Autosomal dominant dehydrated hereditary stomatocytosis (DHSt) usually presents as a compensated hemolytic anemia with macrocytosis and abnormally shaped red blood cells (RBCs). DHSt is part of a pleiotropic syndrome that may also exhibit pseudohyperkalemia and perinatal edema. We identified PIEZO1 as the disease gene for pleiotropic DHSt in a large kindred by exome sequencing analysis within the previously mapped 16q23-q24 interval. In 26 affected individuals among 7 multigenerational DHSt families with the pleiotropic syndrome, 11 heterozygous PIEZO1 missense mutations cosegregated with disease. PIEZO1 is expressed in the plasma membranes of RBCs and its messenger RNA, and protein levels increase during in vitro erythroid differentiation of CD34⁺ cells. PIEZO1 is also expressed in liver and bone marrow during human and mouse development. We suggest for the first time a correlation between a PIEZO1 mutation and perinatal edema. DHSt patient red cells with the R2456H mutation exhibit increased ion-channel activity. Functional studies of PIEZO1 mutant R2488Q expressed in Xenopus oocytes demonstrated changes in ion-channel activity consistent with the altered cation content of DHSt patient red cells. Our findings provide direct evidence that R2456H and R2488Q mutations in PIEZO1 alter mechanosensitive channel regulation, leading to increased cation transport in erythroid cells. Key Points Dehydrated hereditary stomatocytosis is characterized by abnormal RBC morphology but may involve pseudohyperkalemia and perinatal edema. This syndrome is associated with germline mutations in PIEZO1, encoding a transmembrane protein that induces mechanosensitive currents.

Methemoglobinemia is a rare disorder associated with oxidization of divalent ferro‐iron of hemoglobin (Hb) to ferri‐iron of methemoglobin (MetHb). Methemoglobinemia can result from either inherited or acquired processes. Acquired forms are the most common, mainly due to the exposure to substances that cause oxidation of the Hb both directly or indirectly. Inherited forms are due either to autosomal recessive variants in the CYB5R3 gene or to autosomal dominant variants in the globin genes, collectively known as HbM disease. Our recommendations are based on a systematic literature search. A series of questions regarding the key signs and symptoms, the methods for diagnosis, the clinical management in neonatal/childhood/adulthood period, and the therapeutic approach of methemoglobinemia were formulated and the relative recommendations were produced. An agreement was obtained using a Delphi‐like approach and the experts panel reached a final consensus >75% of agreement for all the questions.

Through negative regulation of gene expression, microRNAs (miRNAs) can function in cancers as oncosuppressors, and they can show altered expression in various tumor types. Here we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many cell-fate-determining stages. MBs occur bimodally, with the peak incidence seen between 3–4 years and 8–9 years of age, although it can also occur in adults. Notch regulates a subset of the MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulated these phenomena, and can be used in anti-cancer therapies. In a screening of MB cell lines, the miRNA miR-199b-5p was seen to be a regulator of the Notch pathway through its targeting of the transcription factor HES1. Down-regulation of HES1 expression by miR-199b-5p negatively regulates the proliferation rate and anchorage-independent growth of MB cells. MiR-199b-5p over-expression blocks expression of several cancer stem-cell genes, impairs the engrafting potential of MB cells in the cerebellum of athymic/nude mice, and of particular interest, decreases the MB stem-cell-like (CD133+) subpopulation of cells. In our analysis of 61 patients with MB, the expression of miR-199b-5p in the non-metastatic cases was significantly higher than in the metastatic cases (P = 0.001). Correlation with survival for these patients with high levels of miR-199b expression showed a positive trend to better overall survival than for the low-expressing patients. These data showing the down-regulation of miR-199b-5p in metastatic MBs suggest a potential silencing mechanism through epigenetic or genetic alterations. Upon induction of de-methylation using 5-aza-deoxycytidine, lower miR-199b-5p expression was seen in a panel of MB cell lines, supported an epigenetic mechanism of regulation. Furthermore, two cell lines (Med8a and UW228) showed significant up-regulation of miR-199b-5p upon treatment. Infection with MB cells in an induced xenograft model in the mouse cerebellum and the use of an adenovirus carrying miR-199b-5p indicate a clinical benefit through this negative influence of miR-199b-5p on tumor growth and on the subset of MB stem-cell-like cells, providing further proof of concept. Despite advances in our understanding of the pathogenesis of MB, one-third of these patients remain incurable and current treatments can significantly damage long-term survivors. Here we show that miR-199b-5p expression correlates with metastasis spread, identifying a new molecular marker for a poor-risk class in patients with MB. We further show that in a xenograft model, MB tumor burden can be reduced, indicating the use of miR199b-5p as an adjuvant therapy after surgery, in combination with radiation and chemotherapy, for the improvement of anti-cancer MB therapies and patient quality of life. To date, this is the first report that expression of a miRNA can deplete the tumor stem cells, indicating an interesting therapeutic approach for the targeting of these cells in brain tumors.

