it>exon and intron analysis in individuals with the A_weakB phenotype reveals a novel <it>O</it>^1<it>v</it>-<it>A</it>²hybrid allele that causes four missense mutations in the A transferase

Abstract Background Since the cloning in 1990 of cDNA corresponding to mRNA transcribed at the blood-group ABO locus, polymorphisms due to ethnic and/or phenotypic variations have been reported. Some subgroups have been explained at the molecular level, but unresolved samples are frequently encountered in the reference laboratory. Results ABO blood grouping discrepancies were investigated serologically and by ABO genotyping [duplex polymerase-chain-reaction (PCR) – restriction-fragment-length-polymorphism (RFLP) and PCR – allele-specific-primer (ASP) across intron 6] and DNA sequencing of the ABO gene and its proposed regulatory elements. Blood samples from five individuals living in Portugal, Switzerland, Sweden and the USA were analysed. These individuals were confirmed to be of Black ethnic origin and had the unusual AweakB phenotype but appeared to have the A2B genotype without previously reported mutations associated with weak A or B expression. Sequencing of this A allele (having 467C>T and 1061delC associated with the common A2 [A201] allele) revealed three mutations regularly encountered in the O1v [O02] allele: 106C>T (Val36Phe), 188G>A (Arg63His), 220C>T (Pro74Ser) in exons 3, 4 and 5, respectively. The additional presence of 46G>A (Ala16Thr) was noted, whilst 189C>T that normally accompanies 188G>A in O1v was missing, as were all O1v-related mutations in exons 6 and 7 (261delG, 297A>G, 646T>A, 681G>A, 771C>T and 829G>A). On screening other samples, 46G>A was absent, but two new O alleles were found, a Jordanian O1 and an African O1v allele having 188G>A but lacking 189C>T. Sequencing of introns 2, 3, 4 and 5 in common alleles (A1 [A101], A2, B [B101], O1, O1vand O2 [O03]) revealed 7, 12, 17 and 8 polymorphic positions, respectively, suggesting that alleles could be defined by intronic sequences. These polymorphic sites allowed definition of a breakpoint in intron 5 where the O1v-related sequence was fused with A2 to form the new hybrid. Intron 6 has previously been sequenced. Four new mutations were detected in the hybrid allele and these were subsequently also found in intron 6 of A2 alleles in other Black African samples. Conclusions A novel O1v-A2 hybrid was defined by ABO exon/intron analysis in five unrelated individuals of African descent with the AweakB blood group phenotype.</p

Investigation into A antigen expression on O-2 heterozygous group O-labeled red blood cell units

BACKGROUND: There are two principal types of group O alleles; deletional alleles feature 261delG leading to nonfunctional truncated protein. Nondeletional alleles have the consensus guanosine at residue 261. The major nondeletional allele, O-2, encodes full-length protein with Gly268Arg. While reports vary, O-2 has been proposed to encode weakly functional A-glycosyltransferase (GTA). The main objective of this study was to evaluate if GTA activity is detectable in O-2 donors. STUDY DESIGN AND METHODS: Donor samples from Pittsburgh and Lund were ABO typed by automated methods. DNA was extracted from 779 group O donors whose red blood cells (RBCs) were available for transfusion. ABO genotyping identified those with O-2 alleles. The following tests were performed on randomly selected O-2 samples (number): adsorption-elution with anti-A (3), flow cytometry (15), plasma enzyme activity (4), and attempts to convert group O RBCs to A (2) with O-2 plasma and titration of plasma anti-A/-A(1) (3). RESULTS: Forty O-2-heterozygous donors were identified (5.1%). Adsorption-elution and sensitive flow cytometry did not reveal A antigens on O-2 RBCs. Plasma enzyme analysis failed to show GTA activity above baseline; O-2 plasma was unable to add measurable A antigens to O RBCs. Titers of anti-A/-A(1) appeared reduced in O-2 plasma but did not cause ABO typing discrepancies. No immediate hemolysis or adverse reactions were reported following transfusion of O-2 RBCs to six evaluable group O recipients. CONCLUSIONS: Other than lower plasma anti-A titers, GTA activity was not found in these O-2 samples. Neither automated blood grouping discrepancies nor clinical problems related to transfusing these O-2 units were observed