MNSs Blood Group Research Articles

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.

Identification of recombination events resulting in three hybrid genes encoding human MiV, MiV(J.L.), and Sta glycophorins

MiV, MiV(J.L.), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between alpha and delta glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L.), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing- over site and showed that MiV and MiV(J.L.) genes are arranged in the same 5′ alpha-delta 3′ frame whereas Sta gene is in a reciprocal 5′delta-alpha 3′ configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The alpha-delta breakpoints in MiV and MiV(J.L.) genes were localized to the 3′ end of the HindIII site downstream from exon 3 and to the 5′ end immediately upstream from exon 4, respectively, whereas the delta-alpha breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L.) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the delta glycophorin alleles that must have participated in the recombination.

Identification of Recombination Events Resulting in Three Hybrid Genes Encoding Human MiV, MiV(J.L.), and Sta Glycophorins

AbstractMiV, MiV(J.L), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between α and δ glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing-over site and showed that MiV and MiV(J.L) genes are arranged in the same 5'α-δ3' frame whereas Sta gene is in a reciprocal 5'δ-α3' configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The α-δ breakpoints in MiV and MiV(J.L) genes were localized to the 3' end of the Hind III site downstream from exon 3 and to the 5' end immediately upstream from exon 4, respectively, whereas the δ-α breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the δ glycophorin alleles that must have participated in the recombination.

Molecular genetics of human erythrocyte MiIII and MiVI glycophorins. Use of a pseudoexon in construction of two delta-alpha-delta hybrid genes resulting in antigenic diversification.

Human glycophorins alpha and delta (or A and B) specify the MNSs blood group antigens; they exhibit considerable structural variation among populations. We show that two variant phenotypes of Miltenberger class III and VI are encoded by similar hybrid glycophorin genes in a delta-alpha-delta arrangement. Restriction mapping identified altered fragments unique to the MiIII and MiVI genes. Genomic sequences spanning exons 2 to 4 of the two genes were obtained by allele-specific polymerase chain reaction. Restriction analysis and direct sequencing of the amplified DNA revealed that MiIII and MiVI genes are identical to the delta gene except that, in both, an internal segment of the delta gene has been replaced by its homologous counterpart of the alpha gene, resulting in a delta-alpha-delta hybrid structure. In the process of hybrid formation a portion of alpha exon 3 and intron 3, that carries a functional 5' splicing signal, has been fused to an exon-like sequence in the delta gene that retains a 3' but lacks a 5' splicing signal. These rearrangements created a composite exon resulting in the expression of the ordinarily unexpressed delta gene sequence and conferred the hybrid proteins with new antigenic specificities. The expression of this sequence in MiIII glycophorin is directly demonstrated by protein sequencing. MiIII and MiVI genes differ in the location of upstream (delta-alpha) and downstream (alpha-delta) breakpoints and in the length of sequence replacement. The delta-alpha breakpoints of the two genes occur at different locations within a 35-base pair sequence of exon 3 that is clustered with multiple inverted repeats, whereas the alpha-delta breakpoints reside downstream in two dissimilar blocks of sequences of intron 3. The minimal length of the delta gene sequence that has been replaced by the alpha gene is 55 base pairs in the MiIII gene and 131 base pairs in the MiVI gene. Such segmental DNA transfers may have proceeded unidirectionally through the mechanisms of gene conversion.

Typing of MNSs blood group specific sequences in the human genome and characterization of a restriction fragment tightly linked to S-s- alleles

