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Purpose To date, heterozygous or homozygous COL12A1 variants have been reported in 13 patients presenting with a clinical phenotype overlapping with collagen VI–related myopathies and Ehlers–Danlos syndrome (EDS). The small number of reported patients limits thorough investigation of this newly identified syndrome, currently coined as myopathic EDS. Methods DNA from 78 genetically unresolved patients fulfilling the clinical criteria for myopathic EDS was sequenced using a next-generation panel of COL12A1 , COL6A1 , COL6A2 , and COL6A3 . Results Among this cohort, we identified four pathogenic heterozygous in-frame exon skipping (∆) defects in COL12A1 , clustering to the thrombospondin N-terminal region and the adjacent collagenous domain (Δ52, Δ53, Δ54, and Δ56 respectively), one heterozygous COL12A1 arginine-to-cysteine substitution of unclear significance (p.(Arg1863Cys)), and compound heterozygous pathogenic COL6A1 variants (c.[98–6G>A];[301C>T]) in one proband. Variant-specific intracellular accumulation of collagen XII chains, extracellular overmodification of the long isoform and near-absence of the short isoform of collagen XII, and extracellular decrease of decorin and tenascin-X were observed for the COL12A1 variants. In contrast, the COL6A1 variants abolished collagen VI and V deposition and increased tenascin-X levels. Conclusion Our data further support the significant clinical overlap between myopathic EDS and collagen VI–related myopathies, and emphasize the variant-specific consequences of collagen XII defects.

TANGO1 (transport and Golgi organization‐1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER‐Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild‐to‐moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near‐total bone loss and phenocopies the situation of Mia3−/− mice. Whole‐exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4‐base pair (4‐bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1‐related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe‐to‐lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Genomic variants of uncertain significance (VUS) impede clinical decision-making. In this study, we employ a knock-in strategy in zebrafish to evaluate the COL1A2 c.2123G>A VUS, identified in a 78-year-old female with atypical femoral fractures. Using prime editing, we generated different col1a2 zebrafish lines respectively harboring the VUS, a known pathogenic variant, and a known benign variant. Comprehensive skeletal phenotyping revealed no significant abnormalities in zebrafish harboring the VUS. In contrast, zebrafish with the pathogenic variant showed an increased eye diameter, scoliosis, vertebral fusions, vertebral compressions, fractures, and increased mineralization of the notochord and intervertebral ligament compared to wild type controls. Our findings represent the first demonstration that COL1A2 variant modeling via prime editing in zebrafish not only aids in functional validation, but also holds promise for uncovering the underlying pathogenic mechanisms. This approach can be applied to investigate VUS in other genes as well.

Purpose Infantile Caffey disease is a rare disorder characterized by acute inflammation with subperiosteal new bone formation, associated with fever, pain, and swelling of the overlying soft tissue. Symptoms arise within the first weeks after birth and spontaneously resolve before the age of two years. Many, but not all, affected individuals carry the heterozygous pathogenic COL1A1 variant (c.3040C>T, p.(Arg1014Cys)). Methods We sequenced COL1A1 in 28 families with a suspicion of Caffey disease and performed ultrastructural, immunocytochemical, and biochemical collagen studies on patient skin biopsies. Results We identified the p.(Arg1014Cys) variant in 23 families and discovered a novel heterozygous pathogenic COL1A1 variant (c.2752C>T, p.(Arg918Cys)) in five. Both arginine to cysteine substitutions are located in the triple helical domain of the proα1(I) procollagen chain. Dermal fibroblasts (one patient with p.(Arg1014Cys) and one with p.(Arg918Cys)) produced molecules with disulfide-linked proα1(I) chains, which were secreted only with p.(Arg1014Cys). No intracellular accumulation of type I procollagen was detected. The dermis revealed mild ultrastructural abnormalities in collagen fibril diameter and packing. Conclusion The discovery of this novel pathogenic variant expands the limited spectrum of arginine to cysteine substitutions in type I procollagen. Furthermore, it confirms allelic heterogeneity in Caffey disease and impacts its molecular confirmation.

Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.

Background Proteoglycans are large and structurally complex macromolecules which can be found in abundancy in the extracellular matrix and on the surface of all animal cells. Mutations in the genes encoding the enzymes responsible for the formation of the tetrasaccharide linker region between the proteoglycan core protein and the glycosaminoglycan side chains lead to a spectrum of severe and overlapping autosomal recessive connective tissue disorders, collectively coined the ‘glycosaminoglycan linkeropathies’. Results We report the clinical findings of two novel patients with a complex linkeropathy due to biallelic mutations in B3GAT3 , the gene that encodes glucuronosyltransferase I, which catalyzes the addition of the ultimate saccharide to the linker region. We identified a previously reported c.667G > A missense mutation and an unreported homozygous c.416C > T missense mutation. We also performed a genotype and phenotype-oriented literature overview of all hitherto reported patients harbouring B3GAT3 mutations. A total of 23 patients from 10 families harbouring bi-allelic mutations and one patient with a heterozygeous splice-site mutation in B3GAT3 have been reported. They all display a complex phenotype characterized by consistent presence of skeletal dysplasia (including short stature, kyphosis, scoliosis and deformity of the long bones), facial dysmorphology, and spatulate distal phalanges. More variably present are cardiac defects, joint hypermobility, joint dislocations/contractures and fractures. Seven different B3GAT3 mutations have been reported, and although the number of patients is still limited, some phenotype-genotype correlations start to emerge. The more severe phenotypes seem to have mutations located in the substrate acceptor subdomain of the catalytic domain of the glucuronosyltransferase I protein while more mildly affected phenotypes seem to have mutations in the NTP-sugar donor substrate binding subdomain. Conclusions Loss-of-function mutations in B3GAT3 are associated with a complex connective tissue phenotype characterized by disproportionate short stature, skeletal dysplasia, facial dysmorphism, spatulate distal phalanges and -to a lesser extent- joint contractures, joint hypermobility with dislocations, cardiac defects and bone fragility. Based on the limited number of reported patients, some genotype-phenotype correlations start to emerge.

Purpose: The Ehlers–Danlos syndrome (EDS), dermatosparaxis type, is a recessively inherited connective tissue disorder caused by deficient activity of ADAMTS-2, an enzyme that cleaves the aminoterminal propeptide domain of types I, II, and III procollagen. Only 10 EDS dermatosparaxis patients have been reported, all presenting a recognizable phenotype with characteristic facial gestalt, extreme skin fragility and laxity, excessive bruising, and sometimes major complications due to visceral and vascular fragility. Methods: We report on five new EDS dermatosparaxis patients and provide a comprehensive overview of the current knowledge of the natural history of this condition. Results: We identified three novel homozygous loss-of-function mutations (c.2927_2928delCT, p.(Pro976Argfs*42); c.669_670dupG, p.(Pro224Argfs*24); and c.2751-2A>T) and one compound heterozygous mutation (c.2T>C, p.? and c.884_887delTGAA, p.(Met295Thrfs26*)) in ADAMTS2 in five patients from four unrelated families. Three of these displayed a phenotype that was strikingly milder than that of previously reported patients. Conclusion: This study expands the clinical and molecular spectrum of the dermatosparaxis type of EDS to include a milder phenotypic variant and stresses the importance of good clinical criteria. To address this, we propose an updated set of criteria that accurately captures the multisystemic nature of the dermatosparaxis type of EDS. Genet Med 18 9, 882–891.

