Project description:Wiskott-Aldrich syndrome (WAS) is a rare X-linked primary immunodeficiency characterized by microthrombocytopenia, eczema, recurrent infections, and increased incidence of autoimmune disorders and malignancies. WAS is caused by mutations in the was gene, which is expressed exclusively in hematopoietic cells, and the spleen plays an important role in hematopoiesis and red blood cell clearance. However, to date, detailed comparative analyses of the spleen between WASp-deficient (WAS-KO) mice and WT mice, particularly at the transcriptomic level, have not been reported. Here, we investigated the differences in the transcriptomes of spleen tissues of 10-week-old WAS-KO mice. Comparison of the gene expression profiles of WAS-KO and WT mice revealed 1964 differentially expressed genes (DEGs). Among these genes, 996 DEGs were upregulated, and 968 were downregulated in WAS-KO mice. To determine the functions of the DEGs, GO and KEGG enrichment analyses were performed for significantly upregulated and downregulated DEGs, respectively. The results showed that the levels of cell senescence and apoptosis-related genes were increased, that the antigen processing and presentation mechanisms involved in the immune response were damaged, and that signal transduction processes were impaired in the spleens of WAS-KO mice. Thus, was gene deletion may lead to anemia and hemolysis-related disease, mainly due to increased osmotic fragility of red blood cells, low hemoglobin, increased bilirubin levels and increased serum ferritin. These results indicate that senescence and apoptosis of blood cells also play an important role in the occurrence of WAS. However, most studies have focused only on the immune response. Therefore, the interesting findings of this paper can provide a stronger theoretical basis for further study and help improve the treatment of WAS. We performed gene expression profiling analysis using data obtained from RNA-seq of 2 different group of WT and WAS-KO mice.

Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aν actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future.

Project description:Wiskott-Aldrich syndrome (WAS) is characterized by X-linked thrombocytopenia, eczema, immunodeficiency, recurrent infections and increased risk of autoimmunity and malignancies. WAS is caused by mutations in the WAS gene, which encodes the exclusively hematopoietic WAS protein (WASp) that is classically characterized as aÎ½ actin nucleator. However, disruption of F-actin polymerization by WAS mutations can not account for many aspects of WAS pathogenesis. Ignorance of other functions of WASP precludes in-depth understanding of the pathogenic effects of mutant WASP, and therefore hampers development of effective therapy. Here we generated induced pluripotent stem cells (iPSCs) from WAS patients (WAS-iPSC) bearing different mutations and corresponding isogenic iPSCs in which the pathogenic mutations had been corrected by targeted genome editing. Hematopoietic cells differentiated from WAS-iPSCs not only recapitulated known disease phenotypes, but also revealed novel defects of WASP deficient cells. WASP co-localized with nuclear pores, nucleoli, nuclear speckles and PML bodies by immunocytochemistry and/or serial block face scanning microscopy (SBF-SEM). MudPIT (multi-dimensional protein identification technology) analysis revealed that WASP physically interacted with nuclear body components, nuclear structural proteins, chromatin modifying complexes, and many RNA-binding proteins including major components of the spliceosome. Next-generation sequencing captured a dramatic global change of alternative splicing in WAS patient cells. WAS mutation impacted splicing of multiple genes frequently mutated in myelodysplastic syndrome and other cancers. RNA sequencing showed that WAS-iPSC derived immune cells misregulated many cell cycle regulators, tumor suppressors, immune function genes and splicing factors, and activated gene networks that drive cancer development and inflammatory diseases. Together these data uncovered previously unappreciated functions of the WASP and provided a mechanistic understanding of the pathogenesis of malignancy and autoimmunity in the most severe form of WAS. These new knowledge could help develop targeted therapy for WAS in the future. Human WAS-iPSCs (p.Phe35*) and gene corrected WAS-iPSCs (cWAS-iPSC) were differentiated into macrophages. WAS patient derived B-lymphocyte line ID00003 (p.Glu133Lys) and a wile-type B-lymphocyte line GM11518 were cultured using standard protocol. Total RNAs were extracted and been analyzed by RASL-seq.

			Action	DRS
	SRR21799517_1.fastq.gz	Fastqsanger.gz
	SRR21799517_2.fastq.gz	Fastqsanger.gz
	SRR21799518_1.fastq.gz	Fastqsanger.gz
	SRR21799518_2.fastq.gz	Fastqsanger.gz
	SRR21799519_1.fastq.gz	Fastqsanger.gz

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Transcriptomic insights into the role of the spleen in a mouse model of Wiskott-Aldrich Syndrome

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