Uncovering the Genetic Mechanisms Behind Joint-Specific Osteoarthritis

项目摘要/摘要 在美国，关节疾病骨关节炎(OA)使超过三分之一的65岁以上的人虚弱 已有多年历史，每年导致数十万例膝关节置换。尽管它的流行率很高，但几乎没有 已知调节膝关节形成和骨性关节炎风险的分子机制，以及一个人如何遗传风险 在特定的关节(例如，膝盖对臀部)。最近的GWAS已经确定了至少17个显著 与膝骨性关节炎风险相关。然而，这些基因座的随机变异还没有被识别出来，因为它们的 关联信号跨越大的基因组区间，包含未被研究的非编码调节区。的 这些是生长分化因子5基因(GDF5)的常见变体，GDF5是关节的关键调节因子 发展，可重复地与人类人群中的膝骨性关节炎风险相关联。GDF5的OA变体驻留在 高频130kb单倍型具有大量突变，每个突变都可能导致OA风险，但 有趣的是，还没有发现可以解释这种联系的蛋白质编码突变。在一个 补充研究，我们发现了10个GDF5调控元件(例如，启动子、增强子) 这一时间间隔，并表明它们的功能具有极大的关节特异性(例如，膝盖对髋关节)。这些 增强剂最初是在小鼠胚胎中GDF5表达的早期阶段进行测试的，但我们也知道 GDF5有助于膝关节结构(韧带、肌腱、半月板、关节表面)的分化 在这个发展阶段之后。我们的研究将汇集患者膝骨性关节炎的风险变量 以及我们对GDF5如何在DNA水平上进行控制的知识。我们首先的目标是评估 关节特异性GDF5增强剂对出生前膝关节发育和成人关节发育的功能贡献 使用CRISPR-Cas9编辑在体外人体细胞和体内去除这些元件的动态平衡 老鼠。引人注目的是，我们的试点分析还显示，这些GDF5增强子的子集具有共同的 罕见的人类变异与区间内关联最高的骨关节炎变异处于强烈连锁不平衡状态。 我们的第二个目标是进一步探索该基因座的遗传变异与膝骨性关节炎的关系 使用从完整的骨关节炎倡议MR数据库中获得的关于成人膝盖的数据。我们的预赛 数据显示，与骨性关节炎膝关节形状相关的几种变体存在于几种功能增强剂中，这些增强剂 GDF5在膝关节中的表达控制。最后，我们的第三个目标是从功能上测试这些人类监管 通过使用基因转染法研究和分析其对增强剂活性、膝关节形成和骨关节炎风险的影响 CRISPR-Cas9在人软骨细胞和小鼠模型中的表达。这些研究的完成将揭示 GDF5中的功能变异是其在膝关节形状和骨关节炎风险中的基础作用。

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.