ABO Subgroups Research Articles

Novel glycolipid variations revealed by monoclonal antibody immunochemical analysis of weak ABO subgroups of A

The chemical basis of the subgroups of A is largely unknown. We used thin-layer chromatography immunochemical staining techniques together with a range of characterized monoclonal reagents to analyse glycolipids isolated from a variety of weak subgroups. Glycolipids isolated from red cells collected from nine genetically defined individuals of the rare subgroups of A, including a novel A(3) allele (A(2) 539G>A) not described previously, were subjected to a highly sensitive thin-layer chromatographic immunochemical analysis. Semicharacterized monoclonal antibodies revealed that, in addition to the expected quantitative differences between common phenotypes and the weak subgroups, qualitative glycolipid differences (or at least an apparent qualitative basis), caused by major changes in the ratios of different structures exist. Specifically it was found that the weakest A-expressing samples (A(el) phenotype) appeared to express an unusual A structure in the 8-12 sugar region. Variable expression of several structures in one of the A weak samples were suggestive of novel blood group A structures. Although no structural characterization could be undertaken, the results are clearly indicative that the variant glycosyltransferases of the rare ABO subgroups are not only inefficient, but they may potentially synthesize novel ABO structures.

The Abantu phenotype in the ABO blood group system is due to a splice‐site mutation in a hybrid between a new O1‐like allelic lineage and the A2 allele

Many phenotypic variations in the expression of blood group A have been explained by variations in gene structure, but unresolved samples are frequently encountered in the reference laboratory. Among ABO subgroups, A(bantu) has the highest frequency in a specified population. The molecular basis of this phenotype is now described. Blood from Black donors phenotyped as A(bantu) was subjected to genomic ABO screening and direct sequencing of polymerase chain reaction (PCR)-amplified ABO exons 1-7 and introns 2-6. Total RNA was extracted and ABO cDNA was synthesized by reverse transcription (RT)-PCR. Control material comprised Black South African, Swedish, Jordanian and Brazilian blood samples with common phenotypes. Genomic ABO typing indicated the presence of an A(2) allele in each A(bantu) donor, in combination with an O allele. No previously reported mutations associated with weak A or B expression were found. Direct sequencing indicated the common A(2) sequence with a single nucleotide deletion (AGGT>AGT) at the exon 4/intron 4 junction, predicted either to disrupt the reading frame (resulting in a premature stop codon) or to cause erroneous splicing (resulting in the exclusion of exon 4 from the mRNA). O mRNA, but no transcripts from the A(bantu) allele, could be detected. Surprisingly, the splice-site mutation was also found in approximately 5% of O alleles in Black South Africans, but not in other blood donors, or in non-O(1) alleles. Utilizing intron polymorphisms, the A(bantu) allele was shown to be a recombination between a new allelic lineage (O(1bantu)) and A(2), with a cross-over region near exon 5. The A(bantu) phenotype is caused by an O(1bantu)-A(2) hybrid at the ABO locus.

Importance of Weak ABO Subgroups

Importance of Weak ABO Subgroups

Importance of Weak ABO Subgroups

Importance of Weak ABO Subgroups

High Frequency of Unusual ABO Alleles in Patients with Leukemia.

Background: Mutations critical for ABO group phenotypes which encode the catalytic domain of ABO glycosyltransferases have been studied in healthy blood donors. Weakening of A and B transferases have been reported in patients with leukemia, particularly those in which the myeloid lineage is involved. The purpose of this study was to use sequence specific PCR (PCR-SSP) to perform ABO genotyping of leukemic patients.Material and Methods: A total of 108 unrelated leukemic patients were initially classified serologically using tube test technique and gel test for ABO typing and subgroup detection. Genomic DNA was prepared from peripheral blood cells. All samples were then screened by 32 different PCR-SSP, each specific for a single nucleotide variation to screen 16 known polymorfic sites of exons 6 and 7 (modified from Seltsam, Transfusion 2003,43(4):428–39.Results: The results of PCR screening were conclusive and consistent with the ABO phenotypes in 71 patients (65.07%). Common ABO alleles were found in only 15 (13.8%) of patients studied. Unusual ABO alleles were found in 26 of 28 blood group O patients (ABO*O05, ABO*O21, ABO*O30, ABO*O33, ABO*O35, ABO*O36, ABO*O43 and ABO*O45), in 20 of 43 blood group A patients (ABO*A103, ABO*A104, ABO*A106, ABO*A202, ABO*A203), in 8 of 10 blood group B patients (ABO*B104, ABO*B105) and in 2 of 4 AB blood group patients (ABO*Bx01, ABO*B103). In 37 patients (34.2%), PCR screening revealed allele combinations that were incompatible with known ABO allele combinations or subgroups predicted by serologic analysis.Conclusion: This is the first report of using PCR-SSP for ABO genotyping of leukemic patients. The pathophysiologic role of unusual ABO allele combinations in patients with leukemia warrants further investigation.

