Endoplasmic Reticulum Stress-Unfolding Protein Response-Apoptosis Cascade Causes Chondrodysplasia in a col2a1 p.Gly1170Ser Mutated Mouse Model

The collagen type II alpha 1 (COL2A1) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a col2a1 p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The col2a1 p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies.     

A type II collagen mutation also results in oto-spondylo-megaepiphyseal dysplasia

Oto-spondylo-megaepiphyseal dysplasia (OSMED) is a skeletal dysplasia characterized by severe sensorineural hearing loss, enlarged epiphyses and early onset of osteoarthritis. COL11A2 has been reported as a causative gene for OSMED. We have identified a novel COL2A1 mutation at a splice-acceptor site within intron 10 (c.709–2A>G) in an OSMED patient. This mutation caused the skipping of exon 11, and of exons 11 and 13. These exon-skipping events are presumed to cause an in-frame deletion of the triple helical region of the COL2A1 product. Thus, our findings highlight the genetic heterogeneity of OSMED and extend the phenotypic spectrum of type II collagenopathy, as well as confirming the overlap between type II and type XI collagenopathies.     

Depletion of cartilage collagen fibrils in mice carrying a dominant negative Col2a1 transgene affects chondrocyte differentiation

We have generated transgenic mice harboring the deletion of exon 48 in the mouse 1(II) procollagen gene (Col2a1). This was the first dominant negative mutation identified in the human 1(II) procollagen gene (COL2A1). Patients carrying a single allele with this mutation suffer from a severe skeletal disorder called spondyloepiphyseal dysplasia congenita (SED). Transgenic mice phenotype was neonatally lethal with severe respiratory failure, short bones, and cleft palate. Transgene mRNA was expressed at high levels. Growth plate cartilage of transgenic mice presented morphological abnormalities and reduced number of collagen type II fibrils. Chondrocytes carrying the mutation showed altered expression of several differentiation markers, like fibroblast growth factor receptor 3 (Fgfr3), Indian hedgehog (Ihh), runx2, cyclin-dependent kinase inhibitor P21^CIP/WAF (Cdkn1a), and collagen type X (Col10a1), suggesting that a defective extracellular matrix (ECM) depleted of collagen fibrils affects chondrocytes differentiation and that this defect participates in the reduced endochondral bone growth observed in chondrodysplasias caused by mutations in COL2A1. skeletal dyplasias; growth plate; cartilage extracellular matrix; spondyloepiphyseal dysplasia congenita     

Identification of a Novel Mutation in the COL2A1 Gene in a Chinese Family with Spondyloepiphyseal Dysplasia Congenita

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant chondrodysplasia characterized by disproportionate short-trunk dwarfism, skeletal and vertebral deformities. Exome sequencing and Sanger sequencing were performed in a Chinese Han family with typical SEDC, and a novel mutation, c.620G>A (p.Gly207Glu), in the collagen type II alpha-1 gene (COL2A1) was identified. The mutation may impair protein stability, and lead to dysfunction of type II collagen. Family-based study suggested that the mutation is a de novo mutation. Our study extends the mutation spectrum of SEDC and confirms genotype-phenotype relationship between mutations at glycine in the triple helix of the alpha-1(II) chains of the COL2A1 and clinical findings of SEDC, which may be helpful in the genetic counseling of patients with SEDC.     

Col10a1 gene expression and chondrocyte hypertrophy during skeletal development and disease

The type X collagen gene, COLIOA1, is specifically expressed by hypertrophic chondrocytes during endochondral ossification. Endochondral ossification is a well-coordinated process that involves a cartilage intermediate and leads to formation of most of the skeleton in vertebrates during skeletogenesis. Chondrocyte hypertrophy is a critical stage of endochondral ossification linking both bone and cartilage development. Given its specific association with chondrocyte hypertrophy, type X collagen plays essential roles in endochondral ossification. It was previously shown that transgenic mice with mutant type X collagen develop variable skeleton-hematopoietic abnormalities indicating defective endochondral ossification, while mutations and abnormal expression of human COLIOA1 cause abnormal chondrocyte hypertrophy that has been seen in many skeletal disorders, including skeletal chondrodysplasia and osteoarthritis. In this review, we summarized the skeletal chondrodysplasia with COLIOA1 gene mutation that shows growth plate defect. We also reviewed recent studies that correlate the type X collagen gene expression and chondrocyte hypertrophy with osteoarthritis. Due to its significant clinical relevance, the type X collagen gene regulation has been extensively studied over the past two decades. Here, we focus on recent progress characterizing the cis-enhancer elements and their binding factors that together confer hypertrophic chondroeyte-specific murine type X collagen gene （CollOal） expression. Based on literature review and our own studies, we surmise that there are multiple factors that contribute to hypertrophic chondrocyte-specific CoHOal expression. These factors include both transactivators （such as Runx2, MEF2C etc.） and repressors （such as AP1, NFATcl, Sox9 etc.）, while other co-factors or epigenetic control of CollOal expression may not be excluded.     

