Wiskott-Aldrich Syndrome Protein Gene Research Articles

Mutational screening of WASP gene in patients with Wiskott-Aldrich syndrome

The Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive immunodeficiency disorder characterized by thrombocytopenia and small-sized platelets, eczema, recurrent bacterial and viral infections, higher incidence of autoimmunity and an increased risk of malignancies. WAS occurs due to the mutation or loss of Wiskott-Aldrich Syndrome Protein (WASP) gene located on Xp11.22 – p11.23 of the short arm of the X chromosome. The absence of functional WASP leads to severe clinical symptoms that results in the deaths of patients if they are not diagnosed and treated early. The objective of the study was to identify mutations in the WASP gene of families with children diagnosed with WAS.The whole coding sequence and the intron-exon flanking regions of the WASP were sequenced by Sanger method. Two cases of children who has WAS were found tocarrymutations in the WASP gene. A c.702insAC mutation leadeda frameshift at position of codon 236 and terminated the protein at the position of codon 262 was identified in patient WA007 and a c.91G>A mutation that transformed glutamic acid to lysineat codon 31 was determined in patient WA010.This study provides a data set and screening of mutations in theWASP gene inVietnamese patientsto further identify the genetic causes and contribute to the clinical management and genetic counseling for the affected families.

Orphan designation: Autologous CD34+ cells transfected with lentiviral vector containing the Wiskott-Aldrich syndrome protein gene, Treatment of Wiskott-Aldrich syndrome

Orphan designation: Autologous CD34+ cells transfected with lentiviral vector containing the Wiskott-Aldrich syndrome protein gene, Treatment of Wiskott-Aldrich syndrome

Orphan designation: Autologous CD34+ cells transfected with lentiviral vector containing the Wiskott-Aldrich syndrome protein gene, Treatment of Wiskott-Aldrich syndrome

Orphan designation: Autologous CD34+ cells transfected with lentiviral vector containing the Wiskott-Aldrich syndrome protein gene, Treatment of Wiskott-Aldrich syndrome

Childhood-onset inflammatory bowel diseases associated with mutation of Wiskott-Aldrich syndrome protein gene.

AIMTo screen primary immunodeficiency, Wiskott-Aldrich syndrome (WAS), and chronic granulomatous disease (CGD) among children with inflammatory bowel disease (IBD).METHODSThis was a single-center retrospective study. Eighteen children with IBD were investigated. We analyzed their expression of Wiskott-Aldrich syndrome protein (WASP) in lymphocytes and superoxide generation in phagocytes using flow cytometry. When the expression of WASP or superoxide generation was low or absent, we performed genetic analysis to determine the cause of this.RESULTSEighteen patients were classified as having ulcerative colitis (n = 10), Crohn’s disease (n = 5), or IBD-unclassified (n = 3). In total, three patients revealed low expression of WASP associated with a WAS gene c.1378 C>T p.Pro460Ser mutation, which has previously been reported as a pathogenic mutation in WAS and X-linked thrombocytopenia. However, with respect to the major symptoms of WAS, none of these three patients showed either thrombocytopenia or increased susceptibility to infection, but one patient showed generalized eczema. No CGD patients were discovered in this study.CONCLUSIONDespite the lack of typical clinical manifestations of WAS, low expression of WASP could be associated with the pathogenesis of a subtype of IBD patients.

Whole Wiskott‑Aldrich syndrome protein gene deletion identified by high throughput sequencing.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive immunodeficiency disorder, characterized by thrombocytopenia, small platelets, eczema and recurrent infections associated with increased risk of autoimmunity and malignancy disorders. Mutations in the WAS protein (WASP) gene are responsible for WAS. To date, WASP mutations, including missense/nonsense, splicing, small deletions, small insertions, gross deletions, and gross insertions have been identified in patients with WAS. In addition, WASP-interacting proteins are suspected in patients with clinical features of WAS, in whom the WASP gene sequence and mRNA levels are normal. The present study aimed to investigate the application of next generation sequencing in definitive diagnosis and clinical therapy for WAS. A 5 month-old child with WAS who displayed symptoms of thrombocytopenia was examined. Whole exome sequence analysis of genomic DNA showed that the coverage and depth of WASP were extremely low. Quantitative polymerase chain reaction indicated total WASP gene deletion in the proband. In conclusion, high throughput sequencing is useful for the verification of WAS on the genetic profile, and has implications for family planning guidance and establishment of clinical programs.

