Uncovering the Genetic Mechanisms Behind Joint-Specific Osteoarthritis

项目总结/摘要 在美国，超过三分之一的六十五岁以上的人因关节疾病骨关节炎而衰弱 每年都有成千上万的膝关节置换手术。尽管发病率很高， 已知调节膝关节形成和OA风险的分子机制以及风险如何遗传 在特定关节处（例如，膝关节对髋关节）。最近的GWAS已经确定了至少17个基因座， 与膝关节OA风险相关。然而，这些基因座的偶然变异尚未被鉴定，因为它们的 关联信号跨越大的基因组间隔，其包含未研究的非编码调节区。的 这些，生长分化因子5基因（GDF 5）的常见变体，GDF 5是关节炎的关键调节因子， 在人群中，与膝关节OA风险可重复相关。GDF 5 OA变体位于 高频率的130 kb单倍型，具有许多突变，每个突变都可能是OA风险的原因，但 有趣的是，没有发现蛋白质编码突变可以解释这种关联。中 在一项补充研究中，我们发现了10个GDF 5调控元件（例如，启动子、增强子）跨越 这一间隔并揭示了它们以巨大的关节特异性发挥作用（例如，膝盖对臀部）。这些 增强子最初在小鼠胚胎中GDF 5表达的初始阶段进行了测试，但我们也知道， GDF 5有助于膝关节结构（韧带、肌腱、韧带、关节面）的分化 在这个发展阶段之后。我们的研究将汇集患者中膝关节OA的风险变体 群体和我们对GDF 5如何在DNA水平上控制的知识。我们首先要评估 关节特异性GDF 5增强子对出生前膝关节发育和成人关节的功能贡献 使用CRISPR-Cas9编辑来在体外人类细胞中和体内人类细胞中切除这些元件， 老鼠.引人注目的是，我们的初步分析还显示，这些GDF 5增强子的一个子集具有共同的 和罕见的人类变异体与该区间内最高相关的OA变异体处于强连锁不平衡。 我们的第二个目的是进一步探讨基因座中的遗传变异与OA膝关节形状之间的关联 使用从成人膝关节完整的骨关节炎倡议MR数据库中获得的数据。我们的初步 数据显示，与OA膝关节形状相关的几种变体存在于几种功能增强子中， 控制膝关节中GDF 5的表达。最后，我们的第三个目标是功能测试这些人类调节 通过转染研究，了解其对增强子活性、膝关节形成和OA风险的影响， CRISPR-Cas9在人类软骨细胞和小鼠模型中的应用这些研究的完成将揭示 GDF 5的功能变异，是其在膝关节形状和OA风险中作用的基础。

Project Summary/Abstract In the United States, the joint disease osteoarthritis (OA) debilitates over one-third of people over sixty-five years old and causes hundreds of thousands of knee replacements annually. Despite its high prevalence, little is known about the molecular mechanisms that regulate knee formation and OA risk and how one inherits risk at specific joints (e.g., knee versus hip). Recent GWAS have identified at least seventeen loci that significantly associate with knee OA risk. However, the casual variants for these loci have not been identified because their association signals span large genomic intervals harboring uninvestigated non-coding regulatory regions. Of these, common variants in the Growth Differentiation Factor Five gene (GDF5), a critical regulator of joint development, reproducibly associate with knee OA risk in human populations. GDF5 OA variants reside on a high frequency 130 kb haplotype possessing numerous mutations that each may be causal for OA risk, but interestingly, no protein coding mutations have been uncovered that explain the associations. In a complementary study, we discovered ten GDF5 regulatory elements (e.g., promoters, enhancers) spanning this interval and revealed that they function with tremendous joint specificity (e.g., knee vs. hip). These enhancers were initially tested at incipient stages of GDF5 expression in mouse embryos, but we also know that GDF5 contributes to the differentiation of knee structures (ligaments, tendons, menisci, articular surfaces) well after this stage of development. Our research will bring together knee OA risk variants in patient populations and our knowledge of how GDF5 is controlled at the DNA level. We first aim to assess the functional contributions of joint-specific GDF5 enhancers to pre-natal knee development and adult joint homeostasis using CRISPR-Cas9 editing to excise these elements in vitro in human cells and in vivo in the mouse. Strikingly, our pilot analyses also revealed that a subset of these GDF5 enhancers possesses common and rare human variants in strong linkage disequilibrium with the highest associated OA variants in the interval. Our second aim is to further explore the association between genetic variants in the locus and OA knee shape using data acquired from the complete Osteoarthritis Initiative MR database on adult knees. Our preliminary data reveal that several variants associated with OA knee shape reside in several functional enhancers that control expression of GDF5 in the knee. Finally, our third aim is to functionally test these human regulatory variants for their impact on enhancer activity, knee formation, and OA risk by using transfection studies and CRISPR-Cas9 in human cartilage cells and in the mouse model. Completion of these studies will reveal functional variants in GDF5 that underlie its role in knee shape and OA risk.