Glycophorin Variants Research Articles

Erythroid anion transport, nitric oxide, and blood pressure.

Glycophorin A and glycophorin B are structural membrane glycoproteins bound in the band 3 multiprotein complexes on human red blood cells (RBCs). Band 3 is an erythroid-specific anion exchanger (AE1). AE1-mediated HCO3 - transport provides the substrate for the enzyme-catalyzed conversion HCO3 - (aq) ⇌ CO2(g), which takes place inside the RBCs. Bicarbonate transport via AE1 supports intravascular acid-base homeostasis and respiratory excretion of CO2. In the past decade, we conducted several comparative physiology studies on Taiwanese people having the glycophorin variant GPMur RBC type (which accompanies greater AE1 expression). We found that increased anion transport across the erythrocyte membrane not only enhances gas exchange and lung functions but also elevates blood pressure (BP) and reduces nitric oxide (NO)-dependent vasodilation and exhaled NO fraction (FeNO) in healthy individuals with GP.Mur. Notably, in people carrying the GPMur blood type, the BP and NO-dependent, flow-mediated vasodilation (FMD) are both more strongly correlated with individual hemoglobin (Hb) levels. As blood NO and nitrite (NO2 -) are predominantly scavenged by intraerythrocytic Hb, and NO2 - primarily enters RBCs via AE1, could a more monoanion-permeable RBC membrane (i.e., GPMur/increased AE1) enhance NO2 -/NO3 - permeability and Hb scavenging of NO2 - and NO to affect blood pressure? In this perspective, a working model is proposed for the potential role of AE1 in intravascular NO availability, blood pressure, and clinical relevance.

PCR-based screening of MNS glycophorin variants in patients suspected of having anti-Mia

Background:MNS glycophorin variants such as glycoprotein (GP.) Mur, GP.Hop, GP.Bun, GP.HF, GP.Hut, and GP.Vw are known to carry multiple antigens, including Mia (MNS7). In East and Southeast Asians, an antibody to Mia is not only commonly found, but also clinically significant. Purpose and Methods: In this study, deoxyribonucleic acid (DNA) samples from 47 patients suspected of having anti-Mia by serological screening were genotyped for GP.Mur, GP.Hop, GP.Bun, GP.HF, GP.Hut, and GP.Vw variants using polymerase chain reaction (PCR) assay. Results: All patients were found to be negative for the presence of Mia-positive glycophorin variants. Conclusions: The presence of anti-Mia cannot be excluded in patients studied, as genotyping shows that Mia-positive MNS glycophorin variants are not present. Bangladesh Journal of Medical Science Vol. 21 No. 02 April’22 Page : 432-437

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

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. 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. The GP.HF, GP.Mur, GP.Hut, GP.Vw, GP.Kip and GP.Bun frequencies in 1005594 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 13546 individuals (0·0295%). Of 137370 donors, 10 had anti-Hil (0·0073%) and three had anti-MINY (0·0022%). 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.

Molecular Detection of Glycophorins A and B Variant Phenotypes and their Clinical Relevance.

Crossover or conversion between the homologous regions of glycophorin A (GYPA) and glycophorin B (GYPB) gives rise to several different hybrid glycophorin genes encoding a number of different glycophorin variant phenotypes which bear low prevalence antigens in the MNS blood group system. GP.Mur is the main glycophorin variant phenotype which causes hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN) in East and Southeast Asians. The detection of glycophorin variant phenotypes using serological methods is limited to phenotyping reagents that are not commercially available. Moreover, the red blood cells used for antibody identification are usually of the GP.Mur phenotype. The current Polymerase Chain Reaction (PCR)-based methods and loop-mediated isothermal amplification (LAMP) are available alternatives to phenotyping that allow for the specific detection of glycophorin variant phenotypes. This review highlights the molecular detection method for glycophorins A and B variant phenotypes and their clinical relevance.

An update on the MNS blood group system

This update of the MNS blood group system (Reid ME. MNS blood group system: a review. Immunohematology 2009;25:95-101) reports three new antigens of the MNS system numbered MNS47, MNS48, and MNS49; new glycophorin (GP) variants associated with silent and weak expression of MNS antigens; and the results of new studies on associations of MNS antigens with band 3, Rh proteins, and malaria. The addition of these three antigens brings the total number of antigens in the MNS system (International Society of Blood Transfusion system 2) to 49.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of 36 blood group alleles among 396 Thai samples reveals region-specific variants.

