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Complementary Therapies↗
Biomedical subjects
E Kajii
Publications and source records attributed to E Kajii.
Identification of 5' flanking sequence of RH50 gene and the core region for erythroid-specific expression.
The Rh blood group antigens are carried by two distinct but homologous membrane proteins encoded by two closely related genes, RCHE and RHD. Rh50 glyco-protein is the membrane protein tightly associated with Rh polypeptides and is critical for expression of Rh antigens. The amino acid sequence and predicted membrane topology of Rh50 glycoprotein are significantly homologous with those of the Rh proteins. Northern blot analysis of leukemic cell lines showed that expression of RH50 gene is restricted to cells with erythroid features. HEL and K562 cells showed a transcription levels ratio of 1 to 9.9 for Rh50, and 12.3 to 1 for Rh. The nucleotide sequence of 5' flanking region of RH50 gene and functional promoter assays also supported the erythroid-specific regulation of the gene, whereas the sequence had lower homology with the promoter sequence of RH genes. Seven GATAs, nine E-boxes, two CACCCs, one YY1, and one October motif were identified in the 1868bp 5' flanking sequence. The core promoter of RH50 gene was located within 68bp length from the translation start position, which included an inverse GATA motif, although obvious motifs for Sp1 or erythroid Krüppel-like factor were lacking. The inverse GATA motif was the target sequence of GATA-1 protein, and disruption of the motif abolished the transactivating activity of erythroid cells. These studies confirm the erythroid-specific expression of Rh antigens, but suggest distinct regulatory mechanisms for RH vs RH50 genes.
Amino Acid Sequence↗
A novel missense mutation of the tissue-nonspecific alkaline phosphatase gene detected in a patient with hypophosphatasia.
Hypophosphatasia is a rare heritable inborn error of metabolism characterized by abnormal bone mineralization associated with a deficiency of alkaline phosphatase. The clinical expression of hypophosphatasia is highly variable, ranging from death in utero to pathologic fractures first presenting in adulthood. We investigated the tissue-nonspecific alkaline phosphatase (TNSALP) gene from a Japanese female patient with hypophosphatasia. By a quantitative polymerase chain reaction (PCR) method, the amount of TNSALP mRNA appeared to be almost equal to that in normal individuals. Gene analysis clarified that the hypophosphatasia originated from a missense mutation and a nucleotide deletion. The missense mutation, a C--> T transition at position 1041 of cDNA, results in an amino acid change from Leu to Phe at codon 272, which has not yet been reported. The previously reported deletion of T at 1735 causes a frame shift mutation downstream from Leu at codon 503. Family analysis showed that the mutation 1041T and the deletion 1735T had been inherited from the proband's father and mother, respectively. An expression experiment revealed that the mutation 1041T halved the expression of alkaline phosphatase activity. Using homology analysis, the Leu-272 was confirmed to be highly conserved in other mammals.
Adolescent↗
Expression analysis of human Rhesus blood group antigens by gene transduction into erythroid and non-erythroid cells.
Rh blood group antigens are associated with non-glycosylated human erythrocyte membrane proteins encoded by two closely related genes, RHCE and RHD, and with a glycoprotein, a critical co-expressing factor encoded by the RH50 gene. The sequence analysis of RHCE transcripts has revealed that RhE/e and C/c serological phenotypes are associated with a nucleotide substitution in exon 5 and six substitutions in exons 1 and 2 of RHCE gene, respectively. Smythe et al. have shown that the full length transcript of RhcE gene expressed c and E antigens and the transcript of RhD gene expressed D and G antigens, using retroviral-mediated gene transduction into K562 cells. We performed an epitope analysis of Rh antigen by constructing retroviral gene coding six RH cDNAs, which contain RhcE, ce, CE and D cDNAs, and CE-D, D-CE chimera cDNAs. The cDNAs were introduced into KU812E cells and the expressed antigens were analyzed by flow cytometry. These studies revealed that the C/c and E/e associated substitutions actually participated in respective polymorphic epitopes. However, the C antigen was not detected on the KU812E cells introduced with CE cDNA, despite E antigen being expressed. The study with the chimera gene between CE and D cDNAs also indicated that the Rh epitopes were not constructed with short polymorphic exofacial peptide loops only but also with other peptide fragments and membrane components. Co-expression studies of Rh50 and RhD or cE gene in non-erythroid cells, 293, and expression studies of Rh50 in another erythroid cell, HEL, did not show any Rh antigens on the transduced cells, despite the Northern blot study showing both transcripts in the cells. It was suggested that at least a second co-expressing factor was needed to express RhCE or D antigens on the plasma membrane.
Cell Line↗
Direct antiglobulin test negative autoimmune hemolytic anemia associated with autoimmune hepatitis.
