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: 10572822
• 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/10572822
• 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; Child; 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; Genome; Genome engineering; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Heterogeneous-Nuclear Ribonucleoproteins; Hospitals; Human; Immunoprecipitation; Impairment; In Vitro; Individual; Inherited; Institution; K-Series Research Career Programs; Length; Liver Cirrhosis; Location; Maintenance; Mass Spectrum Analysis; Mentors; Methods; Missense Mutation; Molecular; Mosaicism; Multiprotein Complexes; Mus; Mutation; Nuclear Proteins; 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; gain of function mutation; 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; skills; 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 控制端粒维持的基因中的遗传突变会导致端粒生物学疾病（TBD），一组 肺部纤维化，肝硬化，疾病的疾病和预后不良的疾病 和骨髓衰竭（BMF）。了解有关端粒保存的分子机制和 识别与TBD相关的基因是建立差距。我们最近发表了小说种系杂合子 rpa1中的错义变体出现了TBD的四个无关问题。我们还证明了RPA1 突变体对随机和端粒单链DNA的亲和力升高，这会导致独特的收益 功能（GOF）效应，由于RPA1突变体IPSC细胞中短端粒而导致的造血缺陷和广泛 躯体救援镶嵌。 RPA1 GOF突变引起端粒的分子机制 缩短和TBD尚不清楚。我假设RPA1突变通过胜任必不可少的引起TBD 端粒维持蛋白与ssDNA结合，从而限制了干细胞的复制潜力。这 拟议的研究将通过两个具体目的研究这些问题：1）确定分子 GOF RPA1E240K引起端粒功能障碍并确定RPA1E240K对端粒功能障碍的机制 端粒维持和体内造血。 指导的临床科学家研究职业发展奖（K08）将为候选人提供 实现她作为物理科学家的独立目标所需的受保护时间数量 通过理解BMF和DNA修复障碍患者的临床结局 疾病发病机理的机制。一个强大的职业发展计划，包括经验和 成功的教职顾问兼联合顾问委员会，明确的进度评估计划以及出席 许多课程，讲座和研讨会以提高技术和管理技能的水平 伴随着这些研究目标，以确保成为独立的物理科学家的成功。这个奖项将是 在世界领先的学术机构之一的圣裘德儿童研究医院（SJCRH）完成 专注于小儿灾难性疾病的研究和治疗，使其成为 早期的职业生理科学家发展了自己的职业生涯。除了强大的机构支持，圣裘德 提供无与伦比的研究支持，包括流式细胞仪和分类分类的设施 资源，晚期基因组工程中心（CAGE），细胞和组织的光显微镜分裂 成像中心，动物资源中心，细胞遗传学核心和蛋白质组学中心。