X-linked Neutropenia Research Articles

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Wiskott‐Aldrich syndrome: a comprehensive review

Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and an increased incidence of autoimmunity and malignancies. The disease is caused by mutations in the WAS gene expressed exclusively in hematopoietic cells. WAS protein (WASp) is a multidomain protein that exists in complex with several partners that play important roles in its function. WASp belongs to a family of proteins that relay signals from the surface of the cell to the actin cytoskeleton. Mutations in the WAS gene have various effects on the level of WASp, which, in turn, correlates with the severity of the disease. In addition to WAS, mutations in the WAS gene can result in the mild variant X-linked thrombocytopenia, or in X-linked neutropenia, characterized by neutropenia with myelodysplasia. The absence of functional WASp leads to a severe clinical phenotype that can result in death if not diagnosed and treated early in life. The treatment of choice with the best outcome is hematopoietic stem cell transplantation, preferably from a matched related donor.

Two cases of wiskott–Aldrich syndrome in neonates due to gene mutations

Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive disease characterized by eczema, thrombocytopenia and immune deficiency. WAS gene mutations impair WAS protein function which cause WAS. The WAS-related disorders of X-linked thrombocytopenia (XLT) and X-linked congenital neutropenia (XLN) may have similar but less severe symptoms those are also caused by mutations of the same gene. We present two cases of WAS in neonates with WAS gene mutations. Early genetic diagnosis can help to the treatment and prevention this disease.

Modelling human wiskott aldrich syndrome protein mutants in zebrafish larvae using live in vivo imaging

SummaryWiskott–Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are immunodeficiencies in which the function of several haematopoietic cell lineages is perturbed as a result of mutations in the actin regulator WASp. From in vitro cell biology experiments, and biochemical and structural approaches, we know much about the functional domains of WASp and how WASp might regulate the dynamic actin cytoskeleton downstream of activators such as Cdc42, but in vivo experiments are much more challenging. In patients, there is a correlation between clinical disease and genotype, with severe reductions in WASp expression or function associating with complex multilineage immunodeficiency, whereas specific mutations that cause constitutive activation of WASp result in congenital neutropenia. Here, we take advantage of the genetic tractability and translucency of zebrafish larvae to first characterise how a null mutant in zfWASp influences the behaviour of neutrophils and macrophages in response to tissue damage and to clearance of infections. We then use this mutant background to study how leukocyte lineage-specific transgenic replacement with human WASp variants (including normal wild type and point mutations that either fail to bind Cdc42 or cannot be phosphorylated, and a constitutively active mutant equivalent to that seen in XLN patients) alter the capacity for generation of neutrophils, their chemotactic response to wounds and the phagocytic clearance capacity of macrophages. This model provides a unique insight into WASp-related immunodeficiency at both a cellular and whole organism level.

Excess F-actin mechanically impedes mitosis leading to cytokinesis failure in X-linked neutropenia by exceeding Aurora B kinase error correction capacity

AbstractThe constitutively active mutant of the Wiskott-Aldrich Syndrome protein (CA-WASp) is the cause of X-linked neutropenia and is linked with genomic instability and myelodysplasia. CA-WASp generates abnormally high levels of cytoplasmic F-actin through dysregulated activation of the Arp2/3 complex leading to defects in cell division. As WASp has no reported role in cell division, we hypothesized that alteration of cell mechanics because of increased F-actin may indirectly disrupt dynamic events during mitosis. Inhibition of the Arp2/3 complex revealed that excess cytoplasmic F-actin caused increased cellular viscosity, slowed all phases of mitosis, and perturbed mitotic mechanics. Comparison of chromosome velocity to the cytoplasmic viscosity revealed that cells compensated for increased viscosity by up-regulating force applied to chromosomes and increased the density of microtubules at kinetochores. Mitotic abnormalities were because of overload of the aurora signaling pathway as subcritical inhibition of Aurora in CA-WASp cells caused increased cytokinesis failure, while overexpression reduced defects. These findings demonstrate that changes in cell mechanics can cause significant mitotic abnormalities leading to genomic instability, and highlight the importance of mechanical sensors such as Aurora B in maintaining the fidelity of hematopoietic cell division.

