Variant Glycophorin Research Articles

Glycophorins and the MNS blood group system: a narrative review

: The MNS blood group system, International Society of Blood Transfusion (ISBT) 002, is second after the ABO system. GYPA and GYPB genes encode MNS blood group antigens carried on glycophorin A (GPA), glycophorin B (GPB), or on variant glycophorins. A third gene, GYPE, produce glycophorin E (GPE) but is not expressed. MNS antigens arise from several genetic mechanisms. Single nucleotide variants (SNVs) contribute to the diversity of the MNS system. A new antigen SUMI (MNS50), p.Thr31Pro on GPA has been described in the Japanese population. Unequal crossing-over and gene conversion are the mechanisms forming hybrid glycophorins, usually from parent genes GYPA and GYPB. GYPE also contributes to gene recombination previously only described with GYPA. Recently, however, GYPE was shown to recombine with GYPB to form a GYP(B-E-B) hybrid. A GYP(B-E-B) hybrid allele encodes a mature GP(E-B) molecule expressing a trypsin-resistant M antigen but no S/s. Another novel glycophorin GP.MOT has been described carrying Mia, Mur, MUT, and KIPP antigens. GP.MOT is encoded by a GYP(B-A) hybrid allele. Newly reported cases of haemolytic transfusion reaction (HTR) or haemolytic disease of the fetus and newborn (HDFN) due to antibodies to MNS antigens is a constant reminder of the clinical significance of the MNS system. In one HDFN case, anti-U and anti-D were detected in an Indian D–, S–s–U– mother. The S–s–U– phenotype is rare in Asians and Caucasians but it is more commonly found in the African populations. Several types of novel GYPB deletion alleles that drive the S–s–U– phenotype have been recently described. Two large GYPB deletion alleles, over 100 kb, were identified as the predominant alleles in the African population. The use of advanced DNA sequencing techniques and bioinformatic analysis has helped uncover these large gene-deletion variants. Molecular typing platforms used for MNS genotyping are also discussed in this review. In conclusion, this review considers currently recognised MNS antigens and variants, new hybrid alleles and GYPB gene deletion alleles as well as clinical case studies. These new discoveries contribute to our understanding of the complexity of the MNS system to guide decision-making in genetic analysis and transfusion medicine.

An unusual variant glycophorin expressing protease‐resistant M antigen encoded by the GYPB‐E(2‐4)‐B hybrid gene

MNS is a highly polymorphic blood group comprising 49 antigens recognized by International Society of Blood Transfusion, some of which may have been generated by genomic recombination among the closely linked genes GYPA, GYPB and GYPE. The GYPE gene has an almost identical sequence to GYPA*01 allele in exon 2 (99% homology), which accounts for M antigen. We investigated an unusual glycophorin molecule with protease-resistant M antigen. Blood samples were screened by an automated blood typing system (PK7300) using bromelain-treated red blood cells (RBCs) and murine monoclonal anti-M. The M-positive RBC samples were analysed by immunoblotting using anti-M as the primary antibody. GYPA, GYPB and GYPE genes were analysed by polymerase chain reaction (PCR), cloning and sequencing using reticulocyte mRNA and genomic DNA. Serological tests and immunoblotting revealed that 103 of the 193009 individuals (0·0534%) expressed protease-resistant M-active glycophorin having a molecular weight of 20kDa. All the 103 individuals were S+s- or S-s+. When reticulocyte mRNA from the individuals with M-active glycophorin (20kDa) was examined by PCR and cloning followed by sequencing, a novel GYPE-B hybrid transcript was identified. Long-range PCR and sequencing using genomic DNA revealed that the individuals had a GYPB-E(2-4)-B hybrid gene. This hybrid gene was predicted to encode a 59-amino-acid mature glycoprotein that expresses no S or s antigens CONCLUSIONS: The prevalence of the M-active glycophorin (20kDa) in the Japanese population is 0·0534%. This glycophorin is predicted to be a 59 amino acids polypeptide encoded by the novel GYPB-E(2-4)-B hybrid gene.

