Deep RNA profiling identified CLOCK and molecular clock genes as pathophysiological signatures in collagen VI myopathy.

Collagen VI myopathies are genetic disorders caused by mutations in collagen 6 A1, A2 and A3 genes, ranging from the severe Ullrich congenital muscular dystrophy to the milder Bethlem myopathy, which is recapitulated by collagen-VI-null (Col6a1(-/-)) mice. Abnormalities in mitochondria and autophagic pathway have been proposed as pathogenic causes of collagen VI myopathies, but the link between collagen VI defects and these metabolic circuits remains unknown. To unravel the expression profiling perturbation in muscles with collagen VI myopathies, we performed a deep RNA profiling in both Col6a1(-/-)mice and patients with collagen VI pathology. The interactome map identified common pathways suggesting a previously undetected connection between circadian genes and collagen VI pathology. Intriguingly, Bmal1(-/-)(also known as Arntl) mice, a well-characterized model displaying arrhythmic circadian rhythms, showed profound deregulation of the collagen VI pathway and of autophagy-related genes. The involvement of circadian rhythms in collagen VI myopathies is new and links autophagy and mitochondrial abnormalities. It also opens new avenues for therapies of hereditary myopathies to modulate the molecular clock or potential gene-environment interactions that might modify muscle damage pathogenesis.

Mutations in the collagen XII gene define a new form of extracellular matrix-related myopathy.

Bethlem myopathy (BM) [MIM 158810] is a slowly progressive muscle disease characterized by contractures and proximal weakness, which can be caused by mutations in one of the collagen VI genes (COL6A1, COL6A2 and COL6A3). However, there may be additional causal genes to identify as in ∼50% of BM cases no mutations in the COL6 genes are identified. In a cohort of -24 patients with a BM-like phenotype, we first sequenced 12 candidate genes based on their function, including genes for known binding partners of collagen VI, and those enzymes involved in its correct post-translational modification, assembly and secretion. Proceeding to whole-exome sequencing (WES), we identified mutations in the COL12A1 gene, a member of the FACIT collagens (fibril-associated collagens with interrupted triple helices) in five individuals from two families. Both families showed dominant inheritance with a clinical phenotype resembling classical BM. Family 1 had a single-base substitution that led to the replacement of one glycine residue in the triple-helical domain, breaking the Gly-X-Y repeating pattern, and Family 2 had a missense mutation, which created a mutant protein with an unpaired cysteine residue. Abnormality at the protein level was confirmed in both families by the intracellular retention of collagen XII in patient dermal fibroblasts. The mutation in Family 2 leads to the up-regulation of genes associated with the unfolded protein response (UPR) pathway and swollen, dysmorphic rough-ER. We conclude that the spectrum of causative genes in extracellular matrix (ECM)-related myopathies be extended to include COL12A1.

Characterization of a rare case of Ullrich congenital muscular dystrophy due to truncating mutations within the COL6A1 gene C-terminal domain: a case report.

BACKGROUND: Mutations within the C-terminal region of the COL6A1 gene are only detected in Ullrich/Bethlem patients on extremely rare occasions. CASE PRESENTATION: Herein we report two Brazilian brothers with a classic Ullrich phenotype and compound heterozygous for two truncating mutations in COL6A1 gene, expected to result in the loss of the α1(VI) chain C2 subdomain. Despite the reduction in COL6A1 RNA level due to nonsense RNA decay, three truncated alpha1 (VI) chains were produced as protein variants encoded by different out-of-frame transcripts. Collagen VI matrix was severely decreased and intracellular protein retention evident. CONCLUSION: The altered deposition of the fibronectin network highlighted abnormal interactions of the mutated collagen VI, lacking the α1(VI) C2 domain, within the extracellular matrix, focusing further studies on the possible role played by collagen VI in fibronectin deposition and organization.

Macrophages: a minimally invasive tool for monitoring collagen VI myopathies.

INTRODUCTION: Collagen VI expression was tested in peripheral blood macrophages from patients with collagen VI-related myopathies and compared with muscle biopsy. METHODS: RNA and protein studies were performed in blood macrophages from 5 patients previously diagnosed with either Ullrich congenital muscular dystrophy (UCMD) or Bethlem myopathy (BM). The full spectrum of possible genotypes was considered, including both dominant and recessive UCMD and BM cases. RESULTS: In the dominant BM patient, no collagen VI alterations were detectable in macrophages or muscle biopsy. In the remaining patients, the protein defect caused by the selected mutations, as well as the transcriptional abnormalities, were readily detectable in macrophages, at levels comparable to those observed in muscle biopsy samples and cultured skin fibroblasts. CONCLUSIONS: Our data support the suitability of peripheral blood macrophages as a reliable, minimally invasive tool for supplementing or replacing muscle/skin biopsies in the diagnosis and monitoring of collagen VI-related myopathies.

Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies.

BACKGROUND: Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. METHODS: We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. RESULTS: A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. CONCLUSIONS: Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes.

Cationic PMMA nanoparticles bind and deliver antisense oligoribonucleotides allowing restoration of dystrophin expression in the mdx mouse.

