Mutations In PIEZO1 Research Articles

Trans-acting genetic modifiers of clinical severity in heterozygous β-Thalassemia trait.

There is a group of beta (β)-thalassemia trait 'carriers' (with heterozygous mutations) who should be asymptomatic with minor abnormalities in their hematological parameters, but experience more severe disease manifestations than predicted based solely on their β-globin genotype. This review focuses on literature describing trans-acting genetic modifiers outside of the α- and β-globin gene clusters that could cause this phenomenon. These genetic modifiers are categorized into: mutations affecting the quantity of alpha-globin products, non-globin mutations affecting erythropoiesis, membranopathies, enzymopathies and erythrocyte-independent modifiers of complications relating to β-thalassemia. Although some genetic determinants seem to correlate more directly with β-thalassemia trait severity, such as mutations in SUPT5H, PIEZO1 and hereditary elliptocytosis, the difficulties of linking the contribution of other modulating factors are elucidated in this review. Targeted next generation sequencing of hemolytic anemias can be helpful but also raises another quandary in interpreting variants of uncertain significance. The accrual of knowledge, along with the increased availability of genetic testing for genetic modifiers has considerable potential for clinical applications such as genetic counselling, decision-making for clinical interventions and prognostication, and perhaps generating new therapeutic targets.

PIEZO1 loss-of-function compound heterozygous mutations in the rare congenital human disorder Prune Belly Syndrome

Prune belly syndrome (PBS), also known as Eagle-Barret syndrome, is a rare, multi-system congenital myopathy primarily affecting males. Phenotypically, PBS cases manifest three cardinal pathological features: urinary tract dilation with poorly contractile smooth muscle, wrinkled flaccid ventral abdominal wall with skeletal muscle deficiency, and intra-abdominal undescended testes. Genetically, PBS is poorly understood. After performing whole exome sequencing in PBS patients, we identify one compound heterozygous variant in the PIEZO1 gene. PIEZO1 is a cation-selective channel activated by various mechanical forces and widely expressed throughout the lower urinary tract. Here we conduct an extensive functional analysis of the PIEZO1 PBS variants that reveal loss-of-function characteristics in the pressure-induced normalized open probability (NPo) of the channel, while no change is observed in single-channel currents. Furthermore, Yoda1, a PIEZO1 activator, can rescue the NPo defect of the PBS mutant channels. Thus, PIEZO1 mutations may be causal for PBS and the in vitro cellular pathophysiological phenotype could be rescued by the small molecule, Yoda1. Activation of PIEZO1 might provide a promising means of treating PBS and other related bladder dysfunctional states.

Newly identified roles for PIEZO1 mechanosensor in controlling normal megakaryocyte development and in primary myelofibrosis.

Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in mouse and human Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Yet, a role for PIEZO1 in megakaryopoiesis and proplatelet formation has never been investigated. Here, we show that activation of PIEZO1 increases the number of immature Mks in mice, while the number of mature Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its activation reduces Mk size, ploidy, maturation, and proplatelet extension. Resulting effects of PIEZO1 activation on Mks resemble the profile in Primary Myelofibrosis (PMF). Intriguingly, Mks derived from Jak2V617F PMF mice show significantly elevated PIEZO1 expression, compared to wild-type controls. Accordingly, Mks isolated from bone marrow aspirates of JAK2V617F PMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2V617F PMF patients are rescued upon PIEZO1 inhibition. Together, our data suggest that PIEZO1 places a brake on Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease.

Newly Identified Roles for PIEZO1 Mechanosensor in Controlling Normal Megakaryocyte Development and in Primary Myelofibrosis

