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.

Summary The most consistent feature of Wiskott Aldrich syndrome (WAS) is profound thrombocytopenia with small platelets. The responsible gene encodes WAS protein (WASP), which functions in leucocytes as an actin filament nucleating agent –yet– actin filament nucleation proceeds normally in patient platelets regarding shape change, filopodia and lamellipodia generation. Because WASP localizes in the platelet membrane skeleton and is mobilized by αIIbβ3 integrin outside‐in signalling, we questioned whether its function might be linked to integrin. Agonist‐induced αIIbβ3 activation (PAC‐1 binding) was normal for patient platelets, indicating normal integrin inside‐out signalling. Inside‐out signalling (fibrinogen, JON/A binding) was also normal for wasp‐deficient murine platelets. However, adherence/spreading on immobilized fibrinogen was decreased for patient platelets and wasp‐deficient murine platelets, indicating decreased integrin outside‐in responses. Another integrin outside‐in dependent response, fibrin clot retraction, involving contraction of the post‐aggregation actin cytoskeleton, was also decreased for patient platelets and wasp‐deficient murine platelets. Rebleeding from tail cuts was more frequent for wasp‐deficient mice, suggesting decreased stabilisation of the primary platelet plug. In contrast, phosphatidylserine exposure, a pro‐coagulant response, was enhanced for WASP‐deficient patient and murine platelets. The collective results reveal a novel function for WASP in regulating pro‐aggregatory and pro‐coagulant responses downstream of integrin outside‐in signalling.

IntroductionWiskott-Aldrich syndrome (WAS) is an X-linked disease characterized by thrombocytopenia, small platelets, eczema, and immunodeficiency. The responsible gene encodes Wiskott-Aldrich syndrome protein (WASP), which functions in actin remodeling during the activation of blood cells. 1 WASP is decreased or absent in blood cells of patients with WAS. The absence of WASP is associated with more severe disease, including higher frequency of infections, autoimmune disease, and malignancies. 2 Several recent reports describe patients with WAS who have spontaneous reversion of mutations or second-site mutations that restore function. [3][4][5] These rare reversions/corrections, which can be studied at the DNA and protein levels, become evident if the corrected cells have a selective advantage leading to increased frequency in peripheral-blood cells. Although reversions/corrections have been found both (and only) in T-and B-cell lineages, only T cells demonstrate selective advantage. The frequency of corrected cells was highest for memory T cells, suggesting that selection results from an advantage in proliferation, activation, or both. These studies left unanswered the identity of the reverted progenitor cell and whether reversion, if it were to occur in a multipotent hematopoietic progenitor, would confer proliferative advantage outside the T-cell lineage.Here we report a novel case of somatic mosaicism resulting from spontaneous reversion of an inherited WASP mutation. The revertant sequence was confined to the patient's NK cells, which were a mixture of WASP-negative and WASP-expressing cells and contained the inherited and the revertant gene sequences. Enrichment of WASP-positive cells arising from one or few revertant NK progenitor cells indicates that WASP confers proliferative advantage to the NK-cell compartment and predicts the success of gene therapy in restoring this compartment. Patients, materials, and methods PatientsThe patient P1 was evaluated as an infant in 1994 because of thrombocytopenia (platelet count, 20 ϫ 10 9 to 50 ϫ 10 9 /L) and mild eczema. He did not have recurrent infections. A diagnosis of X-linked thrombocytopenia was considered. Bone marrow aspirate revealed normal trilineage hematopoiesis. Peripheral-blood immunophenotype at age 8 showed a decrease in T cells (22% CD4; 11% CD8) and an increase in NK cells (45% CD16 ϩ CD56 ϩ ). After the birth in 2001 of a brother (P2) with thrombocytopenia and eczema, both boys were reevaluated and diagnosed with WAS based on thrombocytopenia (70 ϫ 10 9 /L or less) and small platelet size (P1, 1.89 m; P2, 1.81 m; normal platelet size, 2.2 Ϯ 0.12 m). 6 Diagnosis was confirmed by identifying the mutation, as described. 7 Cell, DNA, and protein analysisBlood was collected in acid-citrate-dextrose under protocols approved by the Institutional Review Board of the CBR Institute for Biomedical Research (Boston, MA), including informed consent, and was fractionated immediately or after overnight shipment at ambient temperature. Peripheralblood mononucle...

Wiskott-Aldrich syndrome (WAS) is an