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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
Zhonghua Yi Xue Yi Chuan Xue Za Zhi ; 35(2): 207-209, 2018 Apr 10.
Artigo em Chinês | MEDLINE | ID: mdl-29652993

RESUMO

OBJECTIVE: To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome. METHODS: Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members. RESULTS: A hemizygous mutation (c.257G>A) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF. CONCLUSION: The c.257G>A mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.


Assuntos
Mutação , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Fator Ativador de Células B/sangue , Pré-Escolar , Heterozigoto , Humanos , Masculino
8.
ACS Chem Biol ; 13(1): 100-109, 2018 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-29215267

RESUMO

Wiskott-Aldrich syndrome protein (WASp) is exclusively expressed in hematopoietic cells and responsible for actin-dependent processes, including cellular activation, migration, and invasiveness. The C-terminal domain of WASp-Interacting Protein (WIP) binds to WASp and regulates its activity by shielding it from degradation in a phosphorylation dependent manner as we previously demonstrated. Mutations in the WAS-encoding gene lead to the primary immunodeficiencies Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). Here, we shed a first structural light upon this function of WIP using nuclear magnetic resonance (NMR) and in vivo molecular imaging. Coexpression of fragments WASp(20-158) and WIP(442-492) allowed the purification and structural characterization of a natively folded complex, determined to form a characteristic pleckstrin homology domain with a mixed α/ß-fold and central two-winged ß-sheet. The WIP-derived peptide, unstructured in its free form, wraps around and interacts with WASp through short structural elements. Förster resonance energy transfer (FRET) and biochemical experiments demonstrated that, of these elements, WIP residues 454-456 are the major contributor to WASp affinity, and the previously overlooked residues 449-451 were found to have the largest effect upon WASp ubiquitylation and, presumably, degradation. Results obtained from this complementary combination of technologies link WIP-WASp affinity to protection from degradation. Our findings about the nature of WIP·WASp complex formation are relevant for ongoing efforts to understand hematopoietic cell behavior, paving the way for new therapeutic approaches to WAS and XLT.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/química , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Actinas/metabolismo , Sítios de Ligação , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/imunologia , Epitopos , Transferência Ressonante de Energia de Fluorescência , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Células Jurkat , Espectroscopia de Ressonância Magnética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Imagem Molecular/métodos , Complexos Multiproteicos , Mutação , Domínios Proteicos , Dobramento de Proteína , Ubiquitinação , Proteína da Síndrome de Wiskott-Aldrich/genética
10.
Pediatr Hematol Oncol ; 34(5): 286-291, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29200320

RESUMO

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is X-linked recessive disorder associated with microplatelet thrombocytopenia, eczema, infections, and an increased risk of autoimmunity and lymphoid neoplasia. The originally described features of WAS include susceptibility to infections, microthrombocytopenia, and eczema. AIM: In this case report, we present our experience about two cases diagnosed with a new mutation. METHODS: We report phenotypical and laboratory description of two cases with WAS. RESULTS: We, for the first time, detected a new hemizygote mutation of WAS gene (NM_000377.2 p.M393lfs*102 (c.1178dupT)) in two patients. The first case was an 11-month-old boy presenting with complaints of recurrent soft tissue infection, ear infection, anemia, and thrombocytopenia with a low platelet volume. The second case was a 2-month-old boy presenting with thrombocytopenia and a low platelet volume. Both cases were the first-degree relatives: they were cousins and their mothers were sisters. CONCLUSION: Herein, we report two cases of WAS and a new gene mutation which would disrupt the WAS protein function within the Polyproline (PPP) domain. This report adds to the growing number of mutations which cause complex clinical manifestations associated with WAS.