Introduction: Mechanisms through which mature megakaryocytes (Mks) and their progenitors sense the bone marrow extracellular matrix (ECM) to promote lineage differentiation in health and disease are still partially understood. We found PIEZO1, a mechanosensitive cation channel, to be expressed in Mks. Human mutations in PIEZO1 have been described to be associated with blood cell disorders. Primary Myelofibrosis (PMF) is a Chromosome Philadelphia-negative Myeloproliferative Neoplasm (MPN), characterized by altered stiffer ECM and megakaryocytosis, often driven by the gain-of-function mutation in JAK2 V617F. Here, we study a role for PIEZO1 in megakaryopoiesis and proplatelet formation under normal physiological conditions and in the context of PMF. Methods: Bone marrow Mks from C57BL/6J control and myelofibrotic mice carrying the human JAK2 V617F+ mutation, or Mks derived from stem cells of patients carrying the same mutation were analyzed at mRNA and protein levels for Piezo1 expression. Effects of Piezo1 activation or inhibition on Mk maturation and ploidy were assessed by flow cytometry, and proplatelet formation by fluorescent microscopy. Human peripheral blood samples from PMF patients, diagnosed according to established criteria, or healthy individuals were used to compare the two categories for Piezo1 relative expression on mRNA and protein levels and platelet biogenesis. Mk-specific double knockout mice of Piezo1/2 were generated to compare platelet counts under normal conditions or bone marrow ablation with 5-FU challenge. Culture of mouse and human Mks in different 3D microenvironments was used to demonstrate in vitro association of Piezo1 expression levels with different ECM stiffness substrates. Results: Mouse Mks express PIEZO1 and negligible levels of PIEZO2. Pharmacological activation of mouse PIEZO1 increases the number of immature CD41 + Mks, while the number of mature CD41 +CD42 + Mks and Mk ploidy level are reduced. Piezo1/2 knockout mice show an increase in Mk size and platelet count, both at basal state and upon marrow regeneration. Similarly, in human samples, PIEZO1 is expressed during megakaryopoiesis. Its pharmacological activation reduces Mk size, ploidy, maturation, and proplatelet development. Resulting effects of PIEZO1 activation on Mks resemble the profile in PMF. Intriguingly, Mks derived from PMF mice bearing the Jak2 V617F mutation show significantly elevated PIEZO1 expression, compared to wild-type controls. Pharmacological activation of PIEZO1 in these cells significantly augments the number of immature CD41 + Mks, and sharply reduces the number of mature CD41 +CD42 + cells and ploidy level. Accordingly, Mks isolated from bone marrow aspirates of JAK2 V617FPMF patients show increased PIEZO1 expression compared to Essential Thrombocythemia. Additionally, PIEZO1 was overexpressed in 3D culture of human and mouse Mks in stiffer microenvironments as compared to softer ones and liquid medium. Most importantly, PIEZO1 expression in bone marrow Mks is inversely correlated with patient platelet count. The ploidy, maturation, and proplatelet formation of Mks from JAK2 V617FPMF patients are rescued upon PIEZO1 inhibition with GsMTx4. Discussion: Our data show that PIEZO1 might serve as a break of Mk maturation and platelet formation in physiology, and its upregulation in PMF Mks might contribute to aggravating some hallmarks of the disease. Our finding that inhibition of PIEZO1 activity partially rescues the aberrant Mk phenotype associated with PMF suggests that GsMTx4 holds potential as a therapeutic strategy to mitigate the pathological effects observed in Mk development in PMF patients.

Unraveling the Role of PIEZO1 in Stressed Erythropoiesis: Implications for Dyserythropoiesis and Potential Therapeutic Targets in Dehydrated Hereditary Stomatocytosis

PIEZO1 is a mechanosensitive cation channel that plays a crucial role in various physiological processes as a mechanical force sensor. In erythrocytes, it is responsible for regulating cellular volume and hydration. Gain-of-function (GoF) mutations in PIEZO1 lead to dehydrated hereditary stomatocytosis (DHS) by slowing down the channel's inactivation kinetics. DHS patients often exhibit a range of clinical presentations, including mild to severe hemolytic anemia, and iron overload. In addition, studies on erythroid progenitor cells from DHS patients have indicated a mutation-dependent delay in erythroid differentiation. Furthermore, studies on constitutive GoF Piezo1 mice have revealed stressed erythropoiesis characterized by increased erythrocyte turnover. The primary aim of this study is to investigate the role of PIEZO1 during stressed erythropoiesis in DHS and identify potential druggable targets. A total of 80 DHS patients were analysed, and their BMRI (reticulocyte count × patient's Hb/normal Hb), EPO, and ERFE levels were measured. The findings revealed mutation-dependent dyserythropoietic features similar to those observed in CDAII patients (characterized by dyserythropoiesis). The engineered erythroid model of DHS, Hudep2-PIEZO1-KI (Hudep2-KI), was subjected to erythroid differentiation for 12 days and compared to Hudep2-WT. A notable reduction in CD235a expression was detected on the last day of differentiation, indicating that PIEZO1-mediated alteration occurs during the late stages of differentiation. Morphological analysis of differentiating cells indicated that KI cells experienced a significant reduction in proliferation rate (1.5-fold increase vs. day 0) compared to Hudep2-WT (3.1-fold increase vs. day 0) starting from day 7. By day 12 of differentiation, KI cells displayed only half the cell count of WT cells, with a substantial decrease in the percentage of orthochromatic normoblasts (13.7% vs. WT 54.5%). This was accompanied by a relative increase in reticulocytes (Hudep2-WT: 45.5% vs. Hudep2-KI: 86.3%). To identify the altered signalling pathways involved in differentiation deregulation, RNAseq was performed at various time points during differentiation. The gene ontology analysis indicated that the most enriched biological processes were related to apoptosis, cellular response to hypoxia, and glycolysis. During the differentiation process, various apoptosis-related processes were found significantly enriched on different days. Upon conducting a single gene analysis, an imbalance between pro-apoptotic and antiapoptotic signals became evident. Specifically, in Hudep2-KI cells compared to the WT, we observed that 70.8% of proapoptotic genes were upregulated. Conversely, the regulation of apoptosis did not seem to impact antiapoptotic signals, with 57.9% of them being upregulated and 42.1% downregulated. This observation led us to suggest that PIEZO1 GoF induces apoptosis by activating pro-apoptotic signals. The findings of this study demonstrate the role of PIEZO1 GOF mutation in stressed erythropoiesis in both DHS patients and the erythroid cellular system. PIEZO1 GOF variants, through their effect on intracellular calcium concentration, influence late-stage differentiation and disrupt several critical pathways, including apoptosis, response to hypoxia, and glycolysis. Ongoing research involves confirming the data in CD34+ cells from DHS patients and PIEZO1 GOF mice. Additionally, the identified deregulated markers hold promise as potential druggable targets for dyserythropoiesis treatment.

