Characterization of Proteome Changes in Aged and Collagen VI-Deficient Human Pericyte Cultures.

Pericytes are a distinct type of cells interacting with endothelial cells in blood vessels and contributing to endothelial barrier integrity. Furthermore, pericytes show mesenchymal stem cell properties. Muscle-derived pericytes can demonstrate both angiogenic and myogenic capabilities. It is well known that regenerative abilities and muscle stem cell potential decline during aging, leading to sarcopenia. Therefore, this study aimed to investigate the potential of pericytes in supporting muscle differentiation and angiogenesis in elderly individuals and in patients affected by Ullrich congenital muscular dystrophy or by Bethlem myopathy, two inherited conditions caused by mutations in collagen VI genes and sharing similarities with the progressive skeletal muscle changes observed during aging. The study characterized pericytes from different age groups and from individuals with collagen VI deficiency by mass spectrometry-based proteomic and bioinformatic analyses. The findings revealed that aged pericytes display metabolic changes comparable to those seen in aging skeletal muscle, as well as a decline in their stem potential, reduced protein synthesis, and alterations in focal adhesion and contractility, pointing to a decrease in their ability to form blood vessels. Strikingly, pericytes from young patients with collagen VI deficiency showed similar characteristics to aged pericytes, but were found to still handle oxidative stress effectively together with an enhanced angiogenic capacity.

Whole exome sequencing identifies a novel variant in the COL12A1 gene in a family with Ullrich congenital muscular dystrophy 2.

BACKGROUND: Mutations within the COL12A1 gene have been linked with the onset of congenital Ullrich muscular dystrophy 2 (UCMD2) and Bethlem myopathy. The severity of the symptoms exhibited is dependent on the mutation's type and whether it is heterozygous or homozygous. METHODS: We used whole-exome sequencing to identify disease-causing variants in a nine-year-old Iranian patient who had weakness, joint contractures, delayed motor development, and other symptoms. We confirmed the pathogenicity of the identified variant using in silico tools and verified its novelty using various databases. We also performed a co-segregation study and confirmed the presence of the variant in the patient's parents by Sanger sequencing. RESULTS: Our analysis identified a novel homozygous missense variant in the affected patient in COL12A1 (c.8828 C > T; p.Pro2943Leu). This is the second reported family with UCMD2 caused by a mutation in COL12A1. Our findings confirm that this mutation results in significantly more severe symptoms than Bethlem myopathy. CONCLUSION: Our investigation contributes to the expanding body of evidence that links mutations in COL12A1 with UCMD2. Our findings confirm that the homozygous mutation in COL12A1 caused this condition and suggest that genetic testing for this mutation may be useful for diagnosing patients with this disease.

New Clinical and Immunofluoresence Data of Collagen VI-Related Myopathy: A Single Center Cohort of 69 Patients.

Pathogenetic mechanism recognition and proof-of-concept clinical trials were performed in our patients affected by collagen VI-related myopathies. This study, which included 69 patients, aimed to identify innovative clinical data to better design future trials. Among the patients, 33 had Bethlem myopathy (BM), 24 had Ullrich congenital muscular dystrophy (UCMD), 7 had an intermediate phenotype (INTM), and five had myosclerosis myopathy (MM). We obtained data on muscle strength, the degree of contracture, immunofluorescence, and genetics. In our BM group, only one third had a knee extension strength greater than 50% of the predicted value, while only one in ten showed similar retention of elbow flexion. These findings should be considered when recruiting BM patients for future trials. All the MM patients had axial and limb contractures that limited both the flexion and extension ranges of motion, and a limitation in mouth opening. The immunofluorescence analysis of collagen VI in 55 biopsies from 37 patients confirmed the correlation between collagen VI defects and the severity of the clinical phenotype. However, biopsies from the same patient or from patients with the same mutation taken at different times showed a progressive increase in protein expression with age. The new finding of the time-dependent modulation of collagen VI expression should be considered in genetic correction trials.

Collagen VI in the Musculoskeletal System.

