WIP is a chaperone for Wiskott-Aldrich syndrome protein (WASP).

Wiskott-Aldrich syndrome protein (WASP) is in a complex with WASP-interacting protein (WIP). WASP levels, but not mRNA levels, were severely diminished in T cells from WIP(-/-) mice and were increased by introduction of WIP in these cells. The WASP binding domain of WIP was shown to protect WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP levels in T cells from WIP(-/-) mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupt WIP binding. The calpain inhibitor calpeptin increased WASP levels in activated T and B cells from the WASP patients, but not in primary T cells from the patients or from WIP(-/-) mice. Despite its ability to increase WASP levels proteasome inhibition did not correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient. These results demonstrate that WIP stabilizes WASP and suggest that it may also be important for its function.

Genotype-proteotype linkage in the Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a platelet/immunodeficiency disease arising from mutations of WAS protein (WASP), a hemopoietic cytoskeletal protein. Clinical symptoms vary widely from mild (X-linked thrombocytopenia) to life threatening. In this study, we examined the molecular effects of individual mutations by quantifying WASP in peripheral lymphocytes of 44 patients and identifying the molecular variant (collectively called proteotype). Nonpredicted proteotypes were found for 14 genotypes. These include WASP-negative lymphocytes found for five missense genotypes and WASP-positive lymphocytes for two nonsense, five frameshift, and two splice site genotypes. Missense mutations in the Ena/VASP homology 1 (EVH1) domain lead to decreased/absent WASP but normal mRNA levels, indicating that proteolysis causes the protein deficit. Because several of the EVH1 missense mutations alter WIP binding sites, the findings suggest that abrogation of WIP binding induces proteolysis. Whereas platelets of most patients were previously shown to lack WASP, WASP-positive platelets were found for two atypical patients, both of whom have mutations outside the EVH1 domain. WASP variants with alternative splicing and intact C-terminal domains were characterized for eight nonsense and frameshift genotypes. One of these, a nonsense genotype in a mild patient, supports expression of WASP lacking half of the proline-rich region. With one notable exception, genotype and proteotype were linked, indicating that a genotype-proteotype registry could be assembled to aid in predicting disease course and planning therapy for newly diagnosed infants. Knowledge of the molecular effect of mutations would aid also in identifying disease-modifying genes.

Mosaicism of NK cells in a patient with Wiskott-Aldrich syndrome.

Rare cases of somatic mosaicism resulting from reversion of inherited mutations can lead to the attenuation of blood-cell disorders, including Wiskott-Aldrich syndrome (WAS). The impact of the revertant hematopoietic stem or progenitor cells, particularly their representation in blood-cell populations, is of interest because it predicts the outcome of gene therapy. Here we report an 8-year-old patient with WAS caused by a single nucleotide insertion in the WASP gene that abrogates protein expression. The patient nonetheless had mild disease. We found reversion of the mutation in a fraction of patient lymphocytes. Forty percent of natural killer (NK) cells expressed Wiskott-Aldrich syndrome protein (WASP), and NK cells contained both mutated and revertant (normal) sequences. WASP was not expressed in patient T or B cells; T cells contained only the mutated sequence. The selective advantage of WASP+ NK cells was also demonstrated for carrier females. The enrichment of WASP+-revertant NK cells indicates that WASP provides a selective advantage in this lineage and predicts the success of gene therapy for reconstituting the NK-cell compartment. The importance of reconstituting the NK-cell lineage is discussed.

Deficiencies of C1 inhibitor.

Hereditary and acquired deficiencies of the C1 inhibitor result in a single prominent symptom, namely angioedema. Angioedema may involve the skin, the gastrointestinal tract or the upper airway. Genetically determined defects in C1INH cause hereditary angioedema. The defect may be acquired as the result of an auto-antibody to C1INH or be due to the generation of anti-idiotypic antibody to monoclonal immunoglobulins as occurs in various B cell lymphoproliferative diseases. Androgens provide prophylaxis against attacks of angioedema. There is no widely approved treatment for acute attacks of angioedema although several promising drugs are now in the final stages of clinical trials.

