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Background and Summary: We review the transcriptional regulation of ABO expression and discuss variants in the promoter and erythroid cell-specific regulatory region in individuals with weak ABO phenotypes such as Bm, Am, B3, and A3. We also review the molecular mechanisms responsible for variations in ABO expression in development and disease including the cell type-specific expression of ABO during erythroid cell differentiation, and reduction of A- or B-antigens in cancer cells or on red blood cells in patients with leukemia. Although the relationship between ABO blood group antigens and diseases has been characterized, the physiological significance of the ABO blood group system remains unclear. Key Messages: This review discusses accumulated knowledge of the ABO gene regulation and potential reasons for conservation of ABO during evolution. [ABSTRACT FROM AUTHOR]

Background: The Kidd blood group gene SLC14A1 (JK) accounts for approximately 20 Kb from initiation codon to stop codon in the genome. In genomic DNA analysis using Sanger sequencing or short‐read‐based next generation sequencing, it is difficult to determine the cis or trans positions of single nucleotide variations (SNVs), which are occasionally more than 1 Kb away from each other. We aimed to determine the complete nucleotide sequence of a 20‐Kb genomic DNA amplicon to characterize the JK allelic variants associated with Kidd antigen silencing in a blood donor. Study Design and Methods: The Jk(a–b–) phenotype was identified in this donor by standard serological typing. A DNA sample obtained from whole blood was amplified by long‐range PCR to obtain a 20‐Kb fragment of the SLC14A1 gene, including the initiation and stop codons. The fragment was then analyzed by Sanger sequencing and single‐molecule sequencing. Transfection and expression studies were performed in CHO cells using the expression vector construct of JK alleles. Results: Sanger sequencing and single‐molecule sequencing revealed that the donor was heterozygous with JK*01 having c.276G>A (rs763262711, p.Trp92Ter) and JK*02 having c.499A>G (rs2298719, p.Met167Val), c.588A>G (rs2298718, p.Pro196Pro), and c.743C>A (p.Ala248Asp). The two JK alleles identified have not been previously described. Transfection and expression studies indicated that the CHO cells transfected with JK*02 having c.743C>A did not express the Jkb and Jk3 antigens. Conclusions: We identified new JK silencing alleles and their critical SNVs by single‐molecule sequencing and the findings were confirmed by transfection and expression studies. [ABSTRACT FROM AUTHOR]

Background and Objectives: The Xg blood group is composed of two antigens, Xga (XG1) and CD99 (XG2 and MIC2). The XG and CD99 are homologous genes located on pseudoautosomal region 1 of the X and Y chromosomes. The expressions of Xga and CD99 are co‐regulated by a single nucleotide polymorphism (rs311103) in the GATA‐1 binding region. Another mechanism of the Xg(a−) phenotype is the genomic deletion of approximately 114 kb, including the XG gene. Anti‐Xga seems to be naturally occurring by detection in males who have never been transfused. Materials and Methods: In this study, we identified 23 anti‐Xga producers among 580,115 donors (0.004%). Additional 12 anti‐Xga producers were also identified from a separate cohort. Results: All 35 anti‐Xga producers were male. Genomic DNA was obtained from 34 of 35 producers, and all 34 producers were confirmed to carry the XG‐gene‐deficient allele (XGdel). The breakpoints of all 34 producers were identical. The XGdel was also identified in 12 non‐producers of anti‐Xga among 860 donors who have no antibodies against RBCs, and the breakpoints were also identical with the anti‐Xga producers. Conclusion: Our results will serve as the basis for a more complete understanding of Xg blood group polymorphisms. [ABSTRACT FROM AUTHOR]

Background and Objectives: Antigens of the MNS blood group system are expressed on the red blood cell (RBC) membrane on glycophorin A (GPA) and glycophorin B (GPB) or on hybrid molecules of GPA and GPB. This study investigated the distribution of glycophorin variants and alloantibodies against Hil and MINY among Japanese individuals. Methods: Mi(a+) or Hil+ RBCs were screened using an automated blood grouping machine (PK7300) with monoclonal anti‐Mia or polyclonal anti‐Hil. Glycophorin variants were defined by serology with monoclonal antibodies against Mia, Vw, MUT and Mur, and polyclonal antibodies against Hil, MINY and Hop + Nob (KIPP). The glycophorin variants were further confirmed by immunoblotting and Sanger sequencing. Alloanti‐Hil and alloanti‐MINY in the plasma were screened using GP.Hil RBCs in an antiglobulin test. The specificity of anti‐Hil or anti‐MINY was assessed using GP.Hil (Hil+MINY+) and GP.JL (Hil‒MINY+) RBCs. Results: The GP.HF, GP.Mur, GP.Hut, GP.Vw, GP.Kip and GP.Bun frequencies in 1 005 594 individuals were 0·0357%, 0·0256%, 0·0181%, 0·0017%, 0·0009% and 0·0007%, respectively. GP.Hil was found in as four of the 13 546 individuals (0·0295%). Of 137 370 donors, 10 had anti‐Hil (0·0073%) and three had anti‐MINY (0·0022%). Conclusions: Glycophorin variants were relatively rare in Japanese individuals, with the major variants being GP.HF (0·0357%), GP.Hil (0·0295%) and GP.Mur (0·0256%). Only one example of anti‐MINY was previously reported, but we found three more in this study. [ABSTRACT FROM AUTHOR]