A case of direct antiglobulin test(DAT)-negative autoimmune hemolytic anemia (AIHA) associated with autoimmune hepatitis (AIH) is presented. A 54-year-old female was admitted with liver dysfunction and anemia. AIH was diagnosed based on the diagnostic criteria of the International Autoimmune Hepatitis Group. The patient was DAT-negative, and exhibited all the clinical features of warm type AIHA and elevated levels of red blood cell-associated IgG. Marked improvement of subjective symptoms, the return to normal of hematological and liver test values, and a decrease in the level of RBC-associated IgG were observed after the start of corticosteroid therapy. Although an association of AIH and AIHA is rarely reported, the measurement of RBC-associated IgG is recommended in cases of AIH with DAT-negative anemia, as both disorders are autoimmune in nature.
Anemia, Hemolytic, Autoimmune↗
A splicing mutation of the RHAG gene associated with the Rhnull phenotype.
Rhnull is a syndrome serologically characterized by the deficiency of all Rh antigens on human red blood cells. Rhnull is divided into two types: regulator and amorph. Recently, Cherif-Zahar et al. proposed that the RHAG gene encoding the Rh50 glycoprotein is a candidate for inducing regulator type Rhnull. We investigated both the RH and RHAG genes in an Rhnull individual. The reticulocytes from the propositus had RHD, RHcE, and RHCe transcripts without any mutation. However, the sequence analysis of RHAG cDNA showed a deletion of 122 bp from nucleotide 946 to 1067. This deletion was revealed to be due to a homozygous splicing mutation, which is a single base substitution at the consensus sequence of the splicing acceptor site (AG-->AT). The mutation appeared to break the 'GT-AG' splicing rule and to cause 122 bp exon skipping accompanied by a frameshift. This study confirms that the RHAG gene is the most likely candidate for the 'regulator' gene of Rhnull cases.
Adolescent↗
Advance in the Rh blood group system.
The Rh blood group antigens are associated with non-glycosylated red blood cell membrane proteins encoded by two closely related genes, RHD and RHCE. Both RH genes have 10 exons and the open reading frames of their transcripts are composed of 1251 nucleotides. The sequence analyses of RHCE transcripts revealed that RhC/c and RhE/e are related to six nucleotide substitutions and a single substitution, respectively. Molecular analyses have elucidated the genetic background of various RhD and RhCE variants including the Rhnull phenotype with neither RhD nor RhCE antigen. The Rh polypeptides require some co-molecules for their assembly in the plasma membranes. In fact, several unrelated proteins other than those of the Rh polypeptides are reduced or absent on Rhnull red blood cells. This group includes the Rh50 glycoprotein, LW glycoprotein, glycophorin B, Duffy glycoprotein, and CD47 glycoprotein. The mutation of the RH50 gene is inducible of the Rhnull phenotypes. Although the Rh50 glycoprotein is a critical co-expressing factor, one or more factors seem to be required to express RhD or RhC(c)E(e) antigens on the plasma membranes. Additionally, polymerase chain reaction-based tests for Rh genotyping were developed and some have been put in practice. Much information obtained from the molecular approach to the Rh blood group system has shed considerable light on the Rh polypeptides. In the near future, the mechanism of expression, the function, and the evolution of Rh polypeptides will be clarified further.
Anemia, Hemolytic, Autoimmune↗
The RHD gene is highly detectable in RhD-negative Japanese donors.
Recent molecular studies on the Rh blood group system have shown that the Rh locus of each haploid RhD-positive chromosome is composed of two structural genes: RHD and RHCE, whereas the locus is made of a single gene (RHCE) on each haploid RhD-negative chromosome. We analyzed the presence or absence of the RHD gene in 130 Japanese RhD-negative donors using the PCR method. The RhD-negative phenotypes consisted of 34 ccEe, 27 ccee, 17 ccEE, 26 Ccee, 19 CcEe, 1 CcEE, and 6 CCee. Among them, 36 (27.7%) donors demonstrated the presence of the RHD gene. Others showed gross or partial deletions of the RHD gene. These results were confirmed by Southern blot analysis. Additionally, the RHD gene detected in the RhD-negative donors seemed to be intact through sequencing of the RhD polypeptide cDNA and the promoter region of RHD gene. The phenotypes of these donors with the RHD gene were CC or Cc, but not cc. It suggested that there is some relationship between the RHD gene and the RhC phenotypes in RhD-negative individuals. In Caucasian RhD-negative individuals, the RHD gene has not been found outside of the report of Hyland et al. (Hyland, C.A., L.C. Wolter, and A. Saul. 1994. Blood. 84:321-324). The discrepant data on the RHD gene in RhD-negative donors between Japanese and Caucasians appear to be derived from the difference of the frequency of RhD-negative and RhC-positive phenotypes. Careful attention is necessary for clinicians in applying RhD genotyping to clinical medicine.
