Ullrich Congenital Muscular Dystrophy Patients Research Articles

Recessive COL6A2 C-globular Missense Mutations in Ullrich Congenital Muscular Dystrophy: ROLE OF THE C2a SPLICE VARIANT

Ullrich congenital muscular dystrophy (UCMD) is a disabling and life-threatening disorder resulting from either recessive or dominant mutations in genes encoding collagen VI. Although the majority of the recessive UCMD cases have frameshift or nonsense mutations in COL6A1, COL6A2, or COL6A3, recessive structural mutations in the COL6A2 C-globular region are emerging also. However, the underlying molecular mechanisms have remained elusive. Here we identified a homozygous COL6A2 E624K mutation (C1 subdomain) and a homozygous COL6A2 R876S mutation (C2 subdomain) in two UCMD patients. The consequences of the mutations were investigated using fibroblasts from patients and cells stably transfected with the mutant constructs. In contrast to expectations based on the clinical severity of these two patients, secretion and assembly of collagen VI were moderately affected by the E624K mutation but severely impaired by the R876S substitution. The E624K substitution altered the electrostatic potential of the region surrounding the metal ion-dependent adhesion site, resulting in a collagen VI network containing thick fibrils and spots with densely packed microfibrils. The R876S mutation prevented the chain from assembling into triple-helical collagen VI molecules. The minute amount of collagen VI secreted by the R876S fibroblasts was solely composed of a faster migrating chain corresponding to the C2a splice variant with an alternative C2 subdomain. In transfected cells, the C2a splice variant was able to assemble into short microfibrils. Together, the results suggest that the C2a splice variant may functionally compensate for the loss of the normal COL6A2 chain when mutations occur in the C2 subdomain.

Genetic ablation of cyclophilin D rescues mitochondrial defects and prevents muscle apoptosis in collagen VI myopathic mice

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy are inherited muscle disorders caused by mutations of genes encoding the extracellular matrix protein collagen VI (ColVI). Mice lacking ColVI (Col6a1(-/-)) display a myopathic phenotype associated with ultrastructural alterations of mitochondria and sarcoplasmic reticulum, mitochondrial dysfunction with abnormal opening of the permeability transition pore (PTP) and increased apoptosis of muscle fibers. Treatment with cyclosporin (Cs) A, a drug that desensitizes the PTP by binding to cyclophilin (Cyp)-D, was shown to rescue myofiber alterations in Col6a1(-/-) mice and in UCMD patients, suggesting a correlation between PTP opening and pathogenesis of ColVI muscular dystrophies. Here, we show that inactivation of the gene encoding for Cyp-D rescues the disease phenotype of ColVI deficiency. In the absence of Cyp-D, Col6a1(-/-) mice show negligible myofiber degeneration, rescue from mitochondrial dysfunction and ultrastructural defects, and normalized incidence of apoptosis. These findings (i) demonstrate that lack of Cyp-D is equivalent to its inhibition with CsA at curing the mouse dystrophic phenotype; (ii) establish a cause-effect relationship between Cyp-D-dependent PTP regulation and pathogenesis of the ColVI muscular dystrophy and (iii) validate Cyp-D and the PTP as pharmacological targets for the therapy of human ColVI myopathies.

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.

Dysfunction of Mitochondria and Sarcoplasmic Reticulum in the Pathogenesis of Collagen VI Muscular Dystrophies

Ullrich Congenital Muscular Dystrophy (UCMD) and Bethlem Myopathy (BM) are muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen VI. Generation of a dystrophic mouse model where collagen VI synthesis was prevented by genetic ablation of the Col6a1 gene allowed an investigation of pathogenesis, which revealed the existence of a Ca(2+)-mediated dysfunction of mitochondria and the sarcoplasmic reticulum. A key event appears to be inappropriate opening of the mitochondrial permeability transition pore, an inner membrane high-conductance channel. Consistently, the Col6a1(-/-) myopathic mice could be cured with cyclosporin A through inhibition of cyclophilin D, a matrix protein that sensitizes the pore to opening. Studies of myoblasts from UCMD and BM patients demonstrated the existence of a latent mitochondrial dysfunction irrespective of the genetic lesion responsible for the lack or the alteration of collagen VI. These studies suggest that PTP opening may represent the final common pathway for skeletal muscle fiber death; and provided a rationale for a pilot clinical trial with cyclosporin A in patients affected by UCMD and BM, a study that holds great promise for the future treatment of collagen VI myopathies.