Proteome alterations in erythrocytes with PIEZO1 gain-of-function mutations.

Gain-of-function mutations in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis, an autosomal dominant hemolytic anemia characterized by high reticulocyte count, a tendency to macrocytosis, and mild jaundice, as well as by other variably penetrant clinical features, such as perinatal edema, severe thromboembolic complications after splenectomy, and hepatic iron overload. PIEZO1 mutations in DHS lead to slowed inactivation kinetics of the ion channel and/or facilitation of channel opening in response to physiological stimuli. To characterize the alterations of red blood cell proteome in patients with mutated PIEZO1, we used a differential approach to compare the proteome of patients with DHS (16 patients from 13 unrelated ancestries) vs healthy individuals. We identified new components in the regulation of the complex landscape of erythrocytes ion and volume balance mediated by PIEZO1. Specifically, the main impaired processes in patients with DHS were ion homeostasis, transmembrane transport, regulation of vesicle-mediated transport, and the proteasomal catabolic process. Functional assays demonstrated coexpression of PIEZO1 and band 3 when PIEZO1 was activated. Moreover, the alteration of the vesicle-mediated transport was functionally demonstrated by an increased vesiculation rate in patients with DHS compared with healthy controls. This finding also provides an explanation of the pathogenetic mechanism underlying the increased thrombotic rate observed in these patients. Finally, the newly identified proteins, involved in the intracellular signaling pathways altered by PIEZO1 mutations, could be used in the future as potential druggable targets in DHS.

JAK2 unmutated erythrocytosis: 2023 Update on diagnosis and management.

JAK2 unmutated or non-polycythemia vera (PV) erythrocytosis encompasses a heterogenous spectrum of hereditary and acquired entities. Foremost in the evaluation of erythrocytosis is the exclusion of PV through JAK2 (inclusive of exons 12-15) mutation screening. Initial assessment should also include gathering of previous records on hematocrit (Hct) and hemoglobin (Hgb) levels, in order to streamline the diagnostic process by first distinguishing longstanding from acquired erythrocytosis; subsequent subcategorization is facilitated by serum erythropoietin (Epo) measurement, germline mutation screening, and review of historical data, including comorbid conditions and medication list. Hereditary erythrocytosis constitutes the main culprit in the context of longstanding erythrocytosis, especially when associated with a positive family history. In this regard, a subnormal serum Epo level suggests EPO receptor mutation. Otherwise, considerations include those associated with decreased (high oxygen affinity Hgb variants, 2,3-bisphosphoglycerate deficiency, PIEZO1 mutations, methemoglobinemia) or normal oxygen tension at 50% Hgb saturation (P50). The latter include germline oxygen sensing pathway (HIF2A-PHD2-VHL) and other rare mutations. Acquired erythrocytosis commonly results from central (e.g., cardiopulmonary disease, high-altitude habitat) or peripheral (e.g., renal artery stenosis) hypoxia. Other noteworthy conditions associated with acquired erythrocytosis include Epo-producing tumors (e.g., renal cell carcinoma, cerebral hemangioblastoma) and drugs (e.g., testosterone, erythropoiesis stimulating agents, sodium-glucose cotransporter-2 inhibitors). Idiopathic erythrocytosis is an ill-defined terminology that presumes the existence of an increased Hgb/Hct level without an identifiable etiology. Such classification often lacks accounting for normal outliers and is marred by truncated diagnostic evaluation. Current consensus treatment guidelines are not supported by hard evidence and their value is further undermined by limited phenotypic characterization and unfounded concerns for thrombosis. We are of the opinion that cytoreductive therapy and indiscriminate use of phlebotomy should be avoided in the treatment of non-clonal erythrocytosis. However, it is reasonable to consider therapeutic phlebotomy if one were to demonstrate value in symptom control, with frequency determined by symptoms rather than Hct level. In addition, cardiovascular risk optimization and low dose aspirin is often advised. Advances in molecular hematology might result in better characterization of "idiopathic erythrocytosis" and expansion of the repertoire for germline mutations in hereditary erythrocytosis. Prospective controlled studies are needed to clarify potential pathology from JAK2 unmutated erythrocytosis, as well as to document the therapeutic value of phlebotomy.

