Molecular genetics and pathogenic mechanisms of hereditary anemias due to altered permeability of erythrocyte membrane

Genetic defects of erythrocyte transport proteins cause disorders of red blood cell volume that are characterized by abnormal permeability to the cation and, consequently, by changes in red cell hydration. Within this group of hereditary anemias we focused on familial pseudohyperkalemia and dehydrated hereditary stomatocytosis. The main aims of the project thesis are to study both the molecular genetics and the pathogenic mechanisms of these two disorders. Isolated Familial Pseudohyperkalemia (FP) is a dominant red cell trait characterized by cold-induced ‘passive leak’ of red cell K+ into plasma. The causative gene of this condition is ABCB6, encoding an erythrocyte membrane ABC transporter protein bearing the Langereis blood group antigen system. Dehydrated hereditary stomatocytosis (DHS) is an autosomal dominant congenital hemolytic anemia with moderate splenomegaly and often compensated hemolysis. Red cells are characterized by cation leak of the red cell membrane, reflected in elevated sodium content, decreased potassium content, elevated MCHC and MCV, and decreased osmotic fragility. The majority of symptomatic DHS cases reported to date have been associated with gain-of-function mutations in the mechanosensitive cation channel gene, PIEZO1. Our study started with the recruitment of 97 patients affected by both FP and DHS from 41 unrelated families of Italian and foreign countries. Regarding familial pseudohyperkalemia, analyzing three new families, we reported the first functional characterization of ABCB6 mutants, including homozygous mutation V454A, heterozygous mutation R276W, and compound heterozygous mutations R276W and R723Q. All these mutations are annotated in public databases, suggesting that FP could be common in the general population. Indeed, we identified variant R276W in one of 327 random blood donors (0.3%). Measurement of cation flux demonstrated greater loss of K+ or Rb+ from HEK-293 cells expressing ABCB6 mutants than from cells expressing ABCB6 WT. The R276W/R723Q mutations elicited greater cellular K+ efflux than did the other mutants tested. Regarding dehydrated hereditary stomatocytosis by whole exome sequencing analysis of two previously undiagnosed DHS families we identified the second causative gene of DHS, the KCNN4 gene, encoding the Gardos channel (KCa3.1), the erythroid Ca2+-sensitive K+ channel of intermediate conductance. We characterized the expression of KCNN4 in the mutated patients and during erythroid differentiation of hematopoietic progenitor cell CD34+ and K562 cells. We also analyzed KCNN4 expression during mouse embryonic development. Finally, we demonstrated that the mutations in KCNN4, as for PIEZO1, cause a gain of function, by increasing potassium efflux. Moreover, by analysing the genotype of the patients here collected, we characterized a new interesting mutation in PIEZO1, that is a duplication of two aminoacids localized in the pore of the channel, found in two families with different phenotype. We further analysed the modified effect of an additional PIEZO1 missense variant carried by the family exhibiting the more severe phenotype. We found that the missense variant co-inherited with the duplication cause an augmented potassium efflux. In conclusion, ABCB6 missense mutations in FP erythrocytes show elevated K+ efflux. The patients are present at moderate frequency in the blood donor population. Storage of blood of these patients leads to significantly increased K+ levels, with serious clinical implications for neonates and infants receiving large-volume transfusions of whole blood. Genetic tests for FP could be added to blood donor pre-screening. Further study of ABCB6 function and trafficking could be informative for the study of other pathologies of red blood cell hydration. The identification of KCNN4 mutations in DHS patients supports recent studies that indicate it plays a critical role in normal erythrocyte deformation in the microcirculation and participates in maintenance of erythrocyte volume homeostasis. The characterization of PIEZO1 and KCNN4 mutations in DHS has contributed to the understanding of DHS pathogenesis that will be useful for the prognosis, the management, the follow-up, and the treatment of these patients