The study of variant s antigen expression revealing a novel c.160C>T (p.Arg54Cys) variant on GYPB*s allele associated with partial s phenotype.

BACKGROUND: Little s antigen is mainly defined by a single nucleotide polymorphism at c.143C (p.Thr48) on the GYPB gene. Several variants on GYPB can alter the expression of s antigen. The aim of this study was to investigate the molecular basis of variant s antigen expression in the Chinese population. STUDY DESIGN AND METHODS: A total of 4983 whole blood samples were collected to screen the individuals with discrepant s typing results using two different monoclonal anti-s. Then, the sequence of GYPB exon 4 was analyzed by Sanger sequencing. Flow cytometry analysis was performed to quantify s antigen expression on red blood cells (RBCs). In vitro expression study was performed to verify the effect of the GYPB variants identified on the expression of s antigen. RESULTS: Four donors were identified to have discrepant s typing results. Sanger sequencing showed that three donors carried the c.173C > G variant (p.Pro58Arg) specific for sD antigen, the other one carried a novel GYPB (c.160C > T, p.Arg54Cys) variant. Flow cytometry identified a partial and weak expression of s antigen on the RBCs of the four donors. Furthermore, in vitro expression study confirmed the effect of the two variants on the s antigen expression. CONCLUSION: The results demonstrated that in addition to p.Thr48, the two extra amino acids p.Arg54 and p.Pro58 are also important for full expression of s antigen. Since the individuals with partial s antigen are at risk for the development of alloanti-s, it is important to select at least two different monoclonal anti-s for correct s typing.

Radiofrequency dielectric spectroscopy study: Effects of pH, hydrogen bond donors and acceptors on the attachment of spectrin skeleton to the lipid membrane of erythrocytes.

Band 3 protein and glycophorin C are the two major integral proteins of the lipid membrane of human red blood cells (RBCs). They are attached from below to a network of elastic filamentous spectrin, the third major RBC membrane protein. The binding properties of the attachments to spectrin affect the shape and deformability of RBCs. We addressed band 3 and glycophorin C attachments to spectrin by measuring the strength of two recently discovered radiofrequency dielectric relaxations, ßsp (1.4 MHz) and γ1sp (9 MHz), that are observable as changes in the complex admittance of RBCs in medium. In medium at pH 5.2, and also in media with protic substances (formamide, methylformamide, or urea), the ßsp relaxation became inhibited that is attributable to detachment of glycophorin C from spectrin. In medium at pH 9.2, we observed inhibition of γ1sp relaxation attributable to detachment of band 3 from spectrin, as also was seen in media with aprotic substances difluoropyridine, dimethylsolfoxide, dimethylformamide, acetone, sodium tetrakis(4-fluorophenyl)borate), chlorpromazine, thioridazine and trifluopiperazine. The viscogenic cosolvents (glycerol, ethylene glycol, or i-erythritol) inhibited both the ßsp and γ1sp relaxations and significantly lowered their characteristic frequencies. Our observations indicate that the glycophorin C attachment to spectrin has nucleophilic centers whose saturation disconnects this attachment and inhibits the ßsp relaxation, whereas at band 3-spectrin attachment site, it is the saturation of electrophilic centers that weakens this attachment and inhibits the γ1sp relaxation.

Genetic variation of glycophorins and infectious disease.

Glycophorins are transmembrane proteins of red blood cells (RBCs), heavily glycosylated on their external-facing surface. In humans, there are four glycophorin proteins, glycophorins A, B, C and D. Glycophorins A and B are encoded by two similar genes GYPA and GYPB, and glycophorin C and glycophorin D are encoded by a single gene, GYPC. The exact function of glycophorins remains unclear. However, given their abundance on the surface of RBCs, it is likely that they serve as a substrate for glycosylation, giving the RBC a negatively charged, complex glycan "coat". GYPB and GYPE (a closely related pseudogene) were generated from GYPA by two duplication events involving a 120-kb genomic segment between 10 and 15 million years ago. Non-allelic homologous recombination between these 120-kb repeats generates a variety of duplication alleles and deletion alleles, which have been systematically catalogued from genomic sequence data. One allele, called DUP4, encodes the Dantu NE blood type and is strongly protective against malaria as it alters the surface tension of the RBC membrane. Glycophorins interact with other infectious pathogens, including viruses, as well as the malarial parasite Plasmodium falciparum, but the role of glycophorin variation in mediating the effects of these pathogens remains underexplored.