Assuntos
Hemizigoto , Mutação , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/genética , Humanos , Lactente , Masculino , Domínios Proteicos , Síndrome de Wiskott-Aldrich/patologia
11.
J Cell Biol ; 216(12): 4073-4090, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29150539

RESUMO

The antimicrobial defense activity of neutrophils partly depends on their ability to form neutrophil extracellular traps (NETs), but the underlying mechanism controlling NET formation remains unclear. We demonstrate that inhibiting cytoskeletal dynamics with pharmacological agents or by genetic manipulation prevents the degranulation of neutrophils and mitochondrial DNA release required for NET formation. Wiskott-Aldrich syndrome protein-deficient neutrophils are unable to polymerize actin and exhibit a block in both degranulation and DNA release. Similarly, neutrophils with a genetic defect in NADPH oxidase fail to induce either actin and tubulin polymerization or NET formation on activation. Moreover, neutrophils deficient in glutaredoxin 1 (Grx1), an enzyme required for deglutathionylation of actin and tubulin, are unable to polymerize either cytoskeletal network and fail to degranulate or release DNA. Collectively, cytoskeletal dynamics are achieved as a balance between reactive oxygen species-regulated effects on polymerization and glutathionylation on the one hand and the Grx1-mediated deglutathionylation that is required for NET formation on the other.


Assuntos
Citoesqueleto/imunologia , Armadilhas Extracelulares/imunologia , Glutationa/imunologia , Neutrófilos/imunologia , Espécies Reativas de Oxigênio/imunologia , Actinas/genética , Actinas/imunologia , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Citoesqueleto/ultraestrutura , DNA Mitocondrial/imunologia , DNA Mitocondrial/metabolismo , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/imunologia , Glutationa/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Proteínas de Homeodomínio/imunologia , Humanos , Camundongos , Camundongos Transgênicos , NADPH Oxidases/genética , NADPH Oxidases/imunologia , Neutrófilos/citologia , Neutrófilos/efeitos dos fármacos , Oxirredução , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Tubulina (Proteína)/genética , Tubulina (Proteína)/imunologia , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/imunologia
12.
Nat Commun ; 8(1): 1576, 2017 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-29146903

RESUMO

Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott-Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention.


Assuntos
Citoesqueleto de Actina/metabolismo , Autofagia/imunologia , Inflamassomos/imunologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Síndrome de Wiskott-Aldrich/imunologia , Animais , Autofagia/genética , Carga Bacteriana/imunologia , Linhagem Celular Tumoral , Células Dendríticas/imunologia , Escherichia coli Enteropatogênica/imunologia , Humanos , Imunidade Inata/imunologia , Interferon Tipo I/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Nigericina/farmacologia , Septinas/metabolismo , Shigella flexneri/imunologia , Células THP-1 , Receptor 4 Toll-Like/imunologia , Síndrome de Wiskott-Aldrich/metabolismo
13.
Cell Death Dis ; 8(10): e3114, 2017 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-29022901

RESUMO

Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the BCR-ABL1 tyrosine kinase (TK). The development of TK inhibitors (TKIs) revolutionized the treatment of CML patients. However, TKIs are not effective to those at advanced phases when amplified BCR-ABL1 levels and increased genomic instability lead to secondary oncogenic modifications. Wiskott-Aldrich syndrome protein (WASP) is an important regulator of signaling transduction in hematopoietic cells and was shown to be an endogenous inhibitor of the c-ABL TK. Here, we show that the expression of WASP decreases with the progression of CML, inversely correlates with the expression of BCR-ABL1 and is particularly low in blast crisis. Enforced expression of BCR-ABL1 negatively regulates the expression of WASP. Decreased expression of WASP is partially due to DNA methylation of the proximal WASP promoter. Importantly, lower levels of WASP in CML advanced phase patients correlate with poorer overall survival (OS) and is associated with TKI response. Interestingly, enforced expression of WASP in BCR-ABL1-positive K562 cells increases the susceptibility to apoptosis induced by TRAIL or chemotherapeutic drugs and negatively modulates BCR-ABL1-induced tumorigenesis in vitro and in vivo. Taken together, our data reveal a novel molecular mechanism that operates in BCR-ABL1-induced tumorigenesis that can be used to develop new strategies to help TKI-resistant, CML patients in blast crisis (BC).


