Two new JK silencing alleles identified by single molecule sequencing with 20-Kb long-reads.

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.

DSLK and Kg: Antithetical antigens in the RHAG blood group system, and characterization of anti-DSLK antibody.

BACKGROUND AND OBJECTIVES: The RHAG blood group system contains five antigens: Duclos (RHAG001), Ola (RHAG002), DSLK (RHAG003), Kg (RHAG005) and SHER (RHAG006). Individuals who are DSLK-negative and Kg-positive have the same allele RHAG*01.-3, with a single-nucleotide variation (rs144305805), c.490A>C (p.Lys164Gln), in exon 3 of the RHAG gene. We aimed to confirm whether DSLK and Kg are antithetical antigens. MATERIALS AND METHODS: Blood samples of the original DSLK-negative proband with anti-DSLK, her son and another DSLK-negative individual were examined. The RHAG gene was analysed by polymerase chain reaction and Sanger sequencing. Immunocomplex capture fluorescence assays (ICFAs) and monocyte phagocytosis assays were performed to characterize the anti-DSLK antibody. Cross-testing of alloanti-DSLK and monoclonal anti-Kg (OSK46) was performed using transduced HEK293 cells by inducing the construct of expression vectors encoding wild-type RHAG*01 or the variant RHAG*01.-3. RESULTS: ICFA using monoclonal anti-RHAG (LA18.18) revealed that the anti-DSLK and anti-Kg antibodies reacted with the wild-type and variant RhAG (Rh-associated glycoprotein), respectively. The proband and a DSLK-negative individual appeared to be homozygous for variant RHAG*01.-3, and the proband's son was typed as RHAG*01/RHAG*01.-3 heterozygote. HEK293 cells with wild-type RhAG reacted with the anti-DSLK but not anti-Kg antibody, whereas HEK293 cells expressing the variant RhAG reacted with the anti-Kg but not anti-DSLK antibody. Monocyte phagocytosis assays indicated that 64% of red cells sensitized with anti-DSLK were phagocytosed by monocytes. CONCLUSION: Our results demonstrate that DSLK and Kg are antithetical antigens in the RHAG blood group system. Anti-DSLK may be a clinically significant antibody.

Frequencies of glycophorin variants and alloantibodies against Hil and MINY antigens in Japanese.

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.

Genetic background of anti-Xga producers in Japanese blood donors.

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.

GP.MOT: A novel glycophorin variant identified in a Japanese blood donor.

BACKGROUND: In this study, we identified a novel glycophorin variant (GP.MOT) in a Mia -positive Japanese blood donor. The proband with this glycophorin variant was discovered by antigen screening of samples from 475,493 Japanese blood donors using monoclonal anti-Mia . STUDY DESIGN AND METHODS: Standard serological techniques and flow cytometry were performed. GP.MOT RBCs were examined by immunoblotting using anti-GPA, anti-MUT or anti-Mur. Genome DNA was extracted from whole blood, and the GYPA/GYPB was analyzed by polymerase chain reactions and Sanger sequencing. RESULTS: The MNS blood group of the proband was M + N + w S-s + with the presence of other low-frequency antigens including Mia , Mur, MUT, and KIPP. A 43-kDa molecule, which is almost equivalent in size to glycophorin A (GPA), was identified by immunoblotting using monoclonal anti-MUT and anti-Mur. Sanger sequencing clearly indicated that the proband had two different GYPA*M alleles at SNP rs62334651 (GYPA*M232 + 55A and GYPA*M232 + 55G), as well as a GYP(B-A) hybrid allele (GYP*MOT) with breakpoints located on pseudoexon 3 of GYPB from c.210 to c.219. DISCUSSION: We identified a hybrid glycophorin GP.MOT with the deduced unique amino acid sequence GPB (20-45)-GPΨB (46-70)-GPA (71-149), which has not been previously reported.

The Kg-antigen, RhAG with a Lys164Gln mutation, gives rise to haemolytic disease of the newborn.

The Kg-antigen was first discovered in an investigation of a mother whose infant had haemolytic disease of the newborn (HDN). The antibody against the Kg-antigen is believed to be responsible for HDN. The Kg-antigen is provisionally registered under the number 700045, according to the Red Cell Immunogenetics and Blood Group Terminology. However, the molecular nature of the Kg-antigen has remained a mystery for over 30 years. In this study, a monoclonal antibody against the Kg-antigen and the recombinant protein were developed that allowed for the immunoprecipitation analysis. Immunoprecipitants from the propositus' red blood cell ghosts were subjected to mass spectrometry analysis, and DNA sequence analysis of the genes was also performed. A candidate for the Kg-antigen was molecularly isolated and confirmed to be a determinant of the Kg-antigen by cell transfection and flow cytometry analyses. The Kg-antigen and the genetic mutation were then screened for in a Japanese population. The molecular nature of the Kg-antigen was shown to be RhAG with a Lys164Gln mutation. Kg phenotyping further clarified that 0.22% of the Japanese population studied was positive for the Kg-antigen. These findings provide important information on the Kg-antigen, which has been clinically presumed to give rise to HDN.

