Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

Although DEAD-box helicase 41 (DDX41) is implicated in oncogenic and innate immune mechanisms, there are many unanswered questions about how the ever-increasing spectrum of genetic variants impacts DDX41 activity. Here, we describe a facile genetic rescue assay that discriminates activities of DDX41 from those of human myeloid malignancy-linked germline and somatic DDX41 mutants. Our analyses revealed that the variants were impaired in their intrinsic RNA-regulatory activities and to induce monocytic differentiation markers. It will be instructive to extend these analyses to more broadly conduct structure/function assessments for clinical genetic curation and leverage the quantitative assay to elucidate mechanisms and interventions that promote and/or oppose DDX41 function, thereby influencing DDX41-linked pathogenicity. Overall design: Hoxb8-immortalized wild type hematopoietic progenitors (hi-Ddx41+/+) infected with control (empty) retrovirus, Ddx41 heterozygotic KO progenitors (hi-Ddx41+/-) infected with empty retrovirus or empty , wild type (WT) or human disease mutants of DDX41 -expressing retrovirus were FACS isolated for GFP+.