Whole transcriptome expression profiling in collagen VI myopathies KO mice reveals muscle-specific fingerprints and arises the role of circadian clock genes as myopathy biomarkers

Collagen VI is an extracellular matix protein that forms a microfilamentous network in skeletal muscles and other organs. In humans, mutations in the collagen VI alpha1, 2 and 3 genes cause congenital muscular dystrophies as Bethlem , Ullrich and Myosclerosis. Mitochondrial pore abnormalities and defect in the autophagic pathways have been identified both in patients and in Col6a2KO mouse model. In order to bridging these findings to the transcriptome, we have performed whole expression profile in both wild type and KO mice. The transcritpomic data were also analysed by an ad hoc designed software enabling us to design an interactome map of the de-regulated transcripts highlighted by the array studies. The mice muscles analysed were diaphragm, gastrocnemious and tibilias. Various transcripts belonging to the muscle development and regeneration, also including genes involved in the apoptosis and autophagy control are consistently de-regulated in KO mice, supporting the role that these processes play in the disease pathogenesis. Interestingly, six genes acting within the circadian clock mechanisms are constantly down-regulated in KO mice. This behavior is constant in all muscles analysed, though more prominent in tibialis. The pathway scan analysis revealed connections between clock genes and apoptosis and autophagy as well as with muscle regeneration and muscle signaling, opening a previously undisclosed role of circadian rhythm in muscle diseases. These finding make clock genes down-regulation exploratory biomarkers of collagen VI pathology

Macrophages: A minimally invasive tool for monitoring collagen VI myopathies

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

<p>Abstract</p> <p>Background</p> <p>The commonest pathogenic <it>DMD </it>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 <it>DMD </it>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 <it>DMD </it>gene and spans from 100 kb upstream to 100 kb downstream of the 2.2 Mb <it>DMD </it>gene.</p> <p>Results</p> <p>We studied 12 DMD/BMD patients who either had no detectable mutations or carried previously identified quantitative pathogenic changes in the <it>DMD </it>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.</p> <p>Conclusion</p> <p>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.</p

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

Self-Care for Nurses and Midwives: Findings from a Scoping Review

Self-care for health care professionals is essential in order to optimize the care they provide and to prevent serious consequences for their health. This scoping review aimed to identify (a) the concepts used in the literature to describe self-care; (b) interventions that influence self-care. The scoping review was conducted according to the criteria and methodology by Arksey and O&rsquo;Malley, from November 2020 to January 2021, by consulting the following databases: Pubmed, CINAHL, Scopus, PsycInfo, Cochrane Library, Joanna Briggs Library. Various keywords and MesH terms were used for the search, including self-care, nurses, midwives, nursing, midwifery, self-compassion, and self-awareness. Eighteen studies were included. The concept of self-care is related to three constructs: (a) Mindfulness; (b) Compassion; and (c) Resilience. In the literature, self-care interventions can be distinguished as (a) mindfulness-based; (b) educational; (c) multimodal approach; and (d) mind-body interventions. In recent years, the concept of self-care is a topic of great interest in the literature; dealing with self-care from both a theoretical and a practical&mdash;personal and professional&mdash;perspective has become more important in order to promote practitioners&rsquo; well-being. This scoping review helps to clarify the terms related to self-care and looks at tested interventions to improve the well-being of caregivers

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

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

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

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