Differential enzymatic deglycosylation reveals attachment of red cell B antigen onto the carbohydrate moiety of glycophorin A and glycophorin B.

BACKGROUND AND OBJECTIVES: Early studies indicate that red cell A and B antigens are attached primarily onto band 3 and GLUT1 on the erythrocyte membrane and little onto glycophorin A (GPA) and glycophorin B (GPB). But as GPA and band 3 form stable protein complexes and GPA is much more heavily glycosylated than band 3, this study re-examined the association between ABO antigens and GPA/GPB. MATERIALS AND METHODS: Band 3/GPA-associated protein complexes were first immunoprecipitated, followed by differential enzymatic deglycosylation that removed sialic acids, N-glycans and O-glycans. Serological anti-A (BIRMA 1) and anti-B IgM (GAMA 110) could be used for western blot (WB); however, only the anti-B IgM showed significant reactivity for the immunoprecipitates isolated by anti-band 3. The expression of the B antigen in un-deglycosylated and differentially deglycosylated band 3 immunoprecipitates was thus compared. RESULTS: Besides attachment to band 3, red cell B antigen expressed substantially on GPA monomer and homodimer, GPA*GPB heterodimer, and GPB monomer and dimer via attachments through the N- and O-glycans. CONCLUSION: Immunoprecipitation (IP), as a means of protein separation and concentration, was used in combination with a WB to differentiate glycosylation on different proteins and oligomers. This study implemented differential enzymatic deglycosylation during IP of the band 3 complexes. This combined approach allowed separate identification of the B antigen on GPA/GPB monomer and dimer and GPA*GPB heterodimer, and band 3 on the WB and verified non-trivial expression of the B antigen on GPA and GPB on the erythrocyte surface.

Folding and modulation of the helical conformation of Glycophorin A by point mutations.

Transmembrane helix folding and self-association play important roles in biological signaling and transportation pathways across biomembranes. With molecular simulations, studies to explore the structural biochemistry of this process have been limited to focusing on individual fragments of this process - either helix formation or dimerization. While at an atomistic resolution, it can be prohibitive to access long spatio-temporal scales, at the coarse grained (CG) level, current methods either employ additional constraints to prevent spontaneous unfolding or have a low resolution on sidechain beads that restricts the study of dimer disruption caused by mutations. To address these research gaps, in this work, we apply our recent, in-house developed CG model (ProMPT) to study the folding and dimerization of Glycophorin A (GpA) and its mutants in the presence of Dodecyl-phosphocholine (DPC) micelles. Our results first validate the two-stage model that folding and dimerization are independent events for transmembrane helices and found a positive correlation between helix folding and DPC-peptide contacts. The wild type (WT) GpA is observed to be a right-handed dimer with specific GxxxG contacts, which agrees with experimental findings. Specific point mutations reveal several features responsible for the structural stability of GpA. While the T87L mutant forms anti-parallel dimers due to an absence of T87 interhelical hydrogen bonds, a slight loss in helicity and a hinge-like feature at the GxxxG region develops for the G79L mutant. We note that the local changes in the hydrophobic environment, affected by the point mutation, contribute to the development of this helical bend. This work presents a holistic overview of the structural stability of GpA in a micellar environment, while taking secondary structural fluctuations into account. Moreover, it presents opportunities for applications of computationally efficient CG models to study conformational alterations of transmembrane proteins that have physiological relevance.

Effects of cholesterol and PIP2 on interactions between glycophorin A and Band 3 in lipid bilayers.

In the erythrocyte membrane, the interactions between glycophorin A (GPA) and Band 3 are associated strongly with the biological function of the membrane and several blood disorders. In this work, using coarse-grained molecular-dynamics simulations, we systematically investigate the effects of cholesterol and phosphatidylinositol-4,5-bisphosphate (PIP2) on the interactions of GPA with Band 3 in the model erythrocyte membranes. We examine the dynamics of the interactions of GPA with Band 3 in different lipid bilayers on the microsecond time scale and calculate the binding free energy between GPA and Band 3. The results indicate that cholesterols thermodynamically favor the binding of GPA to Band 3 by increasing the thickness of the lipid bilayer and by producing an effective attraction between the proteins due to the depletion effect. Cholesterols also slow the kinetics of the binding of GPA to Band 3 by reducing the lateral mobility of the lipids and proteins and may influence the binding sites between the proteins. The anionic PIP2 lipids prefer binding to the surface of the proteins through electrostatic attraction between the PIP2 headgroup and the positively charged residues on the protein surface. Ions in the solvent facilitate PIP2 aggregation, which promotes the binding of GPA to Band 3.

