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

BACKGROUND: 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. STUDY DESIGN AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.     

编码MNS血型抗原的GYP基因组研究进展

MNS血型系统包括大约40多种抗原,其中最为重要的是M、N、S、s 4种抗原。M和N抗原决定簇位于红细胞膜的血型糖蛋白A(GPA)上。GPA是一种红细胞上的主要唾液酸糖蛋白。S和s抗原决定簇位于红细胞膜上血型糖蛋白B(GPB)的唾液酸糖蛋白上。编码GPA的基因GYPA和编码GPB的基因GYPB位于4号染色体的长臂,两者具有95%的同源序列,同时又与不表达产物的同源基因GYPE紧邻,构成GYPA-GYPB-GYPE结构,为杂交基因的形成创造了条件。这些杂交基因的表达产物具有不同的抗原性。本综述简要介绍GYP基因组的分子基础,特别对Miltenberger抗原系统杂交基因的多态性做了较为深入的阐述。     

Expression and quantitative variation of the low-incidence blood group antigen He on some S-s-red cells

BACKGROUND: Red cells devoid of glycophorin B (GPB)-borne S, s, and U antigens are classified as an S-s-U- or S-s-U variant (U+var) and can arise from deletion and nondeletion genetic backgrounds. In nondeletion forms of S-s-U-, little information is available on whether the altered GPB gene (GYPB) is expressed in red cells. STUDY DESIGN AND METHODS: Red cells classified as S-s-U- or S-s-U+var were tested with anti-U, anti-U/GPB, anti-He, and anti-N by hemagglutination. Selected samples were tested by flow cytometry, immunoblotting, and polymerase chain reaction amplification using allele-specific primers. RESULTS: He (MNS6) was found on 23 percent (20/87) of samples. These and another 21 of the 87 samples were agglutinated by an anti-U/GPB reagent; this indicated that approximately 50 percent of S-s-samples possessed GPB variants. The strength of He varied among the samples. Genomic polymerase chain reaction with allele-specific primers showed the presence of expected DNA GPB-like products encoding He. Immunoblotting showed that He was carried on a membrane component with a relative molecular mass indistinguishable from that of GPB. CONCLUSION: The finding of He on S-s- red cells provides direct evidence for the presence of an altered form of GPB in red cells previously thought to be devoid of this glycophorin. Quantitative variation in He antigen expression was observed in a subset of S-s- red cells.     

The low-frequency MNS blood group antigens Ny(a) (MNS18) and Os(a) (MNS38) are associated with GPA amino acid substitutions

BACKGROUND: Antigens of the MNS blood group system are located on two sialoglycoproteins, GPA and GPB, encoded by GYPA and GYPB. The molecular backgrounds of the low-frequency antigens Ny(a) and Os(a) are not known. STUDY DESIGN AND METHODS: Immunoblotting and a monoclonal antibody-specific immobilization of erythrocyte antigens (MAIEA) assay were used to analyze Os(a). PCR-amplified products of the coding exons of GYPA were studied by single-strand conformation polymorphism analysis, and exon 3 was sequenced. Synthetic peptides were used in hemagglutination-inhibition tests. RESULTS: Sequencing of GYPA exon 3 of two unrelated Ny(a+) persons revealed heterozygosity for a T194A base change encoding an Asp27Glu substitution. Immunoblotting with anti-Os(a) and an MAIEA assay with MoAbs to GPA showed that Os(a) is on GPA. Sequencing exon 3 of an Os(a+) person from the only family with Os(a) revealed heterozygosity for a C273T base change encoding a Pro54Ser substitution. A synthetic peptide representing part of GPA with the Os(a) mutation (VRTVYPSEEETGE) completely inhibited anti-Os(a), whereas the control peptide (VRTVYPPEEETGE) did not inhibit anti-Os(a). CONCLUSION: Ny(a) and Os(a) are low-frequency antigens of the MNS blood group system that represent Asp27Glu and Pro54Ser substitutions in GPA, respectively.     