Blood group phenotype variation has been attributed to potential resistance to pathogen invasion. Variation was mapped in blood donors from Lampang (northern region) and Saraburi (central region), Thailand, where malaria is endemic. The previously unknown blood group allele profiles were characterized and the data were correlated with phenotypes. The high incidence of the Vel-negative phenotype previously reported in Thais was investigated. DNA from 396 blood donors was analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. Outliers were investigated by serology and DNA sequencing. Allele discrimination assays for SMIM1 rs1175550A/G and ACKR1 rs118062001C/T were performed and correlated with antigen expression. All samples were phenotyped for Rh, MNS, and K. Genotyping/phenotyping for RhD, K, and S/s showed 100% concordance. Investigation of three RHCE outliers revealed an e-variant antigen encoded by RHCE*02.22. Screening for rs147357308 (RHCE c.667T) revealed a frequency of 3.3%. MN typing discrepancies in 41 samples revealed glycophorin variants, of which 40 of 41 were due to Mia . Nine samples (2.3%) were heterozygous for FY*01W.01 (c.265C > T), and six samples (1.5%) were heterozygous for JK*02N.01. All samples were wildtype SMIM1 homozygotes with 97% homozygosity for rs1175550A. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is an efficient method for rapid routine genotyping and investigation of outliers identified novel variation among our samples. The expected high prevalence of the Mi(a+) phenotype was observed from both regions. Of potential clinical relevance in a region where transfusion-dependent thalassemia is common, we identified two RHCE*02 alleles known to encode an e-variant antigen.

A direct blood polymerase chain reaction approach for the determination of GP.Mur (Mi.III) and other Hil+ Miltenberger glycophorin variants

GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs. To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR. Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing. This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.

Miltenberger phenotypes are glycophorin variants: a review

Miltenberger phenotypes are glycophorin variants: a review

Prevalence and specificity of red‐blood‐cell antibodies in a multiethnic South and East Asian patient population and influence of using novel MUT+Mur+ kodecytes on its detection

Appropriate screening for irregular red-cell antibodies is essential for ensuring transfusion compatibility and for antenatal management of mothers at risk of haemolytic disease of the foetus and newborn. Screening for all relevant antibodies is, however, limited by screening cells that do not express antigens present in the patient and donor population. Technology to artificially incorporate antigens into red cells is currently available and may be an option for customizing screening cells. We sought to identify retrospectively the changing patterns of alloantibody prevalence in our multiethnic population on change of screening cells. Antibody screening records of 143 501 patients tested from 2004 to 2010 were retrieved and divided into two groups: period-1 (2004-2008) and period-2 (2009-2010). During period-1, standard screening cells were used while in period-2, MUT+Mur+ KODE(™) transformed red cells (kodecytes) were used. Four per cent of samples tested during period-2 were positive on antibody screening compared to 3·2% in period-1. Specific antibodies, excluding anti-D, were identified in 1·66% and 1·52% of patients in period-2 and -1, respectively. When confined to antibodies of clinical significance only, period-2 showed higher alloantibody prevalence of 1·16% as compared to 0·66% in period-1. Antibodies to glycophorin variants of MNS (vMNS) were more commonly detected while antibodies to Lewis antigens declined during period-2. Antibodies to vMNS antigens are common in South and East Asian populations and are often missed when using standard screening cells. Use of specifically engineered screening cells to express red-cell antigens artificially is beneficial in detecting the diverse alloantibodies present in our population.

Genomic typing of human red cell miltenberger glycophorins in a Taiwanese population.

Antigens in the human red cell Miltenberger series are glycophorin variants of the MN (MNS) blood group system that are due to the rearrangement of glycophorin A (GPA) and glycophorin B (GPB) genes. Taking advantage of the differences between the GPA and GPB genes, a polymerase chain reaction-based method was developed to detect all the Miltenberger glycophorin variants and St(a) subtype. GPA- and GPB-specific primers were used to amplify the GPA or GPB gene, and the amplified products were used to recognize the different hybrid genes after restriction enzyme digestions. Among 264 Taiwanese subjects studied, Mi.III and St(a) are the most common types of Miltenberger variants found. Mi.III was present in 13 (4.92%) of 264, and St(a) was found in 8 (3. 03%) of 264; 1 case (0.4%) of Mi.V was also identified from the study group. This is the first polymerase chain reaction-based method of detecting most of the Miltenberger variants and St(a). The genomic typing results were confirmed by control DNA of identified Miltenberger phenotypes. The prevalence rates of Mi. III and St(a) in this study were also consistent with other previous reports using different methods.