Joint efficacy of the three biomarkers SNCA, GYPB and HBG1 for atrial fibrillation and stroke: Analysis via the support vector machine neural network.

Atrial fibrillation (AF) is the most common type of persistent arrhythmia. Although its incidence has been increasing, the pathogenesis of AF in stroke remains unclear. In this study, a total of 30 participants were recruited, including 10 controls, 10 patients with AF and 10 patients with AF and stroke (AF + STROKE). Differentially expressed genes (DEGs) were identified, and functional annotation of DEGs, comparative toxicogenomic database analysis associated with cardiovascular diseases, and predictions of miRNAs of hub genes were performed. Using RT-qPCR, biological process and support vector machine neural networks, numerous DEGs were found to be related to AF. HBG1, SNCA and GYPB were found to be upregulated in the AF group. Higher expression of hub genes in AF and AF + STROKE groups was detected via RT-PCR. Upon training the biological process neural network of SNCA and GYPB for HBG1, only small differences were detected. Based on the support vector machine, the predicted value of SNCA and GYPB for HBG1 was 0.9893. Expression of the hub genes of HBG1, SNCA and GYPB might therefore be significantly correlated to AF. These genes are involved in the incidence of AF complicated by stroke, and may serve as targets for early diagnosis and treatment.

Human Cell-Camouflaged Nanomagnetic Scavengers Restore Immune Homeostasis in a Rodent Model with Bacteremia.

Bloodstream infection caused by antimicrobial resistance pathogens is a global concern because it is difficult to treat with conventional therapy. Here, scavenger magnetic nanoparticles enveloped by nanovesicles derived from blood cells (MNVs) are reported, which magnetically eradicate an extreme range of pathogens in an extracorporeal circuit. It is quantitatively revealed that glycophorin A and complement receptor (CR) 1 on red blood cell (RBC)-MNVs predominantly capture human fecal bacteria, carbapenem-resistant (CR) Escherichia  coli, and extended-spectrum beta-lactamases-positive (ESBL-positive) E. coli, vancomycin-intermediate Staphylococcus aureus (VISA), endotoxins, and proinflammatory cytokines in human blood. Additionally, CR3 and CR1 on white blood cell-MNVs mainly contribute to depleting the virus envelope proteins of Zika, SARS-CoV-2, and their variants in human blood. Supplementing opsonins into the blood significantly augments the pathogen removal efficiency due to its combinatorial interactions between pathogens and CR1 and CR3 on MNVs. The extracorporeal blood cleansing enables full recovery of lethally infected rodent animals within 7 days by treating them twice in series. It is also validated that parameters reflecting immune homeostasis, such as blood cell counts, cytokine levels, and transcriptomics changes, are restored in blood of the fatally infected rats after treatment.

Autoantibodies to red blood cell surface Glycophorin A impact the activation poise of circulating leukocytes.

BACKGROUND: Both M and N alleles encode antigens on Glycophorin A (GPA), a red blood cell (RBC) surface sialoglycoprotein. Interaction between RBC GPA and leukocyte surface lectins may downregulate their activation. The current study investigates if RBC autoantibodies against GPA, such as auto-anti-M/N, prime an activated phenotype in peripheral blood leukocytes. METHODS: Leukocyte activation was assessed in whole blood from patients with auto-anti-GPA (anti-M/N) and compared to those with allo-anti-M/N and healthy subjects. Control samples from healthy subjects with no antibodies incubated in vitro with either anti-GPA or anti-Rh were analyzed for neutrophil and monocyte surface activation marker expression, reactive oxygen species (ROS) content, and formation of aggregates with RBCs. Samples incubated with an IgG1 isotype antibody served as controls. RESULTS: Ex vivo, neutrophil CD66b and monocyte CD63 surface expression was increased in patients with auto-anti-M/N compared to those with allo anti-M/N (p = .1757; p = .0698) and to healthy subjects (p = .0186; p = .013). In vitro, neutrophil CD66b and monocyte CD63 surface expression was increased following incubation with anti-GPA compared to anti-Rh (p = .0003; p = .0328) and isotype control (p = .000; p = .0062). Intracellular ROS content increased in both neutrophils and monocytes incubated with anti-GPA compared to anti-Rh (p = .0012; p = .0693) and isotype control (p = .001; p = .0021). Percentage of neutrophil-RBC aggregates was decreased when incubated with anti-GPA compared to isotype control (p < .01). CONCLUSIONS: Neutrophils and monocytes in peripheral blood exposed to an antibody directed against GPA on RBC surfaces, such as M or N antigens, may be primed towards an activated phenotype.

