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Hereditary Xerocytosis: Differential Behavior of PIEZO1 Mutations in the N-Terminal Extracellular Domain Between Red Blood Cells and HEK Cells

ABSTRACT: Hereditary Xerocytosis, a rare hemolytic anemia, is due to gain of function mutations in PIEZO1, a non-selective cation channel activated by mechanical stress. How these PIEZO1 mutations impair channel function and alter red blood cell (RBC) physiology, is not completely understood. Here, we report the characterization of mutations in the N-terminal part of the protein (V598M, F681S and the double mutation G782S/R808Q), a part of the channel that was subject of many investigations to decipher its role in channel gating. Our data show that the electrophysiological features of these PIEZO1 mutants expressed in HEK293T cells are different from previously characterized PIEZO1 mutations that are located in the pore or at the C-terminal extracellular domain of the protein. Although RBC with PIEZO1 mutations showed a dehydrated phenotype, the activity of V598M, F681S or R808Q in response to stretch was not significantly different from the WT channels. In contrast, the G782S mutant showed larger currents compared to the WT PIEZO1. Interestingly, basal activity of all the mutated channels was not significantly altered at the opposite of what was expected according to the decreased water and cation contents of resting RBC. In addition, the features of mutant PIEZO1 expressed in HEK293 cells do not always correlate with the observation in RBC where PIEZO1 mutations induced a cation leak associated with an increased conductance. Our work emphasizes the role of the membrane environment in PIEZO1 activity and the need to characterize RBC permeability to assess pathogenicity to PIEZO1 mutants associated with erythrocyte diseases.

SUBMITTER: Yamaguchi Y

PROVIDER: S-EPMC8562563 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Proteome of purified erythrocytes from blood samples from patients diagnosed with a PIEZO1-HX based on familial history, non-spherocytic chronic hemolysis, typical osmotic gradient ektacytometry and molecular testing and from healthy donors were analyzed by a label free quantification approach.

2023-03-11 | PXD031963 | Pride

Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis.

Project description:Mutations in PIEZO1 are the primary cause of hereditary xerocytosis, a clinically heterogeneous, dominantly inherited disorder of erythrocyte dehydration. We used next-generation sequencing-based techniques to identify PIEZO1 mutations in individuals from 9 kindreds referred with suspected hereditary xerocytosis (HX) and/or undiagnosed congenital hemolytic anemia. Mutations were primarily found in the highly conserved, COOH-terminal pore-region domain. Several mutations were novel and demonstrated ethnic specificity. We characterized these mutations using genomic-, bioinformatic-, cell biology-, and physiology-based functional assays. For these studies, we created a novel, cell-based in vivo system for study of wild-type and variant PIEZO1 membrane protein expression, trafficking, and electrophysiology in a rigorous manner. Previous reports have indicated HX-associated PIEZO1 variants exhibit a partial gain-of-function phenotype with generation of mechanically activated currents that inactivate more slowly than wild type, indicating that increased cation permeability may lead to dehydration of PIEZO1-mutant HX erythrocytes. In addition to delayed channel inactivation, we found additional alterations in mutant PIEZO1 channel kinetics, differences in response to osmotic stress, and altered membrane protein trafficking, predicting variant alleles that worsen or ameliorate erythrocyte hydration. These results extend the genetic heterogeneity observed in HX and indicate that various pathophysiologic mechanisms contribute to the HX phenotype.

| S-EPMC5649553 | biostudies-literature

Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis.

Project description:Hereditary xerocytosis (HX, MIM 194380) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte dehydration. Copy number analyses, linkage studies, and exome sequencing were used to identify novel mutations affecting PIEZO1, encoded by the FAM38A gene, in 2 multigenerational HX kindreds. Segregation analyses confirmed transmission of the PIEZO1 mutations and cosegregation with the disease phenotype in all affected persons in both kindreds. All patients were heterozygous for FAM38A mutations, except for 3 patients predicted to be homozygous by clinical and physiologic studies who were also homozygous at the DNA level. The FAM38A mutations were both in residues highly conserved across species and within members of the Piezo family of proteins. PIEZO proteins are the recently identified pore-forming subunits of channels that mediate mechanotransduction in mammalian cells. FAM38A transcripts were identified in human erythroid cell mRNA, and discovery proteomics identified PIEZO1 peptides in human erythrocyte membranes. These findings, the first report of mutation in a mammalian mechanosensory transduction channel-associated with genetic disease, suggest that PIEZO proteins play an important role in maintaining erythrocyte volume homeostasis.

| S-EPMC3448561 | biostudies-literature

Increased Red Cell KCNN4 Activity in Sporadic Hereditary Xerocytosis Associated With Enhanced Single Channel Pressure Sensitivity of PIEZO1 Mutant V598M.

Project description:Supplemental Digital Content is available in the text.

| S-EPMC6407803 | biostudies-literature

Piezo1-xerocytosis red cell metabolome shows impaired glycolysis and increased hemoglobin oxygen affinity.

Project description: Not available

| S-EPMC7805316 | biostudies-literature

Clinical and biological features in <i>PIEZO1</i>-hereditary xerocytosis and Gardos channelopathy: a retrospective series of 126 patients.

