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An alloantibody in a homozygous GYP*Mur individual defines JENU (MNS49), a new high-frequency antigen on glycophorin B: JENU (MNS49) A NEW HIGH-FREQUENCY ANTIGEN ON GPB
... The MNS blood group system is highly polymorphic, which comprises 50 distinct antigens. [1,2] The antigens of the MNS system are expressed in the red blood cell (RBC) membrane on glycophorin A (GPA), glycophorin B (GPB), or hybrids of both and fully developed at birth. [3] GPA and GPB are encoded by the homologous glycophorin genes GYPA and GYPB, respectively. ...
... [29] Besides that, a detailed description of the GYP*Mur zygosity is beyond the scope of this study. However, several studies have defined the zygosity of GYP*Mur by molecular methods, such as Sanger sequencing, [12,21] high-resolution melting (HRM), [1,21] and multiplex ligation-dependent probe amplification (MLPA). [11] The sequence chromatograms for the homozygous GYP*Mur (+/+) showed a single peak, while the heterozygous GYP*Mur (+/-) showed double peaks of comparable intensities at the seven nucleotide positions where GYP*Mur differed from GYPB. ...
... [12,21] The results of the HRM curve analysis showed a single peak for GYP*Mur (+/+) and double peaks for GYP*Mur (+/-). [1,21] In the same fashion, MLPA analysis for the GYP*Mur (+/+) exhibited two copies of GYP*Mur-specific signal peak and the absence of the signal peak for the wild-type 5´ splice site of the GYPB intron 3, while the GYP*Mur (+/-) exhibited one copy of GYP*Mur-specific signal peak and one copy of the signal peak for the wild-type 5´ splice site of the GYPB intron 3. [11] In a word, the GYP*Mur (+/+) expresses only GYP*Mur, while the GYP*Mur (+/-) expresses both GYP*Mur and GYPB. ...
BACKGROUND AND OBJECTIVE
A number of glycophorin variant phenotypes or hybrid glycophorin variants of the MNS blood group system bear multiple immunogenic antigens such as Mia, Mur, and MUT. In the East and Southeast Asian populations, glycoprotein (GP.) Mur is the most common glycophorin variant phenotype expressing those three immunogens. The aim of this study was to detect MNS system glycophorin variant phenotypes (GP. Mur, GP. Hop, GP. Bun, GP. HF, and GP. Hut) among Malaysian blood donors.
MATERIALS AND METHODS
In this cross-sectional study, 144 blood donors were selected under stratified random sampling. The deoxyribonucleic acid was extracted from whole blood samples, followed by a polymerase chain reaction assay. Sanger sequencing was used to identify the specific MNS variants and then validated by a serological crossmatch with known anti-Mur and anti-MUT.
RESULTS
GP. Mur was identified among Malaysian blood donors with a prevalence of 6.94%, and no other variants of the MNS system were found.
CONCLUSION
The present study substantiates that GP. Mur is the main variant of the MNS system glycophorin (B-A-B) hybrid in Malaysian blood donors. GP. Mur-negative red blood cells must therefore be considered in the current transfusion policy in order to prevent alloimmunization and immune-mediated transfusion reactions, particularly in transfusion-dependent patients.
... anti-s D , haemolytic disease of the fetus and newborn, MNS blood group system, prevalence of s D in Thai blood donors, s D antigen 1 | INTRODUCTION MNS blood group antigens are carried on glycophorin A (GPA), glycophorin B (GPB), or hybrids of GPA/GPB. 1 GPB, encoded by GYPB gene, express high-prevalence antigens 'N', U, JENU and polymorphic antigens S and s. 1,2 The genetic basis for the s antigen is GYPB c.143C (p.Thr48). 1,2 Expression of the s antigen (MNS4) has been reported to be affected qualitatively as observed in hybrid glycophorins GP.Mur and GP.Bun, [3][4][5] and quantitativelyweak s expression was associated with the s D (MNS23) antigen first observed in the Dreyer family. 6,7 The molecular basis for the s D antigen is GYPB c.173C > G (rs374811215). 7,8 In Caucasian and mixed-race populations in South Africa, the prevalence of s D was 0.1%. ...