Hemolytic disease of the fetus and newborn caused by anti-sD antibody in a GP.Mur/Mur Thai mother and review of the prevalence of sD in Thai blood donors.

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.

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

BACKGROUND AND OBJECTIVES: Antigens of the MNS blood group system are expressed on the red blood cell (RBC) membrane on glycophorin A (GPA) and glycophorin B (GPB) or on hybrid molecules of GPA and GPB. This study investigated the distribution of glycophorin variants and alloantibodies against Hil and MINY among Japanese individuals. METHODS: Mi(a+) or Hil+ RBCs were screened using an automated blood grouping machine (PK7300) with monoclonal anti-Mia or polyclonal anti-Hil. Glycophorin variants were defined by serology with monoclonal antibodies against Mia , Vw, MUT and Mur, and polyclonal antibodies against Hil, MINY and Hop + Nob (KIPP). The glycophorin variants were further confirmed by immunoblotting and Sanger sequencing. Alloanti-Hil and alloanti-MINY in the plasma were screened using GP.Hil RBCs in an antiglobulin test. The specificity of anti-Hil or anti-MINY was assessed using GP.Hil (Hil+MINY+) and GP.JL (Hil-MINY+) RBCs. RESULTS: The GP.HF, GP.Mur, GP.Hut, GP.Vw, GP.Kip and GP.Bun frequencies in 1 005 594 individuals were 0·0357%, 0·0256%, 0·0181%, 0·0017%, 0·0009% and 0·0007%, respectively. GP.Hil was found in as four of the 13 546 individuals (0·0295%). Of 137 370 donors, 10 had anti-Hil (0·0073%) and three had anti-MINY (0·0022%). CONCLUSIONS: Glycophorin variants were relatively rare in Japanese individuals, with the major variants being GP.HF (0·0357%), GP.Hil (0·0295%) and GP.Mur (0·0256%). Only one example of anti-MINY was previously reported, but we found three more in this study.

Characterization of alternatively spliced transcript variants of glycophorin A and glycophorin B genes in Chinese blood donors.

BACKGROUND AND OBJECTIVES: The molecular basis of MNS blood group variants is not fully clear yet. In this study, we have characterized mRNA variants of GYPA and GYPB genes to reveal whether alternative RNA splicing may cause antigenic diversity of the MNS system. MATERIALS AND METHODS: Total RNA was extracted from peripheral blood of Chinese blood donors and full-length cDNA products were generated. A nested polymerase chain reaction (PCR)-based method was established for fragment amplification and Sanger sequencing. Resulted full-length mRNA sequences were aligned with GYPA or GYPB genomic sequences respectively for exon identification. Amino acid (AA) sequences of GPA and GPB proteins were extrapolated and GYPA-EGFP, GYPB-EGFP fusion genes were generated to monitor subcellular distribution of the encoded glycophorin (GP) proteins. RESULTS: Totally 10 blood samples were analysed. GYPB mRNAs of all the subjects demonstrated frequent exon insertion or deletion whereas this kind of variation was only observed in 3 of 10 GYPA mRNA samples. None of the reported Miltenberger hybrids was detected in any of the mRNA samples. The alternative splicing resulted in changes of AA sequences in N-terminal domains where the MNS antigenic motifs resided; however, subcellular localizations of GP-EGFP fusion proteins showed that the above-mentioned AA changes did not affect cell surface distribution of the encoded GP proteins. CONCLUSIONS: Alternative RNA splicing may influence the antigenic features of GP proteins but not their cell surface distribution. Therefore, GYPA and GYPB mRNA characterization might be an invaluable supplement to serological phenotyping and DNA-based genotyping in MNS blood grouping.

