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The purpose of this study was to investigate the molecular genetic basis of A2 subgroup and identify the novel alleles at ABO locus in Chinese Han population. All seven exons and their flanking sequences, enhancer and promoter in the ABO gene of five samples from individuals with serological discrepancies were amplified by polymerase chain reaction (PCR); the PCR products were screened by directly sequencing; the haplotypes of exon 6 and 7 were analyzed by TOPO cloning sequencing. The results showed that five samples were identified as A2 or A2B subgroup by serological technology. The A201 and A205 alleles were confirmed in one A2B individual and one A2 individual, respectively. A novel A2 variant allele was identified in three A2B individuals. The two nucleotide acid alterations (467C > T and 539G > C) at the exon 7 resulting in two amino acid substitutions (P156L and R180P) in this novel allele were observed, when compared with A101 allele. It is concluded that the polymorphism of A2 allele is found to be relatively variable in Chinese population, and a novel A208 allele responsible for A2 subgroup is firstly reported.

Background and Objectives: The A2 is a very rare phenotype in the ABO blood group system in the Oriental population. It corresponds to a special ABO allele encoding a glycosyltransferase that is capable of synthesizing A2 antigens, which is weaker than the typical A antigen. In this study, we report a novel A2 allele in two unrelated Taiwanese individuals., Materials and Methods: Two individuals were identified as the A2 phenotype based on the standard ABO serological test. For analysing the A2 allele, both direct sequencing and gene cloning of the ABO gene were performed., Results: The ABO gene of the two A2 individuals was composed of O1 and A2 alleles, and the novel A2 allele has a 539G > C that results in the amino acid change Arg180Pro. The mutation was not detected in the general group A population., Conclusion: We report for the first time that a 539G > C mutation represents a new molecular basis for the A2 blood type. The amino acid substitution from arginine to proline may have effect on the expression of A antigen.

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.

Cell surface complex carbohydrate structures that are synthesized through the actions of glycosyltransferases play an important role in cell-to-cell and cell-to-extracellular matrix interactions. To examine the feasibility of phage display technique to clone cDNAs encoding glycosyltransferases, we performed biopanning experiments using human histo-blood group A transferase as a model enzyme and its substrate, blood group H-specific glycoproteins, as a bait ligand. Our attempts have been unsuccessful, possibly because of the enzyme's weak affinity with the target. However, we have selectively enriched several phage clones that expressed capsid proteins fused with galectin-3, a galactose/lactose-specific animal lectin of the galectin family. These results demonstrate that this novel approach of phage display is useful in cDNA cloning of proteins with carbohydrate-binding property., (Copyright 1999 Academic Press.)

We have identified a possible mutation which characterizes A2 alleles (a minor subtype of A) at the human histo-blood group ABO locus based on polymerase chain reaction (PCR) of genomic DNA, followed by nucleotide sequencing of the amplified fragments. The A2 subtype has a single base deletion near the carboxyl terminal. As a result of frame-shifting, A2 transferase possesses an extra domain. Introduction of this single base deletion into the A1 transferase cDNA expression construct drastically decreased the A transferase activity in DNA-transfected HeLa cells.

Based on the partial amino acid sequence, the cDNA encoding UDP-GalNAc:Fuc alpha 1----2Gal alpha 1----3GalNAc transferase, the specific primary gene product of histo-blood group A gene (A transferase), was cloned and sequenced. Poly(A)+ RNA from human stomach cancer cell line MKN45, expressing high levels of A antigen, was used for construction of a lambda gt10 cDNA library. Degenerate synthetic oligodeoxynucleotides were used for polymerase chain reactions to detect the presence of the sequence of interest in cDNA (presence test) and to identify the correct clones (identification test) after screening the library with a radiolabeled polymerase chain reaction amplified fragment. Nucleotide sequence analysis revealed a coding region of 1062 base pairs encoding a protein of 41 kDa. Hydrophobicity plot analysis shows the existence of three domains: N-terminal short stretch, transmembranous hydrophobic region, and a long C-terminal domain (a feature common to all glycosyltransferases cloned so far). Southern hybridization analysis has shown that this DNA does not represent a multigene family. No restriction fragment length polymorphism was found to correlate with ABO blood group type. Bands were detected in Northern hybridization of mRNAs from cell lines expressing A, B, AB, or H antigens. These results suggest that sequences of ABO genes are essentially very similar (with minimal differences), and the inability of the O gene to encode A or B transferases is probably due to structural differences rather than A or B transferase expression failure.