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

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.

Novel variants in critical domains of ATP8A2 and expansion of clinical spectrum.

ATP8A2 is a P4-ATPase that flips phosphatidylserine across membranes to generate and maintain transmembrane phospholipid asymmetry. Loss-of-function variants cause severe neurodegenerative and developmental disorders. We have identified three ATP8A2 variants in unrelated Iranian families that cause intellectual disability, dystonia, below-average head circumference, mild optic atrophy, and developmental delay. Additionally, all the affected individuals displayed tooth abnormalities associated with defects in teeth development. Two variants (p.Asp825His and p.Met438Val) reside in critical functional domains of ATP8A2. These variants express at very low levels and lack ATPase activity. Inhibitor studies indicate that these variants are misfolded and degraded by the cellular proteasome. We conclude that Asp825, which coordinates with the Mg2+ ion within the ATP binding site, and Met438 are essential for the proper folding of ATP8A2 into a functional flippase. We also provide evidence on the association of tooth abnormalities with defects in ATP8A2, thereby expanding the clinical spectrum of the associated disease.

ATP8A2-related disorders as recessive cerebellar ataxia.

ATP8A2-related disorders are autosomal recessive conditions that associate encephalopathy with or without hypotonia, psychomotor delay, abnormal movements, chorea, tremor, optic atrophy and cerebellar atrophy (CARMQ4). Through a multi-centric collaboration, we identified six point mutations (one splice site and five missense mutations) involving ATP8A2 in six individuals from five families. Two patients from one family with the homozygous p.Gly585Val mutation had a milder presentation without encephalopathy. Expression and functional studies of the missense mutations demonstrated that protein levels of four of the five missense variants were very low and lacked phosphatidylserine-activated ATPase activity. One variant p.Ile215Leu, however, expressed at normal levels and displayed phospholipid-activated ATPase activity similar to the non-mutated protein. We therefore expand for the first time the phenotype related to ATP8A2 mutations to less severe forms characterized by cerebellar ataxia without encephalopathy and suggest that ATP8A2 should be analyzed for all cases of syndromic or non-syndromic recessive or sporadic ataxia.

The Combination of IFN ß and TNF Induces an Antiviral and Immunoregulatory Program via Non-Canonical Pathways Involving STAT2 and IRF9.

Interferon (IFN) ß and Tumor Necrosis Factor (TNF) are key players in immunity against viruses. Compelling evidence has shown that the antiviral and inflammatory transcriptional response induced by IFNß is reprogrammed by crosstalk with TNF. IFNß mainly induces interferon-stimulated genes by the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway involving the canonical ISGF3 transcriptional complex, composed of STAT1, STAT2, and IRF9. The signaling pathways engaged downstream of the combination of IFNß and TNF remain elusive, but previous observations suggested the existence of a response independent of STAT1. Here, using genome-wide transcriptional analysis by RNASeq, we observed a broad antiviral and immunoregulatory response initiated in the absence of STAT1 upon IFNß and TNF costimulation. Additional stratification of this transcriptional response revealed that STAT2 and IRF9 mediate the expression of a wide spectrum of genes. While a subset of genes was regulated by the concerted action of STAT2 and IRF9, other gene sets were independently regulated by STAT2 or IRF9. Collectively, our data supports a model in which STAT2 and IRF9 act through non-canonical parallel pathways to regulate distinct pool of antiviral and immunoregulatory genes in conditions with elevated levels of both IFNß and TNF.