cis-AB Allele Research Articles

A novel cisAB allele with a missense variant (c.971T>C) in the ABO gene of a Brazilian family.

Missense variants in exon 7 of the ABO gene can lead to the formation of cisAB alleles. These alleles encode glycosyltransferases (GTs) capable of synthesizing both A and B antigens. In this study, we report the discovery of a novel cisAB allele and characterize it at molecular, protein and serological levels. Blood and DNA samples from the proband and seven relatives were examined using standard and modified ABO phenotyping, polymerase chain reaction-restriction fragment length polymorphism and ABO gene sequencing. We assessed the impact of the p.Leu324Ser variant on the protein structure of the mutant GT using bioinformatics tools. Molecular tests revealed a c.971T>C (p.Leu324Ser) variant in the ABO gene in five of the eight individuals. This variant results in a GT that produces more A antigens and fewer B antigens. Bioinformatics analysis suggests that the amino acid substitution (p.Leu324Ser) could potentially affect enzymatic activity and specificity of the GT. We identified a novel cisAB allele resulting from a c.971T>C variant in the ABO gene. This variant led to the expression of an ABweak phenotype.

Analysis of ABO grouping discrepancies among patients from a tertiary hospital in Korea

BackgroundAccurate ABO typing is essential for preventing ABO incompatibility reactions. However, the causes of ABO grouping discrepancy has not been sufficiently studied, and it may vary among different ethnic populations. Thus, the aim of this retrospective study was to investigate the causes of ABO discrepancy in the East Asian population. Materials and methodsA retrospective observational study on ABO typing discrepancy among patients in a tertiary hospital was carried out using the electronic medical record database of Samsung Medical Center (Seoul, Korea) between July 2016 and May 2019. ResultsABO grouping was performed on 551,959 blood samples during the study period; 1468 events of serologic ABO discrepancy were determined from 1334 (0.24 %) samples. A total of 134 samples (0.02 %) presented multiple causes of ABO discrepancy. Weak/missing serum reactivity (594, 40.5 %) was the most frequent reason for ABO discrepancy, followed by extra serum reactivity (370, 25.2 %), weak/missing red cell reactivity (267, 18.2 %), mixed-field red cell reactivity (176, 12.0 %), and extra red cell reactivity (61, 4.2 %). In the category of weak/missing red cell reactivity, ABO subgroup was the most common reason, and using ABO genotyping, 26.2 % of the cases genotyped were found to be related to the cis-AB allele. ConclusionsOur results suggest that the incidence and cause of ABO typing discrepancies vary among institutes and ethnic groups. Our data helps to better understand and facilitate the resolution of ABO typing discrepancies in patients.

A Novel c.796 A>C Mutation in the ABO*B.01 Allele Responsible for CisAB Phenotype

Background: Individuals with the CisAB phenotype are rare in the Chinese population. In the present study, we investigated the sequence of the ABO gene and family members of a newborn suspected to have the CisAB phenotype. Methods: The ABO phenotype was detected using conventional serological tests. The full coding region of exons 1 to 7 of the ABO gene was amplified by polymerase chain reaction and was sequenced. The ABO haplotype was determined by the allele-specific primer sequencing method. Results: The proband and his father and grandfather were assigned the CisAB phenotype according to the results of the serological tests and family investigation. A novel CisAB allele was identified in the proband and his father and grandfather, which has only one nucleotide difference at position 796 from A to C (c.796A>C) compared with the ABO*B.01 allele. Conclusion: A novel CisAB (c.796A>C mutation in ABO*B.01) allele is the first identified in the Chinese population.