Fatal haemolytic transfusion reaction due to anti-Ena and identification of a novel GYPA c.295delG variant in a Thai family.

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.

Influence of hemoglobin on blood pressure among people with GP.Mur blood type☆.

BACKGROUND/PURPOSE: GP.Mur is a clinically important red blood cell (RBC) type. GP.Mur and band 3 interact on the RBCs. We previously observed that healthy adults with GP.Mur type present slightly higher blood pressure (BP). Because band 3 and Hb comodulate nitric oxide (NO)-dependent vasodilation and hemoglobin (Hb) is positively associated with BP, we aimed to test whether these could contribute to higher BP in GP.Mur+ people. METHODS: We recruited 989 non-elderly adults (21% GP.Mur) free of catastrophic illness and not on cardiovascular or anti-hypertensive medication. Their body indices, blood lab data and lifestyle data were collected for analyses of potential BP-related factors (BMI, age, smoking, Hb, and GP.Mur). RESULTS: BMI and age remained the most significant contributors to BP. GP.Mur slightly increased systolic BP (SBP). The direct correlation between Hb and BP was only found in Taiwanese non-anemic men, not women. After age and BMI adjusted, we estimated an increase of 1.8 mmHg and 2.6 mmHg of SBP by 1 g/dL Hb among men without and with GP.Mur type, respectively. Hb was generally lower among people expressing GP.Mur, which likely limited their larger impact on BP. CONCLUSION: GP.Mur contributed to BP in both Hb-dependent and Hb-independent fashion. A pronounced impact of hemoglobin on BP likely requires sufficient Hb, as GP.Mur increased the sensitivity of SBP to Hb only in non-anemic Taiwanese men, and not in Taiwanese women or anemic men. The mechanism through which GP.Mur affected BP independent of Hb is unknown.

Comprehensive transcriptome analysis of erythroid differentiation potential of olive leaf in haematopoietic stem cells.

Anaemia is one of the leading causes of disability in young adults and is associated with increased morbidity and mortality in elderly. With a global target to reduce the disease burden of anaemia, recent researches focus on novel compounds with the ability to induce erythropoiesis and regulate iron homeostasis. We aimed to explore the biological events and potential polypharmacological effects of water-extracted olive leaf (WOL) on human bone marrow-derived haematopoietic stem cells (hHSCs) using a comprehensive gene expression analysis. HPLC analysis identifies six bioactive polyphenols in the WOL. Treatment with WOL for 12 days regulated gene expressions related to erythroid differentiation, oxygen homeostasis, iron homeostasis, haem metabolism and Hb biosynthesis in hHSCs. Functional clustering analysis reveals several major functions of WOL such as ribosomal biogenesis and mitochondrial translation machinery, glycolytic process, ATP biosynthesis and immune response. Additionally, the colonies of both primitive and mature erythroid progenitors, CFU-E and BFU-E, were significantly increased in WOL-treated hHSCs. The expressions of erythroid markers, CD47, glycophorin A (GYPA), and transferrin receptor (TFRC) and adult Hb subunits-HBA and HBB were also confirmed in immunofluorescent staining and flow cytometer analysis in WOL-treated hHSCs. It is well known that induction of lineage-specific differentiation, as well as the maturation of early haematopoietic precursors into fully mature erythrocytes, involves multiple simultaneous biological events and complex signalling networks. In this regard, our genome-wide transcriptome profiling with microarray study on WOL-treated hHSCs provides general insights into the multitarget prophylactic and/or therapeutic potential of WOL in anaemia and other haematological disorders.

Genotyping complex structural variation at the malaria-associated human glycophorin locus using a PCR-based strategy.

Structural variation in the human genome can affect risk of disease. An example is a complex structural variant of the human glycophorin gene cluster, called DUP4, which is associated with a clinically significant level of protection against severe malaria. The human glycophorin gene cluster harbours at least 23 distinct structural variants, and accurate genotyping of this complex structural variation remains a challenge. Here, we use a polymerase chain reaction-based strategy to genotype structural variation at the human glycophorin gene cluster, including the alleles responsible for the U- blood group. We validate our approach, based on a triplex paralogue ratio test, on publically available samples from the 1000 Genomes project. We then genotype 574 individuals from a longitudinal birth cohort (Tori-Bossito cohort) using small amounts of DNA at low cost. Our approach readily identifies known deletions and duplications, and can potentially identify novel variants for further analysis. It will allow exploration of genetic variation at the glycophorin locus, and investigation of its relationship with malaria, in large sample sets at minimal cost, using standard molecular biology equipment.

