NC1 Domain Research Articles

304 Type VII collagen NC2 domain expression differentiates severe from milder recessive dystrophic epidermolysis bullosa subtypes

Recessive dystrophic epidermolysis bullosa (RDEB) results from type VII collagen (C7) deficiency due to COL7A1 gene mutations. C7 contains collagenous domains (COL) flanked by amino (NC1) and carboxyl (NC2) non-collagenous domains. The genotype spectrum of RDEB is well studied but does not entirely explain the highly variable clinical phenotype. In this retrospective study, we aimed to identify a disease severity and prognostic indicator, from an extensively characterized RDEB cohort. 41 RDEB patients were studied with mild, intermediate and severe clinical phenotypes in 7, 11 and 23 patients respectively. 22 of 23 severe RDEB patients showed negative NC2 expression, with variable NC1 staining and variable AF morphology. 7 of 7 mild RDEB patients showed at least one allele with missense mutations in COL domains and positive NC2 and NC1 expression. 5 of 11 intermediate patients showed positive NC2 expression with missense and/or splice site COL domain mutations. 6 intermediate patients with negative NC2 expression showed bi-allelic PTC mutations and very low NC1 staining. Given their paucity of C7 NC2 expression, its possible these latter 6 patients may evolve into a more severe clinical phenotype over time or other factors outside of C7 expression may be playing a role in dictating disease severity. This remains to be further studied. We conclude that genotype or anchoring fibril morphology do not entirely explain the phenotypic variability and C7 NC1 expression did not prove to be a good indicator of disease severity. However, of all the tests utilized, C7 NC2 expression proved the most useful in distinguishing mild from severe RDEB clinical phenotypes.

Serum Type XIX Collagen is Significantly Elevated in Non-Small Cell Lung Cancer: A Preliminary Study on Biomarker Potential.

Type XIX collagen is a poorly characterized collagen associated with the basement membrane. It is abnormally regulated during breast cancer progression and the NC1 (XIX) domain has anti-tumorigenic signaling properties. However, little is known about the biomarker potential of collagen XIX in cancer. In this study, we describe a competitive ELISA, named PRO-C19, targeting the C-terminus of collagen XIX using a monoclonal antibody. PRO-C19 was measured in serum of patients with a range of cancer types and was elevated in non-small cell lung cancer (NSCLC) (p < 0.0001), small cell lung cancer (p = 0.0081), breast (p = 0.0005) and ovarian cancer (p < 0.0001) compared to healthy controls. In a separate NSCLC cohort, PRO-C19 was elevated compared to controls when evaluating adenocarcinoma (AD) (p = 0.0003) and squamous cell carcinoma (SCC) (p < 0.0001) patients but was not elevated in chronic obstructive pulmonary disease patients. SCC also had higher PRO-C19 levels than AD (p = 0.0457). PRO-C19 could discriminate between NSCLC and healthy controls (AUROC:0.749 and 0.826 for AD and SCC, respectively) and maintained discriminatory performance in patients of tumor stages I+II (AUROC:0.733 and 0.818 for AD and SCC, respectively). Lastly, we confirmed the elevated type XIX collagen levels using gene expression data from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) initiatives. In conclusion, type XIX collagen is released into circulation and is significantly elevated in the serum of cancer patients and PRO-C19 shows promise as a cancer biomarker.

Human α1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by α1β1 integrin.

Human α1 type IV collagen NC1 domain exhibits distinct antiangiogenic activity mediated by α1β1 integrin.

Type XIX collagen: a promising biomarker from the basement membranes.