Clinical spectrum, pathophysiology and treatment of the Wiskott–Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS), caused by mutations in the WAS gene, is a complex and diverse disorder with X-linked inheritance. This review focuses on recent developments in the understanding of its basic pathophysiology, diverse clinical phenotypes and optimal patient management including novel therapies. The protein encoded by the WAS gene is a multifunctional signaling element expressed in immune and hematopoietic cells that plays a critical role in cytoskeletal reorganization, immune synapse formation and intracellular signaling. The type of specific mutation, its location within the gene and its effect on protein expression play a major role in determining an individual patient's clinical phenotype. Recent clinical observations and molecular studies have created a sophisticated picture of the disease spectrum. The improved outcome of stem cell transplantation from related and unrelated matched donors and promising early results from the first clinical gene therapy trial have added new therapeutic options for these patients. Classic WAS, X-linked thrombocytopenia and X-linked neutropenia are caused by WAS gene mutations, each having a distinct pattern of clinical symptoms and disease severity. New developments in the understanding of these syndromes and novel therapeutic options will have a major impact on the treatment of individuals with WAS mutations.

Tyrosine Phosphorylation of WASP Promotes Calpain-Mediated Podosome Disassembly In Myeloid Cells.

AbstractAbstract 1498We have previously shown that a point mutation (Ile294Thr) in the Wiskott Aldrich Syndrome Protein (WASP) detected in a Wiskott Aldrich Syndrome (WAS) patient led to enhanced actin polymerising activity of WASP 1 and well as increased instability2. We also showed that macrophages from this patient displayed an increased number of actin based adhesion structures called podosomes. Additionally, podosomes in macrophages from this patient were extremely dynamic with a high rate of turnover. Based on these results we proposed that the active open conformation of WASP promotes actin polymerisation but it also induces podosome disassembly and adhesion turnover. Although the idea that the same open conformation of WASP leads to both assembly and disassembly of podosomes may be counterintuitive at first, it is possible that for termination of podosomes actin polymerisation and integrin recruitment have to be discontinued and the same constituents of growing podosomes such as active WASP may contribute to the disassembly process. We have also shown that the rapid turn-over of podosomes involves cleavage of WASP by the protease calpain, further supporting a role of WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown and need further clarification. Phosphorylation of WASP Y291 (human) or Y293 (mouse) promotes the open conformation of WASP and results in enhanced actin polymerisation. We now report that in myeloid cells, tyrosine phosphorylation of WASP negatively regulates the stability of podosomes, leading to their calpain-dependent disassembly. Additionally, we found that constitutive phosphorylation of WASP results in extensive degradation in a process that involves calpain. Our data also indicate that phosphorylation of WASP sustains the open/active conformation that promotes cleavage of WASP by calpain. Interestingly, phosphorylated WASP can bind to the WASP interacting protein (WIP) and other proteins that form a complex with WASP in podosomes such as Nck, cortactin. Taken together, our data indicate that in myeloid cells, tyrosine phosphorylation sustains the open conformation of WASP and it enhances its susceptibility to calpain-mediated cleavage preventing accumulation of actin filaments and integrin associated proteins in podosomes. This process facilitates podosome disassembly and cell translocation. These new findings support the key role of WASP as a protein that integrates actin polymerisation and cell adhesion required for mobilisation of myeloid cells during the immune response.Reference List1. Ancliff PJ, Blundell MP, Cory GO et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006;108:2182-2189.2. Moulding DA, Blundell MP, Spiller DG et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp. Med 2007;204:2213–2224. Disclosures:No relevant conflicts of interest to declare.

