The Molecular Basis of CMD Types Ullrich and Bethlem

描述(由申请人提供)：这项拨款提案的目的是阐明严重的先天性肌营养不良Ullrich(UCMD)和其轻度对应的Bethlem肌病(BM)的分子机制。UCMD和BM结合了肌肉营养不良和结缔组织紊乱的特征，导致明显的虚弱、进行性痉挛和关节松弛。VI型胶原是一种微纤维成分，存在于大多数组织的细胞外基质中，通常分布在细胞膜上。它的基本杂三聚体单元由三个α链组成。VI型胶原基因(COL6A1、COL6A2、COL6A3)突变已在BM和UCMD中被发现。虽然BM通常由显性突变引起，UCMD通常由隐性突变引起，但我们最近也证实了UCMD的显性和负性机制。VI型胶原的相关功能尚不清楚，在不同的培养系统中，它已被证明能促进细胞的黏附、分化、增殖和存活，而在Col6al失活的小鼠模型中，有证据表明肌肉中的细胞凋亡增加。我们假设成纤维细胞是UCMD和BM的主要细胞类型，通过产生异常的细胞外基质(ECM)，导致肌肉(虚弱)和肌腱(松弛和痉挛)的疾病。我们进一步假设，肌肉与其细胞外基质(ECM)之间的界面的特定干扰会导致肌肉细胞凋亡和虚弱。在这个项目中，我们将全面描述UCMD和BM患者的特征，以确定他们的临床、形态和生化表型，并将这些表型与VI型胶原基因相关联。这将使我们能够描绘UCMD/BM的整个表型、生化和遗传谱。接下来，L将检测VI型胶原基因突变患者真皮成纤维细胞培养中细胞外基质基因表达和细胞凋亡的变化。明确细胞外基质成分和成纤维细胞活性的变化将使我们了解先天性和进行性肌挛缩的基础，关节松弛，并预测异常细胞外基质沉积在肌肉中的性质。为了研究肌肉无力的分子基础，我们正在开发一种新的共培养系统，在该系统中，不同患者来源的成纤维细胞培养物为正常肌肉细胞提供细胞外基质。该系统将用于检测正常和突变的VI型胶原基质对培养的肌管的影响。这将使我们能够确定导致肌肉功能障碍和肌肉细胞死亡的途径。最终，我们希望了解细胞外基质对肌营养不良症发展的影响。对这些途径的了解是在这一新的和重要的肌肉疾病组中制定有效治疗策略的先决条件。

DESCRIPTION (provided by applicant): The goal of this grant proposal is to elucidate the molecular mechanisms underlying the severe congenital muscular dystrophy type Ullrich (UCMD) and its milder counterpart Bethlem myopathy (BM). UCMD and BM combine features of muscular dystrophies and disorders of connective tissue resulting in significant weakness, progressive contractures, and joint laxity. Collagen VI is a microfibrillar component found in the extracellular matrix of most tissues, often in a pericellula r distribution . Its basic heterotrimeric unit is composed of three alpha chains. Mutations in the collagen VI genes (COL6A1, COL6A2, COL6A3) have been identified in BM and more recently also in UCMD. Althoug h BM usuall y is caused by dominan t mutations and UCMD by recessive mutations, we recently demonstrated dominan t negative mechanisms in UCMD also. The relevant functions of collagen VI are unclear, in various culture systems it has been shown to promote cell adhesion, differentiation , proliferation , and survival , whereas in a mouse model of Col6al inactivatio n there was evidence for increased apoptosis in the muscle. We hypothesize that fibroblasts are the primary cell type involved in UCMD and BM causing disease both in muscle (weakness) and in tendon (laxity and contractures) by producing an abnormal extracellular matrix (ECM). We further hypothesize that a specific disturbance of the interface between muscle and its extracellular matrix (ECM) leads to muscle apoptosis and weakness. In this project we will comprehensively characterize patients with UCMD and BM to define their clinical, morphological, and biochemical phenotypes and correlate these with the collagen VI genotypes. This will enable us to delineate the entire phenotypical, biochemical, and genetic spectrum of UCMD/BM. We wil l next determine changes in ECM gene expression as well as apoptosis in dermal fibroblasts cultures from the characterized patients with mutations in the collagen VI genes. Defining changes in ECM composition and fibroblast viability will allow us to understand the basis of the congenital and progressive contractures, the joint laxity, and to predict the nature of the abnormal ECM deposited in muscle. To study the molecular basis of the muscle weakness we are developing a novel co-culture system in which various patient derived fibroblast cultures supply an ECM for normal muscle cells. This system will be used to examine the effects of normal and mutant collagen VI matrices on the myotubes in culture. It will allow us to define the pathways that lead to muscle dysfunction and muscle cell death. Ultimately, we hope to understand the influence of ECM on the development of muscular dystrophy. Knowledge about these pathways is a prerequisite for developing effective treatment strategies in this new and important group of muscle disorders.