Among collagen members in the collagen superfamily, type XIX collagen has raised increasing interest in relation to its structural and biological roles. Type XIX collagen is a Fibril-Associated Collagen with Interrupted Triple helices member, one main subclass of collagens in this superfamily. This collagen contains a triple helix composed of three polypeptide segments aligned in parallel and it is associated with the basement membrane zone in different tissues. The molecular structure of type XIX collagen consists of five collagenous domains, COL1 to COL5, interrupted by six non-collagenous domains, NC1 to NC6. The most relevant domain by which this collagen exerts its biological roles is NC1 domain that can be cleavage enzymatically to release matricryptins, exerting anti-tumor and anti-angiogenic effect in murine and human models of cancer. Under physiological conditions, type XIX collagen expression decreases after birth in different tissues although it is necessary to keep its basal levels, mainly in skeletal muscle and hippocampal and telencephalic interneurons in brain. Notwithstanding, in amyotrophic lateral sclerosis, altered transcript expression levels show a novel biological effect of this collagen beyond its structural role in basement membranes and its anti-tumor and anti-angiogenic properties. Type XIX collagen can exert a compensatory effect to ameliorate the disease progression under neurodegenerative conditions specific to amyotrophic lateral sclerosis in transgenic SOD1G93A mice and amyotrophic lateral sclerosis patients. This novel biological role highlights its nature as prognostic biomarker of disease progression in and as promising therapeutic target, paving the way to a more precise prognosis of amyotrophic lateral sclerosis.

Identification of two novel COL10A1 heterozygous mutations in two Chinese pedigrees with Schmid-type metaphyseal chondrodysplasia

BackgroundSchmid-type metaphyseal chondrodysplasia (MCDS) is an autosomal dominant disorder caused by COL10A1 mutations, which is characterized by short stature, waddling gait, coxa vara and bowing of the long bones. However, descriptions of the expressivity of MCDS are rare.MethodsTwo probands and available family members affected with MCDS were subjected to clinical and radiological examination. Genomic DNA of all affected individuals was subjected to whole-exome sequencing, and candidate mutations were verified by Sanger sequencing in all available family members and in 250 healthy donors. A spatial model of the type X collagen (α1) C-terminal noncollagenous (NC1) domain was further constructed.ResultsWe found that the phenotype of affected family members exhibited incomplete dominance. Mutation analysis indicated that there were two novel heterozygous missense mutations, [c.1765 T > A (p.Phe589Ile)] and [c.1846A > G (p.Lys616Glu)] in the COL10A1 gene in family 1 and 2, respectively. The two novel substitution sites were highly conserved and the mutations were predicted to be deleterious by in silico analysis. Furthermore, protein modeling revealed that the two substitutions were located in the NC1 domain of collagen X (α1), which potentially impacted the trimerization of collagen X (α1) and combination with molecules in the pericellular matrix.ConclusionTwo novel mutations were identified in the present study, which will facilitate diagnosis of MCDS and further expand the spectrum of the COL10A1 mutations associated with MCDS patients. In addition, our research revealed the phenomenon of incomplete dominance in MCDS.

068 Serological characterization of anti-p200 pemphigoid: Epitope spreading as a common phenomenon

Anti-p200 pemphigoid is an orphan subepidermal blistering disease characterized by autoantibody binding to the dermal site of human salt-split skin (SSS) and reactivity against a 200 kDa protein in the extract of human dermis by immunoblotting. Here, we aimed at the thorough serological characterization of anti-p200 pemphigoid. This retrospective study included all 243 patients with anti-p200 pemphigoid diagnosed between 2008 and 2017 in our routine autoimmune laboratory. Patients were diagnosed based on a compatible clinical picture and reactivity with the p200 protein in dermal extract by immunoblotting as described previously. In addition, all sera were subjected to indirect immunofluorescence microscopy on SSS and immunoblotting with the extracellular matrix of cultured keratinocytes for reactivity against laminin 332 and ELISA or immunoblotting applying the recombinant NC1 domain of collagen type VII. Autoantibodies against the dermal side of SSS were found in 91% (221/243) of patients and against the p200 protein and laminin γ1 in 100% (243/243) and 88% (209/238) of sera, respectively. IgG reactivity to laminin 332 was observed in 21% (48/233) of patients, in 98% of those (47/48) against the α3 chain of laminin 332. IgG autoantibodies to type VII collagen were detected in 2% (4/243) of patients. 30% (73/243) of sera showed additional reactivity to the epidermal site of SSS. In 58% (42/73) of epidermal binding sera, antibodies against BP180 or BP230 were detected by ELISA, while in 42% (31/73) of these sera, the target antigen remained unclear. In 37% (89/243) of sera, reactivity against one antigen in addition to p200/laminin γ1 and in 7% (16/243) against 2 additional antigens was detected. In summary, in this so far largest cohort of anti-p200 pemphigoid patients, epitope spreading was a common phenomenon and most frequently included laminin 332 followed by BP180, BP230, and type VII collagen.

