Spectrum of WAS gene mutations in Vietnamese patients with Wiskott-Aldrich syndrome.

BACKGROUND: WAS gene mutational analysis is crucial to establish a definite diagnosis of Wiskott-Aldrich syndrome (WAS). Data on the genetic background of WAS in Vietnamese patients have not been reported. METHODS: We recruited 97 male, unrelated patients with WAS and analyzed WAS gene mutation using Sanger sequencing technology. RESULTS: We identified 36 distinct hemizygous pathogenic mutations, with 17 novel variants, from 38 patients in the entire cohort (39.2%). The mutational spectrum included 14 missense, 12 indel, five nonsense, four splicing, and one non-stop mutations. Most mutations appear only once, with the exception of c.37C>T (p.R13X) and c.374G>A (p.G125E) each of which occurs twice in unrelated patients. CONCLUSION: Our data enrich the mutational spectrum of the WAS gene and are crucial for understanding the genetic background of WAS and for supporting genetic counseling.

Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

Wiskott-Aldrich syndrome: a study of 577 patients defines the genotype as a biomarker for disease severity and survival.

ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

Wiskott Aldrich syndrome protein (WASp)-deficient Th1 cells promote R-loop-driven transcriptional insufficiency and transcription-coupled nucleotide excision repair factor (TC-NER)-driven genome-instability in the pathogenesis of T cell acute lymphoblastic leukemia.

BACKGROUND: T-ALL is an aggressive hematological tumor that develops as the result of a multi-step oncogenic process which causes expansion of hematopoietic progenitors that are primed for T cell development to undergo malignant transformation and growth. Even though first-line therapy has a significant response rate, 40% of adult patients and 20% of pediatric patients will relapse. Therefore, there is an unmet need for treatment for relapsed/refractory T-ALL to develop potential targeted therapies. METHODS: Pediatric T-ALL patient derived T cells were grown under either nonskewingTh0 or Th1-skewing conditions to further process for ChIP-qPCR, RDIP-qPCR and other RT-PCR assays. Endogenous WASp was knocked out using CRISPR-Cas9 and was confirmed using flow cytometry and western blotting. LC-MS/MS was performed to find out proteomic dataset of WASp-interactors generated from Th1-skewed, human primary Th-cells. DNA-damage was assessed by immunofluorescence confocal-imaging and single-cell gel electrophoresis (comet assay). Overexpression of RNaseH1 was also done to restore normal Th1-transcription in WASp-deficient Th1-skewed cells. RESULTS: We discovered that nuclear-WASp is required for suppressing R-loop production (RNA/DNA-hybrids) at Th1-network genes by ribonucleaseH2 (RNH2) and topoisomerase1. Nuclear-WASp is associated with the factors involved in preventing and dissolving R-loops in Th1 cells. In nuclear- WASp-reduced malignant Th1-cells, R-loops accumulate in vivo and are processed into DNA-breaks by transcription-coupled-nucleotide-excision repair (TC-NER). Several epigenetic modifications were also found to be involved at Th1 gene locus which are responsible for active/repressive marks of particular genes. By demonstrating WASp as a physiologic regulator of programmed versus unprogrammed R-loops, we suggest that the transcriptional role of WASp in vivo extends also to prevent transcription-linked DNA damage during malignancy and through modification of epigenetic dysregulations. CONCLUSION: Our findings present a provocative possibility of resetting R-loops as a therapeutic intervention to correct both immune deficiency and malignancy in T-cell acute lymphoblastic leukemia patients and a novel role of WASp in the epigenetic regulation of T helper cell differentiation in T-ALL patients, anticipating WASp's requirement for the suppression of T-ALL progression.

Controlled WASp activity regulates the proliferative response for Treg cell differentiation in the thymus.

