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 that 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 A-glycophorin B fusion gene. By developing a simple, specific, PCR-based assay for DUP4 we show the DUP4 variant reaches a frequency of 13% in a 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 it is associated with a malarial-related phenotype in a non-hospitalized population.Significance statementPrevious work has identified a human complex genomic structural variant called DUP4, which includes two novel glycophorin A-glycophorin B fusion genes, is associated with a profound protection against severe malaria. In this study, we present data showing the molecular basis of this complex variant. We also show evidence of somatic variation in the copy number of the fusion genes. We develop a simple robust assay for this variant and demonstrate that DUP4 is at an appreciable population frequency in Tanzania and that it is associated with higher hemoglobin levels in a malaria-endemic village. We suggest that DUP4 is therefore protective against malarial anemia.

Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes

ABSTRACT Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum.

Hereditary Stomatocytosis Associated with a Loss of Function Mutation In Rh-Associated Glycoprotein (RhAG)

Abstract 2040 The erythroid Rh family of proteins includes RhCE and RhD which carry the common Rh antigens, and the related Rh-associated glycoprotein, RhAG. RhAG is required for trafficking of the blood group proteins to the membrane and forms the core of a macro-complex in the membrane which includes glycophorin B, Band 3, CD47, and LW. The Rh proteins are structurally and functionally related to the Amt superfamily of NH3/NH4+ transport proteins, and RhAG and its nonerythroid paralogs, RhCG and RhBG, have been shown to mediate NH3/NH4+ transport. RhCG is responsible for part of renal collecting duct epithelial cell NH3/NH4+ secretion, and Rhcg-/- mice exhibit incomplete distal renal tubular acidosis due to impaired urinary NH4+ excretion. The Rhag-/- mouse is grossly normal, and the significance of RhAG-mediated NH3/NH4+ transport in human erythrocytes remains unclear. Over-hydrated hereditary stomatocytosis (OHSt) is a rare dominant disorder characterized by moderate hemolytic anemia, increased mean red cell volumes, stomatocytes and echinocytes, and increased red cell permeability to the monovalent cations, Na+ and K+. Six of the seven OHSt kindred studied by Bruce et al. (Blood. 2009;113:1350) displayed a heterozygous Phe65Ser mutation in RhAG. Expression studies of the mutant 65Ser-RhAG in Xenopus oocytes induced a monovalent cation flux compatible with the cation leak seen in RBCs. The increased Na+ and decreased K+ contents of mutant RhAG-expressing oocytes suggested that F65S is a gain-of-function mutation that opens a cation leak, likely within the RhAG polypeptide. In this study the ammonia transport properties of the OHSt mutant 65Ser-RhAG were investigated. Xenopus oocytes were injected with cRNA encoding wild-type RhAG, the OHSt mutant 65Ser-RhAG, and 65Val-RhAG, an engineered mutation with a smaller hydrophobic side chain at position 65. Wild-type and mutant RhAG polypeptides were well-expressed in the oocyte membrane as measured by quantitative immunoblotting. Uptake of the NH3/NH4+ substrate analog 14C-methylammonium (MA), was assayed in oocytes previously injected with water (control) or with cRNA. Expression of wild-type RhAG mediated MA uptake at rates 6-fold greater than that of water-injected controls. Uptake of MA by oocytes expressing 65Val-RhAG was equivalent to that of wild type RhAG. However, MA uptake by oocytes expressing OHSt mutant 65Ser-RhAG was greatly reduced to less than 20% that of oocytes expressing wild-type RHAG or 65Val-RhAG, and was only 1.5-fold greater than that of water-injected control oocytes. Co-expression with other, individual Rh complex members glycophorin B, RhD, RhCE, or Band 3 did not alter MA-mediated uptake by RhAG-expressing oocytes. Importantly, this study reveals that the RhAG mutation Phe65Ser found in patients with type 1 over-hydrated stomatocytosis is a loss of function mutation. Further study is required to define the relationship between loss of NH3/NH4+ transport and erythrocyte Na+ and K+ cation content. Disclosures: Westhoff: Immucor: Scientific Advisor.

Miltenberger blood group antigen type III (Mi.III) enhances the expression of band 3

The special blood group antigen Mi.III exhibits a characteristic hybrid structure of glycophorin A (GPA) and glycophorin B, termed Gp.Mur. This phenotype has exceptionally high occurrence rates in several indigenous tribes in Taiwan (∼21.2%-88.4%). Because glycophorin/Miltenberger begins interaction with anion exchanger-1 (AE1) in the endoplasmic reticulum, we hypothesized that the AE1-based macrocomplexes on erythrocyte membranes obtained from Mi.III+ people could be differentiated from those obtained from non-Miltenberger people. Quantitative mass spectrometric comparison of the AE1-based complexes by iTRAQ™ (Applied Biosystems) revealed 25% to 67% higher expression of AE1 in Mi.III+ erythrocytes. In accordance with the higher AE1 level, the Mi.III+ erythrocytes exhibited superior HCO3− capacities, pH homeostasis, and osmotic resistance. Cotransfection experiments in HEK293 cells showed that Gp.Mur, like GPA, enhanced trafficking of AE1 to the plasma membrane. In summary, the increased surface expression of AE1 in Mi.III+ erythrocytes could be attributed to the additive effect of GPA and Gp.Mur coexpression.