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Biomedical subjects

M E Reid

Publications and source records attributed to M E Reid.

At least 109 records · Page 6Linked to original sources
PubMed1998

Rhnull disease: the amorph type results from a novel double mutation in RhCe gene on D-negative background.

Rhnull disease, which includes the amorph and regulator types, is a rare genetic disorder characterized by stomatocytosis and chronic hemolytic anemia. We studied here a German family transmitting a putative amorph Rhnull disease gene and identified a rare mutation causing the loss-of-function phenotype. We analyzed the genomic and transcript structure of RH30, RH50, and CD47, the three loci thought to be most critical for expression of the Rh complex in the red blood cell membrane. We showed that in this family the Rh50 and CD47 transcripts were normal in primary sequence. However, the RH30 locus contained an unusual double mutation in exon 7 of the RhCe gene, in addition to a deletion of the RhD gene. The mutation targeted two adjacent codons in multiple arrangements probably via the mechanism of microgene conversion. One scheme entails a noncontiguous deletion of two nucleotides, [ATT(Ile322)-->AT] and [CAC(His323)-->CC], whereas the other involves a T-->C transition [ATT(Ile322)--> ATC] and a dinucleotide deletion [CAC(His323)-->C]. They caused the same shift in open reading frame predicted to encode a shortened protein with 398 amino acids. The loss of two transmembrane domains and gain of a new C-terminal sequence are likely to alter the protein conformation and impair the Rh complex assembly. Our findings establish the molecular identity of an amorph Rhnull disease gene, showing that Rh30 and Rh50 are both essential for the functioning of the Rh structures as a multisubunit complex in the plasma membrane.

Amino Acid Sequence↗
PubMed1998

Chromosome location of genes encoding human blood groups.

The chromosomal locations of genes controlling the expression of some 200 antigens constituting the 23 established human blood group systems have been reviewed. Twenty-one of the these genes are located on 12 autosomes, and two are located on the X chromosome. Refined chromosomal positions, to a single cytogenetically distinguishable band, have been established for 13 of the 23 genes. For the remainder, continued investigation will achieve the same result. The genes (RD, MER2, and OK) controlling the expression of one low-incidence and two high-incidence erythrocyte antigens have also been presented. Of these, OK is the most likely candidate for blood group system status, because its chromosomal location distinguishes it from all established system genes except LE and LW, and, the product of the OK gene is different from those of LE and LW (Table 3). This issue will be considered at the next meeting (scheduled for July 1998) of the ISBT Working Party. Alternatively, RD and MER2 are not good candidates for blood group system status because RD and MER2 reside in chromosomal regions containing genes for other blood group systems. In addition, the products of RD and SC have similar biochemical characteristics, and the product of MER2 has not yet been defined (Table 3). The challenge remaining for blood group scientists is characterization of genes that control expression of the approximately 50 other known erythrocyte antigens. Most of these are members of the ISBT's 700 (low-incidence) or 901 (high-incidence) series. Because the current genetic information for each of these antigens (attained by serologic investigation) varies considerably, future studies will have to rely on "tools" from related disciplines to provide the additional information. Use of resources such as molecular biological protocols and GBD should facilitate the effort.

Blood Group Antigens↗
PubMed1998

Molecular insights into blood groups and implications for blood transfusion.

In recent years technologic advances have led to the understanding of the molecular genetics of many blood group antigens. This review highlights the molecular basis of selected antigens and describes implications for transfusion medicine. The knowledge can be used in the following clinical settings to genotype a patient: 1) to identify a fetus at risk for hemolytic disease of the newborn 2) to genotype patients who have been recently transfused or whose erythrocytes have a positive direct antiglobulin test 3) to determine which phenotypically Fy(b-) patients have the FY*B gene, 4) to genotype when an antigen has a depressed expression on erythrocytes, 5) to mass screen for anitgen-negative donors, 6) to resolve blood group A, B, and D typing discrepancies, 7) to determine the zygosity of RhD, 8) to determine the origin of engrafted leukocytes in a stem cell recipient, 9) to determine the origin of lymphocytes in a patient with graft-versus-disease, 10) for tissue typing, 11) for paternity testing, and 12) for forensic testing.