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.

Transfusion-related problems following major ABO-incompatible bone marrow transplantation

A case of acute myelocytic leukemia has been reported in which the patient’s surviving original B lymphocytes after pretransplant-conditioning chemotherapy probably reproduced hemagglutinins that reacted with red blood cells (RBCs) derived from engrafted donor marrow for a prolonged period of time. Although the direct antiglobulin test was negative and hemagglutinins were not detectable in the patient’s sera but only in the eluate, the antibodies reappeared in the sera. Therefore, it is important to confirm that the eluate does not contain antibodies that would react with donor-derived RBCs when the type of red cell used for transfusion is switched from the patient’s type to the donor’s type in a major ABO-mismatched bone marrow transplantation (BMT). Testing of ABO subgroups using lectins is also recommended to avoid a delayed hemolytic transfusion reaction following BMT.

Aspectos moleculares do Sistema Sangüíneo ABO

O sistema ABO é o mais importante grupo sangüíneo na medicina transfusional. O gene ABO codifica as glicosiltransferases responsáveis pela transferência dos resíduos específicos de açúcar, GalNaca1-3 e Gala 1-3, ao substrato H e os convertem ao antígeno A ou B respectivamente. A estrutura do DNA dos três principais alelos do sistema ABO, A¹, B e O foi primeiramente descrita em 1990. Os avanços da genética molecular permitiram o entendimento da base molecular dos genes ABO e o conhecimento do polimorfismo dos alelos comuns a esse locus. Essa revisão tem como objetivo o estudo dos alelos variantes desse sistema, assim como a compreensão das mutações, deleções ou rearranjo de genes responsáveis pela ocorrência de alguns dos subgrupos do sistema ABO. As técnicas mais comumente utilizadas para a genotipagem ABO também são avaliadas, bem como suas vantagens e limitações.

Sequence variation at the human ABO locus.

The ABO blood group is the most important blood group system in transfusion medicine. Since the ABO gene was cloned and the molecular basis of the three major alleles delineated about 10 years ago, the gene has increasingly been examined by a variety of DNA-based genotyping methods and analysed in detail by DNA sequencing. A few coherent observations emerge from these studies. First, there is extensive sequence heterogeneity underlying the major ABO alleles that produce normal blood groups A, B, AB and O when in correct combination with other alleles. Second, there is also extensive heterogeneity underlying the molecular basis of various alleles producing ABO subgroups such as A2, Ax and B3. There are over 70 ABO alleles reported to date and these alleles highlight the extensive sequence variation in the coding region of the gene. A unifying system of nomenclature is proposed to name these alleles. Third, extensive sequence variation is also found in the non-coding region of the gene, including variation in minisatellite repeats in the 5' untranslated region (UTR), 21 single nucleotide polymorphisms (SNPs) in intron 6 and one SNP in the 3' UTR. The haplotypes of these variations reveal a specific relationship with the major ABO alleles. Fourth, excluding the common alleles, about half of the remaining alleles are due to new mutations and the other half can better be explained by intragenic recombination (both crossover and gene conversion) between common alleles. In particular, the recombination sites in hybrid alleles can be quite precisely defined through haplotype analysis of the SNPs in intron 6. This indicates that recombination is equally as important as point mutations in generating the genetic diversity of the ABO locus. Finally, a large number of ABO genotyping methods are available and are based on restriction analysis, allele specific amplification, mutation screening techniques or their combinations.

