Second-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes somatic mosaicism in two WAS siblings
Taizo Wada, Akihiro Konno, Shepherd H. Schurman, Elizabeth K. Garabedian, Stacie M. Anderson, Martha Kirby, David L. Nelson, Fabio Candotti
Taizo Wada, Akihiro Konno, Shepherd H. Schurman, Elizabeth K. Garabedian, Stacie M. Anderson, Martha Kirby, David L. Nelson, Fabio Candotti
View: Text | PDF
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

×

Figure 6

Options: View larger image (or click on image) Download as PowerPoint
In vitro reconstitution experiments. (a) Retrovirus-mediated expression ...
In vitro reconstitution experiments. (a) Retrovirus-mediated expression of WASP. Western blot analysis of WASP was performed using lysates of WAS T cell lines transduced with the indicated retroviral vectors. Arrows indicate the position of the WASP bands. (b) Analysis of the inserted proviral genome by semiquantitative PCR. WASP exons 3 and 4 were amplified using DNA obtained from WAS T cell lines transduced with the indicated retroviral vector. The ratio of the band intensity of the endogenous WASP gene and the inserted transgene is shown. (c) In vitro binding of WASP to SH3 domain-containing proteins. Lysates were incubated and precipitated with SH3-GST fusion proteins. Complexes were then resolved by SDS-PAGE, and WASP was detected by Western blot analysis. (d) Analysis of TCR/CD3 downregulation. T cells were subjected to CD3 cross-linking at 37°C for 60 min and then stained with PE-conjugated streptavidin to analyze CD3 expression. Data are expressed as the percentage of mean fluorescent intensity of unstimulated control cells and represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01. UT, untransduced; Neo, Neo control vector; IB, immunoblotting; End, endogenous; Tg, transgene.