Blood Group Antigens↗
PubMed1998

Use of LOR-15C9 monoclonal anti-D to differentiate erythrocytes with the partial DvI antigen from those with either partial D antigens or weak D antigens.

Historically, red blood cells (RBCs) with partial D antigens have been defined serologically by their pattern of reactivity with polyclonal and monoclonal anti-D. Although numerous variants have been described in tests with well-characterized monoclonal anti-D, definition remains difficult to ascertain serologically. RBCs of known partial D type were tested with LOR-15C9 (a monoclonal anti-D) and commercial anti-D by the tube indirect antiglobulin test (IAT), by micro typing system IgG gel cards, and by immunoblotting. By IAT, LOR-15C9 reacted strongly with DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs in addition to RBCs with common D antigens; weakly with DII, DNU, and DIIIb RBCs; and not at all with DIVa, DIVb, DBT, or R0 Har RBCs. Reactivity was variable (1+ to 4+), with RBCs classified as weak D (Du). As expected, the commercial anti-D agglutinated all D variants and weak D RBC samples by the IAT and by using IgG gel cards; however, the reactivity with DVI RBCs was weaker than with LOR- 15C9. By immunoblotting, LOR-15C9 detected a band with an apparent molecular mass of approximate Mr 30,000-34,000 in membranes prepared from D-positive, DIIIa, DIIIc, DVa, DVI, DVII, and DFR RBCs and an additional band of Mr 20,000-22,000 in membranes prepared from DVI RBCs. No band(s) was detected in membranes from DII, DNU, DIIIb, DIVa, DIVb, DBT, R0 Har, weak D, or D-negative samples. LOR-15C9 provides a useful tool to identify positively DVI samples and thereby differentiate this partial D from other D variants and from weak D samples.

Journal Article↗
PubMed1998

Characterization of a macaque anti-Rh17-like monoclonal antibody.

In order to produce macaque monoclonal antibodies (mAbs) against human red blood cell (RBC) antigens, macaques were immunized with human and gorilla RBCs and their spleen lymphocytes were fused with man-mouse heteromyeloma cells. One macaque-mouse heterohybridoma produced a macaque IgGx (Cvn2-4D5) which agglutinated all human RBCs but not rare human variants Dc-,D-, and Rhnull. Thus, Cyn2-4D5 exhibited RH17-like reactivity. The specificity of Cyn2-4D5 for RHCE-encoded polypeptides was confirmed by specific immunoprecipitation of RhcE and RhCe polypeptides from human RBCs and the absence of immunoprecipitation of the RhD polypeptides extracted from D-RBCs. This study demonstrates that it is possible to produce macaque mAbs against human RBC blood group antigens.

Animals↗
PubMed1998

Design and baseline characteristics of study participants in the Wheat Bran Fiber trial.

The Wheat Bran Fiber (WBF) trial is a Phase III clinical trial designed to assess the effect of a WBF intervention for 3 years on the recurrence of adenomatous polyps. Men and women, 40-80 years of age, who had removal of one or more colorectal adenoma(s) 3 mm or larger within 3 months prior to study entry were recruited from three sites in the Phoenix metropolitan area. After meeting eligibility criteria, 1509 individuals entered a 6-week run-in period, consisting of a low WBF (2 g/day) intervention. Participants (n = 1429) successfully completed this phase and were randomized to a high (13.5 g/day) or low (2 g/day) WBF intervention. Various data and specimens were collected at baseline and throughout the intervention phase, which included dietary intake, physical activity, other risk factor information, blood specimens, rectal biopsies, and polyp tissues. The study design called for a colonoscopy at approximately 1 year after the qualifying colonoscopy; thus, the period between the first year and the final colonoscopy will be used to assess the effect of the intervention, which is expected to be completed in the latter part of 1998.