Collagen VI exerts several functions in the tissues in which it is expressed, including mechanical roles, cytoprotective functions with the inhibition of apoptosis and oxidative damage, and the promotion of tumor growth and progression by the regulation of cell differentiation and autophagic mechanisms. Mutations in the genes encoding collagen VI main chains, COL6A1, COL6A2 and COL6A3, are responsible for a spectrum of congenital muscular disorders, namely Ullrich congenital muscular dystrophy (UCMD), Bethlem myopathy (BM) and myosclerosis myopathy (MM), which show a variable combination of muscle wasting and weakness, joint contractures, distal laxity, and respiratory compromise. No effective therapeutic strategy is available so far for these diseases; moreover, the effects of collagen VI mutations on other tissues is poorly investigated. The aim of this review is to outline the role of collagen VI in the musculoskeletal system and to give an update about the tissue-specific functions revealed by studies on animal models and from patients' derived samples in order to fill the knowledge gap between scientists and the clinicians who daily manage patients affected by collagen VI-related myopathies.

Keratosis pilaris in collagen type VI-related disorders.

Keratosis pilaris is a common skin condition associated with a number of syndromes, including collagen type VI-related disorders. Our patient, recently diagnosed with Ullrich congenital muscular dystrophy, presented with severe keratosis pilaris, hypotonia, and velvety skin on the palms and soles. We present this case to highlight the importance of including cutaneous findings, such as keratosis pilaris, to aid in the diagnosis when evaluating patients with syndromic features.

The Presentation of Two Unrelated Clinical Cases from the Republic of North Ossetia-Alania with the Same Previously Undescribed Variant in the COL6A2 Gene.

Here, we described three affected boys from two unrelated families of Ossetian-Digor origin from the Republic of North Ossetia-Alania who were admitted to the Research Centre for Medical Genetics with unspecified muscular dystrophy. High-throughput sequencing was performed and revealed two novel frameshift variants in the COL6A2 gene (NM_001849.3) in a heterozygous state each in both cases: c.508_535delinsCTGTGG and c.1659_1660del (case 1) and c.1689del and c.1659_1660del (case 2). In two cases, the same nucleotide variant in the COL6A2 gene (c.1659_1660del) was observed. We have suggested that the variant c.1659_1660del may be common in the Ossetian-Digor population because two analyzed families have the same ancestry from the same subethnic group of Ossetians). The screening for an asymptomatic carriage of the nucleotide variant c.1659_1660del in 54 healthy donors from Ossetian-Digor population revealed that the estimated carrier frequency is 0.0093 (CI: 0.0002-0.0505), which is high for healthy carriers of the pathogenic variant. Molecular genetic, anamnestic data and clinical examination results allowed us to diagnose Ullrich muscular dystrophy in those affected boys. Genetic heterogeneity and phenotypic diversity of muscular dystrophies complicate diagnosis. It is important to make a differential diagnosis of such conditions and use HTS methods to determine the most accurate diagnosis.

COL6A1 related muscular dystrophy in Landseer dogs: A canine model for Ullrich congenital muscular dystrophy.

BACKGROUND: Collagen VI related myopathies are congenital diseases of variable phenotype. The severe phenotype is referred to as Ullrich congenital muscular dystrophy. In this study, we describe analoguos clinical signs and histopathological alterations in Landseer dogs. MATERIALS: We collected clinical data from two affected dogs and investigated the neuromuscular changes in five dogs from two different litters with immunohistochemistry and immunofluorescence. All affected dogs were homozygous for the p.Glu97* nonsense variant in the COL6A1 gene encoding the alpha-1 chain of collagen VI. RESULTS: Muscle biopsies revealed alterations similar to those in human patients with Ullrich congenital muscular dystrophy including the virtual absence of collagen VI in skeletal muscles. CONCLUSIONS: The clinical and pathological characterization of the affected Landseer dogs enhances the value of this animal model for human Ullrich congenital muscular dystrophy.

A novel variant in the COL6A1 gene causing Ullrich congenital muscular dystrophy in a consanguineous family: a case report.