Molecular Basis of ABO Variants Including Identification of 16 Novel ABO Subgroup Alleles in Chinese Han Population

Introduction: The characteristic of ABO blood subgroup is crucial for elucidating the mechanisms of such variant phenotypes and offering useful information in blood transfusion. Methods: In total, 211 ABO variants including part of available family members were investigated in this study. The phenotypes of these individuals were typed with serologic methods. The full coding regions of ABO gene and the erythroid cell-specific regulatory elements in intron 1 of them were amplified with polymerase chain reaction and then directly sequenced. The novel alleles were confirmed by cloning and sequencing. Phylogenetic tree was made using CLUSTAL W software. 3D structural analyses of the glycosyltransferases (GTs) with some typical mutations were performed by PyMOL software. Results: Forty-eight distinctly rare ABO alleles were identified in 211 Chinese variant individuals, including 16 novel ABO alleles. All of the alleles were categorized as 5 groups: 16 ABO*A alleles, 23 ABO*B alleles, 4 ABO*BA alleles, 4 ABO*cisAB alleles, and 1 ABO*O alleles. ABO*A2.08 and ABO*BA.02 were the relatively predominant A and B subgroup alleles, respectively. According to the phylogenetic tree, 28 alleles (5 common alleles and 23 alleles identified in our laboratory) were classified into 3 major allelic lineages. The structural analysis of 3D homology modeling predicted reduced protein stability of the mutant GTs and may explain the reduced ABO antigen expression. Conclusions: The molecular basis of ABO variants was analyzed, and 16 novel ABO alleles were identified. The results extended the information of ABO variants and provided a basis for better transfusion strategies and helped to improve blood transfusion safety.

ABO Mistyping of cis-AB Blood Group by the Automated Microplate Technique

Background: The cis-AB phenotype, although rare, is the relatively most frequent of ABO subgroups in Koreans. To prevent ABO mistyping of cis-AB samples, our hospital has applied a combination of the manual tile method with automated devices. Herein, we report cases of ABO mistyping detected by the combination testing system. Methods: Cases that showed discrepant results by automated devices and the manual tile method were evaluated. These samples were also tested by the standard tube method. The automated devices used in this study were a QWALYS-3 and Galileo NEO. Exons 6 and 7 of the ABO gene were sequenced. Results: 13 cases that had the cis-AB allele showed results suggestive of the cis-AB subgroup by manual methods, but were interpreted as AB by either automated device. This happened in 87.5% of these cases by QWALYS-3 and 70.0% by Galileo NEO. Genotyping results showed that 12 cases were ABO*cis-AB01/ABO*O01 or ABO*cis-AB01/ABO*O02, and one case was ABO*cis-AB01/ ABO*A102. Conclusion: Cis-AB samples were mistyped as AB by the automated microplate technique in some cases. We suggest that the manual tile method can be a simple supplemental test for the detection of the cis-AB phenotype, especially in countries with relatively high cis-AB prevalence.

The serological and genetic characterization of CisAB blood group in a Chinese family

BackgroundThe rare CisAB blood genotype results in the inheritance of both the A and B blood types from a single parent. Several CisAB blood type phenotypes have been characterized that differ in their serological reactions and in the activities of the gene-encoded blood group A and B transferases. In this study, we conducted serological and genetic analyses of a Chinese family with four CisAB-carrying members. Study design and methodsA 10-year-old girl was suspected to have the CisAB blood type when forward blood typing indicated that she was blood type AB but reverse blood typing indicated that she was blood type A. An examination of this family identified that four of ten family members across three generations had a pattern of AB inheritance that could be explained only by the inheritance of the CisAB genotype. Blood samples from the family were tested serologically and genetically using PCR–SSP for the relevant CisAB alleles. The samples that were suitable for DNA analysis were sequenced for potential mutations. ResultsOf the ten family members included in this study, four members showed mismatched forward and reverse blood grouping results, four members typed as blood group O and the remaining two members typed as blood group B. Among the four mismatched individuals, the propositus and her mother's mother were serologically typed as having the A2B3 phenotype. The propositus' mother and her mother's brother were typed as having the A2B phenotype, and had the genotypes CisAB01/O02 and CisAB01/B101, respectively. Further sequencing analysis of the four samples with the CisAB blood type revealed that a G(261) deletion occurred along with 297A > G, 467C > T, 646T > A, 681G > A, 771C > T, 803G > C and 829G > C substitutions in exons 6 and 7 of CisAB01/O02, while a 297A > G substitution occurred in exon 6 and 467C > T, 803G > C, 526C > G, 657C > T, 703G > A, 796C > A, 930G > A substitutions occurred in exon 7 of CisAB01/B101. ConclusionA family was identified in which 4/10 family members across three generations had inherited the CisAB blood group. Two of the CisAB genotypes were further identified as CisAB01/O02 and CisAB01/B101. The CisAB heterozygous alleles contributed to the different phenotypes that were observed.

