Searching for a New Fanconi Anemia-BRCA Pathway Gene on Chromosome 11p

描述(申请人提供)：Fanconi贫血(FA)是一种罕见的遗传性疾病，其特征是身体出生缺陷、骨髓衰竭和癌症易感性。FA患者细胞对DNA交联剂如丝裂霉素C(MMC)高度敏感。到目前为止，已鉴定出13个FA基因，这些基因的蛋白产物在FA-BRCA途径中协同作用，修复受损的DNA。然而，有几个FA患者的潜在遗传缺陷尚不清楚。因此，可以得出结论，新的FA基因仍有待发现。FA-BRCA途径激活的关键步骤是Fanconi贫血D2(FANCD2)蛋白的单一泛素化。FANCD2蛋白的单一泛素化信号转位到细胞核，在那里它起到DNA修复的作用。FANCD2蛋白的单一泛素化是由至少八个“上游”FA核心复合体蛋白的协同活性促进的。我们最近发现了一个中国仓鼠卵巢突变株CHO UV-1，它的细胞特征与FA患者细胞在八种FA核心复合体成分中的任何一种都缺陷无法区分。例如，CHO UV-1细胞对MMC高度敏感，FANCD2蛋白的单一泛素化存在缺陷。此外，我们还发现在CHO UV-1细胞中引入人类11号染色体片段可以修复细胞缺陷。这些发现有力地表明在该染色体区域存在一个新的FA-BRCA途径基因。这项提议的目标是识别这种基因。提出了两个目标：首先，利用生物信息学的方法，我们将分析该区域所有已知的基因，并编制候选FA-BRCA基因列表。利用RNA干扰，我们将去除正常细胞中的这些基因，并确定对细胞对MMC的敏感性和对FANCD2单一泛素化的影响。其次，我们将尝试通过感染逆转录病毒全基因组文库来挽救CHO UV-1细胞的FA样表型。感染的CHO UV-1细胞将在预先确定的致死浓度的MMC存在下生长，并分离出MMC抗性克隆。随后将回收并鉴定校正后的cDNA.我们的结果可能导致在染色体11p上发现一个新的FA-BRCA途径基因。更深入地了解FA-BRCA通路的调控将有助于改进对FA的诊断和治疗，并提高我们对FA和普通(非FA)人群中白血病易感性的理解。公共卫生相关性：更多地了解Fanconi贫血(FA)-BRCA途径的调节可能有助于改进FA的诊断和治疗方法，并提高我们对FA和普通(非FA)人群中白血病易感性的理解。

DESCRIPTION (provided by applicant): Fanconi anemia (FA) is a rare genetic disease characterized by physical birth defects, bone marrow failure, and cancer susceptibility. FA patient cells are hypersensitive to DNA crosslinking agents, e.g. mitomycin C (MMC). To date, thirteen FA genes have been identified, and the protein products of these genes function cooperatively in a pathway, the FA-BRCA pathway, to repair damaged DNA. Several FA patients exist, however, for whom the underlying genetic defect is unknown. Therefore, it can be concluded that new FA genes remain to be discovered. A critical step in the activation of the FA-BRCA pathway is the mono- ubiquitination of the Fanconi anemia D2 (FANCD2) protein. Mono-ubiquitination of the FANCD2 protein signals its translocation to the nucleus where it functions in DNA repair. The mono-ubiquitination of the FANCD2 protein is facilitated by the concerted activity of at least eight `upstream' FA core complex proteins. We have recently identified a Chinese Hamster Ovary mutant CHO UV-1, which has cellular characteristics indistinguishable from FA patient cells defective in any of the eight FA core complex components. For example, the CHO UV-1 cells are hypersensitive to MMC and are defective in the mono-ubiquitination of the FANCD2 protein. Furthermore, we have determined that the cellular defects of the CHO UV-1 cells can be rescued by the introduction of a fragment of human chromosome 11. These findings strongly suggest that a new FA-BRCA pathway gene resides in this chromosomal region. The goals of this proposal are to identify this gene. Two aims are proposed: First, using a bioinformatics approach we will analyze all the known genes in this region and compile a list of candidate FA-BRCA genes. Using RNA interference we will deplete these genes from normal cells and determine the effects on cellular sensitivity to MMC and on the mono- ubiquitination of FANCD2. Second, we will attempt to rescue the FA-like phenotypes of the CHO UV-1 cells by infection with a retroviral whole genome cDNA library. The infected CHO UV-1 cells will be grown in the presence of a pre-determined lethal concentration of MMC, and MMC-resistant clones will be isolated. The correcting cDNA will subsequently be recovered and identified. Our results could lead to the identification of a new FA-BRCA pathway gene on chromosome 11p. A greater understanding of the regulation of the FA-BRCA pathway will lead to improved diagnostic and therapeutics to FA, and improve our understanding of leukemia susceptibility in the FA and general (non-FA) populations. PUBLIC HEALTH RELEVANCE: A greater understanding of the regulation of the Fanconi anemia (FA)-BRCA pathway may lead to improved diagnostic and therapeutic approaches to FA, as well as improve our understanding of leukemia susceptibility in the FA and general (non-FA) populations.