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

描述（由申请人提供）：范可尼贫血（Fanconi anemia， FA）是一种罕见的遗传性疾病，其特征是身体先天缺陷、骨髓衰竭和癌症易感性。FA患者细胞对DNA交联剂过敏，如丝裂霉素C （MMC）。迄今为止，已经鉴定了13个FA基因，这些基因的蛋白产物在FA- brca通路中协同起作用，修复受损的DNA。然而，存在一些FA患者，其潜在的遗传缺陷是未知的。因此，新的FA基因仍有待发现。FA-BRCA通路激活的一个关键步骤是Fanconi贫血D2 （FANCD2）蛋白的单泛素化。FANCD2蛋白的单泛素化信号表明其易位到细胞核，在细胞核中起DNA修复作用。FANCD2蛋白的单泛素化是由至少8个“上游”FA核心复合物蛋白的协同活性促进的。我们最近发现了一种中国仓鼠卵巢突变体CHO UV-1，其细胞特征与FA患者细胞在8种FA核心复合物成分中的任何一种缺陷都无法区分。例如，CHO UV-1细胞对MMC过敏，并且在FANCD2蛋白的单泛素化中存在缺陷。此外，我们已经确定CHO UV-1细胞的细胞缺陷可以通过引入人类11号染色体片段来挽救。这些发现强烈提示一个新的FA-BRCA通路基因存在于该染色体区域。这项提议的目标是识别这种基因。我们提出了两个目标：首先，我们将使用生物信息学方法分析该区域的所有已知基因，并编制候选FA-BRCA基因列表。利用RNA干扰，我们将从正常细胞中去除这些基因，并确定对细胞对MMC的敏感性和对FANCD2单泛素化的影响。其次，我们将尝试通过逆转录病毒全基因组cDNA文库感染CHO UV-1细胞来挽救fa样表型。受感染的CHO UV-1细胞将在预先确定的致死浓度的MMC中生长，并分离出抗MMC的克隆。校正cDNA随后将被恢复和鉴定。我们的结果可能导致在11p染色体上鉴定一个新的FA-BRCA通路基因。对FA- brca通路调控的更深入了解将有助于改善FA的诊断和治疗方法，并提高我们对FA和一般（非FA）人群白血病易感性的认识。公共卫生相关性：对Fanconi贫血(FA)-BRCA通路调控的更深入了解可能会改善FA的诊断和治疗方法，并提高我们对FA和一般（非FA）人群白血病易感性的认识。

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.