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M E Reid

Publications and source records attributed to M E Reid.

At least 73 records · Page 4Linked to original sources
PubMed2000

The Rh blood group system: a review.

The Rh blood group system is one of the most polymorphic and immunogenic systems known in humans. In the past decade, intense investigation has yielded considerable knowledge of the molecular background of this system. The genes encoding 2 distinct Rh proteins that carry C or c together with either E or e antigens, and the D antigen, have been cloned, and the molecular bases of many of the antigens and of the phenotypes have been determined. A related protein, the Rh glycoprotein is essential for assembly of the Rh protein complex in the erythrocyte membrane and for expression of Rh antigens. The purpose of this review is to provide an overview of several aspects of the Rh blood group system, including the confusing terminology, progress in molecular understanding, and how this developing knowledge can be used in the clinical setting. Extensive documentation is provided to enable the interested reader to obtain further information. (Blood. 2000;95:375-387)

Animals↗
PubMed2000

High-level, stable expression of blood group antigens in a heterologous system.

The detection and identification of blood group antibodies in patients is crucial for successful allogeneic blood transfusions. Current methods are highly subjective and rely on red blood cells (RBCs), which simultaneously express many blood group antigens, have a short shelf-life, and carry potential biohazard risks. To overcome these problems, we have used the approach of expressing individual blood group antigen-bearing proteins in a heterologous system. We report here the high-level surface expression of type I (Knops), type II (Kell), and type III/multi-pass (Duffy) membrane proteins that carry blood group antigens in mouse erythroleukaemic (MEL) cells using a vector containing the beta-globin locus control region. Importantly, the antigens expressed were detected specifically by a panel of patients' sera containing alloantibodies at sensitivities that are comparable to antigen-positive RBCs. Furthermore, in contrast to other mammalian expression systems, antigen expression was stable following freezing and thawing of the cell lines. Thus, this system has the potential both to replace the current use of RBCs by providing a one step method to detect and identify blood group antibodies and to allow the automation of antibody identification for the clinical laboratory.

Animals↗
PubMed2000

Applications of molecular biology techniques to transfusion medicine.

Other articles in this issue of Seminars in Hematology have reviewed the results of basic research in relation to the understanding of the genes, the molecular basis of blood group variants, and structural and functional aspects of the proteins carrying blood group antigens. Although molecular techniques are currently being used in a limited fashion in clinical laboratories, their application has far-reaching possibilities and undoubtedly will be soon applied more generally. We focus on two general areas: molecular genotyping for blood group antigens and their expression analysis in heterologous systems.

Blood Group Antigens↗
PubMed2000

Summary of the clinical significance of blood group alloantibodies.

The knowledge of the potential clinical significance of alloantibodies to blood group antigens is essential for selecting appropriate red blood cell (RBC) products for transfusion. This article provides a summary of clinical and technical aspects of many alloantibodies.

Anemia, Hemolytic↗
PubMed2000

DNA from blood samples can be used to genotype patients who have recently received a transfusion.

BACKGROUND: The use of hemagglutination to phenotype red cells from recently transfused patients or of red cells that are coated with IgG can be time-consuming and difficult to interpret. Because the molecular bases of many blood group antigens are known, it was investigated whether polymerase chain reaction (PCR) analysis of DNA, from white cells in blood from transfused patients, could be used to predict the blood group antigen profile of a patient. STUDY DESIGN AND METHODS: To prevent problems arising from potentially poor-quality DNA in clinical samples, primers that flanked the polymorphism of interest and that replicated a relatively short PCR amplicon were used. The PCR products, with or without digestion with the appropriate restriction enzyme, were analyzed on gels. Samples were collected from 60 patients who had received from 2 to 50 units of RBCs in the 7 days before sample collection. RBCs from some of these patients were coated with IgG. Analyses for RHD/non-D, RHE/RHe, KEL1/KEL2, FYA/FYB, FY-GATA, JKA/JKB, and GYPA M/N were performed by using assays that had been validated with DNA prepared from untransfused volunteers of known phenotype. The genotyping assays were performed without knowledge of the expected result. RESULTS: The predicted genotype after analysis of the 60 patient samples was that expected from the results of phenotyping. In all cases, the molecular analysis gave a single result; no evidence of chimerism was obtained. CONCLUSION: In each case, the molecular genotype results were in agreement with the blood group antigen as determined by historical phenotyping, phenotyping after hypotonic washing, detection of alloantibodies in the patient's serum, or elution of alloantibody(ies). Under the conditions of these assays, reliable determination of a blood group allele can be made by PCR-restriction fragment length polymorphism testing.

Blood Transfusion↗
PubMed2000

Molecular basis of Cromer blood group antigens.