Red blood cell proteomics reveal remnant protein biosynthesis and folding pathways in PIEZO1-related hereditary xerocytosis.

Hereditary xerocytosis is a dominant red cell membrane disorder characterized by an increased leak of potassium from the inside to outside the red blood cell membrane, associated with loss of water leading to red cell dehydration and chronic hemolysis. 90% of cases are related to heterozygous gain of function mutations in PIEZO1, encoding a mechanotransductor that translates a mechanical stimulus into a biological signaling. Data are still required to understand better PIEZO1-HX pathophysiology. Recent studies identified proteomics as an accurate and high-input tool to study erythroid progenitors and circulating red cell physiology. Here, we isolated red blood cells from 5 controls and 5 HX patients carrying an identified and pathogenic PIEZO1 mutation and performed a comparative deep proteomic analysis. A total of 603 proteins were identified among which 56 were differentially expressed (40 over expressed and 16 under expressed) between controls and HX with a homogenous expression profile within each group. We observed relevant modifications in the protein expression profile related to PIEZO1 mutations, identifying two main "knots". The first contained both proteins of the chaperonin containing TCP1 complex involved in the assembly of unfolded proteins, and proteins involved in translation. The second contained proteins involved in ubiquitination. Deregulation of proteins involved in protein biosynthesis was also observed in in vitro-produced reticulocytes after Yoda1 exposure. Thus, our work identifies significant changes in the protein content of PIEZO1-HX erythrocytes, revealing a "PIEZO1 signature" and identifying potentially targetable pathways in this disease characterized by a heterogeneous clinical expression and contra-indication of splenectomy.

Global PIEZO1 Gain-of-Function Mutation Causes Cardiac Hypertrophy and Fibrosis in Mice.

PIEZO1 is a subunit of mechanically-activated, nonselective cation channels. Gain-of-function PIEZO1 mutations are associated with dehydrated hereditary stomatocytosis (DHS), a type of anaemia, due to abnormal red blood cell function. Here, we hypothesised additional effects on the heart. Consistent with this hypothesis, mice engineered to contain the M2241R mutation in PIEZO1 to mimic a DHS mutation had increased cardiac mass and interventricular septum thickness at 8–12 weeks of age, without altered cardiac contractility. Myocyte size was greater and there was increased expression of genes associated with cardiac hypertrophy (Anp, Acta1 and β-MHC). There was also cardiac fibrosis, increased expression of Col3a1 (a gene associated with fibrosis) and increased responses of isolated cardiac fibroblasts to PIEZO1 agonism. The data suggest detrimental effects of excess PIEZO1 activity on the heart, mediated in part by amplified PIEZO1 function in cardiac fibroblasts.

PIEZO1 E756del gain of function mutation makes human erythrocytes less habitable for malaria parasites

PIEZO1 is a mechanoreceptor cation channel that regulates erythrocyte volume and, as a consequence, modulates cell density. In humans, some PIEZO1 gain of function (GoF) variants are associated with a disease called hereditary xerocytosis, characterized by increased erythrocyte density, shape distortion and compensated hemolytic anemia. E765del is a prevalent GoF PIEZO1 mutation and carriers of this mutation have variable protection from severe malaria. Mice with the same mutation have a lower rate of parasite replication and delayed death in a rodent malaria model.

Loss-of-Function Piezo1 Mutations Display Altered Stability Driven by Ubiquitination and Proteasomal Degradation.

