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Objective Mutations in one of the 3 genes encoding collagen VI (COLVI) are responsible for a group of heterogeneous phenotypes of which Bethlem myopathy (BM) represents the milder end of the spectrum. Genotype-phenotype correlations and long-term follow-up description in BM remain scarce. Methods We retrospectively evaluated the long-term clinical evolution, and genotype-phenotype correlations in 35 genetically identified BM patients (23 index cases). Results Nineteen patients showed a typical clinical picture with contractures, proximal weakness and slow disease progression while 11 presented a more severe evolution. Five patients showed an atypical presentation, namely a limb girdle muscle weakness in 2 and a congenital myopathy pattern with either no contractures, or only limited to ankles, in 3 of them. Pathogenic COL6A1-3 mutations were mostly missense or in frame exon-skipping resulting in substitutions or deletions. Twenty one different mutations were identified including 12novel ones. The mode of inheritance was, autosomal dominant in 83% of the index patients (including 17% (N=4) with a de novo mutation), recessive in 13%, and undetermined in one patient. Skipping of exon 14 of COL6A1 was found in 35% of index cases and was mostly associated with a severe clinical evolution. Missense mutations were detected in 39% of index cases and associated with milder forms of the disease. Conclusions Long-term follow-up identified important phenotypic variability in this cohort of 35 BM patients. However, worsening of the functional disability appeared typically after the age of 40 in 47% of our patients, and was frequently associated with COL6A1 exon 14 skipping.

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.

Mutations in the SEPN1 gene encoding the selenoprotein N (SelN) have been described in different congenital myopathies. Here, we report the first mutation in the selenocysteine insertion sequence (SECIS) of SelN messenger RNA, a hairpin structure located in the 3′ untranslated region, in a patient presenting a classical although mild form of rigid spine muscular dystrophy. We detected a significant reduction in both mRNA and protein levels in the patient's skin fibroblasts. The SECIS element is crucial for the insertion of selenocysteine at the reprogrammed UGA codon by recruiting the SECIS-binding protein 2 (SBP2), and we demonstrated that this mutation abolishes SBP2 binding to SECIS in vitro, thereby preventing co-translational incorporation of selenocysteine and SelN synthesis. The identification of this mutation affecting a conserved base in the SECIS functional motif thereby reveals the structural basis for a novel pathological mechanism leading to SEPN1-related myopathy.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single-nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single-nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple-helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype–phenotype correlation demonstrating that heterozygous glycine substitutions in the triple-helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes. Ann Neurol 2005;58:400 – 410

Multiple sclerosis (MS) is an autoimmune demyelinating disease of the central nervous system. A complex genetic etiology is thought to underlie susceptibility to this disease. The present study was designed to analyze whether differences in genes that encode myelin proteins influence susceptibility to MS. We performed linkage analysis of MS to markers in chromosomal regions that include the genes encoding myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMGP), and myelin oligodendrocyte glycoprotein (MOG) in a well-characterized population of 65 multiplex MS families consisting of 399 total individuals, 169 affected with MS and 102 affected sibpairs. Physical mapping data permitted placement of MAG and PLP genes on the Genethon genetic map; all other genes were mapped on the Genethon genetic map by linkage analysis. For each gene, at least one marker within the gene and/or two tightly linked flanking markers were analyzed. Marker data analysis employed a combination of genetic trait model-dependent (parametric) and model-independent linkage methods. Results indicate that MAG, MBP, OMGP, and PLP genes do not have a significant genetic effect on susceptibility to MS in this population. As MOG resides within the MHC, a potential role of the MOG gene could not be excluded.

In this study, we characterized five Ullrich scleroatonic muscular dystrophy patients (two Italians, one Belgian, and two Turks) with a clinical phenotype showing different degrees of severity, all carrying mutations localized in COL6A1. We sequenced the three entire COL6 complementary DNA. Three of five patients have recessive mutations: two patients (P1and P3) have homozygous single-nucleotide deletions, one in exon 9 and one in exon 22; one patient (P2) has a homozygous single-nucleotide substitution leading to a premature termination codon in exon 31. The nonsense mutation of P2 also causes a partial skipping of exon 31 with the formation of a premature termination codon in exon 32 in 15% of the total COL6A1 messenger RNA. The remaining two patients carry a heterozygous glycine substitution in exons 9 and 10 inside the triple-helix region; both are dominant mutations because the missense mutations are absent in the DNA of their respective parents. As for the three homozygous recessive mutations, the apparently healthy consanguineous parents all carry a heterozygous mutated allele. Here, for the first time, we report a genotype-phenotype correlation demonstrating that heterozygous glycine substitutions in the triple-helix domain of COL6A1 are dominant and responsible for a milder Ullrich scleroatonic muscular dystrophy phenotype, and that recessive mutations in COL6A1 correlate with more severe clinical and biochemical Ullrich scleroatonic muscular dystrophy phenotypes. Ann Neurol 2005;58:400-410

