Abstract
Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD) sit at opposite ends of a clinical spectrum caused by mutations in the extracellular matrix protein collagen VI. Bethlem myopathy is relatively mild, and patients remain ambulant in adulthood while many UCMD patients lose ambulation by their teenage years and require respiratory interventions. Dominant and recessive mutations are found across the entire clinical spectrum; however, recessive Bethlem myopathy is rare, and our understanding of the molecular pathology is limited. We studied a patient with Bethlem myopathy. Electron microscopy of his muscle biopsy revealed abnormal mitochondria. We identified a homozygous COL6A2 p.D871N amino acid substitution in the C-terminal C2 A-domain. Mutant α2(VI) chains are unable to associate with α1(VI) and α3(VI) and are degraded by the proteasomal pathway. Some collagen VI is assembled, albeit more slowly than normal, and is secreted. These molecules contain the minor α2(VI) C2a splice form that has an alternative C terminus that does include the mutation. Collagen VI tetramers containing the α2(VI) C2a chain do not assemble efficiently into microfibrils and there is a severe collagen VI deficiency in the extracellular matrix. We expressed wild-type and mutant α2(VI) C2 domains in mammalian cells and showed that while wild-type C2 domains are efficiently secreted, the mutant p.D871N domain is retained in the cell. These studies shed new light on the protein domains important for intracellular and extracellular collagen VI assembly and emphasize the importance of molecular investigations for families with collagen VI disorders to ensure accurate diagnosis and genetic counseling.
Highlights
Collagen VI amino acid substitutions are common, and it is difficult to determine if they are pathogenic
Around 60% of the collagen VI microfibrils in the medium of control cells contained more than 5 tetramers (Fig. 6e); there were very few of these large microfibrils in UCMD65 medium and around 40% of the microfibrils contained only a single tetramer (Fig. 6, b– e), indicating that end-to-end assembly of the tetramers secreted by UCMD65 fibroblasts was severely compromised. ␣2(VI) p.D871N Chains Are Degraded by the Proteasome—We looked at the fate of the mutant ␣2(VI) p.D871N
As this protein is in the medium it is likely that one or both of the high mannose N-linked oligosaccharides that were added in the endoplasmic reticulum have been modified by the addition of further sugars as the protein was transported through the Golgi. This form was not seen in the D871N cells and this is further confirmation that the C2 D871N domain is recognized as abnormal and retained in the endoplasmic reticulum. To our knowledge this is the first patient with a molecular diagnosis of Bethlem myopathy shown to have mitochondria with paracristalline inclusions by electron microscopy
Summary
Collagen VI amino acid substitutions are common, and it is difficult to determine if they are pathogenic. Much less is known about the consequences of amino acid substitutions in the N- and C-terminal globular A-domains Some of these are recessive disease causing mutations [3], some are dominant mutations [17], and some are found in unaffected individuals and are unlikely to be pathogenic. We have identified a homozygous recessive COL6A2 C2 domain p.D871N mutation in a Bethlem myopathy patient and have done detailed studies in patient muscle biopsy and fibroblasts as well as transfected cells to understand the effect of the mutation on the mutant C2 domain and the mutant ␣2(VI) chain and the consequences for collagen VI intracellular and extracellular protein assembly
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