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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: 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]

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]

Background The ABO blood group is important in blood transfusion. Recently, an erythroid cell-specific regulatory element has been identified in the first intron of ABO using luciferase reporter assays with K562 cells. The erythroid cell-specific regulatory activity of the element was dependent upon GATA-1 binding. In addition, partial deletion of Intron 1 including the element was observed in genomic DNAs obtained from 111 Bm and ABm individuals, except for one, whereas the deletion was never found among 1005 individuals with the common phenotypes. Study Design and Methods In this study, further investigation was performed to reveal the underlying mechanism responsible for reduction of B antigen expression in the exceptional Bm individual. Peptide nucleic acid-clamping polymerase chain reaction was carried out to amplify the B-related allele, followed by sequence determination. Electrophoretic mobility assays and promoter assays were performed to examine whether a nucleotide substitution reduced the binding of a transcription factor and induced loss of function of the element. Results Sequence determination revealed one point mutation of the GATA motif in the element. The electrophoretic mobility shift assays showed that the mutation abolished the binding of GATA transcription factors, and the promoter assays demonstrated complete loss of enhancer activity of the element. Conclusion These observations suggest that the mutation in the GATA motif of the erythroid-specific regulatory element may diminish the binding of GATA transcription factors and down regulate transcriptional activity of the element on the B allele, leading to reduction of B antigen expression in erythroid lineage cells of the Bm individual. [ABSTRACT FROM AUTHOR]

The ABO blood group is of great importance in blood transfusion and organ transplantation. However, the mechanisms regulating human ABO gene expression remain obscure. On the basis of DNase l-hypersensitive sites in and upstream of ABO in K562 cells, in the present study, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays indicated a novel positive regulatory element in intron 1. This element was shown to enhance ABO promoter activity in an erythroid cell-specific manner. Electrophoretic mobility-shift assays demonstrated that it bound to the tissue-restricted transcription factor GATA-1. Mutation of the GATA motifs to abrogate binding of this factor reduced the regulatory activity of the element. Therefore, GATA-1 appears to be involved in the cell-specific activity of the element. Furthermore, we found that a partial deletion in intron 1 involving the element was associated with Bm phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the Bm allele, leading to reduction of B-antigen expression in cells of erythroid lineage, but not in mucus-secreting cells. These results support the contention that the enhancer-like element in intron 1 of ABO has a significant function in erythroid cells. [ABSTRACT FROM AUTHOR]

Background: The low-incidence antigen St a 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 St a 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 St a antigen provides evidence for further complexity in the MNS system., (© 2020 International Society of Blood Transfusion.)

The severity of the hemolytic disease of the fetus and newborn (HDFN) due to Jr a mismatch ranges from no symptoms to severe anemia that requires intrauterine and exchange transfusions. We encountered a newborn, born to a healthy mother having anti-Jr a at 38 weeks of pregnancy, who had moderate anemia, a positive direct antiglobulin test (DAT) result, no increased erythropoiesis, and no jaundice at birth. Flow cytometry revealed that the Jr a antigen of red cells in the infant was nearly negative at birth, biphasic at 5 weeks, and lowly expressed at 7 months of life. We searched online for previous case reports on HDFN due to Jr a incompatibility. Among 63 reported cases, excluding 25 cases, 38 were included with the present case for analysis. Of 39 newborns, 10 developed clear anemia (hemoglobin <10.0 g/dL), and 1 died, 5 developed hydrops fetalis, 4 needed intrauterine transfusion and/or exchange transfusion, and 3 received red cell transfusion after birth; overlaps were included. Among 29 neonates with no anemia, 8 needed interventions including phototherapy and γ-globulin infusion, and the remaining 21 received conservative supports only. The maternal anti-Jr a titer, ranging between 4 and 2048, did not correlate with the severity of anemia, levels of bilirubin, or any interventions required. The DAT of red cells was positive in 29 of 36 fetuses/newborns tested, whereas it was often negative among anemic neonates (4 of 9) (P < .05). Hematopoiesis did not increase effectively, as indicated by reticulocyte ratios between 1.7% and 22.3%, even with the increase in reticulocytes in anemic neonates compared with nonanemic neonates (P < .05). Total bilirubin levels ranged broadly between 0.2 and 14.3 mg/dL but were generally low. The maternal anti-Jr a titer and IgG3 subclass did not correlate with the morbidity of the newborns. Being identical/compatible between mothers and their infants may possibly enhance infants' morbidity, as a weak tendency was observed (P = .053). Maternal anti-Jr a may suppress erythropoiesis in fetuses via a mechanism different from the established HDFN, such as anti-D, as evidenced by the lower reticulocyte count and small increase in bilirubin in neonates. As the anti-Jr a titer, IgG subclass, and DAT were not correlated with the severity, the mechanism of anti-Jr a -induced HDFN remains to be elucidated., (Copyright © 2019 Elsevier Inc. All rights reserved.)