The Lutheran Blood Groups: A Progress Report with Observations on the Development of the Antigens and Characteristics of the Antibodies

Scoring titrations with anti-Lu^a on 81 members of the families of seven Lu(a+b—) propositi did not produce any evidence for the presence of Lu genes. In the Lu(a+b+) heterozygotes the Lu^a antigen was only weakly expressed at birth increasing progressively during the first 15 years. The red cells of Lu(a+b—) children gave scores comparable to those of adults. The reactions of Lu(a—b+) cord cells were somewhat weaker than those of adults but the red cells of Lu(a+b+) infants reacted very weakly with anti-Lu^a. Four infants born to mothers with anti-Lu^b had no evidence of hemolytic disease due to this antibody. Serologic, immunochemical and ultrafiltration studies suggest that the examples of anti-Lu^b studied are mainly IgA. These observations can explain why no unequivocal examples of hemolytic disease of the newborn due to anti-Lu^b have been encountered.     

The MiIII phenotype among Chinese donors in Hong Kong: immunochemical and serological studies

Summary. The incidence of the MiIII phenotype among Chinese blood donors in Hong Kong was found to be 6·28%. Eleven individuals apparently homozygous for the MiIII gene were detected by immunoblotting with monoclonal antibodies Rl.3 and R18. Rl.3 detects an identical epitope on both glycophorins A and B and Rl 8 detects a different epitope on glycophorin A. Immunoblotting with R1.3 showed an absence of bands corresponding to normal glycophorin B. Immunoblotting with R18 showed an absence of a 58 K band, which corresponds to a heterodimer of normal glycophorin B complexing with the MiIII component, found in MiIII heterozygotes. In two families with apparent MiIII homozygous individuals, both parents of the propositi had the MiIII phenotype which implies normal autosomal inheritance of the MiIII gene. In another family, only one parent had the MiIII phenotype and the presence of an S^u gene is postulated to explain the immunochemical and serological findings.     

Novel single‐nucleotide change in GYP*A in a person who made an alloantibody to a new high‐prevalence MNS antigen called ENEV

BACKGROUND: Alloantibodies that define some high‐prevalence MNS antigens are made by people with glycophorin A (GPA) altered by a single‐amino‐acid change or replacement of amino acids from part of the Pseudoexon 3 of GYP*B. The finding of a patient whose plasma contained a novel alloanti‐En^aFR prompted this study. RESULTS: The patient's serum contained an alloantibody to a high‐prevalence antigen, resistant to papain, ficin, trypsin, α‐chymotrypsin, or dithiothreitol. The antibody was strongly reactive with all panel red blood cells (RBCs) tested, showed reduced reactivity with ENEP? and ENAV? RBCs, and was nonreactive with M^kM^k, En(a?), GP.Hil/GP.Hil, and GP.JL/M^k RBCs. The patient's RBCs typed M+N?S+s?, Wr(a?b+^w), ENEP?, and ENAV?. These results indicated that the antibody recognized a new high‐prevalence antigen in the MNS system. Sequencing of DNA prepared from the patient's white blood cells revealed a GYP*A nucleotide substitution of 242T>G (predicted to change Val62 of GPA to Gly). This change ablates an RsaI restriction enzyme site and polymerase chain reaction–restriction fragment length polymorphism confirmed that the proband was homozygous for Nucleotide 242G. CONCLUSIONS: We describe a novel high‐prevalence MNS antigen, characterized by Val62 in GPA and named ENEV. The absence of the antigen is associated with Gly62. The change explains the weakened reactivity of the patient's serum with ENEP? and ENAV? RBCs and nonreactivity with anti‐ENEP and anti‐ENAV against her RBCs. The ENEV antigen has been assigned the ISBT number MNS45.     

抗人红细胞血型糖蛋白A非多态性表位单克隆抗体的制备和鉴定

目的制备抗人红细胞血型糖蛋白A(Glycophorin A,GPA)非多态性表位单克隆抗体并鉴定其特性。方法用小鼠B淋巴细胞杂交瘤技术获得分泌单抗-GPA非多态性表位的杂交瘤细胞株;鉴定抗体特异性和亚型;通过和各种酶处理细胞的反应确定单抗结合抗原位点的特性。结果得到的2株抗-GPA非多态性表位的单克隆细胞株Q6D7和Q7C9,均属IgG1亚类、Kappa型轻链,所针对的抗原位点均抗胰蛋白酶、胰凝乳蛋白酶处理,对无花果酶、木瓜酶、唾液酸酶敏感。结论制备获得2株抗-GPA非多态性表位单克隆抗体。     

Murine monoclonal anti-s and other anti-glycophorin B antibodies resulting from immunizations with a GPB.s peptide