Background In humans, mutations in the SEPN1 gene, encoding selenoprotein N (SelN), are involved in early onset recessive neuromuscular disorders, referred to as SEPN1-related-myopathies. The mechanisms behind these pathologies are poorly understood since the function of SelN remains elusive. However, previous results obtained in humans and more recently in zebrafish pointed to a potential role for SelN during embryogenesis. Using qRT-PCR, Western blot and whole mount in situ hybridization, we characterized in detail the spatio-temporal expression pattern of the murine Sepn1 gene during development, focusing particularly on skeletal muscles. Results In whole embryos, Sepn1 transcripts were detected as early as E5.5, with expression levels peaking at E12.5, and then strongly decreasing until birth. In isolated tissues, only mild transcriptional variations were observed during development, whereas a striking reduction of the protein expression was detected during the perinatal period. Furthermore, we demonstrated that Sepn1 is expressed early in somites and restricted to the myotome, the sub-ectodermal mesenchyme and the dorsal root ganglia at mid-gestation stages. Interestingly, Sepn1 deficiency did not alter somitogenesis in embryos, suggesting that SelN is dispensable for these processes in mouse. Conclusion We characterized for the first time the expression pattern of Sepn1 during mammalian embryogenesis and we demonstrated that its differential expression is most likely dependent on major post-transcriptional regulations. Overall, our data strongly suggest a potential role for selenoprotein N from mid-gestation stages to the perinatal period. Interestingly, its specific expression pattern could be related to the current hypothesis that selenoprotein N may regulate the activity of the ryanodine receptors.

BACKGROUND: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. METHODS: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. RESULTS: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. CONCLUSIONS: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein. Copyright (c) 2007 John Wiley & Sons, Ltd.

We have identified highly similar heterozygous COL6A1 genomic deletions, spanning from intron 8 to exon 13 or intron 13, in two patients with Ullrich congenital muscular dystrophy and the milder Bethlem myopathy. The 5' breakpoints of both deletions are located within a minisatellite in intron 8. The mutations cause in-frame deletions of 66 and 84 amino acids in the amino terminus of the triple-helical domain, leading to intracellular accumulation of mutant polypeptides and reduced extracellular collagen VI microfibrils. Our studies identify a deletion-prone region in COL6A1 and suggest that similar mutations can lead to congenital muscle disorders of different clinical severity.

Multiple sclerosis (MS), an inflammatory autoimmune demyelinating disorder of the central nervous system, is the most common cause of acquired neurological dysfunction arising in the second to fourth decades of life1,2. A genetic component to MS is indicated by an increased relative risk of 20–40 to siblings compared to the general population (γs)3–6, and an increased concordance rate in monozygotic compared to dizygotic twins7. Association and/or linkage studies to candidate genes have produced many reports of significant genetic effects including those for the major histocompatability complex (MHC; particularly the HLA-DR2 allele), immunoglobulin heavy chain (IgH), T-cell receptor (TCR) and myelin basic protein (MBP) loci8–17. With the exception of the MHC, however, these results have been difficult to replicate and/or apply beyond isolated populations. We have therefore conducted a two-stage, multi-analytical genomic screen to identify genomic regions potentially harbouring MS susceptibility genes. We geno-typed 443 markers and 19 such regions were identified. These included the MHC region on 6p, the only region with a consistently reported genetic effect. However, no single locus generated overwhelming evidence of linkage. Our results suggest that a multifactorial aetiology, including both environmental and multiple genetic factors of moderate effect, is more likely than an aetiology consisting of simple mendelian disease gene(s)

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.

The classical phenotypes of collagen VI-associated myopathies are well described. Little is known, however, about the progression of patients at the mildest end of the clinical spectrum. In this report, we describe the clinical findings and the results of MRI, muscle biopsy, collagen VI expression in cultured skin fibroblasts and genetic tests of a series of patients with Bethlem myopathy. Our series highlights the existence of mild presentations of this disorder that progresses only slightly and can easily be overlooked. Analysis of the genetic studies suggests that missense mutations can be associated to a milder clinical presentation. Muscle MRI is extremely useful as it shows a pathognomonic pattern in most patients, especially those with some degree of muscle weakness.