Novel WASP mutation in a patient with Wiskott–Aldrich syndrome: Case report and review of the literature

BackgroundThe Wiskott–Aldrich syndrome (WAS) is a rare X-linked recessive immunodeficiency disorder, caused by mutations in the WAS protein (WASP) gene and characterised by thrombocytopenia, small platelets, eczema, and recurrent infections associated with increased risk of autoimmunity and malignancy disorders. The gene for WAS has been mapped to the short arm of the X chromosome at Xp 11.22-23 and early detection of patients and diagnosis of new mutation might reduce related complications and increase their life expectancy. Method and resultWe found a novel mutation by sequence analysis of genomic DNA coding of a 9-month old boy suffering from WAS. The mutation was insertion G in exon 10 of WASP gene. The consequence of the G insertion is a premature stop immediately at amino acid 335 (N335X or p.G334GfsX1) and truncated protein. ConclusionThe mutation analysis is helpful for the diagnosis of WAS patients and also expanding the spectrum of WASP mutations for carrier detection and prenatal diagnosis.

The identification of hematopoietic-specific regulatory elements for WASp gene expression

Chromosome Conformation Capture (3C) technology was used to identify physical interactions between the proximal Wiskott-Aldrich Syndrome protein (WASp) promoter and its distant DNA segments in Jurkat-T cells. We found that two hematopoietic specific DNase I hypersensitive (DHS) sites (proximal DHS-A, and distal DHS-B) which had high interaction frequencies with the proximal WASp promoter indicating potential regulatory activity for these DHS sites. Subsequently, we cloned several DNA fragments around the proximal DHS-A site into a luciferase reporter vector. Interestingly, no fragments showed enhancer activity, but two fragments exhibited strong silencing activity in Jurkat-T cells. After aligning the chromatin state profiling for hematopoietic and nonhematopoietic cells using the human genome browser (UCSC), we found a 5 kb putative hematopoietic specific enhancer region located 250 kb downstream of the WAS gene. This putative enhancer region contains two hematopoietic cell specific DHS sites. Subsequently, the hematopoietic specific DHS sites enhanced luciferase expression from the proximal WASp promoter in all hematopoietic cells we tested. Finally, using a lentiviral vector stable expression system, the hematopoietic specific-enhancer(s) increased GFP reporter gene expression in hematopoietic cells, and increased WASp gene expression in WASp deficient cells. This enhancer may have the potential to be used in gene therapy for hematological diseases.

An Update from the United States National Heart, Lung, and Blood Institute‐funded Production Assistance for Cellular Therapies (PACT) Program: A Decade of Cell Therapy

Recognizing the challenges faced by researchers and clinicians working in the field of cellular therapy, the National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health, established the Production Assistance for Cellular Therapies (PACT) program in 2003 and expanded it in 2010. The PACT program provides both clinical product manufacturing support that furthers the mission of NHLBI in the areas of cardiac, lung, and blood diseases and broad support of translational development across all disease areas to serve the entire cell therapy community. The program also provides access to expertise in project management, regulatory affairs, and quality assurance and control. Education initiatives include webinars, cell processing facility-hosted workshops, national workshops, and active participation and leadership within the cell therapy community through collaboration with other cell therapy organizations and academia. So far, over 650 PACT-manufactured cell therapy products have been administered in 32 clinical trials for a range of illnesses and diseases such as acute myocardial infarction, sickle cell disease, and graft-versus-host disease.