Human erythrocyte membrane alpha and delta glycophorins (glycophorins A and B) carry the antigens for the M,N,S,s blood group system. Synthetic oligonucleotides spanning coding regions for M,N,S, and s epitopes were used to examine DNAs from 50 individuals selected at random and from individuals known to exhibit S(-)s(-)U- or S(-)s(-)U+ blood group phenotypes. We showed that M,N,S,s, blood group-specific sequences occur as multicopies in the human genome and reside within the alpha and delta glycophorin genes and also within the third glycophorin gene (glycophorin E gene). DNA typing with M- and N-epitope-specific probes showed distinct patterns that allowed correlation of the genotypes with the blood group phenotypes. The correlation using S- and s-specific probes was less definite owing to cross-hybridization. An Mspl restriction fragment length polymorphism (RFLP) residing in the E gene was detected in the black population. This RFLP is also carried by all individuals tested who exhibit the S(-)s(-)U- and S(-)s(-)U+ blood groups phenotypes, thereby serving as a useful marker for the S-s- alleles. The site of cleavage resulting in this RFLP was localized to the second intron of the E gene, and cleavage could occur through differential methylation of its two alleles.

Typing of MNSs Blood Group Specific Sequences in the Human Genome and Characterization of a Restriction Fragment Tightly Linked to S-s- Alleles

Human erythrocyte membrane alpha and delta glycophorins (glycophorins A and B) carry the antigens for the M,N,S,s blood group system. Synthetic oligonucleotides spanning coding regions for M,N,S, and s epitopes were used to examine DNAs from 50 individuals selected at random and from individuals known to exhibit S(-)s(-)U- or S(-)s(-)U+ blood group phenotypes. We showed that M,N,S,s, blood group-specific sequences occur as multicopies in the human genome and reside within the alpha and delta glycophorin genes and also within the third glycophorin gene (glycophorin E gene). DNA typing with M- and N-epitope-specific probes showed distinct patterns that allowed correlation of the genotypes with the blood group phenotypes. The correlation using S- and s-specific probes was less definite owing to cross-hybridization. An Mspl restriction fragment length polymorphism (RFLP) residing in the E gene was detected in the black population. This RFLP is also carried by all individuals tested who exhibit the S(-)s(-)U- and S(-)s(-)U+ blood groups phenotypes, thereby serving as a useful marker for the S-s- alleles. The site of cleavage resulting in this RFLP was localized to the second intron of the E gene, and cleavage could occur through differential methylation of its two alleles.

The Red Cell Antigen JAL in the Swiss Population: Family Studies Showing That JAL Is an Rh Antigen (RH48)

The JAL antigen was found to have an overall frequency of 0.004% in the Swiss population and 0.06% in French-speaking Swiss. Family studies of 5 JAL+ individuals have shown that the JAL antigen is not part of the ABO, MNSs, Fy, Jk and Co blood group systems, or the Se system, nor is it X- or Y-linked. JAL is encoded by the RH locus or by a very closely linked locus. The number RH48 (4.48) has been assigned for JAL by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens.

The Red Cell Antigen JAL in the Swiss Population: Family Studies Showing That JAL Is an Rh Antigen (RH48)

The JAL antigen was found to have an overall frequency of 0.004% in the Swiss population and 0.06% in French-speaking Swiss. Family studies of 5 JAL+ individuals have shown that the JAL antigen is not part of the ABO, MNSs, Fy, Jk and Co blood group systems, or the Se system, nor is it X- or Y-linked. JAL is encoded by the RH locus or by a very closely linked locus. The number RH48 (4.48) has been assigned for JAL by the International Society of Blood Transfusion Working Party on Terminology for Red Cell Surface Antigens.

The MzVariety of the St(a+) Phenotype — a Variant of Glycophorin A Exhibiting a Deletion

The NeuNAc level of erythrocyte membranes from two related donors exhibiting the Mz variety of St(a+) phenotype within the MNSs blood group system was found to be decreased by about 16%. The quantity of glycophorin A was decreased by about 38%, whereas that of glycophorin B was not significantly different from normal. Mz erythrocyte membranes were also found to contain an abnormal component (molar ratio to glycophorin A about 0.89:1.0) with an apparent molecular mass of about 24,000 Da. Immunoblotting experiments and amino-acid sequence analysis revealed that the novel component (and glycophorin A in one of the donors) carries blood group M activity. Blood group N activity was demonstrable for glycophorin A and glycophorin B from both donors. Amino-acid sequence analysis of chymotryptic, tryptic and cyanogen bromide peptides demonstrated that the novel molecule exhibits the typical structure of a Sta-active molecule. However, since it exhibits blood group M activity, it appears to represent a variant of glycophorin A lacking the residues 27-58 (encoded by exon three of the glycophorin A gene) rather than a glycophorin B-glycophorin A-hybrid molecule of the anti-Lepore type. Since one of the Mz heterozygotes was found to exhibit both M- and N activity on glycophorin A, the Mz gene complex appears to encode a blood group N-active glycophorin A apart from the novel component and a blood group s-active glycophorin B, although the level of glycophorin A in the erythrocyte membranes is decreased by about half.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Age differences between distributions of genotypes among pregnant women: evidence of fertility selection

