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PIEZO1 gain-of-function mutations delay reticulocyte maturation in hereditary xerocytosis

Dehydrated hereditary stomatocytosis 1 or hereditary xerocytosis (HX, OMIM 194380) is a rare hereditary autosomal dominant disorder characterized by hemolytic anemia and red blood cell (RBC) dehydration. The occurrence of HX is linked with gain-of-function mutations in PIEZO1, the gene encoding for the mechanosensitive non-specific cation channel PIEZO1 which is activated by shear-stress and in concert with other ion channels (particularly the Gardos potassium calcium-activated channel, KCNN4) regulates cell volume homeostasis and metabolic activity in the RBC. Intriguingly, PIEZO1 gainof-function mutations have recently been reported to occur at a much higher frequency within the population than had been previously described and have also been implicated in malaria resistance, suggesting that the mechanisms underpinning HX may merit further investigation. Since reticulocytosis is one of the hallmarks of HX, we sought to determine whether altered reticulocyte maturation could be a causative agent of this phenotype. We characterize reticulocytes and erythrocytes from 10 HX patients in comparison to healthy controls, revealing alterations in deformability and vesicle content that implicate a maturational defect in HX. We further demonstrate that HX patients suffer from impaired reticulocyte maturation as assayed through differences in the extent and rate of loss of CD71 and RNA content over time and that this effect can be recapitulated in healthy reticulocytes upon chemically-induced PIEZO1 overactivation, providing a functional link to the reticulocytosis phenotype present in HX. A total of 10 samples were investigated in this study, constituting nine patients from six families with one sample in duplicate. The corresponding hematological parameters can be found in Table 1. All patients under investigation suffered from mutations in PIEZO1, with the majority displaying reticulocytosis and abnormal mean cell volume values (MCV). Several patients also displayed anomalous serum ferritin content, which comprised both iron overload and deficiency. Moreover, 3 of 9 patients had previously been subjected to splenectomy. Further detail on individual patients is provided in the Online Supplementary Patient Case Histories. Splenectomy leads to a partial loss of the body’s quality assurance system for ensuring the removal of physiologically-altered circulating RBC, the effects of which can be readily observed on the deformability index (DI) and cross-sectional area profiles obtained upon examination of the cells with the Automated Rheoscope and Cell Analyzer (ARCA), shown in Figure 1A. Individual scatter plots for each sample are shown in the Online Supplementary Figure S1. Both RBC (CD71) and reticulocytes (CD71) from splenectomized PIEZO1-defective patients display decreased deformability (CD71 median DI: 1.33 [Interquartile range (IQR): 1.08-1.58], CD71 median DI: 1.80 [IQR: 1.61-1.99]) in comparison with non-splenectomized patients (CD71 median DI: 1.70 [IQR: 1.461.94], CD71 median DI: 1.91 [IQR: 1.76-2.06]), with their RBC displaying an enriched proportion of microcytic cells (Cross-sectional area =~40 μm). Interestingly, while RBC from non-splenectomized patients also display lowered deformability and macrocytosis in comparison with reference values (ref: CD71median DI: 1.94 [IQR: 1.78-2.11]), their reticulocytes display a relatively normal DI profile (ref: CD71 median DI: 1.93 [IQR: 1.84-2.02]). Whilst a potential influence of CD71 reticulocytes on the total RBC population cannot be excluded, these results nonetheless illustrate that the effect of PIEZO1 overactivation on deformability only manifests in the transition from the reticulocyte to the RBC as defined by the loss of CD71, implicating a potential defect in reticulocyte maturation of HX patients. Using mitochondrial content as a surrogate measure for intracellular vesicle content, as previously described, we proceeded to investigate whether physiological vesicle loss is altered in reticulocytes from HX patients, observ-