Genetic variation impacting the airway smooth muscle in children with asthma

遗传变异影响哮喘儿童气道平滑肌

• 批准号: 10044061
• 负责人: Walter Eckalbar
• 金额: $ 24.9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10044061
• 关键词: ATAC-seq; Acute; Address; Affect; Albuterol; Asthma; Binding; Biological Assay; Bronchodilation; Bronchodilator Agents; CRISPR/Cas technology; Cell Culture Techniques; Cell Line; Cells; ChIP-seq; Child; Childhood; Chromatin; Chronic Disease; Clinical; Collaborations; Communities; Data; Data Analyses; Disease; Encyclopedias; Enhancers; Environment; Ethnic Origin; Ethnic group; Gene Expression; Gene Expression Profiling; Gene Mutation; Genes; Genetic; Genetic Variation; Genomic Segment; Genomics; Goals; Haplotypes; Heritability; Individual; Laboratories; Laboratory Research; Lead; Maps; Measures; Mediating; Medical Genetics; Mexican; Modeling; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacogenomics; Phase; Phenotype; Play; Population; Prevalence; Protocols documentation; Puerto Rican; Quantitative Trait Loci; Race; Regulator Genes; Regulatory Element; Relaxation; Research; Resolution; Respiratory physiology; Risk; Role; SNP genotyping; Severities; Smooth Muscle Myocytes; Symptoms; Technology; Testing; Training; Treatment outcome; Untranslated RNA; Variant; Whole Blood; Work; asthmatic patient; beta-2 Adrenergic Receptors; career; cell type; epigenomics; experience; experimental study; functional genomics; genetic variant; genome editing; genome wide association study; genome-wide; improved; induced pluripotent stem cell; minority children; multidisciplinary; novel; phenotypic data; precision medicine; racial and ethnic; respiratory smooth muscle; response; skills; trait; transcriptome sequencing

PROJECT SUMMARY Albuterol is the most commonly prescribed medication in the world for short-term relief of asthma symptoms, and functions to control these symptoms through relaxation of the airway smooth muscle. Response to albuterol varies widely between individuals and by race/ethnicity. Genome wide association studies (GWAS) have uncovered many loci associated with the genetic factors of asthma risk and the pharmacogenomics of albuterol response. However, these loci reside primarily in uncharacterized noncoding genomic regions, and the genetic basis of asthma and albuterol response remains largely unknown. I hypothesize that rare, population- specific variants inside gene regulatory elements active in airway smooth muscle cells contribute to racial/ethnic and interindividual differences in asthma severity and albuterol response. In order to test this hypothesis, I will build on my functional genomics training and add to them novel skills I will develop in the training phase of this proposal in cellular reprograming, CRISPR/Cas9 genome editing and quantitative trait loci (QTL) analysis. With these new skills, I will utilize functional genomic technologies of RNA-seq, ChIP-seq and ATAC-seq to characterize the gene expression and gene regulatory elements active in primary bronchial smooth muscle cells (BSMCs), generate a robust protocol for creation of induced pluripotent stem cell (iPSC) derived BSMCs, and characterize their gene regulatory environment relative to primary BSMCs (Aim K1). This aim will provide an encyclopedia of active genes, pathways and regulatory elements in a critical cell type relevant to asthma and albuterol response. Through this aim, I will also provide the asthma research community with a well characterized protocol to create patient-specific, iPSC-derived BSMCs (iBSMCs) which could be used for individual genetic and drug assays. I will also identify variants that alter regulatory element activity and gene expression through differential enhancer assays and CRISPR/Cas9 genome editing followed by RNA-seq and ATAC-seq (Aim K2). In the independent phase of this project, I will create iBSMC lines from 100 asthmatic patients with deep genetic and phenotypic data relating to lung function and albuterol response. I will then use these iBSMC lines to carry out expression QTL and chromatin accessibility QTL mapping to identify genetic variants that alter gene expression and enhancer activity, contributing to asthma severity and albuterol response (Aim R1). Finally, I will functionally characterize these genomic variants for their general and ethnic-specific alterations to the gene regulatory environment through CRISPR/Cas9 genome editing of patient-specific iBSMCs and followed by RNA- seq and ATAC-seq (Aim K2). This study will advance our understanding of asthma and albuterol response by creating a functional annotation in a critical cell type and providing a model for carrying out functional experiments in cell lines derived from the patients themselves, thereby advancing precision medicine and improving asthma treatment outcomes. Through this proposed training and research, I will gain the necessary skills to achieve my ultimate career goal of leading a successful and independent research laboratory.

项目摘要 沙丁胺醇是世界上最常用的短期缓解哮喘症状的处方药， 并通过松弛气道平滑肌来控制这些症状。响应于 沙丁胺醇在个体之间和种族/民族之间差异很大。全基因组关联研究（GWAS） 已经发现了许多与哮喘风险遗传因素相关的基因座以及哮喘的药物基因组学 沙丁胺醇反应。然而，这些基因座主要位于未表征的非编码基因组区域， 哮喘和沙丁胺醇反应的遗传基础仍然很不清楚。我假设这种罕见的，人口- 在气道平滑肌细胞中活跃的基因调控元件内的特定变体有助于种族/民族 以及哮喘严重程度和沙丁胺醇反应的个体间差异。为了验证这个假设，我将 在我的功能基因组学培训的基础上，我将在本培训阶段开发新的技能， 在细胞重编程、CRISPR/Cas9基因组编辑和数量性状基因座（QTL）分析方面的建议。与 这些新技能，我将利用RNA-seq，ChIP-seq和ATAC-seq的功能基因组技术， 表征在原代支气管平滑肌细胞中有活性的基因表达和基因调控元件 （BSMC），产生用于产生诱导多能干细胞（iPSC）衍生的BSMC的稳健方案，以及 表征其相对于原代BSMC的基因调控环境（Aim K1）。这一目标将提供一个 与哮喘相关的关键细胞类型中的活性基因、途径和调控元件的百科全书， 沙丁胺醇反应。通过这一目标，我也将提供一个良好的特点哮喘研究社区 产生患者特异性iPSC衍生的BSMC（iBSMC）的方案，其可用于个体遗传和 药物测定。我还将通过以下方式鉴定改变调节元件活性和基因表达的变体： 差异增强子测定和CRISPR/Cas9基因组编辑，然后是RNA-seq和ATAC-seq（Aim K2）。 在这个项目的独立阶段，我将从100名哮喘患者中建立iBSMC系， 以及与肺功能和沙丁胺醇反应相关的表型数据。然后，我将使用这些IBSMC行来携带 外表达QTL和染色质可及性QTL作图，以鉴定改变基因的遗传变体 表达和增强子活性，有助于哮喘的严重程度和沙丁胺醇反应（Aim R1）。最后我 将从功能上描述这些基因组变异的一般和种族特异性基因改变 通过对患者特异性iBSMC进行CRISPR/Cas9基因组编辑，然后进行RNA- seq和ATAC-seq（Aim K2）。这项研究将通过以下方式促进我们对哮喘和沙丁胺醇反应的理解： 在关键细胞类型中创建功能注释，并提供用于执行功能注释的模型， 来自患者自身的细胞系实验，从而推进精准医疗， 改善哮喘治疗效果。通过这次培训和研究，我将获得必要的 我的最终职业目标是领导一个成功和独立的研究实验室。