R-loops cause genomic instability in Wiskott-Aldrich syndrome Thelper lymphocytes

Publication date: Available online 15 December 2017
Source:Journal of Allergy and Clinical Immunology
Author(s): Koustav Sarkar, Seong-Su Han, Kuo-Kwang Wen, Hans D. Ochs, Loïc Dupré, Michael M. Seidman, Yatin M. Vyas
BackgroundWiskott-Aldrich syndrome (WAS), X-linked thrombocytopenia (XLT), and X-linked neutropenia (XLN), caused by WAS mutations affecting WASp expression or activity, manifest in immunodeficiency, autoimmunity, genomic-instability, and lymphoid-cancer. 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.ObjectiveTo define how dysfunctional gene transcription is causally linked to the degree of Thelper (Th) cell deficiency and genomic instability in XLT/WAS clinical spectrum.MethodsIn human Th1- or Th2-skewing cell culture systems, co-transcriptional R-loops (RNA:DNA duplex and displaced single-stranded DNA) and DNA double-strand breaks (DSBs) were monitored in multiple XLT and WAS patient samples, and in normal T cells depleted of WASp.ResultsWASp-deficiency provokes increased R-loops and R-loop-mediated DSBs in Th1-cells relative to Th2-cells. Mechanistically, chromatin-occupancy of Serine2-unphosphorylated-RNA Polymerase II is increased and that of topoisomerase-1, a R-loop preventing factor, is decreased at R-loop-enriched regions of IFNG and TBX21 (Th1 genes) in Th1-cells. These aberrations accompany increased unspliced (intron-retained) and decreased spliced mRNA of IFNG and TBX21 but not of IL13 (Th2-gene). Significantly, increased cellular load of R-loops and DSBs, which are normalized upon RNaseH1-mediated suppression of ectopic R-loops, inversely correlates with disease severity scores.ConclusionTranscriptional R-loop imbalance is a novel molecular defect etiologic in Th1-immunodeficiency and genomic-instability in WAS. The study proposes that cellular R-loop load could be used as a potential biomarker for monitoring symptom severity in the XLT-WAS clinical spectrum, and could be targeted therapeutically.

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