Low prevalence red blood cell antigens: transfusions, babies, and changing demographics

See article on page 870–874, in this issue

IMMUNOHEMATOLOGY: Novel antibody screening cells, MUT+Mur kodecytes, created by attaching peptides onto red blood cells

BACKGROUND: Antibody screening and identification panels are generally limited by the natural antigenic phenotypes present in their source donor population. However, the recent ability to attach peptides to the surface of cells has opened up the opportunity to create red blood cells (RBCs) with antigen profiles specifically designed for antibody screening and identification in a target population.STUDY DESIGN AND METHODS: Clinically significant antibodies to variant glycophorins (GPs) such as GP.Mur are more commonly seen in certain Asian populations. Using peptides representative of the MNS antigens MUT and Mur, RBC antibody screening cells were created using KODE cell surface engineering constructs. MUT‐, Mur‐, and MUT+Mur‐modified RBCs, known as kodecytes, were tested against monoclonal reagents and polyclonal sera with specificity for epitopes on GP.Mur‐positive RBCs.RESULTS: Kodecytes retained their normal expression of intrinsic blood group antigens while expressing the new epitopes attached by KODE technology. The MUT, Mur, and MUT+Mur kodecytes, although unreactive with the various monoclonal reagents, were appropriately reactive with polyclonal sera containing antibodies reactive with GP.Mur‐positive RBCs.CONCLUSIONS: This study used selected MUT and Mur peptides and KODE cell surface engineering technology to create MUT+Mur kodecytes suitable for the detection and identification of RBC antibodies in human serum or plasma. This technology has the potential to create a large range of specialized RBCs for antibody screening and identification.

Antigenic properties of human glycophorins--an update.

Glycophorins are complex heavily glycosylated antigens carrying peptidic and glycopeptidic epitopes. Detailed immunochemical studies showed that GPA/GPB and GPC/GPD molecules have defined sites which are particularly immunogenic. These sites include N-terminal portions of all glycophorins, internal fragments of their extracellular domains, and cytoplasmic tails. The extracellular epitopes involve directly oligosaccharide chains (e.g. blood group M- and N-related epitopes, or N-terminal epitopes of GPC) or have peptidic character, shown by the reaction of respective antibodies with synthetic peptides. Peptidic eitopes are independent of glycosylation, or are variably affected by adjacent O-glycans which may mask the epitopes or may be required for a proper exposure of an antibody binding site. Several low incidence epitopes are present on variant glycophorin molecules. Among anti-glycophorin antibodies there are the 'bispecific' ones, or antibodies recognizing an epitope formed by an interaction of two proteins (Wr(b)). Alltogether, the glycophorins serve as convenient model antigens for studying Ag-Ab interaction and a role of O-glycosylation in protein antigenic properties. Moreover, well defined specificty of monoclonal anti-glycophorin antibodies makes them more precise tools in serological investigation and identification of normal and variant antigens. Last but not least, elucidation of antigenic properties of glycophorins is important for identification and characterization of human anti-glycophorin antibodies, which in some cases create medical problems at transfusion or pregnancy.

Identification of variant glycophorins of human red cells by lectinoblotting: application to the Mi.III variant that is relatively frequent in the Taiwanese population.

Detection of normal and variant glycophorin electrophoretic bands with T- and Tn-specific lectins is based on the possibility of glycophorin transformation into T or Tn antigens by simple chemical modifications in the blot. Human red cell membrane proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted onto nitrocellulose. The blots were submitted to mild acid hydrolysis (desialylation of glycophorins exposing T antigens) and then to Smith degradation (degalactosylation of asialo-glycophorins exposing Tn antigens). The modified glycophorin bands were detected with biotinylated lectins and horseradish peroxidase-conjugated avidin. The lectins from Artocarpus integrifolia (jacalin, anti-T/Tn), Amaranthus hybridus (anti-T), Salvia sclarea (anti-Tn), and Vicia villosa (anti-Tn) were used. The lectins detected normal glycophorin bands in control and variant red cells and characteristic additional bands in Mi.III (GP.Mur) red cells. The sensitivity of the method is comparable to that obtained by immunoblotting with glycophorin monoclonal antibodies. Comparison of the electrophoretic mobility of normal and variant bands is helpful in the classification of glycophorin variants. Lectinoblotting, based on carbohydrate recognition, enables the detection in a red cell sample, with high sensitivity, of all normal and variant glycophorin bands. The method can be also applied to other purposes, such as the identification of poly-O-glycosylated glycoproteins in other cells or the characterization of glycosylation of glycophorins and other poly-O-glycosylated proteins.