Endoplasmic reticulum stress and proteasome pathway involvement in human podocyte injury with a truncated COL4A3 mutation.

Background:Collagen type IV (COL4)-related nephropathy includes a variety of kidney diseases that occur with or without extra-renal manifestations caused by COL4A3-5 mutations. Previous studies revealed several novel mutations, including three COL4A3 missense mutations (G619R, G801R, and C1616Y) and the COL4A3 chr:228172489delA c.4317delA p.Thr1440ProfsX87 frameshift mutation that resulted in a truncated NC1 domain (hereafter named COL4A3 c.4317delA); however, the mutation mechanisms that lead to podocyte injury remain unclear. This study aimed to further explore the mutation mechanisms that lead to podocyte injury.Methods:Wild-type (WT) and four mutant COL4A3 segments were constructed into a lentiviral plasmid, then stably transfected into human podocytes. Real-time polymerase chain reaction and Western blotting were applied to detect endoplasmic reticulum stress (ERS)- and apoptosis-related mRNA and protein levels. Then, human podocytes were treated with MG132 (a proteasome inhibitor) and brefeldin A (a transport protein inhibitor). The human podocyte findings were verified by the establishment of a mus-Col4a3 knockout mouse monoclonal podocyte using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technology.Results:Our data showed that COL4A3 mRNA was significantly overexpressed in the lentivirus stably transfected podocytes. Moreover, the COL4A3 protein level was significantly increased in all groups except the COL4A3 c.4317delA group. Compared to the other test groups, the COL4A3 c.4317delA group showed excessive ERS and apoptosis. Podocytes treated with MG132 showed remarkably increased intra-cellular expression of the COL4A3 c.4317delA mutation. MG132 intervention improved higher ERS and apoptosis levels in the COL4A3 c.4317delA group. Mouse monoclonal podocytes with COL4A3 chr:82717932insA c.4852insA p.Arg1618ThrfsX4 were successfully acquired; this NC1-truncated mutation suggested a higher level of ERS and relatively remarkable level of apoptosis compared to that of the WT group.Conclusions:We demonstrated that excessive ERS and ERS-induced apoptosis were involved in the podocyte injury caused by the NC1-truncated COL4A3 mutation. Furthermore, proteasome pathway intervention might become a potential treatment for collagen type IV-related nephropathy caused by a severely truncated COL4A3 mutation.

A chloride ring is an ancient evolutionary innovation mediating the assembly of the collagen IV scaffold of basement membranes

Collagen IV scaffold is a principal component of the basement membrane (BM), a specialized extracellular matrix that is essential for animal multicellularity and tissue evolution. Scaffold assembly begins with the trimerization of α-chains into protomers inside the cell, which then are secreted and undergo oligomerization outside the cell. For the ubiquitous scaffold composed of α1- and α2-chains, both intracellular and extracellular stages are mediated by the noncollagenous domain (NC1). The association of protomers is chloride-dependent, whereby chloride ions induce interactions of the protomers' trimeric NC1 domains leading to NC1 hexamer formation. Here, we investigated the mechanisms, kinetics, and functionality of the chloride ion-mediated protomer assembly by using a single-chain technology to produce a stable NC1 trimer comprising α1, α2, and α1 NC1 monomers. We observed that in the presence of chloride, the single-chain NC1-trimer self-assembles into a hexamer, for which the crystal structure was determined. We discovered that a chloride ring, comprising 12 ions, induces the assembly of and stabilizes the NC1 hexamer. Furthermore, we found that the chloride ring is evolutionarily conserved across all animals, first appearing in cnidarians. These findings reveal a fundamental role for the chloride ring in the assembly of collagen IV scaffolds of BMs, a critical event enabling tissue evolution and development. Moreover, the single-chain technology is foundational for generating trimeric NC1 domains of other α-chain compositions to investigate the α121, α345, and α565 collagen IV scaffolds and to develop therapies for managing Alport syndrome, Goodpasture's disease, and cancerous tumor growth.