... Anti-s (P3BER) reacts with s expressed on GPB but not with s expressed on GP.Mur. 3,4,14 Twofold serial dilutions of anti-s P3BER (Merck, Millipore) and anti-s polyclonal (CSL) reagents, diluted out to 1/1024 dilution, were prepared. Each dilution was tested against RBCs from the father, baby, control S À s +, and S + s + by IAT. ...
... This reactivity pattern is consistent with the profile reported for GP.Mur/Mur. 3,4 The baby's cells were Mi(a+). ...
Background:
Low-prevalence antigen sD (MNS23) is encoded by GYPB c.173C > G. Hemolytic disease of the fetus and newborn (HDFN) due to anti-sD is rare. A mother delivered a newborn whose red blood cells (RBCs) were DAT-positive and was later diagnosed with HDFN. Serum from the mother was incompatible with the father's RBCs and was used to screen 184 Thai blood donors. This study aimed to investigate the cause of HDFN in a Thai family and determine the prevalence of sD in Thai blood donors.
Materials and methods:
Three family members and four blood donors were investigated in the study. Massively Parallel Sequencing (MPS) was used for genotyping. Standard hemagglutination techniques were used in titration studies, phenotyping, and enzyme/chemical studies. Anti-s, anti-Mia , anti-JENU, and anti-sD reagents were used in serological investigations.
Results:
The mother was GYP*Mur/Mur. The father and the four donors were GYPB*s/sD predicting S - s + sD +. The baby was GYP*Mur/sD and his RBCs were Mia +, s + w with anti-s (P3BER) and JENU+w . RBCs from two GYPB*sD -positive blood donors reacted with anti-sD (Dreyer). Proteolytic enzyme α-chymotrypsin-treated sD + cells did not react with anti-sD (Wat) produced by the GP.Mur/Mur mother but reacted with the original anti-sD (Dreyer).
Discussion:
This is the first report of HDFN due to anti-sD in the Asian population. The genotype frequency for GYPB*sD in a selected Thai blood donor population is 2.2% (4/184). Anti-sD should be considered in mothers with Southeast Asian or East Asian background when antibody identification is unresolved in pregnancies affected by HDFN.
... A Thai individual with thalassemia was transfused with RBCs (46). Following transfusion, anti-E, anti-c, anti-Jk b , anti-S and an antibody to a high-frequency antigen on GPB were identified in the patient's serum (46). ...
... A Thai individual with thalassemia was transfused with RBCs (46). Following transfusion, anti-E, anti-c, anti-Jk b , anti-S and an antibody to a high-frequency antigen on GPB were identified in the patient's serum (46). Epitope mapping analysis using 12-mer peptides, representing the extracellular domain of GPB, showed that an antibody in the patient's plasma recognised an epitope with the sequence SYISSQTNGETG (46). ...
... Following transfusion, anti-E, anti-c, anti-Jk b , anti-S and an antibody to a high-frequency antigen on GPB were identified in the patient's serum (46). Epitope mapping analysis using 12-mer peptides, representing the extracellular domain of GPB, showed that an antibody in the patient's plasma recognised an epitope with the sequence SYISSQTNGETG (46). This sequence is encoded by GYPB Exon 2 and Exon 4 producing 38 SYISSQTN 45 and 46 GETG 49 , respectively. ...
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(A-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.
... A polymerase chain reaction (PCR) and high-resolution melting (HRM) genotyping assay was used to genotype DNA from Mi a serology-positive RBCs. One pair of primers, forward primer: P7-F22TT, 5′-ACGCAGTCACCTCATTCTTGTT-3′, and reverse primer: P9-R23GG, 5′-GGCTTTGGAGTAAAAGAGTTGGG-3′, was designed to amplify GYPB pseudoexon 3 and the GYP(B-A-B) exon 3 hybrid gene [30]. A 270-bp PCR product is expected for GYPB, GYP*Mur, and GYP*Bun. ...
