A Novel Mutant Wasp in X-Linked Neutropenia Partially Recapitulates Defective Neutrophil Production through Its Expression in WASP-Deficient NB4 Cells

Background

Severe congenital neutropenia (SCN) is characterized by prolonged severe neutropenia (absolute neutrophil count < 500/µL) commencing in early infancy, and recurrent bacterial and/or fungal infections. X-linked neutropenia (XLN) is a very rare type of SCN caused by a gain-of-function mutation in the Wiskott-Aldrich syndrome gene (WAS) located on the short arm of the X chromosome. To the best of our knowledge, only three WAS protein (WASp) mutations (L270P, S272P, and I294T) have been described to date. Herein, we report an identification and functional analysis of novel WASP mutation (I290T), which was identified in the first Asian XLN case by phenotype-based gene analysis of next-generation sequencing (NGS) data.

Case

Patient was 29-year-old male who had been clinically diagnosed with SCN at 6 years of age, however the cause of SCN remained unknown. We performed targeted exome sequencing of 4813 disease-relevant genes using a TruSight One Sequencing Panel (Illumina, San Diego, CA). The sequencing data were analyzed in terms of somatic mutations using our in-house pipeline, "Genomon" (http://genomon.hgc.jp/exome/en/). The resulting gene list, including 925 single nucleotide variants (SNVs), was subjected to examination by Phenolyzer (http://phenolyzer.usc.edu/), a web-based computational tool that analyzes clinical phenotypes on the basis of free-text term(s) and predicts the most likely candidate gene(s) by integrating information from multiple databases. The terms entered were 'Congenital neutropenia', 'Familial', 'Myelodysplastic syndrome', and 'Autosomal'. Phenolyzer prioritized the WAS gene as the most probable candidate. Additionally, we identified a missense mutation in exon 9 of the patient's WAS gene (c.T869C, p.I290T) using the Integrative Genomics Viewer (IGV).

Materials and Methods

To investigate the molecular mechanisms of WASp-I290T mutation in XLN, we cloned WASp to a human cell line that can differentiate to neutrophils (NB4). Endogenous WASp in NB4 cells was fully knocked out by utilizing CRISPR/Cas9 system. We constructed a lentiviral AcGFP-tagged WASp overexpression plasmid controlled by CMV promoter, and AcGFP-tagged WASp was induced to NB4 cells. Cell viability evaluation, immunofluorescence imaging, and gene expression analysis were performed to compare NB4 cells treated with/without ATRA harboring wild type and mutant WASp.

Results

Western blot analysis of AcGFP-WASp clearly showed a molecular mass of 84 kD for both wild type and mutant proteins. The viable cell growth assay showed no significant differences between mutant and wild type WASp-transduced NB4^ΔWASPcells (NB4^WASp-WT and NB4^WASp-I290T cells, respectively) (Figure 1A). In contrast, upon ATRA-induced neutrophilic differentiation, the viable cell number significantly decreased in NB4^WASp-I290T cells after 72 hours, compared to NB4, NB4^ΔWASP and NB4^WASp-WT cells. This result was further confirmed by FACS analysis, whereas CD11b expression was induced in surviving NB4^WASp-I290T cells, comparably to other sublines of NB4 cells. Confocal laser microscopy revealed that WASp-I290T preferentially localized in nucleus rather than cytoplasm, compared to wild-type WASP, which was compatible with the previous findings reported for other WASp mutants in XLN. Expression of myeloid differentiation-related genes including C/EBP-alpha, -beta, -epsilon, CSF3R and PU.1 was examined at regular intervals by quantitative RT-PCR, indicating that C/EBP-epsilon transcripts were upregulated in WASp-I290T cells to higher extent than other sublines of NB4 cells after 24 hours of ATRA treatment, and that this difference was more prominent after 72 hours (Figure 1B).

Discussion

In this study, a novel WASp-I290T mutant identified in an adult XLN patient partially recapitulated defective neutrophil production through its expression in ATRA-treated NB4 cells lacking endogenous WASp. Our results suggest that premature upregulation of C/EBP-epsilon may be involved in impaired growth of ATRA-treated NB4WASp-I290T cells. A number of reports suggest the putative role of nuclear WASp in transcriptional regulation. Considering the preferential nuclear localization of WASp-I290T, further investigation of its interaction with nuclear proteins such as histone modifying enzymes is required.

View largeDownload slide

View largeDownload slide

Close modal