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We report on a 5-year-old girl who presented with an association of symptoms reminiscent of an Ullrich-like congenital muscular dystrophy including congenital hypotonia, proximal joint contractures, hyperlaxity of distal joints, normal cognitive development, and kyphoscoliosis. There was an excess of neuromuscular spindles on the skeletal muscle biopsy. This very peculiar feature on muscle biopsy has been reported only in patients with mutations in the HRAS gene. Sequence analysis of the subject's HRAS gene from blood leukocytes and skeletal muscle revealed a previously described heterozygous missense mutation (c.187G>A, p. Glu63Lys). The present report thus extends the differential diagnosis of congenital muscular dystrophy with major "retractile" phenotypes and adds congenital muscular dystrophy to the clinical spectrum of HRAS-related disorders.

Item does not contain fulltext Complete deficiency of the extracellular matrix glycoprotein tenascin-X (TNX) leads to recessive forms of Ehlers-Danlos syndrome, clinically characterized by hyperextensible skin, easy bruising and joint hypermobility. Clinical and pathological studies, immunoassay, and molecular analyses were combined to study a patient suffering from progressive muscle weakness. Clinical features included axial and proximal limb muscle weakness, subclinical heart involvement, minimal skin hyperextensibility, no joint abnormalities, and a history of easy bruising. Skeletal muscle biopsy disclosed striking muscle consistency and the abnormal presence of myotendinous junctions in the muscle belly. TNX immunostaining was markedly reduced in muscle and skin, and serum TNX levels were undetectable. Compound heterozygous mutations were identified: a previously reported 30kb deletion and a non-synonymous novel missense mutation in the TNXB gene. This study identifies a TNX-deficient patient presenting with a primary muscle disorder, thus expanding the phenotypic spectrum of TNX-related abnormalities. Biopsy findings provide evidence that TNX deficiency leads to muscle softness and to mislocalization of myotendinous junctions.

Ullrich congenital muscular dystrophy (UCMD) is a severe congenital dystrophy caused by collagen VI mutations and resulting in absent or aberrant collagen VI secretion in fibroblasts, the main collagen VI secreting cells. ColVI is a ubiquitous extracellular matrix (ECM) protein that forms a microfibrillar network in close association with the basement membrane in many tissues. ColVI was shown to interact with several ECM components. Although an increased apoptotic rate, a mitochondrial defect and an impairment of the autophagocytic flux have been described in myoblasts and muscle cells from ColVI-deficient mice, the molecular pathways that link ColVI to the basal membrane protein network (fibronectin, etc.) and the downstream intracellular mechanisms are not fully elucidated. We present the results of a proteomic comparison between cultured skin fibroblasts from four unrelated UCMD patients, all carrying the heterozygous de novo p.G284R mutation in the COL6A1 gene, and fibroblasts from controls. Some of the dysregulated proteins observed in the proteomic profiles were confirmed by western blot, qPCR analysis, and fibroblast immunohistochemical staining. A detailed description of the new protein networks will be presented. Our results point to the involvement of two major cellular pathways that have up to now not been described in UCMD fibroblasts, which besides their role in the muscle disease, display typical abnormalities in other tissues like skin and tendons. We propose new insights in the pathophysiology of this matrix disease.