Missense mutations in the gene that encodes for the mechanically-gated ion channel Piezo1 have been linked to a number of diseases. Gain-of-function variants are linked to a hereditary anaemia and loss-of-function variants have been linked to generalized lymphatic dysplasia and bicuspid aortic valve. Two previously characterized mutations, S217L and G2029R, both exhibit reduced plasma membrane trafficking. Here we show that both mutations also display reduced stability and higher turnover rates than wild-type Piezo1 channels. This occurs through increased ubiquitination and subsequent proteasomal degradation. Congruent with this, proteasome inhibition using N-acetyl-l-leucyl-l-leucyl-l-norleucinal (ALLN) reduced the degradation of both mutant proteins. While ALLN treatment could not rescue the function of S217L we show via multiple complementary methodologies that proteasome inhibition via ALLN treatment can not only prevent G2029R turnover but increase the membrane localized pool of this variant and the functional Piezo1 mechanosensitive currents. This data in combination with a precision medicine approach provides a new potential therapeutic avenue for the treatment of Piezo1 mediated channelopathies.

Efficient Screening Tests and Gene Mutation Spectrum for Hereditary Stomatocytosis in Japan

Background: Hereditary stomatocytosis (HSt) is a group of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. The most common subtype is dehydrated HSt (DHSt), and heterozygous mutations of the mechanosensitive calcium channel gene PIEZO1 have been associated with it most frequently. DHSt is suspected by screening tests such as erythrocyte morphology, cation concentration measurements inside and outside the erythrocyte membrane, or osmotic gradient ektacytometry; target-captured sequencing (TCS) is used for definitive diagnosis. We have shown that an increase in the residual red cells (%RRC) in a quantitative osmotic fragility test using a flow cytometer (FCM-OF) is useful as a screening test for DHSt.Purpose: We report the clinical findings and mutation spectrum of Japanese patients with DHSt confirmed by genetic testing.Methods: From April 2015 to June 2021, 27 patients who had a clinical diagnosis of DHSt and provided written consent were genetically tested. The clinical indications were hemolytic anemia with stomatocytes, accompanied by hemochromatosis, a family history, perinatal edema, and severe jaundice. Laboratory tests showed increased MCV, and subjects with an increased %RRC in FCM-OF were analyzed. TCS was performed using a hemolytic anemia-related gene panel.Results: Of the 27 patients, 14 had PIEZO1 variants, 3 had KCNN4 variants, and 2 had ABCB6 variants, for a total of 19 cases diagnosed as HSt. There was 1 SPTB mutation, 1 GCLC mutation, and 6 cases without mutations in genes known to be related to hemolytic anemia. There were 12 previously reported mutations (KCNN4: R352H, PIEZO1: V598M, T2014I, R2488Q, E2496ELE) and 5 novel mutations (KCNN4: P204R, A279T, PIEZO1: 427_428ins9AA, A1457V, K2323T).Notably, 5 E2496ELE mutations were found in unrelated individuals. There were no differences in age at diagnosis and severity of anemia between the E2496ELE mutation and other PIEZO1 mutations, but jaundice was more severe in patients with the E2496ELE mutation (p=0.007).The median age at diagnosis of the DHSt patients was 28 years (range: 2 months to 89 years); there were 6 men and 11 women. Three patients underwent splenectomy, and 2 patients with PIEZO1 mutations had postoperative thrombosis; 1 KCNN4 mutation had no complications, but no improvement in hemolytic anemia. Six patients had gallstones, 3 had fetal ascites, and 11 received red blood cell transfusions. Laboratory test results showed median Hb 10.4 g/dL (6.9-15.6), median MCV 99.7 fL (85-127.4), median MCHC 35.6% (33-39), and median T-Bil 3.4 mg/dL (0.5-37.9). The median ferritin level was 569.3 ng/mL (87.1-3895); of the 14 patients whose ferritin was measured at the time of diagnosis, 6 had already exceeded 1,000 ng/mL. The FCM-OF showed high values in all 16 cases tested.Discussion: Genetic testing was performed on cases in which DHSt was suspected based on clinical findings, smears, and FCM-OF; the diagnosis of DHSt was confirmed at a high percentage. As previously reported, the severity of hemolytic anemia was wide-ranging, and many cases of hemochromatosis were observed. The PIEZO1 mutation is the most common in the Japanese population, and the number of E2496ELE mutations is particularly conspicuous. Patients carrying E2496ELE mutations are reported to have a younger age at diagnosis and more severe hematological findings than other mutations; however, our results showed no significant differences in age at diagnosis or degree of anemia. Since the correlation between PIEZO1 gene mutation and phenotype has not yet been clarified, further research is considered necessary. DisclosuresNo relevant conflicts of interest to declare.

