Pathogenesis of ELANE-Associated Neutropenia

摘要

Abstract Autosomal dominant hereditary neutropenia (cyclic neutropenia and severe congenital neutropenia (SCN)) is usually caused by heterozygous mutations in ELANE (formerly known as ELA2), encoding the neutrophil granule serine protease, neutrophil elastase (NE). Two competing theories had been proposed to explain how mutant neutrophil elastase causes neutropenia. The “mislocation” hypothesis holds that mutations disrupting NE's subcellular trafficking misroute proteolytic activity to the wrong destination. A competing “misfolding” hypothesis posits that mutations activate the ER stress and unfolded protein response (UPR), leading to cell death. Both theories are supported by molecular and genetic data. Recent identification of ELANE translational start site mutations suggests a third, “mistranslation” hypothesis, in which mutations force translation, either by loss of the canonical start site or by activating an internal ribosome entry site (IRES), to initiate from downstream in-frame ATG codons, producing amino-terminally truncated proteins bypassing `pre-pro' sequences directing trafficking and regulation of proteolytic activity. Thus, aberrant transport, protein folding, and proteolysis may contribute to pathogenesis. Heretofore, experimentally distinguishing among hypotheses has proven challenging, because patient samples are scarce, the affected cell type (neutrophil) is in low abundance, and mouse knock-in models fail to develop neutropenia. Here we show that patient-derived, induced pluripotent stem cells (iPSC) faithfully recapitulate clinical and molecular phenotypes and that CRISPR/Cas9 gene-editing of ELANE corrects patient germline mutations while reversing biochemical and developmental defects in iPSC, thus providing the first direct human model in which to study ELANE mutations after more than 15 years following their discovery. We propose to employ gene-editing of iPSC to target alterations in ELANE that each theory predicts would complement germline mutations. In the first aim, we specifically focus on removing internal translation initiation sites from ELANE in order to test the mistranslation hypothesis. In the second aim, we will inactivate NE's catalytic site in order to determine how aberrant proteolysis, central to at least two of the three hypotheses, contributes to pathogenesis. These studies will help elucidate mechanisms responsible for hereditary neutropenia, as well as normal granulopoiesis.