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: 10621903
• 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/10621903
• 关键词: ATP phosphohydrolase; Address; Alleles; 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; Heterozygote; 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; Syndrome; Testing; Transplantation; Validation; candidate identification; 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和 表征其疾病发病机理的分子机制。严重的先天性中性粒细胞减少症（SCN）是 颗粒状的先天疾病以出生早期死亡为特征 继发于传染性并发​​症，转化为髓样恶性肿瘤。大约三分之一 病例没有已知的遗传原因。我们进行了85例先天性案例的整个外显子组测序 中性粒细胞减少。在CLPB，编码酪蛋白肽酶B的杂合错义突变中被鉴定 45个（13％）的Elane阴性SCN病例中有6例；随后，我们确定了杂合的CLPB突变 另外3例SCN属于我们原始队列的一部分。 CLPB编码酪蛋白水解肽酶B， 与蛋白质折叠和线粒体功能有关的ATPase。先前的研究表明双重突变 CLPB的3-甲基谷歌尿尿，白内障，神经系统疾病和 可变中性粒细胞减少症。但是，这些突变与我们系列中看到的突变不同， ATP结合口袋附近的杂合和簇。初步数据表明CRISPR-CAS9基因编辑 使人CD34+细胞中突变体CLPB的CLPB或慢病毒介导的过表达导致 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的新原因。在此提案中，我们将通过 突变的CLPB破坏了颗粒。我们还将使用类似的 方法，其他潜在的引起中性粒细胞减少的基因突变，通过电流和持续的测序确定 先天性中性粒细胞减少病例。提出了以下特定目标。 目标1：定义破坏粒状粒子的CLPB突变的频谱。我们将使用慢病毒 - 介导的人CD34+细胞中介导的过表达，以系统地评估对颗粒的影响 先天性中性粒细胞减少症患者发现的CLPB突变，包括在 综合症病例。 目的2。表征clpb突变破坏粒状粒子的机制。我们将测试 突变体Clpb以主导方式破坏CLPB的伴侣功能的假设，导致 线粒体展开的蛋白质反应（UPRMT）的激活受损和凋亡诱导 粒细胞前体。 目的3。确定和验证先天性中性粒细胞减少的新遗传原因。我们将执行外显型 对至少100例其他先天性中性粒细胞减少症进行测序，没有已知原因。这些数据将是 询问以识别候选基因以进行功能验证。