Role of Damage Response in Bone Marrow Failure in Fanconi Anemia

描述（由申请人提供）：该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，出生后不久表现为进行性全血细胞减少症，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，在疾病的后期，他们患某些癌症的风险很高。FA通路涉及基因组稳定性的维持，特别是在细胞周期的S期支持受损DNA的复制。然而，目前尚不清楚该途径究竟是如何发挥这一功能的。这在一定程度上是由于有限的可用性，直接的，在体内的功能测定，以测量复制叉代谢在人类细胞。研究人员将使用一种新的、定量的、敏感的技术，微流体辅助显示拉伸DNA，以确定FA途径的缺陷如何影响细胞在体内复制病变DNA的能力。

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.