New molecular mechanism for Ullrich congenital muscular dystrophy: a heterozygous in-frame deletion in the COL6A1 gene causes a severe phenotype.
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Recessive mutations in two of the three collagen VI genes, COL6A2 and COL6A3, have recently been shown to cause Ullrich congenital muscular dystrophy (UCMD), a frequently severe disorder characterized by congenital muscle weakness with joint contractures and coexisting distal joint hyperlaxity. Dominant mutations in all three collagen VI genes had previously been associated with the considerably milder Bethlem myopathy. Here we report that a de novo heterozygous deletion of the COL6A1 gene can also result in a severe phenotype of classical UCMD precluding ambulation. The internal gene deletion occurs near a minisatellite DNA sequence in intron 8 that removes 1.1 kb of genomic DNA encompassing exons 9 and 10. The resulting mutant chain contains a 33-amino acid deletion near the amino-terminus of the triple-helical domain but preserves a unique cysteine in the triple-helical domain important for dimer formation prior to secretion. Thus, dimer formation and secretion of abnormal tetramers can occur and exert a strong dominant negative effect on microfibrillar assembly, leading to a loss of normal localization of collagen VI in the basement membrane surrounding muscle fibers. Consistent with this mechanism was our analysis of a patient with a much milder phenotype, in whom we identified a previously described Bethlem myopathy heterozygous in-frame deletion of 18 amino acids somewhat downstream in the triple-helical domain, a result of exon 14 skipping in the COL6A1 gene. This deletion removes the crucial cysteine, so that dimer formation cannot occur and the abnormal molecule is not secreted, preventing the strong dominant negative effect. Our studies provide a biochemical insight into genotype-phenotype correlations in this group of disorders and establish that UCMD can be caused by dominantly acting mutations. (+info)
Genomewide linkage and linkage disequilibrium analyses identify COL6A1, on chromosome 21, as the locus for ossification of the posterior longitudinal ligament of the spine.
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Ossification of the posterior longitudinal ligament (OPLL) of the spine is a subset of "bone-forming" diseases, characterized by ectopic ossification in the spinal ligaments. OPLL is a common disorder among elderly populations in eastern Asia and is the leading cause of spinal myelopathy in Japan. We performed a genomewide linkage study with 142 affected sib pairs, to identify genetic loci related to OPLL. In multipoint linkage analysis using GENEHUNTER-PLUS, evidence of linkage to OPLL was detected on chromosomes 1p, 6p, 11q, 14q, 16q, and 21q. The best evidence of linkage was detected near D21S1903 on chromosome 21q22.3 (maximum Zlr=3.97); therefore, the linkage region was extensively investigated for linkage disequilibrium with single-nucleotide polymorphisms (SNPs) covering 20 Mb. One hundred fifty positional candidate genes lie in the region, and 600 gene-based SNPs were genotyped. There were positive allelic associations with seven genes (P<.01) in 280 patients and 210 controls, and four of the seven genes were clustered within a region of 750 kb, approximately 1.2 Mb telomeric to D21S1903. Extensive linkage disequilibrium and association studies of the four genes indicated that SNPs in the collagen 6A1 gene (COL6A1) were strongly associated with OPLL (P=.000003 for the SNP in intron 32 [-29]). Haplotype analysis with three SNPs in COL6A1 gave a single-point P value of.0000007. Identification of the locus of susceptibility to OPLL by genomewide linkage and linkage disequilibrium studies permits us to investigate the pathogenesis of the disease, which may lead to the development of novel therapeutic tools. (+info)
Within tendon, between collagen fascicles, cells are organized in linear arrays surrounded by a specialized environment of extracellular matrix (ECM) proteins that are largely unidentified. Our goal was to identify interfascicular, pericellular ECM components and provide additional resolution to the organization of the pericellular matrix. To this end, we employed a combination of enzymatic digestion, mechanical disruption, and differential sedimentation to demonstrate for the first time that it possible to liberate living linear tendon cell arrays from whole tendon. Here, we identify type VI collagen, versican, and fibrillin-2 as components of the immediate pericellular ECM of linearly arrayed tendon cells. Additionally, a unique fibrillin-2-containing macromolecular assembly is described in detail for the first time. This new structure is unlike any previously described fibrillin-containing macromolecular assembly. Having a largely constant diameter, it runs axially along tendon cell arrays and can exceed 1000 microm in length. (+info)
Collagen type VI expression during cardiac development and in human fetuses with trisomy 21.
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The role played by specific extracellular matrix molecules in normal endocardial cushion differentiation into valves and septa remains to be established. In this respect, type collagen VI is of particular interest because genes encoding the alpha1 and alpha2 chains are located on chromosome 21, and defects involving the atrioventricular (AV) cushions are frequent in trisomy 21. Collagen VI expression was studied in normal human embryonic and fetal hearts (5-18 weeks of development) and compared by immunohistochemistry with results from fetuses (10-16 weeks of development) with trisomy 21. During normal endocardial cushion differentiation (5-8 weeks) there was marked collagen VI expression in the AV cushions, whereas only minor expression was seen in the outflow tract cushions. In the normal fetuses (10-18 weeks), collagen VI in the AV cushions had condensed into a marked zone on the atrial side of the leaflets, as well as subendocardially in other regions of high shear stress. Morphological defects involving the endocardial cushion-derived structures were present in all trisomy 21 cases. An abnormally large membranous septum was observed in three cases. An AV septal defect (AVSD) was present in two, while one had a ventricular septal defect (VSD). Two cases presented with a secondary atrial septal defect (ASDII), and one had an AVSD. Mild to moderate valve dysmorphia was found in all cases. Collagen VI staining in trisomy 21 was more intense than in the normal subjects; however, there were no differences in the spatial expression patterns. We conclude that collagen VI is expressed in the AV cushions and persists during valve differentiation. Collagen VI is more prominent in fetal trisomy 21 hearts than in normal hearts. We hypothesise that collagen VI has a role in the development of heart defects involving endocardial cushion differentiation-specifically in the AV canal, the most common site of malformations affecting children with trisomy 21. (+info)
TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI).