A new antigen SUMI carried on glycophorin A encoded by the GYPA*M with c.91A>C (p.Thr31Pro) belongs to the MNS blood group system.

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.

RUNX1 mutation in a patient with myelodysplastic syndrome and decreased erythrocyte expression of blood group A antigen.

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.

An unusual variant glycophorin expressing protease-resistant M antigen encoded by the GYPB-E(2-4)-B hybrid gene.

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.

Novel hybrid genes and a splice site mutation encoding the Sta antigen among Japanese blood donors.

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.

Production of RBC autoantibody mimicking anti-D specificity following transfusion in a patient with weak D Type 15.

BACKGROUND: Weak D-type RBCs have fewer D epitopes, but it remains unclear whether individuals with certain types of weak D produce alloanti-D directed at D epitopes absent from the RBCs, and whether it is an alloantibody or an autoantibody. We report the first case of a patient with a weak D Type 15 who produced autoantibodies mimicking alloanti-D. CASE REPORT: A 52-year-old Japanese male with weak D developed anti-D 3 months after transfusion of D-negative and -positive RBCs, and the antibody persisted for 24 months with a consistently negative direct antiglobulin test. Eluates from the patient's RBCs demonstrated anti-D specificity. The recipient did not exhibit any signs of delayed hemolytic transfusion reaction. As his anti-D was removed by the different adsorbing cells of weak D Type 15 and autologous as well as D positive, D negative, weak D Type 24, and partial DVa, it was thought to be an autoantibody mimicking anti-D rather than an alloantibody. The patient's RBCs reacted weakly with the 13 anti-D reagents used in the study. Polymerase chain reaction and nucleotide sequencing revealed that the patient had an RHD genotype of RHD*01N.01/RHD*15. CONCLUSION: Anti-D, produced in a patient with weak D Type 15 after transfusion, was found to be mimicking autoanti-D. Alloanti-D was excluded by an adsorption study with different RBC types.

Integrative genome analysis identified the KANNO blood group antigen as prion protein.

BACKGROUND: Anti-KANNO, a broadly reactive RBC alloantibody, is found among some Japanese pregnant women, but the genetic basis of the corresponding antigen remains unclear. STUDY DESIGN AND METHODS: We integrated a statistical approach to identify the coding gene for KANNO antigen by conducting a genome-wide association study (GWAS) on four KANNO-negative individuals and 415 healthy Japanese. We also applied whole-exome sequencing to them and performed a replication study to confirm the identified genome variation using independent 14 KANNO-negative individuals. A monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay was used to locate KANNO antigen on RBC-specific membrane protein. In vivo and in vitro binding assays of anti-KANNO were further applied to the cells expressing a candidate protein. RESULTS: The GWAS revealed a genome-wide significant association of chromosome 20p13 locus (p = 2.76E-08; odds ratio > 1000 [95% confidence interval = 48-23,674]). The identified single-nucleotide polymorphism located in an intronic region of the prion protein (PRNP) gene. Whole-exome sequencing revealed a missense variant in the PRNP gene (rs1800014, E219K), which is in linkage disequilibrium with the single-nucleotide polymorphism identified in the GWAS. All 18 KANNO-negative individuals possessed the homozygous genotype of the missense variant. The MAIEA assay using anti-KANNO and mouse antihuman prion protein showed a clear difference between KANNO-positive and KANNO-negative RBCs. Anti-KANNO showed direct binding to CHO-K1 cells expressing wild-type PRNP but not to those expressing E219K PRNP. CONCLUSION: We first identified the coding gene of the high-frequency antigen KANNO located in PRNP and the missense variation (E219K) that affects the seropositivity of the KANNO antigen, which were confirmed by PRNP overexpressed cells.

Rhnull phenotype caused by a novel RHAG mutation, c.945+1G>A, in the Japanese population.

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.

A novel c.166A>T (p.Thr56Ser) mutation in GYPB*S accounting for unusual S antigen expression.

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.

Application of immortalized human erythroid progenitor cell line in serologic tests to detect red blood cell alloantibodies.