Molecular genetic analysis of rare <I>cis</I>AB variants in Chinese population

We investigated the molecular genetic basis of rare cisAB variants at the ABO locus in Chinese population. Serological techniques were performed to characterize erythrocyte phenotype of 12 discrepant samples and 116 randomly selected samples. Mutations of complete exon 6 and 7 including flanking intron in the ABO genes were screened by PCR and directly sequencing. The haplotypes of diverse genotypes were also analyzed by cloning sequencing. Twelve samples were identified as AweakB or ABweak phenotype by serological technology, and were also identified as cisAB variants including four genotypes by directly sequencing. Two cisAB alleles were found by haplotype sequencing. One allele was cisAB01, in which four nucleotide acid alterations were observed (467C>T and 803G>C in exon 7, 163T>C and 179C>T in intron 6); the other allele maintained a nucleotide acid of A101 allele (803G) compared with B101 allele, which resulted in a polypep-tide containing 176G, 235S, 266M, and 268G four amino acids. This is a novel allele, which has been named cisAB05 by Blood Group Antigen Gene Mutation Database. According to systematically investigation of the molecular genetic basis of the cisAB variants in Chinese population, we found a novel cisAB05 allele and presumed that the cisAB01 allele is derived from homologues exchange of A101-B101 combination.

Three Cases of Cis-AB without B Antigen Expression

The cis-AB bood type is a rare phenomenon in which both the A and B blood types are inherited from a single parent. The cis-AB persons are not homogeneous with respect to reactivity of their red cells to anti-A and anti-B reagents, and are split into three groups with on the basis of the strength and characteristics of the serologic reactions； these reactivities are A2B3, A1B3 and A2B. These differences of representative types is the effect of alleles coupled with that of cis-AB allele, and A2B3 , A1B3, and A2B phenotypes can be applicable to cis-AB/O, cis-AB/A1, and cis-AB/B genotypes, respectively.We report three variant cis-AB cases whose genotype were all cis-AB/A1 but B antigen was not expressed and reactions. All these cases were problematic because the paternity among parents and children blood types could not be explained with pedigree analysis through simple serological test. ABO genotyping was the very efficient and unique tool to investigate the inheritance mode of ABO blood type in these cases. Many questions about its mechanism, frequency, etc. raised their heads, which will become interesting subjects of studies.

Analysis for Eight ABO Alleles in Korean Population

ABO genotyping is a useful tool in case of ABO discrepancies and in legal medicine. Recent knowledge of various alleles in the ABO gene has led to the need of a different method that can cover numerous polymorphisms. We performed a polymerase chain reaction using sequencespecific priming (PCR-SSP) with 12 primer sets and evaluated its value in the detection of 8 ABO alleles. Genomic DNA was isolated from peripheral blood of 222 unrelated Koreans. Sequencespecific primer sets for the nucleotides 261, 297, 467, 802, 803, and 1059 were selected, and 12 PCR reactions were performed for each sample. Direct sequencing was performed to evaluate the accuracy of discrimination between A(1)(Pro) and A(1)(Leu) and between O(1) and O(1v). All the ABO genotype patterns were in an exact match with the ABO phenotypes. The results from sequencing and PCR-SSP were equivalent. The allele frequencies of A(1), B, O(1), and O(1v) were 27.25%, 19.82%, 27.25%, and 25.68% respectively. Out of total 121 A(1) alleles, 6 (4.96%) were A(1)(Pro) alleles and 115 (95.04%) were A(1)(Leu) alleles. No A(2), O(2), or CisAB alleles were found in this study. The proportion of O(1v) allele was similar to that of O(1) allele. This was an unexpected result. We developed a method for detecting 8 ABO alleles by PCR-SSP; the method was accurate and was able to discriminate between A(1)(Pro) and A(1)(Leu) and between O(1) and O(1v).

