P1481: SYSTEMATICALLY REDUCED RED CELL PHOSPHATIDYLSERINE FLIPPASE ACTIVITY IS SUFFICIENT TO INDUCE HEREDITARY HEMOLYTIC ANEMIA

Topic: 28. Enzymopathies, membranopathies and other anemias Background: The Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C (ATP11C) gene encodes the major phosphatidylserine (PS) flippase in human red blood cells (RBCs). Flippases actively transport phospholipids (e.g. PS) from the outer to the inner leaflet of the lipid bilayer to maintain phospholipid asymmetry. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Aims: To study pathophysiological consequences of altered flippase activity based on a case of hemolytic anemia associated with a novel missense mutation in ATP11C. Methods: Targeted next generation sequencing (46 genes, including ATP11C) was performed as part of the diagnostic work up for hereditary hemolytic anemia. Recombinant mutant ATP11C protein was generated by site directed mutagenesis of the equivalent residue in murine Atp11c cDNA. Mutant and wild type (WT) protein were transiently expressed in HEK293T cells, and expression levels were determined by immunoblotting. Recombinant protein was investigated for PS-stimulated ATPase activity, and kinetic parameters Vmax and Michaelis-Menten constant (Km). After obtaining informed consent (Medical Research Ethics Committee Utrecht no. 17/450), further functional analysis was performed using blood of the patient and healthy controls. ATP11C protein expression was determined on RBC ghosts by immunoblotting. Flippase activity and PS surface exposure were measured by flow cytometry on purified RBCs (NBD-PS and Annexin V, respectively). Membrane lipid composition and distribution were determined by lipidomics and confocal live cell imaging HPLC-MS. An extensive evaluation of RBC properties and life span was performed using RBC morphology, ektacytometry, age-fractionation, age-markers and Erysense (Cysmic). Results: A 37-year-old male presented with severe abdominal pain (left upper quadrant), mild splenomegaly, chronic fatigue and reduced exercise capacity. Laboratory parameters showed mild normocytic hemolytic anemia and mild hyperbilirubinemia. Acquired causes of hemolytic anemia were excluded. Mutation analysis showed hemizygosity for a novel c.2365C>T p.(Leu789Phe) mutation in ATP11C, predicted to be pathogenic by in silico tools. In addition, the patient was heterozygous for α-spectrin (SPTA1) αLEPRA mutation, a relatively common cause of autosomal recessive hereditary spherocytosis. Recombinant ATP11C protein expression of the equivalent murine Leu786Phe mutation was reduced to 27% compared to WT. In addition, Vmax was 57% lower compared to WT. In patient-derived RBC ghosts, ATP11C protein expression was similarly reduced. Flippase activity was only 26% compared to controls (Figure 1A). PS surface exposure in the total RBC population was increased compared to control (Figure 1B), more pronounced after aging of the cells. This increase was not due to a higher PS global membrane content. In contrast, membrane lipid lateral distribution showed increased abundance of GM1 ganglioside/cholesterol-enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow were observed. Summary/Conclusion: This study shows that substantially reduced flippase activity is associated with increased PS surface exposure, and thereby the c.2365C>T p.(Leu789Phe) mutation in ATP11C as the likely cause of mild hemolytic anemia in this patient. The observed changes in membrane lipid distribution may underly the changes in RBC flow behavior, suggesting additional functional effects of ATP11C-deficiency.Keywords: Red blood cell, Phosphatidylserine, Hemolytic anemia

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