... lowed by 40 cycles of denaturation (95 ° C for 10 s) and annealing/extension (65 ° C for 30 s). At the end of the PCR step, the temperature was gradually increased by 0.1 ° C every 2 s from 73 ° C to 83 ° C [30]. ...
... GYPB homozygote, GYP*Mur homozygote, and GYP*Mur/ GYPB DNA controls used in the HRM assay were genotyped by matrix-assisted laser desorption/ionisation, time-of-flight mass spectrometry (MALDI-TOF MS) and DNA was fully sequenced [33]. These DNA controls have been used in previous publications [20,30]. ...
Background:
MNS blood group system genes GYPA and GYPB share a high degree of sequence homology and gene structure. Homologous exchanges between GYPA and GYPB form hybrid genes encoding hybrid glycophorins GP(A-B-A) and GP(B-A-B). Over 20 hybrid glycophorins have been characterised. Each has a distinct phenotype defined by the profile of antigens expressed including Mi<sup>a</sup>. Seven hybrid glycophorins carry Mi<sup>a</sup> and have been reported in Caucasian and Asian population groups. In Australia, the population is diverse; however, the prevalence of hybrid glycophorins in the population has never been determined. The aims of this study were to determine the frequency of Mi<sup>a</sup> and to classify Mi<sup>a</sup>-positive hybrid glycophorins in an Australian blood donor population.
Method:
Blood samples from 5,098 Australian blood donors were randomly selected and screened for Mi<sup>a</sup> using anti-Mi<sup>a</sup> monoclonal antibody (CBC-172) by standard haemagglutination technique. Mi<sup>a</sup>-positive red blood cells (RBCs) were further characterised using a panel of phenotyping reagents. Genotyping by high-resolution melting analysis and DNA sequencing were used to confirm serology.
Result:
RBCs from 11/5,098 samples were Mi<sup>a</sup>-positive, representing a frequency of 0.22%. Serological and molecular typing identified four types of Mi<sup>a</sup>-positive hybrid glycophorins: GP.Hut (n = 2), GP.Vw (n = 3), GP.Mur (n = 5), and 1 GP.Bun (n = 1). GP.Mur was the most common.
Conclusion:
This is the first comprehensive study on the frequency of Mi<sup>a</sup> and types of hybrid glycophorins present in an Australian blood donor population. The demographics of Australia are diverse and ever-changing. Knowing the blood group profile in a population is essential to manage transfusion needs.
... The MNS blood group system (ISBT 002) was discovered after the ABO blood group system (ISBT 001)and before the Rhesus (Rh) blood group system (ISBT 004) 1 . The MNS system consists of 49 antigens that are the products of glycophorin A (GPA), glycophorin B (GPB) or hybrids thereof and are fully developed at birth [1][2][3] .The genes encoding GPA (GYPA) and GPB (GYPB) are highly homologous with 95.5% sequence similarity 4 . In blood transfusion practices, the main antibodies of the MNS system considered are anti-M, anti-N, anti-S, anti-s, and anti-U. ...
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
... This correlates with serological studies showing that antibodies raised against the s antigen on Glycophorin B do not necessarily recognise the s antigen on GP.Mur [6]. It was further shown that the predictive model for homodimeric GP.Mur has an interface which obscures the external presentation of the U epitope, this can be correlated to a study by Lopez et al., [27] showing that GP.Mur homozygous individuals are serologically U negative, despite the presence of the U antigen sequence. In the instance of both the loss of the U antigen and the altered s antigen, the amino acid sequence for the U and s epitope remained identical to that on Glycophorin B, and thus genotyping alone would not identify the presence of an altered epitope. ...
Introduction: Blood group antigens are defined by an immune response that generates antibodies against a red blood cell molecule. Antibodies against these antigens can be associated with haemolytic transfusion reactions. However, difficulties can arise when developing antibodies against antigens through the use of peptide sequences alone. Three-dimensional representations (models) of the molecular structure of antigen-bearing proteins can provide valuable insights into tertiary structures and their consequent antigenicity. This can be achieved through predictive computational modelling to produce both structural and molecular dynamics models of blood group proteins.