Assuntos
Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Azacitidina/uso terapêutico , Carcinogênese/genética , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Resistencia a Medicamentos Antineoplásicos , Epigênese Genética , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Mesilato de Imatinib/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/mortalidade , Regiões Promotoras Genéticas/genética , Inibidores de Proteínas Quinases/uso terapêutico , Transdução de Sinais/fisiologia , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/genética
14.
MBio ; 8(5)2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28874467

RESUMO

Many fundamental biological discoveries have been made in Caenorhabditis elegans The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for Orsay virus infection, we designed a forward genetic screen that utilizes a virally induced green fluorescent protein (GFP) reporter. Following chemical mutagenesis, two Viro (virus induced reporter off) mutants that failed to express GFP were mapped to sid-3, a nonreceptor tyrosine kinase, and B0280.13 (renamed viro-2), an ortholog of human Wiskott-Aldrich syndrome protein (WASP). Both mutants yielded Orsay virus RNA levels comparable to that of the residual input virus, suggesting that they are not permissive for Orsay virus replication. In addition, we demonstrated that both genes affect an early prereplication stage of Orsay virus infection. Furthermore, it is known that the human ortholog of SID-3, activated CDC42-associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that VIRO-2 may be a substrate for SID-3 in C. elegans A targeted RNA interference (RNAi) knockdown screen further identified the C. elegans gene nck-1, which has a human ortholog that interacts with TNK2 and WASP, as required for Orsay virus infection. Thus, genetic screening in C. elegans identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway.IMPORTANCE Orsay virus is the only known virus capable of naturally infecting the model organism Caenorhabditis elegans, which shares many evolutionarily conserved genes with humans. We exploited the robust genetic tractability of C. elegans to identify three host genes, sid-3, viro-2, and nck-1, which are essential for Orsay virus infection. Mutant animals that lack these three genes are highly defective in viral replication. Strikingly, the human orthologs of these three genes, activated CDC42-associated kinase (TNK2), Wiskott-Aldrich syndrome protein (WASP), and noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) are part of a known signaling pathway in mammals. These results suggest that TNK2, WASP, and NCK1 may play important roles in mammalian virus infection.


Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/virologia , Evolução Molecular , Nodaviridae/fisiologia , Replicação Viral , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Proteínas de Fluorescência Verde/genética , Interações Hospedeiro-Patógeno , Mutagênese , Nodaviridae/genética , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Interferência de RNA , RNA Viral/genética , Viroses , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo
15.
Mol Med Rep ; 16(5): 6526-6531, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28901403

RESUMO

Wiskott­Aldrich syndrome (WAS) is a rare X­linked 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, WASP­interacting 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 month­old 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.


Assuntos
Deleção de Sequência/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Adulto , Códon sem Sentido/genética , Análise Mutacional de DNA/métodos , Éxons/genética , Feminino , Deleção de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Lactente , Masculino , Mutação/genética , Fenótipo , Trombocitopenia/genética , Síndrome de Wiskott-Aldrich/metabolismo
16.
Elife ; 62017 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-28813247

RESUMO

Actin-related protein 2/3 (Arp2/3) complex activation by nucleation promoting factors (NPFs) such as WASP, plays an important role in many actin-mediated cellular processes. In yeast, Arp2/3-mediated actin filament assembly drives endocytic membrane invagination and vesicle scission. Here we used genetics and quantitative live-cell imaging to probe the mechanisms that concentrate NPFs at endocytic sites, and to investigate how NPFs regulate actin assembly onset. Our results demonstrate that SH3 (Src homology 3) domain-PRM (proline-rich motif) interactions involving multivalent linker proteins play central roles in concentrating NPFs at endocytic sites. Quantitative imaging suggested that productive actin assembly initiation is tightly coupled to accumulation of threshold levels of WASP and WIP, but not to recruitment kinetics or release of autoinhibition. These studies provide evidence that WASP and WIP play central roles in establishment of a robust multivalent SH3 domain-PRM network in vivo, giving actin assembly onset at endocytic sites a switch-like behavior.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Microscopia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína da Síndrome de Wiskott-Aldrich/genética
17.
Integr Biol (Camb) ; 9(8): 695-708, 2017 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-28678266

