Combining genome, function, and phenotype to define the cell type specific gene regulatory architecture of idiopathic pulmonary fibrosis

项目摘要 特发性肺纤维化(IPF)是间质性肺疾病中最常见和最严重的形式。IPF 发生在中老年人中，每年影响超过50,000美国人。大多数IPF患者死亡 在确诊后五年内死于呼吸衰竭。目前的疗法针对的是下游疾病 虽然它们略微减缓了肺功能的下降，但并未显示出改善的迹象 IPF患者的生存或生活质量。特发性肺间质纤维化患者的临床结果有相当大的异质性。 患者，我们认为这种异质性是由于疾病涉及的不同机制和程序造成的 最终导致终末期肺纤维化的一种常见病理。因此，发展 变革性的治疗取决于我们更好地理解和定位“上游”疾病的能力 机械装置。然而，由于对细胞类型的研究有限，这方面的进展受到阻碍。 IPF发病机制的分子变化。最近开发的新技术能够使 以并行、高通量方式对单个细胞中的mRNA水平进行量化(scRNA- SEQ)。我们提议的研究将利用这些技术和疾病在 以确定IPF早期发病的机制和介质。我们将使用 ScRNA-seq以确定非纤维化对照肺(n=50)的基因表达谱和程序，以及 成对的、不同的IPF肺受累区域(n=100，成对的远端，纤维化程度更高，与近端相比，纤维化程度较低 样本)。我们将使用计算方法根据转录水平将细胞分组为假定的细胞类型 相似性和规范标记基因的表达。然后我们将量化每种细胞类型的相对丰度 在这些不同的疾病状态下，使用创新的生物信息学方法来确定基因 驱动疾病发病机制不同阶段的表达程序。然后，要确定 在调控这些疾病途径时，我们将利用个体间的遗传变异 在我们的样本中存在以确定与基因表达相关的单核苷酸多态性 每种独立细胞类型中的变化(EQTL)。接下来，开始询问潜在的机制 疾病的异质性，我们将确定与以下相关的细胞类型特异性基因表达变化 疾病结局的遗传预测因素(MUC5B基因、外周血端粒长度)。最后，我们会 根据细胞类型特定的基因表达模式定义新的疾病内型。本地化和地方化 已识别基因的空间模式将使用匹配的FFPE样本来确定，关键发现将是 在原代细胞/类器官培养系统中得到验证。这项工作将产生最全面的分子 健康和IPF肺的特征，并承诺回答关于细胞类型的基本问题， 基因变异和基因表达改变推动了特发性肺纤维化的发病机制。

Project Summary Idiopathic pulmonary fibrosis (IPF) is the most common and severe form of interstitial lung disease. IPF occurs in middle-aged and older adults and affects over 50,000 Americans each year. Most IPF patients die from respiratory failure within five years of diagnosis. The current therapies target downstream disease mechanisms, and while they modestly slow the decline in lung function, they have not been shown to improve survival or quality of life for IPF patients. There is considerable heterogeneity of clinical outcomes among IPF patients, and we believe this heterogeneity is due to distinct mechanisms and programs involved in disease initiation that culminate in a common a pathology of end-stage lung fibrosis. As such, the development of transformative treatments hinges on our ability to better understand and target “upstream” disease mechanisms. However, progress to this end has been held back by the limited study of the cell types and molecular changes initiating IPF pathogenesis. Novel technologies have recently been developed that enable quantification of mRNA levels in individual cells to be performed in a parallel, high throughput manner (scRNA- seq). Our proposed studies will leverage these technologies and the heterogeneity of the disease within the IPF lung to determine the mechanisms and mediators that underlie the early pathogenesis of IPF. We will use scRNA-seq to determine the gene expression profiles and programs in non-fibrotic control lungs (n=50), and paired, differentially affected regions of IPF lungs (n=100, paired distal, more fibrotic, vs. proximal, less fibrotic samples). We will use computational methods to group cells into putative cell types based on transcriptional similarity and canonical marker gene expression. We will then quantify the relative abundance of each cell type in these different disease states, and use innovative bioinformatic approaches to determine the gene expression programs that drive different phases of disease pathogenesis. Then, to determine the role of genetic variation in regulating these disease pathways, we will utilize the inter-individual genetic variation present in our sample to identify single nucleotide polymorphisms that are associated with gene expression changes (eQTLs) in each independent cell type. Next, to begin to interrogate the mechanisms underlying disease heterogeneity, we will determine cell-type specific gene expression changes that are associated with genetic predictors of disease outcome (MUC5B genotype, peripheral blood telomere length). Finally, we will define novel disease endotypes based on cell type specific gene expression patterns. The localization and spatial patterns of identified genes will be determined using matched FFPE samples, and key findings will be validated in primary cell/organoid culture systems. This work will generate the most comprehensive molecular characterization of healthy and IPF lungs, and promises to answer fundamental questions about cell types, genetic variants, and gene expression changes driving the idiopathic pulmonary fibrosis pathogenesis.