FLI1 Induces Megakaryopoiesis Gene Expression Through WAS/WIP-Dependent and Independent Mechanisms; Implications for Wiskott-Aldrich Syndrome.

Wiskott-Aldrich Syndrome, WAS/WAVE, is a rare, X-linked immune-deficiency disease caused by mutations in the WAS gene, which together with its homolog, N-WASP, regulates actin cytoskeleton remodeling and cell motility. WAS patients suffer from microthrombocytopenia, characterized by a diminished number and size of platelets, though the underlying mechanism is not fully understood. Here, we identified FLI1 as a direct transcriptional regulator of WAS and its binding partner WIP. Depletion of either WAS or WIP in human erythroleukemic cells accelerated cell proliferation, suggesting tumor suppressor function of both genes in leukemia. Depletion of WAS/WIP also led to a significant reduction in the percentage of CD41 and CD61 positive cells, which mark committed megakaryocytes. RNAseq analysis revealed common changes in megakaryocytic gene expression following FLI1 or WASP knockdown. However, in contrast to FLI1, WASP depletion did not alter expression of late-stage platelet-inducing genes. N-WASP was not regulated by FLI1, yet its silencing also reduced the percentage of CD41+ and CD61+ megakaryocytes. Moreover, combined knockdown of WASP and N-WASP further suppressed megakaryocyte differentiation, indicating a major cooperation of these related genes in controlling megakaryocytic cell fate. However, unlike WASP/WIP, N-WASP loss suppressed leukemic cell proliferation. WASP, WIP and N-WASP depletion led to induction of FLI1 expression, mediated by GATA1, and this may mitigate the severity of platelet deficiency in WAS patients. Together, these results uncover a crucial role for FLI1 in megakaryocyte differentiation, implicating this transcription factor in regulating microthrombocytopenia associated with Wiskott-Aldrich syndrome.

IL-18: A potential inflammation biomarker in Wiskott-Aldrich syndrome.

Analysis of serum cytokine levels in Wiskott-Aldrich syndrome patients pre- and post- treatment reveals IL-18 as a stable and reliable marker of inflammation. Definitive stem cell treatment with good myeloid correction correlates with resolution of inflammation and reduction of circulating IL-18, highlighting the importance of actin cytoskeletal regulation of myeloid cells in control of inflammation.

A Novel Mutation in WAS Gene Causing a Phenotypic Presentation of Wiskott-Aldrich Syndrome: A Case Report.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by immunodeficiency, thrombocytopenia, and atopic dermatitis. OBSERVATIONS: This infant presented at birth with petechiae and bruising, with severe neonatal thrombocytopenia. Genetic testing for WAS revealed a variant of unknown significance hemizygous missense mutation in the WAS gene. This variant has not previously been reported. On the basis of the patient's clinical course including bleeding, infection, abnormal immune evaluation, and dermatologic sequelae, he was diagnosed with WAS and underwent allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: We report a novel mutation in the WAS gene that causes a phenotypic presentation of Wiskott-Aldrich Syndrome.

WASp Deficiency Selectively Affects the TCR Diversity of Different Memory T Cell Subsets in WAS Chimeric Mice.

Background: The T cell receptor (TCR) diversity is essential for effective T cell immunity. Previous studies showed that TCR diversity in Wiskott-Aldrich Syndrome (WAS) patients was severely impaired, especially in the memory T cell populations. Whether this defect was caused by intrinsic WASp deficiency or extrinsic reasons is still unclear. Methods: We sorted different T cell subsets from the bone marrow chimeric mice model using both magnetic beads and flow cytometry. TCR repertoires of memory T cells, especially CD4+ effector memory T (TEM) cells and CD8+ central memory T (TCM) cells, were analyzed using the UMI quantitative high-throughput sequencing (HTS). Results: An average of 5.51 million sequencing reads of 32 samples was obtained from the Illumina sequencing platform. Bioinformatic analyses showed that compared with wild type (WT), WAS knock out (KO)-CD4+ TEM cells exhibited increased Simpson index and decreased D50 index (P <0.05); The rank abundance curve of KO-CD4+ TEM cells was shorter and steeper than that of WT, and the angle of qD and q in KO-CD4+ TEM cells was lower than that of WT, while these indexes showed few changes between WT and KO chimeric mice in the CD8+TCM population. Therefore, it indicated that the restriction on the TCRVß repertoires is majorly in KO-CD4+ TEM cells but not KO- CD8+ TCM cells. Principal Component Analysis (PCA), a comprehensive parameter for TCRVß diversity, successfully segregated CD4+ TEM cells from WT and KO, but failed in CD8+ TCM cells. Among the total sequences of TRB, the usage of TRBV12.2, TRBV30, TRBV31, TRBV4, TRBD1, TRBD2, TRBJ1.1, and TRBJ1.4 showed a significant difference between WT-CD4+ TEM cells and KO-CD4+ TEM cells (P <0.05), while in CD8+ TCM cells, only the usage of TRBV12.2 and TRBV20 showed a substantial difference between WT and KO (P <0.05). No significant differences in the hydrophobicity and sequence length of TCRVß were found between the WT and KO groups. Conclusion: WASp deficiency selectively affected the TCR diversity of different memory T cell subsets, and it had more impact on the TCRVß diversity of CD4+ TEM cells than CD8+ TCM cells. Moreover, the limitation of TCRVß diversity of CD4+ TEM cells and CD8+ TCM cells in WAS was not severe but intrinsic.