BACKGROUND: Collagen VI-related dystrophies are a subtype of congenital muscular dystrophy caused by pathogenic variants in COL6A1, COL6A2 or COL6A3 genes affecting skeletal muscles and connective tissue. The clinical phenotype ranges from the milder Bethlem myopathy to the severe Ullrich congenital muscular dystrophy (UCMD). Herein, we report the first consanguineous Sri Lankan family with two children affected with UCMD due to a novel variant in the COL6A1 gene. CASE PRESENTATION: Two sisters, aged 10-years and 7-years, presented with progressive, bilateral proximal muscle weakness. Both probands had delayed motor milestones and demonstrated difficulty in standing from a squatting position, climbing stairs and raising arms above the shoulders. Cognitive, language and social development were age appropriate. Examination showed proximal muscle weakness of the upper and lower extremities and hyperlaxity of the wrist and fingers in both with some variability in clinical severity noted between the two siblings. Serum creatine kinase levels were elevated, and electromyography showed low polyphasic motor unit potentials in the 10-year-old and myopathic features with short duration motor unit potentials with no polyphasia in the 7-year-old. Whole exome sequencing (WES) was performed and a novel, homozygous missense, likely pathogenic variant in exon 25 of COL6A1 gene [NM_001848: c.1667G > T;NP_001839.2:p.Gly556Val] was identified in both probands. This variant was validated by Sanger sequencing in proband 1 as well as proband 2, and the parents and an unaffected sibling were found to be heterozygote carriers for the same variant. CONCLUSIONS: The findings in this family add to the expanding number of COL6A1 variants identified and provides a better understanding of the genotype-phenotype correlations associated with UCMD.

Collagen VI Muscle Disorders: Mutation Types, Pathogenic Mechanisms and Approaches to Therapy.

Mutations in the genes encoding the major collagen VI isoform, COL6A1, COL6A2 and COL6A3, are responsible for the muscle disorders Bethlem myopathy and Ullrich congenital muscular dystrophy. These disorders form a disease spectrum from mild to severe. Dominant and recessive mutations are found along the entire spectrum and the clinical phenotype is strongly influenced by the way mutations impede collagen VI protein assembly. Most mutations are in the triple helical domain, towards the N-terminus and they compromise microfibril assembly. Some mutations are found outside the helix in the C- and N-terminal globular domains, but because these regions are highly polymorphic it is difficult to discriminate mutations from rare benign changes without detailed structural and functional studies. Collagen VI deficiency leads to mitochondrial dysfunction, deficient autophagy and increased apoptosis. Therapies that target these consequences have been tested in mouse models and some have shown modest efficacy in small human trials. Antisense therapies for a common mutation that introduces a pseudoexon show promise in cell culture but haven't yet been tested in an animal model. Future therapeutic approaches await new research into how collagen VI deficiency signals downstream consequences.

Genetic and clinical findings in a Chinese cohort of patients with collagen VI-related myopathies.

Collagen VI-related myopathy, caused by pathogenic variants in the genes encoding collagen VI, represents a clinical continuum from Ullrich congenital muscular dystrophy (UCMD) to Bethlem myopathy (BM). Clinical data of 60 probands and their family members were collected and muscle biopsies of 26 patients were analyzed. COL6A1, COL6A2 and COL6A3 exons were analyzed by direct sequencing or next generation sequencing (NGS). Sixty patients were characterized by delayed motor milestones, muscle weakness, skin and joint changes with 40 UCMD and 20 BM. Muscle with biopsies revealed dystrophic changes and showed completely deficiency of collagen VI or sarcolemma specific collagen VI deficiency. We identified 62 different pathogenic variants in these 60 patients, with 34 were first reported while 28 were previously known; 72 allelic pathogenic variants in COL6A1 (25/72, 34.7%), COL6A2 (33/72, 45.8%) and COL6A3 (14/72, 19.4%). We also found somatic mosaic variant in the parent of 1 proband by personal genome machine amplicon deep sequencing for mosaicism. Here we provide clinical, histological and genetic evidence of collagen VI-related myopathy in 60 Chinese patients. NGS is a valuable approach for diagnosis and accurate diagnosis provides useful information for genetic counseling of related families.