Genomic analysis of clinical samples with serologic ABO blood grouping discrepancies: identification of 15 novel A and B subgroup alleles

Since the cloning in 1990 of complementary DNA corresponding to messenger RNA transcribed at the blood group ABO locus, polymorphisms and phenotype-genotype correlations have been reported by several investigators. Exons 6 and 7, constituting 77% of the gene, have been analyzed previously in samples with variant phenotypes but for many subgroups the molecular basis remains unknown. This study analyzed 324 blood samples involved in ABO grouping discrepancies and determined their ABO genotype. Samples from individuals found to have known subgroup alleles (n = 53), acquired ABO phenotypes associated with different medical conditions (n = 65), probable chimerism (n = 3), and common red blood cell phenotypes (n = 109) were evaluated by ABO genotype screening only. Other samples (n = 94) from apparently healthy donors with weak expression of A or B antigens were considered potential subgroup samples without known molecular background. The full coding region (exons 1-7) and 2 proposed regulatory regions of the ABO gene were sequenced in selected A (n = 22) or B (n = 12) subgroup samples. Fifteen novel ABO subgroup alleles were identified, 2 of which are the first examples of mutations outside exon 7 associated with weak subgroups. Each allele was characterized by a missense or nonsense mutation for which screening by allele-specific primer polymerase chain reaction was performed. The novel mutations were encountered in 28 of the remaining 60 A and B subgroup samples but not among normal donors. As a result of this study, the number of definable alleles associated with weak ABO subgroups has increased from the 14 previously published to 29.

Evidence for a new type of O allele at the ABO locus, due to a combination of the A2 nucleotide deletion and the Ael nucleotide insertion.

Using a recently introduced multiplex polymerase chain reaction and restriction fragment length polymorphism ABO genotype screening method we have found an anomalous ABO genotype (A2O1variant) not correlating with the serological phenotype (blood group O). The blood group was confirmed by absorption/elution and detection of blood group substances in saliva. Sequencing of exons 6 and 7 in the ABO genes of the propositus indicated an A2 gene (C467T and C1060-) apparently inactivated by the same single nucleotide insertion recently reported in individuals with the ABO subgroup Ael. Investigation of relatives confirmed the inheritance of this new inactive hybrid allele.

Evidence for a New Type of O Allele at the ABO Locus, due to a Combination of the A^2 Nucleotide Deletion and the A^el Nucleotide Insertion

Using a recently introduced multiplex polymerase chain reaction and restriction fragment length polymorphism ABO genotype screening method we have found an anomalous ABO genotype (A^2O^1variant) not correlating with the serological phenotype (blood group O).The blood group was confirmed by absorption/elution and detection of blood group substances in saliva. Sequencing of exons 6 and 7 in the ABO genes of the propositus indicated an A^2 gene (C467T and C1060-) apparently inactivated by the same single nucleotide insertion recently reported in individuals with the ABO subgroup A(cl). Investigation of relatives confirmed the inheritance of this new inactive hybrid allele.

Successful Transplantation of Blood Group A2 Kidneys Into Non-A Recipients

The ABO subgroup A2 has been reported to be less reactive with the anti-A1 antibody naturally occurring in the serum of group O and B recipients and to occur in approximately 20% of group A individuals. Between March 1986 and February 1987, the Midwest Organ Bank (MOB) in Kansas City, screened all group A renal donors for the A2 subgroup. A total of 190 cadaverdonor kidneys were retrieved during this time, of which 68 were subgroup A1 and 16 were subgroup A2 (incidence of A2 = 19% of As and 8.5% of all donors). Of the subgroup A2 kidneys, 13 were transplanted into 9 group O and 4 group B recipients. One group O recipient received an HLA-identical A2 living-related graft. Recipients were not preselected or modified by splenectomy, plasmapheresis, or other means, and were treated with cyclosporine, steroids--and, in most cases, azathioprine, after transplantation. There was one hyperacute rejection and there were 5 acute cellular rejection episodes, 3 of which were reversed. One additional patient died at 2.5 months with a functioning graft. Including the successful living-related graft, 10 of 14 patients (71%) have functioning grafts, with a follow-up of 5 to 14 months, and a mean creatinine of 1.7 mg/dl. We find that the A2 subgroup represents a small but important minority of A donors, and that transplantation into non-A recipients can generally, but not universally, be safely accomplished. We recommend the screening of A donors for the A2 subgroup in both the cadaver-donor and living-related groups, and suggest that the utilization of A2 donors in non-A patients may contribute to the transplantation of group O and highly sensitized patients--and, in some cases, improve the degree of HLA matching.