Adenomatous Polyps↗
PubMed1998

Two examples of an inseparable antibody that reacts equally well with DW+ and Rh32+ red blood cells.

BACKGROUND AND OBJECTIVES: Anti-DW is a rare specificity that detects an antigen on DVa red blood cells (RBCs). Some anti-DW contain an inseparable component that cross-reacts weakly with RBCs expressing the low-incidence Rh antigen Rh32. RH32 is expressed by RBCs with either the R=Nor the DBT phenotype. CASE REPORT: We describe here an antibody found in the serum of 2 patients that reacts equally well with RBCs possessing either DVa, R=N, or DBT phenotypes. The reactivity for DW and Rh32 antigens could not be separated by adsorption onto and elution from DW+Rh32- or from DW-Rh32+ RBCs. CONCLUSIONS: We suggest that amino acids encoded by nucleotides at the junction of exon 4 of RHD to exon 5 of RHCE may induce a conformation that is recognized by these equally reactive inseparable antibodies. Until such time that the epitope recognized by these antibodies is defined, we recommend use of the descriptive name anti-DW/Rh32.

Agglutinins↗
PubMed1997

Alternative splicing of a novel glycophorin allele GPHe(GL) generates two protein isoforms in the human erythrocyte membrane.

The Henshaw antigen (synonym: He or MNS6) is carried by an altered form of glycophorin B (GPB), but the molecular basis for its variable expression or quantitative polymorphism remains largely undefined. We report here the identification and analysis of a novel glycophorin He allele, GPHe(GL), which gives rise to the expression of two protein isoforms in the erythrocyte membrane. In addition to the nucleotide changes defining the epitopic sequence of He, a single C-to-G nucleotide transversion in exon V coding for the membrane domain was found to cause aberrant RNA splicings by creating a new acceptor splice site. In addition, a T-to-G transversion at -6 position of the acceptor splice site for exon IV was identified. Both full-length and truncated transcripts of GPHe(GL) were detected as the result of partial activation of the new acceptor splice site and partial inactivation of the normal splice sites. The full-length cDNA encoded He, S, and U antigens, whereas the three truncated ones lacked either the sequence for S and U antigens or a large portion of the membrane domain or both. The GPB gene on the other chromosome was apparently normal and its transcript encoded N, s, and U antigens. These results correlate alternative RNA splicing with the expression of two GPHe isoforms and thus delineate a new mechanism for the phenotypic diversity of membrane glycophorins.

Alleles↗
PubMed1997

The glycophorin A gene family in gorillas: structure, expression, and comparison with the human and chimpanzee homologues.

Homologues of MN blood group antigens, encoded by members of the glycophorin A (GPA) gene family, are expressed in man, anthropoid apes, and some species of Old World monkeys. Previous studies had shown that a three-gene framework, most closely related to that in man, is present in the chimpanzee. Here we report the genomic structure, transcript map, and protein expression of the GYPA locus in gorillas. Compared to the corresponding human and chimpanzee homologues, gorilla GPA, GPB, and GPB/E genes each showed a high degree of sequence identity, with the same exon-intron organization. However, the expression of exons III, IV or V encoding the extracellular or membrane domains of homologous glycophorins varied among the three species. Gorilla GPA and GPB/E genes were unique in that the former occurred in two allelic forms with or without the expression of exon III, whereas the latter contained one (psi exon III) instead of two silenced exons (psi exons III and IV). Differences from human but not chimpanzee GPA also included the presence of a hybrid M/N epitope and the absence of the sequon for N-glycosylation. Owing to the retention of a functional exon III, gorilla GPB was more similar to chimpanzee GPB than human GPB. A transspecies allele was identified in the gorilla that gave rise to the Henshaw (He)-like antigen similar to that found in man. These results provide further insight into the model for evolution of the GPA gene family, indicating that the mechanisms underlying inter- and intraspecific polymorphism of glycophorins could predate the divergence of gorillas as the consequence of gene duplication and diversification.

