Diagnosis and aetiology of congenital muscular dystrophy: we are halfway there

OBJECTIVES: To evaluate the diagnostic outcomes in a large cohort of congenital muscular dystrophy (CMD) patients using traditional and Next Generation Sequencing (NGS) technologies. METHODS: 123 CMD patients were investigated using the traditional approaches of histology, immunohistochemical analysis of muscle biopsy and candidate gene sequencing. Undiagnosed patients available for further testing were investigated using NGS. RESULTS: Muscle biopsy and immunohistochemical analysis found deficiencies of laminin α2, α-dystroglycan or collagen VI in 50% of patients. Candidate gene sequencing and chromosomal microarray established a genetic diagnosis in 32% (39/123). Of 85 patients presenting in the last 20 years, 28 of 51 who lacked a confirmed genetic diagnosis (55%) consented to NGS studies, leading to confirmed diagnoses in a further 11 patients. Using the combination of approaches, a confirmed genetic diagnosis was achieved in 51% (43/85). The diagnoses within the cohort were heterogeneous. 45/59 probands with confirmed or probable diagnoses had variants in genes known to cause CMD (76%), and 11/59 (19%) had variants in genes associated with congenital myopathies, reflecting overlapping features of these conditions. One patient had a congenital myasthenic syndrome and two had microdeletions. Within the cohort, five patients had variants in novel (PIGY and GMPPB) or recently published genes (GFPT1 and MICU1) and seven had variants in TTN or RYR1; large genes that are technically difficult to Sanger sequence. INTERPRETATION: These data support NGS as a first-line tool for genetic evaluation of patients with a clinical phenotype suggestive of CMD, with muscle biopsy reserved as a second-tier investigation. This article is protected by copyright. All rights reserved

Type II and VI collagen in nasal and articular cartilage and the effect of IL-1α on the distribution of these collagens

The distribution of type II and VI collagen was immunocytochemically investigated in bovine articular and nasal cartilage. Cartilage explants were used either fresh or cultured for up to 4 weeks with or without interleukin 1α (IL-1α). Sections of the explants were incubated with antibodies for both types of collagen. Microscopic analyses revealed that type II collagen was preferentially localized in the interchondron matrix whereas type VI collagen was primarily found in the direct vicinity of the chondrocytes. Treatment of the sections with hyaluronidase greatly enhanced the signal for both types of collagen. Also in sections of explants cultured with IL-1α a higher level of labeling of the collagens was found. This was apparent without any pre-treatment with hyaluronidase. Under the influence of IL-1α the area positive for type VI collagen that surrounded the chondrocytes broadened. Although the two collagens in both types of cartilage were distributed similarly, a remarkable difference was the higher degree of staining of type VI collagen in articular cartilage. Concomitantly we noted that digestion of this type of cartilage hardly occurred in the presence of IL-1α whereas nasal cartilage was almost completely degraded within 18 days of culture. Since type VI collagen is known to be relatively resistant to proteolysis we speculate that the higher level of type VI collagen in articular cartilage is important in protecting cartilage from digestion

Surviving Endoplasmic Reticulum Stress Is Coupled to Altered Chondrocyte Differentiation and Function

In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia

Peptide analysis of collagen produced from cDNA by transcription and translation in vitro.

When collagen CNBr-cleavage peptides are analysed by two-dimensional gel electrophoresis each peptide is resolved into a reproducible set of charged forms. To test whether this peptide heterogeneity resulted from polymorphic mRNA, collagen was produced by transcription and translation in vitro of a collagen cDNA clone, and the peptides were mapped by two-dimensional gel electrophoresis. A cDNA construct was produced by ligation of the 5' end of the rat phenylalanine hydroxylase cDNA [Dahl & Mercer (1986) J. Biol. Chem. 261, 4148-4153], containing the translation-initiation codon, to a human alpha 1(I) cDNA [Chu, Myers, Bernard, Ding & Ramirez (1982) Nucleic Acids Res. 10, 5925-5934] coding for a large portion of helical region including the complete CB7 and CB3 CNBr-cleavage peptides. This cDNA construct was ligated into the transcription vector pSP65, and cell-free translation of the mRNA transcribed from the pSP65 plasmid was performed with a rabbit reticulocyte lysate system. After CNBr cleavage of the hybrid protein translation products, the collagen CB7 and CB3 peptides were resolved by two-dimensional electrophoresis into the same multiple charged forms whether the mRNA was produced from the cDNA construct or was extracted from normal fibroblast cultures. This result demonstrated that the multiple peptide spots were not due to polymorphic mRNA species. The heterogeneity must result from some uncharacterized specific post-translational modification or chemical alterations during sample preparation. This method of expression and analysis of proteins from cDNA clones should be of considerable use in the identification and characterization of clones that code for mutant proteins.link_to_subscribed_fulltex

Biochemical heterogeneity of type I collagen mutations in osteogenesis imperfecta.

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Regulation of procollagen synthesis and processing during ascorbate-induced extracellular matrix accumulation in vitro.

Procollagen biosynthesis and matrix deposition were studied in long-term human skin fibroblast cultures exposed to ascorbic acid. Ascorbic acid specifically stimulated types I and III collagen synthesis, reaching a maximum at day 2 and maintaining a specific high rate of production until day 10 of ascorbate exposure, after which collagen production declined. The increased level of collagen synthesis after different exposure times could also be achieved by only brief treatment (10 h) of parallel scorbutic (ascorbic-acid-deficient) cultures with ascorbic acid. This brief exposure did not result in increased collagen mRNA, thus demonstrating that the ascorbate-induced increase in collagen synthesis at all stages of ascorbic acid exposure was due to post-transcriptional mechanisms, most likely a rapid increase in type 1 collagen mRNA translational efficiency. This mechanism, rather than the transcriptional activation, was the primary response and is adequate to explain the ascorbate-induced increase in collagen synthesis. These data also demonstrate that the presence of a collagenous extracellular matrix was not involved in this collagen biosynthetic regulation. During long-term exposure (18 days) to ascorbic acid, a substantial cross-linked collagenous matrix formed, following an approximately sigmoidal time course. The most rapid matrix deposition occurred during the later days of exposure when the rate of collagen synthesis was decreasing, suggesting that the presence of a pre-existing matrix is important for further collagen accumulation. Procollagen was also efficiently processed to collagen during this phase, demonstrating that efficient procollagen processing is an important regulatory event in collagen matrix deposition

Correlation of clinical and molecular biological abnormalities in osteogenesis imperfecta.

Substitution of a glycine residue in the triple helix of the alpha 1(I) chain by either arginine, valine or alanine was associated with the type II lethal perinatal osteogenesis imperfecta phenotype. This phenotype was also produced by a frameshift mutation that resulted in an abnormal amino acid sequence of the carboxy-terminal propeptide of the pro-alpha 1(I) chain. The latter baby, however, showed some clinical and radiographic differences from the other babies with type II OI. The severity of the clinical and radiographic phenotypes are likely to be determined by both the type and site of the mutation as well as by the intra-uterine environment.link_to_subscribed_fulltex