PIEZO1 Mutation May Determine Early Onset of Clinical Manifestation of Anemia of Myelodysplastic Syndromes

BACKGROUNDAnemia is the most frequent cytopenia in myelodysplastic syndromes (MDS), neoplastic diseases characterized by ineffective hematopoiesis and bone marrow dysplasia. MDS usually affect elderly individuals (median age in Italy 74 years), and quite rarely concern younger patients. It is of increasing interest to verify whether “young”, not therapy-related MDS patients carry germline (GM) predisposition, in order to optimize therapeutic and transplant choices. The best approach is to perform Whole Exome Sequencing (WES) instead of targeted next-generation sequencing (t-NGS) panel in cases in which MDS patients have no syndromic signs or any indicative sign of predisposing alterations. AIMWe considered a cohort of 21 consecutive MDS cases with age at diagnosis < 60 years and anemia, referred to our MDS Unit for a second opinion. METHODSAfter bone marrow (BM) re-evaluation and morphological confirmation of dysplasia compatible with MDS, we screened the 21 MDS patients by WES on BM-DNA or peripheral blood DNA (PB-DNA). For DNA sequencing, DNA libraries were sequenced using the NextSeq 550 Illumina System. Variant calling was carried out using GATK caller. T-NGS myeloid panel was in some cases applied to confirm somatic nature of mutations. Samples of 5/21 MDS patients and their relatives were further analyzed after WES by applying high-coverage t-NGS 86-gene custom panel for hereditary anemias on PB-DNA. WES of DNA extracted from saliva (S-DNA) samples was performed to provide a GM confirmation. In order to confirm pathogenic role in red blood cell (RBC) membrane defects of identified variants, we also performed the ektacytometry analysis that evaluates the erythrocyte deformability and the hydration status by subjecting them to an increasing osmotic gradient with constant shear stress. RESULTSFive of 21 MDS cases (23.8%) with symptomatic anemia (3 MDS-MLD, 1 MDS-SLD, 1 MDS with isolated del5q) were identified as carriers of variants suspected responsible of RBC membrane defects in both BM/PB-DNA and S-DNA samples, where no other germline myeloid predisposing alterations were demonstrated. MDS-associated somatic variants were present in 4/5 patients (KRAS, TET2, CBL, SRSF2, U2AF1, IDH2, with VAF >25 %). High coverage t-NGS analysis confirmed the presence of a heterozygous rare missense variant in PIEZO1 in probands 1 and 5; a heterozygous rare missense variant in PIEZO1 and ANK1 in proband 2; a heterozygous rare missense variant in PIEZO1 and SPTA1 in proband 3; a complex mode of inheritance in proband 4 with causative variants in G6PD, KCCN4, SPTB, and ABCB6 genes. PIEZO1 or KCNN4 gene variants result in erythrocyte dehydration and determine dehydrated hereditary stomatocytosis (DHS). Ektacytometry analysis showed a significant difference in the osmolarity curve of these patients, confirming the pathogenic role of the variants identified. Among these MDS suspected cases, only proband 4 was diagnostically re-categorized because of lack of acquired recurrent somatic mutations and presence of multiple alterations in erythroid genes causing dysplasia of erythroid precursors, masquerading as MDS. None of the patients presented a history of unexplained anemia before the onset of MDS, consistent with what shown for DHS1 patients with well-compensated hemolysis. Inheritance pattern of the identified variants according the study of the families confirmed the segregation of the pathogenic variants. CONCLUSIONOnset of de novo MDS in unusually young age should always prompt investigation of predisposing conditions. We suggest here that co-existence of inherited RBC membrane defects with MDS may determine earlier clinical manifestation of the clonal disease. Hereditary anemias, in particular xerocytosis, may mimic MDS morphology. In our study, among the 5 “young” MDS with concomitant inherited RBC membrane defect, one case did not finally confirm MDS diagnosis by karyotype or NGS myeloid mutation analysis. This case fulfilled the diagnostic criteria for combined hereditary hemolytic anemia: DHS2, G6PD deficiency, spherocytosis, familial pseudohyperkalemia 2, with multi-locus mode of inheritance that justifies the clinical condition (splenomegaly, hemolytic crisis, ascites). Addition of genetic testing for hereditary anemias beside assessment of germline predisposing variants may allow a more precise diagnosis in controversial cases of “young” MDS. DisclosuresSanna: Janssen: Consultancy; Abbvie: Consultancy; Astra Zeneca: Consultancy. Iolascon: Celgene: Other: Advisory Board; Bluebird Bio: Other: Advisory Board. Santini: Menarini: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Geron: Membership on an entity's Board of Directors or advisory committees; BMS/Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astex: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

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

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.

