Ryuichi Yabe

Extensive polymorphism of ABO blood group gene: three major lineages of the alleles for the common ABO phenotypes

Polymorphism of the ABO blood group gene was investigated in 262 healthy Japanese donors by a polymerase chain reactions-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A1 (provisionally called ABO*A101, *A102, *A103 and *A104 according to the guidelines for human gene nomenclature), 3 for B (ABO*B101, *B102 and *B103), and 6 for O (ABO*O101, *O102, *O103, *O201, *O202 and *O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. Phylogenetic network analysis revealed that these alleles could be classified into three major lineages, *A/*O1, *B and *O2. In Japanese, *A102 and *13101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, *O101 (43%) and *O201 (53%), were observed. These results may be useful for the establishment of ABO genotyping, and these newly described ABO alleles would be advantageous indicators for population studies.

Recombination and gene conversion-like events may contribute to ABO gene diversity causing various phenotypes

Abstract. We identified five different alleles, tentatively named ABO*O301, *O302, *R102, *R103, and *A110, in Japanese individuals possessing the blood group O phenotype. These alleles lack the guanine deletion at nucleotide position 261 which is shared by a majority of O alleles. Nucleotide sequence analysis revealed that *O301 and *O302 had single nonsynonymous substitutions compared with *A101 or *A102 responsible for the A1 phenotype. Analysis of intron 6 at the ABO gene by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing revealed that *R102 and *R103 had chimeric sequences of A-O2 and B-O2, respectively, from exons 6 to 7. In the analysis of five other chimeric alleles detected in the same manner, we identified a total of four different recombination-breakpoints within or near intron 6. When 510 unrelated Japanese were examined, the frequency of the chimeric alleles generated by recombination in intron 6 or exon 7 was estimated to be 1.7%. In addition, we found that *O301, *A110, *C101, *A111, and 35% of *A102 had a unique A-B-A chimeric sequence at intron 6, presumed to originate from a gene conversion-like event. We had previously established that *A110 also had an A-O2-A chimeric sequence around nucleotide position 646 in exon 7. Thus this allele has an A-B-A-O2-A chimeric sequence from intron 6 to exon 7 probably generated by two different gene conversions. Similar patchwork sequences around nucleotide position 646 in exon 7 were observed in two other new alleles responsible for the Ax and B3 phenotypes. Thus, the site is presumably a hotspot for gene conversion. These results indicate that both recombination and gene conversion-like events play important roles in generating ABO gene diversity.

Different alleles cause an imbalance in A2 and A2B phenotypes of the ABO blood group

Background and Objectives: In several populations, including the Japanese, the frequency of the A2B phenotype is significantly higher than expected based on the A2 phenotype frequency. To understand the genetic basis of this ‘excess’ of A2B, we examined ABO alleles in individuals with A2‐related phenotypes. Materials and Methods: ABO alleles were identified by means of polymerase chain reaction single‐strand conformation polymorphism (SSCP) and nucleotide sequence analyses. Results: The frequencies of A2‐related alleles (*A105, *A106, *A107, *A111 and *R101) were clearly different between the A2 and A2B phenotypes. In particular, a putative recombinant allele, *R101, was uncommon in the A2 but common in the A2B phenotype individuals. This allele was also detected in 4 of 401 (1%) unrelated A1 phenotype (AO genotype) individuals. Conclusion: *R101 is presumably expressed as phenotype A1 in *R101/*O heterozygous individuals, but as phenotype A2 in *R101/*B heterozygotes, thus giving rise to a high A2B phenotype frequency.