Characterization of a novel B(A) allele with BBBA type at the ABO blood group

The ABO blood group is one of the main blood group systems, and it plays an important role in transfusion medicine and transplantation. To date, most of the many ABO subgroups with a weak expression of the A or B antigen on red blood cells have been elucidated to have specific molecular genetic background with respect to the ABO gene. The ABO*B(A) allele or CisAB allele is a type of dual ABO allele which can encode glycosyltransferases responsible for the conversion of H substance to both A and B antigen. We report here our characterization of a novel B(A) allele which differs from those reported previously.

Resolution of ABO Discrepancies by ABO Genotyping

Before a blood transfusion, both red cell and serum typing need to be matched for ABO tests on the donor and patient (recipient). When a mismatch exists in the tests, additional ABO genotyping and serological tests are required for the resolution of the discrepancy. We performed ABO genotyping on a series of blood donors and patients with ABO discrepancies to assist in resolving their blood groups. We examined 46 samples with ABO discrepancies from a random pool of donors recruited at Gwangju-Chonnam Red Cross Blood Center and from patients at Chonnam National University Hospital between May 2004 and July 2005. ABO genotyping was performed on all samples with an allele specific polymerase chain reaction for differentiation of A, B,O, cis-AB, A(var) (784 G>A), and B(var) (547 G>A) alleles; routine serologic tests were also performed. Exon 6 and 7 of ABO gene from five samples were sequenced. The genotypes of 18 donors/patients with weakened A or B antigen expressions consisted of 4 cases of cis-AB/O (3 A(2)B(3), 1 A(2)B); 5 cases of cis-AB/A (5 A(1)B(x or el)); 2 cases of A/O (1 O, 1 A(m or x)); 1 case of B/O (1 B(m or x)); 4 cases of A/B (1 A(2)B , 1 A(1)B(x or el), 2 A(1)B(3)); and 2 cases of A(var)/B (2 A(w)B). On the other hand, the genotypes of 28 samples with unexpected serum reactions included 18 cases of A/O (16 A(1), 2 A(int)); 7 cases of A/A (5 A(1), 1 A(1)B(x or el), 1 A(1)B(w)); and 3 cases of O/O (1 O, 2 B(w)). ABO genotyping is useful for differentiating the ABO discrepancies that were difficult to resolve by serological tests. The most frequent unusual red cell reactions were weak A and B antigen expressions, which were resulted from the ABO subgroup alleles including cis-AB allele, whereas the most frequent unusual serum reactions were caused by decreased anti-B titers.

A novel cis‐AB allele derived from a unique 796C>A mutation in exon 7 of ABO gene

The cis-AB phenotype is very rare, and only three genotypes that correspond to specific ABO allele changes have been reported. Cis-AB01 involves the A102 allele with a nonsynonymous substitution G803C in exon 7, whereas cis-AB02 and cis-AB03 involve different nonsynonymous substitutions A796C and C700T, respectively, on the B101 allele background. The nucleotide substitutions give rise to a change of the respective glycosyltransferase, resulting in varying bifunctional AB transferase activities. Two cis-AB phenotypes were identified in a Taiwanese C. family and two unrelated individuals, respectively. Serologic studies, molecular cloning, and sequencing of exon 6 and exon 7 were carried out to determine their respective phenotypic characteristics and cis-AB alleles. A cohort of 300 AB-phenotype, healthy random individuals served as controls. A novel cis-AB allele is uncovered out of the three family members, of which a 796C>A substitution occurs predicting an amino acid change at residue 266 of leucine to methionine on the background of A102 allele. It is serologically like cis-AB03, an A2B phenotype, but molecularly different. Both of the two unrelated individuals are of cis-AB01 allele, and all of the 300 AB blood group controls are excluded cis-AB phenotype. The C. family described carries a novel cis-AB allele that differs molecularly from all previously reported cis-AB alleles.

