Wiskott-Aldrich Syndrome/X-Linked Thrombocytopenia: WASP Gene Mutations, Protein Expression, and Phenotype

Abstract

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT), caused by mutations of the WAS protein (WASP) gene, represent different phenotypes of the same disease. To demonstrate a phenotype/genotype correlation, we determined WASP gene mutations in 48 unrelated WAS families. Mutations included missense (20 families) and nonsense (eight) mutations located mostly in exons 1 to 4, and splice-site mutations (seven) and deletions and insertions (13) located preferentially in exons 7 to 11. Both genomic DNA and cDNA were sequenced and WASP expression was measured in cell lysates using peptide-specific rabbit anti-WASP antibodies. WASP was expressed in hematopoietic cell lines including bone marrow–derived CD34+ cells. Missense mutations located in exons 1 to 3 caused mild disease in all but one family and permitted WASP expression, although frequently at decreased concentration. Missense mutations affecting exon 4 were associated with classic WAS and, with one exception, barely detectable WASP. Nonsense mutations caused classic WAS and lack of protein. Insertions, deletions, and splice-site mutations resulted in classic WAS and absent, unstable, truncated, or multiply spliced protein. Using affinity precipitation, WASP was found to bind to Src SH3-containing proteins Fyn, Lck, PLC-γ, and Grb2, and mutated WASP, if expressed, was able to bind to Fyn-glutathione S-transferase (GST) fusion protein. We conclude that missense mutations affecting the PH domain (exons 1 to 3) of WASP inhibit less important functions of the protein and result in a mild phenotype, and that missense mutations affecting exon 4 and complex mutations affecting the 3′ portion of WASP interfere with crucial functions of the protein and cause classic WAS.

Protein assays for diagnosis of Wiskott–Aldrich syndrome and X‐linked thrombocytopenia

“…Such disorders are conventionally diagnosed using genetic analysis, but it may take several weeks or months to identify a particular gene mutation. In the case of WAS, several groups have established that WASp expression is absent or abnormal in the majority of patients found to have gene mutations (Remold-O'Donnell et al, 1997;Zhu et al, 1997). Indeed, we have previously reported the use of two anti-WASp sera to confirm the absence of WASp in PBMCs of patients suffering from the disease (MacCarthy-Morrogh et al, 1998).…”

“…From a clinical perspective, one might speculate that, if the abnormal protein in P12 retained partial function, the grandson P11 who has absent protein may suffer a more severe clinical course. Indeed, other studies have suggested that less severe phenotypes may retain residual WASp expression (Zhu et al, 1997;Shcherbina et al, 1999), although in both reports expression was studied in Epstein-Barr virus (EBV)-immortalized B-cell lines that may affect protein stability. The detection of abnormal-sized bands following immunoblot analysis for WASp has not been previously reported or used in the establishment of a diagnosis in WAS and XLT.…”

Wiskott–Aldrich syndrome protein is involved in αIIbβ3‐mediated cell adhesion

“…In classic WAS patients, WASP expression is not detected. Most XLT patients have missense mutations in the WASP amino terminus (within residues 1-137), and express the mutant proteins at a lower concentration than normal subjects (Zhu et al, 1997;Imai et al, 2004).…”

A novel Wiskott–Aldrich syndrome protein mutation in an infant with thrombotic thrombocytopenic purpura

“…Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder with variable clinical phenotypes that correlate with the type of mutations in the WAS protein (WASP) gene (1). The WASP gene is composed of 12 exons containing 1823 base pairs and encodes a 502-amino acid protein that appears to be of central importance for the function of hematopoietic stem cells (2). Mutations of WASP gene are located throughout the gene, although some hot spots have been identified (3).…”

“…Mutations, on the other hand, result in residual expression of a full-length point-mutated WASP, are often associated with X-linked thrombocytopenia (XLT) (5), corresponding to a longer life expectancy (6). A scoring system based on clinical symptoms has been developed to differentiate these distinct clinical phenotypes caused by WASP gene mutations (2,3,7). Autoimmune complications are frequently observed in WAS and patients who develop autoimmune diseases are assigned to a high-risk group with poor prognosis (1).…”