Three new collagen VI genes; COL6A4, COL6A5 and COL6A6

描述（由申请人提供）：胶原VI是一种细胞外基质成分，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）按1:1:1的比例组合。我们在人类3q23染色体的单个位点上发现了另外三个胶原VI基因，分别编码a4（VI）、a5（VI）和a6（VI）链，并将它们命名为COL6A4、COL6A5和COL6A6。COL6A4基因在人类中被破坏，很可能失去功能。COL6A1、COL6A2和COL6A3基因突变可导致两种儿童肌肉疾病；相对较轻的Bethlem肌病（BM）和较严重的Ullrich先天性肌营养不良（UCMD）。我们在COL6A6中发现了一个纯合子移码突变，该突变导致一个过早的终止密码子，并预测会导致a6（VI）蛋白的完全缺失。受影响的个体作为新生儿死于肌肉萎缩症样疾病，比BM或UCMD更严重。我们假设a6（VI）链对发育至关重要。此外，我们假设COL6A6基因在与BM和UCMD不同的严重先天性肌萎缩症患者中存在突变。另外三个胶原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要复杂得多。我们假设这三条新链可以替代a3（VI）与a1（VI）和a2（VI）链共组装，形成胶原蛋白VI的新分子组件。具体目的是：1)鉴定和表征先天性肌营养不良患者COL6A6突变。在确定突变后，将在体外评估患者成纤维细胞中对胶原VI组装和细胞外分泌的生化影响。2)分析a6（VI）链在体内的功能。我们将生成一组col6a6缺失的小鼠，作为我们发现的人类突变的模型。我们将通过生化、免疫组织化学和超微结构分析来研究a6（VI）链缺失对小鼠发育的影响。3)为了了解a4(VI), a5（VI）和a6（VI）组装成胶原VI的机制，将在体外评估新链与a1和a2共组装形成胶原VI异源三聚体的潜力。然后，系统地删除单个结构域，并确定对VI型胶原生物合成、细胞内组装和分泌的影响。我们发现，a6（VI）蛋白导联的缺失在人类中是致命的，扩展了胶原蛋白VI疾病谱系的严重端，并证明a6（VI）链对正常发育至关重要。该基金将通过分析患者细胞和col6a6基因缺失小鼠，探索a6（VI）的发育和病理生理作用。此外，我们将明确新链的组装机制，并探索它们是否可以形成异质胶原VI结构。公共卫生相关性：我们在一名患有严重肌肉萎缩症的患者中发现了一种新的VI型胶原蛋白基因突变。这一发现将改变这类先天性肌肉萎缩症患者及其家人接受的遗传咨询。此外，如果新的治疗方法（如基因治疗）可行，了解与肌肉萎缩症有关的基因和突变的全谱是至关重要的。

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. PUBLIC HEALTH RELEVANCE: We have identified a mutation in a new gene for collagen VI in a patient with a severe muscular dystrophy. This discovery will change the genetic counseling patients and their families with these type of congenital muscular dystrophies receive. In addition, understanding the full spectrum of genes and mutations involved in muscular dystrophy is vital if novel therapeutic approaches, such as gene therapy, are feasible.