Red cell alterations in ENT1_null individuals. (A) Western blot analysis of ENT1 expression in ENT1_null RBC membranes from the 3 siblings. β-actin was used as loading control. (B) Blood smears of ENT1_null individuals showing a macrocytosis and marked anisopoikilocytosis, characterized by the presence of RBCs with abnormal sizes and morphologies, including elliptocytes (black arrow), dacrocytes (red arrow), and schizocytes (blue arrow). Scale bars, 10 µm. (C) Quantification of the different RBC populations was calculated from 300 RBCs per slide. (D) RBC deformability was assessed by an ektacytometry approach and elongation index relative to shear stress is shown for controls, P1, P2, and P3, and 7 controls. (E) Osmoscan test of ENT1_null RBCs from the 3 patients and a healthy control was monitored by ektacytometry. (F) The phosphoproteome of ENT1_null (n = 3) and control (n = 5) RBC membranes was evaluated. Each dot represents a phosphorylated protein, with blue dots corresponding to significantly hypophosphorylated (left) and hyperphosphorylated (right) proteins. (G) Nucleotide-targeted metabolomic analysis of ENT1_null (n = 3; P1, blue; P2, green; P3, red triangles) and control (n = 3; black circles) RBCs were performed and intracellular levels of deoxynucleotides and cyclic nucleotides (cAMP, cGMP) are shown. (H) Schematic representation of the variant filter strategy used to analyze the exome sequencing of ENT1_null patients. Common mutations between P1 and P2, but absent in P3 (blue filled area) were filtered, candidate genes identified, and their chromosomes are shown in the table. (I) Sanger sequencing confirmation of the c.559 G>T; p.Gly187Trp mutation on the ABCC4 gene, showing the absence of the mutation in P3, and its presence in the heterozygous state in P1 and P2.