Identification of new genetic causes of congenital neutropenia

先天性中性粒细胞减少症新遗传原因的鉴定

• 批准号: 10399626
• 负责人: Daniel C Link
• 金额: $ 39.38万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-05-15 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10399626
• 关键词: ATP phosphohydrolase; Address; Bacterial Infections; Binding; Binding Sites; Biological Assay; Birth; Blood; CD34 gene; CRISPR/Cas technology; Candidate Disease Gene; Cataract; Cell model; Cells; Cessation of life; Chronic; Collaborations; Data; Disease; Dominant-Negative Mutation; Frequencies; Gene Mutation; General Population; Genes; Genetic; Genetic Transcription; Goals; Granulopoiesis; Hand; Homo; Human; Impairment; In Vitro; Induction of Apoptosis; International; Missense Mutation; Mitochondria; Molecular; Molecular Chaperones; Mus; Mutation; Myeloproliferative disease; Neutropenia; Pathogenesis; Pathogenicity; Patients; Peptide Hydrolases; Production; Proteins; Recovery; Registries; Research; Respiration; Risk; Secondary to; Sequence Analysis; Series; Stress; Suggestion; Syndrome; Testing; Transplantation; Validation; base; cohort; exome sequencing; granulocyte; improved; lentiviral-mediated; misfolded protein; monomer; mutant; nervous system disorder; neutrophil; overexpression; premature; prevent; protein folding; rapid diagnosis; repaired; response

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.

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遗传原因， 描述其疾病发病机制的分子机制。严重先天性中性粒细胞减少症（SCN）是一种 先天性粒细胞生成障碍，特征为从出生起严重慢性中性粒细胞减少症，过早死亡 继发于感染性并发症和转化为骨髓恶性肿瘤。大约三分之一 这些病例没有已知的遗传原因。我们对85例先天性巨噬细胞进行了全外显子组测序， 中性粒细胞减少症。CLPB编码酪蛋白分解肽酶B的杂合错义突变，在 45例ELANE阴性SCN病例中的6例（13%）;我们随后在一个非ELANE阴性SCN病例中鉴定了杂合CLPB突变。 另外3例SCN病例不属于我们的原始队列。CLPB编码酪蛋白分解肽酶B， 涉及蛋白质折叠和线粒体功能的ATP酶。先前的研究表明双等位基因突变 与3-甲基戊烯二酸尿综合征、白内障、神经系统疾病和 可变中性粒细胞减少症。然而，这些突变与我们系列中看到的突变不同， 杂合的并且聚集在ATP结合口袋附近。初步数据显示，CRISPR-Cas9基因编辑 突变CLPB或慢病毒介导的突变CLPB在人CD 34+细胞中的过表达导致 粒细胞分化受损。总的来说，这些数据表明CLPB的杂合突变 是SCN的一个新的和相对常见的原因。在这个建议中，我们将通过以下方式研究分子机制： 突变的CLPB会破坏粒细胞生成我们还将使用类似的 方法，通过当前和正在进行的测序确定的其他潜在的引起血小板减少症的基因突变， 先天性中性粒细胞减少症病例。提出了以下具体目标。 目的1：确定破坏粒细胞生成的CLPB突变谱。我们会用慢病毒- 介导的人CD 34+细胞过表达，以系统评估 在先天性中性粒细胞减少症患者中发现的CLPB突变，包括在 综合征病例。 目标2.描述CLPB突变破坏粒细胞生成的机制。我们将测试 假设突变CLPB以显性方式破坏CLPB的伴侣蛋白功能， 线粒体未折叠蛋白反应（UPRMT）的活化受损和细胞凋亡的诱导， 粒细胞前体 目标3.确定和验证先天性中性粒细胞减少症的新遗传原因。我们将执行外显子组 对至少100例其他原因不明的先天性中性粒细胞减少症病例进行测序。这些数据将 询问以鉴定用于功能验证的候选基因。