The Wiskott-Aldrich syndrome protein (WASp) regulates actin cytoskeletal dynamics and function of hematopoietic cells. Mutations in the WAS gene lead to two different syndromes; Wiskott-Aldrich syndrome (WAS) caused by loss-of-function mutations, and X-linked neutropenia (XLN) caused by gain-of-function mutations. We previously showed that WASp-deficient mice have a decreased number of regulatory T (Treg) cells in the thymus and the periphery. We here evaluated the impact of WASp mutations on Treg cells in the thymus of WAS and XLN mouse models. Using in vitro Treg differentiation assays, WAS CD4 single-positive thymocytes have decreased differentiation to Treg cells, despite normal early signaling upon IL-2 and TGF-ß stimulation. They failed to proliferate and express CD25 at high levels, leading to poor survival and a lower number of Foxp3+ Treg cells. Conversely, XLN CD4 single-positive thymocytes efficiently differentiate into Foxp3+ Treg cells following a high proliferative response to IL-2 and TGF-ß, associated with high CD25 expression when compared with WT cells. Altogether, these results show that specific mutations of WASp affect Treg cell development differently, demonstrating a critical role of WASp activity in supporting Treg cell development and expansion.

Role of Wiskott Aldrich syndrome protein in haematological malignancies: genetics, molecular mechanisms and therapeutic strategies.

As patients continue to suffer from lymphoproliferative and myeloproliferative diseases known as haematopoietic malignancies can affect the bone marrow, blood, lymph nodes, and lymphatic and non-lymphatic organs. Despite advances in the current treatment, there is still a significant challenge for physicians to improve the therapy of HMs. WASp is an important regulator of actin polymerization and the involvement of WASp in transcription is thought to be linked to the DNA damage response and repair. In some studies, severe immunodeficiency and lymphoid malignancy are caused by WASp mutations or the absence of WASp and these mutations in WAS can alter the function and/or expression of the intracellular protein. Loss-of-function and Gain-of-function mutations in WASp have an impact on cancer malignancies' incidence and onset. Recent studies suggest that depending on the clinical or experimental situation, WASPs and WAVEs can operate as a suppressor or enhancers for cancer malignancy. These dual functions of WASPs and WAVEs in cancer likely arose from their multifaceted role in cells that could be targeted for anticancer drug development. The significant role and their association of WASp in Chronic myeloid leukaemia, Juvenile myelomonocytic leukaemia and T-cell lymphoma is discussed. In this review, we described the structure and function of WASp and its family mechanism, analysing major regulatory effectors and summarising the clinical relevance and drugs that specifically target WASp in disease treatment in various hematopoietic malignancies by different approaches.

Association of Wiskott-Aldrich syndrome protein (WASp) in epigenetic regulation of B cell differentiation in non-small-cell lung cancer (NSCLC).

Non-small-cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancer which is the deadliest type of cancer for both men and women. Previous studies already showed that cell-intrinsic loss of WASp causes B cell tolerance and WASp deficiency in T helper (TH) cells is linked to negative effects on cytokine gene transcription necessary for TH1 differentiation. In the current study, we investigated the molecular mechanisms involved in WASp-mediated epigenetic regulation of B cell differentiation during NSCLC. Our ChIP-qPCR data suggest the less percentage enrichment of the B cell differentiating factors (Ikaros, Pax5, PU.1, BATF) and WASp across the WAS gene in the B cells of NSCLC patients in comparison with normal healthy donors and overexpression of WASp showed the reverse effects. WASp-depleted B cells while co-culturing with respective PBMCs isolated from normal healthy donors and NSCLC patients, we observed upregulation of TH2-, TH17-, and Treg-specific cytokines (IL4, ILI7A, IL10) & transcription factors (GATA3, RORC, FOXP3) and downregulation of TH1-specific cytokine (IFNγ) & transcription factor (TBX21). Our study showed that the overexpression of WASp resulted into upregulation of B cell differentiating factors, tumor suppressor protein (p53), histone methylation marker (H3K4me3) with concomitant downregulation of tumor-promoting factors (Notch 1, ß-Catenin, DNAPKcs) and histone deacetylation marker (HDAC2) and increase in percentage cytotoxicity of NSCLC-specific cells (A549). Successful overexpression of WASp not only helps in epigenetic regulation of B cell differentiation but also supports tumor suppression in NSCLC. Thus, WASp can be targeted for therapeutic intervention of NSCLC.

