The role of microhomology-mediated end joining in Fanconi anemia pathogenesis

微同源介导的末端连接在范可尼贫血发病机制中的作用

基本信息

批准号：
10367981
负责人：
Neil Johnson
金额：
$ 45.41万
依托单位：
RESEARCH INST OF FOX CHASE CAN CTR
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10367981
关键词：
Address Adult Alleles Amino Acids Anemia Animal Model Ashkenazim BRCA1 gene BRCA2 gene Biological Biological Assay Bone marrow failure CRISPR/Cas technology Cell Line Cells Chemicals Child Coiled-Coil Domain Complex DNA DNA Double Strand Break DNA Repair DNA Repair Gene DNA Repair Pathway DNA-Directed DNA Polymerase Data Defect Development Disease Embryo Embryonic Development Equilibrium Etiology Excision Fanconi&apos s Anemia Flow Cytometry Functional disorder Genomic Instability Health Hematology Hematopoiesis Hematopoietic stem cells Hereditary Breast and Ovarian Cancer Syndrome Human Hyperactivity Immunofluorescence Microscopy Incidence Individual Knockout Mice Laboratories Life Measures Mediating Molecular Mus Musculoskeletal Mutation Oncogenes Organism PALB2 gene Pathogenesis Pathologic Pathway interactions Patients Phenotype Population Process Proteins Reporter Resected Role Severities Single-Stranded DNA Subgroup Symptoms Testing Transgenic Animals Transplantation Work cancer predisposition developmental disease experimental study genetic approach homologous recombination inhibitor insight leukemia mouse model mutant mouse model neonatal mice nuclease p53-binding protein 1 pre-clinical protein expression recombinational repair recruit repaired therapeutic development tool

项目摘要

PROJECT SUMMARY Children with Fanconi anemia (FA) demonstrate developmental disorders that include short stature, musculoskeletal defects, cancer predisposition, bone marrow failure (BMF) and anemia. In a significant proportion of cases, FA is associated with biallelic mutations in the hereditary breast and ovarian cancer (HBOC) genes. Pre-clinical FA studies have largely relied on transgenic animal models. However, currently available FA mouse models are born without developmental defects and hematological abnormalities have to be chemically induced. Our laboratory has developed a new Brca1CC mutant mouse model with a 3-amino acid deletion in the coiled-coil (CC) domain of Brca1 that specifically disrupts the Brca1-Palb2 association, resulting in loss of Rad51 loading and HR deficiency. Notably, Brca1CC homozygous mice are born at sub-Mendelian ratios, and neo-natal mice demonstrate a range of phenotypes analogous to FA in humans, including short stature, BMF with severe anemia, and adult mice develop leukemia. Therefore, Brca1CC mice closely resemble human FA and provide a new tool to gain unprecedented insight into biological pathways that underpin FA etiology. Both homologous recombination (HR) and microhomology-mediated end joining (MMEJ) are double stranded DNA break (DSB) repair pathways that are initiated by DNA end resection, a process where DSBs are resected by nucleases to form single stranded (ss)DNA regions. In preliminary data, we examined human FA patient cells, as well as Brca1CC MEFs, for HR and MMEJ activity. Interestingly, while HR was lowered, MMEJ was hyperactivated in FA cell lines. We now hypothesize that hyperactive DNA end resection and MMEJ promote the molecular pathogenesis of FA. We will address the following Specific Aims: 1) identify DNA repair pathways that are hyperactive in FA; 2) uncover mechanisms that promote FA embryonic development and pathogenesis; and 3) examine the effects of MMEJ inhibition on FA pathogenesis. Collectively, the proposed experiments will yield new insight into DNA repair mechanisms that promote genome instability and FA.

项目概要患有范可尼贫血 (FA) 的儿童表现出发育障碍，包括身材矮小、肌肉骨骼缺陷、癌症倾向、骨髓衰竭（BMF）和贫血。在一个重大的部分病例中，FA 与遗传性乳腺癌和卵巢癌 (HBOC) 的双等位基因突变相关基因。临床前 FA 研究很大程度上依赖于转基因动物模型。然而，目前可用的FA 小鼠模型出生时没有发育缺陷，血液学异常必须进行化学检测诱发。我们实验室开发了一种新的Brca1CC突变小鼠模型，其基因组中存在3个氨基酸缺失 Brca1 的卷曲螺旋 (CC) 结构域，专门破坏 Brca1-Palb2 关联，导致 Rad51 丢失负荷和人力资源不足。值得注意的是，Brca1CC 纯合小鼠以亚孟德尔比例出生，并且新生儿小鼠表现出一系列与人类 FA 类似的表型，包括身材矮小、严重的 BMF 贫血，成年小鼠会患上白血病。因此，Brca1CC 小鼠与人类 FA 非常相似，并提供了获得对 FA 病因学基础的生物学途径前所未有的了解的新工具。两者同源重组 (HR) 和微同源介导的末端连接 (MMEJ) 是双链 DNA 断裂 (DSB) 由 DNA 末端切除启动的修复途径，这是一个由核酸酶切除 DSB 的过程，形成单链（ss）DNA 区域。在初步数据中，我们检查了人类 FA 患者细胞，以及 Brca1CC MEF，用于 HR 和 MMEJ 活动。有趣的是，虽然 HR 降低，但 FA 中 MMEJ 过度激活细胞系。我们现在假设过度活跃的 DNA 末端切除和 MMEJ 促进分子 FA 的发病机制。我们将实现以下具体目标：1）确定 DNA 修复途径 FA 过度活跃； 2）揭示促进FA胚胎发育和发病机制；和 3) 检查 MMEJ 抑制对 FA 发病机制的影响。总的来说，所提出的实验将产生对促进基因组不稳定性和 FA 的 DNA 修复机制的新见解。