Vastatin (the NC1 domain of human type VIII collagen a1 chain) is linked to stromal reactivity and elevated in serum from patients with colorectal cancer

ABSTRACTVastatin, a fragment derived from type VIII collagen, is one of the least studied collagen-derived matrikines. Vastatin can be detected in serum but little is known regarding the relevance of serum vastatin in colorectal cancer (CRC). In this study, serum vastatin was measured (ELISA) in 67 healthy controls and 48 CRC patients prior to resection and compared to clinicopathological parameters and serum biomarkers of stromal reactivity (C3M, VICM). Impact of resection and chemotherapy were evaluated by comparing baseline values with a 3-month follow-up sample (n = 23). Serum vastatin was detectable in 114 of 115 subjects. At baseline vastatin was elevated in CRC compared to controls (P < 0.001) with a diagnostic accuracy (AUROC) of 0.865, p < 0.0001. Vastatin correlated with age in controls but not in patients with CRC; no association was seen with clinicopathological parameters. Vastatin was independently associated with C3M (stepwise linear regression coefficient 0.25, p = 0.046). Overall, no difference was seen in vastatin levels between baseline and follow-up. In conclusion, vastatin is elevated in serum from patients with CRC and correlate with interstitial matrix degradation (C3M). This indicates that vastatin is linked to stromal reactivity and suggests that vastatin has biomarker potential in CRC. The association with clinicopathological parameters and treatment effect needs further evaluation.

Inhibitory Anti-Peroxidasin Antibodies in Pulmonary-Renal Syndromes.

Goodpasture syndrome (GP) is a pulmonary-renal syndrome characterized by autoantibodies directed against the NC1 domains of collagen IV in the glomerular and alveolar basement membranes. Exposure of the cryptic epitope is thought to occur via disruption of sulfilimine crosslinks in the NC1 domain that are formed by peroxidasin-dependent production of hypobromous acid. Peroxidasin, a heme peroxidase, has significant structural overlap with myeloperoxidase (MPO), and MPO-ANCA is present both before and at GP diagnosis in some patients. We determined whether autoantibodies directed against peroxidasin are also detected in GP. We used ELISA and competitive binding assays to assess the presence and specificity of autoantibodies in serum from patients with GP and healthy controls. Peroxidasin activity was fluorometrically measured in the presence of partially purified IgG from patients or controls. Clinical disease severity was gauged by Birmingham Vasculitis Activity Score. We detected anti-peroxidasin autoantibodies in the serum of patients with GP before and at clinical presentation. Enriched anti-peroxidasin antibodies inhibited peroxidasin-mediated hypobromous acid production in vitro. The anti-peroxidasin antibodies recognized peroxidasin but not soluble MPO. However, these antibodies did crossreact with MPO coated on the polystyrene plates used for ELISAs. Finally, peroxidasin-specific antibodies were also found in serum from patients with anti-MPO vasculitis and were associated with significantly more active clinical disease. Anti-peroxidasin antibodies, which would previously have been mischaracterized, are associated with pulmonary-renal syndromes, both before and during active disease, and may be involved in disease activity and pathogenesis in some patients.

Peroxidasin-mediated crosslinking of collagen IV is independent of NADPH oxidases

Collagen IV is a major component of the basement membrane in epithelial tissues. The NC1 domains of collagen IV protomers are covalently linked together through sulfilimine bonds, the formation of which is catalyzed by peroxidasin. Although hydrogen peroxide is essential for this reaction, the exact source of the oxidant remains elusive. Members of the NOX/DUOX NADPH oxidase family are specifically devoted to the production of superoxide and hydrogen peroxide. Our aim in this study was to find out if NADPH oxidases contribute in vivo to the formation of collagen IV sulfilimine crosslinks. We used multiple genetically modified in vivo model systems to provide a detailed assessment of this question. Our data indicate that in various peroxidasin-expressing tissues sulfilimine crosslinks between the NC1 domains of collagen IV can be readily detected in the absence of functioning NADPH oxidases. We also analyzed how subatmospheric oxygen levels influence the collagen IV network in collagen-producing cultured cells with rapid matrix turnover. We showed that collagen IV crosslinks remain intact even under strongly hypoxic conditions. Our hypothesis is that during collagen IV network formation PXDN cooperates with a NOX/DUOX-independent H2O2 source that is functional also at very low ambient oxygen levels.