Base Sequence↗
Dinucleotide repeat in the 3' flanking region provides a clue to the molecular evolution of the Duffy gene.
The Duffy blood group system consists of three alleles, FYA, FYB, and FY. To study the molecular evolution of the three alleles, we established the polymorphism of a dinucleotide (GT) repeat sequence (designated FyGT/C) in the 3' flanking region of the Duffy gene, and studied the relationship between FyGT/C and Duffy polymorphism in Japanese, people of African origin, and chimpanzee. By single-strand conformation polymorphism and sequence analysis, five and two alleles were identified in Japanese and Africans, respectively. In 110 random Japanese, the FyGT/C genotypes observed were in agreement with Hardy-Weinberg law. From the sequence of the chimpanzee Duffy gene, including both flanking regions, FYB was identified as the ancestral gene of the human alleles. The FyGT/C sequences associated with the FY allele of Africans were distinct from those of Duffy positives, whereas the FYB and FYA alleles shared common FyGT/C sequences. Thus, it is suggested that the first split took place between the FYB and FY alleles, and the second between the FYB and FYA alleles.
Alleles↗
Identification of the truncated Duffy mRNAs in erythroid cells.
Chaudhruri et al. reported the Duffy glycoprotein cDNA (Fy71-81) (Proc Natl Acad Sci USA 1990; 90; 10793), and we cloned a novel Duffy cDNA named Fy0.1 (Blood 1996; 87: 378). Reverse transcription-polymerase chain reaction analysis revealed the presence of the four truncated mRNAs associated with both transcripts. The truncated Duffy mRNAs is predominantly present in reticulocytes but is not detected in the erythroblasts. The sequencing data indicated that the truncated Duffy mRNAs were processed by splicing. Further study is needed to clarify the biological role of the truncated Duffy mRNA in reticulocytes.
Animals↗
Genotyping for RhC/c and RhE/e by PCR using allele-specific oligonucleotide primers.
The Rh blood group system has five major antigens D, C/c, and E/e. These antigens are encoded in RHD and RHCE genes. In this report, we describe a systemic method for RhC/c and RhE/e genotyping by PCR using allele-specific oligonucleotide primers (ASO-PCR). The ASO-PCR was carried out to determine the RhC/c and RhE/e genotypes in DNA samples from 513 Japanese donors. Genotypes of RhC, RhE, and Rhe were in full concordance with serological phenotypes in 511 donors. However, in two cases with the phenotype of ccdee, the C-specific ASO-PCR product was also detected in addition to the c-specific one. This method is simple and quite useful for the RhC/c and RhE/c genotyping, although further investigation on the 2 exceptional ccdee cases is needed.
Base Sequence↗
[Recent advances in blood typing with DNA analysis].
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ABO Blood-Group System↗
Molecular genetic basis of red cell markers and its forensic application.
(1) The polymerase chain reaction (PCR) was used to amplify Rh-related cDNAs from erythroid cells cultured by the selective two-phase liquid culture system for human erythroid progenitors in peripheral blood. Two Rh polypeptide cDNAs have been isolated from the PCR products and tentatively designated RhPI cDNA and RhPII cDNA. Both cDNA clones have an open reading frame composed of 1251 nucleotides. The RhPI cDNA clone shows a single nucleotide substitution with no amino acid substitution compared with the published sequence. The RhPII cDNA clone differs from the above by 41 nucleotide substitutions in the open reading frame, resulting in 31 amino acid substitutions. Besides these cDNA clones, eleven and five truncated isoforms of the RhPI and RhPII cDNAs, have been isolated, respectively. (2) The promoter region of the Duffy gene was cloned by IPCR of 1.1 kb SacI fragment and the 3' flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The IPCR products contained the known Duffy cDNA sequence without introns. By comparing the coding area of the Duffy gene in 28 Duffy positive individuals, we elucidated that one base change that results in an amino acid substitution (GAT(Asp44)-->GGT(Gly)) is in accordance with the Fya/Fyb polymorphism. This fact proves that the Duffy cDNA and its gene encode the Duffy blood group system. (3) Two common alleles in Esterase D (EsD) polymorphism, EsD1 and EsD2 were characterized by the substitution of one amino acid (Gly-Glu) caused by the point mutation of one nucleotide (G-A). The point mutation between cDNAs of EsD1 and EsD2 alleles was detectable as restriction fragment length polymorphism (RFLP) using Ssp1. The RFLP makes it possible to determine the EsD phenotypes using DNA samples from forensic materials without EsD enzymatic activity. (4) The authors report studies on 19 pairs of donors and recipients in bone marrow transplantation. A broad range of genetic markers at 42 gene loci, including one DNA marker 11 red blood cell markers, five human lymphocyte antigen types, 12 serum protein markers, five red cell enzyme markers, and eight salivary markers was evaluated before and after BMT over about 2 months. As a result, 11 out of 42 gene loci of genetic markers in recipients were transformed into the donor type.