Collagen VI is required for the structural and functional integrity of the neuromuscular junction.

The synaptic cleft of the neuromuscular junction (NMJ) consists of a highly specialized extracellular matrix (ECM) involved in synapse maturation, in the juxtaposition of pre- to post-synaptic areas, and in ensuring proper synaptic transmission. Key components of synaptic ECM, such as collagen IV, perlecan and biglycan, are binding partners of one of the most abundant ECM protein of skeletal muscle, collagen VI (ColVI), previously never linked to NMJ. Here, we demonstrate that ColVI is itself a component of this specialized ECM and that it is required for the structural and functional integrity of NMJs. In vivo, ColVI deficiency causes fragmentation of acetylcholine receptor (AChR) clusters, with abnormal expression of NMJ-enriched proteins and re-expression of fetal AChRγ subunit, both in Col6a1 null mice and in patients affected by Ullrich congenital muscular dystrophy (UCMD), the most severe form of ColVI-related myopathies. Ex vivo muscle preparations from ColVI null mice revealed altered neuromuscular transmission, with electrophysiological defects and decreased safety factor (i.e., the excess current generated in response to a nerve impulse over that required to reach the action potential threshold). Moreover, in vitro studies in differentiated C2C12 myotubes showed the ability of ColVI to induce AChR clustering and synaptic gene expression. These findings reveal a novel role for ColVI at the NMJ and point to the involvement of NMJ defects in the etiopathology of ColVI-related myopathies.

Collagen VI-related myopathy: Expanding the clinical and genetic spectrum.

INTRODUCTION: We aimed to analyze the clinical and genetic characteristics of collagen VI-related myopathy. METHODS: We analyzed the clinical course and mutation spectrum in patients with collagen VI gene mutations among our congenital muscular dystrophy cohort. RESULTS: Among 24 patients with mutations in collagen VI coding genes, 13 (54.2%) were categorized as Ullrich type, and 11 (45.8%) as non-Ullrich type. Congenital orthopedic problems were similarly observed in both types, yet multiple joint contractures were found only in the Ullrich type. Clinical courses and pathology findings varied between patients. Mutations in COL6A1, COL6A2, and COL6A3 were found in 15 (65%), 3 (13%), and 5 (22%) patients, respectively, without genotype-phenotype association. Five novel variants were detected. DISCUSSION: We verified clinical heterogeneity of collagen VI-related myopathy, which emphasizes the importance of genetic testing. Genotype-phenotype association or early predictors for progression were not identified. Multiple joint contractures predict rapid deterioration. Muscle Nerve 58: 381-388, 2018.

Novel Col12A1 variant expands the clinical picture of congenital myopathies with extracellular matrix defects.

INTRODUCTION: Mutations in the COL12A1 (collagen, type XII, alpha 1) gene have been described in a milder Bethlem-like myopathy in 6 patients from 3 families (dominant missense), and in a severe congenital form with failure to attain ambulation in 2 patients in a single pedigree (recessive loss-of-function). METHODS: We describe an 8-year-old girl of Polish origin who presented with profound hypotonia and joint hyperlaxity at birth after a pregnancy complicated by oligohydramnios and intrauterine growth retardation. RESULTS: We identified a novel, potentially pathogenic heterozygous missense COL12A1 c.8329G>C (p.Gly2777Arg) variant using a targeted sequencing panel. Patient fibroblast studies confirmed intracellular retention of the COL12A1 protein, consistent with a dominant-negative mutation. CONCLUSIONS: As our patient showed a more intermediate phenotype, this case expands the phenotypic spectrum for COL12A1 disorders. So far, COL12A1 disorders seem to cover much of the severity range of an Ehlers-Danlos/Bethlem-like myopathy overlap syndrome associated with both connective tissue abnormalities and muscle weakness. Muscle Nerve 55: 277-281, 2017.

Mosaicism for dominant collagen 6 mutations as a cause for intrafamilial phenotypic variability.