BACKGROUND: The Cromer blood group system consists of 10 antigens located on decay-accelerating factor (DAF). Previous molecular genetic analysis has determined the basis for four of these antigens. The present study was undertaken to identify the mutations that determine the remaining antigens. STUDY DESIGN AND METHOD: Existing or new data were used to localize each Cromer system antigen to a specific short consensus repeat (SCR) domain of DAF. The exon encoding that SCR domain was amplified by using the polymerase chain reaction (PCR) on genomic DNA obtained from individuals of that Cromer phenotype, and the DNA product was subjected to DNA sequence analysis. RESULTS: The Tc(a)/Tc(c) polymorphism is due to an R18P amino acid substitution in SCR1 of DAF. The Es(a+)/Es(a-) polymorphism is due to an I46N mutation in SCR1 of DAF. The WES(b)/WES(a) polymorphism is due to an L48R mutation in SCR1 of DAF. The UMC+/UMC- polymorphism is due to a T216M substitution in SCR4 of DAF. CONCLUSIONS: With information from previous reports and the findings of this study, the molecular genetic basis of all known alleles of the Cromer blood group system has been elucidated. Single amino acid substitutions are responsible for 9 of the 10 antigens (all except the multiple-epitope antigen IFC).

Base Sequence↗
PubMed2000

Molecular mechanisms that lead to reduced expression of duffy antigens.

BACKGROUND: In the Duffy blood group system, the null phenotype Fy(a-b-) has been classically associated with a mutated GATA box, while the Fy(x) phenotype weak Fy(b) is associated with Arg89Cys and Ala100Thr mutations. This report assesses the prevalence of the Duffy GATA box and the Fy(x)-associated mutations in white and African American (black) donors and investigates the molecular mechanism underlying the Fy(x) phenotype. STUDY DESIGN AND METHODS: PCR RFLP Duffy genotyping was performed on blood samples from blacks and whites. Duffy antigen expression (Fy(a), Fy(b), Fy6, Fy3) on RBCs was measured by flow cytometry. By site-directed mutagenesis, the relevance of each Fy(x)-associated mutation to Duffy (mRNA, antigen, and protein) expression was analyzed in transfectants by Northern blotting, flow cytometry, and immunoblotting. RESULTS: The mutated GATA box occurred at a high allele frequency (0.8) in blacks and was rare among whites. Conversely, the Fy(x)-associated mutations were absent in blacks, but present in 3.5 percent of whites. By flow cytometry, Duffy antigens (Fy(a) or Fy(b), Fy6 and Fy3) showed a dosage effect in RBC samples that were transcriptionally silenced by the GATA box mutation in one allele. By contrast, the reduced (10%) Duffy protein in Fy(x) RBCs was shown by heterologous expression analysis not to be due to reduced RNA levels, but to protein instability caused by Arg89Cys. CONCLUSIONS: Reduced Duffy expression can result from mutations affecting transcription (mutated GATA box in one allele) or instability of the translated protein (Arg89Cys). The frequencies of these mutations vary among populations.

Black People↗
PubMed2000

Identification of a novel hybrid glycophorin gene encoding GP.Hop.

BACKGROUND: The GP.Hop (Mi.IV) phenotype expresses the MNS low-incidence antigens Mur, Hop, TSEN, MINY, and MUT. Because serologically similar MNS phenotypes expressing some or all of these antigens were shown to be carried by hybrid GP(B-A-B) proteins, it was proposed that a similar protein would be found for GP.Hop. The identification of a second GP.Hop propositus (ES) initiated a study to determine the molecular basis of this phenotype. STUDY DESIGN AND METHODS: Serologic tests and immunoblotting analysis with glycophorin-specific antibodies were performed. GYPB, the gene encoding the GPB protein, was cloned and sequenced after reverse transcription PCR amplification of total RNA isolated from ES. GYPB-specific primers encompassing GYPB pseudoexon 3, intron 3, and exon 4 were also used to clone and sequence genomic DNA from ES and MH, the original GP.Hop proband. RESULTS: Serologic and immunochemical data confirmed that ES's RBCs carried antigens associated with the GP.Hop phenotype. Sequencing of ES's cDNA demonstrated the presence of genes predicted to encode s-specific GPB and an S-specific GP(B-A-B) hybrid in which the 3' end of GYPB pseudoexon 3 had been replaced by a short nucleotide sequence from exon 3 of the GPA gene (GYPA). The hybrid nucleotide sequence contained sequence motifs previously shown to be required for the expression of the Mur, Hop, TSEN, MINY, and MUT, which is consistent with their presence as detected serologically. Genomic DNA analysis found that the crossover point in GYPB pseudoexon 3 was identical in ES and MH. CONCLUSIONS: The GP.Hop phenotype is produced by a hybrid GP(B-A-B) protein caused by a DNA insertion of GYPA into GYPB. The composition of the hybrid protein is GPB(1-26)-GPpsiB(27-50)-GPA(51-58)-GPB(S)(59-103).