Molecular genetics of the glycophorin gene family, the antigens for MNSs blood groups: Multiple gene rearrangements and modulation of splice site usage result in extensive diversification

The purpose of the review is to describe a system of human erythrocyte membrane glycoproteins exhibiting extensive diversity. Glycophorins A and B (GPA and GPB) are the antigens of the MNSs blood groups; thus individuals bearing variant glycophorins can be readily identified by serological typing. Examination of the wide array of variants of these antigens showed that they include many forms, possibly made evident by lack of constraints due to the apparent dispensability of the parent molecules. This article reviews the molecular genetics of 25 variants of the glycophorin gene family, whose common denominator is that they arise from unequal gene recombinations or gene conversions coupled to splice-site mutations. Most rearrangements occurred within a 2-kb region mainly within GPA and GPB of the gene family and only rarely within the third member, GPE. The key feature is the shuffling of sequences within two specific exons (one of which is silent), homologous in the two parent genes. This has resulted in expression of a mosaic of sequences within this region, leading to polymorphism. The common pattern of recombinations coupled to pre-mRNA splicing was the predominant mechanism of the origin of glycophorin diversity. Thus far this mechanism appears to be unique among human gene families. It could have occurred by chance rearrangements among closely linked genes and been driven by a biological advantage, not as yet identified. This remains to be established. Nevertheless, gene rearrangements observed here are akin to those reported for the major histocompatibility complex (MHC). In the glycophorin family the small size of the region within which gene interactions have occurred and the participation of essentially only two alleles makes this relatively simpler system more focused and easier to dissect and describe molecularly.

Some concepts relating to the molecular genetic basis of certain MNS blood group antigens

The unfolding story of genes encoding variant glycophorin molecules is already known to be more complicated than described here. The principles outlined provide a basis for understanding the fundamental events that occur in genes encoding the glycophorins as well as genes encoding unrelated proteins carrying other blood group antigens. Over 20 different genes involving the GYPA and GYPB family have been described. These genes arise from gene rearrangements within a relatively short region. This hot spot of activity has inverted palindromic sequences, which are known to be sites for DNA recombination. Similar structures exist in the major histocompatibility complex (MHC) where allelic diversity is a functional requisite. However, the significance of allelic diversity in the glycophorin gene family is not understood. The GYPA, GYPB and GYPE gene cluster is known to be prone to mutation by radiation because there is a high incidence of somatic mutation events in atomic bomb survivors, in people exposed to accidental radiation, in patients with Bloom's syndrome and in patients receiving radiation therapy. The mutation events were dose dependent: the greater the exposure, the greater proportion of red blood cells exhibited mutations. While it is known that MHC diversity protects against infection, the reason for glycophorin rearrangements remains to be determined.

Alteration of splice site selection by an exon mutation in the human glycophorin A gene.

We report the identification in human glycophorin A gene of a novel exon mutation that affects splice site selection. DNA mapping showed that the mutation has abolished a unique MspI site marking the exon III-intron 3 splice junction (ACCG/GT). Genomic sequencing confirmed the occurrence of a single G-->A transition at the terminal nucleotide position of exon III. Analysis of the mRNA composition demonstrated a partial inactivation of the altered 5' splice site as well as skipping of exons involving the alternative use of other constitutive splice sites. The full-length transcript with the cognate A change encodes a variant glycophorin with an arginine replacing a glycine at position 59 and defining the ERIK epitope, whereas the exon III-deleted transcript specifies a shorter glycophorin carrying the Sta antigen. Also identified were the misspliced mRNA species with exon I-IV and exon I-V connections generated by selection of 5' splice sites far distant from the mutated site. Although having a correct translation frame, the predicted polypeptides of such exon-skipping products were not assembled on the erythrocyte membrane probably due to the severe truncation of the signal sequence required for targeting and translocation. These observations reveal a new mechanism for antigenic diversity of human glycophorins.