BACKGROUND: The blood group antigens S and s are defined by amino acids Met or Thr at position 29, respectively, on glycophorin B (GPB). Commercial anti-s reagents are expensive to produce because of the scarcity of human anti-s serum. Our aim was to develop hybridoma cell lines that secrete reagent-grade anti-s monoclonal antibodies (MoAbs) to supplement the supply of human anti-s reagents.
STUDY DESIGN AND METHODS: Mice were immunized with the GPB^s peptide sequence TKSTISSQTNGE T GQLVHRF. Hybridomas were produced by fusing mouse splenocytes with mouse myeloma cells (X63.Ag8.653). Screening for antibody production was done on microtiter plates by hemagglutination. Characterization of the MoAbs was done by hemagglutination, immunoblotting, and epitope mapping.
RESULTS: Eight immunoglobulin G MoAbs were identified. Five antibodies are specific by hemagglutination for s and two MoAbs, when diluted, are anti-S–like, but additional analyses shows a broad range of reactivity for GPB. Typing red blood cells (RBCs) for s from 35 donors was concordant with molecular analyses as were tests on RBCs with a positive direct antiglobulin test (DAT) from 15 patients. The anti-s MoAbs are most reactive with peptides containing the ³¹QLVHRF³⁶ motif, with ²⁹Thr. By Pepscan analyses, the anti-S–like MoAbs reacted within the same regions as did anti-s, but independently of ²⁹Met. One antibody was defined serologically as anti-U; however, its epitope was identified as ²¹ISSQT²⁵, a sequence common for both GPA and GPB.
CONCLUSION: In addition to their value as typing reagents, these MoAbs can be used to phenotype RBCs with a positive DAT without pre-test chemical modification.     

SAT, a 'new' low frequency blood group antigen, which may be associated with two different MNS variants

Summary. A new private blood group antigen, SAT, was identified in an NFLD-Japanese woman as a result of testing 10,480 blood donors with a serum containing anti-NFLD and anti-SAT. Three other sera were subsequently also shown to contain anti-SAT. The donor's family showed that SAT is inherited as a dominant character and may be associated with a weak M antigen. Serological and immunochemical analysis revealed no other aberrations in the MNS system.
Study of a second SAT+ Japanese blood donor and his family suggested that SAT is associated with an unusual MNS variant resulting from a hybrid glycophorin comprising the N-terminus of glycophorin A and the C-terminus of glycophorin B. The propositus appears to be homozygous for the gene that produces the putative hybrid, which differs from previously described glycophorin (A-B) hybrids by expressing no S, s or U antigen. SAT antigen, therefore, may be associated with two different MNS variants in the only two families in which it has been identified.     

Novel GYP(A-B-A) hybrid gene in a DANE+ person who made an antibody to a high-prevalence MNS antigen

BACKGROUND: The glycophorin (GP) molecule associated with the GP.Dane phenotype is a GP(A‐B‐A) hybrid that contains some amino acids encoded by the Pseudoexon 3 of GYPB and Asn⁴⁵ of GPA and carries the low‐prevalence MNS antigens DANE and Mur. Serum from a woman of English ancestry contained an immunoglobulin M alloantibody to a high‐prevalence MNS antigen, and the purpose of this study was to identify the molecular basis of her phenotype. STUDY DESIGN AND METHODS: Hemagglutination, Western blotting, and DNA analyses were performed by standard methods. RESULTS: Tests of the proband's RBCs with monoclonal antibodies indicated a change of amino acids between positions 27 and 55 of GPA. Her RBCs expressed M, s, Mur, and DANE antigens and were M^g‐negative. The antigen recognized by her antibody was sensitive to treatment with papain, ficin, and trypsin and resistant to α‐chymotrypsin and dithiothreitol. Sequencing of DNA from the proband revealed a sequence of nucleotides identical to the GYP(A‐B‐A) encoding GP.Dane but without the adenyl nucleotide substitution, which has been predicted to change Ile⁴⁶ of GPA to Asn⁴⁵. Testing of her immediate family revealed the presence of an M^k gene. CONCLUSION: The proband had a novel GYP(A‐B‐A) encoding a DANE+ GP that is in cis to GYPB^s and in trans to M^k. The high‐prevalence antigen lacking from this GP.Dane phenotype and recognized by the proband's serum is called ENDA (ISBT Number MNS44). Our results indicate that the change of Ile⁴⁶ of GPA to Asn⁴⁵ of GP.Dane is not required for expression of the DANE antigen.     