Expression, in cartilage, of a 7-amino-acid deletion in type II collagen from two unrelated individuals with Kniest dysplasia.

Kniest dysplasia is a heritable chondrodysplasia that severely affects skeletal growth. Recent evidence suggests that the etiology is based on mutations in COL2A1, the gene for collagen type II. We report the detection and partial characterization of an identical defect in type II collagen in two unrelated patients with Kniest dysplasia. Analysis of cyanogen bromide (CB)-digested cartilage samples from both probands by SDS-PAGE revealed an abnormal band for peptide alpha 1(II)CB12. The peptide was purified and digested with endoproteinase Asp-N. Fragments unique to the Kniest tissues were identified by reverse-phase high-pressure liquid chromatography and by sequence analysis. The results established a deletion of amino acids 102-108 of the alpha 1(II) triple-helical domain, which disrupted the (gly-X-Y)n repeat needed for helix formation. This was confirmed by sequence analysis of DNA amplified from both probands, revealing the molecular basis to be a single nucleotide mutation at a CpG dinucleotide (GCG-->GTG) in the codon for alanine 102. The mutation created a new splice donor site, which would account for the absence of the last seven amino acids from the 3' end of exon 12 in alpha 1(II)CB12. Light and electron micrographs of the probands' cartilage showed the perilacunar foamy matrix ("Swiss cheese") characteristic of Kniest dysplasia and chondrocytes containing dilated rough endoplasmic reticulum, which earlier studies had shown were filled with type II procollagen. These two cases strengthen the concept that Kniest dysplasia is based on mutations of COL2A1 and belongs within the broad spectrum of chondrodysplasias caused by type II collagenopathies.     

The effect of carbamazepine on bone structure and strength in control and osteogenesis imperfecta (Col1a2 +/p.G610C ) mice

The inherited brittle bone disease osteogenesis imperfecta (OI) is commonly caused by COL1A1 and COL1A2 mutations that disrupt the collagen I triple helix. This causes intracellular endoplasmic reticulum (ER) retention of the misfolded collagen and can result in a pathological ER stress response. A therapeutic approach to reduce this toxic mutant load could be to stimulate mutant collagen degradation by manipulating autophagy and/or ER‐associated degradation. Since carbamazepine (CBZ) both stimulates autophagy of misfolded collagen X and improves skeletal pathology in a metaphyseal chondrodysplasia model, we tested the effect of CBZ on bone structure and strength in 3‐week‐old male OI Col1a2 ^+/p.G610C and control mice. Treatment for 3 or 6 weeks with CBZ, at the dose effective in metaphyseal chondrodysplasia, provided no therapeutic benefit to Col1a2 ^+/p.G610C mouse bone structure, strength or composition, measured by micro‐computed tomography, three point bending tests and Fourier‐transform infrared microspectroscopy. In control mice, however, CBZ treatment for 6 weeks impaired femur growth and led to lower femoral cortical and trabecular bone mass. These data, showing the negative impact of CBZ treatment on the developing mouse bones, raise important issues which must be considered in any human clinical applications of CBZ in growing individuals.     

The Mouse MC13 Mutant Is a Novel ENU Mutation in Collagen Type II,Alpha 1

Phenotype-driven mutagenesis experiments are a powerful approach to identifying novel alleles in a variety of contexts. The traditional disadvantage of this approach has been the subsequent task of identifying the affected locus in the mutants of interest. Recent advances in bioinformatics and sequencing have reduced the burden of cloning these ENU mutants. Here we report our experience with an ENU mutagenesis experiment and the rapid identification of a mutation in a previously known gene. A combination of mapping the mutation with a high-density SNP panel and a candidate gene approach has identified a mutation in collagen type II, alpha I (Col2a1). Col2a1 has previously been studied in the mouse and our mutant phenotype closely resembles mutations made in the Col2a1 locus.     

Identification of three novel mutations in the COL2A1 gene in four unrelated Chinese families with spondyloepiphyseal dysplasia congenita

Introduction

Spondyloepiphyseal dysplasia congenita (SEDC) is an autosomal dominant skeletal dysplasia characterized by short stature, abnormal epiphyses, and flattened vertebral bodies. The condition occurs through a mutation in the COL2A1 gene that encodes the type II procollagen alpha1 chain (proalpha1 (II)).

