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.
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.