RESUMO

Dendritic cell migration to the T-cell-rich areas of the lymph node is essential for their ability to initiate the adaptive immune response. While it has been shown that the actin cytoskeleton is required for normal DC migration, the role of many of the individual cytoskeletal molecules is poorly understood. In this study, we investigated the contribution of the Arp2/3 complex binding protein, haematopoietic lineage cell-specific protein 1 (HS1), to DC migration and force generation. We quantified the random migration of HS1-/- DCs on 2D micro-contact printed surfaces and found that in the absence of HS1, DCs have greatly reduced motility and speed. This same reduction in motility was recapitulated when adding Arp2/3 complex inhibitor to WT DCs or using DCs deficient in WASP, an activator of Arp2/3 complex-dependent actin polymerization. We further investigated the importance of HS1 by measuring the traction forces of HS1-/- DCs on micropost array detectors (mPADs). In HS1 deficient DCs, there was a significant reduction in force generation (3.96 ± 0.40 nN per cell) compared to WT DCs (13.76 ± 0.84 nN per cell). Interestingly, the forces generated in DCs lacking WASP were only slightly reduced compared to WT DCs. Taken together, these findings show that HS1 and Arp2/3 complex-mediated actin polymerization are essential for the most efficient DC random migration and force generation.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/fisiologia , Células Dendríticas/fisiologia , Fator Estimulador de Colônias de Granulócitos/fisiologia , Complexo 2-3 de Proteínas Relacionadas à Actina/antagonistas & inibidores , Actinas/metabolismo , Animais , Bioengenharia , Fenômenos Biofísicos , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , Células Dendríticas/imunologia , Fator Estimulador de Colônias de Granulócitos/deficiência , Fator Estimulador de Colônias de Granulócitos/genética , Indóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteína da Síndrome de Wiskott-Aldrich/deficiência , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/fisiologia
18.
BMC Pediatr ; 17(1): 151, 2017 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-28641574

RESUMO

BACKGROUND: Thrombocytopenia can occur in different circumstances during childhood and although immune thrombocytopenia is its most frequent cause, it is important to consider other conditions, especially when there is a persistent or recurrent low platelet count. We report two cases of intermittent thrombocytopenia, previously misdiagnosed as immune thrombocytopenia. CASES PRESENTATION: Both cases described were boys who presented with an intermittent pattern of thrombocytopenia, with a persistently low mean platelet volume. In both patients, peripheral blood smear revealed small platelets and flow cytometry showed low expression of Wiskott-Aldrich syndrome protein (WASP) in leucocytes. Molecular analysis of the first case identified a mutation in exon 2 of the gene coding for WASP, leading to a p.Thr45Met amino acid change and confirming the diagnosis of X-linked thrombocytopenia. In the second case, a novel missense mutation in exon 2 of the gene coding for WASP was detected, which resulted in a p.Pro58Leu amino acid change. CONCLUSION: These two rare presentations of thrombocytopenia highlight the importance of evaluating the peripheral blood smear in the presence of recurrent or persistent thrombocytopenia and show that failing to do so can lead to misdiagnoses. Since thrombocytopenia may be found in pediatric outpatient clinic, increased awareness among general pediatricians will help to improve the differential diagnosis of this condition.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/diagnóstico , Trombocitopenia/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética , Pré-Escolar , Erros de Diagnóstico , Doenças Genéticas Ligadas ao Cromossomo X/sangue , Doenças Genéticas Ligadas ao Cromossomo X/genética , Marcadores Genéticos , Humanos , Lactente , Masculino , Mutação , Contagem de Plaquetas , Trombocitopenia/sangue , Trombocitopenia/genética
19.
Allergy ; 72(12): 1916-1924, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-28600891