An immunoblotting procedure for screening glycophorins and band 3 protein in the same blots: Identification of glycophorin and band 3 variant forms

An immunoblotting procedure is described which makes it possible to screen multiple blood samples for the presence of glycophorin and band 3 variant forms with altered electrophoretic mobility. The procedure can be simplified by using whole red blood cell hemolysates instead of membranes for SDS-polyacrylamide gel electrophoresis. The use of hemolysates also has the advantage that antigens sensitive to proteolysis are not degraded in vitro. The same nitrocellulose blots were used for immunoenzymatic detection of glycophorins with a set of anti-glycophorin monoclonal antibodies, and for autoradiographic detection of band 3-derived bands with 125I-labeled anti-band 3 monoclonal antibody. The screening of 157 Caucasian blood samples revealed the presence of a slower-migrating form of band 3 in seven cases and variant glycophorin in one case. The variant glycophorin exhibited the features of hybrid glycophorin of B-A type.

Gene conversion confined to a direct repeat of the acceptor splice site generates allelic diversity at human glycophorin (GYP) locus.

The glycophorin locus (GYP) on the long arm of chromosome 4 encodes antigens of the MNSs blood group system and displays considerable allelic variation among human populations. The genomic structure and organization of a variant glycophorin allele specifying a novel Miltenberger (Mi)-related phenotype, MiX, were examined. This variant probably arose from a gene conversion event involving a direct repeat of the acceptor splice site. Southern blot analysis indicated that MiX gene derived its 5' and 3' portions from glycophorin B or delta gene but its internal part from glycophorin A or alpha gene. Genomic sequences encompassing the rearranged regions of the MiX gene were amplified by single copy polymerase chain reaction. Direct DNA sequencing showed that during the formation of MiX gene, a short stretch of alpha exon III with a donor splice site has replaced a silent sequence in the delta gene containing a cryptic acceptor splice site. The upstream delta-alpha breakpoint is flanked by the direct repeats of the acceptor splice site, whereas the down-stream alpha-delta breakpoint is located in the adjacent intron. This segmental transfer produced a new composite exon whose expression not only transactivated a portion of silent sequence but also created intraexon and interexon hybrid junctions that characterize the antigenic specificities of MiX glycophorin. The identification of MiX as yet another delta-alpha-delta hybrid different from MiIII and MiVI in gene conversion sites suggests that shuffling of expressed and unexpressed sequences through particular genomic DNA motifs has been an important mechanism for shaping the antigenic diversity of MNSs blood group system during evolution.

Synthetic peptides homologous to human glycophorins of the Miltenberger complex of variants of MNSs blood group system specify the epitopes for Hil, SJL, Hop, and Mur antisera

The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, “Hil”, “SJL”, “Mur”, and “Hop” to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha- delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta- alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur.

Synthetic Peptides Homologous to Human Glycophorins of the Miltenberger Complex of Variants of MNSs Blood Group System Specify the Epitopes for Hil, SJL, Hop, and Mur Antisera