Method and Results

We investigated nine affected individuals from four unrelated Chinese families with SEDC. We screened for COL2A1 gene mutations, and identified found four missense mutations (G447A, G456A, R789C and G1152D). The G447A, G456A and G1152D mutations are novel and the R789C mutation has been reported previously in several other studies with a strikingly similar phenotype.

Conclusions

Our study extends the mutation spectrum of SEDC and is helpful in early molecular diagnoses of SEDC.     

Identification of a novel COL1A1 frameshift mutation, c.700delG,in a Chinese osteogenesis imperfecta family

Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI.     

Deletions in the COL10A1 gene are not associated with skeletal changes in dogs

Type 10 collagen alpha 1 (COL10A1) is a short-chain collagen of cartilage synthesized by chondrocytes during the growth of long bones. COL10A1 mutations, which frequently result in COL10A1 haploinsufficiency, have been identified in patients with Schmid metaphyseal chondrodysplasia (SMCD), a cartilage disorder characterized by short-limbed short stature and bowed legs. Similarities between SMCD and short stature in various dog breeds suggested COL10A1 as a candidate for canine skeletal dysplasia. We report the sequencing of the exons and promoter region of the COL10A1 gene in dog breeds fixed for a specific type of skeletal dysplasia known as chondrodysplasia, breeds that segregate the skeletal dysplasia phenotype, and control dogs of normal stature. Thirteen single nucleotide polymorphisms (SNPs), one insertion, and two deletions, one of which introduces a premature stop codon and likely results in nonsense-mediated decay and the degradation of the mutant allele product, were identified in the coding region. There appear to be no causal relationships between the polymorphisms identified in this study and short stature in dogs. Although COL10A1 haploinsufficiency is an important cause of SMCD in humans, it does not seem to be responsible for the skeletal dysplasia phenotype in these dog breeds. In addition, homozygosity for the nonsense allele does not result in the observed canine skeletal dysplasia phenotype. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users.     

Novel gain-of-function mutation of TRPV4 associated with accelerated chondrogenic differentiation of dental pulp stem cells derived from a patient with metatropic dysplasia

Metatropic dysplasia is a congenital skeletal dysplasia characterized by severe platyspondyly, dumbbell-like deformity of long tubular bones, and progressive kyphoscoliosis with growth. It is caused by mutations in the gene TRPV4, encoding the transient receptor potential vanilloid 4, which acts as a calcium channel. Many heterozygous single base mutations of this gene have been associated with the disorder, showing autosomal dominant inheritance. Although abnormal endochondral ossification has been observed by histological examination of bone in a patient with lethal metatropic dysplasia, the etiology of the disorder remains largely unresolved. As dental pulp stem cells (DPSCs) are mesenchymal stem cells that differentiate into bone lineage cells, DPSCs derived from patients with congenital skeletal dysplasia might be useful as a disease-specific cellular model for etiological investigation. The purpose of this study was to clarify the pathological association between TRPV4 mutation and chondrocyte differentiation by analyzing DPSCs from a patient with non-lethal metatropic dysplasia. We identified a novel heterozygous single base mutation, c.1855C>T in TRPV4. This was predicted to be a missense mutation, p.L619F, in putative transmembrane segment 5. The mutation was repaired by CRISPR/Cas9 system to obtain isogenic control DPSCs for further analysis. The expression of stem cell markers and fibroblast-like morphology were comparable between patient-derived mutant and control DPSCs, although expression of TRPV4 was lower in mutant DPSCs than control DPSCs. Despite the lower TRPV4 expression in mutant DPSCs, the intracellular Ca²⁺ level was comparable at the basal level between mutant and control DPSCs, while its level was markedly higher following stimulation with 4α-phorbol 12,13-didecanoate (4αPDD), a specific agonist for TRPV4, in mutant DPSCs than in control DPSCs. In the presence of 4αPDD, we observed accelerated early chondrocyte differentiation and upregulated mRNA expression of SRY-box 9 (SOX9) in mutant DPSCs. Our findings suggested that the novel missense mutation c.1855C>T of TRPV4 was a gain-of-function mutation leading to enhanced intracellular Ca²⁺ level, which was associated with accelerated chondrocyte differentiation and SOX9 upregulation. Our results also suggest that patient-derived DPSCs can be a useful disease-specific cellular model for elucidating the pathological mechanism of metatropic dysplasia.     