The Malaria-Protective Human Glycophorin Structural Variant DUP4 Shows Somatic Mosaicism and Association with Hemoglobin Levels.

Glycophorin A and glycophorin B are red blood cell surface proteins and are both receptors for the parasite Plasmodium falciparum, which is the principal cause of malaria in sub-Saharan Africa. DUP4 is a complex structural genomic variant that carries extra copies of a glycophorin A-glycophorin B fusion gene and has a dramatic effect on malaria risk by reducing the risk of severe malaria by up to 40%. Using fiber-FISH and Illumina sequencing, we validate the structural arrangement of the glycophorin locus in the DUP4 variant and reveal somatic variation in copy number of the glycophorin B-glycophorin A fusion gene. By developing a simple, specific, PCR-based assay for DUP4, we show that the DUP4 variant reaches a frequency of 13% in the population of a malaria-endemic village in south-eastern Tanzania. We genotype a substantial proportion of that village and demonstrate an association of DUP4 genotype with hemoglobin levels, a phenotype related to malaria, using a family-based association test. Taken together, we show that DUP4 is a complex structural variant that may be susceptible to somatic variation and show that DUP4 is associated with a malarial-related phenotype in a longitudinally followed population.

Molecular genetic analysis of Mia -positive hybrid glycophorins revealed two novel alleles of GP.Vw and multiple variant transcripts of GYPB existing in both the homozygous GP.Mur and wild-type GPB individuals.

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.

GP.MOT: A novel glycophorin variant identified in a Japanese blood donor.

BACKGROUND: In this study, we identified a novel glycophorin variant (GP.MOT) in a Mia -positive Japanese blood donor. The proband with this glycophorin variant was discovered by antigen screening of samples from 475,493 Japanese blood donors using monoclonal anti-Mia . STUDY DESIGN AND METHODS: Standard serological techniques and flow cytometry were performed. GP.MOT RBCs were examined by immunoblotting using anti-GPA, anti-MUT or anti-Mur. Genome DNA was extracted from whole blood, and the GYPA/GYPB was analyzed by polymerase chain reactions and Sanger sequencing. RESULTS: The MNS blood group of the proband was M + N + w S-s + with the presence of other low-frequency antigens including Mia , Mur, MUT, and KIPP. A 43-kDa molecule, which is almost equivalent in size to glycophorin A (GPA), was identified by immunoblotting using monoclonal anti-MUT and anti-Mur. Sanger sequencing clearly indicated that the proband had two different GYPA*M alleles at SNP rs62334651 (GYPA*M232 + 55A and GYPA*M232 + 55G), as well as a GYP(B-A) hybrid allele (GYP*MOT) with breakpoints located on pseudoexon 3 of GYPB from c.210 to c.219. DISCUSSION: We identified a hybrid glycophorin GP.MOT with the deduced unique amino acid sequence GPB (20-45)-GPΨB (46-70)-GPA (71-149), which has not been previously reported.

Atomistic mechanism of transmembrane helix association.

Transmembrane helix association is a fundamental step in the folding of helical membrane proteins. The prototypical example of this association is formation of the glycophorin dimer. While its structure and stability have been well-characterized experimentally, the detailed assembly mechanism is harder to obtain. Here, we use all-atom simulations within phospholipid membrane to study glycophorin association. We find that initial association results in the formation of a non-native intermediate, separated by a significant free energy barrier from the dimer with a native binding interface. We have used transition-path sampling to determine the association mechanism. We find that the mechanism of the initial bimolecular association to form the intermediate state can be mediated by many possible contacts, but seems to be particularly favoured by formation of non-native contacts between the C-termini of the two helices. On the other hand, the contacts which are key to determining progression from the intermediate to the native state are those which define the native binding interface, reminiscent of the role played by native contacts in determining folding of globular proteins. As a check on the simulations, we have computed association and dissociation rates from the transition-path sampling. We obtain results in reasonable accord with available experimental data, after correcting for differences in native state stability. Our results yield an atomistic description of the mechanism for a simple prototype of helical membrane protein folding.