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Tumor endothelial marker (TEM)8 was uncovered as a gene expressed predominantly in tumor endothelium, and its protein product was recently identified as the receptor for anthrax toxin. Here, we demonstrate that TEM8 protein is preferentially expressed in endothelial cells of neoplastic tissue. We used the extracellular domain of TEM8 to search for ligands and identified the alpha 3 subunit of collagen VI as an interacting partner. The TEM8-interacting region on collagen alpha 3(VI) was mapped to its COOH-terminal C5 domain. Remarkably, collagen alpha 3(VI) is also preferentially expressed in tumor endothelium in a pattern concordant with that of TEM8. These results suggest that the TEM8/C5 interaction may play an important biological role in tumor angiogenesis. (+info)
Transient adenoviral gene transfer of Smad7 prevents injury-induced epithelial-mesenchymal transition of lens epithelium in mice.
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We examined the effect of adenovirus-mediated transient expression of Smad7, an inhibitory Smad in TGFbeta/activin signaling, on injury-induced epithelial-mesenchymal transition (EMT) of lens epithelium in mice. A volume of 3 microl of adenoviral solution was injected into the right lens of adult male C57BL/6 mice (n=56) at the time of capsular injury made using a hypodermic needle under general anesthesia. A mixture of recombinant adenovirus carrying CAG promoter-driven Cre (Cre adv) and mouse Smad7 complementary DNA (Smad7 adv) was administered to induce Smad7 expression, while control lenses were treated with Cre adv alone. After healing intervals of 2, 3, 5, and 10 days, animals were killed 2 h after labeling with bromodeoxyuridine (BrdU) and eyes were processed for histology. During healing, marked expression of Smad7 was observed in lens epithelial cells in the Smad7 adv group with loss of nuclear translocation of Smads2/3, while little Smad7 and abundant nuclear Smads2/3 were seen in cells in the Cre adv group. Lens epithelial cells in the Cre adv control group exhibited a fibroblastic appearance at days 5 and 10 and the capsular break was sealed with fibrous tissue, while Smad7 adv-treated cells around the capsular break retained their epithelial morphology and the break was not sealed. Expression of snail mRNA, and alpha-smooth muscle actin, lumican, and collagen VI proteins, markers of EMT, was observed in control-treated eyes, but not in cells of the Smad7 adv group at day 5 with minimal expression at day 10. Additionally, cell proliferation increased in epithelium infected with Smad7 adv consistent with suppression of injury-induced upregulation of TGFbeta1 in epithelium. We conclude that gene transfer of Smad7 in mice prevents injury-induced EMT of lens epithelial cells and sealing of the capsular break with fibrous tissue. (+info)
Smad3 is required for dedifferentiation of retinal pigment epithelium following retinal detachment in mice.
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Retinal pigment epithelial (RPE) cells dedifferentiate and undergo epithelial-mesenchymal transition (EMT) following retinal detachment, playing a central role in formation of fibrous tissue on the detached retina and vitreous retraction (proliferative vitreoretinopathy (PVR)). We have developed a mouse model of subretinal fibrosis with implications for PVR in which retinal detachment is induced without direct damage to the RPE cells. Transforming growth factor-beta (TGF-beta) has long been implicated both in EMT of RPEs and the development of PVR. Using mice null for Smad3, a key signaling intermediate downstream of TGF-beta and activin receptors, we show that Smad3 is essential for EMT of RPE cells induced by retinal detachment. De novo accumulation of fibrous tissue derived from multilayered RPE cells was seen following experimental retinal detachment in eyes of wild type, but not Smad3-null mice. Expression of alpha-smooth muscle actin, a hallmark of EMT in this cell type, and extracellular matrix components, lumican and collagen VI, were also not observed in eyes of Smad3-null mice. Our data show that induction of PDGF-BB by Smad3-dependent TGF-beta signaling is likely an important secondary proliferative component of the disease process. The results suggest that blocking the Smad3 pathway might be beneficial in prevention/treatment of PVR. (+info)
Differential interactions of decorin and decorin mutants with type I and type VI collagens.
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The small leucine-rich proteoglycan decorin can bind via its core protein to different types of collagens such as type I and type VI. To test whether decorin can act as a bridging molecule between these collagens, the binding properties of wild-type decorin, two full-length decorin species with single amino acid substitutions (DCN E180K, DCN E180Q), which previously showed reduced binding to collagen type I fibrils, and a truncated form of decorin (DCN Q153) to the these collagens were investigated. In a solid phase assay dissociation constants for wild-type decorin bound to methylated, therefore monomeric, triple helical type I collagen were in the order of 10(-10) m, while dissociation constants for fibrillar type I collagen were approximately 10(-9) m. The dissociation constant for type VI was approximately 10(-7) m. Using real-time analysis for a more detailed investigation DCN E180Q and DCN E180K exhibited lower association and higher dissociation constants to type I collagen, compared to wild-type decorin, deviating by at least one order of magnitude. In contrast, the affinities of these mutants to type VI collagen were 10 times higher than the affinity of wild-type decorin (K(D) approximately 10(-8) m). Further investigations verified that complexes of type VI collagen and decorin bound type I collagen and that the affinity of collagen type VI to type I was increased by the presence of decorin. These data show that decorin not only can regulate collagen fibril formation but that it also can act as an intermediary between type I and type VI collagen and that these two types of collagen interact via different binding sites. (+info)