Glycophorin He(Sta) of the human red blood cell membrane is encoded by a complex hybrid gene resulting from two recombinational events

A complex glycophorin (GP) variant of the human red blood cell membrane exhibiting both He and Sta antigens was characterized at the molecular level. Restriction mapping identified two novel Msp I fragments derived from the 5′ and 3′ portions of the GPHe(Sta) gene, respectively. Genomic DNA, including exons II-V and their splice junctions, was amplified by polymerase chain reaction, and the nucleotide sequences were determined. Comparison with the GPA and GPB sequences showed the presence in GPHe(Sta) of multiple recombinational breakpoints. In the 5′ region of the variant gene, a sequence covering a portion of exon II to intron 2 had been transferred from GPA to GPB, resulting in a B-A-B hybrid structure. Such a gene conversion-like event introduced a number of templated and untemplated nucleotide replacements and was the direct cause for the expression of the He antigen. In the 3′ region of the variant gene, an unequal crossover from GPB to GPA took place in the third intron at a recombination site apparently identical to that observed in the B-A hybrid GPSta type A gene. These results indicated that GPHe(Sta) occurs as a B-A-B-A hybrid gene, most likely originating from a two-step mechanism of homologous recombination. Transcript analysis showed the maturation from the GPHe(Sta) pre-mRNA of two shortened mRNAs of which the exon III-deleted species encodes both the He and Sta antigens.

Glycophorin He(Sta) of the Human Red Blood Cell Membrane Is Encoded by a Complex Hybrid Gene Resulting From Two Recombinational Events

A complex glycophorin (GP) variant of the human red blood cell membrane exhibiting both He and Sta antigens was characterized at the molecular level. Restriction mapping identified two novel Msp I fragments derived from the 5' and 3' portions of the GPHe(Sta) gene, respectively. Genomic DNA, including exons II-V and their splice junctions, was amplified by polymerase chain reaction, and the nucleotide sequences were determined. Comparison with the GPA and GPB sequences showed the presence in GPHe(Sta) of multiple recombinational breakpoints. In the 5' region of the variant gene, a sequence covering a portion of exon II to intron 2 had been transferred from GPA to GPB, resulting in a B-A-B hybrid structure. Such a gene conversion-like event introduced a number of templated and untemplated nucleotide replacements and was the direct cause for the expression of the He antigen. In the 3' region of the variant gene, an unequal crossover from GPB to GPA took place in the third intron at a recombination site apparently identical to that observed in the B-A hybrid GPSta type A gene. These results indicated that GPHe(Sta) occurs as a B-A-B-A hybrid gene, most likely originating from a two-step mechanism of homologous recombination. Transcript analysis showed the maturation from the GPHe(Sta) pre-mRNA of two shortened mRNAs of which the exon III-deleted species encodes both the He and Sta antigens.

A novel gene member of the human glycophorin A and B gene family

A new gene closely related to the glycophorin A (GPA) and glycophorin B (GPB) genes has been identified in the normal human genome as well as in that of persons with known alterations of GPA and/or GPB expression. This gene, called glycophorin E (GPE), is transcribed into a 0.6-kb message which encodes a 78-amino-acid protein with a putative leader peptide of 19 residues. The first 26 amino acids of the mature protein are identical to those of M-type glycophorin A (GPA), but the C-terminal domain (residues 27-59) differs significantly from those of glycophorins A and B (GPA and GPB). The GPE gene consists of four exons distributed over 30 kb of DNA, and its nucleotide sequence is homologous to those of the GPA and GPB genes in the 5' region, up to exon 3. Because of branch and splice site mutations, the GPE gene contains a large intron sequence partially used as exons in GPA and GPB genes. Compared to its counterpart in the GPB gene, exon 3 of the GPE gene contains several point mutations, an insertion of 24 bp, and a stop codon which shortens the reading frame. Downstream from exon 3, the GPE and the GPB sequences are virtually identical and include the same Alu repeats. Thus, it is likely that the GPE and GPB genes have evolved by a similar mechanism. From the analysis of the GPA, GPB and GPE genes in glycophorin variants [En(a-), S-s-U- and Mk], it is proposed that the three genes are organized in tandem on chromosome 4. Deletion events within this region may remove one or two structural gene(s) and may generate new hybrid structures in which the promoter region of one gene is positioned upstream from the body of another gene of the same family. This model of gene organization provides a basis with which to explain the diversity of the glycophorin gene family.

Molecular analysis of a hybrid gene encoding human glycophorin variant Miltenberger V-like molecule.

