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：
10059180
负责人：
JEFFREY D PARVIN
金额：
$ 52.34万
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 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10059180
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 Gene Proteins 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错义变体的多重功能分析抽象的一般而言，未知意义的变体（VUS）是错义变体的解释表型冲击被困在致病性和良性之间的空隙中。这些氨基酸取代可能对编码蛋白的结构或分子功能造成重大损害，因此显着影响疾病的风险，否则可能没有任何作用。 VUS的问题没有什么比更明显的了与遗传性乳腺癌和卵巢癌的基因检测相比，发表的VUS率范围为2-42％根据公司进行测试/变体调用和面板上包含的基因数量的不同。为了仅BRCA1，临床遗传学数据库Clinvar中就有1020个VU。解决的问题 VUS解释，这需要使遗传测试结果对更多患者更有用，功能性测定可用于了解每个变体如何影响蛋白质功能。但是，执行事后在当前积累速率下，每个变体的功能测定是不可能的。我们在这里提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在BRCA1和BARD1中，它们在人类细胞中DNA修复中的功能。我们发展的方法我们在俄亥俄州立大学和华盛顿大学实验室之间的合作，衡量同源性DNA双链断裂中平行数百种蛋白质变体的功能能力维修测定法。该项目的结果将有两个可交付成果：第一个是对在单个氨基酸分辨率下，两种肿瘤补充剂的序列 - 功能关系保护基因组免受DNA双链的断裂。第二个结果是 BRCA1和BARD1中任何可能的错义变体的功能影响可以由临床使用仿制家为以前已经确定的变种而辅助解释他在诊所被发现。