Vancomycin Mediates IgA Autoreactivity in Drug-Induced Linear IgA Bullous Dermatosis

Vancomycin (VCM) is known to induce linear IgA bullous dermatosis (LAD). However, in contrast to conventional LAD, in which circulating IgA autoantibodies against basement membrane proteins are commonly detected, patient sera from VCM-induced LAD yields negative results in indirect immunofluorescence microscopy, and the targeted autoantigen remains undetermined. By using sera from a typical patient with VCM-induced LAD, we identified that co-incubation of sera with VCM resulted in linear IgA deposition at thebasement membrane zone by indirect immunofluorescence. Patient sera reacted with the dermal side of 1mol/L NaCl-split skin and with the recombinant noncollagenous (i.e., NC1) domain of type VII collagen by both immunoblot and ELISA in the presence of VCM. The investigation of an additional 13 patients with VCM-induced LAD showed that 10 out of the 14 sera (71.4%) reacted with the NC1 domain of type VII collagen by ELISA when spiked with VCM, whereas only 4 (28.6%) tested positive without it. The enhancement of reactivity to NC1 by VCM, as determined by optical density via ELISA, was observed in 10 out of the 14 sera (71.4%). These findings indicate that type VII collagen is a target autoantigen in VCM-induced LAD and that VCM mediates IgA autoreactivity against type VII collagen, providing an insight into mechanisms involved in drug-induced autoimmune disease.

Enhanced expression of Vastatin inhibits angiogenesis and prolongs survival in murine orthotopic glioblastoma model

BackgroundAntiangiogenic therapies are considered promising for the treatment of glioblastoma (GB). The non-collagenous C-terminal globular NC1 domain of type VIII collagen a1 chain, Vastatin, is an endogenous antiangiogenic polypeptide. Sustained enhanced expression of Vastatin was shown to inhibit tumour growth and metastasis in murine hepatocellular carcinoma models. In this study, we further explored the efficacy of Vastatin in the treatment of GB xenografts.MethodTreatment of Vastatin was carried out using a nanopolymer gene vector PEI600-CyD-Folate (H1). Antiangiogenic effect of Vastatin was tested in vitro by using co-culture system and conditioned medium. An orthotopic GB murine model was established to examine the in vivo therapeutic effect of Vastatin alone treatment and its combination with temozolomide.ResultsVastatin gene transfection mediated by H1 could target tumour cells specifically and suppress the proliferation of microvessel endothelial cells (MECs) through a paracrine inhibition manner. Enhancing Vastatin expression by intracerebral injection of H1-Vastatin significantly prolonged animal survival from 48 to 75 days in GB murine model, which was comparable to the effect of Endostatin, the most studied endogenous antiangiogenic polypeptide. The diminished presence of CD34 positive cells in the GB xenografts suggested that Vastatin induced significant antiangiogenesis. Moreover, a synergistic effect in extending survival was detected when H1-Vastatin was administered with temozolomide (TMZ) in GB chemoresistant murine models.ConclusionOur results suggest, for the first time, that Vastatin is an antiangiogenic polypeptide with significant potential therapeutic benefit for GB. H1-Vastatin gene therapy may have important implications in re-sensitizing recurrent GB to standard chemotherapeutic agents.

Lysyl Oxidase-like-2 Cross-links Collagen IV of Glomerular Basement Membrane

The 7S dodecamer is recognized as an important structural cross-linking domain of collagen IV networks that provide mechanical stability to basement membranes, a specialized form of extracellular matrix essential for the development and maintenance of tissue architecture. Although the 7S dodecamer is stabilized by covalent cross-linking, the molecular mechanism by which such cross-links are formed has not been revealed. Here, we aimed to identify the enzyme(s) that cross-links the 7S dodecamer and characterize its expression in the kidney glomerulus. Pharmacological inhibition of candidate extracellular matrix enzymes revealed that lysyl oxidase activity is required for cross-linking of 7S polypeptides. Among all lysyl oxidase family members, lysyl oxidase-like-2 (LOXL2) was identified as the isoform cross-linking collagen IV in mouse embryonal PFHR-9 cells. Biochemical analyses revealed that LOXL2 readily promoted the formation of lysyl-derived cross-links in the 7S dodecamer but not in the NC1 domain. We also established that LOXL2 is the main lysyl oxidase family member present in the glomerular extracellular matrix. Altogether, we demonstrate that LOXL2 is a novel component of the molecular machinery that forms cross-linked collagen IV networks, which are essential for glomerular basement membrane stability and molecular ultrafiltration function.