Collagen VI-myopathies regroup several forms of neuromuscular disorders caused by the deficiency of collagen type VI (COLVI), a protein that contributes to the architectural structure of muscle, its maintenance and survival. COLVI-myopathies constitute a spectrum of clinical presentations ranging from severe, early-onset phenotypes, to intermediate and milder phenotypes, with Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy at each extremity. To investigate in details the pathogenesis and to evaluate therapeutic strategies, we developed a novel mouse model carrying a nonsense mutation in the Col6a2 gene, based on the mutation identified in a patient with classical UCMD. Overall, homozygous KI- Col6a2 animals (KI) are viable and fertile, but males fail to thrive and display a significantly reduced body weight compared to wild type and heterozygous littermates, detectable as early as 4 weeks of age. We examined in detail the skeletal muscle phenotype of the KI- Col6a2 mice in terms of histology, morphology and function. The mutation introduced leads to a premature termination codon which triggers the specific degradation of the Col6a2 transcript through the nonsense-mediated RNA decay and the absence of COLVI in skeletal muscle from KI animals. These mice develop a myopathic phenotype with pronounced fiber size variability, increased internal nuclei and fibrosis. In addition, Tibialis anterior (TA) muscles of KI animals display significant hypoplasia, associated with an increase in type IIA fibers and a decrease in type IIB fibers. Accordingly, SDH and COX assays revealed more abundant oxidative fibers. At the functional level, the maximal specific force of TA muscle is significantly diminished in 9 week-old KI males. Taken together, these data validate the KI- Col6a2 mouse as a model of COLVI-myopathy and identify quantifiable markers of muscle dysfunction that will be extremely useful to assess the potential benefit of therapeutic approaches.

Ullrich congenital muscular dystrophy (UCMD) is a severe congenital dystrophy caused by collagen VI (COL VI) mutations and resulting in absent or aberrant collagen VI secretion in fibroblasts. The link between the mutated collagen VI, and Col VI synthesis, secretion into the ECM, interaction with the cell, mitochondrial defect, cell death, ... is not well characterized. The objective of our study was to provide additional proteomics based insight into the cellular impact of the defective ColVI in cultured skin fibroblasts. To reach this objective, a quantitative iTRAQ based LC-MALDI method was used to compare the proteome in cultured skin fibroblasts from UCMD patients with control fibroblasts. We used western blot as a confirmatory method to assess the dysregulation of proteome identified candidates. The most important findings were the differences in protein expression patterns linked to ER collagen-chaperone functioning and collagen I synthesis (alpha 1 chain, alpha 2 chain) together with fibronectin. A number of proteins involved in the formation of adhesions points that form an important link between the ECM and the intracellular space were found dysregulated. Additionally, Immunofluorescence (IF) for vinculin evaluated by several independent observers showed an increased number of focal adhesions on Col VI deficient fibroblasts. Finally IF collagen V evidenced disturbed extracellular network formation with intracellular retention of the protein. Our findings suggest that the dysregulation of the extracellular network in Col VI deficient fibroblasts possibly leads to intracellular stress and compensatory mechanisms. Proteins involved in molecular pathways such us the mitochondrial defect and the impairment of the autophagocytic flux, implicated in UCMD muscle fibers, were not found as differentially regulated in our proteomics experiment on fibroblasts, suggesting different pathophysiological mechanisms at play in these two important cell types in UCMD.

Mutations in the LAMA2 gene underlie a severe congenital type of muscular dystrophy (MDC1A). We report the clinical, histological and genotypic features of 2 patients aged respectively 35 and 31 years-old, presenting unusual phenotypes. The first patient, aged 36, exhibit a late-onset muscle weakness predominantly in the limb girdle. Contractures of the elbow, wriest, fingers and Achilles tendons were the first symptoms with an onset at 15 years of age. Pelvic muscle weakness was observed at 25 years of age. The CK was elevated (2000 UI/L). In addition, he presented at age 30 a dilated cardiomyopathy with ventricular arrhythmias, requiring the implantation of a cardioverter-defibrillator. The second patient, aged 31 years, presented axial weakness at age 1, followed by the occurrence of rigid spine around 10 years of age associated to contractures at elbow, fingers, hips and Achilles tendons. At age 30, he had predominantly axial and limb girdle muscle weakness associated with upward gaze deficit. He has also generalized and partial complex seizures, beginning at age 6. The brain MRI disclosed bilateral occipital polymicrogyria and lissencephaly. Muscle histology showed dystrophic features, rimmed vacuoles, and partial loss of laminin a immunoreactivity. Nerve conduction study was normal in both patients. High-throughput analyses using “NMD-Chips” DNA capture and next generation sequencing identified novel nonsense mutations of LAMA2 gene at homozygous stage in the two patients. Previous sequencing of LMNA, EMD, DES, FHL1, MYH7, FKRP, FKTN, COL6A1,2,3, SEPN1 genes disclosed no mutation. In summary, these 2 patients disclosed a mild limb-girdle-type pattern of weakness and contractures associated respectively in each patient with dilated cardiomyopathy and cortical dysplasia. Work partly supported by the NMD-CHIP Consortium, an European Commission FP7 HEALTH project (HEALTH-F5-2008-223026).