Therapy of Collagen VI-Related Myopathies (Bethlem and Ullrich)

The collagen VI-related myopathies comprise two major forms, Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), which show a variable combination of muscle wasting and weakness, joint contractures, distal laxity, and respiratory compromise. Specific diagnosis requires molecular genetic testing showing mutation in one of the three genes involved. This review summarizes current treatments, in particular indication for physiotherapy, orthopedic treatment for correction of foot deformity, scoliosis, and flexion contractures of elbows, and treatment of respiratory failure. The turning point in basic research on collagen VI myopathies was the discovery of an unexpected mitochondrial dysfunction as a pathogenetic mechanism underlying the myopathic syndrome seen in Col6a1 null mice. Treatment of Col6a1(-/-) mice with cyclosporin A (CsA) rescued the mitochondrial dysfunction and decreased apoptosis. Similar mitochondrial defects were revealed in cultures of UCMD patients. The results of an open pilot trial with CsA in five patients with collagen VI-related myopathies are summarized and discussed. With the availability of new potential effective treatments, several challenges must be addressed in conducting trials in orphan diseases and in neuromuscular disorders in particular. Outcome measures are discussed in the context of the expected effect of the cure. Randomized clinical trials often are not feasible for rare diseases, and sometimes would be ethically inappropriate. The need to develop alternative outcome measures or biomarkers using platforms such as genomics and proteomics is stressed in this context.

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.

Secretion and Assembly of Type IV and VI Collagens Depend on Glycosylation of Hydroxylysines

Most lysines in type IV and VI collagens are hydroxylated and glycosylated, but the functions of these unique galactosylhydroxylysyl and glucosylgalactosylhydroxylysyl residues are poorly understood. The formation of glycosylated hydroxylysines is catalyzed by multifunctional lysyl hydroxylase 3 (LH3) in vivo, and we have used LH3-manipulated mice and cells as models to study the function of these carbohydrates. These hydroxylysine-linked carbohydrates were shown recently to be indispensable for the formation of basement membranes (Ruotsalainen, H., Sipilä, L., Vapola, M., Sormunen, R., Salo, A. M., Uitto, L., Mercer, D. K., Robins, S. P., Risteli, M., Aszodi, A., Fässler, R., and Myllylä, R. (2006) J. Cell Sci. 119, 625-635). Analysis of LH3 knock-out embryos and cells in this work indicated that loss of glycosylated hydroxylysines prevents the intracellular tetramerization of type VI collagen and leads to impaired secretion of type IV and VI collagens. Mice lacking the LH activity of LH3 produced slightly underglycosylated type IV and VI collagens with abnormal distribution. The altered distribution and aggregation of type VI collagen led to similar ultrastructural alterations in muscle to those detected in collagen VI knockout and some Ullrich congenital muscular dystrophy patients. Our results provide new information about the function of hydroxylysine-linked carbohydrates of collagens, indicating that they play an important role in the secretion, assembly, and distribution of highly glycosylated collagen types.

C.P.2.03 Predictive factors of severity and management of respiratory and orthopaedic complications in 16 Ullrich CMD patients

Ullrich congenital muscular dystrophy (UCMD) is due to mutations in one of the three collagen VI genes. The disease course is very variable and optimization of management is required before therapeutic trials are considered. We analyzed motor, respiratory and orthopaedic data in a series of 16 UCMD patients (8M, 8F, mean age 14 years) to identify predictive parameters of severity and therapeutic interventions with an impact in the course of complications. All patients had at least one mutation in one of the COL6 genes: COL6A1 (7), COL6A2 (8), COL6A3 (2) including nine dominant de novo mutations.