Areas Covered: Authors discuss the use of molecular dynamic simulations on existing structures, as well as the use of computational modelling techniques in the development of protein models lacking pre-existing data. Lastly, the authors discuss the specific example of the use of computationally derived models of the MNS blood group system and its use in attempts to produce antibodies against MNS proteins. The NCBI PubMed Database was searched as well as Google Scholar (published in peer review journals judged by author discretion) across July 2020 – February 2021.
Expert Opinion: Although in silico techniques have limitations, computer-based predictive models can inform the direction of research into blood group proteins. It is to be expected that as computer-based techniques grow more powerful these contributions will be even more significant.
... Dahr has shown that in addition to GPB p.Thr48, some anti-s may require p.Thr44, p.Glu47, p.His53 and p.Arg54 for s expression [10]. Recent evidence for altered s antigen on the GP.Mur hybrid was suggested by Lopez et al. who described the high prevalence GPB antigen, JENU which is abolished on GP.Mur [11]. In their study, red blood cells (RBCs) from GYP*Mur homozygotes failed to react with a monoclonal anti-s but still reacted with a polyclonal anti-s. ...
Background and objectives
The Mi(a+) GP(B‐A‐B) hybrid phenotypes occur with a prevalence of 2%–23% across Southeast Asia. While the s antigen is alleged to be altered, no evidence for specific variants is known. Screening using a monoclonal IgM anti‐s mistyped six S‒s+ RBC units as S‒s‒. Further, alloanti‐s was identified in an S+s+ patient. Our objective was to investigate the s antigen further.
Materials and methods
DNA from 63 Thai blood donor samples PCR‐positive for a GYP(B‐A‐B) hybrid was sequenced with primers spanning GYPB exons 3–4. Flow cytometry was used for semiquantitative analysis of s expression and correlated with the glycophorin genotype.
Results
DNA sequencing showed that GYP*Mur was carried by 56/63 samples (88·9%) of which 5/56 lacked normal GYPB: three of these were GYP*Mur homozygotes, one was a compound heterozygote carrying GYP*Mur and a GYP*Bun‐like allele (designated GYP*Thai), and the fifth sample carried GYP*Mur and another GYP*Bun‐like allele. Seven samples (7/63) were GYP*Thai heterozygotes. IgM monoclonal anti‐s (P3BER) did not react with the s antigen carried by GP.Mur or GP.Bun, whereas two IgG anti‐s showed enhanced reactivity.
Conclusions
We confirmed that GYP*Mur is the most frequent variant in Thai blood donors and also identified GYP*Thai with a frequency of 1·1%. We showed that s antigen on Mi(a+) GP(B‐A‐B) hybrids is qualitatively altered and should be considered when selecting reagents for phenotyping where such hybrids are prevalent, endemically and in blood centres worldwide.
... Forty-nine MNS antigens are carried on glycophorin A, glycophorin B, and hybrids of the glycophorins. [1][2][3] The M and N antigens are on glycophorin A, and the S and s antigens are on glycophorin B, which are encoded by Exon 2 of GYPA and Exon 4 of GYPB genes, respectively. The GYPA and GYPB genes are highly homologous, with more than 95% DNA sequence identity. ...
Background:
The GP.Mur glycophorin with Mia phenotype is relatively common and clinically significant in the Southeast Asian populations. The aim of this study is to genotype Mia -positive Asian American type O blood donors. Red blood cell (RBC) minor antigens were also determined in the same cohort.
