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1.
Nat Commun ; 11(1): 4034, 2020 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-32788576

RESUMO

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency with severe platelet abnormalities and complex immunodeficiency. Although clinical gene therapy approaches using lentiviral vectors have produced encouraging results, full immune and platelet reconstitution is not always achieved. Here we show that a CRISPR/Cas9-based genome editing strategy allows the precise correction of WAS mutations in up to 60% of human hematopoietic stem and progenitor cells (HSPCs), without impairing cell viability and differentiation potential. Delivery of the editing reagents to WAS HSPCs led to full rescue of WASp expression and correction of functional defects in myeloid and lymphoid cells. Primary and secondary transplantation of corrected WAS HSPCs into immunodeficient mice showed persistence of edited cells for up to 26 weeks and efficient targeting of long-term repopulating stem cells. Finally, no major genotoxicity was associated with the gene editing process, paving the way for an alternative, yet highly efficient and safe therapy.


Assuntos
Edição de Genes , Terapia Genética , Células-Tronco Hematopoéticas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Plaquetas/metabolismo , Sistemas CRISPR-Cas/genética , Linhagem da Célula , Códon/genética , Feminino , Loci Gênicos , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Macrófagos/metabolismo , Masculino , Camundongos , Testes de Mutagenicidade , Células Mieloides/metabolismo , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
2.
Oncogene ; 39(35): 5743-5755, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32704133

RESUMO

LIM and SH3 protein 1 (LASP1) is a metastasis-related protein reported to enhance tumour progression in colorectal cancer (CRC). However, the underlying mechanism is still elusive. As the major biological and pathological functions of LASP1 are accomplished by its LIM and SH3 domains via protein-protein interactions, a yeast two-hybrid system was employed to screen novel LASP1-interacting proteins. N-WASP, a member of the Wiskott-Aldrich syndrome protein (WASP) family, was screened and identified as a LASP1-interacting protein overexpressed in CRC tissues. N-WASP could stimulate the migration and invasion of CRC cells in vitro and increase the formation of subcutaneous tumours, mesenteric implanted tumours and hepatic metastatic tumours. N-WASP could interact with and activate the Arp2/3 complex to stimulate actin polymerization, thus changing the migratory and invasive capabilities of CRC cells. The interaction of LASP1 with N-WASP did not influence the expression of N-WASP but recovered the reduced actin polymerization induced by N-WASP silencing. High N-WASP expression was detected in most clinical colorectal samples, and it was positively correlated with the expression of LASP1 and ARP3, as well as the tumour budding and pattern of invasion, but negatively correlated with host lymphocytic response. Our study suggests a new mechanism for LASP1-mediated CRC metastasis determined by exploring LASP1-interacting proteins and identifies N-WASP as a potential therapeutic target for CRC.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Proteínas do Citoesqueleto/genética , Proteínas com Domínio LIM/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Neoplasias Colorretais/patologia , Humanos , Metástase Neoplásica , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
3.
BMC Med Genet ; 21(1): 124, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503528

RESUMO

BACKGROUND: The X-linked recessive primary immunodeficiency disease (PIDD) Wiskott-Aldrich syndrome (WAS) is identified by an extreme susceptibility to infections, eczema and thrombocytopenia with microplatelets. The syndrome, the result of mutations in the WAS gene which encodes the Wiskott-Aldrich protein (WASp), has wide clinical phenotype variation, ranging from classical WAS to X-linked thrombocytopaenia and X-linked neutropaenia. In many cases, the diagnosis of WAS in first affected males is delayed, because patients may not present with the classic signs and symptoms, which may intersect with other thrombocytopenia causes. CASE PRESENTATION: Here, we describe a three-year-old HIV negative boy presenting with recurrent infections, skin rashes, features of autoimmunity and atopy. However, platelets were initially reported as normal in numbers and morphology as were baseline immune investigations. An older male sibling had died in infancy from suspected immunodeficiency. Uncertainty of diagnosis and suspected severe PIDD prompted urgent further molecular investigation. Whole exome sequencing identified c. 397 G > A as a novel hemizygous missense mutation located in exon 4 of WAS. CONCLUSION: With definitive molecular diagnosis, we could target treatment and offer genetic counselling and prenatal diagnostic testing to the family. The identification of novel variants is important to confirm phenotype variations of a syndrome.