Novel PAX9 and COL1A2 Missense Mutations Causing Tooth Agenesis and OI/DGI without Skeletal Abnormalities

Inherited dentin defects are classified into three types of dentinogenesis imperfecta (DGI) and two types of dentin dysplasia (DD). The genetic etiology of DD-I is unknown. Defects in dentin sialophosphoprotein (DSPP) cause DD type II and DGI types II and III. DGI type I is the oral manifestation of osteogenesis imperfecta (OI), a systemic disease typically caused by defects in COL1A1 or COL1A2. Mutations in MSX1, PAX9, AXIN2, EDA and WNT10A can cause non-syndromic familial tooth agenesis. In this study a simplex pattern of clinical dentinogenesis imperfecta juxtaposed with a dominant pattern of hypodontia (mild tooth agenesis) was evaluated, and available family members were recruited. Mutational analyses of the candidate genes for DGI and hypodontia were performed and the results validated. A spontaneous novel mutation in COL1A2 (c.1171G>A; p.Gly391Ser) causing only dentin defects and a novel mutation in PAX9 (c.43T>A; p.Phe15Ile) causing hypodontia were identified and correlated with the phenotypic presentations in the family. Bone radiographs of the proband’s dominant leg and foot were within normal limits. We conclude that when no DSPP mutation is identified in clinically determined isolated DGI cases, COL1A1 and COL1A2 should be considered as candidate genes. PAX9 mutation p.Phe15Ile within the N-terminal β-hairpin structure of the PAX9 paired domain causes tooth agenesis.     

Col6a1 Null Mice as a Model to Study Skin Phenotypes in Patients with Collagen VI Related Myopathies: Expression of Classical and Novel Collagen VI Variants during Wound Healing

Patients suffering from collagen VI related myopathies caused by mutations in COL6A1, COL6A2 and COL6A3 often also display skin abnormalities, like formation of keloids or “cigarette paper” scars, dry skin, striae rubrae and keratosis pilaris (follicular keratosis). Here we evaluated if Col6a1 null mice, an established animal model for the muscle changes in collagen VI related myopathies, are also suitable for the study of mechanisms leading to the skin pathology. We performed a comprehensive study of the expression of all six collagen VI chains in unwounded and challenged skin of wild type and Col6a1 null mice. Expression of collagen VI chains is regulated in both skin wounds and bleomycin-induced fibrosis and the collagen VI α3 chain is proteolytically processed in both wild type and Col6a1 null mice. Interestingly, we detected a decreased tensile strength of the skin and an altered collagen fibril and basement membrane architecture in Col6a1 null mice, the latter being features that are also found in collagen VI myopathy patients. Although Col6a1 null mice do not display an overt wound healing defect, these mice are a relevant animal model to study the skin pathology in collagen VI related disease.     

Characterization of a murine type IIB procollagen-specific antibody

Type II collagen is the major collagenous component of the cartilage extracellular matrix; formation of a covalently cross-linked type II collagen network provides cartilage with important tensile properties. The Col2a1 gene is encoded by 54 exons, of which exon 2 is subject to alternative splicing, resulting in different isoforms named IIA, IIB, IIC and IID. The two major procollagen protein isoforms are type IIA and type IIB procollagen. Type IIA procollagen mRNA contains exon 2 and is generated predominantly by chondroprogenitor cells and other non-cartilaginous tissues. Differentiated chondrocytes generate type IIB procollagen, devoid of exon 2. Although type IIA procollagen is produced in certain non-collagenous tissues during development, this developmentally-regulated alternative splicing switch to type IIB procollagen is restricted to cartilage cells. Though a much studied and characterized molecule, the importance of the various type II collagen protein isoforms in cartilage development and homeostasis is still not completely understood. Effective antibodies against specific epitopes of these isoforms can be useful tools to decipher function. However, most type II collagen antibodies to date recognize either all isoforms or the IIA procollagen isoform. To specifically identify the murine type IIB procollagen, we have generated a rabbit antibody (termed IIBN) directed to a peptide sequence that spans the murine exon 1–3 peptide junction. Characterization of the affinity-purified antibody by western blotting of collagens extracted from wild type murine cartilage or cartilage from Col2a1^+ ex2 knock-in mice (which generates predominantly the type IIA procollagen isoform) demonstrated that the IIBN antibody is specific to the type IIB procollagen isoform. IIBN antibody was also able to detect the native type IIB procollagen in the hypertrophic chondrocytes of the wild type growth plate, but not in those of the Col2a1^+ ex2 homozygous knock-in mice, by both immunofluorescence and immunohistochemical studies. Thus the IIBN antibody will permit an in-depth characterization of the distribution of IIB procollagen isoform in mouse skeletal tissues. In addition, this antibody will be an important reagent for characterizing mutant type II collagen phenotypes and for monitoring type II procollagen processing and trafficking.