Study design and methods:
Asian American blood donors of the Gulf Coast Regional Blood Center (Houston, TX) were screened using a typing reagent (NOVACLONE Anti-Mia Monoclonal IgG Typing Reagent, Dominion Biologicals Ltd) from March 2016 to July 2018. Aliquots of Mia -positive blood from type O donors were subjected to serologic confirmation using Mia - and/or Mur-specific GAMA210 and 64D6 monoclonal antibodies, and two human antisera. Extracted genomic DNA was amplified by polymerase chain reaction (PCR) using GYP hybrid gene/allele-specific primers followed by bidirectional Sanger sequencing. Zygosity for GYP*Mur and GYP*Bun was determined using TaqMan real-time PCR assay. Phenotypes of 35 RBC antigens and three phenotypic variants were determined with use of an in vitro diagnostic test, PreciseType HEA Molecular BeadChip Test (Immucor).
Results:
By screening 4600 blood donations in the Houston metropolitan area, 209 samples from 103 unique donors were identified to be Mia -positive. By PCR and sequencing analysis, 97 of the 103 Mia -positive donors carried hybrid genes GYP*Mur (89.7% including two homozygotes), GYP*Bun (6.2%), GYP*Vw (3.1%) and GYP*Hut (1.0%). Concordance between serology and DNA analysis was 98%, 99%, and 100% for the GAMA210, 64D6, and human antisera, respectively. Genotyping of RBC antigens showed that the Mia -positive donors were predominantly associated M+ N- S- s+ (48.5%) and M+ N+ S- s+ (38.1%) phenotypes.
Conclusions:
The GP.Mur glycophorin is most prevalent in the Mia -positive Asian American type O blood donors.
... 5,6 MNS48, also named KIPP, is a low-prevalence antigen encoded by the same GYP(B-A-B) hybrid that produces Mur, Hil, MUT, and MINY, but the resulting GP(B-A-B) hybrid has p.Ser51, which distinguishes this protein from other known GP(B-A-B) hybrids that have p.Tyr51. 6,7 MNS49, also named JENU, is a high-prevalence antigen on GPB encoded by GYPB, defined as an epitope within the amino acid sequence between positions 38 and 49 ( 38 SYISSQTNGETG 49 ) that is absent from the GP.Mur hybrid as it is disrupted by the insertion of hybrid exon 3. 1,8 New alleles and hybrid genes associated with MNS antigen expressions have also been identified, and the complete list of MNS antigens and alleles is available at http://www.isbtweb. org/working-parties/red-cell-immunogenetics-and-bloodgroup-terminology. ...
Conclusions:
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.
... The antibody maker was a thalassemia patient of Thai origin who was shown to lack normal GPB, but who was homozygous for the GYP.Mur hybrid (GYP*501). Thus, JENU is a high-prevalence antigen on GPB that is absent from the GP.Mur hybrid [1]. The epitope defined above is interrupted in the hybrid protein. ...
Background and objectives
The International Society of Blood Transfusion (ISBT) Working Party for Red Cell Immunogenetics and Blood Group Terminology meets in association with the ISBT congress and has met three times since the last report: at the international meetings held in Dubai, United Arab Emirates, September 2016 and Toronto, Canada, June 2018; and at a regional congress in Copenhagen, Denmark, June 2017 for an interim session.
Methods
As in previous meetings, matters pertaining to blood group antigen nomenclature and classification were discussed. New blood group antigens were approved and named according to the serologic and molecular evidence presented.
Results and conclusions
Fifteen new blood group antigens were added to eight blood group systems. One antigen was made obsolete based on additional data. Consequently, the current total of blood group antigens recognized by the ISBT is 360, of which 322 are clustered within 36 blood groups systems. The remaining 38 antigens are currently unassigned to a known system. Clinically significant blood group antigens continue to be discovered, through serology/sequencing and/or recombinant or genomic technologies.
Background and objectives:
High-frequency antigen Ena (MNS 28) is expressed on glycophorin A (GPA). En(a-) individuals can form anti-Ena when exposed to GPA. A Thai patient formed an antibody that reacted against all reagent red blood cells (RBCs). The patient received incompatible blood resulting in a fatal haemolytic transfusion reaction (HTR). This study aimed to characterize the antibody detected in the patient and investigate the cause of HTR.
Materials and methods:
Blood samples from the patient and three of his family members were investigated. Massively parallel sequencing (MPS) and DNA-microarray were used for genotyping. Standard haemagglutination techniques were used for phenotyping and antibody investigations.