Background: The low‐incidence antigen Sta of the MNS system is usually associated with the GP(B‐A) hybrid molecule, which carries the 'N' antigen at the N terminus. The GP(A‐A) molecule with trypsin‐resistant M antigen has been found in a few St(a+) individuals. Materials and Methods: Among Japanese blood donors, we screened 24 292 individuals for the presence of St(a+) with trypsin‐resistant 'N' antigen and 193 009 individuals for the presence of St(a+) with trypsin‐resistant M antigen. The breakpoints responsible for the Sta antigen were analysed by sequencing the genomic DNAs. Results: A total of 1001 (4·1%) individuals were identified as St(a+) with trypsin‐resistant 'N' antigen. Out of 1001 individuals, 115 were selected randomly for sequencing. Two novel GYP*Sch (GYP*401) variants with new intron 3 breakpoints of GYPA were detected in three cases. Twenty‐five (0·013%) individuals were identified as St(a+) with trypsin‐resistant M antigen. Five individuals had the GYP(A‐ψB‐A) hybrid allele; two of these five individuals were GYP*Zan (GYP*101.01), and the remaining three had a novel GYP(A‐ψB‐A) allele with the first breakpoint in GYPA exon A3 between c.178 and c.203. Nine individuals had a novel GYP(A‐E‐A) allele with GYPE exon E2 and pseudoexon E3 instead of GYPA exon A2 and A3. The 11 remaining individuals had a novel GYP(A‐A) allele with a 9‐bp deletion that included the donor splice site of intron 3 of GYPA. Conclusion: Our finding on diversity of glycophorin genes responsible for Sta antigen provides evidence for further complexity in the MNS system. [ABSTRACT FROM AUTHOR]

Background and objectives: MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease‐resistant M antigen. Methods: Blood samples were screened by an automated blood typing system (PK7300) using bromelain‐treated red blood cells (RBCs) and murine monoclonal anti‐M. The M‐positive RBC samples were analysed by immunoblotting using anti‐M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA. Results: Serological tests and immunoblotting revealed that 103 of the 193 009 individuals (0·0534%) expressed protease‐resistant M‐active glycophorin having a molecular weight of 20 kDa. All the 103 individuals were S+ s− or S− s+. When reticulocyte mRNA from the individuals with M‐active glycophorin (20 kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE‐B hybrid transcript was identified. Long‐range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB‐E(2‐4)‐B hybrid gene. This hybrid gene was predicted to encode a 59‐amino‐acid mature glycoprotein that expresses no S or s antigens Conclusions: The prevalence of the M‐active glycophorin (20 kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB‐E(2‐4)‐B hybrid gene. [ABSTRACT FROM AUTHOR]

Background: MNS is one of the highly polymorphic blood groups comprising many antigens generated by genomic recombination among the GYPA, GYPB, and GYPE genes as well as by single-nucleotide changes. We report a patient with red blood cell (RBC) antibody against an unknown low-frequency antigen, tentatively named SUMI, and investigated its carrier molecule and causal gene.Study Design and Methods: Standard serologic tests, including enzyme tests, were performed. Monoclonal anti-SUMI-producing cells (HIRO-305) were established by transformation and hybridization methods using lymphocytes from a donor having anti-SUMI. SUMI+ RBCs were examined by immunocomplex capture fluorescence analysis (ICFA) using HIRO-305 and murine monoclonal antibodies against RBC membrane proteins carrying blood group antigens. Genomic DNA was extracted from whole blood, and the GYPA gene was analyzed by polymerase chain reactions and Sanger sequencing.Results: Serologic screening revealed that 23 of the 541,522 individuals (0.0042%) were SUMI+, whereas 1351 of the 10,392 individuals (13.0%) had alloanti-SUMI. SUMI antigen was sensitive to ficin, trypsin, pronase, and neuraminidase, but resistant to α-chymotrypsin and sulfydryl-reducing agents. ICFA revealed that the SUMI antigen was carried on glycophorin A (GPA). According to Sanger sequencing and cloning, the SUMI+ individuals had a GYPA*M allele with c.91A>C (p.Thr31Pro), which may abolish the O-glycan attachment site.Conclusions: The new low-frequency antigen SUMI is carried on GPA encoded by the GYPA*M allele with c.91A>C (p.Thr31Pro). Neuraminidase sensitivity suggests that glycophorin around Pro31 are involved in the SUMI determinant. [ABSTRACT FROM AUTHOR]