Quantification of glycophorin A and glycophorin B on normal human RBCs by flow cytometry

BACKGROUND: The quantification of antigens and proteins on RBCs has been achieved by different approaches. Flow cytometry allows the results of the earliest studies to be to reappraised because it offers the possibility of measuring the immunofluorescence intensity of single cells and integrating the individual data of a large number of cells within a very short time. STUDY DESIGN AND METHODS: Flow cytometry was used in this work to analyze the binding of four MoAbs to glycophorin A (GPA) and glycophorin B (GPB). RBCs in their native state (nonfixed) were utilized. To avoid the agglutination problem, cells were disaggregated before measurements, dates were taken on 20,000 events on the single-cell region, and the fluorescence intensity of the principal peak present in the fluorescence histograms was used for the analysis. The quantification of sites per RBC was estimated by applying the Langmuir adhesion model. RESULTS: The numbers of GPA and GPB sites obtained for samples from healthy donors were similar to those found in the literature (1.86-4.9) x 10(5) and (0.48-1.61) x 10(5) for GPA and (0.21-1.14) x 10(5) and (0.47-0.88) x 10(5) for GPB. Differences between antibodies were found that depend on the binding site of each one. CONCLUSION: A simple method to quantify antigen sites on RBCs was developed. It could be applied whenever one antibody is assumed to bind exactly one antigen.     

Seven Vea (Vel) Negative Members in Three Generations of a Family

A family with seven Vel negative ( VeVe ) members in three generations was found because the propositus had a hemolytic transfusion reaction. She had had four full-term pregnancies and two miscarriages and one previously uneventful transfusion. The anti-Vel antibody in her serum gave both hemolysis and agglutination. Study of the blood groups of the family shows that Ve^a is not in the MNS system. Six of the VeVe individuals were tested with anti-Fy^a and all were found to be Fy(a+). Some Ve(a+) bloods were noted to react more strongly with anti-Ve^a than others. The explanation of van Loghem and van der Hart that the differences in the intensity of Ve(a+) reactions is due to subgroups, the stronger termed Ve(a¹) and the weaker Ve(a²), is accepted by the authors.     

On the Blood Group Antigens Bua and Sm

Three Bu(a+)Sm+ x Bu(a+)Sm+ families with three Bu(a+)Sm—, 11 Bu(a+)Sm+ and seven Bu(a—)Sm+ children are reported, supporting the hypothesis that Bu^a and Sm are products of allelic genes. The inheritance of Bu^a has been shown to be independent of sex and of all blood group systems reported prior to 1962 except Diego, Yt and Auberger. It is now reported to be independent of the antigens Do^a and Cs^a.     

Human erythrocyte glycophorins: protein and gene structure analyses.

Human RBCs glycophorins are integral membrane proteins rich in sialic acids that carry blood group antigenic determinants and serve as ligands for viruses, bacteria, and parasites. These molecules have long been used as a general model of membrane proteins and as markers to study normal and pathological differentiation of the erythroid tissue. The RBC glycophorins known as GPA, GPB, GPC, GPD, and GPE have recently been fully characterized at both the protein and the DNA levels, and these studies have demonstrated conclusively that these molecules can be subdivided into two groups that are distinguished by distinct properties. The first group includes the major proteins GPA and GPB, which carry the MN and Ss blood group antigens, respectively, and a recently characterized protein, GPE, presumably expressed at a low level on RBCs. All three proteins are structurally homologous and are essentially erythroid specific. The respective genes are also strikingly homologous up to a transition site defined by an Alu repeat sequence located about 1 Kb downstream from the exon encoding the transmembrane regions. Downstream of the transition site, the GPB and GPE sequences are still homologous, but diverge completely from those of GPA. The three glycophorin genes are organized in tandem on chromosome 4q28-q31, and define a small gene cluster that presumably evolved by duplication from a common ancestral gene. Most likely two sequential duplications occurred, the first, about 9 to 35 million years ago, generated a direct precursor of the GPA gene, and the second, about 5 to 21 million years ago, generated the GPB and GPE genes and that involved a gene that acquired its specific 3' end by homologous recombination through Alu repeats. Numerous variants of GPA and GPB usually detected by abnormal expression of the blood group MNSs antigens are known. An increasing number of these variants have been structurally defined by protein and molecular genetic analyses, and have been shown to result from point mutations, gene deletions, hybrid gene fusion products generated by unequal crossing-over (not at Alu repeats), and microconversion events. The second group of RBC membrane glycophorins includes the minor proteins GPC and GPD both of which carry blood group Gerbich antigens. Protein and nucleic acid analysis indicated that GPD is a truncated form of GPC in its N-terminal region, and that both proteins are produced by a unique gene called GE (Gerbich), which is present as a single copy per haploid genome and is located on chromosome 2q14-q21.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three Examples of Anti-Lub and Related Data

