FANCJ (BRIP1/BACH1) is a hereditary breast and ovarian cancer (HBOC) gene encoding a DNA helicase. Similar to HBOC genes, BRCA1 and BRCA2, FANCJ is critical for processing DNA inter-strand crosslinks (ICL) induced by chemotherapeutics, such as cisplatin. Consequently, cells deficient in FANCJ or its catalytic activity are sensitive to ICL-inducing agents. Unfortunately, the majority of FANCJ clinical mutations remain uncharacterized, limiting therapeutic opportunities to effectively use cisplatin to treat tumors with mutated FANCJ. Here, we sought to perform a comprehensive screen to identify FANCJ loss-of-function (LOF) mutations. We developed a FANCJ lentivirus mutation library representing approximately 450 patient–derived FANCJ nonsense and missense mutations to introduce FANCJ mutants into FANCJ knockout (K/O) HeLa cells. We performed a high-throughput screen to identify FANCJ LOF mutants that, as compared with wild-type FANCJ, fail to robustly restore resistance to ICL-inducing agents, cisplatin or mitomycin C (MMC). On the basis of the failure to confer resistance to either cisplatin or MMC, we identified 26 missense and 25 nonsense LOF mutations. Nonsense mutations elucidated a relationship between location of truncation and ICL sensitivity, as the majority of nonsense mutations before amino acid 860 confer ICL sensitivity. Further validation of a subset of LOF mutations confirmed the ability of the screen to identify FANCJ mutations unable to confer ICL resistance. Finally, mapping the location of LOF mutations to a new homology model provides additional functional information. Implications: We identify 51 FANCJ LOF mutations, providing important classification of FANCJ mutations that will afford additional therapeutic strategies for affected patients. This article is featured in Highlights of This Issue, [p. 933][1] [1]: /lookup/volpage/19/933?iss=6

中文翻译：

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BRCA1 相关 DNA 解旋酶和肿瘤抑制因子 FANCJ/BACH1/BRIP1 的综合突变分析

FANCJ (BRIP1/BACH1) 是编码 DNA 解旋酶的遗传性乳腺癌和卵巢癌 (HBOC) 基因。与 HBOC 基因 BRCA1 和 BRCA2 类似，FANCJ 对于处理由顺铂等化疗药物诱导的 DNA 链间交联 (ICL) 至关重要。因此，FANCJ 或其催化活性缺陷的细胞对 ICL 诱导剂敏感。不幸的是，大多数 FANCJ 临床突变仍然没有特征，限制了有效使用顺铂治疗 FANCJ 突变肿瘤的治疗机会。在这里，我们试图进行全面筛选以识别 FANCJ 功能丧失 (LOF) 突变。我们开发了一个 FANCJ 慢病毒突变库，代表大约 450 个患者衍生的 FANCJ 无义和错义突变，以将 FANCJ 突变体引入 FANCJ 敲除 (K/O) HeLa 细胞。我们进行了高通量筛选以鉴定 FANCJ LOF 突变体，与野生型 FANCJ 相比，这些突变体未能有效地恢复对 ICL 诱导剂、顺铂或丝裂霉素 C (MMC) 的抗性。基于未能赋予对顺铂或 MMC 的抗性，我们确定了 26 个错义和 25 个无义 LOF 突变。无义突变阐明了截断位置与 ICL 敏感性之间的关系，因为氨基酸 860 之前的大多数无义突变赋予 ICL 敏感性。对 LOF 突变子集的进一步验证证实了筛选能够识别无法赋予 ICL 抗性的 FANCJ 突变的能力。最后，将 LOF 突变的位置映射到新的同源模型提供了额外的功能信息。启示：我们确定了 51 个 FANCJ LOF 突变，提供 FANCJ 突变的重要分类，为受影响的患者提供额外的治疗策略。这篇文章被收录在本期的亮点中，[p. 933][1][1]：/lookup/volpage/19/933?iss=6