Role of Damage Response in Bone Marrow Failure in Fanconi Anemia

描述（由申请人提供）：本项目的目标是获得与范可尼贫血（FA）相关的DNA代谢功能缺陷的机制理解。 FA是一种高度可变的遗传性疾病，出生后不久即表现为进行性全血细胞减少，然后是骨髓衰竭。 患有FA的儿童也可能有骨骼和其他系统的先天性异常，并且在疾病的后期，他们有患某些癌症的高风险。 FA途径与基因组稳定性维持有关，特别是在细胞周期的S期期间支持受损DNA的复制。 然而，目前还不清楚该途径究竟是如何执行这一功能的。 这部分是由于测量人细胞中复制叉代谢的直接体内功能测定的有限可用性。 研究人员将使用一种新的、定量的、敏感的技术，即拉伸DNA的微流体辅助显示，来确定FA途径的缺陷如何影响细胞在体内复制含有病变的DNA的能力。 研究人员将首先分析从FA患者细胞建立的细胞系，然后将他们的实验方案应用于临床上与FA病因学最相关的细胞类型-造血干细胞。 这种细胞类型的选择性耗竭是与FA相关的死亡率和发病率的主要原因，并且可以假设这种表型至少部分是由于这些细胞对DNA损伤诱导的复制应激的敏感性增加，和/或在对这种应激的响应中对FA途径的需求增加。 通过一方面测量FA蛋白缺陷和对照造血干细胞中的体内复制，另一方面将其与FA缺陷的原代成纤维细胞进行比较，他们将能够测试这一假设，并为进一步机械解剖干细胞中的FA通路功能奠定基础。 作为实现这一机制见解的第一步，研究人员将建立一个筛选在造血干细胞中存在DNA损伤的情况下改善复制的化合物的方案。 项目叙述：该项目使用一种独特的工具来查询人类骨髓衰竭和癌症易感性疾病范可尼贫血的分子缺陷。 这项工作的结果将有助于我们了解这种疾病的病因，并可能了解其他遗传性基因组不稳定综合征的病因。

DESCRIPTION (Provided by Applicant): The goal of this project is to gain mechanistic understanding of the functional defects of DNA metabolism associated with Fanconi anemia (FA). FA is a highly variable genetic disorder that manifests soon after birth with progressive pancytopenia and then bone marrow failure. Children with FA can also have congenital abnormalities of skeletal and other systems, and later in the course of the disease they are at high risk of developing certain cancers. FA pathway is implicated in genome stability maintenance and specifically in supporting replication of damaged DNA during S phase of the cell cycle. However, it is not known how exactly the pathway performs this function. This is in part due to the limited availability of direct, in vivo functional assays to measure replication fork metabolism in human cells. The investigators will use a novel, quantitative, and sensitive technology, microfluidics-assisted display of stretched DNA, to determine how deficiency in FA pathway affects the ability of cells to replicate lesion-containing DNA in vivo. The investigators will first analyze cell lines established from FA patient cells, and then apply their experimental scheme to the cell type that is clinically most relevant to the etiology of FA -hematopoietic stem cells. Selective depletion of this cell type is a leading cause of mortality and morbidity associated with FA, and it can be hypothesized that this phenotype is at least in part due to the increased sensitivity of these cells to DNA damage-induced replication stress, and/or increased requirement for the FA pathway in the response to this stress. By measuring replication in vivo in FA protein-deficient and control hematopoietic stem cells on the one hand, and comparing it with FA-deficient primary fibroblasts on the other, they will be able to test this hypothesis and set the stage for further mechanistic dissection of the FA pathway function in stem cells. As a first step towards this mechanistic insight, the investigators will establish a protocol for a screen for compounds that improve replication in the presence of DNA damage in hematopoietic stem cells. PROJECT NARRATIVE: This project uses a unique tool to query molecular defects of a human bone marrow failure and cancer predisposition disorder, Fanconi anemia. The results of this work will contribute to our understanding of the etiology of this disease and potentially of other heritable genomic instability syndromes.