Three new collagen VI genes; COL6A4, COL6A5 and COL6A6

描述（由申请人提供）：胶原蛋白VI是一种细胞外基质组分，在几乎所有组织中形成微纤维结构。迄今为止，已经描述了三种胶原VI链; a1（VI）、a2（VI）和a3（VI），它们以1：1：1的比例组装。我们已经在人染色体3q 23上的单个位点鉴定了另外三个胶原VI基因，并将它们命名为COL 6A 4、COL 6A 5和COL 6A 6，它们分别编码α 4（VI）、α 5（VI）和α 6（VI）链。COL 6A 4基因在人类中被破坏，可能无功能。COL 6A 1、COL 6A 2和COL 6A 3中的突变导致两种儿童肌肉疾病;相对轻度的Bethlem肌病（BM）和更严重的Ullrich先天性肌营养不良症（UCMD）。我们鉴定了COL 6A 6中的纯合移码突变，该突变导致提前终止密码子，并预测导致α 6（VI）蛋白的完全缺失。受影响的个体作为比BM或UCMD更严重的肌营养不良样疾病的新生儿死亡。我们假设α 6（VI）链对于发育是必不可少的。此外，我们假设COL 6A 6基因在患有与BM和UCMD不同的严重先天性肌营养不良症的个体中具有突变。三个额外的胶原VI链的发现表明，胶原VI组装和细胞外微纤维组成比以前认为的更复杂。我们假设这三条新链可以取代α 3（VI）与α 1（VI）和α 2（VI）链共组装形成新的VI型胶原分子组装体。具体目的是：1）鉴定和表征先天性肌营养不良症患者中的COL 6A 6突变。在鉴定突变后，然后在患者成纤维细胞中体外评估对胶原VI组装和细胞外分泌的生化后果。2)分析α 6（VI）链在体内的功能。我们将产生一系列col 6a 6-null小鼠，作为我们鉴定的人类突变的模型。将在生物化学、免疫组织化学和超微结构分析中检查缺乏α 6（VI）链对小鼠发育的影响。3)了解α 4（VI）、α 5（VI）和α 6（VI）组装成胶原VI的机制。将在体外评估新链与a1和a2共组装以形成胶原VI异源三聚体的潜力。然后，将系统地删除单个结构域，并确定对胶原VI生物合成、细胞内组装和分泌的影响。我们的发现，α 6（VI）蛋白导致的损失在人类中是致命的，扩展了胶原VI疾病谱的严重端，并表明α 6（VI）链是正常发育所必需的。该基金将通过对患者细胞和col 6a 6基因缺陷小鼠的分析，探索a6（VI）的发育和病理生理作用。此外，我们将定义新链的组装机制，并探索它们是否可以形成异质性胶原VI结构。

DESCRIPTION (provided by applicant): Collagen VI is an extracellular matrix component that forms microfibrillar structures in virtually all tissues. To date, three collagen VI chains have been described; a1(VI), a2(VI) and a3(VI) which assemble in a 1:1:1 ratio. We have identified three additional collagen VI genes at a single locus on human chromosome 3q23 and named them COL6A4, COL6A5 and COL6A6 which encode the a4(VI), a5(VI) and a6(VI) chains, respectively. The COL6A4 gene is disrupted in humans and likely to be non-functional. Mutations in COL6A1, COL6A2 and COL6A3, result in two childhood muscle disorders; the relatively mild Bethlem myopathy (BM) and the more severe Ullrich congenital muscular dystrophy (UCMD). We identified a homozygous frameshift mutation in COL6A6 that results in a premature stop codon and is predicted to result in the complete absence of a6(VI) protein. The affected individual died as a neonate of a muscular dystrophy-like disorder that is more severe than BM or UCMD. We hypothesize that the a6(VI) chain is essential for development. Further, we hypothesize that the COL6A6 gene harbors mutations in individuals with severe congential muscular dystrophy distinct from BM and UCMD. The discovery of three additional collagen VI chains indicates that collagen VI assembly and extracellular microfibril composition is more complicated than previously thought. We hypothesize that the three new chains can substitute for a3(VI) to co-assemble with the a1(VI) and a2(VI) chains to form new molecular assemblies of collagen VI. The Specific Aims are: 1) To identify and characterize COL6A6 mutations in patients with congenital muscular dystrophy. Following identification of mutations, the biochemical consequences on collagen VI assembly and extracellular secretion will then be assessed in vitro in patient fibroblasts. 2) To analyze the function of the a6(VI) chain in vivo. We will generate a line of col6a6-null mice to serve as a model for the human mutation we identified. The effect of the lack of a6(VI) chains on mouse development will be examined in biochemical, immunohistochemical and ultrastructural analyses. 3) To understand the mechanism of a4(VI), a5(VI) and a6(VI) assembly into collagen VI. The potential for the new chains to co-assemble with a1 and a2 to form collagen VI heterotrimers will be assessed in vitro. Then, individual domains will be deleted systematically, and the effect on collagen VI biosynthesis, intracellular assembly and secretion determined. Our finding that the loss of a6(VI) protein leads is lethal in humans extends the severe end of the collagen VI disease spectrum and demonstrates that the a6(VI) chain is essential for normal development. This grant will explore the developmental and pathophysiological role of a6(VI) through the analysis of patient cells and of mice deficient for the col6a6 gene. In addition, we will define the mechanism of assembly of the new chain and explore whether they can form heterogeneous collagen VI structures.