Activating WASP mutations associated with X-linked neutropenia result in enhanced actin polymerization, altered cytoskeletal responses, and genomic instability in lymphocytes

Activating WASP mutations associated with X-linked neutropenia result in enhanced actin polymerization, altered cytoskeletal responses, and genomic instability in lymphocytes

Activating WASP mutations associated with X-linked neutropenia result in enhanced actin polymerization, altered cytoskeletal responses, and genomic instability in lymphocytes

X-linked neutropenia (XLN) is caused by activating mutations in the Wiskott-Aldrich syndrome protein (WASP) that result in aberrant autoinhibition. Although patients with XLN appear to have only defects in myeloid lineages, we hypothesized that activating mutations of WASP are likely to affect the immune system more broadly. We generated mouse models to assess the role of activating WASP mutations associated with XLN (XLN-WASP) in lymphocytes. XLN-WASP is expressed stably in B and T cells and induces a marked increase in polymerized actin. XLN-WASP–expressing B and T cells migrate toward chemokines but fail to adhere normally. In marked contrast to WASP-deficient cells, XLN-WASP–expressing T cells proliferate normally in response to cell-surface receptor activation. However, XLN-WASP–expressing B cells fail to proliferate and secrete lower amounts of antibodies. Moreover, XLN-WASP expression in lymphocytes results in modestly increased apoptosis associated with increased genomic instability. These data indicate that there are unique requirements for the presence and activation status of WASP in B and T cells and that WASP-activating mutations interfere with lymphocyte cell survival and genomic stability.

The Wiskott-Aldrich Syndrome: The Actin Cytoskeleton and Immune Cell Function

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by immune dysregulation, microthrombocytopaenia, eczema and lymphoid malignancies. Mutations in the WAS gene can lead to distinct syndrome variations which largely, although not exclusively, depend upon the mutation. Premature termination and deletions abrogate Wiskott-Aldrich syndrome protein (WASp) expression and lead to severe disease (WAS). Missense mutations usually result in reduced protein expression and the phenotypically milder X-linked thrombocytopenia (XLT) or attenuated WAS [1-3]. More recently however novel activating mutations have been described that give rise to X-linked neutropenia (XLN), a third syndrome defined by neutropenia with variable myelodysplasia [4-6]. WASP is key in transducing signals from the cell surface to the actin cytoskeleton, and a lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration.

The Wiskott-Aldrich syndrome: The actin cytoskeleton and immune cell function.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive primary immunodeficiency characterised by immune dysregulation, microthrombocytopaenia, eczema and lymphoid malignancies. Mutations in the WAS gene can lead to distinct syndrome variations which largely, although not exclusively, depend upon the mutation. Premature termination and deletions abrogate Wiskott-Aldrich syndrome protein (WASp) expression and lead to severe disease (WAS). Missense mutations usually result in reduced protein expression and the phenotypically milder X-linked thrombocytopenia (XLT) or attenuated WAS [1-3]. More recently however novel activating mutations have been described that give rise to X-linked neutropenia (XLN), a third syndrome defined by neutropenia with variable myelodysplasia [4-6]. WASP is key in transducing signals from the cell surface to the actin cytoskeleton, and a lack of WASp results in cytoskeletal defects that compromise multiple aspects of normal cellular activity including proliferation, phagocytosis, immune synapse formation, adhesion and directed migration.

G-CSF receptor (CSF3R) mutations in X-linked neutropenia evolving to acute myeloid leukemia or myelodysplasia

X-linked neutropenia (XLN) is a rare form of Congenital Neutropenia (CN) caused by inherited gain-of-function mutations of WAS. Here we report 2 cases of the original L270P X-linked neutropenia kindred that evolved to MDS or AML, with acquisition of G-CSFR (CSF3R) mutations and monosomy 7. Thus, leukemic transformation with acquisition of CSF3R mutations and monosomy 7 is not restricted to classical congenital neutropenia with autosomal inheritance, but can also occur in other genotypes of inherited neutropenia.