The antigenic epitopes of the MNSs blood groups are localized on alpha and delta glycophorins (glycophorins A and B) of the erythrocyte surface. Hil, SJL, Mur, and Hop antisera define the Miltenberger (Mi) complex of MiV, MiJ.L., MiIII, and MiVI variant serologic phenotypes of this blood group system. We report here the location of the epitopes for antibodies in these antisera. The antigens of these Mi classes are variant glycophorins that are hybrids of alpha and delta glycophorins in alpha-delta and delta-alpha-delta arrangements. The hybrid junctions give rise to novel polypeptide sequences not present in the parent glycophorins; in MiIII and MiVI this also includes an expressed sequence of the delta pseudoexon. These sequences are identical in the above Mi-glycophorins occurring in erythrocytes that share a common Mi determinant. Four peptides of 10 to 14 amino acids each were constructed to be homologous to the identical sequences; they were designated, "Hil", "SJL", "Mur", and "Hop" to reflect the common determinant. The peptides were tested for inhibition of reaction of appropriate cells with the relevant antisera. The Hil peptide, outlining the alpha-delta s junction region in MiIII, MiV, and MiVI glycophorins, inhibited the reaction of respective erythrocytes (red blood cells [RBCs]) with anti-Hil. The SJL peptide, which differs from the Hil peptide by a single Thr----Met substitution, was specific for inhibition of the reaction of MiJ.L. RBCs with anti-SJL (an example of anti-S specific for such RBCs). The Hop peptide, which corresponds to the delta-alpha junction in MiVI glycophorin, inhibited the hemagglutination of MiVIII RBCs by anti-Hop. MiVI and MiVIII glycophorins share an identical sequence at that site. The Mur peptide, corresponding to a portion of the expressed pseudoexon sequence in MiIII and MiVI glycophorins, was specific for inhibition of the reaction of MiIII and MiVI RBCs with anti-Mur. The peptides had no effect on the hemagglutination of control MNSs RBCs by their respective antisera nor of unrelated Mi classes RBCs by antisera that distinguish these classes. We conclude that the alpha-delta junction in MiIII, MiV, and MiVI glycophorins outlines the epitopes for anti-Hil, the alpha-delta junction in MiJ.L. outlines the epitope for anti-SJL, the delta-alpha junction in MiVI constitutes the epitope for anti-Hop, and the expressed delta pseudoexon sequence in MiIII and MiVI constitutes the epitope for anti-Mur.

Amino Acid Sequence of an α-δ-Glycophorin Hybrid: A structure reciprocal to Sta δ α-A-glyc0ph0rin hybrid

In this report we examine the primary sequence of a variant glycophorin obtained from erythrocytes of an individual who exhibits an unusual MNSs blood group phenotype. We show that this protein is a hybrid molecule constructed from sequences of α- and α-glycophorins (glycophorins A and B) in a α-δ arrangement. Serological typing revealed that the donor's phenotype was M+N+S+s+U+; yet his erythrocytes reacted with some but not all examples of anti-S antisera. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a variant glycophorin band, and immunoblotting and reaction with N-glycanase suggested that its amino terminus resembled that of M-α-glycophorin but that its carboxyl terminus did not. A preparation highly enriched in the variant was obtained and used to generate peptide fragments for sequencing. The sequence revealed that the variant was a hybrid molecule whose amino terminus corresponded to M-α-glycophorin and whose carboxyl terminus corresponded to S-δ-glycophorin. CNBr cleavage of the variant glycophorin yielded four peptides. The sequence of the amino-terminal CNBr peptide (residues 1–8) was identical to the amino-terminal octapeptide of M-α-glycophorin. The proceeding peptide (residues 9–61) contained a segment identical to residues 9–58 of α glycophorin, but its carboxyl-terminal sequence had the Gly-Glu-Met sequence from S-δ-glycophorin (residues 27–29). The other two peptides, insoluble in aqueous solutions, contained highly hydrophobic sequences, identical to residues 30–52 and 53–68 of δ-glycophorin. Sequences of overlapping peptides generated by trypsin and V8 protease confirmed the hybrid nature of the variant glycophorin: residues 1–58 were identical to residues 1–58 of M-α-glycophorin, and residues 59–100 were entirely identical to residues 27–68 of S-δ-glycophorin. The variant glycophorin is expected to have 4 additional residues at its carboxyl terminus that correspond to the carboxyl-terminal residues 69–72 of δ-glycophorin. The amino acid sequence arrangement of the variant α-δ-glycophorin is an exact reciprocal of that found in another hybrid glycophorin, Sta, that is a δ-α hybrid. We propose that the two hybrid glycophorins represent the two possible products resulting from a reciprocal recombination event.

