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Bethlem myopathy and engineered collagen VI triple helical deletions prevent intracellular multimer assembly and
S R Lamandé1, K A Shields, A J Kornberg
1Orthopaedic Molecular Biology Research Unit, Department of Paediatrics, University of Melbourne, Royal Children's Hospital, Parkville, Victoria 3052, Australia. lamandes@cryptic.rch.unimelb.edu.au
Insights
Structural mutations in collagen VI genes cause Bethlem myopathy. This study reveals that triple helical deletions prevent collagen VI assembly and secretion, leading to reduced functional protein levels.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Bethlem myopathy is an autosomal dominant disorder caused by mutations in collagen VI genes (COL6A1, COL6A2, COL6A3).
- The precise effects of collagen VI structural mutations on protein assembly, structure, and function remain largely unknown.
Observation:
- A novel Bethlem myopathy mutation was identified, causing exon 14 skipping in COL6A1 pre-mRNA and an 18-amino acid deletion in the alpha1(VI) chain's triple helix.
- This mutation, a G to A transition at the intron 14 splice donor site, led to intracellular monomer formation but prevented dimer/tetramer assembly and secretion.
Findings:
- Triple helical deletions in both alpha1(VI) and alpha3(VI) chains inhibit intracellular assembly into dimers and tetramers.
- Mutant collagen VI molecules containing these deletions are not secreted, resulting in approximately 50% reduction in functional collagen VI.
- Experimental expression of a mutant alpha3(VI) cDNA with a 202-amino acid triple helical deletion confirmed these findings in SaOS-2 cells.
Implications:
- This research provides the first evidence detailing the biosynthetic consequences of structural collagen VI mutations.
- Functional protein haploinsufficiency is suggested as a primary pathogenic mechanism underlying Bethlem myopathy.
- Understanding these molecular defects is crucial for developing targeted therapies for collagen VI-related myopathies.
Abstract:
Mutations in the genes that code for collagen VI subunits, COL6A1, COL6A2, and COL6A3, are the cause of the autosomal dominant disorder, Bethlem myopathy. Although three different collagen VI structural mutations have previously been reported, the effect of these mutations on collagen VI assembly, structure, and function is currently unknown. We have characterized a new Bethlem myopathy mutation that results in skipping of COL6A1 exon 14 during pre-mRNA splicing and the deletion of 18 amino acids from the triple helical domain of the alpha1(VI) chain. Sequencing of genomic DNA identified a G to A transition in the +1 position of the splice donor site of intron 14 in one allele. The mutant alpha1(VI) chains associated intracellularly with alpha2(VI) and alpha3(VI) to form disulfide-bonded monomers, but further assembly into dimers and tetramers was prevented, and molecules containing the mutant chain were not secreted. This triple helical deletion thus resulted in production of half the normal amount of collagen VI. To further explore the biosynthetic consequences of collagen VI triple helical deletions, an alpha3(VI) cDNA expression construct containing a 202-amino acid deletion within the triple helix was produced and stably expressed in SaOS-2 cells. The transfected mutant alpha3(VI) chains associated with endogenous alpha1(VI) and alpha2(VI) to form collagen VI monomers, but dimers and tetramers did not form and the mutant-containing molecules were not secreted. Thus, deletions within the triple helical region of both the alpha1(VI) and alpha3(VI) chains can prevent intracellular dimer and tetramer assembly and secretion. These results provide the first evidence of the biosynthetic consequences of structural collagen VI mutations and suggest that functional protein haploinsufficiency may be a common pathogenic mechanism in Bethlem myopathy.