Loïc Garçon

A unique clonal JAK2 mutation leading to constitutive signalling causes polycythaemia vera

Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels.

Dehydrated hereditary stomatocytosis (DHS) is a genetic condition with defective red blood cell (RBC) membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations inthe mechanically activated PIEZO1(FAM38A) ion channel were associated with DHS. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated DHS cases, we identifythree novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for DHS. All the DHS-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in RBCs of DHS patients. Our findings also suggest a new role for mechanotransduction in RBC biology and pathophysiology.

Usefulness of the eosin‐5′‐maleimide cytometric method as a first‐line screening test for the diagnosis of hereditary spherocytosis: comparison with ektacytometry and protein electrophoresis

This project was supported by the Institutional Research Program of the Texas Tech University Health Sciences Center and Southwest Cancer Treatment and Research Center Program. We thank Teri Fields for her assistance in editing this manuscript and Drs Janet Dertien and Jose-Louis Redondo for their assistance and support in using the confocal microscope. W. Martin Kast holds the Walter A. Richter Cancer Research Chair.

Molecular analysis of 42 patients with congenital dyserythropoietic anemia type II: new mutations in the SEC23B gene and a search for a genotype-phenotype relationship

Background The most frequent form of congenital dyserythropoietic anemia is the type II form. Recently it was shown that the vast majority of patients with congenital dyserythropoietic anemia type II carry mutations in the SEC23B gene. Here we established the molecular basis of 42 cases of congenital dyserythropoietic anemia type II and attempted to define a genotype-phenotype relationship. Design and Methods SEC23B gene sequencing analysis was performed to assess the diversity and incidence of each mutation in 42 patients with congenital dyserythropoietic anemia type II (25 described exclusively in this work), from the Italian and the French Registries, and the relationship of these mutations with the clinical presentation. To this purpose, we divided the patients into two groups: (i) patients with two missense mutations and (ii) patients with one nonsense and one missense mutation. Results We found 22 mutations of uneven frequency, including seven novel mutations. Compound heterozygosity for a missense and a nonsense mutation tended to produce a more severe clinical presentation, a lower reticulocyte count, a higher serum ferritin level, and, in some cases, more pronounced transfusion needs, than homozygosity or compound heterozygosity for two missense mutations. Homozygosity or compound heterozygosity for two nonsense mutations was never found. Conclusions This study allowed us to determine the most frequent mutations in patients with congenital dyserythropoietic anemia type II. Correlations between the mutations and various biological parameters suggested that the association of one missense mutation and one nonsense mutation was significantly more deleterious that the association of two missense mutations. However, there was an overlap between the two categories.

Sideroblastic anemia: molecular analysis of the ALAS2 gene in a series of 29 probands and functional studies of 10 missense mutations.

X‐linked Sideroblastic Anemia (XLSA) is the most common genetic form of sideroblastic anemia, a heterogeneous group of disorders characterized by iron deposits in the mitochondria of erythroid precursors. XLSA is due to mutations in the erythroid‐specific 5‐aminolevulinate synthase (ALAS2) gene. Thirteen different ALAS2 mutations were identified in 16 out of 29 probands with sideroblastic anemia. One third of the patients were females with a highly skewed X‐chromosome inactivation. The identification of seven novel mutations in the ALAS2 gene, six missense mutations, and one deletion in the proximal promoter extends the allelic heterogeneity of XSLA. Most of the missense mutations were predicted to be deleterious, and 10 of them, without any published functional characterization, were expressed in Escherichia coli. ALAS2 activities were assayed in vitro. Five missense mutations resulted in decreased enzymatic activity under standard conditions, and two other mutated proteins had decreased activity when assayed in the absence of exogenous pyridoxal phosphate and increased thermosensitivity. Although most amino acid substitutions result in a clearly decreased enzymatic activity in vitro, a few mutations have a more subtle effect on the protein that is only revealed by in vitro tests under specific conditions. Hum Mutat 32:1–8, 2011.

Frequency of congenital dyserythropoietic anemias in Europe

Congenital dyserythropoietic anemias (CDAs) are rare hereditary disorders characterized by ineffective erythropoiesis and striking abnormalities of erythroblast morphology. The mutated genes are known for the most frequent types, CDA I and II, but data about their frequency do not exist. The objective of this retrospective study was to estimate the frequency of CDA I and II, based on all cases reported in the last 42 yr in publications and identified registries or surveys. Reports were collected of 124 and 377 confirmed cases of CDA I and CDA II cases, respectively. The cumulated incidence of both types combined varied widely between European regions, with minimal values of 0.08 cases/million in Scandinavia and 2.60 cases/million in Italy. CDA II is more frequent than CDA I, with an overall ratio of approximately 3.2, but the ratio also varied between different regions. The most likely explanations for the differences are both differences in the availability of advanced diagnostic procedures and different levels of the awareness for the diagnosis of the CDAs. The estimations reported here are most probably below the true incidence rates, because of failure to make the correct diagnosis and to underreporting. Limited data do not suggest differing levels of risk in identified ethnic groups.

Recommendations regarding splenectomy in hereditary hemolytic anemias.

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.

