A systematic approach to understanding how patient variants affect the activity of Wiskott-Aldrich syndrome protein
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
This article is not in any list yet, why not save it to one of your lists.Abstract
Wiskott-Aldrich syndrome (WAS) and X-linked neutropenia (XLN) are caused by genetic variants in the WAS gene. How WAS variants lead to clinical disease remains unsolved in many cases. We expressed human WASp using a spider silk inspired solubility tag (NT*-tag) and inserted patient’s variants. Native mass spectrometry and pyrene actin assays showed that five variants (L270P, F271S, S272P, I290T, I294T) predicted to cause XLN led to open protein conformation and high actin polymerization rate in the absence of the WASp activator, Cdc42. One previously reported XLN variant (R268W), two loss-of-function WAS variants (A236G, D485N), and one variant of unknown significance (R431W) behaved similarly to wildtype WASp in terms of structural conformation and actin polymerization. Patient CD4 + T cells were used for analysis of WASp expression and phosphorylation, actin polymerization, anti-CD3 induced proliferation capacity, and upregulation of high affinity LFA-1, distinguishing loss-of-function and gain-of-function variants from benign WAS variants. This systematic approach reveals how WAS genetic variants cause severe human disease and stratify variants to guide clinical decision for definitive therapy.
Key Points
-
Gain-of-function WASp variant has extended protein conformation probed by native mass spectrometry and raised pyrene actin polymerization.
-
Functional analysis of patients CD4 + T cells classifies WASp variants as loss-of-function, reduced-function, gain-of-function, and benign.
