Functional phenotyping of human genetic variation

描述(由申请人提供)：拟议研究的目标是开发新的工具和系统的方法，以高通量的方式捕捉和实验确定人类突变的功能后果(即表型)。这项工作直接解决了确定突变功能后果的日益增长的挑战，特别是对于大量未知功能意义(VU)的变体。我们将分析15个成员的Fanconi贫血(或FANC)基因家族的错义突变，该家族在基因组稳定性和对DNA损伤的反应以及疾病风险和治疗方面发挥着关键作用。该基因家族为本研究提供了一个具有生物学意义和临床意义的靶点。意义：基因组计划正在识别更多未知功能意义(VUS)的突变。这项拟议的研究将建立一种简便的方法，使用定义明确的临床相关终点，在基于人类细胞的等基因分析系统中快速和系统地确定大量(数十到数百)并行突变的功能后果。方法：我们正在整合计算工具和新的实验工具，在基于人类细胞的检测系统中确定人类FANC基因所有错义突变的功能表型。我们的目标是：目标1：捕获所有人类Fanconi贫血/FANC基因突变并进行电子分析。目的2：对所有FANC基因错义突变进行高通量人类细胞表型分析。目的3：对FANC蛋白在DNA损伤中的功能进行集成的、数据驱动的建模。创新：拟议的研究有四个创新特征：1.全球性的、无偏见地捕获FANC基因家族中的所有遗传变异；2.使用6种不同的预测算法进行系统的突变功能预测和排名评分；3.一种新的、高通量的基于细胞的功能表型分析，它是定量的，并且建立在临床相关的细胞端点的基础上；以及4.使用突变表型和分子数据来建立对DNA损伤作出反应的Fanconi蛋白质功能的综合动态模型。预期结果：拟议的研究将建立一种系统的方法来确定人类突变的功能表型，包括越来越多的VU。这项工作的结果将极大地提高我们对Fanconi基因功能的理解，同时为其他人类生物学和遗传病研究提供一般方法和新的工具。

DESCRIPTION (provided by applicant): The goal of the proposed research is to develop new tools and a systematic approach to capture and experimentally determine the functional consequences (i.e., the phenotype) of human mutations in high-throughput fashion. This work directly addresses the growing challenge of determining mutation functional consequences, especially for the large number of variants of unknown functional significance (VUS's). We will analyze missense mutations in the 15-member Fanconi anemia (or FANC) gene family that plays key roles in genomic stability and the response to DNA damage, as well as in disease risk and therapy. This gene family provides a biologically interesting and clinically relevant target fo this research. Significance: Genome projects are identifying ever larger numbers of mutations of unknown functional significance (VUS's). The proposed research will establish a facile way to rapidly and systematically determine the functional consequences of large numbers (dozens to hundreds) of mutations in parallel in an isogenic, human cell-based assay system using well-defined, clinically relevant endpoints. Approach: We are integrating computational and new experimental tools to determine in a human cell-based assay system the functional phenotype of all missense mutations in the human Fanconi anemia (FANC) genes. Our Aims are: Aim 1: Capture and in silico analysis of all human Fanconi anemia/FANC gene mutations. Aim 2: High-throughput human cell phenotyping of all FANC gene missense mutations. Aim 3: Integrated, data-driven modeling of FANC protein function in response to DNA damage. Innovation: The proposed research has four innovative features: 1. global, ascertainment bias-free capture of all genetic variation in the FANC gene families; 2. systematic mutation functional prediction and rank-scoring using six different prediction algorithms; 3. a new, high-throughput cell-based functional phenotyping assay that is quantitative, and built on clinically relevant cellular endpoints; and 4. the use of mutation phenotyping and molecular data to build an integrated dynamic model of Fanconi protein function in response to DNA damage. Anticipated outcomes: The proposed research will establish a systematic approach for determining the functional phenotype of human mutations including the growing number of VUS's. Results of this work should substantially improve our understanding of Fanconi gene function, while providing a general approach and new tools to enable other human biology and genetic disease research.