Results:
DNA sequencing showed the patient was homozygous for GYPA*M c.295delG (p.Val99Ter) predicting En(a-). Three family members were heterozygous for GYPA c.295delG. MPS and DNA-microarray predicted the patient was N- discordant with the N+ RBC phenotype. The patient's plasma was positive with enzyme/chemical-treated reagent RBCs but failed to react with En(a-) and Mk Mk RBCs.
Conclusion:
The GYPA c.295delG variant prevented GPA expression on RBCs resulting in En(a-) phenotype. The N+ phenotype result was probably due to the anti-N typing reagent detecting 'N' (MNS30) on GPB. The patient's alloantibody has anti-Ena specificity.
Globally, everyone has gone through an unusual and remarkable period during the COVID-19 pandemic. The pandemic has affected various sectors in the country and implicated society. Important services including the health care system, particularly in surgery, have raised challenges and issues that need to be sorted out. This action is prudent to justify the balance in between care on preventing the spread of COVID-19 infection and at the same time providing surgical services. In this article are the perspectives on how we elicit the issues and the solutions in providing surgical services during the COVID-19 pandemic in our state, Sabah.
Background
The hybrid glycophorins of MNS blood group system express a series of low incidence antigens including Mia, which are commonly found in Southeast Asian populations. In this study, the molecular basis of Mia-positive hybrid glycophorins was firstly clarified in the Chinese Southern Han population. RNA transcripts of GYPB gene in the homozygous GP.Mur individuals were also analyzed.
Study design and methods
DNAs were extracted from the whole blood samples of 111 Mia-positive donors. Then, high-resolution melting (HRM) analysis for GYP(B-A-B) was used to analyze the genotypes. Sequencing of GYPB pseudoexon 3 was conducted in the samples with variant melting curves. TA-cloning and subsequent sequencing of GYPA exons 2–4 were performed in the Mia-positive samples with normal GYPB/GYPB genotype by HRM. The transcript analysis of GYPB was conducted in homozygous GP.Mur and wild-type glycophorin B (GPB) individuals using RNA extracted from the cultured erythroblast.
Results
The heterozygous GYP*Mur/GYPB (n = 101), homozygous GYP*Mur/GYP*Mur (n = 7) including one novel GYP*Mur allele with an extra GYPA/GYPE specific nucleotide substitution (c.229+110A>T), heterozygous GYP*Bun/GYPB (n = 1) and GYP*Vw/GYPA (n = 2) with two novel GYP*Vw alleles were identified. RNA transcript analysis revealed multiple transcripts of GYPB existing in both homozygous GP.Mur and normal GPB individuals.
Conclusion
The results showed the genetic diversity of hybrid glycophorins in the Chinese population. Besides, the successful analysis of GYPB transcripts indicates that the cultured erythroblast is a good source for RNA transcript analysis for the protein only expressed on the red blood cells.
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.
Background:
S and s antigens of the MNS system are of clinical importance because alloanti-S and -s have usually caused delayed hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Various red cell genotyping has been established to predict the phenotypes to solve serological test limitations.
Objectives and methods:
This study aimed to determine S and s genotype frequencies and to estimate the alloimmunization risks among central, northern and southern Thai populations. Altogether, 1237 blood samples from Thai blood donors were included. Only 150 samples were tested with anti-S and anti-s by indirect antiglobulin test. All samples were genotyped for GYPB*S and GYPB*s alleles using inhouse PCR with sequence-specific primer. Additionally, the allele frequencies were used to estimate alloimmunization risks and compare with other populations.
Results:
The phenotyping and genotyping results in 150 samples were in 100% concordance. The allele frequencies of GYPB*S in central, northern and southern Thais were 0.061, 0.040 and 0.097, and GYPB*s were 0.939, 0.960 and 0.903, respectively. The frequencies among central Thais were similar to those among northern Thai and Korean populations (P > 0.05) but significantly differed from those of Asian, Caucasian African American and Hispanic populations (P < 0.05). In addition, the risk of S alloimmunization among southern Thais (0.1566) was higher than those among central (0.1038) and northern Thais (0.0736).
