A novel missense variant in ATP11C is associated with reduced red blood cell phosphatidylserine flippase activity and mild hereditary hemolytic anemia

Abstract

Adenosine Triphosphatase (ATPase) Phospholipid Transporting 11C gene (ATP11C) 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 to establish and maintain phospholipid asymmetry of the lipid bilayer of cell membranes. This asymmetry is crucial for survival since externalized PS triggers phagocytosis by splenic macrophages. Here we report on pathophysiological consequences of decreased flippase activity, prompted by a patient with hemolytic anemia and hemizygosity for a novel c.2365C > T p.(Leu789Phe) missense variant in ATP11C. ATP11C protein expression was strongly reduced by 58% in patient‐derived RBC ghosts. Furthermore, functional characterization showed only 26% PS flippase activity. These results were confirmed by recombinant mutant ATP11C protein expression in HEK293T cells, which was decreased to 27% compared to wild type, whereas PS‐stimulated ATPase activity was decreased by 57%. Patient RBCs showed a mild increase in PS surface exposure when compared to control RBCs, which further increased in the most dense RBCs after RBC storage stress. The increase in PS was not due to higher global membrane content of PS or other phospholipids. In contrast, membrane lipid lateral distribution showed increased abundance of cholesterol‐enriched domains in RBC low curvature areas. Finally, more dense RBCs and subtle changes in RBC morphology under flow hint toward alterations in flow behavior of ATP11C‐deficient RBCs. Altogether, ATP11C deficiency is the likely cause of hemolytic anemia in our patient, thereby underlining the physiological role and relevance of this flippase in human RBCs.

Functional and in silico analysis of ATP8A2 and other P4-ATPase variants associated with human genetic diseases

“…ATP11C and ATP11A have also been linked to inherited diseases ( Arashiki et al, 2016 ; Pater et al, 2022 ; Segawa et al, 2021 ; van Dijk et al, 2023 ). To date, 11 missense mutations in ATP11C have been implicated in congenital hemolytic anemia.…”

“…Of these mutations, five were predicted to be destabilizing by most of the programs, whereas the remaining six were predicted to be either neutral or stabilizing ( Table S3 ). The T418N and L789F ATP11C variants are the only ones that have been characterized at a molecular level ( Arashiki et al, 2016 ; Liou et al, 2019 ; van Dijk et al, 2023 ). These mutations reduced expression levels of ATP11C, consistent with the destabilizing effects of these mutations predicted by all three programs.…”

“…Genetic defects in ATP8B1 cause progressive familial intrahepatic cholestasis 1 (PFIC1) or benign recurrent intrahepatic cholestasis 1 (BRIC1) ( Bull et al, 1998 ; Folmer et al, 2009b ). Mutations in ATP11C are responsible for congenital hemolytic anemia ( Arashiki et al, 2016 ; van Dijk et al, 2023 ), and mutations in ATP11A have been linked to a hypomyelinating leukodystrophy ( Segawa et al, 2021 ) and hearing loss ( Pater et al, 2022 ). Many of these disease phenotypes have been replicated in knockout or transgenic mice ( Coleman et al, 2014 ; Segawa et al, 2021 ; Siggs et al, 2011 ; Stapelbroek et al, 2009 ; Zhu et al, 2012 ).…”

Modeling Red Blood Cell Metabolism in the Omics Era

“…While beneficial for acclimatization to hypoxia, this mechanism is actually deleterious in the context of sickle cell disease [52], as it promotes sickle hemoglobin crystallization, driving cardiorenal dysfunction [53,54]. Among other lipid classes, phosphatidylserines participate in RBC removal from the bloodstream upon loss of phospholipid asymmetry, a process that is maintained by ATP-dependent flippases like ATP11C (EC 7.6.2.1) [55,56]. Other phospholipids, like phosphatidylcholines and phosphatidylethanolamines, can contribute methyl group for oxidant stress-induced isoaspartyl protein damage repair via methylation [57] by protein L-isospartyl O-methyltransferase (EC 2.1.1.77) [58].…”