Amino Acid Sequence↗
PubMed1997

A human monoclonal anti-D antibody which detects a nonconformation-dependent epitope on the RhD protein by immunoblot.

We describe the first human monoclonal anti-D (LOR-15C9) which reacts with a D-specific motif exposed either on a native form on intact D-positive red cells or on a denatured form of the RhD protein (33 kD), and detected by immunoblotting. LOR-15C9 was able to precipitate RhD but not RhcE proteins produced by in vitro transcription-translation assays. The reactivity of the antibody, using panels of red cells with various partial D phenotypes known to lack some D epitopes and corresponding in RHD gene variants, suggested that LOR-15C9 reactivity depends on the portion of the RhD polypeptide encoded by the exon 7 (amino acids 314-358). These findings correlate well with the reactivity of LOR-15C9 with erythrocytes of some nonhuman primates (D(gor)-positive gorillas), but not of chimpanzee and Old or New World monkeys. In membrane proteins from partial D(VI) red cells, LOR-15C9 detected two proteins of molecular weight 33 and 21 kD: the presence of the latter was specific for category D(VI) and presumably represented the product of an alternatively spliced RHD(VI) transcript in these cells. This is consistent with the finding that LOR-15C9 can precipitate a shortened D protein mutant resulting from in vitro transcription-translation and lacking amino-acids 163-313 encoded by exons 4-6. In addition, a 21 kD band polypeptide was detected by immunoblot in all red cell samples but D--, using a rabbit anti-Rh polypeptide antibody (MPC8) raised against the C-terminal domain of Rh proteins. This 21 kD polypeptide most probably results from the translation of an alternatively spliced RHCE gene transcript. This study demonstrates that LOR-15C9 detects an epitope on the RhD protein that is independent of the membrane environment, and therefore could be a useful tool for the study of RhD polypeptides.

Antibodies, Monoclonal↗
PubMed1997

Rh haplotypes that make e but not hrB usually make VS.

BACKGROUND AND OBJECTIVES: The Rh phenotypes hrB- and VS+ are both rare in Whites but more common in Blacks. The high-incidence antigen hrB is present on most red cells that are e+. The presence of VS on red cells is associated with an aberrant expression of e, often called eS. MATERIALS AND METHODS: Using conventional serologic methods, including a monoclonal anti-hrB-like antibody, we studied 65 e+ samples that were apparently hrB-. RESULTS: Of the 65, we found that 59 (91%) were VS+. Recent findings have indicated that in VS+ persons a change from leucine to valine occurs at amino acid 245 of the RHCE-encoded polypeptide. While this residue is predicted to lie within the red cell membrane bilayer, the change presumably affects alanine 226 (that is present when e is expressed) in such a way that eS is seen. CONCLUSIONS: Our findings suggest that the change from e to eS may result in nonexpression or marked depression of expression of hrB that is, perhaps, an epitope of e. While the molecular basis of the hrB-phenotype is not known, it is unlikely that the leucine-to-valine change at residue 245, resulting in the aberrant from of e, explains all hrB-samples. First, hrB-VS+ and hrB- VS- samples must differ. Second, some hrB- VS+ samples are C+, some are C-. Presumably diverse molecular bases are involved in hrB-phenotypes.

Black People↗
PubMed1997

Co-presence of a point mutation and a deletion of exon 3 in the glycophorin C gene and concomitant production of a Gerbich-related antibody.