The number of children produced by a modern woman is usually below her total reproductive capacity and is determined by circumstances other than natural selection. It is, therefore, practically impossible to detect differences in natural fertilities associated with different types (e.g. phenotypes, genotypes) of women. This does not mean, however, that natural selection at the reproductive level cannot at all be detected today. If women of a particular type have high natural fertility, this usually means that they reproduce (become pregnant) at a higher rate than women of a type with lower natural fertility. Hence, when there is a limit on the number of children, women of the first type will reach the limit at an earlier age than women of the second type. As a result, types that have a higher natural fertility should be overrepresented among pregnant women of younger ages and, consequently, underrepresented among older ones, as compared to types with a lower natural fertility. Based on this notion, a model of age-related differences between distributions of types among pregnant women is suggested. The model is applied to data on MNSs-blood group and PGM1 (phosphoglucomutase) types in a sample of pregnant women and an evidence of natural selection at the reproduction level associated with these genetic markers is obtained.

Characterization of a genomic hybrid specifying the human erythrocyte antigen Dantu: Dantu gene is duplicated and linked to a delta glycophorin gene deletion.

Dantu is a rare antigen of the human MNSs blood group system carried by a hybrid glycophorin on the erythrocyte membrane. To delineate its structure and relationship to alpha and delta glycophorins at the genomic level, we analyzed DNA from a three-generation family, in which both the presence of Dantu and Mi-III (another rare MNSs antigen) and the absence of delta glycophorin were seen. Three gross alterations in the gene cluster correlated with the observed phenotypes. Differential hybridization, secondary restriction analysis, and sequence-specific oligonucleotide probing established that Dantu glycophorin is encoded by a distinct hybrid gene derived from fusion of delta and alpha genes. The junction point of the Dantu gene was assigned to the region spanning codons 31-39 of delta and codons 72-79 of alpha genes. Dosage quantitation suggested that Dantu gene is duplicated and tandemly arranged. Identification of delta gene deletion disclosed the molecular basis for the absence of delta glycophorin in two Dantu and Mi-III double heterozygotes. The DNA inheritance pattern together with DNA typing of MN blood groups verified the genotypes and established that the duplicated Dantu hybrid gene is linked to alpha M gene as well as a delta gene deletion. The origin of such a haplotype in a segment of Black population would require two recombination events via either unequal homologous crossing-over or gene conversion.

Status of the MNSs antigens on human platelets.

A sensitive and specific radioimmunoassay was used to determine whether human platelets possess antigens of the MNSs blood group. Mouse monoclonal IgG anti-M and anti-N were purified by Staphylococcus protein A chromatography, labeled with 125I, and incubated with platelet pellets from donors of various MN phenotypes. Human IgG anti-M, -S, and -s were purified by absorption-elution, incubated with platelet pellets from donors of different MNSs phenotypes, washed, and incubated with 125I-labeled mouse monoclonal anti-human IgG. In both assays, the platelet pellets were centrifuged through phthalate ester oils and the radioactivity in the pellets was counted. Dose-response curves and ligand bound per cell indicated no significant difference in the binding of mouse or human anti-M and anti-N to platelets from donors of the MM, MN, or NN phenotype or of human anti-S and anti-s to platelets from donors of the Ss or ss phenotype. Contrary to many previous studies, our data indicate that the MNSs antigens are not expressed on the circulating human platelet. Therefore, antibodies to these antigens probably do not play a role in refractoriness to platelet transfusion.

