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的遗传突变导致端粒生物学障碍（TBD），这是一组 以肺纤维化，肝硬化， 骨髓衰竭（BMF）了解端粒保存的分子机制， 鉴定TBD相关基因是已建立的缺口。我们最近发表了新型种系杂合 在四个不相关的TBD先证者中RPA1的错义变异。我们还证明了RPA 1 突变体对随机和端粒单链DNA的亲和力提高，这导致了独特的 功能（GoF）效应、由于RPA 1突变iPSC细胞中的短端粒导致的造血缺陷以及广泛的 体细胞拯救嵌合体RPA 1 GoF突变导致端粒的分子机制 缩短，TBD未知。我假设RPA1突变通过竞争必需的 端粒维持蛋白结合ssDNA，从而限制干细胞复制潜力。的 拟议的研究将通过两个具体目标调查这些问题：1）确定分子 GoF RPA 1E240 K引起端粒功能障碍的机制和2）确定RPA 1E240 K对端粒功能障碍的影响。 端粒维持和体内造血。 指导临床科学家研究职业发展奖（K 08）将为候选人提供 实现她作为一名医生科学家的独立职业目标所需的受保护的时间量， 通过了解BMF和DNA修复障碍患者的临床结局， 疾病的发病机制。一个强大的职业发展计划，包括经验丰富， 成功的教师顾问和联合顾问委员会，明确的进度评估计划，并出席 许多课程，讲座和研讨会，以提高熟练的技术和管理技能将 伴随着这些研究目标，以确保作为一个独立的医生科学家的成功。此奖项将 在圣裘德儿童研究医院（SJCRH），世界领先的学术机构之一完成 专注于儿科灾难性疾病的研究和治疗，使其成为 一个早期的职业医生科学家来发展他的职业生涯。除了强大的机构支持，圣裘德 提供无与伦比的研究支持，设施包括流式细胞术和细胞分选共享 资源，先进基因组工程中心（CAGE），细胞和组织光学显微镜部 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。