BACKGROUND: Antigens of the human blood group system Gerbich (Ge) are located on sialoglycoproteins glycophorin C (GPC) and glycophorin D (GPD). CASE REPORT: The Ge+ proposita (RW) produced an alloanti-Ge after receiving 2 units of red cells (RBCs) during surgery. Further studies were carried out to characterize the antibody specificity, RBC GPC and/or GPD (GPC/GPD), and the glycophorin C gene (GYPC) from RW and her compatible siblings. RW's serum contained an alloanti-Ge that did not react with RBCs from RW or four of her siblings or with RBCs with Ge-negative phenotypes. An eluate of RW's antibody reacted weakly with GPC in Western blotting. RW's RBCs were positive with 20 alloanti-Ge2, 1 autoanti-Ge2, 4 alloanti-Ge3, and 1 alloanti-Ge4. Titrations revealed weak expression of these antigens on her RBCs and those of her compatible siblings as compared with controls. In contrast, titrations of mouse and rat monoclonal antibodies specific for GPC/GPD showed no differences. Western blotting of RBC membranes using GPC/GPD specific monoclonal antibodies showed a broad diffuse band corresponding to GPC.Ge in addition to GPC and GPD. Blotting of membranes from trypsin-treated RBCs from these individuals revealed an increase of 1500 in M(r) of membrane-bound tryptic fragment over that in the membranes from typsin-treated RBCs from persons with normal GPC/GPD. In RT-PCR, two products were obtained for RW and her compatible siblings: one had a complete deletion of exon 3 and the other had a base change (A-->T) in nucleotide 173 in exon 3 (confirmed by genomic DNA sequencing of exon 3). This point mutation has resulted in the loss of restriction enzyme Tth111 I-sensitive site in the mutant GYPC. CONCLUSION: The specificity of antibody in RW's serum was serologically anti-Ge2. Two genetic events occurred in exon 3 in GYPC of RW and her compatible siblings. The exon 3 deletion confirmed a Ge:-2,-3,4 haplotype. The abnormal tryptic fragment obtained was due to the (A173-->T) base change in exon 3 that resulted in Asp58-->Val in the deduced amino acid sequence at the membrane boundary.

Animals↗
PubMed1997

Practical method for determination of the U status of S-s- erythrocytes.

Red blood cells (RBCs) lacking S and s blood group antigens are classified as S-s-U- or S-s-U+var but the classification may vary due to the characteristics of anti-U and to the technique used. Tests on RBCs known to lack glycophorin B (GPB) or to possess an altered form of GPB showed that polyethylene glycol-indirect antiglobulin testing or MicroTyping Systems (MTS)-gel techniques can be used as a simple and reliable way to detect RBCs with variant forms of GPB. This approach can be important in the selection of rare RBCs for transfusion to patients with undifferentiated anti-U.

Journal Article↗
PubMed1996

Human red blood cell Wright antigens: a genetic and evolutionary perspective on glycophorin A-band 3 interaction.

The Wright (Wra/Wrb) blood group polymorphism is defined by an allelic change (Lys658Glu) in the band 3 protein; nevertheless, the Wrb antigen apparently requires glycophorin A (GPA) for surface presentation. To gain insight into the structural basis for this protein-protein interaction and delineate its relationship with Wrb antigen expression, we investigated GPA and band 3 sequence polymorphisms occurring in rare humans and nonhuman primates. The lack of GPA or amino acid residues 59 through 71 of GPA results in the absence of Wrb from human red blood cells (RBCs) exhibiting the MkMk, En(a-), or MiV phenotype. However, the SAT homozygous cells carried a Glu658 form of band 3 and a hybrid glycophorin with the entire GPA extramembrane domain from residues 1 through 71, yet expressed no Wrb antigen. This finding suggests that formation of the Wrb antigenic structure is dependent on protein folding and that the transmembrane junction of GPA is important in maintaining the required conformation. Comparative analyses of GPA and band 3 homologues led to the identification in the interacting regions of conserved and dispensable amino acid residues that correlated with the Wrb positive or negative status on nonhuman primates. In particular, the chimpanzee RBCs cells expressed Wrb and the Glu658 form of band 3, which is identical to humans, but their GPA contained the Gly rather than Arg residue at position 61. Taken together, the results suggest that (1) Arg61 of GPA and the proposed Arg61-Glu658 charge pair are not crucial for Wrb antigen exhibition and (2) the role of GPA for interaction with band 3, including Glu658, probably involves a number of amino acid residues located in the alpha-helical region and transmembrane junction.

Amino Acid Sequence↗