Red Blood Cell Membrane Conductance in Hereditary Haemolytic Anaemias.

Congenital haemolytic anaemias are inherited disorders caused by red blood cell membrane and cytoskeletal protein defects, deviant hemoglobin synthesis and metabolic enzyme deficiencies. In many cases, although the causing mutation might be known, the pathophysiology and the connection between the particular mutation and the symptoms of the disease are not completely understood. Thus effective treatment is lagging behind. As in many cases abnormal red blood cell cation content and cation leaks go along with the disease, by direct electrophysiological measurements of the general conductance of red blood cells, we aimed to assess if changes in the membrane conductance could be a possible cause. We recorded whole-cell currents from 29 patients with different types of congenital haemolytic anaemias: 14 with hereditary spherocytosis due to mutations in α-spectrin, ß-spectrin, ankyrin and band 3 protein; 6 patients with hereditary xerocytosis due to mutations in Piezo1; 6 patients with enzymatic disorders (3 patients with glucose-6-phosphate dehydrogenase deficiency, 1 patient with pyruvate kinase deficiency, 1 patient with glutamate-cysteine ligase deficiency and 1 patient with glutathione reductase deficiency), 1 patient with ß-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with ß-thalassemia and metabolic enzyme deficiencies showed no changes in their membrane conductance, the patients with hereditary spherocytosis and hereditary xerocytosis showed largely variable results depending on the underlying mutation.

'Gardos Channelopathy': a variant of hereditary Stomatocytosis with complex molecular regulation.

The Gardos channel is a Ca2+ sensitive, K+ selective channel present in several tissues including RBCs, where it is involved in cell volume regulation. Recently, mutations at two different aminoacid residues in KCNN4 have been reported in patients with hereditary xerocytosis. We identified by whole exome sequencing a new family with two members affected by chronic hemolytic anemia carrying mutation R352H in the KCNN4 gene. No additional mutations in genes encoding for RBCs cytoskeletal, membrane or channel proteins were detected. We performed functional studies on patients' RBCs to evaluate the effects of R352H mutation on the cellular properties and eventually on the clinical phenotype. Gardos channel hyperactivation was demonstrated in circulating erythrocytes and erythroblasts differentiated ex-vivo from peripheral CD34+ cells. Pathological alterations in the function of multiple ion transport systems were observed, suggesting the presence of compensatory effects ultimately preventing cellular dehydration in patient's RBCs; moreover, flow cytometry and confocal fluorescence live-cell imaging showed Ca2+ overload in the RBCs of both patients and hypersensitivity of Ca2+ uptake by RBCs to swelling. Altogether these findings suggest that the 'Gardos channelopathy' is a complex pathology, to some extent different from the common hereditary xerocytosis.