Homozygous HAX1 mutations in severe congenital neutropenia patients with sporadic disease: a novel mutation in two unrelated British kindreds

Patients with autosomal dominant (AD), sporadic and X-linked severe congenital neutropenia (SCN) may have mutations in the elastase 2 (ELA2) or Wiskott-Aldrich syndrome (WAS) genes. Homozygous mutations in the HAX1 gene have recently been reported in autosomal recessive (AR) cases of primarily Middle-Eastern descent and the original Kostmann family. We screened 109 predominantly Caucasian SCN kindreds for mutations in these genes; 33 (30%) had 24 different ELA2 mutations, five of them novel, two kindreds (2%) had WAS mutations and four kindreds (4%) had three different HAX1 mutations, two of them novel. One HAX1 mutation (p.Ser43LeufsX11) was found in an AR Ashkenazi Jewish kindred, the other (p.Glu31LysfsX54) in two unrelated British patients with sporadic disease. Microsatellite analysis of the HAX1 locus revealed a common haplotype (maximum distance 4.1 Megabases) for the p.Glu31LysfsX54 patients, suggesting a possible ancestral founder. In functional assays, the level of spontaneous and staurosporine-induced apoptosis was increased in neutrophils from both p.Ser43LeufsX11 patients but not a p.Glu31LysfsX54 patient, suggesting the possible presence of modifying factors. The low incidence of HAX1 mutations in our study suggests that the frequency may vary between racial groups but suggests that irrespective of inheritance or racial origin, SCN patients should be screened for HAX1 mutations.

New insights into the biology of Wiskott-Aldrich syndrome (WAS)

The Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease with a characteristic clinical phenotype that includes thrombocytopenia with small platelets, eczema, recurrent infections due to immunodeficiency, and an increased incidence of autoimmune manifestations and malignancies. The identification of the molecular defect in the WAS gene has broadened the clinical spectrum of disease to include chronic or intermittent X-linked thrombocytopenia (XLT), a relatively mild form of WAS, and X-linked neutropenia (XLN) due to an arrest of myelopoiesis. The pathophysiological mechanisms relate to defective actin polymerization in hematopoietic cells as a result of deficient or dysregulated activity of the WAS protein (WASp). The severity of disease is variable and somewhat predictable from genotype. Treatment strategies therefore range from conservative through to early definitive intervention by using allogeneic hematopoietic stem cell transplantation and potentially somatic gene therapy. All aspects of the condition from clinical presentation to molecular pathology and basic cellular mechanisms have been reviewed recently.

Wiskott-Aldrich Syndrome: Diagnosis, Clinical and Laboratory Manifestations, and Treatment

Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency disorder that has a variable clinical phenotype that correlates with the type of mutation in WASP, the gene encoding the WAS protein (WASP). WASP is a key regulator of actin polymerization in hematopoietic cells and has well-defined domains that are involved in signaling, cell locomotion, and immune synapse formation. Classic WAS often results from mutations that cause the absence of WASP expression, associated with thrombocytopenia with small platelets, sinopulmonary infections, and eczema in young males. Other phenotypes associated with expression of mutated WASP are X-linked thrombocytopenia and neutropenia. To date, the only curative therapy for WAS is hematopoietic cell transplantation (HCT) although gene therapy for WAS is under study. At least 2 retrospective studies of HCT for WAS have indicated that although HLA-matched sibling donors have the best outcomes (81% to 88%), when such a donor is not available, a matched unrelated donor should be considered (71% event free survival), although results are best in patients age < 5 years. Whereas most of the experience to date in Asia, Europe, and North America has been with myeloablative conditioning regimens, more recently, reduced-intensity conditioning (RIC) regimens also have been used with success. The issue of whether mixed chimerism post-HCT (which has a higher incidence in RIC transplantation) is associated with increased autoimmune manifestations in patients with WAS remains to be resolved.

Mutations of the Wiskott–Aldrich Syndrome Protein affect protein expression and dictate the clinical phenotypes

Mutations of the Wiskott-Aldrich Syndrome Protein (WASP) are responsible for classic Wiskott-Aldrich Syndrome (WAS), X-linked thrombocytopenia (XLT), and in rare instances congenital X-linked neutropenia (XLN). WASP is a regulator of actin polymerization in hematopoietic cells with well-defined functional domains that are involved in cell signaling and cell locomotion, immune synapse formation, and apoptosis. Mutations of WASP are located throughout the gene and either inhibit or disregulate normal WASP function. Analysis of a large patient population demonstrates a strong phenotype-genotype correlation. Classic WAS occurs when WASP is absent, XLT when mutated WASP is expressed and XLN when missense mutations occur in the Cdc42-binding site. However, because there are exceptions to this rule it is difficult to predict the long-term prognosis of a given affected boy solely based on the analysis of WASP expression.