Molecular genetic analysis of a hybrid gene encoding Sta glycophorin of the human erythrocyte membrane

Sta is an antigen of the human MNSs blood group system carried by a variant glycophorin residing in the erythrocyte membrane. We examined the structure, organization, and inheritance of Sta gene identified in genomic DNA from an Oriental family. Southern blotting detected a useful genetic marker tightly linked to the Sta gene. Differential hybridization and secondary restriction analyses showed that Sta gene is a fusion hybrid of delta and alpha glycophorin genes. Genomic mapping by extensive use of synthetic oligonucleotides, with overlapping sequence specificity, allowed us to define the delta-alpha junction site and disclose the organization of the variant gene. The junction point of Sta hybrid gene is encompassed by an unexpressed exonlike sequence of the delta gene at the 5′ site, and an expressed sequence of the alpha gene spanning codons 59 through 71, at the 3′ site. Dosage quantification demonstrated the occurrence of Sta gene as a single copy in the genome. Blood group inheritance, evaluated by DNA typing, established the tight linkage of Sta to the alpha M and delta S genes. The data support a single unequal crossing-over event between misaligned delta and alpha genes on the homologous chromosomes as the mechanism for the origin of Sta gene. The Sta gene is similar in overall structure to another delta-alpha hybrid gene, Dantu, but differs from it in junction structure, copy number, gene linkage, and antigen specificity.

Molecular Genetic Analysis of a Hybrid Gene Encoding St* Glycophorin of the Human Erythrocyte Membrane

Sta is an antigen of the human MNSs blood group system carried by a variant glycophorin residing in the erythrocyte membrane. We examined the structure, organization, and inheritance of Sta gene identified in genomic DNA from an Oriental family. Southern blotting detected a useful genetic marker tightly linked to the Sta gene. Differential hybridization and secondary restriction analyses showed that Sta gene is a fusion hybrid of delta and alpha glycophorin genes. Genomic mapping by extensive use of synthetic oligonucleotides, with overlapping sequence specificity, allowed us to define the delta-alpha junction site and disclose the organization of the variant gene. The junction point of Sta hybrid gene is encompassed by an unexpressed exonlike sequence of the delta gene at the 5' site, and an expressed sequence of the alpha gene spanning codons 59 through 71, at the 3' site. Dosage quantification demonstrated the occurrence of Sta gene as a single copy in the genome. Blood group inheritance, evaluated by DNA typing, established the tight linkage of Sta to the alpha M and delta S genes. The data support a single unequal crossing-over event between misaligned delta and alpha genes on the homologous chromosomes as the mechanism for the origin of Sta gene. The Sta gene is similar in overall structure to another delta-alpha hybrid gene, Dantu, but differs from it in junction structure, copy number, gene linkage, and antigen specificity.

A family study of multiple mutations of alpha and delta glycophorins (glycophorins A and B).

Glycophorins alpha and delta are the carriers of the antigens of the MNSs blood system; this report documents the presence of three glycophorin mutations in two individuals of a 16 member family. Erythrocytes were examined by serology, sodium dodecyl sulfate electrophoresis, and immunoblotting. The inheritance pattern and immunoblot profile revealed: (1) A variant Dantu glycophorin showed properties consistent with a delta-alpha glycophorin hybrid structure, previously noted in other individuals. The gene responsible for the Dantu glycophorin in this family is linked to a gene coding for an M-specific alpha glycophorin. (2) Another variant glycophorin, Mi-III glycophorin, was first revealed by immunoblotting and subsequently confirmed by erythrocyte antigen typing. This autosomal dominant trait is associated with N blood group activity and the inheritance pattern indicates that it could be a variant of delta glycophorin. (3) In the individuals with both Dantu and Mi-III glycophorins a delta glycophorin deficiency was observed suggesting that a deletion or alteration of delta gene may exist cis to the Dantu gene. Our findings that document clustering of multiple mutations in MNSs gene loci in the propositus family are very unusual as such variants are relatively rare.

Membrane glycophorins of Dantu blood group erythrocytes.