Assuntos
Mutação/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Sequência de Aminoácidos , Sequência de Bases , Feminino , Humanos , Lactente , Masculino , Volume Plaquetário Médio , Linhagem , África do Sul , Síndrome de Wiskott-Aldrich/sangue , Proteína da Síndrome de Wiskott-Aldrich/química
4.
EMBO J ; 39(5): e102783, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31894880

RESUMO

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact.


Assuntos
Células Apresentadoras de Antígenos/metabolismo , Sinapses Imunológicas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Animais , Células Apresentadoras de Antígenos/imunologia , Movimento Celular , Citoesqueleto/metabolismo , Humanos , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/imunologia , Linfócitos T/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética
5.
FASEB J ; 34(2): 3305-3317, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31916311

RESUMO

Pulmonary edema associated with increased vascular permeability is a severe complication of Pseudomonas (P.) aeruginosa-induced acute lung injury. The mechanisms underlying P aeruginosa-induced vascular permeability are not well understood. In the present study, we investigated the role of neuronal Wiskott Aldrich syndrome protein (N-WASP) in modulating P aeruginosa-induced vascular permeability. Using lung microvascular endothelial and alveolar epithelial cells, we demonstrated that N-WASP downregulation attenuated P aeruginosa-induced actin stress fiber formation and prevented paracellular permeability. P aeruginosa-induced dissociation between VE-cadherin and ß-catenin, but increased association between N-WASP and VE-cadherin, suggesting a role for N-WASP in promoting P aeruginosa-induced adherens junction rupture. P aeruginosa increased N-WASP-Y256 phosphorylation, which required the activation of Rho GTPase and focal adhesion kinase. Increased N-WASP-Y256 phosphorylation promotes N-WASP and integrin αVß6 association as well as TGF-ß-mediated permeability across alveolar epithelial cells. Inhibition of N-WASP-Y256 phosphorylation by N-WASP-Y256F overexpression blocked N-WASP effects in P aeruginosa-induced actin stress fiber formation and increased paracellular permeability. In vivo, N-WASP knockdown attenuated the development of pulmonary edema and improved survival in a mouse model of P aeruginosa pneumonia. Together, our data demonstrate that N-WASP plays an essential role in P aeruginosa-induced vascular permeability and pulmonary edema through the modulation of actin cytoskeleton dynamics.


Assuntos
Citoesqueleto de Actina/metabolismo , Permeabilidade Capilar , Pulmão/metabolismo , Pneumonia/metabolismo , Infecções por Pseudomonas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Junções Aderentes/metabolismo , Animais , Antígenos de Neoplasias/metabolismo , Caderinas/metabolismo , Células Cultivadas , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Integrinas/metabolismo , Pulmão/microbiologia , Camundongos , Pseudomonas aeruginosa/patogenicidade , Ratos , Fator de Crescimento Transformador beta/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , beta Catenina/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo
6.
Zhonghua Er Ke Za Zhi ; 57(8): 631-635, 2019 Aug 02.
Artigo em Chinês | MEDLINE | ID: mdl-31352750