WASP deficiency leads to global defects of directed leukocyte migration in vitro and in vivo.

Intact cellular migration is critically important for the induction and regulation of the immune response. The Wiskott-Aldrich syndrome protein (WASP) regulates surface receptor signaling to the actin cytoskeleton in hematopoietic cells and thus plays a pivotal role in cellular locomotion. WASP deficiency causes the Wiskott-Aldrich syndrome (WAS), characterized by immunodeficiency, thrombocytopenia, and eczema. Cell migration defects may contribute to the pathophysiology of WAS. In this study, we used a variety of in vitro and in vivo assays to comprehensively analyze migration properties of lymphocytes, dendritic cells (DC), and neutrophils from WASP-deficient mice. We provide evidence that WASP-deficient lymphocytes show a marked reduction in tethering in an in vitro flow chamber assay as well as decreased migration of T cells in response to the CC chemokine ligand 19 (CCL19). In vivo, compared with wild-type lymphocytes, WASP-deficient lymphocytes showed significantly impaired homing to Peyer's patches upon adoptive transfer into recipient mice. In addition, bone marrow-derived DC migrated less efficiently in response to CCL19. In vivo studies showed decreased migration of DC from skin to draining lymph nodes in WASP-deficient animals. Finally, we also document decreased neutrophil migration in vitro and in vivo. In summary, our studies suggest that WASP plays an important role in the locomotion of lymphocytes, DC, and granulocytes in vitro and in vivo and thus, reveal a crucial role of WASP in physiological trafficking of various hematopoietic cell lineages. These results further delineate immunological abnormalities in WASP-deficient mice, which will be useful to assess preclinical gene therapy studies.

Vav1/2/3-null mice define an essential role for Vav family proteins in lymphocyte development and activation but a differential requirement in MAPK signaling in T and B cells.

The Vav family of Rho guanine nucleotide exchange factors is thought to orchestrate signaling events downstream of lymphocyte antigen receptors. Elucidation of Vav function has been obscured thus far by the expression of three highly related family members. We generated mice lacking all Vav family proteins and show that Vav-null mice produce no functional T or B cells and completely fail to mount both T-dependent and T-independent humoral responses. Whereas T cell development is blocked at an early stage in the thymus, immature B lineage cells accumulate in the periphery but arrest at a late "transitional" stage. Mechanistically, we show that the Vav family is crucial for both TCR and B cell receptor (BCR)-induced Ca2+ signaling and, surprisingly, is only required for mitogen-activated protein kinase (MAPK) activation in developing and mature T cells but not in B cells. Thus, the abundance of immature B cells generated in Vav-null mice may be due to intact Ras/MAPK signaling in this lineage. Although the expression of Vav1 alone is sufficient for normal lymphocyte development, our data also reveal lineage-specific roles for Vav2 and Vav3, with the first demonstration that Vav3 plays a critical compensatory function in T cells. Together, we define an essential role for the entire Vav protein family in lymphocyte development and activation and establish the limits of functional redundancy both within this family and between Vav and other Rho-guanine nucleotide exchange factors.

WAVE2 deficiency reveals distinct roles in embryogenesis and Rac-mediated actin-based motility.

The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement.

Gene therapy for Wiskott-Aldrich syndrome: rescue of T-cell signaling and amelioration of colitis upon transplantation of retrovirally transduced hematopoietic stem cells in mice.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency that is caused by mutations in the recently identified WASP gene. WASP plays an important role in T-cell receptor-mediated signaling to the actin cytoskeleton. In these studies we assessed the feasibility of using retroviral gene transfer into WASP-deficient hematopoietic stem cells (HSCs) to rescue the T-cell signaling defect that is characteristic of WAS. Upon transplantation of WASP-deficient (WKO) HSCs that have been transduced with WASP-expressing retroviruses, mature B and T cells developed in normal numbers. Most importantly, the defect in antigen receptor-induced proliferation was significantly improved in T cells. Moreover, the susceptibility of colitis by WKO HSCs was prevented or ameliorated in recipient bone marrow chimeras by retrovirus-mediated expression of WASP. A partial reversal of the T-cell signaling defect could also be achieved following transplantation of WASP-deficient HSCs expressing the WASP-homologous protein N-WASP. Furthermore, we have documented a selective advantage of WT over WKO cells in lymphoid tissue using competitive repopulation experiments and Southern blot analysis. Our results provide proof of principle that the WAS-associated T-cell signaling defects can be improved upon transplantation of retrovirally transduced HSCs without overt toxicity and may encourage clinical gene therapy trials.

