Multiplexed functional analysis of BRCA1and BARD1 missense variants in DNA repair

BRCA1和BARD1错义突变体在DNA修复中的多重功能分析 摘要 未知意义的变体(VU)通常是错误的变体，对其的解释 表型影响被困在致病和良性之间的空隙中。这些氨基酸替换中的任何一个 可能会对编码蛋白质的结构或分子功能造成重大损害，因此 显著影响疾病风险，否则可能完全没有效果。VU的问题没有比这更明显的了 而在遗传性乳腺癌和卵巢癌的基因检测中，发表的VUS率从2%到42%不等 这取决于进行测试/变体调用的公司以及面板上包含的基因数量。为 仅BRCA1一项，在临床遗传学数据库ClinVar中就列出了1020个VU。解决……的问题 VUS解释，这是使基因检测结果对更多患者更有用所必需的，功能 分析可以用来理解每个变异是如何影响蛋白质功能的。但是，执行后自组织 按照目前的积累速度，对每一种变异进行功能性分析是不可能的。在这里我们 建议使用深度突变扫描来确定所有可能的错义变异对功能的影响 在BRCA1和BARD1中研究了它们在人类细胞DNA修复中的作用。我们的方法是在 我们在俄亥俄州立大学和华盛顿大学的实验室之间的合作衡量了 数百种蛋白质变体在同源定向DNA双链断裂中平行的功能能力 修复试验。该项目的成果将有两个可交付成果：第一个是对 两种肿瘤抑制因子在单一氨基酸分辨率下的序列-功能关系及其作用 保护基因组免受DNA双链断裂的影响。第二个结果是一个“查询表” BRCA1和BARD1中任何可能的错义变异对功能的影响 遗传学家帮助解释以前已经识别的和尚未识别的变异 有人在诊所看到过。

Multiplexed functional analysis of BRCA1 and BARD1 missense variants in DNA repair ABSTRACT Variants of unknown significance (VUS) are, in general, missense variants for which the interpretation of phenotypic impact is trapped in the void between pathogenic and benign. Any of these amino acid substitutions could cause major damage to the structure or molecular function of the encoded protein and therefore significantly impact disease risk, or it could have no effect all. Nowhere is the problem of VUS more apparent than in genetic testing for hereditary breast and ovarian cancer where published VUS rates range from 2-42% depending on the company doing testing/variant calling and number of genes included on the panel. For BRCA1 alone, there are 1020 VUS listed in the clinical genetics database, ClinVar. To address the problem of VUS interpretation, which is required to make genetic test results more useful for more patients, functional assays could be used to understand how each variant affects protein function. However, performing a post hoc functional assay for each variant as it is discovered is impossible at the current rate of accumulation. Here we propose to use deep mutational scanning to determine the functional impact of all possible missense variants in BRCA1 and BARD1 on their function in DNA repair in human cells. Our approach, developed in collaboration between our labs at Ohio State University and the University of Washington, measures the functional capacity of hundreds of protein variants in parallel in a homology directed DNA double strand break repair assay. The outcome of this project will have two deliverables: The first is an understanding of the sequence–function relationships, at single amino acid resolution, of two tumor suppressors in their role protecting the genome from DNA double strand breaks. The second outcome is a “look up table” for the functional impact of any possible missense variant in BRCA1 and BARD1 that can be used by clinical geneticists to aid interpretation for variants that have been identified previously and those that have not yet been seen in the clinic.