Clinical Features and Outcomes of 23 Patients with Wiskott-Aldrich Syndrome: A Single-Center Experience

Objective: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immune deficiency characterized by microthrombocytopenia, eczema, and recurrent infections. We aimed to evaluate the clinical features and outcomes of a WAS cohort. Materials and Methods: We retrospectively evaluated the clinical courses, immunological features, treatments, and outcomes in a total of 23 WAS patients together with data related to 11 transplanted cases among them between 1982 and 2019. Results: Before admission, 11 patients (48%) were misdiagnosed with immune thrombocytopenia. WAS scores were mostly 4 or 5. Eleven patients were transplanted and they had an overall survival rate of 100% during a median follow-up period of 8.5 years (range: 8 months to 20 years). Five patients who were not transplanted died at a median of 7 years (range: 2-26 years). Nontransplanted patients had high morbidity due to organ damage, mostly caused by autoimmunity, bleeding, and infections. Two novel mutations were also defined. Conclusion: All male babies with microthrombocytopenia should be evaluated for WAS. Hematopoietic stem cell transplantation should be performed at the earliest age with the best possible donors.

Faecal microbial dysbiosis in children with Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by a mutation in the WAS gene that encodes the WAS protein (WASp); up to 5-10% of these patients develop inflammatory bowel disease (IBD). The mechanisms by which WASp deficiency causes IBD are unclear. Intestinal microbial dysbiosis and imbalances in host immune responses play important roles in the pathogenesis of polygenetic IBD; however, few studies have conducted detailed examination of the microbial alterations and their relationship with IBD in WAS. Here, we collected faecal samples from 19 children (all less than 2 years old) with WAS and samples from WASp-KO mice with IBD and subjected them to 16S ribosomal RNA sequencing. We found that microbial community richness and structure in WAS children were different from those in controls; WAS children revealed reduced microbial community richness and diversity. Relative abundance of Bacteroidetes and Verrucomicrobiain in WAS children was significantly lower, while that of Proteobacteria was markedly higher. WASp-KO mice revealed a significantly decreased abundance of Firmicutes. Faecal microbial dysbiosis caused by WASp deficiency is similar to that observed for polygenetic IBD, suggesting that WASp may play crucial function in microbial homoeostasis and that microbial dysbiosis may contribute to IBD in WAS. These microbial alterations may be useful targets for monitoring and therapeutically managing intestinal inflammation in WAS.

A novel mutation of WAS gene in a boy with Mycobacterium bovis infection in spleen.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by mutations of the gene encoding WAS protein (WASp). A scoring system has been used to grade severity of the disease. However, the phenotype of the disease may progress over time, especially in children younger than 2 years of age. Here, we report a male child who presented with X-linked thrombocytopenia (XLT). Mutation analysis revealed a novel hemizygous 13-bp deletion (c.181_193delGCTGAGCACTGGA) on exon 2 of the WAS gene. This frameshift mutation resulted in a premature terminating codon at position 71 (p.A61fsX10). Molecular analysis of maternal DNA revealed a heterozygosity of the same mutation. The disease progressed to classic WAS within 8 months. Later, gastric varices as a consequence of Mycobacterium bovis infection in the spleen was detected. The rapid worsening of the disease may be due to the severe genotype of this patient.

