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.
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.
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.
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.
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.
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.
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.
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.
The severity of the hemolytic disease of the fetus and newborn (HDFN) due to Jr
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
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
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
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
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
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
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
titer, IgG subclass, and DAT were not correlated with the severity, the mechanism of anti-Jr
-induced HDFN remains to be elucidated.
Abstract
Background and Objectives
The Xg blood group is composed of two antigens, Xg
a
(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 Xg
a
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‐Xg
a
seems to be naturally occurring by detection in males who have never been transfused.
Materials and Methods
In this study, we identified 23 anti‐Xg
a
producers among 580,115 donors (0.004%). Additional 12 anti‐Xg
a
producers were also identified from a separate cohort.
Results
All 35 anti‐Xg
a
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‐Xg
a
among 860 donors who have no antibodies against RBCs, and the breakpoints were also identical with the anti‐Xg
a
producers.
Conclusion
Our results will serve as the basis for a more complete understanding of Xg blood group polymorphisms.