RESUMO

Objective: To investigate the clinical and genotypic manifestations of X-linked neutropenia caused by gain-of-function mutation in WAS gene. Methods: The clinical history of two patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene in Shenzhen Children's Hospital were analyzed."X-linked neutropenia" and "WAS mutation" were used as key words to search related literatures published from January 2000 to December 2018 in CNKI,Wanfang, and Pubmed databases. Results: The first case was male,1 year old, admitted for 1 year of neutropenia combined with 5 days of cough and 3 days of fever. Persistent neutropenia (0.1×10(9)-0.3×10(9)/L) was reported before admission and during hospitalization (0.4×10(9)-0.5×10(9)/L). The patient was treated with Ciprofloxacin, cefoperazone sulbactam and Vancomycin,and relieved from fever after 4 weeks of hospitalization,yet the neutropenia (0.1×10(9)-0.6×10(9)/L) continued after discharge. Variant in WAS gene (c.T869C (p.I290T) ) was identified, and the percentage of WAS protein on lymphocyte was 97.7%. The second case was male, 42 days old,admitted for fever and neutropenia (0.5×10(9)/L). Similarly,he relieved from fever after 4 weeks of treatment with amoxicillin sulbactam,vancomysin,meropenem,rifampin and isoniacid,yet was discharged with continued neutropenia. Variant in WAS gene (c.T881C (p.I294T)) was identified and the percentage of WAS protein on lymphocyte was 92%. Published literature reported four variants,including I290T, L270P, S272P and I294T, as the pathogenic mutation of X-linked neutropenia in 18 patients from five families. Neutropenia (0.1×10(9)-1.0×10(9)/L) were reported in 15 patients,while normal neutrophil number was found in the rest. Recurrent infection,mainly pneumonia and otitis media,was the most common clinical manifestation. Conclusions: Neutropenia is the prominent presentation in the patients with X-linked neutropenia caused by gain-of-function mutation in WAS gene, but it unnecessarily correlates with the clinical severity in terms of infection. Gene sequencing should be considered for the male patients with persistent neutropenia.


Assuntos
Cromossomos Humanos X , Mutação com Ganho de Função/genética , Neutropenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Febre/etiologia , Humanos , Lactente , Masculino , Mutação
7.
Zhonghua Er Ke Za Zhi ; 57(6): 429-433, 2019 Jun 02.
Artigo em Chinês | MEDLINE | ID: mdl-31216799

RESUMO

Objective: To explore the clinical value of genetic screening for early identification of WAS gene-related disorders in newborns. Methods: This was a retrospective study. Neonatal Genome Project from Children's Hospital of Fudan University collected 5 800 high-risk newborns in the neonatal intensive care unit to study the patients' genetic causes using high-throughput sequencing from January 2016 to December 2017. Eleven newborns (all were boys) with pathogenic or likely pathogenic variants in WAS gene were enrolled. Data of clinical characteristics,gene variants and genotype-phenotype correlation were collected and summarized. Results: Eleven patients included 5 cases with Wiskott-Aldrich syndrome (WAS) and 6 cases with X-linked thrombocytopenia (XLT).Two patients with WAS developed clinical manifestations in the early neonatal period,and 3 patients in 5-8 weeks after birth. Three neonates with XLT were hospitalized for other diseases in the first place.Their platelet count was found to be reduced after admission to hospital, and diagnosis was made after genetic testing. Eleven pathogenic or likely pathogenic variants in WAS gene were identified. Among them, 7 were first reported in this study, including 2 frame shift variants c.138delG and c.388_390del, 4 splicing variants c.1453+1G>A,c.734+1G>C,c.135G>A and c.1453+3G>C, and 1 missense variant c.1118C>T. The other 4 reported variants were c.777+1G>A,c.107_108delTT, c.436delC and c.1509_*3delAGTG. Conclusions: The clinical features of WAS gene-related disorders in neonatal period lack specificity. Genetic screening in newborns plays an important role in the early diagnosis of diseases and provides providing evidence for the early intervention.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Testes Genéticos/métodos , Trombocitopenia/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/diagnóstico , Criança , Análise Mutacional de DNA , Diagnóstico Precoce , Humanos , Recém-Nascido , Masculino , Mutação , Estudos Retrospectivos , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética
8.
Cell Immunol ; 341: 103919, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31047647

RESUMO

Wiskott-Aldrich syndrome (WAS) is a form of primary immunodeficiency (PIDs) resulting from mutations of the gene that encodes Wiskott-Aldrich syndrome protein (WASp). WASp is the first identified and most widely studied protein belonging to the actin nucleation-promoting factor family and plays significant role in integrating and transforming signals from critical receptors on the cell surface to actin remodeling. WASp functions in immune defense and homeostasis through the regulation of actin cytoskeleton-dependent cellular processes as well as processes uncoupled with actin polymerization like nuclear transcription programs. In this article, we review the mechanisms of WASp activation through an understanding of its structure. We further discuss the role of WASp in adaptive immunity, paying special attention to some recent findings on the crucial role of WASp in the formation of immunological synapse, the regulation of T follicular helper (Tfh) cells and in the prevention of autoimmunity.