Amino Acid Sequence↗
PubMed2000

First example of anti-Kx in a person with the McLeod phenotype and without chronic granulomatous disease.

BACKGROUND: Kx is lacking in the RBCs of patients with the McLeod syndrome. This condition is sometimes associated with chronic granulomatous disease (CGD). If given allogeneic RBCs, CGD patients with the McLeod phenotype may produce anti-Kx and anti-Km, and only phenotypically matched McLeod blood would be compatible. McLeod phenotype persons without CGD have made anti-Km but not anti-Kx (2 examples), and thus both McLeod and K(O) blood would be compatible. CASE REPORT: RBCs from a transfused patient with the McLeod phenotype but not with CGD (non-CGD McLeod) were typed for the Kell blood group antigens, and the plasma was analyzed for the presence of antibody by agglutination. The molecular basis was determined by analyzing for XK protein on RBC membranes by Western immunoblotting, by sequencing the XK gene, and by RFLP. RESULTS: The RBCs did not react with anti-Kx + anti-Km and showed weakening of Kell system antigens. The patient's plasma reacted moderately (2+) with RBCs of common Kell type and strongly (4+) with K(O) RBCs and RBCs of common Kell type treated with dithiothreitol, and did not react with McLeod RBCs. XK protein was absent from the RBC membranes. The XK gene had a point mutation in the donor splice site of intron 1 (G>C). CONCLUSION: This is the first report describing the molecular alteration in a non-CGD McLeod patient who has made anti-Kx. The immune response of people with the McLeod phenotype can vary, and K(O) blood may not always be compatible.

Antibody Formation↗
PubMed2000

Blood groups and their function.

The function(s) assigned to red blood cell membrane components is based on an observed effect in the red cells that lack the component, comparison of the protein sequence (predicted from the nucleotide sequence of the gene) to proteins of known function, and extrapolation of function of the component in other cells. The functions are varied and include membrane structure, transport, receptor, adhesion, enzyme activity, complement components, complement regulation and glycocalyx formation. Several components have more than one function.

Biological Transport↗
PubMed2000

Evans antigen: a new hybrid structure occurring on background of D.. and D-- Rh complexes.

BACKGROUND AND OBJECTIVES: Previously, the Evans antigen (RH37) of the Rh blood group system was shown to be specified by a novel CE-D-CE hybrid gene. We studied further the heterogeneity of Evans and report here its new molecular type resulting from a novel intraexon fusion event on the background of D. and D-- complexes. MATERIALS AND METHODS: A white family with 2 Evans+ and 2 Evans- members was analyzed by serological methods and molecular techniques. RESULTS: The Evans+ proband (JD) typed D+C-c-E-e- and showed a partial loss of RHCE but an increased dose of RHD on DNA blots. On sequencing of Rh cDNAs, a normal D and 3 hybrid transcripts were detected. The D-CE hybrid is characterized by a single breakpoint located in exon 6. The CE-D hybrid derived its exon 1 (or 1 and 2) from RHCE and exons 2-10 (or 3-10) from RHD. The CE-D-CE hybrid had its internal exons 2-7 (or 3-7) from RHD. Family studies showed that the D-CE and CE-D hybrids were linked and cotransmitted from JD to his son, whereas RHD and CE-D-CE hybrid were transmitted from JD to his daughter. CONCLUSION: In this family, Evans is specified by the novel D-CE intraexon fusion gene which occurs in cis to CE-D and trans to CE-D-CE interexon fusion genes. The observed large duplication of RHD reflects a convergent mosaicism underlying the enhancement of D and ablation of CcEe antigens.

Amino Acid Sequence↗
PubMed2000

The MNS blood group antigens, Vr (MNS12) and Mt(a) (MNS14), each arise from an amino acid substitution on glycophorin A.

BACKGROUND AND OBJECTIVES: The antigens, Vr (MNS12) and Mt(a) (MNS14), are low-incidence antigens of the MNS blood group system. The Vr antigen has been found only on the red blood cells (RBCs) of persons of Dutch ancestry whereas the Mt(a) antigen has been found on the RBCs of persons from a wide geographic distribution. The objective of this study was to determine the molecular basis of Vr and Mt(a). MATERIALS AND METHODS: Following RT-PCR amplification of total RNA isolated from one Vr+ person (G488) and one Mt(a+) person (GH), the genes encoding glycophorin A (GYPA) and glycophorin B (GYPB) were cloned and sequenced. To confirm the point mutation observed in the cDNA from G488 (Vr+), GYPA exon 3 was cloned and sequenced from the genomic DNA of G488 and a second unrelated Vr+ person (MU). A restriction fragment length polymorphism (RFLP) assay was used to analyze genomic DNA from 11 Mt(a+) persons (10 unrelated) following PCR amplification of GYPA exon 3. RESULTS: The coding sequence of GYPB was normal in both G488 (Vr+) and GH (Mt(a+)). Sequencing data from GYPA clones derived from G488 showed to full length GYPA sequences: A normal GYPA M allele and a GYPA M allele with a point mutation 197C-->A. Sequencing of GYPA exon 3 from G488 and MU confirmed the point mutation. Sequencing data drom GYPA clones derived from GH showed two full length GYPA sequences: a normal GYPA M allele and a GYPA N allele with a point mutation 230C-->T. RFLP analysis based on the point mutation showed that DNA from 11 Mt(a+) samples were heterozygous for the point mutation. CONCLUSION: The Vr antigen arises from a point mutation 197C-->A on GYPA which is predicted to change serine at position 47 to tyrosine. This change introduces a new alpha-chymotrypsin cleavage site. The Mt(a) antigen arises from a point mutation 230C-->T which is predicted to change threonine at position 58 to isoleucine.