Use of positive pressure in preoperative and intraoperative of bariatric surgery and its effect on the time of extubation / Utilização da pressão positiva no pré e no intraoperatório de cirurgia bariátrica e seus efeitos sobre o tempo de extubação / Utilización de la presión positiva en el pre- y en el intraoperatorio de cirugía bariátrica y sus efectos sobre el tiempo de extubación

BACKGROUND AND OBJECTIVE: To investigate the influence of intraoperative and preoperative positive pressure in the time of extubation in patients undergoing bariatric surgery. METHOD: Randomized clinical trial, in which 40 individuals with a body mass index between 40 and 55 kg/m2, age between 25 and 55 years, nonsmokers, underwent bariatric surgery type Roux-en-Y gastric bypass by laparotomy and with normal preoperative pulmonary function were randomized into the following groups: G-pre (n = 10): individuals who received treatment with noninvasive positive pressure before surgery for 1 h; G-intra (n = 10): individuals who received positive end-expiratory pressure of 10 cm H2O throughout the surgical procedure; and G-control (n = 20): not received any preoperative or intraoperative intervention. Following were recorded: time between induction of anesthesia and extubation, between the end of anesthesia and extubation, duration of mechanical ventilation, and time between extubation and discharge from the post-anesthetic recovery. RESULTS: There was no statistical difference between groups. However, when applied to the Cohen coefficient, the use of positive end-expiratory pressure of 10 cm H2O during surgery showed a large effect on the time between the end of anesthesia and extubation. About this same time, the treatment performed preoperatively showed moderate effect. CONCLUSION: The use of positive end-expiratory pressure of 10 cm H2O in the intraoperative and positive pressure preoperatively, influenced the time of extubation of patients undergoing bariatric surgery. .

JUSTIFICATIVA E OBJETIVO: investigar a influência do uso da pressão positiva nas vias aéreas intraoperatória e pré-operatória no tempo de extubação de pacientes submetidos à cirurgia bariátrica. MÉTODO: Trata-se de ensaio clínico randomizado, no qual 40 indivíduos com índice de massa corporal entre 40 e 55 kg/m2, idade entre 25 e 55 anos, não tabagistas, submetidos à cirurgia bariátrica do tipo derivação gástrica em Y de Roux por laparotomia e com prova de função pulmonar pré-operatória dentro da normalidade foram randomizados nos seguintes grupos: G-pré (n = 10): indivíduos que receberam tratamento com pressão positiva não invasiva antes da cirurgia, durante uma hora, G-intra (n = 10): indivíduos que receberam Positive End-expiratory Pressure de 10 cm H2O durante todo o procedimento cirúrgico e G-controle (n = 20): não receberam qualquer tipo de intervenção pré ou intraoperatória. foram anotados os seguintes tempos: tempo decorrido entre a indução anestésica e a extubação, entre o término da anestesia e extubação, tempo de ventilação mecânica, e tempo entre a extubação e a alta da Recuperação Pós-Anestésica. RESULTADOS: Não houve diferença estatística entre os grupos, porém quando aplicado ao Coeficiente de Cohen, o uso da Positive End-expiratory Pressure de 10 cm H2O no intraoperatório mostrou um efeito grande sobre o tempo entre o término da anestesia e a extubação. Sobre este mesmo tempo, o tratamento realizado no pré-operatório apresentou efeito moderado. CONCLUSÃO: O uso da Positive End-expiratory Pressure de 10 cm H2O no intraoperatório e da pressão positiva no pré-operatório, pode influenciar o tempo de extubação de pacientes submetidos à cirurgia bariátrica. .