Consequences of Nucleic Acid Amplification Testing for Blood Transfusion Centres

This article is also accessible online at: http://BioMedNet.com/karger Blood banks and transfusion centres are faced with the imminent introduction of nucleic acid amplification testing (NAT), or genomic amplification testing of plasma pools used by the plasma industry. The Committee for Proprietary Medicinal Products (CPMP) in Europe requires that all manufactured plasma pools should be tested for HCV RNA by NAT by July 1, 1999. To avoid the destruction of large NAT-reactive plasma pools, the CPMP strongly advises to implement a system for the screening of minipools of plasma by NAT. In future, genomic screening of individual donations for blood-borne viruses is expected to become obligatory. At present, genomic screening of individual donations cannot be routinely performed, and NAT minipool screening (i.e. a pool of plasma of 100 donations) is not (well) standardized, is costly and time-consuming, especially when the individual positive donors from a positive pool have to be sorted out. An especially difficult and ethical question is what should be decided concerning the release of red cell products and especially platelets when minipool screening is implemented. Either these cellular products will be blocked for at least several days, creating shortage and loss of product, or the results of minipool screening tests will not affect these products. This may create different levels of safety and serious ethical problems by informing (or not informing) recipients of these products after a positive result has been obtained. Fourteen experts in the field were asked for their opinion, answers were obtained from 10 of them on the following questions.

Prevalence of RHD alleles in Japanese individuals with weak D phenotype: Identification of 20 new RHD alleles

We identified 46 different RHD alleles from 226 Japanese individuals with weak D phenotype, 26 of which had been previously described and 20 that were novel. Among these weak D individuals, the alleles with c.960G>A, c.845G>A (RHD*15) or c.1013T>C (RHD*01W.24) mutations were most prevalent with relative occurrences of 36·7%, 15·9% and 9·7%, respectively. These findings demonstrate that the prevalence of common weak D alleles in the Japanese population significantly differs from that of Caucasian populations.

Presence of nucleotide substitutions in the ABO promoter in individuals with phenotypes A3 and B3

Recently, the involvement of mutation and deletion of transcription regulatory elements in the Bm, Am, A3 and B3 phenotypes has been reported. In the present study, we carried out genetic analysis of individuals with A3 and B3 using peptide nucleic acid‐clamping PCR to exclude amplification of O alleles. Two single‐point mutations, −76G>C and −68G>T, were found in the ABO promoter on the A‐allele in three A3 individuals and on the B allele in a B3 individual, respectively. Transient transfection of luciferase reporter plasmids carrying the same mutations into K562 cells revealed decreased luciferase activity in comparison with that carrying the wild‐type promoter. These observations suggest that the mutations downregulate the promoter activity, leading to reduction in A‐ or B‐antigen expression on red blood cells in individuals with the A3 and B3 phenotypes.

The B allele with a 5·8 kb deletion in intron 1 of the ABO gene is the major allele in Japanese individuals with Bm and A1Bm phenotypes

Bm and A1Bm phenotypes are the most frequent ABO variants in the Japanese population. The B antigen on Bm red blood cells is only detectable by adsorption and elution tests, and plasma B‐transferase activity is usually detected at half or less levels compared with that of common B. Recently, a B allele lacking an erythroid cell‐specific transcription enhancer in intron 1 of the ABO gene was identified from individuals with Bm and A1Bm phenotypes, which could explain the unique serologic properties of Bm. In the Japanese Red Cross Society, eight Blood Centers tested blood samples from donors throughout Japan and collected blood samples from 888 Bm and 415 A1Bm individuals. DNA analysis revealed that 1300 of 1303 (99·77%) individuals had the B allele with a 5·8 kb deletion (c.28 + 5110_10889del), which included the enhancer element.

Application of immortalized human erythroid progenitor cell line in serologic tests to detect red blood cell alloantibodies: ANTIBODY SCREENING USING CULTURED CELLS

Antibody screening in pretransfusion tests is necessary to avoid critical complications of blood transfusion. Although red blood cells (RBCs) expressing relevant alloantigen(s) have been used for serologic antibody screening, little attention has been given to the use of cell lines, in which blood group antigen gene(s) are transduced, as reagent RBCs for antibody screening.