A Single Point Mutation Reverses the Donor Specificity of Human Blood Group B-synthesizing Galactosyltransferase

Blood group A and B antigens are carbohydrate structures that are synthesized by glycosyltransferase enzymes. The final step in B antigen synthesis is carried out by an alpha1-3 galactosyltransferase (GTB) that transfers galactose from UDP-Gal to type 1 or type 2, alphaFuc1-->2betaGal-R (H)-terminating acceptors. Similarly the A antigen is produced by an alpha1-3 N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-GalNAc to H-acceptors. Human alpha1-3 N-acetylgalactosaminyltransferase and GTB are highly homologous enzymes differing in only four of 354 amino acids (R176G, G235S, L266M, and G268A). Single crystal x-ray diffraction studies have shown that the latter two of these amino acids are responsible for the difference in donor specificity, while the other residues have roles in acceptor binding and turnover. Recently a novel cis-AB allele was discovered that produced A and B cell surface structures. It had codons corresponding to GTB with a single point mutation that replaced the conserved amino acid proline 234 with serine. Active enzyme expressed from a synthetic gene corresponding to GTB with a P234S mutation shows a dramatic and complete reversal of donor specificity. Although this enzyme contains all four "critical" amino acids associated with the production of blood group B antigen, it preferentially utilizes the blood group A donor UDP-GalNAc and shows only marginal transfer of UDP-Gal. The crystal structure of the mutant reveals the basis for the shift in donor specificity.

A novel cis AB allele derived from a B allele through a single point mutation

The very rare cis AB phenotype, first described in 1964, corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing both A and B substances. Until now, gene sequences of only two cis AB alleles were partially characterized. One involved the A1*02 allele with a single nonsynonymous substitution at codon 268, whereas the second arose from a single nonsynonymous substitution at codon 266 in exon 7 of a B1*01 allele. A cis AB phenotype was identified in a French family. The serologic characteristics of this phenotype were determined. The cis AB allele was characterized from exon 6 to exon 7 by cloning and sequencing. The cis AB.tlse(*)01 allele is identical to B(1*)01 except for a single point mutation at nucleotide position 700, where a T replaces a C, implying a change of amino acid 234 (the B(1*)01 proline being replaced by a serine). The cis AB.tlse(*)01 allele clearly differs from all previously reported ABO, including the two previous cis AB described.

Rapid detection of the cisAB allele consisting of a chimera of normal A and B alleles by PCR-RFLPs.

DNA samples were analysed from Japanese individuals with the very rare ABO variant phenotype, cisAB (A2B3), which is characterized by the apparent inheritance of both A and B genes on one chromosome. The nature of the bases present at nucleotide positions (nps) 261, 526, 703, 796 and 803 is important for the specificity of the alleles at the ABO locus and the DNA from the cis AB donors was analysed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to determine which nucleotides are present at these positions. The results indicated that the cisAB allele had the AAAB-structure, which was a chimera of normal A and B alleles, when the expression 'AAAA' and 'BBBB' indicated the nucleotides of normal A (C, G, C and G) and B (G, A, A and C) genes at nps 526, 703, 796 and 803, respectively. The AAAB allele was found in all 27 individuals (17 families) with the cisAB including three phenotypes A2B3, A1B3 and A2B and no other chimeric gene was found. The causative gene of cisAB was the AAAB allele, and the A and B alleles were not on one chromosome. The cisAB allele appeared to be a product of the normal A allele due to a point mutation at nucleotide position 803, from G to C. The AAAB allele is thought to be normally transcribed and translated to produce an unusual transferase polypeptide, which has weak A- and weaker B-specific activity. PCR-RFLP is a rapid and useful means of detecting the cisAB allele (the AAAB allele) without a family study, even when they have A1B3 and A2B phenotypes, because trans-type A1B3 and A2B samples have obviously different PCR-RFLP profiles.

Molecular genetic analysis of the ABO blood group system: 2. cis-AB alleles.

We have determined the nucleotide sequence of the coding region in the last two coding exons of ABO genes from two cis-AB individuals (genotype cis-AB/O) with no consanguinity. In this region, cis-AB alleles from these 2 individuals were identical to one another while different from the A1 allele by two nucleotide substitutions. Both of these nucleotide substitutions result in amino acid substitutions. The first substitution is identical to the one previously found in the A2 allele. The other substitution is found at the fourth position of the four amino acid substitutions which discriminate A1 and B transferases.