Assuntos
Citoesqueleto de Actina/imunologia , Linfócitos B/imunologia , Homeostase/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Proteína da Síndrome de Wiskott-Aldrich/imunologia , Síndrome de Wiskott-Aldrich/imunologia , Citoesqueleto de Actina/genética , Imunidade Adaptativa , Animais , Autoimunidade/genética , Linfócitos B/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Imunidade Inata , Sinapses Imunológicas/genética , Camundongos , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/patologia , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética
9.
Cell Immunol ; 338: 43-50, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30981413

RESUMO

Wiskott-Aldrich syndrome (WAS) patients are characterized by immunodeficiency and viral infections. T cells derived from WAS patients and WAS protein (WASP)-deficient mice have various defects. However, whether WASP plays a role in immune control of cytomegalovirus (CMV) infection remains unclear. We analyzed the distribution of CD8+ T subsets and the pathological damage to various organs and tissues in MCMV infected Was knockout (KO) mice. A relatively high number of MCMV-specific cytotoxic T cells (CTLs) were observed in the spleen of Was KO mice. In MCMV infected Was KO mice, the late differentiated CD8+ T subset (CD27-CD28-) decreased in lungs, compared with those in the spleen and peripheral blood. Additionally, we found that the most severe pathological lesions occurred in the lungs, the main target organ of MCMV infection. By stimulating the spleen-derived CD8+ T lymphocytes of Was KO mice, we found that IL-2 and granzyme B production declined compared with that in wild- type mice. Moreover, the number of apoptotic CD8+ T cells increased in Was KO mice compared with the number in wild-type mice. Therefore, our results demonstrate that WASP may be involved in regulating cytotoxic function and apoptosis in CD8+ T cells following MCMV infection, which is supported by the distribution and memory compartment of MCMV-specific T cells in MCMV infected WAS mice.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por Herpesviridae/imunologia , Pulmão/patologia , Muromegalovirus/fisiologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Animais , Apoptose , Células Cultivadas , Citotoxicidade Imunológica , Modelos Animais de Doenças , Feminino , Granzimas/metabolismo , Humanos , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Knockout , Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/genética
10.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
11.
Zhongguo Shi Yan Xue Ye Xue Za Zhi ; 27(1): 246-252, 2019 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-30738478

RESUMO

OBJECTIVE: To investigate the gene mutation of patients with WAS gene defect and its correlation with clinical manifestations. METHODS: Thirty-one patients consulted in Children's Hospital of Soochow University from January 2013 to February 2018 were enrolled in this study. The hot pot mutations of WAS gene in 31 patients were detected and related clinical phenotypes were analyzed retrospectively. RESULTS: All patients were male. The median onset age was 1 month (range, 0-83 months). Nine mutants were reported as novel mutations among 25 mutants detected in 31 patients, including c.1234_1235dupCC, c.1093-1097delG, c.28-30dupC, c.436G>T, c.273 + 10_273 + 11dupCC, c.995_996insG, c.1010T>A, c.332_333delCC and c.683C>T mutations. There were 25 cases of classic WAS which mutations included missense mutation, deletion mutation, insertion mutation, splicing mutation and nonsense mutation, 2 cases of X-linked thrombocytopenia (XLT) were induced by missense mutation, 1 case of intermittent X-linked thrombocytopenia (IXLT) was induced by splicing mutation, 2 cases of X-linked pancytopenia were induced by missense mutation. Intravenous immunoglobulin (IVIG) and glucocorticoid therapy in IXLT patient was effective, and remission could be sustained, platelets could be increased in the short-term in treated XLT patients, but only a small part of classic WAS patients(8.0%) showed transient response to it, the IVIG and glucocorticoid therapy did not improve the status of platelet in XLP patients. Immune laboratory examination showed that CD3+ was decreased in 60.0% patients, CD19+ was decreased in 12.0% patients, and CD56+CD16+ in 4 patients was decreased, accounting for 16.0%. Out of 24 patients, 22 patients were alive after treated with hematopoietic stem cell transplantation (HSCT), 4 patients who were not given HSCT died of brain bleeding and severe infection, 1 patient diagnosed as IXLT got remission and survived. CONCLUSION: WAS gene defect is an important basis for the diagnosis of WAS and related diseases. IVIG plus glucocorticoid therapy is less effective for fewer patients, the HSCT is an effective treatment for WAS.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X , Trombocitopenia , Proteína da Síndrome de Wiskott-Aldrich/genética , Humanos , Masculino , Mutação , Fenótipo , Estudos Retrospectivos
12.
J Cell Biol ; 218(4): 1138-1147, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30659101

