Consequences of gain-of-function RPA1 mutations on telomere function and hematopoiesis

功能获得性 RPA1 突变对端粒功能和造血的影响

• 批准号: 10704735
• 负责人: Richa Sharma
• 金额: $ 16.7万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-15 至 2027-09-14
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10704735
• 关键词: Affinity; Area; Award; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biology; Bone marrow failure; CRISPR/Cas technology; Cell Maintenance; Cell Separation; Cell division; Cells; Childhood; Chromosomes; Clinical; Complex; Cytogenetics; DNA; DNA Binding; DNA Damage; DNA Repair Disorder; DNA Repair Gene; DNA biosynthesis; Data; Development Plans; Digestion; Disease; Disease model; Educational workshop; Ensure; Exhibits; Faculty; Family; Flow Cytometry; Functional disorder; Generations; Genes; Genetic Induction; Genome; Genome engineering; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneous-Nuclear Ribonucleoproteins; Heterozygote; Human; Immunoprecipitation; Impairment; In Vitro; Individual; Inherited; Institution; K-Series Research Career Programs; Length; Liver Cirrhosis; Location; Maintenance; Maps; Mass Spectrum Analysis; Mentors; Methods; Missense Mutation; Molecular; Mosaicism; Multiprotein Complexes; Mus; Mutation; Nuclear Proteins; Organ; Orthologous Gene; Pathogenesis; Pathologic; Pathology; Patient-Focused Outcomes; Phenotype; Physicians; Process; Prognosis; Proteins; Proteome; Proteomics; Publishing; Pulmonary Fibrosis; Research; Research Support; Resource Sharing; Role; S phase; Saint Jude Children' s Research Hospital; Scientist; Single-Stranded DNA; Stress; System; Telomerase; Telomere Maintenance; Telomere Shortening; Testing; Time; Tissue imaging; Variant; animal resource; career; career development; causal variant; cellular imaging; clinical care; clinical phenotype; experience; gain of function; gene therapy; genetic pedigree; imaging facilities; improved; in vivo; induced pluripotent stem cell; lectures; light microscopy; mouse model; mutant; novel; nuclease; premature; preservation; proband; recruit; repaired; replication factor A; replication factor C; replication stress; response; segregation; skills; stem; stem cell model; stem cells; success; telomere

Project Summary Inherited mutations in genes governing telomere maintenance cause Telomere biology disorders (TBD), a group of diseases with a progressive course and poor prognosis characterized by pulmonary fibrosis, liver cirrhosis, and bone marrow failure (BMF). Understanding the molecular mechanisms governing telomere preservation and identifying TBD associated genes are established gaps. We recently published novel germline heterozygous missense variants in RPA1 in four unrelated probands presenting with TBD. We also demonstrated that RPA1 mutants have elevated affinity to random and telomere single strand DNA, which causes a unique gain-of- function (GoF) effect, defective hematopoiesis due to short telomeres in RPA1 mutant iPSC cells and extensive somatic rescue mosaicism. The molecular mechanism through which RPA1 GoF mutations cause telomere shortening, and TBD is unknown. I hypothesize that RPA1 mutations cause TBD by outcompeting essential telomere maintenance proteins for binding to ssDNA, thereby limiting stem cell replicative potential. The proposed studies will investigate these questions through two specific aims: 1) determine the molecular mechanism by which GoF RPA1E240K causes telomere dysfunction and 2) determine the effect of RPA1E240K on telomere maintenance and hematopoiesis in vivo. A Mentored Clinical Scientist Research Career Development Award (K08) will provide the candidate with the amount of protected time needed to achieve her career goal of independence as a physician scientist and focus on improving the clinical outcomes of patients with BMF and DNA repair disorders through understanding the mechanisms of their disease pathogenesis. A strong career development plan including experienced and successful faculty advisor and co-advisor committee, clear plans for progress assessments, and attendance at numerous courses, lectures, and workshops to increase proficiency in technical and management skills will accompany these research goals to ensure success as an independent physician scientist. This award will be completed at St. Jude Children’s Research Hospital (SJCRH), one of the world’s leading academic institutions focused on the research and treatment of pediatric catastrophic diseases, making it an exemplary location for an early career physician scientist to develop his career. In addition to the strong institutional support, St. Jude offers unmatched research support with facilities including the Flow Cytometry and Cell Sorting Shared Resource, Center for Advanced Genome Engineering (CAGE), Light Microscopy Division of Cell and Tissue Imaging Center, Animal Resource Center, Cytogenetics Core, and Center for Proteomics.

项目摘要 一组控制端粒维持的基因的遗传突变导致端粒生物学紊乱(TBD) 病程进展和预后不良的疾病的特点是肺纤维化，肝硬变， 和骨髓衰竭(BMF)。了解影响端粒保存和修复的分子机制 识别与慢性阻塞性肺疾病相关的基因是已有的空白。我们最近出版了一本新的种系杂合体 四名无血缘关系的先证者中RPA1基因的错义变异。我们还演示了RPA1 突变体对随机和端粒单链DNA的亲和力提高，这导致了一种独特的- RPA1突变的iPSC细胞的功能(GoF)效应、端粒缩短所致的造血缺陷以及广泛的 躯体救援马赛克。RPA1 Gof突变引起端粒的分子机制 缩短，待定是未知的。我假设RPA1突变是通过竞争超过Essential而引起的 端粒维持蛋白与单链DNA结合，从而限制干细胞的复制潜力。这个 拟议的研究将通过两个具体目的来研究这些问题：1)确定分子 Gof RPA1E240K导致端粒功能障碍的机制2)确定RPA1E240K对端粒功能的影响 体内端粒的维持和造血。 指导临床科学家研究职业发展奖(K08)将为候选人提供 作为一名内科医生、科学家和专科医生，实现她独立的职业目标所需的保护时间 通过对BMF和DNA修复障碍的认识改善患者的临床结局 他们的疾病发病机制。一个强有力的职业发展计划，包括有经验和 成功的教师顾问和联合顾问委员会，明确的进度评估计划，以及出席 为了提高技术和管理技能的熟练程度，大量的课程、讲座和研讨会将 遵循这些研究目标，以确保作为一名独立的内科科学家取得成功。这个奖项将是 在圣犹大儿童研究医院(SJCRH)完成，该医院是世界领先的学术机构之一 专注于儿科灾难性疾病的研究和治疗，使其成为 一位职业生涯较早的内科科学家来发展他的职业生涯。除了强有力的机构支持外，圣犹大 提供无与伦比的研究支持，包括共享的流式细胞仪和细胞分类 资源，高级基因组工程中心(CAGE)，细胞和组织光学显微镜分部 影像中心、动物资源中心、细胞遗传学核心和蛋白质组学中心。