Genetic variation impacting the airway smooth muscle in children with asthma

项目总结 沙丁胺醇是世界上最常用的短期缓解哮喘症状的处方药， 并通过松弛呼吸道平滑肌来控制这些症状。对以下问题的回应 沙丁胺醇在不同个体和不同种族/民族之间差异很大。全基因组关联研究 已经发现了许多与哮喘风险的遗传因素和药物基因组学有关的基因座 沙丁胺醇反应。然而，这些基因座主要位于未确定特征的非编码基因组区域，并且 哮喘和沙丁胺醇反应的遗传基础在很大程度上仍不清楚。我假设稀有的人口- 呼吸道平滑肌细胞中活跃的基因调控元件内的特定变异与种族/民族有关 以及哮喘严重程度和沙丁胺醇反应的个体差异。为了检验这一假设，我将 在我的功能基因组学培训的基础上，添加我将在此培训阶段开发的新技能 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。使用 这些新技能，我将利用rna-seq、Chip-seq和atac-seq的功能基因组技术来 原代支气管平滑肌细胞基因表达及活性基因调控元件的研究 (BSMCs)，生成用于创建诱导多能干细胞(IPSC)来源的BSMCs的可靠协议，以及 描述它们相对于原代BSMC的基因调控环境(目标K1)。这一目标将提供一个 与哮喘和哮喘相关的关键细胞类型的活性基因、途径和调节元件的百科全书 沙丁胺醇反应。通过这个目标，我也将为哮喘研究界提供一个有特色的 协议，以创建患者特定的，IPSC来源的BSMCs(IBSMCs)，可用于个体遗传和 毒品化验。我还将确定通过以下途径改变调控元件活性和基因表达的变体 差异增强子分析和CRISPR/Cas9基因组编辑，然后是RNA-seq和atac-seq(目标K2)。 在这个项目的独立阶段，我将从100名具有深层基因的哮喘患者中创建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线路来携带 OUT表达QTL和染色质可及性QTL定位以识别改变基因的遗传变异 表达和增强子活性，有助于哮喘严重程度和沙丁胺醇反应(目标R1)。最后，我 将从功能上描述这些基因组变异对基因的一般性和种族特异性改变 通过CRISPR/Cas9对患者特定的iBSMCs进行基因组编辑并随后进行RNA- SEQ和ATAC-SEQ(目标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 在关键单元类型中创建功能注释，并提供执行功能的模型 在来自患者自己的细胞系中进行实验，从而提高了精准医学和 改善哮喘治疗结果。通过这项拟议的培训和研究，我将获得必要的 实现我的最终职业目标--领导一个成功而独立的研究实验室的技能。

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.