RESUMO

The actin cytoskeleton generates forces on membranes for a wide range of cellular and subcellular morphogenic events, from cell migration to cytokinesis and membrane trafficking. For each of these processes, filamentous actin (F-actin) interacts with membranes and exerts force through its assembly, its associated myosin motors, or both. These two modes of force generation are well studied in isolation, but how they are coordinated in cells is mysterious. During clathrin-mediated endocytosis, F-actin assembly initiated by the Arp2/3 complex and several proteins that compose the WASP/myosin complex generates the force necessary to deform the plasma membrane into a pit. Here we present evidence that type I myosin is the key membrane anchor for endocytic actin assembly factors in budding yeast. By mooring actin assembly factors to the plasma membrane, this myosin organizes endocytic actin networks and couples actin-generated forces to the plasma membrane to drive invagination and scission. Through this unexpected mechanism, myosin facilitates force generation independent of its motor activity.


Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Membrana Celular/metabolismo , Clatrina/metabolismo , Endocitose , Miosina Tipo I/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Sítios de Ligação , Membrana Celular/genética , Regulação Fúngica da Expressão Gênica , Mutação , Miosina Tipo I/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
13.
Clin Lab ; 64(11)2018 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-30549999

RESUMO

BACKGROUND: X-linked thrombocytopenia (XLT) is a milder form of Wiskott-Aldrich syndrome (WAS), characterized predominantly by thrombocytopenia with small-sized platelets. Mutations in the WAS gene are responsible for the disease. We herein detected a new mutation in the WAS gene responsible for XLT in a 3-generation Chinese pedigree. METHODS: Peripheral blood samples were collected from 7 members in the family. WAS gene was amplified from genomic DNA isolated from leucocytes, and then direct sequencing was performed. RESULTS: Three male members of this family (the proband, his younger brother and maternal uncle) had thrombocytopenia and decreased mean platelet volume. A homozygous mutation (T>C) was found at nucleotide position 319 in exon 3, causing the amino acid Tyr (T) to be abnormally changed to His (H) at position 107. Two female members (the proband's mother and grandmother) were carriers of the mutation. CONCLUSIONS: XLT is easy to misdiagnose as immune thrombocytopenic purpura (ITP). The diagnosis of XLT should be considered in any male with congenital microthrombocytopenia or early onset of microthrombocytope-nia (< 7 fL). This article adds to the growing number of known mutations associated with XLT.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação de Sentido Incorreto , Trombocitopenia/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Adolescente , Grupo com Ancestrais do Continente Asiático/genética , Sequência de Bases , China , Análise Mutacional de DNA , Éxons/genética , Feminino , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/etnologia , Humanos , Masculino , Volume Plaquetário Médio , Linhagem , Trombocitopenia/sangue , Trombocitopenia/etnologia , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/etnologia
14.
Front Immunol ; 9: 2475, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30410494

RESUMO

Inflammatory bowel disease (IBD) is a chronic inflammatory condition caused by an aberrant immune response to microbial components of the gastrointestinal tract. Plasmacytoid dendritic cells (pDCs) are innate immune cells specialized in the production of type I interferons and were recently implicated in the pathogenesis of autoimmune disorders such as lupus and scleroderma. While pDCs were shown to infiltrate intestinal mucosa of IBD patients and proposed to participate in intestinal inflammation, their net contribution to the disease remains unclear. We addressed this question by targeting the pDC-specific transcription factor TCF4 (E2-2) in experimental IBD caused by deficiency of Wiskott-Aldrich syndrome protein (WASP) or of interleukin-10 (IL-10). Monoallelic Tcf4 deletion, which was previously shown to abrogate experimental lupus, did not affect autoimmunity manifestations or colitis in WASP-deficient animals. Furthermore, conditional biallelic Tcf4 targeting resulted in a near-complete pDC ablation, yet had no effect on the development of colitis in IL-10-deficient mice. Our results suggest that, in contrast to other inflammatory and autoimmune diseases, pDCs do not play a major role in the pathogenesis of intestinal inflammation during IBD.