Collagen 6-related dystrophies and myopathies (COL6-RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter- and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6-RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild-type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild-type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6-RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected.

Muscle proteomics reveals novel insights into the pathophysiological mechanisms of collagen VI myopathies.

Mutations in the collagen VI genes cause the Ullrich congenital muscular dystrophy (UCMD), with severe phenotype, and Bethlem myopathy (BM) with mild to moderate phenotype. Both, UCMD and BM patients show dystrophic features with degeneration/regeneration and replacement of muscle with fat and fibrous connective tissue. At molecular level, UCMD patients show autophagic impairment and increased PTP opening; these features are less severe in BM. To elucidate the biochemical mechanisms adopted by the muscle to adapt to collagen VI deficiency in BM and UCMD patients, a proteome analysis was carried out on human muscle biopsies. Qualitative and quantitative differences were assessed by 2D-DIGE coupled to MALDI-ToF/ToF MS. Proteomics results, coupled with immunoblotting, indicate changes in UPR, hexosamine pathway, and amino acid and fatty acid metabolism, suggesting an association of ER stress, metabolic dysregulation, autophagic impairment, and alteration in mechanotransduction signaling. Overall, these results indicate that despite the common downregulation of hexosamine pathway in UCMD and BM, in BM the protein quality control system is sustained by a metabolic adaptation supporting energy requirements for the maintenance of autophagy, counteracting ER misfolded protein overload. In UCMD, this multilayered system may be disrupted and worsened by the metabolic rewiring, which leads to lipotoxicity.

Clinical and Molecular Profiles of a Cohort of Egyptian Patients with Collagen VI-Related Dystrophy.

Collagen VI-related dystrophies (COL6-RD) display a wide spectrum of disease severity and genetic variability ranging from mild Bethlem myopathy (BM) to severe Ullrich congenital muscular dystrophy (UCMD) and the intermediate severities in between with dual modes of inheritance, dominant and recessive. In the current study, next-generation sequencing demonstrated potential variants in the genes coding for the three alpha chains of collagen VI (COL6A1, COL6A2, or COL6A3) in a cohort of Egyptian patients with progressive muscle weakness (n = 23). Based on the age of disease onset and the patient clinical course, subjects were diagnosed as follows: 12 with UCMD, 8 with BM, and 3 with intermediate disease form. Fourteen pathogenic variants, including 5 novel alterations, were reported in the enrolled subjects. They included 3 missense, 3 frameshift, and 6 splicing variants in 4, 3, and 6 families, respectively. In addition, a nonsense variant in a single family and an inframe variant in 3 different families were also detected. Recessive and dominant modes of inheritance were recorded in 9 and 8 families, respectively. According to ACMG guidelines, variants were classified as pathogenic (n = 7), likely pathogenic (n = 4), or VUS (n = 3) with significant pathogenic potential. To our knowledge, the study provided the first report of the clinical and genetic findings of a cohort of Egyptian patients with collagen VI deficiency. Inter- and intra-familial clinical variability was evident among the study cohort.

Distinct muscle regenerative capacity of human induced pluripotent stem cell-derived mesenchymal stromal cells in Ullrich congenital muscular dystrophy model mice.