JUSTIFICACIÓN Y OBJETIVO: Investigar la influencia del uso de la presión positiva en las vías aéreas intraoperatoria y preoperatoria en el tiempo de extubación de pacientes sometidos a la cirugía bariátrica. MÉTODO: Se trata de un ensayo clínico aleatorizado, en el cual 40 individuos con IMC entre 40 y 55 kg/m2, edad entre 25 y 55 años, no fumadores, sometidos a cirugía bariátrica del tipo derivación gástrica en Y de Roux por laparotomía y con prueba de función pulmonar preoperatoria dentro de la normalidad fueron aleatorizados en los siguientes grupos: G-pre (n = 10): individuos que recibieron tratamiento con presión positiva no invasiva antes de la cirugía durante una hora; G-intra (n = 10): individuos que recibieron PEEP de 10 cm H2O durante todo el procedimiento quirúrgico y G-control (n = 20): no recibieron ningún tipo de intervención pre- o intraoperatoria. Fueron anotados los siguientes tiempos: tiempo trascurrido entre la inducción anestésica y la extubación, entre el fin de la anestesia y la extubación, tiempo de ventilación mecánica, y tiempo entre la extubación y el alta de la sala de recuperación postanestésica. RESULTADOS: No hubo diferencia estadística entre los grupos, sin embargo cuando se aplicó el coeficiente de Cohen, el uso de la PEEP de 10 cm H2O en el intraoperatorio mostró un efecto importante sobre el tiempo entre el término de la anestesia y la extubación. Sobre ese mismo tiempo, el tratamiento realizado en el preoperatorio presentó un efecto moderado. CONCLUSIÓN: El uso de la PEEP de 10 cm H2O en el intraoperatorio y de la presión positiva en el preoperatorio puede influir en el tiempo de extubación de pacientes sometidos a cirugía bariátrica. .

Phenotypic and Genotypic Correction of WASP Gene Mutation in Wiskott-Aldrich Syndrome by Unrelated Cord Blood Stem Cell Transplantation

We present two cases of Wiskott-Aldrich syndrome (WAS), in which nonsense mutations in the WASP gene were corrected phenotypically as well as genotypically by unrelated cord blood stem cell transplantation (CBSCT). Two male patients were diagnosed with WAS at the age of 5-month and 3-month and each received unrelated CBSCT at 16-month and 20-month of age, respectively. The infused cord blood (CB) units had 4/6 and 5/6 HLA matches and the infusion doses of total nucleated cells (TNC) and CD34+ cells were 6.24x10(7)/kg and 5.08x10(7)/kg for TNC and 1.33x10(5)/kg and 4.8x10(5)/kg for CD34+ cells, for UPN1 and UPN2, respectively. Complete donor cell chimerism was documented by variable number tandem repeat (VNTR) with neutrophil engraftment on days 31 and 13 and platelets on days 58 and 50, respectively. Immunologic reconstitution demonstrated that CBSCT resulted in consistent and stable T-, B-, and NK-cell development. Flow cytometric analysis for immunologic markers and sequence analysis of the WASP gene mutation revealed a normal pattern after CBSCT. These cases demonstrate that CBs can be an important source of stem cells for the phenotypical and genotypical correction of genetic diseases such as WAS.

A Familial Case of Wiskott-Aldrich Syndrome with a Hotspot Mutation in Exon 2 of the WAS Gene

The Wiskott-Aldrich syndrome (WAS) is a severe X-linked disorder characterized classically by thrombocytopenia, immunodeficiency, and eczema. The phenotype observed in this syndrome is caused by mutation in the WAS gene. Peripheral blood DNAs were isolated from an 18-month-old boy with WAS and his mother, maternal uncle, and maternal grandmother. Genetic analysis for the detection of a mutation of WAS gene was performed by polymerase chain reaction-single strand conformational polymorphism analysis (PCR-SSCP) and direct sequencing of the PCR product. In PCR-SSCP, the patient and his maternal uncle had an abnormal shift band, which was not found in normal controls, and his mother and maternal grandmother showed heterozygous bands. In direct sequencing analysis, the patient with WAS had CGC-->CAC point mutation in exon 2 that resulted in an amino acid change in codon 86 (Arg86His). The present study identified a gene mutation responsible for WAS at a mutation hotspot of the WAS gene in a Korean family.