C.P.2.06 Spectrum of COL6A1 mutations in patients with Ullrich congenital muscular dystrophy

Mutations in the genes COL6A1, COL6A2, COL6A3 have been associated with Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathies. UCMD is characterized by an early onset generalized muscle weakness, contractures of joints and hyper-extensibility in distal joints. It was usually considered that Ullrich’s disease is inherited in an autosomal recessive manner and Bethlem myopathy in an autosomal dominant manner. This work focused on the analysis in term of mode of inheritance, nature of mutations, and putative consequences, of 12 UCMD patients with mutations in COL6A1.

Ullrich myopathy phenotype with secondary ColVI defect identified by confocal imaging and electron microscopy analysis

Ullrich congenital muscular dystrophy (UCMD) is clinically characterized by muscle weakness, proximal contractures and distal hyperlaxity and morphologically branded by absence or reduction of collagen VI (ColVI), in muscle and in cultured fibroblasts. The ColVI defect is generally related to COL6 genes mutations, however UCDM patients without COL6 mutations have been recently reported, suggesting genetic heterogeneity. We report comparative morphological findings between a UCMD patient harboring a homozygous COL6A2 mutation and a patient with a typical UCMD phenotype in which mutations in COL6 genes were excluded. The patient with no mutations in COL6 genes exhibited a partial ColVI defect, which was only detected close to the basal membrane of myofibers. We describe how confocal microscopy and rotary-shadowing electron microscopy may be useful to identify a secondary ColVI defect.

Variable penetrance of COL6A1 null mutations: Implications for prenatal diagnosis and genetic counselling in Ullrich congenital muscular dystrophy families

Collagen VI mutations cause mild Bethlem myopathy and severe, progressive Ullrich congenital muscular dystrophy (UCMD). We identified a novel homozygous COL6A1 premature termination mutation in a UCMD patient that causes nonsense-mediated mRNA decay. Collagen VI microfibrils cannot be detected in muscle or fibroblasts. The parents are heterozygous carriers of the mutation and their fibroblasts produce reduced amounts of collagen VI. The molecular findings in the parents are analogous to those reported for a heterozygous COL6A1 premature termination mutation that causes Bethlem myopathy. However, the parents of our UCMD proband are clinically normal. The proband’s brother, also a carrier, has clinical features consistent with a mild collagen VI phenotype. Following a request for prenatal diagnosis in a subsequent pregnancy we found the fetus was a heterozygous carrier indicating that it would not be affected with severe UCMD. COL6A1 premature termination mutations exhibit variable penetrance necessitating a cautious approach to genetic counselling.

A comparative analysis of collagen VI production in muscle, skin and fibroblasts from 14 Ullrich congenital muscular dystrophy patients with dominant and recessive COL6A mutations

Ullrich congenital muscular dystrophy (UCMD) is caused by recessive and dominant mutations in COL6A genes. We have analysed collagen VI expression in 14 UCMD patients. Sequencing of COL6A genes had identified homozygous and heterozygous mutations in 12 cases. Analysis of collagen VI in fibroblast cultures derived from eight of these patients showed reduced extracellular deposition in all cases and intracellular collagen VI staining in seven cases. This was observed even in cases that showed normal collagen VI labelling in skin biopsies. Collagen VI immunolabelling was reduced in all the available muscle biopsies. When comparisons were possible no correlation was seen between the extent of the reduction in the muscle and fibroblast cultures, the mode of inheritance or the severity of the clinical phenotype. Mutations affecting glycine substitutions in the conserved triple helical domain were common and all resulted in reduced collagen VI. This study expands the spectrum of collagen VI defects and shows that analysis of skin fibroblasts may be a useful technique for the detection of collagen VI abnormalities. In contrast, immunohistochemical analysis of skin biopsies may not always reveal an underlying collagen VI defect.

