Functional Analysis of Variants Identified in Human Breast Cancer Susceptibility

人类乳腺癌易感性中鉴定的变异的功能分析

• 批准号: 8349373
• 负责人: SHYAM SHARAN
• 金额: $ 71.92万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8349373
• 关键词: Accounting; Age; Alleles; BRCA1 gene; BRCA2 gene; Bacterial Artificial Chromosomes; Biological Assay; Complement; Defect; Development; Disease; Dissection; Engineering; Genes; Genetic; Germ-Line Mutation; Hereditary Breast Carcinoma; High Prevalence; Human; Individual; Inherited; Malignant Neoplasms; Mus; Mutation; Population; Predisposition; Proteins; Reporting; Risk; Risk Factors; Sequence Analysis; Testing; United States; Variant; Woman; base; breast cancer diagnosis; cancer diagnosis; clinically significant; embryonic stem cell; high risk; malignant breast neoplasm; mutant; segregation; tumorigenesis

To evaluate the functional significance of human BRCA1 and BRCA2 variants and to carry out functional dissection of these proteins, we have developed a simple, reliable and physiologically relevant functional assay using mouse embryonic stem (ES) cells. The assay is based on the fact that Brca1 and Brca2-null ES cells cannot survive. Therefore we engineered ES cells to express a conditional allele of Brca1 or Brca2. Desired mutations are generated in the human BRCA1 or BRCA2 gene in bacterial artificial chromosomes (BAC) and introduced into the ES cells. Functional analysis is performed after deletion of the conditional allele. The ability of and the extent to which specific variants complement the lethality of ES cells associated with Brca1 or Brca2 deficiency is used to evaluate their functional significance. Variants that can rescue lethality are then screened for a defect in the known functions of BRCA1 and BRCA2.

为了评估人类 BRCA1 和 BRCA2 变体的功能意义并对这些蛋白质进行功能解剖，我们使用小鼠胚胎干 (ES) 细胞开发了一种简单、可靠且生理相关的功能测定方法。该测定基于 Brca1 和 Brca2 缺失 ES 细胞无法存活的事实。因此，我们工程化 ES 细胞来表达 Brca1 或 Brca2 的条件等位基因。细菌人工染色体 (BAC) 中的人类 BRCA1 或 BRCA2 基因产生所需的突变，并将其引入 ES 细胞中。删除条件等位基因后进行功能分析。特定变体补充与 Brca1 或 Brca2 缺陷相关的 ES 细胞致死性的能力和程度用于评估其功能意义。然后筛选能够挽救致死率的变体，以确定 BRCA1 和 BRCA2 已知功能是否存在缺陷。