Primary immunodeficiences – options for the future

Primary immunodeficiences – options for the future

The open conformation of WASP regulates its nuclear localization and gene transcription in myeloid cells

Mutations in the gene encoding the Wiskott-Aldrich syndrome protein (WASP) are responsible for Wiskott-Aldrich syndrome and WASP is a major actin regulator in the cytoplasm. Although rare gain-of-function mutations in the WASP gene are known to result in X-linked neutropenia (XLN), the molecular pathogenesis of XLN is not fully understood. In this study, we showed that all reported constitutively activating mutants (L270P, S272P and I294T) of WASP were hyperphosphorylated by Src family tyrosine kinases and demonstrated higher actin polymerization activities compared with wild-type (WT) WASP. Further analysis showed a tendency of activating WASP mutants to localize in the nucleus compared with WT or the Y291F mutant of WASP. In addition, we found that WASP could form a complex with nuclear RNA-binding protein, 54 kDa (p54nrb) and RNA polymerase II (RNAP II). ChIP assays revealed that WASP associated with DNA, although the affinity was relatively weaker than RNAP II. To determine whether gene transcription was affected by WASP mutation in myeloid cells, we performed microarray analysis and found different expression profiles between WT and L270P WASP-transfected K562 cells. Among the genes affected, granulocyte colony-stimulating factor receptor, Runx1, and protein tyrosine phosphatase receptor c were included. ChIP on chip analysis of genomic DNA showed WT and L270P WASP had a highly similar DNA-binding pattern but differed in binding affinity at the same locus. Therefore, our results suggest that the open conformation of WASP regulates its nuclear localization and plays requisite roles in regulating gene transcription that would contribute to the outcome in the nucleus of myeloid cells.

Analysis of novel gene mutation in a case with Wiskott-Aldrich syndrome and its clinical characteristics

Objective To explore one novel gene mutation case suffering from Wiskott-Aldrich syndrome(WAS) and its clinical characteristics. Methods Based on the typical clinical manifestations of WAS including thrombocytopenia, eczema, and recurrent infections, the patient was suspected of having WAS. The 12 exons of Wiskott-Aldrich syndrome protein(WASP) gene of the patient and his parents were detected by PCR direct sequencing analysis, and their phenotypes were analysed. Results By sequence analysis using primer separately, the author found one novel WASP gene mutation, a homozygous mutation, which might cause amino acid mutation. The novel mutation with nucleotide substitution of G to T at position 224(c. 224T>G) was found in exon 2 of WASP gene. The 224T>G resulted in the substitution of valine with glycine at position 75(p. V75G), his parents were not found to have WASP gene mutation. Conclusions One novel WASP gene mutation was found. The case had a mild phenotype. Genetic sequence analysis is useful in the diagnosis of WAS and WASP carrier detection. Key words: Wiskott-Aldrich syndrome; Proteins; Mutation

A novel Wiskott–Aldrich syndrome protein mutation in an infant with thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) has not yet been reported to be associated with mutations in the Wiskott-Aldrich syndrome (WAS) gene. WAS is an X-linked recessive disorder characterized by thrombocytopenia, small platelet size, eczema, recurrent infections, and increased risk of autoimmune disorders and malignancies. A broad spectrum of mutations in the WAS protein (WASP) gene have been identified as causing the disease. In this study, we report on a 2-month-old Japanese boy who presented with cytomegalovirus (CMV) infection and TTP. The activity of von Willebrand factor cleaving metalloproteinase, ADAMTS13 was low and the antibody against ADAMTS13 was positive (3.6Bethesda U/mL). Although TTP was improved by plasma exchange and steroid pulse therapy, thrombocytopenia persisted and regular transfusions of irradiated platelets were needed. Tiny platelets were found on a peripheral blood smear. CMV genome was positive in peripheral blood by polymerase chain reaction and the CMV viremia continued to persist despite intravenous gancyclovir therapy. Through direct sequencing of genomic DNA of the WASP gene in the patient, we identified a novel mutation of WASP gene: the seventh nucleotide in exon 11 (G) had been deleted (1345delG). This mutation causes a frameshift and a stop codon at amino acid 470. Western blotting demonstrated a truncated WAS protein. To our knowledge, this is the first report describing TTP in WAS patients with novel mutation in the WASP gene.

Detection of six novel mutations in WASP gene in fifteen Iranian Wiskott-Aldrich patients.

Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked recessive immunodeficiency disease described as a clinical triad of thrombocytopenia, eczema, and recurrent infections, caused by mutations of the WAS protein (WASP) gene. The milder form of this disease is X-linked thrombocytopenia (XLT) that presents only as platelet abnormalities. Mutation analysis for 15 boys with Wiskott-Aldrich syndrome was performed. Five previously reported mutations and six novel mutations including G8X, R41X, D283E, P412fsX446, E464X, and AfsX358 were detected.