A large kindred with X-linked neutropenia with an I294T mutation of the Wiskott-Aldrich syndrome gene.

X-linked neutropenia (XLN, OMIM #300299) is a rare form of severe congenital neutropenia. It was originally described in a three-generation family with five affected members that had an L270P mutation in the GTP-ase binding domain (GBD) of the Wiskott-Aldrich syndrome protein (WASP) [Devriendt et al (2001) Nature Genetics, Vol. 27, 313-317]. Here, we report and describe a large three-generation family with XLN, with 10 affected males and eight female carriers. A c.882T>C mutation was identified in the WAS gene, resulting in an I294T mutation. The infectious course is variable and mild in view of the profound neutropenia. In addition to the original description, low-normal IgA levels, low to low-normal platelet counts and reduced natural killer (NK)-cell counts also appear as consistent XLN features. However, inverted CD4/CD8 ratios were not found in this family, nor were cases identified with myelodysplastic syndrome or acute myeloid leukaemia. Female carriers exhibited a variable attenuated phenotype. Like L270P WASP, I294T WASP is constitutively active towards actin polymerization. In conclusion, this largest XLN kindred identified to date provides new independent genetic evidence that mutations disrupting the auto-inhibitory GBD of WASP are the cause of XLN. Reduced NK cells, low to low normal platelet counts and low to low-normal IgA levels are also features of XLN.

Wiskott-Aldrich syndrome

Wiskott-Aldrich syndrome is caused by mutations of the Wiskott-Aldrich syndrome protein gene, which codes for a cytoplasmic protein with multiple functions. This review will focus on recent progress in understanding the molecular basis of Wiskott-Aldrich syndrome and its ramifications for the cure of this lethal disease. The discovery of the causative gene has revealed a spectrum of clinical phenotypes demonstrating a strong genotype/phenotype correlation. The discovery of unique functional domains of Wiskott-Aldrich syndrome protein has been instrumental in defining mechanisms that control activation of Wiskott-Aldrich syndrome protein. Long-term follow up of patients undergoing hematopoietic stem cell transplantation has led to important modifications of the procedure. Studies of Wiskott-Aldrich syndrome protein-deficient cell lines and wasp-knockout mice have paved the way for possible gene therapy. Wiskott-Aldrich syndrome protein gene mutations result in four clinical phenotypes: classic Wiskott-Aldrich syndrome and X-linked thrombocytopenia, intermittent thrombocytopenia and neutropenia. Wiskott-Aldrich syndrome protein is a signaling molecule and instrumental for cognate and innate immunity, cell motility and protection against autoimmune disease. The success of hematopoietic stem cell transplantation is related to the recipient's age, donor selection, the conditioning regimen and the extent of reconstitution. Since Wiskott-Aldrich syndrome protein is expressed exclusively in hematopoietic stem cells, and because Wiskott-Aldrich syndrome protein exerts a strong selective pressure, gene therapy is expected to cure the disease.

Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia

Specific mutations in the human gene encoding the Wiskott-Aldrich syndrome protein (WASp) that compromise normal auto-inhibition of WASp result in unregulated activation of the actin-related protein 2/3 complex and increased actin polymerizing activity. These activating mutations are associated with an X-linked form of neutropenia with an intrinsic failure of myelopoiesis and an increase in the incidence of cytogenetic abnormalities. To study the underlying mechanisms, active mutant WASpI294T was expressed by gene transfer. This caused enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability. Live cell imaging demonstrated a delay in mitosis from prometaphase to anaphase and confirmed that multinucleation was a result of aborted cytokinesis. During mitosis, filamentous actin was abnormally localized around the spindle and chromosomes throughout their alignment and separation, and it accumulated within the cleavage furrow around the spindle midzone. These findings reveal a novel mechanism for inhibition of myelopoiesis through defective mitosis and cytokinesis due to hyperactivation and mislocalization of actin polymerization.