Mutations in COL6A1, COL6A2, and COL6A3 genes encoding collagen VI (ColVI) are responsible for a wide spectrum of muscle diseases, also termed collagen VI myopathies. At one end of the spectrum, Bethlem myopathy (BM) represent a mild proximal myopathy beginning within the first or second decade of life and characterized by joint contractures, a hallmark of this condition. We report the clinical presentation, the course and the genetic analysis of 35 BM patients followed at the Institute of Myology. Onset of the disease was noticed before the age of 10 (80%). Early onset was not correlated with a severe clinical course. The majority of patients presented a retractile phenotype. However, three patients showed atypical phenotype: a LGMD muscle weakness pattern without contractures was observed in two patients and a third one had a diagnosis of minicore myopathy. Finger hyperlaxity and scoliosis were observed in 23% and 26% of patients, respectively. The vast majority of patients had a slow disease progression, with the exception of eight patients who used clippers or rolling chair. On long-term follow-up, only four patients presented a vital capacity (VC) lower than 70% of the theoretical value (record age of 18–50 years) which was not correlated with age disease onset. The pattern of involved muscles on muscle imaging was characteristic of ColVI-myopathies. Pathogenic mutations, including missense or exon skipping mutations affecting the triple helical domains, were identified in the three COL6A genes. The majority of the patients (91%) harbored autosomal dominant mutations (14% with de novo mutations), while three patients presented a recessive inheritance. In conclusion, we presented a series of 35 BM patients, three of them presenting an atypical phenotype namely without retractions misdiagnosed as LGMD or minicore myopathy. However, in these patients, muscle imaging was in accordance with the pattern of COLVI-related myopathies orienting towards molecular study of COL6 genes.

Collagen VI (COLVI) is a heterotrimeric protein, ubiquitously expressed in connective tissues. COLVI plays different roles such as maintenance of structural integrity, cellular migration and survival. To date, six COLVI chains are recognized with two short (α1–2(VI)) and three long chains (α3–6(VI)). Little is known regarding the possible assembly of the newly characterized α4–6(VI) polypeptides with the short chains, and a putative compensation between the different chains. In humans, deficiency in α1–3(VI) due to mutations in the COL6A1–3 genes causes COLVI-myopathies a heterogeneous group of neuromuscular disorders. To study the development of muscular tissue as well as the early steps of the disease, we turned to the zebrafish model where we identified only two orthologs of the α 4–6(VI) chains: col6a4a and col6a4b . We showed that the genes encoding the short chains are expressed at higher levels, and that only col6a4b was differentially expressed during development. We created COLVI deficient zebrafish embryos using morpholinos that block splicing of col6a2 , col6a4a and col6a4b , thereby creating premature termination codons. By qPCR, we demonstrated that the targeted transcripts were degraded, likely by the nonsense mediated RNA decay. At the morphological level, all morphant embryos had a curved body and showed severely impaired motility. Birefringence analysis and whole mount immunohistochemistry showed alteration of the muscle structure, with disorganized fibers and U-shaped myosepta. These alterations were confirmed at the ultrastructural level by electron microscopy. In conclusion, col6a2 deficient embryos recapitulate the severe end of the COLVI-myopathy phenotypical spectrum, thereby confirming the importance of col6 genes in muscle development. Furthermore, the phenotypes associated with α4a and α4b deficiency may help orientate group(s) of patients to screen for mutations in the human genes.