ISOLATION OF THE DEB-SENSITIVITY GENE OF FANCONI ANEMIA

范可尼贫血（FA）是一种常染色体隐性遗传病

Fanconi anemia (FA) FA is an autosomal recessive disorder of childhood which is characterized by multiple developmental anomalies that affect the skeletal and renal systems, an invariable and lethal pancytopenia, and an increased predisposition to neoplastic disease, especially myeloid leukemia. Cultured cells from FA patients exhibit increased spontaneous chromosome breakage compared to normal cells. They also are hypersensitive to the cell killing and clastogenic effect of DNA cross-linking and alkylating agents such as mitomycin C (MMC) and diepoxybuate (DEB). We have developed a clonogenic survival system that allows unambiguous discrimination between normal and FA cells based on DEB hypersensitivity. Using this as the selective system, we have demonstrated that both the cellular and chromosomal hypersensitivity of FA cells to DEB treatment can be completely corrected by transfection of human placental or Chinese hamster DNA as calcium phosphate precipitates. Thus, a gene or genes that complement the key cellular phenotypes of FA are present in both human and Chinese hamster DNA. Furthermore, they stably integrate and express upon transfection into mutant cells. If the repair defect turns out to be the primary cellular lesion of this disorder, then cloning and characterization of the responsible gene(s) is significant not only for understanding the molecular basis of the disorder, but also for planning strategies for eventual in vivo correction of the hematopoietic defect. The overall aim of this project is to clone and characterize the gene(s) defective in FA that lead to DNA cross-linking and alkylating agent hypersensitivity with the following Specific Aims: (i) Isolate linked transfections involving the resistance genes of human and hamster derivation and the co- transfect neo- gpt genes using the latter as phenotypic and genotypic markers in second and subsequent rounds of transfection. (ii) Isolate double minute chromosome (DM) DNA from resistant cell lines that express DMs using the in-gel renaturation technique and transfect the amplified sequences back into mutant cells to determine if these indeed represent amplified resistance genes. (iii) Isolate resistance determining sequences of hamster origin by screening the genomic library of a DEB strain derived from hamster DNA transfection with labelled hamster DNA as the probe. (iv) Isolate resistance genes by using subtractive hybridization of a cDNA library constructed from normal cells subjected to chronic DEB exposure and excess poly A+ mRNA from untreated normal and FA cells. (v) Undertake detailed molecular characterization of the structure and function of the resistance gene(s).