Assuntos
Colite/imunologia , Células Dendríticas/imunologia , Doenças Inflamatórias Intestinais/imunologia , Nefrite Lúpica/imunologia , Fator de Transcrição 4/metabolismo , Animais , Colite/genética , Humanos , Imunidade Inata , Interleucina-10/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fator de Transcrição 4/genética , Proteína da Síndrome de Wiskott-Aldrich/genética
15.
JCI Insight ; 3(21)2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30385722

RESUMO

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21-null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-ß1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-ß1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-ß1 secretion, was identified as a direct target of miR-21. miR-21-null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-ß1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors.


Assuntos
Matriz Extracelular/efeitos dos fármacos , Fibrose/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/farmacologia , Idoso , Idoso de 80 Anos ou mais , Animais , Plaquetas/efeitos dos fármacos , Plaquetas/metabolismo , Ensaios Clínicos como Assunto , Matriz Extracelular/genética , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrose/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL/genética , MicroRNAs/genética , Pessoa de Meia-Idade , Miocárdio/patologia , Estudos Prospectivos , Proteômica/métodos , RNA não Traduzido/genética , Fator de Crescimento Transformador beta1/genética , Proteína da Síndrome de Wiskott-Aldrich/efeitos dos fármacos , Proteína da Síndrome de Wiskott-Aldrich/genética
17.
J Biol Chem ; 293(39): 15136-15151, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30104412

RESUMO

Wiskott-Aldrich syndrome protein (WASP) activates the actin-related protein 2/3 homolog (Arp2/3) complex and regulates actin polymerization in a physiological setting. Cell division cycle 42 (Cdc42) is a key activator of WASP, which binds Cdc42 through a Cdc42/Rac-interactive binding (CRIB)-containing region that defines a subset of Cdc42 effectors. Here, using site-directed mutagenesis and binding affinity determination and kinetic assays, we report the results of an investigation into the energetic contributions of individual WASP residues to both the Cdc42-WASP binding interface and the kinetics of complex formation. Our results support the previously proposed dock-and-coalesce binding mechanism, initiated by electrostatic steering driven by WASP's basic region and followed by a coalescence phase likely driven by the conserved CRIB motif. The WASP basic region, however, appears also to play a role in the final complex, as its mutation affected both on- and off-rates, suggesting a more comprehensive physiological role for this region centered on the C-terminal triad of positive residues. These results highlight the expanding roles of the basic region in WASP and other CRIB-containing effector proteins in regulating complex cellular processes and coordinating multiple input signals. The data presented improve our understanding of the Cdc42-WASP interface and also add to the body of information available for Cdc42-effector complex formation, therapeutic targeting of which has promise for Ras-driven cancers. Our findings suggest that combining high-affinity peptide-binding sequences with short electrostatic steering sequences could increase the efficacy of peptidomimetic candidates designed to interfere with Cdc42 signaling in cancer.


Assuntos
Neoplasias/genética , Proteína da Síndrome de Wiskott-Aldrich/química , Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/química , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Humanos , Cinética , Neoplasias/química , Neoplasias/patologia , Ligação Proteica , Sequências Reguladoras de Ácido Nucleico/genética , Transdução de Sinais , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas ras/química , Proteínas ras/genética
18.
J Clin Invest ; 128(9): 4115-4131, 2018 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-30124469