The extracellular matrix (ECM) is a dynamic structure of molecules that can be divided into six different categories and are collectively called the matrisome. The ECM plays pivotal roles in physiological processes in many tissues, including the nervous system. Intriguingly, alterations in ECM molecules/pathways are associated with painful human conditions and murine pain models. Nevertheless, mechanistic insight into the interplay of normal or defective ECM and pain is largely lacking. The goal of this study was to integrate bulk, single-cell, and spatial RNA sequencing (RNAseq) datasets to investigate the expression and cellular origin of matrisome genes in male and female murine and human dorsal root ganglia (DRG). Bulk RNAseq showed that about 65% of all matrisome genes were expressed in both murine and human DRG, with proportionally more core matrisome genes (glycoproteins, collagens, and proteoglycans) expressed compared to matrisome-associated genes (ECM-affiliated genes, ECM regulators, and secreted factors). Single cell RNAseq on male murine DRG revealed the cellular origin of matrisome expression. Core matrisome genes, especially collagens, were expressed by fibroblasts whereas matrisome-associated genes were primarily expressed by neurons. Cell–cell communication network analysis with CellChat software predicted an important role for collagen signaling pathways in connecting vascular cell types and nociceptors in murine tissue, which we confirmed by analysis of spatial transcriptomic data from human DRG. RNAscope in situ hybridization and immunohistochemistry demonstrated expression of collagens in fibroblasts surrounding nociceptors in male and female human DRG. Finally, comparing human neuropathic pain samples with non-pain samples also showed differential expression of matrisome genes produced by both fibroblasts and by nociceptors. This study supports the idea that the DRG matrisome may contribute to neuronal signaling in both mouse and human, and that dysregulation of matrisome genes is associated with neuropathic pain.

The spondylodysplastic type of Ehlers–Danlos syndrome (spEDS) is caused by genetic defects in the B4GALT7 or B3GALT6 genes both deranging the biosynthesis of the glycosaminoglycan linkage region of chondroitin/dermatan sulfate and heparan sulfate proteoglycans. In this study, we have analyzed the linkage regions of urinary chondroitin sulfate proteoglycans of three siblings, diagnosed with spEDS and carrying biallelic pathogenic variants of the B3GALT6 gene. Proteoglycans were digested with trypsin, glycopeptides enriched on anion‐exchange columns, depolymerized with chondroitinase ABC, and analyzed by nLC‐MS/MS. In urine of the unaffected mother, the dominating glycopeptide of bikunin/protein AMBP appeared as only one dominating (99.9%) peak with the canonical tetrasaccharide linkage region modification. In contrast, the samples of the three affected siblings contained two different glycopeptide peaks, corresponding to the canonical tetrasaccharide and to the non‐canonical trisaccharide linkage region modifications in individual ratios of 61/38, 73/27, and 59/41. We propose that the relative distribution of glycosaminoglycan linkage regions of urinary bikunin glycopeptides may serve as a phenotypic biomarker in a diagnostic test but also as a biomarker to follow the effect of future therapies in affected individuals.

Ehlers-Danlos syndromes (EDS) comprise a genetically and clinically heterogenous group of rare diseases that cause severe, often fatal, damage to connective tissue. The molecular basis of EDS implicates defects in extracellular matrix components, including various fibrillar collagens and glycosaminoglycans (GAGs). However, the precise pathogenic mechanisms behind EDS remain elusive. Here, we have implemented a multi-tiered approach to demonstrate the functional impact of B3GALT6 mutations on biochemical and developmental processes, ultimately leading to the spondylodysplastic subtype of EDS (spEDS), characterized by severe musculoskeletal symptoms. We show that the loss of function of β1,3-galactosyltransferase 6 (β3GalT6) is partially compensated by β1,3-glucuronosyltransferase 3 (GlcAT-I), the next enzyme in the GAG biosynthetic pathway. In addition, results from transcriptomics, collagen analysis, and biophysical experiments revealed that impaired collagen maturation, including defective glycosylation of collagen XII, contributes to altered tissue structure and biomechanics, the hallmarks of spEDS. Our findings unravel a new pathogenic mechanism of spEDS and bring us one step closer to therapeutic strategies, including cell and tissue engineering.