Role of Damage Response in Bone Marrow Failure in Fanconi Anemia

描述(由申请人提供)：本项目的目标是从机制上了解与范可尼贫血(FA)相关的DNA代谢功能缺陷。FA是一种高度可变的遗传性疾病，出生后不久表现为进行性全血细胞减少，然后是骨髓衰竭。患有FA的儿童也可能有先天性骨骼和其他系统的异常，在疾病过程中，他们有很高的风险发展成某些癌症。FA途径参与维持基因组的稳定性，特别是在细胞周期的S期支持受损DNA的复制。然而，目前还不知道该通路到底是如何执行这一功能的。这在一定程度上是由于用于测量人类细胞复制分叉新陈代谢的直接体内功能分析的局限性。研究人员将使用一种新的、定量的和敏感的技术-微流控辅助显示拉伸的DNA，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缺失是FA相关死亡和发病的主要原因，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/或在这种应激反应中对FA途径的需求增加。通过一方面测量FA蛋白缺陷的造血干细胞和对照造血干细胞的体内复制，并将其与FA缺陷的原代成纤维细胞进行比较，他们将能够检验这一假说，并为进一步从机制上剖析干细胞中FA途径的功能奠定基础。作为这一机制洞察的第一步，研究人员将建立一种方案，用于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。 项目简介：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障碍的分子缺陷，范科尼贫血。这项工作的结果将有助于我们理解这种疾病的病因学，并潜在地了解其他可遗传的基因组不稳定综合征。

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.