Pathogenesis of ELANE-Associated Neutropenia

摘要 常染色体显性遗传性中性粒细胞减少症（周期性中性粒细胞减少症和重度先天性中性粒细胞减少症（SCN））是 通常由编码中性粒细胞的ELANE（以前称为ELA 2）的杂合突变引起 颗粒丝氨酸蛋白酶、中性粒细胞弹性蛋白酶（NE）。有两种相互竞争的理论被提出来解释 突变的中性粒细胞弹性蛋白酶引起中性粒细胞减少症。“错位”假说认为， NE的亚细胞运输将蛋白水解活性错误地传递到错误的目的地。一个竞争性的“错误折叠” 假设突变激活ER应激和未折叠蛋白反应（UPR），导致细胞 死亡这两种理论都得到了分子和基因数据的支持。ELANE翻译蛋白的鉴定 起始位点突变提出了第三种“误译”假说，即突变迫使翻译， 失去经典起始位点或通过激活内部核糖体进入位点（IRES）， 下游框内ATG密码子，产生绕过“pre-pro”的氨基末端截短的蛋白质 指导运输和调节蛋白水解活性的序列。因此，异常转运，蛋白质折叠， 蛋白质水解可能参与发病机制。因此，通过实验区分假设 已经证明具有挑战性，因为患者样本稀缺，受影响的细胞类型（中性粒细胞）处于低水平， 丰度，并且小鼠基因敲入模型不能发展中性粒细胞减少症。在这里，我们展示了患者衍生的， 诱导多能干细胞（iPSC）忠实地再现了临床和分子表型， ELANE的CRISPR/Cas9基因编辑纠正了患者的生殖系突变，同时逆转了生物化学和遗传学。 iPSC的发育缺陷，从而提供了第一个研究ELANE突变的直接人类模型 在他们被发现后的15年里，我们建议使用iPSC的基因编辑来靶向 每个理论预测的ELANE中的改变将补充种系突变。第一个目标，我们 特别关注从ELANE中去除内部翻译起始位点，以测试误译 假说.在第二个目标中，我们将研究NE的催化位点，以确定如何异常 蛋白质水解是三种假说中至少两种假说的核心，对发病机制有贡献。这些研究将有助于 阐明遗传性中性粒细胞减少症以及正常粒细胞生成的机制。

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.