Digenic Inheritance of a FOXC2 Mutation and Two PIEZO1 Mutations Underlies Congenital Lymphedema in a Multigeneration Family

BackgroundThe lymphatic system is essential for maintaining the balance of interstitial fluid in tissues and for returning protein-rich fluids (lymph) to the bloodstream. Congenital lymphatic defects lead to accumulation of lymph in peripheral tissues and body cavities, termed primary lymphedema. To date, only a limited number of individual genes have been identified in association with primary lymphedema. However, variability of age of onset and severity of lymphatic abnormalities within some families suggests that multiple mutations or genes may be responsible, thus hampering efforts to identify individual associated genes. MethodsWhole exome sequencing (WES) was performed in 4 members of a large multigeneration family with highly variable lymphedema and followed by Sanger sequencing for identified mutations in 34 additional family members. Genotypes were correlated with clinical and lymphangioscintigraphic phenotypes. ResultsWES uncovered 2 different mechanotransducer PIEZO1 mutations and one FOXC2 transcription factor mutation in various combinations. Sanger sequencing confirmed the presence/absence of the 3 variants in affected and unaffected family members and co-segregation of one or more variants with disease. Genetic profiles did not clearly correlate with the highly variable severity of lymphatic abnormalities. ConclusionsWES in lymphedema families can uncover unexpected combinations of several lymphedema-associated mutations. These findings provide essential information for genetic counseling and reveal complex gene interactions in lymphatic developmental pathways. These can offer insights into the complex spectrum of clinical and lymphatic lymphedema phenotypes and potential targets for treatment.

Rapid Gardos Hereditary Xerocytosis Diagnosis in 8 Families Using Reticulocyte Indices.

Gardos channelopathy (Gardos-HX) or type 2 stomatocytosis/xerocytosis is a hereditary hemolytic anemia due to mutations in the KCNN4 gene. It is rarer than inherited type 1 xerocytosis due to PIEZO1 mutations (Piezo1-HX) and its diagnosis is difficult given the absence of a specific clinical or biological phenotype. We report here that this diagnosis can be sped up using red blood cell (RBC) indices performed on an ADVIA 2120 (Siemens®) analyzer, which measures reticulocyte mean corpuscular volume (rMCV) and mean corpuscular hemoglobin concentration (rMCHC). We studied reticulocyte indices in 3 new and 12 described patients (8 families) with Gardos-HX, 11 subjects presented the recurrent p.Arg352His mutation, 4 cases (two families) carried a private KCNN4 mutation. They were compared to 79 described patients (49 families) with Piezo1-HX. Surprisingly, in Gardos-HX cases, rMCV revealed to be smaller than MCV and rMCHC higher than MCHC, in contrast with normal or Piezo1-HX RBC. Consequently, ΔMCV (rMCV-MCV) was −0.9 ± 5 fL vs. 19.8 ± 3 fL (p < 0.001) in Gardos compared with Piezo1-HX and ΔMCHC (rMCHC-MCHC) was 18.7 ± 13 vs. −50 ± 8.7 g/L (p < 0.001). A threshold of 8.6 fL for ΔMCV and −5.5 g/L for ΔMCHC could discriminate between Gardos and Piezo1-HX with 100% sensitivity and specificity, regardless of age, mutation or splenectomy status. Consequently, we showed that reticulocytes indices are useful to suggest Gardos-HX on blood count results, allowing to rapidly target these patients for gene analysis. In addition, these parameters may prove useful as a ‘functional tool’ in interpreting new KCNN4 variants.

A novel PIEZO1 mutation in a patient with dehydrated hereditary stomatocytosis: a case report and a brief review of literature

BackgroundDehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis is a rare, autosomal dominant hemolytic anemia characterized by macrocytosis, presence of stomatocytes and dehydration of red blood cells (RBCs). The dehydration is caused by a defect in cellular cation content. The most frequent expression of the pathology is hemolytic well-compensated anemia with high reticulocyte count, a tendency to macrocytosis, increased mean corpuscular hemoglobin concentration (MCHC) and mild jaundice. We here describe a new mutation of PIEZO1 gene, the most frequent mutated gene in DHS, in a family affected by hereditary hemolytic anemia.Case presentationWe describe the case of a 12-years-old girl with well-compensated chronic hemolysis, increased MCHC and a father who had the same hematological characteristics. After excluding secondary causes of chronic hemolysis and enzymatic defects of the RBCs, microscopic observation of the peripheral blood smear, tests of RBC lysis, ektacytometry, SDS-PAGE and in last instance genetic analysis has been performed. This complex diagnostic workup identified a new variant in the PIEZO1 gene, never described in literature, causative of DHS. This pathogenetic variant was also detected in the father.ConclusionsThis case report highlights the importance of a correct and exhaustive diagnostic-workup in patients with clinical suspicious for hemolytic anemia in order to make a differential diagnosis. This is relevant for the management of these patients because splenectomy is contraindicated in DHS due to high thrombotic risk.

Gain-of-function mutations in PIEZO1 directly impair hepatic iron metabolism via the inhibition of the BMP/SMADs pathway.

Dehydrated hereditary stomatocytosis (DHS), or xerocytosis, is an autosomal dominant hemolytic anemia. Most patients with DHS carry mutations in the PIEZO1 gene encoding a mechanosensitive cation channel. We here demonstrate that patients with DHS have low levels of hepcidin and only a slight increase of ERFE, the erythroid negative regulator of hepcidin. We demonstrated that at the physiological level, PIEZO1 activation induced Ca2+ influx and suppression of HAMP expression in primary hepatocytes. In two hepatic cellular models expressing PIEZO1 WT and two PIEZO1 gain-of-function mutants (R2456H and R2488Q), we highlight altered expression of a few genes/proteins involved in iron metabolism. Mutant cells showed increased intracellular Ca2+ compared to WT, which was correlated to increased phosphorylation of ERK1/2, inhibition of the BMP-SMADs pathway, and suppression of HAMP transcription. Moreover, the HuH7 cells, treated with PD0325901, a potent inhibitor of ERK1/2 phosphorylation, reduced the phosphorylation of ERK1/2 with the consequent increased phosphorylation of SMAD1/5/8, confirming the link between the two pathways. Another "proof of concept" for the mechanism that links PIEZO1 to HAMP regulation was obtained by mimicking PIEZO1 activation by cell Ca2+ overload, by the Ca2+ ionophore A23187. There was strong down-regulation of HAMP gene expression after this Ca2+ overload. Finally, the inhibition of PIEZO1 by GsMTx4 leads to phenotype rescue. This is the first demonstration of a direct link between PIEZO1 and iron metabolism, which defines the channel as a new hepatic iron metabolism regulator and as a possible therapeutic target of iron overload in DHS and other iron-loading anemias.

PIEZO1 activation delays erythroid differentiation of normal and hereditary xerocytosis-derived human progenitor cells.

Hereditary xerocytosis is a dominantly inherited red cell membrane disorder caused in most cases by gain-of-function mutations in PIEZO1, encoding a mechanosensitive ion channel that translates a mechanic stimulus into calcium influx. We found that PIEZO1 was expressed early in erythroid progenitor cells, and investigated whether it could be involved in erythropoiesis, besides having a role in the homeostasis of mature red cell hydration. In UT7 cells, chemical PIEZO1 activation using YODA1 repressed glycophorin A expression by 75%. This effect was PIEZO1-dependent since it was reverted using specific short hairpin-RNA knockdown. The effect of PIEZO1 activation was confirmed in human primary progenitor cells, maintaining cells at an immature stage for longer and modifying the transcriptional balance in favor of genes associated with early erythropoiesis, as shown by a high GATA2/GATA1 ratio and decreased α/β-globin expression. The cell proliferation rate was also reduced, with accumulation of cells in G0/G1 of the cell cycle. The PIEZO1-mediated effect on UT7 cells required calcium-dependent activation of the NFAT and ERK1/2 pathways. In primary erythroid cells, PIEZO1 activation synergized with erythropoietin to activate STAT5 and ERK, indicating that it may modulate signaling pathways downstream of erythropoietin receptor activation. Finally, we studied the in-vitro erythroid differentiation of primary cells obtained from 14 PIEZO1-mutated patients, from 11 families, carrying ten different mutations. We observed a delay in erythroid differentiation in all cases, ranging from mild (n=3) to marked (n=8). Overall, these data demonstrate a role for PIEZO1 during erythropoiesis, since activation of PIEZO1 - both chemically and through activating mutations - delays erythroid maturation, providing new insights into the pathophysiology of hereditary xerocytosis.

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, β-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 β-thalassemia and 2 patients, carriers of several mutations and a complex genotype. While the patients with β-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.