Conclusion:
This was the first study to report S and s predicted phenotypes and estimate alloimmunization risks among Thais, which is beneficial to prevent transfusion-induced alloimmunization among donors and patients.
BACKGROUND
MNS hybrid GP(B‐A‐B) glycophorins are more commonly found in Southeast Asians and alloantibodies to antigens they carry are clinically significant. Detection of hybrid glycophorins by serologic techniques is limited due to lack of commercial reagents. In this study, a genotyping method for GP(B‐A‐B) hybrid glycophorins based on high‐resolution melting (HRM) analysis was applied for genotyping analysis in the Chinese Southern Han population.
STUDY DESIGN AND METHODS
DNA samples from 3104 Chinese Southern Han blood donors were collected. GYP(B‐A‐B) genotypes were analyzed by HRM assay. Parts of samples (n = 106) were also tested by multiplex ligation‐dependent probe amplification (MLPA) assay. Direct sequencing was conducted in samples with variant melting curve profiles.
RESULTS
A total of five GYP(B‐A‐B) genotypes (201/3104, 6.5%) were identified, which were GYP*Mur heterozygote (n = 194), GYP*Mur homozygote (n = 3), GYP*Bun heterozygote (n = 2), GYP*HF heterozygote (n = 1), and a novel GYP(B‐A‐B) hybrid allele (n = 1). Genotyping results for GYP*Mur and wild‐type GYPB samples obtained by HRM were consistent with MLPA, while GYP*Bun and GYP*HF heterozygote identified by HRM could only be identified to have one copy of 5′ inactive splice site of GYPB Pseudoexon 3 by MLPA. In addition, 10 single‐nucleotide polymorphisms (SNPs) including four known and six novel SNPs were identified in 31 samples. One sample was identified carrying both GYP*Mur and GYP*Sch alleles.
CONCLUSION
The HRM assay could distinguish the GYP(B‐A‐B) hybrid alleles successfully. Polymorphisms identified within the GYPB gene should be taken into consideration when developing GYP(B‐A‐B) genotyping kits for the Chinese population.
Molecular immunohematological techniques have been well applied in the transfusion medicine. Here, we review the major applications for blood group genotyping in China. Its application mainly focuses on the typing of blood group antigens in complex cases, such as ABO subgroups, D variants or the Asian specific and common antigens typing (such as the antigens expressed by the MNS hybrid glycophorins), also because of lacking of the corresponding commercial antibodies. The comprehensive antigens profile in some specific samples could be obtained using medium- or high-throughput genotyping platforms. Although, foetal RHD genotyping in D– pregnant women in Europe is highly relevant to avoid unnecessary antenatal RhIg, it is of comparatively less importance in the Chinese D– pregnant women because of the low probability (3–4%) to have a D– baby. In contrast, the application of routine molecular testing for the ‘Asia type’ DEL, which having a frequency up to 30% in East Asian people serologically typed as D-negative, is more necessary to avoid unnecessary administration of antenatal RhIg, because there is increasing evidence that individuals carrying the ‘Asia type’ DEL allele (c.1227G>A, p.Lys409Lys) do not produce alloanti-D after exposure to D+ red blood cells. The molecular methods to detect the RHD*1227A are more accurate and applicable for routine testing of ‘Asia type’ DEL.
This report provides the molecular evidence to support the existing serological evidence for GP.Kip. This hybrid is formed by a unique crossover with an insertion of three amino acids encoded by GYPA, consistent with the unique antigen specificity detected by Hop+Nob antisera, the KIPP antigen. This antigen arises from a GYP(B-A-B) crossover differing from the other GP(B-A-B) hybrids (p.Tyr51) by the retention of a codon from the GYPB pseu-doexon sequence encoding p.Ser51 in the GP.Kip hybrid glycophorin.