A type VII collagen subdomain mutant is thermolabile and shows enhanced proteolytic degradability — Implications for the pathogenesis of recessive dystrophic epidermolysis bullosa?

Type VII collagen is the major constituent of anchoring fibrils. It has a central collagenous domain that is surrounded by a small C-terminal non-collagenous domain (NC2) and a large N-terminal non-collagenous (NC1) domain. Mutations in type VII collagen can lead to hereditary skin blistering disease dystrophic epidermolysis bullosa (DEB). Most of the pathogenic missense mutations are within the collagenous domain. NC1 domain mediates interactions with other extracellular matrix molecules and only very few missense mutations within NC1 causing DEB have been reported. Interestingly, fibronectin III like (FNIII) domain 8 in the human protein can harbour different mutations at position 886 with one (R886P) leading to recessive DEB, whereas the others do not.We characterized subdomains of murine NC1, the FNIII domains 7–8, and the individual domains FNIII7 and FNIII8 by NMR- and CD-spectroscopy. We analysed the influence on stability for a mutation causing DEB and a non-pathogenic mutation. Whereas the silent mutation behaves as the wild type, the pathogenic mutation leads to a dramatic decrease in thermal stability of the FNIII8 domain. The melting temperature lowered from 77°C to 40°C compared to the wild type protein. This renders the domain susceptible to protease cleavage which could be shown by degradation tests with cathepsin G, cathepsin K, and MMP9. Our data show partial unfolding of type VII collagen due to the mutation causes an increased degradation. This could lead to skin blistering and opens new concomitant treatment options in some types of type VII collagen related skin blistering diseases.

Vastatin, an Endogenous Antiangiogenesis Polypeptide That Is Lost in Hepatocellular Carcinoma, Effectively Inhibits Tumor Metastasis

Hepatocellular carcinoma (HCC) is a hypervascular cancer without effective treatment. Here we report that polypeptide of NC1 domain of type VIII collagen (Vastatin) is an endogenous polypeptide expressed in normal liver tissue but lost in the liver of most HCC patients (73.1%). Its expression level is negatively associated with tumor size (P = 0.035) and metastasis (P = 0.016) in HCC patients. To evaluate its potential use as a therapeutic, we constructed a recombinant adeno-associated virus carrying Vastatin (rAAV-Vastatin) to treat HCC in an orthotopic Buffalo rat model. rAAV-Vastatin treatment significantly prolonged the median survival, inhibited tumor growth, and completely prevented metastasis in HCC-bearing rats by decreasing microvessel density and increasing tumor necrosis. No detectable toxicity in nontumor-bearing mice was observed. To investigate its molecular mechanisms, we performed DNA microarray, western blotting assays, and bioinformatic analysis to determine its effect on global gene expression patterns and signal transduction pathways. Our results indicated that rAAV-Vastatin significantly reduced the expressions of Pck1, JAG2, and c-Fos, thus inhibiting the cellular metabolism, Notch and AP-1 signaling pathways, respectively. Hence, we demonstrated for the first time that Vastatin is a novel, safe, and effective antiangiogenic therapeutic and a potential biomarker for HCC.

Atypical anti-glomerular basement membrane disease: lessons learned

Anti-glomerular basement membrane (GBM) disease usually pursues a self-limited course, at least from the immunological perspective. In addition, circulating antibodies to cryptic, conformational epitopes within the NC1 domain of the alpha 3 chain of Type IV Collagen are commonly found at the zenith of the clinical disease. However, exceptions to these general rules do occur, as exemplified by two remarkable cases reported in this issue of the Clinical Kidney Journal. The possible explanations for and the lessons learned from these uncommon occurrences are discussed in this short commentary.

Analysis of the functional consequences of targeted exon deletion in COL7A1 reveals prospects for dystrophic epidermolysis bullosa therapy.

