Not so Rare, Not so Mild Disease: Defects of Flippase Activity Due to ATP11C Mutations. Description of Three New Cases

Introduction. The red blood cell (RBC) lipid bilayer is composed of equal proportions of cholesterol and phospholipids, these latter asymmetrically disposed through the activity of transport proteins: the flippases family (from the outer to the inner monolayer), the floppases (from inner to outside) and the scramblases (bi-directional movement). Lipidic asymmetry is crucial for RBCsurvival since externalized phosphatidylserine (PS) triggers phagocytosis by splenic macrophages.

ATPase-phospholipid transporting11C gene (ATP11C), localized on chromosome X, encodes the major PS flippase in human RBCs. Only 3 cases have been so far reported with defective ATP11C, displaying mild hemolytic anemia and reduced flippase activity.

Aim Here we report three new Italian cases presenting novel variants in the ATP11C gene and impaired flippase activity.

Methods. Routine hematologic tests were performed to investigate patient's hemolysis: complete blood count and reticulocytes, hemolysis markers, osmotic fragility and EMA-binding test, ektacytometry and RBC membrane protein content by SDS-PAGE analysis. In the absence of a definite diagnosis, a next-generation sequencing (NGS)-targeted panel containing 62 genes associated with congenital hemolytic anemias was applied (SureSelect XT HS Reagent Kit, on Illumina MiSeq platform). Flippase activity was measured by PS internalization on RBCs using flow cytometry. X-chromosomal inactivation was assessed using methylation-based analysis of the human AR and ZMYM3 genes.

Results. A 16yrs old Italian boy (Case1) from non-consanguineous parents was studied. He was treated by phototherapy for neonatal jaundice, followed by normal growth and psychomotor development. Due to scleral jaundice, laboratory and clinical investigations revealed compensated hemolysis (Hb 14.8 g/dL, reticulocytes 4%, unconjugated bilirubin 1.81 mg/dL) and splenomegaly. RBC morphology showed mild anisopoikilocytosis with stomatocytes (8%). Screening tests for congenital hemolytic anemias were all negative. Two novel hemizygous variants of unknown significance (VOUS) (p.Tyr295Asp, and p.Asn273Lys) in the ATP11C gene, both transmitted by the healthy mother were identified. The same mutations were present in the clinically healthy 8 yrs old brother, who was consequently studied and also found to have compensated hemolysis (Hb 13.2g/dL, reticulocytes 202x10⁹/L).

A 7yrs old boy (Case 2), born at term from non-consanguineous Italian parents was studied due to mild jaundice. Laboratory parameters showed: Hb 11.9 g/dL, reticulocytes 380 × 10⁹/L, increased unconjugated bilirubin, LDH, and reduced haptoglobin. Peripheral blood smear showed mild anisopoikilocytosis with rare ovalocytes and spherocytes. At the age of 8, during Parvovirus B19 infection, the child experienced pancytopenia (Hb 5.3g/dL, reticulocytes 0.4× 10⁹/L, WBC 2.0×10⁹/L, platelets 84×10⁹/L), requiring RBC transfusion.

Osmotic fragility tests were normal, as well as RBC membrane protein content and enzyme activities. Slightly abnormality of EMA-binding test (16%, n.v.< 11%) and Osmoscan curve were noted. A novel ATP11C13nt insertion (p.Leu448Tyrfs*9) transmitted by the mother was detected. In addition, a de novo ANK1 VOUS variant (p.Thr75Ile) was found, possibly explaining, the slight decrease of EMA-binding and borderline Osmoscan results.

Patients' RBCs displayed a clear decrease of PS flipping activity measured as % of NBD-PS internalized after 20 min incubation: 17.6% and 5.8% (Case 1 and his brother), and 18.6% in Case 2. Both mothers showed 35%, whereas the two healthy fathers were comparable to normal subjects (36.1%, and 48% respectively). Maternal RBCs displayed at flow cytometry two populations, with normal and impaired flippase activity, although in different proportions. To test the hypothesis that X-chromosomal inactivation could influence the expression of flippase activity in females, methylation-based assays was performed showing a mean skewed X-chromosomal inactivation pattern of the wild-type allele, with 69.5% and 70% inactivated respectively.

Conclusions. Defects of flippase activity seem not so rare as so far reported and should be suspected in males with compensated hemolysis of unknown origin. The molecular defect is heterogeneous, cytofluorimetric assay of PS internalization is a rapid an easy method to assess pathogenicity of novel ATP11C variants.

Ferrari:Novartis: Consultancy. Barcellini:Novartis: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Alexion, AstraZeneca Rare Disease: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sobi: Consultancy. Corti:Novartis: Consultancy. Bianchi:Agios Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.