Project description:We describe the clinical, hematologic and genetic characteristics of a retrospective series of 126 subjects from 64 families with hereditary xerocytosis. Twelve patients from six families carried a KCNN4 mutation, five had the recurrent p.Arg352His mutation and one had a new deletion at the exon 7-intron 7 junction. Forty-nine families carried a PIEZO1 mutation, which was a known recurrent mutation in only one-third of the cases and private sequence variation in others; 12 new probably pathogenic missense mutations were identified. The two dominant features leading to diagnosis were hemolysis that persisted after splenectomy and hyperferritinemia, with an inconstant correlation with liver iron content assessed by magnetic resonance imaging. PIEZO1-hereditary xerocytosis was characterized by compensated hemolysis in most cases, perinatal edema of heterogeneous severity in more than 20% of families and a major risk of post-splenectomy thrombotic events, including a high frequency of portal thrombosis. In KCNN4-related disease, the main symptoms were more severe anemia, hemolysis and iron overload, with no clear sign of red cell dehydration; therefore, this disorder would be better described as a 'Gardos channelopathy'. These data on the largest series to date indicate that PIEZO1-hereditary xerocytosis and Gardos channelopathy are not the same disease although they share hemolysis, a high rate of iron overload and inefficient splenectomy. They demonstrate the high variability in clinical expression as well as genetic bases of PIEZO1-hereditary xerocytosis. These results will help to improve the diagnosis of hereditary xerocytosis and to provide recommendations on the clinical management in terms of splenectomy, iron overload and pregnancy follow-up.

| S-EPMC6669138 | biostudies-literature

PIEZO1 Interactome in HEK cells

Project description:The propeller-shaped blades of the PIEZO1 and PIEZO2 ion channels partition into the plasma membrane and respond to indentation or stretching of the lipid bilayer, thus converting mechanical forces into signals that can be interpreted by cells, in the form of calcium flux and changes in membrane potential. While PIEZO channels participate in diverse physiological processes, from sensing the shear stress of blood flow in the vasculature to detecting a light touch on the surface of the skin, the molecular details that enable these mechanosensors to tune their responses over a vast dynamic range of forces remain largely uncharacterized. To survey the molecular landscape surrounding PIEZO channels at the cell surface, we employed a mass spectrometry-based proteomic approach to capture and identify extracellularly exposed proteins in the vicinity of PIEZO1. This PIEZO1-proximal interactome was enriched in surface proteins localized to cell junctions and signaling hubs within the plasma membrane. Functional screening of these interaction candidates by calcium imaging and electrophysiology in an overexpression system identified the adhesion molecule CADM1/SynCAM that slows the inactivation kinetics of PIEZO1 with little effect on PIEZO2. Conversely, we found that CADM1 knockdown accelerates inactivation of endogenous PIEZO1 in Neuro-2a cells. Systematic deletion of CADM1 domains indicate that the transmembrane region is critical for the observed effects on PIEZO1, suggesting that modulation of inactivation is mediated by interactions in or near the lipid bilayer.

2024-10-30 | PXD054618 | Pride

Hereditary Xerocytosis due to Mutations in <i>PIEZO1</i> Gene Associated with Heterozygous Pyruvate Kinase Deficiency and Beta-Thalassemia Trait in Two Unrelated Families.

Project description:Hereditary xerocytosis (HX) is a rare disorder caused by defects of RBC permeability, associated with haemolytic anaemia of variable degree and iron overload. It is sometimes misdiagnosed as hereditary spherocytosis or other congenital haemolytic anaemia. Splenectomy is contraindicated due to increased risk of thromboembolic complications. We report the clinical, haematological, and molecular characteristics of four patients from two unrelated Italian families affected by HX, associated with beta-thalassemia trait and heterozygous pyruvate kinase deficiency, respectively. Two patients had been splenectomised and displayed thrombotic episodes. All patients had iron overload in the absence of transfusion, two of them requiring iron chelation. The diagnosis of HX was confirmed by LoRRca Osmoscan analysis showing a left-shifted curve. PIEZO1 gene sequencing revealed the presence of mutation p.E2496ELE, showing that this is one of the most frequent mutations in this disease. The concomitant defects did not aggravate the clinical phenotype; however, in one patient, the initial diagnosis of pyruvate kinase deficiency delayed the correct diagnosis of HX for many years and resulted in splenectomy followed by thrombotic complications. The study underlines the importance of a precise diagnosis in HX, particularly in view of splenectomy, and the need of a molecular confirmation of suspected RBC enzymopathy.

| S-EPMC5358460 | biostudies-literature

Hemoglobin C trait accentuates erythrocyte dehydration in hereditary xerocytosis.

Project description:A 17-year-old male presented with acute hemolysis with stomatocytosis, elevated mean corpuscular hemoglobin concentration (MCHC), and osmotic gradient ektacytometry consistent with marked erythrocyte dehydration. Erythrocytes from both parents also demonstrated evidence of dehydration with elevated MCHC and abnormal ektacytometry, but neither to the degree of the patient. Genetic studies revealed the patient had hereditary xerocytosis (HX) due to a novel PIEZO1 mutation inherited from his mother and hemoglobin C (HbC) trait inherited from his father. HbC trait accentuated the erythrocyte dehydration of HX. Coinheritance of interrelated disorders and/or modifier alleles should be considered whenever severe erythrocyte dehydration is observed.

| S-EPMC5858911 | biostudies-literature

Xerocytosis is caused by mutations that alter the kinetics of the mechanosensitive channel PIEZO1.

Project description:Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant's loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P1/2, for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ?6-20 nm(2) during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (?200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.

| S-EPMC3606986 | biostudies-literature

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