Amino Acid Substitution↗
PubMed2000

Four examples of anti-TSEN and three of TSEN-positive erythrocytes.

BACKGROUND: TSEN (MNS33) is a low-incidence antigen in the MNS blood group system encoded by hybrid glycophorin genes. TSEN is expressed by a unique amino acid sequence that results from the junction of GPA(58) to GPB(27), if the GPB carries S antigen. Until this study, only one example of anti-TSEN had been found. Antibody screening red blood cells (RBCs) positive for both S and s (ref. No. C873) reacted with four patient sera. Initially, the RBCs had been typed as S+s+, but later were typed as S-s+ in another laboratory. Two other RBC samples, one from a volunteer blood donor (D.L.), the other from a patient whose serum contained anti-En(a)FR (J.S.), also gave anomalous results when tested with anti-S. We suspected the presence of TSEN-positive hybrids on all three RBC samples. MATERIALS AND METHODS: Reactive sera (O.B., E.C., S.K., R.F.) were tested against RBCs with normal MNS phenotypes and with TSEN-positive RBCs. The RBCs of D.L., J.S. and C873 were tested with anti-S whose reactivity with S+s+ TSEN+ RBCs had been established previously, and with the original example of anti-TSEN. Immunoblotting was performed on the C873, D.L. and J.S. RBC membranes using a monoclonal antibody to an epitope common to both glycophorin A and glycophorin B. RESULTS: The sera from O.B., E.C., S.K. and R.F. were strongly reactive on the indirect antiglobulin test with TSEN+ RBCs. The RBCs of C873, D.L. and J.S. were typed as TSEN+. Immunoblotting pattern of D.L. and C873 were consistent with TSEN heterozygotes, while that of J.S. was consistent with a TSEN homozygote. CONCLUSIONS: Based on the estimated number of screening events with C873 RBCs, the incidence of anti-TSEN is approximately 1 in 20,000 sera. The antibody is found in patients with and without documented exposure to allogeneic RBCs. All known examples of anti-TSEN are IgG, but their clinical significance is not known.

Adult↗
PubMed2000

First example of hemolytic disease of the newborn caused by anti-Or and confirmation of the molecular basis of Or.

BACKGROUND AND OBJECTIVES: The rare MNS antigen Or (MNS31) is sensitive to ficin, papain and sialidase, but partially resistant to trypsin (0.05%); the effect of alpha-chymotrypsin is not known. A point mutation, 204C --> T in exon 3 of GYPA, is associated with the Or+ phenotype. We report here the first case of hemolytic disease of the newborn (HDN) caused by anti-Or, and expand the information on the nature of the Or determinant. MATERIALS AND METHODS: A woman, gravida 4, para 0, delivered a baby whose red blood cells (RBCs) were positive (2+) on the direct antiglobulin test (DAT). The mother's serum, an eluate made from the baby's RBCs and the RBCs of the baby's father were investigated. Exon 3 of GYPA, extracted from the father's genomic DNA, was amplified and sequenced. RESULTS: The mother's serum reacted at room temperature, 37 degrees C and on the indirect antiglobulin test with RBCs from the baby's father. The father's RBCs were M+N+S-s+Or+. The antibody in the mother's serum and in the baby's eluate was identified as anti-Or. The serum did not react with the father's RBCs treated with trypsin (180,000 U/ml), but did react with his alpha-chymotrypsin-treated RBCs. Amplification and sequencing of DNA from the father revealed a single point mutation, 204C --> T, in GYPA exon 3. At birth, the baby had clinical symptoms of HDN and was transfused with 36 ml of packed RBCs and received phototherapy for eight days. At week 11, the baby's M+N+S+s+Or+ RBCs were negative on the DAT. CONCLUSION: This is the first case of HDN caused by anti-Or. The observed point mutation, 204C --> T, confirms that of a previous report and predicts a change of Arg (Or-) to Trp (Or+) at amino acid 31.

Adult↗