Glycophorins of erythrocytes of two unrelated individuals who exhibit the Dantu blood group phenotype were studied. Immunoblots indicated that erythrocytes of each individual contained a complement of a normal alpha-glycophorin (glycophorin A) and a variant N-glycophorin. delta-Glycophorin (glycophorin B) was present in one donor's cells but not the other's; the s and N phenotypes of the latter's erythrocytes may derive from the variant glycophorin. The variant glycophorin is of a smaller size, does not bind to Lens culinaris lectin agarose, and lacks residues approximately 40-60 of alpha-glycophorin and its single asparagine-linked carbohydrate; it contains approximately 2 less O-glycosidically bound units whose structures are identical to those found in alpha-glycophorins. All these properties are characteristic of delta-glycophorin. The variant is related to alpha-glycophorin in the carboxyl-terminal region as shown by reaction with a specific antiserum. Sequence analyses of a mixture of chymotryptic peptides of a CNBr fragment of the variant glycophorin identified the sequence Val-His-Arg-Phe-Thr-Val-Pro-Glu-Ile-Thr-Leu-Ile-Ile that contains the junction point of delta- and alpha-glycophorins spanning residues 33-38/39 of delta-glycophorin and residues 71/72-77 of alpha-glycophorin. Sequence analysis of a mixture of CNBr fragments allowed us to conclude that the variant originates from delta-s- rather than delta-S-glycophorin. The quantity of the variant Dantu glycophorin when compared to alpha-glycophorin differed in the two individuals, the ratio being 2/1 in one individual's cells and 0.5/1 in the other's. This may reflect that the two donors belong to different varieties of Dantu phenotypes. Together, the evidence indicates that both donors' erythrocytes contain a (delta-alpha) variant glycophorin, whose amino terminus originates from delta-s-glycophorin and the carboxyl end from alpha-glycophorin with a junction point around residues 39 of delta- and 71 of alpha-glycophorins. The results suggest that the unique junction region may be characteristic of the Dantu phenotype.

Severe Hemolytic Disease of the Newborn due to Anti‐Vw and Detection of Glycophorin A Antigens on the Miltenberger I Sialoglycoprotein by Western Blotting

Anti-Vw detecting an antigen on Miltenberger I (Mi I) variant glycophorin A (GPA) has rarely been reported as a cause of hemolytic disease of the newborn (HDN). We report an infant with severe HDN due to anti-Vw. Examination of the Vw+ erythrocytes of the father and paternal grandmother by sodium dodecylsulphate polyacrylamide gel electrophoresis showed an extra trypsin-sensitive, periodic-acid-Schiff staining band, consistent with Mi I variant GPA. Staining of Western blots by monoclonal antibodies showed that normal paternal GPA expressed blood group M, while Mi I variant GPA expressed blood group N. Mi I variant GPA expressed the trypsin-sensitive antigenic determinant detected by MoAb 10F7, indicating that the alterations known to occur in the trypsin-sensitive fragment of Mi I variant GPA do not affect expression of the antigen detected by 10F7.

Severe Hemolytic Disease of the Newborn due to Anti-Vw and Detection of Glycophorin A Antigens on the Miltenberger I Sialoglycoprotein by Western Blotting

Anti-Vw detecting an antigen on Miltenberger I (Mi I) variant glycophorin A (GPA) has rarely been reported as a cause of hemolytic disease of the newborn (HDN). We report an infant with severe HDN due to anti-Vw. Examination of the Vw^+ erythrocytes of the father and paternal grandmother by sodium dodecylsulphate polyacrylamide gel electrophoresis showed an extra trypsin-sensitive, periodic-acid-Schiff staining band, consistent with Mi I variant GPA. Staining of Western blots by monoclonal antibodies showed that normal paternal GPA expressed blood group M, while Mi I variant GPA expressed blood group N. Mi I variant GPA expressed the trypsin-sensitive antigenic determinant detected by MoAb 10F7, indicating that the alterations known to occur in the trypsin-sensitive fragment of Mi I variant GPA do not affect expression of the antigen detected by 10F7.