An integrative study of circulating leukocyte composition, epigenetic patterns, and functional consequences in knee osteoarthritis

项目概要/摘要 我以前在表观遗传学方面的研究和作为风湿病学研究员的临床培训为我做好了准备， 未来的职业是医生科学家。这里描述的K 08提案将使我能够获得必要的保护， 时间和指导，以扩大我的技能，并实现我的主要目标，成为领导者在该领域 骨关节炎（OA）表观遗传学。我将参加正式的课程，特别侧重于生物信息学， 统计学和研究交流在俄克拉荷马州健康科学中心的大学。我将扩大我的 通过教学培训和研究成果的展示来提高演讲技巧。我的职业生涯 我被任命为俄克拉荷马州共享临床和翻译专家， 资源学者，以及与我的主要导师和咨询委员会定期会议。 在这一关键时期，我的研究将集中在OA，一种慢性，使人衰弱的肌肉骨骼疾病， 40%的美国成年人超过70岁。了解基因与环境相互作用的机制， 关节外组织对OA的贡献是开发临床生物标志物和治疗方法的关键。 剂.我发表的数据显示OA软骨中DNA甲基化模式发生了显著改变， 软骨下骨，并从OA患者外周血单核细胞的初步数据 证明了几种免疫相关途径的明显一致的表观遗传失调。基于 根据这些数据，我假设OA是一种以低水平全身性炎症为特征的疾病， 表观基因组失调，改变基因转录，导致疾病的发病机制。 为了评估这一点，我将招募有放射学进展风险的膝关节OA患者， 抽血和膝盖X光片以备日后分析我会确定循环中的炎症细胞数量的变化 和/或它们对刺激的反应存在于膝OA患者中， 关节炎对照（目标1），并检查炎症细胞组成的模式是否是关节炎的预测指标。 放射学OA随时间的进展，使用最先进的cyTOF分析。然后我会确认一份修改过的 使用来自大型OA队列的库存DNA的DNA甲基化模式（Aim 2a）。随后，我将研究DNA 白细胞亚群中的甲基化和基因转录：CD 4+、CD 8+和单核细胞（目的2b）。最后我 将使用一种新的靶向dCas 9-TET 1融合蛋白来评估特定DNA的功能后果， 我之前在OA软骨工作中发现的去甲基化事件（目标3）。 通过这些目标实现的发现将形成强R 01应用的初步数据，以证实新的 诊断生物标志物和开发新的治疗策略来治疗这种毁灭性的疾病。如果成功，我 将提供关于循环OA炎性细胞亚群的第一个数据。利用核心 在OMRF的设施，我将另外提供一个公开的标本和数据的生物储存库， 收集以供将来学习。

Project Summary / Abstract My previous research in epigenetics and clinical training as a rheumatology fellow have prepared me well for a future career as a physician scientist. The K08 proposal described herein will allow me the necessary protected time and mentoring to expand my skills and achieve my primary goals of becoming leader in the field of osteoarthritis (OA) epigenetics. I will participate in formal coursework with a particular focus on bioinformatics, statistics, and research communication at the University of Oklahoma Health Sciences Center. I will expand my presentation skills through both didactic training and presentation of my research findings. My career development will be greatly advanced by my appointment as an Oklahoma Shared Clinical and Translational Resources Scholar, as well as regular meetings with my primary mentor and advisory committee. My research during this pivotal period will focus on OA, a chronic, debilitating musculoskeletal disease affecting 40% of US adults over the age of 70. Understanding the mechanisms of gene-environmental interaction and the contributions of extraarticular tissues to OA are key to the development of clinical biomarkers and therapeutic agents. I have published data showing significantly altered DNA methylation patterns in OA cartilage and subchondral bone, and have preliminary data from peripheral blood mononuclear cells of OA patients demonstrating remarkably consistent epigenetic dysregulation of several immune-related pathways. Based on these data, I hypothesize that OA is a disease characterized by low-level systemic inflammation, associated with dysregulated epigenomes that alter gene transcription, leading to disease pathogenesis. To evaluate this, I will recruit knee OA patients at risk of radiographic progression and perform serial blood draws and knee X-rays for later analysis. I will determine if changes in circulating inflammatory cell populations and/or their response to stimulation are present in knee OA patients compared to both healthy and rheumatoid arthritis controls (Aim 1), and examine whether patterns in inflammatory cell composition are predictive of radiographic OA progression over time, using state-of-the-art cyTOF analysis. I will then confirm an altered DNA methylation pattern using banked DNA from a large OA cohort (Aim 2a). Subsequently, I will study DNA methylation and gene transcription in leukocyte subsets: CD4+, CD8+, and monocyte cells (Aim 2b). Finally, I will use a novel, targetable dCas9-TET1 fusion protein to evaluate the functional consequences of specific DNA demethylation events I previously identified in my OA cartilage work (Aim 3). Discoveries achieved by these aims will form the preliminary data for strong R01 applications to confirm novel diagnostic biomarkers and develop new therapeutic strategies to treat this devastating disease. If successful, I will provide the first data regarding circulating OA inflammatory cell subpopulations. Leveraging the core facilities at OMRF, I will additionally provide a publically-available biorepository of the specimens and data I collect for future study.