RESUMO

Congenital neutropenia is characterized by low absolute neutrophil numbers in blood, leading to recurrent bacterial infections, and patients often require life-long granulocyte CSF (G-CSF) support. X-linked neutropenia (XLN) is caused by gain-of-function mutations in the actin regulator Wiskott-Aldrich syndrome protein (WASp). To understand the pathophysiology in XLN and the role of WASp in neutrophils, we here examined XLN patients and 2 XLN mouse models. XLN patients had reduced myelopoiesis and extremely low blood neutrophil number. However, their neutrophils had a hyperactive phenotype and were present in normal numbers in XLN patient saliva. Murine XLN neutrophils were hyperactivated, with increased actin dynamics and migration into tissues. We provide molecular evidence that the hyperactivity of XLN neutrophils is caused by WASp in a constitutively open conformation due to contingent phosphorylation of the critical tyrosine-293 and plasma membrane localization. This renders WASp activity less dependent on regulation by PI3K. Our data show that the amplitude of WASp activity inside a cell could be enhanced by cell-surface receptor signaling even in the context in which WASp is already in an active conformation. Moreover, these data categorize XLN as an atypical congenital neutropenia in which constitutive activation of WASp in tissue neutrophils compensates for reduced myelopoiesis.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/metabolismo , Neutropenia/genética , Neutropenia/metabolismo , Neutrófilos/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Animais , Síndrome Congênita de Insuficiência da Medula Óssea , Feminino , Mutação com Ganho de Função , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neutropenia/congênito , Neutrófilos/ultraestrutura , Fagocitose , Fosforilação , Conformação Proteica , Proteína da Síndrome de Wiskott-Aldrich/química
19.
BMJ Case Rep ; 20182018 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-29991546

RESUMO

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder, described as a clinical triad of microthrombocytopenia, eczema and recurrent infections. Different mutations in WAS gene have been identified, resulting in various phenotypes and a broad range of disease severity, ranging from classic WAS to X-linked thrombocytopenia and X-linked neutropenia. WAS in some cases can be fatal without haematopoietic stem cell transplantation early in life. In this particular case, we present a novel mutation with a unique presentation. An 18-year-old man incidentally found to have macrothrombocytopenia and neutropenia at 16 years of age later found to be hemizygous for c. 869T>C (p.Ile290Thr) mutation in WAS gene. The late presentation, absence of other manifestations of WAS and presence of macrothrombocytopenia, rather than microthrombocytopenia, which is usually a characteristic finding in WAS, misled the initial diagnosis. On review of literature, this mutation has not been reported as causing WAS.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Mutação , Neutropenia/genética , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/genética , Adolescente , Humanos , Masculino , Síndrome de Wiskott-Aldrich/complicações , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
20.
BMC Med Genet ; 19(1): 123, 2018 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-30029636

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is an X-linked recessive immunodeficiency due to mutations in Wiskott-Aldrich syndrome (WAS) gene. WAS gene is encoded for a multifunctional protein with key roles in actin polymerization, signaling pathways, and cytoskeletal rearrangement. Therefore, the impaired protein or its absence cause phenotypic spectrum of the disease. Since identification of novel mutations in WAS gene can help uncover the exact pathogenesis of Wiskott-Aldrich syndrome, the purpose of this study was to investigate disease causing-mutation in an Iranian male infant suspicious of this disorder. CASE PRESENTATION: The patient had persistent thrombocytopenia from birth, sepsis, and recurrent gastrointestinal bleeding suggestive of both Wiskott-Aldrich syndrome and chronic colitis in favor of inflammatory bowel disease (IBD). To find mutated gene in the proband, whole exome sequencing was performed for the patient and its data showed a novel, private, hemizygous splice site mutation in WAS gene (c.360 + 1G > C). CONCLUSIONS: Our study found a novel, splice-site mutation in WAS gene and help consider the genetic counselling more precisely for families with clinical phenotypes of both Wiskott-Aldrich syndrome and inflammatory bowel disease and may suggest linked pathways between these two diseases.


Assuntos
Colite/genética , Mutação/genética , Sítios de Splice de RNA/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Éxons/genética , Humanos , Lactente , Doenças Inflamatórias Intestinais/genética , Irã (Geográfico) , Masculino , Proteínas/genética , Trombocitopenia/genética
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