After the first proposed model of the red blood cell membrane skeleton 36 years ago, several additional proteins have been discovered during the intervening years, and their relationship with the pathogenesis of the related disorders have been somewhat defined. The knowledge of erythrocyte membrane structure is important because it represents the model for spectrin-based membrane skeletons in all cells and because defects in its structure underlie multiple hemolytic anemias. This review summarizes the main features of erythrocyte membrane disorders, dividing them into structural and altered permeability defects, focusing particularly on the most recent advances. New proteins involved in alterations of the red blood cell membrane permeability were recently described. The mechanoreceptor PIEZO1 is the largest ion channel identified to date, the fundamental regulator of erythrocyte volume homeostasis. Missense, gain-of-function mutations in the PIEZO1 gene have been identified in several families as causative of dehydrated hereditary stomatocytosis or xerocytosis. Similarly, the KCNN4 gene, codifying the so called Gardos channel, has been recently identified as a second causative gene of hereditary xerocytosis. Finally, ABCB6 missense mutations were identified in different pedigrees of familial pseudohyperkalemia. New genomic technologies have improved the quality and reduced the time of diagnosis of these diseases. Moreover, they are essential for the identification of the new causative genes. However, many questions remain to solve, and are currently objects of intensive studies.

Hereditary hemolytic anemias are a group of disorders with a variety of causes, including red cell membrane defects, red blood cell enzyme disorders, congenital dyserythropoietic anemias, thalassemia syndromes and hemoglobinopathies. As damaged red blood cells passing through the red pulp of the spleen are removed by splenic macrophages, splenectomy is one possible therapeutic approach to the management of severely affected patients. However, except for hereditary spherocytosis for which the effectiveness of splenectomy has been well documented, the efficacy of splenectomy in other anemias within this group has yet to be determined and there are concerns regarding short- and long-term infectious and thrombotic complications. In light of the priorities identified by the European Hematology Association Roadmap we generated specific recommendations for each disorder, except thalassemia syndromes for which there are other, recent guidelines. Our recommendations are intended to enable clinicians to achieve better informed decisions on disease management by splenectomy, on the type of splenectomy and the possible consequences. As no randomized clinical trials, case control or cohort studies regarding splenectomy in these disorders were found in the literature, recommendations for each disease were based on expert opinion and were subsequently critically revised and modified by the Splenectomy in Rare Anemias Study Group, which includes hematologists caring for both adults and children.

Through negative regulation of gene expression, microRNAs (miRNAs) can function as oncosuppressors in cancers, and can themselves show altered expression in various tumor types. Here, we have investigated medulloblastoma tumors (MBs), which arise from an early impairment of developmental processes in the cerebellum, where Notch signaling is involved in many of the cell-fate-determining stages. Notch regulates a subset of MB cells that have stem-cell-like properties and can promote tumor growth. On the basis of this evidence, we hypothesized that miRNAs targeting the Notch pathway can regulate these phenomena, and can be used in anti-cancer therapies. In a screening of potential targets within Notch signaling, miR-34a was seen to be a regulator of the Notch pathway through its targeting of Notch ligand Delta-like 1 (Dll1). Down-regulation of Dll1 expression by miR-34a negatively regulates cell proliferation, and induces apoptosis and neural differentiation in MB cells. Using an inducible tetracycline on-off model of miR-34a expression, we show that in Daoy MB cells, Dll1 is the first target that is regulated in MB, as compared to the other targets analyzed here: Cyclin D1, cMyc and CDK4. MiR-34a expression negatively affects CD133⁺/CD15⁺ tumor-propagating cells, then we assay through reverse-phase proteomic arrays, Akt and Stat3 signaling hypo-phosphorylation. Adenoviruses carrying the precursor miR-34a induce neurogenesis of tumor spheres derived from a genetic animal model of MB (Patch1^+/- p53^-/-), thus providing further evidence that the miR-34a/Dll1 axis controls both autonomous and non autonomous signaling of Notch. In vivo, miR-34a overexpression carried by adenoviruses reduces tumor burden in cerebellum xenografts of athymic mice, thus demonstrating an anti-tumorigenic role of miR-34a in vivo. Despite advances in our understanding of the pathogenesis of MB, one-third of patients with MB remain incurable. Here, we show that stable nucleic-acid-lipid particles carrying mature miR-34a can target Dll1 in vitro and show equal effects to those of adenovirus miR-34a cell infection. Thus, this technology forms the basis for their therapeutic use for the delivery of miR-34a in brain-tumor treatment, with no signs of toxicity described to date in non-human primate trials.