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: 10364603
• 负责人: Euan A Ashley
• 金额: $ 67.77万
• 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/10364603
• 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; induced pluripotent stem cell derived cardiomyocytes; 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以用来推断关键基因的结构-功能关系 疾病基因我们以前已经证明，群体规模的遗传变异数据可以 利用它来阐明导致孟德尔疾病的基因的结构-功能关系 肥厚型心肌病然而，由于个别致病变异的罕见性，群体遗传学是 最终限制于理解任何肿瘤的整个编码区的功能重要性的目标， 特定基因迫切需要实验性的替代品。在这里，我们建议有针对性地引入 将遗传变异大规模地引入人诱导多能干细胞衍生的心肌细胞（iPSC-CM）中（目标1）。 我们提出了两种互补的策略，用于对最常见的基因进行深度突变扫描， 肥厚型心肌病、MYH 7、MYBPC 3和TNNT 2。第一个CRISPR-X是一个融合了 在一些实施方案中，该方法包括将脱氨酶（AID）与核酸酶失活的Cas9（dCas 9）结合，并提供原位靶向突变覆盖。的 第二种是POPcode，使用尿嘧啶化基因模板和一组突变寡核苷酸来创建等位基因文库， 然后使用双整合酶盒交换（DICE）整合到基因组中。来刻画这种 细胞，我们进一步开发了一种定制的基于微流体的快速光学方法，以在真实的中表型单细胞。 时间（目标2）。根据细胞大小和荧光标记预测致病性 肥大表达程序将使用我们的空间扫描方法映射到3D蛋白质结构 并针对金标准判定的患者变异数据进行测试。最后，我们将研究变体特异性 使用单细胞RNA测序来评估每种变体对等位基因的影响， 化学计量和转录编程，以及蛋白质生物化学来评估肌节蛋白 相互作用和发电（目标3）。总之，我们计划全面评估所有潜在的 最常见的孟德尔心血管疾病的最常见致病基因的编码变异 疾病使用创新的表型分析工具和新的统计方法整合人口 和细胞数据，我们的目标是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实验基础。