Red cells exchanges in sickle cells disease lead to a selective reduction of erythrocytes-derived blood microparticles

Red cell exchange (RCE) is a highly effective procedure in the management of complications of sickle cell disease (SCD) (Swerdlow, 2006). During acute vaso-occlusive complications, RCE permits both the input of normal red cells and the removal of sickle cells without increasing blood viscosity and is a recommended part of the initial treatment of vaso-occlusive stroke in children (National Institutes of Health, National Heart, Lung and Blood Institute, 2002). RCE can also be used as a long-term therapy to maintain a low level of HbS in primary and secondary stroke prevention (Platt, 2006), with the major advantage of avoiding the risk of iron overload observed with simple transfusions (Kim et al, 1994). Microparticles (MPs) are small vesicles released from cells after activation or apoptosis (Piccin et al, 2007). In SCD, their total number is robustly increased at steady state or during crisis (Shet et al, 2003; van Beers et al, 2009; van Tits et al, 2009). Of interest, the number of erythrocyte-derived MPs is particularly high in SCD. These MPs seem to play a specific role in the activation of coagulation and thrombin generation and, consequently, could participate in the global pro-coagulant state observed in this pathology (Horne et al, 2006; van Beers et al, 2009). This study aimed to compare the level of total, plateletsderived and erythrocytes-derived MPs before and after RCE in SCD patients. After informed consent, 37 blood samples were collected from 27 patients with SS genotype undergoing a long-term RCE program using apheresis. Two patients underwent RCE before surgery; all the others were included in a RCE program for pregnancy (n = 2), recurrent vaso occlusive complications (n = 7) or chronic complications, such as leg ulcers or vasculopathy (n = 16). For each patient, a blood sample was drawn before and just after the exchange. Samples from donors without neither anaemia nor coagulation disorders were used as controls (n = 19). MPs were isolated with a protocol with no pre-analytic artifacts related to any ultracentrifugation or filtration step (Robert et al, 2009). MPs were

MplK, a natural variant of the thrombopoietin receptor with a truncated cytoplasmic domain, binds thrombopoietin but does not interfere with thrombopoietin-mediated cell growth

OBJECTIVE Interaction of thrombopoietin (TPO) with its receptor c-Mpl is responsible for the formation of megakaryocytes and platelets. In humans, there are two major c-mpl molecules, MplP and MplK, which are generated by alternative splicing. In contrast to MplP, MplK has none of the intracellular sequences required for typical signal transduction but instead has a unique 27 amino acid sequence that is coded by intron 10. We tested to determine if MplK exerts a negative effect on TPO Mpl signal transduction by interfering with the normal homodimerization of MplP. MATERIALS AND METHODS A cassette coding for MplK cDNA was introduced into parental and MplP-expressing BaF3 cells and TPO-mediated cell growth studied. RESULTS Cells expressing MplK alone did not respond to TPO compared to cells that expressed MplP. When MplK was coexpressed with MplP on the cell surface of BaF3, no modification in cell growth was observed when compared to those expressing MplP alone. To determine if the normal homodimerization process was negatively influenced, two genetically engineered variants of c-Mpl, one lacking the box1 sequence and the other containing only the first nine amino acids of the intracellular domain, were introduced into MplP-expressing cells. In contrast to MplK, these mutants had a dominant negative effect on TPO-mediated cell growth. CONCLUSIONS MplK does not influence TPO-mediated growth of Mpl-expressing cells. Our data suggest that the absence of a dominant negative effect of MplK most probably is due to the inability of MplK to dimerize with the MplP receptor.

Red blood cell Gardos channel (KCNN4):the essential determinant of erythrocyte dehydration in Hereditary Xerocytosis

Recent advances have been made in the identification of molecular determinants of the rare hemolytic disease, dehydrated hereditary stomatocytosis (DHSt), also called hereditary xerocytosis (HX). This well-known red blood cell (RBC) pathology is characterized by an abnormal cation leak resulting in KCl loss and red blood cell dehydration. It leads to cell fragility and hemolytic anemia. Two different genes have been linked to this phenotype: PIEZO1 and KCNN4 coding, respectively, for Piezo1, a non-selective cation channel activated by mechanical forces, and a calcium activated K channel (KCNN4) also known the Gardos channel in RBCs. So far, three different point mutations in KCNN4 have been linked to HX. 6 The mutated KCNN4 is over-activated in patient RBCs leading to an increased K loss and, hence, water loss and cell dehydration. About a dozen mutations in Piezo1 are linked to HX, and some of them have been shown to change the kinetics of channel gating. In normal RBCs, Piezo1 appears to be a major factor in cell response to mechanical stress (by controlling calcium influx), and there is a functional connection between Piezo1 and KCNN4 through the modification of intracellular calcium concentration. Our present study was designed to evaluate in HX the functional link between mutated Piezo1 and KCNN4 and to assess the efficiency of a KCNN4 blocker, Senicapoc, to treat HX whatever the molecular cause. Our study focused on three independent index cases with a typical HX clinical and biological phenotype (Online Supplementary Table S1 and Figure 1); two were unreported cases (patients 1 and 2), and one was previously described (patient 3). Patient 1 was a 35-year-old man presenting with undiagnosed compensated hemolytic anemia and iron overload. He was investigated due to an unexplained fatal hydrops history during his wife’s second pregnancy; his first son was well and unaffected. Patient 2 was a 38-year-old women investigated for undiagnosed compensated hemolytic anemia. PIEZO1 sequencing for patient 1 and 2 revealed two new missense mutations : a c.1792G>A mutation in exon 14