Successful transplantation of blood group A2 kidneys into non-A recipients.

The ABO subgroup A2 has been reported to be less reactive with the anti-A1 antibody naturally occurring in the serum of group O and B recipients and to occur in approximately 20% of group A individuals. Between March 1986 and February 1987, the Midwest Organ Bank (MOB) in Kansas City, screened all group A renal donors for the A2 subgroup. A total of 190 cadaverdonor kidneys were retrieved during this time, of which 68 were subgroup A1 and 16 were subgroup A2 (incidence of A2 = 19% of As and 8.5% of all donors). Of the subgroup A2 kidneys, 13 were transplanted into 9 group O and 4 group B recipients. One group O recipient received an HLA-identical A2 living-related graft. Recipients were not preselected or modified by splenectomy, plasmapheresis, or other means, and were treated with cyclosporine, steroids--and, in most cases, azathioprine, after transplantation. There was one hyperacute rejection and there were 5 acute cellular rejection episodes, 3 of which were reversed. One additional patient died at 2.5 months with a functioning graft. Including the successful living-related graft, 10 of 14 patients (71%) have functioning grafts, with a follow-up of 5 to 14 months, and a mean creatinine of 1.7 mg/dl. We find that the A2 subgroup represents a small but important minority of A donors, and that transplantation into non-A recipients can generally, but not universally, be safely accomplished. We recommend the screening of A donors for the A2 subgroup in both the cadaver-donor and living-related groups, and suggest that the utilization of A2 donors in non-A patients may contribute to the transplantation of group O and highly sensitized patients--and, in some cases, improve the degree of HLA matching.

Heterogeneity of the ABO subgroups among Chinese in Taiwan

Heterogeneity of the ABO subgroups among Chinese in Taiwan

Use of the Fluorescent Antibody Technic to Identifying Weak Subgroups of Erythrocytes

Erythrocytes, which appeared to belong to a weak A subgroup, were studied by the indirect immunofluorescence technic utilizing anti-A typing serum and fluorescein-conjugated anti-IgG/IgM. Rare, single fluorescing cells, similar to Ax control cells, were observed in preparations of erythrocytes from three members of the family studied, the father and two sons. The mode of inheritance of the weak A (Aw) gene, presumably allelic at the ABO locus, was elucidated by direct observation of fluorescent patterns in erythrocytes of family members. This technic can be useful in detecting weak subgroups of the ABO blood group system. However, it is not possible to classify these weak subgroups into specific categories, e.g., A3, Am, or Ax, using this technic, since they do not segregate into well-defined subgroups but show considerable degree of overlap. The fluorescent antibody technic, as opposed to the absorption and elution methods commonly used to identify weak subgroups, allows direct observation of the antigens present on erythrocytes. Furthermore, saliva studies used for identifying ABO subgroups can be employed only for those individuals who secrete soluble blood-group substances. Since the fluorescent antibody technic is not dependent on secretor status, it is valuable for investigating weak subgroups in these persons.

Blood group differentiation using fluorescent antibodies.

SummaryHuman antisera against isologous blood groups have been combined with fluorescent chemicals and used in the study of red cell-antiserum reactions. The decrease in fluorescence of the antiserum measured after addition of red cells is used as an indication of the amount of antibody combining with the cells. By this technic, cells have been differentiated by ABO group, subgroup and Rh type.