A Rare Blood Group Antigen, Ola (Oldeide), Associated with Weak Rh Antigens1

A 'new', rare blood group antigen, Ola (Oldeide), is described. Only one Ol(a+) person was found among the 7,151 blood donors tested. Examination of the family of the proposita revealed 10 additional Ol(a+) persons. The study indicated that Ola is inherited as a Mendelian dominant character and segregates independently of the ABO, MNSs, P, Rh and Kidd blood group systems, of the ABH secretor genes, and of sex. Ol(a+) family members had depressed Rh antigens, while the Ol(a-) members had Rh antigens of normal strength. The depression was particularly pronounced for the C and E antigens, less marked for the D antigen. Individual antisera differed in their ability to demonstrate the presence of the antigens. Depression of the c and e antigens could not be demonstrated conclusively with the antisera available.

Comparison of Human and Siamang ABH and MN Blood Groups Using Monoclonal Antibodies

Comparison of human and siamang ABHIi and MNSs blood groups using monoclonal antibodies and enzyme-modified erythrocytes showed a different organization of ABHIi in the siamang, and a greatly altered expression of the sialoglycoproteins that express MNSs. While several monoclonal antibodies to human MNSs sialoglycoproteins failed to agglutinate siamang erythrocytes, MoAb145, a monoclonal antibody that reacts with the MN sialoglycoprotein, agglutinated siamang erythrocytes to almost the same titer as human red blood cells. These studies suggest the potential usefulness of monoclonal antibodies in seroprimatology.

Studies on Mv red cells. II. Immunochemical investigations.

The properties of the Mv antigen, a low incidence receptor of the MNSs blood group system, were investigated by serological tests with protease treated red cells and inhibition assays with glycoproteins or peptides from normal and Mv erythrocytes. Our data demonstrate that the Mv receptor represents an allelomorphic form of the 'N' antigen on the Ss sialoglycoprotein, rather than variant of the M receptor on the MN sialoglycoprotein. Anti-Mv plus -N (serum Arm.) reacts with the N, 'N' and Mv antigens, whereas anti-Mv (serum Arch.) is specifically directed against the latter receptor.

ABO, MNSs and rhesus blood groups in bladder cancer.

Data are presented on a group of cases of primary carcinoma of the bladder detailing their ABO, MNSs and rhesus blood antigenic phenotypes. Differences, either between all cases and controls or between particular stages of presentation and controls, have been found in the ABO red cell system as well as the MNSs red cell system. In the case of the ABO system an excess of the A gene is present but detailed study shows this to be only in patients with T1 and T2 tumours. No excess was seen in patients with carcinoma in situ or T3 or T4 stage disease. The S gene of the MNSs system is also in excess in cases of bladder cancer.

Rh Mosaicism and Aberrant MNSs Antigen Expression in a Patient with Chronic Myelogenous Leukemia

This is, to our knowledge, the first report of combined Rh and MNSs antigen alteration in a leukemic patient. Red blood cells of a chronic myelogenous leukemia patient demonstrated mixed field reactions with anti-Rho (D) reagents in August 1980. Earlier tests indicated he was Rho (D) positive in 1967, but Rho (D) and Du negative from 1973 to 1980. Titration studies with anti-hr' (c) and anti-hr" (e) indicated depressed expression, and there was also very weak s expression. One hundred per cent of cells studied from 1967 to 1980 were Philadelphia chromosome (Ph1) positive. No abnormalities in chromosomes associated with the Rh or MNSs blood groups 1 and 4, respectively, were noted. Phenotypes from August 1980 through September 1981 revealed normalization of red blood cell antigen status with an increase of Rho (D) positive cells from 35% to 100%. Chromosomal studies in October, 1980 and June, 1981 revealed Ph1 mosaicism with 25 and 75% Ph1 negative cells, respectively. These findings suggest that normalization of previously altered red blood cell antigen expression may reflect resurgence of normal stem cell lines.