Second-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes somatic mosaicism in two WAS siblings

Genetics and Molecular Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4442, USA.
Journal of Clinical Investigation (Impact Factor: 13.22). 06/2003; 111(9):1389-97. DOI: 10.1172/JCI15485
Source: PubMed
Revertant mosaicism due to true back mutations or second-site mutations has been identified in several inherited disorders. The occurrence of revertants is considered rare, and the underlying genetic mechanisms remain mostly unknown. Here we describe somatic mosaicism in two brothers affected with Wiskott-Aldrich syndrome (WAS). The original mutation causing disease in this family is a single base insertion (1305insG) in the WAS protein (WASP) gene, which results in frameshift and abrogates protein expression. Both patients, however, showed expression of WASP in a fraction of their T cells that were demonstrated to carry a second-site mutation causing the deletion of 19 nucleotides from nucleotide 1299 to 1316. This deletion abrogated the effects of the original mutation and restored the WASP reading frame. In vitro expression studies indicated that mutant protein encoded by the second-site mutation was expressed and functional, since it was able to bind to cellular partners and mediate T cell receptor/CD3 downregulation. These observations were consistent with evidence of in vivo selective advantage of WASP-expressing lymphocytes. Molecular analysis revealed that the sequence surrounding the deletion contained two 4-bp direct repeats and that a hairpin structure could be formed by five GC pairs within the deleted fragment. These findings strongly suggest that slipped mispairing was the cause of this second-site mutation and that selective accumulation of WASP-expressing T lymphocytes led to revertant mosaicism in these patients.

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    • "In vivo models of gene therapy using Was gene knockout mice have shown that T-cell numbers and responses can be improved in animals treated with different viral vector-mediated gene correction systems [24,6465666768697071. In addition, competitive repopulation experiments demonstrated that wild-type cells have a selective advantage of over Was knockout populations [72], which is consistent with the observations of in vivo selective survival advantage of revertant cells in WAS patients737475 and suggests that gene-corrected cells could similarly have a selective advantage over unmodified populations after gene therapy. The first clinical trial of gene therapy for WAS started in Germany in 2006 and used a gamma-retroviral vector to transduce mobilized peripheral blood CD34? cells of ten patients who received conditioning with busulfan (8 mg/ kg) prior to infusion of gene-corrected cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Gene transfer into the hematopoietic stem cell has shown curative potential for a variety of hematological disorders. Primary immunodeficiency diseases have led to the way in this field of gene therapy as an example and a model. Clinical results from the past 15 years have shown that significant improvement and even cure can be achieved for diseases such as X-linked severe combined immunodeficiency, adenosine deaminase deficiency, chronic granulomatous disease and Wiskott-Aldrich syndrome. Unfortunately, with the initial clear clinical benefits, the first serious complications of gene therapy have also occurred. In a significant number of patients treated using vectors based on murine gamma-retroviruses and carrying powerful viral enhancer elements, insertional oncogenesis events have resulted in acute leukemias that, in some cases, have had fatal outcomes. These serious adverse events have sparked a revision of the assessment of risks and benefits of integrating gene transfer for hematological diseases and prompted the development and application of new generations of viral vectors with recognized superior safety characteristics. This review summarizes the clinical experience of gene therapy for primary immunodeficiencies and discusses the likely avenues of progress in the future development of this expanding field of clinical investigations.
    Preview · Article · Feb 2014 · International journal of hematology
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    • "Hck mediated tyrosine phosphorylation of WASP-PRR mutations was reduced compared to WT WASP (Fig. 8) indicating critical effect of both the WASP mutations on Jurkat T-cell motility, thus, causing defective homing of T-cells and being responsible for WAS. Expression of WASP confers a selective advantage for T-cells [46] and a number of reports have shown that patients with mutations which cause severe WAS exhibit revertant mosaicism where a second-site mutation leads to altered but functional gene products [47,48]. We have shown that though WAS patients had two mutations together , WASP P373S mutation alone compromised all activity of WASP in T-cells. "
    [Show abstract] [Hide abstract] ABSTRACT: Wiskott Aldrich Syndrome (WAS) is caused by mutations in WASP and majority of the mutations are found in the WH1 domain which mediates interaction with WIP (WASP Interacting Protein), a WASP chaperone. Two point mutations together in the PRR domain of WASP (S339Y/P373S) have been reported to cause WAS however the molecular defect has not been characterized. Expression of these mutants separately (WASPR(S339Y), WASPR(P373S)) or together (WASPR(SP/YS)) did not rescue the chemotaxis defect or membrane projections defect of Jurkat(WKD) T-cells (WASP Knockdown). This is not due to the inability of WASP-PRR mutants to form functional WASP-WIP complex in growth rescue experiments in las17Δ yeast strain. Expression of WASPR(S339Y) but not WASPR(P373S) or WASPR(SP/YS) rescued the IL-2 expression defect of Jurkat(WKD) T-cells, suggesting that Pro373Ser mutation alone is sufficient to inhibit WASP functions in T-cell activation. The diffused localization of WASP-PRR mutants in activated Jurkat T-cells suggests that Ser339 and Pro373 are critical for WASP localization. WASP-PRR mutations either together or individually did not abolish interaction of WASP with sixteen WASP binding proteins including Hck, however they caused reduction in Hck mediated tyrosine phosphorylation of WASP which is critical for WASP activity. The auto-inhibitory conformation of WASP(P373S) mutant was not relieved by the binding of Toca-1 or Nck1. Thus, our results suggest that Pro373Ser mutation reduce Tyr291 phosphorylation and prevents conformational changes required for WASP activity in chemotaxis and T-cell activation. Thus Pro3373Ser is probably responsible for all the defects associated with WAS in the patients.
    Full-text · Article · Jan 2014 · Biochimica et Biophysica Acta
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    • "WAS patients' cells were checked for their mutation rate and it was not increased (). In WAS, X-linked SCID, and adenosine deaminase deficiency , a selective growth advantage of revertant lymphocytes has been observed (Hirschhorn et al., 1996; Stephan et al., 1996; Wada et al., 2003; Stewart et al., 2007 ). It should be noted that enzyme replacement therapy can counteract this selective advantage of revertant cells, as illustrated by the negative effect of this therapy on the abundance of revertant cells in the periphery of mosaic individuals (Ariga et al., 2001; Arredonda-Vega et al., 2002). "
    [Show abstract] [Hide abstract] ABSTRACT: Epidermolysis bullosa (EB) is a group of autosomal genetic bullous skin disorders, which can be caused by mutations in as many as 18 different genes. More than 30 subtypes have been described. Revertant mosaicism in EB refers to the presence of healthy skin areas without blistering surrounded by affected skin. The blister formation in the affected skin is the result of the hereditary mutations in an EB gene. In the healthy skin areas an additional mutation has occurred in the diseased gene that reverts the phenotype back to normal. Revertant mosaicism is common in EB, and in some subtypes all patients have multiple clinically healthy areas. Restoration of gene function can be obtained by different genetic mechanisms, including gene conversion, mitotic recombination, and a second-site mutation in the same gene. Typical characteristics of revertant patches in junctional EB-non-Herlitz are the presence of hairs and (hyper)pigmentation. In dystrophic EB, the absence of atrophy is an important criterion. There are still unanswered questions, such as when do the reversion mutations occur and which factors determine the outgrowth of the revertant stem cells. Further research is important to study this "natural gene therapy" phenomenon, especially in using the corrected cells for regenerative medicine.
    Full-text · Article · Sep 2013 · Nederlands Tijdschrift voor Dermatologie en Venereologie
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