Structure function relationships from deep mutational scanning in human cardiomyopathy

人类心肌病深度突变扫描的结构功能关系

• 批准号: 9884435
• 负责人: Euan A Ashley
• 金额: $ 72.28万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9884435
• 关键词: 3-Dimensional; ATP phosphohydrolase; Alleles; Amino Acid Sequence; Arrhythmia; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell Size; Cell model; Cells; Chemicals; Classification; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Custom; Cytidine Deaminase; Data; Data Set; Disease; Disease model; Engineering; Evaluation; Fluorescence; Generations; Genes; Genetic Techniques; Genetic Transcription; Genetic Variation; Genome; Genomics; Goals; Gold; Health; Heart; Heart failure; Human; Human Genetics; Hypertrophic Cardiomyopathy; Hypertrophy; In Situ; Individual; Inherited; Integrase; Libraries; Maps; Measurement; Mendelian disorder; Methods; Microfluidics; Molecular; Molecular Motors; Molecular Structure; Mutagenesis; Mutation; Natural experiment; Oligonucleotides; Optical Methods; Pathogenicity; Patients; Pattern; Phenotype; Population; Population Genetics; Protein Biochemistry; Protein Chemistry; Proteins; Reporter; Sarcomeres; Scanning; Sorting - Cell Movement; Statistical Models; Structure; Structure-Activity Relationship; Sudden Death; Suggestion; System; Technology; Test Result; Testing; Time; Transcript; Variant; adjudicate; base; causal variant; cell motility; deep sequencing; gene function; genetic testing; genetic variant; heart cell; human disease; individual patient; induced pluripotent stem cell; innovation; mutant; mutation screening; novel; nuclease; patient population; programs; protein function; protein protein interaction; protein structure; single-cell RNA sequencing; stoichiometry; sudden cardiac death; tool; transcriptomics; 3-Dimensional; ATP phosphohydrolase; Alleles; Amino Acid Sequence; Arrhythmia; Biological Assay; Biological Models; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cell Size; Cell model; Cells; Chemicals; Classification; Clinic; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Custom; Cytidine Deaminase; Data; Data Set; Disease; Disease model; Engineering; Evaluation; Fluorescence; Generations; Genes; Genetic Techniques; Genetic Transcription; Genetic Variation; Genome; Genomics; Goals; Gold; Health; Heart; Heart failure; Human; Human Genetics; Hypertrophic Cardiomyopathy; Hypertrophy; In Situ; Individual; Inherited; Integrase; Libraries; Maps; Measurement; Mendelian disorder; Methods; Microfluidics; Molecular; Molecular Motors; Molecular Structure; Mutagenesis; Mutation; Natural experiment; Oligonucleotides; Optical Methods; Pathogenicity; Patients; Pattern; Phenotype; Population; Population Genetics; Protein Biochemistry; Protein Chemistry; Proteins; Reporter; Sarcomeres; Scanning; Sorting - Cell Movement; Statistical Models; Structure; Structure-Activity Relationship; Sudden Death; Suggestion; System; Technology; Test Result; Testing; Time; Transcript; Variant; adjudicate; base; causal variant; cell motility; deep sequencing; gene function; genetic testing; genetic variant; heart cell; human disease; individual patient; induced pluripotent stem cell; innovation; mutant; mutation screening; novel; nuclease; patient population; programs; protein function; protein protein interaction; protein structure; single-cell RNA sequencing; stoichiometry; sudden cardiac death; tool; transcriptomics

PROJECT SUMMARY The natural experiment of human genetic variation can be used to infer structure-function relationships for key disease genes. We have previously demonstrated that population-scale genetic variation data can be harnessed to illuminate structure-function relationships for genes causative of the Mendelian disease hypertrophic cardiomyopathy. However, due to the rarity of individual causative variants, population genetics is ultimately limiting to the goal of understanding the functional importance of the entire coding region of any specific gene. There is an urgent need for experimental alternatives. Here, we propose to introduce targeted genetic variation into human induced pluripotent stem cell derived cardiomyocytes (iPSC-CM) at scale (Aim 1). We propose two complementary strategies for deep mutational scanning of the most common genes causing hypertrophic cardiomyopathy, MYH7, MYBPC3 and TNNT2. The first, CRISPR-X, is a fusion of a cytidine deaminase (AID) with nuclease-inactive Cas9 (dCas9), and provides targeted mutational coverage in situ. The second, POPcode, uses a uracilated gene template and a set of mutant oligos to create an allelic library, which is then integrated into the genome using a Dual-Integrase Cassette Exchange (DICE). To characterize these cells, we further develop a custom microfluidics-based, fast optical method to phenotype single cells in real time (Aim 2). Predictions of pathogenicity according to both cell size and a fluorescence marker of the hypertrophy expression program will be mapped to 3D protein structures using our spatial scanning approach and tested against gold standard adjudicated patient variant data. Finally, we will investigate variant-specific mechanisms of disease using single cell RNA sequencing to assess the effect of each variant on allelic stoichiometry and transcriptional programming, as well as protein biochemistry to assess sarcomere protein interaction and power generation (Aim 3). In summary, we plan comprehensive evaluation of all potential coding variation in the most frequently causative genes for the most common Mendelian cardiovascular disease. Using innovative phenotyping tools and novel statistical approaches to the integration of population and cellular data, we aim to understand the structure and function of these genes in health and disease, providing an experimental basis for the classification of genetic variants in the clinical setting.

项目摘要 人类遗传变异的自然实验可用于推断钥匙的结构功能关系 疾病基因。我们以前已经证明，人口规模的遗传变异数据可以是 利用Mendelian疾病的基因启动结构 - 功能关系 肥厚性心肌病。但是，由于个体原因变异的稀有性，人口遗传学是 最终限制了了解任何任何编码区域的功能重要性的目标 特定基因。迫切需要实验替代方案。在这里，我们建议介绍目标 人类诱导多能干细胞衍生的心肌细胞（IPSC-CM）的遗传变异（AIM 1）。 我们提出了两种互补的策略，以对最常见的基因进行深层突变扫描 肥厚的心肌病，MYH7，MYBPC3和TNNT2。第一个是CRISPR-X，是胞苷的融合 脱氨酶（AID）与核酸酶 - 活性CAS9（DCAS9），并在原位提供靶向突变覆盖率。这 其次，爆米法使用尿液的基因模板和一组突变寡寡核武物来创建一个等位基因库，该库 然后使用双积聚盒交换（DICE）集成到基因组中。描述这些 细胞，我们进一步开发了一种基于自定义的基于微流体的快速光学方法 时间（目标2）。根据细胞大小和荧光标记的致病性预测 肥大表达程序将使用我们的空间扫描方法映射到3D蛋白结构 并针对金标准裁决的患者变体数据进行了测试。最后，我们将研究特定于变体的 使用单细胞RNA测序的疾病机制来评估每个变异对等位基因的影响 化学计量和转录编程，以及评估肌动蛋白的蛋白质生物化学 互动和发电（AIM 3）。总而言之，我们计划对所有潜力的全面评估 最常见的Mendelian心血管的最常见病变基因的编码变化 疾病。使用创新的表型工具和新型统计方法来整合人口 和细胞数据，我们旨在了解这些基因在健康和疾病中的结构和功能， 为临床环境中遗传变异的分类提供了实验基础。