Identification of new genetic causes of congenital neutropenia

这项研究的长期目标是确定先天性中性粒细胞减少症的新的遗传原因和 研究其致病的分子机制。严重先天性中性粒细胞减少症是一种 先天的粒细胞生成障碍，特征是从出生起就有严重的慢性中性粒细胞减少症，过早死亡 继发于感染性并发症，并转化为髓系恶性肿瘤。约三分之一的 病例没有已知的遗传原因。我们对85例先天性心脏病患者进行了外显子全序列测定。 中性粒细胞减少症。编码酪蛋白溶解多肽酶B的ClpB杂合性错义突变在 在45例Elane阴性的SCN病例中，有6例(13%)；随后我们发现在 另外3例不在我们最初队列中的SCN病例。ClpB编码酪蛋白溶解多肽酶B， 一种与蛋白质折叠和线粒体功能有关的ATPase。先前的研究表明，双等位基因突变 ClpB的缺失与3-甲基谷氨酸尿症、白内障、神经系统疾病和 变异性中性粒细胞减少症。然而，这些突变与我们系列中看到的不同，这些突变是 杂合子并聚集在ATP结合口袋附近。初步数据显示，CRISPR-Cas9基因编辑 灭活ClpB或慢病毒介导的突变ClpB在人CD34+细胞中的过表达导致 粒细胞分化受损。总而言之，这些数据表明ClpB的杂合突变 是一种新的、相对常见的SCN病因。在这项提案中，我们将通过以下方式研究分子机制 哪种突变的ClpB会扰乱粒细胞的生成。我们还将鉴定和生物验证，使用类似的 方法，通过当前和正在进行的测序确定的其他潜在的导致中性粒细胞减少症的基因突变 先天性中性粒细胞减少症7例。提出了以下具体目标。 目的1：确定扰乱粒系生成的ClpB基因突变谱。我们将使用慢病毒- 人CD34+细胞介导性过表达对粒细胞生成的影响 在先天性中性粒细胞减少症患者中发现的ClpB突变，包括在 症候群病例。 目的2.研究ClpB基因突变破坏粒系造血细胞的机制。我们将测试 假设突变的ClpB以显性方式作用于破坏ClpB的伴侣功能，从而导致 在线粒体未折叠蛋白反应(UPRMT)激活受损和诱导细胞凋亡中 粒细胞前体细胞。 目的3.鉴定和验证先天性中性粒细胞减少症的新的遗传原因。我们将表演exome 对至少100例原因不明的先天性中性粒细胞减少症患者进行测序。这些数据将是 被询问以确定用于功能验证的候选基因。

The long-term goal of this of this research is to identify new genetic causes of congenital neutropenia and characterize their molecular mechanisms of disease pathogenesis. Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by severe chronic neutropenia from birth, premature death secondary to infectious complications, and transformation to myeloid malignancy. Approximately one-third of cases do not have a known genetic cause. We performed whole exome sequencing of 85 cases of congenital neutropenia. Heterozygous missense mutations of CLPB, encoding caseinolytic peptidase B, were identified in 6 of 45 (13%) ELANE-negative SCN cases; we subsequently identified heterozygous CLPB mutations in an additional 3 cases of SCN that were not part of our original cohort. CLPB encodes for caseinolytic peptidase B, an ATPase implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, these mutations are distinct from those seen in our series, which are heterozygous and cluster near the ATP binding pocket. Preliminary data show that CRISPR-Cas9 gene editing to inactivate CLPB or lentiviral-mediated overexpression of mutant CLPB in human CD34+ cells results in impaired granulocytic differentiation. Collectively, these data suggest that heterozygous mutations of CLPB are a new and relatively common cause of SCN. In this proposal, we will examine molecular mechanisms by which mutant CLPB disrupts granulopoiesis. We also will identify and biologically validate, using a similar approach, other potential neutropenia-causing gene mutations identified by current and ongoing sequencing of congenital neutropenia cases. The following specific aims are proposed. Aim 1: To define the spectrum of CLPB mutations that disrupt granulopoiesis. We will use lentiviral- mediated overexpression in human CD34+ cells to systematically assess the impact on granulopoiesis of CLPB mutations identified in patients with congenital neutropenia, including the biallelic mutations found in syndromic cases. Aim 2. To characterize mechanisms by which mutations of CLPB disrupt granulopoiesis. We will test the hypothesis that mutant CLPB acts in a dominant fashion to disrupt the chaperone function of CLPB, resulting in impaired activation of the mitochondrial unfolded protein response (UPRMT) and induction of apoptosis in granulocytic precursors. Aim 3. To identify and validate new genetic causes of congenital neutropenia. We will perform exome sequencing of at least 100 additional cases of congenital neutropenia with no known cause. These data will be interrogated to identify candidate genes for functional validation.