BACKGROUND: Ullrich congenital muscular dystrophy (UCMD) is caused by a deficiency in type 6 collagen (COL6) due to mutations in COL6A1, COL6A2, or COL6A3. COL6 deficiency alters the extracellular matrix structure and biomechanical properties, leading to mitochondrial defects and impaired muscle regeneration. Therefore, mesenchymal stromal cells (MSCs) that secrete COL6 have attracted attention as potential therapeutic targets. Various tissue-derived MSCs exert therapeutic effects in various diseases. However, no reports have compared the effects of MSCs of different origins on UCMD pathology. METHODS: To evaluate which MSC population has the highest therapeutic efficacy for UCMD, in vivo (transplantation of MSCs to Col6a1-KO/NSG mice) and in vitro experiments (muscle stem cell [MuSCs] co-culture with MSCs) were conducted using adipose tissue-derived MSCs, bone marrow-derived MSCs, and xeno-free-induced iPSC-derived MSCs (XF-iMSCs). RESULTS: In transplantation experiments on Col6a1-KO/NSG mice, the group transplanted with XF-iMSCs showed significantly enhanced muscle fiber regeneration compared to the other groups 1 week after transplantation. At 12 weeks after transplantation, only the XF-iMSCs transplantation group showed a significantly larger muscle fiber diameter than the other groups without inducing fibrosis, which was observed in the other transplantation groups. Similarly, in co-culture experiments, XF-iMSCs were found to more effectively promote the fusion and differentiation of MuSCs derived from Col6a1-KO/NSG mice than the other primary MSCs investigated in this study. Additionally, in vitro knockdown and supplementation experiments suggested that the IGF2 secreted by XF-iMSCs promoted MuSC differentiation. CONCLUSION: XF-iMSCs are promising candidates for promoting muscle regeneration while avoiding fibrosis, offering a safer and more effective therapeutic approach for UCMD than other potential therapies.

Collagen VI Deficiency Impairs Tendon Fibroblasts Mechanoresponse in Ullrich Congenital Muscular Dystrophy.

The pericellular matrix (PCM) is a specialized extracellular matrix that surrounds cells. Interactions with the PCM enable the cells to sense and respond to mechanical signals, triggering a proper adaptive response. Collagen VI is a component of muscle and tendon PCM. Mutations in collagen VI genes cause a distinctive group of inherited skeletal muscle diseases, and Ullrich congenital muscular dystrophy (UCMD) is the most severe form. In addition to muscle weakness, UCMD patients show structural and functional changes of the tendon PCM. In this study, we investigated whether PCM alterations due to collagen VI mutations affect the response of tendon fibroblasts to mechanical stimulation. By taking advantage of human tendon cultures obtained from unaffected donors and from UCMD patients, we analyzed the morphological and functional properties of cellular mechanosensors. We found that the length of the primary cilia of UCMD cells was longer than that of controls. Unlike controls, in UCMD cells, both cilia prevalence and length were not recovered after mechanical stimulation. Accordingly, under the same experimental conditions, the activation of the Hedgehog signaling pathway, which is related to cilia activity, was impaired in UCMD cells. Finally, UCMD tendon cells exposed to mechanical stimuli showed altered focal adhesions, as well as impaired activation of Akt, ERK1/2, p38MAPK, and mechanoresponsive genes downstream of YAP. By exploring the response to mechanical stimulation, for the first time, our findings uncover novel unreported mechanistic aspects of the physiopathology of UCMD-derived tendon fibroblasts and point at a role for collagen VI in the modulation of mechanotransduction in tendons.

Optimized allele-specific silencing of the dominant-negative COL6A1 G293R substitution causing collagen VI-related dystrophy.

Collagen VI-related dystrophies (COL6-RDs) are a group of severe, congenital-onset muscular dystrophies for which there is no effective causative treatment. Dominant-negative mutations are common in COL6A1, COL6A2, and COL6A3 genes, encoding the collagen α1, α2, and α3 (VI) chains. They act by incorporating into the hierarchical assembly of the three α (VI) chains and consequently produce a dysfunctional collagen VI extracellular matrix, while haploinsufficiency for any of the COL6 genes is not associated with disease. Hence, allele-specific transcript inactivation is a valid therapeutic strategy, although selectively targeting a pathogenic single nucleotide variant is challenging. Here, we develop a small interfering RNA (siRNA) that robustly, and in an allele-specific manner, silences a common glycine substitution (G293R) caused by a single nucleotide change in COL6A1 gene. By intentionally introducing an additional mismatch into the siRNA design, we achieved enhanced specificity toward the mutant allele. Treatment of patient-derived fibroblasts effectively reduced the levels of mutant transcripts while maintaining unaltered wild-type transcript levels, rescuing the secretion and assembly of collagen VI matrix by reducing the dominant-negative effect of mutant chains. Our findings establish a promising treatment approach for patients with the recurrent dominantly negative acting G293R glycine substitution.