Determing the genetic basis of responses to biomechanical strain in an in vitro model of osteoarthritis

确定骨关节炎体外模型中生物力学应变反应的遗传基础

• 批准号: 10348171
• 负责人: Anthony Hung
• 金额: $ 5.18万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10348171
• 关键词: ATAC-seq; Abbreviations; Accounting; Affect; Biochemical; Biological; Biology; Biomechanics; Bone structure; Cell Culture Techniques; Cell Line; Cells; Chondrocytes; Chromatin; Chronic; Complex; DNA Methylation; Data; Degenerative polyarthritis; Development; Disease; Disease Progression; Environmental Risk Factor; Epigenetic Process; Gene Expression; Gene Expression Profile; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genetic Variation; Genomics; Genotype; Genotype-Tissue Expression Project; Health; Heritability; Heterogeneity; Homeostasis; Human; In Vitro; Individual; Individual Differences; Joints; Link; Maps; Measures; Mediating; Mentors; Methods; Modeling; Nature; Onset of illness; Pain; Pathogenesis; Pathway interactions; Pattern; Periodicity; Phenotype; Pilot Projects; Process; Publishing; Quantitative Trait Loci; Replacement Arthroplasty; Research; Resort; Risk; Sampling; Specificity; Stimulus; Stress; Structure; System; Testing; Time; Tissue-Specific Gene Expression; Tissues; Trauma; Treatment Protocols; United States; Variant; Work; arthropathies; articular cartilage; cartilage cell; disability; experimental study; gene environment interaction; genetic association; genome wide association study; genomic locus; improved; in vitro Model; induced pluripotent stem cell; insight; inter-individual variation; joint inflammation; joint loading; mechanical load; molecular phenotype; response; risk variant; single-cell RNA sequencing; skills; subchondral bone; tissue culture; transcriptome sequencing

Osteoarthritis (OA), a degenerative joint disorder characterized by articular cartilage damage and alterations to the structure of subchondral bone, is the most common joint disease worldwide1. Numerous genetic loci2 and environmental factors such as biomechanical stress3–8 have been associated with joint health and may modulate the regulation of gene expression on the road to mediate disease. However, how these factors interact to initiate OA pathogenesis is still unclear. To evaluate the effects of gene-by-environment interactions on gene regulation in the context of human OA development, the work proposed here will study inter-individual variation in gene expression responses to biomechanical stress in chondrocytes, the primary cells of cartilage. Specifically, in Aim 1, I will characterize gene expression in stressed and control chondrocytes using an iPSC-derived biomechanical strain model of OA. I have optimized a cyclic tensile strain treatment regimen model of OA9–12 for use on iPSC-derived chondrocytes13. I have further applied these methods to three individuals from a panel of 58 Yoruba (abbreviation: YRI) human iPSC lines14. Using bulk and single-cell RNA-seq data from this experiment, I have identified patterns of differential gene expression between biomechanical strain conditions. In the continuation of this work, I will differentiate all 58 YRI iPSC lines into chondrocytes and characterize bulk and single-cell transcription in strained and control chondrocytes. In Aim 2, I will identify biomechanical strain dynamic expression quantitative trait loci (eQTLs) in differentiated chondrocytes. I have used data from a small-scale pilot study to establish the viability of this strain model of OA for mapping eQTLs. I will use RNA- seq data collected in Aim 1 to identify dynamic eQTLs that vary in effect between treatment conditions while assessing and accounting for disparities in differentiation efficiency and heterogeneity in the response to cyclic tensile strain. In Aim 3, I will integrate mapped dynamic eQTLs with genome-wide association study (GWAS) and epigenetic data to better understand the functional consequences of variation at genetic loci associated with OA. I will test for enrichment and colocalization of my dynamic eQTLs among published significant OA GWAS loci15 to determine if OA genetic associations could influence OA risk through context- specific gene regulation. I will also evaluate the tissue-specificity of my dynamic eQTLs using data from the Genotype-Tissue Expression Project16. Finally, I will collect DNA methylation and chromatin accessibility data from my in vitro system to assess how these molecular phenotypes change in response to biomechanical stress and potentially mediate transcriptional changes. Overall, my work will elucidate the genetic basis of how biomechanical stress impacts human joint health. More broadly, my project will deepen our understanding of how genetic associations with complex diseases may be mediated through gene-by-environment interactions. At the same time, this work will provide me abundant opportunities to grow my wet lab and computational research skills while benefitting from the support of a wide group of collaborators and mentors.

骨关节炎（OA），一种退化性关节疾病，其特征是关节软骨损伤和改变 软骨下骨的结构是全球最常见的关节疾病。许多遗传基因座和 生物力学压力等环境因素3-8与关节健康有关，并可能调节 调节基因表达在介导疾病的道路上。但是，这些因素如何相互作用以启动 OA发病机理尚不清楚。评估基因逐环境相互作用对基因调节的影响 在人类OA发展的背景下，这里提出的工作将研究基因的个体间变化 软骨细胞（软骨的原代细胞）对生物力学应力的表达反应。具体来说，目的 1，我将使用IPSC衍生的压力和控制软骨细胞中的基因表达表征 OA的生物力学应变模型。我优化了OA9–12的环状拉伸应变治疗方案模型 用于IPSC衍生的软骨细胞13。我进一步将这些方法应用于面板的三个人 58 Yoruba（缩写：YRI）人IPSC行14。从此使用批量和单细胞RNA-seq数据 实验，我已经确定了生物力学应变条件之间差异基因表达的模式。 在继续这项工作的过程中，我将把所有58 YRI IPSC线区分为软骨细胞并表征散装。 在紧张和对照软骨细胞中的单细胞转录。在AIM 2中，我将确定生物力学菌株 分化软骨细胞中的动态表达定量性状位置（EQTL）。我使用了来自 一项小规模的试点研究，以建立OA的这种菌株模型用于映射EQTL的生存能力。我将使用RNA- 在AIM 1中收集的SEQ数据以识别治疗条件之间有效不同的动态eqTL，而 评估和核算差异效率和异质性的差异 拉伸应变。在AIM 3中，我将将映射的动态EQTL与全基因组关联研究（GWAS）集成 和表观遗传数据，以更好地了解遗传基因座变异的功能后果 与OA相关。我将测试我的动态eqtls的富集和共定位 大量的OA GWAS基因座15确定OA遗传关联是否可以通过环境影响OA风险 - 特定基因调节。我还将使用来自 基因型 - 组织表达项目16。最后，我将收集DNA甲基化和染色质可及性数据 从我的体外系统评估这些分子表型如何响应生物力学应力而变化 并可能介导转录变化。总体而言，我的工作将阐明如何 生物力学压力会影响人类关节健康。更广泛地，我的项目将加深我们对 如何通过环境相互作用如何介导与复杂疾病的遗传关联。 同时，这项工作将为我提供丰富的机会来发展我的湿实验室和计算 研究技能，同时受益于广泛的合作者和导师的支持。