Wiskott–Aldrich syndrome presenting with a clinical picture mimicking juvenile myelomonocytic leukaemia

Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency caused by defects of the WAS protein (WASP) gene. Patients with WAS typically demonstrate micro-thrombocytopenia. The report describes seven male infants with WAS that initially presented with leukocytosis, monocytosis, and myeloid and erythroid precursors in the peripheral blood (PB) and dysplasia in the bone marrow (BM), which was initially indistinguishable from juvenile myelomonocytic leukaemia (JMML). The median age of affected patients was 1 month (range, 1-4 months). Splenomegaly was absent in four of these patients, which was unusual for JMML. A mutation analysis of genes in the RAS-signalling pathway did not support a diagnosis of JMML. Non-haematological features, such as eczema (n = 7) and bloody stools (n = 6), ultimately led to the diagnosis of WAS at a median age of 4 months (range, 3-8 months), which was confirmed by absent (n = 6) or reduced (n = 1) WASP expression in lymphocytes by flow cytometry (FCM) and a WASP gene mutation. Interestingly, mean platelet volume (MPV) was normal in three of five patients and six of seven patients demonstrated occasional giant platelets, which was not compatible with WAS. These data suggest that WAS should be considered in male infants presenting with JMML-like features if no molecular markers of JMML can be detected.

Ubiquitylation-Dependent Negative Regulation of WASp Is Essential for Actin Cytoskeleton Dynamics

The Wiskott-Aldrich syndrome protein (WASp) is a key regulator of actin dynamics during cell motility and adhesion, and mutations in its gene are responsible for Wiskott-Aldrich syndrome (WAS). Here, we demonstrate that WASp is ubiquitylated following T-cell antigen receptor (TCR) activation. WASp phosphorylation at tyrosine 291 results in recruitment of the E3 ligase Cbl-b, which, together with c-Cbl, carries out WASp ubiquitylation. Lysine residues 76 and 81, located at the WASp WH1 domain, which contains the vast majority of WASp gene mutations, serve as the ubiquitylation sites. Disruption of WASp ubiquitylation causes WASp accumulation and alters actin dynamics and the formation of actin-dependent structures. Our data suggest that regulated degradation of activated WASp might be an efficient strategy by which the duration and localization of actin rearrangement and the intensity of T-cell activation are controlled.

Foamy Virus Vector-mediated Gene Correction of a Mouse Model of Wiskott–Aldrich Syndrome

The Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by eczema, thrombocytopenia and immunodeficiency. Hematopoietic cell transplantation can cure the disease and gene therapy is being tested as an alternative treatment option. In this study, we assessed the use of foamy virus (FV) vectors as a gene transfer system for WAS, using a Was knockout (KO) mouse model. Preliminary experiments using FV vectors expressing the green fluorescent protein under the transcriptional control of the endogenous WAS promoter or a ubiquitously acting chromatin opening element allowed us to define transduction conditions resulting in high (>40%) and long-term in-vivo marking of blood cells after transplantation. In following experiments, Was KO mice were treated with FV vectors containing the human WAS complementary DNA (cDNA). Transplanted animals expressed the WAS protein (WASp) in T and B lymphocytes, as well as platelets and showed restoration of both T-cell receptor-mediated responses and B-cell migration. We also observed recovery of platelet adhesion and podosome formation in dendritic cells (DCs) of treated mice. These data demonstrate that FV vectors can be effective for hematopoietic stem cell (HSC)-directed gene correction of WAS.

Impaired cell adhesion, apoptosis, and signaling in WASP gene-disrupted Nalm-6 pre-B cells and recovery of cell adhesion using a transducible form of WASp