Genetically evoked deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB)-a debilitating disease characterized by chronic skin fragility and progressive fibrosis. Removal of exons carrying frame-disrupting mutations can reinstate protein expression in genetic diseases. The therapeutic potential of this approach is critically dependent on gene, protein, and disease intrinsic factors. Naturally occurring exon skipping in COL7A1, translating collagen VII, suggests that skipping of exons containing disease-causing mutations may be feasible for the treatment of DEB. However, despite a primarily in-frame arrangement of exons in the COL7A1 gene, no general conclusion of the aptitude of exon skipping for DEB can be drawn, since regulation of collagen VII functionality is complex involving folding, intra- and intermolecular interactions. To directly address this, we deleted two conceptually important exons located at both ends of COL7A1, exon 13, containing recurrent mutations, and exon 105, predicted to impact folding. The resulting recombinantly expressed proteins showed conserved functionality in biochemical and in vitro assays. Injected into DEB mice, the proteins promoted skin stability. By demonstrating functionality of internally deleted collagen VII variants, our study provides support of targeted exon deletion or skipping as a potential therapy to treat a large number of individuals with DEB.

A novel COL4A1 frameshift mutation in familial kidney disease: the importance of the C-terminal NC1 domain of type IV collagen.

BackgroundHereditary microscopic haematuria often segregates with mutations of COL4A3, COL4A4 or COL4A5 but in half of families a gene is not identified. We investigated a Cypriot family with autosomal dominant microscopic haematuria with renal failure and kidney cysts.MethodsWe used genome-wide linkage analysis, whole exome sequencing and cosegregation analyses.ResultsWe identified a novel frameshift mutation, c.4611_4612insG:p.T1537fs, in exon 49 of COL4A1. This mutation predicts truncation of the protein with disruption of the C-terminal part of the NC1 domain. We confirmed its presence in 20 family members, 17 with confirmed haematuria, 5 of whom also had stage 4 or 5 chronic kidney disease. Eleven family members exhibited kidney cysts (55% of those with the mutation), but muscle cramps or cerebral aneurysms were not observed and serum creatine kinase was normal in all individuals tested.ConclusionsMissense mutations of COL4A1 that encode the CB3 [IV] segment of the triple helical domain (exons 24 and 25) are associated with HANAC syndrome (hereditary angiopathy, nephropathy, aneurysms and cramps). Missense mutations of COL4A1 that disrupt the NC1 domain are associated with antenatal cerebral haemorrhage and porencephaly, but not kidney disease. Our findings extend the spectrum of COL4A1 mutations linked with renal disease and demonstrate that the highly conserved C-terminal part of the NC1 domain of the α1 chain of type IV collagen is important in the integrity of glomerular basement membrane in humans.

A Japanese familial case of Schmid metaphyseal chondrodysplasia with a novel mutation in &lt;i&gt;COL10A1&lt;/i&gt;

Schmid metaphyseal chondrodysplasia (SMCD; OMIM #156500) is an autosomal dominant skeletal dysplasia, characterized by short stature with short legs, bowing of the long bones, and waddling gait. Radiographic features include metaphyseal irregularities with fraying and splaying, and coxa vara, which is defined as a deformity of the hip, wherein the angle between the head and the shaft of the femur is reduced to less than 120 degrees (1). SMCD is caused by heterozygous mutations in COL10A1 (MIM #120110), encoding the α1(X) chains of type X collagen, which is a homotrimeric molecule that consists of three identical α1(X) chains. Each α1(X) chain contains a core triple helical domain, composed of uninterrupted repeats of the Gly-Xaa-Yaa tripeptide, flanked by two globular domains (NC2 and NC1) at both the amino and carboxyl-terminal ends (2). To date, 48 COL10A1 mutations have been reported in families with SMCD of different ethnic origin (Human Gene Mutation Database; http://www.hgmd.cf.ac.uk/ac). Most COL10A1 mutations identified to date were located in the NC1 domain, which contains motifs that promote trimerization of α1(X) chains and subsequent formation of the triple helix to yield stable collagen X molecules. Here, we report a Japanese familial case of SMCD with a novel mutation in COL10A1.