Multiplexed functional analysis of BRCA1and BARD1 missense variants in DNA repair

BRCA 1和BARD 1错义突变体在DNA修复中的多重功能分析 摘要 意义未知的变体（VUS）通常是错义变体，对于这些错义变体， 表型影响被困在致病性和良性之间的空隙中。这些氨基酸的替换 可能对编码蛋白的结构或分子功能造成重大损害， 显著影响疾病风险，也可能完全没有影响。VUS的问题在任何地方都没有 而在遗传性乳腺癌和卵巢癌的基因检测中，已公布的VUS率范围为2-42% 这取决于进行测试/变异调用的公司和面板上包含的基因数量。为 仅BRCA 1，临床遗传学数据库ClinVar中就列出了1020个VUS。缓解因 VUS解释，这是必要的，使基因检测结果更有用的更多的病人，功能 可以使用分析来了解每个变体如何影响蛋白质功能。然而，执行一个事后 在目前的积累速度下，不可能对所发现的每种变异体进行功能性测定。这里我们 我建议使用深度突变扫描来确定所有可能的错义变体的功能影响 BRCA 1和BARD 1在人类细胞DNA修复中的功能。我们的方法， 我们在俄亥俄州州立大学和华盛顿大学的实验室之间的合作， 在同源性指导的DNA双链断裂中平行的数百种蛋白质变体的功能能力 修复试验该项目的成果将有两个交付成果：第一个是对 两种肿瘤抑制因子在其作用中的序列-功能关系，单个氨基酸分辨率 保护基因组免受DNA双链断裂。第二个结果是一个“查找表”， BRCA 1和BARD 1中任何可能的错义变体的功能影响， 遗传学家帮助解释以前已经确定的变异和尚未确定的变异 在诊所见过。

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.