A homozygous COL6A2 intron mutation causes in-frame triple-helical deletion and nonsense-mediated mRNA decay in a patient with Ullrich congenital muscular dystrophy

Ullrich congenital muscular dystrophy (UCMD) is a severe disorder caused, in most cases, by a deficiency in collagen VI microfibrils. Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have been identified in eight of the nine UCMD patients reported thus far. A heterozygous COL6A1 gene deletion, resulting in a mutant protein that exerts a dominant negative effect, has recently been described in a severely affected UCMD patient. Here we describe a patient in whom reverse transcription-PCR analysis of fibroblast RNA suggested a heterozygous in-frame deletion of exon 13 in the triple-helical domain of COL6A2, which is predicted to be dominantly acting. However, a homozygous A --> G mutation at -10 of intron 12 was found in the genomic DNA. The intron mutation activated numerous cryptic splice acceptor sites, generating normal and exon 13-deleted COL6A2 mRNA, and multiple aberrant transcripts containing frameshifts that were degraded through a nonsense-mediated decay mechanism. Northern analysis indicated diminished COL6A2 mRNA expression as the primary pathogenic mechanism in this UCMD patient. Our results underscore the importance of multifaceted analyses in the accurate molecular diagnosis and interpretation of genotype-phenotype correlations of UCMD.

Muscle MRI in Ullrich congenital muscular dystrophy and Bethlem myopathy

The aim of this study was to evaluate the spectrum of muscle involvement on Magnetic Resonance Imaging (MRI) in patients with collagen VI related disorders. Nineteen patients with genetically confirmed collagen VI related disorders, 10 with Bethlem myopathy and 9 with Ullrich congenital muscular dystrophy (CMD), had muscle MRI of their legs using T1 sequences through calves and thighs. In patients with Bethlem myopathy the vasti muscles appeared to be the most frequently and most strikingly affected thigh muscles, with a rim of abnormal signal at the periphery of each muscle and relative sparing of the central part. Another frequent finding was the presence of a peculiar involvement of the rectus femoris with a central area of abnormal signal within the muscle. Patients with Ullrich CMD had a more diffuse involvement of the thigh muscles with relative sparing of sartorius, gracilis and adductor longus. In 8 of the 9 patients with Ullrich CMD, we also observed the peripheral rim of the vastus lateralis and the central area in the rectus femoris observed in patients with Bethlem myopathy. At calf level the results were more variable but a significant proportion of patients with both Bethlem myopathy (8/10) and Ullrich CMD (6/9) showed a rim of abnormal signal at the periphery of soleus and gastrocnemii. Bethlem myopathy and Ullrich CMD patients have distinct patterns of muscle involvement on MRI with some overlap between the two forms. Our results suggest that muscle MR may be used, as an additional tool, to identify patients with collagen VI related disorders. This information is even more important in the patients with a typical Ullrich CMD clinical phenotype but with normal collagen expression of VI in muscle and/or skin.

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...

Collagen VI status and clinical severity in Ullrich congenital muscular dystrophy: phenotype analysis of 11 families linked to the COL6 loci.

Ullrich's congenital muscular dystrophy (UCMD) is an autosomal recessive myopathy characterised by neonatal muscle weakness, proximal joint contractures and distal hyperlaxity. Mutations in the COL6A1, COL6A2 (21 q22.3) and COL6A3 (2 q37) genes, encoding the alpha 1, alpha 2 and alpha 3 chains of collagen VI, respectively, have been recently identified as responsible for UCMD in a total of 9 families. We investigated in detail the clinical and morphological phenotype of 15 UCMD patients from 11 consanguineous families showing potential linkage either to 21 q22.3 (6 families) or to 2 q37 (5 families). Collagen VI deficiency was confirmed on muscle biopsies or skin fibroblasts in 8 families. Although all patients shared a common phenotype, a great variability in severity was observed. Collagen VI deficiency in muscle or cultured fibroblasts was complete in the severe cases and partial in the milder ones, which suggests a correlation between the degree of collagen VI deficiency and the clinical severity in UCMD. No significant phenotypical differences were found between the families linked to each of the 2 loci, which confirms UCMD as a unique entity with underlying genetic heterogeneity.