Wiskott-Aldrich syndrome in a female.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema, and various degrees of immune deficiency. Carriers of mutated WASP have nonrandom X chromosome inactivation in their blood cells and are disease-free. We report data on a 14-month-old girl with a history of WAS in her family who presented with thrombocytopenia, small platelets, and immunologic dysfunction. Sequencing of the WASP gene showed that the patient was heterozygous for the splice site mutation previously found in one of her relatives with WAS. Sequencing of all WASP exons revealed no other mutation. Levels of WASP in blood mononuclear cells were 60% of normal. Flow cytometry after intracellular staining of peripheral blood mononuclear cells with WASP monoclonal antibody revealed both WASP(bright) and WASP(dim) populations. X chromosome inactivation in the patient's blood cells was found to be random, demonstrating that both maternal and paternal active X chromosomes are present. These findings indicate that the female patient has a defect in the mechanisms that lead in disease-free WAS carriers to preferential survival/proliferation of cells bearing the active wild-type X chromosome. Whereas the patient's lymphocytes are skewed toward WASP(bright) cells, about 65% of her monocytes and the majority of her B cells (CD19(+)) are WASP(dim). Her naive T cells (CD3(+)CD45RA(+)) include WASP(bright) and WASP(dim) populations, but her memory T cells (CD3(+)CD45RA(-)) are all WASP(bright). After activation in vitro of T cells, all cells exhibited CD3(+)CD45RA(-) phenotype and most were WASP(bright) with active paternal (wild-type) X chromosome, suggesting selection against the mutated WASP allele during terminal T-cell maturation/differentiation.

Wiskott-Aldrich syndrome protein is required for NK cell cytotoxicity and colocalizes with actin to NK cell-activating immunologic synapses.

The Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency disorder caused by a mutation in WAS protein (WASp) that results in defective actin polymerization. Although the function of many hematopoietic cells requires WASp, the specific expression and function of this molecule in natural killer (NK) cells is unknown. Here, we report that WAS patients have increased percentages of peripheral blood NK cells and that fresh enriched NK cells from two patients with a WASp mutation have defective cytolytic function. In normal NK cells, WASp was expressed and localized to the activating immunologic synapse (IS) with filamentous actin (F-actin). Perforin also localized to the NK cell-activating IS but at a lesser frequency than F-actin and WASp. The accumulation of F-actin and WASp at the activating IS was decreased significantly in NK cells that had been treated with the inhibitor of actin polymerization, cytochalasin D. NK cells from WAS patients lacked expression of WASp and accumulated F-actin at the activating IS infrequently. Thus, WASp has an important function in NK cells. In patients with WASp mutations, the resulting NK cell defects are likely to contribute to their disease.

An Alu-mediated deletion at Xp11.23 leading to Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, eczema and immunodeficiency of varying severity. The WASP gene, mutations of which are responsible for the phenotype, maps to Xp11.23. We describe here a patient with a large deletion in the Xp11.23 region. The deletion, which totals 15.8 kb, begins downstream of DXS1696 and encompasses 13 kb upstream of WASP and includes the distal and proximal promoters and exons 1-6. Analysis of the 5'-boundary region identified sequences missing in the Human Genome database and, as a result, the normal DNA sequence was revised to include 743 bp of novel sequence (AF466616). The patient's upstream breakpoint was localized to an AluSg element within a highly repetitive DNA region containing other Alu elements. A 26-bp recombinogenic element is located downstream of the 5' breakpoint. A 16-bp sequence just upstream of the 5' breakpoint shares close homology with the sequence that spans the 3' breakpoint in intron 6. A heptanucleotide of unknown origin, CAGGGGG, links the 5' and 3' breakpoints. To our knowledge this is the largest deletion in a WAS patient.