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

• 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
• 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' 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

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.

项目摘要 Fanconi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，癌症易感性，骨髓衰竭（BMF）和贫血。重要的是 病例比例，FA与遗传性乳腺癌和卵巢癌（HBOC）中的双重突变有关 基因。临床前的FA研究很大程度上取决于转基因动物模型。但是，目前可用的FA 小鼠模型诞生没有发育缺陷，血液学异常必须是化学上的 诱导。我们的实验室开发了一种新的BRCA1CC突变小鼠模型，并在该模型中使用3-氨基酸缺失 BRCA1的盘绕圈（CC）域，该结构域特异性破坏了BRCA1-PALB2关联，导致RAD51的损失 负载和人力资源缺乏症。值得注意的是，BRCA1CC纯合小鼠出生于亚孟德尔的比例，新生儿 小鼠在人类中表现出类似于FA的一系列表型，包括身材矮小，BMF严重 贫血，成年小鼠患白血病。因此，BRCA1CC小鼠与人FA非常相似，并提供了 新的工具，以获得对基于病因的生物学途径的前所未有的见解。两者都是同源的 重组（HR）和微学介导的终端连接（MMEJ）是双链DNA断裂（DSB） DNA端切除引发的修复途径，该过程是通过核酸酶切除DSB的过程 形成单链（SS）DNA区域。在初步数据中，我们检查了人FA患者细胞，以及 BRCA1CC MEFS，用于HR和MMEJ活动。有趣的是，在降低HR时，MMEJ在FA中过度活化 细胞系。现在，我们假设过度活跃的DNA末端切除和MMEJ促进了分子 FA的发病机理。我们将解决以下具体目的：1）确定DNA修复途径 在FA中多动； 2）发现促进FA胚胎发育和发病机理的机制； 3） 检查MMEJ抑制对FA发病机理的影响。共同提议的实验将产生 对促进基因组不稳定性和FA的DNA修复机制的新见解。