Three new examples of anti-Lu^b, two detected in 1963 and the third in 1965, are described. The first example was responsible for repeated hemolytic transfusion reactions, the second and third though associated with pregnancy do not appear to have been responsible for a clinical problem. 14,802 random donor bloods were typed with the first example and eight Lu(b—) bloods identified, the incidence being 0.057%. Lutheran typings on 95 individuals related to propositi who are Lu(a+b—), Lu(a+b+), and Lu(a—b—) revealed nine new Lu(b—) persons. Medical historical data on 85 known Lu(a+b—) persons are included in an attempt to evaluate the antigenicity of the Lu^b factor. The incidence of the phenotype, Lu(a+b+), in 556 Negroes was found to be 4.86%.     

Partial C antigen in sickle cell disease patients: clinical relevance and prevention of alloimmunization

BACKGROUND: Partial Rh antigens have been widely described in black individuals. Carriers are prone to immunization when exposed to the normal antigens. In sickle cell disease (SCD), patient alloimmunization is a major cause of transfusion failure. The potential of individuals with partial C antigen to make anti-C has not been investigated. We sought partial C status and anti-C production in a cohort of SCD patients with the C+ phenotype, to determine whether exposure to normal C antigen should be avoided.
STUDY DESIGN AND METHODS: We constituted a cohort of 177 randomly selected SCD patients expressing C antigen. We screened for (C)ce^s and R^N haplotypes, presumably associated with partial C antigen in Afro-Caribbeans, and we recorded the number of transfused C+ red blood cell (RBC) units, immunization status, and extended phenotype.
RESULTS: Forty-nine patients carried abnormal C antigen, deduced from the presence of (C)ce^s and/or R^N , not compensated by a normal RHC allele in trans. Among patients with partial C phenotype exposed repeatedly to C+ RBCs, 30% produced anti-C. Two patients experienced hemolysis. In our hospital, with 22% of SCD patients expressing C, prevention of anti-C immunization for all individuals with partial C antigen would require a 7% increase in the use of C– RBC units. These RBCs are already in short supply for SCD patients who are C–.
CONCLUSION: This study demonstrates the need to detect partial C within C+ SCD patients and to prevent immunization. A larger number of Afro-Caribbeans donors is needed to provide these patients with C– RBCs.     

Selected Types of Frozen Blood for Patients with Multiple Blood Group Antibodies

A statistical study of the incidence and specificity of multiple irregular blood group antibodies shows predominance of those within the Rh-Hr, Kell, and Duffy systems. When multiple antibodies included those in the Lewis, Lutheran, MNS, P, or Kidd systems, the other antibodies were usually in the Rh-Hr, Kell, and Duffy systems.
The following categories of donors: 1a Group O Rh-neg. (dce/dce) K-neg. Fy^a-neg. 1b Group O Rh-neg. (dce/dce) K-neg. Fy^b-neg. 2a Group O Rh-pos. (R₁R₁-DCe) K-neg. Fy^a-neg. 2b Group O Rh-pos. (R₁R₁-DCe/DCe) K-neg. Fy^b-neg. 3a Group O Rh-pos. (R₂R₂-DcE7DcE) K-neg. Fy^a-neg. 3b Group O Rh-pos. (R₂R₂-DcE/DcE) K-neg. Fy^b-neg. are further subtyped for Le^a, Le^b, P, Jk^a, Jk^b, M, N, S, s, and Lu^a.
Erythrocytes from these donors, frozen in glycerol, with Anti-A, and Anti-B antibodies removed during deglycerolization simplifies the problem of finding compatible blood for patients with multiple antibodies.     

An Antibody, in the Serum of a Wr(a+) Individual, Reacting with an Antigen of Very High Frequency

The serum of a Wr(a+) woman has been found to contain an antibody reacting with an antigen of very high incidence. Preliminary tests on the strength of the Wr^a antigen on the cells of the antibody former and the variation of reactions of the antibody with the cells of nonrelated Wr(a+) and Wr(a-) individuals suggest that the antibody may be detecting the antithetical antigen to Wr^a.