Base Sequence↗
Characterization of the Duffy gene promoter: evidence for tissue-specific abolishment of expression in Fy(a-b-) of black individuals.
We have previously identified a novel first exon of Duffy gene and two inverse GATA motifs in the 600 bp 5' flanking region. The proximal GATA is positioned downstream from the start position of endothelium and upstream from that of erythroid. One base substitution (-365T --> C) was found in the proximal GATA motif from three black Fy(a-b-) individuals, and was regarded as a common polymorphic mutation in black Fy(a-b-) individuals. The upstream sequence of the novel first exon was inserted in the upstream of chloramphenicol acetyltransferase (CAT) gene and transfected in human erythroleukemia cell line (HEL) and human microvascular endothelial cells (HMvEC). The black type mutation abolished the CAT transcription in HEL cells but not in HMvEC. Deletion mutagenesis study revealed that the proximal GATA motif represent the erythroid regulatory core region for Duffy gene. Gel shift assay showed that the proximal GATA motif is the target sequence of GATA-1. These studies indicate that the black type mutation abolishes Duffy gene expression in erythroid but not in postcapillary venule endothelium, which is compatible with the Northern blot and immunohistochemical observation in black Fy(a-b-) individuals.
Base Sequence↗
Identification of a novel exon and spliced form of Duffy mRNA that is the predominant transcript in both erythroid and postcapillary venule endothelium.
The Duffy gene has been shown not to be split by introns, even in its 5' untranslated region, and to be expressed not only in erythroid but in postcapillary venule endothelium of almost every organ in the body. To further investigate the transcriptional start position in erythroid and postcapillary venule endothelium, we performed 5'-rapid amplification of cDNA ends (5'-RACE). While every positive clone of 5'-RACE encoded the identical sequence of previously identified cDNA downstream from nucleotide 203, the upstream sequences were different. The upstream sequences corresponded to the sequence from nucleotide -279 to -308/-357 in erythroblasts and from -279 to -355/-383 in lung and were regarded as comprising a novel exon. This novel exon encoded seven residues initiated with a methionine, linked to nucleotide 203 in-frame and in agreement with the GT-AG splicing rule. The major erythroid transcriptional start position was identified in human erythroleukemia cells by primer extension and in bone marrow by ribonuclease protection analysis at 34 bases upstream from the first ATG codon. Distinctively, in lung and kidney, the transcription was started at 82 bases upstream from the ATG. Both Northern blotting and reverse transcription-polymerase chain reaction followed by Southern analysis indicated a predominance of the novel spliced form of mRNA of about 50- to 200-fold comparing with the unspliced form, in every studied organ and erythroid lineage cells. The spliced form of cDNA has been transfected into a human erythroleukemic cell line, K562, and the expressed protein reacted with Duffy-specific murine monoclonal antibody Fy6. These studies indicate that the product from the spliced form of mRNA is the major product of the Duffy gene in the erythroid lineage and postcapillary venule endothelium.
Amino Acid Sequence↗
Autoimmune hemolytic anemia in patients with de novo acute myelocytic leukemia.
Autoantibody against erythrocytes has occasionally been observed in patients with de novo acute myelocytic leukemia (AML). However, it is not clear whether this autoantibody in AML patients induces frank hemolysis (autoimmune hemolytic anemia, AIHA), as seen in lymphoid neoplasms. We present two de novo AML patients who showed hemolysis due to antiglobulin test-positive and test-negative AIHA, respectively. AIHA should be considered as one cause of anemia in de novo AML patients, and blood transfusions should be given carefully in such cases to avoid harmful hemolysis.
Adult↗
[Detection and characterization of anti-red cell autoantibodies in autoimmune hemolytic anemias].
Autoimmuine hemolytic anemias (AIHA) are classified into two groups of warm type and cold type according to the thermal properties of the anti-red cell autoantibody. A positive result of the antiglobulin test (DAT) confirms the presence of autoantibodies on red cells. DAT-negative AIHA are diagnosed by means of the elevation of red blood cell-associated IgG. The sera in low titer cold agglutinin disease show a low cold agglutinin titer but a high titer in the presence of bovine albumin. The antigenic specificity of warm reacting autoantibodies has been demonstrated, by using immunoblot and immunoprecipitation, such as Rh-related proteins, band 3, glycophorin A or Wrb antigen.
Anemia, Hemolytic, Autoimmune↗
Present situation of the analysis of Rh genes.
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Cloning, Molecular↗