For subsets of Duchenne muscular dystrophy (DMD) mutations, antisense oligoribonucleotide (AON)-mediated exon skipping has proven to be efficacious in restoring the expression of dystrophin protein. In the mdx murine model systemic delivery of AON, recognizing the splice donor of dystrophin exon 23, has shown proof of concept. Here, we show that using cationic polymethylmethacrylate (PMMA) (marked as T1) nanoparticles loaded with a low dose of 2'-O-methyl-phosphorothioate (2'OMePS) AON delivered by weekly intraperitoneal (IP) injection (0.9 mg/kg/week), could restore dystrophin expression in body-wide striated muscles. Delivery of an identical dose of naked AON did not result in detectable dystrophin expression. Transcription, western, and immunohistochemical analysis showed increased levels of dystrophin transcript and protein, and correct localization at the sarcolemma. This study shows that T1 nanoparticles have the capacity to bind and convoy AONs in body-wide muscle tissues and to reduce the dose required for dystrophin rescue. By immunofluorescence and electron microscopy studies, we highlighted the diffusion pathways of this compound. This nonviral approach may valuably improve the therapeutic usage of AONs in DMD as well as the delivery of RNA molecules with many implications in both basic research and medicine.

Identification and characterization of novel collagen VI non-canonical splicing mutations causing Ullrich congenital muscular dystrophy.

Splicing mutations occurring outside the invariant GT and AG dinucleotides are frequent in disease genes and the definition of their pathogenic potential is often challenging. We have identified four patients affected by Ullrich congenital muscular dystrophy and carrying unusual mutations of COL6 genes affecting RNA splicing. In three cases the mutations occurred in the COL6A2 gene and consisted of nucleotide substitutions within the degenerated sequences flanking the canonical dinucleotides. In the fourth case, a genomic deletion occurred which removed the exon8-intron8 junction of the COL6A1 gene. These mutations induced variable splicing phenotypes, consisting of exon skipping, intron retention and cryptic splice site activation/usage. A quantitative RNA assay revealed a reduced level of transcription of the mutated in-frame mRNA originating from a COL6A2 point mutation at intronic position +3. At variance, the transcription level of the mutated in-frame mRNA originating from a genomic deletion which removed the splicing sequences of COL6A1 exon 8 was normal. These findings suggest a different transcriptional efficiency of a regulatory splicing mutation compared to a genomic deletion causing a splicing defect.

A novel custom high density-comparative genomic hybridization array detects common rearrangements as well as deep intronic mutations in dystrophinopathies.

BACKGROUND: The commonest pathogenic DMD changes are intragenic deletions/duplications which make up to 78% of all cases and point mutations (roughly 20%) detectable through direct sequencing. The remaining mutations (about 2%) are thought to be pure intronic rearrangements/mutations or 5'-3' UTR changes. In order to screen the huge DMD gene for all types of copy number variation mutations we designed a novel custom high density comparative genomic hybridisation array which contains the full genomic region of the DMD gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb DMD gene. RESULTS: We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the DMD gene. We validated the array on patients with previously known mutations as well as unaffected controls, we identified three novel pure intronic rearrangements and we defined all the mutation breakpoints both in the introns and in the 3' UTR region. We also detected a novel polymorphic intron 2 deletion/duplication variation. Despite the high resolution of this approach, RNA studies were required to confirm the functional significance of the intronic mutations identified by CGH. In addition, RNA analysis identified three intronic pathogenic variations affecting splicing which had not been detected by the CGH analysis. CONCLUSION: This novel technology represents an effective high throughput tool to identify both common and rarer DMD rearrangements. RNA studies are required in order to validate the significance of the CGH array findings. The combination of these tools will fully cover the identification of causative DMD rearrangements in both coding and non-coding regions, particularly in patients in whom standard although extensive techniques are unable to detect a mutation.

Expression of collagen VI α5 and α6 chains in human muscle and in Duchenne muscular dystrophy-related muscle fibrosis.

Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1(-/-) mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-ß1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis.

Expression of the collagen VI α5 and α6 chains in normal human skin and in skin of patients with collagen VI-related myopathies.

Collagen VI is an extracellular matrix protein with critical roles in maintaining muscle and skin integrity and function. Skin abnormalities, including predisposition to keratosis pilaris and abnormal scarring, were described in Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) patients carrying mutations in COL6A1, COL6A2, and COL6A3 genes, whereas COL6A5, previously designated as COL29A1, was linked to atopic dermatitis. To gain insight into the function of the newly identified collagen VI α5 and α6 chains in human skin, we studied their expression and localization in normal subjects and in genetically characterized UCMD and BM patients. We found that localization of α5, and to a lesser extent α6, is restricted to the papillary dermis, where the protein mainly colocalizes with collagen fibrils. In addition, both chains were found around blood vessels. In UCMD patients with COL6A1 or COL6A2 mutations, immunolabeling for α5 and α6 was often altered, whereas in a UCMD and in a BM patient, each with a COL6A3 mutation, expression of α5 and α6 was apparently unaffected, suggesting that these chains may substitute for α3, forming α1α2α5 or α1α2α6 heterotrimers.