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Wiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia, which are caused byWASmutations affecting Wiskott-Aldrich syndrome protein (WASp) expression or activity, manifest in immunodeficiency, autoimmunity, genomic instability, and lymphoid and other cancers. WASp supports filamentous actin formation in the cytoplasm and gene transcription in the nucleus. Although the genetic basis for XLT/WAS has been clarified, the relationships between mutant forms of WASp and the diverse features of these disorders remain ill-defined.We sought to define how dysfunctional gene transcription is causally linked to the degree of TH cell deficiency and genomic instability in the XLT/WAS clinical spectrum.In human TH1- or TH2-skewing cell culture systems, cotranscriptional R-loops (RNA/DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple samples from patients with XLT and WAS and in normal T cells depleted of WASp.WASp deficiency provokes increased R-loops and R-loop–mediated DSBs in TH1 cells relative to TH2 cells. Mechanistically, chromatin occupancy of serine 2-unphosphorylated RNA polymerase II is increased, and that of topoisomerase 1, an R-loop preventing factor, is decreased at R-loop–enriched regions ofIFNGandTBX21(TH1 genes) in TH1 cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA ofIFNGandTBX21but notIL13(TH2 gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized on RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores.Transcriptional R-loop imbalance is a novel molecular defect causative in TH1 immunodeficiency and genomic instability in patients with WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity and prognostic outcome in the XLT-WAS clinical spectrum and could be targeted therapeutically.