Pathogenic Mechanisms in Hereditary Multiple Exostoses Syndrome

描述（由申请人提供）：遗传性多发性外生骨疣（HME）是一种常染色体显性遗传疾病，影响约1/20，000的儿童。HME的特征在于邻近生长板形成的带软骨帽的副产物，其突出到周围组织和器官中，并引起生长迟缓、神经压迫和早发性骨关节炎。它们在大约5%的患者中变成恶性的。目前的疗法是姑息性的，患者在痛苦和行动不便中挣扎，一生中要接受多次手术。负责HME病例的基因是编码负责硫酸乙酰肝素（HS）合成的糖基转移酶的EXT 1和EXT 2。患者是EXT 1或EXT 2功能丧失突变的杂合子，他们的细胞产生较低的HS量。富含HS的蛋白聚糖通过各种机制调节关键的生理过程，最显著的是通过限制组织内hedgehog、BMP和其他信号传导因子的地形分布和作用，但尚不清楚这些机制中的缺陷是否会导致HME。在正在进行的研究中，我们发现生长板中的HS缺乏导致印度刺猬（Ihh）的重新分布，其在整个软骨膜上的浸润和软骨膜本身内的外生骨疣样软骨肿块的形成。在HS N-硫酸化缺陷的小鼠生长板中观察到Ihh的类似异位作用。我们还发现，干扰HS功能大大刺激间充质细胞分化为软骨细胞。因此，我们的中心假设是HME中的HS缺乏（i）导致hedgehog和其他促软骨形成因子从生长板重新分布到软骨膜，以及（ii）增强软骨膜细胞对这些和其他局部因子的反应性。由于这种机制的组合，生长板和软骨膜将错误地沟通，软骨膜细胞将失去其正常特性，成为软骨形成细胞并引起外生骨疣。为了验证我们的假设，我们将通过在生长板和/或软骨膜中创建条件性Ext-deficient小鼠并确定促软骨形成信号通路的作用来分析外生骨疣形成的机制（目的1）。我们将确定HS缺陷细胞的软骨形成能力增加的机制，将测试它们对信号传导因子的反应性，并评估它们的HS链的结构和蛋白结合能力（目的2）。然后，我们将进行原理验证实验，以确定促软骨形成信号通路的药理学拮抗剂是否阻断外生骨疣形成（目的3）。该项目将为HME发病机制的细胞和分子机制提供全新的见解，并将基于这些见解测试可能的合理疗法。因此，该项目对HME和相关生长板骨骼发育不良的基础生物医学研究和转化医学具有重要意义。HME患者的数量很少，但他们的家庭社区很大。因此，该项目将为患者和家属带来新的希望，即这种被忽视的疾病将得到积极研究，有朝一日可能找到治愈方法。

DESCRIPTION (provided by applicant): Hereditary Multiple Exostoses (HME) is an autosomal dominant disorder that affects about 1 in 20,000 children. HME is characterized by cartilage-capped outgrowths that form adjacent to the growth plates, protrude into surrounding tissues and organs, and cause growth retardation, compression of nerves and early onset osteoarthritis. They become malignant in about 5% of the patients. Current therapies are palliative, and patients struggle with pain and limited mobility and undergo multiple surgeries through life. The genes responsible for HME cases are EXT1 and EXT2 that encode glycosyltransferases responsible for heparan sulfate (HS) synthesis. Patients are heterozygous for EXT1 or EXT2 loss-of-function mutations and their cells produce lower HS amounts. HS-rich proteoglycans regulate key physiologic processes by various mechanisms and most notably by restricting the topographical distribution and action of hedgehog, BMPs and other signaling factors within tissues, but it is not known whether defects in these mechanisms subtend HME. In ongoing studies, we found that HS deficiency in growth plate leads to re-distribution of Indian hedgehog (Ihh), its infiltration over the entire perichondrium and formation of exostosis-like cartilaginous masses within perichondrium itself. A similar ectopic action of Ihh was seen in mouse growth plates deficient in HS N- sulfation. We found also that interference with HS function greatly stimulates differentiation of mesenchymal cells into chondrocytes. Thus, our central hypothesis is that the HS deficiency in HME (i) causes re-distribution of hedgehog and other pro-chondrogenic factors from growth plate to perichondrium and (ii) enhances responsiveness of perichondrial cells to these and other local factors. As a result of this combination of mechanisms, growth plate and perichondrium would mis-communicate, and perichondrial cells would lose their normal character, become chondrogenic and give rise to exostoses. To test our hypotheses, we will analyze the mechanisms of exostosis formation by creating conditional Ext-deficient mice in growth plate and/or perichondrium and determining roles of pro-chondrogenic signaling pathways (Aim 1). We will determine the mechanisms for increased chondrogenic capacity of HS-deficient cells will test their responsiveness to signaling factors and assess structure and protein binding capabilities of their HS chains (Aim 2). We will then carry out proof-of-principle experiments to determine whether pharmacologic antagonists of pro-chondrogenic signaling pathways block exostosis formation (Aim 3). The project will provide fundamentally new insights into the cellular and molecular mechanisms of HME pathogenesis and will test possible rational therapies based on those insights. The project thus has significant importance for both basic biomedical research and translational medicine in HME and related growth plate-based skeletal dysplasias. The number of HME patients is small, but the community of their families is large. This project will thus provide a renewed sense of hope to patients and families alike that this neglected disease will actively be studied and a cure may one day be found.