The contribution of mouse models to the understanding of constitutional thrombocytopenia.

Constitutional thrombocytopenias result from platelet production abnormalities of hereditary origin. Long misdiagnosed and poorly studied, knowledge about these rare diseases has increased considerably over the last twenty years due to improved technology for the identification of mutations, as well as an improvement in obtaining megakaryocyte culture from patient hematopoietic stem cells. Simultaneously, the manipulation of mouse genes (transgenesis, total or conditional inactivation, introduction of point mutations, random chemical mutagenesis) have helped to generate disease models that have contributed greatly to deciphering patient clinical and laboratory features. Most of the thrombocytopenias for which the mutated genes have been identified now have a murine model counterpart. This review focuses on the contribution that these mouse models have brought to the understanding of hereditary thrombocytopenias with respect to what was known in humans. Animal models have either i) provided novel information on the molecular and cellular pathways that were missing from the patient studies; ii) improved our understanding of the mechanisms of thrombocytopoiesis; iii) been instrumental in structure-function studies of the mutated gene products; and iv) been an invaluable tool as preclinical models to test new drugs or develop gene therapies. At present, the genetic determinants of thrombocytopenia remain unknown in almost half of all cases. Currently available high-speed sequencing techniques will identify new candidate genes, which will in turn allow the generation of murine models to confirm and further study the abnormal phenotype. In a complementary manner, programs of random mutagenesis in mice should also identify new candidate genes involved in thrombocytopenia.

Clinical spectrum, pathophysiology and treatment of the Wiskott-Aldrich syndrome.

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

Alternative control: what's WASp doing in the nucleus?

Wiskott-Aldrich syndrome (WAS) is a rare X-linked recessive immunodeficiency disorder of childhood that is caused by mutations in the WAS gene. WAS encodes WASp, a protein that is known to function in the cytoplasm of hematopoietic cells and is required for the induced differentiation of CD4+ T helper type 1 (TH1) lymphocytes. Now, a paper in Science Translational Medicine describes another mechanism for impaired immunity in WAS by showing that WASp localizes in the nucleus and regulates histone modifications and chromatin structure, thereby modulating expression of the TH1 master gene TBX21 (TBET).

Wiskott-Aldrich syndrome: diagnosis, clinical and laboratory manifestations, and treatment.

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

Abnormal O-glycosylation of CD43 may account for some features of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency disorder characterized by eczema, recurrent infections, thrombocytopenia and small platelets. There is an increased incidence of autoimmune phenomena particularly autoimmune haemolytic anaemias and vasculitic disorders. Mutations in the WASP gene encoding the cytoskeleton regulatory protein WASp (Wiskott-Aldrich syndrome protein) result in abnormal protein activity with defective cytoplasmic signaling and actin polymerization. This accounts for abnormal T cell responses to proliferation and susceptibility to infections, but does not fully explain the autoimmune phenomena nor the progressive lymphopenia seen in these patients. Wiskott Aldrich patients also demonstrate abnormal O-glycosylation of a highly conserved transmembrane glycoprotein CD43 that is expressed on most haemopoeitic cells. The altered glycosylation pattern on WAS lymphocytes is due to increased beta1-->6 GlcNACtransferase activity which leads to branched core 2 glycans or lower molecular forms of CD43 glycoprotein. The clinical hypothesis put forward is that abnormal O-glycosylation of CD43 may underlie the development of the autoimmune disorders and the progressive lymphopenia observed in WAS patients. Regulation of glycosylation of CD43 is important in the selection process of T cells within the thymus and abnormalities of glycosylation may cause many immune perturbations, such as the escape of self-reactive T cells into the periphery and subsequent development of autoimmune disease in these patients.

Combined immune deficiencies in children.

Combined immune deficiencies are a heterogeneous group of congenital disorders usually attributable to a single gene defect. The hallmark of these disorders is recurrent infections resulting from defects in both T- and B-cell function. This article reviews the basic components of the immune system as well as the clinical presentation, laboratory findings, and treatment for 3 distinct combined immune deficiencies.

The interaction between Cdc42 and WASP is required for SDF-1-induced T-lymphocyte chemotaxis.

In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell-derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1-induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1-responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus-TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1-induced chemotaxis of T cells.