Second-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes somatic mosaicism in two WAS siblings
Taizo Wada, … , David L. Nelson, Fabio Candotti
Taizo Wada, … , David L. Nelson, Fabio Candotti
Published May 1, 2003
Citation Information: J Clin Invest. 2003;111(9):1389-1397. https://doi.org/10.1172/JCI15485.
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Article Genetics

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

Abstract

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.

Authors

Taizo Wada, Akihiro Konno, Shepherd H. Schurman, Elizabeth K. Garabedian, Stacie M. Anderson, Martha Kirby, David L. Nelson, Fabio Candotti

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Characterization of WASP gene mutations and expression. (a) The WASP gen...
Characterization of WASP gene mutations and expression. (a) The WASP gene exon 10 sequence was amplified from DNA extracted from normal PBMCs, as well as WASP– T and WASP+ T lymphocytes of patient II-1. Direct sequencing was performed using an automated sequencer. A thick bar highlights the position of the original insG mutation and thin bars show the 4-bp repeat sequences. (b) Model for the generation of hairpin structure facilitating the second-site mutation by slipped mispairing and deletion of 19 bp. (c) Predicted structures of mutated WASP molecules. PH, pleckstrin homology; WH, Wiskott homology; GBD, GTPase-binding domain; PPPP, proline rich. del, deletion. (d) Western blot analysis of WASP was performed using lysates of cultured T lymphocytes obtained from a healthy control and the patients. Arrows indicate the position of the WASP band and of a nonspecific protein reactive with the secondary Ab (NS). IB, immunoblotting.