Pathogenic Mechanisms in Hereditary Multiple Exostoses Syndrome

描述(申请人提供)：遗传性多发性骨软骨病(HME)是一种常染色体显性遗传性疾病，大约每20,000名儿童中就有1名患病。HME的特征是在生长板附近形成软骨覆盖的突起，突入周围组织和器官，导致生长迟缓、神经受压和早发性骨关节炎。在大约5%的患者中，它们变得恶性。目前的治疗方法是姑息治疗，患者一生中都在与疼痛和活动受限作斗争，并接受多次手术。导致HME病例的基因是EXT1和EXT2，它们编码负责合成硫酸乙酰肝素(HS)的糖基转移酶。患者是EXT1或EXT2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的蛋白多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Hedgehog、BMPs和其他信号因子在组织中的地形分布和作用，但这些机制中的缺陷是否抑制了HME尚不清楚。在正在进行的研究中，我们发现生长板中HS缺乏会导致印度刺猬(IHH)的重新分布，它渗透到整个软骨膜，并在软骨膜内形成外骨样软骨肿块。IHH在缺乏HS-N-硫酸盐的小鼠生长板上也有类似的异位作用。我们还发现，干扰HS功能可以极大地刺激间充质细胞向软骨细胞分化。因此，我们的中心假设是，HME中HS缺乏(I)导致Hedgehog和其他促软骨生成因子从生长板重新分布到软骨膜，(Ii)增强软骨膜细胞对这些和其他局部因素的反应性。由于这种机制的结合，生长板和软骨膜将错误地沟通，软骨膜细胞将失去正常特性，成为软骨化，并产生外生骨病。为了验证我们的假设，我们将通过在生长板和/或软骨膜中建立条件性Ext缺陷小鼠并确定促软骨形成信号通路的作用来分析外生性骨疣的形成机制(目标1)。我们将确定HS缺陷细胞软骨形成能力增强的机制，测试它们对信号因子的反应，并评估其HS链的结构和蛋白质结合能力(目标2)。然后，我们将进行原则验证实验，以确定促软骨信号通路的药理拮抗剂是否阻止外生骨疣的形成(目标3)。该项目将从根本上为HME发病的细胞和分子机制提供新的见解，并将基于这些见解测试可能的合理疗法。因此，该项目对HME和相关的生长板型骨骼发育不良的基础生物医学研究和转化医学都具有重要意义。HME患者数量不多，但他们的家庭社区很大。因此，该项目将给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，有朝一日可能会找到治疗方法。

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.