Multiplexed functional analysis of BRCA1and BARD1 missense variants in DNA repair

DNA 修复中 BRCA1 和 BARD1 错义变异的多重功能分析

基本信息

批准号：
10520020
负责人：
JEFFREY D PARVIN
金额：
$ 43.92万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-12-01 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10520020
关键词：
Access to Information Activities of Daily Living Address Affect Alleles Amino Acid Substitution Amino Acids Ankyrin Repeat Atlases BARD1 gene BRCA1 Protein BRCA1 gene BRCA2 gene BRCT Domain Benign Biochemistry Biological Assay C-terminal Cell Line Cell physiology Cells Classification ClinVar Clinic Clinical Collaborations DNA Double Strand Break DNA Repair DNA sequencing Data Databases Disease Distress Double Strand Break Repair Family Gene Frequency Genes Genetic Genetic Databases Genome Growth Health Hereditary Breast and Ovarian Cancer Syndrome Human Industry Information Dissemination Legal patent Length Libraries Mammalian Cell Measurement Measures Medical Genetics Methods Molecular N-terminal Nucleotides Ohio Oncogenes Outcome PALB2 gene Pathogenicity Patients Phenotype Physicians Plasmids Population Prevention strategy Proteins Protocols documentation Publishing RNA Splicing Reporter Reporting Resolution Resources Role Sensitivity and Specificity Serine Structure System Test Result Testing Tumor Suppressor Proteins Universities Validation Variant Washington Woman Work analytical tool cancer prevention cancer risk cost density disorder risk experimental study gene product genetic testing genome sequencing improved insight mutation screening new technology novel protein function repair function technology development tool tumor user-friendly variant of unknown significance web portal web site

项目摘要

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.

DNA 修复中 BRCA1 和 BARD1 错义变异的多重功能分析抽象的一般来说，意义不明的变体（VUS）是错义变体，其解释为表型影响被困在致病性和良性氨基酸替代之间的空白中。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 VUS 的问题最为明显，但它可能对疾病风险产生显着影响，也可能没有任何影响。高于遗传性乳腺癌和卵巢癌的基因检测，其中公布的 VUS 率范围为 2-42% 取决于进行测试/变异调用的公司以及面板中包含的基因数量。仅BRCA1，临床遗传学数据库ClinVar就列出了1020个VUS来解决这一问题。 VUS 解释是使基因检测结果对更多患者更有用所必需的分析可用于了解每种变体如何影响蛋白质功能，但需要进行事后分析。以目前的积累速度，对每个发现的变体进行功能分析是不可能的。建议使用深度突变扫描来确定所有可能的错义变异的功能影响 BRCA1 和 BARD1 在人类细胞 DNA 修复中的功能。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，测量了数百个蛋白质变体在同源定向 DNA 双链断裂中并行的功能能力该项目的成果将有两个可交付成果：第一个是对修复分析的理解。在单个氨基酸分辨率下，两种肿瘤抑制因子在其作用中的序列-功能关系保护基因组免受 DNA 双链断裂的影响。 BRCA1 和 BARD1 中任何可能的错义变异对临床可使用的功能影响遗传学家帮助解释之前已发现和尚未发现的变异在诊所见过。