Background: Loss of blood group ABO antigens on red blood cells (RBCs) is well known in patients with leukemias, and such decreased ABO expression has been reported to be strongly associated with hypermethylation of the ABO promoter. We investigated the underlying mechanism responsible for A-antigen reduction on RBCs in a patient with myelodysplastic syndrome.Study Design and Methods: Genetic analysis of ABO was performed by PCR and sequencing using peripheral blood. RT-PCR were carried out using cDNA prepared from total bone marrow (BM) cells. Bisulfite genomic sequencing was performed using genomic DNA from BM cells. Screening of somatic mutations was carried out using a targeted sequencing panel with genomic DNA from BM cells, followed by transient transfection assays.Results: Genetic analysis of ABO did not reveal any mutation in coding regions, splice sites, or regulatory regions. RT-PCR demonstrated reduction of A-transcripts when the patient's RBCs were not agglutinated by anti-A antibody and did not indicate any significant increase of alternative splicing products in the patient relative to the control. DNA methylation of the ABO promoter was not obvious in erythroid cells. Targeted sequencing identified somatic mutations in ASXL1, EZH2, RUNX1, and WT1. Experiments involving transient transfection into K562 cells showed that the expression of ABO was decreased by expression of the mutated RUNX1.Conclusion: Because the RUNX1 mutation encoded an abnormally elongated protein without a transactivation domain which could act as dominant negative inhibitor, this frame-shift mutation in RUNX1 may be a genetic candidate contributing to A-antigen loss on RBCs. [ABSTRACT FROM AUTHOR]

Background: The Rh complex contributes to cell membrane structural integrity of erythrocytes. Rhnull syndrome is characterized by the absence of the Rh antigen on the erythrocyte membrane, resulting in chronic hemolytic anemia. We recently came across 3 Rhnull phenotype probands within two families with the same novel RHAG mutation in the Japanese population.Materials and Methods: Detailed Rh phenotyping by hemagglutination was performed using monoclonal and polyclonal anti-D, -C, -c, -E, and -e; monoclonal and polyclonal anti-Rh17 antibodies; and polyclonal anti-Rh29 antibodies. RHAG mRNA transcripts were analyzed by reverse transcription-polymerase chain reaction, and the mutation was verified by genomic sequencing.Results: The genomic region spanning exon 6 contained a G > A transition in the invariant GT motif of the 5' donor splice-site of Intron 6 (c.945+1G>A). The Rhnull phenotype was caused by an autosomal recessive mutation in Probands 1 and 2, determined by family history. Regarding clinical features, the degree of hemolysis varied slightly between these individuals, with Proband 3 displaying acute hemolytic anemia with an infection. While no standard therapy has been established, the condition of the patient in this study improved with conservative treatment, including hydration and antibiotics.Conclusion: The mechanisms of hemolysis due to the Rhnull phenotype can vary, but our findings indicate that acute hemolytic crisis caused by the Rhnull syndrome could be associated with infection. [ABSTRACT FROM AUTHOR]

Background: In(Lu) is characterized by a reduced expression of antigens in the Lutheran blood group system as well as other blood group antigens. Mutations of the erythroid transcription factor, KLF1, have been reported to cause the In(Lu) phenotype, and we investigated Japanese In(Lu) to estimate the prevalence of the phenotype and KLF1 polymorphism.Study Design and Methods: Blood samples were screened by monoclonal anti-CD44 and the In(Lu) phenotype was confirmed by tube tests including adsorption and elution tests using anti-Lua and anti-Lub . KLF1, LU, and A4GALT genes were analyzed by polymerase chain reaction and sequencing.Results: We identified 100 of 481,322 blood donors (0.02%), and the previously characterized 20 donors, who had the In(Lu) phenotype with the LUB/LUB genotype. A total of 100 of the 120 In(Lu) individuals had mutant KLF1 alleles, and we identified 13 known and 21 novel alleles. The mutant KLF1 alleles with c.947G>A (p.Cys316Tyr), c.862A>G (p.Lys288Glu), or c.968C>G (p.Ser323Trp) were major in the In(Lu) individuals. The P1 antigen of 29 In(Lu) (two P1 /P1 , 27 P1 /P2 ) showed significantly weakened expression by hemagglutination.Conclusions: The prevalence of the In(Lu) phenotype in the Japanese population was 0.02%, and we identified 13 known and 21 novel KLF1 alleles. The KLF1 mutations cause the reduced expression of the P1 antigen. [ABSTRACT FROM AUTHOR]

We found an individual with weakened S antigen expression on red blood cells (RBCs) during routine blood grouping. The proband was typed S+s+ by polyclonal antibodies, but the RBCs demonstrated different reactivity with three monoclonal anti‐S. The proband did not have alloanti‐S. Cloning and Sanger sequencing revealed that the proband had a c.166A>T (p.Thr56Ser) mutation in exon 4 of GYPB*S. When antibody screening of 60 455 blood donors was performed using the proband RBCs, no antibodies were detected. GYPB*S with c.166T should encode an unusual S antigen but the creation of a novel antigen remains to be investigated. [ABSTRACT FROM AUTHOR]