Background and objectivesDuffy blood group phenotypes can be predicted by genotyping for single nucleotide polymorphisms (SNPs) responsible for the Fya/Fyb polymorphism, for weak Fyb antigen, and for the red cell null Fy(a−b−) phenotype. This study correlates Duffy phenotype predictions with serotyping to assess the most reliable procedure for typing.Materials and methodsSamples, n = 155 (135 donors and 20 patients), were genotyped by high-resolution melt PCR and by microarray. Samples were in three serology groups: 1) Duffy patterns expected n = 79, 2) weak and equivocal Fy(b) patterns n = 29 and 3) Fy(a−b−) n = 47 (one with anti-Fy3 antibody).ResultsDiscrepancies were observed for five samples. For two, SNP genotyping predicted weak Fyb expression discrepant with Fy(b−) (Group 1 and 3). For three, SNP genotyping predicted Fya, discrepant with Fy(a−b−) (Group 3). DNA sequencing identified silencing mutations in these FY*A alleles. One was a novel FY*A 719delG. One, the sample with the anti-Fy3, was homozygous for a 14-bp deletion (FY*01N.02); a true null.Conclusion
Both the high-resolution melting analysis and SNP microarray assays were concordant and showed genotyping, as well as phenotyping, is essential to ensure 100% accuracy for Duffy blood group assignments. Sequencing is important to resolve phenotype/genotype conflicts which here identified alleles, one novel, that carry silencing mutations. The risk of alloimmunisation may be dependent on this zygosity status.
The Miltenberger (Mi) subsystem, which originally consisted of four phenotypes, now has 11 phenotypes. The antigens of this subsystem belong to the MNS blood group system. The Mia antigen has been reported to be present on red blood cells with several Miltenberger phenotypes, namely: Mi.I, Mi.II, Mi.III, Mi.IV, Mi.VI and Mi.X. However, the existence of the Mia antigen as a separate entity has been in question and difficult to prove with polyclonal reagents. We report the first monoclonal anti-Mia (GAMA210), whose epitope is TNDKHKRD or QTNDMHKR, and thereby confirm the existence of the Mia antigen.
The MNS system was the second blood group system discovered and at least 16 of the 46 antigens in the MNS system result from genetic recombination, producing a hybrid glycophorin. The incidence of these hybrid glycophorins is highest in East Asian populations. MNS system antigens defined by hybrid glycophorins are immunogenic with alloimmune IgG responses developing after transfusion or pregnancy; with reports originating from Asia, Europe, the Americas, and Australia. This demonstrates the global nature of problems associated with these antibodies. Since the initial report that production of anti-Mi(a) was a cause of hemolytic disease of the fetus and newborn (HDFN), antibodies to antigens defined by hybrid glycophorins have been reported in 27 cases of HDFN (1 fatal) and 8 cases of hemolytic transfusion reaction (HTR) (1 fatal). In at least 40% of these clinical cases, the disease was reported as severe. Hyporegenerative fetal anemia is a common feature of the reported HDFN cases. In all published cases, the causative antibodies were identified by reference laboratory investigative tests following clinical presentation. The failure to detect these antibodies by routine testing highlights the need for consideration of the medical importance of these antibodies when defining antibody screening practices and reagents. The aim of this review is to raise awareness of severe disease caused by antibodies to MNS antigens defined by hybrid glycophorins and, thus, to improve diagnosis and patient management.
The MNS blood group system is second only to the Rh blood group system in its complexity. Many alloantibodies to antigens in the MNS system are not generally clinically significant although antibodies to low-prevalence and high-prevalence MNS antigens have caused hemolytic disease of the fetus and newborn. The MNS antigens are carried on glycophorin A (GPA), glycophorin B (GPB), or hybrids thereof, which arise from single-nucleotide substitution, unequal crossing over, or gene conversion between the glycophorin genes. Antigens in the MNS system are fully developed at birth. This review summarizes aspects of the MNS system, including the molecular basis of some antigens in the MNS blood group system. Readers are referred to existing excellent reviews for background information. Throughout this document, information given without references can be found in the reviews listed previously, and the reader is referred to these reviews for references to original reports.