BACKGROUND: 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. STUDY DESIGN AND METHODS: The use of an erythroid progenitor cell line for serologic tests was studied. The expression of blood group antigens of erythroid progenitor cells was analyzed by genotyping and flow cytometry. Serologic analysis including hemagglutination was performed using erythroid progenitor cells to evaluate their sensitivity for antibody detection. Overexpression of exogenous erythroid antigen by lentiviral transduction was carried out and investigated for antibody detection sensitivity. RESULTS: Erythroid progenitor cells contained a substantial amount of hemoglobin and expressed sufficient levels of blood group antigens to detect corresponding monoclonal antibodies. Furthermore, the cell line could acquire an exogenous RBC antigen after lentiviral transduction and detected corresponding monoclonal and alloantibodies with equal sensitivity to antigen-positive RBCs. CONCLUSION: Application of erythroid progenitor cell lines for screening for unexpected antibodies could be helpful in solving issues such as reagent availability associated with the conventional RBC-based assay. The genetic expandability of erythroid progenitor cell lines by gene modification techniques could lead to the development of more convenient reagent RBCs.

Epithelial Expression of Human ABO Blood Group Genes Is Dependent upon a Downstream Regulatory Element Functioning through an Epithelial Cell-specific Transcription Factor, Elf5.

The human ABO blood group system is of great importance in blood transfusion and organ transplantation. The ABO system is composed of complex carbohydrate structures that are biosynthesized by A- and B-transferases encoded by the ABO gene. However, the mechanisms regulating ABO gene expression in epithelial cells remain obscure. On the basis of DNase I-hypersensitive sites in and around ABO in epithelial cells, we prepared reporter plasmid constructs including these sites. Subsequent luciferase assays and histone modifications indicated a novel positive regulatory element, designated the +22.6-kb site, downstream from ABO, and this was shown to enhance ABO promoter activity in an epithelial cell-specific manner. Expression of ABO and B-antigen was reduced in gastric cancer KATOIII cells by biallelic deletion of the +22.6-kb site using the CRISPR/Cas9 system. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay demonstrated that the site bound to an epithelial cell-specific transcription factor, Elf5. Mutation of the Ets binding motifs to abrogate binding of this factor reduced the regulatory activity of the +22.6-kb site. Furthermore, ELF5 knockdown with shRNA reduced both endogenous transcription from ABO and B-antigen expression in KATOIII cells. Thus, Elf5 appeared to be involved in the enhancer potential of the +22.6-kb site. These results support the contention that ABO expression is dependent upon a downstream positive regulatory element functioning through a tissue-restricted transcription factor, Elf5, in epithelial cells.

Mutations of the KLF1 gene detected in Japanese with the In(Lu) phenotype.

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.

A new ABCG2 null allele with a 27-kb deletion including the promoter region causing the Jr(a-) phenotype.

BACKGROUND: The high-prevalence antigen Jr(a) is carried on the ATP-binding cassette transporter ABCG2. The ABCG2 gene consists of 16 exons and its translation start codon is located on the second exon. Although the occurrence of the Jr(a-) phenotype is rare, several ABCG2 null alleles have been reported. We report a new ABCG2 null allele having a large deletion in this study. STUDY DESIGN AND METHODS: The Jr(a) status was determined by standard serologic tests and genomic DNA was isolated from whole blood. Exons 1 to 16 and the 5'-untranslated region of the ABCG2 gene were analyzed by polymerase chain reaction and sequencing. Expression of the ABCG2 protein on red blood cells was examined by immunoblotting. RESULTS: A Jr(a-) blood donor had a novel allele having a 27-kb deletion including noncoding Exon 1 and the promoter region of ABCG2, and the donor was apparently homozygous for the allele. In addition, we found three more individuals having heterozygosity for the same allele, with ABCG2*01N.01 having c.376C>T (p.Q126X), but did not find the allele having the 27-kb deletion in 3000 Jr(a+) individuals. Immunoblotting revealed that the ABCG2 protein was not found to be expressed in the individual with homozygosity for the ABCG2 27-kb deleted and in two individuals with an ABCG2 27-kb deleted/ABCG2*01N.01 genotype, which indirectly allows to conclude that the 27-kb deletion is responsible for a null ABCG2 allele. CONCLUSION: We first identified an ABCG2 null allele (provisional ISBT allele number ABCG2*01N.23) having a large deletion including the promoter region.

Expression of ABO blood-group genes is dependent upon an erythroid cell-specific regulatory element that is deleted in persons with the B(m) phenotype.

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 I-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 B(m) phenotypes. Therefore, it is plausible that deletion of the erythroid cell-specific regulatory element could down-regulate transcription in the B(m) 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.