Study of molecular pathogenesis of Fanconi anemia

范可尼贫血是一种常染色体隐性造血疾病，至少有11个遗传不同的群体，其特征是细胞对DNA交联体过敏和染色体不稳定。到目前为止，已经确定了8个基因。包括这些基因产物的多蛋白复合物FANCAICJE/F/G/L是激活FANCD2为单泛素化形式所必需的。这种活性形式与BRCA1和BRCA2IFANCD1共同影响基因组的不稳定性。在本项目中，我们研究了n端氨基酸取代的FANCA突变体的功能，包括核定位信号和FANCA结合位点。我们的研究结果表明，包括FANCAICIE/FIGIL在内的稳定复合物的形成对于FANCD2的激活并不是必需的。由此可见，FANCA/L复合物的核进口在该通路中起核心作用，FANCG负向调节FANCA的核进口。此外，我们还鉴定出hsc70是一种fanca结合蛋白。hsc70的显性阴性形式和hsp90特异性抑制剂17-AAG抑制了FANCA的核定位，这表明FANCA的核导入需要包括hsc70在内的伴侣复合物。我们在27名日本FA患者中发现了FANCA突变，并发现了该基因大缺失产生的新分子机制。我们发现，骨髓衰竭的长期缓解与患者骨髓谱系选择性细胞扩增和逆转有关。这个病例提示了基因治疗的临床应用价值。

Fanconi anemia is an autosomal recessive disorder of hematopoiesis with at least 11 genetically different groups, characterized by cellular hypersensitivity to DNA cross-linkers and chromosome instability. To date, 8 genes have been identified. A multiprotein complex including these gene products, FANCAICJE/F/G/L is required for activation of FANCD2 into a monoubiquitinated form. This active form affects genomic instability in collaboration with BRCA1 and BRCA2IFANCD1. In this project, we studied functions of FANCA mutants with amino acid substitution in its N-terminal region including nuclear localization signal and FANCG-binding sites. Our results indicate that formation of a stable complex including FANCAICIE/FIGIL is not essential for FANCD2 activation. It seems that nuclear import of FANCA/L complex palys a central role in this pathway, and that FANCG negatively regulates nuclear import of FANCA. Furthermore, we identified Hsc7O as a FANCA-binding protein. A dominant-negative form of Hsc7O and 17-AAG, a specific inhibitor of Hsp9O, inhibited nuclear localization of FANCA, suggesting that a chaperone complex including Hsc7O is required for nuclear import of FANCA. We identified FANCA mutations in 27 Japanese FA patients and found novel molecular mechanisms of generation of large deletions in this gene. We found that long-term remission of bone marrow failurewas associated with myeloid lineage-selective expansion of cells with reversion in a patient. This case suggests the clinical usefulness of gene therapy.