Cyclosporine A treatment for Ullrich congenital muscular dystrophy: a cellular study of mitochondrial dysfunction and its rescue.

Mutations in COL6A1, COL6A2 and COL6A3, the genes which encode the extra-cellular matrix component collagen VI, lead to Bethlem myopathy and Ullrich congenital muscular dystrophy (UCMD). Although the Col6a1(-/-) null mouse has an extremely mild neuromuscular phenotype, a mitochondrial defect has been demonstrated, linked to dysregulation of the mitochondrial permeability transition pore (PTP) opening. This finding has been replicated in UCMD muscle cells in culture, providing justification for a clinical trial using cyclosporine A, an inhibitor of PTP opening. We investigated whether PTP dysregulation could be detected in UCMD fibroblasts (the predominant source of muscle collagen VI), in myoblast cells from patients with other diseases and its response to rescue agents other than collagen VI. Although we confirm the presence of PTP dysregulation in muscle-derived cultures from two UCMD patients, fibroblasts from the same patients and the majority of fibroblasts from other well-characterized UCMD patients behave normally. PTP dysregulation is found in limb girdle muscular dystrophy (LGMD) type 2B myoblasts but not in myoblasts from patients with Bethlem myopathy, merosin-deficient congenital muscular dystrophy, LGMD2A, Duchenne muscular dystrophy and Leigh syndrome. In addition to rescue by cyclosporine A and collagen VI, this cellular phenotype was also rescued by other extra-cellular matrix constituents (laminin and collagen I). As the muscle derived cultures demonstrating PTP dysregulation shared poor growth in culture and lack of desmin labelling, we believe that PTP dysregulation may be a particular characteristic of the state of these cells in culture and is not specific to the collagen VI defect, and can in any case be rescued by a range of extra-cellular matrix components. Further work is needed on the relationship of PTP dysregulation with UCMD pathology.

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.

Collagen VI related muscle disorders.

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two conditions which were previously believed to be completely separate entities. BM is a relatively mild dominantly inherited disorder characterised by proximal weakness and distal joint contractures. UCMD was originally described as an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. Here we review the clinical phenotypes of BM and UCMD and their diagnosis and management, and provide an overview of the current knowledge of the pathogenesis of collagen VI related disorders.

Automated genomic sequence analysis of the three collagen VI genes: applications to Ullrich congenital muscular dystrophy and Bethlem myopathy.

INTRODUCTION: Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD). BM is a relatively mild dominantly inherited disorder with proximal weakness and distal joint contractures. UCMD is an autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. METHODS: We developed a method for rapid direct sequence analysis of all 107 coding exons of the COL6 genes using single condition amplification/internal primer (SCAIP) sequencing. We have sequenced all three COL6 genes from genomic DNA in 79 patients with UCMD or BM. RESULTS: We found putative mutations in one of the COL6 genes in 62% of patients. This more than doubles the number of identified COL6 mutations. Most of these changes are consistent with straightforward autosomal dominant or recessive inheritance. However, some patients showed changes in more than one of the COL6 genes, and our results suggest that some UCMD patients may have dominantly acting mutations rather than recessive disease. DISCUSSION: Our findings may explain some or all of the cases of UCMD that are unlinked to the COL6 loci under a recessive model. The large number of single nucleotide polymorphisms which we generated in the course of this work may be of importance in determining the major phenotypic variability seen in this group of disorders.

Characterisation of the dysferlin skeletal muscle promoter.

Deficiency of the skeletal muscle membrane protein dysferlin causes the related and overlapping neuromuscular disorders limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. This paper describes the preliminary characterisation of the human dysferlin promoter. The transcriptional start site of dysferlin has been mapped using 5' RACE PCR, which extended the length of the known 5' UTR to 914 bp. Promoter elements have been mapped by assessing the ability of fragments from this region to activate the expression of a luciferase reporter gene borne on a plasmid transfected into differentiated and undifferentiated C2C12 mouse myoblast cells. Finally, the core promoter region has been screened for mutations in suspected dysferlinopathy patients.

Investigation of mitochondrial function in hereditary spastic paraparesis.

Following the association of hereditary spastic paraparesis (HSP) with mutation in the paraplegin gene (SPG7) and mitochondrial dysfunction, we wished to investigate whether mitochondrial dysfunction might be associated with other forms of HSP. Five cases of HSP caused by mutation in the spastin gene (SPG4) and nine cases with HSP with mutation in the spastin and paraplegin genes excluded (non-SPG4/SPG7), were investigated for mitochondrial dysfunction. Muscle tissue from the HSP groups and a control group was analysed histochemically and spectrophotometrically for mitochondrial dysfunction. A significant decrease in mitochondrial respiratory chain complexes I and IV was demonstrated in the non-SPG4/SPG7 group. No abnormality was detected in the SPG4 group. We therefore conclude that there is evidence for mitochondrial dysfunction in non-SPG4/SPG7 HSP. There is no evidence for mitochondrial dysfunction in the pathogenesis of spastin-related HSP.

Limb-girdle muscular dystrophies--from genetics to molecular pathology.

The limb-girdle muscular dystrophies are a diverse group of muscle-wasting disorders characteristically affecting the large muscles of the pelvic and shoulder girdles. Molecular genetic analyses have demonstrated causative mutations in the genes encoding a disparate collection of proteins involved in all aspects of muscle cell biology. Muscular dystrophy includes a spectrum of disorders caused by loss of the linkage between the extracellular matrix and the actin cytoskeleton. Within this are the forms of limb-girdle muscular dystrophy caused by deficiencies of the sarcoglycan complex and by aberrant glycosylation of alpha-dystroglycan caused by mutations in the fukutin-related protein gene. However, other forms of this disease have distinct pathophysiological mechanisms. For example, deficiency of dysferlin disrupts sarcolemmal membrane repair, whilst loss of calpain-3 may exert its pathological influence either by perturbation of the IkappaBalpha/NF-kappaB pathway, or through calpain-dependent cytoskeletal remodelling. Caveolin-3 is implicated in numerous cell-signalling pathways and involved in the biogenesis of the T-tubule system. Alterations in the nuclear lamina caused by mutations in laminA/C, sarcomeric changes in titin, telethonin or myotilin at the Z-disc, and subtle changes in the extracellular matrix proteins laminin-alpha2 or collagen VI can all lead to a limb-girdle muscular dystrophy phenotype, although the specific pathological mechanisms remain obscure. Differential diagnosis of these disorders requires the careful application of a broad range of disciplines: clinical assessment, immunohistochemistry and immunoblotting using a panel of antibodies and extensive molecular genetic analyses.