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disease affecting cell morphology and signal transduction in hematopoietic cells. The function of Wiskott-Aldrich syndrome protein (WASp) and its partners in protein interaction have been studied intensively in mice; however, detailed biochemical characterization of its signal transduction and assessment of its functional consequence in human WASp-deficient lymphocytes remain difficult. In this study, we generated Nalm-6 cells in which the WAS protein gene (WASP) was disrupted by homologous recombination-based gene targeting and a cell-permeable form of recombinant WASp for functional study. The WASP⁻/⁻ cells showed impaired adhesive capacity and polarization to plate-bound anti-CD47 mAb, anti-CD9 mAb, or to fibronectin. The defective morphological changes were accompanied by impaired intracellular signaling. In addition, the WASp-deficient cells displayed augmented apoptosis induced by CD24 cross-linking. A recombinant fusion protein composed of Hph-1 cell-permeable peptide and WASp prepared in Escherichia coli. Hph-1-WASp was efficiently transduced and expressed in WASP⁻/⁻ Nalm-6 cells in a dose-dependent manner. The wild-type WASp, but not the mutant restored adhesion capacity, spreading morphology, and cytoskeletal reorganization. Additionally, the recombinant protein was successfully transduced into normal lymphocytes. These findings suggest that gene-disrupted model cell lines and cell-permeable recombinant proteins may serve as important tools for the detailed analysis of intracellular molecules involved in PID.

Advances in basic and clinical immunology in 2010

Reports in basic and clinical immunology in 2010 reflected the use of state-of-the-art genetic and immunologic tools to characterize the pathogenesis of immunologic diseases and the development of novel therapies directed to these conditions. B-cell biology has been explained in greater detail, significantly with lessons from the genetic defects found in the humoral immunodeficiencies. Therapeutic mAbs are given for an increasing number of indications, such as anti-CD20 antibodies or rituximab, which was initially developed for non-Hodgkin lymphomas and is currently used in diverse autoimmune and inflammatory disorders. The report of an infant with severe combined immunodeficiency (SCID) in Massachusetts detected by means of newborn screening and successfully treated with hematopoietic stem cell transplantation validated recent efforts toward newborn screening for SCID. Improvement of survival outcomes for patients with primary immunodeficiencies treated with hematopoietic stem cell transplantation was demonstrated in a large European cohort, with significant appreciation of the type of donor graft, particularly the use of HLA-matched unrelated donors for patients with non-SCID. Progress in cellular mechanisms of drug hypersensitivity included the characterization of nitroso-modified drug metabolites as potent T-cell activators and the identification of the relocation of plasmacytoid dendritic cells from blood to skin as a potential risk factor for reactivation of viral disease.

A novel WASP gene mutation in a Chinese boy with Wiskott–Aldrich syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, increased susceptibility to infection, and immunodeficiency. Mutations of the Wiskott-Aldrich syndrome protein (WASP) gene are responsible for this severe congenital disease. In this study, we report on a 2-year-old Chinese boy who presented with classic clinical WAS manifestations. By direct sequencing of cDNA and genomic DNA of the patient, we identified a novel mutation: the first nucleotide in exon 8 (G) had been deleted (769delG). This mutation results in two kinds of aberrant mRNA with abnormal splicing and causes frameshift and a stop codon at amino acid 260. Western blotting demonstrated a 28-kDa truncated WAS protein. A maternal study revealed that his mother had a heterozygous genotype, but showed normal WASP expression.

Analysis of clinical and molecular characteristics of Wiskott–Aldrich syndrome in 24 patients from 23 unrelated Chinese families

The clinical data of 24 children with Wiskott-Aldrich syndrome (WAS) from 23 unrelated Chinese families were reviewed in the present study. WAS protein (WASP) expression in peripheral blood mononuclear cells was examined by flow cytometry (FCM); WASP gene was amplified by PCR and directly sequenced to analyze mutations in the WASP gene in patients and their female relatives. FCM analysis of 21 patients showed that 18 cases were WASP-negative, and three had partially WASP expression. WASP gene analysis revealed mutations in 23 patients, including five missense mutations, four nonsense mutations, four deletion mutations, three insertion mutations, six splice site mutations, and one complex mutation, among which, 20 unique mutations were detected, including seven novel mutations (168 C>A, 747-748del T, 793-797del C, 1185 ins C, Dup 1251-1267, 1277 insA and 1266 C>G; 1267-1269del C). Five WAS children underwent stem cell transplantation. After 2 months of transplantation, WASP expression was restored to normal in all five patients whereas one patient died of cytomegalovirus-induced interstitial lung disease. WASP gene analysis can make a definite diagnosis of WAS and identify mutation carriers, beneficial for timely treatment and genetic counseling for children with WAS.