RESUMO

BACKGROUND: Food allergies are a growing health problem, and the development of therapies that prevent disease onset is limited by the lack of adjuvant-free experimental animal models. We compared allergic sensitization in patients with food allergy or Wiskott-Aldrich syndrome (WAS) and defined whether spontaneous disease in Was-/- mice recapitulates the pathology of a conventional disease model and/or human food allergy. METHODS: Comparative ImmunoCAP ISAC microarray was performed in patients with food allergy or WAS. Spontaneous food allergy in Was-/- mice was compared to an adjuvant-based model in wild-type mice (WT-OVA/alum). Intestinal and systemic anaphylaxis was assessed, and the role of the high-affinity IgE Fc receptor (FcεRI) in allergic sensitization was evaluated using Was-/- Fcer1a-/- mice. RESULTS: Polysensitization to food was detected in both WAS and food-allergic patients which was recapitulated in the Was-/- model. Oral administration of ovalbumin (OVA) in Was-/- mice induced low titers of OVA-specific IgE compared to the WT-OVA/alum model. Irrespectively, 79% of Was-/- mice developed allergic diarrhea following oral OVA challenge. Systemic anaphylaxis occurred in Was-/- mice (95%) with a mortality rate >50%. Spontaneous sensitization and intestinal allergy occurred independent of FcεRI expression on mast cells (MCs) and basophils. CONCLUSIONS: Was-/- mice provide a model of food allergy with the advantage of mimicking polysensitization and low food-antigen IgE titers as observed in humans with clinical food allergy. This model will facilitate studies on aberrant immune responses during spontaneous disease development. Our results imply that therapeutic targeting of the IgE/FcεRI activation cascade will not affect sensitization to food.


Assuntos
Hipersensibilidade Alimentar/etiologia , Hipersensibilidade Alimentar/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Receptores de IgE/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Adulto , Alérgenos/imunologia , Anafilaxia , Animais , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/diagnóstico , Expressão Gênica , Humanos , Imunização , Imunoglobulina E/imunologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Síndrome de Wiskott-Aldrich/diagnóstico , Síndrome de Wiskott-Aldrich/imunologia , Adulto Jovem
20.
J Cell Biol ; 216(6): 1673-1688, 2017 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-28473602

RESUMO

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled pseudopods that we call "α-motility." Mining genomic data reveals a clear trend: only organisms with both WASP and SCAR/WAVE-activators of branched actin assembly-make actin-filled pseudopods. Although SCAR has been shown to drive pseudopod formation, WASP's role in this process is controversial. We hypothesize that these genes collectively represent a genetic signature of α-motility because both are used for pseudopod formation. WASP depletion from human neutrophils confirms that both proteins are involved in explosive actin polymerization, pseudopod formation, and cell migration. WASP and WAVE also colocalize to dynamic signaling structures. Moreover, retention of WASP together with SCAR correctly predicts α-motility in disease-causing chytrid fungi, which we show crawl at >30 µm/min with actin-filled pseudopods. By focusing on one migration mode in many eukaryotes, we identify a genetic marker of pseudopod formation, the morphological feature of α-motility, providing evidence for a widely distributed mode of cell crawling with a single evolutionary origin.


Assuntos
Actinas/metabolismo , Movimento Celular , Quitridiomicetos/metabolismo , Evolução Molecular , Proteínas Fúngicas/metabolismo , Neutrófilos/metabolismo , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Quimiotaxia , Quitridiomicetos/genética , Biologia Computacional , Mineração de Dados , Bases de Dados Genéticas , Proteínas Fúngicas/genética , Genômica/métodos , Células HL-60 , Humanos , Microscopia de Fluorescência , Microscopia de Vídeo , Filogenia , Interferência de RNA , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteína da Síndrome de Wiskott-Aldrich/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética
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