The genomic structure of a human glycophorin variant, Miltenberger class V-like molecule (MiV*), was examined. Southern blot analysis of total genomic DNA revealed that the 5' half of the MiV* gene derived from glycophorin A (GPA) gene whereas the 3' half derived from glycophorin B (GPB) gene. This structure is reciprocal to another glycophorin variant, Sta, which has a GPB-GPA hybrid structure. The genomic sequences around the crossing-over point were amplified by polymerase chain reaction, and the sequences were determined. Comparison of the nucleotide sequences of the GPA, GPB, and MiV* genes indicates that the crossing-over point is located in the region around the 3' end of intron 3 of the GPA gene. This place is different from the crossing-over point for Sta, which was found to be highly homologous to that for haptoglobin-related genes. However, the nucleotide sequences within the presumptive crossing-over point for the MiV* gene were found to be homologous in a reverse orientation to the crossing-over point proposed for haptoglobin-related genes. These results suggest strongly that homologous recombination through unequal crossing over can be facilitated by specific genomic elements such as those in common for formation of MiV*, Sta, and haptoglobin-related genes. The present study also localized the gene of the third glycophorin, GPE, at chromosome 4, q31.1 band, the same locus as for the GPA and GPB genes. The results indicate that GPE was not involved in generating MiV* or Sta hybrid gene despite the fact that it is localized adjacent to the GPA and GPB genes.

Identification of the crossing-over point of a hybrid gene encoding human glycophorin variant Sta. Similarity to the crossing-over point in haptoglobin-related genes.

One of the human glycophorin variants, Stones (Sta), has been shown to be the product of a hybrid gene of which the 5'-half derived from the glycophorin B (GPB) gene whereas the 3'-half derived from the glycophorin A (GPA) gene. The present study reveals the crossing-over point of this hybrid gene from the analysis of polymerase chain reaction products. The genomic sequences encompassing the region corresponding to exon 3 to exon 4 of GPA were amplified by polymerase chain reaction with oligonucleotide primers synthesized according to GPA and GPB genomic sequences (Kudo, S., and Fukuda, M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4619-4623). After subcloning the products, the nucleotide sequences derived from GPA, GPB, and putative Sta genes were determined. Comparison of the nucleotide sequences of GPA, GPB, and Sta genes indicate that the crossing-over took place 200 base pairs upstream from the first nucleotide of exon 4. Intriguingly, the nucleotide sequence surrounding the putative crossing-over point is homologous to the crossing-over point proposed for haptoglobin genes (Maeda, N., McEvoy, S.M., Harris, H.F., Huisman, T.H.J., and Smithies, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 7395-7399). These results suggest strongly that homologous recombination through unequal crossing-over can be facilitated by specific genomic elements, such as those in common in these two crossing-over events. The present study also revealed that this Sta individual has a variant GPA gene; substitution of adenine for guanine at the nucleotide for codon 39 results in substitution of lysine for arginine at amino acid 39, and loss of an SstI restriction site.

Structural Polymorphism of Glycophorins Demonstrated by Immunoblotting Techniques

We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82-131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.

Structural polymorphism of glycophorins demonstrated by immunoblotting techniques

We have explored the polymorphism of the glycophorin system in the human erythrocyte membrane using the immunoblotting techniques and examining 52 individuals selected without prior bias as to their serologic state and ten documented serologic variants of M, N, S, s blood group system. Polyclonal antisera to alpha glycophorin and to alpha glycophorin CNBr carboxyl terminal fragment C (residues 82–131) and M and N specific monoclonal antibodies (MoAbs) were used. The first two reagents detect specific regions of the alpha glycophorin molecule and all electrophoretically resolved species of glycophorins immunologically related to alpha and delta glycophorins (delta glycophorin, [alpha-delta] hybrids and other glycophorins with an alteration in the carboxyl terminal segment); the M and N MoAbs identified the glycophorin species containing or lacking the M or N determinant in the amino terminal octapeptide structures. We find that immunoblotting confirmed in all cases the serologically determined phenotype; we also find that polymorphic forms of the glycophorin system are relatively infrequent; immunoblotting, independent from serologic testing, was capable of detecting five mutants, two most likely S-s-U-phenotypes; a new glycophorin species was detected in normal red cells with both antiglycophorin and antipeptide C sera, which is not evident with MoAbs; immunoblots of known glycophorin variants (En(a-), U-, Mg, Mi I, II, III, V, and Sta) confirmed but also extended our knowledge of the abnormal glycophorins involved; and the He+ and Wrb(-) cells showed normal patterns.